US20190194682A1

US20190194682A1 - Yield improvement in plants

Info

Publication number: US20190194682A1
Application number: US16/299,232
Authority: US
Inventors: Graham J. Hymus; T. Lynne Reuber; Colleen M. Marion; Oliver J. Ratcliffe; Jeffrey M. Libby
Original assignee: Koch Biological Solutions LLC
Current assignee: Plant Response Inc
Priority date: 2013-10-14
Filing date: 2019-03-12
Publication date: 2019-06-27
Also published as: US20190010511A1; WO2015057571A2; US10266841B2; US20160333369A1; WO2015057571A3; US10113177B2

Abstract

Polynucleotides and polypeptides incorporated into expression vectors are introduced into plants and were ectopically expressed. These polypeptides may confer at least one regulatory activity and increased yield, increased light use efficiency, increased photosynthetic capacity, increased photosynthetic rate, increased photosynthetic resource use efficiency, greater vigor, and/or greater biomass as compared to a control plant.

Description

FIELD OF THE INVENTION

The present invention relates to plant genomics and plant improvement.

BACKGROUND OF THE INVENTION

A plant's phenotypic characteristics that enhance photosynthetic resource use efficiency may be controlled through a number of cellular processes. One important way to manipulate that control is by manipulating the characteristics or expression of regulatory proteins, proteins that influence the expression of a particular gene or sets of genes. For example, transformed or transgenic plants that comprise cells with altered levels of at least one selected regulatory polypeptide may possess advantageous or desirable traits, and strategies for manipulating traits by altering a plant cell's regulatory polypeptide content or expression level can result in plants and crops with commercially valuable properties. Examples of such trait manipulation include:
Increasing Canopy Photosynthesis to Increase Crop Yield.
Recent studies by crop physiologists have provided evidence that crop-canopy photosynthesis is correlated with crop yield, and that increasing canopy photosynthesis can increase crop yield (Long et al., 2006. Plant Cell Environ. 29:315-33; Murchie et al., 2009 New Phytol. 181:532-552; Zhu et al., 2010. Ann. Rev. Plant Biol. 61:235-261). Two overlapping strategies for increasing canopy photosynthesis have been proposed. The first recognizes great potential to increase canopy photosynthesis by improving multiple discrete reactions that currently limit photosynthetic capacity (reviewed in Zhu et al., 2010. supra). The second focuses upon improving plant physiological status during environmental conditions that limit the realization of photosynthetic capacity. It is important to distinguish this second goal from recent industry and academic screening for genes to improve stress tolerance. Arguably, these efforts may have identified genes that improve plant physiological status during severe stresses not typically experienced on productive acres (Jones, 2007. J. Exp. Bot. 58:119-130; Passioura, 2007. J. Exp. Bot. 58:113-117). In contrast, improving the efficiency with which photosynthesis operates relative to the availability of key resources of water, nitrogen and light, is thought to be more appropriate for improving yield on productive acres (Long et al., 1994. Ann. Rev. Plant Physiol. Plant Molec. Biol. 45:633-662; Morison et al., 2008. Philosophical Transactions of the Royal Society B: Biological Sciences 363:639-658; Passioura, 2007, supra).
Increasing Nitrogen Use Efficiency (NUE) to Increase Crop Yield.
There has been a large increase in food productivity over the past 50 years causing a decrease in world hunger despite a significant increase in population (Godfray et al., 2010. Science 327:812-818). A significant contribution to this increased yield was a 20-fold increase in the application of nitrogen fertilizers (Glass, 2003. Crit. Rev. Plant Sci. 22:453-470). About 85 million to 90 million metric tons of nitrogen are applied annually to soil, and this application rate is expected to increase to 240 million metric tons by 2050 (Good et al., 2004. Trends Plant Sci. 9:597-605). However, plants use only 30 to 40% of the applied nitrogen and the rest is lost through a combination of leaching, surface run-off, denitrification, volatilization, and microbial consumption (Frink et al., 1999. Proc. Natl. Acad. Sci. USA 96:1175-1180; Glass, 2003, supra; Good et al., 2004, supra; Raun and Johnson, 1999. Agron. J. 91:357-363). The loss of more than 60% of applied nitrogen can have serious environmental effects, such as groundwater contamination, anoxic coastal zones, and conversion to greenhouse gases. In addition, while most fertilizer components are mined (such as phosphates), inorganic nitrogen is derived from the energy intensive conversion of gaseous nitrogen to ammonia. Thus, the addition of nitrogen fertilizer is typically the highest single input cost for many crops, and since its production is energy intensive, the cost is dependent on the price of energy (Rothstein, 2007. Plant Cell 19:2695-2699). With an increasing demand for food from an increasing human population, agriculture yields must be increased at the same time as dependence on applied fertilizers is decreased. Therefore, to minimize nitrogen loss, reduce environmental pollution, and decrease input cost, it is crucial to develop crop varieties with higher nitrogen use efficiency (Garnett et al., 2009. Plant Cell Environ. 32:1272-1283; Hirel et al., 2007. J. Exp. Bot. 58:2369-2387; Lea and Azevedo, 2007. Ann. Appl. Biol. 151:269-275; Masclaux-Daubresse et al., 2010. Ann. Bot. 105:1141-1157; Moll et al., 1982. Agron. J. 74:562-564; Sylvester-Bradley and Kindred, 2009. J. Exp. Bot. 60:1939-1951).
Improving Water Use Efficiency (WUE) to Improve Yield.
Freshwater is a limited and dwindling global resource; therefore, improving the efficiency with which food and biofuel crops use water is a prerequisite for maintaining and improving yield (Karaba et al., 2007. Proc. Natl. Acad. Sci. USA. 104:15270-15275). WUE can be used to describe the relationship between water use and crop productivity over a range of time integrals. The basic physiological definition of WUE equates the ratio of photosynthesis (A) to transpiration (T) at a given moment in time, also referred to as transpiration efficiency. However, the WUE concept can be scaled significantly, for example, over the complete lifecycle of a crop, where biomass or yield can be expressed per cumulative total of water transpired from the canopy. Thus far, the engineering of major field crops for improved WUE with single genes has not yet been achieved (Karaba et al., 2007. supra). Regardless, increased yields of wheat cultivars bred for increased transpiration efficiency (the ratio of photosynthesis to transpiration) have provided important support for the proposition that crop yield can be increased over broad acres through improvement in crop water-use efficiency (Condon et al., 2004. J. Exp. Bot. 55:2447-2460).
Estimates of water-use efficiency integrated over the life of plant tissues can be derived from analysis of the ratio of the ¹³C carbon isotope to the ¹²C carbon isotope in those tissues. The theory that underlies this means to estimating WUE is that during photosynthesis, incorporation of ¹³C into the products of photosynthesis is slower than the lighter isotope ¹²C. Effectively, ¹³C is discriminated against relative to ¹²C during photosynthesis, an effect that is integrated over the life of the plant resulting in biomass with a distinct ¹³C/¹²C signature. Of the many steps in the photosynthetic process during which this discrimination occurs, discrimination at the active site of Rubisco is of most significance, a consequence of kinetic constraints associated with the ¹³CO₂molecule being larger. Significantly, the discrimination by Rubisco is not constant, but varies depending on the CO₂concentration within the leaf. At high CO₂concentration discrimination by Rubisco is highest, however as CO₂concentration decreases discrimination decreases. Because the CO₂concentration within the leaf is overwhelmingly dependent on the balance between CO₂influx through the stomatal pore and the rate of photosynthesis, and because the stomatal pore controls the rate of transpiration from the leaf, the ¹³C/¹²C isotopic signature of plant material provides an integrated record of the balance between transpiration and photosynthesis during the life of the plant and as such a surrogate measure of water-use efficiency (Farquhar et al. 1989. Annu. Rev. Plant Physiol. Plant Mol. Biol. 40:503-537).
With these needs in mind, new technologies for yield enhancement are required. In this disclosure, a phenotypic screening platform that directly measures photosynthetic capacity, water use efficiency, and nitrogen use efficiency of mature plants was used to discover advantageous properties conferred by ectopic expression of the described regulatory proteins in plants.

SUMMARY

The instant description is directed to a transgenic plant or plants that have increased photosynthetic resource use efficiency with respect to a control plant, or a plant part derived from such a plant, e.g., shoot vegetative organs/structures (e.g., leaves, stems and tubers), roots, flowers and floral organs/structures (e.g., bracts, sepals, petals, stamens, carpels, anthers and ovules), seed (including embryo, endosperm, and seed coat) and fruit (the mature ovary), plant tissue (e.g., vascular tissue, ground tissue, and the like), pulped, pureed, ground-up, macerated or broken-up tissue, and cells (e.g., guard cells, egg cells, etc.)). In this regard, the transgenic plant or plants comprise a recombinant polynucleotide comprising a promoter of interest. The choice of promoter may include a constitutive promoter or a promoter with enhanced activity in a tissue capable of photosynthesis (also referred to herein as a “photosynthetic promoter” or a “photosynthetic tissue-enhanced promoter”) such as a leaf tissue or other green tissue. Examples of photosynthetic promoters include for example, an RBCS3 promoter, an RBCS4 promoter or others such as the At4g01060 (also referred to as “G682”) promoter, the latter regulating expression in a guard cell. The promoter regulates a polypeptide that is encoded by the recombinant polynucleotide or by a second (or target) recombinant polynucleotide (in which case expression of the polypeptide may be regulated by a trans-regulatory element). The promoter may also regulate expression of a polypeptide to an effective level of expression in a photosynthetic tissue, that is, to a level that, as a result of expression of the polypeptide to that level, improves photosynthetic resource use efficiency in a transgenic plant relative to a control plant. The recombinant polynucleotide may comprise the promoter and also encode the polypeptide or alternatively, the polynucleotide may comprise the promoter and drive expression of the polypeptide that is encoded by the second recombinant polynucleotide. In an exemplary embodiment, the polypeptide comprises a sequence listed in the sequence listing, or a sequence that is homologous, paralogous or orthologous to said polypeptide, being structurally-related to said polypeptide and having a function similar to said polypeptide as described herein. Expression of the polypeptide under the regulatory control of the constitutive or leaf-enhanced or photosynthetic tissue-enhanced promoter in the transgenic plant confers greater photosynthetic resource use efficiency to the transgenic plants, and may ultimately increase yield that may be obtained from the plants.
The instant description also pertains to methods for increasing photosynthetic resource use efficiency in, or increasing yield from, a plant or plants including the method conducted by growing a transgenic plant comprising and/or transformed with an expression cassette comprising the recombinant polynucleotide that comprises a constitutive promoter or a promoter expressed in photosynthetic tissue, which may be a leaf-enhanced or green tissue-enhanced promoter, such as for example, the RBCS3, RBCS4, At4g01060, or another photosynthetic tissue-enhanced promoter. Examples of photosynthetic tissue-enhanced promoters are found in the Sequence Listing or in Table 22. The promoter regulates expression of a polypeptide that comprises a polypeptide listed in the Sequence Listing. Recombinant polynucleotides encoding these clade polypeptides are described in the following paragraphs (a)-(c), and exemplary polypeptides within the clade are described in the following paragraphs (d)-(f) and are shown in the instant sequence alignments and Figures.
The recombinant polynucleotide that is introduced into a transgenic plant may encode a listed polypeptide sequence or encodes a polypeptide that is phylogenetically-related to a listed polypeptide sequence, including sequences that include:
(a) nucleic acid sequences that are at least 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95% or 96%, 97%, 98%, 99%, or about 100% identical to any of the listed polypeptides; and/or
(b) nucleic acid sequences that encode polypeptide sequences that are at least 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95% or 96%, 97%, 98%, 99%, or about 100% identical in their amino acid sequences to the entire length to any of the listed polypeptides; and/or
(c) nucleic acid sequences that hybridize under stringent conditions (e.g., hybridization followed by one, by two, or by more than two wash steps of 6× saline-sodium citrate buffer (SSC) and 65° C. for ten to thirty minutes per step) to any of the listed polynucleotides.
The listed polypeptides and polypeptides member of their protein clade may include:
(d) polypeptide sequences encoded by the nucleic acid sequences of (a), (b) and/or (c); and/or
(e) polypeptide sequences that have at least 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65f %, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95% or 96%, 97%, 98%, 99%, or about 100% amino acid identity to any of the listed polypeptides, including SEQ ID NO: 2n, where n=1 to 241 (i.e., even integers 2, 4, 6, 8, . . . 482);
and/or at least 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65f %, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95% or 96%, 97%, 98%, 99%, or about 100 amino acid identity to a conserved domain of any of the listed polypeptides, including SEQ ID NO: 483 to 841; and/or
(f) polypeptide sequences that comprise a subsequence that are at least 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95% or 96%, 97%, 98%, 99%, or about 100% identical to any of the consensus sequences provided in the Sequence Listing, including SEQ ID NO: 842 to 861.
Expression of these polypeptides in the transgenic plant may confer increased photosynthetic resource use efficiency relative to a control plant. The transgenic plant may be selected for increased photosynthetic resource use efficiency or greater yield relative to the control plant. The transgenic plant may also be crossed with itself, a second plant from the same line as the transgenic plant, a non-transgenic plant, a wild-type plant, or a transgenic plant from a different line of plants, to produce a transgenic seed.
The instant description also pertains to methods for producing and selecting a crop plant with a greater yield than a control plant, the method comprising producing a transgenic plant by introducing into a target plant a recombinant polynucleotide that comprises a promoter, such as a leaf- or photosynthetic tissue-enhanced promoter that regulates a polypeptide encoded by the recombinant polynucleotide or a second recombinant polynucleotide, wherein the polypeptide comprises a polypeptide listed in the Sequence Listing, or a member of a clades of polypeptides phylogenetically related to a polypeptide listed in the Sequence Listing. A plurality of the transgenic plants is then grown, and a transgenic plant is selected that produces greater yield or has greater photosynthetic resource use efficiency than a control plant. The expression of the polypeptide in the selected transgenic plant confers the greater photosynthetic resource use efficiency and/or greater yield relative to the control plant. Optionally, the selected transgenic plant may be crossed with itself, a second plant from the same line as the transgenic plant, a non-transgenic plant, a wild-type plant, or a transgenic plant from a different line of plants, to produce a transgenic seed. A plurality of the selected transgenic plants will generally have greater cumulative canopy photosynthesis than the canopy photosynthesis of an identical number of the control plants.
The transgenic plant(s) described herein and produced by the instantly described methods may also possess one or more altered traits that result in greater photosynthetic resource use efficiency. The altered trait may include: increased photosynthetic capacity, increased photosynthetic rate, a decrease in leaf chlorophyll content, a decrease in percentage of nitrogen in leaf dry weight, increased leaf transpiration efficiency, an increase in resistance to water vapor diffusion from the leaf exerted by stomata, an increased rate of relaxation of photoprotective reactions operating in the light harvesting antennae, a decrease in the ratio of the carbon isotope ¹²C to ¹³C in above-ground biomass, and/or an increase in the total dry weight of above-ground plant material.
At least one advantage of greater photosynthetic resource use efficiency is that the transgenic plant, or a plurality of the transgenic plants, will have greater cumulative canopy photosynthesis than the canopy photosynthesis of an identical number of the control plants, or produce greater yield than an identical number of the control plants. A wide variety of transgenic plants are envisioned, including corn, wheat, rice, Setaria, Miscanthus, switchgrass, ryegrass, sugarcane, miscane, barley, sorghum, turfgrass, soy, cotton, canola, rapeseed, Crambe, Camelina, sugar beet, alfalfa, tomato, Eucalyptus, poplar, willow, pine, birch and other woody plants.
The instant description also pertains to expression vectors that comprise a recombinant polynucleotide that comprises a promoter expressed in photosynthetic tissue, for example, a constitutive promoter, or a leaf- or green tissue-enhanced promoter including the RBCS3, RBCS4, or At4g01060 promoters, or another photosynthetic tissue-enhanced promoter, for example, such a promoter found in the Sequence Listing or in Table 22, and a subsequence that encodes a polypeptide comprising a polypeptide sequence provided in the Sequence Listing or a member of the polypeptide clades of the polypeptide sequences listed in the Sequence Listing, or, alternatively, two expression constructs, one of which encodes a promoter such as a constitutive promoter, or a leaf-enhanced promoter or other photosynthetic tissue-enhanced promoter, and the second encodes a polypeptide sequence provided in the Sequence Listing or a member of the polypeptide clades of the polypeptide sequences listed in the Sequence Listing. In either instance, whether the polypeptide is encoded by the first or second expression constructs, the promoter regulates expression of the polypeptide by being responsible for production of cis- or trans-regulatory elements, respectively. In some embodiments, the expression vectors or cassettes comprise a promoter of the present application, and a gene of interest, wherein the promoter and the gene of interest do not link to each other under natural conditions, e.g., the linkage between the promoter and the gene of interest does not exist in nature.
The instant description is also directed to a method for producing a monocot plant with increased grain yield by providing a monocot plant cell or plant tissue with stably integrated, exogenous, recombinant polynucleotide comprising a promoter (for example, a constitutive, a non-constitutive, an inducible, a tissue-enhanced, or a photosynthetic tissue-enhanced promoter) that is functional in plant cells and that is operably linked to an exogenous or an endogenous nucleic acid sequence that encodes a listed polypeptide, that is expressed in a photosynthetic tissue of the transgenic plant to a level effective in conferring greater photosynthetic resource use efficiency relative to a control plant that does not contain the recombinant polynucleotide. A plant is generated from the plant cell or the plant tissue that comprises the recombinant polynucleotide, the plant is then grown and an increase in photosynthetic resource use efficiency or grain yield is measured relative to the control plant.
In the above paragraphs, the control plant may be exemplified by a plant of the same species as the plant comprising the recombinant polynucleotide, but the control plant does not comprise the recombinant polynucleotide that encodes a listed polypeptide of interest.

BRIEF DESCRIPTION OF THE SEQUENCE LISTING AND DRAWINGS

The Sequence Listing provides exemplary polynucleotide and polypeptide sequences of the instant description. The traits associated with the use of the sequences are included in the Examples.

Incorporation of the Sequence Listing.

The Sequence Listing provides exemplary polynucleotide and polypeptide sequences. The copy of the Sequence Listing being submitted electronically with this patent application under 37 CFR § 1.821-1.825, is a read-only memory computer-readable file in ASCII text format. The Sequence Listing is named “MPS-0216P_ST25.txt”, the electronic file of the Sequence Listing was created on Oct. 2, 2013, and is (1,651,577 bytes in size (157 megabytes in size as measured in MS-WINDOWS). The Sequence Listing is herein incorporated by reference in its entirety.

In FIG. 1, a phylogenetic tree of ATMYB27 or AT3G53200 (also referred to as G1311) clade members and related full length proteins were constructed using TreeBeST (Ruan et al., 2008. Nucleic Acids Res. 36 (suppl. 1): D735-D740) using the best command to identify the best tree from maximum likelihood and neighbor joining methods. ATMYB27 (AT3G53200.1) appears in the rounded rectangle. An ancestral sequence of ATMYB27 and closely-related sequences is represented by the node of the tree indicated by the arrow “A” in FIG. 1. ATMYB27 clade members are considered those proteins that descended from ancestral sequence “A”, including the exemplary sequences shown in this figure that are bounded by AT3G53200.1 and GSVIVT01033670001_VITVI.

FIGS. 2A-2D show an alignment of ATMYB27 and representative clade-related proteins. The alignment was generated with MUSCLE (3.8) with default parameters. SEQ ID NOs: appear in parentheses after each Gene Identifier (GID). The conserved first and second Myb domains appear in boxes in FIGS. 2A-2B and FIG. 2B, respectively (for which the consensus sequences are SEQ ID NOs 842 and 843).

FIG. 3: Plot showing increased light saturated photosynthesis (A_sat) over a range of leaf sub-stomatal CO₂concentration (C_i) in three ATMYB27 overexpression lines, compared to a control line. Data was collected over a range of C_iover which the activity of Rubisco is known to limit A_sat. The solid line shown is a regression fitted to the data for the control line only. All data are the means±1 standard error for data collected on at least six replicate plants for each line.

Legend for FIG. 3:

Control

□ Line 1

Δ Line 2

X Line 4

⋄ Line 5

◯ Line 6

FIG. 4: Plot showing increased light saturated photosynthesis (A_sat) over a range of leaf sub-stomatal CO₂concentration (C_i) in four ATMYB27 overexpression lines, compared to a control line. Data was collected over a range of C_iover which the capacity to regenerate RuBP is known to limit A_sat. The solid line shown is a regression fitted to the data for the control line only. All data are the means±1 standard error for data collected on at least six replicate plants for each line.

Legend for FIG. 4:

Control

□ Line 1

Δ Line 2

X Line 4

⋄ Line 5

◯ Line 6

In FIGS. 5A and 5B, a phylogenetic tree of RPB45A or AT5G54900 (also referred to as G1940) clade members and related full length proteins were constructed using TreeBeST (Ruan et al., 2008. Nucleic Acids Res. 36 (suppl. 1): D735-D740) using the best command to identify the best tree from maximum likelihood and neighbor joining methods. RPB45A (AT5G54900.1) appears in the rounded rectangle in FIG. 5B. An ancestral sequence of RPB45A and closely-related sequences is represented by the node of the tree indicated by the arrow “A” in FIG. 5B. RPB45A clade members are considered those proteins that descended from ancestral sequence “A”, including the exemplary sequences shown in this figure that are bounded by Bradi5g22410.1_BRADI and Solyc03g031720.2.1_SOLLY.

FIGS. 6A-6AF show an alignment of RPB45A and representative clade-related proteins. The alignment was generated with MUSCLE (3.8) with default parameters. SEQ ID NOs: appear in parentheses after each Gene Identifier (GID). The conserved first, second, and third RNA Recognition Motif (RRM) domains appear in boxes in FIGS. 6I-6N, FIGS. 6M-6R, and 6S-6X, respectively (for which the consensus sequences are

SEQ ID NOs

844, 845 and 846, respectively).

In FIG. 7, a phylogenetic tree of TCP6 or AT5G41030 (also referred to as G1936) clade members and related full length proteins were constructed using TreeBeST (Ruan et al., 2008. Nucleic Acids Res. 36 (suppl. 1): D735-D740) using the best command to identify the best tree from maximum likelihood and neighbor joining methods. TCP6 (AT5G41030.1) appears in the rounded rectangle. An ancestral sequence of TCP6 and closely-related sequences is represented by the node of the tree indicated by the arrow “A” in FIG. 7. TCP6 clade members are considered those proteins that descended from ancestral sequence “A”, including the exemplary sequences shown in this figure that are bounded by Bradi2g59240.1_BRADI and Solyc02g094290.1.1_SOLLY.

FIGS. 8A-8L show an alignment of TCP6 and representative clade-related proteins. The alignment was generated with MUSCLE (3.8) with default parameters. SEQ ID NOs: appear in parentheses after each Gene Identifier (GID). The conserved TCP domain appears in boxes in FIGS. 8C-8E (for which the consensus sequence is SEQ ID NO 847).

In FIG. 9, a phylogenetic tree of PIL1 or AT2G46970 (also referred to as G1649) clade members and related full length proteins were constructed using TreeBeST (Ruan et al., 2008. Nucleic Acids Res. 36 (suppl. 1): D735-D740) using the best command to identify the best tree from maximum likelihood and neighbor joining methods. The PIL1 clade members appear in the large box with the solid line boundary. PIL1 (AT2G46970.1) appears in the rounded rectangle. An ancestral sequence of PIL1 and closely-related sequences is represented by the node of the tree indicated by the arrow “A” in FIG. 9. PIL1 clade members are considered those proteins that descended from ancestral sequence “A”, including the exemplary sequences shown in this figure that are bounded by AT2G46970.1_ARATH and POPTR_0014 s10700.1_POPTR.

FIGS. 10A-10F show an alignment of PIL1 and representative clade-related proteins. The alignment was generated with MUSCLE (3.8) with default parameters. SEQ ID NOs: appear in parentheses after each Gene Identifier (GID). The conserved bHLH domain appears in boxes in FIGS. 10D-10E (for which the consensus sequence is SEQ ID NO 848).

In FIG. 11, a phylogenetic tree of PCL1 or AT3G46640 (also referred to as G2741) clade members and related full length proteins were constructed using TreeBeST (Ruan et al., 2008. Nucleic Acids Res. 36 (suppl. 1): D735-D740) using the best command to identify the best tree from maximum likelihood and neighbor joining methods. PCL1 (AT3G46640.3) appears in the rounded rectangle. An ancestral sequence of PCL1 and closely-related sequences is represented by the node of the tree indicated by the arrow “A” in FIG. 11. PCL1 clade members are considered those proteins that descended from ancestral sequence “A”, including the exemplary sequences shown in this figure that are bounded by Bradi2g62067.1_BRADI and GSVIVT01024916001_VITVI.

FIGS. 12A-12N show an alignment of PCL1 and representative clade-related proteins. The alignment was generated with MUSCLE (3.8) with default parameters. SEQ ID NOs: appear in parentheses after each Gene Identifier (GID). The conserved SANT (aka Myb-related or GARP) domain appears in boxes in FIGS. 12H-12I (for which the consensus sequence is SEQ ID NO 849).

FIG. 13: Plot showing increased light saturated photosynthesis (A_sat) over a range of leaf sub-stomatal CO₂concentration (C_i) in four PCL1 overexpression lines, compared to a control line. Data was collected over a range of C_iwhere the activity of Rubisco is known to limit A_sat. The solid line shown is a regression fitted to the data for the control line only. All data are the means±1 standard error for data collected on at least seven replicate plants for each line.

Legend for FIG. 13:

Control

□ Line 1

Δ Line 2

X Line 3

⋄ Line 4

◯ Line 5

In FIG. 14, a phylogenetic tree of GTL1 or AT1G33240 (also referred to as G634) clade members and related full length proteins were constructed using TreeBeST (Ruan et al., 2008. Nucleic Acids Res. 36 (suppl. 1): D735-D740) using the best command to identify the best tree from maximum likelihood and neighbor joining methods. GTL1 (AT1G33240.1) appears in the rounded rectangle. An ancestral sequence of GTL1 and closely-related sequences is represented by the node of the tree indicated by the arrow “A” in FIG. 14. GTL1 clade members are considered those proteins that descended from ancestral sequence “A”, including the exemplary sequences shown in this figure that are bounded by Bradi5g17150.1_BRADI and POPTR_0005 s21420.1_POPTR.

FIGS. 15A-15X show an alignment of GTL1 and representative clade-related proteins. The sequence denoted G634_P₇₇₅₉₁was the splice variant of GTL1 that was expressed in plants. The alignment was generated with MUSCLE (3.8) with default parameters. SEQ ID NOs: appear in parentheses after each Gene Identifier (GID). The conserved first and second trihelix (aka GT or Myb/SANT-related) domains appear in boxes in FIGS. 15C-15E and FIGS. 15O-15Q, respectively (for which the consensus sequences are SEQ ID NOs 850 and 851).

In FIG. 16, a phylogenetic tree of DREB2H or AT2G40350 (also referred to as G1755) clade members and related full length proteins were constructed using TreeBeST (Ruan et al., 2008. Nucleic Acids Res. 36 (suppl. 1): D735-D740) using the best command to identify the best tree from maximum likelihood and neighbor joining methods. DREB2H (AT2G40350.1) appears in the rounded rectangle. An ancestral sequence of DREB2H and closely-related sequences is represented by the node of the tree indicated by the arrow “A” in FIG. 16. DREB2H clade members are considered those proteins that descended from ancestral sequence “A”, including the exemplary sequences shown in this figure that are bounded by Bradi2g04000.1_BRADI and Solyc06g050520.1.1_SOLLY.

FIGS. 17A-17N show an alignment of DREB2H and representative clade-related proteins. The sequence denoted G1755_P4407 was the sequence of the DREB2H clone expressed in plants. The alignment was generated with MUSCLE (3.8) with default parameters. SEQ ID NOs: appear in parentheses after each Gene Identifier (GID). The conserved AP2 domain appears in boxes in FIGS. 17E-17F (for which the consensus sequence is SEQ ID NO 852).

In FIG. 18, a phylogenetic tree of ERF087 or AT1G28160 (also referred to as G2292) clade members and related full length proteins were constructed using TreeBeST (Ruan et al., 2008. Nucleic Acids Res. 36 (suppl. 1): D735-D740) using the best command to identify the best tree from maximum likelihood and neighbor joining methods. ERF087 (AT1G28160.1) appears in the rounded rectangle. An ancestral sequence of ERF087 and closely-related sequences is represented by the node of the tree indicated by the arrow “A” in FIG. 18. ERF087 clade members are considered those proteins that descended from ancestral sequence “A”, including the exemplary sequences shown in this figure that are bounded by Bradi3g44470.1_BRADI and Glyma16g05070.1_GLYMA.

FIGS. 19A-19J show an alignment of ERF087 and representative clade-related proteins. The alignment was generated with MUSCLE (3.8) with default parameters. SEQ ID NOs: appear in parentheses after each Gene Identifier (GID). The conserved AP2 domain appears in boxes in FIGS. 19C-19D, respectively (for which the consensus sequence is SEQ ID NO 853).

FIG. 20: Non-photochemical quenching (NPQ) dynamics at 22° C. Plot showing decreased NPQ in multiple ERF087 overexpression lines, during short term acclimation to high light. NPQ was calculated from an initial measurement of maximal, dark adapted fluorescence (F_m) and subsequent measurements of fluorescence made under varying incident light (F_m), as NPQ=(F_m/F′_m)−1. During the nine minute assay F′_mwas measured at 30 second intervals: initially after exposure to 700 μmol PAR m⁻²s⁻¹beginning immediately after F_mwas measured; then, after a decrease to 0 μmol PAR m⁻²s⁻¹after 3 minutes; then, after an increase to 2000 μmol PAR m⁻²s⁻¹after 4 minutes. All symbols are the mean±1 standard error of measurements made on at least five replicate leaves for a given line (TAR′ refers to photosynthetically active radiation).

Legend for FIG. 20:

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FIG. 21: Non-photochemical quenching (NPQ) dynamics at 35° C. Plot showing decreased NPQ in multiple ERF087 overexpression lines, during short term acclimation to high light. NPQ was calculated from an initial measurement of maximal, dark adapted fluorescence (F_m) and subsequent measurements of fluorescence made under varying incident light (F′_m), as NPQ=(F_m/F′_m)−1. During the nine minute assay F′_mwas measured at 30 second intervals: initially after exposure to 700 μmol PAR m⁻²s⁻¹beginning immediately after F_mwas measured; then, after a decrease to 0 μmol PAR m⁻²s⁻¹after 3 minutes; then, after an increase to 2000 μmol PAR m⁻²s⁻¹after 4 minutes. All symbols are the mean±1 standard error of measurements made on at least five replicate leaves for a given line.

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In FIG. 22, a phylogenetic tree of BBX18 or AT2G21320 (also referred to as G1881) clade members and related full length proteins were constructed using TreeBeST (Ruan et al., 2008. Nucleic Acids Res. 36 (suppl. 1): D735-D740) using the best command to identify the best tree from maximum likelihood and neighbor joining methods. BBX18 (AT2G21320.1) appears in the rounded rectangle. An ancestral sequence of BBX18 and closely-related sequences is represented by the node of the tree indicated by the arrow “A” in FIG. 22. BBX18 clade members are considered those proteins that descended from ancestral sequence “A”, including the exemplary sequences shown in this figure that are bounded by Bradi4g35950.1_BRADI and Solyc02g084420.2.1_SOLLY.

FIGS. 23A-23G show an alignment of BBX18 and representative clade-related proteins. The alignment was generated with MUSCLE (3.8) with default parameters. SEQ ID NOs: appear in parentheses after each Gene Identifier (GID). The conserved first and second B-box domains appear in boxes in FIGS. 23A-23B and FIGS. 23B-23C, respectively (for which the consensus sequences are SEQ ID NOs 854 and 855).

FIG. 24: Plot showing increased light saturated photosynthesis (A_sat) over a range of leaf sub-stomatal CO₂concentration (C_i) in three BBX18 overexpression lines, compared to a control line. Data was collected over a range of C_i, from low, where the activity of Rubisco is known to limit A_sat, to high, where the capacity to regenerate RuBP limits A_sat. The solid line shown is a regression fitted to the data for the control line only. All data are the means±1 standard error for data collected on at least seven replicate plants for each line.

Legend for FIG. 24:

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In FIG. 25, a phylogenetic tree of bHLH60 or AT3G57800 (also referred to as G2144) clade members and related full length proteins were constructed using TreeBeST (Ruan et al., 2008. Nucleic Acids Res. 36 (suppl. 1): D735-D740) using the best command to identify the best tree from maximum likelihood and neighbor joining methods. bHLH60 (AT3G57800.1) appears in the rounded rectangle. An ancestral sequence of bHLH60 and closely-related sequences is represented by the node of the tree indicated by the arrow “A” in FIG. 25. bHLH60 clade members are considered those proteins that descended from ancestral sequence “A”, including the exemplary sequences shown in this figure that are bounded by Bradi1g35990.1_BRADI and Solyc10g079070.1.1_SOLLY.

FIGS. 26A-26N show an alignment of bHLH60 and representative clade-related proteins. The alignment was generated with MUSCLE (3.8) with default parameters. SEQ ID NOs: appear in parentheses after each Gene Identifier (GID). The conserved bHLH domain appears in boxes FIGS. 26H-26I (for which the consensus sequence is SEQ ID NO 856).

In FIG. 27, a phylogenetic tree of NF-YC6 or AT5G50480 (also referred to as G1820) clade members and related full length proteins were constructed using TreeBeST (Ruan et al., 2008. Nucleic Acids Res. 36 (suppl. 1): D735-D740) using the best command to identify the best tree from maximum likelihood and neighbor joining methods. NF-YC6 (AT5G50480.1) appears in the rounded rectangle. An ancestral sequence of NF-YC6 and closely-related sequences is represented by the node of the tree indicated by the arrow “A” in FIG. 27. NF-YC6 clade members are considered those proteins that descended from ancestral sequence “A”, including the exemplary sequences shown in this figure that are bounded by Bradi3g17790.1_BRADI and Solyc03g111450.1.1_SOLLY.

FIGS. 28A-28T show an alignment of NF-YC6 and representative clade-related proteins. The alignment was generated with MUSCLE (3.8) with default parameters. SEQ ID NOs: appear in parentheses after each Gene Identifier (GID). The conserved NF-Y/histone-like domain appears in boxes in FIGS. 28J-28L (for which the consensus sequence is SEQ ID NO 857).

In FIG. 29, a phylogenetic tree of bHLH121 or AT3G19860 (also referred to as G782) clade members and related full length proteins were constructed using TreeBeST (Ruan et al., 2008. Nucleic Acids Res. 36 (suppl. 1): D735-D740) using the best command to identify the best tree from maximum likelihood and neighbor joining methods. bHLH121 (AT3G19860.2) appears in the rounded rectangle. An ancestral sequence of bHLH121 and closely-related sequences is represented by the node of the tree indicated by the arrow “A” in FIG. 29. bHLH121 clade members are considered those proteins that descended from ancestral sequence “A”, including the exemplary sequences shown in this figure that are bounded by Bradi3g11520.1_BRADI and Solyc01g111130.2.1_SOLLY.

FIGS. 30A-30K show an alignment of bHLH121 and representative clade-related proteins. The alignment was generated with MUSCLE (3.8) with default parameters. SEQ ID NOs: appear in parentheses after each Gene Identifier (GID). The conserved bHLH domains appear in boxes in FIGS. 30B-30D, respectively (SEQ ID NO 858). A distinct putative leucine zipper motif and its consensus sequence that is found with these clade members comprising is found in FIG. 30D (SEQ ID NO: 859), enclosed in a dotted line box.

FIG. 31: Plot showing increased light saturated photosynthesis (A_sat) over a range of leaf sub-stomatal CO₂concentration (C_i) in four out of five bHLH121 overexpression lines, compared to a control line. Data was collected over a range of C_iover which the capacity to regenerate RuBP is known to limit A_sat. The solid line shown is a regression fitted to the data for the control line only. All data are the means±1 standard error for data collected on at least five replicate plants for each line.

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In FIG. 32, a phylogenetic tree of BBX26 or AT1G60250 (also referred to as G1486) clade members and related full length proteins were constructed using TreeBeST (Ruan et al., 2008. Nucleic Acids Res. 36 (suppl. 1): D735-D740) using the best command to identify the best tree from maximum likelihood and neighbor joining methods. BBX26 (AT1G60250) appears in the rounded rectangle. An ancestral sequence of BBX26 and closely-related sequences is represented by the node of the tree indicated by the arrow “A” in FIG. 32. BBX26 clade members are considered those proteins that descended from ancestral sequence “A”, including the exemplary sequences shown in this figure that are bounded by AT3G53200 ARATH and Solyc04007470.2 SOLLY.

FIGS. 33A-33E show an alignment of BBX26 and representative clade-related proteins. The alignment was generated with MUSCLE (3.8) with default parameters. SEQ ID NOs: appear in parentheses after each Gene Identifier (GID). The conserved B-box domain appears in boxes in FIG. 33A (for which the consensus sequence is SEQ ID NO 860).

FIG. 34: Plot showing increased light saturated photosynthesis (A_sat) over a range of leaf sub-stomatal CO₂concentration (C_i) in four BBX26 overexpression lines, compared to a control line. Data was collected over a range of C_iover which the capacity to regenerate RuBP is known to limit A_sat. The solid line shown is a regression fitted to the data for the control line only. All data are the means±1 standard error for data collected on at least six replicate plants for each line.

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In FIGS. 35A and 35B, a phylogenetic tree of PMT24 or AT1G29470 (also referred to as G837) clade members and related full length proteins were constructed using TreeBeST (Ruan et al., 2008. Nucleic Acids Res. 36 (suppl. 1): D735-D740) using the best command to identify the best tree from maximum likelihood and neighbor joining methods. PMT24 (AT1G29470.1) appears in the rounded rectangle in FIG. 35B. An ancestral sequence of PMT24 and closely-related sequences is represented by the node of the tree indicated by the arrow “A” in FIG. 35B. PMT24 clade members are considered those proteins that descended from ancestral sequence “A”, including the exemplary sequences shown in this figure that are bounded by Bradi2g57087.1_BRADI and GSVIVT01026451001_VITVI.

FIGS. 36A-36X show an alignment of PMT24 and representative clade-related proteins. The alignment was generated with MUSCLE (3.8) with default parameters. SEQ ID NOs: appear in parentheses after each Gene Identifier (GID). The conserved putative methyltransferase domain appears in boxes in FIGS. 36M-36S (for which the consensus sequence is SEQ ID NO 861).

FIG. 37: Plot showing increased light saturated photosynthesis (A_sat) over a range of leaf sub-stomatal CO₂concentration (C_i) in four out of five PMT24 overexpression lines, compared to a control line. Data was collected over a range of C_iover which the capacity to regenerate RuBP is known to limit A_sat. The solid line shown is a regression fitted to the data for the control line only. All data are the means±1 standard error for data collected on at least five replicate plants for each line.

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DETAILED DESCRIPTION

The present description relates to polynucleotides and polypeptides for modifying phenotypes of plants, particularly those associated with increased photosynthetic resource use efficiency and increased yield with respect to a control plant. Throughout this disclosure, various information sources are referred to and/or are specifically incorporated. The information sources include scientific journal articles, patent documents, textbooks, and internet entries. While the reference to these information sources clearly indicates that they can be used by one of skill in the art, each and every one of the information sources cited herein are specifically incorporated in their entirety, whether or not a specific mention of “incorporation by reference” is noted. The contents and teachings of each and every one of the information sources can be relied on and used to make and use embodiments of the instant description.
As used herein and in the appended claims, the singular forms “a”, “an”, and “the” include the plural reference unless the context clearly dictates otherwise. Thus, for example, a reference to “a host cell” includes a plurality of such host cells, and a reference to “a plant” is a reference to one or more plants, and so forth.
A “recombinant polynucleotide” is a polynucleotide that is not in its native state, e.g., the polynucleotide comprises a nucleotide sequence not found in nature, or the polynucleotide is in a context other than that in which it is naturally found, e.g., separated from nucleotide sequences with which it typically is in proximity in nature, or adjacent (or contiguous with) nucleotide sequences with which it typically is not in proximity. For example, the sequence at issue can be cloned into a vector, or otherwise recombined with one or more additional nucleic acid.
A “polypeptide” is an amino acid sequence comprising a plurality of consecutive polymerized amino acid residues e.g., at least about 15 consecutive polymerized amino acid residues. In many instances, a polypeptide comprises a polymerized amino acid residue sequence that is a regulatory polypeptide or a domain or portion or fragment thereof. Additionally, the polypeptide may comprise: (i) a localization domain; (ii) an activation domain; (iii) a repression domain; (iv) an oligomerization domain; (v) a protein-protein interaction domain; (vi) a DNA-binding domain; or the like. The polypeptide optionally comprises modified amino acid residues, naturally occurring amino acid residues not encoded by a codon, or non-naturally occurring amino acid residues.
“Protein” refers to an amino acid sequence, oligopeptide, peptide, polypeptide or portions thereof whether naturally occurring or synthetic.
In the instant description, “exogenous” refers to a heterologous nucleic acid or polypeptide that may not be naturally expressed in a plant of interest. Exogenous nucleic acids may be introduced into a plant in a stable or transient manner via, for example, transformation or breeding, and may thus serve to produce in planta a homologous RNA molecule and an encoded and functional polypeptide. Exogenous nucleic acids and polypeptides introduced thusly may comprise sequences that are wholly or partially identical or homologous to sequences that naturally occur in (i.e., are endogenous with respect to) the plant.
A “recombinant polypeptide” is a polypeptide produced by translation of a recombinant polynucleotide. A “synthetic polypeptide” is a polypeptide created by consecutive polymerization of isolated amino acid residues using methods well known in the art. An “isolated polypeptide,” whether a naturally occurring or a recombinant polypeptide, is more enriched in (or out of) a cell than the polypeptide in its natural state in a wild-type cell, e.g., more than about 5% enriched, more than about 10% enriched, or more than about 20%, or more than about 50%, or more, enriched, i.e., alternatively denoted: 105%, 110%, 120%, 150% or more, enriched relative to wild type standardized at 100%. Such an enrichment is not the result of a natural response of a wild-type plant. Alternatively, or additionally, the isolated polypeptide is separated from other cellular components with which it is typically associated, e.g., by any of the various protein purification methods herein.
“Identity” or “similarity” refers to sequence similarity between two polynucleotide sequences or between two polypeptide sequences, with identity being a more strict comparison. The phrases “percent identity” and “% identity” refer to the percentage of sequence similarity found in a comparison of two or more polynucleotide sequences or two or more polypeptide sequences. “Sequence similarity” refers to the percent similarity in base pair sequence (as determined by any suitable method) between two or more polynucleotide sequences. Two or more sequences can be anywhere from 0-100% similar or identical, or any integer value between 0-100%. Identity or similarity can be determined by comparing a position in each sequence that may be aligned for purposes of comparison. When a position in the compared sequence is occupied by the same nucleotide base or amino acid, then the molecules are identical at that position. A degree of similarity or identity between polyBLAST nucleotide sequences is a function of the number of identical, matching or corresponding nucleotides at positions shared by the polynucleotide sequences. A degree of identity of polypeptide sequences is a function of the number of identical amino acids at corresponding positions shared by the polypeptide sequences. A degree of homology or similarity of polypeptide sequences is a function of the number of amino acids at corresponding positions shared by the polypeptide sequences. The fraction or percentage of components in common is related to the homology or identity between the sequences. Alignments such as those of FIG. 2A-2E may be used to identify conserved domains and relatedness within these domains. An alignment may suitably be determined by means of computer programs known in the art, such as MACVECTOR software, (1999; Accelrys, Inc., San Diego, Calif.).
“Homologous sequences” refers to polynucleotide or polypeptide sequences that are similar due to common ancestry and sequence conservation. The terms “ortholog” and “paralog” are defined below in the section entitled “Orthologs and Paralogs”. In brief, orthologs and paralogs are evolutionarily related genes that have similar sequences and functions. Orthologs are structurally related genes in different species that are derived by a speciation event. Paralogs are structurally related genes within a single species that are derived by a duplication event.
“Functional homologs” are polynucleotide or polypeptide sequences, including orthologs and paralogs, that are similar due to common ancestry and sequence conservation and have identical or similar function at the catalytic, cellular, or organismal levels. The presently disclosed polypeptides, clade members and phylogenetically related sequences are “functionally-related and/or closely-related” by having descended from common ancestral sequences, and/or by being sufficiently similar to the sequences and domains listed in the instant Tables and Sequence Listing that they confer the same function to plants of increased photosynthetic resource use efficiency, increased yield, increased grain yield, increased light use efficiency, increased photosynthetic capacity, increased photosynthetic rate, greater vigor, and/or greater biomass as compared to a control plant.
Functionally-related and/or closely-related polypeptides may be created artificially, semi-synthetically, or may occur naturally by having descended from the same ancestral sequence as the disclosed closely-related sequences, where the polypeptides have the function of conferring increased photosynthetic resource use efficiency to plants.
“Conserved domains” are recurring units in molecular evolution, the extents of which can be determined by sequence and structure analysis. A “conserved domain” or “conserved region” as used herein refers to a region in heterologous polynucleotide or polypeptide sequences where there is a relatively high degree of sequence identity between the distinct sequences. Conserved domains contain conserved sequence patterns or motifs that allow for their detection in, and identification and characterization of, polypeptide sequences. A Myb domain is an example of a conserved domain.
A transgenic plant is expected to have improved or increased photosynthetic resource use efficiency relative to a control plant when the transgenic plant is transformed with a recombinant polynucleotide encoding any of the listed polypeptide sequences or polypeptide found in polypeptide clade of any of the listed polypeptide sequences, or when the transgenic plant contains or expresses a listed polypeptide or a member of any of the same polypeptide clades sequence in which the listed polypeptides may be found.
The terms “highly stringent” or “highly stringent condition” refer to conditions that permit hybridization of DNA strands whose sequences are highly complementary, wherein these same conditions exclude hybridization of significantly mismatched DNAs. Polynucleotide sequences capable of hybridizing under stringent conditions with the polynucleotides of the present description may be, for example, variants of the disclosed polynucleotide sequences, including allelic or splice variants, or sequences that encode orthologs or paralogs of presently disclosed polypeptides. Nucleic acid hybridization methods are disclosed in detail by Kashima et al., 1985. Nature 313: 402-404; Sambrook et al., 1989. Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., and by Haymes et al., 1985. Nucleic Acid Hybridization: A Practical Approach, IRL Press, Washington, D.C., which references are incorporated herein by reference.
In general, stringency is determined by the temperature, ionic strength, and concentration of denaturing agents (e.g., formamide) used in a hybridization and washing procedure (for a more detailed description of establishing and determining stringency, see the section “Identifying Polynucleotides or Nucleic Acids by Hybridization”, below). The degree to which two nucleic acids hybridize under various conditions of stringency is correlated with the extent of their similarity. Thus, similar nucleic acid sequences from a variety of sources, such as within a plant's genome (as in the case of paralogs) or from another plant (as in the case of orthologs) that may perform similar functions can be isolated on the basis of their ability to hybridize with known related polynucleotide sequences. Numerous variations are possible in the conditions and means by which nucleic acid hybridization can be performed to isolate related polynucleotide sequences having similarity to sequences known in the art and are not limited to those explicitly disclosed herein. Such an approach may be used to isolate polynucleotide sequences having various degrees of similarity with disclosed polynucleotide sequences, such as, for example, encoded regulatory polypeptides listed in the Sequence Listing, or polypeptides that are phylogenetically related to the polypeptides listed in the Sequence Listing.
“Fragment”, with respect to a polynucleotide, refers to a clone or any part of a polynucleotide molecule that retains a usable, functional characteristic. Useful fragments include oligonucleotides and polynucleotides that may be used in hybridization or amplification technologies or in the regulation of replication, transcription or translation. A “polynucleotide fragment” refers to any subsequence of a polynucleotide, typically, of at least about nine consecutive nucleotides, preferably at least about 30 nucleotides, more preferably at least about 50 nucleotides, of any of the sequences provided herein. Exemplary polynucleotide fragments are the first sixty consecutive nucleotides of the polynucleotides listed in the Sequence Listing. Exemplary fragments also include fragments that comprise a region that encodes an conserved domain of a polypeptide. Exemplary fragments also include fragments that comprise a conserved domain of a polypeptide. Exemplary fragments include fragments that comprise an conserved domain of a polypeptide, for example, any of the domains listed in in the instant Tables or in the Sequence Listing.
Fragments may also include subsequences of polypeptides and protein molecules, or a subsequence of the polypeptide. Fragments may have uses in that they may have antigenic potential. In some cases, the fragment or domain is a subsequence of the polypeptide which performs at least one biological function of the intact polypeptide in substantially the same manner, or to a similar extent, as does the intact polypeptide. For example, a polypeptide fragment can comprise a recognizable structural motif or functional domain such as a DNA-binding site or domain that binds to a DNA promoter region, an activation domain, or a domain for protein-protein interactions, and may initiate transcription. Fragments can vary in size from as few as three amino acid residues to the full length of the intact polypeptide, but are preferably at least about 30 amino acid residues in length and more preferably at least about 60 amino acid residues in length.
Fragments may also refer to a functional fragment of a promoter region. For example, a recombinant polynucleotide capable of modulating transcription in a plant may comprise a nucleic acid sequence with similarity to, or a percentage identity to, a promoter region exemplified by a promoter sequence provided in the Sequence Listing (also see promoters listed in Example II), a fragment thereof, or a complement thereof, wherein the nucleic acid sequence, or the fragment thereof, or the complement thereof, regulates expression of a polypeptide in a plant cell.
The term “plant” includes whole plants, shoot vegetative organs/structures (for example, leaves, stems and tubers), roots, flowers and floral organs/structures (for example, bracts, sepals, petals, stamens, carpels, anthers and ovules), seed (including embryo, endosperm, and seed coat) and fruit (the mature ovary), plant tissue (for example, vascular tissue, ground tissue, and the like), pulped, pureed, ground-up, macerated or broken-up tissue, and cells (for example, guard cells, egg cells, and the like), and progeny of same. The class of the plants that can be transformed using the methods provided of the instant description is generally as broad as the class of higher and lower plants amenable to transformation techniques, including angiosperms (monocotyledonous and dicotyledonous plants), gymnosperms, ferns, horsetails, psilophytes, lycophytes, and bryophytes. These plant parts, organs, structures, cells, tissue, or progeny may contain a recombinant polynucleotide of interest, such as one that comprises a described or listed polynucleotide or one that encodes a described or listed polypeptide or a polypeptide that is phylogenetically-related to a listed polypeptide, and is thus a member of the same polypeptide clade.
A “control plant” as used in the present description refers to a plant cell, seed, plant component, plant tissue, plant organ or whole plant used to compare against transgenic or genetically modified plant for the purpose of identifying an enhanced phenotype in the transgenic or genetically modified plant. A control plant may in some cases be a transgenic plant line that comprises an empty vector or marker gene, but does not contain the recombinant polynucleotide of the present description that is expressed in the transgenic or genetically modified plant being evaluated. In general, a control plant is a plant of the same line or variety as the transgenic or genetically modified plant being tested. A suitable control plant would include a genetically unaltered or non-transgenic plant of the parental line used to generate a transgenic plant herein.
A “transgenic plant” refers to a plant that contains genetic material not found in a wild-type plant of the same species, variety or cultivar. The genetic material may include a transgene, an insertional mutagenesis event (such as by transposon or T-DNA insertional mutagenesis), an activation tagging sequence, a mutated sequence, a homologous recombination event or a sequence modified by chimeraplasty. Typically, the foreign genetic material has been introduced into the plant by human manipulation, but any method can be used as one of skill in the art recognizes.
A transgenic line or transgenic plant line refers to the progeny plant or plants deriving from the stable integration of heterologous genetic material into a specific location or locations within the genome of the original transformed cell.
A transgenic plant may contain an expression vector or cassette. The expression vector or cassette typically comprises a polypeptide-encoding sequence operably linked (i.e., under regulatory control of) to appropriate inducible, tissue-enhanced, tissue-specific, or constitutive regulatory sequences that allow for the controlled expression of the polypeptide. The expression vector or cassette can be introduced into a plant by transformation or by breeding after transformation of a parent plant. A plant refers to a whole plant as well as to a plant part, such as seed, fruit, leaf, or root, plant tissue, plant cells or any other plant material, e.g., a plant explant, as well as to progeny thereof, and to in vitro systems that mimic biochemical or cellular components or processes in a cell. In some other embodiments, the expression vectors or cassettes do not occur naturally. In some embodiments, the expression vectors or cassettes comprise a promoter of the present application, and a gene of interest, wherein the promoter and the gene of interest do not link to each other under natural conditions, e.g., the linkage between the promoter and the gene of interest does not exist in nature. For example, in some embodiments, the promoter and the gene of interest are derived from a same plant species, but are not linked to each other under natural conditions. In some embodiments, the promoter and the gene of interest are derived from two different species, e.g., the promoter and the gene of interest are heterologous to each other. In some embodiments, the gene of interest is derived from a different plant species, a bacteria species, a fungal species, a viral species, an algae species, or an animal species. In some embodiments, the expression vectors or cassettes comprise synthetic sequences.
“Germplasm” refers to a genetic material or a collection of genetic resources for an organism from an individual plant, a group of related individual plants (for example, a plant line, a plant variety or a plant family), or a clone derived from a plant line, plant variety, plant species, or plant culture.
A constitutive promoter is active under most environmental conditions, and in most plant parts. Regulation of protein expression in a constitutive manner refers to the control of expression of a gene and/or its encoded protein in all tissues regardless of the surrounding environment or development stage of the plant.
Alternatively, expression of the disclosed or listed polypeptides may be under the regulatory control of a promoter that is not a constitutive promoter. For example, tissue-enhanced (also referred to as tissue-preferred), tissue-specific, cell type-specific, and inducible promoters constitute non-constitutive promoters; that is, these promoters do not regulate protein expression in a constitutive manner. Tissue-enhanced or tissue-preferred promoters facilitate expression of a gene and/or its encoded protein in specific tissue(s) and generally, although perhaps not completely, do not express the gene and/or protein in all other tissues of the plant, or do so to a much lesser extent. Promoters under developmental control include promoters that preferentially initiate transcription in certain tissues, such as xylem, leaves, roots, or seeds. Such promoters are examples of tissue-enhanced or tissue-preferred promoters (see U.S. Pat. No. 7,365,186). Tissue-specific promoters generally confine transgene expression to a single plant part, tissue or cell-type, although many such promoters are not perfectly restricted in their expression and their regulatory control is more properly described as being “tissue-enhanced” or “tissue-preferred”. Tissue-enhanced promoters primarily regulate transgene expression in a limited number of plant parts, tissues or cell-types and cause the expression of proteins to be overwhelming restricted to a few particular tissues, plant parts, or cell types. An example of a tissue-enhanced promoter is a “photosynthetic tissue-enhanced promoter”, for which the promoter preferentially regulates gene or protein expression in photosynthetic tissues (e.g., leaves, cotyledons, stems, etc.). Tissue-enhanced promoters can be found upstream and operatively linked to DNA sequences normally transcribed in higher levels in certain plant tissues or specifically in certain plant tissues, respectively. “Cell-enhanced”, “tissue-enhanced”, or “tissue-specific” regulation thus refer to the control of gene or protein expression, for example, by a promoter that drives expression that is not necessarily totally restricted to a single type of cell or tissue, but where expression is elevated in particular cells or tissues to a greater extent than in other cells or tissues within the organism, and in the case of tissue-specific regulation, in a manner that is primarily elevated in a specific tissue. Tissue-enhanced or preferred promoters have been described in, for example, U.S. Pat. No. 7,365,186, or 7,619,133.
Another example of a promoter that is not a constitutive promoter is a “condition-enhanced” promoter, the latter term referring to a promoter that activates a gene in response to a particular environmental stimulus. This may include, for example, an abiotic stress, infection caused by a pathogen, light treatment, etc., and a condition-enhanced promoter drives expression in a unique pattern which may include expression in specific cell and/or tissue types within the organism (as opposed to a constitutive expression pattern in all cell types of an organism at all times).
“Wild type” or “wild-type”, as used herein, refers to a plant cell, seed, plant component, plant tissue, plant organ or whole plant that has not been genetically modified or treated in an experimental sense. Wild-type cells, seed, components, tissue, organs or whole plants may be used as controls to compare levels of expression and the extent and nature of trait modification with cells, tissue or plants of the same species in which a polypeptide's expression is altered, e.g., in that it has been knocked out, overexpressed, or ectopically expressed.
When two or more plants have “similar morphologies”, “substantially similar morphologies”, “a morphology that is substantially similar”, or are “morphologically similar”, the plants have comparable forms or appearances, including analogous features such as overall dimensions, height, width, mass, root mass, shape, glossiness, color, stem diameter, leaf size, leaf dimension, leaf density, internode distance, branching, root branching, number and form of inflorescences, and other macroscopic characteristics at a particular stage of growth. It may be difficult to distinguish two plants that are genotypically distinct but morphologically similar based on morphological characteristics alone. If the plants are morphologically similar at all stages of growth, they are also “developmentally similar”.
With regard to gene knockouts as used herein, the term “knockout” (KO) refers to a plant or plant cell having a disruption in at least one gene in the plant or cell, where the disruption results in a reduced expression or activity of the polypeptide encoded by that gene compared to a control cell. The knockout can be the result of, for example, genomic disruptions, including transposons, tilling, and homologous recombination, antisense constructs, sense constructs, RNA silencing constructs, or RNA interference. A T-DNA insertion within a gene is an example of a genotypic alteration that may abolish expression of that gene.
“Ectopic expression” or “altered expression” in reference to a polynucleotide indicates that the pattern of expression in, e.g., a transgenic plant or plant tissue, is different from the expression pattern in a wild-type plant or a reference plant of the same species. The pattern of expression may also be compared with a reference expression pattern in a wild-type plant of the same species. For example, the polynucleotide or polypeptide is expressed in a cell or tissue type other than a cell or tissue type in which the sequence is expressed in the wild-type plant, or by expression at a time other than at the time the sequence is expressed in the wild-type plant, or by a response to different inducible agents, such as hormones or environmental signals, or at different expression levels (either higher or lower) compared with those found in a wild-type plant. The term also refers to altered expression patterns that are produced by lowering the levels of expression to below the detection level or completely abolishing expression. The resulting expression pattern can be transient or stable, constitutive or inducible. In reference to a polypeptide, the term “ectopic expression or altered expression” further may relate to altered activity levels resulting from the interactions of the polypeptides with exogenous or endogenous modulators or from interactions with factors or as a result of the chemical modification of the polypeptides.
The term “overexpression” as used herein refers to a greater expression level of a gene in a plant, plant cell or plant tissue, compared to expression of that gene in a wild-type plant, cell or tissue, at any developmental or temporal stage. Overexpression can occur when, for example, the genes encoding one or more polypeptides are under the control of a strong promoter (e.g., the cauliflower mosaic virus 35S transcription initiation region). Overexpression may also be achieved by placing a gene of interest under the control of an inducible or tissue specific promoter, or may be achieved through integration of transposons or engineered T-DNA molecules into regulatory regions of a target gene. Other means for inducing overexpression may include making targeted changes in a gene's native promoter, e.g. through elimination of negative regulatory sequences or engineering positive regulatory sequences, though the use of targeted nuclease activity (such as zinc finger nucleases or TAL effector nucleases) for genome editing. Elimination of micro-RNA binding sites in a gene's transcript may also result in overexpression of that gene. Additionally, a gene may be overexpressed by creating an artificial transcriptional activator targeted to bind specifically to its promoter sequences, comprising an engineered sequence-specific DNA binding domain such as a zinc finger protein or TAL effector protein fused to a transcriptional activation domain. Thus, overexpression may occur throughout a plant, in specific tissues of the plant, or in the presence or absence of particular environmental signals, depending on the promoter or overexpression approach used.
Overexpression may take place in plant cells normally lacking expression of polypeptides functionally equivalent or identical to the present polypeptides. Overexpression may also occur in plant cells where endogenous expression of the present polypeptides or functionally equivalent molecules normally occurs, but such normal expression is at a lower level. Overexpression thus results in a greater than normal production, or “overproduction” of the polypeptide in the plant, cell or tissue.
“Photosynthetic resource-use efficiency” is defined as the rate of photosynthesis achieved per unit use of a given resource. Consequently, increases in photosynthesis relative to the use of a given resource will improve photosynthetic resource-use efficiency. Photosynthesis is constrained by the availability of various resources, including light, water and nitrogen. Improving the efficiency with which photosynthesis makes use of light, water and nitrogen is a means for increasing plant productivity, crop growth, and yield. For the purposes of comparing a plant of interest to a reference or control plant, the ratio of photosynthesis to use of a given resource is often determined for a fixed unit of leaf area. Examples of increased photosynthetic resource-use efficiency would be an increase in the ratio of the rate of photosynthesis for a given leaf relative to, for example, the rate of transpiration from the same leaf area, nitrogen or chlorophyll invested in that leaf area, or light absorbed by that same leaf area. Increased photosynthetic resource use efficiency may result from increased photosynthetic rate, photosynthetic capacity, a decrease in leaf chlorophyll content, a decrease in percentage of nitrogen in leaf dry weight, increased transpiration efficiency, an increase in resistance to water vapor diffusion exerted by leaf stomata, an increased rate of relaxation of photoprotective reactions operating in the light harvesting antennae, a decrease in the ratio of the carbon isotope ¹²C to ¹³C in above-ground biomass, and/or an increase in the total dry weight of above-ground plant material.
“Photosynthetic rate” refers to the rate of photosynthesis achieved by a leaf, and is typically expressed relative to a unit of leaf area. The photosynthetic rate at any given time results from the photosynthetic capacity of the leaf (see below) and the biotic or abiotic environmental constraints prevailing at that time.
“Photosynthetic capacity” refers to the capacity for photosynthesis per unit leaf area and is set by the leafs investment in the components of the photosynthetic apparatus. Key components, among many, would be the pigments and proteins required to regulate light absorption and transduction of light energy to the photosynthetic reaction centers, and the enzymes required to operate the C3 and C4 dark reactions of photosynthesis. Increasing photosynthetic capacity is seen as an important means of increasing leaf and crop-canopy photosynthesis, and crop yield.
“Rubisco (ribulose-1,5-bisphosphate carboxylase oxygenase) activity” refers to the activation state of Rubisco, the most abundant protein in the chloroplast and a key limitation to C3 photosynthesis. Increasing Rubisco activity by: increasing the amount of Rubisco in the chloroplast; impacting any combination of specific reactions that regulate Rubisco activity; or increasing the concentration of CO₂in the chloroplast, is seen as an important means to improving C3 leaf and crop-canopy photosynthesis and crop yield.
The “capacity for RuBP (ribulose-1,5-bisphosphate) regeneration” refers to the rate at which RuBP, a key photosynthetic substrate is regenerated in the Calvin cycle. Increasing the capacity for RuBP regeneration by increasing the activity of enzymes in the regenerative phase of the Calvin cycle is seen as an important means to improving C3 leaf and crop-canopy photosynthesis and crop yield that will become progressively more important as atmospheric CO₂concentrations continue to rise.
“Leaf chlorophyll content” refers to the chlorophyll content of the leaf expressed either per unit leaf area or unit weight. Sun leaves in the upper part of crop canopies are thought to have higher leaf chlorophyll content than is required for photosynthesis. The consequence is that these leaves: invest more nitrogen in chlorophyll than is required for photosynthesis; are prone to photodamage associated with absorbing more light energy than can be dissipated via photosynthesis; and impair the transmission of light into the leaf and lower canopy where photosynthesis is light limited. Consequently, decreasing leaf chlorophyll content of upper canopy leaves is considered an effective means to improving photosynthetic resource-use efficiency.
“Non-photochemical quenching” is a term that covers photoprotective processes that dissipate absorbed light energy as heat from the light-harvesting antenna of photosystem II. Non-photochemical quenching is a key regulator of the efficiency with which electron transport is initiated by PSII and the efficiency of photosynthesis at low light. Decreasing the level of non-photochemical quenching, or increasing the speed with which it relaxes is expected to confer cumulative gains in photosynthesis every time the light intensity to which the canopy is exposed transitions from high to low, and is considered a means to improving canopy photosynthesis when integrated over a growing season.
“Nitrogen limitation” or “nitrogen-limiting” refers to nitrogen levels that act as net limitations on primary production in terrestrial or aquatic biomes. Much of terrestrial growth, including much of crop growth, is limited by the availability of nitrogen, which can be alleviated by nitrogen input through deposition or fertilization.
“Water use efficiency”, or WUE, measured as the biomass produced per unit transpiration, describes the relationship between water use and crop production. The basic physiological definition of WUE equates to the ratio of photosynthesis (A) to transpiration (T), also referred to as transpiration efficiency (Karaba et al. 2007, supra; Morison et al., 2008, supra).
“Stomatal conductance” refers to a measurement of the limitation that the stomatal pore imposes on CO₂diffusion into, and H₂O diffusion out of, the leaf. Decreasing stomatal conductance will decrease water loss from the leaf and crop canopy via transpiration. This will conserve soil water, delay the onset and reduce the severity of drought effects on canopy photosynthesis and other physiology. Decreasing stomatal conductance will also decrease photosynthesis. However, the magnitude of the decrease in photosynthesis will typically be less than the decrease in transpiration, and transpiration efficiency will increase as a result. Conversely, increasing stomatal conductance can increase the diffusion of CO₂into the leaf and increase photosynthesis in a C3 leaf. Typically, transpiration will increase to a greater extent than photosynthesis, and transpiration efficiency will therefore decrease.
“Yield” or “plant yield” refers to increased plant growth, increased crop growth, increased biomass, and/or increased plant product production (including grain), and is dependent to some extent on temperature, plant size, organ size, planting density, light, water and nutrient availability, and how the plant copes with various stresses, such as through temperature acclimation and water or nutrient use efficiency. For grain crops, yield generally refers to an amount of grain produced or harvested per unit of land area, such as bushels or tons per acre or tonnes per hectare. Increased or improved yield may be measured as increased seed yield, increased plant product yield (plant products include, for example, plant tissue, including ground or otherwise broken-up plant tissue, and products derived from one or more types of plant tissue), or increased vegetative yield.

DESCRIPTION OF THE SPECIFIC EMBODIMENTS

Regulatory Polypeptides Modify Expression of Endogenous Genes.
A regulatory polypeptide may include, but is not limited to, any polypeptide that can activate or repress transcription of a single gene or a number of genes. As one of ordinary skill in the art recognizes, regulatory polypeptides can be identified by the presence of a region or domain of structural similarity or identity to a specific consensus sequence or the presence of a specific consensus DNA-binding motif (see, for example, Riechmann et al., 2000a. supra).
Generally, regulatory polypeptides control the manner in which information encoded by genes is used to produce gene products and control various pathways, and may be involved in diverse processes including, but not limited to, cell differentiation, proliferation, morphogenesis, and the regulation of growth or environmental responses. Accordingly, one skilled in the art would recognize that by expressing the present sequences in a plant, one may change the expression of autologous genes or induce the expression of introduced genes. By affecting the expression of similar autologous sequences in a plant that have the biological activity of the present sequences, or by introducing the present sequences into a plant, one may alter a plant's phenotype to one with improved traits related to photosynthetic resource use efficiency. The sequences of the instant description may also be used to transform a plant and introduce desirable traits not found in the wild-type cultivar or strain. Plants may then be selected for those that produce the most desirable degree of over- or under-expression of target genes of interest and coincident trait improvement.
The sequences of the present description may be from any species, particularly plant species, in a naturally occurring form or from any source whether natural, synthetic, semi-synthetic or recombinant. The sequences of the instant description may also include fragments of the present amino acid sequences. Where “amino acid sequence” is recited to refer to an amino acid sequence of a naturally occurring protein molecule, “amino acid sequence” and like terms are not meant to limit the amino acid sequence to the complete native amino acid sequence associated with the recited protein molecule.
In addition to methods for modifying a plant phenotype by employing one or more polynucleotides and polypeptides of the instant description described herein, the polynucleotides and polypeptides of the instant description have a variety of additional uses. These uses include their use in the recombinant production (i.e., expression) of proteins; as regulators of plant gene expression, as diagnostic probes for the presence of complementary or partially complementary nucleic acids (including for detection of natural coding nucleic acids); as substrates for further reactions, e.g., mutation reactions, PCR reactions, or the like; as substrates for cloning e.g., including digestion or ligation reactions; and for identifying exogenous or endogenous modulators of the regulatory polypeptides. The polynucleotide can be, e.g., genomic DNA or RNA, a transcript (such as an mRNA), a cDNA, a PCR product, a cloned DNA, a synthetic DNA or RNA, or the like. The polynucleotide can comprise a sequence in either sense or antisense orientations.
Expression of genes that encode polypeptides that modify expression of endogenous genes, polynucleotides, and proteins are well known in the art. In addition, transgenic plants comprising polynucleotides encoding regulatory polypeptides may also modify expression of endogenous genes, polynucleotides, and proteins. Examples include Peng et al., 1997. Genes Development 11: 3194-3205, and Peng et al., 1999. Nature 400: 256-261. In addition, many others have demonstrated that an Arabidopsis regulatory polypeptide expressed in an exogenous plant species elicits the same or very similar phenotypic response. See, for example, Fu et al., 2001. Plant Cell 13: 1791-1802; Nandi et al., 2000. Curr. Biol. 10: 215-218; Coupland, 1995. Nature 377: 482-483; and Weigel and Nilsson, 1995. Nature 377: 482-500.
In another example, Mandel et al., 1992b. Cell 71-133-143, and Suzuki et al., 2001. Plant J. 28: 409-418, teach that a transcription factor expressed in another plant species elicits the same or very similar phenotypic response of the endogenous sequence, as often predicted in earlier studies of Arabidopsis transcription factors in Arabidopsis (see Mandel et al., 1992a. Nature 360: 273-277; Suzuki et al., 2001. supra). Other examples include Müller et al., 2001. Plant J. 28: 169-179; Kim et al., 2001. Plant J. 25: 247-259; Kyozuka and Shimamoto, 2002. Plant Cell Physiol. 43: 130-135; Boss and Thomas, 2002. Nature, 416: 847-850; He et al., 2000. Transgenic Res. 9: 223-227; and Robson et al., 2001. Plant J. 28: 619-631.
In yet another example, Gilmour et al., 1998. Plant J. 16: 433-442 teach an Arabidopsis AP2 transcription factor, CBF1, which, when overexpressed in transgenic plants, increases plant freezing tolerance. Jaglo et al., 2001. Plant Physiol. 127: 910-917, further identified sequences in Brassica napus which encode CBF-like genes and that transcripts for these genes accumulated rapidly in response to low temperature. Transcripts encoding CBF proteins were also found to accumulate rapidly in response to low temperature in wheat, as well as in tomato. An alignment of the CBF proteins from Arabidopsis, B. napus, wheat, rye, and tomato revealed the presence of conserved consecutive amino acid residues which bracket the AP2/EREBP DNA binding domains of the proteins and distinguish them from other members of the AP2/EREBP protein family (Jaglo et al., 2001. supra).
Regulatory polypeptides mediate cellular responses and control traits through altered expression of genes containing cis-acting nucleotide sequences that are targets of the introduced regulatory polypeptide. It is well appreciated in the art that the effect of a regulatory polypeptide on cellular responses or a cellular trait is determined by the particular genes whose expression is either directly or indirectly (e.g., by a cascade of regulatory polypeptide binding events and transcriptional changes) altered by regulatory polypeptide binding. In a global analysis of transcription comparing a standard condition with one in which a regulatory polypeptide is overexpressed, the resulting transcript profile associated with regulatory polypeptide overexpression is related to the trait or cellular process controlled by that regulatory polypeptide. For example, the PAP2 gene and other genes in the Myb family have been shown to control anthocyanin biosynthesis through regulation of the expression of genes known to be involved in the anthocyanin biosynthetic pathway (Bruce et al., 2000. Plant Cell 12: 65-79; and Borevitz et al., 2000. Plant Cell 12: 2383-2393). Further, global transcript profiles have been used successfully as diagnostic tools for specific cellular states (e.g., cancerous vs. non-cancerous; Bhattacharjee et al., 2001. Proc. Natl. Acad. Sci. USA 98: 13790-13795; and Xu et al., 2001. Proc. Natl. Acad. Sci. USA 98: 15089-15094). Consequently, it is evident to one skilled in the art that similarity of transcript profile upon overexpression of different regulatory polypeptides would indicate similarity of regulatory polypeptide function.
Polypeptides and Polynucleotides of the Present Description.
The present description includes putative regulatory polypeptides, and isolated or recombinant polynucleotides encoding the polypeptides, or novel sequence variant polypeptides or polynucleotides encoding novel variants of polypeptides derived from the specific sequences provided in the Sequence Listing; the recombinant polynucleotides of the instant description may be incorporated in expression vectors for the purpose of producing transformed plants.
Because of their relatedness at the nucleotide level, the claimed sequences will typically share at least about 30% nucleotide sequence identity, or at least 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95% or 96%, 97%, 98%, 99%, or about 100% sequence identity to one or more of the listed full-length sequences, or to a listed sequence but excluding or outside of the region(s) encoding a known consensus sequence or consensus DNA-binding site, or outside of the region(s) encoding one or all conserved domains. The degeneracy of the genetic code enables major variations in the nucleotide sequence of a polynucleotide while maintaining the amino acid sequence of the encoded protein.
Because of their relatedness at the protein level, the claimed nucleotide sequences will typically encode a polypeptide that is at least 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95% or 96%, 97%, 98%, 99%, or about 100% identical in its amino acid sequence to the entire length of any of the polypeptides listed in the Sequence Listing or the instant Tables, or closely- or phylogenetically-related sequences.
Also provided are methods for modifying yield from a plant by modifying the mass, size or number of plant organs or seed of a plant by controlling a number of cellular processes, and for increasing a plant's photosynthetic resource use efficiency. These methods are based on the ability to alter the expression of critical regulatory molecules that may be conserved between diverse plant species. Related conserved regulatory molecules may be originally discovered in a model system such as Arabidopsis and homologous, functional molecules then discovered in other plant species. The latter may then be used to confer increased yield or photosynthetic resource use efficiency in diverse plant species.
Sequences in the Sequence Listing, derived from diverse plant species, may be ectopically expressed in overexpressor plants. The changes in the characteristic(s) or trait(s) of the plants may then be observed and found to confer increased yield and/or increased photosynthetic resource use efficiency. Therefore, the polynucleotides and polypeptides can be used to improve desirable characteristics of plants.
The polynucleotides of the instant description are also ectopically expressed in overexpressor plant cells and the changes in the expression levels of a number of genes, polynucleotides, and/or proteins of the plant cells observed. Therefore, the polynucleotides and polypeptides can be used to change expression levels of genes, polynucleotides, and/or proteins of plants or plant cells.
The data presented herein represent the results obtained in experiments with polynucleotides and polypeptides that may be expressed in plants for the purpose of increasing yield that arises from improved photosynthetic resource use efficiency.
Variants of the Disclosed Sequences.
Also within the scope of the instant description is a variant of a nucleic acid listed in the Sequence Listing, that is, one having a sequence that differs from the one of the polynucleotide sequences in the Sequence Listing, or a complementary sequence, that encodes a functionally equivalent polypeptide (i.e., a polypeptide having some degree of equivalent or similar biological activity) but differs in sequence from the sequence in the Sequence Listing, due to degeneracy in the genetic code. Included within this definition are polymorphisms that may or may not be readily detectable using a particular oligonucleotide probe of the polynucleotide encoding polypeptide, and improper or unexpected hybridization to allelic variants, with a locus other than the normal chromosomal locus for the polynucleotide sequence encoding polypeptide.
Differences between presently disclosed polypeptides and polypeptide variants are limited so that the sequences of the former and the latter are closely similar overall and, in many regions, identical. Presently disclosed polypeptide sequences and similar polypeptide variants may differ in amino acid sequence by one or more substitutions, additions, deletions, fusions and truncations, which may be present in any combination. These differences may produce silent changes and result in a functionally equivalent polypeptides. Thus, it will be readily appreciated by those of skill in the art, that any of a variety of polynucleotide sequences is capable of encoding the polypeptides and homolog polypeptides of the instant description. A polypeptide sequence variant may have “conservative” changes, wherein a substituted amino acid has similar structural or chemical properties.
Conservative substitutions include substitutions in which at least one residue in the amino acid sequence has been removed and a different residue inserted in its place. Such substitutions generally are made in accordance with the Table 1 when it is desired to maintain the activity of the protein. Table 1 shows amino acids which can be substituted for an amino acid in a protein and which are typically regarded as conservative substitutions.

TABLE 1

Possible conservative amino acid substitutions

	Amino Acid	Conservative
	Residue	substitutions

	Ala	Ser
	Arg	Lys
	Asn	Gln; His
	Asp	Glu
	Gln	Asn
	Cys	Ser
	Glu	Asp
	Gly	Pro
	His	Asn; Gln
	Ile	Leu, Val
	Leu	Ile; Val
	Lys	Arg; Gln
	Met	Leu; Ile
	Phe	Met; Leu; Tyr
	Pro	Gly
	Ser	Thr; Gly
	Thr	Ser; Val
	Trp	Tyr
	Tyr	Trp; Phe
	Val	Ile; Leu

The polypeptides provided in the Sequence Listing have a novel activity, such as, for example, regulatory activity. Although all conservative amino acid substitutions (for example, one basic amino acid substituted for another basic amino acid) in a polypeptide will not necessarily result in the polypeptide retaining its activity, it is expected that many of these conservative mutations would result in the polypeptide retaining its activity. Most mutations, conservative or non-conservative, made to a protein but outside of a conserved domain required for function and protein activity will not affect the activity of the protein to any great extent.
Deliberate amino acid substitutions may thus be made on the basis of similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity, and/or the amphipathic nature of the residues, as long as a significant amount of the functional or biological activity of the polypeptide is retained. For example, negatively charged amino acids may include aspartic acid and glutamic acid, positively charged amino acids may include lysine and arginine, and amino acids with uncharged polar head groups having similar hydrophilicity values may include leucine, isoleucine, and valine; glycine and alanine; asparagine and glutamine; serine and threonine; and phenylalanine and tyrosine. More rarely, a variant may have “non-conservative” changes, e.g., replacement of a glycine with a tryptophan Similar minor variations may also include amino acid deletions or insertions, or both. Related polypeptides may comprise, for example, additions and/or deletions of one or more N-linked or O-linked glycosylation sites, or an addition and/or a deletion of one or more cysteine residues. Guidance in determining which and how many amino acid residues may be substituted, inserted or deleted without abolishing functional or biological activity may be found using computer programs well known in the art, for example, DNASTAR software (see U.S. Pat. No. 5,840,544).
Conserved Domains.
Conserved domains are recurring functional and/or structural units of a protein sequence within a protein family (for example, a family of regulatory proteins), and distinct conserved domains have been used as building blocks in molecular evolution and recombined in various arrangements to make proteins of different protein families with different functions. Conserved domains often correspond to the 3-dimensional domains of proteins and contain conserved sequence patterns or motifs, which allow for their detection in polypeptide sequences with, for example, the use of a Conserved Domain Database (for example, at www.ncbi.nlm.nih.gov/cdd). The National Center for Biotechnology Information Conserved Domain Database defines conserved domains as recurring units in molecular evolution, the extents of which can be determined by sequence and structure analysis. Conserved domains contain conserved sequence patterns or motifs, which allow for their detection in polypeptide sequences (Conserved Domain Database; www.ncbi.nlm.nih.gov/Structure/cdd/cdd.shtml).
Conserved domains may also be identified as regions or domains of identity to a specific consensus sequence (see, for example, Riechmann et al., 2000a. Science 290, 2105-2110; Riechmann et al., 2000b. Curr Opin Plant Biol 3: 423-434). Thus, by using alignment methods well known in the art, the conserved domains of the plant polypeptides, for example, for the Myb domain polypeptides may be determined. The polypeptides of the instant Tables have conserved domains associated with the disclosed functions of the proteins in which they are found and specifically indicated by amino acid coordinate start and stop sites. A comparison of the regions of these polypeptides allows one of skill in the art (see, for example, Reeves and Nissen, 1990. J. Biol. Chem. 265, 8573-8582; Reeves and Nissen, 1995. Prog. Cell Cycle Res. 1: 339-349) to identify domains or conserved domains for any of the polypeptides listed or referred to in this disclosure.
Conserved domain models are generally identified with multiple sequence alignments of related proteins spanning a variety of organisms (for example, conserved domains of the disclosed sequences can be found in the instant Figures, Tables, and the Sequence Listing). These alignments reveal sequence regions containing the same, or similar, patterns of amino acids. Multiple sequence alignments, three-dimensional structure and three-dimensional structure superposition of conserved domains can be used to infer sequence, structure, and functional relationships (Conserved Domain Database, supra). Since the presence of a particular conserved domain within a polypeptide (prophetically including any of the instantly listed polypeptides) is highly correlated with an evolutionarily conserved function, a conserved domain database may be used to identify the amino acids in a protein sequence that are putatively involved in functions such as binding or catalysis, as mapped from conserved domain annotations to the query sequence. For example, the presence in a protein of Myb domain that is structurally and phylogenetically similar to one or more domains shown in the instant Tables would be a strong indicator of a related function in plants (e.g., the function of regulating and/or improving yield, light use efficiency, photosynthetic capacity, photosynthetic rate, photosynthetic resource use efficiency, vigor, and/or biomass as compared to a control plant; i.e., a polypeptide with such a domain is expected to confer altered yield, light use efficiency, photosynthetic capacity, photosynthetic rate, photosynthetic resource use efficiency, vigor, and/or biomass as compared to a control plant when its expression level is altered). Sequences herein referred to as functionally-related and/or closely-related to the sequences or domains listed in the instant Tables, including polypeptides that are closely related to the polypeptides of the instant description, may have conserved domains that share at least at least nine base pairs (bp) in length and at least 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95% or 96%, 97%, 98%, 99%, or about 100% amino acid identity to the sequences provided in the Sequence Listing or in the instant Tables, and have similar functions in that the polypeptides of the instant description. Said polypeptides may, when their expression level is altered by suppressing their expression, knocking out their expression, or increasing their expression, confer at least one regulatory activity selected from the group consisting of increased yield, increased light use efficiency, increased photosynthetic capacity, increased photosynthetic rate, increased photosynthetic resource use efficiency, greater vigor, and/or greater biomass as compared to a control plant.
Methods using manual alignment of sequences similar or homologous to one or more polynucleotide sequences or one or more polypeptides encoded by the polynucleotide sequences may be used to identify regions of similarity and conserved domains or other motifs. Such manual methods are well-known of those of skill in the art and can include, for example, comparisons of tertiary structure between a polypeptide sequence encoded by a polynucleotide that comprises a known function and a polypeptide sequence encoded by a polynucleotide sequence that has a function not yet determined. Such examples of tertiary structure may comprise predicted alpha helices, beta-sheets, amphipathic helices, leucine zipper motifs, zinc finger motifs, proline-rich regions, cysteine repeat motifs, and the like.
With respect to polynucleotides encoding presently disclosed polypeptides, a conserved domain refers to a subsequence within a polypeptide family (for example, in any of the instantly listed polypeptides or members of the listed polypeptide families) the presence of which is correlated with at least one function exhibited by members of the polypeptide family, and which exhibits a high degree of sequence homology, such as at least 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95% or 96%, 97%, 98%, 99%, or about 100% identity to a conserved domain of a polypeptide of the Sequence Listing or listed in the instant Tables that show the instant polypeptides and closely-related or phylogenetically-related sequences. Sequences that possess or encode for conserved domains that meet these criteria of percentage identity, and that have comparable biological and regulatory activity to the present polypeptide sequences, thus being members of the clade polypeptides or sequences listed in the sequence Listing or in Example I, are described. Sequences having lesser degrees of identity but comparable biological activity are considered to be equivalents.
Orthologs and Paralogs.
Homologous sequences as described above can comprise orthologous or paralogous sequences. Several different methods are known by those of skill in the art for identifying and defining these functionally homologous sequences. General methods for identifying orthologs and paralogs, including phylogenetic methods, sequence similarity and hybridization methods, are described herein; an ortholog or paralog, including equivalogs, may be identified by one or more of the methods described below.
As described by Eisen, 1998. Genome Res. 8: 163-167, evolutionary information may be used to predict gene function. It is common for groups of genes that are homologous in sequence to have diverse, although usually related, functions. However, in many cases, the identification of homologs is not sufficient to make specific predictions because not all homologs have the same function. Thus, an initial analysis of functional relatedness based on sequence similarity alone may not provide one with a means to determine where similarity ends and functional relatedness begins. Fortunately, it is well known in the art that protein function can be classified using phylogenetic analysis of gene trees combined with the corresponding species. Functional predictions can be greatly improved by focusing on how the genes became similar in sequence (i.e., by evolutionary processes) rather than on the sequence similarity itself (Eisen, supra). In fact, many specific examples exist in which gene function has been shown to correlate well with gene phylogeny (Eisen, supra). Thus, “[t]he first step in making functional predictions is the generation of a phylogenetic tree representing the evolutionary history of the gene of interest and its homologs. Such trees are distinct from clusters and other means of characterizing sequence similarity because they are inferred by techniques that help convert patterns of similarity into evolutionary relationships . . . . After the gene tree is inferred, biologically determined functions of the various homologs are overlaid onto the tree. Finally, the structure of the tree and the relative phylogenetic positions of genes of different functions are used to trace the history of functional changes, which is then used to predict functions of [as yet] uncharacterized genes” (Eisen, supra).
Within a single plant species, gene duplication may cause two copies of a particular gene, giving rise to two or more genes with similar sequence and often similar function known as paralogs. A paralog is therefore a similar gene formed by duplication within the same species. Paralogs typically cluster together or in the same clade (a group of similar genes) when a gene family phylogeny is analyzed using programs such as CLUSTAL (Thompson et al., 1994. Nucleic Acids Res. 22: 4673-4680; Higgins et al., 1996. Methods Enzymol. 266: 383-402). Groups of similar genes can also be identified with pair-wise BLAST analysis (Feng and Doolittle, 1987. J. Mol. Evol. 25: 351-360). For example, a clade of very similar MADS domain transcription factors from Arabidopsis all share a common function in flowering time (Ratcliffe et al., 2001. Plant Physiol. 126: 122-132), and a group of very similar AP2 domain transcription factors from Arabidopsis are involved in tolerance of plants to freezing (Gilmour et al., 1998. supra). Analysis of groups of similar genes with similar function that fall within one clade can yield sub-sequences that are particular to the clade. These sub-sequences, known as consensus sequences, can not only be used to define the sequences within each clade, but define the functions of these genes; genes within a clade may contain paralogous sequences, or orthologous sequences that share the same function (see also, for example, Mount, 2001, in Bioinformatics: Sequence and Genome Analysis, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., p. 543).
Regulatory polypeptide gene sequences are conserved across diverse eukaryotic species lines (Goodrich et al., 1993. Cell 75:519-530; Lin et al., 1991. Nature 353:569-571; Sadowski et al., 1988. Nature 335: 563-564). Plants are no exception to this observation; diverse plant species possess regulatory polypeptides that have similar sequences and functions. Speciation, the production of new species from a parental species, gives rise to two or more genes with similar sequence and similar function. These genes, termed orthologs, often have an identical function within their host plants and are often interchangeable between species without losing function. Because plants have common ancestors, many genes in any plant species will have a corresponding orthologous gene in another plant species. Once a phylogenic tree for a gene family of one species has been constructed using a program such as CLUSTAL (Thompson et al., 1994. supra; Higgins et al., 1996. supra) potential orthologous sequences can be placed into the phylogenetic tree and their relationship to genes from the species of interest can be determined. Orthologous sequences can also be identified by a reciprocal BLAST strategy. Once an orthologous sequence has been identified, the function of the ortholog can be deduced from the identified function of the reference sequence.
By using a phylogenetic analysis, one skilled in the art would recognize that the ability to deduce similar functions conferred by closely-related polypeptides is predictable. This predictability has been confirmed by our own many studies in which we have found that a wide variety of polypeptides have orthologous or closely-related homologous sequences that function as does the first, closely-related reference sequence. For example, distinct regulatory polypeptides, including:
(i) AP2 family Arabidopsis G47 (found in U.S. Pat. No. 7,135,616), a phylogenetically-related sequence from soybean, and two phylogenetically-related homologs from rice all can confer greater tolerance to drought, hyperosmotic stress, or delayed flowering as compared to control plants;
(ii) CAAT family Arabidopsis G481 (found in PCT patent publication no. WO2004076638), and numerous phylogenetically-related sequences from eudicots and monocots can confer greater tolerance to drought-related stress as compared to control plants;
(iii) Myb-related Arabidopsis G682 (found in U.S. Pat. Nos. 7,223,904 and 7,193,129) and numerous phylogenetically-related sequences from eudicots and monocots can confer greater tolerance to heat, drought-related stress, cold, and salt as compared to control plants;
(iv) WRKY family Arabidopsis G1274 (found in U.S. Pat. No. 7,196,245) and numerous closely-related sequences from eudicots and monocots have been shown to confer increased water deprivation tolerance, and
(v) AT-hook family soy sequence G3456 (found in U.S. patent publication no. 20040128712A1) and numerous phylogenetically-related sequences from eudicots and monocots, increased biomass compared to control plants when these sequences are overexpressed in plants.
Examples of Methods for Identifying Identity, Similarity, Homology and Relatedness.
Percent identity can be determined electronically, e.g., by using the MEGALIGN program (DNASTAR, Inc. Madison, Wis.). The MEGALIGN program can create alignments between two or more sequences according to different methods, for example, the clustal method (see, for example, Higgins and Sharp, 1988. Gene 73: 237-244). The clustal algorithm groups sequences into clusters by examining the distances between all pairs. The clusters are aligned pairwise and then in groups. Other alignment algorithms or programs may be used for preparing alignments and/or determining percentage identities, including Accelrys Gene, FASTA, BLAST, or ENTREZ, FASTA and BLAST, some of which may also be used to calculate percent similarity. Accelrys Gene is available from Accelrys, Inc., San Diego, Calif. Other programs are available as a part of the GCG sequence analysis package (University of Wisconsin, Madison, Wis.), and can be used with or without default settings. ENTREZ is available through the National Center for Biotechnology Information. In one embodiment, the percent identity of two sequences can be determined by the GCG program with a gap weight of 1, e.g., each amino acid gap is weighted as if it were a single amino acid or nucleotide mismatch between the two sequences (see U.S. Pat. No. 6,262,333).
Software for performing BLAST analyses is publicly available, e.g., through the National Center for Biotechnology Information (see internet website at www.ncbi.nlm.nih.gov/). This algorithm involves first identifying high scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence, which either match or satisfy some positive-valued threshold score T when aligned with a word of the same length in a database sequence. T is referred to as the neighborhood word score threshold (Altschul, 1990. J. Mol. Biol. 215: 403-410; Altschul, 1993. J. Mol. Evol. 36: 290-300). These initial neighborhood word hits act as seeds for initiating searches to find longer HSPs containing them. The word hits are then extended in both directions along each sequence for as far as the cumulative alignment score can be increased. Cumulative scores are calculated using, for nucleotide sequences, the parameters M (reward score for a pair of matching residues; always >0) and N (penalty score for mismatching residues; always <0). For amino acid sequences, a scoring matrix is used to calculate the cumulative score. Extension of the word hits in each direction are halted when: the cumulative alignment score falls off by the quantity X from its maximum achieved value; the cumulative score goes to zero or below, due to the accumulation of one or more negative-scoring residue alignments; or the end of either sequence is reached. The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment. The BLASTN program (for nucleotide sequences) uses as defaults a wordlength (W) of 11, an expectation (E) of 10, a cutoff of 100, M=5, N=−4, and a comparison of both strands. For amino acid sequences, the BLASTP program uses as defaults a wordlength (W) of 3, an expectation (E) of 10, and the BLOSUM62 scoring matrix (see Henikoff and Henikoff, 1989. supra; Henikoff and Henikoff, 1991. supra). Unless otherwise indicated for comparisons of predicted polynucleotides, “sequence identity” refers to the % sequence identity generated from a tBLASTx using the NCBI version of the algorithm at the default settings using gapped alignments with the filter “off” (see, for example, internet website at www.ncbi.nlm.nih.gov).
Other techniques for alignment are described by Doolittle, ed., 1996. Methods in Enzymology, vol. 266: “Computer Methods for Macromolecular Sequence Analysis” Academic Press, Inc., San Diego, Calif., USA. Preferably, an alignment program that permits gaps in the sequence is utilized to align the sequences. The Smith-Waterman is one type of algorithm that permits gaps in sequence alignments (see Shpaer, 1997. Methods Mol. Biol. 70: 173-187). Also, the GAP program using the Needleman and Wunsch alignment method can be utilized to align sequences. An alternative search strategy uses MPSRCH software, which runs on a MASPAR computer. MPSRCH uses a Smith-Waterman algorithm to score sequences on a massively parallel computer. This approach improves ability to pick up distantly related matches, and is especially tolerant of small gaps and nucleotide sequence errors. Nucleic acid-encoded amino acid sequences can be used to search both protein and DNA databases.
The percentage similarity between two polypeptide sequences, e.g., sequence A and sequence B, is calculated by dividing the length of sequence A, minus the number of gap residues in sequence A, minus the number of gap residues in sequence B, into the sum of the residue matches between sequence A and sequence B, times one hundred. Gaps of low or of no similarity between the two amino acid sequences are not included in determining percentage similarity. Percent identity between polynucleotide sequences can also be counted or calculated by other methods known in the art, e.g., the Jotun Hein method (see, for example, Hein, 1990. Methods Enzymol. 183: 626-645). Identity between sequences can also be determined by other methods known in the art, e.g., by varying hybridization conditions (see U.S. patent publication no. 20010010913).
The percent identity between two polypeptide sequences can also be determined using Accelrys Gene v2.5, 2006 with default parameters: Pairwise Matrix: GONNET; Align Speed: Slow; Open Gap Penalty: 10.000; Extended Gap Penalty: 0.100; Multiple Matrix: GONNET; Multiple Open Gap Penalty: 10.000; Multiple Extended Gap Penalty: 0.05; Delay Divergent: 30; Gap Separation Distance: 8; End Gap Separation: false; Residue Specific Penalties: false; Hydrophilic Penalties: false; Hydrophilic Residues: GPSNDQEKR. The default parameters for determining percent identity between two polynucleotide sequences using Accelrys Gene are: Align Speed: Slow; Open Gap Penalty: 10.000; Extended Gap Penalty: 5.000; Multiple Open Gap Penalty: 10.000; Multiple Extended Gap Penalty: 5.000; Delay Divergent: 40; Transition: Weighted.
In addition, one or more polynucleotide sequences or one or more polypeptides encoded by the polynucleotide sequences may be used to search against a BLOCKS (Bairoch et al., 1997. Nucleic Acids Res. 25: 217-221), PFAM, and other databases which contain previously identified and annotated motifs, sequences and gene functions. Methods that search for primary sequence patterns with secondary structure gap penalties (Smith et al., 1992. Protein Engineering 5: 35-51) as well as algorithms such as Basic Local Alignment Search Tool (BLAST; Altschul, 1990, supra; Altschul et al., 1993, supra), BLOCKS (Henikoff and Henikoff, 1991, supra), Hidden Markov Models (HMM; Eddy, 1996. Curr. Opin. Str. Biol. 6: 361-365; Sonnhammer et al., 1997. Proteins 28: 405-420), and the like, can be used to manipulate and analyze polynucleotide and polypeptide sequences encoded by polynucleotides. These databases, algorithms and other methods are well known in the art and are described in Ausubel et al., 1997. Short Protocols in Molecular Biology, John Wiley & Sons, New York, N.Y., unit 7.7, and in Meyers, 1995. Molecular Biology and Biotechnology, Wiley VCH, New York, N.Y., p 856-853.
Thus, the instant description provides methods for identifying a sequence similar or paralogous or orthologous or homologous to one or more polynucleotides as noted herein, or one or more target polypeptides encoded by the polynucleotides, or otherwise noted herein and may include linking or associating a given plant phenotype or gene function with a sequence. In the methods, a sequence database is provided (locally or across an internet or intranet) and a query is made against the sequence database using the relevant sequences herein and associated plant phenotypes or gene functions.
A further method for identifying or confirming that specific homologous sequences control the same function is by comparison of the transcript profile(s) obtained upon overexpression or knockout of two or more related polypeptides. Since transcript profiles are diagnostic for specific cellular states, one skilled in the art will appreciate that genes that have a highly similar transcript profile (e.g., with greater than 50% regulated transcripts in common, or with greater than 70% regulated transcripts in common, or with greater than 90% regulated transcripts in common) will have highly similar functions. Fowler and Thomashow, 2002. Plant Cell 14, 1675-1690, have shown that three paralogous AP2 family genes (CBF1, CBF2 and CBF3) are induced upon cold treatment, each of which can condition improved freezing tolerance, and all have highly similar transcript profiles. Once a polypeptide has been shown to provide a specific function, its transcript profile becomes a diagnostic tool to determine whether paralogs or orthologs have the same function.
Identifying Polynucleotides or Nucleic Acids by Hybridization.
Polynucleotides homologous to the sequences illustrated in the Sequence Listing and tables can be identified, e.g., by hybridization to each other under stringent or under highly stringent conditions. Stringency is influenced by a variety of factors, including temperature, salt concentration and composition, organic and non-organic additives, solvents, etc. present in both the hybridization and wash solutions and incubations, and the number of washes, as described in more detail in the references cited below (e.g., Sambrook et al., 1989. supra; Berger and Kimmel, eds., 1987. Methods Enzymol. 152: 507-511; Anderson and Young, 1985. “Quantitative Filter Hybridisation”, In: Hames and Higgins, ed., Nucleic Acid Hybridisation, A Practical Approach. Oxford, IRL Press, 73-111), each of which are incorporated herein by reference. Conditions that are highly stringent, and means for achieving them, are also well known in the art and described in, for example, Sambrook et al., 1989. supra; Berger and Kimmel, eds., 1987. Meth. Enzymol. 152:467-469; and Anderson and Young, 1985. supra.
Also provided in the instant description are polynucleotide sequences that are capable of hybridizing to the claimed polynucleotide sequences, including any of the polynucleotides within the Sequence Listing, and fragments thereof under various conditions of stringency (see, for example, Wahl and Berger, 1987. Methods Enzymol. 152: 399-407; Berger and Kimmel, ed., 1987. Methods Enzymol. 152:507-511). In addition to the nucleotide sequences listed in the Sequence Listing, full length cDNA, orthologs, and paralogs of the present nucleotide sequences may be identified and isolated using well-known methods. The cDNA libraries, orthologs, and paralogs of the present nucleotide sequences may be screened using hybridization methods to determine their utility as hybridization target or amplification probes.
Stability of DNA duplexes is affected by such factors as base composition, length, and degree of base pair mismatch. Hybridization conditions may be adjusted to allow DNAs of different sequence relatedness to hybridize. The melting temperature (T_m) is defined as the temperature when 50% of the duplex molecules have dissociated into their constituent single strands. The melting temperature of a perfectly matched duplex, where the hybridization buffer contains formamide as a denaturing agent, may be estimated by the following equations:
T _m(° C.)=81.5+16.6(log [Na+])+0.41(% G+C)−0.62(% formamide)−500/L (I) DNA-DNA:
T _m(° C.)=79.8+18.5(log [Na+])+0.58(% G+C)+0.12(% G+C)²−0.5(% formamide)−820/L (II) DNA-RNA:
T _m(° C.)=79.8+18.5(log [Na+])+0.58(% G+C)+0.12(% G+C)²−0.35(% formamide)−820/L (III) RNA-RNA:
where L is the length of the duplex formed, [Na+] is the molar concentration of the sodium ion in the hybridization or washing solution, and % G+C is the percentage of (guanine+cytosine) bases in the hybrid. For imperfectly matched hybrids, approximately 1° C. is required to reduce the melting temperature for each 1% mismatch.
Hybridization experiments are generally conducted in a buffer of pH between 6.8 to 7.4, although the rate of hybridization is nearly independent of pH at ionic strengths likely to be used in the hybridization buffer (Anderson and Young, 1985. supra). In addition, one or more of the following may be used to reduce non-specific hybridization: sonicated salmon sperm DNA or another non-complementary DNA, bovine serum albumin, sodium pyrophosphate, sodium dodecylsulfate (SDS), polyvinyl-pyrrolidone, ficoll and Denhardt's solution. Dextran sulfate and polyethylene glycol 6000 act to exclude DNA from solution, thus raising the effective probe DNA concentration and the hybridization signal within a given unit of time. In some instances, conditions of even greater stringency may be desirable or required to reduce non-specific and/or background hybridization. These conditions may be created with the use of higher temperature, lower ionic strength and higher concentration of a denaturing agent such as formamide.
Stringency conditions can be adjusted to screen for moderately similar fragments such as homologous sequences from distantly related organisms, or to highly similar fragments such as genes that duplicate functional enzymes from closely related organisms. The stringency can be adjusted either during the hybridization step or in the post-hybridization washes. Salt concentration, formamide concentration, hybridization temperature and probe lengths are variables that can be used to alter stringency (as described by the formula above). As a general guideline, high stringency is typically performed at T_m−5° C. to T_m−20° C., moderate stringency at T_m−20° C. to T_m−35° C. and low stringency at T_m−35° C. to T_m−50° C. for duplex >150 base pairs. Hybridization may be performed at low to moderate stringency (25-50° C. below T_m), followed by post-hybridization washes at increasing stringencies. Maximum rates of hybridization in solution are determined empirically to occur at T_m−25° C. for DNA-DNA duplex and T_m−15° C. for RNA-DNA duplex. Optionally, the degree of dissociation may be assessed after each wash step to determine the need for subsequent, higher stringency wash steps.
High stringency conditions may be used to select for nucleic acid sequences with high degrees of identity to the disclosed sequences. An example of stringent hybridization conditions obtained in a filter-based method such as a Southern or Northern blot for hybridization of complementary nucleic acids that have more than 100 complementary residues is about 5° C. to 20° C. lower than the thermal melting point (T_m) for the specific sequence at a defined ionic strength and pH. Conditions used for hybridization may include about 0.02 M to about 0.15 M sodium chloride, about 0.5% to about 5% casein, about 0.02% SDS or about 0.1% N-laurylsarcosine, about 0.001 M to about 0.03 M sodium citrate, at hybridization temperatures between about 50° C. and about 70° C. More preferably, high stringency conditions are about 0.02 M sodium chloride, about 0.5% casein, about 0.02% SDS, about 0.001 M sodium citrate, at a temperature of about 50° C. Nucleic acid molecules that hybridize under stringent conditions will typically hybridize to a probe based on either the entire DNA molecule or selected portions, e.g., to a unique subsequence, of the DNA.
Stringent salt concentration will ordinarily be less than about 750 mM NaCl and 75 mM trisodium citrate. Increasingly stringent conditions may be obtained with less than about 500 mM NaCl and 50 mM trisodium citrate, to even greater stringency with less than about 250 mM NaCl and 25 mM trisodium citrate. Low stringency hybridization can be obtained in the absence of organic solvent, e.g., formamide, whereas high stringency hybridization may be obtained in the presence of at least about 35% formamide, and more preferably at least about 50% formamide. Stringent temperature conditions will ordinarily include temperatures of at least about 30° C., more preferably of at least about 37° C., and most preferably of at least about 42° C. with formamide present. Varying additional parameters, such as hybridization time, the concentration of detergent, e.g., sodium dodecyl sulfate (SDS) and ionic strength, are well known to those skilled in the art. Various levels of stringency are accomplished by combining these various conditions as needed.
The washing steps that follow hybridization may also vary in stringency; the post-hybridization wash steps primarily determine hybridization specificity, with the most critical factors being temperature and the ionic strength of the final wash solution. Wash stringency can be increased by decreasing salt concentration or by increasing temperature. Stringent salt concentration for the wash steps will preferably be less than about 30 mM NaCl and 3 mM trisodium citrate, and most preferably less than about 15 mM NaCl and 1.5 mM trisodium citrate.
Thus, high stringency hybridization and wash conditions that may be used to bind and remove polynucleotides with less than the desired homology to the nucleic acid sequences or their complements that encode the present polypeptides include, for example:
6×SSC at 65° C.;
50% formamide, 4×SSC at 42° C.; or
0.5×SSC, 0.1% SDS at 65° C.;
with, for example, two wash steps of 10-30 minutes each. Useful variations on these conditions will be readily apparent to those skilled in the art.
A person of skill in the art would not expect substantial variation among polynucleotide species provided with the present description because the highly stringent conditions set forth in the above formulae yield structurally similar polynucleotides.
If desired, one may employ wash steps of even greater stringency, including about 0.2×SSC, 0.1% SDS at 65° C. and washing twice, each wash step being about 30 minutes, or about 0.1×SSC, 0.1% SDS at 65° C. and washing twice for 30 minutes. The temperature for the wash solutions will ordinarily be at least about 25° C., and for greater stringency at least about 42° C. Hybridization stringency may be increased further by using the same conditions as in the hybridization steps, with the wash temperature raised about 3° C. to about 5° C., and stringency may be increased even further by using the same conditions except the wash temperature is raised about 6° C. to about 9° C. For identification of less closely related homologs, wash steps may be performed at a lower temperature, e.g., 50° C.
An example of a low stringency wash step employs a solution and conditions of at least 25° C. in 30 mM NaCl, 3 mM trisodium citrate, and 0.1% SDS over 30 minutes. Greater stringency may be obtained at 42° C. in 15 mM NaCl, with 1.5 mM trisodium citrate, and 0.1% SDS over 30 minutes. Even higher stringency wash conditions are obtained at 65° C.-68° C. in a solution of 15 mM NaCl, 1.5 mM trisodium citrate, and 0.1% SDS. Wash procedures will generally employ at least two final wash steps. Additional variations on these conditions will be readily apparent to those skilled in the art (see, for example, U.S. patent publication no. 20010010913).
Stringency conditions can be selected such that an oligonucleotide that is perfectly complementary to the coding oligonucleotide hybridizes to the coding oligonucleotide with at least about a 5-10× higher signal to noise ratio than the ratio for hybridization of the perfectly complementary oligonucleotide to a nucleic acid encoding a polypeptide known as of the filing date of the application. It may be desirable to select conditions for a particular assay such that a higher signal to noise ratio, that is, about 15× or more, is obtained. Accordingly, a subject nucleic acid will hybridize to a unique coding oligonucleotide with at least a 2× or greater signal to noise ratio as compared to hybridization of the coding oligonucleotide to a nucleic acid encoding known polypeptide. The particular signal will depend on the label used in the relevant assay, e.g., a fluorescent label, a colorimetric label, a radioactive label, or the like. Labeled hybridization or PCR probes for detecting related polynucleotide sequences may be produced by oligolabeling, nick translation, end-labeling, or PCR amplification using a labeled nucleotide.
The present description also provides polynucleotide sequences that are capable of hybridizing to the claimed polynucleotide sequences, including any of the polynucleotides within the Sequence Listing, and fragments thereof under various conditions of stringency (see, for example, Wahl and Berger, 1987, supra, pages 399-407; and Kimmel, 1987. Meth. Enzymol. 152, 507-511). In addition to the nucleotide sequences in the Sequence Listing, full length cDNA, orthologs, and paralogs of the present nucleotide sequences may be identified and isolated using well-known methods. The cDNA libraries, orthologs, and paralogs of the present nucleotide sequences may be screened using hybridization methods to determine their utility as hybridization target or amplification probes.

Examples

It is to be understood that this description is not limited to the particular devices, machines, materials and methods described. Although particular embodiments are described, equivalent embodiments may be used to practice the claims.
The specification, now being generally described, will be more readily understood by reference to the following examples, which are included merely for purposes of illustration of certain aspects and embodiments of the present description and are not intended to limit the claims or description. It will be recognized by one of skill in the art that a polypeptide that is associated with a particular first trait may also be associated with at least one other, unrelated and inherent second trait which was not predicted by the first trait.

Example I. The Instant Polynucleotides and their Encoded or Predicted Polypeptides

The instant polypeptides sequences belong to distinct clades of polypeptides that include members from diverse species. In each case, clade member sequences derived from both eudicots and monocots may be shown to confer increased yield or tolerance to one or more abiotic stresses when the sequences were overexpressed. These studies can demonstrate that evolutionarily conserved genes from diverse species are likely to function similarly (i.e., by regulating similar target sequences and controlling the same traits), and that polynucleotides from one species may be transformed into closely-related or distantly-related plant species to confer or improve traits.
The listed polypeptide sequences may be found within the polypeptide clades of Myb Domain Protein 27 (“AtMYB27”; AT3G53200; G1311), ACBF-like family member RNA-binding protein 45A (“RBP45A”; AT5G54900; G1940), PCF family member TEOSINTE BRANCHED1 //CYCLOIDEA//PCF 6 transcription factor 6 (“TCP6”; AT5G41030; G1936), Basic helix-loop-helix protein (bHLH) family member Phytochrome Interacting Factor 3-like 1 (“PIL1”: AT2G46970; G1649), GARP family member PHYTOCLOCK 1 (“PCL1”; AT3G46640.3; G2741), TH family member GT-2-Like1 (“GTL1”; AT1G33240; G634), AP2 family members Dehydration-Responsive Element-Binding Protein 2H (“DREB2H”; AT2G40350; G1755), and ethylene-responsive transcription factor ERF087 (“ERF087”; AT1G28160; G2292), CCAAT family member Nuclear Transcription Factor Y subunit C-6 (“NF-YC6”; AT5G50480; G1820), Z—CO-like family member CONSTANS-like B-box zinc finger protein (“BBX18”, “F3K23.8”; AT2G21320; G1881), HLH/MYC family member Basic Helix-Loop-Helix 60 (“bHLH60”; AT3G57800.2; G2144), Z—CO-like family member CONSTANS-like B-box zinc finger protein (“BBX26”, AT1G60250, G1486), bHLH family member bHLH121 (At3g19860, G782), and Putative MethylTransferase 24 (“PMT24” NP_174240, G837).
Orthologs and paralogs of presently disclosed polypeptides may be cloned using compositions provided by the present description according to methods well known in the art. cDNAs can be cloned using mRNA from a plant cell or tissue that expresses one of the present sequences. Appropriate mRNA sources may be identified by interrogating Northern blots with probes designed from the present sequences, after which a library is prepared from the mRNA obtained from a positive cell or tissue. Polypeptide-encoding cDNA is then isolated using, for example, PCR, using primers designed from a presently disclosed gene sequence, or by probing with a partial or complete cDNA or with one or more sets of degenerate probes based on the disclosed sequences. The cDNA library may be used to transform plant cells. Expression of the cDNAs of interest is detected using, for example, microarrays, Northern blots, quantitative PCR, or any other technique for monitoring changes in expression. Genomic clones may be isolated using similar techniques to those.
Examples of orthologs of the Arabidopsis polypeptide sequences and their functionally similar orthologs are listed in the instant Tables and the Sequence Listing. In addition to the sequences in the instant Tables and the Sequence Listing, the claimed nucleotide sequences are phylogenetically and structurally similar to sequences listed in the Sequence Listing and can function in a plant by increasing yield, light use efficiency, photosynthetic capacity, photosynthetic rate, photosynthetic resource use efficiency, vigor, and/or biomass as compared to a control plant when ectopically expressed, or overexpressed, in a plant. Since a significant number of these sequences are phylogenetically and sequentially related to each other and may be shown to increase yield from a plant and/or photosynthetic resource use efficiency, one skilled in the art would predict that other similar, phylogenetically related sequences falling within the present clades of polypeptides, including AtMYB27, RBP45A, TCP6, PIL1, PCL1, GTL1, DREB2H, ERF087, NF-YC6, BBX18, bHLH60, BBX26, bHLH121, or PMT24 clade polypeptide sequences, would also perform similar functions when ectopically expressed.
Background Information for AtMYB27, RBP45A, TCP6, PIL1, PCL1, GTL1, DREB2H, ERF087, NF-YC6, BBX18, bHLH60, BBX26, bHLH121, or PMT24, and their Clade Member Sequences
A number of phylogenetically-related sequences have been found in other plant species. The instant Tables list a number of AtMYB27, RBP45A, TCP6, PIL1, PCL1, GTL1, DREB2H, ERF087, NF-YC6, BBX18, bHLH60, BBX26, bHLH121, or PMT24 clade sequences from diverse species. The tables include the SEQ ID NO: (Column 1), the species from which the sequence was derived and the Gene Identifier (“GID”; Column 2), the percent identity of the polypeptide in Column 1 to the first listed full length polypeptide (SEQ ID NO: 2, 42, 86, 108, 126, 156, 192, 246, 278, 318, 356, 388, 410, or 444), as determined by a BLASTp analysis, for example, with a wordlength (W) of 3, an expectation (E) of 10, and the BLOSUM62 scoring matrix (Henikoff and Henikoff, 1989. Proc. Natl. Acad. Sci. USA 89:10915; Henikoff and Henikoff, 1991. Nucleic Acids Res. 19: 6565-6572) (Column 3), the amino acid residue coordinates for the conserved domains in amino acid coordinates beginning at the N-terminus, of each of the sequences (Column 4), the conserved domain sequences of the respective polypeptides (Column 5); the SEQ ID NO: of each of the domains (Column 6), and the percentage identity of the conserved domain in Column 5 to the conserved domain of the first listed sequence (as determined by a BLASTp analysis, wordlength (W) of 3, an expectation (E) of 10, and the BLOSUM62 scoring matrix, and with the proportion of identical amino acids in parentheses; Column 7).
Species abbreviations that appear in Columns 2 of the following Tables include: At—Arabidopsis thaliana; Bd—Brachypodium distachyon; Cc—Citrus clementina; Eg—Eucalyptus grandis; Gm—Glycine max; Os—Oryza sativa; Pt—Populus trichocarpa; Si—Setaria italica; Sl—Solanum lycopersicum; Vv—Vitus vinifera; Zm—Zea mays.

AtMYB27 Clade Polypeptides

TABLE 2

Conserved ‘Myb domain 1’ of AtMYB27 and closely related sequences

						Col. 7
						Percent
						identity
		Col. 3	Col. 4			of the
		Percent	Myb		Col. 6	Myb domain
Col. 1		identity of	domain 1		SEQ ID	in Col. 5
SEQ	Col. 2	polypeptide	in amino	Col. 5	NO: of	to the Myb
ID	Species/	in Col. 1	acid co-	Conserved Myb	Myb do-	domain 1
NO:	Identifier	to AtMyb27	ordinates	domain 1	main 1	of AtMyb27

2	At/AtMyb27 or	100%	11-58	RGPWLEEEDERLVKVI	483	100%
	AT3G53200.1	(238/238)		SLLGERRWDSLAIVSG		(48/48)
				LKRSGKSCRLRWMNY
				L

14	Vv/GSVIVT010	72%	8-55	KGSWLEEEDERLTAF	489	77%
	33670001	(83/115)		VGLLGERRWDSIARA		(37/48)
				SGLKRSGKSCRLRWL
				NYL

4	Gm/Glyma10g0	44%	8-55	KGTWLQEEDEQLTSF	484	75%
	6680.1	(106/236)		VTRLGERRWDSLAKV		(36/48)
				AGLKRSGKSCRLRWM
				NYL

6	Gm/Glyma13g2	70%	8-55	KGTWLQEEDEQLTSF	485	75%
	0880.1	(77/110)		VARLGERRWDSLAKV		(36/48)
				AGLKRSGKSCRLRWM
				NYL

8	Cc/clementine0.	49%	38-85	KGPWHEEEDELLVTF	486	72%
	9_029544m	(90/182)		VTLFGERRWDYIAKA		(35/48)
				SGLKRSGKSCRLRWL
				NYL

10	Eg/Eucgr.A016	43%	15-62	KGPWIEQEDEILTAFV	487	68%
	48.1	(102/234)		TVLGERRWDYIAKTS		(33/48)
				GLKRSGKSCRLRWKN
				YL

12	Pt/POPTR_000	48%	10-57	KGSWQEEEDERLTAS	488	68%
	6s12400.1	(98/203)		ATLLGERKWDSIARLS		(33/48)
				GLMRSGKSCRMRWL
				NYL

TABLE 3

Conserved ‘Myb domain 2’ of AtMYB27 and closely related sequences

						Col. 7
						Percent
						identity
		Col. 3	Col. 4			of second
		Percent	Myb		Col. 6	Myb domain
Col. 1		identity of	domain 2		SEQ ID	in Col. 5
SEQ	Col. 2	polypeptide	in amino	Col. 5	NO: of	to Myb do-
ID	Species/	in Col. 1	acid co-	Conserved Myb	Myb do-	main 2 of
NO:	Identifier	to AtMyb27	ordinates	domain 2	main 2	AtMyb27

2	At/AtMyb27 or	100%	64-109	RGPMSQEEERIIFQLH	490	100%
	AT3G53200.1	(238/238)		ALWGNKWSKIARRL		(46/46)
				PGRTDNEIKNYWRTH
				Y

12	Pt/POPTR_000	48%	63-108	RGHISAEEEQIIIQFHG	495	77%
	6s12400.1	(98/203)		QWGNKWARIARRLP		(35/45)
				GRTDNEIKNYWRTH
				M

14	Vv/GSVIVT01	72%	61-106	RCQISAEEEQIILQLH	496	76%
	033670001	(83/115)		KRWGNKWSWIARSL		(35/46)
				PGRTDNEIKNYWRTH
				L

10	Eg/Eucgr.A016	43%	68-113	HGPISPEEERIIIKFHE	494	72%
	48.Eg/1	(102/234)		QWGNKWSRIAEKLP		(32/44)
				GRTDNEIKNFWKTHL

4	Gm/Glyma10g0	44%	61-106	HGHFSVEEEQLIVQL	491	70%
	6680.1	(106/236)		QQQLGNKWAKIARK		(31/44)
				LPGRTDNEIKNFWRT
				HL

6	Gm/Glyma13g2	70%	61-106	HGHFSVEEEQLIVQL	492	68%
	0880.1	(77/110)		QQELGNKWAKIARK		(31/45)
				LPGRTDNEIKNYWKT
				HL

8	Cc/clementine0.	49%	91-139	HGYISTEEEQIIIQLHK	493	63%
	9_029544m	(90/182)		NIKIYLHGWSRIARSL		(29/46)
				PGRTDNEIKNCWRTR
				I

Sequences that are functionally-related and/or closely-related to the polypeptides in the above Tables may be created artificially, semi-synthetically, or may occur naturally by having descended from the same ancestral sequence as the disclosed closely-related sequences, where the polypeptides have the function of conferring increased photosynthetic resource use efficiency to plants.
These functionally-related and/or closely-related AtMYB27 clade polypeptides may be identified by a consensus first Myb domain (Myb domain 1) consensus sequence, SEQ ID NO: 842:

	X¹GX³WX⁵X⁶X⁷EDEX¹¹LX¹³X¹⁴X¹⁵X¹⁶X¹⁷X¹⁸X¹⁹GERX²³

	WDX²⁶X²⁷AX²⁹X³⁰X³¹GLX³⁴RSGKSCRX⁴²RWX⁴⁵NYL

where X¹=K or R; X³=any amino acid; X⁵=any amino acid; X⁶=Q or E; X⁷=Q or E; X¹¹=any amino acid; X¹³=T or V; X¹⁴=any amino acid; X¹⁵=any amino acid; X¹⁶=any amino acid; X¹⁷=S, A, T or G; X¹⁸=any amino acid; X¹⁹=F, I, L, V or M; X²³=R or K; X²⁶=any amino acid; X²⁷=I, L, V or M; X²⁹=any amino acid; X³⁰=any amino acid; X³¹=S or A; X³⁴=any amino acid; X⁴²=I, L, V or M; and X⁴⁵=any amino acid.
These functionally-related and/or closely-related AtMYB27 clade polypeptides also may be identified by a consensus second Myb domain (Myb domain 2) consensus sequence, SEQ ID NO: 843:
X¹X²X³X⁴SX⁶EEEX¹⁰X¹¹IX¹³X¹⁴X¹⁵X¹⁶X¹⁷X¹⁸X¹⁹X²⁰X²¹

X²²X²³X²⁴X²⁵WX²⁷X²⁸IAX³¹X³²LPGRTDNEIKNX⁴⁴WX⁴⁶TX⁴⁸

X⁴⁹

where X¹=any amino acid; X²=any amino acid; X³=any amino acid; X⁴=F, I, L, V or M; X⁶=any amino acid; X¹⁰=any amino acid; X¹¹=I, L, V or M; X¹³=F, I, L, V or M; X¹⁴=Q or K; X¹⁵=F, I, L, V or M; X¹⁶=H or Q; X¹⁷=any amino acid; X¹⁸=any amino acid; X¹⁹=any amino acid; X²⁰=any amino acid; X²¹=any amino acid; X²²=any amino acid; X²³=L or absent; X²⁴=H or absent; X²⁵=G or absent; X²⁷=S or A; X²⁸=any amino acid; X³¹=any amino acid; X³²=any amino acid; X⁴⁴=any amino acid; X⁴⁶=K or R; X⁴⁸=any amino acid; and X⁴⁹=any amino acid.

RBP45A Clade Polypeptides

TABLE 4

Conserved ‘RRM1 domain’ of RBP45A and closely related sequences

						Col. 7
						Percent
						identity
						of RRM1
		Col. 3				domain
Col.		Percent	Col. 4		Col. 6	in Col.
1		identity of	RRM1	Col. 5	SEQ ID	5 to
SEQ	Col. 2	polypeptide	domain in	Conserved	NO: of	RRM1 do-
ID	Species/	in Col. 1	amino acid	RRM1	RRM1	main of
NO:	Identifier	to RBP45A	coordinates	domain	domain	RBP45A

42	At/RBP45A or	100%	61-141	SLWIGDLQQWMDE	510	100%
	AT5G54900.1	(387/387)		NYIMSVFAQSGEAT		(81/81)
				SAKVIRNKLTGQSE
				GYGFIEFVSHSVAER
				VLQTYNGAPMPSTE
				QTFRLNWAQAG

40	At/AT4G27000	71%	81-161	SLWIGDLQPWMDEN	509	79%
	.1	(282/394)		YLMNVFGLTGEATA		(64/81)
				AKVIRNKQNGYSEG
				YGFIEFVNHATAER
				NLQTYNGAPMPSSE
				QAFRLNWAQLG

44	Pt/POPTR_000	70%	68-148	SLWIGDLQQWMDE	511	76%
	1s45000.1	(228/323)		NYILSIFSTTGEVVQ		(62/81)
				AKVIRNKQTGYPEG
				YGFIEFVSHAAAERI
				LQTYNGTPMPNSEQ
				TFRLNWATLG

46	Pt/POPTR_001	69%	71-151	SLWIGDLQQWMDE	512	72%
	1s14150.1	(231/331)		NYLLSIFSATGEIVQ		(59/81)
				AKVIRNKQTGYPEG
				YGFIEFVSRAAAERI
				LQTYNGTPMPNSEQ
				AFRLNWATLG

66	S1/Solyc02g080	66%	80-160	SLWIGDLQFWMDEQ	522	72%
	420.2.1	(216/325)		YLLNCFAQTGEVTS		(59/81)
				AKVIRNKQSGQSEG
				YGFIEFISHAAAERN
				LQAYNGTLMPNIEQ
				NFRLNWASLG

68	Sl/Solyc10g005	65%	77-157	TLWIGDLQFWMDE	523	71%
	260.2.1	(218/333)		QYLYSCFAQTGEVV		(58/81)
				SAKVIRNKQTQQSE
				GYGFIEFNSHAAAER
				NLQAYNGTLMPNIE
				QNFRLNWASLG

70	Sl/Solyc03g031	64%	73-153	SLWIGDLQFWMDEQ	524	70%
	720.2.1	(215/335)		YIQNCFAHTGEVAS		(57/81)
				VKVIRNKQSGQSEG
				YGFVEFISHAAAERN
				LQTYNGSMMPNSEQ
				PFRLNWASLG

48	Sl/Solyc07g064	67%	82-162	SLWIGDLQYWMDES	513	69%
	510.2.1	(218/324)		YLSTCFYHTGELVS		(56/81)
				AKVIRNKQSGQSEG
				YGFLEFRSHAAAET
				VLQTYNGALMPNVE
				QNFRMNWASLG

62	Gm/Glymal3g2	64%	67-147	TLWIGDLQYWMDE	520	69%
	7570.1	(214/331)		NYLYTCFAHTGEVT		(56/81)
				SVKVIRNKQTSQSEG
				YGFIEFNSRAGAERI
				LQTYNGAIMPNGGQ
				SFRLNWATFS

24	Os/LOC_Os08g	63%	96-174	TLWIGDLQYWMDE	501	69%
	09100.1	(206/325)		NYISACFAPTGELQS		(56/81)
				VKLIRDKQTGQLQG
				YGFIEFTSHAGAERV
				LQTYNGAMMPNVE
				QTYRLNWAS

54	Pt/POPTR_000	61%	78-158	TLWIGDLQYWMDE	516	69%
	4s01690.1	(213/349)		NYIASCFAHTGEVAS		(56/81)
				VKIIRNKQTSQIEGY
				GFIEMTSHGAAERIL
				QTYNGTPMPNGEQN
				FRLNWASFS

52	At/AT1G11650	63%	63-144	TLWIGDLQYWMDE	515	68%
	.2	(206/325)		NFLYGCFAHTGEMV		(56/82)
				SAKVIRNKQTGQVE
				GYGFIEFASHAAAER
				VLQTFNNAPIPSFPD
				QLFRLNWASLS

60	Gm/Glyma17g0	66%	66-146	TLWIGDLQYWMDE	519	67%
	1800.1	(218/327)		NYLYTCFAHTGELA		(55/81)
				SVKVIRNKQTSQSEG
				YGFIEFTSRAGAERV
				LQTYNGTIMPNGGQ
				NFRLNWATFS

64	Gm/Glyma15g1	64%	68-148	TLWIGDLQYWMDE	521	67%
	1380.1	(215/333)		NYLYTCFAHTGEVS		(55/81)
				SVKVIRNKQTSQSEG
				YGFIEFNSRAGAERI
				LQTYNGAIMPNGGQ
				SFRLNWATFS

38	Zm/GRMZM2	68%	71-151	TLWIGDLQYWMDE	508	66%
	G002874_T01	(219/322)		NYLYSCFSQAGEVIS		(54/81)
				VKIIRNKQTGQPEGY
				GFIEFSNHAVAEQVL
				QNYNGQMMPNVNQ
				PFKLNWATSG

58	Gm/Glyma07g3	66%	62-142	TLWIGDLQYWMDE	518	66%
	8940.1	(217/326)		NYLYTCLAHTGEVA		(54/81)
				SVKVIRNKQTSQSEG
				YGFIEFTSRAGAERV
				LQTYNGTIMPNGGQ
				NFRLNWATLS

50	Eg/Eucgr.F034	69%	65-145	SLWIGDLQPHMDET	514	65%
	62.1	(226/323)		YLLNCFAHSGEVLS		(53/81)
				AKVIRNKQTALPEG
				YGFIEFMTRAAAERI
				LQTYNGTLMPNSDQ
				NFRLNWATLG

36	Os/LOC_Os03g	67%	68-148	TLWIGDLQFWMEEN	507	65%
	37270.1	(218/323)		YLYNCFSQAGELISA		(53/81)
				KIIRNKQTGQPEGYG
				FIEFGSHAIAEQVLQ
				GYNGQMMPNGNQV
				FKLNWATSG

56	Eg/Eucgr.D013	63%	93-171	TLWIGDLQYWMDE	517	64%
	10.1	(193/303)		AYLGTCFAATGEVA		(52/81)
				NVKVIRNKQTMQPE
				GYGFIEFYTRAAAER
				VLQTYNGAIMPNGG
				QSFRLNWAS

26	Zm/GRMZM2	62%	122-200	TLWIGDLQYWMDD	502	62%
	G426591_T01	(204/324)		NYIYGCFASTGEVQ		(51/81)
				NVKLIRDKHTGQLQ
				GYGFIEFISRAAAER
				VLQTYNGTMMPNV
				ELPFRLNWAS

22	Bd/Bradi3g151	56%	99-177	TLWIGDLQYWMDE	500	61%
	80.1	(213/376)		NYVYGCFAHTGEVQ		(50/81)
				SVKLIRDKQTGQLQ
				GYGFVEFTTRAGAE
				RVLQTYNGATMPN
				VEMPYRLNWAS

16	Bd/Bradi5g224	61%	89-167	TLWIGDLQYWMDE	497	60%
	10.1	(201/327)		TYIHGCFASTGELQS		(49/81)
				VKLIRDKQTGQLQG
				YGFVEFTSHAAAER
				VLQGYNGHAMPNV
				DLAYRLNWAS

30	Os/LOC_Os07g	58%	129-210	SLWIGDLQYWMDES	504	59%
	33330.1	(205/348)		YLSNAFAPMGQQVT		(49/82)
				SVKVIRNKQSGHSE
				GYGFIEFQSHAAAE
				YALANFNGRMMLN
				VDQLFKLNWASSG

18	Zm/GRMZM2	58%	93-171	TLWIGDLQYWMDE	498	59%
	G012628_T01	(191/329)		NYVFGCFSNTGEVQ		(48/81)
				NVKLIRDKNSGQLQ
				GYGFVEFTSRAAAE
				RVLQTYNGQMMPN
				VDLTFRLNWAS

20	Zm/GRMZM2	58%	87-165	TLWIGDLQYWMDD	499	58%
	G058098_T02	(192/331)		NYVFGCFSNTGEVQ		(47/81)
				NVKLIRDKNSGQLQ
				GYGFVEFTSRAAAE
				RVLQTYNGQMMPN
				VDLTFRLNWAS

32	Zm/GRMZM2	53%	115-197	TLWIGDLQHWMDE	505	57%
	G127510_T01	(191/354)		NYLHYNAFAAVAQ		(48/83)
				QIASVKIIRNKQTGH
				SEGYGFIEFYSRAAA
				EHTLMNFNGQMMP
				NVEMTFKLNWASAS

34	Zm/GRMZM2	55%	147-229	TLWIGDLQYWMDE	506	56%
	G169615_T01	(188/338)		NYLHYNAFAPVAQQ		(47/83)
				IASVKIIRNKQTGHS
				EGYGFIEFYSQAAAE
				HTLMNFNGQMMPNI
				EMAFKLNWASAS

28	Bd/Bradi1g262	55%	115-197	SLWIGDLQYWMDE	503	56%
	10.1	(181/324)		AYLHNAFAPMGPQQ		(47/83)
				VASVKIIRNKQTGQP
				EGYGFIEFHSRAAAE
				YALASFNGHAMPNV
				DLPFKLNWASAS

TABLE 5

Conserved ‘RRM2 domain’ of RBP45A and closely related sequences

						Col. 7
						Percent
						identity
						of RRM2
		Col. 3				domain
Col.		Percent	Col. 4		Col. 6	in Col.
1		identity of	RRM2	Col. 5	SEQ ID	5 to
SEQ	Col. 2	polypeptide	domain in	Conserved	NO: of	RRM2 do-
ID	Species/	in Col. 1	amino acid	RRM2	RRM2	main of
NO:	Identifier	to RBP45A	coordinates	domain	domain	RBP45A

42	At/RBP45A or	100%	153-232	DHTIFVGDLAPEVTD	538	100%
	AT5G54900.1	(387/387)		YMLSWITKNVYGSV		(80/80)
				KGAKVVLDRTTGRS
				KGYGFVRFADENEQ
				MRAMTEMNGQYCS
				TRPMRIGPAA

40	At/AT4G27000	71%	172-251	EHTVFVGDLAPDVT	537	83%
	.1	(282/394)		DHMLTETFKAVYSS		(67/80)
				VKGAKVVNDRTTG
				RSKGYGFVRFADES
				EQIRAMTEMNGQYC
				SSRPMRTGPAA

44	Pt/POPTR_000	70%	159-238	DYTVFIGDLAADVN	539	82%
	1s45000.1	(228/323)		DYLLQETFRNVYSS		(66/80)
				VKGAKVVTDRVTG
				RSKGYGFVRFADEN
				EQMRAMVEMNGQY
				CSTRPMRIGPAA

46	Pt/POPTR_001	69%	162-241	DFTVFVGDLAADVN	540	81%
	1s14150.1	(231/331)		DYLLQETFRNVYPS		(65/80)
				VKGAKVVTDRVTG
				RSKGYGFIRFADENE
				QRRAMVEMNGQYC
				STRPMRIGPAA

50	Eg/Eucgr.F034	69%	156-235	DYTIFVGDLAADVT	542	78%
	62.1	(226/323)		DHMLQETFRAHYPS		(63/80)
				VKGAKIVIDRTTGRS
				KGYGFVRFGDETEQ
				LRAMTEMNGMYCS
				SRPMRIGPAA

38	Zm/GRMZM2	68%	162-241	DYTIFVGDLASDVT	536	77%
	G002874_T01	(219/322)		DFILQDTFKSRYPSV		(62/80)
				KGAKVVFDRTTGRS
				KGYGFVKFADSDEQ
				TRAMTEMNGQYCSS
				RAMRLGPAS

36	Os/LOC_Os03g	67%	159-238	DYTIFVGDLASDVT	535	77%
	37270.1	(218/323)		DLILQDTFKAHYQS
				VKGAKVVFDRSTGR		(62/80)
				SKGYGFVKFGDLDE
				QTRAMTEMNGQYC
				SSRPMRIGPAS

52	At/AT1G11650	63%	154-233	DYTIFVGDLAADVT	543	77%
	.2	(206/325)		DYILLETFRASYPSV		(62/80)
				KGAKVVIDRVTGRT
				KGYGFVRFSDESEQI
				RAMTEMNGVPCSTR
				PMRIGPAA

48	Sl/Solyc07g064	67%	172-251	EYTIFVGDLAADVT	541	76%
	510.2.1	(218/324)		DYVLQETFKPVYSS		(61/80)
				VKGAKVVTDRITGR
				TKGYGFVKFSDESE
				QLRAMTEMNGVLC
				SSRPMRIGPAA

70	Sl/Solyc03g031	64%	164-243	EYTIFVGDLAADVT	552	76%
	720.2.1	(215/335)		DYMLQETFRANYPS		(61/80)
				VKGAKVVTDRVTG
				RTKGYGFVKFADES
				EQLHAMTEMNGKF
				CSTRPMRIGPAA

66	Sl/Solyc02g080	66%	171-250	EYTIFVGDLAADVS	550	75%
	420.2.1	(216/325)		DYMLQETFRANYPS		(60/80)
				VKGAKVVTDKATG
				RTKGYGFVKFGDES
				EQLRAMTEMNGQF
				CSTRPMRIGPAA

58	Gm/Glyma07g3	66%	153-232	DHTIFVGDLAADVT	546	75%
	8940.1	(217/326)		DYLLQETFRARYPSI		(60/80)
				KGAKVVIDRLTGRT
				KGYGFVRFGDESEQ
				VRAMTEMQGVLCS
				TRPMRIGPAS

68	Sl/Solyc10g005	65%	168-247	EYTIFVGDLAADVT	551	75%
	260.2.1	(218/333)		DYMLQETFRPNYPSI		(60/80)
				KGAKVVTDRATGH
				TKGYGFVRFGDESE
				QLRAMTEMNGKFCS
				TRPMRIGPAA

62	Gm/Glyma13g2	64%	159-238	DYTIFVGDLAADVT	548	75%
	7570.1	(214/331)		DYLLQETFRARYNS		(60/80)
				VKGAKVVIDRLTGR
				TKGYGFVRFSDESE
				QVRAMTEMQGVLC
				STRPMRIGPAS

64	Gm/Glyma15g1	64%	160-239	DYTIFVGDLAADVT	549	75%
	1380.1	(215/333)		DYLLQETFRARYNS		(60/80)
				VKGAKVVIDRLTGR
				TKGYGFVRFSEESEQ
				MRAMTEMQGVLCS
				TRPMRIGPAS

60	Gm/Glyma17g0	66%	157-236	DHTIFVGDLAADVT	547	73%
	1800.1	(218/327)		DYLLQETFRARYPS		(59/80)
				AKGAKVVIDRLTGR
				TKGYGFVRFGDESE
				QVRAMSEMQGVLC
				STRPMRIGPAS

30	Os/LOC_Os07g	58%	222-301	EHTIFVGDLASDVTD	532	73%
	33330.1	(205/348)		SMLEEAFKTSYPSVR		(59/80)
				GAKVVFDKVTGRSK
				GYGFVRFGDENEQT
				RAMTEMNGATLSTR
				QMRLGPAA

24	Os/LOC_Os08g	63%	184-263	DYTIFVGDLAADVT	529	72%
	09100.1	(206/325)		DYILQETFRVHYPSV		(58/80)
				KGAKVVTDKMTMR
				SKGYGFVKFGDPSE
				QARAMTEMNGMVC
				SSRPMRIGPAA

26	Zm/GRMZM2	62%	210-289	DYTIFVGDLAADVT	530	72%
	G426591_T01	(204/324)		DYVLQETFRAHYPS		(58/80)
				VKGAKVVTDKLTM
				RTKGYGFVKFGDPN
				EQARAMTEMNGML
				CSSRPMRIGPAA

16	Bd/Bradi5g224	61%	177-256	DYTIFVGDLAADVT	525	72%
	10.1	(201/327)		DYILQETFRVHYPSV		(58/80)
				KGAKVVTDKMTMR
				SKGYGFVKFGDPTE
				QARAMTEMNGMPC
				SSRPMRIGPAA

54	Pt/POPTR_000	61%	168-247	DFTIFVGDLAADVT	544	72%
	4s01690.1	(213/349)		DFMLQETFRAHFPS		(58/80)
				VKGAKVVIDRLTGR
				TKGYGFVRFGDESE
				QLRAMTEMNGAFCS
				TRPMRVGLAS

22	Bd/Bradi3g151	56%	187-266	DYTIFVGDLAADVT	528	72%
	80.1	(213/376)		DYILQETFRVHYPSV		(58/80)
				KGAKVVTDKLTMR
				SKGYGFVKFSDPTE
				QTRAMTEMNGMVC
				SSRPMRIGPAA

34	Zm/GRMZM2	55%	240-319	DHAIFVGDLAPDVT	534	72%
	G169615_T01	(188/338)		DSMLEDVFRANYPS		(58/80)
				VRGAKVVVDRITGR
				PKGYGFVHFGDLNE
				QARAMTEMNGMML
				STRKMRIGAAA

28	Bd/Bradi1g262	55%	208-287	DHTIFVGDLASDVT	531	72%
	10.1	(181/324)		DSMLQEIFKASYPSV		(58/80)
				RGANVVTDRATGRS
				KGYGFVRFGDVNEQ
				TRAMTEMNGVTLSS
				RQLRIGPAA

20	Zm/GRMZM2	58%	175-254	DYTIFVGDLAADVT	527	71%
	G058098_T02	(192/331)		DYLLQETFRVHYPS		(57/80)
				VKGAKVVTDKLTM
				RTKGYGFVKFGDPT
				EQARAMTEMNGMP
				CSSRPMRIGPAA

18	Zm/GRMZM2	58%	181-260	EYTIFVGDLAADVT	526	70%
	G012628_T01	(191/329)		DYLLQETFRVHYPS		(56/80)
				VKGAKVVTDKLTM
				RTKGYGFVKFGDPT
				EQARAMTEMNGMP
				CSSRPMRIGPAA

32	Zm/GRMZM2	53%	208-287	DRTIFVGDLAHDVT	533	68%
	G127510_T01	(191/354)		DSMLEDVFRAKYPS		(55/80)
				VRGANVVVDRMTG
				WPKGFGFVRFGDLN
				EQARAMTEMNGML
				LSTRQMRIGAAA

56	Eg/Eucgr.D013	63%	182-261	DYTIFVGDLASDVT	545	67%
	10.1	(193/303)		DYMLQEMFRGRYPS		(54/80)
				VRSAKVVMDRLTSR
				TKGYGFVKFGDESE
				QIRAMSEMNGVFLS
				TRPMRIGLAT

TABLE 6

Conserved ‘RRM3 domain’ of RBP45A and closely related sequences

						Col. 7
						Percent
						identity
		Col. 3				of RRM3
Col.		Percent	Col. 4		Col. 6	domain in
1		identity of	RRM3	Col. 5	SEQ ID	Col. 5 to
SEQ	Col. 2	polypeptide	domain in	Conserved	NO: of	RRM3
ID	Species/	in Col. 1	amino acid	RRM3	RRM3	domain of
NO:	Identifier	to RBP45A	coordinates	domain	domain	RBP45A

42	At/RBP45A or	100%	260-332	TTIFVGGLDANVTD	566	100%
	AT5G54900.1	(387/387)		DELKSIFGQFGELLH		(73/73)
				VKIPPGKRCGFVQY
				ANKASAEHALSVLN
				GTQLGGQSIRLSWG
				RS

40	At/AT4G27000	71%	278-350	TTIFVGAVDQSVTED	565	83%
	.1	(282/394)		DLKSVFGQFGELVH		(61/73)
				VKIPAGKRCGFVQY
				ANRACAEQALSVLN
				GTQLGGQSIRLSWG
				RS

50	Eg/Eucgr.F034	69%	265-337	TTIFVGGLDPSVSDD	570	75%
	62.1	(226/323)		LLRQVFSQYGELHH		(55/73)
				VKIPPGKRCGFVQFT
				SRACAEQALLMLNG
				TQLGGQSIRLSWGR
				S

38	Zm/GRMZM2	68%	271-343	TTVFVGGLDPSVTD	564	75%
	G002874_T01	(219/322)		ELLKQTFSPYGELLY		(55/73)
				VKIPVGKRCGFVQY
				SNRASAEEAIRVLNG
				SQLGGQSIRLSWGRS

66	Sl/Solyc02g080	66%	278-350	TTIFVGNLDSNITDE	578	73%
	420.2.1	(216/325)		HLRQIFGHYGQLLH		(54/73)
				VKIPVGKRCGFIQFA
				DRSCAEEALRVLNG
				TQLGGQSIRLSWGR
				S

60	Gm/Glyma17g0	66%	265-337	TTIFVGNLDPNVTDD	575	73%
	1800.1	(218/327)		HLRQVFGQYGELVH		(54/73)
				VKIPAGKRCGFVQF
				ADRSCAEEALRVLN
				GTLLGGQNVRLSWG
				RS

70	Sl/Solyc03g031	64%	271-343	TTIFVGNLDANVTD	580	73%
	720.2.1	(215/335)		DHLRQVFGNYGQLL		(54/73)
				HVKIPVGKRCGFVQ
				FADRSCAEEALRAL
				SGTQLGGQTIRLSW
				GRS

46	Pt/POPTR_001	69%	270-342	TTIFVGALDPSVTDD	568	72%
	1s14150.1	(231/331)		TLRAVFSKYGELVH		(53/73)
				VKIPAGKRCGFVQF
				ANRTSAEQALSMLN
				GTQIAGQNIRLSWG
				RS

36	Os/LOC_Os03g	67%	268-340	TTVFVGGLDPSVTD	563	72%
	37270.1	(218/323)		EVLKQAFSPYGELV		(53/73)
				YVKIPVGKRCGFVQ
				YSNRASAEEAIRML
				NGSQLGGQSIRLSW
				GRS

58	Gm/Glyma07g3	66%	261-333	TTIFVGNLDPNVTDD	574	72%
	8940.1	(217/326)		HLRQVFGHYGELVH		(53/73)
				VKIPAGKRCGFVQF
				ADRSCAEEALRVLN
				GTLLGGQNVRLSWG
				RS

62	Gm/Glyma13g2	64%	269-341	TTIFVGNLDPNVTDD	576	72%
	7570.1	(214/331)		HLRQVFSQYGELVH		(53/73)
				VKIPAGKRCGFVQF
				ADRSCAEEALRVLN
				GTLLGGQNVRLSWG
				RS

64	Gm/Glyma15g1	64%	270-342	TTIFVGNLDPNVTDD	577	72%
	1380.1	(215/333)		HLRQVFSQYGELVH		(53/73)
				VKIPAGKRCGFVQF
				ADRSCAEEALRVLN
				GTLLGGQNVRLSWG
				RS

56	Eg/Eucgr.D013	63%	291-363	KTVFVGGLDPNVTD	573	72%
	10.1	(193/303)		DHLRQVFGQYGEIV		(53/73)
				QVKIPPGKRCGFVQF
				ADRSCAEEALRMLN
				GTQLGGQNIRLSWG
				RS

54	Pt/POPTR_000	61%	276-348	TTIFVGNLDSNVMD	572	72%
	4s01690.1	(213/349)		DHLKELFGQYGQLL		(53/73)
				HVKIPAGKRCGFVQ
				FADRSSAEEALKML
				NGAQLSGQNIRLSW
				GRN

44	Pt/POPTR_000	70%	267-339	TTIFVGALDPSVTDD	567	71%
	1s45000.1	(228/323)		TLRAVFSKYGELVH		(52/73)
				VKIPAGKRCGFVQF
				ANRTCAEQALSMLN
				GTQIAGQNIRLSWG
				RS

48	Sl/Solyc07g064	67%	279-351	TTIFVGGLDPSVAEE	569	71%
	510.2.1	(218/324)		HLRQVFSPYGELVH		(52/73)
				VKIVAGKRCGFVQF
				GSRASAEQALSSLN
				GTQLGGQSIRLSWG
				RS

68	Sl/Solyc10g005	65%	274-346	TTIFVGNLDASVTDD	579	71%
	260.2.1	(218/333)		HLRQVFGNYGQLLH		(52/73)
				VKIPLGKRCGFVQFT
				DRSCAEEALNALSG
				TQLGGQTIRLSWGR
				S

52	At/AT1G11650	63%	261-333	TTVFVGGLDASVTD	571	69%
	.2	(206/325)		DHLKNVFSQYGEIV		(51/73)
				HVKIPAGKRCGFVQ
				FSEKSCAEEALRML
				NGVQLGGTTVRLSW
				GRS

26	Zm/GRMZM2	62%	315-387	TTIFVGGLDPNVTED	558	69%
	G426591_T01	(204/324)		MLKQVFTPYGDVV		(51/73)
				HVKIPVGKRCGFVQ
				YANRSSAEEALVILQ
				GTLVGGQNVRLSW
				GRS

24	Os/LOC_Os08g	63%	289-361	TTIFVGGLDPSVTDD	557	68%
	09100.1	(206/325)		MLKQVFTPYGDVV		(50/73)
				HVKIPVGKRCGFVQ
				FANRASADEALVLL
				QGTLIGGQNVRLSW
				GRS

16	Bd/Bradi5g224	61%	284-356	TTIFVGGLDPNVTED	553	68%
	10.1	(201/327)		ALKQVFAPYGEVIH		(50/73)
				VKIPVGKRCGFVQF
				VNRPSAEQALQMLQ
				GTPIGGQNVRLSWG
				RS

22	Bd/Bradi3g151	56%	293-365	TTIFVGGLDPNVTED	556	68%
	80.1	(213/376)		MLKQVFAPYGEVV		(50/73)
				HVKIPVGKRCGFVQ
				YASRSSSEEALLML
				QGTVIGGQNVRLSW
				GRS

30	Os/LOC_Os07g	58%	332-404	TTIFVGGLDSNVNED	560	64%
	33330.1	(205/348)		HLKQVFTPYGEIGY		(47/73)
				VKIPLGKRCGFVQFT
				SRSSAEEAIRVLNGS
				QIGGQQVRLSWGRT

18	Zm/GRMZM2	58%	288-360	TTIFVGGLDPNVTED	554	64%
	G012628_T01	(191/329)		TLKQVFSPYGEVVH		(47/73)
				VKIPVGKRCGFVQF
				VTRPSAEQALLMLQ
				GALIGAQNVRLSWG
				RS

20	Zm/GRMZM2	58%	282-354	TTIFVGGLDPNVTED	555	64%
	G058098_T02	(192/331)		VLKQAFSPYGEVIH		(47/73)
				VKIPVGKRCGFVQF
				VTRPSAEQALLMLQ
				GALIGAQNVRLSWG
				RS

34	Zm/GRMZM2	55%	351-423	TTVFVGGLDSNVDE	562	58%
	G169615_T01	(188/338)		EYLRQIFTPYGEISY		(43/73)
				VKIPVGKHCGFVQF
				TSRSCAEEAIQMLN
				GSQIGGQKARLSWG
				RS

32	Zm/GRMZM2	53%	319-391	TTVFVGGLDSNVNE	561	58%
	G127510_T01	(191/354)		EYLRQIFTPYGEISY		(43/73)
				VKIPVGKHCGFVQF
				TSRSCAEEAIRMLNG
				SQVGGQKVRLSWG
				RS

28	Bd/Bradi1g262	55%	320-392	TTIFVGGLDSNIDEN	559	57%
	10.1	(181/324)		YLRQVFTPYGEVGY		(42/73)
				VKIPVGKRCGFVQF
				TSRSCAEEAINALNG
				TPIGGNNVRLSWGR
				S

These functionally-related and/or closely-related RBP45A clade polypeptides may be identified by a consensus first RRM domain (RRM1 domain) sequence, SEQ ID NO: 844:

	X¹LWIGDLQX⁹X¹⁰MX¹²X¹³X¹⁴X¹⁵X¹⁶X¹⁷X¹⁸X¹⁹X²⁰X²¹X²²

	X²³X²⁴X²⁵X²⁶X²⁷X²⁸X²⁹X³⁰X³¹X³²KX³⁴IRX³⁷KX³⁹X⁴⁰

	X⁴¹X⁴²X⁴³X⁴⁴GYGFX⁴⁹EX⁵¹X⁵²X⁵³X⁵⁴X⁵⁵X⁵⁶AEX⁵⁹X⁶⁰

	LX⁶²X⁶³X⁶⁴NX⁶⁶X⁶⁷X⁶⁸X⁶⁹X⁷⁰X⁷¹X⁷²X⁷³X⁷⁴X⁷⁵X⁷⁶X⁷⁷

	X⁷⁸X⁷⁹NWAX⁸³X⁸⁴X⁸⁵

where X¹=S or T; X⁹=any amino acid; X¹⁰=H or W; X¹²=D or E; X¹³=D or E; X¹⁴=any amino acid; X¹⁵=F or Y; X¹⁶=I, L, V or M; X¹⁷=H or absent; X¹⁸=any amino acid; X¹⁹=any amino acid; X²⁰=any amino acid; X²¹=F, I, L, V or M; X²²=any amino acid; X²³=any amino acid; X²⁴=any amino acid; X²⁵=A or G; X²⁶=P or absent; X²⁷=Q or absent; X²⁸=E or Q; X²⁹=any amino acid; X³⁰=any amino acid; X³¹=any amino acid; X³²=any amino acid; X³⁴=I, L, V or M; X³⁷=N or D; X³⁹=any amino acid; X⁴⁰=any amino acid; X⁴¹=any amino acid; X⁴²=any amino acid; X⁴³=any amino acid; X⁴⁴=E or Q; X⁴⁹=I, L, V or M; X⁵¹=F, I, L, V or M; X⁵²=any amino acid; X⁵³=any amino acid; X⁵⁴=H, Q or R; X⁵⁵=A, S or G; X⁵⁶=any amino acid; X⁵⁹=any amino acid; X⁶⁰=any amino acid; X⁶²=any amino acid; X⁶³=any amino acid; X⁶⁴=F or Y; X⁶⁶=any amino acid; X⁶⁷=any amino acid; X⁶⁸=any amino acid; X⁶⁹=I, L, V or M; X⁷⁰=any amino acid; X⁷¹=any amino acid; X⁷²=any amino acid; X⁷³=P or absent; X⁷⁴=any amino acid; X⁷⁵=any amino acid; X⁷⁶=any amino acid; X⁷⁷=F or Y; X⁷⁸=K or R; X⁷⁹=I, L, V or M; X⁸³=any amino acid or absent; X⁸⁴=any amino acid or absent; and X⁸⁵=S or G.

These functionally-related and/or closely-related RBP45A clade polypeptides also may be identified by a consensus second RRM domain (RRM2 domain) sequence, SEQ ID NO: 845:

	X¹X²X³X⁴FX⁶GDLAX¹¹X¹²VX¹⁴DX¹⁶X¹⁷LX¹⁹X²⁰X²¹FX²³X²⁴

	X²⁵X²⁶X²⁷SX²⁹X³⁰X³¹AX³³X³⁴VX³⁶DX³⁸X³⁹TX⁴¹X⁴²X⁴³

	KGX⁴⁶GFX⁴⁹X⁵⁰FX⁵²X⁵³X⁵⁴X⁵⁵EQX⁵⁸X⁵⁹AMX⁶²EMX⁶⁵GX⁶⁷

	X⁶⁸X⁶⁹SX⁷¹RX⁷³X⁷⁴RX⁷⁶GX⁷⁸AX⁸⁰

where X¹=E or D; X²=any amino acid; X³=A or T; X⁴=I, L, V or M; X⁶=I, L, V or M; X¹¹=any amino acid; X¹²=E or D; X¹⁴=any amino acid; X¹⁶=any amino acid; X¹⁷=I, L, V or M; X¹⁹=any amino acid; X²⁰=E or D; X²¹=any amino acid; X²³=K or R; X²⁴=any amino acid; X²⁵=any amino acid; X²⁶=F or Y; X²⁷=any amino acid; X²⁹=any amino acid; X³⁰=K or R; X³¹=S or G; X³³=Nor K; X³⁴=I, L, V or M; X³⁶=any amino acid; X³⁸=K or R; X³⁹=any amino acid; X⁴¹=any amino acid; X⁴²=any amino acid; X⁴³=any amino acid; X⁴⁶=F or Y; X⁴⁹=I, L, V or M; X⁵⁰=any amino acid; X⁵²=A, S or G; X⁵³=E or D; X⁵⁴=any amino acid; X⁵⁵=any amino acid; X⁵⁸=any amino acid; X⁵⁹=any amino acid; X⁶²=any amino acid; X⁶⁵=N or Q; X⁶⁷=any amino acid; X⁶⁸=any amino acid; X⁶⁹=any amino acid; X⁷¹=S or T; X⁷³=any amino acid; X⁷⁴=I, L, V or M; X⁷⁶=any amino acid; X⁷⁸=any amino acid; and X⁸⁰=A, S or T.

These functionally-related and/or closely-related RBP45A clade polypeptides also may be identified by a consensus third RRM domain (RRM3 domain) sequence, SEQ ID NO: 846:

	X¹TX³FVGX⁷X⁸DX¹⁰X¹¹X¹²X¹³X¹⁴X¹⁵X¹⁶LX¹⁸X¹⁹X²⁰FX²²

	X²³X²⁴GX²⁶X²⁷X²⁸X²⁹VKIX³³X³⁴GKX³⁷CGFX⁴¹QX⁴³X⁴⁴

	X⁴⁵X⁴⁶X⁴⁷X⁴⁸X⁴⁹X⁵⁰X⁵¹AX⁵³X⁵⁴X⁵⁵LX⁵⁷GX⁵⁹X⁶⁰X⁶¹X⁶²

	X⁶³X⁶⁴X⁶⁵X⁶⁶RLSWGRX⁷³

where X¹=any amino acid; X³=I, L, V or M; X⁷=any amino acid; X⁸=I, L, V or M; X¹⁰=any amino acid; X¹¹=any amino acid; X¹²=I, L, V or M; X¹³=any amino acid; X¹⁴=E or D; X¹⁵=N, E or D; X¹⁶=any amino acid; X¹⁸=K or R; X¹⁹=any amino acid; X²⁰=any amino acid; X²²=any amino acid; X²³=any amino acid; X²⁴=F or Y; X²⁶=E, Q or D; X²⁷=I, L, V or M; X²⁸=any amino acid; X²⁹=any amino acid; X³³=any amino acid; X³⁴=any amino acid; X³⁷=any amino acid; X⁴¹=I, L, V or M; X⁴³=F or Y; X⁴⁴=any amino acid; X⁴⁵=any amino acid; X⁴⁶=K or R; X⁴⁷=any amino acid; X⁴⁸=S or C; X⁴⁹=A or S; X⁵⁰=E or D; X⁵¹=any amino acid; X⁵³=I, L, V or M; X⁵⁴=any amino acid; X⁵⁵=any amino acid; X⁵⁷=any amino acid; X⁵⁹=any amino acid; X⁶⁰=any amino acid; X⁶¹=I, L, V or M; X⁶²=S, A or G; X⁶³=A or G; X⁶⁴=any amino acid; X⁶⁵=any amino acid; X⁶⁶=any amino acid; and X⁷³=any amino acid.

TCP6 Clade Polypeptides

TABLE 7

Conserved ‘TCP domain’ of TCP6 and closely related sequences

						Col. 7
						Percent
						identity
		Col. 3				of TCP
Col.		Percent	Col. 4		Col. 6	domain in
1		identity of	TCP	Col. 5	SEQ ID	Col. 5 to
SEQ	Col. 2	polypeptide	domain in	Conserved	NO: of	TCP
ID	Species/	in Col. 1	amino acid	TCP	TCP	domain of
NO:	Identifier	to TCP6	coordinates	domain	domain	TCP6

86	At/TCP6 or	100%	64-125	KKKPNKDRHLKVEG	588	100%
	AT5G41030.1	(243/243)		RGRRVRLPPLCAARI		(62/62)
				YQLTKELGHKSDGE
				TLEWLLQHAEPSILS
				ATVN

106	SL/soLyc02g094	76%	33-94	KRKSNKDRHTKVEG	598	75%
	290.1.1	(48/63)		RGRRIRMPALCAARI		(47/62)
				FQLTRELGHKSDGE
				TIQWLLQKAEPSIIA
				ATGH

96	Gm/Glyma16g0	48%	68-129	KRSSNKDRHTKVEG	593	74%
	5840.1	(67/138)		RGRRIRMPALCAARI		(46/62)
				FQLTRELGHKSDGE
				TIQWLLQQAEPSIIA
				ATGT

84	At/AT3G27010	41%	74-135	KRSSNKDRHTKVEG	587	74%
	.1	(128/311)		RGRRIRMPALCAARI		(46/62)
				FQLTRELGHKSDGE
				TIQWLLQQAEPSIIA
				ATGS

94	Pt/POPTR_001	37%	83-144	KRSSNKDRHTKVEG	592	74%
	7s09820.1	(113/300)		RGRRIRMPALCAARI		(46/62)
				FQLTRELGHKSDGE
				TIQWLLQQAEPSIIA
				ATGT

88	Cc/clementine0.	36%	66-127	KRSSNKDRHTKVEG	589	74%
	9_016144m	(114/312)		RGRRIRMPALCAARI		(46/62)
				FQLTRELGHKSDGE
				TIQWLLQQAEPSIIA
				ATGT

90	Cc/clementine0.	36%	66-127	KRSSNKDRHTKVEG	590	74%
	9_016174m	(114/312)		RGRRIRMPALCAARI		(46/62)
				FQLTRELGHKSDGE
				TIQWLLQQAEPSIIA
				ATGT

92	Pt/POPTR_000	34%	78-139	KRSSNKDRHTKVEG	591	74%
	1s33470.1	(115/332)		RGRRIRMPALCAARI		(46/62)
				FQLTRELGHKSDGE
				TIQWLLQQAEPSIIA
				ATGT

98	Gm/Glyma19g2	34%	68-129	KRSSNKDRHTKVEG	594	74%
	6560.1	(100/290)		RGRRIRMPALCAARI		(46/62)
				FQLTRELGHKSDGE
				TIQWLLQQAEPSIIA
				ATGT

100	Cc/clementine0.	38%	30-91	KRSSNKDRHKKVDG	595	73%
	9_018374m	(95/245)		RGRRIRMPALCAARI		(45/62)
				FQLTRELGHKSDGE
				TIQWLLQQAEPSIIA
				ATGT

104	Pt/POPTR_000	35%	60-121	KRSSNKDRHKKVEG	597	72%
	3s16630.1	(99/280)		RGRRIRIPALCAARIF		(45/62)
				QLTRELEHKSDGETI
				QWLLQQAEPSIIAAT
				GT

72	Bd/Bradi2g592	39%	84-145	KRSSNKDRHTKVDG	581	70%
	40.1	(86/220)		RGRRIRMPALCAARI		(44/62))
				FQLTRELGHKSDGE
				TVQWLLQQAEPAIV
				AATGS

74	Os/LOC_Os01g	39%	83-144	KRSSNKDRHTKVDG	582	70%
	69980.1	(76/194)		RGRRIRMPALCAARI		(44/62)
				FQLTRELGHKSDGE
				TVQWLLQQAEPAIV
				AATGT

78	Zm/GRMZM2	38%	98-159	KRSSNKDRHTKVDG	584	70%
	G092214_T01	(85/218)		RGRRIRMPALCAARI		(44/62)
				FQLTRELGHKSDGE
				TVQWLLQQAEPAIV
				AATGT

80	Zm/GRMZM2	38%	98-159	KRSSNKDRHTKVDG	585	70%
	G092214_T02	(85/218)		RGRRIRMPALCAARI		(44/62))
				FQLTRELGHKSDGE
				TVQWLLQQAEPAIV
				AATGT

76	Zm/GRMZM2	37%	88-149	KRSSNKDRHTKVDG	583	70%
	G034638_T01	(82/216)		RGRRIRMPALCAARI		(44/62)
				FQLTRELGHKSDGE
				TVQWLLQQAEPAIV
				AATGT

102	Pt/POPTR_000	36%	60-121	KRSSNKDRHKKVDG	596	70%
	1s13500.1	(85/236)		RGRRIRMPALCAARI		(44/62)
				FQLTRELGNKSDGE
				TIQWLLQQAEPSIIA
				ATGT

82	Eg/Eucgr.B035	36%	40-101	KRSSNKDRHKKVDG	586	70%
	29.1	(105/286)		RGRRIRMPALCAARI		(44/62)
				FQLTRELGHKTDGE
				TIQWLLQQAEPSIVA
				ATGT

These functionally-related and/or closely-related TCP6 clade polypeptides may be identified by a consensus TCP domain sequence, SEQ ID NO: 847:
KX²X³X⁴NKDRHX¹⁰KVX¹³GRGRRX¹⁹RX²¹PX²³LCAARIX³⁰QLTX³⁴

ELX³⁷X³⁸KX⁴⁰DGETX⁴⁵X⁴⁶WLLQX⁵¹AEPX⁵⁵IX⁵⁷X⁵⁸ATX⁶¹X⁶²

where X²=K or R; X³=any amino acid; X⁴=S or P; X¹⁰=any amino acid; X¹³=D or E; X¹⁹=I, L, V or M; X²¹=I, L, V or M; X²³=A or P; X³⁰=F or Y; X³⁴=K or R; X³⁷=any amino acid; X³⁸=H or N; X⁴⁰=S or T; X⁴⁵=I, L, V or M; X⁴⁶=Q or E; X⁵¹=H, Q or K; X⁵⁵=S or A; X⁵⁷=I, L, V or M; X⁵⁸=S or A; X⁶¹=any amino acid; and X⁶²=any amino acid.

PIL1 Clade Polypeptides

TABLE 8

Conserved ′bHLH domain′ of PIL1 and closely related sequences

						Col. 7
						Percent
						identity of
		Col. 3				bHLH
		Percent	Col. 4		Col. 6	domain in
Col. 1		identity of	bHLH		SEQ ID	Col. 5 to
SEQ	Col. 2	polypeptide	domain in	Col. 5	NO: of	bHLH
ID	Species/	in Col. 1	amino acid	Conserved bHLH	bHLH	domain of
NO:	Identifier	to PIL1	coordinates	domain	domain	PIL1

108	At/PIL1 pr	100%	227-283	RKRSTEVHKLYER	599	100%
	AT2G46970.1	(416/416)		KRRDEFNKKMRAL		(57/57)
				QDLLPNCYKDDKA
				SLLDEAIKYMRTLQ
				LQVQ


112	Cc/clementine	41%	309-365	KKRTPEVHKRYER	601	70%
	0.9_007946m	(96/231)		KRRDKINKKMRAL		(40/57)
				QELIPNCNKVDKAS
				VLEEAIDYLKTLQF
				QVM


116	Pt/POPTR_00	40%	379-435	RRRAIEIHNLSERK	603	70%
	14s10700.1	(104/254)		RRDRINKKMRALQ		(40/57)
				DLIPNSNKVDKAS
				MLGEAIDYLKSLQL
				QVQ


114	Gm/Glyma10g	39%	187-243	RSRNAEVHNLCER	602	63%
	27910.1	(104/262)		KRRDKINKRMRILK		(36/57)
				ELIPNCNKTDKASM
				LDDAIEYLKTLKLQ
				LQ


110	At/AT3G6209	43%	186-242	RKRNAEAYNSPER	600	61%
	0.2	(173/399)		NQRNDINKKMRTL		(35/57)
				QNLLPNSHKDDNE
				SMLDEAINYMTNL
				QLQVQ

These functionally-related and/or closely-related PIL1 clade polypeptides may be identified by a consensus bHLH domain sequence, SEQ ID NO: 848:
X¹X²RX⁴X⁵EX⁷X⁸X⁹X¹⁰X¹¹ERX¹⁴X¹⁵RX¹⁷X¹⁸X¹⁹NRX²²MRX²⁵LX²⁷

X²⁸LX³⁰PNX³³X³⁴RX³⁶DX³⁸X³⁹SX⁴¹LX⁴³X⁴⁴AIX⁴⁷YX⁴⁹X⁵⁰X⁵¹

LX⁵³X⁵⁴QX⁵⁶X⁵⁷

where X¹=R or K; X²=any amino acid; X⁴=any amino acid; X⁵=any amino acid; X⁷=any amino acid; X⁸=H or Y; X⁹=N or K; X¹⁰=any amino acid; X¹¹=any amino acid; X¹⁴=N or K; X¹⁵=any amino acid; X¹⁷=D or N; X¹⁸=any amino acid; X¹⁹=F, I, L, V or M; X²²=R or K; X²⁵=any amino acid; X²⁷=Q or K; X²⁸=N, D or E; X³⁰=I, L, V or M; X³³=S or C; X³⁴=any amino acid; X³⁶=any amino acid; X³⁸=N or K; X³⁹=any amino acid; X⁴¹=I, L, V or M; X⁴³=any amino acid; X⁴⁴=D or E; X⁴⁷=any amino acid; X⁴⁹=I, L, V or M; X⁵⁰=any amino acid; X⁵¹=any amino acid; X⁵³=Q or K; X⁵⁴=F, I, L, V or M; X⁵⁶=I, L, V or M; and X⁵⁷=any amino acid.

PCL1 Clade Polypeptides

TABLE 9

Conserved ′SANT domain′ of PCL1 and closely related sequences

						Col. 7
						Percent
						identity of
		Col. 3				bHLH
		Percent	Col. 4		Col. 6	domain in
Col. 1		identity of	SANT		SEQ ID	Col. 5 to
SEQ	Col. 2	polypeptide	domain in	Col. 5	NO: of	SANT
ID	Species/	in Col. 1	amino acid	Conserved SANT	SANT	domain of
NO:	Identifier	to PCL1	coordinates	domain	domain	PCL1

126	At/PCL1 or	100%	146-196	RLVWTPQLHKRFVD	608	100%
	AT3G46640.3	(324/324)		VVAHLGIKNAVPKTI		(51/51)
				MQLMNVEGLTREN
				VASHLQKYR

128	At/	63%	143-193	RLVWTPQLHKRFVD	609	100%
	AT5G59570.1	(181/286)		VVAHLGIKNAVPKTI		(51/51)
				MQLMNVEGLTREN
				VASHLQKYR

148	Gm/Glyma11g1	62%	146-196	RLVWTPQLHKRFVD	619	100%
	4490.1	(156/249)		VVAHLGIKNAVPKTI		(51/51)
				MQLMNVEGLTREN
				VASHLQKYR

150	Gm/Glyma11g1	62%	146-196	RLVWTPQLHKRFVD	620	100%
	4490.2	(156/249)		VVAHLGIKNAVPKTI		(51/51)
				MQLMNVEGLTREN
				VASHLQKYR

152	Gm/Glyma12g0	60%	145-195	RLVWTPQLHKRFVD	621	100%
	6410.1	(148/245)		VVAHLGIKNAVPKTI		(51/51)
				MQLMNVEGLTREN
				VASHLQKYR

144	S1/Solyc06g005	59%	148-198	RLVWTPQLHKRFVD	617	100%
	680.2.1	(145/242)		VVAHLGIKNAVPKTI		(51/51)
				MQLMNVEGLTREN
				VASHLQKYR

130	Cc/clementine0.	58%	158-208	RLVWTPQLHKRFVD	610	100%
	9_013078m	(146/251)		VVAHLGIKNAVPKTI		(51/51)
				MQLMNVEGLTREN
				VASHLQKYR

132	Cc/clementine0.	58%	158-208	RLVWTPQLHKRFVD	611	100%
	9_013095m	(146/251)		VVAHLGIKNAVPKTI		(51/51)
				MQLMNVEGLTREN
				VASHLQKYR

134	Cc/clementine0.	58%	158-208	RLVWTPQLHKRFVD	612	100%
	9_013088m	(146/251)		VVAHLGIKNAVPKTI		(51/51)
				MQLMNVEGLTREN
				VASHLQKYR

136	Eg/Eucgr.B023	53%	170-220	RLVWTPQLHKRFVD	613	100%
	13.1	(150/281)		VVAHLGIKNAVPKTI		(51/51)
				MQLMNVEGLTREN
				VASHLQKYR

124	Si/Si002653m	47%	129-179	RLVWTPQLHKRFVD	607	100%
		(137/291)		VVAHLGIKNAVPKTI		(51/51)
				MQLMNVEGLTREN
				VASHLQKYR

142	Pt/POPTR_000	56%	133-183	RLVWTPQLHKRFVD	616	98%
	9s03990.2	(167/297)		VVSHLGIKNAVPKTI		(50/51)
				MQLMNVEGLTREN
				VASHLQKYR

154	Vv/GSVIVT01	54%	233-283	RLVWTPQLHKRFVD	622	98%
	024916001	(160/291)		VVGHLGIKNAVPKTI		(50/51)
				MQLMNVEGLTREN
				VASHLQKYR

140	Pt/POPTR_000	53%	161-211	RLVWTPQLHKRFVD	615	98%
	9s03990.1	(170/315)		VVSHLGIKNAVPKTI		(50/51)
				MQLMNVEGLTREN
				VASHLQKYR

120	Os/LOC_Os01g	55%	120-170	RLVWTPQLHKRFVE	605	96%
	74020.1	(129/233)		VVAHLGMKNAVPK		(49/51)
				TIMQLMNVEGLTRE
				NVASHLQKYR

122	Zm/GRMZM2	51%	118-168	RLVWTPQLHKRFVD	606	96%
	G067702_T01	(118/229)		VVAHLGIKKAVPKTI		(49/51)
				MELMNVEGLTREN
				VASHLQKYR

146	Sl/Solyc06g076	53%	152-202	RLVWTPQLHKRFIE	618	94%
	350.2.1	(129/241)		VVAHLGIKGAVPKTI		(48/51)
				MQLMNVEGLTREN
				VASHLQKYR

138	Pt/POPTR_000	50%	118-168	RLVWTPQLHKRFVD	614	94%
	1s25040.1	(155/310)		VVGHLGMKNAVPK		(48/51)
				TIMQWMNVEGLTRE
				NVASHLQKYR

118	Bd/Bradi2g620	47%	116-166	RMVWNPQLHKRFV	604	94%
	67.1	(142/301)		DVVAHLGIKSAVPK		(48/51)
				TIMQLMNVEGLTRE
				NVASHLQKYR

These functionally-related and/or closely-related PCL1 clade polypeptides may be identified by a consensus SANT domain sequence, SEQ ID NO: 849:
RX²VWX⁵PQLHKRFX¹³X¹⁴VVX¹⁷HLGX²¹KX²³AVPKTIMX³¹X³²MNVEGLTRE

NVASHLQKYR

where X²=I, L, V, or M; X⁵=any amino acid; X¹³=I, L, V, or M; X¹⁴=D or E; X¹⁷=A, S or G; X²¹=I, L, V, or M; X²³=any amino acid; X³¹=Q or E; and X³²=any amino acid.

GTL1 Clade Polypeptides

TABLE 10

Conserved ′Tribelix domain l′ of GTL1 and closely related sequences

						Col. 7
						Percent
						identity of
						trihelix
		Col. 3	Col. 4			domain 1
		Percent	Trihelix		Col. 6	in Col. 5
Col. 1		identity of	domain 1	Col. 5	SEQ ID	to first
SEQ	Col. 2	polypeptide	in amino	Conserved	NO: of	trihelix
ID	Species/	in Col. 1	acid	trihelix	trihelix	domain of
NO:	Identifier	to GTL1	coordinates	domain 1	domain 1	GTL1

168	At/GTL1 or	100%	60-143	GNRWPREETLALLRI	629	100%
	AT1G33240.1	(669/669)		RSDMDSTFRDATLK		(84/84)
				APLWEHVSRKLLEL
				GYKRSSKKCKEKFE
				NVQKYYKRTKETRG
				GRHDGKAYKFFSQ

156	At/GTL1 or	100%	60-143	GNRWPREETLALLRI	623	100%
	G634	(152/152)		RSDMDSTFRDATLK		(84/84)
				APLWEHVSRKLLEL
				GYKRSSKKCKEKFE
				NVQKYYKRTKETRG
				GRHDGKAYKFFSQ

158	Bd/Bradi5g171	72%	85-168	GNRWPREETLALIRI	624	82%
	50.1	(66/91)		RSEMDATFRDATLK		(69/84)
				GPLWEEVSRKLAEL
				GYKRNAKKCKEKFE
				NVHKYYKRTKEGRT
				GRQDGKSYRFFSE

166	Pt/POPTR_000	75%	104-187	GNRWPRQETLALLQ	628	80%
	1s45870.1	(70/93)		IRSEMDAAFRDATL		(68/84)
				KGPLWEDVSRKLAE
				MGYKRSAKKCKEK
				FENVHKYYKRTKEG
				RAGRQDGKSYRFFS
				Q

172	Gm/Glyma20g3	68%	63-146	GNRWPRQETLALLR	631	77%
	0640.1	(73/107)		IRSDMDVAFRDASV		(65/84)
				KGPLWEEVSRKMAE
				LGYHRSSKKCKEKF
				ENVYKYHKRTKEGR
				SGKQDGKTYRFFDQ

186	Gm/Glyma20g3	75%	66-149	GNRWPRQETLALLK	638	76%
	0650.1	(66/87)		IRSDMDAVFRDSSL		(64/84)
				KGPLWEEVARKLSE
				LGYHRSAKKCKEKF
				ENVYKYHKRTKESR
				SGKHEGKTYKFFDQ

162	Bd/Bradi3g304	52%	86-169	GNRWPRQETLVLLK	626	76%
	57.1	(143/275)		IRSDMDAAFRDATL		(64/84)
				KGPLWEEVSRKLAE
				EGYRRNAKKCKEKF
				ENVHKYYKRTKDSR
				AGRNDGKTYRFFQQ

164	Si/Si034382m	63%	74-157	GNRWPRQETLALLK	627	75%
		(74/116)		IRSEMDAAFREAAL		(63/84)
				KGPLWEQVSRKLEA
				MGYKRSAKKCREKF
				ENVDKYYKRTKDG
				RAGRGDGKAYRFFS
				E

160	Zm/GRMZM2	58%	98-181	GNRWPREETLALIRI	625	75%
	G169580_T01	(80/136)		RTEMDADFRNAPLK		(63/84)
				APLWEDVARKLAGL
				GYHRSAKKCKEKFE
				NVHKYYKRTKDAH
				AGRQDGKSYRFFSQ

188	Pt/POPTR_000	71%	58-141	ANRWPRQETLALLK	639	73%
	2s06900.1	(68/95)		IRSDMDAVFRDSGL		(62/84)
				KGPLWEEVSRKLAE
				LGYHRSAKKCKEKF
				ENVYKYHKRTKEGR
				TGKSEGKSYKFFDE

184	Gm/Glyma16g2	41%	53-136	GNRWPRQETLALLK	763	73%
	8240.1	(108/260)		IRSDMDTVFRDSSLK		(62/84)
				GPLWEEVSRKLAEL
				GYQRSAKKCKEKFE
				NVYKYNKRTKDNK
				SGKSHGKTYKFFDQ

190	Pt/POPTR_000	72%	61-144	ANRWPRQETLALLK	640	71%
	5s21420.1	(63/87)		IRSAMDAVFRDSSL		(60/84)
				KGPLWEEVSRKLAE
				LGYHRSAKKCKEKF
				ENLYKYHKRTKEGR
				TGKSEGKTYKFFDE

178	Pt/POPTR_000	70%	40-123	GNRWPKQETLALLK	634	71%
	1s31660.1	(64/91)		IRSDMDVAFKDSGL		(60/84)
				KAPLWEEVSKKLNE
				LGYNRSAKKCKEKF
				ENIYKYHRRTKEGR
				SGRPNGKTYRFEEQ

170	Pt/POPTR_000	44%	64-147	GSRWPRQETLALLKI	630	71%
	5s21410.1	(117/262)		RSGMDVAFRDASVK		(60/84)
				GPLWEEVSRKLAEL
				GYNRSGKKCKEKFE
				NVYKYHKRTKDGR
				TGKQEGKTYRFFDQ

176	Sl/Solyc12g056	40%	70-153	GNRWPRQETLALLK	633	70%
	510.1.1	(212/519)		IRSEMDVVFKDSSLK		(59/84)
				GPLWEEVSRKLAEL
				GYHRSAKKCKEKFE
				NVYKYHRRTKDGR
				ASKADGKTYRFFDQ

180	Pt/POPTR_001	69%	40-123	ANRWPKQETLALLE	635	69%
	9s02650.1	(64/92)		IRSDMDVAFRDSVV		(58/84)
				KAPLWEEVSRKLNE
				LGYNRSAKKCKEKF
				ENIYKYHRRTKGSQ
				SGRPNGKTYRFIAEQ

174	Sl/Solyc04g071	42%	58-141	GNRWPRQETIALLKI	632	69%
	360.2.1	(74/175)		RSEMDVIFRDSSLKG		(58/84)
				PLWEEVSRKMADLG
				FHRSSKKCKEKEEN
				VYKYHKRTKDGRA
				SKADGKNYRFFEQ

182	Sl/Solyc11g005	69%	52-135	GNRWPHEETLALLK	636	66%
	380.1.1	(64/92)		IRSEMDVAFRDSNL		(56/84)
				KSPLWDEISRKMAE
				LGYNRNAKKCREKF
				ENIYKYHKRTKDGR
				SGRQTGKNYRFFEQ

TABLE 11

Conserved ′Tribelix domain 2′ of GTL1 and closely related sequences

						Col. 7
						Percent
						identity of
						trihelix
		Col. 3	Col. 4			domain 2
		Percent	Trihelix		Col. 6	in Col. 5
Col. 1		identity of	domain 2	Col. 5	SEQ ID	to second
SEQ	Col. 2	polypeptide	in amino	Conserved	NO: of	trihelix
ID	Species/	in Col. 1	acid	trihelix	trihelix	domain of
NO:	Identifier	to GTL1	coordinates	domain 2	domain 2	GTL1

168	At/GTL1 or	100%	433-517	SSRWPKAEILALINL	647	100%
	AT1G33240.1	(669/669)		RSGMEPRYQDNVPK		(85/85)
				GLLWEEISTSMKRM
				GYNRNAKRCKEKW
				ENINKYYKKVKESN
				KKRPQDAKTCPYFH
				R

156	At/GTL1 or	100%	187-259	SSRWPKAEILALINL	641	82%
	G634	(152/152)		RSGMEPRYQDNVPK		(70/85)
				GLLWEEISTSMKRM
				GYNRNAKRCKEKW
				ENINKYYKKVKESN
				NSN

166	Pt/POPTR_000	75%	520-604	SSRWPKPEVLALIKL	646	76%
	1s45870.1	(70/93)		RSGLETRYQEAGPK		(65/85)
				GPLWEEISAGMLRL
				GYKRSSKRCKEKWE
				NINKYFKKVKESNK
				KRFEDAKTCPYFHE

172	Gm/Glyma20g3	68%	457-541	SSRWPKVEVQALIK	649	76%
	0640.1	(73/107)		LRTSMDEKYQENGP		(65/85)
				KGPLWEEISASMKK
				LGYNRNAKRCKEK
				WENINKYFKKVKES
				NKRRPEDSKTCPYF
				HQ

174	Sl/Solyc04g071	42%	459-543	SSRWPKAEVEALIKL	650	76%
	360.2.1	(74/175)		RTNLDVKYQENGPK		(65/85)
				GPLWEEISSGMKKIG
				YNRNAKRCKEKWE
				NINKYFKKVKESNK
				KRPEDSKTCPYFHQ

158	Bd/Bradi5g171	72%	496-580	SSRWPKTEVHALIQL	642	75%
	50.1	(66/91)		RMDMDNRYQENGP		(64/85)
				KGPLWEEISSGMRR
				LGYNRNPKRCKEK
				WENINKYFKKVKES
				NKRRPEDSKTCPYF
				HQ

162	Bd/Bradi3g304	52%	453-537	SSRWPKAEVHALIQ	644	75%
	57.1	(143/275)		LRSNLDTRYQEAGP		(64/85)
				KGPLWEEISAGMRR
				MGYSRSSKRCKEK
				WENINKYFKKVKES
				NKKRPEDSKTCPYF
				HQ

176	Sl/Solyc12g056	40%	455-539	SSRWPKEEIEALISLR	651	75%
	510.1.1	(212/519)		TCLDLKYQENGPKG		(64/85)
				PLWEEISSGMRKIGY
				NRNAKRCKEKWENI
				NKYFKKVKESNKKR
				PEDSKTCPYFHQ

164	Si/Si034382m	63%	458-542	PSRWPKAEVHALIQ	645	74%
		(74/116)		LRTELEARYQDSGP		(63/85)
				KGPLWEDISAGMRR
				LGYNRSAKRCKEK
				WENINKYFKKVKES
				NKKRPEDSKTCPYY
				HQ

188	Pt/POPTR_000	71%	405-489	SSRWPKVEVQALIN	657	70%
	2s06900.1	(68/95)		LRANLDVKYQENG		(60/85)
				AKGPLWEDISAGMQ
				KLGYNRSAKRCKEK
				WENINKYFKKVKES
				NKKRPEDSKTCPYF
				DQ

182	Sl/Solyc11g005	69%	363-448	SSRWPKAEVEALIKL	654	70%
	380.1.1	(64/92)		RTNVDLQYQDNGSS		(61/86)
				KGPLWEDISCGMKK
				LGYDRNAKRCKEK
				WENINKYYRRVKES
				QKKRPEDSKTCPYF
				HQ

180	Pt/POPTR_001	69%	331-415	SSRWPKEEIESLIKIR	653	70%
	9s02650.1	(64/92)		TYLEFQYQENGPKG		(60/85)
				PLWEEISTSMKNLG
				YDRSAKRCKEKWE
				NMNKYFKRVKDSN
				KKRPGDSKTCPYFQ
				Q

170	Pt/POPTR_000	44%	404-488	PSRWPKVEVEALIRI	648	70%
	5s21410.1	(117/262)		RTNLDCKYQDNGPK		(60/85)
				GPLWEEISARMRKL
				GYNRNAKRCKEKW
				ENINKYFKKVKESK
				KKRPEDSKTCPYFQ
				Q

186	Gm/Glyma20g3	75%	442-526	SSRWPKTEVHALIRL	656	69%
	0650.1	(66/87)		RTSLEAKYQENGPK		(59/85)
				APFWEDISAGMLRL
				GYNRSAKRCKEKW
				ENINKYFKKVKESN
				KQRREDSKTCPYFH
				E


178	Pt/POPTR_000	70%	337-421	PSRWPKEEIEALIGL	652	69%
	1s31660.1	(64/91)		RTKLEFQYEENGPK		(59/85)
				GPLWEEISASMKKL
				GYDRSAKRCKEKW
				ENMNKYFKRVKESN
				KRRPGDSKTCPYFQ
				Q


160	Zm/GRMZM2	58%	411-495	SSRWPKEEVEALIQV	643	68%
	G169580_T01	(80/136)		RNEKDEQYHDAGG		(58/85)
				KGPLWEDIAAGMRR
				IGYNRSAKRCKEKW
				ENINKYYKKVKESN
				KRRPEDSKTCPYFH
				Q


190	Pt/POPTR_000	72%	406-490	SSRWPKVEVQALISL	658	67%
	5s21420.1	(63/87)		RADLDIKYQEHGAK		(57/85)
				GPLWEDISAGMQKL
				GYNRSAKRCKEKW
				ENINKYFKKVKESN
				RKRPGDSKTCPYFD
				Q

184	Gm/Glyma16g2	41%	412-496	SSRWPKAEVHALIRI	655	67%
	8240.1	(108/260)		RTSLETKYQENGPK		(57/85)
				APLWEDISIAMQRL
				GYNRSAKRCKEKW
				ENINKYFKRVRESSK
				ERREDSKTCPYFHE

These functionally-related and/or closely-related GTL1 clade polypeptides may be identified by a consensus first Trihelix domain sequence (Trihelix 1), SEQ ID NO: 850:
X¹X²RWPX⁶X⁷ETX¹⁰X¹¹LX¹³X¹⁴IRX¹⁷X¹⁸MDX²¹X²²PX²⁴X²⁵X²⁶X²⁷X²⁸

RX³⁰PLWX³⁴X³⁵X³⁶X³⁷X³⁸RX⁴⁰X⁴¹X⁴²X⁴³GX⁴⁵X⁴⁶RX⁴⁸X⁴⁹RRCX⁵³EK

FENX⁵⁹X⁶⁰KYX⁶³X⁶⁴RTKX⁶⁸X⁶⁹X⁷⁰X⁷¹X⁷²X⁷³X⁷⁴X⁷⁵GKX⁷⁸YX⁸⁰FFX⁸³X⁸⁴

where X¹=A or G; X²=any amino acid; X⁶=any amino acid; X⁷=Q or E; X¹⁰=I, L, V or M; X¹¹=any amino acid; X¹³=I, L, V or M; X¹⁴=any amino acid; X¹⁷=S or T; X¹⁸=any amino acid; X²¹=any amino acid; X²²=any amino acid; X²⁴=R or K; X²⁵=N, D or E; X²⁶=A or S; X²⁷=any amino acid; X²⁸=I, L, V or M; X³⁰=A, S or G; X³⁴=D or E; X³⁵=any amino acid; X³⁶=I, L, V or M; X³⁷=A or S; X³⁸=R or K; X⁴⁰=I, L, V or M; X⁴¹=any amino acid; X⁴²=any amino acid; X⁴³=any amino acid; X⁴⁵=F or Y; X⁴⁶=H, Q, N, R or K; X⁴⁸=any amino acid; X⁴⁹=A, S or G; X⁵³=K or R; X⁵⁹=I, L, V or M; X⁶⁰=any amino acid; X⁶³=any amino acid; X⁶⁴=K or R; X⁶⁸=any amino acid; X⁶⁹=any amino acid; X⁷⁰=H, Q, K or, R; X⁷¹=any amino acid; X⁷²=S or G; X⁷³=R or K; X⁷⁴=any amino acid; X⁷⁵=any amino acid; X⁷⁸=any amino acid; X⁸⁰=R or K; X⁸³=any amino acid; and X⁸⁴=Q or E.
These functionally-related and/or closely-related GTL1 clade polypeptides may also be identified by a consensus first Trihelix domain sequence (Trihelix 2), SEQ ID NO: 851:

X¹SRWPKX⁷EX⁹X¹⁰X¹¹LIX¹⁴X¹⁵RX¹⁷X¹⁸X¹⁹X²⁰X²¹X²²YX²⁴X²⁵X²⁶X²⁷X²⁸

X²⁹KX³¹X³²X³³WEX³⁶IX³⁸X³⁹X⁴⁰MX⁴²X⁴³X⁴⁴GYX⁴⁷RX⁴⁹X⁵⁰KRCKEKWEN

X⁶⁰NKYX⁶⁴X⁶⁵X⁶⁶VX⁶⁸X⁶⁹SX⁷¹X⁷²X⁷³X⁷⁴X⁷⁵X⁷⁶X⁷⁷X⁷⁸X⁷⁹X⁸⁰X⁸¹X⁸²X⁸³

X⁸⁴X⁸⁵X⁸⁶

where X¹=S or P; X⁷=any amino acid; X⁹=I, L, V or M; X¹⁰=any amino acid; X¹¹=A or S; X¹⁴=any amino acid; X¹⁵=I, L, V or M; X¹⁷=any amino acid; X¹⁸=any amino acid; X¹⁹=any amino acid; X²⁰=E or D; X²¹=any amino acid; X²²=K, Q or R; X²⁴=any amino acid; X²⁵=E or D; X²⁶=any amino acid; X²⁷=any amino acid; X²⁸=S or absent; X²⁹=any amino acid; X³¹=A or G; X³²=any amino acid; X³³=F or L; X³⁶=E or D; X³⁸=A or S; X³⁹=any amino acid; X⁴⁰=any amino acid; X⁴²=any amino acid; X⁴³=N, K or R; X⁴⁴=I, L, V or M; X⁴⁷=any amino acid; X⁴⁹=any amino acid; X⁵⁰=A, S or P; X⁶⁰=I, L, V or M; X⁶⁴=F or Y; X⁶⁵=K or R; X⁶⁶=K or R; X⁶⁸=K or R; X⁶⁹=E or D; X⁷¹=any amino acid; X⁷²=N, K or R; X⁷³=any amino acid; X⁷⁴=N or R; X⁷⁵=any amino acid or absent; X⁷⁶=any amino acid or absent; X⁷⁷=D or absent; X⁷⁸=S, A or absent; X⁷⁹=K or absent; X⁸⁰=T or absent; X⁸¹=C or absent; X⁸²=P or absent; X⁸³=Y or absent; X⁸⁴=F, Y or absent; X⁸⁵=H, Q, D or absent; X⁸⁶=any amino acid or absent.

DREB2H Clade Polypeptides

TABLE 12

Conserved ′AP2 domain′ of DREB2H and closely related sequences

						Col. 7
						Percent
						identity of
		Col. 3				AP2
		Percent	Col. 4		Col. 6	domain in
Col. 1		identity of	AP2	Col. 5	SEQ ID	Col. 5 to
SEQ	Col. 2	polypeptide	domain in	Conserved	NO: of	AP2
ID	Species/	in Col. 1	amino acid	AP2	AP2	domain of
NO:	Identifier	to DREB2H	coordinates	domain	domain	DREB2H

218	At/DREB2H or	100%	65-122	CDYTGVRQRTWGK	672	100%
	AT2G40350.1	(157/157)		WVAEIREPGRGAKL		(58/58)
				WLGTFSSSYEAALA
				YDEASKAIYGQSAR
				LNL

192	At/G1755	100%	72-129	CDYTGVRQRTWGK	659	100%
		(155/155)		WVAEIREPGRGAKL		(58/58)
				WLGTFSSSYEAALA
				YDEASKAIYGQSAR
				LNL

216	At/AT2G40340.1	86%	70-127	CDYRGVRQRRWGK	671	89%
		(108/125)		WVAEIREPDGGARL		(52/58)
				WLGTFSSSYEAALA
				YDEAAKAIYGQSAR
				LNL

232	Sl/Solyc05g052	65%	72-129	CKYRGVRQRTWGK	679	75%
	410.1.1	(82/125)		WVAEIREPHRGRRL		(44/58)
				WLGTFDTAIEAALA
				YDEAARAMYGPCA
				RLNL

230	Pt/POPTR_001	65%	78-135	CNYRGVRQRTWGK	678	75%
	0s19100.1	(79/121)		WVAEIREPNRGPRL		(44/58)
				WLGTFPTAYEAALA
				YDEAARAMYGPYA
				RLNV

226	Gm/Glyma14g0	64%	78-135	CNYRGVRQRTWGK	676	75%
	6080.1	(80/125)		WVGEIREPNRGSRL		(44/58)
				WLGTFSSAQEAALA
				YDEAARAMYGPCA
				RLNF

224	Gm/Glyma02g4	62%	78-135	CNYRGVRQRTWGK	675	75%
	2960.1	(79/127)		WVGEIREPNRGSRL		(44/58)
				WLGTFSSAQEAALA
				YDEAARAMYGPCA
				RLNF

228	Pt/POPTR_000	65%	78-135	CNYRGVRQRTWGK	677	74%
	8s07360.1	(79/121)		WVAEIREPNRGPRL		(43/58)
				WLGTFPTAYEAALA
				YDNAARAMYGSCA
				RLNI

234	Sl/Solyc06g050	63%	80-137	CKYRGVRQRIWGK	680	72%
	520.1.1	(79/125)		WVAEIREPKRGSRL		(42/58)
				WLGTFGTAIEAALA
				YDDAARAMYGPCA
				RLNL

214	Gm/Glyma13g3	61%	63-120	CNYRGVRQRTWGK	670	72%
	8030.1	(76/123)		WVAEIREPNRGNRL		(42/58)
				WLGTFPTAIGAALA
				YDEAARAMYGSCA
				RLNF

208	Gm/Glyma06g4	61%	65-122	CNYRGVRQRTWGK	667	72%
	5680.1	(72/118)		WVAEIREPNRGSRL		(42/58)
				WLGTFPTAISAALA
				YDEAARAMYGSCA
				RLNF

212	Gm/Glyma12g3	60%	63-120	CNYRGVRQRTWGK	669	72%
	2400.1	(75/123)		WVAEIREPNRGNRL		(42/58)
				WLGTFPTAIGAALA
				YDEAARAMYGSCA
				RLNF

210	Gm/Glyma12g1	60%	65-122	CNYRGVRQRTWGK	668	72%
	1150.1	(72/119)		WVAEIREPNRGSRL		(42/58)
				WLGTFPTAISAALA
				YDEAAMAMYGFCA
				RLNF

206	Eg/Eucgr.G030	61%	69-126	FNYRGVRQRTWGK	666	70%
	94.1	(74/121)		WVAEIREPNRGSRL		(41/58)
				WLGTFPTAIEAAKA
				YDEAATAMYGPCA
				RLNF

198	Si/Si002067m	60%	167-224	CPYRGVRQRTWGK	662	70%
		(72/120)		WVAEIREPNRGKRL		(41/58)
				WLGSFPTAVEAAHA
				YDEAAKAMYGPKA
				RVNF


194	Bd/Bradi2g040	56%	76-133	CAYRGVRQRTWGK	660	68%
	00.1	(67/119)		WVAEIREPNRGKRL		(40/58)
				WLGSFPTAVEAAHA
				YDEAARAMYGAKA
				RVNF


196	Os/LOC_Os01g	55%	81-138	CAYRGVRQRTWGK	661	68%
	07120.1	(66/119)		WVAEIREPNRGRRL		(40/58)
				WLGSFPTALEAAHA
				YDEAARAMYGPTA
				RVNF

202	Si/Si022619m	58%	83-140	CGYRGVRQRTWGK	664	65%
		(73/125)		WVAEIREPNRANRL		(38/58)
				WLGTFPTAEDAARA
				YDQAARAMYGEVA
				RTNF


204	Si/Si022621m	58%	82-139	CGYRGVRQRTWGK	665	65%
		(73/125)		WVAEIREPNRANRL		(38/58)
				WLGTFPTAEDAARA
				YDQAARAMYGEVA
				RTNF


200	Bd/Bradi2g299	56%	130-187	CKFRGVRQRTWGK	663	65%
	60.1	(70/123)		WVAEIREPNRVSRL		(38/58)
				WLGTFPTAETAACA
				YDEAARAMYGPLA
				RTNF


220	Gm/Glyma07g1	57%	65-129	CKFRGVRQRIWGK	673	63%
	9220.1	(73/128)		WVAEIREPINGKLV		(41/65)
				GEKANRLWLGTFST
				ALEAALAYDEAAKA
				MYGPCARLNF

222	Gm/Glyma18g4	57%	65-129	CKFRGVRQRIWGK	674	63%
	3750.1	(73/127)		WVAEIREPINGKLV		(41/65)
				GEKANRLWLGTFST
				ALEAALAYDEAAKA
				LYGPCARLNF

These functionally-related and/or closely-related DREB2H clade polypeptides may be identified by a consensus AP2 domain sequence, SEQ ID NO: 852:
X¹X²X³GVRQRX⁹WGKWVX¹⁵EIREPX²¹X²²X²³X²⁴X²⁵X²⁶X²⁷X²⁸X²⁹X³⁰X³¹

X³²LWWX³⁷FX³⁹X⁴⁰X⁴¹X⁴²X⁴³AAX⁴⁶AYDX⁵⁰AX⁵²X⁵³AX⁵⁵YGX⁵⁸X⁵⁹AR

X⁶²NX⁶⁴

where X¹=any amino acid; X²=F or Y; X³=any amino acid; X⁹=any amino acid; X¹⁵=A or G; X²¹=I or absent; X²²=Nor absent; X²³=G or absent; X²⁴=K or absent; X²⁵=L or absent; X²⁶=V or absent; X²⁷=G or absent; X²⁸=any amino acid; X²⁹=any amino acid; X³⁰=any amino acid; X³¹=any amino acid; X³²=R or K; X³⁷=S or T; X³⁹=any amino acid; X⁴⁰=S or T; X⁴¹=A or S; X⁴²=any amino acid; X⁴³=any amino acid; X⁴⁶=any amino acid; X⁵⁰=Q, D, N or E; X⁵²=A or S; X⁵³=any amino acid; X⁵⁵=I, L, V or M; X⁵⁸=any amino acid; X⁵⁹=any amino acid; X⁶²=any amino acid; and X⁶⁴=F, I, L, V or M

ERF087 Clade Polypeptides

TABLE 13

Conserved ′AP2 domain′ of ERF087and closely related sequences

						Col. 7
						Percent
						identity of
		Col. 3				AP2
		Percent	Col. 4			domain in
Col. 1		identity of	AP2	Col. 5	Col. 6	Col. 5 to
SEQ	Col. 2	polypeptide	domain in	Conserved	SEQ ID	AP2
ID	Species/	in Col. 1	amino acid	AP2 NO: of	AP2	domain of
NO:	Identifier	to ERF087	coordinates	domain	domain	ERF087

246	At/ERF087 or	100%	38-101	KYVGVRRRPWGRY	686	100%
	AT1G28160.1	(245/245)		AAEIRNPTTKERYW		(64/64)
				LGTFDTAEEAALAY
				DRAARSIRGLTART
				NFVYSDMPR

254	At/AT5G13910.1	83%	19-82	RFLGVRRRPWGRYA	690	85%
		(64/77)		AEIRDPTTKERHWL		(55/64)
				GTFDTAEEAALAYD
				RAARSMRGTRARTN
				FVYSDMPP

248	Eg/Eucgr.B035	47%	46-109	RFLGVRRRPWGRYA	687	85%
	65.1	(104/221)		AEIRDPTTKERHWL		(55/64)
				GTFDTAEEAALAYD
				RAARSMRGAKART
				NFVYSDMPP

266	Vv/GSVIVT01	84%	21-84	RFLGVRRRPWGRYA	696	84%
	032961001	(65/77)		AEIRDPSTKERHWL		(54/64)
				GTFDTAEEAALAYD
				RAARSMRGSRARTN
				FVYSDMPP

256	Pt/POPTR_000	81%	25-88	RFLGVRRRPWGRYA	691	84%
	is 15710.1	(64/79)		AEIRDPSTKERHWL		(54/64)
				GTFDTAEEAALAYD
				RAARSMRGSRARTN
				FVYSDMPA

258	Pt/POPTR_000	81%	25-88	RFLGVRRRPWGRYA	692	84%
	3s07540.1	(64/79)		AEIRDPSTKERHWL		(54/64)
				GTFDTAEEAALAYD
				RAARSMRGPRARTN
				FVYSDMPA

252	Pt/POPTR_000	70%	39-102	RFLGVRRRPWGRYA	689	84%
	3s15940.1	(84/120)		AEIRDPSTKERHWL		(54/64)
				GTFDTAEEAALAYD
				RAARSMRGSKARTN
				FVYSDMPP

250	Pt/POPTR_000	45%	37-100	RFLGVRRRPWGRYA	688	84%
	1s12820.1	(109/240)		AEIRDPSTKERHWL		(54/64)
				GTFDTAEEAALAYD
				RAARSMRGSKARTN
				FVYSDMPP

262	Gm/Glyma02g0	72%	31-94	RYLGVRRRPWGRY	694	82%
	7460.1	(69/95)		AAEIRDPSTKERHW		(53/64)
				LGTFDTAEEAALAY
				DRAARSMRGSRART
				NFVYPDTPP

260	Eg/Eucgr.I0157	51%	27-90	RFLGVRRRPWGRYA	693	82%
	6.1	(81/157)		AEIRDPSTKERHWL		(53/64)
				GTFDTAEEAALAYD
				RAARSMRGSRARTN
				FVYSDLPA

238	Os/LOC_Os02g	75%	39-102	RYLGVRRRPWGRY	682	79%
	32040.1	(60/79)		AAEIRDPATKERHW		(51/64)
				LGTFDTAEEAAVAY
				DRAARTIRGAAART
				NFAYPDLPP

264	Gm/Glyma16g2	70%	31-94	RYLGVRRRPWGRY	695	79%
	6460.1	(67/95)		AAEIRDPSTKERHW		(51/64)
				LGTFDTAEEAALAY
				DKAARSMRGSRART
				NFIYPDTPP

240	Os/LOC_Os04g	69%	51-114	RYLGVRRRPWGRY	683	79%
	32790.1	(62/89)		AAEIRDPATKERHW		(51/64)
				LGTFDTAEEAAVAY
				DRAARSLRGARART
				NFAYPDLPP

242	Zm/GRMZM2	69%	41-104	RYLGVRRRPWGRY	684	79%
	G023708_T01	(62/89)		AAEIRDPATKERHW		(51/64)
				LGTFDTAEEAAVAY
				DRAARSLRGARART
				NFAYPDLPP

236	Zm/GRMZM2	64%	73-136	RYLGVRRRPWGRY	681	79%
	G047999_T01	(73/114)		AAEIRDPATKERHW		(51/64)
				LGTFDTAEEAAIAY
				DRAARNIRGANART
				NFAYPDLPP

244	Zm/GRMZM2	63%	37-100	RYLGVRRRPWGRY	685	79%
	G079825_T01	(73/115)		AAEIRDPATKERHW		(51/64)
				LGTFDTAEEAAVAY
				DRAARSLRGARART
				NFAYPDLPP

268	Gm/Glyma16g0	49%	14-77	RYLGVRRRPWGRY	697	78%
	5070.1	(74/150)		AAEIRDPSTKERHW		(50/64)
				LGTFDTADEAALAY
				DRAARAMRGSRAR
				TNFVYADTTP

These functionally-related and/or closely-related ERF087 clade polypeptides may be identified by a consensus AP2 domain sequence, SEQ ID NO: 853:
X¹X²X³GVRRRPWGRYAAEIRX¹⁹PX²¹TKERX²⁶WLGTFDTAX³⁵EAAX³⁹AY

DX⁴³AARX⁴⁷X⁴⁸RGX⁵¹X⁵²ARTNFX⁵⁸YX⁶⁰DX⁶²X⁶³X⁶⁴

where X¹=K or R; X²=F or Y; X³=I, L, V or M; X¹⁹=N or D; X²¹=A, S or T; X²⁶=H or Y; X³⁵=D or E; X³⁹=I, L, V or M; X⁴³=R or K; X⁴⁷=any amino acid; X⁴⁸=I, L, V or M; X⁵¹=any amino acid; X⁵²=any amino acid; X⁵⁸=any amino acid; X⁶⁰=S, A or P; X⁶²=any amino acid; X⁶³=T or P; and X⁶⁴=any amino acid.

BBX18 Clade Polypeptides

TABLE 14

Conserved first ′BBX domain′ of BBX18 and closely related sequences

						Col. 7
						Percent
						identity of
		Col. 3	Col. 4			first BBX
		Percent	BBX		Col. 6	domain in
Col. 1		identity of	domain 1	Col. 5	SEQ ID	Col. 5 to
SEQ	Col. 2	polypeptide	in amino	Conserved	NO: of	first BBX
ID	Species/	in Col. 1	acid	BBX	BBX	domain of
NO:	Identifier	to BBX18	coordinates	domain	1	domain 1	BBX18

278	At/BBX18 or	100%	5-42	CDACESAAAIVFCA	702	100%
	AT2G21320.1	(172/172)		ADEAALCCSCDEKV		(38/38)
				HKCNKLASRH

290	Pt/POPTR_000	61%	5-42	CDACESAAAIVFCA	708	92%
	7s13830.1	(114/184)		ADEAALCLACDEKV		(35/38)
				HMCNKLASRH

284	Gm/Glyma01g3	60%	5-42	CDACESAAAIVFCA	705	92%
	7370.1	(111/183)		ADEAALCRACDEKV		(35/38)
				HMCNKLASRH

286	Gm/Glyma11g0	59%	5-42	CDACESAAAIVFCA	706	92%
	7930.1	(112/189)		ADEAALCRACDEKV		(35/38)
				HMCNKLASRH

296	Pt/POPTR_000	60%	5-42	CDVCESAAAILFCA	711	89%
	4s16950.1	(111/184)		ADEAALCRSCDEKV		(34/38)
				HMCNKLASRH

298	Pt/POPTR_000	60%	5-42	CDVCESAAAILFCA	712	89%
	9s12730.1	(111/184)		ADEAALCRSCDEKV		(34/38)
				HLCNKLASRH

300	Vv/GSVIVT01	59%	5-42	CDACESAAAILFCA	713	89%
	024173001	(117/198)		ADEAALCRACDEKV		(34/38)
				HMCNKLASRH

302	Sl/Solyc01g110	58%	5-42	CDVCESAAAILFCA	714	89%
	370.2.1	(114/196)		ADEAALCRSCDEKV		(34/38)
				HLCNKLASRH

280	At/AT4G38960.1	76%	5-42	CDACENAAAIIFCAA	703	86%
		(131/171)		DEAALCRPCDEKVH		(33/38)
				MCNKLASRH

288	Pt/POPTR_000	61%	5-42	CDACESAFAIVFCAA	707	86%
	5s11900.1	(114/184)		DEAALCLACDKKVH		(33/38)
				MCNKLASRH

292	Vv/GSVIVT01	60%	5-42	CDVCESAAAILFCA	709	86%
	018818001	(110/183)		ADEAALCRVCDEKV		(33/38)
				HMCNKLASRH

282	Eg/Eucgr.I0236	57%	5-42	CDACESAAAVVFCA	704	86%
	8.1	(107/185)		ADEAALCSACDDKV		(33/38)
				HMCNKLASRH

306	Gm/Glyma12g0	63%	5-42	CDVCESAAAIVFCA	716	84%
	4130.1	(110/172)		ADEAALCSACDHKI		(32/38)
				HMCNKLASRH

294	Eg/Eucgr.I0132	57%	5-42	CDVCENAAAIFFCA	710	84%
	8.1	(111/193)		ADEAALCRACDEKV		(32/38)
				HLCNKLASRH

270	Bd/Bradi4g359	55%	5-42	CDVCESAVAVLFCA	698	84%
	50.1	(108/196)		ADEAALCRSCDEKV		(32/38)
				HLCNKLASRH

274	Zm/GRMZM2	57%	5-42	CDVCESAPAVLFCA	700	81%
	G143718_T01	(109/191)		ADEAALCRPCDEKV		(31/38)
				HMCNKLASRH

276	Zm/GRMZM2	57%	5-42	CDVCESAPAVLFCA	701	81%
	G422644_T01	(109/190)		ADEAALCRPCDEKV		(31/38)
				HMCNKLASRH

304	Gm/Glyma11g1	57%	5-42	CDVCESAAAILFCA	715	81%
	1850.1	(112/196)		ADEAALCSACDHKI		(31/38)
				HMCNKLASRH

272	Os/LOC_Os09g	57%	5-42	CDVCESAPAVLFCV	699	78%
	35880.1	(113/197)		ADEAALCRSCDEKV		(30/38)
				HMCNKLARRH

TABLE 15

Conserved second ′BBX domain of BBX18 and closely related sequences

						Col. 7
						Percent
						identity of
						second
						BBX
		Col. 3	Col. 4			domain in
		Percent	BBX		Col. 6	Col. 5 to
Col. 1		identity of	domain 2		SEQ ID	second
SEQ	Col. 2	polypeptide	in amino	Col. 5	NO: of	BBX
ID	Species/	in Col. 1	acid	Conserved BBX	BBX	domain of
NO:	Identifier	to BBX18	coordinates	domain	2	domain 2	BBX18

278	At/BBX18 or	100%	56-91	CDICENAPAFFYCEI	721	100%
	AT2G21320.1	(172/172)		DGSSLCLQCDMVVH		(36/36)
				VGGKRTH

280	At/AT4G38960.1	76%	56-91	CDICENAPAFFYCEI	722	100%
		(131/171)		DGSSLCLQCDMVVH		(36/36)
				VGGKRTH

306	Gm/Glyma12g0	63%	56-91	CDICENAPAFFYCEI	735	97%
	4130.1	(110/172)		DGSSLCLQCDMIVH		(35/36)
				VGGKRTH

298	Pt/POPTR_000	60%	56-91	CDICENAPAFFYCEI	731	97%
	9s12730.1	(111/184)		DGSSLCLQCDMIVH		(35/36)
				VGGKRTH

302	Sl/Solyc01g110	58%	56-91	CDICENAPAFFYCEI	733	97%
	370.2.1	(114/196)		DGSSLCLQCDMIVH		(35/36)
				VGGKRTH

294	Eg/Eucgr.I0132	57%	56-91	CDICENAPAFFYCEI	729	97%
	8.1	(111/193)		DGSSLCLQCDMLVH		(35/36)
				VGGKRTH

304	Gm/Glyma11g1	57%	56-91	CDICENAPAFFYCEI	734	97%
	1850.1	(112/196)		DGSSLCLQCDMIVH		(35/36)
				VGGKRTH

288	Pt/POPTR_000	61%	56-91	CDICENAPAFFYCET	726	94%
	5s11900.1	(114/184)		DGSSLCLQCDMTVH		(34/36)
				VGGKRTH

290	Pt/POPTR_000	61%	56-91	CDICENAPAFFYCET	727	94%
	7s13830.1	(114/184)		DGSSLCLQCDMTVH		(34/36)
				VGGKRTH

296	Pt/POPTR_000	60%	56-91	CDICEKAPAFFYCEI	730	94%
	4s16950.1	(111/184)		DGSSLCLQCDMIVH		(34/36)
				VGGKRTH

284	Gm/Glyma01g3	60%	56-91	CDICENAPAFFYCET	724	94%
	7370.1	(111/183)		DGSSLCLQCDMIVH		(34/36)
				VGGKRTH

292	Vv/GSVIVT01	60%	56-91	CDICENAPAFFYCEI	728	94%
	018818001	(110/183)		DGTSLCLQCDMIVH		(34/36)
				VGGKRTH

286	Gm/Glyma11g0	59%	56-91	CDICENAPAFFYCET	725	94%
	7930.1	(112/189)		DGSSLCLQCDMIVH		(34/36)
				VGGKRTH

300	Vv/GSVIVT01	59%	56-91	CDICENAPAFFYCEV	732	91%
	024173001	(117/198)		DGTSLCLQCDMIVH		(33/36)
				VGGKRTH

272	Os/LOC_Os09g	57%	56-91	CDICENAPAFFYCEI	718	91%
	35880.1	(113/197)		DGTSLCLSCDMTVH		(33/36)
				VGGKRTH

282	Eg/Eucgr.I0236	57%	56-91	CDICENAPAFFYCEV	723	91%
	8.1	(107/185)		DGTSLCLQCDMIVH		(33/36)
				VGGKRTH

274	Zm/GRMZM2	57%	56-91	CDICENSPAFFYCEI	719	88%
	G143718_T01	(109/191)		DGTSLCLSCDMTVH		(32/36)
				VGGKRTH

276	Zm/GRMZM2	57%	56-91	CDICENSPAFFYCEI	720	88%
	G422644_T01	(109/190)		DGTSLCLSCDMTVH		(32/36)
				VGGKRTH

270	Bd/Bradi4g359	55%	56-91	CDICENSPAFFYCDI	717	86%
	50.1	(108/196)		DGTSLCLSCDMAVH		(31/36)
				VGGKRTH

These functionally-related and/or closely-related BBX18 clade polypeptides may be identified by a first consensus BBX domain sequence (BBX1), SEQ ID NO: 854:
CDX³CEX⁶AX⁸AX¹⁰X¹¹FCX¹⁴ADEAALCX²²X²³CDX²⁶KX²⁸HX³⁰CNKLAX³⁶RH

where X³=any amino acid; X⁶=any amino acid; X⁸=any amino acid; X¹⁰=I, L, V or M; X¹¹=F, I, L, V or M; X¹⁴=any amino acid; X²²=any amino acid; X²³=any amino acid; X²⁶=any amino acid; X²⁸=I, L, V or M; X³⁰=any amino acid; and X³⁶=any amino acid.
These functionally-related and/or closely-related BBX18 clade polypeptides may also be identified by a second consensus BBX domain sequence (BBX2), SEQ ID NO: 855:
CDICEX⁶X⁷PAFFYCX¹⁴X¹⁵DGX¹⁸SLCLX²³CDMX²⁷VHVGGKRTH

X⁶=N or K; X⁷=S or A; X¹⁴=D or E; X¹⁵=T, I, L, V or M; X¹⁸=S or T; X²³=I, L, V or M; and X²⁷=I, L, V or M.
bHLH60 Clade Polypeptides

TABLE 16

Conserved ′bHLH domain′ of bHLH60 and closely related sequences

						Col. 7
						Percent
						identity of
		Col. 3				bHLH
		Percent	Col. 4		Col. 6	domain in
Col. 1		identity of	bHLH	Col. 5	SEQ ID	Col. 5 to
SEQ	Col. 2	polypeptide	domain in	Conserved	NO: of	bHLH
ID	Species/	in Col. 1	amino acid	bHLH	bHLH	domain of
NO:	Identifier	to bHLH60	coordinates	domain	domain	bHLH60

318	At/bHLH60 or	100%	208-265	RGQATDSHSLAER	741	100%
	AT3G57800.2	(379/379)		ARREKINARMKLL		(58/58)
				QELVPGCDKIQGTA
				LVLDEIINHVQSLQ
				RQVE

316	At/AT2G4230	68%	189-246	RGQATDNHSLAER	740	96%
	0.1	(261/380)		ARREKINARMKLL		(56/58)
				QELVPGCDKIQGTA
				LVLDEIINHVQTLQ
				RQVE

330	Cc/clementine	55%	242-299	RGQATDSHSLAER	747	96%
	0.9_015567m	(178/321)		ARREKINARMKLL		(56/58)
				QELVPGCNKISGTA
				LVLDEIINHVQSLQ
				RQVE

322	Gm/Glyma03g	53%	210-267	RGQATDSHSLAER	743	96%
	29710.1	(190/352)		ARREKINARMKLL		(56/58)
				QELVPGCDKISGTA
				MVLDEIINHVQSLQ
				RQVE

324	Gm/Glyma19g	52%	204-261	RGQATDSHSLAER	744	96%
	32570.1	(203/388)		ARREKINARMKLL		(56/58)
				QELVPGCDKISGTA
				MVLDEIINHVQSLQ
				RQVE

328	Cc/clementine	51%	242-299	RGQATDSHSLAER	746	96%
	0.9_011877m	(214/418)		ARREKINARMKLL		(56/58)
				QELVPGCNKISGTA
				LVLDEIINHVQSLQ
				RQVE

334	Vv/GSVIVTO	55%	201-258	RGQATDSHSLAER	749	94%
	1033350001	(218/396)		ARREKINARMKLL		(55/58)
				QELVPGCNKISGTA
				LVLDEIISHVQSLQR
				QVE

320	Eg/Eucgr.A02	52%	185-242	RGQATDSHSLAER	742	94%
	413.1	(179/338)		ARREKINARMKLL		(55/58)
				QELVPGCSKISGTA
				SVLDEIINHVQSLQ
				RQVE

336	Sl/Solyc10g07	47%	198-255	RGQATDSHSLAER	750	94%
	9070.1.1	(163/342)		ARREKINARMKLL		(55/58)
				QELVPGCNKISGTA
				MVLDEIINHVQSLQ
				RQVE

332	Pt/POPTR_00	53%	181-238	RGQATDSHSLAER	748	93%
	06s05600.1	(188/354)		ARREKINQRMKLL		(54/58)
				QELVPGCNKISGTA
				LVLDEIINHVQSLQ
				CQVE

326	Gm/Glyma10g	46%	196-253	RGQATDSHSLAER	745	91%
	12210.1	(179/386)		ARREKINARMKLL		(53/58)
				QELVPGCNKISGTA
				LVLDKIINHVQSLQ
				NEVE

310	Zm/GRMZM2	43%	161-218	RGQATDSHSLAER	737	91%
	G074438_T01	(134/309)		ARREKINARMELL		(53/58)
				KELVPGCSKVSGTA
				LVLDEIINHVQSLQ
				RQVE

314	Si/Si006781m	43%	177-234	RGQATDSHSLAER	739	91%
		(132/307)		ARREKINARMELL		(53/58)
				KELVPGCSKVSGTA
				LVLDEIINHVQSLQ
				RQVE

312	Zm/GRMZM2	43%	184-241	RGQATDSHSLAER	738	91%
	G378653_T01	(135/309)		ARREKINARMELL		(53/58)
				KELVPGCSKVSGTA
				LVLDEIINHVQSLQ
				RQVE

308	Bd/Bradi1g35	42%	145-202	RGQATDSHSLAER	736	91%
	990.1	(147/348)		ARREKINARMELL		(53/58)
				KELVPGCSKVSGTA
				LVLDEIINHVQSLQ
				RQVE

These functionally-related and/or closely-related bHLH60 clade polypeptides may be identified by a consensus bHLH domain sequence, SEQ ID NO: 856:
RGQATDX⁷HSLAERARREKINX²¹RMX²⁴LLX²⁷ELVPGCX³⁴KX³⁶X³⁷GTAX⁴¹

VLDX⁴⁵IIX⁴⁸HVQX⁵²LQX⁵⁵X⁵⁶VE

where X⁷=any amino acid; X²¹=any amino acid; X²⁴=K or E; X²⁷=Q or K; X³⁴=any amino acid; X³⁶=I, L, V or M; X³⁷=any amino acid; X⁴¹=any amino acid; X⁴⁵=K or E; X⁴⁸=any amino acid; X⁵²=S or T; X⁵⁵=any amino acid; and X⁵⁶=Q or E.

NF-YC6 Clade Polypeptides

TABLE 17

Conserved ′NF-Y/histone-like domain′ of NF-YC6 and closely related sequences

						Col. 7
						Percent
						identity of
						NF-
						Y/histone-
						like
			Col. 4		Col. 6	domain in
		Col. 3	NF-		SEQ ID	Col. 5 to
		Percent	Y/histone-		NO: of	NF-
Col. 1		identity of	like domain	Col. 5	NF-	Y/histone-
SEQ	Col. 2	polypeptide	in amino	Conserved NF-	Y/histone-	like
ID	Species/	in Col. 1	acid	Y/histone-like	like	domain of
NO:	Identifier	to NF-YC6	coordinates	domain	domain	NF-YC6

356	At/NF-YC6 or	100%	53-117	RQLPLARIKKIMKA	760	100%
	AT5G50480.1	(202/202)		DPDVHMVSAEAPII		(65/65)
				FAKACEMFIVDLT
				MRSWLKAEENKRH
				TLQKSDISNAV


348	Si/Si015775m	55%	55-119	HSLPLARIKKIMKA	756	67%
		(57/103)		DEDVKMIAAEAPV		(44/65)
				VFAKACEMFILELT
				LRSWLHTEGTKRR
				TMQRSDVSAAI

350	At/AT5G2791	54%	35-99	HDLPITRIKKIMKY	757	67%
	0.1	(92/168)		DPDVTMIASEAPIL		(44/65)
				LSKACEMFIMDLT
				MRSWLHAQESKRV
				TLQKSNVDAAV

338	Bd/Bradi3g17	52%	77-141	HSLPLARIKKIMKA	751	67%
	790.1	(55/105)		DEDVQMIAGEAPA		(44/65)
				VFAKACEMFILELT
				LRSWLQTRENNRN
				TLQKNDIATVV

346	Os/LOC_Os08	47%	380-444	PNLPLARIKKIMKA	755	63%
	g10560.1	(59/125)		DEDVKMIAGEAPA		(41/65)
				LFAKACEMFILDMT
				LRSWQHFLEGRRR
				TLQRSDVEAVI

340	Bd/Bradi3g17	48%	240-305	HSLPLARIKKIMKA	752	60%
	800.1	(56/116)		SGENVQMIAGEAH		(40/66)
				GLLAKACEIFIQELT
				LRSWLQTRENNRR
				TLQKNDIAAAV

344	Bd/Bradi3g17	51%	99-164	HSLPLARIKKIMKA	754	59%
	810.1	(51/100)		SGEDIRMIASEAPG		(39/66)
				LLAKASEIFIQELTL
				RSWLETRDNNRRT
				LQKNDIGAAV

352	At/AT5G5049	45%	35-99	HEFPISRIKRIMKED	758	58%
	0.1	(78/172)		PDVSMIAAEAPNLL		(38/65)
				SKACEMFVMDLTM
				RSWLHAQESNRLTI
				RKSDVDAVV

368	Sl/Solyc03g11	43%	64-128	HSLPISRIKKIMKSD	766	58%
	1460.1.1	(69/158)		KEVRMISAESPILLA		(38/65)
				KACELFIQELTHRS
				WLKAQECQRQTLK
				KIDLFTVL

342	Bd/Bradi3g17	52%	7-72	HSLPLERIKKIMKA	753	56%
	820.1	(45/85)		SGENVQVIAGEAPG		(37/66)
				VLTKACEIFIQELTL
				RSWLQTREKNRRT
				LQKNDIAAAV

370	Sl/Solyc03g11	48%	74-138	HSLPIFRIKKIMKSD	767	56%
	1470.1.1	(45/92)		KEVRMISAESPILLD		(37/65)
				KACELFIQELTHRS
				WLKAQECQRRTLK
				KIDFFTTE

354	At/AT5G5047	48%	62-132	HAFPLTRIKKIMKS	759	56%
	0.1	(92/189)		NPEVNMVTAEAPV		(40/71)
				LISKACEMLILDLT
				MRSWLHTVEGGRQ
				TLKRSDTLTRSDIS
				AAT

362	Sl/Solyc03g11	49%	52-117	RLLLPPTRIKKIMK	763	53%
	0840.1.1	(51/104)		KNEDVRMVAGESP		(35/66)
				VLLAKACELFIQDL
				TLRSSIHAQENHRRI
				LKKDDLTDVI

364	Sl/Solyc03g11	46%	53-118	NLLPRIHRIKKIMKT	764	53%
	0850.1.1	(54/116)		DKDVRMIATESPVL		(35/66)
				LAKACELFIQELTL
				RSWFKAEENHRRIL
				KKDDVTDVI

366	Sl/Solyc11g01	45%	53-118	NLLPSINRIKKIMKT	765	50%
	6920.1.1	(53/116)		DKDVRMIATESPVL		(33/66)
				LAKACELFIQELTL
				RSWFKTEKNHRRIL
				KKDDVTDVI

360	Sl/Solyc02g02	40%	54-119	NHLLPPNLIKKLMK	762	46%
	1330.1.1	(55/137)		TDEDDQMIAAESPV		(31/66)
				LLAKTCELFIQELTL
				RSWLNAQEKHQHI
				LKKDDVTDVI


358	Sl/Solyc00g10	42%	55-120	NLLVSPNRIKNIMK	761	45%
	7050.1.1	(40/94)		TNKDVRRITSESPV		(30/66)
				LLAKACDFFIQELT
				LRSWLNAQENHRR
				ILKKKDVTDVI

372	Sl/Solyc03g11	40%	102-166	HHFPISRIKRIIKSEN	768	44%
	1450.1.1	(69/171)		NAIKLSAETPILFSK		(29/65)
				ACELFVLELTLRSW
				FHAQQNNRGSLKK
				TDFAAAI

These functionally-related and/or closely-related NF-YC6 clade polypeptides may be identified by a consensus NF-Y/histone-like domain sequence, SEQ ID NO: 857:

X¹X²X³X⁴X⁵X⁶X⁷X⁸IKX¹¹X¹²X¹³KX¹⁵X¹⁶X¹⁷X¹⁸X¹⁹X²⁰X²¹X²²X²³X²⁴X²⁵

EX²⁷X²⁸X²⁹X³⁰X³¹X³²KX³⁴X³⁵X³⁶X³⁷X³⁸X³⁹X⁴⁰X⁴¹X⁴²TX⁴⁴RSX⁴⁷X⁴⁸X⁴⁹

X⁵⁰X⁵¹X⁵²X⁵³X⁵⁴X⁵⁵X⁵⁶X⁵⁷X⁵⁸X⁵⁹X⁶⁰X⁶¹X⁶²X⁶³X⁶⁴X⁶⁵X⁶⁶X⁶⁷X⁶⁸X⁶⁹X⁷⁰

X⁷¹X⁷²X⁷³

where X¹=any amino acid; X²=any amino acid; X³=F, I, L, V or M; X⁴=any amino acid or absent; X⁵=any amino acid; X⁶=any amino acid; X⁷=any amino acid; X⁸=any amino acid; X¹¹=N, K or R; X¹²=I, L, V or M; X¹³=I, L, V or M; X¹⁵=any amino acid; X¹⁶=any amino acid; X¹⁷=G or absent; X¹⁸=any amino acid; X¹⁹=N, E or D; X²⁰=any amino acid; X²¹=any amino acid; X²²=any amino acid; X²³=I, L, V or M; X²⁴=S, A or T; X²⁵=A, S, T or G; X²⁷=A, S or T; X²⁸=any amino acid; X²⁹=any amino acid; X³⁰=I, L, V or M; X³¹=F, I, L, V or M; X³²=any amino acid; X³⁴=any amino acid; X³⁵=S or C; X³⁶=E or D; X³⁷=F, I, L, V or M; X³⁸=I, L, V or M; X³⁹=I, L, V or M; X⁴⁰=any amino acid; X⁴¹=E or D; X⁴²=I, L, V or M; X⁴⁴=any amino acid; X⁴⁷=any amino acid; X⁴⁸=any amino acid; X⁴⁹=any amino acid; X⁵⁰=A or T; X⁵¹=any amino acid; X⁵²=any amino acid; X⁵³=any amino acid; X⁵⁴=any amino acid; X⁵⁵=Q, R; X⁵⁶=any amino acid; X⁵⁷=any amino acid; X⁵⁸=I, L, V or M; X⁵⁹=K, Q, R; X⁶⁰=R or absent; X⁶¹=S or absent; X⁶²=D or absent; X⁶³=T or absent; X⁶⁴=L or absent; X⁶⁵=T or absent; X⁶⁶=K, R; X⁶⁷=any amino acid; X⁶⁸=N, D; X⁶⁹=F, I, L, V or M; X⁷⁰=any amino acid; X⁷¹=any amino acid; X⁷²=A, T, V; and X⁷³=any amino acid.
bHLH121 Clade Polypeptides

TABLE 18

Conserved ′bHLH domain′ of bHLH121 and closely related sequences

						Col. 7
						Percent
						identity of
		Col. 3				bHLH
		Percent	Col. 4		Col. 6	domain in
Col. 1		identity of	bHLH	Col. 5	SEQ ID	Col. 5 to
SEQ	Col. 2	polypeptide	domain in	Conserved	NO: of	bHLH
ID	Species/	in Col. 1	amino acid	bHLH	bHLH	domain of
NO:	Identifier	to bHLH121	coordinates	domain	domain	bHLH121

388	At/bHLH121	100%	6-60	RKSQKAGREKLRR	813	100%
	or	(284/284)		EKLNEHFVELGNV		(55/55)
	AT3G19860.1			LDPERPKNDKATIL
				TDTVQLLKELTSEV
				N


390	Cc/clementine	64%	60-114	RKMQKADREKLRR	814	80%
	0.9_014901m	(185/288)		DRLNEHFFELGNAL		(44/55)
				DPDRPKNDKATILA
				DTVQLLKDLTSQV
				E


392	Cc/clementine	64%	60-114	RKMQKADREKLRR	815	80%
	0.9_014926m	(185/288)		DRLNEHFFELGNAL		(44/55)
				DPDRPKNDKATILA
				DTVQLLKDLTSQV
				E


400	Pt/POPTR_00	62%	42-96	RKIQKADREKLRR	819	80%
	04s17540.1	(156/249)		DRLNEHFVELGNTL		(44/55)
				DPDRPKNDKATILA
				DTIQLLKDLTSQVD

402	Pt/POPTR_00	61%	38-92	RKIQKADREKLRR	820	80%
	09s13220.1	(176/288)		DRLNEHFVELGNTL		(44/55)
				DPDRPKNDKATILA
				DTVQLLKDLNSKV
				D

386	Vv/GSVIVTO	56%	50-104	RKVQKADREKLRR	812	78%
	1018777001	(141/248)		DRLNEHFLELGNTL		(43/55)
				DPDRPKNDKATILA
				DTIQMLKDLTAEV
				N


382	Gm/Glyma07g	53%	56-110	RKVLKADREKLRR	810	78%
	26910.1	(123/232)		DRLNEHFQELGNA		(43/55)
				LDPDRPKNDKATIL
				TETVQMLKDLTAE
				VN

394	Gm/Glyma08g	65%	7-61	RKTQKADREKLRR	816	76%
	15740.1	(157/238)		DRLNEQFVELGNIL		(42/55)
				DPDRPKNDKATIIG
				DTIQLLKDLTSQVS

398	Gm/Glyma12g	57%	7-61	RKTQKADREKLRR	818	76%
	02740.1	(137/239)		DRFNVQFVELGNIL		(42/55)
				DPDRPKNDKATILG
				DTIQLLKDLTSEVS


396	Gm/Glyma15g	63%	21-75	RKTQKADREKLRR	817	74%
	29630.1	(151/238)		DRINEQFVELGNIL		(41/55)
				DPDRPKNDKATILC
				DTIQLLKDLISQVS


404	Vv/GSVIVTO	57%	46-100	RKVQKADREKLRR	821	74%
	1024084001	(166/288)		DRLNEQFIELGNAL		(41/55)
				DPDRPKNDKATILS
				DTIQLLKDLTAQVE


384	Pt/POPTR_00	51%	61-115	KKVQKADREKLRR	811	74%
	05s11550.1	(116/227)		DNLNEQFLELGTTL		(41/55)
				DPDRPKNDKATILT
				DTIQVLKDLTAEVN


378	Si/Si017804m	48%	36-90	RKVQKADREKMRR	808	74%
		(130/268)		DKLNEQFQELGNT		(41/55)
				LDPDRPRNDKATIL
				GDTIQMLKDLTSH
				VN

406	Sl/Solyc01g11	61%	57-111	RKVQKADREKLRR	822	70%
	1130.2.1	(151/247)		DRLNEQFMELGKT		(39/55)
				LDPDRPKNDKASIL
				SDTVQILKDLTAQV
				S


408	Zm/GRMZM2	50%	30-84	RKVQKADREKMRR	823	70%
	G114444_T02	(119/236)		DKLNEQFQDLGNA		(39/55)
				LDPDRPRNDKATIL
				GDTIQMLKDLTTQ
				VN

374	Bd/Bradi3g11	49%	40-94	RKVQKADRERMRR	806	69%
	520.1	(127/256)		DKLNEQFQELGTTL		(38/55)
				DPDRPRNDKATILG
				DTIQMLKDLSSQVN

376	Os/LOC_Os02	48%	40-94	RKVQKADREKMRR	807	69%
	g23823.1	(116/241)		DRLNEQFQELGSTL		(38/55)
				DPDRPRNDKATILS
				DAIQMLKDLTSQV
				N


380	At/AT4G3606	65%	41-99	KKEAVCSQKAERE	809	61%
	0.1	(68/104)		KLRRDKLKEQFLEL		(36/59)
				GNALDPNRPKSDK
				ASVLTDTIQMLKD
				VMNQVD

TABLE 19

Conserved ′putative leucine zipper domain′ of bHLH121 and closely related sequences

						Col. 7
						Percent
						identity of
						putative
						leucine
						zipper
			Col. 4		Col. 6	domain in
		Col. 3	Putative		SEQ ID	Col. 5 to
		Percent	leucine	Col. 5	NO: of	putative
Col. 1		identity of	zipper	Conserved	putative	leucine
SEQ	Col. 2	polypeptide	domain in	putative	leucine	zipper
ID	Species/	in Col. 1	amino acid	leucine zipper	zipper	domain of
NO:	Identifier	to bHLH121	coordinates	domain	domain	bHLH121

388	At1bHLH121	100%	61-97	KLKSEYTALTDESR	831	100%
	or	(284/284)		ELTQEKNDLREEKT		(37/37)
	AT3G19860.1			SLKSDIENL

406	Sl/Solyc01g11	61%	112-148	RLKSEYAALTDESR	840	89%
	1130.2.1	(151/247)		ELTQEKNDLREEK		(33/37)
				ASLKSDIESL

394	Gm/Glyma08g	65%	62-98	KLKDEYATLNEESR	834	81%
	15740.1	(157/238)		ELTQEKNDLREEK		(30/37)
				ASLKSDIGNL

390	Cc/clementine	64%	115-151	KLKTEHAALTEESR	832	81%
	0.9_014901m	(185/288)		ELTQEKNDLREEKL		(30/37)
				SLRSEIENL

392	Cc/clementine	64%	115-151	KLKTEHAALTEESR	833	81%
	0.9_014926m	(185/288)		ELTQEKNDLREEKL		(30/37)
				SLRSEIENL

400	Pt/POPTR_00	62%	97-133	KLKAEYATLSEESL	837	81%
	04s17540.1	(156/249)		ELTQEKNDLREEK		(30/37)
				ASLKSDIENL

402	Pt/POPTR_00	61%	93-129	KLKAEHAALSEESR	838	78%
	09s13220.1	(176/288)		ELTLEKNDLREEKA		(29/37)
				SLKSDVENL

396	Gm/Glyma15g	63%	6-112	KLKDEYAMLNEES	835	75%
	29630.1	(151/238)		RELTLEKTDLREEK		(28/37)
				ASLKSDIDNL

404	Vv/GSVIVTO	57%	101-137	KLKAENASLNEESR	839	75%
	1024084001	(166/288)		ELTQEKNDLREEK		(28/37)
				ASLKSATENL

382	Gm/Glyma07g	53%	111-147	RLKTEHKTLSEESR	828	75%
	26910.1	(123/232)		ELMQEKNELREEK		(28/37)
				TSLKSDIENL

374	Bd/Bradi3g11	49%	95-131	KLKAEYSSLSEEER	824	72%
	520.1	(127/256)		ELTQEKNELRDEK		(27/37)
				ASLKSDIDNL

398	Gm/Glyma12g	57%	62-98	KLKDEYATLNEESC	836	70%
	02740.1	(137/239)		ELAQEKNELREEK		(26/37)
				ASLKSDILKL

386	Vv/GSVIVTO	56%	105-141	RLKVECAALSEESR	830	70%
	1018777001	(141/248)		ELVQEKNELREEK		(26/37)
				VALKSDIDNL

378	Si/Si017804m	48%	91-127	KLKAEYTSLSEEAR	826	70%
		(130/268)		ELTQEKNELRDEK		(26/37)
				ASLKSEVDNL

380	At/AT4G3606	65%	100-136	RLKAEYETLSQESR	827	67%
	0.1	(68/104)		ELIQEKSELREEKA		(25/37)
				TLKSDIEIL

408	Zm/GRMZM2	50%	85-121	KLKAEYTSLSEEAC	841	67%
	G114444_T02	(119/236)		ELTQEKNELRDEK		(25/37)
				ASLKSEVDNL

376	Os/LOC_Os02	48%	95-131	KLKAEYTSLSEEAR	825	67%
	g23823.1	(116/241)		ELTQEKNELRDEK		(25/37)
				VSLKFEVDNL

384	Pt/POPTR_00	51%	116-152	RLKAECATLSEETH	829	62%
	05s11550.1	(116/227)		ELMQEKNELREEK		(23/37)
				ASLKADTENL

These functionally-related and/or closely-related bHLH121 clade polypeptides may be identified by a consensus bHLH domain sequence, SEQ ID NO: 858:
X¹KX³X⁴X⁵X⁶X⁷X⁸KAX¹¹REX¹⁴X¹⁵RRX¹⁸X¹⁹X²⁰X²¹X²²X²³FX²⁵X²⁶LGX²⁹

X³⁰LDPX³⁴RPX³⁷X³⁸DKAX⁴²X⁴³X⁴⁴X⁴⁵X⁴⁶X⁴⁷X⁴⁸QX⁵⁰LKX⁵³X⁵⁴X⁵⁵X⁵⁶X⁵⁷

VX⁵⁹

where X¹=K or R; X³=E or absent; X⁴=A or absent; X⁵=V or absent; X⁶=C or absent; X⁷=any amino acid; X⁸=any amino acid; X¹¹=any amino acid; X¹⁴=K or R; X¹⁵=I, L, V or M; X¹⁸=D or E; X¹⁹=N, K or R; X²⁰=F, I, L, V or M; X²¹=N or K; X²²=any amino acid; X²³=H or Q; X²⁵=any amino acid; X²⁶=D or E; X²⁹=any amino acid; X³⁰=any amino acid; X³⁴=N, D or E; X³⁷=K or R; X³⁸=any amino acid; X⁴²=S or T; X⁴³=I, L, V or M; X⁴⁴=I, L, V or M; X⁴⁵=any amino acid; X⁴⁶=D, E; X⁴⁷=A or T; X⁴⁸=I, L, V or M; X⁵⁰=I, L, V or M; X⁵³=D or E; X⁵⁴=I, L, V or M; X⁵⁵=any amino acid; X⁵⁶=any amino acid; X⁵⁷=any amino acid; and X⁵⁹=any amino acid.
These functionally-related and/or closely-related bHLH121 clade polypeptides may also be identified by a consensus putative leucine zipper domain sequence, SEQ ID NO: 859:
X¹LKX³X⁴EX⁶X⁷X⁸LX¹⁰X¹¹EX¹³X¹⁴ELX¹⁷X¹⁸EKX²¹X²²LRX²⁵EKX²⁸X²⁹

LX³¹X³²X³³X³⁴X³⁵X³⁶L

where X¹=R or K; X⁴=any amino acid; X⁶=any amino acid; X⁷=any amino acid; X⁸=any amino acid; X¹⁰=any amino acid; X¹¹=Q, D or E; X¹³=any amino acid; X¹⁴=any amino acid; X¹⁷=A, I, L, V or M; X¹⁸=any amino acid; X²¹=any amino acid; X²²=D or E; X²⁵=D or E; X²⁸=any amino acid; X²⁹=S, A or T; X³¹=K or R; X³²=any amino acid; X³³=any amino acid; X³⁴=T, I, L, V or M; X³⁵=any amino acid; and X³⁶=any amino acid.

BBX26 Clade Polypeptides

TABLE 20

Conserved ′BBX domain′ of BBX26 and closely related sequences

						Col. 7
						Percent
						identity of
		Col. 3				BBX
		Percent	Col. 4		Col. 6	domain in
Col. 1		identity of	BBX	Col. 5	SEQ ID	Col. 5 to
SEQ	Col. 2	polypeptide	domain in	Conserved	NO: of	BBX
ID	Species/	in Col. 1	amino acid	BBX	BBX	domain of
NO:	Identifier	to BBX26	coordinates	domain	domain	BBX26

410	At/BBX26 or	100%	5-41	CHTCRHVTAVIHC	769	100%
	AT1G60250.1	(251/251)		VTEALNFCLTCDNL		(37/37)
				RHHNNIHAEH

412	At/AT1G6819	33%	14-51	CEFCKAYRAVVYCI	770	36%
	0.1	(28/84)		ADTANLCLTCDAK		(14/38)
				VHSANSLSGRH

414	Pt/POPTR_00	33%	5-42	CEFCMALRPVVYC	771	31%
	08s12410.1	(24/71)		NADAAYLCLSCDA		(12/38)
				KVHSANALFNRH

420	Sl/Solyc04g00	26%	7-44	CEFCMLLKPVVYC	774	31%
	7470.2	(25/94)		EADAAHLCLSCDA		(12/38)
				KVHSANALSNRH

416	Gm/Glyma10g	27%	5-42	CEFCTALRPLVYCK	772	28%
	41540.1	(22/79)		ADAAYLCLSCDAK		(11/38)
				VHLANAVSGRH

418	Gm/Glyma20g	27%	5-42	CEFCTALRPLVYCK	773	28%
	25700.1	(22/79)		ADAAYLCLSCDSK		(11/38)
				VHLANAVSGRH

These functionally-related and/or closely-related BBX26 clade polypeptides may be identified by a consensus BBX domain sequence, SEQ ID NO: 860:
CX²X³CX⁵X⁶X⁷X⁸X⁹X¹⁰X¹¹X¹²CX¹⁴X¹⁵X¹⁶X¹⁷X¹⁸X¹⁹X²⁰CLX²³CDX²⁶X²⁷

X²⁸HX³⁰X³¹NX³³X³⁴X³⁵X³⁶X³⁷H

where X²=any amino acid; X³=any amino acid; X⁵=any amino acid; X⁶=any amino acid; X⁷=Y, I, L, V, or M; X⁸=any amino acid; X⁹=A or P; X¹⁰=I, L, V, or M; X¹¹=I, L, V, or M; X¹²=any amino acid; X¹⁴=any amino acid; X¹⁵=A or T; X¹⁶=D or E; X¹⁷=A or T; X¹⁸=any amino acid; X¹⁹=any amino acid; X²⁰=F, I, L, V, or M; X²³=S or T; X²⁶=any amino acid; X²⁷=any amino acid; X²⁸=any amino acid; X³⁰=any amino acid; X³¹=A or absent; X³³=any amino acid; X³⁴=I, L, V, or M; X³⁵=any amino acid; X³⁶=any amino acid; and X³⁷=any amino acid.
bHLH121 Clade Polypeptides

TABLE 2

Conserved ′Methyltransferase domain′ of PMT24 and closely related sequences

						Col. 7
						Percent
						identity of
						methyl-
						transferase
		Col. 3	Col. 4		Col. 6	domain in
		Percent	Methyl	Col. 5	SEQ ID	Col. 5 to
Col. 1		identity of	trans-	Conserved	NO: of	methyl-
SEQ	Col. 2	polypeptide	ferase	methyl-	methyl-	transferase
ID	Species/	in Col. 1	amino acid	transferase	transferase	domain
NO:	Identifier	to PMT24	coordinates	domain	domain	of PMT24

444	At1bHLH121	100%	367-584	VILDVGCGVASFGG	786	100%
	or	(770/770)		YLFDRDVLALSFAP		(218/218)
	AT1G29470.1			KDEHEAQVQFALE
				RGIPAMSNVMGTK
				RLPFPGSVFDLIHC
				ARCRVPWHIEGGK
				LLLELNRALRPGGF
				FVWSATPVYRKTE
				EDVGIWKAMSKLT
				KAMCWELMTIKKD
				ELNEVGAAIYQKP
				MSNKCYNERSQNE
				PPLCKDSDDQNAA
				WNVPLEACIHKVT
				EDSSKRGAVWPES
				WPERVETVPQWLD
				SQEGVY

446	At/AT2G3430	81%	367-584	VILDVGCGVASFGG	787	92%
	0.1	(638/783)		YLFERDVLALSFAP		(202/218)
				KDEHEAQVQFALE
				RGIPAMLNVMGTK
				RLPFPGSVFDLIHC
				ARCRVPWHIEGGK
				LLLELNRALRPGGF
				FVWSATPVYRKNE
				EDSGIWKAMSELT
				KAMCWKLVTIKKD
				KLNEVGAAIYQKPT
				SNKCYNKRPQNEPP
				LCKDSDDQNAAW
				NVPLEACMHKVTE
				DSSKRGAVWPNM
				WPERVETAPEWLD
				SQEGVY

450	Pt/POPTR_00	66%	423-640	VILDVGCGVASFGG	789	81%
	05s20670.1	(548/826)		YLFERDVLAMSFAP		(177/218)
				KDEHEAQVQFALE
				RGIPAMLAVMGTK
				RLPFPSSVFDVVHC
				ARCRVPWHVEGGK
				LLLELNRVLRPGGY
				FVWSATPVYQKLP
				EDVGIWKAMSKLT
				KSMCWDLVVIKKD
				KLNGVGAAIFRKPT
				SNDCYNNRPQNEPP
				LCKESDDPNAAWN
				VPLEACMHKVPED
				ASVRGSRWPEQWP
				QRLEKPPYWLNSQ
				VGVY

448	Pt/POPTR_00	64%	412-629	VILDVGCGVASFGG	788	80%
	02s07640.1	(526/815)		YLLEKDVLAMSFA		(176/218)
				PKDEHEAQVQFAL
				ERGIPAMLAVMGT
				KRLPFPNSVFDLVH
				CARCRVPWHIEGG
				KLLLELNRVLRPGG
				YFVWSATPVYRKR
				PEDVGIWKAMSKL
				TKSMCWDLVVIKT
				DTLNGVGAAIYRK
				PTSNDCYNNRPQN
				EPPLCKESDDPNAA
				WNVLLEACMHKVP
				VDASVRGSHWPEQ
				WPKRLEKPPYVVLN
				SQVGVY

478	Pt/POPTR_00	62%	392-610	VILDVGCGVASFGG	803	75%
	05s06640.1	(499/803)		YLFDRDVLAMSFA		(166/219)
				PKDEHEAQIQFALE
				RGIPAISAVMGTKR
				LPYPGRVFDAVHC
				ARCRVPWHIEGGK
				LLLELNRVLRPGGF
				FVWSATPVYQKLA
				EDVEIWQAMTELT
				KAMCWELVSINKD
				TLNGVGVATYRKP
				TSNDCYEKRSKQEP
				PLCEASDDPNAAW
				NVPLQACMHKVPV
				GSLERGSQWPEQW
				PARLDKTPYWMLS
				SQVGVY

468	Gm/Glyma04g	62%	390-608	VILDVGCGVASFGG	798	75%
	38870.1	(494/794)		FLFDRDVLAMSLAP		(165/219)
				KDEHEAQVQFALE
				RGIPAISAVMGTKR
				LPFPGKVFDVVHC
				ARCRVPWHIEGGK
				LLLELNRVLRPGGF
				FVWSATPIYQKLPE
				DVEIWKAMKTLTK
				AMCWEVVSISKDQ
				VNGVGVAVYKKPT
				SNECYEQRSKNEPP
				LCPDSDDPNAAWN
				IKLQACMHKVPASS
				KERGSKLPELWPA
				RLTKVPYWLLSSQ
				VGVY

480	Pt/POPTR_00	61%	420-638	VILDVGCGVASFGG	804	75%
	07s04340.1	(506/829)		YLFDRDVLTMSFAP		(165/219)
				KDEHEAQVQFALE
				RGIPAISAVMGTKR
				LPYPGRVFDAVHC
				ARCRVPWHIEGGK
				LLLELNRVLRPGGL
				FVWSATPVYQKLA
				EDVEIWQAMTELT
				KAMCWELVSINKD
				TINGVGVATYRKPT
				SNDCYEKRSKQEPP
				LCEASDDPNAAWN
				VPLQACMHKVPVD
				SLERGSQWPEQWP
				ARLGKTPYWMLSS
				QVGVY


428	Si/Si000354m	60%	397-615	VILDVGCGVASFGG	778	75%
		(486/810)		YMFDRDVLTMSFA		(166/219)
				PKDEHEAQVQFAL
				ERGIPAISAVMGTK
				RLPYPSRVFDVIHC
				ARCRVPWHIEGGM
				LLLELNRLLRPGGY
				FVWSATPVYQKLP
				EDVEIWNAMSALT
				KSMCWKMVNKTK
				DKLNQVGMAIYQK
				PMDNNCYEKRSEN
				NPPLCKDSDDADA
				AWNVPLEACMHKL
				PAGPTVRGAKWPE
				SWPQRLEKTPFVVL
				NGSQVGVY

466	Eg/Eucgr.I001	59%	410-628	VILDVGCGVASFGG	797	75%
	86.1	(487/819)		YLFDRDVLAMSLA		(165/219)
				PKDEHEAQVQFAL
				ERGIPAISAVMGTT
				RLPFPSRVFDIVHC
				ARCRVPWHIEGGK
				LLLELNRLLRPGGF
				FVWSATPVYQKIPD
				DVAIWKAMSALLK
				SMCWELISINKDTL
				NGVGVATYRKPMS
				NECYEKRSQNDPP
				MCADSDDSNAAW
				YVPLQTCMHKIPID
				SAERGSQWPEEWP
				ARLVKTPYWLLSS
				QVGVY

470	Gm/Glyma06g	62%	402-620	VILDVGCGVASFGG	799	74%
	16050.1	(503/810)		FLFDRDVLAMSLAP		(164/219)
				KDEHEAQVQFALE
				RGIPAISAVMGTKR
				LPFPGKVFDVVHC
				ARCRVPWHIEGGK
				LLLELNRVLRPGGF
				FVWSATPIYQKLPE
				DVEIWKAMKALTK
				AMCWEVVSISKDP
				VNGVGVAVYRKPT
				SNECYEQRSKNEPP
				LCPDSDDPNAAWN
				IQLQACLHKAPVSS
				KERGSKLPELWPA
				RLIKVPYWLSSSQV
				GVY

482	Vv/GSVIVTO	61%	357-575	VVLDVGCGVASFG	805	74%
	1026451001	(480/775)		GYLFDKDVLTMSF		(164/219)
				APKDEHEAQVQFA
				LERGIPGISAVMGT
				KRLPFPAMVFDVV
				HCARCRVPWHIEG
				GKLLLELNRVLRPG
				GFFVWSATPVYQK
				LADDVAIWNAMTE
				LMKSMCWELVVIK
				RDVVNRVAAAIYK
				KPTSNDCYEKRSQ
				NEPPICADSEDANA
				AWNVPLQACMHK
				VPVDASKRGSQWP
				ELWPARLDKSPYW
				LTSSQVGVY

452	Eg/Eucgr.F042	55%	423-639	VILDVGCGVASFGG	790	74%
	85.1	(465/832)		YLFERDVLTMSFAP		(162/218)
				KDVHEAQVQFALE
				RGIPAILGVMGTKR
				LPFPGGVFDVIHCA
				RCRVPWHIEGGKL
				LLELNRVLRPGGYF
				LWSATPIYRRDQED
				IGIWKEMSKLTMA
				MCWDLVMIKKDK
				LNKVAIAMYRKPT
				SNECYEKRPQNEPP
				LCDNFDDPNSAWN
				VTLQACMHKVPVD
				MSKRGSNWPEKWP
				VRLEKPPYWLNEL
				GVY

460	Vv/GSVIVTO	73%	150-368	VILDVGCGVASFGG	794	73%
	1008776001	(409/554)		YIFERDVLAMSFAP		(162/219)
				KDEHEAQVQFALE
				RGIPAISAVMGTTR
				LPFPSRVFDVVHCA
				RCRVPWHIEGGKL
				LLELNRVLRPGGYF
				VWSATPVYRKVPE
				DVGIWNAMSEITK
				KICWDLVAMSKDS
				LNGIGAAIYRKPTS
				NECYEKRPRNEPPL
				CEESDNADAAWNI
				PLQACMHKVPVLT
				SERGSQWPEQWPL
				RVEKAPNWLKSSQ
				VGVY

462	Sl/Solyc04g06	59%	364-582	VILDVGCGVASFGG	795	73%
	3230.2.1	(470/784)		YLFERDVLAMSLA		(162/219)
				PKDEHEAQVQFAL
				ERGIPAISAVMGTK
				RLPFPGKVFDAVHC
				ARCRVPWHIEGGK
				LLLELNRVLRPGGH
				FIWSATPVYRKDEE
				NVGIWEAMSELTK
				SMCWELLEINEDKL
				NEVGVAIFRKPTTN
				DCYQSRTQNDPPM
				CEEADDPDAAWNI
				TLQACLHKAPADA
				SARGAKWPAKWPL
				RSEKLPYWLKSSQ
				VGVY

426	Zm/GRMZM2	59%	392-610	VILDVGCGVASFGG	777	73%
	G049269_T01	(479/801)		YMFDRDALTMSFA		(160/219)
				PKDEHEAQVQFAL
				ERGIPAISAVMGTK
				RLPYPSRVFDVIHC
				ARCRVPWHIEGGM
				LLLELNRLLRPGGY
				FVWSATPVYQKLP
				EDVEIWNAMSTLT
				KSMCWKMVNKTK
				DKLNQVGMVIYQK
				PMDNICYEKRSENS
				PPLCKESDDADAA
				WNVPLEACMHKLP
				GGSKVRGSKWPEL
				WPQRLEKTPFWID
				GSKVGVY

476	Sl/Solyc05g05	59%	409-627	VILDVGCGVASFGG	802	73%
	6580.2.1	(486/818)		YLFERDVLAMSLA		(160/219)
				PKDEHEAQVQFAL
				ERGIPAISAVMGTK
				RLPFPSRVFDVVHC
				ARCRVPWHIEGGK
				LLLELNRVLRPGGL
				FVWSATPVYQKLP
				EDVEIWEAMQKLT
				KAMCWDLVSKTK
				DRVNGVGVAVYR
				KPTSNECYEQRSKD
				APPICQGSDDPNAA
				WNVPLQACMHKA
				PVATSERGSQWPEP
				WPARLSKSPYWLL
				SSQVGVY

474	Gm/Glyma08g	58%	438-656	VILDVGCGVASFGG	801	73%
	00320.1	(493/847)		FLEERDVLTMSLAP		(162/219)
				KDEHEAQVQFALE
				RGIPAISAVMGTKR
				LPYPGRVFDVVHC
				ARCRVPWHIEGGK
				LLLELNRVLRPGGF
				FVWSATPIYQKLPE
				DVEIWNEMKALTK
				AMCWEVVSISKDK
				LNGVGIAVYKKPTS
				NECYEKRSQNQPPI
				CPDSDDPNAAWNV
				PLQACMHKVPVSS
				TERGSQWPEKWPA
				RLTNIPYWLTNSQV
				GVY

472	Gm/Glyma05g	58%	427-645	VILDVGCGVASFGG	800	73%
	32670.1	(492/837)		FLEERDVLTMSLAP		(161/219)
				KDEHEAQVQFALE
				RGIPAISAVMGTKR
				LPYPGRVFDVVHC
				ARCRVPWHIEGGK
				LLLELNRVLRPGGF
				FVWSATPIYQKLPE
				DVEIWNEMKALTK
				AMCWEVVSISKDK
				LNGVGIAVYKKPTS
				NECYEKRSQNQPPI
				CPDSDDPNAAWNIP
				LQACMHKVPVSST
				ERGSQWPEKWPAR
				LTNTPYWLTNSQV
				GVY

456	Gm/Glyma04g	56%	429-646	VILDVGCGVASFGG	792	73%
	42270.1	(473/835)		YLFEKDVLTMSFAP		(160/218)
				KDVHEAQVQFALE
				RGIPATLGVMGTV
				RLPYPGSVFDLVHC
				ARCRVPWHIEGGK
				LLLELNRVLRPGGH
				FVWSATPVYQKDP
				EDVEIWKAMGEIT
				KSMCWDLVVIAKD
				KLNGVAAAIYRKP
				TDNECYNNRIKHEP
				PMCSESDDPNTAW
				NVSLQACMHKVPV
				DASERGSIWPEQWP
				LRLEKPPYWIDSQA
				GVY


422	Bd/Bradi2g57	59%	394-612	VILDVGCGVASFGG	775	72%
	087.1	(485/810)		YMFDRDVLTMSFA		(158/219)
				PKDEHEAQVQFAL
				ERGIPAISAVMGTK
				RLPYPSRVFDVIHC
				ARCRVPWHIEGGK
				LLLELNRLLRPGGY
				FVWSATPVYQKLP
				EDVEIWNAMSSLT
				KSMCWKMVKKTK
				DTLNQVGMAIYQK
				PMDNNCYEKRSED
				SPPLCKETDDADAS
				WNITLQACIHKLPV
				GPSVRGSKWPEFW
				PQRLEKTPFVVIDGS
				HVGVY


424	Os/LOC_Os01	58%	406-624	VILDVGCGVASFGG	776	72%
	g66110.1	(479/812)		YMFERDVLTMSFA		(159/219)
				PKDEHEAQVQFAL
				ERGIPAISAVMGTK
				RLPYPSRVFDVIHC
				ARCRVPWHIEGGM
				LLLELNRLLRPGGY
				FVWSATPVYQKLP
				EDVEIWNAMSSLT
				KAMCWKMVNKTK
				DKLNQVGMAIYQK
				PMDNSCYEKRPEN
				SPPLCKETDDADAA
				WNVPLQACMHKLP
				AGQSVRGSKWPET
				WPQRLEKTPYWID
				DSHVGIY

464	At/AT5G6403	58%	425-643	VVLDVGCGVASFG	796	72%
	0.1	(490/834)		GFLFDRDVITMSLA		(159/219)
				PKDEHEAQVQFAL
				ERGIPAISAVMGTT
				RLPFPGRVFDIVHC
				ARCRVPWHIEGGK
				LLLELNRVLRPGGF
				FVWSATPVYQKKT
				EDVEIWKAMSELIK
				KMCWELVSINKDTI
				NGVGVATYRKPTS
				NECYKNRSEPVPPI
				CADSDDPNASWKV
				PLQACMHTAPEDK
				TQRGSQWPEQWPA
				RLEKAPFVVLSSSQT
				GVY

458	Gm/Glyma06g	57%	406-623	VILDVGCGVASFGG	793	72%
	12540.1	(464/811)		YLFEKDVLTMSFAP		(159/218)
				KDVHEAQVQFALE
				RGIPATLGVMGTV
				RLPYPGSVFDLLHC
				ARCRVPWHVEGGK
				LLLELNRVLRPGGY
				FVWSATPVYQKDP
				EDVEIWKAMGEIT
				KSMCWDLVVIAKD
				KLNGVAAAIYRKP
				TDNECYNNRIKNEP
				SMCSESDDPNTAW
				NVSLQACMHKVPV
				DASERGSIWPEQWP
				LRLEKPPYWIDSQA
				GVY

438	Bd/Bradi4g23	50%	469-688	VVLDVGCGVASFG	783	70%
	610.1	(440/868)		GFLFDRGALTMSFA		(155/220)
				PKDEHEAQVQFAL
				ERGIPALSAVMGTK
				RLPFPAGVFDVVHC
				ARCRVPWHIDGGM
				LLLELNRLLRPGGF
				FVWSATPVYQKLP
				EDVEIWDDMVKLT
				KAMCWEMVKKIL
				DTLDQVGLVIFRKP
				KSNRCYETRRQKEP
				PLCDGSDDPNAAW
				NIKLRACMHRAPA
				DYPSVRGSRWPAP
				WPERAEAVPYWLN
				NSQVGVY

434	Zm/GRMZM2	69%	271-489	VVLDVGCGVASFG	781	69%
	G002642_T02	(387/557)		GYLFDRDVITMSFA		(152/219)
				PKDEHEAQVQFAL
				ERGIPAISAVMGTK
				RLPFPSRVFDVVHC
				ARCRVPWHIEGGK
				LLLELDRLLRPGGY
				FVWSATPVYQKLP
				EDVEIWQAMSALT
				SSMCWKMVNKVK
				DRVNRVGIAIYRKP
				TDNSCYEARSETNP
				PLCGEYDDPDAAW
				NISLGACMHKLPV
				DPTVRGSQWPELW
				PLRLEKPPYWLRGS
				EAGVY

440	Os/LOC_Os11	57%	468-686	VALDVGCGVASFG	784	69%
	g08314.1	(418/726)		GYLFDHDVLTMSL		(153/219)
				APKDEHEAQVQFA
				LERGIPAISAVMGT
				RRLPFPSNVFDAVH
				CARCRVPWHIEGG
				MLLLELNRLLRPGG
				FFVWSATPVYQELP
				EDVEIWGEMVKLT
				KAMCWEMVSKTS
				DTVDQVGLVTFRK
				PADNACYMKRRQK
				EPPLCEPSDDPNAA
				WNITLRACMHWVP
				TDPSVRGSWVVPER
				WPERMEKTPYWLN
				SSQVGVY

430	Bd/Bradi5g27	55%	319-537	VVLDVGCGVASFG	779	69%
	590.1	(429/773)		GYLFDRDVLTMSF		(153/219)
				APKDEHEAQVQFA
				LERGIPAISAVMGT
				KRLPFPGRVFDAVH
				CARCRVPWHIEGG
				KLLLELDRLLRPGG
				YFVWSATPAYQKL
				PEDVEIWQAMSAL
				TRSMCWKMVNKV
				KDRLNRVGVAIFQ
				KPIDNRCYDGRSAA
				NLPLCGEYDNVDA
				AWNVSLESCIHKLP
				VDPAIRSSRWPEEW
				PLRLERAPYWLKSS
				EPGVY

454	Eg/Eucgr.F042	54%	407-624	VILDVGCGVGSFGG	791	69%
	86.1	(441/813)		YLFERDVLTMSFAP		(152/219)
				KDEHEAQVQFALE
				RGIPAMLAVMGTK
				RLPFPSGVFDAIHC
				ARCRVPWHIEGGK
				LLLELNRLLRPGGY
				FVWSATPIYRKGPE
				DLGIWKEMSKLTT
				AMCWNFTLIKRKD
				KMNKVSIALYRKP
				TSNECIESRTKNEPP
				LCNGLDDANSTWN
				VTLQACMHKVPTD
				MSERGSQWPENWL
				HRLGKPPYWLNKV
				AVN

442	Si/Si028042m	51%	434-654	VVLDVGCGVASFG	785	69%
		(436/840)		GYLFDRDVLTMSL		(154/221)
				APKDEHEAQVQFA
				LERGIPAISAVMGT
				RRLPFPGGVFDVVH
				CARCRVPWHIDGG
				MLLLELNRLLRPGG
				VFVWSATPVYQKL
				PDDVEIWDEMAKL
				TKAMCWEMVAKT
				KHTVVDDQVGVAI
				FRKPERNGCYEKRP
				EKAPPLCEPSDDPN
				AAWNIKLRACMHR
				VPEDPSERGARWPE
				PWPERLGKAPYWL
				DGSQTGVY

432	Os/LOC_Os04	65%	275-493	VVLDVGCGVASFG	780	68%
	g59590.1	(393/599)		GYLFDRDVLTMSF		(151/219)
				APKDEHEAQVQFA
				LERGIPAMSAVMG
				TKRLPFPGRVFDVV
				HCARCRVPWHIEG
				GKLLLELDRLLRPG
				GYFVWSATPVYQK
				LPEDVEIWEAMSTL
				TRSMCWEMVNKV
				KDRVNRVGIAIFRK
				PTDNSCYEARSAA
				NPPICGEYDDPDAA
				WNISLQSCVHRLPT
				DPAIRGSQWPVEW
				PLRLEKPPYWLKNS
				EAGVY

436	Si/Si021320m	55%	337-555	VVLDVGCGVASFG	782	68%
		(432/776)		GYLFDRDVITMSFA		(150/219)
				PKDEHEAQVQFAL
				ERGIPAISAVMGTK
				RLPFPSRVFDVVHC
				ARCRVPWHIEGGK
				LLLELDRLLRPGGY
				FVWSATPVYQKLP
				EDVEIWEAMSALT
				RSMCWKMVNKVK
				DRVNRVGIAIFRKP
				TDNSCYEERSEANS
				PICGEYDDPDAAW
				NVSLRTCMHKLPV
				DLTIRGSKWPELWP
				LRLEKPPYWLKSSE
				AGVY

These functionally-related and/or closely-related PMT24 clade polypeptides may be identified by a consensus methyltransferase domain sequence, SEQ ID NO: 861:

VX²LDVGCGVASFGGX¹⁵X¹⁶FX¹⁸X¹⁹X²⁰X²¹X²²X²³X²⁴SX²⁶AFKDX³¹HEA

QVQFALERGIPAX⁴⁷X⁴⁸X⁴⁹VMGTX⁵⁴RLPX⁵⁸PX⁶⁰X⁶¹VFDX⁶⁵X⁶⁶HCARC

RVPWHX⁷⁷X⁷⁸GGX⁸¹LLLELX⁸⁷RX⁸⁹LRPGGX⁹⁵FX⁹⁷WSATPX¹⁰³YX¹⁰⁵X¹⁰⁶

X¹⁰⁷X¹⁰⁸X¹⁰⁹X¹¹⁰X¹¹¹X¹¹²IWX¹¹⁵X¹¹⁶MX¹¹⁸X¹¹⁹X¹²⁰X¹²¹X¹²²X¹²³MCWX¹²⁷

X¹²⁸X¹²⁹X¹³⁰X¹³¹X¹³²X¹³³X¹³⁴X¹³⁵X¹³⁶X¹³⁷X¹³⁸X¹³⁹X¹⁴⁰VX¹⁴²X¹⁴³X¹⁴⁴

X¹⁴⁵X¹⁴⁶X¹⁴⁷KPX¹⁵⁰X¹⁵¹NX¹⁵³CYX¹⁵⁶X¹⁵⁷RX¹⁵⁹X¹⁶⁰X¹⁶¹X¹⁶²X¹⁶³X¹⁶⁴X¹⁶⁵

CX¹⁶⁷X¹⁶⁸X¹⁶⁹DX¹⁷¹X¹⁷²X¹⁷³X¹⁷⁴X¹⁷⁵WX¹⁷⁷X¹⁷⁸X¹⁷⁹LX¹⁸¹X¹⁸²CX¹⁸⁴HX¹⁸⁶

X¹⁸⁷X¹⁸⁸X¹⁸⁹X¹⁹⁰X¹⁹¹X¹⁹²X¹⁹³X¹⁹RX¹⁹⁶X¹⁹⁷X¹⁹⁸X¹⁹⁹PX²⁰¹X²⁰²WPX²⁰⁵R

X²⁰⁷X²⁰⁸X²⁰⁹X²¹⁰FX²¹²WX²¹⁴X²¹⁵X²¹⁶SX²¹⁸X²¹⁹GX²²¹Y

where X²=any amino acid; X¹⁵=F or Y; X¹⁶=I, L, V or M; X¹⁸=D or E; X¹⁹=H, K or R; X²⁰=D or G; X²¹=any amino acid; X²²=I, L, V or M; X²³=A or T; X²⁴=I, L, V or M; X²⁶=F, I, L, V or M; X³¹=any amino acid; X⁴⁷=T, I, L, V or M; X⁴⁸=any amino acid; X⁴⁹=any amino acid; X⁵⁴=I, L, V or M; X⁵⁸=F or Y; X⁶⁰=A, S or G; X⁶¹=any amino acid; X⁶⁵=I, L, V or M; X⁶⁶=I, L, V or M; X⁷⁷=I, L, V or M; X⁷⁸=D or E; X⁸¹=any amino acid; X⁸⁷=N or D; X⁸⁹=A, I, L, V or M; X⁹⁵=any amino acid; X⁹⁷=I, L, V or M; X¹⁰³=A, I, L, V, or M; X¹⁰⁵=Q or R; X¹⁰⁶=any amino acid; X¹⁰⁷=any amino acid; X¹⁰⁸=any amino acid; X¹⁰⁹=D or E; X¹¹⁰=D or N; X¹¹¹=any amino acid; X¹¹²=any amino acid; X¹¹⁵=any amino acid; X¹¹⁶=any amino acid; X¹¹⁸=any amino acid; X¹¹⁹=any amino acid; X¹²⁰=I, L, V or M; X¹²¹=T, I, L, V or M; X¹²²=any amino acid; X¹²³=any amino acid; X¹²⁷=any amino acid; X¹²⁸=I, L, V or M; X¹²⁹=I, L, V or M; X¹³⁰=any amino acid; X¹³¹=any amino acid; X¹³²=any amino acid; X¹³³=any amino acid; X¹³⁴=H, D; X¹³⁵=any amino acid; X¹³⁶=I, L, V or M; X¹³⁷=V or absent; X¹³⁸=D or absent; X¹³⁹=I, L, V or M; X¹⁴⁰=any amino acid; X¹⁴²=A or G; X¹⁴³=any amino acid; X¹⁴⁴=any amino acid; X¹⁴⁵=T, I, L, V or M; X¹⁴⁶=F or Y; X¹⁴⁷=Q, R or K; X¹⁵⁰=any amino acid; X¹⁵¹=any amino acid; X¹⁵³=any amino acid; X¹⁵⁶=any amino acid; X¹⁵⁷=any amino acid; X¹⁵⁹=any amino acid; X¹⁶⁰=any amino acid; X¹⁶¹=any amino acid; X¹⁶²=any amino acid; X¹⁶³=any amino acid; X¹⁶⁴=S or P; X¹⁶⁵=I, L, V or M; X¹⁶⁷=any amino acid; X¹⁶⁸=any amino acid; X¹⁶⁹=any amino acid; X¹⁷¹=N or D; X¹⁷²=any amino acid; X¹⁷³=N or D; X¹⁷⁴=A or T; X¹⁷⁵=A or S; X¹⁷⁷=any amino acid; X¹⁷⁸=I, L, V or M; X¹⁷⁹=any amino acid; X¹⁸¹=any amino acid; X¹⁸²=A, S or T; X¹⁸⁴=I, L, V or M; X¹⁸⁶=any amino acid; X¹⁸⁷=A, I, L, V or M; X¹⁸⁸=any amino acid; X¹⁸⁹=any amino acid; X¹⁹⁰=any amino acid; X¹⁹¹=Y or absent; X¹⁹²=any amino acid; X¹⁹³=any amino acid; X¹⁹⁴=any amino acid; X¹⁹⁶=S or G; X¹⁹⁷=S or A; X¹⁹⁸=any amino acid; X¹⁹⁹=any amino acid; X²⁰¹=any amino acid; X²⁰²=any amino acid; X²⁰⁵=any amino acid; X²⁰⁷=any amino acid; X²⁰⁸=any amino acid; X²⁰⁹=any amino acid; X²¹⁰=any amino acid; X²¹²=any amino acid; X²¹⁴=I, L, V or M; X²¹⁵=any amino acid; X²¹⁶=any amino acid or absent; X²¹⁸=K, E, H or Q; X²¹⁹=any amino acid; and X²²¹=I, L, V or M.

Alternative consensus sequences comprising the above with conservative substitutions found in the instant Tables are also envisaged and may be expected to provide equivalent function(s).
The presence of one or more of these consensus sequences and/or these amino acid residues is correlated with conferring of improved or increased photosynthetic resource use efficiency to a plant when the expression level of the polypeptide is altered in a plant by being reduced, knocked-out, or overexpressed. An AtMYB27, RBP45A, TCP6, PIL1, PCL1, GTL1, DREB2H, ERF087, NF-YC6, BBX18, bHLH60, BBX26, bHLH121, or PMT24 clade polypeptide sequence that is “functionally-related and/or closely-related” to the listed full length protein sequences or domains provided in the instant Tables may also have, to any of the listed sequences found in the Sequence Listing or to the entire length of a listed sequence, at least 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95% or 96%, 97%, 98%, 99%, or about 100% amino acid identity to any of SEQ ID NOs: 2, 42, 86, 108, 126, 156, 192, 246, 278, 318, 356, 388, 410, 444 or SEQ ID NOs: 2n where n=1 to 241, and/or at least 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95% or 96%, 97%, 98%, 99%, or about 100% amino acid identity to a domain of any of SEQ ID NOs: 483, 490, 510, 538, 566, 588, 599, 608, 623, 629, 659, 686, 702, 721, 741, 760, 769, 786, 813, and/or at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or about 100% identity to any of consensus sequences SEQ ID NOs: 842-861. The presence of the listed domains in a listed polypeptide sequence is correlated with the conferring of improved or increased photosynthetic resource use efficiency to a plant when the expression level of the polypeptide is altered in a plant by being reduced, knocked-out, or overexpressed. All of the sequences that adhere to these functional and sequential relationships are herein referred to as AtMYB27, RBP45A, TCP6, PIL1, PCL1, GTL1, DREB2H, ERF087, NF-YC6, BBX18, bHLH60, BBX26, bHLH121, or PMT24 clade polypeptides, or which fall within the AtMYB27, RBP45A, TCP6, PIL1, PCL1, GTL1, DREB2H, ERF087, NF-YC6, BBX18, bHLH60, BBX26, bHLH121, or PMT24 clade exemplified in the phylogenetic trees presented in the Figures.

Example II. Plant Genotypes and Vector and Cloning Information

A variety of constructs may be used to modulate the activity of regulatory polypeptides (RPs), and to test the activity of orthologs and paralogs in transgenic plant material. This platform provides the material for all subsequent analysis.
An individual plant “genotype” refers to a set of plant lines containing a particular construct or knockout (for example, this might be 35S lines for a given gene sequence (GID, Gene Identifier) being tested, 35S lines for a paralog or ortholog of that gene sequence, lines for an RNAi construct, lines for a GAL4 fusion construct, or lines in which expression of the gene sequence is driven from a particular promoter that enhances expression in particular cell, tissue or condition). For a given genotype arising from a particular transformed construct, multiple independent transgenic lines may be examined for morphological and physiological phenotypes. Each individual “line” (also sometimes known as an “event”) refers to the progeny plant or plants deriving from the stable integration of the transgene(s), carried within the T-DNA borders contained within a transformation construct, into a specific location or locations within the genome of the original transformed cell. It is well known in the art that different lines deriving from transformation with a given transgene may exhibit different levels of expression of that transgene due to so called “position effects” of the surrounding chromatin at the locus of integration in the genome, and therefore it is necessary to examine multiple lines containing each construct of interest.
(1) Overexpression/Tissue-Enhanced/Conditional Expression.
Expression of a given regulatory protein from a particular promoter, for example a photosynthetic tissue-enhanced promoter (e.g., a green tissue- or leaf-enhanced promoter), is achieved either by a direct-promoter fusion construct in which that regulatory protein is cloned directly behind the promoter of interest or by a two component system.
The Two-Component Expression System.
For the two-component system, two separate constructs are used: Promoter::LexA-GAL4TA and opLexA::RP. The first of these (Promoter::LexA-GAL4TA) comprises a desired promoter cloned in front of a LexA DNA binding domain fused to a GAL4 activation domain. The construct vector backbone (pMEN48, also known as P5375) also carries a kanamycin resistance marker, along with an opLexA::GFP (green fluorescent protein) reporter. Transgenic lines are obtained containing this first component, and a line is selected that shows reproducible expression of the reporter gene in the desired pattern through a number of generations. A homozygous population is established for that line, and the population is supertransformed with the second construct (opLexA::RP) carrying the regulatory protein of interest cloned behind a LexA operator site. This second construct vector backbone (pMEN53, also known as P5381) also contains a sulfonamide resistance marker.
Conditional Expression.
Various promoters can be used to overexpress disclosed polypeptides in plants to confer improved photosynthetic resource use efficiency. However, in some cases, there may be limitations in the use of various proteins that confer increased photosynthetic resource use efficiency when the proteins are overexpressed. Negative side effects associated with constitutive overexpression such as small size, delayed growth, increased disease sensitivity, and development and alteration in flowering time are not uncommon. A number of stress-inducible promoters can be used promote protein expression during the periods of stress, and therefore may be used to induce overexpression of polypeptides that can confer improved stress tolerance when they are needed without the adverse developmental or morphological effects that may be associated with their constitutive overexpression.
Promoters that drive protein expression in response to stress can be used to regulate the expression of the disclosed polypeptides to confer photosynthetic resource use efficiency to plants. The promoter may regulate expression of a disclosed polypeptide to an effective level in a photosynthetic tissue. Effective level in this regard refers to an expression level that confers greater photosynthetic resource use efficiency in the transgenic plant relative to the control plant that, for example, does not comprise a recombinant polynucleotide that encodes the disclosed polypeptide. Optionally, the promoter does not regulate protein expression in a constitutive manner.
Such promoters include, but are not limited to, the sequences located in the promoter regions of At5g52310 (RD29A), At5g52300, AT1G16850, At3g46230, AT1G52690, At2g37870, AT5G43840, At5g66780, At3g17520, and At4g09600.
In addition, promoters with expression specific to or enhanced in particular cells or tissue types may be used to express a given regulatory protein only in these cells or tissues. Examples of such promoter types include but are not limited to promoters expressed in green tissue, guard cell, epidermis, whole root, root hairs, vasculature, apical meristems, and developing leaves.
Table 22 lists a number of photosynthetic tissue-enhanced promoters, specifically, mesophyll tissue-enhanced promoters from rice, that may be used to regulate expression of polynucleotides and polypeptides found in the Sequence Listing and structurally and functionally-related sequences. Promoters that may be used to drive expression of polynucleotides and polypeptides found in the Sequence Listing and structurally and functionally-related sequences included, but are not limited to, promoter sequences SEQ ID NO: 862-864 and the following promoters listed in Table 22, as well as promoters that are at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95% or 96%, 97%, 98%, 99%, or about 100% identical to SEQ ID NO: 862-888, or comprise a functional fragment of promoters that are at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95% or 96%, 97%, 98%, 99%, or about 100% identical to SEQ ID NO: 862-888.

TABLE 22

Rice Genes with Photosynthetic Tissue-Enhanced Promoters

		Rice Gene Identifier of Photosynthetic
	SEQ ID NO:	Tissue-Enhanced Promoter

	865	Os02g09720
	866	Os05g34510
	867	Os11g08230
	868	Os01g64390
	869	Os06g15760
	870	Os12g37560
	871	Os03g17420
	872	Os04g51000
	873	Os01g01960
	874	Os05g04990
	875	Os02g44970
	876	Os01g25530
	877	Os03g30650
	878	Os01g64910
	879	Os07g26810
	880	Os07g26820
	881	Os09g11220
	882	Os04g21800
	883	Os10g23840
	884	Os08g13850
	885	Os12g42980
	886	Os03g29280
	887	Os03g20650
	888	Os06g43920

Tissue-enhanced promoters that may be used to drive expression of polynucleotides and polypeptides found in the Sequence Listing and structurally and functionally-related sequences have also been described in U.S. patent publication no. 20110179520A1, incorporated herein by reference. Such promoters include, but are not limited to, Arabidopsis sequences located in the promoter regions of AT1G08465, AT1G10155, AT1G14190, AT1G24130, AT1G24735, AT1G29270, AT1G30950, AT1G31310, AT1G37140, AT1G49320, AT1G49475, AT1G52100, AT1G60540, AT1G60630, AT1G64625, AT1G65150, AT1G68480, AT1G68780, AT1G69180, AT1G77145, AT1G80580, AT2G03500, AT2G17950, AT2G19910, AT2G27250, AT2G33880, AT2G39850, AT3G02500, AT3G12750, AT3G15170, AT3G16340, AT3G27920, AT3G30340, AT3G42670, AT3G44970, AT3G49950, AT3G50870, AT3G54990, AT3G59270, AT4G00180, AT4G00480, AT4G12450, AT4G14819, AT4G31610, AT4G31615, AT4G31620, AT4G31805, AT4G31877, AT4G36060, AT4G36470, AT4G36850, AT4G37970, AT5G03840, AT5G12330, AT5G14070, AT5G16410, AT5G20740, AT5G27690, AT5G35770, AT5G39330, AT5G42655, AT5G53210, AT5G56530, AT5G58780, AT5G61070, and AT5G6491.
In addition to the sequences provided in the Sequence Listing or in this Example, a promoter region may include a fragment of the promoter sequences provided in the Sequence Listing or in this Example, or a complement thereof, wherein the promoter sequence, or the fragment thereof, or the complement thereof, regulates expression of a polypeptide in a plant cell, for example, in response to a biotic or abiotic stress, or in a manner that is enhanced or preferred in certain plant tissues.
(2) Knock-Out/Knock-Down
In some cases, lines mutated in a given regulatory protein may be analyzed. Where available, T-DNA insertion lines in a given gene are isolated and characterized. In cases where a T-DNA insertion line is unavailable, an RNA interference (RNAi) strategy is sometimes used.

Example III. Transformation Methods

Crop species that overexpress polypeptides of the instant description may produce plants with increased photosynthetic resource use efficiency and/or yield. Thus, polynucleotide sequences listed in the Sequence Listing recombined into, for example, one of the expression vectors of the instant description, or another suitable expression vector, may be transformed into a plant for the purpose of modifying plant traits for the purpose of improving yield, quality, and/or photosynthetic resource use efficiency. The expression vector may contain a constitutive, tissue-enhanced or inducible promoter operably linked to the polynucleotide. The cloning vector may be introduced into a variety of plants by means well known in the art such as, for example, direct DNA transfer or Agrobacterium tumefaciens-mediated transformation.
Transformation of Monocots.
Cereal plants including corn, wheat, rice, sorghum, barley, or other monocots may be transformed with the present polynucleotide sequences, including monocot or eudicot-derived sequences such as those presented in the present Tables, cloned into a vector such as pGA643 and containing a kanamycin-resistance marker, and expressed constitutively under, for example, the CaMV35S or COR15 promoters, or with tissue-enhanced or inducible promoters. The expression vectors may be one found in the Sequence Listing, or any other suitable expression vector may be similarly used. For example, pMEN020 may be modified to replace the NptII coding region with the BAR gene of Streptomyces hygroscopicus that confers resistance to phosphinothricin. The KpnI and BglII sites of the Bar gene are removed by site-directed mutagenesis with silent codon changes.
The cloning vector may be introduced into a variety of cereal plants by means well known in the art including direct DNA transfer or Agrobacterium tumefaciens-mediated transformation. The latter approach may be accomplished by a variety of means, including, for example, that of U.S. Pat. No. 5,591,616, in which monocotyledon callus is transformed by contacting dedifferentiating tissue with the Agrobacterium containing the cloning vector.
The sample tissues are immersed in a suspension of 3×10⁻⁹cells of Agrobacterium containing the cloning vector for 3-10 minutes. The callus material is cultured on solid medium at 25° C. in the dark for several days. The calli grown on this medium are transferred to a Regeneration Medium. Transfers are continued every two to three weeks (two or three times) until shoots develop. Shoots are then transferred to Shoot-Elongation Medium every 2-3 weeks. Healthy looking shoots are transferred to Rooting Medium and after roots have developed, the plants are placed into moist potting soil.
The transformed plants are then analyzed for the presence of the NPTII gene/kanamycin resistance by ELISA, using the ELISA NPTII kit from 5Prime-3Prime Inc. (Boulder, Colo.).
It is also routine to use other methods to produce transgenic plants of most cereal crops (Vasil, 1994. Plant Mol. Biol. 25: 925-937) such as corn, wheat, rice, sorghum (Cassas et al., 1993. Proc. Natl. Acad. Sci. USA 90: 11212-11216), and barley (Wan and Lemeaux, 1994. Plant Physiol. 104: 37-48). DNA transfer methods such as the microprojectile method can be used for corn (Fromm et al., 1990. Bio/Technol. 8: 833-839; Gordon-Kamm et al., 1990. Plant Cell 2: 603-618; Ishida, 1990. Nature Biotechnol. 14:745-750), wheat (Vasil et al., 1992. Bio/Technol. 10:667-674; Vasil et al., 1993. Bio/Technol. 11:1553-1558; Weeks et al., 1993. Plant Physiol. 102:1077-1084), and rice (Christou, 1991. Bio/Technol. 9:957-962; Hiei et al., 1994. Plant J. 6:271-282; Aldemita and Hodges, 1996. Planta 199: 612-617; and Hiei et al., 1997. Plant Mol. Biol. 35:205-218). For most cereal plants, embryogenic cells derived from immature scutellum tissues are the preferred cellular targets for transformation (Hiei et al., 1997. supra; Vasil, 1994. supra). For transforming corn embryogenic cells derived from immature scutellar tissue using microprojectile bombardment, the A188XB73 genotype is the preferred genotype (Fromm et al., 1990. Bio/Technol. 8: 833-839; Gordon-Kamm et al., 1990. supra). After microprojectile bombardment the tissues are selected on phosphinothricin to identify the transgenic embryogenic cells (Gordon-Kamm et al., 1990. supra). Transgenic plants from transformed host plant cells may be regenerated by standard corn regeneration techniques (Fromm et al., 1990. Bio/Technol. 8: 833-839; Gordon-Kamm et al., 1990. supra).
Transformation of Dicots.
It is now routine to produce transgenic plants using most eudicot plants (see U.S. Pat. No. 8,273,954 (Rogers et al.) issued Sep. 25, 2012; Weissbach and Weissbach, 1989. Methods for Plant Molecular Biology, Academic Press; Gelvin et al., 1990. Plant Molecular Biology Manual, Kluwer Academic Publishers; Herrera-Estrella et al., 1983. Nature 303: 209; Bevan, 1984. Nucleic Acids Res. 12: 8711-8721; and Klee, 1985. Bio/Technology 3: 637-642). Methods for analysis of traits are routine in the art and examples are disclosed above.
Numerous protocols for the transformation of tomato and soy plants have been previously described, and are well known in the art. Gruber et al., in Glick and Thompson, 1993. Methods in Plant Molecular Biology and Biotechnology. eds., CRC Press, Inc., Boca Raton, describe several expression vectors and culture methods that may be used for cell or tissue transformation and subsequent regeneration. For soybean transformation, methods are described by Miki et al., 1993. in Methods in Plant Molecular Biology and Biotechnology, p. 67-88, Glick and Thompson, eds., CRC Press, Inc., Boca Raton; and U.S. Pat. No. 5,563,055, (Townsend and Thomas), issued Oct. 8, 1996.
There are a substantial number of alternatives to Agrobacterium-mediated transformation protocols, other methods for the purpose of transferring exogenous genes into soybeans or tomatoes. One such method is microprojectile-mediated transformation, in which DNA on the surface of microprojectile particles is driven into plant tissues with a biolistic device (see, for example, Sanford et al., 1987. Part. Sci. Technol. 5:27-37; Sanford, 1993. Methods Enzymol. 217: 483-509; Christou et al., 1992. Plant. J. 2: 275-281; Klein et al., 1987. Nature 327: 70-73; U.S. Pat. No. 5,015,580 (Christou et al), issued May 14, 1991; and U.S. Pat. No. 5,322,783 (Tomes et al.), issued Jun. 21, 1994).
Alternatively, sonication methods (see, for example, Zhang et al., 1991. Bio/Technology 9: 996-997); direct uptake of DNA into protoplasts using CaCl₂precipitation, polyvinyl alcohol or poly-L-ornithine (see, for example, Hain et al., 1985. Mol. Gen. Genet. 199: 161-168; Draper et al., 1982. Plant Cell Physiol. 23: 451-458); liposome or spheroplast fusion (see, for example, Deshayes et al., 1985. EMBO J., 4: 2731-2737; Christou et al., 1987. Proc. Natl. Acad. Sci. USA 84: 3962-3966); and electroporation of protoplasts and whole cells and tissues (see, for example, Donn et al. 1990. in Abstracts of VIIth International Congress on Plant Cell and Tissue Culture IAPTC, A2-38: 53; D'Halluin et al., 1992. Plant Cell 4: 1495-1505; and Spencer et al., 1994. Plant Mol. Biol. 24: 51-61) have been used to introduce foreign DNA and expression vectors into plants.
After a plant or plant cell is transformed (and the transformed host plant cell then regenerated into a plant), the transformed plant may propagated vegetatively or it may be crossed with itself or a plant from the same line, a non-transformed or wild-type plant, or another transformed plant from a different transgenic line of plants. Crossing provides the advantages of producing new and often stable transgenic varieties. Genes and the traits they confer that have been introduced into a tomato or soybean line may be moved into distinct line of plants using traditional backcrossing techniques well known in the art. Transformation of tomato plants may be conducted using the protocols of Koornneef et al, 1986. In Tomato Biotechnology: Alan R. Liss, Inc., 169-178, and in U.S. Pat. No. 6,613,962, the latter method described in brief here. Eight day old cotyledon explants are precultured for 24 hours in Petri dishes containing a feeder layer of Petunia hybrida suspension cells plated on MS medium with 2% (w/v) sucrose and 0.8% agar supplemented with 10 μM α-naphthalene acetic acid and 4.4 μM 6-benzylaminopurine. The explants are then infected with a diluted overnight culture of Agrobacterium tumefaciens containing an expression vector comprising a polynucleotide of the instant description for 5-10 minutes, blotted dry on sterile filter paper and cocultured for 48 hours on the original feeder layer plates. Culture conditions are as described above. Overnight cultures of Agrobacterium tumefaciens are diluted in liquid MS medium with 2% (w/v/) sucrose, pH 5.7, to an OD₆₀₀of 0.8.
Following cocultivation, the cotyledon explants are transferred to Petri dishes with selective medium comprising MS medium with 4.56 μM zeatin, 67.3 μM vancomycin, 418.9 μM cefotaxime and 171.6 μM kanamycin sulfate, and cultured under the culture conditions described above. The explants are subcultured every three weeks onto fresh medium. Emerging shoots are dissected from the underlying callus and transferred to glass jars with selective medium without zeatin to form roots. The formation of roots in a kanamycin sulfate-containing medium is a positive indication of a successful transformation.
Transformation of soybean plants may be conducted using the methods found in, for example, U.S. Pat. No. 5,563,055 (Townsend et al., issued Oct. 8, 1996), described in brief here. In this method soybean seed is surface sterilized by exposure to chlorine gas evolved in a glass bell jar. Seeds are germinated by plating on 1/10 strength agar solidified medium without plant growth regulators and culturing at 28° C. with a 16 hour day length. After three or four days, seed may be prepared for cocultivation. The seedcoat is removed and the elongating radicle removed 3-4 mm below the cotyledons.
Eucalyptus is now considered an important crop that is grown for example to provide feedstocks for the pulp and paper and biofuel markets. This species is also amenable to transformation as described in PCT patent publication WO/2005/032241.
Crambe has been recognized as a high potential oilseed crop that may be grown for the production of high value oils. An efficient method for transformation of this species has been described in PCT patent publication WO 2009/067398 A1.
Overnight cultures of Agrobacterium tumefaciens harboring the expression vector comprising a polynucleotide of the instant description are grown to log phase, pooled, and concentrated by centrifugation. Inoculations are conducted in batches such that each plate of seed was treated with a newly resuspended pellet of Agrobacterium. The pellets are resuspended in 20 ml inoculation medium. The inoculum is poured into a Petri dish containing prepared seed and the cotyledonary nodes are macerated with a surgical blade. After 30 minutes the explants are transferred to plates of the same medium that has been solidified. Explants are embedded with the adaxial side up and level with the surface of the medium and cultured at 22° C. for three days under white fluorescent light. These plants may then be regenerated according to methods well established in the art, such as by moving the explants after three days to a liquid counter-selection medium (see U.S. Pat. No. 5,563,055).
The explants may then be picked, embedded and cultured in solidified selection medium. After one month on selective media transformed tissue becomes visible as green sectors of regenerating tissue against a background of bleached, less healthy tissue. Explants with green sectors are transferred to an elongation medium. Culture is continued on this medium with transfers to fresh plates every two weeks. When shoots are 0.5 cm in length they may be excised at the base and placed in a rooting medium.
Experimental Methods; Transformation of Arabidopsis.
Transformation of Arabidopsis is performed by an Agrobacterium-mediated protocol based on the method of Bechtold and Pelletier, 1998. Unless otherwise specified, all experimental work is performed using the Columbia ecotype.
Plant Preparation.
Arabidopsis seeds are gas sterilized and sown on plates with media containing 80% MS with vitamins, 0.3% sucrose and 1% Bacto™ agar. The plates are placed at 4° in the dark for the days then transferred to 24 hour light at 22° for 7 days. After 7 days the seedlings are transplanted to soil, placing individual seedlings in each pot. The primary bolts are cut off a week before transformation to break apical dominance and encourage auxiliary shoots to form. Transformation is typically performed at 4-5 weeks after sowing.
Bacterial Culture Preparation.
Agrobacterium stocks are inoculated from single colony plates or from glycerol stocks and grown with the appropriate antibiotics until saturation. On the morning of transformation, the saturated cultures are centrifuged and bacterial pellets are re-suspended in Infiltration Media (0.5×MS, 1× Gamborg's Vitamins, 5% sucrose, 200 μl/L Silwet® L77) until an A₆₀₀reading of 0.8 is reached.
Transformation and Harvest of Transgenic Seeds.
The Agrobacterium solution is poured into dipping containers. All flower buds and rosette leaves of the plants are immersed in this solution for 30 seconds. The plants are laid on their side and wrapped to keep the humidity high. The plants are kept this way overnight at 22° C. and then the pots are turned upright, unwrapped, and moved to the growth racks. In most cases, the transformation process is repeated one week later to increase transformation efficiency.
The plants are maintained on the growth rack under 24-hour light until seeds are ready to be harvested. Seeds are harvested when 80% of the siliques of the transformed plants are ripe (approximately five weeks after the initial transformation). This seed is deemed T₀seed, since it is obtained from the T₀generation, and is later plated on selection plates (either kanamycin or sulfonamide). Resistant plants that are identified on such selection plates comprise the T1 generation, from which transgenic seed comprising an expression vector of interest may be derived.

Example IV. Primary Screening Materials and Methods

Plant Growth Conditions.
Seeds from Arabidopsis lines are chlorine gas sterilized using a standard protocol and spread onto plates containing a sucrose-based media augmented with vitamins (80% MS+Vit, 1% sucrose, 0.65% PhytoBlend™ Agar; Caisson Laboratories, Inc., North Logan, Utah) and appropriate kanamycin or sulfonamide concentrations where selection is required. Seeds are stratified in the dark on plates, at 4° C. for 3 days then moved to a walk-in growth chamber (Conviron MTW120, Conviron Controlled Environments Ltd, Winnipeg, Manitoba, Canada) running at a 10 hour photoperiod at a photosynthetic photon flux of approximately 200 μmol m⁻²s⁻¹at plant height and a photoperiod/night temperature regime of 22° C./19° C. After seven days of light exposure seedlings are transplanted into 164 ml volume pots containing autoclaved ProMix® soil. All pots are returned to the same growth-chamber where they are stood in water and covered with a lid for the first seven days. This protocol keeps the soil moist during this period. Seven days after transplanting lids are removed and a watering and nutrition regime begun. All plants receive water three times a week, and a weekly a fertilizer treatment (80% Peter's NPK fertilizer).
Primary Screening.
Between 35 and 38 days after being transferred to lighted conditions on plates, and after between 28 and 31 days growth in soil, a suite of leaf-physiological parameters are measured using an infrared gas analyzer (LI-6400XT, LI-COR® Biosciences, Lincoln, Neb., USA) integrated with a fluorimeter that measures fluorescence from Chlorophyll A (LI-6400-40, LI-COR Biosciences). This technique involves clamping a leaf between two gaskets, effectively sealing it inside a chamber, then measuring the exchange of carbon dioxide and water vapor between the leaf and the air flowing through the chamber. This gas exchange is monitored simultaneously with the fluorescence levels from the chlorophyll a molecules in the leaf. The growth conditions used, and plant age and leaf selection criteria for measurement are designed to maximize the chance that the leaves sampled fill the 2 cm²leaf chamber of the gas-exchange system and that plants show no visible signs of having transitioned to reproductive growth.
Screening High-Light Leaf Physiology at Two Air Temperatures.
Leaf physiology is screened after plants have been acclimated to high light (700 μmol photons m⁻²s⁻¹) under LED light banks emitting visible light (400-700 nm, Photon Systems Instruments, Brno, Czech Republic), for 40 minutes. Other than the change in light level, the atmospheric environment is the same as that in which the plants have been grown, and the LI-6400 leaf chamber is set to reflect this, being set to deliver a photosynthetic photon flux of 700 μmol photons m⁻²s⁻¹and operate at an air temperature of 22° C. Forty minutes acclimation to a photosynthetic photon flux of 700 μmol photons m⁻²s⁻¹has repeatedly been shown to be sufficient to achieve a steady-state rate of light-saturated photosynthesis and stomatal conductance in control plants. Gas exchange and fluorescence data are logged simultaneously two minutes after the leaf has been closed in the chamber. Two minutes is found to be long enough for the leaf chamber CO₂and H₂O concentrations to stabilize after closing a new leaf inside, and thereby minimizing leaf physiological adjustment to small differences between the growth environment and the LI-6400 chamber. Screening at the growth air temperature of 22° C. is begun one hour into the photoperiod and is typically completed in two hours. After being screened at 22° C., plants are returned to growth-light levels prior to being screened again at 35° C. later in the photoperiod. The higher-temperature screening begins six hours into the photoperiod and measurements are made after the rosettes have been acclimated to the same high light dose as described above, but this time in a controlled environment with an air temperature set to 35° C. Measurements are again made in a leaf chamber set to match the warmer air temperature and logged using the protocol described above for the 22° C. measurements. Data generated at both 22° C. and 35° C. are used to calculate: rates of CO₂assimilation by photosynthesis (A, μmol CO₂m⁻²s⁻¹); rates of H₂O loss through transpiration (Tr, mmol H₂O m⁻²s⁻¹); the conductance to CO₂and H₂O movement between the leaf and air through the stomatal pore (g mol. H₂O m⁻²s⁻¹); the sub-stomatal CO₂concentration (C_i, μmol CO₂mol⁻¹); transpiration efficiency, the instantaneous ratio of photosynthesis to transpiration, (TE=A/Tr (μmol CO₂mmol H₂O m⁻²s⁻¹)); the rate of electron flow through photosystem two (ETR μmol e-m⁻²s⁻¹). Derivation of the parameters described above followed established published protocols (Long & Bernacchi, 2003. J. Exp. Botany; 54:2393-24)
Leaves from up to 10 replicate plants are screened for a given line of interest. Data generated from these lines are compared with that from an empty vector control line planted at the same time, grown within the same flats, and screened at the same time.
For control lines, data are collected not only at an atmospheric CO₂concentration of 400 μmol CO₂mol⁻¹, but also after stepwise changes in CO₂concentration to 350, 300, 450 and 500 μmol CO₂mol⁻¹. These measurements underlay screening for more complex physiological traits of: (1) photosynthetic capacity; (2) Non-photochemical quenching; and (3) non-photosynthetic metabolism.
Screening Photosynthetic Capacity.
Under most conditions, the rate of light-saturated photosynthesis in a C3 leaf is a product of the biochemical capacity of the Calvin cycle and the transfer conductance of CO₂concentration to the sites of carboxylation (Farquhar et al., 1980. Planta: 149, 78-90). Plotting the rate of photosynthesis against an estimate of the sub-stomatal CO₂concentration (C_i) provides a means to identify changes in photosynthetic capacity of the Calvin cycle independent of changes in stomatal conductance, a key component of the total transfer conductance to CO₂of the leaf. Consequently, for lines being screened, rates of photosynthesis are plotted against a regression plot of A vs. C_igenerated for the control lines over a range of atmospheric CO₂concentration, as described above. This technique enables visual confirmation of changes in photosynthetic capacity in lines of interest.
Screening Non-Photochemical Quenching.
During acclimation to high light, the efficiency with which photosystem PSII operates will reach a steady state regulated largely by the feedback between non-photochemical quenching (NPQ) in the antenna and the metabolic demand for energy produced in the chloroplast (Genty et al., 1989. Biochim. Biophys. Acta 990:87-92; Baker et al., 2007. Plant Cell Environ. 30:1107-1125). This understanding is used in this screen to identify lines in which the limitation that non-photochemical quenching exerts on the efficiency with which photosystem II operates is decreased or increased. A decrease in non-photochemical quenching may be the consequence of a decrease in the capacity for NPQ. This would result in lower levels of non-photochemical quenching and a higher efficiency of photosynthesis over a range of light levels, but importantly, higher rates of photosynthesis at low light where light-use efficiency is important. However, changes in rate at which NPQ responds to light could also underlie any increases or decreases in NPQ. Of these, an increase in the rate at which NPQ relaxes has the potential to increase rates of photosynthesis as leaves in crop canopies transition from high to low light, and is therefore relevant to increasing crop-canopy photosynthesis (Zhu et al., 2010. Plant Biol. 61:235-261). In keeping with the A/Ci analysis described above, a regression of the operating efficiency of PSII against non-photochemical quenching is generated for the control line from data collected over a range of atmospheric CO₂concentration. This technique enables visual confirmation of changes in the regulation of PSII operation that are driven by changes in non-photochemical quenching in lines of interest.
Screening for Non-Photosynthetic Metabolism.
Measurement of the ratio of the rate of electron flow through PSII (ETR) to the rate of photosynthesis (A) is used to screen for changes in non-photosynthetic metabolism. This screen is based upon the understanding that the transport of four μmol of electrons from PSII to photosystem one PSI will supply the NADPH and ATP required to fix one μmol of CO₂in the Calvin cycle. For a C3 leaf operating in an atmosphere with 21% oxygen, the ratio of electron flow to photosynthesis should be higher than four, reflecting photorespiratory and other metabolism. However, because the rate of photorespiration in a C3 leaf is dependent upon the concentration of CO₂at the active site of Rubisco, a regression of the ratio of electron flow to photosynthesis, generated over the range of CO₂concentrations described above, provides the reference regression against which lines being screened can be compared to controls. Changes in the ratio of ETR to A, when observed at the same C_ias the control line, could indicate changes in the specificity of the Rubisco active site for O₂relative to CO₂and or other metabolic sinks which would be expected to have important implications for crop productivity and/or stress tolerance.
Surrogate Screening for Growth-Light Physiology.
Rosette biomass: the dry weight of whole Arabidopsis rosettes (i.e., above-ground biomass) is measured after being dried down at 80° C. for 24 hours, a time found to be sufficient to reach constant weight. Samples are taken after 35-38 days growth, and used as an assay of above-ground productivity at growth light. Typically, five replicate rosettes are sampled per Arabidopsis line being screened.
Rosette chemical and isotopic C and N analysis: after weighing, the five rosettes sampled for each line screened are pooled together and ground to a fine powder. The pooled sample generated is sub-sampled and approximately 4 μg samples are prepared for analysis.
Chlorophyll content index (CCI): measurements of light transmission through the leaf are made for plants being screened using a chlorophyll content meter (CCM-200, Apogee Instruments, Logan, Utah, USA). The first is made within the first hour of the photoperiod prior to any acclimation to high light on leaves of plants samples for rosette analysis. The second is made later in the photoperiod on leaves of plants that had undergone the high-temperature screening.
Light absorption: measurements of CCI are used as a surrogate for leaf light absorption, based upon a known relationship between the two. The estimates of light absorption by the leaf, required to construct this relationship, were made by placing the leaf on top of a quantum sensor (LI-190, LI-COR Biosciences) with both the leaf and quantum sensor then pressed firmly up to the foam gasket underneath the LI-6400 light source. This procedure provides an estimate of the transmission of a known light flux through the leaf and is used to estimate the fraction of light absorbed by the leaf.

Example V. Experimental Results

This Example provides experimental observations for transgenic plants overexpressing AtMYB27, RBP45A, TCP6, PIL1, PCL1, GTL1, DREB2H, ERF087, NF-YC6, BBX18, bHLH60, BBX26, bHLH121, or PMT24 related polypeptides assayed for improved photosynthetic resource use efficiency.
The ability of a crop canopy to photosynthesize, and the rate at which it can do this relative to the availability of resources, is an important determinant of crop yield. Consequently, increasing the rate of photosynthesis relative to resources that can limit productivity and yield is considered a pathway to improving crop yield across broad acres.
The biochemical capacity for photosynthesis is a key determinant of the efficiency with which photosynthesis operates relative to resources required for plant growth. The biochemical capacity for photosynthesis is the product of plant resource investment in numerous pigments and proteins required to absorb light and couple it to the enzymatic reduction of carbon in the air to sugars, in the chloroplast. This capacity for photosynthesis sets limits upon the rate of photosynthesis that can be achieved by a leaf, and ultimately the yield potential of crops. Consequently, increasing photosynthetic capacity is considered a pathway to improving crop yield across broad acres.
Tables 23-35 and the instant Figures provide evidence for improved photosynthetic resource use efficiency observed in Arabidopsis plants overexpressing AtMYB27, RBP45A, TCP6, PIL1, PCL1, GTL1, DREB2H, ERF087, NF-YC6, BBX18, bHLH60, BBX26, bHLH121, or PMT24 in experiments conducted to date. All experimental observations of greater photosynthetic resource use efficiency were made by comparison to control plants (e.g., plants that did not comprise a recombinant construct encoding an AtMYB27, RBP45A, TCP6, PIL1, PCL1, GTL1, DREB2H, ERF087, NF-YC6, BBX18, bHLH60, BBX26, bHLH121, or PMT24-related polypeptide or overexpress an AtMYB27, RBP45A, TCP6, PIL1, PCL1, GTL1, DREB2H, ERF087, NF-YC6, BBX18, bHLH60, BBX26, bHLH121, or PMT24 clade or phylogenetically-related regulatory protein). Where a numerical value was determined, the percentage increases (+%) or decreases (−%) relative to control plants are shown in parentheses.
Table 23 describes an increased capacity for photosynthesis and increased photosynthetic rate in five independent lines overexpressing AtMYB27. Table 23 describes increased photosynthesis in five out of six independent lines overexpressing MYB27. When averaged for these five MYB27 overexpression lines, photosynthetic rate was increased by 23%. Table 5 also details how for four of these MYB27 overexpression lines this increase in photosynthetic rate is clearly linked to an increase in capacity for photosynthesis. Of the numerous steps that can limit photosynthesis, the activity of Rubisco and the capacity to regenerate RuBP in the Calvin cycle are key constraints. FIGS. 3 and 4 display evidence of increases in both Rubisco activity and the capacity to regenerate RuBP in multiple MYB27 overexpression lines (Long & Bernacchi 2003 already cited above, describe the basis for assaying Rubisco activity and RuBP regeneration capacity). For lines 1, 2 and 6, both the activity of Rubisco and the capacity to regenerate RuBP were increased by MYB27 overexpression. For line 5, only an increase in the capacity for RuBP regeneration was observed. Photosynthetic resource use efficiency was also increased in five of the six lines assayed. When averaged for these five lines, the 23% increase in photosynthetic rate was observed in tandem with a smaller, 13% increase in the nitrogen content of the rosette tissue.

TABLE 23

Components of increased photosynthetic resource use efficiency in AtMYB27 overexpression
lines. Effects and relative effect size are displayed for leaf photosynthetic rate (photosynthesis) and
rosette nitrogen concentration (rosette [N]). Effects on photosynthetic capacity are also described and
where known the biochemical basis for the effect is described as either due to effects on Rubisco activity
(Rubisco) or the capacity to regenerate RuBP (RuBP).

Poly-
peptide	SEQ
Sequence/	ID				Photosynthetic
Line	NO:	Driver	Target	Photosynthesis	Capacity	Rosette [N]

AtMYB	2	35S::m35S::oEnh:LexA:	opLexA::	Increased	Increased	Increased
27/		GAL4_opLexA::GFP	G1311	(43%)	Rubisco and	(25%)
Line 1					RuBP
AtMYB	2	35S::m35S::oEnh:LexA:	opLexA::	Increased	Increased	Increased
27/		GAL4_opLexA::GFP	G1311	(30%)	Rubisco and	(30%)
Line 2					RuBP
AtMYB
	2	35S::m35S::oEnh:LexA:	opLexA::	No effect	No effect	No effect
27/		GAL4_opLexA::GFP	G1311
Line 3
AtMYB	2	35S::m35S::oEnh:LexA:	opLexA::	Increased	No effect	Increased
27/		GAL4_opLexA::GFP	G1311	(14%)		(8%)
Line 4
AtMYB	2	35S::m35S::oEnh:LexA:	opLexA::	Increased	Increased	No effect
27/		GAL4_opLexA::GFP	G1311	(8%)	RuBP
Line 5
AtMYB	2	35S::m35S::oEnh:LexA:	opLexA::	Increased	Increased	Increased
27/		GAL4_opLexA::GFP	G1311	(20%)	Rubisco and	(3%)
Line 6					RuBP

The results presented in Table 23 were determined after screening six independent transgenic events. Lines 1 and 2 were screened twice and the effect size reported for a given parameter is the mean of the two screening runs. For both lines the direction of the effect was the same in both runs. Lines 3, 4, 5 and 6 were screened once.
Table 24 details indicators of photosynthetic resource use efficiency observed in Arabidopsis plants overexpressing RBP45A in experiments conducted to date. Table 24 describes increased photosynthesis in five out of six independent lines overexpressing RBP45A. When averaged for these six lines, photosynthetic rate was increased by 11% in the RBP45A overexpression lines. Leaf chlorophyll absorbs light energy utilized for photosynthesis, and was increased by 5% when average across all six lines. Rosette nitrogen content was reduced by 3% in the five lines for which it was measured. That photosynthesis is increased in RBP45A overexpression lines to a greater extent than the investment in chlorophyll, while rosette nitrogen content is decreased, provides evidence that RBP45A overexpression improves the efficiency with which photosynthesis operates relative to availability of the key resources of light and nitrogen.

TABLE 24

Components of increased photosynthetic resource use efficiency in RBP45A overexpression
lines. Effects and relative effect size are displayed for leaf photosynthetic rate (photosynthesis), leaf
chlorophyll content and rosette nitrogen concentration (rosette [N]).

Poly-
peptide	SEQ
Sequence/	ID				Leaf
Line	NO:	Driver	Target	Photosynthesis	Chlorophyll	Rosette [N]

RBP45A/	42	35S::m35S::oEnh:LexA:	opLexA::	Increased	Increased	No effect
Line
1		GAL4_opLexA::GFP	G1940	(15%)	(8%)	(<1%)
RBP45A/	42	35S::m35S::oEnh:LexA:	opLexA::	Increased	Decreased	—
Line 2		GAL4_opLexA::GFP	G1940	(12%)	(10%)
RBP45A/	42	35S::m35S::oEnh:LexA:	opLexA::	Increased	Increased	Decreased
Line 3		GAL4_opLexA::GFP	G1940	(11%)	(4%)	(3%)
RBP45A/	42	35S::m35S::oEnh:LexA:	opLexA::	Increased	Increased	Decreased
Line 4		GAL4_opLexA::GFP	G1940	(14%)	(16%)	(4%)
RBP45A/	42	35S::m35S::oEnh:LexA:	opLexA::	Increased	Decreased	Decreased
Line 5		GAL4_opLexA::GFP	G1940	(15%)	(1%)	(5%)
RBP45A/	42	35S::m35S::oEnh:LexA:	opLexA::	Decreased	Increased	Increased
Line 6		GAL4_opLexA::GFP	G1940	(3%)	(11%)	(1%)

The results presented in Table 24 were determined after screening six independent transgenic events. Photosynthetic rate and leaf chlorophyll were screened in two independent experiments for lines 1, and 3, and the effect size reported for is the mean of the two screening runs. The direction of the effect was the same in each screening run. Lines 2, 4, 5, and 6 were screened once. Rosette nitrogen data was collected in one experiment.
Table 25 details indicators of photosynthetic resource use efficiency observed in Arabidopsis plants overexpressing TCP6 in experiments conducted to date. Table 25 describes increased photosynthesis in eight independent lines overexpressing TCP6. When averaged for these eight lines, photosynthetic rate was increased by 14% in the TCP6 overexpression lines. Leaf chlorophyll absorbs light energy utilized for photosynthesis, and was increased by 17% across the eight lines studied. Leaf chlorophyll and photosynthetic enzymes are a major sink for plant nitrogen. However, rosette nitrogen content increased by only 3% when averaged across the six lines for which data is available in Table 7. That photosynthesis and leaf chlorophyll content can be increased with negligible effects on rosette nitrogen content is evidence that TCP6 overexpression improves the efficiency with which photosynthesis operates relative to nitrogen availability.
All experimental observations of greater photosynthetic resource use efficiency were made by comparison to control plants (e.g., plants that did not comprise a recombinant construct encoding a TCP6 related polypeptide or overexpress a TCP6 clade or phylogenetically-related regulatory protein). Where a numerical value was determined, the percentage increases (+%) or decreases (−%) relative to control plants are shown in parentheses.

TABLE 25

Components of increased photosynthetic resource use efficiency in TCP6 overexpression lines.
Effects and relative effect size are displayed for leaf photosynthetic rate (photosynthesis), leaf
chlorophyll content and rosette nitrogen concentration (rosette [N]).

Poly-
peptide	SEQ
Sequence	ID				Leaf
Line	NO:	Driver	Target	Photosynthesis	Chlorophyll	Rosette [N]

TCP6/	86	35S::m35S::oEnh:LexA:	opLexA::	Increased	Increased	Increased
Line 1		GAL4_opLexA::GFP	G1936	(9%)	(10%)	(2%)
TCP6/	86	35S::m35S::oEnh:LexA:	opLexA::	Increased	Increased	Increased
Line 2		GAL4_opLexA::GFP	G1936	(14%)	(15%)	(4%)
TCP6/	86	35S::m35S::oEnh:LexA:	opLexA::	Increased	Increased	Increased
Line 3		GAL4_opLexA::GFP	G1936	(10%)	(17%)	(18%)
TCP6/	86	35S::m35S::oEnh:LexA:	opLexA::	Increased	Increased	Decreased
Line 4		GAL4_opLexA::GFP	G1936	(19%)	(14%)	(4%)
TCP6/	86	35S::m35S::oEnh:LexA:	opLexA::	Increased	Increased	No effect
Line
5		GAL4_opLexA::GFP	G1936	(20%)	(10%)
TCP6/	86	35S::m35S::oEnh:LexA:	opLexA::	Increased	Increased	Decreased
Line 6		GAL4_opLexA::GFP	G1936	(17%)	(24%)	(4%)
TCP6/	86	35S::m35S::oEnh:LexA:	opLexA::	Increased	Increased	—
Line 7		GAL4_opLexA::GFP	G1936	(21%)	(42%)
TCP6/	86	35S::m35S::oEnh:LexA:	opLexA::	Increased	Increased	—
Line 8		GAL4_opLexA::GFP	G1936	(3%)	(6%)

The results presented in Table 25 were determined after screening eight independent transgenic events. Lines 1, 2 and 3 were screened in three independent experiments and the effect size reported for a given parameter is the mean of the three screening runs. Lines 4, 5, 6, 7 and 8 were screened once.
Table 26 details indicators of photosynthetic resource use efficiency observed in Arabidopsis plants overexpressing PIL1 in experiments conducted to date. Table 26 describes increased photosynthesis in six independent lines overexpressing PIL1. When averaged for these six lines, photosynthetic rate was increased by 15% in the PIL1 overexpression lines. Leaf chlorophyll absorbs light energy utilized for photosynthesis, and was decreased by 2% across three lines for which data was collected. Rosette nitrogen content was increased by 1% in the same three lines. That photosynthesis could be increased in PIL1 overexpression lines while decreasing investment in chlorophyll and for a much smaller relative increase in nitrogen is evidence that PIL1 overexpression improves the efficiency with which photosynthesis operates relative to availability of the key resources of light and nitrogen.
All experimental observations of greater photosynthetic resource use efficiency were made by comparison to control plants (e.g., plants that did not comprise a recombinant construct encoding a PIL1-related polypeptide or overexpress a PIL1 clade or phylogenetically-related regulatory protein). Where a numerical value was determined, the percentage increases (+%) or decreases (−%) relative to control plants are shown in parentheses.

TABLE 26

Components of increased photosynthetic resource use efficiency in PIL1 overexpression lines.
Effects and relative effect size are displayed for leaf photosynthetic rate (photosynthesis), leaf
chlorophyll content and rosette nitrogen concentration (rosette [N]).

Poly-
peptide	SEQ
Sequence/	ID				Leaf
Line	NO:	Driver	Target	Photosynthesis	Chlorophyll	Rosette [N]

PIL1/	108	35S::m35S::oEnh:LexA:	opLexA::	Increased	Decreased	Decreased
Line 1		GAL4_opLexA::GFP	G1649	(17%)	(2%)	(3%)
PIL1/	108	35S::m35S::oEnh:LexA:	opLexA::	Increased	Decreased	Decreased
Line 2		GAL4_opLexA::GFP	G1649	(9%)	(6%)	(2%)
PIL1/	108	35S::m35S::oEnh:LexA:	opLexA::	Increased	Decreased	Increased
Line 3		GAL4_opLexA::GFP	G1649	(16%)	(4%)	(3%)
PIL1/	108	35S::m35S::oEnh:LexA:	opLexA::	Increased	—	—
Line 4		GAL4_opLexA::GFP	G1649	(23%)
PIL1/	108	35S::m35S::oEnh:LexA:	opLexA::	Increased	—	—
Line 5		GAL4_opLexA::GFP	G1649	(19%)
PIL1/	108	35S::m35S::oEnh:LexA:	opLexA::	Increased	—	—
Line 6		GAL4_opLexA::GFP	G1649	(18%)

The results presented in Table 26 were determined after screening six independent transgenic events. Photosynthetic rate was screened in two independent experiments for lines 1, and 2, and the effect size reported for is the mean of the two screening runs. The direction of the effect was the same in each screening run. Lines 3, 4, 5, and 6 were screened once.
Table 27 details indicators of photosynthetic resource use efficiency observed in Arabidopsis plants overexpressing PCL1 in experiments conducted to date.
The biochemical capacity for photosynthesis is a key determinant of the efficiency with which photosynthesis operates relative to resources required for plant growth. The biochemical capacity for photosynthesis is the product of plant resource investment in numerous pigments and proteins required to absorb light and couple it to the enzymatic reduction of carbon in the air, to sugars in the chloroplast. This capacity for photosynthesis sets limits upon the rate of photosynthesis that can be achieved by a leaf, and ultimately the yield potential of crops. Consequently, increasing photosynthesis and photosynthetic capacity is considered a pathway to improving crop yield across broad acres. Table 27 describes increased photosynthesis in four out of five independent lines overexpressing PCL1. When averaged for these five lines photosynthetic rate was increased by 14% in the PCL1 overexpression lines. Table 27 also details how for four of these PCL1 overexpression lines this increase in photosynthetic rate is observed in lines that also displayed an increase in the capacity for photosynthesis. Of the numerous steps that can limit photosynthesis, the activity of Rubisco is a key constraint. FIG. 13 displays evidence of increased in both Rubisco activity in four out of five PCL1 overexpression lines (Long & Bernacchi 2003 already cited above, describe the basis for assaying Rubisco activity and RuBP regeneration capacity).

TABLE 27

Components of increased photosynthetic resource use efficiency in PCL1 overexpression lines.
The effects and relative effect size is displayed for leaf photosynthetic rate (photosynthesis) Effects on
photosynthetic capacity are also described and where known the biochemical basis for the effect is
described as either due to effects on Rubisco activity (Rubisco) or the capacity to regenerate RuBP
(RuBP).

Poly-
peptide	SEQ
Sequence/	ID				Photosynthetic
Line	NO:	Driver	Target	Photosynthesis	Capacity

PCL1/	126	35S::m35S::oEnh:LexA:	opLexA::	Increased	Increased
Line 1		GAL4_opLexA::GFP	G2741	(16%)	Rubisco
PCL1/	126	35S::m35S::oEnh:LexA:	opLexA::	Increased	Increased
Line 2		GAL4_opLexA::GFP	G2741	(30%)	Rubisco
PCL1/	126	35S::m35S::oEnh:LexA:	opLexA::	Increased	Increased
Line 3		GAL4_opLexA::GFP	G2741	(9%)	Rubisco
PCL1/	126	35S::m35S::oEnh:LexA:	opLexA::	Decreased (3%)	No effect
Line
4		GAL4_opLexA::GFP	G2741
PCL1/	126	35S::m35S::oEnh:LexA:	opLexA::	Increased	Increased
Line 5		GAL4_opLexA::GFP	G2741	(17%)	Rubisco

The results presented in Table 27 were determined after screening five independent transgenic events. Lines 1 and 2 were screened three times, lines 3, 4 and 5 were screened twice. For all lines the effect size shown is the mean of the individual effects recorded in each independent screening run.
Table 28 details indicators of photosynthetic resource use efficiency observed in Arabidopsis plants overexpressing GTL1 in experiments conducted to date.
The ability of a crop canopy to photosynthesize, and the rate at which it can do this relative to the availability of resources, is an important determinant of crop yield. Consequently, increasing the rate of photosynthesis relative to resources that can limit productivity and yield is considered a pathway to improving crop yield across broad acres. Table 28 describes an increase in leaf photosynthetic rate in GTL1 overexpression lines for plants that had decreased leaf chlorophyll content and rosette nitrogen concentration. When averaged for the four lines studied, photosynthetic rate was increased by 12% and leaf chlorophyll content decreased by 20%. Rosette nitrogen content was decreased by 6%, when averaged over the three out of the four lines for which it was measured. Increasing photosynthesis while decreasing both chlorophyll and nitrogen in the rosette provides evidence that GTL1 overexpression improves the efficiency with which photosynthesis operates relative to availability of the key resources of light and nitrogen. This combination of phenotypes would be expected to increase light-limited photosynthesis in the crop canopy while providing protection against photodamage to the photosynthetic apparatus, associated with excess light absorption, in upper canopy leaves.

TABLE 28

Components of increased photosynthetic resource use efficiency in GTL1 overexpression lines.
Effects and relative effect size are displayed for leaf chlorophyll content, photosynthetic rate
(photosynthesis) and rosette nitrogen concentration (rosette [N]).

Poly-
peptide	SEQ
Sequence/	ID			Leaf		Rosette
Line	NO:	Driver	Target	Chlorophyll	Photosynthesis	[N]

GTL1/	156	prRBCS4::LexA:GAL4_	opLexA::	Decreased	Increased	Decreased
Line
1		opLexA::GFP,	G634	(18%)	(10%)	(7%)
		Col_Wt
GTL1/	156	prRBCS4::LexA:GAL4_	opLexA::	Decreased	Decreased	Decreased
Line
2		opLexA::GFP,	G634	(12%)	(3%)	(6%)
		Col_Wt
GTL1/	156	prRBCS4::LexA:GAL4_	opLexA::	Decreased	Increased	Decreased
Line
3		opLexA::GFP,	G634	(22%)	(14%)	(5%)
		Col_Wt
GTL1/	156	prRBCS4::LexA:GAL4_	opLexA::	Decreased	Increased	—
Line 4		opLexA::GFP,	G634	(26%)	(22%)
		Col_Wt

The results presented in Table 28 were determined after screening four independent transgenic events. Lines 1 and 3 were screened twice, and the effect shown is the mean of the effect observed in both experiments. Lines 2 and 4 were screened once.
Table 29 details indicators of photosynthetic resource use efficiency observed in Arabidopsis plants overexpressing DREB2H in experiments conducted to date. Table 29 describes a decrease in leaf chlorophyll in G1755 overexpression lines that has no effect on photosynthetic rate. When averaged for the six lines studied, leaf chlorophyll content was decreased by 19%, while photosynthetic rate was increased by 2%.
Increasing photosynthesis while decreasing chlorophyll provides evidence that DREB2H overexpression improves the efficiency with which photosynthesis operates relative to light availability. This combination of phenotypes would be expected to increase light-limited photosynthesis in the crop canopy while providing protection against photodamage to the photosynthetic apparatus, associated with excess light absorption, in upper canopy leaves.
All experimental observations of greater photosynthetic resource use efficiency were made by comparison to control plants (e.g., plants that did not comprise a recombinant construct encoding a DREB2H-related polypeptide or overexpress a DREB2H clade or phylogenetically-related regulatory protein). Where a numerical value was determined, the percentage increases (+%) or decreases (−%) relative to control plants are shown in parentheses.

TABLE 29

Components of increased photosynthetic resource use efficiency in DREB2H overexpression
lines. Effects and relative effect size are displayed for leaf chlorophyll content and photosynthetic rate
(photosynthesis).

	SEQ
Polypeptide	ID			Leaf
Sequence/Line	NO:	Driver	Target	Chlorophyll	Photosynthesis

DREB2H/	192	35S::m35S::oEnh:LexA:GAL4_	opLexA::G1755	Decreased	Increased
Line 1		opLexA::GFP, Col_Wt		(18%)	(6%)
DREB2H/	192	35S::m35S::oEnh:LexA:GAL4_	opLexA::G1755	Decreased	No effect
Line
2		opLexA::GFP, Col_Wt		(19%)	(<1%)
DREB2H/	192	35S::m35S::oEnh:LexA:GAL4_	opLexA::G1755	Decreased	Decreased
Line 3		opLexA::GFP, Col_Wt		(17%)	(9%)
DREB2H/	192	35S::m35S::oEnh:LexA:GAL4_	opLexA::G1755	Decreased	Decreased
Line 4		opLexA::GFP, Col_Wt		(21%)	(9%)
DREB2H/	192	35S::m35S::oEnh:LexA:GAL4_	opLexA::G1755	Decreased	Increased
Line 5		opLexA::GFP, Col_Wt		(14%)	(32%)
DREB2H/	192	35S::m35S::oEnh:LexA:GAL4_	opLexA::G1755	Decreased	Increased
Line 6		opLexA::GFP, Col_Wt		(24%)	(4%)

The results presented in Table 29 were determined after screening six independent transgenic events. Lines 2 and 3 were screened twice, and the effect shown is the mean of the effect observed in both experiments. Lines 1, 4, 5 and 6 were screened once.
FIGS. 20 and 21 detail indicators of photosynthetic resource use efficiency observed in Arabidopsis plants overexpressing ERF087 in experiments conducted to date.
The ability of a crop canopy to photosynthesize, and the rate at which it can do this relative to the availability of resources, is an important determinant of crop yield. Consequently, increasing the rate of photosynthesis relative to resources that can limit productivity and yield is considered a pathway to improving crop yield across broad acres. FIGS. 20 and 21 show lower levels of non-photochemical quenching in five out of six ERF087 overexpression lines, as plants acclimated to a sudden increase in light incident on the leaves. The decrease in the ERF087 overexpression lines was most pronounced for plants acclimated to an air temperature of 35° C. (FIG. 21), but was also seen for measurements made at a growth temperature of 22° C. (FIG. 20). Non-photochemical quenching is a term that covers a range of processes that collectively dissipate absorbed light energy as heat from the light harvesting antenna, and thereby regulating the supply of light energy to photosystem two. Decreasing non-photochemical quenching would be expected to increase the efficiency of light energy transfer to the photosynthetic reaction centers and increase the light-use efficiency of photosynthesis.
Table 30 details indicators of photosynthetic resource use efficiency observed in Arabidopsis plants overexpressing BBX18 in experiments conducted to date.
The biochemical capacity for photosynthesis is a key determinant of the efficiency with which photosynthesis operates relative to resources required for plant growth. The biochemical capacity for photosynthesis is the product of plant resource investment in numerous pigments and proteins required to absorb light and couple it to the enzymatic reduction of carbon in the air, to sugars in the chloroplast. This capacity for photosynthesis sets limits upon the rate of photosynthesis that can be achieved by a leaf, and ultimately the yield potential of crops. Consequently, increasing photosynthesis and photosynthetic capacity is considered a pathway to improving crop yield across broad acres. Table 30 describes increased photosynthesis in six out of six independent lines overexpressing BBX18. When averaged for these six lines photosynthetic rate was increased by 28% in the BBX18 overexpression lines. Table 30 also details how for all six of these BBX18 overexpression lines this increase in photosynthetic rate is observed in with an increase in the capacity for photosynthesis. Of the numerous steps that can limit photosynthesis, the activity of Rubisco and the capacity to regenerate RuBP, in the Calvin cycle are key constraints. FIG. 24 displays evidence of an increase in both Rubisco activity and the capacity to regenerate RuBP in the three of the six BBX18 overexpression lines that were assayed for insights into the biochemical basis for increased photosynthetic capacity (Long & Bernacchi 2003 already cited above, describe the basis for assaying Rubisco activity and RuBP regeneration capacity). When averaged over the six lines assayed, the increase in photosynthetic capacity and photosynthetic rate observed in the BBX18 overexpression lines was achieved with no increase in leaf chlorophyll content, providing evidence of optimization of resources within the photosynthetic apparatus.

TABLE 30

Components of increased photosynthetic resource use efficiency in BBX18 overexpression
lines. The effects and relative effect size is displayed for leaf photosynthetic rate (photosynthesis) Effects
on photosynthetic capacity are also described and, where known the biochemical basis for the effect is
described as either due to effects on Rubisco activity (Rubisco) or the capacity to regenerate RuBP
(RuBP).

Poly-
peptide	SEQ					Chlorophyll
Sequence/	ID				Photosynthetic	Content
Line	NO:	Driver	Target	Photosynthesis	Capacity	Index

BBX18/	278	35S::m35S::oEnh:LexA:	opLexA::	Increased	Increased	Decreased
Line
1		GAL4_opLexA::GFP	G1881	(3%)		(7%)
BBX18/	278	35S::m35S::oEnh:LexA:	opLexA::	Increased	Increased	Decreased
Line
2		GAL4_opLexA::GFP	G1881	(16%)		(10%)
BBX18/	278	35S::m35S::oEnh:LexA:	opLexA::	Increased	Increased	Decreased
Line
3		GAL4_opLexA::GFP	G1881	(16%)		(6%)
BBX18/	278	35S::m35S::oEnh:LexA:	opLexA::	Increased	Increased	Increased
Line
4		GAL4_opLexA::GFP	G1881	(30%)	Rubisco/RuBP	(8%)
BBX18/	278	35S::m35S::oEnh:LexA:	opLexA::	Increased	Increased	Increased
Line
5		GAL4_opLexA::GFP	G1881	(72%)	Rubisco/RuBP	(5%)
BBX18/	278	35S::m35S::oEnh:LexA:	opLexA::	Increased	Increased	Increased
Line
6		GAL4_opLexA::GFP	G1881	(30%)	Rubisco/RuBP	(10%)

Table 31 details indicators of photosynthetic resource use efficiency observed in Arabidopsis plants overexpressing bHLH60 in experiments conducted to date. Table 31 describes a decrease in leaf chlorophyll in bHLH60 overexpression lines that has no effect on photosynthetic rate. When averaged for the six lines studied, leaf chlorophyll content was decreased by 15%, while photosynthetic rate was decreased by 7%.
Decreasing chlorophyll provides evidence that bHLH60 overexpression improves the efficiency with which photosynthesis operates relative to light availability. This combination of phenotypes would be expected to increase light-limited photosynthesis in the crop canopy while providing protection against photodamage to the photosynthetic apparatus, associated with excess light absorption, in upper canopy leaves.

TABLE 31

Components of increased photosynthetic resource use efficiency in bHLH60 overexpression
lines. Effects and relative effect size are displayed for leaf chlorophyll
content and photosynthetic rate (photosynthesis).

	SEQ
Polypeptide	ID			Leaf
Sequence/Line	NO:	Driver	Target	Chlorophyll	Photosynthesis

bHLH60/	318	35S::m35S::oEnh:LexA:GA	opLexA::G2144	Decreased	Decreased
Line 1		L4_opLexA::GFP, Col_Wt		(16%)	(8%)
bHLH60/	318	35S::m35S::oEnh:LexA:GA	opLexA::G2144	Decreased	Increased
Line 2		L4_opLexA::GFP, Col_Wt		(14%)	(2%)
bHLH60/	318	35S::m35S::oEnh:LexA:GA	opLexA::G2144	Decreased	Decreased
Line 3		L4_opLexA::GFP, Col_Wt		(17%)	(4%)
bHLH60/	318	35S::m35S::oEnh:LexA:GA	opLexA::G2144	Decreased	Decreased
Line 4		L4_opLexA::GFP, Col_Wt		(11%)	(17%)

The results presented in Table 31 were determined after screening four independent transgenic events. Line 1 and 2 were screened twice and the data presented is the mean of the results of those two experiments. Lines 3 and 4 were screened once.
Table 32 details indicators of photosynthetic resource use efficiency observed in Arabidopsis plants overexpressing NF-YC6 in experiments conducted to date. Table 32 describes a decrease in leaf chlorophyll in NF-YC6 overexpression lines that has no effect on photosynthetic rate. When averaged for the five lines studied, leaf chlorophyll content was decreased by 13%, while photosynthetic rate was unaffected (<1% change). Rosette nitrogen content was measured for three of the five lines studied. Averaged for these three lines, rosette nitrogen content was decreased by 7% from 7.3 to 6.7% of rosette dry weight. Increasing photosynthesis while decreasing both chlorophyll and nitrogen in the rosette provides evidence that NF-YC6 overexpression improves the efficiency with which photosynthesis operates relative to the availability of the key resources of light and nitrogen. This combination of phenotypes would be expected to increase light-limited photosynthesis in the crop canopy while providing protection against photodamage to the photosynthetic apparatus, associated with excess light absorption, in upper canopy leaves.

TABLE 32

Components of increased photosynthetic resource use efficiency in NF-YC6 overexpression
lines. Effects and relative effect size are displayed for leaf chlorophyll content, photosynthetic rate
(photosynthesis) and rosette nitrogen concentration (rosette [N]).

Poly-
peptide	SEQ
Sequence/	ID			Leaf		Rosette
Line	NO:	Driver	Target	Chlorophyll	Photosynthesis	[N]

NF-YC6/	356	35S::m35S::oEnh:LexA:GA	opLexA::	Decreased	Decreased	Decreased
Line
1		L4_opLexA::GFP, Col_Wt	G1820	(12%)	(2%)	(2%)
NF-YC6/	356	35S::m35S::oEnh:LexA:GA	opLexA::	Decreased	Increased	Decreased
Line
2		L4_opLexA::GFP, Col_Wt	G1820	(17%)	(8%)	(9%)
NF-YC6/	356	35S::m35S::oEnh:LexA:GA	opLexA::	Decreased	Decreased	Decreased
Line
3		L4_opLexA::GFP, Col_Wt	G1820	(11%)	(3%)	(11%)
NF-YC6/	356	35S::m35S::oEnh:LexA:GA	opLexA::	Decreased	No effect
Line
4		L4_opLexA::GFP, Col_Wt	G1820	(20%)	(<1%)
NF-YC6/	356	35S::m35S::oEnh:LexA:GA	opLexA::	Decreased	No effect
Line
5		L4_opLexA::GFP, Col_Wt	G1820	(4%)	(<1%)

The results presented in Table 32 were determined after screening five independent transgenic events. Lines 1, 2 and 3 were screened twice and the effect size reported for a given parameter is the mean of the two screening runs. Lines 4 and 5 were screened once.
Table 33 details indicators of photosynthetic resource use efficiency observed in Arabidopsis plants overexpressing bHLH121 in experiments conducted to date.
The biochemical capacity for photosynthesis is a key determinant of the efficiency with which photosynthesis operates relative to resources required for plant growth. The biochemical capacity for photosynthesis is the product of plant resource investment in numerous pigments and proteins required to absorb light and couple it to the enzymatic reduction of carbon in the air, to sugars in the chloroplast. This capacity for photosynthesis sets limits upon the rate of photosynthesis that can be achieved by a leaf, and ultimately the yield potential of crops. Consequently, increasing photosynthesis and photosynthetic capacity is considered a pathway to improving crop yield across broad acres. Table 33 describes increased photosynthesis in five out of five independent lines overexpressing bHLH121. When averaged for these five bHLH121 overexpression lines, photosynthetic rate was increased by 11%. Table 33 also details how for four of the five lines, the increase in photosynthetic rate is linked to an increase in capacity for photosynthesis. Of the numerous steps that can limit photosynthesis, the capacity to regenerate RuBP in the Calvin cycle is a key constraint. FIG. 31 displays evidence of an increase in the capacity to regenerate RuBP in four of the five bHLH121 overexpression lines (Long & Bernacchi 2003 already cited above, describe the basis for RuBP regeneration capacity). All the bHLH121 lines screened were grown in the same environment as the control lines, consequently the increase in photosynthetic capacity and photosynthetic rate observed has been achieved through an increase in photosynthetic resource-use efficiency in these lines.

TABLE 33

Components of increased photosynthetic resource use efficiency in bHLH121 overexpression
lines. Effects and relative effect size are displayed for leaf photosynthetic rate (photosynthesis),
photosynthetic capacity and leaf chlorophyll content. Where known, the biochemical basis for increase in
photosynthetic capacity is described as either due to effects on Rubisco activity (Rubisco) or the capacity
to regenerate RuBP (RuBP).

Polypeptide	SEQ					Leaf
Sequence/	ID				Photosynthetic	Chlorophyll
Line	NO:	Driver	Target	Photosynthesis	capacity	content

bHLH121/	388	35S::m35S::oEnh:LexA:	opLexA::	Increased	Increased	Increased
Line
1		GAL4_opLexA::GFP	G782	(18%)	RuBP	(21%)
bHLH121/	388	35S::m35S::oEnh:LexA:	opLexA::	Increased	No effect	Increased
Line 2		GAL4_opLexA::GFP	G782	(12%)		(18%)
bHLH121/	388	35S::m35S::oEnh:LexA:	opLexA::	Increased	Increased	Increased
Line
3		GAL4_opLexA::GFP	G782	(8%)	RuBP	(8%)
bHLH121/	388	35S::m35S::oEnh:LexA:	opLexA::	Increased	Increased	Increased
Line
4		GAL4_opLexA::GFP	G782	(2%)	RuBP	(9%)
bHLH121/	388	35S::m35S::oEnh:LexA:	opLexA::	Increased	Increased	Increased
Line
5		GAL4_opLexA::GFP	G782	(14%)	RuBP	(21%)

The results presented in Table 33 were determined after screening six independent transgenic events. Lines 1 and 2 were screened twice and the effect size reported for a given parameter is the mean of the two screening runs. For both lines the direction of the effect was the same in both runs. Lines 3, 4 and 5 were screened once.
Table 34 details indicators of photosynthetic resource use efficiency observed in Arabidopsis plants overexpressing BBX26 in experiments conducted to date. Table 34 describes increased photosynthesis in five out of five independent lines overexpressing BBX26 When averaged for these five BBX26 overexpression lines, photosynthetic rate was increased by 14%. Table 34 also details how for all five lines, the increase in photosynthetic rate is linked to an increase in capacity for photosynthesis. Of the numerous steps that can limit photosynthesis, the capacity to regenerate RuBP in the Calvin cycle is a key constraint. FIG. 34 displays evidence of an increase in the capacity to regenerate RuBP in four of the five BBX26 overexpression lines (Long & Bernacchi 2003 already cited above, describe the basis for RuBP regeneration capacity). Photosynthetic resource use efficiency was also increased in lines overexpressing BBX26. When averaged for these five lines, the 14% increase in photosynthetic rate was observed in tandem with a 13% increase in leaf chlorophyll content, but also with an 8% decrease in rosette nitrogen content from 7.0% to 6.4%, evidence that leaf nitrogen was being preferentially apportioned to the photosynthetic apparatus.

TABLE 34

Components of increased photosynthetic resource use efficiency in BBX26
overexpression lines. Effects and relative effect size are displayed for leaf photosynthetic rate
(photosynthesis), leaf chlorophyll content and rosette nitrogen concentration (rosette [N]). Effects on
photosynthetic capacity (P. Cap) are also described and where known, the biochemical basis for the effect
is described as either due to effects on Rubisco activity (Rubisco) or the capacity to regenerate RuBP
(RuBP).

Poly-
peptide	SEQ					Leaf
Sequence/	ID					Chlorophyll	Rosette
Line	NO:	Driver	Target	Photosynthesis	P. Cap	content	[N]

BBX26/	410	35S::m35S::oEnh:Le	opLexA::	Increased	Increased	Increased	Decreased
Line
1		xA:GAL4_opLexA::	G1486	(15%)	RuBP	(9%)	(6%)
		GFP
BBX26/	410	35S::m35S::oEnh:Le	opLexA::	Increased	Increased	Increased	Decreased
Line
2		xA:GAL4_opLexA::	G1486	(13%)		(24%)	(5%)
		GFP
BBX26/	410	35S::m35S::oEnh:Le	opLexA::	Increased	Increased	Increased	Decreased
Line
3		xA:GAL4_opLexA::	G1486	(17%)	RuBP	(20%)	(4%)
		GFP
BBX26/	410	35S::m35S::oEnh:Le	opLexA::	Increased	Increased	Increased	Decreased
Line
4		xA:GAL4_opLexA::	G1486	(15%)	RuBP	(23%)	(2%)
		GFP
BBX26/	410	35S::m35S::oEnh:Le	opLexA::	Increased	Increased	Decreased	Decreased
Line
5		xA:GAL4_opLexA::	G1486	(10%)	RuBP	(10%)	(2%)
		GFP

The results presented in Table 34 were determined after screening six independent transgenic events. Lines 1 and 2 were screened twice and the effect size reported for a given parameter is the mean of the two screening runs. For both lines the direction of the effect was the same in both runs. Lines 3, 4, 5 and 6 were screened once.
Table 35 details indicators of photosynthetic resource use efficiency observed in Arabidopsis plants overexpressing PMT24 in experiments conducted to date.
The biochemical capacity for photosynthesis is a key determinant of the efficiency with which photosynthesis operates relative to resources required for plant growth. The biochemical capacity for photosynthesis is the product of plant resource investment in numerous pigments and proteins required to absorb light and couple it to the enzymatic reduction of carbon in the air, to sugars in the chloroplast. This capacity for photosynthesis sets limits upon the rate of photosynthesis that can be achieved by a leaf, and ultimately the yield potential of crops. Consequently, increasing photosynthesis and photosynthetic capacity is considered a pathway to improving crop yield across broad acres. Table 35 describes increased photosynthesis in five out of seven independent lines overexpressing PMT24. When averaged for these seven PMT24 overexpression lines, photosynthetic rate was increased by 18%. Table 35 also details how for five out of seven lines, the increase in photosynthetic rate is linked to an increase in capacity for photosynthesis. Of the numerous steps that can limit photosynthesis, the capacity to regenerate RuBP in the Calvin cycle is a key constraint. FIG. 34 displays evidence of an increase in the capacity to regenerate RuBP in four of the five PMT24 overexpression lines run through a focused secondary screen (Long & Bernacchi 2003 already cited above, describe the basis for RuBP regeneration capacity). This increase in photosynthetic capacity was achieved without any increase in leaf chlorophyll content, which was increased by less than 1% when averaged across all seven lines. These findings suggest that PMT24 overexpression changes resource investment in different components of the photosynthetic apparatus and, because all the PMT24 lines screened were grown in the same environment as the control lines, that the increase in photosynthetic capacity and photosynthetic rate observed has been achieved through an increase in photosynthetic resource-use efficiency in these lines.

TABLE 35

Components of increased photosynthetic resource use efficiency in PMT24 overexpression
lines. Effects and relative effect size are displayed for leaf photosynthetic rate (photosynthesis),
photosynthetic capacity and leaf chlorophyll content. Where know the biochemical basis for increase in
photosynthetic capacity is described as either due to effects on Rubisco activity (Rubisco) or the capacity
to regenerate RuBP (RuBP).

Poly-
peptide	SEQ					Leaf
Sequence/	ID				Photosynthetic	chlorophyll
Line	NO:	Driver	Target	Photosynthesis	capacity	content

PMT24/	444	35S::m35S::oEnh:LexA:	opLexA::	Increased	Increased	Decreased
Line 1		GAL4_opLexA::GFP	G837	(46%)		(3%)
PMT24/	444	35S::m35S::oEnh:LexA:	opLexA::	Increased	No effect	Increased
Line 2		GAL4_opLexA::GFP	G837	(26%)		(2%)
PMT24/	444	35S::m35S::oEnh:LexA:	opLexA::	Increased	Increased	Increased
Line 3		GAL4_opLexA::GFP	G837	(34%)	RuBP	(2%)
PMT24/	444	35S::m35S::oEnh:LexA:	opLexA::	No effect	Increased	Increased
Line 4		GAL4_opLexA::GFP	G837	(<1%)	RuBP	(4%)
PMT24/	444	35S::m35S::oEnh:LexA:	opLexA::	Decreased	Increased	No effect
Line 5		GAL4_opLexA::GFP	G837	(6%)	RuBP	(<1%)
PMT24/	444	35S::m35S::oEnh:LexA:	opLexA::	Increased	Increased	Decreased
Line 6		GAL4_opLexA::GFP	G837	(14%)	RuBP	(4%)
PMT24/	444	35S::m35S::oEnh:LexA:	opLexA::	Increased	No effect	Increased
Line 7		GAL4_opLexA::GFP	G837	(11%)		(3%)

The results presented in Table 35 were determined after screening seven independent transgenic events. Line 3 was screened twice and the effect size reported for a given parameter is the mean of the two screening runs. All other lines were screened once.
The present disclosure thus describes how the transformation of plants, which may include monocots and/or dicots, with an AtMYB27, RBP45A, TCP6, PIL1, PCL1, GTL1, DREB2H, ERF087, NF-YC6, BBX18, bHLH60, BBX26, bHLH121, or PMT24 clade polypeptide can confer to the transformed plants greater photosynthetic resource use efficiency than the level of photosynthetic resource use efficiency exhibited by control plants. In one embodiment, expression of AtMYB27, RBP45A, TCP6, PIL1, PCL1, GTL1, DREB2H, ERF087, NF-YC6, BBX18, bHLH60, BBX26, bHLH121, or PMT24 is driven by a constitutive promoter. In another embodiment, expression of AtMYB27, RBP45A, TCP6, PIL1, PCL1, GTL1, DREB2H, ERF087, NF-YC6, BBX18, bHLH60, BBX26, bHLH121, or PMT24 is driven by a promoter with enhanced activity in a tissue capable of photosynthesis (also referred to herein as a “photosynthetic promoter” or a “photosynthetic tissue-enhanced promoter”) such as a leaf tissue or other green tissue. Examples of photosynthetic tissue-enhanced promoters include for example, an RBCS3 promoter (SEQ ID NO: 862), an RBCS4 promoter (SEQ ID NO: 863) or others such as the At4g01060 (also referred to as “G682”) promoter (SEQ ID NO: 864), the latter regulating expression in guard cells, or promoters listed in Table 4. Other photosynthetic tissue-enhanced promoters have been taught by Bassett et al., 2007. BMC Biotechnol. 7: 47, specifically incorporated herein by reference in its entirety. Other photosynthetic tissue-enhanced promoters of interest include those from the maize aldolase gene FDA (U.S. patent publication no. 20040216189, specifically incorporated herein by reference in its entirety), and the aldolase and pyruvate orthophosphate dikinase (PPDK) (Taniguchi et al., 2000. Plant Cell Physiol. 41:42-48, specifically incorporated herein by reference in its entirety). Other tissue enhanced promoters or inducible promoters are also envisioned that may be used to regulate expression of the disclosed clade member polypeptides and improve photosynthetic resource use efficiency in a variety of plants.

Example VI. Utilities of AtMYB27, RBP45A, TCP6, PIL1, PCL1, GTL1, DREB2H, ERF087, NF-YC6, BBX18, bHLH60, BBX26, bHLH121, or PMT24 Clade Sequences for Improving Photosynthetic Resource Use Efficiency, Yield or Biomass

By expressing the present polynucleotide sequences in a commercially valuable plant, the plant's phenotype may be altered to one with improved traits related to photosynthetic resource use efficiency or yield. The sequences may be introduced into the commercially valuable plant, by, for example, introducing the polynucleotide in an expression vector or cassette to produce a transgenic plant, or by crossing a target plant with a second plant that comprises said polynucleotide. The transgenic or target plant may be any valuable species of interest, including but not limited to a crop or model plant such as a wheat, Setaria, corn (maize), rice, barley, rye, millet, sorghum, turfgrass, sugarcane, miscane, turfgrass, Miscanthus, switchgrass, soybean, cotton, rape, oilseed rape including canola, Eucalyptus, or poplar plant. The present polynucleotide sequences encode an AtMYB27, RBP45A, TCP6, PIL1, PCL1, GTL1, DREB2H, ERF087, NF-YC6, BBX18, bHLH60, BBX26, bHLH121, or PMT24 clade polypeptide sequence and the ectopic expression or overexpression in the transgenic or target plant of any of said polypeptides, for example, a polypeptide comprising any of SEQ ID NOs: 2, 42, 86, 108, 126, 156, 192, 246, 278, 318, 356, 388, 410, 444, or SEQ ID NOs: 2n where n=1 to 241, or at least 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95% or 96%, 97%, 98%, 99%, or about 100% amino acid identity to any of SEQ ID NOs: 2, 42, 86, 108, 126, 156, 192, 246, 278, 318, 356, 388, 410, or 444, and/or SEQ ID NOs: 2n, where n=1 to 241, and/or at least 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95% or 96%, 97%, 98%, 99%, or about 100% amino acid identity to a domain of any of SEQ ID NOs: 483, 490, 510, 538, 566, 588, 599, 608, 623, 629, 659, 686, 702, 721, 741, 760, 769, 786, 813, and/or at least 90%, 91%, 92%, 93%, 94%, 95% m 96% m 97%, 98%, 99%, or about 100% identity to any of consensus sequences SEQ ID NOs: 842-861, can confer improved photosynthetic resource use efficiency or yield in the plant. For plants for which biomass is the product of interest, increasing the expression level of AtMYB27, RBP45A, TCP6, PIL1, PCL1, GTL1, DREB2H, ERF087, NF-YC6, BBX18, bHLH60, BBX26, bHLH121, or PMT24 clade of polypeptide sequences may increase yield, light use efficiency, photosynthetic capacity, photosynthetic rate, photosynthetic resource use efficiency, vigor, and/or biomass as compared to a control plant of the plants. Thus, it is thus expected that these sequences will improve yield, light use efficiency, photosynthetic capacity, photosynthetic rate, photosynthetic resource use efficiency, vigor, and/or biomass as compared to a control plant in non-Arabidopsis plants relative to control plants. This yield improvement may result in yield increases of at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 25%, 30% or greater yield relative to the yield that may be obtained with control plants.
It is expected that the same methods may be applied to identify other useful and valuable sequences that are functionally-related and/or closely-related to the listed sequences or domains provided in the instant Tables, and the sequences may be derived from diverse species. Because of morphological, physiological and photosynthetic resource use efficiency similarities that may occur among closely-related sequences, the disclosed clade sequences are expected to increase yield, light use efficiency, photosynthetic capacity, photosynthetic rate, photosynthetic resource use efficiency, vigor, and/or biomass as compared to a control plant to a variety of crop plants, ornamental plants, and woody plants used in the food, ornamental, paper, pulp, lumber or other industries.

Example VII: Expression and Analysis of Increased Yield or Photosynthetic Resource Use Efficiency in Non-Arabidopsis or Crop Species

Northern blot analysis, RT-PCR or microarray analysis of the regenerated, transformed plants may be used to show expression of a polypeptide or the instant description and related genes that are capable of inducing improved photosynthetic resource use efficiency, and/or larger size.
After a eudicot plant, monocot plant or plant cell has been transformed (and the latter plant host cell regenerated into a plant) and shown to have or produce increased yield, increased light use efficiency, increased photosynthetic capacity, increased photosynthetic rate, photosynthetic resource use efficiency, greater vigor, and/or greater biomass as compared to a control plant relative to a control plant, the transformed monocot plant may be crossed with itself or a plant from the same line, a non-transformed or wild-type monocot plant, or another transformed monocot plant from a different transgenic line of plants.
The function of one or more specific polypeptides of the instant description has been analyzed and may be further characterized and incorporated into crop plants. The ectopic overexpression of one or more of the disclosed clade polypeptide sequences may be regulated using constitutive, inducible, or tissue-enhanced regulatory elements. Genes that have been examined have been shown to modify plant traits including increasing yield and/or photosynthetic resource use efficiency. It is expected that newly discovered polynucleotide and polypeptide sequences closely related, as determined by the disclosed hybridization or identity analyses, to polynucleotide and polypeptide sequences found in the Sequence Listing can also confer alteration of traits in a similar manner to the sequences found in the Sequence Listing, when transformed into any of a considerable variety of plants of different species, and including dicots and monocots. The polynucleotide and polypeptide sequences derived from monocots (e.g., the rice sequences) may be used to transform both monocot and dicot plants, and those derived from dicots (e.g., the Arabidopsis and soy genes) may be used to transform either group, although it is expected that some of these sequences will function best if the gene is transformed into a plant from the same group as that from which the sequence is derived.
As an example of a first step to determine photosynthetic resource use efficiency, seeds of these transgenic plants may be grown as described above or methods known in the art.
Closely-related homologs of AtMYB27, RBP45A, TCP6, PIL1, PCL1, GTL1, DREB2H, ERF087, NF-YC6, BBX18, bHLH60, BBX26, bHLH121, or PMT24 derived from various diverse plant species may be overexpressed in plants and have the same functions of conferring increased photosynthetic resource use efficiency. It is thus expected that structurally similar orthologs of the disclosed polypeptide clades, including SEQ ID NOs: 2n where n=1 to 241, orthologs that comprise any of consensus sequences SEQ ID NOs: 842-861, can confer increased yield, increased light use efficiency, increased photosynthetic capacity, increased photosynthetic rate, increased photosynthetic resource use efficiency, greater vigor, greater biomass, and/or size, relative to control plants. As at least one sequence of the instant description has increased photosynthetic resource use efficiency in Arabidopsis, it is expected that the sequences provided in the Sequence Listing, or polypeptide sequences comprising one of or any of the conserved domains provided in the instant Tables, will increase the photosynthetic resource use efficiency and/or yield of transgenic plants including transgenic non-Arabidopsis (plant species other than Arabidopsis species) crop or other commercially important plant species, including, but not limited to, non-Arabidopsis plants and plant species such as monocots and dicots, wheat, Setaria, corn (maize), teosinte (Zea species which is related to maize), rice, barley, rye, millet, sorghum, turfgrass, sugarcane, miscane, turfgrass, Miscanthus, switchgrass, soybean, cotton, rape, oilseed rape including canola, tobacco, tomato, tomatillo, potato, sunflower, alfalfa, clover, banana, blackberry, blueberry, strawberry, raspberry, cantaloupe, carrot, cauliflower, coffee, cucumber, eggplant, grapes, honeydew, lettuce, mango, melon, onion, papaya, peas, peppers, pineapple, pumpkin, spinach, squash, sweet corn, watermelon, rosaceous fruits including apple, peach, pear, cherry and plum, and brassicas including broccoli, cabbage, cauliflower, Brussels sprouts, and kohlrabi, currant, avocado, citrus fruits including oranges, lemons, grapefruit and tangerines, artichoke, cherries, endive, leek, roots such as arrowroot, beet, cassava, turnip, radish, yam, and sweet potato, beans, woody species including pine, poplar, Eucalyptus, mint or other labiates, nuts such as walnut and peanut. Within each of these species the closely-related homologs of AtMYB27, RBP45A, TCP6, PIL1, PCL1, GTL1, DREB2H, ERF087, NF-YC6, BBX18, bHLH60, BBX26, bHLH121, or PMT24 may be overexpressed or ectopically expressed in different varieties, cultivars, or germplasm.
The instantly disclosed transgenic plants comprising the disclosed recombinant polynucleotides can be enhanced with other polynucleotides, resulting in a plant or plants with “stacked” or jointly introduced traits, for example, the traits of increased photosynthetic resource use efficiency and improved yield combined with an enhanced trait resulting from expression of a polynucleotide that confers herbicide, insect or and/or pest resistance in a single plant or in two or more parental lines. The disclosed polynucleotides may thus be stacked with a nucleic acid sequence providing other useful or valuable traits such as a nucleic acid sequence from Bacillus thuringensis that confers resistance to hemiopteran, homopteran, lepidopteran, coliopteran or other insects or pests.
Thus, the disclosed sequences and closely related, functionally related sequences may be identified that, when ectopically expressed or overexpressed in plants, confer one or more characteristics that lead to greater photosynthetic resource use efficiency. These characteristics include, but are not limited to, the embodiments listed below.
1. A dicot or monocot transgenic plant that has greater or increased photosynthetic resource use efficiency^†relative to a control plant;
wherein the transgenic plant comprises an exogenous recombinant polynucleotide comprising a promoter selected from the group consisting of:
a constitutive promoter, a non-constitutive promoter, an inducible promoter, a tissue-enhanced promoter, and a photosynthetic tissue-enhanced promoter;
wherein the promoter regulates expression of a polypeptide having a percentage identity to an amino acid sequence comprising an AtMYB27, RBP45A, TCP6, PIL1, PCL1, GTL1, DREB2H, ERF087, NF-YC6, BBX18, bHLH60, BBX26, bHLH121, or PMT24 clade polypeptide in a photosynthetic or green tissue of the transgenic plant;
wherein the percentage identity is at least:

- 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95% or 96%, 97%, 98%, 99%, or about 100% identity to the entire length of any of SEQ ID NOs: 2n, where n=1-241; and/or
- at least 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95% or 96%, 97%, 98%, 99%, or about 100% identity to a domain of any of SEQ ID NOs: 483 to 841; and/or
- at least 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95% or 96%, 97%, 98%, 99%, or about 100% identical to a consensus sequence of any of SEQ ID NO: 842-861; and/or
- the exogenous recombinant polynucleotide hybridizes with any of SEQ ID NO: 1, 41, 85, 107, 125, 155, 191, 245, 277, 317, 355, 387, 409, or 443 under stringent hybridization conditions followed by one, two, or more wash steps of 6×SSC and 65° C. for ten to thirty minutes per step;

wherein expression of the polypeptide under the regulatory control of the promoter confers greater or increased photosynthetic resource use efficiency in the transgenic plant relative to the control plant;
wherein the control plant does not comprise the recombinant polynucleotide; and/or
2. The transgenic plant of embodiment 1, wherein the photosynthetic tissue-enhanced promoter is an RBCS3 promoter, an RBCS4 promoter, an At4g01060 promoter, an Os02g09720 promoter, an Os05g34510 promoter, an Os11g08230 promoter, an Os01g64390 promoter, an Os06g15760 promoter, an Os12g37560 promoter, an Os03g17420 promoter, an Os04g51000 promoter, an Os01g01960 promoter, an Os05g04990 promoter, an Os02g44970 promoter, an Os01g25530 promoter, an Os03g30650 promoter, an Os01g64910 promoter, an Os07g26810 promoter, an Os07g26820 promoter, an Os09g11220 promoter, an Os04g21800 promoter, an Os10g23840 promoter, an Os08g13850 promoter, an Os12g42980 promoter, an Os03g29280 promoter, an Os03g20650 promoter, or an Os06g43920 promoter (SEQ ID NO: 862-888, respectively), or a functional variant thereof, or a functional fragment thereof, or a promoter sequence that is at least 80% identical to SEQ ID NO: 862-888; and/or
3. The transgenic plant of embodiments 1 or 2, wherein:
the recombinant polynucleotide encodes the polypeptide which comprises any of SEQ ID NOs: 2n, where n=1-241; and/or
any of SEQ ID NOs: 483, 490, 510, 538, 566, 588, 599, 608, 623, 629, 659, 686, 702, 721, 741, 760, 769, 786, 813; and/or
any of SEQ ID NO: 842-861; and/or
4. The transgenic plant of any of embodiments 1 to 3, wherein the polypeptide is encoded by
(a) the exogenous recombinant polynucleotide, or
(b) a second exogenous recombinant polynucleotide and expression of the polypeptide is regulated by a trans-regulatory element; and/or
5. The transgenic plant of any of embodiments 1 to 4, wherein a plurality of the transgenic plants have greater cumulative canopy photosynthesis than the canopy photosynthesis of the same number of the control plants grown under the same conditions and at the same density; and/or
6. The transgenic plant of any of embodiments 1 to 5, wherein the transgenic plant produces a greater yield than the control plant, including, but not limited to, a greater yield of: vegetative biomass, plant parts, whole plants, shoot vegetative organs/structures (for example, leaves, stems and tubers), roots, flowers and floral organs/structures (for example, bracts, sepals, petals, stamens, carpels, anthers and ovules), seed (including embryo, endosperm, and seed coat) and fruit (the mature ovary), plant tissue (for example, vascular tissue, ground tissue, pulped, pureed, ground-up, macerated or broken-up tissue, and the like) and cells (for example, guard cells, egg cells, and the like); and/or
7. The transgenic plant of any of embodiments 1 to 6, wherein the transgenic plant is selected from the group consisting of a corn, wheat, rice, Setaria, Miscanthus, switchgrass, ryegrass, sugarcane, miscane, barley, sorghum, turfgrass, soy, cotton, canola, rapeseed, Crambe, Camelina, sugar beet, alfalfa, tomato, Eucalyptus, poplar, willow, pine, birch and a woody plant; and/or
8. The transgenic plant of any of embodiments 1 to 7, wherein the transgenic plant is morphologically similar to the control plant at one or more stages of growth, and/or developmentally similar to the control plant.
9. A method for increasing photosynthetic resource use efficiency^†in a dicot or monocot plant, the method comprising:
(a) providing one or more dicot or monocot plants that comprise an exogenous recombinant polynucleotide comprising a promoter selected from the group consisting of:
a constitutive promoter, a non-constitutive promoter, an inducible promoter, a tissue-enhanced promoter, and a photosynthetic tissue-enhanced promoter;
wherein the promoter regulates expression of a polypeptide having a percentage identity to an amino acid sequence comprising an AtMYB27, RBP45A, TCP6, PIL1, PCL1, GTL1, DREB2H, ERF087, NF-YC6, BBX18, bHLH60, BBX26, bHLH121, or PMT24 clade polypeptide in a photosynthetic or green tissue of the dicot or monocot plant;
wherein the percentage identity is at least:

- 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95% or 96%, 97%, 98%, 99%, or about 100% identity to the entire length of any of SEQ ID NOs: 2n, where n=1-241; and/or
- at least 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95% or 96%, 97%, 98%, 99%, or about 100% identity to a domain of any of SEQ ID NOs: 483 to 841; and/or
- at least 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95% or 96%, 97%, 98%, 99%, or about 100% identical to a consensus sequence of any of SEQ ID NO: 842-861; and/or
- the exogenous recombinant polynucleotide hybridizes with any of SEQ ID NO: 1, 41, 85, 107, 125, 155, 191, 245, 277, 317, 355, 387, 409, or 443 under stringent hybridization conditions followed by one, two, or more wash steps of 6×SSC and 65° C. for ten to thirty minutes per step;
- wherein expression of the polypeptide in the one or more dicot or monocot plants confers greater or increased photosynthetic resource use efficiency relative to a control plant that does not comprise the recombinant polynucleotide; and

(b) growing the one or more dicot or monocot plants; and/or
10. The method of embodiment 9, wherein the photosynthetic tissue-enhanced promoter is an RBCS3 promoter, an RBCS4 promoter, an At4g01060 promoter, an Os02g09720 promoter, an Os05g34510 promoter, an Os11g08230 promoter, an Os01g64390 promoter, an Os06g15760 promoter, an Os12g37560 promoter, an Os03g17420 promoter, an Os04g51000 promoter, an Os01g01960 promoter, an Os05g04990 promoter, an Os02g44970 promoter, an Os01g25530 promoter, an Os03g30650 promoter, an Os01g64910 promoter, an Os07g26810 promoter, an Os07g26820 promoter, an Os09g11220 promoter, an Os04g21800 promoter, an Os10g23840 promoter, an Os08g13850 promoter, an Os12g42980 promoter, an Os03g29280 promoter, an Os03g20650 promoter, or an Os06g43920 promoter (SEQ ID NO: 862-888, respectively), or a functional variant thereof, or a functional fragment thereof, or a promoter sequence that is at least 80% identical to SEQ ID NO: 862-888; and/or
11. The method of embodiments 9 or 10, wherein an expression cassette comprising the recombinant polynucleotide is introduced into a target plant to produce the dicot or monocot plant comprising the exogenous recombinant polynucleotide; and/or
12. The method of any of embodiments 9 to 11, wherein the polypeptide is encoded by
(a) the exogenous recombinant polynucleotide, or
(b) a second exogenous recombinant polynucleotide and expression of the polypeptide is regulated by a trans-regulatory element; and/or
13. The method of any of embodiments 9 to 12, wherein the dicot or monocot plant is selected for having the increased photosynthetic resource use efficiency relative to the control plant; and/or
14. The method of any of embodiments 9 to 13, wherein the dicot or monocot plant produces a greater yield relative to the control plant; and/or
15. The method of any of embodiments 9 to 14, wherein the dicot or monocot plant is selected for having the greater yield relative to the control plant; and/or
16. The method of any of embodiments 9 to 15, wherein a plurality of the dicot or monocot plants have greater cumulative canopy photosynthesis than the canopy photosynthesis of the same number of the control plants grown under the same conditions and at the same density; and/or
17. The method of any of embodiments 9 to 16, wherein the dicot or monocot plant is selected from the group consisting of a corn, wheat, rice, Setaria, Miscanthus, switchgrass, ryegrass, sugarcane, miscane, barley, sorghum, turfgrass, soy, cotton, canola, rapeseed, Crambe, Camelina, sugar beet, alfalfa, tomato, Eucalyptus, poplar, willow, pine, birch and a woody plant; and/or
18. The method of any of embodiments 9 to 17, the method steps further including:
crossing the dicot or monocot plant with itself, a second plant from the same line as the dicot or monocot plant, a non-transgenic plant, a wild-type plant, or a transgenic plant from a different line of plants, to produce a transgenic seed.
19. A method for producing and selecting a dicot or monocot crop plant with greater yield or greater photosynthetic resource use efficiency^†than a control plant, the method comprising:

- (a) providing one or more dicot or monocot transgenic plants that comprise an exogenous recombinant polynucleotide comprising a promoter selected from the group consisting of:
  - a constitutive promoter, a non-constitutive promoter, an inducible promoter, a tissue-enhanced promoter, and a photosynthetic tissue-enhanced promoter;
  - wherein the promoter regulates expression of a polypeptide having a percentage identity to an amino acid sequence comprising an AtMYB27, RBP45A, TCP6, PIL1, PCL1, GTL1, DREB2H, ERF087, NF-YC6, BBX18, bHLH60, BBX26, bHLH121, or PMT24 clade polypeptide in a photosynthetic or green tissue of the dicot or monocot transgenic plant;
  - wherein the percentage identity is:
  - at least 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95% or 96%, 97%, 98%, 99%, or about 100% identity to the entire length of any of SEQ ID NOs: 2n, where n=1-241; and/or
  - at least 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95% or 96%, 97%, 98%, 99%, or about 100% identity to a domain of any of SEQ ID NOs: 483 to 841; and/or
  - at least 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95% or 96%, 97%, 98%, 99%, or about 100% identical to a consensus sequence of any of SEQ ID NO: 842-861; and/or
- the exogenous recombinant polynucleotide hybridizes with any of SEQ ID NO: 1, 41, 85, 107, 125, 155, 191, 245, 277, 317, 355, 387, 409, or 443 under stringent hybridization conditions followed by one, two, or more wash steps of 6×SSC and 65° C. for ten to thirty minutes per step;
  - wherein the photosynthetic tissue-enhanced promoter does not regulate protein expression in a constitutive manner;
- (b) growing a plurality of the dicot or monocot transgenic plants; and
- (c) selecting a dicot or monocot transgenic plant that:
  - has greater photosynthetic resource use efficiency than the control plant, wherein the control plant does not comprise the recombinant polynucleotide; and/or
  - comprises the recombinant polynucleotide;
- wherein expression of the polypeptide in the selected dicot or monocot transgenic plant confers the greater photosynthetic resource use efficiency or the greater yield relative to the control plant; and/or
  20. The method of embodiment 19, the method steps further including:
- (d) crossing the selected dicot or monocot transgenic plant with itself, a second plant from the same line as the selected transgenic plant, a non-transgenic plant, a wild-type plant, or a transgenic plant from a different line of plants, to produce a transgenic seed; and/or
  21. The method of embodiment 19 or 20, wherein the dicot or monocot transgenic plant is selected for having the increased photosynthetic resource use efficiency relative to the control plant; and/or
  22. The method of any of embodiments 19 to 21, wherein a plurality of the selected dicot or monocot transgenic plants have greater cumulative canopy photosynthesis than the canopy photosynthesis of the same number of the control plants grown under the same conditions and at the same density; and/or
  23. The method of any of embodiments 19 to 22, wherein the selected dicot or monocot transgenic plant has an altered trait that confers the greater photosynthetic resource use efficiency.
  24. A method for producing a dicot or monocot crop plant with greater photosynthetic resource use efficiency^†than a control plant, the method comprising:
- (a) providing a dicot or monocot transgenic plant that comprises an exogenous recombinant polynucleotide that comprises a promoter selected from the group consisting of:
  - a constitutive promoter, a non-constitutive promoter, an inducible promoter, a tissue-enhanced promoter, or a photosynthetic tissue-enhanced promoter;
  - wherein the promoter regulates expression of a polypeptide comprising SEQ ID NO: 2, 42, 86, 108, 126, 156, 192, 246, 278, 318, 356, 388, 410, or 444 in a photosynthetic or green tissue of the transgenic plant to a level that is effective in conferring greater photosynthetic resource use efficiency in the transgenic plant relative to the control plant; and
- (b) measuring an altered trait that confers the greater photosynthetic resource use efficiency,
  - wherein expression of the polypeptide in the selected dicot or monocot transgenic plant confers the greater photosynthetic resource use efficiency of the transgenic plant relative to the control plant, thereby producing the crop plant with greater photosynthetic resource use efficiency than the control plant; and/or
    25. The method of embodiment 24, wherein the transgenic dicot or monocot plant is selected for having the increased photosynthetic resource use efficiency relative to the control plant.
    26. A method for producing a monocot plant with increased grain yield, said method including:
- (a) providing a monocot plant cell or plant tissue with stably integrated, exogenous recombinant polynucleotide comprising a promoter (for example, a constitutive, a non-constitutive, an inducible, a tissue-enhanced, or a photosynthetic tissue-enhanced promoter) that is functional in plant cells and that is operably linked to an exogenous or an endogenous nucleic acid sequence that encodes a polypeptide that has a percentage identity to an amino acid sequence comprising an AtMYB27, RBP45A, TCP6, PIL1, PCL1, GTL1, DREB2H, ERF087, NF-YC6, BBX18, bHLH60, BBX26, bHLH121, or PMT24 clade polypeptide, wherein the percentage identity is:
  - at least 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95% or 96%, 97%, 98%, 99%, or about 100% identity to the entire length of any of SEQ ID NOs: 2n, where n=1-241; and/or
  - at least 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95% or 96%, 97%, 98%, 99%, or about 100% identity to a domain of any of SEQ ID NOs: 483 to 841; and/or
  - at least 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95% or 96%, 97%, 98%, 99%, or about 100% identical to a consensus sequence of any of SEQ ID NO: 842-861; and/or
  - the exogenous recombinant polynucleotide hybridizes with any of SEQ ID NO: 1, 41, 85, 107, 125, 155, 191, 245, 277, 317, 355, 387, 409, or 443 under stringent hybridization conditions followed by one, two, or more wash steps of 6×SSC and 65° C. for ten to thirty minutes per step;
- (b) generating a monocot plant from the plant cell or the plant tissue, wherein the monocot plant comprises the exogenous recombinant polynucleotide, wherein the polypeptide is expressed in a photosynthetic or green tissue of the monocot plant to a level that is effective in conferring greater photosynthetic resource use efficiency† in the monocot plant relative to a control plant that does not contain the recombinant polynucleotide;
- (c) growing the monocot plant; and
- (d) measuring an increase in photosynthetic resource use efficiency of at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 2%, 28%, 29%, or 30% relative to the control plant, or an increase in grain yield of at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 2%, 28%, 29%, or 30% or at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 bushels per acre;
- thereby producing the monocot plant with increased grain yield relative to the control plant; and/or
  27. The method of embodiment 26, wherein the AtMYB27, RBP45A, TCP6, PIL1, PCL1, GTL1, DREB2H, ERF087, NF-YC6, BBX18, bHLH60, BBX26, bHLH121, or PMT24 clade polypeptide comprises a consensus sequence of one or more of any of SEQ ID NOs: 842-861; and/or
  28. A transgenic monocot plant produced by the method of embodiment 26; and/or
  29. The transgenic monocot plant of embodiment 28, wherein transgenic monocot plant is a corn, wheat, rice, Miscanthus, Setaria, switchgrass, ryegrass, sugarcane, miscane, barley, sorghum or turfgrass plant; and/or
  30. The method of embodiment 26, wherein the promoter is an RBCS3 promoter, an RBCS4 promoter, an At4g01060 promoter, an Os02g09720 promoter, an Os05g34510 promoter, an Os11g08230 promoter, an Os01g64390 promoter, an Os06g15760 promoter, an Os12g37560 promoter, an Os03g17420 promoter, an Os04g51000 promoter, an Os01g01960 promoter, an Os05g04990 promoter, an Os02g44970 promoter, an Os01g25530 promoter, an Os03g30650 promoter, an Os01g64910 promoter, an Os07g26810 promoter, an Os07g26820 promoter, an Os09g11220 promoter, an Os04g21800 promoter, an Os10g23840 promoter, an Os08g13850 promoter, an Os12g42980 promoter, an Os03g29280 promoter, an Os03g20650 promoter, or an Os06g43920 promoter (SEQ ID NO: 862-888, respectively) or a Cauliflower Mosaic 35S promoter, or a functional variant thereof, or a functional fragment thereof, or a promoter sequence that is at least 80% identical to SEQ ID NO: 862-888; and/or
  31. The method of embodiment 28, wherein the clade polypeptide comprises any of SEQ ID NO: 2, 42, 86, 108, 126, 156, 192, 246, 278, 318, 356, 388, 410, or 444. † In the above embodiments 1, 9, 19, 24, and 26, greater photosynthetic resource use efficiency may be characterized by or measured as, but is not limited to, any one or more of following measurements or characteristics relative to a control plant. The measured or altered trait may be selected from the group consisting of:(a) increased photosynthetic capacity, measured as an increase in the rate of light-saturated photosynthesis of at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, or 40% when compared to the rate of light-saturated photosynthesis of a control leaf at the same leaf-internal CO₂concentration. Optionally, measurements are made after 40 minutes of acclimation to a light intensity that is saturating for photosynthesis; and/or(b) increased photosynthetic rate, measured as an increase in the rate of light-saturated photosynthesis of at least 5%, 10%, 15%, 19%, 20%, 22%, 23%, 25%, 30%, 32%, 35%, or 40%. Optionally, measurements are made after 40 minutes of acclimation to a light intensity known to be saturating for photosynthesis; and/or(c) a decrease in the chlorophyll content of the leaf of at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, or 40%, observed in the absence of a decrease in photosynthetic capacity; and/or(d) a decrease in the percentage of the leaf dry weight that is nitrogen of at least 0.5%, 1.0%, 1.5%, 2.0%, 2.5%, 3.0%, 3.5%, or 4.0% observed in the absence of a decrease in photosynthetic capacity or increase in dry weight; and/or(e) increased transpiration efficiency, measured as an increase in the rate of light-saturated photosynthesis relative to water loss via transpiration from the leaf, of at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, or 40%; optionally, measurements are made after 40 minutes of acclimation to a light intensity of 700 μmol PAR m⁻²s⁻¹; and/or(f) an increase in the resistance to water vapor diffusion out of the leaf that is exerted by the stomata, measured as a decrease in stomatal conductance to H₂O loss from the leaf of at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, or 40%; optionally, measurements were are after 40 minutes of acclimation to a light intensity of 700 μmol PAR m-2 s-1; and/or(g) a decrease in the resistance to carbon dioxide diffusion into the leaf that is exerted by the stomata, measured as an increase in stomatal conductance of at least 5%, 10%, 13%, 15%, 20%, 25%, 27%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, or 68%; optionally, measurements were are after 40 minutes of acclimation to a light intensity of 700 μmol PAR m-2 s-1; and/or(h) a decrease in non-photochemical quenching of at least 2%, at least 3%, at least 4%, at least 5%, at least 6%, at least 7%, at least 8%, at least 9%, or at least 10%, for leaf measurements made after 40 minutes of acclimation to a light intensity of 700 μmol PAR m⁻²s⁻¹; and/or(i) a decrease in the ratio of the carbon isotope ¹²C to ¹³C found in either all the dried above-ground biomass, or specific components of the above-ground biomass, e.g., leaves or reproductive structures, of at least 0.5‰ (0.5 per mille), or at least 1.0‰, 1.5‰, 2.0‰, 2.5‰, 3.0‰, 3.5‰, or 4.0‰ measured as a decrease in the ratio of ¹²C to ¹³C relative to the controls with both ratio being expressed relative to the same standard; and/or(j) an increase in the total dry weight of above-ground plant material of at least 5%, 10%, 15%, 20%, 23%, 25%, 30%, 32%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, or 75%.

All publications and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference.
The present invention is not limited by the specific embodiments described herein. The invention now being fully described, it will be apparent to one of ordinary skill in the art that many changes and modifications can be made thereto without departing from the spirit or scope of the appended claims. Modifications that become apparent from the foregoing description and accompanying figures fall within the scope of the claims.

Claims

1. A transgenic plant having greater photosynthetic resource use efficiency than a control plant;

wherein the transgenic plant comprises an exogenous recombinant polynucleotide comprising a photosynthetic tissue-enhanced promoter which is operably linked to a nucleic acid sequence that encodes a polypeptide comprising the amino acid sequence of SEQ ID NO: 156;

wherein the photosynthetic tissue-enhanced promoter regulates expression of the polypeptide in a photosynthetic tissue to a level that is effective in conferring greater photosynthetic resource use efficiency in the transgenic plant relative to the control plant;

wherein the control plant does not comprise the recombinant polynucleotide;

wherein the photosynthetic tissue-enhanced promoter does not regulate protein expression in a constitutive manner; and

wherein expression of the polypeptide under the regulatory control of the photosynthetic tissue-enhanced promoter confers greater photosynthetic resource use efficiency in the transgenic plant relative to the control plant.

2. (canceled)

3. The transgenic plant of claim 1, wherein the photosynthetic tissue-enhanced promoter is an RBCS4 promoter as set forth in SEQ ID NO: 863.

4. (canceled)

5. The transgenic plant of claim 1, wherein the transgenic plant has an altered trait, relative to the control plant, that confers the greater photosynthetic resource use efficiency, wherein the altered trait is selected from the group consisting of:

(a) increased photosynthetic capacity, measured as an increase in the rate of light-saturated photosynthesis of at least 10% when compared to the rate of light-saturated photosynthesis of a control leaf at the same leaf-internal CO₂concentration, with measurements made after 40 minutes of acclimation to a light intensity that is saturating for photosynthesis;

(b) increased photosynthetic rate, measured as an increase in the rate of light-saturated photosynthesis of at least 10%, with measurements made after 40 minutes of acclimation to a light intensity that is saturating for photosynthesis;

(c) a decrease in the chlorophyll content of the leaf of at least 10%, observed in the absence of a decrease in photosynthetic capacity;

(d) a decrease in the percentage of the leaf dry weight that is nitrogen of at least 0.5%, observed in the absence of a decrease in photosynthetic capacity or increase in dry weight;

(e) increased transpiration efficiency, measured as an increase in the rate of light-saturated photosynthesis relative to water loss via transpiration from the leaf, of at least 10%, with measurements made after 40 minutes of acclimation to a light intensity of 700 μmol PAR m⁻²s⁻¹;

(f) an increase in the resistance to water vapor diffusion out of the leaf that is exerted by the stomata, measured as a decrease in stomatal conductance to H₂O loss from the leaf of at least 10%, with measurements made after 40 minutes of acclimation to a light intensity of 700 μmol PAR m⁻²s⁻¹;

(g) a decrease in the resistance to carbon dioxide diffusion into the leaf that is exerted by the stomata, measured as an increase in stomatal conductance of at least 10%, with measurements made after 40 minutes of acclimation to a light intensity of 700 μmol PAR m⁻²s⁻¹;

(h) a decrease in the relative limitation that non-photochemical quenching exerts on the operation of PSII measured as a decrease in leaf non-photochemical quenching of at least 2% after 40 minutes of acclimation to a light intensity of 700 μmol PAR m⁻²s⁻¹;

(i) a decrease in the ratio of the carbon isotope ¹²C to ¹³C found in either all the dried above-ground biomass, or specific components of the above-ground biomass, leaves, or reproductive structures, of at least 0.5‰ (0.5 per mille), measured as a decrease in the ratio of ¹²C to ¹³C relative to the controls with both ratio being expressed relative to the same standard; and

(j) an increase in the total dry weight of above-ground plant material of at least 5%.

6. The transgenic plant of claim 1, wherein a plurality of the transgenic plants have greater cumulative canopy photosynthesis than the canopy photosynthesis of the same number of the control plants grown under the same conditions and at the same density.

7. The transgenic plant of claim 1, wherein the transgenic plant produces a greater yield than the control plant.

8. The transgenic plant of claim 1, wherein the transgenic plant is selected from the group consisting of a dicot plant, monocot plant, corn, wheat, rice, Setaria, Miscanthus, switchgrass, ryegrass, sugarcane, miscane, barley, sorghum, turfgrass, soybean, cotton, canola, rapeseed, Crambe, Camelina, sugar beet, alfalfa, tomato, Eucalyptus, poplar, willow, pine, birch, and a woody plant.

9. A method for increasing photosynthetic resource use efficiency in a plant, the method comprising:

(a) providing one or more transgenic plants that comprise an exogenous recombinant polynucleotide comprising a photosynthetic tissue-enhanced promoter operably linked to a nucleic acid sequence that encodes a polypeptide comprising the amino acid sequence of SEQ ID NO: 156;

wherein the photosynthetic tissue-enhanced promoter regulates expression of the polypeptide in a non-constitutive manner; and

(b) growing the one or more transgenic plants;

wherein expression of the polypeptide in the one or more transgenic plants confers increased photosynthetic resource use efficiency relative to a control plant that does not comprise the recombinant polynucleotide.

10. The method of claim 9, wherein the photosynthetic tissue-enhanced promoter is an RBCS4 promoter as set forth in SEQ ID NO: 863.

11. The method of claim 9, wherein an expression cassette comprising the recombinant polynucleotide is introduced into a target plant to produce the transgenic plant.

12. The method of claim 9, wherein the transgenic plant has an altered trait, relative to the control plant, that confers the greater photosynthetic resource use efficiency, wherein the altered trait is selected from the group consisting of:

(f) an increase in the resistance to water vapor diffusion out of the leaf that is exerted by the stomata, measured as a decrease in stomatal conductance of at least 10%, with measurements made after 40 minutes of acclimation to a light intensity of 700 μmol PAR m⁻²s⁻¹;

(i) a decrease in the ratio of the carbon isotope ¹²C to ¹³C found in either all the dried above-ground biomass, or specific components of the above-ground biomass, leaves, or reproductive structures, of at least 0.5 ‰ (0.5 per mille), measured as a decrease in the ratio of ¹²C to ¹³C relative to the controls with both ratio being expressed relative to the same standard;

(j) an increase in the total dry weight of above-ground plant material of at least 5%; and

(k) increased yield.

13. The method of claim 9, wherein the transgenic plant is selected for having the increased photosynthetic resource use efficiency relative to the control plant.

14. (canceled)

15. The method of claim 9, wherein a plurality of the transgenic plants have greater cumulative canopy photosynthesis than the canopy photosynthesis of the same number of the control plants grown under the same conditions and at the same density.

16. The method of claim 9, wherein the transgenic plant is selected from the group consisting of a dicot plant, monocot plant, corn, wheat, rice, Setaria, Miscanthus, switchgrass, ryegrass, sugarcane, miscane, barley, sorghum, turfgrass, soybean, cotton, canola, rapeseed, Crambe, Camelina, sugar beet, alfalfa, tomato, Eucalyptus, poplar, willow, pine, birch, and a woody plant.

17. The method of claim 9, the method steps further including: crossing the target plant with itself, a second plant from the same line as the target plant, a non-transgenic plant, a wild-type plant, or a transgenic plant from a different line of plants, to produce a transgenic seed, wherein the transgenic seed comprises the exogenous recombinant polynucleotide.

18. A method for producing and selecting a crop plant with greater yield or photosynthetic resource use efficiency than a control plant, the method comprising:

(a) providing one or more transgenic plants that comprise an exogenous recombinant polynucleotide that comprises a photosynthetic tissue-enhanced promoter operably linked to a nucleic acid sequence that encodes a polypeptide comprising the amino acid sequence of SEQ ID NO: 156, and wherein the photosynthetic tissue-enhanced promoter does not regulate protein expression in a constitutive manner;

(b) growing a plurality of the transgenic plants; and

(c) selecting a transgenic plant from the step (b) that has greater photosynthetic resource use efficiency than the control plant, wherein the control plant does not comprise the recombinant polynucleotide;

and wherein expression of the polypeptide in the selected transgenic plant confers the greater yield of the selected transgenic plant relative to the control plant.

19. The method of claim 18, the method steps further including:

(d) crossing the selected transgenic plant with itself, a second plant from the same line as the selected transgenic plant, a non-transgenic plant, a wild-type plant, or a transgenic plant from a different line of plants, to produce a transgenic seed, wherein the transgenic seed comprises the exogenous recombinant polynucleotide.

20. The method of claim 18, wherein a plurality of the selected transgenic plants have greater cumulative canopy photosynthesis than the canopy photosynthesis of the same number of the control plants grown under the same conditions and at the same density.

21. The method of claim 18, wherein the selected transgenic plant has an altered trait, relative to the control plant, that confers the greater photosynthetic resource use efficiency, wherein the altered trait is selected from the group consisting of:

(i) a decrease in the ratio of the carbon isotope ¹²C to ¹³C found in either all the dried above-ground biomass, or specific components of the above-ground biomass, leaves, or reproductive structures, of at least 0.5 ‰ (0.5 per mille), measured as a decrease in the ratio of ¹²C to ¹³C relative to the controls with both ratio being expressed relative to the same standard; and