Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
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Stress tolerant plants
The present invention relates to a method for obtaining stress tolerant
plants, for
example tolerant to salinity, to vectors comprising genetic information
capable of
conferring said tolerance to the plants, to muteins encoded by the said
genetic
information and to plants and plant materials obtainable by the said method.
to
Several documents are cited throughout the text of this specification. Each of
the
documents cited herein (including any manufacturer's specifications,
instructions,
etc.) are hereby incorporated by reference; however, there is no admission
that any
document cited is indeed prior art as to the present invention.
Abiotic stress conditions, such as shortage or excess of solar energy, water
and
nutrients, salinity, high and low temperature and pollution (e.g., heavy
metals), can
have a major impact on plant growth and can significantly reduce the yield of,
e.g.,
cultivars. In the state of the art it is known that, under conditions of
abiotic stress, the
2 o growth of plant cells is inhibited by arresting the cell cycle in late G,,
before DNA
synthesis, andlor at the GZIM boundary; see reviews of Dudits, 1997, Plant
Cell
Division, Portland Press Research, Monograph, Francis, D., Dudits, D. and
Inz~, D.
eds, ch 2, pp 21, and Bergounioux, Protoplasma 142 (1988), 127-136.
The regulation of the cell cycle in plant cells is however poorly understood:
WO 92109685 generally describes a method for controlling plant cell growth
comprising modulating the level of cell cycle control proteins in said plant
cells. The
method disclosed in W092I09685 is described to be applicable for improvements
of
plant growth behavior in the presence of one or more environmental conditions.
In
particular, WO 92109685 describes the presence of a p34°'''~ protein in
plants, a
so protein which is known to play a key role in the cell cycle of yeasts and
vertebrates
(see, e.g., the review by Lew and Kombluth, in Curr. Op. Cell Biol. 8 (1996),
795-804,
herein incorporated by reference), wherein an indication is made that the
amount of
plant p34'~ protein becomes limiting for cell division in plant tissue.
However, no
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2
clear indication was given as to the role of plant p34'~ protein or other
putative plant
cell cycle control proteins in arresting the cell cycle under conditions of
abiotic in
particular salt stress nor related to the onset of cell cycle progress after
the said cell
cycle arrest.
Different approaches for the generation of stress tolerant plants have been
described
in the prior art. For example, WO 97113843 describes the production of water
stress
or salt stress tolerant transgenic cereal plants by transforming the cereal
plant cell or
protoplast with a nucleic acid encoding a late embryogenesis abundant protein.
Furthermore, the production of disease and stress tolerant plants by
increasing the
to juvenility and antioxidant capacity was suggested; see Bama, Novenytermeles
44
(1995), 561-567. However, the above-described approaches have not been shown
to
be generally applicable and means that can be used to confer stress tolerance
to
plants without otherwise substantially affecting phenotype of the plant, e.g.,
growth
characteristics, were hitherto not available.
Thus, the technical problem underlying the present invention is to provide
means and
methods for conferring or enhancing stress tolerance to plants which are
particularly
useful in agriculture.
Zo The solution to the technical problem is achieved by providing the
embodiments
characterized in the claims.
Accordingly, the present invention relates to a method for obtaining plants,
tolerant to abiotic stress conditions, comprising introducing into a plant
cell, plant
tissue or plant a nucleic acid molecule andlor regulatory sequence, wherein
the
introduction of said nucleic acid molecule or regulatory sequence results in
the
presence of a Cyclin Dependent Kinase (CDK) protein that is not susceptible to
inhibitory phosphorylation under abiotic stress conditions.
so Control of CDK activity can be achieved by cyclin association and
phosphoryfation. The phosphorylation of CDK can either have an inhibitory
effect
or an activating effect on its activity depending on the position of the
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3
phosphorylation site. p34'd'2 is regulated by an activating phosphorylation
during
G2 at Thr 167 and by inhibitory phosphorylations at Thr-14 andlor Tyr-15
(Jacobs, Annu. Rev. Plant Physiol. Plant Mol Biol. 46 (1995), 317-339). The
teen
"not susceptible to inhibitory phosphorylation under abiotic stress
conditions" as
s used herein means that the CDK protein that is usually phosphorylated under
stress conditions in the plant cell, is under-phosphorylated, i.e., non-
phosphorylated at certain inhibitory phosphorylation sites which are otherwise
phosphorylated under stress conditions. Thus, if, e.g., the CDK protein
comprises
four inhibitory phosphorylation sites which are usually phosphorylated in the
plant
io cell under stress conditions, said CDK is present in a non-phosphorylated
form in
accordance with the present invention if at least one of said phosphorylation
sites
is unphosphorylated. The terms "not susceptible to inhibitory phosphorylation
under abiotic stress conditions" and "non-phosphorylated form of a CDK
protein"
are used interchangeable herein.
is The term "abiotic stress" as used herein refers to any adverse effect on
metabolism, growth or viability of the cell, tissue, organ or whole plant
which is
produced by an non-living or non-biological (i.e., not biotic: insect,
bacteria,
fungal, virus) environmental stressor, e.g., environmental factors such as
water
(flooding, drought, dehydration), anaerobic (low level of oxygen, COz etc.),
20 osmotic (salt), temperature (hotlheat, cold, freezing, frost),
nutrientslpollutants, or
by a hormone, second messenger or other molecule which is related to or
induced by said stressor.
The term "anaerobic stress" means any reduction in oxygen levels sufficient to
produce a stress as hereinbefore defined, including hypoxia and anoxia.
2s The term "flooding stress" refers to any stress which is associated with or
induced
by prolonged or transient immersion of a plant, plant part, tissue or isolated
cell in
a liquid medium such as occurs during monsoon, wet season, flash flooding or
excessive irrigation of plants, etc.
"Cold stress" and "heat stress" are stresses induced by temperatures which are
so respectively, below or above, the optimum range of growth temperatures for
a
particular plant species. Such optimum growth temperature ranges are readily
determined or known to those skilled in the art.
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PCT/EP99/02696
"Dehydration stress" is any stress which is associated with or induced by the
loss
of water, reduced turgor or reduced water content of a cell, tissue, organ or
whole
plant.
"Drought stress" refers to any stress which is induced by or associated with
the
s deprivation of water or reduced supply of water to a cell, tissue, organ or
organism.
The terms "salinity-induced stress", "salt-stress" or similar term refer to
any stress
which is associated with or induced by a perturbation in the osmotic potential
of
the intracellular or extracellular environment of a cell.
io The transgenic plant obtained in accordance with the method of the present
invention, upon the presence of the nucleic acid molecule and/or regulatory
sequence introduced into said plant, attains tolerance or improved tolerance
against abiotic stress which the corresponding wild-type plant was susceptible
to.
The terms "tolerance" and "tolerant" cover the range of protection from a
delay to
is complete inhibition of alteration in cellular metabolism, reduced cell
growth andlor
cell death caused by the abiotic stress defined hereinbefore. Preferably, the
transgenic plant obtained in accordance with the method of the present
invention
is tolerant to abiotic stress in the sense that said plant is capable of
growing
substantially normal under environmental conditions where the corresponding
ao wild-type plant shows reduced growth, metabolism, viability andlor male or
female
sterility.
Progression through the cell cycle is dependent on the activity of cycfin
dependent kinases (CDKs) in all eukaryotes. In fission (Nurse and Bisset,
Nature
292 {1981 ), 558-560) and budding yeast (Nasmyth, Curr. Opin. Cell. Biol. 5,
zs (1993), 166-179) the CDC2 and respectively the CDC28 protein kinase are the
central regulators of the cell cycle while in higher eukaryotes multiple CDKs
with
distinct roles are present (Mironov, V., De Veylder, V., Van Montagu, M. and
Inze,
D. (1999), Molecular Control of the Cell Division Cycle in Higher Plants. The
Plant Cell; and Pines, Sem. Cell. Biol. 5 (1994), 399-408). Dephosphorylation
of
3o CDC2 at tyrosine 15 in yeast (could, and Nurse, Nature 342 (1989), 39-45)
and
simultaneously at threonine 14 in animal cells by a CDC25 tyrosine phosphatase
(Norbury, EMBO J. (1991), 3321-3329) is a prerequisite for cell cycle
progression
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into mitosis. substitution of the Tyr15 residue to the non-phosphorylatable
Phe15
results in fission yeast in small cells, the wee phenotype, as a consequence
of
premature mitotical entry (Russell and Nurse, Cell 49 (1987), 559-567). In
Arabidopsis, the Thr14 and Tyr15 phosphorylation sites are conserved in the
s protein kinase CDC2aAt (Mironov (1999)). No phenotypic changes were however
detected in transgenic Arabidopsis lines overexpressing a dominant negative
mutant form of CDC2aAt with substituted Thr14 and Tyr15, except for a tendency
to loose apical dominance (Hemerly, EMBO J. 14 (1995), 3925-3936).
io In accordance with the present invention it was found that at the onset of
the cell
cycle arrest under abiotic stress conditions, the plant CDK protein, being
functionally
equivalent to the known CDC2a of Arabidopsis thaiiana, was phosphorylated at a
tyrosine and optionally also at a threonine residue, corresponding to the
tyrosine of
position 15 and the threonine of position 14 of said CDC2a respectively.
Moreover, it
is was surprisingly found that the expression of non-phosphorylatable mutants
of CDKs
results in abiotic stress tolerant plants. The present invention is based on
the finding
that the transgenic plants overexpressing a mutant CDK, i.e., CDC2aAt with non-
phosphorylatable A1a14 and Phe15 residues show increased tolerance to abiotic
stress, in particular salt stress. Compared to wild type plants (WT) and
transgenic
ao Arabidopsis plants (YF2 and YFS, Example 2) ectopically expressing the wild
type
form of CDC2aAt (CDC2aWT), the YF lines displayed an enhanced shoot growth
after cultivation in the presence of NaCI. Additionally the YF lines recovered
faster upon release from salinity than the CDC2aWT and WT plants (Fig. 1 ).
The
results obtained in accordance with the present invention strongly suggest
that
25 the said phosphorylation in particular of plant CDK proteins appears to be
one of the
key events in abiotic stress-induced cell cycle arrest.
The terms "CDK" or "plant CDK" are meant to encompass all plant CDK proteins
having a cell cycle regulatory function in plants or plant cells having the
above-
mentioned phosphorylatable tyrosine residue, and optionally in addition
thereto, the
3o said threonine residue. Examples of these CDICs are the members of the CDC2
family, as identified in Arabidopsis thaliana, such as CDC2a and CDC2b.
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While the findings described above have been obtained with the CDK protein
CDC2a
of Arabidopsis thaliana, the present invention can be performed with any CDK
protein
that is functional in plants, i.e., plant CDK proteins, functionally
equivalent to the
known CDC2a of Arabidopsis thaliana. With "plant CDK protein, functionally
equiva-
s lent to the known CDC2a of Arabidopsis thaliana" is meant each CDK protein
having
a similar regulatory function as CDC2a of Arabidopsis thaliana in plants or
plant cells
respectively, e.g., having the PSTAIRE conserved cyclin binding motif, and the
above-mentioned phosphorylatable tyrosine and threonine residues. Intensive
cloning efforts have identified a large number of CDK proteins in diverse
plant
io species, among which at least five types can be distinguished on the basis
of
their sequences (for a compilation see Segers, In: Plant cell proliferation
and its
regulation in growth and development. Bryant JA, Chiatante D, editors.
Chichester: John Wiley & Sons (1997), 1-19). In the model plant, Arabidopsis
thaliana, two CDKs, each belonging to a different family, have been
is characterized. One such example is the CDC2aAt gene, which contains the
conserved PSTAIRE amino acid motif, and is constitutively expressed during the
cell cycle at transcriptional and protein level. However, the associated
kinase
activity is maximal at the G1lS and G2IM transitions, suggesting a role at
both
checkpoints (Hemerly, Plant Cell 5 (1993) 1711-1723; Burssens, Plant Physioi.
Zo Biochem. 36 (1998), 9-19; Segers, Plant J. 10 (1996), 601-612). CDC2bAt
contains a PPTALRE motif and its mRNA levels are preferentially present during
S and G2 phase (Segers, 1996 and references cited therein). The protein
follows
the transcriptional level but the CDC2bAt kinase activity becomes only maximal
during mitosis, implying a role during the M phase. Furthermore, CDKs or
Zs mutants thereof that can be employed in accordance with the present
invention
can be tested for their ability to confer abiotic stress tolerance to plants
according
to methods well-known in the art, see, e.g., Physical Stresses in Plants:
Genes
and Their Products for Tolerance. S. Grillo (Editor), A. Leone (Editor) (June
1996)
Springer Verlag; ISBN: 3540613471; Handbook of Plant and Crop Stress.
so Mohammad Pessarakli (Editor), Marcel Dekker; ISBN: 0824789873; The
Physiology of Plants Under Stress: Abiotic Factors. Erik T. Nilsen, David M.
Orcutt (Contributor), Eric T. Nilsen. 2nd edition (October 1996), John Wiley &
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Sons; ISBN: 0471031526; Drought, Salt, Cold, and Heat Stress: Molecular
Responses in Higher Plants (Biotechnology Intelligence Unit). Kazuo Shinozaki
(Editor), Kazuko Yamaguchi-Shinozaki (Editor) {1999). R G Landes Co; ISBN:
1570595631; Plants Under Stress: Biochemistry, Physiology and Ecology and
s Their Application to Plant Improvement {Society for Experimental Biology
Seminar Serie). Hamlyn G. Jones, T.J. Flowers, M.B. Jones (Editor). (September
1989). Cambridge Univ. Pr. (Short); ISBN: 0521344239; Plant Adaptation to
Environmental Stress. Leslie Fowden, Terry Mansfield, John Stoddart (Editor)
(October 1993) Chapman & Hall; ISBN: 0412490005; or as described in the
io appended examples.
Determination of phosphorylation sites in CDKs corresponding to tyrosine at
position 15 and the threonine of position 14 of CDC2a can be done, for
example, by
computer-assisted identification of such sites in the amino acid sequence of a
given CDK using, e.g., BLAST2, which stands for Basic Local Alignment Search
is Tool (Altschul, 1997; Altschul, J. Mol. Evol. 36 {1993), 290-300; Altschul,
J. Mol.
Biol. 215 (1990), 403-410), which can be used to search for local sequence
alignments. BLAST produces alignments of both nucleotide and amino acid
sequences to determine sequence similarity. Because of the local nature of the
alignments, BLAST is especially useful in determining exact matches or in
ao identifying homologues. Phosphorylating sites can also be determined using
anti-
phospho-tyrosine and anti-phospho-threonine antibodies as described by Zhang,
Planta 200 (1996), 2-12.
The introduction of the nucleic acid molecule in the method of the present
as invention enhances the amount or results in de novo production of said non-
phosphorylated form of CDK protein. For example, said nucleic acid molecule
comprises a coding sequence of the mentioned protein or of a regulatory
protein,
e.g., a transcription factor, capable of inducing the expression of said CDK
protein in its non-phosphorylated form or, e.g., of a CDK dephosphorylating
so enzyme or for example antisense to enzymes phosphorylating CDKs.
A "coding sequence" is a nucleotide sequence which is transcribed into mRNA
andlor translated into a polypeptide when placed under the control of
appropriate
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regulatory sequences. The boundaries of the coding sequence are determined by
a translation start codon at the 5'-terminus and a translation stop codon at
the 3'-
terminus. A coding sequence can include, but is not limited to mRNA, cDNA,
recombinant nucleotide sequences or genomic DNA, while introns may be
s present as well under certain circumstances.
The terms "gene(s)", "polynucleotide", "nucleic acid sequence", "nucleotide
sequence", "DNA sequence" or "nucleic acid molecule(s)" as used herein refers
to a polymeric form of nucleotides of any length, either ribonucleotides or
deoxyribonucieotides. This term refers only to the primary - structure of the
io molecule. Thus, this term includes double- and single-stranded DNA, and
RNA. It
also includes known types of modifications, for example, methylation, "caps"
substitution of one ar more of the naturally occurring nucleotides with an
analog.
The term "regulatory sequence" as used herein denotes a nucleic acid molecule
increasing the expression of the said protein, e.g., the above-mentioned
protein,
is due to its integration into the genorne of a plant cell in close proximity
to the
gene, e.g., encoding inhibitors of phosphorylation. Such regulatory sequences
comprise promoters, enhancers, inactivated silencer intron sequences, 3'UTR
andlor 5'UTR coding regions, protein andlor RNA stabilizing elements or other
gene expression control elements which are known to activate gene expression
ao and/or increase the amount of the gene products.
The introduction of said nucleic acid molecule leads to de novo expression, or
if
the mentioned regulatory sequence is used to increase andlor induction of
expression of said proteins, resulting in the end in an increased amount of a
non-
2s phosphorylated form of CDK protein in the cell. Thus, the present invention
is
aiming at providing de novo and/or increased expression of non-phosphorylated
CDKs. In a preferred embodiment of the method of the present invention said
CDK is a PSTAIRE type CDK, preferably said CDK is CDC2a.
so As is demonstrated in the appended examples, it was found that in plants,
at the
onset of the cell cycle arrest under abiotic stress conditions, the endogenous
Cyclin
Dependent Kinases (CDICs) were phosphorylated at a tyrosine at position 15 and
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9
optionally also at a threonine residue at position 14. In most plants for
which CDK
sequences have been identified, the positions of the said tyrosine and
threonine
residues are at positions 15 and 14, respectively; this is, e.g., the case for
CDC2a of
Arabidopsis thaliana. it is however possible that, e.g., during the course of
evolution,
s in some plant CDICs the respective positions of these consensus Y-15 and T-
14 have
been shifted somewhat, i.e., as a result of one or more deletions or additions
at the
N-terminus of the protein.
The terms "tyrosine at position 15" and "threonine residue at position 14" as
used
herein, are therefor meant to encompass the positions 14 and 15 of the
respective
io CDK, as well as such positional changes of the said tyrosine and threonine
residues
within the plant CDK protein, wherein the characteristic of these residues
being
phosphorylated at the onset of the stress-induced cell cycle arrest is
retained. This
means that the said positions as defined herein correspond to the tyrosine at
position
15 and the threonine at position 14 of CDC2a of Arabidopsis thaiiana,
respectively.
is
Thus, in a preferred embodiment of the method of any one of the present
invention,
the CDK is free of phosphate at the tyrosine at a position that corresponds to
position 15 in the amino acid sequence of CDC2a of Arabidopsis thaliana.
Particularly preferred is that the COK protein is free of phosphate groups at
both
Zo the tyrosine and the threonine, corresponding to the tyrosine at position
15 and
the threonine at position 14, respectively, in the amino acid sequence of
CDC2a
of Arabidopsis thaiiana.
In one embodiment of the method of the present invention, said non-
as phosphorylated CDK protein is a non-phosphorylatable CDK mutein. A
preferred
embodiment of the present invention is by conferring to the plant the capacity
to
produce, under stress conditions, a CDK mutein, of which Y-15 is substituted
to a
non-phosphorylatable residue. When the plant is able to produce such a CDK
mutein, said mutein will substantially not be sensitive for the
phosphorylation system,
3o triggering the stress-induced cell cycle arrest. In this way, the plant
circumvents the
downregulation of the cell cycle, being more tolerant to said stress
conditions.
The term "CDK mutein", used herein, is defined as a CDK fragment or CDK
protein
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comprising at least one mutation, e.g., an amino acid substitution, deletion
or additi-
on. Additionally, phosphorylation of T-14 may play a potentiating or mediating
role in
the above-discussed downregulation mechanism. Therefore, in another preferred
embodiment of the method according to the present invention, the CDK mutein
also
s comprises a non-phosphorylatabie amino acid residue at position 14.
Preferably, the said mutein is derived from endogenous CDK of the stress
tolerant
plant to be obtained. By starting from the endogenous CDK, the risk of
malfunctioning
muteins is minimized. However, in view of homology between different plant
CDKs, it
to will be obvious to the skilled person that it is also possible to use CDK
from another
plant species. CDK, of, e.g., yeast or vertebrate origin may, dependent on the
homology with the endogenous plant CDK, as well be suitable in the present
invention; the suitability can easily be determined by the skilled person.
As a non-phosphorylatable amino acid residue substituting Y-15 (i.e., the
tyrosine of
the CDK, corresponding to the tyrosine on position 15 of CDC2a of Arabidopsis
thaliana), the CDK mutein preferably comprises a Y-15 -> F-15 mutation, F
being
phenylalanine. In all plants investigated so far, the expression of said
mutein led to
enhanced stress tolerance. Similarly, as a non-phosphorylatable amino acid
residue
2o substituting T-14 (i.e., the threonine of the CDK, corresponding to the
threonine on
position 14 of CDC2a of Arabidopsis thaiiana), the CDK mutein preferably
comprises
a T-14 -> A-14 mutation, A being alanine. Expression of such a mutein led to
improved stress tolerance.
2s As has been explained above, the method of the present invention can be
performed in various ways. Thus, one could use, e.g., a plant cell that
already
comprises in its genome a nucleic acid molecule encoding a non-
phosphorylatable form of CDK as described above, but does not express the
same in an appropriate manner due to, e.g., a weak promoter. in such a case it
so would be sufficient to introduce into the plant cell a regulatory sequence
such as
a strong promoter in close proximity to the endogenous nucleic acid molecule
encoding said non-phosphorylatable form of CDK so as to induce expression of
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m
the same. Usually, however, a wild-type plant cell will not have an endogenous
gene encoding a non-phosphoryiatable form of CDK. Therefore, in a preferred
embodiment of the present invention said nucleic acid molecule to be
introduced
into the plant cell or plant tissue or plant encodes said non-phosphorylatable
form
s of CDK.
Alternatively, the method of the present invention can be performed wherein
said
non-phosphorylated form of CDK is due to dephosphorylation andlor inhibition
of
phosphorylation of CDK.
to As has been discussed above, it could surprisingly be shown that the
downregulation of the cell division of plants, exposed to abiotic stress, was
effectively
counteracted by the presence of a CDK, in particular CDK, equivalent to CDC2a
of
Arabidopsis thaliana, being free of a phosphate at the Y-15 position. A
preferred
embodiment of the present invention therefore relates to conferring to the
plant the
is capacity to provide, at the stress conditions, CDK protein, being
functionally equiva-
lent to CDC2a of Arabidopsis thaliana, a substantial portion thereof being
free of
phosphate at the tyrosine, corresponding to the tyrosine of position 15 of
said
CDC2a. A "substantial portion" in this respect is defined herein as the amount
of
CDK, being free of phosphate at the Y-15 position, that is sufficient to
confer to the
a o plant improved growth during abiotic stress conditions. The person skilled
in the art
will understand that not all of the corresponding CDK present in the plant or
plant cell
has to be phosphate free at the Y-15 position to improve said stress
tolerance. This
may, e.g., be accomplished by conferring to the plant the capacity of
preventing
phosphorylation of the said tyrosine, or of activating the dephosphorylation
2s mechanism for the said tyrosine. As this counteraction may further be
improved upon
T-14 being additionally free of phosphate, the CDK protein is preferably free
of
phosphate groups at both the tyrosine and the threonine, con-esponding to the
tyrosi-
ne on position 15 and the threonine on position 14 of said CDC2a,
respectively.
so An attractive way to obtain stress tolerant plants according to the present
invention is
therefore by conferring to the plant the capacity to provide under stress
conditions,
CDC25 or a functional analogue thereof, capable of dephosphorylating at least
the
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tyrosine at position 15 of the endogenous CDK of the said plant. The
dephosphorylating activity of CDC25 is described in Lew and Kombluth, supra.
By
enabling the plant to produce, at the stress conditions, functional CDC25
protein, i.e.,
capable of dephosphorylating the above-mentioned tyrosine, and optionally also
the
s adjacent threonine of the endogenous CDK, the phosphorylation of CDK as a
result
of the stress conditions is effectively counteracted.
As well in mammals as in yeast the function of the WEE1 protein kinase is
antagonistic to CDC25, acting as a mitotic inhibitor by phosphorylation of
CDC2
to on Tyr15 (Igarashi, Nature 353 (1991 ), 80-3; Russell and Nurse, Cell 49
(1987),
559-567; Labib and Nurse, Current Biology, 3 (1993), 164-166). A Wee 1 plant
homologue from maize, ZmWee1 has recently been identified (Sun, Proc. Natl.
Acad. Sci. USA 96 {1999), 4180-4185). In fission yeast MIK1 acts cooperatively
with the WEE1 protein kinase in the inhibitory Tyr15 phosphorylation of CDC2
is (Lundgren, Cell 64 (1991), 1111-1122). In Xenopus a MYT1 kinase has been
identified that phosphorylates CDC2 at both Tyr15 and Thr 14 to keep the CDC2
complex in a mitotic inactive state (Mueller, Science 270 (1998), 86-89).
Thus, another attractive route to obtain stress tolerant plants according to
the present
2 o invention is by conferring to the plant the capacity to inhibit, under
stress conditions,
the expression or activity of at least Wee-kinase, MIK1 or MYT or a functional
equivalent thereof, thereby inhibiting or reducing the endogenous
phosphorylation of
CDK of the said plant at least the tyrosine at position 15. Wee-kinase is
reviewed in,
e.g., Lew and Kombluth, supra. This kinase phosphorylates the above-discussed
Y-
2s 15 of CDK and may also be responsible for the phosphorylation of the T-14.
With
"functional equivalent of Wee-kinase" is meant any endogenous kinase of the
plant
having the function of known Wee-kinase in phosphorylating the respective
tyrosine
residue and optionally the threonine residue of the endogenous plant CDK The
recently identified Myt1 kinase (Mueller, Science 270 (1995), pp 86) may
therefore be
3o regarded as such a functional equivalent. By inhibiting the expression of
the Wee-
kinase under abiotic stress conditions, the phosphorylation of CDK will be
inhibited,
reducing the downregulation of cell division (mitotic activity) and growth,
thus
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13
obtaining stress tolerance.
Thus, engineering of transgenic plants in accordance with the present
invention
comprises the use of the animal or yeast CDC25, WEE9, MYT9 or MlK1 genes or
s more preferably their plant homologues such as Wee1 form maize; see Sun,
supra.
Strategies include overexpressing CDC25 homologue by use of a regulatory
sequence described herein and knock out of protein kinases (WEE, MYT and
MIK) by, e.g., RNA antisense constructs, t-DNA insertion, cosuppression,
io dominant negative mutants, homologous recombination technology, etc.
described in more detail below.
As will be appreciated by the person skilled in the art, the expression of the
above-described phosphatases or inhibition of said protein kinases can be
i5 achieved in different ways. For example, the expression of the plant
homologue
of CDC25 can be induced by introduction of a regulatory sequence as defined
above so as to induce the expression of the endogenous phosphatase gene.
Furthermore, it is possible to inhibit phosphorylation by the mentioned
kinases
through inhibition of gene expression of the said protein kinases or
inactivation of
ao their gene products. In a preferred method of the present invention, said
nucleic
acid molecule introduced into the plant cell, plant tissue or plant encodes
said
CDC25 or its functional analog. In humans, three phosphatases have been
identified in : CDC25a, b, and c. CDC25a plays a role at the G1IS transition
while
CDC25b and c are considered to be functional at the G2lM transition (Sahdu,
2s Proc. Natl. Acad. Sci. USA 87 (1990), 5139-5143; Galaktiniov, Cell 67
(1991),
1181-1194; Nagata, New Biol. 10 (1991), 959-968; Jinno, EMBO J. 13 (1994),
1549-1556). In fission yeast (S.pombe) one CDC25 phosphatase has been
isolated (could and Nurse, Nature 342 (1989), 39-45; Labib and Nurse (1993)).
Although no CDC25 cognate has yet been isolated in plants, considering the
so evolutionary character of the cell cycle, similar phosphatases might
however exist
in plants (cfr. Tyr15 phosphorylation of CDC2 like protein kinases is
suggested by
the work of Zhang, Planta 200 (1996), 2-12; Schuppler, Plant Physiol. 117
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14
(1998), 667-678, and demonstrated by the analysis of the YF plants upon
salinity
stress as presented in the examples). The phenotypic analysis of transgenic
plants that overexpress the fission yeast CDC25 by McKibbin, Plant Mol. Biol.
36
(1996), 601-612 confirms this hypothesis. These plants produced more lateral
s roots, implying that pericycle cells, from which lateral roots are
initiated, bypass a
checkpoint, which is relieved by the action of the fission yeast CDC25.
in a further preferred embodiment, said nucleic acid molecule to be introduced
into the plant cell or plant tissue or plant encodes an antisense RNA of said
WEE
kinase, MYT, MIK or functional analogue or equivalent thereof.
to
In case the above-described proteins or at least one of them are to be
expressed
de novo, it is preferred to employ in the method of the present invention
genes
encoding such proteins, e.g., CDK muteins, wherein said gene is expressible in
plant cells. Thus, in another embodiment the method of the present invention
said
is nucleic acid molecule is operatively linked to regulatory sequences
allowing the
expression of the nucleic acid molecule in the plant. Said regulatory
sequences
comprise a promoter, enhancer, silencer, intron sequences, 3'UTR andlor 5'UTR
regions, protein and/or RNA stabilizing elements. Preferably, said regulatory
sequence is a chimeric, tissue specific, constitutive or inducible promotor.
The
Zo term "operably linked" refers to a juxtaposition wherein the components so
described are in a relationship permitting them to function in their intended
manner. A control sequence "operably linked" to a coding sequence is ligated
in
such a way that expression of the coding sequence is achieved under conditions
compatible with the control sequences. In case the control sequence is a
Zs promoter, it is obvious for a skilled person that double-stranded nucleic
acid is
preferably used. Furthermore, the nucleic acid molecule to be used in
accordance with the present invention can be operably linked to poly-A signals
ensuring termination of transcription and stabilization of the transcript, for
example, those of the 35S RNA from Cauliflower Mosaic Virus (CaMV) and from
3o the Nopaline Synthase promoter. Additional regulatory elements may include
transcriptional as well as translational enhancers. A plant translational
enhancer
often used is the tobaccos mosaic virus (TMV) omega sequences, the inclusion
of
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an intron (Intron-1 from the Shrunken gene of maize, for example) has been
shown to increase expression levels by up to 100-fold. (Malt, Transgenic
Research 6 (1997), 143-156; Ni, Plant Journal 7 (1995), 661-676).
s Any promoter that functions in the target cells can be used. The use of the
CaMV35S
promoter, per se known to the skilled person, resulted in plants with improved
tolerance to salt stress (i.e., to a salt concentration in the growth medium
of, e.g., 1
w/v % NaCI). It is preferable to use a promoter, that can be induced upon the
abiotic
stress conditions. Such promoters can be taken for example from stress-related
to genes which are regulated directly or indirectly by an environmental, i.e.,
preferably
abiotic, stress in a plant cell, including genes for which expression is
increased,
reduced or otherwise altered. These stress-related genes comprise genes the
expression of which is either induced or repressed by anaerobic stress,
flooding
stress, cold stress, dehydration stress, drought stress, heat stress or
salinity,
15 amongst others. For example, the stress-related gene may encode an ANP
selected
from the group consisting of sucrose synthase, phosphoglucomutase,
phosphoglucose isomerase, fructose-1,6-diphosphate aldolase, glyceraldehyde-3-
phosphate dehydrogenase, phosphoglycerate mutase, enolase, pyruvate
decarboxylase, alcohol dehydrogenase and alanine amino transferase, amongst
others. Such promoters are known in the art (see also, e.g., Lang, and Palva,
Plant
Mol. Biol. 20 (1992), 951 and Table 1 below).
Table 1
Name Stress Reference
PSCS (delta(1 )-pyrroline-5-salt, water Zhang, Plant Science
carboxylate synthase) 129
(1997), 81-89
cor15a cold Hajela, Plant Physiol.
93
(1990), 1246-1252
cor15b cold Wilhelm, Plant Mol
Biol. 23
(1993), 'f 073-7
cor15a (-305 to +78 cold, drought Baker, Plant Mol Biol.
nt) 24 ~,
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16
Name Stress Reference
(1994), 701-13
rd29 salt, drought, Kasuga, Nature Biotechno-
cold
logy 18 (1999); 287-291
heat shock proteins, heat Barros, Plant Mol.
Biol.
including artificial 19(4) (1992), 665-75;
promoters
containing the heat Marrs, Dev Genet. 14(1
shock )
element (HSE) (1993), 27-41; Schoffl,
Mol.
Gen. Genet. 217(2-3)
(1989), 246-53
smHSP (small heat shockheat Waters, J. Experimental
proteins) Botany 47, 296 (1996),
325-
338,
wcs120 cold Ouellet, FEES Lett.
423
(1998), 324-328
ci7 cold Kirch, Plant Mol. Biol.
33
(1997), 897-909
Adh cold, drought, Dolferus, Plant Physiol,
hypoxia 105(4) (1994), 1075-87
pwsi18 water: salt Joshee, Plant Cell
and Physiol.
drought 39 (1998), 64-72
ci21A cold Schneider, Plant Physiol.
113 (1997), 335-45
Trg-31 drought Chaudhary, Plant Mol.
Biol.
30 (1996), 1247-57
osmotin osmotic Raghothama, Plant Mol.
Biol. 23 (1993), 1117-28
In a particularly preferred embodiment of the method of the present invention
said
inducible promoter is inducible by abiotic stress, preferably, said abiotic
stress is
osmotic stress, preferably caused by salt.
Preferably, the above-described nucleic acid molecules are comprised in an
expression vector. An "expression vector" is a construct that can be used to
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17
transform a selected host cell and provides for expression of a coding
sequence
or antisense in the selected host. Expression vectors can for instance be
cloning
vectors, binary vectors or integrating vectors. Expression comprises
transcription
of the nucleic acid molecule preferably into a translatable mRNA.
s Advantageously, the above-described vectors of the invention comprise a
selectable and/or scorable marker. Selectable marker genes useful for the
selection of transformed plant cells, callus, plant tissue and plants are well
known
to those skilled in the art and comprise, for example, antimetabolite
resistance as
the basis of selection for dhfr, which confers resistance to methotrexate
(Reiss,
io Plant Physiol. (Life Sci. Adv.) 13 (1994), 143-149); npt, which confers
resistance
to the aminoglycosides neomycin, kanamycin and paromycin (Herrera-Estrella,
EMBO J. 2 (1983), 987-995) and hygro, which confers resistance to hygromycin
(Marsh, Gene 32 (1984), 481-485). Additional selectable genes have been
described, namely trp8, which allows cells to utilize indole in place of
tryptophan;
15 hisD, which allows cells to utilize histinol in place of histidine
(Hartman, Proc.
Natl. Acad. Sci. USA 85 (1988), 8047); mannose-6-phosphate isomerase which
allows cells to utilize mannose (WO 94/20627) and ODC {ornithine
decarboxylase) which confers resistance to the ornithine decarboxylase
inhibitor,
2-{difluoromethyl)-DL-ornithine, DFMO (McConiogue, 1987, In: Current
2o Communications in Molecular Biology, Cold Spring Harbor Laboratory ed.) or
deaminase from Aspergillus terreus which confers resistance to Blasticidin S
(Tamura, Biosci. Biotechnol. Biochem. 59 (1995), 2336-2338).
Useful scorable markers are also known to those skilled in the art and are
25 commercially available. Advantageously, said marker is a gene encoding
luciferase (Giacomin, PI. Sci. 116 (1996), 59-72; Scikantha, J. Bact. 178
(1996),
121 ), green fluorescent protein (Gerdes, FEES Lett. 389 (1996), 44-47) or f3
glucuronidase (Jefferson, EMBO J. 6 (1987), 3901-3907). This embodiment is
particularly useful for simple and rapid screening of cells, tissues and
organisms
so containing a vector of the invention.
The present invention also relates to vectors, particularly plasmids, cosmids,
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18
viruses, bacteriophages and other vectors used conventionally in genetic
engineering that contain at least one nucleic acid molecules andlor regulatory
sequences according to the invention. In particular, the present invention
relates to
a vector, at least comprising a stress-inducible promoter, preferably, a salt
stress-
s inducible promoter, functional in plant cells, operably linked to a DNA
sequence,
coding for a mutated cdc2a gene of Arabidopsis thaliana or functionally
equivalent
gene of another species, preferably a plant species, the gene product thereof
being a
CDK mutein functional in the said plant cells and comprising, in the CDK
mutein, a
non-phosphorylatable amino acid residue at the position, corresponding to the
1o tyrosine on position 15 of CDC2a. Preferably, the mutein also comprises a
non-
phosphorylatable amino acid residue at the position of the mutein,
corresponding to
the threonine on position 14 of said CDC2a. In order to minimize the
possibility of
malfunctioning, it is preferred that a plant CDK gene is used. Being
transformed with
a vector of this type, plant cells are capable of producing, at abiotic stress
conditions,
i5 CDK muteins that are not susceptible to the above-discussed regulatory
phospho-
rylation events, therefor leading to stress tolerant plants or plant cells. In
a further
embodiment, the vector comprises a promoter as defined above, functional in
plant
cells, operably finked to a DNA sequence, coding for CDC25 or a functional
analogue
thereof, capable of dephosphorylating at least the tyrosine of at least one
plant CDK,
Zo corresponding with the tyrosine on position 15 of CDC2a of Arabidopsis
thaliana.
Such a vector can be used to transform plants in order to, as is discussed
above,
express CDC25 in plants, resulting in dephosphorylation of Y-15 and optionally
the
T-14 of the endogenous plant CDK, leading to improved stress tolerance.
2s Methods which are well known to those skilled in the art can be used to
construct
various plasrnids and vectors; see, for example, the techniques described in
Sambrook, Molecular Cloning A Laboratory Manual, Cold Spring Harbor
Laboratory (1989) N.Y. and Ausubel, Current Protocols in Molecular Biology,
Green Publishing Associates and Wiley Interscience, N.Y. (1989), (1994).
so Plasmids and vectors to be preferably employed in accordance with the
present
invention include those well known in the art. Alternatively, the nucleic acid
molecules and vectors of the invention can be reconstituted into liposomes for
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19
delivery to target cells. The present invention furthermore relates to host
cells
comprising a vector as described above wherein the nucleic acid molecule is
foreign to the host cell.
s By "foreign" it is meant that the nucleic acid molecule is either
heterologous with
respect to the host cell, this means derived from a cell or organism with a
different genomic background, or is homologous with respect to the host cell
but
located in a different genomic environment than the naturally occurring
counterpart of said nucleic acid molecule. This means that, if the nucleic
acid
io molecule is homologous with respect to the host cell, it is not located in
its natural
location in the genome of said host cell, in particular it is surrounded by
different
genes. In this case the nucleic acid molecule may be either under the control
of
its own promoter or under the control of a heterologous promoter. The vector
or
nucleic acid molecule according to the invention which is present in the host
cell
i5 may either be integrated into the genome of the host cell or it may be
maintained
in some form extrachromosomally.
The host cell can be any prokaryotic or eukaryotic cell, such as bacterial,
insect,
fungal, plant or animal cells. Preferred fungal cells are, for example, those
of the
Zo genus Saccharomyces, in particular those of the species S. cerevisiae.
In a preferred method for obtaining stress tolerant plants according to the
present
invention, the said capacity is conferred to one or more cells of said plant
by a) trans-
forming one or more plant cells with a vector, at least comprising, under the
control of
zs a promoter functional in the said plant cells, a DNA sequence, coding for a
mutated
cdk gene of Arabidopsis thaliana or functional equivalent gene of another
species,
the gene product thereof being a CDK mutein functional in the said plant cells
and
comprising a non-phosphorylatable amino acid residue at the position of the
CDK
mutein, corresponding to the tyrosine on position 15 of CDC2a of Arabidopsis
3o thaliana, preferably, the mutein also comprises a non-phosphorylatable
amino acid
residue at position 14 of the CDK mutein b) by regenerating a plant from one
or more
of the transformed plant cells, e.g., by the Agrobacterium tumefaciens
transformation
CA 02326689 2000-10-23
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system. However, other transformation methods known in the field may be used.
With
"mutein, functional in plant cells", muteins are meant, which, when expressed
in the
said plant cells, lead to improved stress tolerance of the said cells.
s Methods for the introduction of foreign DNA into plants are also well known
in the art.
These include, for example, the transformation of plant cells or tissues with
T-DNA
using Agrobacterium tumefaciens or Agrobacterium rhizogenes, the fusion of
protoplasts, direct gene transfer (see, e.g., EP-A 164 575), injection,
electroporation,
biolistic methods like particle bombardment, pollen-mediated transformation,
plant
io RNA virus-mediated transformation, liposome-mediated transformation,
transformation using wounded or enzyme-degraded immature embryos, or
wounded or enzyme-degraded embryogenic callus and other methods known in
the art. The vectors used in the method of the invention may contain further
functional elements, for example "left border"- and "right border"-sequences
of
is the T-DNA of Agrobacterium which allow for stably integration into the
plant
genome. Furthermore, methods and vectors are known to the person skilled in
the art which permit the generation of marker free transgenic plants, i.e.,
the
selectable or scorable marker gene is lost at a certain stage of plant
development
or plant breeding. This can be achieved by, for example co-transformation
zo (Lyznik, Plant Mol. Biol. 13 (1989), 151-161; Peng, Plant Mol. Biol. 27
(1995), 91-
104) andlor by using systems which utilize enzymes capable of promoting
homologous recombination in plants (see, e.g., W097I08331; Bayley, Plant Mol.
Biol. 18 (1992), 353-361 ); Lloyd, Mol. Gen. Genet. 242 (1994), 653-657;
Maeser,
Mol. Gen. Genet. 230 (1991 ), 170-176; Onouchi, Nucl. Acids Res. 19 (1991 ),
6373-6378). Methods for the preparation of appropriate vectors are described
by,
e.g., Sambrook (Molecular Cloning; A Laboratory Manual, 2nd Edition (1989),
Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY).
Suitable strains of Agrobacterium tumefaciens and vectors as well as
transformation of Agrobacteria and appropriate growth and selection media are
so well known to those skilled in the art and are described in the prior art
(GV3101
(pMK90RK), Koncz, Mol. Gen. Genet. 204 (1986), 383-396; C58C1 (pGV
3850kan), Deblaere, Nucl. Acid Res. 13 (1985), 4777; Bevan, Nucleic Acid Res.
CA 02326689 2000-10-23
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21
12 ( 1984), 8711; Koncz, Proc. Natl. Acad. Sci. USA 86 (1989), 846?-8471;
Koncz,
Plant Mol. Biol. 20 (1992), 963-976; Koncz, Specialized vectors for gene
tagging and
expression studies. In: Plant Molecular Biology Manual Vol 2, Gelvin and
Schilperoort
(Eds.), Dordrecht, The Netherlands: Kluwer Academic Publ. (1994), 1-22; EP-A-
120
s 516; Hoekema: The Binary Plant Vector System, Offsetdrukkerij Kanters B.V.,
Alblasserdam (1985), Chapter V, Fraley, Crit. Rev. Plant. Sci., 4, 1-46; An,
EMBO
J. 4 (1985), 277-287). Although the use of Agrobacterium tumefaaens is
preferred in
the method of the invention, other Agrobacterium strains, such as
Agrobacterium
rhizogenes, may be used, for example if a phenotype conferred by said strain
is
io desired.
Methods for the transformation using biolistic methods are well known to the
person
skilled in the art; see, e.g., Wan, Plant Physiol. 104 (1994), 37-48; Vasil,
Biolfechnology 11 (1993), 1553-1558 and Christou, Trends in Plant Science 1
(1996), 423-431. Microinjection can be performed as described in Potrykus and
is Spangenberg (eds.), Gene Transfer To Plants. Springer Verlag, Berlin, NY
(1995).
The transformation of most dicotyledonous plants is possible with the methods
described above. But also for the transformation of monocotyledonous plants
several
successful transformation techniques have been developed. These include the
transformation using biolistic methods as, e.g., described above as well as
protoplast
ao transformation, electroporation of partially permeabilized cells,
introduction of DNA
using glass fibers, Agrobacterium mediated transformation etc.
The term "transformation" as used herein, refers to the transfer of an
exogenous
polynucleotide into a host cell, irrespective of the method used for the
transfer.
2s The polynucleotide may be transiently or stably introduced into the host
cell and
may be maintained non-integrated, for example, as a plasmid, or alternatively,
may be integrated into the host genome. The resulting transformed plant cell
or
plant tissue can then be used to regenerate a transformed plant in a manner
known by a skilled person.
In general, the plants which can be modified according to the invention and
which
show over- and/or de novo expression of a non-phosphorylated form of a CDK
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22
protein can be derived from any desired plant species. They can be
monocotyledonous plants or dicotyledonous plants, preferably they belong to
plant
species of interest in agriculture, wood culture or horticulture interest,
such as a crop
plant, root plant, oil producing plant, wood producing plant, agricultured
s bioticultured plant, fruit-producing plant, fodder or forage legume,
companion
plant, or horticultured plant e.g., such a plant is wheat, barley, maize,
rice, carrot,
sugar beet, chicory, cotton, sunflower, tomato, cassava, grapes, soybean,
sugar
cane, flax, oilseed rape, tea, canola, onion, asparagus, carrot, celery,
cabbage,
lentil, broccoli, cauliflower, brussel sprout, artichoke, okra, squash, kale,
collard
io greens, rye, sorghum, oats, tobacco, pepper, grape or potato. Additional
species
are not excluded. Crops grown on cultivated lands in arid or semi-arid areas
in
which irrigation with ground water is needed may advantageously benefit from
the
invention.
is Thus, the present invention relates also to transgenic plant cells which
contain a
nucleic acid molecule or regulatory sequence as defined above or vector
according
to the invention wherein the nucleic acid molecule or regulatory sequence is
foreign
to the transgenic plant cell. For the meaning of the term "foreign"; see
supra.
2 o In one aspect the present invention relates to a transgenic plant cell
comprising
stably integrated into the genome a nucleic acid molecule, regulatory
sequence, or a
vector in accordance with the present invention or obtainable according to the
method of the invention wherein the expression of the nucleic acid molecule or
conferred by the regulatory sequence results in an increased or de novo
expression
25 Of a non-phosphorylated form of CDK or of a dephosphorylating enzyme
described
above in transgenic plants compared to wild-type plants. Alternatively, a
plant cell
having a nucleic acid molecule encoding a CDK mutein or corresponding
dephosphorylating enzyme present in its genome can be used and modified such
that said plant cell expresses the endogenous gene corresponding to this
nucleic
so acid molecule under the control of regulatory sequences described above
such
as heterologous promoter andlor enhancer elements. The introduction of the
heterologous promoter and mentioned elements which do not naturally control
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23
the expression of a nucleic acid molecule encoding, e.g., a CDK mutein using,
e.g., gene targeting vectors can be done according to standard methods, see
supra and, e.g., Hayashi, Science 258 (1992), 1350-1353; Fritze and Walden,
Gene activation by T-DNA tagging. In Mefhods in Molecular Biology 44
(Gartland,
s K.M.A. and Davey, M.R., eds). Totowa: Human Press (1995), 281-294) or
transposon tagging (Chandlee, Physiologic Plantarum 78~ (1990), 105-115).
Suitable promoters and other regulatory elements such as enhancers include
those mentioned herein before.
io In another aspect, the present invention relates to a transgenic plant cell
which
contains stably integrated into the genome a nucleic acid molecule, regulatory
sequence or vector described above or obtainable according to the method of
the
invention, wherein the presence, transcription and/or expression of the
nucleic acid
molecule, regulatory sequences or part thereof leads to reduction of the
synthesis or
is the activity of proteins phosphorylating CDKs under abiotic stress
conditions in
transgenic plants compared to wild type plants. Preferably, said reduction is
achieved
by an antisense, sense, ribozyme, co-suppression, in vivo mutagenesis,
antibody
expression and/or dominant mutant effect. Therefore, the use of nucleic acid
molecules encoding an antisense RNA which is complementary to transcripts of
an
2o enzyme phosphorylating CDK in a plant is also the subject matter of the
present
invention. Thereby, complementarity does not signify that the encoded RNA has
to
be 100% complementary. A low degree of complementarity is sufficient, as long
as it
is high enough in order to inhibit the expression of a protein phosphorylating
CDK
upon expression in plant cells. The transcribed RNA is preferably at least 90%
and
25 most preferably at least 95% complementary to the transcript of the nucleic
acid
molecule encoding such a phosphorylating enzyme. In order to cause an
antisense-
effect during the transcription in plant cells such DNA molecules have a
length of at
least 15 bp, preferably a length of more than 100 by and most preferably a
length or
more than 500 bp, however, usually less than 5000 bp, preferably shorter than
2500
3o bp. Also DNA molecules can be employed which, during expression in plant
cells,
lead to the synthesis of an RNA which in the plant cells due to a co-
suppression-
effect reduces the expression of the nucleic acid molecules encoding the
described
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24
phosphorylating protein. The principle of the co-suppression as well as the
production of corresponding DNA sequences is precisely described, for example,
in
WO 90/12084. Such DNA molecules preferably encode an RNA having a high
degree of homology to transcripts of the genes encoding phosphorylating
enzymes. It
s is, however, not absolutely necessary that the coding RNA is translatable
into a
protein. The principle of co-suppression effect is known to the person skilled
in the
art and is, for example, described in Jorgensen, Trends Biotechnol. 8 (1990),
340-
344; Niebel, Curr. Top. Microbiol. Immunol. 197 (1995), 91-103; Flavell, Curr.
Top.
Microbiol. Immunol. 197 (1995), 43-36; Palaqui and Vaucheret, Plant. Mol.
Biol. 29
io (1995), 149-159; Vaucheret, Mol. Gen. Genet. 248 (1995), 311-317; de Bome,
Mol.
Gen. Genet. 243 (1994), 613-621 and in other sources.
Likewise, DNA molecules encoding an RNA molecule with ribozyme activity which
specifically cleaves transcripts of a gene encoding the dephosphorylating
enzyme
i5 can be used. Ribozymes are catalytically active RNA molecules capable of
cleaving RNA molecules and specific target sequences. By means of
recombinant DNA techniques it is possible to after the specificity of
ribozymes.
There are various classes of ribozymes. For practical applications aiming at
the
specific cleavage of the transcript of a certain gene, use is preferably made
of
2o representatives of two different groups of ribozymes. The first group is
made up
of ribozymes which belong to the group I intron ribozyme type. The second
group
consists of ribozymes which as a characteristic structural feature exhibit the
so-
called "hammerhead" motif. The specific recognition of the target RNA molecule
may be modified by altering the sequences flanking this motif. By base pairing
2s with sequences in the target molecule these sequences determine the
position at
which the catalytic reaction and therefore the cleavage of the target molecule
takes place. Since the sequence requirements for an efficient cleavage are
low, it
is in principle possible to develop specific ribozymes for practically each
desired
RNA molecule.
In order to produce DNA molecules encoding a ribozyme which spec~cally cleaves
transcripts of a gene encoding a kinase for CDK, for example a DNA sequence
CA 02326689 2000-10-23
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encoding a catalytic domain of a ribozyme is bilaterally linked with DNA
sequences
which are homologous to sequences encoding the target protein. Sequences
encoding the catalytic domain may for example be the catalytic domain of the
satellite
DNA of the SCMo virus (Davies, Virology 177 (1990), 216-224 and Steinecke,
EMBO
s J. 11 (1992), 1525-1530) or that of the satellite DNA of the TobR virus
(Haseloff and
Geriach, Nature 334 (1988), 585-591). The DNA sequences flanking the catalytic
domain are preferably derived from the above-described DNA molecules of the
invention. The expression of ribozymes in order to decrease the activity in
certain
proteins in cells is also known to the person skilled in the art and is, for
example,
io described in EP-B1 0 321 201. The expression of ribozymes in plant cells
was,
for example, described, in Feyter et al. (Mol. Gen. Genet. 250 (1996), 329-
338).
Furthermore, the kinase activity of enzymes capable of phosphorylating CDK in
the plant cells of the invention can also be decreased by the so-called "in
vivo
is mutagenesis", for which a hybrid RNA-DNA oligonucleotide ("chimeroplast")
is
introduced into cells by transformation of cells TIBTECH 15 (1997), 441-447;
W095115972; Kren, Hepatology 25 (1997), 1462-1468; Cole-Strauss, Science 273
(1996), 1386-1389). Part of the DNA component of the RNA-DNA oligonucleotide
is homologous to a nucleic acid sequence of an endogenous enzyme capable of
2o phosphorylating CDK, in comparison to the said nucleic acid sequence
protease it
displays, however, a mutation or contains a heterologous region which is
surrounded by the homologous regions. By means of base pairing of the
homologous regions of the RNA-DNA oligonucleotide and of the endogenous
nucleic acid molecule followed by a homologous recombination the mutation
2s contained in the DNA component of the RNA-DNA oligonucleotide or the
heterologous region can be transferred to the genome of a plant cell. This
results
in a decrease of the activity.
Furthermore, nucleic acid molecules encoding antibodies specifically
recognizing an
3o enzyme capable of phosphorylating a CDK in a plant or parts, i.e., specific
fragments
or epitopes, of such a protein can be used for inhibiting the activity of the
protein in
plants. These antibodies can be monoclonal antibodies, polyclonal antibodies
or
CA 02326689 2000-10-23
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26
synthetic antibodies as well as fragments of antibodies, such as Fab, Fv or
scFv
fragments etc. Monoclonal antibodies can be prepared, for example, by the
techniques as originally described in Kbhler and Milstein, Nature 256 (1975),
495,
and Galfr~, Meth. Enzymol. 73 (1981 ), 3, which comprise the fusion of mouse
s myeloma cells to spleen cells derived from immunized mammals. Furthermore,
antibodies or fragments thereof to the aforementioned peptides can be obtained
by using methods which are described, e.g., in Harlow and Lane "Antibodies, A
Laboratory Manual", CSH Press, Cold Spring Harbor, 1988. Expression of
antibodies or antibody-like molecules in plants can be achieved by methods
well
io known in the art, for example, full-size antibodies (During, Plant. Mol.
Biol. 15 (1990),
281-293; Hiatt, Nature 342 (1989), 469170; Voss, Mol. Breeding 1 (1995), 39-
50),
Fab-fragments (De Neve, Transgenic Res. 2 (1993), 227-237), scFvs (Owen,
BioITechnoiogy 10 (1992), 790-794; Zimmermann, MoI. Breeding 4 (1998), 369-
379;
Tavladoraki, Nature 366 (1993), 469-472) and dAbs (Benvenuto, Plant Mol. Biol.
17
is (1991 ), 865-874) have been successfully expressed in Tobacco, Potato
(Schouten,
FEBS Lett. 415 (1997), 235-241 ) or Arabidopsis, reaching expression levels as
high
as 6.8% of the total protein (Fiedler, Immunotechnology 3 (1997), 205-216).
In addition, nucleic acid molecules encoding mutant forms of a protein capable
of
2o phosphorylating a CDK in a plant protease can be used to interfere with the
activity of
the wild type protein. Such mutant forms preferably have lost their biological
activity,
e.g., kinase activity and may be derived from the corresponding wild-type
protein by
way of amino acid deletion(s), substitution(s), andlor additions in the amino
acid
sequence of the protein. Mutant forms such proteins also encompass hyper-
active
2s mutant forms of such proteins which display, e.g., an increased substrate
affinity
and/or higher substrate turnover of the same. Furthermore, such hyper-active
forms may be more stable in the cell due to the incorporation of amino acids
that
stabilize proteins in the cellular environment. These mutant forms may be
naturally
occurring or genetically engineered mutants, see also supra.
The nucleic acid and amino acid sequences for proteins capable of
phosphorylating CDK in a plant can be arrived, for example, from the above-
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27
described Wee-kinase MIK or MYT proteins. Furthermore, it is immediately
evident to the person skilled in the art that the above-described antisense,
ribozyme, co-suppression, in-vivo mutagenesis, antibody expression and
dominant mutant effects can also be used for the reduction of the expression
of
s genes that encode a regulatory protein such as transcription factors that
control
the expression of enzymes capable of phosphorylating CDK in plant cells.
Likewise the described methods can be used, for example, to knock-out the
activity of regulatory proteins that, for example, are necessary for CDK
phosphorylating enzymes to become active. Furthermore, the above-described
io methods can be used to knock-out the expression or activity of the
endogenous
wild-type forms of CDKs in plant cells. This would have the advantage that a
CDK
mutein in the plant cell does not have to compete with the wild-type form and
that
therefore, lower levels of CDK muteins may be sufficient so as to achieve the
desired phenotype.
It is also evident from the disclosure of the present invention, that any
combination of the above-identified strategies can be used for the generation
of
transgenic plants, which due to the presence of non-phosphorylated form of CDK
display a novel or enhanced abiotic stress tolerance. Such combinations can be
2o made, e.g., by (co-transformation of corresponding nucleic acid molecules
into
the plant cell, plant tissue or plant, or may be achieved by crossing
transgenic
plants that have been generated by different embodiments of the method of the
present invention. Likewise, the plants obtainable by the method of the
present
invention can be crossed with other transgenic plants so as to achieve a
2s combination of abiotic stress tolerance and another genetically engineered
trait,
see also infra.
In addition, the present invention also relates to transgenic plants and plant
tissue
comprising transgenic plant cells according to the invention. Said transgenic
plant
3o cell comprises at least one nucleic acid molecule or regulatory sequence as
defined above or obtainable by the method of the present invention.
Furthermore,
the present invention relates to transgenic plants and plant tissue obtainable
by
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the method of the present invention. As mentioned above, said transgenic
plants
may display various idiotypic modifications due to their abiotic stress
tolerance,
preferably display accelerated and/or enhanced plant growth, root growth
andlor
yield compared to the corresponding wild type plant.
As described before, the plant cells, plant tissue, in particular, transgenic
plants
of the invention display a certain degree of (higher) abiotic stress
resistance
compared to the corresponding wild-type plants. For the meaning of "abiotic
stress"; see supra. In a preferred embodiment of the present invention, the
io transgenic plant displays increased tolerance to osmotic stress, preferably
to salt
stress. An increase in tolerance to salt stress is understood to refer to the
capability of the transgenic plant to grow on a medium such as soil,
comprising a
higher content of salt in the order of at least about 10% compared to a medium
the corresponding non-transformed wild-type plants is capable to grow on,
which
is already provides for beneficial effects on the vitality of the plant such
as, e.g.,
improved growth. Advantageously, the transgenic plant of the invention is
capable of growing on a medium or soil comprising at least about 50%,
preferably
more than about 75%, particularly preferred at least about more than 100% and
still more preferable more than about 200% salt than medium or soil the
2o corresponding wild-type plant is capable of growing on.
In a particular preferred embodiment of the present invention, the above-
described transgenic plants are capable of growing on medium or soil
containing
40, more preferably 100, still more preferably 200, ~ and even more
2s advantageously 300 mM salt. Said salt can be for example, water soluble
inorganic salts such as sodium sulfate, magnesium sulfate, calcium sulfate,
sodium chloride, magnesium chloride, calcium chloride, potassium chloride
etc.,
salts of agricultural fertilizers and salts associated with alkaline or acid
soil
conditions. Preferably, said salt is NaCI.
Any transformed plant obtained according to the invention can be used in a
conventional breeding scheme or in in vitro plant propagation to produce more
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29
transformed plants with the same characteristics andlor can be used to
introduce
the same characteristic in other varieties of the same or related species.
Furthermore, the characteristic of the transgenic plants of the present
invention to
maintain rapidlhigh growth rates under stress conditions can be combined with
s various approaches to confer biotic or abiotic stress tolerance with the use
of
other stress tolerance genes. Some examples of such stress tolerant genes are
provided in Table 2 and see generally Holmberg and Bulow, Trends Plant Sci. 3,
61-66 (1998). Most prior art approaches which include the introduction of
various
stress tolerance genes have the drawback that they result in reduced growth
io (compared to non-transgenic controls) under normal, non-stressed
conditions,
namely stress tolerance comes at the expense of growth and productivity. This
correlation between constitutive expression of stress-responsive genes and
reduced growth rate under normal growth conditions indicates the presence of
cross talk mechanisms between stress response control and growth control.
is Therefore, making cell division (and growth) insensitive to stress control
by
preventing or removing inhibitory phosphorylation of CDK protein will also
result
in faster growth of plants that constitutively express stress-tolerance
mechanisms
under non-stressed conditions.
Furthermore, the characteristic of the transgenic plants of the present
invention to
2o display abiotic stress tolerance can be combined with various approaches to
confer to plants other stress tolerance genes, e.g., osmotic protectants such
as
mannitol, proline; glycine-betaine, water-channeling proteins, etc. Thus, the
approach of the present invention to confer abiotic stress tolerance to plants
can
be combined with prior art approaches which include introduction of various
2s stress tolerance genes; see, e.g., Table 2.
Table 2
Stress tolerance Reference
gene
pyrroline-5-carboxylateKishor, Plant Physiol. 108 (1995), 1387-1394.
synthetase
mannitol Tarczynski, Science 259 (1993), 508-510
CA 02326689 2000-10-23
WO 99/54489 PCT/EP99/02696
Stress tolerance Reference
gene
Holmstrom, Nature (1996), 683-684
fructan Pilon-Smith, Plant Physiol. 107(1995),
125-130
COR Jaglo-Ottensen, Science 280 (1998), 104-106
codA Hayashi, Plant J. 12 (1997), 133-142
w-3 fatty acid Kodama, Plant Physiol. 105 (1994), 601-605
desaturase
Delta9 desaturase Ishizaki-Nishizawa, Nat. Biotechnol. 14
(1996}, 1003
-1006
rd29A, rd17, cor6.6,Liu-Q, Plant Cell. Aug. 10 (8) (1998),
cor15a, erd10, kin11391-1406
MYB2 W099/16878
CaN-calcineurin W099I05902
water channel proteinsW098/17803
late embryogenesis W097/13843
abundant protein
HVA1
mannitol US 5,780,709
Thus, due to the findings of the present invention, it is now also possible to
produce transgenic plants which have the ability to grow under abiotic stress
s conditions and display further new phenotype characteristics compared to
naturally occurring wild-type plants, for example, due to the presence of
another
transgene. Hence, the above-described nucleic acid molecules and regulatory
sequences can be used in combination with other transgenes that confer another
phenotype to the plant. Likewise, it is possible to first confer abiotic
stress
is tolerance to a plant in accordance with the method of the invention and to
then in
an additional step transform such plant in accordance thereof with a further
nucleic acid molecule, the presence of which results in another new phenotype
characteristic of said plant. Irrespective of the actual performance of
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31
transformation, the result of the present invention displays at least two new
properties compared to a naturally occurring wild-type plant, that is
increased
tolerance to abiotic stress, in particular osmotic stress preferably to high
salinity
and; a phenotype that is due to the presence of a further nucleic acid
molecule in
s said plants. For example, said phenotype is conferred by the
(over)expression of
homologous or heterolagous genes or suppression of endogenous genes of the
plant or their gene products.
Some examples for the (over)expression of homologous or heterologous genes
and antisense inhibition and co-suppression aiming at manipulating certain
io metabolic pathways in transgenic plants are reviews in Herbers (TIBTECH 14
(1996), 198-205): Ribozymes of different kinds which are capable of
specifically
cleaving the (pre)-mRNA of a target gene are described in, e.g., EP-B1 0 291
533, EP-A10 321 201 and EP-A2 0 360 257. Selection of appropriate target sites
and corresponding ribozymes can be done as described for example in
is Steinecke, Ribozymes, Methods in Cell Biology 50, Galbraith et al. eds
Academic
Press, Inc. (1995), 449-460. An example for ribozyme mediated virus resistance
is described in Feyter (Mol. Gen. Genet. 250 {1996), 329-228). Thus, it is
immediately evident to the person skilled in the art that the method of the
present
invention can be employed to produce transgenic stress tolerant plant with any
2o further desired trait (see for review TIPTEC Plant Product & Crop
Biotechnology
13 (1995), 312-397) comprising (i) herbicide tolerance (DE-A-3701623; Stalker,
Science 242 (1988), 419), (ii) insect resistance (Vaek, Plant Cell 5 (1987),
159-
169), (iii) virus resistance (Powell, Science 232 (1986), 738-743; Pappu,
World
Journal of Microbiology & Biotechnology 11 (1995), 426-437; t_awson,
Zs Phytopathology 86 (1996) 56 suppl.), {vi) ozone resistance (Van Camp,
Biotech.
12 (1994), 165-168), {v) improving the preserving of fruits (Oeller, Science
254
(1991 ), 437.439), (vi) improvement of starch composition and/or production
(Stark, Science 242 (1992), 419; Visser, Mol. Gen. Genet. 225 (1991 ), 289-
296),
(vii) altering lipid composition (Voelker, Science 257 (1992), 72-74), (viii)
3o production of (bio)polymers (Poirer, Science 256 (1992), 520-523), (ix)
alteration
of the flower color, e.g., by manipulating the anthocyanin and flavonoid
biosynthetic pathway (Meyer, Nature 330 (1987), 667-678, WO 90/ 12084), {x)
CA 02326689 2000-10-23
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32
resistance to bacteria, insects and fungi (Duering, Molecular Breeding 2
(1996),
297-305; Strittmatter, Bio/Technology 13 ( 1995), 1085-1089; Estruch, Nature
Biotechnology 15 (1997), 137-141 ), {xi) alteration of alkaloid andlor cardiac
glycoside composition, (xii) inducing maintaining male and/or female sterility
(EP-
s A1 0 412 006; EP-A1 0 223 399; WO 93/25695); (xiii) higher longevity of the
inflorescences/flowers, and (xvi) stress resistance; see references supra,
e.g.,
those mentioned in Table 2.
Thus, the present invention relates to any plant cell, plant tissue, or plant
which
io due to genetic engineering displays abiotic stress tolerance obtainable in
accordance with the method of the present invention and comprising a further
nucleic acid molecule conferring a novel phenotype to the plant such as one of
those described above.
is As mentioned before, the combination of the approaches can be done by
crossing plants displaying the individual phenotypes referred to above. Such
plants are also part of the invention. Seeds obtained from the transformed
plants
genetically also contain the same characteristic and are part of the
invention. As
mentioned before, the present invention is in principle applicable to any
plant and
2o crop that can be transformed with any of the transformation method known to
those skilled in the art and includes those mentioned hereinbefore for
instance
corn, wheat, barley, rice, oilseed crops, cotton, tree species, sugar beet,
cassava,
tomato, potato, numerous other vegetables, fruits.
2s In yet another aspect, the invention also relates to harvestable parts and
to
propagation material of the transgenic plants according to the invention which
contain
transgenic plant cells according to the invention. Harvestable parts can be in
principle any useful parts of a plant, for example, flowers, pollen,
seedlings, tubers,
leaves, stems, fruit, seeds, roots etc. Propagation material includes, for
example,
3o seeds, fruits, cuttings, seedlings, tubers, rootstocks etc.
It is to be understood that the skilled person, aware of the above teaching,
will be
CA 02326689 2000-10-23
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33
able to apply numerous techniques to confer to a plant the capacity to
counteract the
stress-induced downregulation of cell cycle progress as is discussed above.
Instead
of or in addition to transforming plant cells with a gene coding for a CDK
mutein, it is
possible to overexpress in the plant cells CDC25 or functional analogue
thereof by
s transforming the said cells with a functional cdc25 gene under the control
of a
suitable promoter, e.g., the CaMV35S promoter, or, e.g., to transform the
plants with
nucleic acids coding for anti-sense RNA, capable to basepair with, and leading
to
cleavage of, the mRNA coding for any protein that is desired to be knocked
out, like
Wee-kinase-mRNA.
io
Thus, the present invention generally relates to the use of the above
described
nucleic acid molecules, regulatory sequences, and vectors for conferring
abiotic
stress tolerance to a plant andlor as a selectable marker for plants.
The above described nucleic acid molecules, regulatory sequences and vectors
is in accordance with the method of the invention for conferring abiotic
stress
tolerance can be used as selectable markers in plants according to other
systems
which for example employ (over)expression of enzymes or muteins thereof
capable of conferring tolerance (i.e., resistance) to plant cell killing
effects of,
e.g., herbicides. An example for such a system is the overexpression of the
Zo enzyme 5-enolpyruvlyshikimate-3-phosphate (EPSP) synthase that confers
tolerance to the herbicide glyphosphate. In a similar way, the nucleic acid
molecules, regulatory sequences and vectors described above can be used for
conferring tolerance against abiotic stress, in particular salt stress as
shown in
the appended examples. For example, transgenic plants obtained in accordance
is with the method of the present invention can be easily selected for in the
green
house on soil, which contains, for example 40 to 300 mM salt, e.g., NaCI.
As has been discussed hereinbefore, in investigating the behavior of plant
cells and
plants under conditions of abiotic stress, it could be shown in accordance
with the
3o present invention that stress-dependent downregulation of the cell division
(cell
cycle) is mediated by endogenous cellular components. Said components may
comprise cell cycle regulatory proteins that may undergo stress induced
alterations,
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34
thereby being activated or deactivated. Moreover, it could surprisingly be
shown that
one could confer to the plant the capacity to counteract or even avoid the
downregu-
lation of the cell division under conditions of abiotic stress, in particular
osmotic stress
due to, e.g., high salinity of the soil, thus enabling the plant to be
tolerant to the said
s stress conditions, by, e.g., altering, or inhibiting, or competing with, or
circumventing
the regulatory actions of, the above-mentioned endogenous cellular components.
Thus, in a still further embodiment, the present invention relates to the use
of a
nucleic acid molecule or regulatory sequence capable of counteracting stress-
induced down-regulation of cell division for the production for osmotic stress
io tolerant plants.
Furthermore, the present invention relates to the use of a plant obtainable by
the
method of the invention or a plant as described hereinbefore for culturing on
soil
with 40 mM to 300 mM salt content.
These and other embodiments are disclosed and encompassed by the
description and examples of the present invention. Further literature
concerning
any one of the methods, uses and compounds to be employed in accordance with
the present invention may be retrieved from public libraries, using for
example
2o electronic devices. For example the public database "Medline" may be
utilized
which is available on the Internet, for example under
http://www.ncbi.nlm.nih.gov/PubMed/medline.html. Further databases and
addresses, such as http:/lwww.ncbi.nlm.nih.gov/, http:l/www.infobiogen.frl,
http://www.fmi.chlbiology/research tools.html, http://www.tigr.orgl, are known
to
2s the person skilled in the art and can also be obtained using, e.g.,
http:l/www.lycos.com. An overview of patent information in biotechnology and a
survey of relevant sources of patent information useful for retrospective
searching
and for current awareness is given in Berks, TIBTECH 12 (1994), 352-364.
so The figures show:
Fig. 1: Recovery from salt stress of wild type (WT), CDC2aWT, YF2 and YF5
CA 02326689 2000-10-23
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I I neS.
(A) Control plants after seven days transfer to K1 medium
(B) Salt stressed plants after seven days transfer to K1 medium
containing 1 %NaCI
5 (C) Control plants after fourteen days transfer to K1 medium
(D) Salt stressed plants after seven days recovery from a 1 % containing
K7 medium
Fig. 2: Differential growth upon salt stress of the WT, CDC2aWT, YF2 and YF5
~o lines.
(a) Adaxial epidermal layer of the third leaf of non-stressed WT (A),
CDC2aIM' (C), YF2 (E), YF5 (G) and stressed WT (B), CDC2aWT
(D), YF2 (F), YF5 (H) plants
(b) Decrease of total surface of the third leaf upon salt stress
is (c) Epidermal cell number per leaf area unit in normal and stress
conditions
(d) Mean cell size of the third leaf in normal and salt stress conditions
(e) Elongation rates of hypocotyl upon salt stress
2o Fig. 3 Histone H1 CDK activity of stressed and non-stressed WT, CDC2aWT
and YF2 lines.
Fig. 4 Percentage of G2 Arabidopsis cells with a 4C content with and without
addition of NaCI to the medium (---. 0% NaCI; -- 0.5% NaCI).
The present invention is further illustrated by reference to the following non-
limiting examples.
so Unless stated otherwise in the examples, all recombinant DNA techniques are
pertormed according to protocols as described in Sambrook et al. (1989),
Molecular Cloning : A Laboratory Manual. Cold Spring Harbor Laboratory Press,
NY or in Volumes 1 and 2 of Ausubel et al. (1994), Current Protocols in
Molecular
Biology, Current Protocols. Standard materials and methods for plant molecular
3s work are described in Plant Molecular Biology Labfase (1993} by R.D.D.
Croy,
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36
jointly published by BIOS Scientific Publications Ltd (UK) and Blackwell
Scientific
Publications (UK).
s Example 1: Morphological alterations in response to salt stress and
correlation to the expression of cell cycle regulatory genes
In order to investigate morphological alterations in response to salt stress,
an
histochemical analysis of three known plant cell cycle regulating proteins was
io performed. Therefor, time course experiments were performed on transgenic
plants
transformed with cycfin (CycA2;1, CycB1;1) and CDK (CDC2aAt) promoter-gus
fusions respectively. Both cyclin and CDK promoters originated from
Arabidopsis
fhaliana. Ten days old Arabidopsis plants were transferred to solid media
containing
1 % NaCI ; GUS activity and morphological changes were observed after 12hrs,
i s 36hrs, 4 days, 1 week and two weeks. After 12hrs treatment the promoter
activities
declined in the apical meristem, before any morphological change was visible.
After
36 hrs growth in the presence of salt the swollen roottips showed a decrease
in
expression of all cell cycle genes concomitant with a shrinkage of the root
apical
meristem. After four days, morphological alterations in the aerial part of the
stressed
zo plant were clearly visible when compared to control. The stressed plants
were shorter
due to a less elongated hypocotyl and the leaves were smaller. During
adaptation to
stress an induction of CycA2;1 and CycB 1;1 expression in the shoot apical
meristem
could be noticed. Measurements of the length of the leaves and of the length
of the
meristematic region in the roots made after two weeks growth on salt
containing
z5 medium, demonstrated a strong reduction in comparison to control plants.
The
number of leaves initiated was significantly lower in salt stressed plants
than in
control plants. In expanding leaves of salt stressed plants no GUS staining
for
CycB1;1 nor CDC2aAt expression could be detected contrarily to control plants,
illustrating the decline in mitotic activity in these organs.
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37
Example 2: Improved tolerance to salt stress of Arabidopsis thaliana
containing a CDC2a-Y15F1T14A mutant gene under the control of a
CaMV35S promoter
s A comparative study was made between transgenic Arabidopsis plants
containing a
CDC2a-Y15F/T14A mutant gene under the control of a CaMV35S promoter (see
Hemerly, EMBO J. 14 (1995), 3925-3936) and wild-type plants, in response to
salt
stress. Arabidopsis plants (ecotype C24) were engineered containing a mutated
form
of the CDC2aAt gene in which the phosphorylation sites T14 (Threonine at amino
io acid position number 14) and the Y15 (Tyrosine at amino acid position
number 15)
were changed in A14 (Alanine at amino acid position number 14) and F15
(Phenylalanine at amino acid position number 15) under control of a CaMV35S
promoter. The overexpression of mutant CDC2a-Y15FIT14A in Arabidopsis lines
did
not show drastic changes in development. Only a tendency for loss for apical
is dominance could be noticed. Two independent transgenic Arabidopsis lines
(YF2
and YF5) overexpressing mutant CDC2aAt with non-phosphorylatable A1a14 and
Phe15 residues CDC2a Y15FIT14A were selected to study their response on salt
stress. As controls non-transformed Arabidopsis plants (C24) and transgenic
plants
carrying a construct of a non-mutated CDC2aAt gene under the control of a
2o CaMV35S promoter were included in the experiment. Plants of ten days old,
grown
on solid germination medium, were transferred to the same medium containing 1
NaCI and their further growth and development was observed compared to the
control plants. Both mutant lines displayed an improved tolerance to salinity
which
was phenotypically visible. The salt stressed mutant plants had bigger and
more
2s elongated leaves than control non transformed plants and plants
overexpressing the
wild type CDC2aAt gene. Furthermore, compared to wild type plants (WT) and
transgenic Arabidopsis plants ectopically expressing the wild type form of
CDC2aAt (CDC2aWT), the YF lines displayed an enhanced shoot growth after
cultivation in the presence of NaCI. Additionally the YF lines recovered
faster
3o upon release from salinity than the CDC2aWT and WT plants (Fig. 1 ). As
mentioned before, CDC2aWT and YF lines contain respectively the wild type and
mutated CDC2aAt form in which Thr14 and Tyr15 were substituted for A1a14 and
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38
Phe15 residues, placed under control of a constitutive CaMV 35S promoter
(Hemerly, EMBO J. 14 (1995), 3925-3936). Arabidopsis seedlings (ecotype C24),
grown for ten days in sterile conditions on solid K1 germination medium
(Valvekens, Proc. Natl. Acad. Sci. USA 85 (1988), 5536-5540), were transferred
s for seven days to the same medium to which 1 % NaCI was added. To release
them from salinity they were transferred again to a solid K1 medium without
NaCI.
The picture was taken after seven days recovery.
To quantify growth, leaf epidermal cell numbers and hypocotyl lengths were
io measured. Ten days old Arabidopsis seedlings, grown in vitro on solid K1
medium, were transferred to the same medium with or without the addition of 1
NaCI respectively for seven days. Measurements of epidermal cell numbers and
individual cell surfaces were obtained from digitafized camera-lucida drawings
made from the adaxial leaf surface of the third leaf, using D!C optics on a
Leitz
i5 Diaplan microscope (Leitz, Wetzlar, Germany). The third leaf was chosen
based
on the expression of the CycB9; l:gus marker, representative for actively
dividing
cells (Ferreira, Plant Cell 6 (1994), 1763-1774), at the moment of transfer to
the
saline environment. A monolayer of epidermal cells was visualized in whole
mounted leaves that had been fixed in 100% methanol, and cleared in lactic
acid.
Zo Image analyses were performed with the public domain Scion Image Program
(version f3-3b, Scion Corporation). The reported means for each genotype are
the
mean value of three independent measurements from three different leaves.
Hypocotyllengths of at least ten plants per genotype were measured by means of
a Zeiss stereomicroscope. All data were incorporated in histograms by use of
the
25 Excel) Microsoft Program. In all tested lines, salt stress caused a
decrease in the
total leaf area and epidermal cell number (Fig. 2a,b). Interestingly, the
epidermal
cell density was significantly higher in YF2 and YF5 leaves (Fig. 2c),
implying that
more cell divisions had occurred during stress. In agreement, the mean
epidermal
cell size was smaller in YF than in WT and CDC2aWT fines (Fig. 2d),
3o comparable to the yeast wee phenotype. Moreover, the stomata) complexes,
formed by division from meristemoids (Yang, Plant Cell 7 (1995), 2227-2239),
were more developed in stressed YF than in control lines (CDC2aAtWT and WT)
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39
(Fig. 2a). Also the hypocotyl growth was reduced after transfer of the
seedlings to
a saline environment in 1NT and CDC2aWT lines while this was not the case in
the YF lines (Fig. 2e). These data suggest that, contrarily to WT and CDC2aWT,
elongation is not inhibited or retarded upon salt stress in the YF lines since
cell
s divisions are not significantly involved in hypocotyl growth of Arabidopsis
seedlings (Gendreau, Plant Physiol. 114(1 ) (1997), 295-305).
In order to correlate the observed phenotypes with CDC2 activity, the H1
kinase
activities of the CDK complexes were determined (Fig. 3). Arabidopsis
seedlings
io were grown for ten days in sterile conditions on ~Iters on solid K1 medium
and
subsequently transferred to liquid K1 medium for two hours. The plants were
then
transferred to fresh liquid K1 medium with or without the addition of 1 %
NaCI.
Samples were taken after 3 and 24hrs and H1 kinase activities were measured
(Azzi, Eur. J. Biochem. 203 (1992), 353-360). Protein concentrations were
is determined using the Protein Assay kit (Bio-Rad, Munich, Germany), using
Bovine Albumin Serum as a standard. Histone H1 kinase assays were performed
with the CDK complexes purred from crude extracts by p13SUC1 affinity (Azzi,
Eur. J. Biochem. 203 (1992), 353-360), using 50~,g of total proteins and 201
of a
50% suspension of p13s"°'-agarose beads. Phosphorylated histone H1 was
ao visualized through phosphorimager scanning (Molecular, Eugene, OR). CDC2
like kinase activities were rapidly decreasing upon salt stress in WT, but
remained high in the CDC2aWT and YF2 lines. The growth discrepancies
between the CDC2aWT and YF plants upon salinity were however not reflected
in the kinase activity measurements. The quantitative detection limit of the
2s method used for the determination of the kinase activities (Azzi, Eur. J.
Biochem.
203 (1992), 353-360), may be restrictive to visualize the differences in
kinase
activities between the transgenic lines.
Hence, the enhanced growth of the YF plants demonstrates the importance of a
regulatory control mechanism upon abiotic stress such as salt stress that
inhibits
3o CDC2aAt activity by alteratian of phosphorylation status of the CDC2aAt
complex. The activity of CDC2aAt is maximal at the G1IS and G2IM transitions,
suggesting a functional involvement at both checkpoints.
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In order to find out at which transition point this stress-induced control
mechanism may be operative, the nuclear content of Arabidopsis cell
suspensions was analyzed after addition of NaCI. The Arabidopsis cell line
s (Axelos, Mol. Gen. Genet. 219 (1989), 106-112) was subcultured every seven
days in Gamborg B5 medium (Sigma) supplemented with 0.2 mgll a-naphtalenic
acid. NaCI (0.5 %) was added 48hrs after subculturing in fresh medium. To
determine the DNA nuclear content of the cells, 1 ml of the cell suspensions
was
centrifuged and the nuclei were released (Glab, FEES Lett. 353(2) (1994), 207-
io 211 ) before flow cytometry analysis (Biorad, Bryte HS). It was found that
salinity
caused a G2 phase arrest in Arabidopsis cell suspensions cultures (Fig. 4). A
G2/M arrest has also been reported in dehydrated wheat leaves (Schuppler,
Plant Physiol. 117 (1998), 667-678) and in yeast upon hyperosmolarity
(Shiozaki
and Russet, Nature 378 (1995), 739-743). In wheat leaves Tyr phosphorylation
of
is CDC2 like proteins has been speculated (Schuppler, Plant Physiol. 117
(1998),
667-678) as a consequence of water stress but no clue whatsoever has been
made about specific CDC2 proteins nor phosphorylation sites involved in this
process.
2o As has been demonstrated above, for the first time genetic evidence is
provided
for a link between the regulation of cell cycle progression and growth
inhibition by
abiotic stress, in particular salt stress. The data obtained in accordance
with the
present invention demonstrate that plants have conserved the CDC2 Tyr15
checkpoint control at the G2/M border coupled to environmental signals.
2s Mammals contain at least three distinct CDC25 Tyr15 phosphatases of which
two
are functionally implicated in the G2/M transition (Sadhu, Proc. Natl. Acad.
Sci.
USA 87 (1990), 5139-5143; Galaktionov, Cell 67 (1991 ), 1181-1194. Cell 57,
1181-1194; Nagata, New Biol. 10 { 1991 ), 959-968; Suto, EMBO ,!. 13 ( 1994),
1549-1556). In fission yeast MIK1 acts cooperatively with the WEE1 protein
so kinase in the inhibitory Tyr15 phosphorylation of CDC2 (Lundgren, Celi 64
(1991), 1111-1122). Besides WEE1, a MYT1 protein kinase that phosphorylates
CDC2 on both Thr14 and Tyr15, has been discovered in Xenopus (Mueller,
CA 02326689 2000-10-23
WO 99/544$9 PCT/EP99/02696
41
Science 270 (1998), 86-89). Considering the evolutionary conservative
character
of the cell cycle regulation, similar phosphatase and kinase relatives are
expected to exist in plants. Genetic manipulation of these protein
phosphatases
and kinases that regulate CDK activity might contribute to engineer abiotic
stress
s tolerant an in particular osmotolerant plants in the future.
In summary, the experiments performed in accordance with the present invention
demonstrate that a stress-induced phosphorylation of a cyclin dependent kinase
results in an inhibitory growth response. As has been discussed in the
to embodiments hereinbefore, this finding opens up the way for several
benef;cial
applications in plant science and agriculture.
Example 3: Experimental setup to defne other stress conditions
To verify if plants are also responding differently to other environmental
stresses
than salinity stress, experiments can be designed by the person skilled in the
art
in accordance with methods known in the art (see, e.g., references cited
hereinbefore), for example exposure of the plants to
a o - Cold stress: 2-5°C
- Heat stress: 28-40°C
- Drought stress: withholding water for 5-14 days or withhold water for 5
days,
supply water for 2 days and withhold water for another 5 days
- Freezing stress: -6 to -4°C
2s - Growth under stress conditions: Transfer of ten-days-old Arabidopsis
seedlings (WT, CDC2aAtWT, YF2. and YF5) grown in sterile conditions on
solid K1 medium to a growth chamber where the temperature is lowered to, for
example, 4°C (cold shock) or increased to 28°C (heat shock).
Observations
can be made daily after 4 to 10 days transfer to observe growth differences.
so - Recovery of stress conditions: Release of stressed plants (cold or heat
shock)
described above after 4 to ten days to normal growth conditions. Observations
can made daily up to 15 days after the moment of release.
CA 02326689 2000-10-23
WO 99/54489 PCT/EP99/02696
42
The period of exposure to a stress and the empirical values of the stress
(e.g., 40°C
vs. 6°C) will depend upon the species of plant being tested, however, a
person
skilled in the art is able to easily determined these periods or values.
s
It will be clear that the invention may be practiced otherwise than as
particularly
described in the foregoing description and examples. Numerous mod~cations and
variations of the present invention are possible in light of the above
teachings and,
therefore, are within the scope of the appended claims.
io
The entire disclosure of each document cited (including patents, patent
applications,
journal articles, abstracts, laboratory manuals, books, or other disclosures)
in the
Background of the Invention, Detailed Description, and Examples is hereby
incorporated by reference.
