Methods of Modulating Seed and Organ Size in Plants
Field of Invention
This invention relates to methods of altering the size of the seeds
and organs of plants, for example to improve plant yield.
Background of Invention
The size of seeds and organs is an agronomically and ecologically
important trait that is under genetic control (Alonso-Blanco, C .
PNAS USA 96, 4710-7 (1999); Song, X.J. Nat Genet 39, 623-30 (2007);
Weiss, J . Int J Dev Biol 49, 513-25 (2005); Dinneny, J.R.
Development 131, 1101-10 (2004); Disch, S . Curr Biol 16, 272-9
(2006) ;Science 289, 85-8 (2000) ;Horiguchi, G . Plant J 43, 68-78
(2005) ; Hu, Y Plant J 47, 1-9 (2006); Hu, Y .Plant Cell 15, 1951-61
(2003); Krizek, B.A. Dev Genet 25, 224-36 (1999) ;Mizukami, Y . PNAS
USA 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. PNAS USA 103, 13233-43 (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,
litrle 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 locus (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. PNAS USA 102, 3123-3128 (2005); Jofuku, K.D. PNAS
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 . . PNAS 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)).
Several factors involved in ubiquitin-related activities have been
known to influence seed size. A growth-restricting factor, DAI, is a
ubiquitin receptor and contains two ubiquitin interaction motifs
(UIMs) that bind ubiquitin in vitro, and dal-1 mutant forms large
seeds by influencing the maternal integuments of ovules (Li et al.,
2008). Mutations in an enhancer of dal-1 {EOD1) , which encodes the
E3 ubiquitin ligase BIG BROTHER (BB) (Disch et al., 2006; Li et al .,
2008), synergistically enhance the seed size phenotype of dal-1,
indicating that DAI acts synergistically with EODl/BB to control
seed size.
Identification of further 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 unexpectedly discovered that disruption
of the LIM domain and/or the LIM-like domain in plant DAI proteins
does not abolish DA homodimerisation or activity but instead confers
a dominant-negative phenotype.
An aspect of the invention provides a method of increasing the yield
of a plant or enhancing a yield-related trait in a plant; comprising
expressing a DAI protein having an inactivated LIM domain or LIMlike
domain within cells of said plant.
The DAI protein may comprise one or more mutations relative to the
wild-type sequence that disrupt or inactivate the LIM domain or LIMlike
domain of the DAI protein.
Expression of a DAI protein with a disrupted or inactivated LIM
domain or LIM-like domain enhances one or more yield related traits
and increases the yield of the plant.
The DAI protein having an inactivated LIM domain or LIM-like domain
may be expressed from a heterologous nucleic acid coding sequence in
one or more cells of the plant or may be expressed from an
endogenous nucleic acid coding sequence in one or more cells of the
plant ,
Another aspect of the invention provides a method of producing a
plant with an increased yield and/or one or more enhanced yieldrelated
traits comprising:
introducing into a plant cell a heterologous nucleic acid
which encodes a DAI protein having an inactivated LIM domain or LIMlike
domain, or
introducing a mutation into the nucleotide sequence of a plant
cell which encodes the DAI protein, such that the LIM domain or LIMlike
domain of the DAI protein is inactivated, and
regenerating the plant from the plant cell.
Another aspect of the invention provides a plant cell comprising a
heterologous nucleic acid encoding a DAI protein having an
inactivated LIM domain or LIM-like domain.
Another aspect of the invention provides a plant comprising one or
more plant cells that comprise a heterologous nucleic acid encoding
a DAI protein having an inactivated L M domain or LIM-like domain.
The plant may display increased yield or an enhanced a yield-related
trait relative to controls.
Brief Description of Drawings
Figure 1 shows the spacing and identity of the eight zinc binding
residues (1-8) in the LIM domain based on an analysis of 135 human
LIM sequences. Infrequently observed patterns (<10%) of conserved
sequence and topography of the LIM domain.
Figure 2 shows the topology of Zn coordination of a LIM domain.
Purple circles indicate Zn binding residues. Semi conserved
aliphatic/bulky residues are shown as green, non-conserved residues
with invariant spacing are red. Dashed yellow circles indicate a
variable number of residues (X) that are possible within the space.
Figure 3 shows in vitro immunoprecipitation that shows the binding
of dallim8 to wild-type DAI. E . coli expressed GST-tagged bait
proteins were incubated with E . coli expressed FLAG-tagged prey
proteins before purification on glutathione sepharose beads and
immunoblotting for GST and FLAG. FLAG-DAI and FLAG-dallim8 copurified
with GST-DAl and GST-dallim8 (lanes 5,6,8,9) but not with
the negative control GST-GUS (lanes 2,3); revealing that mutating
the LIM domain in DAI is not sufficient to abolish the physical
interaction between DAI proteins.
Figure 4 shows the effect of the lim8 mutation on seed size in a Col
background.
Detailed Description of Embodiments of the Invention
This invention relates the expression in plants of DAI proteins in
which the LIM or LIM-like domain is disrupted or inactivated
(collectively termed LIM-disrupted DAI proteins herein) . This may be
useful in altering plant traits which affect yield, such as seed and
organ size.
DAI is a plant ubiquitin receptor that is described in detail in Li
et al (2008), Wang, et al (2012) and WO2009/047525.
DAI proteins are characterised by the presence of a LIM domain, a
LIM-like domain, a conserved C terminal domain and one or more UIM
domains .
A LIM domain comprises two Zn finger motifs and may have the amino
acid sequence (SEQ ID NO:l);
C(X) C(X)i6-23 H /C) (X )2/, (C/H/E) (X) 2C (X) C (X) 14-21 (C/H) (X) 2/1/3 (C/H/D/E) X
where X is any amino acid and Zn coordinating residues are
underlined.
The Z coordinating residues in the LIM domain may be C , H , D or E ,
preferably C .
In some preferred embodiments, a LIM domain may comprise CXXC,
HXXCXXCXXC and HxxC motifs, where X is any amino acid. For example,
a LIM domain may comprise the amino acid sequence (SEQ ID NO :2)
C(X) C[X) 1 -23 ( ) (X (X) C(X) C(X)u-2iH(X) CX
where X is any amino acid and Zn coordinating residues are
underlined
In some emboiments, a LIM domain nay comprise the amino acid
sequence of the AtDAl LIM domain;
CAGCNMEIGHGRFLNCLNSLWHPECFRCYGCSQPISEYEFSTSGNYPFHKACY
(SEQ ID NO: 3 ; Zn coordinating residues are underlined)
Other LIM domains include the L domain of an DAI amino acid
sequence shown in Table 1 (dashed box) , for example residues 141 to
193 of SEQ ID NO: 4 (Si_GI-514815267 .pro) , residues 123 to 175 of
SEQ ID NO: 5 (Bd_GI-357157184 .pro > , residues 155 to 207 of SEQ ID
NO: (Br_DAlb.pro) , residues 172 to 224 of SEQ ID NO: 7
(Br_DAla.pro) , residues 172 to 224 of SEQ ID NO: 8 (At_GI-
15221983. pro) , residues 117 to 169 of SEQ ID NO: 9 (Tc_GI-
508722773 .pro) , residues 117 to 169 of SEQ ID NO: 10 (Gm_ GI-
356564241 .pro) , residues 121 to 173 of SEQ ID NO: 11 (G G -
356552145 .pro) , residues 119 to 171 of SEQ ID NO: 12 (Vv_ GI-
302142429 -pro) , residues 122 to 174 of SEQ ID NO: 13 (Vv_ GI-
359492104 p ro ), residues 125 to 177 of SEQ ID NO: 14 (Sl_ GI-
460385048 -pro) , residues 516 to 563 of SEQ ID NO: 15 (Os__GI-
218197709 -pro) , residues 124 t 17 of SEQ ID NO: 16 (Os_ GI-
115466772 .pro) , residues 150 to 202 of SEQ ID NO: 17 (Bd__GI-
357160893 -pro) , residues 132 to 184 of SEQ ID NO: 18 (Bd__GI-
357164660 p ro ), residues 124 to 176 of SEQ ID NO: 19 (Sb__GI-
242092232 .pro) , residues 147 to 199 of SEQ ID NO: 20 (Zm__GI-
212275448 .pro) , residues 190 to 242 of SEQ ID NO: 21 (At_
240256211 .pro) , residues 162 to 214 of SEQ ID NO: 22 (At__GI-
145360806. ro ), residues 1240 to 1291 o f SEQ ID NO: 23 (At_GI-
22326876. ro), residues 80 to 122 of SEQ ID NO: 24 (At_GI-
30698242. pro) , residues 347 to 402 of SEQ ID NO: 25 (At_GI-
30698240. pro) , residues 286 to 341 of SEQ ID NO: 26 (At_GI-
15240018 .pro) or residues 202 to 252 of SEQ ID NO: 27 (At_GI-
334188680. ro ) .
LIM domain sequences may be identified using standard sequence
analysis techniques (e.g. Simple Modular Architecture Research Tool
(SMART) ; EMBL Heidelberg, DE) .
A LIM-like domain comprises two Zn finger motifs and may comprise
CXXC, HXXXXXXXCXXH and CxxC motifs, where X is any amino acid. For
example, a LIM-like domain may comprise the amino acid sequence (SEQ
ID NO:28) ;
CX2CXl6-23HX7_CX2HX7CX2CXl9CX C
where X is any amino acid, Zn coordinating residues are solid
underlined and putative Zn coordinating residues are dotted
underlined
Preferably, a LIM-like domain may comprise the amino acid sequence
(SEQ ID NO: 29) ;
CXVCXi6-23HPFWX3YCPX_HX7CCSCERXEX YX2LXDXRXLCXXC
where X is any amino acid, Zn coordinating residues are solid
underlined and putative Zn coordinating residues are dotted
underlined.
More preferably, a LIM-like domain may comprise the amino acid
sequence (SEQ ID NO: 30) ;
C(D/E/Y/H) VCXX(F/K) (I/K/F) (P/S/Absent) (T/R/V/Absent )(N/T/absent) XX (G/
Absent) (L/I/M/G) (R/K/I) (E/G/K/T) (Y/F) (R/H/S/N/K) (A/C/E/I/N) HPF (Q/E)
(K/T/R) YCP (F/V/I/S/T) H (E/D) D (G/K/R/S/A) T (P/T/A) (R/K) CCSCER(M/L) E (P/S
/H)X YX2LXD (G/F/N)R(R/K/S/W) LC (L/R/V) (E/K)C
where X is any amino acid, Zn coordinating residues are solid
underlined and putative Zn coordinating residues are dotted
underlined .
In some embodiments, a LIM-like domain may comprise the amino acid
sequence of the AtDAI LIM-like domain;
CDVCSHFI PTNHAGLIEYRAHPFWVQKYCPSHEHDATPRCCSCERMEPRNTRYVELNDGRKLCLEC (SEQ D
NO: 31)
Other LIM-like domains include the LIM domain of an DAI amino acid
sequence shown in Table 1 (solid box) , for example residues 200 to
266 of SEQ ID NO: 4 (Si_GI -514815267 .pro ), residues 182 to 248 of
SEQ ID NO: 5 (Bd_GI -357157184 .pro ) , residues 214 to 280 of SEQ ID
NO: 6 (Br_DAlb.pro) , residues 231 to 297 of SEQ ID NO: 7
(Br_DAla.pro) , residues 231 to 297 of SEQ ID NO: 8 (At_GI15221983.
ro ), residues 176 to 242 of SEQ ID NO: 9 (Tc_GI-
508722773 pro) residues 176 to 242 of SEQ ID NO: 10 (Gm_ GI-
356564241 .pro) residues 180 to 246 of SEQ ID NO: 11 (Gm__GI-
356552145 pro) residues 178 to 244 of SEQ ID NO: 12 (Vv_ GI-
302142429 .pro) residues 181 to 247 of SEQ ID NO: 13 (Vv_ GI-
359492104 .pro) residues 184 to 250 of SEQ ID NO: 14 (Sl__GI-
460385048 .pro) residues 575 to 641 of SEQ ID NO: 15 (Os__GI-
218197709 .pro) , residues 183 to 149 of SEQ ID NO: 16 (Os__GI-
115466772 .pro) residues 209 to 275 of SEQ ID NO: 17 (Bd__GI-
357160893 .pro) residues 191 to 257 of SEQ ID NO: 18 (Bd__GI-
357164660 .pro) residues 183 to 249 of SEQ ID NO: 19 (Sb__GI-
242092232 .pro) residues 206 to 272 of SEQ ID NO: 20 (Zm_ GI-
212275448 -pro) residues 249 to 315 of SEQ ID NO: 21 (At__GI-
240256211 -pro) residues 221 to 28^ of SEQ ID NO: 22 (At_ GI-
145360806 -pro) residues 129£ to 1363 4SEEQ or EK (X) 8R (X) SEQ motif and a HEMMH motif,
a UIM domain of SEQ ID NO: 35, and
a UIM domain of SEQ ID NO: 36.
A DAI protein may comprise an amino acid sequence of a plant DAI
protein shown in Table 1 (SEQ ID NOS: 4 to 27) or may be an allele
or variant of one of these sequences which has DAI activity.
For example, a DAI protein may comprise the amino acid sequence of
AtDAl, AtDARl, AtDAR2, AtDAR3, AtDAR4, AtDAR5, AtDAR6, AtDAR7,
BrDAla, BrDAlb, BrDARl, BrDAR2, BrDAR3-7, BrDALl, BrDAL2, BrDAL3,
OsDAl, OSDAR2, OsDAL3, OSDAL5, PpDALl, PpDAL2, PpDAL3, PpDAL4,
PpDAL5, PpDAL6, PpDAL7, PpDAL8, SmDALl , SmDAL2 or Z DA (ACR35367.1
GI:238008664) , preferably AtDAl, AtDARl BrDAla, BrDAlb, OsDAl or
ZmDAl or an allele or variant of one of these sequences.
In some preferred embodiments, a DAI protein may comprise the amino
acid sequence of AtDAl (SEQ ID NO: 8 ; AT1G19270; NP_173361.1 G :
15221983) or may be an allele or variant of this sequence which has
DAI activity.
Other DAI protein sequences which include the characteristic
features set out above and encoding DAI nucleic acid sequences may
be identified using standard sequence analysis tools in any plant
species of interest.
A DAI protein in a plant species of interest may have an amino acid
sequence which is a variant of a DAI protein reference amino acid
sequence set out herein.
A DA protein which is a homologue or variant of a reference plant
DAI sequence, such as any one of SEQ ID NOS : 4-27, 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.
Particular amino acid sequence variants that occur in a plant
species may differ from a reference sequence set out herein 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 DAI polypeptide which is a variant of AtDAl
sequence of any one of SEQ NOS: 4 to 27 may comprise a LIM domain
having the sequence of SEQ ID NO: 3 and a LIM-like domain having the
sequence of SEQ ID NO: 31.
A nucleic acid encoding a DAI protein may comprise a nucleotide
sequence set out in a database entry selected from the group
consisting of NM_101785. 3 GI:42562:170 (AtDAl); NM_001057237 .1
GI: 115454202 (OsDAl) ; BT085014.1 GI : 238008663 (ZmDAl) or may be an
allele or variant of one of these sequences which encodes an active
DAI protein.
In some preferred embodiments, a nucleic acid encoding a DAI protein
may comprise the nucleotide sequence cf AtDAl (NM_101785.3 GI :
42562170), ZmDAl (BT085014.1 G : 238008663), OsDAl (NM_001057237 .1
G :115454202) or may be an allele or variant of any one of these
sequences which encodes a protein with DAI activity.
A nucleic acid that encodes a DAI protein in a plant species of
interest may have a nucleotide sequence which is a variant of a DAI
reference nucleotide sequence set out herein.
DAI polypeptides and encoding nucleic acids may be identified in
plant species, in particular crop plants, such as wheat, barley,
maize, rice, and another agricultural plants, using routine sequence
analysis techniques.
For example, variant nucleotide sequence may be a homologue of a
reference DAI sequence set out herein, and may differ from the
reference DAI nucleotide sequence by one or more of addition,
insertion, deletion or substitution of one or more nucleotides in
the nucleic acid, for example 2 3 , 4 , 5-10, 10-20 20-30, 30-50, or
more than 50, leading to the addition, insertion, deletion or
substitution of one or more amino acids in the encoded protein. Of
course, changes to the nucleic acid that make no difference to the
encoded amino acid sequence are included. A nucleic acid encoding a
DAI protein may comprise a sequence having at least 20% or at least
30% sequence identity with the reference nucleic acid sequence,
preferably at least 40%, at least 50%, at least 60%, at least 65%,
at least 70%, at least 80%, at least 90%, at least 95% or at least
98%. Sequence identity is described herein.
Sequence identity is 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), FAS A (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.
A DAI nucleotide sequence which is a variant of a reference DAI
nucleic acid sequence set out herein, may selectively hybridise
under stringent conditions with this reference 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 a2HP0 , pH 7.2, 6.5% SDS, 10% dextran sulfate and a final wash
at 55°C in 0 .IX 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 Na2HP0 , 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 65°C or at 50-60°C in lx SSC/0.1%
SDS, as required.
DAI proteins and encoding nucleic acids may be identified in plant
species, in particular crop plants, such as wheat, barley, maize,
rice, and another agricultural plants, using routine sequence
analysis techniques and/or comparison with the reference sequences
set out herein.
The LIM domain, the LIM-like domain or both the LIM domain and the
LIM-like domain of a DAI protein for use as described herein may be
inactivated or disrupted ("LIM disrupted-DAl protein") .
LIM domains and LIM-like domains are described in detail above and
may be identified within any DAI protein using standard sequence
analysis techniques.
A DAI protein with an inactivated or disrupted LIM domain or LIMlike
domain may display aberrant, for example increased or activated,
peptidase activity. For example, inactivation or disruption of the
LIM domain or LIM-like domain may reduce or prevent the domain from
interacting with the C terminal region of the DAI protein and
inhibiting DAI peptidase activity.
In some embodiments, a DAI protein with an inactivated or disrupted
LIM domain or LIM-like domain may be display reduced stability in a
plant cell following ubiquitinylation compared to wild-type DAI
protein.
A disrupted or inactivated LIM domain or LIM-like domain may be
unable to coordinate Zn or form Zn finger motifs, such that the
function of the domain is abolished i.e. the disrupted LIM or LIMlike
domain is unable to mediate protein :protein interactions. For
example, a disrupted LIM domain or LIM-like domain may be unable to
interact intramolecularly with the C terminal region of the DAI
protein to inhibit peptidase activity.
An inactivated or disrupted LIM domain or LIM-like domain may
comprise a sequence alteration or mutation which abolishes one or
more Zn finger motifs in the LIM or LIM-like domain.
The amino acid sequence of a DAI protein may be altered or mutated
by insertion, substitution or deletion of one or more amino acids
relative to the wild-type amino acid sequence in order to inactivate
the LIM domain or LIM-like domain. For example, 1 , 2 , 3 , 4 , 5 , 6 , 7 ,
8 , 9 or 10 or more amino acids may be altered, for example deleted
or substituted, relative to the wild-type amino acid sequence. In
some embodiments, 1 to 30, 1 to 20 or 1 to 10 residues may be
altered.
Single amino acid substitutions within LIM domains and LIM-like
domains are sufficient to elicit a LIM knockout phenotype. LIM
domains, for example, may be inactivated by mutations in the Zn
coordinating residues or other residues within the LIM domain
(Mcintosh et al (1998) Am J Human Genet 63 1651-16581; Clough et al
(1999) Human mutation 14 459-465; Hamlington et al (2001) Human
mutation 18 458-464; Taira et al., Nature 1994 372, 677-9; Agulnick
et al., Nature 1996 384, 270-2). LIM-like domains may also be
inactivated by mutations in the Zn coordinating residues (i.e.
conserved Cys residues), or other residues within the LIM-like
domain (Yang et al The Plant Journal, (2010), 63, 283-296).
Suitable inactivating or disrupting mutations are preferably within
the LIM domain or LIM-like domain or adjacent thereto.
An inactivated or disrupted LIM domain or LIM-like domain may
comprise a mutation of one or more Zn coordinating residues, or
putative Zn coordinating residues, for example a cysteine or
histidine residue in a CxxC or CXXH context, and/or a mutation of
one or more non-Zn coordinating amino acid residues.
An inactivated or disrupted LIM domain may comprise a mutation at
one or more of the first, second, third, fourth, fifth, sixth,
seventh and eighth Zn coordinating residues in the LIM domain as
shown in SEQ ID NO: 1 or SEQ ID NO: 2 above. For example, a
inactivated or disrupted LIM domain may comprise a mutation at one
or more of the cysteine residues in the CXXC motifs or a cysteine or
histidine residue in the HXXC motif, for example the
cysteine/histidine residues shown in positions 1 , 4 , 22, 25, 28,31,
49 and 52 of SEQ ID NO: 3 and underlined in SEQ ID NOS 1 and 2
above. The LIM domain of said DAI protein may comprise a mutation
at one or more of the underlined residues of the DAI LIM domain
shown above, preferably C141, C144, H162, C165, C168, C171, H189 and
C192 of the DAI sequence of SEQ ID NO: 4 , C123, C126, H144, C147,
C150, C153, H171 and C174 of the DAI sequence of SEQ ID NO: 5 , C155,
C158, H176, C179, C182, C185, H203 and C206 of the DAI sequence of
SEQ ID NO: 6 , C172, C175, H193, C196, C199, C202, H220 and C223 of
the DAI sequence of SEQ ID NO: 7 , C172, C175, H193, C196, C199,
C202, H220 and C223 of the AtDAI sequence of SEQ ID NO: 8 , C117,
C120, H138, C141, C144, C147, H165 and C168 of the DAI sequence of
SEQ ID NO: 9 , C177, C180, H198, C201, C204, C207, H225 and C228 of
the DAI sequence of SEQ ID NO: 10, C121, C124, H142, C145, C148,
C151, H169 and C172 of the DAI sequence of SEQ ID NO: 11, C119,
C122, H140, C143, C146, C149, H167 and C170 of the DAI sequence of
SEQ ID NO: 12, C122, C125, H143, C146, C149, C152, H170 and C173 of
the DAI sequence of SEQ ID NO: 13, C125, C128, H146, C149, C152,
C155, H173 and C176 of the DAI sequence of SEQ ID NO: 14, C516,
C519, H537, C540, C543, C546, H564 and C567 of the DAI sequence of
SEQ ID NO: 15, C124, C127, H145, C148, C151, C154, H172 and C175 of
the DAI sequence of SEQ ID NO: 16, C150, C153, H171, C174, C177,
C180, H198 and C201 of the DAI sequence of SEQ ID NO: 17, C132,
C135, H153, C156, C159, C162, H180 and C183 of the DAI sequence of
SEQ ID NO: 18, C124, C127, H145, C148, C151, C154, H172 and C175 of
the DAI sequence of SEQ ID NO: 19, C147, C150, H168, C172, C175,
C178, H196 and C199 of the DAl sequence of SEQ ID NO: 20, C190,
C193, H211, C204, C207, C210, H228 and C231 of the DAl sequence of
SEQ ID NO: 21, C162, C165, H183, C186, C189, C192, H210 and C213 of
the DAl sequence of SEQ ID NO: 22, C1240, C1243, H1261, C1264,
C1267, C1270, H1287 and C1290 of the DAl sequence of SEQ ID NO: 23,
C347, C350, H368, C371, C374, C377, H398 and C401 of the DAl
sequence of SEQ ID NO: 25, C286, C289, H307, C310, C313, C316, H337
and C340 of the DAl sequence of SEQ ID NO: 26, C201, C204, H222,
C225, C228, C231, H248 and C251 of the DAl sequence of SEQ ID NO:
27, or the equivalent cysteine residues in other DAl protein
sequences .
For example the LIM disrupted DAl protein may have a C to Y , C to G
or other substitution at one or more of these positions.
Zn coordinating residues within the LIM domain of a DAl protein may
be identified by standard sequence analysis. Cysteine and histidine
residues equivalent to C172, C175, H193, C196, C199, C202, H220 and
C223 of SEQ ID NO: 8 are sequence residues in the same sequence
context in a different DAI protein sequence and may be identified by
standard sequence analysis, as shown in Table 1 .
An inactivated or disrupted LI domain may comprise a mutation at
one or more non-Zn coordinating residues in the LIM domain as shown
in SEQ ID NO:l or SEQ ID N0:2 above. A non-Zn coordinating residue
may be located within 4 residues of a Zn coordinating residue in the
LIM domain sequence or may be located 4 or more residues away from a
Zn coordinating residue.
An inactivated or disrupted LIM-like domain may comprise a mutation
at one or more of the first, second, third, fourth, fifth, sixth,
seventh and eighth Zn coordinating residues or putative Zn
coordinating residues in the LIM-like domain as shown in any one of
SEQ ID NOS: 28 to 31 above. For example, a inactivated or disrupted
LIM-like domain may comprise a mutation at one or more of the
cysteine residues in the CXXC motifs or a cysteine or histidine
residue in the CXXH motif, for example the cysteine/histidine
residues shown in positions 1 , 4 , 29, 32, 40, 43, 63 or 66 of SEQ ID
NO: 31 and underlined in SEQ ID NOS 28 to 31 above. Two of the
three putative Zn coordinating residues H252, C260, H263 in the LIMlike
domain are responisible for Zn coordination (i.e. H252 and
C260; H252 and H263; or C260 and H263) . The LIM-like domain of said
DAI protein may comprise a mutation at one or more of the underlined
residues of the AtDAI LIM-like domain shown above, preferably C232,
C235, H252, C260, H263, C271, C274, C294 and/or C297 of the AtDAI
sequence of SEQ ID NO: 8 , or the equivalent cysteine residues in
other DAI protein sequences. For example the LIM disrupted DAI
protein may have a C to Y , C to G or other substitution at one or
more of these positions.
Cysteine residues equivalent to C232, C235, H252, C260, H263, C271,
C274, C294 and C297 of SEQ ID NO: 8 are sequence residues in the
same sequence context in a different DAI protein sequence and may be
identified by standard sequence analysis, as shown in Table 1 .
An inactivated or disrupted LIM-like domain may comprise a mutation
at one or more residues in the LIM-like domain other than conserved
cysteine or histidine residues as shown in SEQ ID NO: 28 to SEQ ID
NO: 31 above. Suitable residues may be located within 4 residues of a
conserved cysteine or histidine residue in the LIM-like domain
sequence or may be located 4 or more residues away from a conserved
cysteine or histidine residue.
Some preferred mutations include the conversion of a Zn coordinating
residue in a LIM or LIM-like domain, such as cysteine or histidine,
to a neutral amino acid, such as glycine.
Other mutations that disrupt Zn finger motifs and are suitable for
abolishing LIM or LIM-like function in a DAl protein will be readily
apparent to the skilled person. Unlike mutations in other domains
within the DAl protein, LIM domain and LIM-like domain mutations
destabilise the DAl protein in the presence of its interacting
partner EODl in the plant cell. Suitable LIM domain and LIM-like
domain mutations may therefore be identified by determining the
stability the mutant DAl protein in the presence of EODl using
standard experimental techniques. Reduce stability relative to the
wild-type DAl is indicative that a mutation disrupts the LIM or LIMlike
domain.
A LIM-disrupted DAl protein as described herein may comprise a
conserved R residue located at a position in the DAl amino acid
sequence which is equivalent to position 358 of SEQ ID NO: 8 of A .
thaliana DAl, position 333 of SEQ ID NO: 8 of the Z . mays DAl or the
equivalent position in another DAl amino acid sequence, for example
a DAl sequence of Table 1 (conserved R residue shown by arrow) . The
conserved R residue that is located at a position in a DAl amino
acid sequence which is equivalent to position 358 of SEQ ID NO: 8 of
A . thaliana DAl or position 333 of the Z . mays DAl of SEQ ID NO: 20
is located at the position within the DAl amino acid sequence which
corresponds to R333 of SEQ ID NO: 20 and R358 of SEQ ID NO: 8 i.e. it
is in the same position relative to to the other motifs and domains
of the DAl protein. The conserved R residue is located between the
L domain and the HE H (SEQ ID NO: 32) peptidase motif of the C
terminal region and is completely conserved in the same sequence
context in DAI proteins. The conserved R residue may be contained in
a EK (X 8R(X) 4SEEQ (SEQ ID NO: 33) or EK (X) gR (X) ,SEQ (SEQ ID NO: 34)
motif within the C terminal region.
The data herein shows that the LIM domain and the LIM-like domain do
not mediate DAI homodimersation and a LIM disrupted-DAl protein
retains the ability to bind to wild-type DAI.
Expression of a LIM-disrupted DAI protein in one or more cells of a
plant reduces DAI activity in the cells and enhances yield-related
plant traits, such as seed or organ size (see for example Li et al
(2008); WO2009/047525; Wang et al 2012) thereby increasing plant
yield. A plant expressing a LIM-disrupted DAI protein may have a
dal-1 or a dal-1 like phenotype.
In some embodiments, a LIM-disrupted DAI protein may be expressed
from heterologous nucleic acid in the one or more plant cells.
The LIM-disrupted DAI protein may be expressed in one or more cells
of a plant by any convenient technique and suitable techniques are
well-known in the art.
Nucleic acid encoding the LIM-disrupted DAI protein may be
recombinantly 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).
Nucleic acids provided may 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 .
"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 .
Nucleic acid encoding the LIM-disrupted DAI protein 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 nucleic acid encoding the LIM-disrupted DAI protein 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 Agrobactarium 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, chlorsulf uron, methotrexate, gentamycin,
spectinomycin, imidazolinones, glyphosate and d-amino acids.
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.
It is desirable to use a construct and transformation method which
enhances expression of the nucleic acid encoding the LIM or LIM-like
disrupted DAI protein. Integration of a single copy of the gene
into the genome of the plant cell may b e beneficial to minimize gene
silencing effects. Likewise, control o f 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 plant species. 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) 5¾eor 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, 1B9-200; 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-434 616) , 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 raicroparticles (EP-A-486234)
or irdcroprojectile bombardment to induce wounding followed by cocultivation
with Agrobacterium (EP-A-48 6233) .
Following transformation, a plant ay 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.
Following transformation, a plant cell that expresses the L -
disrupted DAI protein may be identified and/or selected. A plant may
be regenerated from the plant cell.
In other embodiments, a mutation may be introduced into a nucleic
acid sequence within the genome of a plant cell which encodes a DAI
protein, such that the nucleic acid encodes a LIM-disrupted DAI
protein. For example, a mutation nay be introduce into the sequence
encoding the LIM domain or LIM-like domain of the DAI protein. A
plant may then be regenerated from the mutated cell.
The nucleic acid encoding the DAI protein may be mutated by
insertion, substitution or deletion of one or more nucleotides
relative to the wild-type nucleotide sequence. For example, 1,2, 3 ,
4,5,6, 7 , 8 , 9 or 10 or more nucleotides may be altered relative to
the wild-type nucleotide sequence n order to inactivated the
encoded LIM or LIM-like domain. The mutations inactivate or knock
out the LIM domain and/or the LIM-like domain and are preferably in
the region of the nucleic acid sequence encoding the LIM domain or
the LIM-like domain. Prefered mutations do not cause frameshifts.
Techniques for the mutagenesis, inactivation or knockout of target
genes are well-known in the art (see for example In Vitro
Mutagenesis Protocols; Methods in Molecular Biology (2nd edition) Ed
Jeff Braman; Sambrook J et al . 2012. Molecular Cloning: A Laboratory
Manual (4th Edition) CSH Press; Current Protocols in Molecular
Biology; Ed Ausubel et al (2013) Wiley) . In some embodiments,
mutations may be introduced into a target DAI coding sequence by
genome editing techniques, for example RNA guided nuclease
techniques such as CRISPR, Zinc-finger nucleases (ZFNs) and
transactivator-like effector nucleases (TALENs) (Urnov, F.D. et al
Nature reviews. Genetics 11, 636-646 (2010); Joung, J . . et al.
Nature reviews. Molecular cell biology 14, 49-55 (2013); Gasiunas,
G . et al PNAS USA 109, E2579-2586 (2012); Cong, L . et al . Science
339, 819-823 (2013) ).
A plant that expresses a LIM-disrupted DAI protein as described
above (i.e. a DAI protein with an inactivated or disrupted LIM
domain or LIM-like domain) 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.
The plant or its off-spring or descendents may be tested for seed
size, organ size and/or plant yield relative to controls.
A plant which expresses a LIM-disrupted DAI protein as described
herein may display increased seed and/or organ size relative to the
controls and may have higher plant yields.
The effect of dominant-negative DAI alleles on yield-associated
traits in plants is increased in plants that are deficient in EODl
expression or activity (Li et al (2008), WO2009/047525) .
A LIM-disrupted DAI protein may be expressed as described above in a
plant that is deficient in EODl expression or activity.
EODl proteins are plant E3 ubiquitin ligases (Disch et al. (2006),
Li et al (2008), WO2009/047525) . EODl proteins comprise an EOD
domain. A plant EOD domain may consist of the amino acid sequence of
SEQ ID NO: 37;
E / )RCVICQ (L/M) (K/R/G/T/E) Y (K/R) (R/I) (G/K) (D/N/E) (R/Q/K/L) Q (I/M/V) (K
/N/T/A)L(L/P)C(K/S)H(V/A)YH(S/T/G/A) (E/Q/D/S/G) C ( /G/T/V) (S/T) (K/R)WL
(G/T/S) INK(V/I/A/K)CP(V/I)C (SEQ ID NO: 37)
In some preferred embodiments, an EODl protein may comprise a EOD
domain having an amino acid sequence of residues 150 to 192 of SEQ
ID NO: 38, residues 187 to 229 of SEQ ID NO: 39, residues 192 to 234
of SEQ ID NO: 40, residues 189 to 231 of SEQ ID NO: 41, residues 194
to 236 of SEQ ID NO: 42, residues 194 to 236 of SEQ ID NO: 43,
residues 194 to 236 of SEQ ID NO: 44, residues 195 to 237 of SEQ ID
NO: 45, residues 189 to 231 of SEQ ID NO: 46, residues 195 to 237 of
SEQ ID NO: 47, residues 195 to 237 of SEQ ID NO: 48, residues 195
to 237 of SEQ ID NO: 49, residues 218 to 260 of SEQ ID NO: 50,
residues 196 to 238 of SEQ ID NO: 51, residues 197 to 239 of SEQ ID
NO: 52, or residues 193 to 235 of SEQ ID NO: 53.
Further suitable EOD 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 EODl protein whose expression or activity is reduced in the plant
cell expressing the LIM disrupted DAI protein may comprise an amino
acid sequence of any one of SEQ ID NOS 38 to 53 as set out in Table
2 . In some preferred embodiments, a EODl protein may comprise the
amino acid sequence of SEQ ID NO: 45 (AtEODl) or SEQ ID NOS: 50 or
51 (OsEODl) or may be a variant of this sequence which retains E3
ubiquitin ligase activity.
A EODl protein which is a variant of any one of SEQ ID NOS: 38 to 53
or other reference EODl sequence 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 EODl sequence.
A EOD protein which is a variant of any one of SEQ ID NOS: 38 to 53
may further comprise a EOD domain having the sequence of SEQ ID NO:
37. Examples of suitable sequences are set out above.
A nucleic acid encoding a EODl protein may comprise a nucleotide
sequence set out in a database entry selected from the group
consisting of XM_002299911 .1 GI:224059639 (PtEODl) ; XM_002531864 .1
GI:255582235 (RcEODl) ; XM_002279758 .2 GI:359487285 (VvEODl) ;
XM_003542806.1 GI:356548934 (GmEODla) XM_003540482 .1 GI:356544175
(GmEODlb) ; XM_0024 68372 .1 GI.242042044 (SbEODl); NM_001147247 .1
GI: 226496788 (ZmEODl) ; or NP_001030922 .1 G : 79316205 (AtEODl;
At3g63530) or may be variant of one of these sequences.
In some preferred embodiments, a nucleotide sequence encoding a EODl
protein in a plant may encode AtEODl or OsEODl or may be a variant
thereof .
EODl proteins and encoding nucleic acids whose expression or
activity may be reduced as described herein may be readily
identified in any plant species of interest, in particular a crop
plant, such as wheat, barley, maize, rice, and another agricultural
plants, using routine sequence analysis techniques.
Suitable methods for reducing EODl expression or activity are wellknown
in the art.
For example, the activity of EODl may be reduced, preferably
abolished, by introducing a mutation, such as a deletion, insertion
or substitution, at a position corresponding to position 44 of SEQ
ID NO: 45, for example, an A to T substitution. A position in a EODl
protein sequence which is equivalent to position 44 of SEQ ID NO: 45
may be identified using standard sequence analysis and alignment
tools.
In some embodiments, the expression of a EODl protein may be reduced
in a plant cell by expressing a heterologous nucleic acid which
encodes or transcribes a suppressor nucleic acid, for example a
suppressor RNA or RNAi molecule, within cells of said plant. The
suppressor RNA suppresses the expression of EODl protein in the
plant cells that express LIM-disrupted DAI.
An suitable RNAi sequence may correspond to a fragment of a
reference EODl nucleotide sequence set out herein or may be a
variant thereof.
In other embodiments, a knock out or knock down mutation may be
introduced into a nucleic acid sequence within the genome of a plant
cell which encodes an EODl protein, such that expression or activity
of EODl is reduced. A plant may then be regenerated from the mutated
cell.
The nucleic acid encoding EOD1 may be mutated by insertion,
substitution or deletion of one or more nucleotides relative to the
wild-type nucleotide sequence. For example, 1 , 2 , 3 , 4 , 5 , 6 , 7 , 8 ,
9 or 10 or more nucleotides may be altered relative to the wild-type
nucleotide sequence.
LIM-disrupted DAI proteins may be expressed as described herein in
any plant species. Examples of suitable plants for use in accordance
with any aspect of the invention described herein include
monocotyledonous and dicotelydonous higher plants, for example
agricultural or crop plants, such as plants 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 transgenic plant which
expresses a LIM-disrupted DAI protein, as described above.
The plant may comprise an exogenous nucleic acid which encodes the
LIM-disrupted DAI protein.
One or more yield-related traits in the plant may be improved,
increased or enhanced in the plant relative to control plants which
do not express LIM-disrupted DAI protein. Yield-related traits may
include life-span, organ size and seed size.
The plant may have increased yield relative to control wild-type
plants (i.e. identical plants which do not express a LIM-disrupted
DAI protein) . For example, the mass of seeds (e.g. grain) or other
plant product per unit area may be increased relative to control
plants .
A suitable plant may be produced by a method described above.
In addition to 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.
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.
"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) 3 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.
All documents mentioned in this specification are incorporated
herein by reference in their entirety for all purposes.
The contents of all database entries mentioned in this specification
are also incorporated herein by reference in their entirety for all
purposes. This includes the versions of any sequences which are
current at the filing date of this application.
Experiments
1. Methods
1.1 Co-Immunoprecipitation analysis
All bait proteins for these studies were GST-tagged and glutathione
sepharose beads (GE Life Science 17-0756-01) were used for their
pull-down.
A flask of 10ml LB with appropriate antibiotics was inoculated with
a BL21 glycerol stock of the appropriate expression construct and
left to grow overnight at 37 °C and 220rpm. The following morning the
10ml preculture was used to inoculate an 100ml LB flask (at a ratio
of 1:100), and this culture was incubated at 37°C for two hours at
220rpm. The flask was removed from the incubator, IPTG (Melford
MB1008) was added to a final concentration of lmM before the culture
was incubated at 28°C (and 220rpm) for another three hours.
Following this growth phase, the cultures were centrifuged at 4500xg
for 10 minutes, the supernatants were discarded and the pellets
resuspended at 4°C in 2.5ml TGH Buffer (50mM HEPES (pH7.5), 150mM
NaCl, 1% Triton-X-100, 10% Glycerol, lmM DTT, 1 complete EDTA-free
protease inhibitor tablet (per 50ml) (Roche 11873580001) ). The
bacterial suspension was then sonicated (on ice) for four bursts of
ten seconds, separated by 20-second intervals, before being
centrifuged at 12 OOOx g for 20minutes to pellet any cellular
debris. Cleared sonicates were then stored on ice while a 50% slurry
of washed glutathione sepharose beads (GE Life Sciences 17-0756-01)
was prepared according to the manufacturer's instructions. 201 of
the 50% glutathione sepharose slurry was then combined with 2.5ml of
protein extract from bait protein (GST-tagged) expressing cells and
2.5ml of protein extract from prey protein (HA-/FLAG-/HIS-tagged)
expressing cells. This mixture was incubated for 30 minutes at 4°C
on a rotating wheel and then the glutathione sepharose beads were
washed five times with an excess (5001 ) of TGH buffer (following
manufacturer's instructions). After washing, proteins were eluted
with 351 GST-elution buffer (50mM TRIS-glycine (pH8.0), lOmM
reduced glutathione) over 30 minutes at 4°C before being analysed by
western blot analysis.
1.2 Western Blots
20%, 12% or 4-20% precast SDS-polyacrylamide gels (RunBlue NXG02012,
NXG01227, NXG42027) were submerged in RunBlue SDS-TRIS-tricine run
buffer (RunBlue NXB0500) , in a gel tank (Atto Japan AE6450) Samples
were mixed with 2x Laemmli sample buffer (Bio-Rad Ltd 161-0737)
placed in a heat block for 10 minutes at 96°C and then loaded into
rinsed wells in the gel in either or 201 aliquots. The gels
were run at 160V for 60 minutes along with a 3ul aliquot of
PageRuler Plus Prestained Protein Ladder, 10 to 250kDa (Fermentas
26619). If appropriate, gels were stained at this stage.
Transfers were carried out using the Bio-Rad Mini Trans-Blot® Cell
kit (Bio-Rad 170-3836) . Gels were removed from their glass casing
and laid on top of a sponge (from Bio-Rad Mini Trans-Blot® Cell
kit), two pieces of chromatography paper (VWR WHAT3030-917) and a
methanol-washed PVDF membrane (Roche Diagnostics 03010040001) . Air
bubbles were removed from between the gel and membrane and then two
further pieces of Whatman paper and a sponge were applied to the
gel. This was enclosed in a gel holder cassette (from Bio-Rad Mini
Trans-Blot® Cell kit) , submerged in transfer buffer (25mM TRIS,
192mM glycine, 10% (v/v) methanol) and run at 90V for 70 minutes at
4°C.
Following the transfer the membrane was washed for 10 minutes in
50ml PBS (140mM NaCl, 2 .7mM KC1, lOmM Na2HPO,, 1 .8mM KH2PO4, pH 7.3)
at room temperature, before being agitated in 50ml blocking solution
(5% (w/v) milk powder, 0.1% (v/v) Tween-20) for either one hour at
room temperature or overnight at 4°C. Primary antibodies were
diluted to their appropriate concentration (see Table 2.9) in
blocking solution and incubated with the membrane (10ml per membrane
with gentle agitation) for one hour before five washes with 50ml
PBST (140mM NaCl, 2.7mM C1, lOmM Na HP0 , 1 .8mM H2P0 , 0.1% (v/v)
Tween-20, pH 7.3) at room temperature. If secondary antibody was
required, staining and washing steps were repeated.
The washed membrane was held with forceps and carefully one corner
was blotted onto blue-roll to remove excess moisture. It was then
laid in a petri dish and treated with peroxidise substrate
(SuperSignal West FE TO Max. Sensitivity substrate (Fisher
Scientific PN34095) ) at a rate of 8001 substrate per membrane.
Membranes were left in this substrate for five minutes, dried as
before and placed in an X-ray cassette under a piece of X-ray film
(Fuji Film X-RAY 18x24cm - (FujiFilm 497772RXNO) ). X-ray films were
developed using a Konica SRX-101 Table Top X-ray film developer
(Konica 106931659) .
Subsequent to analysis, if required, membranes were washed in 50ml
PBST and stained with 10ml Ponceau S solution (Sigma -Aldrich P7170)
for 30 minutes, followed by a single wash in 50ml PBST and drying at
room temperature.
1.3 Seed Size Determination
Seed area was used as a proxy measurement of seed size. Seeds were
scattered in a petri dish and scanned against a white background
using a desktop scanner (Hewlett Packard Scanjet 4370) at a high
resolution (<3600dpi) . Images were stored as black and white 8-bit
images, and subjected to image analysis using the Image software.
ImageJ was opened and the threshold (Ctrl+Shif t+T) set such that
all seeds are completely red, the select all seeds with the
"rectangular selection" tool and chose the analyse option (Analyze >
Analyze Particles) . In the dialog box set a size threshold to
exclude smaller (non-seed) structures and large structures such as
aggregations of seeds. Seed lengths and widths were calculated by
fitting an ellipse to each seed (Analyze > Set measurements > Fit
ellipse) . When this option is selected the analysis outputs a
"Major" and "Minor" value corresponding to length and width of the
ellipse, representing the longest and widest parts of the seed. [Jl]
2 . Results
L M domains (Prosite: PS00478) are a tandem zinc finger domains that
act as a platform for protein:protein interactions (fig 1 ) .
Web-based domain prediction software (Pfam, SMART, PROSITE) predict
the presence of a single LIM domain in DAI (AtDAl 170aa-230aa) ,
which was assumed to be involved i mediating putative DAl-DAl
homo-dimerisation (Li et al ., 2008) .
Surprisingly however, a variant of DAI with a mutated L domain
(henceforth ') induced a dominant negative organ size
phenotype equivalent to the da-1 mutant when introduced into a Col
background in Arabidopsis (fig 4 ) . This shows that the LIM domain of
DAI is not involved in DAI homo-dimerisation.
4 key zinc coordinating amino acids (C172, C175, C199 and C202) were
converted to glycines to produce the DAllim8 mutant. These mutations
were predicted to abolish the Zn finger motifs, which are due to Zn
coordination by patterns of cysteine (C) residues.
Recombinant GST-tagged bait proteins were incubated with recombinant
FLAG-tagged prey proteins before precipitation of GST-tagged bait
proteins on glutathione sepharose beads. The purified proteins were
then eluted and subjected to SDS-PAGE and immunoblot analysis. The
ability of -glucuronidase (GUS) tc form a homo-tetramer was
utilised to design a positive control of GST-GUS vs FLAG-GUS. Two
sets of negative controls were alsc used; these were GST-GUS vs
FLAG-prey, and GST-bait vs FLAG-GUS.
These in vitro co-immunoprecipitation experiments showed that
Dallim8 is able to bind to wild-type DAI protein (Figure 3 ) and that
the lim8 seed size phenotype in a Col background is equivalent to
that of dal-1 (Figure 4 ) .
The sequences of DAI proteins were further analysed using a two-step
domain prediction analysis. First, an initial homology detection
screen (HHpred) was carried out to identify proteins with similar
domains and structures. This was then followed by a domain
prediction screen (Pfam, SMART, PROSITE) , which used these proteins
as query sequences. This strategy revealed that the region 230aa-
297aa of AtDAl shared significant structural homology with the LIM
domains of other proteins (including the mouse LIM/homeobox protein
LHX3) . This new putative domain was termed the LIM-like domain.
The purported second pair of zinc coordinating amino acids in the
LIM-like domain of DAI was not detected by classical domain
prediction software (Pfam, SMART, PROSITE) because of significant
sequence divergence from the canonical LIM pattern. By considering a
CxxH pairing at position 261aa-264aa in the AtDAl sequence, it was
apparent that an insertion in the first zinc finger domain and the
inter-finger region causes the sequence to deviate significantly
from the LIM consensus pattern. This results in a finger length of
24aa and an inter-finger region of 7aa (rather that 16-23aa and 2aa
respectively) .
The LIM-like domain therefore represents a second Zn finger
containing LIM domain within the DAI protein.
Si G I-514815267. ro
B __GI- 357157184 .pro
Br_ DAlb.pro
Br" DAla.pro
t _GI- 15221983. pro
Tc""GI- 508722773 .pro
G _ 356564241 .pro
G ~GI- 356552145 .pro
~GI- 302142429 .pro
Vv""GI- 359492104 .pro
Sl "GI- 460385048 .pro
Os""GI- 218197709 .pro
Os "GI- 115466772 .pro
Bd"^GI- 357160893 .pro
Bd""GI- 357164660 .pro
Sb_ GI- 242092232 .pro
Zm GI- 212275448 .pro
At_ GI- 240256211 .pro
At""GI- 145360806 .pro
At_ G I-22326876 .pro MEPPAARVTPSIKADCSHSVNIICEETVLHSLVSHLSAALRREGISVFVDACGLQETKFF 60
At""GI- 30698242 .pro
At" G I- 30698240. pro
At_ GI- 15240018. pro
334188680 .pro
Si_GI-514815267.pro
Bd_GI-357157184.pro
Br_DAlb.pro
Br_DAla .pro
At_GI-15221983 .pro
Tc_GI-508722773.pro
Gm_GI-356564241 .pro
Gm_GI-356552145.pro
Vv_GI-302142429.pro
Vv_GI-359492104 .pro
Sl_GI-460385048 .pro
Os_GI-218197709.pro
Os_GI-115466772 .pro
Bd_GI-357160893.pro
Bd_GI-357164660 .pro
Sb_GI-242092232.pro
Zm_GI-212275448.pro
At_GI-240256211.pro
At_GI-145360806.pro
At_GI-2232 687 6.pro SIKQNQPLTDGARVLVVVISDSVEFYDPWFPKFLKVIQGWQNNGHVVVPVFYGVDSLTRV 120
At_GI-30698242.pro
At_GI-30698240.pro
At_GI-15240018.pro
At_GI-334188680.pro
Si_GI-514815267.pro
Bd_GI-357157184.pro
Br_DAlb.pro
Br_DAla.pro
At_GI-15221983 .pro
Tc_GI-508722773.pro
Gm_GI-356564241.pro
Gm_GI-356552145.pro
Vv_GI-302142429.pro
W_GI-359492104.pro
Sl_GI-460385048.pro
Os_GI-218197709.pro
Os_GI-115466772.pro
Bd_GI-357160893.pro
Bd_GI-357164660.pro
Sb_GI-242092232.pro
Zm_GI-212275448.pro
At_GI-240256211.pro
At_GI-145360806.pro
At_GI-2232687 6.pro YGWANSWLEAEKLTSHQSKIL3NNVLTDSELVEEIVRDVYGKLYPAERVGIYARLLEIEK 1
At_GI-30698242.pro
At_GI-30698240.pro
At_GI-15240018.pro
At_GI-3.34188680.pro
Si_GI-514815267.pro
Bd_GI-357157184.pro
Br_DAlb.pro
Br_DAla.pro
At_GI-15221983.pro
Tc_GI-508722773.pro
Gm_GI-356564241.pro
Gm_GI-356552145.pro
_GI-302142429.pro
Vv_GI-359492104 .pro
Sl_GI -460385048 .pro
Os_GI-218197709.pro
Os_GI -115466772 .pro
Bd_GI-357160893.pro
Bd_GI-357164660.pro
Sb_GI-242092232.pro
Zm_GI -212275448 .pro
At_GI-240256211.pro
At_GI-145360806.pro
At_GI-22326876 .pro LLYKQHRDIRSIGIWGMPGIGKTTLRKAVFNHMSTDYDASCFIENFDEAFHKEGLHRLLK 240
At_GI-30698242.pro
At_GI-30698240.pro
At_GI-15240018.pro
At_GI-334188680.pro
Si_GI-514815267.pro
Bd_GI-357157184.pro
Br_DAlb.pro
Br_DAla.pro
At_GI-15221983.pro
Tc_GI-508722773.pro
Gm_GI-356564241.pro
Gm_GI-356552145.pro
W_GI-302142429.pro
Vv_GI-359492104 .pro
Sl_GI-460385048.pro
Os_GI-218197709.pro
Os_GI-115466772.pro
Bd_GI-357160893.pro
Bd_GI-357164660.pro
Sb_GI-242092232.pro
Zm_GI-212275448.pro
At_GI-240256211.pro
At_GI-145360806.pro
At_GI-22326876.pro ERIGKILKDEFDIESSYIMRPTLHRDKLYDKRILWLDDVRDSLAAESFLKRLDWFGSGS 300
At_GI-30698242.pro
At_GI-30698240.pro
At_GI-15240013.pro
At_GI-334188680.pro
LIIITSVDKQVFAFCQINQIYTVQGLNVHEALQLFSQSVFGINEPEQNDRKLSMKVIDYV 360
At_GI-15240018 .pro
At_GI-334188 680.pro
Si GI--514815267 .pro
Bd GI--357157184 .pro
Br_ DAlb .pro
Br DAla .pro
At" GI--15221983. pro
Tc "GI--508722773 .pro
G '-356564241 .pro
Gm_"GI--356552145 .pro
Vv_ GI--302142429 .pro
Vv _GI--359492104 .pro
si]"GI--460385048 .pro
Os GI--218197709 .pro
Os G I -115466772 .pro
Bd GI--357160893 .pro
Bd ] '-357164660 .pro
Sb]"GI--242092232 .pro
Z _ -212275448 .pro
At""GI--240256211 .pro
t] -145360806 .pro
At]"G I -22326876.;pro NGNPLALSIYGRELMGKKSEMETAFFELKHCPPLKIQDVLKNAYSALSDNEKNIVLDIAF 420
At_ G I -30698242.;pro
At""G I -30698240.;pro
At G I -15240018.;pro
At""G I -334188680 .pro
Si GI- 514815267 .pro
Bd_ G I- 357157184 .pro
Br_ DAlb.pro
B r DAla .pro
At" GI- 15221983. pro
Tc_ GI- 5C8722773 .pro
Gm_ GI- 356564241 .pro
Gm GI- 356552145 .pro
]G --3C2142429 .pro
Vv""GI- 359492104 .pro
Sl_ G I- 460385048 .pro
Os"]GI- 218197709 .pro
Os _GI- 115466772 .pro
Bd""GI- 357160893 .pro
Bd Gl- 357164660 .pro
Sb_ G I- 242092232 .pro
Zm_ G I- 212275448 .pro
t G I- 240256211 .pro
A t""GI- 145360806 .pro
At_ G - 22326876. pro FFKGETVNYVMQLLEESHYFPRLAI DVLVDKCVLTI SENTVQMNNLIQDTCQEIFNGEIE 480
t _GI- 30698242 . ro
A t ]GI- 30698240. pro
At__GI- 15240018. ro
At_ G I- 334188680 .pro
Si GI- 514815267 .pro
Bd "G - 357157184 .pro
Br_ DAlb.pro
Br_ DAI a . ro
At_ GI- 15221983. pro
Tc_"GI- 508722773 .pro
G _]GI- 356564241.pro
Gm_]GI- 356552145 .pro
Vv_ GI- 302142429.pro
Vv GI- 359492104 .pro
Si""GI- 460385048 .pro
Os GI- 218197709.pro
Os""GI- 115466772 .pro
Bd""GI- 357160893 .pro
Bd"]GI- 357164660 .pro
Sb] GI- 242092232 .pro
Zm_]GI- 212275448 .pro
At]]GI- 240256211 .pro
At GI- 145360806 .pro
At]_GI--22326876.;pro TCTRMWEPSRIRYLLEYDELEGSGETKAMPKSGLVAEHIESIFLDTSNVKFDVKHDAFKN 5 0
At""GI--30698242 . ;pro
At_GI-30698240.pro
At_GI-15240018.pro
At_GI-334188680.pro
Si GI--514815267,.pro
Bd""GI-357157184 .pro
Br__DAlb.pro
Br"_DAla.pro
At" GI--15221983. ro
c]"GI--508722773 .pro
G _"GI--356564241 .pro
G _]GI--356552145 .pro
Vv_"GI--302142429 .pro
Vv "GI--359492104 .pro
Si GI--460385048 .pro
Os ] -218197709,.pro
Os""GI--115466772 .pro
Bd"] -357160893 .pro
Bd""GI--357164660 .pro
Sb]]G -242092232 .pro
Zm "GI-212275448 .pro
At" GI-240256211 .pro
At ]G --145360806 .pro
At_GI-22326876.pro MFNLKFLKI YNSCSKYISGLNFPKGLDSLPYELRLLHWENYPLQSLPQDFDFGHLVKLSM 600
At_GI-30698242.pro
At_GI-30698240.pro
At_GI-15240018.pro
At_GI-334188680.pro
Si _GI--514815267 .pro
B ]GI--357157184 .pro
Br__DAlb.pro
Br" DA . ro
At GI--15221983. pro
Tc""GI--5D8722773 .pro
Gm__GI--356564241 .pro
Gm_]GI -356552145 .pro
Vv"]GI -302142429 .pro
Vv""GI--359492104 .pro
.]GI -460385048 .pro
OS "GI--218197709 .pro
Os"]GI--115466772 .pro
Bd""GI--357160893 .pro
Bd GI--357164660 .pro
Sb GI--242092232 .pro
Zm "GI--212275448 .pro
A t ]GI -240256211 .pro
At""G -145360806 .pro
A t ]GI -22326876. ro PYSQLHKLGTRVKDLVMLKRLILSHSLQLVECDILIYAQNIELIDLQGCTGLQRFPDTSQ 660
At G -30698242. ro
ft ]GI -30698240. ro
At""GI-15240018. ro
At" GI-334188680 .pro
Si_GI-5148152 67.pro
Bd_GI-357157134.pro
Br_DAlb.pro
Br_DAla.pro
At_- -15221983. ro
Tc]]GI--508722773,.pro
Gm_]GI--356564241,.pro
Gm _GI--356552145,.pro
V GI--302142429,.pro
Vv"]GI--359492104,.pro
S "]GI--460385048,.pro
Os"]GI--218197709,.pro -MGDRP 5
Os"]GI--115466772,.pro
Bd"]GI--357160893,.pro
Bd""GI--357164660 .pro
Sb] GI--242092232 .pro
Zm_"G -212275448 .pro
At""GI--240256211 .pro
At G -145360806 .pro
At""GI--22326876. ro LQNLRVVNLSGCTEIKCFSGVPPNIEELHLQGTRIREIPIFNATHPPKVKLDRKKLWNLL 720
At_GI-30698242.pro
At_GI-30698240.pro
At_GI-15240018.pro
At_GI-334188680.pro
Si_GI-514815267.pro
Bd_GI-357157184.pro
Br_DAlb.pro
Br_DAla.pro
At_GI-15221983.pro
Tc_GI-508722773.pro
Gm_GI- 35 65 642- 1 .pro
G _GI-3 6552 145. pro
Vv_GI-302142429.pro
Vv_GI-359492104 .pro
Sl_GI-460385048.pro
Os_GI-218197709.pro DMGAGVALRFSHNDWTLEEDSKALHFLQPDLVLFTGDYGNENVQLVKSISDLQLPKAAIL 65
Os_GI- 1154 66772. pro
Bd_GI-357160893.pro
Bd_GI-357164660.pro
Sb_GI-242092232.pro
Zm_GI-212275448 .pro
At_GI-240256211.pro
At_GI-145360806.pro
At_GI-22326876.pro ENFSDVEHI DLECVTNLATVT SNNHVMGKLVCLNMKYCSNLRGLPDMVSLESLKVL LSG 780
At_GI-30698242.pro
At_GI-30698240.pro
At_GI-15240018.pro
At_GI-334188680.pro
Si -514815267. ro
Bd_ GI--357157184. ro
Br" DAlb.pro
Br DAla.pro
At GI--15221983. ro
[ GI--508722773. pro
G "G --356564241. pro
Gm_ -356552145. pro
Vv[ -302142429. pro
V G I --359492104. pro
[GI -460385048. pro
Os_[GI -218197709. pro GNHDCWHT YQFSEKKVDRVQLQLESLGEQHVGYKCLDFPTIKLSWGGRPFSCGGNRI FR 125
Os G -115466772. pro
Bd G I -357160893. ro
Bd""G I -357164660. pro
Sb["G -242092232. ro
Z "G I -212275448. pro
A t""G I -240256211. pro
At"[GI -145360806. pro
At""G I -22326876. pro CSELEKIMGFERNLKKLYVGGTAIRELPQLPNSLEFLNAHGCKHLKSINLDFEQLPRHFI 8 0
ft [[ -30698242. pro
" I -30698240. ro
A t""GI--15240018. ro
At G I -334188680. ro
Si GI--514B15267..pro
Bd["GI--3571571B4..pro
Br[ DAlb .pro
Br _DAla.pro
ft t[_GI--15221983. pro
Tc[[GI--508722773,.pro
Gm_[GI--356564241,.pro
G GI--356552145,.pro
Vv [GI--302142429,.pro
Vv "GI--359492104,.pro
S ["G -460385048 .pro
Os"[GI -218197709 .pro PKLLSKWYGVNDMAESAKRIYDAATNAPKEHAVILLAHNGPTGLGSRMEDICGRDWVAGG 185
Os"[GI -115466772 .pro
Bd"[GI -357160893 .pro
Bd [GI -357164660 .pro
Sb[_G -242092232 .pro
Zm "G -212275448 .pro
At[_GI -240256211 .pro
At["G -145360806 .pro
_GI- 22326876. pro FSNCYRFSSQVIAEFVEKGLVASLARAKQEELIKAPEVIICIPMDTRQRSSFRLQAGRNA 900
At"~GI- 30698242. pro
At GI- 30698240. pro MPISDVASLVGGAALGAPLSE 21
At""GI- 15240018. pro MASDYYSSDDEGFGEKVGLIG 21
At""GI- 334188680. ro MWCLSCFKPSTKHDP 15
Si_GI-514815267.pro
Bd_GI-357157184.pro
Br_DAlb.pro
Br_DAla.pro
At_GI-15221983.pro
Tc_GI-508722773.pro
Gm_GI-356564241.pro
Gm_GI-356552145.pro
Vv_GI-302142429.pro
Vv_GI-359492104.pro
Sl_GI-460385048.pro
Os_GI-218 197709. ro GDHGDPDLEQAISDLQRETGVSIPLVVFGHMHKSLAYGRGLRKMIAFGANRTIYLNGAW 245
Os_GI-115466772.pro
Bd_GI-357160893.pro
Bd_GI-357164 660.pro
Sb_GI-242092232.pro
Zm_GI-212275448.pro
At_GI- 40256211.pro
At_GI-1453 60805.pro
At_GI-22326876.pro MTDLVPWMQKPISGFSMSVVVSFQDDYHNDVGLRIRCVGTWKTWNNQPDRIVERFFQCWA 960
At_GI-30698242.pro
At_GI-30698240.pro KLV EEAKKVKDFKP- X 39
At_GI-15240018.pro EKDRFEAETIHVIEVSQ- H 39
At_GI-334188 680.pro SEDRFEEETNIVTGIS- - 31
Si GI--514815267 .pro
Bd GI--357157184 .pro
B r _DAlb.pro
Br"_DAla.pro
A _GI--15221983. ]pro
Tc]]GI--508722773 .pro
Gm_]GI--356564241 .pro
Gm_"GI--356552145 .pro
Vv_"GI--302142429 .pro
Vv "GI--359492104 .pro
Si""GI--460335043 .pro
Os GI--218197709 .pro PRVNHAQSSRQPAISTSEKTGLEGLTGLMVPTSRAFTIVDLFEGAVEKISEVWVTVGDAR 305
Os"]GI--115466772 .pro
Bd"]GI--357160893 .pro
Bd"]GI--357164660 .pro
]GI--242092232 .pro
Zm_]GI--212275448 .pro
At]]GI--240256211 .pro
At""GI--145360806 .pro MDSSSSSSSSSPSSSYGVARVS 22
At_ GI--22326876. pro PTEAPKVVADHIFVLYDTKMHPSDSEENHISMWAHEVKFEFHTVSGENNPLGASCKVTEC 1020
At GI--30698242. pro
At] GI--30698240. pro SQDLASTMERLVPIFNEIDMMQQGSNRGTSELKVLTETMERAGEMVHKCSRIQWYSIAKK 99
At""GI--15240018. pro EADIQKAKQRSLATHEAEKLDLATHEAEQLDLAIQEFSRQEEEEERRRTRELENDAQIAN 99
At""GI--334188680 .pro LYEDVILRQRRSEADQIEWAIQDS FNPQE TSRCRQREEDDQIAR 75
Si GI--514815267 .pro MGWLSKIFKGSVN-RVSRGHYNGNSHE GYS 29
Bd]"GI--357157184 .pro MGWLNKIFKGSVN-RVSRGNYDGNWHD GNS 2
Br__DAlb.pro MGWLNKIFKGSNQ-RHPLGNEHYHHNGGYYENYP 33
Br] DAla.pro MGWFNKIFKGSTQ-RFRLGNDHDHN — GYYQSYP 31
At" GI--15221983. ro MGWFNKIFKGSNQ-RLRVGNNKHNHN-VYYDNYP 32
Tc_ G -508722773 .pro MDWIKKIFKGCAH-KFSEG HHHG NYV 25
Gm_"GI--356564241 .pro MGWLSRIFKGSDHNKLSEGHYYKEDA GYY 29
Gm_]GI--356552145 .pro MGWLSRIFKGSDHNKLSEGHYYKEDA GYY 2
Vv""GI--302142429 .pro MGWLNKIFKGSSH-KISEGNYHGRYQ GDT 28
Vv_]GI--359492104 .pro MGWLNKIFKGSSH-KISEGNYHGRYQ GDT 2
S ]]GI--460385048 .pro MGWLNKIFRGSSH-KISEGQYDWRCE GHT 28
Os""GI--218197709 .pro TELEQELVLYKQPHKSVPSNIAIWSTMGWLTKFFRGSTH-KISEGQYHSKPAEETIWNGP 364
Os "GI--115466772 .pro MGWLTKFFRGSTH-KISEGQYHSKPAEETIWNGP 33
Bd G I -357160893 .pro MGWLTKIFRGSTY-KISEGQRQSRPAEEAVWNEP 33
Bd -357164660 .pro MGWLTKFFRGSTH-NISEGQDQSKPAEETVWNEP 33
Sb_ GI--242092232 .pro MGWLTKFFRGSTH-NISEGQYHSRPAEDTAWNEP 33
Zm ]GI -212275448 .pro MGWLTKFFRGSTH-NISEEQYHSRPAEDTAWNEP 33
At]]GI--240256211 .pro MGWLTKILKGSSH-KFSDGQCNGRYREDRNLEGP 33
At_GI- 1453 60806. pro HISNPCIFGEVGSSSSSTYRDKKWKLMKWVSKLFKSGSNGGGSGAHTNHHPPQFQEDENM 82
At_GI-22326876.pro GVEVITAATGDTSVSGIIRESETITIIEKEDTHDEEDTPLLSRKPEETNRSRSSSELQK 1080
At_GI-30698242.pro
At_GI-30698240.pro ALYTREIKA--INQDFLKFCQIELQLIQHRNQLQYMRSMGMASVSTKADLLSDIGNEFSK 1.57
At_GI-15240018.pro VLQHEERE RLINKKTALEDEEDELLARTLEESLKENNRRKMFEEQVNKDEQ 150
At_GI-334188680.pro GLQYVEET ELDKSVVDEED QQ 96
Si_GI-514815267.pro TQHTKSY 36
Bd_GI-357157184.pro SENIR 34
Br_DAlb.pro -HEHS EPSAETDA DHT 48
Br_DAla.pro -HDEPSADTDPDPDPDPDE THT 52
At_GI-15221983.pro TASHDDEPSAADTDADNDEP HHT 55
Tc_GI-508722773.pro EDPHP QF 32
Gm_GI-356564241.pro LPSTS 34
Gm_GI-356552145.pro LPSTS 34
Vv_GI-302142429.pro VQNEP 33
Vv_GI-359492104.pro VQNEP 33
Sl_GI-460385048.pro EEDDP 33
Os_GI-218197709.pro SNSAVVTMVYPLESTFGQLDLLLLATDLRQLVIDDVDCCKLRQQAQPVLHLMYSQLQLLQ 424
Os_GI-115466772.pro SNSAVVT 40
Bd_GI-357160893.pro SSSTVVT 40
Bd_GI-357164660.pro SSSTAVN 40
Sb_GI-242092232.pro SSSPVVT 40
Zm_GI-212275448.pro SSSPVVT 40
At_GI-240256211.pro RYSAEGSDFDKEEIECAIALSLS- EQEHVIPQDDKGKKIIE 73
At_GI-145360806.pro VFPLPPS
At_GI-22326876.pro LSSTSSKVRSKGNVFWKWLGCFP- -LQPKNLRSRSRRTTALEEA 1122
At_GI-30698242.pro
At_GI-30698240.pro LCLVAQPEVVTKFWLKRPLMELKKMLFEDGV- -VTWVSAPYALGKTTLVTK 207
At_GI-15240018.pro LALIVQESLNMEEYPIR-LEEYK — SISRRAPLDVDEQ-FAKA 189
At_GI-334188 680.pro LSKIVEESLKE 7
Si_GI-514815267.pro GAHGNED-E DMDHAIALSLSEQDQRKGKAIDTEHHLD — ED 74
Bd_GI-357157184.pro GAYDESDNE DIDRAIALSLAEEDPNKGKAIIDPDYS 70
Br_DAlb.pro QEPSTSEEETWNGKENE EVDRVIALSILEE-ENQRPETNTG 88
Br_DAla.pro QEPSTSEEDTS-GQENE DIDRAIALSLIENSQGQTNNTCAAN 93
At_GI-15221983.pro QEPSTSEDNTSNDQENE DIDRAIALSLLEE — NQEQTSISG 94
Tc_GI-508722773.pro NAPSVS-GDAWQELENE DVDRAIALSLLGE — SQKGRKVID 70
Gm_GI-356564241.pro GVTN NQNENE DIDRAIALSLVEESRRANNNVNGER 69
Gm_GI-35 6552 145. pro GVTNDAWNQSQNQNENE DIDRAIALSLVEETQKANNNVN 73
Vv_GI-302142429.pro SCSGDVWAETENE DIDRAIALSLSEE — EQKGKKVID 68
Vv_GI-359492104.pro SCSGDVWAETENE DIDRAIALSLSEE — EQKGKKVIDE 69
Sl_GI-460385043.pro STAEDSWSEIE EIDRAIAISLSEE — EQKGKIVID 66
Os_GI-218197709.pro TSHAHQHGDVPS EFDNE DIARAISLSLLEEEQRKAKAIEKD 465
Os_GI-115466772.pro DVPSEFDNE DIARAISLSLLEEEQRKAKAIEKD 73
Bd_GI-357160893.pro DVLSEFDNE DIDRAIALSLSEE-QRKSKGTGKD 72
Bd_GI-357164660.pro YALSEFDNE DIDRAIALSLSEEEQRKSKGTGKD 73
Sb_GI-242092232.pro DIFSEFNNE DIDRAIALSLSEEEQRKAKTIDKD 73
Zm_GI-21227544 8.pro DILSEFNNE DIDRAIALSLSEEEQRKEKAIDKD 73
At_GI-240256211.pro YKSETEEDDDDDEDEDEEYMPAQLEAAEEEERRVAQAQIEEEEKRRAEAQLEETEKLLAK 133
At_GI-145360806.pro SLDDRSRGARDKE ELDRSISLSLADN-TKRPHGYGWS 125
At_GI-22326876.pro LEEALKEREKLEDTREL QIALIESKKIKKIKQADERDQIKHADER 1167
At_GI-30698242.pro MVRRKRQEEDEKI EIERVKEESLKLAKQAEEKRRLEESKEQ 1
At_GI-30698240.pro LCHDADVKEKFKQI FF SVSKFPNVRLIGHKLLEHIGCKANEYEN 252
At_GI-15240018.pro VKESLKNKGKGKQFEDE QVKKDEQLALIVQESLNMVESPPRLEEN 234
At_GI-334188680.pro KGKSKQFEDD QVENDEQQALMVQESLYMVELSAQLEED 1 5
Si_GI-514815267.pro EQLARALQENTSPTLDEDEQLAR- -ALQESMNDEHP 108
Bd_GI-357157184.pro LEEDEOLAR -ALHESLNTGSP 90
Br_DAlb.pro AWKHAM-MDDDEQLAR- -AIQESMIARN- 113
Br DAla.pro AGKYAM-VDEDEQLAR -AIQESMVVGNT 119
At_ G -152219B3.pro KYSMPVDEDEQLAR -ALQESMVVGNS 119
Tc "G -508722773 .pro DEYQLEEDEQLAR -ALQESLNFEPP 94
Gm ~GI -356564241 .pro LSLQTLLEEDEQLAR -AIEQSLNLESP 96
Gm " I-356552145 .pro DYRSQLEEDEQLAR -AIEQSLNLESP 98
Vv""G -302142429 .pro NEFQLEEDEQLAR -AIQESLNIESP 92
Vv""G I-359492104 .pro L-DNEFQLEEDEQLAR -AIQESLNIESP 95
S ""G I-460385048 .pro SESQLKEDEQLAR -ALQESLNVESP 90
OS "G I-218197709 .pro MHLEEDEQLAR -AIQESLNVESP 487
Os -115466772 .pro MHLEEDEQLAR -AIQESLNVESP 95
Bd""G I-357160893 .pro LHLDEDEQLAR -AIHESLNVESP 94
Bd G I-357164660 .pro QHLDEDEQLAR -AIQESLNVESP 95
Sb""G -242092232 .pro MHLEEDEQLAR -AIQESLNVESP 95
Z "G I-212275448 .pro MHLEEDEQLAR -AIQESLNVESP 95
At_GI-240256211.pro ARLEEEEMRRSKAQLEEDELLAK ALQES VGSP 167
At_GI-145360806.pro MDNNRDFPR PFHGGLNESSF 145
At_GI-2232 687 6.pro EQRKHSKDHEEEEIESNEKEERRHSKDYVIEELVLKGKGKRKQLDDDKADEKEQ 1221
At_GI-33698242 .pro GKRIQVDDD QLAKTTSKDKGQ 62
At_GI -30698240.pro DLDAMLYIQQLLKQLGRNGSILLVLDDV WAEEESLLQKFL 2 2
At_GI-15240018.pro N ISTRAPVDEDEQLAK AVEESLKGKGQ 262
At_GI-334188680.pro KNISTIPPLNEDAQLQK VIWESAKGKGQ 173
Si_GI-514815267.pro -DHAYHRSCY^ERF-H 198
Bd_GI-357157184.pro -NQPYHKSCYkDFF-H 180
Br_DAlb.pro -NYPFHKACY}¾ERF-H 212
Br_DAla . ro -NYPFHKACYftERY-H 229
At_GI-15221983.pro -NYPFHKACY{¾ERY-H 229
Tc_GI- 50872277 3 .pro -NYRFHKSCYf(ERY-H 1 4
Gm_GI-35 42 1.pro JGCYTEIGFGRYLNCLNAFWHPECFRCRACNLPISDYEFSTSG- 174
Gm_GI-35 6552145. pro 178
Vv_GI-302142429.pro 176
Vv_GI-3594 92104.pro GCNTEIGHGRFLSCMGAVWHPECFRCHGCGYPISDYEYSMNG- 179
Sl_GI-4 60385048. ro GCSTEIGHGRFLSCMGAVWHPECFRCHACNQPISDYEFSMSG- -NYPYHKTCYfcEHY-H 182
Os_GI-218197709.pro GCHSEIGHGRFLSCMGAVWHP3CFRCHACNQPIYDYEFSMSG- -NHPYHKTCY^ERF-H 573
Os_GI-l 154 66772. ro GCHSEIGHGRFLSCMGAVWHPZCFRCHACNQPIYDYEFSMSG- -NHPYHKTCYfcERF-H 181
Bd_GI-357160893.pro GCHSEIGHGRFLSCMGAVWHP3CFCCHACSQPIYDYEFSMSG- 207
Bd_GI-357164 660.pro GCHSEIGHGRFLSCMGAVWHPECFCCHGCSQPIYDYEFSMSG- 189
Sb_GI-242092232 .pro GCHREIGHGRFLSCMGAVWHPECFRCHACSQPIYDYEFSMSG- 181
Zm_GI-2.2275448.pro GCHREIGHGRFLSCMGAVWHPECFRCHACSQPIYDYEFSMSG- -NHPYHKTCYkEQF-H 204
At_GI-240256211.pro GCQAEIGHGRFLSCMGGVWHPECFCCNACDKPIIDYEFSMSG- -NRPYHKLCYkEQH-H 247
At_GI-145360B06.pro GCNSDIGSGNYLGCMGTFFHPECFRCHSCGYAITEHEFSLSG- -TKPYHKLCFkELT-H 219
At_GI-22326876.pro DCKSAIEDGISINAYGSVWHPQCFCCLRCREPIAMNEISDLR- — GMYHKPCYKELR-H 1296
At_GI-30698242.pro DGKSEIGDGTSVN PRCLCCFHCHRPFVMHEILKK GKFHIDCYKEYYRN 128
At_GI-30698240.pro GCNSAVKHEESVNILGVLWHPGCFCCRSCDKPIAIHELENHVSNSRGKFHKSCYfeR 404
At_GI-1524001B.pro GCNFAVEHGGSVNILGVLWHPGCFCCRACHKPIAIHDIENHVSNSRGKFHKSC 343
At_GI-334188680.pro GCKSAIEYGRSVHALGVNWHPECFCCRYCDKPIAMHEFS NTKGRCHITCYERSH— 256
Zm_GI-212275448.pro P :DVCKQFIPTNMNGLIEYI AHPFWVQKYCPSHEMDGTPRCCSCERMEPRESKYVLLDD 264
At_GI-24 02562 11. pro P :DVCHNFIPTNPAGLIEYRAHPFWMQKYCPSHERDGTPRCCSCERMEPKDTKYLILDD 307
At_GI-145360806.pro P ;EVCHHFIPTNDAGLIEYRCHPFWNQKYCPSHEYDKTARCCSCERLESWDVRYYTLED 279
At_GI-22326B76.pro P ;YVCEKKIPRTAEGL-KYHEHPFWMETYCPSHDGDGTPKCCSCERLEHCGTQYVMLAD 1355
At_GI-30698242.pro R ;YVCQQKI PVNAEGIRKFSEHPFWKEKYCPIHDEDGTAKCCSCERLEPRGTNYVMLGD 188
At_GI-30698240.pro -Y;YVCKEKK MKTYNIHPFWEERYCPVHEADGTPKCCSCERLEPRGTKYGKLSD 457
At_GI-1524 0 1 .pro - YVCKEKK MKTYNNHPFWEERYCPVHEADGTEKCCSCERLEPRESNYVMLAD 396
At_GI-334188680.pro PK :HVCKKKFP GRKYKEHPFWKEKYCPFHEVDGTPKCCSCERLEPWGTKYVMLAD 311
Si G I-514815267 .pro GRRLCLEC LHTAIMDTNECQPLYIDIQEFYEGMNMKVEQQVPLLLVERQALNEAMEAEKI 318
Bd[[ -357157184 .pro GRKLCLEC LTSATMDSPECQHLYMDIQEFFEGLNMKVEQQVPLLLVERQALNEALEAEKS 300
B _DAlb .pro GRKLCLEC L.DSSVMDTFQCQPLYLQIQEFYEGLNMTVEQEVPLLLVERQALNEAREGERN 332
B " DAla.pro GRKLCLEC LDSAVMDTFQCQPLYLQIQEFYEGLFMKVEQDVPLLLVERQALNEAREGEKN 349
At_ G I-15221983. pro GRKLCLEC LDSAVMDTMQCQPLYLQIQNFYEGLNMKVEQEVPLLLVERQALNEAREGEKN 349
T "G I-508722773 .pro GRKLCLEC LDSAVMDTKQCQPLYLDILEFYEGLNMKVEQQVPLLLVERQALNEAREGEKN 294
Gm_ -356564241 .pro GRKLCLEC LDSSIMDTNECQPLHADIQRFYDSLNMKLDQQIPLLLVERQALNEAREGEKN 294
Gm "G I-356552145 .pro GRKLCLEC LDSAIMDTNECQPLHADIQRFYESLNMKLDQQIPLLLVERQALNEAREGEKN 298
Vv[[GI -302142429 .pro GRKLCLEC LDSAIMDTNECQPLYLDIQEFYEGLNMKVQQQVPLLLVERQALNEAMEGEKS 296
Vv G I -359492104 .pro GRKLCLEC LDSAIMDTNECQPLYLDIQEFYEGLNMKVQQQVPLLLVERQALNEAMEGEKS 299
S " I -460385048 .pro GRKLCLEC LDSAIMDTSQCQPLYYDIQEFYEGLNMKVEQKVPLLLVERQALNEAMDGERH 302
Os""G I-218197709 .pro GRKLCLEC LDSAVMDTSECQPLYLEIQEFYEGLNMKVEQQVPLLLVERQALNEAMEGEKT 693
Os"~-115466772 .pro GRKLCLEC DSAV DTSECQPLYLEIQEFYEGLNMKVEQQVPLLLVERQALNEAMEGEKT 301
B "[G -357160893 .pro GRKLCLEC DSAVMDTTECQPLYLEIQEFYEGL KVEQQVPLLLVERQALNEAMEGEKT 327
Bd G I-357164660 .pro GRKLCLEC -DSAVMDTTECQPLYLEIQEFYEGLNMKVEQQVPLLLVERQALNEAMEGEKT 309
Sb_ G -242092232 .pro GRKLCLEC LDSAVMDTNECQPLYLEIQEFYEGLNMKVEQQVPLLLVERQALNEAMEGEKA 301
Zm "G -212275448 .pro GRKLCLEC LDSAVMDTNDCQPLYLEIQEFYEGLNMKVEQQVPLLLVERQALNEAMEGEKA 324
At" G I -240256211 .pro GRKLCLEC LDSAIMDTHECQPLYLEIREFYEGLHMKVEQQIPMLLVERSALNEAMEGEKH 367
At""G I-145360806 .pro GRSLCLECHETAITDTGECQPLYHAIRDYYEGMYMKLDQQIPMLLVQREALNDAIVGEKN 339
A t [G -22326B76.]pro FRWLCREC ^DSAIMDSDECQPLHFEIREFFEGLHMKIEEEFPVYLVEKNALNKAEKEEKI 1415
At"[GI -30698242. ro FRWLCIEC yiGSAVMDTNEVQPLHFEIREFFEGLFLKVDKEFALLLVEKQALNKAEEEEKI 2 8
At_[GI -3069B240.]pro GRWLCLEC -KSAMDSDECQPLYFDMRDFFESLNMKIEKEFPLILVRKELLNK--KEEKI 514
At" -15240018.]pro GRWLCLEC4NSAVMDSDECQPLHFDMRDFFEGLNMKIEKEFPFLLVEKQALNKAEKEEKI 456
At"[GI -334188680 .pro NRWLCVKC tfECAVMDTYECQPLHFEIREFFGSLNMKVEKEFPLLLVEKEALKKAEAQEKI 371
Si_GI-514815267.pro G-HHLP ETRGLCLSEEQIVRTILRRPI -GPGNRI IDMITG YKLVRRCEVTAILIL 3 3
Bd_GI-357157184.pro G-HHLP ETRGLCLSEEQIVRTILRRPTI -GPGNRI DMITG YKLVRRCEVTAILIL 355
Br_DAlb.pro GHYHMP ETRGLCLSEEQTVRTVRKRSK GNWSGNMITEQFKLTRRCEVTAI L L 385
Br_DAla.pro GHYH P ETRGLCLSEEQTVSTVRKRSKH-GTGNWAGNMITEPYKLTRQCEVTAILIL 405
At_GI-15221983.pro GHYHMP ETRGLCLSEEQTVSTVRKRSKH-GTGKWAGN-ITEPYKLTRQCEVTAILIL 404
Tc_GI-508722773.pro GHYHMP ETRGLCLSEEQTVSTILRQPRF-GTGNRAMDMITEPCKLTRRCEVTAILIL 350
Gm_GI-356564241.pro GHYHMP ETRGLCLSEE — LSTFSRRPRL-G TAMDMRAQPYRPTTRCDVTAILVL 345
Gm_GI-356552145.pro GHYHMP ETRGLCLSEE — LSTFSRRPRL-G TTMDMRAQPYRPTTRCDVTAILIL 349
Vv_GI-302142429.pro GHHHMP ETRGLCLSEEQTVSTILRRPKI-GTGNRVMNMITEPCKLTRRCDVTAVLIL 352
Vv_GI-359492104.pro GHHHMP ETRGLCLSEEQTVSTILRRPKI-GTGNRVMNMITEPCKLTRRCDVTAVLIL 355
Sl_GI-460385048.pro GYHHMP ETRGLCLSEEQTISTIQRRPRI-GAGNRVMDMRTEPYKLTRRCEVTAILIL 358
Os_GI-218197709.pro GHHHLP ETRGLCLSEEQTVSTILRRPRM-AGN-KVMEMITEPYRLTRRCEVTAILIL 48
Os_GI-115466772.pro GHHHLP ETRGLCLSEEQTVSTILRRPRM-AGN-KVMEMITEPYRLTRRCEVTAILIL 356
Bd_GI-357160893.pro GHHHLP ETRGLCLSEEQTVSTILRRPRM-TGN-KIMEMITEPYRLTRRCEVTAILIL 382
Bd_GI-357164660.pro GHHHLP ETRGLCLSEEQTVSTILRRPRM-AGN-KIMEMRTEPYRLTRRCEVTAILIL 364
Sb_GI-242092232.pro GHHHLP ETRGLCLSEEQTVSTILRRPRM-AGN-KIMGMITEPYRLTRRCEVTAILIL 356
Zm_GI-212275448.pro GHHHLP ETRGLCLSEEQTVSTILR-PRM-AGN-KIMGMITEPYRLTRRCEVTAILIL 378
At_GI-240256211.pro GHHHLP ETRGLCLSEEQTVTTVLRRPRI-GAGYKLIDMITEPCRLIRRCEVTAILIL 423
At_GI-145360806.pro GYHHMP ETRGLCLSEEQTVTSVLRRPRL-G-AHRLVGMRTQPQRLTRKCEVTAILVL 394
At_GI-22326876.pro DKQGDQCLMWRGICLSEEQIVTSVSQGVRR-MLNKQILDTVTESQRVVRKCEVTAILIL 1474
At_GI-30698242.pro DYHR AAVTRGLCMSEEQIVPSIIKGPRMGPDNQLITDIVTESQRVS-GFEVTGILII 304
At_GI-30698240.pro DNHY EVLIRAYCMSEQKIMTYVSEEPRT-GQNKQLIDMDTEPQGVVHECKVTAILIL 570
At_GI-15240018.pro DYQY EVVTRGICLSEEQI VDSVSQRPVR-GPNNKLVGMATESQKVTRECEVTAI L 512
At_GI-334188680.pro DNQH GWTRGICLSEGQIVNSVFKKPTM-GPNGELVSLGTEPQKVVGGCEVTAILIL 427
Si_GI-514815267.pro YGLPRLLTGSILAHEMMHAYLRLK GYRTLSPEV 406
Bd_GI-357157184.pro YGLPRLQTGS ILAHEMMHAYLRLK GYRSLSPQV 388
Br_DAlb.pro FGLPRLLTGSILAHEMMHAWMRLK GFRPLSQDV 418
Br_DAla.pro FGLPRLLTGSILAHEMMHAWMRLK GFRTLSQDV 438
At_GI-15221983 .pro FGLPRLLTGSILAHEMMHAWMRLK GFRTLSQDV 437
Tc_GI-508722773.pro YGLPRLLTGS LAHEMMHAWMRLQ GFRTLSQDV 383
Gm_GI-356564241.pro YGLPRLLTGSILAHEMMHAWLRLK GYRTLSQDV 378
Gm_GI-356552145.pro YGLPRLLTGSILAHEMMHAWLRLK GYRTLSQDV 382
Vv_GI-302142429.pro YGLPRLLTGS LAHEMMHAWLRLN GYRTLAQDV 385
Vv_GI-359492104 .pro YGLPRLLTGS ILAHEMMHAWLRLN GYRTLAQDV 388
Sl_GI-460385048.pro YGLPRLLTGSILAHEMMHAWLRLR GYRTLSQDV 391
Os_GI-218197709.pro YGLPRLLTGSILAHEMMHAWLRLK GYRTLSPDV 781
Os_GI-115466772.pro YGLPRLLTGSILAHEMMHAWLRLK GYRTLSPDV 389
Bd_GI-357160893.pro YGLPRLLTGSILAHEMMHAWLRLK GYRTLSPEI 415
Bd G I-357164660 .pro YGLPRLLTGSI LAHEMMHAWLRLK GYRTLSPDI 397
Sb__G I -242092232 .pro YGLPRLLTGSI LAHEMMHAWLRLK GYRTLSPDV 389
Zm _GI -212275448 .pro YGLPRLLTGSI LAHEMMHAWLRLK GYRTLSPDV 411
At""G I-240256211 .pro YGLPRLLTGSI LAHEMMHAWLRLN GYPNLRPEV 456
-145360806 .pro YGLPRLLTGAILAHELMHGWLRLN GFRNLNPEV 427
At "G I-22326876. pro YGLPRLLTGYILAHEMMHAYLRLN GYRNLNMVL 1507
At""G -30698242. pro YGLPRLLTGYI LAHEMMHAWLRLN GYKNLKLEL 337
At" -30698240. pro YGLPRLLTGYI LAHEMMHAWLRLN GHMNLNNIL 603
At""G I-15240018. pro YGLPRLLTGYILAHEMMHAYLRLN GHRNLNNIL 545
. -334188680 .pro YGLPRLLTGYILAHEMMHAWLRLNGTTSTQFVFANQYGESSQLKVLFGLITGYRNLKLEL 487
Si_GI-514815267.pro EEGICQVLAHLWLESEITSGSGSMATTSAASSS SSTS— SSSKKGA-KTEFEKRL 458
Bd_GI-357157184.pro EEGICQVLSHMWLESEIIAGASGNTASTSVPSS SSAP— TSSKKGA-KTEFEKRL 440
Br_DAlb.pro EEGICQVMAHKWLEAELAAGSRNSNAASSSSSS Y GGVKKGP-RSQYERKL 467
Br_DAla.pro EEGICQVMAHKWLEAELAAGSRNSNVASSSSS RGVKKGP-RSQYERKL 485
At_ GI- 15221983.'pro EEGICQVMAHKWLDAELAAGSTNSNAASSSSSS QGLKKGP-RSQYERKL 485
Tc "GI- 508722773 .pro EEGICQVLAHMWLLTQLEYAS-SSNVASASSSA S SRLQKGK-RPQFEGKL 431
Gm_ GI- 356564241 .pro EEGICOVLAHMWLESELSSASGSNFVSASSSSA S HTSRKGK-RPQFERKL 427
Gm_ 3I- 356552145 .pro EEGICQVLSHMWLESELSSASGSNFVSASSSSA S HTSRKGK-RPQFERKL 431
Vv_ GI- 302142429 .pro EEGICQVLAYMWLDAELTSGSGR SQCERKL 415
Vv GI- 359492104 .pro EEGICQVLAYMWLDAELTSGSGSNV-PSTSSAS TSSKKGA-GSQCERKL 435
Si""GI- 460385048 .pro EEGICQVLAHMWLETQIASISSSNGGASTSSGM SSSKQGI-RSPFERKL 439
Os GI- 218197709 .pro EEGICQVLAHMWIESEIIAGSGSNGASTSSSSS AS TSSKKGG-RSQFERKL 831
Os""GI- 115466772 .pro EEGICQVLAHMWIESEIIAGSGSNGASTSSSSS AS TSSKKGG-RSQFERKL 439
Bd G I- 357160893 .pro EEGICQVLAHMWIESEIMAGSSSNAASTSSSSS SS ISSKKGG-RSQFERKL 465
Bd""GI- 357164660 .pro EEGICQVLAHMWIESEITAGSGSNAASTSSSST S SKKGG-RSQFERKL 444
Sb'_"GI- 242092232 .pro EEGICQVLAHLWIESEIMAGSGSGAASSSSGSS SS MSSKKAG-RSQFEHKL 439
Zm_ GI- 212275448 .pro EEGICQVLAHMWIESEIMAGSGSSAASSSSGSS SS TSSKKGG-RSQFEHRL 461
t _GI- 240256211 .pro EEGICQVLAHMWLESETYAGSTLVDIASSSSSA W S ASSKKGE-RSDFEKKL 507
At_ GI- 145360806 .pro EEGICQVLSYMWLESEVLSDPSTRNLPSTSSVA TSSSSSFSNKKGG-KSNVEKKL 481
At_]GI- 22326876. pro EEGLCQVLGYMWLECQTYVFD TATIASSS — SSSRTPLSTTTSKKVD-PSDFEKRL 1560
At "GI- 30698242. pro EEGLCQALGLRWLESQTFASTDAAAAAAVASSSSFSSSTAPPAAITSKKSDDWSIFEKKL 3 7
At__GI- 30698240. pro EEGICQVLGHLWLESQTYATADTTADAASASSS SSRTPPAASASKKGE -WSDFDKKL 659
At GI- 15240018. pro EEGICQVLGHLWLDSQTYATADATADASSSASS SSRTPPAASASKKGE -WSDFDKKL 601
At GI- 334188680 .pro EEGICQVLGHMWLESQTYS SSAAASSASS SSRTP-AANASKKGA-QSDYEKKL 538
Si_GI-514815267.pro GEFFKHQIETDPSVAYGDGFRAGMRAVERYG — LRSTLDHIKLTGSFP 504 SEQ 4
Bd_GI-357157184.pro GAFIKNQIETDSSVEYGDGFRAGNRAVERYG— LRSTLDHMKITGSFPY 487 SEQ 5
Br_DAlb.pro GEFFKHQIESDASPVYGDGFRAGRLAVNKYG— LWRTLEHIQMTGRFPV 514 SEQ 6
Br_DAla.pro GEFFKHQIESDASPVYGDGFRAGRLAVNKYG— LPKTLEHIQMTGRFPV 532 SEQ 7
At_GI-15221983.pro GEFFKHQIESDASPVYGDGFRAGRLAVHKYG — LRKTLEH QMTGRFPV 532 SEQ 8
Tc_GI-508722773.pro GEFFKHQIESDTSPVYGDGFRAGHQAVYKYG— LRRTLEHIRMTGRFPY 478 SEQ 9
Gm_GI-356564241.pro GEFFKHQI ESDI SPVYGDGFRAGQKAVRKYG — LQRTLHHIRMTGTFPY 474 SEQ 10
Gm_GI-356552145.pro GEFFKHQIESDISPVYGGGFRAGQKAVSKYG— LQRTLHHIRMTGTFPY 478 SEQ 11
Vv_GI-302 142429. ro GQFFKHQIESDTSLVYGAGFRAGHQAVLKYG — LPATLKHIHLTGNFPY 462 SEQ 12
Vv_GI-359492104.pro GQFFKHQIESDTSLVYGAGFRAGHQAVLKYG — LPATLKHIHLTGNFPY 482 SEQ 13
Sl_GI-460385048.pro GDFFKHQIESDTSPIYGNGFRAGNQAVLKYG — LERTLDHIRMTGTFPY 486 SEQ 14
Os_GI-218197709.pro GDFFKHQIESDTSMAYGDGFRAGNRAVLQYG— LKRTLEHIRLTGTFPF 878 SEQ 15
Os_GI- 15 66772. pro GDFFRHQIESDTSMAYGDGFRAGNRAVLQYG — LKRTLEHIRLTGTFPF 486 SEQ 16
Bd_GI-357160893.pro GDFFKHQIESDTSVAYGNGFRSGNQAVLQYG — LKRTLEHIWLTGTWPF 512 SEQ 17
Bd_GI-357164660.pro GDFFKHQIESDTSVAYGDGFRAGNQAVLQYG — LKRTLEHIRLTGTLPF 491 SEQ 18
Sb_GI-242092232.pro GDFFKHQIETDTSMAYGEGFRAGNRAVLQYG — LKRTLEHIRLTGTFPF 486 SEQ 19
Zm_GI-212275448.pro GDFFKHQIETDTSMAYGDGFRTGNRAVLHYG — LKRTLEHIRLTGTFPF 508 SEQ 20
At_GI-240256211.pro GEFFKHQI ESDSSSAYGDGFRQGNQAVLKHG — LRRTLDHIRLTGTFP 553 SEQ 21
At_GI-145360806.pro GEFFKHQI AHDASPAYGGGFRAANAAACKYG — LRRTLDHIRLTGTFPL 528 SEQ 22
At_GI-2232687 6.pro VNFCKHQIETDESPFFGDGFRKVNKMMASNNHSLKDTLKEIISISKTPQYSKL 1613 SEQ 23
At_GI-30698242.pro VEFCMNQIKEDDSPVYGLGFKQVYEMMVSNNYNIKDTLKDIVSASNATPDSTV 450 SEQ 24
At_GI-30698240.pro VEFCKNQIETDESPVYGLGFRTVNEMVTNS— SLQETLKEILRRR 702 SEQ 25
At_GI-15240018.pro VEFCKNQIETDDSPVYGLGFRTVNEMVTNS — SLQETLKEILRQR 644 SEQ 26
At_GI-334188680.pro VEFCKDQIETDDSPVYGVGFRKVNQMVSDS--SLHKILKSIQHWTKPDSNL-587 SEQ 27
Table 1 Alignment o f DAI proteins (SEQ ID OS : 4 to 2 7 )
Pt_GI-224059640.pro MEVHYMNTDFPYTTTESFMDFFEGLTHAPV 30
Rc_GI-255582236.pro MEVHYINTGFPYTVTESFLDFFEGLSHVPV 30
Pp_GI-462414664 .pro MNGN — GQMDVHYIDTDFPYTPTESFMDFFGGVTHVPM 36
Tc_GI-508704801.pro MNGN — RQMEVHYIDTGFPYTATESFMDFFEGLTHVPV 36
Vv GI-359487286.pro M — RQMEVHYINTGFPYTITESFMDFFEGLGHVPV 36
Gm_GI-356548935.pro MNDG — RQMGVHYVDAGFPYAVNDNFVDFFQGFTHVPV 36
Gm_GI-35 4 1 6.pro MNDG — RQMGVNYVDAGFPYAVNENFVDFFQGFTPVPV 36
At_EODl.pro MNGDNRPVEDAHYTETGFPYAATGSYMDFYGGAAQGPL 38
Cr_GI-482561003.pro MNGD-RPVEDAHYTEAEFPYAASGS DFYGGAPQGPL 37
Sb_GI-242042045 .pro MNSC — RQMELHYINTGFPYTITESFMDFFEGLTYAHA 36
Zm_GI-223973923.pro MNSS — RQMELHYINTGFPYTITESFMDFFEGLTYAHA 36
Zm_GI-226496789.pro MTSS — RQMELHYINTGFPYTITESFMDFFEGLTYAHA 36
Os_GI-222624282 .pro MTESHERDTEVTRWQVHDPSEGMNGS — RQMELHYINTGFPYTITESFMDFFEGLTYAHA 58
Os_GI- 115451045. pro MNGS — RQMELHYINTGFPYTITESFMDFFEGLTYAHA 36
Bd_GI-357113826.pro MNGS — RQMELHYINTGFPYTITESFMDFFEGLTYAHA 36
Sl_GI-460410949.pro MNW — QQTEIYYTNGAMPYNSIGSFMDFFGGVTYDHV 36
* : :** .::**: *
Pt_GI-224059640.pro YAH GPMHD QDNAYWSMN-MNAYKFGFSGLGSTSYYSP YEVNDNLPRMDVSRM 83
Rc_GI - 5 582236. pro HYAHTGQVLDQ-VQENAYWSMN-MNAYKYGFSGPGST-YYDP YEVNDNLPRMDVSRS 8
Pp_GI-462414664 .pro NYGHAMPMHD QETAYWSMN-MHSYKFGPSGPGSNSYYGNY — YEVNDHLPRMDVSRR 90
Tc_GI - 08704801. ro YTHTVPMQD QEN YWSMS-MNAYKFGFSGPEST-FYSP YEVSDHLPRMDVSRR 88
Vv GI-359 872 86 . ro NYAQAEAMHNQSIQENFYWTMN-MNSYKFGFSGPGST-YYGP YDVNEHVPGIEVSRR 91
G _G -3 8 35.pro NYAFAGS D QESVYWSMN-MNPYKFGLSGPGSTSYYSS YEVNGHLPRMEIDRA
Gm_GI-35654417 6.pro NYAFAGSIPD QESVYWSMN-MNPYKFGLSGPGSTSYYSS YEVNGHL RME RA 89
At_E0Dl . ro NYDHAATMHP QDNLYWTMN-TNAYKFGFSGSDNASFYGS YDMNDHLSRMSIGRT 91
Cr_GI -4825 61003. ro NYAHAGTM DNLYWTMN-TNAYKFGFSGSDNPSFYNS YDMTDHLSRMSIGRT 87
Sb_GI-242042045.pro DFALMDGFQD QGNPYWAMMHTNSYKYGYSGPG — NYYTYAHVYDIDDYMHRADGGRR 91
Zm_GI-223973923.pro DFALTDGFQD QGNPYWAMMHTNSYKYGYSGPG — NYYSYAHVYDIDDYMRRADGGRR 91
Zm_GI -22 4 78 . ro DFALMDGFQD QGNPYWTMMHTNSYKYGYSGSG — NYYSYAHAYDIDDYMHRTDGGRR 91
Os_GI -222624282. ro DFAIADAFHD QANP YWAMMHTNS YKYGYSGAG — NYYSYGHVYDMNDYMHRADGGRR 113
Os_GI- 115451 045 .pro DFAIADAFHD QANPYWAMMHTNSYKYGYSGAG — NYYSYGHVYDMNDYMHRADGGRR 1
Bd_GI- 357113826. pro DFALADAFQD QANP YWTMMQTNSYKYGYSGAS — NYYSYGHVYDMNDYMHRADGGRR 1
Sl_GI-460410949.pro NYIFADPPYA QES-LYPSIST YKFGYSEAGS SYYDYDRE YVVNDHVSGIEEHDR 92
. **.+ .+ * . .
Pt_GI-224059640.pro AWEYPSVV IKALWQDDVDPDT 104
Rc_GI-255582236.pro TWEYPSVVN-MEEATTTDTQSEGDAVVGVHASPEECIPN-HT-SGDSPQGVWQDDVDPDN 141
Pp_GI -462414664. pro TWEHPSVMN-SEEPANI DSHPEEED-AVAEAAPEECIQN-QQ-NTNTSQVVWQEDIDPDN 1 6
Tc_GI-508704801.pro TWDYPSTLN-SEEPATIDMQPGGEAVVGIHAI PEECITN-HQ-SNSNSQVVWQDNIDPDN 145
Vv G -359 8728 6 . ro PWEYPSSMI-VEEPTTIETQPTGNEVMNVHAIPEECSPN-HY-SATSSQAIWQDNVDPDN 148
Gm_GI -356548935. pro EWEYPSTITTVEEPATTDSPPRRDGVTSMQTI PEECSPN-HHESNSSSQVIWQDNI YPDD 148
Gm_GI-35654417 6.pro EWEYPSTITTVEEPATTDSPPRRDGVTNMQTIPEECSPN-HHESNSSSQVIWQDNIDPDN 148
At_EODl .pro NWDYHPMVNVADDPENTVARSVQIGDTDEHSEAEECIAN-EH-DPDSPQVSWQDDIDPDT 149
Cr_GI-4825 61003. pro NWEYHPMVNVDD-PDITLARSVQIGDSDEHSEAEDCIAN-EH-DPDSPQVSWQDDIDPDT 144
Sb_GI -242042045. ro VWDNTTPANNVDSANVVLQGS-EAPRTTANTTTEECIQQ-VHQSPGSPHVVWQDNIDPDN 149
Zm_GI-223973923.pro IWDNTTPVNNVDSANVVLQGG-EAPHTTTNTINKECIQQ-VHQSPGSPQVVWQDNIEPDN 149
Zm_GI -226496789. ro TWDNTTPVNNVDSANVVLQGG-EAPRTTANTTSEDCIQQ-VHQSPGSPQVVWQDNIDPDN 149
OS_GI-222624282 .pro IWDNATPVNNTESPNVVLQGG-ETPHANTSSTTEECIQQQVHQNSSSPQVIWQDNIDPDN 172
OS_GI-115451045.pro IWDNATPVNNTESPNVVLQGG-ETPHANTSSTTEECIQQQVHQNSSSPQVIWQDNIDPDN 150
Bd_GI-3 57113826.pro IWDNPTPASNTDSPNVVLQGAAEAPHPRASSTTEECIQQPVHQNSSSPQVVWQDNVDPDN 151
Sl_GI-4 60410949.pro HLENPSTTTVNVAANVHRE EISGSNSLTNSVECPRG — QINTRDSEVVWQDNIDPDN 147
Pt_GI 24059640 .pro MTYEELVDLGETVGTQSKGLSPELISLLPTSKCKFGSFFSRKRSG-ER:VICQMKYKRGD
Rc_GI-2 55582236 .pro MTYEELLDLGETVGTQSRGLSQELISLLPTSKCKFRSFFLRKKAG-ER;VICQMRYKRGD
Pp_GI-4 62414664 .pro MTYEELLDLGEAVGTQSRGLSDELISLLPTSKYKCGSFFSRKKSG-ER :VICQMRYKRGD
Tc_GI-5 08704801 .pro MTYEELLDLGETIGSQSRGLSQELIDLLPTSKCKFGSFFSTKR — E ;VICQMRYKRGE
Vv G 35948728 6 .pro MTYEELLDLGEAVGTQSRGLSQEHINLLPTCRYKSGRLFSRKRSA-ER:VICQMGYKRGD
Gm_GI- 56548935 .pro MTYEELLDLGEAVGTQSRGLSQELIDMLPTSKYKFGSLFKRKNSG-KR ;VICQMTYRRGD
Gm_GI-3 56544176 .pro MTYEELLDLGEAVGTQSRGLSQELIDMLPTSKYKFGNLFKRKNSG-KR:VICQMTYRRGD
At SODl pro MTYEELVELGEAVGTESRGLSQELIETLPTKKYKFGSIFSRKRAG-ER:VICQLKYKIGE
Cr_GI-4 82561003 .pro MTYEELVELGEAVGTESRGLSQELIETLPTRKFKFGSIFSRKRAG-ER ;VICQLKYKIGE
Sb_GI- 42042045 .pro MTYEELLDLGEVVGTQSRGLSQERISSLPVTKYKCG-FFSRKKTRRER :VICQMEYRRGN
Zm_GI-2 23973923 .pro MTYEELLDLGEAVGTQSRGLSQERISSLPVTKYKCG-FFSRKKTRRER :VICQMEYRRGN
Zm_GI-2 264967B9 .pro MTYEELLDLGEAVGTQSRGLSQECISLLPITKYKCG-FFSRKKTRRER ;VICQMEYRRGN
Os_GI- 226242B2 .pro MTYEELLDLGEAVGTQSRGLSQERISLLPVTKYKCG-FFSRKKTRRER ;VICQMEYRRGN
Os_GI-l 15451045 .pro YEELLDLGEAVGTQSRGLSQERI SLLPVTKYKCG-FFSRKKTRRER :VICQMEYRRGN
Bd_GI-3 57113B26 .pro MTYEELLDLGEAVGTQSRGLSQERI SSLPVTKYKCG-FFSRKKTRRER ;VICQMEYRRGD
SI GI 60410949 .pro M YEELLELGEAVGTQSRGLSQNQI SLLPVTKFKCG-FFSRKKSRKER :VICQMEYKRKD
****** . .*** ★**.
Pt_GI -22405 9640. ro KQIKLLCKHAYHSECITKWLGINKVCPVC DEVFGEESR 203
Rc_GI-255582236.pro KQMKLPCKHVYHSECISKWLGINKVCPVC tfNEVFGEDSRH 240
Pp_GI-462414664.pro RQINLPCKHVYHSECISKWLGINKVCPVC tfLEVSGEESRH 245
Tc_GI-5 08704 01.pr QQMKLPCKHVYHSQCITKWLSINKICPVC NEVFGEESR 242
Vv GI- 35948728 6 . ro RQIKLPCKHVYHTDCGTKWLTINKVCPVC SIIEVFGEESRH 2 7
Gm_GI-356548935.pro QQMKLPCSHVYHGECITKWLSINKKCPVC NTEVFGEESTH 247
G _GI- 65 176.pro QQMKLPCSHVYHGECITKWLSINKKCPVC STEVFGEESTH 247
At_EODl .pro RQMNLPCKHVYHSECISKWLSINKVCPVC iJSEVFGEPSIH 248
Cr_GI-482561003.pro RQMNLPCKHVYHSECISKWLSINKVCPVC UTEVFGDPSIH 2 3
Sb_GI-2 2042045. pro LQMTLPCKHVYHASCVTRWLSINKVCPVC FAEVPGDEPKRQ 249
Zm_GI-223973923.pro LQMTLPCKHVYHASCVTRWLGINKVCPVC FAEVPGEDPEAMSQQL 253
Zm_GI-22 64967 89.pro LQITLPCKHVYHASCVTRWLSINKVCPVC FAEVPGEDSLRQ 249
Os_GI-222624282.pro LQMTLPCKHVYHASCVTRWLSINKVCPVC FAEVPGDEPKRQ 272
Os_GI- 115451045. ro LQMTLPCKHVYHASCVTRWLSINKVCPVqFAEVPGDEPKRQ 250
Bd_GI-35711382 6.pro 251
Sl_GI-460410949.pro 246
Table 2 (SEQ ID NOS 3 8 to 5 3 )
Claims :
1 . A method of increasing the yield of a plant or enhancing a
yield-related trait in a plant comprising;
expressing a DAI protein within cells of said plant,
wherein said the amino acid sequence of the DAI protein
comprises a mutation that disrupts or inactivates the LIM domain or
the LIM-like domain of the DAI protein.
2 . A method according to claim 1 wherein the DAI protein is
expressed from a heterologous nucleic acid coding sequence in one or
more cells of the plant.
3 . A method of producing a plant with an increased yield and/or
one or more enhanced yield-related traits comprising:
introducing into a plant cell a heterologous nucleic acid
which encodes a DAI protein,
wherein said the amino acid sequence of the DAI protein
comprises a mutation that disrupts or inactivates the LIM domain or
the LIM-like domain of the DAI protein, or
introducing a mutation into the nucleotide sequence of a plant
cell which encodes a DAI protein, such that the LIM domain or the
LIM-like domain of the DAI protein is disruped or inactivated, and
regenerating the plant from the plant cell.
. A method according to any one of the preceding claims wherein
the plant expressing the DAI protein has increased life-span, organ
size and/or seed size relative to controls.
5 . A method according to any one of the preceding claims wherein
the DAI protein with the inactivated LIM and/or LIM-like domain has
aberrant peptidase activity relative to the wild-type DAI protein.
6 . A method according to any one of the preceding claims wherein
the LIM domain of the DAI protein is inactivated.
7 . A method according to any one of the preceding claims wherein
wherein the inactivated LIM domain of the DAI protein comprises one
or more sequence alterations relative to the wild-type LIM domain
which inactivate LIM domain activity or function.
8 . A method according to claim 6 or 7 wherein the wild-type LIM
domain has the sequence of SEQ ID NO: 1 or SEQ ID NO: 2 .
9 . A method according to any one of claims 6 to 8 wherein the
wild-type LIM domain comprises two Zn finger motifs and the sequence
alterations abolish one or both Zn finger motifs.
10, A method according to any one of claim 7 to 9 wherein the
sequence alterations include a mutation of one or more Zn
coordinating residues in the LIM domain.
11, A method according to any one of claim 7 to 10 wherein the
sequence alterations include a mutation of one or more non-Zn
coordinating residues in the LIM domain.
12, A method according to claim 11 wherein the non-Zn coordinating
residues in the LIM domain are positioned within 4 residues of a Zn
coordinating residue in the LIM domain.
13, A method according to claim 1 wherein the non-Zn coordinating
residues in the LIM domain are positioned 4 or more residues from a
Zn coordinating residue in the LIM domain.
14, A method according to any one of the preceding claims wherein
the LIM-like domain of the DAI protein is inactivated.
15. A method according to claim 14 wherein wherein the inactivated
LIM-like domain of the DAI protein comprises one or more sequence
alterations relative to the wild-type LIM-like domain which
inactivate LIM-like domain activity or function.
16. A method according to claim 14 or claim 15 wherein the wildtype
LIM-like domain has the sequence of SEQ ID NO: 28, 29, 30 or
31..
17. A method according to any one of claims 14 to 16 wherein the
wild-type LIM-like domain comprises two Zn finger motifs and the
sequence alterations abolish one or both Zn finger motifs.
18. A method according to any one of claim 14 to 17 wherein the
sequence alterations include a mutation of one or more Zn
coordinating residues in the LIM-like domain.
19. A method according to any one of claim 14 to 16 wherein the
sequence alterations include a mutation of one or more non-Zn
coordinating residues in the LIM-like domain.
20. A method according to claim 19 wherein the non-Zn coordinating
residues in the LIM-like domain are positioned within 4 residues of
a Zn coordinating residue in the LIM domain.
21. A method according to claim 19 wherein the other residues in
the LIM-like domain are positioned 4 or more residues from a
conserved cysteine residues in the LIM-like domain.
22. A method according to any one of the preceding claims wherein
the DAI protein comprises a C terminal region having at least 20%
sequence identity to residues 229 to 532 of SEQ ID NO: 8 .
23. A method according to claim 22 wherein the C terminal region
comprises the metallopeptidase motif HEMMH (SEQ ID NO: 32) .
24. A method according to claim 22 or claim 23 wherein the C
terminal region comprises the amino acid sequence EK (X) R (X) ,SEEQ
(SEQ ID NO: 33) or EK (X) R (X) 4SEQ (SEQ ID NO: 34).
25. A method according to any one of the preceding claims wherein
the wherein the DAI protein comprises a UIM1 domain of SEQ ID NO: 35
and a UIM2 domain of SEQ ID NO: 36.
26. A method according to any one of the preceding claims wherein
the DAI protein comprises one or more sequence alterations relative
to the wild-type DAI sequence which disrupt or inactivate LIM domain
activity or function.
27. A method according to claim 26 wherein the wild-type DAI
sequence comprises the amino acid sequence of any one of SEQ ID NOS :
4 to 27 or is a variant thereof.
28. A method according to claim 27 wherein the wild-type DAI
sequence comprises an amino acid sequence having at least 50%
sequence identity to any one of SEQ ID NOS: 4 to 27.
29. A method according to any one of the preceding claims wherein
the nucleic acid encoding the DAI protein is operably linked to a
heterologous promoter.
30. A method according to claim 29 wherein the promoter is a
tissue-specific promoter or an inducible promoter.
31. A method according to any one of claims 29 to 30 wherein the
nucleic acid encoding the DA protein acid is comprised in one or
more vectors.
32. A method according to any one of the preceding claims wherein
the plant or plant cell is deficient in EOD1 expression or activity.
33. A method according to any one of the preceding claims
comprising selecting a plant or plant cell having increased yield or
one or more enhanced yield-related traits compared to control
plants.
34. A method according to any one of the preceding claims
comprising comprising sexually or asexually propagating or growing
off-spring or descendants of the plant expressing the DAI protein.
35. A method according to any one of the preceding claims wherein
the plant is a higher plant.
36. A method according to claim 35 wherein the plant is an
agricultural plant selected from the group consisting of
osp erra u 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.
37. A plant cell comprising a heterologous nucleic acid encoding a
DAl protein having a disrupted or inactivated L domain.
38. A plant comprising a plant cell according to claim 37.
39. A plant according to claim 38 which is produced by a method
according to any one of claims 1 tc 36.