# Patent

## Title
Nucleotide sequences and corresponding polypeptides conferring modulated plant growth rate and biomass in plants

## Abstract

The present invention relates to isolated nucleic acid molecules and their corresponding encoded polypeptides able confer the trait of modulated plant size, vegetative growth, organ number, plant architecture, growth rate, seedling vigor, growth rate, fruit and seed yield, tillering and/or biomass in plants. The present invention further relates to the use of these nucleic acid molecules and polypeptides in making transgenic plants, plant cells, plant materials or seeds of a plant having plant size, vegetative growth, organ number, plant architecture, growth rate, seedling vigor and/or biomass that are altered with respect to wild type plants grown under similar conditions.

## Background
CROSS-REFERENCE TO RELATED APPLICATION
This application is a Continuation-In-Part of and claims priority under 35 U.S.C. §120 on co-pending application Ser. No. 11/172,740 filed on Jun. 30, 2005, which claims priority under 35 U.S.C. §119(e) on U.S. Provisional Application Nos. 60/583,621; 60/584,829 and 60/584,800 filed on Jun. 30, 2004. The entire contents of application Ser. Nos. 11/172,740, 60/583,621; 60/584,829 and 60/584,800 are hereby incorporated by reference.


FIELD OF THE INVENTION
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.
BACKGROUND OF THE INVENTION
Plants specifically improved for agriculture, horticulture, biomass conversion, and other industries (e.g. paper industry, plants as production factories for proteins or other compounds) can be obtained using molecular technologies. As an example, 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.
Similarly, 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. For example, reductions in the height of specific crops and tree species can be beneficial by allowing easier harvesting. Alternatively, 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.
Availability and maintenance of a reproducible stream of food and animal feed to feed animals and people has been a high priority throughout the history of human civilization and lies at the origin of agriculture. Specialists and researchers in the fields of agronomy science, agriculture, crop science, horticulture, and forest science are even today constantly striving to find and produce plants with an increased growth potential to feed an increasing world population and to guarantee a supply of reproducible raw materials. The robust level of research in these fields of science indicates the level of importance leaders in every geographic environment and climate around the world place on providing sustainable sources of food, feed, chemicals and energy for the population.
Manipulation of crop performance has been accomplished conventionally for centuries through plant breeding. The breeding process is, however, both time-consuming and labor-intensive. Furthermore, appropriate breeding programs must be specially designed for each relevant plant species.
On the other hand, great progress has been made in using molecular genetics approaches to manipulate plants to provide better crops. Through introduction and expression of recombinant nucleic acid molecules in plants, researchers are now poised to provide the community with plant species tailored to grow more efficiently and produce more product despite unique geographic and/or climatic environments. These new approaches have the additional advantage of not being limited to one plant species, but instead being applicable to multiple different plant species (Zhang et al. (2004) Plant Physiol. 135:615).
Despite this progress, today there continues to be a great need for generally applicable processes that improve forest or agricultural plant growth to suit particular needs depending on specific environmental conditions. To this end, 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.
SUMMARY OF THE INVENTION
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.
Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.



BRIEF DESCRIPTION OF THE FIGURES
FIG. 1. Amino acid sequence alignment of homologues of Lead 29 (ME04717), SEQ ID NO. 93: Ceres Gdna 1460991, SEQ ID NO: 94; Lead clone 123905, SEQ ID NO: 93; gil51536200, SEQ ID NO: 97; CeresClone 1494990, SEQ ID NO: 99; CeresClone 634402, SEQ ID NO: 96. Conserved regions are enclosed in a box. A consensus sequence, comprised of SEQ ID NOs. 122-134, is shown below the alignment.
FIG. 2. Amino acid sequence alignment of homologues of Lead 36 (ME03195), SEQ ID NO. 99: gil50941583, SEQ ID NO: 102; Lead clone 679923, SEQ ID NO: 99; Ceres Gdna 14719788, SEQ ID NO: 100; Ceres Gdna 1533259, SEQ ID NO: 101. Conserved regions are enclosed in a box. A consensus sequence, comprised of SEQ ID NOs. 135-144, is shown below the alignment.
FIG. 3. Amino acid sequence alignment of homologues of Lead 15 (ME04077), SEQ ID NO. 81: gil31580813, SEQ ID NO: 83; gil34591565, SEQ ID NO: 84; gil71834745, SEQ ID NO: 82. Conserved regions are enclosed in a box. A consensus sequence, comprised of SEQ ID NOs. 145-158, is shown below the alignment.
FIG. 4. Amino acid sequence alignment of homologues of Lead ME04012, SEQ ID NO. 110: ME04012, SEQ ID NO: 110; gil3582021, SEQ ID NO: 115; gil469-47673, SEQ ID NO: 116; gil25282608, SEQ ID NO: 121 gil134904242, SEQ ID NO: 118. Conserved regions are enclosed in a box. A consensus sequence, comprised of SEQ ID NOs. 159-198, is shown below the alignment.
FIG. 5. Amino acid sequence alignment of homologues of Lead Clone 691319, SEQ ID NO. 104: Lead clone 691319, SEQ ID NO: 104; Ceres Gdna 1443093, SEQ ID NO: 105; and Ceres Gdna 1452324, SEQ ID NO: 106. Conserved regions are enclosed in a box. A consensus sequence, comprised of SEQ ID NOs. 199-222, is shown below the alignment.



DETAILED DESCRIPTION OF THE INVENTION
1. The Invention
The invention of the present application may be described by, but not necessarily limited to, the following exemplary embodiments.
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.
More particularly, 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. 80, 90, 92, 98, 109, and 103, (d) a 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 temperature from about 40° C. to about 48° C. below a melting temperature of the hybridized nucleic acid duplex, and (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. More particularly, the first and second nucleic acids may be operably linked. Even more particularly, 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. Most particularly, the first and second organisms may be different species.
In a further embodiment of the present invention, 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.
In another embodiment 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.
Other embodiments of the present invention 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. Furthermore, 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. More preferably, 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. In another embodiment of the present invention 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. In another embodiment of the present invention 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 FIGS. 1-5.
2. Definitions
The following terms are utilized throughout this application:
Biomass: As used herein, “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-mediated transformation (of dicots (Needleman and Wunsch (1970) J. Mol. Biol. 48:443; Pearson and Lipman (1988) Proc. Natl. Acad. Sci. (USA) 85: 2444), of monocots (Yamauchi et al. (1996) Plant Mol. Biol. 30:321-9; Xu et al. (1995) Plant Mol. Biol. 27:237; Yamamoto et al. (1991) Plant Cell 3:371), and biolistic methods (P. Tijessen, “Hybridization with Nucleic Acid Probes” In Laboratory Techniques in Biochemistry and Molecular Biology, P. C. vand der Vliet, ed., c. 1993 by Elsevier, Amsterdam), electroporation, in planta techniques, and the like. Such a plant containing the exogenous nucleic acid is referred to here as a T0 for the primary transgenic plant and T1 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.
Functionally comparable proteins will give rise to the same characteristic to a similar, but not necessarily the same, degree. Typically, comparable proteins give the same characteristics where the quantitative measurement due to one of the comparables is at least 20% of the other; more typically, between 30 to 40%; even more typically, between 50-60%; even more typically between 70 to 80%; even more typically between 90 to 100% of the other.
Heterologous sequences: “Heterologous sequences” are those that are not operatively linked or are not contiguous to each other in nature. For example, a promoter from corn is considered heterologous to an Arabidopsis coding region sequence. Also, 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. On the other hand, these same elements remain operatively linked but become heterologous if other filler sequence is placed between them. Thus, 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: The term “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: As used herein, the term “percent 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). “Percentage of sequence identity,” as used herein, is determined by comparing two optimally aligned sequences over a comparison window, where the fragment of the polynucleotide or amino acid sequence in the comparison window may comprise additions or deletions (e.g., gaps or overhangs) as compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences. The percentage is calculated by determining the number of positions at which the identical nucleic acid base or amino acid residue occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison and multiplying the result by 100 to yield the percentage of sequence identity. Optimal alignment of sequences for comparison may be conducted by the local homology algorithm of Smith and Waterman Add. APL. Math. 2:482 (1981), by the homology alignment algorithm of Needleman and Wunsch J. Mol. BioL 48:443 (1970), by the search for similarity method of Pearson and Lipman Proc. Natl. Acad. Sci. (USA) 85: 2444 (1988), by computerized implementations of these algorithms (GAP, BESTFIT, BLAST, PASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group (GCG), 575 Science Dr., Madison, Wis.), or by inspection. Given that two sequences have been identified for comparison, GAP and BESTFIT are preferably employed to determine their optimal alignment. Typically, the default values of 5.00 for gap weight and 0.30 for gap weight length are used. The term “substantial sequence identity” between polynucleotide or polypeptide sequences refers to polynucleotide or polypeptide comprising a sequence that has at least 80% sequence identity, preferably at least 85%, more preferably at least 90% and most preferably at least 95%, even more preferably, at least 96%, 97%, 98% or 99% sequence identity compared to a reference sequence using the programs.
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. For fast pairwise alignment of nucleic acid sequences, the following default parameters are used: word size: 2; window size: 4; scoring method: percentage; number of top diagonals: 4; and gap penalty: 5. For multiple alignment of nucleic acid sequences, the following parameters are used: gap opening penalty: 10.0; gap extension penalty: 5.0; and weight transitions: yes. For fast pairwise alignment of protein sequences, the following parameters are used: word size: 1; window size: 5; scoring method: percentage; number of top diagonals: 5; gap penalty: 3. For multiple alignment of protein sequences, the following parameters are used: weight matrix: blosum; gap opening penalty: 10.0; gap extension penalty: 0.05; hydrophilic gaps: on; hydrophilic residues: Gly, Pro, Ser, Asn, Asp, Gln, Glu, Arg, and Lys; residue-specific gap penalties: on. 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.
In case of the functional homolog searches, to ensure a subject sequence having the same function as the query sequence, 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. To determine a percent identity between a query sequence and a 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. For example, 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 Regions: The term “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. As used herein, the term “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: As used herein, “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: “Stringency,” as used herein 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 Tm, 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 Tm. High stringency conditions are those providing a condition of Tm−5° C. to Tm−10° C. Medium or moderate stringency conditions are those providing Tm−20° C. to Tm−29° C. Low stringency conditions are those providing a condition of Tm−40° C. to Tm−48° C. The relationship between hybridization conditions and Tm (in ° C.) is expressed in the mathematical equation:

Tm=81.5−16.6(log10[Na+])+0.41(% G+C)−(600/N)  (I)

where 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 Tm 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):

Tm=81.5+16.6 log {[Na+]/(1+0.7[Na+])}+0.41(% G+C)−500/L0.63(% formamide)  (II)

where L represents the number of nucleotides in the probe in the hybrid (21). The Tm of Equation II is affected by the nature of the hybrid: for DNA-RNA hybrids, Tm is 10-15° C. higher than calculated; for RNA-RNA hybrids, Tm is 20-25° C. higher. Because the Tm decreases about 1° C. for each 1% decrease in homology when a long probe is used (Frischauf et al. (1983) J. Mol Biol, 170: 827-842), stringency conditions can be adjusted to favor detection of identical genes or related family members.

Equation II is derived assuming the reaction is at equilibrium. Therefore, 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 Tm, medium or moderate stringency is 26-29° C. below Tm and low stringency is 45-48° C. below Tm.
T0: The term “T0” refers to the whole plant, explant or callus tissue, inoculated with the transformation medium.
T1: The term T1 refers to either the progeny of the T0 plant, in the case of whole-plant transformation, or the regenerated seedling in the case of explant or callous tissue transformation.
T2: The term T2 refers to the progeny of the T1 plant. T2 progeny are the result of self-fertilization or cross-pollination of a T1 plant.
T3: The term T3 refers to second generation progeny of the plant that is the direct result of a transformation experiment. T3 progeny are the result of self-fertilization or cross-pollination of a T2 plant.
3. Important Characteristics of the Polynuceotides and Polypeptides of the Invention
The 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. For example, 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.
Because the disclosed sequences and methods increase vegetative growth, and growth rate, the disclosed methods can be used to enhance biomass production. For example, 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. Examples of 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. In 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. In rice, this results in plants with an increase number of tillers. The sequences of Leads 15 and 28 of the present invention and their functional homologs in particular provide transformed plants with enhanced yield, including fruit yield and yield per acre. In tomatoes, this results in plants with increased fruit yield on more compact plants. In rice, this results in plants with an increase number of tillers.
The life cycle of flowering plants in general can be divided into three growth phases: vegetative, inflorescence, and floral (late inflorescence phase). In the vegetative phase, the shoot apical meristem (SAM) generates leaves that later will ensure the resources necessary to produce fertile offspring. Upon receiving the appropriate environmental and developmental signals 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. During this 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. Once established, the plant enters the late inflorescence phase (Xu et al. (1995) Plant Mol. 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.
For example, increased seedling vigor would be advantageous for cereal plants such as rice, maize, wheat, etc. production. For these crops, growth can often be slowed or stopped by cool environmental temperatures during the planting season. In addition, 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, Dec. 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 in 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 in nonplant eukaryotes and have been identified as important regulatory components in diverse biological processes. Many different functions have been identified for those proteins in 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 (bHLH) is a protein structural motif that characterizes a family of transcription factors. The motif is characterized by two α 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 in development or cell activity.
4. The Polynucleotides/Polypeptides of the Invention
The polynucleotides 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.
5. Use of the Polypeptides to Make Transgenic Plants
To use the sequences of the present invention or a combination of them or parts and/or mutants and/or fusions and/or variants of them, 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-mediated 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. Sci. USA, 89: 8794-8797; Hamilton et al. (1996) Proc. Natl. Acad. Sci. USA, 93: 9975-9979; Burke et al. (1987) Science, 236:806-812; Sternberg N. et al. (1990) Proc Natl Acad Sci USA., 87:103-7; Bradshaw et al. (1995) Nucl Acids Res, 23: 4850-4856; Frischauf et al. (1983) J. Mol Biol, 170: 827-842; Huynh et al., Glover N M (ed) DNA Cloning: A practical Approach, Vol. 1 Oxford: IRL Press (1985); Walden et al. (1990) Mol Cell Biol 1: 175-194).
Typically, 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.
It will be understood that more than one 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.
To “operably link” a promoter sequence to a 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). For example, a suitable enhancer is a cis-regulatory element (−212 to −154) from the upstream region of the octopine synthase (ocs) gene. Fromm et al., The Plant Cell 1:977-984 (1989).
A 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.
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.
Some suitable promoters initiate transcription only, or predominantly, in certain cell types. For example, 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, zygote, endosperm, integument, or seed coat) can be used. Thus, as used herein 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).
Examples of various classes of promoters are described below. Some of the promoters indicated below are described in more detail in U.S. Patent Application Ser. Nos. 60/505,689 (expired); 60/518,075 (expired); 60/544,771 (expired); 60/558,869 (expired); 60/583,691 (expired); 60/619,181 (expired); 60/637,140 (expired); 10/950,321 (U.S. Pat. No. 7,173,121); 10/957,569 (pending); 11/058,689 (pending); 11/172,703 (issued as U.S. Pat. No. 7,173,121); 11/208,308 (abandoned); 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.
Other Regulatory Regions: A 5′ untranslated region (UTR) 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. For example, 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. As another example, 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), p13879 (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). Additional examples include the cauliflower mosaic virus (CaMV) 35S promoter, the mannopine synthase (MAS) promoter, the 1′ or 2′ promoters derived from T-DNA of Agrobacterium tumefaciens, the figwort mosaic virus 34S promoter, actin promoters such as the rice actin promoter, and ubiquitin promoters such as the maize ubiquitin-1 promoter. In some cases, 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. In some embodiments, 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). Other 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 al., Proc. Natl. Acad. Sci. USA 86:7890-7894 (1989)), root cell specific promoters reported by Conkling et al., Plant Physiol. 93:1203-1211 (1990), and the tobacco RD2 gene promoter.
In some embodiments, 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 al. (1989) Plant Cell 1(9):839-853), the soybean trypsin inhibitor promoter (Riggs et al. (1989) Plant Cell 1(6):609-621), the ACP promoter (Baerson et al. (1993) Plant Mol Biol, 22(2):255-267), the stearoyl-ACP desaturase gene (Slocombe et al. (1994) Plant Physiol 104(4):167-176), the soybean α′ subunit of β-conglycinin promoter (Chen et al. (1986) Proc Natl Acad Sci USA 83:8560-8564), the oleosin promoter (Hong et al. (1997) Plant Mol Biol 34(3):549-555), and zein promoters, such as the 15 kD zein promoter, the 16 kD zein promoter, 19 kD zein promoter, 22 kD zein promoter and 27 kD zein promoter. Also suitable are the Osgt-1 promoter from the rice glutelin-1 gene (Zheng et al. (1993) Mol. 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. Other such promoters that drive gene expression preferentially in ovules are YP0007 (SEQ ID NO: 30), YP0111 (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), YP0115 (SEQ ID NO: 47), YP0119 (SEQ ID NO: 49), YP0120 (SEQ ID NO: 50) and YP0374 (SEQ ID NO: 68).
In some other embodiments of the present invention, 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, GenBank No. 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. Pat. No. 6,906,244). Other promoters that may be suitable include those derived from the following genes: maize MAC1 (see, Sheridan (1996) Genetics, 142:1009-1020); maize Cat3 (see, GenBank No. L05934; Abler (1993) Plant Mol. Biol., 22:10131-1038). Other promoters include the following Arabidopsis promoters: YP0039 (SEQ ID NO: 34), YP0101 (SEQ ID NO: 41), YP0102 (SEQ ID NO: 42), YP0110 (SEQ ID NO: 45), YP0117 (SEQ ID NO: 48), YP0119 (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: p530c10, pOsFIE2-2, pOsMEA, pOsYp102, 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 (Ltp1) 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).
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-1,5-bisphosphate carboxylase (RbcS) promoters such as the RbcS promoter from eastern larch (Larix laricina), 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 Mol. Biol. 15:921-932), the CAB-1 promoter from spinach (Lubberstedt et al. (1994) Plant Physiol. 104:997-1006), the cab1R promoter from rice (Luan et al. (1992) Plant Cell 4:971-981), the pyruvate orthophosphate dikinase (PPDK) promoter from corn (Matsuoka et al. (1993) Proc Natl Acad Sci USA 90:9586-9590), the tobacco Lhcb1*2 promoter (Cerdan et al. (1997) Plant Mol. Biol. 33:245-255), the Arabidopsis thaliana SUC2 sucrose-H+ symporter promoter (Truernit et al. (1995) Planta 196:564-570), and thylakoid membrane protein promoters from spinach (psaD, psaF, psaE, PC, FNR, atpC, atpD, cab, rbcS. Other 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).
In some other embodiments of the present invention, inducible promoters may be desired. Inducible promoters drive transcription in response to external stimuli such as chemical agents or environmental stimuli. For example, inducible promoters can confer transcription in response to hormones such as giberellic acid or ethylene, or in response to light or drought. Examples of 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). Examples of 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).
Other Promoters: Other classes of 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. Promoters designated YP0086 (SEQ ID NO: 36), YP0188 (SEQ ID NO: 58), YP0263 (SEQ ID NO: 62), PT0758 (SEQ ID NO: 22), PT0743 (SEQ ID NO: 21), PT0829 (SEQ ID NO: 23), YP0119 (SEQ ID NO: 49), and YP0096 (SEQ ID NO: 39), as described in the above-referenced patent applications, may also be useful.
Alternatively, 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. In another alternative embodiment of the present invention, 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. The term “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, while “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).
Thus, for example, 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. Alternatively, 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.
In another method, a nucleic acid can be transcribed into a ribozyme, or catalytic RNA, that affects expression of an mRNA. (See, U.S. Pat. No. 6,423,885). 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′-UG-3′ nucleotide sequence. The construction and production of hammerhead ribozymes is known in the art. See, for example, U.S. Pat. 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. Perriman, et al. (1995) Proc. Natl. Acad. Sci. USA, 92(13):6175-6179; de Feyter and Gaudron, Methods in Molecular Biology, Vol. 74, Chapter 43, “Expressing Ribozymes in Plants”, Edited by Turner, P. C, Humana Press Inc., Totowa, N.J. RNA endoribonucleases such as the one that occurs naturally in Tetrahymena thermophila, and which have been described extensively by Cech and collaborators can be useful. See, for example, U.S. Pat. No. 4,987,071.
Methods based on RNA interference (RNAi) can be used. 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 in 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.
In some nucleic-acid based methods for inhibition of gene expression in plants, 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 al., 1996, Bioorgan. Med. Chem., 4: 5-23. In addition, the deoxyphosphate backbone can be replaced with, for example, a phosphorothioate or phosphorodithioate backbone, a phosphoroamidite, or an alkyl phosphotriester backbone.
Transformation
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. Sci. USA 82:5824), PEG (Paszkowski et al. (1984) EMBO J. 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 (H. J. Rehm, G. Reed, A. Püler, P. Stadler, eds., Vol. 2, 627-659, VCH Weinheim-New York-Basel-Cambridge), and via T-DNA using Agrobacterium tumefaciens (Crit. Rev. Plant. Sci. 4:146; Fromm et al. (1990) Biotechnology 8:833-844) or Agrobacterium rhizogenes (Cho et al. (2000) Planta 210:195-204) or other bacterial hosts (Brootghaerts et al. (2005) Nature 433:629-633), for example.
In addition, a number of 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 Mol. Biol. Rep. 16:1-4) and viral transfection (Lacomme et al. (2001), “Genetically Engineered Viruses” (C. J. 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.
The nucleic acid molecules of the present invention may be used to confer the trait of an altered flowering time.
The 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. Dicotyledonous plants belonging to the orders of the Magniolales, Illiciales, Laurales, Piperales Aristochiales, Nymphaeales, Ranunculales, Papeverales, Sarraceniaceae, Trochodendrales, Hamamelidales, Eucomiales, Leitneriales, Myricales, Fagales, Casuarinales, Caryophyllales, Batales, Polygonales, Plumbaginales, Dilleniales, Theales, Malvales, Urticales, Lecythidales, Violales, Salicales, Capparales, Ericales, Diapensales, Ebenales, Primulales, Rosales, Fabales, Podostemales, Haloragales, Myrtales, Cornales, Proteales, Santales, Rafflesiales, Celastrales, Euphorbiales, Rhamnales, Sapindales, Juglandales, Geraniales, Polygalales, Umbellales, Gentianales, Polemoniales, Lamiales, Plantaginales, Scrophulariales, Campanulales, Rubiales, Dipsacales, and Asterales, for example, are also suitable. Monocotyledonous plants belonging to the orders of the Alismatales, Hydrocharitales, Najadales, Triuridales, Commelinales, Eriocaulales, Restionales, Poales, Juncales, Cyperales, Typhales, Bromeliales, Zingiberales, Arecales, Cyclanthales, Pandanales, Arales, Lilliales, and Orchidales also may be useful in embodiments of the present invention. Further examples include, but are not limited to, plants belonging to the class of the Gymnospermae are Pinales, Ginkgoales, Cycadales and Gnetales.
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, Servicea 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 like alfalfa, hemp, Jerusalem artichoke and grasses such as sorgum, switchgrass, Johnson grass and the likes.
Homologues Encompassed by the Invention
It is known in the art that one or more 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.
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.
Biologically 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. In a preferred embodiment of the present invention, 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.
Identification of Useful Nucleic Acid Molecules and Their Corresponding Nucleotide Sequences
The 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. One or more of the following screens were, therefore, utilized to identify the nucleotide (and amino acid) sequences of the present invention.
The present invention is further exemplified by the following examples. The examples are not intended to in any way limit the scope of the present application and its uses.
6. Experiments Confirming the Usefulness of the Polynucleotides and Polypeptides of the Invention
General Protocols
Agrobacterium-Mediated Transformation of Arabidopsis 
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.
Ten independently transformed events are typically selected and evaluated for their qualitative phenotype in the T1 generation.
Preparation of Soil Mixture: 24 L SunshineMix #5 soil (Sun Gro Horticulture, Ltd., Bellevue, Wash.) is mixed with 16 L Therm-O-Rock vermiculite (Therm-O-Rock West, Inc., Chandler, Ariz.) 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. Generally, 4-inch diameter pots are filled with soil mixture. Pots are then covered with 8-inch squares of nylon netting.
Planting: Using a 60 mL syringe, 35 mL of the seed mixture is aspirated. 25 drops are added to each pot. Clear propagation domes are placed on top of the pots that are then placed under 55% shade cloth and subirrigated by adding 1 inch of water.
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.
Preparation of Agrobacterium: To 150 mL fresh YEB is added 0.1 mL each of carbenicillin, spectinomycin and rifampicin (each at 100 mg/ml stock concentration). Agrobacterium starter blocks are obtained (96-well block with Agrobacterium cultures grown to an OD600 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 shaking at 27° C. Cultures are spun down after attaining an OD600 of approximately 1.0 (about 24 hours). 200 mL infiltration media is added to resuspend Agrobacterium pellets. Infiltration media is prepared by adding 2.2 g MS salts, 50 g sucrose, and 5 μl 2 mg/ml benzylaminopurine to 900 ml water.
Dipping Infiltration: The pots are inverted and submerged for 5 minutes so that the aerial portion of the plant is in the Agrobacterium suspension. Plants are allowed to grow normally and seed is collected.
High-throughput Phenotypic Screening of Misexpression Mutants: Seed is evenly dispersed into water-saturated soil in pots and placed into a dark 4° C. cooler for two nights to promote uniform germination. Pots are then removed from the cooler and covered with 55% shade cloth for 4-5 days. Cotyledons are fully expanded at this stage. FINALE® (Sanofi Aventis, Paris, France) is sprayed on plants (3 ml FINALES 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.

    
    
        Seedling—the time after the cotyledons have emerged, but before the 3rd true leaf begins to form.
        Rosette—the time from the emergence of the 3rd true leaf through just before the primary bolt begins to elongate.
        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).
        Senescence—the time following the onset of senescence (with the exception of “delayed senescence”, most observations should be made after the plant has completely dried). Seeds are then collected.
    
    


Screens: Screening for increased size, vegetative growth and/or biomass is performed by taking measurements, specifically T2 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 ((L×W)*3.14)/4.
        Height=length of longest inflorescence from base to apex. This measurement was taken at the termination of flowering/onset of senescence.
        Primary Inflorescence Thickness=diameter of primary inflorescence 2.5 cm up from base. This measurement was taken at the termination of flowering/onset of senescence.
        Inflorescence Number=total number of unique inflorescences. This measurement was taken at the termination of flowering/onset of senescence.
    
    


PCR was used to amplify the cDNA insert in one randomly chosen T2 plant. This PCR product was then sequenced to confirm the sequence in the plants.
Results:
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) are noted by an entry in the “miscellaneous features” field for each respective sequence. 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).
For some of the polynucleotides/polypeptides of the invention, the 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 1






PHENOTYPE





TISSUE
QUALIFIER
PHENOTYPE
DESCRIPTION
TRANSLATION







WHOLE
Senescence Time
Early
the plant senesces
Useful for accelerating


PLANT

Senescence
significantly early
crop development and





(note the approximate
harvest





number of days early





it started to senesce





in the comments)


INFLORESCENCE
Flowering Time
Early Flowering
the plant flowers
Useful for accelerating





significantly early
flowering time





(note the approximate





number of days early





it flowered in the





comments)


INFLORESCENCE
Flowering Time
Late Flowering
the plant flowers
Useful for delaying





significantly late
flowering time





(note the approximate





number of days late it





flowered in the





comments)


INFLORESCENCE
Flowering Time
Dtb
days to bolt
Useful for delaying






flowering time


WHOLE
Senescence Time
Late Senescence
the plant senesces
Useful for delaying


PLANT


significantly late
senescence





(note the approximate





number of days late it





started to senesce in





the comments)


COTYLEDONS
Silver
Silver
cotyledons have a
Useful for drought or





gray/silver colored
stress tolerance





surface; This





phenotype is often





accompanied by a





small size mutation,





but not always


WHOLE
Dark Green
Dark Green
plant is visibly darker
Useful for increasing


SEEDLING


green
chlorophyll and






photosynthetic capacity


WHOLE
Color
Dark Green
the plant is
Useful for increasing


PLANT


abnormally dark
chlorophyll and





green
photosynthetic capacity


WHOLE
High
High
the plant is purple in
Useful for increasing


SEEDLING
Anthocyanin
Anthocyanin
color
increasing anthocyanin






content


WHOLE
Color
High
the plant is purple in
Useful for increasing


PLANT

Anthocyanin
color
increasing anthocyanin






content


ROOT
No Growth in
No Growth in
roots grow along the
Useful for increasing root



Soil
Soil
soil surface instead of
growth eg to enhance





into the soil
nutrient uptake


ROOT
Other
Other
this correlates with
Useful for increasing root





any root mutant
growth eg to enhance





phenotypes which do
nutrient uptake





not fit into the above





categories (a picture





should be taken for





documentation)


LATERAL
Number
Less Lateral
there is an
Useful for increasing root


ROOTS

Roots
abnormally low
growth eg to enhance





number of lateral
nutrient uptake





roots


LATERAL
Other
Other
this correlates with
Useful for increasing root


ROOTS


any lateral root
growth eg to enhance





mutant phenotypes
nutrient uptake





which do not fit into





the above categories





(a picture should be





taken for





documentation)


ROOT
Classic
Classic
there is a lack of
Useful for increasing root





lateral roots (buds
growth eg to enhance





may appear but do
nutrient uptake





not elongate)


ROOT
Dwarf
Dwarf
there is a stunted root
Useful for increasing root





system
growth eg to enhance






nutrient uptake


ROOT
Mid-Section
Mid-Section
there are lateral roots
Useful for increasing root





in the top and bottom
growth eg to enhance





quarters of the whole
nutrient uptake





root, but none in the





middle


ROOT
Split
Split
appears as “classic”
Useful for increasing root





but with two primary
growth eg to enhance





roots, both
nutrient uptake





originating from the





hypocotyl base


ROOT
Other
Other
this correlates with
Useful for increasing root





any overall root
growth eg to enhance





structure mutant
nutrient uptake





phenotypes which do





not fit into the above





categories (a picture





should be taken for





documentation)


PRIMARY
Other
Other
this correlates with
Useful for increasing root


ROOT


any primary root
growth eg to enhance





mutant phenotypes
nutrient uptake





which do not fit into





the above categories





(a picture should be





taken for





documentation)


ROOT
Length
Longer Root
the root hairs are
Useful for increasing root


HAIRS

Hair
abnormally long
growth eg to enhance






nutrient uptake


ROOT
Length
Smaller Root
the root hairs are
Useful for increasing root


HAIRS

Hair
abnormally short
growth eg to enhance






nutrient uptake


ROOT
Number
Less root hairs
there is an
Useful for increasing root


HAIRS


abnormally low
growth eg to enhance





number of root hairs
nutrient uptake


ROOT
Other
Other
this correlates with
Useful for increasing root


HAIRS


any root hair mutant
growth eg to enhance





phenotypes which do
nutrient uptake





not fit into the above





categories (a picture





should be taken for





documentation)


ROOT
Bulbous Root
Bulbous Root
Bulbous Root Hairs
Useful for increasing root


HAIRS
Hairs
Hairs

growth eg to enhance






nutrient uptake


ROOT
Bearded
Bearded
the lateral roots are
Useful for increasing root



(Nitrogen)
(Nitrogen)
long in high nitrogen,
growth eg to enhance





and they are short in
nutrient uptake





low nitrogen


PRIMARY
Thickness
Thicker Primary
the primary root is
Useful for increasing root


ROOT

Root
abnormally thick
growth eg to enhance






nutrient uptake


WHOLE
Stress
Root
Identify plants with
Useful for increasing root


PLANT

Architecture
increased root mass
growth eg to enhance






nutrient uptake


PRIMARY
Thickness
Thinner Primary
the primary root is
Useful for increasing root


ROOT

Root
abnormally thin
growth eg to enhance






nutrient uptake


PRIMARY
Wavy
Wavy
there is a consistent
Useful for increasing root


ROOT


and gentle wavy
growth eg to enhance





appearance
nutrient uptake


LATERAL
Length
Longer Lateral
the lateral roots are
Useful for increasing root


ROOTS

Root
abnormally long
growth eg to enhance






nutrient uptake


LATERAL
Number
More Lateral
there is an
Useful for increasing root


ROOTS

Roots
abnormally high
growth eg to enhance





number of lateral
nutrient uptake





roots


ROOT
Number
More root hairs
there is an
Useful for increasing root


HAIRS


abnormally high
growth eg to enhance





number of root hairs
nutrient uptake






Useful for increasing seed






carbon or nitrogen


SEED
Seed Weight
Weight
weight of seed
Useful for increasing seed






weight


SILIQUES
Length
Long
siliques are
Useful for increasing





abnormally long (the
seed/fruit yield or





percent difference in
modifying fruit content





length compared to





the control should be





noted in the





comments)


SILIQUES
Length
Short
siliques are
Useful for increasing





abnormally short
seed/fruit yield or





(the percent
modifying fruit content





difference in length





compared to the





control should be





noted in the





comments)


SILIQUES
Other
Other
this correlates with
Useful for increasing





any silique mutant
seed/fruit yield or





phenotypes which do
modifying fruit content





not fit into the above





categories (a picture





should be taken for





documentation)


ROSETTE
Size
Large
rosette leaves are
Useful for increasing


LEAVES


abnormally large
vegetative growth and





(the percent
enhancing foliage





difference in size





compared to the





control should be





noted in the





comments)






Useful for making






nutraceuticals/pharmaceuticals






in plants


HYPOCOTYL
Other
Other
this correlates with
Useful for making larger





any hypocotyl mutant
plants





phenotypes which do





not fit into the above





categories (a picture





should be taken for





documentation)


WHOLE
Other
Other
this correlates with
Useful for making larger


SEEDLING


any whole plant
plants





mutant phenotypes





which do not fit into





the above categories





(a picture should be





taken for





documentation)


WHOLE
Other
Other
this correlates with
Useful for making larger


PLANT


any whole plant
plants





mutant phenotypes





which do not fit into





the above categories





(a picture should be





taken for





documentation)


CAULINE
Petiole Length
Long Petioles
the cauline petioles
Useful for making larger


LEAVES


are abnormally long
plants





(the percent





difference in size





compared to the





control should be





noted in the





comments)


WHOLE
Size
Large
plant is abnormally
Useful for making larger


SEEDLING


large (the percent
plants





difference in size





compared to the





control should be





noted in the





comments)


WHOLE
Size
Large
plant is abnormally
Useful for making larger


PLANT


large (the percent
plants





difference in size





compared to the





control should be





noted in the





comments)


SEED
Lethal
Lethal
the seed is inviable
Useful for making lethal





and appears as a
plants for genetic





small, dark, raisin-
confinement systems





like seed in the





mature silique


WHOLE
Germination
No Germination
none of the seed
Useful for making lethal


SEEDLING


germinates
plants for genetic






confinement systems


WHOLE
Germination
Poor
a portion of the seed
Useful for making lethal


SEEDLING

Germination
never germinates
plants for genetic






confinement systems


WHOLE
Germination
Slow
a portion of the seed
Useful for making lethal


SEEDLING

Germination
germinates
plants for genetic





significantly later
confinement systems





than the rest of the





seed in the pot


ROSETTE
Vitrified
Vitrified
leaves are somewhat
Useful for making lethal


LEAVES


translucent or ?water
plants for genetic





soaked?
confinement systems


CAULINE
Vitrified
Vitrified
leaves are somewhat
Useful for making lethal


LEAVES


translucent or ?water
plants for genetic





soaked?
confinement systems


COTYLEDONS
Albino
Opaque Albino
plant is opaque and
Useful for making lethal





devoid of pigment
plants for genetic






confinement systems


COTYLEDONS
Albino
Translucent
plant is translucent
Useful for making lethal




Albino
and devoid of
plants for genetic





pigment
confinement systems


WHOLE
Lethal
Seedling Lethal
cotyledons emerge
Useful for making lethal


SEEDLING


(although they are
plants for genetic





often small), but then
confinement systems





the plant ceases to





develop further; No





true leaves appear





and the plant dies





early (These differ





from yellow-green





lethals in that the





cotyledons are wild-





type in color and may





not look differ


WHOLE
Lethal
Yellow-Green
cotyledons are small
Useful for making lethal


SEEDLING

Lethal
and pale yellow-
plants for genetic





green in color, but
confinement systems





NOT totally devoid





of pigment; In





addition to yellow-





green cotyledons,





these plants produce





no or severely





reduced size true





leaves, which, if





present, are also





yellow-green; These





plants die prem


WHOLE
Meristem Mutant
Meristem Mutant
this term
Useful for making lethal


SEEDLING


encompasses a
plants for genetic





variety of
confinement systems





phenotypes, all of





which have one thing





in common, i.e., they





all have something





significantly wrong





with how the





meristem is





producing its leaves;





Depending on the





severity of the





phenotype, the plants





in this category


WHOLE
Seedling
Seedling
this term
Useful for making lethal


SEEDLING
Defective
Defective
encompasses a
plants for genetic





variety of phenotypes
confinement systems





which share similar





characteristics, i.e.,





they are small, have





distorted structures,





and are prone to early





death; For example,





patterning mutants





would be a class of





mutants which fall





under this category


WHOLE
Color
Yellow-Green
the leaves and
Useful for making lethal


PLANT

Viable 1
cotyledons are
plants for genetic





yellow-green in
confinement systems





color, but this is not a





lethal phenotype


WHOLE
Color
Yellow-Green
the leaves are yellow-
Useful for making lethal


PLANT

Viable 2
green in color but the
plants for genetic





cotyledons are a
confinement systems





wild-type green in





color


WHOLE
Color
Yellow-Green
the leaves start out
Useful for making lethal


PLANT

Viable 3
wild-type green and
plants for genetic





gradually turn
confinement systems





yellow-green in





color, while the





cotyledons stay wild-





type green


WHOLE
Color
Yellow-Green
the leaves appear
Useful for making lethal


PLANT

Viable 4
wild-type green, but
plants for genetic





slowly turn yellow-
confinement systems





green over time,





while the cotyledons





appear and remain





yellow-green


WHOLE
Stress
Seed Bleaching
Identify plants whose
Useful for making low


PLANT


seed coats do not
fiber seeds with increased





bleach out under long
digestability





bleach soaking


ROSETTE
Fused
Leaf Fused to
the leaf is fused to an
Useful for making


LEAVES

Inflorescence
inflorescence
ornamental plants with






flowers and leaves fused


ROSETTE
Interveinal
Interveinal
the leaf tissue is
Useful for making


LEAVES
Chlorosis
Chlorosis
chlorotic between its
ornamental plants with





veins
modified color


CAULINE
Interveinal
Interveinal
the leaf tissue is
Useful for making


LEAVES
Chlorosis
Chlorosis
chlorotic between its
ornamental plants with





veins
modified color


FLOWER
Organ
Fused Sepals
the sepals are fused
Useful for making



Morphology

together and won?t
ornamental plants with





open naturally, but
modified flowers





the flower is





otherwise wild-type


FLOWER
Organ
Narrow Petals
the petals are
Useful for making



Morphology

abnormally narrow
ornamental plants with






modified flowers


FLOWER
Organ
Narrow Sepals
the sepals are
Useful for making



Morphology

abnormally narrow
ornamental plants with






modified flowers


FLOWER
Organ
Short Petals
the petals are
Useful for making



Morphology

abnormally short
ornamental plants with






modified flowers


FLOWER
Organ
Short Sepals
the sepals are
Useful for making



Morphology

abnormally short
ornamental plants with






modified flowers


FLOWER
Size
Large
flower is abnormally
Useful for making





large (the percent
ornamental plants with





difference in size
modified flowers





compared to the





control should be





noted in the





comments)


FLOWER
Size
Small
flower is abnormally
Useful for making





small (the percent
ornamental plants with





difference in size
modified flowers





compared to the





control should be





noted in the





comments)


FLOWER
Other
Other
this correlates with
Useful for making





any flower mutant
ornamental plants with





phenotypes which do
modified flowers





not fit into the above





categories (a picture





should be taken for





documentation)


INFLORESCENCE
Aerial Rosette
Aerial Fosette
rosette forms at or
Useful for making





above the first
ornamental plants with





internode
modified flowers


INFLORESCENCE
Appearance
Corkscrew
the inflorescence is
Useful for making




Appearance
really twisted, almost
ornamental plants with





like a corkscrew, but
modified flowers





somewhat more





irregular


INFLORESCENCE
Appearance
Curved
the inflorescence has
Useful for making




Appearance
a slight, irregular
ornamental plants with





curve upwards,
modified flowers





greater than that of





the control plants


INFLORESCENCE
Appearance
Multi-
the inflorescence is
Useful for making




Inflorescence
fused to another
ornamental plants with




Fusion
inflorescence,
modified flowers





creating a celery-like





appearance


INFLORESCENCE
Appearance
Undulate
the inflorescence is
Useful for making




Appearance
wavy in appearance
ornamental plants with






modified flowers


INFLORESCENCE
Branching
Acauline
first branching is not
Useful for making




Branching
subtended by a
ornamental plants with





cauline leaf
modified flowers


INFLORESCENCE
Wax
Glaucous
inflorescence is
Useful for making





abnormally dull in
ornamental plants with





appearance
modified flowers


INFLORESCENCE
Wax
Glossy
inflorescence is
Useful for making





shiny/glossy in
ornamental plants with





appearance
modified flowers


INFLORESCENCE
Other
Other
this correlates with
Useful for making





any inflorescence
ornamental plants with





mutant phenotypes
modified flowers





which do not fit into





the above categories





(a picture should be





taken for





documentation)


COTYLEDONS
Asymmetric
Asymmetric
the shape of the
Useful for making





cotyledon is
ornamental plants with





asymmetric in
modified foliage





reference to the





vertical axis


ROSETTE
Other
Other
this correlates with
Useful for making


LEAVES


any leaf mutant
ornamental plants with





phenotypes which do
modified leaves





not fit into the above





categories (a picture





should be taken for





documentation)


CAULINE
Other
Other
this correlates with
Useful for making


LEAVES


any cauline mutant
ornamental plants with





phenotypes which do
modified leaves





not fit into the above





categories (a picture





should be taken for





documentation)


FLOWER
Homeotic
Homeotic
the flower has one or
Useful for making plants



Mutant
Mutant
more of its organs
sterile and for genetic





converted to another
confinement





type of organ





(specific details





should be noted in





the comments)


FLOWER
Organ
Aberrant Organ
there is an abnormal
Useful for making plants



Morphology
Number
number of some or
sterile and for genetic





all of the flowers
confinement





organs


FLOWER
Organ
Short Stamens
the stamens are
Useful for making plants



Morphology

abnormally short;
sterile and for genetic





This often leads to
confinement





mechanical problems





with fertility


FLOWER
Fertility
Aborted fertility
the ovule is
Useful for making plants





unfertilized and
sterile and for genetic





appears as a brown or
confinement





white speck in the





mature silique


FLOWER
Fertility
Female-sterile
there is a problem
Useful for making plants





with the ovules such
sterile and for genetic





that no fertilization is
confinement





occurring


FLOWER
Fertility
Male-sterile
there is a problem
Useful for making plants





with the pollen such
sterile and for genetic





that no fertilization is
confinement





occurring


FLOWER
Fertility
Reduced fertility
a reduced number of
Useful for making plants





successful
sterile and for genetic





fertilization events,
confinement





and therefore seeds,





are being produced





by the plant


FLOWER
Fertility
Sterile
no successful
Useful for making plants





fertilization events,
sterile and for genetic





and therefore no seed
confinement





is being produced by





the plant; The reason





for this sterility is not





known at the time of





the observation


FLOWER
Fertility
Other
this correlates with
Useful for making plants





any fertility mutant
sterile and for genetic





phenotypes which do
confinement





not fit into the above





categories (a picture





should be taken for





documentation)


WHOLE
Stress
Early Flowering
Identify plants that
Useful for making plants


PLANT


flower early
that flower early


COTYLEDONS
Petiole Length
Long Petioles
the cotyledon petioles
Useful for making plants





are abnormally long
that grow and better in





(the percent
shade





difference in size





compared to the





control should be





noted in the





comments)


ROSETTE
Petiole Length
Varying Petiole
the leaf petioles vary
Useful for making plants


LEAVES

Lengths
in length throughout
that grow better in shade





the rosette


ROSETTE
Petiole Length
Long Petioles
the leaf petioles are
Useful for making plants


LEAVES


abnormally long (the
that grow better in shade





percent difference in





size compared to the





control should be





noted in the





comments)






Useful for making plants






tolerant to biotic stress


WHOLE
Stress

Identify plants able to
Useful for making plants


PLANT


tolerate high density
tolerant to density and





and no phosphate and
low fertilizer





nitrogen, possible





lead assay for vigor





under population





density and low





nutrient conditions


WHOLE
Stress
pH (high)
Identify plants
Useful for making plants


PLANT


tolerant to high pH,
tolerant to high pH or low





and possibly low
phosphate





phosphate


WHOLE
Stress
Low Nitrate
Identify plants
Useful for making plants


PLANT


tolerant to low
tolerant to low nitrogen





nitrogen/nitrate





growth media


WHOLE
Stress
LNABA
Identify plants
Useful for making plants


PLANT


tolerant to low
tolerant to low nitrogen





nitrogen and high





ABA concentrations


WHOLE
Stress
No Nitrogen
Identify plants with
Useful for making plants


PLANT


increased vigor under
tolerant to low nitrogen





no nitrogen





conditions


WHOLE
Stress
MSX
Identify plants
Useful for making plants


PLANT


tolerant to nitrogen
tolerant to low nitrogen





assimilation inhibitor,





and possibly low





nitrogen tolerance





and/or seed nitrogen





accumulation


WHOLE
Stress
No N, No PO4
Identify plants
Useful for making plants


PLANT


tolerant to no
tolerant to low





nitrogen and no
nitrogen/low phosphate





phosphate growth





media


WHOLE
Stress
Oxidative
Identify plants
Useful for making plants


PLANT


tolerant to oxidative
tolerant to oxidative





stress
stresses


ROSETTE
Trichomes
Few Trichomes
trichomes are sparse
Useful for making plants


LEAVES


but present on the
with enhanced chemical





leaves
composition


ROSETTE
Trichomes
Glabrous
trichomes are totally
Useful for making plants


LEAVES


absent
with enhanced chemical






composition


ROSETTE
Trichomes
Abnormal
the trichomes are
Useful for making plants


LEAVES

Trichome Shape
abnormally shaped
with enhanced chemical






composition


CAULINE
Trichomes
Few Trichomes
trichomes are sparse
Useful for making plants


LEAVES


but present on the
with enhanced chemical





leaves
composition


CAULINE
Trichomes
Glabrous
trichomes are totally
Useful for making plants


LEAVES


absent
with enhanced chemical






composition


CAULINE
Trichomes
Abnormal
the trichomes are
Useful for making plants


LEAVES

Trichome Shape
abnormally shaped
with enhanced chemical






composition


INFLORESCENCE
Trichomes
Glabrous
trichomes are totally
Useful for making plants





absent
with enhanced chemical






composition


INFLORESCENCE
Trichomes
Abnormal
the trichomes are
Useful for making plants




Trichome Shape
abnormally shaped
with enhanced chemical






composition


ROSETTE
Curled
Corkscrew
leaves appear as
Useful for making plants


LEAVES


“Curled 5”, with the
with altered leaf shape eg





additional attribute of
curled leaves





twisting like a





corkscrew, instead of





uniformly curling





from both sides of the





leaf


ROSETTE
Curled
Cup-shaped
leaves are curled up
Useful for making plants


LEAVES


at the leaf margins
with altered leaf shape eg





such that they form a
curled leaves





cup or bowl-like





shape


ROSETTE
Curled
Curled 1
leaves are abnormally
Useful for making plants


LEAVES


curled slightly up or
with altered leaf shape eg





down at the leaf
curled leaves





margins, but do not





fall under the “cup-





shaped” description





(least severe type)


ROSETTE
Curled
Curled 2
leaves are abnormally
Useful for making plants


LEAVES


curled up or down at
with altered leaf shape eg





the leaf margins, but
curled leaves





do not fall under the





“cup-shaped”





description (more





severe than Curled 1,





but less severe than





Curled 3)


ROSETTE
Curled
Curled 3
leaves are abnormally
Useful for making plants


LEAVES


curled up or down at
with altered leaf shape eg





the leaf margins, but
curled leaves





do not fall under the





“cup-shaped”





description (more





severe than Curled 2,





but less severe than





Curled 4)


ROSETTE
Curled
Curled 4
leaves are abnormally
Useful for making plants


LEAVES


curled/rolled up or
with altered leaf shape eg





down at the leaf
curled leaves





margins (more





severe than Curled 3,





but less severe than





Curled 5)


ROSETTE
Curled
Curled 5
leaves are completely
Useful for making plants


LEAVES


curled/rolled up or
with altered leaf shape eg





down at the leaf
curled leaves





margins (most severe





type)


CAULINE
Curled
Corkscrew
leaves appear as
Useful for making plants


LEAVES


“Curled 5”, with the
with altered leaf shape eg





additional attribute of
curled leaves





twisting like a





corkscrew, instead of





uniformly curling





from both sides of the





leaf


CAULINE
Curled
Cup-shaped
the cauline leaves are
Useful for making plants


LEAVES


curled up at the leaf
with altered leaf shape eg





margins such that
curled leaves





they form a cup or





bowl-like shape


CAULINE
Curled
Curled 1
the cauline leaves are
Useful for making plants


LEAVES


abnormally curled
with altered leaf shape eg





slightly up or down at
curled leaves





the leaf margins, but





do not fall under the





“cup-shaped”





description (least





severe type)


CAULINE
Curled
Curled 2
the cauline leaves are
Useful for making plants


LEAVES


abnormally curled up
with altered leaf shape eg





or down at the leaf
curled leaves





margins, but do not





fall under the “cup-





shaped” description





(more severe than





Curled 1, but less





severe than Curled 3)


CAULINE
Curled
Curled 3
the cauline leaves are
Useful for making plants


LEAVES


abnormally curled up
with altered leaf shape eg





or down at the leaf
curled leaves





margins, but do not





fall under the “cup-





shaped” description





(more severe than





Curled 2, but less





severe than Curled 4)


CAULINE
Curled
Curled 4
the cauline leaves are
Useful for making plants


LEAVES


abnormally
with altered leaf shape eg





curled/rolled up or
curled leaves





down at the leaf





margins (more





severe than Curled 3,





but less severe than





Curled 5)


CAULINE
Curled
Curled 5
the cauline leaves are
Useful for making plants


LEAVES


completely
with altered leaf shape eg





curled/rolled up or
curled leaves





down at the leaf





margins (most severe





type)


ROSETTE
Size
Small
rosette leaves are
Useful for making plants


LEAVES


abnormally small
with decreased vegetative





(the percent
growth





difference in size





compared to the





control should be





noted in the





comments)


COTYLEDONS
Wilted
Wilted
cotyledons appear
Useful for making plants





wilted, i.e., they look
with enhanced abiotic





as though they have
stress tolerance





suffered from





drought conditions


ROSETTE
Wax
Glaucous
leaves are abnormally
Useful for making plants


LEAVES


dull in appearance
with enhanced abiotic






stress tolerance


ROSETTE
Wax
Glossy
leaves are
Useful for making plants


LEAVES


shiny/glossy in
with enhanced abiotic





appearance
stress tolerance


CAULINE
Wax
Glaucous
leaves are abnormally
Useful for making plants


LEAVES


dull in appearance
with enhanced abiotic






stress tolerance


CAULINE
Wax
Glossy
leaves are
Useful for making plants


LEAVES


shiny/glossy in
with enhanced abiotic





appearance
stress tolerance


WHOLE
Stress
Metabolic
Identify plants with
Useful for making plants


PLANT

Profiling
altered metabolic
with enhanced metabolite





profiles as defined in
accumulation





4a


WHOLE
Stress
Plant
Identify plants with
Useful for making plants


PLANT

Architecture
improved architecture
with enhanced plant






architecture


WHOLE
Stress
ABA
Identify plants
Useful for making plants


PLANT


tolerant to ABA, and
with enhanced tolerance





possibly drought
to drought





and/or other stresses


WHOLE
Stress
Mannitol
Identify plants
Useful for making plants


PLANT


tolerant to mannitol,
with enhanced tolerance





and possibly drought
to drought





stress


WHOLE
Stress
Dessication
Identify plants
Useful for making plants


PLANT


tolerant to water loss,
with enhanced tolerance





possibly drought
to drought





stress tolerant


WHOLE
Stress
High Sucrose
Identify plants
Useful for making plants


PLANT


tolerant to high
with enhanced tolerance





sucrose conditions
to drought





(possible Lead assay





for C/N partitioning)


WHOLE
Stress
Heat
Identify plants with
Useful for making plants


PLANT


thermotolerance
with enhanced tolerance






to heat


WHOLE
Stress
High Nitrogen
Identify plants
Useful for making plants


PLANT


tolerant to high
with enhanced tolerance





nitrogen conditions
to high nitrogen


WHOLE
Stress
Etiolation
Identify plants with
Useful for making plants


PLANT


increased vigor in the
with enhanced tolerance





dark
to light stress


ROSETTE
Disorganized
Disorganized
rosette leaves do not
Useful for making plants


LEAVES
Rosette
Rosette
appear in the normal
with increased biomass





fashion, i.e., their





phyllotaxy may be





abnormal or too





many leaves may be





emerging in





comparison to the





control


INFLORESCENCE
Phyllotaxy
Even Phyllotaxy
a phyllotaxy mutant
Useful for making plants





whose new branches
with increased biomass





emerge at exactly the





same height as each





other, i.e., there is no





internode between





them


COTYLEDONS
Shape
Elliptic Shape
cotyledons are quite
Useful for making plants





narrow and pointed,
with increased biomass





more so than
and foliage





lanceolate


ROSETTE
Fused
Leaf Fused to
the leaf is fused to its
Useful for making plants


LEAVES

Petiole
petiole
with increased biomass






and foliage


ROSETTE
Shape
Cordate Shaped
similar to ovate,
Useful for making plants


LEAVES


except the leaf is not
with increased biomass





rounded at its base
and foliage


ROSETTE
Shape
Elliptic Shaped
leaves are quite
Useful for making plants


LEAVES


narrow and pointed,
with increased biomass





more so that
and foliage





lanceolate


ROSETTE
Shape
Lanceolate
leaves are narrow and
Useful for making plants


LEAVES

Shaped
come to a dull point
with increased biomass





at the apex
and foliage


ROSETTE
Shape
Lobed Shaped
leaves have very deep
Useful for making plants


LEAVES


and rounded
with increased biomass





serrations, giving an
and foliage





appearance of many





lobes forming the





margins of the leaves


ROSETTE
Shape
Oval Shaped
leaves are much
Useful for making plants


LEAVES


rounder than wild-
with increased biomass





type
and foliage


ROSETTE
Shape
Ovate Shaped
leaves are wider at
Useful for making plants


LEAVES


base than at apex,
with increased biomass





otherwise similar to
and foliage





wild-type


ROSETTE
Shape
Serrate Margins
leaf margins have
Useful for making plants


LEAVES


little ?teeth? on them,
with increased biomass





i.e., they are serrated
and foliage


ROSETTE
Shape
Trident Shaped
leaves look
Useful for making plants


LEAVES


somewhat like a
with increased biomass





trident, i.e., they have
and foliage





a sharp point at the





apex, and a sharp





point on each side


ROSETTE
Shape
Undulate Shaped
leaves are wavy
Useful for making plants


LEAVES



with increased biomass






and foliage


WHOLE
Rosette Shape
Bushy Rosette
the different petioles
Useful for making plants


PLANT

Shaped
have very varied
with increased biomass





liminal angles, giving
and foliage





the plant a very





bushy appearance;





This is often





accompanied by a





“Disorganized





Rosette” phenotype


WHOLE
Rosette Shape
Flat Rosette
the petioles have a
Useful for making plants


PLANT

Shaped
very small liminal
with increased biomass





angle, i.e., the rosette
and foliage





appears flat instead of





having its usual slight





vertical angle


WHOLE
Rosette Shape
Standing Rosette
the petioles have a
Useful for making plants


PLANT

Shaped
very large liminal
with increased biomass





angle, i.e., it appears
and foliage





as though the leaves





are standing up





instead of having





their usual small





vertical angle from





the soil


CAULINE
Fused
Leaf Fused to
the cauline leaf is
Useful for making plants


LEAVES

Inflorescence
fused to an
with increased biomass





inflorescence or
and foliage





branch


CAULINE
Fused
Leaf Fused to
the cauline leaf is
Useful for making plants


LEAVES

Leaf
fused to itself or
with increased biomass





another cauline leaf
and foliage


CAULINE
Shape
Cordate Shaped
similar to ovate,
Useful for making plants


LEAVES


except the leaf is not
with increased biomass





rounded at its base
and foliage


CAULINE
Shape
Elliptic Shaped
leaves are quite
Useful for making plants


LEAVES


narrow and pointed,
with increased biomass





more so that
and foliage





lanceolate


CAULINE
Shape
Lanceolate
leaves are narrow and
Useful for making plants


LEAVES

Shaped
come to a dull point
with increased biomass





at the apex
and foliage


CAULINE
Shape
Lobed Shaped
leaves have very deep
Useful for making plants


LEAVES


and rounded
with increased biomass





serrations, giving an
and foliage





appearance of many





lobes forming the





margins of the leaves


CAULINE
Shape
Oval Shaped
leaves are much
Useful for making plants


LEAVES


rounder than wild-
with increased biomass





type
and foliage


CAULINE
Shape
Ovate Shaped
leaves are wider at
Useful for making plants


LEAVES


base than at apex,
with increased biomass





otherwise similar to
and foliage





wild-type


CAULINE
Shape
Serrate Margins
leaf margins have
Useful for making plants


LEAVES


little ?teeth? on them,
with increased biomass





i.e., they are serrated
and foliage


CAULINE
Shape
Trident Shaped
leaves look
Useful for making plants


LEAVES


somewhat like a
with increased biomass





trident, i.e., they have
and foliage





a sharp point at the





apex, and a sharp





point on each side


CAULINE
Shape
Undulate Shaped
leaves are wavy
Useful for making plants


LEAVES



with increased biomass






and foliage


CAULINE
Size
Large
cauline is abnormally
Useful for making plants


LEAVES


large (the percent
with increased biomass





difference in size
and foliage





compared to the





control should be





noted in the





comments)


CAULINE
Size
Small
cauline is abnormally
Useful for making plants


LEAVES


small (the percent
with increased biomass





difference in size
and foliage





compared to the





control should be





noted in the





comments)


LATERAL
Length
Smaller Lateral
the lateral roots are
Useful for making plants


ROOTS

Root
abnormally short
with increased root






growth to prevent lodging






or enhance nutrient






uptake


PRIMARY
Length
Long Primary
the primary root is
Useful for making plants


ROOT

Root
abnormally long
with increased root





(the percent
growth to prevent lodging





difference in size
or enhance nutrient





compared to the
uptake





control should be





noted in the





comments)


PRIMARY
Length
Short Primary
the primary root is
Useful for making plants


ROOT

Root
abnormally short
with increased root





(the percent
growth to prevent lodging





difference in size
or enhance nutrient





compared to the
uptake





control should be





noted in the





comments)


WHOLE
Stress
Plant Size
Identify plants of
Useful for making plants


PLANT


increased size
with increased size and





compared to wild
biomass





type


WHOLE
Stress
Starch
Identify plants with
Useful for making plants


PLANT


increased starch
with increased starch





accumulation
content


WHOLE
Stress
Cold
Identify plants that
Useful for making plants


PLANT

Germination
germinate better at
with increased tolerance





cold temperatures
to cold stress


WHOLE
Stress
Cold Growth
Identify plants that
Useful for making plants


PLANT


grow faster at cold
with increased tolerance





temperatures
to cold stress


WHOLE
Stress
Soil Drought
Identify plants with
Useful for making plants


PLANT


increased tolerance to
with increased tolerance





soil drought
to drought


WHOLE
Stress
Soil Drought —
Identify plants that
Useful for making plants


PLANT

Desiccation
are tolerant to low
with increased tolerance




tolerance
soil moisture and
to drought





resist wilting


WHOLE
Stress
PEG
Identify plants
Useful for making plants


PLANT


tolerant to PEG, and
with increased tolerance





possibly drought
to drought





stress


SEED
Size
Large
the seed is
Useful for making plants





abnormally large
with larger seeds





(the percent





difference in size





compared to the





control should be





noted in the





comments)


INFLORESCENCE
Branching
Asecondary
the plant does not
Useful for making plants




Branching
form any secondary
with modified flowers





inflorescences


SEED
Size
Small
the seed is
Useful for making plants





abnormally small
with smaller seeds or no





(the percent
seeds





difference in size





compared to the





control should be





noted in the





comments)


WHOLE
Stress
C/N Content
Identify plants/seeds
Useful for making seeds


PLANT


with altered
with altered





carbon/nitrogen
carbon/nitrogen levels





levels


INFLORESCENCE
Internode Length
Short Internode
the internode is
Useful for making shorter





abnormally short
plants and plants with





(the percent
modified flowers





difference in length





compared to the





control should be





noted in the





comments)


WHOLE
Dwarf
Brassino-Steroid
these plants are small
Useful for making smaller


PLANT

Dwarf
in stature, dark green,
plants





have oval leaves,





strong bolts, and are





often sterile


WHOLE
Dwarf
Misc. Dwarf
these are dwarf plants
Useful for making smaller


PLANT


the do not fall under
plants





the brassino-steroid





dwarf category


HYPOCOTYL
Length
Short
hypocotyl is visibly
Useful for making smaller





shorter than in wild-
plants





type (the percent





difference in size





compared to the





control should be





noted in the





comments)


INFLORESCENCE
Height
Short
the inflorescences of
Useful for making smaller





the plants are
plants





abnormally short





(plant height is





encompassed under





the whole plant size





category, but this





entry would be used





if the height of the





plant is abnormal, but





is otherwise of





normal size) (the





percent difference in





size


WHOLE
Size
Small
plant is abnormally
Useful for making smaller


SEEDLING


small (the percent
plants





difference in size





compared to the





control should be





noted in the





comments)


ROSETTE
Petiole Length
Short Petioles
the leaf petioles are
Useful for making smaller


LEAVES


abnormally short
plants





(the percent





difference in size





compared to the





control should be





noted in the





comments)


WHOLE
Size
Small
plant is abnormally
Useful for making smaller


PLANT


small (the percent
plants





difference in size





compared to the





control should be





noted in the





comments)


CAULINE
Petiole Length
Short Petioles
the cauline petioles
Useful for making smaller


LEAVES


are abnormally short
plants





(the percent





difference in size





compared to the





control should be





noted in the





comments)


INFLORESCENCE
Strength
Strong
the primary
Useful for making





inflorescence appears
stronger plants





significantly stronger,





whether by thickness





or rigidity


INFLORESCENCE
Strength
Weak
the primary
Useful for making





inflorescence appears
stronger plants





significantly weaker,





whether by thickness





or rigidity


INFLORESCENCE
Inflorescence
Thickness
thickness of the
Useful for making





primary inflorescence
stronger plants


HYPOCOTYL
Length
Long
hypocotyl is visibly
Useful for making taller





longer than in wild-
plants





type (the percent





difference in size





compared to the





control should be





noted in the





comments)


INFLORESCENCE
Internode Length
Long Internode
the internode is
Useful for making taller





abnormally long (the
plants and plants with





percent difference in
longer flowers





length compared to





the control should be





noted in the





comments)


INFLORESCENCE
Height
Tall
the inflorescences of
Useful for making taller





the plants are
plants and plants with





abnormally long
longer inflorescences





(plant height is





encompassed under





the whole plant size





category, but this





entry would be used





if the height of the





plant is abnormal, but





is otherwise of





normal size) (the





percent difference in





size


SEED
Color
Dark Color
the seed is
Useful for modifying





abnormally dark
fiber content in seed


SEED
Color
Light Color
the seed is
Useful for modifying





abnormally light;
fiber content in seed





Transparent Testa is





an example of this





phenotype


SILIQUES
Shape
Bent
the silique has sharp
Useful for modifying fruit





bend to it part of the
shape, composition and





way down the length
seed yield





of the silique; this





bend can be as much





as approaching 90





degrees


SILIQUES
Shape
Bulging
the seeds in the
Useful for modifying fruit





silique appears
shape, composition and





“shrink-wrapped”,
seed yield





giving the silique a





bulging appearance


SILIQUES
Shape
Clubbed
the silique is
Useful for modifying fruit





somewhat bulbous at
shape, composition and





its terminal end
seed yield


SILIQUES
Shape
Sickle
the silique is curved,
Useful for modifying fruit





much like the blade
shape, composition and





of a sickle
seed yield


INFLORESCENCE
Branching
No Branching
there is no branching
Useful for modifying





at all
plant architecture, ie






amount of branching


INFLORESCENCE
Branching
Horizontal
new branches arise at
Useful for modifying




Branching
a 90 degree angle
plant architecture, ie





from the bolt they are
branch angle





emerging from


COTYLEDONS
Horizontally
Horizontally
cotyledon is visibly
Useful for modifying



Oblong
Oblong
wider than it is long,
plant architecture, ie leaf





and it is also
structure





symmetrical (or very





close to it) when cut





along its horizontal





axis


INFLORESCENCE
Branching
Two Leaf
two cauline leaves
Useful for modifying




Branching
subtend branches
plant architecture, ie





instead of one
reducing foliage


INFLORESCENCE
Branching
Reduced Apical
the dominance of the
Useful for modifying




Dominance
primary inflorescence
plant structure, ie





is diminished, with
increased branching





the secondaries





appearing as





dominant or nearly as





dominant


SEED
Seed
Stacked
the seeds/embryos
Useful for modifying seed



Arrangement
Arrangement
are stacked one on
content





top of the other





within the silique,





instead of having the





usual side-by-side





distribution


SEED
Other
Other
this correlates with
Useful for modifying seed





any seed mutant
content





phenotypes which do





not fit into the above





categories (a picture





should be taken for





documentation)


SEED
Shape
Oval Shape
the seeds are much
Useful for modifying seed





more rounded on the
structure and composition





ends, giving the seed





a true oval





appearance


SEED
Shape
Ridged Shape
the seeds have small
Useful for modifying seed





ridges or bumps on
structure and composition





them


SEED
Shape
Tapered Shape
the ends of the seeds
Useful for modifying seed





narrow down to a
structure and composition





much sharper point





than usual


COTYLEDONS
Cotyledon
Single Cotyledon
Only one cotyledon
Useful for modifying seed



Number

appears after
structure and content





germination; This is





simply one cotyledon





that had formed





instead of two, and is





not related to the





fused phenotype;





With this exception,





the plant is often





otherwise wild-type





in appearance


COTYLEDONS
Cotyledon
Tricot
three cotyledons
Useful for modifying seed



Number

emerge instead of
structure and content





two; With this





exception, the plant is





often otherwise wild-





type in appearance


COTYLEDONS
Curled
Cup-shaped
cotyledons are curled
Useful for modifying seed





up at the cotyledon
structure and content





margins such that





they form a cup or





bowl-like shape


COTYLEDONS
Curled
Curled 1
cotyledons are
Useful for modifying seed





abnormally curled
structure and content





slightly up or down at





the cotyledon





margins, but do not





fall under the “cup-





shaped” description





(least severe type)


COTYLEDONS
Curled
Curled 2
cotyledons are
Useful for modifying seed





abnormally curled up
structure and content





or down at the





cotyledon margins,





but do not fall under





the “cup-shaped”





description (more





severe than Curled 1,





but less severe than





Curled 3)


COTYLEDONS
Curled
Curled 3
cotyledons are
Useful for modifying seed





abnormally curled up
structure and content





or down at the





cotyledon margins,





but do not fall under





the “cup-shaped”





description (more





severe than Curled 2,





but less severe than





Curled 4)


COTYLEDONS
Curled
Curled 4
cotyledons are
Useful for modifying seed





abnormally
structure and content





curled/rolled up or





down at the





cotyledon margins





(more severe than





Curled 3, but less





severe than Curled 5)


COTYLEDONS
Curled
Curled 5
cotyledons are
Useful for modifying seed





completely
structure and content





curled/rolled up or





down at the





cotyledon margins





(most severe type)


COTYLEDONS
Dimorphic
Dimorphic
one cotyledon is
Useful for modifying seed



Cotyledons
Cotyledons
significantly larger
structure and content





than the other


COTYLEDONS
Fused
Fused 1
cotyledons are fused
Useful for modifying seed





to each other,
structure and content





creating one





cotyledon structure





(least severe type)


COTYLEDONS
Fused
Fused 2
cotyledons are fused
Useful for modifying seed





to each other,
structure and content





creating one





cotyledon structure





(more severe than





Fused 1, but less





severe than Fused 3)


COTYLEDONS
Fused
Fused 3
cotyledons are fused
Useful for modifying seed





to each other,
structure and content





creating one





cotyledon structure





(more severe than





Fused 2, but less





severe than Fused 4)


COTYLEDONS
Fused
Fused 4
cotyledons are fused
Useful for modifying seed





to each other,
structure and content





creating one





cotyledon structure





(more severe than





Fused 3, but less





severe than Fused 5)


COTYLEDONS
Fused
Fused 5
cotyledons are fused
Useful for modifying seed





to each other,
structure and content





creating one





cotyledon structure





(most severe type)


COTYLEDONS
Other
Other
this correlates with
Useful for modifying seed





any cotyledon mutant
structure and content





phenotypes which do





not fit into the above





categories (a picture





should be taken for





documentation)


ROSETTE
Fused
Leaf Fused to
the leaf is fused to
Useful for plants with


LEAVES

Leaf
itself or another leaf
fused leaves eg






ornamentals


COTYLEDONS
Petiole Length
Short Petioles
the cotyledon petioles
Useful for shade





are abnormally short
avoidance and for making





(the percent
smaller plants





difference in size





compared to the





control should be





noted in the





comments)


PRIMARY
Agravitropic
Agravitropic
the primary root does


ROOT


not appear to have a





gravitropic response


PRIMARY
Kinked
Kinked
there is a sharp bend


ROOT


in the root


ROSETTE
Rosette Diameter
Diameter
diameter of rosette


LEAVES


WHOLE
Plant Weight
Plant Weight
weight of whole plant


PLANT


WHOLE
Plant Height
Height
height of whole plant


PLANT


WHOLE
Plant DTH
Dth
days to harvest of


PLANT


plant


WHOLE
Plant Harvest
Harvest Index
harvest index of plant


PLANT
Index


CAULINE
Fused
Leaf Fused to
the cauline leaf is


LEAVES

Petiole
fused to its petiole


N/A
N/A
N/A
N/A


WHOLE
HERBICIDE
HERBICIDE
herbicide segregation


PLANT
SEGREGATION
SEGREGATION
ratio


WHOLE
N/A
No Mutant
The plants were


PLANT

Phenotype
screened at all




Observed
appropriate stages





and showed no





mutant phenotype,





i.e., they looked like





normal, wild type





Arabidopsis plants









From the results reported in Table 1 and the Sequence Listing, it can be seen that the 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;
        l. increased chlorophyll and photosynthetic capacity;
        m. increased anthocyanin content;
        n. increased root growth, and increased nutrient uptake;
        o. increased or decreased seed weight or size, increased seed carbon or nitrogen content;
        p. modified, including increased, seed/fruit yield or modified fruit content;
        q. enhanced foliage;
        r. usefulness for making nutratceuticals/pharmaceuticals in plants;
        s. plant lethality;
        t. decrease seed fiber content to provide increased digestability;
        u. modified ornamental appearance with modified leaves, flowers, color or foliage;
        v. modified sterility in plants;
        w. enhanced ability to grow in shade;
        x. enhanced biotic stress tolerance;
        y. increased tolerance to density and low fertilizer;
        z. 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;
        ll. 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 (SEQ II) NO: 90)
Qualitative Analysis of the T1 Plants:
All 10 of the events produced rosettes with more leaves and more inflorescences than the control. The plants were also slightly smaller than the control (Table 1-1). The transgenic “control” was a set of plants expressing a different 35S::cDNA but which were indistinguishable from the untransformed WS wildtype. This method of scoring phenotypes is typical for our large-scale morphological phenotyping project.







TABLE 1-1







Qualitative phenotypes observed in 35S::cDNA 13499809 T1 events











Increased Rosette Leaf




Number, Increased




Inflorescence Number,



Event
& Slightly Smaller







ME04701-01
x



ME04701-02
x



ME04701-03
x



ME04701-04
x



ME04701-05
x



ME04701-06
x



ME04701-07
x



ME04701-08
x



ME04701-09
x



ME04701-10
x










Quantitative Analysis of the T2 Plants:

Events ME04701-08 and ME04701-09 were evaluated in greater detail in the T2 generation. These two events were selected because they had the most advantageous phenotypes. Eighteen individuals were sown and observed for both events. The transgenic plants showed an increased number of inflorescences to a 0.05 level of statistical significance (Table 1-2). The T2 plants did not have significantly more leaves than the controls, unlike in the T1. ME04701-08 was slightly later flowering than the control. ME04701-09 had significantly larger rosettes than the control. All plants noted in the table as ME04701-08 and ME04701-09 were segregating progeny of the T1 which exhibited the phenotype of interest. All plants noted in the table as -08 or -09 Control were T2 segregating progeny which did not exhibit the phenotype and did not contain the transgene (internal controls; Table 1-2).

Segregation frequencies of the plants under test suggest that each event contains a single insert, as calculated by a Chi-square test (Table 1-2 and data not shown).
The increase in the inflorescence number for the two events was much less than the increase observed when the 35S promoter was used to express this cDNA (data not shown). This evidence further supports our hypothesis that the degree of expression/dosage of the gene product is highly relevant to the strength of the observed phenotype. By using a promoter with a different expression pattern, we were able to keep the positive phenotype of the previously observed 35S phenotype, while removing the negative aspects of infertility previously observed. Of course, the trade-off is to lessen the positive phenotype, although keeping it significant.







TABLE1-2







Quantitative phenotypes observed in p326F::cDNA 13499809 T2 events


(PIT = Primary Inflorescence Thickness)
















Rosette
Number


Days




Number of
Area
of
Height
PIT
to
Number of


Event/Control
Observations
(mm2)
Leaves
(cm)
(mm)
Bolt
Inflorescences

















ME04701-08
14
1241.8
6.0
42.1
0.99
17.8*
4.3*


-08 Control
4
1419.1
5.8
38.8
1.02
16.5
2.8


ME04701-09
14
1620.0*
5.9
40.1
1.01
16.8
4.8*


-09 Control
4
996.5
6.0
40.4
0.93
16.6
2.8





*significantly different from control at 0.05 level, via t-test


Although all of the plants in this experiment had fewer inflorescences than the general greenhouse population, the plants were healthy. The transgenics had significantly greater number of inflorescences than the control, so the overall decrease - which was due to greenhouse conditions prevailing at the time of the experiment - in the number of inflorescences did not affect the conclusions of the experiment.






Lead Summary/Discussion:

    
    
        Over-expression of Lead 28/cDNA 13499809 with an appropriate promoter results in an increase in the number of inflorescences. As this is a glycine-rich protein (GRP) there is a likely effect on cell wall structure affecting cell expansion or adhesion, different positioning of cell planes, and/or different opportunities for inflorescence initiation. It would be interesting to combine this gene with the gene encoding an unknown protein with an AP2 which also affects plant growth and development.
        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.
    
    
























Event
Percent



Event 1
Event 4
Event 5
Event 7
Average
Increase























Plant weight
1952
1888
1423
1682
1523
1629
110%



Transgenic



1952 Control
1516
1471
1383
1559
1482


Fruit weight
1952
5892
3704
5131
5814
5135
105%


per plant
Transgenic



1952 Control
4746
4826
4601
5343
4879


Percent red
1952
40.1
42.4
36.5
47.2
42
107%


fruit
Transgenic



1952 Control
42.4
46.7
28.7
37.8
39


Harvest index
1952
75.7%
72.2%
75.3%
79.2%
76%
99%



Transgenic



1952 Control
75.8%
76.6%
76.9%
77.4%
77%









    
    
        Increased vegetative biomass can give an improved source:sink ratio and improved fixation of carbon to sucrose and starch, leading to improved yield.
        More inflorescences gives the opportunity for more flowers and therefore more seeds. The combination of improved biomass and inflorescence number can give a significant improvement in yield.

Tomato Field Trial Results

    
    


Clone 1952 was transformed into tomato under the control of the plasmid p326. 4 independent transgenic events were selected for field testing. Results are shown in the following Table 1-3. On the average, there is an increase in total plant weight, fruit weight and percent red fruit per plant. Event 4 did not show an improvement in performance. If event 4 is not considered in the analysis the average plant weight, fruit weight and percent red fruit each increase to approximately 115% of control.
Table 1-3-Results from Tomato Field Trials
Example 2
Lead 29-ME04717-Clone 123905-cDNA 12562634 (SEQ ID NO: 92)


    
    
        Ectopic expression of Ceres cDNA 12562634 under the control of the 326D promoter induces a number of phenotypes including:
        
            Increased number of inflorescences
            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.

Qualitative Analysis of the T1 Plants:

    
    


Using the 326D promoter, 9 of the 10 events produced rosettes with more leaves and more inflorescences than the control (Table 1). One of the 9 events also had fertility defects, much like what was seen using 35S::cDNA 12562634. The transgenic “control” was a set of plants expressing different 35S::cDNA constructs and which were indistinguishable from the untransformed WS wildtype. This method of scoring phenotypes is typical for our large-scale morphological phenotyping project.







TABLE 2-1







Qualitative phenotypes observed in p326D::cDNA 12562634


T1 events (2 events with the most advantageous


phenotypes were chosen for T2 evaluation)










Increased Rosette Leaf




Number & Increased


Event
Inflorescence Number
Fertility Defects





ME04717-02
x
x


ME04717-03
x


ME04717-04
x


ME04717-05
x


ME04717-06
x


ME04717-07
x


ME04717-08
x


ME04717-09
x


ME04717-10
x









Quantitative Analysis of the T2 Plants:

Events ME04717-03 and ME04717-05 were evaluated in greater detail in the T2 generation. Eighteen individuals were sown and observed for both events. The transgenic plants showed an increased number of inflorescences to a 0.05 level of statistical significance. ME04717-03 also had significantly larger rosettes than the control. All plants noted in Table 2-2 as ME04717-03 and ME04717-05 were segregating progeny of the T1 which exhibited the phenotype of interest. All plants noted in the Table 2-2 as -03 or -05 Control were T2 segregating progeny which did not exhibit the phenotype and did not contain the transgene (internal controls; Table 2-2).

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).
It should be noted that the increase in the inflorescence number for the events documented below was less than the increase observed in the 35S::cDNA 12532634 events (data not shown). Other p326D::cDNA 12532634 T2 events, not shown in this report, contained multiple inserts. Some of the T2 progeny of these multiple insert-containing events exhibited some negative effects (fertility defects and dwarfing) similar to the T2 progeny of the 35S::cDNA 12532634 events. This evidence further supports our hypothesis that the degree of expression/dosage of the gene product is highly relevant to the strength of the observed phenotype. By using a new promoter, and creating transgenics with a single insert, we were able to keep the positive phenotype of the previously observed 35S phenotype, while removing the negative aspects previously seen. A consequence of accomplishing this goal is a lessening of the degree of the positive phenotype, although keeping it at a very significant level.







TABLE 2-2







Quantitative phenotypes observed in p326D::cDNA 12562634 T2 events


(PIT = Primary Inflorescence Thickness)
















Rosette
Number


Days




Number of
Area
of
Height
PIT
to
Number of


Event/Control
Observations
(mm2)
Leaves
(cm)
(mm)
Bolt
Inflorescences

















ME04717-03
13
2701.5*
7.7*  
35.2
0.94
18.0
8.4*


-03 Control
5
1086.9
6.6
32.7
0.99
18.6
3.8


ME04717-05
14
1057.6
5.7
35.0
0.91
16.1
7.3*


-05 Control
4
504.6
5.0
29.3
0.71
16.0
4.0





*significantly different from control at 0.05 level, via t-test


The decrease in stature and flowering time is accurate. The plants were healthy, but may have been flowering earlier than other plants grown in the greenhouse at that time. This is especially the case for the flat containing ME04717-05 and its controls. All plants were treated equally within the flat Our goal was only to assay for inflorescence number.






Lead Summary/Discussion:

    
    
        Ectopic expression of Ceres cDNA 12562634 under the control of the 326D promoter induces a number of phenotypes including increased number of inflorescences and more 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.
        There is also likely to be a positive impact on harvest index although it has not yet been measured.
        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.
        Increased vegetative biomass can give an improved source:sink ratio and improved fixation of carbon to sucrose and starch, leading to improved yield.
        More inflorescences gives the opportunity for more flowers and therefore more seeds. The combination of improved biomass and inflorescence number can give a significant improvement in yield.

Tomato Yield Trial Results

    
    


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, their harvest index improved. These events also had increases in percent red fruit per plant. Event 14 had increased biomass and yield.







TABLE 2-3







Tomato Field Trial Results













Per plant

1
14
21
26
average
















Leaf/stem
C5
1410.0
1537.1
1294.4
1564.1
1451.4


weight
C5 control
1866.4
1215.9
1738.8
1766.0
1646.8


fruit weight
C5
4300.5
4936.5
4122.5
4159.0
4379.6



C5-control
4293.5
4608.5
4098.0
4877.0
4469.3


Percent
C5
35.3
33.2
56.2
36.9
40.4


red fruit
C5-control
16.7
33.0
45.8
34.8
32.6


Harvest
C5
75%
76%
76%
73%
75%


index
C5-control
70%
79%
70%
73%
73%









In summary, tomato plants transformed with gene 123905 tended to have more branches and leaves, and more fruit as compared to control.
Rice Field Trial Results:
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

Days to
Days to
Approx.



of plants
Tillers
first
mid
leaf



Plants
per plant
flower
flower
angle















123905-1
1060
7.1
22
32
33.1


123905-4-6
200
8.2
19
28
45


123905-8-3
650
7.4
28
33
32.1


123905-12-3
300
8.9
22
33
38.6


Kitaake control
1200
5.4
22
31
31.2









Several events showed significant reductions in height. 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.







TABLE 2-5







Results from Rice Field Trials












Plant
Biomass
Yield
Total Yield



Height
(grams per
(grams per
per plot



(cm)
plant)
plant)
(gms)














123905-1
54.0
25.3
12.52
417.0


123905-4-6
37.0
19.6
7.82
116.5


123905-8-3
65.5
30.6
14.9
668.8


123905-12-3
48.8
23.3
10.02
312.3


Kitaake
61.2
26.7
13.59
537.5









Observations on Reduced Stature in Rice

Gene 123905 was transformed into rice cultivar Kitaake under the control of p326. Measurements were conducted to determine which internodes were reduced in 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.







TABLE 2-6







Results from Rice Field Trials















Plant









height
No.

Internode
Internode
Internode
Internode



(cm)
panicle
Internode I
II
III
IV
V


















123905-1
89.0
8.8
35.2
20.0
7.9
3.0
0.1


123905-4-6
68.2
18.3
30.4
16.4
4.6
1.7
0.1


123905-8-3
111.6
9.8
38.4
24.8
19.8
10.6
0.8


123905-12-3
82.6
12.2
32.4
21.4
7.0
4.1
0.3


Kitaake
110.6
10.0
36.6
24.5
19.8
11.6
0.4


Control









Observations on Germination in Rice

Transgenic lines 123905-1 and 123905-12-3 germinate 1 to 2 days faster than Kitaake control seed.
Example 3
Lead 36-ME03195-Clone 679923-cDNA 13594332 (SEQ ID NO:98)
Clone 679923 in the 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).
Qualitative Analysis of the T1 Plants:
All 5 events produced larger rosettes with slightly curled leaves with little to no petiole elongation, and very short inflorescences compared to the controls. These plants were also delayed in flowering time by several days and had no fertility defects (Table 3-1). The transgenic “control” was a set of plants expressing a different 35S::cDNA fusion and which were indistinguishable from the untransformed WS wildtype. This method of scoring phenotypes is typical for our large-scale morphological phenotyping project. After seed collection, it was also apparent that these plants produced a significantly higher number of seeds relative to typical mutants of their height.







TABLE 3-1







Qualitative phenotypes observed in 35S::cDNA 13594332 T1 events











Large rosettes with curled leaves with




short/no petioles, short inflorescences,



Event
delayed flowering time







ME03195-01
x



ME03195-02
x



ME03195-03
x



ME03195-04
x



ME03195-05
x










Quantitative Analysis of the T2 Plants:

The original hypothesis formulated from the T1 observations was that the 35S::cDNA 13594332 plants may have a significantly increased harvest index. Events ME03195-02 and ME03195-04 were evaluated in greater detail in the T2 generation to test this hypothesis. Eighteen individuals were sown and observed for both events. 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).
After detailed T2 analyses, we determined the following regarding the transgenics (results below are statistically significant to a 0.05 level or better via t-test unless otherwise noted):

    
    
        Flowering time (days to bolt) was 5-8 days later than controls.
        Rosette leaf number at bolt was increased by approximately 2.5 leaves.
        Rosette area was 2-3 times larger than controls.
        Height was approximately 1⁄2 that of controls.
        Total seed weight was not significantly different than controls.
        Total plant dry weight was slightly greater for event -04, and no different than the controls for event −02.
        Harvest index was slightly lower than the controls.
        Twice as much seed was produced per unit height of plant than in controls.

Details can be found in Tables 3-2 and 3-3.

    
    









TABLE 3-2







Quantitative phenotypes observed in p35S::cDNA


13594332 T2 events
















Rosette



Event/
Number of
Day to
Number of
Area
Height


Control
Observations
Bolt
Leaves
(mm2)
(cm)















ME03195-02
12
 26*
9.6*
6735.43*
18.63*


-02 Control
3
18
7
1802.62
42.33


ME03195-04
8
  24.9*
10.3*
7758.16*
25.06*


-04 Control
7
19
7.7
2884.43
44.71





*significantly different from control at 0.05 level, via t-test













TABLE 3-3







Quantitative phenotypes observed in p35S::cDNA 13594332


T2 events














Seed
Plant

Seed Weight



Number of
Weight
Weight
Harvest
(g) per Unit


Event/Control
Observations
(g)
(g)
Index
Height (cm)















ME03195-02
12
0.375
0.73
53.25*
0.0204*


-02 Control
3
0.376
0.58
64.47
0.0090


ME03195-04
8
0.431
0.8258*
53.26*
0.0171*


-04 Control
7
0.397
0.6119
64.90
0.0089





*significantly different from control at 0.05 level, via t-test






Events -02 and -04 each had three T2 plants which exhibited a much more severe form of the above-described phenotype. These plants were severely late bolting, had little inflorescence elongation, and were nearly sterile. From other experiments using these plant lines (data not shown), we determined that the detrimental phenotype is due to a dosage/homozygous insert effect, suggesting that hemi/heterozygous plants gave a beneficial trait of increased seed production per unit height, but that the homozygous lines gave the negative phenotype. Our statistical analyses compared the internal controls to the plants which contained the transgene and beneficial phenotype. All transgene-containing plants with the detrimental phenotype were omitted from the statistical analyses in Tables 3-2 and 3-3.
Example 4
ME04012-Gemini ID 5000F6 (SEQ 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.
Example 5
Lead 15-ME04077-Clone 92459-cDNA 12561537 (SEQ ID NO: 80)
Clone 92459 in the Ceres Arabidopsis cDNA library, contains cDNA 12561537, encoding Arabidopsis MADS Affecting Flowering 1 (MAF1). 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 phenotype in Arabidopsis when overexpressed.

    
    
        Ectopic expression of Ceres cDNA 12561537 under the control of the 35S promoter induces a number of phenotypes including:
        
            Taller plants
            Thicker inflorescences
            Larger rosettes
            Increased rosette leaf number
            Delayed flowering
        
        
        Misexpression of Ceres cDNA 12561537 can be useful to increase overall plant size/biomass.

Qualitative Analysis of the T1 Plants:

    
    


All ten events were late flowering, produced larger rosettes with more leaves and tall, thick inflorescences compared to the controls (Table 5-1). The transgenic “control” was a set of different 35S::cDNA expressing plants which were indistinguishable from the untransformed WS wild type. This method of scoring phenotypes is typical for our large-scale morphological phenotyping project.







TABLE 5-1







Qualitative phenotypes observed in 35S::cDNA 12561537 T1 events











Increased Rosette Size
Late



Event
Increased Rosette Leaf Number
Flowering
Tall & Thick





ME04077-01
X
X
X


ME04077-02
X
X
X


ME04077-03
X
X
X


ME04077-04
X
X
X


ME04077-05
X
X
X


ME04077-06
X
X
X


ME04077-07
X
X
X


ME04077-08
X
X
X


ME04077-09
X
X
X


ME04077-10
X
X
X









Quantitative Analysis of the T2 Plants:

Events ME04077-06 and ME04077-10 were evaluated in greater detail in the T2 generation. Eighteen individuals were sown and observed for event 06, whereas 17 individuals were sown and observed for event 10. The transgenic plants for both events showed increased primary inflorescence thickness, increased number of rosette leaves, a larger rosette, and delay of flowering time to a 0.05 level of statistical significance (Table 5-2). The plants of both events were visibly much taller than the controls, but only event -10 was quantitatively taller to a 0.05 level of statistical significance via t-test. If a greater number of internal controls were available for event -06, this event would very likely fall under the same degree of significance via the same test. Both events had normal fertility. All plants noted in the table as ME04077-06 and ME04077-10 were segregating progeny of the T1 event which we had confirmed to contain the transgene under test. All plants noted in the table as -06 Control or -10 Control were T2 segregating progeny which did not contain the transgene under test (internal controls).
Both events produce significantly more seeds than the control, as would be expected for a typical, fertile, late flowering plant.
Event ME04077-06 had 12 transgene-containing pl 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-containing 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 T2 seeds segregate 3R:1S for both events (data not shown).







TABLE 5-2







Quantitative phenotypes observed in 35S::cDNA 12561537 T2 events














Number of
Rosette
Number
Height
Primary Inflorescence



Event/Control
Observations
Area (mm2)
of Leaves
(cm)
Thickness (inches)
Days to Bolt
















ME04077-06
12
7302.7*
15.6*
72.8
0.068*
23.9


-06 Control
3
1666.3
6.7
55.9
0.046
18.3


ME04077-10
9
9343.9*
19.6*
73.0*
0.086*
24.4*


-10 Control
7
2696.1
8.9
52.3
0.053
18.6





*significantly different from control at 0.05 level, via t-test






Lead Summary/Discussion:

    
    
        The ectopic expression of cDNA 12561537 with a strong constitutive promoter (35S) results in taller plants, with thicker inflorescences, a larger rosette, and more rosette leaves.
        The increase in plant size seen by this expression is accompanied by a delay in flowering time, but no reduction in fertility.
        It can also be a useful gene to increase root growth, given the similar expression pattern in shoot meristems and root tip cells.
        Increased vegetative biomass can give an improved source:sink ratio and improved fixation of carbon to sucrose and starch, leading to improved yield.
        Taller inflorescences give the opportunity for more flowers and therefore more seeds. The combination of improved biomass and inflorescence stature can give a significant improvement in yield.
        Thicker inflorescences may prevent against “snap” against wind, rain or drought.
        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”.

Tomoto Field Trial Results

    
    


This Lead 15 (clone 92459) was transformed into tomato under the control of plasmid p13879. 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.







TABLE 5-3







Tomato Field Trial Results












Event-13
Percent Increase













Plant weight
92459 Transgenic
2042.54
120%



92459 Control
1707.50


Fruit weight per plant
92459 Transgenic
4932
100%



92459 Control
4956


Percent red fruit
92459 Transgenic
28.9
 93%



92459 Control
31.0


Harvest index
92459 Transgenic
71%
 95%



92459 Control
74%









Example 6
Determination of Functional Homolog Sequences
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. Sci. 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.
Before starting a Reciprocal BLAST process, 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. In the forward search step, a query polypeptide sequence, “polypeptide A,” from source species SA 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.
In the reverse search round, the 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 SA. 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 FIGS. 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 FIGS. 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 FIGS. 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. Thus, useful polypeptides include those which comprise one or more of the numbered conserved regions in each alignment table in an individual Figure depicted in FIGS. 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 FIGS. 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 FIGS. 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 FIGS. 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 FIGS. 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.
The invention being thus described, it will be apparent to one of ordinary skill in the art that various modifications of the materials and methods for practicing the invention can be made. Such modifications are to be considered within the scope of the invention as defined by the following claims.
Each of the references from the patent and periodical literature cited herein is hereby expressly incorporated in its entirety by such citation.
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## Claims

1. A method of increasing size, vegetative growth, architecture, seedling vigor, growth rate, fruit and seed yield or biomass of a plant, said method comprising the steps of:
(a) transforming plant cells with an isolated nucleic acid molecule comprising a nucleotide sequence encoding a polypeptide having an amino acid sequence that is at least 95% identical to the amino acid sequence set forth in SEQ ID NO: 93, and wherein the nucleotide sequence is operably linked to the promoter p326 which has the nucleotide sequence according to SEQ ID NO. 76;
(b) expressing said polypeptide in the transformed plant cells;
(c) regenerating transgenic plants from said transformed plant cells; and
(d) identifying a transgenic plant from said transgenic plants, which exhibits increase in size, vegetative growth, architecture, seedling vigor, growth rate, fruit and seed yield or biomass as compared to an untransformed plant of the same plant species.

2. The method of claim 1, wherein said plant is a tomato plant.

3. The method of claim 1, wherein said nucleic acid molecule comprises a nucleotide sequence encoding a polypeptide having an amino acid sequence that is at least 97% identical to the amino acid sequence set forth in SEQ ID NO: 93.

4. The method of claim 1, wherein said nucleic acid molecule comprises a nucleotide sequence encoding a polypeptide having the amino acid sequence set forth in SEQ ID NO: 93.

5. A method of increasing size, vegetative growth, seedling vigor, growth rate, fruit and seed yield, biomass, fruit weight, percent red fruit or harvest index in a tomato plant, said method comprising the steps of:
(a) transforming tomato plant cells with an isolated nucleic acid molecule comprising a nucleotide sequence encoding an amino acid sequence that is at least 95% identical to the amino acid sequence set forth in SEQ ID NO: 93, and wherein the nucleotide sequence is operably linked to the promoter p326 which has the nucleotide sequence according to SEQ ID NO. 76;
(b) expressing said polypeptide in the transformed tomato plant cells;
(c) regenerating transgenic tomato plants from said transformed tomato plant cells; and
(d) identifying a transgenic tomato plant from said transgenic tomato plants, which exhibits increase in size, vegetative growth, seedling vigor, growth rate, fruit and seed yield, biomass, fruit weight, percent red fruit or harvest index as compared to an untransformed tomato plant.

6. The method of claim 5, wherein said nucleic acid molecule comprises a nucleotide sequence encoding a polypeptide having an amino acid sequence that is at least 97% identical to the amino acid sequence set forth in SEQ ID NO: 93.

7. The method of claim 5, wherein said nucleic acid molecule comprises a nucleotide sequence encoding a polypeptide having the amino acid sequence set forth in SEQ ID NO: 93.

8. A method for increasing tillers per plant or increasing seed yield in a rice plant, said method comprising the steps of:
(a) transforming rice plant cells with an isolated nucleic acid molecule comprising a nucleotide sequence encoding an amino acid sequence that is at least 95% identical to the amino acid sequence set forth in SEQ ID NO: 93, and wherein the nucleotide sequence is operably linked to the promoter p326 which has the nucleotide sequence according to SEQ ID NO. 76;
(b) expressing said polypeptide in the transformed rice plant cells;
(c) regenerating transgenic rice plants from said transformed rice plant cells; and
(d) identifying a transgenic rice plant from said transgenic rice plants, which exhibits increase in tillers per plant or increase in seed yield as compared to an untransformed plant of the same plant species.

9. The method of claim 8, wherein said nucleic acid molecule comprises a nucleotide sequence encoding a polypeptide having an amino acid sequence that is at least 97% identical to the amino acid sequence set forth in SEQ ID NO: 93.

10. The method of claim 8, wherein said nucleic acid molecule comprises a nucleotide sequence encoding a polypeptide having the amino acid sequence set forth in SEQ ID NO: 93.