Classifications

• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
  • C12N15/09—Recombinant DNA-technology
  • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
  • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
  • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
  • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
  • C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/415—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from plants
• • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
  • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
  • Y02A40/00—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production
  • Y02A40/10—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production in agriculture
  • Y02A40/146—Genetically Modified [GMO] plants, e.g. transgenic plants

Definitions

• the present invention relates to isolated nucleic acid molecules and their corresponding encoded polypeptides able to modulate plant growth rate, vegetative growth, organ size, architecture seedling vigor and/or biomass in plants.
• the present invention further relates to using the nucleic acid molecules and polypeptides to make transgenic plants, plant cells, plant materials or seeds of a plant having modulated growth rate, vegetative growth, organ number, architecture, seedling vigor and/or biomass as compared to wild-type plants grown under similar conditions.
• Plants specifically improved for agriculture, horticulture, biomass conversion, and other industries can be obtained using molecular technologies.
• great agronomic value can result from modulating the size of a plant as a whole or of any of its organs or the number of any of its organs.
• modulation of the size and stature of an entire plant, or a particular portion of a plant, or growth rate, or seedling vigor allows production of plants better suited for a particular industry.
• reductions in the height of specific crops and tree species can be beneficial by allowing easier harvesting.
• increasing height, thickness or organ size, organ number may be beneficial by providing more biomass useful for processing into food, feed, fuels and/or chemicals (see the US Department of Energy website for Energy Efficiency and Renewable Energy).
• Other examples of commercially desirable traits include increasing the length of the floral stems of cut flowers, increasing or altering leaf size and shape or enhancing the size of seeds and/or fruits. Changes in organ size, organ number and biomass also result in changes in the mass of constituent molecules such as secondary products and convert the plants into factories for these compounds.
• the present invention is directed to advantageously manipulating plant size, organ number, plant growth rate, plant architecture and/or biomass to maximize the benefits of various crops depending on the benefit sought and the particular environment in which the crop must grow, characterized by expression of recombinant DNA molecules in plants.
• These molecules may be from the plant itself, and simply expressed at a higher or lower level, or the molecules may be from different plant species.
• the present invention therefore, relates to isolated nucleic acid molecules and polypeptides and their use in making transgenic plants, plant cells, plant materials or seeds of plants having life cycles, particularly plant size, vegetative growth, plant growth rate, organ number, plant architecture and/or biomass, that are altered with respect to wild-type plants grown under similar or identical conditions.
• the present invention discloses novel isolated nucleic acid molecules, nucleic acid molecules that interfere with these nucleic acid molecules, nucleic acid molecules that hybridize to these nucleic acid molecules, and isolated nucleic acid molecules that encode the same protein due to the degeneracy of the DNA code. Additional embodiments of the present application further include the polypeptides encoded by the isolated nucleic acid molecules of the present invention.
• the nucleic acid molecules of the present invention comprise: (a) a nucleotide sequence encoding an amino acid sequence that is at least 85% identical to any one of Leads 15, 28, 29, 36, ME04012 and Clone 691319, corresponding to SEQ ID Nos. 80, 90, 92, 98, 109, and 103, respectively, (b) a nucleotide sequence that is complementary to any one of the nucleotide sequences according to (a), (c) a nucleotide sequence according to any one of SEQ ID Nos.
• nucleotide sequence that is in reverse order of any one of the nucleotide sequences according to (c) when read in the 5' to 3' direction, (e) a nucleotide sequence able to interfere with any one of the nucleotide sequences according to (a), (f) a nucleotide sequence able to form a hybridized nucleic acid duplex with the nucleic acid according to any one of paragraphs (a) - (e) at a
• nucleic acid duplex (g) a nucleotide sequence encoding any one of amino acid sequences of Leads 15, 28, 29, 36, ME04012 and Clone 691319 corresponding to SEQ ID Nos. 81, 91, 93, 99, 110, and 104, respectively.
• Additional embodiments of the present invention include those polypeptide and nucleic acid molecule sequences disclosed in SEQ ID Nos. 80, 81, 90, 91, 92, 93, 98, 99, 109, 110, 103 and 104.
• the present invention further embodies a vector comprising a first nucleic acid having a nucleotide sequence encoding a plant transcription and/or translation signal, and a second nucleic acid having a nucleotide sequence according to the isolated nucleic acid molecules of the present invention.
• the first and second nucleic acids may be operably linked.
• the second nucleic acid may be endogenous to a first organism, and any other nucleic acid in the vector may be endogenous to a second organism.
• the first and second organisms may be different species.
• a host cell may comprise an isolated nucleic acid molecule according to the present invention. More particularly, the isolated nucleic acid molecule of the present invention found in the host cell of the present invention may be endogenous to a first organism and may be flanked by nucleotide sequences endogenous to a second organism. Further, the first and second organisms may be different species. Even more particularly, the host cell of the present invention may comprise a vector according to the present invention, which itself comprises nucleic acid molecules according to those of the present invention.
• the isolated polypeptides of the present invention may additionally comprise amino acid sequences that are at least 85% identical to any one of Leads 15, 28, 29, 36, ME04012 and Clone 691319, corresponding to SEQ ID Nos. 81, 91, 93, 99, 110, and 104, respectively.
• inventions include methods of introducing an isolated nucleic acid of the present invention into a host cell. More particularly, an isolated nucleic acid molecule of the present invention may be contacted to a host cell under conditions allowing transport of the isolated nucleic acid into the host cell. Even more particularly, a vector as described in a previous embodiment of the present invention, may be introduced into a host cell by the same method.
• Methods of detection are also available as embodiments of the present invention. Particularly, methods for detecting a nucleic acid molecule according to the present invention in a sample. More particularly, the isolated nucleic acid molecule according to the present invention may be contacted with a sample under conditions that permit a comparison of the nucleotide sequence of the isolated nucleic acid molecule with a nucleotide sequence of nucleic acid in the sample. The results of such an analysis may then be considered to determine whether the isolated nucleic acid molecule of the present invention is detectable and therefore present within the sample.
• a further embodiment of the present invention comprises a plant, plant cell, plant material or seeds of plants comprising an isolated nucleic acid molecule and/or vector of the present invention. More particularly, the isolated nucleic acid molecule of the present invention may be exogenous to the plant, plant cell, plant material or seed of a plant.
• a further embodiment of the present invention includes a plant regenerated from a plant cell or seed according to the present invention. More particularly, the plant, or plants derived from the plant, plant cell, plant material or seeds of a plant of the present invention preferably has increased size (in whole or in part), increased vegetative growth, increased organ number and/or increased biomass (sometimes hereinafter collectively referred to as increased biomass), lethality, sterility or ornamental characteristics as compared to a wild-type plant cultivated under identical conditions.
• the transgenic plant may comprise a first isolated nucleic acid molecule of the present invention, which encodes a protein involved in modulating growth and phenotype characteristics, and a second isolated nucleic acid molecule which encodes a promoter capable of driving expression in plants, wherein the growth and phenotype modulating component and the promoter are operably linked.
• the first isolated nucleic acid may be mis-expressed in the transgenic plant of the present invention, and the transgenic plant exhibits modulated characteristics as compared to a progenitor plant devoid of the gene, when the transgenic plant and the progenitor plant are cultivated under identical environmental conditions.
• the modulated growth and phenotype characteristics may be due to the inactivation of a particular sequence, using for example an interfering RNA.
• a further embodiment consists of a plant, plant cell, plant material or seed of a plant according to the present invention which comprises an isolated nucleic acid molecule of the present invention, wherein the plant, or plants derived from the plant, plant cell, plant material or seed of a plant, has the modulated growth and phenotype characteristics as compared to a wild-type plant cultivated under identical conditions.
• the polynucleotide conferring increased biomass or vigor may be mis- expressed in the transgenic plant of the present invention, and the transgenic plant exhibits an increased biomass or vigor as compared to a progenitor plant devoid of the polynucleotide, when the transgenic plant and the progenitor plant are cultivated under identical environmental conditions.
• increased biomass or vigor phenotype may be due to the inactivation of a particular sequence, using for example an interfering RNA.
• Another embodiment consists of a plant, plant cell, plant material or seed of a plant according to the present invention which comprises an isolated nucleic acid molecule of the present invention, wherein the plant, or plants derived from the plant, plant cell, plant material or seed of a plant, has increased biomass or vigor as compared to a wild- type plant cultivated under identical conditions.
• Another embodiment of the present invention includes methods of enhancing biomass or vigor in plants. More particularly, these methods comprise transforming a plant with an isolated nucleic acid molecule according to the present invention. Preferably, the method is a method of enhancing biomass or vigor in the transformed plant, whereby the plant is transformed with a nucleic acid molecule encoding the polypeptide of the present invention.
• Polypeptides of the present invention include consensus sequences.
• the consensus sequences are those as shown in Figures 1-5.
• Biomass refers to useful biological material including a product of interest, which material is to be collected and is intended for further processing to isolate or concentrate the product of interest.
• Biomass may comprise the fruit or parts of it or seeds, leaves, or stems or roots where these are the parts of the plant that are of particular interest for the industrial purpose.
• Biomass as it refers to plant material, includes any structure or structures of a plant that contain or represent the product of interest.
• Transformation Examples of means by which this can be accomplished are described below and include Agrobacterium-medi&ted transformation (of dicots (Needleman and Wunsch (1970) J MoI. Biol 48:443; Pearson and Lipman (1988) Proc. Natl. Acad. Sd. (USA) 85: 2444), of monocots (Yamauchi et al. (1996) Plant MoI Biol. 30:321- 9; Xu et al. (1995) Plant MoI. Biol. 27:237; Yamamoto et al. (1991) Plant Cell 3:371), and biolistic methods (P.
• Such a plant containing the exogenous nucleic acid is referred to here as a To for the primary transgenic plant and T 1 for the first generation.
• Functionally Comparable Proteins or Functional Homologs This term describes those proteins that have at least one functional characteristic in common. Such characteristics include sequence similarity, biochemical activity, transcriptional pattern similarity and phenotypic activity. Typically, the functionally comparable proteins share some sequence similarity or at least one biochemical. Within this definition, analogs are considered to be functionally comparable. In addition, functionally comparable proteins generally share at least one biochemical and/or phenotypic activity.
• Heterologous sequences are those that are not operatively linked or are not contiguous to each other in nature.
• a promoter from corn is considered heterologous to an Ar ⁇ bidopsis coding region sequence.
• a promoter from a gene encoding a growth factor from corn is considered heterologous to a sequence encoding the corn receptor for the growth factor.
• Regulatory element sequences such as UTRs or 3' end termination sequences that do not originate in nature from the same gene as the coding sequence, are considered heterologous to said coding sequence.
• Elements operatively linked in nature and contiguous to each other are not heterologous to each other.
• these same elements remain operatively linked but become heterologous if other filler sequence is placed between them.
• the promoter and coding sequences of a corn gene expressing an amino acid transporter are not heterologous to each other, but the promoter and coding sequence of a corn gene operatively linked in a novel manner are heterologous.
• misexpression refers to an increase or a decrease in the transcription of a coding region into a complementary RNA sequence as compared to the wild-type. This term also encompasses expression and/or translation of a gene or coding region or inhibition of such transcription and/or translation for a different time period as compared to the wild-type and/or from a non-natural location within the plant genome, including a gene or coding region from a different plant species or from a non-plant organism.
• Percentage of sequence identity refers to the degree of identity between any given query sequence and a subject sequence.
• a query nucleic acid or amino acid sequence is aligned to one or more subject nucleic acid or amino acid sequences using the computer program ClustalW (version 1.83, default parameters), which allows alignments of nucleic acid or protein sequences to be carried out across their entire length (global alignment).
• ClustalW calculates the best match between a query and one or more subject sequences, and aligns them so that identities, similarities and differences can be determined. Gaps of one or more residues can be inserted into a query sequence, a subject sequence, or both, to maximize sequence alignments.
• word size 2; window size: 4; scoring method: percentage; number of top diagonals: 4; and gap penalty: 5.
• gap opening penalty 10.0; gap extension penalty: 5.0; and weight transitions: yes.
• the output is a sequence alignment that reflects the relationship between sequences.
• ClustalW can be run, for example, at the Baylor College of Medicine Search Launcher website and at the European Bioinformatics Institute website on the World Wide Web.
• the alignment has to be along at least 80% of the length of the query sequence so that the majority of the query sequence is covered by the subject sequence.
• ClustalW divides the number of identities in the best alignment by the number of residues compared (gap positions are excluded), and multiplies the result by 100.
• the output is the percent identity of the subject sequence with respect to the query sequence. It is noted that the percent identity value can be rounded to the nearest tenth.
• 78.11, 78.12, 78.13, and 78.14 are rounded down to 78.1, while 78.15, 78.16, 78.17, 78.18, and 78.19 are rounded up to 78.2.
• regulatory region refers to nucleotide sequences that, when operably linked to a sequence, influence transcription initiation or translation initiation or transcription termination of said sequence and the rate of said processes, and/or stability and/or mobility of a transcription or translation product.
• operably linked refers to positioning of a regulatory region and said sequence to enable said influence.
• Regulatory regions include, without limitation, promoter sequences, enhancer sequences, response elements, protein recognition sites, inducible elements, protein binding sequences, 5' and 3' untranslated regions (UTRs), transcriptional start sites, termination sequences, polyadenylation sequences, and introns. Regulatory regions can be classified in two categories, promoters and other regulatory regions.
• Seedling vigor refers to the plant characteristic whereby the plant emerges from soil faster, has an increased germination rate (i.e., germinates faster), has faster and larger seedling growth and/or germinates faster under cold conditions as compared to the wild type or control under similar conditions. Seedling vigor has often been defined to comprise the seed properties that determine "the potential for rapid, uniform emergence and development of normal seedlings under a wide range of field conditions".
• Stringency is a function of nucleic acid molecule probe length, nucleic acid molecule probe composition (G + C content), salt concentration, organic solvent concentration and temperature of hybridization and/or wash conditions. Stringency is typically measured by the parameter T n , which is the temperature at which 50% of the complementary nucleic acid molecules in the hybridization assay are hybridized, in terms of a temperature differential from T m . High stringency conditions are
• N is the number of nucleotides of the nucleic acid molecule probe. This equation works well for probes 14 to 70 nucleotides in length that are identical to the target sequence.
• the equation below, for T m of DNA-DNA hybrids, is useful for probes having lengths in the range of 50 to greater than 500 nucleotides, and for conditions that include an organic solvent (formamide):
• T m 81.5+16.6 log ([Na + ]Z(HOJ[Na + ]))+ 0.41(%G+C)-500/L 0.63(%formamide)
• Equation II is affected by the nature of the hybrid: for DNA-RNA hybrids, T m is 10-15 0 C
• T m is 20-25 0 C higher. Because the T m
• Equation II is derived assuming the reaction is at equilibrium.
• hybridizations according to the present invention are most preferably performed under conditions of probe excess and allowing sufficient time to achieve equilibrium.
• the time required to reach equilibrium can be shortened by using a hybridization buffer that includes a hybridization accelerator such as dextran sulfate or another high volume polymer.
• Stringency can be controlled during the hybridization reaction, or after hybridization has occurred, by altering the salt and temperature conditions of the wash solutions.
• the formulas shown above are equally valid when used to compute the stringency of a wash solution.
• Preferred wash solution stringencies lie within the ranges stated above;
• high stringency is 5-8°C below T m
• medium or moderate stringency is 26-29°C below T m
• low stringency is 45-48°C below T m .
• T 0 refers to the whole plant, explant or callus tissue, inoculated with the transformation medium.
• T 1 refers to either the progeny of the T 0 plant, in the case of whole-plant transformation, or the regenerated seedling in the case of explant or callous tissue transformation.
• T 2 refers to the progeny of the T 1 plant. T 2 progeny are the result of self-fertilization or cross-pollination of a T 1 plant.
• T 3 refers to second generation progeny of the plant that is the direct result of a transformation experiment. T 3 progeny are the result of self-fertilization or cross-pollination of a T 2 plant 3.
• nucleic acid molecules and polypeptides of the present invention are of interest because when the nucleic acid molecules are mis-expressed (i.e., when expressed at a non-natural location or in an increased or decreased amount relative to wild- type) they produce plants that exhibit modulated biomass, growth rate, or seedling vigor as compared to wild-type plants, as evidenced by the results of various experiments disclosed below. This trait can be used to exploit or maximize plant products.
• the nucleic acid molecules and polypeptides of the present invention are used to increase the expression of genes that cause the plant to have modulated biomass, growth rate or seedling vigor.
• the disclosed sequences and methods increase vegetative growth, and growth rate
• the disclosed methods can be used to enhance biomass production.
• plants that grow vegetatively have an increase biomass production, compared to a plant of the same species that is not genetically modified for substantial vegetative growth.
• increases in biomass production include increases of at least 5%, at least 20%, or even at least 50%, when compared to an amount of biomass production by a plant of the same species not growing vegetatively.
• the sequence of Lead 36 of the present invention and its functional homologs in particular provide transformed plants with enhanced yield, including fruit yield and yield per acre, somewhat early maturity, and a more compact stature (20%, 30%, 40% or 60% more compact) with shorter stems, but without proportionally reduced biomass. In tomatoes, this results in plants with increased fruit yield on more compact plants, hi rice, this results in plants with an increase number of tillers.
• the sequence of Lead 29 of the present invention and its functional homologs in particular provide transformed plants with enhanced yield, including fruit yield and yield per acre, somewhat early maturity, and a more compact stature (20%, 30%, 40% or 60% more compact) with shorter stems. In tomatoes, this results in plants with increased fruit yield on more compact plants.
• the life cycle of flowering plants in general can be divided into three growth phases: vegetative, inflorescence, and floral (late inflorescence phase).
• vegetative phase the shoot apical meristem (SAM) generates leaves that later will ensure the resources necessary to produce fertile offspring.
• SAM shoot apical meristem
• the plant switches to floral, or reproductive, growth and the SAM enters the inflorescence phase (I) and gives rise to an inflorescence with flower primordia.
• I inflorescence phase
• the fate of the SAM and the secondary shoots that arise in the axils of the leaves is determined by a set of meristem identity genes, some of which prevent and some of which promote the development of floral meristems.
• the plant Once established, the plant enters the late inflorescence phase (Xu et al. (1995) Plant MoI. Biol. 27:237) where the floral organs are produced. If the appropriate environmental and developmental signals the plant switches to floral, or reproductive, growth are disrupted, the plant will not be able to enter reproductive growth, therefore maintaining vegetative growth.
• Seed or seedling vigor is an important characteristic that can greatly influence successful growth of a plant, such as crop plants.
• Adverse environmental conditions such as dry, wet, cold or hot conditions, can affect a plant growth cycle, and the vigor of seeds (i.e. vitality and strength under such conditions can differentiate between successful and failed crop growth).
• Seedling vigor has often been defined to comprise the seed properties that determine "the potential for rapid, uniform emergence and development of normal seedlings under a wide range of field conditions". Hence, it would be advantageous to develop plant seeds with increased vigor.
• increased seedling vigor would be advantageous for cereal plants such as rice, maize, wheat, etc. production.
• growth can often be slowed or stopped by cool environmental temperatures during the planting season.
• rapid emergence and tillering of rice would permit growers to initiate earlier flood irrigation which can save water and suppress weak growth.
• Genes associated with increased seed vigor and/or cold tolerance in rice have therefore been sought for producing improve rice varieties. See e.g., Pinson, S., "Molecular Mapping of Seedling Vigor QTLs in Tropical Rice", USDA Agricultural Research Service, December 16, 2000.
• Seedling vigor has been measured by different tests and assays, including most typically a cold tolerance test and an accelerated aging test.
• Some of the nucleotide sequences of the invention code for basic-helix- loop (bHCH) transcription factors. It is known that transcription factors often control the expression of multiple genes hi a pathway.
• the basic/helix-loop-helix (BHLH) proteins are a superfamily of transcription factors that bind as dimers to specific DNA target sites.
• the bHLH transcription factors have been well characterized hi nonplant eukaryotes and have been identified as important regulatory components in diverse biological processes. Many different functions have been identified for those proteins hi animals, including the control of cell proliferation and transcription often involves homo- or hetero-dimerization.
• Members of the R/B basic helix-loop-helix (bHLH) family of plant transcription factors are involved in a variety of growth and differentiation processes.
• a basic-helix-loop-helix is a protein structural motif that characterizes a family of transcription factors.
• the motif is characterized by two ⁇ helices connected by a loop. Transcription factors of this type are typically dimeric, each with one helix containing basic amino acid residues that facilitate DNA binding.
• One helix is typically smaller and due to the flexibility of the loop allows dimerization by folding and packing against another helix.
• the larger helix typically contains the DNA binding regions.
• bHLH proteins typically bind to a consensus sequence called an E-box, CANNTG.
• the canonical E- box is CACGTG, however some bHLH transcription factors bind to different sequences, which are often similar to the E-box.
• bHLH transcription factors are often important hi development or cell activity.
• polypeptides of the present invention and the proteins expressed via translation of these polynucleotides are set forth in the Sequence Listing, specifically SEQ ID NOS. 80, 81, 90, 91, 92, 93, 98, 99, 109, 110, 103, and 104.
• the Sequence Listing also consists of functionally comparable proteins.
• Polypeptides comprised of a sequence within and defined by one of the consensus sequences can be utilized for the purposes of the invention, namely to make transgenic plants with modulated biomass, growth rate and/or seedling vigor.
• recombinant DNA constructs are prepared that comprise the polynucleotide sequences of the invention inserted into a vector and that are suitable for transformation of plant cells.
• the construct can be made using standard recombinant DNA techniques (see, Sambrook et al., Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press, 1989, New York.) and can be introduced into the plant species of interest by, for example, Agrobacterium-medi&ted transformation, or by other means of transformation, for example, as disclosed below.
• the vector backbone may be any of those typically used in the field such as plasmids, viruses, artificial chromosomes, BACs, YACs, PACs and vectors such as, for instance, bacteria-yeast shuttle vectors, lambda phage vectors, T-DNA fusion vectors and plasmid vectors (see, Shizuya et al. (1992) Proc. Natl. Acad. Sd. USA, 89: 8794-8797; Hamilton et al. (1996) Proc. Natl. Acad. Sd. USA, 93: 9975-9979; Burke et al. (1987) Science, 236:806-812 ; Sternberg N. et al.
• the construct comprises a vector containing a nucleic acid molecule of the present invention with any desired transcriptional and/or translational regulatory sequences such as, for example, promoters, UTRs, and 3' end termination sequences.
• Vectors may also include, for example, origins of replication, scaffold attachment regions (SARs), markers, homologous sequences, and introns.
• the vector may also comprise a marker gene that confers a selectable phenotype on plant cells.
• the marker may preferably encode a biocide resistance trait, particularly antibiotic resistance, such as resistance to, for example, kanamycin, bleomycin, or hygromycin, or herbicide resistance, such as resistance to, for example, glyphosate, chlorosulfuron or phosphinotricin.
• antibiotic resistance such as resistance to, for example, kanamycin, bleomycin, or hygromycin
• herbicide resistance such as resistance to, for example, glyphosate, chlorosulfuron or phosphinotricin.
• a regulatory region may be present in a recombinant polynucleotide, e.g., introns, enhancers, upstream activation regions, transcription terminators, and inducible elements. Thus, more than one regulatory region can be operably linked to said sequence.
• the translation initiation site of the translational reading frame of said sequence is typically positioned between one and about fifty nucleotides downstream of the promoter.
• a promoter can, however, be positioned as much as about 5,000 nucleotides upstream of the translation initiation site, or about 2,000 nucleotides upstream of the transcription start site.
• a promoter typically comprises at least a core (basal) promoter.
• a promoter also may include at least one control element, such as an enhancer sequence, an upstream element or an upstream activation region (UAR).
• a suitable enhancer is a cis-regulatory element (-212 to -154) from the upstream region of the octopine synthase (ocs) gene. Fromm et ah, The Plant Cell 1:977-984 (1989).
• Basal promoter is the minimal sequence necessary for assembly of a transcription complex required for transcription initiation. Basal promoters frequently include a "TATA box” element that may be located between about 15 and about 35 nucleotides upstream from the site of transcription initiation. Basal promoters also may include a "CCAAT box” element (typically the sequence CCAAT) and/or a GGGCG sequence, which can be located between about 40 and about 200 nucleotides, typically about 60 to about 120 nucleotides, upstream from the transcription start site.
• TATA box typically the sequence CCAAT
• GGGCG sequence typically the sequence CCAAT
• promoters The choice of promoters to be included depends upon several factors, including, but not limited to, efficiency, selectability, inducibility, desired expression level, and cell- or tissue-preferential expression. It is a routine matter for one of skill in the art to modulate the expression of a sequence by appropriately selecting and positioning promoters and other regulatory regions relative to said sequence.
• Suitable promoters initiate transcription only, or predominantly, in certain cell types.
• a promoter that is active predominantly in a reproductive tissue e.g., fruit, ovule, pollen, pistils, female gametophyte, egg cell, central cell, nucellus, suspensor, synergid cell, flowers, embryonic tissue, embryo sac, embryo, 2ygote, endosperm, integument, or seed coat
• a cell type- or tissue-preferential promoter is one that drives expression preferentially in the target tissue, but may also lead to some expression in other cell types or tissues as well.
• Methods for identifying and characterizing promoter regions in plant genomic DNA include, for example, those described in the following references: Jordano, et al, Plant Cell, 1:855-866 (1989); Bustos, et al, Plant Cell, 1:839-854 (1989); Green, et al, EMBO J. 7, 4035-4044 (1988); Meier, et al, Plant Cell, 3, 309-316 (1991); and Zhang, et al, Plant Physiology 110: 1069-1079 (1996).
• promoters examples include various classes of promoters. Some of the promoters indicated below are described in more detail in U.S. Patent Application Ser. Nos. 60/505,689; 60/518,075; 60/544,771; 60/558,869; 60/583,691; 60/619,181; 60/637,140; 10/950,321; 10/957,569; 11/058,689; 11/172,703; 11/208,308; and PCT/US05/23639. It will be appreciated that a promoter may meet criteria for one classification based on its activity in one plant species, and yet meet criteria for a different classification based on its activity in another plant species.
• a 5' untranslated region can be included in nucleic acid constructs described herein.
• a 5' UTR is transcribed, but is not translated, and lies between the start site of the transcript and the translation initiation codon and may include the +1 nucleotide.
• a 3' UTR can be positioned between the translation termination codon and the end of the transcript.
• UTRs can have particular functions such as increasing mRNA stability or attenuating translation. Examples of 3' UTRs include, but are not limited to, polyadenylation signals and transcription termination sequences, e.g., a nopaline synthase termination sequence.
• Various promoters can be used to drive expression of the genes of the present invention. Nucleotide sequences of such promoters are set forth in SEQ ID NOS: 1-79. Some of them can be broadly expressing promoters, others may be more tissue preferential.
• a promoter can be said to be "broadly expressing” when it promotes transcription in many, but not necessarily all, plant tissues or plant cells.
• a broadly expressing promoter can promote transcription of an operably linked sequence in one or more of the shoot, shoot tip (apex), and leaves, but weakly or not at all in tissues such as roots or stems.
• a broadly expressing promoter can promote transcription of an operably linked sequence in one or more of the stem, shoot, shoot tip (apex), and leaves, but can promote transcription weakly or not at all in tissues such as reproductive tissues of flowers and developing seeds.
• Non-limiting examples of broadly expressing promoters that can be included in the nucleic acid constructs provided herein include the p326 (SEQ ID NO: 76), YP0144 (SEQ ID NO: 55), YP0190 (SEQ ID NO: 59), pl3879 (SEQ ID NO: 75), YP0050 (SEQ ID NO: 35), p32449 (SEQ ID NO: 77), 21876 (SEQ ID NO: 1), YP0158 (SEQ ID NO: 57), YP0214 (SEQ ID NO: 61), YP0380 (SEQ ID NO: 70), PT0848 (SEQ ID NO: 26), and PT0633 (SEQ ID NO: 7).
• CaMV 35S promoter the cauliflower mosaic virus (CaMV) 35S promoter
• MAS mannopine synthase
• figwort mosaic virus 34S promoter actin promoters such as the rice actin promoter
• ubiquitin promoters such as the maize ubiquitin- 1 promoter.
• the CaMV 35S promoter is excluded from the category of broadly expressing promoters.
• Root-active promoters drive transcription in root tissue, e.g., root endodermis, root epidermis, or root vascular tissues.
• root-active promoters are root-preferential promoters, i.e., drive transcription only or predominantly in root tissue.
• Root-preferential promoters include the YP0128 (SEQ ID NO: 52), YP0275 (SEQ ID NO: 63), PT0625 (SEQ ID NO: 6), PT0660 (SEQ ID NO: 9), PT0683 (SEQ ID NO: 14), and PT0758 (SEQ ID NO: 22).
• root-preferential promoters include the PT0613 (SEQ ID NO: 5), PT0672 (SEQ ID NO: 11), PT0688 (SEQ ID NO: 15), and PT0837 (SEQ ID NO: 24), which drive transcription primarily in root tissue and to a lesser extent in ovules and/or seeds.
• Other examples of root-preferential promoters include the root-specific subdomains of the CaMV 35S promoter (Lam et ah, Proc. Natl. Acad. ScL USA 86:7890- 7894 (1989)), root cell specific promoters reported by Conkling et ah, Plant Physiol. 93:1203-1211 (1990), and the tobacco RD2 gene promoter.
• promoters that drive transcription in maturing endosperm can be useful. Transcription from a maturing endosperm promoter typically begins after fertilization and occurs primarily in endosperm tissue during seed development and is typically highest during the cellularization phase. Most suitable are promoters that are active predominantly in maturing endosperm, although promoters that are also active in other tissues can sometimes be used.
• Non-limiting examples of maturing endosperm promoters that can be included in the nucleic acid constructs provided herein include the napin promoter, the Arcelin-5 promoter, the phaseolin gene promoter (Bustos et a (1989) Plant Cell l(9):839-853), the soybean trypsin inhibitor promoter (Riggs et a (1989) Plant Cell l(6):609-621), the ACP promoter (Baerson et a (1993) Plant MoI Biol, 22(2):255-267), the stearoyl-ACP desaturase gene (Slocombe et a (1994) Plant Physiol 104(4): 167- 176), the soybean ⁇ ' subunit of ⁇ -conglycinin promoter (Chen et a (1986) Proc Natl Acad Sd USA 83:8560-8564), the oleosin promoter (Hong et a (1997) Plant MoI Biol 34(3):
• Osgt-1 promoter from the rice glutelin-1 gene (Zheng et a (1993) MoI. Cell Biol. 13:5829-5842), the beta-amylase gene promoter, and the barley hordein gene promoter.
• Other maturing endosperm promoters include the YP0092 (SEQ ID NO: 38), PT0676 (SEQ ID NO: 12), and PT0708 (SEQ ID NO: 17).
• Promoters that drive transcription in ovary tissues such as the ovule wall and mesocarp can also be useful, e.g., a polygalacturonidase promoter, the banana TRX promoter, and the melon actin promoter.
• promoters that drive gene expression preferentially in ovules are YP0007 (SEQ ID NO: 30), YPOlIl (SEQ ID NO: 46), YP0092 (SEQ ID NO: 38), YP0103 (SEQ ID NO: 43), YP0028 (SEQ ID NO: 33), YP0121 (SEQ ID NO: 51), YP0008 (SEQ ID NO: 31), YP0039 (SEQ ID NO: 34), YPOl 15 (SEQ ID NO: 47), YPOl 19 (SEQ ID NO: 49), YP0120 (SEQ ID NO: 50) and YP0374 (SEQ ID NO: 68).
• embryo sac/early endosperm promoters can be used in order drive transcription of the sequence of interest in polar nuclei and/or the central cell, or in precursors to polar nuclei, but not in egg cells or precursors to egg cells. Most suitable are promoters that drive expression only or predominantly in polar nuclei or precursors thereto and/or the central cell.
• a pattern of transcription that extends from polar nuclei into early endosperm development can also be found with embryo sac/early endosperm-preferential promoters, although transcription typically decreases significantly in later endosperm development during and after the cellularization phase. Expression in the zygote or developing embryo typically is not present with embryo sac/early endosperm promoters.
• Promoters that may be suitable include those derived from the following genes: Arabidopsis viviparous-1 (see, GenBankNo. U93215); Arabidopsis atmycl (see, Urao (1996) Plant MoL Biol, 32:571-57; Conceicao (1994) Plant, 5:493-505); Arabidopsis FIE (GenBank No. AF129516); Arabidopsis MEA; Arabidopsis FIS2 (GenBank No. AF096096); and FIE 1.1 (U.S. Patent 6,906,244).
• Arabidopsis viviparous-1 see, GenBankNo. U93215
• Arabidopsis atmycl see, Urao (1996) Plant MoL Biol, 32:571-57; Conceicao (1994) Plant, 5:493-505
• Arabidopsis FIE GeneBank No. AF129516
• Arabidopsis MEA Arabidopsis FIS2
• FIE 1.1
• promoters that may be suitable include those derived from the following genes: maize MACl (see, Sheridan (1996) Genetics, 142:1009- 1020); maize Cat3 (see, GenBank No. L05934; Abler (1993) Plant MoI. Biol, 22:10131- 1038).
• promoters include the following Arabidopsis promoters: YP0039 (SEQ ID NO: 34), YPOlOl (SEQ ID NO: 41), YP0102 (SEQ ID NO: 42), YPOIlO (SEQ ID NO: 45), YPOl 17 (SEQ ID NO: 48), YPOl 19 (SEQ ID NO: 49), YP0137 (SEQ ID NO: 53), DME, YP0285 (SEQ ID NO: 64), and YP0212 (SEQ ID NO: 60).
• Other promoters that may be useful include the following rice promoters: p530cl0, pOsFIE2-2, pOsMEA, pOsYpl02, and pOsYp285.
• Promoters that preferentially drive transcription in zygotic cells following fertilization can provide embryo-preferential expression and may be useful for the present invention. Most suitable are promoters that preferentially drive transcription in early stage embryos prior to the heart stage, but expression in late stage and maturing embryos is also suitable.
• Embryo-preferential promoters include the barley lipid transfer protein (Ltpl) promoter ⁇ Plant Cell Rep (2001) 20:647-654, YP0097 (SEQ ID NO: 40), YP0107 (SEQ ID NO: 44), YP0088 (SEQ ID NO: 37), YP0143 (SEQ ID NO: 54), YP0156 (SEQ ID NO: 56), PT0650 (SEQ ID NO: 8), PT0695 (SEQ ID NO: 16), PT0723 (SEQ ID NO: 19), PT0838 (SEQ ID NO: 25), PT0879 (SEQ ID NO: 28) and PT0740 (SEQ ID NO: 20).
• Ltpl barley lipid transfer protein
• Promoters active in photosynthetic tissue in order to drive transcription in green tissues such as leaves and stems are of particular interest for the present invention. Most suitable are promoters that drive expression only or predominantly such tissues. Examples of such promoters include the ribulose-l,5-bisphosphate carboxylase (RbcS) promoters such as the RbcS promoter from eastern larch ⁇ Larix laricin ⁇ ), the pine cab6 promoter (Yamamoto et al. (1994) Plant Cell Physiol. 35:773-778), the Cab-1 gene promoter from wheat (Fejes et al. (1990) Plant MoI. Biol.
• RbcS ribulose-l,5-bisphosphate carboxylase
• promoters that drive transcription in stems, leafs and green tissue are PT0535 (SEQ ID NO: 3), PT0668 (SEQ ID NO: 2), PT0886 (SEQ ID NO: 29), PR0924 (SEQ ID NO: 78), YP0144 (SEQ ID NO: 55), YP0380 (SEQ ID NO: 70) and PT0585 (SEQ ID NO: 4).
• inducible promoters may be desired.
• Inducible promoters drive transcription in response to external stimuli such as chemical agents or environmental stimuli.
• external stimuli such as chemical agents or environmental stimuli.
• inducible promoters can confer transcription hi response to hormones such as giberellic acid or ethylene, or in response to light or drought.
• drought inedible promoters are YP0380 (SEQ ID NO: 70), PT0848 (SEQ ID NO: 26), YP0381 (SEQ ID NO: 71), YP0337 (SEQ ID NO: 66), YP0337 (SEQ ID NO: 66), PT0633 (SEQ ID NO: 7), YP0374 (SEQ ID NO: 68), PT0710 (SEQ ID NO: 18), YP0356 (SEQ ID NO: 67), YP0385 (SEQ ID NO: 73), YP0396 (SEQ ID NO: 74), YP0384 (SEQ ID NO: 72), YP0384 (SEQ ID NO: 72), PT0688 (SEQ ID NO: 15), YP0286 (SEQ ID NO: 65), YP0377 (SEQ ID NO: 69), and PD1367 (SEQ ID NO: 79).
• promoters induced by nitrogen are PT0863 (SEQ ID NO: 27), PT0829 (SEQ ID NO: 23), PT0665 (SEQ ID NO: 10) and PT0886 (SEQ ID NO: 29).
• An example of a shade inducible promoter is PR0924 (SEQ ID NO: 78).
• Promoters include, but are not limited to, leaf-preferential, stem/shoot-preferential, callus-preferential, guard cell-preferential, such as PT0678 (SEQ ID NO: 13), and senescence-preferential promoters.
• misexpression can be accomplished using a two component system, whereby the first component consists of a transgenic plant comprising a transcriptional activator operatively linked to a promoter and the second component consists of a transgenic plant that comprise a nucleic acid molecule of the invention operatively linked to the target- binding sequence/region of the transcriptional activator.
• the two transgenic plants are crossed and the nucleic acid molecule of the invention is expressed in the progeny of the plant.
• the misexpression can be accomplished by having the sequences of the two component system transformed in one transgenic plant line.
• Another alternative consists in inhibiting expression of a biomass or vigor-modulating polypeptide in a plant species of interest.
• expression refers to the process of converting genetic information encoded in a polynucleotide into RNA through transcription of the polynucleotide (i.e., via the enzymatic action of an RNA polymerase), and into protein, through translation of mRNA.
• Up-regulation or “activation” refers to regulation that increases the production of expression products relative to basal or native states
• down-regulation or “repression” refers to regulation that decreases production relative to basal or native states.
• a number of nucleic-acid based methods including anti-sense RNA, ribozyme directed RNA cleavage, and interfering RNA (RNAi) can be used to inhibit protein expression in plants.
• Antisense technology is one well-known method. In this method, a nucleic acid segment from the endogenous gene is cloned and operably linked to a promoter so that the antisense strand of RNA is transcribed. The recombinant vector is then transformed into plants, as described above, and the antisense strand of RNA is produced.
• the nucleic acid segment need not be the entire sequence of the endogenous gene to be repressed, but typically will be substantially identical to at least a portion of the endogenous gene to be repressed. Generally, higher homology can be used to compensate for the use of a shorter sequence. Typically, a sequence of at least 30 nucleotides is used (e.g., at least 40, 50, 80, 100, 200, 500 nucleotides or more).
• an isolated nucleic acid provided herein can be an antisense nucleic acid to one of the aforementioned nucleic acids encoding a biomass- modulating polypeptide.
• a nucleic acid that decreases the level of a transcription or translation product of a gene encoding a biomass-modulating polypeptide is transcribed into an antisense nucleic acid similar or identical to the sense coding sequence of the biomass- or growth rate-modulating polypeptide.
• the transcription product of an isolated nucleic acid can be similar or identical to the sense coding sequence of a biomass growth rate-modulating polypeptide, but is an RNA that is unpolyadenylated, lacks a 5' cap structure, or contains an unsplicable intron.
• a nucleic acid in another method, can be transcribed into a ribozyme, or catalytic RNA, that affects expression of an mRNA.
• Ribozymes can be designed to specifically pair with virtually any target RNA and cleave the phosphodiester backbone at a specific location, thereby functionally inactivating the target RNA.
• Heterologous nucleic acids can encode ribozymes designed to cleave particular mRNA transcripts, thus preventing expression of a polypeptide.
• Hammerhead ribozymes are useful for destroying particular mRNAs, although various ribozymes that cleave mRNA at site-specific recognition sequences can be used.
• Hammerhead ribozymes cleave mRNAs at locations dictated by flanking regions that form complementary base pairs with the target mRNA. The sole requirement is that the target RNA contain a 5 f -UG-3' nucleotide sequence.
• the construction and production of hammerhead ribozymes is known in the art. See, for example, U.S. Patent No. 5,254,678 and WO 02/46449 and references cited therein.
• Hammerhead ribozyme sequences can be embedded in a stable RNA such as a transfer RNA (tRNA) to increase cleavage efficiency in vivo.
• tRNA transfer RNA
• RNA endoribonucleases such as the one that occurs naturally hi Tetrahymena thermophila, and which have been described extensively by Cech and collaborators can be useful. See, for example, U.S. Patent No. 4,987,071.
• RNA interference is a cellular mechanism to regulate the expression of genes and the replication of viruses. This mechanism is thought to be mediated by double-stranded small interfering RNA molecules. A cell responds to such a double-stranded RNA by destroying endogenous mRNA having the same sequence as the double-stranded RNA.
• Methods for designing and preparing interfering RNAs are known to those of skill in the art; see, e.g., WO 99/32619 and WO 01/75164. For example, a construct can be prepared that includes a sequence that is transcribed into an interfering RNA.
• Such an RNA can be one that can anneal to itself, e.g., a double stranded RNA having a stem-loop structure.
• One strand of the stem portion of a double stranded RNA comprises a sequence that is similar or identical to the sense coding sequence of the polypeptide of interest, and that is from about 10 nucleotides to about 2,500 nucleotides hi length.
• the length of the sequence that is similar or identical to the sense coding sequence can be from 10 nucleotides to 500 nucleotides, from 15 nucleotides to 300 nucleotides, from 20 nucleotides to 100 nucleotides, or from 25 nucleotides to 100 nucleotides.
• the other strand of the stem portion of a double stranded RNA comprises an antisense sequence of the biomass-modulating polypeptide of interest, and can have a length that is shorter, the same as, or longer than the corresponding length of the sense sequence.
• the loop portion of a double stranded RNA can be from 10 nucleotides to 5,000 nucleotides, e.g., from 15 nucleotides to 1,000 nucleotides, from 20 nucleotides to 500 nucleotides, or from 25 nucleotides to 200 nucleotides.
• the loop portion of the RNA can include an intron. See, e.g., WO 99/53050.
• a suitable nucleic acid can be a nucleic acid analog.
• Nucleic acid analogs can be modified at the base moiety, sugar moiety, or phosphate backbone to improve, for example, stability, hybridization, or solubility of the nucleic acid. Modifications at the base moiety include deoxyuridine for deoxythymidine, and 5-methyl-2'-deoxycytidine and 5-bromo-2'- deoxycytidine for deoxycytidine. Modifications of the sugar moiety include modification of the 2' hydroxyl of the ribose sugar to form 2'-O-methyl or 2'-O-allyl sugars.
• the deoxyribose phosphate backbone can be modified to produce morpholino nucleic acids, in which each base moiety is linked to a six-membered morpholino ring, or peptide nucleic acids, in which the deoxyphosphate backbone is replaced by a pseudopeptide backbone and the four bases are retained. See, for example, Summerton and Weller, 1997, Antisense Nucleic Acid Drug Dev., 7:187-195; Hyrup et ah, 1996, Bioorgan. Med. Chem., 4: 5-23.
• the deoxyphosphate backbone can be replaced with, for example, a phosphorothioate or phosphorodithioate backbone, a phosphoroamidite, or an alkyl phosphotriester backbone.
• Nucleic acid molecules of the present invention may be introduced into the genome or the cell of the appropriate host plant by a variety of techniques. These techniques, able to transform a wide variety of higher plant species, are well known and described in the technical and scientific literature (see, e.g., Weising et al. (1988) Ann. Rev. Genet, 22:421 and Christou (1995) Euphytica, 85:13-27).
• a variety of techniques known in the art are available for the introduction of DNA into a plant host cell. These techniques include transformation of plant cells by injection (Newell (2000)), microinjection (Griesbach (1987) Plant ScI 50:69-77), electroporation of DNA (Fromm et al. (1985) Proc. Natl. Acad. Sd. USA 82:5824), PEG (Paszkowski et al. (1984) EMBOJ. 3:2717), use of biolistics (Klein et al. (1987) Nature 327:773), fusion of cells or protoplasts (Willmitzer, L. (1993) Transgenic Plants. In: Iotechnology, A Multi-Volume Comprehensive treatise (HJ.
• non-stable transformation methods that are well known to those skilled in the art may be desirable for the present invention.
• Such methods include, but are not limited to, transient expression (Lincoln et al. (1998) Plant MoI. Biol. Rep. 16:1-4) and viral transfection (Lacomme et al. (2001), "Genetically Engineered Viruses” (CJ.A. Ring and E.D. Blair, Eds). Pp. 59-99, BIOS Scientific Publishers, Ltd. Oxford, UK).
• Seeds are obtained from the transformed plants and used for testing stability and inheritance. Generally, two or more generations are cultivated to ensure that the phenotypic feature is stably maintained and transmitted.
• a person of ordinary skill in the art recognizes that after the expression cassette is stably incorporated in transgenic plants and confirmed to be operable, it can be introduced into other plants by sexual crossing. Any of a number of standard breeding techniques can be used, depending upon the species to be crossed.
• nucleic acid molecules of the present invention may be used to confer the trait of an altered flowering time.
• nucleic acid molecules of the present invention encode appropriate proteins from any organism, but are preferably found in plants, fungi, bacteria or animals.
• the methods according to the present invention can be applied to any plant, preferably higher plants, pertaining to the classes of Angiospermae and Gymnospermae. Plants of the subclasses of the Dicotylodenae and the Monocotyledonae are particularly suitable.
• the methods of the present invention are preferably used in plants that are important or interesting for agriculture, horticulture, biomass for bioconversion and/or forestry.
• Non-limiting examples include, for instance, tobacco, oilseed rape, sugar beet, potatoes, tomatoes, cucumbers, peppers, beans, peas, citrus fruits, avocados, peaches, apples, pears, berries, plumbs, melons, eggplants, cotton, soybean, sunflowers, roses, poinsettia, petunia, guayule, cabbages, spinach, alfalfa, artichokes, sugarcane, mimosa, Serviced lespedera, corn, wheat, rice, rye, barley, sorghum and grasses such as switch grass, giant reed, Bermuda grass, Johnson grasses or turf grass, millet, hemp, bananas, poplars, eucalyptus trees and conifers.
• Of interest are plates grown for energy production, so called energy crops, such as broadleaf plants
• amino acids in a sequence can be substituted with other amino acid(s), the charge and polarity of which are similar to that of the substituted amino acid, i.e. a conservative amino acid substitution, resulting in a biologically/functionally silent change.
• Conservative substitutes for an amino acid within the polypeptide sequence can be selected from other members of the class to which the amino acid belongs.
• Amino acids can be divided into the following four groups: (1) acidic (negatively charged) amino acids, such as aspartic acid and glutamic acid; (2) basic (positively charged) amino acids, such as arginine, histidine, and lysine; (3) neutral polar amino acids, such as serine, threonine, tyrosine, asparagine, and glutamine; and (4) neutral nonpolar (hydrophobic) amino acids such as glycine, alanine, leucine, isoleucine, valine, proline, phenylalanine, tryptophan, cysteine, and methionine.
• acidic (negatively charged) amino acids such as aspartic acid and glutamic acid
• basic (positively charged) amino acids such as arginine, histidine, and lysine
• neutral polar amino acids such as serine, threonine, tyrosine, asparagine, and glutamine
• neutral nonpolar (hydrophobic) amino acids such
• Nucleic acid molecules of the present invention can comprise sequences that differ from those encoding a protein or fragment thereof selected from the group consisting of Leads 15, 28, 29, 36, ME04012 and Clone 691319, SEQ ID Nos. 80, 90, 92, 98, 109, and 103, respectively, due to the fact that the different nucleic acid sequence encodes a protein having one or more conservative amino acid changes.
• Bioly functional equivalents of the polypeptides, or fragments thereof, of the present invention can have about 10 or fewer conservative amino acid changes, more preferably about 7 or fewer conservative amino acid changes, and most preferably about 5 or fewer conservative amino acid changes.
• the polypeptide has between about 5 and about 500 conservative changes, more preferably between about 10 and about 300 conservative changes, even more preferably between about 25 and about 150 conservative changes, and most preferably between about 5 and about 25 conservative changes or between 1 and about 5 conservative changes.
• nucleic acid molecules, and nucleotide sequences thereof, of the present invention were identified by use of a variety of screens that are predictive of nucleotide sequences that provide plants with altered size, vegetative growth, growth rate, organ number, plant architecture and/or biomass.
• Screens that are predictive of nucleotide sequences that provide plants with altered size, vegetative growth, growth rate, organ number, plant architecture and/or biomass.
• One or more of the following screens were, therefore, utilized to identify the nucleotide (and amino acid) sequences of the present invention.
• Wild-type Arabidopsis thaliana Wassilewskija (WS) plants are transformed with Ti plasmids containing clones in the sense orientation relative to the 35S promoter.
• a Ti plasmid vector useful for these constructs, CRS 338 contains the Ceres- constructed, plant selectable marker gene phosphinothricin acetyltransferase (PAT), which confers herbicide resistance to transformed plants.
• PAT phosphinothricin acetyltransferase
• Ten independently transformed events are typically selected and evaluated for their qualitative phenotype in the T 1 generation.
• Horticulture, Ltd., Bellevue, WA is mixed with 16L Therm-O-Rock vermiculite (Therm-O- Rock West, Inc., Chandler, AZ) in a cement mixer to make a 60:40 soil mixture.
• To the soil mixture is added 2 Tbsp Marathon 1% granules (Hummert, Earth City, MO), 3 Tbsp OSMOCOTE ® 14-14-14 (Hummert, Earth City, MO) and 1 Tbsp Peters fertilizer 20-20-20 (J.R. Peters, Inc., Allentown, PA), which are first added to 3 gallons of water and then added to the soil and mixed thoroughly.
• 4-inch diameter pots are filled with soil mixture. Pots are then covered with 8-inch squares of nylon netting.
• Plant Maintenance 3 to 4 days after planting, lids and shade cloth are removed. Plants are watered as needed. After 7-10 days, pots are thinned to 20 plants per pot using forceps. After 2 weeks, all plants are subirrigated with Peters fertilizer at a rate of 1 Tsp per gallon of water. When bolts are about 5-10 cm long, they are clipped between the first node and the base of stem to induce secondary bolts. Dipping infiltration is performed 6 to 7 days after clipping.
• Agrobacterium starter blocks are obtained (96-well block with Agrobacterium cultures grown to an OD 6O0 of approximately 1.0) and inoculated one culture vessel per construct by transferring 1 mL from appropriate well in the starter block. Cultures are then incubated with
• infiltration media is prepared by adding 2.2 g MS salts, 5O g sucrose, and 5 ⁇ l 2 mg/ml benzylaminopurine to 900 ml water.
• Seed is evenly dispersed into water-saturated soil in pots and placed into a dark 4°C cooler
• FINALE ® (Sanofi Aventis, Paris, France) is sprayed on plants (3 ml FINALE ® diluted into 48 oz. water) and repeated every 3-4 days until only transformants remain.
• Screening Screening is routinely performed at four stages: Seedling,
• Rosette, Flowering, and Senescence o Seedling - the time after the cotyledons have emerged, but before the 3 rd true leaf begins to form. o Rosette - the time from the emergence of the 3 rd true leaf through just before the primary bolt begins to elongate. o Flowering - the time from the emergence of the primary bolt to the onset of senescence (with the exception of noting the flowering time itself, most observations should be made at the stage where approximately 50% of the flowers have opened). o Senescence - the time following the onset of senescence (with the exception of
• Screens Screening for increased size, vegetative growth and/or biomass is performed by taking measurements, specifically T 2 measurements were taken as follows:
• Days to Bolt number of days between sowing of seed and emergence of first inflorescence.
• Rosette Leaf Number at Bolt number of rosette leaves present at time of emergence of first inflorescence.
• Rosette Area area of rosette at time of initial inflorescence emergence, using formula ((LxW)*3.14)/4.
• PCR was used to amplify the cDNA insert in one randomly chosen T 2 plant. This PCR product was then sequenced to confirm the sequence in the plants.
• Plants transformed with the genes of interest were screened as described above for modulated growth and phenotype characteristics.
• the observations include those with respect to the entire plant, as well as parts of the plant, such as the roots and leaves.
• the observations for transformants with each polynucleotide sequence are noted in the Sequence listing for each of the tested nucleotide sequences and the corresponding encoded polypeptide.
• the modulated characteristics i.e. observed phenotypes
• the "Phenotype" noted in the Sequence Listing for each relevant sequence further includes a statement of the useful utility of that sequence based on the observations.
• the observations made for the various transformants can be categorized, depending upon the relevant plant tissue for the observation and the consequent utility/usefulness of the nucleotide sequence/polypeptide used to make that transformant.
• Table 1 correlates the shorthand notes in the sequence listing to the observations noted for each tranformant (the "description” column), the tissue of the observation, the phenotype thereby associated with the transformant, and the consequent utility/usefulness of the inserted nucleotide sequence and encoded polypeptide (the "translation” column).
• sequence listing further includes (in a "miscellaneous feature" section) an indication of important identified dominant(s) and the corresponding function of the domain or identified by comparison to the publicly available pfam database. TABLE l
• nucleotides/polypeptides of the inventions are useful, depending upon the respective individual sequence, to make plants with modified growth and phenotype characteristics, including: a. modulated plant size, including increased and decreased height or length; b. modulated vegetative growth (increased or decreased); c. modulated organ number; d. increased biomass; e. sterility; f. seedling lethality; g. accelerated crop development or harvest; h. accelerated flowering time; i. delayed flowering time; j. delayed senescence; k. enhanced drought or stress tolerance;
• enhanced tolerance to high or low pH, to low or high nitrogen or phosphate aa. enhanced tolerance to oxidative stress; bb. enhanced chemical composition; cc. altered leaf shape; dd. enhanced abiotic stress tolerance; ee. increased tolerance to cold stress; ff. increased starch content; gg. reduced number or no seeds; hh. enhanced plant strength; ii. modified flower length; jj. longer inflorescences; kk. modified seed fiber content;
• modified fruit shape is modified fruit shape; mm. modified fruit composition; nn. modified seed yield; oo. modified plant architecture, such as modified amount or angle of branching, modified leaf structure, or modified seed structure; and pp. enhanced shade avoidance.
• Example 1 Lead 28 - ME04701 - Clone 1952 - cDNA 13499809 ( SEO ID NO: 90) Qualitative analysis of the Ti plants:
• This polynucleotide/protein can be an especially useful one for controlling the number/rate of cell division in meristems without disturbing overall plant morphology. It can be developed in crops with an appropriate promoter to regulate size and growth rate of many individual organs.
• Exanmle2 Lead 29 -ME04717- Clone 123905 - cDNA 12562634 fSEO ID NO: 92) Ectopic expression of Ceres cDNA 12562634 under the control of the 326D promoter induces a number of phenotypes including: o Increased number of inflorescences o Continuation of rosette leaf initiation after flowering to generate an overall increased number of leaves.
• Misexpression of Ceres cDNA 12562634 can be useful to increase branching and the number of inflorescences. This can have a significant impact on seed number.
• Segregation frequencies of the plants under test suggest that each event contains a single insert, as calculated by a chi-square test (data not shown).
• This gene/protein can be an especially useful one for controlling the rate of cell division in the meristems without disturbing overall plant morphology. It can be developed in crops with an appropriate promoter to regulate size and growth rate of many individual organs.
• Gene 123905 was also transformed into tomato under the control of the promoter p326.
• 4 independent transgenic events were characterized in the field. A number of independent events were originally evaluated and 4 were selected for further analysis based on expression of the gene, presence of a simple insert and the phenotype of the plants observed in the greenhouse. Homozygous T2 seeds were planted in the field in a randomized complete block design. Each event had a corresponding control line. Results of plant weight, the total weight of individual plants, total fruit weight per plant, percent red fruit per plant and harvest index are shown in the Table 2-3 below. The results indicate that events 1 and 21 had substantially reduced leaf mass while retaining yields comparable to controls. Hence, thek harvest index improved. These events also had increases in percent red fruit per plant. Event 14 had increased biomass and yield.
• tomato plants transformed with gene 123905 tended to have more branches and leaves, and more fruit as compared to control.
• Gene 123905 was transformed into rice cultivar Kitaake under the control of p326.
• Five (5) independent transgenic events were evaluated in the field in a randomized complete block design.
• the traits evaluated were tillers per plant, days to flowering, leaf angle, plant height, biomass in grams per plant, yield in grams per plant and total plot yield in grams, the results for which are shown below in Tables 2-4, 2-5 and 2-6. Each event resulted in an increase in the number of tillers per plant.
• Table 2 -4 Results from Rice Field Trials Number of plants Days to Days to Approx.
• Event 8-3 showed an increase in height, biomass and yield relative to control. While generally lower in yield, and significantly reduced in stature, event 1 and event 12 produce biomass similar to controls indicating an increase in biomass density relative to controls.
• Gene 123905 was transformed into rice cultivar Kitaake under the control of p326. Measurements were conducted to determine which internodes were reduced hi length, where internode I is the uppermost internode and internode V is the lowermost internode. In events 1, 4 and 12 which have significantly reduced stature relative to control, internodes III and IV are significantly reduced in length, while internodes I and II are reduced only slightly or not at all.
• Transgenic lines 123905-1 and 123905-12-3 germinate 1 to 2 days faster than Kitaake control seed.
• Clone 679923 intibie Ceres soy cDNA library contains cDNA 13594332, encoding a transcription factor similar to the Arabidopsis LEAFY PETIOLE (LEP) gene. This protein sequence contains an AP2 domain.
• the cDNA was placed into the Ceres Misexpression Pipeline because it was determined to be a putative ortholog of a known Arabidopsis gene (LEP).
• Example 4 ME04012 - Gemini ID 5000F6 (SEO ID NO: 109)
• ME04012 contains a genomic clone which encodes a putative Cytochrome P450. Plant line ME04012 was being assayed for drought tolerance when it was observed that 15/20 plants in event -03 showed a plant architecture phenotype. 6/15 were a weaker version showing only a wavy stem. 9/15 were strong and showed a wavy stem, decreased height and decreased branch and pedicel angles.
• ExanwleS Leadl5-ME04077- Clone 92459 - cDNA 12561537(SEO H) NO: 80) Clone 92459 in the Ceres Arabidopsis cDNA library, contains cDNA 12561537, encoding Arabidopsis MADS Affecting Flowering 1 (MAFl).
• the cDNA was placed into the Ceres Misexpression Pipeline because it is a transcription factor. Transcription factors are of particular interest because they can affect many genes simultaneously, and they therefore have an increased likelihood of producing an altered phenorype in Arabidopsis when overexpressed.
• AU plants noted in the table as ME04077-06 and ME04077-10 were segregating progeny of the T 1 event which we had confirmed to contain the transgene under test. All plants noted in the table as -06 Control or -10 Control were T 2 segregating progeny which did not contain the transgene under test (internal controls).
• Event ME04077-06 had 12 transgene-containing pi ants which exhibited the beneficial phenotype and 3 transgene-containing plants which appeared wild-type (these three were omitted from statistical analyses in Table 5-2).
• Event ME04077-10 had 9 transgene- containing plants which exhibited the beneficial phenotype and 1 transgene-containmg plant which appeared wild-type.
• Our statistical analyses compared the internal controls to those plants with the beneficial phenotype which contained the transgene. Segregation frequencies of the transgene under test suggest that each event contains a single insert, as calculated by a Chi-square test. The T 2 seeds segregate 3R:1S for both events (data not shown).
• Biomass advantage and presumed photosynthesis advantage should be useful in corn and soybean.
• This gene/protein can be an especially useful one for controlling the number/rate of cell division in meristems without disturbing overall plant morphology. It can be developed in crops with an appropriate promoter to regulate size and growth rate of many individual organs. The protein can be useful for creating sturdier stems in corn and preventing against "snap".
• This Lead 15 (clone 92459) was transformed into tomato under the control of plasmid pl3879. 1 transgenic event was selected for field testing. This event shows an increase in biomass, as shown below in the results of Table 5-3.
• the "Lead" sequences described above in Examples 1 - 5 are utilized to identify functional homologs of the lead sequences and, together with those sequences, are utilized to determine a consensus sequence for a given group of lead and functional homolog sequences.
• a subject sequence is considered a functional homolog of a query sequence if the subject and query sequences encode proteins having a similar function and/or activity.
• a process known as Reciprocal BLAST (Rivera et al, Proc.Natl Acad. ScL USA, 1998, 95:6239-6244) is used to identify potential functional homolog sequences from databases consisting of all available public and proprietary peptide sequences, including NR from NCBI and peptide translations from Ceres clones.
• a specific query polypeptide is searched against all peptides from its source species using BLAST in order to identify polypeptides having sequence identity of 80% or greater to the query polypeptide and an alignment length of 85% or greater along the shorter sequence in the alignment.
• the query polypeptide and any of the aforementioned identified polypeptides are designated as a cluster.
• the main Reciprocal BLAST process consists of two rounds of BLAST searches; forward search and reverse search.
• a query polypeptide sequence, "polypeptide A” from source species S A is BLASTed against all protein sequences from a species of interest.
• Top hits are determined using an E- value cutoff of 10 ⁇ 5 and an identity cutoff of 35%. Among the top hits, the sequence having the lowest E- value is designated as the best hit, and considered a potential functional homolog. Any other top hit that had a sequence identity of 80% or greater to the best hit or to the original query polypeptide is considered a potential functional homolog as well. This process is repeated for all species of interest.
• top hits identified in the forward search from all species are used to perform a BLAST search against all protein or polypeptide sequences from the source species S A .
• a top hit from the forward search that returned a polypeptide from the aforementioned cluster as its best hit is also considered as a potential functional homolog.
• Functional homologs are identified by manual inspection of potential functional homolog sequences. Representative functional homologs are shown in Figures 1 - 5. Each Figure represents a grouping of a lead/query sequence aligned with the corresponding identified functional homolog subject sequences. Lead sequences and their corresponding functional homolog sequences are aligned to identify conserved amino acids and to determine a consensus sequence that contains a frequently occurring amino acid residue at particular positions in the aligned sequences, as shown in Figures 1-5.
• Each consensus sequence then is comprised of the identified and numbered conserved regions or domains, with some of the conserved regions being separated by one or more amino acid residues, represented by a dash (-), between conserved regions.
• Useful polypeptides of the inventions therefore, include each of the lead and functional homolog sequences shown in Figures 1-5, as well as the consensus sequences shown in those Figures.
• the invention also encompasses other useful polypeptides constructed based upon the consensus sequence and the identified conserved regions.
• useful polypeptides include those which comprise one or more of the numbered conserved regions in each alignment table in an individual Figure depicted in Figures 1-5, wherein the conserved regions may be separated by dashes.
• Useful polypeptides also include those which comprise all of the numbered conserved regions in an individual alignment table selected from Figures 1-5, alternatively comprising all of the numbered conserved regions in an individual alignment table and in the order as depicted in an individual alignment table selected from Figures 1-5.
• Useful polypeptides also include those which comprise all of the numbered conserved regions in an individual alignment table and in the order as depicted in an individual alignment table selected from Figures 1-5, wherein the conserved regions are separated by dashes, wherein each dash between two adjacent conserved regions is comprised of the amino acids depicted in the alignment table for lead and/or functional homolog sequences at the positions which define the particular dash.
• Such dashes in the consensus sequence can be of a length ranging from length of the smallest number of dashes in one of the aligned sequences up to the length of the highest number of dashes in one of the aligned sequences.
• Such useful polypeptides can also have a length (a total number of amino acid residues) equal to the length identified for a consensus sequence or of a length ranging from the shortest to the longest sequence in any given family of lead and functional homolog sequences identified in an individual alignment table selected from Figures 1-5.
• the present invention further encompasses nucleotides that encode the above described polypeptides, as well as the complements thereof, and including alternatives thereof based upon the degeneracy of the genetic code.

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