U.S. patent application number 13/543370 was filed with the patent office on 2012-11-01 for shine clade of transcription factors and their use.
This patent application is currently assigned to Stichting Dienst Landbouwkundig Onderzoek. Invention is credited to Asaph AHARONI, Chital Dixit, Andy Pereira.
Application Number | 20120278949 13/543370 |
Document ID | / |
Family ID | 34928288 |
Filed Date | 2012-11-01 |
United States Patent
Application |
20120278949 |
Kind Code |
A1 |
AHARONI; Asaph ; et
al. |
November 1, 2012 |
SHINE CLADE OF TRANSCRIPTION FACTORS AND THEIR USE
Abstract
The present invention relates to the field of transgenic plants
with novel phenotypes, especially plants with enhanced drought
tolerance. Provided are SHINE proteins and nucleic acid sequences
encoding these, which are useful in conferring novel phenotypes to
plants.
Inventors: |
AHARONI; Asaph; (Tel Aviv,
IL) ; Dixit; Chital; (Wageningen, NL) ;
Pereira; Andy; (Ede, NL) |
Assignee: |
Stichting Dienst Landbouwkundig
Onderzoek
Wageningen
NL
|
Family ID: |
34928288 |
Appl. No.: |
13/543370 |
Filed: |
July 6, 2012 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11629080 |
Nov 12, 2008 |
8252978 |
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PCT/NL2005/000418 |
Jun 9, 2005 |
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13543370 |
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60579325 |
Jun 14, 2004 |
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Current U.S.
Class: |
800/290 ;
435/320.1; 536/23.6; 800/278; 800/298; 800/320; 800/320.1;
800/320.2; 800/320.3 |
Current CPC
Class: |
C12N 15/8273 20130101;
C07K 14/415 20130101 |
Class at
Publication: |
800/290 ;
800/298; 800/320.1; 800/320.2; 800/320.3; 800/320; 536/23.6;
435/320.1; 800/278 |
International
Class: |
C12N 15/29 20060101
C12N015/29; C12N 15/82 20060101 C12N015/82; A01H 5/08 20060101
A01H005/08; A01H 5/00 20060101 A01H005/00; A01H 5/10 20060101
A01H005/10 |
Foreign Application Data
Date |
Code |
Application Number |
Jun 11, 2004 |
NL |
04076757.6 |
Claims
1. A transgenic monocotyledonous crop plant comprising a chimeric
gene, integrated in its genome, which chimeric gene comprises a
transcription regulatory sequence active in plant cells, operably
linked to a nucleic acid sequence encoding a SHN protein that
comprises the sequence SEQ ID NO:15.
2. The plant according to claim 1, wherein said SHN protein further
comprises the sequence SEQ ID NO:16.
3. The plant according to claim 1, the phenotype of which is one or
more of: (i) enhanced drought tolerance without modification of its
epicuticular wax layer, (ii) enhanced wound healing properties,
(iii) enhanced suberization, (iv) enhanced salinity tolerance, and
(v) fruit with an enhanced weight percentage of soluble solids.
4. The plant according to claim 1, wherein the plant is male
sterile or has enhanced pod shatter resistance, and wherein said
SHN protein further comprises a fused repressor domain.
5. The plant according to claim 4, wherein the repressor domain is
the EAR repressor domain the sequence of which is SEQ ID NO:21.
6. The plant according to claim 1, wherein said transcription
regulatory sequence is selected from the group consisting of: a
constitutive promoter, an inducible promoter, a tissue-specific
promoter and a developmentally-regulated promoter.
7. The plant according to claim 1, wherein the plant is selected
from a genus Zea, Oryza, Triticum, Hordeum, Avena or Sorghum.
8. The plant according to claim 1, wherein the sequence of the SHN
protein is selected from the group consisting of SEQ ID NO:11, SEQ
ID NO:12, SEQ ID NO:13, SEQ ID NO:14 and SEQ ID NO:24.
9. A seed or a fruit of a plant according to claim 1, which seed or
fruit comprises said chimeric gene.
10. A chimeric gene comprising a tissue specific-, inducible- or
developmentally-regulated promoter active in plant cells, operably
linked to a nucleic acid sequence encoding a SHN protein that
comprises the amino acid sequence SEQ ID NO:15.
11. A nucleic acid vector comprising the chimeric gene according to
claim 10.
12. A method of generating a transgenic plant according to claim 1,
comprising introducing and expressing in said plant a nucleic acid
sequence encoding said SHN protein, thereby generating said
plant.
13. The method of claim 12, wherein: (a) the plant has one or more
of the following properties: enhanced drought tolerance and an
unmodified epicuticular wax layer, enhanced male sterility,
enhanced pod shatter resistance, enhanced wound healing, enhanced
suberization, enhanced salinity tolerance and fruit with an
enhanced weight percentage of soluble solids, and (b) the
introduced SHN protein comprises the amino acid sequence SEQ ID
NO:15.
Description
FIELD OF THE INVENTION
[0001] The present invention relates to a novel class of
transcription factors, referred herein to as the SHINE clade of
transcription factors, and their use to confer various novel
phenotypes onto plants, such as drought tolerance and other water
use related phenotypes, indehiscence of plant dehiscence zones
(conferring for example male sterility or pod shatter resistance)
and modification of other cell layers involved in cell separation
or in the cell--environment interface. The invention provides
nucleic acid sequences encoding SHINE (SHN) proteins, or functional
fragments thereof, which are useful for modifying, or newly
conferring, one or more novel plant phenotypes. Further provided
are isolated SHINE proteins, chimeric genes, nucleic acid vectors,
recombinant microorganisms and plants, as well as methods and means
for using SHINE (SHN) nucleic acid sequence to confer novel plant
phenotypes.
BACKGROUND OF THE INVENTION
[0002] The interface between plants and the environment plays a
dual role as a protective barrier, as well as a medium for the
exchange of gases, water and nutrients. The primary aerial plant
surfaces (including leaves, stems, flowers, fruit) are covered by a
cuticle, acting as a protective layer, which plays a role in
regulating water loss and protects the plant against the
surrounding environment (e.g., pathogen damage, insect damage,
mechanical damage, UV radiation, frost) (Sieber et al. 2000, Plant
Cell 12:721-737). It is a heterogeneous layer composed mainly of
lipids, namely cutin and intracuticular wax, with epicuticular
waxes deposited on the surface and has an important role in
regulating epidermal permeability and non-stomatal water loss.
Without the protective cuticle, transpiration of most land plants
would be so rapid that death would result. Cuticle metabolism and
the structure of the epidermal surfaces are, therefore, crucial
factors in determining plant water management and in protecting
plants from environmental stress, both abiotic stresses (such as
drought, freezing, salinity, wind, metals, etc.) and biotic
stresses (such as plant pathogens or insects). In addition the
cuticular layer also has a role in normal plant development
processes including the prevention of post-genital organ fusion and
pollen-pistil interactions and it has been suggested that cuticle
permeability in such processes will also influence cell-to-cell
communication by enhancing or attenuating the passage of signal
molecules (Pruitt et al. 2000, PNAS USA 97, 1311-1316; Sieber at
al. 2000, supra). Such signals could be, for example, required for
organ adhesion (moving across the cuticle), or mediating signalling
between trichomes and stomata (moving within the developing
epidermis) (Lolle et al., 1997, Dev. Biol. 189:311-321; Krolikowski
et al., 2003, Plant J. 35:501-511).
[0003] As tolerance to biotic and abiotic stresses has a direct
impact on plant productivity (yield and product quality),
mechanisms for conferring or enhancing stress tolerance have been
widely studied and various approaches for conferring environmental
stress tolerance have been described in the art. One of the most
serious abiotic stresses plants have to cope with world-wide is
drought stress or dehydration stress. Four-tenths of the world's
agricultural land lies in arid or semi-arid regions. Apart from
that, also plants grown in regions with relatively high
precipitation may suffer spells of drought throughout the growing
season. Many agricultural regions, especially in developing
countries, have consistently low rain-fall and rely on irrigation
to maintain yields. Water is scarce in many regions and its value
will undoubtedly increase with global warming, resulting in an even
greater need for drought tolerant crop plants, which maintain yield
levels (or even have higher yields) and yield quality under low
water availability. It has been estimated that the production of 1
kg of cotton requires about 15,000 liters of water in irrigated
agriculture, while 1 kg of rice requires 4000 liters. Conferring or
enhancing the tolerance of crop plants to short and long spells of
drought and reducing the water requirement of crops grown in
irrigated agriculture is clearly an important objective.
[0004] Although breeding (e.g., marker assisted) for drought
tolerance is possible and is being pursued for a range of crop
species (mainly cereals, such as maize, upland rice, wheat,
sorghum, pearl millet, but also in other species such as cowpea,
pigeon pea and Phaseolus bean), it is extremely difficult and
tedious because drought tolerance or resistance is a complex trait,
determined by the interaction of many loci and gene-environment
interactions. Single, dominant genes, which confer or improve
drought tolerance and which can be easily transferred into high
yielding crop varieties and breeding lines are therefore sought
after. Most water is lost through the leaves, by transpiration, and
many transgenic approaches have focused on modifying the water loss
through changing the leaves. For example WO00/73475 describes the
expression of a C4 NADP+-malic enzyme from maize in tobacco
epidermal cells and guard cells, which, according to the
disclosure, increases water use efficiency of the plant by
modulating stomatal aperture. Other approaches involve, for
example, the expression of osmo-protectants, such as sugars (e.g.,
trehalose biosynthetic enzymes) in plants in order to increase
water-stress tolerance, see e.g., WO99/46370. Yet other approaches
have focused on changing the root architecture of plants.
[0005] To date another promising approach to enhance drought
tolerance is the overexpression of CBF/DREB genes (DREB refers to
dehydration response element binding; DRE binding), encoding
various AP2/EREBP (ethylene response element binding protein)
transcription factors (WO98/09521). Overexpression of the CBF/DREB1
proteins in Arabidopsis resulted in an increase in freezing
tolerance (also referred to as freeze-induced dehydration
tolerance) (Jaglo-Ottosen et al., Science 280:104-106, 1998; Liu et
al., Plant Cell 10:1391-1406, 1998; Kasuga et al., Nat. Biotechnol.
17:287-291, 1999; Gilmour et al. Plant Physiol. 124:1854-1865,
2000) and enhanced the tolerance of the recombinant plants to
dehydration caused either by water deficiency or exposure to high
salinity (Liu et al., 1998, supra; Kasuga et al., 1999, supra).
Another CBF transcription factor, CBF4, has been described to be a
regulator of drought adaptation in Arabidopsis (Haake et al. 2002,
Plant Physiology 130:639-648).
[0006] Despite the availability of some genes which have been shown
to enhance drought tolerance in a number of plant species, such as
Brassicaceae and Solanaceae, there is a need for the identification
of other genes with the ability to confer or improve drought
tolerance when expressed in crop plants. In one embodiment, the
present invention provides a new family of genes and proteins which
fulfil this need.
[0007] Apart from the cuticle, forming a protective layer between
the leaves and the environment, plants form a range of other
protective or cell-separating layers, such as "dehiscence zones"
and suberin layers. Dehiscence zones are cell layers formed during
cell wall separation processes, such as the abscission of leaves,
flowers, fruits (e.g., pods or siliques) or in anther dehiscence.
Brassicaceae produces fruits in the form of pods (siliques) in
which the two carpel valves (ovary walls) are joined to the replum,
a visible suture that divides the two carpels. The dehiscence zone
is a layer of only one to three cells in width that extends along
the entire length of the valve/replum boundary (Meakin and Roberts,
1990, J. Exp. Botany 41:995-1002). As the cells in the dehiscence
zone separate from one another, the valves detach from the replum,
allowing seeds to be dispersed (often prematurely), which is
referred to as podshatter or seedshatter. Premature shattering
causes significant yield losses in Brassica species, such as
Brassica napus (oilseed rape or "canola" if erucic acid and
glucosinolate levels are below a certain threshold value). As
breeding for shatter resistance is virtually impossible, due to
lack of genetic variation in this trait, transgenic approaches are
being explored in order to confer shatter resistance to pod-bearing
plants, such as Brassica napus or soybean. To date such approaches
involve for example a gene referred to as "indehiscent 1" (IND1),
identified in Arabidopsis (see WO017951), MADS-Box genes AGL1, AGL5
and AGL8 (FUL) (WO99/00503), or the SGT10166 gene (WO01/59122). One
of the difficulties in transgenic podshatter approaches is that on
the one hand it is desired to prevent easy separation of the two
pod valves, on the other hand it must still remain possible to
separate the valves in order to harvest the seeds.
[0008] Another dehiscence process in flowering plants is anther
dehiscence, whereby the anther opens to release pollen grains into
the environment. Two processes are believed to contribute to anther
dehiscence, namely splitting of the anther wall which occurs at the
stomium, a specialized group of cell types running the length of
the anther, and the inversion of the anther walls which exposes the
pollen. Splitting of the anther wall involves cell-to-cell
separation at the stomium. Anther development and dehiscence
involves many genes, see for an overview Goldberg et al., 1993 (The
Plant Cell 5:1217-1229). The reduction or prevention of pollen
release from plants, or a change in the time point of pollen
release, has significant benefits, such as the production of male
sterile plants (useful, for example, for hybrid seed production,
see WO96/26283; Mariani et al. 1990, Nature 347:737-741; Mariani et
al. 1992, Nature 357:384-387) or prevention (or reduction) of
pollen release where this is undesirable, as for example because of
risks of allergenicity or risks of releasing pollen of transgenic
plants into the environment. Recombinant approaches used to date to
confer male sterility involve for example the tissue specific
expression of genes encoding cytotoxic proteins, such as the
barnase gene (Mariani et al. 1990 and 1992, supra), leading to a
selective destruction of specific cell types during anther
development (e.g., the tapetum layer).
[0009] However, there is still a need to identify novel genes which
are suitable to confer shatter resistance or male sterility to
plants, especially to crop plants. In one embodiment, the present
invention provides a new family of genes and proteins which fulfill
this need.
[0010] As mentioned above, another protective layer formed in
plants is the suberin layer, which is functionally related to the
cutin layer and also prevents water loss from specific tissues,
blocks pathogen invasion and strengthens the cell wall. Suberin is
formed as a protective layer on underground plant cell surfaces
such as the root endodermis and also as a strengthening component
in cell walls, for example in the root as a Casparian strip in the
cell wall of the root endodermis and in bundle sheath cells of
grasses. It also covers the cork cells formed in tree bark and is
deposited as scar tissue after wounding, for example as a
protective layer after leaf abscission or on the surface of wounded
potato tubers (Kolattukudy 1981, Ann. Rev. Plant Physiol.; Nawrath
2002, The biopolymers cutin and suberin, The Arabidopsis Book, Eds.
C. R. Sommerville et al., American Society of Plant Biologists,
Rockville, Md.). Similar to cutin, suberin consists of a complex
mixture of fatty acids and further contains phenolic compounds,
such as ferulic acid. Genes involved in suberization and which are
useful in modifying suberin formation in plants are generally
desirable, for example for improving wound healing properties of
tubers or strengthening root formation.
[0011] The prior art shows that there is a continuous need for
novel genes and methods which are useful for the modification of
plant protective layers (epidermis and cuticle, suberin layers) and
cell layers involved in cell-to-cell separation processes. The
present invention provides a novel class of genes which influence
the formation and metabolism of the interface between the plant
surface and the environment (wounding sites, root cap cells and
some organs at the epidermal layer) and of the interface between
cells and cell layer above ground (e.g., dehiscence zones and
abscission zones) or below ground (e.g., the endodermis). In
addition, the present invention discloses how to use this class of
genes to generate plants with novel phenotypes, especially drought
tolerance or resistance, male sterility, seed shatter resistance,
fruit (e.g., tomatoes) with more solid flesh and a higher
concentration of soluble solids, plants (especially tubers) with
improved wound healing properties or woody trees with enhanced
suberization of cork cells.
GENERAL DEFINITIONS
[0012] The term "nucleic acid sequence" (or nucleic acid molecule)
refers to a DNA or RNA molecule in single or double stranded form,
particularly a DNA encoding a protein or protein fragment according
to the invention. An "isolated nucleic acid sequence" refers to a
nucleic acid sequence which is no longer in the natural environment
from which it was isolated, e.g., the nucleic acid sequence in a
bacterial host cell or in the plant nuclear or plastid genome. The
terms "protein" or "polypeptide" are used interchangeably and refer
to molecules consisting of a chain of amino acids, without
reference to a specific mode of action, size, 3 dimensional
structure or origin. A "fragment" or "portion" of a SHINE protein
may thus still be referred to as a "protein". An "isolated protein"
is used to refer to a protein which is no longer in its natural
environment, for example in vitro or in a recombinant bacterial or
plant host cell. The term "gene" means a DNA sequence comprising a
region (transcribed region), which is transcribed into an RNA
molecule (e.g., an mRNA) in a cell, operably linked to suitable
regulatory regions (e.g., a promoter). A gene may thus comprise
several operably linked sequences, such as a promoter, a 5' leader
sequence comprising e.g., sequences involved in translation
initiation, a (protein) coding region (cDNA or genomic DNA) and a
3' non-translated sequence comprising e.g., transcription
termination sites.
[0013] A "chimeric gene" (or recombinant gene) refers to any gene,
which is not normally found in nature in a species, in particular a
gene in which one or more parts of the nucleic acid sequence are
present that are not associated with each other in nature. For
example the promoter is not associated in nature with part or all
of the transcribed region or with another regulatory region. The
term "chimeric gene" is understood to include expression constructs
in which a promoter or transcription regulatory sequence is
operably linked to one or more coding sequences or to an antisense
(reverse complement of the sense strand) or inverted repeat
sequence (sense and antisense, whereby the RNA transcript forms
double stranded RNA upon transcription).
[0014] "Expression of a gene" refers to the process wherein a DNA
region, which is operably linked to appropriate regulatory regions,
particularly a promoter, is transcribed into an RNA, which is
biologically active, i.e., which is capable of being translated
into a biologically active protein or peptide (or active peptide
fragment) or which is active itself (e.g., in post-transcriptional
gene silencing or RNAi). An active protein in certain embodiments
refers to a protein having a dominant-negative function due to a
repressor domain being present. The coding sequence is preferably
in sense-orientation and encodes a desired, biologically active
protein or peptide, or an active peptide fragment. In gene
silencing approaches, the DNA sequence is preferably present in the
form of an antisense DNA or an inverted repeat DNA, comprising a
short sequence of the target gene in antisense or in sense and
antisense orientation. "Ectopic expression" refers to expression in
a tissue in which the gene is normally not expressed.
[0015] A "transcription regulatory sequence" is herein defined as a
nucleic acid sequence that is capable of regulating the rate of
transcription of a (coding) sequence operably linked to the
transcription regulatory sequence. A transcription regulatory
sequence as herein defined will thus comprise all of the sequence
elements necessary for initiation of transcription (promoter
elements), for maintaining and for regulating transcription,
including e.g., attenuators or enhancers. Although mostly the
upstream (5') transcription regulatory sequences of a coding
sequence are referred to, regulatory sequences found downstream
(3') of a coding sequence are also encompassed by this
definition.
[0016] As used herein, the term "promoter" refers to a nucleic acid
fragment that functions to control the transcription of one or more
genes, located upstream with respect to the direction of
transcription of the transcription initiation site of the gene, and
is structurally identified by the presence of a binding site for
DNA-dependent RNA polymerase, transcription initiation sites and
any other DNA sequences, including, but not limited to
transcription factor binding sites, repressor and activator protein
binding sites, and any other sequences of nucleotides known to one
of skill in the art to act directly or indirectly to regulate the
amount of transcription from the promoter. A "constitutive"
promoter is a promoter that is active in most tissues under most
physiological and developmental conditions. An "inducible" promoter
is a promoter that is physiologically (e.g., by external
application of certain compounds) or developmentally regulated. A
"tissue specific" promoter is only active in specific types of
tissues or cells.
[0017] As used herein, the term "operably linked" refers to a
linkage of polynucleotide elements in a functional relationship. A
nucleic acid is "operably linked" when it is placed into a
functional relationship with another nucleic acid sequence. For
instance, a promoter, or rather a transcription regulatory
sequence, is operably linked to a coding sequence if it affects the
transcription of the coding sequence. Operably linked means that
the DNA sequences being linked are typically contiguous and, where
necessary to join two protein encoding regions, contiguous and in
reading frame so as to produce a "chimeric protein". A "chimeric
protein" or "hybrid protein" is a protein composed of various
protein "domains" (or motifs) which is not found as such in nature
but which a joined to form a functional protein, which displays the
functionality of the joined domains (for example DNA binding or
repression leading to a dominant negative function). A chimeric
protein may also be a fusion protein of two or more proteins
occurring in nature. The term "domain" as used herein means any
part(s) or domain(s) of the protein with a specific structure or
function that can be transferred to another protein for providing a
new hybrid protein with at least the functional characteristic of
the domain. Specific domains can also be used to identify protein
members belonging to the SHINE clade of transcription factors, such
as SHINE orthologs from other plant species. Examples of domains
found in SHINE proteins are the AP2 domain, the "mm" domain and the
"cm" domain.
[0018] The terms "target peptide" refers to amino acid sequences
which target a protein to intracellular organelles such as
plastids, preferably chloroplasts, mitochondria, or to the
extracellular space (secretion signal peptide). A nucleic acid
sequence encoding a target peptide may be fused (in frame) to the
nucleic acid sequence encoding the amino terminal end (N-terminal
end) of the protein.
[0019] A "nucleic acid construct" or "vector" is herein understood
to mean a man-made nucleic acid molecule resulting from the use of
recombinant DNA technology and which is used to deliver exogenous
DNA into a host cell. The vector backbone may for example be a
binary or superbinary vector (see e.g., U.S. Pat. No. 5,591,616,
US2002138879 and WO9506722), a co-integrate vector or a T-DNA
vector, as known in the art and as described elsewhere herein, into
which a chimeric gene is integrated or, if a suitable transcription
regulatory sequence is already present, only a desired nucleic acid
sequence (e.g., a coding sequence, an antisense or an inverted
repeat sequence) is integrated downstream of the transcription
regulatory sequence. Vectors usually comprise further genetic
elements to facilitate their use in molecular cloning, such as
e.g., selectable markers, multiple cloning sites and the like (see
below).
[0020] A "host cell" or a "recombinant host cell" or "transformed
cell" are terms referring to a new individual cell (or organism)
arising as a result of at least one nucleic acid molecule,
especially comprising a chimeric gene encoding a desired protein or
a nucleic acid sequence which upon transcription yields an
antisense RNA or an inverted repeat RNA (or hairpin RNA) for
silencing of a target gene/gene family, having been introduced into
said cell. The host cell is preferably a plant cell or a bacterial
cell. The host cell may contain the nucleic acid construct as an
extra-chromosomally (episomal) replicating molecule, or more
preferably, comprises the chimeric gene integrated in the nuclear
or plastid genome of the host cell.
[0021] The term "selectable marker" is a term familiar to one of
ordinary skill in the art and is used herein to describe any
genetic entity which, when expressed, can be used to select for a
cell or cells containing the selectable marker. Selectable marker
gene products confer for example antibiotic resistance, or more
preferably, herbicide resistance or another selectable trait such
as a phenotypic trait (e.g., a change in pigmentation) or a
nutritional requirements. The term "reporter" is mainly used to
refer to visible markers, such as green fluorescent protein (GFP),
eGFP, luciferase, GUS and the like.
[0022] The term "ortholog" of a gene or protein refers herein to
the homologous gene or protein found in another species, which has
the same function as the gene or protein, but (usually) diverged in
sequence from the time point on when the species harbouring the
genes diverged (i.e., the genes evolved from a common ancestor by
speciation). Orthologs of the Arabidopsis shn1, shn2 and shn3 genes
may thus be identified in other plant species based on both
sequence comparisons (e.g., based on percentages sequence identity
over the entire sequence or over specific domains) and functional
analysis.
[0023] The terms "homologous" and "heterologous" refer to the
relationship between a nucleic acid or amino acid sequence and its
host cell or organism, especially in the context of transgenic
organisms. A homologous sequence is thus naturally found in the
host species (e.g., a tomato plant transformed with a tomato gene),
while a heterologous sequence is not naturally found in the host
cell (e.g., a tomato plant transformed with a sequence from potato
plants). Depending on the context, the term "homolog" or
"homologous" may alternatively refer to sequences which are
descendent from a common ancestral sequence (e.g., they may be
orthologs).
[0024] "Stringent hybridization conditions" can be used to identify
nucleotide sequences, which are substantially identical to a given
nucleotide sequence. Stringent conditions are sequence dependent
and will be different in different circumstances. Generally,
stringent conditions are selected to be about 5.degree. C. lower
than the thermal melting point (T.sub.m) for the specific sequences
at a defined ionic strength and pH. The T.sub.m is the temperature
(under defined ionic strength and pH) at which 50% of the target
sequence hybridizes to a perfectly matched probe. Typically
stringent conditions will be chosen in which the salt concentration
is about 0.02 molar at pH 7 and the temperature is at least
60.degree. C. Lowering the salt concentration and/or increasing the
temperature increases stringency. Stringent conditions for RNA-DNA
hybridizations (Northern blots using a probe of e.g., 100 nt) are
for example those which include at least one wash in 0.2.times.SSC
at 63.degree. C. for 20 min, or equivalent conditions. Stringent
conditions for DNA-DNA hybridization (Southern blots using a probe
of e.g., 100 nt) are for example those which include at least one
wash (usually 2) in 0.2.times.SSC at a temperature of at least
50.degree. C., usually about 55.degree. C., for 20 min, or
equivalent conditions. See also Sambrook et al. (1989) and Sambrook
and Russell (2001).
[0025] "Sequence identity" and "sequence similarity" can be
determined by alignment of two peptide or two nucleotide sequences
using global or local alignment algorithms. Sequences may then be
referred to as "substantially identical" or "essentially similar"
when they (when optimally aligned by for example the programs GAP
or BESTFIT using default parameters) share at least a certain
minimal percentage of sequence identity (as defined below). GAP
uses the Needleman and Wunsch global alignment algorithm to align
two sequences over their entire length, maximizing the number of
matches and minimizes the number of gaps. Generally, the GAP
default parameters are used, with a gap creation penalty=50
(nucleotides)/8 (proteins) and gap extension penalty=3
(nucleotides)/2 (proteins). For nucleotides the default scoring
matrix used is nwsgapdna and for proteins the default scoring
matrix is Blosum62 (Henikoff & Henikoff, 1992, PNAS
89:915-919). Sequence alignments and scores for percentage sequence
identity may be determined using computer programs, such as the GCG
Wisconsin Package, Version 10.3, available from Accelrys Inc., San
Diego, Calif. Alternatively percent similarity or identity may be
determined by searching against databases such as FASTA, BLAST,
etc.
[0026] In this document and in its claims, the verb "to comprise"
and its conjugations is used in its non-limiting sense to mean that
items following the word are included, but items not specifically
mentioned are not excluded. In addition, reference to an element by
the indefinite article "a" or "an" does not exclude the possibility
that more than one of the element is present, unless the context
clearly requires that there be one and only one of the elements.
The indefinite article "a" or "an" thus usually means "at least
one". It is further understood that, when referring to "sequences"
herein, generally the actual physical molecules with a certain
sequence of subunits (e.g., amino acids) are referred to.
DETAILED DESCRIPTION OF THE INVENTION
[0027] Using activation tagging, the inventors isolated and
characterized an Arabidopsis gene, referred to as SHN1, the
overexpression of which resulted in a number of changes in plant
surface structure compared to the wild type. The activation of SHN1
resulted in leaves having a deep shiny green appearance, with a
curled structure and an altered cuticle permeability, cuticular wax
load/structure and epidermal differentiation. The SHN1 gene was
cloned and sequenced and was found to be similar to transcriptional
factors defined as AP2/EREBP (Alonso et al. 2003, Science
301:653-657). This gene has also recently been described in the art
to encode a transcriptional activator of epidermal wax accumulation
in Arabidopsis (Broun et al. 2004, PNAS 101:4706-4711). However no
other functions of SHN1 have been described and no uses for the
SHN1 gene suggested other than activation of wax deposition.
Although leaf wax load and composition plays some role in
protecting the plant from water loss, the inventors surprisingly
found that shn1 expression resulted in an altered cuticle
structure, which resulted in an increase in cuticular water loss.
This finding was contrary to what might be expected from the
phenotype described by Broun et al. (2004, supra). The cuticular
water loss of leaves continued beyond the time when stomata close,
indicating that non-stomatal water loss was significantly
increased. In addition an increase in cuticle permeability due to
SHN1 activation was illustrated by a higher elution of chlorophyll
when conducting chlorophyll leaching experiments.
[0028] It was further surprisingly found that SHN overexpression in
monocots, e.g., rice, lead to plants which did not show any changes
in epicuticular wax, but were still drought tolerant, proving that
the changes to the epicuticular wax layer observed are not
functional with respect to generating drought tolerance, but that
it is the modified epidermal and cuticle properties which provide
the drought tolerant phenotype. This surprising finding could not
have been foreseen from Broun et al. (supra) or WO03/013228. From
these disclosures one would not expect the SHN gene to be able to
confer drought tolerance in plants or plant parts without modifying
the epicuticular wax layer, as the mechanism would be expected to
be completely dependent on changing the wax composition or content.
In contrast to what would be concluded from the prior art, the
present invention shows, therefore, that drought tolerant plants
can be made, which do not have a modified epicuticular wax layer
(i.e., the epicuticular wax remains unchanged in SHN overexpressing
plants), i.e., wherein the wax composition and content is
unchanged/as in the wild type. Thus, this finding enables the
generation of drought tolerant plants, especially monocotyledonous
plants but also dicotyledonous plants, having a modified cuticle
and epidermis (and therefore being drought tolerant), but wherein
the epicuticular wax is not changed (wild type). Similarly, organ
specific or tissue specific expression results in drought
tolerance/dehydration tolerance of those parts without modifying
the epicuticular wax composition and content.
[0029] Constitutive expression of SHN1 cDNA in transgenic
Arabidopsis plants showed the same phenotype as the original
activation tag line, although the phenotype was more severe. In
addition flower morphology was also affected, which was not the
case in the original tagged line, resulting in petals which were
folded and in part "hidden" in-between the sepals and the flower
interior organs. In addition trichome number and shape was
significantly changed in transgenic 35S::SHN1 plants. Most
interestingly, epidermal cell differentiation in transgenic lines
was altered in two ways. Firstly, pavement cell density on the
abaxial side of the leaves was significantly reduced and secondly
stomatal density was significantly reduced compared to the wild
type. However, cuticle permeability (as determined by water loss
and chlorophyll leaching) was again increased, as seen in the
original tagged line, with this phenotype being more dramatic than
in the original line.
[0030] Based on the finding that the SHN1 expression resulted in an
increase in cuticular water loss, it was even more surprising to
find that 35S::SHN1 transformants showed enhanced drought tolerance
and recovery. Thus apparently the increased non-stomatal water loss
through the altered cuticle was outweighed by the effect of the
reduced stomatal index.
[0031] Using in silico analysis two homologs of SHN1 were
identified, herein referred to as SHN2 and SHN3 (encoding proteins
whose function had not yet been disclosed in the art).
Overexpression of SHN2 and SHN3 resulted in similar phenotypes as
SHN1 overexpression, confirming the functional relationship between
SHN1-SHN3. The SHINE clade of proteins consists, thus, of three
members in Arabidopsis, defined by their sequence (especially by
unique sequence motifs) and function. The SHINE proteins belong to
the plant-specific family of AP2/EREBP transcription factors. This
super-family of transcription factors contains 141 members in
Arabidopsis thaliana (Alonso et al. 2003, Science 301,
653-657).
[0032] Spatio-temporal expression of SHN1, SHN2 and SHN3 was
analyzed by generating transformation vectors comprising about 2 kb
of the genomic DNA upstream (5') of the ATG codon of SHN1 (SEQ ID
NO: 17), SHN2 (SEQ ID NO: 18) and SHN3 (SEQ ID NO: 19),
respectively. The GUS expression pattern showed that SHN1, SHN2 and
SHN3 differ in their spatio-temporal expression pattern, although
some overlap was observed, as described elsewhere herein.
Nucleic Acid Sequences and Proteins According to the Invention
[0033] In one embodiment of the invention nucleic acid sequences
and amino acid sequences of members of the SHINE Glade of
transcription factors are provided (including orthologs), as well
as methods for isolating or identifying orthologs of the SHINE
Glade of other plant species.
[0034] The "SHINE Glade" of transcription factors is defined herein
by the presence of specific amino acid sequence domains in
combination with a related in vivo function of the proteins in the
formation of plant protective layers or plant cell separation
processes. The SHINE Glade encompasses, therefore, orthologs of the
Arabidopsis SHN proteins (SHN1, SHN2 and SHN3), such as but not
limited to orthologs from monocotyledonous species (rice, maize,
wheat, sorghum, pearl millet, barley and other cereals) or from
dicotyledonous plants such as for example Brassicaceae (e.g.,
Brassica napus), cotton, bean, pea, tomato, potato, other vegetable
species, etc. Two ortholog member of the SHINE clade have been
identified in rice (Oryza sativa cv japonica) and are herein
referred to as OsSHN1 (amino acid SEQ ID NO: 14, encoded by the
cDNA sequence of SEQ ID NO: 10) and OsSHN2 (amino acid SEQ ID NO:
24, encoded by the cDNA sequence of SEQ ID NO: 23). OsSHN1 and
OsSHN2 are used herein to exemplify how other members of the SHINE
clade can be identified in other species (especially in other plant
species) and used.
[0035] In order to provide guidance as to which proteins are
members of the SHINE clade, the essential structural and functional
features of members of the SHINE clade is described below. Firstly
the amino acid sequences of SHN1 (SEQ ID NO: 11), SHN2 (SEQ ID NO:
12), SHN3 (SEQ ID NO: 13) and OsSHN1 (SEQ ID NO: 14) and OsSHN2
(SEQ ID NO: 24) are described.
[0036] SHN1, SHN2 and SHN3 are proteins of 199, 189 and 186 amino
acids in length, respectively, while OsSHN1 is 205 amino acids long
and OsSHN2 243 amino acids. Each comprises a single AP2 DNA binding
domain, a conserved middle domain "mm" and a conserved C-terminal
domain "cm". The consensus sequences of these domains are as
follows:
TABLE-US-00001 Consensus middle domain "mm" (61 amino acids)- SEQ
ID NO: 15 S-X-X-X-S-X-X- S/N-L-S-X- I/L-L- S/N-A-K-L-R-K-X-C-K-X
-X- S/T-P- S/Y-L-T-C-L-R-L-D-X-X- S/K-S-H-I-G-V-W-Q-K-R- A-G- S/A-
K/R-X-X-X-X-W-V- M/K-X- V/L-E-L Consensus C-terminal domain "cm"
(10 amino acids)- SEQ ID NO: 16 V/L/M/I-A- L/M- Q/E-M-I-E-E-L-L (X
refers to any amino acid and consensus sequences are presented in
N- to C-terminal order).
[0037] The presence of the "mm" domain is one of the distinguishing
features of SHINE clade members. Especially the presence of an "mm"
domain in combination with a "cm" domain and/or an AP2 domain is
characteristic. In one embodiment SHN proteins are defined as
comprising at least one "mm" domain and having a function in the
formation of protective layers and/or cell separation layers. It is
understood that the "mm" domain may be modified without losing its
function. For example single amino acid substitutions, deletions or
replacements (e.g., conservative amino acid replacements) may be
present in the "mm" domain according to the invention. The "mm"
domain of SHN proteins can also be defined in terms of sequence
identity, whereby domains having a sequence identity of at least
55%, preferably at least 60% or more are encompassed herein (see
Table 2).
[0038] Alternatively or additionally, SHN proteins may be defined
by their amino acid sequence identity over their entire length. SHN
proteins have a sequence identity of 50% or more over their entire
length (see Table 1) (such as but not limited to 55%, 60%, 70%,
80%, 90% or more), and a sequence identity of 45% or more,
preferably at least 50%, 55%, 57%, 58%, 59%, 60%, 70%, 80%, 90%,
95% or more over the middle domain region "mm" (see Table 2).
[0039] To illustrate the distinction between SHINE members and
non-SHINE members, the Arabidopsis sequence with Accession number
At5g25190 and the tomato LeERF1 sequence (Accession number
AY077626) are included in Table 1, both of which are non-SHINE
proteins. Both lack the consensus middle domain "mm", as a result
of which the overall sequence identity is much lower (generally
below 40% sequence identity with SHINE proteins). In addition the
At5g25190 overexpression showed that the function of this protein
was not essentially similar to that of SHN1, SHN2 and SHN3, maybe
due to the absence of the middle domain. The overexpression lines
did not display the phenotypic characteristics of the SHN gene
overexpression lines, but exhibited other distinct phenotypes
suggesting a different function.
TABLE-US-00002 TABLE 1 Amino acid sequence identity over entire
length LeERF1 SHN1 SHN2 SHN3 OsSHN1 At5g25190 (AY077626) SHN1 100%
55.9% 50.2% 59.3% 40.2% 36.6% SHN2 100% 66.8% 50.7% 38.8% 32.7%
SHN3 100% 51.5% 39.7% 34.4% (GAP opening = 8, GAP extension = 2,
Blosum62)
TABLE-US-00003 TABLE 2 Amino acid sequence identity over "mm"
domain SHN1 SHN2 SHN3 OsSHN1 SHN1 100% 68.9% 65.6% 75.4% SHN2 100%
83.6% 60.7% SHN3 100% 60.7% (GAP opening = 8, GAP extension = 2,
Blosum62)
[0040] The SHINE clade members can thus be defined as comprising at
least one consensus middle domain and preferably further comprising
at least one consensus C-terminal domain and/or at least one AP2
binding domain in addition to an in vivo function which is
essentially similar to that of SHN1, SHN2, SHN3 and/or OsSHN1
and/or OsSHN2 when expressed in a host plant. A "function which is
essentially similar to the function of SHN1, SHN2, SHN3 and/or
OsSHN1 and/or OsSHN2" refers herein to the protein having a proven
function in the development/formation of plant protective layers
(cuticle layers and/or suberin layers) and/or cell separation
processes (dehiscence and/or abscission).
[0041] The function of a protein can be tested using a variety of
known methods, preferably by comparing the phenotype of
transformants constitutively expressing the protein being tested to
the phenotype of SHN1, SHN2, SHN3 and/or OsSHN1 and/or OsSHN2
over-expressing transformants of the same host species (and
variety) (preferably comprising a chimeric SHN encoding gene stably
integrated into the host's genome), allowing a direct comparison of
the functional effect on the phenotype of the transformants. It is
understood that in any transformation experiments a certain degree
of variation in the phenotype of transformants is seen, normally
due to position effects in the genome and/or due to copy number. A
skilled person will know how to compare transformants to one
another, e.g., by selecting single copy number events and analyzing
their phenotypes. Other methods of determining or confirming in
vivo gene/protein function include the generation of knock-out
mutants or transient expression studies. Promoter-reporter gene
expression studies may also provide information as to the
spatio-temporal expression pattern and the role of the protein.
[0042] Constitutive (over)expression of a SHINE clade member should
result in one or more of the following phenotypic changes compared
to the wild type or control transformants: [0043] increased cuticle
permeability, especially non-stomatal permeability [0044] reduced
stomatal index/density due to altered epidermal cell
differentiation, [0045] increased (absolute) cuticular wax load
and/or altered wax composition (relative wax composition) [0046]
reduced number of trichomes and/or altered trichome structure
[0047] shiny green leaves and/or curled leaves.
[0048] In a preferred embodiment, however, overexpression results
in an epidermal change leading to reduced stomatal index/density,
but no change to the epicuticular wax layer. By generating or
selecting such plants or plant parts, the plant tissue appearance
remains unchanged (i.e., leaves are not shiny and/or curled and
have no increased wax load and/or altered wax composition), while
the plant (or plant part) has one or more of the novel phenotypes
described elsewhere herein. In a preferred embodiment these plants
(or plant parts) are monocotyledonous plants, but generation and
selection of dicotyledonous plants (or plant parts) which have a
novel phenotype but which have an unmodified epicuticular wax layer
is also possible. The expression "the epicuticular wax layer is
unmodified" refers to the layer being essentially as in the wild
type, i.e., if the wild type has no layer, the transformant also
has not layer, and if the wild type has a very thin layer, the
transformant also has a very thin layer. Especially, the
epicuticular wax content and composition is essentially as in the
wild type.
[0049] An "increased cuticle permeability" refers to the
(non-stomatal) water loss occurring through the cuticle and can be
measured by, for example, carrying out fresh weight loss
experiments or Chlorophyll Leaching Assays, as described in the
Examples. The average rate of water loss per gram fresh weight of
the transformants, and the total amount of water lost after e.g., 1
hour, is significantly increased compared to controls, especially
at least about 3 fold, 5 fold, 10 fold, or more, preferably at
least about 5-10 fold. Chlorophyll leaching of transformants are
carried out by adding alcohol (e.g., 80% ethanol) to the tissue
samples and measuring the absorbance of the samples after a certain
period of incubation (see Examples and Lolle et al. 1997, Dev Biol
189, 311-321). The rate of chlorophyll leaching per fresh weight of
the transformants, and the total amount of chlorophyll leached
after e.g., 1 hour, is significantly increased compared to
controls, especially at least about 3 fold, 5 fold, 10 fold, 12
fold, 15 fold or more, preferably at least about 5-10 fold. For
example, for 1 mmol Chlorophyll/mg fresh weight leached after 1
hour in the control about 12 mmol Chlorophyll/mg fresh weight
leached in the transformant (see Examples).
[0050] An "altered epidermal differentiation" refers to a
significantly reduced stomatal density (number of stomata per
mm.sup.2) and stomatal index, compared to that of control plants or
tissues. Stomatal density is reduced by at least about 15%, 20%,
30% or more in tissue of transformants compared to suitable
controls. The stomatal index is reduced by at least 25%, more
preferably by at least 30%, 40%, 45% or more compared to the
stomatal index of controls. The stomatal index can be determined by
making imprints of leaf (abaxial) surfaces and counting pavement
cells and stomata under a microscope, as described in the examples.
The stomatal index can be calculated according to Mishra 1997 (Ann.
Bot. 80:689-692).
[0051] An "increased cuticular wax load" refers to an increase of
the amount of total extractable cuticular lipids per surface area
compared to that of control tissue samples. The total cuticular wax
load of the transformant shows an average fold increase of at least
4.times., 5.times., 6.times., 7.times. (or more) over the control.
An increase in cuticular wax load can be determined e.g., by
Scanning Electron Microscopy (SEM) or by extraction and chemical
analysis as known in the art and as described in the Examples.
[0052] An "altered wax composition" refers herein to a change in
the relative amounts (i.e., a qualitative change) of the individual
components making up the wax layer. Especially the relative amounts
of alkanes, secondary alcohols and ketones are increased at least
5, 6, 7, 8, 9, 10, 11 fold or more in the transformants.
[0053] A "reduced number of trichomes" and/or "altered trichome
structure" refers to a significant reduction (by at least 20%, 30%,
40%, 50% or more) of trichome numbers and/or a change in trichome
structure (in particular branching) in transformants compared to
wild type epidermal surfaces and is also indicative of an
alteration in epidermal cell differentiation.
[0054] These phenotypes can be utilized in creating transgenic
plants or plant tissues/organs with modified and improved
agronomical characteristics, such as enhanced drought tolerance
and/or enhanced salinity tolerance and others as described
elsewhere herein.
[0055] Other putative members of the SHINE clade can be identified
in silico, e.g., by identifying nucleic acid or protein sequences
in existing nucleic acid or protein database (e.g., GENBANK,
SWISSPROT, TrEMBL) and using standard sequence analysis software,
such as sequence similarity search tools (BLASTN, BLASTP, BLASTX,
TBLAST, FASTA, etc.). Especially the screening of plant sequence
databases, such as the rice genome database, the wheat genome
database, etc. for the presence of amino acid sequences or nucleic
acid sequences encoding the consensus "mm" domain or a sequence
essentially similar to the "mm" domain is desired. Putative amino
acid sequences or nucleic acid sequences comprising or encoding at
least one "mm" domain are selected, cloned or synthesized de novo
and tested for in vivo functionality by e.g., overexpression in a
plant host.
[0056] In accordance with the invention "SHN1", "SHN2", "SHN3" and
"OsSHN1" and "OsSHN2" refers to any protein comprising the smallest
biologically active fragment of SEQ ID NO's 11, 12, 13, 14, and 24
respectively, which retains a function in the formation of plant
protective layers and/or cell separation layers. This includes
hybrid and chimeric proteins comprising the smallest active
fragment. Preferably, at least one "mm" consensus domain is
present. More preferably additionally at least one consensus "cm"
domain is present. Also included in this definition are variants of
SHN1, SHN2, SHN3 and OsSHN1 and OsSHN2, such as amino acid
sequences essentially similar to SEQ ID NO's 11, 12, 13, 14 or 24
respectively, having a sequence identity of at least 50%, 55%, 60%,
65%, 70%, 75%, 80%, 85%, 90%, 97%, 99%, 99.6%, 99.8% or more at the
amino acid sequence level, as determined using pairwise alignment
using the GAP program (with a gap creation penalty of 8 and an
extension penalty of 2). Preferably proteins having some,
preferably 5-10, particularly less than 5, amino acids added,
replaced or deleted without significantly changing the protein
activity are included in this definition. For example conservative
amino acid substitutions within the categories basic (e.g., Arg,
His, Lys), acidic (e.g., Asp, Glu), nonpolar (e.g., Ala, Val, Trp,
Leu, Ile, Pro, Met, Phe, Trp) or polar (e.g., Gly, Ser, Thr, Tyr,
Cys, Asn, Gln) fall within the scope of the invention as long as
the activity of the SHN protein is not significantly, preferably
not, changed, at least not changed in a negative way. In addition
non-conservative amino acid substitutions fall within the scope of
the invention as long as the activity of the SHN protein is not
changed significantly, preferably not, or at least is not changed
in a negative way.
[0057] The SHN proteins according to the invention may be isolated
from natural sources, synthesized de novo by chemical synthesis
(using e.g., a peptide synthesizer such as supplied by Applied
Biosystems) or produced by recombinant host cells. The SHN proteins
according to the invention may be used to raise mono- or polyclonal
antibodies, which may for example be used for the detection of SHN
proteins in samples (immunochemical analysis methods and kits).
[0058] Chimeric or hybrid SHN proteins comprise at least one "mm"
domain, but may further comprise a "cm" domain and/or an AP2 domain
or other domains from other proteins. Domains may thus be exchanged
(domain swapping) between SHN proteins or between SHN proteins and
other, unrelated proteins, as long as the functionality of the
resulting chimeric protein essentially similar to that of SHN1,
SHN2, SHN3 or OsSHN1 or OsSHN2. A chimeric SHN protein may thus,
for example, comprise an AP2 domain from SHN1, an "mm" domain from
SHN2 and a "cm" domain from OsSHN1. Similarly, a chimeric SHN
protein may comprise at least one "mm" domain in addition to one or
more protein domains not normally found in SHN proteins, such as
stabilizing domains, binding domains (e.g., hormone binding
domains, such as found in the glucocorticoid receptor, resulting in
inducibility), etc. In another embodiment chimeric SHN proteins are
provided which comprise a SHN-repressor domain fusion, such as the
SHN-EAR fusion described below. In transgenic plants,
overexpression of these chimeric proteins result in a dominant
negative phenotype, as described further below. SHN-repressor
domain fusion may also comprise additional domains fused thereto,
such as e.g., a hormone binding domain (see e.g., Markel et al.
2002, Nucl. Acid Res. 30, 4709-4719).
[0059] The function of specific domains, such as the "mm" or "cm"
domain, can be analyzed by deleting all or part of the domain(s) in
a SHN protein or the introduction of mutations into the domain, and
analysis of the resulting effect on the function of the SHN
protein.
[0060] Also provided are nucleic acid sequences (genomic DNA, cDNA,
RNA) encoding SHN Glade proteins, such as for example SHN1, SHN2,
SHN3, OsSHN1 and OsSHN2 as defined above (including any chimeric or
hybrid SHN proteins), or any SHN protein from another species. In
addition, the nucleic acid sequences encoding "mm" domains or "cm"
domains are provided. Due to the degeneracy of the genetic code
various nucleic acid sequences may encode the same amino acid
sequence. Any nucleic acid sequence encoding SHN1, SHN2, SHN3 or
OsSHN1 or OsSHN2 is herein referred to as "SHN1","SHN2","SHN3", and
"OsSHN1" and "OsSHN2". The nucleic acid sequences provided include
naturally occurring, artificial or synthetic nucleic acid
sequences. Examples of nucleic acid sequences encoding SHN1-SHN3
and OsSHN1 are provided for in SEQ ID NO: 1, 2 and 3 (genomic SHN1,
SHN2 and SHN3 sequences from Arabidopsis, respectively), SEQ ID NO:
4, 5 and 6 (RNA transcripts of SHN1, SHN2 and SHN3 from
Arabidopsis, respectively) and SEQ ID NO: 7, 8, 9, 10 and 23 (cDNA
of SHN1, SHN2, SHN3, OsSHN1 and OsSHN2, respectively). It is
understood that when sequences are depicted in as DNA sequences
while RNA is referred to, the actual base sequence of the RNA
molecule is identical with the difference that thymine (T) is
replace by uracil (U).
[0061] Also included are variants and fragments of SHN nucleic acid
sequences, such as nucleic acid sequences hybridizing to SHN
nucleic acid sequences, e.g., to SHN1, SHN2, SHN3 and/or OsSHN1
and/or OsSHN2, under stringent hybridization conditions as defined.
Variants of SHN nucleic acid sequences also include nucleic acid
sequences which have a sequence identity to SEQ ID NO: 7, 8, 9 or
10 or 23 of at least 50% or more, preferably at least 55%, 60%,
70%, 80%, 90%, 95%, 99%, 99.5%, 99.8% or more. It is clear that
many methods can be used to identify, synthesize or isolate
variants or fragments of SHN nucleic acid sequences, such as
nucleic acid hybridization, PCR technology, in silico analysis and
nucleic acid synthesis, and the like.
[0062] The nucleic acid sequence, particularly DNA sequence,
encoding the SHN proteins of this invention can be inserted in
expression vectors to produce high amounts of SHN proteins (or
e.g., chimeric SHN proteins), as described below. For optimal
expression in a host the SHN DNA sequences can be codon-optimized
by adapting the codon usage to that most preferred in plant genes,
particularly to genes native to the plant genus or species of
interest (Bennetzen & Hall, 1982, J. Biol. Chem. 257:3026-3031;
Itakura et al., 1977 Science 198:1056-1063.) using available codon
usage tables (e.g., more adapted towards expression in cotton,
soybean corn or rice). Codon usage tables for various plant species
are published for example by Ikemura (1993, In Plant Molecular
Biology Labfax, Croy, ed., Bios Scientific Publishers Ltd.) and
Nakamura et al. (2000, Nucl. Acids Res. 28, 292.) and in the major
DNA sequence databases (e.g., EMBL at Heidelberg, Germany).
Accordingly, synthetic DNA sequences can be constructed so that the
same or substantially the same proteins are produced. Several
techniques for modifying the codon usage to that preferred by the
host cells can be found in patent and scientific literature. The
exact method of codon usage modification is not critical for this
invention.
[0063] Small modifications to a DNA sequence such as described
above can be routinely made, i.e., by PCR-mediated mutagenesis (Ho
et al., 1989, Gene 77:51-59., White et al., 1989, Trends in Genet.
5:185-189). More profound modifications to a DNA sequence can be
routinely done by de novo DNA synthesis of a desired coding region
using available techniques.
[0064] Also, the SHN nucleic acid sequences can be modified so that
the N-terminus of the SHN protein has an optimum translation
initiation context, by adding or deleting one or more amino acids
at the N-terminal end of the protein. Often it is preferred that
the proteins of the invention to be expressed in plants cells start
with a Met-Asp or Met-Ala dipeptide for optimal translation
initiation. An Asp or Ala codon may thus be inserted following the
existing Met, or the second codon, Val, can be replaced by a codon
for Asp (GAT or GAC) or Ala (GCT, GCC, GCA or GCG). The DNA
sequences may also be modified to remove illegitimate splice
sites.
[0065] In one embodiment of the invention SHN gene expression is
downregulated in a host cell, plant or specific tissue(s), by,
e.g., RNAi approaches, as described elsewhere. In yet another
embodiment SHN loss-of-function phenotypes (of host cells, tissues
or whole plants) are generated by expressing a nucleic acid
sequence encoding a protein fusion of a SHN protein (as defined)
with a (dominant) repressor domain. "Loss-of-function" refers
herein to the loss of SHN protein function in a host tissue or
organisms, and encompasses the function at the molecular level
(e.g., loss of transcriptional activation of downstream target
genes of the SHN transcription factor) and preferably also at the
phenotypic level (e.g., podshatter resistance or male sterility).
For example, in order to provide loss-of-function, SHN protein
fusions are made with a 12 amino acid `EAR` repressor domain as
described by Hiratsu et al., 2003 (Plant J. 34:733-739),
incorporated herein by reference. These repressor domain fusions to
any one of the SHN proteins (as defined), termed herein `SHN-EAR`
fusion proteins, are able to cause repression of the downstream
target genes and thus result in an effective loss-of-function
mutant (dominant negative effect). These repressor fusions also
effect repression in heterologous plants where the orthologous
genes have not yet been identified. In one embodiment a nucleic
acid sequence is provided which encodes a chimeric repressor
domain-SHN protein fusion protein, especially a SHN-EAR fusion
protein. In addition a vector comprising said nucleic acid sequence
and a host cell, tissue and/or organism comprising the chimeric
gene is provided. To generate a SHN-repressor domain fusion
protein, the nucleic acid sequence encoding the repressor domain is
translationally fused to the nucleic acid sequence comprising the
SHN coding sequence. The SHN-repressor domain fusion protein
encoding nucleic acid sequence (especially SHN-EAR) is placed under
control of constitutive or specific promoters (e.g., tissue
specific or developmentally regulated). Constitutive expression
provides a loss-of-function in all host tissues where SHN1, SHN2
and SHN3 or including the orthologs e.g., OsSHN1 or OsSHN2, are
expressed and required for function. Specific expression of the
SHN-EAR protein provides a loss-of-function in the specific tissue
or condition, e.g., when a dehiscence zone specific promoter is
operably linked to a nucleic acid encoding a SHN-EAR fusion
protein, e.g., the SHN2 promoter, loss of SHN function in the
dehiscent zones of anther and silique results.
[0066] To generate a SHN-EAR fusion protein, the following 12
specific amino acids are added in frame to the C-terminal of a SHN
protein: LDLDLELRLGFA (SEQ ID NO: 21). To generate a SHN-EAR fusion
protein, the EAR domain encoding nucleic acid sequences, such as
SEQ ID NO: 22, may be added in frame to the 3' end of the SHN
coding sequence, followed by a stop codon (e.g., TAA).
TABLE-US-00004 SEQ ID NO: 22 (EAR repressor coding sequence):
5'-CTG GAT CTG GAT CTA GAA CTC CGT TTG GGT TTC GCT (TAA)-3'
[0067] It is understood that SHN proteins may be operably fused to
other repression domain available in the art which function in
plant cells. These include repressor domains of animal proteins,
such as the Drosophila ENGRAILED (En) repressor domain. For example
the N-terminal 298 amino acids may be fused to a SHN protein
according to the invention, creating a dominant-negative chimeric
protein (see Markel et al. 2002, Nucleic Acid Research 30:4709-4719
and Chandler and Werr 2003, Trends Plant Sci 8:279-285, both
incorporated by reference). It is noted that repressor domains may
be fused to the SHN protein at the C-terminus or at the N-terminus,
depending on the domain. The nucleic acid sequence encoding the
dominant-negative fusion protein may be referred to as a
"dominant-negative chimeric gene" and when transferred into a host
genome as a "dominant-negative transgene" (either stably integrated
in the host genome or transiently expressed). Other plant repressor
domains are for example the LEUNG and SEUSS co-repressors of
AGAMOUS, FLC and polycomb proteins. Other animal repressor domains
include for example the WT1, eve, c-ErbA and v-ErbA and Kruppel
associated box (see Chandler and Werr, 2003, supra and references
therein).
[0068] In another embodiment of the invention PCR primers and/or
probes and kits for detecting the SHN DNA sequences are provided.
Degenerate or specific PCR primer pairs to amplify SHN DNA from
samples can be synthesized based on SEQ ID NO's 1-10 as known in
the art (see Dieffenbach and Dveksler (1995) PCR Primer: A
Laboratory Manual, Cold Spring Harbor Laboratory Press, and
McPherson et al. (2000) PCR-Basics: From Background to Bench,
1.sup.st Ed., Springer Verlag, Germany). Likewise, DNA fragments of
SEQ ID NO's 1-10 can be used as hybridization probes. An SHN
detection kit may comprise either SHN specific primers and/or SHN
specific probes, and an associated protocol to use the primers or
probe to detect SHN DNA in a sample. Such a detection kit may, for
example, be used to determine, whether a plant has been transformed
with an SHN gene (or part thereof) of the invention. Because of the
degeneracy of the genetic code, some amino acid codons can be
replaced by others without changing the amino acid sequence of the
protein.
[0069] In another embodiment antibodies that bind specifically to a
SHN protein according to the invention are provided. In particular
monoclonal or polyclonal antibodies that bind to SHN1, SHN2, SHN3
or OsSHN1 or OsSHN2, or to fragments or variants thereof, are
encompassed herein. An antibody can be prepared by using a SHN
protein according to the invention as an antigen in an animal using
methods known in the art, as e.g., described in Harlow and Lane
Using Antibodies: A Laboratory Manual, New York: Cold Spring Harbor
Press 1998; and in Liddell and Cryer, A Practical Guide to
Monoclonal Antibodies (Wiley and Sons, 1991). The antibodies can
subsequently be used to isolate, identify, characterize or purify
the SHN protein to which it binds, for example to detect the SHN
protein in a sample, allowing the formation of an immune complex
and detecting the presence of the immune complex by e.g., ELISA
(enzyme linked immunoassay) or immunoblot analysis. Also provided
are immunological kits, useful for detecting the SHN proteins,
protein fragments or epitopes in a sample provided. Samples may be
cells, cell supernatants, cell suspensions, tissues, etc. Such a
kit comprises at least an antibody that binds to a SHN protein and
one or more immunodetection reagents. The antibodies can also be
used to isolate/identify other SHN proteins, for example by ELISA
or Western blotting.
[0070] In addition, nucleic acid sequences comprising SHN1, SHN2,
SHN3 and OsSHN1 promoters are provided herein. The transcription
regulatory sequences are found in the about 2 kb sequence upstream
of the ATG codon of SEQ ID NO: 1, 2 and 3. The transcription
regulatory sequences of SHN1, SHN2 and SHN3 are provided herein in
SEQ ID NO: 17, 18 and 19, respectively, and the transcription
regulatory sequence of OsSHN1 is provided as SEQ ID NO: 20. These
transcription regulatory sequences may be used for the construction
of chimeric genes and for expressing operably linked nucleic acid
sequences in hosts or host cells. Especially the SHN1 transcription
regulatory sequence may be used for expression in inflorescence
tissues, root tissue and abscission zone of siliques. The SHN2
transcription regulatory sequence may be used to direct expression
in dehiscence zones of anthers and siliques and may thus be useful
for generating male sterility or podshatter resistance. The
transcription regulatory region of SHN3 is active in many tissues
and may thus be used for directing broader expression in
essentially all organs and tissues (see Examples). It is understood
that the tissue specificity of the transcription regulatory
sequences can be improved or specified by analyzing deletion
fragments of the sequences provided for their ability to direct
expression of nucleic acid sequences operably linked thereto. Such
deletion analysis allows the removal of nucleic acid parts which
cause non-specific (background) expression. Similarly, the
transcription regulatory sequences of other SHN genes can be
isolated by sequencing the genomic DNA upstream of the ATG codon,
using known methods such as TAIL-PCR.
Chimeric Genes, Vectors and Recombinant Microorganisms According to
the Invention
[0071] In one embodiment of the invention nucleic acid sequences
encoding SHN proteins (including e.g., fusion proteins such as
SHN-EAR), as described above, are used to make chimeric genes, and
vectors comprising these for transfer of the chimeric gene into a
host cell and production of the SHN protein(s) in host cells, such
as cells, tissues, organs or organisms derived from transformed
cell(s). Host cells are preferably plant cells and, but microbial
hosts (bacteria, yeast, fungi, etc.) are also envisaged. Any crop
plant may be a suitable host, such as monocotyledonous plants or
dicotyledonous plants, for example maize/corn (Zea species, e.g.,
Z. mays, Z. diploperennis (chapule), Zea luxurians (Guatemalan
teosinte), Zea mays subsp. huehuetenangensis (San Antonio Huista
teosinte), Z. mays subsp. mexicana (Mexican teosinte), Z. mays
subsp. parviglumis (Balsas teosinte), Z. perennis (perennial
teosinte) and Z. ramosa), wheat (Triticum species), barley (e.g.,
Hordeum vulgare), oat (e.g., Avena sativa), sorghum (Sorghum
bicolor), rye (Secale cereale), soybean (Glycine spp, e.g., G.
max), cotton (Gossypium species, e.g., G. hirsutum, G. barbadense),
Brassica spp. (e.g., B. napus, B. juncea, B. oleracea, B. rapa,
etc.), sunflower (Helianthus annus), tobacco (Nicotiana species),
alfalfa (Medicago sativa), rice (Oryza species, e.g., O. sativa
indica cultivar-group or japonica cultivar-group), forage grasses,
pearl millet (Pennisetum spp. e.g., P. glaucum), tree species,
vegetable species, such as Lycopersicon ssp (e.g., Lycopersicon
esculentum), potato (Solanum tuberosum, other Solanum species),
eggplant (Solanum melongena), peppers (Capsicum annuum, Capsicum
frutescens), pea, bean (e.g., Phaseolus species), fleshy fruit
(grapes, peaches, plums, strawberry, mango) ornamental species
(e.g., Rose, Petunia, Chrysanthemum, Lily, Gerbera species), woody
trees (e.g., species of Populus, Salix, Quercus, Eucalyptus), fiber
species e.g., flax (Linum usitatissimum) and hemp (Cannabis
sativa). In one embodiment monocotyledonous crop plants are
preferred.
[0072] A "crop plant" refers herein to a plant species which is
cultivated and bred by humans and excludes weeds such as
Arabidopsis thaliana. A crop plant may be cultivated for food
purposes (e.g., field crops), or for ornamental purposes (e.g.,
production of flowers for cutting, grasses for lawns, etc.). A crop
plant as defined herein also includes plants from which non-food
products are harvested, such as oil for fuel, plastic polymers,
pharmaceutical products, cork and the like.
[0073] The construction of chimeric genes and vectors for,
preferably stable, introduction of SHN protein encoding nucleic
acid sequences into the genome of host cells is generally known in
the art. To generate a chimeric gene the nucleic acid sequence
encoding a SHN protein (or e.g., a SHN-repressor domain fusion
protein) is operably linked to a promoter sequence, suitable for
expression in the host cells, using standard molecular biology
techniques. The promoter sequence may already be present in a
vector so that the SHN nucleic sequence is simply inserted into the
vector downstream of the promoter sequence. The vector is then used
to transform the host cells and the chimeric gene is inserted in
the nuclear genome or into the plastid, mitochondrial or
chloroplast genome and expressed there using a suitable promoter
(e.g., McBride et al., 1995 Bio/Technology 13 362; U.S. Pat. No.
5,693,507). In one embodiment a chimeric gene comprises a suitable
promoter for expression in plant cells or microbial cells (e.g.,
bacteria), operably linked thereto a nucleic acid sequence encoding
a SHN protein or fusion protein according to the invention,
optionally followed by a 3' nontranslated nucleic acid
sequence.
[0074] The SHN nucleic acid sequence, preferably the SHN chimeric
gene, encoding an functional SHN protein (or in certain embodiments
a functional SHN-repressor domain fusion protein), can be stably
inserted in a conventional manner into the nuclear genome of a
single plant cell, and the so-transformed plant cell can be used in
a conventional manner to produce a transformed plant that has an
altered phenotype due to the presence of the SHN protein in certain
cells at a certain time. In this regard, a T-DNA vector, comprising
a nucleic acid sequence encoding a SHN protein, in Agrobacterium
tumefaciens can be used to transform the plant cell, and
thereafter, a transformed plant can be regenerated from the
transformed plant cell using the procedures described, for example,
in EP 0116718, EPO 270822, PCT publication WO84/02913 and published
European Patent application EP 0242246 and in Gould et al. (1991,
Plant Physiol. 95:426-434). The construction of a T-DNA vector for
Agrobacterium mediated plant transformation is well known in the
art. The T-DNA vector may be either a binary vector as described in
EP 0 120 561 and EP 0 120 515 or a co-integrate vector which can
integrate into the Agrobacterium Ti-plasmid by homologous
recombination, as described in EP 0116718.
[0075] Preferred T-DNA vectors each contain a promoter operably
linked to SHN encoding nucleic acid sequence between T-DNA border
sequences, or at least located to the left of the right border
sequence. Border sequences are described in Gielen et al. (1984,
EMBO J. 3:835-845). Of course, other types of vectors can be used
to transform the plant cell, using procedures such as direct gene
transfer (as described, for example in EP 0223247), pollen mediated
transformation (as described, for example in EP 0270356 and
WO85/01856), protoplast transformation as, for example, described
in U.S. Pat. No. 4,684,611, plant RNA virus-mediated transformation
(as described, for example in EP 0067553 and U.S. Pat. No.
4,407,956), liposome-mediated transformation (as described, for
example in U.S. Pat. No. 4,536,475), and other methods such as
those described methods for transforming certain lines of corn
(e.g., U.S. Pat. No. 6,140,553; Fromm et al., 1990, Bio/Technology
8:833-839; Gordon-Kamm et al., 1990, The Plant Cell 2:603-618) and
rice (Shimamoto et al., 1989, Nature 338:274-276; Datta et al.
1990, Bio/Technology 8:736-740) and the method for transforming
monocots generally (PCT publication WO92/09696). For cotton
transformation see also WO 00/71733, and for rice transformation
see also the methods described in WO92/09696, WO94/00977 and
WO95/06722. For sorghum transformation see e.g., Jeoung J M et al.
2002, Hereditas 137: 20-8 or Zhao Z Y et al. 2000, Plant Mol. Biol.
44:789-98). Likewise, selection and regeneration of transformed
plants from transformed cells is well known in the art. Obviously,
for different species and even for different varieties or cultivars
of a single species, protocols are specifically adapted for
regenerating transformants at high frequency.
[0076] Besides transformation of the nuclear genome, also
transformation of the plastid genome, preferably chloroplast
genome, is included in the invention. One advantage of plastid
genome transformation is that the risk of spread of the
transgene(s) can be reduced. Plastid genome transformation can be
carried out as known in the art, see e.g., Sidorov V A et al. 1999,
Plant J. 19:209-216 or Lutz K A et al. 2004, Plant J.
37:906-13.
[0077] The resulting transformed plant can be used in a
conventional plant breeding scheme to produce more transformed
plants with the same characteristics or to introduce the gene part
into other varieties of the same or related plant species. Seeds,
which are obtained from the transformed plants, contain the
chimeric SHN gene as a stable genomic insert. Cells of the
transformed plant can be cultured in a conventional manner to
produce the SHN protein, which can be recovered for other use e.g.,
antibody production.
[0078] The SHN nucleic acid sequence is inserted in a plant cell
genome so that the inserted coding sequence is downstream (i.e.,
3') of, and under the control of, a promoter which can direct the
expression in the plant cell. This is preferably accomplished by
inserting the chimeric gene in the plant cell genome, particularly
in the nuclear or plastid (e.g., chloroplast) genome.
[0079] Preferred promoters include: the strong constitutive 35S
promoters or enhanced 35S promoters (the "35S promoters") of the
cauliflower mosaic virus (CaMV) of isolates CM 1841 (Gardner et
al., 1981, Nucleic Acids Res 9:2871-2887), CabbB-S (Franck et al.,
1980, Cell 21:285-294) and CabbB-JI (Hull and Howell, 1987,
Virology 86:482-493); the 35S promoter described by Odell et al.
(1985, Nature 313:810-812) or in U.S. Pat. No. 5,164,316, promoters
from the ubiquitin family (e.g., the maize ubiquitin promoter of
Christensen et al., 1992, Plant Mol. Biol. 18:675-689, EP 0342926,
see also Cornejo et al. 1993, Plant Mol. Biol. 23:567-581), the
gos2 promoter (de Pater et al., 1992 Plant J. 2:834-844), the emu
promoter (Last et al., 1990, Theor. Appl. Genet. 81:581-588),
Arabidopsis actin promoters such as the promoter described by An et
al. (1996, Plant J. 10:107.), rice actin promoters such as the
promoter described by Zhang et al. (1991, The Plant Cell
3:1155-1165) and the promoter described in U.S. Pat. No. 5,641,876
or the rice actin 2 promoter as described in WO07/0067; promoters
of the Cassaya vein mosaic virus (WO 97/48819, Verdaguer et al.
1998, Plant Mol. Biol. 37:1055-1067), the pPLEX series of promoters
from Subterranean Clover Stunt Virus (WO 96/06932, particularly the
S7 promoter), a alcohol dehydrogenase promoter, e.g., pAdh1S
(GenBank accession numbers X04049, X00581), and the TR1' promoter
and the TR2' promoter (the "TR1' promoter" and "TR2' promoter",
respectively) which drive the expression of the 1' and 2' genes,
respectively, of the T-DNA (Velten et al., 1984, EMBO J.
3:2723-2730), the Figwort Mosaic Virus promoter described in U.S.
Pat. No. 6,051,753 and in EP426641, histone gene promoters, such as
the Ph4a748 promoter from Arabidopsis (Plant Mol. Biol. 8:179-191),
or others.
[0080] Alternatively, a promoter can be utilized which is not
constitutive but rather is specific for one or more tissues or
organs of the plant (tissue preferred/tissue specific, including
developmentally regulated promoters), for example leaf preferred,
epidermis preferred, root preferred, flower tissue e.g., tapetum or
anther preferred, seed preferred, pod preferred, etc.), whereby the
SHN gene (including e.g., the SHN-repressor fusion protein encoding
gene) is expressed only in cells of the specific tissue(s) or
organ(s) and/or only during a certain developmental stage. For
example, the SHN gene(s) can be selectively expressed in the leaves
of a plant by placing the coding sequence under the control of a
light-inducible promoter such as the promoter of the
ribulose-1,5-bisphosphate carboxylase small subunit gene of the
plant itself or of another plant, such as pea, as disclosed in U.S.
Pat. No. 5,254,799 or Arabidopsis as disclosed in U.S. Pat. No.
5,034,322. The choice of the promoter is determined by the
phenotype one aims to achieve, as will be described in more detail
below. For example, to achieve fruits (e.g., tomatoes) with an
increased water loss and therefore a more solid fruit flesh and
enhanced taste, a fruit specific or fruit preferred promoter is the
most suitable.
[0081] To achieve drought tolerance a constitutive, a leaf
specific, epidermis specific or light-inducible promoter would be
suitable. Suitable epidermal specific promoters, such as for
example the Arabidopsis LTP1 promoter (Thoma et al. 1994, Plant
Physiol. 105:35-45), the CER1 promoter (Aarts et al 1995. Plant
Cell 7:2115-27), and the CER6 promoter (Hooker et al., 2002, Plant
Physiol 129:1568-80.) and the orthologous tomato LeCER6 (Vogg et
al., 2004, J. Exp Bot. 55:1401-10), provide specific expression in
above ground epidermal surfaces. To achieve male sterility an
anther/anther tissue or anther development specific promoter such
as e.g., the SHN2 promoter provided herein, the tapetum specific
promoters TA13 and TA29 from tobacco (U.S. Pat. No. 6,562,354;
Koltunow et al., 1990, Plant Cell 2:1201-1224; Seurinck et al.,
1990 Nucleic Acids Res. 18:3403), the tapetum specific promoter
CA55 from Zea mays (EP570422), tapetum specific MS2 promoter from
Arabidopsis (Aarts et al., 1997, Plant J. 12:615-23), anther
specific TAA promoters from wheat (Wang et al., 2002, Plant J. 30:
613-623), tapetum specific promoter from rice (e.g., PE1, T42, T72
from rice), a microspore development specific promoter such as
NTM19 from tobacco (EP 790311) or a male germline specific promoter
(e.g., LGC1 from 111y, WO99/05281) or others may be used.
[0082] For certain phenotypes such as potatoes (i.e., tubers) with
enhanced wound healing and/or peel quality a tuber or peel specific
promoter is the most suitable such as the class II patatin promoter
(Nap et al., 1992, Plant Mol. Biol. 20:683-94.) that specifies
expression in the outer layer of the tuber, or a promoter with leaf
and tuber peel expression such as the potato UBI7 promoter
(Garbarino et al., 1995, Plant Physiol. 109:1371-8).
[0083] For phenotypes in root tissue a promoter preferentially
active in roots is described in WO00/29566. Another promoter for
root preferential expression is the ZRP promoter (and modifications
thereof) as described in U.S. Pat. No. 5,633,363.
[0084] To confer expression to fruits, a tomato fruit and peel
specific promoter e.g., beta-Galactosidase II (Smith et al., 1998,
Plant Physiol 117:417-23) or tomato Epicuticular wax promoter
LeCER6 (Vogg et al., 2004, supra) can be used to induce water loss
from fruit peel through the cuticle. A fruit skin or epidermal
promoter can be identified and isolated by one skilled in the art,
using microarrays and confirmation by transformation of promoter
reporter gene fusions.
[0085] Another alternative is to use a promoter whose expression is
inducible. Examples of inducible promoters are wound-inducible
promoters, such as the MPI promoter described by Cordera et al.
(1994, Plant J. 6:141), which is induced by wounding (such as
caused by insect or physical wounding), or the COMPTII promoter
(WO00/56897) or the promoter described in U.S. Pat. No. 6,031,151.
Alternatively the promoter may be inducible by a chemical, such as
dexamethasone as described by Aoyama and Chua (1997, Plant J. 11:
605-612) and in U.S. Pat. No. 6,063,985 or by tetracycline (TOPFREE
or TOP 10 promoter, see Gatz, 1997, Annu Rev Plant Physiol Plant
Mol. Biol. 48:89-108 and Love et al., 2000, Plant J. 21:579-88).
Other inducible promoters are for example inducible by a change in
temperature, such as the heat shock promoter described in U.S. Pat.
No. 5,447,858, by anaerobic conditions (e.g., the maize ADH1S
promoter), by light (U.S. Pat. No. 6,455,760), by pathogens (e.g.,
EP759085 or EP309862) or by senescence (SAG12 and SAG13, see U.S.
Pat. No. 5,689,042). Obviously, there are a range of other
promoters available. A podwall specific promoter from Arabidopsis
is the FUL promoter (also referred to as AGL8 promoter, WO99/00502;
WO99/00503; Liljegren et al., 2004 Cell. 116:843-53)), the
Arabidopsis IND1 promoter (Lijegren et al., 2004, supra.;
WO99/00502; WO99/00503) or the dehiscence zone specific promoter of
a Brassica polygalacturonase gene (WO97/13856). A petal specific
promoter has been described in WO99/15679. Seed specific promoters
are described in EP723019, EP255378 or WO98/45461.
[0086] The SHN coding sequence (or a chimeric SHN protein encoding
sequence) is inserted into the plant genome so that the coding
sequence is upstream (i.e., 5') of suitable 3' end transcription
regulation signals ("3' end") (i.e., transcript formation and
polyadenylation signals). Polyadenylation and transcript formation
signals include those of the CaMV 35S gene ("3' 35S"), the nopaline
synthase gene ("3' nos") (Depicker et al., 1982 J. Molec. Appl.
Genetics 1:561-573.), the octopine synthase gene ("3' ocs") (Gielen
et al., 1984, EMBO J. 3:835-845) and the T-DNA gene 7 ("3' gene 7")
(Velten and Schell, 1985, Nucleic Acids Res 13:6981-6998), which
act as 3'-untranslated DNA sequences in transformed plant cells,
and others.
[0087] Introduction of the T-DNA vector into Agrobacterium can be
carried out using known methods, such as electroporation or
triparental mating.
[0088] A SHN encoding nucleic acid sequence can optionally be
inserted in the plant genome as a hybrid gene sequence whereby the
SHN sequence is linked in-frame to a (U.S. Pat. No. 5,254,799;
Vaeck et al., 1987, Nature 328:33-37) gene encoding a selectable or
scorable marker, such as for example the neo (or nptII) gene (EP
0242236) encoding kanamycin resistance, so that the plant expresses
a fusion protein which is easily detectable.
[0089] Transformation of plant cells can also be used to produce
the SHN protein(s) of the invention in large amounts in plant cell
cultures to induce activated precursors of suberin, cutin and wax
biosynthesis that might be channeled for cross-linking into
bio-polymers. When reference to a transgenic plant cell is made
herein, this refers to a plant cell (or also a plant protoplast) as
such in isolation or in tissue culture, or to a plant cell (or
protoplast) contained in a plant or in a differentiated organ or
tissue, and both possibilities are specifically included herein.
Hence, a reference to a plant cell in the description or claims is
not meant to refer only to isolated cells in culture, but refers to
any plant cell, wherever it may be located or in whatever type of
plant tissue or organ it may be present.
[0090] All or part a SHN nucleic acid sequence, encoding a SHN
protein (or a chimeric SHN protein), can also be used to transform
microorganisms, such as bacteria (e.g., Escherichia coli,
Pseudomonas, Agrobacterium, Bacillus, etc.), fungi, viruses, algae
or insects. Transformation of bacteria, with all or part of a SHN
nucleic acid sequence of this invention, incorporated in a suitable
cloning vehicle, can be carried out in a conventional manner,
preferably using conventional electroporation techniques as
described in Maillon et al., (1989) FEMS Microbiol. Letters
60:205-210.) and WO 90/06999. For expression in prokaryotic host
cell, the codon usage of the nucleic acid sequence may be optimized
accordingly (as described for plants above). Intron sequences
should be removed and other adaptations for optimal expression may
be made as known.
[0091] For obtaining enhanced expression in monocot plants such as
grass species, e.g., corn or rice, an intron, preferably a monocot
intron, can be added to the chimeric gene. For example the
insertion of the intron of the maize Adh1 gene into the 5'
regulatory region has been shown to enhance expression in maize
(Callis et. al., 1987, Genes Develop. 1:1183-1200). Likewise, the
HSP70 intron, as described in U.S. Pat. No. 5,859,347, may be used
to enhance expression. The DNA sequence of the SHN nucleic acid
sequence can be further changed in a translationally neutral
manner, to modify possibly inhibiting DNA sequences present in the
gene part by means of site-directed intron insertion and/or by
introducing changes to the codon usage, e.g., adapting the codon
usage to that most preferred by plants, preferably the specific
relevant plant genus, as described above.
[0092] In accordance with one embodiment of this invention, the SHN
proteins (or chimeric proteins) are targeted to intracellular
organelles such as plastids, preferably chloroplasts, mitochondria,
or are secreted from the cell, potentially optimizing protein
stability and/or expression. Similarly, the protein may be targeted
to vacuoles. For this purpose, in one embodiment of this invention,
the chimeric genes of the invention comprise a coding region
encoding a signal or target peptide, linked to the SHN protein
coding region of the invention. Particularly preferred peptides to
be included in the proteins of this invention are the transit
peptides for chloroplast or other plastid targeting, especially
duplicated transit peptide regions from plant genes whose gene
product is targeted to the plastids, the optimized transit peptide
of Capellades et al. (U.S. Pat. No. 5,635,618), the transit peptide
of ferredoxin-NADP+oxidoreductase from spinach (Oelmuller et al.,
1993, Mol. Gen. Genet. 237:261-272), the transit peptide described
in Wong et al., 1992, Plant Molec. Biol. 20:81-93) and the
targeting peptides in published PCT patent application WO 00/26371.
Also preferred are peptides signalling secretion of a protein
linked to such peptide outside the cell, such as the secretion
signal of the potato proteinase inhibitor11 (Keil et al., 1986,
Nucl. Acids Res. 14:5641-5650), the secretion signal of the
alpha-amylase 3 gene of rice (Sutliff et al., 1991, Plant Molec.
Biol. 16:579-591) and the secretion signal of tobacco PR1 protein
(Cornelissen et al., 1986, EMBO J. 5:37-40). Particularly useful
signal peptides in accordance with the invention include the
chloroplast transit peptide (e.g., Van Den Broeck et al., 1985,
Nature 313: 358), or the optimized chloroplast transit peptide of
U.S. Pat. No. 5,510,471 and U.S. Pat. No. 5,635,618 causing
transport of the protein to the chloroplasts, a secretory signal
peptide or a peptide targeting the protein to other plastids,
mitochondria, the ER, or another organelle. Signal sequences for
targeting to intracellular organelles or for secretion outside the
plant cell or to the cell wall are found in naturally targeted or
secreted proteins, preferably those described by Klosgen et al.,
1989, Mol. Gen. Genet. 217:55-161; Klosgen and Weil, 1991, Mol.
Gen. Genet. 225:297-304; Neuhaus & Rogers (1998, Plant Mol.
Biol. 38:127-144; Bih et al., 1999, J. Biol. Chem. 274:22884-94;
Morris et al., 1999, Biochem. Biophys. Res. Commun. 255:328-333;
Hesse et al., 1989, EMBO J. 8:453-461; Tavladoraki et al., 1998,
FEBS Lett. 426:62-66; Terashima et al., 1999, Appl. Microbiol.
Biotechnol. 52:516-523; Park et al. 1997, J. Biol. Chem.
272:6876-81; Shcherban et al., 1995, Proc. Natl. Acad. Sci. USA
92:9245-9249).
[0093] To allow secretion of the SHN proteins to the outside of the
transformed host cell, an appropriate secretion signal peptide may
be fused to the amino terminal end (N-terminal end) of the SHN
protein. Putative signal peptides can be detected using computer
based analysis, using programs such as the program Signal Peptide
search (SignalP V1.1 or 2.0)(Von Heijne, Gunnar, 1986; Nielsen et
al., 1996).
[0094] In one embodiment, several SHN encoding nucleic acid
sequences are co-expressed in a single host. A co-expressing host
plant is easily obtained by transforming a plant already expressing
SHN protein of this invention, or by crossing plants transformed
with different SHN proteins of this invention. Alternatively,
several SHN protein encoding nucleic acid sequences can be present
on a single transformation vector or be co-transformed at the same
time using separate vectors and selecting transformants comprising
both chimeric genes. Similarly, one or more SHN encoding genes may
be expressed in a single plant together with other chimeric genes,
for example encoding other proteins which enhance drought
tolerance, such as CBF1, DREB1A, the rice OsDREB genes (Dubouzet et
al. 2003, Plant J. 33:751) or others.
[0095] It is understood that the different proteins can be
expressed in the same plant, or each can be expressed in a single
plant and then combined in the same plant by crossing the single
plants with one another. For example, in hybrid seed production,
each parent plant can express a single protein. Upon crossing the
parent plants to produce hybrids, both proteins are combined in the
hybrid plant.
[0096] Preferably, for selection purposes but also for weed control
options, the transgenic plants of the invention are also
transformed with a DNA encoding a protein conferring resistance to
herbicide, such as a broad-spectrum herbicide, for example
herbicides based on glufosinate ammonium as active ingredient
(e.g., Liberty.RTM. or BASTA; resistance is conferred by the PAT or
bar gene; see EP 0242236 and EP 0242246) or glyphosate (e.g.,
RoundUp.RTM.; resistance is conferred by EPSPS genes, see e.g., EPO
508909 and EP 0507 698). Using herbicide resistance genes (or other
genes conferring a desired phenotype) as selectable marker further
has the advantage that the introduction of antibiotic resistance
genes can be avoided.
[0097] Alternatively, other selectable marker genes may be used,
such as antibiotic resistance genes. As it is generally not
accepted to retain antibiotic resistance genes in the transformed
host plants, these genes can be removed again following selection
of the transformants. Different technologies exist for removal of
transgenes. One method to achieve removal is by flanking the
chimeric gene with lox sites and, following selection, crossing the
transformed plant with a CRE recombinase-expressing plant (see
e.g., EP506763B1). Site specific recombination results in excision
of the marker gene. Another site specific recombination systems is
the FLP/FRT system described in EP686191 and U.S. Pat. No.
5,527,695. Site specific recombination systems such as CRE/LOX and
FLP/FRT may also be used for gene stacking purposes. Further,
one-component excision systems have been described, see e.g.,
WO9737012 or WO9500555).
Transformed Plant Cells/Plants/Seeds and Uses of the Nucleic Acid
Sequence and Proteins According to the Invention
[0098] In the following part the use of the SHN sequences according
to the invention to generate transgenic plant cells, plants, plant
seeds and any derivatives/progeny thereof, with one or more
modified phenotypes is described.
[0099] A) Plants with Enhanced Drought Tolerance
[0100] A transgenic, drought tolerant plant can be generated by
transforming a plant host cell with a nucleic acid sequence
encoding at least one SHN protein under the control of a suitable
promoter, as described above, and regenerating a transgenic plant
from said cell. Preferred promoters are promoters which are active
specifically in above-ground parts of the plant, such as in the
leaves, leaf epidermis or upon light induction or following
application of chemical compounds. In particular the following
promoters are preferred: leaf epidermal specific promoters such as
the Arabidopsis LTP1 (Thoma et al. 1994, supra), the CER1 promoter
(Aarts et al. 1995, supra), the CER6 promoter (Hooker et al 2002,
supra) and the orthologous tomato LeCER6 promoter (Vogg et al.
2004, supra); leaf or photosynthetic tissue specific promoters,
such as the light inducible ribulose 1,5-bisphosphate carboxylase
small subunit promoter (Pssu) from Arabidopsis as described in U.S.
Pat. No. 5,034,322 or from sunflower, from pea (U.S. Pat. No.
5,254,799) or from Zea mays; the potato ST-LS1 promoter which is
stem and leaf specific (Stockhaus et al. 1987, Nucleic Acids
Res.15:3479-91); the promoter of the chlorophyll a/b binding
protein (CAB).
[0101] As the promoter of the SHN3 gene is active in all plant
organs analyzed, the SHN3 promoter (SEQ ID NO: 19) according to the
invention, or the smallest active fragment thereof, may also be
used.
[0102] "Drought tolerance" or "increased/enhanced drought
tolerance" is used herein to refer to an enhanced ability of
transformants (compared to wild type or control transformants) to
tolerate a period of drought (water deprivation/depletion leading
to e.g., visible leaf wilting symptoms in control plants) and to
recover subsequently, thereby leading to a reduced overall yield
loss, as more plants per m.sup.2 survive and/or the yield of the
surviving plants is not significantly reduced. Drought tolerance
can be assessed in controlled environments (green house or growth
chambers) by placing at least about 10 transformants per
transformation event and at least 10 control plants for various
time periods (ranging from 1-4 weeks or more) into the environment
without watering them, until leaf wilting or loss of turgor is
caused on control plants, and subsequently watering the plants
again for 1-2 weeks, while their recovery phenotype is analyzed.
Transformants with drought tolerance survive at least 2, 3, 4, 5,
6, 7 days, preferably at least 2-5 days longer without water than
control-transformants (e.g., transformed with an empty vector) or
wild type plants do under the same conditions, and which show
irreversible tissue damage. In another method of estimating
tolerance the recovery of transformants is at least about 2-5 times
higher than that of the control plants (e.g., with 20% control
recovery, 40-100% survival in transformants).
[0103] Transformants expressing high levels of the SHN protein are
selected by e.g., analyzing copy number (Southern blot analysis),
mRNA transcript levels (e.g., RT-PCR using SHN primer pairs or
flanking primers) or by analyzing the presence and level of SHN
protein in various tissues (e.g., SDS-PAGE; ELISA assays, etc.).
For regulatory reasons, preferably single copy transformants are
selected and the sequences flanking the site of insertion of the
chimeric gene is analyzed, preferably sequenced to characterize the
"event". High SHN expressing transgenic events are selected for
further crossing/backcrossing/selfing until a high performing elite
event with a stable SHN transgene is obtained. Generally, SHN gene
expression levels and SHN protein levels will correlate with the
drought tolerance phenotype. In one embodiment especially the
transgenic seeds derived from such plants are provided, which may
be sold as being "drought tolerant".
[0104] Transformants expressing one or more SHN genes according to
the invention may also comprise other transgenes, such as other
genes conferring drought tolerance or conferring tolerance to other
biotic or abiotic stresses. To obtain such plants with "stacked"
transgenes, other transgenes may either be introgressed into the
SHN transformants, or the SHN transformants may be transformed
subsequently with one or more other genes, or alternatively several
chimeric genes may be used to transform a plant line or variety.
For example, several chimeric genes may be present on a single
vector, or may be present on different vectors which are
co-transformed.
[0105] In one embodiment the following genes are combined with one
or more SHN genes according to the invention: Genes encoding other
AP2/EREBP type transcription factors, preferably ones which have a
role in the plant's response to environmental stresses, such as for
example the CBF1, CBF2, CBF3 and/or CBF4 encoding genes from
Arabidopsis (Jaglo-Ottosen et al 1998, Kasuga et al 1999, supra) or
orthologs thereof from other species (Dubouzet et al 2003, supra),
with insect resistance genes such as Bacillus thuringiensis toxin
genes (encoding insecticidal proteins, such as cry genes, vip
genes, etc. see http://www.biols.susx.ac.uk/home/for a list of
available genes), fungal resistance genes, or other genes.
[0106] The stacked transformants may thus have an even broader
environmental stress tolerance, to for example salinity, cold
stress, insect resistance, pathogen resistance, heat stress, water
stress, etc.
It is also possible to introduce or introgress the SHN gene into a
plant breeding line which already has a relatively high drought
tolerance, whereby this tolerance may be due to a different
underlying molecular mechanism (e.g., root architecture).
[0107] In a preferred embodiment the transformants are drought
tolerant, but have an unmodified epicuticular wax layer and thus
the leaves have unmodified appearance compared to wild type plants.
In this embodiment monocotyledonous plants, such as rice and maize,
are especially preferred.
[0108] In one embodiment, SEQ ID NO: 144 of WO03/013228 and/or the
WIN1 gene described by Broun et al. (supra) are excluded
herein.
[0109] B) Podshatter Resistant Plants
[0110] In another embodiment podshatter resistant plants are
provided, which overexpress a SHN-repressor domain fusion protein
according to the invention (e.g., a SHN1-EAR, SHN2-EAR, SHN3-EAR or
OsSHN1-EAR fusion protein, or another SHN ortholog-EAR fusion) or
which express a nucleic acid sequence which causes silencing of the
endogenous SHN gene(s). "Podshatter resistance" refers herein to
the plant's pods having an increased resistance to pod valve
separation at maturity, resulting in a reduced seed loss during
harvest. However, the increase in resistance to valve separation
preferably does not result in an inability to separate the pod
valves, which would make the harvesting of seeds very difficult or
impossible. This "fine-tuning" of the ease/difficulty of separating
the pod valves may be achieved by selecting a suitable
promoter/coding sequence combination.
[0111] A number of tests exist which can be used to assess the
podshatter resistance of a plant, such as the Random Impact Test
(RIT) (see Summers et al., 2003, J. Agric Science 140:43-52 and
Bruce et al., 2002, Biosystems Eng. 81:179-184). The RIT involves
collecting fully mature pods from plants and placing them for a
number of days in a controlled environment (e.g., 3 days at 25C and
50% RH). Twenty undamaged pods are then placed together with six
steel balls of 12.5 mm diameter in a 20 cm diameter cylindrical
container. The container is mechanically shaken at a frequency of
4.98 Hz and a stroke length of 51 mm for two 10s periods, followed
where required by one period each of 20, 40 and 80s. At the end of
each period pods are examined and classed as shattered if at least
one of the valves had detached. Statistical analysis is then used
to calculate the time (s) taken for 50% of the pods to shatter
(RIT.sub.50 value). In such a test a shatter susceptible plant line
will result in mean RIT.sub.50 values of around 18 seconds with a
narrow distribution around the mean. A shatter resistant plant can
be defined by having an RIT50 value which is significantly larger
than the RIT.sub.50 value of the control (e.g., the wild type or
control transformant), for example a mean RIT.sub.50 of 1.5.times.,
2.times., 3.times., 4.times. (or more) the value of the control.
Alternatively, seed loss in the field can be assessed, for example
by placing trays underneath the plants and collecting the shattered
seeds.
[0112] Podshatter resistant plants according to the invention may
be generated by repressing the formation of the dehiscence zone by
silencing the SHN gene or by expressing a SHN-repressor domain
fusion protein, especially a SHN-EAR fusion protein (as described
above). This can be achieved by transforming a plant cell with a
chimeric construct comprising a pod- or fruit-specific promoter or
a promoter which is preferentially active in a specific tissue of
the pod or during a specific stage of pod-development, operably
linked to either a SHN-repressor domain fusion protein (e.g., a
SHN-EAR fusion protein) encoding nucleic acid sequence or a gene
silencing SHN fragment (e.g., a sense and/or antisense SHN DNA
fragment, see below) and suitably a 3' sequence. Suitable promoters
are for example the SHN2 promoter (SEQ ID NO: 18) or an active
fragment thereof, the promoter of the Arabidopsis or Brassica napus
FRUITFUL gene (also referred to as AGL8) (see U.S. Pat. No.
6,198,024), the Arabidopsis or Brassica dehiscence zone specific
regulatory elements of genes AGL1 or AGL5 (see U.S. Pat. No.
6,198,024), the promoter of the Arabidopsis INDEHISCENT1 gene
(IND1; see WO01/7951) or of the Brassica napus homolog of IND1, or
a dehiscence zone specific promoters such as the Brassica
polygalacturonase promoter described in WO97/13856, or derivatives
thereof. Alternatively a constitutive promoter may be used.
[0113] As pod shattering and the associated yield loss is a problem
in pod-bearing plants, mainly members of the Brassicaceae such as
of Brassica napus, but also members of the Fabaceae, such as
soybeans, peas, lentils and beans such as soybean, the host plant
is preferably selected from these plants. The host may also be a
synthetic B. napus or a double haploid B. napus line.
[0114] The transgenic, shatter resistant plant according to the
invention may also be a double haploid plant. The double haploid
plant can be generated, e.g., by culturing microspores obtained
from the transformed plant, followed by chromosome doubling (e.g.,
induced by colchicine treatment) and regeneration.
[0115] In addition the use of SHN transcription regulatory
elements, especially SHN2 transcription regulatory element (SEQ ID
NO: 18 or the smallest active fragment thereof) or the
transcription regulatory element of a nucleotide sequence encoding
a SHN2 ortholog, may be used to confer dehiscence zone specific
expression and may thus be used to confer pod shatter resistance.
For this purpose a nucleic acid sequence which modulate the pod
structure, especially the anatomical structure of the pod
dehiscence zone, may be operable linked downstream of the
transcription regulatory element. Examples nucleic acid sequences
suitable are for example the Arabidopsis FRUITFUL gene (FUL or
AGL8; EP 1002087) or homologs thereof. Alternatively the promoter
may be used in gene silencing constructs, resulting in pod shatter
resistance. For example a short antisense fragment of the
Arabidopsis IND1 gene or a sense/antisense fragment (inverted
repeat) may be operably linked downstream of the transcription
regulatory element. For gene silencing constructs, see below.
Likewise, a nucleic acid sequence encoding a SHN-repressor domain
fusion protein may be operably linked to a SHN transcription
regulatory element, such as the SHN2 promoter.
[0116] C) Male Sterile Plants
[0117] Further provided are transgenic male sterile plants and
method for making these using a SHN nucleic acid sequence according
to the invention. Transgenic male sterile plants can be generated
by transforming a host plant cell with a vector comprising a
suitable promoter operably linked to a SHN-repressor domain fusion
protein (preferably a SHN-EAR protein) encoding DNA sequence and
optionally a suitable 3' nontranslated nucleic acid region. The
promoter sequence is suitably selected from a dehiscence zone
specific promoter active during anther dehiscence, an anther
specific promoter or a tapetum specific promoter (for all see
above), or the SHN2 promoter (SEQ ID NO: 18) or an active fragment
thereof. A chemically inducible promoter may also be used. If the
chemical is sprayed at the right stage of flower development the
sprayed plants will be sterile.
[0118] Overexpression of the SHN-EAR protein (or of another
SHN-repressor domain fusion protein) during anther and/or pollen
development leads to male sterility. "Male sterility" is herein
defined as a significantly reduced release of mature pollen grains
from the anther, preferably the complete absence of pollen
release.
[0119] Transgenic male sterile plants may be used for producing
hybrid seeds, for example by growing male sterile (MS) and male
fertile plants (fertility restorer lines, RF) in rows next to each
other allowing cross pollination of the male sterile plants. The
seed collected from the male sterile plants are pure hybrid seeds.
To maintain the pure male sterile line, anther dehiscence can be
achieved mechanically from isolated anthers and used for brush or
blow pollination on the same line. The hybrids are produced by
crossing the MS lines to RF lines for seed crop production. The RF
line comprises for example a homologous (from same crop) SHN gene,
optionally encoding a SHN protein with a fusion to an Activation
domain, such as the transcriptional activation domain of the VP16
protein from Herpes simplex virus or the yeast GAL4 (see Wilde et
al. 1994, Plant Mol. Biol. 24, 381-388 and Moore et al. 1998, Proc.
Natl. Acad. Sci. 95, 376-381), under control of a strong promoter
expressed in the anther dehiscent zone. The promoter in the RF line
should have higher expression levels (preferably 10 times) than
that of the promoter driving the Repressor SHN-EAR gene in the MS
line. The high expression of the homologous SHN (preferably with an
Activation domain) will out-compete the Repressor SHN-EAR and allow
anther dehiscence and pollen release that will pollinate the crop
plant by natural cross-pollination, e.g., by wind or bees.
[0120] Male sterile plants may also be used for other purposes,
such as reducing pollen dispersal into the environment and
allergenicity problems caused by pollen. In one embodiment the male
sterile plants are plants which can be propagated by vegetative
propagation, such as grasses. Male sterile plants according to the
invention may also be used to produce pharmaceutically active
molecules in such transgenic plants. The male sterility reduces the
risk of the transgenes spreading to other plants. A plant according
to the invention may therefore additionally comprise a chimeric
gene encoding a pharmaceutical protein or protein fragment, such as
antigens, antibodies or antibody chains, and the like.
[0121] In addition the use of SHN transcription regulatory
elements, especially SHN2 transcription regulatory element (SEQ ID
NO: 18 or the smallest active fragment thereof) or the
transcription regulatory element of a nucleotide sequence encoding
a SHN2 ortholog, may be used to confer dehiscence zone specific
expression and may thus be used to confer male sterility. For this
purpose genes for mutants involved in anther dehiscence can be
used, e.g., AtMYB26 (Steiner-Lange, 2003, Plant J. 34:519-528),
delayed dehiscence] (Sanders et al., 2000, Plant Cell 12:1041-61).
To create specific loss-of-function in the anther dehiscence zone
an antisense or RNAi strategy can be followed, or a chimeric
transcriptional factor repressor as described using e.g., the EAR
repressor domain (Hiratsu et al 2003, supra). Another way is to use
the SHN2 promoter to specifically disrupt the dehiscent zone using
a nucleic acid sequence encoding for example a cytotoxic protein or
a RNA may be operably linked downstream of the transcription
regulatory element. Examples of nucleic acid sequences suitable are
the gene encoding the ribonuclease barnase from Bacillus
amyloliquefaciens (see EP 0344029B1), diphtheria toxin, RNase-T1
from Aspergillus oryzae (Quaas et al., 1988, Eur J Biochem
173:617-622) or others.
[0122] In another embodiment a SHN gene silencing construct,
whereby a sense and or antisense SHN RNA is transcribed in the host
cell is used to generate male sterile plants (see below).
[0123] D) Postharvest/Processing Fleshy Fruit Improvement: Texture,
Firmness, Soluble Solids of Whole Fruit and Juice
During fruit development (e.g., of tomato) the ovary wall becomes
the pericarp, which is covered by a thin cuticle. The skin of the
pericarp consists of an epidermal cell layer and three to four
layers of collenchymous tissue. The outer epidermal cells contain
no stomata, so that water content is regulated via cuticle
permeability. Due to the fact that SHN proteins were found to
result in an increased water loss through the cuticle, the
production of SHN proteins in fruit or fruit cells/tissues
(especially the outer epidermal cells) results in an increased
cuticular water loss of the developing fruit and in fruit with a
higher % weight soluble solids than found in the fruit of control
plants. The percentage of soluble solids is increased by at least
1%, 2%, 3%, more preferably by at least 5%, 6%, 7% or more,
compared to controls. Soluble-solids concentration are defined in
.sup.0Brix, that is a standard refractometric measure primarily
detecting reducing sugars, but also affected by other soluble
constituents. .sup.0Brix can be measured by a hand-held
refractometer (e.g., American Optical Corp., Buffalo, N.Y.), where
a 1.degree. Brix is approximately 1% w/w.
[0124] Soluble solids are an important quality trait, especially
for the fruit processing industries. Other important traits are
fruit texture and firmness, as well as flavor, which are also
influenced by fruit water content and can therefore be modified by
overexpressing one or more SHN proteins according to the
invention.
In one embodiment transgenic plants are provided, comprising within
their genome a chimeric gene which comprises a fruit peel specific
promoter operably linked to a SHN protein encoding DNA sequence
according to the invention. Also provided are the mature fruit of
those plants, as well as seeds and progeny thereof. In one
embodiment the phenotype of the transgenic fruit is modified
compared to the fruit of non-transgenic plants in that the
percentage soluble solids is increased, and/or the fruit texture
and/or firmness is increased, and/or the fruit flavor is improved.
In a preferred embodiment the host plant is a tomato plant
(Lycopersicon species) and the modified fruit is a tomato.
Processing tomatoes require a higher percentage of soluble solids
than fresh market tomatoes and the fruit according to the invention
are therefore particularly suitable for the processing industry
(tomato pastes, canned tomatoes, cooked tomatoes, etc.). In one
embodiment the processed pure/juice is be improved for one or more
processing characteristics, including pH, titratable acidity,
precipitate weight ratio, total solids, serum viscosity, efflux
viscosity and color. The fruit will also be easier and cost
effective to transport with less damage and spoilage.
[0125] Lycopersicon species include L. cheesmanii, L. chilense, L.
chmielewskii, L. esculentum (tomato), Lycopersicon esculentum var.
cerasiforme (cherry tomato), L. esculentum x L. peruvianum, L.
glandulosum, L. hirsutum, L. minutum, L. parviflorum Lycopersicon
pennellii, L. peruvianum (Peruvian tomato), L. peruvianum var.
humifusum and L. pimpinellifolium (currant tomato).
[0126] The modified phenotype can be generated by transforming any
plant host producing fleshy fruit, for example grape, peach, plum,
cherry, mango, strawberry can be transformed in order to
concentrate the soluble solids and reduced post-harvest damage
prior to processing for fruit concentrate products and/or improve
fruit flavor and fruit juices.
[0127] Suitable fruit specific promoters or promoters specifically
expressed during fruit development and/or in a certain
cells/tissues of the fruit (especially the outer epidermal cells)
are known in the art. Examples are the promoter of the tomato
cuticular wax gene LeCER6 (Vogg et al. 2004, J. Exp Bot. 55:
1401-10) or for example provided in U.S. Pat. No. 5,753,475
(describing e.g., a tomato polygalacturonase promoter, which is
active in at least the breaker through red fruit stage in tomato
fruit). Other suitable promoters can be easily identified by a
person skilled in the art. For example, for each fleshy fruit, a
fruit skin or epidermis specific promoter can be identified.
[0128] In a preferred embodiment the transgenic fruit are more
solid in texture and/or have an improved flavor and/or improved
processing characteristics compared to controls.
[0129] E) Plants with Enhance Wound Healing Properties and/or
Enhanced Suberization
[0130] In yet a further embodiment transgenic plants, expressing
one or more SHN proteins according to the invention, are provided,
which have an enhanced wound healing phenotype. "Enhanced wound
healing" refers to the enhanced ability to form a protective layer
on the wounded tissue surface following wounding. The protective
layer may be either produced more rapidly than in control plants
(e.g., non-transgenic plants) or it may be altered in thickness
and/or chemical composition.
[0131] Wounding may occur during processing of plants (e.g., during
harvest) or naturally by wind, animals feeding on tissue, etc.
Often wounding may result in yield loss and in quality loss of crop
plants. In a preferred embodiment the host plant is potato (Solanum
tuberosum). Preferably the SHN coding sequence is expressed under a
tuber-peel specific promoter. Tubers of transgenic plants
preferably comprise a protective shiny outer tuber layer that would
protect the tubers from mechanical damage and display an attractive
tuber quality for consumer preference. In addition, damage to
tubers during harvest and post-harvest transport is reduced by
enhanced wound healing, thus preventing further spoilage to the
rest of the tubers stored along with the damaged tubers. This also
contributes to improved general tuber quality and reduction in
post-harvest yield losses.
[0132] In another embodiment woody tree species (e.g., Populus,
Salix, Quercus, Eucalyptus species) are transformed with a vector
according to the invention, whereby one or more SHN proteins are
produced by the transgenic tree, leading to cork cells with
enhanced suberin formation. The high production of woody biomass as
renewable energy use, as well as traditional uses for timber and
paper is being addressed by development of genomics and
biotechnological resources (Taylor, 2002, Annals Botany 90:
681-689). Transformation systems and specific promoters are
identified that enable the expression of the SHN genes to regulate
the deposition of increased suberin in the cork of woody species.
The natural production of suberin in the cork of Quercus can be
enhanced, and also more suberin produced in the other woody trees.
Cork is a natural defensive mechanism against drought, brush fires
and temperature fluctuations in the natural habitat where the cork
trees grow. Thus producing an enhanced suberin cork layer in other
trees would provide similar properties to the other woody tree
species. Cork is actually made of water-resistant cells that
separate the outer bark from the delicate interior bark. It has a
unique set of properties not found in any other naturally existing
material. It is lightweight, rot resistant, fire resistant, termite
resistant, impermeable to gas and liquid, soft and buoyant. Thus
these qualities would improve the wood quality of other woody trees
providing new applications. Other uses of processed corkboard are
for soundproofing and as insulation in refrigerators and cold
storage plants; gaskets and washers in engines and motors; pipe
coverings; polishing wheels; floor and wall coverings in addition
to the traditional beverage bottle caps (including wine and
champagne).
[0133] Whole plants, seeds, cells, tissues and progeny (such as F1,
F2 seeds/plants, etc.) of any of the transformed plants described
above are encompassed herein and can be identified by the presence
of the transgene in the DNA, for example by PCR analysis using
total genomic DNA as template and using SHN specific PCR primer
pairs. Also "event specific" PCR diagnostic methods can be
developed, where the PCR primers are based on the plant DNA
flanking the inserted chimeric gene, see U.S. Pat. No. 6,563,026.
Similarly, event specific AFLP fingerprints or RFLP fingerprints
may be developed which identify the transgenic plant or any plant,
seed, tissue or cells derived there from.
[0134] It is understood that the transgenic plants according to the
invention preferably do not show non-desired phenotypes, such as
yield reduction, enhanced susceptibility to diseases or undesired
architectural changes (dwarfing, deformations) etc. and that, if
such phenotypes are seen in the primary transformants, these can be
removed by normal breeding and selection methods
(crossing/backcrossing/selfing, etc.). Any of the transgenic plants
described herein may be homozygous or hemizygous for the
transgene.
[0135] F) Gene Silencing and the Generation of Loss-of-Function
Phenotypes by SHN-Repressor Domain Fusions Proteins
For certain applications it is desired to generate transgenic
plants in which a SHN gene or the SHN gene family is silenced or is
silenced in specific cells or tissues of the plant. "Gene
silencing" refers to the down-regulation or complete inhibition of
gene expression of one or more target genes. The use of inhibitory
RNA to reduce or abolish gene expression is well established in the
art and is the subject of several reviews (e.g., Baulcombe 1996,
Stam et al. 1997, Depicker and Van Montagu, 1997). There are a
number of technologies available to achieve gene silencing in
plants, such as chimeric genes which produce antisense RNA of all
or part of the target gene (see e.g., EP 0140308B1, EP 0240208B1
and EP 0223399B1), or which produce sense RNA (also referred to as
co-suppression), see EP 0465572B1.
[0136] The most successful approach so far has however been the
production of both sense and antisense RNA of the target gene
("inverted repeats"), which forms double stranded RNA (dsRNA) in
the cell and silences the target gene. Methods and vectors for
dsRNA production and gene silencing have been described in EP
1068311, EP 983370A1, EP 1042462A1, EP 1071762A1 and EP
1080208A1.
[0137] A vector according to the invention may therefore comprise a
transcription regulatory region which is active in plant cells
operably linked to a sense and/or antisense DNA fragment of a SHN
gene according to the invention. Generally short (sense and
antisense) stretches of the target gene sequence, such as 17, 18,
19, 20, 21, 22 or 23 nucleotides of cording or non-coding sequence
are sufficient. Longer sequences can also be used, such as 100, 200
or 250 nucleotides. Preferably, the short sense and antisense
fragments are separated by a spacer sequence, such as an intron,
which forms a loop (or hairpin) upon dsRNA formation. Any short
stretch of SEQ ID NO: 1-10 may be used to make a SHN gene silencing
vector and a transgenic plant in which one or more SHN genes are
silenced in all or some tissues or organs. A convenient way of
generating hairpin constructs is to use generic vectors such as
pHANNIBAL and pHELLSGATE, vectors based on the Gateway.RTM.
technology (see Wesley et al. 2004, Methods Mol. Biol. 265:117-30;
Wesley et al. 2003, Methods Mol. Biol. 236:273-86 and Helliwell
& Waterhouse 2003, Methods 30:289-95.), all incorporated herein
by reference.
[0138] By choosing conserved nucleic acid sequences all SHN gene
family members in a host plant can be silenced. Encompassed herein
are also transgenic plants comprising a transcription regulatory
element operably linked to a sense and/or antisense DNA fragment of
a SHN gene and exhibiting a SHN gene silencing phenotype. Gene
silencing constructs may also be used in reverse genetic
approaches, to elucidate or confirm the function of a SHN gene or
gene family in a host species.
[0139] In one embodiment SHN gene silencing is used to generate
podshatter resistance and/or male sterility in host plants.
However, due to structural and functional redundancy, gene
silencing approaches may not always be successful and may show no
phenotypic change or only a subtle phenotype, possibly revealed
only under extreme environmental conditions, when knocked-out. A
preferred approach is, therefore, to generate male sterile plants
and/or podshatter resistant plants by over-expressing a
SHN-repressor domain fusion protein in the host cells, as described
above. In a preferred embodiment this chimeric protein is a SHN-EAR
fusion protein or a En-SHN fusion protein, e.g., a En.sup.298-SHN
fusion protein.
[0140] G. Transgenic Plants Having Enhanced Salinity Tolerance
[0141] A transgenic, salinity tolerant (salt tolerant) plant can be
generated by transforming a plant host cell with a nucleic acid
sequence encoding at least one SHN protein under the control of a
suitable promoter, as described above and in the Examples, and
regenerating a transgenic plant from said cell. Preferred promoters
are promoters are constitutive, inducible or root specific
promoters.
[0142] "Salinity tolerance" or "enhanced salinity tolerance" refers
to the ability to grow and survive on saline soil or growth medium,
especially without yield loss or only with minimal yield loss.
Preferably, a salinity tolerant plant has a percentage of survival
on saline soil, which is at least 10, 20, 30, 40, 50, 80, 90 or
100% higher than that of the control plants.
[0143] Salinity tolerance can be determined as described in the
Examples (by assessing the number of plants surviving when
subjected to saline medium) or by growing the plants and controls
on soils with various salinity levels, such as soils having an
EC.sub.e value (Electrical Conductivity of the extract) of 2-4 dS/m
(deciSiemens per meter), 4-8 dS/m, 8-16 dS/m or above 16 dS/m (very
saline). A plant is salinity tolerant if it can grow on soil with a
higher EC.sub.e value than the control plant, without yield loss or
with only minimal yield loss. Preferably, SHN overexpressing plants
are able to grow without yield loss (or only with minimal yield
loss) on soil with an ECe value which is at least one, preferably
at least 2, more preferably at least 3 or more dS/m units higher
than that of the control.
[0144] In a preferred embodiment the plant is both salinity
tolerant and drought tolerant.
[0145] H. Non-Transgenic Plants Comprising a Modified Phenotype
[0146] It is also an embodiment of the invention to use
non-transgenic methods, e.g., mutagenesis systems such as TILLING
(Targeting Induced Local Lesions IN Genomics; McCallum et al.,
2000, Nat Biotech 18:455, and McCallum et al. 2000, Plant Physiol.
123, 439-442, both incorporated herein by reference) and selection
to generate plant lines which produce higher levels of one or more
SHN proteins according to the invention. Without limiting the scope
of the invention, it is believed that such plants could comprise
point/deletion mutations in the promoter that are binding sites for
repressor proteins that would make the host SHN gene constitutive
or higher in expression. Preferably SHN protein levels in the
mutant or parts of the mutant are at least about 2, 5, 10, 15% or
more increased in the mutant compared to non-mutant plants. TILLING
uses traditional chemical mutagenesis (e.g., EMS mutagenesis)
followed by high-throughput screening for mutations (e.g., using
Cel 1 cleavage of mutant-wildtype DNA heteroduplexes and detection
using a sequencing gel system), see e.g., Henikoff et al. Plant
Physiology Preview May 21, 2004. Thus, non-transgenic plants, seeds
and tissues comprising an enhanced SHN gene expression in one or
more tissues and comprising one or more of the SHN phenotypes
according to the invention (e.g., enhanced drought tolerance,
enhanced salinity tolerance, enhanced suberization, etc., all as
described above) and methods for generating and identifying such
plants is encompassed herein.
[0147] The method comprises in one embodiment the steps of
mutagenizing plant seeds (e.g., EMS mutagenesis), pooling of plant
individuals or DNA, PCR amplification of a region of interest,
heteroduplex formation and high-throughput detection,
identification of the mutant plant, sequencing of the mutant PCR
product. It is understood that other mutagenesis and selection
methods may equally be used to generate such mutant plants. Seeds
may for example be radiated or chemically treated and the plants
screened for a modified SHN phenotype, such as enhanced drought
tolerance.
[0148] In another embodiment of the invention, the plant materials
are natural populations of the species or related species that
comprise polymorphisms or variations in DNA sequence at the SHN
orthologous coding and/or regulatory sequence. Mutations at the SHN
gene target can be screened for using a ECOTILLING approach
(Henikoff et al 2004, supra). In this method natural polymorphisms
in breeding lines or related species are screened for by the above
described TILLING methodology, in which individual or pools of
plants are used for PCR amplification of the SHN target,
heteroduplex formation and high-throughput analysis. This can be
followed up by selecting of individual plants having the required
mutation that can be used subsequently in a breeding program to
incorporate the desired SHN-orthologous allele to develop the
cultivar with desired trait.
[0149] In a further embodiment non-transgenic mutant plants which
produce lower levels of SHN protein in one or more tissues are
provided, or which completely lack SHN protein in specific tissues
or which produce a non-functional SHN protein in certain tissues,
e.g., due to mutations in one or more endogenous SHN alleles. For
this purpose also methods such as TILLING may be used. Seeds may be
mutagenized using e.g., radiation or chemical mutagenesis and
mutants may be identified by detection of DNA polymorphisms using
for example CEL 1 cleavage. Especially, mutants which comprise
mutations in one or more SHN alleles and which are shatter
resistant and or male sterile are provided. Non-functional SHN
alleles may be isolated and sequenced or may be transferred to
other plants by breeding methods.
[0150] Mutant plants can be distinguished from non-mutants by
molecular methods, such as the mutation(s) present in the DNA, SHN
protein levels, SHN RNA levels etc., and by the modified phenotypic
characteristics.
[0151] The non-transgenic mutants may be homozygous or heterozygous
for the mutation conferring the enhanced expression of the
endogenous SHN gene(s) or for the mutant SHN allele(s).
SEQUENCES
[0152] SEQ ID NO 1: Arabidopsis thaliana genomic DNA encoding SHN1
[0153] SEQ ID NO 2: Arabidopsis thaliana genomic DNA encoding SHN2
[0154] SEQ ID NO 3: Arabidopsis thaliana genomic DNA encoding SHN3
[0155] SEQ ID NO 4: Arabidopsis thaliana SHN1 transcript [0156] SEQ
ID NO 5: Arabidopsis thaliana SHN2 transcript [0157] SEQ ID NO 6:
Arabidopsis thaliana SHN3 transcript [0158] SEQ ID NO 7:
Arabidopsis thaliana SHN1 coding sequence [0159] SEQ ID NO 8:
Arabidopsis thaliana SHN2 coding sequence [0160] SEQ ID NO 9:
Arabidopsis thaliana SHN3 coding sequence [0161] SEQ ID NO 10:
Oryza sativa OsSHN1 coding sequence [0162] SEQ ID NO 11:
Arabidopsis thaliana SHN1 amino acid sequence [0163] SEQ ID NO 12:
Arabidopsis thaliana SHN2 amino acid sequence [0164] SEQ ID NO 13:
Arabidopsis thaliana SHN3 amino acid sequence [0165] SEQ ID NO 14:
Oryza sativa OsSHN1 amino acid sequence [0166] SEQ ID NO 15: SHINE
"mm" consensus domain [0167] SEQ ID NO 16: SHINE "cm" consensus
domain [0168] SEQ ID NO 17: transcription regulatory sequence of
SHN1 [0169] SEQ ID NO 18: transcription regulatory sequence of SHN2
[0170] SEQ ID NO 19: transcription regulatory sequence of SHN3
[0171] SEQ ID NO 20: transcription regulatory sequence of OsSHN1
[0172] SEQ ID NO 21: EAR repressor domain [0173] SEQ ID NO 22:
coding sequence of EAR repressor domain [0174] SEQ ID NO 23: cDNA
of OsSHN2 [0175] SEQ ID NO 24: amino acid sequence of OsSHN2
BRIEF DESCRIPTION OF THE DRAWINGS
[0176] FIG. 1--Chain length distribution [% of compound class] for
the four major fractions in the leaf cuticular wax of wild type and
shn.
[0177] FIG. 2--The shn Mutant and 35S::SHN1 Plants Phenotype and
Surface Permeability. (A) Chlorophyll leaching assays with mature
rosette leaves of shn and wild-type Ws immersed in 80% ethanol for
different time intervals. The results are derived from three
independent experiments and depicted with standard error of the
mean for each time point. (B) Chlorophyll leaching assays as
described above but using mature rosette leaves derived from
35S::SHN1 (#2-2) progeny and wild-type plants. (C) Rate of water
loss from the progeny of the activation tag shn mutant, two
35S::SHN1 primary transformants (#2-2 and #2-5) and wild type Ws.
Four rosette explants (root system and inflorescence stem detached)
were weighed during the time intervals depicted. The results are
derived from three independent experiments and depicted with
standard error of the mean for each time point.
[0178] FIG. 3--The SHINE Clade of Arabidopsis AP2/EREBP
Transcription Factor Family. Sequence alignment of the four SHN
proteins. SNH1--SEQ ID NO:11; SNHN2--SEQ ID NO:12; SHN3--SEQ ID
NO:13; Oryza sativa (rice)--SEQ ID NO:14). SHN Glade members
contain a single AP2 domain at their N-termini, a conserved middle
domain (termed "mm"; SEQ ID NO:15) and a most conserved C-terminal
domain (termed "cm"; SEQ ID NO:16). Black background indicates 100%
conservation, gray is 75% and light gray is 50% conservation.
[0179] FIG. 4--Drought Tolerance Experiment with shn and 35S::SHN1
Lines. Fifteen days old seedlings of either wild-type Ws, progenies
of shn, two 35S::SHN1 lines (#2-2 and #2-5) and a positive control
rd29-DREB1A line (providing drought tolerance; Kasuga et al. 1999,
supra) were exposed for a period of 9 to 12 days of dehydration.
Subsequently, seedlings were watered and their appearance after a
week (recovery) is presented in the image (apart from the first row
at 9 DOD, in which pictures were taken directly at the end of the
dehydration period). DOD, Days of dehydration.
EXAMPLES
[0180] The following non-limiting Examples describe the use of SHN
genes for modifying plant phenotypes. Unless stated otherwise in
the Examples, all recombinant DNA techniques are carried out
according to standard protocols as described in Sambrook et al.
(1989) Molecular Cloning: A Laboratory Manual, Second Edition, Cold
Spring Harbor Laboratory Press, and Sambrook and Russell (2001)
Molecular Cloning: A Laboratory Manual, Third Edition, Cold Spring
Harbor Laboratory Press, NY; and in Volumes 1 and 2 of Ausubel et
al. (1994) Current Protocols in Molecular Biology, Current
Protocols, USA. Standard materials and methods for plant molecular
work are described in Plant Molecular Biology Labfax (1993) by
R.D.D. Croy, jointly published by BIOS Scientific Publications Ltd
(UK) and Blackwell Scientific Publications, UK.
Example 1
Material and Methods
1.1 Plant Material and Drought Tolerance Experiment
[0181] All plants, including the activation tag population
(Marsch-Martinez et al., 2002, Plant Physiol. 129:1544-1556) and
transgenic lines were grown in the greenhouse at around 22.degree.
C. and were in the Arabidopsis ecotype Wassilewskija (Ws). For the
drought tolerance experiments, soil mixture comprised 1 part of
sand and perlite and 2 parts of compost. Seeds were sown (after 3
nights at 4.degree. C.) at density of six plants per 4 cm pot in a
tray with 51 pots (Aracon containers, BetaTech, Belgium). Mineral
nutrients were supplied 10 days after germination and at two weeks
after germination the plants were subjected to drought (for 9, 10,
11 or 12 days) by transferring the pots to dry trays (after drying
each pot from outside). Every 2 days in drought, the plants were
moved within the tray to nullify pot position effects.
Subsequently, plants were rehydrated and observed for recovery
after one week. The drought experiments were conducted with 4
replications and the whole experiment repeated 5 times.
1.2 Isolation of Flanking DNA and Sequence Analysis
[0182] DNA was isolated according to Pereira and Aarts (1998,
Transposon tagging with the En-I system, Totowa, N.J., Humana
Press), from two leaves or young flower buds, and 10 ng of genomic
DNA was used for Thermal Asymmetric Interlaced-PCR (TAIL PCR) as
described by (Marsch-Martinez et al., 2002, supra). A re-PCR was
generally performed before sequencing the amplified fragments, and
identifying the insert position in the Arabidopsis genome using a
BlastN algorithm (Altschul et al. 1990, J. Mol. Biol. 215:403-410).
Multiple sequence alignments were performed using CLUSTAL X
(Thompson et al. 1997, Nucl. Acid Res. 25, 4876-4882) and DNASTAR
(DNASTAR Inc. Madison, Wis.) while the GENEDOC (Nicholas et al.
1997, EMBNET News 4, 1-4) and TreeView (Page, 1996, Comp. Applic.
Biosci. 12: 357-358) programs were used for editing the alignment
and producing the phylogenetic tree, respectively. Phylogenetic
analysis including bootstrapping was conducted as described by
Lucker et al. (2002, Eur. J. Biochem. 269:3160-3171).
1.3 Generation of Plant Transformation Constructs and Transgenic
Arabidopsis
[0183] Fragments encompassing the full length coding regions were
amplified (using pfu DNA polymerase) from flower buds cDNA (for
SHN1, At1g15360) or genomic DNA (for At5g11190, SHN2 and At5g25390,
SHN3) to generate the three overexpression constructs. The cDNA
(produced as described below in Gene Expression Analysis) and
genomic DNA used for amplification were from the Arabidopsis
ecotype Columbia. Oligonucleotides AP35 and AP36 were used to
amplify SHN1, while oligonucleotides AP69 and AP70 were used to
amplify SHN2.
[0184] Both pairs of oligonucleotides introduced BamHI and SstI
restriction sites to the amplified fragments at their 5' and 3',
respectively, which were utilized to ligate the coding region
fragments to the BamHI and SstI sites in the pBI121 binary vector
(Clontech, Palo Alto, Calif.) in between a .sup.35S promoter of the
cauliflower mosaic virus (CaMV) and a nopaline synthase (NOS)
terminator. Oligonucleotides AP71 and AP72 were used to amplify
SHN3 and introduced BglII and XhoI restriction sites to the
amplified fragment at the 5' and 3', which were utilized to ligate
the coding region fragment to the BamHI and SalI sites in the pNEW
binary vector (a modified pBI121 binary vector, Nayelli
Marsch-Martinez, unpublished) in between the .sup.35S CaMV promoter
and the NOS terminator. For generating the promoter::GUS
constructs, fragments upstream to the ATG codon of each gene (2 kb
of SHN1 and SHN3 and 1.857 kb of SHN2) were amplified from genomic
DNA (ecotype Columbia) using Taq DNA polymerase and
oligonucleotides which introduced XbaI NcoI restriction sites at
the 5' and 3', respectively. Only in the case of SHN3 the amplified
fragment contained already an endogenous XbaI site at the 5' end.
This allowed ligation of the fragments to the XbaI and NcoI sites
in a modified pBinPlus vector (Raffaella Greco, unpublished)
upstream of the .beta.-glucuronidase (GUS) reporter gene. The
oligonucleotides AP61 and AP62 were used to amplify the SHN1
upstream region, AP147 and AP148 for SHN2 and AP149 and AP150 for
SHN3. In all cases fragments were A-tailed and introduced to the
pGEM-T Easy vector as described by the manufacturer (Promega) and
subsequently sequenced from both sides before digestion and
ligation to the Binary vector. PCR, restriction digests, plasmid
DNA isolation and gel electrophoresis were performed using standard
protocols. The rd29A-DREB1A construct was similar to that described
(Kasuga et al., 1999, Nat. Biotech. 17:287-291), except that the
gene fusion was inserted into pBinPlus (van Engelen et al., 1995,
Trans. Res. 4:288-290). The constructs were introduced into the
plants using the floral dipping transformation method (Clough and
Bent, 1998, Plant J. 16:735-743). The seeds were plated on
one-half-strength Murashige and Skoog medium (1/2MS; Murashige and
Skoog, 1962, Physiol. Plant. 15:473-497) and seedlings selected on
50 mg/L kanamycin were subsequently transferred to the
greenhouse.
Oligonucleotides:
TABLE-US-00005 [0185] AP35 (5'- CGGATCCATGGTACAGACGAAGAGTTCAG -3')
AP36 (5'- CGAGCTCGATTTAGTTTGTATTGAGAAGC- 3') AP69 (5'-
CGGATCCATGGTACATTCGAGGAAGTTCCG -3') AP70 (5'-
CGAGCTCTCAATCCAATTCAGCAACTCC -3') AP71 (5'-
CAGATCTGAAGAATGGTACATTCGAAG -3') AP72 (5'-
CTCGAGCCTTTAGACCTGTGCAATGG -3') AP61 (5'-
CTCTAGAACGAATGGCCGTTGATCAGAG -3') AP62 (5'- CCCATGGTTACTTACTCTGTG
-3') AP147 (5'- CTCTAGAGATTGGGTACTAGGTTAAGG -3') AP148 (5'-
CCCATGGTTTAGTTTCCTTCA -3') AP149 (5'- ATCGTGTGAAACGTCAATCG -3')
AP150 (5'- CCCATGGCTTCGAATGTACCATGGTTCTG -3') AP151 (5'-
CTGGATCTGGATCTAGAACTCCGTTTGGGTTTCGCT TAA -3') (AP151 is an EAR
repressor primer)
1.4 Gene Expression Analyses
[0186] Total RNA for Reverse Transcriptase-PCR (RT-PCR) was
isolated from mature, green, rosette leaves derived from 4 weeks
old shn activation tag mutant and wild type (ecotype WS) plants
using the Trizol Reagent as described by the manufacturer
(Invitrogen, Life technologies). Approximately 1 .mu.g of total RNA
was used for DNase I treatment and cDNA synthesis (using
SuperScriptII reverse transcriptase) as described by the supplier
(Invitrogen, Carlsbad, Calif.). The cDNA was diluted 50 times and
used for amplification using specific oligonucleotides for the
actin gene
TABLE-US-00006 RACTP1, 5'- GCGGTTTTCCCCAGTGTTGTTG -3' RACTP2, 5'-
TGCCTGGACCTGCTTCATCATACT -3'
to equalize the concentrations of the cDNA samples. Subsequently
the diluted cDNA was utilized to perform a PCR reaction using
specific oligonucleotides designed to amplify the two genes
flanking the insertion site. Oligonucleotides AP8 and AP9, to
amplify the At1g15350 gene and AP6 and AP7, to amplify At1g15360
(SHN1). The reaction conditions for PCR included a denaturing step
of 95.degree. C. for 3 min, followed by 35 cycles of 1 min at
95.degree. C., 1 min at 55.degree. C., and 1.5 min at 72.degree.
C., ending with an elongation step of 5 min at 72.degree. C. For
the control PCR with actin oligonucleotides, 30 amplification
cycles were used.
TABLE-US-00007 AP8 5'- CAAACGCTCAAGGGTCTCGTC -3' AP9 5'-
CTGAGCACAACCAAGTCCACCA -3' AP6 5'- CTTCATCGCTCTCTTCCATCC -3' AP7,
5'- CCAATACTTCTTCTCTGCTGC -3'
1.5 Wax Extraction and Chemical Analysis
[0187] Cuticular wax was extracted exhaustively by dipping intact
leaves twice for 30 sec into 20 mL of chloroform (>99%; Fisher
Scientific, Nepean, Ontario, Canada) at room temperature.
Tetracosane (Sigma-Aldrich, Oakville, Ontario, Canada) was added as
internal standard, the extracts were filtered, and the solvent was
removed by a gentle stream of N2 while heating the solution to
50.degree. C. Then all samples were treated with
bis-N,N-(trimethylsilyl)trifluoroacetamide (BSTFA, Sigma-Aldrich)
in pyridine (Fluka, Buchs, Switzerland, 30 min at 70.degree. C.) to
transform all hydroxyl-containing compounds into the corresponding
trimethylsilyl derivatives. The extracted surface area was
subsequently measured digitally by scanning photocopies of the
leaves. The qualitative composition was studied with capillary GC
(6890N, Agilent, Palo Alto, Calif., USA) with He carrier gas inlet
pressure constant at 30 kPa and mass spectrometric detector (70 eV,
m/z 50-750, 5973N, Agilent). GC was carried out with
temperature-programmed injection at 50.degree. C. oven for 2 min at
50.degree. C., raised by 40.degree. C. min.sup.-1 to 200.degree.
C., held for 2 min at 200.degree. C., then raised again by
3.degree. C. min.sup.-1 to 320.degree. C. and held for 30 min at
320.degree. C. The quantitative composition of the mixtures was
studied by capillary GC (Agilent; 30 m HP-1, 0.32 mm i.d., df=1
.mu.m) and flame ionization detection under the same gas
chromatographic conditions as above, but H2 carrier gas inlet
pressure was programmed for 50 kPa at injection, held for 5 min,
then raised with 3 kPa min.sup.1 to 150 kPa and held for 40 min at
150 kPa. Single compounds were quantified against the internal
standard by manually integrating peak areas.
1.6 Chlorophyll Leaching Assay, Fresh Weight and Stomata
Analyses
[0188] For chlorophyll leaching assays, roots and inflorescence
stems of 4 weeks old plants were cut off, and the remaining rosette
was rinsed with tap water, weighed and put in tubes containing 30
ml of 80% ethanol at room temperature (gently agitating in the
dark). Four hundred microliter were removed from each sample every
ten minutes during the first hour, and then after 90 and 120 min
Absorbance of each sample was measured at 664 and 647 and the
following formula (Lolle et al., 1997, Dev. Biol. 189:311-321), was
used to calculate the micromolar concentration of total chlorophyll
per gram of fresh weight of tissue: Total micromoles
chlorophyll=7.93 (A664)+19.53 (A647).
[0189] Seed from wild type and the mutant lines were stratified in
cold (4.degree. C.) for 3 nights and sown in 9-cm diameter pots, at
a density of approximately 12 seeds/pot. The plants were given
nutrition on the 10th day after germination, allowed to grow to 4
weeks then used for water-loss analysis. The rosette and emerging
stems of plants were detached from the roots and weighed
immediately for the fresh weight. All samples maintained at room
temperature (22 degrees C.) were weighed at several regular time
intervals. Initial observations were taken at short time intervals
of 2 minutes and then later gradually increased to longer intervals
of 1 hour. The samples were weighed for 7 hours or more.
Observations were taken from 4 different plants of wild type and
mutants, and the experiment was repeated in 3 batches at different
days. The average fresh weight, average dry weight (samples were
kept at 60 degrees for 2 days and then weighed), average rate of
water loss per unit fresh weight and the standard deviation were
calculated. A graph was plotted with average rate of water loss per
unit fresh weight against time in minutes.
[0190] For stomatal density, pavement cell density and stomatal
index measurements we used similar size and age mature green
rosette leaves, derived from 6 weeks old plants of wild type and
35S::SHN1 line #2-2. Two leaves from four different plants (from
each of the two genotypes) were used to generate imprints of their
abaxial surface. A xylene-thermocol mixture made by dissolving
thermocol in xylene until the solution becomes viscous was applied
uniformly on the abaxial surface of the leaves and allowed to dry.
Subsequently, the imprints were detached from the leaf surface, and
pieces derived from the region in between the main vein and the
leaf blade edge were mounted on glass microscope slides with 50%
glycerol and observed under 20.times. magnification using a light
microscope (Zeiss). Numbers of epidermal pavement cells and stomata
were counted per mm.sup.2 (two different regions per leaf) and
stomatal index was calculated (Mishra, 1997, Ann Bot. 80,
689-692).
1.7 GUS Staining and Microscopy
[0191] Tissues from various organs either from soil grown plants or
seedlings grown on 1/2MS in vitro were analyzed for their GUS
expression patterns. The GUS solution contained 100 Mm sodium
phosphate buffer, pH 7.0, 0.5 mg/ml 5-bromo-4-chloro-3-indolyl
.beta.-D glucoronic acid (X-Gluc, Duchefa, The Netherlands), 0.1%
Triton, and 0.5 mM each of potassium ferri/ferrocyanide. Samples
were vacuum infiltrated and incubated at 37.degree. C. for 16 to 24
h and depleted from chlorophyll in 70% ethanol. Observation were
conducted either under the binocular (WILD M3Z of Heerbrugg
Switzerland, type-S), or with a light microscope (Zeiss) and an RS
Photometrics CoolSNAP camera (MediaCybernetics.RTM.) was used to
take the digital images, with the corresponding CoolSNAP
software.
[0192] For Scanning Electron Microscopy (SEM) samples were glued on
a sample holder with conductive carbon cement (Leit-C, Neubauer
Chemikalien, Germany) and subsequently frozen in liquid nitrogen.
The samples were transferred under vacuum to a dedicated
cryo-preparation chamber (Oxford cryo-system, CT 1500 HF, Eynsham,
UK) onto a sample stage at -90.degree. C. Cryo-fractures were made
at approx -150.degree. C. using a cold (-196.degree. C.) scalpel
blade. The fractured samples were freeze dried for 3 min at
-90.degree. C. in vacuum (3.times.10-7 Pa) to remove water vapor
contamination. After the sample surface was sputter-coated with 10
nm Platinum it was transferred to the cold sample stage
(-190.degree. C.) inside the Cryo-FESEM (JEOL 6300F Field Emission
SEM, Japan, Tokyo) and subsequently analyzed with an accelerating
voltage of 5 kV. Images were digitally recorded (Orion,
Belgium).
Example 2
Identification of the Shine Mutant
[0193] By screening a collection of 2000 Arabidopsis transposon
activation tag lines (Marsch-Martinez et al., 2002) a mutant plant
was identified which showed leaf surface alterations (not shown).
Both rosette and cauline leaves of the mutant (termed shine, shn)
had a more brilliant, shiny green color when compared to wild type
plants and often had curved-down edges (not shown). The stem of
mature plants was often bowed-down, siliques were slightly smaller
than wild type and also showed a more brilliant surface. Structure
of other floral organs and plant fertility did not seem to be
affected in shn. Progeny analysis of the self-pollinated shn mutant
line suggested a dominant mutation (three quarters of the plants
exhibited the shn phenotype).
Example 3
Alterations to Wax Load in the shn Mutant
[0194] Scanning electron microscopy (SEM) was utilized for a
detailed comparison between the surfaces of wild type plant organs
and those of shn. The surfaces of stems and siliques of Arabidopsis
are covered by a dense mixture of different types of wax crystals
while leaf surfaces normally exhibit only small numbers of
epicuticular wax crystals. In contrast to wild type we detected
more wax crystals on both adaxial and abaxial sides of rosette and
cauline leaves of shn (data not shown). The leaf surface was not
entirely covered by crystals, as in the case of wild type siliques
and stems, but rather had irregular patches of plate-like wax
crystals. An additional characteristic of the shn mutant was the
presence of cuticular ridges on the surface of both cauline leaves
and siliques, which were not detected in the wild type (data not
shown). Such cuticular ornamentation was not visible on either the
adaxial or abaxial surfaces of shn rosette leaves. Freeze
fractionation of siliques and cauline leaf tissues further
demonstrated the presence of the cuticular ridges in shn tissues,
which showed similarity to the cuticular ridges present normally on
surfaces of wild type Arabidopsis petals (data not shown). In this
analysis the cuticle thickness did not seem to be drastically
altered. Neither an increase in wax crystal numbers nor cuticular
ridges were detected on surfaces of shn sepals, anther filament and
petals.
[0195] A detailed chemical analysis of total wax mixtures was
conducted in both shn and wild type leaf cuticles in order to
quantify the changes in wax load detected by SEM. The shn mutant
wax phenotype was characterized by a six-fold increase in wax
coverage over the wild type, expressed as mass of extractable
cuticular lipids per surface area (Table 3).
TABLE-US-00008 TABLE 3 Composition of cuticular wax on leaves of
wild type and shn. Wild type WS Mutant shn Average fold
[.mu.g/cm.sup.2] [.mu.g/cm.sup.2] increase Fatty acids 0.13 .+-.
0.02 0.50 .+-. 0.30 3.8 Aldehydes 0.05 .+-. 0.03 0.11 .+-. 0.12 2.2
prim. Alcohols 0.18 .+-. 0.03 0.50 .+-. 0.28 2.8 Alkyl esters tr*
0.07 .+-. 0.05 1.4 Alkanes 0.23 .+-. 0.06 2.08 .+-. 1.38 9.0 sec.
Alcohols tr 0.10 .+-. 0.03 11.9 Ketones 0.01 .+-. 0.01 0.11 .+-.
0.08 11.0 Steroids 0.08 .+-. 0.05 0.34 .+-. 0.27 4.3 Isoalcohols
0.05 .+-. 0.04 0.11 .+-. 0.09 2.2 Unidentified 0.07 .+-. 0.07 0.84
.+-. 0.77 12.0 Total 0.80 .+-. 0.26 4.78 .+-. 2.35 6.0 Coverages of
total extracted lipids and of individual compound classes are given
as mean values with standard deviation. *traces, i.e., less than
0.05 .mu.g/cm.sup.2 detectable.
[0196] Wild type leaf wax was found to contain approximately equal
amounts of compounds from the acyl reduction pathway (primary
alcohols, alkyl esters) and from the decarbonylation pathway
(alkanes, secondary alcohols, ketones). In sharp contrast, the shn
mutant wax was characterized by differences in amounts of compounds
resulting from both pathways. While primary alcohols and alkyl
esters showed only 2.8- and 1.4-fold increases, the alkanes,
secondary alcohols and ketones were increased by 9.0-, 11.9- and
11.0-fold, respectively. Aldehydes, regarded as intermediates of
the decarbonylation pathway, showed 2.2-fold higher levels in the
mutant wax mixture. Similarly, other compound classes (fatty acids,
branched alcohols and steroids) were also found at elevated levels
in the mutant wax, albeit only with moderate increases.
[0197] In both wild type and mutant leaf waxes the fatty acids,
aldehydes and primary alcohols were dominated by constituents with
even carbon numbers, as expected for acyl derivatives resulting
from C2 elongation cycles (FIG. 1). The alkanes, secondary alcohols
and ketones showed a clear preponderance of odd-numbered
representatives, typical for metabolites from the
elongation/decarbonylation route. The wild type wax showed chain
length distributions dominated by C32/C34 for fatty acids and
aldehydes, by C31 for alkanes, and by C26/C28 for primary alcohols.
Only C29 secondary alcohol and ketone, with functional groups both
in the C14 and C15 position, could be detected. As compared to
these wild type patterns, the mutant leaf wax contained much higher
concentrations of C30 fatty acid, C30 aldehyde and C27/C29 alkanes,
compensating for lower relative amounts of C34 fatty acid, C34
aldehyde and C33 alkane, respectively (FIG. 1). The chain length
distribution of secondary alcohols, ketones, and primary alcohols
were similar in the wild type and the mutant.
Example 4
Alterations to Cuticle Permeability in the shn Mutant
[0198] To investigate whether the shn cuticular membrane properties
were altered a chlorophyll leaching experiment was conducted in
which rosette leaves from both shn and wild type plants were
submerged in 80% ethanol for different time periods and the
chlorophyll concentration in the solution was determined
Chlorophyll was extracted much faster from leaves of shn leaves as
compared to wild type (FIG. 2A) and therefore the higher elution of
chlorophyll from shn leaves indicates an increase in cuticle
permeability.
[0199] To assay cuticular water loss, fresh weight changes of
detached rosettes were monitored. Roots and emerging inflorescence
stem of four-week old seedlings were detached from the rosettes,
which were used to examine loss of water over time. The results
(FIG. 2C) show that fresh weight loss from the rosette tissues was
increased in shn when compared to wild type rosette tissues. As
this water loss in shn continues beyond the time when stomata close
(Yoshida et al., 2002, Plant Cell Physiol. 43, 1473-1483), it is
the increased cuticular water loss in shn that is revealed.
Example 5
A Member of the AP2/EREBP Transcription Factor Family is
Responsible for the shn Mutant Phenotype
[0200] DNA gel blot analysis showed that shn contains a single
insertion (data not shown). Isolation and sequence analysis of DNA
flanking the insertion site further indicated that the insertion is
located in an intergenic region on chromosome 1. The location of
the 35S enhancer tetramer is between a gene encoding an unknown
protein (4025 base pairs upstream of the promoter) and a gene
encoding a member of the plant specific AP2/EREBP family of
transcription factors (620 base pairs upstream of the promoter). To
examine if these two genes were induced in expression in shn
compared to wild type, we conducted a Reverse Transcription PCR
(RT-PCR) experiment using cDNA isolated from shn and wild type leaf
tissues. The results showed that the genes from both sides of the
35S enhancer tetramer were induced in the shn mutant leaves
compared to wild type leaves (data not shown).
Example 6
Transgenic Plants Overexpressing SHN1
[0201] The downstream gene (At1g15360), encoding the AP2/EREBP
transcription factor, was chosen as primary candidate determining
the shn mutant phenotype. Consequently, the coding region of the
gene (termed SHINE1 or SHN1) was cloned and constitutively
expressed in Arabidopsis under the control of the 35S CaMV
promoter. In fact, all the transgenic plants raised (20
individuals) showed a phenotype resembling the original activation
tag line, in particular the shn brilliant green leaf and silique
surface and downward curling of the leaves (data not shown). The
phenotype of most of the 35S::SHN1 lines (both primary
transformants and subsequent generations) was more severe compared
to the original shn mutant. In most cases plants were smaller, and
in some cases even dwarfed (3 to 5 cm in size upon maturity), and
their leaves were very strongly curved, even rolled (data not
shown). Further chemical analyses showed that the transformant
leaves had cuticular wax load, relative compositions of compound
classes, and chain length distributions within these classes
similar to the original shn tag mutant.
[0202] In contrast to the activation tag shn mutant, flower
morphology was also affected, particularly in petals which were
folded and in part "hidden" in-between the sepals and the flower
interior organs (data not shown). Scanning electron microscopy was
used to investigate the surface petals derived from the SHN1
overexpressing lines (data not shown). The anterior and distal
parts of the adaxial surface of wild type Arabidopsis petals
normally show a uniform spread of conical epidermal cells, which
exhibit a typical cuticular ornamentation (data not shown). On the
other hand, in shn petals one could identify a mix of both typical,
conical cells and much longer cells, often more than doubled in
size.
[0203] The number and structure of trichomes was analyzed in the
first true leaves of 35S::SHN1 seedlings compared to wild type. The
adaxial side of the first true leaf of wild type (ecotype
Wassilewskija) contained approximately 25 of mainly triple-branched
trichomes, spread on its surface. In contrast, the first true
leaves of 35S::SHN1 seedlings contained much lower numbers of
trichomes, ranging from leaves with no trichomes at all up to a
maximum of 8-10 trichomes (data not shown). When trichomes were
present on the first leaves of 35S::SHN1 they were nearly all
single-branched and located on leaf blade margins. The same
observations were also detected in leaves derived from older
plants.
[0204] Two other features of epidermal cell differentiation were
also altered by the overexpression of SHN1. Both pavement cell
density and stomatal density on the abaxial side of the 35S::SHN1
lines were reduced compared to wild type leaves (see Table 4).
Calculating the stomatal index revealed that it was reduced by 41%
in the 35S::SHN1 leaves compared to wild type (Table 4).
TABLE-US-00009 TABLE 4 Stomatal Density, Pavement Cell Density and
Stomatal Index of Mature shn and Wild-type Rosette Leaf Blades
Stomatal Density Pavement Cell Density (cells/mm.sup.2 .+-. SD)
(cells/mm.sup.2 .+-. SD) Stomatal Index Wild-type 27.03 .+-. 9.63
80.16 .+-. 19.88 25.22 .+-. 4.48 35S::SHN1 8.91 .+-. 3.76 51.56
.+-. 15.35 14.73 .+-. 3.96
[0205] Leaching assays with progeny of two 35S::SHN1 primary
transformants (#2-2 and #2-5) showed that their cuticle was more
permeable to ethanol, since chlorophyll could be extracted easier
(FIG. 2B). In line with the overall stronger phenotype of the
35S::SHN1 lines, the difference in chlorophyll leaching compared to
wild type leaves was more dramatic than initially observed for the
activation tag shn mutant. The two 35S::SHN1 primary transformants
(#2-2 and #2-5) showed also an increased rate of water loss
compared to wild type (FIG. 2C).
Example 7
Overexpression of Two Other Members of the SHINE Clade Results in
Similar Phenotype
[0206] The plant AP2/EREBP super-family of transcription factors
contains 141 members in Arabidopsis (Alonso et al., 2003, Science
301, 653-657). Sequence homology searches and phylogenetic analysis
across the entire AP2/EREBP family showed that SHN1 is part of a
small, distinct group of four proteins, 199, 189, 186 and 205 amino
acid residues long (SHN1, SHN2, SHN3 and OsSHN1 respectively; FIG.
3). They contain the highly conserved AP2 domain and share two
other conserved motifs in their central portion ("mm", positions 87
to 147 in FIG. 3) and C-termini ("cm", positions 189 to 198 in FIG.
3). The At5g25190 protein is more distant in sequence from SHINE
proteins.
[0207] The genomic regions encompassing the coding regions of SHN2
and SHN3 were used for overexpression (using the double-enhanced
35S CaMV promoter) of both genes in Arabidopsis plants.
Interestingly, plants overexpressing SHN2 and SHN3 showed an
identical phenotype to the one obtained when overexpressing the
SHN1 gene (data not shown).
Example 8
Spatial and Temporal Expression of the SHN Clade Members
[0208] In order to examine the expression of SHN1, SHN2 and SHN3
three plant transformation constructs were generated, which linked
2.0-kb DNA sequences upstream of the predicted ATG codon of each
gene to the .beta.-glucuronidase (GUS) reporter gene. In general
GUS expression was detected in most plant organs, in some cases
overlapping patterns were detected while in others very specific
expression was evident in certain cell layers.
[0209] SHN1 expression was detected in the inflorescence and root
tissues, but not in stem, rosette or cauline leaves (data not
shown). Expression could be detected in sepals of very young closed
buds (stage 6; Smyth et al., 1990, Plant Cell 2, 755-767), and
later at stage 10. At that time, expression could also be detected
in petals and developing gynoecium, but not in stamens. In petals
and sepals, veins were stained stronger than the rest of the organ,
in which it was restricted to the epidermis. At anthesis (stage 13)
the expression of SHN1 was reduced in the gynoecium, commenced in
the anther and showed weaker expression in the anther filament.
When petals and sepals withered (stage 16), strong expression could
be detected at the bottom of the silique, in the abscission zone
and in the pedicel region below it, while later, at silique
maturity, it was detected in the same region, but only at the
nectaries. Additional GUS expression was observed at the branch
points of pedicels of most young flowers in the inflorescence, in
small lateral inflorescences (including the small bract adjacent to
them), and in a patchy pattern in roots of mature plants and very
young leaves in the rosette, including support cells of their
trichomes.
[0210] The SHN2 gene shows a pattern of expression associated with
anther and silique dehiscence. At stage 12, when petals level with
long stamens and tapetum degeneration is initiated in the anther
(stage 10 of anther development; Sanders et al. 1999, Sexual Plant
Rep. 11, 297-322), expression could be detected in the stomium
region. Up to anthesis, during which the septum is degenerated, a
bilocular anther is formed, the stomium splits and pollen is
released, expression of SHN2 became more specific to the dehiscence
zone and continued until stamens fell off the senescing flower
(data not shown). Subsequently, when petals and sepals withered
(stage 16), GUS expression could be detected as an intense spot at
the bottom of each valve. One stage later, i.e., in the growing
phase of the green silique as it reached final length and the
dehiscence zone differentiated, SHN2 was strongly expressed along
the valve margin-redplum boundary, the region where pod shatter
occurs, allowing seed dispersal.
[0211] The SHN3 gene was most broadly expressed and was active in
all plant organs. It showed expression in the vasculature and in
the lateral root tip (data not shown). When staining young 10
day-old seedlings, expression was detected in the support cells of
trichomes present on the most newly formed leaves. In older leaves
(rosette) as well as in cauline leaves, SHN3 was mainly expressed
in the central vein with lower expression in the entire blade. It
was not expressed in a uniform manner in stems, showing mostly weak
epidermal expression. Expression of SHN3 in the inflorescence and
young rosette leaves overlapped to a large extent with that
observed for SHN1 (see above). Most interestingly, it showed an
organ--specific wound induction. While wounding did not induce it
in rosette leaves, it did activate it in cauline leaves, stems and
siliques.
Example 9
Plants Overexpressing SHN1 show Enhanced Drought Tolerance
[0212] In order to examine to what extent the change in plant
surface, as a result from SHN1 over-expression, affected its
drought tolerance capacity. To do so, 15 day-old seedlings of the
original activation tag lines, two of the 35S::SHN1 transformant
lines (lines #2-5 and #2-2) and wild type (ecotype Wassilewskija)
were exposed to a period of 9-11 days of dehydration (FIG. 4).
Subsequently, seedlings were watered and their recovery monitored
for a week. While wild type plants did not recover from the
dehydration treatments longer than 9 days and completely dried out,
all seedlings derived from lines expressing the SHN1 gene recovered
to become greener and stronger. Consistent with the phenotype
characteristics described above, seedlings derived from the
activation tagged line were relatively weak in recovery when
compared to the two transgenic 35S::SHN1 lines.
[0213] Similarly, overexpression of SHN1 in rice also leads to
plants with an increased drought tolerance. Transformants with a
35S::SHN1 construct are able to withstand prolonged leaf wilting
under water deprivation compared to control plants, as assessed by
recovery following rehydration. See also further Example below.
Example 10
Plants Expressing the SHN-EAR Fusion Show Loss of Function
[0214] Transgenic plants expressing the SHN-EAR repressor fusions
were generated by transformation. Transgenic plants expressing
either SHN1-EAR, SHN2-EAR or SHN3-EAR displayed similar loss of
function phenotypes, as expected for redundant genes coding for
proteins having similar DNA binding and protein interaction
properties. Expression of the SHN-EAR under specific promoters
could specify the loss of function to a specific tissue, e.g.,
conferring non-dehiscence of anthers or reduced podshatter. See
also further Examples below.
Example 11
Overexpression of the Rice OsSHINE Gene in Arabidopsis Reveals a
Conserved Function
[0215] In comparison of the amino acid sequences of the three
similar Arabidopsis SHN-related proteins, high similarity was found
in the central portion (middle `nm`) and C termini (`cm`) as well
as the AP2 DNA-binding domains. Using these consensus domains
(`nm`, `cm` and `AP2`) to screen the sequence databases, members of
the SHN Glade of proteins could be defined as those that show high
similarity to the Arabidopsis SHN proteins in these conserved
domains. We searched the rice genome database for proteins with
amino acid sequences similar to the SHN protein conserved regions,
and found two genomic clones with predicted amino acids that showed
high homology in these conserved regions (accession number BAD15859
and BAD35470). We named these two genes OsSHN1 and OsSHN2. OsSHN1
and OsSHN2 contained an open reading frame of 206 and 244 amino
acids, respectively, with an amino acid sequence of 205 and 243
amino acids (SEQ ID NO: 14 and SEQ ID NO: 24). These proteins are
42.3-62.4% similar to the Arabidopsis proteins and 68.3% similar to
each other.
[0216] Fragments encompassing the full length coding region and the
upstream region of OsSHN1 were amplified (using pfu DNA polymerase)
from young leaf genomic DNA of rice cv. Nipponbare.
Oligonucleotides
TABLE-US-00010 OsSHN1F (5'-AATAAGGATCCATGGTACAGCCAAAGAAG-3') and
OsSHN1R (5'-AATAAGTCGACTCAGATGACAAAGCTACC-3')
were used to amplify 0.76 kb fragment containing the full length
coding region of OsSHN1. The pair of oligonucleotides introduced
BamHI and SalI restriction sites to the amplified fragments at
their 5' and 3', respectively, which were utilized for ligation. In
all cases fragments were A-tailed and introduced to the pGEM-T Easy
vector as described by the manufacturer (Promega) and subsequently
sequenced from both sides before digestion and ligation to the
binary vector. The overexpression and chimeric repressor constructs
were assembled by multi-point ligations, in which the individual
fragments (promoter, OsSHN1 gene, terminator) with appropriate
compatible cohesive ends were ligated together to the binary vector
in one reaction. A CaMV35S promoter fragment extending from -526 to
the transcription start site, was obtained as a 0.55 kb
HindIII-BamHI fragment from a pBS-SK+ derivative of pDH51 (Pietrzak
et al., 1986). A CaMV35S terminator fragment was obtained as a 0.21
kb SalI-EcoRI fragment from a pBS-SK+ derivative of pDH51 (Pietrzak
et al., 1986). The construct was made in the binary vector pMOG22
(ZENECA-MOGEN, NL) which contains a chimeric CaMV 35S-hygromycin
phosphotransferase-tNos for selection during transformation. PCR,
restriction digestions, plasmid DNA isolation and gel
electrophoresis were performed using standard protocols. The
constructs were introduced into the plants using the floral dipping
transformation method (Clough and Bent, 1998). The seeds were
plated on one-half-strength Murashige and Skoog medium (1/2MS;
Murashige and Skoog, 1962) and 15 sucrose. Seedlings selected on 20
mg/L hygromycin were subsequently transferred to the
greenhouse.
[0217] All plants were grown in the greenhouse at around 22.degree.
C. and were in the Arabidopsis ecotype Wassilewskij a (Ws). For the
drought tolerance experiments, soil mixture comprised 1 part of
sand and perlite and 2 parts of compost [a mixture made up of 25%
clay and 75% turf with EC=1 (NPK); Hortimea, Netherlands]. Seeds
were sown (after 3 nights at 4.degree. C.) at density of six plants
per 4 cm pot in a tray with 51 pots (Aracon containers, BetaTech,
Belgium). Nutrients (Hydroagri, Rotterdam, The Netherlands; 2.6 EC)
were supplied 10 days after germination and at two weeks after
germination the plants were subjected to drought (for 13, 14, or 16
days) by transferring the pots to dry trays (after drying each pot
from outside). Every 2 days in drought, the plants were moved
within the tray to nullify pot position effects. Subsequently,
plants were rehydrated and observed for recovery after one week.
Experiment was conducted to compare drought tolerance between
wild-type and 35S::AtSHN1(#2-2), 35S::OsSHN1(#1) and
35S::OsSHN1(#16) plants.
[0218] Plants overexpressing OsSHN1 showed an identical visual
phenotype to the one obtained when overexpressing the Arabidopsis
SHN1 gene, including the brilliant, shiny green color of both
rosette and cauline leaves, leaf curling, and altered silique
length.
[0219] To investigate whether the cuticular membrane properties of
OsSHN1 overexpressor were altered, we conducted a chlorophyll
leaching experiment in which rosette leaves from both OsSHN1
overexpressor and wild-type plants were submerged in 80% ethanol
for different time periods, and the chlorophyll concentration in
the solution was determined Chlorophyll was extracted much faster
from leaves of OsSHN1 overexpressor as compared with the wild type;
therefore, the higher elution of chlorophyll from OsSHN1
overexpressor indicates an increase in cuticle permeability to
organic solvents.
[0220] We tested whether two other features of epidermal cell
differentiation were also altered by overexpression of OsSHN1. Both
pavement cell density and stomatal density on the abaxial side of
the OsSHN1 overexpressor was reduced compared with wild-type
leaves. Calculating the stomatal index revealed that it was reduced
by 40% in the OsSHN1 overexpressor leaves compared with the
wild-type (Table 5).
TABLE-US-00011 TABLE 5 Stomatal Density, Pavement Cell Density, and
Stomatal Index of Mature 35S::OsSHN1 and Wild-Type Rosette Leaf
Blades Pavement Cell Stomatal Density Density Plant line
(cells/mm.sup.2 .+-. SD) (cells/mm.sup.2 .+-. SD) Stomatal Index
Wild Type 25.39 .+-. 3.59 83.20 .+-. 10.13 30.55 .+-. 2.88
35S:OsSHN1 10.94 .+-. 3.61 59.38 .+-. 9.41 18.16 .+-. 4.52
[0221] To investigate whether the OsSHN1 has the same downstream
target genes as that as the Arabidopsis SHN1 we conducted RT-PCR
for the CER1 gene using leaf rosette RNA samples from both the
OsSHN1 overexpressor and wild-type plants. We found that the CER1
gene was significantly overexpressed in the 35S-OsSHN1 plants.
[0222] The Arabidopsis transformants overexpressing the OsSHN1 gene
were used in a pot assay for drought tolerance as described above.
Whereas wild-type plants did not recover from the dehydration
treatments longer than 13 days and completely dried out, all
seedlings derived from lines expressing the OsSHN1 gene recovered
after rehydration to become greener and stronger. The drought
tolerance revealed in this test is equivalent to that shown by the
Arabidopsis SHN1 gene.
[0223] Over-expression of the SHN1 in transgenic Arabidopsis plants
resulted in higher tolerance to drought, probably related to the
reduced stomatal density. Over-expression of OsSHN1 in transgenic
Arabidopsis also enhanced drought tolerance. It is probable that
the reduction in the number of stomata that we also found in the
OsSHN1 overexpressors is responsible for this drought tolerance.
But we also found that overexpression of OsSHN1 induced expression
of rd22, a gene responsive to dehydration stress
(Yamaguchi-Shinozaki and Shinozaki, 1993), as detected by RT-PCR.
This indicated that another mechanism is probably also involved in
enhancement of drought tolerance in OsSHN1 overexpressor. In our
microarray data, rd22 is one of many abiotic stress-inducible genes
up-regulated in transgenic 35S::SHN1 Arabidopsis (unpublished
data).
Example 12
Overexpression of the Arabidopsis SHINE Gene in Rice Confers
Drought Tolerance
[0224] The SHINE overexpression construct for rice transformation
was assembled by multi-point ligation, in which the individual
fragments (promoter, AtSHN2 gene, terminator) with appropriate
compatible cohesive ends were ligated together to the binary vector
in one reaction. A CaMV35S promoter fragment extending from -526 to
the transcription start site, was obtained as a 0.55 kb
HindIII-BamHI fragment from a pBS-SK+ derivative of pDH51 (Pietrzak
et al., 1986). The full length coding region of AtSHN2 was obtained
as BamHI-NotI fragment from Aharoni et al. (2004). A CaMV35S
terminator fragment was obtained as a 0.21 kb NotI-EcoRI fragment
from a pBS-SK+ derivative of pDH51 (Pietrzak et al., 1986). The
construct was made in the binary vector pMOG22 (ZENECA-MOGEN, NL)
which contains a chimeric CaMV 35S-hygromycin
phosphotransferase-tNos for selection during transformation.
[0225] Agrobacterium-mediated transformation of Oryza sativa ssp.
japonica cv. Nipponbare, plant regeneration and growth were
performed following as described in Greco et al. (2001). The
Agrobacterium strain AGL-1 was used for transformation. For growing
progeny seeds, the seeds were dehusked, surface-sterilized (1 min
in 70% ethanol, followed by 20 min in 1% NaOCl, and four rinses
with sterile water) and sown on 50 mg/l hygromycin in sterile MQ
water. Plants were grown in a climate chamber under long-day
conditions (16 h light, 8 h dark, 280C) for about two weeks, before
being transferred to the greenhouse.
[0226] Transformation of rice yielded fifteen independent
transgenic lines. None of the rice transformants revealed any
obvious leaf wax increase or plant leaf phenotype, unlike that
observed in Arabidopsis. RT-PCR analysis, however, confirmed high
level expression of the SHN2 gene. Lines with high expression and
enough seed were used for further experimentation.
[0227] We tested whether other features of epidermal cell
differentiation were also altered by the overexpression of SHN2.
Stomatal density on the abaxial side of the 35S::SHN2 leaves was
reduced to 3/4 compared with wild-type leaves (Table 6).
TABLE-US-00012 TABLE 6 Stomatal Density (cells/mm.sup.2 .+-. SD)
Wild Type 40.62 .+-. 3.61 Transgenic 29.69 .+-. 3.12
[0228] A Drought resistance experiment was conducted with 35S::SHN2
lines and the wild type. For this 14 days old seedlings (5
seedlings per pot) of either wild-type or 35S::SHN2 lines were
exposed to dehydration stress by withholding water for 9 days. At
this stage the wild-type were wilted completely while the 35S-SHN2
lines were still green and had water. The seedlings were then
watered and their appearance noted after a week. There was a clear
difference between wild-type and 35S::SHN2, in which there is 100%
recovery of the overexpression line which turned into light green
and no recovery of the wild-type was visible.
[0229] The above results showed that all rice transformants reveal
no obvious leaf wax increase or modified plant phenotype. Thus,
overexpression of SHINE in rice does not increase the leaf
epicuticular wax or induce downstream target genes involved in
epicuticular wax biosynthesis. Neither does it cause a change in
leaf morphology like curling. However overexpression does cause a
change in cuticular and epidermal properties, like permeability and
reduction in stomatal density. In other words the expression of
SHINE in rice is able to dissect and distinguish between the
epidermal and cuticular changes from the epicuticular wax
changes.
[0230] As some monocots like rice have very low wax and as no
changes in epicuticular wax were found in SHINE overexpressing
plants, it was very surprising to find that SHINE overexpression
resulted in drought tolerant monocot plants. This example clearly
showed that wax synthesis is not required for generating drought
tolerant plants and that the SHINE clade genes can, therefore, be
also used to generate drought tolerant plants without an alteration
of the epicuticular wax layers and may, therefore, also be used to
generate drought tolerance without modifying the epicuticular wax
layer or properties, e.g., in plants or plant organs which have
very low or no epicuticular wax (e.g., monocots like rice). The
alteration in epicuticular wax appears, thus, to be a phenotype
which is irrelevant with respect to generating drought tolerance
and it is only the change in the epidermal and cuticular properties
which are effective in generating drought tolerance in plants.
[0231] The drought resistance is, therefore, not dependent on the
leaf epicuticular wax and thus epicuticular wax and the leaf
phenotype do not need to be modified in order to provide drought
resistance in crop plants.
Example 13
Overexpression of the Arabidopsis SHINE Genes Shows Salinity
Tolerance in Arabidopsis and Rice
[0232] To conduct a Salinity Tolerance assay, the Arabidopsis
plants overexpressing the SHINE gene (35S-SHN1) and appropriate
wild-type controls were grown in the greenhouse at
.about.22.degree. C. For salt tolerant assays, plants were grown in
potting soil (Hortimea, Elst, The Netherlands). Seeds were sown
(after three nights at 4.degree. C.) at density of 1-2 plants per
4-cm pot in a tray with 51 pots (Aracon containers; BetaTech, Gent,
Belgium). Nutrients (Hydroagri, Rotterdam, The Netherlands; 2.6 EC)
were supplied 2 weeks after germination, and after 3 weeks of
germination the plants were subjected to 300 mM NaCl solution at
the interval of 3 days for three applications and subsequently
monitored for bleaching for the next 2 weeks. Photographs were
taken and survival rates were counted on the 10th day after third
application of NaCl. The experiment was repeated three times.
[0233] The 35S-SHN1 line showed enhanced salt tolerance compared to
its wild type (WT), ecotype Ws. The WT plant gradually bleach out
and do not survive approximately 1 week under salt stress, whereas
35S-SHN1 not only survives the salt stress but is also able to
function normally (Table 7).
TABLE-US-00013 TABLE 7 Percent survival rate in 300 mM salt treated
plants No. of plants tested % survival Ws 20 15 35S-SHN1 20 85
[0234] Samples were collected from the NaCl treated plants and the
non-treated plants. There FW's (fresh weight) were measured
immediately after harvesting and samples were dried for 5 days at
65.degree. C. in an oven and later there DW's (dry weights) were
measured. The samples were then used for analysis of sodium
(Na.sup.+), calcium (Ca.sup.++) and potassium (K.sup.+) content.
About 15 to 50 mg of dry material was digested with 1 ml of the
digestion mixture (sulfuric acid--salicylic acid and selenium) and
2 carborundum beads and swirled carefully until all the plant
material was moistened and treated overnight. Temperature was
increased gradually in small steps to about 330.degree. C. and
later on cooling 0.1 ml of hydrogen peroxide was added and heated
again. This step was repeated 3 times until the digest had turned
colorless. On cooling down to room temperature 5 ml of demi-water
was added to make up to the mark and left overnight. The Na.sup.++
Ca.sup.++ and K.sup.+ ion content were determined by using an
Atomic Emission Spectrophotometer (Elex, Eppendorf, Hamburg,
Germany).
TABLE-US-00014 TABLE 8 Mineral Analysis Sample Na K Ca Nr. mmol/kg
mmol/kg mmol/kg Non-treated WT 55 .+-. 3 1249 .+-. 42 578 .+-. 19
35S-SHN1 61 .+-. 6 1093 .+-. 105 626 .+-. 58 Treated WT 4882 .+-.
960.74 534 .+-. 63.09 .sup. 410 .+-. 0.45 35S-SHN1 4940 .+-. 796.09
716 .+-. 27.72 657 .+-. 73.72 Na.sup.+, Ca.sup.++ and K.sup.+
contents were measured and their standard errors measured based on
two independent experiments.
[0235] The analysis shows that under non-treated conditions both
35S-SHN1 and WT shows no difference in the content of Na.sup.+,
Ca.sup.++ and K.sup.+ (Table 8), however some of these components
were found to be altered under salt stress conditions. Salt treated
35S-SHN1 and WT showed increased accumulation of Na.sup.+ compared
to the non treated plants and this increase in Na.sup.+
accumulation was found to be the same in WT and 35S-SHN1. The level
of K.sup.+ was decreased both in 35S-SHN1 and WT, however this
decrease was found to be significantly more in WT compared to the
35S-SHN1. The levels of Ca.sup.++ was decreased in salt treated WT
compared to the non treated WT whereas it was maintained in salt
treated 35S-SHN1 compared to non treated.
[0236] The results indicate that under salt stress condition the
35S-SHN1 is able to maintain its calcium levels, which in turn
helps to maintain the level of K.sup.+ in the plant by enhancing
the selectivity of the root K.sup.+ transport system (Lauchli,
1990). It is known that calcium is one of the important factors
which are involved in the regulation of K.sup.+/Na.sup.+
selectivity of K.sup.+ transport during NaCl stress (Lauchli,
1990).
[0237] Microarray results of 35S-SHN1 showed induction of calcium
binding proteins like Calreticulin 3 (CRT3), Calnexin 1 (CNX1),
Calreticulin 2 (CRT2). In addition genes involved in stress
responses like LEAS (late embryogenesis abundant), RD22 and Protein
kinase family proteins. This indicates that overexpression of SHINE
triggers a signal which results in overexpression of calcium
binding genes, which then activates the transport system that has
higher affinity for the selectivity of K.sup.+ over Na.sup.+ in
salt stress condition (Liu & Zhu, 1997).
Example 14
A Dominant Negative Mutant Using a SHINE-EAR Repressor Fusion
Displays Loss of Function with Reduction in Stem Wax, Change in
Inflorescence Phenotype and Siliques with Reduction in
Shattering
[0238] Dominant negative mutant phenotypes using a SHINE repressor
fusion protein
[0239] Mutant Phenotypes: reduction in stem wax, change in
inflorescence phenotype and siliques with reduction in
shattering.
[0240] To assess the role of the SHINE genes by making a loss of
function mutant, we modified the SHINE protein to be a chimeric
repressor (SHN-SRDX) by fusing it to the EAR repression domain
(Hiratsu et al., 2003) and overexpressed it in Arabidopsis. Other
studies using RNAi constructs of the Arabidopsis SHN genes did not
reveal mutant phenotypes, therefore this alternative option was
taken to avoid functional redundancy. We made constructs with both
the Arabidopsis and rice SHINE genes that showed similar results,
however the example of the rice SHN gene will be demonstrated
here.
[0241] To make the dominant repressor SHINE-EAR gene fusion
construct, PCR fragments were isolated using specific primers.
Fragments encompassing the full length coding region and the
upstream region of OsSHN1 were amplified (using pfu DNA polymerase)
from young leaf genomic DNA of rice cv. Nipponbare.
Oligonucleotides
TABLE-US-00015 OsSHN1F (5'-AATAAGGATCCATGGTACAGCCAAAGAAG-3') and
OsSHN1::SRDXR (5'-CGTCGACTCAAGCGAAACCCAAACGGAGTTCTAGATCCAGATCCA
GGATGACAAAGCTACCCTCTCCCTCTC-3')
were used to amplify 0.8 kb fragment containing chimeric fusion of
the full length coding region of OsSHN1 and SRDX (LDLDLELRLGFA) at
the 3' end. Oligunucleotidel OsSHN1::SRDXR introduced a SalI
restriction site to the amplified fragment at its 3' and OsSHN1F
introduced an BamHI restriction site at the 5' end of the fragment.
The introduced BamHI and SalI restriction sites to the amplified
fragments at their 5' and 3', respectively, were utilized for
ligation. In all cases fragments were A-tailed and introduced to
the pGEM-T Easy vector as described by the manufacturer (Promega)
and subsequently sequenced from both sides before digestion and
ligation to the binary vector. The overexpression and chimeric
repressor constructs were assembled by multi-point ligations, in
which the individual fragments (promoter, OsSHN1::SRDX gene,
terminator) with appropriate compatible cohesive ends were ligated
together to the binary vector in one reaction. A CaMV35S promoter
fragment extending from -526 to the transcription start site, was
obtained as a 0.55 kb HindIII-BamHI fragment from a pBS-SK+
derivative of pDH51 (Pietrzak et al., 1986). A CaMV35S terminator
fragment was obtained as a 0.21 kb SalI-EcoRI fragment from a
pBS-SK+ derivative of pDH51 (Pietrzak et al., 1986). The construct
was made in the binary vector pMOG22 (ZENECA-MOGEN, NL) which
contains a chimeric CaMV 35S-hygromycin phosphotransferase-tNos for
selection during transformation. PCR, restriction digestions,
plasmid DNA isolation and gel electrophoresis were performed using
standard protocols. The constructs were introduced into the plants
using the floral dipping transformation method (Clough and Bent,
1998). The seeds were plated on one-half-strength Murashige and
Skoog medium (1/2MS; Murashige and Skoog, 1962) and 15 sucrose.
Seedlings selected on 20 mg/L hygromycin were subsequently
transferred to the greenhouse.
[0242] Forty-five primary transformants were generated from the
transformation experiments. From these, eighteen primary
transformants showed a loss-of-function mutant stem phenotype with
reduced epicuticular wax (glossy green stem). Some of the primary
transformants did not set seed showing very short empty siliques
indicating sterility. Some of the sterile primary transformants
were covered in a plastic bag for a few days during flowering and
showed good seed set, indicating a conditional male semi-sterile
phenotype as is seen for some Arabidopsis cer mutants lacking wax
in the pollen coat (Aarts et al., 1995).
[0243] We also found some primary transformants had flat siliques
that is due to change in structure of the silique replum and valves
making the silique more extended laterally. The glossy green stem
phenotype was not very obvious on primary transformants transferred
to the greenhouse from selection media primarily due to the thin
stem structure. The T2 progeny, however, revealed the glossy
thinner stems inherited as a dominant allele (about 3/4 progeny).
The OsSHN1--SRDX overexpressors also showed smaller rosette leaves
and shorter siliques in the progeny.
[0244] To prove that downregulation of CER1 transcript is
responsible for the glossy `cer` stem phenotype we conducted RT-PCR
for the CER1 gene using stem RNA samples from both 35S:OsSHN1-SRDX
and wild-type plants. We found that CER1 gene was significantly
repressed in the 35S:OsSHN1--SRDX plants.
[0245] The phenotype of the SHN-repressor plants reveal the role of
the different SHN genes in Arabidopsis. The glossy `cer` stem is
probably due to repression of the Epicuticular wax pathway leading
to reduction in stem wax. The short siliques are due to conditional
pollen sterility, due to lack of a wax coat requiring high-humidity
for making the pollen fertile and thus seed formation in the
silique. The change in flower inflorescence structure reflects the
expression pattern of the Arabidopsis SHN1 and SHN3 that are
probably required for function in this tissue. The flat silique
shape is indicative of the expression of the AtSHN2 that is
expressed in the valve margin, alteration or malfunction of this
layer cell separation layer inhibits silique opening and
shattering. Thus SHN proteins are required for opening or
shattering of the silique or pod.
REFERENCES
[0246] Aarts M G M, Keizer C J, Stiekema W J and Pereira A (1995)
Molecular characterization of the CER1 gene of Arabidopsis involved
in epicuticular wax biosynthesis and pollen fertility. Plant Cell
7:2115-2127 [0247] Clough S J and Bent A F (1998) Floral dip: a
simplified method for Agrobacterium-mediated transformation of
Arabidopsis thaliana. Plant J 16:735-743 [0248] Greco R, Ouwerkerk
P B F, Taal A J C, Favalli C, Beguiristain T, Puigdomenech P,
Colombo L, Hoge J H C and Pereira A (2001) Early and multiple Ac
transpositions in rice generated by an adjacent strong enhancer.
Plant Mol Biol 46:215-227 [0249] Hiratsu K, Matsui K, Koyama T,
Ohme-Takagi M (2003) Dominant repression of target genes by
chimeric repressors that include the EAR motif, a repression
domain, in Arabidopsis. Plant J 34:733-739 [0250] Lauchli, A.
(1990) in Calcium in Plant Growth and Development, eds. Leonard, R.
T., Hepler, P. K. & The American Society of Plant Physiologists
Symposium Series (American Society of Plant Physiologists.
Rockville, Md.), Vol. 4, pp. 26-35. [0251] Liu, J and Zhu, J-K
(1997) An Arabidopsis mutant that requires increased calcium for
potassium nutrition and salt tolerance. Proc Natl Acad Sci USA.
94:14960-4. [0252] Murashige T and Skoog F (1962) A revised medium
for rapid growth and bioassays with tobacco tissue cultures.
Physiol Plant 15:473-497 [0253] Pietrzak M, Shillito R D, Hohn T,
Potrykus I (1986) Expression in plants of two bacterial antibiotic
resistance genes after protoplast transformation with a new plant
expression vector. Nucleic Acids Res 14:5857-5868 [0254]
Yamaguchi-Shinozaki K, Shinozaki K (1993) The plant hormone
abscisic acid mediates the drought-induced expression but not the
seed-specific expression of rd22, a gene responsive to dehydration
stress in Arabidopsis thaliana. Mol Gen Genet. 238: 17-25
Sequence CWU 1
1
2411133DNAunknownArabidopsis thaliana genomic DNA encoding SHN1
1atcttacata tattactcat catcaagttc ctactttctc tctgacaaac atcacagagt
60aagtaagaat ggtacagacg aagaagttca gaggtgtcag gcaacgccat tggggttctt
120gggtcgctga gattcgtcat cctctcttgt acctttcttc tttctttcct
ttttctctgc 180gttgttcatt tatttcctct ctctgatcca ttaaagtaca
caaccacaca tacatatata 240tacatgatcg tagctaactc atttctggtt
tatcttcctt tgttgttttc ctcttcttag 300ctaacacata aaatcatttt
aacatctaga tacgtaaata tcaataagtt attgcttaaa 360aattctacta
tatatatata tatatatata ttcaccatgc ttaattatta attttgacat
420gcgtgatgat ctttcaaaat aaaaaggaaa cggaggattt ggctagggac
gttcgagacc 480gcagaggagg cagcaagagc atacgacgag gccgccgttt
taatgagcgg ccgcaacgcc 540aaaaccaact ttcccctcaa caacaacaac
accggagaaa cttccgaggg caaaaccgat 600atttcagctt cgtccacaat
gtcatcctca acatcatctt catcgctctc ttccatcctc 660agcgccaaac
tgaggaaatg ctgcaagtct ccttccccat ccctcacctg cctccgtctt
720gacacagcca gctcccatat cggcgtctgg cagaaacggg ccggttcaaa
gtctgactcc 780agctgggtca tgacggtgga gctaggtccc gcaagctcct
cccaagagac tactagtaaa 840gcttcacaag acgctattct tgctccgacc
actgaagttg aaattggtgg cagcagagaa 900gaagtattgg atgaggaaga
aaaggttgct ttgcaaatga tagaggagct tctcaataca 960aactaaatct
tatttgctta tatatatgta cctattttca ttgctgattt acagccaaaa
1020taatcaatta taccgtgtat tttatagatg ttttatatta aaaggttgtt
agctatatat 1080tgtttctctt tttccacatt tgtatctaat aaagtattgg
tgtttgtaac taa 113321076DNAunknownArabidopsis thaliana genomic DNA
encoding SHN2 2atggtacatt cgaggaagtt ccgaggtgtc cgccagcgac
aatggggttc ttgggtctct 60gagattcgcc atcctctatt gtaagtaatt ggctctctat
atacatatat gatctccatt 120gtttactgtt ttcctgctta ggattacata
ttatataatg ttgtagttgc ttttctcata 180tgaagctact atctttattc
tttagtatat ttcatcaaaa ctttatttgt atattaaaac 240aaacgctcta
ctattgctta acaatctaca aaatattaaa aggaattaaa agttaaaacc
300aaccatgcat gctaatcaaa gttaagttat cgactgattt tcatctactt
gattctttga 360ttatttttgt aaattaaatg tggtaatttc taaggttagt
ttggatttca gctcaaaact 420taattgaaat gtttacttgg attttgtaac
taatataaaa attactaaaa tcctttttta 480acgtgaatgt tatatgatac
caaaacgtta aaaattatat ttcaaaattc atatcatcat 540atgtagtgta
tttagtattt acacgtgtgt gtgatatatt aatcaggaag agaagagtgt
600ggcttggaac tttcgaaacg gcagaagcgg ctgcaagagc atacgaccaa
gcggctcttc 660taatgaacgg ccaaaacgct aagaccaatt tccctgtcgt
aaaatcagag gaaggctccg 720atcacgttaa agatgttaac tctccgttga
tgtcaccaaa gtcattatct gagcttttga 780acgctaagct aaggaagagc
tgcaaagacc taacgccttc tttgacgtgt ctccgtcttg 840atactgacag
ttcccacatt ggagtttggc agaaacgggc cgggtcgaaa acaagtccga
900cttgggtcat gcgcctcgaa cttgggaacg tagtcaacga aagtgcggtt
gacttagggt 960tgactacgat gaacaaacaa aacgttgaga aagaagaaga
agaagaagaa gctattatta 1020gtgatgagga tcagttagct atggagatga
tcgaggagtt gctgaattgg agttga 107631517DNAunknownArabidopsis
thaliana genomic DNA encoding SHN3 3atcagtagag aggacgtggg
aaaagcagag agttaagtga gtagttggag atagaaagat 60cagagacgag gaatctctct
cccactctca ctttctctcc tattcttagt tcgtgtcaga 120aacacacaga
gaaattaaga accctaattt aaaacagaag aatggtacat tcgaagaagt
180tccgaggtgt ccgccagcgt cagtggggtt cttgggtttc tgagattcgt
catcctctct 240tgtcagttct ctctccctct ctatctatta atagagacaa
cagtataaat ctgtttatgc 300aagtccatgc tatgagttaa gtttacattt
ttggtgtacg tgtagggcag ttttctgatc 360attgattata ttttcgaaga
ttcatgcaaa gctcttataa tatcatgcat ttttgtttaa 420gtcgttttcc
ttattttttt ccaattaaaa aacccacgtt tattaacata tacatatgtt
480gagtatttgt actttttgtg ttgtattttg aaattttgag tgtacataaa
tttaaatgct 540ttgtcatata attgatactg atgtattttc tatgtcttag
ttggattata gaaaatcata 600tagcaaagaa ttgggaagga acgaacacta
ctcactataa actttcctca aaaagaaaac 660gacccaattt attatcagta
acaatttata acaaatttgt tgtgtgtttg ttttagtact 720ttgaagtttg
aataaaatgt gtgttttttg cttacgtaac ttatgtgagc tatcaggaag
780agaagagtgt ggctaggaac attcgacacg gcggaaacag cggctagagc
ctacgaccaa 840gccgcggttc taatgaacgg ccagagcgcg aagactaact
tccccgtcat caaatcgaac 900ggttcaaatt ccttggagat taactctgcg
ttaaggtctc ccaaatcatt atcggaacta 960ttgaacgcta agctaaggaa
gaactgtaaa gaccagacac cgtatctgac gtgtctccgc 1020ctcgacaacg
acagctcaca catcggcgtc tggcagaaac gcgccgggtc aaaaacgagt
1080ccaaactggg tcaagcttgt tgaactaggt gacaaagtta acgcacgtcc
cggtggtgat 1140attgagacta ataagatgaa ggtacgaaac gaagacgttc
aggaagatga tcaaatggcg 1200atgcagatga tcgaggagtt gcttaactgg
acctgtcctg gatctggatc cattgcacag 1260gtctaaagga gaatcattga
attatatgat caagataata atatagttga gggttaataa 1320taatcgaggg
taagtaattt acgtgtagct aataattaat ataattttcg aacatatata
1380tgaatatatg atagctctag aaatgagtac gtatatatac gtaaacattt
ttcctcaaat 1440atagtatatg tgttgtgatt ctaagacttg taaactgata
tggcctactg tttaaagagt 1500agttgatatt ttctatt
15174834DNAunknownArabidopsis thaliana shn1 transcript 4atcttacata
tattactcat catcaagttc ctactttctc tctgacaaac atcacagagt 60aagtaagaat
ggtacagacg aagaagttca gaggtgtcag gcaacgccat tggggttctt
120gggtcgctga gattcgtcat cctctcttga aacggaggat ttggctaggg
acgttcgaga 180ccgcagagga ggcagcaaga gcatacgacg aggccgccgt
tttaatgagc ggccgcaacg 240ccaaaaccaa ctttcccctc aacaacaaca
acaccggaga aacttccgag ggcaaaaccg 300atatttcagc ttcgtccaca
atgtcatcct caacatcatc ttcatcgctc tcttccatcc 360tcagcgccaa
actgaggaaa tgctgcaagt ctccttcccc atccctcacc tgcctccgtc
420ttgacacagc cagctcccat atcggcgtct ggcagaaacg ggccggttca
aagtctgact 480ccagctgggt catgacggtg gagctaggtc ccgcaagctc
ctcccaagag actactagta 540aagcttcaca agacgctatt cttgctccga
ccactgaagt tgaaattggt ggcagcagag 600aagaagtatt ggatgaggaa
gaaaaggttg ctttgcaaat gatagaggag cttctcaata 660caaactaaat
cttatttgct tatatatatg tacctatttt cattgctgat ttacagccaa
720aataatcaat tataccgtgt attttataga tgttttatat taaaaggttg
ttagctatat 780ttgtttctct ttttccacat ttgtatctaa taaagtattg
gtgtttgtaa ctaa 8345570DNAunknownArabidopsis thaliana shn2
transcript 5atggtacatt cgaggaagtt ccgaggtgtc cgccagcgac aatggggttc
ttgggtctct 60gagattcgcc atcctctatt gaagagaaga gtgtggcttg gaactttcga
aacggcagaa 120gcggctgcaa gagcatacga ccaagcggct cttctaatga
acggccaaaa cgctaagacc 180aatttccctg tcgtaaaatc agaggaaggc
tccgatcacg ttaaagatgt taactctccg 240ttgatgtcac caaagtcatt
atctgagctt ttgaacgcta agctaaggaa gagctgcaaa 300gacctaacgc
cttctttgac gtgtctccgt cttgatactg acagttccca cattggagtt
360tggcagaaac gggccgggtc gaaaacaagt ccgacttggg tcatgcgcct
cgaacttggg 420aacgtagtca acgaaagtgc ggttgactta gggttgacta
cgatgaacaa acaaaacgtt 480gagaaagaag aagaagaaga agaagctatt
attagtgatg aggatcagtt agctatggag 540atgatcgagg agttgctgaa
ttggagttga 5706944DNAunknownArabidopsis thaliana shn3 transcript
6agagagttaa gtgagtagtt ggagatagaa agatcagaga cgaggaatct ctctcccact
60ctcactttct ctcctattct tagttcgtgt cagaaacaca cagagaaatt aagaacccta
120atttaaaaca gaagaatggt acattcgaag aagttccgag gtgtccgcca
gcgtcagtgg 180ggttcttggg tttctgagat tcgtcatcct ctcttagtgt
ggctaggaac attcgacacg 240gcggaaacag cggctagagc ctacgaccaa
gccgcggttc taatgaacgg ccagagcgcg 300aagactaact tccccgtcat
caaatcgaac ggttcaaatt ccttggagat taactctgcg 360ttaaggtctc
ccaaatcatt atcggaacta ttgaacgcta agctaaggaa gaactgtaaa
420gaccagacac cgtatctgac gtgtctccgc ctcgacaacg acagctcaca
catcggcgtc 480tggcagaaac gcgccgggtc aaaaacgagt ccaaactggg
tcaagcttgt tgaactaggt 540gacaaagtta acgcacgtcc cggtggtgat
attgagacta ataagatgaa ggtacgaaac 600gaagacgttc aggaagatga
tcaaatggcg atgcagatga tcgaggagtt gcttaactgg 660acctgtcctg
gatctggatc cattgcacag gtctaaagga gaatcattga attatatgat
720caagataata atatagttga gggttaataa taatcgaggg taagtaattt
acgtgtagct 780aataattaat ataattttcg aacatatata tgaatatatg
atagctctag aaatgagtac 840gtatatatac gtaaacattt ttcctcaaat
atagtatatg tgttgtgatt ctaagacttg 900taaactgata tggcctactg
tttaaagagt agttgatatt ttct 9447600DNAunknownArabidopsis thaliana
SHN1 coding sequence 7atggtacaga cgaagaagtt cagaggtgtc aggcaacgcc
attggggttc ttgggtcgct 60gagattcgtc atcctctctt gaaacggagg atttggctag
ggacgttcga gaccgcagag 120gaggcagcaa gagcatacga cgaggccgcc
gttttaatga gcggccgcaa cgccaaaacc 180aactttcccc tcaacaacaa
caacaccgga gaaacttccg agggcaaaac cgatatttca 240gcttcgtcca
caatgtcatc ctcaacatca tcttcatcgc tctcttccat cctcagcgcc
300aaactgagga aatgctgcaa gtctccttcc ccatccctca cctgcctccg
tcttgacaca 360gccagctccc atatcggcgt ctggcagaaa cgggccggtt
caaagtctga ctccagctgg 420gtcatgacgg tggagctagg tcccgcaagc
tcctcccaag agactactag taaagcttca 480caagacgcta ttcttgctcc
gaccactgaa gttgaaattg gtggcagcag agaagaagta 540ttggatgagg
aagaaaaggt tgctttgcaa atgatagagg agcttctcaa tacaaactaa
6008570DNAunknownArabidopsis thaliana SHN2 coding sequence
8atggtacatt cgaggaagtt ccgaggtgtc cgccagcgac aatggggttc ttgggtctct
60gagattcgcc atcctctatt gaagagaaga gtgtggcttg gaactttcga aacggcagaa
120gcggctgcaa gagcatacga ccaagcggct cttctaatga acggccaaaa
cgctaagacc 180aatttccctg tcgtaaaatc agaggaaggc tccgatcacg
ttaaagatgt taactctccg 240ttgatgtcac caaagtcatt atctgagctt
ttgaacgcta agctaaggaa gagctgcaaa 300gacctaacgc cttctttgac
gtgtctccgt cttgatactg acagttccca cattggagtt 360tggcagaaac
gggccgggtc gaaaacaagt ccgacttggg tcatgcgcct cgaacttggg
420aacgtagtca acgaaagtgc ggttgactta gggttgacta cgatgaacaa
acaaaacgtt 480gagaaagaag aagaagaaga agaagctatt attagtgatg
aggatcagtt agctatggag 540atgatcgagg agttgctgaa ttggagttga
5709561DNAunknownArabidopsis thaliana SHN3 coding sequence
9atggtacatt cgaagaagtt ccgaggtgtc cgccagcgtc agtggggttc ttgggtttct
60gagattcgtc atcctctctt agtgtggcta ggaacattcg acacggcgga aacagcggct
120agagcctacg accaagccgc ggttctaatg aacggccaga gcgcgaagac
taacttcccc 180gtcatcaaat cgaacggttc aaattccttg gagattaact
ctgcgttaag gtctcccaaa 240tcattatcgg aactattgaa cgctaagcta
aggaagaact gtaaagacca gacaccgtat 300ctgacgtgtc tccgcctcga
caacgacagc tcacacatcg gcgtctggca gaaacgcgcc 360gggtcaaaaa
cgagtccaaa ctgggtcaag cttgttgaac taggtgacaa agttaacgca
420cgtcccggtg gtgatattga gactaataag atgaaggtac gaaacgaaga
cgttcaggaa 480gatgatcaaa tggcgatgca gatgatcgag gagttgctta
actggacctg tcctggatct 540ggatccattg cacaggtcta a
56110618DNAunknownOryza sativa OsSHN1 coding sequence 10atggtacagc
caaagaagaa gtttcgtgga gtcaggcagc ggcactgggg ctcctgggtc 60tctgagatca
gacaccccct ccttaaaagg agggtgtggc tgggcacctt tgagacggcc
120gaggaggctg cgcgagccta cgatgaggct gctgtgctga tgagtggccg
caacgccaag 180accaacttcc ccgtgcagag gaactccacc ggtgatctcg
ccacggccgc agaccaggac 240gcccgtagca atggcggtag caggaactcc
tccgcgggca acctgtcaca gattctcagt 300gctaagctcc gcaagtgctg
caaggcgcca tctccgtcct taacctgcct ccgcctcgac 360cccgagaagt
cccacattgg cgtgtggcaa aagcgcgcag gggcccgtgc tgactccaac
420tgggtgatga cggtggagct caacaaagag gtagaaccaa ctgaacctgc
agctcagccc 480acatcaacag caacagcttc gcaagtgaca atggatgatg
aggaaaagat tgcgctgcaa 540atgatcgagg agttgctgag caggagcagt
ccagcttcac cctcacatgg agagggagag 600ggtagctttg tcatctga
61811199PRTunknownArabidopsis SHN1 amino acid sequence 11Met Val
Gln Thr Lys Lys Phe Arg Gly Val Arg Gln Arg His Trp Gly1 5 10 15Ser
Trp Val Ala Glu Ile Arg His Pro Leu Leu Lys Arg Arg Ile Trp 20 25
30Leu Gly Thr Phe Glu Thr Ala Glu Glu Ala Ala Arg Ala Tyr His Glu
35 40 45Ala Ala Val Leu Met Ser Gly Arg Asn Ala Lys Thr Asn Phe Pro
Leu 50 55 60Asn Asn Asn Asn Thr Gly Glu Thr Ser Glu Gly Lys Thr Asp
Ile Ser65 70 75 80Ala Ser Ser Thr Met Ser Ser Ser Thr Ser Ser Ser
Ser Leu Ser Ser 85 90 95Ile Leu Ser Ala Lys Leu Arg Lys Cys Cys Lys
Ser Pro Ser Pro Ser 100 105 110Leu Thr Cys Leu Arg Leu Asp Thr Ala
Ser Ser His Ile Gly Val Trp 115 120 125Gln Lys Arg Ala Gly Ser Lys
Ser Asp Ser Ser Trp Val Met Thr Val 130 135 140Glu Leu Gly Pro Ala
Ser Ser Ser Gln Glu Thr Thr Ser Lys Ala Ser145 150 155 160Gln Asp
Ala Ile Leu Ala Pro Thr Thr Glu Val Glu Ile Gly Gly Ser 165 170
175Arg Glu Glu Val Leu Asp Glu Glu Glu Lys Val Ala Leu Gln Met Ile
180 185 190Glu Glu Leu Leu Asn Thr Asn
19512189PRTunknownArabidopsis SHN2 amino acid sequence 12Met Val
His Ser Arg Lys Phe Arg Gly Val Arg Gln Arg Gln Trp Gly1 5 10 15Ser
Trp Val Ser Glu Ile Arg His Pro Leu Leu Lys Arg Arg Val Trp 20 25
30Leu Gly Thr Phe Glu Thr Ala Glu Ala Ala Ala Arg Ala Tyr Asp Gln
35 40 45Ala Ala Leu Leu Met Asn Gly Gln Asn Ala Lys Thr Asn Phe Pro
Val 50 55 60Val Lys Ser Glu Glu Gly Ser Asp His Val Lys Asp Val Asn
Ser Pro65 70 75 80Leu Met Ser Pro Lys Ser Leu Ser Glu Leu Leu Asn
Ala Lys Leu Arg 85 90 95Lys Ser Cys Lys Asp Leu Thr Pro Ser Leu Thr
Cys Leu Arg Leu Asp 100 105 110Thr Asp Ser Ser His Ile Gly Val Trp
Gln Lys Arg Ala Gly Ser Lys 115 120 125Thr Ser Pro Thr Trp Val Met
Arg Leu Glu Leu Gly Asn Val Val Asn 130 135 140Glu Ser Ala Val Asp
Leu Gly Leu Thr Thr Met Asn Lys Gln Asn Val145 150 155 160Glu Lys
Glu Glu Glu Glu Glu Glu Ala Ile Ile Ser Asp Glu Asp Gln 165 170
175Leu Ala Met Glu Met Ile Glu Glu Leu Leu Asn Trp Ser 180
18513186PRTunknownArabidopsis SHN3 amino acid sequence 13Met Val
His Ser Lys Lys Phe Arg Gly Val Arg Gln Arg Gln Trp Gly1 5 10 15Ser
Trp Val Ser Glu Ile Arg His Pro Leu Leu Val Trp Leu Gly Thr 20 25
30Phe Asp Thr Ala Glu Thr Ala Ala Arg Ala Tyr Asp Gln Ala Ala Val
35 40 45Leu Met Asn Gly Gln Ser Ala Lys Thr Asn Phe Pro Val Ile Lys
Ser 50 55 60Asn Gly Ser Asn Ser Leu Glu Ile Asn Ser Ala Leu Arg Ser
Pro Lys65 70 75 80Ser Leu Ser Glu Leu Leu Asn Ala Lys Leu Arg Lys
Asn Cys Lys Asp 85 90 95Gln Thr Pro Tyr Leu Thr Cys Leu Arg Leu Asp
Asn Asp Ser Ser His 100 105 110Ile Gly Val Trp Gln Lys Arg Ala Gly
Ser Lys Thr Ser Pro Asn Trp 115 120 125Val Lys Leu Val Glu Leu Gly
Asp Lys Val Asn Ala Arg Pro Gly Gly 130 135 140Asp Ile Glu Thr Asn
Lys Met Lys Val Arg Asn Glu Asp Val Gln Glu145 150 155 160Asp Asp
Gln Met Ala Met Gln Met Ile Glu Glu Leu Leu Asn Trp Thr 165 170
175Cys Pro Gly Ser Gly Ser Ile Ala Gln Val 180
18514205PRTunknownOryza sativa OsSHN1 amino acid sequence 14Met Val
Gln Pro Lys Lys Lys Phe Arg Gly Val Arg Gln Arg His Trp1 5 10 15Gly
Ser Trp Val Ser Glu Ile Arg His Pro Leu Leu Lys Arg Arg Val 20 25
30Trp Leu Gly Thr Phe Glu Thr Ala Glu Glu Ala Ala Arg Ala Tyr Asp
35 40 45Glu Ala Ala Val Leu Met Ser Gly Arg Asn Ala Lys Thr Asn Phe
Pro 50 55 60Val Gln Arg Asn Ser Thr Gly Asp Leu Ala Thr Ala Ala Asp
Gln Asp65 70 75 80Ala Arg Ser Asn Gly Gly Ser Arg Asn Ser Ser Ala
Gly Asn Leu Ser 85 90 95Gln Ile Leu Ser Ala Lys Leu Arg Lys Cys Cys
Lys Ala Pro Ser Pro 100 105 110Ser Leu Thr Cys Leu Arg Leu Asp Pro
Glu Lys Ser His Ile Gly Val 115 120 125Trp Gln Lys Arg Ala Gly Ala
Arg Ala Asp Ser Asn Trp Val Met Thr 130 135 140Val Glu Leu Asn Lys
Glu Val Glu Pro Thr Glu Pro Ala Ala Gln Pro145 150 155 160Thr Ser
Thr Ala Thr Ala Ser Gln Val Thr Met Asp Asp Glu Glu Lys 165 170
175Ile Ala Leu Gln Met Ile Glu Glu Leu Leu Ser Arg Ser Ser Pro Ala
180 185 190Ser Pro Ser His Gly Glu Gly Glu Gly Ser Phe Val Ile 195
200 2051561PRTunknownconsensus middle domain of SHN proteins 15Ser
Xaa Xaa Xaa Ser Xaa Xaa Xaa Leu Ser Xaa Xaa Leu Xaa Ala Lys1 5 10
15Leu Arg Lys Xaa Cys Lys Xaa Xaa Xaa Pro Xaa Leu Thr Cys Leu Arg
20 25 30Leu Asp Xaa Xaa Xaa Ser His Ile Gly Val Trp Gln Lys Arg Ala
Gly 35 40 45Xaa Xaa Xaa Xaa Xaa Xaa Trp Val Xaa Xaa Xaa Glu Leu 50
55 601610PRTunknownconsensus "cm" domain of SHN proteins 16Xaa Ala
Xaa Xaa Met Ile Glu Glu Leu Leu1 5 10171998DNAunknowntranscription
regulatory sequence of SHN1 17atttatggag aagttttgaa agtgaacaca
aaacaaacat ctttgaattt agtaaatttg 60aacgaatggc cgttgatcag agttgaatac
agaagaagga aacggtttct tgtggagaaa 120tttaaggtgg catttgctgg
catttgatca cttctatttc ggggttgact gttcttcgcg 180ccgttgctgt
aaaacctaac ttctttacat tcattgtgga catcagtatg gcccaataaa
240ggcccatttt gaaataatcg ggctatttgg tccacacatg aggacacgtg
catacgtaag 300agagtaccag
aaaaggagat tcataaaccc gttaaattcg cggttaggaa acttttgcct
360tttggttgaa acatcgttgg cctcttgcaa gttaattttc ttattctatc
gactatttcg 420gacattcaat cacgtgattt tcgtgtttga aaagatcaac
aatttttttt ttcgttaaaa 480tatctggcat ttaaataaac actatacgta
aattttagca gtatgaaatt aactaggctg 540atggattcga aaaataatac
aataaaacca aaaagaaaag aaaaacatgt attccaaatt 600acaggagtac
tatcatcaat cattcaatct ttaagtagct acaaaagtct acaacaaaaa
660taaagagaaa caaaataata atttaaaggc tagataattg aaaggaatag
tgatatgcaa 720gttccaaaca caagtatgaa caagagttaa tggaagagac
tataattatg aaatatcggt 780catggactca gggaagtggg gaccctatga
taacaagcca ccaccaaatt agcacctcaa 840aaacaaaaat gtataaacca
tattatattg tatattaaat ataagcatac attaggttaa 900agatgtttgc
tcaaaaatta aaagcaaata gatatgagat tgacacctaa acagccttaa
960tctggaccca atctgacagt tagggcttag gctatgggta cgacaatcaa
ctagatatgt 1020ggacaaaaca ttctactaat tacacaatat acatgtacat
ggaccacctt ttaattttta 1080ttttttttgt ccttttactt tttaacttgt
acatgcatgt tgtcaatgtc taattatata 1140atcacttttc ccctctatgt
atgttgtttt atacattgca tgtgttatat ttgatcgcga 1200gtgaacaacc
acttttctca gcgcttatta cgtgttttct ttttgcggca aatcatttta
1260cgattgatta atcctattca aattcactac ttaaacttac tcccactgaa
caaagtccca 1320actaaagctt ataaattgta taatgtttac attaagtctt
ggggattgga ttgacaatga 1380tgattttggc cgatggttag tgctggttgt
taaccaacat gatagtataa tataaaatga 1440aacattaata tggactgatg
ttcggtctta attaccactc cactcttaga tataaaattt 1500tgcagttaat
ggtgaacgca aatttgctta tacgtacgta cgaagctgta tacaaatata
1560agtaagcatc aaattaaaca gagagagaga ggcgtagtac ttacgtagct
atatgatgtg 1620gacgattgat taggatgtac gtatgctaaa aacaatatac
acggccatgt gatacttgta 1680ccacttgcgc atctctacat atatacgatc
caacctttgg aatttatatt gtttaccttt 1740ataataacca tccttcacat
tagcaatcaa tctacaccaa caactacaac taaattctat 1800ctctctctct
ataataaact agcgagcgag gacgtcaaat gtaataagag taagaacaag
1860tacacctttt catccaccaa aatttaacct atgtatataa atatacaaca
ttaccattta 1920ccaaacatcc atcttacata tattactcat catcaagttc
ctactttctc tctgacaaac 1980atcacagagt aagtaaga
1998181848DNAunknowntranscription regulatory sequence of SHN2
18gattgggtac taggttaagg tcaaatatgg ttcaaataaa agttaatatt aaaagatgga
60aagtatgaag tttatactag tgagttactt gttctttagt tttagaatgg atatatttta
120ttttattttt atactttaga ccattaagca ggaaaaatat gtaagcaata
ctctaatagg 180tagaagaaac aagagataca agtaaagtag tgcataaaga
gatgaaaaca ctgtctttgg 240tacctctaat tgttaccggc cgttttaata
attgataatg ttatagtata tttgtttatt 300tgacaaatat ctcaaacttt
aaaaattaca caaagacaaa gatgtaaaac tatttgcagc 360atttcaacgg
atgcgataga ttacccatta aaaaaaaaaa aaaaaaaaaa aacacgaatg
420gaatagtctt ttcaatggct aatattagtt ttcacttctg atcacttagt
aaaatcatga 480aagacacata cattagaatt gtgtccatat tgaggaaaag
agaaagagaa tggtgtacat 540acataaattt aatacatgtt tattatgaga
aagcaacgta cgtaatacat tactttgtct 600ctaggtaaca aattgtggat
ttccattggt tggtaacaga aaaaagaacg attgggggat 660aaactacaat
atgtgagttt ttttgttcaa actagatgtg aactatatat tggtgaatac
720ctttgaaaat attacaaaca ttactatcca tcccatagcg tcgtcacctt
actcatgact 780tcaattatag tctgacaata taggactgaa aaacatgatt
gcaaatgttg tgaatacatt 840tattttagca caaagtttta aaagttattg
gtctattata cgttgaacct taaatgaacg 900ttgaaatatc tacgcccttt
caccacccga cccaccgaca tttctttggt ttttgttttt 960ttagatatat
atatatatat atttttaatt caaacatgta aattgaatta aactggaaga
1020taaacaaagc tgcaccagga attcgagccg aacttacgag gtggccttag
agctatgtta 1080ttatgtctac gtacacccaa agtttgaagt acagaattag
gttatagatc tgtcttgtgg 1140aaagaaaaac ttattgctaa tttaggttga
ctcagattat aactgtagtt gagatgagac 1200agtcaatgga gacagatcta
taacaaattg atgttttggc aaataaaggt atgatatata 1260atgtagttat
atgataataa aaaaaagcta tgatatatcc tatatatata atgcatgttg
1320atgtttaaat aaaagaaagc tatatatgtt ttagtttctt tgtgggtatt
aaaactcagg 1380catgagtccc tatatgggtc gatcgtggga aaaaaaaaaa
aaaagagagt ccctatatgg 1440catgaaagta acgttagaaa cgaaggtacg
agtcgtgtct tcactagatt cattctaata 1500aagtaaggaa gttgtattca
taaataaaga atagatatct attgatgtta cgtccctcac 1560taaggcttgt
gagacaaagt agtgggctca aatatcttaa ttaccaacgt gaatcgaatt
1620tatatagatc atcgaagaaa ggtcaattta caaacaaaga gaaacttcaa
attaaggccc 1680tatataaata gttagttggg gacgaaccac agtaggatca
gggactatat cagtctctat 1740ctttctctct ctcttctgat tgtccgagtt
gtgtctgcta agaagagaga gaaacttaaa 1800accctaaatt tcaaatcaga
aaatatagag tttgaaggaa actaaaag 1848192004DNAunknowntranscription
regulatory sequence of SHN3 19tctagaccaa tattttatta ttattcatat
aattaaacgt ttattgaatt attttagctc 60taaaaaatat aatgaaattt agaattgtta
tagaaacttt aaatgactca aaactgatct 120ttgttttttt gtcaaccaca
cattaattaa aaactgatct ttgttaaaca tctaaatatt 180gaaaaacaaa
aggattacca ccgcacctat cctgcatcgt cttgtaccat ttgcattcat
240cagagccatt atatattcat gtggctgtca catccttata tacatttcaa
cttataaatt 300taattttgta cgtttagcag atgttggtga attcatttat
tcaagcttaa ggtttaactc 360gttcttgtct cctttaggtt cataacattt
aatggacaac aactactcca aatatccgtg 420gactttacca cgcgacgcac
cgacaattct ctagcctttt cattaattga tcagtaaaaa 480cacttctata
attttaatta ttctcagaaa ttttaaccgg tagtaacata aacgtagatg
540atcgaaaaag atgaaatcga ggtctctgtt ttgtgtcgaa ttaatagatc
tataagaaat 600caaacaatga aaattaaata gatgaatgaa atatgttttg
acgacgtttt cacgtcactg 660tttaagttat ctgttagtaa aatttgatag
catttaattt gaattgctat gtaactcgga 720ataaattcga cggcaagagg
tgtgggatgt gatcagtctt ctaattaagt gtaatcttta 780caaaaaatca
gtatgagtta atacaggttt ataaatagaa agacatctac agtaaaacct
840ctataaatta ataatgttgg gactgcaaaa ttttattaat ttagaaagat
tttaatttat 900cgataaatta atatttatta atttatagag agattttcgt
aatttagtaa cttttcaaaa 960atttctataa aatttttttc attatagaaa
atatctttca aaaatcaatt acaagtaaat 1020aataatatca tgtttagaaa
acaacaccaa aagtttttga taaagtaaaa tactataact 1080aaatttaaat
aagaataaat acaaatttta agcaaaaaaa tattagaatt atatttcaaa
1140ataatttctt acatataatg tatatatagt tgatatattt ataaaattat
taatttatga 1200tattgatggg accatatatt tacataagat tttttaaaaa
gttattatct tattaattta 1260tcgatttgtg tcacttttta cactgatccc
aactcgggac tggaagaatt tattaattta 1320tagaggttat taatttatcg
agtattaatt tatagaggtt taactgtata tgaataattg 1380aatacacatg
ggacatcatt atagttacct tagtacgatt ttatgcattt ggttaccaaa
1440atccactagg cttcgtaatt ggttgtgggt cagtgtccat tcgatagttt
gcattagcga 1500taaacactta aaacagtaaa acgtgatcta tttttaaaca
ataacatttc ttcttatcaa 1560atattcacat aattaatgaa gaaaagagac
gaattgatag gcaataagaa atatgatcac 1620ttggtaccat tgtatgcatg
gtatgaactg acatcaagta tttgtacgtt gtagggtaat 1680tattaacatt
gtagaatgac aagtatgatc aaatggaaaa ggattagtta tatgtagcaa
1740aactagtatt tgtacttgga ctatcttgtc atgtagtttt cctaacgcta
tatataatcc 1800aataacccca tgacacggaa gagatagggg acatacaata
aatatcagta gagaggacgt 1860gggaaaagca gagagttaag tgagtagttg
gagatagaaa gatcagagac gaggaatctc 1920tctcccactc tcactttctc
tcctattctt agttcgtgtc agaaacacac agagaaatta 1980agaaccctaa
tttaaaacag aaga 2004201362DNAunknowntranscription regulatory
sequence of OsSHN1 20actcagcagt gagcacacac catcatcata accaggctgt
gtacatggag ctaccttaga 60caagcttaaa tgccaaccaa ctccaagcag tcccagatca
aaaagctcat gacaacggaa 120aacttgaaaa agaaaaaaaa aactttacaa
gtcttaacca aaaaaactct aaatttacac 180acacacacac acacacacta
aaggcaacac attcttacac atttcaaact ccagttctat 240agcgcaaaac
aagacttgag tgttgaaata tgaagaaagt ttcccaataa gacgagagaa
300aactgaacac gtcatcacaa ataaagcacg agatattccc aaacgcagtt
tgcagcataa 360gtcttgccat atgagacgcc tagttaagaa caacgtccat
gcatccactg gcactcaaag 420ctcatgactg gaccatatcc acaccccacc
atagctgtgg aagaattatg gccatggagt 480tgatctgaca caaatgcaca
attgctttgc agaacaacta gcctagctta tactaatgct 540ctaggagcta
atgtgctaat gtgaatcaac ccggggttgc cccacaatta atgtggctac
600catgggcgta ggtgagagtg ttaattacca atgtcctagc agagaaggtg
gttatgctaa 660tacccgcaca gttcaccgac agcccccact gcgggccttg
tggactggac caccttcacc 720ttcagttgcc cctcccctga attctctctt
cacctctact acctctgtcc cgaaataact 780ttatttttca cctatcccgt
acataccaat acaaagacaa aaataccata atgtcctcta 840ttttaacaaa
tcacaatgca attttacccc actttaacaa actccaatgc atttgtctcc
900cacttccata gattcaatat aatgatttac ttaaaagata aaagttattt
tgagacaaat 960aagatggtaa aaaatgaagt tattttggga tagagggaag
tatctccagt cctgccttac 1020ttttatccct tggcacacac ctgctagttg
ctactgcttg tgaacccagc ccttggtgat 1080gttcagtgaa aactaggcca
aacacaatct ctttgattct ctctttctat ctctgtatct 1140ctgatacgta
ctatttgacc acctatacgt ctcaccacat ttaacgcggc actgtagacg
1200caagtacagg ccgcagcagt ttatattcac tcaaacaagt gctttcctcc
tcccccacac 1260ctcctccgtt cagttcagag gcgcctagca atagcagctc
attgcctcat ctctgcctcc 1320cctgtccttc tgggggcaga gaatctctcc
actgctggaa aa 13622112PRTunknownEAR repressor domain 21Leu Asp Leu
Asp Leu Glu Leu Arg Leu Gly Phe Ala1 5 102236DNAunknownnucleic acid
sequence encoding the EAR repressor domain 22ctggatctgg atctagaact
ccgtttgggt ttcgct 3623732DNAunknownOryza sativa cDNA OsSHN2
23atgggacagt cgaagaagaa gttccgcgga gtcaggcagc gccactgggg ctcctgggtc
60tccgagatca ggcaccctct ccttaagagg agggtgtggc tgggtacctt tgagacggcg
120gaggaggcgg cgcgggcgta cgacgaggcc gccatcctga tgagcggccg
caacgccaag 180accaacttcc cagtcgcgag gaacgccacg ggggagctca
caccggcggc tgcggtggca 240gggcgggatg gccgtgtcgg cggcggcagc
ggcagctcgt cctcaatgac ggccaacggc 300ggcgggaaca gcctgtctca
gatcctcagc gccaagctcc gcaagtgctg caagacgccg 360tcgccgtcgc
tcacctgcct ccgccttgac ccggagaagt cccacattgg cgtctggcag
420aagcgcgccg gcgcacgcgc tgactccagc tgggtcatga ccgtcgagct
caacaaggac 480acggccgtgt cgtcggctgc gacggtggca gcagcaacag
cagtgtcgtc cagcgaccag 540ccgactccga gtgacagcac agtcacaacg
acgtccacgt ccaccacggg ctcgccgtcg 600ccaccacctc cggcaatgga
cgacgaggag aggatcgcgc tgcagatgat cgaggagctg 660ctgggcagga
gcggcccggg ctcgccgtca catgggctgc tgcacggtgg tgaaggtagc
720ctcgtcatct ga 73224243PRTunknownOsSHN2 amino acid sequence 24Met
Gly Gln Ser Lys Lys Lys Phe Arg Gly Val Arg Gln Arg His Trp1 5 10
15Gly Ser Trp Val Ser Glu Ile Arg His Pro Leu Leu Lys Arg Arg Val
20 25 30Trp Leu Gly Thr Phe Glu Thr Ala Glu Glu Ala Ala Arg Ala Tyr
Asp 35 40 45Glu Ala Ala Ile Leu Met Ser Gly Arg Asn Ala Lys Thr Asn
Phe Pro 50 55 60Val Ala Arg Asn Ala Thr Gly Glu Leu Thr Pro Ala Ala
Ala Val Ala65 70 75 80Gly Arg Asp Gly Arg Val Gly Gly Gly Ser Gly
Ser Ser Ser Ser Met 85 90 95Thr Ala Asn Gly Gly Gly Asn Ser Leu Ser
Gln Ile Leu Ser Ala Lys 100 105 110Leu Arg Lys Cys Cys Lys Thr Pro
Ser Pro Ser Leu Thr Cys Leu Arg 115 120 125Leu Asp Pro Glu Lys Ser
His Ile Gly Val Trp Gln Lys Arg Ala Gly 130 135 140Ala Arg Ala Asp
Ser Ser Trp Val Met Thr Val Glu Leu Asn Lys Asp145 150 155 160Thr
Ala Val Ser Ser Ala Ala Thr Val Ala Ala Ala Thr Ala Val Ser 165 170
175Ser Ser Asp Gln Pro Thr Pro Ser Asp Ser Thr Val Thr Thr Thr Ser
180 185 190Thr Ser Thr Thr Gly Ser Pro Ser Pro Pro Pro Pro Ala Met
Asp Asp 195 200 205Glu Glu Arg Ile Ala Leu Gln Met Ile Glu Glu Leu
Leu Gly Arg Ser 210 215 220Gly Pro Gly Ser Pro Ser His Gly Leu Leu
His Gly Gly Glu Gly Ser225 230 235 240Leu Val Ile
* * * * *
References