Manipulating BS1 for plant seed yield

The present invention provides methods for increasing plant seed yield or seed size through reduced expression of a BS1 gene. Also provided are plants with increased seed yield or seed size comprising reduced expression of a BS1 gene produced by such methods.

BACKGROUND OF THE INVENTION

Field of the Invention

The present invention relates generally to the field of molecular biology. More specifically, the invention relates to plant genes involved in plant morphology and methods of use thereof.

INCORPORATION OF SEQUENCE LISTING

The sequence listing that is contained in the file named “NBLE090US-revised_ST25.txt”, which is 252 kilobytes as measured in Microsoft Windows operating system and was created on Mar. 31, 2016, is filed electronically herewith and incorporated herein by reference.

BACKGROUND OF THE INVENTION

Genetic modification of plants has, in combination with conventional breeding programs, led to significant increases in agricultural yield over the last decades. Genetically modified plants may be selected for a single agronomic trait, for example by expression of a single enzyme coding sequence (e.g., enzymes that provide herbicide resistance). Genetic manipulation of genes involved in plant growth and yield may enable increased production of valuable commercial crops, resulting in benefits in agriculture and development of alternate energy sources such as biofuels. Accordingly, methods capable of increasing seed yield and/or seed size through gene regulation are described.

SUMMARY OF THE INVENTION

In one aspect, the invention provides a method of increasing seed yield comprising reducing expression of a BS1 gene in a plant, wherein the seed yield of the plant is increased when compared to a plant that lacks the reduced expression. In one embodiment, the BS1 gene is a gene set forth in Table 1. In another embodiment, the plant is a dicotyledonous plant, such as a plant selected from the group consisting ofArabidopsis thaliana, Arabidopsis lyrata, Carica papaya, Ricinus communis, Cucumis sativus, Prunus persica, Vitis vinifera, Manihot esculenta, Citrus sinensis, Eutrema salsugineum, Citrus clementina, Capsella rubella, Aquilegia coerulea, Malus domestica, Linum usitatissimum, Eucalyptus grandis, Solanum lycopersicum, Solanum tuberosum, Gossypium raimondii, Populus trichocarpa, Phaseolus vulgaris, Solanum lycopersicum, Theobroma cacao, Conradina grandiflora, Mimulus guttatus, Brassica rapa, Boechera stricta, Arachis hypogaea, Medicago trunculata, Medicago sativa, Glycine max, cotton, carrot, canola, tomato, potato, alfalfa, grape, clover, poplar, willow,eucalyptus, hemp, aLotussp., aVincasp., aNicotianasp., aVitissp., or aRicinussp. In other embodiments, the plant has altered morphology when compared to a plant that lacks the increased expression, such as increased plant biomass or increased seed yield. In another embodiment, reducing expression of the BS1 gene comprises use of an antisense or RNAi construct targeting the BS1 gene, or mutation of the BS1 gene.

In another aspect, the invention provides a plant comprising reduced expression of a BS1 gene, wherein the seed yield of the plant is increased when compared to a plant that lacks the reduced expression. In certain embodiments, the invention also provides a seed that produces such a plant, or a seed produced by such a plant. In another embodiment, the invention also provides a DNA-containing plant part of such a plant, which may be further defined a protoplast, cell, meristem, root, leaf, node, pistil, anther, flower, seed, embryo, stalk or petiole.

In another aspect, the invention provides a plant comprising reduced expression of a BS1 gene, wherein the seed yield of the plant is increased when compared to a plant that lacks the reduced expression. In certain embodiments, the invention also provides a seed that produces such a plant or a seed produced by such a plant. In another embodiment, the invention also provides a DNA-containing plant part of such a plant, which may be further defined a protoplast, cell, meristem, root, leaf, node, pistil, anther, flower, seed, embryo, stalk or petiole

In another aspect, the invention provides a method of producing a plant comprising increased seed yield, the method comprising: (a) obtaining a plant comprising reduced expression of a BS1 gene, wherein the seed yield of the plant is increased when compared to a plant that lacks the reduced expression; (b) growing said plant under plant growth conditions to produce plant tissue from the plant; (c) crossing said plant with itself or another, distinct plant to produce progeny plants; and (d) selecting a progeny plant comprising reduced expression of a BS1 gene, wherein said progeny plant comprises increased seed yield when compared to a plant that lacks the reduced expression.

In another aspect, the invention provides a transgenic plant comprising a selected DNA, wherein the selected DNA down regulates a cell proliferation factor, wherein said down-regulation increases seed yield or seed size. In an embodiment, the selected DNA comprises a BS1 sequence, such as set forth in Table 1. In another embodiment, the selected DNA is an antisense or RNAi construct. In another embodiment, the transgenic plant is further defined as a legume or an R0 transgenic plant. In another embodiment, the transgenic plant is further defined as a progeny plant of any generation of an R0 transgenic plant, wherein the transgenic plant has inherited the selected DNA from the R0 transgenic plant.

BRIEF DESCRIPTION OF THE SEQUENCE LISTING

SEQ ID NOs:1-2—DNA sequences of the forward and reverse primers for genotyping and cloning BS1 inMedicago truncatula.

SEQ ID NOs:3-4—DNA sequence of the forward and reverse primers for amplification of the deletion border inMedicago truncatula.

SEQ ID NOs:5-6—DNA sequence of primers for amplification of the BS1 genomic sequence inMedicago truncatula.

SEQ ID NOs:7-10—DNA sequence of primers for genotyping for PPD inArabidopsis thaliana.

SEQ ID NOs:11-14—DNA sequence of primers for genotyping for AN3 inArabidopsis thaliana.

SEQ ID NOs:15-21—DNA sequence of primers for performing tail PCR of the deletion ends of BS1 inM. truncatula.

SEQ ID NOs:22-45—DNA sequence of primers for performing qRT-PCR.

SEQ ID NOs:46-60—DNA sequence of primers for construct components.

SEQ ID NOs:61-68—DNA sequence of primers for performing ChIP-qPCR.

SEQ ID NOs:69-118—DNA sequences of BS1 genes listed in Table 1.

SEQ ID NOs:119-168—Protein sequences of BS1 genes listed in Table 1.

SEQ ID NOs:169-170—genomic DNA sequences of additional orthologs ofM. sativaBS1.

SEQ ID NOs:171-172—Protein sequences of additional orthologs ofM. sativaBS1.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides a method of increasing the seed yield in a plant by reducing or eliminating expression of a BS1 gene. Plants of the present invention that exhibit reduced expression of a BS1 gene demonstrate beneficial traits including increased seed yield, increased seed size and weight, increased pod size, increased leaf size, and increased plant biomass when compared to a plant that lacks the reduced expression.

The size of plant lateral organs such as seeds and leaves is controlled by a plant's genetic make-up and also by the environmental conditions under which plants are grown. Plant lateral organs are primary sources of food and feed and as such, methods for increasing these would be beneficial. To facilitate an improvement in crop yield, the inventors provide for the first time a conserved BIG SEEDS1 (BS1) regulatory module that controls plant organ size inMedicago truncatula, soybean (Glycine max), andArabidopsis thaliana. BS1 encodes a plant-specific transcription regulator that determines lateral organ growth by negatively targeting factors that mediate cell proliferation. By downregulating BS1 orthologs in soybean (Glycine max) using microRNA (miRNA), the inventors have been able to significantly increase seed size and weight in these plants, thus providing a powerful strategy for increasing soybean seed yield. Thus, the present invention comprises methods for reducing or eliminating expression of a BS1 gene in a plant comprising introducing into the plant a nucleic acid sequence that when expressed in the plant produces reduces or eliminates expression of a BS1 gene. Such a sequence may comprise a nucleic acid sequence such as a dsRNA or miRNA and may result in increased seed yield or seed size in the plant.

Thus, in one embodiment, a plant in accordance with the invention having increased seed yield or seed size may comprise reduced expression of a BS1 gene sequence, such as a sequence set forth herein in Table 1. In another embodiment, a plant with increased seed yield or seed size may lack expression of a BS1 gene sequence, such as a sequence set forth herein in Table 1. In another embodiment, a plant in accordance with the invention having increased seed yield or seed size may comprise reduced expression of a BS1 gene, such as a gene set forth in Table 1. In other embodiments, the invention provides primers which may be useful for detection or amplification of a sequence as described herein. Such sequences are set forth herein as SEQ ID NOs: 1-68. In another embodiment, such primers may be useful for detecting the presence of absence of a gene or sequence of the invention. In accordance with the invention, nucleic acid and/or protein sequences may share sequence identity at the nucleic acid or amino acid level. For example, such sequences may share 100%, 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 89%, 88%, 87%, 86%, 85%, 84%, 83%, 82%, 81%, 80% sequence identity, or the like.

I. Nucleic Acids, Polypeptides and Plant Transformation Constructs

Certain embodiments of the current invention concern isolated nucleic acid sequences comprising a BS1 coding sequence, set forth herein in Table 1. The invention also provides sequences complementary to such sequences. Also provided are primers for detecting or amplifying a sequence in accordance with the invention, which are set forth herein as SEQ ID NOs:1-68. Complements to any nucleic acid sequences described herein are also provided.

“Identity,” as is well understood in the art, is a relationship between two or more polypeptide sequences or two or more polynucleotide sequences, as determined by comparing the sequences. In the art, “identity” also means the degree of sequence relatedness between polypeptide or polynucleotide sequences, as determined by the match between strings of such sequences. Methods to determine “identity” are designed to give the largest match between the sequences tested. Moreover, methods to determine identity are codified in publicly available programs. “Identity” can be readily calculated by known methods including, but not limited to, those described in Lesk, ed., (1988); Smith, ed., (1993); Griffin, and Griffin, eds., (1994); von Heinje, (1987); Gribskov and Devereux, eds., (1991); and Carillo and Lipman, (1988). Computer programs that can be used to determine “identity” between two sequences may include but are in no way limited to, GCG (Devereux, 1984); suite of five BLAST programs, three designed for nucleotide sequences queries (BLASTN, BLASTX, and TBLASTX) and two designed for protein sequence queries (BLASTP and TBLASTN) (Coulson, 1994; Birren, et al., 1997). The BLASTX program is publicly available from NCBI and other sources (BLAST Manual, Altschul et al., NCBI NLM NIH, Bethesda, Md. 20894; Altschul et al., 1990). The well known Smith Waterman algorithm can also be used to determine identity.

Parameters for polypeptide sequence comparison include the following: Algorithm: Needleman and Wunsch (1970); Comparison matrix: BLOSUM62 from Hentikoff and Hentikoff, (1992); Gap Penalty: 12; and Gap Length Penalty: 4. A program which can be used with these parameters is publicly available as the “gap” program from Genetics Computer Group, Madison Wis. The above parameters along with no penalty for end gap may serve as default parameters for peptide comparisons.

Parameters for nucleic acid sequence comparison are known in the art and may include the following: Algorithm: Needleman and Wunsch (1970); Comparison matrix: matches=+10; mismatches=0; Gap Penalty: 50; and Gap Length Penalty: 3. A program which can be used with these parameters is publicly available as the “gap” program from Genetics Computer Group, Madison Wis. The above parameters may serve as the default parameters for nucleic acid comparisons.

As used herein, “hybridization,” “hybridizes,” or “capable of hybridizing” is understood to mean the forming of a double- or triple-stranded molecule or a molecule with partial double- or triple-stranded nature. Such hybridization may take place under relatively high-stringency conditions, including low salt and/or high temperature conditions, such as provided by a wash in about 0.02 M to about 0.15 M NaCl at temperatures of about 50° C. to about 70° C. for 10 min. In one embodiment of the invention, the conditions are 0.15 M NaCl and 70° C. Stringent conditions tolerate little mismatch between a nucleic acid and a target strand. Such conditions are well known to those of ordinary skill in the art, and are preferred for applications requiring high selectivity. Non-limiting applications include isolating a nucleic acid, such as a gene or a nucleic acid segment thereof, or detecting at least one specific mRNA transcript or a nucleic acid segment thereof, and the like.

Coding sequences, such as a BS1 coding sequence, or complements thereof, may be provided in a recombinant vector or construct operably linked to a heterologous promoter functional in plants, in either sense or antisense orientation. Expression constructs may also be provided comprising these sequences, including antisense oligonucleotides thereof. In one embodiment, such a recombinant vector or construct may encode an RNA molecule such as a miRNA that reduces or eliminates the expression of a gene involved in cell proliferation in the plant, such as a BS1 gene. In other embodiments, plants and plant cells transformed with the sequences may be provided. The construction of vectors which may be employed in conjunction with plant transformation techniques using these or other sequences according to the invention will be known to those of skill of the art in light of the present disclosure (see, for example, Sambrook et al., 1989; Gelvin et al., 1990). The techniques of the current invention are thus not limited to any particular nucleic acid sequences.

The choice of any additional elements used in conjunction with the BS1 sequences may depend on the purpose of the transformation. One of the major purposes of transformation of crop plants is to add commercially desirable, agronomically important traits to the plant, as described herein. Such traits may include, but are not limited to increased seed yield, increased seed size and weight, increased pod size, increased leaf size, and increased plant biomass, pesticide resistance, herbicide tolerance, drought tolerance, and the like.

Vectors or constructs used for plant transformation may include, for example, plasmids, cosmids, YACs (yeast artificial chromosomes), BACs (bacterial artificial chromosomes) or any other suitable cloning system known in the art, as well as fragments of DNA therefrom. Thus, when the term “vector” or “expression vector” is used, all of the foregoing types of vectors, as well as nucleic acid sequences isolated therefrom, are included. It is contemplated that utilization of cloning systems with large insert capacities will allow introduction of large DNA sequences comprising more than one selected gene. In accordance with the invention, this could be used to introduce genes corresponding to, e.g., an entire biosynthetic pathway, into a plant.

Particularly useful for transformation are expression cassettes which have been isolated from such vectors. DNA segments used for transforming plant cells will generally comprise the cDNA, gene, or genes which one desires to introduce into and have expressed in the host cells. These DNA segments can further include structures such as promoters, enhancers, polylinkers, or even regulatory genes as desired. The DNA segment or gene chosen for cellular introduction will often encode a protein which will be expressed in the resultant recombinant cells resulting in a screenable or selectable trait and/or which will impart an improved phenotype to the resulting transgenic plant. In an embodiment, introduction of such a construct into a plant may result in increased expression of a particular gene in the plant. In another embodiment, introduction of such a construct may result in reduction or elimination of expression of a particular gene. Preferred components likely to be included with vectors used in the current invention are as follows.

A. Regulatory Elements

As used herein, “gene suppression” or “reduced expression” or “decreased expression” may refer to any of the well-known methods for reducing the levels of protein produced as a result of gene transcription to mRNA and subsequent translation of the mRNA. Gene suppression also refers to the reduction of protein expression from a gene or a coding sequence including posttranscriptional gene suppression and transcriptional suppression. Posttranscriptional gene suppression is mediated by homology between all or a part of an mRNA molecule transcribed from a gene or coding sequence targeted for suppression and the corresponding double stranded RNA used for suppression, and refers to the substantial and measurable reduction of the amount of mRNA in the cell. The transcribed RNA can be in the sense orientation, in the anti-sense orientation, or in both orientations. Transcriptional suppression is mediated by the presence in the cell of a nucleic acid molecule such as a double-stranded RNA (dsRNA) exhibiting substantial sequence identity to a desired DNA sequence or the complement thereof. Such suppression may be effective against a native plant gene associated with a trait, or a gene that may be introduced into the plant.

Post-transcriptional gene suppression by anti-sense or sense oriented RNA to regulate gene expression in plant cells is disclosed in, for example, U.S. Pat. Nos. 5,107,065, 5,759,829, 5,283,184, and 5,231,020. The use of dsRNA or miRNA to suppress genes in plants is disclosed in WO 99/53050, WO 99/49029, U.S. Patent Application Publication No. 2003/0175965, and 2003/0061626, U.S. patent application Ser. No. 10/465,800, and U.S. Pat. Nos. 6,506,559, and 6,326,193.

A preferred method of posttranscriptional gene suppression in plants employs both sense-oriented and anti-sense-oriented, transcribed RNA which is stabilized, e.g., as a hairpin and stem and loop structure. A preferred DNA construct for effecting post transcriptional gene suppression may be one which encodes an RNA exhibiting an anti-sense orientation exhibiting substantial identity to a segment of a gene targeted for suppression. Such a sequence may be linked to a second segment encoding an RNA exhibiting substantial complementarity to the first segment. Such a construct would be expected to form a stem and loop structure by hybridization of the first segment with the second segment and a loop structure from the nucleotide sequences linking the two segments (see WO94/01550, WO98/05770, US 2002/0048814, and US 2003/0018993). In particular embodiments, the present invention thus provides methods of reducing or eliminating expression of a BS1 gene through expression of a dsRNA or miRNA targeting a BS1 gene in the plant, as well as plants produced by such methods.

Exemplary promoters for expression of a nucleic acid sequence include plant promoters such as the CaMV 35S promoter (Odell et al., 1985), or others such as CaMV 19S (Lawton et al., 1987), nos (Ebert et al., 1987), Adh (Walker et al., 1987), sucrose synthase (Yang and Russell, 1990), α-tubulin, actin (Wang et al., 1992), cab (Sullivan et al., 1989), PEPCase (Hudspeth and Grula, 1989) or those promoters associated with the R gene complex (Chandler et al., 1989). Tissue-specific promoters such as leaf specific promoters, or tissue selective promoters (e.g., promoters that direct greater expression in leaf primordia than in other tissues), and tissue-specific enhancers (Fromm et al., 1986) are also contemplated to be useful, as are inducible promoters such as ABA- and turgor-inducible promoters. Any suitable promoters known in the art may be used to express a nucleic acid sequence in accordance with the invention in a plant. In an embodiment, such a nucleic acid sequence may encode an RNA molecule such as a miRNA or dsRNA that results in reduction or elimination of expression of a BS1 gene in a plant. In an embodiment of the invention, the CaMV35S promoter or a native promoter may be used to express an RNA molecule that results in reduction or elimination of expression of a BS1 gene in a plant.

The DNA sequence between the transcription initiation site and the start of the coding sequence, i.e., the untranslated leader sequence, can also influence gene expression. One may thus wish to employ a particular leader sequence with a transformation construct of the invention. In an embodiment, leader sequences are contemplated to include those which comprise sequences predicted to direct optimum expression of the attached gene, i.e., to include a consensus leader sequence which may increase or maintain mRNA stability and prevent inappropriate initiation of translation. The choice of such sequences will be known to those of skill in the art in light of the present disclosure. In some embodiments, sequences that are derived from genes that are highly expressed in plants may be used for expression of nucleic acid sequences targeting a BS1 gene in a plant.

It is envisioned that nucleic acid sequences targeting a BS1 gene may be introduced under the control of novel promoters, enhancers, etc., or homologous or tissue-specific or tissue-selective promoters or control elements. Vectors for use in tissue-specific targeting of genes in transgenic plants will typically include tissue-specific or tissue-selective promoters and may also include other tissue-specific or tissue-selective control elements such as enhancer sequences. Promoters which direct specific or enhanced expression in certain plant tissues will be known to those of skill in the art in light of the present disclosure. These include, for example, the rbcS promoter, specific for green tissue; the ocs, nos and mas promoters, which have higher activity in roots.

Transformation constructs prepared in accordance with the invention may include a 3′ end DNA sequence that acts as a signal to terminate transcription and allow for the polyadenylation of the mRNA produced by coding sequences operably linked to a promoter. In one embodiment of the invention, the native terminator of a BS1 sequence may be used. Alternatively, a heterologous 3′ end may enhance the expression of sense or antisense BS1 sequences. Examples of such sequences that may be used in this context include those from the nopaline synthase gene ofAgrobacterium tumefaciens(nos 3′ end) (Bevan et al., 1983), the terminator sequence for the T7 transcript from the octopine synthase gene ofAgrobacterium tumefaciens, and the 3′ end of the protease inhibitor I or II gene from potato or tomato. Regulatory elements such as an Adh intron (Callis et al., 1987), sucrose synthase intron (Vasil et al., 1989) or TMV omega element (Gallie et al., 1989), may further be included where desired.

C. Transit or Signal Peptides

Sequences that are joined to the coding sequence of an expressed gene, which are removed post-translationally from the initial translation product and which facilitate the transport of the protein into or through intracellular or extracellular membranes, are termed transit (usually into vacuoles, vesicles, plastids and other intracellular organelles) and signal sequences (usually to the endoplasmic reticulum, Golgi apparatus, and outside of the cellular membrane). By facilitating the transport of the protein into compartments inside and outside the cell, these sequences may increase the accumulation of gene products by protecting them from proteolytic degradation. These sequences also allow for additional mRNA sequences from highly expressed genes to be attached to the coding sequence of the genes. Since mRNA being translated by ribosomes is more stable than naked mRNA, the presence of translatable mRNA in front of the gene may increase the overall stability of the mRNA transcript from the gene and thereby increase synthesis of the gene product. Since transit and signal sequences are usually post-translationally removed from the initial translation product, the use of these sequences allows for the addition of extra translated sequences that may not appear on the final polypeptide. It further is contemplated that targeting of certain proteins may be desirable in order to enhance the stability of the protein (U.S. Pat. No. 5,545,818, incorporated herein by reference in its entirety).

Additionally, vectors may be constructed and employed in the intracellular targeting of a specific gene product within the cells of a transgenic plant or in directing a protein to the extracellular environment. This generally will be achieved by joining a DNA sequence encoding a transit or signal peptide sequence to the coding sequence of a particular gene. The resultant transit or signal peptide will transport the protein to a particular intracellular or extracellular destination, respectively, and will then be post-translationally removed.

By employing a selectable or screenable marker, one can provide or enhance the ability to identify transformants. “Marker genes” are genes that impart a distinct phenotype to cells expressing the marker protein and thus allow such transformed cells to be distinguished from cells that do not have the marker. Such genes may encode either a selectable or screenable marker, depending on whether the marker confers a trait which one can “select” for by chemical means, i.e., through the use of a selective agent (e.g., a herbicide, antibiotic, or the like), or whether it is simply a trait that one can identify through observation or testing, i.e., by “screening” (e.g., β-glucuronidase ‘GUS,’ green fluorescent protein). Of course, many examples of suitable marker proteins are known to the art and can be employed in the practice of the invention.

Many selectable marker coding regions are known and could be used with the present invention including, but not limited to, neo (Potrykus et al., 1985), which provides kanamycin resistance and can be selected for using kanamycin, G418, paromomycin, etc.; bar, which confers bialaphos or phosphinothricin resistance; a mutant EPSP synthase protein (Hinchee et al., 1988) conferring glyphosate resistance; a nitrilase such as bxn fromKlebsiella ozaenaewhich confers resistance to bromoxynil (Stalker et al., 1988); a mutant acetolactate synthase (ALS) which confers resistance to imidazolinone, sulfonylurea or other ALS inhibiting chemicals (European Patent Application 154, 204, 1985); a methotrexate resistant DHFR (Thillet et al., 1988), a dalapon dehalogenase that confers resistance to the herbicide dalapon; or a mutated anthranilate synthase that confers resistance to 5-methyl tryptophan.

An illustrative embodiment of selectable marker capable of being used in systems to select transformants are those that encode the enzyme phosphinothricin acetyltransferase, such as the bar gene fromStreptomyces hygroscopicusor the pat gene fromStreptomyces viridochromogenes. The enzyme phosphinothricin acetyl transferase (PAT) inactivates the active ingredient in the herbicide bialaphos, phosphinothricin (PPT). PPT inhibits glutamine synthetase, (Murakami et al., 1986; Twell et al., 1989) causing rapid accumulation of ammonia and cell death.

One beneficial use of the sequences provided by the invention may be in the alteration of plant phenotypes by genetic transformation with nucleic acid molecules, such as miRNA or dsRNA molecules, which are complementary to a BS1 coding sequence. Such nucleic acid molecules may be provided with other sequences. Where an expressible coding region that is not necessarily a marker coding region is employed in combination with a marker coding region, one may employ the separate coding regions on either the same or different DNA segments for transformation. In the latter case, the different vectors are delivered concurrently to recipient cells to maximize cotransformation.

Additionally provided herein are transgenic plants transformed with a recombinant vector as described herein encoding or producing a BS1 sequence, or a sequence modulating expression thereof. In an embodiment, a recombinant vector as described herein may encode a dsRNA or miRNA complementary to a BS1 sequence.

In one embodiment, the disclosure provides a transgenic plant or plant cell comprising a polynucleotide molecule or a recombinant DNA construct as described herein, wherein the polynucleotide molecule or recombinant DNA construct encodes or produces a BS1 sequence, or a variant or homologue thereof. In an embodiment, the polynucleotide molecule or a recombinant DNA construct may result in the downregulation or elimination of BS1 expression in the plant. The disclosure therefore also provides progeny of these plants, vegetative, propagative, and reproductive parts of the plants comprising a transgene encoding a BS1 sequence. In an embodiment, a plant in accordance with the present disclosure comprises increased seed yield or seed size relative to a plant not comprising such a polynucleotide molecule or DNA construct.

Suitable methods for transformation of plant or other cells for use with the current invention are believed to include virtually any method by which DNA can be introduced into a cell, such as by direct delivery of DNA, byAgrobacterium-mediated transformation (U.S. Pat. No. 5,591,616 and U.S. Pat. No. 5,563,055; both specifically incorporated herein by reference) by acceleration of DNA coated particles (U.S. Pat. No. 5,550,318; U.S. Pat. No. 5,538,877; and U.S. Pat. No. 5,538,880; each specifically incorporated herein by reference in its entirety), etc. Through the application of techniques such as these, the cells of virtually any plant species may be stably transformed, and these cells developed into transgenic plants.

Agrobacterium-mediated transfer is a widely applicable system for introducing genes into plant cells because the DNA can be introduced into whole plant tissues, thereby bypassing the need for regeneration of an intact plant from a protoplast. The use ofAgrobacterium-mediated plant integrating vectors to introduce DNA into plant cells is well known in the art. See, for example, the methods described by Fraley et al., (1985), Rogers et al., (1987) and U.S. Pat. No. 5,563,055, specifically incorporated herein by reference in its entirety.

Agrobacterium-mediated transformation is most efficient in dicotyledonous plants and is the preferable method for transformation of dicots, includingArabidopsis, tobacco, tomato, alfalfa and potato. Indeed, whileAgrobacterium-mediated transformation has been routinely used with dicotyledonous plants for a number of years, including alfalfa (Thomas et al., 1990), it has only recently become applicable to monocotyledonous plants. Advances inAgrobacterium-mediated transformation techniques have now made the technique applicable to nearly all monocotyledonous plants. For example,Agrobacterium-mediated transformation techniques have now been applied to rice (Hiei et al., 1997; U.S. Pat. No. 5,591,616, specifically incorporated herein by reference in its entirety), wheat (McCormac et al., 1998), barley (Tingay et al., 1997; McCormac et al., 1998) and maize (Ishidia et al., 1996).

ModernAgrobacteriumtransformation vectors are capable of replication inE. colias well asAgrobacterium, allowing for convenient manipulations as described (Klee et al., 1985). Moreover, recent technological advances in vectors forAgrobacterium-mediated gene transfer have improved the arrangement of genes and restriction sites in the vectors to facilitate the construction of vectors capable of expressing various polypeptide coding genes. The vectors described (Rogers et al., 1987) have convenient multi-linker regions flanked by a promoter and a polyadenylation site for direct expression of inserted polypeptide coding genes and are suitable for present purposes. Gateway™ and other recombination-based cloning technology is also available in vectors useful for plant transformation. In addition,Agrobacteriumcontaining both armed and disarmed Ti genes can be used for the transformations. In those plant strains whereAgrobacterium-mediated transformation is efficient, it is the method of choice because of the facile and defined nature of the gene transfer.

One also may employ protoplasts for electroporation transformation of plants (Bates, 1994; Lazzeri, 1995). For example, the generation of transgenic soybean plants by electroporation of cotyledon-derived protoplasts is described by Dhir and Widholm in Intl. Patent Appl. Publ. No. WO 9217598 (specifically incorporated herein by reference). Other examples of species for which protoplast transformation has been described include barley (Lazerri, 1995), sorghum (Battraw et al., 1991), maize (Bhattacharjee et al., 1997), wheat (He et al., 1994) and tomato (Tsukada, 1989).

Another method for delivering transforming DNA segments to plant cells in accordance with the invention is microprojectile bombardment (U.S. Pat. No. 5,550,318; U.S. Pat. No. 5,538,880; U.S. Pat. No. 5,610,042; and PCT Application WO 94/09699; each of which is specifically incorporated herein by reference in its entirety). In this method, particles may be coated with nucleic acids and delivered into cells by a propelling force. Exemplary particles include those comprised of tungsten, platinum, and preferably, gold. It is contemplated that in some instances DNA precipitation onto metal particles would not be necessary for DNA delivery to a recipient cell using microprojectile bombardment. However, it is contemplated that particles may contain DNA rather than be coated with DNA. Hence, it is proposed that DNA-coated particles may increase the level of DNA delivery via particle bombardment but are not, in and of themselves, necessary.

An illustrative embodiment of a method for delivering DNA into plant cells by acceleration is the Biolistics Particle Delivery System, which can be used to propel particles coated with DNA or cells through a screen, such as a stainless steel or Nytex screen, onto a filter surface covered with monocot plant cells cultured in suspension. The screen disperses the particles so that they are not delivered to the recipient cells in large aggregates. Microprojectile bombardment techniques are widely applicable, and may be used to transform virtually any plant species. Examples of species for which have been transformed by microprojectile bombardment include monocot species such as maize (PCT Application WO 95/06128), barley (Ritala et al., 1994; Hensgens et al., 1993), wheat (U.S. Pat. No. 5,563,055, specifically incorporated herein by reference in its entirety), rice (Hensgens et al., 1993), oat (Torbet et al., 1995; Torbet et al., 1998), rye (Hensgens et al., 1993), sugarcane (Bower et al., 1992), and sorghum (Casa et al., 1993; Hagio et al., 1991); as well as a number of dicots including tobacco (Tomes et al., 1990; Buising and Benbow, 1994), soybean (U.S. Pat. No. 5,322,783, specifically incorporated herein by reference in its entirety), sunflower (Knittel et al. 1994), peanut (Singsit et al., 1997), cotton (McCabe and Martinell, 1993), tomato (VanEck et al. 1995), and legumes in general (U.S. Pat. No. 5,563,055, specifically incorporated herein by reference in its entirety).

The transgenic plants of the present invention comprising reduced or eliminated BS1 expression can be of any species. In some embodiments, the transgenic plant is a dicotyledonous plant, for example an agronomically important plant such as soybean,Medicago truncatula, a poplar, a willow, aeucalyptus, a hemp, aMedicagosp., aLotussp., aTrifoliumsp., aMelilotussp., aVincasp., aNicotianasp., aVitissp., aRicinussp., or anArabidopsisspecies. The plant can be an R0transgenic plant (i.e., a plant derived from the original transformed tissue). The plant can also be a progeny plant of any generation of an R0transgenic plant, wherein the transgenic plant has the nucleic acid sequence from the R0transgenic plant.

Seeds of the any above-described transgenic plants may also be provided, particularly where the seed comprises the nucleic acid sequence. Additionally contemplated are host cells transformed with the above-identified recombinant vector. In some embodiments, the host cell is a plant cell.

Also contemplated herein is a plant genetically engineered to exhibit reduced expression of a BS1, wherein the BS1 comprises a protein product of a sequence set forth in Table 1, wherein the protein product (e.g. a polypeptide) alters plant morphology. In an embodiment, the plant may lack expression of a BS1 gene. In an embodiment, the altered plant morphology may be increased seed yield, increased seed size, increased leaf size, or increased biomass. Such plants are described in the Examples, and may be useful, e.g., as commercial plants, due to their increased plant size and seed number.

The plants of these embodiments having decreased or a lack of expression of BS1 may be of any species. The species may be any monocotyledonous or dicotyledonous plant, such as those described herein. One of skill in the art will recognize that the present invention may be applied to plants of other species by employing methods described herein and others known in the art.

Application of these systems to different plant strains depends upon the ability to regenerate that particular plant strain from protoplasts. Illustrative methods for the regeneration of cereals from protoplasts have been described (Toriyama et al., 1986; Yamada et al., 1986; Abdullah et al., 1986; Omirulleh et al., 1993 and U.S. Pat. No. 5,508,184; each specifically incorporated herein by reference in its entirety). Examples of the use of direct uptake transformation of cereal protoplasts include transformation of rice (Ghosh-Biswas et al., 1994), sorghum (Battraw and Hall, 1991), barley (Lazerri, 1995), oat (Zheng and Edwards, 1990) and maize (Omirulleh et al., 1993).

Tissue cultures may be used in certain transformation techniques for the preparation of cells for transformation and for the regeneration of plants therefrom. Maintenance of tissue cultures requires use of media and controlled environments. “Media” refers to the numerous nutrient mixtures that are used to grow cells in vitro, that is, outside of the intact living organism. A medium usually is a suspension of various categories of ingredients (salts, amino acids, growth regulators, sugars, buffers) that are required for growth of most cell types. However, each specific cell type requires a specific range of ingredient proportions for growth, and an even more specific range of formulas for optimum growth. The rate of cell growth also will vary among cultures initiated with the array of media that permit growth of that cell type.

Tissue that can be grown in a culture includes meristem cells, Type I, Type II, and Type III callus, immature embryos and gametic cells such as microspores, pollen, sperm, and egg cells. Type I, Type II, and Type III callus may be initiated from tissue sources including, but not limited to, immature embryos, seedling apical meristems, root, leaf, microspores and the like. Those cells which are capable of proliferating as callus also are recipient cells for genetic transformation.

Somatic cells are of various types. Embryogenic cells are one example of somatic cells which may be induced to regenerate a plant through embryo formation. Non-embryogenic cells are those which typically will not respond in such a fashion. Certain techniques may be used that enrich recipient cells within a cell population. For example, Type II callus development, followed by manual selection and culture of friable, embryogenic tissue, generally results in an enrichment of cells. Manual selection techniques which can be employed to select target cells may include, e.g., assessing cell morphology and differentiation, or may use various physical or biological means. Cryopreservation also is a possible method of selecting for recipient cells.

III. Production and Characterization of Stably Transformed Plants

After effecting delivery of exogenous DNA to recipient cells, the next steps generally concern identifying the transformed cells for further culturing and plant regeneration. In order to improve the ability to identify transformants, one may desire to employ a selectable or screenable marker gene with a transformation vector prepared in accordance with the invention. In this case, one would then generally assay the potentially transformed cell population by exposing the cells to a selective agent or agents, or one would screen the cells for the desired marker gene trait.

It is believed that DNA is introduced into only a small percentage of target cells in any one study. In order to provide an efficient system for identification of those cells receiving DNA and integrating it into their genomes one may employ a means for selecting those cells that are stably transformed. One exemplary embodiment of such a method is to introduce, into the host cell, a marker gene which confers resistance to some normally inhibitory agent, such as an antibiotic or herbicide. Examples of antibiotics which may be used include the aminoglycoside antibiotics neomycin, kanamycin and paromomycin, or the antibiotic hygromycin. Resistance to the aminoglycoside antibiotics is conferred by aminoglycoside phosphostransferase enzymes such as neomycin phosphotransferase II (NPT II) or NPT I, whereas resistance to hygromycin is conferred by hygromycin phosphotransferase.

Potentially transformed cells then are exposed to the selective agent. In the population of surviving cells will be those cells where, generally, the resistance-conferring gene has been integrated and expressed at sufficient levels to permit cell survival. Cells may be tested further to confirm stable integration of the exogenous DNA.

One herbicide which constitutes a desirable selection agent is the broad-spectrum herbicide bialaphos. Another example of a herbicide which is useful for selection of transformed cell lines in the practice of the invention is the broad-spectrum herbicide glyphosate. Glyphosate inhibits the action of the enzyme EPSPS which is active in the aromatic amino acid biosynthetic pathway. Inhibition of this enzyme leads to starvation for the amino acids phenylalanine, tyrosine, and tryptophan and secondary metabolites derived therefrom. U.S. Pat. No. 4,535,060 describes the isolation of EPSPS mutations which confer glyphosate resistance on the EPSPS ofSalmonella typhimurium, encoded by the gene aroA. The EPSPS gene fromZea mayswas cloned and mutations similar to those found in a glyphosate resistant aroA gene were introduced in vitro. Mutant genes encoding glyphosate resistant EPSPS enzymes are described in, for example, International Patent WO 97/4103.

To use the bar-bialaphos or the EPSPS-glyphosate selective system, transformed tissue is cultured for 0-28 days on nonselective medium and subsequently transferred to medium containing from 1-3 mg/l bialaphos or 1-3 mM glyphosate as appropriate. While ranges of 1-3 mg/l bialaphos or 1-3 mM glyphosate will typically be preferred, it is proposed that ranges of 0.1-50 mg/l bialaphos or 0.1-50 mM glyphosate will find utility.

Cells that survive the exposure to the selective agent, or cells that have been scored positive in a screening assay, may be cultured in media that supports regeneration of plants. In an exemplary embodiment, MS and N6 media may be modified by including further substances such as growth regulators. One such growth regulator is dicamba or 2,4-D. However, other growth regulators may be employed, including NAA, NAA+2,4-D or picloram. Media improvement in these and like ways has been found to facilitate the growth of cells at specific developmental stages. Tissue may be maintained on a basic media with growth regulators until sufficient tissue is available to begin plant regeneration efforts, or following repeated rounds of manual selection, until the morphology of the tissue is suitable for regeneration, at least 2 weeks, then transferred to media conducive to maturation of embryoids. Cultures are transferred every 2 weeks on this medium. Shoot development will signal the time to transfer to medium lacking growth regulators.

The transformed cells, identified by selection or screening and cultured in an appropriate medium that supports regeneration, will then be allowed to mature into plants. Developing plantlets are transferred to soilless plant growth mix, and hardened, e.g., in an environmentally controlled chamber, for example, at about 85% relative humidity, 600 ppm CO2, and 25-250 microeinsteins m−2s−1of light. Plants may be matured in a growth chamber or greenhouse. Plants can be regenerated in from about 6 weeks to 10 months after a transformant is identified, depending on the initial tissue. During regeneration, cells are grown on solid media in tissue culture vessels. Illustrative embodiments of such vessels are Petri dishes and Plant Cons. Regenerating plants can be grown at about 19 to 28° C. After the regenerating plants have reached the stage of shoot and root development, they may be transferred to a greenhouse for further growth and testing.

To confirm the presence of the exogenous DNA or “transgene(s)” in the regenerating plants, a variety of assays may be performed. Such assays include, for example, “molecular biological” assays, such as Southern and northern blotting and PCR™; “biochemical” assays, such as detecting the presence of a protein product, e.g., by immunological means (ELISAs and western blots) or by enzymatic function; plant part assays, such as leaf or root assays; and also, by analyzing the phenotype of the whole regenerated plant.

Positive proof of DNA integration into the host genome and the independent identities of transformants may be determined using the technique of Southern hybridization. Using this technique specific DNA sequences that were introduced into the host genome and flanking host DNA sequences can be identified. Hence the Southern hybridization pattern of a given transformant serves as an identifying characteristic of that transformant. In addition it is possible through Southern hybridization to demonstrate the presence of introduced genes in high molecular weight DNA, i.e., confirm that the introduced gene has been integrated into the host cell genome. The technique of Southern hybridization provides information that is obtained using PCR™, e.g., the presence of a gene, but also demonstrates integration into the genome and characterizes each individual transformant.

Both PCR™ and Southern hybridization techniques can be used to demonstrate transmission of a transgene to progeny. In most instances the characteristic Southern hybridization pattern for a given transformant will segregate in progeny as one or more Mendelian genes (Spencer et al., 1992) indicating stable inheritance of the transgene.

Whereas DNA analysis techniques may be conducted using DNA isolated from any part of a plant, RNA will only be expressed in particular cells or tissue types and hence it will be necessary to prepare RNA for analysis from these tissues. PCR™ techniques also may be used for detection and quantitation of RNA produced from introduced genes. In this application of PCR™ it is first necessary to reverse transcribe RNA into DNA, using enzymes such as reverse transcriptase, and then through the use of conventional PCR™ techniques amplify the DNA. In most instances PCR™ techniques, while useful, will not demonstrate integrity of the RNA product. Further information about the nature of the RNA product may be obtained by Northern blotting. This technique will demonstrate the presence of an RNA species and give information about the integrity of that RNA. The presence or absence of an RNA species also can be determined using dot or slot blot northern hybridizations. These techniques are modifications of northern blotting and will only demonstrate the presence or absence of an RNA species.

The expression of a gene product is often determined by evaluating the phenotypic results of its expression. These assays also may take many forms including but not limited to analyzing changes in the chemical composition, morphology, or physiological properties of the plant. Chemical composition may be altered, for instance, by expression of genes encoding enzymes or storage proteins which change amino acid composition and may be detected by amino acid analysis, or by enzymes that change starch quantity which may be analyzed by near infrared reflectance spectrometry. Morphological changes may include, for instance, larger seeds, larger seed pods, larger leaves, greater stature, thicker stalks, and altered leaf-stem ratio, among others. Most often changes in response of plants or plant parts to imposed treatments are evaluated under carefully controlled conditions termed bioassays.

IV. Evaluation of Increased Seed Production or Size

A plant useful for the present invention may be an R0transgenic plant. Alternatively, the plant may be a progeny plant of any generation of an R0transgenic plant, where the transgenic plant has the nucleic acid sequence from the R0transgenic plant.

Plants in accordance with the invention exhibiting reduced or a lack of BS1 expression may also be used to produce increased seed size or numbers, increased plant biomass, for example by obtaining the above-identified plant comprising reduced or a lack of BS1 expression, growing said plant under plant growth conditions to produce plant tissue from the plant; and preparing biomass from said plant tissue. The increased seed production or biomass can be subsequently used for any purpose, for example for food or commodity products, or to produce biofuel.

V. Breeding Plants of the Invention

In addition to direct transformation of a particular plant genotype with a construct prepared according to the current invention, transgenic plants may be made by crossing a plant having a selected DNA of the invention to a second plant lacking the construct. For example, a selected nucleic acid sequence producing a dsRNA or miRNA targeting a BS1 coding sequence can be introduced into a particular plant variety by crossing, without the need for ever directly transforming a plant of that given variety. Therefore, the current invention not only encompasses a plant directly transformed or regenerated from cells which have been transformed in accordance with the current invention, but also the progeny of such plants. As used herein, the term “progeny” denotes the offspring of any generation of a parent plant prepared in accordance with the instant invention, wherein the progeny comprises a selected DNA construct prepared in accordance with the invention. “Crossing” a plant to provide a plant line having one or more added transgenes relative to a starting plant line, as disclosed herein, is defined as the techniques that result in a transgene of the invention being introduced into a plant line by crossing a plant of a starting line with a plant of a donor plant line that comprises a transgene of the invention. To achieve this one could, for example, perform the following steps:

(a) plant seeds of the first (starting line) and second (donor plant line that comprises a transgene of the invention) parent plants;

(b) grow the seeds of the first and second parent plants into plants that bear flowers;

(c) pollinate a flower from the first parent plant with pollen from the second parent plant; and

(d) harvest seeds produced on the parent plant bearing the fertilized flower.

Backcrossing is herein defined as the process including the steps of:

(a) crossing a plant of a first genotype containing a desired gene, DNA sequence or element to a plant of a second genotype lacking the desired gene, DNA sequence or element;

(b) selecting one or more progeny plant containing the desired gene, DNA sequence or element;

(c) crossing the progeny plant to a plant of the second genotype; and

(d) repeating steps (b) and (c) for the purpose of transferring a desired DNA sequence from a plant of a first genotype to a plant of a second genotype.

Introgression of a DNA element into a plant genotype is defined as the result of the process of backcross conversion. A plant genotype into which a DNA sequence has been introgressed may be referred to as a backcross converted genotype, line, inbred, or hybrid. Similarly a plant genotype lacking the desired DNA sequence may be referred to as an unconverted genotype, line, inbred, or hybrid.

Expression: The combination of intracellular processes, including transcription and translation, undergone by a coding DNA molecule such as a structural gene to produce a polypeptide. A plant in accordance with the invention may exhibit altered expression of a gene set forth herein. Such altered expression may include increased expression, decreased expression, or complete absence of expression.

Genetic Transformation: A process of introducing a DNA sequence or construct (e.g., a vector or expression cassette) into a cell or protoplast in which that exogenous DNA is incorporated into a chromosome or is capable of autonomous replication.

Heterologous: A sequence which is not normally present in a given host genome in the genetic context in which the sequence is currently found. In this respect, the sequence may be native to the host genome, but be rearranged with respect to other genetic sequences within the host sequence. The sequence may also be altered, i.e. mutated, with respect to the native regulatory sequence. For example, a regulatory sequence may be heterologous in that it is linked to a different coding sequence relative to the native regulatory sequence.

Obtaining: When used in conjunction with a transgenic plant cell or transgenic plant, obtaining means either transforming a non-transgenic plant cell or plant to create the transgenic plant cell or plant, or planting transgenic plant seed to produce the transgenic plant cell or plant. Such a transgenic plant seed may be from an R0transgenic plant or may be from a progeny of any generation thereof that inherits a given transgenic sequence from a starting transgenic parent plant.

Promoter: A recognition site on a DNA sequence or group of DNA sequences that provides an expression control element for a structural gene and to which RNA polymerase specifically binds and initiates RNA synthesis (transcription) of that gene.

R0transgenic plant: A plant that has been genetically transformed or has been regenerated from a plant cell or cells that have been genetically transformed.

Regeneration: The process of growing a plant from a plant cell (e.g., plant protoplast, callus or explant).

Selected DNA: A DNA segment which one desires to introduce or has introduced into a plant genome by genetic transformation.

Transformation construct: A chimeric DNA molecule which is designed for introduction into a host genome by genetic transformation. Preferred transformation constructs will comprise all of the genetic elements necessary to direct the expression of one or more exogenous genes. In particular embodiments of the instant invention, it may be desirable to introduce a transformation construct into a host cell in the form of an expression cassette.

Transformed cell: A cell in which the DNA complement has been altered by the introduction of an exogenous DNA molecule into that cell.

Transgene: A segment of DNA which has been incorporated into a host genome or is capable of autonomous replication in a host cell and is capable of causing the expression of one or more coding sequences. Exemplary transgenes will provide the host cell, or plants regenerated therefrom, with a novel phenotype relative to the corresponding non-transformed cell or plant. Transgenes may be directly introduced into a plant by genetic transformation, or may be inherited from a plant of any previous generation which was transformed with the DNA segment.

Transgenic plant: A plant or progeny plant of any subsequent generation derived therefrom, wherein the DNA of the plant or progeny thereof contains an introduced exogenous DNA segment not naturally present in a non-transgenic plant of the same strain. The transgenic plant may additionally contain sequences which are native to the plant being transformed, but wherein the “exogenous” gene has been altered in order to alter the level or pattern of expression of the gene, for example, by use of one or more heterologous regulatory or other elements.

Vector: A DNA molecule designed for transformation into a host cell. Some vectors may be capable of replication in a host cell. A plasmid is an exemplary vector, as are expression cassettes isolated therefrom.

EXAMPLES

The following examples are included to demonstrate preferred embodiments of the invention. It should be appreciated by those of skill in the art that the techniques disclosed in the examples which follow represent techniques discovered by the inventors to function well in the practice of the invention, and thus can be considered to constitute preferred modes for its practice. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the concept, spirit and scope of the invention. More specifically, it will be apparent that certain agents which are both chemically and physiologically related may be substituted for the agents described herein while the same or similar results would be achieved. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the invention as defined by the appended claims.

Plant Materials

Homozygous big seeds1-1 (bs1-1; M477), bs1-2 (FN 1860-III) and bs1-3 (FN 2876) mutants were isolated from a fast neutron bombardment (FNB)-induced deletion mutant collection ofMedicago truncatulacv. Jemalong A17 background. The bs1-1 allele was backcrossed to wild type (A17). BC1 mutant and its descendants were used for phenotypic characterization. F2 mapping populations were generated from crosses between bs1-1 and a polymorphic ecotypeM. truncatulacv. Jemalong A20. A total of 518 individuals (432 wild-type like and 86 mutants) was used in a bulk segregant and fine mapping analyses to construct a linkage map of the bs1 locus.Arabidopsis thalianaΔppd (CS16548) and an3-1 (CS241) mutant lines were obtained from theArabidopsisStock Center at Ohio State University. AN3pro::uidA line was described previously (Horiguchi et al.,Plant J43:68-78, 2005).

Isolation and Phenotypic Analysis of bs1 Mutant

From a forward genetic screen of mutants with an increased seed size from a fast neutron bombardment (FNB)-induced deletion mutant collection ofMedicago truncatula(cv. Jemalong A17) (Peng et al.,Plant Cell23:3929-3943, 2011; Chen et al.,Proc Natl Acad Sci USA107:10754-10759, 2010), a unique mutant named big seeds1 (bs1) was isolated. The bs1 mutant exhibited large seeds, seed pods, and leaves, along with several other phenotypes compared with wild-type plants (FIG. 1a-g). To measure leaf and seed parameters, digital images of leaves and seeds were obtained using a high-resolution scanner (EPSON PERFECTION V700 PHOTO, EPISON) and analyzed using ImageJ and Tomato Analyzer (Brewer et al.,Plant Physiology141:15-25, 2006). To accurately measure bs1-1 mutant leaves, leaves were flattened by carefully cutting the leaf margin before scanning.

Time-course experiments show that developing seeds 11 DPA (days post anthesis) and older were significantly larger in the bs1 mutant than in wild-type plants (FIG. 1a, b, andh). Seed pods were already larger at earlier stages of development in the mutant (FIG. 1a). Maturation of seeds and seed pods appeared to be slightly delayed in the bs1 mutant compared with wild-type plants (FIG. 1a, b). In line with an increase in seed size, mature seeds were more than 20% heavier in the bs1 mutant than wild-type (FIG. 1i).

In the bs1 mutant, both terminal and lateral leaflets of its trifoliate leaves were dramatically increased in length, width, perimeter, and size compared with wild-type counterparts (FIG. 1c-g;FIG. 2a, b). In addition, the shape of leaflets was also altered. Leaflets of the bs1 mutant had a dome-shaped curvature, in contrast to the flat leaflets of wild-type plants. The dome-shaped leaflets could be flattened by introducing cuts in the edges (FIG. 1e-g). Petioles of the bs1 mutant were longer than wild-type petioles (FIG. 2a, b). Further, the size of stipules was increased by more than 10 times in the bs1 mutant compared with wild-type stipules (FIG. 1c-g;FIG. 2a, b).

Although the size and identity of floral organs was not altered in the bs1 mutant, floral patterning was altered (FIGS. 3 and 4). As a member of the Papilionoideae subfamily of legumes (Fabaceae),M. truncatulaproduces flowers resembling those of garden pea (Pisum sativum) with three types of petals: one dorsal petal with bilateral symmetry, two asymmetric wing petals, and one symmetric ventral petal derived from fusion of two ventral petal primordia during early floral development (FIG. 4) (Wang et al.,Plant Physiol146:1759-1772, 2008). In the bs1 mutant, the ventral petal primordia failed to fuse together, resulting in two separate ventral petals with an acquired internal asymmetry (FIG. 4).

An increase in organ size may result from an increase in cell proliferation and/or cell expansion, two successive processes contributing to the final organ size (Hepworth, et al.,Curr Opin Plant Biol17:36-42, 2014; Gonzalez, et al.,Plant Physiol153:1261-1279, 2010). Measurements show that epidermal cells of fully-expanded leaves were similar in size between the bs1 mutant and wild-type plants, suggesting that cell proliferation rather than cell expansion is altered in the bs1 mutant (FIG. 5a-e). Leaf primordia are initiated from the periphery of the shoot apical meristem (SAM) and initially consist of cells that undergo coordinated division without changes in cell size. As the leaf grows, cells at the distal tip cease dividing and differentiate, resulting in a cell cycle arrest front moving from the tip to base. Eventually, all cells cease dividing and the leaf reaches its final size (Nath et al.,Science299:1404-1407, 2003; Donnelly et al.,Developmental Biology215:407-419, 1999; Poethig et al.,Planta165:170-184 1985).

To further examine the developmental processes affected in the bs1 mutant, leaf development was followed over time. For the time lapse analysis of leaf size, the youngest emerging leaf of six-week-old plants was labeled and imaged daily during a 12-day time course. For the time lapse analysis of seed size, flowers were labeled and seed pods were collected at different time points after anthesis. Seeds were carefully removed from seed pods and imaged. To measure seed weight, wild type (A17) and the bs1-1 mutant, and soybean seeds were weighed on a digital balance. Fresh weights of fully-expanded leaves from six-week-old plants were also measured using a digital balance.

In the first 4 days after emerging from the shoot apex, young leaves (P5; P for plastochron) of both wild-type and the bs1 mutant similarly expanded along the proximodistal (length) and mediolateral (width) axes (FIG. 2c-f). The expansion of wild-type leaves along the length and width axes reached a plateau approximately 2 days later (FIG. 2c-f). By contrast, leaves of the bs1 mutant continuously expanded and did not reach a plateau until approximately 6 days later (FIG. 2c-f). These results show that the enlarged organ phenotype of the bs1 mutant was caused by prolonged cell proliferation. This is consistent with the observation that seed and seed pod maturation was delayed in the bs1 mutant (FIG. 1a, b).

Forage Quality Analysis of Bs1 Mutant

Wild-type and the bs1-1 mutant plants were grown in 1-gallon pots in the greenhouse. Arial portions of three-month-old plants were harvested and dried in a 50° C. oven for three days. The samples were then ground in a Thomas-Wiley model 4 Laboratory Mill (Lehman Scientific, Wrightsville, Pa.) with 1-mm sieves. Acid detergent fiber (ADF) and neutral detergent fiber (NDF) were estimated by standard protocols (Reddy et al.,Proc Natl Acad Sci USA102:16573-16578, 2005). To determine NDF, 0.5 grams of ground samples were transferred to a F57 ANKOM filter bag (ANKOM Technology, Fairport, N.Y.) and heated at 100° C. for 1 h in an ANKOM Fiber Analyzer. The samples were washed in near-boiling water, dried at 100° C. for 2 h, and weighed to determine fiber loss. To determine ADF, the material remaining after NDF analysis was incubated in an acid detergent solution (2% cetyltrimethylammonium bromide in 0.5M H2SO4) for 1 h, filtered and rinsed sequentially with near-boiling water and acetone, and dried at 100° C. for 2 h to determine fiber loss.

Forage quality analyses showed that acid detergent fiber (ADF) and neutral detergent fiber (NDF) were significantly decreased in three-month-old bs1-1 mutant compared with wild-type plants (FIG. 6). These results show that an increase in cell proliferation and leaf growth due to loss-of-function of BS1 improved the forage quality of the plants.

Mapping of BS1

Total genomic DNA was isolated from fresh leaf tissues of individual plants grown in the greenhouse using the CTAB method (Saghai-Maroof et al.,Proc Natl Acad Sci USA81:8014-8018, 1984). PCR amplification and PCR products separation using a Li-Cor 4300 DNA sequencer were carried out as previously described (Yu et al.,Theor Appl Genet113:308-320, 2006). A total of 267 SSR (simple sequence repeat) markers distributed across the eight chromosomes ofM. truncatula(available at medicago.org/genome/downloads.php) were used to construct the linkage map using a bulked-extreme and recessive-class approach as previously described (Zhang et al.,Proc Natl Acad Sci USA91:8675-8679, 1994). Two bulked DNA samples from 15 mutant plants were made each by pooling equal amounts of DNA samples prepared from individual plants. DNA samples from polymorphic wild-type plants (M. truncatulacv. Jemalong A17 and Jemalong A20) were included in the analysis to validate SSR markers. Recombination between markers and the big seeds1 locus was calculated using the maximum likelihood estimator, r=(N1+N2/2)/N, where N is the total number of mutant plants, N1 is the number of recombinant homozygotes and N2 is the number of recombinant heterozygotes. The recombination ratio variance was given by Vr=r(1−r)/2N. The Kosambi mapping function was used to estimate the genetic distances between the markers and the big seeds1 locus and the recombination ratio was converted to map distance in centiMorgans (Koornneef et al.,Heredity74:265-272, 1983). A bulk segregant analysis revealed that 20 SSR markers located on chromosome 1 showed co-segregation with the big seeds1 locus. Segregation data obtained from the mutant plants were used in the color map method (Kiss et al.,Acta Biol Acad Sci Hung19:125-142, 1998), which employed a comparison of graphical genotypes for mapping. According to theMedicago truncatulagenetic map (available at medicago.org/genome/map.php), recombination frequencies were calculated with 9 of the 20 SSR markers using 86 mutant plants and determined map positions of these markers. Based on this analysis, the big seeds1 locus was mapped to a region on chromosome 1 flanked by SSR markers, h2-49g13b and 005E04, and tightly linked to the SSR marker, MTIC61 (Table 2 andFIGS. 7 and 8a) (Choi et al.,Genetics166:1463-1502, 2004).

Cloning of BS1

Due to the presence of sequence gaps in the mapped region (FIG. 8b), alternative approaches were used to clone the gene. First, the transcript profiles in vegetative shoot buds and young leaves were compared between wild type and the mutant.Medicago truncatulatranscriptome analysis was performed as previously described (Tadege et al.,Plant Cell23:2125-2142, 2011; Uppalapati et al.,Plant Cell24:353-370, 2012). Total RNA was isolated from vegetative shoot buds and young leaves of six week-old plants using the RNeasy Plant Mini Kit (Qiagen). Three biological replicates for wild-type (A17) and the bs1-1 mutant were analyzed using AffymetrixMedicagoGeneChip (Affymetrix). Probe labelling, hybridization and scanning were carried out according to manufacturer's instructions. Raw data were normalized with Robust Multichip Average (Irizarry et al.,Nucleic acids research31:e15, 2003). Presence and absence calls of probesets were obtained using dCHIP. Probesets with expression ratios of bs1-1/A17 greater than 2 or less than 0.5 were selected and analyzed for differentially expressed genes between A17 and bs1-1 using associative analyses (Dozmorov et al.,Bioinformatics19:204-211, 2003).

The microarray approach revealed that several probesets, which match to unanchored BAC (bacterial artificial chromosome) sequences containing the flanking h2-49g13b and 005E 04 markers (available at medicago.org/genome/downloads.php), were similarly expressed in the mutant and wild-type plants, suggesting that their sequences are intact in the mutant (FIG. 8c). Next, genome analysis identified that a region on chromosome 5 inLotus japonicusand regions on chromosomes 10 and 20 in soybean (Glycine max), two closely-related species, are syntenic to the mapped BS1 region (FIG. 8b). Using the syntenic sequences, one microarray probeset, Mtr.44029.1.S1_at, was mapped to the syntenic location and was found to be significantly downregulated in both shoot buds and young leaves of the bs1 mutant compared with wild-type plants (FIG. 8c, d). Reverse transcription (RT)-PCR amplification detected the corresponding transcript in wild-type plants but not in the bs1 mutant (FIG. 9b), suggesting that Mtr.44029.1.S1_at was deleted in the mutant. The deletion borders were recovered using PCR-based chromosomal walking and thermal asymmetric interlaced (TAIL)-PCR (FIG. 9a). Sequencing results showed that the right deletion border occurred in the first exon at 398 bp downstream from the translation initiation codon ATG of the candidate gene. However, the left border did not match to any available sequences and was likely located in the gap region.

Confirmation of BS1 Candidate Gene

To confirm the candidate gene, two additional alleles named bs1-2 (FN 1860) and bs1-3 (FN 2876) were isolated from the FNB mutant collection (FIG. 10a). Genomic PCR and RT-PCR showed that the candidate BS1 locus was completely deleted in both alleles (FIG. 10b, c). In addition, introducing the BS1 coding sequence under the control of its own promoter completely rescued the bs1 mutant phenotypes, confirming that Mtr.44029.1.S1_at corresponds to BS1 (FIG. 11a-d). Sequence analysis shows that BS1 encodes a protein of 331 amino acids in length, belonging to the group II of the TIFY family of plant-specific transcription factors and regulators (FIG. 9h) (Bai et al.,Genomics98:128-136, 2011; Vanholme et al.,Trends Plant Sci12:239-244, 2007). When transiently expressed in tobacco leaves or stably expressed inArabidopsis thaliana, BS1-GFP fusion proteins were localized to the nucleus, in contrast to free GFP that exhibited cytoplasmic localization (FIGS. 9c-fand12). The GFP signal was imaged, using the Leica SP2 laser confocal microscope as previously described (Ge et al.,Plant Physiol164:216-228, 2014; Peng et al.,Plant Cell23:3929-3943, 2011). Database searches and PCR amplification identified BS1 homologous sequences from diverse eudicot species such as alfalfa (M. sativa),L. japonicas, soybean (G. max), tomato (Solanum lycopersicum) and potato (Solanum tuberosumL.). However, no homologous sequences were identified from monocot species including rice and maize (FIG. 9l).

Expression Analysis of BS1

Reverse transcription was performed using Qiagen SuperScript II Kit (Qiagen). Quantitative PCR was conducted on 7900HT Fast Real-Time PCR system (Applied Biosystems) as previously described (Ge et al.,Plant Physiol164:216-228, 2014). RT-PCR analyses showed that BS1 was expressed in all major organs including seeds, seed pods, flowers, shoot buds, and leaves (FIG. 9g). RNA in situ hybridization was performed as previously described (Ge et al.,Plant Physiol164:216-228, 2014), with minor modifications. The BS1 riboprobes correspond to a 751-bp sequence from the BS1 coding region. Eight micrometer sections from shoot apices of 2-4-week-old seedlings and flowers were processed and hybridized with digoxigenin-labeled sense and antisense probes. RNA in situ hybridization showed that BS1 transcripts were present in the shoot apical meristem (SAM), leaf primordia as early as P0 and lamina tissues (FIG. 9h, i). In reproductive tissues, BS1 transcripts were detected in petal primordia, carpel and embryos (FIG. 9j, k).

Analysis of Transcript Profiles of bs1 and Wild Type Plants

To dissect the mechanisms by which BS1 regulates cell proliferation, transcript profiles were analyzed in both vegetative shoot buds and young leaves of wild type and the bs1 mutant (FIG. 13a). A large number of core cell cycle (CCC) genes were significantly upregulated in young leaves, but not in the shoot buds of the bs1 mutant compared with wild-type (Table 3), consistent with an increase in cell proliferation observed in bs1 mutant leaves.

TABLE 3Upregulation of core cell cycle genes in the bs1 mutant.Fold changesArabidopsis(bs1/A17)homologsProbesetsShoot budsYoung leavesMedicago geneLocusGeneMtr.27379.1.S1_at1.052.02AC235748_1029.1At5g43080.1CYCA3;1Mtr.29458.1.S1_at1.021.68AC235748_1029.1At5g43080.1CYCA3;1Mtr.5292.1.S1_at1.051.79contig_57417_1.1At5g43080.1CYCA3;1Mtr.31859.1.S1_at1.142.39At1g16330.1CYCB3;1Msa.1804.1.S1_s_at1.122.52At4g34160.1CYCD3;1Mtr.12785.1.S1_at1.152.63contig_83623_1.1At4g34160.1CYCD3;1Msa.913.1.S1_at1.051.62contig_85803_1.1At3g54180.1CDKB1;1Mtr.38989.1.S1_at0.971.81contig_85803_1.1At3g54180.1CDKB1;1Mtr.27402.1.S1_at1.101.53Medtr1g011470.1At5g11300.1CYCA2;2Mtr.50839.1.S1_at0.982.14Medtr1g075610.1At1g20930.1CDKB2;2Mtr.32437.1.S1_at1.021.58At2g36010.3E2FaMtr.3426.1.S1_at1.112.11Medtr2g102530.1At1g15570.1CYCA2;3Mtr.41123.1.S1_at1.143.11Medtr3g102310.1At3g50070.1CYCD3;3Mtr.11388.1.S1_at1.101.80Medtr4g052000.1At5g22220.1E2FbMtr.42269.1.S1_at1.131.70At5g22220.1E2FbMtr.16199.1.S1_at0.821.51Medtr4g086450.1At1g49620.1KRP7Mtr.17064.1.S1_s_at1.122.12Medtr4g106540.2At3g48160.2DEL1Mtr.21277.1.S1_at0.782.32Medtr5g015670.1At4g37630.1CYCD5;1Msa.3007.1.S1_at1.131.60Medtr5g023790.1At1g20610.1CYCB2;3Mtr.32176.1.S1_at1.081.83Medtr5g088980.1At3g11520.1CYCB1;3Mtr.4712.1.S1_s_at1.051.79Medtr7g089080.1At5g06150.1CYCB1;2Mtr.31360.1.S1_at1.051.79Medtr7g089080.1At5g06150.1CYCB1;2Mtr.24738.1.S1_at1.031.98Medtr8g074000.1At4g35620.1CYCB2;2Mtr.32067.1.S1_s_at0.941.51At4g35620.1CYCB2;2Mtr.31170.1.S1_at1.101.61At1g76310.1CYCB2;4Mtr.33796.1.S1_at1.111.64At1g76310.1CYCB2;4Mtr.33796.1.S1_s_at1.041.77At1g76310.1CYCB2;4Mtr.39505.1.S1_at1.071.99Medtr8g095930.1At1g44110.1CYCA1;1

Comparisons of transcript levels between shoot buds and young leaves show that the expression of CCC genes was dramatically downregulated in leaf tissues of both wild-type and the bs1 mutant plants (FIG. 12). However, the downregulation of CCC genes was significantly less in the bs1 mutant than in wild-type plants (FIG. 14), indicating an insufficient suppression of CCC gene expression in expanding leaves of the bs1 mutant.

The expression levels of all known organ size regulatory genes from the transcript profiling data were examined. Interestingly, five probesets related to cell proliferation were most significantly upregulated in bs1 leaves, three of which were also upregulated in bs1 shoot buds, compared with wild-type plants (Table 4). These microarray probesets are related toArabidopsisGrowth Regulating Factor5 (GRF5) and GRF-Interacting Factor1 (GIF1, also known as ANGUSTIFOLIA3 or AN3). GRF5 and GIF1 are interacting transcription factor and co-activator, respectively; and, AN3 is known to regulate transcription of GRF5 (Horiguchi et al.,Plant J43:68-78, 2005; Kim et al.,Proc Natl Acad Sci USA101:13374-13379, 2004; Vercruyssen et al.,Plant Cell26:210-229, 2014; Kawade et al.,Curr Biol23:788-792, 2013; Horiguchi et al.,Plant Cell Physiol52:112-124, 2011). Overexpression of either gene increases leaf size and alters leaf shape, resembling the bs1 leaf phenotypes.

Using the availableMedicagosequence (Mt4.0v1) (Young et al.,Nature,2011), two GRF5 probesets were mapped to Medtr8g020560 (MtGRF5) and one GIF1 probeset to Medtr1g080590 (MtGIF1; Table 4). Quantitative RT-PCR confirms that MtGRF5 and MtGIF1 were significantly upregulated in leaves of the bs1-1 mutant compared with wild-type plants (FIG. 13b).

TABLE 4Upregulation of organ size regulatory genes in the bs1 mutant.Fold ChangesArabidopsis(bs1/A17)homologProbesetsShoot budsYoung leavesMedicago geneLocusGeneMtr.29046.1.S1_at1.76582621831.91493284Medtr1g080590.1At5g28640.1GIF1Mtr.50542.1.S1_at3.44073716711.289024Medtr8g020550.1At3g13960.1GRF5Mtr.39218.1.S1_at1.48145813310.1833997At5g28640.1GIF1Mtr.50543.1.S1_at3.2865456817.359077706Medtr8g020560.1At3g13960.1GRF5Mtr.13651.1.S1_at0.9377872514.382843705contig_237721_1.1At3g13960.1GRF5

Plasmids and Plant Transformation

BS1 promoter was cloned into the SacI and NcoI sites of pCAMBIA3301 to generate the BS1pro::uidA reporter construct. To generate the complementation construct, the uidA (GUS) reporter gene was replaced by the BS1 cDNA at the NcoI and BstEII sites. To generate the BS1 overexpression construct, the BS1 cDNA without the stop codon was first cloned into pENTRY-D (Invitrogen), according to the manufacture's instructions, and then was cloned into the pMDC83 gateway destination vector, using LR reaction. To silence soybean BS1 orthologs, an artificial microRNA (pre-amiR) was designed to target both GmBS1 and GmBS2, using Web MicroRNA Designer (WMD, available at wmd3.weigelworld.org/cgi-bin/webapp.cgi). The soybean native microRNA319 backbone was used to generate pre-amiR. Using overlapping PCR, pre-amiR was generated and cloned into the pTF101 vector (Paz et al.,Plant Cell Rep25:206-213, 2006). To generate BS1 and PPD2 yeast two hybrid bait constructs, BS1 and PPD2 cDNAs were cloned into the NdeI and SalI sites of pGBKT7 vector. To generate theM. truncatulaMYC2 prey construct, MtMYC2 cDNA was cloned into the NdeI and BamHI sites of pGADT7 vector. To generate the bait constructs forM. truncatulaJAZ3 and JAZ3-JAZ domain, the full-length CDS and the JAZ domain of MtJAZ3 were first cloned into pENTRY-D (Invitrogen), and then cloned into the pGBKT7-GATEWAY vector, using LR reactions.M. truncatulaNINJA andArabidopsisMYC2 prey constructs were cloned into the pGADT7-GATEWAY vector, using the gateway cloning strategy.Medicago(R108), soybean (G. maxcv. Williams 82) andArabidopsis(Columbia-0) were transformed as previously described (Paz et al.,Plant Cell Rep25:206-213, 2006; Wang et al.,Plant Physiol146:1759-1772, 2008). Primers used in this study are listed in Table 5.

The extent of functional conservation of BS1/GRF5/AN3 inA. thalianawas evaluated. BS1 is related to two tandem-repeat genes, At4g14713 (PPD1) and At4g14720 (PPD2) inArabidopsis. In ppd1 ppd2 deletion mutant (Δppd), leaves are dome-shaped and larger than wild-type (FIG. 15a) (White et al.,Proc Natl Acad Sci USA103, 13238-13243, 2006). Siliques of the Δppd mutant were wider at the top and shorter in length than wild-type siliques (FIG. 15b, c, h) (White et al.,Proc Natl Acad Sci USA103, 13238-13243, 2006). However, the seed size was normal in the Δppd mutant, suggesting partially diverged function of PPD genes in reproductive development inArabidopsis. Single ppd mutants displayed weaker phenotypes than the double deletion mutant due to a functional redundancy (FIG. 16a, b). Overexpression of theMedicagoBS1 gene (35S::BS1) rescued the phenotypes of Δppd and ppd2 mutants (FIG. 16a-d), confirming that BS1 and PPDs are functional orthologs. PPDs have been reported to play a role in secondary cell cycle arrest by negatively regulating proliferation of dispersed meristematic cells (DMCs) in leaves. By contrast, GRF5 and GIF1 (AN3) are known to play a role in primary cell cycle arrest inArabidopsisleaves (Horiguchi et al.,Plant J43:68-78, 2005; Kim et al.,Proc Natl Acad Sci USA101:13374-13379, 2004).

To further examine the role of PPDs, the AN3pro::uidA reporter was introduced into the Δppd mutant by crossing. Histochemical staining shows that the GUS activity was greatly upregulated in siliques of the Δppd mutant, compared with that of wild-type plants (FIG. 15i-l). Furthermore, the silique and leaf phenotypes of the Δppd mutant was restored to wild type- and an3-like phenotypes, respectively, in Δppd an3 double mutant (FIG. 15a-c, h); the epistatic interaction confirms that AN3 (GIF1) acts downstream of PPDs in controlling leaf expansion and silique shape. These results demonstrate a novel and conserved role of BS1/PPDs in regulating lateral organ size and shape inMedicagoandArabidopsisthrough negatively regulating AN3 expression.

Evaluation of BS1 Protein Interactions

As a group II member of the TIFY family of proteins, BS1 and its orthologs do not contain DNA-binding domains. Some TIFY family members are known to interact with the adaptor protein, NINJA, which, in turn, interacts with the transcription co-repressors, TOPLESS (TPL) and TOPLESS-RELATED PROTEINs (TPRs), via its ERF-associated amphiphilic repressor (EAR) domain (Cuellar Perez et al.,PLoS One9:e84891, 2014; Pauwels et al.,Nature464:788-791, 2010).

Bait and prey constructs were co-transformed into the yeast two hybrid strain GOLD (Clontech), using Frozen-EZ Yeast Transformation II Kit (Zymo Research). Protein-protein interactions were tested on the SD medium containing X-a-Gal (40 ng/ml) and Aureobasidin A (125 ng/ml), without leucine, tryptophan, adenine and histidine.

Using yeast two-hybrid assays, we show that BS1 interacted strongly with NINJA, but not with MYC2, a transcription factor involved in jasmonic acid (JA) signaling25 (FIG. 15m). Similarly, PPD2 also did not interact with MYC2 (FIG. 15m). As a control,MedicagoJAZ3 was shown to interact strongly with both MYC2 and NINJA via its JAZ domain (FIG. 17). These results suggest that BS1/PPD forms a repressor complex with NINJA and TPL to negatively control expression of specific downstream targets such as GIF1 (AN3)18. To confirm this in vivo, chromatin immunoprecipitation coupled was performed with quantitative PCR (ChIP-qPCR), usingArabidopsisplants overexpressing BS1-GFP. Because 35S::BS1-GFP completely rescued the Δppd mutant phenotypes, this suggests that the fusion protein is functional. ChIP-qPCR results show that GIF1 (AN3) promoter sequences were specifically enriched in the BS1-GFP chromatin (FIG. 15n), supporting that GIF1 (AN3) is a direct downstream target of BS1.

Downregulation of BS1 in Soybean

Because of the functional conservation seen inMedicagoandArabidopsis, downregulation of BS1 orthologs in soybean (G. maxcv. Williams 82) was attempted. Based on genome syntenies and sequence similarities, we isolated two soybean BS1 orthologs named GmBS1 (Glyma10g38970) and GmBS2 (Glyma20g28840) (FIG. 9l). To downregulate their expression, an artificial microRNA was designed to target a highly conserved coding sequence of GmBS1 and GmBS2 (FIG. 18). RT-PCR analyses show that GmBS gene expression was significantly downregulated in three out of five independent transgenic soybean plants transformed with the artificial microRNA under the control of 35S promoter (35S::pre-amiR). Consistent with this, these transgenic plants (lines #9, #19 and #30) displayed dramatically increased sizes and weights of seeds, seed pods and leaves compared with wild-type plants (FIG. 18). Gene expression analysis shows that soybean GRF5, AN3, and several cell cycle-related genes such as CYCD3 and HISTONE4 were all upregulated in the transgenic plants, similar to the bs1 mutants (FIG. 19).

Taken together, the results show that downregulation of the soybean BS1 orthologues results in plants with significantly enlarged lateral organs, including seeds, seed pods and leaves similar to theMedicagobs1 mutants, confirming a conserved molecular mechanism in controlling plant organ size and shape in multiple species. In addition, our work presents an effective strategy to increase soybean seed yield. Soybean is a major crop worldwide. Identification of the BS1/AN3/GRF5 module in the control of plant organ size from multiple species provides novel opportunities to develop an optimal strategy for improving crop yield.

Seed Amino Acid Contents of Wild Type and Transgenic Soybean Lines

The amino acid content of dried soybean seeds from wild type (w.t.; Williams 82) and the three independent transgenic lines (#9, #19 and #30) in which the BIG SEEDS1 gene is downregulated was measured. For each line, five biological replicates were determined with one seed for each measurements. 16 amino acids (Asp, Thr, Ser, Gly, Pro, Gly, Ala, Val, Met, Ile, Leu, Tyr, Phe, His, Lys and Arg) were measured using a HITACHI L8900 Amino Acid Analyzer (http://www.aaaservicelab.comcastbiz.net/index.html), with proline (Pro) and lysine (Lys) measured as hydroxyproline and hydroxylysine, respectively. No deleterious effects on total amino acid or individual amino acid contents were detected in the transgenic soybean lines when compared with wild type plants (FIG. 20A). The results show, however, that in the transgenic lines #19 and #30, total seed amino acid and individual amino acid contents were significantly increased compared to wild type plants (FIG. 20B; Student's t-test, p<0.05, n=5). In the transgenic line #9, total seed amino acid, and ASP, THR, SER, GLU, PRO, GLY, ALA, VAL, MET, TYR, HIS, LYS and ARG were also increased (FIG. 20B; Student's t-test, p<0.1, n=5) compared to wild type plants.

Seed Amino Acid Contents of Wild Type and Transgenic Soybean Lines Measurements of Seed Lipid Contents of Wild Type and Transgenic Soybean Lines

Total seed lipid and fatty aid content was measured of dried soybean seeds from wild type (w.t.; Williams 82) and the three independent transgenic lines (#9, #19 and #30), in which the BIG SEEDS1 gene is downregulated, using GC-MS (gas chromatography-mass spectrometry) by the Noble Foundation Analytical Chemistry Core Facility. Five biological replicates were measured with one seed for each measurements. Five major seed fatty acids were measured as Methyl Palmitate, Methyl Stearate, Methyl Oleate, Methyl Linoleate and Methyl Linolenate, respectively. No deleterious effects on total seed lipid and fatty acid contents were detected in the transgenic lines compared with wild type plants (FIGS. 21A, 21B).

Measurements of Leaf Stem Ratios ofMedicago truncatulabs1 Mutant Plants

Forage quality analyses show that mutations of theMedicago truncatulaBIG SEEDS1 gene significantly increased the forage quality of the mutant plants. To confirm whether this was be due to an increase in the leaf stem ratio of the mutant, the leaf stem ratios of two-months-old wild type (Jemalong A17) and bs1 mutant plants were measured.FIG. 22shows that the leaf stem ratios based on fresh weights or dry weights were significantly increased in the bs1 mutant compared to wild type plant (Student's t-test, p<0.05, n=10).

Identification of Alfalfa (Medicago sativa) BIG SEEDS1 Orthologous Sequences and their Use

In order to introduce theMedicago truncatulabs1 mutant phenotype into alfalfa (Medicago sativa) for improvements of its forage quality, a total of three potential alfalfa BIG SEEDS1 orthologous sequences were identified based on their high degree of sequence similarity with theM. truncatulaBIG SEEDS1 sequence of SEQ ID NO:117, using the available alfalfa genome sequences. These genes are designated as MsBs1 (Table 1; SEQ ID NOs:118, 168 for nucleotide and predicted protein sequences, respectively), MsBS2 (Table 1; SEQ ID NOs:169, 171 for genomic nucleotide and predicted protein sequences, respectively) and MsBS3 (Table 1; SEQ ID NOs:170, 172 for genomic nucleotide and predicted protein sequences, respectively). Expression of MsBs1, MsBS2, and/or MsBS3 may be altered inM. sativato achieve beneficial agronomic qualities such as increased seed yield, increased plant biomass, enhanced forage quality, altered seed amino acid content etc.