Method For Speeding Up Plant Growth And Improving Yield By Introducing Phosphatases In Transgenic Plant

Abstract

Transgenic plants having increased growth rate, increased sugar content, and increase yield are disclosed, and methods for making the same. The transgenic plants have a gene coding for a phosphatase having a C-terminal motif under control of a heterologous promoter incorporated into the genomic DNA of the plant.

Description

RELATED APPLICATIONS

[0001] This application claims priority to provisional application Ser. No. 61/138,918, filed on Dec. 18, 2008, which is incorporated herein by reference.

1. TECHNICAL FIELD

[0002] The present disclosure provides methods that speeds up plant growth and elevates plant yields by introducing phosphatases with a C-terminal motif into plants. The present disclosure relates to phosphatases with a C-terminal motif, and their respectively encoded protein products, as well as fragments, derivatives, homologues, and variants thereof. Methods for introducing these genes into plants to (1) speed up the growth rate of plants, (2) to increase the sugar contents of plants, and (3) to increase of yield of plants, are provided.

2. BACKGROUND

[0003] Purple acid phosphatases (PAPs) catalyze the hydrolysis of a wide range of activated phosphoric acid mono- and di-esters and anhydrides (Klabunde et al., 1996). The PAP proteins are characterized by seven conserved amino acid residues (shown in bold face) in the five conserved motifs XDXX, XDXXY, GNH(D/E), XXXH, XHXH, which are involved in the coordination of the dimetal nuclear center (Fe.sup.3+-Me.sup.2+) in the active site (Li et al., 2002), where Me is a transition metal and Me.sup.2+ is mostly found to be Fe.sup.2+ in mammalian, and Zn.sup.2+, or Mn.sup.2+ in plants (Klabunde and Krebs, 1997; Schenk et al., 1999).

[0004] Purple acid phosphatases are distinguished from the other phosphatases by their characteristic purple color, which is caused by a charge transfer transition at 560 nm from a metal-coordinating tyrosine to the metal ligand Fe.sup.3+ (Klabunde and Krebs, 1997; Schenk et al., 2000). Different from the other acid phosphatases, PAPs are insensitive to inhibition by tartrate, so they are also known as tartrate-resistant acid phosphatases (TRAPs).

[0005] The biochemical properties of some plant PAPs have been characterized, firstly in red kidney bean, and later in soybean suspension cell, soybean seedlings, rice culture cells, spinach leaves, sweet potato tubers, tomato, yellow lupin seeds, medicago and Arabidopsis, etc. (Schenk et al., 1999). Plant PAPs are generally considered to mediate phosphorus acquisition and redistribution based on their ability to hydrolyze phosphate compounds (Cashikar et al., 1997; Bozzo et al., 2004; Lung et al., 2008). Regulation of some plant PAPs transcripts by external phosphate level in medium or soil, strongly suggest their involving in phosphate acquisition. For example, the transcription level of Medicago MtPAP1 in roots was increased under P stress, implicating a role in P acquisition or internal mobilization (Xiao et al., 2005; Xiao et al., 2006). Some plant PAPs could be secreted from root cells to extracellular environment, then hydrolyze various phosphate esters. Lung et al. purified a secreted PAP phosphatase from tobacco, which could hydrolyze broad substrates and help to alleviate P starvation (Lung et al., 2008). Certain plant PAPs can also hydrolyze phytate, a major storage compound of phosphorus in plants. Hegeman and Grabau (2001) purified a novel PAPs (GmPhy) from the cotyledon of the germinating soybean seedlings. GmPhy was introduced into soybean tissue culture and was assayed to show phosphatase activity. Most recently, AtPAP15 and 23 in Arabidopsis sharing high sequence homology (73-52%) with this soybean PAP, were found to exhibit phytase activity (Zhu et al., 2005; Zhang et al., 2008).

[0006] Besides involvement in P acquisition, plant PAPs may perform some other physiological roles. For example, the PAPs AtACP5 (AtPAP17), SAP1, and SAP2 (del Pozo et al., 1999; Bozzo et al., 2002) display not only phosphatase but also peroxidase activity, suggesting their involvement in the removal of reactive oxygen compounds in plant organs. A pollen-specific PAP from Ester lily was suggested to function as an iron carrier in mature pollen (Kim and Gynheung, 1996). Other studies indicate that plant PAPs may also be involved in NaCl stress adaption or cell regeneration (Kaida, 2003; Liao et al., 2003).

[0007] In the Arabidopsis genome, twenty-nine potential PAP genes were identified based on sequence comparison. Twenty-four of these putative enzymes contain seven conserved amino-acids residues involved in metal binding. One (AtPAP13) lacked four of these seven residues, and the other four (AtPAP14, 16, 28 and 29) lacked either the first, the second, or both motifs of the five conserved motifs. Twenty-eight are actively transcribed in Arabidopsis (Zhu et al., 2005).

[0008] To date, relatively little is known about AtPAPs biochemical properties and physiological roles, though several members have been characterized (del Pozo et al., 1999). AtPAP17 (AtACP5) was first known to be induced by phosphorus starvation. The transcription of AtPAP17 was also responsive to ABA, salt stress (NaCl), oxidative stress (H.sub.2O.sub.2) and leaves senescence, according to GUS activity assay. No alteration in the expression of AtPAP17 was observed during the nitrogen or potassium starvation, and paraquat or salicylic acid. Like the other type5 acid phosphatases, AtPAP17 displayed peroxidation activity, which may be involved in the metabolism of reactive oxygen species in stressed or senescent parts of plants.

[0009] Besides AtPAP17, several AtPAPs were found to be involved in phosphorus metabolism in Arabidopsis. Root secretion of AtPAP12 was induced by P stress, and its regulation was mainly at transcriptional level (Patel et al., 1998; Coello, 2002/11). AtPAP4, as well as AtPAP10, AtPAP11 and AtPAP12 were involved in phosphorus starvation response since their transcription levels increased during phosphate deprivation (Li et al., 2002; Wu et al., 2003). In contrast, AtPAP20, 21 and 22 were irrespective to P starvation and expressed constitutively in Pi sufficient or deficient condition. Fluorescent signals were detected in the cytoplasm via the baculovirus expression system, indicating that they may function in the cytoplasm (Li and Wang, 2003).

[0010] AtPAP26 was purified and characterized from Pi-starved Arabidopsis suspension cell culture (Veljanovski et al., 2006). It exists as a homodimer with 55 kDa glycosylated protein, showing wide substrate specificity with the highest activity against phosphoenolpyruvate (PEP) and polypeptide phosphate. AtPAP26 also displayed alkaline peroxidase activity with the probable roles in the metabolism of reactive oxygen species. Proteomic study suggested that it may be localized in vacuole, and involved in recycling Pi from intracellular P metabolites (Shimaoka et al., 2004).

[0011] PAPs can act on a wide range of substrates, but not all of them exhibit phytase activity. An enzyme assay involving the GST-AtPAP23 fusion protein revealed that AtPAP23 exhibits phytase activity. A GUS study showed that AtPAP23 is exclusively expressed in the flower of the Arabidopsis, and may play certain roles in flower development (Zhu et al., 2005). In a recent report, a recombinant AtPAP15 expressed and partial purified in E. coli and yeast was also found to exhibit phytase activity (Zhang et al., 2008). It was proposed that AtPAP15 may be involved in ascorbic acid biosynthesis with the end product myo-inositol of phytate hydrolysis as the precursor of ascorbic acid synthesis.

[0012] As stated above, most of the functions of characterized plant PAPs are related to phosphorus metabolism. None of the functionally or biochemically characterized plant PAPs carry transmembrane motif, and none of them were shown to be associated with membrane. Furthermore, to date, no AtPAPs or any plant PAPs, have been showed to affect sugar signalling and carbon metabolism in plant.

[0013] The first report of transgenic expression of plant PAP in plant was reported in 2005 (Xiao et al., 2005). The PAP-phosphatase gene from Medicago (MtPHY1) was expressed in transgenic Arabidopsis, resulting in increased capacity of P acquisition from phytate in agar culture (Xiao et al., 2005). Nonetheless, the growth performance of the plants was not reported to be different under normal growth.

3. SUMMARY

[0014] The present disclosure provides a method that speeds up plant growth and elevates plant yields by introducing phosphatases with a C-terminal motif into plants. Phosphatases with a C-terminal motif, and their respectively encoded protein products, as well as fragments, derivatives, homologues, and variants thereof are disclosed. Methods for introducing this class of genes into plants to speed up the growth rate of plants, to increase the sugar contents of plants, and to increase of yield of plants, are provided. Without wishing to be bound by any particular theory, the C-terminal motif is believed to function as a transmembrane structural element (transmembrane motif).

[0015] As stated above in the Background section, most of the functions of characterized plant PAPs are related to phosphorus metabolism. None of the functionally or biochemically characterized plant PAPs carry transmembrane motif, and none of them were shown to be associated with membrane. Furthermore, to date, no AtPAPs or any plant PAPs, have been showed to affect sugar signalling and carbon metabolism in plant.

[0016] The first report of transgenic expression of plant PAP in plant was reported in 2005 (Xiao et al., 2005). The PAP-phosphatase gene from Medicago (MtPHY1) was expressed in transgenic Arabidopsis, resulting in increased capacity of P acquisition from phytate in agar culture. Nonetheless, the growth performance of the plants was not reported to be different under normal growth.

[0017] We also produced transgenic tobacco and Arabidopsis that overexpressed AtPAP15, a PAP with phosphatase activity, which does not carry any C-terminal motif equivalent to that of AtPAP2; phosphatase activity was secreted into extracellular growth medium. Significant secretion of phosphatase activity was observed in the transgenic plants and the transgenic plants showed larger biomass than the control plants in agar and soil supplemented with exogenous phytate. Higher P content was also obtained in overexpressed transgenic lines in phytate treatment. However, the growth of transgenic plants overexpressing AtPAP15 did not show any difference in growth phenotypes when it was compared with the wild-type, under treatments of K--P or No--P, or in soil.

[0018] Here, we have developed a technology to speed up plant growth and improve seed yield by overexpressing a phosphatase with a C-terminal motif in plants. An example is the use of a purple acid phosphatase (PAP). This disclosure is the first report to show that overexpressing a phosphatase with a C-terminal motif in transgenic plant is able to speed up the growth of the plants, to increase the sugar contents of plants, and to increase the yield of plants, by altering the carbon metabolism of the plants.

[0019] The present advances are based, in part, on the characterization of a group of purple acid phosphatases (SEQ ID NOS: 1-8 and 18-47) from plants and the observations that overexpression of a purple acid phosphatase (AtPAP2, SEQ ID NO:1) of this group in plants resulted in rapid plant growth, higher sugar content, and higher yield. Accordingly, nucleotide sequences of a group of purple acid phosphatase genes (SEQ ID NOs:1, 3, 5, 7, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44 and 46), which share a C-terminal motif/domain, from plants and amino acid sequences of their encoded proteins (SEQ ID NOS:2, 4, 6, 8, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45 and 47), as well as fragments, derivatives, homologues, and variants thereof, as defined herein, are disclosed. Furthermore, nucleic acid molecules encoding the polypeptides of interest and include cDNA, genomic DNA, and RNA, are disclosed.

[0020] As used herein, italicizing the name of a gene shall indicate the gene, in contrast to its encoded protein or polypeptide product which is indicated by the name of the gene in the absence of any italicizing. For example, "Gene" shall mean the Gene gene, whereas "Gene" shall indicate the protein or polypeptide product of the Gene gene.

[0021] In one embodiment, isolated nucleic acid molecules hybridize under stringent conditions, as defined herein, to nucleic acids having the sequence of SEQ ID NOS: 1, 3, 5, 7, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44 or 46, or homologues thereof, wherein the nucleic acid molecules encode proteins or polypeptides which exhibit at least one structural and/or functional feature of the polypeptides of the invention.

[0022] Another embodiment includes, nucleic acid molecules, which are suitable for use as primers or hybridization probes for the detection of nucleic acids encoding one of the disclosed phosphatase polypeptides or other sequences.

[0023] Yet another embodiment includes vectors, e.g., recombinant expression vectors, comprising a nucleic acid molecule of the invention. Furthermore, host cells containing such a vector or engineered to contain and/or express a nucleic acid molecule of the invention and host cells containing a nucleotide sequence of the invention operably linked to a heterologous promoter are disclosed.

[0024] A further embodiment includes methods for preparing a polypeptide of the invention by a recombinant DNA technology in which the host cells containing a recombinant expression vector encoding a polypeptide of the invention or a nucleotide sequence encoding a polypeptide of the invention operably linked to a heterologous promoter, are cultured, and the polypeptide of the invention are produced.

[0025] In still further another embodiment, a transgenic plant contains a nucleic acid molecule which encodes an isolated polypeptides or proteins comprising the five conserved motifs of purple acid phosphatases, including XDXX, XDXXY, GNH(D/E), XXXH, XHXH, and linked to a C-terminal motif.

[0026] Embodiments further provide antibodies that immunospecifically bind a polypeptide of the invention. Such antibodies include, but are not limited to, antibodies from various animals, humanized, chimeric, polyclonal, monoclonal, bi-specific, multi-specific, single chain antibodies, Fab fragments, F(ab').sub.2 fragments, disulfide-linked Fvs, fragments containing either a VL or VH domain or even a complementary determining region (CDR), that immunospecifically binds to a polypeptide of the invention.

[0027] In an additional embodiment, method for detecting the presence, activity or expression of a polypeptide of the invention or similar polypeptide in a biological material, such as cells, culture media, and so forth are provided. The increased or decreased activity or expression of the polypeptide in a sample relative to a control sample can be determined by contacting the biological material with an agent that can detect directly or indirectly the presence, activity or expression of the polypeptide of the invention. In a particular embodiment, such an agent is an antibody or a fragment thereof which immunospecifically binds to a one of the disclosed polypeptides.

[0028] In a still another embodiment, a fusion protein comprising a bioactive molecule and one or more domains of a disclosed polypeptide or fragment thereof is provided. In particular, fusion proteins comprising a bioactive molecule recombinantly fused or chemically conjugated (including both covalent and non-covalent conjugations) to one or more domains of a disclosed polypeptide or fragments thereof.

[0029] We also produced transgenic tobacco and Arabidopsis that overexpressed AtPAP15, a PAP with phosphatase activity, which does not carry any C-terminal motif and was found to be secreted into extracellular growth medium. Significant secretion of phosphatase activity was observed in the transgenic plants and the transgenic plants showed larger biomass than the control plants in agar and soil supplemented with exogenous phytate. Higher P content was also obtained in overexpressed transgenic lines in phytate treatment. However, the growth of transgenic plants overexpressing AtPAP15 did not show any difference in growth phenotypes when it was compared with the wild-type, under treatments of K--P or No--P, or in soil.

[0030] In conclusion, this disclosure is the first report to show that overexpressing a phosphatase with a C-terminal motif in transgenic plant is able to speed up the growth of the plants, to increase the sugar contents of plants, and to increase the yield of plants, by altering the carbon metabolism of the plants.

3.1 DEFINITIONS

[0031] The term "acidic" or "acid pH" as used herein refers to a pH value of less than about 6.0.

[0032] The term "homologue" as used herein refers to a polypeptide that possesses a similar or identical function to polypeptides encoded by SEQ ID NOS: 2, 4, 6, 8, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45 and 47, and/or a fragment of these polypeptides, that do not have an identical amino acid sequence of these polypeptides and/or a fragment of these polypeptides. A polypeptide that has a similar amino acid sequence included in the definition of the term "homologue" includes a polypeptide that satisfied at least one of the following: (i) polypeptide having an amino acid sequence that is one or more of at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, and at least about 98% identical. (ii) a polypeptide encoded by a nucleotide sequence that is one or more of at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, and at least about 98% identical and/or conservatively substituted to one or more of the nucleotide sequences encoding the polypeptides of SEQ ID NOS: 2, 4, 6, 8, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45 and 47, and/or a fragment of the these polypeptides; (iii) a polypeptide encoded by a nucleotide sequence that hybridizes under stringent conditions as defined herein to one or more of nucleotide sequences SEQ ID NOS: 1, 3, 5, 7, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44 and 6; (iv) a polypeptide having an amino acid sequence that is one or more of at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, and at least about 98% identical and/or conservatively substituted; (v) a nucleic acid sequence encoding an amino acid sequence that is one or more of at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, and at least about 98% identical and/or conservatively substituted; (vi) a fragment of any of the polypeptides or nucleic acid sequences described in (i) through (v) having one of at least 20 amino acid residues, at least 25 amino acid residues, at least 40 amino acid residues, at least 80 amino acid residues, at least 90 amino acid residues, at least 100 amino acid residues, at least 125 amino acid residues, at least 150 amino acid residues, at least 175 amino acid residues, at least 200 amino acid residues, at least 225 amino acid residues, at least 250 amino acid residues, at least 275 amino acid residues, at least 300 amino acid residues, at least 325 amino acid residues, at least 350 amino acid residues, or at least 375 amino acid residues; (vii) a polypeptide with similar structure and function or a nucleotide sequence encoding a polypeptide with similar structure and function, exhibiting the antigenicity, immunogenicity, catalytic activity, and other readily assayable activities, to polypeptides encoded by SEQ ID NOS: 2, 4, 6, 8, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45 and 47, and/or a fragment of these polypeptides, refers to a polypeptide that has a similar secondary, tertiary, or quaternary structure of these polypeptides, or a fragment of these polypeptides. The structure of a polypeptide can be determined by methods known to those skilled in the art, including but not limited to, X-ray crystallography, nuclear magnetic resonance, and crystallographic electron microscopy. The term "homologue" is used herein to describe a sequence that has sequence homology. A sequence having sequence homology can be made using standard molecular biology techniques including site-directed mutagenesis including insertion or deletion of sequences. The term "homologue" is not limited to homologous genes or proteins originating from different species and expressly includes artificial modification to the sequences disclosed herein.

[0033] The term "conservatively substituted variant" refers to a polypeptide or a nucleic acid sequence encoding a homologue polypeptide in which one or more amino acid residues or codons have been modified by conservative substitution with an amino acid residue or a codon coding for an amino acid residue of similar chemical-type, as described below.

[0034] The term "an antibody or an antibody fragment which immunospecifically binds to polypeptides encoded by SEQ ID NOS: 2, 4, 6, 8, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45 and 47," as used herein refers to an antibody or a fragment thereof that immunospecifically binds to polypeptides encoded by SEQ ID NOS: 2, 4, 6, 8, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45 and 47, or a fragment of these polypeptide and does not non-specifically bind to other polypeptides. An antibody or a fragment thereof that immunospecifically binds to polypeptides encoded by SEQ ID NOS: 2, 4, 6, 8, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45 and 47, or a fragment of these polypeptide, may cross-react with other antigens. Preferably, an antibody or a fragment thereof that immunospecifically binds to polypeptides encoded by SEQ ID NOS: 2, 4, 6, 8, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45 and 47, or a fragment of these polypeptides, does not cross-react with other antigens. An antibody or a fragment thereof that immunospecifically binds to polypeptides encoded by SEQ ID NOS: 2, 4, 6, 8, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45 and 47, or a fragment of these polypeptide, can be identified by, for example, immunoassays or other techniques known to those skilled in the art. An antibody or an antibody fragment which immunospecifically binds polypeptides encoded by SEQ ID NOS: 2, 4, 6, 8, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45 and 47, may be interchangeably referred to as "anti-PAP antibody".

[0035] The term "derivative" as used herein refers to a given peptide or protein that is otherwise modified, e.g., by covalent attachment of any type of molecule, preferably having bioactivity, to the peptide or protein, including the incorporation of non-naturally occurring amino acids. The resulting bioactivity retains one or more biological activities of the peptide protein.

[0036] The term "fragment" as used herein refers to a fragment of a nucleic acid molecule containing one of at least about 25, at least about 30, at least about 35, at least about 40, at least about 45, at least about 100, at least about 150, at least about 200, at least about 250, at least about 300, at least about 350, at least about 400, at least about 450, at least about 500, at least about 550, at least about 600, at least about 650, at least about 700, at least about 750, at least about 800, at least about 850, at least about 900, at least about 950, at least about 1000, at least about 1050, at least about 1100, at least about 1150, at least about 1200, at least about 1250, at least about 1300, at least about 1350, from about 500 to about 2000, from about 1000 to about 2000 from about 200 to about 500, from about 500 to about 1000, form about 1000 to about 1500, and from about 1500 to about 2000 nucleic acid bases in length of the relevant nucleic acid molecule and having at least one functional feature of the nucleic acid molecule (or the encoded protein has one functional feature of the protein encoded by the nucleic acid molecule); or a fragment of a protein or a polypeptide containing one or more of at least about 5, at least about 10, at least about 15, at least about 20, at least about 25, at least about 30, at least about 35, at least about 40, at least about 45, at least about 50, at least about 55, at least about 60, at least about 65, at least about 70, at least about 75, at least about 80, at least about 90, at least about 100, at least about 120, at least about 140, at least about 160, at least about 180, at least about 200, at least about 220, at least about 240, at least about 260, at least about 280, at least about 300, at least about 320, at least about 340, at least about 360, from about 250 to about 660, from about 350 to about 660, form about 450 to about 660, and form about 550 to about 660 amino acid residues in length of the relevant protein or polypeptide and having at least one functional feature of the protein or polypeptide, such functional features include ability to bind a Fe.sup.3+-Me.sup.2+ dimetal nuclear center and form a C-terminal motif.

[0037] An "isolated" nucleic acid molecule is one which is separated from other nucleic acid molecules which are present in the natural source of the nucleic acid molecule. Moreover, an "isolated" nucleic acid molecule, such as a cDNA molecule, can be substantially free of other cellular materials, or culture medium when produced by recombinant techniques, or substantially free of chemical precursors or other chemicals when chemically synthesized, but excludes nucleic acid molecules present in recombinant DNA libraries. In a preferred embodiment, nucleic acid molecules encoding the disclosed polypeptides/proteins are isolated or purified.

[0038] The term "operably linked" as used herein refers to when transcription under the control of the "operably linked" promoter produces a functional messenger RNA, translation of which results in the production of the polypeptide encoded by the DNA operably linked to the promoter.

[0039] The term "under stringent condition" refers to hybridization and washing conditions under which nucleotide sequences having homology to each other remain hybridized to each other. Such hybridization conditions are described in, for example but not limited to, Current Protocols in Molecular Biology, John Wiley & Sons, N.Y. (1989), 6.3.1-6.3.6.; Basic Methods in Molecular Biology, Elsevier Science Publishing Co., Inc., N.Y. (1986), pp. 75-78, and 84-87; and Molecular Cloning, Cold Spring Harbor Laboratory, N.Y. (1982), pp. 387-389, and are well known to those skilled in the art. A preferred, non-limiting example of stringent hybridization conditions is hybridization in 6.times. sodium chloride/sodium citrate (SSC), 0.5% SDS at about 68.degree. C. followed by one or more washes in 2.times.SSC, 0.5% SDS at room temperature. Another preferred, non-limiting example of stringent hybridization conditions is hybridization in 6.times.SSC at about 45.degree. C. followed by one or more washes in 0.2.times.SSC, 0.1% SDS at about 50-65.degree. C.

[0040] The term "variant" as used herein refers either to a naturally occurring allelic variation of a given peptide or a recombinantly prepared variation of a given peptide or protein in which one or more amino acid residues have been modified by amino acid substitution, addition, or deletion.

[0041] The term "aligned" as used herein refers to a homology alignment between two or more sequences using a standard algorithm such as BLAST (http://blast.ncbi.nlm.nih.gov/Blast.cgi).

[0042] The term "predicted to form a transmembrane motif by TMHMM analysis" or "predicted to form a C-terminal motif by TMHMM analysis" (http://www.cbs.dtu.dk/services/TMHMM/) herein refers to a probability that is equal to or greater than about 0.5.

BRIEF DESCRIPTION OF THE FIGURES

[0043] The following figures illustrate the embodiments and are not meant to limit the scope of the invention encompassed by the claims.

[0044] FIG. 1 shows the phylogenetic tree of PAP-like sequences in the Arabidopsis genome. Twenty-nine PAPs were aligned using ClustalX and the phylogenetic tree was created by the neighbor-joining algorithm of the MEGA4 program. The accession numbers of the PAP-like, transmembrane-like C-terminal motif containing, polypeptide from Zea mays (ZmPAP2) and Oryza sativa (OsPAP2) were ACG47621 and BAC15853.1, respectively.

[0045] FIG. 2A is the amino acid alignment of the C-terminal transmembrane-like motifs in AtPAP2 with other PAP sequences.

[0046] FIG. 2B is the amino acid alignment of AtPAP2 with other PAP sequences, showing the full length of each sequence. These sequences include homologous sequences from B. napus (BnPAP2), G. max (GmPAP2) and Z. may (ZmPAP2). The five conserved motifs (XDXX, XDXXY, GNH(D/E), XXXH, XHXH) are boxed. Residues in shades have low or no homology. Hydrophobic motifs at the C-termini of these polypeptides are underlined by a bar (614.sup.th-636.sup.th amino acid), which is absent from the sequence of AtPAP15. As shown, AtPAP15 does not have a C-terminal region corresponding to the other PAP sequences.

[0047] FIG. 3 shows that a unique hydrophobic motif is present at the C-termini of AtPAP2 and ZmPAP2 by TMHMM analysis. This transmembrane-like C-terminal motif is absent from AtPAP15.

[0048] FIG. 4 shows the characteristics of the T-DNA lines. The T-DNA line (Salk.sub.--013567) was obtained from TAIR. The AtPAP2 genomic sequence carries two exons and the T-DNA was inserted in exon 2 and causes a disruption of the AtPAP2 mRNA (a). Three PCR primers (A, B and C) were designed for the differentiation of the wild-type (WT) and the T-DNA line (atpap2-8) and they were used for PCR screening of genomic DNA extracted from WT and the T-DNA line (b). Total RNA was extracted from 10-day-old seedlings grown on MS with 2% sucrose using the TRIzol RNA isolation method and were used for RT-PCR (c). 50 .mu.g of seedlings proteins were loaded for Western blotting studies, using the anti-AtPAP2 specific antiserum (Section 6.3).

[0049] FIG. 5 is the schematic diagram of the expression vector pBV-AtPAP2. CaMV 35S: 35S promoter of the cauliflower mosaic virus; NOS: polyadenylation signal of nopaline synthase gene; aadA: bacterial streptomycin/spectinomycin resistance gene encoding aminoglycoside-3''-adenyltransferase; pNOS:BAR: bialaphos resistance gene under the control of the nopaline synthase promoter; bom: basis of mobility from pBR322; ColE1: replication origin from pBR322; pVS1-REP: replication origin from pVS1; pVS1-STA: STA region from pVS1 plasmid; LB: left border T-DNA repeat; RB: right border T-DNA repeat. (Hajdukiewicz et al., 1994).

[0050] FIG. 6A shows the results of the Western blot analysis of the overexpression lines (OE), wild-type (WT), T-DNA and the complementation lines (CP) of AtPAP2 and FIG. 6B shows the results of the Western blot analysis of the overexpression lines (C-15) and wild-type (WT) of AtPAP15.

[0051] FIG. 7 shows the expression analysis of AtPAP2. The mRNA expression profile was analysed by the Spot History program of NASC (a). The protein expression profiles of 30 day old, soil-grown plant (b), seedlings germinated on MS agar (c) and 2 week old plants transferred to Pi-sufficient/Pi-deficient MS agar for 3 days (d), were analyzed by Western blotting using the anti-PAP2 antiserum.

[0052] FIG. 8 shows the growth performance of the wild-type, T-DNA and overexpression lines in soil. Seeds were germinated in MS agar with 2% sucrose for 10 days. Seedlings with 2 small visible rosette leaves (.about.1 mm) were transferred to soil and grown under 16 h/8 h light/dark cycles.

[0053] FIG. 9 shows the levels of sucrose and glucose in the rosette leaves of 21-day-old, soil grown seedlings.

[0054] FIG. 10 shows the recovery of various lines after prolonged darkness treatment. Seeds were germinated in MS agar with 2% sucrose for 10 days. Seedlings with 2 small visible rosette leaves (.about.1 mm) were transferred to soil and grown for 12 days under 16 h/8 h light/dark cycles. The lights of the growth chamber were then switched off for 12 days and the plants were allowed to recover under 16 h/8 h light/dark cycles for 1 week. n=9-12 per line.

[0055] FIG. 11. Detection of AtPAP2 protein in subcellular fractions by Western blotting. Mito.: Mitochondria; Chlorop.: Chloroplasts.

[0056] FIG. 12. shows a schematic representation of two vector constructs incorporating the AtPAP2 gene.

[0057] FIG. 13. shows Western blot analysis results for overexpression of AtPAP2 proteins missing the C-terminal motif.

DETAILED DESCRIPTION

5.1 Method of Speeding Up Plant Growth and Improving Crop Yield

[0058] The present disclosure provides a method that speeds up plant growth and elevates plant yields by introducing phosphatases with a C-terminal motif into plants. In an embodiment, the present disclosure relates to a class of genes of purple acid phosphatases, and their respectively encoded protein products, as well as fragments, derivatives, homologues, and variants thereof. Methods for introducing this class of genes into plants to speed up the growth rate of plants, to increase the sugar contents of plants, and to increase of yield of plants, are provided.

[0059] A group of purple acid phosphatases (PAPs) which carry seven conserved amino acid residues (shown in bold face) in the five conserved motifs XDXX (example GDXG (SEQ ID NO: 48)), XDXXY (SEQ ID NO: 49), GNH(D/E) (SEQ ID NOS: 50-51), XXXH (example ZXGH (SEQ ID NO: 52)), XHXH (SEQ ID NO: 53), where X is any amino acid and Z is any amino acid selected from L, I, V, F, and M, and a transmembrane-like motif at their C-termini were identified in the genomes of a number of plants (FIGS. 1, 2A, and 2B). The presence of the C-terminal transmembrane-like motif enables the localization of this group of PAPs to the membrane fraction (FIGS. 3 and 11). This property makes this group of PAPs differ from the other previously characterized PAPs because all previously characterized PAPs did not carry any C-terminal motif (FIGS. 2A, 2B, and 3). By using the protein sequence of a representative gene of this group, AtPAP2, to blast the NCBI database and various EST databases, a number of genomic or cDNA sequences were identified to encodes polypeptides that carry the five conserved motifs XDXX, XDXXY, GNH(D/E), XXXH, XHXH of PAPs and a transmembrane motif at their C-termini (FIG. 2B).

[0060] The introduction of a representative gene of this group of phosphatases, AtPAP2, into the genome of Arabidopsis by transgenic technology produced transgenic Arabidopsis that grew faster than the wild-type plants (FIG. 8), and the yield of seeds were elevated by approximately 40% (Table 3). However, transgenic plant that expressed AtPAP15 did not show these phenotypes. The sugar contents, including glucose and sucrose, in the leaf of the transgenic lines, were also found to be higher than that of the wild-types (FIG. 9).

[0061] Thus, this disclosure provides a method that speeds up plant growth and elevates plant yields by introducing phosphatases into plants. In an embodiment, a group of genes of purple acid phosphatases, and their respectively encoded protein products, as well as fragments, derivatives, homologues, and variants thereof are described.

5.2 Homologues, Derivatives, and Variants of Phosphatases

[0062] In addition to the nucleic acid molecules (SEQ ID NOS: 1, 3, 5, 7, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44 and 46) and polypeptides (SEQ ID NOS: 2, 4, 6, 8, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45 and 47) described in claims 9-16, the nucleic acid molecules and polypeptides also encompass those nucleic acid molecules and polypeptides having a common biological activity, similar or identical structural domain and/or having sufficient nucleotide sequence or amino acid identity (homologues) to those of the nucleic acid molecules and polypeptides described above.

[0063] Such common biological activities of the polypeptides include antigenicity, immunogenicity, catalytic activity especially phosphatase activity, ability to bind a Fe.sup.3+-Me.sup.2+ dimetal nuclear center, fold into or form a transmembrane-like C-terminal motif and other activities readily assayable by the skilled artisan.

[0064] A polypeptide that has a similar amino acid sequence (homologue) refers to a polypeptide that satisfied at least one of the following: (i) a polypeptide having an amino acid sequence that is one of at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, and at least about 95%, and at least about 98% identical and/or conservatively substituted to the amino acid sequence of a AtPAP2 (SEQ ID NO: 2) and/or other PAPs with a transmembrane-like C-terminal motif including SEQ ID NOS: 4, 6, 8, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45 and/or 47, a fragment of AtPAP2, and having at least one biological feature of the described polypeptides; (ii) a polypeptide encoded by a nucleotide sequence that is one of at least about 30%, at least about 40%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, and at least about 98% identical to the nucleotide sequence encoding AtPAP2 (SEQ ID NO: 1) and/or other PAPs with a transmembrane-like C-terminal motif including SEQ ID NOS: 3, 5, 7, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44 and/or 46, a fragment of AtPAP2 and having at least one structural and/or biological feature of AtPAP2; (iii) a polypeptide encoded by a nucleotide sequence that hybridizes under stringent conditions as defined herein to a nucleotide sequence encoding AtPAP2 (SEQ ID NO: 1) and/or other PAPs with a motif including SEQ ID NOS: 3, 5, 7, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44 and/or 46, a fragment of AtPAP2 and having at least one structural and/or biological feature of AtPAP2. A polypeptide with similar structure to AtPAP2, or a fragment of AtPAP2, refers to a polypeptide that has a similar secondary, tertiary, or quaternary structure of AtPAP2, a fragment of AtPAP2 and has at least one functional feature of a AtPAP2, including one or more of ability to bind a Fe.sup.3+-Me.sup.2+ dimetal nuclear center and fold into or form a transmembrane-like C-terminal motif. The structure of a polypeptide can be determined by methods known to those skilled in the art, including but not limited to, X-ray crystallography, nuclear magnetic resonance, and crystallographic electron microscopy.

[0065] Those having skill in the art will readily recognized that mutations, deletions or insertions can be made in any of the sequences disclosed herein, including SEQ ID NOS: 1-8 and 18-47, without affecting function. Sequences useful in practicing the embodiments include sequences having homology to SEQ ID NOS: 1-8 and 18-47 and being a protein, polypeptide, or polynucleotide coding for such protein or peptide having functionality to bind a dimetal nuclear center (Fe.sup.3+-Me.sup.2+) and being a protein, polypeptide, or polynucleotide coding for such protein or peptide having a C-terminal motif. That is, those skilled in the art will recognize that many mutations can be made to any of SEQ ID NOS: 1-8 and 18-47 without affecting the catalytic functionality nor interrupting the transmembrane-like C-terminal motif. Such modified sequences that maintain catalytic activity and a transmembrane-like C-terminal motif are defined as homologues to SEQ ID NOS: 1-8 and 18-47 and are including within the scope of useful sequences.

[0066] In one embodiment, such homologues can have about 30% or more identity to the sequences disclosed herein. In another embodiment, such homologues can have about 40% or more identity to the sequences disclosed herein. In yet another embodiment, such homologues can have about 50% or more identity to the sequences disclosed herein. In sill yet another embodiment, such homologues can have about 60% or more identity to the sequences disclosed herein. In even sill yet another embodiment, such homologues can have about 70% or more identity to the sequences disclosed herein. In a further embodiment, such homologues can have about 80% or more identity to the sequences disclosed herein. In yet a still further embodiment, homologues can have about 90% or more identity to the sequences disclosed herein. In a still further embodiment, homologues can have about 98% or more identity to the sequences disclosed herein.

[0067] Those having skill in the art will recognize that mutations can be made to proteins and peptides and/or to polynucleotides coding for protein and peptides or complementary thereto that substitute amino acid residue for other amino acids residues having similar chemical properties (conservative substitutions) and that such mutations are less likely to cause structural changes that affect functionality including catalytic activity and/or the function of a transmembrane-like C-terminal motif. Conservatively substituting amino acids are substituting an amino acid residue belong to any of the following 11 chemical groups with another amino acid from the same chemical group: (1) acidic (negatively charged) amino acids such as aspartic acid and glutamic acid; (2) basic (positively charged) amino acids such as arginine, histidine, and lysine; (3) neutral polar amino acids such as glycine, serine, threonine, cysteine, tyrosine, asparagine, and glutamine; (4) neutral nonpolar (hydrophobic) amino acids such as alanine, leucine, isoleucine, valine, proline, phenylalanine, tryptophan, and methionine; (5) amino acids having aliphatic side chains such as glycine, alanine, valine, leucine, and isoleucine; (6) amino acids having aliphatic-hydroxyl side chains such as serine and threonine; (7) amino acids having amide-containing side chains such as asparagine and glutamine; (8) amino acids having aromatic side chains such as phenylalanine, tyrosine, and tryptophan; (9) amino acids having basic side chains such as lysine, arginine, and histidine; (10) amino acids having sulfur-containing side chains such as cysteine and methionine; (11); amino acids having similar geometry and hydrogen bonding patterns such as aspartic acid, asparagine, glutamic acid and glutamine.

[0068] In one embodiment, homologues can have about 30% or more identity and/or conservative substitutions to the sequences disclosed herein. In another embodiment, homologues can have about 40% or more identity and/or conservative substitutions to the sequences disclosed herein. In yet another embodiment, homologues can have about 50% or more identity and/or conservative substitutions to the sequences disclosed herein. In still yet another embodiment, homologues can have about 60% or more identity and/or conservative substitutions to the sequences disclosed herein. In a further embodiment, homologues can have about 70% or more identity and/or conservative substitutions to the sequences disclosed herein. In a still further embodiment, homologues can have about 80% or more identity and/or conservative substitutions to the sequences disclosed herein. In still another embodiment, homologues can have about 90% or more identity and/or conservative substitutions to the sequences disclosed herein. In still another further embodiment, homologues can have about 98% or more identity and/or conservative substitutions to the sequences disclosed herein.

[0069] Embodiments further provide isolated nucleic acid molecules which comprise or consist of one or more of at least about 25, at least about 30, at least about 35, at least about 40, at least about 45, at least about 100, at least about 150, at least about 200, at least about 250, at least about 300, at least about 350, at least about 400, at least about 450, at least about 500, at least about 550, at least about 600, at least about 650, at least about 700, at least about 750, at least about 800, at least about 850, at least about 900, at least about 950, at least about 1000, at least about 1050, at least about 1100, at least about 1150, at least about 1200, at least about 1250, at least about 1300, at least about 1350, from about 500 to about 2000, from about 1000 to about 2000, from about 200 to about 500, from about 500 to about 1000, form about 1000 to about 1500, and from about 1500 to about 2000 nucleotides of the nucleotide sequences of SEQ ID NOS: 1, 3, 5, 7, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44 and 46, or a complement thereof encoding a protein or polypeptide having one or more activity of the amino acid sequences of their encoded proteins (SEQ ID NOS: 2, 4, 6, 8, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45 and 47). The activity includes one or more of antigenicity, immunogenicity, catalytic activity (e.g., phosphatase activity), ability to bind a Fe.sup.3+-Me.sup.2+ dimetal nuclear center, fold into or form a transmembrane-like C-terminal motif, and other activities readily assayable.

[0070] Embodiments provide isolated polypeptides or proteins consisting of an amino acid sequence that contains one of about 5, at least about 10, at least about 15, at least about 20, at least about 25, at least about 30, at least about 35, at least about 40, at least about 45, at least about 50, at least about 55, at least about 60, at least about 65, at least about 70, at least about 75, at least about 80, at least about 90, at least about 100, at least about 120, at least about 140, at least about 160, at least about 180, at least about 200, at least about 220, at least about 240, at least about 260, at least about 280, at least about 300, at least about 320, at least about 340, at least about 360, from about 250 to about 660, from about 350 to about 660, form about 450 to about 660, and form about 550 to about 660 amino acid bases in length of the relevant protein or polypeptide and having at least one functional feature of the protein or polypeptide, such functional features including ability to bind a Fe.sup.3+-Me.sup.2+ dimetal nuclear center and form a transmembrane-like C-terminal motif.

[0071] Additional embodiments are any of the phosphatases and homologues thereof with the identity and/or conservative substitutions to SEQ ID NOS: 1-8 and 18-47 described above that additionally consist of a protein, polypeptide, or polynucleotide encoding a protein having the five conserved motifs in purple acid phosphatases, including XDXX, XDXXY, GNH(D/E), XXXH, XHXH, where X is any amino acid. In one embodiment, the described phosphatases and homologues thereof consist of a protein, polypeptide, or polynucleotide encoding one of the sequences YHVCIGNHEYDF (SEQ ID NO: 54) and YHVCIGNHEYDW (SEQ ID NO: 55). In one embodiment, the described phosphatases and homologues thereof consist of a protein, polypeptide, or polynucleotide encoding a sequence of amino acid residues having one of the sequences YHVCIGNFIEYD(W/F) (SEQ ID NO: 54) and YHVCIGNHEYN(W/F) (SEQ ID NO: 55) or a protein, polypeptide, or polynucleotide encoding a homologue to one of the foregoing sequences with only conservative substitutions, as described above, to those sequences. In yet another embodiment, the described phosphatases and homologues thereof consist of a protein, polypeptide, or polynucleotide encoding a sequence of amino acid residues having one of the sequences GNHE (SEQ ID NO: 51) and GNHD (SEQ ID NO: 50). In still yet another embodiment, the described phosphatases and homologues thereof consist of a protein, polypeptide, or polynucleotide encoding a sequence of amino acid residues having one of the sequences GNHE (SEQ ID NO: 51) and GNHD (SEQ ID NO: 50) or a protein, polypeptide, or polynucleotide encoding a protein having a homologous sequence to one of the foregoing sequences SEQ 1N NOS: 50-51 with only conservative substitutions.

[0072] Additional embodiments are any of the phosphatases and homologues thereof with the identity and/or conservative substitutions to SEQ ID NOS: 1-8 and 18-47 described above that additionally consist of a protein, polypeptide, or polynucleotide encoding a sequence having at least about 70% or more identity and/or conservative substitutions to amino acid residues 302-315 of SEQ ID NO: 2 when such sequence is aligned with SEQ ID NO: 2. In another embodiment, the described phosphatases and homologues thereof consist of a protein, polypeptide, or polynucleotide encoding a protein having about 80% or more identity and/or conservative substitutions to amino acid residues 302-315 of SEQ ID NO: 2 when such sequence is aligned with SEQ ID NO: 2. In another embodiment, the described phosphatases and homologues consist of a protein, polypeptide, or polynucleotide encoding a sequence of amino acid residues having at least about 70% or more identity to the sequence HIGDISYARGYSW (SEQ ID NO: 56). In another embodiment, the described phosphatases and homologues thereof consist of a protein, polypeptide, or polynucleotide encoding a sequence of amino acid residues having the sequence HIGDISYARGYSW (SEQ ID NO: 56) or a protein, polypeptide, or polynucleotide encoding a protein having a homologous sequence to the foregoing sequences with only conservative substitutions, as described above, to those sequences.

[0073] In another embodiment, the described phosphatases and homologues thereof consist of a protein, polypeptide, or polynucleotide encoding a sequence of amino acid residues having at least about 70% or more identity to the sequences KEKLTVSFVGNHDGEVHD (SEQ ID NO: 57), KERLTLSYVGNHDGEVHD (SEQ ID NO: 58), REKLTLTYVGNHDGQVHD (SEQ ID NO: 59), and KEKLTLTYIGNHDGQVHD (SEQ ID NO: 60). In still yet another embodiment, the described phosphatases and homologues thereof consist of a protein, polypeptide, or polynucleotide encoding a sequence of amino acid residues having one or more of the sequences KEKLTVSFVGNHDGEVHD (SEQ ID NO: 57), KERLTLSYVGNHDGEVHD (SEQ ID NO: 58), REKLTLTYVGNHDGQVHD (SEQ ID NO: 59), and KEKLTLTYIGNHDGQVHD (SEQ ID NO: 60) or a protein, polypeptide, or polynucleotide encoding a protein having a homologous sequence to one of the foregoing sequences with only conservative substitutions, as described above, to those sequences.

[0074] In a further embodiment, the described phosphatases and homologues thereof consist of a protein, polypeptide, or polynucleotide encoding a sequence of amino acid residues having the sequence (F/Y)(V/I)GNHDGXXH (SEQ ID NOS: 61-64), where the first residue of the sequence can be F or Y and the second residue of the sequence can be V or I. In a still further embodiment, the described phosphatases and homologues thereof consist of a protein, polypeptide, or polynucleotide encoding a sequence of amino acid residues having the sequence (F/Y)(V/I)GNHDGXXH (SEQ ID NOS: 61-64), where the first residue of the sequence can be F or Y and the second residue of the sequence can be V or I, or a protein, polypeptide, or polynucleotide encoding a protein having a homologous sequence to the foregoing sequence with only conservative substitutions, as described above, to the foregoing sequence. In a yet still further embodiment, the described phosphatases and homologues thereof consist of a protein, polypeptide, or polynucleotide encoding a sequence of amino acid residues having the sequence (F/Y)(V/I)GNHDGXXH (SEQ ID NOS: 61-64), where the first residue of the sequence can be F or Y and the second residue of the sequence can be V or I, or a protein, polypeptide, or polynucleotide encoding a protein having a homologous sequence having at least about 70% identity and/or conservative substitution, as described above, to the foregoing sequence.

[0075] In one embodiment, the described phosphatases and homologues thereof consist of a protein, polypeptide, or polynucleotide encoding a sequence of amino acid residues having at least about 60% or more identity and/or conservative substitutions to amino acid residues 614-636 of SEQ ID NO: 2 (SEQ ID NO: 65) and/or having at least about 60% or more identity and/or conservative substitutions to the sequence of 23 amino acid residues of SEQ ID NOS: 4, 6, 8, 19, 21, 23, 25, 27, 29, 31, 33 and 47 aligned with residues 614-636 of SEQ ID NO: 2 (SEQ ID NO: 65), and where amino acid residues aligned with amino acid residues 614-636 of SEQ ID NO: 2 are predicted to form a transmembrane-like C-terminal motif by TMHMM analysis (http://www.cbs.dtu.dk/services/TMHMM/). In one embodiment, the described phosphatases and homologues thereof consist of a protein, polypeptide, or polynucleotide encoding a sequence of amino acid residues having at least about 70% or more identity and/or conservative substitutions to amino acid residues 614-636 of SEQ ID NO: 2 (SEQ ID NO: 65) and/or having at least about 60% or more identity and/or conservative substitutions to the sequence of 23 amino acid residues of SEQ ID NOS: 4, 6, 8, 19, 21, 23, 25, 27, 29, 31, 33 and 47 aligned with residues 614-636 of SEQ ID NO: 2 (SEQ ID NO: 65), and where amino acid residues aligned with amino acid residues 614-636 of SEQ ID NO: 2 (SEQ ID NO: 65) are predicted to form a transmembrane-like C-terminal motif by TMHMM analysis (http://www.cbs.dtu.dk/services/TMHMM/). In one embodiment, the described phosphatases and homologues thereof consist of a protein, polypeptide, or polynucleotide encoding a sequence of amino acid residues having at least about 80% or more identity and/or conservative substitutions to amino acid residues 614-636 of SEQ ID NO: 2 and/or having at least about 60% or more identity and/or conservative substitutions to the sequence of 23 amino acid residues of SEQ ID NOS: 4, 6, 8, 19, 21, 23, 25, 27, 29, 31, 33 and 47 aligned with residues 614-636 of SEQ ID NO: 2 (SEQ ID NO: 65), and where amino acid residues aligned with amino acid residues 614-636 of SEQ ID NO: 2 (SEQ ID NO: 65) are predicted to form a transmembrane-like C-terminal motif by TMHMM analysis (http://www.cbs.dtu.dk/services/TMHMM/). In one embodiment, the described phosphatases and homologues thereof consist of a protein, polypeptide, or polynucleotide encoding a sequence of amino acid residues having at least about 90% or more identity and/or conservative substitutions to amino acid residues 614-636 of SEQ ID NO: 2 and/or having at least about 90% or more identity and/or conservative substitutions to the sequence of 23 amino acid residues of SEQ ID NOS: 4, 6, 8, 19, 21, 23, 25, 27, 29, 31, 33 and 47 aligned with residues 614-636 of SEQ ID NO: 2, and where amino acid residues aligned with amino acid residues 614-636 of SEQ ID NO: 2 are predicted to form a transmembrane-like C-terminal motif by TMHMM analysis (http://www.cbs.dtu.dk/services/TMHMM/).

[0076] In one embodiment, the described phosphatases or phosphatase genes consist of a protein, polypeptide, or polynucleotide encoding the sequence (L/M/V)-(L/M/V)-Z-(G/A)-(V/A/L)-Z-Z-G-(F/Y)-X-Z-G (SEQ ID NO: 66), where Z is any of the hydrophobic residues L, I, V, F, and M. In another embodiment, the described phosphatase or phosphatase genes consist of a protein, polypeptide, or polynucleotide encoding the sequence (L/M/V)-(L/M/V)-Z-(G/A)-(V/A/L)-Z-Z-G-(F/Y)-X-Z-G (SEQ ID NO: 66), or a protein, polypeptide, or polynucleotide encoding a sequence having at least 70% identity and/or conservative substitution to the foregoing sequence.

[0077] Embodiments also encompass derivatives of the disclosed polypeptides. For example, but not by way of limitation, derivatives may include peptides or proteins that have been modified, e.g., by glycosylation, acetylation, pegylation, phosphorylation, amidation, derivatization by known protecting/blocking groups, proteolytic cleavage, linkage to a cellular ligand or other protein, etc. Any of numerous chemical modifications may be carried out by known techniques, including, but not limited to, specific chemical cleavage, acetylation, formylation, etc. Additionally, the derivative may contain one or more non-classical amino acids.

[0078] In another aspect, an isolated nucleic acid molecule encodes a variant of a polypeptide in which the amino acid sequences have been modified by genetic engineering so that biological activities of the polypeptides are either enhanced or reduced, or the local structures thereof are changed without significantly altering the biological activities. In one aspect, these variants can act as either agonists or as antagonists. An agonist can retain substantially the same or a portion of the biological activities of the polypeptides and an antagonist can inhibit one or more of the activities of the polypeptides. Such modifications include amino acid substitution, deletion, and/or insertion. Amino acid modifications can be made by any method known in the art and various methods are available to and routine for those skilled in the art.

[0079] For example, mutagenesis may be performed in accordance with any of the techniques known in the art including, but not limited to, synthesizing an oligonucleotide having one or more modifications within the sequence of a given polypeptide to be modified. Site-specific mutagenesis can be conducted using specific oligonucleotide sequences which encode the nucleotide sequence containing the desired mutations in addition to a sufficient number of adjacent nucleotides in the polypeptide. Such oligonucleotides can serve as primers which can form a stable duplex on both sides of the deletion junction being traversed. Typically, a primer of about 15 to about 75 nucleotides or more in length is preferred, with about 10 to about 25 or more residues on both sides of the junction of the sequence being altered. A number of such primers introducing a variety of different mutations at one or more positions can be used to generate a library of mutants.

[0080] The technique of site-specific mutagenesis is well known in the art, as described in various publications (e.g., Kunkel et al., Methods Enzymol., 154:367-82, 1987, which is hereby incorporated by reference in its entirety). In general, site-directed mutagenesis is performed by first obtaining a single-stranded vector or melting apart of two strands of a double stranded vector which includes within its sequence a DNA sequence which encodes the desired peptide. An oligonucleotide primer bearing the desired mutated sequence is prepared, generally synthetically. This primer is then annealed with the single-stranded vector, and subjected to DNA polymerizing enzymes such as T7 DNA polymerase, in order to complete the synthesis of the mutation-bearing strand. Thus, a heteroduplex is formed wherein one strand encodes the original non-mutated sequence and the second strand bears the desired mutation. This heteroduplex vector is then used to transform or transfect appropriate cells, such as E. coli cells, and clones are selected which include recombinant vectors bearing the mutated sequence arrangement. As will be appreciated, the technique typically employs a phage vector which exists in both a single stranded and double stranded form. Typical vectors useful in site-directed mutagenesis include vectors such as the M13 phage. These phages are readily commercially available and their use is generally well known to those skilled in the art. Double stranded plasmids are also routinely employed in site directed mutagenesis which eliminates the step of transferring the gene of interest from a plasmid to a phage.

[0081] Alternatively, the use of PCR with commercially available thermostable enzymes such as Taq DNA polymerase may be used to incorporate a mutagenic oligonucleotide primer into an amplified DNA fragment that can then be cloned into an appropriate cloning or expression vector. See, e.g., Tomic et al., Nucleic Acids Res., 18(6):1656, 1987, and Upender et al., Biotechniques, 18(1):29-30, 32, 1995, for PCR-mediated mutagenesis procedures, which are hereby incorporated in their entireties. PCR employing a thermostable ligase in addition to a thermostable polymerase may also be used to incorporate a phosphorylated mutagenic oligonucleotide into an amplified DNA fragment that may then be cloned into an appropriate cloning or expression vector (see e.g., Michael, Biotechniques, 16(3):410-2, 1994, which is hereby incorporated by reference in its entirety).

[0082] Other methods known to those skilled in art of producing sequence variants of a given polypeptide or a fragment thereof can be used. For example, recombinant vectors encoding the amino acid sequence of the polypeptide or a fragment thereof may be treated with mutagenic agents, such as hydroxylamine, to obtain sequence variants.

[0083] Optionally, the amino acid residues to be modified are surface exposed residues. Additionally, in making amino acid substitutions, preferably the amino acid residue to be substituted is a conservative amino acid substitution, for example, a polar residue is substituted with a polar residue, a hydrophilic residue with a hydrophilic residue, hydrophobic residue with a hydrophobic residue, a positively charged residue with a positively charged residue, or a negatively charged residue with a negatively charged residue. Moreover, the amino acid residue that can be modified is not highly or completely conserved across strains or species and/or is critical to maintain the biological activities of the protein.

[0084] Accordingly, included in the scope of the disclosure are nucleic acid molecules encoding a polypeptide of the invention that contains amino acid modifications that are not critical to its biological activity.

5.3 Fusion Proteins

[0085] The present disclosure further encompasses fusion proteins in which the polypeptides or fragments thereof, are recombinantly fused or chemically conjugated (e.g., covalent and non-covalent conjugations) to heterologous polypeptides (i.e., an unrelated polypeptide or portion thereof, preferably at least 10, at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90 or at least 100 amino acids of the polypeptide) to generate fusion proteins. The fusion can be direct, but may occur through linker sequences.

[0086] In one aspect, the fusion protein comprises a polypeptide which is fused to a heterologous signal sequence at its N-terminus. For example, the signal sequence naturally found in the polypeptide can be replaced by a signal sequence which is derived from a heterologous origin. Various signal sequences are commercially available.

[0087] In another embodiment, a polypeptide can be fused to tag sequences, e.g., a hexa-histidine peptide, among others, many of which are commercially available. As described in Gentz et al., 1989, Proc. Natl. Acad. Sci. USA, 86:821-824, for instance, hexa-histidine provides for convenient purification of the fusion protein. Other examples of peptide tags are the hemagglutinin "HA" tag, which corresponds to an epitope derived from the influenza hemagglutinin protein (Wilson et al., 1984, Cell, 37:767) and the "flag" tag (Knappik et al., 1994, Biotechniques, 17(4):754-761). These tags are especially useful for purification of recombinantly produced polypeptides.

[0088] Fusion proteins can be produced by standard recombinant DNA techniques or by protein synthetic techniques, e.g., by use of a DNA synthesizer. For example, a nucleic acid molecule encoding a fusion protein can be synthesized by conventional techniques including automated DNA synthesizers. Alternatively, PCR amplification of gene fragments can be carried out using anchor primers which give rise to complementary overhangs between two consecutive gene fragments which can subsequently be annealed and reamplified to generate a chimeric gene sequence (see, e.g., Current Protocols in Molecular Biology, Ausubel et al., eds., John Wiley & Sons, 1992).

[0089] The nucleotide sequence coding for a fusion protein can be inserted into an appropriate expression vector, i.e., a vector which contains the necessary elements for the transcription and translation of the inserted protein-coding sequence.

[0090] In a specific embodiment, the expression of a fusion protein is regulated by an inducible promoter.

5.4 Preparation of Transgenic Plants

[0091] Carbon flow is a key process in plant biology and high energy carbon molecules (e.g. glucose) were harvested by plant through photosynthesis. The carbon molecules were then converted into more complicated carbohydrate molecules such as starch, cellulose, etc. Cellulose is the major component of cell wall and starch is the major storage form of glucose in plant cells and plant seeds. Therefore, the efficiency and/or the equilibrium of the carbon flow process become a limiting factor for plant growth and crop yield.

[0092] The present disclosure is based upon the discovery that overexpression of a membrane-bound phosphatase can enhance the growth performance of plants by altering its carbon metabolism, as indicated by, for example, a faster growth rate, a higher sugar contents, and a higher seed yield.

[0093] In an embodiment, the present disclosure provides a transgenic plant containing a nucleic acid molecule that encodes and expresses a phosphatase having a C-terminal transmembrane-like domain. The transgenic plants disclosed herein have faster growth rate, and higher seed yield to comparable unengineered plants i.e. same species (strain). In a specific embodiment, such a phosphatase is from a plant species having a phosphatase activity and a C-terminal motif. In another embodiment, a transgenic plant disclosed herein comprises a nucleic acid molecule encoding phosphatase and expresses AtPAP2 (SEQ ID NO: 2) and/or other PAPs with a C-terminal motif including one or more of SEQ ID NOS: 4, 6, 8, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45 and 47. In another embodiment, the phosphatase is expressed on cellular membrane, for example, the ER or the Golgi apparatus. Such a membrane expression of a phosphatase in plants can be achieved by fusing onto the C-terminus with a nucleotide sequence encoding a C-terminal motif peptide which can efficiently attach the phosphatase upon translation thereof from the cells of a given plant. Accordingly, in another embodiment, a transgenic plant comprises a nucleic acid molecule encoding phosphatase and expresses AtPAP2 (SEQ ID NO: 2) and/or other PAPs with a C-terminal motif including SEQ ID NOS: 4, 6, 8, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45 and 47, except that all or a portion, particularly an N-terminal portion, of amino acid residues 1 to 80, preferably all or a portion of amino acid residues 1 to 30, of SEQ ID NO: 2 or all or a portion, particularly an N-terminal portion, of amino acid residues 1 to 80, preferably all or a portion of amino acid residues 1 to 30, of SEQ ID NOS: 4, 6, 8, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45 or 47, are replaced by a heterologous plant signal peptide by genetic engineering. In such a transgenic plant, the phosphatases are directed to various organelles/compartments of the cells. In another embodiment, a transgenic plant comprises a nucleic acid molecule encoding phosphatase and expresses homologues, derivatives, and/or fragments thereof having at least one functional feature and/or structural feature of a phosphatase polypeptide. In all embodiments where all or a portion of the N-terminal portion of SEQ ID NOS: 4, 6, 8, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45 and/or 47 are replaced, the embodiments include homologues to such sequences, as described above, having at least one functional feature and/or structural feature of a phosphatase polypeptide. In yet another embodiment, a transgenic plant comprises a nucleic acid molecule that hybridizes under stringent conditions, as defined herein, to a nucleic acid molecule having the sequence of SEQ ID NOS: 1, 3, 5, 7, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44 or 46, or a complement thereof, and encodes a protein or polypeptide that exhibits at least one structural and/or functional feature of the disclosed phosphatase polypeptides. Specifically, the production of transgenic plant that overexpressed a membrane-bound phosphatase, which contributes to improving plant physiology, such as plant growth rate and characteristics, for example, in seed yield, is provided.

[0094] Accordingly, also provided are chimeric gene constructs for genetic modification of plants to increase their growth rate and improve the yield. The chimeric gene constructs comprise a sequence that encodes substantially solely for a phosphatase enzyme that carry a C-terminal transmembrane-like motif. Such a phosphatase enzyme can be derived from the purple acid phosphatase family. In a specific embodiment, the chimeric gene constructs comprise a nucleic acid having the sequence of SEQ ID NOS: 1, 3, 5, 7, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44 or 46. In another embodiment, the chimeric gene constructs comprise a nucleic acid molecule that encodes a homologue or fragment thereof having at least one functional feature and/or structural feature of a phosphatase polypeptide. In another specific embodiment, the chimeric gene constructs comprise a sequence that hybridizes under stringent conditions, as defined herein, to a nucleic acid having the sequence of SEQ ID NOS: 1, 3, 5, 7, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44 or 46, or a complement thereof, wherein the sequence encodes a protein or a polypeptide that exhibits at least one structural and/or functional feature of the phosphatase polypeptides. Furthermore, the phosphatases encoded by the nucleic acid molecules contained in the chimeric gene constructs can be any other phosphatases that have similar structural characteristics, such as having a C-terminal transmembrane-like motif, to those of the phosphatases described herein. Such phosphatase include, but not limited to, the following polypeptides: Purple acid phosphatases from Zea mays (Accession No: ACG47621); and Oryza sativa (Accession No: BAC15853.1).

[0095] The phosphatase-coding sequence is operatively linked to upstream and downstream regulatory components, preferably heterologous to the phosphatase sequence; for example CMV 35S promoter, which acts to cause expression of the gene (production of the enzyme) in plant cells (see Section 6.2). When a construct containing a gene for a phosphatase according to this disclosure, is introduced into plant cells by a conventional transformation method, such as microparticle bombardment, Agrobacterium infection, or microinjection, the gene is expressed in the cells under the control of the regulatory sequences. The expressed phosphatase successfully interacts with the biosynthetic machinery that is naturally present in the plant cells to alter the carbon metabolism. By altering the carbon metabolism, the method described herein also favors the growth rate of the plant, resulting in faster growth rate and higher yield. Thus, the time required for the maturation of the plant and the time required for flowering is shortened. Also provided are methods for increasing growth rate and yield of plants, comprising the step of inserting into such plant cells or the cells of such whole plants a chimeric gene construct.

[0096] In specific embodiments, Arabidopsis (see Section 6) was adopted as the model system. An overexpression construct the gene coding for phosphatase were introduced into Arabidopsis.

[0097] In an embodiment, the phosphatase from Arabidopsis is used. The results obtained with this disclosure indicate that the growth rate and the seed yield of transgenic Arabidopsis were enhanced by overexpressing this gene (see Section 6.5 and FIG. 8 and Table 3).

[0098] While any plant species can be modified using the expression cassette and methods described herein, preferably included without limitation are species from the following genera with representative species in parentheses:

[0099] Monocots: genera Asparagus (asparagus), Bromus (cheatgrass), Hemerocallis (daylily), Hordeum (barley), Lolium (ryegrass), Oryza (rice), Panicum (Switchgrass), Pennisetum (fountaingrass), Saccharum (Sugar cane), Sorghum, Trigonella (fenu grass), Triticum (wheat), Zea (corn); and

[0100] Dicots: genera Antirrhinum (flower sp.), Arabidopsis (thaliana), Arachis (peanut), Atropa (deadly nightshade), Brassica (rapeseed), Browallia, Capsicum (pepper), Carthamus (safflower), Cichorium (chicory), Citrus (orange, lemon), Chrysanthemum, Cucumis (cucumber), Datura (thorn apple), Daucus (carrot), Digitalis (foxglove), Fragaria (strawberry), Geranium (flower sp.), Glycine (soybean), Helianthus (sunflower), Hyscyamus, Ipomoea (morning glory), Latuca (lettuce), Linum (linseed), Lotus (flower sp.), Lycopersicon (tomato), Majorana, Malva (cotton), Manihot, Medicago (alfalfa), Nemesia, Nicotiana (tobacco), Onobrychis, Pelargonium (citrosa), Petunia (flower sp.), Ranunculus (flower sp.), Raphanus (radishes), Salpiglossis, Senecio (flower sp.), Sinapis (albae semen), Solanum (potato), Trifolium (clovers), Vigna (mungbean, faba bean), Vitis (grape).

[0101] Genetic engineering of plants can be achieved in several ways. The most common method is Agrobacterium-mediated transformation. In this method, A. tumefaciens, which in nature infects plants by inserting tumor causing genes into a plant's genome, is altered. Selected genes are engineered into the T-DNA of the bacterial Ti (tumor-inducing) plasmid of A. tumefaciens in laboratory conditions so that they become integrated into the plant chromosomes when the T-DNA is transferred to the plant by the bacteria's own internal transfer mechanisms. The only essential parts of the T-DNA are its two small (25 base pair) border repeats, at least one of which is needed for plant transformation. The bacterial genes encoding for plant hormones that promote tumor growth are excised from the T-DNA and replaced with a sequence of DNA that typically contains: a selectable marker (e.g. an antibiotic-resistance gene; usually kanamycin resistance), a restriction site--a site with a specific sequence of nucleotides where a restriction enzyme will cut the DNA, and the desired genes to be incorporated into the plant (B. Tinland, 1996. The integration of T-DNA into plant genomes. Trends in Plant Science 1, 178-184; D. Grierson (ed.) 1991. Plant Genetic Engineering. Blackie, Glasgow). Agrobacterium can be added to plant protoplasts (plant cells with cell walls removed) in culture, that are then allowed to regenerate cell walls at which point non-transformed plants are killed with antibiotics for which the transformed plants have been given resistance genes. Plantlets are then regenerated from the surviving transformed cells using standard plant tissue culture techniques. In an alternative technique, sterile disks or fragments of vegetative portions of plants are place in liquid culture medium with Agrobacterium, then hormones are used to induce rooting thereby regenerate plantlets which are grown on selection media. A third technique for delivering genes is possible for some plants such as Arabidopsis where the Agrobacterium or even "naked" DNA can be infused through the seed coat to cause transformation (Clough S J and Bent A F, 1998. Floral dip: a simplified method for Agrobacterium-mediated transformation of Arabidopsis thaliana. Plant J 16:735-43).

[0102] The biolistic method for genetic engineering of plants was developed more recently and is becoming more widely employed. In this method, very small particles (microprojectiles) of tungsten or gold coated with biologically active DNA are propelled at high-velocities into plant cells using an electrostatic pulse, air pressure, or gunpowder percussion. As the particles pass through the cell, the DNA dissolves and can then integrate into the genome of that cell and its progeny. It has been demonstrated this method can produce stable transformants (Christou, P., et al., 1988. Stable transformation of soybean callus by DNA-coated gold particles, Plant Physiology 87:671-674). The method can be practiced on whole plants and is particularly effective on meristematic tissue. It is also capable of delivering DNA either to the nucleus or into mitochondria (Johnston, S. A., et al., 1988. Mitochondrial transformation in yeast by bombardment with microprojectiles (Science 240, 1538-41) and chloroplasts (Svab, Z., et al., 1990, Stable transformation of plastids in higher plants, Proc Natl Acad. Sci. USA 87, 8526-8530).

[0103] The electroporation method of plant genetic engineering has met with less success. In this technique, protoplasts in culture take up pure DNA when treated with certain membrane-active agents or with electroporation, a rapid pulse of high-voltage direct current. Once the DNA has entered the protoplast it can be integrated into the cells genome. Standard tissue culture techniques are then used to regenerate transgenic plants.

[0104] The microinjection method of plant genetic engineering is perhaps the most difficult. In this method, DNA is microinjected into target plant cells using very thin glass needles in a method similar to that used with animals. The technique is laborious, ineffective, and impractical for generating large numbers of transgenic plants.

[0105] The method chosen for genetically engineering plants is most often dependent on the targeted plant species and which methods have been proven effective therein.

5.5 Preparation of Antibodies

[0106] Antibodies which specifically recognize one of the described phosphatase polypeptides or fragments thereof can be used for detecting, screening, and isolating the polypeptide of the invention or fragments thereof, or similar sequences that might encode similar enzymes from the other organisms. For example, in one specific embodiment, an antibody which immunospecifically binds AtPAP2 or fragments thereof can be used for various in vitro detection assays, including enzyme-linked immunosorbent assays (ELISA), radioimmunoassays, Western blot, etc., for the detection of the polypeptide of the invention or fragments, derivatives, homologues, or variants thereof, or similar molecules having the similar enzymatic activities as the phosphatase polypeptides, in samples, for example, a biological material, including plant cells, plants, food, drinks, or any materials derived from plants.

[0107] Antibodies specific for the described phosphatase polypeptides can be generated by any suitable method known in the art. Polyclonal antibodies to an antigen-of-interest can be produced by various procedures well known in the art. For example, an antigen derived from the phosphatase polypeptide can be administered to various host animals including, but not limited to, rabbits, mice, rats, etc., to induce the production of antisera containing polyclonal antibodies specific for the antigen. Various adjuvants may be used to increase the immunological response, depending on the host species, and include but are not limited to, Freund's (complete and incomplete) adjuvant, mineral gels such as aluminum hydroxide, surface active substances such as lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpet hemocyanins, dinitrophenol, and potentially useful adjuvants for humans such as BCG (Bacille Calmette-Guerin) and Corynebacterium parvum. Such adjuvants are also well known in the art.

[0108] Monoclonal antibodies can be prepared using a wide variety of techniques known in the art including the use of hybridoma, recombinant, and phage display technologies, or a combination thereof. For example, monoclonal antibodies can be produced using hybridoma techniques including those known in the art and taught, for example, in Harlow et al., Antibodies: A Laboratory Manual, (Cold Spring Harbor Laboratory Press, 2nd ed. 1988); Hammerling, et al., in: Monoclonal Antibodies and T-Cell Hybridomas, pp. 563-681 (Elsevier, N.Y., 1981) (both of which are incorporated by reference in their entireties). The term "monoclonal antibody" as used herein is not limited to antibodies produced through hybridoma technology. The term "monoclonal antibody" refers to an antibody that is derived from a single clone, including any eukaryotic, prokaryotic, or phage clone, and not the method by which it is produced.

[0109] Methods for producing and screening for specific antibodies using hybridoma technology are routine and well known in the art. In a non-limiting example, mice can be immunized with an antigen of interest or a cell expressing such an antigen. Once an immune response is detected, e.g., antibodies specific for the antigen are detected in the mouse serum, the mouse spleen is harvested and splenocytes isolated. The splenocytes are then fused by well known techniques to any suitable myeloma cells. Hybridomas are selected and cloned by limiting dilution. The hybridoma clones are then assayed by methods known in the art for cells that secrete antibodies capable of binding the antigen. Ascites fluid, which generally contains high levels of antibodies, can be generated by inoculating mice intraperitoneally with positive hybridoma clones.

[0110] Antibody fragments which recognize specific epitopes may be generated by known techniques. For example, Fab and F(ab').sub.2 fragments may be produced by proteolytic cleavage of immunoglobulin molecules, using enzymes such as papain (to produce Fab fragments) or pepsin (to produce F(ab').sub.2 fragments). F(ab').sub.2 fragments contain the complete light chain, and the variable region, the CH1 region and the hinge region of the heavy chain.

[0111] The antibodies or fragments thereof can be also produced by any method known in the art for the synthesis of antibodies, in particular, by chemical synthesis or preferably, by recombinant expression techniques.

[0112] The nucleotide sequence encoding an antibody may be obtained from any information available to those skilled in the art (i.e., from Genbank, the literature, or by routine cloning). If a clone containing a nucleic acid encoding a particular antibody or an epitope-binding fragment thereof is not available, but the sequence of the antibody molecule or epitope-binding fragment thereof is known, a nucleic acid encoding the immunoglobulin may be chemically synthesized or obtained from a suitable source (e.g., an antibody cDNA library, or a cDNA library generated from, or nucleic acid, preferably poly A+ RNA, isolated from any tissue or cells expressing the antibody, such as hybridoma cells selected to express an antibody) by PCR amplification using synthetic primers hybridizable to the 3' and 5' ends of the sequence or by cloning using an oligonucleotide probe specific for the particular gene sequence to identify, e.g., a cDNA clone from a cDNA library that encodes the antibody. Amplified nucleic acids generated by PCR may then be cloned into replicable cloning vectors using any method well known in the art.

[0113] Once the nucleotide sequence of the antibody is determined, the nucleotide sequence of the antibody may be manipulated using methods well known in the art for the manipulation of nucleotide sequences, e.g., recombinant DNA techniques, site directed mutagenesis, PCR, etc. (see, for example, the techniques described in Sambrook et al., supra; and Ausubel et al., eds., 1998, Current Protocols in Molecular Biology, John Wiley & Sons, NY, which are both incorporated by reference herein in their entireties), to generate antibodies having a different amino acid sequence by, for example, introducing amino acid substitutions, deletions, and/or insertions into the epitope-binding domain regions of the antibodies or any portion of antibodies which may enhance or reduce biological activities of the antibodies.

[0114] Recombinant expression of an antibody requires construction of an expression vector containing a nucleotide sequence that encodes the antibody. Once a nucleotide sequence encoding an antibody molecule or a heavy or light chain of an antibody, or portion thereof has been obtained, the vector for the production of the antibody molecule may be produced by recombinant DNA technology using techniques well known in the art as discussed in the previous sections. Methods which are well known to those skilled in the art can be used to construct expression vectors containing antibody coding sequences and appropriate transcriptional and translational control signals. These methods include, for example, in vitro recombinant DNA techniques, synthetic techniques, and in vivo genetic recombination. The nucleotide sequence encoding the heavy-chain variable region, light-chain variable region, both the heavy-chain and light-chain variable regions, an epitope-binding fragment of the heavy- and/or light-chain variable region, or one or more complementarity determining regions (CDRs) of an antibody may be cloned into such a vector for expression. Thus-prepared expression vector can be then introduced into appropriate host cells for the expression of the antibody. Accordingly, embodiments include host cells containing a polynucleotide encoding an antibody specific for the disclosed phosphatase polypeptides or fragments thereof.

[0115] The host cell can be co-transfected with two expression vectors, the first vector encoding a heavy chain derived polypeptide and the second vector encoding a light chain derived polypeptide. The two vectors may contain identical selectable markers which enable equal expression of heavy and light chain polypeptides or different selectable markers to ensure maintenance of both plasmids. Alternatively, a single vector may be used which encodes, and is capable of expressing, both heavy and light chain polypeptides. In such situations, the light chain should be placed before the heavy chain to avoid an excess of toxic free heavy chain (Proudfoot, 1986, Nature, 322:52; and Kohler, 1980, Proc. Natl. Acad. Sci. USA, 77:2197). The coding sequences for the heavy and light chains may comprise cDNA or genomic DNA.

[0116] In another embodiment, antibodies can also be generated using various phage display methods known in the art. In phage display methods, functional antibody domains are displayed on the surface of phage particles which carry the polynucleotide sequences encoding them. In a particular embodiment, such phage can be utilized to display antigen binding domains, such as Fab and Fv or disulfide-bond stabilized Fvs, expressed from a repertoire or combinatorial antibody library (e.g., human or murine). Phage expressing an antigen binding domain that binds the antigen of interest can be selected or identified with antigen, e.g., using labeled antigen or antigen bound or captured to a solid surface or bead. Phages used in these methods are typically filamentous phage, including fd and M13. The antigen binding domains are expressed as a recombinantly fused protein to either the phage gene III or gene VIII protein. Examples of phage display methods that can be used to make the immunoglobulins, or fragments thereof, include those disclosed in Brinkman et al., 1995, J. Immunol. Methods 182:41-50; Ames et al., 1995, J. Immunol. Methods 184:177-186; Kettleborough et al., 1994, Eur. J. Immunol., 24:952-958; Persic et al., 1997, Gene, 187:9-18; Burton et al., 1994, Advances in Immunology 57:191-280; PCT application No. PCT/GB91/01134; PCT publications WO 90/02809; WO 91/10737; WO 92/01047; WO 92/18619; WO 93/11236; WO 95/15982; WO 95/20401; and U.S. Pat. Nos. 5,698,426; 5,223,409; 5,403,484; 5,580,717; 5,427,908; 5,750,753; 5,821,047; 5,571,698; 5,427,908; 5,516,637; 5,780,225; 5,658,727; 5,733,743 and 5,969,108; each of which is incorporated herein by reference in its entirety.

[0117] As described in the above documents, after phage selection, the antibody coding regions from the phage can be isolated and used to generate whole antibodies, including human antibodies, or any other desired fragments, and expressed in any desired host, including mammalian cells, insect cells, plant cells, yeast, and bacteria, e.g., as described in detail below. For example, techniques to recombinantly produce Fab, Fab' and F(ab').sub.2 fragments can also be employed using methods known in the art such as those disclosed in PCT publication WO 92/22324; Mullinax et al., 1992, BioTechniques 12(6):864-869; and Sawai et al., 1995, AJRI 34:26-34; and Better et al., Science, 240:1041-1043, 1988 (each of which is incorporated by reference in its entirety). Examples of techniques which can be used to produce single-chain Fvs and antibodies include those described in U.S. Pat. Nos. 4,946,778 and 5,258,498; Huston et al., 1991, Methods in Enzymology 203:46-88; Shu et al., 1993, PNAS 90:7995-7999; and Skerra et al., 1988, Science 240:1038-1040.

[0118] Once an antibody molecule has been produced by any methods described above, it may then be purified by any method known in the art for purification of an immunoglobulin molecule, for example, by chromatography (e.g., ion exchange, affinity, particularly by affinity for the specific antigen after Protein A or Protein G purification, and sizing column chromatography), centrifugation, differential solubility, or by any other standard techniques for the purification of proteins. Further, the antibodies or fragments thereof may be fused to heterologous polypeptide sequences described herein or otherwise known in the art to facilitate purification.

[0119] Antibodies fused or conjugated to heterologous polypeptides may be used in in vitro immunoassays and in purification methods (e.g., affinity chromatography) well known in the art. See e.g., PCT publication Number WO 93/21232; EP 439,095; Naramura et al., 1994, Immunol. Lett. 39:91-99; U.S. Pat. No. 5,474,981; Gillies et al., 1992, PNAS 89:1428-1432; and Fell et al., 1991, J. Immunol. 146:2446-2452, which are incorporated herein by reference in their entireties.

[0120] Antibodies may also be attached to solid supports, which are particularly useful for immunoassays or purification of the described polypeptides or fragments, derivatives, homologues, or variants thereof, or similar molecules having the similar enzymatic activities as the polypeptide of the invention. Such solid supports include, but are not limited to, glass, cellulose, polyacrylamide, nylon, polystyrene, polyvinyl chloride or polypropylene.

5.6 Detection Assays

[0121] An exemplary method for detecting the presence or absence of an over-expressed phosphatase polypeptide or an inserted phosphatase-encoding nucleic acid in a biological sample involves obtaining a biological sample from various sources and contacting the sample with a compound or an agent capable of detecting a polypeptide or nucleic acid (e.g., mRNA, genomic DNA) such that the presence of a heterologous polypeptide or nucleic acid is detected in the sample. An exemplary agent for detecting mRNA or genomic DNA encoding an inserted phosphatase polypeptide is a labeled nucleic acid probe capable of hybridizing to mRNA or genomic DNA encoding any of the described phosphatase polypeptides. The nucleic acid probe can be, for example, a full-length cDNA, such as the nucleic acid of SEQ ID NOS: 1, 3, 5, 7, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44 or 46, or a portion thereof, such as an oligonucleotide of at least one of at least about 15, at least about 20, at least about 25, at least about 30, at least about 50, at least about 100, at least about 250, at least about 500, or more nucleotides in length and sufficient to specifically hybridize under stringent conditions to a mRNA or genomic DNA encoding a polypeptide of the invention.

[0122] An exemplary agent for detecting an over-expressed phosphatase polypeptide is an antibody capable of binding to a phosphatase polypeptide product of an inserted phosphatase gene, preferably an antibody with a detectable label. Antibodies can be polyclonal, or more preferably, monoclonal. An intact antibody, or a fragment thereof. (e.g., Fab or F(ab').sub.2) can be used. See also the detailed descriptions about antibodies in Section 5.5.

[0123] The term "labeled", with regard to the probe or antibody, is intended to encompass direct labeling of the probe or antibody by coupling (i.e., physically linking) a detectable substance to the probe or antibody, as well as indirect labeling of the probe or antibody by reactivity with another reagent that is directly labeled. Examples of indirect labeling include detection of a primary antibody using a fluorescently labeled secondary antibody and end-labeling of a DNA probe with biotin such that it can be detected with fluorescently labeled streptavidin. The detection method can be used to detect mRNA, protein, or genomic DNA in a sample in vitro as well as in vivo. For example, in vitro techniques for detection of mRNA include Northern hybridizations and in situ hybridizations. In vitro techniques for detection of a heterologous polypeptide include enzyme linked immunosorbent assays (ELISAs), Western blots, immunoprecipitations and immunofluorescence. In vitro techniques for detection of genomic DNA include Southern hybridizations. Furthermore, in vivo techniques for detection of a heterologous polypeptide include introducing into a subject organism a labeled antibody directed against the polypeptide. For example, the antibody can be labeled with a radioactive marker whose presence and location in the subject organism can be detected by standard imaging techniques, including autoradiography.

[0124] In a specific embodiment, the methods further involve obtaining a control sample from a control subject, contacting the control sample with a compound or agent capable of detecting an over-expressed polypeptide product or the mRNA transcription product or genomic DNA encoding an inserted phospatase gene, such that the presence of the polypeptide or mRNA or genomic DNA encoding the phosphatase polypeptide is detected in the sample, and comparing the presence of the phosphatase polypeptide or mRNA or genomic DNA encoding the polypeptide in the control sample with the presence of the polypeptide or mRNA or genomic DNA encoding endogenous phosphatase polypeptides in the test sample.

[0125] Embodiments also encompasse kits for detecting the presence of a heterologous polypeptide or nucleic acid in a test sample.

[0126] The kit, for example, can comprise a labeled compound or agent capable of detecting the polypeptide or mRNA encoding the polypeptide in a test sample and means for determining the amount of the polypeptide or mRNA in the sample (e.g., an antibody which binds the polypeptide or an oligonucleotide probe which binds to DNA or mRNA encoding the polypeptide). Kits can also optionally include instructions for use.

[0127] For antibody-based kits, the kit can comprise, for example: (1) a first antibody (e.g., attached to a solid support) which binds to a phosphatase polypeptide; and, optionally, (2) a second, different antibody which binds to either the polypeptide or the first antibody and is conjugated to a detectable agent.

[0128] For oligonucleotide-based kits, the kit can comprise, for example: (1) an oligonucleotide, e.g., a detectably labeled oligonucleotide, which hybridizes to a nucleic acid sequence encoding an inserted phosphatase polypeptide or (2) a pair of primers useful for amplifying a nucleic acid molecule encoding an inserted phosphatase polypeptide. The kit can also comprise, e.g., a buffering agent, a preservative, or a protein stabilizing agent. The kit can also comprise components necessary for detecting the detectable agent (e.g., an enzyme or a substrate). The kit can also contain a control sample or a series of control samples which can be assayed and compared to the test sample contained. Each component of the kit is usually enclosed within an individual container and all of the various containers are within a single package along with instructions for use.

5.7 Commercial Application of Transgenic Plants

[0129] The transgenic plants generated can have many useful applications, including food, feed, biomass, biofuels (starch, cellulose, seed lipids) and wood pulp. The enhanced growth rate of the transgenic plants may provide additional carbon dioxide fixation per hectare of land per year and thus generate carbon credits.

6. EXAMPLES

[0130] The following examples illustrate the cloning of AtPAP2, its overexpression in transgenic Arabidopsis, and the characterization of the transgenic plants. These examples should not be construed as limiting. The following examples illustrate some embodiments. Unless otherwise indicated in the following examples and elsewhere in the specification and claims, all parts and percentages are by weight, all temperatures are in degrees Centigrade, and pressure is at or near atmospheric pressure.

6.1 Sequence Alignment and Phylogenetic Analysis

[0131] PAP2 locus and its genomic organization, including its intron/exon boundaries, were identified in the Arabidopsis Col-0 ecotype (http://www.arabidopsis.org). Sequence alignment and phylogenetic tree were conducted using MEGA4 (Kumar et al., 2004) and ClustalW program (http://www.ebi.ac.uk/Tools/clustalw2/index.html). Amino acid sequence comparisons were performed using CLC Sequence Viewer 5.1.1 (www.cicbio.com).

[0132] Twenty nine PAP-like sequences were identified from the Arabidopsis genome and a phylogenetic tree was produced by neighbor-joining algorithm (FIG. 1). The gene locus of AtPAP2 (At1g13900) composes of two exons and the coding region is 1971 bp in length (SEQ ID NO: 1), which is predicted to encode a polypeptide of .about.73.7-KD. Among the twenty nine PAP-like protein sequences, only AtPAP2 and AtPAP9 carry a unique hydrophobic motif at their C-termini by TMHMM analysis (http://www.cbs.dtu.dk/services/TMHMM-2.0/) (FIG. 3). AtPAP2 was found to share 72% sequence identity in amino acid sequence with AtPAP9. Two sequences from Zea mays (Accession No: ACG47621) and Oryza sativa (Accession No: BAC15853.1) were found to share 58% and 57% a.a. identity with AtPAP2, respectively. Their sequences were aligned in FIG. 2.

[0133] AtPAP2-like sequences from other plant species that carry a hydrophobic motif at their C-termini were retrieved by tblastn program from Plant GDB database (http://www.plantgdb.org/) and NCBI database (http://blast.ncbi.nlm.nih.gov/Blast.cgi) using the amino acid sequence of AtPAP2 as the search sequence. cDNA and protein sequences that share high homology with that of AtPAP2 were identified in Zea mays (SEQ ID NOs: 7 and 8), Brassica rapa (SEQ ID NOs: 18 and 19), Hordeum vulgare (SEQ ID NOs: 20 and 21), Medicago truncatula (SEQ ID NOs: 22 and 23), Physcomitrella patens (SEQ ID NOs: 24 and 25), Populus trichocarpa (SEQ ID NOs: 26 and 27), Saccharum officinarum (SEQ ID NOs: 28 and 29), Solanum tuberosum (SEQ ID NOs: 30 and 31), Vitis vinifera (SEQ ID NOs: 32 and 33), Oryza sativa (SEQ ID NOs: 34 and 35), Gossypium hirsutum (SEQ ID NOs: 36 and 37) Panicum virgatum (SEQ ID NOs: 38 and 39), Solanum lycopersicum (SEQ ID NOs: 40 and 41), Sorghum bicolor (SEQ ID NOs: 42 and 43) and Triticum aestivum (SEQ ID NOs: 44 and 45).

[0134] The cDNA sequences of AtPAP-like sequences were amplified from a local Glycine max variety (SEQ ID NO: 5) and the Brassica napus cultivar Westar (SEQ ID NO: 46) by RT-PCR using primers designed from corresponding EST sequences, which were retrieved from the Plant GDB database (http://www.plantgdb.org/).

6.2 Screening of T-DNA Line and Production of Overexpression Lines and Complementation Lines in Arabidopsis

[0135] T-DNA insertion lines of PAP2 gene (Arabidopsis genomic locus name: Salk.sub.--013567), in the Col ecotype were obtained from Arabidopsis Biological Resources Center (Alonso et al., 2003). Homologous T-DNA lines were identified by genomic PCR screening from SIGnAl database (http://signal.salk.edu/cgi-bin/tdnaexpress) by using the primers (LBa-1,5'-TGGTTCACGTAGTGGGCCATCG-3', SEQ ID NO: 9) and PAP2 specific forward primer (P2LP, 5'-TTGAAGTTTAACATGCCTGGG-3, SEQ ID NO: 10) and reverse primer (P2RP, 5'-TCCAATGCTCGA TTGATTAGC-3', SEQ ID NO: 11). The PCR product was sequenced and the T-DNA insertion site was confirmed. To exclude the possibility that another T-DNA locus interferes with the PAP2 mutant site, homologous pap2 mutant lines were backcrossed to the wild-type to dilute the potential T-DNA sites. The produced heterozygous pap2 mutants were grown on the MS plates containing 50 mg/ml Kanamycin. The ratio of the resistant to sensitive plants was about 3:1. These results demonstrated a single insertion locus site of the T-DNA line (pap2-8) lines.

[0136] The inability of the T-DNA line to express full length AtPAP2 mRNA was confirmed by RT-PCR. Total RNA was extracted from 10-day-old seedlings grown on MS with 2% (w/v) sucrose using the TRIzol RNA isolation method (Invitrogen) with DNase I treatment. cDNAs were generated using Superscript III reverse transcriptase (Invitrogen, Carlsbad, Calif., USA) using an oligo dT primer. Two gene-specific primers, P2YF (5'-GGCCGTCGACATGATCGTTAATT TCTCTTTC-3' SEQ ID NO: 12) and P2NR (5'-CCGGACTAGTTCATGTCTCCTCGTTCTTGAC-3' SEQ ID NO: 13), were used to amplify a 1971 bp coding region of AtPAP2. For each sample, 1 .mu.g of cDNA was amplified for 30 cycles, with an annealing temperature of 50.degree. C. and using elongation factor (EF) primers, EF-1 (5'-GTTTCACATCAACATTGTGGTCA TTGG-3, SEQ ID NO: 14) and EF-2 (5'-GAGTACTTGGGGGTAGTGGCATCC-3, SEQ ID NO: 15) (Axelos et al., 1989) for control experiment.

[0137] The inability of the T-DNA line to express protein was confirmed by Western blotting analysis (FIG. 4). Antiserum specific to AtPAP2 was raised in rabbit as described in Section 6.3.

[0138] To create transgenic AtPAP2 overexpressing lines or expressing this gene in the knockout mutants, the full length coding region of the AtPAP2 cDNA was amplified by PCR using primers P2YF (SEQ ID NO: 12) and P2NR (SEQ ID NO: 13). A SalI site and a SpeI site were engineered into P2YF and P2NR, respectively. The resulting product (1976 bp) was inserted into the XhoI/Spe I sites of a binary vector, immediately downstream to the cauliflower mosaic virus (CaMV) 35S promoter (FIG. 5).

[0139] The vector was introduced into Agrobacterium tumefaciens strain GV3101 and then transformed by the floral dip method (Clough and Bent, 1998), into wild-type Col-0 to generate PAP2-overexpressing lines or into homologous pap2 plants (T-DNA lines) to generate complementation lines. Through 2 generations of selection on MS agar plate with 50 mg/l Basta (Riedel-deHaen), homologous 35S:PAP2 transgenic lines were obtained. The resistant plants were transferred to soil to grow to maturity, and their transgenic status was further confirmed by PCR and immunoblot analyses. As shown in FIG. 6A, AtPAP2 protein was overexpressed in OE lines but was absence from the T-DNA line. The homozygous T3 seeds of the transgenic plants were used for further analysis.

[0140] To create transgenic AtPAP15 overexpression lines, the cDNA of AtPAP15 was also amplified by RT-PCR and then subcloned into a plant binary vector which bared a kanamycin-resistant gene and a cauliflower mosaic virus 35S promoter (CaMV). This expression construct named was then mobilized into Agrobacterium tumefaciens strain EHA105 by freeze-thaw transformation (Hofgen and Willmitzer, 1988) and transformed into Arabidopsis. Transgenic status was further confirmed by PCR and immunoblot analyses using an anti-AtPAP15 antiserum. As shown in FIG. 6B, AtPAP15 protein was overexpressed in OE lines. The homozygous T3 seeds of the transgenic plants were used for further analysis.

6.3 Production of PAP2 Polycolonal Antiserum and Western Blots Analysis

[0141] A fragment of AtPAP2 cDNA corresponding to the N terminal 120 amino acids (from 21 to 141) was amplified using forward primer P2AF (5'-GGTTGAGCTCGATTCTAAAGCGACCATTTC-3', SEQ ID NO: 16) and reverse primer P2AR (5'-TTTTGGTACCTCAGGATCCGAA AGTCAGC-3', SEQ ID NO: 17). The PCR product was cleaved by Sad and KpnI and cloned into the pRsetA vector (Invitrogen) so that the coding sequence of the first 120 a.a. of AtPAP2 was fused to a His-tag sequence. The resulting plasmid was transformed into Escherichia coli strain BL21 (DE3). The BL21 cells were induced at 30.degree. C. by 0.1 mM isopropylthio-.beta.-D-galactoside for 4 h and resuspended in 100 mM NaCl and 50 mM Tris-HCl, pH 7.5, 2 mM phenylmethylsulphonyl fluoride (PMSF). The lysates were sonicated 5 times for 30 s each. The overexpressed His-AtPAP2 fusion proteins in inclusion bodies were centrifuged at 5000.times.g for 15 mM, and the pellets were solubilized in 150 mM NaCl, 8 M urea, and 20 mM Tris-HCl, pH 7.5. The fusion proteins were purified on a HisTrap FF (GE Healthcare) column and were used for standard immunization protocols in rabbits.

6.4 Expression Analysis of AtPAP2 mRNA and its Protein Levels

[0142] The mRNA expression level of AtPAP2 was analyzed by the Spot History program (http://affymetrix.arabidopsis.info/narrays/spothistory.pl) that presented the expression levels of a given gene in thousands of microarray (Affymetrix ATH1 microarray) database. Spot history analysis indicated that the expression of AtPAP2 was constitutive but is relative low in most experimental circumstances (FIG. 7a). To determine AtPAP2 expression levels, different tissues of wild-type A. thaliana (Col-0) were collected.

[0143] The expression level of proteins were also studied by western blotting, using the anti-AtPAP2 antiserum generated from Section 6.3. Total plant soluble protein was extracted from wild-type A. thaliana, T-DNA line, AtPAP2-overexpress lines in grinding buffer (Tris-HCl 50 mM, pH7.4 containing 150 mM NaCl, 1 mM EDTA, 0.2 mM PMSF) on ice. Protein extracts were centrifuged at 16000.times.g and supernatants were collected for Bradford protein concentration determination assay. Equal amount of protein samples (50-90 .mu.g/lane in different experiments) were loaded and separated in 12% (w/v) SDS-PAGE. The separated proteins were transferred to Hybond C-Extra membranes (Amersham Biosciences) (400 mA, 1 h). Membranes were blocked with 5% (w/v) non-fat milk in TTBS washing buffer (pH 7.6) for 2 hours and probed with specific anti-AtPAP2 antiserum for 3 hours or overnight at an 1:1000 dilution at 4.degree. C. After rinsing the membrane with three changes of TTBS washing buffer (20 mM Tris-HCl, pH7.6, 136 mM NaCl, 0.1% Tween20) in half an hour, HRP-labeled secondary antibody, diluted 1:10,000 in TTBS washing buffer was added. After 2 hours, the membrane was washed thrice before the bands were visualized by Enhanced Chemiluminescence method (Amersham Biosciences). As shown in FIG. 7b, AtPAP2 protein was expressed in all tissues tested (Leaf, Flower, Stem, Root, Silique) at equal levels. The protein expression level of AtPAP2 during germination was very stable too (FIG. 7c) and was independent of phosphorus status (FIG. 7d).

[0144] 6.5 Growth Phenotypes of WT, T-DNA Line and OE Lines

[0145] Arabidopsis seeds were soaked in water at 4.degree. C. for 3 days. The seeds were surface sterilized and sown on Murashige and Skoog (MS) medium supplemented with 2% (w/v) sucrose for 10 days. Seedlings with 2 rosette leaves of the same size were transferred to soil under Long Day (16 h light at 22.degree. C./8 h dark at 18.degree. C.) or Short Day (8 h light at 22.degree. C./16 h dark at 18.degree. C.) conditions in a plant growth chamber. Flowering time was started to be measured by scoring the number of rosette leaves and cauline leaves when the primary inflorescence florescence reached 1 cm above the rosette leaves. Ten to 20 plants were scored for each line (Liu et al., 2008; Wu et al., 2008).

[0146] The inflorescences of OE lines of AtPAP2 emerged earlier (5-6 days for Long Day, 14-16 days for Short Day) than that of the WT and T-DNA lines (Table 1). Under Long Day conditions, the number of rosette leaves of the OE lines were less (5-6 leaves) than the WT during the emergence of inflorescence (Table 1 and FIG. 8). At day 28 (Long Day), the OE lines of AtPAP2 had more cauline leaves and inflorescences than the WT and T-DNA lines, but had less rosette leaves (Table 2.). This phenotype observation was repeated at least four times and the results of one of the experiments were shown here.

TABLE-US-00001 TABLE 1 AtPAP2 OE lines flowered at an earlier developmental stage. Long Day (16 h/8 h) Short Day (8 h/16 h) Lines AEI SD NRL SD AEI SD NRL SD Col-0 26.9 1.2 13.0 0.8 41.0 4.7 18.0 3.0 T-DNA 25.7 0.7 11.6 1.1 40.7 4.9 15.0 3.0 OE7 20.0* 1.1 6.4* 0.5 25.6* 1.3 5.3* 0.5 OE21 20.8* 0.6 6.5* 0.7 26.0* 1.1 5.4* 0.5 AEI: Average date of emergence of inflorescence NRL: No. of rosette leaves at the first appearance of inflorescence *Statistically (p < 0.001) different from the wild-type (n = 15).

TABLE-US-00002 TABLE 2 Phenotypes of AtPAP2 OE lines at Day 28 (Long Day). No. of No. of No. of Lines Rosette Leaf SD Cauline Leaf SD Inflorescence SD Col-0 14.5 1.2 1.6 0.5 1.0 0.0 T-DNA 16.7 1.7 1.9 0.6 1.0 0.0 OE7 9.9* 1.0 6.0* 1.2 3.6* 0.7 OE21 10.2* 1.8 7.2* 1.6 3.7* 1.1 *Statistically (p < 0.001) different from the wild-type (n = 15).

[0147] At maturity (Long Day), the number of siliques and the total weight of seeds harvested from each line were recorded. Two separate experimental trials are shown in Tables 3A and 3B. Our results showed that overexpression of AtPAP2 resulted in increase number of siliques per plant and the seed yield per plant. Compared to that of the wild-type, the seed yield of the two overexpression lines shown in Table 3A increased 38-40%. Compared to that of the wild-type, the seed yield of the two overexpression lines shown in Table 3B increased 54-58%.

TABLE-US-00003 TABLE 3A OE lines produced more siliques and seeds (Trial 1). Weight of seeds Lines No. of siliques/plant SD (g)/plant SD N Col-0 327.4 53.3 0.188 0.047 5 T-DNA 236.6* 60.2 0.121* 0.040 7 OE7 453.2** 62.1 0.264** 0.039 5 OE21 498.2.sup.# 52.5 0.260** 0.049 7 Statistically (p < 0.02*, p < 0.01**, p < 0.001.sup.#) different from the wild-type.

TABLE-US-00004 TABLE 3B OE lines produced more siliques and seeds (Trial 2). Weight of seeds Lines No. of siliques/plant SD (g)/plant SD N Col-0 396.4 89.5 0.225 0.058 13 T-DNA 386.3 70.4 0.240 0.049 12 OE7 610.9* 76.6 0.351* 0.050 7 OE21 624.9* 94.7 0.355* 0.066 11 Statistically (p < 0.0001*) different from the wild-type.

[0148] However, the OE lines of AtPAP15 grew normally and were not different from the wild-types. Therefore, the enhanced growth performance was due to the overexpression of AtPAP2, which bears a transmembrane-like motif at its C-terminus (FIGS. 2 and 3).

6.6 Growth Phenotypes of Truncated AtPAP2 Constructs

[0149] An alternate vector construct employing the sequence for AtPAP2 was also constructed using analogous techniques to those described above. As shown in FIG. 12, a construct equivalent to the OE lines of AtPAP2 missing the C-terminal motif (residues 614-636 of SEQ ID NO: 2) was constructed (P2C lines). Transgenic plants were generated using substantially identical techniques to those described above. Western blot analysis was used to confirm the over-expression of the AtPAP2 fragment proteins in transformed plant lines. Performance of Western blot analysis was identical to that reported above. As show in FIG. 13, the P2C lines were strongly overexpressed. The growth phenotype of the P2C lines appeared to be indifferent from the wild-type, which is indicative of the importance of the C-terminal domain of AtPAP2 in developing an increased growth phenotype.

6.7 MS/MS Analysis of Sucrose and Glucose Levels in Leaf

[0150] Rosette leaves of plants of various developmental stages were harvested at the end of the light period of 21-day-old plants. Soluble sugars were extracted from Arabidopsis using chloroform/methanol method (Lunn et al., 2006; Antonio et al., 2007; Luo et al., 2007). 100 mg plant tissues were ground to a fine powder in liquid nitrogen and mixed and vortexed with 250 .mu.l ice-cold chloroform:methanol (3:7, v/v). Soluble metabolites were then extracted at -20.degree. C. overnight. 200 .mu.l water was added to the mixture with repeated shaking. The extracts were centrifuged at 16000.times.g for 10 min and the supernatant was collected. The pellet was re-extracted by 204.1 water and the supernatant was collected by centrifugation as described above. The combined supernatant was evaporated to dryness using a SpeedVac and the pellet was re-dissolved in 200 .mu.l water. Finally, debris was removed by centrifugation at 16000.times.g for 30 min.

[0151] 20 .mu.l filtered samples were analyzed by an API-3000 triple-quadrupole mass spectrometer (Applied Biosystems) via an electrospray ionization source. The parameters, optimized by 0-40 .mu.g/ml glucose and sucrose standards, were as following: curtain gas (CUR) 25, nebulizer gas (GS1) 50, auxiliary gas (GS2) 30, ionspray voltage -4.5 k V, temperature 400.degree. C., declustering potential (DP) -106 V, entrance potential (EP) -8.5 V, collision cell entrance potential (CEP) -46.7 V, collision energy (CE) 20 V. The peaks were identified by comparison with glucose and sucrose standards and the amount of sugars were quantified by standard curves of these sugars. The Analyst 1.3.1 software (Applied Biosystems) was used for data acquisition, peak integration, and calculation. The amount of sucrose and glucose at the end of day in the shoots of 21-day-old soil grown plants were shown in FIG. 9. It was found that the levels of both sugars were significantly higher than that in WT.

6.8 Recovery of Plants after Prolonged Darkness Treatment

[0152] Seeds of wild-type, T-DNA, OE7 and OE21 lines were germinated in MS (2% sucrose) medium for 10 days. Seedlings with 2 small visible rosette leaves (1 mm) of the same size were transferred to soil for another 12 days in normal growth conditions (LDs, 16 h/light (22.degree. C.)/8 h darkness (18.degree. C.)). The light source of the growth chamber was then switched off for 12 days. Then the plants were allowed to recover under the 16 h/light (22.degree. C.)/8 h darkness (18.degree. C.) cycle for 10 days. The plants that stayed green and that continued to emerge inflorescence were recorded in Table 4.

TABLE-US-00005 TABLE 4 Surviving rate and flowering ratio after prolonged darkness treatment. Flowering after Recovery (leaf recovery greening) Wild-type 8/12 11/12 T-DNA 5/12 8/12 OE7 9/9 9/9 OE21 12/12 12/12

[0153] Extended darkness could induce carbohydrate starvation (Thompson et al., 2005). Our data showed that the OE lines exhibited 100% recovery rate under prolonged (12 days) darkness treatment, which was higher than that of the WT and the T-DNA line (FIG. 10). This could be attributed to a higher endogenous sugar levels (FIG. 9) in the OE lines.

6.9 Phenotypes of Plants Under NaCl and ABA Treatments

[0154] 5-day-old seedlings grown on MS agar were transferred to MS agar with NaCl (50 mM, 100 mM, 150 mM), ABA (0.1 uM, 0.2 uM. 0.5 uM, 1 uM, 2 uM) or sorbitol (300 mM, 400 mM, 500 mM). Alternatively, seeds were directly germinated on the treatment media. Wild-type, T-DNA and OE lines did not show remarkable phenotypic differences under the above conditions.

6.10 Subcellular Fractionation

[0155] Rosette leaves of three-week-old wild-type (Col-0) Arabidopsis were harvested and stored at -80.degree. C. freezer until use. Tissues (4-5 g) were ground to fine powder in liquid nitrogen using a mortar with a pestle. The powder was transferred into 10 ml grinding buffer (0.3 M sucrose, 40 mM Tris-HCl (pH 7.8), 5 mM MgCl.sub.2, 1 mM PMSF) and swelled on ice for 5 min. Homogenization was performed for two 30-second pulses at high-speed setting. The homogenate was filtered through two layers of Miracloth (Tetko, Elmsford, N.Y., USA). Subsequently, the homogenate was separated by centrifugation at 350 g for 10 min at 4.degree. C. The pellet (crude nuclear) was further layered onto 1 ml of 2.3 M sucrose, 50 mM Tris-HCl (pH 8.8), 5 mM MgCl.sub.2 in an Eppendorf tube for centrifugation at 15,000 g 10 min at 4.degree. C., to obtain the nuclear fraction in the derived pellet. Supernatants from the first low-speed centrifugation (350 g) were centrifuged at 12,000.times.g for 20 min at 4.degree. C. The pellet contained large particles including mitochondria, chloroplasts and peroxisomes. The supernatant was further centrifuged at 100,000.times.g for 1 h at 4.degree. C. to yield the soluble cytosol fraction in the resulting supernatant. The pellet representing the membrane fraction was resuspended in 0.1 ml grinding buffer. Protein concentration in the extract was determined following the method of Bradford (Bradford, 1976) using the Bio-Rad Protein Assay Kit I.

[0156] To isolate cell wall, leaf tissues were homogenized in grinding buffer (62.5 mM Tris-HCl, pH 7.5, 5 mM DTT, 1% (v/v) bovine serum albumin, 2 mM phenylmethylsulfonyl fluoride, 2 .mu.g/ml leupeptin, 2 .mu.g/ml E-64, 2 .mu.g/ml pepstatin A) using a Polytron (full speed, 3.times.10 s). The homogenate was centrifuged at 1,000.times.g for 3 min. The pellet was washed with ice-cold grinding buffer (without 1% BSA) 10 times. Finally the (cell wall) pellet was washed by resuspending in 500 mM CaCl.sub.2, 20 mM NaCl, 62.5 mM Tris-HCl, pH 7.5, and spinning at 10,000.times.g for 15 min (He et al., 1996).

[0157] The subcellular fractions were run in a SDS-PAGE gel and were probed with anti-AtPAP2 antiserum. AtPAP2 was detected in membrane and soluble protein fractions but not in nucleus, mitochondria nor chloroplasts (FIG. 11).

[0158] In summary, Arabidopsis plants transformed with the AtPAP2 gene have the following phenotypes when they were compared with the wild-type: (1) Faster growth rate (Tables 1 and 2); (2) Higher sucrose content (FIG. 9); (3) Higher glucose content (FIG. 9); and (4) Higher crop yield (Table 3).

[0159] Those skilled in the art will recognize, or be able to ascertain many equivalents to the specific embodiments described herein using no more than routine experimentation. Such equivalents are intended to be encompassed by the following claims.

[0160] With respect to any figure or numerical range for a given characteristic, a figure or a parameter from one range may be combined with another figure or a parameter from a different range for the same characteristic to generate a numerical range.

[0161] All publications, patents and patent applications mentioned in this specification are herein incorporated by reference into the specification to the same extent as if each individual publication, patent or patent application was specifically and individually indicated to be incorporated herein by reference.

[0162] Citation or discussion of a reference herein shall not be construed as an admission that such is prior art to the present patent application.

[0163] While the embodiments have been explained in relation to certain embodiments, it is to be understood that various modifications thereof will become apparent to those skilled in the art upon reading the specification. Therefore, it is to be understood that the embodiments disclosed herein are intended to cover such modifications as fall within the scope of the appended claims. Features of two or more of any of the above embodiments can be combined to form additional embodiments.

[0164] Other than in the operating examples, or where otherwise indicated, all numbers, values and/or expressions referring to quantities of ingredients, reaction conditions, etc., used in the specification and claims are to be understood as modified in all instances by the term "about."

6.11 Assays of Enzymes Involved in Sucrose Metabolism

[0165] Sucrose phosphate synthesis (SPS), sucrose synthesis (SuSy), cytosolic invertase and cell wall invertase activities in the shoot of 20-day-old plants were determined. Samples were collected 8 h after the light and dark period (Long Day). SPS activity was measured under both optimal (Vmax) and limiting (V limit) assay conditions (Park et al., 2008). SuSy, cytosolic invertase and insoluble cell wall invertase activities were also determined (Doehlert, 1987). The assays were repeated three times and the SPS (Vmax and V limit) activities of both independent lines were significantly higher than that of the wild-type and T-DNA lines in all three repeated experiments. The data of a representative experiment is shown in table 5. In contrast to SPS, SuSy, cytosolic invertase and cell wall invertase activities were not different among the lines.

TABLE-US-00006 TABLE 5 Enzyme assays Plant line WT T-DNA OE7 OE21 Sucrose phosphate synthase (.mu.M sucrose/.mu.g enzyme extracts/hour) Vmax (Day) 108.8 .+-. 18.1 116.1 .+-. 11.9 157.9 .+-. 21.7** 159.5 .+-. 19.0** Vlimit (Day) 63.6 .+-. 6.4 69.5 .+-. 2.5 90.8 .+-. 18.5** 79.8 .+-. 13.7* Vmax (Night) 118.3 .+-. 11.4 104.9 .+-. 14.4 150.9 .+-. 19.4** 136.5 .+-. 15.1* Vlimit (Night) 74.9 .+-. 6.3 56.7 .+-. 3.3 93.4 .+-. 3.6** 97.3 .+-. 10.9** Sucrose synthase (.mu.M glucose/.mu.g enzyme extracts/hour) Day 249.2 .+-. 4.6 247.0 .+-. 24.0 248.6 .+-. 4.5 255.2 .+-. 5.4 Night 249.4 .+-. 7.2 252.7 .+-. 8.8 250.8 .+-. 8.7 258.8 .+-. 5.5 Cytosolic invertase (.mu.M glucose/.mu.g enzyme extracts/hour) Day (Acid) 14.3 .+-. 2.3 12.1 .+-. 5.4 18.3 .+-. 9.1 18.0 .+-. 0.8 Night (Acid) 14.0 .+-. 6.8 26.1 .+-. 10.9 17.6 .+-. 4.3 13.8 .+-. 0.8 Day (Alkaline) 169.7 .+-. 9.8 161.2 .+-. 32.3 160.9 .+-. 27.9 178.8 .+-. 16.9 Night (Alkaline) 130.0 .+-. 8.2 105.1 .+-. 12.6 136.1 .+-. 13.6 136.6 .+-. 1.4 Cell wall invertase (.mu.M sucrose/.mu.g enzyme extracts/hour) Day (Acid) 22.3 .+-. 3.9 18.0 .+-. 4.9 23.5 .+-. 6.3 24.0 .+-. 6.6 Night (Acid) 21.6 .+-. 10.1 17.0 .+-. 4.3 28.3 .+-. 4.2 22.3 .+-. 3.3 Day (Alkaline) 121.7 .+-. 2.8 127.1 .+-. 2.2 101.8 .+-. 6.4 105.5 .+-. 2.0 Night (Alkaline) 138.9 .+-. 6.3 151.5 .+-. 8.7 123.4 .+-. 5.6 135.4 .+-. 14.7 (**P < 0.01; *P < 0.05)

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(2007) Simultaneous determination of multiple intracellular metabolites in glycolysis, pentose phosphate pathway and tricarboxylic acid cycle by liquid chromatography-mass spectrometry. J Chromatogr A, 1147, 153-164. [0192] Park, J. Y., Canam, T., Kang, K. Y., Ellis, D. D. and Mansfield, S.D. (2008) Over-expression of an arabidopsis family A sucrose phosphate synthase (SPS) gene alters plant growth and fibre development. Transgenic Res, 17, 181-192. [0193] Patel, K., Lockless, S., Thomas, B. and McKnight, T. D. (1998) Secreted purple acid phosphatase from Arabidopsis thaliana. American Society of Plant Physiogists, 373-374. [0194] Schenk, G., Ge, Y., Carrington, L. E., Wynne, C. J., Searile, I. R., Carroll, B. J., Hamilton, S, and de-Jersey, J. (1999) Binuclear metal centers in plant purple acid phosphatases: Fe--Mn in sweet potato and Fe--Zn in soybean. Arch. Biochem Biophys, 370, 183-189. [0195] Schenk, G., Korsinczky, M. L., Hume, D. A., Hamilton, S, and DeJersey, J. (2000) Purple acid phosphatases from bacteria: similarities to mammalian and plant enzymes. Gene, 255, 419-424. [0196] Shimaoka, T., Ohnishi, M., Sazuka, T., Mitsuhashi, N., Hara-Nishimura I, Shimazaki, K., Maeshima, M., Yokota, A., Tomizawa, K. and Mimura, T. (2004) solation of intact vacuoles and proteomic analysis of tonoplast from suspension-cultured cells of Arabidopsis thaliana. Plant Cell Physiol, 45, 672-683. [0197] Thompson, A. R., Doelling, J. H., Suttangkakul, A. and Vierstra, R. D. (2005) Autophagic nutrient recycling in Arabidopsis directed by the ATG8 and ATG12 conjugation pathways. Plant Physiol, 138, 2097-2110. [0198] Veljanovski, V., Vanderbeld, B., Knowles, V. L., Snedden, W. A. and Plaxton, W. C. (2006) Biochemical and molecular characterization of AtPAP26, a vacuolar purple acid phosphatase up-regulated in phosphate-deprived Arabidopsis suspension cells and seedlings. Plant Physiol, 142, 1282-1293. [0199] Wu, J. F., Wang, Y. and Wu, S. H. (2008) Two New Clock Proteins, LWD1 and LWD2, Regulate Arabidopsis Photoperiodic Flowering. Plant Physiol, 148, 948-959. [0200] Wu, P., Ma, L., Hou, X., Wang, M., Wu, Y., Liu, F. and Deng, X. W. (2003) Phosphate starvation triggers distinct alterations of genome expression in Arabidopsis roots and leaves. Plant Physiol, 132, 1260-1271. [0201] Xiao, K., Harrison, M. J. and Wang, Z. Y. (2005) Transgenic expression of a novel M. truncatula phytase gene results in improved acquisition of organic phosphorus by Arabidopsis. Planta, 222, 27-36. [0202] Xiao, K., Katagi, H., Harrison, M. and Wang, Z. Y. (2006) Improved phosphorus acquisition and biomass production in Arabidopsis by transgenic expression of a purple acid phosphatase gene from M. truncatula. Plant Science, 170, 191-202. [0203] Zhang, W., Gruszewski, H. A., Chevone, B. I. and Nessler, C. L. (2008) An Arabidopsis purple Acid phosphatase with phytase activity increases foliar ascorbate. Plant Physiol, 146, 431-440. [0204] Zhu, H. F., Qian, W. Q., Lu, X. Z., Li, D. P., Liu, X., Liu, K. F. and Wang, D. W. (2005) Expression patterns of purple acid phosphatase genes in Arabidopsis organs and functional analysis of AtPAP23 predominantly transcribed in flower. Plant Molecular Biology, 59, 581-594.

Sequence CWU 1

1

6611971DNAArabidopsis thaliana 1atgatcgtta atttctcttt cttcctcctt ctcttcgtct ccgtcttcgt ttcctctgct 60gattctaaag cgaccatttc aatttcccct aatgctctca atcgatctgg cgattccgtt 120gtgatacaat ggtccggtgt cgattctccg tcagatctcg attggttagg actctactcg 180ccgccggagt ctcctaatga tcactttatt ggttacaaat tcctcaatga atcgtccact 240tggaaagatg gtttcggttc gatttctctt cctttaacca atctccgatc aaattacaca 300ttccggatct tccgttggag cgaatccgag attgatccga aacataagga tcatgatcag 360aatcctttac caggaactaa acatcttcta gctgaatcgg agcagctgac tttcggatcc 420ggtgttggta tgccggagca gatccatttg tcgttcacaa atatggttaa cacgatgcgt 480gttatgtttg tagctggaga tggtgaagaa cgttttgtta gatacggtga atcgaaggat 540ttgttaggta attccgcggc ggcgcgtggg atgaggtacg agagagagca catgtgtgat 600tcgccggcga attccactat tggttggaga gatcctggtt ggatttttga taccgtcatg 660aagaatttga atgatggcgt tagatactat tatcaggttg ggagtgattc taagggatgg 720agtgagatcc atagctacat tgctcgagat gtgactgcag aagaaaccgt agctttcatg 780tttggagata tgggttgtgc tacaccatac acgacattta tccgcacaca agatgagagc 840atatctacag tgaagtggat cctccgtgac attgaagctc ttggtgataa gccagctatg 900atttcacaca ttggagatat aagttatgct cgtggttact cgtgggtatg ggatgagttc 960tttgctcagg ttgagcctat tgcctcgaca gttccttacc atgtttgcat tggtaaccat 1020gagtatgatt tctctactca gccgtggaaa cctgattggg cagcttctat ttatggaaac 1080gatggtggtg gcgaatgtgg tgtgccgtat agcttgaagt ttaacatgcc tgggaattct 1140tcagagtcta caggaatgaa agctcctccg acaaggaatt tatattattc ttatgatatg 1200ggaacggtcc atttcgttta tatctccaca gagacgaatt ttcttaaagg aggtagtcaa 1260tatgaattca taaagcgaga tctagagtct gtagacagga agaaaacacc gtttgttgtt 1320gtgcaaggac atagaccaat gtacactacg agcaacgagg ttagagacac tatgattcga 1380caaaagatgg ttgagcatct agaacctttg tttgtgaaaa acaatgtcac acttgctcta 1440tggggacatg ttcatagata cgaaaggttt tgtcccataa gcaacaacac ttgcggcaca 1500cagtggcaag gaaatccggt tcatcttgtg atcggtatgg ctggtcaaga ttggcaaccg 1560atttggcagc ctagaccaaa ccatccagat cttcctatat tccctcagcc tgaacaatca 1620atgtatcgta caggtgagtt tggttacact cgtttagttg caaacaaaga aaagctcact 1680gtttcttttg tgggtaatca cgatggcgaa gttcatgata ctgttgagat gttagcatct 1740ggggtagtaa tcagtgggag caaagagagt actaaaatcc caaatctgaa aaccgttcct 1800gcttctgcta cacttatggg aaaatcagaa tctaatgctt tgtggtatgc caaaggagca 1860ggcttgatgg ttgtgggtgt gcttttaggg ttcattatcg ggttttttac ccggggaaag 1920aaatcttcgt ctggaaaccg ttggatccca gtcaagaacg aggagacata a 19712656PRTArabidopsis thaliana 2Met Ile Val Asn Phe Ser Phe Phe Leu Leu Leu Phe Val Ser Val Phe1 5 10 15Val Ser Ser Ala Asp Ser Lys Ala Thr Ile Ser Ile Ser Pro Asn Ala 20 25 30Leu Asn Arg Ser Gly Asp Ser Val Val Ile Gln Trp Ser Gly Val Asp 35 40 45Ser Pro Ser Asp Leu Asp Trp Leu Gly Leu Tyr Ser Pro Pro Glu Ser 50 55 60Pro Asn Asp His Phe Ile Gly Tyr Lys Phe Leu Asn Glu Ser Ser Thr65 70 75 80Trp Lys Asp Gly Phe Gly Ser Ile Ser Leu Pro Leu Thr Asn Leu Arg 85 90 95Ser Asn Tyr Thr Phe Arg Ile Phe Arg Trp Ser Glu Ser Glu Ile Asp 100 105 110Pro Lys His Lys Asp His Asp Gln Asn Pro Leu Pro Gly Thr Lys His 115 120 125Leu Leu Ala Glu Ser Glu Gln Leu Thr Phe Gly Ser Gly Val Gly Met 130 135 140Pro Glu Gln Ile His Leu Ser Phe Thr Asn Met Val Asn Thr Met Arg145 150 155 160Val Met Phe Val Ala Gly Asp Gly Glu Glu Arg Phe Val Arg Tyr Gly 165 170 175Glu Ser Lys Asp Leu Leu Gly Asn Ser Ala Ala Ala Arg Gly Met Arg 180 185 190Tyr Glu Arg Glu His Met Cys Asp Ser Pro Ala Asn Ser Thr Ile Gly 195 200 205Trp Arg Asp Pro Gly Trp Ile Phe Asp Thr Val Met Lys Asn Leu Asn 210 215 220Asp Gly Val Arg Tyr Tyr Tyr Gln Val Gly Ser Asp Ser Lys Gly Trp225 230 235 240Ser Glu Ile His Ser Tyr Ile Ala Arg Asp Val Thr Ala Glu Glu Thr 245 250 255Val Ala Phe Met Phe Gly Asp Met Gly Cys Ala Thr Pro Tyr Thr Thr 260 265 270Phe Ile Arg Thr Gln Asp Glu Ser Ile Ser Thr Val Lys Trp Ile Leu 275 280 285Arg Asp Ile Glu Ala Leu Gly Asp Lys Pro Ala Met Ile Ser His Ile 290 295 300Gly Asp Ile Ser Tyr Ala Arg Gly Tyr Ser Trp Val Trp Asp Glu Phe305 310 315 320Phe Ala Gln Val Glu Pro Ile Ala Ser Thr Val Pro Tyr His Val Cys 325 330 335Ile Gly Asn His Glu Tyr Asp Phe Ser Thr Gln Pro Trp Lys Pro Asp 340 345 350Trp Ala Ala Ser Ile Tyr Gly Asn Asp Gly Gly Gly Glu Cys Gly Val 355 360 365Pro Tyr Ser Leu Lys Phe Asn Met Pro Gly Asn Ser Ser Glu Ser Thr 370 375 380Gly Met Lys Ala Pro Pro Thr Arg Asn Leu Tyr Tyr Ser Tyr Asp Met385 390 395 400Gly Thr Val His Phe Val Tyr Ile Ser Thr Glu Thr Asn Phe Leu Lys 405 410 415Gly Gly Ser Gln Tyr Glu Phe Ile Lys Arg Asp Leu Glu Ser Val Asp 420 425 430Arg Lys Lys Thr Pro Phe Val Val Val Gln Gly His Arg Pro Met Tyr 435 440 445Thr Thr Ser Asn Glu Val Arg Asp Thr Met Ile Arg Gln Lys Met Val 450 455 460Glu His Leu Glu Pro Leu Phe Val Lys Asn Asn Val Thr Leu Ala Leu465 470 475 480Trp Gly His Val His Arg Tyr Glu Arg Phe Cys Pro Ile Ser Asn Asn 485 490 495Thr Cys Gly Thr Gln Trp Gln Gly Asn Pro Val His Leu Val Ile Gly 500 505 510Met Ala Gly Gln Asp Trp Gln Pro Ile Trp Gln Pro Arg Pro Asn His 515 520 525Pro Asp Leu Pro Ile Phe Pro Gln Pro Glu Gln Ser Met Tyr Arg Thr 530 535 540Gly Glu Phe Gly Tyr Thr Arg Leu Val Ala Asn Lys Glu Lys Leu Thr545 550 555 560Val Ser Phe Val Gly Asn His Asp Gly Glu Val His Asp Thr Val Glu 565 570 575Met Leu Ala Ser Gly Val Val Ile Ser Gly Ser Lys Glu Ser Thr Lys 580 585 590Ile Pro Asn Leu Lys Thr Val Pro Ala Ser Ala Thr Leu Met Gly Lys 595 600 605Ser Glu Ser Asn Ala Leu Trp Tyr Ala Lys Gly Ala Gly Leu Met Val 610 615 620Val Gly Val Leu Leu Gly Phe Ile Ile Gly Phe Phe Thr Arg Gly Lys625 630 635 640Lys Ser Ser Ser Gly Asn Arg Trp Ile Pro Val Lys Asn Glu Glu Thr 645 650 65531956DNAArabidopsis thaliana 3atgatcgccg ccgtttacac tctcttcttc ttcttcctct taatctcctc tgtttattcc 60aaagccacga tttcaatctc tccccaaacc ttaaaccgat ccggcgacat agtcgtgatc 120aaatggtccg gcgtcgaatc accgtccgat ctcgactggt taggaatcta ctcgccaccg 180gactctcctc acgatcactt catcggctac aaattcctct ccgattcacc cacgtggcaa 240tccgggtcgg gttcgatttc acttccctta accaatctcc gatcaaatta cactttccgg 300atctttcatt ggacccaatc cgaaataaac ccgaaacacc aagaccatga tcacaatcct 360ttacccggaa ctcgtcatct cttaaccgaa tcaaaccagt taaatttccg gttcgctgtt 420aaccgaccgg agcagattca tttaagttac acagataaca tcaacgagat gagagtagtg 480tttgtaaccg gagatggaga agaacgagaa gctcgctacg gtgaggttaa ggacaagctc 540gataacatag cggtggcgcg tggagttagg tacgagatag aacatatgtg tcacgcgccg 600gcgaattcta cagtcggatg gagagatcca ggttggacat tcgatgccgt gatgaagaat 660ctaaaacaag ggattaggta ttattatcag gttgggagtg atttaaaagg atggagtgag 720attcatagct ttgtttctcg aaatgagggt tcagaagaaa cattagcttt catgtttggt 780gatatgggat gttatacacc ttatacaaca tttatccgtg gagaagaaga aagtttatca 840actgtgaaat ggattttaag agacattgaa gctttaggtg atgataagcc tgtgattgta 900tcacatatcg gagatataag ttatgcacga ggttactcgt ggatttggga tgagttcttt 960actcagattg agcctattgc ttctaaagtg ccttaccatg tatgtattgg taaccatgag 1020tatgattggc ctaaccagcc ttggaaacca gattgggctg cttatgttta tggtaaagat 1080agtggtggtg aatgtggtgt accgtatagt gttaagttca acatgcctgg taattcaacg 1140gaagctactg gtatggttaa gggacctcaa agtcggaacc tttactattc ttatgatatg 1200ggttctgttc atttcgttta tatttcgaca gagactgatt ttcttaaagg tgggaagcag 1260tatagttttt tgaagagtga tttggagtct gttaatagga gtaagacacc gtttgttgtt 1320gtccaagggc atagacctat gtacactacg agtaggaaga tcagagacgc tgctataaga 1380gagaagatga tcgagcattt ggaaccgttg ttagtgaaga acaatgtgac ggttgcttta 1440tggggacatg tacatagata tgaaaggttt tgtgcgatta gtaacaatac ttgtggtgaa 1500cgttggcaag gaaatccagt tcatcttgtg attggtatgg ctggaaaaga ctcacaaccg 1560atgtgggaac cgagagctaa tcacgaggat gtcccgatct ttcctcagcc tgctaactca 1620atgtaccgtg gaggcgagtt tgggtacatt cgtttggttg ctaataagga aagacttact 1680ctttcttatg tgggaaacca tgacggagaa gttcatgatg ttgttgagat tttggcttct 1740ggggaagtta ttagcggtag tgatgatggt actaaagact caaactttgg atcagaatct 1800gactttgcag tcttgtggta cattgaagga gcaagtgtga tggttgtggg agtgattttt 1860gggtactttg tcggttttct tagtcgtaaa aagaaagaat ctggagttgg atcatctaat 1920cgtagttgga tccaagtgaa aaacgaggag acatga 19564651PRTArabidopsis thaliana 4Met Ile Ala Ala Val Tyr Thr Leu Phe Phe Phe Phe Leu Leu Ile Ser1 5 10 15Ser Val Tyr Ser Lys Ala Thr Ile Ser Ile Ser Pro Gln Thr Leu Asn 20 25 30Arg Ser Gly Asp Ile Val Val Ile Lys Trp Ser Gly Val Glu Ser Pro 35 40 45Ser Asp Leu Asp Trp Leu Gly Ile Tyr Ser Pro Pro Asp Ser Pro His 50 55 60Asp His Phe Ile Gly Tyr Lys Phe Leu Ser Asp Ser Pro Thr Trp Gln65 70 75 80Ser Gly Ser Gly Ser Ile Ser Leu Pro Leu Thr Asn Leu Arg Ser Asn 85 90 95Tyr Thr Phe Arg Ile Phe His Trp Thr Gln Ser Glu Ile Asn Pro Lys 100 105 110His Gln Asp His Asp His Asn Pro Leu Pro Gly Thr Arg His Leu Leu 115 120 125Thr Glu Ser Asn Gln Leu Asn Phe Arg Phe Ala Val Asn Arg Pro Glu 130 135 140Gln Ile His Leu Ser Tyr Thr Asp Asn Ile Asn Glu Met Arg Val Val145 150 155 160Phe Val Thr Gly Asp Gly Glu Glu Arg Glu Ala Arg Tyr Gly Glu Val 165 170 175Lys Asp Lys Leu Asp Asn Ile Ala Val Ala Arg Gly Val Arg Tyr Glu 180 185 190Ile Glu His Met Cys His Ala Pro Ala Asn Ser Thr Val Gly Trp Arg 195 200 205Asp Pro Gly Trp Thr Phe Asp Ala Val Met Lys Asn Leu Lys Gln Gly 210 215 220Ile Arg Tyr Tyr Tyr Gln Val Gly Ser Asp Leu Lys Gly Trp Ser Glu225 230 235 240Ile His Ser Phe Val Ser Arg Asn Glu Gly Ser Glu Glu Thr Leu Ala 245 250 255Phe Met Phe Gly Asp Met Gly Cys Tyr Thr Pro Tyr Thr Thr Phe Ile 260 265 270Arg Gly Glu Glu Glu Ser Leu Ser Thr Val Lys Trp Ile Leu Arg Asp 275 280 285Ile Glu Ala Leu Gly Asp Asp Lys Pro Val Ile Val Ser His Ile Gly 290 295 300Asp Ile Ser Tyr Ala Arg Gly Tyr Ser Trp Ile Trp Asp Glu Phe Phe305 310 315 320Thr Gln Ile Glu Pro Ile Ala Ser Lys Val Pro Tyr His Val Cys Ile 325 330 335Gly Asn His Glu Tyr Asp Trp Pro Asn Gln Pro Trp Lys Pro Asp Trp 340 345 350Ala Ala Tyr Val Tyr Gly Lys Asp Ser Gly Gly Glu Cys Gly Val Pro 355 360 365Tyr Ser Val Lys Phe Asn Met Pro Gly Asn Ser Thr Glu Ala Thr Gly 370 375 380Met Val Lys Gly Pro Gln Ser Arg Asn Leu Tyr Tyr Ser Tyr Asp Met385 390 395 400Gly Ser Val His Phe Val Tyr Ile Ser Thr Glu Thr Asp Phe Leu Lys 405 410 415Gly Gly Lys Gln Tyr Ser Phe Leu Lys Ser Asp Leu Glu Ser Val Asn 420 425 430Arg Ser Lys Thr Pro Phe Val Val Val Gln Gly His Arg Pro Met Tyr 435 440 445Thr Thr Ser Arg Lys Ile Arg Asp Ala Ala Ile Arg Glu Lys Met Ile 450 455 460Glu His Leu Glu Pro Leu Leu Val Lys Asn Asn Val Thr Val Ala Leu465 470 475 480Trp Gly His Val His Arg Tyr Glu Arg Phe Cys Ala Ile Ser Asn Asn 485 490 495Thr Cys Gly Glu Arg Trp Gln Gly Asn Pro Val His Leu Val Ile Gly 500 505 510Met Ala Gly Lys Asp Ser Gln Pro Met Trp Glu Pro Arg Ala Asn His 515 520 525Glu Asp Val Pro Ile Phe Pro Gln Pro Ala Asn Ser Met Tyr Arg Gly 530 535 540Gly Glu Phe Gly Tyr Ile Arg Leu Val Ala Asn Lys Glu Arg Leu Thr545 550 555 560Leu Ser Tyr Val Gly Asn His Asp Gly Glu Val His Asp Val Val Glu 565 570 575Ile Leu Ala Ser Gly Glu Val Ile Ser Gly Ser Asp Asp Gly Thr Lys 580 585 590Asp Ser Asn Phe Gly Ser Glu Ser Asp Phe Ala Val Leu Trp Tyr Ile 595 600 605Glu Gly Ala Ser Val Met Val Val Gly Val Ile Phe Gly Tyr Phe Val 610 615 620Gly Phe Leu Ser Arg Lys Lys Lys Glu Ser Gly Val Gly Ser Ser Asn625 630 635 640Arg Ser Trp Ile Gln Val Lys Asn Glu Glu Thr 645 65051989DNAGlycine max 5atgattcccg atctacccct ccccttcctc ttctccttat tcatcatctt cttccacctc 60gctgaatcca aaccctccct caccgccacg ccaaccaccc tcccagcctc cggcgccacc 120gtcaatctcc gctggtccgg catcccttcc ccctccgacc tcgacttcct cgccatctat 180tcccctccga cctccccgca cgacaacttc atcggatacc tcttcctctc gcagtccgcc 240acgtggcgca ccggctccgg caacctctcc ctccccctcg tcgacctccg ctccaactac 300tccttccgca tcttcagctg gacccgcgcc gagatcaacc ccaagcgcca ggaccacgat 360cacaaccctc tcccggtcac acgccacctc ctcgcgtttt cggaggaggt ctccttcgcc 420cctcaccgtg ggccccaaca gatccacctg gcgttcgttg gggcccacgg caaggaggag 480gatatgcgcg tgatgtacat cgcgcgcgat ccgagagaaa cctacgtaag gtacggggag 540agggaggata agcttgatgg gattgcggtc gcacgtgtgg agaggtacga gagggagcac 600atgtgcgacg cgcctgccaa cacgagtgtt gggtggaggg accctgggtt tatcaacgac 660gccgttctca taggtttgaa gaagggacag aggtattatt acaaggttgg aaatgataac 720ggaggttgga gtgcaactca aagttttgtg tcgaggaata gtgattcaga cgaaacaata 780gctttcctgt ttggtgacat gggaacagct gtaccataca atacgtttct gcgaacgcag 840gatgaaagca tatcaaccat gaagtggatc ctccgtgatg ttgaagctct aggcgacaag 900ccagcctttg tgtcgcacat tggagacatt agttatgcaa gaggttattc ctggttgtgg 960gaccattttt ttgcccagat tgaacctgtt gcctcccaag tggcatacca tgtttgcatt 1020ggcaatcatg agtatgactg gcctttgcag ccatggaaac ctgattgggc cagttatgga 1080aaagatgggg gtggtgagtg tggtgtgcct tacagtttaa ggtttaacat gcccggaaac 1140tcttcagaac tcactggaaa tgctgcagcc ccaccaacta ggaatcttta ttactcattt 1200gatatgggag cagtacactt tgtgtatatt tccacggaga ccaattttgt tcctgggagc 1260aaacagtacg acttcttgaa gcatgatttg gaatcggtta acaggagcaa gactcctttt 1320gtggtggtgc aagggcacag gcccatgtac actaccagcc atgaaaatag ggatgctgct 1380ttaagaggaa agatgcttga gcacttggaa cctctgttgg tgaataacaa tgtgacactt 1440gccctttggg gtcatgttca tagatacgag agattttgtc cactgaataa cttcacttgt 1500ggtgttaatg cgggtcacaa tgcaggggac aaaaaaggat atactgttca cattgtgatc 1560ggcatggcag ggcaagactg gcaacctgtc tgggaaccaa ggccagacca tcccgatgat 1620ccaatctttc cacagccaaa atggtctctg taccgcggag gcgagtttgg gtacacaaga 1680ctcgtcgcta caaagcagaa gctcgtgctt tcttatgtgg gaaaccatga cggtgaggtg 1740catgatcagt tggaaattct ggcatctggg gaagttgtca gtggtgacgg aggctgtagt 1800attgctgatg ctaattctaa agctggaaat gtgattgtgg aatccacatt gtcttggtat 1860gtcaagggag gaagtgtgct gctgcttggt gcattcatgg gttacgtttt tggttacgtt 1920acaagtgcaa ggaagaagtc tgaggtgcca gagagcaatt ggactccggt gaagactgag 1980gaaacttga 19896662PRTGlycine max 6Met Ile Pro Asp Leu Pro Leu Pro Phe Leu Phe Ser Leu Phe Ile Ile1 5 10 15Phe Phe His Leu Ala Glu Ser Lys Pro Ser Leu Thr Ala Thr Pro Thr 20 25 30Thr Leu Pro Ala Ser Gly Ala Thr Val Asn Leu Arg Trp Ser Gly Ile 35 40 45Pro Ser Pro Ser Asp Leu Asp Phe Leu Ala Ile Tyr Ser Pro Pro Thr 50 55 60Ser Pro His Asp Asn Phe Ile Gly Tyr Leu Phe Leu Ser Gln Ser Ala65 70 75 80Thr Trp Arg Thr Gly Ser Gly Asn Leu Ser Leu Pro Leu Val Asp Leu 85 90 95Arg Ser Asn Tyr Ser Phe Arg Ile Phe Ser Trp Thr Arg Ala Glu Ile 100 105 110Asn Pro Lys Arg Gln Asp His Asp His Asn Pro Leu Pro Val Thr Arg 115 120 125His Leu Leu Ala Phe Ser Glu Glu Val Ser Phe Ala Pro His Arg Gly 130 135 140Pro Gln Gln Ile His Leu Ala Phe Val Gly Ala His Gly Lys Glu Glu145 150 155

160Asp Met Arg Val Met Tyr Ile Ala Arg Asp Pro Arg Glu Thr Tyr Val 165 170 175Arg Tyr Gly Glu Arg Glu Asp Lys Leu Asp Gly Ile Ala Val Ala Arg 180 185 190Val Glu Arg Tyr Glu Arg Glu His Met Cys Asp Ala Pro Ala Asn Thr 195 200 205Ser Val Gly Trp Arg Asp Pro Gly Phe Ile Asn Asp Ala Val Leu Ile 210 215 220Gly Leu Lys Lys Gly Gln Arg Tyr Tyr Tyr Lys Val Gly Asn Asp Asn225 230 235 240Gly Gly Trp Ser Ala Thr Gln Ser Phe Val Ser Arg Asn Ser Asp Ser 245 250 255Asp Glu Thr Ile Ala Phe Leu Phe Gly Asp Met Gly Thr Ala Val Pro 260 265 270Tyr Asn Thr Phe Leu Arg Thr Gln Asp Glu Ser Ile Ser Thr Met Lys 275 280 285Trp Ile Leu Arg Asp Val Glu Ala Leu Gly Asp Lys Pro Ala Phe Val 290 295 300Ser His Ile Gly Asp Ile Ser Tyr Ala Arg Gly Tyr Ser Trp Leu Trp305 310 315 320Asp His Phe Phe Ala Gln Ile Glu Pro Val Ala Ser Gln Val Ala Tyr 325 330 335His Val Cys Ile Gly Asn His Glu Tyr Asp Trp Pro Leu Gln Pro Trp 340 345 350Lys Pro Asp Trp Ala Ser Tyr Gly Lys Asp Gly Gly Gly Glu Cys Gly 355 360 365Val Pro Tyr Ser Leu Arg Phe Asn Met Pro Gly Asn Ser Ser Glu Leu 370 375 380Thr Gly Asn Ala Ala Ala Pro Pro Thr Arg Asn Leu Tyr Tyr Ser Phe385 390 395 400Asp Met Gly Ala Val His Phe Val Tyr Ile Ser Thr Glu Thr Asn Phe 405 410 415Val Pro Gly Ser Lys Gln Tyr Asp Phe Leu Lys His Asp Leu Glu Ser 420 425 430Val Asn Arg Ser Lys Thr Pro Phe Val Val Val Gln Gly His Arg Pro 435 440 445Met Tyr Thr Thr Ser His Glu Asn Arg Asp Ala Ala Leu Arg Gly Lys 450 455 460Met Leu Glu His Leu Glu Pro Leu Leu Val Asn Asn Asn Val Thr Leu465 470 475 480Ala Leu Trp Gly His Val His Arg Tyr Glu Arg Phe Cys Pro Leu Asn 485 490 495Asn Phe Thr Cys Gly Val Asn Ala Gly His Asn Ala Gly Asp Lys Lys 500 505 510Gly Tyr Thr Val His Ile Val Ile Gly Met Ala Gly Gln Asp Trp Gln 515 520 525Pro Val Trp Glu Pro Arg Pro Asp His Pro Asp Asp Pro Ile Phe Pro 530 535 540Gln Pro Lys Trp Ser Leu Tyr Arg Gly Gly Glu Phe Gly Tyr Thr Arg545 550 555 560Leu Val Ala Thr Lys Gln Lys Leu Val Leu Ser Tyr Val Gly Asn His 565 570 575Asp Gly Glu Val His Asp Gln Leu Glu Ile Leu Ala Ser Gly Glu Val 580 585 590Val Ser Gly Asp Gly Gly Cys Ser Ile Ala Asp Ala Asn Ser Lys Ala 595 600 605Gly Asn Val Ile Val Glu Ser Thr Leu Ser Trp Tyr Val Lys Gly Gly 610 615 620Ser Val Leu Leu Leu Gly Ala Phe Met Gly Tyr Val Phe Gly Tyr Val625 630 635 640Thr Ser Ala Arg Lys Lys Ser Glu Val Pro Glu Ser Asn Trp Thr Pro 645 650 655Val Lys Thr Glu Glu Thr 66071965DNAZea mays 7atgtaccccg aaaaccccca cctccgcttc ctcctcttcc tcgccgtcgc ggcagttgcc 60gccggcgggg ctgcggcgaa caccaccctc accgcgtccc tctccggcaa ccagatcaag 120atcatctggt ccggactccc ggccccggac ggcctcgact acgttgccat ctactcgccg 180ccgtcctccc tcgaccgcga cttcctcggc tatctcttcc tcaacggctc cgcctcctgg 240cgcggcggct ccggggagct ctccctcccg ctcctcccga cgctccgcgc gccctaccag 300ttccgtctct ttcgctggcc cgccaaggag tactcctacc accacgtcga ccacgaccag 360aacccgctcc cccacggcaa gcaccgcgtc gccgtctccg ccgacgtctc cgtcggcgac 420cccgcccgcc ccgagcagct gcacctcgcg tttgcggatg aggtcgacga gatgcgggtc 480ctgttcgtgt gcggcgaccg cggggagagg gtcgtcaggt acgggctgca gaaggaggac 540gacaaggagt ggaaggaggt gggcacggat gtgagcacgt acgagcagag gcacatgtgc 600gattggccgg ccaacagcag cgtcgcctgg agggatccgg gattcgtctt cgacggcctc 660atgaagggat tggagcccgg aaggaggtac ttttacaagg ttggtagtga cacaggagga 720tggagtgaga tatacagctt catttcacgt gacagtgaag ccagtgagac caatgctttt 780ctatttggtg acatgggaac ttatgtgcct tataacacct acattcgcac acaatctgag 840agcttgtcca ctgtaaagtg gatccttcgt gatattgaag cccttggaga taaacccgcc 900tttatttcac acattgggga catcagctat gctagaggtt attcttgggt ctggtatcat 960ttcttcagcc agatcgagcc tattgctgcc aatactccat accatgtctg tataggaaat 1020catgagtatg attggccatc acaaccctgg aaaccatggt gggctacata tggaacggac 1080ggtggaggcg aatgtggaat accttatagt gtcaggttca gaatgccagg caattctatt 1140ctacctacag gtaatggtgg cccagacacc aggaatcttt attactcctt cgactcaggc 1200gtggtgcatt tcgtctacat gtcgaccgaa acaaattttg ttcagggaag tgagcagcac 1260aacttcttga aagcggacct tgagaaggtg aaccgaagta gaacaccctt tgttgttttt 1320cagggccacc gccccatgta cacctcgagc gatgaaacca gggacgcggc tttgaaacag 1380cagatgctcc agaatctgga accgctgctg gtgacataca atgtgaccct cgcgctatgg 1440ggacatgtcc acaggtacga gaggttctgc ccgatgcaga attcccaatg tgtcaacact 1500tcatcaagct tccagtactc tggcgctcct gtgcatcttg tgatcgggat gggcggccaa 1560gactggcaac ctgtatggca accgaggcct gatcacccag acgtccctat ctttcctcag 1620cctgagcgtt ccatgtaccg cggtggcgag tttggatacg ccagacttgt ggcaacaagg 1680gagaagctaa cattgactta tgtggggaac catgatgggc aggtccatga tatggtggag 1740atattttctg gcctggtatc ccccagtaac agtagtgttg ctgaggcggt ggatggaacc 1800aaacttggca caggagtcag caccgtgcgg aaaatttctc cgctgtactt ggaaattgga 1860ggcagtgtga tgtttgcgct gctcctggga ttttcctttg ggatacttgt caggagaaag 1920aaagaagctg cacagtggac tcaagtaaag aatgaggaat cgtag 19658654PRTZea mays 8Met Tyr Pro Glu Asn Pro His Leu Arg Phe Leu Leu Phe Leu Ala Val1 5 10 15Ala Ala Val Ala Ala Gly Gly Ala Ala Ala Asn Thr Thr Leu Thr Ala 20 25 30Ser Leu Ser Gly Asn Gln Ile Lys Ile Ile Trp Ser Gly Leu Pro Ala 35 40 45Pro Asp Gly Leu Asp Tyr Val Ala Ile Tyr Ser Pro Pro Ser Ser Leu 50 55 60Asp Arg Asp Phe Leu Gly Tyr Leu Phe Leu Asn Gly Ser Ala Ser Trp65 70 75 80Arg Gly Gly Ser Gly Glu Leu Ser Leu Pro Leu Leu Pro Thr Leu Arg 85 90 95Ala Pro Tyr Gln Phe Arg Leu Phe Arg Trp Pro Ala Lys Glu Tyr Ser 100 105 110Tyr His His Val Asp His Asp Gln Asn Pro Leu Pro His Gly Lys His 115 120 125Arg Val Ala Val Ser Ala Asp Val Ser Val Gly Asp Pro Ala Arg Pro 130 135 140Glu Gln Leu His Leu Ala Phe Ala Asp Glu Val Asp Glu Met Arg Val145 150 155 160Leu Phe Val Cys Gly Asp Arg Gly Glu Arg Val Val Arg Tyr Gly Leu 165 170 175Gln Lys Glu Asp Asp Lys Glu Trp Lys Glu Val Gly Thr Asp Val Ser 180 185 190Thr Tyr Glu Gln Arg His Met Cys Asp Trp Pro Ala Asn Ser Ser Val 195 200 205Ala Trp Arg Asp Pro Gly Phe Val Phe Asp Gly Leu Met Lys Gly Leu 210 215 220Glu Pro Gly Arg Arg Tyr Phe Tyr Lys Val Gly Ser Asp Thr Gly Gly225 230 235 240Trp Ser Glu Ile Tyr Ser Phe Ile Ser Arg Asp Ser Glu Ala Ser Glu 245 250 255Thr Asn Ala Phe Leu Phe Gly Asp Met Gly Thr Tyr Val Pro Tyr Asn 260 265 270Thr Tyr Ile Arg Thr Gln Ser Glu Ser Leu Ser Thr Val Lys Trp Ile 275 280 285Leu Arg Asp Ile Glu Ala Leu Gly Asp Lys Pro Ala Phe Ile Ser His 290 295 300Ile Gly Asp Ile Ser Tyr Ala Arg Gly Tyr Ser Trp Val Trp Tyr His305 310 315 320Phe Phe Ser Gln Ile Glu Pro Ile Ala Ala Asn Thr Pro Tyr His Val 325 330 335Cys Ile Gly Asn His Glu Tyr Asp Trp Pro Ser Gln Pro Trp Lys Pro 340 345 350Trp Trp Ala Thr Tyr Gly Thr Asp Gly Gly Gly Glu Cys Gly Ile Pro 355 360 365Tyr Ser Val Arg Phe Arg Met Pro Gly Asn Ser Ile Leu Pro Thr Gly 370 375 380Asn Gly Gly Pro Asp Thr Arg Asn Leu Tyr Tyr Ser Phe Asp Ser Gly385 390 395 400Val Val His Phe Val Tyr Met Ser Thr Glu Thr Asn Phe Val Gln Gly 405 410 415Ser Glu Gln His Asn Phe Leu Lys Ala Asp Leu Glu Lys Val Asn Arg 420 425 430Ser Arg Thr Pro Phe Val Val Phe Gln Gly His Arg Pro Met Tyr Thr 435 440 445Ser Ser Asp Glu Thr Arg Asp Ala Ala Leu Lys Gln Gln Met Leu Gln 450 455 460Asn Leu Glu Pro Leu Leu Val Thr Tyr Asn Val Thr Leu Ala Leu Trp465 470 475 480Gly His Val His Arg Tyr Glu Arg Phe Cys Pro Met Gln Asn Ser Gln 485 490 495Cys Val Asn Thr Ser Ser Ser Phe Gln Tyr Ser Gly Ala Pro Val His 500 505 510Leu Val Ile Gly Met Gly Gly Gln Asp Trp Gln Pro Val Trp Gln Pro 515 520 525Arg Pro Asp His Pro Asp Val Pro Ile Phe Pro Gln Pro Glu Arg Ser 530 535 540Met Tyr Arg Gly Gly Glu Phe Gly Tyr Ala Arg Leu Val Ala Thr Arg545 550 555 560Glu Lys Leu Thr Leu Thr Tyr Val Gly Asn His Asp Gly Gln Val His 565 570 575Asp Met Val Glu Ile Phe Ser Gly Leu Val Ser Pro Ser Asn Ser Ser 580 585 590Val Ala Glu Ala Val Asp Gly Thr Lys Leu Gly Thr Gly Val Ser Thr 595 600 605Val Arg Lys Ile Ser Pro Leu Tyr Leu Glu Ile Gly Gly Ser Val Met 610 615 620Phe Ala Leu Leu Leu Gly Phe Ser Phe Gly Ile Leu Val Arg Arg Lys625 630 635 640Lys Glu Ala Ala Gln Trp Thr Gln Val Lys Asn Glu Glu Ser 645 650922DNAArtificial SequenceSynthetic primer 9tggttcacgt agtgggccat cg 221021DNAArtificial SequenceSynthetic primer 10ttgaagttta acatgcctgg g 211121DNAArtificial sequenceSynthetic primer 11tccaatgctc gattgattag c 211231DNAArtificial SequenceSynthetic primer 12ggccgtcgac atgatcgtta atttctcttt c 311331DNAArtificial SequenceSynthetic primer 13ccggactagt tcatgtctcc tcgttcttga c 311427DNAArtificial SequenceSynthetic primer 14gtttcacatc aacattgtgg tcattgg 271524DNAArtificial SequenceSynthetic primer 15gagtacttgg gggtagtggc atcc 241630DNAArtificial SequenceSynthetic primer 16ggttgagctc gattctaaag cgaccatttc 301729DNAArtificial SequenceSynthetic primer 17ttttggtacc tcaggatccg aaagtcagc 29181959DNABrassica rapa 18atgatcgtcg agttctctac cttcatcctc ttcctctccg tcttcgtctc ctcagctaac 60gccaaagcaa ccttatccat ctcccccaaa actctaagcc gatccggcga ttccatcctc 120atcaaatggt ccaacctcga ctctccctcc gatctcgact ggctaggcat ctactccccc 180ccagcctctc cccacgacca cttcatcggc tacaagttcc tcaacgcctc ccccacgtgg 240caatccggct ccggcgcgat ctccctcccc ctcaccaacc tccgatcgaa ctacacgttc 300cgtatcttcc gatggacgca gtccgagatc aatccgaagc acaaggacca cgaccagaat 360cccttaccgg gaacgaagca ccttctggcg gaatcggagc aggtggggtt cggatccgcc 420ggcgtgggga ggccggagca gatccatttg gcgttcgagg ataaggttaa caggatgcag 480gtcacgttcg tagctgggga tggggaagaa aggttcgtga ggtacggaga ggcggaggat 540gcgttggcga actccgcggc ggcgcgcggg attaggtacg agagggagca tatgtgtaat 600gctccggcta attccaccgt gggatggaga gatcccgggt ggatttttca taccgttatg 660aagaatttga acggtggcgt taggtattat tatcaggttg ggagtgattc aaagggatgg 720agtgagatcc acagcttcat cgctcgagat atctactcag aagaaaccat agctttcatg 780ttcggagaca tgggttgcgc tacaccttac aataccttta tccggacgca ggacgagagt 840atgtccacag tgaagtggat actccgcgac atcgaagctc ttggtgacaa gccggctctc 900gtttcgcaca ttggagatat aagctacgct cgtggttact cctgggtgtg ggatgagttc 960tttgctcaga tcgagcctat tgcctcgaga gttccttacc acgtctgcat tggtaaccac 1020gagtatgact tccctactca gccgtggaaa cctgattggg gaacttacgg taatgacggt 1080gggggagagt gcggtgtgcc gtatagtctc aagttcaaca tgcctggaaa ctcgtcggaa 1140ccaacgggaa cgaaagctcc tcctacaagg aatttgtatt actcttacga catggggtcg 1200gttcatttcc tttacatctc caccgagacg aactttctca aaggagggag gcaatacgag 1260tttataaagc gagatcttga gtctgtgaac agggagaaaa caccgtttgt tgtcgtgcaa 1320ggacacagac cgatgtacac cacgagcaac gaggtgagag acgcgatgat taggcaaaag 1380atggtggagc atttggagcc gctgtttgtg gagaacaacg tgacgcttgc tctgtgggga 1440catgttcata gatacgagag gttttgtccg ataagcaaca acacgtgtgg gaaacagtgg 1500agaggaagcc cggttcatct tgtgatcggt atgggtggtc aagactggca accgatttgg 1560cagccgagac cgaaccatcc gggtcttcct atattccctc agcctgaaca gtcgatgtac 1620aggacgggtg agtttgggta cactcgtttg gttgcgaaca aagagaagct cactgtttcg 1680tttgtgggta accatgatgg agaagttcat gatagtgttg agatgtttgc gtctggggaa 1740gtaatcagtg ggaggaaaga ggaaactatt aagaccgttc ctgtatctgc aacacttgtg 1800gggaaacctg agtctgatgt cttatggtat gttaaaggag caggcttgtt ggttattggt 1860gtgcttttag ggttcattat agggtttttt acaaggggga agaaaggatc ttcttcatct 1920gataaccgtt ggatcccagt caagaacgag gagacatga 195919652PRTBrassica rapa 19Met Ile Val Glu Phe Ser Thr Phe Ile Leu Phe Leu Ser Val Phe Val1 5 10 15Ser Ser Ala Asn Ala Lys Ala Thr Leu Ser Ile Ser Pro Lys Thr Leu 20 25 30Ser Arg Ser Gly Asp Ser Ile Leu Ile Lys Trp Ser Asn Leu Asp Ser 35 40 45Pro Ser Asp Leu Asp Trp Leu Gly Ile Tyr Ser Pro Pro Ala Ser Pro 50 55 60His Asp His Phe Ile Gly Tyr Lys Phe Leu Asn Ala Ser Pro Thr Trp65 70 75 80Gln Ser Gly Ser Gly Ala Ile Ser Leu Pro Leu Thr Asn Leu Arg Ser 85 90 95Asn Tyr Thr Phe Arg Ile Phe Arg Trp Thr Gln Ser Glu Ile Asn Pro 100 105 110Lys His Lys Asp His Asp Gln Asn Pro Leu Pro Gly Thr Lys His Leu 115 120 125Leu Ala Glu Ser Glu Gln Val Gly Phe Gly Ser Ala Gly Val Gly Arg 130 135 140Pro Glu Gln Ile His Leu Ala Phe Glu Asp Lys Val Asn Arg Met Gln145 150 155 160Val Thr Phe Val Ala Gly Asp Gly Glu Glu Arg Phe Val Arg Tyr Gly 165 170 175Glu Ala Glu Asp Ala Leu Ala Asn Ser Ala Ala Ala Arg Gly Ile Arg 180 185 190Tyr Glu Arg Glu His Met Cys Asn Ala Pro Ala Asn Ser Thr Val Gly 195 200 205Trp Arg Asp Pro Gly Trp Ile Phe His Thr Val Met Lys Asn Leu Asn 210 215 220Gly Gly Val Arg Tyr Tyr Tyr Gln Val Gly Ser Asp Ser Lys Gly Trp225 230 235 240Ser Glu Ile His Ser Phe Ile Ala Arg Asp Ile Tyr Ser Glu Glu Thr 245 250 255Ile Ala Phe Met Phe Gly Asp Met Gly Cys Ala Thr Pro Tyr Asn Thr 260 265 270Phe Ile Arg Thr Gln Asp Glu Ser Met Ser Thr Val Lys Trp Ile Leu 275 280 285Arg Asp Ile Glu Ala Leu Gly Asp Lys Pro Ala Leu Val Ser His Ile 290 295 300Gly Asp Ile Ser Tyr Ala Arg Gly Tyr Ser Trp Val Trp Asp Glu Phe305 310 315 320Phe Ala Gln Ile Glu Pro Ile Ala Ser Arg Val Pro Tyr His Val Cys 325 330 335Ile Gly Asn His Glu Tyr Asp Phe Pro Thr Gln Pro Trp Lys Pro Asp 340 345 350Trp Gly Thr Tyr Gly Asn Asp Gly Gly Gly Glu Cys Gly Val Pro Tyr 355 360 365Ser Leu Lys Phe Asn Met Pro Gly Asn Ser Ser Glu Pro Thr Gly Thr 370 375 380Lys Ala Pro Pro Thr Arg Asn Leu Tyr Tyr Ser Tyr Asp Met Gly Ser385 390 395 400Val His Phe Leu Tyr Ile Ser Thr Glu Thr Asn Phe Leu Lys Gly Gly 405 410 415Arg Gln Tyr Glu Phe Ile Lys Arg Asp Leu Glu Ser Val Asn Arg Glu 420 425 430Lys Thr Pro Phe Val Val Val Gln Gly His Arg Pro Met Tyr Thr Thr 435 440 445Ser Asn Glu Val Arg Asp Ala Met Ile Arg Gln Lys Met Val Glu His 450 455 460Leu Glu Pro Leu Phe Val Glu Asn Asn Val Thr Leu Ala Leu Trp Gly465 470 475 480His Val His Arg Tyr Glu Arg Phe Cys Pro Ile Ser Asn Asn Thr Cys 485 490 495Gly Lys Gln Trp Arg Gly Ser Pro Val His Leu Val Ile Gly Met Gly 500

505 510Gly Gln Asp Trp Gln Pro Ile Trp Gln Pro Arg Pro Asn His Pro Gly 515 520 525Leu Pro Ile Phe Pro Gln Pro Glu Gln Ser Met Tyr Arg Thr Gly Glu 530 535 540Phe Gly Tyr Thr Arg Leu Val Ala Asn Lys Glu Lys Leu Thr Val Ser545 550 555 560Phe Val Gly Asn His Asp Gly Glu Val His Asp Ser Val Glu Met Phe 565 570 575Ala Ser Gly Glu Val Ile Ser Gly Arg Lys Glu Glu Thr Ile Lys Thr 580 585 590Val Pro Val Ser Ala Thr Leu Val Gly Lys Pro Glu Ser Asp Val Leu 595 600 605Trp Tyr Val Lys Gly Ala Gly Leu Leu Val Ile Gly Val Leu Leu Gly 610 615 620Phe Ile Ile Gly Phe Phe Thr Arg Gly Lys Lys Gly Ser Ser Ser Ser625 630 635 640Asp Asn Arg Trp Ile Pro Val Lys Asn Glu Glu Thr 645 650201965DNAHordeum vulgare 20atgcacccca aaaccccgcc tctcctcctc gtcctcctct tcttcgccgc cggcgaggcc 60gcggggacca ccctcacggc caccccggcg aagctcaccc aatccgacca agaaatcacg 120atccggtggt ccgacctccc gtccccggat ggcctcgacc acgtggcgat ctactccccg 180ccgtcctcca gcgaccgcga cttcctaggc tacatcttcc tcaatggctc cgcctcctgg 240cgcagcggcc gcggagagct caccctccca cggctcccca acctgcgggc gccctaccag 300ttccgcctct tccgctggcc cgccagagag tactcctacc accacgtcga ccacgacggc 360aacccgctcc cccacggcca ccaccgcgtc gccctatccg gcgaggtcgc tttcgcgggc 420tcggccgcgc ggcccgagca ggtgcacctc gcgttcgccg atagggccga cgagatgcgg 480gtgatgttcg tgtgcgcgga cgccggcaag agggccgtga ggtacgggct tgagaaggag 540gaggagaagg gctggacgga agtgggcacg gaggtgagga cgtacgagca gaagcacatg 600tgcgacacgc cggcgaacga caccgtaggg tggagggatc cgggcttcgt cttcgatggc 660ctcatgaatg ggttggagcc cggaaggagg tacttttaca aggtcggtag tgacctggga 720ggatggagcg agacatacag ctttatttca cgtgacagtg aggccaatga gaccattgct 780tttctcttcg gtgatatggg cacttatgta ccatacaaca cctacatccg cacacaagat 840gagagcttgt caacggtgaa gtggatcctc cgtgatattg aagcccttgg agataagcct 900gcatttattt cgcacattgg ggacatcagt tatgccagag gctatgcttg ggtgtgggat 960catttcttca gccagattga gcctattgca gccaatactc cataccatgt ctgcatagga 1020aatcatgagt atgattggcc ttcacaacct tggaaacctt catggtctac atatgggaag 1080gatggtggag gtgaatgtgg aataccatac agtgtcaagt tcagaatgcc tggggattct 1140gttctaccta ctggcaatgg agctccggac acacggaatc tctactactc ttttgattca 1200ggcgtcgtgc attttgtgta catgtcgact gaaactaatt tcgttcaggg cagcgaccaa 1260cacaatttcc taaaagctga tctggagaag gtgaaccgaa gcagaacccc atttgttgtg 1320tttcagggcc accggcccat gtatacctcg agcaacgaag ccagggattc tgccatgaga 1380cagcagatgg tccagcatct tgaaccgctc ttggtgatat acaatgtgac gcttgccctg 1440tggggacatg tccataggta tgagaggttc tgccccatga agaattcaca gtgtctgaac 1500acatcatcaa gcttcgtata ccctggtgcc cctgttcatg ttgtgatcgg gatggctgga 1560caagattggc aaccgatctg gcaaccaagg cgtgatcatc caaatgttcc catctttcca 1620cagcctggga tctccatgta ccgtggtggt gagttcgggt acacaaagct cgcagctaac 1680agggagaagc taacgctgat gtacgttggg aaccacgatg gacaagtcca tgacatggtg 1740gaaatattct ctggacaaac atctactgaa gctagcgcta ctgaggcggt caatcaaaca 1800aagctcagct cgggagccag cgccaagctg aagatttccc caatatactt ggaaattgga 1860ggtagtgtga tgtttgccct aatgcttggt tttgccttgg gatttctcct caggaagaag 1920agagaagctg cacaatggac tccggtcaag aacgaggaat cctaa 196521654PRTHordeum vulgare 21Met His Pro Lys Thr Pro Pro Leu Leu Leu Val Leu Leu Phe Phe Ala1 5 10 15Ala Gly Glu Ala Ala Gly Thr Thr Leu Thr Ala Thr Pro Ala Lys Leu 20 25 30Thr Gln Ser Asp Gln Glu Ile Thr Ile Arg Trp Ser Asp Leu Pro Ser 35 40 45Pro Asp Gly Leu Asp His Val Ala Ile Tyr Ser Pro Pro Ser Ser Ser 50 55 60Asp Arg Asp Phe Leu Gly Tyr Ile Phe Leu Asn Gly Ser Ala Ser Trp65 70 75 80Arg Ser Gly Arg Gly Glu Leu Thr Leu Pro Arg Leu Pro Asn Leu Arg 85 90 95Ala Pro Tyr Gln Phe Arg Leu Phe Arg Trp Pro Ala Arg Glu Tyr Ser 100 105 110Tyr His His Val Asp His Asp Gly Asn Pro Leu Pro His Gly His His 115 120 125Arg Val Ala Leu Ser Gly Glu Val Ala Phe Ala Gly Ser Ala Ala Arg 130 135 140Pro Glu Gln Val His Leu Ala Phe Ala Asp Arg Ala Asp Glu Met Arg145 150 155 160Val Met Phe Val Cys Ala Asp Ala Gly Lys Arg Ala Val Arg Tyr Gly 165 170 175Leu Glu Lys Glu Glu Glu Lys Gly Trp Thr Glu Val Gly Thr Glu Val 180 185 190Arg Thr Tyr Glu Gln Lys His Met Cys Asp Thr Pro Ala Asn Asp Thr 195 200 205Val Gly Trp Arg Asp Pro Gly Phe Val Phe Asp Gly Leu Met Asn Gly 210 215 220Leu Glu Pro Gly Arg Arg Tyr Phe Tyr Lys Val Gly Ser Asp Leu Gly225 230 235 240Gly Trp Ser Glu Thr Tyr Ser Phe Ile Ser Arg Asp Ser Glu Ala Asn 245 250 255Glu Thr Ile Ala Phe Leu Phe Gly Asp Met Gly Thr Tyr Val Pro Tyr 260 265 270Asn Thr Tyr Ile Arg Thr Gln Asp Glu Ser Leu Ser Thr Val Lys Trp 275 280 285Ile Leu Arg Asp Ile Glu Ala Leu Gly Asp Lys Pro Ala Phe Ile Ser 290 295 300His Ile Gly Asp Ile Ser Tyr Ala Arg Gly Tyr Ala Trp Val Trp Asp305 310 315 320His Phe Phe Ser Gln Ile Glu Pro Ile Ala Ala Asn Thr Pro Tyr His 325 330 335Val Cys Ile Gly Asn His Glu Tyr Asp Trp Pro Ser Gln Pro Trp Lys 340 345 350Pro Ser Trp Ser Thr Tyr Gly Lys Asp Gly Gly Gly Glu Cys Gly Ile 355 360 365Pro Tyr Ser Val Lys Phe Arg Met Pro Gly Asp Ser Val Leu Pro Thr 370 375 380Gly Asn Gly Ala Pro Asp Thr Arg Asn Leu Tyr Tyr Ser Phe Asp Ser385 390 395 400Gly Val Val His Phe Val Tyr Met Ser Thr Glu Thr Asn Phe Val Gln 405 410 415Gly Ser Asp Gln His Asn Phe Leu Lys Ala Asp Leu Glu Lys Val Asn 420 425 430Arg Ser Arg Thr Pro Phe Val Val Phe Gln Gly His Arg Pro Met Tyr 435 440 445Thr Ser Ser Asn Glu Ala Arg Asp Ser Ala Met Arg Gln Gln Met Val 450 455 460Gln His Leu Glu Pro Leu Leu Val Ile Tyr Asn Val Thr Leu Ala Leu465 470 475 480Trp Gly His Val His Arg Tyr Glu Arg Phe Cys Pro Met Lys Asn Ser 485 490 495Gln Cys Leu Asn Thr Ser Ser Ser Phe Val Tyr Pro Gly Ala Pro Val 500 505 510His Val Val Ile Gly Met Ala Gly Gln Asp Trp Gln Pro Ile Trp Gln 515 520 525Pro Arg Arg Asp His Pro Asn Val Pro Ile Phe Pro Gln Pro Gly Ile 530 535 540Ser Met Tyr Arg Gly Gly Glu Phe Gly Tyr Thr Lys Leu Ala Ala Asn545 550 555 560Arg Glu Lys Leu Thr Leu Met Tyr Val Gly Asn His Asp Gly Gln Val 565 570 575His Asp Met Val Glu Ile Phe Ser Gly Gln Thr Ser Thr Glu Ala Ser 580 585 590Ala Thr Glu Ala Val Asn Gln Thr Lys Leu Ser Ser Gly Ala Ser Ala 595 600 605Lys Leu Lys Ile Ser Pro Ile Tyr Leu Glu Ile Gly Gly Ser Val Met 610 615 620Phe Ala Leu Met Leu Gly Phe Ala Leu Gly Phe Leu Leu Arg Lys Lys625 630 635 640Arg Glu Ala Ala Gln Trp Thr Pro Val Lys Asn Glu Glu Ser 645 650221980DNAMedicago truncatula 22atgatccctc acacaatact cttcacctta ctcttatcct caaccttcac ctctaccctc 60gcccaatcaa aacccaccct aacagtaacc ccaaccaccc tcacaaaatc tggcgacacc 120gtcaccctcc ggtggtccgg tatccaatcc ccctccgatc tcgacttcct cgcaatctac 180tccccaccta cctccgccca caaaaactac atcggctacc tcttcctctc caaatccccc 240acctggcaat ccggctccgg caacctctct cttcctctca tcaacctccg ttccaactac 300tccttccgta tcttccactg gtcccaatcc gaaatcaacc ctaaacgtca agatcacgat 360cataatccct taccacaaac gcatcacctt cttgctttct ctgatgaagt ttcttttcca 420tcccttcgac cggagcagat tcatcttgct tttgcagatg aagaagatgc tatgagggtg 480atgtatgtga cgggggttcc gaagaagacg tatgtgagat atggagaaag agaggatatg 540atggatagat tggttgttgc gaatgtgaag agatatgaga gagagcatat gtgtgatgct 600cctgctaatc agagtgttgg ttggagggat cctggtttta ttcatgatgc tttgattact 660ggtttggaca aaggaagaag atattactac caggttggaa atgataatgg aggttggagt 720gcaacccata gctttgtgtc gaggaatagt gattcaaatg aaacaatagc tttccttttc 780ggtgacatgg gaacatttac agcatacaat acgtatttgc gtacacaaga tgagagcata 840tcaaccatga agtggatcct gcgtgatgtt gaagctctag gaaacaagcc cgcctttata 900tcacacattg gagacacaag ttatgcaaga ggttatgcgt ggttgtggga tcattttttc 960gcacagattg aacctgttgc aaccaaagtg gcataccatg tatgcattgg caatcacgag 1020tataactggc ctttacagcc gtggaaacct gattgggcta attatagaac agatggaggt 1080ggtgaatgtg gtgtacccta cagtttaagg ttcaacatgc caggaaactc ttcagaaccc 1140actggaactg tagctccagc cactaggaat ctttattact catttgatat gggagcagta 1200cattttgttt atatttccac agagaccaat ttccttcctg ggagcaatca gtataacttc 1260ttaaagcgtg atttggaatc agttgacagg aacaagactc cttttgtagt agtccaaggg 1320caccgaccca tgtacacaac aagcaatgaa tttagggatg ctgcgttaag aggaaagatg 1380gttgagcacc tggaacctct attggtgaat aaccatgtaa cccttgccct ttggggtcat 1440gttcataggt acgagagatt ttgtccacta aacaacttta cttgtggaaa tggtgtgggt 1500cggagagcag gggaaaaagg tcataccatt catcttgtga tcggcatggc agggcaagac 1560tggcaaccca tgtggcgacc aagaccggat catcccgatg tcccaatcta tccacaacca 1620aaacgatctt tgtaccgcgg gggtgagttc ggatacatta gattgatggc tacaaagcag 1680aatctcgtga tttcttatgt tggtaatcat gatggcgagg tgcatgacac attggagatt 1740ctggaatctg gagaagttgt tagtggtggt ggtggtaacg ataatgttaa tggcggtatt 1800ggtagtgcta aacctgaagg tcagattaaa gaatccacgt tgtcgtggta tgtccaggga 1860ggaagtgtac tagtgcttgg ggcctttatg ggctacattc ttggtttcgt ttcacatgct 1920aggaagaagc agcccgagtc caggagtggt tttagccccg tgaagactga ggaaacatga 198023659PRTMedicago truncatula 23Met Ile Pro His Thr Ile Leu Phe Thr Leu Leu Leu Ser Ser Thr Phe1 5 10 15Thr Ser Thr Leu Ala Gln Ser Lys Pro Thr Leu Thr Val Thr Pro Thr 20 25 30Thr Leu Thr Lys Ser Gly Asp Thr Val Thr Leu Arg Trp Ser Gly Ile 35 40 45Gln Ser Pro Ser Asp Leu Asp Phe Leu Ala Ile Tyr Ser Pro Pro Thr 50 55 60Ser Ala His Lys Asn Tyr Ile Gly Tyr Leu Phe Leu Ser Lys Ser Pro65 70 75 80Thr Trp Gln Ser Gly Ser Gly Asn Leu Ser Leu Pro Leu Ile Asn Leu 85 90 95Arg Ser Asn Tyr Ser Phe Arg Ile Phe His Trp Ser Gln Ser Glu Ile 100 105 110Asn Pro Lys Arg Gln Asp His Asp His Asn Pro Leu Pro Gln Thr His 115 120 125His Leu Leu Ala Phe Ser Asp Glu Val Ser Phe Pro Ser Leu Arg Pro 130 135 140Glu Gln Ile His Leu Ala Phe Ala Asp Glu Glu Asp Ala Met Arg Val145 150 155 160Met Tyr Val Thr Gly Val Pro Lys Lys Thr Tyr Val Arg Tyr Gly Glu 165 170 175Arg Glu Asp Met Met Asp Arg Leu Val Val Ala Asn Val Lys Arg Tyr 180 185 190Glu Arg Glu His Met Cys Asp Ala Pro Ala Asn Gln Ser Val Gly Trp 195 200 205Arg Asp Pro Gly Phe Ile His Asp Ala Leu Ile Thr Gly Leu Asp Lys 210 215 220Gly Arg Arg Tyr Tyr Tyr Gln Val Gly Asn Asp Asn Gly Gly Trp Ser225 230 235 240Ala Thr His Ser Phe Val Ser Arg Asn Ser Asp Ser Asn Glu Thr Ile 245 250 255Ala Phe Leu Phe Gly Asp Met Gly Thr Phe Thr Ala Tyr Asn Thr Tyr 260 265 270Leu Arg Thr Gln Asp Glu Ser Ile Ser Thr Met Lys Trp Ile Leu Arg 275 280 285Asp Val Glu Ala Leu Gly Asn Lys Pro Ala Phe Ile Ser His Ile Gly 290 295 300Asp Thr Ser Tyr Ala Arg Gly Tyr Ala Trp Leu Trp Asp His Phe Phe305 310 315 320Ala Gln Ile Glu Pro Val Ala Thr Lys Val Ala Tyr His Val Cys Ile 325 330 335Gly Asn His Glu Tyr Asn Trp Pro Leu Gln Pro Trp Lys Pro Asp Trp 340 345 350Ala Asn Tyr Arg Thr Asp Gly Gly Gly Glu Cys Gly Val Pro Tyr Ser 355 360 365Leu Arg Phe Asn Met Pro Gly Asn Ser Ser Glu Pro Thr Gly Thr Val 370 375 380Ala Pro Ala Thr Arg Asn Leu Tyr Tyr Ser Phe Asp Met Gly Ala Val385 390 395 400His Phe Val Tyr Ile Ser Thr Glu Thr Asn Phe Leu Pro Gly Ser Asn 405 410 415Gln Tyr Asn Phe Leu Lys Arg Asp Leu Glu Ser Val Asp Arg Asn Lys 420 425 430Thr Pro Phe Val Val Val Gln Gly His Arg Pro Met Tyr Thr Thr Ser 435 440 445Asn Glu Phe Arg Asp Ala Ala Leu Arg Gly Lys Met Val Glu His Leu 450 455 460Glu Pro Leu Leu Val Asn Asn His Val Thr Leu Ala Leu Trp Gly His465 470 475 480Val His Arg Tyr Glu Arg Phe Cys Pro Leu Asn Asn Phe Thr Cys Gly 485 490 495Asn Gly Val Gly Arg Arg Ala Gly Glu Lys Gly His Thr Ile His Leu 500 505 510Val Ile Gly Met Ala Gly Gln Asp Trp Gln Pro Met Trp Arg Pro Arg 515 520 525Pro Asp His Pro Asp Val Pro Ile Tyr Pro Gln Pro Lys Arg Ser Leu 530 535 540Tyr Arg Gly Gly Glu Phe Gly Tyr Ile Arg Leu Met Ala Thr Lys Gln545 550 555 560Asn Leu Val Ile Ser Tyr Val Gly Asn His Asp Gly Glu Val His Asp 565 570 575Thr Leu Glu Ile Leu Glu Ser Gly Glu Val Val Ser Gly Gly Gly Gly 580 585 590Asn Asp Asn Val Asn Gly Gly Ile Gly Ser Ala Lys Pro Glu Gly Gln 595 600 605Ile Lys Glu Ser Thr Leu Ser Trp Tyr Val Gln Gly Gly Ser Val Leu 610 615 620Val Leu Gly Ala Phe Met Gly Tyr Ile Leu Gly Phe Val Ser His Ala625 630 635 640Arg Lys Lys Gln Pro Glu Ser Arg Ser Gly Phe Ser Pro Val Lys Thr 645 650 655Glu Glu Thr 242007DNAPhyscomitrella patens 24atgggatcgc aagtattcca ttttcttctg gtgttctttg ggtactttct gcatggagct 60tcatcagaat ctgtgatttt ggacgcgaga cctacaatat tacaacattc aggagaaaat 120atcactcttg cttggaaggg tgtgaattta ccgacgaaat acgattggct gggtatatat 180acgcctccta cttctcctga cgaccagcat atcgggtaca tacttctctc ttcctgttca 240acatggacaa caggcgcctg ctccttgcag atccccttgg tcaacatgcg tgctccttac 300agtttccgaa ttttcagagg cgtgttcgta aatgtatctg caagtacaaa tgtgactgga 360tcaaacaatg gggctacaac gatatcattg gatcgggagg gtaatcctct accagatgtc 420acgaaacggt tagctgcaag cccagttgtt caattctcca attacaacga gccaacacaa 480attcatctag ctctttcctc ggacgagact gctgttaggg ttatgtttgt cactagggat 540cctctgagaa gccaagtaag attcggggaa gatggagatg aactgggcaa cacagttgat 600gctacatcag tcacatactc tcaaattgat atgtgcgatg aacctgcaag ttcttatggg 660tggagatctc cgggatacat acataatgtt gtgatggggg ggctgaatcc tgggagtcgc 720tatttctatc gggtaggaag caatgtagga ggatggagct cgacctatag cttcatcgct 780ccacatcctc gtgctgatga aacaaatgct ctcatattcg gtgacatggg tacttcgatt 840ccttattcaa cgtatcaata cacgcagagc gagagcaaga ataccgtgaa gtggctcaca 900cgggacctag aacaaatagg tgacaaacct agcttcgtag cgcacattgg tgacataagc 960tatgctcgtg gtttatcttg gctctgggac aacttcttca cccaaatcga gcccgtagct 1020gcaagatcac catatcatgt ttgcatggga aaccacgaat atgattggcc tgggcaacct 1080ttcaagccag actggtcacc ataccaaaca gatggaggcg gagaatgtgg cgtgccatat 1140agcttacgct tcatcatgcc gggaaactcc tccttaccca ctggaactac ctccccagcc 1200accaaaaacc tctattattc cattgatgtt ggggttgtgc atttcctctt ctattctacc 1260gaaaccgatt tccaggtagg ctccccccag tacactttta tagccaacga cttgagaaca 1320gttgacagga acaagacgcc ctttgtggta tttttgggcc atcggccgct ctatacaacc 1380gattaccgag ccttgttaga cacgatgaca cagaaattag ttcaaacttt tgagcctttg 1440ttgatagata ccaatgtcac tgtagccttt tgtggccatg tccataagta cgagcgaatg 1500tgccccttga aaaattacac ctgtattgaa ccatctaagg caaacggtga gcttccaatt 1560catatggtgg tgggaatggg aggtgctgat caccaaccca ttgatgaccc tctccccagt 1620caaagtcagc ctatctttcc tcagcccagc tggtcagtat ttcgaacatt tgaatgggga 1680tatatcaggc tacatgcaac gagacatctc atgacgattt catatgttgg taaccacgat 1740gggaaggtgc atgatgttgt cgaaattcca gttctggatg atatcaagtc tggagcatat 1800gttgagtcga gggagtcttt ttttgacact gccagcggag tgcaaatacc ttgtggcagg 1860tctgagaata ttgtagcatt cctgtttgtt ttagcgttgg gttgtggatg cggggcggct 1920gctactcttt ttttcatgcg gaggcagcag aggaagcaga tttggcagcc tgtcaaccgt 1980gaggaagcta gttcttctca attataa 200725668PRTPhyscomitrella patens 25Met Gly Ser Gln Val Phe His Phe Leu Leu Val Phe Phe Gly Tyr Phe1

5 10 15Leu His Gly Ala Ser Ser Glu Ser Val Ile Leu Asp Ala Arg Pro Thr 20 25 30Ile Leu Gln His Ser Gly Glu Asn Ile Thr Leu Ala Trp Lys Gly Val 35 40 45Asn Leu Pro Thr Lys Tyr Asp Trp Leu Gly Ile Tyr Thr Pro Pro Thr 50 55 60Ser Pro Asp Asp Gln His Ile Gly Tyr Ile Leu Leu Ser Ser Cys Ser65 70 75 80Thr Trp Thr Thr Gly Ala Cys Ser Leu Gln Ile Pro Leu Val Asn Met 85 90 95Arg Ala Pro Tyr Ser Phe Arg Ile Phe Arg Gly Val Phe Val Asn Val 100 105 110Ser Ala Ser Thr Asn Val Thr Gly Ser Asn Asn Gly Ala Thr Thr Ile 115 120 125Ser Leu Asp Arg Glu Gly Asn Pro Leu Pro Asp Val Thr Lys Arg Leu 130 135 140Ala Ala Ser Pro Val Val Gln Phe Ser Asn Tyr Asn Glu Pro Thr Gln145 150 155 160Ile His Leu Ala Leu Ser Ser Asp Glu Thr Ala Val Arg Val Met Phe 165 170 175Val Thr Arg Asp Pro Leu Arg Ser Gln Val Arg Phe Gly Glu Asp Gly 180 185 190Asp Glu Leu Gly Asn Thr Val Asp Ala Thr Ser Val Thr Tyr Ser Gln 195 200 205Ile Asp Met Cys Asp Glu Pro Ala Ser Ser Tyr Gly Trp Arg Ser Pro 210 215 220Gly Tyr Ile His Asn Val Val Met Gly Gly Leu Asn Pro Gly Ser Arg225 230 235 240Tyr Phe Tyr Arg Val Gly Ser Asn Val Gly Gly Trp Ser Ser Thr Tyr 245 250 255Ser Phe Ile Ala Pro His Pro Arg Ala Asp Glu Thr Asn Ala Leu Ile 260 265 270Phe Gly Asp Met Gly Thr Ser Ile Pro Tyr Ser Thr Tyr Gln Tyr Thr 275 280 285Gln Ser Glu Ser Lys Asn Thr Val Lys Trp Leu Thr Arg Asp Leu Glu 290 295 300Gln Ile Gly Asp Lys Pro Ser Phe Val Ala His Ile Gly Asp Ile Ser305 310 315 320Tyr Ala Arg Gly Leu Ser Trp Leu Trp Asp Asn Phe Phe Thr Gln Ile 325 330 335Glu Pro Val Ala Ala Arg Ser Pro Tyr His Val Cys Met Gly Asn His 340 345 350Glu Tyr Asp Trp Pro Gly Gln Pro Phe Lys Pro Asp Trp Ser Pro Tyr 355 360 365Gln Thr Asp Gly Gly Gly Glu Cys Gly Val Pro Tyr Ser Leu Arg Phe 370 375 380Ile Met Pro Gly Asn Ser Ser Leu Pro Thr Gly Thr Thr Ser Pro Ala385 390 395 400Thr Lys Asn Leu Tyr Tyr Ser Ile Asp Val Gly Val Val His Phe Leu 405 410 415Phe Tyr Ser Thr Glu Thr Asp Phe Gln Val Gly Ser Pro Gln Tyr Thr 420 425 430Phe Ile Ala Asn Asp Leu Arg Thr Val Asp Arg Asn Lys Thr Pro Phe 435 440 445Val Val Phe Leu Gly His Arg Pro Leu Tyr Thr Thr Asp Tyr Arg Ala 450 455 460Leu Leu Asp Thr Met Thr Gln Lys Leu Val Gln Thr Phe Glu Pro Leu465 470 475 480Leu Ile Asp Thr Asn Val Thr Val Ala Phe Cys Gly His Val His Lys 485 490 495Tyr Glu Arg Met Cys Pro Leu Lys Asn Tyr Thr Cys Ile Glu Pro Ser 500 505 510Lys Ala Asn Gly Glu Leu Pro Ile His Met Val Val Gly Met Gly Gly 515 520 525Ala Asp His Gln Pro Ile Asp Asp Pro Leu Pro Ser Gln Ser Gln Pro 530 535 540Ile Phe Pro Gln Pro Ser Trp Ser Val Phe Arg Thr Phe Glu Trp Gly545 550 555 560Tyr Ile Arg Leu His Ala Thr Arg His Leu Met Thr Ile Ser Tyr Val 565 570 575Gly Asn His Asp Gly Lys Val His Asp Val Val Glu Ile Pro Val Leu 580 585 590Asp Asp Ile Lys Ser Gly Ala Tyr Val Glu Ser Arg Glu Ser Phe Phe 595 600 605Asp Thr Ala Ser Gly Val Gln Ile Pro Cys Gly Arg Ser Glu Asn Ile 610 615 620Val Ala Phe Leu Phe Val Leu Ala Leu Gly Cys Gly Cys Gly Ala Ala625 630 635 640Ala Thr Leu Phe Phe Met Arg Arg Gln Gln Arg Lys Gln Ile Trp Gln 645 650 655Pro Val Asn Arg Glu Glu Ala Ser Ser Ser Gln Leu 660 665261944DNAPopulus trichocarpa 26atgaagctcc ctatcttcct cctcctcctc ctcctctccc tcatcactca aacttccctc 60tccaaagtca ccatctccgt gactccaaca accctccaga aatccggtga cacagtaacc 120atttcctggt ccaacgttga ttcaccttcc aaactcgact ggctcgggct ctattcacct 180cctgactcac ctcacgacca cttcattggc tacaagttcc tttcttcctc tccttcatgg 240caatccgggt cgggttccat ttccttgccc atcaccaacc tccgctccaa ttactctttc 300cggatcttcc actggaccga atccgaaatc aaccccaaac gccatgacca tgatcacaac 360cccctccctg ggacggccca ttttctggcg gagtcggatg ttgtcgggtt cgagtcgggt 420catgggccag agcagatcca tttggcatat acggatgatg aggacgagat gagggtgatg 480tttgtggtgg gtgatggaga ggagaggggt gtgaagtggg gagagaggga cggggagtgg 540agtcacgtga gtggggcacg tgtggtgagg tatgaaaggg aggatatgtg tgatgctccg 600gcaaatggga gtattgggtg gagagatccg ggttggatcc atgatggggt aatgaaggat 660ttgaagaaag gtgttaggta ttattatcag gttggaagcg actctaaggg ttggagcaca 720actaggagct ttgtctctcg gaatggagac tcggatgaaa caatagcctt cctgtttggg 780gacatgggaa cttcaacacc atatgctacc tttatccgta cacaagatga aagcatatca 840accatgaagt ggatcctccg agacatagaa gctattggtg acaagcatgc ttttgtttct 900catataggag atatcagcta tgcaagaggg tactcatggt tgtgggacca tttttttacc 960caagtggaac ctgttgcttc caaagtgcca taccatgtgt gcattggtaa tcatgagtac 1020gattggccct tacagccctg gaaaccagat tgggccaatg cagtttacgg aactgatggt 1080ggtggtgaat gtggggttcc ttacagcctt aaatttaaca tgccagggaa ctcttcagac 1140tcaactggga cccgtgctcc tgcaacccga aacctttact actcttttga cacaggggct 1200gtacattttg tgtacatatc aactgagacc aattttgttg ctgggagcag ccaatataac 1260tttataaagc aagatctgga atcagttgac cggagcaaga ctccttttgt ggtagtccaa 1320gggcacagac caatgtatac tactagcaat gaaaacaggg atgccccaat gaggaacaaa 1380atgcttgagc acttggaacc tttgtttacg aaatacaatg ttacccttgc actgtggggt 1440catgtgcata gatacgaaag gttttgtcca gtgaataact tcatctgcgg aagcacttgg 1500aagggatttc cagtccatgc tgtgattggc atggcaggac aagactggca gcccatctgg 1560gagccaagat cagaccaccc aaatgatcca atttttccac agccagccag gtctatgttc 1620cgtggggggg agttcgggta caccaaattg gttgccacaa aggagaagct aacacttact 1680tatgtaggta accatgatgg aaagatgcac gatatggttg agtttttggc atctggagaa 1740gttctcagtg gtgatgacag cattagtgtg gatgctggag ccaggattgg ggtggttgat 1800tctacgttct catggtatgt caagggggca agtgttcttg tccttggggc ttttgtgggc 1860tatactcttg gctacgcatc ccattccagg aagcaaaatg gtaacaaggc cagctggact 1920cctgtgaaaa gtgaggatat atga 194427647PRTPopulus trichocarpa 27Met Lys Leu Pro Ile Phe Leu Leu Leu Leu Leu Leu Ser Leu Ile Thr1 5 10 15Gln Thr Ser Leu Ser Lys Val Thr Ile Ser Val Thr Pro Thr Thr Leu 20 25 30Gln Lys Ser Gly Asp Thr Val Thr Ile Ser Trp Ser Asn Val Asp Ser 35 40 45Pro Ser Lys Leu Asp Trp Leu Gly Leu Tyr Ser Pro Pro Asp Ser Pro 50 55 60His Asp His Phe Ile Gly Tyr Lys Phe Leu Ser Ser Ser Pro Ser Trp65 70 75 80Gln Ser Gly Ser Gly Ser Ile Ser Leu Pro Ile Thr Asn Leu Arg Ser 85 90 95Asn Tyr Ser Phe Arg Ile Phe His Trp Thr Glu Ser Glu Ile Asn Pro 100 105 110Lys Arg His Asp His Asp His Asn Pro Leu Pro Gly Thr Ala His Phe 115 120 125Leu Ala Glu Ser Asp Val Val Gly Phe Glu Ser Gly His Gly Pro Glu 130 135 140Gln Ile His Leu Ala Tyr Thr Asp Asp Glu Asp Glu Met Arg Val Met145 150 155 160Phe Val Val Gly Asp Gly Glu Glu Arg Gly Val Lys Trp Gly Glu Arg 165 170 175Asp Gly Glu Trp Ser His Val Ser Gly Ala Arg Val Val Arg Tyr Glu 180 185 190Arg Glu Asp Met Cys Asp Ala Pro Ala Asn Gly Ser Ile Gly Trp Arg 195 200 205Asp Pro Gly Trp Ile His Asp Gly Val Met Lys Asp Leu Lys Lys Gly 210 215 220Val Arg Tyr Tyr Tyr Gln Val Gly Ser Asp Ser Lys Gly Trp Ser Thr225 230 235 240Thr Arg Ser Phe Val Ser Arg Asn Gly Asp Ser Asp Glu Thr Ile Ala 245 250 255Phe Leu Phe Gly Asp Met Gly Thr Ser Thr Pro Tyr Ala Thr Phe Ile 260 265 270Arg Thr Gln Asp Glu Ser Ile Ser Thr Met Lys Trp Ile Leu Arg Asp 275 280 285Ile Glu Ala Ile Gly Asp Lys His Ala Phe Val Ser His Ile Gly Asp 290 295 300Ile Ser Tyr Ala Arg Gly Tyr Ser Trp Leu Trp Asp His Phe Phe Thr305 310 315 320Gln Val Glu Pro Val Ala Ser Lys Val Pro Tyr His Val Cys Ile Gly 325 330 335Asn His Glu Tyr Asp Trp Pro Leu Gln Pro Trp Lys Pro Asp Trp Ala 340 345 350Asn Ala Val Tyr Gly Thr Asp Gly Gly Gly Glu Cys Gly Val Pro Tyr 355 360 365Ser Leu Lys Phe Asn Met Pro Gly Asn Ser Ser Asp Ser Thr Gly Thr 370 375 380Arg Ala Pro Ala Thr Arg Asn Leu Tyr Tyr Ser Phe Asp Thr Gly Ala385 390 395 400Val His Phe Val Tyr Ile Ser Thr Glu Thr Asn Phe Val Ala Gly Ser 405 410 415Ser Gln Tyr Asn Phe Ile Lys Gln Asp Leu Glu Ser Val Asp Arg Ser 420 425 430Lys Thr Pro Phe Val Val Val Gln Gly His Arg Pro Met Tyr Thr Thr 435 440 445Ser Asn Glu Asn Arg Asp Ala Pro Met Arg Asn Lys Met Leu Glu His 450 455 460Leu Glu Pro Leu Phe Thr Lys Tyr Asn Val Thr Leu Ala Leu Trp Gly465 470 475 480His Val His Arg Tyr Glu Arg Phe Cys Pro Val Asn Asn Phe Ile Cys 485 490 495Gly Ser Thr Trp Lys Gly Phe Pro Val His Ala Val Ile Gly Met Ala 500 505 510Gly Gln Asp Trp Gln Pro Ile Trp Glu Pro Arg Ser Asp His Pro Asn 515 520 525Asp Pro Ile Phe Pro Gln Pro Ala Arg Ser Met Phe Arg Gly Gly Glu 530 535 540Phe Gly Tyr Thr Lys Leu Val Ala Thr Lys Glu Lys Leu Thr Leu Thr545 550 555 560Tyr Val Gly Asn His Asp Gly Lys Met His Asp Met Val Glu Phe Leu 565 570 575Ala Ser Gly Glu Val Leu Ser Gly Asp Asp Ser Ile Ser Val Asp Ala 580 585 590Gly Ala Arg Ile Gly Val Val Asp Ser Thr Phe Ser Trp Tyr Val Lys 595 600 605Gly Ala Ser Val Leu Val Leu Gly Ala Phe Val Gly Tyr Thr Leu Gly 610 615 620Tyr Ala Ser His Ser Arg Lys Gln Asn Gly Asn Lys Ala Ser Trp Thr625 630 635 640Pro Val Lys Ser Glu Asp Ile 645281962DNASaccharum officinarum 28atgtcccccg aaaaccccca cctccgcttc ctcctcttcc tcgccgtcgc ggccgtcgcc 60gccggcgggg ctgcggcggg caccaccctc accgcgaccc tctccagcga ccagatcaag 120atccgctgga caggcctccc ggccccggac ggcctcgact acgtcggcat ctactcgccg 180ccgtcctccc gcgaccgcga cttcctcggc tacctcttcc tcaacggctc cgcctcctgg 240cgcggcggct caggggagct ctccctcccg cgcctcccga ccctgcgcgc gccctaccag 300ttccgcctct tccgctggcc cgccaacgag tactcctacc accacatcga ccatgaccgg 360aacccgctcc cccacggcaa gcaccgcgtc gccgtctccg ccgacgtctc cgtcggcgac 420cccgcgcgcc ccgagcaggt gcacctcgcg ttcgcggatg ggatcgacga gatgcgggtc 480ctgttcgtgt gcggcgaccg cgggaagagg gtcgtcaggt acgggctgca aaaggaagac 540gagaaggagt ggaaggaggt gggcacggat gtgagcacgt acaagcaaaa gcacatgtgc 600gattggccgc cgaacagcag cgtcgcctgg agggatcccg gattcgtctt cgacgggctc 660atgaagggat tggagcctgg aaggaggtac ttttacaagg ttggtagtga cactggagga 720tggagtgaga tatacagctt tatttcacgt gacagtgaag ccaatgagac caacacattt 780ctgtttggtg acatgggaac ttatgtgcct tatcacacct acattcgcac acaagatgag 840agcttgtcca ctgtaaagtg gatccttcgt gatattgaag cccttgggga taaacccgcc 900tttatttcac acattgggga catcagctat gctagaggtt attcttgggt atgggatcat 960ttcttcagtc agattgagcc aattgctgcc aataccccat accatgtctg tataggaaat 1020catgagtatg attggccatc tcaaccttgg aaaccatggt gggctacata tggaaaggat 1080ggtggaggcg aatgtggaat accgtatagc gtcaagttca gaatgcctgg caattctatt 1140ctaccaactg gtaatggtgg cccagacacc aggaatcttt attactcctt tgactcaggt 1200gtggtgcatt tcgtctacat gtcaaccgaa acaaattttg ttcagggcag tgatcagtac 1260aacttcttga aagcggacct tgagaaggtg aaccgaagta gaacaccatt tgttgttttt 1320cagggccacc gccccatgta cacctcaagt gatgaaacca gggacgcggc cttgagacag 1380cagatgctcc agcatttgga accgctgctg gtgacataca gtgtgaccct tgcactatgg 1440ggacatgtcc acaggtacga gaggttctgc ccgatgaaga acttccaatg tgtcaacact 1500tcatcaagct tccaatactc tggtgctcct gtgcatcttg tgattgggat gggcggggca 1560gactgggcaa ccatatggca accgaggcct gatcacccag acgtccccat ctttccacag 1620cctgagaggt ccatgtaccg tggcggtgag tttggataca caaggcttgc agcaacaagg 1680gagaagctaa cattaaccta tgtggggaac catgatgggc aagtccatga tataatggag 1740atattttccg gcctggtatc tagtaacagt agtgttgctg aggtggtgga tgatactaaa 1800catggcacag gagtcagcac cgtgcgaaaa atatctccgt tgtacttgga aatcggaggc 1860agtgtattgt ttgcactgct tctgggattt tcctttggat ttcttatcag gagaaagaaa 1920gaagctgcac agtggactcc agtaaagaac gaggaatcgt aa 196229653PRTSaccharum officinarum 29Met Ser Pro Glu Asn Pro His Leu Arg Phe Leu Leu Phe Leu Ala Val1 5 10 15Ala Ala Val Ala Ala Gly Gly Ala Ala Ala Gly Thr Thr Leu Thr Ala 20 25 30Thr Leu Ser Ser Asp Gln Ile Lys Ile Arg Trp Thr Gly Leu Pro Ala 35 40 45Pro Asp Gly Leu Asp Tyr Val Gly Ile Tyr Ser Pro Pro Ser Ser Arg 50 55 60Asp Arg Asp Phe Leu Gly Tyr Leu Phe Leu Asn Gly Ser Ala Ser Trp65 70 75 80Arg Gly Gly Ser Gly Glu Leu Ser Leu Pro Arg Leu Pro Thr Leu Arg 85 90 95Ala Pro Tyr Gln Phe Arg Leu Phe Arg Trp Pro Ala Asn Glu Tyr Ser 100 105 110Tyr His His Ile Asp His Asp Arg Asn Pro Leu Pro His Gly Lys His 115 120 125Arg Val Ala Val Ser Ala Asp Val Ser Val Gly Asp Pro Ala Arg Pro 130 135 140Glu Gln Val His Leu Ala Phe Ala Asp Gly Ile Asp Glu Met Arg Val145 150 155 160Leu Phe Val Cys Gly Asp Arg Gly Lys Arg Val Val Arg Tyr Gly Leu 165 170 175Gln Lys Glu Asp Glu Lys Glu Trp Lys Glu Val Gly Thr Asp Val Ser 180 185 190Thr Tyr Lys Gln Lys His Met Cys Asp Trp Pro Pro Asn Ser Ser Val 195 200 205Ala Trp Arg Asp Pro Gly Phe Val Phe Asp Gly Leu Met Lys Gly Leu 210 215 220Glu Pro Gly Arg Arg Tyr Phe Tyr Lys Val Gly Ser Asp Thr Gly Gly225 230 235 240Trp Ser Glu Ile Tyr Ser Phe Ile Ser Arg Asp Ser Glu Ala Asn Glu 245 250 255Thr Asn Thr Phe Leu Phe Gly Asp Met Gly Thr Tyr Val Pro Tyr His 260 265 270Thr Tyr Ile Arg Thr Gln Asp Glu Ser Leu Ser Thr Val Lys Trp Ile 275 280 285Leu Arg Asp Ile Glu Ala Leu Gly Asp Lys Pro Ala Phe Ile Ser His 290 295 300Ile Gly Asp Ile Ser Tyr Ala Arg Gly Tyr Ser Trp Val Trp Asp His305 310 315 320Phe Phe Ser Gln Ile Glu Pro Ile Ala Ala Asn Thr Pro Tyr His Val 325 330 335Cys Ile Gly Asn His Glu Tyr Asp Trp Pro Ser Gln Pro Trp Lys Pro 340 345 350Trp Trp Ala Thr Tyr Gly Lys Asp Gly Gly Gly Glu Cys Gly Ile Pro 355 360 365Tyr Ser Val Lys Phe Arg Met Pro Gly Asn Ser Ile Leu Pro Thr Gly 370 375 380Asn Gly Gly Pro Asp Thr Arg Asn Leu Tyr Tyr Ser Phe Asp Ser Gly385 390 395 400Val Val His Phe Val Tyr Met Ser Thr Glu Thr Asn Phe Val Gln Gly 405 410 415Ser Asp Gln Tyr Asn Phe Leu Lys Ala Asp Leu Glu Lys Val Asn Arg 420 425 430Ser Arg Thr Pro Phe Val Val Phe Gln Gly His Arg Pro Met Tyr Thr 435 440 445Ser Ser Asp Glu Thr Arg Asp Ala Ala Leu Arg Gln Gln Met Leu Gln 450 455 460His Leu Glu Pro Leu Leu Val Thr Tyr Ser Val Thr Leu Ala Leu Trp465 470 475 480Gly His Val His Arg Tyr Glu Arg Phe Cys Pro Met Lys Asn Phe Gln 485 490 495Cys Val Asn Thr Ser Ser Ser Phe Gln Tyr Ser Gly

Ala Pro Val His 500 505 510Leu Val Ile Gly Met Gly Gly Ala Asp Trp Ala Thr Ile Trp Gln Pro 515 520 525Arg Pro Asp His Pro Asp Val Pro Ile Phe Pro Gln Pro Glu Arg Ser 530 535 540Met Tyr Arg Gly Gly Glu Phe Gly Tyr Thr Arg Leu Ala Ala Thr Arg545 550 555 560Glu Lys Leu Thr Leu Thr Tyr Val Gly Asn His Asp Gly Gln Val His 565 570 575Asp Ile Met Glu Ile Phe Ser Gly Leu Val Ser Ser Asn Ser Ser Val 580 585 590Ala Glu Val Val Asp Asp Thr Lys His Gly Thr Gly Val Ser Thr Val 595 600 605Arg Lys Ile Ser Pro Leu Tyr Leu Glu Ile Gly Gly Ser Val Leu Phe 610 615 620Ala Leu Leu Leu Gly Phe Ser Phe Gly Phe Leu Ile Arg Arg Lys Lys625 630 635 640Glu Ala Ala Gln Trp Thr Pro Val Lys Asn Glu Glu Ser 645 650301950DNASolanum tuberosum 30atgatgatcc cttttgtcac cttcacctta ctcatcttct tcaacttgat ttcatcatct 60tcttcttcac aaatctccat ttctgtaacc ccaaaaacct tatcaaaatc tggtgatttt 120gttacaatca aatggactgg tatcccctca ccttctaaac tcgatttctt aggaatttac 180tctccaccca gttcactcca cgacaatttc attggctata ttttcctatc ttcaacaccc 240gaatgggaat ctgggtcggg ttcaatttcc atccctttag tcaatcttcg atctgggtat 300cagtttcgga tattcagatg gacggaatcg gagattgtac cggatctagt ggatcatgac 360cacaatccat tgccgcagac gaagcatatt cttgcggtgt cggaggaggt tgggtttgtt 420tcgggtcggg gacccgaaca ggttcatttg gctttaacgg gttttgaaga tgagatgcgg 480gttatgtttg ttacgcctga cgggaaagag agttatgtga gatatgggtt gacccggggt 540agattgggtc gggttgtgaa aactcgggtt gtgaggtatg agaaggaaga tttatgtgat 600gcaccagcta atagtagtat tggatggaga gatcctgggt atatacatga tggtgttatg 660cttaatttga aaaagggaaa gaagtattat tatcaggttg gcagtgattc agggggctgg 720agcaccattt acagctttgt gtcacagaat agagactcag gtgaaacatt tgctttcttg 780tttggagaca tggggactgc taccccatac ttgacatttc ttcgtacaca ggacgaaagt 840aaatcaacga ttaagtggat tagccgtgat attgaagctc ttggtaataa gcctgccctt 900atctcacata ttggagatat cagctacgct agaggatact cttggttgtg ggacaacttt 960tttactcagg tagaacctgt tgcatccaga gttccatacc atgtatgcat cggaaaccat 1020gaatatgatt ggccacttca accttggaag cctgattggt caagctacgg gaaagatggg 1080ggaggtgaat gtggtgtacc ctacaggtca tacttccata tgccaagaaa ctcttcagtg 1140ccgactggaa tgcatgctcc tgcaactcgg aatctttatt actcatttga ttctgggccc 1200gttcactttg tctatatgtc aactgaaacc aatttccttc caggtagtaa ccagtatgac 1260tttttaaagc atgacttgga atcagttgat cgagtaaaaa ctccttttgt tgtctttcaa 1320gggcacagac caatgtacag ttcaagtagc ggagcaaaag atatatcttt gaggaagaga 1380atgatggagt atttggaacc tcttcttgtg aagaacaatg tgaatcttgt attgtggggg 1440catgttcata ggtatgagag gttttgccct ttgaataact tcacctgtgg aagcttggcc 1500ttgaatggga aggagcaaaa ggctttccca gttcaaattg tgattgggat ggcaggacag 1560gactggcagc ctatctgggc accaagagaa gaccacccta cggatcctat tttcccacag 1620cctctgcaat ctctgtaccg tgggagtgaa tttggctacg tgaggctgca tgccacaaag 1680aaaaagctta cactttctta tgtaggaaac catgacggag aggtgcatga taaggtggag 1740ttcctagctt caggactact tctcagtgct ggtatccgtg atggtcctgc agatgcagta 1800cacatggagt ctaagttctc atggtatgta aaggttggaa gtgtgctaat gcttggagct 1860tttatgggtt acatagttgg attcttatct catgctcgga aaaattctgc tgataaagga 1920tggagaccta taaaaactga ggaaatatga 195031649PRTSolanum tuberosum 31Met Met Ile Pro Phe Val Thr Phe Thr Leu Leu Ile Phe Phe Asn Leu1 5 10 15Ile Ser Ser Ser Ser Ser Ser Gln Ile Ser Ile Ser Val Thr Pro Lys 20 25 30Thr Leu Ser Lys Ser Gly Asp Phe Val Thr Ile Lys Trp Thr Gly Ile 35 40 45Pro Ser Pro Ser Lys Leu Asp Phe Leu Gly Ile Tyr Ser Pro Pro Ser 50 55 60Ser Leu His Asp Asn Phe Ile Gly Tyr Ile Phe Leu Ser Ser Thr Pro65 70 75 80Glu Trp Glu Ser Gly Ser Gly Ser Ile Ser Ile Pro Leu Val Asn Leu 85 90 95Arg Ser Gly Tyr Gln Phe Arg Ile Phe Arg Trp Thr Glu Ser Glu Ile 100 105 110Val Pro Asp Leu Val Asp His Asp His Asn Pro Leu Pro Gln Thr Lys 115 120 125His Ile Leu Ala Val Ser Glu Glu Val Gly Phe Val Ser Gly Arg Gly 130 135 140Pro Glu Gln Val His Leu Ala Leu Thr Gly Phe Glu Asp Glu Met Arg145 150 155 160Val Met Phe Val Thr Pro Asp Gly Lys Glu Ser Tyr Val Arg Tyr Gly 165 170 175Leu Thr Arg Gly Arg Leu Gly Arg Val Val Lys Thr Arg Val Val Arg 180 185 190Tyr Glu Lys Glu Asp Leu Cys Asp Ala Pro Ala Asn Ser Ser Ile Gly 195 200 205Trp Arg Asp Pro Gly Tyr Ile His Asp Gly Val Met Leu Asn Leu Lys 210 215 220Lys Gly Lys Lys Tyr Tyr Tyr Gln Val Gly Ser Asp Ser Gly Gly Trp225 230 235 240Ser Thr Ile Tyr Ser Phe Val Ser Gln Asn Arg Asp Ser Gly Glu Thr 245 250 255Phe Ala Phe Leu Phe Gly Asp Met Gly Thr Ala Thr Pro Tyr Leu Thr 260 265 270Phe Leu Arg Thr Gln Asp Glu Ser Lys Ser Thr Ile Lys Trp Ile Ser 275 280 285Arg Asp Ile Glu Ala Leu Gly Asn Lys Pro Ala Leu Ile Ser His Ile 290 295 300Gly Asp Ile Ser Tyr Ala Arg Gly Tyr Ser Trp Leu Trp Asp Asn Phe305 310 315 320Phe Thr Gln Val Glu Pro Val Ala Ser Arg Val Pro Tyr His Val Cys 325 330 335Ile Gly Asn His Glu Tyr Asp Trp Pro Leu Gln Pro Trp Lys Pro Asp 340 345 350Trp Ser Ser Tyr Gly Lys Asp Gly Gly Gly Glu Cys Gly Val Pro Tyr 355 360 365Arg Ser Tyr Phe His Met Pro Arg Asn Ser Ser Val Pro Thr Gly Met 370 375 380His Ala Pro Ala Thr Arg Asn Leu Tyr Tyr Ser Phe Asp Ser Gly Pro385 390 395 400Val His Phe Val Tyr Met Ser Thr Glu Thr Asn Phe Leu Pro Gly Ser 405 410 415Asn Gln Tyr Asp Phe Leu Lys His Asp Leu Glu Ser Val Asp Arg Val 420 425 430Lys Thr Pro Phe Val Val Phe Gln Gly His Arg Pro Met Tyr Ser Ser 435 440 445Ser Ser Gly Ala Lys Asp Ile Ser Leu Arg Lys Arg Met Met Glu Tyr 450 455 460Leu Glu Pro Leu Leu Val Lys Asn Asn Val Asn Leu Val Leu Trp Gly465 470 475 480His Val His Arg Tyr Glu Arg Phe Cys Pro Leu Asn Asn Phe Thr Cys 485 490 495Gly Ser Leu Ala Leu Asn Gly Lys Glu Gln Lys Ala Phe Pro Val Gln 500 505 510Ile Val Ile Gly Met Ala Gly Gln Asp Trp Gln Pro Ile Trp Ala Pro 515 520 525Arg Glu Asp His Pro Thr Asp Pro Ile Phe Pro Gln Pro Leu Gln Ser 530 535 540Leu Tyr Arg Gly Ser Glu Phe Gly Tyr Val Arg Leu His Ala Thr Lys545 550 555 560Lys Lys Leu Thr Leu Ser Tyr Val Gly Asn His Asp Gly Glu Val His 565 570 575Asp Lys Val Glu Phe Leu Ala Ser Gly Leu Leu Leu Ser Ala Gly Ile 580 585 590Arg Asp Gly Pro Ala Asp Ala Val His Met Glu Ser Lys Phe Ser Trp 595 600 605Tyr Val Lys Val Gly Ser Val Leu Met Leu Gly Ala Phe Met Gly Tyr 610 615 620Ile Val Gly Phe Leu Ser His Ala Arg Lys Asn Ser Ala Asp Lys Gly625 630 635 640Trp Arg Pro Ile Lys Thr Glu Glu Ile 645321959DNAVitis vinifera 32atgtttccaa ttttatcttt ctgcctcttc ttcgtcctcg ctcctcctct cctcgcatct 60tcttctccag tctccataac cctaaccgcc aaaatcttag ccaaatcggg cgacccgatc 120cgaatcaaat ggtcggggat cgactccccg tccgacctcg actggctcgg catctactcg 180ccgccgtcct ccgcccacga caacttcatt ggctatgttt ttctgtcgtc atgtcccaca 240tgggaatctg gatcgggttc gatcagctta cccctggtta atctccgtgc taactactct 300tttcggatat tccggtggtc ccggtccgag gtcgacccga cccggatgga ccacgaccac 360aatcccttgc cggggacaac gcatctggtg gcggagtccg gggaggtggg gttcgggggc 420ggcggggggc cggagcagat ccatttggcg tacacggata gggaggatga gatgcgggtg 480atgttcgtga cgggggacgc gggcgtgagg actgtgaggt atggcttgag cagggacgcg 540atgcacaggg tggtgacggc ggcggtgggg agatatgaga gggaggacat gtgtgactcg 600ccagcgaatg agagtgttgg gtggagagat ccgggtttta ttcaagatgc ggtgatgagg 660aatttgaaga aagggaagag atattattat aaggttggaa gtgattcagg aggttggagc 720gcaattcaca actttatgtc acgggatatg gactctgaaa aaacaatagc ttttctattt 780ggtgacatgg ggacagcaac accatactca acctttcttc gtacacaaga ggaaagcaag 840tcaaccgtta aatggatcct ccgtgacatt gaggctcttg atgacaaccc tgccttcatc 900tcgcatattg gagatattag ctatgctaga ggttattcat ggttgtggga caattttttc 960actcaggttg aacctatcgc ctccagactc ccataccatg tgtgtattgg taatcatgaa 1020tatgattggc cattgcagcc ttggaaacct gattggtcct ccacagttta tggaacagat 1080ggtggcggtg aatgtggagt gccctacagc cttaagttca aaatgcctgg aaactcttca 1140gaactaactg gaacccgtgc cccagccact cgaaacctct tctactcatt tgatacgaag 1200gcagtgcatt ttgtgtacat atcaactgag accaatttcc ttccagggag cagccaatat 1260gactttataa agcaggattt ggagtcagtt gatcggaaaa aaaccccttt tgtggttgtc 1320caagggcaca gaccaatgta cacaacaagc aatgaactta gagatgcccc agtgagggag 1380aggatgctca agtatttgga acctcttttt gtgaagaaca atgtgaccct tgcactctgg 1440ggtcatgtcc acagatatga gaggttttgc ccaataaata acttcacttg tggaaacatg 1500ggattgaatg gggaatacct ggggggattg cctgttcata tcgtgattgg gatggcaggg 1560caagactggc agcccacatg ggaaccaaga ccagaccacc cgaaggaccc tgtctaccca 1620caacctaaat ggtcattgta ccgtgggggt gagtttgggt acactaggtt ggttgccacc 1680aaagagaagc taactctttc ttatgtagga aaccatgatg gtgaggtgca tgatactgtt 1740gagattctgg catctggaca agttctcagt ggtgttggag aggatgatgc tcaacccaga 1800gttgaggtgg cagagtacac attttcatgg tatgttaagg gggcaagtat cttggtgctg 1860ggggctttta tgggctatgt tattgggttc gtatcacatg ccaggagaga agctgccttg 1920agaaagaact ggactccagt gaagatcgaa gatagctga 195933652PRTVitis vinifera 33Met Phe Pro Ile Leu Ser Phe Cys Leu Phe Phe Val Leu Ala Pro Pro1 5 10 15Leu Leu Ala Ser Ser Ser Pro Val Ser Ile Thr Leu Thr Ala Lys Ile 20 25 30Leu Ala Lys Ser Gly Asp Pro Ile Arg Ile Lys Trp Ser Gly Ile Asp 35 40 45Ser Pro Ser Asp Leu Asp Trp Leu Gly Ile Tyr Ser Pro Pro Ser Ser 50 55 60Ala His Asp Asn Phe Ile Gly Tyr Val Phe Leu Ser Ser Cys Pro Thr65 70 75 80Trp Glu Ser Gly Ser Gly Ser Ile Ser Leu Pro Leu Val Asn Leu Arg 85 90 95Ala Asn Tyr Ser Phe Arg Ile Phe Arg Trp Ser Arg Ser Glu Val Asp 100 105 110Pro Thr Arg Met Asp His Asp His Asn Pro Leu Pro Gly Thr Thr His 115 120 125Leu Val Ala Glu Ser Gly Glu Val Gly Phe Gly Gly Gly Gly Gly Pro 130 135 140Glu Gln Ile His Leu Ala Tyr Thr Asp Arg Glu Asp Glu Met Arg Val145 150 155 160Met Phe Val Thr Gly Asp Ala Gly Val Arg Thr Val Arg Tyr Gly Leu 165 170 175Ser Arg Asp Ala Met His Arg Val Val Thr Ala Ala Val Gly Arg Tyr 180 185 190Glu Arg Glu Asp Met Cys Asp Ser Pro Ala Asn Glu Ser Val Gly Trp 195 200 205Arg Asp Pro Gly Phe Ile Gln Asp Ala Val Met Arg Asn Leu Lys Lys 210 215 220Gly Lys Arg Tyr Tyr Tyr Lys Val Gly Ser Asp Ser Gly Gly Trp Ser225 230 235 240Ala Ile His Asn Phe Met Ser Arg Asp Met Asp Ser Glu Lys Thr Ile 245 250 255Ala Phe Leu Phe Gly Asp Met Gly Thr Ala Thr Pro Tyr Ser Thr Phe 260 265 270Leu Arg Thr Gln Glu Glu Ser Lys Ser Thr Val Lys Trp Ile Leu Arg 275 280 285Asp Ile Glu Ala Leu Asp Asp Asn Pro Ala Phe Ile Ser His Ile Gly 290 295 300Asp Ile Ser Tyr Ala Arg Gly Tyr Ser Trp Leu Trp Asp Asn Phe Phe305 310 315 320Thr Gln Val Glu Pro Ile Ala Ser Arg Leu Pro Tyr His Val Cys Ile 325 330 335Gly Asn His Glu Tyr Asp Trp Pro Leu Gln Pro Trp Lys Pro Asp Trp 340 345 350Ser Ser Thr Val Tyr Gly Thr Asp Gly Gly Gly Glu Cys Gly Val Pro 355 360 365Tyr Ser Leu Lys Phe Lys Met Pro Gly Asn Ser Ser Glu Leu Thr Gly 370 375 380Thr Arg Ala Pro Ala Thr Arg Asn Leu Phe Tyr Ser Phe Asp Thr Lys385 390 395 400Ala Val His Phe Val Tyr Ile Ser Thr Glu Thr Asn Phe Leu Pro Gly 405 410 415Ser Ser Gln Tyr Asp Phe Ile Lys Gln Asp Leu Glu Ser Val Asp Arg 420 425 430Lys Lys Thr Pro Phe Val Val Val Gln Gly His Arg Pro Met Tyr Thr 435 440 445Thr Ser Asn Glu Leu Arg Asp Ala Pro Val Arg Glu Arg Met Leu Lys 450 455 460Tyr Leu Glu Pro Leu Phe Val Lys Asn Asn Val Thr Leu Ala Leu Trp465 470 475 480Gly His Val His Arg Tyr Glu Arg Phe Cys Pro Ile Asn Asn Phe Thr 485 490 495Cys Gly Asn Met Gly Leu Asn Gly Glu Tyr Leu Gly Gly Leu Pro Val 500 505 510His Ile Val Ile Gly Met Ala Gly Gln Asp Trp Gln Pro Thr Trp Glu 515 520 525Pro Arg Pro Asp His Pro Lys Asp Pro Val Tyr Pro Gln Pro Lys Trp 530 535 540Ser Leu Tyr Arg Gly Gly Glu Phe Gly Tyr Thr Arg Leu Val Ala Thr545 550 555 560Lys Glu Lys Leu Thr Leu Ser Tyr Val Gly Asn His Asp Gly Glu Val 565 570 575His Asp Thr Val Glu Ile Leu Ala Ser Gly Gln Val Leu Ser Gly Val 580 585 590Gly Glu Asp Asp Ala Gln Pro Arg Val Glu Val Ala Glu Tyr Thr Phe 595 600 605Ser Trp Tyr Val Lys Gly Ala Ser Ile Leu Val Leu Gly Ala Phe Met 610 615 620Gly Tyr Val Ile Gly Phe Val Ser His Ala Arg Arg Glu Ala Ala Leu625 630 635 640Arg Lys Asn Trp Thr Pro Val Lys Ile Glu Asp Ser 645 650341962DNAOryza sativa 34atgcttctct tcctcctctt cctcctcgcc gccggcgagg ccgcggcggc ggcggcggcc 60accacgctca ccgcgacgcc ggcgaagctc acccagtccg accgcgagat cacgatccgg 120tggtcgggcc tcccggaccc ggacggcctc gactacgtcg gcatctactc gccgccgacc 180tcctccgacc gcgacttcct cggctacctc ttcctcaacg gctcggccac ctggcgcacg 240ggcaccggcg agctcaccct cccgcgcctc cccaacctgc gcgcgcccta ccagttccgc 300ctcttccgct ggcccgcgag ggagtactcc taccaccaca tcgaccacga cgggaacccg 360ctcccccacg gccgccaccg cgtcgccgcc tccggtgagg tcgccttcga ctccccctcc 420cgccccgacc aggtgcacct ctcgttcgcc gacggggtcg acgagatgcg ggtcatgttc 480gtctgcggcg acggcgggag gagggtggtg aggtacgggc cggcgaagga ggagggggag 540ggctggaagg aggtggccgc ggaggtgagg acgtacgagc agaagcacat gtgcgactcg 600ccggcgaact cctccgtcgg gtggagggat ccagggttcg tcttcgatgg actcatgaag 660ggattggagc ccgggaggag gtacttctac aaggttggta gcaactcttc aggatggagc 720gatacgtaca gcttcatttc acgtgacaac gaagccaatg aaactattgc atttctcttt 780ggtgacatgg gcacttatat accatataac acctatgtcc gcacgcaaga tgaaagcttg 840tcgactgtaa agtggatact tcgtgatatt caagcccttg gagataagcc tgcatttatt 900tcacacattg gggacatcag ctatgctaga ggttatgctt gggtatggga tcacttcttc 960aaccagattg agcctattgc tgccaatacc ccataccatg tctgcatagg aaatcatgaa 1020tatgattggc cattgcaacc ttggaaacct tggtgggcaa ctggtatata tggaacagat 1080ggtggaggtg aatgtggcat accttacagc gtaaagttca gaatgcctgg caattctttt 1140gtgcctactg gcaatggagc tcccgacacc cgaaatcttt actactcctt cgattcaggg 1200gttgtgcatt ttgtttacat gtcaactgag actaattttg ttcagggcag tgaccaatac 1260aacttcataa aagctgacct agagaaggtc aaccgaagta gaactccttt cattgtgttt 1320cagggccacc ggccaatgta tacatcaagc aatgaagcta gggattttgc tcatagacag 1380cagatgctcc agaacctgga accactcttg gtaacataca aagtgaccct tgcactctgg 1440ggacatgtcc acaggtacga gaggttctgc cccatgaaaa acttccaatg tgtcaacatg 1500tcatcaagct tcgtataccc tggtgcccct gttcatcttg tgatcgggat gggtggtcaa 1560gattatcaac cattctggca gccaaggaag gatcaccctg atgtacctgt ctatccgcag 1620cctgagaggt ctatgtaccg tggtggggag tttggataca caaaacttgt agctacaaag 1680gagaagttga cactaacgta catcggcaac catgatgggc aagtccatga tatggtggag 1740atattctctg ggcaagtatc taataacaat ggtgttcctg aggtgatcga tgatacaaag 1800ctcagcacag gggtcagcac caaactgaaa atccctctgt tctccttgga aattgtaggc 1860agcgtgatgt ttgcactggt tctgggtttc tctcttggat ttctgatcag aaggaagaaa 1920gaagctgcac agtggacccc agtgaagaac gaggagacct aa 196235653PRTOryza sativa 35Met Leu Leu Phe Leu Leu Phe Leu Leu Ala Ala Gly Glu Ala Ala Ala1 5 10 15Ala Ala Ala Ala Thr Thr Leu Thr Ala Thr Pro Ala Lys Leu Thr Gln 20 25

30Ser Asp Arg Glu Ile Thr Ile Arg Trp Ser Gly Leu Pro Asp Pro Asp 35 40 45Gly Leu Asp Tyr Val Gly Ile Tyr Ser Pro Pro Thr Ser Ser Asp Arg 50 55 60Asp Phe Leu Gly Tyr Leu Phe Leu Asn Gly Ser Ala Thr Trp Arg Thr65 70 75 80Gly Thr Gly Glu Leu Thr Leu Pro Arg Leu Pro Asn Leu Arg Ala Pro 85 90 95Tyr Gln Phe Arg Leu Phe Arg Trp Pro Ala Arg Glu Tyr Ser Tyr His 100 105 110His Ile Asp His Asp Gly Asn Pro Leu Pro His Gly Arg His Arg Val 115 120 125Ala Ala Ser Gly Glu Val Ala Phe Asp Ser Pro Ser Arg Pro Asp Gln 130 135 140Val His Leu Ser Phe Ala Asp Gly Val Asp Glu Met Arg Val Met Phe145 150 155 160Val Cys Gly Asp Gly Gly Arg Arg Val Val Arg Tyr Gly Pro Ala Lys 165 170 175Glu Glu Gly Glu Gly Trp Lys Glu Val Ala Ala Glu Val Arg Thr Tyr 180 185 190Glu Gln Lys His Met Cys Asp Ser Pro Ala Asn Ser Ser Val Gly Trp 195 200 205Arg Asp Pro Gly Phe Val Phe Asp Gly Leu Met Lys Gly Leu Glu Pro 210 215 220Gly Arg Arg Tyr Phe Tyr Lys Val Gly Ser Asn Ser Ser Gly Trp Ser225 230 235 240Asp Thr Tyr Ser Phe Ile Ser Arg Asp Asn Glu Ala Asn Glu Thr Ile 245 250 255Ala Phe Leu Phe Gly Asp Met Gly Thr Tyr Ile Pro Tyr Asn Thr Tyr 260 265 270Val Arg Thr Gln Asp Glu Ser Leu Ser Thr Val Lys Trp Ile Leu Arg 275 280 285Asp Ile Gln Ala Leu Gly Asp Lys Pro Ala Phe Ile Ser His Ile Gly 290 295 300Asp Ile Ser Tyr Ala Arg Gly Tyr Ala Trp Val Trp Asp His Phe Phe305 310 315 320Asn Gln Ile Glu Pro Ile Ala Ala Asn Thr Pro Tyr His Val Cys Ile 325 330 335Gly Asn His Glu Tyr Asp Trp Pro Leu Gln Pro Trp Lys Pro Trp Trp 340 345 350Ala Thr Gly Ile Tyr Gly Thr Asp Gly Gly Gly Glu Cys Gly Ile Pro 355 360 365Tyr Ser Val Lys Phe Arg Met Pro Gly Asn Ser Phe Val Pro Thr Gly 370 375 380Asn Gly Ala Pro Asp Thr Arg Asn Leu Tyr Tyr Ser Phe Asp Ser Gly385 390 395 400Val Val His Phe Val Tyr Met Ser Thr Glu Thr Asn Phe Val Gln Gly 405 410 415Ser Asp Gln Tyr Asn Phe Ile Lys Ala Asp Leu Glu Lys Val Asn Arg 420 425 430Ser Arg Thr Pro Phe Ile Val Phe Gln Gly His Arg Pro Met Tyr Thr 435 440 445Ser Ser Asn Glu Ala Arg Asp Phe Ala His Arg Gln Gln Met Leu Gln 450 455 460Asn Leu Glu Pro Leu Leu Val Thr Tyr Lys Val Thr Leu Ala Leu Trp465 470 475 480Gly His Val His Arg Tyr Glu Arg Phe Cys Pro Met Lys Asn Phe Gln 485 490 495Cys Val Asn Met Ser Ser Ser Phe Val Tyr Pro Gly Ala Pro Val His 500 505 510Leu Val Ile Gly Met Gly Gly Gln Asp Tyr Gln Pro Phe Trp Gln Pro 515 520 525Arg Lys Asp His Pro Asp Val Pro Val Tyr Pro Gln Pro Glu Arg Ser 530 535 540Met Tyr Arg Gly Gly Glu Phe Gly Tyr Thr Lys Leu Val Ala Thr Lys545 550 555 560Glu Lys Leu Thr Leu Thr Tyr Ile Gly Asn His Asp Gly Gln Val His 565 570 575Asp Met Val Glu Ile Phe Ser Gly Gln Val Ser Asn Asn Asn Gly Val 580 585 590Pro Glu Val Ile Asp Asp Thr Lys Leu Ser Thr Gly Val Ser Thr Lys 595 600 605Leu Lys Ile Pro Leu Phe Ser Leu Glu Ile Val Gly Ser Val Met Phe 610 615 620Ala Leu Val Leu Gly Phe Ser Leu Gly Phe Leu Ile Arg Arg Lys Lys625 630 635 640Glu Ala Ala Gln Trp Thr Pro Val Lys Asn Glu Glu Thr 645 65036738DNAGossypium hirsutum 36gcacgagcgg gaagcagcca atatgacttt ctgaagcatg atctagagtc ggttgatcgg 60atgaagaccc cttttgttgt agttcaaggg catagaccaa tgtacactac aagtttcgaa 120agtagggacg ccccattgag agagaaaatg cttgagcatt tggaaccttt atttgtgaaa 180aacaatgtga accttgcatt atggggccat gttcatcggt acgagaggtt ttgtccattg 240aagaacttca catgtggaag catggggcag aaggggaagg attgggaggc atttccagtt 300catgttgtga ttgggatggc aggacaagac tggcaaccaa catgggaacc tcgaccagac 360catccaacga tcccgtctac ccacaacccc aagaggtctt tgtaccgcac aggcgagttt 420gggtacacta gattaattgc tacaaaagag aaacttacac tatcgttcgt aggaaaccat 480gacggggagg tgcatgacat ggttgagatt ttggcatctg ggcaagttct caatggtggt 540gatgataaca atggtaaagt cggagcagtc cataaggttg atgatgtgac acggtactca 600ttttcacact atgtctgggg tggtagtgtc ttggtgcttg gtggttttgt tggctatgtt 660ctgggtttcg tttcacatgc taggagacaa attgcaacag aaagaggctg gacttccttg 720aaaaccgagg agcaatga 73837245PRTGossypium hirsutum 37Ala Arg Ala Gly Ser Ser Gln Tyr Asp Phe Leu Lys His Asp Leu Glu1 5 10 15Ser Val Asp Arg Met Lys Thr Pro Phe Val Val Val Gln Gly His Arg 20 25 30Pro Met Tyr Thr Thr Ser Phe Glu Ser Arg Asp Ala Pro Leu Arg Glu 35 40 45Lys Met Leu Glu His Leu Glu Pro Leu Phe Val Lys Asn Asn Val Asn 50 55 60Leu Ala Leu Trp Gly His Val His Arg Tyr Glu Arg Phe Cys Pro Leu65 70 75 80Lys Asn Phe Thr Cys Gly Ser Met Gly Gln Lys Gly Lys Asp Trp Glu 85 90 95Ala Phe Pro Val His Val Val Ile Gly Met Ala Gly Gln Asp Trp Gln 100 105 110Pro Thr Trp Glu Pro Arg Pro Asp His Pro Thr Ile Pro Ser Thr His 115 120 125Asn Pro Lys Arg Ser Leu Tyr Arg Thr Gly Glu Phe Gly Tyr Thr Arg 130 135 140Leu Ile Ala Thr Lys Glu Lys Leu Thr Leu Ser Phe Val Gly Asn His145 150 155 160Asp Gly Glu Val His Asp Met Val Glu Ile Leu Ala Ser Gly Gln Val 165 170 175Leu Asn Gly Gly Asp Asp Asn Asn Gly Lys Val Gly Ala Val His Lys 180 185 190Val Asp Asp Val Thr Arg Tyr Ser Phe Ser His Tyr Val Trp Gly Gly 195 200 205Ser Val Leu Val Leu Gly Gly Phe Val Gly Tyr Val Leu Gly Phe Val 210 215 220Ser His Ala Arg Arg Gln Ile Ala Thr Glu Arg Gly Trp Thr Ser Leu225 230 235 240Lys Thr Glu Glu Gln 24538930DNAPanicum virgatum 38tggccatcac aaccttggaa accatggtgg gctacatatg gaaaggacgg tgggggtgaa 60tgtggaatac catacagtgt caagttcaga atgcctggca attcagttct acctactggt 120aatggtggtc cagacaccag gaatctttat tactcctttg attcaggtgt ggtgcatttc 180gtgtacatgt caactgaaac taattttctt cagggcagtg accagtacaa cttcttaaaa 240gcggaccttg agaaggtgaa ccgaactaga acaccattcg ttgtttttca gggccaccgt 300cccatgtaca cctcaagtga tgaaaccagg gatgctgctt tgaaacagca gatgctccag 360aatttggaac cactgctggt gacatacaat gtgacccttg cactctgggg acatgtccac 420aggtatgaga ggttctgccc catgaagaac ttccaatgtg ttaacacttc gtcaagcttc 480caataccctg gcgcccctgt gcatcttgtg atcgggatgg gtggtcaaga ctggcaacct 540atatggcaac caaggcctga tcaccctgat gttcccatct ttccgcagcc tgagaggtct 600atgtaccgtg gtggtgtgtt tggatacaca agacttgtag ctacaaggga gaagctaaca 660ctaacgtatg tggggaacca tgatgggcaa gtccatgata tggtggagat attttctggc 720caagtatcca gcaacagcag tgttgctgag gctgttgatg gtgcaaaact cagcacagga 780gtcagcaccg tgcgaaaaat gcctcctttg tacttggaaa tcggaggcag tgtgatgttt 840gcactactgc tggggtttgg ttttggattt cttgtcagga gaaagaaaga agctgcacaa 900tgggctccgg taaagaacga ggaatcttaa 93039309PRTPanicum virgatum 39Trp Pro Ser Gln Pro Trp Lys Pro Trp Trp Ala Thr Tyr Gly Lys Asp1 5 10 15Gly Gly Gly Glu Cys Gly Ile Pro Tyr Ser Val Lys Phe Arg Met Pro 20 25 30Gly Asn Ser Val Leu Pro Thr Gly Asn Gly Gly Pro Asp Thr Arg Asn 35 40 45Leu Tyr Tyr Ser Phe Asp Ser Gly Val Val His Phe Val Tyr Met Ser 50 55 60Thr Glu Thr Asn Phe Leu Gln Gly Ser Asp Gln Tyr Asn Phe Leu Lys65 70 75 80Ala Asp Leu Glu Lys Val Asn Arg Thr Arg Thr Pro Phe Val Val Phe 85 90 95Gln Gly His Arg Pro Met Tyr Thr Ser Ser Asp Glu Thr Arg Asp Ala 100 105 110Ala Leu Lys Gln Gln Met Leu Gln Asn Leu Glu Pro Leu Leu Val Thr 115 120 125Tyr Asn Val Thr Leu Ala Leu Trp Gly His Val His Arg Tyr Glu Arg 130 135 140Phe Cys Pro Met Lys Asn Phe Gln Cys Val Asn Thr Ser Ser Ser Phe145 150 155 160Gln Tyr Pro Gly Ala Pro Val His Leu Val Ile Gly Met Gly Gly Gln 165 170 175Asp Trp Gln Pro Ile Trp Gln Pro Arg Pro Asp His Pro Asp Val Pro 180 185 190Ile Phe Pro Gln Pro Glu Arg Ser Met Tyr Arg Gly Gly Val Phe Gly 195 200 205Tyr Thr Arg Leu Val Ala Thr Arg Glu Lys Leu Thr Leu Thr Tyr Val 210 215 220Gly Asn His Asp Gly Gln Val His Asp Met Val Glu Ile Phe Ser Gly225 230 235 240Gln Val Ser Ser Asn Ser Ser Val Ala Glu Ala Val Asp Gly Ala Lys 245 250 255Leu Ser Thr Gly Val Ser Thr Val Arg Lys Met Pro Pro Leu Tyr Leu 260 265 270Glu Ile Gly Gly Ser Val Met Phe Ala Leu Leu Leu Gly Phe Gly Phe 275 280 285Gly Phe Leu Val Arg Arg Lys Lys Glu Ala Ala Gln Trp Ala Pro Val 290 295 300Lys Asn Glu Glu Ser305401179DNASolanum lycopersicum 40gctttcttgt ttggagacat ggggactgct acgccatact tgacatttct tcgtacacag 60gaagaaagta aatcaacgat taagtggata agccgtgata ttgaagctct tggtaataag 120cctgccctta tctcacatat tggagatatc agctacgcca gaggatactc ttggttgtgg 180gacaactttt ttactcaggt ggaacctgtt gcatccagag ttccatacca tgtatgcatc 240ggaaaccatg aatatgattg gccacttcaa ccttggaagc ctgattggtc aagctacggg 300aaagatgggg gaggtgaatg tggtgtaccc tacagtcata agttccatat gccaggaaac 360tcttcagtgc cgactggaat gcatgctcct gcaactcgga atctttatta ctcatttgat 420tctgggcccg ttcactttgt ctatatgtca actgaaacaa atttcctgcc aggtagtaac 480cagtatgact ttttaaagca tgacttggaa tcagttgatc gagtaaaaac tccttttgtc 540gtctttcaag ggcacagacc aatgtacagt tcaagtagcg gaacaaaaga tatatctttg 600aggaagagaa tggttgagta tttggaacct cttcttgtga agaacaatgt gaatcttgta 660ttgtgggggc atgttcatag gtatgagagg ttttgccctt tgaataactt cacctgtgga 720agcttggcct tgaacgggaa ggagcaaaag gctttccctg ttcaaattgt gatcgggatg 780gcaggacagg actggcagcc tatctgggca ccaagagaag accaccctac ggatcctatt 840ttcccacagc ctctgcaatc tctgtaccgt gggagtgaat ttggatacat gaggctgcat 900gccacaaagg aaaagcttac actttcttat gtaggaaacc atgacggaga ggtgcatgat 960aaggtggagt tcctagcttc aggacaactt ctcaatgctg gtatccgtga tggtcctgca 1020gatacagtac acatggagtc taacttctca tggtatgtaa aggttggaag tgtgctaatg 1080cttggagctt tgatgggtta catagttgga ttcatatctc atgctcggaa aaattctgct 1140gataatggtt ggaggcctat aaaaactgag gtaatatga 117941392PRTSolanum lycopersicum 41Ala Phe Leu Phe Gly Asp Met Gly Thr Ala Thr Pro Tyr Leu Thr Phe1 5 10 15Leu Arg Thr Gln Glu Glu Ser Lys Ser Thr Ile Lys Trp Ile Ser Arg 20 25 30Asp Ile Glu Ala Leu Gly Asn Lys Pro Ala Leu Ile Ser His Ile Gly 35 40 45Asp Ile Ser Tyr Ala Arg Gly Tyr Ser Trp Leu Trp Asp Asn Phe Phe 50 55 60Thr Gln Val Glu Pro Val Ala Ser Arg Val Pro Tyr His Val Cys Ile65 70 75 80Gly Asn His Glu Tyr Asp Trp Pro Leu Gln Pro Trp Lys Pro Asp Trp 85 90 95Ser Ser Tyr Gly Lys Asp Gly Gly Gly Glu Cys Gly Val Pro Tyr Ser 100 105 110His Lys Phe His Met Pro Gly Asn Ser Ser Val Pro Thr Gly Met His 115 120 125Ala Pro Ala Thr Arg Asn Leu Tyr Tyr Ser Phe Asp Ser Gly Pro Val 130 135 140His Phe Val Tyr Met Ser Thr Glu Thr Asn Phe Leu Pro Gly Ser Asn145 150 155 160Gln Tyr Asp Phe Leu Lys His Asp Leu Glu Ser Val Asp Arg Val Lys 165 170 175Thr Pro Phe Val Val Phe Gln Gly His Arg Pro Met Tyr Ser Ser Ser 180 185 190Ser Gly Thr Lys Asp Ile Ser Leu Arg Lys Arg Met Val Glu Tyr Leu 195 200 205Glu Pro Leu Leu Val Lys Asn Asn Val Asn Leu Val Leu Trp Gly His 210 215 220Val His Arg Tyr Glu Arg Phe Cys Pro Leu Asn Asn Phe Thr Cys Gly225 230 235 240Ser Leu Ala Leu Asn Gly Lys Glu Gln Lys Ala Phe Pro Val Gln Ile 245 250 255Val Ile Gly Met Ala Gly Gln Asp Trp Gln Pro Ile Trp Ala Pro Arg 260 265 270Glu Asp His Pro Thr Asp Pro Ile Phe Pro Gln Pro Leu Gln Ser Leu 275 280 285Tyr Arg Gly Ser Glu Phe Gly Tyr Met Arg Leu His Ala Thr Lys Glu 290 295 300Lys Leu Thr Leu Ser Tyr Val Gly Asn His Asp Gly Glu Val His Asp305 310 315 320Lys Val Glu Phe Leu Ala Ser Gly Gln Leu Leu Asn Ala Gly Ile Arg 325 330 335Asp Gly Pro Ala Asp Thr Val His Met Glu Ser Asn Phe Ser Trp Tyr 340 345 350Val Lys Val Gly Ser Val Leu Met Leu Gly Ala Leu Met Gly Tyr Ile 355 360 365Val Gly Phe Ile Ser His Ala Arg Lys Asn Ser Ala Asp Asn Gly Trp 370 375 380Arg Pro Ile Lys Thr Glu Val Ile385 39042406DNASorghum bicolor 42accatgacca gaacccatat ggcaaccgag gcccgatcac ccagatgtcc ccatctttcc 60acagcctgag aggtccatgt accgtggcgg tgagtttgga tacacaaggc ttgtagcaac 120aagggagaag ctaacattaa cctatgtggg gaaccatgat gggcaagtcc atggtatggt 180ggagatattt tctggcctgg tatccagtaa cagtagtgtt gctgtggcag tgcatgacac 240caaacttggc acagaagtca gcaccgtgcg aaaaatatct ccattgtact tggaaatcgg 300aggcagtgta ttgtttgcac tgctcctggg attttccttt ggatttctta tcaggagaaa 360ggaagaagct gcacagtgga ctccagtaaa gaacgaggaa tcataa 40643130PRTSorghum bicolor 43Pro Ile Trp Gln Pro Arg Pro Asp His Pro Asp Val Pro Ile Phe Pro1 5 10 15Gln Pro Glu Arg Ser Met Tyr Arg Gly Gly Glu Phe Gly Tyr Thr Arg 20 25 30Leu Val Ala Thr Arg Glu Lys Leu Thr Leu Thr Tyr Val Gly Asn His 35 40 45Asp Gly Gln Val His Gly Met Val Glu Ile Phe Ser Gly Leu Val Ser 50 55 60Ser Asn Ser Ser Val Ala Val Ala Val His Asp Thr Lys Leu Gly Thr65 70 75 80Glu Val Ser Thr Val Arg Lys Ile Ser Pro Leu Tyr Leu Glu Ile Gly 85 90 95Gly Ser Val Leu Phe Ala Leu Leu Leu Gly Phe Ser Phe Gly Phe Leu 100 105 110Ile Arg Arg Lys Glu Glu Ala Ala Gln Trp Thr Pro Val Lys Asn Glu 115 120 125Glu Ser 13044960DNATriticum aestivum 44catgtctgca taggaaatca tgagtatgat tggccttcac aaccttggaa accttcgtgg 60tctacatatg gaaaggatgg tggaggtgaa tgcggaatac catacagtgt caagttcagg 120atgcctggga attctgttct acctactggc aatggagctc cggacacacg gaatctctat 180tactcttttg attcaggtgt tgtgcatttt gtgtacatgt cgactgaaac taatttcgtt 240cagggcagcg accaacacaa tttcctaaaa gctgacctag agaaggtgaa ccgaagtaga 300accccatttg ttgtgtttca gggccaccgg cccatgtata cctcgagcaa cgaagccagg 360gattttgcca tgagacagca gatgatccag catcttgaac tgctcttggt gatgtacaat 420gtgacccttg ccctgtgggg acatgtccat aggtatgaga ggttctgccc catgaagaat 480tcacagtgtc tgaacacatc atcaagcttc atataccctg gtgcccctgt tcatgttgtg 540atcgggatgg ccggacaaga ctggcaaccg atctggcaac caaggcgtga tcatccagat 600gttcccatct ttccacagcc tgggatctcc atgtaccgtg gtggtgagtt cgggtacaca 660aaactggtag ctaccaggga gaagctaacg ctgatgtacg tcgggaacca tgacggacaa 720gtccatgaca tggtggagat attctctgga caaacatcta ctgaagctag tgctaccgag 780gcggtcaatc aaacaaagct cggctcggga accagcgcca agctgaagat ttccccatta 840tacttggaaa ttggaggtag tgtgatgttg gcattgctgc ttggttttgc cttgggattt 900ctcctcagga agaagagaga agcggcacaa tggactccgg tgaagaacga ggaatcctaa 96045319PRTTriticum aestivum 45His Val Cys Ile Gly Asn His Glu Tyr Asp Trp Pro Ser Gln Pro Trp1 5 10 15Lys Pro Ser Trp Ser Thr Tyr Gly Lys Asp Gly Gly Gly Glu Cys Gly 20 25 30Ile Pro

Tyr Ser Val Lys Phe Arg Met Pro Gly Asn Ser Val Leu Pro 35 40 45Thr Gly Asn Gly Ala Pro Asp Thr Arg Asn Leu Tyr Tyr Ser Phe Asp 50 55 60Ser Gly Val Val His Phe Val Tyr Met Ser Thr Glu Thr Asn Phe Val65 70 75 80Gln Gly Ser Asp Gln His Asn Phe Leu Lys Ala Asp Leu Glu Lys Val 85 90 95Asn Arg Ser Arg Thr Pro Phe Val Val Phe Gln Gly His Arg Pro Met 100 105 110Tyr Thr Ser Ser Asn Glu Ala Arg Asp Phe Ala Met Arg Gln Gln Met 115 120 125Ile Gln His Leu Glu Leu Leu Leu Val Met Tyr Asn Val Thr Leu Ala 130 135 140Leu Trp Gly His Val His Arg Tyr Glu Arg Phe Cys Pro Met Lys Asn145 150 155 160Ser Gln Cys Leu Asn Thr Ser Ser Ser Phe Ile Tyr Pro Gly Ala Pro 165 170 175Val His Val Val Ile Gly Met Ala Gly Gln Asp Trp Gln Pro Ile Trp 180 185 190Gln Pro Arg Arg Asp His Pro Asp Val Pro Ile Phe Pro Gln Pro Gly 195 200 205Ile Ser Met Tyr Arg Gly Gly Glu Phe Gly Tyr Thr Lys Leu Val Ala 210 215 220Thr Arg Glu Lys Leu Thr Leu Met Tyr Val Gly Asn His Asp Gly Gln225 230 235 240Val His Asp Met Val Glu Ile Phe Ser Gly Gln Thr Ser Thr Glu Ala 245 250 255Ser Ala Thr Glu Ala Val Asn Gln Thr Lys Leu Gly Ser Gly Thr Ser 260 265 270Ala Lys Leu Lys Ile Ser Pro Leu Tyr Leu Glu Ile Gly Gly Ser Val 275 280 285Met Leu Ala Leu Leu Leu Gly Phe Ala Leu Gly Phe Leu Leu Arg Lys 290 295 300Lys Arg Glu Ala Ala Gln Trp Thr Pro Val Lys Asn Glu Glu Ser305 310 315461959DNABrassica napus 46atgatcgtcg acttctctac cttcatcctc ttcatctccg tcttcatttc ctcagctaac 60gccaaagcaa ccttatccat ctcccccaaa actctaagcc gatccggcga ttccatcctc 120atcaaatggt ccaacgtcga ctctccctcc gatctcgact ggctaggcat ctactccccc 180ccagactctc cccacgacca cttcatcggc tacaaattcc tcaacgtctc ccccacgtgg 240caatccggct ccggcgcgat ctccctcccc ctcaccaacc tccgatcgaa ctacacgttc 300cgtatcttcc gatggacgca gtccgagatc aatccgaagc acaaggacca cgaccagaat 360cccttaccgg gaacgaagca ccttctggcg gaatcggagc aggtggggtt cggatccgcc 420ggcgtgggga ggccggagca gatccatttg gcgttcgagg ataaggttaa caggatgcgg 480gtcacgttcg tagctgggga tggggaagaa aggttcgtga ggtacggaga ggggaaggac 540gcgttggcga actccgcggc ggcgcgcggg attaggtacg agagggagca tatgtgtaat 600gctccggcta attccaccgt gggatggaga gatcccgggt ggatttttca taccgttatg 660aagaatttga acggtggcgt taggtattat tatcaggttg ggagtgattc aaagggatgg 720agtgagatcc acagctttat cgctcgagat atctactcag aagaaaccat agctttcatg 780ttcggagaca tgggttgcgc tacaccttac aataccttta tccggacgca ggacgagagt 840atctcaacag ttaagtggat actccgcgac atcgaagctc ttggtgacaa gccagctctt 900gtttcgcaca ttggtgatat aagctacgct cgtggttact cgtgggtgtg ggatgagttc 960ttcgctcaga tcgagcctat tgcctcgaga gttccttacc acgtctgcat tggtaaccac 1020gagtatgact tccctactca gccgtggaaa cctgattggg gaacttacgg taatgacggt 1080gggggagagt gcggtgtgcc gtatagtctc aagttcaaca tgcctggaaa ctcgtcggaa 1140ccaacgggaa cgaaagctcc tcctacaagg aatttgtatt actcttacga catggggtcg 1200gttcatttcc tttacatctc caccgagacg aactttctca aaggagggag gcaatacgag 1260tttataaagc gagatcttga gtctgtgaac agggagaaaa caccgtttgt tgtcgtgcaa 1320ggacacagac cgatgtacac cacgagcaac gaggtgagag acgcgatgat taggcaaaag 1380atggtggagc atttggagcc gctgtttgtg gagaacaacg tgacgcttgc tctgtgggga 1440catgttcata gatacgagag gttttgtccg ataagcaaca acacgtgtgg gaaacagtgg 1500agaggaagcc cggttcatct tgtgatcggt atgggcggtc aagactggca accgatttgg 1560cagccgagac cgaaccatcc gggtcttcct atattccctc agcctgaaca gtcgatgtac 1620aggacgggtg agtttgggta cactcgtttg gttgcgaaca aagagaagct tactgtttcg 1680tttgtgggta accatgatgg agaagttcat gatagtgttg agatcttagc gtctggggaa 1740gtaatcagtg ggaggaaaga ggaaactatt aagaccgttc ctgtatctgc aacacttgtg 1800gggaaacctg agtctgatgt cttatggtat gttaaaggag caggcttgtt ggttatgggt 1860gtgcttttag ggttccttat agggtttttt acaaggggga agaaaggatc ttcttcatct 1920gataaccgtt ggatcccagt caagaacgag gagacatga 195947652PRTBrassica napus 47Met Ile Val Asp Phe Ser Thr Phe Ile Leu Phe Ile Ser Val Phe Ile1 5 10 15Ser Ser Ala Asn Ala Lys Ala Thr Leu Ser Ile Ser Pro Lys Thr Leu 20 25 30Ser Arg Ser Gly Asp Ser Ile Leu Ile Lys Trp Ser Asn Val Asp Ser 35 40 45Pro Ser Asp Leu Asp Trp Leu Gly Ile Tyr Ser Pro Pro Asp Ser Pro 50 55 60His Asp His Phe Ile Gly Tyr Lys Phe Leu Asn Val Ser Pro Thr Trp65 70 75 80Gln Ser Gly Ser Gly Ala Ile Ser Leu Pro Leu Thr Asn Leu Arg Ser 85 90 95Asn Tyr Thr Phe Arg Ile Phe Arg Trp Thr Gln Ser Glu Ile Asn Pro 100 105 110Lys His Lys Asp His Asp Gln Asn Pro Leu Pro Gly Thr Lys His Leu 115 120 125Leu Ala Glu Ser Glu Gln Val Gly Phe Gly Ser Ala Gly Val Gly Arg 130 135 140Pro Glu Gln Ile His Leu Ala Phe Glu Asp Lys Val Asn Arg Met Arg145 150 155 160Val Thr Phe Val Ala Gly Asp Gly Glu Glu Arg Phe Val Arg Tyr Gly 165 170 175Glu Gly Lys Asp Ala Leu Ala Asn Ser Ala Ala Ala Arg Gly Ile Arg 180 185 190Tyr Glu Arg Glu His Met Cys Asn Ala Pro Ala Asn Ser Thr Val Gly 195 200 205Trp Arg Asp Pro Gly Trp Ile Phe His Thr Val Met Lys Asn Leu Asn 210 215 220Gly Gly Val Arg Tyr Tyr Tyr Gln Val Gly Ser Asp Ser Lys Gly Trp225 230 235 240Ser Glu Ile His Ser Phe Ile Ala Arg Asp Ile Tyr Ser Glu Glu Thr 245 250 255Ile Ala Phe Met Phe Gly Asp Met Gly Cys Ala Thr Pro Tyr Asn Thr 260 265 270Phe Ile Arg Thr Gln Asp Glu Ser Ile Ser Thr Val Lys Trp Ile Leu 275 280 285Arg Asp Ile Glu Ala Leu Gly Asp Lys Pro Ala Leu Val Ser His Ile 290 295 300Gly Asp Ile Ser Tyr Ala Arg Gly Tyr Ser Trp Val Trp Asp Glu Phe305 310 315 320Phe Ala Gln Ile Glu Pro Ile Ala Ser Arg Val Pro Tyr His Val Cys 325 330 335Ile Gly Asn His Glu Tyr Asp Phe Pro Thr Gln Pro Trp Lys Pro Asp 340 345 350Trp Gly Thr Tyr Gly Asn Asp Gly Gly Gly Glu Cys Gly Val Pro Tyr 355 360 365Ser Leu Lys Phe Asn Met Pro Gly Asn Ser Ser Glu Pro Thr Gly Thr 370 375 380Lys Ala Pro Pro Thr Arg Asn Leu Tyr Tyr Ser Tyr Asp Met Gly Ser385 390 395 400Val His Phe Leu Tyr Ile Ser Thr Glu Thr Asn Phe Leu Lys Gly Gly 405 410 415Arg Gln Tyr Glu Phe Ile Lys Arg Asp Leu Glu Ser Val Asn Arg Glu 420 425 430Lys Thr Pro Phe Val Val Val Gln Gly His Arg Pro Met Tyr Thr Thr 435 440 445Ser Asn Glu Val Arg Asp Ala Met Ile Arg Gln Lys Met Val Glu His 450 455 460Leu Glu Pro Leu Phe Val Glu Asn Asn Val Thr Leu Ala Leu Trp Gly465 470 475 480His Val His Arg Tyr Glu Arg Phe Cys Pro Ile Ser Asn Asn Thr Cys 485 490 495Gly Lys Gln Trp Arg Gly Ser Pro Val His Leu Val Ile Gly Met Gly 500 505 510Gly Gln Asp Trp Gln Pro Ile Trp Gln Pro Arg Pro Asn His Pro Gly 515 520 525Leu Pro Ile Phe Pro Gln Pro Glu Gln Ser Met Tyr Arg Thr Gly Glu 530 535 540Phe Gly Tyr Thr Arg Leu Val Ala Asn Lys Glu Lys Leu Thr Val Ser545 550 555 560Phe Val Gly Asn His Asp Gly Glu Val His Asp Ser Val Glu Ile Leu 565 570 575Ala Ser Gly Glu Val Ile Ser Gly Arg Lys Glu Glu Thr Ile Lys Thr 580 585 590Val Pro Val Ser Ala Thr Leu Val Gly Lys Pro Glu Ser Asp Val Leu 595 600 605Trp Tyr Val Lys Gly Ala Gly Leu Leu Val Met Gly Val Leu Leu Gly 610 615 620Phe Leu Ile Gly Phe Phe Thr Arg Gly Lys Lys Gly Ser Ser Ser Ser625 630 635 640Asp Asn Arg Trp Ile Pro Val Lys Asn Glu Glu Thr 645 650484PRTArabidopsis thalianaMISC_FEATURE(3)..(4)Xaa is any amino acid 48Gly Asp Xaa Gly1495PRTArtificial SequenceSequence from plant purple acid phosphatases found in plant species such as Arabidopsis thaliana 49Xaa Asp Xaa Xaa Tyr1 5504PRTArabidopsis thaliana 50Gly Asn His Asp1514PRTArabidopsis thaliana 51Gly Asn His Glu1524PRTArtificial SequenceSequence from plant purple acid phosphatases found in plant species such as Arabidopsis thaliana 52Xaa Xaa Gly His1534PRTArtificial SequenceSequence from plant purple acid phosphatases found in plant species such as Arabidopsis thaliana 53Xaa His Xaa His15412PRTArabidopsis thalianaMISC_FEATURE(12)..(12)Xaa is Phe or Trp 54Tyr His Val Cys Ile Gly Asn His Glu Tyr Asp Xaa1 5 105512PRTArtificial SequenceSequence from plant purple acid phosphatases found in plant species such as Arabidopsis thaliana 55Tyr His Val Cys Ile Gly Asn His Glu Tyr Asn Xaa1 5 105613PRTArabidopsis thaliana 56His Ile Gly Asp Ile Ser Tyr Ala Arg Gly Tyr Ser Trp1 5 105718PRTArabidopsis thaliana 57Lys Glu Lys Leu Thr Val Ser Phe Val Gly Asn His Asp Gly Glu Val1 5 10 15His Asp5818PRTArtificial SequenceSequence derived from plant purple acid phosphatases found in plant species such as Arabidopsis thaliana 58Lys Glu Arg Leu Thr Leu Ser Tyr Val Gly Asn His Asp Gly Glu Val1 5 10 15His Asp5918PRTZea mays 59Arg Glu Lys Leu Thr Leu Thr Tyr Val Gly Asn His Asp Gly Gln Val1 5 10 15His Asp6018PRTArtificial SequenceSequence derived from plant purple acid phosphatases found in plant species such as Arabidopsis thaliana 60Lys Glu Lys Leu Thr Leu Thr Tyr Ile Gly Asn His Asp Gly Gln Val1 5 10 15His Asp6110PRTArabidopsis thalianaMISC_FEATURE(8)..(9)Xaa is any amino acid 61Phe Val Gly Asn His Asp Gly Xaa Xaa His1 5 106210PRTArtificial SequenceSequence derived from plant purple acid phosphatases found in plant species such as Arabidopsis thaliana 62Phe Ile Gly Asn His Asp Gly Xaa Xaa His1 5 106310PRTZea maysMISC_FEATURE(8)..(9)Xaa is any amino acid 63Tyr Val Gly Asn His Asp Gly Xaa Xaa His1 5 106410PRTArtificial SequenceSequence derived from plant purple acid phosphatases found in plant species such as Arabidopsis thaliana 64Tyr Ile Gly Asn His Asp Gly Xaa Xaa His1 5 106523PRTArabidopsis thaliana 65Leu Trp Tyr Ala Lys Gly Ala Gly Leu Met Val Val Gly Val Leu Leu1 5 10 15Gly Phe Ile Ile Gly Phe Phe 206612PRTArtificial SequenceSequence derived from plant purple acid phosphatases found in plant species such as Arabidopsis thaliana 66Xaa Xaa Xaa Xaa Xaa Xaa Xaa Gly Xaa Xaa Xaa Gly1 5 10

Claims

1. A method to make a transgenic plant having increased rate of plant growth and elevate plant yields comprising: introducing a gene coding for a phosphatase into a plant, the phosphatase gene encodes for a polypeptide comprising a C-terminal motif having the sequence of SEQ ID NO: 66 or a homologue thereof, and one or more sequences selected from the group consisting of SEQ ID NOS: 48-53.

2. The method of claim 1, wherein the phosphatase gene is a purple acid phosphatase gene comprising a nucleotide sequence encoding a polypeptide comprising an amino acid sequence of one or more selected from SEQ ID NOS: 2, 6, 8, 19, 21, 23, 25, 27, 29, 31, 33, 35, and 47, and homologues thereof.

3. The method of claim 1, wherein the phosphatase gene is a purple acid phosphatase gene comprising a nucleotide sequence selected from SEQ ID NOS: 1, 5, 7, 18, 20, 22, 24, 26, 28, 30, 32, 34, and 46 and homologues thereof.

4. The method of claim 2, wherein said nucleotide sequence comprises one or more selected from the group consisting of SEQ ID NOs 1, 5, 7, 18, 20, 22, 24, 26, 28, 30, 32, 34, and 46 and homologues thereof.

5. The method of claim 2, wherein said nucleotide sequence comprises a nucleotide sequence encoding a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 2 and conservatively substituted variants thereof.

6. The method of claim 2, wherein said nucleotide sequence comprises a nucleotide sequence encoding a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NOS: 54 and 55 and conservatively substituted variants thereof.

7. The method of claim 6, wherein said nucleotide sequence comprises a nucleotide sequence encoding a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NOS: 50 and 51.

8. The method of claim 2, wherein said nucleotide sequence comprises a nucleotide sequence encoding a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NOS: 57-60 and conservatively substituted variants thereof.

9. The method of claim 8, wherein said nucleotide sequence comprises a nucleotide sequence encoding a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NOS: 50 and 51.

10. The method of claim 2, wherein said nucleotide sequence comprises a nucleotide sequence encoding a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NOS: 61-64.

11. The method of claim 1, wherein introducing the phosphatase gene up-regulates the enzymatic activity of sucrose phosphate synthase in the transgenic plant relative to the wild-type plant.

12. The method of claim 1, wherein introducing the phosphatase gene up-regulates the sucrose and/or glucose level in the transgenic plant relative to the wild-type plant.

13. The method of claim 1, wherein introducing the phosphatase gene increases the growth rate of the transgenic plant relative to the wild-type plants.

14. The method of claim 1, wherein introducing the phosphatase gene results in a higher crop yield of the transgenic plant relative to the wild-type plants.

15. The method of claim 1, wherein the plant is a species selected from one of the group consisting of the following genera: Asparagus, Bromus, Hemerocalli, Hordeum, Loliu, Panicum, Pennisetum, Saccharum, Sorghum, Trigonell, Triticum, Zea, Antirrhinum, Arabidopsis, Arachis, Atropa, Brassica, Browallia, Capsicum, Carthamus, Cichorium, Citrus, Chrysanthemum, Cucumis, Datura, Daucus, Digitalis, Fragaria, Geranium, Glycine, Helianthus, Hyscyamus, Ipomoea, Latuca, Linum, Lotus, Solanum lycopersicon, Majorana, Malva, Gossypium, Manihot, Medicago, Nemesia, Nicotiana, Onobrychis, Pelargonium, Petunia, Ranunculus, Raphanus, Salpiglossis, Senecio, Sinapis, Solanum, Trifolium, Vigna, and Vitis.

16. The method of claim 1, wherein the plant is a species selected from the family Brassica.

17. A transformed plant, comprising: a plant comprising at least one additional gene coding for a phosphatase relative to a corresponding wild-type plant, the phosphatase gene encodes for a polypeptide comprising a C-terminal motif having the sequence of SEQ ID NO: 66 or a homologue thereof, and one or more sequences selected from the group consisting of SEQ ID NOS: 48-53.

18. The transformed plant of claim 17, wherein the phosphatase gene is a purple acid phosphatase gene comprising a nucleotide sequence encoding a polypeptide comprising an amino acid sequence of one or more selected from SEQ ID NOS: 2, 6, 8, 19, 21, 23, 25, 27, 29, 31, 33, 35, and 47, and homologues thereof.

19. The transgenic plant of claim 17, wherein the phosphatase gene is a purple acid phosphatase gene comprising a nucleotide sequence selected from SEQ ID NOS: 1, 5, 7, 18, 20, 22, 24, 26, 28, 30, 32, 34, and 46 and homologues thereof.

20. The transgenic plant of claim 17, wherein the phosphatase gene is a purple acid phosphatase gene comprising a nucleotide sequence selected from SEQ ID NO: 1 or a homologue thereof.

21. The transformed plant of claim 17, wherein the phosphatase gene is a purple acid phosphatase gene comprising a nucleotide sequence encoding a polypeptide comprising an amino acid sequence of one or more selected from SEQ ID NOS: 2 or a homologue thereof.

22. The transformed plant of claim 17, wherein said plant is of a monocotyledonous species.

23. The transformed plant of claim 17, wherein said plant is of a dicotyledonous species.

24. The transformed plant of claim 17, wherein the plant is a species selected from one of the group consisting of the following genera: Asparagus, Bromus, Hemerocalli, Hordeum, Loliu, Panicum, Pennisetum, Saccharum, Sorghum, Trigonell, Triticum, Zea, Antirrhinum, Arabidopsis, Arachis, Atropa, Brassica, Browallia, Capsicum, Carthamus, Cichorium, Citrus, Chrysanthemum, Cucumis, Datura, Daucus, Digitalis, Fragaria, Geranium, Glycine, Helianthus, Hyscyamus, Ipomoea, Latuca, Linum, Lotus, Solanum lycopersicon, Majorana, Malva, Gossypium, Manihot, Medicago, Nemesia, Nicotiana, Onobrychis, Pelargonium, Petunia, Ranunculus, Raphanus, Salpiglossis, Senecio, Sinapis, Solanum, Trifolium, Vigna, and Vitis.

25. The transformed plant of claim 17, wherein the plant is a species selected from the family Brassica.

26. A vector comprising a plasmid comprising a phosphatase gene comprising a nucleic acid sequence selected from the group consisting of SEQ ID NOS: 1, 5, 7, 18, 20, 22, 24, 26, 28, 30, 32, 34, and 46 and homologues thereof, wherein the plasmid is capable of transforming a bacterial cell.

27. The vector of claim 26, wherein the bacterial cell is Agrobacterium tumefaciens.

28. The vector of claim 26, wherein said phosphatase gene encodes an amino acid sequence comprising one or more selected from the group consisting of SEQ ID NOS: 2, 6, 8, 19, 21, 23, 25, 27, 29, 31, 33, 35, and 47 and homologues thereof, except that all or an N-terminal portion of amino acid residues 1 to 50 of these amino acid sequences are replaced by a plant signal peptide such that said polypeptide is sorted to various organelles or compartments of plant cells upon expression of the phosphatase gene in a transformed host plant cell.

29. The vector of claim 26, wherein the phosphatase gene comprises a nucleic acid sequence selected from SEQ ID NO: 1 or a homologue thereof.

30. A host cell comprising the vector of claim 26.

31. The host cell of claim 30, wherein the phosphatase gene comprising the vector is operably linked to a heterologous promoter.

32. The host cell of claim 31, wherein the heterologous promoter is a plant promoter.

33. The host cell of claim 31, wherein the heterologous promoter is a promoter derived from cauliflower mosaic virus.

34. The host cell of claim 30, wherein the host cell is a plant species.

35. A method for preparing a cell or progeny thereof capable of expressing a purple acid phosphatase in a host cell, comprising: transforming a bacterial cell with the vector of claim 26 and transforming the host cell by transfer of DNA from the bacterial cell to the host cell.

36. The method of claim 35, wherein the bacterial cell is Agrobacterium tumefaciens and the host cell is a plant cell.

37. The method of claim 36, wherein the host cell is from the family Brassica.

38. An expression cassette comprising a phosphatase gene comprising a nucleotide sequence encoding an enzyme having phosphatase activity, wherein said nucleotide sequence hybridizes to a nucleotide sequences selected from the group consisting of SEQ ID NOS: 1, 5, 7, 18, 20, 22, 24, 26, 28, 30, 32, 34, and 46, under stringent condition and is operably linked to regulatory nucleotide sequences such that said regulatory nucleotide sequences cause expression of the nucleotide sequence in plant cells.

39. The expression cassette of claim 38, wherein said nucleotide sequence hybridizes to the nucleotide sequence of SEQ ID NO: 1.

40. The Expression cassette of claim 38, wherein said regulator nucleotide sequence is a cauliflower mosaic virus promoter.

41. A method of feeding animals, comprising: providing a feed comprising matter derived from the transformed plant of claim 17 to an animal for consumption by the animal.