CN105612171A - Methods of modulating seed and organ size in plants - Google Patents

Abstract

The present invention relates to plant E3 ubiquitin ligase (designated DA2) which acts synergistically with DA1 to control seed and organ size. Methods for increasing yield in a plant are provided, the methods comprising reducing expression or activity of DA2 in a plant lacking expression or activity of DA 1. Plants having increased yield and methods of producing such plants are also provided.

Description

Methods of regulating seed and organ size in plants

technical field

The present invention relates to methods of altering the size of seeds and organs of plants, for example in order to increase plant yield.

Background technique

Seed and organ size are agronomically and ecologically important traits under genetic control (Alonso-Blanco, C. PNAS USA 96, 4710-7 (1999); Song, X. J. Nat Genet 39, 623-30 (2007); Weiss , J. Int J Dev Biol 49, 513-25 (2005); Dinneny, J. R. Development 131, 1101-10 (2004); Disch, S. Curr Biol 16, 272-9 (2006); Science 289, 85-8 (2000); Horiguchi, G. Plant J 43, 68-78 (2005); Hu, Y Plant J 47, 1-9 (2006); Hu, Y. Plant Cell 15, 1951-61 (2003); Krizek, B.A. Dev Genet 25, 224-36 (1999); Mizukami, Y. PNASUSA 97 , 942-7(2000); Nath, U.Science299, 1404-7(2003); Ohno, C.K.Development131, 1111-22(2004); Szecsi, J.EmboJ25, 3912-20(2006); White, D.W.PNASUSA103 , 13238-43(2006); Horvath, B.M.EmboJ25, 4909-20(2006); Garcia, D.PlantCell17,52-60(2005). The final size of seeds and organs is constant within a given species, while species The size of seeds and organs varies greatly between the two species, suggesting that plants have regulatory mechanisms to control the growth of seeds and organs in a coordinated and timely manner. However, despite the importance of seed and organ size, the The molecular and genetic mechanisms are poorly understood.

Genetic regulation of seed size has been studied in plants including tomato, soybean, maize and rice using quantitative trait loci (QTL) mapping. So far, in published literature, two genes have been identified (Song, X.J. Nat Genet 39, 623-30 (2007); Fan, C. Theor. Appl. Genet. 112, 1164-1171 (2006)), both Two genes are potential two major QTLs for grain size in rice; however, the molecular mechanisms of these genes remain to be elucidated. In Arabidopsis, eleven loci affecting seed weight and/or length in crosses between accessions Ler and Cvi have been mapped {Alonso-Blanco, 1999 supra} but have not yet been identified corresponding gene. Recent studies have revealed that AP2 and ARF2 are involved in controlling seed size. Unfortunately, however, ap2 and arf2 mutants are less fertile than wild type (Schruff, M.C. Development 137, 251-261 (2006); Ohto, M.A. PNASUSA102, 3123-3128 (2005); Jofuku, K.D. PNASUSA102 , 3117-3122 (2005)). In addition, studies using mutant plants have identified several positive and negative regulators that affect organ size by acting on cell proliferation or enlargement {Krizek, B.A. Dev Genet 25, 224-36 (1999); Mizukami, Y. ProcNatlAcadSciUSA97, 942- 7(2000); Nath, U.Science299, 1404-7(2003); Ohno, C.K.Development131, 1111-22(2004); Szecsi, J.EmboJ25, 3912-20(2006); White, D.W.PNASUSA103, 13238- 43 (2006); Horvath, B.M. Embo J 25, 4909-20 (2006); Garcia, D. Plant Cell 17, 52-60 (2005). Horiguchi, G. Plant J 43, 68-78 (2005); Hu, Y Plant J 47, 1-9 (2006) Dinneny, J.R. Development 131, 1101-10 (2004)).

Several factors involved in ubiquitin-related activities are known to affect seed size. The growth limiting factor DA1 is a ubiquitin receptor and includes two ubiquitin-interacting motifs (UIMs) that bind ubiquitin in vitro, and the da1-1 mutant forms large seeds by affecting the maternal integument of the ovule (Li et al. , 2008). Mutations in one enhancer of da1-1 (EOD1), which encodes the E3 ubiquitin ligase BIGBROTHER (BB) (Disch et al., 2006; Li et al., 2008), synergistically enhance the seed size phenotype of da1-1, thereby indicated that DA1 cooperates with EOD1/BB to control seed size. In rice, the quantitative trait locus (QTL) encoding E3 ubiquitin ligase of grain width and grain weight 2 (GW2) controls grain size by restricting cell division (Song et al., 2007). A GW2 homolog in wheat has been identified (Ta-GW2; Bednarek et al. 2012). An unknown protein encoded by rice qSW5/GW5 is required to limit grain size in rice (Shomura et al., 2008; Weng et al., 2008). GW5 physically interacts with polyubiquitin in a yeast two-hybrid assay, thus suggesting that GW5 may be involved in the ubiquitin-proteasome pathway (Weng et al., 2008). However, it is unclear whether the two Whether the factors function in maternal and/or zygotic tissues in rice.

Identifying other factors that control the final size of both seeds and organs would not only improve the understanding of the mechanisms of size control in plants, but could have numerous practical applications, for example, in increasing crop yields and plant biomass for biofuel production .

Contents of the invention

The present inventors have identified a plant E3 ubiquitin ligase (termed DA2) that regulates the final size of seeds and organs by limiting cell proliferation in the integument of developing seeds. It was unexpectedly found that DA2 acts synergistically with DA1 and independently of EOD1 to control seed and organ size. Targeting DA2 and DA1 and/or EOD1 can thus be used to increase plant yield.

One aspect of the present invention provides a method of increasing the yield of plants, comprising:

reducing the expression or activity of the DA2 polypeptide in cells of said plant,

wherein said plant lacks DAl expression or activity.

Another aspect of the present invention provides a method of increasing the yield of plants, comprising:

reducing the expression or activity of the DA2 polypeptide in cells of said plant,

wherein said plant lacks EOD1 expression or activity.

Another aspect of the present invention provides a method of increasing the yield of plants, comprising:

reducing the expression or activity of the DA2 polypeptide in cells of said plant,

wherein said plant lacks DA1 and EOD1 expression or activity.

Another aspect of the present invention provides a method of increasing the yield of plants, comprising:

reducing or eliminating the expression or activity of the DA2 polypeptide in cells of said plant, and;

i) reducing or eliminating the expression or activity of the DAl polypeptide in said cell,

ii) reducing or eliminating the expression or activity of EOD1 in said cell, and/or

iii) expressing a dominant-negative DA polypeptide in said cell.

Another aspect of the present invention provides a method of producing plants with increased yield, comprising:

providing a plant cell lacking the expression or activity of DA1, EOD1, or both DA1 and EOD1,

Incorporating a heterologous nucleic acid that eliminates or inhibits the expression or activity of the DA2 polypeptide into said plant cell by transformation, and;

The plants are regenerated from one or more transformed cells.

Description of drawings

Figure 1 shows seed and organ sizes in da2-1 mutants. 1A shows the projected areas of Col-0, da2-1 and 35S::DA2#1 seeds. Seeds were divided into three groups (>0.13, 0.12-0.13 and <0.12mm2). Values for each group are expressed as a percentage of the total number of seeds analyzed. 1B shows the number of seeds per silique for Col-0, da2-1 and 35S::DA2#1. The siliques on the main stem (from the fourth to the tenth silique) were used to measure the number of seeds per silique. 1C shows seed weight per plant for Col-0, da2-1 and 35S::DA2#1. ID shows the number of seeds per plant for Col-0, da2-1 and 35S::DA2#1. 1E shows the height of Col-0, da2-1 and 35S::DA2#1 plants. Values (B-E) are given as mean ± SE relative to wild-type values, set to 100%. **, P<0.01 and *, P<0.05 compared to wild type (Student's t-test). Ruler: F, 1cm; G, 1mm

Figure 2 shows 4-day-old plants (F) of Col-0 (left), da2-1 (middle) and 35S::DA2#1 (right) and Col-0 (top), da2-1 (middle) and 35S::DA2#1 (bottom) flower (G).

Figure 3 shows that DA1 and DA2 act synergistically to control seed size. 3A shows dry seeds of Col-0, da1-1, da2-1 and da1-1 da2-1. 3B shows 10-day-old seedlings of Col-0, da2-1, dal-1, and dal-1 da2-1 (from left to right). 3C shows the seed weights of Col-0, da1-1, da2-1 and da1-1 da2-1. 3D shows the seed weight of Col-0, dal-ko1, da2-1 and dal-ko1da2-1. Values are given as mean ± SE relative to the corresponding wild-type value, set to 100%. **, P<0.01 and *, P<0.05 compared to wild type (Student's t-test). Ruler: A, 0.1mm; B, 1m

Figure 4 shows that DA1 and DA2 act synergistically to control seed size. The upper left panel shows the cotyledon area of 10-day-old Col-0, da1-1, da2-1 and da1-1 da2-1 seedlings. The upper right panel shows the cotyledon area of 10-day-old Col-0, da1-ko1, da2-1 and da1-ko1da2-1 seedlings. The lower left panel shows the average area of palisade cells in cotyledons of Col-0, da1-1, da2-1 and da1-1 da2-1 embryos. The bottom right panel shows the projected areas of Col-0, da1-1, da1-1da2-1, da1-ko1da2-1, da1-ko1dar1-1 and da1-ko1dar1-1da2-1 seeds. Values are given as mean ± SE relative to the corresponding wild-type value, set to 100%. **, P<0.01 and *, P<0.05 compared to wild type (Student's t-test). Ruler: A, 0.1mm; B, 1m

Figure 5 shows that DA1 and DA2 act synergistically to control cell proliferation in the maternal integument of developing seeds. (5A-5D) show mature ovules of Col-0, da1-1, da2-1 and da1-1 da2-1, respectively. The da2-1 mutation synergistically enhances the ovule size of da1-1.

Figure 6 shows (left panel) Col-0×Col-0(c/c)F1, da2-1×da2-1(d2/d2)F1, Col-0×da2-1(c/d2)F1 and Projected area of da2-1×Col-0(d2/c)F1 seeds and (middle panel) Col-0×Col-0(c/c)F1, da1-ko1da2-1×da1-ko1da2-1(dd/ dd) Projected area of F1, Col-0×da1-ko1da2-1(c/dd)F1, da1-ko1da2-1×Col-0(dd/c)F1 seeds. Right panel shows da1-ko1/+da2-1/+ plants after pollination with da1-ko1da2-1 double mutant pollen resulting in da1-ko1/+ within the da1-ko1/+da2-1/+ seed coat After development of da2-1/+(a), da1-ko1/+da2-1da2-1(b), da1-ko1/da1-ko1da2-1/+(c), and da1-ko1da2-1(d) embryos The projected seed area of . The projected area of individual seeds from da1-ko1/+da2-1/+ plants fertilized with pollen of the da1-ko1 da2-1 double mutant was measured. These seeds were further genotyped for the da1-ko1 and da2-1 mutations. The data showed that da1-ko1 and da2-1 mutations were not associated with changes in the size of these seeds (P>0.05, Student's t-test). Values are given as mean ± SE relative to the corresponding wild-type value, set to 100%. Compared with wild type, **, P<0.01 (Student's t-test). Ruler: A-D, 0.5mm.

Figure 7 shows (left panel) the projected areas of mature ovules of Col-0, da1-1, da2-1 and dal-1da2-1; (middle panel) Col-0, da1-1, da2- at 6DAP and 8DAP Number of cells in the outer integument of 1 and da1-1da2-1 seeds; and (right panel) Col-0, da1-1, da2- Average length of cells in the outer integument of 1 and da1-1da2-1 seeds.

Figure 8A shows the DA2 gene structure. Start codon (ATG) and stop codon (TAA) are indicated. Closed boxes indicate coding sequences, open boxes indicate 5' and 3' untranslated regions, and lines between boxes indicate introns. The T-DNA insertion site (da2-1) in the DA2 gene is shown. Figure 8B shows that the DA2 protein includes a predicted RING domain.

Figure 9 shows the E3 ubiquitin ligase activity of DA2. MBP-DA2 and mutated DA2 (MBP-DA2C59S and MBP-DA2N91L) fusion proteins were assayed for E3 ubiquitin ligase activity in the presence of El, E2 and His-ubiquitin (His-Ub). Ubiquitinated proteins were detected by immunoblotting (IB) with anti-His antibody (anti-His) and anti-MBP antibody (anti-MBP), respectively. The lower arrow indicates MBP-DA2 protein and the upper arrow shows ubiquitinated MBP-DA2 protein.

Figure 10 shows the projected areas of Col-0, da2-1, COM#6, COM#8, and COM#10 seeds (upper panel), where COM is da2 transformed with the DA2 coding sequence driven by its own promoter -1; Petal area of Col-0, da2-1, COM#6, COM#8 and COM#10 plants (middle panel) and Col-0, da2-1, COM#6, COM#8 and COM#10 Quantitative real-time RT-PCR analysis of DA2 gene expression in seedlings (lower panel). Values (D and E) are given as mean ± SE relative to the da2-1 value, set to 100%. **, P<0.01 compared to da2-1 mutant (Student's t-test).

Figure 11 shows the expression pattern of DA2. 11A shows quantitative real-time RT-PCR analysis of DA2 gene expression. Total RNA was isolated from roots (R), stems (S), leaves (L), seedlings (Se) and inflorescences (In). 11B-11N show DA2 expression activity monitored by pDA2:GUS transgene expression. Four GUS expressing lines were observed, and all showed a similar pattern, although they differed slightly in the intensity of staining. 4-day-old seedlings (11B), 10-day-old seedlings (11C), flower inflorescences (11D), developing petals (11E-11G), developing stamens (11H and 11I), developing carpels (11J - 11L) and histochemical analysis of GUS activity in developing ovules (11M and 11N). Ruler: B-D, 1mm; E-N, 0.1mm.

Figure 12 shows that DA1 directly interacts with DA2 in vitro. GST-DA1, GST-DA1R358K, GST-DA1-UIM, GST-DA1-LIM, GST-DA1-LIM+C, and GST-DA1-C were pulled down by MBP-DA2 immobilized on amylose resin (PD ) and analyzed by immunoblotting (IB) using an anti-GST antibody.

Figure 13 shows a schematic diagram of DAl and its derivatives containing specific protein domains. The predicted DA1 protein includes two UIM motifs, a single LIM domain and a C-terminal region.

Figure 14 shows that DA1 interacts with DA2 in vivo. Nicotiana benthamiana leaves were transformed by injection of Agrobacterium tumefaciens GV3101 cells harboring 35S:Myc-DA1 and 35S:GFP-DA2 plasmids. Total protein was immunoprecipitated with GFP-Trap-A, and the immunoblots were probed with anti-GFP antibody and anti-Myc antibody, respectively. Myc-DA1 was detected in the immunoprecipitated GFP-DA2 complex, indicating a physical association between DA1 and DA2 in the plants.

Figure 15 shows that da2-1 mutants exhibit increased organ size. 15A shows the petal length (PL), petal width (PW), petal area (PA), sepal area (SA), carpel length (CL), long Stamen length (LSL) and short stamen length (SSL). 15B shows the fifth leaf area of Col-0, da2-1 and 35S::DA2#1 plants. 15C shows the weight of Col-0, da2-1 and 35S::DA2#1 flowers. 15D shows the size of the adaxial epidermal cells in the region of maximum width of Col-0 and da2-1 petals. 15E shows the size of palisade cells in the fifth leaf of Col-0 and da2-1. Open flowers (stage 14) were used to measure petal size (15A), flower weight (C) and epidermal cell size (15D). Values (A-E) are given as mean ± SE relative to the corresponding wild-type value, set to 100%. Compared with wild type, **, P<0.01 (Student's t-test).

Figure 16 shows that DA1 and DA2 act synergistically to control seed size. 16D shows the petal area of Col-0, da1-ko1, da2-1 and da1-ko1 da2-1 flowers. 16E shows the size of the adaxial epidermal cells in the region of maximum width of Col-0, dal-ko1, da2-1 and dal-ko1da2-1 petals. 16F shows the seed weights of Col-0, eod1-2, da2-1 and eod1-2da2-1. 16G shows the petal area of Col-0, eod1-2, da2-1 and eod1-2da2-1. Open flowers (stage 14) were used to measure petal size (16D and 16G) and epidermal cell size (16E). Values (16D-G) are given as mean ± SE relative to the corresponding wild-type value, set to 100%. **, P<0.01 and *, P<0.05 compared to wild type (Student's t-test). Ruler: 0.1mm.

Figure 17 shows that overexpression of DA2 limits organ growth. 17A shows the petal area of Col-0, 35S:DA2#2 and 35S:DA2#4. 17B shows the expression level of DA2 in Col-0, 35S:DA2#2 and 35S:DA2#4 seedlings. Values (A and B) are given as mean ± SE relative to Col-0 values, set to 100%. Compared with wild type, **, P<0.01 (Student's t-test).

Figure 18 shows that overexpression of DA2L limits organ growth. 18A shows 20 day old plants of Col-0, 35S:DA2L#1, 35S:DA2L#3, 35S:DA2L#4, 35S:DA2L#5 and 35S:DA2L#6. 18B shows 30 day old plants of Col-0, 35S:DA2L#1, 35S:DA2L#3, 35S:DA2L#4, 35S:DA2L#5 and 35S:DA2L#6. 18C shows RT-PCR analysis of DA2L expression in Col-0, 35S:DA2L#1, 35S:DA2L#3, 35S:DA2L#4, 35S:DA2L#5 and 35S:DA2L#6 seedlings. RT-PCR was performed on first-strand cDNA prepared from 2-week-old seedlings. The cDNA was normalized by reference to ACTIN2 standards. Ruler: A, 1cm, B, 1cm

Figure 19 shows that overexpression of GW2 limits seed and organ growth. 19A shows 30 day old plants of Col-0, 35S:GW2#1, 35S:GW2#2, 35S:GW2#3, 35S:GW2#6 and 35S:GW2L#7. 19B shows the projected areas of Col-0, 35S:GW2#1, 35S:GW2#2, 35S:GW2#3, 35S:GW2#6 and 35S:GW2L#7 seeds. 19C shows quantitative real-time RT-PCR analysis of GW2 gene expression in Col-0, 35S:GW2#1, 35S:GW2#2, 35S:GW2#3, 35S:GW2#6 and 35S:GW2L#7 seedlings. Values (B) are given as mean ± SE relative to Col-0 values, set to 100%. Compared with wild type, **, P<0.01 (Student's t-test). Ruler: A, 1cm

detailed description

The present invention relates to methods for altering plant traits affecting yield, such as seed and organ size, by altering the expression or activity of the plant E3 ubiquitin ligase DA2 in combination with altering the expression or activity of DAl and/or EODl. Preferably, the expression or activity of DA2 and DA1 is altered in a plant.

The expression or activity of DA2 can be altered before, simultaneously with, or after the expression or activity of DA1 and/or EOD1 is altered. For example, in some embodiments, the expression or activity of a DA2 polypeptide can be altered in one or more plant cells that already have one of: altered DAl expression or activity, altered EODl expression or activity, or Altered DA1 and EOD1 expression or activity.

Provided herein is a method of increasing the yield of a plant, e.g., by increasing organ or seed size, the method comprising providing a plant lacking DA1 and/or EOD1 expression or activity, and reducing the expression of DA2 in one or more cells of the plant . In other embodiments, the expression or activity of DA1 and/or EOD1 can be reduced in one or more plant cells having reduced expression or activity of a DA2 polypeptide.

Other methods may include reducing the expression of DA2 in one or more cells of a plant and reducing the expression or activity of DAl, EOD1, or both DAl and EOD1 in one or more cells.

Also provided herein are methods of producing plants having increased yield relative to wild-type plants, comprising:

(a) incorporating into plant cells by transformation:

(i) a first heterologous nucleic acid that reduces expression of a DA2 polypeptide,

(ii) a second heterologous nucleic acid that reduces the expression of one of the DAl polypeptide and the EOD1 polypeptide, and optionally,

(iii) a third heterologous nucleic acid that reduces expression of the other of the DAl polypeptide and the EOD1 polypeptide, and

(b) regenerating said plant from one or more transformed cells.

Other methods of producing plants with increased yield may include:

providing plant cells lacking DAl and/or EOD1 expression or activity, preferably DAl activity,

incorporating a heterologous nucleic acid that reduces the activity or expression of the DA2 polypeptide into said plant cell by transformation, and;

The plants are regenerated from the transformed cells.

Following regeneration, plants can be selected for reduced DA2 polypeptide activity or expression and reduced DA1 and/or EOD1 activity or expression relative to wild-type plants.

The combination of reduced DA2 expression and reduced DA1 and/or EOD1 expression synergistically increases the size of seeds and/or organs of the plant, thereby increasing plant yield.

One or more yield-related traits in plants may be improved by reduced expression or activity of DA2 in combination with reduced expression or activity of DA1 and/or EOD1. For example, one or more of lifespan, organ size, and seed size of a plant can be increased relative to control or wild-type plants in which expression of the DA2 polypeptide has not been reduced.

The expression or activity of DA2, DA1 or EOD1 may be reduced by at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 98% relative to wild-type plants in the methods described herein. In some preferred embodiments, expression or activity is reduced to zero or substantially zero (ie, expression or activity is eliminated).

The methods of the invention involve altering the expression or activity of a DA2 polypeptide in one or more cells of a plant.

DA2 polypeptides are E3 ubiquitin ligases found in plants. A DA2 polypeptide whose expression or activity is reduced as described herein may comprise a RING domain (Stone, S.L. et al. (2005)), preferably a C5HC2, C5NC2 or C5TC2 RING domain. A suitable RING domain may consist of the amino acid sequence of SEQ ID NO: 1;

C(X) ₂ C(X) ₁₁ CC(X) ₄ CX ₂ CX ₇ (H/N/T)X ₆ CX ₂ C. (SEQ ID NO: 1)

For example, a suitable RING domain may consist of the amino acid sequence of SEQ ID NO: 2;

CPICFL(Y/F)YPSLNRS(K/R)CC(S/M/T/A)K(G/S)ICTECFL(Q/R)MK(P/N/S/V/T/N)(T /P)(H/N/T)(T/S)(A/T/C)(R/Q/K)PTQCP(F/Y)C

(SEQ ID NO: 2)

In some embodiments, the H/N/T residue at position 33 in the RING domain of SEQ ID NO: 2 may be T or N.

In some preferred embodiments, the DA2 polypeptide may include a RING domain, and the RING domain has an amino acid sequence (SEQ ID NO: 3-19) shown in Table 1, such as Arabidopsis DA2 (SEQ ID NO: 11), Arabidopsis A. thaliana DAL2 (SEQ ID NO: 13) or rice GW2 (SEQ ID NO: 7) or a variant thereof. For example, the RING domain may have the amino acid sequence of: residues 59 to 101 of SEQ ID NO: 20 (Pt_GI-224061326.pro), residues 59 to 101 of SEQ ID NO: 21 (Rc_GI-255578534.pro), SEQ ID NO: Residues 59 to 101 of 22 (Vv_GI-147817790.pro), Residues 59 to 101 of SEQ ID NO:23 (Gm_GI-356549538.pro), Residues 59 to 101 of SEQ ID NO:24 (At_GI-18411948.pro), SEQ ID NO :25 (Ta_GI408743661.pro) residues 61 to 103, SEQ ID NO:26 (Hv_GI-164371454.pro) residues 61 to 103, SEQ ID NO:27 (Bd_GI-357140854.pro) residues 61 to 103, SEQ ID NO: Residues 62 to 104 of 28 (Os_GI-115445269.pro), Residues 63 to 105 of SEQ ID NO:29 (Sb_GI-242064618.pro), Residues 65 to 107 of SEQ ID NO:30 (Zm_GI-220961719.pro), SEQ ID NO :31 (Ta_GI-408743658.pro) residues 61 to 103, SEQ ID NO:32 (Bd_GI-357125256.pro) residues 43 to 85, SEQ ID NO:33 (Os_GI-218197613.pro) residues 62 to 104, Residues 62 to 104 of SEQ ID NO: 34 (Zm_GI-260935347.pro) or residues 62 to 104 of SEQ ID NO: 35 (Sb_GI-242092026.pro).

Other suitable RING domain sequences can be identified using standard sequence analysis techniques as described herein (eg, Simple Modular Construction Research Tool (SMART); EMBL Heidelberg, DE).

The DA2 polypeptide can also include a first consensus domain. The first consensus domain may be located upstream (ie, on the N-terminal side) of the RING domain. A suitable first consensus domain may consist of the amino acid sequence of SEQ ID NO:36.

Q(Q/Not Existing)GLY(P/M/N/V/Q/L/V/E)(H/S/N)(P/K/R)D(I/V)D(L/I /H/V/Q)(K/R)KL

(R/K)(R/K)LI(V/L)(E/D)(A/S/T)KLAPC

(SEQ ID NO: 36)

In some preferred embodiments, the DA2 polypeptide may comprise a first consensus domain of the DA2 amino acid sequence shown in Table 2, such as residues 20 to 45 of SEQ ID NO: 20, residues 20 to 45 of SEQ ID NO: 21, Residues 20 to 45 of SEQ ID NO: 22, Residues 20 to 45 of SEQ ID NO: 23, Residues 20 to 45 of SEQ ID NO: 24, Residues 21 to 46 of SEQ ID NO: 25, Residues 21 to 46 of SEQ ID NO: 26, Residues 21 to 46 of SEQ ID NO: 27, Residues 21 to 46 of SEQ ID NO: 28, Residues 21 to 46 of SEQ ID NO: 29, Residues 21 to 46 of SEQ ID NO: 30, Residues 21 to 46 of SEQ ID NO: 31, Residues 4 to 29 of SEQ ID NO:32, residues 23 to 48 of SEQ ID NO:33, residues 23 to 48 of SEQ ID NO:34 or residues 23 to 48 of SEQ ID NO:35.

DA2 polypeptides may also include a second consensus domain. The second consensus domain may be located downstream (ie, on the C-terminal side) of the RING domain. The second consensus domain may consist of the amino acid sequence of SEQ ID NO:37.

(N/S)YAVEYRG(V/G)K(T/S)KEE(K/R)(G/S)(V/T/I/F/L/M)EQ(L/I/V/F )EEQ(R/L/K)VIEA(Q/K)(I/M)RMR(H/Q)(K/Q)(E/A)

(SEQ ID NO: 37).

In some preferred embodiments, the DA2 polypeptide may comprise a second consensus domain of the DA2 amino acid sequence shown in Table 2, such as residues 106 to 141 of SEQ ID NO: 20, residues 106 to 141 of SEQ ID NO: 21, residues 106 to 141 of SEQ ID NO: 21, : 22 residues 106 to 141, SEQ ID NO: 23 residues 106 to 141, SEQ ID NO: 24 residues 106 to 141, SEQ ID NO: 25 residues 107 to 143, SEQ ID NO: 26 residues 107 to 143, SEQ ID NO: : 27 residues 107 to 143, SEQ ID NO: 28 residues 108 to 144, SEQ ID NO: 29 residues 109 to 145, SEQ ID NO: 30 residues 111 to 147, SEQ ID NO: 31 residues 107 to 143, SEQ ID NO: : residues 90 to 125 of 32, residues 108 to 143 of SEQ ID NO: 33, residues 108 to 143 of SEQ ID NO: 34, or residues 108 to 143 of SEQ ID NO: 35.

Other examples of suitable first and second domain sequences can be identified using standard sequence analysis techniques as described herein (eg, Simple Modular Construction Research Tool (SMART); EMBL Heidelberg, DE).

In some preferred embodiments, the DA2 polypeptide whose expression or activity is reduced as described herein may comprise the RING domain of SEQ ID NO:2, the first consensus domain of SEQ ID NO:36, and the second consensus structure of SEQ ID NO:37 area.

For example, a DA2 polypeptide can include any combination of the RING domain sequence, first consensus domain sequence, and second consensus domain sequence listed above.

A suitable DA2 polypeptide may comprise the amino acid sequence of any one of SEQ ID NOs 20 to 35 as set forth in Table 2 or may be a variant of one of these sequences. In some preferred embodiments, the DA2 polypeptide may comprise the amino acid sequence of SEQ ID NO: 28 or 33 (OsGW2), SEQ ID NO: 24 (AtDA2), SEQ ID NO: 25 or SEQ ID NO: 31 (TaGW2) or may be a protein having an E3 ubiquitin ligase Active variants of any of these sequences.

A DA2 polypeptide that is a variant of any one of SEQ ID NOs: 20 to 35 or other reference DA2 sequences may comprise at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, Amino acid sequences having at least 70%, at least 80%, at least 90%, at least 95%, or at least 98% sequence identity.

The DA2 polypeptide that is a variant of any one of SEQ ID NO: 20 to 35 may further comprise a RING domain having the sequence of SEQ ID NO: 2, a first consensus domain having the sequence of SEQ ID NO: 36, and a RING domain having the sequence of SEQ ID NO: 37. Second consensus domain. Examples of suitable sequences are listed above. In some preferred embodiments, the DA2 polypeptide may comprise the RING domain, the first consensus domain and the second consensus domain of any one of SEQ ID NO: 20 to 35.

A nucleic acid encoding a DA2 polypeptide may comprise or may be a nucleotide sequence listed in a database entry selected from the group consisting of JN896622.1GI:408743658 (TaGW2-A); and JN896623.1GI:408743660 (TaGW2-B) A variant of one of the .

In some preferred embodiments, the nucleic acid encoding a DA2 polypeptide may comprise a nucleotide sequence encoding AtDA2, AtDAL2, OsGW2, TaGW2-A or TaGW2-B or may be a variant of any of these DA2 sequences encoding A polypeptide having DA2 activity.

Routine sequence analysis techniques can be used to identify DA2 polypeptides and encoding nucleic acids in any plant species of interest, particularly crop plants such as wheat, barley, corn, rice, soybean; and additional agricultural plants.

Reduction of DA2 expression or activity in plants is herein shown to synergistically enhance the effect of mutations reducing the activity or expression of DA1 on yield-related traits in plants. In preferred embodiments, the methods described herein may comprise reducing DA2 expression in a plant lacking DAl expression or activity or reducing both DAl and DA2 expression in a plant.

The DAl polypeptide is a ubiquitin receptor found in plants and described in detail in Li et al. (2008), Wang et al. (2012) and WO2009/047525. A DAl polypeptide whose expression or activity is reduced as described herein may include a LIM domain, a conserved C-terminal domain, and one or more UIM domains.

The LIM domain includes two Zn zinc finger motifs and may have an amino acid sequence (SEQ ID NO: 38);

C (X) ₂ C (X) _16-23 (H/C) (X) _2/4 (C/H/E) (X) ₂ C (X) ₂ C (X) _14-21 (C/H ) (X) _2/1/3 (C/H/D/E) X

where X is any amino acid and the Zn coordinating residue is underlined.

The Zn coordinating residue in the LIM domain can be C, H, D or E, preferably C.

In some preferred embodiments, the LIM domain may comprise CXXC, HXXCXXCXXC and HxxC motifs, wherein X is any amino acid. For example, a LIM domain can comprise the amino acid sequence (SEQ ID NO: 39);

C (X) ₂ C (X) _16-23 (H) (X) ₂ (C) (X) ₂ C (X) ₂ C (X) _14-21 H (X) ₂ C X

where X is any amino acid and the Zn coordinating residue is underlined.

In some embodiments, the LIM domain can comprise the amino acid sequence of an AtDA1 LIM domain;

C AG C NMEIGHGRFLNCLNSLW H PE C FR C YG C SQPISEYEFSTSGNYPF H KA C Y

(SEQ ID NO:40; Zn coordinating residues are underlined)

Other LIM domains include those of the DA1 amino acid sequence shown in Table 3, for example residues 141 to 193 of SEQ ID NO:41 (Si_GI-514815267.pro), residues of SEQ ID NO:42 (Bd_GI-357157184.pro) 123 to 175, residues 155 to 207 of SEQ ID NO:43 (Br_DA1b.pro), residues 172 to 224 of SEQ ID NO:44 (Br_DA1a.pro), residues 172 to 224 of SEQ ID NO:45 (At_GI-15221983.pro) , residues 117 to 169 of SEQ ID NO:46 (Tc_GI-508722773.pro), residues 117 to 169 of SEQ ID NO:47 (Gm_GI-356564241.pro), residues 121 to 169 of SEQ ID NO:48 (Gm_GI-356552145.pro) 173. Residues 119 to 171 of SEQ ID NO: 49 (Vv_GI-302142429.pro), Residues 122 to 174 of SEQ ID NO: 50 (Vv_GI-359492104.pro), Residue 125 of SEQ ID NO: 51 (Sl_GI-460385048.pro) to 177, residues 516 to 568 of SEQ ID NO:52 (Os_GI-218197709.pro), residues 124 to 176 of SEQ ID NO:53 (Os_GI-115466772.pro), residues of SEQ ID NO:54 (Bd_GI-357160893.pro) 150 to 202, residues 132 to 184 of SEQ ID NO:55 (Bd_GI-357164660.pro), residues 124 to 176 of SEQ ID NO:56 (Sb_GI-242092232.pro), residues of SEQ ID NO:57 (Zm_GI-212275448.pro) Bases 147 to 199, residues 190 to 242 of SEQ ID NO:58 (At_GI-240256211.pro), residues 162 to 214 of SEQ ID NO:59 (At_GI-145360806.pro), residues of SEQ ID NO:60 (At_GI-22326876.pro) Residues 1240 to 1291, residues 80 to 122 of SEQ ID NO:61 (At_GI-30698242.pro), residues 347 to 402 of SEQ ID NO:62 (At_GI-30698240.pro), SEQ ID NO:63 (At_GI-15240018.pro) Residues 286 to 341 of, SEQ ID NO:64 (At_GI-334 188680.pro) from residues 202 to 252.

LIM domain sequences can be identified using standard sequence analysis techniques (eg, Simple Modular Construction Research Tool (SMART); EMBL Heidelberg, DE).

In addition to the LIM domain, the DAl protein may also include a carboxy-terminal region having a sequence at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, Amino acid sequences of at least 80%, at least 90%, at least 95%, or at least 98% amino acid identity: residues 198 to 504 of SEQ ID NO: 41, residues 180 to 487 of SEQ ID NO: 42, residues 212 to 43 of SEQ ID NO: 43 514, residues 229 to 532 of SEQ ID NO:44, residues 229 to 532 of SEQ ID NO:45, residues 174 to 478 of SEQ ID NO:46, residues 174 to 474 of SEQ ID NO:47, residues 178 to 474 of SEQ ID NO:48 478, residues 176 to 462 of SEQ ID NO:49, residues 179 to 482 of SEQ ID NO:50, residues 182 to 486 of SEQ ID NO:51, residues 573 to 878 of SEQ ID NO:52, residues 181 to 878 of SEQ ID NO:53 486, residues 207 to 512 of SEQ ID NO:54, residues 189 to 491 of SEQ ID NO:55, residues 181 to 486 of SEQ ID NO:56, residues 204 to 508 of SEQ ID NO:57, residues 247 to 58 of SEQ ID NO:58 553, residues 219 to 528 of SEQ ID NO:59, residues 1296 to 1613 of SEQ ID NO:60, residues 128 to 450 of SEQ ID NO:61, residues 404 to 702 of SEQ ID NO:62, residues 343 to 702 of SEQ ID NO:63 644. Residues 256 to 587 of SEQ ID NO:64.

The carboxy-terminal region of the DAl protein may include the metallopeptidase motif HEMMH (SEQ ID NO: 65).

The carboxy-terminal region may also include EK(X) ₈ R(X) ₄ SEEQ (SEQ ID NO: 66) or EK(X) ₈ R(X) ₄ SEQ (SEQ ID NO: 67) between the LIM domain and the HEMMH motif motif.

In addition to the LIM domain and the conserved carboxy-terminal region, the DA1 protein may include a UIM1 domain and a UIM2 domain. The UIM1 and UIM2 domains can be located between the N-terminus of the DAl protein and the LIM domain.

The UIM1 domain may consist of the sequence of SEQ ID NO:68 and the UIM2 domain may consist of the sequence of SEQ ID NO:69.

p---pLpbAlpb.Sbp-.ppp (SEQ ID NO: 68)

p---pLpbAlpb.Sbp-sppp (SEQ ID NO: 69)

in;

P is a polar amino acid residue, for example, C, D, E, H, K, N, Q, R, S or T;

b is a large amino acid residue, for example, E, F, H, I, K, L, M, Q, R, W or Y;

s is a small amino acid residue, for example, A, C, D, G, N, P, S, T or V;

l is an aliphatic amino acid residue, such as I, L or V;

.is the absence or any amino acid, and

- is any amino acid.

Additional examples of UIM1 and UIM2 domain sequences can be identified using standard sequence analysis techniques as described herein (eg, Simple Modular Construction Research Tool (SMART); EMBL Heidelberg, DE).

In some preferred embodiments, the DAl polypeptide may include;

the LIM domain of SEQ ID NO: 39,

A C-terminal region having at least 20% sequence identity to an equivalent region of any one of residues 229 to 532 of SEQ ID NO: 45 or SEQ NOs 41 to 44 or 46 to 64, as listed above and comprising EK(X) ₈ R(X) ₄ SEEQ or EK(X) ₈ R(X) ₄ SEQ motif and HEMMH motif,

the UIM domain of SEQ ID NO:66, and

The UIM domain of SEQ ID NO:67.

The DAl protein may comprise the amino acid sequence (SEQ ID NO: 41 to 64) of the plant DAl protein shown in Table 3 or may be a homologue or variant of one of these sequences, said homologue or variant having DAl activity . For example, a DAl polypeptide may comprise the amino acid sequences shown in Table 3 (SEQ ID NO: 41 to 64) or may be a variant of one of these sequences, which variant has DAl activity.

For example, a DAl polypeptide can include AtDAR1, AtDAR1, AtDAR2, AtDAR3, AtDAR4, AtDAR5, AtDAR6, AtDAR7, BrDA1a, BrDA1b, BrDAR1, BrDAR2, BrDAR3-7, BrDAL1, BrDAL2, BrDAL3, OsDA1, OsDAR2, OsDAL3, OsDAL5, PpDAL1, The amino acid sequence of PpDAL2, PpDAL3, PpDAL4, PpDAL5, PpDAL6, PpDAL7, PpDAL8, SmDAL1, SmDAL2 or ZmDA1, preferably the amino acid sequence of AtDA1, AtDAR1, BrDA1a, BrDA1b, OsDA1 or ZmDA1, or a homologue of one of these sequences or variants.

In some preferred embodiments, the DAl polypeptide may include the amino acid sequence of AtDA1 (AT1G19270; NP_173361.1GI: 15221983) or may be a variant of this sequence, and the variant has DAl activity.

Other DAl protein sequences that include the characteristic features listed above can be identified using standard sequence analysis tools. A skilled artisan can readily identify a nucleic acid sequence encoding a DAl protein in any plant species of interest.

The DAl protein in the plant species of interest may have an amino acid sequence that is a variant of the reference amino acid sequence of the DAl protein listed herein.

A DAl polypeptide that is a variant of a reference DAl sequence, such as any one of SEQ ID NOs 41 to 64, may comprise at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70% Amino acid sequences having %, at least 80%, at least 90%, at least 95%, or at least 98% sequence identity.

Specific amino acid sequence variants occurring in plant species may differ from the reference sequences listed herein by 1 amino acid, 2, 3, 4, 5-10, 10-20, 20-30, 30-50, or more. Insertion, addition, substitution or deletion of 50 amino acids.

In some embodiments, a DAl polypeptide that is a variant of the AtDAl sequence of SEQ ID NO: 45 can include a UIM1 domain having the sequence QENEDIDRAIALSLLEENQE (SEQ ID NO: 70) and a UIM2 domain having the sequence DEDEQIARALQESMVVGNSP (SEQ ID NO: 71).

A DAl polypeptide that is a variant of the AtDA1 sequence of SEQ ID NO: 45 may comprise a LIM domain having the following sequence:

ICAGCNMEIGHGRFLNCLNSLWHPECFRCYGCSQPISEYEFSTSGNYPFHKAC

(SEQ ID NO: 72)

A nucleic acid encoding a DAl polypeptide may comprise a nucleotide sequence listed in a database entry selected from the group consisting of NM_101785.3GI:42562170 (AtDA1); NM_001057237.1GI:115454202 (OsDA1); BT085014.1GI:238008663 (ZmDA1) or There may be a variant of one of these sequences, which variant encodes an active DAl polypeptide.

In some preferred embodiments, the nucleic acid encoding the DAl polypeptide may include the nucleotide sequence of AtDA1 (NM_101785.3GI: 42562170), ZmDA1 (BT085014.1GI: 238008663), OsDA1 (NM_001057237.1GI: 115454202) or may be these A variant of any of the sequences that encodes a polypeptide that retains DAl activity.

Routine sequence analysis techniques can be used to identify DAl polypeptides and encoding nucleic acids in plant species, particularly crop plants such as wheat, barley, corn, rice, and other agricultural plants.

In some preferred embodiments, DAl activity in one or more cells of a plant can be reduced by expressing a dominant-negative DAl polypeptide in said one or more cells (see, e.g., Li et al. (2008); WO2009 /047525; Wang et al. 2012). A plant expressing a dominant-negative DA1 polypeptide can have a dal-1 phenotype.

A dominant negative allele of a DAl polypeptide may include a DAl polypeptide having a mutation (e.g., a substitution or deletion) at a conserved R residue at position 358 of the Arabidopsis DAl amino acid sequence, the maize DAl amino acid sequence or the equivalent position in another DAl amino acid sequence. For example, a dominant negative allele of a DAl polypeptide can include a mutation of a conserved R residue at a position equivalent to position 358 of the Arabidopsis DAl amino acid sequence or position 333 of the maize DAl amino acid sequence. In preferred embodiments, conserved R residues may be substituted with K.

The conserved R residue at a position in the DAl amino acid sequence equivalent to position 358 of SEQ ID NO: 45 of Arabidopsis DA1 or position 333 of maize DAl of SEQ ID NO: 57 is located within the DAl amino acid sequence corresponding to SEQ ID NO: 57 At the position of R333 and R358 of SEQ ID NO: 45, ie it is in the same position relative to other motifs and domains of the DAl protein. The conserved R residue is located between the LIM domain in the C-terminal region and the HEMMH peptidase motif and is fully conserved in the context of the same sequence in the DAl protein. Conserved R residues can be included in the EK(X) ₈ R(X) ₄ SEEQ (SEQ ID NO: 66) or EK(X) ₈ R(X) ₄ SEQ (SEQ ID NO: 67) motifs within the C-terminal region.

Conserved R residues can be identified by aligning these conserved C-terminal regions using standard sequence analysis and alignment tools and are indicated by arrows in the sequences in Table 3.

Nucleic acids encoding dominant negative alleles of the DA protein can be produced by any convenient technique. For example, site-directed mutagenesis can be employed on a nucleic acid encoding a DAl polypeptide to change a conserved R residue, eg, to a K, at an equivalent position to R358 of Arabidopsis DAl or R333 of maize DAl. Reagents and kits for in vitro mutagenesis are commercially available.

In some embodiments, a nucleic acid encoding a dominant-negative DAl polypeptide as described herein may be operably linked to a heterologous regulatory sequence, such as a promoter, e.g., a constitutive, inducible, tissue-specific or developmental-specific promoter son. A nucleic acid encoding a dominant-negative DAl polypeptide can be included in one or more vectors. For example, a mutated nucleic acid encoding a dominant negative allele of the DAl protein can also be cloned into an expression vector and expressed in plant cells as described below to alter the plant phenotype.

In other embodiments, a mutation can be introduced into an endogenous DAl nucleic acid in a plant such that the DAl polypeptide encoded by the mutant DAl nucleic acid has a dominant negative activity.

A nucleic acid encoding a dominant-negative DAl polypeptide may be expressed in the same plant species or variety from which it was originally isolated or in a different plant species or variety (ie, a heterologous plant).

Reduction or elimination of DA2 expression in plants It is also shown herein that enhancing the effect of mutations reducing the expression or activity of EOD1 on yield-related traits in plants.

The methods described herein can comprise reducing DA2 expression or activity in a plant lacking EOD1 expression or activity or reducing the expression or activity of both DA2 and EOD1 in a plant. In preferred embodiments, the plant may also lack DAl activity or the method may additionally comprise reducing or eliminating DAl expression in said plant.

EOD1 polypeptides are E3 ubiquitin ligases found in plants and described in detail in Disch et al. (2006), Li et al. (2008) and WO2009/047525.

An EOD1 polypeptide having reduced expression or activity as described herein may include an EOD domain. A suitable EOD domain may consist of the amino acid sequence of SEQ ID NO:73;

(E/K)RCVICQ(L/M)(K/R/G/T/E)Y(K/R)(R/I)(G/K)(D/N/E)(R/Q/ K/L)Q(I/M/V)(K/N/T/A)L(L/P)C(K/S)H(V/A)YH(S/T/G/A)( E/Q/D/S/G)C(I/G/T/V)(S/T)(K/R)WL(G/T/S)INK(V/I/A/K)CP( V/I) C (SEQ ID NO: 73)

In some preferred embodiments, the EOD1 polypeptide may comprise an EOD domain having the amino acid sequence of: SEQ ID NO:74 (Zm_GI-223973923.pro) residues 195 to 237, SEQ ID NO:75 (Sb_GI-242042045.pro ), residues 195 to 237 of SEQ ID NO: 76 (Zm_GI-226496789.pro), residues 195 to 237 of SEQ ID NO: 77 (Os_GI-222624282.pro), residues 218 to 260 of SEQ ID NO: 78 (Os_GI-115451045. pro), residues 196 to 238 of SEQ ID NO:79 (Bd_GI-357113826.pro), residues 197 to 239 of SEQ ID NO:80 (Sl_GI-460410949.pro), residues 193 to 235 of SEQ ID NO:81 (Rc_GI-255582236 .pro) residues 187 to 229, SEQ ID NO:82 (Pt_GI-224059640.pro) residues 150 to 192, SEQ ID NO:83 (Gm_GI-356548935.pro) residues 194 to 236, SEQ ID NO:84 (Gm_GI- 356544176.pro), residues 194 to 236 of SEQ ID NO:85 (Vv_GI-359487286.pro), residues 189 to 231 of SEQ ID NO:86 (Tc_GI-508704801.pro), SEQ ID NO:87 (Pp_GI -462414664.pro) residues 192 to 234, SEQ ID NO:88 (Cr_GI-482561003.pro) residues 190 to 232, SEQ ID NO:89 (At_GI-22331928.pro) residues 195 to 237 or SEQ ID NO:90 Residues 195 to 237 (Sl_GI-460370551.pro), as shown in Table 4.

Other suitable EOD domain sequences can be identified using standard sequence analysis techniques as described herein (eg, Simple Modular Construction Research Tool (SMART); EMBL Heidelberg, DE).

The EOD1 polypeptide whose expression or activity is reduced as described herein may comprise the amino acid sequence of any one of SEQ ID NOs 74 to 90 as listed in Table 4. In some preferred embodiments, the EOD1 polypeptide may comprise the amino acid sequence of SEQ ID NO: 89 (AtEOD1 ) or SEQ ID NO: 77 or 78 (OsEOD1 ) or may be a variant of this sequence, which retains E3 ubiquitin ligase activity.

An EOD1 polypeptide that is a variant of any one of SEQ ID NOs: 74 to 90 or other reference EOD1 sequences may comprise at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, Amino acid sequences having at least 70%, at least 80%, at least 90%, at least 95%, or at least 98% sequence identity.

An EOD polypeptide that is a variant of any one of SEQ ID NO:74 to 90 may also include an EOD domain having the sequence of SEQ ID NO:73. Examples of suitable sequences are listed above.

A nucleic acid encoding an EOD1 polypeptide may comprise a nucleotide sequence listed in a database entry selected from the group consisting of: XM_002299911.1GI:224059639 (PtEOD1); XM_002531864.1GI:255582235 (RcEOD1); XM_002279758.2GI:359487285 (VvEOD1)；XM_003542806.1GI:356548934(GmEOD1a)；XM_003540482.1GI:356544175(GmEOD1b)；XM_002468372.1GI:242042044(SbEOD1)；NM_001147247.1GI:226496788(ZmEOD1)；或NP_001030922.1GI:79316205(AtEOD1；At3g63530 ), or may be a variant of one of these sequences.

In some preferred embodiments, the nucleic acid encoding an EOD1 polypeptide may include a nucleotide sequence encoding AtEOD1 or OsEOD1 or may be a variant of any of these sequences, and the variant encodes a polypeptide having EOD1 activity.

EOD1 polypeptides and encoding nucleic acids whose expression or activity is reduced as described herein can be identified using conventional sequence analysis techniques in any plant species of interest, particularly crops such as wheat, barley, corn, rice, soybean; and other agricultural plants.

DA2 mutations in plants are also shown herein to synergistically enhance the effect of the combination of DA1 and EOD1 mutations on yield-related traits in plants.

The methods described herein are not limited to a particular plant species, and the expression or activity of DA2, DA1 and/or EOD1 can be reduced in any plant species of interest, as described herein.

A DAl, DA2 or EODl polypeptide in a plant species of interest may have an amino acid sequence that is a variant of the corresponding DAl, DA2 or EODl reference amino acid sequence listed herein. A DA1, DA2 or EOD1 polypeptide that is a variant of a reference sequence listed herein may comprise at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least Amino acid sequences having 80%, at least 90%, at least 95%, or at least 98% sequence identity.

Specific amino acid sequence variants occurring in plant species may differ from the reference sequences listed herein by 1 amino acid, 2, 3, 4, 5-10, 10-20, 20-30, 30-50, or more. Insertion, addition, substitution or deletion of 50 amino acids.

A DAl, DA2 or EODl nucleic acid in a plant species of interest may have a nucleotide sequence that is a variant of the corresponding DAl, DA2 or EODl reference nucleotide sequence listed herein. For example, a variant nucleotide sequence may be a homologue or allele of a reference DAl, DA2 or EOD1 sequence listed herein, and may differ from said reference DAl, DA2 or EOD1 nucleotide sequence in that One of addition, insertion, deletion or substitution of one or more (eg 2, 3, 4, 5-10, 10-20, 20-30, 30-50 or more than 50) nucleotides in a nucleic acid or more, resulting in the addition, insertion, deletion or substitution of one or more amino acids in the encoded polypeptide. Of course, changes made to the nucleic acid that have no effect on the encoded amino acid sequence are included. A DA1, DA2 or EOD1 encoding nucleic acid may comprise a sequence having at least 20% or at least 30% sequence identity to the reference nucleic acid sequence, preferably at least 40%, at least 50%, at least 60%, at least 65%, at least 70%, at least 80% %, at least 90%, at least 95%, or at least 98%. Sequence identity is described above.

Sequence similarity and identity are usually defined with reference to the algorithm GAP (Wisconsin Package, Accelerys, San Diego USA). GAP aligns two complete sequences using the Needleman and Wunsch algorithm, which maximizes the number of matches and minimizes the number of gaps. Typically, the default parameters are used with Gap Creation Penalty=12 and Gap Extension Penalty=4. Using GAP may be preferred, but other algorithms such as BLAST (which uses the method of Altschul et al. (1990) J. Mol. Biol. 215:405-410), FASTA (which uses the method of Pearson and Lipman (1988) The method of PNAS USA 85:2444-2448) or the Smith-Waterman algorithm (Smith and Waterman (1981) J. Mol Biol. 147:195-197) or the TBLASTN program of Altschul et al. (1990) supra, usually with default parameters. Specifically, the psi-Blast algorithm (Nucl. Acids Res. (1997) 253389-3402) can be used.

Sequence comparisons can be made over the full length of related sequences described herein.

Suitable variant amino acid and nucleotide sequences can be identified in any plant species of interest using standard sequence analysis techniques.

A DAl, DA2 or EODl nucleotide sequence that is a variant of a reference DAl, DA2 or EODl nucleic acid sequence listed herein selectively hybridizes under stringent conditions to the nucleic acid sequence or its complement.

Stringent conditions include, for example, for sequences that hybridize to about 80%-90% identity, hybridization overnight at 42°C in 0.25M Na ₂ HPO ₄ (pH 7.2), 6.5% SDS, 10% dextran sulfate and at 55°C. Final wash in 0.1XSSC, 0.1% SDS at °C. For sequences detected to be greater than about 90% identical, suitable conditions include hybridization overnight at 65°C in 0.25M Na ₂ HPO ₄ , pH 7.2, 6.5% SDS, 10% dextran sulfate and at 60°C at 0.1 Final wash in XSSC, 0.1% SDS.

An alternative that may be particularly suitable in the case of plant nucleic acid preparations is a solution of 5x SSPE (final 0.9M NaCl, 0.05M sodium phosphate, 0.005 MEDTA pH 7.7), 5X Denhardt's solution, 0.5% SDS at 50°C or 65°C overnight. Washing can be performed at 65°C in 0.2xSSC/0.1% SDS or at 50°C-60°C in 1xSSC/0.1%SDS, as desired.

Nucleic acids as described herein may be wholly or partially synthetic. In particular, they may be recombinant in that nucleic acid sequences (discontinuous stretches) not found together in nature have been joined or otherwise combined artificially. Alternatively, they may have been directly synthesized, for example using an automated synthesizer.

Expression of DA2 nucleic acid and DA1 and/or EOD1 nucleic acid may be reduced or eliminated in one or more cells of a plant by any convenient technique.

Methods for reducing the expression or activity of DA2 polypeptides and DA1 and/or EOD1 polypeptides in plants are well known in the art and are described in more detail below. In some embodiments, the expression of an active DA2, DA1 and/or EOD1 polypeptide can be reduced, preferably eliminated, by introducing a mutation into a nucleic acid sequence in a plant cell that encodes said polypeptide or regulates said nucleic acid sequence expression. Such mutations can disrupt the expression or function of the DA2, DA1 and/or EOD1 polypeptides. Suitable mutations include knockouts and knockdown mutations. In some embodiments, the mutation produces a dominant negative allele of DAl. Plants can then be regenerated from the mutated cells. Nucleic acids can be mutated by insertion or deletion of one or more nucleotides. Techniques for mutating, inactivating or knocking out target genes are well known in the art (see e.g. InVitro Mutagenesis Protocols; Methods in Molecular Biology (2nd Edition) Ed Jeff Braman; Sambrook J et al 2012. Molecular Cloning: A Laboratory Manual (4th Edition) CSHPress; Current Protocols in Molecular Biology; Ed Ausubel et al. (2013) Wiley). In some embodiments, mutations can be introduced into the target EOD1, DA2, or DAl gene by genome editing techniques, such as RNA-guided nuclease technologies such as CRISPR, zinc finger nucleases (ZFNs), and transcription activator-like effector nucleases ( TALEN) (Urnov, F.D. et al. Naturereviews. Genetics 11, 636-646 (2010); Joung, J.K. et al. Naturereviews. Molecular cell biology 14, 49-55 (2013); Gasiunas, G. et al. PNASUSA 109, E2579-2586 (2012); Cong, L. et al. Science 339, 819-823 (2013)).

Sequence mutations that reduce expression or activity may include deletion, insertion or substitution of one or more nucleotides, increase or decrease in gene amplification or methylation, such as hypermethylation, relative to the wild-type nucleotide sequence. The one or more mutations may be in coding or non-coding regions of the nucleic acid sequence. Mutations in the coding region of a gene encoding an element may prevent translation of a full-length active protein, ie, a truncation mutation, or allow translation of a full-length but inactive or impaired functional protein, ie, a missense mutation. A mutation or an epigenetic change, such as methylation, in a non-coding region of a gene encoding an element, for example in a regulatory element, can prevent transcription of said gene. A nucleic acid comprising one or more sequence mutations may encode a variant polypeptide with reduced or eliminated activity, eg, through altered activity of regulatory elements, or may encode a wild-type polypeptide with little or no expression in the cell. A nucleic acid comprising one or more sequence mutations may have one, two, three, four or more mutations relative to the unmutated sequence.

For example, the activity of EOD1 can be reduced, preferably eliminated, by introducing mutations such as deletions, insertions or substitutions, eg, A to T substitutions, at a position corresponding to position 44 of SEQ ID NO:89. A position in the EOD1 polypeptide sequence equivalent to position 44 of SEQ ID NO:89 can be identified using standard sequence analysis and alignment tools, as shown in Table 4.

DA2, DA1 and EOD1 coding sequences can be identified in any plant species of interest using standard sequence analysis techniques, eg, by comparison to the reference sequences listed herein.

Mutations suitable for abrogating expression of active DA2, DA1 and/or EOD1 polypeptides will be apparent to the skilled artisan.

In some preferred embodiments, mutations that reduce or eliminate DA2 expression or activity can be introduced into plant cells that express a dominant-negative DAl polypeptide and optionally include i) a heterozygous EOD1 inhibitor nucleic acid encoding A source nucleic acid or ii) a mutation that reduces EOD1 expression or activity.

In some embodiments, the expression of a DAl, DA2 and/or EOD1 polypeptide in a plant cell can be reduced by expressing a heterologous nucleic acid encoding or a transcriptional repressor nucleic acid, e.g., an inhibitor of daughter RNA or RNAi molecule. The suppressor RNA suppresses the expression of its target polypeptide (ie DA1, DA2 or EOD1) in plant cells.

Nucleic acids as described herein may be wholly or partially synthetic. In particular, they may be recombinant in that nucleic acid sequences (discontinuous stretches) not found together in nature have been joined or otherwise combined artificially. Alternatively, they may have been directly synthesized, for example using an automated synthesizer.

The nucleic acid may of course be double-stranded or single-stranded, cDNA or genomic DNA or RNA. Nucleic acids may be wholly or partially synthetic, depending on design. In general, the skilled artisan will understand that when the nucleic acid includes RNA, references to the sequence shown are to be construed as references to the RNA equivalent, wherein U is replaced by T.

"Heterologous" indicates that the gene/sequence of the nucleotide in question or the sequence regulating the gene/sequence in question has been introduced into said cell of the plant or its ancestors using genetic engineering or recombinant methods, ie by human intervention. A nucleotide sequence heterologous to a plant cell may be non-naturally occurring (i.e., exogenous or foreign) in a cell of that type, variety or species or may be present in a cell of that cell. A sequence that does not naturally occur in a subcellular or genomic environment or may be a sequence that is not naturally regulated in the cell, ie, is operably linked to a non-native regulatory element.

Inhibiting the expression of target polypeptides in plant cells is well known in the art. A suitable suppressor nucleic acid may be a copy of all or part of a target DAl, DA2, and/or EODl gene inserted in antisense or sense orientation, or both, relative to the DAl, DA2, and/or EODl gene in order to achieve said target Decreased gene expression. See, eg, van der Krol et al., (1990) The Plant Cell 2, 291-299; Napoli et al., (1990) The Plant Cell 2, 279-289; Zhang et al., (1992) The Plant Cell 4, 1575-1588 and US-A-5,231,020. Additional refinements of this method can be found in WO95/34668 (Biosource); Angell & Baulcombe (1997) The EMBO Journal 16, 12:3675-3684; and Voinnet & Baulcombe (1997) Nature 389: p.553.

In some embodiments, the suppressor nucleic acid can be a sense suppressor of expression of a DA1, DA2 and/or EOD1 polypeptide.

A suitable sense suppressor nucleic acid may be double-stranded RNA (Fire A. et al. Nature, Vol 391, (1998)). dsRNA-mediated silencing is gene-specific and is often referred to as RNA interference (RNAi). RNAi is a two-step process. First, dsRNA is cleaved intracellularly to produce short interfering RNA (siRNA) of about 21-23 nt length with a 5' terminal phosphate and a short 3' overhang (about 2 nt). siRNAs target corresponding mRNA sequences specific for destruction (Zamore P.D. Nature Structural Biology, 8, 9, 746-750, (2001 ).

siRNAs (sometimes called microRNAs) downregulate gene expression by binding complementary RNA and triggering mRNA depletion (RNAi) or preventing translation of mRNA into protein. siRNAs can be obtained by processing longer double-stranded RNAs, and when found in nature often have exogenous origin. Micro-interfering RNAs (miRNAs) are endogenously encoded small non-coding RNAs obtained by processing short hairpins. siRNAs and miRNAs can inhibit translation of mRNAs carrying partially complementary target sequences without RNA cleavage, and degrade mRNAs carrying fully complementary sequences.

Therefore, the present invention provides the use of RNAi sequences based on DAl, DA2 and/or EOD1 nucleic acid sequences for inhibiting the expression of DAl, DA2 and/or EOD1 polypeptides. For example, the RNAi sequence may correspond to a fragment of a reference DA2, DA1 or EOD1 nucleotide sequence listed herein or may be a variant thereof.

siRNA molecules are usually double-stranded, and in order to optimize the effectiveness of RNA-mediated down-regulation of the function of a target gene, it is preferable to select the length and sequence of the siRNA molecule to ensure proper recognition of the siRNA by the RISC complex (the RISC complex The recognition is mediated by siRNA of the mRNA target) and such that the siRNA is short enough to reduce the host response.

miRNA ligands are typically single-stranded and have a partially complementary region that enables the ligand to form a hairpin. miRNAs are RNA sequences that are transcribed from DNA but not translated into protein. The DNA sequence encoding the miRNA is longer than the miRNA. This DNA sequence includes the miRNA sequence and the approximate reverse complement. When this DNA sequence is transcribed into a single-stranded RNA molecule, the miRNA sequence and its reverse complement base pair to form a partially double-stranded RNA segment. The design of microRNA sequences is discussed in John et al., PLoS Biology, 11(2), 1862-1879, 2004.

Typically, RNA molecules intended to mimic the effects of siRNA or miRNA have between 10 and 40 ribonucleotides (or synthetic analogs thereof), more preferably between 17 and 30 ribonucleotides, more preferably 19 and between 25 ribonucleotides and most preferably between 21 and 23 ribonucleotides. In some embodiments of the invention employing double-stranded siRNA, the molecule may have, for example, a symmetrical 3' overhang of one or two (ribose) nucleotides, typically the UU of the dTdT 3' overhang. Based on the disclosure provided herein, the skilled artisan can readily design suitable siRNA and miRNA sequences, eg, using resources such as siRNA Discoverer (Ambion). siRNA and miRNA sequences can be produced synthetically and added exogenously to cause gene downregulation or produced using expression systems such as vectors. In preferred embodiments, the siRNA is synthesized synthetically.

Longer double-stranded RNAs can be processed in cells to produce siRNAs (see, eg, Myers (2003) Nature Biotechnology 21 :324-328). Longer dsRNA molecules may have symmetrical 3' or 5' overhangs of eg one or two (ribo)nucleotides, or may have blunt ends. Longer dsRNA molecules can be 25 nucleotides or longer. Preferably, the longer dsRNA molecule is between 25 and 30 nucleotides in length. More preferably, the longer dsRNA molecule is between 25 and 27 nucleotides in length. Most preferably, the longer dsRNA molecule is 27 nucleotides in length. The vector pDECAP can be used to express dsRNAs with a length of 30 nucleotides or more (Shinagawa et al., Genes and Dev., 17, 1340-5, 2003).

Another alternative is to express short hairpin RNA molecules (shRNA) in cells. shRNA is more stable than synthetic siRNA. shRNA consists of short inverted repeats separated by minicircle sequences. An inverted repeat is complementary to the gene target. In cells, shRNA is processed by DICER into siRNA that degrades target gene mRNA and inhibits expression. In preferred embodiments, the shRNA is produced endogenously (inside the cell) by transcription from the vector. The shRNA can be produced in the cell by transfecting the cell with a vector encoding the shRNA sequence under the control of an RNA polymerase III promoter, such as the human H1 or 7SK promoter or the RNA polymerase II promoter. Alternatively, shRNA can be synthesized exogenously (in vitro) by transcription from a vector. The shRNA can then be introduced directly into the cells. Preferably, the shRNA molecule includes partial sequences of DA1, DA2 and/or EOD1. For example, shRNA sequences are between 40 and 100 bases in length, more preferably between 40 and 70 bases in length. The length of the stem of the hairpin is preferably between 19 and 30 base pairs. The stem may include G-U pairing to stabilize the hairpin structure.

siRNA molecules, longer dsRNA molecules or miRNA molecules can be produced recombinantly by transcription of a nucleic acid sequence preferably included in a vector. Preferably, the siRNA molecule, longer dsRNA molecule or miRNA molecule comprises a partial sequence of the reference DA2, DA1 or EOD1 nucleotide sequence listed herein or a variant thereof.

In other embodiments, the suppressor nucleic acid can be an antisense suppressor of expression of a DA1, DA2 and/or EOD1 polypeptide. In the use of antisense sequences to downregulate gene expression, a nucleotide sequence is placed under the control of a promoter in a "reverse orientation" such that transcription produces RNA that is identical to the "sense" strand from the target gene The transcribed normal mRNA is complementary. See eg, Rothstein et al., 1987; Smith et al., (1988) Nature 334, 724-726; Zhang et al., (1992) The Plant Cell 4, 1575-1588; English et al., (1996) The Plant Cell 8, 179-188. Antisense technology is also reviewed in Bourque, (1995), Plant Science 105, 125-149 and Flavell (1994) PNASUSA 91, 3490-3496.

An antisense suppressor nucleic acid may include an antisense sequence of at least 10 nucleotides from a nucleotide sequence that is a fragment of a reference DA2, DA1 or EOD1 nucleotide sequence listed herein, or a variant thereof.

It may be preferred that there be complete sequence identity in the sequence used to down-regulate the expression of the target sequence and said target sequence, but complete complementarity or similarity of sequences is not required. One or more nucleotides in the sequence used may differ from the target gene. Thus, the sequences employed in the down-regulation of gene expression according to the invention may be wild-type sequences (eg genes) selected from those available or variants of such sequences.

The sequence does not have to include an open reading frame or specify RNA to be translatable. It may be preferred that there is sufficient homology of corresponding antisense and sense RNA molecules to hybridize. There may be downregulation of gene expression even in cases where there are about 5%, 10%, 15% or 20% or more mismatches between the sequence used and the target gene. Effectively, homology should be sufficient for downregulation of gene expression to occur.

A suppressor RNA molecule may comprise 10-40 nucleotides of the sense or antisense strand of a nucleic acid sequence encoding a DA2, DA1 and/or EOD1 polypeptide.

A suppressor nucleic acid can be operably linked to a heterologous promoter, such as a tissue-specific or inducible promoter. For example, integuments and seed-specific promoters can be used to specifically downregulate two or more DA1, DA2 and/or EOD1 nucleic acids in developing ovules and seeds to increase final seed size.

In some preferred embodiments, a DA2 inhibitor nucleic acid can be expressed in a plant cell having a nucleic acid encoding a dominant negative DAl polypeptide and optionally an EOD1 inhibitor nucleic acid.

Nucleic acids encoding suppressor nucleic acids and/or dominant-negative DAl polypeptides can be included in one or more vectors.

A nucleic acid encoding a suppressor nucleic acid as described herein and/or a dominant-negative DAl polypeptide may be operably linked to a heterologous regulatory sequence, such as a promoter, e.g., a constitutive, inducible, tissue-specific or Developmental specific promoter.

A nucleic acid encoding a suppressor nucleic acid and/or a dominant-negative DAl polypeptide as described herein may be included on a nucleic acid construct or vector. The construct or vector is preferably suitable for transformation into and/or expression in plant cells. A vector is in particular any plasmid, cosmid, phage or Agrobacterium binary vector in double-stranded or single-stranded linear or circular form, which may or may not be self-transferable or mobile, and which Vectors can transform prokaryotic or eukaryotic hosts, in particular plant hosts, by integrating into the genome of the cell or existing extrachromosomally (eg, autonomously replicating a plasmid with an origin of replication).

Specifically included is a shuttle vector, which means a DNA vehicle capable of replicating in two different organisms, either naturally or by design, which can be selected from the group consisting of Actinomycetes and related species, bacteria and eukaryotes (eg higher plant, mammalian, yeast or fungal) cells.

A construct or vector including a nucleic acid as described above need not include a promoter or other regulatory sequences, especially if the vector is not used to introduce the nucleic acid into a cell for recombination into the genome.

Constructs and vectors may also include selectable genetic markers consisting of genes conferring selectable phenotypes such as resistance to antibiotics such as kanamycin, hygromycin, phosphinotricin , chlorsulfuron, methotrexate, gentamicin, spectinomycin, imidazolinones, glyphosate and d-amino acids.

Those skilled in the art are able to construct vectors and design protocols for recombinant gene expression, for example in microorganisms or plant cells. Suitable vectors can be selected or constructed to contain appropriate regulatory sequences, including promoter sequences, terminator fragments, polyadenylation sequences, enhancer sequences, marker genes, and other sequences as appropriate. For further details, see, eg, Molecular Cloning: a Laboratory Manual: 3rd Edition, Sambrook et al., 2001, Cold Spring Harbor Laboratory Press and Protocols in Molecular Biology, 2nd Edition, Ausubel et al., eds. John Wiley & Sons, 1992. Specific procedures and vectors previously used with wide success in plants are given by Bevan, Nucl. Acids Res. (1984) 12, 8711-8721) and Guerineau and Mullineaux, (1993) Plant transformation and expression vectors. In: Plant Molecular Biology Labfax (Ed. Croy RRD) Oxford, BIOS Scientific Publishers , described on pages 121-148.

Certain considerations well known to those skilled in the art must be taken into account when introducing a genetic construct of choice into a cell. The nucleic acid to be inserted should be assembled within a construct that includes effective regulatory elements that will drive transcription. There must be a method available to transport the construct into the cell. Once the construct is within the cell membrane, integration into endogenous chromosomal material will or will not occur. Finally, the target cell type is preferably such that the cells can be regenerated into whole plants.

It is desirable to employ constructs and transformation methods that enhance expression of nucleic acids encoding suppressor nucleic acids or dominant-negative DAl polypeptides. Integration of a single copy of a gene into the genome of a plant cell may be beneficial to minimize gene silencing effects. Again, controlling the complexity of integration can be beneficial in this regard. Of particular interest in this regard is the transformation of plant cells with a minimal gene expression construct according to eg European Patent No. EP1407000B1, which is hereby incorporated by reference for this purpose.

Techniques well known to those skilled in the art can be used to introduce nucleic acid constructs and vectors into plant cells to produce transgenic plants having the properties described herein.

Agrobacterium transformation is a method widely used by those skilled in the art to transform plant species. The generation of stable fertile transgenic plants is now routine in the art (see for example Toriyama, et al. (1988) Bio/Technology 6, 1072-1074; Zhang, et al. (1988) Plant Cell Rep. 7, 379-384; Zhang, et al. ( 1988) TheorAppl Genet 76, 835-840; Shimamoto, et al. (1989) Nature 338, 274-276; Datta, et al. (1990) Bio/Technology 8, 736-740; Christou, et al. (1991) Bio/Technology 9, 957-962 ; Peng, et al. (1991) International Rice Research Institute, Manila, Philippines 563-574; Cao, et al. (1992) Plant Cell Rep. 11, 585-591; Li, et al. (1993) Plant Cell Rep. 12, 250-255; Rathore, et al. , 871-884; Fromm, et al. (1990) Bio/Technology 8, 833-839; Gordon-Kamm, et al. (1990) PlantCell 2, 603-618; D'Halluin, et al. (1992) PlantCell 4, 1495-1505; Walters, et al. (1992) Plant Molecular Biology 18, 189-200; Koziel, et al. (1993) Biotechnology 11, 194-200; Vasil, I.K. (1994) Plant Molecular Biology 25, 925-937; Weeks, et al. (1993) Plant Physiology 102, 8477-10 ; Somers, et al. (1992) Bio/Technology 10, 1589-1594; WO 92/14828; Nilsson, O. et al. (1992) Transgenic Research 1, 209-220).

Other methods, such as microparticle or particle bombardment (US5100792, EP-A-444882, EP-A-434616), electroporation (EP290395, WO8706614), microinjection (WO92/09696, WO94/00583, EP331083, EP175966, Green, etc. Human (1987) Plant Tissue and Cell Culture, Academic Press), direct DNA uptake (DE4005152, WO9012096, US4684611), liposome-mediated DNA uptake (e.g. Freeman et al. Plant Cell Physiol. 29:1353 (1984)) or vortex methods (e.g. Kindle, PNA SU.S.A. 87:1228 (1990d)) may be preferred in cases where Agrobacterium transformation is inefficient or ineffective, for example in some gymnosperm species. Physical methods for transformation of plant cells are reviewed in Oard, 1991, Biotech. Adv. 9:1-11.

Alternatively, a combination of different techniques can be used to enhance the efficiency of the transformation process, such as bombardment with Agrobacterium-coated microparticles (EP-A-486234) or microparticle bombardment to induce wounding followed by co-cultivation with Agrobacterium (EP-A-486234). -486233).

Following transformation, plants can be regenerated, eg, from single cells, callus or leaf discs, as is standard in the art. Almost any plant can be completely regenerated from plant cells, tissues and organs. Available techniques are reviewed in Vasil et al., Cell Culture and Somatic Cell Genetics of Plants, Vol. I, II and III, Laboratory Procedures and Their Applications, Academic Press, 1984 and Weissbach and Weissbach, Methods for Plant Molecular Biology, Academic Press, 1989.

The particular choice of transformation technique will be determined by its efficiency in transforming certain plant species as well as the experience and preference of the practitioner of the invention and the particular method of choice. It will be clear to the skilled person that the particular choice of transformation system for introducing nucleic acid into plant cells is not a requirement for or a limitation of the invention, nor is it a choice of technique for regeneration of plants.

Following transformation, plant cells with reduced expression of DA2 and reduced expression or activity of DA1 and/or EOD1 can be identified and/or selected. Plants can be regenerated from the plant cells.

Plants as described above having reduced DA2 activity or expression that also lack DAl, EODl, or both DAl and EODl can be reproduced or grown sexually or vegetatively to produce progeny or descendants. Progeny or descendants of a plant that can reproduce sexually or asexually or grow regenerated from one or more cells. Plants or their progeny or descendants can be crossed with other plants or with themselves.

DA1, DA2 and/or EOD1 amino acid or nucleic acid sequences can be used as molecular markers to determine one or more of the above-listed DA1, DA2 and/or in plants before, during or after growth or sexual or vegetative reproduction or the expression or activity of an EOD1 polypeptide. One approach may include:

Provides groups of plants,

determining the amount of expression of a DA1, DA2 and/or EOD1 polypeptide in one or more plants of the population, and

One or more plants of the population having reduced expression of a DA1, DA2 and/or EOD1 polypeptide relative to other members of the population are identified.

The plant population can be produced as described above.

In some embodiments, a method may include:

crossing the first and second plants to produce a population of progeny plants;

determining the expression of one or more of the DA1, DA2 and EOD1 polypeptides in the progeny plants in the population, and

Progeny plants in the population having reduced expression of the DA1, DA2 and/or EOD1 polypeptide relative to a control are identified.

One or both of the first and second plants may be produced as described above.

Progeny plants having reduced expression of DA2 and DA1 and/or EOD1 polypeptides relative to a control (e.g., other members of the population) may exhibit increased seed and/or organ size relative to the control and may have higher plant yield .

In some embodiments, the DAl and EOD1 amino acid or nucleic acid sequences can be used as molecular markers to determine the expression or activity of one or more DAl and/or EOD1 polypeptides in plants to identify plants lacking DAl and/or EOD1 or plant cells in which the expression or activity of the DA2 polypeptide can be reduced as described above. One approach may include:

Provides groups of plants,

determining the amount of expression of the DAl and/or EOD1 polypeptide in one or more plants of the population, and

One or more plants of the population having reduced expression of a DAl and/or EOD1 polypeptide relative to other members of the population are identified.

DA2 expression or activity can be reduced in identified plants using the methods described above.

A plant or progeny plant can be identified by i) measuring the amount of DAl, DA2 and/or EOD1 polypeptide in one or more cells of said plant, ii) measuring DAl, DA2 and/or EOD1 polypeptide in one or more cells of said plant, The amount of DA2 and/or EOD1 mRNA, or iii) sequencing nucleic acid encoding a DA1, DA2 and/or EOD1 polypeptide in one or more cells of said plant and identifying the presence of one or more mutations.

The identified plants can be further propagated or crossed, eg, crossed or self-crossed with other plants having reduced expression of DA1, DA2 and/or EOD1 to produce inbred lines. A population of progeny plants can be assayed for expression or activity of a DAl, DA2 and/or EOD1 polypeptide and one or more progeny plants having reduced DAl, DA2 and/or EOD1 expression or activity can be identified.

In some embodiments, the amount of expression of DAl, DA2, and/or EODl can be determined at the protein level. One approach may include:

Provides groups of plants,

Determining the amount of a DAl, DA2 and/or EOD1 polypeptide in one or more plants of the population, and identifying a reduced amount of a DAl, DA2 and/or EOD1 polypeptide in the population relative to other members of the population of one or more plants.

Conveniently, immunological techniques such as Western blot may be employed using antibodies that bind to the DA1, DA2 or EOD1 polypeptide and show little or no binding to other antigens in the plant. For example, the amount of a DAl, DA2 and/or EOD1 polypeptide in a plant cell can be determined by combining a sample comprising the plant cell with an antibody or other specific binding member directed against the DAl, DA2 or EOD1 polypeptide contacting, and determining the binding of the DA1, DA2 or EOD1 polypeptide to the sample. The amount of binding of the specific binding member is indicative of the amount of DAl, DA2 or EODl polypeptide expressed in the cell.

The DA1, DA2 and/or EOD1 polypeptides may be assayed in one or more cells of the plant, preferably in cells from aerial parts or tissues of the plant, such as the vasculature in shoots and primary and secondary meristems amount.

In other embodiments, expression of a DAl, DA2, or EODl polypeptide can be determined at the nucleic acid level. For example, the amount of nucleic acid encoding a DA1, DA2 or EOD1 polypeptide can be determined. A method of producing plants having increased yield-related traits may comprise:

Provides groups of plants,

determining the level or amount of nucleic acid, e.g., mRNA, encoding a DA1, DA2 or EOD1 polypeptide in cells of one or more plants of the population, and

One or more plants in the population are identified that have a reduced amount of a DAl, DA2, or EODl encoding nucleic acid relative to other members of the population.

The level or amount of encoding nucleic acid in a plant cell can be determined, for example, by detecting the amount of transcribed encoding nucleic acid in said cell. This can be done using standard techniques such as Northern blot or RT-PCR.

Alternatively, the presence of sequence variations that affect the expression or activity of a DA1, DA2 or EOD1 polypeptide can be assayed. Another method of producing plants with increased growth and/or biomass may include:

Provides groups of plants,

Determining one or more sequence variations (e.g., polymorphisms, mutations, or hypermethylated regions) in nucleic acids encoding DAl, DA2, and/or EOD1 polypeptides in cells in one or more plants of the population The presence,

wherein said one or more sequence variations reduce the expression or activity of said encoded DA1, DA2 and/or EOD1 polypeptide, and

One or more plants in the population are identified that have one or more sequence variations that reduce the expression or activity of DAl, DA2 and/or EODl relative to other members of the population.

DA1, DA2 and/or EOD1 polypeptides and encoding nucleic acids are described in more detail above.

The presence of one or more sequence variations in a nucleic acid can be determined by detecting the presence of a variant nucleic acid sequence in one or more plant cells or by detecting the presence of a variant polypeptide encoded by said nucleic acid sequence. Preferred nucleic acid sequence variation detection techniques include ARMS ^™ - Allele Specific Amplification, OLA, ALEX ^™ , COPS, Taqman, Molecular Beacons, RFLP, and restriction site based PCR and FRET techniques.

A number of suitable methods for determining the amount of a DAl, DA2 or EOD1 polypeptide-encoding nucleic acid or the presence or absence of sequence variation in a DAl, DA2 or EOD1 polypeptide-encoding nucleic acid in a plant cell are available in the art ( See e.g. (see e.g. Molecular Cloning: a Laboratory Manual: 3rd Edition, Sambrook & Russell (2001) Cold Spring Harbor Laboratory Press NY; Current Protocols in Molecular Biology, edited by Ausubel et al. John Wiley & Sons (1992); DNA Cloning, The Practical Approach Series (1995), series editors D. UK and PCR Protocols: A Guide to Methods and Applications (Innis, et al. 1990. Academic Press, San Diego, Calif.)). Many current methods for detecting sequence variation are described by Nollau et al., Clin. Chem. 43, 1114-1120, 1997; and in standard textbooks such as "Laboratory Protocols for Mutation Detection", edited by U. Landegren, Oxford University Press, 1996 and "PCR", 2nd ed. Newton & Graham, BIOS Scientific Publishers Limited, 1997 reviewed.

Preferred polypeptide sequence variation techniques include immunoassays, which are well known to those skilled in the art, for example APractical Guide to ELISA, DM Kemeny, Pergamon Press 1991; Principles and Practice of Immunoassay, 2nd Edition, CPPrice & DJ Newman, 1997, published by Stockton Press in the US and Canada and Macmillan Reference in the UK.

In some embodiments, a nucleic acid or amplified region thereof can be sequenced to identify or determine the presence of polymorphisms or mutations therein. Polymorphisms or mutations can be identified by comparing the obtained sequence to the known sequence of DAl, DA2 or EODl (eg, as listed in a sequence database). Alternatively, the obtained sequence can be compared to the sequence of the corresponding nucleic acid from a control cell. Specifically, the presence of one or more polymorphisms or mutations that cause a reduction in function, but not complete elimination, can be determined. Sequencing can be performed using any of a range of standard techniques. Sequencing of the amplified product may eg involve precipitation with isopropanol, resuspension and sequencing using the TaqFS+Dye Terminator Sequencing Kit (eg from GE Healthcare UK Ltd UK). Extension products can be electrophoresed on an ABI377 DNA sequencer and the data analyzed using SequenceNavigator software.

Progeny plants identified as having reduced expression of DA1, DA2 and/or EOD1 can be tested for increased or enhanced yield-related traits relative to controls, such as increased seed or organ size.

The identified progeny plants can be further propagated or crossed, eg, crossed (ie, backcrossed) with a first or second plant, or self-crossed to produce an inbred line.

The identified progeny plants can be tested against controls for seed size, organ size and/or plant yield.

Plants produced as described herein may lack DA2 expression or activity and may also lack DAl expression or activity, EODl expression or activity, or both DAl and EODl expression or activity.

The expression or activity of DA2, DA1 and EOD1 in plants can be reduced or eliminated by mutation of one or more nucleotides in the plant coding sequence and/or by expression of a heterologous nucleic acid encoding a suppressor nucleic acid. In some preferred embodiments, the activity of DAl in plants can be reduced or eliminated by expressing a heterologous nucleic acid encoding a dominant-negative DAl polypeptide.

A plant may thus include a heterologous nucleic acid that encodes a suppressor nucleic acid, such as siRNA or shRNA, that reduces expression of one or more of DAl, DA2, and EODl or that encodes a dominant-negative DAl polypeptide.

Any combination of mutations, suppressor nucleic acids can be employed in plants as described herein. For example, a plant can include i) a mutation that reduces DA2 activity or expression, a heterologous nucleic acid encoding a suppressor nucleic acid that reduces EOD1 expression, and a heterologous nucleic acid that encodes a nucleic acid that encodes a dominant-negative DAl polypeptide; Heterologous nucleic acids of suppressor nucleic acids, mutations that reduce expression of EOD1, and heterologous nucleic acids encoding nucleic acids encoding dominant-negative DA1 polypeptides; iii) heterologous nucleic acids encoding suppressor nucleic acids that reduce expression of EOD1 and DA2 and encoding dominant-negative A heterologous nucleic acid of a nucleic acid that is negative for a DAl polypeptide; or iv) a mutation that reduces the activity or expression of EOD1 and DA2 and a heterologous nucleic acid encoding a nucleic acid encoding a dominant negative DAl polypeptide.

In other embodiments, the plant may comprise i) a mutation that reduces DA2 activity or expression, a heterologous nucleic acid encoding a suppressor nucleic acid that reduces DAl expression; ii) a heterologous nucleic acid encoding a suppressor nucleic acid that reduces DA2 expression, that reduces DAl expression iii) a heterologous nucleic acid encoding a suppressor nucleic acid that reduces the expression of DA1 and DA2; iv) a mutation that reduces the activity or expression of DA1 and DA2; or v) a mutation that reduces the activity or expression of DA2 or encodes a suppressor that reduces the expression of DA2 Heterologous nucleic acids of nucleic acids and heterologous nucleic acids encoding nucleic acids encoding dominant-negative DAl polypeptides.

The heterologous nucleic acid encoding the dominant-negative DAl polypeptide and/or suppressor nucleic acid can be on the same or different expression vectors and can be incorporated into plant cells by conventional techniques.

Examples of suitable plants for use according to any aspect of the invention described herein include monocotyledonous and dicotyledonous higher plants, such as agricultural plants or crops, such as plants selected from the group consisting of Lithospermumerythrorhizon ), yew, tobacco, gourd, carrot, brassica vegetable, melon, pepper, vine, lettuce, strawberry, brassica oilseed, sugar beet, wheat, barley, corn, rice, soybean, pea, sorghum, Sunflowers, tomatoes, potatoes, peppers, chrysanthemums, carnations, linseed, hemp and rye. Plants produced as described above can be propagated or grown sexually or vegetatively to produce progeny or descendants. Progeny or descendants of a plant that can reproduce sexually or asexually or grow regenerated from one or more cells. Plants or their progeny or descendants can be crossed with other plants or with themselves.

Another aspect of the present invention provides a transgenic plant having reduced or eliminated expression or activity of a DA2 polypeptide in one or more cells thereof, wherein the plant lacks expression of DAl, EODl, or both DAl and EODl or activity.

The plant may include exogenous nucleic acid that reduces or eliminates the expression or activity of one or more of DA2, DA1 and EOD1. In some embodiments, the transgenic plant expresses a dominant-negative DAl polypeptide that reduces the activity of DAl.

In some embodiments, the plant may have reduced or eliminated expression of DA1, DA2 and EOD1 or may have reduced or eliminated expression of DA2 and EOD1 and may express a dominant negative DAl.

In addition to plants produced by the methods described herein, the invention encompasses any clone of said plant, seed, selfed or hybrid progeny or descendants, and any part or propagule of any of these, such as cuttings and Seed, which can be used for breeding or propagation, sexual or asexual. The invention also encompasses plants that are sexually or asexually propagated progeny, clones or descendants of such plants, or any part or propagule of such plants, progeny, clones or progeny.

Suitable plants can be produced by the methods described above.

Said plants may have increased yield relative to control wild-type plants (ie the same plants in which the expression or activity of DA2 and optionally DA1 and/or EOD1 has not been reduced). For example, the mass of seed (eg, grain) or other plant product per unit area can be increased relative to control plants.

For example, one or more yield-related traits in a plant may be improved. Yield-related traits may include longevity, organ size, and seed size.

Yield-related traits The yield-related traits in plants may be improved, increased or enhanced relative to control plants in which expression of a nucleic acid encoding a DA2 polypeptide has not been eliminated or reduced (i.e. wherein DA2 and optionally DA1 and/or The same plants whose expression of EOD1 has not been reduced or eliminated).

Plants according to the invention may be plants which are not pure in one or more traits. Plant varieties may be excluded, in particular registrable plant varieties under plant growers' rights.

DA1 is shown herein to physically interact with DA2 in vivo. Compounds that disrupt or interfere with such interactions are useful for increasing seed or organ size and increasing plant yield.

Methods of identifying compounds that increase plant yield may include:

determining the effect of the test compound on the binding of the DA2 polypeptide to the DA1 polypeptide,

A reduction or elimination of binding indicates that the compound is useful for increasing plant yield.

DA1 and DA2 polypeptides are described in more detail above.

DA1 and DA2 polypeptides may be isolated or expressed recombinantly or endogenously in plant cells.

Compounds that reduce or eliminate DA1/DA2 binding are useful in the treatment of plants to increase yield.

"And/or" when used herein is to be read as a specific disclosure of each of the two specified features or components with or without the other. For example, "A and/or B" should be considered a specific disclosure of each of (i) A, (ii) B, and (iii) A and B, as if each were individually listed herein.

Unless the context dictates otherwise, the descriptions and definitions of the features listed above are not limited to any particular aspect or embodiment of the invention and apply equally to all aspects and embodiments described.

Further aspects and embodiments of the present invention provide the aspects and embodiments described above wherein the term "comprising" is replaced by the term "consisting of" and wherein the term "comprising" is replaced by the term "consisting essentially of" Aspects and embodiments described above.

All documents mentioned in this specification are hereby incorporated by reference in their entirety for all purposes.

The contents of all database entries mentioned in this specification are hereby incorporated by reference in their entirety for all purposes. This includes any serial versions current as of the filing date of this application.

experiment

1. Method

1.1 Plant material and growth conditions

Arabidopsis ecotype Columbia (Col-0) was the wild type line used. All mutants are on the Col-0 background. da2-1 (SALK_150003) was obtained from the NASC and ABRC deposits of Arabidopsis stock centers. T-DNA insertion was confirmed by PCR and sequencing. Seeds were surface sterilized with 100% isopropanol for 1 min and 10% (v/v) household bleach for 10 min, washed at least three times with sterile water, stratified in the dark at 4°C for 3 days, Disperse on GM medium with 0.9% agar and 1% glucose, and then grow at 22°C. Plants were grown at 22°C under long-day conditions (16 hours light/8 hours dark).

1.2 Constructs and transformation

The pDA2:DA2 construct was prepared by using the PCR-based Gateway system. Primers DA2proGW-F and DA2proGW-R were used to amplify the 1960 bp promoter sequence of DA2. The PCR product was then cloned into the pCR8/GW/TOPOTA cloning vector (Invitrogen). DA2CDS was amplified and the PCR product was then cloned into the AscI and KpnI sites of Gateway vector pMDC110 to obtain the DA2CDS-pMDC110 plasmid. The DA2 promoter was then subcloned into DA2CDS-pMDC110 by LR reaction to generate the pDA2:DA2 construct. Plasmid pDA2:DA2 was introduced into da2-1 mutant plants using Agrobacterium tumefaciens GV3101 and transformants were selected on media containing hygromycin (30 μg/ml).

The 35S:DA2 construct was prepared using the PCR-based Gateway system. The PCR product was subcloned into pCR8/GW/TOPOTA cloning vector (Invitrogen) using TOPO enzyme. The DA2 gene was then subcloned into the Gateway binary vector pMDC32 containing the 35S promoter (Curtis and Grossniklaus, 2003). Plasmid 35S:DA2 was introduced into Col-0 plants using Agrobacterium tumefaciens GV3101 and transformants were selected on media containing hygromycin (30 μg/ml).

The 1960 bp promoter sequence of DA2 was amplified and the PCR product was cloned into pGEM-T vector (Promaga) using T4 DNA ligase and sequenced. The DA2 promoter was then inserted into the SacI and NcoI sites of the binary vector pGreen-GUS (Curtis and Grossniklaus, 2003) to generate the transformation plasmid pDA2:GUS. Plasmid pDA2:GUS was introduced into Col-0 plants using Agrobacterium tumefaciens GV3101 and transformants were selected on medium containing kanamycin (50 μg/ml). The 35S:GW2 construct was prepared using the PCR-based Gateway system. The PCR product was subcloned into pCR8/GW/TOPOTA cloning vector (Invitrogen) using TOPO enzyme. The GW2 gene was then subcloned into the Gateway binary vector pMDC32 containing the 35S promoter (Curtis and Grossniklaus, 2003). Plasmid 35S:GW2 was introduced into Col-0 plants using Agrobacterium tumefaciens GV3101 and transformants were selected on media containing hygromycin (30 μg/ml).

1.3 Morphological and cellular analysis

The average seed weight was determined by weighing mature dry seeds in batches of 500 using an electronic analytical balance (METTLERMOLEDOAL 104CHINA). The weight of five sample batches was measured for each seed batch. Seeds were photographed under a Leica microscope (LEICAS8APO) using a Leica CCD (DFC420) and the seed size was measured by using ImageJ software. Area measurements of petals (Stage 14), leaves and cotyledons were performed by scanning the organs to generate digital images, and area, length and width were then calculated by using ImageJ software. Leaf, petal and germ cell sizes were measured from DIC images. Biomass accumulation in flowers (stage 14) was measured by weighing the organs.

1.4 GUS staining

Samples (pDA2:GUS) were stained in a solution of 1 mMX-gluc, 100 mM Na3PO4 buffer, 3 mM K3Fe(CN)6/K4Fe(CN)6, 10 mM EDTA and 0.1% Nodidet-P40 each, and incubated at room temperature for 6 hours. After GUS staining, chlorophyll was removed using 70% ethanol.

1.5 RNA isolation, RT-PCR and quantitative real-time RT-PCR analysis

Total RNA was extracted from Arabidopsis roots, stems, leaves, seedlings and inflorescences using RNeasy Plant Mini kit (TIANGEN, China). Reverse transcription (RT)-PCR was performed as described (Li et al., 2006). The cDNA samples were normalized based on the amount of actin transcripts using primers ACTIN2-F and ACTIN2-R. Quantitative real-time RT-PCR analysis was performed with lightcycler480engine (Roche) using lightcycler480SYBRGreenMaster (Roche). ACTIN7 mRNA was used as an internal control, and the comparative threshold cycle method was used to calculate the relative amount of mRNA.

1.6E3 ubiquitin ligase activity assay

The coding sequence of DA2 was cloned into the BamHI and PstI sites of the pMAL-C2 vector to generate construct MBP-DA2. Mutant DA2 (DA2C59S and DA2N91L) were generated by following the instruction manual of the Multi-site Directed Mutagenesis Kit (Stratagene).

Bacterial lysates expressing MBP-DA2 and mutated MBP-DA2 were prepared from E. coli BL21 induced with 0.4 mM IPTG for 2 hours. Bacteria were lysed in TGH lysis buffer (50 mM HEPES [pH 7.5], 150 mM NaCl, 1.5 mM MgCl2, 1 mMEGTA, 1% TritonX-100, 10% glycerol, and protease inhibitor cocktail [Roche]) and sonicated. Lysates were clarified by centrifugation and incubated with amylose resin (New England Biolabs) for 30 minutes at 4°C. Beads were washed by column buffer (20 mM Tris pH 7.4, 200 mM NaCl, 1 mM EDTA) and equilibrated by reaction buffer (50 mM Tris pH 7.4, 20 mM DTT, 5 mM MgCl2, 2 mM ATP). 110 ng E1 (Boston Biochem), 170 ng E2 (Boston Biochem), 1 μg His-ubiquitin (Sigma-Aldrich) and 2 μg DA2-MBP or mutated DA2-MBP fusion protein were incubated in 20 μl reaction buffer at 30° C. for 2 hours.

Polyubiquitinated proteins were detected by immunoblotting with antibodies against His (Abmart) and antibodies against MBP (New England Biolabs).

1.7 In vitro protein-protein interactions

The coding sequences of DAl, dal-1 and DAl derivatives containing specific protein domains were cloned into the BamHI and NotI sites of the pGEX-4T-1 vector to generate GST-DA1, GST-DA1R358K, GST-DA1-UIM and the GST-DA1-LIM+C construct, and cloned into the EcoRI and XhoI sites of the pGEX-4T-1 vector to generate the GST-DA1-LIM and GST-DA1-C constructs.

To test protein-protein interactions, bacterial lysates containing approximately 15 μg of MBP-DA2 fusion protein were mixed with approximately 30 μg of GST-DA1, GST-DA1R358K, GST-DA1-UIM, GST-DA1-LIM, GST-DA1 - Lysates of LIM+C or GST-DA1-C fusion proteins pooled. 20 μl of amylose resin (New England Biolabs) was added to each combined solution and shaking was continued for 1 hour at 4°C. The beads were washed several times with TGH buffer, and the separated proteins were separated on a 10% SDS-polyacrylamide gel and analyzed by Western blot with anti-GST antibody (Abmart) and anti-MBP antibody (Abmart), respectively to test.

1.8 Co-immunoprecipitation

The coding sequences of DA1 and DA1-C were cloned into the KpnI and BamHI sites of the pCAMBIA1300-221-Myc vector to generate transformation plasmids 35S::Myc-DA1 and 35S::Myc-DA1-C. The PCR product was subcloned into pCR8/GW/TOPOTA cloning vector (Invitrogen) using TOPO enzyme. The DA2 gene was then subcloned into the Gateway binary vector pMDC43 containing the 35S promoter and GFP gene (Curtis and Grossniklaus, 2003). The PCR product was subcloned into pCR8/GW/TOPOTA cloning vector (Invitrogen) using TOPO enzyme. The PEX10 gene was then subcloned into the Gateway binary vector pH7FWG2 containing the 35S promoter and the GFP gene.

N. benthamiana leaves were transformed by injection of Agrobacterium tumefaciens GV3101 cells harboring the 35S:Myc-DA1 and 35S:GFP-DA2 plasmids, as previously described (Voinnet et al., 2003). Total protein was extracted with extraction buffer (50 mM Tris/HCl (pH7.5), 150 mM NaCl, 20% glycerol, 2% TritonX-100, 1 mM EDTA, 1× complete protease inhibitor cocktail (Roche) and MG13220 ug/ml) and combined with GFP- Trap-A (Chromotek) was incubated at 4°C for 1 hour. Beads were washed 3 times with wash buffer (50 mM Tris/HCl (pH 7.5), 150 mM NaCl, 0.1% TritonX-100, and 1X complete protease inhibitor cocktail (Roche)). Immunoprecipitates were separated in 10% SDS-polyacrylamide gels and detected by Western blot analysis with anti-GFP antibody (Beyotime) and anti-Myc antibody (Abmart), respectively.

1.9 Accession number

The Arabidopsis Genome Project locus identifiers for the Arabidopsis genes referred to herein are as follows: At1g19270 (NP_173361.1GI: 15221983) (DA1), At4g36860 (NP_195404.6GI: 240256211) (DAR1), At1g78420 (NP_001185425.1GI: 334183988) (DA2), At1g17145 (NP_564016.1GI: 18394446) (DA2L) and At3g63530 (NP_001030922.1GI:79316205) (EOD1/BB).

2. Results

2.1da2-1 mutant produces large seeds

To further understand the mechanism of ubiquitin-mediated seed size control, we collected publicly available T- DNA insertion lines and their seed growth phenotypes were studied. From this screen, we identified several T-DNA insertion mutants with altered seed size. One of these mutants was designated da2-1 (DA means "big" in Chinese) with reference to the order of discovery of the large seed size mutants. Seeds produced by da2-1 were larger and heavier than wild-type seeds (Fig. 1A, 3C and 3D). The number of seeds per silique and seed yield per plant in da2-1 were slightly higher than those in the wild type (Fig. 1B and 1C). In contrast, the total number of seeds per plant was not significantly increased in da2-1 compared to the total number of seeds per plant in wild type (Fig. ID). da2-1 plants were higher than wild-type plants at maturity (Fig. 1E). Furthermore, da2-1 mutant plants developed larger flowers and leaves and increased biomass compared to wild-type plants (Fig. 2; Fig. 15). The increased petal and leaf size of da2-1 mutants was not caused by larger cells (Figure 15), indicating a higher number of petal and leaf cells.

2.2 DA2 cooperates with DA1 to control seed size but is independent of EOD1 to control seed size

The da2-1 mutant displayed a weak but similar seed size phenotype to dal-1 (Li et al., 2008), providing an indication that DA1 and DA2 can function in a common pathway. To test the genetic interaction between DA1 and DA2, we generated a dal-1da2-1 double mutant and determined its seed size. Although the da2-1 mutant has slightly larger and heavier seeds than the wild type (Fig. 1A, 3C and 3D), the da2-1 mutation synergistically enhances the seed size and weight phenotype of da1-1 (Fig. 3A and 3C) , thus revealing a synergistic genetic interaction between DA1 and DA2 in terms of seed size. Changes in seed size were reflected in the size of the embryo and resulting seedlings (Fig. 3B). The cotyledon area of 10-day-old seedlings was further measured. A synergistic enhancement of cotyledon size by the da2-1 mutation da1-1 was also observed (Figures 3B and 4). The mutant protein encoded by the dal-1 allele has negative activity against DA1 and DA1-related protein (DAR1), the most closely related family members (Li et al., 2008).

The double da1-ko1 dar1-1 T-DNA insertion mutant exhibited a da1-1 phenotype, whereas the da1-ko1 and dar1-1 single mutants did not display an obvious seed size phenotype (Li et al., 2008). Because da1-1 and da2-1 act synergistically to increase seed size, it would be expected that da1-ko1 could synergistically enhance the da2-1 phenotype. To test this, we generated a da1-ko1da2-1 double mutant. As shown in Figure 3D, the seed size and weight phenotypes of da2-1 were also synergistically enhanced by the da1-ko1 mutation. The cotyledon area of 10-day-old seedlings was further measured. The da1-ko1 mutation synergistically enhanced the cotyledon size phenotype of da2-1 (Fig. 4 upper right). Similarly, a synergistic enhancement of da2-1 petal size was also observed by the da1-ko1 mutation (Fig. 16D). These results further demonstrate the synergy of the simultaneous disruption of both DA1 and DA2.

The size of germ cells and petal epidermal cells was further measured. The cell size of the dal-1da2-1 and dal-ko1da2-1 double mutants was not increased compared to the cell size measured in their parental lines (Fig. 4 bottom left; Fig. 16E), thus providing an indication that DA1 and DA2 act synergistically to Limit cell proliferation process.

The da1-1da2-1 double mutant has larger seeds than the da1-ko1da2-1 double mutant (Figures 3C, 3D, and 4), which is consistent with our previous report that the dal-1 allele has a stronger The phenotype is consistent with (Li et al., 2008). The size of the dal-1 seeds was similar to that of the dal-ko1 dar1-1 double mutant seeds because the dal-1 allele was negatively active against both DA1 and DAR1 (Fig. 4 bottom right) (Li et al., 2008). Therefore, it would be expected that the size of the dal-lda2-1 double mutant seeds may be similar to the size of the dal-koldarl-lda2-1 triple mutant seeds. So we produced the da1-ko1dar1-1da2-1 triple mutant and studied its seed size. As shown in Figure 4, the size of the da1-ko1dar1-1da2-1 triple mutant seeds was comparable to that of the da1-1da2-1 double mutant seeds, but larger than that of the da1-ko1da2-1 double mutant seeds. Therefore, these genetic analyzes further support that the dal-1 allele has a negative effect on both DA1 and DAR1 (Li et al., 2008).

We have previously identified an enhancer for dal-1 (EOD1 ) that is allelic to BIGBROTHER (BB) (Disch et al., 2006; Li et al., 2008). Mutations in eod1 synergistically enhance the seed size phenotype of da1-1 (Li et al., 2008). Similarly, the seed size and weight phenotypes of da2-1 were synergistically enhanced by dal-1 and dal-ko1 (Figures 3A, 3C and 3D). therefore asked whether DA2 and EOD1 could function in a common pathway. To determine the genetic relationship between DA2 and EOD1, we analyzed the eod1-2da2-1 double mutant. The genetic interaction between eod1-2 and da2-1 was substantially additive for both seed weight and petal size compared to its parental line (Figure 16), providing an indication that DA2 functions separately from EOD1 to affect seed and organ growth.

2.3 DA2 parent body acts to affect seed size

Considering that seed size is influenced by maternal and/or zygotic tissue, it was asked whether DA2 functions maternally or zygotically. To test this, we performed a reciprocal cross between wild type and da2-1. As shown in Figure 6, the effect of da2-1 on seed size was only observed when the maternal plants were homozygous for the da2-1 mutation. Regardless of the genotype of the pollen donor, the seeds produced by the maternal da2-1 plants were consistently larger than those produced by the maternal wild-type plants. This result indicates that da2-1 can act maternally to affect seed size. We have previously demonstrated that DA1 also functions maternally to control seed size (Li et al. 2008). Since the da1-ko1 mutations synergistically enhanced the seed size phenotype of da2-1 (Fig. 3D), we further conducted a reciprocal cross experiment between the wild type and the da1-ko1da2-1 double mutant. Similarly, the effect of dal-ko1da2-1 on seed size was only observed when dal-ko1da2-1 served as the parent plant (Fig. 6).

Pollination of da1-ko1/+da2-1/+ plants with pollen of the da1-ko1da2-1 double mutant results in da1-ko1da2-1, da1-ko1/da1- Development of ko1da2-1/+, da1-ko1/+da2-1da2-1 and da1-ko1/+da2-1/+ embryos. Individual seeds from da1-ko1/+da2-1/+ plants fertilized with pollen of the da1-ko1 da2-1 double mutant were further measured for size and genotyped for the da1-ko1 and da2-1 mutations. The results showed that da1-ko1 and da2-1 mutations were not associated with changes in the size of these seeds (Figure 6). Taken together, these analyzes indicate that genotypes of DA1 and DA2 in the germ and endosperm do not affect seed size and that DA1 and DA2 are required in the sporophyte tissue of the mother plant to control seed growth.

2.4 DA2 cooperates with DA1 to affect cell proliferation in the maternal integument

Reciprocal crosses showed that DA1 and DA2 function maternally to determine seed size (Figure 6) (Li et al., 2008). The integument surrounding the ovule is maternal tissue and after fertilization forms the seed coat, which physically restricts seed growth. Several studies have shown that the integument size of the ovule determines the seed size (Schruff et al., 2006; Adamski et al., 2009). It was therefore asked whether DA1 and DA2 control seed size through maternal integumentary action. To test this, we studied mature ovules from wild type, da1-1, da2-1 and da1-1da2-1 at 2 days after emasculation. Consistent with our previous findings (Li et al., 2008), the size of da1-1 ovules was significantly larger than that of wild-type ovules (Figures 5 and 7).

da2-1 ovules were also larger than wild-type ovules (Figures 5 and 7). The da2-1 mutation synergistically enhances the ovule size phenotype of da1-1, consistent with its synergistic interaction on seed size.

We investigated the outer integumentary cell numbers of developing seeds in wild type, da1-1, da2-1 and da1-1da2-1 at 6DAP and 8DAP. In wild-type seeds, the number of cells in the outer integument at 6 DAP was similar to the number of cells in the outer integument at 8 DAP (Fig. 7 middle panel), indicating that the cells in the outer integument of wild-type seeds had completely ceased at 6 DAP Split. Similarly, cells in the outer integument of dal-1, da2-1 and dal-1da2-1 seeds completely ceased cell proliferation at 6 DAP. The number of outer integumentary cells was significantly increased in dal-1 and da2-1 seeds compared to the number of outer integumentary cells in wild type seeds (Figure 7). The da2-1 mutation synergistically enhances the outer integument cell number of da1-1. We further investigated the outer integument cell length of wild-type, da1-1, da2-1 and da1-1da2-1 seeds at 6 and 8 days after pollination. The cells in the outer integument of da1-1, da2-1, and da1-1da2-1 were significantly shorter than those in the wild-type outer integument (Fig. indication of the compensation mechanism. Thus, these results suggest that DA2 acts synergistically with DA1 to limit cell proliferation in the maternal integument.

2.5DA2 encodes a functional E3 ubiquitin ligase

The da2-1 mutation was identified in the case of a T-DNA insertion in the seventh exon of the gene At1g78420 (Fig. 8A). The T-DNA insertion site was further confirmed by PCR using T-DNA specific and flanking primers and sequencing the PCR product. The full-length mRNA of At1g78420 could not be detected by semi-quantitative RT-PCR in the da2-1 mutant. We expressed the At1g78420CDS under the control of its own promoter in da2-1 plants and isolated 62 transgenic plants. Almost all transgenic lines showed complementation of the da2-1 phenotype (Figure 10), indicating that At1g78420 is the DA2 gene.

To further characterize DA2 function, specifically the gain-of-function phenotype, the coding region of DA2 was expressed under the control of the CaMV35S promoter in wild-type plants and 77 isolated transgenic plants. Overexpression of DA2 caused a decrease in seed size, seed yield per plant, and number of seeds per plant (FIGS. 1A, 1C and ID). Furthermore, most transgenic plants overexpressing DA2 had smaller flowers and leaves, shorter siliques, reduced plant height, and reduced biomass compared to wild type (Fig. 1E, 2 and 15). These results further support a role for DA2 in limiting seed and organ growth.

The DA2 gene was predicted to encode a 402 amino acid protein containing a predicted RING domain (59-101) (Fig. 8B; Table 1). To investigate whether DA2 has E3 ubiquitin ligase activity, we expressed DA2 in E. coli as a fusion protein with maltose-binding protein (MBP) and purified the MBP-DA2 protein from the soluble fraction. In the presence of El ubiquitin activating enzyme, E2 conjugating enzyme, His-ubiquitin and MBP-DA2, polyubiquitination signal was observed by Western blot using anti-His antibody (Fig. 9, fifth lane from the left). Anti-MBP blot analysis also indicated that MBP-DA2 was ubiquitinated (Figure 9, fifth lane from the left). However, in the absence of any of E1, E2, His-ubiquitin, or MBP-DA2, no polyubiquitination was detected (Figure 9, first to fourth lanes from the left), thus demonstrating that DA2 is a functional Sexual E3 ubiquitin ligase. The RING motif is essential for the E3 ubiquitin ligase activity of RING zinc finger proteins (Xie et al., 2002). We therefore examined whether the full RING zinc finger domain is required for DA2E3 ligase activity. A single amino acid substitution allele was generated by mutating cysteine-59 to serine (C59S), as this mutation was predicted to disrupt the RING domain (Tables 1 and 2). In vitro ubiquitination assays indicated that E3 ligase activity was abolished in the C59S mutant of DA2 (Figure 9, sixth lane from the left), indicating that the intact RING domain is required for DA2E3 ubiquitin ligase activity. We further overexpressed DA2C59S (35S:DA2C59S) in wild-type Col-0 plants and isolated 69 transgenic plants. The seed size of transgenic plants was comparable to that of wild-type plants, but transgenic plants had high DA2C59S expression levels, indicating that the DA2C59S mutation affects the function of DA2 in seed growth.

Three RING types RING-H2, RING-Hca, and RING-HCb and five modified RING types RING-C2, RING-v, RING-D, RING-S/T, and RING-HCb have been described in Arabidopsis. G (Stone et al., 2005). A new type of RING domain (C5HC2) found in rice GW2 was proposed (Song et al., 2007). Although the spacing of cysteines in the predicted RING domain of DA2 is similar to the spacing of cysteines in the RING domain of rice GW2 (C5HC2), the RING domain of DA2 lacks replacement by asparagine residues The conserved histidine residue (Asn-91) (Tables 1 and 2). This amino acid substitution was also observed in the predicted RING domain of DA2 homologs in dicots such as soybean and canola (Table 1). It was therefore asked whether this asparagine residue (Asn-91 ) is critical for its E3 ubiquitin ligase activity. Single amino acid substitution alleles were generated by mutating Asn-91 to leucine (N91L). In vitro ubiquitination assays indicated that the N91L mutant of DA2 has E3 ligase activity (Figure 9, seventh lane from the left), suggesting that Asn-91 may not be required for DA2 E3 ligase activity. These results suggest that the RING domain of DA2 may be a variant of the RING domain found in GW2. We further overexpressed DA2N91L (35S:DA2N91L) in wild-type plants and isolated 26 transgenic plants. Seeds of transgenic plants were smaller than wild-type seeds, suggesting that DA2N91L can limit seed growth.

2.6 Homologs of Arabidopsis DA2

Proteins sharing significant homology with DA2 outside the RING domain were found in Arabidopsis and crops including rapeseed, soybean, rice, maize and barley (Table 2). A predicted protein in Arabidopsis shared extensive amino acid similarity with DA2 and was named DA2-like protein (DA2L; At1g17145). Like the 35S:DA2 plants, the DA2L overexpression line exhibited smaller plants and organs (Figure 18), providing an indication that DA2 and DA2L have similar functions. Similar proteins in other plant species showed 39.2% - 84.5% amino acid sequence identity to DA2 (Table 2). The homologue in Brassica napus had the highest amino acid sequence identity (84.5%) with DA2 (Table 2). Rice GW2 has 43.1% amino acid sequence identity with Arabidopsis DA2 (Table 2). Since overexpression of GW2 reduces grain width in rice (Song et al., 2007), it was asked whether DA2 and GW2 perform similar functions in seed size control. GW2 is thus overexpressed in wild-type plants. Like the 35S:DA2 and 35S:DA2L transgenic lines, Arabidopsis transgenic plants overexpressing GW2 produced smaller seeds and organs than wild-type plants, indicating the role of Arabidopsis DA2 and rice GW2 in seed and organ growth control. Conservative function.

2.7 DA2 and DA1 show similar expression patterns

To determine the expression pattern of DA2, RNA from roots, stems, leaves, seedlings and inflorescences were analyzed by quantitative real-time RT-PCR analysis. DA2 mRNA was detected in all plant organs tested (Fig. 11A). The tissue-specific expression pattern of DA2 was investigated using histochemical assays of GUS activity in transgenic plants containing a DA2 promoter:GUS (pDA2:GUS) fusion. GUS activity was detected in roots, cotyledons, leaves, and inflorescences (Figures 11B and 11C). Relatively high GUS activity was detected in leaf primordia and roots (Figures 1 IB and 11C). In flowers, relatively stronger DA2 expression was observed in young floral organs than in old floral organs (Figs. 11D-11L). Similarly, higher GUS activity was detected in younger ovules than in older ovules (Figures 11M and 11N). This suggests that DA2 expression is temporally and spatially regulated.

2.8 Interaction between DA1 and DA2 in vitro and in vivo

Our genetic analysis suggests that DA1 cooperates with DA2 to limit seed and organ growth. An in vitro interaction/pull-out assay was therefore used to assess whether DA1 interacts with the E3 ubiquitin ligase DA2. DA1 was expressed as a GST fusion protein, while DA2 was expressed as a MBP fusion protein. As shown in Figure 12 (first and second lanes from the left), GST-DA1 bound to MBP-DA2, while GST-DA1 did not bind to the negative control (MBP). This result indicates that DA1 and DA2 physically interact in vitro.

DA1 includes two ubiquitin-interacting motifs (UIMs), a single LIM domain, and a highly conserved C-terminal region (Figure 13) (Li et al., 2008). It was further asked which domain of DA1 is required for the interaction between DA1 and DA2. A series of D1 derivatives containing specific protein domains to be expressed in E. coli: DA1-UIM with only two UIM domains, DA1-LIM with only LIM domain, only LIM domain and C-terminal region DA1-LIM+C and DA1-C with only the C-terminal region were expressed as GST fusion proteins ( FIG. 13 ).

DA2 was expressed as an MBP fusion protein and used in pull-out experiments. As shown in Figure 12, GST-DA1-LIM+C and GST-DA1-C interacted with MBP-DA2, but GST-DA1-UIM and GST-DA1-LIM did not bind to MBP-DA2. This result indicates that the conserved C-terminal region of DAl interacts with DA2.

Considering that the mutant protein encoded by the dal-1 allele (DA1R358K) has a mutation in the C-terminal region (Figure 13) (Li et al., 2008), it was asked whether the DA1R358K mutation affects the interaction with DA2. Using the GST-DA1R358K fusion protein in pull-out experiments with MBP-DA2, we showed that the mutation in DA1R358K did not affect the interaction between DA1 and DA2 (Figure 12, third lane from the left).

To further investigate the possible correlation between DA1 and DA2 in plants, co-immunoprecipitation analysis was used to detect their in vivo interactions. Transient coexpression of 35S:Myc-DA1 and 35S:GFP-DA2 in N. benthamiana leaves. Transient co-expression of 35S:GFP and 35S:Myc-DA1 in N. benthamiana leaves was used as a negative control. Total protein was isolated and incubated with GFP-Trap-A agarose beads to immunoprecipitate GFP-DA2 or GFP. Precipitates were detected with anti-GFP and anti-Myc antibodies, respectively. As shown in Figure 14, Myc-DA1 was detected in the immunoprecipitated GFP-DA2 complex, but not in the negative control (GFP), indicating that there is a physical association between DA1 and DA2 in plants . Since the C-terminal region of DAl interacts with DA2 in the pull-out assay (Figure 12), it was further asked whether the C-terminal end of DAl interacts with DA2 in plants. Co-immunoprecipitation analysis revealed that the C-terminal region of DA1 (Myc-DA1-C) was detected in the GFP-DA2 complex but not in the negative control (PEX10-GFP, a RING-type E3 ubiquitin ligase) (Platta et al., 2009; Kaur et al., 2013). Therefore, these results indicate that the C-terminal region of DAl is required for interaction with DA2 in vitro and in vivo.

Seed size in higher plants is a key determinant of evolutionary fitness and is also an important agronomic trait in crop domestication (Gomez, 2004; Orsi and Tanksley, 2009).

Several factors have been identified that act maternally to control seed size, such as ARF2/MNT, AP2, KLU/CYP78A5, EOD3/CYP78A6 and DA1. However, the genetic and molecular mechanisms of these factors in seed size control are almost completely unknown. We previously demonstrated that the ubiquitin receptor DA1 cooperates with the E3 ubiquitin ligase EOD1/BB to control seed size (Li et al., 2008).

In this study, we identified Arabidopsis DA2 as another RINGE3 ubiquitin ligase involved in the control of seed size. Genetic analysis revealed that DA2 acts cooperatively with DA1 to control final seed size, but independently of the E3 ubiquitin ligase EOD1. We further reveal that DA1 physically interacts with DA2. Our results define a ubiquitin-based system involving DA1, DA2 and EOD1 that controls the final seed size of Arabidopsis.

2.9 DA2 matrix acts to control seed size

da2-1 loss-of-function mutants formed large seeds and organs, whereas plants overexpressing DA2 produced small seeds and organs (Fig. 1A), indicating that DA2 is a negative factor in the control of seed and organ size. Surprisingly, Arabidopsis DA2 has recently been proposed as a positive regulator of organ growth, although nothing is known about how DA2 controls seed and organ growth (VanDaele et al., 2012). In this study, we have sufficient evidence to demonstrate that DA2 acts as a negative factor in seed and organ growth control. da2-1 loss-of-function mutants formed large seeds and organs (Figures 1 to 4). This is supported by the synergistic enhancement of the seed and organ size phenotypes of da1-1 and da1-ko1 by the da2-1 mutation (Figures 1 to 4). The da2-1 mutation also enhanced the seed and organ size phenotypes of eod1-2, further indicating that the da2-1 mutation promotes seed and organ growth. The da2-1 mutant formed large ovules with more cells in the integument, and the da2-1 mutation synergistically enhanced the ovule size phenotype of da1-1 (Figure 6).

Furthermore, most transgenic plants overexpressing DA2 and DA2L were smaller than wild-type plants (Fig. 2; Fig. S9). The organ growth phenotypes of these transgenic plants correlated with their corresponding expression levels (Figures S4 and S9). Thus, our data clearly demonstrate that DA2 acts as a negative regulator of seed and organ size. Several Arabidopsis mutants with large organs also form large seeds (Krizek, 1999; Mizukami and Fischer, 2000; Schruff et al., 2006; Li et al., 2008; Adamski et al., 2009), thus suggesting a relationship between organ size and seed A possible link between growth. In contrast, several other mutants with large organs exhibited normal sized seeds (Hu et al., 2003; White, 2006; Xu and Li, 2011), indicating that organ and seed size are not always positively correlated. These results suggest that seeds and organs have shared and distinct pathways to control their corresponding sizes.

Reciprocal cross experiments showed that DA2 acts maternally to affect seed growth, and the germ and endosperm genotypes of DA2 did not affect seed size (Fig. 6). The integument surrounding the ovule is maternal tissue and forms the seed coat after fertilization. Changes in maternal integument size (such as those observed in arf2, dal-1 and klu ovules) are known to contribute to changes in seed size (Schruff et al., 2006; Li et al., 2008; Adamski et al., 2009). Mature da2-1 ovules were also larger than mature wild-type ovules (Figures 5 and 7). The da2-1 mutation also synergistically enhanced the integument size of da1-1 ovules. Thus, the general theme emerging from these studies is that control of parent integument size is one of the key mechanisms for determining final seed size. Consistent with this notion, so far isolated seed size-controlling plant maternal factors such as KLU, ARF2 and DA1 affect integument size (Schruff et al., 2006; Li et al., 2008; Adamski et al., 2009).

The size of the integument, or seed coat, is determined by the two coordinated processes of cell proliferation and cell expansion. The number of cells in the integument of a mature ovule sets the growth potential of the testa after fertilization. For example, arf2 mutants produce large ovules with more cells, resulting in large seeds (Schruff et al. 2006), while klu mutants have small ovules with fewer cells, resulting in small seeds (Adamski et al., 2009). Our results indicated that the integument of dal-1 and da2-1 seeds had more cells than that of wild-type seeds, and that dal-1 and da2-1 acted synergistically to promote cell proliferation in the integument. We also observed that cells in the outer integument of dal-1, da2-1, and da1-1da2-1 seeds were shorter than those in the wild-type integument, thus suggesting a link between cell proliferation and cell elongation in the maternal integument possible compensation mechanisms. Thus, the maternal integument or seed coat, potentially acting as a physical constraint on seed growth, can set an upper limit on final seed size.

2.10 Genetic framework for ubiquitin-mediated seed size control

DA2 encodes a protein with a predicted RING domain distinct from any of the previously described plant RING domains. The RING domain of DA2 shares the highest homology with that of rice GW2 (C5HC2), but it lacks a conserved metal ligand amino acid (histidine residue) replaced by an asparagine residue (Song et al. People, 2007). It remains possible that the RING domain of DA2 could be a variant of the RING domain found in GW2. Many RING-type domains are found in E3 ubiquitin ligases, and ubiquitinated substrates are often targeted to them for subsequent proteasomal degradation (Smalle and Vierstra, 2004). We tested the E3 activity of recombinant DA2 in an in vitro ubiquitin ligase assay and demonstrated that DA2 is a functional E3 ubiquitin ligase, thereby suggesting that DA2 can target positive regulators of cell proliferation for ubiquitination by the 26S proteasome dependent degradation. Proteins sharing homology with DA2 outside the RING domain are found in Arabidopsis and other plant species. In Arabidopsis, DA2-like proteins (DA2L) share extensive amino acid similarity with DA2. Like the 35S:DA2 plants, the DA2L overexpression line exhibited smaller plants (Figure 18), indicating that DA2 and DA2L may perform similar functions. The homologue of DA2 in rice is the RING-type (C5HC2) protein GW2 (Song et al., 2007), which is known to act as a negative regulator of seed size. However, the genetic and molecular mechanisms of GW2 in seed size control are largely unknown in rice.

We previously identified DA1, a ubiquitin receptor with ubiquitin-binding activity, as a negative regulator of seed size (Li et al., 2008). A modifier gene screen identified an enhancer of dal-1 (EOD1) (Li et al., 2008) that is BB allelic to the E3 ubiquitin ligase (Disch et al., 2006). Analysis of double eod1-2da1-1 mutants revealed a synergistic genetic interaction between DA1 and EOD1 (Li et al., 2008), suggesting that they can control seed growth by modulating the activity of shared targets. Although the genetic interaction between da1-1 and eod1-2 also synergistically enhances seed and organ size, genetic analysis suggests that DA2 acts independently of EOD1 to affect seed growth, thus suggesting that DA2 and EOD1 may target distinct growth for degradation stimuli, which are co-regulated via DA1. Thus, our findings establish a framework for the control of seed and organ size by the three ubiquitin-associated proteins DA1, DA2 and EOD1. Furthermore, we observed that overexpression of GW2 limits seed and organ growth in Arabidopsis, providing an indication of a possible conserved function in Arabidopsis and rice. It may be interesting to study the effect of the combination of GW2 and the rice homologues of DA1 and EOD1 on grain size in rice.

2.11 Possible molecular mechanisms of DA1 and DA2 in seed size control

Our results demonstrate that the E3 ubiquitin ligase DA2 interacts with the ubiquitin receptor DA1 in vitro and in vivo (Figures 12-14). However, it is unlikely that DA2 targets DA1 for proteasomal degradation, since a T-DNA inserted mutant of the DA1 gene (da1-ko1) synergistically enhances the seed size phenotype of da2-1 (Figs 3 and 4) . However, many other types of ubiquitin modifications regulate proteins in a proteasome-independent manner (Schnell and Hicke, 2003). For example, monoubiquitination has been implicated in the activation of signaling proteins, endocytosis and histone modification (Schnell and Hicke, 2003). In animals, monoubiquitination of the ubiquitin receptor EPS15 depends on the interaction between EPS15 and the Nedd4 family of E3 ligases (Woelk et al., 2006). In contrast, E3-independent monoubiquitination of ubiquitin receptors has also been reported (Hoeller et al., 2007). Given that DA1 interacts with DA2, we tested whether DA2 is able to ubiquitinate or monoubiquitinate DA1. In the presence of E1, E2 and ubiquitin, DA2-His has E3 ubiquitin ligase activity. However, no ubiquitinated DA1-HA was detected under our reaction conditions in the presence of E1, E2, ubiquitin and DA2-His (E3). Ubiquitin receptors are able to interact with polyubiquitinated substrates of E3 via the domain UIM and promote their degradation by the proteasome (Verma et al., 2004). We previously demonstrated that the UIM domain of DA1 is capable of binding ubiquitin (Li et al., 2008).

In conclusion, where DAl interacts with DA2 through its C-terminal region (Figures 12 and 14), DAl may be involved in mediating the degradation of ubiquitinated substrates of DA2 by the proteasome. One mechanism may involve the interaction of DA1 with DA2, which facilitates the specific recognition of ubiquitinated substrates of DA2 by DA1. DAl can then bind the polyubiquitin chains of ubiquitinated substrates through its UIM domain and mediate the degradation of said polyubiquitinated substrates. Improving seed yield is an important goal of crop breeding worldwide, and seed size is an important component of total seed yield. We identified DA2 as an important regulator of seed size, which acts synergistically with DA1 to affect seed size.

DA1 also cooperates with EOD1 to affect seed growth. Overexpression of a dominant-negative da1-1 mutation (Zmda1-1) was reported to increase seed quality in cereals (Wang et al., 2012), suggesting the role of combining DA1, DA2 and EOD1 from different seed Prospects for engineering large seed sizes.

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Table 1: Alignment of DA2RING domains (SEQ ID NO: 3-19).

Table 2: Alignment of DA2 polypeptides (SEQ ID NO:20-35)

* indicates the same residue

: indicates a conserved residue

.indicates semi-conserved residues

The RING domain and the first and second consensus domains are boxed.

Table 3: Alignment of DA1 proteins (SEQ ID NO: 41-64)

Table 4: Alignment of EOD1 proteins (SEQ ID NO:74-90)

Claims (44)

1. A method of increasing the yield of a plant, said method comprising: reducing the expression or activity of the DA2 polypeptide in cells of said plant, wherein said plant has reduced expression or activity of DA1 and/or EOD1. 2. The method of claim 1, wherein the plant has reduced EODl activity. 3. The method of claim 1 or claim 2, wherein the plant has reduced DAl expression or activity. 4. The method of claim 3, wherein the plant expresses a dominant-negative DAl polypeptide. 5. The method according to any one of claims 1 to 4, wherein the expression or activity of the DA2 polypeptide is abolished in cells of the plant. 6. A method of increasing the yield of a plant, said method comprising: reducing the expression or activity of the DA2 polypeptide, and; Decreasing the expression or activity of the DAl polypeptide and/or the EOD polypeptide in the cells of said plant. 7. The method of claim 6, wherein the method comprises reducing EODl polypeptide expression or activity in cells of the plant. 8. The method according to claim 7, wherein the expression or activity of the EOD1 polypeptide is reduced by introducing a mutation into the nucleotide sequence of the plant cell and regenerating the plant from the mutated cell, the nucleoside The acid sequence encodes the EOD1 polypeptide or regulates its expression. 9. The method according to claim 7, wherein the expression or activity of the EOD1 polypeptide is reduced by incorporating a heterologous nucleic acid into the plant cell, and the expression of the heterologous nucleic acid reduces the inhibition of the expression of the EOD1 polypeptide sub-nucleic acid. 10. The method according to any one of claims 6 to 9, wherein the method comprises reducing DAl polypeptide expression or activity in cells of the plant. 11. The method according to claim 10, wherein the expression or activity of the DAl polypeptide is reduced by introducing a mutation into the nucleotide sequence of the plant cell and regenerating the plant from the mutated cell, the nucleoside The acid sequence encodes the DAl polypeptide or regulates its expression. 12. The method according to claim 10, wherein the expression or activity of the DAl polypeptide is reduced by incorporating a heterologous nucleic acid into the plant cell, the expression of the heterologous nucleic acid reducing the inhibition of the expression of the DAl polypeptide sub-nucleic acid. 13. The method of claim 10, wherein the expression or activity of the DAl polypeptide is decreased by expressing a dominant-negative DAl polypeptide in cells of the plant. 14. The method according to any one of claims 6 to 13, wherein the expression of the DA2 polypeptide is reduced by introducing a mutation into the nucleotide sequence of the plant cell and regenerating the plant from the mutated cell or activity, the nucleotide sequence encodes the DA2 polypeptide or regulates its expression. 15. The method according to any one of claims 6 to 13, wherein the expression or activity of the DA2 polypeptide is reduced by incorporating a heterologous nucleic acid into the plant cell, the heterologous nucleic acid expression reducing the A suppressor nucleic acid for the expression of a DA2 polypeptide. 16. The method according to any one of claims 6 to 16, wherein the expression or activity of the DA2 polypeptide is abolished in cells of the plant. 17. A method of producing plants with increased yield, said method comprising: providing a plant cell lacking the expression or activity of DA1, EOD1, or both DA1 and EOD1, incorporating a heterologous nucleic acid that reduces the expression or activity of a DA2 polypeptide; or introducing into said plant cell a mutation that reduces the expression or activity of a DA2 polypeptide, and; The plants are regenerated from one or more transformed cells. 18. The method of claim 17, wherein the plant cell lacks EODl polypeptide expression or activity. 19. The method according to claim 18, wherein said plant cell comprises a mutation in the nucleotide sequence of said plant cell, said nucleotide sequence encodes said EOD1 polypeptide or regulates its expression; or another isotropic A source nucleic acid that expresses a suppressor nucleic acid that reduces expression of said EOD1 polypeptide in said plant cell. 20. The method of any one of claims 17-19, wherein the plant cell lacks DAl polypeptide expression or activity. 21. The method according to claim 20, wherein said plant cell comprises a mutation in a nucleotide sequence of said plant cell that encodes said DAl polypeptide or regulates its expression; another heterologous A nucleic acid that expresses a suppressor nucleic acid that reduces expression of the DAl polypeptide in the plant cell; or another heterologous nucleic acid that expresses a dominant-negative expression in a cell of the plant DA1 polypeptide. 22. The method of any one of claims 17-21, wherein the heterologous nucleic acid expresses into the plant cell an inhibitor nucleic acid that reduces expression of the DA2 polypeptide. 23. The method of any one of claims 17-22, wherein the heterologous nucleic acid abrogates the expression or activity of the DA2 polypeptide in cells of the plant. 24. The method according to any one of the preceding claims, wherein the plant has increased plant size, seed size and/or organ size relative to a wild type plant. 25. The method according to any one of the preceding claims, wherein the nucleic acid encoding the dominant negative DA polypeptide and/or the suppressor nucleic acid is operably linked to a heterologous promoter. 26. The method of claim 25, wherein the promoter is a tissue-specific promoter. 27. The method of claim 26, wherein the promoter is an inducible promoter. 28. The method according to any one of the preceding claims, wherein the heterologous nucleic acid is comprised in one or more vectors. 29. The method according to any one of claims 1 to 15, comprising sexually or asexually propagating or growing progeny or descendants of said plant having reduced DA2 expression or activity. 30. The method according to any one of the preceding claims, wherein the DA2 polypeptide comprises the RING domain of SEQ ID NO:1. 31. The method of claim 30, wherein the DA2 polypeptide comprises the RING domain of SEQ ID NO:2. 32. The method of claim 31, wherein the DA2 polypeptide comprises the first consensus domain of SEQ ID NO:36. 33. The method of claim 31 or claim 32, wherein the DA2 polypeptide comprises the second consensus domain of SEQ ID NO:37. 34. The method of any one of claims 30-33, wherein the DA2 polypeptide comprises an amino acid sequence having at least 20% sequence identity to any one of SEQ ID NOs: 20-35. 35. The method according to any one of the preceding claims, wherein the DAl polypeptide comprises the UIM1 domain of SEQ ID NO:68 and the UIM2 domain of SEQ ID NO:69. 36. The method of claim 35, wherein the DAl polypeptide comprises the LIM domain of SEQ ID NO: 38 or 39. 37. The method of claim 35 or claim 36, wherein the DAl polypeptide comprises a C-terminal region having at least 20% sequence identity to residues 229 to 532 of SEQ ID NO:45. 38. The method of any one of claims 35-37, wherein the DAl protein comprises a sequence having at least 20% sequence identity to any one of SEQ ID NOs: 41-64. 39. The method according to any one of the preceding claims, wherein the dominant-negative DAl comprises an R to K substitution at a position equivalent to position 358 of the DAl polypeptide of SEQ ID NO: 45 in the amino acid sequence of the DAl polypeptide . 40. The method of any one of the preceding claims, wherein the EOD1 polypeptide comprises a sequence having at least 20% sequence identity to any one of SEQ ID NOs: 74-90. 41. The method of any one of claims 1 to 40, wherein the plant is a higher plant. 42. The method of claim 41, wherein the plant is an agricultural plant selected from the group consisting of comfrey, yew, tobacco, gourd, carrot, Brassica vegetable, melon, pepper, Grapevine, lettuce, strawberry, Brassica oilseed, sugar beet, wheat, barley, corn, rice, soybean, pea, sorghum, sunflower, tomato, potato, pepper, chrysanthemum, carnation, flaxseed, hemp, and rye. 43. A plant having reduced expression or activity of a DA2 polypeptide and reduced expression or activity of a DA1 polypeptide and/or EOD1 polypeptide, wherein the expression or activity of one or more of said DA2 polypeptide, said DAl polypeptide and said EOD1 polypeptide is reduced by incorporating a heterologous nucleic acid into one or more cells of said plant. 44. The plant of claim 43 produced by the method of any one of claims 1-42.