WO2001023530A1 - Genes expressed in germinating seeds and their uses - Google Patents

Abstract

The present invention provides sequences isolated from germinating seeds that can be used to control seed germination in plants. Such enzymes include seed-specific polygalacturonases, cellulases arabinosidases, xyloglucan endotransglycosylases (XET) and expansins.

Description

GENES EXPRESSED IN GERMINATING SEEDS AND THEIR USES

CROSS-REFERENCE TO RELATED APPLICATIONS This application is a continuation in part of U.S. Patent Application No. 09/410, 191, filed September 30, 1999, which is hereby incorporated by reference.

STATEMENT AS TO RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSORED RESEARCH AND DEVELOPMENT

This invention was made with Government support under Grant Nos. IBN- 9407264 and UBN-9722978 awarded by the National Science Foundation. The

Government has certain rights in this invention.

FIELD OF THE INVENTION This invention relates to plant genetic engineering. In particular, it relates to methods of modulating seed germination in plants

BACKGROUND OF THE INVENTION In most seeds, radicle extension through the structures surrounding the embryo is the event that terminates germination and marks the commencement of seedling growth (Bewley, Plant Cell 9:1055-1066 (1997)). In seeds whose embryos are embedded in a rigid endosperm, the micropylar portion of the endosperm, termed the endosperm cap, presents a physical restraint to radicle extension. This restraint must be lessened through the weakening of the endosperm cap to allow radicle emergence (Groot and Karssen, Planta 171: 525-531 (1987)). Endosperm cap weakening is thought to be the result of cell wall hydrolysis. As mannose-containing polysaccharides are a major component of the endosperm cell walls of seeds of tomato and other Solanaceae (Sanchez et al, Plant Physiol 93: 89-97 (1990); Dahal et al, Plant Physiol 113: 1243-1252 (1997)), endo-β-mannanase has been regarded as a good candidate to control the weakening process (Groot et al, Planta 174: 500-504 (1988); Nomaguchi et al, Physiol Plant 94: 105-109 (1995)). Increased mannanase activity is consistently associated with radicle emergence (Nonokaki et al, Physiol Plant 102: 236-242 (1998)), but there are also conditions where emergence does not occur even though high enzyme activity is present (Still and Bradford, Plant Physiol 113: 21-29 (1997)). Thus, while endo-β- mannanase may be necessary for germination, it does not appear to be sufficient in all cases.

In addition to endo-β-mannanase, mannosidase, galactosidase, cellulase, pectin methylesterase, polygalacturonase, arabinosidase, xyloglucan endotransglycosylase, β-l,3-glucanase and chitinase are also expressed during tomato seed germination (Groot et al, Planta 174: 500-504 (1988); Leviatov et al, Ann Bot 76: 1-6 (1995); Downie et al, Anal Biochem 264: 149-157 (1998)). Since some of these hydrolases are associated with cell separation or cell wall disassembly in other developmental processes, such as abscission zones and fruit ripening (Del Campillo and Lewis, Plant Physiol 98: 955-961 (1992); Lashbrook et al, Plant Cell 6: 1484-1493 (1994)), it is reasonable to expect that they also may be involved in endosperm weakening. Additional factors may also be involved in controlling this process (Bewley, Trends Plant Sci 2: 464-469 (1997)).

Expansins are extracellular proteins that facilitate cell wall extension, possibly by disrupting hydrogen bonding between hemicellulosic wall components and cellulose microfibrils. In addition, some expansins are expressed in non-growing tissues such as ripening fruits, where they may contribute to cell wall disassembly associated with tissue softening.

Expansin was first identified from cucumber hypocotyls by its ability to induce stress relaxation in killed cell walls (McQueen-Mason et al, Plant Cell 4: 1425- 1433 (1992). Expansins are proposed to function as cell wall loosening factors by disrupting noncovalent linkages, such as hydrogen bonds, at the cellulose-hemicellulose interface, thereby relaxing an important constraint to turgor-driven cell expansion (McQueen-Mason and Cosgrove, Proc Natl Acad Sci USA 91: 6574-6578 (1994); Cosgrove, Plant Physiol 118: 333-339 (1998)). Expansins have been highly conserved throughout plant evolution, as homologous genes have been identified in gymnosperms and in both monocots and dicots among the angiosperms. Expansins occur as multi-gene families in Arabidopsis, rice, cucumber, tomato, and other species where they have been examined in detail. The large number of expansin-like genes (e.g., at least 22 in Arabidopsis) suggests multiple developmental or tissue-specific roles for these proteins, possibly in addition to vegetative growth per se. Expansins are expressed in shoot meristems during the early stages of leaf initiation and also in ripening fruits at a time when cell wall disassembly associated with fruit softening is occurring. During ripening, extensive cell wall degradation and solubilization of wall components occurs, resulting in tissue softening and cell separation without cell enlargement. Preliminary results with expansin promoters linked to the GUS reporter also indicate that expression of specific expansin genes occurs in germinating seeds, in the root cap, and in association with abscission zones or tissues where cell separation will occur. Thus, in addition to their role in cell growth, specific expansins may also contribute to cell wall processes associated with developmental events such as ripening, abscission, and cell separation (Cosgrove, Proc Natl Acad Sci USA 94: 5504-5505 (1997).

The prior art lacks means for controlling seed germination by controlling expression of genes associated with weakening tissues surrounding the embryo. The present invention addresses these and other needs.

SUMMARY OF THE INVENTION The present invention provides sequences isolated from germinating seeds that can be used to control seed germination in plants. In particular, the present invention provides nucleic acid molecules which encode polypeptides having greater than about 70% identity to SEQ ID NO: 4, 6, 8, 10, 14, 18, 20, and 22. The polypeptides of the invention include a number of proteins and enzymes associated with weakening tissues surrounding the embryo and/or initiating radicle growth. In some embodiments, the nucleic acids of the invention encode expansins that are expressed in seeds. An exemplary expansin is LeExρ4 (SEQ ID NO: 1). The invention further provides nucleic acid molecules which encode polypeptides having 99% or more identity to SEQ ID NO: 16, as well as nucleic acid molecules which encode polypeptides having greater than about 80% identity to SEQ ID NO: 12. The present invention further provides recombinant expression vectors comprising the nucleic acid sequences of the invention. Preferably, the vectors comprise a plant promoter operably linked to the nucleic acid sequence. The promoter can be either a constitutive promoter, or an inducible promoter.

The present invention also provides for transgenic plants comprising a recombinant expression cassette of the invention. The recombinant expression cassettes are useful in methods of modulating seed germination in plants. For example, the nucleic acids of the invention can be used to enhance expression of the endogenous gene and thereby promote seed germination. Alternatively, the nucleic acids can be used to inhibit expression of the endogenous genes and thereby inhibit seed germination. DEFINITIONS The phrase "nucleic acid sequence" refers to a single or double-stranded polymer of deoxyribonucleotide or ribonucleotide bases read from the 5' to the 3' end. It includes chromosomal DNA, self-replicating plasmids, infectious polymers of DNA or RNA and DNA or RNA that performs a primarily structural role.

The term "promoter" refers to regions or sequence located upstream and/or downstream from the start of transcription and which are involved in recognition and binding of RNA polymerase and other proteins to initiate transcription. A "plant promoter" is a promoter capable of initiating transcription in plant cells. Such a promoter can be derived from plant genes or from other organisms, such as viruses capable of infecting plant cells.

The term "plant" includes whole plants, shoot vegetative organs/structures (e.g. leaves, stems and tubers), roots, flowers and floral organs/structures (e.g. bracts, sepals, petals, stamens, carpels, anthers and ovules), seed (including embryo, endosperm, and seed coat) and fruit (the mature ovary), plant tissue (e.g. vascular tissue, ground tissue, and the like) and cells (e.g. guard cells, egg cells, trichomes and the like), and progeny of same. The class of plants that can be used in the method of the invention is generally as broad as the class of higher and lower plants amenable to transformation techniques, including angiosperms (monocotyledonous and dicotyledonous plants), gymnosperms, ferns, and multicellular algae. It includes plants of a variety of ploidy levels, including aneuploid, polyploid, diploid, haploid and hemizygous.

A polynucleotide sequence is "heterologous to" an organism or a second polynucleotide sequence if it originates from a foreign species, or, if from the same species, is modified from its original form. For example, a promoter operably linked to a heterologous coding sequence refers to a coding sequence from a species different from that from which the promoter was derived, or, if from the same species, a coding sequence which is not naturally associated with the promoter (e.g. a genetically engineered coding sequence or an allele from a different ecotype or variety). A polynucleotide "exogenous to" an individual plant is a polynucleotide which is introduced into the plant by any means other than by a sexual cross. Examples of means by which this can be accomplished are described below, and include Agrobacterium-mediated transformation, biolistic methods, electroporation, and the like. Such a plant containing the exogenous nucleic acid is referred to here as a Ti (e.g. in Arabidopsis by vacuum infiltration) or Ro (for plants regenerated from transformed cells in vitro) generation transgenic plant. Transgenic plants that arise from sexual cross or by selfϊng are descendants of such a plant.

"Recombinant" refers to a human manipulated polynucleotide or a copy or complement of a human manipulated polynucleotide. For instance, a recombinant expression cassette comprising a promoter operably linked to a second polynucleotide may include a promoter that is heterologous to the second polynucleotide as the result of human manipulation (e.g., by methods described in Sambrook et al, Molecular Cloning - A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, (1989) or Current Protocols in Molecular Biology Volumes 1-3, John Wiley & Sons, Inc. (1994-1998)) of an isolated nucleic acid comprising the expression cassette. In another example, a recombinant expression cassette may comprise polynucleotides combined in such a way that the polynucleotides are extremely unlikely to be found in nature. For instance, human manipulated restriction sites or plasmid vector sequences may flank or separate the promoter from the second polynucleotide. One of skill will recognize that polynucleotides can be manipulated in many ways and are not limited to the examples above.

The phrase "substantially identical," in the context of two nucleic acids or two polypeptides, refers to a sequence or subsequence that has at least 60% sequence identity with a reference sequence. Alternatively, percent identity can be any integer from 60% to 100%. More preferred embodiments include at least: 60%, 65%, 70%, 75%, 80%), 85%, 90%, 95%), or 99%. compared to a reference sequence when aligned for maximum correspondence over a comparison window as measured using one of the following sequence comparison algorithms or by manual alignment and visual inspection. Preferably, the comparison is made by BLAST using standard parameters, as described below. This definition also refers to the complement of a test sequence, when the test sequence has substantial identity to a reference sequence. With respect to the LeMAN2 polypeptide, it is preferred if the sequence identity to a second polypeptide is any integer between 80% and 100%, with higher percentages of sequence identity being preferred over lower percentages. With respect to the LVA-P1 polypeptide, it is preferred if the sequence identity to a second polypeptide is 99 % or 100%.

Two nucleic acid sequences or polypeptides are said to be "identical" if the sequence of nucleotides or amino acid residues, respectively, in the two sequences is the same when aligned for maximum correspondence as described below. The terms "identical" or percent "identity," in the context of two or more nucleic acids or polypeptide sequences, refer to two or more sequences or subsequences that are the same or have a specified percentage of amino acid residues or nucleotides that are the same, when compared and aligned for maximum correspondence over a comparison window, as measured using one of the following sequence comparison algorithms or by manual alignment and visual inspection. When percentage of sequence identity is used in reference to proteins or peptides, it is recognized that residue positions that are not identical often differ by conservative amino acid substitutions, where amino acids residues are substituted for other amino acid residues with similar chemical properties (e.g., charge or hydrophobicity) and therefore do not change the functional properties of the molecule. Where sequences differ in conservative substitutions, the percent sequence identity may be adjusted upwards to correct for the conservative nature of the substitution. Means for making this adjustment are well known to those of skill in the art. Typically this involves scoring a conservative substitution as a partial rather than a full mismatch, thereby increasing the percentage sequence identity. Thus, for example, where an identical amino acid is given a score of 1 and a non-conservative substitution is given a score of zero, a conservative substitution is given a score between zero and 1. The scoring of conservative substitutions is calculated according to, e.g., the algorithm of Meyers & Miller, Computer Applic. Biol. Sci. 4:11-17 (1988) e.g., as implemented in the program PC/GENE (Intelligenetics, Mountain View, California, USA).

One of skill in the art will recognize that two polypeptides can also be "substantially identical" if the two polypeptides are immunologically similar. Thus, overall protein structure may be similar while the primary structure of the two polypeptides display significant variation. Therefore a method to measure whether two polypeptides are substantially identical involves measuring the binding of monoclonal or polyclonal antibodies to each polypeptide. Two polypeptides are substantially identical if the antibodies specific for a first polypeptide bind to a second polypeptide with an affinity of at least one third of the affinity for the first polypeptide.

For sequence comparison, typically one sequence acts as a reference sequence, to which test sequences are compared. When using a sequence comparison algorithm, test and reference sequences are input into a computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated. The sequence comparison algorithm then calculates the percent sequence identity for the test sequence(s) relative to the reference sequence, based on the designated program parameters.

Optimal alignment of sequences for comparison can be conducted, e.g., by the local homology algorithm of Smith & Waterman, Adv. Appl Math. 2:482 (1981), by the homology alignment algorithm of Needleman & Wunsch, J. Mol. Biol. 48:443 (1970), by the search for similarity method of Pearson & Lipman, Proc. Nat 'I. Acad. Sci. USA 85:2444 (1988), by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, WI), or by visual inspection (see generally, Current Protocols in Molecular Biology, F.M. Ausubel et al, eds., Current Protocols, a joint venture between Greene Publishing Associates, Inc. and John Wiley & Sons, Inc., (1995 Supplement) (Ausubel)).

Examples of algorithms that are suitable for determining percent sequence identity and sequence similarity are the BLAST and BLAST 2.0 algorithms, which are described in Altschul et al. (1990) J. Mol. Biol. 215: 403-410 and Altschuel et al. (1977) Nucleic Acids Res. 25: 3389-3402, respectively. Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information (http://www.ncbi.nlm.nih.gov/). This algorithm involves first identifying high scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence, which either match or satisfy some positive-valued threshold score T when aligned with a word of the same length in a database sequence. T is referred to as the neighborhood word score threshold (Altschul et al, supra). These initial neighborhood word bits act as seeds for initiating searches to find longer HSPs containing them. The word hits are then extended in both directions along each sequence for as far as the cumulative alignment score can be increased. Cumulative scores are calculated using, for nucleotide sequences, the parameters M (reward score for a pair of matching residues; always > 0) and N (penalty score for mismatching residues; always < 0). For amino acid sequences, a scoring matrix is used to calculate the cumulative score. Extension of the word hits in each direction are halted when: the cumulative alignment score falls off by the quantity X from its maximum achieved value; the cumulative score goes to zero or below, due to the accumulation of one or more negative-scoring residue alignments; or the end of either sequence is reached. The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment. The BLASTN program (for nucleotide sequences) uses as defaults a wordlength (W) of 11, an expectation (E) of 10, M=5, N=-4, and a comparison of both strands. For amino acid sequences, the BLASTP program uses as defaults a wordlength (W) of 3, an expectation (E) of 10, and the BLOSUM62 scoring matrix (see Henikoff & Henikoff, Proc. Natl Acad. Sci. USA 89:10915 (1989)).

In addition to calculating percent sequence identity, the BLAST algorithm also performs a statistical analysis of the similarity between two sequences (see, e.g., Karlin & Altschul, Proc. Nat 'l Acad. Sci. USA 90:5873-5787 (1993)). One measure of similarity provided by the BLAST algorithm is the smallest sum probability (P(N)), which provides an indication of the probability by which a match between two nucleotide or amino acid sequences would occur by chance. For example, a nucleic acid is considered similar to a reference sequence if the smallest sum probability in a comparison of the test nucleic acid to the reference nucleic acid is less than about 0.1, more preferably less than about 0.01, and most preferably less than about 0.001. A further indication that two nucleic acid sequences or polypeptides are substantially identical is that the polypeptide encoded by the first nucleic acid is immunologically cross reactive with the polypeptide encoded by the second nucleic acid, as described below. Thus, a polypeptide is typically substantially identical to a second polypeptide, for example, where the two peptides differ only by conservative substitutions. Another indication that two nucleic acid sequences are substantially identical is that the two molecules hybridize to each other under stringent conditions, as described below.

"Conservatively modified variants" applies to both amino acid and nucleic acid sequences. With respect to particular nucleic acid sequences, conservatively modified variants refers to those nucleic acids which encode identical or essentially identical amino acid sequences, or where the nucleic acid does not encode an amino acid sequence, to essentially identical sequences. Because of the degeneracy of the genetic code, a large number of functionally identical nucleic acids encode any given protein. For instance, the codons GCA, GCC, GCG and GCU all encode the amino acid alanine. Thus, at every position where an alanine is specified by a codon, the codon can be altered to any of the corresponding codons described without altering the encoded polypeptide. Such nucleic acid variations are "silent variations," which are one species of conservatively modified variations. Every nucleic acid sequence herein which encodes a polypeptide also describes every possible silent variation of the nucleic acid. One of skill will recognize that each codon in a nucleic acid (except AUG, which is ordinarily the only codon for methionine) can be modified to yield a functionally identical molecule. Accordingly, each silent variation of a nucleic acid which encodes a polypeptide is implicit in each described sequence.

As to amino acid sequences, one of skill will recognize that individual substitutions, in a nucleic acid, peptide, polypeptide, or protein sequence which alters a single amino acid or a small percentage of amino acids in the encoded sequence is a "conservatively modified variant" where the alteration results in the substitution of an amino acid with a chemically similar amino acid. Conservative substitution tables providing functionally similar amino acids are well known in the art.

The following six groups each contain amino acids that are conservative substitutions for one another: 1 ) Alanine (A), Serine (S), Threonine (T);

2) Aspartic acid (D), Glutamic acid (E);

3) Asparagine (N), Glutamine (Q);

4) Arginine (R), Lysine (K);

5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V); and 6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W).

(see, e.g., Creighton, Proteins (1984)).

An indication that two nucleic acid sequences or polypeptides are substantially identical is that the polypeptide encoded by the first nucleic acid is immunologically cross reactive with the antibodies raised against the polypeptide encoded by the second nucleic acid. Thus, a polypeptide is typically substantially identical to a second polypeptide, for example, where the two peptides differ only by conservative substitutions. Another indication that two nucleic acid sequences are substantially identical is that the two molecules or their complements hybridize to each other under stringent conditions, as described below. The phrase "selectively (or specifically) hybridizes to" refers to the binding, duplexing, or hybridizing of a molecule only to a particular nucleotide sequence under stringent hybridization conditions when that sequence is present in a complex mixture (e.g., total cellular or library DNA or RNA). The phrase "stringent hybridization conditions" refers to conditions under which a probe will hybridize to its target subsequence, typically in a complex mixture of nucleic acid, but to no other sequences. Stringent conditions are sequence-dependent and will be different in different circumstances. Longer sequences hybridize specifically at higher temperatures. An extensive guide to the hybridization of nucleic acids is found in Tijssen, Techniques in Biochemistry and Molecular Biology— Hybridization with Nucleic Probes, "Overview of principles of hybridization and the strategy of nucleic acid assays" (1993). Generally, highly stringent conditions are selected to be about 5-10°C lower than the thermal melting point (T_m) for the specific sequence at a defined ionic strength pH. Lower stringency conditions are generally selected to be about 15-30 °C below the T_m. The T_m is the temperature (under defined ionic strength, pH, and nucleic concentration) at which 50% of the probes complementary to the target hybridize to the target sequence at equilibrium (as the target sequences are present in excess, at T_m, 50% of the probes are occupied at equilibrium). Stringent conditions will be those in which the salt concentration is less than about 1.0 M sodium ion, typically about 0.01 to 1.0 M sodium ion concentration (or other salts) at pH 7.0 to 8.3 and the temperature is at least about 30°C for short probes (e.g., 10 to 50 nucleotides) and at least about 60°C for long probes (e.g., greater than 50 nucleotides). Stringent conditions may also be achieved with the addition of destabilizing agents such as formamide. For selective or specific hybridization, a positive signal is at least two times background, preferably 10 time background hybridization.

Nucleic acids that do not hybridize to each other under stringent conditions are still substantially identical if the polypeptides which they encode are substantially identical. This occurs, for example, when a copy of a nucleic acid is created using the maximum codon degeneracy permitted by the genetic code. In such cases, the nucleic acids typically hybridize under moderately stringent hybridization conditions.

In the present invention, genomic DNA or cDNA comprising nucleic acids of the invention can be identified in standard Southern blots under stringent conditions using the nucleic acid sequences disclosed here. For the purposes of this disclosure, suitable stringent conditions for such hybridizations are those which include a hybridization in a buffer of 40% formamide, 1 M NaCl, 1% SDS at 37°C, and at least one wash in 0.2X SSC at a temperature of at least about 50°C, usually about 55°C to about 60°C, for 20 minutes, or equivalent conditions. A positive hybridization is at least twice background. Those of ordinary skill will readily recognize that alternative hybridization and wash conditions can be utilized to provide conditions of similar stringency.

Nucleic acids that do not hybridize to each other under stringent conditions are still substantially identical if the polypeptides that they encode are substantially identical. This occurs, for example, when a copy of a nucleic acid is created using the maximum codon degeneracy permitted by the genetic code. In such cases, the nucleic acids typically hybridize under moderately stringent hybridization conditions. Exemplary "moderately stringent hybridization conditions" include a hybridization in a buffer of 40% formamide, 1 M NaCl, 1% SDS at 37°C, and a wash in IX SSC at 45°C. A positive hybridization is at least twice background. Those of ordinary skill will readily recognize that alternative hybridization and wash conditions can be utilized to provide conditions of similar stringency.

A further indication that two polynucleotides are substantially identical is if the reference sequence, amplified by a pair of oligonucleotide primers, can then be used as a probe under stringent hybridization conditions to isolate the test sequence from a cDNA or genomic library, or to identify the test sequence in, e.g., an RNA gel or DNA gel blot hybridization analysis.

DETAILED DESCRIPTION The present invention provides nucleic acids that are expressed in seeds and can be used to control seed germination. As noted above, although endo-β- mannanase activity is consistently associated with germination, there are conditions where enzyme activity is high, yet radicle emergence does not occur. Thus, the present invention is based, at least in part on the identification of cell wall hydrolases and other enzymes that contribute to seed germination. Such enzymes include seed-specific mannanases, polygalacturonases, cellulases, arabinosidases, xyloglucan endotransglycosylases (XET) and expansins. All are dependent upon gibberellin (GA) for expression in GA-deficient seeds, are expressed initially in the endosperm caps, and are associated with cell wall hydrolysis. As LeEXP4 is expressed specifically in endosperm caps prior to radicle emergence, this expansin is involved, possibly in conjunction with wall hydrolases, such as LeMAN2, in effecting tissue weakening.

The polypeptides encoded by the nucleic acids are thus associated with the weakening of tissues surrounding the embryo and/or initiating radicle growth. The control of expression of the endogenous genes is therefore a convenient means for controlling seed germination. Means for controlling polypeptide activity and gene expression in plants are well known and can be used with the nucleic acids of the invention, explained below. Increasing polypeptide activity or gene expression

Any of a number of means well known in the art can be used to increase activity of polypeptides or polynucleotides of the invention in plants. Enhanced expression is useful to promote seed germination. Usually isolated sequences prepared as described herein are used to prepare recombinant expression cassettes in recombinant vectors. The vectors are introduced into plant cells using methods well known to those of skill in the art. Preparation of suitable constructs and means for introducing them into plants are described below.

One of skill will recognize that the polypeptides encoded by the nucleic acids of the invention, like other proteins, have different domains that perform different functions. Thus, gene sequences of the invention need not be full length, so long as the desired functional domain of the protein is expressed.

Modified protein chains can also be readily designed utilizing various recombinant DNA techniques well known to those skilled in the art and described in detail below. For example, the chains can vary from the naturally occurring sequence at the primary structure level by amino acid substitutions, additions, deletions, and the like. These modifications can be used in a number of combinations to produce the final modified protein chain.

Alternatively, endogenous genes can be modified to enhance expression of these genes. Methods for introducing genetic mutations into plant genes and selecting plants with desired traits are well known. For instance, seeds or other plant material can be treated with a mutagenic chemical substance, according to standard techniques. Such chemical substances include, but are not limited to, the following: diethyl sulfate, ethylene imine, ethyl methanesulfonate and N-nitroso-N-ethylurea. Alternatively, ionizing radiation from sources such as, X-rays, fast neutrons or gamma rays can be used. Alternatively, homologous recombination can be used to induce targeted gene modifications by specifically targeting gene of the invention in vivo (see, generally, Grewal and Klar, Genetics 146: 1221-1238 (1997) and Xu et al, Genes Dev. 10: 2411- 2422 (1996)). Homologous recombination has been demonstrated in plants (Puchta et al, Experientia 50: 277-284 (1994), Swoboda et al, EMBOJ. 13: 484-489 (1994); Offringa et al, Proc. Natl. Acad. Sci. USA 90: 7346-7350 (1993); and Kempin et al. Nature 389:802-803 (1997)).

Other means for increasing activity of polynucleotides and polypeptides of the invention can also be used. For example, one method to increase expression of genes of the invention is to use "activation mutagenesis" (see, e.g. Hiyashi et al. Science 258:1350-1353 (1992)). In this method an endogenous gene of the invention can be modified to be expressed constitutively, ectopically, or excessively by insertion of T-DNA sequences that contain strong/constitutive promoters upstream of the endogenous gene. Inhibition of activity or expression of polvnucleotides or polypeptides of the invention Activity of endogenous genes an also be inhibited using well known techniques. Inhibition of expression of these genes can be used to inhibit seed germination and thus control the timing of seed germination, for example, by using an inducible promoter to control expression of the nucleic acids in recombinant expression cassettes of the invention.

The nucleic acid sequences disclosed here can be used to design nucleic acids useful in a number of methods to inhibit expression of genes of the invention in plants. For instance, antisense technology can be conveniently used. To accomplish this, a nucleic acid segment from the desired gene is cloned and operably linked to a promoter such that the antisense strand of RNA will be transcribed. The construct is then transformed into plants and the antisense strand of RNA is produced. In plant cells, it has been suggested that antisense suppression can act at all levels of gene regulation including suppression of RNA translation (see, Bourque, Plant Sci. (Limerick) 105: 125- 149 (1995); Pantopoulos, In Progress in Nucleic Acid Research and Molecular Biology, Vol. 48. Cohn, W. E. and K. Moldave (Ed.). Academic Press, Inc.: San Diego, California, USA; London, England, UK. p. 181-238; Heiser et al, Plant Sci. (Shannon) 127: 61-69 (1997)) and by preventing the accumulation of mRNA which encodes the protein of interest, (see, Baulcombe, Plant Mol. Bio. 32:79-88 (1996); Prins and Goldbach, Arch. Virol. 141: 2259-2276 (1996); Metzlaff et al. Cell 88: 845-854 (1997), Sheehy et al., Proc. Nat. Acad. Sci. USA, 85:8805-8809 (1988), and Hiatt et al., U.S. Patent No. 4,801,340).

The nucleic acid segment to be introduced generally will be substantially identical to at least a portion of the endogenous gene or genes to be repressed. The sequence, however, need not be perfectly identical to inhibit expression. The vectors of the present invention can be designed such that the inhibitory effect applies to other genes within a family of genes exhibiting identity or substantial identity to the target gene.

For antisense suppression, the introduced sequence also need not be full length relative to either the primary transcription product or fully processed mRNA. Generally, higher identity can be used to compensate for the use of a shorter sequence. Furthermore, the introduced sequence need not have the same intron or exon pattern, and identity of non-coding segments may be equally effective. Normally, a sequence of between about 30 or 40 nucleotides to about the full length of a nucleotide should be used, though a sequence of at least about 100 nucleotides is preferred, a sequence of at least about 200 nucleotides is more preferred, and a sequence of about 500 to about 3500 nucleotides is especially preferred.

A number of gene regions can be targeted to suppress expression of genes of the invention. The targets can include, for instance, the coding regions, introns, sequences from exon/intron junctions, 5' or 3' untranslated regions, and the like. Another well-known method of suppression is sense co-suppression.

Introduction of nucleic acid configured in the sense orientation has been recently shown to be an effective means by which to block the transcription of target genes. For an example of the use of this method to modulate expression of endogenous genes (see, Assaad et al, Plant Mol. Bio. 22: 1067-1085 (1993); Flavell, Proc. Natl. Acad. Sci. USA 91 : 3490-3496 (1994); Stam et al, Annals Bot. 79: 3-12 (1997); Napoli et al., The Plant Cell 2:279-289 (1990); and U.S. Patents Nos. 5,034,323, 5,231,020, and 5,283,184).

The suppressive effect may occur where the introduced sequence contains no coding sequence per se, but only intron or untranslated sequences homologous to sequences present in the primary transcript of the endogenous sequence. The introduced sequence generally will be substantially identical to the endogenous sequence intended to be repressed. This minimal identity will typically be greater than about 65%, but a higher identity might exert a more effective repression of expression of the endogenous sequences. Substantially greater identity of more than about 80% is preferred, though about 95% to absolute identity is most preferred. As with antisense regulation, the effect should apply to any other proteins within a similar family of genes exhibiting identity or substantial identity.

For co-suppression, the introduced sequence, needing less than absolute identity, also need not be full length, relative to either the primary transcription product or fully processed mRNA. This may be preferred to avoid concurrent production of some plants that over-express the introduced sequence. A higher identity in a sequence shorter than full-length compensates for a longer, less identical sequence. Furthermore, the introduced sequence need not have the same intron or exon pattern, and identity of non- coding segments will be equally effective. Normally, a sequence of the size ranges noted above for antisense regulation is used. In addition, the same gene regions noted for antisense regulation can be targeted using co-suppression technologies.

Other means of inhibiting expression are known. These methods include formation of triple-helix DNA (see, e.g., Havre and Glazer J. Virology 67:7324-7331

(1993); Scanlon et al. FASEB J. 9:1288-1296 (1995); Giovannangeli et al. Biochemistry 35:10539-10548 (1996); Chan and Glazer J. Mol. Medicine (Berlin) 75: 267-282 (1997)) and ribozymes (Zhao and Pick, Nature 365:448-451 (1993); Eastham and Ahlering, J.

Urology 156:1186-1188 (1996); Sokol and Murray, Transgenic Res. 5:363-371 (1996);

Sun et al, Mol. Biotechnology 7:241-251 (1997); and Haseloff et al, Nature, 334:585-

591 (1988)). Modification of endogenous genes can also be used to inhibit expression.

Methods for introducing genetic mutations described above can also be used to select for plants with decreased expression of genes of the invention.

Other means for inhibiting polynucleotide or polypeptide activity can also be used. Activity of polynucleotides of the invention may be modulated by eliminating the proteins that are required for cell-specific expression of such polynucleotides. Thus, expression of regulatory proteins and/or the sequences that control gene expression can be modulated using the methods described here.

Purification of polypeptides

Naturally occurring or recombinant polypeptides of the invention can be purified for use in functional assays. Naturally occurring polypeptides can be purified, e.g., from plant tissue and any other source of the desired polypeptide. Recombinant polypeptides can be purified from any suitable expression system.

The polypeptides of the invention may be purified to substantial purity by standard techniques, including selective precipitation with such substances as ammonium sulfate; column chromatography, immunopurification methods, and others (see, e.g.,

Scopes, Protein Purification: Principles and Practice (1982); U.S. Patent No. 4,673,641;

Ausubel et al, supra; and Sambrook et al, supra).

A number of procedures can be employed when recombinant polypeptides are being purified. For example, proteins having established molecular adhesion properties (e.g. epitope tags, histidine tags and the like) can be reversibly fused to polypeptides of the invention. With the appropriate ligand, the such polypeptides can be selectively adsorbed to a purification column and then freed from the column in a relatively pure form. Isolation of nucleic acids of the invention

Generally, the nomenclature and the laboratory procedures in recombinant DNA technology described below are those well known and commonly employed in the art. Standard techniques are used for cloning, DNA and RNA isolation, amplification and purification. Generally enzymatic reactions involving DNA ligase, DNA polymerase, restriction endonucleases and the like are performed according to the manufacturer's specifications. These techniques and various other techniques are generally performed according to Sambrook et al, Molecular Cloning - A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, (1989) or Current Protocols in Molecular Biology Volumes 1-3, John Wiley & Sons, Inc. (1994-1998). The isolation of nucleic acids of the invention may be accomplished by a number of techniques. For instance, oligonucleotide probes based on the sequences disclosed here can be used to identify the desired gene in a cDNA or genomic DNA library. To construct genomic libraries, large segments of genomic DNA are generated by random fragmentation, e.g. using restriction endonucleases, and are ligated with vector DNA to form concatemers that can be packaged into the appropriate vector. To prepare a cDNA library, mRNA is isolated from the desired organ, such as seeds, and a cDNA library that contains a gene transcript of the invention is prepared from the mRNA. Alternatively, cDNA may be prepared from mRNA extracted from other tissues in which genes of the invention or homo logs are expressed. The cDNA or genomic library can then be screened using a probe based upon the sequence of a cloned gene of the invention as disclosed here. Probes may be used to hybridize with genomic DNA or cDNA sequences to isolate homologous genes in the same or different plant species. Alternatively, antibodies raised against a polypeptide of the invention can be used to screen an mRNA expression library. Alternatively, the nucleic acids of interest can be amplified from nucleic acid samples using amplification techniques. For instance, polymerase chain reaction (PCR) technology can be used to amplify the sequences of genes of the invention directly from genomic DNA, from cDNA, from genomic libraries or cDNA libraries. PCR and other in vitro amplification methods may also be useful, for example, to clone nucleic acid sequences that code for proteins to be expressed, to make nucleic acids to use as probes for detecting the presence of the desired mRNA in samples, for nucleic acid sequencing, or for other purposes. For a general overview of PCR, see PCR Protocols: A Guide to Methods and Applications. (Innis, M, Gelfand, D., Sninsky, J. and White, T., eds.), Academic Press, San Diego (1990). Appropriate primers and probes for identifying sequences of the invention from plant tissues are generated from comparisons of the sequences provided here (e.g. SEQ ID NO: 1, SEQ ID NO:3, etc.).

Polynucleotides may also be synthesized by well-known techniques as described in the technical literature. See, e.g., Carruthers et al, Cold Spring Harbor Symp. Quant. Biol. 47:411-418 (1982), and Adams et al, J. Am. Chem. Soc. 105:661

(1983). Double stranded DNA fragments may then be obtained either by synthesizing the complementary strand and annealing the strands together under appropriate conditions, or by adding the complementary strand using DNA polymerase with an appropriate primer sequence.

Preparation of recombinant vectors

To use isolated sequences in the above techniques, recombinant DNA vectors suitable for transformation of plant cells are prepared. Techniques for transforming a wide variety of higher plant species are well known and described in the technical and scientific literature. See, for example, Weising et al. Ann. Rev. Genet.

22:421-477 (1988). A DNA sequence coding for the desired polypeptide, for example a cDNA sequence encoding a full length protein, will preferably be combined with transcriptional and translational initiation regulatory sequences which will direct the transcription of the sequence from the gene in the intended tissues of the transformed plant.

For example, for overexpression, a plant promoter fragment may be employed which will direct expression of the gene in all tissues of a regenerated plant. Such promoters are referred to herein as "constitutive" promoters and are active under most environmental conditions and states of development or cell differentiation. Examples of constitutive promoters include the cauliflower mosaic virus (CaMV) 35S transcription initiation region, the 1'- or 2'- promoter derived from T-DNA of Agrobacterium tumefaciens, and other transcription initiation regions from various plant genes known to those of skill. Constitutive promoters and regulatory elements can also be isolated from genes that are expressed constitutively or at least expressed in most if not all tissues of a plant. Such genes include, for example, ACT 11 from Arabidopsis (Huang et al. Plant Mol. Biol. 33:125-139 (1996)), Cat3 from Arabidopsis (GenBank No. U43147, Zhong et al, Mol. Gen. Genet. 251 :196-203 (1996)), the gene encoding stearoyl-acyl carrier protein desaturase from Brassica napus (Genbank No. X74782, Solocombe et al. Plant Physiol. 104: 1167- 1176 ( 1994)), GPcl from maize (GenBank No. X15596, Martinez et al. J. Mol. Biol 208:551-565 (1989)), and Gpc2 from maize (GenBank No. U45855, Manjunath et al, Plant Mol. Biol. 33:97-112 (1997)).

Alternatively, the plant promoter may direct expression of a nucleic acid of the invention in a specific tissue, organ or cell type (i.e. tissue-specific promoters) or may be otherwise under more precise environmental or developmental control (i.e. inducible promoters). Examples of environmental conditions that may effect transcription by inducible promoters include anaerobic conditions, elevated temperature, the presence of light, or application of chemicals/hormones (such promoters can be used, for example, in the chemical induction of antisense sequences for inhibition of seed germination until a desired time). Exemplary promoters for this purpose include promoters from glucocorticoid receptor genes (Aoyama and Chau, Plant J 11 :605-12 (1997)). Tissue- specific promoters may only promote transcription within a certain time frame of developmental stage within that tissue. Other tissue specific promoters may be active throughout the life cycle of a particular tissue. One of skill will recognize that a tissue- specific promoter may drive expression of operably linked sequences in tissues other than the target tissue. Thus, as used herein a tissue-specific promoter is one that drives expression preferentially in the target tissue or cell type, but may also lead to some expression in other tissues as well.

A number of tissue-specific promoters can be used in the invention. Preferred promoters are those that direct expression of nucleic acids in seeds. As used herein a seed-specific promoter is one which directs expression in seed tissues, such promoters may be, for example, ovule-specific, embryo-specific, endosperm-specific, integument-specific, seed coat-specific, or some combination thereof. Examples include a promoter from the ovule-specific BEL1 gene described in Reiser et al Cell 83:735-742 (1995) (GenBank No. U39944). Other suitable seed specific promoters are derived from the following genes: MAC1 from maize (Sheridan et al. Genetics 142:1009-1020 (1996), Cat3 from maize (GenBank No. L05934, Abler et al. Plant Mol. Biol. 22:10131-1038 (1993), the gene encoding oleosin 18kD from maize (GenBank No. J05212, Lee et al. Plant Mol. Biol. 26:1981-1987 (1994)), vivparous-1 from Arabidopsis (Genbank No. U93215), the gene encoding oleosin from Arabidopsis (Genbank No. Z17657), Atmycl from Arabidopsis (Urao et al. Plant Mol. Biol. 32:571-576 (1996), the 2s seed storage protein gene family from Arabidopsis (Conceicao et al. Plant 5:493-505 (1994)) the gene encoding oleosin 20kD from Brassica napus (GenBank No. M63985), napA from Brassica napus (GenBank No. J02798, Josefsson et al. JBL 26:12196-1301 (1987), the napin gene family from Brassica napus (Sjodahl et al. Planta 197:264-271 (1995), the gene encoding the 2S storage protein from Brassica napus (Dasgupta et al. Gene 133:301-302 (1993)), the genes encoding oleosin A (Genbank No. U09118) and oleosin B (Genbank No. U09119) from soybean and the gene encoding low molecular weight sulphur rich protein from soybean (Choi et al. Mol Gen, Genet. 246:266-268 (1995)). Another example of a promoter useful in the present invention is the promoter of the LeXPGl gene provided in SEQ ID NO: 23. One of skill will recognize that the variants of this promoter sequence can also be used. For example, the promoter can be less than full length (e.g. fragments of 500 to about 1000 nucleotides in length) and still provide suitable expression levels.

The vector comprising the sequences (e.g., promoters or coding regions) from genes of the invention will typically comprise a marker gene that confers a selectable phenotype on plant cells. For example, the marker may encode biocide resistance, particularly antibiotic resistance, such as resistance to kanamycin, G418, bleomycin, hygromycin, or herbicide resistance, such as resistance to chlorosulfuron or Basta. Production of transgenic plants

DNA constructs of the invention may be introduced into the genome of the desired plant host by a variety of conventional techniques. For example, the DNA construct may be introduced directly into the genomic DNA of the plant cell using techniques such as electroporation and microinjection of plant cell protoplasts, or the DNA constructs can be introduced directly to plant tissue using ballistic methods, such as DNA particle bombardment.

Microinjection techniques are known in the art and well described in the scientific and patent literature. The introduction of DNA constructs using polyethylene glycol precipitation is described in Paszkowski et al. EMBO. J. 3:2717-2722 (1984). Electroporation techniques are described in Fromm et al Proc. Natl. Acad. Sci. USA 82:5824 (1985). Ballistic transformation techniques are described in Klein et al Nature 327:70-73 (1987). Alternatively, the DNA constructs may be combined with suitable T-DNA flanking regions and introduced into a conventional Agrobacterium tumefaciens host vector. The virulence functions of the Agrobacterium tumefaciens host will direct the insertion of the construct and adjacent marker into the plant cell DNA when the cell is infected by the bacteria. Agrobacterium tumefaciens-mediated transformation techniques, including disarming and use of binary vectors, are well described in the scientific literature. See, for example Horsch et al. Science 233:496-498 (1984), and Fraley et al. Proc. Natl. Acad. Sci. USA 80:4803 (1983) and Gene Transfer to Plants, Pofrykus, ed. (Springer- Verlag, Berlin 1995). Transformed plant cells which are derived by any of the above transformation techniques can be cultured to regenerate a whole plant which possesses the transformed genotype and thus the desired phenotype such as increased seed mass. Such regeneration techniques rely on manipulation of certain phytohormones in a tissue culture growth medium, typically relying on a biocide and/or herbicide marker that has been introduced together with the desired nucleotide sequences. Plant regeneration from cultured protoplasts is described in Evans et al., Protoplasts Isolation and Culture, Handbook of Plant Cell Culture, pp. 124-176, MacMillilan Publishing Company, New York, 1983; and Binding, Regeneration of Plants, Plant Protoplasts, pp. 21-73, CRC Press, Boca Raton, 1985. Regeneration can also be obtained from plant callus, explants, organs, or parts thereof. Such regeneration techniques are described generally in Klee et al. Ann. Rev. of Plant Phys. 38:467-486 (1987).

The nucleic acids of the invention can be used to confer desired traits on essentially any plant. Thus, the invention has use over a broad range of plants, including species from the genera Anacardium, Arachis, Asparagus, Atropa, Avena, Brassica, Citrus, Citrullus, Capsicum, Carthamus, Cocos, Coffea, Cucumis, Cucurbita, Daucus, Elaeis, Fragaria, Glycine, Gossypium, Helianthus, Heterocallis, Hordeum, Hyoscyamus, Lactuca, Linum, Lolium, Lupinus, Lycopersicon, Malus, Manihot, Majorana, Medicago, Nicotiana, Olea, Oryza, Panieum, Pannesetum, Persea, Phaseolus, Pistachia, Pisum, Pyrus, Prunus, Raphanus, Ricinus, Secale, Senecio, Sinapis, Solanum, Sorghum, Theobromus, Trigonella, Triticum, Vicia, Vitis, Vigna, and Zea.

One of skill will recognize 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. Using known procedures one of skill can screen for plants of the invention by detecting the increase or decrease of mRNA or protein of the invention in transgenic plants, particularly in the seed. Means for detecting and quantitating mRNAs or proteins are well known in the art.

Plants with modulated seed germination can be easily be selected by monitoring seed germination using standard techniques.

EXAMPLES

The following examples are offered to illustrate, but not to limit the claimed invention.

Example 1 :

This Example describes the cloning of expansin genes from tomato seeds.

MATERIALS AND METHODS

Plant Materials

Tomato (Lycopersion esculentum Mill.) seeds from either wild-type (cv. Moneymaker) plants or homozygous gibberellin-deficient (gib-1) mutant plants were used throughout the study. The gib-1 mutant and its isogenic parent line were originally obtained from Dr. Cees Karssen, Wageningen Agricultural University, The Netherlands. Mutant plants were sprayed 3 times per week with 100 μM GA to revert the dwarf habit and allow more vigorous growth and fertility. After fruits were harvested, seeds were extracted, treated with 0.25 M HCl, dried to 6% moisture content (fresh basis) and stored at -20°C until used (Ni and Bradford, Plant Physiol 101: 607-617 (1993).

Germination Conditions

Approximately 500 seeds were incubated at 25°C in the dark in 9-cm diameter Petri dishes on top of two layers of filter paper moistened with 12 mL of deionized water, 100 μM GA₄₊ , 100 μM ABA or PEG 8000 solutions having water potentials of -0.5, -1.0 and -2.0 MPa. For far-red (FR) light treatment, seeds were imbibed at 25 °C for 40 h under continuous FR illumination in a custom-made FR chamber (Lagarias et al, Plant Cell 9: 675-688 (1997) where peak transmittance, half- band pass, and fluence rate were 760 nm, 85 nm and 22 μmolnr ¹, respectively, at the level of the seed as measured by a LI-COR LI-8000 portable spectroradiometer (LI-COR, Inc., Lincoln, Nebraska, USA).

RNA Isolation, PCR Amplification, and cDNA Library Screening Samples of 500 whole seeds imbibed for 24 h were pulverized in LN₂ and the frozen material transferred to 2 mL of extraction buffer (10 mM Tris-HCl pH 8.2, 100 raM LiCl, 1 mM EDTA, 1% (w/v) SDS, 25 mM DTT) in a ground glass homogenizer on ice. Extraction followed a modification of the phenol/SDS method of Ausubel et al. Current Protocols in Molecular Biology. (New York: Wiley-Interscience, 1987). One μg of purified total RNA was used as template for RT-PCR. Two degenerate PCR primers, the 5' primer [G(GC)(N)CA(TC)GC(N)AC(N)TT(CT)TA(CT)GG(N)G] and the 3' primer [(TC)TGCCA(AG)TT(TC)TG(N)CCCCA(AG)TT] (N=A, T, G or C) were designed based on two conserved amino acid domains according to the alignment of deduced amino acid sequences of known expansins (Shcherban et al, Proc Natl Acad Sci USA 92: 9245-9249 (1995); Cho and Kende Plant Cell 9:1661-1671 (1997); Rose et al, Proc Natl Acad Sci USA 94: 5955-5960 (1997). After amplification for 36 cycles (94°C for 1 min, 50°C for 1.5 min, and 72°C for 1.5 min), the amplified fragments were cloned into pCR2.1 according to the manufacturer's instructions (Invitrogen). DNA sequences were determined with universal primers (T3 and M13-forward) using an Applied Biosystems model 377 sequencer (Perkin-Elmer) with dye termination chemistry and AmpliTaq DNA polymerase FS (Perkin-Elmer/ Applied Biosystems). The PCR fragments were used to screen a cDNA library prepared from gib-1 seeds imbibed in 100 μM GA₄₊₇ for 24 h. The cDNAs were labeled with enhanced chemiluminescence (ECL) labeling reagents (Amersham Life Science, Inc.), then were added to prehybridization solution at a final concentration of 10 ng/mL. Prehybridization was for 30 min at 42°C, and hybridization was for 3 h at 42°C. Following hybridization, the membranes were washed twice at 42°C with 6 M urea, 0.5% SDS (low stringency) or 0.2% SDS (high stringency) and then washed twice for 5 min each with 2X SSC at room temperature. Independent inserts in the library vector pBK-CMV were sequenced.

DNA Gel Blot Analyses

For cDNA gel blot analysis, 5 ng of PCR product obtained by T3/T7 primers from library vector containing target gene was subjected to electrophoresis, and transferred to a Hybond-N* membrane (Amersham Life Science, Inc.). Prehybridization, hybridization, washing, and autoradiography of the blot were performed as described for cDNA library screening using ECL lit, except that hybridization was kept for overnight. Genomic DNA was isolated from young tomato leaves (cv. Moneymaker) as described by Murray and Thompson Nucleic Acids Res 8: 4321-4325 (1980) and modified by Bernatzky and Tanksley Theor Appl Genet 72: 314-321 (1986). Aliquots (10 μg) were digested with restriction enzymes, fractionated on a 0.8% agarose gel, and transferred to Hybond-N⁺ membranes. Prehybridization, hybridization, washing, and autoradiography of the blots were performed as described for cDNA gel blot analysis.

RNA Gel Blot Analyses

Total RNA was isolated from germinating seeds, seed parts or different tissues according to the above method. For seed part RNA extraction, imbibed seeds were first dissected into three parts: the endosperm cap, the radicle tip removed from within the endosperm cap, and the rest of seed (lateral endosperm and remainder of embryo). Three pools containing 1000 seed parts were used for RNA isolation. Total RNA from each sample (5 μg) was subjected to electrophoresis on 1% (w/v) agarose/ 10% formaldehyde denaturing gels, transferred to Hybond-N^1" membrane and UV cross-linked. The probe for Northern Blots was DIG-labeled DNA probe prepared by using PCR labeling method. Primers corresponding to 3'-terminal untranslation region of the gene were used to incorporate DIG-labeled dNTP into DNA probe. The labeling efficiency was estimated according to the manual (Boehringer Mannheim, Inc.). Final probe concentration used for hybridization is 25ng/ML hybridization buffer. Since DNA probe is used for RNA detection, high SDS buffer (7% SDS) was used for hybridization at 42°C. The washing (60°C) and detection followed the recommended method using the chemiluminescent substrate CPSD (Boehringer Mannheim, Inc.). Exposure time was from 10 min to 2 h depending on the strength of the signal.

Puncture Force Measurements The force required to puncture the micropylar endosperm and testa surrounding the radicle tip was analyzed for both gib-1 and MM seeds following various treatments. Each micropylar tip was sliced from the seed and the radicle tip teased out of the embryo cavity. A food texture analyzer (Stable Micro Systems, Texture Technologies Corp., Scarsdale, New York, USA) fitted with a custom-made probe (0.5 mm diameter) was used to determine puncture force. The endosperm cap without the radicle tip was placed on the texture analyzer probe and the test conducted at an inching speed of 10 mm/min (Downie, et al., 1999). In each test, the background resistance generated by the probe against the side of the cap was subtracted from the peak resistance to puncture force using XT.RA Dimension version 3.7F software supplied by the manufacturer (Stable Micro Systems). Twenty-four individual seeds were measured at each time point and means were expressed as the puncture force in newtons (N).

RESULTS

Cloning and Sequence Analysis of Tomato Seed Expansins Using primers to conserved expansin sequences, a ~540 bp cDNA band was amplified by RT-PCR from germinating tomato seed RNA and subsequent sequence analyses indicated the existence of six expansin homologs (termed TE1 to TE6). These six independent fragments share high amino acid sequence identity among themselves and with two truncated cucumber expansins from hypocotyl. The predicted amino acid sequence of all six tomato expansin homologs exhibit the basic features of expansins, including eight conserved cysteines and conserved tryptophans (Shcherban et al, 1995). The sequence of TE1 was identical to truncated sequence of LeExpl, which is expressed during tomato fruit ripening (Rose et al, 1997), and the sequence of TE2 was identical to truncated sequence of LeExp2, which is expressed in expending tissues (Reinhardt et al, Plant Cell 10: 1427-1438 (1998). TE3 was identical with the truncated sequence of LeExp4 (SEQ ID NO: 1) which is detected in flowers (Brummell et al, Plant Mol Biol 39: 161-169 (1999). The remaining three fragments TE4, TE5 and TE6 were unique in the database. Since the colonies were randomly selected for sequencing, it is possible that additional expansin homologs are expressed in germinating tomato seeds.

A cDNA library prepared from GA-treated gib-1 tomato seeds was screened with TE1 to TE6 respectively. The full length sequences for TE1, TE2 and TE3 were confirmed to be identical with LeExpl, LeExpl and LeExp4. The full length sequences for TE4, TE5 and TE6 were named LeExp8, (SEQ ID NO: 3) LeExp9 and

LeExplO (SEQ ID NO: 5), respectively. Database searching with the deduced amino acid sequences of the full-length cDNAs revealed high degree of homology to expansins from cucumber, Arabidopsis, rice, pea and tomato. LeExp4 is a Member of a Multigene Family with Tissue-Specific Expression in Germinating Seeds

To test expression level of individual expansin genes, a gene-specific probe was designed based on 3'- terminal untranslation region and obtained by PCR and then used for Northern Blot analysis. No signal or very weak signal can be detected from germinating seeds for LeExpl, LeExp2 or LeExp9, while strong signal can be detected for LeExp4, LeExpδ and LeExplO.

Hormonal and Environmental Regulation ofLeExp4 Expression and Endosperm Cap Weakening

Endosperm cap weakening and germination of gibberellin-defϊcient g?^'t 7 tomato seeds is dependent on GA. To determine the expression pattern of LeExp4 in response to GA, total RNA was extracted from gib-1 seeds after imbibition for 2, 12, 24, 48 and 60h in water or in 100 μM GA and hybridized with the EeExp -specific RNA probe. No expression was detected in dry gib-1 seeds or gib-1 seeds imbibed in water. In the presence of GA, LeExp4 transcript accumulated within 12 h and reached a maximal level at 24 h of imbibition. Subsequently, LeExp4 mRNA abundance declined slightly by 48 h. (Approximately 4% of the seeds had completed radicle emergence by 48 h, although only ungerminated seeds were sampled for RNA). Puncture force analysis showed that endosperm caps did not weaken when gib-1 seeds were imbibed in water, but weakening did occur when GA was present in the imbibition solution. Weakening was evident within 12 h of imbibition in GA, a time when LeExp4 mRNA was also detected. Germination of wild-type tomato seeds can be inhibited by ABA. The effect of ABA is similar in MM seeds with gib-1 seeds. When total RNA was extracted from MM seeds after imbibition for 2, 12, 24,48 and 60 h in water or in 100 μM ABA and hybridized with the ZeExp^-specific probe. LeExp4 mRNA could be detected from 12 h of imbibition in both water and ABA, and the pattern of expression was similar regardless of the presence of ABA. ABA also had no effect on the decrease in puncture force of the endosperm cap, although it did prevent radicle emergence.

Low water potential can delay or prevent tomato seed germination. When tomato seeds were imbibed in PEG solutions maintaining -0.5 or -1.0 MPa osmotic potentials, the abundance oϊLeExp4 mRNA slightly decreased as the water potential decreased. Puncture force analysis also showed that low water potential can prevent or delay the endosperm weakening process.

Far-red light can also inhibit tomato seed germination. When MM seeds were imbibed under continuous far-red light, germination was prevented and no expression of LeExp4 was detected. Far-red light also prevented any weakening of the endosperm cap.

DISCUSSION

Expansins comprise a large superfamily of genes sharing conserved sequences both within and among species. The results presented here show here that LeExp4 has a highly specific pattern of expression. Using a gene-specific probe, LeExp4 mRNA was detected in flowers and germinating seeds. In the germinating seed, expression of LeExp4 was restricted to the endosperm cap tissue directly opposite the radicle tip. This endosperm cap tissue is anatomically and physiologically differentiated from the remainder of the endosperm, for example by having thinner cell walls and by the expression of a cap-specific isoform of endo-β-mannanase prior to radicle emergence. Cell wall degradation, vacuolization, and other visible changes occur in the cap region prior to the initiation of such changes in the remainder of the endosperm. Thus, the occurrence of a specific expansin and of unique iso forms of hydrolytic enzymes in the endosperm cap is likely associated with the tissue weakening and cell separation that occurs to allow radicle emergence, rather than being strictly a reserve mobilization phenomenon as in the remainder of the endosperm. This conclusion is supported by the expression pattern of LeExp4 in response to factors that also regulate germination. In gib-1 seeds, which require GA to complete germination, GA induces accumulation of LeExp4 mRNA within 12 h of imbibition. Even though the entire seed is in contact with the imbibition solution containing GA, LeExp4 mRNA is detected only in the endosperm cap. On the other hand, both reduced water potential and far-red light inhibit germination reduce or prevent expression of LeExp4. Furthermore, the extent of endosperm cap weakening was quantitatively related to the abundance of LeExp4 mRNA.

The relationship of LeExp4 expression to ABA at first appears rather anomalous. ABA effectively inhibits germination, and in isolated endosperm caps it also blocks GA-induced weakening. ABA did not prevent LeExp4 expression and had no effect on endosperm weakening. Thus, ABA does not appear to inhibit germination by blocking the expression of genes associated with endosperm cap weakening. It has been suggested that there is a second phase of weakening required for radicle emergence, and that ABA inhibits this second phase. Alternatively, ABA may act primarily on the growth potential of the embryo, reducing it below that required to penetrate even the weakened endosperm cap.

The expansin gene LeExp4 is expressed specifically in the endosperm cap of imbibed tomato seeds. Expression of LeExp4 is initiated within 12 h of imbibition, is regulated by factors that affect germination, and is quantitatively correlated with the extent of weakening of the endosperm cap tissues. Together, these results support the hypothesis that LeExp4, most likely in conjunction with cell wall hydrolases, is involved in the cell wall changes associated with tissue weakening and cell separation in the endosperm cap. If expansin protein is required to loosen hemicellulosic bonds and/or to allow access of hydrolases to the polymer matrix, regulation of its expression could be a critical control point in the germination process.

Example 2

Using the RT-PCR approach described above, other cDNAs from genes associated with germination were isolated. For example polygalacturonase (PG) that is expressed in imbibed tomato seeds was identified. Extracts from seeds have exhibited only exo-PG activity and no endo-PG activity, this enzyme is likely encoded by the cloned gene (LeXPG 1, SEQ ID NO: 21; the encoded polypeptide is set forth in SEQ LD NO:22 cDNAs (Cel55 and Cel68 SEQ ID NO: 17 and \9) having high homology to known β-l,4-glucanases have also been identified. The encoded polypeptide sequences are set forth in SEQ ID NOs:18 and 20, respectively.

A third putative hydrolase cDNA (LeARA 1, SEQ ID NO: 9, the encoded polypeptide is set forth in SEQ ID NO: 10) was isolated from a differential cDNA display screen of gib-1 mutant tomato seeds imbibed in the presence or absence of GA. The corresponding mRNA was initially expressed only in the endosperm caps in response to GA, and subsequently expression spread through the remainder of the endosperm, but not into the embryo. The predicted amino acid sequence showed high homology to bacterial and fungal arabinosidases, and seed extracts exhibited arabinosidase activity. Another candidate hydrolase involved in germination is xyloglucan endotransglycosylase (XET, SEQ ID NO: 13, the encoded polypeptide is set forth as SEQ ID NO: 14). It is believed that xyloglucans, the principal hemicellulose component in the primary cell walls of dicots, can form a tightly bound, non-covalent association with cellulose. XET catalyzes both the endo-type splitting of a xyloglucan molecule and the linking of the newly generated reducing end to a nonreducing end of another xyloglucan molecule or an oligosaccharide. This lengthening and rearrangement of xyloglucans may release tension and accommodate wall expansion during cell growth. Other genes identified in this way include a gibberellin-stimulated/ABA down regulated gene of unknown function (LeGAS2, SEQ ID NO: 7, the encoded polypeptide is set forth as SEQ ID NO:8).

Example 3

This example described the cloning of a mannanase gene (LeMAN2, SEQ ID NO: 11) from tomato seeds.

MATERIALS AND METHODS

Plant Material and Seed Germination

Tomato (Lycopersicon esculentum Mill.) seeds, either from wild-type (cv Moneymaker) plants or homozygous gibberellin-deficient mutant plants (gib-1) were used in this study. Mutant plants were sprayed three times per week with 100 μM GA4₊₇ to allow more vigorous growth and fertility. After fruits were harvested, seeds were collected, treated with 0.25 M HCl, dried to 6% moisture content (fresh weight basis) and stored at 20°C until used (Ni, B.R. et al, Plant Physiol 101:607-617 (1993)). For germination, 100 or 200 tomato seeds were placed on two layers of filter papers moistened with 12 mL of water or test solutions in Petri dishes and incubated at 25°C in the dark. For hormone treatments seeds were imbibed in the presence of 100 μM GA₄₊ and/or 100 μM (±)ABA.

In some cases, seeds were dissected into the micropylar tip and the remainder of the seed as previously described (Nonogaki, H. et al, Physiol Plant 85:167- 172 (1992)), and the embryonic tissues were removed from each part using forceps. The embryo-less micropylar tip and the embryo-less remainder of the seed were denoted as the endosperm cap and lateral endosperm, respectively. Isolation ofcDNAs

A cDNA library was constructed using mRNA from gib-1 seeds imbibed in 100 μM GA_n-₇ for 24 h using a λZAP Express cDNA Synthesis Kit (Stratagene, La Jolla, California) according to the manufacturer's instructions. The cDNA library was screened by hybridization of nitrocellulose filter plaque replicas with a partial length (0.9 kb) cDNA of the postgerminative mannanase cDNA (Bewley et al. 1997) labeled with ECL labeling reagents (Amersham Pharmacia Biotech, Inc., Piscataway, NJ). Hybridization was done at 42°C overnight after 1 h prehybridization at the same temperature. Following hybridization, the membranes were washed twice for 20 min each at 42°C with 6 M urea, 0.5% (w/v) SDS, 0.5 X SSC and twice for 5 min each at room temperature with 2X SSC. Independent inserts in the library vector pBK-CMV were sequenced by the Advanced Plant Genetics Facility, University of California, Davis. Sequence comparisons were made using DNAStar software (DNAStar Inc., Madison, WI). Signal peptide prediction was performed using the Signal IP V 1.1 server (www.cbs.dtu.dk/services/SignalIP).

Expression of Recombinant Protein in Escherichia coli The coding region (without the signal peptide) of the mannanase cDNA (nucleotides 83 to 1264 of SEQ ID NO: 11) was amplified by PCR using BamHI site- linked forward primer (5 ' -CGGGATCCTGTGAAGCTAGGGTT-3 ') and XbaT site- linked reverse primer (5'-CGTCTAGACTAAATCTTAACCAAATG-3'). The product was digested with BamHΪ and XbaT and ligated into the BamHI and XbaT sites of the maltose-binding protein expression vector pMAL-c2 (New England Biolabs, Inc.). The empty vector and the vector containing insertion were transformed into competent cells of a proteinase-deficient strain (BL21) of E. coli and the resulting transformant cells were selected using blue-white screening with IPTG-Xgal plates. After incubation of a 1% overnight culture for 4 h at 37°C, protein expression was induced by addition of JJPTG to a final concentration of 2 mM and further incubation at 37°C for 2 h. The bacterial cells were harvested by centrifugation at 6,000 g and the pellet was dissolved in sonication buffer (50 mM sodium phosphate buffer pH 8.0 containing 0.3 M NaCl, 1 mg/mL lysozyme [Boebringer Mannheim]). After overnight freezing at -20°C, the bacterial lysates were thawed and centrifuged at 10,000 g for 10 min and the supernatants were collected. Expressed proteins were visualized by SDS-PAGE of the supernatants (crude extracts) of induced or uninduced bacterial cultures with or without insertion. For purification of the fusion protein, the supernatant of an induced bacterial culture with insertion was applied to a maltose-binding protein affinity column (amylose resin, New England Biolabs, Inc.). The fusion protein was eluted from the column with 10 mM maltose. The fractions containing the fusion protein were examined by SDS-PAGE, mixed and dialyzed against 10 mM Tris-HCl pH 7.5 overnight at 4°C. The dialysate was stored at -80°C.

PAGE and Immunoblotting

Proteins were separated by SDS-PAGE using 10% (w/v) acrylamide gels according to Laemmli (1970). Native PAGE was done in 7.5% gels according to Davis (1964) except that ammonium peroxydisulfate was used in place of riboflavin in the stacking gel. After electrophoresis, proteins were transferred to PVDF membranes using semidry blotter (TRAN-BLOT SD, BioRad Laboratories, Hercules, CA) and blocked with 5% (w/v) nonfat milk in PBS containing 0.5% (v/v) Tween 20. Anti-maltose binding protein antibody (New England Biolabs) and anti-M3 mannanase antibody were used for immunoblotting at 1 :5000 dilution. Bound antibody was detected using horseradish peroxidase-conjugated goat anti-rabbit immunoglobulin (Sigma Immunochemicals, St. Louis, MO). The bands were visualized on X-ray film after the reaction with a chemiluminescence reagent (Renaissance; DuPont NEN Products, Boston, MA).

Endo-β-Mannanase Extraction and Assays

Endo-β-mannanase activity was extracted from tomato seeds or seed parts by homogenizing the tissues in 50 mM K-phosphate buffer, pH 6.8 with a mortar and pestle. The homogenate was centrifuged at 10,000 g for 5 mm and the supernatant was used as the enzyme solution. The endo-β-mannanase activity was assayed by the modified gel diffusion method (Still, D.W. et al, Plant Physiol 113:21-29 (1997)). Agarose (0.8% [w/v]) plates containing 0.05% (w/v) locust bean galactomannan (Sigma, St. Louis, MO) were solidified and wells were formed on the plates by scoring with a 3- mm cork borer and removing the plug by suction. The extracts (10 μL) from tomato seed parts or purified recombinant protein solution (10 μL) were applied to the wells and the plates were incubated at 25°C for 24 h. After incubation, the agarose gel plates were stained by 0.5% (w/v) Congo red dye (Sigma) as described previously (Still, D.W. et al, Plant Physiol 113:21-29 (1997)). The hydrolyzed areas were visible as clear circles on a dark background. The diameter of the hydrolyzed area is logarithmically related to the enzyme activity. This agarose gel method was also used for activity staining of native PAGE gels. After electrophoresis, a native gel was overlaid on top of the substrate- containing agarose gel and incubated at 25°C for 1 h. The activity band was visualized as an transparent zone on the substrate gel after staining as described above.

DNA Extraction and Southern Hybridization

Genomic DNA was isolated from young tomato leaves (cv. Moneymaker) as described by Murray and Thompson (1980). Genomic DNA (10 μg) was digested with the restriction enzymes BamHI, XbaT and XhoT (New England Biolabs, Beverly, MA), separated on a 1.0% (w/v) agarose gel, and transferred to positively charged membranes (Hybond-N*, Amersham Phaπnacia Biotech Inc.). Prehybridization, hybridization, washing, and detection were performed as described for cDNA library screening. For preparing the DNA probes, the vectors containing the full-length mannanase cDNAs were digested with BamHT and XhoT, and the gel-purified insertion was used to make ECL- labeled probes (Amersham Pharmacia Biotech Inc.).

RNA Extraction and Northern Hybridization Total RNA was extracted from seed parts (endosperm cap, lateral endosperm or whole embryo) of germinating or germinated tomato seeds using a standard phenol extraction method (Sambrook, J. et al, Molecular Cloning. A Laboratory Manual, Second Edition. N Ford, C Nolan, M Ferguson eds, Cold Spring Harbor Laboratory Press, Cold Spring Harbor (1989)). Total RNA (2 to 10 μg) was subjected to electrophoresis on 1.3% (w/v) agarose gels containing 7% (v/v) formaldehyde, transferred to a neutral membrane (Hybond-N, Amersham Pharmacia Biotech) and UV-crosslinked. RNA probes were prepared using a DIG-labeled dNTP mixture (Boehringer Mannheim, Inc., Indianapolis, IN) Hybridization was routinely done at 60°C overnight after 1 h prehybridization at the same temperature. The membranes were washed once for 25 mm with 2X SSC, 0.1 % (w/v) SDS at 70°C and twice for 25 min with 0.2X SSC, 0.1 % (w/v) SDS at 70°C. The membranes were then blocked for 1 h with 5% (w/v) nonfat milk in 0.1 M maleic acid buffer pH 7.5 containing 0.15 M NaCl, 0.3% (v/v) Tween 20 (buffer A) and incubated with alkaline phosphatase conjugated anti-DIG antibody (1:15,000 dilution) for 1 h at 25°C. After washing with buffer A, the membranes were subjected to chemiluminescence detection. The signal was detected on X-ray film after 5 to 20 min exposures. When the signals on two different membranes hybridized with different probes were compared, those membranes were exposed together on the same X-ray film for the same time.

RESULTS Isolation of the Germinative Mannanase cDNA

As the expression of mannanase in tomato seeds before radicle protrusion is induced by GA (Groot, S.P.C. et al, Planta 174:500-504 (1988)), a cDNA library of GA-induced gib-1 seeds was screened with a partial length (0.9 kb) cDNA of the postgerminative mannanase (Bewley et al., Plant Cell 9:1055-1066 (1997)). Four positive clones isolated from cDNA library screening had sequences similar to that of the postgerminative mannanase cDNA. The longest cDNA insert of the putative mannanase was rescued into pBK-CMV vector, then subcloned into pBluescript II KS, and designated LeMAN2 (Lycopersicon esculentum mannanase 2). In this paper, we designate the cDNA of the postgerminative mannanase as LeMANJ because it was the first mannanase cDNA isolated from tomato seeds.

The 1481 bp LeMAN2 cDNA (nucleotide sequence deposited in Genbank as Accession No. API 84238) contained an open reading frame encoding a protein of 415 amino acids. A putative signal peptide sequence of 22 amino acids was identified at the amino terminus of the protein. The mature protein of 393 amino acids was encoded from Cys residue at nucleotide 83 to He at nucleotide 1259, with a predicted molecular weight of 44,379 and pi of pH 5.7. The predicted amino acid sequence of the protein encoded by LeMAN2 was compared with the postgerminative mannanase protein encoded by LeMANl, with an Arabidopsis genomic sequence and with two fungal mannanases. Overall amino acid sequence similarity between LeMANl and LeMAN2 was 77%. While overall sequence similarity was high, LeMAN2 contained additional amino acids compared with LeMANl protein (e.g., amino acids 26-35, 132-159), accounting for the greater predicted size of LeMAN2 (44 kDa versus 38 kDa for LeMANl). Potential catalytic sites and a potential N-glycosylation site (Asn-Gly-Ser) (amino acids 50-52) that have been identified in the postgerminative mannanase were also present in LeMAN2 protein. The amino acid sequence similarity between the tomato mannanases (LeMANl and LeMAN2) and fungal mannanases (Aspergillus aculeatus and Trichoderma reeseii [Accession number AAA34208]) was approximately 30%. Interestingly, the amino acid sequences in LeMAN2 that were deleted in the LeMANl protein still showed identity to the fungal mannanase proteins.

Expression of the Protein Encoded by LeMAN2 cDNA To confirm that the LeMAN2 cDNA encodes endo-β-mannanase protein, the cDNA was inserted into a maltose-binding protein overexpression vector and transformed into E. coli. When the transformed cells were induced for protein expression by adding IPTG, a strong intensity band with an apparent molecular mass of 90 kDa was observed in the bacterial lysates, close to the predicted size of the fusion protein (maltose- binding protein [43 kDa] plus LeMAN2 mannanase [44 kDa]). This protein band was absent in the uninduced cells and in both induced and uninduced cells containing the empty vector. The putative fusion protein band was recognized by both anti-maltose- binding protein antibody and antibody to one of the postgerminative mannanses (anti-M3 mannanase antibody). These results confirm that the overexpressed protein contains the maltose-binding: :mannanase fusion protein. Extracts of the induced bacterial cells containing the LeMAN2 insert showed endo-β-mannanase activity, which could not be detected in bacterial cells that contained empty pMAL vector, indicating that the overexpressed recombinant protein was an active form of mannanase. When the fusion protein was affinity purified to homogeneity by using a maltose-binding protein affinity resin, the fractions containing the fusion protein showed high mannanase activity and were recognized by the anti-mannanase antibody. The gel diffusion assay method for mannohydrolase activity is specific for endo-type enzymes, so LeMAN2 clearly encodes an endo-β-mannanase.

Southern Hybridization The hybridization patterns of the LeMANl and LeMAN2 cDNAs with tomato genomic DNA were compared using Southern hybridization. Both cDNAs hybridized to the same sets of DNA fragments, confirming that multiple mannanase genes are present in the tomato genome. However, some DNA fragments hybridized more strongly to the LeMANl cDNA, while other bands showed a stronger signal with the LeMAN2 cDNA. This supports the sequence data indicating that different genes encode the germinative and postgerminative mannanases. Expression of LeMANl and LeMAN2 mRNA in tomato seeds Since LeMAN2 was isolated from a cDNA library prepared from tomato seeds prior to radicle emergence, the LeMAN2 protein is likely to be a germinative mannanase. Only the Mα germinative mannanase specific to the endosperm cap is present in the endosperm at this time (Nonogaki, H. et al, Plant Physiol 110:555-559 (1996)), but two embryo-specific mannanases are also present in germinating tomato seeds (Nonogaki et al, Physiol. Plant 102:236-242 (1998). To investigate in which tissue(s) of imbibed seeds the LeMAN2 mRNA is expressed, RNA gel blot analyses were performed. When the total RNA from dissected seed parts (endosperm cap, lateral endosperm and embryo) from wild type tomato seeds imbibed in water for 24 h was hybridized with a full-length LeMAN2 RNA probe, the transcript was detected only from the endosperm cap, indicating that LeMAN2 mRNA is specifically expressed in this tissue. The endosperm cap tissue also contained high mannanase activity, while little or no activity was detected from the lateral endosperm. Although some mannanase activity was present in the embryo as well, no hybridization between the LeMAN2 and embryonic mRNA was detected. Since the northern hybridization was performed under relatively high stringency conditions (70°C washing), hybridization at low stringency (55°C) was also examined. However, even at low stringency, no signal could be detected in the embryo. To compare the tissue specificity of expression oϊLeMAN2 (see above) with that of LeMANl, total RNA was extracted from the endosperm caps of seeds prior to radicle emergence and from the lateral endosperms of germinated seedlings at different stages of development. To directly compare the hybridization patterns, the same sets of RNA samples were loaded on the same gel, transferred to the same membrane, processed using the same anti-DIG-antibody solution following hybridization to the different probes, and exposed to the same X-ray film. The patterns of hybridization by LeMANl and LeMAN2 riboprobes to these RNA samples were completely different. When the LeMANl probe was used, hybridization was detected specifically in the RNA samples from postgerminative lateral endosperms after radicle growth had begun, although a faint band could also be seen in the endosperm caps prior to radicle emergence after a longer exposure to the X-ray film. On the other hand, when LeMAN2 was used as a probe, a strong signal of the transcript was detected in the RNA sample from the endosperm cap of seeds prior to radicle emergence, and only faint signals were detected at post-emergence stages. Thus, under the conditions used, there is little cross-hybridization of riboprobes prepared from each cDNA. The results demonstrate that expression of LeMAN2 is specific to the endosperm cap prior to radicle emergence and that LeMANl expression is localized to the lateral endosperm after radicle emergence. Given the timing and location of its expression, it is likely that LeMAN2 is involved in cell wall hydrolysis associated with endosperm cap weakening prior to radicle protrusion. To examine the correlation between the expression of LeMAN2 and germination, the abundance of LeMAN2 message in the endosperm caps of imbibed wild type tomato seeds was analyzed. It is known that there is no detectable mannanase activity in dry tomato seeds and that activity begins to increase 6 to 12 h after imbibition (Groot, S.P.C. et al, Planta 174:500-504 (1988)). LeMAN2 transcript was present in the endosperm cap within 12 h of imbibition and increased markedly by 24 h before declining slightly by 36 h. The timing of LeMAN2 expression corresponded to the appearance of mannanase activity in the same tissue, although the peak of mRNA accumulation occurred earlier than the maximum activity, as would be expected. Radicle protrusion of wild type seeds was first observed 40 to 48 h after imbibition (data not shown), well after the increase in LeMAN2 message and mannanase activity.

Hormonal regulation of the expression of LeMAN2 in the wild type- and gib-1 tomato seeds was also analyzed. While wild type seeds can germinate in water, germination of gib-1 mutant tomato seeds is dependent on application of exogenous GA. LeMAN2 mRNA could not be detected in the endosperm caps of gib-1 mutant seeds incubated in water and mannanase activity was barely detectable in this condition. In contrast, both LeMAN2 mRNA expression and mannanase activity were induced in the endosperm caps of gib-1 seeds imbibed in GA₄₊ . The expression of LeMAN2 mRNA and mannanase activity in the endosperm caps of wild type seeds in water or gib-1 seeds in GA was not inhibited by 100 μM ABA, although radicle protrusion did not occur in either case (data not shown). These findings are consistent with previous observations that germinative mannanase activity in the endosperm cap is insensitive to ABA (Toorop, P.E. et al, Planta 200:153-158 (1996); Dahal P. et al, Plant Physiol 113:1243-1252 (1997); Still, D.W. et al, Plant Physiol 113:13-20 (1997)). Example 4

This example describes cloning of a subunit of the vacuolar proton- translocating ATPase (LVA-P1, SEQ ID NO: 15).

Tomato (Lycopersicon esculentum Mill.) seeds were harvested from field- grown wild type MM (cv. Moneymaker) plants or from homozygous gibberellin- deficient (gib-1) mutant plants grown in a glasshouse (seeds originally obtained from Dr. Cees Karssen, Wageningen Agricultural University, The Netherlands). Plant culture and seed extraction were as described previously (Ni, B.R. et al, Plant Physiol 101:607-617 (1993)). For germination, approximately 200 seeds were incubated at 25°C in the dark in 9 x 100 mm diameter Petri dishes on top of two layers of blotter paper moistened with 12 mL of either distilled, deionized water or 100 μM GA₄₊₇ (Abbott Laboratories, Chicago, IL, USA)

Differential cDNA Display Analysis

Using DCD (Liang, P. et al, Science 257:967-971 (1992)), mRNA from the radicle tips or endosperm caps of gib-1 mutant seeds imbibed in water (which do not germinate) were compared with mRNA from the same tissues of gib-1 seeds imbibed in 100 μM GA₄₊ (in which radicle emergence begins at 36 to 40 h and is completed by most seeds within 60 h). Seeds in both the presence and absence of GA^-? were sampled at 40 h, excluding any seeds from which the radicle had emerged. The micropylar regions were excised from 100 seeds in each treatment and separated into endosperm caps and radicle tips. Tissues were frozen immediately in LN₂ and stored at -80°C. Frozen radicle tips or endosperm caps were pulverized in LN₂ and RNA was extracted and purified by the phenol/SDS method (Ausubel F.M. et al, Current Protocols in Molecular Biology, Wiley-Interscience, New York (1987)). Prior to use, aliquots of RNA were incubated with DNase I for 1 h at 37°C in digestion buffer (40 mM Tris-HCI pH 7.5, 6 mM MgCl₂, 20 U RNasin and 10 mM NaCl) followed by extraction with 1 volume phenol/chloroform isoamyl alcohol (25:24:1). The RNA was precipitated in ethanol and dissolved in the original volume of 2 mM EDTA.

Nine 3' anchor primers were synthesized as 5'-Tι₂MM-3', where M is A, G or C. Anchor primers were then combined at equal concentration to give three sets:

T₁₂,MA, T₁₂MG, and T_!2MC. These three anchor pools were used in reverse transcription reactions (Sambrook, J. et al, Molecular Cloning. A Laboratory Manual, Second Edition. NFord, C Nolan, M Ferguson eds, Cold Spring Harbor Laboratory Press, Cold Spring Harbor (1989)) and then in conjunction with eight specific ten-mers (Am-Aos, Operon Technologies Inc., Alameda, CA, USA) in the differential display PCR reactions (Liang, P. et al, Science 257:967-971 (1992)). Two microliters from the RT reaction were used in each of the subsequent 20 μL differential display reactions (10 mM Tris-HCl pH 8.3, 50 mM KC1, 1.5 mM MgCl₂, 2.5 μM dNTPs, 1 μM of each anchor primer, 0.2 μM 10- mer primer, 1 U AmpliTaq (Perkin-Elmer Cetus, Norwalk, CT, USA), 4 μCi [ -³⁵S]- dATP (1200 Ci/mmol). Cycling conditions were 30 s at 94°C, 2 mm annealing at 40°C and 30 s extension at 72°C for 40 cycles in a Perkin-Elmer Cetus 480 Thermal Cycler. PCR reactions were loaded onto a 40 cm x 40 cm x 0.4 mm 6% native polyacrylamide gel and electrophoresced at 40 watts. Selected cDNA fragment bands exhibiting differential amplification were cut from the DCD gels and recovered by boiling for 30 mm in 50 μL modified TE (10 mM Tris-HCl pH 7.5, 0.1 mM EDTA). The cDNAs were precipitated in ethanol in the presence of 10 μg linear acrylamide as a carrier (Gaillard, C et al, Nuc Acids Res 18:378 (1990)) and dissolved in 20 μL TE. Five microliters of each isolated cDNA was re-amplified using the same conditions as the first amplification except that the (now unlabelled) dNTP concentration was increased to 20 μM.

Fragments re-amplified from DCD gels were tested for differential expression using the reverse-northern technique. Four identical blots of electrophoresced DNA fragments were probed with labeled cDNA products from reverse transcription reactions using 10 μg total RNA from endosperm caps or radicle tips of seeds imbibed for 40 h in water or 100 μM GA₄₊₇ as template and labeled using 200 μM dNTP and 100 μCi (α-³²P) dCTP (3000 Ci/mmol) per 50 μL reaction.

PCP fragments selected on the basis of the reverse-northern results were ligated into the TA cloning vector pCRII (Invitrogen Corp., San Diego, CA, USA) and the resulting plasmids electroporated (Cooley, M.B. et al, J Bacteriol 173:2608-2626 (1991)) into competent E. coli JM109 cells (Stratagene, La Jolla, CA, USA) using the Gene Pulser Electroporator (Bio-Rad Laboratories, Hercules, CA, USA). The cloned DCD fragments were sequenced at the UC Davis Advanced Plant Genetics Facility on an ABI Prism 377 DNA Sequencer (ABI, Perkin-Elmer, Foster City, CA).

Northern Analyses

Total RNA was extracted as described above from intact wild type MM or gib-1 mutant seeds or isolated endosperm caps, radicle tips, and the rest of the seed incubated in water or in 100 μM GA₄₊₇ for the indicated times at 25°C. Additionally, total RNA was isolated from the flowers, leaves and roots of mature MM tomato plants. Total RNA (2 to 20 μg per lane) was electrophoresced (Sambrook, J. et al, Molecular Cloning. A Laboratory Manual, Second Edition. N Ford, C Nolan, M Ferguson eds, Cold Spring Harbor Laboratory Press, Cold Spring Harbor (1989)), transferred onto positively charged nylon membranes and UV crosslinked at 120,000 μJ cm^"2 on a FB-UVXL-1000 Stratalinker (Fisher Scientific, Santa Clara, CA, USA). Hybridization was detected using DIG-labeled RNA probes (Boehringer Mannheim Corp., Indianapolis, IN, USA) synthesized by either Sρ6 (Ambion Inc., Austin, TX, USA) or T7 (Pharmacia Biotechnology, Inc., Alameda, CA, USA) RNA polymerase. Detection of DIG-labeled probes was performed according to instructions in the Genius System (Boehringer Mannheim Corporation. Genius System User 's Guide for Membrane Hybridization. Version 3.0. (1995)) using CSPD (disodium 3-(4-methoxyspiro{l,2-dioxetane-3,2'-(5'- chloro)tricyclo[3.3.1.1³'⁷]decan}-4-yl) phenyl phosphate) as substrate.

cDNA Library Screening

Transcripts hybridizing to PCR fragment G21, which were differentially expressed in seeds in response to GA, were shown by northern analysis to be expressed in tomato roots as well as in seeds. Hence, 5xl0⁵ recombinants from a tomato root cDNA library in the plasmid pCGN 1703 (Ewing, N.N. et al, Plant Physiol 94:1874-1881

(1990)) were screened with G21 labeled in a random priming reaction with α-(³²P)-dATP. cDNA from hybridizing recombinants was recovered from the vector by restriction digestion with Smal and was subcloned into the Smal site in the polycloning region of pBSII KS (Stratagene, La Jolla, CA). The full-length cDNA hybridizing to G21, subsequently termed L VA-Pl, was sequenced at the Center for Engineering Plants for Resistance Against Pathogens (CEPRAP), University of California, Davis.

Protein Extraction and Western Blotting

Two grams of gib-1 mutant tomato seeds imbibed in water or 100 μM GA₄₊₇ for 36 h were homogenized for 1 mm in an Ultra-Turrax T25 (Janke and Kunkel JJ A Labortechnik, Staufel, Germany) at high speed in 5 mL of extraction buffer (70 mM Tris pH 8.0, 250 mM sucrose, 3 mM EDTA, 0.5% PVP-40, 0.1% BSA, 4 mM DTT). The slurry was filtered through cheesecloth and the filtrate was centrifuged at 12,000 g for 15 mm at 4°C and the supernatant again centrifuged at 113,000 g for 30 minutes at 4°C. The resulting pellet was resuspended in buffer (10 mM Tris/Mes pH 7; 250 mM sucrose, 1 mM DTT) and electrophoresced on 12% SDS-polyacrylamide (20 μg of total protein per lane). The proteins were transferred to nitrocellulose and detected as described below for western tissue prints.

Northern and Western Tissue Printing Protocols

For tissue printing (Reid, P.D. et al, Tissue Printing: Tools for the Study of Anatomy, Histochemistry, and Gene Expression. Academic Press, New York (1992)), tomato seeds were sliced longitudinally with a fresh razor blade. Each half-seed was pressed firmly in identical positions on separate nitrocellulose membranes for exactly 60 s (northerns) or 20 s (westerns), then removed with forceps, providing two mirror-image prints of the same seed. The entire procedure was performed using powder-free gloves. For northern prints, the membranes were UV crosslinked and treated with 10 U RNase-free DNase I (Pharmacia Biotechnology, Inc., Alameda, CA, USA) in 10 mL of digestion buffer for 1 h at 37°C in a roller tube. Subsequent steps in prehybridization, hybridization, washing and detection were as described for the DIG-labeled northerns (see above) except that the signal was detected using CDP-Star (disodium 4-chloro-3-(4- methoxyspiro{l,2-dioxetane-3,2'-(5'-chloro)tricyclol[3.3.1 ' ]decan}-4-yl) phenyl phosphate; Boehringer Mannheim) in conjunction with enhancer for chemiluminescent detection on nitrocellulose membranes (Tropix, Inc., Bedford, MA, USA). One membrane was hybridized with an antisense probe to detect the target mRNA while its mirror-image print was hybridized with a sense probe to control for nonspecific binding. Both membranes were then stripped and rehybridized with antisense and sense cDNAs complementary to a constitutively expressed mRNA coding for a ribosomal protein (G46) as a control for RNA bound to the membrane.

For western prints, the printed membranes were blocked using 1% BSA in lx TBS, washed four times for 5 min each in lx TBS-Tween (Sambrook, J. et al, Molecular Cloning. A Laboratory Manual, Second Edition. N Ford, C Nolan, M Ferguson eds, Cold Spring Harbor Laboratory Press, Cold Spring Harbor (1989)) and then incubated with primary antibody diluted 1 :4000 in 1% BSA in lx TBS. Rabbit polyclonal antibodies specific for either subunit A (68 kD) or subunit B (57 kD) of V- ATPase from mung bean (Vigna radiata) were provided by Dr. Masayoshi Maeshima (Matsuura-Endo, C. et al, Plant Physiol 100:718-722 (1992)). The membranes were subsequently washed in lx TBS-Tween and incubated with a 1:10,000 dilution of goat anti-rabbit IgG alkaline phosphatase-conjugated secondary antibody. After washing, the membranes were developed using NBT-BCIP (nitroblue tetrazolium chloride and 5- bromo-4-chloro-3-indolyl-phosphate; Boehringer Mannheim).

RESULTS Cloning of a V-ATPase Subunit c Gene from Germinating Tomato Seeds Pools of total RNA from gib-1 tomato radicle tips and endosperm caps were isolated separately after imbibition in either water or 100 μM GA ₊₇ for 40 h, or just prior to the initiation of radicle emergence in the presence of GA^_? (radicle emergence does not occur in water). cDNA fragments identified by DCD as being differentially expressed were confirmed by northern analysis. One such fragment (G21) identified a transcript that increased in abundance in both endosperm caps and radicle tips in the presence of GA₄₊₇ and was present in untreated roots (data not shown). This fragment was used to recover a homologous full-length cDNA from a tomato root cDNA library. The deduced amino acid sequence is highly homologous to that of the 16 kD hydrophobic subunit c that forms the membrane-spanning, proton conductance pathway of plant vacuolarH⁺-translocating ATPases (Stevens, T.H. et al., Annu Rev Cell Dev Biol 13:779- 808 (1997); Sze, H. et al, J Bioenerg Biomem 24:371-381 (1995)). For example, the tomato cDNA sequence showed the following percentage amino acid identities

(nucleotide identities in parentheses) to other V-ATPase c subunits: 98.2% (84%) to Arabidopsis thaliana (Accession No. L44581; Perera, I.Y. et al, Plant Mol Biol 29:227- 244 (1995)); 98.2% (85%) to Gossypium hirsutum (U13669; Hasenfratz, M-P. et al, Plant Physiol 108:1395-1404 (1995); and 95.8% (79%) to Avena sativa (M73232; Lai, S. et al, JBiol Chem 266: 16078-16084 (1991)). We have therefore named this cDNA LVA- PI Lycopersicon Vacuolar ATPase-Proteolipid 1), in analogy with A VA-Pl and related genes in Arabidopsis (Perera, I.Y. et al, Plant Mol Biol 29:227-244 (1995)). The highly conserved amino acid sequence among species (including over 60% identity to corresponding mouse [M64298] and yeast [LO7105] genes) and the >98% amino acid sequence identity of LVA-Pl to other dicot V-ATPase subunit c genes leave little doubt that LVA-Pl is a tomato homolog of this gene. LVA-Pl Expression Patterns During Germination To determine the expression pattern of LVA-Pl, total RNA was extracted from endosperm caps, radicle tips, and the rest of gib-1 seeds (includes the lateral endosperm and most of the embryo) after imbibition for 1, 12, 24, and 40 h in water or in 100 _μM GA^-7 and hybridized with full-length E VA-Pl cDNA. In the presence of G/ ₊ , LVA-Pl transcript accumulated preferentially in the micropylar region of the seed (endosperm cap and radicle tip) within 12 h of imbibition. Subsequently, L VA-Pl mRNA abundance in the endosperm caps declined by 40 h but remained high in the radicle tips. Approximately 30% of the seeds had completed radicle emergence by 40 h, although RNA was extracted only from ungerminated seeds. Transcripts hybridizing to LVA-Pl were also detected in wild type MM flowers, leaves and roots.

Previous results have documented that a wide range of enzyme activity can exist among individual seeds, even in homozygous inbred tomato lines (Still, D.W. et al, Plant Physiol 113:21 -29 (1997)). Thus, mRNA extracted from pooled seed samples may not accurately reflect individual seed responses to GA. We therefore utilized tissue printing to assay mRNA abundance on a single-seed basis. Individual seeds were bisected after various times of imbibition and each mirror-image half was printed in an ordered array on separate nitrocellulose membranes. The membranes were treated with DNase, then hybridized with riboprobes made from either the antisense or the sense strand of the cDNA. This tissue printing method was specific, with little or no hybridization detected with riboprobes made from the sense strands of LVA-Pl or G46. Hybridization of the antisense riboprobe to the constitutive G46 mRNA, however, showed that approximately equal amounts of RNA were bound to the membrane by each seed. Hybridization of the antisense LVA-Pl riboprobe revealed an increase in LVA-Pl mRNA abundance only in the presence of GA4₊₇. Furthermore, in most seeds, L VA-Pl mRNA was most abundant in the micropylar region, in agreement with the pattern inferred from the northern blots of pooled samples.

As LVA-Pl expression in gib-1 seeds was dependent upon GA₄₊₇, transcript abundance was investigated in wild type MM seeds, which do not require additional GA for germination. LVA-Pl mRNA was present during seed development but declined in seeds from mature green and breaker stages of fruit development, before increasing again in seeds from ripe fruit (0 h of imbibition). Following imbibition, whole seeds were sampled every 12 h and separated into germinated and ungerminated seeds at 48 and 60 h. LVA-Pl mRNA content changed relatively little prior to radicle emergence and remained abundant in germinated seeds. As was observed for gib-1 seeds in the presence of GA₄₊₇, LVA-Pl mRNA was most abundant in the micropylar tissues. Imbibition of MM seeds in 100 μM GA₄₊₇ had no additional effect on mRNA abundance, indicating that the endogenous GA content of wild type seeds is sufficient to saturate the response.

GA-dependent Expression of V-ATPase Protein Subunits in gib-1 Seeds As GA stimulated the expression of LVA-Pl mRNA in gib-1 seeds, we tested whether other protein subunits associated with the V-ATPase complex were induced as well. The membrane-spanning hydrophobic subunit c coded by LVA-Pl is difficult to extract and detect with antibodies, so western blots of proteins from gib-1 seeds imbibed in either water or G ₄+₇ were performed using antibodies specific to the major 68 kDa catalytic nucleo tide-binding subunit A and the 57 kDa noncatalytic nucleotide-binding subunit B of mung bean V-ATPase (Matsuura-Endo, C. et al, Plant Physiol 100:718-722 (1992); Maeshima, M. et al, Plant Physiol 106:61-69 (1994)). The V-ATPase complex requires the membrane-spanning subunit c for assembly of the V₀ membrane sector, to which the cytoplasmic Vi sector containing subunits A and B is attached (Sze, H. et al, J Bioenerg Biomem 24:371-381 (1995)). Thus, detection of both A and B subunits is likely to be a good indicator of the presence of the holoenzyme, including the subunit c protein. Protein bands of the expected size increased in intensity in extracts from gib-1 seeds that had been imbibed in GA₄₊₇. No other proteins were detected, so the antibody for the 57 kDa subunit B was used with tissue prints to determine the localization of the V-ATPase within the seed. In agreement with the expression pattern of LVA-Pl mRNA, the subunit B protein was most abundant in the micropylar region of GA-treated gib-1 seeds, particularly in the endosperm cap, while seeds imbibed in water exhibited only background staining.

It is understood that the examples and embodiments described herein are for illustrative purposes only and that various modifications or changes in light thereof will be suggested to persons skilled in the art and are to be included within the spirit and purview of this application and scope of the appended claims. All publications, patents, and patent applications cited herein are hereby incorporated by reference in their entirety for all purposes.

Claims

WHAT IS CLAIMED IS:

1. An isolated nucleic acid molecule comprising a polynucleotide sequence encoding a polypeptide that modulates seed germination, wherein the polypeptide comprises an amino acid sequence that has at least about 70% identity to SEQ ID NOs: 4, 6, 8, 10, 14, 18, 20, and 22.

2. The nucleic acid molecule of claim 1, wherein the polynucleotide sequence specifically hybridizes to SEQ ID NO: 3, 5, 7, 9, 13, 17, 19, 21 or their complements.

3. The nucleic acid molecule of claim 1, wherein the polypeptide is as shown in SEQ ID NO: 4, 6, 8, 10, 14, 18, 20, and 22.

4. The nucleic acid molecule of claim 1, wherein the polynucleotide sequence is as shown in SEQ ID NO: 3, 5, 7, 9, 13, 17, 19, or 21.

5. A recombinant expression vector comprising the polynucleotide sequence of claim 1.

6. The recombinant expression vector of claim 5, further comprising a promoter operably linked to the polynucleotide sequence.

7. The recombinant expression vector of claim 6, wherein the promoter is a constitutive promoter.

8. The recombinant expression vector of claim 6, wherein the promoter is an inducible promoter.

9. A host cell transformed with the recombinant expression vector of claim 5.

10. A transgenic plant comprising a recombinant expression cassette comprising a promoter operably linked to the polynucleotide sequence of claim 1.

11. The transgenic plant of claim 10, wherein the plant is tomato.

12. A method of modulating seed germination in a plant, the method comprising a) introducing into the plant a recombinant expression vector comprising a promoter operably linked to a polynucleotide sequence encoding a polypeptide that modulates seed germination, wherein the polypeptide comprises an amino acid sequence that has at least about 70% identity to SEQ ID NOs: 2, 4, 6, 8, 10, 14, 18, 20, and 22; and b) selecting a plant with modulated seed germination.

13. The method of claim 12, wherein the polynucleotide specifically hybridizes to SEQ ID NO: 1, 3, 5, 7, 9, 13, 17, 19, 21 or their complements.

14. The method of claim 12, wherein the polypeptide is as shown in SEQ ID NO: 2, 4, 6, 8, 10, 14, 18, 20, and 22.

15. An isolated nucleic acid molecule comprising a promoter sequence from SEQ ID NO: 23 operably linked to heterologous nucleic acid sequence.

16. An isolated nucleic acid molecule comprising a polynucleotide sequence encoding a polypeptide that modulates seed germination, wherein the polypeptide comprises an amino acid sequence that has 99% or greater identity to SEQ ID NO: 16.

17. The nucleic acid molecule of claim 16, wherein the polynucleotide sequence specifically hybridizes to SEQ ID NO: 15 or its complement.

18. The nucleic acid molecule of claim 16, wherein the polypeptide is as shown in SEQ ID NO: 16.

19. The nucleic acid molecule of claim 1 , wherein the polynucleotide sequence is as shown in SEQ ID NO: 15.

20. A recombinant expression vector comprising the polynucleotide sequence of claim 16.

21. The recombinant expression vector of claim 20, further comprising a promoter operably linked to the polynucleotide sequence.

22. The recombinant expression vector of claim 20, wherein the promoter is a constitutive promoter.

23. The recombinant expression vector of claim 20, wherein the promoter is an inducible promoter.

24. A host cell transformed with the recombinant expression vector of claim 20.

25. A transgenic plant comprising a recombinant expression cassette comprising a promoter operably linked to the polynucleotide sequence of claim 16.

26. The transgenic plant of claim 25, wherein the plant is tomato.

27. A method of modulating seed germination in a plant, the method comprising a) introducing into the plant a recombinant expression vector comprising a promoter operably linked to a polynucleotide sequence encoding a polypeptide that modulates seed germination, wherein the polypeptide comprises an amino acid sequence that has 99% or greater identity to SEQ ID NO: 16; and b) selecting a plant with modulated seed germination.

28. The method of claim 27, wherein the polynucleotide specifically hybridizes to SEQ ID NO: 15 or its complement.

29. The method of claim 27, wherein the polypeptide is as shown in SEQ ID NO: 16.

30. An isolated nucleic acid molecule comprising a polynucleotide sequence encoding a polypeptide that modulates seed germination, wherein the polypeptide comprises an amino acid sequence that has 80% or greater identity to SEQ ID NO: 12.

31. The nucleic acid molecule of claim 30, wherein the polynucleotide sequence specifically hybridizes to SEQ ID NO: 11 or its complement.

32. The nucleic acid molecule of claim 30, wherein the polypeptide is as shown in SEQ ID NO: 12.

33. The nucleic acid molecule of claim 30, wherein the polynucleotide sequence is as shown in SEQ ID NO: 12.

34. A recombinant expression vector comprising the polynucleotide sequence of claim 30.

35. The recombinant expression vector of claim 34, further comprising a promoter operably linked to the polynucleotide sequence.

36. The recombinant expression vector of claim 34, wherein the promoter is a constitutive promoter.

37. The recombinant expression vector of claim 34, wherein the promoter is an inducible promoter.

38. A host cell transformed with the recombinant expression vector of claim 34.

39. A transgenic plant comprising a recombinant expression cassette comprising a promoter operably linked to the polynucleotide sequence of claim 30.

40. The transgenic plant of claim 39, wherein the plant is tomato.

41. A method of modulating seed germination in a plant, the method comprising a) introducing into the plant a recombinant expression vector comprising a promoter operably linked to a polynucleotide sequence encoding a polypeptide that modulates seed germination, wherein the polypeptide comprises an amino acid sequence that has 80% or greater identity to SEQ ID NO: 12; and b) selecting a plant with modulated seed germination.

42. The method of claim 41 , wherein the polynucleotide specifically hybridizes to SEQ ID NO: 11 or its complement.

43. The method of claim 41 , wherein the polypeptide is as shown in SEQ ID NO: 12.