Field of Invention
This invention relates to methods of controlling the size of the
seeds and organs of plants.
Background of Invention
The size of seeds and organs is an agronomically and ecologically
important trait that is under genetic control (Alonso-Blanco, C.
Proc Natl Acad Sci U S A 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.Proc
Natl Acad Sci U S A 97, 942-7 (2000); Nath, U. Science 299, 1404-7
(2003);Ohno, C.K. Development 131, 1111-22 (2004); Szecsi, J. Embo J
25, 3912-20 (2006); White, D.W.Proc Natl Acad Sci USA 103, 13238-
43 (2006); Horvath, B.M. Embo J 25, 4909-20 (2006); Garcia, D. Plant
Cell 17, 52-60 (2005). The final size of seeds and organs is
constant within a given species, whereas interspecies seed and organ
size variation is remarkably large, suggesting that plants have
regulatory mechanisms that control seed and organ growth in a
coordinated and timely manner. Despite the importance of seed and
organ size, however, little is known about the molecular and genetic
mechanisms that control final organ and seed size in plants.
The genetic regulation of seed size has been investigated in plants,
including in tomato, soybean, maize, and rice, using quantitative
trait locuc (QTL) mapping. To date, in the published literature, two
genes (Song, X.J. Nat Genet 39, 623-30 (2007); Fan, C. Theor. Appl.
Genet. 112, 1164-1171 (2006)), underlying two major QTLs for rice
grain size, have been identified, although the molecular mechanisms
of these genes remain to be elucidated. In Arabidopsis, eleven loci
affecting seed weight and/or length in crosses between the
accessions Ler and Cvi, have been mapped {Alonso-Blanco, 1999
supra}, but the corresponding genes have not been identified. Recent
studies have revealed that AP2 and ARF2 are involved in control of
seed size. Unfortunately, however, ap2 and arf2 mutants have lower
fertility than wild type (Schruff, M.C. Development 137, 251-261
(2006); Ohto, M.A. Proc. Natnl Acad. Sci USA 102, 3123-3128 (2005);
Jofuku, K.D. Proc. Natnl Acad. Sci. USA 102, 3117-3122 (2005)). In
addition, studies using mutant plants have identified several
positive and negative regulators that influence organ size by acting
on cell proliferation or expansion {Krizek, B.A. Dev Genet 25, 224-
36 (1999); Mizukami, Y.Proc Natl Acad Sci U S A 97, 942-7 (2000);
Nath, U. Science 299, 1404-7 (2003); Ohno, C.K. Development 131,
1111-22 (2004); Szecsi, J. Embo J 25, 3912-20 (2006); White,
D.W.Proc Natl Acad Sci USA 103, 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)).
Identification of a factor or factors that control the final size of
both seeds and organs will not only advance understanding of the
mechanisms of size control in plants, but may also have substantial
practical applications for example in improving crop yield and plant
biomass for generating biofuel.
Summary of Invention
The present inventors have identified a UIM and LIM domain-
containing protein (termed DAI) which is a key regulator in
controlling the final size of seeds and organs by restricting the
duration of proliferative growth. An allele (termed the dal-1
allele) is shown herein to act as a dominant negative interfering
mutation for DARs or DA1-related proteins. Over-expression of the
dal-1 mutant gene (R358K) in wild type causes an increase in seed
and organ size in wild type plants, indicating that the. da1-1 allale
interferes with DARs in a dosage dependent manner. Mutations that
reduce or abolish the function of EOD1/BB, which encodes an E3
ubiquitin ligase, synergistically enhance the phenotypes of dal-1,

indicating that DA1 acts in parallel with E0D1/BB to limit the size
of seeds and organs. The functional characterization of DA1 and
E0D1/BB provides insight into the mechanism of control of the final
seed and organ size and may be a valuable tool for improving crop
yield and increasing plant biomass.
Aspects of the invention provide an isolated protein which is DA1
and an isolated nucleic acid encoding a protein which is DA1. Also
provided are DA1-related proteins and encoding nucleic acid. DA1
and DA1-related proteins (DARs) are collectively referred to herein
as DA proteins.
Other aspects of the invention provide an isolated protein
(DA1R358K) which interferes with the function of DA1 and DA1-
related proteins and an isolated nucleic acid encoding such a
protein.
Another aspect of the invention provides a method for producing
plants having normal fertility but which have one or more features
selected from longer life-span, enlarged organ size, enlarged seed
size.
Another aspect of the invention provides a plant having normal
fertility but which has a feature selected from longer life-span,
enlarged organ size, enlarged seed size, and combinations of these
features
Brief Description of Drawings
Figure 1 shows that da1-1 has large seeds and organs. (A and B) Dry
seeds of Col-0 (A) and da1-1 (B). (C and D) Mature embryos of Col-0
(C) and da1-1(D). (E and F) 9d- old seedlings of Col-0 (E) and da1-
1 (F). da1-1 has larger cotyledons than WT. (G) The fifth leaves of
Col-0 (left) and da1-1 (right). da1-1 has larger and rounder leaves
compared with wild type Col-0. (H and I) Flowers of Col-0 (H) and
dal-1(I). (J and K) Siliques of Col-0 (J) and da1-1 (K). (L) Average
seed weight of Col-0, da1-1, da1-ko1, dar1-1, and da1-ko1dar1-1 is
given in mg per 100 seeds. Standard deviations (SD) are shown (n=5).
Plants were grown under identical conditions. (M-O) stem diameter
(M), epidermal cell number in stem cross sections (N), and petal
area (0) of Col-0, dal-1, DA1C0M#2, and 35S: :DAlR358K#5. (P and Q)
Mass of 5 fresh flowers (stage 14) (P) and leaves (1st -7th ) of 35d-
old plants (Q). (R) Cell area of embryos (E), petals (P) and leaves
(L) in Col-0 and dal-1. Values are given as mean ± SD relative to
the respective wild type value, set at 100%. (S) Relative expression
levels of DA1 in Col-0 and 35S:: DA1R358K # 5 seedlings were measured by
quantitative real-time RT-PCR. Scale bars: 200 µm (A and B), 100µm
(C and D), 1mm (E and F), 0.5cm (G), 1mm (H to K).
Figure 2 shows kinematic analysis of petal and leaf growth. (A)
Growth of Col-0 and da1-1 mutant petals. The largest petals of each
series are from opened flowers (stage 14). (B) Mitotic index in WT
and da1-1 mutant petals. Time axis in (B) corresponds to the one in
(A). (C) Growth of the fifth leaf of Col-0, da1-1, DA1C0M#2, and
35S: :DA1R358K#5 over time. DAE is days after emergence.
Figure 3 shows the identification and expression of the DA1 gene.
(A) DA1 gene structure, showing the mutated sites of da1-1, sod1-1,
sod1-2, and sod1-3 alleles. The start codon (ATG) and the stop codon
(TGA) are indicated. Closed boxes indicate the coding sequence and
lines between boxes indicate introns. T-DNA insertion sites (da1-
ko1, da1-ko2 and da1-ko3) in DA1 gene are shown. (B to G) DA1
promoter activity monitored by pDA1::GUS transgene expression. GUS
staining in seedlings (B and C), an embryo (D), roots (E), and
petals (F and G). (H and I) The flowers of Col-0 (H) and dal-
koldarl-1 double mutant (I). (J) Siliques of Col-0 (left) and dal-
koldarl-1 double mutant (right). (K) Petal area of Col-0, dal-kol,
darl-1, dal-koldarl-1 double mutants. The dal-koldarl-1 double
mutant displays a da1-1 phenotype including large flowers and
petals, wide and flattened siliques, and short styles. (L)
Quantitative RT-PCR analysis revealed that expression of DA1 is
slowly induced by ABA. 7d- old WT seedlings were treated with 10µm
ABA for 2, 4, 6, 18 and 30 hours. (M and N) Wild type Col-0 and dal-
1 seeds were grown on MS medium with 2 µm ABA under constant light
conditions. The da1-1 mutant (N) exhibits ABA- insensitive seedling
establishment compared with wild type Col-0 (M). (0) 4d- old
seedlings of Col-0 (left), da1-1(middle) and DAI COM #2(right) were
transferred to MS medium with 5 µm ABA for 3 weeks, da1-1 mutant
seedlings continue to grow in the presence of low levels of ABA that
inhibit the growth of wild type Col-0 seedlings. Scale bars: 1mm (B,
H, I, M, and N) , 50 µm (D and E), 0.5mm (C and J), 0.1mm (F and G),
0.5cm (0).
Figure 4 show mutations in EOD1/BB synergistically enhance the
phenotypes of da1-1. (A) Flowers of Col-0, da1-1, eod1-2 and eod1-
2da1-1 double mutants. (B) Soil grown plants of Col-0, eod1-2, eod1-
2 and eod1-2da1-1 double mutants. (C) Average seed weights of Col-0,
da1-1, eod1-2and eod1-2da1-1 double mutants are shown as mg per 100
seeds. Standard deviations are shown (n=5). Plants were grown under
identical conditions. (D) Petal area of Col-0, da1-1, eod1-2and
eod1-2da1-1 double mutant. Standard deviation values are shown
(n>50), (E) A model of DAI and EOD1/BB in controlling seed and organ
size. Scale bars: 2mm (A), 50mm (B).
Figure 5 shows that da1-1 has large seeds. Preweighed batches of
wild type Col-0 (A), da1-1 (B) , DA1C0M#2 (C) , 35S: :DA1R358K#5 (D) ,and
da1-ko1dar1-1 mutant seeds from individual plants were passed
through a series of wire sieves of decreasing mesh size (in µm) as
described in Supplementary methods. (E) The average seed weight per
plant. Standard deviation values was given (n=5). Plants were grown
under identical conditions.
Figure 6 shows seed development in wild type and da1-1 plants.
(A to L), Cleared ovules (A,B) and seeds (C to L) of wild type (A,
C, E, G, I and K), and da1-1 (B, D, F, H, J and L) imaged with
differential contrast optics. Scale bars: 50um (A to L).
Figure 7 shows that da1-1 plant has large flower with extra petals
and deformed silique with extra carpels. (A) Wild type flower. (B
and C) da1-1 flowers with extra petals. (D) Wild type silique, (E to
G) da1-1 siliques with extra carpels. Scale bars: 1mm (A to C), 2mm
(D to G).
Figure 8 shows that da1-1 mutant has the prolonged cell
proliferation. (A and B) pCyclinB1;1::GUS activity in the first
leaves (9 days after germination) of wild type (A) and da1-1 (B)
seedlings grown on MS medium containing 1% glucose. (C) Expression
level of SAG12 gene in the fifth leaves of wild type Col-0 and da1-1
plants was detected by using Quantitative real-time RT-PCR analysis.
DAE is days after emergence.
Figure 9 shows map-based cloning of DA1. (A) Fine mapping of the DA1
locus. The DA1 locus was mapped to chromosome 1 (Chr 1) between
markers T16Nlland CER451450. The DA1 locus was further narrowed to a
30-kb genomic DNA region between markers T29M8-26 and F18014-52 and
co-segregated with CAPS marker DA1CAPS. The number of recombinants
identified from F2 plants is shown. (B) The mutation in da1-1 was
identified using the CAPS marker DA1CAPS1. (C to E) Expression
levels of DA1 (C) and DAR1 (D) in wild type and T-DNA lines were
revealed by RT-PCR analysis.
Figure 10 shows the identification of DAl-related proteins in
Arabidopsis and homologs of DAl in other species. DAl-related
proteins in Arabidopsis are shown in Figure 10A and DAl-related
proteins in other species are shown in Figure 10B.
Figure 11 shows that the R358K mutation in DA1 is responsible for
increased seed and organ size. (A) Petal area of Col-0, da1-1, da1-
ko1, da1-ko2, da1-ko3, da1-1/Col-0 F1, dal-kol/da1-1 F1, da1-ko2/da1-
1 F1, da1-ko3/da1-1 F1, da1-ko1/Co1-0 F1, dal-ko2/Col-0 F1, da-
ko3/Col-0 F1, and dal-kol/da1-1 F1. Standard deviation values are

given (n>50). (B) Average seed weight of Col-0, da1-1, da1-ko1, da1-
ko1/da1-1 F1, and da1-ko1/Co1-0 F1 is given in mg per 100 seeds.
Standard deviation values are given (n=5). Plants were grown under
identical conditions.
Figure 12 shows that mutations in an enhancer of da1-1 (EOD1/BB)
synergistically enhance the large seed and organ phenotypes of da1-
1. (A) The eod1-1da1-1 double mutant has an increased seed weight
compared with da1-1. Average seed weight of da1-1 and eod1-1da1-1
double mutant is given in mg per 100 seeds. Standard deviation
values are shown (n=5). Plants were grown under identical
conditions. (B) The eod1-lda1-1 double mutant has larger flower than
da1-1. (C) EOD1/BB gene structure, showing the mutated sites of the
two eod1 alleles. The start codon (ATG) and the stop codon (TGA) are
indicated. Closed boxes indicate the coding sequence and lines
between boxes indicate introns. The mutated site in eod1-1 and T-DNA
insertion site in eod1-2 also are shown. (D) Eight week old plants
of Col-0, da1-1, eod1-2, and eod1-2da1-1 plants are shown. The eod2-
lda1-1 plant has a longer growing period than da1-1. (E) Eight week
old plants of Ler, da1-1Ler, bb-1, and bb-1da1-1Ler plants are shown.
The bb-lda1-1Ler plant has a longer growing period than da1-1Ler.
Scale bars: 1mm (B) , 5cm (D and E) . (F) Petal areas of Ler, da1-1Ler,
bb-1, and bb-1da1-1Ler double mutants. Standard deviation values are
shown (n>50). Mutations in BB synergistically enhance the petal size
phenotype of da1-1, suggesting that DA1 and BB act in parallel
pathways.
Figure 13 shows that genetic analysis between da1-1 and ant-5, axrl-
12, ap2-7, and arf2-7. (A and B) The petal size phenotype of ant-
5da1-1Ler and axrl-12da1-1 double mutant is essentially additive,
compared to their parental lines. (C and D) The seed size phenotype
of ap2-7da1-1 and arl2-/da1-1 double mutants is also essentially
additive, compared to their parental lines.

Figure 14 shows a phylogenetic analysis of da1-1ike proteins. Left
graph: A distance matrix phylogenetic tree was created using PHYLIP
software (VERSION 3.66) with the default settings (the JTT model of
protein sequence evolution and the neighbour-joining algorithm). The
tree was then imported into MEGA 4.0 software to rearrange.
Bootstrap values (the numbers on the branches indicate the number of
times the partition of the species into the two sets which are
separated by that branch occurred among the trees, only shown over
70) were obtained by 100 replicates. The data for the tree was the
C-terminal 250 amino acid region of full length da1-1ike protein
sequences. The right graph shows a simplified overview of plant
evolution based on the hyperbolic tree presented at
(http://ucjeps.berkeley.edu/TreeofLife/ hyperbolic.php). Clades that
are related with text are retained in the graph. Species that were
analysed are underlined.
Figure 15 (A-D) shows siliques of Col-0, BrDAlaCOM (35S:: BrDAla
transgenic line), OsDAlCOM (35S:: OsDAl transgenic line) and da1-1.
(E-H) Rosette leaves of Col-0, da1-1, BrDAlbCOM (35S:: BrDAlb
transgenic line) and 35S: :BrDAlaR/K (overexpressing 35S: :BrDAlaR/K in
Col-0). (I) DA1 gene structure showing the mutated sites of da1-1 and T-DNA insertion sites {dal-kol, dal-ko2 and dal-ko3) .
Detailed Description of Embodiments of the Invention
In various aspects, the invention provides isolated DA polypeptides
encoded by DA genes and nucleic acid sequences described herein.
DA polypeptides include both DA-1 polypeptides and DA-1 related
(DAR) polypeptides, and functional homologues thereof, as described
herein.
DA polypeptides, including DA-1 polypeptides and DA-1 related (DAR)
polypeptides, possess a characteristic domain structure.

A DA polypeptide may comprise a UIM1 domain and a UIM2 domain. A
UIM1 domain may consist of the sequence of SEQ ID NO: 3 and a UIM2
domain may consist of the sequence of SEQ ID NO: 4.
p---pLpbAl pb.Sbp-.pp p (SEQ ID NO: 3)
p---pLpbAl pb.Sbp-spp p (SEQ ID NO:4)
wherein;
p is a polar amino acid residue, for example, C, D, E, H, K,
N, Q, R, S or T;
b is a big 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;
1 is an aliphatic amino acid residue, for example, I, L or V;
. is absent or is any amino acid, and
- is any amino acid.
Examples of suitable UIM1 and UIM2 domain sequences are set out
below. Further examples of UIM1 and UIM2 domain sequences may be
identified using standard sequence analysis techniques as described
herein (e.g. Simple Modular Architecture Research Tool (SMART); EMBL
Heidelberg, DE).
A DA polypeptide may comprise an LIM domain. An LIM domain may
consist of the sequence of SEQ ID NO: 5;
pCs.CscsIh s.....bhlp tb.sp.aH.. .pCFpCs..p CppsLss... .p.ab.pcsp
baCpps... (SEQ ID NO: 5)
wherein;
c is a charged amino acid residue, for example, D, E, H, K,
R;
p is a polar amino acid residue, for example, C, D, E, H, K,
N, Q, R, S or T;

h is a hydrophobic amino acid residue, for example, A, C, F,
G, H, I, L, M, T, V, W and Y;
t is a tiny amino acid residue, for example, A, G or S;
a is an aromatic amino acid residue, for example, F, H, W or
Y;
b is a big 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;
1 is an aliphatic amino acid residue, for example, I, L or V;
. is absent or is any amino acid; and
- is any amino acid.
Examples of suitable LIM domain sequences are set out below. Further
examples of LIM domain sequences may be identified using standard
sequence analysis techniques (e.g. Simple Modular Architecture
Research Tool (SMART); EMBL Heidelberg, DE).
A DA polypeptide may comprise a carboxyl terminal region having at
least 20%, at least 30%, at least 40%, at least 50%, at least 60%,
at least 70%, at least 80%, at least 90%, at least 95%, or at least
98% amino acid identity to residues 250 to 532 of SEQ ID NO: 1 that
define the C terminal domain of DAI.
A DA polypeptide may further comprise R at a position equivalent to
position 358 of SEQ ID NO: 1.
A position in an amino acid sequence which is equivalent to position
358 of SEQ ID NO: 1 can be readily identified using standard
sequence analysis tools. Examples of sequences with an R residue at
a position equivalent to position 358 of SEQ ID NO: 1 are shown
elsewhere herein.
In some preferred embodiments, a DA polypeptide may comprise;
a UIM domain of SEQ ID NO:3

a UIM domain of SEQ ID NO:4
a LIM domain of SEQ ID NO:5, and
a C terminal region having at least 20% sequence identity to
residues 250 to 532 of SEQ ID NO: 1.
A preferred DA polypeptide may further comprise R at a position
equivalent to position 358 of SEQ ID NO: 1.
For example, a DA polypeptide may comprise an amino acid sequence
set out in a database entry selected from the group consisting of
SGN-U317073, SGN-U277808, SGN-U325242, AT4G36860, SGN-U209255,
AB082378.1, AT2G39830, CAN69394.1, OS03G16090, 9234.M000024,
29235.M000021, AT5G66620, AT5G66630, AT5G66610, AT5G66640,
AT5G17890, SGN-U320806, AB096533.1, CAL53532.1, OS06G08400, SGN-
U328968, OS03G42820 and OS12G40490 or may be variant or a fragment
of one of these sequences which retains DA activity.
A DA polypeptide may comprise an amino acid sequence of AtDA1,
AtDAR1, AtDAR2, AtDAR3, AtDAR4, AtDAR5, AtDAR6, AtDAR7, BrDA1a,
BrDA1b, BrDAR1, BrDAR2, BrDAR3-7, BrDAL1, BrDAL2, BrDAL3, OsDA1,
OsDAR2, OsDAL3, OsDAL5, PpDAL1, PpDAL2, PpDAL3, PpDAL4, PpDAL5,
PpDAL6, PpDAL7, PpDAL8, SmDAL1 and SmDAL2 (as shown in Alignment E).
Other examples of database entries of sequences of DA polypeptides
are shown in Table 6 and Table 11. Other DA polypeptide sequences
which include the characteristic features set out above may be
identified using standard sequence analysis tools.
In some preferred embodiments, a DA polypeptide may comprise the
amino acid sequence of SEQ ID NO: 1 (AT1G19270; NP_173361.1 GI:
15221983) or may be a fragment or variant of this sequence which
retains DA activity.
A DA polypeptide which is a variant of a reference DA sequence, such
as SEQ ID NO: 1 or a sequence shown in alignment E, may comprise an
amino acid sequence having at least 20%, at least 30%, at least 40%,
at least 50%, at least 60%, at least 70%, at least 80%, at least
90%, at least 95%, or at least 98% sequence identity to the
reference sequence.
A DA polypeptide which is a variant of SEQ ID NO: 1 may comprise a
UIM1 domain having the sequence QENEDIDRAIALSLLEENQE (SEQ ID NO: 6)
and a UIM2 domain having the sequence DEDEQIARALQESMVVGNSP (SEQ ID
NO: 7).
A DA polypeptide which is a variant of SEQ ID NO: 1 may comprise a
LIM domain having the sequence:
ICAGCNMEIGHGRFLNCLNSLWHPECFRCYGCSQPISEYEFSTSGNYPFHKAC (SEQ ID NO: 8)
Particular amino acid sequence variants may differ from the DA-1
polypeptide of SEQ ID NO:1 by insertion, addition, substitution or
deletion of 1 amino acid, 2, 3, 4, 5-10, 10-20 20-30, 30-50, or more
than 50 amino acids.
Sequence similarity and identity are commonly defined with reference
to the algorithm GAP (Wisconsin Package, Accelerys, San Diego USA) .
GAP uses the Needleman and Wunsch algorithm to align two complete
sequences that maximizes the number of matches and minimizes the
number of gaps. Generally, default parameters are used, with a gap
creation penalty = 12 and gap extension penalty = 4.
Use of GAP may be preferred but other algorithms may be used, e.g.
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) 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, generally
employing default parameters. In particular, the psi-Blast
algorithm (Nucl. Acids Res. (1997) 25 3389-3402) may be used.
Sequence comparison may be made over the full-length of the relevant
sequence described herein.
In various aspects, the invention provides DA genes and nucleic acid
sequences which encode DA polypeptides, as described herein.
For example, a nucleic acid encoding a DA polypeptide may comprise a
nucleotide sequence set out in a database entry selected from the
group consisting of SGN-U317073, SGN-U277808, SGN-U325242,
AT4G36860, SGN-U209255, AB082378.1, AT2G39830, CAN69394.1,
OS03G16090, 9234.M000024, 29235.M000021, AT5G66620, AT5G66630,
AT5G66610, AT5G66640, AT5G17890, SGN-U320806, AB096533.1,
CAL53532.1, OS06G08400, SGN-U328968, OS03G42820 and OS12G40490
or may be variant or a fragment of one of these sequences.
Other database entries of nucleic acid sequences which encode DA
polypeptides are shown in Table 7.
In some preferred embodiments, a nucleic acid encoding a DA
polypeptide may comprise the nucleotide sequence of SEQ ID NO: 2 or
any one of SEQ ID NOS: 11 to 16 or may be a variant or fragment of
this sequence which encodes a polypeptide which retains DA activity.
A variant sequence may be a mutant, homologue, or allele of a
reference DA sequence, such as SEQ ID NO: 2; any one of SEQ ID NOS:
11 to 16; or a sequence having a database entry set out above, and
may differ from the reference DA sequence by one or more of
addition, insertion, deletion or substitution of one or more
nucleotides in the nucleic acid, leading to the addition, insertion,
deletion or substitution of one or more amino acids in the encoded
polypeptide. Of course, changes to the nucleic acid that make no
difference to the encoded amino acid sequence are included. A
nucleic acid encoding a DA polypeptide may comprise a sequence
having at least 20% or at least 30% sequence identity with the
reference DA 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.
A fragment or variant may comprise a sequence which encodes a
functional DA polypeptide i.e. a polypeptide which retains one or
more functional characteristics of the polypeptide encoded by the
wild-type DA gene, for example, the ability to modulate the duration
of proliferative growth.
A nucleic acid comprising a nucleotide sequence which is a variant
of a reference DA nucleic acid sequence, such as SEQ ID NO: 2 or any
one of SEQ ID NOS: 11 to 16, may selectively hybridise under
stringent conditions with this nucleic acid sequence or the
complement thereof.
Stringent conditions include, e.g. for hybridization of sequences
that are about 80-90% identical, hybridization overnight at 42°C in
0.25M Na2HPO4, pH 7.2, 6.5% SDS, 10% dextran sulfate and a final wash
at 55°C in 0.1X SSC, 0.1% SDS. For detection of sequences that are
greater than about 90% identical, suitable conditions include
hybridization overnight at 65°C in 0.25M Na2HPO4, pH 7.2, 6.5% SDS,
10% dextran sulfate and a final wash at 60°C in 0.1X SSC, 0.1% SDS.
An alternative, which may be particularly appropriate with plant
nucleic acid preparations, is a solution of 5x SSPE (final 0.9 M
NaCl, 0.05M sodium phosphate, 0.005M EDTA pH 7.7), 5X Denhardt's
solution, 0.5% SDS, at 50°C or 65°C overnight. Washes may be
performed in 0.2x SSC/0.1% SDS at 65CC or at 50-60°C in 1x SSC/0.1%
SDS, as required.
Nucleic acids as described herein may be wholly or partially
synthetic. In particular, they may be recombinant in that nucleic
acid sequences which are not found together in nature (do not run
contiguously) have been ligated or otherwise combined artificially.
Alternatively, they may have been synthesised directly e.g. using an
automated synthesiser.
The nucleic acid may of course be double- or single-stranded, cDNA
or genomic DNA, or RNA. The nucleic acid may be wholly or partially
synthetic, depending on design. Naturally, the skilled person will
understand that where the nucleic acid includes RNA, reference to
the sequence shown should be construed as reference to the RNA
equivalent, with U substituted for T.
In various aspects, the invention provides dominant negative DA
polypeptides and encoding nucleic acids. A dominant negative DA
polypeptide may increase one or more of organ size, seed size or
longevity without affecting fertility, upon expression in a plant.
A dominant negative allele of a DA polypeptide may comprise a DA
polypeptide having a mutation, e.g. a substitution or deletion, at a
position equivalent to position 358 of SEQ ID NO: 1.
For example, a dominant negative allele of a DA polypeptide may
comprise a mutation of the conserved R residue at a position
equivalent to position 358 of SEQ ID NO: 1. In preferred
embodiments, the conserved R residue may be substituted for K.
Position R358 of SEQ ID NO: 1 is located within the conserved C
terminal region (amino acids 250 to 532 of SEQ ID NO: 1). An R
residue at a position in a DA polypeptide sequence which is
equivalent to position 358 of SEQ ID NO: 1 may be identified by
aligning these conserved C terminal regions using standard sequence
analysis and alignment tools.
Nucleic acid which encodes a dominant negative allele of a DA
protein may be produced by any convenient technique. For example,
site directed mutagenesis may be employed on a nucleic acid encoding
a DA polypeptide to alter the conserved R residue at the equivalent
position to R358 in SEQ ID NO: 1, for example to K. Reagents and
kits for in vitro mutagenesis are commercially available. The
mutated nucleic acid encoding the dominant negative allele of a DA
protein and may be further cloned into an expression vector and
expressed in plant cells as described below to alter plant
phenotype.
The nucleic acid encoding the DA 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 (i.e. a heterologous
plant).
"Heterologous" indicates that the gene/sequence of nucleotides in
question or a sequence regulating the gene/sequence in question, has
been introduced into said cells of the plant or an ancestor thereof,
using genetic engineering or recombinant means, i.e. by human
intervention. Nucleotide sequences which are heterologous to a plant
cell may be non-naturally occurring in cells of that type, variety
or species (i.e. exogenous or foreign) or may be sequences which are
non-naturally occurring in that sub-cellular or genomic environment
of the cells or may be sequences which are non-naturally regulated
in the cells i.e. operably linked to a non-natural regulatory
element. "Isolated" indicate that the isolated molecule (e.g.
polypeptide or nucleic acid) exists in an environment which is
distinct from the environment in which it occurs in nature. For
example, an isolated nucleic acid may be substantially isolated with
respect to the genomic environment in which it naturally occurs. An
isolated nucleic acid may exist in an environment other than the
environment in which it occurs in nature.
A nucleic acid encoding a DA polypeptide as described herein may be
operably linked to a heterologous regulatory sequence, such as a
promoter, for example a constitutive, inducible, tissue-specific or
developmental specific promoter.

Many suitable regulatory sequences are known in the art and may be
used in accordance with the invention. Examples of suitable
regulatory sequences may be derived from a plant virus, for example
the Cauliflower Mosaic Virus 35S. (CaMV 35S) gene promoter that is
expressed at a high level in virtually all plant tissues (Benfey et
al, (1990) EMBO J 9: 1677-1684). Other suitable constitutive
regulatory elements include the cauliflower mosaic virus 19S
promoter; the Figwort mosaic virus promoter; and the nopaline
synthase (nos) gene promoter (Singer et al., Plant Mol. Biol. 14:433
(1990); An, Plant Physiol. 81:86 (1986)).
Constructs for expression of the DA genes under the control of a
strong constitutive promoter (the 35S promoter) are exemplified
below but those skilled in the art will appreciate that a wide
variety of other promoters may be employed to advantage in
particular contexts.
A tissue-specific promoter may be employed to express the dominant
negative form of the DA polypeptide in a specific tissue or organ to
increase size of that tissue or organ relative to tissues or organs
in which the tissue-specific promoter is not active and the dominant
negative form of the DA polypeptide is not expressed. For example,
to increase the size of seeds, the dominant negative form of the DA
polypeptide may be preferentially expressed in seed tissue, using a
seed specific promoter. For example, the polypeptide may be
expressed in developing integument using an integument-specific
promoter such as the INO promoter (Meister R.M., Plant Journal 37:
426-438 (2004)) or in embryos using an embryo specific promoter such
as the histone H4 promoter (Devic M. Plant Journal 9; 205-215
(1996)).
Alternatively, or in addition, one might select an inducible
promoter. In this way, for example, the dominant negative form of
the DA polypeptide may be expressed at specific times or places in
order to obtain desired changes in organ growth. Inducible promoters

include the alcohol inducible AlcA gene-expression system (Roslan et
al., Plant Journal; 2001 Oct; 28(2):225-35) may be employed.
The DA nucleic acid may be contained on a nucleic acid construct or
vector. The construct or vector is preferably suitable for
transformation into and/or expression within a plant cell.
A vector is, inter alia, any plasmid, cosmid, phage or Agrobacterium
binary vector in double or single stranded linear or circular form,
which may or may not be self transmissible or mobilizable, and which
can transform prokaryotic or eukaryotic host, in particular a plant
host, either by integration into the cellular genome or exist
extrachromasomally (e.g. autonomous replicating plasmid with an
origin of replication).
Specifically included are shuttle vectors by which is meant a DNA
vehicle capable, naturally or by design, of replication in two
different organisms, which may be selected from Actinomyces and
related species, bacteria and eukaryotic (e.g. higher plant,
mammalia, yeast or fungal) cells.
A construct or vector comprising nucleic acid as described above
need not include a promoter or other regulatory sequence,
particularly if the vector is to be used to introduce the nucleic
acid into cells for recombination into the genome.
Constructs and vectors may further comprise selectable genetic
markers consisting of genes that confer selectable phenotypes such
as resistance to antibiotics such as kanamycin, hygromycin,
phosphinotricin, chlorsulfuron, methotrexate, gentamycin,
spectinomycin, imidazolinones, glyphosate and d-amino acids.
Those skilled in the art are well able to construct vectors and
design protocols for recombinant gene expression, in particular in a
plant cell. Suitable vectors can be chosen or constructed,
containing appropriate regulatory sequences, including promoter

sequences, terminator fragments, polyadenylation sequences, enhancer
sequences, marker genes and other sequences as appropriate. For
further details see, for example, Molecular Cloning: a Laboratory-
Manual: 3rd edition, Sambrook & Russell, 2001, Cold Spring Harbor
Laboratory Press.
Those skilled in the art can construct vectors and design protocols
for recombinant gene expression, for example in a microbial or plant
cell. Suitable vectors can be chosen or constructed, containing
appropriate regulatory sequences, including promoter sequences,
terminator fragments, polyadenylation sequences, enhancer sequences,
marker genes and other sequences as appropriate. For further details
see, for example, Molecular Cloning: a Laboratory Manual: 3rd
edition, Sambrook et al, 2001, Cold Spring Harbor Laboratory Press
and Protocols in Molecular Biology, Second Edition, Ausubel et al.
eds. John Wiley & Sons, 1992. Specific procedures and vectors
previously used with wide success upon plants are described by Bevan,
Nucl. Acids Res. (1984) 12, 8711-8721), and Guerineau and Mullineaux,
(1993) Plant transformation and expression vectors. In: Plant
Molecular Biology Labfax (Croy RRD ed) Oxford, BIOS Scientific
Publishers, pp 121-148.
When introducing a chosen gene construct into a cell, certain
considerations must be taken into account, well known to those
skilled in the art. The nucleic acid to be inserted should be
assembled within a construct that contains effective regulatory
elements that will drive transcription. There must be available a
method of transporting the construct into the cell. Once the
construct is within the cell membrane, integration into the
endogenous chromosomal material either will or will not occur.
Finally, the target cell type is preferably such that cells can be
regenerated into whole plants.
Those skilled in the art will also appreciate that in producing
constructs for achieving expression of the genes according to this

invention, it is desirable to use a construct and transformation
method which enhances expression of the nucleic acid encoding the
dominant negative form of the DA polypeptide. Integration of a
single copy of the gene into the genome of the plant cell may be
beneficial to minimize gene silencing effects. Likewise, control of
the complexity of integration may be beneficial in this regard. Of
particular interest in this regard is transformation of plant cells
utilizing a minimal gene expression construct according to, for
example, EP Patent No. EP1407000B1, herein incorporated by reference
for this purpose.
Techniques well known to those skilled in the art may be used to
introduce nucleic acid constructs and vectors into plant cells to
produce transgenic plants with the properties described herein.
Agrobacterium transformation is one method widely used by those
skilled in the art to transform woody plant species, in particular
hardwood species such as poplar. Production 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) Theor Appl
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. (1993) Plant Molecular Biology
21, 871-884; Fromm, et al. (1990) Bio/Technology 8, 833-839; Gordon-
Kamm, et al. (1990) Plant Cell 2, 603-618; D'Halluin, et al. (1992)
Plant Cell 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, 1077-1084; Somers, et al. (1992)
Bio/Technology 10, 1589-1594; W092/14828; Nilsson, 0. et al (1992)
Transgenic Research 1, 209-220).

Other methods, such as microprojectile or particle bombardment (US
5100792, EP-A-444882, EP-A-434616), electroporation (EP 290395, WO
8706614), microinjection (WO 92/09696, WO 94/00583, EP 331083, EP
175966, Green et al. (1987) Plant Tissue and Cell Culture, Academic
Press), direct DNA uptake (DE 4005152, WO 9012096, US 4684611),
liposome mediated DNA uptake (e.g. Freeman et al. Plant Cell
Physiol. 29: 1353 (1984)) or the vortexing method (e.g. Kindle,
PNAS U.S.A. 87: 1228 (1990d)) may be preferred where Agrobacterium
transformation is inefficient or ineffective, for example in some
gymnosperm species.
Physical methods for the transformation of plant cells are reviewed
in Oard, 1991, Biotech. Adv. 9: 1-11.
Alternatively, a combination of different techniques may be employed
to enhance the efficiency of the transformation process, e.g.
bombardment with Agrobacterium coated microparticles (EP-A-486234)
or microprojectile bombardment to induce wounding followed by co-
cultivation with Agrobacterium (EP-A-486233).
Following transformation, a plant may be regenerated, e.g. from
single cells, callus tissue or leaf discs, as is standard in the
art. Almost any plant can be entirely regenerated from cells,
tissues and organs of the plant. 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 a transformation technology will be
determined by its efficiency to transform certain plant species as
well as the experience and preference of the person practising the
invention with a particular methodology of choice. It will be
apparent to the skilled person that the particular choice of a

transformation system to introduce nucleic acid into plant cells is
not essential to or a limitation of the invention, nor is the choice
of technique for plant regeneration.
Another aspect of the invention provides a method of altering the
phenotype of a plant comprising;
expressing a nucleic acid encoding a dominant-negative DA
polypeptide within cells of said plant relative to control plants.
Suitable dominant-negative DA polypeptides and methods for
expression in plant cells are described above.
A plant with altered phenotype produced as described above may have
an extended period of proliferative growth and may display one or
more of increased life-span, increased organ size and increased seed
size relative to control plants. Preferably, the fertility of plants
having the altered phenotype is normal. Methods described herein may
be useful, for example, in increasing plant yields, improving grain
yield in crop plants, and/or for increasing plant biomass, for
example, in the production of biofuels.
The effect of dominant negative alleles of DA proteins is shown
herein to be enhanced by reducing or abolishing the expression or
function of the Big Brother (BB) protein in the plant.
Big Brother (BB) is an E3 ubiquitin ligase which is known to repress
plant organ growth {Disch, 2006). A BB protein may comprise the
amino acid sequence of SEQ ID NO: 9 (At3g63530 NP_001030922.1 GI:
79316205) or the sequence of a database entry shown in table 9, or
may be a fragment or variant of any one of these sequences which
retains BB activity or is capable of interfering with the function
of BB.
A BB polypeptide which is a variant of a reference BB sequence, for
example SEQ ID NO: 9 or the sequence of a database entry shown in

Table 9, may comprise an amino acid sequence having at least 20%, at
least 30%, at least 40%, at least 50%, at least 60%, at least 70%,
at least 80%, at least 90%, at least 95%, or at least 98% sequence
identity to the reference sequence. Sequence identity is described
in more detail above.
Particular amino acid sequence variants may differ from the BB
polypeptide of SEQ ID NO:9 by insertion, addition, substitution or
deletion of 1 amino acid, 2, 3, 4, 5-10, 10-20 20-30, 30-50, or more
than 50 amino acids.
In some embodiments, a BB polypeptide may comprise an A at a
position corresponding to position 44 of SEQ ID NO: 9.
A nucleic acid encoding the BB polypeptide may for example comprise
a nucleotide set out in a database entry shown in table 10 or may be
a variant or fragment thereof.
In some preferred embodiments, a nucleic acid encoding a BB
polypeptide may comprise the nucleotide sequence of SEQ ID NO: 10
(NM_001035845.1 GI: 79316204) or may be a variant or fragment of
this sequence which encodes a polypeptide which retains BB activity.
A variant sequence may be a mutant, homologue, or allele of a
reference BB sequence, such as SEQ ID NO: 10 or a sequence having a
database entry set out in table 10, and may differ from the
reference BB sequence by one or more of addition, insertion,
deletion or substitution of one or more nucleotides in the nucleic
acid, leading to the addition, insertion, deletion or substitution
of one or more amino acids in the encoded polypeptide. Of course,
changes to the nucleic acid that make no difference to the encoded
amino acid sequence are included. A nucleic acid encoding a BB
polypeptide may comprise a sequence having at least 20% or at least
30% sequence identity with the reference BB 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.
A fragment or variant may comprise a sequence which encodes a
functional BB polypeptide i.e. a polypeptide which retains one or
more functional characteristics of the polypeptide encoded by the
wild-type BB gene, for example, E3 ubiquitin ligase activity.
A method of altering a plant phenotype as described herein may
further comprise reducing or abolishing the expression or activity
of a BB polypeptide in said plant.
This may enhance or increase the effect of the expression of a
dominant negative DA polypeptide on one or more of organ size, seed
size or longevity.
Methods for reducing or abolishing the expression or activity of a
BB polypeptide in said plant are well known in the art and are
described in more detail below.
The expression of active protein may be abolished by mutating the
nucleic acid sequences in the plant cell which encode the BB
polypeptide and regenerating a plant from the mutated cell. The
nucleic acids may be mutated by insertion or deletion of one or more
nucleotides. Techniques for the inactivation or knockout of target
genes are well-known in the art.
For example, an EOD1 allele of a BB polypeptide may be generated by
introducing a mutation, such as a deletion, insertion or
substitution, at a position corresponding to position 44 of SEQ ID
NO: 9, for example, an A to T substitution. A position in a BB
polypeptide sequence which is equivalent to position 44 of SEQ ID
NO: 9 may be identified using standard sequence analysis and
alignment tools. Others mutations suitable for abolishing expression
of an active protein will be readily apparent to the skilled person.

The expression of active protein may be reduced using suppression
techniques. The suppression of the expression of target polypeptides
in plant cells is well-known in the art. Suitable suppressor
nucleic acids may be copies of all or part of the target BB gene
inserted in antisense or sense orientation or both relative to the
BB gene, to achieve reduction in expression of the BB gene. See,
for example, 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. Further
refinements of this approach may be found in W095/34668 (Biosource);
Angell & Baulcombe (1997) The EMBO Journal 16, 12:3675-3684; and
Voinnet & Baulcombe (1997) Nature 389: pg 553.
In some embodiments, the suppressor nucleic acids may be sense
suppressors of expression of the BB polypeptide.
A suitable sense suppressor nucleic acid may be a double stranded
RNA (Fire A. et al Nature, Vol 391, (1998)). dsRNA mediated
silencing is gene specific and is often termed RNA interference
(RNAi). RNAi is a two step process. First, dsRNA is cleaved within
the cell to yield short interfering RNAs (siRNAs) of about 21-23nt
length with 5' terminal phosphate and 3' short overhangs (~2nt). The
siRNAs target the corresponding mRNA sequence specifically for
destruction (Zamore P.D. Nature Structural Biology, 8, 9, 746-750,
(2001)
siRNAs (sometimes called microRNAs) down-regulate gene expression by
binding to complementary RNAs and either triggering mRNA elimination
(RNAi) or arresting mRNA translation into protein. siRNA may be
derived by processing of long double stranded RNAs and when found in
nature are typically of exogenous origin. Micro-interfering RNAs
(miRNA) are endogenously encoded small non-coding RNAs, derived by
processing of short hairpins. Both siRNA and miRNA can inhibit the
translation of mRNAs bearing partially complementary target

sequences without RNA cleavage and degrade mRNAs bearing fully
complementary sequences.
Accordingly, the present invention provides the use of RNAi
sequences based on the BB nucleic acid sequence for suppression of
the expression of the DA polypeptide. For example, an RNAi sequence
may correspond to a fragment of SEQ ID NO: 10 or other BB nucleic
acid sequence referred to above, or a variant thereof.
siRNA molecules are typically double stranded and, in order to
optimise the effectiveness of RNA mediated down-regulation of the
function of a target gene, it is preferred that the length and
sequence of the siRNA molecule is chosen to ensure correct
recognition of the siRNA by the RISC complex that mediates the
recognition by the siRNA of the mRNA target and so that the siRNA is
short enough to reduce a host response.
miRNA ligands are typically single stranded and have regions that
are partially complementary enabling the ligands to form a hairpin.
miRNAs are RNA sequences which are transcribed from DNA, but are not
translated into protein. A DNA sequence that codes for a miRNA is
longer than the miRNA. This DNA sequence includes the miRNA
sequence and an 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 on John et al, PLoS Biology, 11(2), 1862-
1879, 2004.
Typically, the RNA molecules intended to mimic the effects of siRNA
or miRNA have between 10 and 40 ribonucleotides (or synthetic
analogues thereof), more preferably between 17 and 30
ribonucleotides, more preferably between 19 and 25 ribonucleotides
and most preferably between 21 and 23 ribonucleotides. In some
embodiments of the invention employing double-stranded siRNA, the

molecule may have symmetric 3' overhangs, e.g. of one or two
(ribo)nucleotides, typically a UU of dTdT 3' overhang. Based on the
disclosure provided herein, the skilled person can readily design
suitable siRNA and miRNA sequences, for example using resources such
as Ambion's siRNA finder, see
http://www.ambion.com/techlib/misc/siRNA_finder.html. siRNA and
miRNA sequences can be synthetically produced and added exogenously
to cause gene downregulation or produced using expression systems
(e.g. vectors). In a preferred embodiment, the siRNA is synthesized
synthetically.
Longer double stranded RNAs may be processed in the cell to produce
siRNAs (see for example Myers (2003) Nature Biotechnology 21:324-
328). The longer dsRNA molecule may have symmetric 3' or 5'
overhangs, e.g. of one or two (ribo) nucleotides, or may have blunt
ends. The longer dsRNA molecules may be 25 nucleotides or longer.
Preferably, the longer dsRNA molecules are between 25 and 30
nucleotides long. More preferably, the longer dsRNA molecules are
between 25 and 27 nucleotides long. Most preferably, the longer
dsRNA molecules are 27 nucleotides in length. dsRNAs 30 nucleotides
or more in length may be expressed using the vector pDECAP
(Shinagawa et al., Genes and Dev., 17, 1340-5, 2003).
Another alternative is the expression of a short hairpin RNA
molecule (shRNA) in the cell. shRNAs are more stable than synthetic
siRNAs. A shRNA consists of short inverted repeats separated by a
small loop sequence. One inverted repeat is complementary to the
gene target. In the cell the shRNA is processed by DICER into a
siRNA which degrades the target gene mRNA and suppresses expression.
In a preferred embodiment the shRNA is produced endogenously (within
a cell) by transcription from a vector. shRNAs may be produced
within a cell by transfecting the cell with a vector encoding the
shRNA sequence under control of a RNA polymerase III promoter such
as the human HI or 7SK promoter or a RNA polymerase II promoter.
Alternatively, the shRNA may be synthesised exogenously (in vitro)
by transcription from a vector. The shRNA may then be introduced
directly into the cell. Preferably, the shRNA molecule comprises a
partial sequence of SHR. For example, the shRNA sequence is between
40 and 100 bases in length, more preferably between 40 and 70 bases
in length. The stem of the hairpin is preferably between 19 and 30
base pairs in length. The stem may contain G-U pairings to
stabilise the hairpin structure.
siRNA molecules, longer dsRNA molecules or miRNA molecules may be
made recombinantly by transcription of a nucleic acid sequence,
preferably contained within a vector. Preferably, the siRNA
molecule, longer dsRNA molecule or miRNA molecule comprises a
partial sequence of SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15 or a
variant thereof.
In other embodiments, the suppressor nucleic acids may be anti-sense
suppressors of expression of the two or more DA polypeptides. In
using anti-sense sequences to down-regulate gene expression, a
nucleotide sequence is placed under the control of a promoter in a
"reverse orientation" such that transcription yields RNA which is
complementary to normal mRNA transcribed from the "sense" strand of
the target gene. See, for example, 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) PNAS USA 91, 3490-3496.
An anti-sense suppressor nucleic acid may comprise an anti-sense
sequence of at least 10 nucleotides from a nucleotide sequence is a
fragment of SEQ ID NO: 10 or other BB sequence referred to above, or
a variant thereof.
It may be preferable that there is complete sequence identity in the
sequence used for down-regulation of expression of a target
sequence, and the target sequence, although total complementarity or

similarity of sequence is not essential. One or more nucleotides
may differ in the sequence used from the target gene. Thus, a
sequence employed in a down-regulation of gene expression in
accordance with the present invention may be a wild-type sequence
(e.g. gene) selected from those available, or a variant of such a
sequence.
The sequence need not include an open reading frame or specify an
RNA that would be translatable. It may be preferred for there to be
sufficient homology for the respective anti-sense and sense RNA
molecules to hybridise. There may be down regulation of gene
expression even where there is about 5%, 10%, 15% or 20% or more
mismatch between the sequence used and the target gene. Effectively,
the homology should be sufficient for the down-regulation of gene
expression to take place.
Suppressor nucleic acids may be operably linked to tissue-specific
or inducible promoters. For example, integument and seed specific
promoters can be used to specifically down-regulate two or more DA
nucleic acids in developing ovules and seeds to increase final seed
size.
Nucleic acid which suppresses expression of a BB polypeptide as
described herein may be operably linked to a heterologous regulatory
sequence, such as a promoter, for example a constitutive, inducible,
tissue-specific or developmental specific promoter as described
above.
The construct or vector may be transformed into plant cells and
expressed as described above.
A plant expressing the dominant-negative form of the DA polypeptide
and, optionally having reduced or abolished expression of a BB
polypeptide, may be sexually or asexually propagated or off-spring
or descendants may be grown.
Another aspect of the invention provides a method of producing a
plant with an altered phenotype comprising:
incorporating a heterologous nucleic acid which encodes a
dominant-negative DA polypeptide into a plant cell by means of
transformation, and;
regenerating the plant from one or more transformed cells.
The altered phenotype of the plant produced by the method is
described in more detail above. The method may be useful, for
example, in producing plants having increased yields, for example,
crop plants having improved grain yield, relative to control plants.
In some embodiments, a method may further comprise reducing or
abolishing the expression or activity of a BB polypeptide in the
plant cell or plant.
This may be carried out before, at the same time or after the
incorporation of the nucleic acid which encodes the dominant-
negative DA polypeptide. For example, in some embodiments, the
expression or activity of a BB polypeptide may be abolished or
reduced in one or more plant cells which already incorporate the
nucleic acid encoding the dominant negative DA polypeptide. In other
embodiments, the nucleic acid encoding the dominant negative DA
polypeptide may be incorporated into one or more plant cells which
have abolished or reduced expression of a BB polypeptide.
A plant thus produced may comprise a heterologous nucleic acid which
encodes a dominant-negative DA polypeptide and may possess abolished
or reduced expression or activity of a BB polypeptide in one or more
of its plant cells.
The expression or activity of a BB polypeptide may be reduced or
abolished as described above. For example, a method may comprise
incorporating a heterologous nucleic acid into a plant cell by means

of transformation, wherein the nucleic acid encodes a suppressor
nucleic acid, such as an siRNA or shRNA, which reduces the
expression of a BB polypeptide.
The heterologous nucleic acids encoding the dominant negative DA
polypeptide and BB suppressor nucleic acid may be on the same or
different expression vectors and may be incorporated into the plant
cell by conventional techniques.
Dominant-negative DA polypeptides and BB suppressor nucleic acids
are described in more detail above.
A plant produced as described above may be sexually or asexually
propagated or grown to produce off-spring or descendants. Off-
spring or descendants of the plant regenerated from the one or more
cells may be sexually or asexually propagated or grown. The plant or
its off-spring or descendents may be crossed with other plants or
with itself.
A plant suitable for use in the present methods is preferably a
higher plant, for example an agricultural plant selected from the
group consisting of Lithospermum erythrorhizon, Taxus spp, tobacco,
cucurbits, carrot, vegetable brassica, melons, capsicums, grape
vines, lettuce, strawberry, oilseed brassica, sugar beet, wheat,
barley, maize, rice, soyabeans, peas, sorghum, sunflower, tomato,
potato, pepper, chrysanthemum, carnation, linseed, hemp and rye.
Another aspect of the invention provides a plant which expresses a
dominant negative DA polypeptide and optionally has reduced or
abolished expression of a BB polypeptide, wherein said plant
displays an altered phenotype relative to controls.
The dominant negative DA polypeptide may be heterologous
polypeptides.

A suitable plant may be produced by a method described herein
As described above, the plant may have one or more of increased
life-span, increased organ size, increased duration of proliferative
growth and increased seed size relative to control plants. The
plant may have normal fertility relative to control plants.
A plant according to the present invention may be one which does not
breed true in one or more properties. Plant varieties may be
excluded, particularly registrable plant varieties according to
Plant Breeders Rights.
In addition to a plant expressing a dominant negative DA
polypeptide, for example, a plant produced by a method described
herein, the invention encompasses any clone of such a plant, seed,
selfed or hybrid progeny and descendants, and any part or propagule
of any of these, such as cuttings and seed, which may be used in
reproduction or propagation, sexual or asexual. Also encompassed by
the invention is a plant which is a sexually or asexually propagated
off-spring, clone or descendant of such a plant, or any part or
propagule of said plant, off-spring, clone or descendant.
The present inventors have shown that reducing or abolishing the
expression or activity of two or more DA polypeptides also produces
an altered phenotype characterised by normal fertility and one or
more of increased life-span, increased organ size, increased
duration of proliferative growth and increased seed size.
Another aspect of the invention provides a method of altering the
phenotype of a plant comprising;
reducing or abolishing the expression or activity of two or
more active DA proteins in one or more cells of the plant.
Another aspect of the invention provides a method of producing a
plant with an altered phenotype comprising:

reducing or abolishing the expression or activity of two or
more active DA proteins in a plant cell, and;
regenerating the plant from the plant cell.
The phenotype of the plant following reduction or abolition of
expression is described in more detail above.
The expression of active protein may be abolished by mutating the
nucleic acid sequences in the plant cell which encode the two or
more DA proteins and regenerating a plant from the mutated cell. The
nucleic acids may be mutated by insertion or deletion of one or more
nucleotides. Techniques for the inactivation or knockout of target
genes are well-known in the art.
The expression of target polypeptides in plant cells may be reduced
by suppression techniques. The use of suppressor nucleic acids to
suppress expression of target polypeptides in plant cells is well-
known in the art and is described in more detail above.
Suppressor nucleic acids which reduce expression of two or more DA
polypeptides may be operably linked to tissue-specific or inducible
promoters. For example, integument and seed specific promoters can
be used to specifically down-regulate two or more DA nucleic acids
in developing ovules and seeds to increase final seed size.
Other aspects of the invention relate to the over-expression of DA
polypeptides in plant cells. A method of altering the phenotype of a
plant may comprise;
expressing a nucleic acid encoding a DA polypeptide within
cells of said plant.
The plant may have an altered phenotype characterised by normal
fertility and one or more of reduced life-span, reduced organ size,
reduced duration of proliferative growth and reduced seed size
relative to control plants.

Nucleic acid encoding a DA polypeptide may be expressed in a plant
cell as described above mutatis mutandis for dominant negative DA
polypeptides.
Another aspect of the invention provides a method of identifying a
dominant negative DA polypeptide comprising;
providing an isolated nucleic acid encoding a DA polypeptide,
incorporating one or more mutations into the nucleic acid,
introducing the nucleic acid into a plant cell by means of
transformation;
regenerating the plant from one or more transformed cells and,
identifying the phenotype of the regenerated plant.
An altered phenotype which includes normal fertility and one or more
of increased life-span, increased organ size and increased seed size
relative to control plants is indicative that the mutated nucleic
acid encodes a dominant negative DA allele.
Another aspect of the invention provides a method of producing a
dominant-negative DA polypeptide comprising;
providing a nucleic acid sequence encoding a plant DA
polypeptide,
identifying an R residue in the encoded plant DA polypeptide
at a position equivalent to position 358 of SEQ ID NO: 1 and
mutating the nucleic acid to alter said R residue in the
encoded plant DA polypeptide,
the mutant nucleic acid sequence encoding a dominant negative
DA polypeptide.
Mutated nucleic acid encoding a dominant negative DA polypeptide
which are identified or produced as described above may be used to
produce plants having the altered phenotype, as described above.

"and/or" where used herein is to be taken as specific disclosure of
each of the two specified features or components with or without the
other. For example "A and/or B" is to be taken as specific
disclosure of each of (i) A, (ii) B and (iii) A and B, just as if
each is set out individually herein.
Unless context dictates otherwise, the descriptions and definitions
of the features set out above are not limited to any particular
aspect or embodiment of the invention and apply equally to all
aspects and embodiments which are described.
Having generally described the invention above, certain aspects and
embodiments of the invention will now be illustrated by way of
example to extend the written description and enablement of the
invention, and to ensure adequate disclosure of the best mode of
practicing the invention. Those skilled in the art will appreciate,
however, that the scope of this invention should not be interpreted
as being limited by the specifics of these examples. Rather,
variations, extensions, modifications and equivalents of these
specifics and generic extensions of these details may be made
without departing from the scope of the invention comprehended by
this disclosure. Therefore, for an appreciation of the scope of
this invention and the exclusive rights claimed herein, reference
should be had to the claims appended to this disclosure, including
equivalents thereof.
All documents mentioned in this specification are incorporated
herein by reference in their entirety.
The contents of all database entries mentioned in this specification
are also incorporated herein by reference in their entirety. This
includes the versions of any sequences which are current at the
filing date of this application.
Examples

The data set out below shows that "DAI" is a key regulator in
terminating seed and organ growth, and encodes a novel protein
containing UIM and LIM domains. DA1 is shown to control both seed
and organ size by restricting the duration of proliferative growth,
eod1, an enhancer of da1-1, is allelic to bb, suggesting that the
DA1 and the E3 ubiquitin ligase BB, "Big Brother" {Disch, S. Curr
Biol 16, 272-9 (2006); Science 289, 85-8 (2000)) can act in parallel
pathways to control the final size of seeds and plant organs. It is
possible that DA1 and EOD1/BB may share down stream components that
control seed and organ size.
Previous study has shown that BB acts a negative regulator of organ
growth, most likely by marking cellular proteins for degradation
(Disch, S. Curr. Biol. 16, 272-279 (2006)). DA1 contains two
predicted UIM motifs, which may have the function of binding
ubiquitin and promoting ubiquitination (Hurley, J.H. Biochem. J.
399,361-372 (2006)).
Expression of the DA1 gene is induced by the phytohormone abscisic
acid (ABA), and the da1-1 mutant is insensitive to ABA, providing
indication that ABA negatively regulates organ growth through DAI.
The inhibitory effects of ABA on growth have long been recognized as
resulting from an inhibition of cell division (Lui, J.H. Planta 194,
368-373 (1994), consistent with the fact that ABA can induce the
expression of a cyclin-dependent kinase inhibitor (ICK1), an
important regulator of cell cycle progression (Wang, H. Cell Biol.
Int. 27, 297-299 (2003)). In seed development, the transition from
developing seeds to mature seeds is also correlated with an increase
in seed ABA content (Finkelstein, R.R. Plant Cell 14 Suppl. S15-45
(2002)), which suggests that ABA may be one of environmental cues
sensed by plants to control the final size of seeds and organs, by
inducing negative growth regulators such as DA1. We herein report
that one such negative growth regulator is DA1.

By conducting genetic analysis of abi4-lda1-1 and abi5-lda1-1 double
mutants, we found that the large organ size phenotype of da1-1 is
independent of ABI4 and ABI5 pathways.
We also show herein that suppressors of da1-1 (sod1) are molecules
which have a second site mutation in the da1-1 mutant gene that are
predicted to reduce gene function, indicating that the R358K
mutation in DA1 is responsible for increased seed size and that the
da1-1 allele interferes with activities of DARs.
We also show herein that the da1-1 R358K allele also interferes with
DA1 functions in a dosage dependent manner, as evidenced by the fact
that plants overexpressing da1-1 allele (35S::DA1R358K) in wild type
have large seed and organ size. This result also demonstrates that
the da1-1 mutant gene (DA1R358K) may be used to genetically engineer
significant increases in seed weight and biomass.
To date, some mutants (e.g., ap2 and arf2) exhibiting large seeds
usually have strong negative effects on their fertility and growth
(Schruff, M.C. Development 137, 251-261 (2006); Ohto, M.A. Proc.
Natnl Acad. Sci USA 102, 3123-3128 (2005); Jofuku, K.D. Proc. Natnl
Acad. Sci. USA 102, 3117-3122 (2005)). However, the experiments set
out below show that da1-1 has increased seed mass, large organ size,
but normal fertility, compared with wild type.
Methods
Plant materials
The Arabidopsis thaliana Columbia (Col-0) accession was used as a
wild type. All mutants are in the Col-0 background, except for dal-
1Ler and bb-1, which are in Landsberg erecta background. Before
analysis, da1-1 and da1-1Ler were backcrossed into Col-0 and Ler six
times, respectively. T-DNA insertion lines were obtained from SIGNAL
(Salk Institute) and NASC (Nottingham).
Genetic screen and map-based cloning
da1-1 was identified as a novel seed and organ size mutant from an
ethyl-methanesulphonate (EMS)- treated M2 populations of Col-0
accession, sod1-1, sod1-2, sod1-3 and eod1-1 were identified as
suppressor and enhancer of da1-1 from an ethyl-methanesulphonate
(EMS)- treated M2 populations of da1-1, respectively. F2 mapping
populations were generated from a single cross of Ler/da1-1,
Ler/sod1-3da1-1, and da1-1Ler/eod1-1da1-1. A list of primer
sequences is provided in Table 2.
Plasmids and transgenic plants
The following constructs were generated: DA1C0M, 35S::DA1-HA,
35S::GFP-DA1, 35S::DA1-GFP, 35S::DA1R358K, pDAl::GUS, and 35S::EOD1.
Morphological and cellular analysis
Sample preparation, measurement, microscopy, and histochemical
staining for ß-glucuronidase activity used standard methods
(Jefferson R., EMBO J. Embo J 6, 3901-3907 (1987).
DAI limits the size of seeds and organs
To identify repressors of seed and/or organ growth, we screened for
da mutants (DA means 'large' in Chinese) with large seed and/or
organ size from an EMS mutagenized population in the Col-0 accession
of Arabidopsis thaliana. da1-1 mutant has large seed and organ size,
but normal fertility, compared with wild type (Fig.1a-1p), providing
indication that seed and organ growth share common regulatory
mechanisms. Genetic analysis with reciprocal crosses between da1-1 and wild type plants revealed that da1-1 possesses a mutation in a
single nuclear locus.
To reveal differences in seed size between wild type and da1-1 mutant, we examined da1-1 mutant seed size by fractionating seeds
produced by individual wild type and da1-1 plants by using a series
wire mesh screens. Seeds from wild type were retained only in 180-
300um aperture meshes while the mutant seeds displayed a shift in
range to larger exclusion sizes, 180- 355 µm (Fig. 5a and 5b) . More

than 80% of the wild type seeds were retained in 250um aperture
meshes, whereas about 70% da1-1 seeds were retained in 300µm
aperture meshes. To determine whether the increase in seed size in
da1-1 reflected an alternation in embryo size, we isolated mature
embryos from wild type and da1-1 mutant seeds, da1-1 mature embryos
were significantly fatter and longer than those of wild type (Fig.
1c and 1d). We observed that the seed cavity in da1-1 seeds is
larger throughout development than that in wild type (Fig. 6). In
addition, the average seed mass of da1-1 mutant is increased to 132%
of that of wild type (Table 5).
The fertility of da1-1 plant was found to be normal and the average
seed weight per da1-1 plant is higher than that per wild type plant
(Fig. 5). Therefore, we concluded that DA1 contributes to the
determination of seed size and seed weight in Arabidopsis. We also
identified examples of DA1 and DAR- related genes from crop plants
that demonstrate related genes with related functions can be
targeted by making the R358K dominant interfering mutation, or
reducing expression of selected DA1- and DAR- related proteins using
RNA interference methods described above.
We investigated whether DA1 acts maternally or zygotically. As shown
in Table 1, the effect of the da1-1 mutation on seed mass was
observed only when maternal plants were homozygous for the mutation.
Seeds produced by a da1-1 mother, regardless of the genotype of the
pollen donor, were consistently heavier than those produced by
maternal wild type plants. In contrast, da1-1 mutant and wild type
pollen produced seeds whose weight was comparable to that of wild
type maternal plants. These results show that da1-1 is a maternal
effect mutation that affects seed mass.
In addition to the increased seed mass, the da1-1 mutant exhibited
larger organ size than wild type (Fig. 1e-m and o). Compared with
wild type, da1-1 plants have large flowers (Fig. 1h and i) that
frequently have extra petals and carpels (Fig. 7). The average size
of da1-1 petals was about 1.6-fold that of comparable wild-type
petals (Fig. 1o). Siliques of the da1-1 mutant were wide, deformed
and flattened, compared with the narrow, smooth, cylindrical shape
of wild type siliques (Fig. 1j and 1k). da1-1 mutant also forms
larger cotyledons and leaves, as well as thicker stems than wild
type (Fig. 1e,-g , i, and m). Consistent with this, da1-1 mutant
accumulates more biomass in the form of flowers and leaves than wild
type plants (Fig. 1p and q). Taken together, these results indicated
that DA1 limits the size of seeds and organs in Arabidopsis.
DAI restricts the duration of proliferative growth
Seed and organ size is determined by both cell number and/or size.
To understand which parameter is responsible for large seed and
organ size in da1-1 mutant, we analyzed cell size of embryos, petals
and leaves. As shown in Fig.lr, the size of cells from da1-1
embryos, petals and leaves, were comparable with that of
corresponding wild type cells. The epidermal cell number of stem in
da1-1 mutant is increased to 180% that of wild type stems (Fig. In).
These results indicate that da1-1 induced effects on seed mass and
organ size are due to the increased cell number.
To determine how DA1 limits seed and organ size, we performed a
kinematic analysis of embryos, petals, and leaves in wild type and
the da1-1 mutant. We manually pollinated da1-1 mutant and wild type
plants with their own pollen grains and examined the duration of
seed development. Most of the wild-type seeds developed into
desiccation stage in 8 days after fertilization, whereas most of the
da1-1 seeds developed into mature stage in 10 days after
fertilization in our growth conditions, suggesting that the period
of seed development of da1-1 mutant was prolonged. Plotting the size
of petal primordia and leaves over time revealed that the organ
enlargement in da1-1 mutant is mainly due to a longer growing period
of time (Fig. 2a, c). Consistent with this, da1-1 plants have
younger and fresher organs in early developmental stages (Fig. 1g)
and longer lifespan than wild type (Fig. 12 e, f).
To determine how cell division contributes to the observed growth
dynamic, we measured the mitotic index of petals and leaves in wild
type and mutant. A transgene marker of cell- cycle progression, a
pCYCBl:1::GUS fusion, was used to compare the extent of cell
proliferation in developing petals of wild type and da1-1 plants.
The cells in da1-1 petals continue to proliferate for a longer time
than those in wild- type petals (Fig. 2b). Similarly, the arrest of
cell cycling in the cells of leaves was also delayed (Fig. 8). The
analysis of ploidy level also indicated that da1-1 mutant exits cell
cycle later than wild type. This result provided indication that da1-1 exhibits prolonged cell proliferation.
DAI encodes a novel protein containing UIM and LIM domains
The DA1 locus was fine-mapped to an about 30-kilobase (kb) region
using polymerase chain reaction-based markers (Fig. 9). DNA
sequencing revealed that the da1-1 allele carries a single
nucleotide mutation from G to A in the Atlgl9270 gene which
cosegregated with the da1-1 phenotype and results in a change of an
argine (R) to a lysine (K) at amino acid 358 of the predicted
protein (Fig.3a and Fig. 9a,b). A binary plasmid (DA1C0M) carrying a
6.4-kb wild- type genomic fragment containing the entire ORF and a
plasmid (35S::DA1) carrying 35S promoter plus Atlgl9270 cDNA were
able to rescue the phenotypes of the da1-1 mutant (Fig.1i-q and
Fig.2a), confirming that that Atlgl9270 is indeed the DA1 gene.
DAI is predicted to encode a 532-amino acid protein containing two
ubiquitin interaction motifs (UIM) (Hiyama, H. J. Bio. Chem. 274,
28019-28025 (1999)) and one zinc-binding domain (LIM) present in
Lin-11, Isl-1, Mec-3 (Freyd, G. Nature 344, 876-879 (1990) at the N
terminus (see SequenceS). The UIM is a short peptide motif with the
dual function of binding ubiquitin and promoting ubiquitination.
This motif is conserved throughout eukaryotes and is present in
numerous proteins involved in a wide variety of cellular processes
including endocytosis, protein trafficking, and signal transduction
(Hurley J.H. Biochem. J. 399, 361-372 (2006)). The LIM domain is a
protein-protein interaction motif critically involved in a variety
of fundamental biological process, including cytoskeleton
organization, organ development and signal transduction (Dawid, I.B.
Trends Genet. 14, 156-162 (1998); Dawid, I.B. CR Acad. Sci. III 318,
295-306 (1995); Kadrmas, J.L. Nat. Rev. Mol. Cell Biol. 5, 920-931
(2004)) Seven other predicted proteins in Arabidopsis share
extensive amino acid similarity (>30% identity) with DA1 and have
been named DAl-related proteins (DARs) (see sequence alignment D).
The conserved regions among DA1 and DARs lie in the C terminal
portion of the molecule, indicating that these conserved regions may
be crucial for their function. Proteins that share significant
homology with DA1 outside the UIM and LIM are also found in plants
and green algae, but not animals.
The spatial expression patterns of DA1 were revealed by
histochemical assays of ß-glucuronidase (GUS) activity of transgenic
plants containing DA1 promoter::GUS fusions (pDAl::GUS).
Histochemical staining shows DA1 gene expression throughout the
plant, including cotyledons, true leaves, flowers, and embryos (Fig.
3b-h), consistent with the large size phenotypes of da1-1 mutant
plants. Relatively high levels of GUS activity were detected in
proliferating tissues (Fig. 3c-f). In addition, the DA1 promoter is
also active in roots (Fig. 3b, c). Given the effects of hormones on
organ growth, we tested whether any major classes of phytohormones
(abscisic acid, jasmonic acid, ethylene, auxin, cytokinin,
gibberellin, brassinosteroids and glucose) could influence
transcription of DA1 gene. The expression level of the DA1 gene was
induced slowly by ABA (Fig. 3m), but not by other hormones,
suggesting that the ABA signal may participate in regulation of DAI.
Consistent with this, da1-1 mutant is insensitive to ABA (Fig. 3n),
providing indication that ABA may be one of the environmental cues
that regulate DA1 gene to limit seed and organ growth.

A green fluorescent protein (GFP)-DA1 translational fusion under the
control of 35S promoter rescued the da1-1 phenotype. However, we
could not detect GFP signal. Consistent with this, we also could not
detect DA1 proteins of transgenic plants overexpressing DA1 with HA
(35::DA1-HA) and GFP (35S::DA1-GFP) tags using western blot
providing indication that the DA1 protein is readily degraded or
cleaved in plants.
da1-1 acts as a type of dominant negative mutation for DA1-related
proteins
To identify the novel components of the DA1 pathway that determines
the final size of seed and organ size, we screened for suppressors
of the large seed and organ size phenotypes of da1-1 (sod) and found
three sod1 alleles that were mapped to the original DA1 locus. We
sequenced the DA1 gene from these lines and found that each
harboured a second site mutation that is predicted to reduce or
abolish gene function (Fig. 3A). That second site mutations in the
da1-1 mutant gene suppress the da1-1 phenotype indicates that the
(R358K) mutation within the DA1 coding sequence produces the large
seed and organ size. Consistent with this, disruptions of the DA1
gene via T-DNA insertions (da1-ko1, da1-ko2 and da1-ko3) display no
obvious phenotype (Fig. 10). To determine if one amino acid change
found in the original da1-1 allele was necessary for the da1-1
phenotype, we crossed da1-1 with wild type or da1-ko lines. All
heterozygotic lines (F1) from crosses between Co1-0 and da1-1
exhibited the wild type phenotype, whereas all the Fl plants from
crosses between da1-1 and T-DNA lines (dal-kol, dal-ko2 and dal-ko3)
exhibited similar phenotypes to da1-1 (Fig. S9). In addition, the da1-1 phenotype was also observed in wild type plants carrying the
35S::DA1R358K transgene (Fig.1i-r). Therefore, the R358K mutation in DA1 is necessary and sufficient to cause the da1-1 phenotype.
The loss-of-function alleles display no obvious phenotype. We
therefore postulated that DA1 may act redundantly with DARs and
expression of da1-1 allele interferes with the ability of DARs to

replace DAI. To test this hypothesis, we generated da1-ko1dar1-1
double mutants. The dal-koldarl-1 double mutant displayed the
original da1-1 phenotype (Fig.3i-1, Table 1 and Fig. 4e), indicating
that da1-1 acts as a type of recessive interfering allele for DARs.
Large seed and organ size phenotypes of plants overexpressing the
da1-1 allele in Col-0 suggested that the da1-1 allele also
interferes with the activity of DA1 in dosage-dependent manner (Fig.
1i-q).
DA1 acts in parallel with E0D1/BB, independent of ANT, AXR1, ARF2
and AP2
In enhancer screens, we isolated one allele of a recessive enhancer
of da1-1 (eod1-1). eod1-1da1-1 plants exhibits larger seed and organ
size, more extra petals and longer lifespan than da1-1 (Fig. 12a,b).
We mapped the eod1-1 mutation and found that it was linked to Big
Brother (BB) gene (At3g63530) that encodes an E3 ubiquitin ligase
and represses organ growth in Arabidopsis {Disch, 2006}. Sequencing
revealed that the eod1-1 allele carries a single nucleotide mutation
from G to A in the At3g63530 and resulted in a change of an Alanine
(A) to a Threonine (T) at amino acid 44 of the predicted BB protein
(Fig. 12c). Both T-DNA insertion in the intron (eod1-2) and bb-1
also enhance da1-1 phenotypes (Fig.4 and Fig. 12d, e). A binary
plasmid (35S::E0D1) carrying 35S promoter plus At3g63530 cDNA was
able to rescue the phenotype of the eodl mutant, indicating that
EOD1 is the BB gene. To determine the relationships between EOD1/BB
and DA1 in limiting organ size, we analyzed the mRNA expression
levels of DA1 in a bb-1 mutant and of BB in a da1-1 mutant.
Expression of the DA1 gene in a bb-1 mutant and of the BB gene in a da1-1 plant is not significantly affected, compared with wild type
(Fig. 12a,b).
To understand the genetic relationships between EOD1/BB and DA1, we
measured seed and petal size in eod1-2da1-1 and bb-1da1-1Ler double
mutants and found that mutations in EODl/BB synergistically enhance
the phenotype of da1-1 (Fig. 4, Fig. 11 d, e and 12a), providing
indication that the two genes act in parallel pathways to limit seed
and organ size in plants (Fig. 4e).
aintegumenta (ant) alleles exhibit small petals and plants over-
expressing ANT exhibit organ enlargement because of a prolonged
period of organ growth {Krizek, B.A. Dev Genet 25, 224-36 (1999);
Mizukami, Y.Proc Natl Acad Sci U S A 97, 942-7 (2000)}, providing
indication that DA1 and ANT could function antagonistically in a
common pathway. To test this, we analyzed the mRNA expression levels
of DA1 in ant mutants and of ANT and its downstream target
CyclinD3;l in da1-1 mutant. DA1 mRNA levels do not show robust
changes in ant mutants (Fig. 12b). Similarly, the levels of both ANT
and Cyclin3;l mRNA are not significantly affected by the da1-1 mutation, as is the mRNA level of ARGOS {Hu, Y.Plant Cell 15, 1951-
61 (2003)} (Fig. lie, d, e). Genetic analysis also showed that the
petal size phenotype of ant-5da1-1 mutant was essentially additive,
providing indication that DA1 and ANT act in independent pathways.
We also generated axrl-12da1-1, arf2-7da1-1 and ap2-7da1-1 double
mutants, since axrl, arf2 and ap2 mutants have altered organ and/or
seed size (Lincoln, C. Plant Cell 2, 1071-1080 (1990); Schruff, M.C.
Development 137, 251-261 (2006); Ohto, M.A. Proc. Natnl Acad. Sci
USA 102, 3123-3128 (2005);Jofuku, K.D. Proc. Natnl Acad. Sci. USA
102, 3117-3122 (2005)). Genetic analysis revealed that the petal
size phenotype of axrl-12da1-1 mutant or the seed size phenotype of
arf2-7da1-1 and ap2-7da1-1 were essentially additive (Fig.
12b,c,d,e), compared with their parental lines. Therefore, we
concluded that DA1 appears to act in parallel with EOD1/BB,
independent of ANT, ARX1, ARF2 and AP2.
The DA1 protein family in Arabidopsis thaliana
As described above, the DA1 gene is predicted to encode a 532-amino-
acid protein containing two ubiquitin interaction motifs (UIM)
typical of ubiquitin receptors and a single zinc-binding LIM domain
defined by its conservation with the canonical Lin-11, Isl-1, and
Mec-3 domains(Li et al. 2008). In Arabidopsis, seven other predicted
proteins share extensive C-terminal (outside UIM and LIM domains)
amino acid similarity with DA1 and have been named DAl-related (DAR)
proteins, of which four (DAR3,DAR5-7) are found in a tandem cluster
on chromosome 5. Using SMART (http://smart.embl-heidel
berg.de/smart/show_motifs.pl), the different functional domains were
characterised (see Table 11).
UIM is the ubiquitin-interacting motif with two conserved serine
residues required for binding and forms a short a-helix structure
with ubiquitin(Haglund and Dikic 2005). LIM is a cysteine-rich
protein interaction motif, has zinc-binding ability(Freyd et al.
1990). NB-ARC (stands for "a nucleotide-binding adaptor shared by
APAF-1, certain R gene products and CED-4") links a protein-protein
interaction module to an effector domain, it is a novel signalling
motif shared by plant resistance gene products and regulators of
cell death in animals. LRRs are leucine-rich repeats, they are short
sequence motifs and are thought to be involved in protein-protein
interactions. RPW8 belongs to a family that consists of several
broad-spectrum mildew resistance proteins from Arabidopsis thaliana.
These diverse protein structures provide indication the family has
diverse functions and has functionally diversified recently. Table
11 may be used to guide the formation of double and triple mutants
eg DAI, DAR1 are similar and have been shown to function
redundantly; it is possible that DA6 and DAR7 may also function
redundantly with each other and DA1 and DAR1 because of their
similar structures.
Characterisation of da1-1ike (DAL) proteins in Physcomitrella
patens, Selaginella moellendorffi, Brassica rapa, Brachypodium
distachyon and Oryza sativa
The amino acid sequences of Arabidopsis DA1 and DAR1-7 were used as
queries to screen the available Physcomitrella patens, Selaginella
moellendorffi, Brassica rapa, Brachypodium distachyon and Oryza

sativa databases. Using different BLAST algorithms, candidate genes
were then selected for preliminary phylogenetic analysis.
DAI-like proteins in early land plants, Physcomitrella patens and
Selaginella moellendorffi
The DA1 family orthologs in P. patens were searched by using DA1 amino acid sequence as query in NCBI BLAST, and then revised in JGI
P.patens BLAST(http://genome.jgi-psf.org/ cgi-bin/runAlignment?
db=Phypal _l&advanced=1). Eight genes (PpDAL1-8) were selected based
on scores, E-values and preliminary phylogenetic analysis. All P.
patens da1-1ike proteins are shorter than DA1 amino acid sequences,
due to absence of the two UIM domains at the N-terminal (see Figure
1), according to SMART (Simple Molecular Architecture Research Tool)
program (http://smart.embl-heidelberg.de/). The S. moellendorffi
sequencing project provides us an opportunity to investigate DA1 family orthology in first vascular plant. By using JGI S.
moellendorffi BLAST(http://genome.jgi-psf.org/cgi-
bin/runAlignment?db=Selmol&advanced=l) , two orthologs were found,
and comparing with P.patens da1-1ike proteins, they had similar
amino acid sequences length with Arabidopsis DA1 family proteins.
The regions preceding the LIM domain were predicted to be low-
complexity regions by SMART, and no clear UIM protein sequence
motifs were found. We can therefore conclude that the characteristic DA1 protein structure is not found in lower plants.
da1-1ike proteins in Brassica rapa
Due to the close evolutionary relationships of Arabidopsis and
Brassica, nucleotide BLAST methods for identifying Brassica DA1 family orthologs were used. In the Brassica database
(http://www.brassica.bbsrc.ac.uk/), full length cDNA sequences of
Arabidopsis DA1 and DAR1-7 were used as queries. Because of a recent
entire genome duplication, one Arabidopsis gene probably has 2 or 3
homologous genes in Brassica rapa. Two DA1 orthologs and one DAR2
orthologs were found (Figure 1). These were called DAL {DA- Like)
genes. The DAR3-7 Brassica orthologs were also found in a tandem
cluster. The number of Brassica orthologs found is less than
predicted probably due to incomplete genome sequencing. The partial
sequences of Brassica DAL genes were used to design primers for the
specific amplification of full-length DAL genes from B. rapa.
da1-1ike proteins in the grasses Brachypodium distachyon and Oryza
sativa
In Brachypodium distachyon, three major DA1 family orthologs were
identified, and in Oryza sativa, four da1-1ike proteins were found
using PROTEIN BLAST at NCBI. Rice DA1 and DAR2 orthologs were
identified and named OsDA1 and 0sDAR2. No rice gene was found to
match Arabidopsis DAR1.
In the phylogenetic tree of Figure 14 all DA-like proteins from
vascular plants form one large clade. In that clade, S.moellendorffi
DA-like proteins are highly divergent, but it is possible that DA-
like proteins might originate from bryophytes, and function as size
regulators since the evolution of the first vascular plants
(Lycophytes). In the tree, a clade was formed by AtDAR2, BrDAR2,
BdDAL3 and 0sDAR2. These protein sequences show greater similarity,
suggesting that DAR2 evolved before monocots originated (see right
graph of Figure 14) and is functionally conserve during evolution.
Another clade consists of AtDA1, BrDAla and BrDAlb. The high
similarity between them suggests Brassica rapa DA1 proteins might
have the same function as AtDA1. The clade consisting of OsDAl,
BdDALl and BdDAL2 was placed apart from this clade (see Figure 14),
indicating that grass DA1 proteins may be slightly functionally
divergent from those in the Brassicaceae. All Brassicaceae DAR1 and
DAR3-7 were placed in one clade, indicating these genes probably
arose from DA1 or DAR2 in the genome duplication within
Brassicaceae. This hypothesis has been partially proved by genetic
analysis, which demonstrated, in Arabidopsis, DA1 and DAR1 are
functional redundant.

Functional analysis of DA1-like proteins in Brassica rapa, Oryza
sativa
In silico, two Brassica rapa da1-1ike genes (BrDA1a, BrDA1b) and one
rice da1-1ike gene (OsDA1) were identified. Full length cDNAs were
synthesised and sequenced using directional TOPO vectors. The
predicted biochemical characteristics of AtDAl, BrDAla, BrDAlb and
OsDAl are shown in Table 12. The proteins these three genes encode
have very similar biochemical characteristics, particularly the two
Brassica ones. Interestingly, although analysis based on amino acid
sequences shown BrDAla is more close to AtDAl, BrDAlb was predicted
to have more similar biochemical features to AtDAl (Table 12).
The phenotypes of da1-1 are rescued by BrDAla, BrDAlb and OsDAl
genes
Full-length BrDAla, BrDAlb and OsDAl cDNAs were sub-cloned to TOPO
vectors and transferred to pMDC32 binary destination vectors by LR
recombination. These vectors express cDNAs from the constitutive 35S
promoter. da1-1 plants were transformed to test whether the wild-
type DAL genes from Brassica and rice could complement the large
growth phenotypes of da1-1 plants. The 35S:: BrDAla and 35S:: OsDAl
transgenic plants showed at least partial complementation (see
Figure 15A-D). Interestingly, although BrDAlb is not the closest
homolog to AtDAl, the 35S:: BrDAlb transgenic plants showed full
complementation of the da1-1 phenotype (see Figures 15E-G),
consistent with the high biochemical similarity to AtDAl (see Table
12). Two rounds of transformants were screened. In the first round,
10 out of 40 35S: -.BrDAla and 3 out of 11 35S::OsDAl Tl plants show
the siliques phenotypes in Figure 15A-D. In the second round, 30 out
of 150 35S: -.BrDAlb have shown the rosette leaves phenotypes in
Figure 15E-G. This is convincing data that BrDAlb functions like
Arabidopsis DAI. Consequently we have demonstrated that DA1 and
related genes have similar functions in controlling organ and seed
size in Brassica and rice, and probably many other types of plants.

BrDAlaR358k can interfere with AtDA1 function in Col-0 plants
Site directed mutagenesis was used to generate the equivalent R358K
mutation in the BrDAla cDNA in the TOPO vector and then the mutated
cDNA was transferred to pMDC32 destination vectors using the gateway
system. Typical da1-1 phenotypes were observed in wildtype Col-0
plants expressing 35S:: BrDAla R358K (see Figure 15E,F,H), including
large organ phenotypes. In this transformation experiment, 60 Tl
transgenic plants were screened and 7 of these were found to have
characteristic large organ phenotypes seen in da1-1 plants.
DAI protein stability.
We have observed that transformants expressing fusions of the GFP
protein with the C terminus of the full length DA1 protein
complements the DA1R358K large organ phenotype, demonstrating that the
fusion protein is fully functional. However, we did not detect GFP
fluorescence in many transgenic lines, providing indication that
DA1GFP protein levels are very low. This is supported by the
observation that detection of DA1 protein with a good specific
antibody in plant extracts is very difficult. We therefore tested
the stability of DA1 protein in Arabidopsis using DA1 protein
expressed in E. coli and cell-free protein extracts from
Arabidopsis. Full length DA1 protein expressed and purified as an N-
terminal GST fusion protein, was incubated with Arabidopsis protein
extracts and ATP, and subjected to Western analysis using a specific DA1 antibody. DA1 protein was found to be rapidly degraded under these
conditions. MG132, a specific inhibitor of the proteasome, was found
to abolish this degradation. Therefore, DA1 is rapidly degraded by
the proteasome in Arabidopsis. The UIM motifs of DA1 are predicted,
based on knowledge of UIM function in animal cells, to be involved
in ubiqutination. It may be that DA1 is ubiquitinated and targeted
for degradation as part of the mechanism of growth control.

1: CA061229
unnamed protein product [Vitis vinifera]
giI 157335399|emb|CA061229.1| [157335399]
2: EAZ36049
hypothetical protein OsJ_019532 [Oryza sativa (japonica
cultivar-group)]
gi|125596269|gb|EAZ36049.1| [1255962 69]
3: EAY99923
hypothetical protein OsI_021156 [Oryza sativa (indica
cultivar-group)]
gi|125554318|gb|EAY99923.1| [125554318]
4: NP_001056985
Os06g0182500 [Oryza sativa (japonica cultivar-group)]
gi|1154 66772|ref|NP_001056985.1|[1154 66772]
5: CA022 922
unnamed protein product [Vitis vinifera]
gi |157348212|emb|CA022922.1| [15734 8212]
6: EAZ21100
hypothetical protein OsJ_035309 [Oryza sativa (japonica
cultivar-group)]
gi| 12557 9954|gb|EAZ21100.1| [12557 9954]
7: NP_001067188
Osl2g0596800 [Oryza sativa (japonica cultivar-group)]
gi| 11548 9402|ref|NP_001067188.1| [11548 94 02]
8: CA022 921
unnamed protein product [Vitis vinifera]
gi| 1573482111emb|CA022921.1|[157348211]
9: AAW34243
putative LIM domain containing protein [Oryza sativa
(japonica cultivar-group)]
gi|57164484|gb|AAW34243.1|[57164484]
10: AAW34242
putative LIM domain containing protein [Oryza sativa
(japonica cultivar-group)]
gi|57164483|gb|AAW34242.1| [57164483]
11: NP_001050702
Os03g0626600 [Oryza sativa (japonica cultivar-group)]
gi|115454203|ref|NP_001050702.1| [1154 54203]
12: EAY83760
hypothetical protein OsI_037719 [Oryza sativa (indica
cultivar-group)]
gi|125537272|gb|EAY837 60.1| [125537272]
13: EAZ27845
hypothetical protein OsJ_011328 [Oryza sativa (japonica
cultivar-group)]
gi| 125587181|gb|EAZ27845.1|[125587181]
14: NP_001049668
Os03g0267800 [Oryza sativa (japonica cultivar-group)]
gi|115452135|ref|NP_00104 9668.1|[115452135]
15:
EAY91080
hypothetical protein OsI_012313 [Oryza sativa (indica
cultivar-group)]
gi|125544941|gb|EAY91080.1|[125544941]
16:
AAP06895
hypothetical protein [Oryza sativa (japonica cultivar-group)]
gi|29893641|gb|AAP06895.1|[29893641]
17: EAY89390
hypothetical protein OsI_010623 [Oryza sativa (indica
cultivar-group)]
gi|125543251|gb|EAY89390.1|[12554 3251]
18:
CA016347
unnamed protein product [Vitis vinifera]
gi|157346464|emb|CAO16347.1|[157346464]
19: CAN64300
hypothetical protein [Vitis vinifera]
gi|147817187|emb|CAN64300.1|[147817187]
20: CAN69394
nypotnetical protein [Vitis vinifera]
gi|147768077|emb|CAN69394.1|[1477 68077]
Table 6
Oryza sativa (japonica cultivar-group) Os06g0182500 (Os06g0182500)
mRNA, complete cds
gi|115466771|ref|NM_001063520.1|[115466771]
Oryza sativa (japonica cultivar-group) cDNA clone:001-201-F10, full
insert sequence
gi|32990928|dbj|AK105719.1|[32990928]
Oryza sativa (japonica cultivar-group) cDNA clone:J023004G21, full
insert sequence
gi|32979080|dbj|AK069056.1|[3297 9080]
Oryza sativa (japonica cultivar-group) Os12g0596800 (Os12g0596800)
mRNA, complete cds
gi|115489401|ref|NM_001073720.1|[115489401]
Oryza sativa (japonica cultivar-group) cDNA clone:J013039D10, full
insert sequence
gi|32975778|dbj|AK065760.1|[32 975778]
Oryza sativa (japonica cultivar-group) cDNA clone:J013073O11, full
insert sequence
gi|32976683|dbj|AK066665.1|[32976683]
Oryza sativa (japonica cultivar-group) Os03g0626600 (Os03g0626600)
mRNA, partial cds
gi|115454202|ref|NM_001057237.1|[115454202]
Oryza sativa (japonica cultivar-group) cDNA clone:001-043-H07, full
insert sequence
gi|32972053|dbj|AK062035.1|[32972053]
Oryza sativa (japonica cultivar-group) Os03g0267800 (Os03g0267800)
mRNA, complete cds
gi|115452134|ref|NM_301056203.1|[115452134]
Oryza sativa (japonica cultivar-group) cDNA clone:J023020C05, full
insert sequence
gi|32979610|dbj|AK069586.1|[32979610]
Oryza sativa (japonica cultivar-group) isolate 29050 unknown mRNA
gi|29368349|gb|AY224559.1 [2 936834 9]
Oryza sativa (japonica cultivar-group) isolate 29050 disease
resistance-like protein mRNA, partial cds
gi|29367476|gb|AY224475.1|[29367476]
Oryza sativa (japonica cultivar-group) genomic DNA, chromosome 6
gi|58531193|dbj|AP008212.1|[58531193]
Oryza sativa (japonica cultivar-group) genomic DNA, chromosome 6, BAC
clone:OSJNBb0036B04
gi|50657316|dbj|AP007226.1| [50657316]
Oryza sativa (japonica cultivar-group) genomic DNA, chromosome 12
gi|58531199|dbj|AP008218.1|[58531199]
Oryza sativa chromosome 12, . BAC OSJNBa0056D07 of library OSJNBa from
chromosome 12 of cultivar Nipponbare of ssp. japonica of Oryza sativa
(rice), complete sequence
gi|23897123|emb|AL928754.2|[238 97123]
Oryza sativa chromosome 12, . BAC OJ1306_H03 of library Monsanto from
chromosome 12 of cultivar Nipponbare of ssp. japonica of Oryza sativa
(rice), complete sequence
gi|20513132|emb|AL713904.3|[20513132]
Oryza sativa (japonica cultivar-group) genomic DNA, chromosome 3
gi|5853078 9|dbj|AP008209.1| [5853078 9]
Oryza sativa (japonica cultivar-group) chromosome 3 clone
OSJNBa0002I03 map E1419S, complete sequence
gi|57164481|gb|AC091246.8|[57164 4 81]
Oryza sativa (japonica cultivar-group) chromosome 3 clone OJA1364E02,
complete sequence
gi|27901829|gb|AC139168.1|[27901829]
Oryza sativa (japonica cultivar-group) chromosome 3 clone OJ1364E02,
complete sequence
gi|27 901828|gb|AC135208.3|[27901828]
Oryza sativa chromosome 3 BAC OSJNBb0013K08 genomic sequence, complete
sequence
gi|16356889|gb|AC092390.3|[16356889]
Oryza sativa (indica cultivar-group) clone OSE-97-192-H5 zn ion
binding protein mRNA, partial cds
gi|149390776|gb|EF575818.1|[149390776]
Oryza sativa (indica cultivar-group) cDNA clone:OSIGCRA102J03, full
insert sequence
gi|116633496|emb|CT833300.1|[116633496]
Oryza sativa (japonica cultivar-group) Os01g0916000 (Os01g0916000)
mRNA, complete cds
gi|115441820|ref|NM_001051725.1|[115441820]
Oryza sativa (japonica cultivar-group) genomic DNA, chromosome 1
gi|58530787|dbj|AP008207.1|[58530787]
Oryza sativa (japonica cultivar-group) genomic DNA, chromosome 1, PAC
clone:P0413C03
gi|1938674 4|dbj|AP003451.4|[19386744]
Oryza sativa (japonica cultivar-group) genomic DNA, chromosome 1, PAC
clone:P0004D12
gi|20804980|dbj|AP003433.3|[20804980]
Oryza sativa (japonica cultivar-group) cDNA clone:002-101-C04, full
insert sequence
gi|32991509|dbj|AK106300.1|[32991509]
Oryza sativa (japonica cultivar-group) cDNA, clone: J100024O13, full
insert sequence
gi|116012466|dbj|AK24 3101.1|[116012466]
Zea mays clone EL01N0524A08.d mRNA sequence
gi|54653541|gb|BT018760.1|[54653541]
Zea mays PCO156068 mRNA sequence
gi|21212590|gb|AY109151.1|[21212590]
Zea mays clone EL01N0553E07 mRNA sequence
gi|85540336|gb|BT024085.1|[85540336]
Zea mays clone E04912705F06.c mRNA sequence
gi|54651736|gb|BT016955.1| [54651736]
Zea mays nitrate reductase gene, promoter region
gi|4894 987|gb|AF141939.1|AF141939[4894987]
Hordeum vulgare subsp. vulgare cDNA clone: FLbaf82hl6, mRNA sequence
gi|151419042|dbj|AK250393.1|[151419042]
Vitis vinifera, whole genome shotgun sequence, contig VV78X106678.4,
clone ENTAV 115
gi|123680846|emb|AM4 88121.1|[123680846]
Vitis vinifera contig VV78X222701.5, whole genome shotgun sequence
gi|147817185|emb|AM484789.2|[147817185]
Vitis vinifera, whole genome shotgun sequence, contig VV78X165152.5,
clone ENTAV 115
gi|123703056|emb|AM483648.1|[123703056]
Vitis vinifera contig VV78X263569.4, whole genome shotgun sequence
gi|147790377|emb|AM453516.2|[147790377]
Vitis vinifera contig VV78X179395.3, whole genome shotgun sequence
gi|147769864|emb|AM4 56337.2|[147769864]
Vitis vinifera contig VV78X219892.2, whole genome shotgun sequence
gi|147780236|emb|AM461946.2|[147780236]
Vitis vinifera, whole genome shotgun sequence, contig VV78X014445.8,
clone ENTAV 115
gi|123704690|emb|AM483793.1|[123704690]
Vitis vinifera contig VV78X193742.9, whole genome shotgun sequence
gi|147768076|emb|AM435996.2|[147768076]
Lotus japonicus genomic DNA, chromosome 2, clone:LjB06D23, BM0787,
complete sequence
gi|37591131|dbj|AP006541.1|[37591131]
Agropyron cristatum isolate Bsyl y-type high-molecular-weight glutenin
subunit pseudogene, complete sequence
gi|71159564|gb|DQ073532.1[71159564]
Agropyron cristatum isolate Btyl y-type high-molecular-weight glutenin
subunit pseudogene, complete sequence
gi|71159568|gb|DQ073535.1|[71159568]
Agropyron cristatum isolate Bfy1 y-type high-molecular-weight glutenin
subunit pseudogene, complete sequence
gi|71159563|gb|DQ073531.1|[71159563]
Pinus taeda putative cell wall protein (lp5) gene, complete cds
gi|2317763|gb|AF013805.1| [2317763]
Brassica rapa subsp. pekinensis clone KBrHO11G10, complete sequence
gi|110797257|gb|AC189577.1|[1107 97257]
Brassica rapa subsp. pekinensis clone KBrB032C14, complete sequence
gi|110796986|gb|AC189306.1|[1107 9698 6]
Brassica rapa subsp. pekinensis clone KBrB011P07, complete sequence
gi|110744010|gb|AC189225.1|[110744010]
Poplar cDNA sequences
gi|115416791|emb|CT029673.1|[1154167 91]
Poplar cDNA sequences
gi|115416790|emb|CT029672.1|[115416790]
Coffea arabica microsatellite DNA, clone 26-4CTG
gi|13398992|emb|AJ308799.1|[13398 992]
Medicago truncatula clone mth2-34ml4, complete sequence
gi|61675739|gb|AC126779.19|[61675739]
Medicago truncatula chromosome 5 clone mth2-5p5, COMPLETE SEQUENCE
gi|119359633|emb|CU302347.1|[119359633]
Medicago truncatula chromosome 8 clone mth2-14m21, complete sequence
gi|50355770|gb|AC14 8241.21|[50355770]
Solanum lycopersicum cDNA, clone: LEFL1035BC02, HTC in leaf
gi|148538338|dbj|AK247104.1|[14 8538338]
Mimulus guttatus clone MGBa-44P14, complete sequence
gi|150010729|gb|AC182564.2|[150010729]
Mimulus guttatus clone MGBa-64L10, complete sequence
gi|154350257|gb|AC182570.2|[154 350257]
Selaginella moellendorffii clone JGIASXY-5I19, complete sequence
gi|62510100|gb|AC158187.2|[62510100]
M.truncatula DNA sequence from clone MTH2-170H18 on chromosome 3,
complete sequence
gi|115635794|emb|CT967314.8|[115635794]
Vigna unguiculata glutelin 2 mRNA, partial cds
gi|4973069|gb|AF14 2332.1|AF142332[4973069]
Table 7
Soybean cDNA clones
gb|CX711863.1|CX7118 63
gb|BM525343.1|BM525343
gb|BG156297.1|BG156297
gb|BM30814 8.1|BM30814 8
gb|AI856660.1|AI856660
gb|BF596520.1|BF596520
gb|BI472193.1|B1472193
gb|CO982042.1|CO982042
gb|BM143278.1|BM143278
gb|AW831270.1|AW831270
gb|BE329874.1|BE329874
gb|BG652163.1|BG652163
gb|BI967821.1|BI967821
gb|BI321493.1|BI321493
gb|BU546579.1|BU546579
gb|C0984945.1|C0984945
gb|DW247614.1|DW247614
gb|BG726202.1|BG726202
gb|BI968915.1|BI968915
gb|BG043212.1|BG043212
gb|BG510065.1|BG510065
gb|BG043153.1|BG043153
gb|AW832591.1|AW832591
gb|AI856369.1|AI856369
gb|BI699452.1|BI699452
gb|BG650019.1|BG650019
gb|AW234002.1|AW234002
gb|AW310220.1|AW310220
gb|AW394699.1|AW394 699
gb|AW832462.1|AW8324 62
gb|AW459788.1|AW459788
gb|BM731310.1|BM731310
gb|BI317518.1|BI317518
gb|AI988431.1|AI9884 31
gb|CA801874.1|CA801874
gb|BE057592.1|BE057592
gb|AW102002.1|AW102002
gb|CA938750.1|CA938750
gb|AW397679.1|AW397679
Table 8
emb|CAO40855.1| unnamed protein product [Vitis vinifera]
gb|EAY88740.1| hypothetical protein Osl_009973 [Oryza sativa (indica cultivar-group)]
gb|EAZ25768.1| hypothetical protein OsJ_009251 [Oryza sativa (japonica cultivar-group)]
reflNP_001049123.1| Os03g0173900 [Oryza sativa (japonica cultivar-group)]
emb|CA021927.1| unnamed protein product [Vitis vinifera]
emb|CAD41576.3| OSJNBa0088l22.8 [Oryza sativa (japonica cultivar-group)]
gb|EAY95219.1| hypothetical protein Osl_016452 [Oryza sativa (indica cultivar-group)]
emb|CAH67282.1| OSIGBa0111L12.9 [Oryza sativa (indica cultivar-group)]
ref|NP_001053604.11 Os04g0571200 [Oryza sativa (japonica cultivar-group)]
ref|NP_001063719.1| Os09g0525400 [Oryza sativa (japonica cultivar-group)]
gb|EAZ09811.1| hypothetical protein Osl_031043 [Oryza sativa (indica cultivar-group)]
gb|EAZ45411.1| hypothetical protein OsJ_028894 [Oryza sativa (japonica cultivar-group)]
ref|NP_001062434.11 Os08g0548300 [Oryza sativa (japonica cultivar-group)]
gb|EAZ07897.1| hypothetical protein Osl_029129 [Oryza sativa (indica cultivar-group)]
ref|NP_001063778.11 Os09g0535100 [Oryza sativa (japonica cultivar-group)]
emb|CAC10211.1| hypothetical protein [Cicer arietinum]
emb|CA044394.1| unnamed protein product [Vitis vinifera]
gb|ABG73441.1| zinc finger C3HC4 type family protein [Oryza brachyantha]
ref|NP_001056653.1| Os06g0125800 [Oryza sativa (japonica cultivar-group)]
gb|EAZ09879.1| hypothetical protein Osl_031111 [Oryza sativa (indica cultivar-group)]
gb|EAZ45482.1| hypothetical protein OsJ_028965 [Oryza sativa (japonica cultivar-group)]
dbj|BAD82497.1| RING-H2 finger protein RHG1a-like [Oryza sativa (japonica cultivar-group)]
emb|CAN71989.1| hypothetical protein [Vitis vinifera]
dbj|BAD05399.1| DNA binding zinc finger protein-like [Oryza sativa (japonica
emb|CAH65886.1| OSIGBa0148J22.5 [Oryza sativa (indica cultivar-group)]
emb|CAE02518.2| OSJNBb0003A12.5 [Oryza sativa (japonica cultivar-group)]
ref|NP_001052192.1| Os04g0185500 [Oryza sativa (japonica cultivar-group)]
emb|CA071872.1| unnamed protein product [Vitis vinifera]
emb|CA039354.1| unnamed protein product [Vitis vinifera]
emb|CAE01827.2| OSJNBa0041A02.20 [Oryza sativa (japonica cultivar-group)]
emb|CA071869.1| unnamed protein product [Vitis vinifera]
emb|CAA85320.1| C-terminal zinc-finger [Glycine max]
gb|EAZ08608.1| hypothetical protein Osl_029840 [Oryza sativa (indica cultivar-group)]
gb|EAY75305.1| hypothetical protein Osl_003152 [Oryza sativa (indica cultivar-group)]
ref|NP_001043810.1| Os01g0667700 [Oryza sativa (japonica cultivar-group)]
dbj|BAD73651.1| RING-finger protein-like [Oryza sativa (japonica cultivar-group)]
emb|CA071875.1| unnamed protein product [Vitis vinifera]
ref|NP_001062870.11 Os09g0323100 [Oryza sativa (japonica cultivar-group)]
gb|EAZ35180.1| hypothetical protein OsJ_018663 [Oryza sativa (japonica cultivar-group)]
dbj|BAA74802.1| DNA binding zinc finger protein (Pspzf) [Pisum sativum]
ref|NP_001056239.1| Os05g0550000 [Oryza sativa (japonica cultivar-group)]
gb|EAY98923.1| hypothetical protein Osl_020156 [Oryza sativa (indica cultivar-group)]
emb|CAN83345.1| hypothetical protein [Vitis vinifera]
emb|CA043928.1| unnamed protein product [Vitis vinifera]
emb|CAN79375.1| hypothetical protein [Vitis vinifera]
Table 9 BB polypeptides identified by Blastp
ref|NM_001055658.11 Oryza sativa (japonica cultivar-group) Os03g0173900 (Os03g0173900) mRNA, complete cds
dbj|AK063978.1| Oryza sativa (japonica cultivar-group) cDNA clone:001-124-C08, full insert sequence
dbj|AP006425.1| Lotus japonicus genomic DNA, chromosome 1, clone:LjT39B10, TM0315, complete sequence
gb|AY110224.11 Zea mays CL5837_1 mRNA sequence
ref|NM_001060139.11 Oryza sativa (japonica cultivar-group) Os04g0571200 (Os04g0571200) mRNA, partial cds
dbj|AK071401.1| Oryza sativa (japonica cultivar-group) cDNA clone: J023097G23, full insert sequence
ref|NM_001068969.11 Oryza sativa (japonica cultivar-group) Os08g0548300 (Os08g0548300) mRNA, complete cds
dbj|AK073266.1| Oryza sativa (japonica cultivar-group) cDNA clone:J033029A20, full insert sequence
emb|CT832808.1| Oryza sativa (indica cultivar-group) cDNA clone:OSIGCSN035L02, full insert sequence
ref|NM_001070254.11 Oryza sativa (japonica cultivar-group) Os09g0525400 (Os09g0525400) mRNA, complete cds
dbj|AK104112.1 j Oryza sativa (japonica cultivar-group) cDNA clone:006-202-G09, full insert sequence
dbj|AK066238.11 Oryza sativa (japonica cultivar-group) cDNA clone:J013059J01, full insert sequence
emb|CT832015.11 Oryza sativa (indica cultivar-group) cDNA clone:OSIGCRA126H24,full insert sequence
dbj|AK250973.1| Hordeum vulgare subsp. vulgare cDNA clone: FLbaf101a03, mRNA sequence
dbj|AK249803.1| Hordeum vulgare subsp. vulgare cDNA clone: FLbaf58c16, mRNA sequence
emb|CT832014.11 Oryza sativa (indica cultivar-group) cDNA clone:OSIGCRA115D08, full insert sequence
gb|BT016451.1| Zea mays clone Contig284 mRNA
sequence
emb|AJ299062.1|CAR299062 Cicer arietinum partial mRNA for hypothetical protein (ORF1), clone Can183
gb|AY109631.11 Zea mays CL5026_1 mRNA sequence
gb|AY108288.11 Zea mays PC0148716 mRNA sequence
ref|NM_001070313.1| Oryza sativa (japonica cultivar-group) Os09g0535100 (Os09g0535100) mRNA, complete cds
dbj|AK069888.11 Oryza sativa (japonica cultivar-group) cDNA clone:J023039O04, full insert sequence
gb|AY103990.11 Zea mays PCO093361 mRNA sequence
emb|AM485242.1| Vitis vinifera, whole genome shotgun sequence, contig W78X218805.2, clone ENTAV 115
gb|BT018037.11 Zea mays clone EL01N0530G02.C mRNA sequence
emb|CT829435.1| Oryza sativa (indica cultivar-group) cDNA clone:OSIGCRA107A15, full insert sequence
ref|NM_001063188.11 Oryza sativa (japonica cultivar-group) Os06g0125800 (Os06g0125800) mRNA, complete cds
gb|AY225189.1| Oryza sativa (indica cultivar-group) zinc finger protein mRNA, complete cds
gb|AY207044.1| Oryza sativa (indica cultivar-group) zinc-finger protein mRNA, complete cds
dbj|AK104425.11 Oryza sativa (japonica cultivar-group) cDNAclone:006-205-F10,full insert sequence
dbj|AK068302.1| Oryza sativa (japonica cultivar-group) cDNA clone:J013144A04, full insert sequence
gb|AY112568.11 Zea mays CL32837_1 mRNA sequence
emb|AM453896.2| Vitis vinifera contig W78X100953.6, whole genome shotgun sequence
gb|AC157490.18| Medicago truncatula clone mth2-123f23, complete sequence
gb|AC151824.13| Medicago truncatula clone mth2-45n18, complete sequence
ref|NM_001058727.11 Oryza sativa Oaponica cultivar-group) Os04g0185500 (Os04g0185500) mRNA, complete cds
gb|BT019187.1| Zea mays clone Contig858.F mRNA sequence
dbj|AK064939.1| Oryza sativa (japonica cultivar-group) cDNA clone:J013000P06,
gb|AY110468.1| Zea mays CL16240_2 mRNA sequence
gb|AY110685.1| Zea mays CL9024_1 mRNA sequence
dbj|AK246964.1| Solanum lycopersicum cDNA, clone: LEFL1004CA06, HTC in leaf
dbj|AP008214.1| Oryza sativa (japonica cultivar-group) genomic DNA, chromosome
ref|NM_001050479.11 Oryza sativa (japonica cultivar-group) Os01g0692700 (Os01g0692700) mRNA, partial cds
dbj|AK065626.1| Oryza sativa (japonica cultivar-group) cDNA clone:J013028F14,
dbj|AP004704.3| Oryza sativa (japonica cultivar-group) genomic DNA, chromosome 8, PAC clone:P0544G09
dbj|AP006265.2| Oryza sativa (japonica cultivar-group) genomic DNA, chromosome 8, BAC clone:OJ1112_E06
Table 10 nucleic acid encoding BB polypeptides identified by tBlastn
Sequence Alignments
A. amino acid (SEQ ID NO: 1) and nucleotide (SEQ ID NO: 2) sequences
of DA1 and mutation sites of da1-1, sod1-1, sod1-2 and sod1-3.
The domains predicted by using SMART software are shown.
B. Alignment of UIM motifs among different UIM motif- containing
proteins. UIM motifs were predicted by using SMART software. The
predicted UIM1 (E-value, 6.39e-02) and UIM2 (E-value,7.34e-02)
sequences are shown in A.
C. Alignment of LIM domains among LIM domain- containing proteins.
In the LIM domain, there are seven conserved cysteine residues and
one conserved histidine. The arrangement followed by these conserved
residues is C-x(2)-C-x(16,23)-H-x(2)-[CH]-x(2)-C-x(2)-C-x(16,21)-C-
x(2,3)-[CHD]. The LIM domain (E-value, 3.05e-10) was predicted by
using SMART software and is shown in A.
D. Alignment of DA1 and DAl-related proteins in Arabidopsis. The
conserved regions among DA1 and DARs are in their C-terminal
regions. The da1-1 allele has a single nucleotide G to A transition
in gene Atlgl9270 and is predicted to cause an arginine (R) to
lysine change (K) in a conserved amino acid at position 358. An
asterisk indicates identical amino acid residues in the alignment. A
colon indicates conserved substitutions in the alignment and a
period indicates semi-conserved substitutions in the alignment.
E: Amino acid alignments of da1-like proteins. Full length amino
acid sequences of da1-1ike proteins from Physcomitrella patens (Pp),
Selaginella moellendorffi (Sm), Brassica rapa (Br), Arabidopsis
thaliana (At), Brachypodium distachyon (Bd) and Oryza sativa (Os)
were aligned with default setting ClustalW
(http://www.ebi.ac.uk/Tools/clustalw2/index.html), and edited
display settings in VectorNTi. The red arrow shows the mutation in da1-1 allele. DAL stands for da1-like.
WE CLAIM:
1. A method of altering the phenotype of a plant comprising;
expressing a nucleic acid encoding a dominant-negative DA
polypeptide within cells of said plant.
2. A method according to claim 1 comprising reducing or
abolishing expression of a BB polypeptide within cells of said
plant.
3. A method of producing a plant with an altered phenotype
comprising:
incorporating a heterologous nucleic acid which encodes a
dominant-negative DA polypeptide into a plant cell by means of
transformation, and;
regenerating the plant from one or more transformed cells.
4. A method according to claim 4 further comprising incorporating
a heterologous nucleic acid which expresses a suppressor nucleic
acid which reduces expression of a BB polypeptide into said plant
cell by means of transformation.
5. A method according to any one of claims 1 to 4 wherein the
altered phenotype includes normal fertility and one or more of
increased life-span, increased organ size and increased seed size
relative to control plants.
6. A method according to any one of claims 1 to 5 wherein the
dominant-negative DA polypeptide comprises a UIM1 domain of SEQ ID
NO: 3 and a UIM2 domain of SEQ ID NO: 4.
7. A method according to claim 6 wherein the dominant-negative DA
polypeptide comprises a LIM domain of SEQ ID NO: 5.
8. A method according to claim 6 or 7 wherein the dominant-
negative DA polypeptide comprises a C terminal region having at
least 20% sequence identity to residues 250 to 532 of SEQ ID NO: 1.
9. A method according to any one of claims 1 to 8 wherein the
dominant-negative DA protein comprises a sequence having at least
20% sequence identity to SEQ ID NO: 1.
10. A method according to any one of claims 1 to 9 wherein the
dominant-negative DA protein comprises an R to K mutation at a
position equivalent to position 358 of SEQ ID NO: 1.
11. A method according to claim 2 or claim 4 wherein the BB
polypeptide comprises an amino acid sequence having at least 20%
sequence identity to SEQ ID NO: 9.
12. A method according to any one of claims 1 to 11 wherein
wherein the nucleic acid encoding the dominant negative DA
polypeptide and/or the suppressor nucleic acid is operably linked to
a heterologous promoter.
13. A method according to claim 12 wherein the promoter is a
tissue-specific promoter.
14. A method according to claim 13 wherein the promoter is an
inducible promoter.
15. A method according to any one of claims 12 to 14 wherein the
nucleic acid encoding the dominant negative DA polypeptide and/or
the suppressor nucleic acid is comprised in one or more vectors.
16. A method according to any one of claims 1 to 15 comprising
sexually or asexually propagating or growing off-spring or
descendants of the plant expressing the dominant-negative form of a
DA polypeptide.
17. A method according to any one of claims 1 to 16 wherein the
plant is a higher plant.
18. A method according to claim 17 wherein the plant is an
agricultural plant selected from the group consisting of
Lithospermum erythrorhizon, Taxus spp, tobacco, cucurbits, carrot,
vegetable brassica, melons, capsicums, grape vines, lettuce,
strawberry, oilseed brassica, sugar beet, wheat, barley, maize,
rice, soyabeans, peas, sorghum, sunflower, tomato, potato, pepper,
chrysanthemum, carnation, linseed, hemp and rye.
19. A plant expressing a heterologous nucleic acid encoding a
dominant-negative DA polypeptide within its cells and optionally
having reduced or abolished expression of a BB polypeptide.
20. A plant according to claim 19 which produced by a method
according to claim 3 or claim 4.
21. A plant according to claim 19 or claim 20 which has normal
fertility relative to control plants and one or more of increased
life-span, increased organ size and increased seed size relative to
control plants.
22. A method of altering the phenotype of a plant comprising;
reducing or abolishing the expression of two or more nucleic
acids encoding DA polypeptides in one or more cells of the plant.
23. A method of producing a plant with an altered phenotype
comprising:
reducing or abolishing the expression of two or more nucleic
acids encoding DA polypeptides in a plant cell, and;
regenerating the plant from the plant cell
24. A method according to claim 22 or 23 wherein expression is
abolished by mutating the nucleic acid sequences in the plant cell
which encode the two or more DA proteins and regenerating the plant
from the mutated cell.
25. A method according to claim 22 or 23 wherein expression is
reduced or abolished by expressing two or more heterologous nucleic
acids which suppress expression of said two or more nucleic acids
within cells of said plant.
26. A method according to any one of claims 22 to 25 wherein one
or more DA polypeptides comprise a UIM1 domain of SEQ ID NO: 3, UIM2
domain of SEQ ID NO: 4, a LIM domain of SEQ ID NO: 5, a C terminal
region having at least 20% sequence identity to residues 250 to 532
of SEQ ID NO: 1 and an R residue at a position equivalent to
position 358 of SEQ ID NO: 1.
27. A method according to any one of claims 22 to 26 wherein the
altered phenotype is characterised by normal fertility relative to
control plants and one or more of increased life-span, increased
organ size and increased seed size relative to control plants.
28. A method of altering the phenotype of a plant comprising;
expressing a nucleic acid encoding a DA polypeptide within
cells of said plant relative to control plants.
29. A method according to claim 28 wherein the altered phenotype
is characterised by normal fertility and one or more of reduced
life-span, reduced organ size and reduced seed size relative to
control plants.
30. A method according to claim 28 or claim 29 wherein the DA
polypeptides comprises a UIM1 domain of SEQ ID NO: 3, UIM2 domain of
SEQ ID NO: 4, a LIM domain of SEQ ID NO: 5, a C terminal region
having at least 20% sequence identity to residues 250 to 532 of SEQ
ID NO: 1 and an R residue at a position equivalent to position 358
of SEQ ID NO: 1.
31. A method of identifying a dominant negative DA polypeptide
comprising;
providing an isolated nucleic acid encoding a DA polypeptide,
introducing one or more mutations into the nucleic acid,
incorporating the nucleic acid into a plant cell by means of
transformation;
regenerating the plant from one or more transformed cells and,
identifying the phenotype of the regenerated plant,
wherein the altered phenotype which includes normal fertility
and one or more of reduced life-span, reduced organ size and reduced
seed size relative to control plants is indicative that the mutated
DA polypeptide is a negative plant growth regulator.
32. A method of producing a dominant-negative DA polypeptide
comprising;
providing a nucleic acid sequence encoding a plant DA
polypeptide,
identifying an R residue in the encoded plant DA polypeptide
at a position equivalent to position 358 of SEQ ID NO: 1 and
mutating the nucleic acid to alter said R residue in the
encoded plant DA polypeptide,
the mutant nucleic acid sequence encoding a dominant negative
DA polypeptide.
33. An isolated DA1 or DAR protein.
34. The isolated DA1 or DAR protein according to claim 33 which
comprises at least one UIM and at least one LIM domain and the
extended protein homology found in the C terminal regions of DA1 and
DAR proteins that contain the conserved arginine- at position 358 in
DA1.
35. The isolated DA1 or DAR gene according to claim 34 which is
AT1G19270, SGN-U317073, SGN-U277808, SGN-U325242, AT4G36860, SGN-
U209255, AB082378.1, AT2G39830, CAN69394.1, OS03G16090,
9234.M000024, 29235.M000021, AT5G66620, AT5G66630, AT5G66610,
AT5G66640, AT5G17890, SGN-U320806, AB096533.1, CAL53532.1,
OS06G08400, SGN-U328968, OS03G42820, OS12G40490, or a homologue,
operative variant or portion thereof, wherein an operative portion
or variant is a molecule which has the ability to either limit the
duration of proliferative growth or the ability to interfere with
the limitation of duration of proliferative growth and increase seed
and or organ size.
36. The isolated DA1 or DAR according to any one of claims 33 to
35 comprising a mutation of Arg at position 358 to a Lys at that
position or a position equivalent to that position of said DA1 or
DAR.
37. A plant comprising the DA1 or DAR according to claim 36.
38. An isolated nucleic acid encoding a protein according to any
one of claims 33 to 36.
39. A vector comprising the nucleic acid according to claim 38
operatively linked to a promoter.
40. The vector according to claim 39 further comprising nucleic
acid sequences encoding at least one eod sequence.
41. A method for increasing the size of plant organs, seeds or
both without adversely affecting fertility which comprises
expressing within a plant a variant of DA1 or DAR which interferes
with the limitation of duration of proliferative growth and increase
seed and or organ size.
42. The method according to claim 41 wherein said variant of DA1
or DAR comprises AT1G19270, SGN-U317073, SGN-U277808, SGN-U325242,
AT4G36860, SGN-U209255, AB082378.1, AT2G39830, CAN69394.1,
OS03G16090, 9234.M000024, 29235.M000021, AT5G66620, AT5G66630,
AT5G66610, AT5G66640, AT5G17890, SGN-U320806, AB096533.1,
CAL53532.1, OS06G08400, SGN-U328968, OS03G42820, OS12G40490, or a
homologue, operative variant or portion thereof, wherein said
variant interferes with the limitation of duration of proliferative
growth and increase seed and or organ size.
43. The method according to claim 42 wherein said variant
comprises a mutation equivalent to DA1R358K.
44. A method of prolonging the growth period in a plant which
comprises expressing DA1R358K within said plant.
45. The method according to any one of claims 41 to 44 further
comprising genetic combinations with mutations that disrupt the
functions of EOD genes or a homologue, operative variant or portion
thereof wherein said mutant combinations interfere with the
limitation of duration of proliferative growth and increase seed and
or organ size.
46. The method according to any one of claims 41 to 44 comprising
genetic combinations with transgenes expressing an RNA interference
construct(s) that reduce expression of EOD1 or a homologue,
operative variant or portion thereof wherein said mutant
combinations interfere with the limitation of duration of
proliferative growth and increase seed and or organ size,
47. A plant comprising a knockout in at least a first gene
encoding DA1 and in at least one second gene encoding a DAR.
48. A plant comprising a transgene expressing an RNA interference
construct(s) that reduce expression of DA1, DAR, or a homologue,
operative variant or portion thereof, wherein said variant
interferes with the limitation of duration of proliferative growth
and increase seed and or organ size.
49. A plant containing a transgene expressing an RNA interference
construct of any type that reduces RNA levels of DA1, DAR, or a
homologue, operative variant or portion thereof, in specific organs
and tissues wherein said construct interferes with the limitation of
proliferative growth increase seed and or organ size in specific
tissues and organs
50. A plant according to any one of claims 47 to 49 further
comprising genetic combinations with mutations that disrupt the
functions of EOD genes or a homologue, operative variant or portion
thereof.

This invention relates to the identification of a regulator protein
(termed DA) which controls the size of plant seeds and organs in
Arabidopsis and other plants. Manipulation of DA protein expression
may useful, for example, in improving crop yield and increasing
plant biomass.