WO2010068777A2 - Materials and methods for modulating plant photosynthetic capacity and biomass - Google Patents

Abstract

The subject invention concerns materials and methods for modulating plant photosynthetic capacity and biomass in plants. Increased photosynthetic capacity and biomass is provided by increasing leaf width and total leaf area of a plant. In one embodiment, expression of an ARF gene and/or ARF gene product is inhibited or decreased in a plant. The subject invention also concerns materials and methods for decreasing plant photosynthetic capacity and biomass in plants by decreasing leaf width and total leaf area.

Description

DESCRIPTION

MATERIALS AND METHODS FOR MODULATING PLANT PHOTOSYNTHETIC CAPACITY AND BIOMASS

CROSS-REFERENCE TO RELATED APPLICATION

The present application claims the benefit of U.S. Provisional Application Serial No. 61/201,467, filed December 10, 2008, which is hereby incorporated by reference herein in its entirety, including any figures, tables, nucleic acid sequences, amino acid sequences, and drawings.

GOVERNMENT SUPPORT

The subject matter of this application has been supported by a research grant from the Department of Energy, Office of Science, Office of Biological and Environmental Research under grant number DE-FG02-05ER64114. Accordingly, the government has certain rights in this invention.

BACKGROUND OF THE INVENTION A primary goal of evolutionary and developmental genetics is to understand the molecular basis for morphological and adaptive differences between species or genera. Novel molecular tools and experimental designs, based on interspecific populations, can be applied to identify genes and sequence elements contributing to evolutionarily relevant phenotypic variation. The genus Populus is composed of five diverse evolutionary sections, yet leaf morphological variation is frequently diagnostic of relationships between species. Leaves play the essential role of CO₂ capture and conversion to sucrose through photosynthesis — a key step for biomass production. Leaf area determines in part the capacity of a plant to capture CO₂ and, therefore produce biomass. BRIEF SUMMARY OF THE INVENTION

The subject invention concerns materials and methods for modulating photosynthetic capacity and biomass in plants. In one embodiment, photosynthetic capacity and biomass in plants is increased. Increased photosynthetic capacity and biomass is provided by increasing leaf width and total leaf area of a plant.

In a pseudo-backcross pedigree of narrow-leaf Populns trichocarpa and broad- leaf Populus deltoides, a major quantitative trait locus (QTL) (Likelihood Ratio [LR] = 28.8) explained approximately 40% of the heritable variation in leaf width and spanned an interval encompassing approximately 450 genes. To identify candidate genes in the interval associated with the quantitative trait, a genetical genomics strategy was applied by measuring genome- wide gene expression in expanding leaves from 148 segregants and mapping resulting transcript variation as expression QTL (eQTL). An ADP-ribosylation factor (ARF) gene with a significant cw-eQTL (LR = 30.3) was physically localized within the leaf width QTL interval. Furthermore, ARF transcript abundance strongly correlated (r² = -0.37) with phenotypic variation in leaf width. Sequence-level allelic variation was characterized within and around the ARF gene, and interspecific polymorphisms were identified in the 5' regulatory region that may contribute to the quantitative phenotype. ARFs have been implicated in molecular trafficking of auxin efflux proteins in Arabidopsis, suggesting that differentially established auxin gradients within leaves could underlie evolutionarily relevant phenotypic variation observed in Populus.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 shows examples of leaf morphological variation between P. trichocarpa and P. deltoides, the Fl hybrid, and pseudo-backcross progeny of Family 52-124. Genotypic values (Least-square means estimates of genotype effect for biologically replicated clones) are depicted as LWR measurements. Leaf width was measured at the widest point on the expanded blade. A continuous, quantitative distribution is noted for both LWR (shown) and leaf width (not shown) measurements. Figure 2 shows genome wide QTL analysis of leaf morphological pheno types.

Least-squared means estimates of genotype effects for LWR and leaf width in 396 progeny genotypes were subjected to composite interval mapping QTL analysis in QTL Cartographer (v. 2.5) and the single tree framework SSR map for maternal genotype 52- 225. Significance was judged using a standard permutation threshold procedure (n=1000). QTL targeted for further analysis is designated with an asterisk.

Figure 3 shows LG X fine mapping of leaf width QTL. Procedures were as described above and in Figure 2. Co-localization of QTL was noted for measurements under high (solid line) and low (dotted line) nitrogen treatments independently, as well as LSM based on combined high and low nitrogen data (striped line).

Figure 4 shows that expression QTL for estExt_Genewiseljvl.C_LG_X0744

(ARFl, dotted green line) associates tightly with the phenotypic QTL for leaf blade width on LG_X and the physical location of estExt_Genewisel_vl .C_LG_X0744. Normalized microarray signal at the probe interrogating estExt_Genewisel_vl.C_LG_X0744 from

150 progeny was subjected to quantitative trait analysis as described in Figure 2.

Figure 5 shows that gene expression for estExt_Genewisel_vl.C_LG_X0744 correlates with variation in leaf width phenotypes in family 52-124. LSM estimates for leaf width were correlated (Pearson's) with normalized microarray signal from each genotype in JMP 7.0.

Figure 6 shows the sequence alignment of 500bp upstream of the start codon of estExt_Genewisel_vl.C_LGX0744 for the P. trichocarpa (SEQ ID NO:4) and P. deltoides (SEQ ID NO:3) haplotypes in the Family 52-124 pedigree. DNA was isolated from the parent trees of Family 52-124 and subjected to Genome Walker PCR (Clontech Laboratories). Resulting amplicons were cloned in the pGEM-T Easy vector (Promega) and sequenced bidirectionally from the SP6 and T7 promoters using traditional dye terminator sequencing. Sequence alignments were produced in TCOFFEE using default parameters.

BRIEF DESCRIPTION OF TFIE SEQUENCES

SEQ ID NO:1 is a polynucleotide of the present invention. SEQ ID NO:2 is an amino acid sequence of an ARF protein of the present invention encoded by SEQ ID NO:1. SEQ ID NO:3 is a polynucleotide of the present invention.

SEQ ID NO:4 is a polynucleotide of the present invention. DETAILED DESCRIPTION OF THE INVENTION

The subject invention concerns materials and methods for modulating plant photosynthetic capacity and biomass in plants. In one embodiment, photosynthetic capacity and biomass in plants is increased. Increased photosynthetic capacity and biomass is provided by increasing leaf width and total leaf area of a plant. In one embodiment, expression of an ADP-ribosylation factor (ARF) gene and/or ARF gene product is inhibited or decreased or downregulated in a plant. The subject invention also concerns materials and methods for decreasing plant photosynthetic capacity and biomass in plants by decreasing leaf width and total leaf area. In one embodiment, expression of an ARF gene and/or ARF gene product is increased or upregulated in a plant.

In one embodiment, expression of an ARF gene and/or ARF gene product is inhibited or decreased in a plant, which thereby results in increased leaf width and total leaf area. In one embodiment, an ARF gene of the present invention comprises a nucleotide sequence shown in SEQ ID NO:1, SEQ ID NO:3, or SEQ ID NO:4. In one embodiment, an ARF gene of the present invention comprises in the about 500 bp upstream of the start codon one or more of the nucleotide changes (nucleotide addition, substitution, or deletion) shown in the P. deltoides nucleotide sequence (SEQ ID NO:3) as compared to the nucleotide sequence of P. trichocarpa (SEQ ID NO:4), for example, one or more of the nucleotide changes in one or more of the transcription factor binding motifs. In one embodiment, an ARF gene product comprises the amino acid sequence shown in SEQ ID NO:2. Plants contemplated within the scope of the invention include woody and non-w^roody plants. In a specific embodiment, the plant is a tree. In a more specific embodiment, the plant is a species of Populus.

Any method available in the art for modulating expression of an ARF gene and/or ARF gene product is contemplated within the scope of the present invention. In one embodiment, one or more mutations are introduced into an ARF gene of a plant that provides for results in inhibited or decreased transcription of the ARF gene, or inhibited or decreased translation of ARF mRNA, and/or that results in an ARF protein exhibiting inhibited or decreased enzymatic activity. In one embodiment, a mutation is introduced in the ARF gene upstream of the transcription start site and/or downstream of the transcription start site. Mutations include one or more nucleotide insertions, deletions, or substitutions. Methods for introducing mutations and selecting for mutants are known in the art. In one embodiment, one or more mutations are located in a transcription factor binding motif of an ARF gene. Mutations can provide for inhibited or decreased transcription of an ARF gene. In a further embodiment, inhibition or reduction of expression of an ARF gene of the invention can be obtained by introducing an antisense construct that provides for a sequence that is antisense to the ARF polynucleotide. In another embodiment, expression of an ARF gene is inhibited by providing in the plant a ribozyme that can cleave ARF RNA and thereby lead to inhibition of endogenous ARF gene expression. In a further embodiment, expression of an ARF gene in a plant is inhibited by providing the plant with small interfering RNA (siRNA) that target ARF polynucleotides. In a still further embodiment, ARF gene expression can be inhibited by engineering a plant to contain a mutation in the ARF gene that results in the insertion of one or more premature stop codons or nonsense mutations in the ARF gene transcript. In another embodiment, inhibition or reduction of ARF gene expression can be achieved by eliminating or non-functionalizing one or more endogenous ARF genes in the plant, for example, by homologous recombination.

In one embodiment, a method of the invention comprises introducing a polynucleotide into a plant wherein the polynucleotide, or the expression product thereof, provides for decreased expression of an ARF gene or protein relative to a plant wherein the polynucleotide has not been introduced (e.g., a wild type plant). In one embodiment, a polynucleotide can be introduced that increases degradation of ARF gene transcripts or gene product. In another embodiment, a polynucleotide can be introduced that encodes an ARF protein that exhibits decreased enzymatic activity (for example, via decreased resistance to inhibition of enzyme activity or via decreased affinity of enzyme for substrate). In a further embodiment, a polynucleotide can be introduced that encodes a protein having ARF enzyme activity, wherein the polynucleotide comprises regulatory elements that provide for decreased expression of the polynucleotide and/or the protein encoded thereby. In a still further embodiment, a polynucleotide can be introduced that encodes a protein or peptide that inhibits or reduces enzymatic activity of an ARF protein. For example, the encoded protein can be an antibody, or an antigen binding fragment thereof, that binds to an ARF protein and thereby inhibits or reduces enzymatic activity of the ARF protein. Methods for producing a monoclonal antibody against a specific antigen and methods for transforming a plant with a polynucleotide to express an antibody are known in the art. Plants containing a polynucleotide of the invention, or progeny thereof, optionally can be screened for decreased expression of ARF gene and/or protein, or decreased enzymatic activity of the protein.

The subject invention also concerns plants, plant tissue, and plant cells of the invention that exhibit (relative to a wild type plant) increased or decreased expression of an ARF-encoding polynucleotide or the protein encoded by the polynucleotide, or that express a mutant ARF polynucleotide or a mutant ARF enzyme of the invention that is characterized by increased or decreased expression, or increased or decreased enzymatic activity or function, or a fragment or variant thereof. Plants of the invention have increased or decreased photosynthetic capacity and biomass. Plants of the invention having increased photosynthetic capacity and biomass typically exhibit increased leaf width and/or increased total leaf area. Plant tissue includes, but is not limited to, seed, scion, and rootstock. In a specific embodiment, the plant, plant tissue, or plant cell is a species of a Populus plant. In one embodiment, a plant, plant tissue, or plant cell is a transgenic plant, plant tissue, or plant cell. In another embodiment, a plant, plant tissue, or plant cell is one that has been obtained through a breeding program. In one embodiment, the plant, plant tissue, or plant cell is homozygous for a mutant ARF polynucleotide or gene. In another embodiment, the plant, plant tissue, or plant cell is heterozygous for a mutant ARF polynucleotide or gene. The mutant ARF polynucleotide or gene can be provided by the maternal parent and/or the paternal parent. In one embodiment, a plant, plant tissue, or plant cell of the invention is a hybrid plant, plant tissue, or plant cell obtained from breeding a plant comprising a polynucleotide that encodes a mutant ARF enzyme of the invention with a plant that comprises a polynucleotide that encodes a wild type ARF enzyme. In one embodiment, a plant of the invention is an inbred line that has been transformed or bred to exhibit increased or decreased expression of an ARF encoding polynucleotide or the protein encoded by the polynucleotide, or that expresses a mutant ARF polynucleotide or an ARF enzyme of the invention. In one embodiment, a plant, plant tissue, or plant cell of the invention comprises a polynucleotide comprising the nucleotide sequence of SEQ ID NO:1, 3, or 4, or a sequence that is antisense to all or a portion of an ARF polynucleotide sequence such as a sequence of SEQ ID NO:1, 3, or 4, or an siRNA that targets an ARF polynucleotide sequence such as a sequence of SEQ ID NO:1, 3, or 4. The subject invention also concerns an isolated ARF plant gene or polynucleotide, wherein a plant expressing the gene or polynucleotide exhibits decreased levels of ARF enzyme and/or ARF enzymatic activity. In one embodiment, the ARF-encoding polynucleotide comprises a nucleotide sequence that provides for or results in decreased or inhibited transcription and/or translation of the ARF gene when expressed in a plant (for example, as compared to ARF expression of wild type P. trichocarpa). In one embodiment, the ARF-encoding polynucleotide encodes an ARF enzyme with decreased enzymatic activity relative to a non-mutated or wild type ARF enzyme. In a specific embodiment, the ARF enzyme comprises the amino acid sequence shown in SEQ ID NO:2, or a fragment or variant thereof. In one embodiment, the polynucleotide encoding SEQ ID NO:2 comprises the nucleotide sequence shown in SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:4, or a fragment or variant thereof. Allelic variants of ARF genes and polynucleotides of the invention are included within the scope of the invention.

Polynucleotides useful in the present invention can be provided in an expression construct. Expression constructs of the invention generally include regulatory elements that are functional in the intended host cell in which the expression construct is to be expressed. Thus, a person of ordinary skill in the art can select regulatory elements for use in bacterial host cells, yeast host cells, plant host cells, insect host cells, mammalian host cells, and human host cells. Regulatory elements include promoters, transcription termination sequences, translation termination sequences, enhancers, and polyadenylation elements. As used herein, the term "expression construct" refers to a combination of nucleic acid sequences that provides for transcription of an operably linked nucleic acid sequence. As used herein, the term "operably linked" refers to a juxtaposition of the components described wherein the components are in a relationship that permits them to function in their intended manner. In general, operably linked components are in contiguous relation.

An expression construct of the invention can comprise a promoter sequence operably linked to a polynucleotide sequence encoding a ARF polypeptide of the invention. Promoters can be incorporated into a polynucleotide using standard techniques known in the art. Multiple copies of promoters or multiple promoters can be used in an expression construct of the invention. In a preferred embodiment, a promoter can be positioned about the same distance from the transcription start site in the expression construct as it is from the transcription start site in its natural genetic environment. Some variation in this distance is permitted without substantial decrease in promoter activity. A transcription start site is typically included in the expression construct.

If the expression construct is to be provided in or introduced into a plant cell, then plant viral promoters, such as, for example, a cauliflower mosaic virus (CaMV) 35S (including the enhanced CaMV 35S promoter (see, for example U.S. Patent No. 5,106,739)) or a CaMV 19S promoter or a cassava vein mosaic can be used. Other promoters that can be used for expression constructs in plants include, for example, prolifera promoter, Ap3 promoter, heat shock promoters, T-DNA 1'- or 2'-promoter of A. tumefaciens, polygalacturonase promoter, chalcone synthase A (CHS-A) promoter from petunia, tobacco PR- Ia promoter, ubiquitin promoter, actin promoter, ale A gene promoter, ρin2 promoter (Xu et al, 1993), maize Wipl promoter, maize trpA gene promoter (U.S. Patent No. 5,625,136), maize CDPK gene promoter, and RUBISCO SSU promoter (U.S. Patent No. 5,034,322) can also be used. Tissue-specific promoters can be used. Fruit-specific promoters such as flower organ-specific promoters can be used with an expression construct of the present invention for expressing a polynucleotide of the invention in the flower organ of a plant. Examples of flower organ- specific promoters include any of the promoter sequences described in U.S. Patent Nos. 6,462,185; 5,639,948; and 5,589,610. Seed-specific promoters such as the promoter from a β- phaseolin gene (for example, of kidney bean) or a glycinin gene (for example, of soybean), and others, can also be used. Endosperm-specific promoters include, but are not limited to, MEGl (EPO application No. EP 1528104) and those described by Wu et al. (1998), Furtado et al. (2001), and Hwang et al. (2002). Root-specific promoters, such as any of the promoter sequences described in U.S. Patent No. 6,455,760 or U.S. Patent No. 6,696,623, or in published U.S. patent application Nos. 20040078841 ; 20040067506; 20040019934; 20030177536; 20030084486; or 20040123349, can be used with an expression construct of the invention. Constitutive promoters (such as the CaMV, ubiquitin, actin, or NOS promoter), developmentally-regulated promoters, and inducible promoters (such as those promoters than can be induced by heat, light, hormones, or chemicals) are also contemplated for use with polynucleotide expression constructs of the invention. Expression constructs of the invention may optionally contain a transcription termination sequence, a translation termination sequence, a sequence encoding a signal peptide, and/or enhancer elements. Transcription termination regions can typically be obtained from the 3' untranslated region of a eukaryotic or viral gene sequence. Transcription termination sequences can be positioned downstream of a coding sequence to provide for efficient termination. A signal peptide sequence is a short amino acid sequence typically present at the amino terminus of a protein that is responsible for the relocation of an operably linked mature polypeptide to a wide range of post-translational cellular destinations, ranging from a specific organelle compartment to sites of protein action and the extracellular environment, Targeting gene products to an intended cellular and/or extracellular destination through the use of an operably linked signal peptide sequence is contemplated for use with the polypeptides of the invention. Classical enhancers are cis-acting elements that increase gene transcription and can also be included in the expression construct. Classical enhancer elements are known in the art, and include, but are not limited to, the CaMV 35S enhancer element, cytomegalovirus (CMV) early promoter enhancer element, and the SV40 enhancer element. Intron- mediated enhancer elements that enhance gene expression are also known in the art. These elements must be present within the transcribed region and are orientation dependent. Examples include the maize shrunken- 1 enhancer element (Clancy and Hannah, 2002).

DNA sequences which direct polyadenylation of mRNA transcribed from the expression construct can also be included in the expression construct, and include, but are not limited to, an octopine synthase or nopaline synthase signal. The expression constructs of the invention can also include a polynucleotide sequence that directs transposition of other genes, i. e. , a transposon.

Polynucleotides of the present invention can be composed of either RNA or DNA. Preferably, the polynucleotides are composed of DNA. The subject invention also encompasses those polynucleotides that are complementary in sequence to the polynucleotides disclosed herein. Polynucleotides and polypeptides of the invention can be provided in purified or isolated form.

Polynucleotides and polypeptides contemplated within the scope of the subject invention can also be defined in terms of more particular identity and/or similarity ranges with those sequences of the invention specifically exemplified herein. The sequence identity will typically be greater than 60%, preferably greater than 75%, more preferably greater than 80%, even more preferably greater than 90%, and can be greater than 95%. The identity and/or similarity of a sequence can be 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, 96, 97, 98, or 99% as compared to a sequence exemplified herein. Unless otherwise specified, as used herein percent sequence identity and/or similarity of two sequences can be determined using the algorithm of Karlin and Altschul (1990), modified as in Karlin and Altschul (1993). Such an algorithm is incorporated into the NBLAST and XBLAST programs of Altschul et al. (1990). BLAST searches can be performed with the NBLAST program, score = 100, wordlength = 12, to obtain sequences with the desired percent sequence identity. To obtain gapped alignments for comparison purposes, Gapped BLAST can be used as described in Altschul et al. (1997). When utilizing BLAST and Gapped BLAST programs, the default parameters of the respective programs (NBLAST and XBLAST) can be used. See NCBI/NIH website.

The subject invention also contemplates those polynucleotide molecules having sequences which are sufficiently homologous with the polynucleotide sequences exemplified herein so as to permit hybridization with that sequence under standard stringent conditions and standard methods (Maniatis et al, 1982). As used herein, "stringent" conditions for hybridization refers to conditions wherein hybridization is typically carried out overnight at 20-25 C below the melting temperature (Tm) of the DNA hybrid in 6x SSPE, 5x Denhardt's solution, 0.1% SDS, 0.1 mg/ml denatured DNA. The melting temperature, Tm, is described by the following formula (Beltz et al, 1983): Tm=81.5 C+16.6 Log[Na+]+0.41(%G+C)-0.61(% formamide)-600/length of duplex in base pairs.

Washes are typically carried out as follows:

(1) Twice at room temperature for 15 minutes in Ix SSPE, 0.1% SDS (low stringency wash). (2) Once at Tm-20 C for 15 minutes in 0.2x SSPE, 0.1% SDS (moderate stringency wash). As used herein, the terms "nucleic acid" and "polynucleotide" refer to a deoxyribonucleotide, ribonucleotide, or a mixed deoxyribonucleotide and ribonucleotide polymer in either single- or double-stranded form, and unless otherwise limited, would encompass known analogs of natural nucleotides that can function in a similar manner as naturally-occurring nucleotides. The polynucleotide sequences include the DNA strand sequence that is transcribed into RNA and the strand sequence that is complementary to the DNA strand that is transcribed. The polynucleotide sequences also include both full- length sequences as well as shorter sequences derived from the full-length sequences. Allelic variations of polynucleotide sequences of the invention also fall within the scope of the subject invention. The polynucleotide sequence includes both the sense and antisense strands either as individual strands or in the duplex.

Plants within the scope of the present invention include monocotyledonous plants, such as, for example, rice, wheat, barley, oats, rye, sorghum, maize, sugarcane, pineapple, onion, bananas, coconut, lilies, turfgrasses, millet, and monocot trees such as Agavaceae (Agave family), such as Cabbage tree {Cordyline australis), Dragon tree {Dracaena draco), Joshua tree {Yucca brevifolia); Arecaceae (Palmae) (Palm family), such as Areca Nut {Areca catechu), Coconut {Cocos nucifera), Date Palm {Phoenix dactylifera), Chusan Palm (Trachycarpus fortunei); and Poaceae (grass family), such as Bamboos Poaceae subfamily Bambusoideae. Plants within the scope of the present invention also include dicotyledonous plants, such as, for example, tomato, cucumber, squash, peas, alfalfa, melon, chickpea, chicory, clover, kale, lentil, soybean, beans, tobacco, potato, sweet potato, yams, cassava, radish, broccoli, spinach, cabbage, rape, apple trees, citrus (including oranges, mandarins, grapefruit, lemons, limes and the like), grape, cotton, sunflower, strawberry, lettuce, and dicot trees such as Adoxaceae (Moschatel family), such as Moschatel {Adoxa moschatellina), Elderberry {Sambucus species), Sinadoxa {Sinadoxa corydalifolia), Viburnum {Viburnum species); Altingiaceae (Sweetgum family), such as Sweetgum {Liquidambar species); Anacardiaceae (Cashew family), such as Cashew {Anacardium occidentale), Mango {Mangifera indica), Pistachio {Pistacia vera), Sumac {Rhus species), Lacquer tree {Toxicodendron verniciflua); Annonaceae (Custard apple family), such as Cherimoya {Annona cherimola), Custard apple {Annona reticulata), Pawpaw {Asimina triloba), Soursop {Annona muricata); Apocynaceae (Dogbane family), such as Pachypodium {Pachypodium species); Aquifoliaceae (Holly family), such as Holly (Ilex species); Araliaceae (Ivy family), such as Kalopanax (Kalopanax pictus); Betulaceae (Birch family), such as Alder (Alnus species), Birch (Betula species), Hornbeam (Carpinus species), Hazel (Corylus species); Bignoniaceae (family), such as Catalpa (Catalpa species); Cactaceae (Cactus family), such as Saguaro (Carnegiea gigantea); Cannabaceae (Cannabis family), such as Hackberry (Celtis species); Cornaceae (Dogwood family), such as Dogwood (Cornus species); Dipterocarpaceae family, such as Garjan (Dipterocarpus species), Sal (Shorea species); Ebenaceae (Persimmon family), such as Persimmon (Diospyros species); Ericaceae (Heath family), such as Arbutus (Arbutus species); Eucommiaceae (Eucommia family), such as Eucommia (Eucommia ulmoides); Fabaceae (Pea family), such as Acacia (Acacia species), Honey locust (Gleditsia triacanthos), Black locust (Robinia pseudoacacia), Laburnum (Laburnum species), Pau Brasil, Brazilwood (Caesalpinia echinata); Fagaceae (Beech family), such as Chestnut (Castanea species), Beech (Fagus species), Southern beech (Nothofagus species), Tanoak (Lithocarpus densiflorus), Oak (Quercus species); Fouquieriaceae (Boojum family), such as Boojum (Fouquieria columnaris); Hamamelidaceae (Witch-hazel family), such as Persian Ironwood (Parrotia persica); Juglandaceae (Walnut family), such as Walnut (Juglans species), Hickory (Carya species), Wingnut (Plerocarya species); Lauraceae (Laurel family), such as Cinnamon (Cinnamomum zeylanicum), Bay Laurel (Laurus nobilis), Avocado (Persea americana); Lecythidaceae (Paradise nut family), such as Brazil Nut (Bertholletia excelsa); Lythraceae (Loosestrife family), such as Crape-myrtle (Lager stroemia species); Magnoliaceae (Magnolia family), such as Tulip tree (Liriodendron species), Magnolia (Magnolia species); Malvaceae (Mallow family; including Tiliaceae, Sterculiaceae and Bombacaceae), such as Baobab (Adansonia species), Silk-cotton tree (Bombax species), Bottletrees (Brachychiton species), Kapok (Ceiba pentandrά), Durian (Durio zibethinus), Balsa (Ochroma lagopus), Cacao (cocoa) (Theobroma cacao), Linden (Basswood, Lime) (Tilia species); Meliaceae (Mahogany family), such as Neem (Azadirachta indica), Bead tree (Melia azedarach), Mahogany (Swietenia mahagoni); Moraceae (Mulberry family), such as Fig (Ficus species), Mulberry (Morus species); such as Myristicaceae (Nutmeg family), such as Nutmeg (Mysristica fragrans); Myrtaceae (Myrtle family), such as Eucalyptus (Eucalyptus species), Myrtle (Myrtus species), Guava (Psidium guajava); Nyssaceae (Tupelo family; sometimes included in Cornaceae), such as Tupelo (Nyssa species), Dove tree (Davidia involucrata); Oleaceae (Olive family), such as Olive (Olea europaea), Ash (Fraxinus species); Paulowniaceae (Paulownia family), such as Foxglove Tree (Paulownia species); Platanaceae (Plane family), such as Plane {Plalanus species); Rhizophoraceae (Mangrove family), such as Red Mangrove (Rhizophora mangle); Rosaceae (Rose family), such as Rowans, Whitebeams, Service Trees (Sorbus species), Hawthorn (Crataegus species), Pear (Pyrus species), Apple (Mains species). Almond (Prunus dulcis), Peach (Prunus persica). Apricot (Prunus armeniaca), Plum (Prunus domestica), Cherry (Prunus species); Rubiaceae (Bedstraw family), such as Coffee (Coffea species); Rutaceac (Rue family), such as Citrus (Citrus species), Cork-tree (Phellodendron species), Euodia (Tetradium species); Salicaceae (Willow family), such as Aspen (Populus species), Poplar (Populus species), Willow (Salix species); Sapindaceae (including Aceraceae, Hippocastanaceae) (Soapberry family), such as Maple (Acer species), Buckeye, Horse-chestnut (Aesculus species), Mexican Buckeye (Ungnadia speciosa), Lychee (Litchi sinensis), Golden rain tree (Koelreuteria); Sapotaceae (Sapodilla family), such as Argan (Argania spinosa), Gutta-percha (Palaquium species), Tambalacoque, or "dodo tree" (Sideroxylon grandiflorum), previously Calvaria major; Simaroubaceae family, such as Tree of heaven (Ailanthus species); Theaceae (Camellia family), such as Gordonia (Gordonia species), Stewartia (Stewartia species); Thymelaeaceae (Thymelaea family), such as Ramin (Gonystylus species); Ulmaceae (Elm family), such as Elm (Ulmus species), Zelkova (Zelkova species); and Verbenaceae family, such as Teak, Tectona species. Herb plants containing a polynucleotide of the invention are also contemplated within the scope of the invention. Herb plants include parsley, sage, rosemary, thyme, and the like.

Techniques for transforming plant cells with a gene are known in the art and include, for example, Agrobacterium infection, biolistic methods, electroporation, calcium chloride treatment, PEG-mediated transformation, etc. U.S. Patent No. 5,661,017 teaches methods and materials for transforming an algal cell with a heterologous polynucleotide. Transformed cells can be selected, redifferentiated, and grown into plants that contain and express a polynucleotide of the invention using standard methods known in the art. The seeds and other plant tissue and progeny of any transformed or transgenic plant cells or plants of the invention are also included within the scope of the present invention. The subject invention also concerns methods for producing a plant that exhibits increased or decreased ARF content and/or enzymatic activity relative to a wild type plant. In one embodiment, a polynucleotide exhibiting decreased or inhibited ARF gene transcription or translation, and/or a polynucleotide encoding an ARF or a mutant ARF enzyme of the present invention is introduced into a plant cell and the polypeptide(s) encoded by the polynucleotide(s) is expressed, or wherein a polynucleotide that is antisense to an ARF polynucleotide sequence is introduced into a plant cell, or wherein an siRNA (or a polynucleotide that provides the siRNA) that targets an ARF polynucleotide is introduced into a plant cell. In another embodiment, the polynucleotide encodes a ribozyme that cleaves an ARF polynucleotide, such as ARF RNA. In another embodiment, the polynucleotide encodes a protein or peptide that can bind to and inhibit function of an ARF polypeptide. In a specific embodiment, the protein or peptide is an antibody or an antigen binding fragment thereof. In one embodiment, the polynucleotide or polynucleotides is incorporated into the genome of the plant cell and a plant is grown from the plant cell. In a preferred embodiment, the plant grown from the plant cell stably expresses the incorporated polynucleotide or polynucleotides.

The subject invention also concerns methods and materials for selecting for plants having increased or decreased leaf width and/or total leaf area. In one embodiment, an ARF gene or polynucleotide that exhibits decreased transcription and/or translation, or that encodes an ARF enzyme having decreased enzymatic activity relative to a wild type or non-mutated ARF is utilized as a genetic marker. In a specific embodiment, the ARF enzyme comprises an amino acid sequence of SEQ ID NO:2, or a fragment or variant thereof. In a specific embodiment, the ARF gene or polynucleotide comprises a nucleotide sequence of SEQ ID NO: L SEQ ID NO:3, or SEQ ID NO:4, or a fragment or variant thereof. In one embodiment, the ARF gene or polynucleotide comprises in the about 500 bp upstream of the start codon one or more of the nucleotide changes shown in SEQ ID NO:3 as compared to the sequence in SEQ ID NO:4. Methods of the invention comprise determining whether a plant, plant tissue, or plant cell contains an ARF gene or polynucleotide of the invention, and/or determining whether a plant, plant tissue, or plant cell comprises or expresses an ARF enzyme of the present invention. In one embodiment, the presence of an ARF gene or polynucleotide is determined by screening nucleic acid from the plant, plant tissue, or plant cell for hybridization with a nucleic acid probe (e.g., an oligonucleotide of the invention) that hybridizes with an ARF gene or polynucleotide of the invention. In another embodiment, the presence of an ARF gene or polynucleotide is determined by restriction fragment length polymorphism (RFLP) analysis, by polymerase chain reaction (PCR) amplification of specific ARF nucleic acid sequences, or by sequencing ARF-encoding nucleic acid from the plant, plant tissue, or plant cell and identifying whether the gene or polynucleotide comprises a sequence that provides for decreased ARF mRNA levels or decreased ARF enzymatic activity.

The subject invention also concerns methods for marker assisted selection and breeding in plants using a gene or polynucleotide that provides for modulated expression (increased or decreased) of ARF or the gene product thereof for selecting for plants, plant tissue, or plant cells that exhibit a phenotypic characteristic of interest, e.g., increased or decreased leaf width, total leaf area, etc. Methods for marker assisted selection are known in the art. In one embodiment, a method uses an ARF gene or polynucleotide of the invention that encodes an ARF enzyme that is non-functional or that exhibits decreased enzymatic activity relative to a non-mutant ARF enzyme, or wherein the ARF gene or polynucleotide provides for decreased or no expression of gene transcripts or translation of gene transcripts.

The subject invention also concerns oligonucleotide probes and primers, such as polymerase chain reaction (PCR) primers, that can hybridize to a coding or non-coding sequence of a polynucleotide of the present invention. Oligonucleotide probes of the invention can be used in methods for detecting and quantitating nucleic acid sequences encoding an ARF protein. Oligonucleotide primers of the invention can be used in PCR methods and other methods involving nucleic acid amplification. In a preferred embodiment, a probe or primer of the invention can hybridize to a polynucleotide of the invention under stringent conditions. Probes and primers of the invention can optionally comprise a detectable label or reporter molecule, such as fluorescent molecules, enzymes, radioactive moiety (e.g., ³H, ³⁵S, ¹²⁵I, etc.), and the like. Probes and primers of the invention can be of any suitable length for the method or assay in which they are being employed. Typically, probes and primers of the invention will be 10 to 500 or more nucleotides in length. Probes and primers that are 10 to 20, 21 to 30, 31 to 40, 41 to 50, 51 to 60, 61 to 70, 71 to 80, 81 to 90, 91 to 100 or more nucleotides in length are contemplated within the scope of the invention. Probes and primers of the invention can have complete (100%) nucleotide sequence identity with the polynucleotide sequence, or the sequence identity can be less than 100%. For example, sequence identity between a probe or primer and a sequence can be 99%, 98%, 97%, 96%, 95%, 90%, 85%, 80%, 75%, 70% or any other percentage sequence identity so long as the probe or primer can hybridize under stringent conditions to a nucleotide sequence of a polynucleotide of the invention. In one embodiment, a probe or primer of the invention has 70% or greater, 75% or greater, 80% or greater, 85% or greater, 90% or greater, or 95% to 100% sequence identity with a nucleotide sequence of SEQ ID NO:1, SEQ ID NO:3, or SEQ ID NO:4, or the complement thereof. The subject invention also concerns cells transformed with a polynucleotide of the present invention encoding an ARF protein of the invention. In one embodiment, the cell is transformed with a polynucleotide sequence comprising a sequence encoding the amino acid sequence shown in SEQ ID NO:2, or an enzymatically active fragment or variant thereof. In a specific embodiment, the cell is transformed with a polynucleotide sequence shown in SEQ ID NO: 1, 3, or 4, or a sequence encoding an enzymatically active fragment or variant of SEQ ID NO:2.

Preferably, the polynucleotide sequence is provided in an expression construct of the invention. The transformed cell can be a prokaryotic cell, for example, a bacterial cell such as E. coli or B. sublilis, or the transformed cell can be a eukaryotic cell, for example, a plant cell, including protoplasts, or an animal cell. Plant cells include, but are not limited to, dicotyledonous, monocotyledonous, and conifer cells. In one embodiment, the plant cell is a cell from a species of Populus plant.

An "antisense" nucleic acid sequence (antisense oligonucleotide) can include a nucleotide sequence that is complementary to a "sense" nucleic acid sequence encoding a protein, e.g., complementary to the coding strand of a double-stranded cDNA molecule or complementary to at least a portion of an ARF gene. The antisense nucleic acid sequence can be complementary to an entire coding strand of a target sequence, or to only a portion thereof. In another embodiment, the antisense nucleic acid molecule is antisense to a "noncoding region" of the coding strand of a nucleotide sequence within the gene. An antisense oligonucleotide can be, for example, about 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100 or more nucleotides in length. An antisense nucleic acid sequence can be designed such that it is complementary to the entire gene, but can also be an oligonucleotide that is antisense to only a portion of the gene. For example, the antisense oligonucleotide can be complementary to the region surrounding the translation start site of the target mRNA, e.g., between the -10 and +10 regions of the target gene nucleotide sequence of interest.

An antisense nucleic acid sequence of the invention can be constructed using chemical synthesis and enzymatic ligation reactions using procedures known in the art. For example, an antisense nucleic acid (e.g., an antisense oligonucleotide) can be chemically synthesized using naturally occurring nucleotides or variously modified nucleotides designed to increase the biological stability of the molecules or to increase the physical stability of the duplex formed between the antisense and sense nucleic acids, e.g., phosphorothioate derivatives and acridine substituted nucleotides can be used. The antisense nucleic acid sequence also can be produced biologically using an expression vector into which a nucleic acid sequence has been subcloned in an antisense orientation (i.e., RNA transcribed from the inserted nucleic acid sequence will be of an antisense orientation to a target nucleic acid sequence of interest, described further in the following subsection).

Ribozymes are a type of RNA that can be engineered to enzymatically cleave and inactivate other RNA targets in a specific, sequence-dependent fashion. By cleaving the target RNA, ribozymes inhibit translation, thus preventing the expression of the target gene. Ribozymes can be chemically synthesized in the laboratory and structurally modified to increase their stability and catalytic activity using methods known in the art. Alternatively, ribozyme encoding nucleotide sequences can be introduced into cells through gene-delivery mechanisms known in the art. A ribozyme having specificity for ARF RNA can include one or more sequences complementary to the nucleotide sequence of at least a portion of one or more ARF mRNA, and a sequence having known catalytic sequence responsible for mRNA cleavage (see U.S. Patent No. 5,093,246 or Haselhoff el al. 1988). For example, a derivative of a Tetrahymena L- 19 IVS RNA can be constructed in which the nucleotide sequence of the active site is complementary to the nucleotide sequence to be cleaved in the ARF mRNA (see, e.g., U.S. Patent No. 4,987,071; and U.S. Patent No. 5,116,742). Alternatively, ARF mRNA encoding an ARF protein can be used to select a catalytic RNA having a specific ribonuclease activity from a pool of RNA molecules (see, e.g., Bartel et al. 1993).

As used herein, the term "RNA interference" ("RNAi") refers to a selective intracellular degradation of RNA. RNAi occurs in cells naturally to remove foreign RNAs {e.g., viral RNAs). Natural RNAi proceeds via fragments cleaved from free dsRNA which direct the degradative mechanism to other similar RNA sequences.

Alternatively, RNAi can be initiated by the hand of man, for example, to silence the expression of target genes.

As used herein, the term "small interfering RNA" or "short interfering RNA" ("siRNA") refers to an RNA (or RNA analog) comprising between about 10-50 nucleotides (or nucleotide analogs) which is capable of directing or mediating RNA interference.

As used herein, a siRNA having a "sequence sufficiently complementary to a target mRNA sequence to direct target-specific RNA interference (RNAi)" means that the siRNA has a sequence sufficient to trigger the destruction of the target mRNA by the

RNAi machinery or process. "mRNA", "messenger RNA", and "transcript" each refer to single-stranded RNA that specifies the amino acid sequence of one or more polypeptides.

This information is translated during protein synthesis when ribosomes bind to the mRNA.

All patents, patent applications, provisional applications, and publications referred to or cited herein arc incorporated by reference in their entirety, including all figures and tables, to the extent they are not inconsistent with the explicit teachings of this specification. Following are examples that illustrate procedures for practicing the invention.

These examples should not be construed as limiting. All percentages are by weight and all solvent mixture proportions are by volume unless otherwise noted. EXAMPLE 1

Leaf morphological variation was measured as leaf blade lengthiwidth ratio (LWR) and leaf blade width in the two parents and a pseudo-backcross progeny of 396 biologically triplicated genotypes, grown under two nitrogen treatments (0 mM, 25 mM). From the raw data, we calculated the least-square means genotypic value of leaf width and LWR was calculated for each parent and progeny genotype (Figure 1) for subsequent phenotypic QTL mapping. Additionally, RNA was isolated from expanding leaves in 150 genotypes and hybridized to full-genome 60-mer NimbleGen microarrays. Resulting expression data were background subtracted, quantile normalized, and log₂ transformed. Phenotypic and expression variation were mapped as QTL using a standard composite interval mapping procedure and judged for significance at a permuted threshold (n=1000). Mapping was focused against the hybrid parent SSR map to identify alleles associated to interspecific variation.

Major QTL were detected for both leaf LWR (LR = 64.5) and blade width (LR = 28.8, Figure 2) and the corresponding genomic region was identified as significantly affecting the traits independent of environmental variance in nitrogen abundance (Figure 3). The most significant QTL for the two traits were co-localized in an interval on linkage group 10 that encompassed about 650 genes or 40 cM (Figure 2, marked with asterisk). We focused on the leaf width trait and targeted this interval for fine-mapping, increasing the resolution to an interval comprising about 460 genes or about 25 cM (Figure 3). The candidate gene model, estExt_Genewisel_vl .C_LGJX0744, is contained within this interval (Figure 3) as indicated by the physical orientation of the genetic map to the Populus genome sequence (Tuskan et al, 2006) and the molecular markers identified about 1 kb upstream and downstream of the estExt_Genewisel_vl .C_LG_X0744 locus (Figure 3, ARFlUSl [upstream] and ARFl D S2 [downstream]).

Next, we identified gene expression QTL for transcript abundance estimated from the NimbleGen microarray data. Our primary hypothesis is that genetically controlled variation in transcript abundance may underlie the phenotypic variation in leaf morphology we observed between P. trichocarpa and P. deltoides. Therefore, we were interested in identifying genes whose transcript abundances were controlled by the same region on linkage group 10 that controlled the phenotypic trait variation. We identified eQTL for 119 genes in the phenotypic QTL interval. Among these genes, 88 were physically localized to the region comprising the phenotypic QTL interval as predicted from the poplar genome sequence. The transcript encoded by estExt_Gencwisel_vl.C_LG_X0744 displayed a highly significant eQTL (LR = 30.3) that closely associated with the profile of the phenotypic QTL identified for leaf blade width (Figure 4). We expect that if the transcript encoded by estExt_Genewiseljvl.C_LG_X0744 plays a role in controlling variation in leaf blade width, the abundance of the transcript should exhibit statistical correlation with the phenotypic variance. Pearson's Correlation analysis indicated that abundance of estExt_Genewisel_yl.C_LG_X0744 mRNA correlated well with variation in leaf width (r² = -.37, Figure 5), consistent with the additive effect estimates that the allele decreasing mRNA abundance increases leaf width (not shown).

The full-length genomic region corresponding to estExt_Genewisel_vl .C_LG_X0744 in each parent of pedigree 52-124 was isolated (SEQ ID NO: 1). Furthermore, more than 500 bp upstream of the start codon have been isolated in each parent using a genome walker PCR approach (Figure 6) (SEQ ID NO:3 and SEQ ID NO:4). From this sequence, polymorphisms between P. trichocarpa and P. deltoids were identified in several putative transcription factor binding motifs. We are currently expanding our sequencing effort to include at least 1.5 Kb upstream of the start codon to classify additional candidate polymorphisms. The closest homolog in Arabidopsis thaliana, At2g47170, has been shown to play a direct role in controlling epidermal cell polarity (Xu and Scheres, 2005) providing a potential mechanism by which estExt_Genewisel jvl .C_LG_X0744 may be influencing the leaf phenotype in Populus.

It should be understood that the examples and embodiments described herein are for illustrative purposes only and that various modifications or changes in light thereof will be suggested to persons skilled in the art and are to be included within the spirit and purview of this application and the scope of the appended claims. In addition, any elements or limitations of any invention or embodiment thereof disclosed herein can be combined with any and/or all other elements or limitations (individually or in any combination) or any other invention or embodiment thereof disclosed herein, and all such combinations are contemplated with the scope of the invention without limitation thereto. REFERENCES

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Claims

CLAIMS We claim:

1. A method for modulating plant photosynthetic capacity and/or biomass in a plant, said method comprising modulating the expression of an ADP-ribosylation factor (ARF) gene and/or expression or enzymatic activity of an ARF gene product in a cell of the plant.

2. The method according to claim 1, wherein photosynthetic capacity and/or biomass of the plant is increased.

3. The method according to claim 1 or 2, wherein leaf width and/or total area of a leaf is increased.

4. The method according to claim 1, wherein said ARF gene comprises a nucleotide sequence shown in SEQ ID NO:1.

5. The method according to claim 1, wherein said ARF gene comprises a nucleotide sequence shown in SEQ ID NO:3 or SEQ ID NO:4.

6. The method according to claim 1, wherein said ARF gene product comprises the amino acid sequence shown in SEQ ID NO:2.

7. The method according to claim 2, wherein expression is modulated via one or more mutations in said ARF gene that result in decreased or inhibited transcription of said ARF gene, or decreased or inhibited translation of ARF mRNA, or decreased or inhibited ARF enzymatic activity.

8. The method according to claim 7, wherein said one or more mutations is upstream of the transcription start site.

9. The method according to claim 7, wherein said mutations is downstream of the transcription start site.

10. The method according to claim 2, wherein expression is modulated via an antisense construct that provides a sequence that is antisense to an ARF polynucleotide sequence.

11. The method according to claim 2, wherein expression is modulated via a ribozyme that cleaves ARF RNA.

12. The method according to claim 2, wherein expression is modulated via an siRNA that targets an ARF polynucleotide.

13. The method according to claim 2, wherein expression is modulated by a protein or peptide that binds to and inhibits enzymatic function of said ARF gene product.

14. The method according to claim 13, wherein said protein or peptide is an antibody, or an antigen binding fragment thereof.

15. The method according to any of claims 1 to 14, wherein the plant is rice, wheat, barley, oats, rye, sorghum, maize, sugarcane, pineapple, onion, bananas, coconut, lilies, turfgrasses, millet, and monocot trees such as Agavaceae (Agave family), such as Cabbage tree {Cordyline australis), Dragon tree {Dracaena draco), Joshua tree {Yucca brevifolia); Arecaceae (Palmae) (Palm family), such as Areca Nut {Areca catechu), Coconut {Cocos nucifera). Date Palm {Phoenix dactylifera), Chusan Palm {Trachycarpus fortune!); or Poaceae (grass family), such as Bamboos Poaceae subfamily Bambusoideae.

16. The method according to any of claims 1 to 14, wherein the plant is tomato, cucumber, squash, peas, alfalfa, melon, chickpea, chicory, clover, kale, lentil, soybean, beans, tobacco, potato, sweet potato, yams, cassava, radish, broccoli, spinach, cabbage, rape, apple trees, citrus (including oranges, mandarins, grapefruit, lemons, limes and the like), grape, cotton, sunflower, strawberry, lettuce, and dicot trees such as Adoxaceae (Moschatel family), such as Moschatel (Adoxa mυschatellina), Elderberry {Sambucus species), Sinadoxa (Sinadoxa cυrydalifolia), Viburnum (Viburnum species); Altingiaceae (Sweetgum family), such as Sweetgum (Liquidambar species); Anacardiaceae (Cashew family), such as Cashew {Anacardium occidentale), Mango (Mangifera indica), Pistachio (Pistacia vera), Sumac (Rhus species), Lacquer tree (Toxicodendron verniciflua); Annonaceae (Custard apple family), such as Cherimoya (Annona cherimolά), Custard apple (Annona reticulata), Pawpaw (Asimina triloba), Soursop (Annona muricata); Apocynaceae (Dogbane family), such as Pachypodium (Pachypodium species); Aquifoliaceae (Holly family), such as Holly (Ilex species); Araliaceae (Ivy family), such as Kalopanax (Kalopanax pictus); Betulaceae (Birch family), such as Alder (Alnus species), Birch (Betula species), Hornbeam (Carpinus species), Hazel (Corylus species); Bignoniaceae (family), such as Catalpa (Calalpa species); Cactaceae (Cactus family), such as Saguaro (Carnegiea gigantea); Cannabaceae (Cannabis family), such as Hackberry (Celtis species); Cornaceae (Dogwood family), such as Dogwood (Cornus species); Dipterocarpaceae family, such as Garjan (Dipterocarpus species), Sal (Shorea species); Ebenaceae (Persimmon family), such as Persimmon (Diospyros species); Ericaceae (Heath family), such as Arbutus (Arbutus species); Eucommiaceae (Eucommia family), such as Eucommia (Eucommia ulmoides); Fabaceae (Pea family), such as Acacia (Acacia species), Honey locust (Gleditsia triacanthos), Black locust (Robinia pseudoacacia), Laburnum (Laburnum species), Pau Brasil, Brazilwood (Caesalpinia echinata); Fagaceae (Beech family), such as Chestnut (Castanea species), Beech (Fagus species), Southern beech (Nothofagus species), Tanoak (Lithocarpus densiflorus), Oak (Quercus species); Fouquieriaceae (Boojum family), such as Boojum (Fouquieria columnaris); Hamamelidaceae (Witch-hazel family), such as Persian Ironwood (Parrotia persica); Juglandaceae (Walnut family), such as Walnut (Juglans species), Hickory (Carya species), Wingnut (Pterocarya species); Lauraceae (Laurel family), such as Cinnamon (Cinnamomum zeylanicum), Bay Laurel (Laurus nobilis), Avocado (Persea americana); Lecythidaceae (Paradise nut family), such as Brazil Nut (Bertholletia excelsa); Lythraceae (Loosestrife family), such as Crape-myrtle (Lagerstroemia species); Magnoliaceae (Magnolia family), such as Tulip tree (Liriodendron species), Magnolia (Magnolia species); Malvaceae (Mallow family; including Tiliaceae, Sterculiaceae and Bombacaceae), such as Baobab (Adansonia species), Silk-cotton tree (Bombax species), Bottletrees {Brachychiton species), Kapok (Ceiba pentandra), Durian (Durio zibethinus), Balsa (Ochroma lagopus), Cacao (cocoa) (Theobroma cacao), Linden (Basswood, Lime) {Tilia species); Meliaceae (Mahogany family), such as Neem {Azadirachta indica), Bead tree (Melia a∑edarach), Mahogany (Swietenia mahagoni); Moraceae (Mulberry family), such as Fig (Ficus species), Mulberry (Morus species); such as Myristicaceae (Nutmeg family), such as Nutmeg {Mysristica fragrans); Myrtaceae (Myrtle family), such as Eucalyptus (Eucalyptus species), Myrtle (Myrtus species), Guava (Psidium guajava); Nyssaceae (Tupelo family; sometimes included in Cornaceae), such as Tupelo (Nyssa species), Dove tree (Davidia involucrata); Oleaceae (Olive family), such as Olive (Olea europaea), Ash (Fraxinus species); Paulowniaceae (Paulownia family), such as Foxglove Tree (Paulownia species); Platanaceae (Plane family), such as Plane (Platanus species); Rhizophoraceae (Mangrove family), such as Red Mangrove (Rhi∑ophora mangle); Rosaceae (Rose family), such as Rowans, Whitebeams, Service Trees (Sorbus species), Hawthorn (Crataegus species), Pear (Pyrus species), Apple (Mains species), Almond (Prunus dulcis), Peach (Prunus persica), Apricot (Prunus armeniaca), Plum (Prunus domestica), Cherry (Prunus species); Rubiaceae (Bedstraw family), such as Coffee (Coffea species); Rutaceae (Rue family), such as Citrus (Citrus species), Cork-tree (Phellodendron species), Euodia (Tetradium species); Salicaceae (Willow family), such as Aspen (Populus species), Poplar (Populus species), Willow (Salix species); Sapindaceae (including Aceraceae, Hippocastanaceae) (Soapberry family), such as Maple (Acer species), Buckeye, Horse-chestnut (Aesculus species), Mexican Buckeye (Ungnadia speciosa), Lychee (Litchi sinensis), Golden rain tree (Koelreuteria); Sapotaceae (Sapodilla family), such as Argan (Argania spinosa), Gutta-percha (Palaquium species), Tambalacoque, or "dodo tree" (Sideroxylon grandiflorum), previously Calvaria major, Simaroubaceae family, such as Tree of heaven (Ailanthus species); Theaceae (Camellia family), such as Gordonia (Gordonia species), Stewartia (Stewarlia species); Thymelaeaceae (Thymelaea family), such as Ramin (Gonystylus species); Ulmaceae (Elm family), such as Elm (Ulmus species), Zelkova (Zelkova species); or Verbenaceae family, such as Teak, Tectona species.

17. The method according to claim 1, wherein the plant is a species of the genus Populus.

18. The method according to claim 1, wherein said method comprises incorporating in said plant a polynucleotide that modulates expression or function of said ARF gene and/or said ARF gene product.

19. The method according to claim 18, wherein said polynucleotide is overexpressed or constitutively expressed in said plant.

20. The method according to claim 18, wherein said polynucleotide encodes an ARF gene product and said polynucleotide comprises a nucleotide sequence shown in SEQ ID NO:3, or said polynucleotide comprises in the about 500 bp upstream of the start codon one or more of the nucleotide changes shown in SEQ ID NO.3 as compared to the sequence in SEQ ID NO:4.

21. The method according to claim 18, wherein said polynucleotide provides for an antisense nucleotide sequence that is antisense to an ARF polynucleotide sequence, or said polynucleotide encodes a ribozyme that cleaves an ARF polynucleotide, or said polynucleotide provides for an siRNA that targets an ARF polynucleotide, whereby ARF gene expression and/or ARF enzymatic function is decreased or inhibited.

22. The method according to claim 18, wherein said polynucleotide encodes a protein or peptide that binds to and inhibits enzymatic function of an ARF gene product.

23. The method according to claim 22, wherein said protein or peptide is an antibody, or an antigen binding fragment thereof.

24. A plant having increased or decreased photosynthetic capacity and biomass, relative to a wild type plant, wherein said plant exhibits increased or decreased expression of an ARF gene or an ARF gene product relative to a wild type plant, or that expresses a mutant ARF polynucleotide or a mutant ARF enzyme that is characterized by increased or decreased expression or increased or decreased enzymatic activity or function.

25. The plant according to claim 24, wherein said plant has increased photosynthetic capacity and biomass.

26. The plant according to claim 24, wherein said plant is a transformed or transgenic plant.

27. The plant according to any of claims 24 to 26, wherein the plant is rice, wheat, barley, oats, rye, sorghum, maize, sugarcane, pineapple, onion, bananas, coconut, lilies, turfgrasses, millet, and monocot trees such as Agavaceae (Agave family), such as Cabbage tree {Cordyline australis), Dragon tree {Dracaena draco), Joshua tree {Yucca brevifolia); Arecaceae (Palmae) (Palm family), such as Areca Nut {Areca catechu), Coconut {Cocos nucifera), Date Palm {Phoenix dactylifera), Chusan Palm {Trachycarpus fortiinei); or Poaceae (grass family), such as Bamboos Poaceae subfamily Bambusoideae.

28. The plant according to any of claims 24 to 26, wherein the plant is tomato, cucumber, squash, peas, alfalfa, melon, chickpea, chicory, clover, kale, lentil, soybean, beans, tobacco, potato, sweet potato, yams, cassava, radish, broccoli, spinach, cabbage, rape, apple trees, citrus (including oranges, mandarins, grapefruit, lemons, limes and the like), grape, cotton, sunflower, strawberry, lettuce, and dicot trees such as Adoxaceae (Moschatel family), such as Moschatel {Adoxa moschatellina). Elderberry {Sambucus species), Sinadoxa {Sinadoxa corydalifoliά), Viburnum {Viburnum species); Altingiaceae (Sweetgum family), such as Sweetgum {Liquidambar species); Anacardiaceae (Cashew family), such as Cashew {Anacardium occidentale), Mango {Mangifera indica), Pistachio {Pistacia vera), Sumac {Rhus species), Lacquer tree {Toxicodendron verniciflua); Annonaceae (Custard apple family), such as Cherimoya {Annona cheήmola), Custard apple {Annona reticulata), Pawpaw (Asimina triloba), Soursop {Annona muricata); Apocynaceae (Dogbane family), such as Pachypodium {Pachypodium species); Aquifoliaceae (Holly family), such as Holly {Ilex species); Araliaceae (Ivy family), such as Kalopanax {Kalopanax pictus); Betulaceae (Birch family), such as Alder {Alnus species), Birch {Betula species), Hornbeam {Carpinus species), Hazel {Corylus species); Bignoniaceae (family), such as Catalpa {Catalpa species); Cactaceae (Cactus family), such as Saguaro {Carnegiea gigantea); Cannabaceae (Cannabis family), such as Hackberry {Celtis species); Cornaceae (Dogwood family), such as Dogwood (Cornus species); Dipterocarpaceae family, such as Garjan (Dipterocarpus species), Sal (Shorea species); Ebenaceae (Persimmon family), such as Persimmon (Diospyros species); Ericaceae (Heath family), such as Arbutus (Arbutus species); Eucommiaceae (Eucommia family), such as Eucommia (Eucommia ulmoides); Fabaceae (Pea family), such as Acacia (Acacia species), Honey locust (Gleditsia triacanthos), Black locust (Robinia pseudoacaciά), Laburnum (Laburnum species), Pau Brasil, Brazilwood (Caesalpinia echinata); Fagaceae (Beech family), such as Chestnut (Casianea species), Beech (Fagus species), Southern beech (Nothofagus species), Tanoak (Lithocarpus densiflorus), Oak (Quercus species); Fouquieriaceae (Boojum family), such as Boojum (Fouquieria columnaris); Hamamelidaceae (Witch-hazel family), such as Persian Ironwood (Parrotia persica); Juglandaceae (Walnut family), such as Walnut (Juglans species), Hickory (Carya species), Wingnut (Pterocarya species); Lauraceae (Laurel family), such as Cinnamon (Cinnamomum zeylanicum), Bay Laurel (Laurus nobilis), Avocado (Persea americana); Lecythidaceae (Paradise nut family), such as Brazil Nut (Bertholletia excelsa); Lythraceae (Loosestrife family), such as Crape-myrtle (Lagerstroemia species); Magnoliaceae (Magnolia family), such as Tulip tree (Liriodendron species), Magnolia (Magnolia species); Malvaceae (Mallow family; including Tiliaceae, Sterculiaceae and Bombacaceae), such as Baobab (Adansonia species), Silk-cotton tree (Bombax species), Bottletrees (Brachychiton species), Kapok (Ceiba pentandra), Durian (Durio zibethinus), Balsa (Ochroma lagopus), Cacao (cocoa) (Theobroma cacao), Linden (Basswood, Lime) (Tilia species); Meliaceae (Mahogany family), such as Neem (Azadirachta indica), Bead tree (Melia azedarach), Mahogany (Swietenia mahagoni); Moraceae (Mulberry family), such as Fig (Ficus species), Mulberry (Morus species); such as Myristicaceae (Nutmeg family), such as Nutmeg (Mysristica fragrans); Myrtaceae (Myrtle family), such as Eucalyptus (Eucalyptus species), Myrtle (Myrtus species), Guava (Psidium guajava); Nyssaceae (Tupelo family; sometimes included in Cornaceae), such as Tupelo (Nyssa species), Dove tree (Davidia involucrata); Oleaceae (Olive family), such as Olive (Olea europaea), Ash (Fraxinus species); Paulowniaceae (Paulownia family), such as Foxglove Tree (Paulownia species); Platanaceae (Plane family), such as Plane (Platanus species); Rhizophoraceae (Mangrove family), such as Red Mangrove (Rhizophora mangle); Rosaceae (Rose family), such as Rowans, Whitebeams, Service Trees (Sorbus species), Hawthorn {Crataegus species), Pear (Pyrus species), Apple {Malm species), Almond (Prunus dulcis), Peach (Prunus persica), Apricot (Prunus armeniaca), Plum (Prunus domestica), Cherry (Prunus species); Rubiaceae (Bedstraw family), such as Coffee (Coffea species); Rutaceae (Rue family), such as Citrus {Citrus species), Cork-tree {Phellodendron species), Euodia (Tetradium species); Salicaceae (Willow family), such as Aspen (Populus species), Poplar (Populus species), Willow (Salix species); Sapindaceae (including Aceraceae, Hippocastanaceae) (Soapberry family), such as Maple {Acer species), Buckeye, Horse-chestnut (Aesculus species), Mexican Buckeye {Ungnadia speciosa), Lychee (Litchi sinensis), Golden rain tree {Koelreuteria); Sapotaceae (Sapodilla family), such as Argan (Argania spinosa), Gutta-percha (Palaquium species), Tambalacoque, or "dodo tree" {Sideroxylon grandiflorum), previously Calvaria major; Simaroubaceae family, such as Tree of heaven (Ailanthus species); Theaceae (Camellia family), such as Gordonia (Gordonia species), Stewartia (Stewartia species); Thymelaeaceae (Thymelaea family), such as Ramin {Gonystylus species); Ulmaceae (Elm family), such as Elm (Ulmus species), Zelkova {Zelkova species); or Verbenaceae family, such as Teak, Tectona species.

29. The plant according to claim 24, wherein said plant is a species of Populus.

30. An isolated plant ARF gene or polynucleotide, wherein a plant expressing said ARF gene or polynucleotide exhibits decreased levels of ARF gene transcription, and/or decreased levels of ARF mRNA translation, and/or decreased levels of ARF enzyme and/or ARF enzymatic activity.

31. The isolated plant ARF gene or polynucleotide according to claim 30, wherein said ARF gene or polynucleotide comprises a nucleotide sequence shown in SEQ ID NO:1.

32. The isolated plant ARF gene or polynucleotide according to claim 30, wherein said ARF gene or polynucleotide comprises a nucleotide sequence shown in SEQ ID NO:3 or SEQ ID NO:4.

33. The isolated plant ARF gene or polynucleotide according to claim 30, wherein said ARF gene or polynucleotide encodes an ARF protein comprising the amino acid sequence shown in SEQ ID NO:2.

34. The isolated plant ARF gene or polynucleotide according to claim 30, wherein said ARF gene or polynucleotide comprises in the about 500 bp upstream of the start codon one or more of the nucleotide changes shown in SEQ ID NO:3 as compared to the sequence in SEQ ID NO:4.

35. The isolated plant ARF gene or polynucleotide according to claim 30, wherein said ARF gene or polynucleotide comprises one or more mutations in a transcription factor binding sequence, whereby transcription of said ARF gene or polynucleotide is decreased or inhibited.

36. The method according to claim 18, wherein said polynucleotide is an ARF gene or polynucleotide and wherein said ARF gene or polynucleotide comprises in the about 500 bp upstream of the start codon one or more of the nucleotide changes shown in SEQ ID NO:3 as compared to the sequence in SEQ ID NO:4.

37. The method according to claim 18, wherein said polynucleotide is an ARF gene or polynucleotide and said gene or polynucleotide comprises one or more mutations in a transcription factor binding sequence, whereby transcription of said ARF gene or polynucleotide is decreased or inhibited.

38. A plant obtained from a method of any of claims 1-23 or 36-37.