Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
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METHOD OF MODIFYING PLANT MORPHOLOGY,
BIOCHEMISTRY AND PHYSIOLOGY
FIELD OF THE INVENTION
The present invention relates generally to a method of modifying plant
morphological,
biochemical and physiological properties or characteristics, such as one or
more
environmental adaptive responses and/or developmental processes, including but
not
limited to the initiation, promotion, stimulation or enhancement of cell
division and/or
seed development and/or tuber formation and/or shoot initiation and/or
bushiness
and/or dwarfism and/or pigment synthesis, and/or the modification of
source/sink
relationships, and/or the modification of root growth and/or the inhibition of
apical
dominance and/or the delay of senescence, said method comprising expressing a
cell
cycle control protein, in particular Cdc25 phosphoprotein phosphatase, in the
plant,
operably under the control of a regulatable promoter sequence such as a cell-
specific
promoter, tissue-specific promoter, or organ-specific promoter sequence.
Preferably,
the characteristics modified by the present invention are cytokinin-mediated
and/or
gibberellin-mediated characteristics. The present invention extends to genetic
constructs which are useful for performing the inventive method and to
transgenic
plants produced therewith having altered morphological and/or biochemical
and/or
physiological properties compared to their otherwise isogenic counterparts.
GENERAL
Those skilled in the art will be aware that the invention described herein is
subject to
variations and modifications other than those specifically described. It is to
be
understood that the invention described herein includes all such variations
and
modifications. The invention also includes all such steps, features,
compositions and
compounds referred to or indicated in this specification, individually or
collectively, and
any and all combinations of any two or more of said steps or features.
Throughout this specification, unless the context requires otherwise the word
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"comprise", and variations such as "comprises" and "comprising", will be
understood
to imply the inclusion of a stated integer or step or group of integers or
steps but not
the exclusion of any other integer or step or group of integers or steps.
Bibliographic details of the publications referred to by author in this
specification are
collected at the end of the description.
As used herein, the term "derived from" shall be taken to indicate that a
particular
integer or group of integers has originated from the species specified, but
has not
necessarily been obtained directly from the specified source.
BACKGROUND TO THE INVENTION
Development and environmental adaptation are highly regulated processes in
plants.
These processes are not cell-autonomous but rather involve extensive
communication
between different parts of the plant. Amongst the most important mobile
signals
involved in this long-distance communication are plant hormones such as
auxins,
cytokinins, abscisic acid, gibberellins, and ethylene. Other signals, so far
not defined
as plant hormones, include salicyclic acid, jasrronic acid and
brassinosteroids.
There are plethora of data showing that the external application of plant
hormones has
profound effects on development, metabolism and environmental fitness. For
example, the external application of cytokinins produces a variety of
morphological,
biochemical and physiological effects in plants, including the stimulation of
organogenesis, shoot initiation from callus cultures, release of lateral buds
from apical
dominance, dwarf growth, alteration of source/sink relationships, stimulation
of pigment
synthesis, inhibition of root growth, and delay of senescence. Additionally,
exogenous
cytokinin application following anthesis in cereals enhances grain set and
yield and the
phase of nuclear and cell division in the developing endosperm of cereal
grains is
accompanied by a peak of cytokinin concentration, suggesting a role for
cytokinins in
grain development in cereals (Herzog, 1980; Morgan et al., 1983). Cytokinins
have
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also been implicated in promoting the initiation of tuber formation in potato
(International Patent Publication No. WO 93/07272) and in improving the
resistance
of potato plants to insects (United States Patent No. 5, 496, 732) and in
inducing male
sterility and partial female sterility in tobacco plants (European Patent No.
EP-A-
334,383).
The effect of cytokinin on plant development and morphology may be attributed,
at
least in part, to modified biochemistry of the plant, such as a modification
to the
source/sink relationship in the plant or plant part.
Attempts to modify plant cytokinin-mediated and/or gibberellin-mediated growth
and
developmental responses employ the exogenous application of cytokinins and/or
gibberellins respectively. Such approaches are costly and produce undesirable
pleiotropic side-effects on the plant tissue.
Other approaches to modifying plant cytokinin-mediated growth and
developmental
responses employ the ectopic expression of an introduced bacterial
isopentenyladenosine transferase (IPT) gene (International Patent Publication
No. WO
93/07272; United States Patent No. 5, 496, 732; United States Patent No. 5,
689, 042)
under the control of a strong constitutive promoter sequence, developmentally-
regulated promoter sequence or hormonally-inducible promoter sequence.
Alternatively, plant cytokinin-mediated growth and developmental responses
have
been modified by the ectopic expression of the Agrobacterium rhizogenes RoIC
gene
(European Patent No. EP-A-334,383).
Previously, it had been shown that constitutive expression of yeast Cdc25 in
tobacco
resulted in precocious flowering, more flowers per flowering head and the
presence of
"petalless" flowers alongside normal ones. Other changes in development
included
the positioning of the leaves. When yeast Cdc25 was expressed under control of
an
inducible promoter, a greater frequency of lateral root formation was
observed. Yet,
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the importance of Cdc25 in cytokinin action was so far not recognised (Patent
WO
92/09685; Patent WO 93/122239 and, as a consequence, the presented invention
and
its embodiments could not be envisaged.
SUMMARY OF THE INVENTION
In work leading to the present invention, the present inventors sought to
develop a
method of producing specific targeted modifications to plant morphology,
biochemistry
and physiology, in particular specific target modifications to cytokinin-
mediated and
gibberellin-mediated plant growth and development, thereby avoiding the
problem of
pleiotropy associated with the prior art.
Surprisingly, the inventors discovered that the targeted ectopic expression of
a cell
cycle control protein in particular cells, tissues or organs of the plant
would produce
localised specific modifications to plant morphology, biochemistry and
physiology,
compared to otherwise isogenic non-transformed plants.
More particularly, the inventors have discovered that the cytokinin-mediated
or
gibberellin-mediated induction of mitosis in plants can be obtained by the
expression
of the yeast Cdc25 phosphoprotein phosphatase therein.
The cytokinin-mediated induction of mitosis by Cdc25 is shown in Example 2.
Whilst
not being bound by any theory or mode of action, it is likely that the ectopic
expression
of yeast Cdc25 phosphatase in plants releases the inhibition of cdc2 activity,
which is
key enzyme in the control of the cell cycle, and as a consequence, causes
isolated
cells to enter mitosis. The Cdc25 phosphatase is an intracellular protein,
which, unlike
exogenously-applied cytokinins or cytokinins produced by ectopic expression of
ipt or
rolC genes, will only exert a localised effect at the site of protein
synthesis. This
observation has led the present inventors to develop methods for controlled
expression
of yeast Cdc25 in particular cells, tissues and organs of plants, for the
purposes of
modifying cytokinin-mediated plant morphology and/or biochemistry and/or
physiology,
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and to facilitate the selection of specific cells, tissues and organs which
exhibit
cytokinin-mediated morphological characteristics and/or biochemical
characteristics
and/or physiological characteristics.
Accordingly, one aspect of the invention provides a method of modifying plant
morphology and/or biochemistry and/or physiology comprising expressing in
particular
cells, tissues or organs of a plant, a genetic sequence encoding a cell cycle
control
protein operably under the control of a regulatable promoter sequence selected
from
the list comprising cell-specific promoter sequences, tissue-specific promoter
sequences, and organ-specific promoter sequences.
In a particularly preferred embodiment of the invention, the cell cycle
control protein
is the yeast Cdc25 phosphoprotein phosphatase or a biologically-active
homologue,
analogue or derivative thereof. The present invention clearly contemplates the
use of
functional homologues of the fission yeast Cdc25 protein, based upon the
evidence
provided herein for the presence of Cdc25-like activity and Cdc25-like protein
in
tobacco (Example 3). Accordingly, the present invention is not limited in
application to
the use of nucleotide sequences encoding the fission yeast Cdc25 protein.
Preferred embodiments of the invention relate to the effects) of cytokinins
and/or
gibberellins on plant morphology and architecture. With respect to cytokinins,
the
present invention clearly contemplates the broad application of the inventive
method
to the modification of a range of cellular processes, including but not
limited to the
initiation, promotion, stimulation or enhancement of cell division and/or seed
development and/or tuber formation and/or shoot initiation and/or bushiness
and/or
dwarfism and/or pigment synthesis, and/or the modification of source/sink
relationships, and/or the modification of root growth and/or the inhibition of
apical
dominance and/or the delay of senescence. With respect to gibberellins, the
present
invention clearly contemplates the broad application of the inventive method
to the
modification of a range of cellular processes, including but not limited to
the initiation,
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promotion, stimulation or enhancement of cell division and/or seed development
and/or tuber formation and/or shoot initiation and/or bushiness and/or
dwarfism
and/or pigment synthesis, and/or the modification of source/sink
relationships, and/or
the modification of root growth and/or the inhibition of apical dominance
and/or the
delay of senescence. In this regard, the identification of substrates of Cdc25
phosphatase other than cdc2 will also reveal the mechanism by which Cdc25 is
linked
to many cellular processes other than cell division.
In one preferred embodiment of the present invention, the yeast Cdc25 protein
or a
homologue, analogue or derivative thereof, or a modified substrate of Cdc25
that
mimics the effect of Cdc25 is expressed operably under the control of a
promoter
derived from a stem-expressible gene, to increase the strength and thickness
of a
plant stem to confer improved stability and wind-resistance on the plant.
In another preferred embodiment of the present invention, the yeast Cdc25
protein or
a homologue, analogue or derivative thereof, or a modified substrate of Cdc25
that
mimics the effect of Cdc25 is expressed in a tuber-forming plant operably
under the
control of a promoter derived from a stem-expressible gene or tuber-
expressible gene,
to increase improve tuber production in the plant.
In another embodiment of the present invention, the yeast Cdc25 protein or a
homologue, analogue or derivative thereof, or a modified substrate of Cdc25
that
mimics the effect of Cdc25 is expressed in a tree crop plant such as, but not
limited to,
Eucalyptus spp. or Populus spp., operably under the control of a promoter
derived
from a gene that is expressed in vascular tissue and/or cambium cells, to
increase
lignin content therein. Without being bound by any theory or mode of action,
the
ectopic expression of Cdc25 under control of a promoter that is operable in
vascular
tissue and preferably, in cambial cells, will produce thick-stemmed plants and
a higher
ratio of vascular tissue-to-pith cells within the stem, thereby resulting in
more lignin
production. Within the vascular tissue, cambial cells contain the highest
levels of
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auxins and are therefore the preferential tissue for Cdc25 overproduction.
In yet another preferred embodiment of the present invention, the yeast Cdc25
protein
or a homologue, analogue or derivative thereof, or a modified substrate of
Cdc25 that
mimics the effect of Cdc25 is expressed operably under the control of a
promoter
derived from a seed-expressible gene, to increase seed production in plants,
in
particular to increase seed set and seed yield. More preferably, the promoter
is
operable in the endosperm of the seed, in which case the combination of the
cell cycle-
control protein and endosperm-expressible promoter provides the additional
advantage
of increasing the grain size and grain yield of the plant.
In yet another preferred embodiment of the present invention, the yeast Cdc25
protein
or a homologue, analogue or derivative thereof, or a modified substrate of
Cdc25 that
mimics the effect of Cdc25 is expressed operably under the control of a
promoter
derived from a meristem-expressible gene or a shoot-expressible gene or a root-
expressible gene, to reduce apical dominance and/or to promote bushiness of
the
plant and/or to increase or enhance the production of lateral roots.
In still another preferred embodiment of the present invention, the yeast
Cdc25 protein
or a homologue, analogue or derivative thereof, or a modified substrate of
Cdc25 that
mimics the effect of Cdc25 is expressed operably under the control of a
promoter
derived from a leaf-expressible gene, to prevent or delay or otherwise reduce
leaf
chlorosis and/or leaf necrosis.
In a further preferred embodiment of the present invention, the yeast Cdc25
protein or
a homologue analogue or derivative thereof, or a modified substrate of Cdc25
that
mimics the effect of Cdc25 is expressed under the control of a promoter that
is
operative in meristem tissue of grain crops, to stimulate cell division in the
intercalary
meristem of the youngest stem internode and produce greater elongation of the
stem
and/or to generate a more extensive photosynthetic canopy.
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Those skilled in the art will recognise that the present invention may be
applied to the
selection of any cell, tissue, organ or whole organism that expresses exhibit
cytokinin-
mediated and/or gibberellin-mediated morphological characteristics and/or
biochemical
characteristics and/or physiological characteristics, from a background of
cells, tissues,
organs or whole organisms that do not exhibit such characteristics.
Accordingly, a second aspect of the present invention provides a method of
detecting
or identifying transformed or transfected plant cells, tissues or organs that
are
hormone-dependent, comprising expressing the yeast Cdc25 protein or a
homologue,
analogue or derivative thereof, or a modified substrate of Cdc25 that mimics
the effect
of Cdc25 in said plant cell, tissue or organ operably under the control of an
plant-
expressible inducible promoter, preferably a chemically-inducible promoter for
a time
and under conditions sufficient for hormone-mediated cell division and/or
hormone-
mediated tissue differentiation to occur. In an alternative embodiment, the
Cdc25
protein is expressed under the operable control of a plant-expressible
constitutive
promoter sequence, wherein said promoter sequence in operable connection with
a
nucleotide sequence encoding Cdc25 are integrated into a transposable element
to
facilitate hormone-mediated cell division and/or hormone-mediated tissue
differentiation only in those cells which also contain the transposable
element.
As used herein, the term "hormone-dependent" means any cell, tissue or organ
that
requires the exogenous application of a gibberellin or cytokinin to facilitate
or produce
cell division and/or cell proliferation in primary culture in vitro.
A third aspect of the invention provides a genetic construct or vector
comprising a
nucleotide sequence that encodes a cell cycle control protein operably under
the
control of a regulatable promoter sequence selected from:
(i) a plant-expressible cell-specific promoter sequence, plant-expressible
tissue-specific promoter sequence, or a plant-expressible organ-specific
promoter sequence; and
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(ii) a plant-expressible constitutive promoter sequence, wherein the
nucleotide sequence encoding the Cdc25 protein and the plant-expressible
constitutive promoter sequence are integrated into a transposable element.
Preferably, the genetic construct or vector according to this aspect of the
invention is
suitable for expression in a plant cell, tissue, organ or whole plant and more
preferably,
the subject genetic construct or vector is suitable for introduction into and
maintenance
in a plant cell, tissue, organ or whole plant.
A fourth aspect of the invention provides a plant cell, tissue, organ or whole
plant that
has been transformed or transfected with an isolated nucleic acid molecule
that
comprises a nucleotide sequence which encodes a cell cycle control protein,
wherein
the expression of said nucleotide sequence is placed operably under the
control of a
plant-expressible cell-specific promoter sequence, plant-expressible tissue-
specific
promoter sequence, a plant-expressible organ-specific promoter sequence, or a
plant-
expressible constitutive promoter sequence such that said plant-expressible
constitutive promoter sequence and said nucleotide sequence encoding a cell
cycle
control protein are integrated into a transposable genetic element.
This aspect of the invention extends to cdc2 reproducing said primary
transformants/transfectants.
BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1-1 is a copy of a photographic representation of a northern blot
hybridisation
showing the induction of Cdc25 mRNA in tobacco cells containing a
dexamethasone-
inducible Cdc25 gene, in the absence of exogenous cytokinin. Prior to
induction, cells
were brought to arrest at the cytokinin control point in late G2 phase by
culture without
hormone and then with auxin only. Total RNA was extracted from tobacco cells
either
in the absence of added dexamethasone (lane 0), or after 12 h induction with
0.01 NM,
or 0.10 NM, or 1.00 NM, or 10.00 NM dexamethasone and then loaded onto agarose
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gels (60 ~cg aliquots RNA per lane), transferred to membrane support and
probed with
a Cdc25-specific probe.
Figure 1-2 is a copy of a photographic representation of a western blot
showing the
induction of p67~d°ZS protein in tobacco cells containing a
dexamethasone-inducible
Cdc25 gene, in the absence of exogenous cytokinin. Prior to induction, cells
were
brought to arrest at the cytokinin control point in late G2 phase by culture
without
hormone and then with auxin only. Total protein was extracted from tobacco
cells
either in the absence of added dexamethasone (lane 1), or after 12 h induction
with
0.01 NM dexamethasone (Lane 2), or 0.10 NM dexamethasone (Lane 3), or 1.00 NM
dexamethasone (Lane 4), or 10.00 NM dexamethasone (Lane 5) and then loaded
onto
SDS/polyacrylamide gels (50 ~cg aliquots total soluble protein per lane),
transferred to
membrane support and probed with antibody specific for the Cdc25-specific
probe.
p6~cd~2s was detected by western blot of 50 ~cg aliquots of total soluble
~~6°l
protein.
Figure 1-3 is a copy of a graphical representation showing the induction of
cell division
in culture, as measured by an increase in cell number, for tobacco cells
transformed
with a dexamethasone-inducible Cdc25 gene, in the absence of exogenous
cytokinin.
Prior to induction, cells were brought to arrest at the cytokinin control
point in late G2
phase by culture without hormone and then with auxin only. Cell numbers were
determined either in the absence of added dexamethasone, or after 12 h
induction with
0.01-10.00 NM dexamethasone. Data were also obtained for both transformed
cells (O)
and for control non-transformed cells (o) grown under identical culture
conditions.
Figure 2-1 is a copy of a photographic representation showing the activity of
Cdc25
phosphatase (Cdc25) and cdc2 histone kinase (cdc2) in transgenic tobacco cells
containing a dexamethasone-inducible Cdc25 gene and progressing from the late
G2
phase hormonal control point into division, that have either not been induced
with 0.1
,uM dexamethasone (-D), or alternatively, that have been induced with 0.1 ~cM
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dexamethasone (+D). The activity of Cdc25 was measured by activation of the
tyrosine- phosphorylated cdc2 enzyme substrate as determined by assaying for
phosphorylation of H1 histone by H1 histone kinase. The Cdc25 enzyme from
cells
induced for 6 hours with dexamethasone was purified using antibodies against
authentic fission yeast Cdc25 protein, or alternatively, using preimmune serum
(lane
marked p-i) or an antibody that had been pre-competed with repeat-freeze-thaw
inactivated GST-Cdc25 fusion protein (lane marked p-c). The cdc2 kinase from
cells
induced for 12 h with dexamethasone was purified with antibody, or antibody
that had
been pre-competed with 0.1 mM antigen (lane marked p-c), and assayed by
phosphorylation of H1 histone.
Figure 2-2 is a graphical representation showing the change in activities of
Cdc25
phosphatase (o) and cdc2 histone kinase (O) in transgenic tobacco cells
containing
a dexamethasone-inducible Cdc25 gene progressing from the late G2 phase
hormonal
control point into division and following induction with 0.1 NM dexamethasone.
The
activities of Cdc25 phosphatase and cdc2 histone kinase were measured as
described
for Figure 2-1.
Figure 2-3 is a graphical representation showing the change in cell number
(cells/ml
x 106) of transgenic and non-transgenic tobacco cells containing a
dexamethasone-
inducible Cdc25 gene, progressing from the late G2 phase hormonal control
point into
division and following induction with 0.1 NM dexamethasone or cytokinin. Data
show
cell number for both transgenic cells induced using dexamethasone (D) or
cytokinin
(o), and for non-transgenic cells induced using dexamethasone (O).
Figure 2-4 is a graphical representation showing the change in activities of
Cdc25
phosphatase (o) and cdc2 histone kinase (O) in transgenic tobacco cells
containing
a dexamethasone-inducible Cdc25 gene, progressing from the late G2 phase
hormonal control point into division and following induction with cytokinin in
the
absence of added dexamethasone. The activities of Cdc25 phosphatase and cdc2
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histone kinase were measured as described for Figure 2-1.
Figure 2-5 is a graphical representation showing the change in activity of
cdc2 histone
kinase in transgenic tobacco cells containing a dexamethasone-inducible Cdc25
gene,
progressing from the late G2 phase hormonal control point into division and
following
their stimulation with cytokinin. The cdc2 histone kinase was purified using
p13s~°,
beads and treated with GST-Cdc25 fusion protein that had been produced in
Escherichia coli cells. Data indicate the cdc2 activity before Cdc25 treatment
(O), and
after treatment (~) with cytokinin.
Figure 2-6 is a copy of a photographic representation showing the activation
of cdc2
histone kinase by Cdc25 phosphatase in transgenic tobacco cells containing a
dexamethasone-inducible Cdc25 gene, prior to stimulation with cytokinin (lanes
1-3)
or following 3 hours stimulation with cytokinin (lanes 4-6). Detectable cdc2
activity was
observed in control samples that had been incubated without added Cdc25 (lanes
1
and 4), or following incubation with (i) immunoprecipitated Cdc25 that had
been
derived from non-transgenic tobacco cells induced with cytokinin for 6 hours
(lanes
2 and 5); or (ii) Cdc25 derived from transgenic tobacco cells containing a
dexamethasone-inducible Cdc25 gene that had been induced with dexamethasone
for
6 hours (lanes 3 and 6). The activity of cdc2 histone kinase was measured as
described for Figure 2-1. Detection of Cdc25 activity in the immuno-recovered
fraction
derived from non-transgenic cells indicates the presence of a plant-encoded
Cdc25.
Figure 2-~ is a copy of a photographic representation showing the presence of
phosphorylated tyrosine in cdc2a (arrow) following induction of transgenic
tobacco
cells containing a dexamethasone-inducible Cdc25 gene with dexamethasone. The
cdc2a protein was immuno-precipitated with purified antibody, or with antibody
precompeted with repeat-freeze-thaw inactivated GST-Cdc25 (lane marked p-c).
The
upper band indicated in the Figure represents excess IgG.
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Figure 3 is a copy of a photographic representation of a western blot showing
purified
plant-derived Cdc25 protein. The arrow indicates the plant Cdc25 polypeptide.
Anti-
GST-Cdc25 antibody at a dilution of 1:500 in buffered saline was used to probe
affinity-
purified plant Cdc25 protein alone (lane 1 ) or affinity-purified plant Cdc25
protein
S following incubation for 1 hour with 0.1 mM GST-Cdc25 fusion protein.
Molecular
weight markers indicating the molecular mass (kDa) of proteins are indicated
at the
left of the Figure.
Figure 4 is a copy of a photographic representation showing the cytokinin-
dependent
proliferation of tobacco cells in culture. Cell proliferation was detected by
the
incorporation of BrdU into nuclear DNA of excised tobacco pith tissue primary
culture
on MS medium either without added hormone (panels a,b), or supplemented with
5.4
NM NAA (panels c,d) or with 0.56 NM BAP (panels e,f) or 5.4 NM NAA plus 0.56
pM
BAP (panels g,h). Cell cultures shown in panels a, c, e, and g have been
stained with
DAPI, to detect nuclei. Cell cultures shown in panels b, d, f, and h have been
incubated with BrdU, and BrdU-containing DNA has been detected by fluorescence
of antibody specific for BrdU-containing DNA.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
One aspect of the invention provides a method of modifying one or more plant
morphological and/or biochemical and/or physiological characteristics
comprising
expressing in one or more particular cells, tissues or organs of a plant, a
cell cycle
control protein operably under the control of a regulatable promoter sequence
selected
from the list comprising cell-specific promoter sequences, tissue-specific
promoter
sequences, and organ-specific promoter sequences.
Preferably, the plant morphological, biochemical or physiological
characteristic which
is modified is a cytokinin-mediated or a gibberellin-mediated characteristic.
The word "modify" or variations such as "modifying" or "modified" as used
herein with
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reference to any specified integer or group of integers shall be taken to
indicate that
said integer is altered by the performance of one or more steps pertaining to
the
invention described herein, compared to said integer in the absence of such
performance.
Accordingly, by "modifying one or more plant morphological and/or biochemical
and/or
physiological characteristics" is meant that one or more morphological and/or
biochemical and/or physiological characteristics of a plant is altered by the
performance of one or more steps pertaining to the invention described herein.
"Plant morphology" or the term "plant morphological characteristic" or similar
term will
be understood by those skilled in the art to refer to the external appearance
of a plant,
including any one or more structural features or combination of structural
features
thereof. Such structural features include the shape, size, colour, texture,
arrangement,
and patternation of any cell, tissue or organ or groups of cells, tissues or
organs of a
plant, including the root, leaf, shoot, petiole, trichome, flower, petal,
stigma, style,
stamen, pollen, ovule, seed, embryo, endosperm, seed coat, aleurone, fibre,
cambium,
wood, heartwood, parenchyma, aerenchyma, seive element, phloem or vascular
tissue, amongst others.
"Plant biochemistry" or the term "plant biochemical characteristic" or similar
term will
be understood by those skilled in the art to refer to the metabolic and
catalytic
processes of a plant, including primary and secondary metabolism and the
products
thereof, including any small molecules, macromolecules or chemical compounds,
such
as but not limited to starches, sugars, proteins, peptides, enzymes, hormones,
growth
factors, nucleic acid molecules, celluloses, hemicelluloses, calloses,
lectins, fibres,
pigments such as anthocyanins, vitamins, minerals, micronutrients, or
macronutrients,
that are produced by plants.
"Plant physiology" or the term "plant physiological characteristic" or similar
term will be
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understood to refer to the functional processes of a plant, including
developmental
processes such as growth, expansion and differentiation, sexual development,
sexual
reproduction, seed set, seed development, grain filling, asexual reproduction,
cell
division, dormancy, germination, light adaptation, photosynthesis, leaf
expansion, fibre
production, secondary growth or wood production, amongst others; responses of
a
plant to externally-applied factors such as metals, chemicals, hormones,
growth
factors, environment and environmental stress factors (eg. anoxia, hypoxia,
high
temperature, low temperature, dehydration, light, daylength, flooding, salt,
heavy
metals, amongst others), including adaptive responses of plants to said
externally
applied factors,
The word "express" or variations such as "expressing" and "expression" as used
herein
shall be taken in their broadest context to refer to the transcription of a
particular
genetic sequence to produce sense or antisense mRNA or the translation of a
sense
mRNA molecule to produce a peptide, polypeptide, oligopeptide, protein or
enzyme
molecule. In the case of expression comprising the production of a sense mRNA
transcript, the word "express" or variations such as "expressing" and
"expression" may
also be construed to indicate the combination of transcription and translation
processes, with or without subsequent post-translational events which modify
the
biological activity, cellular or sub-cellular localization, turnover or steady-
state level of
the peptide, polypeptide, oligopeptide, protein or enzyme molecule.
As used herein, the term "cell cycle control protein" shall be taken to refer
to a peptide,
polypeptide, oligopeptide, enzyme or other protein that is involved in
controlling or
regulating the cell cycle of a cell, tissue, organ or whole organism and/or
DNA
replication therein. In this regard, those skilled in the art will recognise
that the "cell
cycle" refers to the growth cycle of an individual cell, including the G1
phase that is
entered after the ploidy of the cell has been halved by mitosis, the S phase
in which
each chromatid is duplicated, the G2 phase in which duplication is complete
but
mitosis has not been initiated, and the M phase in which mitosis occurs. Cell
cycle
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- 16-
control proteins and their role in regulating the cell cycle of eukaryotic
organisms are
reviewed in detail by John (1981) and the contributing papers therein; Nurse
(1990);
Norbury and Nurse (1992); Ormrod and Francis (1993) and the contributing
papers
therein; Francis and Halford, (1995); Elledge (1996); Doerner et al (1996);
and Francis
et al. (1998).
Preferably, the cell cycle control protein is derived from a yeast or plant
cell or
animal cell, more preferably, from the fission yeast (Schizosaccharomyces
pombe)
or from a plant cell, such as a monocotyledonous or dicotyledonous plant cell.
Preferred cell cycle control proteins according to this embodiment of the
invention
include the cdc2 T14Y15 phosphatases such as Cdc25 protein phosphatase or p80
cd~s (Russell and Nurse, 1986; Kumagai and Dunphy, 1991; Bell et al, 1993;
Elledge,
1996) and Pyp3 (Elledge, 1996); cdc2 protein kinase or p34 ~°2 (Nurse
and Bisset,
1981; Lee and Nurse, 1987; John et al., 1989; Feiler et al., 1990; Colasanti
et al.,
1991; Hirt et al. 1991; John et al., 1993); cdc2a protein kinase (Hemerly et
al, 1993);
cdc2 T14Y15 kinases such as wee1 or p107 '"'(Russell and Nurse, 1986; 1987a;
1987b; Elledge, 1996), mik1 (Lundgren et al.,1991 ) and myt1 (Elledge, 1996);
cdc2
T161 kinases such as Cak and Civ (Elledge, 1996); cdc2 T161 phosphatases such
as
Kap1 (Elledge, 1996); cdc28 protein kinase or p34 ~~8 (Reed et al., 1985;
Nasmyth,
1993); p40 ""o,s (Fesquet et al., 1993; Poon et al., 1993); chk1 kinase(Zeng
et al.,
1998); cds1 kinase (Zeng et al., 1998); growth-associated H1 kinase (GAK; Lake
and
Salzman, 1972; Langhan, 1978, Labbe et al., 1989; Arion et al., 1988); cyclins
A, B,
C, D and E (Evans et al. 1983; Swenson et al., 1986; Labbe et al., 1989;
Murray et al.,
1989; Francis et al, 1998); cyclin-dependent kinase inhibitor (CKI) proteins
such as
Sic1, Far1, Rum1, p21, p27, p57, p16, p15, p18, p19 (Pines, 1995; Elledge,
1996),
p14 and p14ARF ; p13 Su°,; (Hayles et al., 1986) and nim-1 (Fantes,
1979; Russell and
Nurse, 1986; 1987a; 1987b).
Other cell cycle control proteins that are involved in cyclin D-mediated entry
of cells
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-17-
into G1 from GO include pRb (Xie et al., 1996; Huntley et al., 1998), E2F,
RIP, MCM7C
and potentially the pRb-like proteins p107 and p130.
Other cell cycle control proteins that are involved in the formation of a pre-
replicative
complex at one or more origins of replication, such as, but not limited to,
ORC, CDC6,
CDC14, RPA and MCM proteins or in the regulation of formation of this pre-
replicative
complex, such as, but not limited to, the CDC7, DBF4 and MBF proteins.
Additional cell cycle control proteins are not excluded. The present invention
clearly
encompasses the use of homologues, analogues or derivatives of any of t"e
above-
mentioned cell cycle control proteins which also function as cell cycle
control proteins,
in modifying plant morphology and/or physiology and/or biochemistry.
For the present purpose, the term "cell cycle control protein" shall further
be taken to
include any one or more of those proteins that are involved in the turnover of
a cell
cycle control protein, or in regulating the half life of a cell cycle control
protein, such as,
but not limited to, proteins that are involved in the proteolysis of one or
more of the
above-mentioned cell cycle control proteins. Particularly preferred proteins
which are
involved in the proteolysis of one or more of the above-mentioned cell cycle
control
proteins include the yeast-derived and animal-derived proteins, Skp1, Skp2,
Rub1,
Cdc20, cullins, CDC23, CDC27, CDC16, and plant-derived homologues thereof
(Cohen-Fix and Koshland, 1997; Hochstrasser, 1998; Krek, 1998; Lisztwan, 1998;
Plesse et al., 1998).
"Homologues" of a cell cycle control protein such as Cdc25 are those peptides.
oligopeptides, polypeptides, proteins and enzymes which contain amino acid
substitutions, deletions and/or additions relative to the Cdc25 polypeptide
without
altering one or more of its cell cycle control properties, in particular
without reducing
the ability of the Cdc25 polypeptide to induce one or more cytokinin-mediated
and/or
CA 02263067 1999-03-30
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-18-
gibberellin-mediated effects in a plant cell, tissue, organ or whole organism.
To produce such homologues of a cell cycle control protein such as Cdc25,
amino
acids present in Cdc25 can be replaced by other amino acids having similar
properties,
for example hydrophobicity, hydrophilicity, hydrophobic moment, antigenicity,
propensity to form or break a-helical structures or (3-sheet structures, and
so on.
Substitutional variants are those in which at least one residue in the Cdc25
amino acid
sequence has been removed and a different residue inserted in its place. Amino
acid
substitutions are typically of single residues, but may be clustered depending
upon
functional constraints placed upon the polypeptide; insertions will usually be
of the
order of about 1-10 amino acid residues. and deletions will range from about 1-
20
residues. Preferably, amino acid substitutions will comprise conservative
amino acid
substitutions, such as those described supra.
Insertional amino acid sequence variants are those in which one or more amino
acid
residues are introduced into a predetermined site in the Cdc25 protein.
Insertions can
comprise amino- terminal and/or carboxyl terminal fusions as well as intra-
sequence
insertions of single or multiple amino acids. Generally, insertions within the
amino acid
sequence will be smaller than amino or carboxyl terminal fusions, of the order
of about
1 to 4 residues.
Deletional variants are characterised by the removal of one or more amino
acids from
the Cdc25 sequence.
Amino acid variants of the Cdc25 polypeptide may readily be made using peptide
synthetic techniques well known in the art, such as solid phase peptide
synthesis and
the like, or by recombinant DNA manipulations. The manipulation of DNA
sequences
to produce variant proteins which manifest as substitutional, insertional or
deletional
variants are well known in the art. For example, techniques for making
substitution
CA 02263067 1999-03-30
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- 19-
mutations at predetermined sites in DNA having known sequence are well known
to
those skilled in the art, such as by M13 mutagenesis or other site-directed
mutagenesis protocol.
"Analogues" of a cell cycle control protein such as Cdc25 are defined as those
peptides, oligopeptides, polypeptides, proteins and enzymes which are
functionally
equivalent to the Cdc25 polypeptide in inducing one or more cytokinin-mediated
and/or
gibberellin-mediated effects in plant cells, tissues, organs or whole
organisms, but
which contain certain non-naturally occurring or modified amino acid residues
as will
be known to those skilled in the art.
"Derivatives" of a cell cycle control protein such as Cdc25 are those
peptides,
oligopeptides, polypeptides, proteins and enzymes which comprise at least
about five
contiguous amino acid residues of a naturally-occurring Cdc25 polypeptide, in
particular the fission yeast p80 ~d°2s polypeptide, but which retain
activity in the
induction of one or more cytokinin-mediated and/or gibberellin-mediated
effects in a
plant cell, tissue, organ or whole organism. A "derivative" may further
comprise
additional naturally-occurring, altered glycosylated, acylated or non-
naturally occurring
amino acid residues compared to the amino acid sequence of a naturally-
occurring
Cdc25 polypeptide. Alternatively or in addition, a derivative may comprise one
or more
non-amino acid substituents compared to the amino acid sequence of a naturally-
occurring Cdc25 polypeptide, for example a reporter molecule or other ligand,
covalently or non-covalently bound to the amino acid sequence such as, for
example,
a reporter molecule which is bound thereto to facilitate its detection.
Other examples of recombinant or synthetic mutants and derivatives of the
Cdc25
polypeptide include those incorporating single or multiple substitutions,
deletions
and/or additions therein, such as carbohydrates, lipids and/or proteins or
polypeptides.
Naturally-occurring or altered glycosylated or acylated forms of the Cdc25
polypeptide
are also contemplated by the present invention. Additionally, homopolymers or
CA 02263067 1999-03-30
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-20-
heteropolymers comprising one or more copies of the Cdc25 polypeptide are
within the
scope of the invention, the only requirement being that such molecules possess
biological activity in inducing one or more cytokinin-mediated and/or
gibberellin-
mediated effects in plant cells, tissues, organs or whole organisms.
Particularly preferred homologues, analogues and derivatives of the fission
yeast
Cdc25 polypeptide contemplated by the present invention are derived from
plants. As
exemplified herein, the present inventors have identified a Cdc25 activity in
tobacco
cells which is contemplated as being of particular use in performing the
various
embodiments described herein.
In a particularly preferred embodiment of the invention, the cell cycle
control protein
is the yeast Cdc25 phosphoprotein phosphatase or a biologically-active
homologue,
analogue or derivative thereof and in particular, a plant-derived homologue of
the yeast
Cdc25 phosphoprotein phosphatase. The present invention clearly contemplates
the
use of functional homologues of the fission yeast Cdc25 protein, based upon
the
evidence provided herein for the presence of Cdc25-like activity and Cdc25-
like protein
in tobacco (Example 3). Accordingly, the present invention is not limited in
application
to the use of nucleotide sequences encoding the fission yeast p8p~d°zs
protein.
To effect expression of the cell cycle control protein in a plant cell, tissue
or organ,
either the protein may be introduced directly to said cell, such as by
microinjection
means or alternatively, an isolated nucleic acid molecule encoding said
protein may
be introduced into the cell, tissue or organ in an expressible format.
By "expressible format" is meant that the isolated nucleic acid molecule is in
a form
suitable for being transcribed into mRNA and/or translated to produce a
protein, either
constitutively or following induction by an intracellular or extracellular
signal, such as
an environmental stimulus or stress (anoxia, hypoxia, temperature, salt,
light,
dehydration, etc) or a chemical compound such as an antibiotic (tetracycline,
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-21 -
ampicillin, rifampicin, kanamycin) hormone (eg. gibberellin, auxin, cytokinin,
glucocorticoid, etc), hormone analogue (iodoacetic acid (IAA), 2,4-D, etc) ,
metal (zinc,
copper, iron, etc), or dexamethasone, amongst others. As will be known to
those
skilled in the art, expression of a functional protein may also require one or
more post-
s translational modifications, such as glycosylation, phosphorylation,
dephosphorylation,
or one or more protein-protein interactions, amongst others. All such
processes are
included within the scope of the term "expressible format".
Preferably, expression of a cell cycle control protein in a specific plant
cell, tissue, or
organ is effected by introducing and expressing an isolated nucleic acid
molecule
encoding said protein, such as a cDNA molecule, genomic gene, synthetic
oligonucleotide molecule, mRNA molecule or open reading frame, to said cell,
tissue
or organ, wherein said nucleic acid molecule is placed operably in connection
with a
suitable plant-expressible promoter sequence.
Reference herein to a "promoter" is to be taken in its broadest context and
includes the
transcriptional regulatory sequences derived from a classical eukaryotic
genomic gene,
including the TATA box which is required for accurate transcription
initiation, with or
without a CCAAT box sequence and additional regulatory elements (i.e. upstream
activating sequences, enhancers and silencers) which alter gene expression in
response to developmental and/or external stimuli, or in a tissue-specific
manner.
The term "promoter" also includes the transcriptional regulatory sequences of
a
classical prokaryotic gene, in which case it may include a -35 box sequence
and/or a
-10 box transcriptional regulatory sequences.
The term "promoter" is also used to describe a synthetic or fusion molecule,
or
derivative which confers, activates or enhances expression of a nucleic acid
molecule
in a cell, tissue or organ.
CA 02263067 1999-03-30
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-22-
Preferred promoters may contain additional copies of one or more specific
regulatory
elements, to further enhance expression and/or to alter the spatial expression
and/or
temporal expression of a nucleic acid molecule to which it is operably
connected. For
example, copper-responsive, glucocorticoid-responsive or dexamethasone-
responsive
regulatory elements may be placed adjacent to a heterologous promoter sequence
driving expression of a nucleic acid molecule to confer copper inducible,
glucocorticoid-
inducible, or dexamethasone-inducible expression respectively, on said nucleic
acid
molecule.
In the context of the present invention, the promoter is a plant-expressible
promoter
sequence. By "plant-expressible" is meant that the promoter sequence,
including any
additional regulatory elements added thereto or contained therein, is at least
capable
of inducing, conferring, activating or enhancing expression in a plant cell,
preferably
a monocotyledonous or dicotyledonous plant cell and in particular a
dicotyledonous
plant cell, tissue, or organ. Accordingly, it is within the scope of the
invention to include
any promoter sequences that also function in non-plant cells, such as yeast
cells,
animal cells and the like.
In the present context, a "regulatable promoter sequence" is a promoter that
is capable
of being expressed in a particular cell, tissue, or organ or group of cells,
tissues or
organs of a plant, optionally under specific conditions, however is generally
not
expressed throughout the plant under all conditions. Accordingly, a
regulatable
promoter sequence may be a promoter sequence that confers expression on a gene
to which it is operably connected in a particular location within the plant or
alternatively,
throughout the plant under a specific set of conditions, such as following
induction of
gene expression by a chemical compound or other elicitor.
Preferably, the regulatable promoter used in the performance of the present
invention
confers expression in a specific location within the plant, either
constitutively or
following induction, however not in the whole plant under any circumstances.
Included
CA 02263067 1999-03-30
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-23-
within the scope of such promoters are cell-specific promoter sequences,
tissue-
specific promoter sequences, organ-specific promoter sequences and
constitutive
promoter sequences that have been modified to confer expression in a
particular part
of the plant at any one time, such as by integration of said constitutive
promoter within
a transposable genetic element (Ac, Ds, Spm, En, or other transposon).
The term "cell-specific" shall be taken to indicate that expression is
predominantly in
a particular plant cell or plant cell-type, albeit not necessarily exclusively
in that plant
cell or plant cell-type.
Similarly, the term "tissue-specific" shall be taken to indicate that
expression is
predominantly in a particular plant tissue or plant tissue-type, albeit not
necessarily
exclusively in that plant tissue or plant tissue-type.
Similarly, the term "organ-specific" shall be taken to indicate that
expression is
predominantly in a particular plant organ albeit not necessarily exclusively
in that plant
organ.
As will be apparent from the preceding description, the present invention does
not
require the exclusive expression of the cell cycle control protein in a cell,
tissue or
organ of a plant, in order to induce non-pleiotropic cytokinin-mediated and/or
gibberellin-mediated effects therein, subject to the proviso that expression
is at least
predominantly localised in a particular cell, tissue or organ of the plant.
Preferably, the
promoter selected for regulating expression of the cell cycle control protein
in the plant
cell, tissue or organ, will confer expression in a range of cell-types or
tissue-types or
organs, sufficient to produce the desired phenotype, whilst avoiding
undesirable
phenotypes produced in other cell-types or tissue-types or organs.
More preferably, the promoter selected for regulating expression of the cell
cycle
control protein in the plant cell, tissue or organ, will confer expression in
a limited
CA 02263067 1999-03-30
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-24-
number of cells or cell-types or tissues or tissue-types or organs of the
plant.
Even more preferably, the promoter selected for regulating expression of the
cell cycle
control protein in the plant cell, tissue or organ, will confer expression in
a single cell-
type or tissue-type or organ of the plant.
Those skilled in the art will readily be capable of selecting appropriate
promoter
sequences for use in regulating appropriate expression of the cell cycle
control protein
from publicly-available or readily-available sources, without undue
experimentation.
Placing a nucleic acid molecule under the regulatory control of a promoter
sequence,
or in operable connection with a promoter sequence, means positioning said
nucleic
acid molecule such that expression is controlled by the promoter sequence.
A promoter is usually, but not necessarily, positioned upstream, or at the 5'-
end, and
within 2 kb of the start site of transcription, of the nucleic acid molecule
which it
regulates.
In the construction of heterologous promoter/structural gene combinations it
is
generally preferred to position the promoter at a distance from the gene
transcription
start site that is approximately the same as the distance between that
promoter and
the gene it controls in its natural setting (i.e., the gene from which the
promoter is
derived). As is known in the art, some variation in this distance can be
accommodated
without loss of promoter function. Similarly, the preferred positioning of a
regulatory
sequence element with respect to a heterologous gene to be placed under its
control
is defined by the positioning of the element in its natural setting (i.e., the
gene from
which it is derived). Again, as is known in the art, some variation in this
distance can
also occur.
Examples of promoters suitable for use in genetic constructs of the present
invention
CA 02263067 1999-03-30
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-25-
include those listed in Table 1, amongst others. The promoters listed in Table
1 are
provided for the purposes of exemplification only and the present invention is
not to be
limited by the list provided therein. Those skilled in the art will readily be
in a position
to provide additional promoters that are useful in performing the present
invention.
In an alternative embodiment, the promoter is a tissue-specific inducible
promoter
sequence, such as but not limited to a light-inducible rbcs-1A or rbcs-3A
promoter,
anoxia-inducible maize Adh1 gene promoter (Howard et al., 1987; Walker et al.,
1987),
hypoxia-inducible maize Adh1 gene promoter (Howard et al., 1987; Walker et
al.,
1987), and the temperature-inducible heat shock promoter. Such environmentally-
inducible promoters are reviewed in detail by Kuhlemeier et al. 1987).
In an alternative embodiment, the promoter is a chemically-inducible promoter,
such
as the 3-~- indoylacrylic acid-inducible Tip promoter; IPTG-inducible lac
promoter;
phosphate-inducible promoter; L-arabinose-inducible ara8 promoter; heavy metal
inducible metallothionine gene promoter; dexamethasone-inducible promoter;
glucocorticoid-inducible promoter; ethanol-inducible promoter (Zeneca); the
N,N-diallyl
2,2-dichloroacetamide-inducible glutathione-S-transferase gene promoter
(Wiegand
et al., 1986); or any one or more of the chemically-inducible promoters
described by
Gatz et al. (1996), amongst others.
In an alternative embodiment, the promoter is a wound-inducible or pathogen-
inducible
promoter, such as the phenylalanine ammonia lyase (PAL) gene promoter (Ebel et
al.,
1984), chalcone synthase gene promoter (Ebel et al., 1984) or the potato wound-
inducible promoter (Cleveland et al., 1987), amongst others.
In a further alternative embodiment, the promoter is a hormone-inducible
promoter,
such as the abscisic acid-inducible wheat 7S globulin gene promoter and the
wheat
Em gene promoter (Marcotte et al.,1988); an auxin-responsive gene promoter; or
a
gibberellin-inducible promoter such as the Amy32b gene promoter (Lanahan et
al.
CA 02263067 1999-03-30
P:\OPER\MRO\CDC25.PRV -26/2/99
-26-
1992), amongst others.
In a further alternative embodiment, the promoter is a constitutive plant-
expressible
promoter sequence such as the CaMV 35S promoter sequence , CaMV 19S promoter
sequence, the octopine synthase (OCS) promoter sequence , or nopaline synthase
(NOS) promoter sequence (Ebert et al. 1987), amongst others.
In the case of constitutive promoters or promoters that induce expression
throughout
the entire plant, it is preferred that such sequences are modified by the
addition of
nucleotide sequences derived from one or more of the tissue-specific promoters
listed
in Table 1, or alternatively, nucleotide sequences derived from one or more of
the
above-mentioned tissue-specific inducible promoters, to confer tissue-
specificity
thereon. For example, the CaMV 35S promoter may be modified by the addition of
maize Adh9 promoter sequence, to confer anaerobically-regulated root-specific
expression thereon, as described previously (Ellis et al., 1987). Such
modifications can
be achieved by routine experimentation by those skilled in the art.
Preferred embodiments of the invention relate to the effects) of cytokinins on
plant
morphology and architecture. The present invention clearly contemplates the
broad
application of the inventive method to the modification of a range of cellular
processes,
including but not limited to the initiation, promotion, stimulation or
enhancement of cell
division and/or seed development and/or tuber formation and/or shoot
initiation
and/or bushiness and/or dwarfism and/or pigment synthesis, and/or the
modification
of source/sink relationships, and/or the inhibition of root growth and/or the
inhibition of
apical dominance and/or the delay of senescence. In this regard, the
identification of
substrates of Cdc25 phosphatase other than cdc2 will also reveal the mechanism
by
which Cdc25 is linked to many cellular processes other than cell division.
CA 02263067 1999-03-30
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y y ip C N ~ ps C N O O O
~ N N f0 ~ ~ .~ p_~N N f0 p
t t p N ~ '~ ~ ~ ~ .O U U U :~
~ uJ fd ~ cn U ~ Q Q Q Z Y
v~ H
m
v
0
E
.c
Q _
o 'o
0
N
v
'vi m
E ~ E E
N O U a~ O a? a: :? ~ N
~ 'O ~ ..o.0 .p 'O ~ N N N N ~-
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N uj a7 ~ us
U > O N N O N N N 'C O O O O
... ..~.
O ~ N N tOnvOsvOSVOsO O O O O N E
O N LVSt~n(n(n
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.~ .tf7 .N U
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C ~_ N w H ~ !~ '+C-..N
o,O a a7 c O "'C
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~' ~' N
CA 02263067 1999-03-30
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In a particularly preferred embodiment of the present invention, there is
provided a
method of increasing the strength and/or thickness and/or stability and/or
wind-
resistance of a plant comprising expressing the yeast Cdc25 protein or a
homologue,
analogue or derivative thereof, or a modified substrate of Cdc25 that mimics
the effect
of Cdc25 operably under the control of a stem-expressible promoter sequence.
Preferably, the stem-expressible promoter sequence is derived from the rhcs-1A
gene,
the rbcs-3A gene, the AtPRP4 gene, the T. bacilliform virus gene, or the
sucrose-
binding protein gene set forth in Table 1, or a stem-specific or stem-
expressible
homologue, analogue or derivative thereof.
In the present context, the term "substrate of Cdc25" shall be taken to refer
to any
protein that interacts with Cdc25 in regulating the plant cell cycle,
including, but not
limited to cyclin-dependent kinases (CDKs), the most significant of which is
cdc2,
which in all cells is the key enzyme driving entry into mitosis. In all cells,
the switch
that raises activity of cdc2 at entry into mitosis is the Cdc25-catalysed
removal of
phosphate from tyrosine-15 in cdc2. In yeasts there is only one CDK (cdc2) and
the
Cdc25-catalysed removal of phosphate from tyrosine-15 in cdc2 occurs only once
in
the cell cycle, at the G2/M phase transition. In contrast, higher eukaryotic
cells (animal
and plant cells) contain several CDKs. In mammals, the molecular switch of
Cdc25-catalysed removal of phosphate from tyrosine-15 in cdc2 is also used at
entry
into the S phase, and a separate CDK (CDK2) and a separate Cdc25 (Cdc25A)
perform this function. In plants, whilst it is known there are several CDKs,
it is not
known if there is a single CDK that is controlled at S phase, like CDK2, by
tyrosine
phosphate.
Without being bound by any theory or mode of action, the substitution or
deletion of
the phosphorylation sites of a protein that is a substrate for a cyclin-
dependent kinase
protein mimics the effect of a constitutive phosphatase activity, such as the
effect of
Cdc25 protein phosphatase ( p80~5) activity, because phosphorylated protein
will not
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be produced at high steady-state concentrations in either the absence of
phosphorylation or when phosphatases are expressed at high levels.
Accordingly, the
Cdc25-induced effects described herein can also be obtained by the regulated
expression of a modified substrate of Cdc25.
The term "modified substrate of Cdc25" refers to a homologue, analogue or
derivative
of a substrate of Cdc25 that mimics the effect of Cdc25 activity, in
particular a non-
phosphorylatable Cdc25 substrate that mimics the effect of Cdc25 activity. For
example, substitution of threonine and tyrosine at positions 14 and 15 of
cyclin-
dependent kinases (CDKs) for alanine and phenylalanine, respectively, can
produce
one or more cytokinin-like effects in the plant, similar to those observed
following
constitutive Cdc25 expression in the plant. Notwithstanding that this may be
the case,
the effects of CDK(A,4F,5 ) expression are inferior to those of naturally-
occurring or
wild-type Cdc25, possibly because plant cells comprise several Cdc25
substrates.
Accordingly, similar effects to the Cdc25-induced effects obtained by
expressing
Cdc25 under control of the regulatable promoter, can be obtained by expressing
the
Cdc25 substrate or a modified form thereof operably under control of the same
or a
functionally-equivalent promoter. The present invention clearly extends to
such
arrangements.
The present invention extends further to the co-expression of Cdc25 and one or
more
Cdc25 substrates and/or one or more modified Cdc25 substrates, operably under
the
control of a regulatable promoter that is selected for a particular
application as
described herein.
In another preferred embodiment of the present invention, there is provided a
method
of increasing tuber formation and/or development in a tuberous crop plant
comprising
expressing the yeast Cdc25 protein or a homologue, analogue or derivative
thereof,
or a modified substrate of Cdc25 that mimics the effect of Cdc25 operably
under the
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control of a tuber-specific promoter sequence.
Preferably, the tuberous crop plant is potato and the tuber-specific promoter
is the
potato patatin gene promoter. Additional species and promoters are not
excluded.
In another preferred embodiment of the present invention, there is provided a
method
of modifying the lignin content of a woody crop plant comprising expressing
the yeast
Cdc25 protein or a homologue, analogue or derivative thereof, or a modified
substrate
of Cdc25 that mimics the effect of Cdc25 operably under the control of a
cambium-
specific or vascular-tissue-specific promoter sequence.
Preferably, the promoter is a cinnamoyl alcohol dehydrogenase (CAD) gene
promoter,
laccase gene promoter, cellulose synthase gene promoter and xyloglucan
endotransglucosylase (XET) gene promoter sequences, amongst others. The T.
bacilliform virus gene promoter and the sucrose-binding protein gene promoter
are
also useful for this application of the invention.
Preferred target plant species according to this embodiment are woody plants
of
economid agronomic value, in particular hardwood crop plants such as, but not
limited
to Eucalyptus spp., Populus spp., Quercus spp., Acer spp., Juglans spp., Fagus
spp.,
Acacia spp., or teak, amongst others. More preferably, this embodiment of the
invention is applicable to modifying the lignin content of Eucalyptus spp., in
particular
E. globulus and E. robusta; or Quercus spp., in particular Q. dentata, Q.
ilex, Q.
incana, and Q. robur Acacia spp., in particular A. brevispica, A. bussei, A.
drepanolobium, A. nilotica, A. pravissima, and A. seyal; Acer spp., in
particular A.
pseudoplatanus and A. saccharum. Additional species are not excluded.
Without being bound by any theory or mode of action, the ectopic expression of
Cdc25
under control of a promoter that is operable in vascular tissue and
preferably, in
cambial cells, will produce thick-stemmed plants and a higher ratio of
vascular tissue-
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to-pith cells within the stem, thereby resulting in more lignin production.
Within the
vascular tissue, cambial cells contain the highest levels of auxins and are
therefore the
preferential tissue for Cdc25 overproduction.
In yet another preferred embodiment of the present invention, there is
provided a
method of increasing seed set and/or seed production and/or grain yield in a
plant
comprising expressing the yeast Cdc25 protein or a homologue, analogue or
derivative
thereof, or a modified substrate of Cdc25 that mimics the effect of Cdc25
operably
under the control of a seed-specific promoter sequence.
Preferably, the seed-specific promoter is operable in the seeds of
monocotyledonous
plants, for example the barley Amy32b gene promoter, Cathepsin (3-like gene
promoter, wheat ADP-glucose pyrophosphorylase gene promoter, maize zein gene
promoter, or rice glutelin gene promoter. In an alternative embodiment, the
seed-
specific promoter is operable in the seeds of dicotyledonous plant species,
for example
the legumin gene promoter, napA gene promoter, Brazil Nut albumin gene
promoter,
pea vicilin gene promoter and sunflower oleosin gene promoter, amongst others.
Those skilled in the art will be aware that grain yield in crop plants is
largely a function
of the amount of starch produced in the endosperm of the seed. The amount of
protein
produced in the endosperm is also a contributing factor to grain yield. In
contrast, the
embryo and aleurone layers contribute little in terms of the total weight of
the mature
grain.
Accordingly, in a preferred embodiment, the Cdc25 gene is placed operably in
connection with a promoter that is operable in the endosperm of the seed, in
which
case the combination of the cell cycle-control protein and endosperm-
expressible
promoter provides the additional advantage of increasing the grain size and
grain yield
of the plant.
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Endosperm-specific promoters that can be used to drive Cdc25 expression have
been
identified. The components of the promoters responsible for specific
expression have
been identified (Grosset et al (1997) and are interchangeable between
agriculturally
important cereals (Olsen et al 1992; Russell and Fromm, 1997). Several
promoters can
S be used, including the zein (ZmZ27) gene promoter, the rice glutelin 1 gene
(osGT1 )
promoter, the rice small subunit ADP-glucose pyrophosphorylase (osAGP)
promoter,
the maize granule-bound starch synthase (Waxy) gene (zmGBS) promoter surveyed
by Russell and Fromm (1997), the Brazil Nut albumin gene promoter, and the pea
vicilin gene promoter, amongst others. Promoters derived from those genes that
are
expressed in the endosperm during nuclear proliferation are also useful for
driving
Cdc25 expression. Promoters derived from those genes that are expressed in the
endosperm at the stage of nuclear proliferation is ending could be ideal for
extending
this period.
A three way correlation exists between cytokinin level in the endosperm, the
number
of endosperm cells formed during seed development and grain size, in which
cytokinin
activates Cdc25 enzyme which in turn activates Cdc2 kinase to drive nuclear
division.
Accordingly, ectopic expression of the Cdc25 gene in the endosperm enhances
Cdc2
activation and nuclear proliferation, resulting in increased grain size,
without incurring
the non-specific side effects that application of cytokinin or expression of
the ipt gene
would produce in the plant.
A further advantage of the present inventive approach is that the activity of
cytokinin
metabolising enzymes is circumvented by the direct raising of Cdc25 activity
in the
endosperm, by the ectopic expression of Cdc25 therein. In cases where
exogenous
cytokinin is used to increase grain size and/or endosperm size, the elevated
cytokinin
levels and nuclear division in the grain are curtailed by an increase in the
activities of
cytokinin degrading enzymes, including cytokinin oxidase (Chaifield and
Armstrong
1987; reviewed by Morris et al 1993).
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In another preferred embodiment of the present invention, there is provided a
method
of inhibiting or reducing apical dominance or increasing the bushiness of a
plant,
comprising expressing the yeast Cdc25 protein or a homologue, analogue or
derivative
thereof, or a modified substrate of Cdc25 that mimics the effect of Cdc25
operably
under the control of a meristem-specific promoter sequence or a stem-specific
promoter sequence.
Without being bound by any theory or mode of action, increased cell division
in the
dormant lateral meristem of plants as a consequence of increased Cdc25
activity
therein results in a higher degree of branch formation in the plant, thereby
alleviating
auxin-induced apical dominance in the plant.
In another preferred embodiment of the present invention, there is provided a
method
of increasing lateral root production in a plant comprising expressing the
yeast Cdc25
protein or a homologue, analogue or derivative thereof, or a modified
substrate of
Cdc25 that mimics the effect of Cdc25 operably under the control of a root-
specific
promoter sequence.
Preferred promoter sequences according to this embodiment of the present
invention
include any one of the root-expressible or root-specific promoters listed in
Table 1 and
in particular, the tobacco auxin-inducible gene promoter described by Van der
Zaal et
al (1991) that confers expression in the root tip of plants, in particular
dicotyledonous
plants.
In yet another preferred embodiment of the present invention, there is
provided a
method of increasing the nitrogen-fixing capability of a plant comprising
expressing the
yeast Cdc25 protein or a homologue, analogue or derivative thereof, or a
modified
substrate of Cdc25 that mimics the effect of Cdc25 operably under the control
of a
nodule-specific promoter sequence.
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Preferred nodule-specific promoter sequences according to this embodiment of
the
present invention are listed in Table 1. Additional promoters that are suited
for this
purpose include the hemoglobin gene promoters derived from Frankia spp., A.
thaiiana
or other plants.
In still another preferred embodiment of the present invention, there is
provided a
method of prevent or delay or otherwise reduce leaf chlorosis and/or leaf
necrosis in
a plant comprising expressing the yeast Cdc25 protein or a homologue, analogue
or
derivative thereof, or a modified substrate of Cdc25 that mimics the effect of
Cdc25
operably under the control of a leaf-specific promoter sequence.
Preferred promoters for use according to this embodiment of the present
invention
include the SAM22 promoter, rbcs-7A and rbcs-3A gene promoters listed in Table
1.
The SAM22 gene promoter is particularly preferred in light of the
developmental
regulation of the SAM22 gene and its induction in senescent leaves.
In a further preferred embodiment of the present invention, the yeast Cdc25
protein or
a homologue analogue or derivative thereof, or a modified substrate of Cdc25
that
mimics the effect of Cdc25 is expressed in one of the specialised minority of
plant
tissues in which the activation of cell cycle progression that is generally
contributed by
cytokinin is in part performed by other hormones. An example of such a tissue
is the
youngest stem internode of cereal plants in which gibberellic acid stimulates
cell
division.
Accordingly, the present invention preferably provides a method of stimulating
cell
division in the intercalary meristem of the youngest stem internode to produce
greater
elongation of the stem and/or to generate a more extensive photosynthetic
canopy of
a plant comprising expressing the yeast Cdc25 protein or a homologue, analogue
or
derivative thereof, or a modified substrate of Cdc25 that mimics the effect of
Cdc25
operably under the control of a meristem specific promoter sequence.
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Without being bound by any theory or mode of action, increas in cell division
in the
intercalary meristem of the youngest stem internode as a consequence of
increased
Cdc25 activity therein results in greater vigour of the plant due to stem
elongation and
the production of a more extensive canopy. It is proposed that this leads to
an
increase in the plant's capacity to support grain production. The stimulatory
effect of
gibberellic acid application is thus obtained without side effects on
flowering time and
seed germination.
Preferred promoters for use according to this embodiment of the invention
include
meristem promoters listed in Table 1 and in particular the Proliferating Cell
Nuclear
Antigen (PCNA) promoter of rice described by Kosugi et al (1991 ).
In each of the preceding embodiments of the present invention, the cell cycle
control
protein is expressed under the operable control of a regulatable promoter
sequence.
As will be known those skilled in the art, this is generally achieved by
introducing a
genetic construct or vector into plant cells by transformation or transfection
means.
The nucleic acid molecule or a genetic construct comprising same may be
introduced
into a cell using any known method for the transfection or transformation of
said cell.
Wherein a cell is transformed by the genetic construct of the invention, a
whole
organism may be regenerated from a single transformed cell, using any method
known
to those skilled in the art.
By "transfect" is meant that the genetic construct or vector or an active
fragment
thereof comprising the Cdc25 gene operably under the control of the
regulatable
promoter sequence is introduced into said cell without integration into the
cell's
genome.
By "transform" is meant that the genetic construct or vector or an active
fragment
thereof comprising the Cdc25 gene operably under the control of the
regulatable
promoter sequence is stably integrated into the genome of the cell.
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Accordingly, in a further preferred embodiment, the present invention provides
a
method of modifying one or more plant morphological and/or biochemical and/or
physiological characteristics comprising
S (i) introducing to a plant cell, tissue or organ a genetic construct or
vector
comprising a nucleotide sequence that encodes a cell cycle control protein
operably in connection with a regulatable promoter sequence selected from the
list comprising cell-specific promoter sequences, tissue-specific promoter
sequences, and organ-specific promoter sequences; and
(ii) expressing said cell cycle control protein in one or more of said cells,
tissues or organs of the plant.
In an alternative embodiment, the present invention provides a method of
modifying
one or more plant morphological and/or biochemical and/or physiological
characteristics comprising
(i) introducing into a plant cell a genetic construct or vector comprising a
nucleotide sequence that encodes a cell cycle control protein operably in
connection with a regulatable promoter sequence selected from the list
comprising cell-specific promoter sequences, tissue-specific promoter
sequences, and organ-specific promoter sequences;
(ii) regenerating a whole plant from said plant cell; and
(iii) expressing said cell cycle control protein in one or more particular
cells,
tissues or organs of the plant.
Means for introducing recombinant DNA into plant tissue or cells include, but
are not
limited to, transformation using CaCl2 and variations thereof, in particular
the method
described by Hanahan (1983), direct DNA uptake into protoplasts (Krens et al,
1982;
Paszkowski et al, 1984), PEG-mediated uptake to protoplasts (Armstrong et al,
1990)
microparticle bombardment, electroporation (Fromm et aL, 1985), microinjection
of
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DNA (Crossway et al., 1986), microparticle bombardment of tissue explants or
cells
(Christou et al, 1988; Sanford, 1988), vacuum-infiltration of tissue with
nucleic acid, or
in the case of plants, T-DNA-mediated transfer from Agrobacterium to the plant
tissue
as described essentially by An et al.(1985), Herrera-Estrella et al. (1983a,
1983b,
1985).
For microparticle bombardment of cells, a microparticle is propelled into a
cell to
produce a transformed cell. Any suitable ballistic cell transformation
methodology and
apparatus can be used in performing the present invention. Exemplary apparatus
and
procedures are disclosed by Stomp et al. (U.S. Patent No. 5,122,466) and
Sanford and
Wolf (U.S. Patent No. 4,945,050). When using ballistic transformation
procedures, the
genetic construct may incorporate a plasmid capable of replicating in the cell
to be
transformed.
Examples of microparticles suitable for use in such systems include 1 to 5 ~cm
gold
spheres. The DNA construct may be deposited on the microparticle by any
suitable
technique, such as by precipitation.
A whole plant may be regenerated from the transformed or transfected cell, in
accordance with procedures well known in the art. Plant tissue capable of
subsequent
clonal propagation, whether by organogenesis or embryogenesis, may be
transformed
with a genetic construct of the present invention and a whole plant
regenerated
therefrom. The particular tissue chosen will vary depending on the clonal
propagation
systems available for, and best suited to, the particular species being
transformed.
Exemplary tissue targets include leaf disks, pollen, embryos, cotyledons,
hypocotyls,
megagametophytes, callus tissue, existing meristematic tissue (e.g., apical
meristem,
axillary buds, and root meristems), and induced meristem tissue (e.g.,
cotyledon
meristem and hypocotyl meristem).
The term "organogenesis", as used herein, means a process by which shoots and
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roots are developed sequentially from meristematic centres.
The term "embryogenesis", as used herein, means a process by which shoots and
roots develop together in a concerted fashion (not sequentially), whether from
somatic
cells or gametes.
The regenerated transformed plants may be propagated by a variety of means,
such
as by clonal propagation or classical breeding techniques. For example, a
first
generation (or T1 ) transformed plant may be selfed to give homozygous second
generation (or T2) transformant, and the T2 plants further propagated through
classical
breeding techniques.
The regenerated transformed organisms contemplated herein may take a variety
of
forms. For example, they may be chimeras of transformed cells and non-
transformed
cells; clonal transformants (e.g., all cells transformed to contain the
expression
cassette); grafts of transformed and untransformed tissues (e.g., in plants, a
transformed root stock grafted to an untransformed scion ).
A further aspect of the present invention clearly provides the genetic
constructs and
vectors designed to facilitate the introduction and/or expression and/or
maintenance
of the cell cycle control protein-encoding sequence and regulatable promoter
into a
plant cell, tissue or organ.
In addition to the cell cycle control protein-encoding sequence and
regulatable
promoter sequence, the genetic construct of the present invention may further
comprise one or more terminator sequences.
The term "terminator" refers to a DNA sequence at the end of a transcriptional
unit
which signals termination of transcription. Terminators are 3'-non-translated
DNA
sequences containing a polyadenylation signal, which facilitates the addition
of
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polyadenylate sequences to the 3'-end of a primary transcript. Terminators
active in
cells derived from viruses, yeasts, moulds, bacteria, insects, birds, mammals
and
plants are known and described in the literature. They may be isolated from
bacteria,
fungi, viruses, animals and/or plants.
Examples of terminators particularly suitable for use in the genetic
constructs of the
present invention include the Agrobacterium tumefaciens nopaline synthase
(NOS)
gene terminator, the Agrobacterium tumefaciens octopine synthase (OCS) gene
terminator sequence, the Cauliflower mosaic virus (CaMV) 35S gene terminator
sequence, the Oryza sativa ADP-glucose pyrophosphorylase terminator sequence
(t3'Bt2), the Zea mays zein gene terminator sequence, the rbcs-7A gene
terminator,
and the rbcs-3A gene terminator sequences, amongst others.
Those skilled in the art will be aware of additional promoter sequences and
terminator
sequences which may be suitable for use in performing the invention. Such
sequences
may readily be used without any undue experimentation.
The genetic constructs of the invention may further include an origin of
replication
sequence which is required for maintenance and/or replication in a specific
cell type,
for example a bacterial cell, when said genetic construct is required to be
maintained
as an episomal genetic element (eg. plasmid or cosmid molecule) in said cell.
Preferred origins of replication include, but are not limited to, the f7-on
and colE1
origins of replication.
The genetic construct may further comprise a selectable marker gene or genes
that
are functional in a cell into which said genetic construct is introduced.
As used herein, the term "selectable marker gene" includes any gene which
confers
a phenotype on a cell in which it is expressed to facilitate the
identification and/or
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selection of cells which are transfected or transformed with a genetic
construct of the
invention or a derivative thereof.
Suitable selectable marker genes contemplated herein include the ampicillin
resistance
(Amp), tetracycline resistance gene (Tc'), bacterial kanamycin resistance gene
(Kan~, phosphinothricin resistance gene, neomycin phosphotransferase gene
(nptll),
hygromycin resistance gene, ~3-glucuronidase (GUS) gene, chloramphenicol
acetyltransferase (CAT) gene, green fluorescent protein (gfp) gene (Haseloff
ef al,
1997), and luciferase gene, amongst others.
In fact, the cell cycle control protein-encoding sequence, in particular the
Cdc25
protein-encoding sequence may also be used as a selectable marker gene as
defined
herein, by virtue of the altered morphology and/or biochemistry and/or
physiology
conferred by its regulated expression in plant cells, tissues, organs or whole
plants.
Those skilled in the art may be aware that plant cells in culture require
cytokinin as well
as auxin for cell proliferation. In a minority of tissues such as the shoot
internode
meristem of the Graminae, a hormone other than cytokinin, in particular
gibberellin,
appears to be required for cell proliferation.
The present inventors have confirmed the cytokinin requirement, for tissues
other than
the shoot internode meristem of the Graminae. In particular, the inventors
have
observed that whilst auxin alone is able to stimulate the enlargement cells
derived from
excised tobacco pith tissue, cytokinin is also required for cell division to
occur (Figure
4). Only in the presence of both auxin and cytokinin do tobacco cells
proliferate and
form callus, as measured by the incorporation of bromodeoxyuridine (BrdU) into
replicating DNA (Figure 4).
With regard to the dependence of cells upon gibberellin, Sauter et al (1995)
showed
that within 4 hours of gibberellin application to rice stems, a synchronous
decline
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occurs in the number of cells that are in G2 phase, and cells move into
mitosis,
indicating that the G2-arrested cells had been stimulated. However, the
application of
gibberellins to plant tissues to promote cell division is also accompanied
undesirable
pleiotropic side-effects, in particular the induction of flowering, stem
elongation, seed
germination, fruit and seed development.
The inability of genetically unmodified plant cells derived directly from the
plant to
divide without added cytokinin or gibberellin in addition to auxin forms the
basis for the
positive selection method of the present invention, which depends upon the
unexpected ability of Cdc25 gene expression to replace the requirement for
these
hormones. In this embodiment of the invention, plant cells that ectopically-
express
Cdc25 under the control of a regulatable promoter are able to proliferate
without added
cytokinin or, in the case of certain tissues such as the shoot meristem
internode,
without added gibberellin, thereby providing a strong positive selection for
transformed
or transfected cells on medium lacking cytokinin or gibberellin, as the case
may be.
The present invention overcomes the disadvantages associated with exogenous
gibberellin and/or cytokinin application, by promoting hormone-mediated cell
division
via the ectopic expression of Cdc25 in particular cells, tissues or organs of
the plant.
This positive selection for transgenic cells is particularly advantageous for
plant
breeding by gene transfer, wherein the Cdc25 gene operably in connection with
a
plant-expressible promoter is introduced into the plant cell at the same time
as a gene-
of-interest. According to this embodiment of the present invention, the Cdc25
gene
may be contained in the same plasmid or virus vector as the gene-of-interest,
or
alternatively, on separate plasmid or virus vector molecules, in which case
these
molecules are generally co-introduced to the plant cell.
Accordingly, a further aspect of the present invention provides a method of
detecting
or identifying transformed or transfected plant cells, tissues or organs that
are
cytokinin-dependent, comprising expressing the yeast Cdc25 protein or a
homologue,
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analogue or derivative thereof, or a modified substrate of Cdc25 that mimics
the effect
of Cdc25 in said plant cell, tissue or organ operably under the control of a
regulatable
plant-expressible promoter, for a time and under conditions sufficient for
cytokinin
mediated cell division and/or cytokinin-mediated tissue differentiation to
occur in the
absence of cytokinin.
As used herein, the term "cytokinin-dependent" shall be taken to refer to a
naturally-
occurring plant cell, tissue or organ that at least requires the application
of exogenous
cytokinin to promote cell division and/or proliferation in vitro.
A further aspect of the present invention provides a method of detecting or
identifying
transformed or transfected plant cells, tissues or organs that are gibberellin-
dependent,
comprising expressing the yeast Cdc25 protein or a homologue, analogue or
derivative
thereof, or a modified substrate of Cdc25 that mimics the effect of Cdc25 in
said plant
cell, tissue or organ operably under the control of a regulatable plant-
expressible
promoter, for a time and under conditions sufficient for gibberellin-mediated
cell
division and/or gibberellin-mediated tissue differentiation to occur in the
absence of
gibberellin.
Preferably, the gibberellin-dependent plant tissue is the meristem shoot
internode or
intercalary meristem of the youngest stem internode derived from a
monocotyledonous
plant species, in particular the Graminae.
As used herein, the term "gibberellin-dependent" shall be taken to refer to a
naturally-
occurring plant cell, tissue or organ that at least requires the application
of exogenous
gibberellin to promote cell division and/or proliferation in vitro.
Preferably, the regulatable promoter used in the transfection/transformation
selection
systems described herein is a promoter listed in Table 1. Wherein the promoter
is
operable in a specific tissue or cell type, selection will only be possible in
that cell or
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tissue following transformation, and optionally, following regeneration
therefrom,
because in other tissues or cells of the transformed plant material the Cdc25
gene will
not be expressed.
S More preferably, the plant-expressible promoter is an inducible promoter,
such as a
tissue-specific inducible promoter sequence, environmentally-inducible
promoter, a
chemically-inducible promoter, a wound-inducible promoter, a pathogen-
inducible
promoter, or a hormone-inducible promoter. In this case, dividing cells are
selected by
switching on the expression of Cdc25 with the appropriate stimulus for the
promoter
of choice.
Wherein the promoter is chemically-inducible, the transformed cells can be
selected
in the presence of the chemical that induces Cdc25 gene expression. In a
particularly
preferred embodiment exemplified herein, the Cdc25 gene is expressed in
tobacco
cells under the control of the dexamethasone-inducible promoter and dividing
cells are
selected on media containing both the synthetic auxin 2,4-D and dexamethasone,
in
the absence of added cytokinin.
In an alternative preferred embodiment, the promoter is a constitutive plant-
expressible
promoter sequence, wherein the regulatable promoter plus cell cycle control
protein-
encoding sequence is modified by the insertion of 5'- and/or 3'-transposable
genetic
element sequences to facilitate movement of genetic construct/vector and
selective
expression of the cell cycle control protein in a sub-set of cells, tissue or
organs of the
plant. According to this embodiment, the cell cycle control protein is
expressed only in
those cells which also contain the transposable element inserted into their
genome.
A further aspect of the invention clearly extends to a plant cell, tissue,
organ or whole
plant that has been transformed or transfected with an isolated nucleic acid
molecule
that comprises a nucleotide sequence which encodes a cell cycle control
protein,
wherein the expression of said nucleotide sequence is placed operably under
the
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control of a plant-expressible cell-specific promoter sequence, plant-
expressible tissue-
specific promoter sequence, a plant-expressible organ-specific promoter
sequence,
or a plant-expressible constitutive promoter sequence such that said plant-
expressible
constitutive promoter sequence and said nucleotide sequence encoding a cell
cycle
control protein are integrated into a transposable genetic element.
The present invention is applicable to any plant, in particular a
monocotyledonous
plants and dicotyledonous plants including a fodder or forage legume,
companion
plant, food crop, tree, shrub, or ornamental selected from the list comprising
Acacia
spp., Acer spp., Actinidia spp.,Aesculus spp., Agathis australis, Albizia
amara,
Alsophila tricolor, Andropogon spp., Arachis spp, Areca catechu, AstEliu
fragrans,
Astragalus cicer, Baikiaea plurijuga, Betula spp., Bruguiera gymnorrhiza,
Burkea
afiicana, Butea frondosa, Cadaba farinosa, Calliandra spp, Camellia sinensis,
Canna
indica, Capsicum spp., Cassia spp., Centroema pubescens, Chaenomeles
spp., Cinnamomum cassia, Coffea arabica, Colophospermum mopane, Coronillia
varia,
Cotoneaster serotina, Crataegus spp., Cucumis spp., Cupressus spp., Cyathea
dealbata, Cydonia oblongs, Cryptomeria japonica, Cymbopogon spp., Cynthea
dealbata, Cydonia oblongs, Dalbergia monetaria, Davallia divaricata, Desmodium
spp.,
Dicksonia squarosa, Diheteropogon amplectens, Dioclea spp, Dolichos spp.,
Dorycnium rectum, Echinochloa pyramidalis, Ehrartia spp., Eleusine coracana,
Eragrestis spp., Erythrina spp., Eucalyptus spp., Euclea schimperi, Eulalia
villosa,
Fagopyrum spp., Feijoa sellowiana, Fragaria spp., Flemingia spp, Freycinetia
banksii,
Geranium thunbergii, Ginkgo biloba, Glycine javanica, Gliricidia spp,
Gossypium
hirsutum, Grevillea spp., Guibourtia coleosperma, Hedysarum spp., Hemarthia
altissima, Heteropogon contortus, Hordeum vulgare, Hyparrhenia rufa, Hypericum
erectum, Hyperthelia dissoluta, Indigo incamata, Iris spp., Leptarrhena
pyrolifolia,
Lespediza spp., Lettuca spp., Leucaena leucocephala, Loudetia simplex, Lotonus
bainesii, Lotus spp., Macrotyloma axillare, Malus spp., Manihot esculenta,
Medicago
sativa, Metasequoia glyptostroboides, Musa sapientum, Nicotianum spp.,
Onobrychis
spp., Omithopus spp., Peltophorum afiicanum, Pennisetum spp., Persea
gratissima,
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Petunia spp., Phaseolus spp., Phoenix canariensis, Phormium cookianum,
Photinia
spp., Picea glauca, Pinus spp., Pisum sativum, Podocarpus totara, Pogonarthria
fleckii,
Pogonarthria squarrosa, Populus spp., Prosopis cineraria, Pseudofsuga
menziesii,
Pterolobium stellatum, Pyrus communis, Quercus spp., Rhaphiolepsis umbellata,
Rhopalostylis sapida, Rhus natalensis, Ribes grossularia, Ribes spp., Robinia
pseudoacacia, Rosa spp., Rubus spp., Salix spp., Schyzachyrium sanguineum,
Sciadopitys verticillata, Sequoia sempervirens, Sequoiadendron giganteum,
Sorghum
bicolor, Spinacia spp., Sporobolus fimbriatus, Stiburus alopecuroides,
Stylosanthos
humiiis, Tadehagi spp, Taxodium distichum, Themeda triandra, Trifolium spp.,
Triticum
spp., Tsuga heterophylla, Vaccinium spp., Vicia spp. Vitis vinifera, Watsonia
pyramidata, Zantedeschia aethiopica, Zea mays, straw, amaranth, onion,
asparagus,
sugar cane, soybean, sugarbeet, sunflower, carrot, celery, cabbage, canola,
tomato,
potato, lentil, flax, broccoli, oilseed rape, cauliflower, brussel sprout,
artichoke, okra,
squash, kale, collard greens, and tea, amongst others, or the seeds of any
plant
specifically named above or a tissue, cell or organ culture of any of the
above species.
Preferably, the plant is a plant that is capable of being transfected or
transformed with
a genetic sequence, or which is amenable to the introduction of a protein by
any art-
recognised means, such as microprojectile bombardment, microinjection,
Agrobacterium-mediated transformation, protoplast fusion, or electroporation,
amongst
others.
This aspect of the invention further extends to plant cells, tissues, organs
and plants
parts, propagules and progeny plants of the primary transformed or transfected
cells,
tissues, organs or whole plants that also comprise the introduced isolated
nucleic acid
molecule operably under control of the cell-specific, tissue-specific or organ-
specific
promoter sequence and, as a consequence, exhibit similar phenotypes to the
primary
transformants/transfectants or at least are useful for the purpose of
replicating or
reproducing said primary transformants/transfectants.
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The present invention is further described with reference to the following non-
limiting
Examples and to the drawings.
EXAMPLE 1
CELL CULTURE, PROTEIN AND ENZYME METHODS
Cell culture
Suspension cultured cells of Nicotiana plumbiginifolia were grown in CS V
medium
supplemented with 9 ,uM 2,4-dichlorophenoxyacetic acid and 0.23 ~cM kinetin,
and
were brought to arrest at the cytokinin control point by the omission of
kinetin from the
culture medium. Arrest of cell cultures was confirmed by cell counting.
Antibodies
Polyclonal antibodies were raised in rabbits using the carboxy terminal amino
acid
sequence of the tobacco cdc2 protein, designated as cdc2a, as an immunogen.
This
peptide has the amino acid sequence KRITARNALEHEYFKDIGYVP and has been
demonstrated by complementation analyses in yeast to be a functional homologue
of
cdc2. The cdc2a peptide was synthesised chemically, purified by HPLC and
conjugated to keyhole limpet haemocyanin. Antibodies were also prepared
against a
recombinant GST-Cdc25 catalytic core fusion protein, that had been synthesised
in
Escherichia coli.
Assay of cdc2 and Cdc25 activities
Both cdc2 and Cdc25 enzyme activities were extracted from tobacco cells, by
grinding
the cells in liquid nitrogen. For cdc2 extraction, NDE buffer containing 25 mM
HEPES
(pH 7.2) with protease and phosphatase inhibitors was used. For Cdc25
extraction,
PDE buffer, containing 25 mM MOPS (pH 7.2), 100 mM NaCI, 10 mM DTT, 5mM
EDTA, 1 mM EGTA, 1 % NP-40, 50 mM NaF, 0.5 mM PMSF, 3 ,ug ml-' leupeptin, and
20 ~g ml~' aprotinin, was used.
Immunoprecipitates of cdc2 and Cdc25 were obtained by reaction with 25 ,ul
protein
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A-purified antibodies against cdc2 and Cdc25 respectively, for 3 h at
4°C, followed by
sedimentation of the antigen-antibody complexes using 35 ul protein A beads
per
sample. The immunoprecipitates were then washed three times, for 10 min per
wash,
using HDW buffer, followed by similar washing using HBK buffer. In the case of
Cdc25
immunoprecipitates, the HBK buffer was supplemented with 2 ,uM spermidine.
To measure cdc2 activity, the phosphorylation of HI histone was followed.
To measure Cdc25 activity, assays were conducted in two stages. First, Cdc25
immunoprecipitates from 500 ,ug total soluble plant protein were incubated for
30 min
at 30°C in Cdc25 assay buffer with 0.25 ~g tyrosine phosphorylated cdc2
substrate
that had been purified with p13s~''-beads from 500 ,ug protein of arrested
Cdc25-22
mutant fission yeast. The phosphatase reaction was stopped by removing the
complexed Cdc25/cdc2 by sedimentation. In the second stage of the Cdc25 assay,
the
supernatant was assayed for yeast cdc2 kinase that had been activated. Assays
to
be compared directly were run and exposed together in a Phosphorimager.
cdc2a phosphotyrosine assay
To assay phosphotyrosine in cdc2a, the cdc2a enzyme fraction was recovered
essentially as described supra for the cdc2 activity assay, except that 5 mg
of
extracted plant protein was used as starting material, and the NDE buffer was
modified
to include 2.5 mM sodium vanadate and 1 mM phosphotyrosine, and the immune
complexes were washed with HDW buffer supplemented with 1 mM with sodium
vanadate.
Western blots of cell-derived protein were probed with anti-phosphotyrosine
mouse
monoclonal (PY99, Santa Cruz Biotechnology, S.C., USA), followed by ['251]-
labelled
second antibody, and the signal obtained was detected by Phosphorimage
analysis.
Northern blots
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RNA was extracted from cells ground in liquid nitrogen, into 2 volumes of 10
mM Tris/
HCI (pH 8.0), 100 mM NaCI, 1 mM EDTA, 1 % (w/v) SDS and 2 volumes of
phenol:chloroform:iso-amylalcohol 25:24:1 at 4°C and fractionated.
RNA was electrophoresed on agarose gels, transferred to membrane and probed
with
the 65- by Bglll-Xbal fragment of the Cdc25 gene, using standard procedures.
EXAMPLE 2
Expression of yeast Cdc25 makes cell division in plant cells independent
of cell division
The effect of ectopic expression of yeast Cdc25 in plant s was investigated
because
cells arrested by lack of cytokinin, whether derived from suspension culture
or excised
freshly from the plant, have abundant cdc2 protein that is enzymically
inactive because
phosphorylated at tyrosine.
Latent cdc2 protein kinase activity can be released in vitro by incubation
with the
phosphoprotein phosphatase Cdc25 that is specific for cdc2. When cytokinin
stimulates entry into mitosis, dephosphorylation of cdc2 is one of the events
that occur,
but it was uncertain whether the hormone might have several effects in the
cell cycle.
To test this possibility we therefore arranged the inducible expression of the
fission
yeast Cdc25 gene in tobacco under the control of a dexamethasone-inducible
promoter. Only if the sole essential action of cytokinin is to cause
dephosphorylation
and activation of cdc2 kinase can the ectopic expression of the Cdc25 gene
substitute
for presence cytokinin at mitosis.
We now report that the sole essential action of cytokinin in sustaining cell
division is
activation of Cdc25 since the hormone can be substituted by expression of this
gene.
Levels of the fission yeast enzyme Cdc25 that removes inhibitory phosphate
from
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tyrosine in cdc2 kinase were brought under genetic control in the plant by
joining the
yeast Cdc25 gene to a modified plant promoter that contained rat
glucocorticoid
response elements (GREs), which are responsive to rat glucocorticoid receptor
protein
(GR) in the presence of dexamethasone and therefore allowed induction without
interference from plant hormones. The GRE-Cdc25, together with the
constitutively
expressed NOS promoter-GR construct, were inserted into the vector pBin19,
which
contains pnos:nptll for kanamycin resistance, and introduced into cells of N.
plumbaginifolia by electroporation into protoplasts. Clones resistant to
kanamycin
were tested for ability to form a colony on solid medium containing
dexamethasone
and auxin but no cytokinin.
At the high concentration of 10 ~cM dexamethasone, cells commonly arrested at
prophase in mitotic catastrophe but lower inducer concentrations allowed
colony
formation and generated cell lines in which inducible expression of Cdc25 was
detected by Western blot analysis using antibody against glutathione-S-
transferase
(GST)-Cdc25 fusion protein.
Inducible cell lines contained yeast Cdc25 DNA (detected in Southern blots,
not
shown) and in 0.01-10 ,uM dexamethasone they accumulated Cdc25 mRNA and
protein (Figure 1-1; Figure 1-2). Effects on division were tested in cells
that had been
arrested at the G2 phase hormonal control point by depletion of auxin and
cytokinin
followed by provision of auxin only. Dexamethasone at 0.01-10 ~cM induced
division
(Figure 1-3) and a sharp optimum concentration of 0.1 ,uM dexamethasone was
observed in independent clones, consistent with requirement for a critical
optimum
Cdc25 activity. No cell division was observed without inducer, or in
untransformed
cells treated with dexamethasone (Figure 1-3). Three independent lines were
analysed biochemically and had similar properties. Results from one line are
shown.
The experimental system used for subsequent experiments involved the prior
arrest
of suspension culture cells, at the cytokinin control point in late G2 phase.
Arrest at
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this point was obtained by incubation without hormone and then with auxin (2,4-
D)
without cytokinin. Mitosis could then be induced by addition of cytokinin, or
alternative
potentially mitogenic treatments could be tested. Progress through prophase is
a little
slower after this arrest than in cells not emerging from hormonal block and is
very
suitable for study of the succession of biochemical events in plant mitosis.
Induced synthesis of Cdc25 in cells at the cytokinin control point in late G2
resulted in
appearance of Cdc25 activity, which was detected by its activation of yeast
cdc2 HI
histone kinase that was provided a substrate in low activity form,
phosphorylated on
tyrosine 15 and amenable to activation by Cdc25 (Figure 2-1 to Figure 2-7).
The
induced Cdc25 phosphatase activity peaked at 6 h and provides an explanation
for the
increase in cdc2 kinase activity, which increased while Cdc25 was active
(Figure 2-2).
Specific recovery of cdc2a and Cdc25 was indicated by precompetition with
cdc2a
peptide antigen and by preimmune anti-Cdc25 serum or anti-Cdc25 antibody
precompeted with inactive GST-Cdc25 (Figure 2-1).
To test whether the effectiveness of ectopically expressed Cdc25 derived from
the
operation of mechanisms present in normal mitosis, transgenic cells induced
with
dexamethasone were monitored for Cdc25 phosphatase and cdc2 kinase activity
(Figure 2-2; Figure 2-3) in parallel with cells induced with cytokinin (Figure
2-3; Figure
2-4). Both showed increase in Cdc25 activity and then cdc2a kinase activity
leading
to division. A control over Cdc25 activity at post-translational level is
indicated by the
absence of a higher Cdc25 catalytic activity when yeast enzyme was expressed
in
addition to the endogenous Cdc25 (Figure 2-2; Figure 2-4). This suggests that
the
additional yeast enzyme comes under homeostatic controls that are conserved
between yeasts and plants. Post-translational control of Cdc25 activity is
known to be
complex, tolerant to different levels of the protein, and to involve
activating
phosphorylations and ubiquitin-directed proteolysis.
The temporal correlation of induced Cdc25 phosphatase activity with increase
in cdc2
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kinase activity (Figure 2-2; Figure 2-4) suggested that the phosphatase is
responsible.
We tested this by investigating whether Cdc25 enzyme could activate cdc2 from
cells
in prophase and whether the extent of activation by Cdc25 declined when
activation
had already occurred in vivo. Data presented in Figure 2-5 show that excess
bacterially-synthesised GST-Cdc25 could activate plant cdc2 enzyme that was
extracted in prophase between 3 hours and 12 hours, and that the extent of
activation
declined in proportion with activation that had previously occurred. These
data are
consistent with the increase in Cdc25 phosphatase driving prophase progression
by
dephosphorylating cdc2. After 12 hours, cdc2 activity declined during anaphase
and
the enzyme then became unresponsive to GST-Cdc25, consistent with the anaphase
decline in activity being due to proteolysis of cyclin, as observed for cyclin
1b in maize
mitosis.
The low level of Cdc25 activity in cells that are arrested by limiting
cytokinin, as at time
zero (i.e. 0 hours) in Figure 2, indicates that down-regulation of Cdc25
activity is part
of the cytokinin control mechanism and that induced Cdc25 therefore provides a
biologically relevant signal. The resulting daughter cells were viable;
indicating that
mitosis driven by induced Cdc25 is functionally normal. These daughter cells
could
proliferate indefinitely with dexamethasone replacing cytokinin and required
nine-fold
dilution every 7 days precisely as in control cultures provided with auxin and
cytokinin.
They are routinely maintained in dexamethasone without cytokinin. Thus,
unexpectedly the data provided herein reveal that the sole essential action of
cytokinin
in sustaining cell division is activation of Cdc25 and the hormone can be
substituted
by expression of the Cdc25 gene.
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EXAMPLE 3
Evidence for the presence of Cdc25 protein in plant cells
The ability of yeast Cdc25 to influence cytokinin-mediated cell division in
plants
suggested to the present inventors that the yeast protein replaces the
activity of an
endogenous plant Cdc25 enzyme that is activated by cytokinin. To demonstrate
that
this is the case, the effectiveness of induced yeast Cdc25 produced in
transformed
plant cells to activate cdc2, was compared to the effectiveness of a putative
plant-
derived Cdc25 from genetically unmodified cells to activate cdc2.
The yeast and putative plant Cdc25 enzymes recovered by immunoprecipitutien
using
anti-Cdc25 antibody were compared in reaction with tyrosine phosphorylated
plant
cdc2 enzyme taken from cells arrested at the G2 control point (Figure 2-6,
lanes 1-3).
Substrate cdc2 was also taken from cells after 3 hours stimulation with
cytokinin
(Figure 2-6, lanes 4-6), when partial cdc2 activation had occurred (Figure 2-
5; Figure
2-6, lanes 1 and 4).
The activation of plant cdc2 by yeast Cdc25 expressed in plant cells (Figure 2-
6, lane
6) demonstrates a mechanism by which Cdc25 can substitute for cytokinin.
Furthermore this activation mechanism is a normal part of plant mitosis,
because non-
transgenic plant cells also contain a Cdc25 activity, unambiguously of plant
origin, that
is both present following cytokinin stimulation and capable of activating
plant cdc2
(Figure 2-6, compare lanes 2 and 5). Moreover, the plant Cdc25 activity is
slightly more
effective than the heterologous yeast Cdc25 in activating plant cdc2 in the
tobacco
cells tested (Figure 2-6, compare lanes 5 and 6).
We also assayed phosphotyrosine in the cdc2a kinase that increased in activity
when
the hormonal block was released. As shown in Figure 2-7, levels of tyrosine
phosphate in cdc2a declined after induction of Cdc25, as the catalytic
activity of cdc2a
increased (Figure 2-2), indicating that a decline in phosphotyrosine caused by
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induction of Cdc25 in transgenic cells stimulates entry of cells into mitosis.
To further test the evidence for Cdc25 presence in genetically unmodified
plant cells,
we tested for immunological cross-reactivity between plant Cdc25 and authentic
fission
yeast Cdc25. In western blot analyses, antibodies against fission yeast Cdc25
detected a protein of 67 kDa in a tobacco cell fraction obtained using the
mitotic
protein p13s~°, as an affinity ligand to purify cell cycle proteins
(Figure 3, lane 1).
Moreover, the binding of antibody to this 67 kDa tobacco protein was
eliminated by
pre-competition with authentic yeast Cdc25 protein (Figure 3, lane 2),
suggesting that
the yeast and plant Cdc25 protein share protein epitopes, such as primary
amino acid
sequences, secondary, or tertiary structures. The size of the 67 kDa tobacco
protein
correlates with the known size of other Cdc25 molecules.
EXAMPLE 4
Expression of Cdc25 under the control of the patatin gene promoter increases
tuber size and number in potato plants
The fission yeast Cdc25 coding sequence is cloned between the promoter of a
class
I patatin gene ( Liu et al.,1991 ) and the transcription termination signals
of the nopaline
synthase (NOS) gene of Agrobacterium tumefaciens. Preferentially, the B repeat
region and the distal region of the A repeat of the patatin promoter is used,
without the
proximal region of the A repeat. The proximal region of the A repeat of the
patatin
promoter confers sucrose-responsiveness in various tissues, which is not a
desirable
characteristic for our purposes (Grierson et al., 1994). This construct is
placed in a
binary vector, mobilized to Agrobacferium tumefaciens, and the introduced into
potato
plants.
The Cdc25 protein is expressed under the control of the Class I patatin
promoter when
the first stolon starts to tuberize, consistent with the expression pattern
for the patatin
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gene (Liu et al., 1991). At this stage, expression is associated with both
internal and
external phloem. After tuber induction has occurred, promoter activity is
found both in
tuberized stolons and in non-tuberized stolons. Expression then expands to the
entire
storage parenchyma, cortex and pith, but remains absent from the periderm.
Because the Class II patatin promoters are expressed in the periderm and as
such are
complementary to the Class I promoters (Koster-Topfer et al.,; Liu et al.,
1991; Nap et
al., 1992), it is beneficial to have Cdc25 expression driven by both Class I
and Class
II promoters within the same plant. Because the Class I patatin promoter is
not
expressed before the first stolon initiates tuberization, no effects of Class
I patatin-
Cdc25 transgenes is seen on tuber initiation. However, the Class I patatin
promoter
drives Cdc25 expression very early after tuber initiation onwards, allowing a
maximal
impact of Cdc25 activity on organ formation and, as a consequence, on tuber
size. The
fact that the Class I patatin promoter activity subsequently also appears in
non-
tuberized stolons implies that the Class I patatin - Cdc25 transgene increases
both the
size and number of tubers.
EXAMPLE 5
Expression of Cdc25 under the control of the SAUR gene promoter or the A.
rhizogenes rolB promoter increases lignin in poplar plants
The fission yeast Cdc25 coding sequence is cloned between the promoter of the
soybean SAUR gene (Li et al., 1992 ) and the transcription termination signals
of the
nopaline synthase (NOS) gene of Agrobacterium tumefaciens. The SAUR promoter
is inducible by auxins. This chimeric genetic construct is introduced between
the T-
DNA borders of the binary vector pB1121 or similar vector and mobilised into
Agrobacterium tumefaciens. Poplar is transformed by Agrobacterium-mediated
transformation using standard procedures.
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Transgenic poplar trees containing this construct show increased lignin
content,
correlated with an increased stem diameter and the higher ratio of vascular
tissue to
pith and cortex cells.
A similar phenotype in poplar is produced when the Cdc25 expression is driven
by the
rol8 promoter of Agrobacterium r~hizogenes (Nilsson et al.,1997), that is
expressed in
cambial cells (i.e. the dividing cells of the vascular tissue).
EXAMPLE 6
Expression of Cdc25 under the control of endosperm-specific promoters
increases grain size and yield of grain crop plants
The fission yeast Cdc25 coding sequence is placed operably in connection with
the
endosperm-specific ltr1 promoter from barley, or a synthetic promoter
containing the
endosperm box (GCN motif) of the barley Hor2 gene (Vicente-Carbajosa et
al.,1998).
In each case, the Cdc25 structural gene is placed upstream of the
transcription
termination signals of the Agrobacterium tumefaciens nopaline synthase (NOS)
gene.
Cereals, in particular rice, maize, wheat and barley, are transformed using
standard
procedures, in particular microprojectile bombardment or Agrobacterium-
mediated
transformation systems, with the genetic constructs.
The grain size and starch storage capacity of the endosperm of the seeds of
transformed plants is increased relative to otherwise isogenic non-transformed
plants.
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EXAMPLE 7
Expression of Cdc25 under the control of meristem-specific promoters
reduces apical dominance in A. thaliana and B. napus plants
The fission yeast Cdc25 coding sequence is placed operably in connection with
the
shoot meristem-specific LEAFY promoter (Weigel et al., 1992), or the KNOTTED-
like
Arabidopsis thaliana knat1 promoter (Accession number AJ131822), or the
KNOTTED-like Malus domestics kn1 promoter ( Accession No. Z71981), or the
Arabidopsis thaliana CLAVATA1 promoter (Accession number AF049870). In each
case, the Cdc25 structural gene is placed upstream of the transcription
termination
signals of the Agrobacterium tumefaciens nopaline synthase (NOS) gene. A.
thaliana
and Brassica napus plants are transformed as described by Bechtold et al.,
1993.
Transformed plants exhibit cytokinin-like effects at the level of the shoot
(and flower)
meristem, resulting in reduced apical dominance.
EXAMPLE 8
Expression of Cdc25 under the control of the cab-6 or ubi7 promoters
reduces leaf necrosis and chlorosis in lettuce plants
The fission yeast Cdc25 coding sequence is placed operably in connection with
the
leaf-specific cab-6 gene promoter derived from Pinus (Yamamoto et al., 1994)
or
senescence-specific ubi7 gene promoter (Garbarino et al., 1995). In each case,
the
Cdc25 structural gene is placed upstream of the transcription termination
signals of the
Agrobacterium tumefaciens nopaline synthase (NOS) gene. Lettuce is transformed
as described by Bechtold et al., 1993.
Leaf deterioration (chlorosis and necrosis) in lettuce, for example as a
consequence
of post-harvest storage, is delayed in transformed lettuce plants compared to
non-
transformed control plants.
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EXAMPLE 9
Cdc25 as a selectable marker gene in transformed plants
In the present example of the application of the present invention, pith
tissue excised
from tobacco plants according to the method described by Zhang et al (1996) is
treated with Agrobacterium tumefaciens containing the binary vector pBIN19
comprising within the T-DNA borders the following genetic constructs:
(1) the gfp gene placed operably under control of the CaMV 35S promoter
and upstream of the NOS terminator;
(2) the fission yeast Cdc25 gene placed operably under the control of a
modified plant promoter that contains rat glucocorticoid response elements
(GREs), which are responsive to rat glucocorticoid receptor protein (GR) in
the
presence of dexamethasone (Schena et al., 1991 ); and
(3) a NOS promoter-GR gene construct that expresses GR constitutively in
plant cells.
The green fluorescent protein is targeted to the endoplasmic reticulum by
virtue of the
inclusion of an appropriate signal peptide sequence being encoded in the
genetic
construct.
The binary vector is introduced into plant cells, essentially according to
Bechtold et al.
(1993).
When the transformed plant tissue is incubated on agar medium that contains
auxin
and 1 ~M dexamethasone, but lacking cytokinin, only cells containing the
binary vector
in a format such that the expression of Cdc25 occurs therein are capable of
proliferating and form transgenic callus. Untransformed cells fail to
proliferate and
eventually are overgrown and die. None of the surviving cells fail to express
green
fluorescent protein, indicating that the frequency of escapes using the
inventive
method is very low to negligible.
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Transformed calli are initially on agar containing medium with dexamethasone
and
auxin but no cytokinin, to obtain selection for transgenic cells, which alone
are able to
proliferate. The calli resulting from sustained rapid proliferation of these
cells are then
transferred to medium without dexamethasone, but with both auxin and cytokinin
to
allow shoot formation. Finally, to allow root formation, shooting calli are
transferred to
medium without auxin, or alternatively or in addition, with the root-promoting
hormone
indole butyric acid.
In broadly applying the inventive method, the gfp reporter gene construct is
substituted
with a gene-of-interest that encodes a desired characteristic, such as a
desired
agronomic trait, for which positive selection for successful transfer is
sought.
An advantage of the inventive method is that, whilst cytokinin expressed from
the ipt
gene or exogenously supplied from the medium allows the proliferation of
neighbouring
non-transgenic cells, the ectopic expression of Cdc25 in transformed cells is
highly
localised and, as a consequence, highly-specific. Accordingly, the present
invention
overcomes the need to carry out extensive genetic crossing to eliminate
progeny that
do not contain the desired gene, in order to establish lines able to transmit
the new
character to future generations according to standard Mendelian inheritance.
Moreover, in the present inventive method, the plantlet relies entirely on
normal
endogenous hormone production during the later regeneration stages and
develops
into a normal plant. The present method generates plants no longer expressing
Cdc25,
completely normal in growth, and having gained the new beneficial trait
conferred by
gene transfer. In contrast, when cytokinin is expressed from the ipt gene or
exogenously supplied from the medium, the continuing raised cytokinin
synthesis alters
development and growth of plantlets.
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