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United States Patent Application 20180010140
Kind Code A1
Hellmann; Hanjo January 11, 2018

Plants Having Altered Expression and Activity of Yield-Related Proteins

Abstract

Transgenic plants that have enhanced yield-related traits, such as increased seed oil production, are produced by genetically engineering the plants to down-regulate the expression of at least one BPM protein. Such transgenic plants can, for example, be cultivated and yield higher seed oil production than control plants which have not been genetically engineered for down regulation of a BPM protein.


Inventors: Hellmann; Hanjo; (Pullman, WA)
Applicant:
Name City State Country Type

Washington State University

Pullman

WA

US
Family ID: 1000002883124
Appl. No.: 15/653950
Filed: July 19, 2017


Related U.S. Patent Documents

Application NumberFiling DatePatent Number
14519577Oct 21, 20149745591
15653950
61895007Oct 24, 2013

Current U.S. Class: 1/1
Current CPC Class: C07K 14/415 20130101; C12N 15/8247 20130101; C12N 15/8261 20130101; C12N 15/8218 20130101
International Class: C12N 15/82 20060101 C12N015/82; C07K 14/415 20060101 C07K014/415

Claims



1-14. (canceled)

15. A transgenic plant genetically engineered to express a nucleic acid which, as compared to a control plant, down-regulates expression of or reduces the activity of one or more proteins characterized as Broad complex, Tramtrack, Bric-a-brac/Pox virus and Zing finger having a Meprin and tumor necrosis factor receptor associated factor homolog (MATH-BTB/POZ), wherein the transgenic plant exhibits enhanced yield-related traits as compared to the control plant, wherein the enhanced yield-related traits are selected from the group consisting of increased seed oil production, increased stress-tolerance, and increased growth rate as compared to the control plant.

16. The transgenic plant of claim 15, wherein said plant is of the Brassicaceae family.

17. The transgenic plant of claim 16, wherein said plant is Arabidopsis Thaliana.

18. The transgenic plant of claim 15, wherein said transgenic plant exhibits increased seed oil production as compared to the control plant.

19. The transgenic plant of claim 15 wherein the one or more proteins includes at least a plurality of proteins.

20. A method for seed oil production comprising: cultivating transgenic plants under conditions promoting plant growth and development, wherein each of the transgenic plants are genetically engineered to express a nucleic acid which, as compared to a control plant, down-regulates expression of or reduces the activity of one or more proteins characterized as Broad complex, Tramtrack, Bric-a-brac/Pox virus and Zing finger having a Meprin and tumor necrosis factor receptor associated factor homolog (MATH-BTB/POZ), wherein the transgenic plant exhibits increased seed oil production compared to the control plant; and recovering said seed oil from said transgenic plant.

21. The method of claim 20 wherein the transgenic plants are of the Brassicaceae family.

22. The method of claim 21 wherein the transgenic plants are Arabidopsis Thaliana.

23. The method of claim 20 wherein the one or more proteins which have down-regulated expression or reduced activity compared to the control plant includes at least a plurality of proteins.

24. A method for enhancing yield-related traits in a plant as compared to a control plant comprising: genetically engineering the plant to express a nucleic acid which, as compared to a control plant, down-regulates expression of or reduces the activity of one or more proteins characterized as Broad complex, Tramtrack, Bric-a-brac/Pox virus and Zing finger having a Meprin and tumor necrosis factor receptor associated factor homolog (MATH-BTB/POZ), wherein the transgenic plant exhibits enhanced yield-related traits as compared to the control plant, wherein the enhanced yield-related traits are selected from the group consisting of increased seed oil production, increased stress-tolerance, and increased growth rate as compared to the control plant.

25. The method of claim 23 wherein said plant exhibits increased seed oil production as compared to a control plant.

26. The method of claim 23 wherein said plant is of the Brassicaceae family.

27. The method of claim 26 wherein said plant is Arabidopsis Thaliana.

28. The method of claim 24 wherein said step of genetically engineering comprises introducing artificial microRNA (amiRNA) to down-regulate the one or more MATH-BTB/POZ proteins.

29. The method of claim 24, wherein the one or more MATH-BTB/POZ proteins includes a plurality of proteins.
Description



BACKGROUND OF THE INVENTION

Field of the Invention

[0001] Embodiments of the invention generally relate to enhanced yield-related traits in plants. In particular, the invention provides plants that have been genetically engineered to down-regulate expression or reduce the activity of BPM proteins, resulting in enhanced yield-related traits including without limitation enhanced seed oil production, as well as products made by or from the plants.

Background of the Invention

[0002] Under field conditions, plant performance, for example in terms of growth, development, biomass accumulation and seed generation, depends on a plant's tolerance and acclimation ability to numerous environmental conditions, changes and stresses. There has always been a need for improving plant traits in crop cultivation. Breeding strategies foster crop properties to withstand biotic and abiotic stresses, to improve nutrient use efficiency and to alter other intrinsic crop specific yield parameters.

[0003] Plants are sessile organisms and consequently need to cope with various environmental stresses. Biotic stresses such as plant pests and pathogens on the one hand, and abiotic environmental stresses on the other hand are major limiting factors for plant growth and productivity (Boyer, 1982; Bohnert et al., 1995), thereby limiting plant cultivation and geographical distribution. Plants exposed to different stresses typically have low yields of plant material, like seeds, fruit or other produces. Crop losses and crop yield losses caused by abiotic and biotic stresses represent a significant economic and political factor and contribute to food shortages, particularly in many underdeveloped countries.

[0004] Conventional means for crop and horticultural improvements today utilize selective breeding techniques to identify plants with desirable characteristics. Advances in molecular biology have allowed for the production of transgenic plants with enhanced yield-related traits. Various yield-related traits in plants are important to many industries worldwide. In particular, plant seed oils are an important source of calories for human nutrition, as feedstocks for non-food uses such as soaps and polymers, and can serve as a high-energy biofuel. World production from oilseed crops in 2011 reached a value near US$120 billion with plant oil consumption expected to double by 2040 (Bates et al., 2013). As a result, methods for increasing seed oil biosynthesis have been an important research topic. However, previous attempts to modulate the transcription levels of factors critical for seed oil biosynthesis, such as WRINKLED1 (WRIT), resulted in relatively low increases in seed oil content (Liu et al., 2010; Shen et al., 2010; Pouvreau et al., 2011).

[0005] Effective regulatory mechanisms to time and control developmental and physiological processes in response to environmental cues are of utmost importance to plants due to their sessile life style. A mechanism that allows plants to quickly and flexibly respond is the ubiquitin (UBQ) proteasome pathway (Hua and Vierstra, 2011). It is highly conserved among eukaryotes and requires the concerted activities of an E1 UBQ activating enzyme, a UBQ conjugating enzyme E2, and an E3 UBQ ligase. While E1 and E2 activate the UBQ to modify target substrates, the E3 ligase binds the E2 and a substrate protein to facilitate transfer of the UBQ moiety. Upon building up a UBQ chain on the substrate, the ubiquitylated protein is marked for degradation via the 26S proteasome (Hua and Vierstra, 2011).

[0006] CUL3-based RING E3 ligases (CRL3) have been described only recently and mainly with respect to their basic architecture (Figueroa et al., 2005; Gingerich et al., 2005; Weber et al., 2005; Gingerich et al., 2007). They are composed of a cullin 3 protein, as the scaffolding subunit, that binds in its C-terminal region the RING-finger protein RBX1, while its N-terminal part is recognized by proteins containing a BTB/POZ (Broad complex, Tramtrack, Bric-a-brac/Pox virus and Zinc finger) fold (Figueroa et al., 2005; Weber et al., 2005). BTB/POZ proteins comprise a diverse group of proteins within Arabidopsis and rice, containing 80 and 149 members, respectively (Gingerich et al., 2007). They have been divided into 12 subgroups based on their secondary domains (Gingerich et al., 2007). While the BTB/POZ fold is required for assembly with the cullin and to interact with other BTB/POZ proteins, the secondary domain may function as an adaptor to allow binding of a substrate and delivery to the CRL3 core for ubiquitylation.

[0007] Based on its role as the central scaffolding subunit that assembles with potentially many BTB/POZ proteins, it is not surprising that the loss of CUL3 causes an embryo lethal phenotype. Reduced amounts of functional cullin 3 protein affects red light and ethylene signaling and impacts plant development (Dieterle et al., 2005; Thomann et al., 2009).

[0008] One BTB/POZ subfamily is the BPM (BTB/POZ-MATH) family that contains a BTB/POZ fold in their C-terminal region, and a MATH (Meprin and TRAF [tumor necrosis factor receptor associated factor] homolog) domain located within the first 200 amino acids of their N-terminal region. BPM proteins are known in the art and may also be referred to as MATH-BTB/POZ proteins. The family comprises six members in Arabidopsis, all of which have molecular weights between 40-50 kDa (Weber et al., 2005). A recent study of Brassica rapa provided a phylogenetic analysis of select BPM proteins, but there has yet to be any functional characterization of these genes in the Brassica species (Zhao et al., 2013). In Zea mays, it was found that the loss of a BPM protein resulted in defects in female gametophyte development (Jurani et al., 2012). However, there has been no study linking the downregulation of BPM proteins to enhanced yield-related traits.

SUMMARY OF THE INVENTION

[0009] Based on the surprising finding that BPM proteins assemble widely with ERF/AP2 transcription factors, and as demonstrated with a selected member of this family, WRI1, that the interaction is a requirement to destabilize WRI1 in plants, embodiments of the invention provide genetically engineered plants having increased yield-related traits, in particular and without limitation, seed oil production as compared to non-transgenic plants or other control plants which have not been genetically engineered as described herein, wherein at least one BPM protein is down-regulated or its activity reduced, and methods of making the same. Aspects of the invention also relate to methods of producing and recovering seed oil in plants. Although increased expression of polypeptides containing BTB/POZ domains has been implicated in modifying plant yield-related traits (U.S. patent application Ser. No. 13/818,858), no study has suggested or shown that down-regulating the expression of such polypeptides comprising a MATH domain can enhance yield-related traits.

[0010] An embodiment the invention provides transgenic plants, wherein the plants are genetically engineered so as to down-regulate expression or reduce the activity of at least one BPM (BTB/POZ-MATH) protein as compared to a control plant such as a non-transgenic plant or a plant in which the expression or activity of a BPM protein has not been reduced through the genetic engineering described herein. In preferred embodiments, the transgenic plant is of the Brassicaceae family, in particular, Arabidopsis thaliana. In some embodiments, the transgenic plant exhibits enhanced yield-related traits as compared to a control plant. In exemplary embodiments, the transgenic plant of the claimed invention exhibits increased seed oil production as compared to a control plant.

[0011] Another aspect of the invention provides a method for recovering seed oil from a transgenic plant comprising cultivating said transgenic plant under conditions promoting plant growth and development, wherein said transgenic plant is genetically engineered to down-regulate expression or reduce the activity of at least one BPM protein as compared to a control plant; and recovering seed oil from the transgenic plant. In preferred embodiments, the transgenic plant is of the Brassicaceae family, in particular, Arabidopsis thaliana. In some embodiments, said step of genetically engineering comprises introducing artificial microRNA (amiRNA) to down-regulate said BPM protein. In still other embodiments, said step of genetically engineering comprises the expression of an exogenous MATH domain to compete with BPM protein.

[0012] Additional aspects of the invention provide a method for enhancing yield-related traits in a plant comprising genetically engineering the plant so as to down-regulate expression or reduce the activity of at least one BPM protein as compared to a control plant. In some embodiments, the method of the invention results in increased seed oil production in the transgenic plant as compared to a control plant. In preferred embodiments, the transgenic plant is of the Brassicaceae family, in particular, Arabidopsis thaliana. In some embodiments, the step of genetically engineering comprises introducing artificial microRNA (amiRNA) to down-regulate the BPM protein. In still other embodiments, the step of genetically engineering comprises the expression of an exogenous MATH domain to compete with BPM protein.

[0013] Further embodiments of the invention relate to a product produced by or from a transgenic plant which is genetically engineered to down-regulate expression or reduce the activity of at least one BPM protein as compared to a control plant.

[0014] Other features and advantages of the present invention will be set forth in the description of invention that follows, and in part will be apparent from the description or may be learned by practice of the invention. The invention will be realized and attained by the compositions and methods particularly pointed out in the written description and claims hereof.

BRIEF DESCRIPTION OF THE DRAWINGS

[0015] FIG. 1: Verification of .alpha.-CUL3 and .alpha.-WRI1 antibodies. (A) Upper sequence: partial alignment of Arabidopsis CUL3a and CUL3b sequences (SEQ ID NO:65 and SEQ ID NO:66, respectively). Lower sequence: Arabidopsis WRI1 (SEQ ID NO:67). The peptide sequences used for antibody generation are highlighted with arrows. (B) Western blot analysis in WT, cul3a, cul3b and cul3.sup.hyp backgrounds with .alpha.-CUL3. (C) The specificity of .alpha.-CUL3 was confirmed by Western blot analysis in transient expression assays with a GFP:CUL3a construct in tobacco. (D) PCR identification of homozygous T-DNA mutant wril-3. A WRI1 gene-specific product was amplified in WT but not in wril-3, while one T-DNA specific product was amplified in wril-3 but not in WT. (E) WRI1 is detectable in WT but not in wril-3.

[0016] FIG. 2: Interaction studies of WRI1 with BPM and CUL3 proteins. (A) BPM1 interacts with ERF4, RAV1 and DREB1a, but only poorly with ERF1 in Y2H assays. SDII, medium for transformation selection; SDIV, medium for test of interaction. Pictures of single spots were taken seven days after transformation. (B) WRI1 can assemble with itself in Y2H assay, as well as with representative members of the BPM family, BPM1, BPM3, BPM4, and BPM5. (C) In vitro translated and [.sup.355]-methionine labeled BPM1 protein was used in pull-down assay with E. coli expressed and GST and GST:WRI1. (D) Pulldown experiments with in E. coli expressed GST: WRI1 results in the precipitation of WRI1 and CUL3, while GST alone was ineffective. Asterisks indicate GST:WRI1 band, while the band below is plant WRI1. Pulldowns were first tested with .alpha.-WRI1, and then with .alpha.-CUL3 after the membrane had been stripped. (E) Silver-stained SDS-PAGE gel to illustrate the IPTG-induced expression of purified GST and GST: WRI1 proteins from E. coli. (F) Pulldown experiments with purified proteins show that His:WRI1 can precipitate GST:CUL3a if GST:BPM1 is present in the assay (left blot), while this is not the case when GST alone is used (right blot). Blots were probed first with a .alpha.-GST antibody, before stripped and subsequently probed with .alpha.-CUL3 and .alpha.-WRI1. PD, pulldown. (G) IP experiments with .alpha.-WRI1 antibody shows co-precipitation of CUL3 with WRI1 from Arabidopsis wild type (WT) protein extract. If not otherwise stated in this and subsequent FIGS. 30 .mu.g of total protein extract were loaded as input, and experiments were done with 14-days old seedlings. (H) WRI1 is present in the plant extract used for the IP shown in (G).

[0017] FIG. 3: FPLC analysis and subcellular localization of WRI1. (A) Western blot analysis of FPLC fractions from two-week-old Arabidopsis WT plantlets tested with either .alpha.-WRI1 or .alpha.-CUL3, showed co-migration of the two proteins in fractions between 70 and 150 kDa. WRI1 is also present in fractions corresponding to proteins of around 40-50 kDa, and in fractions higher than 150 kDa indicating that the protein assembles also in complexes distinct from CUL3. (B) Transient expression analysis of GFP:WRI1 in tobacco shows that the fusion protein is located in the nucleus (green fluorescence derives from GFP; blue fluorescence from DAPI staining).

[0018] FIG. 4: CHX treatment does not reduce the protein level of WRI1 but it induces its transcription level. (A) WRI1 protein levels do not decline 6 h after CHX treatment (both with 50 .mu.M or 100 .mu.M). (B) CHX but not MG132 treatment up-regulates WRI1 expression (p<0.01). (C) IAA5 (auxin-responsive protein IAA5) was used as positive control for CHX treatment. The expression of IAA5 was strongly induced by CHX treatment (p<0.01). In all cases two-week-old seedlings were used. Error bars illustrate standard error.

[0019] FIG. 5: Stability and expression level of WRI1 in WT and cul3.sup.hyp. (A) Treatment of WT plants with ActD2 (6 h) and CHX (3 h) inhibitors shows instability of WRI1. This is blocked by co-treatment with MG132 (6 h). (B) WRI1 protein accumulates in cul3.sup.hyp double mutants in comparison to WT. (C) Expression of WRI1 is comparable to WT in cul3.sup.hyp plants. (D) WRI1 protein is more stable in cul3.sup.hyp P than in WT. Error bars in this and all subsequent figures represent standard deviation.

[0020] FIG. 6: Predicted target sites for artificial microRNA (squares) on the different BPM genes. Numbers indicate base pairs.

[0021] FIG. 7: Generation of 6.times.amiBPM and MATH-overexpressing lines and their impact on WRI1 protein levels. (A, B) qRT-PCR analysis show significantly reduced expression levels for six BPMs is in two representative 6.times.amiBPM lines compared to WT. (C) Expression levels of BPM1.sup.MATH show significant increases in two BPM1.sup.MATH lines, and two BPM1.sup.MATH:NLS lines compared to WT. (D) WRI1 protein content is strongly reduced in MATH-overexpressing lines when compared to WT, while both 6.times.amiBPM lines display increased WRI1 levels. All asterisks in this and subsequent figures indicate the statistical significant difference of at least p<0.05 (One-way ANOVA) to WT.

[0022] FIG. 8: Sub-cellular localization of GFP:BPM1.sup.MATH and GFP:BPM1.sup.MATH:NLS. (A) Transient expression analysis of GFP:BPM1.sup.MATH in tobacco demonstrates that the fusion protein is present in the cytosol and nucleus (bar=5 .mu.m). (B) GFP:BPM1.sup.MATH:NLS fusion protein is strictly localized to the nucleus (bar=10 .mu.m). Arrows indicate nuclei. (C) The expression level of WRI1 in WT and two individual lines of GFP:BPM1.sup.MATH, GFP:BPM1.sup.MATH:NLS.

[0023] FIG. 9: Phenotype analysis of WT, two 6.times.amiBPM lines, and MATH-overexpressing lines. (A) Primary root lengths of 2-weeks old Arabidopsis seedlings is strongly reduced in all transgenic lines when compared to WT (n=45). (B) Specifically 6.times.amiBPM plants have reduced numbers of lateral roots development (2-weeks old seedlings; n=45). (C) Root phenotype of WT and two 6.times.amiBPM lines. Picture was taken 14-days post-germination. (D) Overview of rosette phenotypes from WT and transgenic lines. Picture was taken 33 days after germination. (E) All transgenic lines are late flowering. Data were taken for 33-day-old plants (n=30). (F) Rosette leaf number at time of flowering (n=10). (G) Rosette area of 25-day-old Arabidopsis plants for each genetic background (n=30).

[0024] FIG. 10: Rosette leaf phenotype on the time of primary inflorescence. 6.times.amiBPM and MATH-overexpressing plants develop less and shorter leaves then WT. Leaves of transgenic plants frequently developed wider blades (scale=1 cm).

[0025] FIG. 11: Studies of WRI1's protein level, WRI1's transcriptional activity, and complex assembly at the DNA level. (A) WRI1 protein is stabilized in MATH overexpressing and 6.times.amiBPM lines. (B) CUL3 could be precipitated with .alpha.-WRI1 from WT plant extracts but not from BPM1.sup.MATH (left half) or 6.times.amiBPM (right half) extracts. Input was tested for presence of WRI1 and CUL3 proteins using the respective antibodies. (C) Protein levels of WRI1, and expression of two WRI1 target genes, AtGLB1 and BCCP1, in WT and the different transgenic backgrounds. (D-G) ChIP-qPCR analysis on WT, two 6.times.amiBPM lines, and wril-3 shows enrichment of the two WRI1 target promoters proBCCP1 and proAtGLB1 in WT (D) but not in a wril-3 mutant (G) after IP with either .alpha.-WRI1 or .alpha.-CUL3 antibodies. While no enrichment was detectable in samples derived from .alpha.-CUL3 ChIPs in two 6.times.amiBPM lines (E, F), significant enrichment was detectable in .alpha.-WRI1 ChIP samples when compared to WT (E, F). ChIP-qPCR experiments were repeated at least three times independently. Error bar indicates the value of standard error. Asterisk indicates a statistical significant difference (One-Way ANOVA, p<0.05) compared to individual control.

[0026] FIG. 12: Stability assays of WRI1 in WT, 6.times.amiBPM, and MATH overexpressing lines. In comparison to WT, WRI1 protein is stabilized in both (A) 6.times.amiBPM and (B) MATH overexpressing plants.

[0027] FIG. 13: Seed weight, size, and fatty acid content in WT, wril-3, and 6.times.amiBPM plants. (A, B) In comparison to WT, seed weight and size is significantly reduced in wril-3, while it is increased in both 6.times.amiBPM lines, and to a greater extend in 6.times.amiBPM#1 then it is in #2 (data in (A) represent average of n=5 measurements of 20 seeds. Scale bar in (B) represents 1 mm. (C) Seeds in 6.times.amiBPM lines contain higher WRI1 levels which correlate with expression of the WRI1 target genes BCCP1 and AtGLB1. (D) Differences in total fatty acid contents for WT, wril-3, and the two 6.times.amiBPM lines correlated with seed weights and sizes. Data represent average of n=5 measurements on 30 seeds. The asterisk shows the statistical significant difference to WT (p<0.05, T-test).

[0028] FIGS. 14A-D: Fatty acid profile and metabolic profile in seeds of WT, wril-3, and two 6.times.amiBPM lines. All extractions and measurements were done from mature and desiccated seeds from WT, wril-3, and 6.times.amiBPM lines. Graphs 14A for fatty acids, 14B for sugar-sugar alcohols, 14C for amino acids and 14D for organic acids are shown. In the graph for fatty acids, numbers indicate the type of fatty acid (carbon chain length and number of double bonds). All values are based on five independent samples. Error bar indicates the value of standard deviation.

[0029] FIG. 15: Seed phenotype analyzes for 6.times.amiBPM #3. (A) WRI1 protein is elevated in the mutant line in comparison to WT, which (B,C) correlates with seed size and weight, and (D) increased expression of BCCP1 and AtGLB1. (E) Fatty acid levels are significantly increased in 6.times.amiBPM #3 in comparison to WT (p<0.05, T-test).

[0030] FIGS. 16A-C: Sequence alignment of full length BPM proteins from 27 different plant species (SEQ ID NOs. 1 to 27) and a consensus sequence (SEQ ID NO. 28). Amino acid residues with black background color are fully conserved, those with a dark gray background are highly conserved. Arrows at the top of the alignment indicate start and end of either the predicted MATH (black solid line) or BTB/POZ (black dotted line) domains in the Arabidopsis thaliana BPM3 protein. (A) shows the predicted MATH domain and surrounding sequence. (B) shows the predicted BTB/POZ domain and surrounding sequence. (C) shows the c-terminal sequence of the BPM proteins. For example, in Arabidopsis the predicted MATH domain comprises residues 29-141 and the BTB/POZ domain comprises residues 194-303. The following are the latin names of the different species listed as well as the accession numbers (AccNo) of the corresponding BPM proteins: Arabidopsis: Arabidopsis thaliana; AccNo BAH19418 (SEQ ID NO:1); Polish Canola: Brassica rapa; AccNo XP_009141835 (SEQ ID NO:2). Barbados Nut: Jatropha curcas; AccNo KDP44889.1 (SEQ ID NO:3). California poplar: Populus trichocarpa; AccNo XP_002311186 (SEQ ID NO:4). Cacao tree: Theobroma cacao AccNo XP_007009287 (SEQ ID NO:5). Clementine: Citrus clementina; AccNo XP_006435614 (SEQ ID NO:6). Castor oil plant: Ricinus communis; AccNo XP_002524218 (SEQ ID NO:7). Eucalyptus: Eucalyptus grandis; AccNo KCW65573 (SEQ ID NO:8). Grape vine: Vitis vinfera; AccNo XP_002282536 (SEQ ID NO:9). Peach: Prunus persica; AccNo XP_007218050 (SEQ ID NO:10). String bean: Phaseolus vulgaris; AccNo XP_007163464 (SEQ ID NO:11). Soybean: Glycine max AccNo XP_003552772 (SEQ ID NO:12). Date palm: Phoenix dactylifera; AccNo XP_008785535 (SEQ ID NO:13). Strawberry: Fragaria vesca subsp. Vesca; AccNo XP_004307466 (SEQ ID NO:14). Apple: Malus domestica; AccNo XP_008372026 (SEQ ID NO:15). Tomato: Solanum lycopersicum; AccNo XP_004239913 (SEQ ID NO:16). Potato: Solanum tuberosum; AccNo XP_006355691 (SEQ ID NO:17). Oryza: Oryza brachyantha; AccNo XP_006657384 (SEQ ID NO:18). Brachypodium: Brachypodium distachyon; AccNo XP_003557713 (SEQ ID NO:19). Rice: Oryza sativa Japonica Group; AccNo NP 001058677 (SEQ ID NO:20). Barley: Hordeum vulgare subsp. vulgare; AccNo BAJ94248 (SEQ ID NO:21). Spikemoss: Selaginella moellendorffii; AccNo XP_002961582 (SEQ ID NO:22). Barrel Clover: Medicago truncatula; AccNo KEH23724 (SEQ ID NO:23). Robusta coffee: Coffea canephora; AccNo CDP03595 (SEQ ID NO:24). Corn: Zea mays; AccNo NP_001142069 (SEQ ID NO:25). Sorghum: Sorghum bicolor; AccNo XP_002461292 (SEQ ID NO:26). Muskmelon: Cucumis melo; AccNo XP_008458543 (SEQ ID NO:27). A consensus sequence is also shown (SEQ ID NO:28).

[0031] FIG. 17: Comparison of protein identities and similarities of BPM proteins from 27 different plant species. Arab: Arabidopsis. Cano: Polish Canola. Barb: Barbados Nut. Popl: California poplar. Caca: Cacao tree. Clem: Clementine. Cast: Castor oil plant. Euca: Eucalyptus. Vine: Grape vine. Peac: Peach. Stri: String bean. Soy: Soybean. Palm: Date palm. Stra: Strawberry. Appl: Apple. Toma: Tomato. Pota: Potato. Oryz: Oryza. Brac: Brachypodium. Rice: Rice. Barl: Barley. Moss: Spikemoss. Cloy: Barrel Clover. Coff: Robusta coffee. Corn: Corn. Sorg: Sorghum. Musk: Muskmelon. For specific latin names and accession numbers, see FIG. 16.

[0032] FIGS. 18A-B: Salt stress assay. (A) For salt stress tolerance assays, wild type (WT) and bpm mutants (6.times.amiBPM and BPM.sup.MATH:NLS) were plated on solid minimal culture medium, and grown vertically for five days. Afterwards, they were carefully transferred to plates that were supplemented with 150 mM NaCl. The root length was measured for six days by tracking root tips. n=30 (B) Wild type root elongation growth at day six was significantly more inhibited under salt stress conditions than in bpm mutant plants. Asterisks indicate the statistical significant difference of at least P<0.05 (one-way analysis of variance) of WT plants in comparison to mutants.

[0033] FIGS. 19A-D: Photosynthetic parameters of dark adapted plants exposed to drought stress. Plants were grown for three weeks in soil under standard growth conditions (long day (16 h light: 8 h dark)). Drought stress was applied by withholding water over a period of 5 days which is indicated on the X-axis. At day four significant changes were observed between wild type and 6.times.amiBPM plants (n=12). The changes indicate increased sensitivity of the mutant towards drought stress. (A)_qL; Estimates the fraction of open PSII centers on the basis of the lake model for PSII. (B)_NPQ; Non-photochemical quenching. Monitors the apparent rate constant for heat loss from PSII. (C) Fv/Fm Maximum quantum efficiency of PSII photochemistry. Maximum efficiency at which light absorbed by PSII is used for reduction of Qa. Healthy plants are usually between 0.8 and 0.85. (D) Phi II; PSII operating efficiency. Estimates the efficiency at which light absorbed by PSII is used for Qa reduction, and provides an estimate of linear electron flux through PSII.

[0034] FIGS. 20A-C: Flowering phenotype analysis of WT, and 6.times.amiBPM mutants. (A) Expression level of Flowering Locus T (FT), a key regulator of the flowering time point, in rosette leaves at the end of the third, fourth, fifth, and sixth week after germination in wild type (WT) and 6.times.amiBPM plants. FT expression is significantly down regulated in 6.times.amiBPM plants when compared to WT which is in agreement with the late flowering phenotype of the mutants. (B) Schematic drawing of six different FT promoter regions analyzed via qPCR after .alpha.-CUL3 ChIP experiments. "0" indicates location of the start codon. (C) Significant enrichments were detectable in regions 1, 5 and 6 in WT, but not in a 6.times.amiBPM#1 control, indicating that CRL3.sup.BPM E3 ligases are directly involved in controlling FT expression. "NC" indicates negative control primer set afterwards of FT gene's ATG code. Asterisks indicate significant differences of mutant plants to WT (one-way ANOVA; P<0.05).

[0035] FIGS. 21A-E: Inducible 6.times.amiBPM constructs allow controlled increase in seed size. A, treatment of plants with estradiol over a time period of 24 hours leads to a significant down-regulation of all six BPM genes. B, pMDC7:6.times.amiBPM plants that carry an estradiol (E) inducible construct are indistinguishable from wild type (WT) when not treated with estradiol. Bar represents 5 cm. C-E, Plants carrying an estradiol (E) inducible 6.times.amiBPM construct were sprayed daily for around two weeks, starting at the onset of flowering with 10 mM estradiol (+E). The plants were indistinguishable in development from WT, except that seeds in E-treated 6.times.amiBPM plants were significantly larger then WT seeds (C, E), and heavier (D; data shown represent the average weight of 30 seeds). Bar represents 1 mm.

[0036] FIGS. 22A-J: Nucleotide sequence of full length BPM proteins from 27 different plant species (SEQ ID NOs. 68 to 94). Nucleotide sequences corresponding to the amino acid sequences listed in FIG. 16A-C.

DETAILED DESCRIPTION

[0037] Embodiments of the invention provide transgenic plants, wherein the expression of at least one BPM protein has been down-regulated or its activity reduced, resulting in enhanced yield-related traits, in particular but without limitation, increased seed oil production, as compared to a control plant. Control plants of the invention may be non-transgenic plants or plants wherein the expression or activity of a BPM protein has not been reduced or decreased by genetic engineering. Further embodiments of the invention provide methods for enhancing yield-related traits, in particular producing and recovering the seed oil from transgenic plants, wherein the expression of at least one BPM (BTB/POZ-MATH) protein is down-regulated or its activity reduced.

[0038] The following definitions are used throughout:

[0039] The terms "protein", "polypeptide" and "peptide" refer to contiguous chains of amino acids that are covalently bonded (linked) to each other by peptide (amide) bonds. In general, a peptide contains up to about 50 amino acids and a polypeptide contains about 50 or more amino acids. Proteins may contain one or more than one polypeptide. Those of skill in the art will recognize that these definitions are considered somewhat arbitrary, and these terms may be used interchangeably herein. The terms encompass amino acid polymers that are synthesized (transcribed and translated) in vivo and amino acid polymers that are chemically synthesized using procedures well known to those skilled in the art.

[0040] As used herein, the terms "nucleic acid" or "polynucleotide" or "nucleic acid molecule" refer to deoxyribonucleotides or ribonucleotides and polymers thereof in either single- or double-stranded form. Exemplary nucleic acids include DNA (including cDNA), RNA (e.g. mRNA, tRNA, rRNA, microRNA, amiRNA, antisense RNA, RNAi, etc.), and hybrids thereof.

[0041] The term "gene" means a segment of DNA that encodes a biologically active RNA, which may be further translated into a polypeptide chain. The term may or may not include regions preceding and following the coding region as well as intervening sequences (introns) between individual coding segments (exons). As used herein, a gene may be a recombinant or genetically engineered DNA sequence that encodes a polypeptide of interest from which introns have been eliminated.

[0042] The term "consensus sequence" or "motif" refers to a short conserved region in the sequence of evolutionary related proteins. Motifs are frequently highly conserved parts of domains, but may also include only part of the domain, or be located outside of a conserved domain.

[0043] As used herein, the term "transformation" refers to the transfer of nucleic acid (i.e., a nucleotide polymer) into a cell. As used herein, the term "genetic transformation" refers to the transfer and incorporation of DNA, especially recombinant DNA, into a cell. The term "transformant" refers to a cell, tissue or organism that has undergone transformation.

[0044] As used herein, the term "transgenic" refers to cells, cell cultures, organisms (e.g., plants), and progeny which comprise a modified or foreign (heterologous) gene, wherein the modified or foreign gene is not originally present in the host organism. The term "transgenic" also refers to modification of an endogenous gene through the introduction of a transgene that modifies the endogenous gene in a plant. Transgenic organisms may receive the transgene by one of the various methods of transformation, but may also receive the transgene via conventional breeding techniques whereby at least one of the parent organisms comprises such a transgene.

[0045] "Recombinant" refers to a product of genetic engineering, e.g. a nucleic acid such as recombinant DNA, a protein that results from the expression of recombinant DNA, and recombinant cells or organisms that are transformed with recombinant DNA.

[0046] As used herein, the terms "plant" and "plant tissue" refer to any part of a plant. Examples of plant organs include, but are not limited to the leaf, stem, root, tuber, seed, branch, pubescence, nodule, leaf axil, flower, pollen, stamen, pistil, petal, peduncle, stalk, stigma, style, bract, fruit, trunk, carpel, sepal, anther, ovule, pedicel, needle, cone, rhizome, stolon, shoot, pericarp, endosperm, placenta, berry, stamen, and leaf sheath. Plants also include vegetables and fruit plants. "Lower plants" is a collective term for three main groups of plants (mosses, liverworts and lichens) which do not have roots and produce spores to reproduce, rather than flowers. "Higher plants" refers to plants that have vascular tissue (as known as tracheophytes). "Seed producing plants" is a term referring to those plants that produce seed (Spermatophytes) and includes "Flowering plants", which refers to seed-producing plants, also known as Angiospermae or Magnoliophyta, as well as the Gymnospermae. Plants may be grown (e.g. in a field or a greenhouse) for production of food, fuel or fiber or other uses (e.g. wood, ornamentals). All such plants are encompassed by the present invention.

[0047] Exemplary plants or plant cells that may be utilized in the practice of the invention include but are not limited to: oil seed plants, canola, safflower, camelina, soybean, corn, sunflower, peanut, sesame, cotton rice, wheat, etc. Generally, oil seed plants (which may be trees) are cultivated so that oil, especially edible oil, can be produced from the seeds, nuts, tubers, etc. of the plants. Exemplary oil seed plants include but are not limited to: coconut, corn, cotton, olive, palm, peanut (ground nut), various rapeseed plants including canola, safflower, sesame, flax, soybean, sunflower, and the like. Various plant species that produce nuts from which oils are extracted may also be employed, including those that produce hazelnuts (e.g. from the common hazel), almond, beech (e.g. which produce Fagus sylvatica nuts), cashew macadamia, mongongo (or manketti, seeds of the Schinziophyton rautanenii tree), pecan, pine, pistachio, walnut, etc. Various citrus plants and trees produce seeds which are used to prepare edible oils, e.g. lemon, orange oil, grapefruit, sea-buckthorn, etc. Various melons and gourds may be utilized, e.g. watermelon (e.g. Citrullus vulgaris), members of the Cucurbitaceae family including gourds, melons, pumpkins, and squashes; the bitter gourd (Momordica charantia), bottle gourd (e.g. Lagenaria siceraria), buffalo gourd (Cucurbita foetidissima), butternut squash (e.g. Cucurbita moschata), egusi (Cucumeropsis mannii naudin, pumpkin, etc. Other plants and/or trees that may be utilized include borage (e.g. Borago officinalis), blackcurrant, evening primrose (e.g. Oenothera biennis), acai (e.g. any of several species of the Acai palm (Euterpe), black seed (e.g. from Nigella sativa), blackcurrant (e.g. Ribes nigrum), flax (linseed, e.g. Linum usitatissimum), carob, amaranth (e.g. from Amaranthus cruentus and Amaranthus hypochondriacus), apricot, apple, argan (e.g. from Argania spinosa), avocado, babassu r.g. Attalea speciosa), the seeds of Moringa oleifera, from which "ben" oil is extracted, species of genus Shorea, cape chestnut, the cacao plant, cocklebur (e.g. species of genus Xanthium), poppy, the Attalea cohune (cohune palm), coriander, date, Irvingia gabonensis, Camelina sativa, grape, hemp, Ceiba pentandra, Hibiscus cannabinus, Lallemantia iberica, Trichilia emetica, Sclerocarya birrea, meadowfoam, mustard, nutmeg (e.g. from cogeners of genus Myristica), okra (e.g. Abelmoschus esculentus), papaya, perilla, persimmon (e.g. Diospyros virginiana), Caryocar brasiliense, pili nut (e.g. Canarium ovatum), pomegranate (e.g. Punica granatum), prune quinoa, ramtil (e.g. several species of genus Guizotia abyssinica (Niger pea), rice, Prinsepia utilis, shea, Sacha inchi, sapote (e.g. Jessenia bataua), arugula (e.g. Eruca sativa), tea (Camellia), thistle (e.g. Silybum marianum), Cyperus esculentus, tobacco (e.g. Nicotiana tabacum and other Nicotiana species), tomato, and wheat, among others.

[0048] In some aspects, embodiments of the invention provide products produced by plants or from plants or parts of plants, for example, oils produced from the seeds or nuts of the transgenic plants. Exemplary oils of the invention include but are not limited to: Coconut oil, Corn oil, Cottonseed oil, Olive oil, Palm oil, Peanut oil (Ground nut oil), Rapeseed oil (including Canola oil) Safflower oil, Sesame oil, Soybean oil, and Sunflower oil. Various nut oils are also contemplated, including but not limited to: Almond oil, Beech nut oil, Cashew oil, Hazelnut oil, Macadamia oil, Mongongo nut oil (or manketti oil), Pecan oil, Pine nut oil, Pistachio oil, and Walnut oil. Various Cctrus oils are also contemplated, including but not limited to: Grapefruit seed oil, Lemon oil, Orange oil, and sea-buckthorn oil. Oils from melon and gourd seeds are also contemplated, including but not limited to: Cucurbitaceae oils from e.g. gourds, melons, pumpkins, and squashes such as Watermelon seed oil, Bitter gourd oil, Bottle gourd oil, Buffalo gourd oil, Butternut squash seed oil, Egusi seed oil, and Pumpkin seed oil, Various other plant-derived oils are also encompassed by the invention, including but not limited to: Acai oil, Arabidopsis oil, Black seed oil, Blackcurrant seed oil, Borage seed oil, Evening primrose oil, Flaxseed oil (linseed oil), Carob seed pods, Apricot oil, Apple seed oil, Argan oil, Avocado oil, Babassu oil, Ben oil, Borneo tallow nut oil, Cape chestnut oil, Carob pod oil (Algaroba oil), Cocoa butter, Cocklebur oil, Cohune oil, Coriander seed oil Date seed oil, Dika oil, False flax oil Grape seed oil, Hemp oil, Kapok seed oil, Kenaf seed oil, Lallemantia oil, Mafura oil, Marula oil, Meadowfoam seed oil, Mustard oil (pressed), Poppyseed oil, Nutmeg butter, Okra seed oil, Papaya seed oil, Perilla seed oil, Persimmon seed oil, Pequi oil, Pili nut oil, Pomegranate seed oil, Prune kernel oil, Quinoa oil, Ramtil oil, Rice bran oil Royle oil, Sacha inchi oil, Sapote oil, Seje oil, Shea butter, Taramira oil, Tea seed oil (Camellia oil), Thistle oil, Tigernut oil (or nut-sedge oil) Tobacco seed oil, Tomato seed oil, and Wheat germ oil, etc.

[0049] It is an object of the invention to provide transgenic plants having enhanced yield-related traits as compared to a non-transgenic plant or other control plant which has not been similarly genetically engineered according to the teachings provided herein. Yield-related traits include, but are not limited to, seed oil production, flowering, stress-tolerance, and increased growth rate.

[0050] In exemplary embodiments, the transgenic plants have increased seed oil production as compared to control plants (e.g., non-transgenic plants or plants not genetically engineered as described herein).

[0051] The plants of the present invention have been genetically engineered using molecular biology techniques to down-regulate the expression or reduce the activity of at least one BPM protein. Methods of producing transgenic plants are well known to those of ordinary skill in the art. Transgenic plants can be produced by a variety of different transformation methods including, but not limited to, electroporation; microinjection; microprojectile bombardment, also known as particle acceleration or biolistic bombardment, e.g. using needle-like crystals ("whiskers") of silicon carbide; viral-mediated transformation; Agrobacterium-, Rhizobium-, Mesorhizobium- and Sinorffizobium-mediated transformation. See, for example, U.S. Pat. Nos. 5,405,765; 5,472,869; 5,538,877; 5,538,880; 5,550,318; 5,641,664; 5,736,369; 5,736369; and US patent applications 2005/0289672 and 2005/0289667; each of which is expressly incorporated herein by reference in entirety. Progeny of the transgenic plants of the invention are also encompassed.

[0052] BPM proteins are found in almost all plants and the exemplary amino acid sequences of BPM proteins from 27 different species and a consensus sequence are shown in FIGS. 16A-C. The corresponding nucleotide sequences are shown in FIGS. 22A-J. The species listed in FIGS. 16A-C contain members of the Eudicots as well as monocot groups and one spikemoss. The spikemoss is an ancient plant with members of this family existing before Angio- and Gymnosperms were present. The alignment as shown in FIG. 16 indicates that BPMs are functionally conserved. FIG. 17 compares the full length BPM proteins shown in FIGS. 16A-C for identity and similarity.

[0053] Aspects of the invention related to the down-regulation of at least one BPM protein in a transgenic plant. The term "down-regulation" refers to a decrease in endogenous gene expression and/or polypeptide levels as compared to a control, wherein the expression levels in the control have not been modulated. The reduction may be at least about 10% or more reduced compared to that of a non-transgenic control plant, preferably at least 40% and more preferably at least 60%. Methods of decreasing expression of proteins in plants are well known in the art and include, but are not limited to artificial microRNA (amiRNA), antisense RNA, RNAi or co-suppression, and T-DNA insertion. In exemplary embodiments, amiRNA is introduced into the plant to down-regulate expression of at least one BPM protein. Other methods for down-regulating expression of proteins, for example the use of CRISPR-Cas9 nucleases, can be used in the practice of this invention and the invention encompasses each of these methods (Hsu et al., 2014). One of ordinary skill in the art would be able to adapt the various known biological methods for silencing so as to reduce the expression of a gene in a plant or in parts thereof.

[0054] Additional aspects of the invention relate to the reduced activity of at least one BPM protein. The term "reduced activity" refers to the functional aspects of the BPM protein. For example, "reduced activity" is construed to mean that the ability of the BPM protein to assemble with cullin, to interact with other BTB/POZ proteins, and/or to function as an adaptor to allow binding of a substrate and delivery to the CRL3 core for ubiquitylation is fully or partially inhibited. Methods of altering the activity of a protein are known in the art and include, but are not limited to reducing expression (for example, amiRNA), susbstrate competition (for example, MATH domain overexpression), targeted mutation of amino acid residues required for binding to substrates, and targeted mutation of amino acid residues in substrate proteins required for recognition (by the MATH domain for example). In exemplary embodiments, an exogenous BPM domain, preferably the MATH domain, is expressed in the plant to compete with at least one BPM protein for binding with a substrate.

[0055] Exemplary amino acid sequences are provided, but those of skill in the art will recognize that various other modified forms (variants or derivatives) of the amino acid sequences disclosed herein may be made, and the invention encompasses all such variants/derivatives, as long as the resulting molecule retains a desired level of activity as described herein. Exemplary encoding nucleotide sequences are also provided, but those of skill in the art will recognize that, due to the redundancy of the genetic code, other nucleotide sequences may also encode the same protein/polypeptide.

[0056] The nucleic acid molecules described herein may be modified, for example, by codon optimization to facilitate expression in heterologous cells. This type of modification changes or alters the nucleotide sequence that encodes a protein of interest to use, throughout the sequence, codons that are more-commonly used in the transgenic expression host cell. In addition, changes may be made to the nucleotide sequence that encodes the protein to adjust the relative concentration of A/T and G/C base pairs to ratios that are more similar to those of the expression host.

[0057] In addition, nucleotide sequences encoding MATH domains of the invention may be further modified to encode other sequences such as those described above as being beneficial or desirable for inclusion in the plants of the invention, e.g. sequences which target or direct the polypeptide to a particular location or locations within the expression host cell, etc.

[0058] The invention also encompasses vectors that comprise the nucleic acid sequences described herein. "Vector" refers broadly to any plasmid or virus encoding an exogenous nucleic acid. (However, the term may also be construed to include non-plasmid and non-viral compounds which facilitate transfer of nucleic acid into virions or cells, such as, for example, polylysine compounds and the like.) The vector may be a viral vector that is suitable as a delivery vehicle for delivery of the nucleic acid, or mutant thereof, to a cell, or the vector may be a non-viral vector which is suitable for the same purpose. Examples of viral and non-viral vectors for delivery of DNA to cells and tissues are well known in the art. Examples of viral vectors include, but are not limited to recombinant vaccinia, adeno-, retro-, adeno-associated, avian pox and other viral vectors. Examples of non-viral vectors include, but are not limited to, liposomes, polyamine derivatives of DNA, and the like.

[0059] Embodiments of the invention provide transgenic plants with enhanced yield-related traits as compared to non-transgenic plants and methods for producing the same. In preferred embodiments, the transgenic plants have increased seed oil production. The amount of seed oil that can be recovered from plants of the present invention is more than about 1% in comparison to the oil recovered from non-transgenic plants, preferably more than about 25%, and more preferably more than about 50%. The more severe the down-regulation of BPMs or the reduction of the activity of BPMs, the higher the amount of seed oil that may be recovered from the plant. Methods of recovery of oil from a plant are known in the art and can be performed substantially as described in Focks and Benning, 1998. Methods of cultivating plants under conditions promoting plant growth and development are also known in the art.

[0060] Embodiments of the invention provide novel biotechnological approaches to improve yield-related traits in plants, in particular seed oil production, with beneficial impacts for biofuel or food-related products. Embodiments of the invention have many applications including, but not limited to, producing food, feed, or an industrial product comprising obtaining a plant or a part thereof, as herein described, including plants wherein the expression of at least one BPM protein is down-regulated or its activity reduced and preparing the food, feed or industrial product from the plant or part thereof. The food or feed may be oil, meal, grain, starch, flour or protein; or the industrial product may be biofuel, fiber, industrial chemicals, a pharmaceutical or a nutraceutical.

[0061] Before exemplary embodiments of the present invention are described in greater detail, it is to be understood that this invention is not limited to particular embodiments described, as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present invention will be limited only by the appended claims.

[0062] Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limit of that range and any other stated or intervening value in that stated range, is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges and are also encompassed within the invention, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the invention.

[0063] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present invention, representative illustrative methods and materials are now described.

[0064] All publications and patents cited in this specification are herein incorporated by reference as if each individual publication or patent were specifically and individually indicated to be incorporated by reference and are incorporated herein by reference to disclose and describe the methods and/or materials in connection with which the publications are cited. The citation of any publication is for its disclosure prior to the filing date and should not be construed as an admission that the present invention is not entitled to antedate such publication by virtue of prior invention. Further, the dates of publication provided may be different from the actual publication dates which may need to be independently confirmed.

[0065] It is noted that, as used herein and in the appended claims, the singular forms "a", "an", and "the" include plural referents unless the context clearly dictates otherwise. It is further noted that the claims may be drafted to exclude any optional element. As such, this statement is intended to serve as antecedent basis for use of such exclusive terminology as "solely," "only" and the like in connection with the recitation of claim elements, or use of a "negative" limitation.

[0066] As will be apparent to those of skill in the art upon reading this disclosure, each of the individual embodiments described and illustrated herein has discrete components and features which may be readily separated from or combined with the features of any of the other several embodiments without departing from the scope or spirit of the present invention. Any recited method can be carried out in the order of events recited or in any other order which is logically possible.

EXAMPLES

Example 1. Arabidopsis BPM Proteins Function as Substrate Adaptors to a CUL3-Based E3 Ligase Affecting Fatty Acid Metabolism in Plants

SUMMARY

[0067] Regulation of transcriptional processes is a critical mechanism that enables efficient coordination of the synthesis of required proteins in response to environmental and cellular changes. Transcription factors require accurate activity control because they play a critical role as key mediators assuring specific expression of target genes. In this example, it is shown that Cul3-based E3 ligases can interact with a broad range of ERF/AP2 transcription factors, mediated by MATH-BTB/POZ proteins. The assembly with an E3 ligase causes degradation of their substrates via the 26S proteasome, as demonstrated for the WRINKLED1 ERF/AP2 protein. Furthermore, loss of MATH-BTB/POZ proteins widely affects plant development and causes changed fatty acid contents in mutant seeds. Overall the work provides a novel link between fatty acid metabolism and E3 ligase activities in plants, and establishes Cul3-based E3 ligases as key regulators in transcriptional processes that involve ERF/AP2 family members.

Materials and Methods

Plant Materials and Growth Conditions

[0068] Arabidopsis thaliana wild-type ecotype Columbia plants and plants of the different genetic backgrounds were grown either on Arabidopsis thaliana (AT) medium without supplement of Suc (Estelle and Somerville, 1987) in a growth chamber at 22.degree. C. with 120 .mu.mol/m.sup.2/s light intensity or in soil in a greenhouse at 20.degree. C. under long-day conditions (16 hours light/8 hours dark).

[0069] Clone Constructions

[0070] The cDNAs of BPM1.sup.MATH, BPM1.sup.MATH:NLS, CUL3s, ERF/AP2s, and BPMs were cloned into pDONR221 (Invitrogen). For Y2H studies, the corresponding cDNAs were shuffled into destination vectors pACT2 (prey) and pBTM116-D9 (bait) by Gateway technology (Invitrogen) as described (Weber et al., 2005). pDEST15 (Invitrogen) and pET-58-DEST (Merck) were used to express and purify GST- and His-tagged proteins in Escherichia coli, respectively; where necessary, elution of GST proteins from glutathione-agarose beads was done following standard procedures. pMDC43 was used for expression in plants and for subcellular localization studies as described (Curtis and Grossniklaus, 2003). The NLS sequence was adopted from Howard et al. (1992), extended, and attached to the MATH domain in a four-step-based PCR process. To generate artificial microRNAs, a protocol from the WMD2 microRNA designer Web page; see FIG. 6) was followed using pRS300 as starting vector. For expression in plants, ami constructs were first cloned into pDONR221 before being shuffled into the binary vector pGBW14. For primers used see Table 1 below.

TABLE-US-00001 TABLE 1 Primers used for different PCR-based approaches. Cloning of constructs Name of Primer Sequence of Primer (5'-3') T7BPM1MATHEW (SEQ ID NO: 29) TAATACGACTCACTATAGGGAGAATGTTCAAGATCTGTGGGTA C BPM1MATHRW (SEQ ID NO: 30) CTACATTTCTAGACTGGACCTCCTG BPM1-MATH-RW-NLS (SEQ ID TCGTCCTCAGTGGACGCTTAGAGAGCACTTCTAGACTGGACCT NO: 31) CCTG NLS-1RW (SEQ ID NO: 32) ACGCTTACGCTCAGATGGCTCACCGTCGTCGTCCTCACGTGGA CGCTTAG NLS-RW2 (SEQ ID NO: 33) CACGACCGTCCTTAGAACGCTCGTCACGCTCACGCTTACGCTC AGATGGC Attb2stop-NLS (SEQ ID NO: 34) AGAAAGCTGGGTCACGACGGTTACCACCACGACCGTCC WRI1-FW (SEQ ID NO: 35) ATGAAGAAGCGCTTAACCAC WRI-RW (SEQ ID NO: 36) TCAGACCAAATAGTTACAAG qRT-PCR Name of Primer Sequence of Primer (5'-3') ACTIN2-qRT FW (SEQ ID NO: 37) CCTGCCATGTATGTTGCCATT ACTIN2-qRT RW (SEQ ID NO: 38) AATCGAGCACAATACCGGTTGT BPM1-qRT-FW (SEQ ID NO: 39) ATTGGCGTCTACTCTTGT BPM1-qRT-RW (SEQ ID NO: 40) AATGATGCTGCTCTGCTA BPM2-qRT-FW (SEQ ID NO: 41) TAATCGGCACAGACTTGA BPM2-qRT-RW (SEQ ID NO: 42) ACTCGCATATTGTTCTAAGC BPM3-qRT-FW (SEQ ID NO: 43) CACCAGTTCACGATTCAAG BPM3-qRT-RW (SEQ ID NO: 44) CCACCAACGGAGAAGATAT BPM4-qRT-FW (SEQ ID NO: 45) TCCTGATGGCAAGAATCC BPM4-qRT-RW (SEQ ID NO: 46) CGAAGTGGCTATGAACCT BPM5-qRT-FW (SEQ ID NO: 47) TTAGGCTCAGGTTGTTGT BPM5-qRT-RW (SEQ ID NO: 48) TCATCCTTCATCTGTTGGTA BPM6-qRT-FW (SEQ ID NO: 49) GCATAAGGTTCATAGCCATT BPM6-qRT-RW (SEQ ID NO: 50) AGATGTCTCAAGCAAGGA WRI1-qRT-FW (SEQ ID NO: 51) GAGCAACAAGAAGCAGAG WRI1-qRT-RW (SEQ ID NO: 52) CCACAACGATCCATTTCC BCCP1-qRT-FW (SEQ ID NO: 53) CAGCCAAATCGTCACT BCCP1-qRT-RW (SEQ ID NO: 54) GTTCCGGTATGGTCAG AtGLB1-qRT-FW (SEQ ID NO: 55) CTTTCACCGTCTTAGGAACAAACAG AtGLB1-qRT-RW (SEQ ID NO: 56) TAGGAACAGAGTTTCGATGTCTGAGAAC BPM1-MATH-qRTFW (SEQ ID CGGAGGATAACTCGTCTT NO: 57) BPM1-MATH-qRTRW (SEQ ID AATGGCTATGAACCTTATGC NO: 58) ChIP-qPCR Name of Primer Sequence of Primer (5'-3') EF1-qRT-FW (SEQ ID NO: 59) CTGGAGGTTTTGAGGCTGGTTA EF1-qRT-RW (SEQ ID NO: 60) CCAAGGGTGAAAGCAAGAAGA ProBCCP1-qRT-FW (SEQ ID NO: 61) AAGTGAACTGTTGTTGTT ProBCCP1-qRT-RW (SEQ ID NO: 62) CGTCTTCTTATTGTTATTGG Pro-GLB1-qRT-FW (SEQ ID NO: 63) TTCCAATAATTACCTCCTT Pro-GLB1-qRT-RW (SEQ ID NO: 64) TTTAACACAACTTTCAAAG

Subcellular Localization Studies

[0071] The fluorescent fusion proteins GFP:WRI1, GFP:BPM1.sup.MATH, and BPM1.sup.MATH:NLS were transiently expressed in Nicotiana benthamiana epidermal cells following the method described by Sparkes et al. (2006). GFP expression was detected and documented with a Zeiss LSM 510 Meta confocal microscope.

Interaction and Complex Assembly Studies

[0072] Y2H studies were followed as described by Weber et al. (2005). SDII selection medium supplemented with uracil and His was used as a transformation control, while for interaction studies, SDIV minimal medium was chosen without uracil and His supplements. Photos were taken from single spots 7 days after plating. FPLC was performed with 2 mg protein injections and a flow rate of 50 .mu.L/min using an AKTA FPLC system (GE Healthcare Science) and as described by Leuendorf et al. (2010). Pull-down analysis and IP studies were followed as described before (Hellmann et al., 2003; Bernhardt et al., 2006). Extraction and washing buffers contained at all times 1 mM PMSF (Sigma-Aldrich) and 10 .mu.M MG132 (Sigma-Aldrich) to prevent proteolytic and proteasomal activities, respectively. For pull-down analysis with GST- and His-tagged proteins, GST-containing proteins were first eluted from glutathione-agarose beads before incubated with His: WRI1 that remained attached to tetradentated-chelated nickel resin. In general, proteins were incubated at least 1.5 h at 4.degree. C. under shaking conditions before being centrifuged. Precipitates were washed no less than three times to remove unspecific bindings, before they were taken up in Laemmli buffer (Laemmli, 1970) and boiled (10 min, 95.degree. C.). IPs were followed as described by Bernhardt et al. (2006). In brief, 1 mg of fresh protein extracts from 2-week-old seedlings were precleaned with 30 .mu.L protein-A-agarose beads (Santa Cruz Biotechnology; 1.5 h, 4.degree. C.). The beads were centrifuged, and the supernatant was transferred into a fresh tube and incubated first with .alpha.-WRI1 (1.5 h, 4.degree. C.) before 30 .mu.L protein-A-agarose beads were added (1.5 h, 4.degree. C.). After brief centrifugation, four washing steps followed, after which precipitates were taken up in Laemmli buffer and boiled as described above. For pull-down and IP studies, precipitates were further analyzed by SDS-PAGE and protein gel blotting using standard procedures. Where applicable, membranes were stained with Ponceau S to detect transferred proteins. For immunodetection, custom-made (.alpha.-CUL3 [rabbit] and .alpha.-WRI1 [rabbit]; GeneScript) or commercially available antibodies (GST, secondary .alpha.-rabbit IgG-horseradish peroxidase; Santa Cruz) in combination with an ECL Plus Western Blotting Detection Kit (GE Healthcare Life Science) were used.

Stability Assays

[0073] For stability assays, Arabidopsis seedlings were cultured on solid AT medium for 2 weeks before being transferred to 5 mL AT liquid medium. Plants were incubated for 3 h with the transcriptional inhibitor ActD2 (Sigma-Aldrich; final concentration 10 .mu.g/mL) and/or the proteasomal inhibitor MG132 (Sigma-Aldrich; 20 .mu.M/mL, 6 h) before CHX (Sigma-Aldrich; 100 .mu.M/mL, 3 h) was added to inhibit translation. DMSO was used as mock control and as a dissolvent for all inhibitors. Protein gel blot analysis and protein detection were conducted using standard procedures. The antibodies against WRI1 and CUL3 were designed and produced by GeneScript and used in a 1:1000 dilution. Protein detection was followed as described in the ECL Plus Western Blotting Detection Kit manual (GE Healthcare Life Science).

RNA Isolation and Expression Analysis

[0074] Total Arabidopsis RNA was extracted following the protocol of the Isolate RNA kit from Bioline; reverse transcription was done according to the manual for the high-capacity cDNA reverse transcription kit (Applied Biosystems). qRT-PCR reactions (95.degree. C., 7 min; 95.degree. C., 15 s; 60.degree. C., 1 min; 40 cycles) were performed using the SYBR green method on a 7500 Fast Real-Time PCR system (Applied Biosystems). Relative gene expression analyses were calculated by the full quantification method with ACTIN2 as the internal control gene. Fourteen 2-week-old seedlings were pooled for each replicate. At least three biological replicates were performed for each individual experiment. Primers used for qRT-PCR are shown in Table 1 above.

ChIP Assays

[0075] For ChIP assays, an established protocol was followed (Morohashi et al., 2009). In brief, 60 mg (fresh weight) of 15-day-old seedlings were harvested for each ChIP experiment and cross-linked for 10 min under vacuum in cross-link buffer containing 1% formaldehyde as described by Morohashi et al. (2009). Cross-linked samples were incubated in 100 mM Gly for 5 min under a vacuum, thoroughly washed in double-distilled water, and snap frozen in liquid nitrogen. Frozen tissues were ground into fine powder and dissolved in nuclear isolation buffer (Morohashi et al., 2009) supplemented with 1.times. protease inhibitor cocktail (Sigma-Aldrich). After filtering through single-layered Miracloth (Merck), the samples were centrifuged (10 min, 1200 g, 4.degree. C.). Pellets were resuspended in nuclear isolation buffer supplemented with 0.3% Triton X-100 and centrifuged again (10 min, 10,000 g, 4.degree. C.). After resuspension in lysis buffer, the purified nuclei were then sonicated (three times, 20 s, 9 W; Fisher Scientific Model 100 dismembrator) to yield chromatin fragments of 300 to 500 bps. Sonicated chromatin fragments (2 mg) were first precleared with protein-A-agarose beads (Sigma-Aldrich) (1.5 h, 4.degree. C.) before being incubated with specific antibodies (1 mg/mL; 1.5 h, 4.degree. C.), followed by a fresh batch of protein-A-agarose beads (30 .mu.L; 1.5 h, 4.degree. C.) to IP protein-DNA complexes. After IP, cross-linking was reversed by incubating samples overnight at 65.degree. C. in elution buffer (1% SDS, 0.1 M NaHCO.sub.3, and 0.25 mg/mL proteinase K; Morohashi et al., 2009), after which RNaseA (1 mg/mL) was added (30 min; room temperature). As input control for data normalization, a portion of sonicated, cross-linked, and precleared DNA was treated accordingly except for undergoing an IP. Samples were further cleaned up using a DNA purification kit (NuCleoSpin Extraction II; Macherey-Nagel) and quantified to use equal amounts of template (50 ng/reaction) for qRT-PCR analysis. To amplify promoter sequences that contain an AW-box recognized by WRI1 (Maeo et al., 2009), specific primers were designed (Table 1). EF1 was selected as a reference gene for internal control. qRT-PCR reactions (95.degree. C., 10 min; 95.degree. C., 15 s; 60.degree. C., 1 min; 50 cycles) were done as described above and repeated with at least three independent biological replicates.

Metabolic Analysis

[0076] Metabolic profiling of 2-week-old seedlings grown on ATS plate (100 mg fresh weight) and seed samples (50 mg dry weight) of all backgrounds used in this study were analyzed according to earlier described protocols (Roessner-Tunali et al., 2003). Seed fatty acids were extracted exactly as described before (Focks and Benning, 1998). For quantification with gas chromatography, pentadecanoic acid was used as an internal standard (Browse et al., 1985).

Accession Numbers

[0077] Sequence data from this Example can be found in the GenBank/EMBL data libraries under the following accession numbers: ACTIN2, At3 g18780; IAA5, At1 g15580; BPM1, At5 g19000; BPM2, At3 g06190; BPM3, At2 g39760; BPM4, At3 g03740; BPM5, At5 g21010; BPM6, At3 g43700; DREB1a, At4 g25480; ERF1, At3 g23240; ERF4, At3 g15210; RAV1, At1 g13260; WRI1, At3 g54320; BCCP1, At5 g16390; and GLB1, At2 g16060.

Results

[0078] BPM Proteins Interact Broadly with ERF/AP2 Transcription Factors

[0079] We have earlier described that BPM proteins assemble with several, but not all members, of the A6 group of ERF/AP2 transcription factors (Weber and Hellmann, 2009). According to Sakuma and co-workers, the ERF/AP2 superfamily can be divided into five subgroups: AP-2, RAV, DREB, ERF, and others (Sakuma et al., 2002). The A6 group belongs to the ERF subfamily. To investigate how broadly BPM proteins assemble with ERF/AP2 transcription factors, we also tested in yeast-2-hybrid (Y2H) assays additional members outside the A6 group using BPM1 as prey (FIG. 2A). The ERF subfamily member ERF1 (At3 g23240) showed weak interaction, while the ERF-subfamily members WRI1 (At3 g54320) and ERF4 (At3 g15210), showed a strong interaction with BPM1 in the Y2H assay. WRI1 also tested positively for self-assembly in the yeast assay (FIG. 2B). Finally, DREB1a (At4 g25480), which belongs to the DREB subfamily, and also RAV1 (At1 g13260), a member of the RAV-subfamily, strongly interacted with BPM1 (FIG. 2A). Since BPMs interact also with CUL3 proteins, we tested interaction of the different ERF/AP2 transcription factors with CUL3a, and did not observe any in the yeast system (data not shown). We therefore concluded that a large number of ERF/AP2 transcription factors is recognized by BPM proteins in Arabidopsis.

CUL3 and BPM Proteins Assemble in Planta with WRI1

[0080] To identify and demonstrate basic principles of CRL3.sup.BPM complex assembly with substrates, we focused on a single well-described protein, WRI1, which is a key player in fatty acid and carbohydrate metabolism (Cernac and Benning, 2004; Baud et al., 2009).

[0081] In agreement with findings from the Y2H assays, pulldown experiments using a GST:WRI1 fusion protein can co-precipitate in vitro translated BPM1 from rabbit reticulolysates (FIG. 2C). For further investigation of in planta complex assembly, specific peptide-based antibodies were raised against CUL3 and WRI1 (FIG. 1). The antibody against CUL3 does not distinguish between CUL3a and b (FIG. 1 A,C); however transient expression experiments in tobacco clearly demonstrated specificity of the .alpha.-CUL3 antibody. We only observed a single band of around 85 kDa appearing on Western blots with wild type (WT) or cul3 mutant plant extracts (FIG. 1B). Likewise, only a single band was detectable when the .alpha.-WRI1 antibody was used on total plant extracts from WT plants, and which was missing in a wril-3 mutant when the .alpha.-WRI1 antibody was used on total plant extracts (FIG. 1E).

[0082] Pulldown experiments using GST:WRI1 against WT plant extract showed that the fusion protein precipitates both endogenous WRI1 and CUL3, however, this was not the case with GST alone (FIG. 2D). In addition, pulldown analysis with GST- and His-tag proteins expressed in and purified from E. coli demonstrated that BPM proteins are necessary to bridge assembly between WRI1 and CUL3 proteins. Here His:WRI1 is only capable of co-precipitating GST:CUL3a, if GST:BPM1 protein is present in the assay but not with GST alone (FIG. 2F). Also, immunoprecipitation (IP) studies using the .alpha.-WRI1 antibody successfully precipitated CUL3 from plant extracts in WT background (FIG. 2G). We also observed in FPLC studies co-migration of WRI1 with CUL3 (FIG. 3A), and detected GFP:WRI1 localized to the nucleus, as has been earlier shown for most BPM proteins and CUL3a (FIG. 3B; (Weber and Hellmann, 2009)). Overall these studies demonstrate that WRI1 assembles with CUL3 proteins in planta, and support the working hypothesis that the assembly is mediated by BPM proteins.

WRI1 is a CUL3-Dependent Target of the 26S Proteasome

[0083] One outcome of BPM assembly with CUL3 proteins is the proteolytic degradation of their substrates. Consequently, stability assays were performed using the translational inhibitor cycloheximide (CHX) and the proteasomal inhibitor MG132. In initial experiments, CHX treatments did not point to instability of the WRI1 protein (FIG. 4A), although accumulation of WRI1 was observed when plants were treated with MG132 (FIG. 4A).

[0084] To further characterize this phenomenon, WRI1 expression was tested in plants treated with CHX or with the proteasomal inhibitor. While plants incubated with MG132 did not show any change in WRI1 expression, CHX caused a strong up-regulation of the WRI1 gene (FIG. 4B). It was therefore decided to pre-treat plants with the transcriptional inhibitor actinomycin D2 (ActD2) before CHX was given. Under these conditions WRI1 protein was completely gone after 6 h treatment, and its disappearance was blocked by co-incubation with MG132 (FIG. 5A), demonstrating that WRI1 is unstable in a 26S proteasome-dependent manner. Notably, the accumulation of WRI1 protein in samples treated with all three inhibitors is most likely due to pre-treatment of plants with ActD2 and MG132 for three hours before CHX was supplemented.

[0085] A cul3.sup.hyp double mutant was previously described that is knocked-out for CUL3b and partially functional for CUL3a (Thomann et al., 2009). We took advantage of this mutant to investigate whether WRI1 is stabilized in this genetic background and to prove that the instability is mediated by a CUL3-based complex. Western-blot analysis on WT and cul3.sup.hyp plant extracts showed that WRI1 was present in the mutant in higher amounts than in WT (FIG. 5B). This was not based on increased transcriptional activities in the mutant since no significant difference in WRI1 expression was detectable in either plant (FIG. 5C). Stability assays with ActD2 and CHX showed no considerable change in protein content over six hour treatments in the mutant, while WRI1 was not detectable in WT extracts (FIG. 5D), revealing that WRI1 is instable in a 26S proteasome- and CUL3-dependent manner.

BPM Proteins Bridge the Assembly Between WRI1 and CUL3 and are Broadly Important for Development.

[0086] The results show that BPM proteins assemble with a broad range of ERF/AP2 transcription factors and that, if ERF/AP2 proteins are in complex with CUL3s, the BPMs likely function as their bridging substrate receptors. Since WRI1 is unstable, and because complex formation requires presence of a functional CUL3 protein in the plant, it was necessary to investigate whether loss of BPM proteins is also stabilizing WRI1.

[0087] Two strategies were followed to support the hypothesis that BPM proteins function as substrate receptors and are required for mediating WRI1 instability. First, because of a lack of T-DNA insertion mutants for nearly all BPM genes, a 35S artificial microRNA (amiRNA) construct was designed to down-regulate expression of all six members, based on predictions from the WMD 2--Web MicroRNA Designer; FIG. 6) (the lines are further denoted as 6.times.amiBPM). Second, the MATH domain from BPM1 was cloned under the control of a 35S promoter and behind a GFP reporter, and with (further denoted as BPM1.sup.MATH:NLS) or without (BPM1.sup.MATH) a nuclear localization signal attached to the end of the domain to affect subcellular localization. The MATH construct was generated to impose a competition in the plant where endogenous BPM proteins have reduced access to WRI1 and thus, hypothetically cause its stabilization.

[0088] Several independent plant lines were successfully generated, and two independent lines of the T4 generation were chosen for each construct for further analysis. In both 6.times.amiBPM lines a significant down-regulation in gene expression of all BPMs was measurable (FIG. 7A,B), while the BPM1.sup.MATH and BPM1.sup.MATH:NLS lines showed strong expression of the transgene (FIG. 7C). In addition, based on the GFP reporter, BPM1.sup.MATH constructs were detectable throughout the cell, including the nucleus (FIG. 8A), while BPM1.sup.MATH:NLS was exclusively present in the nucleus (FIG. 8B). Analysis of T4-generation plants showed that the 6.times.amiBPM lines consistently had higher WRI1 protein levels comparable to cul3.sup.hyp mutants, while surprisingly all MATH-overexpression lines had significantly less WRI1 protein (FIG. 7D). Although the absolute degree of WRI1 reduction in MATH-overexpression lines varied among tested plants, we never observed any levels that equaled or exceeded those in WT. Also of note is that this is not based on reduced WRI1 expression, since the gene is actually up-regulated in BPM1.sup.MATH and BPM1.sup.MATH:NLS lines (FIG. 8C).

[0089] The different plant lines were broadly affected in development. Primary root growth was significantly delayed in all six lines, and most strongly in the two 6.times.amiBPM lines (FIG. 9A). While lateral roots emerged at a lower frequency in the BPM1.sup.MATH lines, no significant changes were detectable in plants expressing the BPM1.sup.MATH:NLS construct (FIG. 9B). The 6.times.amiBPM plants developed very low numbers of lateral roots (FIG. 9B,C), and all transgenic lines were affected in shoot development. In addition all were late flowering, most strongly pronounced in BPM1.sup.MATH and 6.times.amiBPM lines (FIG. 9D,E), with less leaves present at the beginning of flowering (FIG. 4F), and a reduced rosette size (FIG. 9D,G). Besides being smaller and present in fewer numbers, the leaves of transgenic plants also had a tendency to develop wider blades then WT (FIG. 10).

[0090] To characterize the extent WRI1 protein stability is affected in the different lines, stability assays were performed on selected plants (FIG. 11 and FIG. 12). The assays consistently showed that in either the 6.times.amiBPM or MATH-overexpressing backgrounds, WRI1 was highly stable in comparison to WT (FIG. 11A; FIG. 12A, B).

[0091] IP experiments were carried out on two MATH-overexpressing and two 6.times.amiBPM lines. As shown in FIG. 11B, CUL3 protein was precipitated from WT plant extracts, while no precipitated CUL3 was detectable in either the MATH-overexpressing or the 6.times.amiBPM lines. These findings together with stability assays demonstrate that the BPM proteins are required (i) for assembly of WRI1 into a complex with CUL3, and (ii) for mediation of the transcription factor's degradation.

WRI1 Activity is Affected by CRL3.sup.BPM

[0092] We showed stabilization of WRI1 and an effect on its protein content in the plant in three different genetic backgrounds. In both cul3.sup.hyp double mutants and 6.times.amiBPM lines, WRI1 levels are increased, while in MATH-lines WRI1 amounts are decreased. In 6.times.amiBPM lines BPM expression is reduced, and in the cul3.sup.hyp and MATH-backgrounds BPM protein levels are likely unchanged. However, based on each genetic background, the assembly of WRI1 into a CUL3-based complex is differently affected by either reduced CUL3 availability and/or functionality (cul3.sup.hyp), reduced BPM content (6.times.amiBPM), or reduced accessibility of BPMs to WRI1 (MATH-overexpressing lines). To show how these situations differently affect WRI1 transcriptional activities, expression of two confirmed WRI1 targets, BCCP1 and AtGLB1, were tested (Baud et al., 2009; Maeo et al., 2009). BCCP1 (At5 g16390), which encodes for a biotin carboxyl carrier protein, and AtGLB1 (At4 g01900), which encodes for a PII protein, are both critical players in fatty acid biosynthesis, but also participate in carbon and nitrogen metabolism (Tissot et al., 1998; Chen et al., 2006).

[0093] qRT-PCR analysis showed loss of BCCP1 and AtGLB1 expression in the wril-3 null mutant compared to WT (FIG. 11C). Expression of both genes in MATH-overexpressing lines was similarly reduced. In contrast, both genes were strongly up-regulated in 6.times.amiBPM lines correlating with changes in WRI1 protein content. Interestingly, no change in BCCP1 and AtGLB1 expression in comparison to WT was noticeable in the cul3.sup.hyp line, despite the fact that WRI1 protein levels were elevated comparable to 6.times.amiBPM lines. These findings indicate, based on the presumed presence (cul3.sup.hyp) or absence (6.times.amiBPM), that BPM proteins also negatively affect transcriptional activity of their target proteins.

CUL3 assembles with WRI1 at the DNA level

[0094] To show whether CUL3 forms a complex with WRI1 at the DNA level we performed chromosomal immunoprecipitation experiments (ChIP) (Morohashi et al. 2009). In WT plants, .alpha.-WRI1 and .alpha.-CUL3 based ChIP experiments resulted in a two to three-fold enrichment of WRI1 binding sites (proBCCP1 and proAtGLB1), respectively (FIG. 11D), while no enrichment was detectable in the wril-3 null mutant, which served as a negative control (FIG. 11G). Interestingly, .alpha.-WRI1 ChIP in the two 6.times.amiBPM lines yielded higher levels of proAtGLB1 and proBCCP1 sites which was in agreement with higher WRI1 protein levels in these plants (FIG. 11E, F), as well as increased transcription of the corresponding genes (FIG. 11C). Finally, ChIP using the .alpha.-CUL3 antibody in 6.times.amiBPM lines did not lead to any enrichment of proAtGLB1 and proBCCP1 sites (FIG. 11E, F), which corroborates the finding that loss of BPMs disrupt the ability of CUL3 to assemble into a complex with WRI1 (FIG. 11B). Overall these results show that CUL3 proteins form a complex with WRI1 at the DNA level, and that this assembly requires BPM proteins.

Reduced BPM Content Affects Fatty Acid Metabolism in Seeds

[0095] The finding that a reduced BPM expression in 6.times.amiBPM lines increases both WRI1 protein content and expression of WRI1 target genes was intriguing as it opened up the possibility that seeds of 6.times.amiBPM lines may also contain elevated levels of fatty acids due to augmented levels of active WRI1 (Baud et al., 2009). In agreement with this idea, both 6.times.amiBPM lines showed significant increases in seed weights and size when compared to WT seeds (FIG. 13A,B). However, changes were much more pronounced in 6.times.amiBPM #1 than in 6.times.amiBPM #2 plants, which may be due to different activities of the 35S promoter in seeds of the two lines. They also showed increased WRI1 content in seeds and elevated expression of the two target genes BCCP1 and AtGLB1 (FIG. 13C). Similar to increases in weight, both lines also showed altered total fatty acid contents (FIG. 13D); and while changes in 6.times.amiBPM #2 plants were only very mild and furthermore non-significant (.about.96 .mu.g/30 seeds in average versus .about.93 .mu.g/30 seeds in WT), the total fatty acid content in 6.times.amiBPM #1 seeds was increased by around 50% (.about.140 .mu.g/30 seeds) when compared to WT. The wril-3 line was used in these experiments as a control, and showed a significant reduction in both seed weight and total fatty acid contents (.about.79 .mu.g/30 seeds) when compared to WT and the two 6.times.amiBPM lines. While changes were observable for total fatty acid content measurements of individual fatty acids did not detect any significant changes (FIG. 14). In addition, no significant changes were observed in a general metabolic profile (amino and organic acids as well as soluble sugar) for wril-3 or the two amiBPM lines when compared to WT (FIG. 14), indicating that the changes in seed size and weight for the mutants are primarily based on aberrant fatty acid contents. Overall these data further underscore that BPM proteins are critical regulators of WRI1 activity, and that their loss positively affects both WRI1 stability as well as its actions.

[0096] Because seeds of the two 6.times.amiBPM lines varied significantly in their weight and fatty acid content, we included a third 6.times.amiBPM line in our analysis to ensure that loss of BPMs is leading in a reproducible manner to increases in fatty acid content and seed size. As observed for the other two lines, 6.times.amiBPM #3 seeds are also increased in size (FIG. 15B) as well as in dry weight (FIG. 15C), and this correlated with elevated WRI1 content (FIG. 15A), as well as up-regulated expression of BCCP1 and AtGLB1 (FIG. 15D). Likewise, we also observed a significant increase in fatty acid content in these seeds in comparison to WT (FIG. 15E), substantiating findings for 6.times.amiBPM #1 plants that reduced BPM activity likely result in higher fatty acid levels in seeds.

Discussion

[0097] This example shows that BPM proteins have the ability to interact with a broad-range of ERF/AP2 proteins. Y2H studies indicate that many ERF/AP2 proteins are targeted in Arabidopsis by a CRL3.sup.BPM complex. IP and pull down studies in this work underscore that WRI1 assembles in vitro and in the plant into a complex with CUL3, and the missing CUL3-WRI1 assembly in 6.times.amiBPM and MATH-overexpressing backgrounds emphasizes that BPM proteins are required for this step. The studies further show that the interaction of BPMs with WRI1 results in the destabilization of their substrate. This is supported by the finding that WRI1 is stabilized in a cul3.sup.hyp background, as well as in MATH overexpression and 6.times.amiBPM plants. Moreover, the ChIP data strongly supports our conclusion that WRI1-CRL3.sup.BPM assembly occurs at the DNA level. Consequently, BPM proteins can be considered as negative regulators of WRI1 activities by mediating assembly with the CRL3 core, and ultimately causing its degradation via the 26S proteasome. This is also supported by the finding that fatty acid levels are significantly increased in seeds of 6.times.amiBPM #1 and #3 plants. These changes resemble earlier descriptions for plants overexpressing WRI1 (Cernac and Benning. 2004). However, it is significant to note that similar changes can be accomplished in Arabidopsis without ectopically expressing a transgenic WRI1 in seeds. The fact that overall metabolic changes were mostly restricted to total fatty acid contents also indicates that the function of BPM proteins in seeds is strongly connected with WRI1 activity. In this context, it is of note that WRI1 has very recently been described as part of a small gene family with a total of four members in Arabidopsis (To et al. 2012). Although WRI1 is the primary member that controls fatty acid biosynthesis in seeds, the other members also contribute to this pathway but in other tissues (To et al. 2012). The current findings also support the earlier suggestion that instability of RAP2.4, another BPM interacting protein, is mediated by a CRL3.sup.BPM ligase (Weber and Hellmann, 2009). Overall, it is likely that a general consequence of BPM interaction with ERF/AP2 transcription factors is degradation of the latter.

[0098] In this context, it is also important to note that the BPM family members have very recently been established as regulators of an ABA response by targeting the Homeodomain-Leucine Zipper transcription factor AtHB6 for degradation (Lechner et al., 2011). Consequently, plants with reduced levels of BPM1, 4, 5, and 6 (amiR-bpm) display aberrant responses in stomatal opening (Lechner et al., 2011); however, germinating amiR-bpm seedlings only display increased ABA resistance when combined with an AtHB6 overexpression background. The finding that a member of another transcription factor family is a substrate of BPM proteins, further increases the number of potential substrate proteins that are targeted by CRL3.sup.BPM for degradation. Furthermore, many members of the ERF/AP2 family have been described in context with stress tolerance including the ones tested in this study. For example DREB1a is a classical regulator of drought and cold tolerance responses in plants (Sakuma et al., 2002; Miura et al., 2007). ERF1 is known to play a role in biotic stress (Lorenzo et al., 2003; Zhang et al., 2011), and RAV1 has been described in context with senescence and different abiotic stress conditions (Sohn et al., 2006; Woo et al., 2010; Yun et al., 2010). It is therefore likely that both MATH overexpression and 6.times.amiBPM plants display different sensitivities towards stress such as cold or drought, and treatments with phytohormones such as ethylene or jasmonic acids, in addition to ABA.

[0099] It is also noteworthy that degradation of WRI1 appears to occur continuously rather than being stimulated by a specific signal, and this also holds true for RAP2.4 and AtHB6 (Weber and Hellmann, 2009; Lechner et al., 2011). It is unlikely that the cell is degrading these proteins always to the same amount since this seems to be a quite inefficient and uneconomical approach to control protein amounts. Rather one would expect that specific signals are in place that slow down turnover of CRL3.sup.BPM substrates similar to ethylene signal transduction, where ethylene disrupts proteasomal degradation of EIN3 mediated by the F-box proteins EBF1 and EBF2 (Guo and Ecker, 2003; Potuschak et al. 2003). In fact, ABA treatment has a stabilizing impact on AtHB6, but the kind of signal that may have a similar impact on WRI1 or RAP2.4 remains unclear.

[0100] The wide-ranging developmental changes in both 6.times.amiBPM, as well as MATH overexpression lines, emphasizes that the BPM family is widely required for plant development. amiR-BPM plants showed a reduced shoot growth (Lechner et al., 2011), which we also observed for 6.times.amiBPM lines. Interestingly, we could not detect any problems in fertility as observed by Lechner and co-workers for amiR-bpm plants. In addition, 6.times.amiBPM plants had a strongly reduced root development and fewer leaves, and it remains open whether these changes were also seen by Lechner et al (2011). Moreover, changes in root development were not apparent in MATH overexpression lines, indicating that reduced BPM expression and binding competition approaches differently affected the developmental program of the root in the corresponding plants.

[0101] The different approaches followed in this work to affect CRL3.sup.BPM-WRI1 interplay revealed two additional interesting aspects about the function of BPM proteins besides being substrate receptors to a CRL3.sup.BPM ligase. First, comparing cul3.sup.hyp and 6.times.amiBPM plants clearly demonstrated that in both genetic backgrounds WRI1 protein content is increased due to greater stability of the transcription factor. However, the transcriptional activity of WRI1 was only elevated in 6.times.amiBPM plants but not in cul3.sup.hyp, as indicated by the changed versus unchanged transcriptional levels of AtGLB1 and BCCP1. Given that in the cul3.sup.hyp mutant BPM protein levels are likely normal, these findings indicate that BPM proteins negatively interfere with WRI1 activity, most likely by binding to the transcription factor, while more active WRI1 is available in 6.times.amiBPM plants. Secondly, the reduced WRI1 amount was quite surprising and unexpected. Because WRI1 expression was up-regulated in MATH overexpressors, one may suggest that the reduced WRI1 content was sensed by the cell, and that changes on the transcriptional level represent a feedback-loop response. In addition, these data also clearly indicate that the MATH domain also interfered with post-transcriptional processes, and thus point out that BPM proteins may have even further diverse roles in addition to targeting ERF/AP2 or AtHB6 transcription factors for ubiquitylation and proteasomal degradation.

[0102] Finally, the ChIP data strongly indicate that CRL3.sup.BPM E3 ligase assembles with WRI1 at the DNA level while the transcription factor is bound to its target sites. In summary, the current work reveals a new link between fatty acid metabolism and CUL3-based E3 ligase activities. The work also confirms that BPM proteins function in planta as substrate receptor proteins to a CRL3.sup.BPM ligase with the purpose to destabilize bound substrates. These findings further indicate that a large number of ERF/AP2 proteins are targets of BPM proteins, and that this complex plays a major role in plant development and stress tolerance by broadly regulating transcriptional, and potentially post-transcriptional, processes in the plant.

Example 2. Modulation of BPM Expression or Activity Enhances Several Yield-Related Traits

[0103] The transgenic plants (6.times.amiBPM and BPM.sup.MATH:NLS) as described in Example 1 were tested under varying conditions to assess the effects on several yield-related traits. For salt stress tolerance assays, wild type (WT) and transgenic plants (6.times.amiBPM and BPM.sup.ATH:NLS) were plated on solid minimal culture medium, and grown vertically for five days. Afterwards, they were carefully transferred to plates that were supplemented with 150 mM NaCl. The transgenic plants had significantly increased root growth from day 3 to 6 after the addition of salt as compared to the WT plants (FIG. 18A). Wild type root elongation growth at day six was significantly more inhibited under salt stress conditions than in transgenic plants (FIG. 18B).

[0104] The 6.times.amiBPM plants were then tested under drought conditions. After withholding water for four days, significant changes were observed between WT and 6.times.amiBPM plants which indicate increased sensitivity of the transgenic plant towards drought stress (FIG. 19).

[0105] The 6.times.amiBPM plants were also found to affect the flowering phenotype as compared to WT plants. Expression of Flowering Locus T (FT), a key regulator of the flowering time point, is significantly down regulated in 6.times.amiBPMplants when compared to WT which is in agreement with the late flowering phenotype of the transgenic plants (FIG. 20A). FIG. 20B shows a schematic drawing of six different FT promoter regions analyzed via qPCR after .alpha.-CUL3 ChIP experiment. Significant enrichments were detectable in regions 1, 5 and 6 in WT, but not in a 6.times.amiBPM#1 control, indicating that CRL3.sup.BPM E3 ligases are directly involved in controlling FT expression (FIG. 20C).

[0106] Inducible 6.times.amiBPM constructs were generated and shown to allow for controlled increase in seed size (FIG. 21). Treatment of plants with estradiol over a time period of 24 hours leads to a significant down-regulation of all six BPM genes (FIG. 21A). pMDC7: 6.times.amiBPM plants that carry an estradiol inducible construct are indistinguishable from wild type plants when not treated with estradiol (FIG. 21B). When the transgenic plants were sprayed with estradiol for about 2 weeks, the seeds in estradiol-treated 6.times.amiBPMplants were significantly larger (FIGS. 21C and E), and heavier (FIG. 21D) than WT seeds.

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[0150] While the invention has been described in terms of its preferred embodiments, those skilled in the art will recognize that the invention can be practiced with modification within the spirit and scope of the appended claims. Accordingly, the present invention should not be limited to the embodiments as described above, but should further include all modifications and equivalents thereof within the spirit and scope of the description provided herein.

Sequence CWU 1

1

941408PRTArabidopsis thaliana 1Met Ser Thr Val Gly Gly Ile Glu Gln Leu Ile Pro Asp Ser Val Ser 1 5 10 15 Thr Ser Phe Ile Glu Thr Val Asn Gly Ser His Gln Phe Thr Ile Gln 20 25 30 Gly Tyr Ser Leu Ala Lys Gly Met Ser Pro Gly Lys Phe Ile Gln Ser 35 40 45 Asp Ile Phe Ser Val Gly Gly Tyr Asp Trp Ala Ile Tyr Phe Tyr Pro 50 55 60 Asp Gly Lys Asn Pro Glu Asp Gln Ser Ser Tyr Ile Ser Leu Phe Ile 65 70 75 80 Ala Leu Ala Ser Asp Ser Asn Asp Ile Arg Ala Leu Phe Glu Leu Thr 85 90 95 Leu Met Asp Gln Ser Gly Lys Gly Lys His Lys Val His Ser His Phe 100 105 110 Asp Arg Ala Leu Glu Gly Gly Pro Tyr Thr Leu Lys Tyr Lys Gly Ser 115 120 125 Met Trp Gly Tyr Lys Arg Phe Phe Lys Arg Ser Ala Leu Glu Thr Ser 130 135 140 Asp Tyr Leu Lys Asp Asp Cys Leu Val Ile Asn Cys Thr Val Gly Val 145 150 155 160 Val Arg Ala Arg Leu Glu Gly Pro Lys Gln Tyr Gly Ile Val Leu Pro 165 170 175 Leu Ser Asn Met Gly Gln Gly Leu Lys Asp Leu Leu Asp Ser Glu Val 180 185 190 Gly Cys Asp Ile Ala Phe Gln Val Gly Asp Glu Thr Tyr Lys Ala His 195 200 205 Lys Leu Ile Leu Ala Ala Arg Ser Pro Val Phe Arg Ala Gln Phe Phe 210 215 220 Gly Pro Ile Gly Asn Asn Asn Val Asp Arg Ile Val Ile Asp Asp Ile 225 230 235 240 Glu Pro Ser Ile Phe Lys Ala Met Leu Ser Phe Ile Tyr Thr Asp Val 245 250 255 Leu Pro Asn Val His Glu Ile Thr Gly Ser Thr Ser Ala Ser Ser Phe 260 265 270 Thr Asn Met Ile Gln His Leu Leu Ala Ala Ala Asp Leu Tyr Asp Leu 275 280 285 Ala Arg Leu Lys Ile Leu Cys Glu Val Leu Leu Cys Glu Lys Leu Asp 290 295 300 Val Asp Asn Val Ala Thr Thr Leu Ala Leu Ala Glu Gln His Gln Phe 305 310 315 320 Leu Gln Leu Lys Ala Phe Cys Leu Glu Phe Val Ala Ser Pro Ala Asn 325 330 335 Leu Gly Ala Val Met Lys Ser Glu Gly Phe Lys His Leu Lys Gln Ser 340 345 350 Cys Pro Thr Leu Leu Ser Glu Leu Leu Asn Thr Val Ala Ala Ala Asp 355 360 365 Lys Ser Ser Thr Ser Gly Gln Ser Asn Lys Lys Arg Ser Ala Ser Ser 370 375 380 Val Leu Gly Cys Asp Thr Thr Asn Val Arg Gln Leu Arg Arg Arg Thr 385 390 395 400 Arg Lys Glu Val Arg Ala Val Ser 405 2399PRTBrassica rapa 2Met Ser Ala Ser His Pro Asn His Asp Ser Val Ser Thr Thr Val Met 1 5 10 15 Glu Thr Val Asn Gly Ser His Gln Phe Thr Ile Lys Gly Tyr Ser Leu 20 25 30 Ala Lys Gly Met Ser Pro Gly Arg Tyr Ile Gln Ser Asp Val Phe Ser 35 40 45 Val Asn Gly Tyr Asp Trp Val Ile Tyr Phe Tyr Pro Asp Gly Lys Asn 50 55 60 Pro Glu Glu Asn Ser Thr Tyr Val Ser Leu Phe Ile Ala Leu Ala Ser 65 70 75 80 Asp Ser Ser Asp Ile Arg Ala Leu Phe Glu Leu Thr Leu Met Asp Gln 85 90 95 Ser Gly Arg Gly Arg His Lys Val His Ser His Phe Asp Arg Ala Leu 100 105 110 Glu Gly Gly Pro Tyr Thr Leu Lys Tyr Lys Gly Ser Met Trp Gly Tyr 115 120 125 Lys Arg Phe Leu Arg Arg Thr Ala Leu Glu Ala Ser Asp Tyr Leu Lys 130 135 140 Asp Asp Cys Leu Ile Ile Asn Cys Thr Val Gly Val Val Arg Ala Arg 145 150 155 160 Leu Glu Gly Pro Lys Gln Phe Gly Ile Val Pro Pro Pro Ser Asn Met 165 170 175 Gly Gln Gly Leu Lys Asp Leu Leu Asp Ser Glu Leu Gly Cys Asp Ile 180 185 190 Ala Phe Gln Val Gly Asp Glu Thr Tyr Lys Ala His Lys Leu Ile Leu 195 200 205 Ala Ala Arg Ser Pro Val Phe Arg Ala Gln Phe Tyr Gly Pro Val Gly 210 215 220 Asn Asn Ser Val Asp Arg Val Val Ile Glu Asp Met Glu Pro Ser Ile 225 230 235 240 Phe Lys Ala Met Leu Ser Phe Ile Tyr Thr Asp Val Leu Pro Asp Val 245 250 255 His Glu Ile Thr Gly Ser Thr Ser Thr Ala Ser Phe Thr Asn Met Ile 260 265 270 Gln His Leu Leu Ala Ala Ala Asp Leu Tyr Asp Leu Gly Arg Leu Lys 275 280 285 Ile Leu Cys Glu Ala Phe Leu Cys Glu Glu Leu Asn Val Asp Asn Val 290 295 300 Ala Thr Thr Leu Ala Leu Ala Asp Gln His Gln Phe Leu Gln Leu Lys 305 310 315 320 Ala Phe Cys Leu Lys Phe Val Ala Ser Pro Ala Asn Leu Arg Ala Val 325 330 335 Met Lys Ser Glu Gly Phe Lys His Leu Asn Gln Ser Cys Pro Ser Val 340 345 350 Leu Pro Glu Leu Leu Asn Thr Val Ala Ala Ala Asp Lys Ser Ser Thr 355 360 365 Ser Ser Ser Gly Gln Ser Ser Lys Lys Arg Ser Val Ser Ser Val Leu 370 375 380 Gly Cys Asp Thr Ser Thr Thr Asn Ala Arg Gln Val Arg Arg Thr 385 390 395 3407PRTJatropha curcas 3Met Val Asp Val Lys Ala Asp Phe Asp Lys Glu Ser Cys Ser Lys Ser 1 5 10 15 Val Asn Glu Thr Val Asn Gly Ser His Gln Phe Thr Ile Lys Gly Tyr 20 25 30 Ser Leu Ala Lys Gly Met Gly Ala Gly Lys Cys Ile Ser Ser Asp Ile 35 40 45 Phe Thr Val Gly Gly Tyr Asp Trp Ala Ile Tyr Phe Tyr Pro Asp Gly 50 55 60 Lys Asn Pro Glu Asp Ser Ser Met Tyr Val Ser Val Phe Ile Ala Leu 65 70 75 80 Ala Ser Glu Gly Thr Asp Val Arg Ala Leu Phe Glu Leu Thr Leu Val 85 90 95 Asp Gln Ser Gly Asn Gly Lys His Lys Val His Ser His Phe Asp Arg 100 105 110 Ala Leu Glu Ser Gly Pro Tyr Thr Leu Lys Tyr Arg Gly Ser Met Trp 115 120 125 Gly Tyr Lys Arg Phe Phe Arg Arg Thr Thr Leu Glu Asn Ser Asp Tyr 130 135 140 Ile Lys Asp Asp Cys Leu Leu Met Asn Cys Thr Val Gly Val Val Arg 145 150 155 160 Thr Arg Leu Val Gly Pro Lys Gln Cys Phe Ile Thr Ile Pro Pro Ser 165 170 175 Asp Met Gly Gln Gly Leu Lys Glu Leu Leu Glu Ser Glu Val Gly Cys 180 185 190 Asp Ile Ala Phe Gln Val Gly Asp Glu Thr Phe Lys Ala His Lys Leu 195 200 205 Ile Leu Ala Ala Arg Ser Pro Val Phe Arg Ala Gln Phe Phe Gly Leu 210 215 220 Phe Gly Asp Pro Asn Leu Asp Lys Val Val Val Lys Asp Ile Asp Pro 225 230 235 240 Ser Ile Phe Lys Ala Met Leu Leu Phe Val Tyr Thr Asp Lys Leu Pro 245 250 255 Asp Val His Glu Ile Thr Gly Thr Thr Ser Met Cys Thr Ser Thr Asn 260 265 270 Met Val Gln His Leu Leu Ala Ala Ala Asp Leu Tyr Asn Leu Asp Arg 275 280 285 Leu Lys Leu Leu Cys Glu Ser Lys Leu Cys Glu Glu Leu Ser Ala Glu 290 295 300 Thr Val Ala Thr Thr Leu Ala Leu Ala Glu Gln His Gln Cys Ser Gln 305 310 315 320 Leu Arg Ala Ile Cys Leu Lys Phe Ala Ala Thr Pro Ala Asn Leu Gly 325 330 335 Ala Val Met Gln Ser Glu Gly Phe Arg His Leu Glu Glu Ser Cys Pro 340 345 350 Ala Leu Leu Cys Glu Met Leu Lys Thr Phe Ala Leu Gly Asp Glu Asn 355 360 365 Ser Asn Gln Ser Gly Arg Lys Arg Ser Gly Ser Ser Ile Tyr Gly Leu 370 375 380 Asp Leu Ala Thr Asp Gly Ala Ala Ala Glu Ser Val Asn Pro Asn Ala 385 390 395 400 Arg Arg Leu Arg Arg Arg Tyr 405 4407PRTPopulus trichocarpa 4Met Asp Asp Phe Lys Gly Asp Val Asp Lys Glu Ser Cys Ser Lys Ser 1 5 10 15 Ile Asn Glu Thr Val Asn Gly Ser His Gln Phe Thr Ile Lys Gly Tyr 20 25 30 Ser Leu Ala Lys Gly Met Gly Ala Gly Arg Cys Ile Pro Ser Asp Val 35 40 45 Phe Asn Val Gly Gly Tyr Asp Trp Ala Ile Tyr Phe Tyr Pro Asp Gly 50 55 60 Lys Asn Pro Glu Asp Ser Ser Met Tyr Val Ser Val Phe Ile Ala Leu 65 70 75 80 Ala Ser Glu Gly Thr Asp Val Arg Ala Leu Phe Glu Leu Thr Leu Val 85 90 95 Asp Gln Ser Gly Lys Gly Lys His Lys Val His Ser His Phe Asp Arg 100 105 110 Ala Leu Glu Ser Gly Pro Tyr Ser Leu Lys Tyr Arg Gly Ser Met Trp 115 120 125 Gly Tyr Lys Arg Phe Phe Arg Arg Thr Thr Leu Glu Thr Ser Asp Tyr 130 135 140 Leu Lys Asp Asp Cys Leu Ile Met Asn Cys Thr Val Gly Val Val Arg 145 150 155 160 Thr Arg Leu Glu Gly Pro Lys Gln Tyr Ser Ile Ser Val Pro Pro Ser 165 170 175 Asp Met Gly Trp Gly Phe Lys Glu Leu Leu Glu Ser Glu Ser Gly Cys 180 185 190 Asp Ile Asp Phe Gln Val Gly Asp Glu Thr Phe Arg Ala His Lys Leu 195 200 205 Ile Leu Ala Ala Arg Ser Pro Val Phe Arg Ala Gln Phe Phe Gly Leu 210 215 220 Val Gly Asp Pro Asn Met Asp Lys Val Val Val Lys Asp Val Asp Pro 225 230 235 240 Leu Ile Phe Lys Ala Met Leu Leu Phe Ile Tyr Thr Asp Lys Leu Pro 245 250 255 Asp Ala His Glu Ile Thr Gly Ser Thr Ser Met Cys Thr Ser Thr Asn 260 265 270 Met Val Gln His Leu Leu Ala Val Ser Asp Leu Tyr Asn Leu Asp Arg 275 280 285 Leu Lys Leu Leu Cys Glu Ala Lys Leu Cys Glu Glu Leu Ser Ala Glu 290 295 300 Asn Val Ala Thr Thr Leu Ala Leu Ala Glu Gln His Gln Cys Met Gln 305 310 315 320 Leu Lys Ala Ile Cys Leu Lys Phe Ala Ala Asn Pro Ala Asn Leu Gly 325 330 335 Ala Val Met Gln Ser Glu Gly Phe Arg His Leu Glu Glu Ser Cys Pro 340 345 350 Ser Met Leu Cys Glu Leu Leu Lys Thr Leu Ala Ser Gly Asp Glu Asn 355 360 365 Ser Ser Leu Leu Ser Gly Arg Lys Arg Ser Gly Ser Ser Leu Leu Gly 370 375 380 Val Asp Leu Ala Asp Gly Ala Pro Ala Glu Ser Ala Asn Pro Asn Gly 385 390 395 400 Arg Arg Leu Arg Arg Arg Phe 405 5402PRTTheobroma cacao 5Met Asp Asp Phe Lys Asp Ser Val Ser Lys Ser Val Ser Glu Thr Val 1 5 10 15 Asn Gly Ser His Gln Phe Thr Ile Lys Gly Tyr Ser Leu Ala Lys Gly 20 25 30 Met Gly Pro Gly Lys Cys Ile Ala Ser Asp Val Phe Thr Val Gly Gly 35 40 45 Phe Asp Trp Val Ile Tyr Phe Tyr Pro Asp Gly Lys Asn Pro Glu Asp 50 55 60 Ser Ala Met Tyr Val Ser Val Phe Ile Ala Leu Ala Ser Glu Gly Thr 65 70 75 80 Asp Val Arg Ala Leu Phe Glu Leu Thr Leu Val Asp Gln Ser Gly Lys 85 90 95 Gly Lys His Lys Val His Ser His Phe Asp Arg Ala Leu Glu Ser Gly 100 105 110 Pro Tyr Thr Leu Lys Tyr Arg Gly Ser Met Trp Gly Tyr Lys Arg Phe 115 120 125 Phe Arg Arg Thr Thr Leu Glu Thr Ser Asp Tyr Ile Lys Asp Asp Cys 130 135 140 Leu Ile Met Asn Cys Thr Val Gly Val Val Arg Thr Arg Leu Glu Gly 145 150 155 160 Pro Lys Gln Cys Ser Ile Ser Val Pro Pro Ser Glu Met Gly Gln Asn 165 170 175 Leu Lys Ala Leu Leu Glu Ser Glu Val Gly Cys Asp Ile Ile Phe Gln 180 185 190 Val Val Asp Glu Lys Phe Lys Ala His Lys Leu Ile Leu Ala Ala Arg 195 200 205 Ser Pro Val Phe Arg Ala Gln Phe Phe Gly Leu Val Gly Asp Pro Asn 210 215 220 Met Asp Lys Val Val Val Glu Asp Phe Glu Pro Ser Ile Phe Lys Ala 225 230 235 240 Met Leu Leu Phe Ile Tyr Thr Asp Lys Leu Pro Asp Val Gln Glu Ile 245 250 255 Thr Gly Ser Thr Ser Met Cys Met Ser Thr Asn Met Val Gln His Leu 260 265 270 Leu Ala Ala Ala Asp Leu Tyr Asn Leu Asp Arg Leu Lys Val Leu Cys 275 280 285 Glu Ala Lys Leu Cys Glu Glu Leu Asn Ala Asp Thr Val Ala Thr Thr 290 295 300 Leu Ala Leu Ala Glu Gln His His Cys Ala Gln Leu Lys Ala Ile Cys 305 310 315 320 Leu Lys Phe Ala Ala Thr Pro Ala Asn Leu Gly Ala Val Met Gln Ser 325 330 335 Glu Gly Phe Arg His Leu Glu Glu Cys Cys Pro Ser Leu Leu Ser Glu 340 345 350 Leu Leu Lys Thr Phe Ala Ser Gly Glu Glu Ser Leu Ser Gln Leu Ser 355 360 365 Ser Arg Lys Arg Ser Gly Ser Ser Val Tyr Gly Met Asp Leu Ala Ala 370 375 380 Glu Gly Pro Val Ala Glu Ser Val Asn Pro Asn Gly Arg Arg Val Arg 385 390 395 400 Arg Arg 6404PRTCitrus clementina 6Met Gly Asn Ser Glu Lys Asp Ser Thr Ser Lys Ser Ile Asn Glu Thr 1 5 10 15 Val Asn Gly Ser His Gln Phe Thr Val Lys Gly Tyr Ser Leu Ala Lys 20 25 30 Gly Met Gly Pro Gly Lys Cys Leu Ser Ser Asp Val Phe Thr Val Gly 35 40 45 Gly Tyr Asp Trp Ala Ile Tyr Phe Tyr Pro Asp Gly Lys Asn Pro Glu 50 55 60 Asp Gly Ala Leu Tyr Val Ser Val Phe Ile Ala Leu Ala Ser Glu Gly 65 70 75 80 Thr Asp Val Arg Ala Leu Phe Glu Leu Thr Leu Val Asp Gln Ser Gly 85 90 95 Lys Gly Lys His Lys Val His Ser His Phe Asp Arg Ala Leu Glu Ser 100 105 110 Gly Pro Tyr Thr Leu Lys Tyr Arg Gly Ser Met Trp Gly Tyr Lys Arg 115 120 125 Phe Phe Lys Arg Thr Ser Leu Glu Thr Ser Asp Tyr Ile Lys Asp Asp 130 135 140 Cys Leu Leu Ile Asn Cys Thr Val Gly Val Val Arg Asn Arg Leu Glu 145 150 155 160 Gly Pro Lys Gln Tyr Ser Ile Pro Val Pro Pro Ser Asp Met Gly Gln 165 170 175 Gly Leu Lys Asp Leu Leu Glu Ser Glu Ile Gly Cys Asp Ile Val Phe 180 185 190 Glu Val Gly Asp Glu Thr Phe Lys Ala His Lys Leu Ile Leu Ala Ala 195 200 205 Arg Ser Pro Val Phe Arg Ala Gln Phe Tyr Gly Leu Val Gly Asp Arg 210 215 220 Asn Leu Asp Lys Val Val Val Lys Asp Val Glu Pro Ser Ile Phe Lys 225 230 235 240 Ala Met Leu Leu Phe Ile Tyr Thr Asp Lys Phe Pro Asp Val Tyr Glu 245 250 255 Ile Thr Gly Thr Thr Ser Met Cys Thr Thr Thr Asn Met Val Gln His 260

265 270 Leu Leu Ala Ala Ala Asp Leu Tyr Asn Val Asp Arg Leu Lys Leu Leu 275 280 285 Cys Glu Ser Lys Leu Cys Glu Glu Leu Asn Ala Glu Thr Val Ala Thr 290 295 300 Thr Leu Ala Leu Ala Glu Gln His Gln Cys Pro Gln Leu Lys Ala Ile 305 310 315 320 Cys Leu Lys Phe Ala Ala Thr Pro Ala Asn Leu Gly Val Ile Met Gln 325 330 335 Ser Glu Gly Phe Lys His Leu Glu Glu Ser Cys Pro Ser Leu Leu Ser 340 345 350 Glu Leu Leu Lys Thr Leu Ala Ser Gly Asp Asp Thr Ser Ser Leu Ser 355 360 365 Ser Asn Arg Lys Arg Ser Gly Ser Ser Ile Tyr Ala Leu Asp Leu Ala 370 375 380 Gly Asp Gly Ala Ala Ala Glu Ser Ala Asn Pro Asn Gly Arg Arg Val 385 390 395 400 Arg Arg Arg Phe 7408PRTRicinus communis 7Met Val Glu Leu Lys Ser Asp Ser Asp Lys Glu Ser Cys Ser Met Ser 1 5 10 15 Ile Asn Glu Thr Val Asn Gly Ser His Gln Phe Ser Ile Lys Gly Tyr 20 25 30 Ser Leu Ala Lys Gly Met Gly Ala Gly Lys Cys Ile Ala Ser Asp Ile 35 40 45 Phe Thr Val Gly Gly Tyr Asp Trp Ala Ile Tyr Phe Tyr Pro Asp Gly 50 55 60 Lys Asn Pro Glu Asp Ser Ser Met Tyr Val Ser Val Phe Val Ala Leu 65 70 75 80 Ala Ser Glu Gly Thr Asp Val Arg Ala Leu Phe Glu Leu Thr Leu Val 85 90 95 Asp Gln Ser Gly Asn Gly Lys His Lys Val His Ser His Phe Asp Arg 100 105 110 Ala Leu Glu Ser Gly Pro Tyr Thr Leu Lys Tyr Arg Gly Ser Met Trp 115 120 125 Gly Tyr Lys Arg Phe Phe Arg Arg Thr Thr Leu Glu Asn Ser Asp Tyr 130 135 140 Ile Lys Asp Asp Cys Leu Ile Met Asn Cys Thr Val Gly Val Val Arg 145 150 155 160 Thr Arg Leu Glu Gly Pro Lys Gln Tyr Ser Ile Ser Leu Pro Pro Ser 165 170 175 Asp Met Gly Gln Gly Leu Lys Glu Leu Leu Glu Ser Glu Val Gly Cys 180 185 190 Asp Ile Val Phe Gln Val Gly Asp Glu Thr Phe Lys Ala His Lys Leu 195 200 205 Ile Leu Ala Ala Arg Ser Pro Val Phe Arg Ala Gln Phe Phe Gly Leu 210 215 220 Val Gly Asp Pro Asn Leu Asp Lys Val Val Val Glu Asp Ile Asp Pro 225 230 235 240 Ser Ile Phe Lys Ala Met Leu Leu Phe Ile Tyr Thr Asp Lys Leu Pro 245 250 255 Asn Val His Glu Ile Thr Gly Thr Thr Ser Met Cys Thr Ser Thr Asn 260 265 270 Met Val Gln His Leu Leu Ala Ala Ala Asp Leu Tyr Asn Leu Asp Gln 275 280 285 Leu Lys Leu Leu Cys Glu Ser Lys Leu Cys Glu Glu Leu Ser Ala Glu 290 295 300 Thr Val Ala Thr Thr Leu Ala Leu Ala Glu Gln His Gln Cys Ser Gln 305 310 315 320 Leu Lys Val Val Cys Leu Lys Phe Ala Ala Asn Pro Ala Asn Leu Gly 325 330 335 Ala Val Met Gln Ser Glu Gly Phe Arg His Leu Glu Glu Ser Cys Pro 340 345 350 Ser Leu Leu Cys Glu Met Leu Lys Thr Phe Ala Ser Gly Asp Glu Asn 355 360 365 Ser Ser Leu Leu Ser Ser Arg Lys Arg Ser Gly Ser Ser Ile Tyr Gly 370 375 380 Leu Asp Ile Ala Ala Asp Gly Ala Ala Ala Glu Ser Ala Asn Pro Met 385 390 395 400 Gly Arg Arg Val Arg Arg Arg Phe 405 8419PRTEucalyptus grandis 8Met Gln Arg Lys Ala Met Cys Ala Pro Ile Gly Gly Gly Gly Gly Asp 1 5 10 15 Gly Gly Gly Glu Cys Gly Ser Thr Ser Ile Ser Arg Thr Val Asn Gly 20 25 30 Ser His Thr Phe Thr Ile Ser Gly Tyr Ser Leu Ala Lys Gly Met Gly 35 40 45 Ala Gly Lys Phe Ile Ala Ser Asp Val Phe Thr Val Gly Gly Tyr Asp 50 55 60 Trp Ala Ile Tyr Phe Tyr Pro Asp Gly Lys Asn Pro Glu Asp Ser Thr 65 70 75 80 Thr Tyr Val Ser Val Phe Ile Ala Leu Ala Ser Asp Gly Ser Asp Val 85 90 95 Arg Ala Leu Phe Glu Leu Thr Leu Val Asp Gln Ser Gly Lys Gly Lys 100 105 110 His Lys Val His Ser His Phe Asp Arg Ala Leu Gln Ser Gly Pro Tyr 115 120 125 Thr Leu Lys Tyr Arg Gly Ser Met Trp Gly Tyr Lys Arg Phe Leu Lys 130 135 140 Arg Val Ala Leu Glu Thr Ser Asp Tyr Ile Lys Asp Asp Cys Leu Val 145 150 155 160 Met His Cys Thr Val Gly Val Val Arg Thr His Thr Glu Gly Pro Lys 165 170 175 Gln Tyr Arg Ile Pro Ile Pro Pro Ser Asp Met Gly Gln Cys Leu Lys 180 185 190 Ala Leu Leu Asp Ser Glu Val Gly Cys Asp Ile Ala Phe Val Val Gly 195 200 205 Asp Glu Thr Phe Arg Ala His Lys Leu Ile Leu Ala Ala Arg Ser Pro 210 215 220 Val Phe Arg Ala Gln Phe Phe Gly Leu Val Gly Asp Cys Asn Ile Glu 225 230 235 240 Lys Val Val Val Glu Asp Val Asp Pro Ser Ile Phe Lys Ala Met Leu 245 250 255 Leu Phe Ile Tyr Met Asp Glu Met Pro Asp Leu Arg Glu Ile Thr Gly 260 265 270 Ser Ser Ser Ser Gly Thr Leu Thr Asn Val Val Gln His Leu Leu Ala 275 280 285 Ala Ala Asp Arg Tyr Asn Leu Glu Arg Leu Lys Leu Leu Cys Glu Ser 290 295 300 Lys Leu Cys Glu Glu Ile Thr Ala Asp Thr Val Ala Thr Thr Leu Ala 305 310 315 320 Leu Ala Glu Gln His Gln Phe Gly Gln Leu Lys Ala Met Cys Leu Lys 325 330 335 Phe Ala Ala His Pro Thr Asn Leu Ala Val Val Met Gln Ser Glu Gly 340 345 350 Phe Arg His Leu Glu Glu Ser Cys Pro Ser Leu Leu Ser Glu Leu Leu 355 360 365 Lys Ala Phe Val Thr Val Asp Asp Ser Ser Asp Arg Phe Ser Asn Lys 370 375 380 Lys Arg Gly Thr Ser Ser Ile Tyr Gly Leu Asp Thr Val Pro Val Val 385 390 395 400 Thr Gly Ala Glu His Gly Asp Ile Asp Gly Arg Arg Val Lys Arg Arg 405 410 415 Asn Leu Glu 9406PRTVitis vinifera 9Met Val Asn Ser Lys Ala Asp Ile Glu Arg Asp Ser Cys Ser Lys Ser 1 5 10 15 Ile Asn Glu Thr Val Asn Gly Ser His His Phe Leu Ile Lys Gly Tyr 20 25 30 Ser Leu Ala Lys Gly Met Gly Ala Gly Lys Tyr Ile Ser Ser Asp Thr 35 40 45 Phe Thr Val Gly Gly Tyr Asp Trp Ala Ile Tyr Phe Tyr Pro Asp Gly 50 55 60 Lys Asn Ala Glu Asp Asn Ser Met Tyr Val Ser Val Phe Ile Ala Leu 65 70 75 80 Ala Ser Glu Gly Thr Asp Val Arg Ala Leu Phe Glu Leu Thr Leu Leu 85 90 95 Asp Gln Ser Gly Lys Gly Lys His Lys Val His Ser His Phe Asp Arg 100 105 110 Ala Leu Glu Ser Gly Pro Tyr Thr Leu Lys Tyr Arg Gly Ser Met Trp 115 120 125 Gly Tyr Lys Arg Phe Phe Arg Arg Thr Thr Leu Glu Thr Ser Asp Phe 130 135 140 Ile Lys Asp Asp Cys Leu Ala Met His Cys Thr Val Gly Val Val Arg 145 150 155 160 Thr Arg Val Glu Gly Pro Lys Gln Tyr Thr Ile Pro Ile Pro Pro Ser 165 170 175 Asp Ile Gly Gln Ser Leu Lys Asp Leu Leu Glu Ser Glu Val Gly Cys 180 185 190 Asp Ile Thr Phe Gln Val Ala Asp Glu Thr Phe Lys Ala His Lys Leu 195 200 205 Ile Leu Ala Ala Arg Ser Pro Val Phe Arg Ala Gln Phe Phe Gly Leu 210 215 220 Val Gly Asn Pro Asn Met Asp Lys Val Val Val Glu Asp Val Glu Pro 225 230 235 240 Ser Ile Phe Lys Ala Met Leu Leu Phe Ile Tyr Ser Asp Lys Leu Pro 245 250 255 Asp Val Asp Glu Ile Thr Gly Ser Ala Ser Val Cys Thr Ser Thr Ile 260 265 270 Met Val Gln His Leu Leu Ala Ala Ala Asp Arg Phe Gly Leu Asp Arg 275 280 285 Leu Lys Leu Leu Cys Glu Ser Lys Leu Cys Lys Glu Val Ser Ala Glu 290 295 300 Thr Val Ala Thr Thr Leu Ala Leu Ala Glu Gln His Arg Cys Pro Gln 305 310 315 320 Leu Lys Ala Ile Cys Leu Lys Phe Ala Ala Thr Pro Ser Ile Leu Gly 325 330 335 Ala Val Met Gln Ser Glu Gly Phe Gly Tyr Leu Glu Glu Cys Cys Pro 340 345 350 Ser Leu Leu Ser Glu Leu Leu Gly Val Ile Ala Ser Val Asp Glu Asn 355 360 365 Leu Thr Met Leu Ser Ser Lys Lys Arg Ser Gly Ser Ser Ile Leu Gly 370 375 380 Leu Asp Leu Pro Ala Asp Gly Ala Pro Ala Glu Ser Ala Ser Gly Arg 385 390 395 400 Arg Ile Arg Arg Arg Phe 405 10425PRTPrunus persica 10Met Pro Asn His Lys Ser Ser Arg Gly Ala Gln Leu Gly Glu Ala Met 1 5 10 15 Ser Asn Ser Lys Pro Gly Val Asp Gln Glu Ser Cys Ser Arg Ser Ile 20 25 30 Ser Glu Thr Val Asn Gly Ser His Arg Phe Thr Ile Lys Gly Tyr Ser 35 40 45 Leu Ala Lys Gly Met Gly Ala Gly Lys Tyr Ile Met Ser Asp Thr Phe 50 55 60 Thr Val Gly Gly Tyr Asp Trp Ala Ile Tyr Phe Tyr Pro Asp Gly Lys 65 70 75 80 Asn Pro Glu Asp Ser Ser Thr Tyr Val Ser Val Phe Ile Ala Leu Val 85 90 95 Ser Glu Gly Thr Asp Val Arg Ala Leu Phe Glu Leu Thr Leu Val Asp 100 105 110 Gln Thr Lys Ser Gly Lys Asp Lys Val His Ser His Phe Asp Arg Ala 115 120 125 Leu Glu Ser Gly Pro Tyr Thr Leu Lys Tyr Arg Gly Ser Met Trp Gly 130 135 140 Tyr Lys Arg Phe Phe Lys Arg Ser Ala Leu Glu Thr Ser Glu Phe Leu 145 150 155 160 Arg Asp Asp Cys Leu Val Leu Asn Cys Thr Val Gly Val Val Arg Thr 165 170 175 Arg Leu Glu Arg Pro Lys Gln Phe Ser Ile Thr Val Pro Ser Ser Asp 180 185 190 Met Gly Gln Asp Leu Lys Asp Phe Leu Asp Ser Glu Ala Gly Cys Asp 195 200 205 Ile Val Phe Gln Val Gly Asp Glu Leu Phe Lys Ala His Lys Leu Ile 210 215 220 Leu Ala Ala Arg Ser Pro Val Phe Arg Ala Gln Phe Phe Gly Leu Val 225 230 235 240 Gly Asp Cys Ser Ile Asp Lys Val Val Val Lys Asp Val Glu Pro Phe 245 250 255 Ile Phe Lys Ala Met Leu Leu Phe Ile Tyr Thr Asp Lys Leu Pro Asp 260 265 270 Val His Glu Val Met Gly Ser Ser Pro Leu Cys Thr Phe Thr Val Met 275 280 285 Val Gln His Leu Leu Ala Ala Ala Asp Leu Tyr Asn Leu Glu Arg Leu 290 295 300 Lys Val Leu Cys Glu Ser Lys Leu Cys Glu Glu Ile Thr Thr Glu Thr 305 310 315 320 Val Ala Thr Thr Leu Ala Leu Ala Glu Gln His His Cys Pro Gln Leu 325 330 335 Lys Ala Val Cys Leu Lys Phe Ala Ala Asn Pro Ala Asn Leu Gly Ala 340 345 350 Val Met Gln Ser Asp Gly Tyr Lys His Leu Glu Glu Ser Cys Pro Ser 355 360 365 Met Leu Leu Glu Leu Leu Glu Thr Phe Ala Ala Val Asp Glu Ser Ser 370 375 380 Ser Leu Leu Ser Ser Arg Lys Arg Ser Gly Ser Ser Ile Tyr Gly Leu 385 390 395 400 Asp Leu Pro Ala Asp Gly Gly Gly Ala Val Ala Glu Ser Ala Asn Pro 405 410 415 Asn Gly Arg Arg Val Arg Arg Arg Tyr 420 425 11414PRTPhaseolus vulgaris 11Met Ala Glu Leu Glu Glu Asp Arg Met Gly Asp Phe Lys Pro Phe Ser 1 5 10 15 Glu Gly Ser Ser Cys Ser Arg Ser Ile Ser Glu Thr Val Asn Gly Ser 20 25 30 His Gln Phe Thr Ile Lys Gly Tyr Ser Leu Ala Lys Gly Met Gly Ala 35 40 45 Gly Lys Tyr Ile Met Ser Asp Ser Phe Ser Val Gly Gly Tyr Asp Trp 50 55 60 Ala Ile Tyr Phe Tyr Pro Asp Gly Lys Asn Pro Glu Asp Asn Ser Met 65 70 75 80 Tyr Val Ser Val Phe Ile Ala Leu Ala Ser Asp Gly Thr Asp Val Arg 85 90 95 Ala Leu Phe Lys Leu Thr Leu Val Asp Gln Ser Glu Lys Gly Asn Asp 100 105 110 Lys Val His Ser His Phe Asp Arg Pro Leu Asp Gly Gly Pro Tyr Thr 115 120 125 Leu Lys Tyr Arg Gly Ser Met Trp Gly Tyr Lys Arg Phe Phe Arg Arg 130 135 140 Asn Leu Leu Glu Ser Ser Glu Tyr Leu Lys Asp Asp Cys Leu Val Met 145 150 155 160 His Cys Thr Val Gly Val Val Lys Thr Arg Phe Glu Gly Ser Lys Gln 165 170 175 Gly Val Thr Val Pro Gln Ser Asp Met Gly Arg Asn Phe Lys Asp Leu 180 185 190 Leu Asp Ser Glu Val Gly Cys Asp Ile Val Phe Lys Val Lys Ser Glu 195 200 205 Ser Phe Lys Ala His Lys Leu Ile Leu Ala Ala Arg Ser Pro Val Phe 210 215 220 Arg Ala Gln Phe Phe Gly Leu Val Gly Asp Pro Ser Leu Glu Glu Val 225 230 235 240 Val Val Glu Asp Ile Glu Pro Phe Ile Phe Lys Ala Met Leu Leu Phe 245 250 255 Ile Tyr Ser Asp Lys Leu Pro Asp Ile Tyr Glu Val Met Asp Ser Met 260 265 270 Asn Val Cys Ser Tyr Ala Val Met Val Gln His Leu Leu Ala Ala Ala 275 280 285 Asp Leu Tyr Asn Leu Asp Arg Leu Lys Leu Leu Cys Glu Ser Lys Leu 290 295 300 Cys Glu Glu Ile Asn Thr Asp Asn Val Ala Thr Thr Leu Ala Leu Ala 305 310 315 320 Glu Gln His Asn Cys Pro Gln Leu Lys Ala Ile Cys Leu Lys Phe Ile 325 330 335 Ala Asn Pro Ala Asn Leu Gly Ala Val Met Gln Ser Glu Ala Phe Val 340 345 350 His Leu Lys Glu Ser Cys Pro Ala Met Leu Leu Glu Leu Leu Glu Thr 355 360 365 Phe Ala Ser Val Asp Asp Asn Ser Ser Leu Thr Leu Ser Arg Lys Arg 370 375 380 Ser Gly Ser Ser Ile Tyr Ala Gln Asp Leu Ala Asp Gly Ala Ala Thr 385 390 395 400 Glu Ser Val Asn Pro Asn Gly Arg Arg Val Arg Arg Arg Thr 405 410 12414PRTGlycine max 12Met Ala Glu Leu Glu Glu Glu Arg Met Gly Asp Phe Lys Pro Phe Ser 1 5 10 15 Glu Gly Ser Ser Cys Ser Arg Ser Ile Ser Glu Thr Val Asn Gly Ser 20 25 30 His Gln Phe Thr Ile Lys Gly Tyr Ser Leu Ala Lys Gly Met Gly Ala 35 40 45 Gly Lys Tyr Ile Met Ser Asp Thr Phe Thr Val Gly Gly Tyr Asp Trp 50 55 60 Ala Ile Tyr Phe Tyr Pro Asp Gly Lys Asn Pro Glu Asp Asn Ser Met 65 70 75

80 Tyr Val Ser Val Phe Ile Ala Leu Ala Ser Asp Gly Thr Asp Val Arg 85 90 95 Ala Leu Phe Lys Leu Thr Leu Val Asp Gln Ser Glu Lys Gly Asn Asp 100 105 110 Lys Val His Ser His Phe Asp Arg Pro Leu Glu Ser Gly Pro Tyr Thr 115 120 125 Leu Lys Tyr Lys Gly Ser Met Trp Gly Tyr Lys Arg Phe Phe Arg Arg 130 135 140 Thr Gln Leu Glu Thr Ser Glu Tyr Leu Lys Asn Asp Cys Leu Val Met 145 150 155 160 His Cys Thr Val Gly Val Val Lys Thr Arg Phe Glu Gly Ser Lys Gln 165 170 175 Gly Val Ile Val Pro Gln Ser Asp Met Gly Arg Asp Phe Lys Asp Leu 180 185 190 Leu Glu Ser Glu Val Gly Cys Asp Ile Leu Phe Lys Val Lys Ser Glu 195 200 205 Ser Phe Lys Ala His Lys Leu Ile Leu Ala Ala Arg Ser Pro Val Phe 210 215 220 Arg Ala Gln Phe Phe Gly Leu Val Gly Asp Pro Thr Leu Glu Glu Val 225 230 235 240 Val Val Glu Asp Ile Glu Pro Phe Ile Phe Lys Ala Met Leu Leu Phe 245 250 255 Val Tyr Ser Asp Lys Leu Pro Gly Ile Tyr Glu Val Met Asp Ser Met 260 265 270 Pro Leu Cys Ser Tyr Thr Val Met Val Gln His Leu Leu Ala Ala Ala 275 280 285 Asp Leu Tyr Asn Leu Asp Arg Leu Lys Leu Leu Cys Glu Ser Lys Leu 290 295 300 Cys Glu Glu Ile Asn Thr Asp Asn Val Ala Thr Thr Leu Ala Leu Ala 305 310 315 320 Glu Gln His His Cys Pro Gln Leu Lys Ala Ile Cys Leu Lys Tyr Ile 325 330 335 Ala Asn Pro Ala Asn Leu Gly Ala Val Met Gln Ser Glu Ala Phe Val 340 345 350 His Leu Lys Glu Ser Cys Pro Ser Met Leu Leu Glu Leu Leu Glu Thr 355 360 365 Phe Ala Ser Val Asp Asp Asn Ser Gly Gln Thr Leu Ser Arg Lys Arg 370 375 380 Ser Gly Ser Ser Ile Tyr Gly Gln Asp Leu Ala Asp Gly Ala Ala Ala 385 390 395 400 Glu Ser Val Asn Pro Asn Gly Arg Arg Val Arg Arg Arg Thr 405 410 13411PRTPhoenix dactylifera 13Met Ala Lys Leu Glu Glu Glu Gln Gly Gly Leu Asn Asn Arg Gln Leu 1 5 10 15 Asn Pro Leu Asn Val Ser Arg Ser Arg Ser Val Cys Glu Thr Val Asn 20 25 30 Gly Ser His Arg Tyr Thr Val Lys Gly Phe Ser Leu Ala Lys Gly Met 35 40 45 Gly Pro Gly Arg Tyr Leu Ser Ser Asp Thr Phe Thr Val Gly Gly Phe 50 55 60 Gln Trp Ala Val Tyr Phe Tyr Pro Asp Gly Lys Asn Pro Glu Asp Asn 65 70 75 80 Ser Leu Tyr Val Ser Val Phe Ile Ala Leu Ala Ser Glu Gly Thr Asp 85 90 95 Val Arg Ala Leu Phe Glu Leu Thr Leu Leu Asp Gln Asn Gly Lys Gly 100 105 110 Arg His Lys Val His Ser His Phe Asp Arg Ala Leu Glu Ala Gly Pro 115 120 125 Tyr Thr Leu Lys Tyr Arg Gly Ser Met Trp Gly Tyr Lys Arg Phe Tyr 130 135 140 Arg Arg Thr Ser Leu Glu Thr Ser Asp Tyr Leu Lys Asp Asp Cys Leu 145 150 155 160 Ile Met Asn Cys Thr Val Gly Val Val Arg Asn His Ile Glu Thr Pro 165 170 175 Thr Gln Leu Ser Ile Ser Val Pro Pro Pro Asp Leu Gly Gln Cys Leu 180 185 190 Lys Glu Leu Phe Ile Ser Gly Ile Gly Ser Asp Ile Asp Phe Glu Val 195 200 205 Gly Asp Glu Thr Phe Lys Ala His Lys Gln Ile Leu Ala Ala Arg Ser 210 215 220 Pro Val Phe Ser Ala Gln Phe Phe Gly Leu Ile Gly Asn Pro Asn Val 225 230 235 240 Asp Lys Ile Val Val Glu Asp Val Glu Pro Pro Ile Phe Lys Ala Met 245 250 255 Leu Leu Phe Ile Tyr Ser Asp Glu Leu Pro Asp Val His Asp Leu Thr 260 265 270 Gly Ser Val Ser Met Cys Thr Ser Thr Ile Met Val Gln His Leu Leu 275 280 285 Ala Ala Ala Asp Arg Tyr Gly Leu Glu Arg Leu Lys Leu Leu Cys Glu 290 295 300 Ala Lys Leu Cys Glu Glu Val Thr Ala Asp Thr Val Ala Thr Thr Leu 305 310 315 320 Ala Leu Ala Glu Gln His Gln Cys Ala Gln Leu Lys Ala Val Cys Leu 325 330 335 Lys Phe Thr Ala Ala Arg Glu Asn Leu Gly Ala Val Met Gln Thr Glu 340 345 350 Gly Phe Asn Tyr Leu Glu Ala Thr Cys Pro Ser Leu Leu Ser Asp Leu 355 360 365 Leu Ala Thr Val Ala Val Ala Asp Asp Asp Ser Ser Pro Ile Ser Arg 370 375 380 Lys Arg Ser Gly Ser Ser Asn Ile Gly Leu Asn Leu Met Asp Ser Val 385 390 395 400 Asp Leu Asn Gly Arg Arg Met Lys Arg Arg Met 405 410 14429PRTFragaria vesca 14Met Pro Pro Ile Gln Lys His Ser Leu Arg Gly Ala Gln Leu Gly Gly 1 5 10 15 Arg Ile Ser Ser Met Lys Ser Lys Leu Glu Asn Asp Glu Ser Cys Ser 20 25 30 Arg Ser Ile Ser Glu Thr Val Asn Gly Ser His Arg Phe Thr Ile Lys 35 40 45 Gly Tyr Ser Leu Ala Lys Gly Met Gly Ala Gly Lys Tyr Ile Leu Ser 50 55 60 Asp Thr Phe Thr Val Gly Gly Tyr Asp Trp Ala Ile Tyr Phe Tyr Pro 65 70 75 80 Asp Gly Lys Asn Pro Glu Asp Ser Ser Val Tyr Val Ser Val Phe Ile 85 90 95 Ala Leu Val Ser Glu Gly Thr Asp Val Arg Ala Leu Phe Glu Leu Thr 100 105 110 Leu Val Asp Gln Ser Asn Ser Gly Lys Asp Lys Val His Ser His Phe 115 120 125 Asp Arg Ala Leu Glu Ser Gly Pro Tyr Thr Leu Lys Tyr Arg Gly Ser 130 135 140 Met Trp Gly Tyr Lys Arg Phe Phe Arg Arg Ser Ala Leu Glu Thr Ser 145 150 155 160 Glu Phe Leu Lys Asp Asp Ser Leu Val Leu Asn Cys Thr Val Gly Val 165 170 175 Val Arg Thr Arg Leu Glu Cys Pro Lys His Phe Ala Ile Thr Val Pro 180 185 190 Pro Ser Asp Met Gly Glu Gly Leu Lys Ala Phe Leu Asp Ser Gly Ala 195 200 205 Gly Cys Asp Leu Val Phe Gln Val Gly Asp Glu Glu Phe Lys Ala His 210 215 220 Lys Leu Ile Leu Ala Ala Arg Ser Pro Val Phe Lys Ala Gln Phe Phe 225 230 235 240 Gly His Leu Gly Asp Ser Ser Val Asp Lys Val Val Val Lys Asp Val 245 250 255 Glu Pro Phe Ile Phe Lys Ala Met Leu Leu Phe Ile Tyr Gly Asp Lys 260 265 270 Leu Pro Asp Ile Arg Glu Val Thr Gly Ser Ser Ser Leu Cys Thr Phe 275 280 285 Thr Val Met Val Gln His Leu Leu Ala Ala Ala Asp Leu Tyr Asp Leu 290 295 300 Glu Arg Leu Lys Leu Leu Cys Glu Ser Met Leu Cys Glu Glu Ile Thr 305 310 315 320 Thr Glu Thr Val Ala Thr Thr Leu Ala Leu Ala Glu Gln His His Cys 325 330 335 Pro Gln Leu Lys Ala Val Cys Leu Lys Phe Ala Ala Lys Ser Thr Asn 340 345 350 Leu Gly Ala Val Met Gln Ser Asp Gly Tyr Lys His Leu Glu Glu Ser 355 360 365 Cys Pro Ser Val Leu Gln Glu Leu Leu Lys Thr Phe Ala Ser Val Asp 370 375 380 Ala Asn Glu Asn Ser Asn Ser Ser Lys Lys Arg Ser Gly Ser Ser Ile 385 390 395 400 Tyr Gly Leu Asp Leu Pro Ala Asp Gly Ser Gly Ala Val Ala Glu Ser 405 410 415 Ala Asn Pro Asn Gly Arg Arg Leu Arg Pro Arg Arg Tyr 420 425 15426PRTMalus domestica 15Met Pro Pro Ile Arg Lys His Ser Arg Gly Ala Lys Ser Gly Glu Ser 1 5 10 15 Met Gly Asn Ser Lys Pro Gly Phe Asp Gln Glu Ser Cys Ser Arg Ser 20 25 30 Ile Ser Glu Thr Val Asn Gly Ser His Arg Phe Thr Ile Lys Gly Tyr 35 40 45 Ser Leu Ala Lys Gly Met Gly Ala Gly Lys Tyr Leu Met Ser Asp Thr 50 55 60 Phe Thr Val Gly Gly Tyr Asp Trp Ala Ile Tyr Phe Tyr Pro Asp Gly 65 70 75 80 Lys Asn Pro Glu Asp Ser Asn Ala Tyr Val Ser Val Phe Ile Ala Leu 85 90 95 Val Ser Glu Gly Thr Asp Val Arg Ala Leu Phe Glu Leu Thr Leu Val 100 105 110 Asp Gln Thr Asp Ser Gly Lys Asp Lys Val His Ser His Phe Asp Arg 115 120 125 Ala Leu Glu Gly Gly Pro Tyr Thr Leu Lys Tyr Arg Gly Ser Met Trp 130 135 140 Gly Tyr Lys Lys Phe Phe Arg Arg Ser Ile Leu Glu Thr Ser Glu Phe 145 150 155 160 Leu Lys Asp Asp Cys Leu Val Leu Asn Cys Thr Val Gly Val Val Arg 165 170 175 Thr Arg Leu Glu Gln Pro Lys Gln Phe Thr Ile Thr Val Pro Ser Ser 180 185 190 Asp Met Gly Arg Asp Leu Lys Asp Phe Leu Asp Ser Glu Ala Gly Cys 195 200 205 Asp Ile Val Phe Gln Val Gly Asp Glu Gln Phe Lys Ala His Lys Leu 210 215 220 Ile Leu Ala Ala Arg Ser Arg Val Phe Arg Ala Gln Phe Tyr Gly Leu 225 230 235 240 Val Gly Asp Cys Asn Val Asp Lys Val Val Val Lys Asp Val Glu Pro 245 250 255 Phe Ile Phe Lys Ala Met Leu Leu Phe Ile Tyr Thr Asp Lys Leu Pro 260 265 270 Asp Thr His Glu Val Met Gly Ser Ser Pro Leu Cys Thr Phe Thr Val 275 280 285 Met Val Gln His Leu Leu Ala Ala Ala Asp Leu Tyr Asn Leu Asp Arg 290 295 300 Leu Lys Leu Leu Cys Glu Ser Lys Leu Cys Glu Glu Ile Thr Thr Glu 305 310 315 320 Thr Val Ala Thr Thr Leu Ala Leu Ala Glu Gln His Gln Cys Arg Gln 325 330 335 Leu Lys Asp Val Cys Leu Lys Phe Thr Ala Asn Pro Ser Asn Leu Gly 340 345 350 Ala Val Met Gln Ser Glu Gly Tyr Lys His Leu Glu Glu Ser Cys Pro 355 360 365 Ser Met Leu Val Glu Leu Leu Glu Thr Phe Ala Ala Val Asp Asp Asn 370 375 380 Ser Ser Leu Leu Ser Ser Arg Lys Arg Ser Gly Ser Ser Ile Tyr Gly 385 390 395 400 Leu Asp Leu Pro Ala Asp Gly Gly Gly Thr Ala Ala Glu Ser Ala Asn 405 410 415 Pro Asn Gly Arg Arg Val Arg Arg Arg Phe 420 425 16408PRTSolanum lycopersicum 16Met Asn Gln Ile Ser Val Asp Arg Ala Gly Lys Asp Ser Ser Ser Lys 1 5 10 15 Ser Val Asn Glu Thr Val Asn Gly Ser His His Phe Thr Ile Arg Gly 20 25 30 Tyr Ser Leu Ala Lys Gly Met Gly Pro Gly Lys Tyr Ile Ser Ser Asp 35 40 45 Ile Phe Thr Val Gly Gly Tyr Asp Trp Ala Ile Tyr Phe Tyr Pro Asp 50 55 60 Gly Lys Asn Ile Glu Asp Ser Ser Met Tyr Val Ser Val Phe Ile Ala 65 70 75 80 Leu Ala Ser Glu Gly Thr Asp Val Arg Ala Leu Phe Glu Leu Thr Met 85 90 95 Leu Asp Gln Ser Gly Lys Val Lys His Lys Val His Ser His Phe Asp 100 105 110 Arg Ala Leu Glu Ser Gly Pro Tyr Thr Leu Lys Tyr Arg Gly Ser Met 115 120 125 Trp Gly Tyr Lys Arg Phe Phe Arg Arg Ala Ser Leu Glu Thr Ser Asp 130 135 140 Tyr Leu Lys Asp Asp Cys Leu Ser Met His Cys Thr Val Gly Val Val 145 150 155 160 Arg Thr Arg Val Glu Gly Pro Lys Asn Tyr Ser Val Thr Ile Pro Pro 165 170 175 Ser Asp Met Gly Gln Ser Leu Lys Tyr Leu Leu Asp Ala Glu Leu Gly 180 185 190 Cys Asp Ile Val Phe Arg Val Gly Glu Glu Ala Phe Lys Gly His Lys 195 200 205 Leu Ile Leu Ala Ala Arg Ser Pro Val Phe Arg Ala Gln Phe Phe Gly 210 215 220 Leu Ile Gly Asn Pro Lys Thr Asp Glu Val Glu Ile Glu Asp Ile Glu 225 230 235 240 Pro Ser Val Phe Lys Ala Met Leu Gln Tyr Ile Tyr Ser Asp Glu Leu 245 250 255 Pro Asp Leu Ile Glu Ile Thr Gly Ser Thr Ser Thr Cys Thr Ser Thr 260 265 270 Ile Val Thr Gln His Leu Leu Ala Ala Ala Asp Arg Phe Gly Val Asp 275 280 285 Arg Leu Lys Glu Leu Cys Glu Ala Lys Leu Cys Glu Glu Val Asn Val 290 295 300 Asp Thr Val Ala Thr Thr Leu Ser Leu Ala Glu Gln His Arg Cys Pro 305 310 315 320 Gln Leu Lys Ala Ile Cys Leu Lys Phe Ala Ala Thr Asn Leu Gly Val 325 330 335 Val Met Gln Lys Asp Gly Phe Lys His Leu Glu Glu Ser Cys Pro Leu 340 345 350 Leu Leu Ser Glu Leu Leu Glu Thr Val Ala Ser Val Asp Glu Lys Pro 355 360 365 Ser Leu Thr Ser Ser Lys Lys Arg Asn Ser Ser Ser Ser Ile Phe Gly 370 375 380 Leu Asp Leu Ala Ala Asp Gly Ala Ala Ala Asp Ser Val Asn Leu Thr 385 390 395 400 Ala Arg Arg Val Arg Arg Arg Met 405 17408PRTSolanum tuberosum 17Met Asn Gln Ile Ser Ile Asp Arg Ala Gly Asn Asp Ser Ser Ser Lys 1 5 10 15 Ser Val Asn Glu Thr Val Asn Gly Ser His His Phe Thr Ile Arg Gly 20 25 30 Tyr Ser Leu Ala Lys Gly Met Gly Pro Gly Lys Tyr Ile Ser Ser Asp 35 40 45 Ile Phe Thr Val Gly Gly Tyr Asp Trp Ala Ile Tyr Phe Tyr Pro Asp 50 55 60 Gly Lys Asn Ile Glu Asp Ser Ser Met Tyr Val Ser Val Phe Ile Ala 65 70 75 80 Leu Ala Ser Glu Gly Thr Asp Val Arg Ala Leu Phe Glu Leu Thr Met 85 90 95 Leu Asp Gln Ser Gly Lys Val Lys His Lys Val His Ser His Phe Asp 100 105 110 Arg Ala Leu Glu Ser Gly Pro Tyr Thr Leu Lys Tyr Arg Gly Ser Met 115 120 125 Trp Gly Tyr Lys Arg Phe Phe Arg Arg Ala Ser Leu Glu Met Ser Asp 130 135 140 Tyr Leu Lys Asp Asp Cys Leu Ser Met His Cys Thr Val Gly Val Val 145 150 155 160 Arg Thr Arg Val Glu Gly Pro Lys Asp Tyr Ser Val Thr Ile Pro Pro 165 170 175 Ser Asp Met Gly Gln Ser Leu Lys Tyr Leu Leu Asp Ala Glu Leu Gly 180 185 190 Cys Asp Ile Val Phe Arg Val Gly Glu Glu Ala Phe Lys Gly His Lys 195 200 205 Leu Ile Leu Ala Ala Arg Ser Pro Val Phe Arg Ala Gln Phe Phe Gly 210 215 220 Leu Ile Gly Asn Pro Lys Thr Asp Glu Val Glu Ile Glu Asp Ile Glu 225 230 235 240 Pro Ser Val Phe Lys Ala Met Leu Gln Tyr Ile Tyr Ser Asp Glu Leu 245 250 255 Pro Asp Leu Ile Glu Ile Thr Gly Ser Thr Ser Thr Cys Thr Ser Thr 260 265 270 Ile Val Met Gln His Leu Leu Ala Ala Ala Asp Arg Phe Gly Leu Asp 275

280 285 Arg Leu Lys Glu Leu Cys Glu Ala Lys Leu Cys Glu Glu Val Asn Val 290 295 300 Asp Thr Val Ala Thr Thr Leu Ser Leu Ala Glu Gln His Arg Cys Pro 305 310 315 320 Gln Leu Lys Ala Ile Cys Leu Lys Phe Ala Ala Thr Asn Leu Gly Val 325 330 335 Val Met Gln Lys Asp Gly Phe Lys His Leu Glu Glu Ser Cys Pro Leu 340 345 350 Leu Leu Ser Glu Leu Leu Glu Thr Val Ala Ser Val Asp Glu Lys Pro 355 360 365 Ser Leu Thr Ser Ser Lys Lys Arg Ser Ser Ser Ser Ser Ile Phe Gly 370 375 380 Leu Asp Leu Ala Ala Asp Gly Ala Ala Ala Asp Ser Val Asn Leu Thr 385 390 395 400 Val Arg Arg Val Arg Arg Arg Met 405 18400PRTOryza brachyantha 18Met Thr Val Pro Pro Pro Thr Pro Pro Pro Ser Trp Ser Arg Ser Val 1 5 10 15 Thr Glu Thr Val Arg Gly Ser His Gln Tyr Thr Val Lys Gly Phe Ser 20 25 30 Met Ala Lys Gly Met Gly Pro Gly Arg Tyr Val Thr Ser Asp Thr Phe 35 40 45 Ala Val Gly Gly Tyr His Trp Ala Val Tyr Leu Tyr Pro Asp Gly Lys 50 55 60 Asn Pro Glu Asp Asn Ala Asn Tyr Val Ser Val Phe Val Ala Leu Ala 65 70 75 80 Ser Asp Gly Ala Asp Val Arg Ala Leu Phe Glu Leu Thr Leu Leu Asp 85 90 95 Gln Ser Gly Arg Gly Arg His Lys Val His Ser His Phe Asp Arg Ser 100 105 110 Leu Gln Ala Gly Pro Tyr Thr Leu Lys Tyr Arg Gly Ser Met Trp Gly 115 120 125 Tyr Lys Arg Phe Tyr Arg Arg Ser Leu Leu Glu Ser Ser Asp Phe Leu 130 135 140 Lys Asp Asp Cys Leu Val Met Asn Cys Thr Val Gly Val Val Lys Asn 145 150 155 160 Arg Leu Glu Thr Pro Lys Asn Ile Gln Ile His Ile Pro Pro Ser Asp 165 170 175 Met Gly Arg Cys Phe Lys Asn Leu Leu Asn Leu Gly Ile Gly Cys Asp 180 185 190 Ile Thr Phe Glu Val Gly Asp Asp Thr Val Gln Ala His Lys Trp Ile 195 200 205 Leu Ala Ala Arg Ser Pro Val Phe Lys Ala Gln Phe Phe Gly Pro Ile 210 215 220 Gly Asn Pro Asp Leu His Ser Val Thr Val Glu Asp Val Glu Pro Val 225 230 235 240 Val Phe Lys Ala Met Val Asn Phe Ile Tyr Ser Asp Glu Leu Pro Ser 245 250 255 Ile His Glu Leu Ala Gly Ser Val Ser Thr Trp Thr Ser Thr Val Val 260 265 270 Val Gln His Leu Leu Ala Ala Ala Asp Arg Tyr Gly Leu Asp Arg Leu 275 280 285 Arg Leu Leu Cys Glu Glu Lys Leu Cys Asp Glu Leu Thr Ala Glu Thr 290 295 300 Val Ala Thr Thr Leu Ala Leu Ala Glu Gln His His Cys Thr Gln Leu 305 310 315 320 Lys Ser Ala Cys Leu Lys Phe Thr Ala Val Arg Glu Asn Leu Gly Ala 325 330 335 Val Met Glu Thr Glu Gly Phe Asn Tyr Leu Glu Glu Thr Cys Pro Ser 340 345 350 Leu Leu Ser Asp Leu Leu Ala Thr Val Ala Val Val Asp Asp Asp Ser 355 360 365 Ala Thr Leu Asn Arg Lys Arg Gly Val Ser Gly Asn Glu Gly Ala Asn 370 375 380 Pro Val Glu Ser Val Glu Ala Ser Glu Arg Arg Ile Arg Arg Arg Val 385 390 395 400 19397PRTBrachypodium distachyon 19Met Ala Ala Val Pro Arg Pro Ser Trp Ser Arg Ser Val Ser Glu Thr 1 5 10 15 Val Arg Gly Ser His Gln Tyr Thr Val Lys Gly Phe Ser Leu Ala Lys 20 25 30 Gly Ile Gly Pro Gly Arg His Leu Ala Ser Asp Thr Phe Ala Val Gly 35 40 45 Gly Tyr Asp Trp Ala Val Tyr Leu Tyr Pro Asp Gly Lys Asn Pro Glu 50 55 60 Asp Asn Ala Ser Tyr Val Ser Val Phe Val Ala Leu Ala Ser Glu Gly 65 70 75 80 Thr Asp Val Arg Ala Leu Phe Glu Leu Thr Leu Leu Asp Gln Ser Gly 85 90 95 Arg Ala Arg His Lys Val His Ser His Phe Asp Arg Ser Met Gln Ala 100 105 110 Gly Pro Tyr Thr Leu Lys Tyr Arg Gly Ser Met Trp Gly Tyr Lys Arg 115 120 125 Phe Tyr Arg Arg Ser Gln Leu Glu Thr Ser Asp Phe Leu Lys Asn Asp 130 135 140 Cys Leu Val Met Asn Cys Thr Val Gly Val Val Lys Thr Arg Leu Glu 145 150 155 160 Thr Pro Lys Asn Ile Gln Ile Asn Val Pro Pro Ser Asp Ile Gly Arg 165 170 175 Cys Phe Lys Glu Leu Leu Arg Leu Arg Ile Gly Cys Asp Ile Thr Phe 180 185 190 Glu Val Gly Asp Glu Lys Val Gln Ala His Lys Trp Ile Leu Ala Ala 195 200 205 Arg Ser Pro Val Phe Lys Ala Gln Phe Phe Gly Pro Ile Gly Lys Ala 210 215 220 Asp Leu Asp Arg Val Val Val Glu Asp Val Glu Pro Ile Val Phe Lys 225 230 235 240 Ala Met Val Asn Phe Ile Tyr Ser Asp Glu Leu Pro Ser Ile His Glu 245 250 255 Leu Ala Gly Ser Phe Ser Met Trp Thr Ser Thr Ala Val Ile Gln His 260 265 270 Leu Leu Ala Ala Ala Asp Arg Tyr Gly Leu Asp Arg Leu Arg Ile Leu 275 280 285 Cys Glu Ala Gln Leu Cys Asp Gly Leu Thr Ala Glu Thr Val Ala Thr 290 295 300 Thr Leu Ala Leu Ala Glu Gln His His Cys Ala Gln Leu Lys Ser Ala 305 310 315 320 Cys Leu Lys Phe Thr Ala Val Arg Glu Asn Leu Gly Val Val Met Glu 325 330 335 Thr Asp Gly Phe Asn Tyr Leu Glu Glu Thr Cys Pro Ser Leu Leu Ser 340 345 350 Asp Leu Leu Ala Thr Val Ala Val Val Asp Asp Asp Pro Thr Ser Val 355 360 365 Asn Arg Lys Arg Gly Val Cys Ile Asn Glu Asp Val Asn Pro Val Glu 370 375 380 Ser Val Glu Ala Ser Asp Arg Arg Ile Arg Arg Arg Val 385 390 395 20395PRTOryza sativa 20Met Thr Ala Ala Ala Ser Trp Ser Arg Ser Val Thr Glu Thr Val Arg 1 5 10 15 Gly Ser His Gln Tyr Thr Val Lys Gly Phe Ser Met Ala Lys Gly Val 20 25 30 Gly Ala Gly Arg Tyr Val Ser Ser Asp Thr Phe Ala Val Gly Gly Tyr 35 40 45 His Trp Ala Val Tyr Leu Tyr Pro Asp Gly Lys Asn Pro Glu Asp Asn 50 55 60 Ala Asn Tyr Val Ser Val Phe Val Ala Leu Ala Ser Asp Gly Ala Asp 65 70 75 80 Val Arg Ala Leu Phe Glu Leu Thr Leu Leu Asp Gln Ser Gly Arg Gly 85 90 95 Arg His Lys Val His Ser His Phe Asp Arg Ser Leu Gln Ala Gly Pro 100 105 110 Tyr Thr Leu Lys Tyr Arg Gly Ser Met Trp Gly Tyr Lys Arg Phe Tyr 115 120 125 Arg Arg Ser Leu Leu Glu Ser Ser Asp Phe Leu Lys Asp Asp Cys Leu 130 135 140 Val Met Asn Cys Thr Val Gly Val Val Lys Asn Arg Leu Glu Thr Pro 145 150 155 160 Lys Asn Ile His Ile Asn Ile Pro Pro Ser Asp Met Gly Arg Cys Phe 165 170 175 Asn Asn Leu Leu Asn Leu Arg Ile Gly Cys Asp Val Ser Phe Glu Val 180 185 190 Gly Asp Glu Arg Val Gln Ala His Lys Trp Ile Leu Ala Ala Arg Ser 195 200 205 Pro Val Phe Lys Ala Gln Phe Phe Gly Pro Ile Gly Asn Pro Asp Leu 210 215 220 His Thr Val Ile Val Glu Asp Val Glu Pro Leu Val Phe Lys Ala Met 225 230 235 240 Val Asn Phe Ile Tyr Ser Asp Glu Leu Pro Ser Ile His Glu Leu Ala 245 250 255 Gly Ser Val Ser Thr Trp Thr Ser Thr Val Val Val Gln His Leu Leu 260 265 270 Ala Ala Ala Asp Arg Tyr Gly Leu Asp Arg Leu Arg Leu Leu Cys Glu 275 280 285 Glu Lys Leu Cys Asp Glu Leu Thr Ala Glu Thr Val Ala Thr Thr Leu 290 295 300 Ala Leu Ala Glu Gln His His Cys Thr Gln Leu Lys Ser Ala Cys Leu 305 310 315 320 Lys Phe Thr Ala Val Arg Glu Asn Leu Gly Ala Val Met Glu Thr Glu 325 330 335 Gly Phe Asn Tyr Leu Glu Glu Thr Cys Pro Ser Leu Leu Ser Asp Leu 340 345 350 Leu Ala Thr Val Ala Val Val Asp Asp Asp Ala Ala Ser Phe Asn Arg 355 360 365 Lys Arg Gly Val Gly Gly Asn Glu Gly Ala Asn Pro Val Glu Ser Val 370 375 380 Glu Ala Ser Asp Arg Arg Ile Arg Arg Arg Val 385 390 395 21396PRTHordeum vulgare 21Met Ala Val Pro Arg Pro Ser Trp Ser Arg Ser Val Thr Glu Thr Val 1 5 10 15 Arg Gly Ser His Gln Tyr Thr Val Lys Gly Phe Ser Leu Ala Lys Gly 20 25 30 Ile Gly Pro Gly Arg His Leu Ser Ser Asp Thr Phe Ala Val Gly Gly 35 40 45 Tyr Asp Trp Ala Val Tyr Leu Tyr Pro Asp Gly Lys Asn Gln Glu Asp 50 55 60 Asn Ala Asn Tyr Val Ser Val Phe Val Ala Leu Ala Ser Glu Gly Thr 65 70 75 80 Asp Val Arg Ala Leu Phe Glu Leu Thr Leu Leu Asp Gln Ser Gly Arg 85 90 95 Ala Arg His Lys Val His Ser His Phe Asp Arg Ser Met Gln Ala Gly 100 105 110 Pro Tyr Thr Leu Lys Tyr Arg Gly Ser Met Trp Gly Tyr Lys Arg Phe 115 120 125 Tyr Arg Arg Thr Gln Leu Glu Ala Ser Asp Phe Leu Lys Asp Asp Cys 130 135 140 Leu Val Met Asn Cys Thr Val Gly Val Val Lys Asn Arg Leu Glu Thr 145 150 155 160 Pro Lys Asn Ile Gln Ile Asn Val Pro Pro Ser Asp Ile Gly Arg Tyr 165 170 175 Phe Lys Glu Leu Leu Lys Leu His Ile Gly Cys Asp Ile Thr Phe Glu 180 185 190 Val Gly Asp Glu Lys Val Gln Ala His Lys Trp Ile Leu Ala Ala Arg 195 200 205 Ser Pro Val Phe Lys Ala Gln Phe Phe Gly Pro Ile Gly Lys Pro Asp 210 215 220 Leu Asp Arg Val Val Val Glu Asp Val Glu Pro Ile Val Phe Lys Ala 225 230 235 240 Met Val Asn Phe Ile Tyr Ser Asp Glu Leu Pro Ser Ile His Glu Val 245 250 255 Ala Gly Ser Phe Ser Met Trp Thr Ser Thr Ala Val Thr Gln His Leu 260 265 270 Leu Ala Ala Ala Asp Arg Tyr Gly Leu Asp Arg Leu Arg Ile Leu Cys 275 280 285 Glu Ala Lys Leu Cys Asp Glu Leu Thr Ser Glu Thr Val Ala Thr Thr 290 295 300 Leu Ala Leu Ala Glu Gln His His Cys Ala Gln Leu Lys Ser Ala Cys 305 310 315 320 Leu Lys Phe Thr Ala Val Arg Gln Asn Leu Gly Ala Val Met Glu Thr 325 330 335 Glu Gly Phe Asn Tyr Leu Glu Glu Thr Cys Pro Ser Leu Leu Ser Asp 340 345 350 Leu Leu Ala Thr Val Ala Val Val Asp Asp Asp Pro Ala Ser Val Asn 355 360 365 Arg Lys Arg Gly Val Cys Ile Asn Glu Asp Ala Asn Pro Val Glu Ser 370 375 380 Val Glu Ala Ser Asp Arg Arg Thr Arg Arg Arg Val 385 390 395 22409PRTSelaginella moellendorffii 22Met Ala Arg Thr Ser Val Val Leu Gln Asp Asp Ser Gly Gln Val Val 1 5 10 15 Gly Ser Pro Thr Ser Thr Ala Thr Pro Ser Arg Ser Arg Cys Ile Thr 20 25 30 Glu Thr Val Asn Gly Ser His His Phe Thr Ile His Gly Tyr Ser Leu 35 40 45 Ala Lys Gly Met Gly Val Gly Lys Tyr Ile Ala Ser Asp Thr Phe Thr 50 55 60 Val Gly Gly Tyr Gln Trp Ala Ile Tyr Phe Tyr Pro Asp Gly Lys Asn 65 70 75 80 Thr Glu Asp Asn Ser Leu Tyr Val Ser Val Phe Ile Ala Leu Ala Ser 85 90 95 Glu Gly Thr Asp Val Arg Ala Leu Phe Glu Leu Thr Leu Leu Asp Gln 100 105 110 Ser Gly Lys Asn Lys His Lys Ile His Ser His Phe Asp Arg Ser Leu 115 120 125 Glu Ser Gly Pro Tyr Thr Leu Lys Tyr Arg Gly Ser Met Trp Gly Tyr 130 135 140 Lys Arg Phe Phe Arg Arg Ala Val Leu Glu Thr Ser Asp Phe Leu Lys 145 150 155 160 Asp Asp Ser Leu Ser Ile Thr Cys Thr Val Gly Val Val Val Ser Ser 165 170 175 Met Gln Ala Leu Lys Gln His Ser Leu Leu Val Pro Glu Ser Asp Ile 180 185 190 Gly Gln His Phe Leu Ser Leu Leu Glu Ser Gly Glu Gly Thr Asp Val 195 200 205 Asn Phe Asn Val Lys Gly Glu Ala Phe Ser Ala His Lys Leu Leu Leu 210 215 220 Ala Ala Arg Ser Pro Val Phe Lys Ala Gln Leu Phe Gly Pro Met Lys 225 230 235 240 Asp Glu Asn Gly Asp Val Ile Glu Ile Asp Asp Met Glu Pro Pro Val 245 250 255 Phe Lys Ala Met Leu His Phe Ile Tyr Lys Asp Ser Leu Pro Asp Thr 260 265 270 Asn Glu Met Thr Gly Ser Ser Ser Gln Ser Thr Ala Thr Met Met Ala 275 280 285 Gln His Leu Leu Ala Ala Ala Asp Arg Phe Cys Leu Asp Arg Leu Arg 290 295 300 Leu Leu Cys Glu Ser Arg Leu Cys Glu Gln Ile Thr Val Asp Thr Val 305 310 315 320 Ala Thr Thr Leu Ala Leu Ala Asp Gln His His Ala Ser Gln Leu Lys 325 330 335 Asn Val Cys Leu Lys Phe Ala Ala Ser Asn Leu Ala Val Val Met Gln 340 345 350 Ser Asp Gly Phe Glu Tyr Leu Arg Glu Ser Cys Pro Ser Leu Gln Ser 355 360 365 Glu Leu Leu Lys Thr Val Ala Gly Val Glu Glu Glu Ala Lys Ala Gly 370 375 380 Thr Lys Asn Arg Thr Val Trp Thr His Val Ala Asp Gly Gly Asp Gly 385 390 395 400 Leu Gly Arg Arg Val Arg Gln Lys Ile 405 23410PRTMedicago truncatula 23Met Gly Lys Ile Leu Arg Glu Thr Ala Lys Pro Ser Ser Asn Pro Ser 1 5 10 15 Ser Pro Ser Ser Ser Ser Glu Pro Ala Thr Thr Ser Ser Thr Ser Ile 20 25 30 Thr Glu Thr Val Lys Gly Ser His Gln Phe Lys Ile Thr Gly Tyr Ser 35 40 45 Leu Ser Lys Gly Ile Gly Ile Gly Lys Tyr Ile Ala Ser Asp Ile Phe 50 55 60 Ser Val Gly Gly Tyr Asp Trp Ala Ile Tyr Phe Tyr Pro Asp Gly Lys 65 70 75 80 Ser Val Glu Asp Asn Ala Thr Tyr Val Ser Leu Phe Ile Ala Leu Ala 85 90 95 Ser Asp Gly Thr Asp Val Arg Ala Leu Phe Glu Leu Thr Leu Leu Asp 100 105 110 Gln Ser Gly Lys Glu Arg His Lys Val His Ser His Phe Glu Arg Thr 115 120 125 Leu Glu Ser Gly Pro Tyr Thr Leu Lys Tyr Arg Gly Ser Met Trp Gly 130 135 140 Tyr Lys Arg Phe Phe Lys Arg Thr Ala Leu Glu Thr Ser Asp Tyr Leu 145 150 155

160 Lys Asp Asp Cys Leu Ser Val Asn Cys Ser Val Gly Val Val Arg Ser 165 170 175 Arg Thr Glu Gly Pro Lys Ile Tyr Ser Ile Ala Ile Pro Pro Ser Asn 180 185 190 Ile Gly His Gln Phe Gly Gln Leu Leu Glu Asn Gly Lys Gly Ser Asp 195 200 205 Val Ser Phe Glu Val Asp Gly Glu Val Phe Thr Ala His Lys Leu Val 210 215 220 Leu Ala Ala Arg Ser Pro Val Phe Arg Ala Gln Leu Phe Gly Pro Met 225 230 235 240 Arg Asp Gln Ser Thr Gln Ser Ile Lys Val Glu Asp Met Glu Ala Pro 245 250 255 Val Phe Lys Ala Leu Leu His Phe Met Tyr Trp Asp Ser Leu Pro Asp 260 265 270 Met Gln Glu Leu Thr Gly Met Asn Thr Lys Trp Ala Thr Thr Leu Met 275 280 285 Ala Gln His Leu Leu Ala Ala Ala Asp Arg Tyr Ala Leu Glu Arg Leu 290 295 300 Arg Leu Ile Cys Glu Ala Ser Leu Cys Glu Asp Val Ala Ile Asn Thr 305 310 315 320 Val Ala Thr Thr Leu Ala Leu Ala Glu Gln His His Cys Phe Gln Leu 325 330 335 Lys Ala Val Cys Leu Lys Phe Ile Ala Thr Ser Glu Asn Leu Arg Ala 340 345 350 Val Met Gln Thr Asp Gly Phe Glu Tyr Leu Lys Glu Ser Cys Pro Ser 355 360 365 Val Leu Thr Glu Leu Leu Glu Tyr Val Ala Arg Phe Thr Glu His Ser 370 375 380 Asp Phe Leu Cys Lys His Arg Asn Glu Ala Ile Leu Asp Gly Ser Asp 385 390 395 400 Ile Asn Gly Arg Arg Val Lys Gln Arg Leu 405 410 24409PRTCoffea canephora 24Met Gly Arg Val Tyr Asn Gly Glu Thr Ser Asn Pro Ser Ser Ser Thr 1 5 10 15 Thr Ala Ser Thr Ser Pro Pro Pro Val Thr Thr Ser Thr Ser Ile Thr 20 25 30 Glu Thr Val Asn Gly Thr His Asp Phe Lys Ile Thr Gly Tyr Ser Leu 35 40 45 Ser Lys Gly Ile Gly Ile Gly Lys Tyr Val Ala Ser Asp Ile Phe Met 50 55 60 Val Gly Gly Tyr Ala Trp Ala Ile Tyr Phe Tyr Pro Asp Gly Lys Ser 65 70 75 80 Val Glu Asp Asn Ala Thr Tyr Val Ser Leu Phe Ile Ala Leu Ala Ser 85 90 95 Glu Gly Thr Asp Val Arg Ala Leu Phe Glu Leu Thr Leu Met Asp Gln 100 105 110 Ser Gly Arg Ala Arg His Lys Ile His Ser His Phe Gly Arg Ala Leu 115 120 125 Glu Ser Gly Pro Tyr Thr Leu Lys Tyr Arg Gly Ser Met Trp Gly Tyr 130 135 140 Lys Arg Phe Phe Lys Arg Thr Ala Leu Glu Thr Ser Asp Tyr Leu Lys 145 150 155 160 Asn Asp Cys Leu Gln Val His Cys Cys Val Gly Val Val Arg Ser Gln 165 170 175 Thr Glu Gly Pro Lys Ile Tyr Ser Ile Pro Leu Pro Pro Ser Asp Ile 180 185 190 Gly Gln His Phe Gly Gln Leu Leu Glu Cys Gly Lys Gly Thr Asp Val 195 200 205 Asn Phe Glu Val Asn Gly Glu Lys Phe Ser Ala His Lys Leu Val Leu 210 215 220 Ala Ala Arg Ser Pro Val Phe Arg Ala Gln Leu Phe Gly Pro Met Lys 225 230 235 240 Asp His Asp Thr Gln Cys Ile Arg Val Glu Asp Met Glu Ala Pro Val 245 250 255 Phe Lys Ala Leu Leu His Phe Ile Tyr Trp Asp Cys Leu Pro Asp Met 260 265 270 Glu Glu Leu Thr Gly Leu Asn Ser Lys Gly Ala Thr Ser Leu Met Ala 275 280 285 Gln His Leu Leu Ala Ala Ala Asp Arg Tyr Gly Leu Asp Arg Leu Arg 290 295 300 Leu Ile Cys Glu Ala Asn Leu Cys Glu Asp Val Ala Ile Asn Thr Val 305 310 315 320 Ala Thr Thr Leu Ala Leu Ala Glu Gln His His Cys Phe Gln Leu Lys 325 330 335 Ser Val Cys Leu Lys Phe Val Ala Met Pro Glu Asn Leu Arg Ala Val 340 345 350 Met Gln Thr Asp Gly Phe Glu Tyr Leu Lys Glu Ser Cys Pro Ser Val 355 360 365 Leu Thr Glu Leu Leu Glu Tyr Val Ala Arg Ile Asn Glu His Ser Val 370 375 380 Ser Val Asn Lys Gln Leu Thr Asp Gly Ile Leu Asp Gly Ser Asp Val 385 390 395 400 Asn Gly Arg Arg Val Lys Gln Arg Leu 405 25399PRTZea mays 25Met Ala Ile Pro Pro Arg Thr Pro Ser Pro Pro Pro Ser Trp Ser Arg 1 5 10 15 Ser Val Thr Glu Thr Val Arg Gly Ser His Gln Phe Thr Val Arg Gly 20 25 30 Tyr Ser Leu Ala Lys Gly Met Gly Pro Gly Arg Tyr Leu Ala Ser Asp 35 40 45 Val Phe Ala Val Gly Gly Tyr His Trp Ala Val Tyr Leu Tyr Pro Asp 50 55 60 Gly Lys Asn Ala Glu Asp Asn Ser Asn Tyr Val Ser Val Phe Val Ala 65 70 75 80 Leu Ala Ser Asp Gly Ile Asp Val Arg Ala Leu Phe Glu Leu Thr Leu 85 90 95 Leu Asp Gln Ser Gly Arg Gly Cys His Lys Val His Ser His Phe Asp 100 105 110 Arg Ser Leu Lys Phe Gly Pro Tyr Thr Leu Lys Tyr Arg Gly Ser Met 115 120 125 Trp Gly Tyr Lys Arg Phe Tyr Lys Arg Thr Leu Leu Glu Glu Ser Asp 130 135 140 Phe Leu Lys Asn Asp Cys Leu Val Met Asn Cys Thr Val Gly Val Val 145 150 155 160 Lys Asn Arg Ile Glu Thr Pro Lys Asp Ile Gln Ile His Val Pro Arg 165 170 175 Ser Asp Met Gly Arg Cys Phe Lys Glu Leu Leu Ser Arg Cys Ile Gly 180 185 190 Cys Asp Ile Thr Phe Glu Val Arg Asp Glu Lys Val Arg Ala His Lys 195 200 205 Trp Ile Leu Ala Ala Arg Ser Pro Val Phe Lys Ala Gln Phe Phe Gly 210 215 220 Pro Ile Gly Lys Pro Asp Leu His Thr Val Val Val Glu Asp Val Glu 225 230 235 240 Pro Val Val Phe Lys Ala Met Val Asn Phe Ile Tyr Ala Asp Glu Leu 245 250 255 Pro Ser Ile Pro Glu Leu Ala Gly Ser Ala Ser Thr Trp Thr Ser Thr 260 265 270 Val Val Val Gln His Leu Leu Ala Ala Ala Asp Arg Tyr Gly Leu Val 275 280 285 Arg Leu Arg Ile Leu Cys Glu Ser Lys Leu Cys Asp Glu Leu Thr Pro 290 295 300 Glu Thr Val Ala Thr Thr Leu Ala Leu Ala Glu Gln His His Cys Ala 305 310 315 320 Glu Leu Lys Ser Ala Cys Leu Lys Phe Ile Ala Leu Arg Gly Asn Leu 325 330 335 Gly Ala Val Met Glu Thr Glu Gly Phe Asp Tyr Leu Glu Asp Thr Cys 340 345 350 Pro Ser Leu Leu Ser Asp Leu Leu Ala Thr Val Ala Val Val Asp Asp 355 360 365 Asp Leu Ala Ser Leu Asn Arg Lys Arg Gly Val Ser Gly Asn Gln Val 370 375 380 Met Ala Leu Val Gly Ser Val Glu Arg Arg Thr Arg Arg Lys Leu 385 390 395 26402PRTSorghum bicolor 26Met Ala Ile Pro Pro Arg Thr Pro Pro Pro Pro Pro Ser Trp Ser Arg 1 5 10 15 Tyr Val Thr Glu Thr Val Lys Gly Ser His Gln Phe Thr Val Arg Gly 20 25 30 Phe Ser Leu Ala Lys Gly Met Gly Pro Gly Arg His Leu Ala Ser Asp 35 40 45 Ile Phe Ala Val Gly Gly Tyr His Trp Ala Val Tyr Phe Tyr Pro Asp 50 55 60 Gly Lys Asn Ala Glu Asp Asn Ser Asn Tyr Val Ser Val Phe Val Ala 65 70 75 80 Leu Ala Ser Asp Gly Ile Asp Val Arg Ala Leu Phe Asp Leu Thr Leu 85 90 95 Leu Asp Gln Ser Gly Arg Gly Arg His Lys Ile His Ser His Phe Gly 100 105 110 Arg Lys Leu Asp Ser Gly Pro Tyr Thr Leu Lys Tyr Arg Gly Ser Met 115 120 125 Trp Gly Tyr Lys Arg Phe Tyr Lys Arg Ser Leu Leu Glu Ala Ser Asp 130 135 140 Phe Leu Lys Asn Asp Cys Leu Val Met Asn Cys Thr Val Gly Val Val 145 150 155 160 Lys Asn Arg Met Glu Thr Pro Lys Asp Ile Gln Ile His Val Pro Arg 165 170 175 Ser Asp Met Gly His Cys Phe Lys Glu Leu Leu Ser Arg Gly Ile Gly 180 185 190 Cys Asp Ile Thr Phe Glu Val Arg Asp Glu Lys Val Arg Ala His Lys 195 200 205 Trp Ile Leu Ala Ala Arg Ser Pro Val Phe Lys Ala Gln Phe Phe Gly 210 215 220 Pro Ile Gly Lys Pro Asp Leu His Thr Val Val Val Glu Asp Val Glu 225 230 235 240 Pro Val Val Phe Lys Ala Met Val Asn Phe Met Tyr Thr Asp Glu Leu 245 250 255 Pro Ser Ile Ser Glu Leu Ala Gly Ser Ala Ser Thr Trp Thr Ser Thr 260 265 270 Val Val Val Gln His Leu Leu Ala Ala Ala Asp Arg Tyr Gly Leu Asp 275 280 285 Arg Leu Arg Ile Leu Cys Glu Ser Lys Leu Cys Asp Glu Leu Thr Pro 290 295 300 Glu Thr Val Ala Thr Thr Leu Ala Leu Ala Glu Gln His His Cys Ala 305 310 315 320 Glu Leu Lys Ser Ala Cys Leu Arg Phe Ala Ala Val Arg Glu Asn Leu 325 330 335 Gly Ala Val Met Gly Thr Glu Gly Phe Asp Tyr Leu Glu Glu Thr Cys 340 345 350 Pro Ser Leu Leu Ser Asp Leu Leu Ala Thr Val Ala Glu Val Asp Asp 355 360 365 Asp Pro Ala Ser Leu Asp Arg Lys Arg Gly Val Cys Gly Asn Gln Val 370 375 380 Leu Ala Pro Val Glu Ser Val Glu Ala Thr Glu Arg Arg Thr Arg Arg 385 390 395 400 Arg Leu 27411PRTCucumis melo 27Met Gly Thr Ile Lys Ser Cys Arg Asp Thr Ser Lys Ser Tyr Ser Asn 1 5 10 15 Leu Arg Ser Pro Thr Pro Pro Pro Val Thr Phe Ser Thr Ser Arg Phe 20 25 30 Glu Thr Val Asn Gly Ser His Glu Phe Lys Ile Asn Gly Tyr Ser Leu 35 40 45 Asn Lys Gly Met Gly Ile Gly Lys Tyr Ile Ala Ser Asp Thr Phe Met 50 55 60 Val Gly Gly Tyr Ala Phe Ala Ile Tyr Phe Tyr Pro Asp Gly Lys Ser 65 70 75 80 Val Glu Asp Asn Ala Ser Tyr Val Ser Val Phe Ile Ala Leu Ala Ser 85 90 95 Glu Gly Thr Asp Val Arg Ala Leu Phe Glu Leu Thr Leu Leu Asp Gln 100 105 110 Ser Gly Lys Glu Asn His Lys Val His Ser His Phe Glu Arg Arg Leu 115 120 125 Glu Ser Gly Pro Tyr Thr Leu Lys Tyr Arg Gly Ser Met Trp Gly Tyr 130 135 140 Lys Arg Tyr Phe Lys Arg Thr Val Leu Glu Thr Ser Asp Phe Leu Lys 145 150 155 160 Asp Asp Cys Leu Glu Ile His Cys Val Val Gly Val Val Lys Ser His 165 170 175 Thr Glu Gly Pro Lys Ile Tyr Ser Ile Thr Pro Pro Pro Ser Asp Ile 180 185 190 Gly Gln His Phe Gly Lys Leu Leu Glu Ser Gly Lys Leu Thr Asp Val 195 200 205 Asn Phe Glu Val Asp Gly Glu Thr Phe Ser Ala His Lys Leu Val Leu 210 215 220 Ala Ala Arg Ser Pro Val Phe Arg Ala Gln Leu Phe Gly Pro Leu Lys 225 230 235 240 Asp Gln Asn Thr Glu Cys Ile Lys Val Glu Asp Met Glu Ala Pro Val 245 250 255 Phe Lys Ala Leu Leu His Phe Ile Tyr Trp Asp Ala Leu Pro Asp Met 260 265 270 Gln Glu Ile Val Gly Leu Asn Ser Lys Trp Ala Ser Thr Leu Met Ser 275 280 285 Gln His Leu Leu Ala Ala Ala Asp Arg Tyr Ala Leu Asp Arg Leu Lys 290 295 300 Leu Leu Cys Glu Ala Lys Leu Cys Glu Asp Val Ala Ile Asn Thr Val 305 310 315 320 Ala Thr Thr Leu Ala Leu Ala Glu Gln His His Cys Phe Gln Leu Lys 325 330 335 Ala Val Cys Leu Lys Val Ile Ala Leu Pro Glu Asn Leu Arg Ala Val 340 345 350 Met Gln Thr Glu Gly Phe Glu Tyr Leu Lys Glu Ser Cys Pro Ser Val 355 360 365 Leu Thr Glu Leu Leu Glu Tyr Val Ala Arg Val Thr Glu His Ala Val 370 375 380 Ile Thr Cys Ser Gly Tyr Gly Asn Gly Thr Val Leu Asp Gly Ser Tyr 385 390 395 400 Val Asn Gly Arg Arg Val Arg Gln Arg Leu Tyr 405 410 28285PRTArtificial SequenceArtificial consensus sequence 28Met Glu Ser Ser Lys Ser Ile Ser Glu Thr Val Asn Gly Ser His Gln 1 5 10 15 Phe Thr Ile Lys Gly Tyr Ser Leu Ala Lys Gly Met Gly Gly Lys Tyr 20 25 30 Ile Ser Asp Ile Phe Thr Val Gly Gly Tyr Asp Trp Ala Ile Tyr Phe 35 40 45 Tyr Pro Asp Gly Lys Asn Pro Glu Asp Tyr Val Ser Val Phe Ile Ala 50 55 60 Leu Ala Ser Glu Gly Thr Asp Val Arg Ala Leu Phe Glu Leu Thr Leu 65 70 75 80 Val Asp Gln Ser Gly Lys Gly Lys His Lys Val His Ser His Phe Asp 85 90 95 Arg Ala Leu Glu Ser Gly Pro Tyr Thr Leu Lys Tyr Arg Gly Ser Met 100 105 110 Trp Gly Tyr Lys Arg Phe Phe Arg Arg Thr Leu Glu Thr Ser Asp Tyr 115 120 125 Leu Lys Asp Asp Cys Leu Ile Met Asn Cys Thr Val Gly Val Val Arg 130 135 140 Leu Glu Gly Pro Lys Gln Tyr Ser Ile Val Pro Pro Ser Asp Met Gly 145 150 155 160 Gln Leu Lys Glu Leu Leu Glu Ser Glu Val Gly Cys Asp Ile Phe Val 165 170 175 Gly Asp Glu Phe Lys Ala His Lys Leu Ile Leu Ala Ala Arg Ser Pro 180 185 190 Val Phe Arg Ala Gln Phe Phe Gly Leu Val Gly Asp Pro Leu Asp Lys 195 200 205 Val Val Val Glu Asp Val Glu Pro Ile Phe Lys Ala Met Leu Phe Thr 210 215 220 Asp Leu Pro Asp Val Glu Ile Thr Gly Ser Thr Ser Met Cys Thr Ser 225 230 235 240 Thr Val Met Val Gln His Leu Leu Ala Ala Ala Asp Arg Tyr Leu Asp 245 250 255 Arg Leu Lys Leu Leu Cys Glu Lys Leu Cys Glu Glu Leu Ser Glu Thr 260 265 270 Val Ala Thr Thr Leu Ala Leu Ala Glu Gln His His Cys 275 280 285 2944DNAArtificial SequenceSynthetic primer 29taatacgact cactataggg agaatgttca agatctgtgg gtac 443025DNAArtificial SequenceSynthetic primer 30ctacatttct agactggacc tcctg 253147DNAArtificial SequenceSynthetic primer 31tcgtcctcag tggacgctta gagagcactt ctagactgga cctcctg 473250DNAArtificial SequenceSynthetic primer 32acgcttacgc tcagatggct caccgtcgtc gtcctcacgt ggacgcttag 503350DNAArtificial SequenceSynthetic primer 33cacgaccgtc cttagaacgc tcgtcacgct cacgcttacg ctcagatggc 503438DNAArtificial SequenceSynthetic primer 34agaaagctgg gtcacgacgg ttaccaccac gaccgtcc 383520DNAArtificial SequenceSynthetic primer 35atgaagaagc gcttaaccac 203620DNAArtificial SequenceSynthetic primer 36tcagaccaaa tagttacaag 203721DNAArtificial SequenceSynthetic primer

37cctgccatgt atgttgccat t 213822DNAArtificial SequenceSynthetic primer 38aatcgagcac aataccggtt gt 223918DNAArtificial SequenceSynthetic primer 39attggcgtct actcttgt 184018DNAArtificial SequenceSynthetic primer 40aatgatgctg ctctgcta 184118DNAArtificial SequenceSynthetic primer 41taatcggcac agacttga 184220DNAArtificial SequenceSynthetic primer 42actcgcatat tgttctaagc 204319DNAArtificial SequenceSynthetic primer 43caccagttca cgattcaag 194419DNAArtificial SequenceSynthetic primer 44ccaccaacgg agaagatat 194518DNAArtificial SequenceSynthetic primer 45tcctgatggc aagaatcc 184618DNAArtificial SequenceSynthetic primer 46cgaagtggct atgaacct 184718DNAArtificial SequenceSynthetic primer 47ttaggctcag gttgttgt 184820DNAArtificial SequenceSynthetic primer 48tcatccttca tctgttggta 204920DNAArtificial SequenceSynthetic primer 49gcataaggtt catagccatt 205018DNAArtificial SequenceSynthetic primer 50agatgtctca agcaagga 185118DNAArtificial SequenceSynthetic primer 51gagcaacaag aagcagag 185218DNAArtificial SequenceSynthetic primer 52ccacaacgat ccatttcc 185316DNAArtificial SequenceSynthetic primer 53cagccaaatc gtcact 165416DNAArtificial SequenceSynthetic primer 54gttccggtat ggtcag 165525DNAArtificial SequenceSynthetic primer 55ctttcaccgt cttaggaaca aacag 255628DNAArtificial SequenceSynthetic primer 56taggaacaga gtttcgatgt ctgagaac 285718DNAArtificial SequenceSynthetic primer 57cggaggataa ctcgtctt 185820DNAArtificial SequenceSynthetic primer 58aatggctatg aaccttatgc 205922DNAArtificial SequenceSynthetic primer 59ctggaggttt tgaggctggt ta 226021DNAArtificial SequenceSynthetic primer 60ccaagggtga aagcaagaag a 216118DNAArtificial SequenceSynthetic primer 61aagtgaactg ttgttgtt 186220DNAArtificial SequenceSynthetic primer 62cgtcttctta ttgttattgg 206319DNAArtificial SequenceSynthetic primer 63ttccaataat tacctcctt 196419DNAArtificial SequenceSynthetic primer 64tttaacacaa ctttcaaag 196550PRTArabidopsis thaliana 65Leu Val Thr Val Arg Asp Val Met Thr Ser His Leu Arg Glu Met Gly 1 5 10 15 Lys Gln Leu Val Thr Asp Pro Glu Lys Ser Lys Asp Pro Val Glu Phe 20 25 30 Val Gln Arg Leu Leu Asp Glu Arg Asp Lys Tyr Asp Lys Ile Ile Asn 35 40 45 Thr Ala 50 6650PRTArabidopsis thaliana 66Leu Val Thr Val Arg Asp Val Met Thr Leu His Leu Arg Glu Met Gly 1 5 10 15 Lys Gln Leu Val Thr Asp Pro Glu Lys Ser Lys Asp Pro Val Glu Phe 20 25 30 Val Gln Arg Leu Leu Asp Glu Arg Asp Lys Tyr Asp Arg Ile Ile Asn 35 40 45 Met Ala 50 6746PRTArabidopsis thaliana 67Cys Ser Ser Ser Pro Ser Ser Ser Val Ser Ser Ser Thr Thr Thr Ser 1 5 10 15 Ser Pro Ile Gln Ser Glu Ala Pro Arg Pro Lys Arg Ala Lys Arg Ala 20 25 30 Lys Lys Ser Ser Pro Ser Gly Asp Lys Ser His Asn Pro Thr 35 40 45 681636DNAArabidopsis thaliana 68gaaggcgaaa acagtttccc ccaaattctc ataattttca caaacaacct ctcgtcttct 60aggttaatcc aatttcgtcg attcatgaag ttcacaattc tcccatcgga aaattcttcg 120taatcgacga cgaagagatc atgagtaccg tcggaggtat agagcagttg atacctgatt 180ccgtttcaac gtcgttcatc gaaacggtga acggttcgca ccagttcacg attcaaggtt 240actctctagc caaaggcatg agccctggga agtttataca gagcgatatc ttctccgttg 300gtggatacga ttgggcgatt tacttctatc ctgatgggaa gaacccggag gaccagtcct 360cgtatatctc tttgttcatc gctttagcga gtgattctaa tgatattagg gctttgtttg 420agcttacgct tatggatcag agtgggaaag ggaaacataa ggtgcatagt cactttgatc 480gggcgcttga aggtggtcct tatacactta agtataaagg aagcatgtgg ggttacaaac 540gctttttcaa acgatcagct ttagaaacct ctgactactt aaaggatgat tgtcttgtca 600tcaattgtac tgttggcgtt gttagagccc gactcgaggg tccaaaacag tatggcattg 660tgctacccct gtcgaatatg ggtcagggat tgaaagactt gttagattct gaagttggtt 720gtgacatagc tttccaagtc ggagatgaaa catacaaagc tcacaaactg attctcgcgg 780cacgctcccc agtttttaga gctcagtttt ttggaccaat tgggaataac aatgtggata 840gaatagtgat agacgacatc gaaccttcta tcttcaaggc tatgcttagc ttcatttaca 900ccgatgtact tcctaatgtg catgaaatta ccgggtcaac ttctgccagt tcgtttacaa 960acatgataca acatctcttg gcagctgctg acctctatga ccttgcaagg ttaaagatat 1020tatgtgaagt tttgctatgc gaaaaacttg atgttgataa tgtggcaaca acacttgcgt 1080tggctgaaca gcaccaattc ttacagctca aagcgttctg cttagaattt gttgcatctc 1140cagcaaactt gggagctgta atgaagtccg aagggttcaa gcacttgaaa cagagctgtc 1200ccactttgtt atctgagttg ctgaacactg ttgcagcagc agataagagc tcgacgagtg 1260gacaatcaaa caagaaaaga agtgcgagca gtgtattagg gtgtgacact acaaatgtga 1320ggcaattgag gaggagaaca cgaaaagaag tgcgagcagt gtcttaggat cattacatac 1380cgtatgcaaa attctagaat tatgcattgt gtttcaagca gagtttatga attccaagtc 1440atcccgtgaa cttttttacc agtgagaatt atagaggcct gaactctgaa ccaaactgtt 1500tgtgtcaatc attttacatt tctggacaaa agaaagtaca atctccacaa agagctgtga 1560gaattgactc aaaacaaatc ctaaaactct gtaccagatt gttcaatttc tcattaaatc 1620ccacaatatg attttc 1636691647DNABrassica rapa 69ctaatcataa ccaaaaccga aaccttagtt aaaatccggt agaatctcca caaaccaaaa 60ccatccgaat actaaaccaa accaaaccaa atccaatcga atttattttg gttcagttcg 120tcacaatttt aaccgaatca aactaacaaa ccaaacttct accgaacccg cagtcctaaa 180gtatcatctc caatcaaacg gagcttttat cattttcaaa atcaaatcga cggcgacgat 240gagcgcatct catccgaatc acgattcggt atcaacaacc gtaatggaga cggtgaacgg 300atcgcaccaa ttcacgatca aaggctactc tctcgccaaa ggcatgagcc cggggaggta 360catacagagc gacgtcttct ccgtgaacgg atacgactgg gtgatctact tctaccccga 420cgggaagaac cccgaggaga actccaccta cgtctctctc ttcatcgcct tggcgagcga 480ttcgagcgac attagggctt tgttcgagct gacgctgatg gatcagagcg ggagagggag 540gcataaggtt catagtcatt tcgatcgggc gcttgaagga gggccttata cgcttaagta 600taaagggagt atgtggggtt acaaacgctt tttaagaaga acagctttgg aagcatctga 660ctacttgaag gatgattgtc ttatcatcaa ctgtactgtt ggcgtcgtta gagctcgcct 720tgagggtcct aaacagtttg gcattgtgcc accaccttca aacatgggtc agggattgaa 780agacttgtta gactctgaac ttggctgcga cattgctttc caagtcggag atgagactta 840caaagctcac aaactgatcc tcgcagcacg ctcgccggtc tttagagctc agttctatgg 900accagttggg aataacagtg tggatagagt agtcatagag gacatggagc cttcaatctt 960taaggctatg cttagcttca tctacacgga tgtacttcct gatgtgcatg agattacagg 1020gtctacttct accgcttcgt tcacgaacat gatacagcat ctattggcag ctgctgacct 1080ctatgacctt gggaggttaa agatactgtg tgaagctttt ctatgtgaag aactaaacgt 1140tgataatgtg gcaacaacac ttgcactagc tgaccaacac cagttcttgc agctcaaagc 1200cttctgctta aaatttgttg catctccagc aaatttgcga gccgtaatga agtcagaagg 1260tttcaagcac ttgaaccaga gctgtccctc tgtgttgcct gagttgctaa acacagttgc 1320agcagcggat aagagctcga cgtcgtcgag tggacagtca agcaagaaaa gaagtgtgag 1380cagtgtgttg ggctgtgata caagcacaac aaatgcgaga caggtgagga ggacgtaggt 1440aggatcgacc caagtgcaag taatgcttta gtctgatgct actttgctag actttttact 1500tattgtaatg aaaataattg tttgtagtat gtctacagtt agtgtaaagc tttaggcaat 1560ggaacatctg ttttgctttg cgtgtttgta aaagctttgg ataatactag gttaaaagct 1620ttggagttaa tagtcttttt tgttgca 1647701224DNAJatropha curcas 70atggtcgacg tcaaagcgga tttcgataaa gagtcgtgtt cgaaatcagt aaacgagaca 60gtgaacgggt cgcaccagtt caccataaag ggatattctt tggcgaaagg gatgggagct 120gggaaatgca tatcgagtga tattttcacg gttggtggtt acgattgggc gatttacttt 180tacccagatg gtaaaaaccc tgaagatagc tccatgtatg tttccgtttt tattgccctg 240gcgagcgaag gaacggatgt tagggctttg tttgagttga cgttggttga tcagagtgga 300aacgggaagc acaaagtgca tagccacttt gatcgtgcat tggagagtgg gccgtacact 360ttgaagtata gagggagcat gtggggttac aagcgtttct ttagaaggac gaccttagaa 420aattctgatt atataaagga tgattgccta ctcatgaact gtactgttgg agttgtcaga 480actcgtcttg taggaccaaa acaatgtttt attaccattc caccctcaga catgggccag 540ggcctcaaag aactcttgga atctgaagtt ggttgtgaca ttgctttcca ggttggggat 600gaaacattta aagctcataa attgatactt gctgctcgct ctccagtttt cagggcccag 660ttttttggac tttttgggga tcctaaccta gataaagtag ttgtgaagga tattgacccc 720tcaatcttca aggcaatgct actattcgta tacacagaca aacttcctga tgtacatgaa 780attactggca cgacgtctat gtgcacatcc accaatatgg tgcagcatct attggctgct 840gctgacctat acaatttaga tcgattgaaa ttgctatgtg aatcgaagtt gtgtgaggaa 900ctgagtgctg agacagtggc gacgacgctt gcattagctg agcagcatca gtgttcgcag 960cttagggcca tctgtttgaa atttgctgca actcctgcaa acttgggagc ggtaatgcaa 1020tcagaaggat tccggcactt agaagaaagc tgcccggcat tgttgtgtga gatgctgaag 1080acatttgcat taggagatga gaattcaaat cagtcaggtc ggaagaggag tgggagcagc 1140atctatgggc tagatctagc aacagatggg gctgcagcag aatcagtaaa tcccaatgcc 1200aggcgtttga ggaggcggta ttag 1224711224DNAPopulus trichocarpa 71atggacgatt tcaagggaga tgtagataag gagtcgtgtt cgaagtcaat aaacgagacg 60gtgaatgggt ctcaccagtt tacgataaaa gggtattcat tagcgaaagg aatgggagct 120gggagatgca taccgagtga tgttttcaac gtgggtggtt atgattgggc gatttatttt 180tacccagatg ggaaaaaccc tgaggatagc tcgatgtatg tgtcggtttt tattgcgtta 240gcgagcgaag gaacggatgt tagggctttg ttcgagttga cgctggtgga tcagagtggg 300aaagggaagc ataaagtaca tagtcatttc gatcgtgcgt tggagagtgg accttattca 360ttgaagtaca gaggcagcat gtggggttac aaacgtttct tccgaaggac aaccttggaa 420acttctgatt atctgaagga tgactgcctt atcatgaact gcactgttgg agttgtcaga 480actcgtcttg aaggaccaaa acagtactcc atttcagttc caccttcaga catgggttgg 540ggttttaaag aactactgga gtctgaatct ggttgtgaca tagatttcca ggttggtgat 600gaaacattta gagctcataa gctgatcctt gctgctcgtt cacctgtttt cagagctcaa 660ttttttggac ttgtcgggga tcctaacatg gataaagtag tagtgaagga tgttgatccc 720ttgatattca aggcaatgct tctgtttata tacacagaca aacttcctga tgcacatgaa 780ataactggct cgacatcaat gtgcacatcc accaatatgg tgcagcatct gttggctgtc 840tctgaccttt acaatttaga tcgattgaaa ttgttatgtg aagcaaagtt gtgtgaggaa 900ctcagtgccg agaatgtggc aacaacactg gcattggctg agcagcatca gtgcatgcaa 960ctgaaggcca tctgtttgaa atttgcagca aatccagcga acttgggagc ggtaatgcag 1020tcagaagggt tccgacactt ggaggagagc tgcccttcaa tgttatgtga gttgctgaag 1080acacttgctt ctggagatga gaactcaagt cttctgtcag gtaggaagag gagtggcagc 1140agtttacttg gggttgatct agcggatggg gctccagcag aatcagcaaa tcccaatggc 1200aggcgtttga ggaggcggtt ttag 1224721209DNATheobroma cacao 72atggacgatt tcaaggactc ggtatcgaaa tcggtgagcg agactgtgaa cgggtcgcac 60cagttcacga tcaagggtta ctcgttggcg aaagggatgg gccctggaaa atgtatagcc 120agcgatgttt tcaccgtcgg aggtttcgat tgggtgattt acttttaccc cgacggtaaa 180aatccggagg atagtgctat gtatgtttcg gttttcattg ctctggccag cgaaggtacc 240gatgtccgtg cacttttcga gctcacgctt gtggaccaga gtgggaaagg gaagcataag 300gttcatagtc actttgatcg ggcgttggag agtggacctt atacgttgaa gtatagaggg 360agcatgtggg gttacaagcg tttctttaga agaacaactt tagaaacttc tgactatatt 420aaggatgatt gcctaatcat gaactgcact gttggagtag tcagaactcg cctcgaggga 480ccaaagcagt gttctatttc tgtaccgcca tcagaaatgg gtcagaatct taaagccttg 540ttggagtctg aagttggttg tgatatcatt ttccaggttg ttgatgagaa atttaaagca 600cataagttga tccttgctgc ccgctcacct gtttttagag cgcagttttt tgggcttgtt 660ggggatccta acatggataa agtagtagtg gaagattttg agccctctat cttcaaggca 720atgcttttgt ttatttatac cgacaagctt cctgatgtac aagagattac aggctcaacg 780tccatgtgta tgtctaccaa catggtgcag catcttttgg ctgctgctga tctgtacaat 840ttagatagac tcaaagtgtt gtgcgaggca aaattgtgtg aagaacttaa tgctgacaca 900gtggcaacaa cccttgcact agctgagcag caccattgcg cacagcttaa ggccatatgt 960ttgaaatttg ctgcaactcc agcaaacttg ggagcggtaa tgcagtcaga agggttcagg 1020cacttggagg aatgttgccc atctttgttg tctgagcttt tgaagacctt tgcatcaggt 1080gaggagagct tgagtcagct gtccagtagg aagaggagtg gcagcagtgt atacgggatg 1140gatctagcag cagaaggtcc tgtggcagaa tcggtgaatc ctaatggcag gcgtgttcgg 1200aggcgttga 1209731215DNACitrus clementina 73atgggcaatt cggagaaaga ttcgacgtcg aagtcaatta acgagacggt gaacgggtcc 60caccagttca cggtaaaagg ttactccctg gcgaagggaa tgggccctgg caagtgctta 120tcgagcgacg tttttaccgt gggcggttac gattgggcga tttactttta ccccgacggc 180aagaacccgg aagatggggc tttgtatgtt tcggtgttta ttgcgttggc gagtgaagga 240acggacgtga gggcgctgtt tgagttaact ttggttgacc aaagtgggaa aggaaagcat 300aaagttcata gtcattttga tcgagcgtta gagagtggcc cgtacacctt gaagtatcgt 360ggaagcatgt ggggctataa gcgcttcttt aaaagaacat ctctggagac ttctgattat 420attaaggatg attgtcttct catcaactgc actgttggag ttgttagaaa ccgccttgag 480ggaccaaaac agtattccat accagtgcca ccgtcagaca tgggccaggg tcttaaggat 540ttgctagagt ctgaaattgg atgtgacata gtttttgagg ttggtgatga aacatttaaa 600gctcataaac tgatacttgc tgctcgctct cctgttttca gagcccaatt ctatgggctt 660gttggagatc gtaacttgga taaagtagtt gtgaaggatg ttgaaccctc aatcttcaag 720gcaatgctcc tgtttatata caccgataaa tttcctgatg tatatgaaat tactggcaca 780acatcaatgt gcacaacaac caacatggta cagcatctac tggctgcagc tgatctttat 840aatgtagatc gattgaaatt gttgtgtgaa tcaaaattat gtgaagaact aaatgctgag 900acagtggcca caacactcgc actggcagaa caacatcagt gtccccagct taaggctatc 960tgcttgaagt ttgctgcaac tccggcgaat ttgggagtga taatgcagtc agaagggttc 1020aagcacttgg aggagagctg cccatcactg ttgtccgagc tcctgaagac attggcttca 1080ggtgatgata cctcaagtct gtcatcaaat aggaaaagaa gtggcagcag tatatatgca 1140ctagatctag ctggagatgg ggcagcagca gagtcagcaa atcccaatgg caggcgtgta 1200cgaaggcggt tttag 1215741227DNARicinus communis 74atggttgaat tgaagtcaga ttctgataaa gagtcatgtt caatgtcaat aaacgagacg 60gtaaatgggt ctcaccaatt ttccataaaa gggtattctt tagcgaaagg aatgggagct 120ggaaaatgta tagcaagtga tattttcact gtgggtggtt atgattgggc gatctatttt 180tacccagatg gtaaaaatcc tgaagatagt tctatgtatg tttctgtttt tgtagctttg 240gctagtgaag gaactgatgt tagggctttg tttgagttga ccttggttga tcaaagcgga 300aatgggaagc ataaagttca cagtcatttc gatcgtgcgt tggaaagtgg gccttatact 360ttgaagtata gagggagcat gtggggttac aagcgtttct ttagaagaac aactcttgaa 420aattctgatt atataaagga tgattgccta atcatgaact gcacagttgg agttgttaga 480acccgtcttg aaggaccaaa gcagtattcc atttcacttc cgccgtcaga catggggcaa 540ggccttaagg aactgttaga atctgaagtt ggttgcgaca ttgttttcca ggttggggat 600gaaacattta aagcgcataa gttgatactt gctgctcgtt cccctgtttt tagagctcaa 660ttctttggac ttgttgggga tccaaactta gataaagtag tagtggagga tattgacccc 720tcaattttca aggcaatgct cctgtttata tacacagaca agcttcctaa tgtacatgag 780attactggca caacatcaat gtgcacatcc accaacatgg tgcagcattt attggctgct 840gctgatcttt acaatttaga tcaattgaaa ttgttatgtg aatcaaaatt gtgcgaggaa 900ctgagtgctg agactgtggc aacaactctt gcattagctg agcagcatca atgttcgcaa 960ctcaaggtcg tctgtctgaa atttgctgca aatccagcaa acttgggagc ggtaatgcag 1020tcagaaggat tccgacactt ggaagagagc tgcccttcat tgttgtgcga gatgctaaag 1080acatttgcgt caggcgatga gaactcaagt cttctatcaa gtcggaagag gagcggaagc 1140agtatatatg ggctagatat agctgcagat ggggctgcag cagaatcagc caatcccatg 1200ggcaggcgag taaggaggcg tttttag 1227751260DNAEucalyptus grandis 75atgcagcgca aagcgatgtg cgctccgatc ggcggcggcg gcggcgacgg cgggggggag 60tgcggctcga cgtcgatcag ccggacggtg aacgggtcgc acacgttcac gatcagcggc 120tactcgctgg ccaaggggat gggggccggg aagttcatcg ccagcgacgt gttcaccgtc 180gggggctacg actgggccat ctacttctac cccgacggga agaacccgga ggacagcacg 240acgtacgtgt ccgtgttcat cgccctggcc agcgacggct ccgacgtcag ggcgctgttc 300gagctgaccc tggtcgacca gagcgggaag gggaagcaca aggtccacag ccacttcgac 360cgcgcgctcc agagcgggcc ttacacgctc aagtaccgcg gcagcatgtg gggttacaag 420cgtttcttga aaagagttgc tttagagact tctgattaca tcaaggacga ttgccttgtg 480atgcactgta ctgtcggggt tgtgagaacc cataccgagg gccccaaaca gtaccgaatt 540cctattccgc cgtctgacat gggccagtgt ctgaaggccc tgttagattc tgaagttggc 600tgcgacatag catttgttgt tggtgacgaa acctttagag ctcataaact gatcctcgct 660gctcgttctc cggtctttcg agcccaattt tttggtcttg ttggtgattg caatatagag 720aaagttgtcg tggaggatgt tgatccctca atttttaagg caatgctcct gttcatttac 780atggacgaaa tgcctgatct acgtgaaatc acgggctcat cctcttctgg tacattgact 840aacgtagtgc agcatctgtt agctgctgcc gaccgctaca atctagaacg attgaaatta 900ttatgtgagt cgaaattatg tgaggagatt actgctgata cagtggctac aacacttgcc 960ctagcagagc agcaccagtt tggacagctg aaggcaatgt gtctaaaatt tgctgcgcat 1020ccaacaaact tggcggtggt aatgcagtca gaaggcttca ggcacttgga ggagagctgc 1080ccttccttgt tgtctgaact gctcaaggct tttgtaacgg tggatgattc ttctgaccga 1140ttttcaaata agaagagagg caccagcagc atttacggac tagatacggt gccagttgtg 1200actggagctg aacatgggga tatagatgga aggcgtgtga agaggcggaa tttagaatga 1260761221DNAVitis vinifera 76atggttaatt ccaaggccga tattgagaga gactcgtgtt cgaagtcgat caacgagacg

60gtgaatggct cgcaccattt cttgataaag ggttattccc tcgcaaaggg aatgggcgcg 120ggcaaataca tctcgagcga cacgtttacc gttggaggat atgattgggc aatttacttc 180tatcctgatg gcaagaacgc ggaggataat tcgatgtatg tgtcggtgtt cattgcgttg 240gcgagcgagg gcactgacgt tagggctttg tttgaattga cgttgttgga tcagagtggg 300aaaggcaagc acaaagtaca cagtcatttt gatcgcgcat tggagagtgg cccatatact 360ttgaaatata gaggaagcat gtggggctac aagcgcttct tcagacggac aactttagaa 420acatctgatt ttatcaagga tgattgcctt gctatgcatt gcactgttgg ggttgtcaga 480actcgtgttg aggggcctaa acagtatacc attcctatac caccttcaga cattggtcag 540agtcttaagg acttgctaga atctgaagtt ggttgtgaca taacttttca ggttgcagat 600gagacattca aagctcataa gttgatactt gctgctcgtt ctcctgtatt tagagctcag 660ttttttggac ttgttggaaa tcctaatatg gataaagttg tagtggagga tgttgaaccc 720tctatcttta aggcgatgct cctgtttatt tactcagaca agcttcctga tgtagacgaa 780attacaggct cagcgtctgt gtgcacatcc acaataatgg ttcagcactt actagctgct 840gctgaccgct ttggtttaga tcgtctgaaa ctattatgtg aatcaaaatt gtgtaaagaa 900gtcagtgctg aaacggtggc cacaacactt gccctagctg agcagcatcg ttgtccacaa 960cttaaagcca tctgtttgaa atttgcagcc actccgtcaa tcttgggagc ggtaatgcaa 1020tcagaagggt ttgggtactt ggaagagtgc tgcccctcat tgttatctga gctgcttgga 1080gtgattgcat cagtagatga aaacttgacg atgctctcga gtaagaagag aagtggcagc 1140agcatattag ggttagatct accagcagat ggagctccag cagaatcagc cagtggcagg 1200cgcataagga ggcggtttta g 1221771278DNAPrunus persica 77atgccgaatc acaaatcgtc cagaggggct caattgggtg aagccatgtc gaattcgaag 60cctggagtcg accaggagtc gtgttcgaga tcgatcagcg agactgtcaa tgggtctcac 120cggttcacga taaaggggta ttctttggcc aaagggatgg gtgccggaaa gtacataatg 180agcgatacgt ttacggtggg tggctacgat tgggcaattt acttctaccc cgacggcaaa 240aatcctgagg atagttccac gtacgtctcc gttttcattg ctctggtcag tgagggtacg 300gatgtgaggg ctttgttcga gctgactttg gtggaccaga ccaagagtgg gaaggacaag 360gtgcatagcc actttgatcg cgcgctcgag agcgggccgt acacgttgaa gtacagaggc 420agcatgtggg gttacaagag atttttcaaa agatcagccc tcgaaacttc tgagtttcta 480agggatgatt gccttgtatt gaactgcact gttggagttg tcagaactcg ccttgagcga 540ccaaaacaat tttcaattac tgtaccatca tcagacatgg gtcaagatct taaggacttt 600ctagactctg aagctggttg tgacatagtt tttcaggttg gcgatgaatt gtttaaagct 660cacaagttga tacttgctgc ccgttctcct gtatttagag cacagttttt tggacttgtc 720ggggattgta gcatagataa agtagttgtg aaggatgttg agccctttat cttcaaggca 780atgcttctgt ttatttacac ggacaaactt cctgatgtac acgaagttat gggctcatca 840ccattgtgca cattcactgt catggtgcag catcttttgg ctgccgcgga cctgtataat 900ctagaacgac tgaaagtatt gtgtgaatca aagttgtgtg aagaaatcac tactgaaaca 960gttgcgacca cacttgctct agctgaacaa catcactgtc cgcagctcaa ggctgtgtgc 1020ttaaaatttg cagcaaatcc tgcaaactta ggagctgtga tgcaatcaga tgggtacaag 1080catctagaag agagctgccc ctcaatgttg ctggagttgc tagagacatt tgcagcagtg 1140gatgagagct caagtcttct gtcaagtagg aagaggagtg gcagcagcat atatgggcta 1200gacttgccag cagatggtgg cggggctgta gcagaatcag caaatcccaa tggaaggcgt 1260gtgaggcggc ggtattag 1278781245DNAPhaseolus vulgaris 78atggcggaat tggaggagga ccggatgggg gatttcaagc ccttctcgga gggctcttcg 60tgctcacgtt cgatcagcga aacggtgaat ggctctcacc aattcacgat aaaggggtac 120tctctcgcaa aggggatggg tgctgggaag tacatcatga gcgacagttt tagcgttggt 180ggttacgatt gggcaattta cttctaccct gatgggaaga accccgagga caattccatg 240tacgtttcgg tcttcatagc tctcgctagc gacggaaccg atgttagggc tctgttcaag 300ttgacgctgg tggatcagag tgagaaggga aacgataagg tccatagcca tttcgatagg 360cctcttgacg gtggaccgta caccttgaag tatagaggca gcatgtgggg ttacaagcgt 420ttcttcagaa gaaatttact tgaatcttca gagtatctaa aagacgattg ccttgtcatg 480cattgcactg ttggtgttgt caaaactcgt tttgagggat ctaaacaagg tgttactgtg 540ccacagtcag acatgggccg aaattttaag gacttgctgg actcagaggt tggttgcgac 600atagttttca aggttaaaag cgaaagcttc aaagctcata agttaatact tgcggcccga 660tctcctgtgt ttagagcaca gttttttgga cttgttgggg atcctagctt agaggaagta 720gtggtagagg atattgagcc ttttatcttc aaggcaatgc ttctcttcat ttattctgac 780aaacttccag acatctatga agttatggac tcaatgaatg tctgctcata tgccgtcatg 840gtgcagcatc tcttggctgc tgctgatctc tataatcttg accggctcaa actgctttgt 900gaatcaaaat tgtgtgaaga aatcaatact gacaatgtag ccacgacact tgccctggca 960gagcaacaca actgtccaca gcttaaggca atctgtttaa aatttattgc caatccagca 1020aatttgggag ctgtaatgca gtcggaagct tttgtgcatt tgaaagagag ctgccccgca 1080atgttgttgg agctgctgga gacatttgcc tcagtggacg ataactcaag cctgacattg 1140agcagaaaga gaagtggcag tagcatatat gctcaagatt tggcagacgg ggcagctact 1200gaatcagtta atccaaatgg caggcgagta aggaggcgaa cataa 1245791245DNAGlycine max 79atggcggaat tggaggagga gcggatgggg gatttcaagc ccttctcgga aggttcttcg 60tgctcgcgtt cgatcagcga aaccgtgaac ggctcgcacc aattcacgat aaagggttac 120tctttggcca aagggatggg tgctggaaag tacatcatga gcgacacttt caccgttggt 180ggttacgatt gggctattta cttctacccc gatgggaaga accctgagga caattccatg 240tacgtttcgg tctttattgc gctcgctagc gacggaaccg atgttagggc tttgttcaag 300ttgacgctgg tggatcagag tgagaagggg aatgataaag ttcatagcca tttcgatcgc 360cctctcgaga gtggacctta taccttgaag tataaaggca gcatgtgggg ttacaaacgc 420ttcttcagaa gaacacaact ggaaacctca gagtatctaa aaaatgattg ccttgtcatg 480cattgcactg ttggtgttgt taaaactcgt tttgagggat ctaaacaggg tgttattgtg 540ccacagtcag acatgggccg ggattttaag gacttgttgg aatctgaggt cggttgtgac 600atacttttca aggtcaaaag tgaaagcttc aaagctcata agttgatact tgcagcccga 660tctcctgtgt ttagagccca gttttttggg cttgttgggg atcctacctt agaggaagta 720gtggtagagg atattgagcc ctttatcttc aaggcaatgc ttctctttgt ttactctgac 780aaacttcctg gcatatatga ggttatggac tcaatgccct tgtgctcata caccgtcatg 840gtgcagcatc tcttggctgc tgctgatctc tataatcttg atcggctcaa actgctttgc 900gaatcaaaat tgtgtgaaga aatcaatact gacaatgtgg ccacaacact tgcgctggca 960gagcaacatc actgtccaca gcttaaggca atctgtttaa aatatattgc aaatcctgca 1020aacttgggag ctgtaatgca gtcagaagct tttgtgcatt tgaaagagag ctgcccctca 1080atgctgttgg aattgctgga gacatttgca tcagtggatg ataactcagg ccagacattg 1140agcagaaaga gaagtggcag tagcatatat gggcaagatt tagcagacgg ggcagctgct 1200gaatcagtta atccaaatgg caggcgagta aggaggcgga cataa 1245801236DNAPhoenix dactylifera 80atggcgaagc tcgaggagga gcagggagga ttgaacaacc gtcagctcaa tccgctgaac 60gtgtcgcggt ctcggtcggt gtgcgagacg gtaaacgggt cgcaccggta cacggtgaag 120gggttctcgc tggcgaaggg gatgggtcct ggaaggtacc tgtccagcga caccttcacc 180gtggggggat tccagtgggc cgtctacttc tatcccgacg gcaagaaccc ggaggacaac 240tccctttatg tctcggtgtt cattgccctg gcgagcgagg ggaccgacgt gagggcgctc 300ttcgaactca ctctgctcga ccagaacggc aaggggaggc acaaggtgca cagccacttc 360gatcgggcgc tggaggccgg gccctacacg ctcaagtacc gggggagcat gtggggttac 420aagcggtttt acaggaggac atccttagaa acatcggatt atctcaagga tgattgtcta 480attatgaact gcacagtggg tgttgttaga aaccatattg aaacaccaac acagctttca 540atttctgtac caccacctga cttgggtcag tgtctcaagg agttgttcat atctggcatt 600ggttctgaca tagattttga ggttggtgat gagacattta aagctcacaa gcagattctt 660gctgctcgct cgccagtttt tagtgcacaa ttttttggtc ttatcgggaa tccaaatgtg 720gacaaaattg ttgtggagga tgttgaacct cctattttca aggccatgct tctgtttata 780tattcagatg aactccctga tgtgcatgat ctaactggat ctgtttctat gtgcacatcc 840acgattatgg tacaacattt attggctgca gcagatagat atggactgga acgtctgaag 900ctgttatgcg aagcaaaact gtgcgaagaa gtcactgctg atactgtagc aacaaccttg 960gccctggcag agcaacacca atgtgctcaa ttgaaggctg tctgcttaaa atttacagca 1020gctcgagaaa acttgggagc tgttatgcag actgaagggt tcaattactt ggaggcgacg 1080tgcccatctt tgctgtcaga cttgttggca actgttgctg tggcggatga tgactctagt 1140cctatcagca ggaagaggag cggtagcagt aacatagggc tcaatttaat ggacagtgtt 1200gatttgaatg ggaggcgtat gaaaaggcgg atgtag 1236811290DNAFragaria vesca 81atgccaccga ttcagaaaca ctccctccgc ggcgcgcaat tgggcggtag aatctcatcc 60atgaagtcga agctcgaaaa cgacgagtcg tgttcgcggt cgatcagcga gaccgtgaac 120ggctcccacc ggttcaccat aaaggggtat tccttggcca aaggaatggg cgccgggaaa 180tacatactca gcgacacttt caccgtcggc ggttacgatt gggcgattta cttttacccc 240gacggtaaaa accccgagga tagctccgtc tacgtctccg tcttcattgc gctggtgagc 300gaaggcaccg acgtgagggc cttgtttgag ctcaccttgg tggaccagag caacagcggc 360aaggacaagg tccatagtca ctttgatcgt gcccttgaga gcgggcctta cacgttgaag 420taccgtggaa gcatgtgggg ttacaagcga ttcttcagaa gatcagccct tgaaacgtcc 480gagtttctaa aggatgattc ccttgtgttg aactgcactg ttggagtcgt cagaactcgc 540ctagagtgtc cgaaacattt tgcaattact gtaccaccat cagacatggg tgaaggtctt 600aaggcctttc tagactctgg agctggttgc gacctggttt ttcaggttgg cgatgaggaa 660ttcaaagctc acaagttgat acttgctgct cgttctcctg tattcaaagc acagtttttt 720ggacatcttg gagattcgag tgtagataaa gtagtcgtga aggatgttga gcccttcatc 780ttcaaggcaa tgcttctttt tatatacggg gacaaacttc ctgatatccg tgaagttaca 840ggttcatcat ctttgtgcac attcactgtc atggtgcagc atctgttggc tgctgcagac 900ctgtatgacc tagagcgact gaagttgttg tgtgaatcaa tgttgtgtga agaaatcacg 960actgaaacag tggcaaccac attggccctt gctgagcagc atcactgtcc acagctgaag 1020gctgtgtgtc taaagtttgc ggcaaagtca acaaacttgg gagctgtaat gcagtcagat 1080ggatacaagc atctagaaga gagctgcccc tcagtgttac aggagctgct gaagacattt 1140gcatctgtcg atgccaatga gaattcaaat tcaagtaaga agaggagtgg cagcagcata 1200tatgggctag acttgccagc agatggcagt ggggcagtag cagaatcagc aaatcccaat 1260ggtaggcggt tgaggccgcg gcgatattaa 1290821281DNAMalus domestica 82atgccgccga ttcggaaaca ttccagaggg gcgaaatcgg gtgaatccat ggggaattcg 60aagcctgggt tcgaccagga atcgtgctcg agatcgatca gcgagactgt gaacggctcc 120caccggttca cgataaaggg gtattcgctg gccaaaggga tgggagccgg gaagtacctg 180atgagcgata cgttcacggt gggcggatac gattgggcaa tttactttta ccccgacggt 240aaaaaccccg aggatagcaa cgcgtacgtc tcggttttca ttgctttggt tagtgagggt 300acggatgtga gggctctgtt cgagctgacg ttggtggatc agacggacag tgggaaggac 360aaggtgcaca gtcactttga tcgcgctctc gagggcgggc cgtacacgct gaagtacaga 420ggcagcatgt ggggttacaa gaaattcttc agaagatcaa tcctagaaac ttctgagttc 480cttaaggatg attgccttgt attgaactgc actgttggag ttgtcagaac tcgccttgag 540caaccaaaac aatttacaat cactgttcca tcatcagaca tgggacgaga cctaaaggac 600tttctagatt ctgaagctgg ttgtgacata gtttttcagg ttggtgatga acagtttaaa 660gctcacaagt tgatacttgc tgctcggtct cgtgtattta gagcgcagtt ttatggactt 720gtcggggatt gtaacgtaga taaagtagtt gtgaaggatg ttgagccctt catcttcaag 780gcaatgcttc tctttattta cacggacaaa cttcctgata cacacgaagt tatgggctca 840tcacctttgt gcacattcac tgtcatggtg cagcatctgt tggcagctgc agacctgtat 900aatctagatc gactgaaatt gttgtgtgaa tcaaagttat gtgaagaaat cactactgag 960acagtggcga ctacacttgc gcttgctgaa cagcatcaat gccgacagct taaggatgtc 1020tgtcttaaat ttacagcaaa tccgtcgaac ttgggagctg taatgcaatc agaagggtac 1080aagcatctag aagagagctg cccatcaatg ttggtagagc tgctggagac atttgcagcg 1140gtggatgaca attctagtct tctgtcaagt cggaagagga gtggcagcag catatatgga 1200ctagatttgc cagcagatgg gggtgggact gcagcagaat cagcaaatcc caatggtagg 1260cgcgtgaggc ggcggtttta g 1281831227DNASolanum lycopersicum 83atgaaccaaa tttccgtcga ccgtgccggg aaggattcat catccaagtc tgtaaacgaa 60acggtgaatg ggtctcacca ttttaccatc aggggttact ctttggccaa aggaatggga 120ccgggaaagt acatatctag cgacattttc accgttggtg ggtatgattg ggcaatttat 180ttctacccag atggtaaaaa catagaggat tcttcaatgt atgtgtctgt ttttatagca 240ttggctagcg aaggaacgga tgttagggcg ttgtttgagt tgacgatgtt ggatcagagt 300ggaaaagtga aacataaagt tcatagccat tttgatcggg cattggaaag tggaccttat 360actttgaaat atagaggaag catgtggggt tacaaacgat tttttagaag agcaagttta 420gaaacttctg actacctgaa ggatgattgc ctttccatgc actgtactgt tggagttgtc 480agaactcgtg ttgaaggccc caaaaattat agtgttacaa ttccaccttc agacatgggt 540caaagtctca aatacttgct ggatgctgaa cttggttgtg atatagtttt ccgggttgga 600gaagaggcat ttaagggtca taagttgata cttgctgctc ggtctcctgt atttagagca 660caattctttg gccttattgg gaatcctaaa acggacgaag tggaaattga ggatattgaa 720ccctcagtct tcaaggctat gcttcagtac atttattctg atgaacttcc agatttgatt 780gaaattactg gctctacttc aacttgcact tctacgatag tgacacagca tctattggca 840gcagccgatc gatttggtgt agataggttg aaagagttat gtgaggcgaa attgtgtgaa 900gaagttaatg tggatactgt ggcaacaact ctttctcttg ctgagcagca tcggtgccca 960caactcaagg ccatctgttt gaaatttgca gctacaaact tgggagtggt catgcagaaa 1020gatggattca agcacttgga agagagttgc cccttattgt tgtcagagct gctggaaaca 1080gtagcatccg tcgatgagaa gccaagtctg acgtctagca agaaaaggaa tagcagcagc 1140agcatctttg gactggatct ggctgcagat ggcgcggcag cagattctgt taaccttacc 1200gctaggcggg tgaggaggag gatgtaa 1227841227DNASolanum tuberosum 84atgaaccaaa tttccatcga ccgtgccgga aacgattcgt catccaagtc tgtaaacgaa 60acggtgaatg ggtctcacca ttttaccatc aggggttact ctttggccaa aggaatggga 120cctggaaagt acatatctag cgacattttc accgttggtg ggtatgattg ggcaatttat 180ttctacccag atggtaaaaa catagaggat tcttccatgt atgtgtctgt ttttatagca 240ttggctagcg aaggaacaga tgttagggcg ttgtttgagt tgacgatgtt ggatcagagt 300ggaaaagtga aacataaagt tcatagccat tttgatcggg cattggaaag tggaccttat 360actttgaaat atagaggaag catgtggggt tacaaacgat tttttagaag agcaagttta 420gaaatgtctg actacctgaa ggatgattgc ctttccatgc actgtactgt tggagttgtc 480agaactcgtg ttgaaggccc aaaagattat agtgttacaa ttccaccatc agacatgggc 540caaagtctca aatacttgct ggatgctgaa cttggttgtg atatagtttt ccgggttgga 600gaagaggcat ttaagggtca taagttgata cttgctgctc ggtctcctgt gtttagagcc 660caattctttg gccttattgg gaatcctaaa acggacgaag tggaaattga ggatattgaa 720ccctcagtct tcaaggctat gctccagtac atttattctg atgagcttcc agatttaatt 780gaaattactg gctctacttc aacttgcact tctacgatag tgatgcagca tttattggca 840gcagctgatc gatttggttt ggataggttg aaagagttat gtgaggcgaa attgtgtgaa 900gaagtcaatg tggatactgt ggcaacaact ctttctcttg ctgagcagca tcgatgccca 960caactcaagg ccatctgttt gaaatttgca gctacaaact tgggagtggt catgcagaaa 1020gatggattca agcacttaga agagagctgc cccttactgt tgtcagagct gctggaaaca 1080gtggcatccg tcgatgagaa gccaagtctg acgtctagca agaaaaggag tagcagcagc 1140agcatctttg gactagatct ggctgcagat ggcgcagcag cagattctgt taaccttacc 1200gttaggcggg tgaggaggag gatgtaa 1227851203DNAOryza brachyantha 85atgacggtgc cgccgccgac gccgcccccc tcgtggtctc gctccgtcac ggagaccgtg 60cggggatctc accagtacac cgtcaagggc ttctccatgg ccaagggcat gggccccggc 120cgctacgtca ccagcgacac cttcgccgtc ggcggctacc actgggccgt ctacctctac 180cccgacggta agaaccccga ggacaacgcc aactacgtct ccgtcttcgt cgccctcgcc 240tccgacgggg ccgacgtccg cgccctcttc gagctcaccc tcctcgacca gtccggccgc 300ggacgccaca aggtccattc ccatttcgac cgatccctgc aggccggacc ctacaccctc 360aagtaccgag gctccatgtg gggttacaag cgcttctaca gaagatcact cctagaatct 420tccgactttc tcaaggacga ttgccttgta atgaactgca cagtaggcgt cgtcaagaac 480cgtctcgaaa ccccaaagaa cattcagatc cacattccgc cttctgacat gggccgttgc 540ttcaagaacc ttctcaacct cggcattgga tgtgacataa ctttcgaggt tggtgatgac 600acagtccagg cacacaagtg gattcttgct gctcgctccc cggtattcaa agcccaattc 660tttggtccta ttgggaatcc tgacctacac tcggtcactg tggaggatgt tgaacctgtt 720gttttcaagg cgatggtgaa tttcatatac tccgatgaac ttcctagtat tcatgaacta 780gctggatctg tctcaacatg gacatcgaca gtagtagtac agcatttgtt ggcagcagct 840gatagatatg gattagatcg gctacgtctc ctatgcgagg aaaagttatg tgatgaactc 900acagctgaaa cagttgcaac aaccttagcc ctagctgaac aacatcattg tactcagctg 960aaatctgctt gcctaaagtt cactgccgtt cgggaaaatc tgggagctgt gatggagaca 1020gaaggattta actacttgga ggagacatgc ccgtccctac tgtccgactt gttggctact 1080gtcgcagtgg tggatgatga ttctgcaaca ttaaaccgga agaggggagt cagtggtaac 1140gaaggagcga atcccgtgga gagcgtggag gctagtgaaa ggcgcatccg caggagggtt 1200tag 1203861194DNABrachypodium distachyon 86atggcggcgg tgccgcggcc gtcgtggtcg cgctcggtca gcgagacggt gcgggggtcg 60caccagtaca ccgtcaaggg cttctccctc gccaagggca tcggtcccgg ccgccacctc 120gccagcgaca ccttcgccgt cggcggctac gactgggccg tctacctcta ccccgacggc 180aagaaccccg aggacaacgc cagctacgtc tccgtcttcg tcgccctcgc ctccgagggc 240accgacgtcc gcgccctctt cgagctcacc ctcctcgacc agtccggccg cgcacgccac 300aaggtccact cccacttcga ccgctccatg caggccggac cgtacaccct caagtacagg 360ggatccatgt ggggttacaa gaggttctac agaaggtcac agttagaaac atcagatttt 420ctaaagaacg attgcctagt aatgaactgc acagtaggtg ttgtcaagac tcggctcgaa 480acaccaaaga acatccagat taacgttcct ccatctgaca tcggccgttg cttcaaggag 540ctcctcagac tccgcattgg ctgtgacata acatttgaag taggtgacga gaaggtccag 600gcacataaat ggattcttgc tgctcgttcc ccagtattca aagcccaatt ctttggacca 660attggtaaag ctgacttgga cagagttgtt gtggaggatg ttgaacctat cgtcttcaag 720gcaatggtga atttcatata ctctgatgag cttcctagta ttcatgaact agctggatct 780ttctcaatgt ggacatcaac tgcagttata cagcatttgt tggcagcagc tgatagatat 840ggattggacc ggctacgaat actatgtgag gcacagttat gtgatgggct tactgctgaa 900acagttgcga caaccttagc cctggctgaa cagcatcatt gtgctcagct caagtcagcc 960tgcttaaagt ttactgctgt ccgagaaaat cttggagttg tgatggagac tgatgggttt 1020aactacttgg aggagacatg cccatccctg ctgtctgatt tgttagcaac cgtcgcggta 1080gtggacgatg atcctacatc tgttaaccgg aaaaggggag tttgtatcaa cgaagatgtg 1140aatccagttg aaagtgttga ggctagtgac aggcgcatcc gcaggagggt ttag 1194871188DNAOryza sativa 87atgacggcgg cggcgtcgtg gtcccggtcg gtgacggaga cggtgcgggg gtctcaccag 60tacacggtga aggggttctc gatggcgaag ggcgtagggg ccgggcggta cgtgagcagc 120gacaccttcg cggtgggcgg ctaccactgg gccgtctacc tctaccccga cggcaagaac 180cccgaggaca acgccaacta cgtctccgtc ttcgtcgccc tcgcctccga cggcgccgac 240gtccgcgccc tcttcgagct caccctcctc gaccagtccg gccgcggccg ccacaaggtc 300cactcccact tcgaccgatc cctccaggcc ggaccctaca ccctcaagta ccgaggctcc 360atgtggggct acaagcgctt ctaccgaaga tcactcttag aatcatccga ctttctcaag 420gacgactgcc tcgttatgaa ctgcactgta ggcgtcgtca agaaccgtct cgaaacacca 480aagaacatcc acatcaatat tcctccatcc gacatgggcc gttgcttcaa caacctcctc 540aatctccgca tcggctgtga cgtatctttt gaggtgggtg atgaaagagt ccaggcgcac 600aagtggattc ttgctgcccg ctcccctgta ttcaaagccc aattctttgg tcctattggg 660aatcctgacc tacacacagt cattgtcgag gatgtagaac ctcttgtctt caaggcaatg 720gtgaatttca tatactctga tgaacttcct agtattcatg aactagctgg atctgtctca 780acttggacat cgacagtagt agtacagcat ttgttggcgg

ctgctgacag atatggacta 840gatcggctac gtctgctatg cgaggaaaag ttatgtgatg aactcactgc tgaaacagtt 900gcaacaactt tagccctagc tgaacaacat cattgtactc agctgaaatc tgcttgtctg 960aagttcactg ctgttcggga aaatctggga gctgtgatgg agacagaagg atttaattac 1020ttggaggaga catgcccgtc cctgctatct gacttgttag ctactgtcgc agtagtggat 1080gatgatgctg cgtcattcaa ccggaagagg ggagtcggtg gtaacgaagg agcgaatcct 1140gtggagagcg tggaggctag tgataggcgc atccgcagga gggtttag 1188881191DNAHordeum vulgare 88atggcggtgc cgcggccgtc atggtcgcgg tcggtcacag agaccgtgcg gggttcgcac 60cagtacaccg tcaagggatt ctccctcgcc aagggcatcg gccccggccg gcacctctcc 120agtgacacct tcgccgtcgg cggctatgac tgggccgtct acctctaccc ggacgggaag 180aaccaagagg acaacgccaa ctacgtctcc gtgttcgtcg ccctcgcctc cgagggtacc 240gacgtccgcg ccctcttcga gctcaccctc ctcgaccagt ccggccgcgc ccgccacaag 300gtccactccc atttcgatcg atccatgcag gccggaccat acaccctcaa gtacagagga 360tccatgtggg gttacaagag attctacaga aggacacagt tagaagcatc agatttttta 420aaggatgatt gcctagtaat gaactgcaca gtaggtgtcg tcaagaaccg tctcgaaaca 480ccgaagaata tccagattaa tgtcccccca tctgatattg gtcgttactt caaggaactc 540ctcaaactcc acattggctg cgacataact tttgaagtag gtgatgagaa agtccaggca 600cataaatgga ttcttgctgc tcgctcccct gtgttcaaag cccaattctt tggacctatt 660ggtaaacctg acttggacag agttgttgtg gaggatgttg aacctatcgt cttcaaggca 720atggtgaatt tcatatattc tgatgagctt cctagtattc atgaagtagc tggatctttc 780tcaatgtgga catctactgc ggtaacacaa catctgttgg cagcagctga tagatatgga 840ttggaccggc tacgaatcct atgtgaggca aagttatgtg atgaactcac ttctgaaaca 900gtagcgacaa ccttagccct agctgaacag caccactgtg ctcagctcaa gtctgcctgt 960ctaaagttca ctgctgttcg acaaaatctg ggagctgtga tggagacaga agggtttaat 1020tacttggagg agacttgccc atccttgctg tctgatttgt tagcaacagt cgcagtagtg 1080gatgatgatc ctgcatctgt taaccggaaa aggggagttt gtatcaatga agatgcgaat 1140cccgtcgaaa gcgttgaggc tagtgacagg cgcacccgca ggagggttta g 1191891230DNASelaginella moellendorffii 89atggcacgga cgtcggtagt cttgcaggac gattcagggc aagtggtcgg gagtcccaca 60tccacggcaa cgccttcccg atctcgatgc atcacagaga ctgtgaatgg atctcaccat 120ttcacgatcc atggctattc cctggccaaa gggatgggcg tagggaagta cattgcgagc 180gacacattca cggttggggg ctaccagtgg gcgatctact tctatccgga tgggaagaac 240accgaggaca actcgctcta cgtgtcggtg ttcatagctc tggcaagtga agggacggat 300gtgagggcgc tgttcgagct gacgcttctg gatcaaagcg gcaagaacaa gcataagatc 360cacagccact ttgatcgttc gctggagagt ggtccttaca cactgaagta tcgaggcagt 420atgtggggtt acaagcgctt cttcagacgg gccgtgctcg agacgtccga ttttctgaaa 480gacgacagtc tttcaatcac ctgcacggtc ggcgtcgtag tttcctccat gcaagccttg 540aagcaacact ctttgttagt tccggaatcc gatattggcc aacatttcct gtctttgttg 600gaaagtggtg aaggaacgga cgttaacttt aacgtaaaag gggaggcatt cagtgctcac 660aagttgttac tggctgcgag atccccagtg ttcaaagcgc agctgtttgg acccatgaag 720gacgagaatg gtgacgtgat cgaaatcgac gacatggaac cacctgtctt caaggccatg 780ctacacttta tatataaaga cagtctgccc gataccaacg agatgacagg gtcttcgtca 840cagtcgacgg cgacgatgat ggctcagcat ttactcgcag ccgcagatag gttttgcctg 900gatcgtttaa gacttttgtg cgagtccagg ctctgtgaac agatcactgt tgacacagtg 960gcgactacgc ttgcgttggc agaccaacac catgcatctc agctcaaaaa tgtctgcctc 1020aagttcgctg cttccaacct tgcagtggtg atgcagtctg atggttttga gtacctgcgt 1080gagagctgcc cgtcattaca atccgagctc ctcaagacgg tcgcgggagt agaagaagaa 1140gccaaggctg gaacaaagaa caggaccgtc tggacgcacg tcgcagatgg tggcgacgga 1200ttgggaaggc gcgtgcggca aaagatctga 1230901233DNAMedicago truncatula 90atgggtaaga ttctccgaga aaccgcgaaa ccatcttcca atccatcatc accatcttcc 60tcatcggaac cggcgacaac ttcttcgaca tcgataaccg aaacagtgaa aggctcgcac 120cagttcaaga tcactgggta ctcgctttcg aaagggatcg ggattgggaa atacatagcg 180tcggatatct tttcggttgg tgggtacgat tgggccattt atttctaccc tgatggaaag 240agtgttgagg ataatgctac ctatgtgtcg cttttcattg cgcttgcgag tgatgggact 300gatgttaggg ctctttttga gttgaccctt ttggatcaga gtgggaaaga gaggcataag 360gttcatagcc attttgagag gactcttgaa agtggacctt ataccttgaa ataccgcggt 420agtatgtggg gttacaagcg gttttttaag aggacagctt tagagacatc tgattacctt 480aaagatgatt gcctttctgt taattgtagt gttggtgttg tgaggtcacg cacggaaggc 540ccaaagatat attccattgc aataccacct tctaacattg gtcaccaatt tggtcaactg 600ctggaaaatg gtaaaggaag tgatgtgagc tttgaagtgg atggggaagt tttcactgct 660cataaattgg tgctagcagc tcgttcacct gttttcagag cccagctttt tggtcctatg 720agagatcaaa gtacccagtc tattaaagtt gaagacatgg aagctccagt ttttaaggca 780ttgcttcatt ttatgtactg ggactcgctg cctgacatgc aagagcttac tgggatgaac 840acaaaatggg caacaacctt gatggcccaa catcttctag cggctgctga tcgttatgcc 900ttagagaggc tcaggcttat atgtgaagcg agtctatgtg aagatgttgc cattaatacc 960gtggctacaa ctttagcctt ggcagagcaa caccactgtt tccagctgaa agcagtctgt 1020ctcaagttta ttgccacctc tgaaaatctc agagctgtga tgcaaactga tggatttgag 1080tacttgaagg aaagttgccc atctgttctg actgagctac tggagtacgt ggctagattt 1140actgagcatt cggacttttt gtgcaagcac aggaatgaag caatacttga tggtagcgac 1200ataaatggaa ggcgggtgaa gcaaaggctt tag 1233911230DNACoffea canephora 91atgggaaggg tttacaatgg agaaacctcc aacccgtcgt cttccacaac ggcgtcaaca 60tcgccgccgc cggtgacgac gtcgacgtcg atcacggaga ctgtgaatgg aacgcacgat 120tttaagatca cggggtattc cttgtccaag ggaattggga ttggcaagta cgtagcgtct 180gatattttca tggtgggagg ctatgcgtgg gcgatctatt tctatcctga tgggaaaagc 240gtggaggaca atgcgacgta tgtttccttg tttattgcgc tagccagcga gggaacggac 300gttagagcgc tgtttgaact gacgcttatg gatcagagcg ggagagcgag gcataagatt 360catagccatt tcggaagggc tttagagagt gggccttaca cgttaaaata ccgcggaagc 420atgtggggct ataagcggtt ttttaagaga actgcactag aaacatcaga ctatctgaag 480aatgattgtc ttcaggttca ttgttgtgtt ggtgtagtta gatcccaaac tgagggaccc 540aaaatctact ctataccgct tccaccttcg gacattggtc aacattttgg gcagctactg 600gaatgtggaa agggaactga tgtaaatttt gaagtcaatg gagaaaaatt ttctgctcac 660aagttggttc ttgctgcgcg ctcacctgta tttagagctc aactatttgg cccaatgaaa 720gatcatgaca cacaatgtat tcgagttgaa gacatggaag ctcctgtttt taaggctcta 780cttcatttca tatactggga ttgcttaccc gatatggaag aacttactgg tttgaactca 840aaaggggcta caagcttgat ggctcaacat ctgcttgctg ctgcagatag atatggtttg 900gataggctca ggttgatatg tgaagctaat ctctgcgagg atgttgccat aaatactgtt 960gctactacgc tggcccttgc agagcagcat cactgtttcc agctgaagtc tgtatgccta 1020aaatttgttg ccatgccaga aaatcttagg gctgttatgc agacagacgg gtttgaatac 1080ctaaaagaaa gttgtccaag cgtgctcaca gaattgttgg agtatgtagc taggatcaat 1140gagcattctg tcagtgtgaa caagcaattg actgatggta tattggacgg gagtgatgtc 1200aatggtcggc gggtgaagca gagattgtag 1230921200DNAZea mays 92atggcgattc cgccgcggac tccttccccg ccgccatcgt ggtcgcgctc tgtaaccgag 60accgttcggg ggtcccacca gttcaccgta cggggctact ccctcgccaa gggcatgggc 120cccggccgct acctcgccag cgacgtcttc gccgtcggag gataccactg ggccgtctac 180ctctaccccg acggcaagaa cgccgaggac aactccaact acgtctccgt tttcgtcgcc 240ctcgcttccg acggcatcga cgtccgagcc ctcttcgagc tcaccctcct cgaccagtcc 300ggccgcggct gccacaaggt tcactcgcac tttgaccgct cgctcaagtt cggcccatac 360accctcaagt acaggggatc catgtggggt tacaagcgct tctacaaaag aacactcttg 420gaagaatctg atttcttaaa gaatgattgc ctagtgatga actgcacagt aggtgttgtc 480aagaaccgta tagaaacacc aaaggacatc cagattcatg ttccacgatc agacatgggc 540cgctgcttca aggagctcct cagccgctgc attggatgtg acataacatt cgaagtgcga 600gatgagaaag tcagggcaca caagtggatt cttgctgctc gctccccagt atttaaagcc 660cagttctttg gtcctattgg aaagcctgac ctgcacacgg ttgttgtgga ggatgtggaa 720cctgttgtct tcaaggcaat ggtgaacttc atttacgctg atgaactccc cagcattcct 780gagctagctg ggtctgcctc aacgtggaca tcaacagtag tagtacagca tttgttggca 840gcagctgata gatatggact ggtccgtctg cgtatcctgt gtgaatcaaa gctctgtgat 900gaactgactc ctgaaactgt cgcaacaact ttagcccttg ctgaacagca ccattgtgct 960gagctgaagt ctgcatgtct aaagttcatt gctttgcgag gaaatttggg agctgttatg 1020gagacggaag gctttgatta cctggaggat acatgcccgt ccctactatc tgacttgtta 1080gctactgtgg cagtcgtgga cgacgatctt gcatccctta accgaaaaag gggagtcagc 1140gggaaccaag tcatggctct agtgggaagc gttgaaaggc gcacccggag gaagctttag 1200931209DNASorghum bicolor 93atggcgattc cgccgcggac tcctcccccg ccgccatcgt ggtcgcgcta cgtcaccgag 60accgtgaagg ggtcccacca gttcaccgtc cggggcttct ccctcgccaa gggcatgggc 120cccggccgcc acctcgccag cgacatcttc gctgtcggag gataccactg ggccgtctac 180ttctaccccg acggcaagaa cgccgaggac aactccaact acgtctccgt cttcgtcgcc 240ctcgcctccg acggcatcga cgtccgagcc ctcttcgacc tcaccctcct cgaccagtcc 300ggccgcggcc gccacaagat tcactcgcac tttggccgca agctagattc cggcccatac 360accctcaagt acaggggctc catgtggggt tacaaacgct tctacaaaag atcactcttg 420gaagcatctg atttcttaaa gaatgattgc ctagtgatga actgcacagt aggtgttgtc 480aagaaccgta tggaaacacc aaaggacatc cagattcatg ttccacgatc agacatgggc 540cactgcttca aggagctcct cagccgcggc attggatgtg acataacctt cgaagtgcgc 600gacgagaaag tcagggcaca caagtggatt cttgctgctc gctccccagt atttaaagcc 660cagttctttg gtcctattgg aaagcctgac ctgcacacgg ttgtcgtgga ggatgtggaa 720cctgtcgtct tcaaggcaat ggtgaacttc atgtacactg atgaactccc cagcatttct 780gagctagctg gatctgcctc aacatggaca tcaacagtag tagtacagca tttgttggca 840gcagctgata gatatggact ggaccgtctt cgtatcctgt gtgaatcaaa gctatgtgat 900gaactgactc ctgaaactgt cgcaacaacc ttagcccttg ctgaacaaca ccattgcgct 960gagctgaagt ctgcctgtct aaggtttgct gctgtgcgag aaaatttggg agctgttatg 1020gggacggaag gctttgatta cttggaagag acatgcccgt ccctactatc cgacttgtta 1080gctactgtgg cagaagtgga cgatgatcct gcatcccttg accgaaaaag gggagtttgc 1140ggtaaccaag tcttggctcc agtggaaagt gtcgaggcta ctgaaaggcg cacccggagg 1200aggctttag 1209941236DNACucumis melo 94atgggcacga ttaaatcttg cagggatacc tctaaatcct actcaaatct tcggtcgccg 60acgcctccac cagtgacttt ttcaacttct cgtttcgaga ccgtcaatgg atcgcatgag 120ttcaagatca atgggtattc ccttaataaa gggatgggga ttgggaaata catcgcgtct 180gataccttta tggttggggg atatgcgttt gctatatatt tttacccaga cgggaagagc 240gtcgaggata acgcatcgta tgtctcggtt tttatagcgt tggctagtga agggactgac 300gttagagccc tttttgaatt gacgttgttg gatcaaagtg ggaaggagaa ccacaaggtg 360cacagccatt tcgagagaag actcgagagt ggtccttata cgcttaaata tcgaggaagc 420atgtgggggt ataaacgtta ttttaaaaga acagttttag aaacatccga cttcctaaag 480gacgactgcc ttgaaatcca ctgtgtagtt ggtgttgtta agtcccatac agagggacca 540aagatttact ccataacacc accaccttct gatataggcc agcattttgg gaagcttttg 600gagagtggga aactaactga tgtgaacttt gaagtagatg gggaaacatt ttctgcccac 660aagttagttc ttgctgcgcg gtcacctgtc tttagggcac aactctttgg ccctctgaag 720gaccagaata ctgagtgtat aaaagtcgaa gatatggaag ccccagtatt taaggcattg 780cttcatttca tatactggga tgctctacca gatatgcaag aaattgtagg tttaaactca 840aaatgggctt ccactctgat gtcccagcat ctacttgcgg cagcagacag atatgcactt 900gacagactca aattgctatg cgaggctaaa ctttgtgagg acgttgctat aaatacagtg 960gcaacgacat tggcattggc tgagcagcat cactgtttcc aactaaaagc tgtatgtttg 1020aaagtcattg cattgccgga gaatttgaga gctgtaatgc aaacggaggg gtttgaatat 1080ttgaaagaga gctgcccatc ggttctcact gaactactag aatatgtagc aagggtgacg 1140gagcatgcag tgattacttg cagcgggtat ggaaatggaa cagtgttaga tggtagttac 1200gtgaatggaa gacgggtaag gcagaggttg tattga 1236

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