Method of enhancing endosperm development in a plant

Abstract

The invention provides methods of controlling endosperm development in plants.

Description

FIELD OF THE INVENTION

The present invention is directed to plant genetic engineering. In particular, it relates to modulation of expression of genes controlling endosperm development in plants.

BACKGROUND OF THE INVENTION

A fundamental problem in biology is to understand how fertilization initiates reproductive development. In higher plants, the ovule generates the female gametophyte which is composed of egg, central, synergid and antipodal cells (Reiser, et al., Plant Cell, 1291-1301 (1993)). All are haploid except the central cell which contains two daughter nuclei that fuse prior to fertilization. One sperm nucleus fertilizes the egg to form the zygote, whereas another sperm nucleus fuses with the diploid central cell nucleus to form the triploid endosperm nucleus (van Went, et al., Embryology of Angiosperms , pp. 273-318 (1984)). The two fertilization products undergo distinct patterns of development. In Arabidopsis , the embryo passes through a series of stages that have been defined morphologically as preglobular, globular, heart, cotyledon and maturation (Goldberg, R. B., et al., Science (1994) 266: 605-614; Mansfield, S. G., et al., Arabidopsis: An Atlas of Morphology and Development , pp. 367-383 (1994)). The primary endosperm nucleus undergoes a series of mitotic divisions to produce nuclei that migrate into the expanding central cell (Mansfield, S. G., et al., Arab Inf Serv 27: 53-64 (1990); Webb, M. C., et al., Planta 184: 187-195 (1991)). Cytokinesis sequesters endosperm cytoplasm and nuclei into discrete cells (Mansfield, S. G., et al., Arab Inf Serv 27: 65-72 (1990)) that produce storage proteins, starch, and lipids which support embryo growth (opes, M. A. et al., Plant Cell 5: 1383-1399 (1993)). Fertilization also activates development of the integument cell layers of the ovule that become the seed coat, and induces the ovary to grow and form the fruit, or silique, in Arabidopsis.

Control of the expression of genes that control egg and central cell differentiation, or those that activate reproductive development in response to fertilization is useful in the production of plants with a range of desired traits. These and other advantages are provided by the present application.

SUMMARY OF THE INVENTION

The present invention provides methods of modulating fruit and seed development and other traits in plants. The methods involve providing a plant comprising a recombinant expression cassette containing an FIE nucleic acid linked to a plant promoter.

In some embodiments, transcription of the FIE nucleic acid inhibits expression of an endogenous FIE gene or activity the encoded protein. This embodiment is particularly useful, for instance, making embryo-less seed and parthenocarpic fruit. Alternatively, expression of the FIE nucleic acid may enhance expression of an endogenous FIE gene or FIE activity

In the expression cassettes, the plant promoter may be a constitutive promoter, for example, the CaMV 35S promoter. Alternatively, the promoter may be a tissue-specific promoter. Examples of tissue specific expression useful in the invention include ovule-specific or embryo-specific expression. For instance, the promoter sequence from the FIE genes disclosed here can be used to direct expression in relevant plant tissues.

The invention also provides seed or fruit produced by the methods described above. The seed or fruit of the invention comprise a recombinant expression cassette containing an FIE nucleic acid.

Definition

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.

A “promoter” is defined as an array of nucleic acid control sequences that direct transcription of an operably linked nucleic acid. As used herein, a “plant promoter” is a promoter that functions in plants. Promoters include necessary nucleic acid sequences near the start site of transcription, such as, in the case of a polymerase II type promoter, a TATA element. A promoter also optionally includes distal enhancer or repressor elements, which can be located as much as several thousand base pairs from the start site of transcription. A “constitutive” promoter is a promoter that is active under most environmental and developmental conditions. An “inducible” promoter is a promoter that is active under environmental or developmental regulation. The term “operably linked” refers to a functional linkage between a nucleic acid expression control sequence (such as a promoter, or array of transcription factor binding sites) and a second nucleic acid sequence, wherein the expression control sequence directs transcription of the nucleic acid corresponding to the second sequence.

The term “plant”, includes whole plants, plant organs (e.g., leaves, stems, flowers, roots, etc.), seeds and plant cells and progeny of same. The class of plants which can be used in the method of the invention is generally as broad as the class of higher plants amenable to transformation techniques, including angiosperms (monocotyledonous and dicotyledonous plants), as well as gymnosperms. It includes plants of a variety of ploidy levels, including 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 different from any naturally occurring allelic variants.

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 an R, generation transgenic plant. Transgenic plants which arise from sexual cross or by selfing are descendants of such a plant.

A “FIE nucleic acid” or “FIE polynucleotide sequence” of the invention is a subsequence or full length polynucleotide sequence of a gene which encodes a polypeptide involved in control of reproductive development and which, when mutated, allows for aspects of fertilization independent reproductive development. In some embodiments, the polypeptides of the invention have substantial sequence identity (as defined below) to a polycomb group gene of Drosophila. An exemplary nucleic acid of the invention is the Arabidopsis FIE1 and FIE3 sequences disclosed below. FIE polynucleotides are defined by their ability to hybridize under defined conditions to the exemplified nucleic acids or PCR products derived from them. An FIE polynucleotide is typically at least about 30-40 nucleotides to about 3000, usually less than about 5000 nucleotides in length. The nucleic acids contain coding sequence of from about 100 to about 2000 nucleotides, often from about 500 to about 1700 nucleotides in length.

FIE nucleic acids are a new class of plant regulatory genes that encode polypeptides with sequence identity to members of the polycomb group genes first identified in Drosophila . Polycomb group gene products and their homologues in other species are responsible for repression of homeotic genes. The proteins are a heterogenous group that interact with each other to form large complexes that bind DNA and thereby control gene expression. For a review of the current understanding of polycomb complex genes see, Pirrotta Cur. Op. Genet. Dev. 7:249-258 (1997). Nine groups of polycomb genes have been identified. FIE1 (SEQ ID NO:1) is related to the group of polycomb genes encoding protein comprising a SET domain (see, e.g., Jenuwein et al. Cell. Mol. Life Sci. 54:80-93 (1998). FIE3 (SEQ ID NO:3) is related to the group encoding proteins comprising WD40 repeats (see, Gutjahr et al. EMBO J. 14:4296-4306 (1995).

In the case of both expression of transgenes and inhibition of endogenous genes (e.g., by antisense, or sense suppression) one of skill will recognize that the inserted polynucleotide sequence need not be identical, but may be only “substantially identical” to a sequence of the gene from which it was derived. As explained below, these substantially identical variants are specifically covered by the term FIE nucleic acid.

In the case where the inserted polynucleotide sequence is transcribed and translated to produce a functional polypeptide, one of skill will recognize that because of codon degeneracy a number of polynucleotide sequences will encode the same polypeptide. These variants are specifically covered by the terms “FIE nucleic acid”. In addition, the term specifically includes those sequences substantially identical (determined as described below) with an FIE polynucleotide sequence disclosed here and that encode polypeptides that are either mutants of wild type FIE polypeptides or retain the function of the FIE polypeptide (e.g., resulting from conservative substitutions of amino acids in the FIE polypeptide). In addition, variants can be those that encode dominant negative mutants as described below.

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, Calif., USA).

The phrase “substantially identical,” in the context of two nucleic acids or polypeptides, refers to sequences or subsequences that have at least 60%, preferably 80%, most preferably 90-95% nucleotide or amino acid residue identity 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. This definition also refers to the complement of a test sequence, which has substantial sequence or subsequence complementarity when the test sequence has substantial identity to a reference sequence.

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 entered into a computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated. Default program parameters can be used, or alternative parameters can be designated. The sequence comparison algorithm then calculates the percent sequence identities for the test sequences relative to the reference sequence, based on the program parameters.

A “comparison window”, as used herein, includes reference to a segment of any one of the number of contiguous positions selected from the group consisting of from 20 to 600, usually about 50 to about 200, more usually about 100 to about 150 in which a sequence may be compared to a reference sequence of the same number of contiguous positions after the two sequences are optimally aligned. Methods of alignment of sequences for comparison are well-known in the art. 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'l. 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, Wis.), or by manual alignment and visual inspection.

One example of a useful algorithm is PILEUP. PILEUP creates a multiple sequence alignment from a group of related sequences using progressive, pairwise alignments to show relationship and percent sequence identity. It also plots a tree or dendogram showing the clustering relationships used to create the alignment. PILEUP uses a simplification of the progressive alignment method of Feng & Doolittle, J. Mol. Evol. 35:351-360 (1987). The method used is similar to the method described by Higgins & Sharp, CABIOS 5:151-153 (1989). The program can align up to 300 sequences, each of a maximum length of 5,000 nucleotides or amino acids. The multiple alignment procedure begins with the pairwise alignment of the two most similar sequences, producing a cluster of two aligned sequences. This cluster is then aligned to the next most related sequence or cluster of aligned sequences. Two clusters of sequences are aligned by a simple extension of the pairwise alignment of two individual sequences. The final alignment is achieved by a series of progressive, pairwise alignments. The program is run by designating specific sequences and their amino acid or nucleotide coordinates for regions of sequence comparison and by designating the program parameters. For example, a reference sequence can be compared to other test sequences to determine the percent sequence identity relationship using the following parameters: default gap weight (3.00), default gap length weight (0.10), and weighted end gaps.

Another example of algorithm that is suitable for determining percent sequence identity and sequence similarity is the BLAST algorithm, which is described in Altschul et al., J. Mol. Biol. 215:403-410 (1990). Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information. 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 hits act as seeds for initiating searches to find longer HSPs containing them. The word hits are extended in both directions along each sequence for as far as the cumulative alignment score can be increased. 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 BLAST program uses as defaults a wordlength (W) of 11, the BLOSUM62 scoring matrix (see Henikoff & Henikoff, Proc. Natl. Acad. Sci. USA 89:10915 (1989)) alignments (B) of 50, expectation (E) of 10, M=5, N=−4, and a comparison of both strands.

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 0.2, more preferably less than about 0.01, and most preferably less than about 0.001.

“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, deletions or additions to a nucleic acid, peptide, polypeptide, or protein sequence which alters, adds or deletes 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. Low 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 cased, the nucleic acids typically hybridize under moderately stringent hybridization conditions.

In the present invention, genomic DNA or cDNA comprising FIE 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 0.2×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.

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., a northern or Southern blot.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A and 1B show the genetic map used to clone the FIE3 gene.

FIG. 2 shows the analysis of the sequence in the DNA shown in FIG. 1 using the GENSCANW program.

FIG. 3 shows the position of primers used to PCR amplify sequences from the FIE3 gene region.

FIG. 4 shows the genetic map used to clone the FIE1 gene.

FIG. 5 shows the results of complementation tests establishing that a single gene (FIE1) was present on the complementing cosmid (6-22) that was not fully encoded on either of the non-complementing cosmids (2-9 and 2-8).

DESCRIPTION OF THE PREFERRED EMBODIMENTS

This invention provides molecular strategies for controlling seed and fruit development.

Reproduction in higher plants is unique because it is initiated by two fertilization events in the haploid female gametophyte. One sperm nucleus fertilizes the egg to form the embryo. A second sperm nucleus fertilizes the central cell to form the endosperm, a unique tissue that supports the growth of the embryo. Fertilization also activates maternal tissue differentiation, the ovule integuments form the seed coat and the ovary forms the fruit.

The present invention is based, at least in part, on the discovery of a set of female-gametophytic mutations, termed fie (fertilization-independent endosperm), and the subsequent cloning of the genes involved. Three mutants are disclosed here fie1, fie2, and fie3, which have been mapped to chromosomes 1, 2, and 3 of Arabidopsis , respectively. The fie mutations affect the central cell, allowing for replication of the central cell nucleus and endosperm development without fertilization. FIE/fie seed coat and fruit undergo fertilization-independent differentiation, showing that the fie female gametophyte is the source of signals that activates sporophytic fruit and seed coat development. Generally, the mutant fie alleles are not transmitted by the female gametophyte. Inheritance of a mutant fie allele (e.g., fie3) by the female gametophyte usually results in embryo abortion, even when the pollen bears the wild-type FIE allele. In the case of fie1 and fie2, however, transmission of the trait occurs in about 1% of the progeny from the female gametophyte. In contrast, the fie1, fie2, and fie3 mutant alleles are passed through the male gametophyte (i.e., pollen) in normal fashion.

The isolated sequences prepared as described herein, can be used in a number of techniques, for example, to suppress or enhance endogenous FIE gene expression. Modulation of FIE gene expression or FIE activity in plants is particularly useful, for example, in producing embryo-less seed, parthenocarpic fruit, or as part of a system to generate apomictic seed.

Isolation of FIE Nucleic Acids

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, N.Y., (1989).

The isolation of FIE nucleic acids 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 ovules, and a cDNA library which contains the FIE gene transcript is prepared from the mRNA. Alternatively, cDNA may be prepared from mRNA extracted from other tissues in which FIE genes or homologs are expressed.

The cDNA or genomic library can then be screened using a probe based upon the sequence of a cloned FIE gene 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 an FIE polypeptide 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 the FIE genes 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 FIE sequences from plant tissues are generated from comparisons of the sequences provided here with other polycomb group genes. For instance, FIE1 can be compared to the other polycomb genes containing the SET domain, such as the Arabidopsis curly leaf gene (Goodrich et al. Nature 386:44-51 (1997)) or the Drosophila enhancer of zeste (E(z)) gene. FIE3 can be compared to genes containing WD40 repeats, such as the extra sex combs (esc) gene from Drosophila . Using these techniques, one of skill can identify conserved regions in the nucleic acids disclosed here to prepare the appropriate primer and probe sequences. Primers that specifically hybridize to conserved regions in FIE1 or FIE3 genes can be used to amplify sequences from widely divergent plant species.

Standard nucleic acid hybridization techniques using the conditions disclosed above can then be used to identify full length cDNA or genomic clones.

Control of FIE Activity or Gene Expression

Since FIE genes are involved in controlling seed, in particular endosperm, development, inhibition of endogenous Fie activity or gene expression is useful in a number of contexts. For instance, inhibition of expression is useful in the development of parthenocarpic fruit (i.e., fruit formed in the absence of fertilization).

In addition, inhibition of FIE activity can be used for production of fruit with small and/or degraded seed (referred to here as “seedless fruit”) after fertilization. In many plants, particularly dicots, the endosperm is not persistent and eventually is degraded. Thus, in plants of the invention in which Fie activity is inhibited, embryo-less seed do not persist and seedless fruit are produced.

Alternatively, plants of the invention can be used to prevent pre-harvest sprouting in seeds, especially those derived from cereals. In these plants, the endosperm persists and is the major component of the mature seed. Premature growth of embryos in stored grain causes release of degradative enzymes which digest starch and other components of the endosperm. Plants of the present invention are useful in addressing this problem because the seeds lack an embryo and thus will not germinate.

In yet another use, nucleic acids of the invention can be used in the development of apomictic plant lines (i.e., plants in which asexual reproductive processes occur in the ovule, see, Koltunow, A. Plant Cell 5: 1425-1437 (1993) for a discussion of apomixis). Apomixis provides a novel means to select and fix complex heterozygous genotypes that cannot be easily maintained by traditional breeding. Thus, for instance, new hybrid lines with desired traits (e.g., hybrid vigor) can be obtained and readily maintained.

In still another use, nucleic acids of the invention can be used to control endosperm production in transgenic plants. In particular, inhibition of FIE activity can be used to produce larger seeds with increased endosperm. This trait is particularly useful in species in which the endosperm persists in the seed (e.g., monocots, particularly grains).

One of skill will recognize that a number of methods can be used to modulate FIE activity or gene expression. FIE activity can be modulated in the plant cell at the gene, transcriptional, posttranscriptional, translational, or posttranslational, levels. Techniques for modulating FIE activity at each of these levels are generally well known to one of skill and are discussed briefly below. Methods for introducing genetic mutations into plant genes 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, for example, X-rays or gamma rays can be used.

Alternatively, homologous recombination can be used to induce targeted gene disruptions by specifically deleting or altering the FIE gene 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., EMBO J. 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)).

In applying homologous recombination technology to the genes of the invention, mutations in selected portions of an FIE gene sequences (including 5′ upstream, 3′ downstream, and intragenic regions) such as those disclosed here are made in vitro and then introduced into the desired plant using standard techniques. Since the efficiency of homologous recombination is known to be dependent on the vectors used, use of dicistronic gene targeting vectors as described by Mountford et al. Proc. Natl. Acad. Sci. USA 91: 4303-4307 (1994); and Vaulont et al. Transgenic Res. 4: 247-255 (1995) are conveniently used to increase the efficiency of selecting for altered FIE gene expression in transgenic plants. The mutated gene will interact with the target wild-type gene in such a way that homologous recombination and targeted replacement of the wild-type gene will occur in transgenic plant cells, resulting in suppression of FIE activity.

Alternatively, oligonucleotides composed of a contiguous stretch of RNA and DNA residues in a duplex conformation with double hairpin caps on the ends can be used. The RNA/DNA sequence is designed to align with the sequence of the target FIE gene and to contain the desired nucleotide change. Introduction of the chimeric oligonucleotide on an extrachromosomal T-DNA plasmid results in efficient and specific FIE gene conversion directed by chimeric molecules in a small number of transformed plant cells. This method is described in Cole-Strauss et al. Science 273:1386-1389 (1996) and Yoon et al. Proc. Natl. Acad. Sci. USA 93: 2071-2076 (1996).

Gene expression can be inactivated using recombinant DNA techniques by transforming plant cells with constructs comprising transposons or T-DNA sequences. FIE mutants prepared by these methods are identified according to standard techniques. For instance, mutants can be detected by PCR or by detecting the presence or absence of FIE mRNA, e.g., by Northern blots. Mutants can also be selected by assaying for development of endosperm in the absence of fertilization.

The isolated nucleic acid sequences prepared as described herein, can also be used in a number of techniques to control endogenous FIE gene expression at various levels. Subsequences from the sequences disclosed here can be used to control, transcription, RNA accumulation, translation, and the like.

A number of methods can be used to inhibit gene expression 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, Calif., USA; London, England, UK. p. 5181-238; 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. Pat. No. 4,801,340).

The nucleic acid segment to be introduced generally will be substantially identical to at least a portion of the endogenous FIE 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 homology or substantial homology 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 homology 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 homology of non-coding segments may be equally effective. Normally, a sequence of between about 30 or 40 nucleotides and about full length nucleotides 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 1700 nucleotides is especially preferred.

A number of gene regions can be targeted to suppress FIE gene expression. The targets can include, for instance, the coding regions, introns, sequences from exon/intron junctions, 5′ or 3′ untranslated regions, and the like. In some embodiments, the constructs can be designed to eliminate the ability of regulatory proteins to bind to FIE gene sequences that are required for its cell- and/or tissue-specific expression. Such transcriptional regulatory sequences can be located either 5′-, 3′-, or within the coding region of the gene and can be either promote (positive regulatory element) or repress (negative regulatory element) gene transcription. These sequences can be identified using standard deletion analysis, well known to those of skill in the art. Once the sequences are identified, an antisense construct targeting these sequences is introduced into plants to control gene transcription in particular tissue, for instance, in developing ovules and/or seed.

Oligonucleotide-based triple-helix formation can be used to disrupt FIE gene expression. Triplex DNA can inhibit DNA transcription and replication, generate site-specific mutations, cleave DNA, and induce homologous recombination (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)). Triple helix DNAs can be used to target the same sequences identified for antisense regulation.

Catalytic RNA molecules or ribozymes can also be used to inhibit expression of FIE genes. It is possible to design ribozymes that specifically pair with virtually any target RNA and cleave the phosphodiester backbone at a specific location, thereby functionally inactivating the target RNA. In carrying out this cleavage, the ribozyme is not itself altered, and is thus capable of recycling and cleaving other molecules, making it a true enzyme. The inclusion of ribozyme sequences within antisense RNAs confers RNA-cleaving activity upon them, thereby increasing the activity of the constructs. Thus, ribozymes can be used to target the same sequences identified for antisense regulation.

A number of classes of ribozymes have been identified. One class of ribozymes is derived from a number of small circular RNAs which are capable of self-cleavage and replication in plants. The RNAs replicate either alone (viroid RNAs) or with a helper virus (satellite RNAs). Examples include RNAs from avocado sunblotch viroid and the satellite RNAs from tobacco ringspot virus, lucerne transient streak virus, velvet tobacco mottle virus, solanum nodiflorum mottle virus and subterranean clover mottle virus. The design and use of target RNA-specific ribozymes is described in 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).

Another methods of suppression is sense cosuppression. 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. Pat. 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 would be most preferred. As with antisense regulation, the effect should apply to any other proteins within a similar family of genes exhibiting homology or substantial homology.

For sense 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 which are overexpressers. A higher identity in a shorter than full length sequence 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 targetted using cosuppression technologies.

Alternatively, FIE activity may be modulated by eliminating the proteins that are required for FIE cell-specific gene expression. Thus, expression of regulatory proteins and/or the sequences that control FIE gene expression can be modulated using the methods described here.

Another method is use of engineered tRNA suppression of FIE mRNA translation. This method involves the use of suppressor tRNAs to transactivate target genes containing premature stop codons (see, Betzner et al. Plant J. 11:587-595 (1997); and Choisne et al. Plant J. 11: 597-604 (1997). A plant line containing a constitutively expressed FIE gene that contains an amber stop codon is first created. Multiple lines of plants, each containing tRNA suppressor gene constructs under the direction of cell-type specific promoters are also generated. The tRNA gene construct is then crossed into the FIE line to activate FIE activity in a targeted manner. These tRNA suppressor lines could also be used to target the expression of any type of gene to the same cell or tissue types.

As noted above, FIE proteins as products of polycomb group genes are believed to form large complexes in vivo. Thus, production of dominant-negative forms of FIE polypeptides that are defective in their abilities to bind to other polycomb group proteins is a convenient means to inhibit endogenous FIE activity. This approach involves transformation of plants with constructs encoding mutant FIE polypeptides that form defective complexes with endogenous polycomb group proteins and thereby prevent the complex from forming properly. The mutant polypeptide may 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. Use of dominant negative mutants to inactivate target genes is described in Mizukami et al. Plant Cell 8:831-845 (1996).

Another strategy to affect the ability of an FIE protein to interact with itself or with other proteins involves the use of antibodies specific to FIE. In this method cell-specific expression of FIE-specific Abs is used inactivate functional domains through antibody:antigen recognition (see, Hupp et al. Cell 83:237-245 (1995)).

Use of Nucleic Acids of the Invention to Enhance FIE Gene Expression

Isolated sequences prepared as described herein can also be used to introduce expression of a particular FIE nucleic acid to enhance or increase endogenous gene expression. For instance, polycomb genes are known to control cell cycling. Enhanced expression can therefore be used to control plant morphology by controlling whether or not cell division takes place in desired tissues or cells. Enhanced expression can also be used, for instance, to increase vegetative growth by preventing the plant from setting seed. Where overexpression of a gene is desired, the desired gene from a different species may be used to decrease potential sense suppression effects.

One of skill will recognize that the polypeptides encoded by the genes of the invention, like other proteins, have different domains which perform different functions. Thus, the gene sequences 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.

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 tumafaciens , and other transcription initiation regions from various plant genes known to those of skill. Such genes include for example, ACT11 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)), GPc1 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 the FIE nucleic acid in a specific tissue or may be otherwise under more precise environmental or developmental control. Examples of environmental conditions that may effect transcription by inducible promoters include anaerobic conditions, elevated temperature, or the presence of light. Such promoters are referred to here as “inducible” or “tissue-specific” promoters. 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, but may also lead to some expression in other tissues as well.

Examples of promoters under developmental control include promoters that initiate transcription only (or primarily only) in certain tissues, such as fruit, seeds, or flowers. Promoters that direct expression of nucleic acids in ovules, flowers or seeds are particularly useful in the present invention. As used herein a seed-specific promoter is one which directs expression in seed tissues, such promoters may be, for example, ovule-specific (which includes promoters which direct expression in maternal tissues or the female gametophyte, such as egg cells or the central cell), 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 18 kD 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), Atmyc1 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 20 kD 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)).

In addition, the promoter sequences from the FIE genes disclosed here can be used to drive expression of the FIE polynucleotides of the invention or heterologous sequences. The sequences of the promoters are identified below.

If proper polypeptide expression is desired, a polyadenylation region at the 3′-end of the coding region should be included. The polyadenylation region can be derived from the natural gene, from a variety of other plant genes, or from T-DNA.

The vector comprising the sequences (e.g., promoters or coding regions) from genes of the invention will typically comprise a marker gene which 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).

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 which 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.

Seed obtained from plants of the present invention can be analyzed according to well known procedures to identify plants with the desired trait. If antisense or other techniques are used to control Fie gene expression, Northern blot analysis can be used to screen for desired plants. In addition, the presence of fertilization independent reproductive development can be detected. Plants can be screened, for instance, for the ability to form embryo-less seed, form seed that abort after fertilization, or set fruit in the absence of fertilization. These procedures will depend, part on the particular plant species being used, but will be carried out according to methods well known to those of skill.

The following Examples are offered by way of illustration, not limitation.

EXAMPLE 1

The following example describes methods used to identify the fie mutants. The methods described here are generally as described in Ohad et al., Proc. Natl. Acad. Sci. USA 93:5319-5324 (1996).

Materials and Methods

Growth and Phenotype of Plants

Plants were grown under low humidity conditions (less than 50%) in glass houses under 16 hr light/8 hr dark photoperiods generated by supplemental lighting. Plants were grown at high humidity (greater than 80%) in a lighted incubator (Percival, Boone, Iowa).

To test for fertilization-independent development, flower buds from plants that had not yet begun to shed pollen (stage 12; (Smyth, D. R, et al., Plant Cell 2: 755-767 (1990))) were opened, immature anthers were removed, and the flower bud was covered with a plastic bag. Seven days later, the silique was measured, dissected, and the number of seed-like structures and degenerating ovules were counted. To determine the frequency of seed abortion following fertilization, siliques were harvested 10 days after self-pollination, dissected, and wild-type and aborted seeds were counted.

Genetic Mapping

Heterozygous FIE/fie (Landsberg erecta ecotype) plants were crossed as males with female plants (Columbia ecotype). Because the mutant fie allele is only transmitted through the male gametophyte, FIE/fie progeny were crossed as males a second time to female g11/g11 (Columbia ecotype) plants. Approximately fifty-five progeny were scored for the segregation of the wild-type FIE and mutant fie alleles and for alleles of molecular markers as described previously (Bell, C., et al., Genomics 19: 137-144 (1994)). This analysis indicated that fie3 is located at approximately position 30 on chromosome three, fie2 is located at approximately position 65 on chromosome two, and fie1 is located at approximately position 2 on chromosome one. Genetic recombination frequencies and map distances were calculated according to Koornneef and Stam (Koornneef, M., et al., Methods in Arabidopsis Research, pp. 83-99 (1992)) and Kosambi (Kosambi, Ann. Eugen., 12: 172-175 (1944)).

Light Microscopy

Nomarski photographs of whole-mount embryos and endosperm were obtained by fixing longitudinally slit siliques in an ethanol:acetic acid (9:1) solution overnight, followed by two washes in 90% and 70% ethanol, respectively. Siliques were cleared with a chloral hydrate:glycerol:water solution (8:1:2, w:v:v) (Berleth, T., et al., Devel 118: 575-587 (1993)). Whole mount preparations were fixed and stained with hematoxylin (Beeckman, T., et al., Plant Mol Biol Rep 12: 37-42 (1994)). Embryo and endosperm were photographed with a Zeiss Axioskop microscope (Carl Zeiss, Inc., Oberkochen, Germany) using Nomarski optics that permits visualization of optical sections within the seed.

GUS Histochemical Assays

GUS activity was detected histochemically as described previously by (Beeckman, T., et al., Plant Mol Biol Rep 12: 37-42 (1994)).

Image Processing

Photographs were scanned using a Microtek scanner. Pictures were processed for publication using Adobe Photoshop 3.0 and printed on a Tektronix Phaser 400 color printer.

Results

Isolation of Mutant Lines

To begin to understand mechanisms that initiate reproductive development, we generated mutant Arabidopsis plants that undergo several reproductive processes in the absence of fertilization. Arabidopsis plants homozygous for the conditional male sterile pop1 mutation (Preuss, D., et al., Genes and Devel 7: 974-985 (1993)) were used as the parental strain (Landsberg erecta ecotype). Fertility in pop1 plants is sensitive to humidity because pop1 pollen do not hydrate properly due to a defect in wax biosynthesis. When grown at permissive condition, high relative humidity (>80%), pop1 plants were male fertile and produced long siliques with many viable seeds. By contrast, when grown at non-permissive condition, low relative humidity (<50%), pop1 plants were male sterile and produced short siliques with no seeds. Thus, silique elongation is a marker for reproductive events. To isolate mutations, homozygous pop1 seeds were mutagenized with ethylmethansulfonate (EMS) and approximately 50,000 M1 plants were screened for silique elongation at non-permissive conditions. Rare M1 plants were identified that displayed heterozygous sectors with elongated siliques. These plants were transferred to permissive conditions to insure the production of viable M2 seed. Plants from M2 and M3 families grown at non-permissive conditions were rechecked for non-sectored silique elongation. To eliminate any effects of the pop1 mutation, or other EMS-induced lesions on the mutant phenotype, mutant plants were backrossed twice, as males, to wild-type plants. After removing the pop1 mutation, fertilization-independent phenotypes were confirmed after manual removal of anthers from immature flowers before pollen was shed. A total of twelve lines were identified that displayed elongated siliques in the absence of fertilization.

Fertilization-Independent Endosperm

Seed Coat and Silique Development In a representative line chosen for further study, heterozygous plants produced by back crosses to wild-type plants generated elongated siliques after anther removal with numerous seed-like structures. These results indicated that heterozygous mutant plants were capable of silique elongation and seed-like structure development in the absence of fertilization. We compared the development of the mutant seed-like structures to that of wild-type seeds. After fertilization, the endosperm nucleus replicated and daughter nuclei migrated into the expanding central cell. Ultimately, a syncytium of endosperm nuclei was produced. Nuclear divisions of the endosperm preceded the zygotic divisions that formed the globular stage embryo. Embryo, endosperm or seed coat development did not occur in wild-type plants in the absence of fertilization. Development of the ovule and female gametophyte in heterozygous mutant plants was normal. Just prior to flower opening, female gametophytes in these plants contained a single, prominent central cell nucleus. Subsequently, in the absence of fertilization, central cells with two large nuclei were detected. Further divisions resulted in the production of additional nuclei that migrated into the expanded central cell. Later in development, a nuclear-syncytium was formed with abundant endosperm nuclei. These results indicated that the central cell in mutant female gametophytes initiated endosperm development in the absence of fertilization. We have named this mutation fie for fertilization-independent endosperm. By contrast, replication of other nuclei in fie female gametophytes (egg, synergid, or antipodal) was not detected. Thus, the fie mutation specifically affects replication of the central cell nucleus.

We analyzed the frequency of multinucleate central cell formation in fie female gametophytes by comparing the percentage of multinucleate central cells at three, five, and six days after emasculation of heterozygous FIE/fie and control wild-type flowers. At each time point, only 3% to 5% of wild-type central cells had more than one nucleus. Because none had more than two nuclei, most likely, these represented central cells with haploid nuclei that had not fused during female gametophyte development. By contrast, the percentage of central cells in female gametophytes from FIE/fie siliques with two or more nuclei increased from 21% to 47% over the same time period. These results indicated that the fie mutation caused a significant increase in formation of multinucleate central cells in the absence of fertilization. The fact that close to 50% of the female gametophytes in heterozygous plants had multinucleate central cells suggested that fie is a gametophytic mutation because a 1:1 segregation of wild-type and mutant fie alleles occurs during meiosis.

We compared the fertilization-independent development of the maternal seed coat in FIE/fie seed-like structures to that of fertilized wild-type seeds. The seed coat in wild-type Arabidopsis is generated by the integuments of the ovule and surrounds the developing embryo and endosperm. Similarly, FIE/fie ovule integuments formed a seed coat that surrounded the developing mutant endosperm. These results indicated that the fie mutation activated both endosperm development and maternal sporophytic seed coat and silique differentiation that support reproduction. No other effects on sporophytic growth and development were detected in FIE/fie plants.

The fie3 Mutant Allele is Not Transmitted by the Female Gametophyte to the Next Generation

To understand the mode of inheritance of the fie mutation, we analyzed the progeny of reciprocal crosses. FIE3/fie3 females, crossed to wild-type males, produced siliques with approximately equal numbers of viable seeds with normal green embryos and nonviable white seeds with embryos aborted at the heart stage (344:375, 1:1, c2=1.3, P>0.2). Viable seeds from this cross were germinated and all 120 F1 progeny generated were wild-type. That is, none of the F1 progeny had significant levels of F2 aborted seeds in their siliques after self-pollination. Nor did the F1 progeny demonstrate fertilization-independent development. This indicated that presence of the fie mutant allele in the female gametophyte, even when the male provided a wild-type allele, resulted in embryo abortion. Thus, the fie mutation is not transmitted by the female gametophyte to the next generation. To study transmission of fie through the male gametophyte, we pollinated female wild-type plants with pollen from male FIE3/fie3 plants. Siliques from these crosses contained no aborted F1 seed. F1 plants were examined and a 1:1 segregation of wild-type and FIE3/fie3 genotype was observed (62:58, c2=0.13, P>0.5). This indicated that wild-type and mutant fie3 alleles were transmitted by the male gametophyte with equal efficiency. That is, fie does not affect male gametophyte, or pollen grain, function. Results from reciprocal crosses were verified by analyzing the progeny from self-pollinated FIE3/fie3 plants. Self-pollinated siliques displayed 1:1 segregation of normal and aborted seeds (282:286, c2=0.03, P>0.8). Viable seed from self-pollinated siliques were germinated and a 1:1 (71:64, c2 0.36, P>0.5) segregation of wild-type and FIE3/fie3 progeny was observed. These results confirmed that inheritance of a fie mutant allele by the female gametophyte resulted in embryo abortion, and that inheritance of a fie mutant allele by the male gametophyte did not affect pollen function. Thus, the wild-type FIE3 allele probably carries out a function unique to the female gametophyte and does not appear to be needed for male fertility.

In contrast, fie1 and fie2 mutant alleles were transmitted at low frequencies (about 1% of normal) through the female gametophyte. In this way, fie1 homozygous mutants and fie2 homozygous mutants were obtained that appeared to display normal vegetative growth and development.

Discussion

In wild-type plants, fertilization initiates embryogenesis and endosperm formation, and activates maternal seed coat and silique development. The results presented here indicate that specific aspects of plant reproductive development can occur in FIE/fie plants in the absence of fertilization. These include silique elongation, seed coat formation, and endosperm development. Morphological analysis shows that early aspects of fertilization-independent fie endosperm development closely resemble fertilized wild-type endosperm development. First, the fie central cell nucleus is stimulated to undergo replication. Second, nuclei that are produced migrate from the micropylar end of the central cell and take up new positions in the central cell. Third, the developing fie central cell expands to form an endosperm cavity. Thus, the requirement for fertilization to initiate these early events in endosperm formation has been eliminated by the fie mutation. This suggests that FIE plays a role in a signal transduction pathway that links fertilization with the onset of central cell nuclear replication and early endosperm development.

Mechanisms for Regulation of Endosperm Development by FIE

One can envision two possible mechanisms for how FIE regulates replication of the central cell nucleus in response to fertilization. The protein encoded by the FIE gene may be involved in a positive regulatory interaction. In this model, FIE is required for the central cell to initiate endosperm development. Normally, fertilization is needed for the presence of active FIE protein. The fie mutation results in the presence of active protein in the absence of fertilization. Alternatively, F1 may by involved in a negative regulatory interaction. In this model, the function of FIE protein is to prevent the central cell from initiating endosperm development, and fertilization results in the inactivation of FIE protein. The fie mutation results in the production of inactive protein, so that fertilization is no longer required to initiate endosperm development However, complementation experiments using transgenic plants indicate that FIE1 and FIE3 alleles are dominant over their respective mutant alleles. This indicates that the wild-type allele is involved in a negative regulatory interaction. Recently, it has been shown that cyclin-dependent kinase complexes, related to those that function in mammals, control the induction of DNA synthesis and mitosis in maize endosperm (Grafi, G. et al., Science 269: 1262-1264 (1995)). Because fie stimulates replication of the central cell, fie may, either directly or indirectly, impinge upon cell cycle control of the central cell nucleus, allowing replication to take place in the absence of fertilization.

Communication Between the fie Female Gametophyte and the Sporophytic Ovule and Carpels

The analysis of FIE/fie mutant plants has provided clues about interactions between endosperm and maternal sporophytic tissues. FIE/fie ovule integuments surrounding a mutant fie female gametophyte initiate seed coat development, whereas FIE/fie integuments in contact with a quiescent wild-type female gametophyte do not develop. This suggests that the FIE/fie ovule integuments initiate seed coat differentiation in response to a signal produced by the fie female gametophyte. We propose that the source of the signal is the mutant fie central cell that has initiated endosperm development, although we cannot rule out the participation of other cells in the fie female gametophyte. In wild-type plants, most likely, fertilization of the central cell produces an endosperm that activates seed coat development. This is consistent with experiments showing that the maize endosperm interacts with nearby maternal cells (Miller, M. E., et al., Plant Cell 4: 297-305 (1992)). FIE/fie plants also display fertilization-independent elongation of the ovary to form the silique. We propose that a signal is produced by the developing seed-like structures to initiate silique elongation. This is in agreement with experiments suggesting that seeds are the source of hormones, auxins and gibberellins, that activate fruit development (Lee, T. D. Plant Reproductive Ecology , pp. 179-202 (1988)). Taken together, these results suggest that the fertilized female gametophyte activates maternal developmental programs.

Relationship Between fie and Apomixis

Certain plant species display aspects of fertilization-independent reproductive development, including apomictic generation of embryo and endosperm, and development of the maternal seed coat and fruit (reviewed in (Koltunow, a. Plant Cell 5: 1425-1437 (1993)). The fie mutation reveals that Arabidopsis , a sexually reproducing plant, has the genetic potential for aspects of fertilization-independent reproductive development. It is not known whether the mechanism of fertilization-independent endosperm development conferred by the fie mutation is the same as autonomous endosperm formation observed in certain apomictic plant species. However, the fact that the fie phenotype is caused by a single genetic locus substantiates the view that the number of genetic differences between sexually and asexually reproducing plants is small (Koltunow, a M., et al., Plant Physiol 108:1345-1352 (1995)).

EXAMPLE 2

This example describes cloning of two Fie genes, Fie1 and Fie3.

Cloning the FIE3 Gene

a. Mapping the Position of the fie3 Gene Genetically

The fie3 mutation was initially mapped to position 30 on chromosome 3, between AXR2 (auxin resistant dwarf) and EMB29 (embryo lethal). Next, two sets of F2 plants with recombination breakpoints in the fie3 gene region were obtained. One set was between emb29 and fie3 and the other set was between axr2 and fie3. As shown in FIG. 1A , these recombinants were used to map the fie3 gene relative to molecular markers (NDR, CH18, CH18S, BO20, AG20, KN1 and E13F12) that were obtained from overlapping YAC (yUP13F12), BAC (T1B4 and T4N1) and cosmid clones (FIG. 1 A). YAC and BAC clones were obtained from the Arabidopsis Stock Center (Ohio State University, USA). Cosmid subclones were generated in my laboratory. As shown in FIGS. 1A and 1B , this genetic analysis indicates that the fie3 gene resides within the 25 Kb region between the BO20 and AG20 markers.

b. Mapping the Position of fie3 Gene by Complementation Experiments

To more precisely localize the fie3 gene, we analyzed a series of overlapping cosmid clones (BO20, GM15, AG20 and EI12) that span the fie3 gene region. Each cosmid clone was tested for its ability to complement the fie3 mutation in transgenic plants. Only cosmid GM15 complemented the fie3 mutation (FIG. 1 A). These results indicate that an essential portion of the fie3 gene is in the 10 Kb region that is unique to cosmid GM15. As shown in FIG. 1B , we have cloned DNA that spans this essential portion of the fie3 gene and have determined its DNA sequence. As shown in FIG. 2 , analysis of the sequence using the GENSCANW program revealed a gene with an open reading frame. The predicted cDNA sequence and predicted amino acid sequence are shown in SEQ ID NO:3 and SEQ ID NO:4, respectively. Comparing the predicted amino acid sequence to those in public data bases revealed significant homology to the WD40 family of Polycomb Group genes, and in particular, the “extra sex combs” gene in Drosophila . FIG. 3 shows the position of primers used to PCR amplify this region. SEQ ID NO:5 provides the genomic DNA sequence of the WD40/Polycomb gene, plus approximately 3.8 Kb of 5′-flanking sequences and 0.3 Kb of 3′-flanking sequences, plus the sequence of primers used to PCR amplify this region. The transcription start site in SEQ ID NO:5 is at position 3,872. Thus, the promoter sequence for FIE3 is located between position 1 and 3,872. The 5′-flanking and 3′-flanking regions contain regulatory DNA sequences that control the expression of this gene.

Cloning the FIE1 Gene

a. Mapping the Position of the FIE1 Gene Genetically

The fie1 mutation was initially mapped to position 3 on chromosome 1, between AXR3 (auxin resistant dwarf) and EMB60 (embryo lethal). Next, two sets of F2 plants with recombination breakpoints in the FIE1 gene region were obtained. One set was between emb60 and fie3 and the other set was between axr3 and fie3. These recombinants were used to map the fie3 gene relative to molecular markers ( FIG. 4 ) that were obtained from an overlapping series of YAC and BAC clones from the Arabidopsis Stock Center (Ohio State University, USA).

b. Mapping the Position of the FIE1 Gene by Complementation Experiments

To more precisely localize the FIE1 gene, a series of overlapping cosmid clones (2-9, 6-22, 2-8) that span the FIE1 gene region were analyzed (FIG. 4 ). Each cosmid clone was tested for its ability to complement the fie1 mutation in transgenic plants. Only cosmid 6-22 complemented the fie1 mutation. The cosmids were analyzed for genes with open reading frames. FIG. 5 shows that a single gene was present on the complementing cosmid (6-22) that was not fully encoded on either of the non-complementing cosmids (2-9 and 2-8). By RTPCR and 5′-race, the cDNA sequence of this gene and predicted amino acid of its protein were obtained (SEQ ID NO:1 and SEQ ID NO:2, respectively). Comparison of the predicted amino acid sequence to those in public data bases revealed significant homology to the SET family of Polycomb Group Genes (e.g., Enhancer of Zeste in Drosophila and Curly Leaf in Arabiopsis ). We compared the wild-type and fie1 mutant sequence in 6-22. The only difference is a single base pair change that creates a premature translation stop codon in the 5′-end of the set/polycomb group gene. The base pair change is at position 823 (C→T) on the cDNA sequence shown in SEQ ID NO:1.

SEQ ID NO:6 shows the genomic sequence of the FIE1 SET/polycomb gene, plus approximately 2 Kb of 5′-flanking sequences and approximately 0.7 Kb of 3′-flanking sequences. The translation start site is located at position 2036 of SEQ ID NO:6. Thus, the promoter sequence is located between position 1 and position 2036.

The above examples are provided to illustrate the invention but not to limit its scope. Other variants of the invention will be readily apparent to one of ordinary skill in the art and are encompassed by the appended claims. All publications, patents, and patent applications cited herein are hereby incorporated by reference.

324 1 2136 DNA Arabidopsis sp. CDS (43)..(2112) fertilization-independent endosperm 1 (FIE1) cDNA 1aacatcagag aagacgagaa aaaaagaaga ggcgagtggt ta atg gag aag gaa 54 Met Glu Lys Glu 1aac cat gag gac gat ggt gag ggt ttg cca ccc gaa cta aat cag ata 102Asn His Glu Asp Asp Gly Glu Gly Leu Pro Pro Glu Leu Asn Gln Ile 5 10 15 20aaa gag caa atc gaa aag gag aga ttt ctg cat atc aag aga aaa ttc 150Lys Glu Gln Ile Glu Lys Glu Arg Phe Leu His Ile Lys Arg Lys Phe 25 30 35gag ctg aga tac att cca agt gtg gct act cat gct tca cac cat caa 198Glu Leu Arg Tyr Ile Pro Ser Val Ala Thr His Ala Ser His His Gln 40 45 50tcg ttt gac tta aac cag ccc gct gca gag gat gat aat gga gga gac 246Ser Phe Asp Leu Asn Gln Pro Ala Ala Glu Asp Asp Asn Gly Gly Asp 55 60 65aac aaa tca ctt ttg tcg aga atg caa aac cca ctt cgt cat ttc agt 294Asn Lys Ser Leu Leu Ser Arg Met Gln Asn Pro Leu Arg His Phe Ser 70 75 80gcc tca tct gat tat aat tct tac gaa gat caa ggt tat gtt ctt gat 342Ala Ser Ser Asp Tyr Asn Ser Tyr Glu Asp Gln Gly Tyr Val Leu Asp 85 90 95 100gag gat caa gat tat gct ctt gaa gaa gat gta cca tta ttt ctt gat 390Glu Asp Gln Asp Tyr Ala Leu Glu Glu Asp Val Pro Leu Phe Leu Asp 105 110 115gaa gat gta cca tta tta cca agt gtc aag ctt cca att gtt gag aag 438Glu Asp Val Pro Leu Leu Pro Ser Val Lys Leu Pro Ile Val Glu Lys 120 125 130cta cca cga tcc att aca tgg gtc ttc acc aaa agt agc cag ctg atg 486Leu Pro Arg Ser Ile Thr Trp Val Phe Thr Lys Ser Ser Gln Leu Met 135 140 145gct gaa agt gat tct gtg att ggt aag aga caa atc tat tat ttg aat 534Ala Glu Ser Asp Ser Val Ile Gly Lys Arg Gln Ile Tyr Tyr Leu Asn 150 155 160ggt gag gca cta gaa ttg agc agt gaa gaa gat gag gaa gat gaa gaa 582Gly Glu Ala Leu Glu Leu Ser Ser Glu Glu Asp Glu Glu Asp Glu Glu165 170 175 180gaa gat gag gaa gaa atc aag aaa gaa aaa tgc gaa ttt tct gaa gat 630Glu Asp Glu Glu Glu Ile Lys Lys Glu Lys Cys Glu Phe Ser Glu Asp 185 190 195gta gac cga ttt ata tgg acg gtt ggg cag gac tat ggt ttg gat gat 678Val Asp Arg Phe Ile Trp Thr Val Gly Gln Asp Tyr Gly Leu Asp Asp 200 205 210ctg gtc gtg cgg cgt gct ctc gcc aag tac ctc gaa gtg gat gtt tcg 726Leu Val Val Arg Arg Ala Leu Ala Lys Tyr Leu Glu Val Asp Val Ser 215 220 225gac ata ttg gaa aga tac aat gaa ctc aag ctt aag aat gat gga act 774Asp Ile Leu Glu Arg Tyr Asn Glu Leu Lys Leu Lys Asn Asp Gly Thr 230 235 240gct ggt gag gct tct gat ttg aca tcc aag aca ata act act gct ttc 822Ala Gly Glu Ala Ser Asp Leu Thr Ser Lys Thr Ile Thr Thr Ala Phe245 250 255 260cag gat ttt gct gat aga cgt cat tgc cgt cgt tgc atg ata ttc gat 870Gln Asp Phe Ala Asp Arg Arg His Cys Arg Arg Cys Met Ile Phe Asp 265 270 275tgt cat atg cat gag aag tat gag ccc gag tct aga tcc agc gaa gac 918Cys His Met His Glu Lys Tyr Glu Pro Glu Ser Arg Ser Ser Glu Asp 280 285 290aaa tct agt ttg ttt gag gat gaa gat aga caa cca tgc agt gag cat 966Lys Ser Ser Leu Phe Glu Asp Glu Asp Arg Gln Pro Cys Ser Glu His 295 300 305tgt tac ctc aag gtg agg agt gtg aca gaa gct gat cat gtg atg gat 1014Cys Tyr Leu Lys Val Arg Ser Val Thr Glu Ala Asp His Val Met Asp 310 315 320aat gat aac tct ata tca aac aag att gtg gtc tca gat cca aac aac 1062Asn Asp Asn Ser Ile Ser Asn Lys Ile Val Val Ser Asp Pro Asn Asn325 330 335 340act atg tgg acg cct gta gag aag gat ctt tac ttg aaa gga att gag 1110Thr Met Trp Thr Pro Val Glu Lys Asp Leu Tyr Leu Lys Gly Ile Glu 345 350 355ata ttt ggg aga aac agt tgt gat gtt gca tta aac ata ctt cgg ggg 1158Ile Phe Gly Arg Asn Ser Cys Asp Val Ala Leu Asn Ile Leu Arg Gly 360 365 370ctt aag acg tgc cta gag att tac aat tac atg cgc gaa caa gat caa 1206Leu Lys Thr Cys Leu Glu Ile Tyr Asn Tyr Met Arg Glu Gln Asp Gln 375 380 385tgt act atg tca tta gac ctt aac aaa act aca caa aga cac aat cag 1254Cys Thr Met Ser Leu Asp Leu Asn Lys Thr Thr Gln Arg His Asn Gln 390 395 400gtt acc aaa aaa gta tct cga aaa agt agt agg tcg gtc cgc aaa aaa 1302Val Thr Lys Lys Val Ser Arg Lys Ser Ser Arg Ser Val Arg Lys Lys405 410 415 420tcg aga ctc cga aaa tat gct cgt tat ccg cct gct tta aag aaa aca 1350Ser Arg Leu Arg Lys Tyr Ala Arg Tyr Pro Pro Ala Leu Lys Lys Thr 425 430 435act agt gga gaa gct aag ttt tat aag cac tac aca cca tgc act tgc 1398Thr Ser Gly Glu Ala Lys Phe Tyr Lys His Tyr Thr Pro Cys Thr Cys 440 445 450aag tca aaa tgt gga cag caa tgc cct tgt tta act cac gaa aat tgc 1446Lys Ser Lys Cys Gly Gln Gln Cys Pro Cys Leu Thr His Glu Asn Cys 455 460 465tgc gag aaa tat tgc ggg tgc tca aag gat tgc aac aat cgc ttt gga 1494Cys Glu Lys Tyr Cys Gly Cys Ser Lys Asp Cys Asn Asn Arg Phe Gly 470 475 480gga tgt aat tgt gca att ggc caa tgc aca aat cga caa tgt cct tgt 1542Gly Cys Asn Cys Ala Ile Gly Gln Cys Thr Asn Arg Gln Cys Pro Cys485 490 495 500ttt gct gct aat cgt gaa tgc gat cca gat ctt tgt cgg agt tgt cct 1590Phe Ala Ala Asn Arg Glu Cys Asp Pro Asp Leu Cys Arg Ser Cys Pro 505 510 515ctt agc tgt gga gat ggc act ctt ggt gag aca cca gtg caa atc caa 1638Leu Ser Cys Gly Asp Gly Thr Leu Gly Glu Thr Pro Val Gln Ile Gln 520 525 530tgc aag aac atg caa ttc ctc ctt caa acc aat aaa aag att ctc att 1686Cys Lys Asn Met Gln Phe Leu Leu Gln Thr Asn Lys Lys Ile Leu Ile 535 540 545gga aag tct gat gtt cat gga tgg ggt gca ttt aca tgg gac tct ctt 1734Gly Lys Ser Asp Val His Gly Trp Gly Ala Phe Thr Trp Asp Ser Leu 550 555 560aaa aag aat gag tat ctc gga gaa tat act gga gaa ctg atc act cat 1782Lys Lys Asn Glu Tyr Leu Gly Glu Tyr Thr Gly Glu Leu Ile Thr His565 570 575 580gat gaa gct aat gag cgt ggg aga ata gaa gat cgg att ggt tct tcc 1830Asp Glu Ala Asn Glu Arg Gly Arg Ile Glu Asp Arg Ile Gly Ser Ser 585 590 595tac ctc ttt acc ttg aat gat cag ctc gaa atc gat gct cgc cgt aaa 1878Tyr Leu Phe Thr Leu Asn Asp Gln Leu Glu Ile Asp Ala Arg Arg Lys 600 605 610gga aac gag ttc aaa ttt ctc aat cac tca gca aga cct aac tgc tac 1926Gly Asn Glu Phe Lys Phe Leu Asn His Ser Ala Arg Pro Asn Cys Tyr 615 620 625gcc aag ttg atg att gtg aga gga gat cag agg att ggt cta ttt gcg 1974Ala Lys Leu Met Ile Val Arg Gly Asp Gln Arg Ile Gly Leu Phe Ala 630 635 640gag aga gca atc gaa gaa ggt gag gag ctt ttc ttc gac tac tgc tat 2022Glu Arg Ala Ile Glu Glu Gly Glu Glu Leu Phe Phe Asp Tyr Cys Tyr645 650 655 660gga cca gaa cat gcg gat tgg tcg cgt ggt cga gaa cct aga aag act 2070Gly Pro Glu His Ala Asp Trp Ser Arg Gly Arg Glu Pro Arg Lys Thr 665 670 675ggt gct tct aaa agg tct aag gaa gcc cgt cca gct cgt tagtttttga 2119Gly Ala Ser Lys Arg Ser Lys Glu Ala Arg Pro Ala Arg 680 685tctgaggaga agcagca 2136 2 689 PRT Arabidopsis sp. 2Met Glu Lys Glu Asn His Glu Asp Asp Gly Glu Gly Leu Pro Pro Glu 1 5 10 15Leu Asn Gln Ile Lys Glu Gln Ile Glu Lys Glu Arg Phe Leu His Ile 20 25 30Lys Arg Lys Phe Glu Leu Arg Tyr Ile Pro Ser Val Ala Thr His Ala 35 40 45Ser His His Gln Ser Phe Asp Leu Asn Gln Pro Ala Ala Glu Asp Asp 50 55 60Asn Gly Gly Asp Asn Lys Ser Leu Leu Ser Arg Met Gln Asn Pro Leu 65 70 75 80Arg His Phe Ser Ala Ser Ser Asp Tyr Asn Ser Tyr Glu Asp Gln Gly 85 90 95Tyr Val Leu Asp Glu Asp Gln Asp Tyr Ala Leu Glu Glu Asp Val Pro 100 105 110Leu Phe Leu Asp Glu Asp Val Pro Leu Leu Pro Ser Val Lys Leu Pro 115 120 125Ile Val Glu Lys Leu Pro Arg Ser Ile Thr Trp Val Phe Thr Lys Ser 130 135 140Ser Gln Leu Met Ala Glu Ser Asp Ser Val Ile Gly Lys Arg Gln Ile145 150 155 160Tyr Tyr Leu Asn Gly Glu Ala Leu Glu Leu Ser Ser Glu Glu Asp Glu 165 170 175Glu Asp Glu Glu Glu Asp Glu Glu Glu Ile Lys Lys Glu Lys Cys Glu 180 185 190Phe Ser Glu Asp Val Asp Arg Phe Ile Trp Thr Val Gly Gln Asp Tyr 195 200 205Gly Leu Asp Asp Leu Val Val Arg Arg Ala Leu Ala Lys Tyr Leu Glu 210 215 220Val Asp Val Ser Asp Ile Leu Glu Arg Tyr Asn Glu Leu Lys Leu Lys225 230 235 240Asn Asp Gly Thr Ala Gly Glu Ala Ser Asp Leu Thr Ser Lys Thr Ile 245 250 255Thr Thr Ala Phe Gln Asp Phe Ala Asp Arg Arg His Cys Arg Arg Cys 260 265 270Met Ile Phe Asp Cys His Met His Glu Lys Tyr Glu Pro Glu Ser Arg 275 280 285Ser Ser Glu Asp Lys Ser Ser Leu Phe Glu Asp Glu Asp Arg Gln Pro 290 295 300Cys Ser Glu His Cys Tyr Leu Lys Val Arg Ser Val Thr Glu Ala Asp305 310 315 320His Val Met Asp Asn Asp Asn Ser Ile Ser Asn Lys Ile Val Val Ser 325 330 335Asp Pro Asn Asn Thr Met Trp Thr Pro Val Glu Lys Asp Leu Tyr Leu 340 345 350Lys Gly Ile Glu Ile Phe Gly Arg Asn Ser Cys Asp Val Ala Leu Asn 355 360 365Ile Leu Arg Gly Leu Lys Thr Cys Leu Glu Ile Tyr Asn Tyr Met Arg 370 375 380Glu Gln Asp Gln Cys Thr Met Ser Leu Asp Leu Asn Lys Thr Thr Gln385 390 395 400Arg His Asn Gln Val Thr Lys Lys Val Ser Arg Lys Ser Ser Arg Ser 405 410 415Val Arg Lys Lys Ser Arg Leu Arg Lys Tyr Ala Arg Tyr Pro Pro Ala 420 425 430Leu Lys Lys Thr Thr Ser Gly Glu Ala Lys Phe Tyr Lys His Tyr Thr 435 440 445Pro Cys Thr Cys Lys Ser Lys Cys Gly Gln Gln Cys Pro Cys Leu Thr 450 455 460His Glu Asn Cys Cys Glu Lys Tyr Cys Gly Cys Ser Lys Asp Cys Asn465 470 475 480Asn Arg Phe Gly Gly Cys Asn Cys Ala Ile Gly Gln Cys Thr Asn Arg 485 490 495Gln Cys Pro Cys Phe Ala Ala Asn Arg Glu Cys Asp Pro Asp Leu Cys 500 505 510Arg Ser Cys Pro Leu Ser Cys Gly Asp Gly Thr Leu Gly Glu Thr Pro 515 520 525Val Gln Ile Gln Cys Lys Asn Met Gln Phe Leu Leu Gln Thr Asn Lys 530 535 540Lys Ile Leu Ile Gly Lys Ser Asp Val His Gly Trp Gly Ala Phe Thr545 550 555 560Trp Asp Ser Leu Lys Lys Asn Glu Tyr Leu Gly Glu Tyr Thr Gly Glu 565 570 575Leu Ile Thr His Asp Glu Ala Asn Glu Arg Gly Arg Ile Glu Asp Arg 580 585 590Ile Gly Ser Ser Tyr Leu Phe Thr Leu Asn Asp Gln Leu Glu Ile Asp 595 600 605Ala Arg Arg Lys Gly Asn Glu Phe Lys Phe Leu Asn His Ser Ala Arg 610 615 620Pro Asn Cys Tyr Ala Lys Leu Met Ile Val Arg Gly Asp Gln Arg Ile625 630 635 640Gly Leu Phe Ala Glu Arg Ala Ile Glu Glu Gly Glu Glu Leu Phe Phe 645 650 655Asp Tyr Cys Tyr Gly Pro Glu His Ala Asp Trp Ser Arg Gly Arg Glu 660 665 670Pro Arg Lys Thr Gly Ala Ser Lys Arg Ser Lys Glu Ala Arg Pro Ala 675 680 685Arg 3 1563 DNA Arabidopsis sp. CDS (199)..(1308) fertilization-independent endosperm 3 (FIE3) cDNA 3aaaggtgagt tgtgtgttgt gtcaggtcca aaataaaagt ttgtcgtgag gtcaaaatct 60acggttacag taattttaat aacctgtgaa tctgtgtcta atcgaaaatt acaaaacacc 120agttgttgtt gcatgagaga cttgtgagct tagattagtg tgcgagagtc agacagagag 180agagatttcg aatatcga atg tcg aag ata acc tta ggg aac gag tca ata 231 Met Ser Lys Ile Thr Leu Gly Asn Glu Ser Ile 1 5 10gtt ggg tct ttg act cca tcg aat aag aaa tcg tac aaa gtg acg aat 279Val Gly Ser Leu Thr Pro Ser Asn Lys Lys Ser Tyr Lys Val Thr Asn 15 20 25agg att cag gaa ggg aag aaa cct ttg tat gct gtt gtt ttc aac ttc 327Arg Ile Gln Glu Gly Lys Lys Pro Leu Tyr Ala Val Val Phe Asn Phe 30 35 40ctt gat gct cgt ttc ttc gat gtc ttc gtt acc gct ggt gga aat cgg 375Leu Asp Ala Arg Phe Phe Asp Val Phe Val Thr Ala Gly Gly Asn Arg 45 50 55att act ctg tac aat tgt ctc gga gat ggt gcc ata tca gca ttg caa 423Ile Thr Leu Tyr Asn Cys Leu Gly Asp Gly Ala Ile Ser Ala Leu Gln 60 65 70 75tcc tat gct gat gaa gat aag gaa gag tcg ttt tac acg gta agt tgg 471Ser Tyr Ala Asp Glu Asp Lys Glu Glu Ser Phe Tyr Thr Val Ser Trp 80 85 90gcg tgt ggc gtt aat ggg aac cca tat gtt gcg gct gga gga gta aaa 519Ala Cys Gly Val Asn Gly Asn Pro Tyr Val Ala Ala Gly Gly Val Lys 95 100 105ggt ata atc cga gtc att gac gtc aac agt gaa acg att cat aag agt 567Gly Ile Ile Arg Val Ile Asp Val Asn Ser Glu Thr Ile His Lys Ser 110 115 120ctt gtg ggt cat gga gat tca gtg aac gaa atc agg aca caa cct tta 615Leu Val Gly His Gly Asp Ser Val Asn Glu Ile Arg Thr Gln Pro Leu 125 130 135aaa cct caa ctt gtg att act gct agc aag gat gaa tct gtt cgt ttg 663Lys Pro Gln Leu Val Ile Thr Ala Ser Lys Asp Glu Ser Val Arg Leu140 145 150 155tgg aat gtt gaa act ggg ata tgt att ttg ata ttt gct gga gct gga 711Trp Asn Val Glu Thr Gly Ile Cys Ile Leu Ile Phe Ala Gly Ala Gly 160 165 170ggt cat cgc tat gaa gtt cta agt gtg gat ttt cat ccg tct gat att 759Gly His Arg Tyr Glu Val Leu Ser Val Asp Phe His Pro Ser Asp Ile 175 180 185tac cgc ttt gct agt tgt ggt atg gac acc act att aaa ata tgg tca 807Tyr Arg Phe Ala Ser Cys Gly Met Asp Thr Thr Ile Lys Ile Trp Ser 190 195 200atg aaa gag ttt tgg acg tac gtc gag aag tca ttc aca tgg act gat 855Met Lys Glu Phe Trp Thr Tyr Val Glu Lys Ser Phe Thr Trp Thr Asp 205 210 215gat cca tca aaa ttc ccc aca aaa ttt gtc caa ttc cct gta ttt aca 903Asp Pro Ser Lys Phe Pro Thr Lys Phe Val Gln Phe Pro Val Phe Thr220 225 230 235gct tcc att cat aca aat tat gta gat tgt aac cgt tgg ttt ggt gat 951Ala Ser Ile His Thr Asn Tyr Val Asp Cys Asn Arg Trp Phe Gly Asp 240 245 250ttt atc ctc tca aag agt gtg gac aac gag atc ctg ttg tgg gaa cca 999Phe Ile Leu Ser Lys Ser Val Asp Asn Glu Ile Leu Leu Trp Glu Pro 255 260 265caa ctg aaa gag aat tct cct ggc gag gga gct tca gat gtt cta tta 1047Gln Leu Lys Glu Asn Ser Pro Gly Glu Gly Ala Ser Asp Val Leu Leu 270 275 280aga tac ccg gtt cca atg tgt gat att tgg ttt atc aag ttt tct tgt 1095Arg Tyr Pro Val Pro Met Cys Asp Ile Trp Phe Ile Lys Phe Ser Cys 285 290 295gac ctc cat tta agt tct gtt gcg ata ggt aat cag gaa gga aag gtt 1143Asp Leu His Leu Ser Ser Val Ala Ile Gly Asn Gln Glu Gly Lys Val300 305 310 315tat gtc tgg gat ttg aaa agt tgc cct cct gtt ttg att aca aag tta 1191Tyr Val Trp Asp Leu Lys Ser Cys Pro Pro Val Leu Ile Thr Lys Leu 320 325 330tca cac aat caa tca aag tct gta atc agg caa aca gcc atg tct gtc 1239Ser His Asn Gln Ser Lys Ser Val Ile Arg Gln Thr Ala Met Ser Val 335 340 345gat gga agc acg att ctt gct tgc tgc gag gac ggg act ata tgg cgc 1287Asp Gly Ser Thr Ile Leu Ala Cys Cys Glu Asp Gly Thr Ile Trp Arg 350 355 360tgg gac gtg att acc aag tagcggtctg agtcttgtag gaattgatga 1335Trp Asp Val Ile Thr Lys 365attaggagtg cgaagaaatg agatatccat tcttttattg taattctgat catgttgcta 1395ctccctgaga ccttgagatg ctctttgtag ccttgttaac gtccaccctt gtaccacagt 1455gtataccctt tctggagatt ttgtcttatt ctcttagttc aatacacaag gctgtatcct 1515ggagctttat tgcaggaacc actctctttc ataagctttc tagtattc 1563 4 369 PRT Arabidopsis sp. 4Met Ser Lys Ile Thr Leu Gly Asn Glu Ser Ile Val Gly Ser Leu Thr 1 5 10 15Pro Ser Asn Lys Lys Ser Tyr Lys Val Thr Asn Arg Ile Gln Glu Gly 20 25 30Lys Lys Pro Leu Tyr Ala Val Val Phe Asn Phe Leu Asp Ala Arg Phe 35 40 45Phe Asp Val Phe Val Thr Ala Gly Gly Asn Arg Ile Thr Leu Tyr Asn 50 55 60Cys Leu Gly Asp Gly Ala Ile Ser Ala Leu Gln Ser Tyr Ala Asp Glu 65 70 75 80Asp Lys Glu Glu Ser Phe Tyr Thr Val Ser Trp Ala Cys Gly Val Asn 85 90 95Gly Asn Pro Tyr Val Ala Ala Gly Gly Val Lys Gly Ile Ile Arg Val 100 105 110Ile Asp Val Asn Ser Glu Thr Ile His Lys Ser Leu Val Gly His Gly 115 120 125Asp Ser Val Asn Glu Ile Arg Thr Gln Pro Leu Lys Pro Gln Leu Val 130 135 140Ile Thr Ala Ser Lys Asp Glu Ser Val Arg Leu Trp Asn Val Glu Thr145 150 155 160Gly Ile Cys Ile Leu Ile Phe Ala Gly Ala Gly Gly His Arg Tyr Glu 165 170 175Val Leu Ser Val Asp Phe His Pro Ser Asp Ile Tyr Arg Phe Ala Ser 180 185 190Cys Gly Met Asp Thr Thr Ile Lys Ile Trp Ser Met Lys Glu Phe Trp 195 200 205Thr Tyr Val Glu Lys Ser Phe Thr Trp Thr Asp Asp Pro Ser Lys Phe 210 215 220Pro Thr Lys Phe Val Gln Phe Pro Val Phe Thr Ala Ser Ile His Thr225 230 235 240Asn Tyr Val Asp Cys Asn Arg Trp Phe Gly Asp Phe Ile Leu Ser Lys 245 250 255Ser Val Asp Asn Glu Ile Leu Leu Trp Glu Pro Gln Leu Lys Glu Asn 260 265 270Ser Pro Gly Glu Gly Ala Ser Asp Val Leu Leu Arg Tyr Pro Val Pro 275 280 285Met Cys Asp Ile Trp Phe Ile Lys Phe Ser Cys Asp Leu His Leu Ser 290 295 300Ser Val Ala Ile Gly Asn Gln Glu Gly Lys Val Tyr Val Trp Asp Leu305 310 315 320Lys Ser Cys Pro Pro Val Leu Ile Thr Lys Leu Ser His Asn Gln Ser 325 330 335Lys Ser Val Ile Arg Gln Thr Ala Met Ser Val Asp Gly Ser Thr Ile 340 345 350Leu Ala Cys Cys Glu Asp Gly Thr Ile Trp Arg Trp Asp Val Ile Thr 355 360 365Lys 5 5801 DNA Arabidopsis sp. CDS (3872)..(5566) fertilization-independent endosperm 3 (FIE3) WD40/polycomb gene genomic sequence 5tctgaagcag ctaatcgatc cactaatctt gtggagatcg tgtgttgctt tggtgcatat 60atatacaaat agacaaatac atatgcgttt acatatatat gtaagcacgt atttagagag 120caacaataag gcatgagaaa tgtgattatc gtcaaatcat gattgctaca tgacaaatcg 180atcttaattt tgaaaaagag acatttaaat attcaaaaaa cggtaaaaat ttctttaaga 240ccaaccatgg aaataacatg agaagactga gagggagatt agaacttaca acaagagaat 300ctttttcctt caatattttt tttaaacact tttcttttgt agggaatttg ataatatgaa 360atggatagat tttactgctt aatttttaat cattttttat cagaaacttt ttcgttttaa 420atctacggct agaattttcg gtcggtttta tactttatat agatgctaga tttttttctt 480ctagtcatcg tttattagta caattttgtt tttatatatt gattacttga atttataata 540ggattggtac aaaggtggta attataaagt gcattttttt ggatattgtt caattcaaat 600atttttactt agattctcaa actattgaaa aatatccaaa atatccggaa aatttcaatt 660taatcgaata aaaaaattag aatggaaaga ataaaaaatt atcgggtaca attagaagag 720taatgtgttt agtttggttt ttactcggat accagttcag ttttcacgta ttattcgatc 780ctataggagc aattgtgaat tagttgtgag attttgggag cattcgcttc cagaacttag 840tgctaggaga aatgctattt tcctataaga gttgtacgag gaagcgagca agtacacaac 900aaccacaaaa gctttcaata cttgtttact cctagggttt aaaactagag gttctataga 960tctctaaatt tttttgaaca aatgtgtttt ccacacgtga tattctacaa taccactcga 1020aaattatcca taattgcttt aaactatttt tttgtttaaa ttatataatt tgtaccgttg 1080taaactgatt atttcaaatt ataattaaag cactataatt tcatatatta cattcaacat 1140atattaaaat aaactataac catgtatttt tttgtcttcc tttcctataa acattgattg 1200gactctatcg taaattttgt cgttatcgca aattttgtcg ttatcgatga gtttctcaaa 1260gtttggacct tgattatctt gtttggagat gttcaaatcg ttatatccaa atagtgaact 1320tctaattttc ttttttgata atgtgactta tttggaaaag tattccaaag tattcaaata 1380aaccctttaa aaatccatta aatacatttt aaataagtaa aatgctctca acgaagagat 1440atcatggtaa ataacaacag tgagaggata aaatgttaaa tcaatttatt tacaacttca 1500aataggcgga catcaaacct acttagcaca ctttctattt tcaaattggt tatggtttgt 1560ctattagttg ttgcatctat gttttttaat tcttatatcg gtgatcttga ttttgttttg 1620gtgtatctaa aatctatttt agttaaagtg caagaaaata aaataaaaac ttaaggtaag 1680agatgaaagt aagctttaaa taaaacagag cacttctatg gtcgattata gagccaagtt 1740cgttcctcca ttttggctta atgcaatatt acaagtaaat cttataaaac tttccataag 1800tatcgtatta cccatggata ctatgatata taaactctcg gaggtgtagt ccagaagaaa 1860tgatccatat ttgcatacag taaacttgat ggaaaaaata tgtggtactg ttggaattgt 1920agctattgag tatcaaattt gagaaaaagg taaaaaaata tgtaaaattt gggtggaaga 1980aaagaattac ataaaattga gaaatgtatg taattgacaa aataatgttt tcaaaacata 2040aaaacgtgat accatttaaa tccaaacctt atatcattta accattttta gtaaaactaa 2100tagtaatgaa tggtcaataa tataagatta catattaaat aattactact ttcagaaaat 2160ttcaatcaaa tctataatat tcctttgaaa aaaaagaaag acaaataggt aaacttcgat 2220cgtatcaatc aaagaatata tttatttttc atcgtaacgt ttaattctaa gtcctattaa 2280aaaacgttaa atttgatttt tcttaccatt tttttctaaa aggtgagttg tgtgttgtgt 2340caggtccaaa ataaaagttt gtcgtgaggt caaaatctac ggttacagta attttaataa 2400cctgtgaatc tgtgtctaat cgaaaattac aaaacaccag ttgttgttgc atgagagact 2460tgtgagctta gattagtgtg cgagagtcag acagagagag agatttcgaa tatcgaatgt 2520cgaagataac cttagggaac gagtcaatag ttgggtcttt gactccatcg aataagaaat 2580cgtacaaagt gacgaatagg attcaggaag ggaagaaacc tttgtatgct gttgttttca 2640acttccttga tgctcgtttc ttcgatgtct tcgttaccgc tggtggaaat cgggtaaaag 2700atctcgactt tcaattcgaa atcactgttt tcaattctgg gtctgtttag gttttgattc 2760agattgattg taacattaag gcctttcctt ttgtgtttga ttttggattc tgatttctag 2820cctttagtga gattaaaaga ttgaaacttt gcttgatgct atagtctaag attatgtaac 2880atttagttca aactttctgg ttttggagat tttgtggaag atatggtttt tgttttctaa 2940tttaaagtga actcattacc ttatacactt gatttgcatt ctgttctaaa aaaaattgaa 3000actttggttg atgttgttag tctgcttatc taaggaggtt ccttttgaaa cggtcatcaa 3060gtgagttatg aagcgtttag tttaagcttt cctgtattgg agattttgtg gaagttattt 3120ttttttctaa ttttgaaact agatagagtg aagtcattac cttatacatt agactgctct 3180attttgtttt caatgtgggt tccgaatgta cctgatagtg gctctttagg ctcatttgta 3240ttcgtcgaaa catcgatcgg atacccgttt gggcttagta ggctctgata ccgcgtaaag 3300ttctcgggtt ccatgaaaaa ccaatcggta atgagtggag ttaatttgta atcgtcttcg 3360gtcgagcatt tgggattagt gggctttgat accatgtgaa agtccttggg gtccaatcgg 3420caatgagtag agttaacttg taatcttaca cacttggtta ggtctcattc tctttataat 3480gttgtgtgcc taacagtttc cgcactaagg ttgtttggtt gctcagtctc aatatactta 3540tcttaactag ttgtagtttt tttcatcttt cctagtttcc gttggatttt aaattgaatg 3600atttactagt tagaaatatt tgagtttctc atagaagctt taaccaaggg gttctttcat 3660ttaaccttta cttagctagt tcatgaatct cattactgcc attggtgtat ctcttattat 3720gtagattact ctgtacaatt gtctcggaga tggtgccata tcagcattgc aatcctatgc 3780tgatgaagat gtaaggaagc atacatatta gcttttccat caaattaaag taagtgatgt 3840ttcactgagg ccatttggtt atattttgtc tatgtcctct ggagagcaga aggaagagtc 3900gttttacacg gtaagttggg cgtgtggcgt taatgggaac ccatatgttg cggctggagg 3960agtaaaaggt ataatccgag tcattgacgt caacagtgaa acgattcata aggtattatt 4020gcatttttat ggatgttcta tgtatcctag caaatgattc tatatctttc ttgtataatc 4080tgtgctcgca aatgtgcaga gtcttgtggg tcatggagat tcagtgaacg aaatcaggac 4140acaaccttta aaacctcaac ttgtgattac tgctagcaag gtatatctct tggctttctt 4200ttcttcctaa agtatcctga cttctttttt atttgttggt gattaagagc tgttacgttt 4260taattgaata aggatgaatc tgttcgtttg tggaatgttg aaactgggat atgtattttg 4320atatttgctg gagctggagg tcatcgctat gaagttctaa gtgtggtgag ccaatattgt 4380tttatctaat tcagttagtt ttctacaata atatatagag acaatgttaa ggggaaccat 4440cttattttga aaattgtagg attttcatcc gtctgatatt taccgctttg ctagttgtgg 4500tatggacacc actattaaaa tatggtcaat gaaaggtacg atcgagcaca tattgtaata 4560aacttccatt ttaaaaaacc ttttgagaaa aatggcttgt ggttcgtttg tatgatcttc 4620ttattctttg gctgtctata gagttttgga cgtacgtcga gaagtcattc acatggactg 4680atgatccatc aaaattcccc acaaaatttg tccaattccc tgtaagtatt ttgttttagc 4740cttgtcttgt aacaacaagt gacatacaaa tattggtgat ggcctttgta aataacatta 4800cttctatatg taggtattta cagcttccat tcatacaaat tatgtagatt gtaaccgttg 4860gtttggtgat tttatcctct caaaggttag taagtcaatg atggttaaga ttaattcatt 4920tggtgtactg ttaaaacact ttactcttgt gttgttctat cggattttag agtgtggaca 4980acgagatcct gttgtgggaa ccacaactga aagagaattc tcctggcgag gttaggatct 5040cattgttgct ccaaacacaa cataatcatt catttcatca catatattta cagttgaact 5100ttttgtggtt tgcagggagc ttcagatgtt ctattaagat acccggttcc aatgtgtgat 5160atttggttta tcaagttttc ttgtgacctc catttaagtt ctgttgcgat aggtaatcag 5220agagctcgtt agatacaaat ttgcattcta tagatagatt acttcaactt ttcttattca 5280ttttgtgaca aattactcgc tggtttgtta tcaggtaatc aggaaggaaa ggtttatgtc 5340tgggatttga aaagttgccc tcctgttttg attacaaagt aagttagttt cggattcaga 5400tacaatgttt gatctttaag aaatgtttta gtcttgacat gattttctgt tgccatatag 5460gttatcacac aatcaatcaa agtctgtaat caggcaaaca gccatgtctg tcgatggaag 5520gtataaatcc atcttctctc tcaccaatgc agtgaaaatt tcttaatgtt atttatgact 5580caatagttac tgtaaatcaa accaaacttt ggattctgac acactgtttc ttccatggga 5640ttgtagcacg attcttgctt gctgcgagga cgggactata tggcgctggg acgtgattac 5700caagtagcgg tctgagtctt gtaggaattg atgaattagg agtgcgaaga aatgagatat 5760ccattctttt attgtaattc tgatcatgtt gctactccct g 5801 6 7015 DNA Arabidopsis sp. CDS (1)..(7014) fertilization-independent endosperm 1 (FIE1) SET/polycomb gene genomic sequence reading frame 1 6gga tcc att att ttt aaa aat caa att ttt tca tat cta tta ttt gtt 48Gly Ser Ile Ile Phe Lys Asn Gln Ile Phe Ser Tyr Leu Leu Phe Val 1 5 10 15tca aag aaa aaa aaa aca cac gac gat tat cca tct gcc ggc tgt gtt 96Ser Lys Lys Lys Lys Thr His Asp Asp Tyr Pro Ser Ala Gly Cys Val 20 25 30cat cgg taa acc tat att tta aaa ctg gtg ggc ttt tca tta cca taa 144His Arg Thr Tyr Ile Leu Lys Leu Val Gly Phe Ser Leu Pro 35 40 45gtt tgg aca tgt ttt tat aat ttg atg tat agt gta gac caa aaa ata 192Val Trp Thr Cys Phe Tyr Asn Leu Met Tyr Ser Val Asp Gln Lys Ile 50 55 60gag aaa taa gaa agg gaa cct ttg tgg tga ttg taa caa aac aga aat 240Glu Lys Glu Arg Glu Pro Leu Trp Leu Gln Asn Arg Asn 65 70 75 80cat tat att gaa tca ttc gaa aag acg aaa aga tca aac ctt tgt agc 288His Tyr Ile Glu Ser Phe Glu Lys Thr Lys Arg Ser Asn Leu Cys Ser 85 90 95tag atg acc ata gac gtg gct gcc aat tac agt ctt aat gct ttt ata 336 Met Thr Ile Asp Val Ala Ala Asn Tyr Ser Leu Asn Ala Phe Ile 100 105 110tag atc ttt ctt aca tcc tct gtt cct tca cat tca aga aac agt atc 384 Ile Phe Leu Thr Ser Ser Val Pro Ser His Ser Arg Asn Ser Ile 115 120 125atc cca ttt tct ttc ttc ttc tca gtg ttt caa tct ttg cga att aag 432Ile Pro Phe Ser Phe Phe Phe Ser Val Phe Gln Ser Leu Arg Ile Lys 130 135 140atg gaa cat gaa gaa aca caa aag aac aca aga aac agc tgg tcc ctg 480Met Glu His Glu Glu Thr Gln Lys Asn Thr Arg Asn Ser Trp Ser Leu145 150 155 160att cga cca ttt caa atg atc tcc att agc ttt ctt agc ctc ctc ctc 528Ile Arg Pro Phe Gln Met Ile Ser Ile Ser Phe Leu Ser Leu Leu Leu 165 170 175cct cta tct ttc ctc ttt ctt tca cgt ctc tct ctc tat acc tcc tca 576Pro Leu Ser Phe Leu Phe Leu Ser Arg Leu Ser Leu Tyr Thr Ser Ser 180 185 190act ccg gtc acc gtc tcc ggc gtt tcc tct gtt att cac cag gca gat 624Thr Pro Val Thr Val Ser Gly Val Ser Ser Val Ile His Gln Ala Asp 195 200 205gtc gga gtc tta tac acg atc ttg ttt ctc atc atc gtc ttc act tta 672Val Gly Val Leu Tyr Thr Ile Leu Phe Leu Ile Ile Val Phe Thr Leu 210 215 220atc cac agt ctc tca gga aaa cca gaa tgc tct gtt ctc cat tcc cat 720Ile His Ser Leu Ser Gly Lys Pro Glu Cys Ser Val Leu His Ser His225 230 235 240ctc tac atc tgc tgg atc gtt ctc ttc atc gcc caa gct tgt gcc ttt 768Leu Tyr Ile Cys Trp Ile Val Leu Phe Ile Ala Gln Ala Cys Ala Phe 245 250 255ggg atc aaa aga acc atg agc acg acc atg tct ata aat cca gac aaa 816Gly Ile Lys Arg Thr Met Ser Thr Thr Met Ser Ile Asn Pro Asp Lys 260 265 270aac ttg ttt ctt gcg aca cat gaa aga tgg atg ttg gtt agg gtt ttg 864Asn Leu Phe Leu Ala Thr His Glu Arg Trp Met Leu Val Arg Val Leu 275 280 285ttc ttt ttg ggg cta cac gaa gtg atg ctg atg tgg ttt aga gtc gtg 912Phe Phe Leu Gly Leu His Glu Val Met Leu Met Trp Phe Arg Val Val 290 295 300gtt aag cct gtg gtt gac aac act ata tat ggg gtc tac gtg gag gag 960Val Lys Pro Val Val Asp Asn Thr Ile Tyr Gly Val Tyr Val Glu Glu305 310 315 320agg tgg tcc gag aga gcc gtt gtg gca gtg acc ttt ggt ata atg tgg 1008Arg Trp Ser Glu Arg Ala Val Val Ala Val Thr Phe Gly Ile Met Trp 325 330 335tgg tgg agg cta aga gat gag gta gaa agt ctt gtg gtg gtg gtt acg 1056Trp Trp Arg Leu Arg Asp Glu Val Glu Ser Leu Val Val Val Val Thr 340 345 350gcg gat aga ctt aac ctc ccc att cgt ttg gag ggt ctc aat ttt gtg 1104Ala Asp Arg Leu Asn Leu Pro Ile Arg Leu Glu Gly Leu Asn Phe Val 355 360 365aac tgg tgt atg tat tac atc tgt gtt gga att ggt tta atg aag atc 1152Asn Trp Cys Met Tyr Tyr Ile Cys Val Gly Ile Gly Leu Met Lys Ile 370 375 380ttc aaa ggg ttt ttg gat ttt gtg aat acg ttg act ttg agc att aag 1200Phe Lys Gly Phe Leu Asp Phe Val Asn Thr Leu Thr Leu Ser Ile Lys385 390 395 400agg tcg aga aaa ggc tgt gaa tca tgt gtt ttt gat gat atg tgt aat 1248Arg Ser Arg Lys Gly Cys Glu Ser Cys Val Phe Asp Asp Met Cys Asn 405 410 415gat gat cat gtg taa gat att tga cat att ata ctc atc tct tga atg 1296Asp Asp His Val Asp Ile His Ile Ile Leu Ile Ser Met 420 425 430ttt ttg aga ttt ttt tat ttt tat ttt cta ttt ctt gct agg aat tta 1344Phe Leu Arg Phe Phe Tyr Phe Tyr Phe Leu Phe Leu Ala Arg Asn Leu 435 440 445acc cgt ata tat gtc aca aaa ata gta gaa tat cag aaa gca aaa ata 1392Thr Arg Ile Tyr Val Thr Lys Ile Val Glu Tyr Gln Lys Ala Lys Ile 450 455 460ttt tat cta aaa ata acc att gaa cat taa ttt aag tct ttt tat aat 1440Phe Tyr Leu Lys Ile Thr Ile Glu His Phe Lys Ser Phe Tyr Asn465 470 475 480tat att ttt ata aca cac cct ttt taa gaa aaa ctt gga gat tta att 1488Tyr Ile Phe Ile Thr His Pro Phe Glu Lys Leu Gly Asp Leu Ile 485 490 495aac gtt ata aat agt aaa aaa tat cgg att tac gta gaa gtt tta aat 1536Asn Val Ile Asn Ser Lys Lys Tyr Arg Ile Tyr Val Glu Val Leu Asn 500 505 510gcg tat aat taa att tac gaa ttg aat aat ata gcc ata tat ata ttt 1584Ala Tyr Asn Ile Tyr Glu Leu Asn Asn Ile Ala Ile Tyr Ile Phe 515 520 525ttg aag att taa act cat ttt gtt tct tcc ata tat gca taa tat ata 1632Leu Lys Ile Thr His Phe Val Ser Ser Ile Tyr Ala Tyr Ile 530 535 540agc tta aat aga aaa cta gct agg aat gaa tac taa tat ata taa tga 1680Ser Leu Asn Arg Lys Leu Ala Arg Asn Glu Tyr Tyr Ile545 550 555 560cat taa tat aag tct tac cgg aca ctc caa aat gta tat att gat cta 1728His Tyr Lys Ser Tyr Arg Thr Leu Gln Asn Val Tyr Ile Asp Leu 565 570 575tca aca ttt ttt cat tgg ttt act aaa cca agt tgt cac ata aat atg 1776Ser Thr Phe Phe His Trp Phe Thr Lys Pro Ser Cys His Ile Asn Met 580 585 590agt taa cgc ctt ttt ttt tat aat att gta tat gaa ttt aaa ctt gag 1824Ser Arg Leu Phe Phe Tyr Asn Ile Val Tyr Glu Phe Lys Leu Glu 595 600 605ctg tca aac gtc aag caa acc caa cat cta cat aca tat agt act ata 1872Leu Ser Asn Val Lys Gln Thr Gln His Leu His Thr Tyr Ser Thr Ile 610 615 620ttt tga aaa tta aaa ttt tct taa att tcc cat att att ttc ctt tta 1920Phe Lys Leu Lys Phe Ser Ile Ser His Ile Ile Phe Leu Leu625 630 635 640aag caa gca agt cca aat acg ttt ctt cca gat tat aat ttt cct taa 1968Lys Gln Ala Ser Pro Asn Thr Phe Leu Pro Asp Tyr Asn Phe Pro 645 650 655taa ggt ttt cta caa aaa aaa atc aac ttc tta ttt aaa aaa ccc ttt 2016 Gly Phe Leu Gln Lys Lys Ile Asn Phe Leu Phe Lys Lys Pro Phe 660 665 670gca tta tcc ttt tca cca aca tca gag aag acg aga aaa aaa gaa gag 2064Ala Leu Ser Phe Ser Pro Thr Ser Glu Lys Thr Arg Lys Lys Glu Glu 675 680 685gcg agt ggt taa tgg aga agg tta gtt tca ctc caa aca tat atg aat 2112Ala Ser Gly Trp Arg Arg Leu Val Ser Leu Gln Thr Tyr Met Asn 690 695 700tga cta ggt tat gaa atc cat ata ttt taa ttg tgt gtt tat gat aga 2160 Leu Gly Tyr Glu Ile His Ile Phe Leu Cys Val Tyr Asp Arg705 710 715 720tca ata aca ttt agg gtt gaa ttt tct tgt gat cta tta tgt tat tcg 2208Ser Ile Thr Phe Arg Val Glu Phe Ser Cys Asp Leu Leu Cys Tyr Ser 725 730 735tcc cat gca tga tcc ata aaa ctt tta ttt ttg aat ttg tct agg aaa 2256Ser His Ala Ser Ile Lys Leu Leu Phe Leu Asn Leu Ser Arg Lys 740 745 750acc atg agg acg atg gtg agg gtt tgc cac ccg aac taa atc aga taa 2304Thr Met Arg Thr Met Val Arg Val Cys His Pro Asn Ile Arg 755 760 765aag agc aaa tcg aaa agg aga gat ttc tgc ata tca agg taa gag aca 2352Lys Ser Lys Ser Lys Arg Arg Asp Phe Cys Ile Ser Arg Glu Thr 770 775 780ttt ggt tgc ttt aat att tta ttc tct tct gta tgt ttt tct gaa aat 2400Phe Gly Cys Phe Asn Ile Leu Phe Ser Ser Val Cys Phe Ser Glu Asn785 790 795 800taa gga gag gag agg act taa tct cat aac tat acg att cca aag aga 2448 Gly Glu Glu Arg Thr Ser His Asn Tyr Thr Ile Pro Lys Arg 805 810 815tgt taa gat aca tct aat aaa cag tta tac att agt cat aat ctt taa 2496Cys Asp Thr Ser Asn Lys Gln Leu Tyr Ile Ser His Asn Leu 820 825 830aac taa aaa gag aaa ttt cca aac ttt taa att aaa aac aga att tag 2544Asn Lys Glu Lys Phe Pro Asn Phe Ile Lys Asn Arg Ile 835 840 845aaa atg cca gcg aat cga taa cga cat cca gat ctg tcg ggt atc caa 2592Lys Met Pro Ala Asn Arg Arg His Pro Asp Leu Ser Gly Ile Gln 850 855 860aac tta gaa taa aaa aat aat taa tat att tat aat ata aag ctg gaa 2640Asn Leu Glu Lys Asn Asn Tyr Ile Tyr Asn Ile Lys Leu Glu865 870 875 880ctt agg tta taa aat aaa att gaa aat aat agt aga ttt ttt tgt ttt 2688Leu Arg Leu Asn Lys Ile Glu Asn Asn Ser Arg Phe Phe Cys Phe 885 890 895tgt caa aca aaa tag taa tac aat ttg ttt ttt tta gta caa aga aac 2736Cys Gln Thr Lys Tyr Asn Leu Phe Phe Leu Val Gln Arg Asn 900 905 910taa ata ggt cca aat tgt ttt ttt ttt aac att cag cca aaa aag cca 2784 Ile Gly Pro Asn Cys Phe Phe Phe Asn Ile Gln Pro Lys Lys Pro 915 920 925aga ttg atg cat ata tca aga aat cga aat caa aac ttt tgt att caa 2832Arg Leu Met His Ile Ser Arg Asn Arg Asn Gln Asn Phe Cys Ile Gln 930 935 940gta ttc tag ttt cac tat ata tag agt cca gtt tct gaa att taa aaa 2880Val Phe Phe His Tyr Ile Ser Pro Val Ser Glu Ile Lys945 950 955 960atc att tac cta tat att act tga tta aca gag aaa att cga gct gag 2928Ile Ile Tyr Leu Tyr Ile Thr Leu Thr Glu Lys Ile Arg Ala Glu 965 970 975ata cat tcc aag tgt ggc tac tca tgc ttc aca cca tca atc gtt tga 2976Ile His Ser Lys Cys Gly Tyr Ser Cys Phe Thr Pro Ser Ile Val 980 985 990ctt aaa cca gcc cgc tgc aga gga tga taa tgg agg aga caa caa atc 3024Leu Lys Pro Ala Arg Cys Arg Gly Trp Arg Arg Gln Gln Ile 995 1000 1005act ttt gtc gag aat gca aaa ccc act tcg tca ttt cag tgc ctc atc 3072Thr Phe Val Glu Asn Ala Lys Pro Thr Ser Ser Phe Gln Cys Leu Ile 1010 1015 1020tga tta taa ttc tta cga aga tca agg tta tgt tct tga tga gga tca 3120 Leu Phe Leu Arg Arg Ser Arg Leu Cys Ser Gly Ser1025 1030 1035 1040aga tta tgc tct tga aga aga tgt acc att att tct tga tga aga tgt 3168Arg Leu Cys Ser Arg Arg Cys Thr Ile Ile Ser Arg Cys 1045 1050 1055acc att att acc aag tgt caa gct tcc aat tgt tga gaa gct acc acg 3216Thr Ile Ile Thr Lys Cys Gln Ala Ser Asn Cys Glu Ala Thr Thr 1060 1065 1070atc cat tac atg ggt ctt cac caa aag gca tgt gtg ttt ttt gtt tcg 3264Ile His Tyr Met Gly Leu His Gln Lys Ala Cys Val Phe Phe Val Ser 1075 1080 1085tac tag ttt caa aat att aat cat ata cta tat agt aat cac tca tag 3312Tyr Phe Gln Asn Ile Asn His Ile Leu Tyr Ser Asn His Ser 1090 1095 1100tgc ata tat aca ttt ctt taa cat tgc agt agc cag ctg atg gct gaa 3360Cys Ile Tyr Thr Phe Leu His Cys Ser Ser Gln Leu Met Ala Glu1105 1110 1115 1120agt gat tct gtg att ggt aag aga caa atc tat tat ttg aat ggt gag 3408Ser Asp Ser Val Ile Gly Lys Arg Gln Ile Tyr Tyr Leu Asn Gly Glu 1125 1130 1135gca cta gaa ttg agc agt gaa gaa gat gag gaa gat gaa gaa gaa gat 3456Ala Leu Glu Leu Ser Ser Glu Glu Asp Glu Glu Asp Glu Glu Glu Asp 1140 1145 1150gag gaa gaa atc aag aaa gaa aaa tgc gaa ttt tct gaa gat gta gac 3504Glu Glu Glu Ile Lys Lys Glu Lys Cys Glu Phe Ser Glu Asp Val Asp 1155 1160 1165cga ttt ata tgg tta gtt ttt gca tta cat atg ttc ttg att att aat 3552Arg Phe Ile Trp Leu Val Phe Ala Leu His Met Phe Leu Ile Ile Asn 1170 1175 1180ttg tag tcc ata ttt aat aaa ctg ctc aag aaa ttt tca gga cgg ttg 3600Leu Ser Ile Phe Asn Lys Leu Leu Lys Lys Phe Ser Gly Arg Leu1185 1190 1195 1200ggc agg act atg gtt tgg atg atc tgg tcg tgc ggc gtg ctc tcg cca 3648Gly Arg Thr Met Val Trp Met Ile Trp Ser Cys Gly Val Leu Ser Pro 1205 1210 1215agt acc tcg aag tgg atg ttt cgg aca tat tgg taa caa tat tcg aat 3696Ser Thr Ser Lys Trp Met Phe Arg Thr Tyr Trp Gln Tyr Ser Asn 1220 1225 1230aaa aac ttc ata cgt cga tca ata act ttc ctg ctt att taa ttt ttg 3744Lys Asn Phe Ile Arg Arg Ser Ile Thr Phe Leu Leu Ile Phe Leu 1235 1240 1245ttg ttt ttc gtc gtg aga aat gtt tta aat ttt caa atc taa tgt agg 3792Leu Phe Phe Val Val Arg Asn Val Leu Asn Phe Gln Ile Cys Arg 1250 1255 1260aaa gat aca atg aac tca agc tta aga atg atg gaa ctg ctg gtg agg 3840Lys Asp Thr Met Asn Ser Ser Leu Arg Met Met Glu Leu Leu Val Arg1265 1270 1275 1280ctt ctg att tga cat cca aga caa taa cta ctg ctt tcc agg att ttg 3888Leu Leu Ile His Pro Arg Gln Leu Leu Leu Ser Arg Ile Leu 1285 1290 1295ctg ata gac gtc att gcc gtc gtt gca tgg taa ctt tga atc ttt ctt 3936Leu Ile Asp Val Ile Ala Val Val Ala Trp Leu Ile Phe Leu 1300 1305 1310ttt taa ttt agc cac aaa aaa ggg aga tga tca tac atg ttt tta ttt 3984Phe Phe Ser His Lys Lys Gly Arg Ser Tyr Met Phe Leu Phe 1315 1320 1325tat ttt atc att tgt ttt aca gat att cga ttg tca tat gca tga gaa 4032Tyr Phe Ile Ile Cys Phe Thr Asp Ile Arg Leu Ser Tyr Ala Glu 1330 1335 1340gta tga gcc cga gtc tag atc cgt aag cat taa att cat tta aat tat 4080Val Ala Arg Val Ile Arg Lys His Ile His Leu Asn Tyr1345 1350 1355 1360ttt gtt agt ttc aca acc ctt ata tat aag gtt aag tga tta act taa 4128Phe Val Ser Phe Thr Thr Leu Ile Tyr Lys Val Lys Leu Thr 1365 1370 1375tta gat tgc ttt ggc ttg tca gag cga aga caa atc tag ttt gtt tga 4176Leu Asp Cys Phe Gly Leu Ser Glu Arg Arg Gln Ile Phe Val 1380 1385 1390gga tga aga tag aca acc atg cag tga gca ttg tta cct caa ggt ctc 4224Gly Arg Thr Thr Met Gln Ala Leu Leu Pro Gln Gly Leu 1395 1400 1405tat ctc tct ccc tct ctc tct caa ttt ttt tgt cta ttc ctt aat tac 4272Tyr Leu Ser Pro Ser Leu Ser Gln Phe Phe Cys Leu Phe Leu Asn Tyr 1410 1415 1420gtt tat tag tta ctg gtt taa tat taa ata ggt gag gag tgt gac aga 4320Val Tyr Leu Leu Val Tyr Ile Gly Glu Glu Cys Asp Arg1425 1430 1435 1440agc tga tca tgt gat gga taa tga taa ctc tat atc aaa caa gat tgt 4368Ser Ser Cys Asp Gly Leu Tyr Ile Lys Gln Asp Cys 1445 1450 1455ggt ctc aga tcc aaa caa cac tat gtg gac gcc tgt aga gaa gga tct 4416Gly Leu Arg Ser Lys Gln His Tyr Val Asp Ala Cys Arg Glu Gly Ser 1460 1465 1470tta ctt gaa agg aat tga gat att tgg gag aaa cag gta aaa aaa taa 4464Leu Leu Glu Arg Asn Asp Ile Trp Glu Lys Gln Val Lys Lys 1475 1480 1485aaa tag att taa tgc att aat ata tat act tac act gta ttc ctt gat 4512Lys Ile Cys Ile Asn Ile Tyr Thr Tyr Thr Val Phe Leu Asp 1490 1495 1500tat gct ggt tcg cag ttg tga tgt tgc att aaa cat act tcg ggg gct 4560Tyr Ala Gly Ser Gln Leu Cys Cys Ile Lys His Thr Ser Gly Ala1505 1510 1515 1520taa gac gtg cct aga gat tta caa tta cat gcg cga aca aga tca atg 4608 Asp Val Pro Arg Asp Leu Gln Leu His Ala Arg Thr Arg Ser Met 1525 1530 1535tac tat gtc att aga cct taa caa aac tac aca aag aca caa tca ggt 4656Tyr Tyr Val Ile Arg Pro Gln Asn Tyr Thr Lys Thr Gln Ser Gly 1540 1545 1550aca cta acc tat gtc gta att att ctc atg aca tgt atg tta aaa aca 4704Thr Leu Thr Tyr Val Val Ile Ile Leu Met Thr Cys Met Leu Lys Thr 1555 1560 1565cat gaa gtt tcc tat atg tgt tga tgg ttt tat cac agg tta cca aaa 4752His Glu Val Ser Tyr Met Cys Trp Phe Tyr His Arg Leu Pro Lys 1570 1575 1580aag tat ctc gaa aaa gta gta ggt cgg tcc gca aaa aat cga gac tcc 4800Lys Tyr Leu Glu Lys Val Val Gly Arg Ser Ala Lys Asn Arg Asp Ser1585 1590 1595 1600gaa aat atg ctc gtt atc cgc ctg ctt taa aga aaa caa cta gtg gag 4848Glu Asn Met Leu Val Ile Arg Leu Leu Arg Lys Gln Leu Val Glu 1605 1610 1615aag cta agt ttt ata agc act aca cac cat gca ctt gca agt caa aat 4896Lys Leu Ser Phe Ile Ser Thr Thr His His Ala Leu Ala Ser Gln Asn 1620 1625 1630gtg gac agc aat gcc ctt gtt taa ctc acg aaa att gct gcg aga aat 4944Val Asp Ser Asn Ala Leu Val Leu Thr Lys Ile Ala Ala Arg Asn 1635 1640 1645att gcg ggt atg tca ttc aat ttt tcc taa gcc gga aga tcc atg aga 4992Ile Ala Gly Met Ser Phe Asn Phe Ser Ala Gly Arg Ser Met Arg 1650 1655 1660ttt aat ttg aac atg agt ttg tat ttt ttg ttc agg tgc tca aag gat 5040Phe Asn Leu Asn Met Ser Leu Tyr Phe Leu Phe Arg Cys Ser Lys Asp1665 1670 1675 1680tgc aac aat cgc ttt gga gga tgt aat tgt gca att ggc caa tgc aca 5088Cys Asn Asn Arg Phe Gly Gly Cys Asn Cys Ala Ile Gly Gln Cys Thr 1685 1690 1695aat cga caa tgt cct tgt ttt gct gct aat cgt gaa tgc gat cca gat 5136Asn Arg Gln Cys Pro Cys Phe Ala Ala Asn Arg Glu Cys Asp Pro Asp 1700 1705 1710ctt tgt cgg agt tgt cct ctt agg taa cac ttt cac ttc aat atc tct 5184Leu Cys Arg Ser Cys Pro Leu Arg His Phe His Phe Asn Ile Ser 1715 1720 1725tta tac aaa ttc tat aat caa agt aat tca aac caa aag tct tat aaa 5232Leu Tyr Lys Phe Tyr Asn Gln Ser Asn Ser Asn Gln Lys Ser Tyr Lys 1730 1735 1740aaa aac ttt ata tat agc tgt gga gat ggc act ctt ggt gag aca cca 5280Lys Asn Phe Ile Tyr Ser Cys Gly Asp Gly Thr Leu Gly Glu Thr Pro1745 1750 1755 1760gtg caa atc caa tgc aag aac atg caa ttc ctc ctt caa acc aat aaa 5328Val Gln Ile Gln Cys Lys Asn Met Gln Phe Leu Leu Gln Thr Asn Lys 1765 1770 1775aag gta atc aac gtc aaa tcc gta ccg aaa att taa aac taa tta tac 5376Lys Val Ile Asn Val Lys Ser Val Pro Lys Ile Asn Leu Tyr 1780 1785 1790gaa aga cat tta act atc att tcc cgt att tta cta gat tct cat tgg 5424Glu Arg His Leu Thr Ile Ile Ser Arg Ile Leu Leu Asp Ser His Trp 1795 1800 1805aaa gtc tga tgt tca tgg atg ggg tgc att tac atg ggt aag caa tca 5472Lys Val Cys Ser Trp Met Gly Cys Ile Tyr Met Gly Lys Gln Ser 1810 1815 1820tgt aaa tat aag aat aag ttt aat agt tat tgg tgc att cat aac act 5520Cys Lys Tyr Lys Asn Lys Phe Asn Ser Tyr Trp Cys Ile His Asn Thr1825 1830 1835 1840ttt ttt ttt tta ata atg ttt tat act tta gac cat taa ata tat tgt 5568Phe Phe Phe Leu Ile Met Phe Tyr Thr Leu Asp His Ile Tyr Cys 1845 1850 1855gtg ata tgg ttt gac ccg tca gga ctc tct taa aaa gaa tga gta tct 5616Val Ile Trp Phe Asp Pro Ser Gly Leu Ser Lys Glu Val Ser 1860 1865 1870cgg aga ata tac tgg aga act gat cac tca tga tga agc taa tga gcg 5664Arg Arg Ile Tyr Trp Arg Thr Asp His Ser Ser Ala 1875 1880 1885tgg gag aat aga aga tcg gat tgg ttc ttc cta cct ctt tac ctt gaa 5712Trp Glu Asn Arg Arg Ser Asp Trp Phe Phe Leu Pro Leu Tyr Leu Glu 1890 1895 1900tga tca ggt aac ttc aga ata att ttg aag taa cgt ttt aat cat tcg 5760 Ser Gly Asn Phe Arg Ile Ile Leu Lys Arg Phe Asn His Ser1905 1910 1915 1920cgg gtt aca cat cta ttc gaa tca aag taa cat tta ttt tac agc tcg 5808Arg Val Thr His Leu Phe Glu Ser Lys His Leu Phe Tyr Ser Ser 1925 1930 1935aaa tcg atg ctc gcc gta aag gaa acg agt tca aat ttc tca atc act 5856Lys Ser Met Leu Ala Val Lys Glu Thr Ser Ser Asn Phe Ser Ile Thr 1940 1945 1950cag caa gac cta act gct acg cca agg tac taa gcc gtt ata ctt tat 5904Gln Gln Asp Leu Thr Ala Thr Pro Arg Tyr Ala Val Ile Leu Tyr 1955 1960 1965ctt gaa caa ata cta aca tta tac aaa caa aaa tac tta tgt tag ttt 5952Leu Glu Gln Ile Leu Thr Leu Tyr Lys Gln Lys Tyr Leu Cys Phe 1970 1975 1980ctt tag tta aat cgt gta tca act tta ctc gtc gtt gat tgg ttt tca 6000Leu Leu Asn Arg Val Ser Thr Leu Leu Val Val Asp Trp Phe Ser1985 1990 1995 2000tat tga aga tat tcc aag aaa ctc aaa ctc att tta aat gat ttt ttc 6048Tyr Arg Tyr Ser Lys Lys Leu Lys Leu Ile Leu Asn Asp Phe Phe 2005 2010 2015ttg tcg aga aaa ttt agg tta cga aaa ttt atg gtt tcg tgt gca gtt 6096Leu Ser Arg Lys Phe Arg Leu Arg Lys Phe Met Val Ser Cys Ala Val 2020 2025 2030gat gat tgt gag agg aga tca gag gat tgg tct att tgc gga gag agc 6144Asp Asp Cys Glu Arg Arg Ser Glu Asp Trp Ser Ile Cys Gly Glu Ser 2035 2040 2045aat cga aga agg tga gga gct ttt ctt cga cta ctg cta tgg acc aga 6192Asn Arg Arg Arg Gly Ala Phe Leu Arg Leu Leu Leu Trp Thr Arg 2050 2055 2060aca tgc gga ttg gtc gcg tgg tcg aga acc tag aaa gac tgg tgc ttc 6240Thr Cys Gly Leu Val Ala Trp Ser Arg Thr Lys Asp Trp Cys Phe2065 2070 2075 2080taa aag gtc taa gga agc ccg tcc agc tcg tta gtt ttt gat ctg agg 6288 Lys Val Gly Ser Pro Ser Ser Ser Leu Val Phe Asp Leu Arg 2085 2090 2095aga agc agc aat tca agc agt cct ttt ttt atg tta tgg tat atc aat 6336Arg Ser Ser Asn Ser Ser Ser Pro Phe Phe Met Leu Trp Tyr Ile Asn 2100 2105 2110taa taa tgt aat gct att ttg tgt tac taa acc aaa act taa gtt tct 6384 Cys Asn Ala Ile Leu Cys Tyr Thr Lys Thr Val Ser 2115 2120 2125gtt tta ttt gtt tta ggg tgt ttt gtt tgt atc ata tgt gtc tta act 6432Val Leu Phe Val Leu Gly Cys Phe Val Cys Ile Ile Cys Val Leu Thr 2130 2135 2140ttc aaa gtt ttc ttt ttg tat ttc aat tta aaa aca atg ttt atg ttg 6480Phe Lys Val Phe Phe Leu Tyr Phe Asn Leu Lys Thr Met Phe Met Leu2145 2150 2155 2160tta gtt tgc ata gac ctt tgg aaa aaa aaa gct ttg cac aac ttt aca 6528Leu Val Cys Ile Asp Leu Trp Lys Lys Lys Ala Leu His Asn Phe Thr 2165 2170 2175ttt att tag tct tca ttt agc gaa aaa tca cat aac aca agt ctg tgg 6576Phe Ile Ser Ser Phe Ser Glu Lys Ser His Asn Thr Ser Leu Trp 2180 2185 2190tac gta atg tac aaa aat gtc aaa ata atg ggt ttt atc att aaa aaa 6624Tyr Val Met Tyr Lys Asn Val Lys Ile Met Gly Phe Ile Ile Lys Lys 2195 2200 2205aaa tat tgg tta tga atg aag tat agt tag aat ttt agg tat tag ctc 6672Lys Tyr Trp Leu Met Lys Tyr Ser Asn Phe Arg Tyr Leu 2210 2215 2220gtt tgg ttt taa aac gtt ttt cga gat tta att ttg tag tct att gag 6720Val Trp Phe Asn Val Phe Arg Asp Leu Ile Leu Ser Ile Glu2225 2230 2235 2240taa tac atg gaa gaa tca tca aca aag tgg ctg tag ctt acg aaa ggt 6768 Tyr Met Glu Glu Ser Ser Thr Lys Trp Leu Leu Thr Lys Gly 2245 2250 2255ttt act tta atg taa ata tgt att tga tgc atc taa cat tta gta tct 6816Phe Thr Leu Met Ile Cys Ile Cys Ile His Leu Val Ser 2260 2265 2270aaa caa ata aaa aca aaa aaa aag aaa aaa gct ctt taa aat ccg aaa 6864Lys Gln Ile Lys Thr Lys Lys Lys Lys Lys Ala Leu Asn Pro Lys 2275 2280 2285gta act att ttc aaa aaa tct aaa tta taa act taa atg ttt gga atc 6912Val Thr Ile Phe Lys Lys Ser Lys Leu Thr Met Phe Gly Ile 2290 2295 2300gcg aac gac tat tgc taa ata taa atg cta aat ata cat gaa gat gtg 6960Ala Asn Asp Tyr Cys Ile Met Leu Asn Ile His Glu Asp Val2305 2310 2315 2320aaa aac atg ttg gat ttg tgg aat cgt taa tga cca cgg tta aat ggc 7008Lys Asn Met Leu Asp Leu Trp Asn Arg Pro Arg Leu Asn Gly 2325 2330 2335ggg atc c 7015Gly Ile 7 34 PRT Arabidopsis sp. 7Gly Ser Ile Ile Phe Lys Asn Gln Ile Phe Ser Tyr Leu Leu Phe Val 1 5 10 15Ser Lys Lys Lys Lys Thr His Asp Asp Tyr Pro Ser Ala Gly Cys Val 20 25 30His Arg 8 12 PRT Arabidopsis sp. 8Thr Tyr Ile Leu Lys Leu Val Gly Phe Ser Leu Pro 1 5 10 9 18 PRT Arabidopsis sp. 9Val Trp Thr Cys Phe Tyr Asn Leu Met Tyr Ser Val Asp Gln Lys Ile 1 5 10 15Glu Lys 10 6 PRT Arabidopsis sp. 10Glu Arg Glu Pro Leu Trp 1 5 11 20 PRT Arabidopsis sp. 11Gln Asn Arg Asn His Tyr Ile Glu Ser Phe Glu Lys Thr Lys Arg Ser 1 5 10 15Asn Leu Cys Ser 20 12 15 PRT Arabidopsis sp. 12Met Thr Ile Asp Val Ala Ala Asn Tyr Ser Leu Asn Ala Phe Ile 1 5 10 15 13 307 PRT Arabidopsis sp. 13Ile Phe Leu Thr Ser Ser Val Pro Ser His Ser Arg Asn Ser Ile Ile 1 5 10 15Pro Phe Ser Phe Phe Phe Ser Val Phe Gln Ser Leu Arg Ile Lys Met 20 25 30Glu His Glu Glu Thr Gln Lys Asn Thr Arg Asn Ser Trp Ser Leu Ile 35 40 45Arg Pro Phe Gln Met Ile Ser Ile Ser Phe Leu Ser Leu Leu Leu Pro 50 55 60Leu Ser Phe Leu Phe Leu Ser Arg Leu Ser Leu Tyr Thr Ser Ser Thr 65 70 75 80Pro Val Thr Val Ser Gly Val Ser Ser Val Ile His Gln Ala Asp Val 85 90 95Gly Val Leu Tyr Thr Ile Leu Phe Leu Ile Ile Val Phe Thr Leu Ile 100 105 110His Ser Leu Ser Gly Lys Pro Glu Cys Ser Val Leu His Ser His Leu 115 120 125Tyr Ile Cys Trp Ile Val Leu Phe Ile Ala Gln Ala Cys Ala Phe Gly 130 135 140Ile Lys Arg Thr Met Ser Thr Thr Met Ser Ile Asn Pro Asp Lys Asn145 150 155 160Leu Phe Leu Ala Thr His Glu Arg Trp Met Leu Val Arg Val Leu Phe 165 170 175Phe Leu Gly Leu His Glu Val Met Leu Met Trp Phe Arg Val Val Val 180 185 190Lys Pro Val Val Asp Asn Thr Ile Tyr Gly Val Tyr Val Glu Glu Arg 195 200 205Trp Ser Glu Arg Ala Val Val Ala Val Thr Phe Gly Ile Met Trp Trp 210 215 220Trp Arg Leu Arg Asp Glu Val Glu Ser Leu Val Val Val Val Thr Ala225 230 235 240Asp Arg Leu Asn Leu Pro Ile Arg Leu Glu Gly Leu Asn Phe Val Asn 245 250 255Trp Cys Met Tyr Tyr Ile Cys Val Gly Ile Gly Leu Met Lys Ile Phe 260 265 270Lys Gly Phe Leu Asp Phe Val Asn Thr Leu Thr Leu Ser Ile Lys Arg 275 280 285Ser Arg Lys Gly Cys Glu Ser Cys Val Phe Asp Asp Met Cys Asn Asp 290 295 300Asp His Val305 14 6 PRT Arabidopsis sp. 14His Ile Ile Leu Ile Ser 1 5 15 42 PRT Arabidopsis sp. 15Met Phe Leu Arg Phe Phe Tyr Phe Tyr Phe Leu Phe Leu Ala Arg Asn 1 5 10 15Leu Thr Arg Ile Tyr Val Thr Lys Ile Val Glu Tyr Gln Lys Ala Lys 20 25 30Ile Phe Tyr Leu Lys Ile Thr Ile Glu His 35 40 16 14 PRT Arabidopsis sp. 16Phe Lys Ser Phe Tyr Asn Tyr Ile Phe Ile Thr His Pro Phe 1 5 10 17 26 PRT Arabidopsis sp. 17Glu Lys Leu Gly Asp Leu Ile Asn Val Ile Asn Ser Lys Lys Tyr Arg 1 5 10 15Ile Tyr Val Glu Val Leu Asn Ala Tyr Asn 20 25 18 15 PRT Arabidopsis sp. 18Ile Tyr Glu Leu Asn Asn Ile Ala Ile Tyr Ile Phe Leu Lys Ile 1 5 10 15 19 9 PRT Arabidopsis sp. 19Thr His Phe Val Ser Ser Ile Tyr Ala 1 5 20 13 PRT Arabidopsis sp. 20Tyr Ile Ser Leu Asn Arg Lys Leu Ala Arg Asn Glu Tyr 1 5 10 21 31 PRT Arabidopsis sp. 21Tyr Lys Ser Tyr Arg Thr Leu Gln Asn Val Tyr Ile Asp Leu Ser Thr 1 5 10 15Phe Phe His Trp Phe Thr Lys Pro Ser Cys His Ile Asn Met Ser 20 25 30 22 31 PRT Arabidopsis sp. 22Arg Leu Phe Phe Tyr Asn Ile Val Tyr Glu Phe Lys Leu Glu Leu Ser 1 5 10 15Asn Val Lys Gln Thr Gln His Leu His Thr Tyr Ser Thr Ile Phe 20 25 30 23 5 PRT Arabidopsis sp. 23Lys Leu Lys Phe Ser 1 5 24 23 PRT Arabidopsis sp. 24Ile Ser His Ile Ile Phe Leu Leu Lys Gln Ala Ser Pro Asn Thr Phe 1 5 10 15Leu Pro Asp Tyr Asn Phe Pro 20 25 34 PRT Arabidopsis sp. 25Gly Phe Leu Gln Lys Lys Ile Asn Phe Leu Phe Lys Lys Pro Phe Ala 1 5 10 15Leu Ser Phe Ser Pro Thr Ser Glu Lys Thr Arg Lys Lys Glu Glu Ala 20 25 30Ser Gly 26 12 PRT Arabidopsis sp. 26Trp Arg Arg Leu Val Ser Leu Gln Thr Tyr Met Asn 1 5 10 27 8 PRT Arabidopsis sp. 27Leu Gly Tyr Glu Ile His Ile Phe 1 5 28 25 PRT Arabidopsis sp. 28Leu Cys Val Tyr Asp Arg Ser Ile Thr Phe Arg Val Glu Phe Ser Cys 1 5 10 15Asp Leu Leu Cys Tyr Ser Ser His Ala 20 25 29 24 PRT Arabidopsis sp. 29Ser Ile Lys Leu Leu Phe Leu Asn Leu Ser Arg Lys Thr Met Arg Thr 1 5 10 15Met Val Arg Val Cys His Pro Asn 20 30 13 PRT Arabidopsis sp. 30Lys Ser Lys Ser Lys Arg Arg Asp Phe Cys Ile Ser Arg 1 5 10 31 18 PRT Arabidopsis sp. 31Glu Thr Phe Gly Cys Phe Asn Ile Leu Phe Ser Ser Val Cys Phe Ser 1 5 10 15Glu Asn 32 5 PRT Arabidopsis sp. 32Gly Glu Glu Arg Thr 1 5 33 10 PRT Arabidopsis sp. 33Ser His Asn Tyr Thr Ile Pro Lys Arg Cys 1 5 10 34 13 PRT Arabidopsis sp. 34Asp Thr Ser Asn Lys Gln Leu Tyr Ile Ser His Asn Leu 1 5 10 35 7 PRT Arabidopsis sp. 35Lys Glu Lys Phe Pro Asn Phe 1 5 36 5 PRT Arabidopsis sp. 36Ile Lys Asn Arg Ile 1 5 37 6 PRT Arabidopsis sp. 37Lys Met Pro Ala Asn Arg 1 5 38 12 PRT Arabidopsis sp. 38Arg His Pro Asp Leu Ser Gly Ile Gln Asn Leu Glu 1 5 10 39 11 PRT Arabidopsis sp. 39Tyr Ile Tyr Asn Ile Lys Leu Glu Leu Arg Leu 1 5 10 40 16 PRT Arabidopsis sp. 40Asn Lys Ile Glu Asn Asn Ser Arg Phe Phe Cys Phe Cys Gln Thr Lys 1 5 10 15 41 10 PRT Arabidopsis sp. 41Tyr Asn Leu Phe Phe Leu Val Gln Arg Asn 1 5 10 42 33 PRT Arabidopsis sp. 42Ile Gly Pro Asn Cys Phe Phe Phe Asn Ile Gln Pro Lys Lys Pro Arg 1 5 10 15Leu Met His Ile Ser Arg Asn Arg Asn Gln Asn Phe Cys Ile Gln Val 20 25 30Phe 43 4 PRT Arabidopsis sp. 43Phe His Tyr Ile 1 44 6 PRT Arabidopsis sp. 44Ser Pro Val Ser Glu Ile 1 5 45 8 PRT Arabidopsis sp. 45Lys Ile Ile Tyr Leu Tyr Ile Thr 1 5 46 23 PRT Arabidopsis sp. 46Leu Thr Glu Lys Ile Arg Ala Glu Ile His Ser Lys Cys Gly Tyr Ser 1 5 10 15Cys Phe Thr Pro Ser Ile Val 20 47 8 PRT Arabidopsis sp. 47Leu Lys Pro Ala Arg Cys Arg Gly 1 5 48 22 PRT Arabidopsis sp. 48Trp Arg Arg Gln Gln Ile Thr Phe Val Glu Asn Ala Lys Pro Thr Ser 1 5 10 15Ser Phe Gln Cys Leu Ile 20 49 9 PRT Arabidopsis sp. 49Phe Leu Arg Arg Ser Arg Leu Cys Ser 1 5 50 6 PRT Arabidopsis sp. 50Gly Ser Arg Leu Cys Ser 1 5 51 7 PRT Arabidopsis sp. 51Arg Arg Cys Thr Ile Ile Ser 1 5 52 13 PRT Arabidopsis sp. 52Arg Cys Thr Ile Ile Thr Lys Cys Gln Ala Ser Asn Cys 1 5 10 53 21 PRT Arabidopsis sp. 53Glu Ala Thr Thr Ile His Tyr Met Gly Leu His Gln Lys Ala Cys Val 1 5 10 15Phe Phe Val Ser Tyr 20 54 13 PRT Arabidopsis sp. 54Phe Gln Asn Ile Asn His Ile Leu Tyr Ser Asn His Ser 1 5 10 55 6 PRT Arabidopsis sp. 55Cys Ile Tyr Thr Phe Leu 1 5 56 74 PRT Arabidopsis sp. 56His Cys Ser Ser Gln Leu Met Ala Glu Ser Asp Ser Val Ile Gly Lys 1 5 10 15Arg Gln Ile Tyr Tyr Leu Asn Gly Glu Ala Leu Glu Leu Ser Ser Glu 20 25 30Glu Asp Glu Glu Asp Glu Glu Glu Asp Glu Glu Glu Ile Lys Lys Glu 35 40 45Lys Cys Glu Phe Ser Glu Asp Val Asp Arg Phe Ile Trp Leu Val Phe 50 55 60Ala Leu His Met Phe Leu Ile Ile Asn Leu 65 70 57 41 PRT Arabidopsis sp. 57Ser Ile Phe Asn Lys Leu Leu Lys Lys Phe Ser Gly Arg Leu Gly Arg 1 5 10 15Thr Met Val Trp Met Ile Trp Ser Cys Gly Val Leu Ser Pro Ser Thr 20 25 30Ser Lys Trp Met Phe Arg Thr Tyr Trp 35 40 58 17 PRT Arabidopsis sp. 58Gln Tyr Ser Asn Lys Asn Phe Ile Arg Arg Ser Ile Thr Phe Leu Leu 1 5 10 15Ile 59 15 PRT Arabidopsis sp. 59Phe Leu Leu Phe Phe Val Val Arg Asn Val Leu Asn Phe Gln Ile 1 5 10 15 60 21 PRT Arabidopsis sp. 60Cys Arg Lys Asp Thr Met Asn Ser Ser Leu Arg Met Met Glu Leu Leu 1 5 10 15Val Arg Leu Leu Ile 20 61 4 PRT Arabidopsis sp. 61His Pro Arg Gln 1 62 17 PRT Arabidopsis sp. 62Leu Leu Leu Ser Arg Ile Leu Leu Ile Asp Val Ile Ala Val Val Ala 1 5 10 15Trp 63 4 PRT Arabidopsis sp. 63Ile Phe Leu Phe 1 64 7 PRT Arabidopsis sp. 64Phe Ser His Lys Lys Gly Arg 1 5 65 20 PRT Arabidopsis sp. 65Ser Tyr Met Phe Leu Phe Tyr Phe Ile Ile Cys Phe Thr Asp Ile Arg 1 5 10 15Leu Ser Tyr Ala 20 66 4 PRT Arabidopsis sp. 66Ile Arg Lys His 1 67 17 PRT Arabidopsis sp. 67Ile His Leu Asn Tyr Phe Val Ser Phe Thr Thr Leu Ile Tyr Lys Val 1 5 10 15Lys 68 12 PRT Arabidopsis sp. 68Leu Asp Cys Phe Gly Leu Ser Glu Arg Arg Gln Ile 1 5 10 69 4 PRT Arabidopsis sp. 69Thr Thr Met Gln 1 70 25 PRT Arabidopsis sp. 70Ala Leu Leu Pro Gln Gly Leu Tyr Leu Ser Pro Ser Leu Ser Gln Phe 1 5 10 15Phe Cys Leu Phe Leu Asn Tyr Val Tyr 20 25 71 8 PRT Arabidopsis sp. 71Ile Gly Glu Glu Cys Asp Arg Ser 1 5 72 4 PRT Arabidopsis sp. 72Ser Cys Asp Gly 1 73 28 PRT Arabidopsis sp. 73Leu Tyr Ile Lys Gln Asp Cys Gly Leu Arg Ser Lys Gln His Tyr Val 1 5 10 15Asp Ala Cys Arg Glu Gly Ser Leu Leu Glu Arg Asn 20 25 74 9 PRT Arabidopsis sp. 74Asp Ile Trp Glu Lys Gln Val Lys Lys 1 5 75 18 PRT Arabidopsis sp. 75Cys Ile Asn Ile Tyr Thr Tyr Thr Val Phe Leu Asp Tyr Ala Gly Ser 1 5 10 15Gln Leu 76 9 PRT Arabidopsis sp. 76Cys Cys Ile Lys His Thr Ser Gly Ala 1 5 77 21 PRT Arabidopsis sp. 77Asp Val Pro Arg Asp Leu Gln Leu His Ala Arg Thr Arg Ser Met Tyr 1 5 10 15Tyr Val Ile Arg Pro 20 78 32 PRT Arabidopsis sp. 78Gln Asn Tyr Thr Lys Thr Gln Ser Gly Thr Leu Thr Tyr Val Val Ile 1 5 10 15Ile Leu Met Thr Cys Met Leu Lys Thr His Glu Val Ser Tyr Met Cys 20 25 30 79 33 PRT Arabidopsis sp. 79Trp Phe Tyr His Arg Leu Pro Lys Lys Tyr Leu Glu Lys Val Val Gly 1 5 10 15Arg Ser Ala Lys Asn Arg Asp Ser Glu Asn Met Leu Val Ile Arg Leu 20 25 30Leu 80 29 PRT Arabidopsis sp. 80Arg Lys Gln Leu Val Glu Lys Leu Ser Phe Ile Ser Thr Thr His His 1 5 10 15Ala Leu Ala Ser Gln Asn Val Asp Ser Asn Ala Leu Val 20 25 81 17 PRT Arabidopsis sp. 81Leu Thr Lys Ile Ala Ala Arg Asn Ile Ala Gly Met Ser Phe Asn Phe 1 5 10 15Ser 82 62 PRT Arabidopsis sp. 82Ala Gly Arg Ser Met Arg Phe Asn Leu Asn Met Ser Leu Tyr Phe Leu 1 5 10 15Phe Arg Cys Ser Lys Asp Cys Asn Asn Arg Phe Gly Gly Cys Asn Cys 20 25 30Ala Ile Gly Gln Cys Thr Asn Arg Gln Cys Pro Cys Phe Ala Ala Asn 35 40 45Arg Glu Cys Asp Pro Asp Leu Cys Arg Ser Cys Pro Leu Arg 50 55 60 83 66 PRT Arabidopsis sp. 83His Phe His Phe Asn Ile Ser Leu Tyr Lys Phe Tyr Asn Gln Ser Asn 1 5 10 15Ser Asn Gln Lys Ser Tyr Lys Lys Asn Phe Ile Tyr Ser Cys Gly Asp 20 25 30Gly Thr Leu Gly Glu Thr Pro Val Gln Ile Gln Cys Lys Asn Met Gln 35 40 45Phe Leu Leu Gln Thr Asn Lys Lys Val Ile Asn Val Lys Ser Val Pro 50 55 60Lys Ile 65 84 20 PRT Arabidopsis sp. 84Leu Tyr Glu Arg His Leu Thr Ile Ile Ser Arg Ile Leu Leu Asp Ser 1 5 10 15His Trp Lys Val 20 85 41 PRT Arabidopsis sp. 85Cys Ser Trp Met Gly Cys Ile Tyr Met Gly Lys Gln Ser Cys Lys Tyr 1 5 10 15Lys Asn Lys Phe Asn Ser Tyr Trp Cys Ile His Asn Thr Phe Phe Phe 20 25 30Leu Ile Met Phe Tyr Thr Leu Asp His 35 40 86 13 PRT Arabidopsis sp. 86Ile Tyr Cys Val Ile Trp Phe Asp Pro Ser Gly Leu Ser 1 5 10 87 12 PRT Arabidopsis sp. 87Val Ser Arg Arg Ile Tyr Trp Arg Thr Asp His Ser 1 5 10 88 17 PRT Arabidopsis sp. 88Ala Trp Glu Asn Arg Arg Ser Asp Trp Phe Phe Leu Pro Leu Tyr Leu 1 5 10 15Glu 89 9 PRT Arabidopsis sp. 89Ser Gly Asn Phe Arg Ile Ile Leu Lys 1 5 90 14 PRT Arabidopsis sp. 90Arg Phe Asn His Ser Arg Val Thr His Leu Phe Glu Ser Lys 1 5 10 91 32 PRT Arabidopsis sp. 91His Leu Phe Tyr Ser Ser Lys Ser Met Leu Ala Val Lys Glu Thr Ser 1 5 10 15Ser Asn Phe Ser Ile Thr Gln Gln Asp Leu Thr Ala Thr Pro Arg Tyr 20 25 30 92 19 PRT Arabidopsis sp. 92Ala Val Ile Leu Tyr Leu Glu Gln Ile Leu Thr Leu Tyr Lys Gln Lys 1 5 10 15Tyr Leu Cys 93 15 PRT Arabidopsis sp. 93Leu Asn Arg Val Ser Thr Leu Leu Val Val Asp Trp Phe Ser Tyr 1 5 10 15 94 50 PRT Arabidopsis sp. 94Arg Tyr Ser Lys Lys Leu Lys Leu Ile Leu Asn Asp Phe Phe Leu Ser 1 5 10 15Arg Lys Phe Arg Leu Arg Lys Phe Met Val Ser Cys Ala Val Asp Asp 20 25 30Cys Glu Arg Arg Ser Glu Asp Trp Ser Ile Cys Gly Glu Ser Asn Arg 35 40 45Arg Arg 50 95 21 PRT Arabidopsis sp. 95Gly Ala Phe Leu Arg Leu Leu Leu Trp Thr Arg Thr Cys Gly Leu Val 1 5 10 15Ala Trp Ser Arg Thr 20 96 5 PRT Arabidopsis sp. 96Lys Asp Trp Cys Phe 1 5 97 28 PRT Arabidopsis sp. 97Gly Ser Pro Ser Ser Ser Leu Val Phe Asp Leu Arg Arg Ser Ser Asn 1 5 10 15Ser Ser Ser Pro Phe Phe Met Leu Trp Tyr Ile Asn 20 25 98 7 PRT Arabidopsis sp. 98Cys Asn Ala Ile Leu Cys Tyr 1 5 99 52 PRT Arabidopsis sp. 99Val Ser Val Leu Phe Val Leu Gly Cys Phe Val Cys Ile Ile Cys Val 1 5 10 15Leu Thr Phe Lys Val Phe Phe Leu Tyr Phe Asn Leu Lys Thr Met Phe 20 25 30Met Leu Leu Val Cys Ile Asp Leu Trp Lys Lys Lys Ala Leu His Asn 35 40 45Phe Thr Phe Ile 50 100 33 PRT Arabidopsis sp. 100Ser Ser Phe Ser Glu Lys Ser His Asn Thr Ser Leu Trp Tyr Val Met 1 5 10 15Tyr Lys Asn Val Lys Ile Met Gly Phe Ile Ile Lys Lys Lys Tyr Trp 20 25 30Leu 101 4 PRT Arabidopsis sp. 101Met Lys Tyr Ser 1 102 4 PRT Arabidopsis sp. 102Asn Phe Arg Tyr 1 103 4 PRT Arabidopsis sp. 103Leu Val Trp Phe 1 104 8 PRT Arabidopsis sp. 104Asn Val Phe Arg Asp Leu Ile Leu 1 5 105 10 PRT Arabidopsis sp. 105Tyr Met Glu Glu Ser Ser Thr Lys Trp Leu 1 5 10 106 8 PRT Arabidopsis sp. 106Leu Thr Lys Gly Phe Thr Leu Met 1 5 107 16 PRT Arabidopsis sp. 107His Leu Val Ser Lys Gln Ile Lys Thr Lys Lys Lys Lys Lys Ala Leu 1 5 10 15 108 12 PRT Arabidopsis sp. 108Asn Pro Lys Val Thr Ile Phe Lys Lys Ser Lys Leu 1 5 10 109 9 PRT Arabidopsis sp. 109Met Phe Gly Ile Ala Asn Asp Tyr Cys 1 5 110 17 PRT Arabidopsis sp. 110Met Leu Asn Ile His Glu Asp Val Lys Asn Met Leu Asp Leu Trp Asn 1 5 10 15Arg 111 7 PRT Arabidopsis sp. 111Pro Arg Leu Asn Gly Gly Ile 1 5 112 38 PRT Arabidopsis sp. 112Asp Pro Leu Phe Leu Lys Ile Lys Phe Phe His Ile Tyr Tyr Leu Phe 1 5 10 15Gln Arg Lys Lys Lys His Thr Thr Ile Ile His Leu Pro Ala Val Phe 20 25 30Ile Gly Lys Pro Ile Phe 35 113 16 PRT Arabidopsis sp. 113Asn Trp Trp Ala Phe His Tyr His Lys Phe Gly His Val Phe Ile Ile 1 5 10 15 114 33 PRT Arabidopsis sp. 114Arg Asn Lys Lys Gly Asn Leu Cys Gly Asp Cys Asn Lys Thr Glu Ile 1 5 10 15Ile Ile Leu Asn His Ser Lys Arg Arg Lys Asp Gln Thr Phe Val Ala 20 25 30Arg 115 59 PRT Arabidopsis sp. 115Thr Trp Leu Pro Ile Thr Val Leu Met Leu Leu Tyr Arg Ser Phe Leu 1 5 10 15His Pro Leu Phe Leu His Ile Gln Glu Thr Val Ser Ser His Phe Leu 20 25 30Ser Ser Ser Gln Cys Phe Asn Leu Cys Glu Leu Arg Trp Asn Met Lys 35 40 45Lys His Lys Arg Thr Gln Glu Thr Ala Gly Pro 50 55 116 5 PRT Arabidopsis sp. 116Phe Asp His Phe Lys 1 5 117 57 PRT Arabidopsis sp. 117Ser Pro Leu Ala Phe Leu Ala Ser Ser Ser Leu Tyr Leu Ser Ser Phe 1 5 10 15Phe His Val Ser Leu Ser Ile Pro Pro Gln Leu Arg Ser Pro Ser Pro 20 25 30Ala Phe Pro Leu Leu Phe Thr Arg Gln Met Ser Glu Ser Tyr Thr Arg 35 40 45Ser Cys Phe Ser Ser Ser Ser Ser Leu 50 55 118 37 PRT Arabidopsis sp. 118Ser Thr Val Ser Gln Glu Asn Gln Asn Ala Leu Phe Ser Ile Pro Ile 1 5 10 15Ser Thr Ser Ala Gly Ser Phe Ser Ser Ser Pro Lys Leu Val Pro Leu 20 25 30Gly Ser Lys Glu Pro 35 119 5 PRT Arabidopsis sp. 119Ala Arg Pro Cys Leu 1 5 120 27 PRT Arabidopsis sp. 120Ile Gln Thr Lys Thr Cys Phe Leu Arg His Met Lys Asp Gly Cys Trp 1 5 10 15Leu Gly Phe Cys Ser Phe Trp Gly Tyr Thr Lys 20 25 121 31 PRT Arabidopsis sp. 121Cys Gly Leu Glu Ser Trp Leu Ser Leu Trp Leu Thr Thr Leu Tyr Met 1 5 10 15Gly Ser Thr Trp Arg Arg Gly Gly Pro Arg Glu Pro Leu Trp Gln 20 25 30 122 5 PRT Arabidopsis sp. 122Cys Gly Gly Gly Gly 1 5 123 23 PRT Arabidopsis sp. 123Lys Val Leu Trp Trp Trp Leu Arg Arg Ile Asp Leu Thr Ser Pro Phe 1 5 10 15Val Trp Arg Val Ser Ile Leu 20 124 12 PRT Arabidopsis sp. 124Thr Gly Val Cys Ile Thr Ser Val Leu Glu Leu Val 1 5 10 125 9 PRT Arabidopsis sp. 125Arg Ser Ser Lys Gly Phe Trp Ile Leu 1 5 126 36 PRT Arabidopsis sp. 126Ala Leu Arg Gly Arg Glu Lys Ala Val Asn His Val Phe Leu Met Ile 1 5 10 15Cys Val Met Met Ile Met Cys Lys Ile Phe Asp Ile Leu Tyr Ser Ser 20 25 30Leu Glu Cys Phe 35 127 13 PRT Arabidopsis sp. 127Asp Phe Phe Ile Phe Ile Phe Tyr Phe Leu Leu Gly Ile 1 5 10 128 7 PRT Arabidopsis sp. 128Pro Val Tyr Met Ser Gln Lys 1 5 129 9 PRT Arabidopsis sp. 129Asn Ile Arg Lys Gln Lys Tyr Phe Ile 1 5 130 14 PRT Arabidopsis sp. 130Pro Leu Asn Ile Asn Leu Ser Leu Phe Ile Ile Ile Phe Leu 1 5 10 131 10 PRT Arabidopsis sp. 131His Thr Leu Phe Lys Lys Asn Leu Glu Ile 1 5 10 132 8 PRT Arabidopsis sp. 132Ile Val Lys Asn Ile Gly Phe Thr 1 5 133 8 PRT Arabidopsis sp. 133Met Arg Ile Ile Lys Phe Thr Asn 1 5 134 5 PRT Arabidopsis sp. 134Pro Tyr Ile Tyr Phe 1 5 135 14 PRT Arabidopsis sp. 135Arg Phe Lys Leu Ile Leu Phe Leu Pro Tyr Met His Asn Ile 1 5 10 136 39 PRT Arabidopsis sp. 136Leu Gly Met Asn Thr Asn Ile Tyr Asn Asp Ile Asn Ile Ser Leu Thr 1 5 10 15Gly His Ser Lys Met Tyr Ile Leu Ile Tyr Gln His Phe Phe Ile Gly 20 25 30Leu Leu Asn Gln Val Val Thr 35 137 35 PRT Arabidopsis sp. 137Val Asn Ala Phe Phe Phe Ile Ile Leu Tyr Met Asn Leu Asn Leu Ser 1 5 10 15Cys Gln Thr Ser Ser Lys Pro Asn Ile Tyr Ile His Ile Val Leu Tyr 20 25 30Phe Glu Asn 35 138 11 PRT Arabidopsis sp. 138Asn Phe Leu Lys Phe Pro Ile Leu Phe Ser Phe 1 5 10 139 55 PRT Arabidopsis sp. 139Ser Lys Gln Val Gln Ile Arg Phe Phe Gln Ile Ile Ile Phe Leu Asn 1 5 10 15Lys Val Phe Tyr Lys Lys Lys Ser Thr Ser Tyr Leu Lys Asn Pro Leu 20 25 30His Tyr Pro Phe His Gln His Gln Arg Arg Arg Glu Lys Lys Lys Arg 35 40 45Arg Val Val Asn Gly Glu Gly 50 55 140 6 PRT Arabidopsis sp. 140Phe His Ser Lys His Ile 1 5 141 15 PRT Arabidopsis sp. 141Val Met Lys Ser Ile Tyr Phe Asn Cys Val Phe Met Ile Asp Gln 1 5 10 15 142 19 PRT Arabidopsis sp. 142His Leu Gly Leu Asn Phe Leu Val Ile Tyr Tyr Val Ile Arg Pro Met 1 5 10 15His Asp Pro 143 4 PRT Arabidopsis sp. 143Asn Phe Tyr Phe 1 144 6 PRT Arabidopsis sp. 144Ile Cys Leu Gly Lys Pro 1 5 145 107 PRT Arabidopsis sp. 145Gly Phe Ala Thr Arg Thr Lys Ser Asp Lys Arg Ala Asn Arg Lys Gly 1 5 10 15Glu Ile Ser Ala Tyr Gln Gly Lys Arg His Leu Val Ala Leu Ile Phe 20 25 30Tyr Ser Leu Leu Tyr Val Phe Leu Lys Ile Lys Glu Arg Arg Gly Leu 35 40 45Asn Leu Ile Thr Ile Arg Phe Gln Arg Asp Val Lys Ile His Leu Ile 50 55 60Asn Ser Tyr Thr Leu Val Ile Ile Phe Lys Thr Lys Lys Arg Asn Phe 65 70 75 80Gln Thr Phe Lys Leu Lys Thr Glu Phe Arg Lys Cys Gln Arg Ile Asp 85 90 95Asn Asp Ile Gln Ile Cys Arg Val Ser Lys Thr 100 105 146 10 PRT Arabidopsis sp. 146Asn Lys Lys Ile Ile Asn Ile Phe Ile Ile 1 5 10 147 30 PRT Arabidopsis sp. 147Ser Trp Asn Leu Gly Tyr Lys Ile Lys Leu Lys Ile Ile Val Asp Phe 1 5 10 15Phe Val Phe Val Lys Gln Asn Ser Asn Thr Ile Cys Phe Phe 20 25 30 148 5 PRT Arabidopsis sp. 148Tyr Lys Glu Thr Lys 1 5 149 15 PRT Arabidopsis sp. 149Val Gln Ile Val Phe Phe Leu Thr Phe Ser Gln Lys Ser Gln Asp 1 5 10 15 150 38 PRT Arabidopsis sp. 150Cys Ile Tyr Gln Glu Ile Glu Ile Lys Thr Phe Val Phe Lys Tyr Ser 1 5 10 15Ser Phe Thr Ile Tyr Arg Val Gln Phe Leu Lys Phe Lys Lys Ser Phe 20 25 30Thr Tyr Ile Leu Leu Asp 35 151 147 PRT Arabidopsis sp. 151Gln Arg Lys Phe Glu Leu Arg Tyr Ile Pro Ser Val Ala Thr His Ala 1 5 10 15Ser His His Gln Ser Phe Asp Leu Asn Gln Pro Ala Ala Glu Asp Asp 20 25 30Asn Gly Gly Asp Asn Lys Ser Leu Leu Ser Arg Met Gln Asn Pro Leu 35 40 45Arg His Phe Ser Ala Ser Ser Asp Tyr Asn Ser Tyr Glu Asp Gln Gly 50 55 60Tyr Val Leu Asp Glu Asp Gln Asp Tyr Ala Leu Glu Glu Asp Val Pro 65 70 75 80Leu Phe Leu Asp Glu Asp Val Pro Leu Leu Pro Ser Val Lys Leu Pro 85 90 95Ile Val Glu Lys Leu Pro Arg Ser Ile Thr Trp Val Phe Thr Lys Arg 100 105 110His Val Cys Phe Leu Phe Arg Thr Ser Phe Lys Ile Leu Ile Ile Tyr 115 120 125Tyr Ile Val Ile Thr His Ser Ala Tyr Ile His Phe Phe Asn Ile Ala 130 135 140Val Ala Ser145 152 6 PRT Arabidopsis sp. 152Trp Leu Lys Val Ile Leu 1 5 153 8 PRT Arabidopsis sp. 153Leu Val Arg Asp Lys Ser Ile Ile 1 5 154 4 PRT Arabidopsis sp. 154Met Val Arg His 1 155 26 PRT Arabidopsis sp. 155Ala Val Lys Lys Met Arg Lys Met Lys Lys Lys Met Arg Lys Lys Ser 1 5 10 15Arg Lys Lys Asn Ala Asn Phe Leu Lys Met 20 25 156 5 PRT Arabidopsis sp. 156Thr Asp Leu Tyr Gly 1 5 157 7 PRT Arabidopsis sp. 157Phe Leu His Tyr Ile Cys Ser 1 5 158 25 PRT Arabidopsis sp. 158Leu Leu Ile Cys Ser Pro Tyr Leu Ile Asn Cys Ser Arg Asn Phe Gln 1 5 10 15Asp Gly Trp Ala Gly Leu Trp Phe Gly 20 25 159 32 PRT Arabidopsis sp. 159Ser Gly Arg Ala Ala Cys Ser Arg Gln Val Pro Arg Ser Gly Cys Phe 1 5 10 15Gly His Ile Gly Asn Asn Ile Arg Ile Lys Thr Ser Tyr Val Asp Gln 20 25 30 160 12 PRT Arabidopsis sp. 160Leu Ser Cys Leu Phe Asn Phe Cys Cys Phe Ser Ser 1 5 10 161 10 PRT Arabidopsis sp. 161Ile Phe Lys Ser Asn Val Gly Lys Ile Gln 1 5 10 162 4 PRT Arabidopsis sp. 162Trp Asn Cys Trp 1 163 14 PRT Arabidopsis sp. 163Phe Asp Ile Gln Asp Asn Asn Tyr Cys Phe Pro Gly Phe Cys 1 5 10 164 59 PRT Arabidopsis sp. 164Thr Ser Leu Pro Ser Leu His Gly Asn Phe Glu Ser Phe Phe Phe Asn 1 5 10 15Leu Ala Thr Lys Lys Gly Asp Asp His Thr Cys Phe Tyr Phe Ile Leu 20 25 30Ser Phe Val Leu Gln Ile Phe Asp Cys His Met His Glu Lys Tyr Glu 35 40 45Pro Glu Ser Arg Ser Val Ser Ile Lys Phe Ile 50 55 165 15 PRT Arabidopsis sp. 165Ile Ile Leu Leu Val Ser Gln Pro Leu Tyr Ile Arg Leu Ser Asp 1 5 10 15 166 56 PRT Arabidopsis sp. 166Ile Ala Leu Ala Cys Gln Ser Glu Asp Lys Ser Ser Leu Phe Glu Asp 1 5 10 15Glu Asp Arg Gln Pro Cys Ser Glu His Cys Tyr Leu Lys Val Ser Ile 20 25 30Ser Leu Pro Leu Ser Leu Asn Phe Phe Val Tyr Ser Leu Ile Thr Phe 35 40 45Ile Ser Tyr Trp Phe Asn Ile Lys 50 55 167 50 PRT Arabidopsis sp. 167Val Arg Ser Val Thr Glu Ala Asp His Val Met Asp Asn Asp Asn Ser 1 5 10 15Ile Ser Asn Lys Ile Val Val Ser Asp Pro Asn Asn Thr Met Trp Thr 20 25 30Pro Val Glu Lys Asp Leu Tyr Leu Lys Gly Ile Glu Ile Phe Gly Arg 35 40 45Asn Arg 50 168 68 PRT Arabidopsis sp. 168Lys Asn Lys Asn Arg Phe Asn Ala Leu Ile Tyr Ile Leu Thr Leu Tyr 1 5 10 15Ser Leu Ile Met Leu Val Arg Ser Cys Asp Val Ala Leu Asn Ile Leu 20 25 30Arg Gly Leu Lys Thr Cys Leu Glu Ile Tyr Asn Tyr Met Arg Glu Gln 35 40 45Asp Gln Cys Thr Met Ser Leu Asp Leu Asn Lys Thr Thr Gln Arg His 50 55 60Asn Gln Val His 65 169 23 PRT Arabidopsis sp. 169Lys His Met Lys Phe Pro Ile Cys Val Asp Gly Phe Ile Thr Gly Tyr 1 5 10 15Gln Lys Ser Ile Ser Lys Lys 20 170 22 PRT Arabidopsis sp. 170Val Gly Pro Gln Lys Ile Glu Thr Pro Lys Ile Cys Ser Leu Ser Ala 1 5 10 15Cys Phe Lys Glu Asn Asn 20 171 41 PRT Arabidopsis sp. 171Ala Leu His Thr Met His Leu Gln Val Lys Met Trp Thr Ala Met Pro 1 5 10 15Leu Phe Asn Ser Arg Lys Leu Leu Arg Glu Ile Leu Arg Val Cys His 20 25 30Ser Ile Phe Pro Lys Pro Glu Asp Pro 35 40 172 108 PRT Arabidopsis sp. 172Val Cys Ile Phe Cys Ser Gly Ala Gln Arg Ile Ala Thr Ile Ala Leu 1 5 10 15Glu Asp Val Ile Val Gln Leu Ala Asn Ala Gln Ile Asp Asn Val Leu 20 25 30Val Leu Leu Leu Ile Val Asn Ala Ile Gln Ile Phe Val Gly Val Val 35 40 45Leu Leu Gly Asn Thr Phe Thr Ser Ile Ser Leu Tyr Thr Asn Ser Ile 50 55 60Ile Lys Val Ile Gln Thr Lys Ser Leu Ile Lys Lys Thr Leu Tyr Ile 65 70 75 80Ala Val Glu Met Ala Leu Leu Val Arg His Gln Cys Lys Ser Asn Ala 85 90 95Arg Thr Cys Asn Ser Ser Phe Lys Pro Ile Lys Arg 100 105 173 17 PRT Arabidopsis sp. 173Ser Thr Ser Asn Pro Tyr Arg Lys Phe Lys Thr Asn Tyr Thr Lys Asp 1 5 10 15Ile 174 7 PRT Arabidopsis sp. 174Leu Ser Phe Pro Val Phe Tyr 1 5 175 39 PRT Arabidopsis sp. 175Ile Leu Ile Gly Lys Ser Asp Val His Gly Trp Gly Ala Phe Thr Trp 1 5 10 15Val Ser Asn His Val Asn Ile Arg Ile Ser Leu Ile Val Ile Gly Ala 20 25 30Phe Ile Thr Leu Phe Phe Phe 35 176 4 PRT Arabidopsis sp. 176Cys Phe Ile Leu 1 177 6 PRT Arabidopsis sp. 177Thr Ile Lys Tyr Ile Val 1 5 178 53 PRT Arabidopsis sp. 178Tyr Gly Leu Thr Arg Gln Asp Ser Leu Lys Lys Asn Glu Tyr Leu Gly 1 5 10 15Glu Tyr Thr Gly Glu Leu Ile Thr His Asp Glu Ala Asn Glu Arg Gly 20 25 30Arg Ile Glu Asp Arg Ile Gly Ser Ser Tyr Leu Phe Thr Leu Asn Asp 35 40 45Gln Val Thr Ser Glu 50 179 28 PRT Arabidopsis sp. 179Ser Asn Val Leu Ile Ile Arg Gly Leu His Ile Tyr Ser Asn Gln Ser 1 5 10 15Asn Ile Tyr Phe Thr Ala Arg Asn Arg Cys Ser Pro 20 25 180 13 PRT Arabidopsis sp. 180Arg Lys Arg Val Gln Ile Ser Gln Ser Leu Ser Lys Thr 1 5 10 181 16 PRT Arabidopsis sp. 181Leu Leu Arg Gln Gly Thr Lys Pro Leu Tyr Phe Ile Leu Asn Lys Tyr 1 5 10 15 182 13 PRT Arabidopsis sp. 182His Tyr Thr Asn Lys Asn Thr Tyr Val Ser Phe Phe Ser 1 5 10 183 24 PRT Arabidopsis sp. 183Ile Val Tyr Gln Leu Tyr Ser Ser Leu Ile Gly Phe His Ile Glu Asp 1 5 10 15Ile Pro Arg Asn Ser Asn Ser Phe 20 184 78 PRT Arabidopsis sp. 184Met Ile Phe Ser Cys Arg Glu Asn Leu Gly Tyr Glu Asn Leu Trp Phe 1 5 10 15Arg Val Gln Leu Met Ile Val Arg Gly Asp Gln Arg Ile Gly Leu Phe 20 25 30Ala Glu Arg Ala Ile Glu Glu Gly Glu Glu Leu Phe Phe Asp Tyr Cys 35 40 45Tyr Gly Pro Glu His Ala Asp Trp Ser Arg Gly Arg Glu Pro Arg Lys 50 55 60Thr Gly Ala Ser Lys Arg Ser Lys Glu Ala Arg Pro Ala Arg 65 70 75 185 37 PRT Arabidopsis sp. 185Gly Glu Ala Ala Ile Gln Ala Val Leu Phe Leu Cys Tyr Gly Ile Ser 1 5 10 15Ile Asn Asn Val Met Leu Phe Cys Val Thr Lys Pro Lys Leu Lys Phe 20 25 30Leu Phe Tyr Leu Phe 35 186 9 PRT Arabidopsis sp. 186Gly Val Leu Phe Val Ser Tyr Val Ser 1 5 187 10 PRT Arabidopsis sp. 187Leu Ser Lys Phe Ser Phe Cys Ile Ser Ile 1 5 10 188 6 PRT Arabidopsis sp. 188Lys Gln Cys Leu Cys Cys 1 5 189 29 PRT Arabidopsis sp. 189Thr Phe Gly Lys Lys Lys Leu Cys Thr Thr Leu His Leu Phe Ser Leu 1 5 10 15His Leu Ala Lys Asn His Ile Thr Gln Val Cys Gly Thr 20 25 190 6 PRT Arabidopsis sp. 190Cys Thr Lys Met Ser Lys 1 5 191 12 PRT Arabidopsis sp. 191Trp Val Leu Ser Leu Lys Lys Asn Ile Gly Tyr Glu 1 5 10 192 19 PRT Arabidopsis sp. 192Ser Ile Val Arg Ile Leu Gly Ile Ser Ser Phe Gly Phe Lys Thr Phe 1 5 10 15Phe Glu Ile 193 24 PRT Arabidopsis sp. 193Phe Cys Ser Leu Leu Ser Asn Thr Trp Lys Asn His Gln Gln Ser Gly 1 5 10 15Cys Ser Leu Arg Lys Val Leu Leu 20 194 10 PRT Arabidopsis sp. 194Cys Lys Tyr Val Phe Asp Ala Ser Asn Ile 1 5 10 195 4 PRT Arabidopsis sp. 195Tyr Leu Asn Lys 1 196 13 PRT Arabidopsis sp. 196Lys Gln Lys Lys Arg Lys Lys Leu Phe Lys Ile Arg Lys 1 5 10 197 24 PRT Arabidopsis sp. 197Leu Phe Ser Lys Asn Leu Asn Tyr Lys Leu Lys Cys Leu Glu Ser Arg 1 5 10 15Thr Thr Ile Ala Lys Tyr Lys Cys 20 198 5 PRT Arabidopsis sp. 198Ile Tyr Met Lys Met 1 5 199 13 PRT Arabidopsis sp. 199Lys Thr Cys Trp Ile Cys Gly Ile Val Asn Asp His Gly 1 5 10 200 4 PRT Arabidopsis sp. 200Met Ala Gly Ser 1 201 4 PRT Arabidopsis sp. 201Ile His Tyr Phe 1 202 48 PRT Arabidopsis sp. 202Lys Ser Asn Phe Phe Ile Ser Ile Ile Cys Phe Lys Glu Lys Lys Asn 1 5 10 15Thr Arg Arg Leu Ser Ile Cys Arg Leu Cys Ser Ser Val Asn Leu Tyr 20 25 30Phe Lys Thr Gly Gly Leu Phe Ile Thr Ile Ser Leu Asp Met Phe Leu 35 40 45 203 24 PRT Arabidopsis sp. 203Cys Arg Pro Lys Asn Arg Glu Ile Arg Lys Gly Thr Phe Val Val Ile 1 5 10 15Val Thr Lys Gln Lys Ser Leu Tyr 20 204 11 PRT Arabidopsis sp. 204Ile Ile Arg Lys Asp Glu Lys Ile Lys Pro Leu 1 5 10 205 12 PRT Arabidopsis sp. 205Leu Asp Asp His Arg Arg Gly Cys Gln Leu Gln Ser 1 5 10 206 34 PRT Arabidopsis sp. 206Cys Phe Tyr Ile Asp Leu Ser Tyr Ile Leu Cys Ser Phe Thr Phe Lys 1 5 10 15Lys Gln Tyr His Pro Ile Phe Phe Leu Leu Leu Ser Val Ser Ile Phe 20 25 30Ala Asn 207 21 PRT Arabidopsis sp. 207Arg Asn Thr Lys Glu His Lys Lys Gln Leu Val Pro Asp Ser Thr Ile 1 5 10 15Ser Asn Asp Leu His 20 208 106 PRT Arabidopsis sp. 208Pro Pro Pro Pro Ser Ile Phe Pro Leu Ser Phe Thr Ser Leu Ser Leu 1 5 10 15Tyr Leu Leu Asn Ser Gly His Arg Leu Arg Arg Phe Leu Cys Tyr Ser 20 25 30Pro Gly Arg Cys Arg Ser Leu Ile His Asp Leu Val Ser His His Arg 35 40 45Leu His Phe Asn Pro Gln Ser Leu Arg Lys Thr Arg Met Leu Cys Ser 50 55 60Pro Phe Pro Ser Leu His Leu Leu Asp Arg Ser Leu His Arg Pro Ser 65 70 75 80Leu Cys Leu Trp Asp Gln Lys Asn His Glu His Asp His Val Tyr Lys 85 90 95Ser Arg Gln Lys Leu Val Ser Cys Asp Thr 100 105 209 5 PRT Arabidopsis sp. 209Lys Met Asp Val Gly 1 5 210 15 PRT Arabidopsis sp. 210Gly Phe Val Leu Phe Gly Ala Thr Arg Ser Asp Ala Asp Val Val 1 5 10 15 211 32 PRT Arabidopsis sp. 211Gln His Tyr Ile Trp Gly Leu Arg Gly Gly Glu Val Val Arg Glu Ser 1 5 10 15Arg Cys Gly Ser Asp Leu Trp Tyr Asn Val Val Val Glu Ala Lys Arg 20 25 30 212 11 PRT Arabidopsis sp. 212Gly Arg Lys Ser Cys Gly Gly Gly Tyr Gly Gly 1 5 10 213 42 PRT Arabidopsis sp. 213Pro Pro His Ser Phe Gly Gly Ser Gln Phe Cys Glu Leu Val Tyr Val 1 5 10 15Leu His Leu Cys Trp Asn Trp Phe Asn Glu Asp Leu Gln Arg Val Phe 20 25 30Gly Phe Cys Glu Tyr Val Asp Phe Glu His 35 40 214 6 PRT Arabidopsis sp. 214Glu Val Glu Lys Arg Leu 1 5 215 4 PRT Arabidopsis sp. 215Ile Met Cys Phe 1 216 27 PRT Arabidopsis sp. 216Ser Cys Val Arg Tyr Leu Thr Tyr Tyr Thr His Leu Leu Asn Val Phe 1 5 10 15Glu Ile Phe Leu Phe Leu Phe Ser Ile Ser Cys 20 25 217 25 PRT Arabidopsis sp. 217Glu Phe Asn Pro Tyr Ile Cys His Lys Asn Ser Arg Ile Ser Glu Ser 1 5 10 15Lys Asn Ile Leu Ser Lys Asn Asn His 20 25 218 16 PRT Arabidopsis sp. 218Leu Tyr Phe Tyr Asn Thr Pro Phe Leu Arg Lys Thr Trp Arg Phe Asn 1 5 10 15 219 12 PRT Arabidopsis sp. 219Lys Ile Ser Asp Leu Arg Arg Ser Phe Lys Cys Val 1 5 10 220 6 PRT Arabidopsis sp. 220Leu Asn Leu Arg Ile Glu 1 5 221 25 PRT Arabidopsis sp. 221Tyr Ser His Ile Tyr Ile Phe Glu Asp Leu Asn Ser Phe Cys Phe Phe 1 5 10 15His Ile Cys Ile Ile Tyr Lys Leu Lys 20 25 222 9 PRT Arabidopsis sp. 222Ile Leu Ile Tyr Ile Met Thr Leu Ile 1 5 223 10 PRT Arabidopsis sp. 223Val Leu Pro Asp Thr Pro Lys Cys Ile Tyr 1 5 10 224 9 PRT Arabidopsis sp. 224Ser Ile Asn Ile Phe Ser Leu Val Tyr 1 5 225 14 PRT Arabidopsis sp. 225Thr Lys Leu Ser His Lys Tyr Glu Leu Thr Pro Phe Phe Leu 1 5 10 226 13 PRT Arabidopsis sp. 226Ala Val Lys Arg Gln Ala Asn Pro Thr Ser Thr Tyr Ile 1 5 10 227 30 PRT Arabidopsis sp. 227Tyr Tyr Ile Leu Lys Ile Lys Ile Phe Leu Asn Phe Pro Tyr Tyr Phe 1 5 10 15Pro Phe Lys Ala Ser Lys Ser Lys Tyr Val Ser Ser Arg Leu 20 25 30 228 15 PRT Arabidopsis sp. 228Phe Ser Leu Ile Arg Phe Ser Thr Lys Lys Asn Gln Leu Leu Ile 1 5 10 15 229 39 PRT Arabidopsis sp. 229Lys Thr Leu Cys Ile Ile Leu Phe Thr Asn Ile Arg Glu Asp Glu Lys 1 5 10 15Lys Arg Arg Gly Glu Trp Leu Met Glu Lys Val Ser Phe Thr Pro Asn 20 25 30Ile Tyr Glu Leu Thr Arg Leu 35 230 9 PRT Arabidopsis sp. 230Asn Pro Tyr Ile Leu Ile Val Cys Leu 1 5 231 4 PRT Arabidopsis sp. 231Ile Asn Asn Ile 1 232 19 PRT Arabidopsis sp. 232Ser Ile Met Leu Phe Val Pro Cys Met Ile His Lys Thr Phe Ile Phe 1 5 10 15Glu Phe Val 233 37 PRT Arabidopsis sp. 233Glu Asn His Glu Asp Asp Gly Glu Gly Leu Pro Pro Glu Leu Asn Gln 1 5 10 15Ile Lys Glu Gln Ile Glu Lys Glu Arg Phe Leu His Ile Lys Val Arg 20 25 30Asp Ile Trp Leu Leu 35 234 9 PRT Arabidopsis sp. 234Tyr Phe Ile Leu Phe Cys Met Phe Phe 1 5 235 10 PRT Arabidopsis sp. 235Lys Leu Arg Arg Gly Glu Asp Leu Ile Ser 1 5 10 236 11 PRT Arabidopsis sp. 236Leu Tyr Asp Ser Lys Glu Met Leu Arg Tyr Ile 1 5 10 237 4 PRT Arabidopsis sp. 237Thr Val Ile His 1 238 13 PRT Arabidopsis sp. 238Ser Leu Lys Leu Lys Arg Glu Ile Ser Lys Leu Leu Asn 1 5 10 239 26 PRT Arabidopsis sp. 239Lys Gln Asn Leu Glu Asn Ala Ser Glu Ser Ile Thr Thr Ser Arg Ser 1 5 10 15Val Gly Tyr Pro Lys Leu Arg Ile Lys Lys 20 25 240 4 PRT Arabidopsis sp. 240Leu Ile Tyr Leu 1 241 5 PRT Arabidopsis sp. 241Tyr Lys Ala Gly Thr 1 5 242 29 PRT Arabidopsis sp. 242Ile Phe Leu Phe Leu Ser Asn Lys Ile Val Ile Gln Phe Val Phe Phe 1 5 10 15Ser Thr Lys Lys Leu Asn Arg Ser Lys Leu Phe Phe Phe 20 25 243 34 PRT Arabidopsis sp. 243His Ser Ala Lys Lys Ala Lys Ile Asp Ala Tyr Ile Lys Lys Ser Lys 1 5 10 15Ser Lys Leu Leu Tyr Ser Ser Ile Leu Val Ser Leu Tyr Ile Glu Ser 20 25 30Ser Phe 244 18 PRT Arabidopsis sp. 244Asn Leu Lys Asn His Leu Pro Ile Tyr Tyr Leu Ile Asn Arg Glu Asn 1 5 10 15Ser Ser 245 17 PRT Arabidopsis sp. 245Asp Thr Phe Gln Val Trp Leu Leu Met Leu His Thr Ile Asn Arg Leu 1 5 10 15Thr 246 100 PRT Arabidopsis sp. 246Thr Ser Pro Leu Gln Arg Met Ile Met Glu Glu Thr Thr Asn His Phe 1 5 10 15Cys Arg Glu Cys Lys Thr His Phe Val Ile Ser Val Pro His Leu Ile 20 25 30Ile Ile Leu Thr Lys Ile Lys Val Met Phe Leu Met Arg Ile Lys Ile 35 40 45Met Leu Leu Lys Lys Met Tyr His Tyr Phe Leu Met Lys Met Tyr His 50 55 60Tyr Tyr Gln Val Ser Ser Phe Gln Leu Leu Arg Ser Tyr His Asp Pro 65 70 75 80Leu His Gly Ser Ser Pro Lys Gly Met Cys Val Phe Cys Phe Val Leu 85 90 95Val Ser Lys Tyr 100 247 4 PRT Arabidopsis sp. 247Ser Tyr Thr Ile 1 248 13 PRT Arabidopsis sp. 248Ser Leu Ile Val His Ile Tyr Ile Ser Leu Thr Leu Gln 1 5 10 249 4 PRT Arabidopsis sp. 249Pro Ala Asp Gly 1 250 4 PRT Arabidopsis sp. 250Phe Cys Asp Trp 1 251 8 PRT Arabidopsis sp. 251Glu Thr Asn Leu Leu Phe Glu Trp 1 5 252 6 PRT Arabidopsis sp. 252Gly Thr Arg Ile Glu Gln 1 5 253 11 PRT Arabidopsis sp. 253Gly Arg Asn Gln Glu Arg Lys Met Arg Ile Phe 1 5 10 254 18 PRT Arabidopsis sp. 254Arg Cys Arg Pro Ile Tyr Met Val Ser Phe Cys Ile Thr Tyr Val Leu 1 5 10 15Asp Tyr 255 5 PRT Arabidopsis sp. 255Phe Val Val His Ile 1 5 256 41 PRT Arabidopsis sp. 256Thr Ala Gln Glu Ile Phe Arg Thr Val Gly Gln Asp Tyr Gly Leu Asp 1 5 10 15Asp Leu Val Val Arg Arg Ala Leu Ala Lys Tyr Leu Glu Val Asp Val 20 25 30Ser Asp Ile Leu Val Thr Ile Phe Glu 35 40 257 30 PRT Arabidopsis sp. 257Lys Leu His Thr Ser Ile Asn Asn Phe Pro Ala Tyr Leu Ile Phe Val 1 5 10 15Val Phe Arg Arg Glu Lys Cys Phe Lys Phe Ser Asn Leu Met 20 25 30 258 51 PRT Arabidopsis sp. 258Glu Arg Tyr Asn Glu Leu Lys Leu Lys Asn Asp Gly Thr Ala Gly Glu 1 5 10 15Ala Ser Asp Leu Thr Ser Lys Thr Ile Thr Thr Ala Phe Gln Asp Phe 20 25 30Ala Asp Arg Arg His Cys Arg Arg Cys Met Val Thr Leu Asn Leu Ser 35 40 45Phe Leu Ile 50 259 36 PRT Arabidopsis sp. 259Pro Gln Lys Arg Glu Met Ile Ile His Val Phe Ile Leu Phe Tyr His 1 5 10 15Leu Phe Tyr Arg Tyr Ser Ile Val Ile Cys Met Arg Ser Met Ser Pro 20 25 30Ser Leu Asp Pro 35 260 9 PRT Arabidopsis sp. 260Ala Leu Asn Ser Phe Lys Leu Phe Cys 1 5 261 6 PRT Arabidopsis sp. 261Phe His Asn Pro Tyr Ile 1 5 262 50 PRT Arabidopsis sp. 262Val Ile Asn Leu Ile Arg Leu Leu Trp Leu Val Arg Ala Lys Thr Asn 1 5 10 15Leu Val Cys Leu Arg Met Lys Ile Asp Asn His Ala Val Ser Ile Val 20 25 30Thr Ser Arg Ser Leu Ser Leu Ser Leu Ser Leu Ser Ile Phe Leu Ser 35 40 45Ile Pro 50 263 12 PRT Arabidopsis sp. 263Leu Arg Leu Leu Val Thr Gly Leu Ile Leu Asn Arg 1 5 10 264 5 PRT Arabidopsis sp. 264Gln Lys Leu Ile Met 1 5 265 23 PRT Arabidopsis sp. 265Trp Ile Met Ile Thr Leu Tyr Gln Thr Arg Leu Trp Ser Gln Ile Gln 1 5 10 15Thr Thr Leu Cys Gly Arg Leu 20 266 5 PRT Arabidopsis sp. 266Arg Arg Ile Phe Thr 1 5 267 19 PRT Arabidopsis sp. 267Lys Glu Leu Arg Tyr Leu Gly Glu Thr Gly Lys Lys Ile Lys Ile Asp 1 5 10 15Leu Met His 268 8 PRT Arabidopsis sp. 268Tyr Ile Tyr Leu His Cys Ile Pro 1 5 269 10 PRT Arabidopsis sp. 269Leu Cys Trp Phe Ala Val Val Met Leu His 1 5 10 270 9 PRT Arabidopsis sp. 270Thr Tyr Phe Gly Gly Leu Arg Arg Ala 1 5 271 15 PRT Arabidopsis sp. 271Arg Phe Thr Ile Thr Cys Ala Asn Lys Ile Asn Val Leu Cys His 1 5 10 15 272 28 PRT Arabidopsis sp. 272Thr Leu Thr Lys Leu His Lys Asp Thr Ile Arg Tyr Thr Asn Leu Cys 1 5 10 15Arg Asn Tyr Ser His Asp Met Tyr Val Lys Asn Thr 20 25 273 95 PRT Arabidopsis sp. 273Ser Phe Leu Tyr Val Leu Met Val Leu Ser Gln Val Thr Lys Lys Val 1 5 10 15Ser Arg Lys Ser Ser Arg Ser Val Arg Lys Lys Ser Arg Leu Arg Lys 20 25 30Tyr Ala Arg Tyr Pro Pro Ala Leu Lys Lys Thr Thr Ser Gly Glu Ala 35 40 45Lys Phe Tyr Lys His Tyr Thr Pro Cys Thr Cys Lys Ser Lys Cys Gly 50 55 60Gln Gln Cys Pro Cys Leu Thr His Glu Asn Cys Cys Glu Lys Tyr Cys 65 70 75 80Gly Tyr Val Ile Gln Phe Phe Leu Ser Arg Lys Ile His Glu Ile 85 90 95 274 22 PRT Arabidopsis sp. 274Phe Glu His Glu Phe Val Phe Phe Val Gln Val Leu Lys Gly Leu Gln 1 5 10 15Gln Ser Leu Trp Arg Met 20 275 16 PRT Arabidopsis sp. 275Leu Cys Asn Trp Pro Met His Lys Ser Thr Met Ser Leu Phe Cys Cys 1 5 10 15 276 11 PRT Arabidopsis sp. 276Met Arg Ser Arg Ser Leu Ser Glu Leu Ser Ser 1 5 10 277 13 PRT Arabidopsis sp. 277Val Thr Leu Ser Leu Gln Tyr Leu Phe Ile Gln Ile Leu 1 5 10 278 6 PRT Arabidopsis sp. 278Phe Lys Pro Lys Val Leu 1 5 279 5 PRT Arabidopsis sp. 279Lys Lys Leu Tyr Ile 1 5 280 7 PRT Arabidopsis sp. 280Leu Trp Arg Trp His Ser Trp 1 5 281 17 PRT Arabidopsis sp. 281Asp Thr Ser Ala Asn Pro Met Gln Glu His Ala Ile Pro Pro Ser Asn 1 5 10 15Gln 282 45 PRT Arabidopsis sp. 282Lys Gly Asn Gln Arg Gln Ile Arg Thr Glu Asn Leu Lys Leu Ile Ile 1 5 10 15Arg Lys Thr Phe Asn Tyr His Phe Pro Tyr Phe Thr Arg Phe Ser Leu 20 25 30Glu Ser Leu Met Phe Met Asp Gly Val His Leu His Gly 35 40 45 283 5 PRT Arabidopsis sp. 283Leu Leu Val His Ser 1 5 284 21 PRT Arabidopsis sp. 284His Phe Phe Phe Phe Asn Asn Val Leu Tyr Phe Arg Pro Leu Asn Ile 1 5 10 15Leu Cys Asp Met Val 20 285 18 PRT Arabidopsis sp. 285Pro Val Arg Thr Leu Leu Lys Arg Met Ser Ile Ser Glu Asn Ile Leu 1 5 10 15Glu Asn 286 11 PRT Arabidopsis sp. 286Ser Leu Met Met Lys Leu Met Ser Val Gly Glu 1 5 10 287 11 PRT Arabidopsis sp. 287Lys Ile Gly Leu Val Leu Pro Thr Ser Leu Pro 1 5 10 288 9 PRT Arabidopsis sp. 288Leu Gln Asn Asn Phe Glu Val Thr Phe 1 5 289 51 PRT Arabidopsis sp. 289Ser Phe Ala Gly Tyr Thr Ser Ile Arg Ile Lys Val Thr Phe Ile Leu 1 5 10 15Gln Leu Glu Ile Asp Ala Arg Arg Lys Gly Asn Glu Phe Lys Phe Leu 20 25 30Asn His Ser Ala Arg Pro Asn Cys Tyr Ala Lys Val Leu Ser Arg Tyr 35 40 45Thr Leu Ser 50 290 26 PRT Arabidopsis sp. 290Thr Asn Thr Asn Ile Ile Gln Thr Lys Ile Leu Met Leu Val Ser Leu 1 5 10 15Val Lys Ser Cys Ile Asn Phe Thr Arg Arg 20 25 291 16 PRT Arabidopsis sp. 291Leu Val Phe Ile Leu Lys Ile Phe Gln Glu Thr Gln Thr His Phe Lys 1 5 10 15 292 7 PRT Arabidopsis sp. 292Phe Phe Leu Val Glu Lys Ile 1 5 293 10 PRT Arabidopsis sp. 293Val Thr Lys Ile Tyr Gly Phe Val Cys Ser 1 5 10 294 57 PRT Arabidopsis sp. 294Glu Glu Ile Arg Gly Leu Val Tyr Leu Arg Arg Glu Gln Ser Lys Lys 1 5 10 15Val Arg Ser Phe Ser Ser Thr Thr Ala Met Asp Gln Asn Met Arg Ile 20 25 30Gly Arg Val Val Glu Asn Leu Glu Arg Leu Val Leu Leu Lys Gly Leu 35 40 45Arg Lys Pro Val Gln Leu Val Ser Phe 50 55 295 21 PRT Arabidopsis sp. 295Ser Glu Glu Lys Gln Gln Phe Lys Gln Ser Phe Phe Tyr Val Met Val 1 5 10 15Tyr Gln Leu Ile Met 20 296 66 PRT Arabidopsis sp. 296Cys Tyr Phe Val Leu Leu Asn Gln Asn Leu Ser Phe Cys Phe Ile Cys 1 5 10 15Phe Arg Val Phe Cys Leu Tyr His Met Cys Leu Asn Phe Gln Ser Phe 20 25 30Leu Phe Val Phe Gln Phe Lys Asn Asn Val Tyr Val Val Ser Leu His 35 40 45Arg Pro Leu Glu Lys Lys Ser Phe Ala Gln Leu Tyr Ile Tyr Leu Val 50 55 60Phe Ile 65 297 4 PRT Arabidopsis sp. 297Arg Lys Ile Thr 1 298 18 PRT Arabidopsis sp. 298His Lys Ser Val Val Arg Asn Val Gln Lys Cys Gln Asn Asn Gly Phe 1 5 10 15Tyr His 299 9 PRT Arabidopsis sp. 299Lys Lys Ile Leu Val Met Asn Glu Val 1 5 300 18 PRT Arabidopsis sp. 300Val Leu Ala Arg Leu Val Leu Lys Arg Phe Ser Arg Phe Asn Phe Val 1 5 10 15Val Tyr 301 32 PRT Arabidopsis sp. 301Val Ile His Gly Arg Ile Ile Asn Lys Val Ala Val Ala Tyr Glu Arg 1 5 10 15Phe Tyr Phe Asn Val Asn Met Tyr Leu Met His Leu Thr Phe Ser Ile 20 25 30 302 22 PRT Arabidopsis sp. 302Thr Asn Lys Asn Lys Lys Lys Glu Lys Ser Ser Leu Lys Ser Glu Ser 1 5 10 15Asn Tyr Phe Gln Lys Ile 20 303 21 PRT Arabidopsis sp. 303Ile Ile Asn Leu Asn Val Trp Asn Arg Glu Arg Leu Leu Leu Asn Ile 1 5 10 15Asn Ala Lys Tyr Thr 20 304 20 PRT Arabidopsis sp. 304Arg Cys Glu Lys His Val Gly Phe Val Glu Ser Leu Met Thr Thr Val 1 5 10 15Lys Trp Arg Asp 20 305 23 DNA Artificial Sequence Description of Artificial Sequenceprimer Nir Cla-73 305ggcggacatc aaacctactt agc 23 306 24 DNA Artificial Sequence Description of Artificial Sequenceprimer cer1n2 306tgtaacatta aggcctttcc tttt 24 307 23 DNA Artificial Sequence Description of Artificial Sequenceprimer Nir-C-2-S-N 307cggtcatcaa gtgagttatg aag 23 308 19 DNA Artificial Sequence Description of Artificial Sequenceprimer cer1s659 308ggtccaatcg gcaatgagt 19 309 22 DNA Artificial Sequence Description of Artificial Sequenceprimer cer1ns10596n 309gtccaatcgg caatgagtag ag 22 310 22 DNA Artificial Sequence Description of Artificial Sequenceprimer Nir La-4Cla-S-S 310gtgtgcctaa cagtttccgc ac 22 311 24 DNA Artificial Sequence Description of Artificial Sequenceprimer cer1ns10265n 311tctcggagat ggtgccatat cagc 24 312 40 DNA Artificial Sequence Description of Artificial Sequenceprimer fie3cds5′.seq 312atgtcctctg gagagcagaa ggaagagtcg ttttacacgg 40 313 24 DNA Artificial Sequence Description of Artificial Sequenceprimer cer1ns10129n 313tctggagagc agaaggaaga gtcg 24 314 22 DNA Artificial Sequence Description of Artificial Sequenceprimer cer1ns10030n 314cgagtcattg acgtcaacag tg 22 315 24 DNA Artificial Sequence Description of Artificial Sequenceprimer cer1ns9922n 315ctcgcaaatg tgcagagtct tgtg 24 316 20 DNA Artificial Sequence Description of Artificial Sequenceprimer cer1n1570 316aggtcatcgc tatgaagttc 20 317 20 DNA Artificial Sequence Description of Artificial Sequenceprimer cer1ns98f9511n 317gctagttgtg gtatggacac 20 318 21 DNA Artificial Sequence Description of Artificial Sequenceprimer cer1ns98f9311s 318cacatggact gatgatccat c 21 319 20 DNA Artificial Sequence Description of Artificial Sequenceprimer cer1s2099 319gtaaccgttg gtttggtgat 20 320 25 DNA Artificial Sequence Description of Artificial Sequenceprimer Nir E-4-N-N 320ggttagtaag tcaatgatgg ttaag 25 321 19 DNA Artificial Sequence Description of Artificial Sequenceprimer cer1ns8795n 321gcgataggta atcagagag 19 322 20 DNA Artificial Sequence Description of Artificial Sequenceprimer cer1ns8517n 322ctgtaatcag gcaaacagcc 20 323 20 DNA Artificial Sequence Description of Artificial Sequenceprimer cer1ns98f8483s 323cagccatgtc tgtcgatgga 20 324 40 DNA Artificial Sequence Description of Artificial Sequenceprimer fie3cds3′.seq 324atccatcttc tctctcacca atgcagtgaa aatttcttaa 40

Claims

1. A method of enhancing endosperm development in a plant, the method comprising introducing into the plant an expression cassette containing a plant promoter operably linked to a polynucleotide that:a. specifically hybridizes to SEQ ID NO:3 in a buffer of 40% formamide, 1M NaCl, 1% SDS at 37° C., followed by one wash for 20 minutes in 0.2X SSC at a temperature of about 50° C.; and b. enhances endosperm development in the absence of fertilization when the polynucleotide is operably linked to a plant promoter to inhibit FIE gene expression and introduced into a plant.

2. The method of claim 1, wherein the polynucleotide encodes a polypeptide that has an amino acid sequence as shown in SEQ ID NO:4.

3. The method of claim 1, wherein the polynucleotide is linked to the promoter in an antisense orientation.

4. The method of claim 1, wherein the polynucleotide is SEQ ID NO:3.

5. The method of claim 1, wherein the plant promoter is from a FIE gene.

6. The method of claim 1, wherein the expression cassette is introduced into the plant through a sexual cross.

7. The method of claim 1, wherein the polynucleotide encodes a polypeptide that is at least 80% identical to SEQ ID NO:4.

8. The method of claim 1, wherein the polynucleotide is at least 100 nucleotides in length.

9. The method of claim 1, wherein the plant promoter is tissue-specific.

10. The method of claim 1, wherein the plant promoter is ovule- or embryo-specific.

11. The method of claim 1, wherein the polynucleotide is operably linked to the plant promoter in a sense orientation.