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 11
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, said method
comprising expressing a cell cycle control protein, in particular cyclin B, in
the plant,
operably under the control of a regulatable promoter sequence. Preferably, the
characteristics modified by the present invention are cytokinin-mediated
and/or
gibberellin-mediated characteristics. The present invention extends to gene
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
"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
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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, jasmonic 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
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.
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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-inducibfe 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). These approaches also produce undesirable
side-effects in the plant and, even in cases where ipf or rolC is expressed
under the
control of tissue-specific promoters, these side-effects are observed in other
tissues,
presumably because the cytokinin is transported readily between cells and
tissues of
the plant.
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 such as cyclin B in particular cells, tissues or organs
of the plant
produces localised specific modifications to cytokinin-mediated cellular
metabolism
and cell fate compared to otherwise isogenic non-transformed plants.
More particularly, the inventors have discovered that the G2 phase of the cell
cycle in
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plants can be shortened by the ectopic expression of the alfalfa CycMs2
protein
therein. This shortening of the G2 phase results in altered cell fate and
sink/source
relationships in plants expressing CycMs2, thereby mimicking many of the
cytokinin-
mediated and/or gibberellin-mediated developmental and biochemical processes
which
can be induced by the exogenous application of phytohormones, without the
undesirable side-effects of the prior art.
The shortening of the G2 phase by ectopic expression of alfalfa CycMs2 in
tobacco is
shown in Example 3. Whilst not being bound by any theory or mode of action, it
is
likely that the ectopic expression of a cyclin B such as alfalfa CycMs2, or
ectopic
expression of a cyclin B-type protein, in plants, advances cell division by
advancing the
entry of cells into mitosis, which modifies cellular metabolism via a
mechanism that
involves modifications to the partitioning of carbon and/or the activities of
one or more
enzymes involved in carbon partitioning (eg. invertase) and/or the levels of
regulatory
molecules such as sucrose, ATP, ADP and inorganic orthophosphate. Because cell
fate is in some way related to the cell cycle, the altered cellular metabolism
which
occurs is able to modify cell fate. For example, roots can be regenerated from
calli in
culture when the cells exit the cell cycle in G2 phase, while shoot formation
is
dependent upon an exit from the cell cycle in the G1 phase, such that the
formation
of roots versus shoots depends upon the phase (G1 or G2) at which cells exit
the cell
cycle. This proposed mechanism may explain the inhibition of root regeneration
observed by the present inventors (see Example 4) when the G2/M transition
and/or
the duration of the G2 phase is shortened by ectopic expression of CycMs2.
Cyclin B 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 alfalfa CycMs2 in
particular
cells, tissues and organs of plants, for the purposes of modifying cytokinin-
mediated
plant morphology and/or biochemistry and/or physiology, and to facilitate the
selection
of specific cells, tissues and organs which exhibit cytokinin-mediated
morphological
characteristics and/or biochemical characteristics andlor physiological
characteristics.
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Accordingly, one aspect of the invention provides a method of modifying cell
fate
and/or plant development and/or 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, and, in particular a
cyclin
protein, operably in connection with a plant-operable promoter sequence.
Expression of the cell cycle control protein is preferably carried out by
introducing an
isolated nucleic acid molecule comprising the protein-encoding nucleotide
sequence
into a cell, tissue or organ of the plant, regenerating plant tissue or whole
plants
therefrom and then culturing those plant parts or whole plants under
conditions
suitable for activity of the promoter sequence to which said nucleotide
sequence is
operably connected.
Preferably, the genetic sequence encoding the cyclin protein is placed
operably under
the control of a plant-expressible promoter sequence selected from the list
comprising
strong constitutive promoter sequences, cell-specific promoter sequences,
tissue-
specific promoter sequences, organ-specific promoter sequences, cell-cycle-
specific
promoter sequences, and inducible promoter sequences (both pathogen-inducible
and
environmentally-inducible promoters are contemplated herein). The present
invention
further encompasses the use of a promoter sequence in a gene construct wherein
an
excisable genetic element is inserted into said construct so as to inactivate
expression
of the cyclin protein during transformation and regeneration steps. According
to this
embodiment, excision of the excisable genetic element in the regenerated plant
or
progeny derived therefrom facilitates ectopic expression of the cyclin
protein. Methods
to induce excision of such genetic elements are known to those skilled in the
art. The
excisable genetic element may be an autonomous or non-autonomous excisable
genetic element.
In a particularly preferred embodiment of the invention, the cyclin protein is
a cyclin B
protein, and, more particularly, the alfalfa CycMs2 protein, or a biologically-
active
homologue, analogue or derivative thereof. The present invention clearly
contemplates
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the use of functional homologues of cyclin B proteins. Accordingly, the
present
invention is not limited in application to the use of nucleotide sequences
encoding the
alfalfa CycMs2 protein.
The present invention clearly extends to the use of modified cyclin B
proteins, or
substrates of a cyclin B protein, or modified substrates of a cyclin B
protein, that
produce the same effects in respect of the present invention as can be
produced using
a cyclin B protein, in particular the alfalfa CycMs2 protein described herein.
The ectopic expression of a cyclin protein or a homologue, analofue or
derivative
thereof in a plant can produce a range of desirable phenotypes in plants, such
as, for
example, by modifying one or more morphological, biochemical, or physiological
characteristics as follows: (i) modifying the length of the G2 phase of the
cell cycle of
a plant; (ii) modifying the G2/M phase transition of a plant cell; (iii)
modification of the
initiation, promotion, stimulation or enhancement of cell division; (iv)
modification of
the initiation, promotion, stimulation or enhancement of DNA replication;(v)
modification of the initiation, promotion, stimulation or enhancement of seed
set and/or
size and/or development; (vi) modification of the initiation, promotion,
stimulation or
enhancement of tuber formation; (vii) modification of the initiation,
promotion,
stimulation or enhancement of shoot initiation andlor development; (viii)
modification
of the initiation, promotion, stimulation or enhancement of root initiation
and/or
development; (ix) modification of the initiation, promotion, stimulation or
enhancement
of lateral root initiation and/or development; (x) modification of the
initiation, promotion,
stimulation or enhancement of nodule formation andlor nodule function; (xi)
modification of the initiation, promotion, stimulation or enhancement of
bushiness of
the plant; (xii) modification of the initiation, promotion, stimulation or
enhancement of
dwarfism in the plant; (xiii) modification of the initiation, promotion,
stimulation or
enhancement of pigment synthesis; (xiv) modification of source/sink
relationships; (xv)
modification of carbon partitioning in the plant; (xvi) modification of the
initiation,
promotion, stimulation or enhancement of senescence; and (xvii) modification
of stem
thickness and/or strength characteristics and/or wind-resistance of the stem.
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As used herein, unless specifically stated otherwise, the term "modification
of the
initiation, promotion, stimulation or enhancement" in relation to a specified
integer shall
be taken as a clear indication that the integer is capable of being enhanced,
increased,
stimulated, or promoted, or alternatively, decreased, delayed, repressed, or
inhibited.
In a preferred embodiment of the present invention, a cyclin B protein or a
homologue,
analogue or derivative thereof, such as, for example, a modified substrate of
cyclin B
that mimics the effect of cyclin B is expressed operably under the control of
a
regulatable promoter that is expressible in a plant cell, to shorten the
duration of the
G2 phase and/or to shorten the G2/M phase transition of said cell.
In another preferred embodiment of the present invention,a cyclin B protein or
a
homologue, analogue or derivative thereof, or a modified substrate of cyclin B
that
mimics the effect of cyclin B is expressed operably under the control of a
regulatable
promoter that is expressible in a plant cell, tissue or organ to modify cell
fate and/or
plant development.
Other preferred embodiments of the invention relate to the effects) of
cytokinins
and/or gibberellins on plant metabolism. With respect to such hormone-mediated
effects, the present invention clearly contemplates the broad application of
the
inventive method to the modification of a range of cellular processes mediated
by
cytokinins and/or gibberellins, including but not limited to cellular
development and/or
cell fate; the advancement of cell division; the initiation, promotion,
stimulation or
enhancement of seed development andlor tuber formation and/or shoot initiation
andlor bushiness and/or dwarfism and/or pigment synthesis, andlor the
modification
of source/sink relationships, and/or the modification of root growth and/or
the inhibition
of root apical dominance and/or the delay of senescence.
Accordingly, in another preferred embodiment of the present invention, a
cyclin B
protein or a homologue, analogue or derivative thereof, or a modified
substrate of
cyclin B that mimics the effect of cyclin B is expressed operably under the
control of
a regulatable promoter that is expressible in a plant cell, tissue or organ to
modify
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carbon partitioning between the cells, tissues, or organs of plants.
In another preferred embodiment of the present invention, a cyclin B protein
or a
homologue, analogue or derivative thereof, or a modified substrate of cyclin B
that
mimics the effect of cyclin B is expressed operably under the control of a
regulatable
promoter that is expressible in a plant cell, tissue or organ to modify
sink/source
relationships in the plant, and, in particular, in the seed of a plant. For
modification of
sink/source relationships in the seed of a plant, the cyclin protein is
preferably
expressed under the control of a promoter sequence that is operable in the
endosperm
of the seed, in which case the seed produced exhibit enhanced grain filling
and higher
levels of starch in the dried seed than the seed of otherwise isogenic plants.
In another preferred embodiment of the present invention, a cyclin B protein
or a
homologue, analogue or derivative thereof, or a modified substrate of cyclin B
that
mimics the effect of cyclin B 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, seed size, and seed yield. More preferably,
the
promoter is operable in the endosperm of the seed or in the storage cotyledon,
in
which cases the combination of the cell cycle-control protein and endosperm-
expressible or cotyledon-expressible promoter provides the additional
advantage of
increasing the grain size and grain yield of the plant.
In still another preferred embodiment of the present invention, a cyclin B or
a
homologue, analogue or derivative thereof, or a modified substrate of cyclin B
that
mimics the effect of cyclin B is expressed operably under the control of a
promoter
derived from a leaf-expressible gene, to prevent or delay or otherwise reduce
leaf
chlorosis andlor leaf necrosis and/or leaf sensecence.
In yet another preferred embodiment of the present invention, a cyclin B
protein or a
homologue, analogue or derivative thereof, or a modified substrate of cyclin B
that
mimics the effect of cyclin B is expressed operably under the control of a
promoter
derived from a meristem-expressible gene or a shoot-expressible gene or root-
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expressible gene, to reduce bushiness of the plant. In a related embodiment,
such
promoter/cyclin B combinations are used to reduce root apical dominance in
plants.
In another preferred embodiment of the present invention, a cyclin B protein
or a
homologue, analogue or derivative thereof, or a modified substrate of cyclin B
that
mimics the effect of cyclin B 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, a cyclin B protein
or a
homologue, analogue or derivative thereof, or a modified substrate of cyclin B
that
mimics the effect of cyclin B 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, a cyclin B protein or a
homologue,
analogue or derivative thereof, or a modified substrate of cyclin B that
mimics the
effect of cyclin B 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 cyclin B 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
auxins and are therefore the preferential tissue for cyclin B overproduction.
In a further preferred embodiment of the present invention, a cyclin B protein
or a
homologue analogue or derivative thereof, or a modified substrate of cyclin B
that
mimics the effect of cyclin B 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
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and/or to generate a more extensive photosynthetic canopy.
A second aspect of the invention provides a gene construct or vector
comprising a
nucleotide sequence that encodes a cyclin protein, such as, for example, a
cyclin B
protein, and in particular, the alfalfa CycMs2 protein, or a homologue,
analogue, or
derivative thereof, placed operably under the control of a plant-expressible
promoter
sequence selected from the group consisting of:
(i) a plant-expressible cell-specific promoter sequence;
(ii) a plant-expressible tissue-specific promoter sequence;
(iii) a plant-expressible organ-specific promoter sequence;
(iv) a plant expressible inducible promoter sequence;
(v) a plant-expressible cell cycle specific gene promoter sequence;
(vi) a plant-expressible constitutive promoter sequence, wherein the
nucleotide sequence encoding said cyclin protein, and the plant-expressible
constitutive promoter sequence, are integrated into an excisable genetic
element; and
(vii) a plant-expressible constitutive promoter sequence, wherein the
nucleotide sequence encoding saidcyclin protein and said promoter sequence
are such that expression of said substrate or modified substrate is capable of
being modulated by an excisable genetic element.
Preferably, the gene 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 gene construct or vector is suitable for introduction into and
maintenance
in a plant cell, tissue, organ or whole plant.
A third 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 cyclin 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, a plant-
expressible
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cell cycle gene specific promoter, or alternatively, 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
an excisable genetic element.
This aspect of the invention extends to plant propagules and plant parts that
contain
the introduced nucleic acid molecule and have the potential to reproduce one
or more
of the phenotypes of the primary transformants/transfectants, either by
inducing gene
expression directly therein or by the application of standard breeding or
recombinant
technologies.
BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1-1 is a copy of a photographic representation of a northern blot
hybridisation
showing the expression of alfalfa CycMs2-HA mRNA in the leaves of six
transgenic
tobacco lines from a tetracyclin-regulatable promoter sequence, either in the
absence
(-) or presence (+) of 1 mg/dm 3 CI-tetracycline. The positions of CycMs2-HA
mRNA
and a control mRNA (pCNT6) are indicated.
Figure 1-2 is a copy of a photographic representation of a northern blot
hybridisation
showing the time-dependent accumulation of alfalfa CycMs2-HA mRNA in a
suspension culture initiated from line 2 (Figure 1-1) on 2,4-D-containing
medium and
subsequently incubated in medium containing 1 mg/dm 3 CI-tetracycline. The
positions
of CycMs2-HA mRNA and a control mRNA (CycM) are indicated. Numbers at the top
of the figure indicate elapsed time (hours) following addition of
tetracycline.
Figure 1-3 is a copy of a photographic representation showing the tetracycline
concentration-dependent expression of CycMs2-HA fusion protein in cultured
tobacco
cells. In the top panel, a western blot of total cellular extracts was probed
with anti-HA
antibody. In the middle panel, a western blot of immunopurified CDK complexes
were
probed with anti-HA antibodies. In the lower panel, CDK complexes were
purified by
binding to the p13 S~°, protein. The position of the CycMs2-HA fusion
protein is
indicated in the upper and middle panels, whilst the position of total p13
S~°, protein-
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binding CDK fraction is indicated in the lower panel (p13 S~°,).
Figure 2 is a copy of a photographic representation of an immunofluorescence
micrograph probed with anti-HA antibody, showing the expression of the CycMs2-
HA
fusion protein in the nuclei of transgenic tobacco cells following induction
by
tetracycline. As a control, cells were stained using DAPI to identify the
nuclear DNA.
Figure 3 is a copy of a photographic representation showing the histone-kinase
activities of CycMs2-HA-associated CDKs (i.e. CycMs2-HA) and p13 S~', protein-
bound CDKs (p13 S~°, ) in total cellular extracts, and in the nuclear
and cytoplasmic
fractions of tobacco cells expressing alfalfa CycMs2 as a fusion protein with
a
haemaglutinin epitope tag (HA).
Figure 4-1 is a copy of a graphical representation showing the percentage of
aphidicolin-released tobacco cells entering mitosis following tetracycline
induction of
CycMs2 gene expression (+) or in the absence of tetracycline induction (-), as
a
function of time.
Figure 4-2 is a copy of a graphical representation showing the percentage of
microtubule structures in aphidicolin-released tobacco cells following
tetracycline
induction of CycMs2 gene expression (+) or in the absence of tetracycline
induction
(-), as a function of time.
Figure 4-3 is a copy of a graphical representation showing the percentage of
aphidicolin-released tobacco cells entering mitosis following tetracycline
induction of
CycMs2 gene expression (+) or in the absence of tetracycline induction (-), as
determined by flow cytometry and expressed as a function of time.
Figure 4-4 is a copy of a graphical representation showing the shortened
duration of
the G2 phase in aphidicolin-released tobacco cells following tetracycline
induction of
CycMs2 gene expression (+) compared to isogenic cells incubated in media
lacking
tetracycline (-).
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Figure 5 is a copy of a photographic representation of a northern blot
hybridisation
showing the expression of CycMs2 mRNA in G2 phase cells ectopically expressing
the
CycMs2-HA fusion protein, compared to the levels of histone H4 mRNA; the
endogenous tobacco CycM mRNA; and the control pCNT6. Cells expressing the
CycMs2-HA fusion protein were incubated in the presence of tetracycline (+
tetracycline), whilst cells not expressing this fusion protein were incubated
in the
absence of tetracycline (- tetracycline).
Figure 6 is a copy of a photographic representation showing the inhibition of
root
regeneration from tobacco leaves of transgenic fines that ectopically express
alfalfa
CycMs2 mitotic cyclin.
Figure 7 is a copy of a graphical representation of a flow cytometric analysis
of the
DNA contents in tetracyclin-treated (+tet) and untreated (-tet) tobacco leaves
or leaf
discs, in the absence of cytokinin and auxin (0/0), or treated with different
cytokinin:auxin ratios (i.e. 0.5/0.1; 0.1/0.5) showing the higher percentage
of cells with
G2 DNA content in leaf cells derived from wild-type plants treated with high
auxin:cytokinin [0.5/0.1 (root)], compared to leaf cells derived from wild-
type plants
treated with low auxin:cytokinin (0.1/0.5 (shoot)] or on equivalent levels of
cytokinins
and auxins [ 0.5/0.5(callus)].
Figure 8 is a copy of a schematic representation of non-limiting cell cycle
model of
shoot/root regeneration.
Figure 9 is a copy of a photographic representation showing the ectopic
expression
of CycMs2 retards plant growth and mimics cytokinin effect in dark-growth
seedlings.
TM 100 2/5 are seedlings transformed with CycMs2. Bin Hyg are transformants
with
the control plasmid pain-HygTX.
Figure 10 is a copy of a photographic representation of a Epifluorescence
microphotograph of GFP fluorescence (A); and DIC phase contrast image of the
cell
(B) transformed with a CycMs2-GFP fusion.
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DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
One aspect of the present invention provides a method of modifying cell fate
and/or
plant development and/or plant morphology and/or biochemistry and/or
physiology
comprising expressing in particular cells, tissues or organs of a plant,
comprising
expressing in said cells, tissues or organs 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.
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
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 "cell fate and/or plant development and/or plant morphology
and/or
biochemistry and/or physiology" is meant that one or more developmental and/or
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.
"Cell fate" refers to the cell-type or cellular characteristics of a
particular cell that are
produced during plant development or a cellular process therefor, in
particular during
the cell cycle or as a consequence of a cell cycle process. Our results
presented
herein indicate that increased mitotic cyclin expression produces a shortened
G2-phase in plant cells. It is likely that mitotic cyclin overexpression has a
cytokinin-like
effect, evidenced by our finding that mitotic cyclin overproduction results in
the
suppression of root formation.
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The shape of a plant is the result of the polarity of auxin and cytokinin.
Auxins are
produced in the shoot tips where they maintain the shoot meristem. They
suppress
further shoot formation (shoot apical dominance) and induce root formation.
Cytokinins are produced in the root tips where they maintain the root
meristem. They
suppress further root formation and induce shoot formation. Thus, root
suppression
by mitotic cyclin overexpression can be seen as a cytokinin effect. A further
finding
was that, at a critical period in root formation from leaf disks (two weeks
after
incubating excised leaf disks on an auxin-containing medium), dividing cell
populations
giving rise to roots had an extended G2 phase. Tetracycline-induced mitotic
cyclin
overexpression abolished this extended G2 phase and suppressed root formation,
suggesting a link between a long G2 phase and root formation. This is the
first time
that it has been shown that the experimentally controlled length of a phase of
the plant
cell cycle dramatically alters the developmental fate of these cells.
The fact that cyclin overexpression throughout the plant affects not only the
length of
a cell cycle phase but suppresses a specific developmental potency of certain
cells
such as root cells, indicates that cyclin overexpression mimics a specific
effect of a
plant hormone and has negative pleiotropic effects, at least in these cells.
Accordingly, a novel strategy for designing novel crops would therefore be to
overexpress or to repress the expression of specific cyclins in specific cell,
tissue or
organ types via the use of appropriate regulatable promoter sequences, in
order to
modify the pleiotropic effects linked to the specific cyclin, and, in
particular to modify
those peiotropic effects that are hormone-mediated (such as, for example
cytokinin-
mediated andlor gibberellin-mediated effects) in the plant. In the case of
ectopically-
expressed cyclins, the pleiotropic effect of the cyclin in question is
enhanced. In the
case of repressed expression of cyclins, the pleiotropic effect of the cyclin
in question
is reversed or suppressed and consequent de-repression of a repressed cyclin,
using
for example an inducible promoter, induces the formation of organs/cell
types/structures/functions that were previously repressed. Persons skilled in
the art are
aware of means for repressing the expression of genes in plants, for example
using
antisense, co-suppression, post-transcriptional gene silencing, targetted gene
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disruption (i.e. gene-targeting), ribozyme-mediated approaches, chemical
mutagenesis, T-DNA-mediated mutagenesis, and transposon-mediated mutagenesis,
amongst others. The present invention clearly extends to all such embodiments.
Moreover, induced downregulation of cyclins provides for controlled
morphogenesis
in the one or the other or both directions. Non-morphogenic cell cultures,
such as
those overexpressing a mitotic cyclin and a G1 cyclin, are useful for biomass
production, particularly when coupled with autotrophy.
"Plant development" or the term "plant developmental characteristic" or
similar term
shall be taken to mean any cellular process of a plant that is involved in
determining
the developmental fate of a plant cell, in particular the specific tissue or
organ type into
which a progenitor cell will develop. Cellular processes relevant to plant
development
will be known to those skilled in the art. Such processes include, for
example,
morphogenesis, photomorphogenesis, shoot development, root development,
vegetative development, reproductive development, stem elongation, flowering,
and
regulatory mechanisms involved in determining cell fate, in particular a
process or
regulatory process involving the cell cycle. For example, in the present
context, the
inventors have shown that the development of roots from tobacco leaf discs in
culture
can be inhibited by shortening the G2 phase of the cell cycle.
"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, number, position,
colour,
texture, arrangement, and patternation of any cell, tissue or organ or groups
of cells,
tissues or organs of a plant, including the root, stem, leaf, shoot, petiole,
trichome,
flower, petal, stigma, style, stamen, pollen, ovule, seed, embryo, endosperm,
seed
coat, aleurone, fibre, cambium, wood, heartwood, parenchyma, aerenchyma, sieve
element, phloem or vascular tissue, amongst others.
The suppression of organ formation by cyclin overexpression may be used to
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specifically design plant architecture or morphology, particularly in crops in
which
apical dominance is weak (e.g. in fruit trees, fruit-bearing crop plants such
as tomato,
vegetable crops, or cereals). Shoot apical dominance has been described as a
mechanism by which the dominant organs repress growth elsewhere in the plant
by
acting as an auxin-induced sink for root-derived cytokinins.
"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.
Because most biosynthetic activities of plant cells, such as in vitro-cultured
cells, are
linked to differentiation, such cell cultures may be of interest for
fermentation. In a first
step, high CDK activity would allow cell mass production, selective
downregulation of
plant cyclins may derepress morphogenesis and specific biosynthetic activities
(e.g.
secondary plant metabolite production) may occur.
"Plant physiology" or the term "plant physiological characteristic" or similar
term will be
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.
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In addition to modifying cell fate, regulating the bioactivity of a cyclin
protein or a
homologue, analogue or derivative thereof in a plant, such as by ectopically
expressing
said cyclin protein, or by reducing the expression of endogenous cyclin
proteins,
preferably produces a wide range of desirable phenotypes in the plant, such
as, for
S example, a morphological, biochemical or physiological characteristic
selected from
the group consisting of:(i) enhanced seed size; (ii) enhanced grain yield;
(iii) enhanced
stem strength; (iv) enhanced stem thickness; (v) enhanced stem stability; (vi)
enhanced wind-resistance of the stem; (vii) enhanced tuber formation; (viii)
enhanced
tuber development; (ix) increased lignin content; (x) enhanced ploidy of the
seed; (xi)
enhanced endosperm size; (xii) reduced apical dominance; (xiii) increased
bushiness;
(xiv) enhanced lateral root formation; (xv) enhanced rate of lateral root
production; (xvi)
enhanced nitrogen-fixing capability; (xvii) enhanced nodulation or nodule
size; (xviii)
reduced or delayed leaf chlorosis; (xix) reduced or delayed leaf necrosis;
(xx) partial
or complete inhibition of the arrest of DNA replication in a plant cell under
growth-
limiting conditions; (xxi) enhanced endoreplication; (xxii) enhanced
endoreduplication,
including enhanced endoreduplication in the seed; and (xxiii) enhanced cell
expansion.
More preferably, the plant morphological, biochemical or physiological
characteristic
which can be modified by modifying the bioactive concentration of cyclin
protein is a
cytokinin-mediated or a gibberellin-mediated characteristic selected from the
group
consisting of: (i) enhanced stem thickness; (ii) enhanced stem stability;
(iii) enhanced
wind-resistance of the stem; (iv) enhanced tuber formation; (v) enhanced tuber
development; (vi) increased lignin content; (vii) enhanced seed set; (viii)
enhanced
seed production; (ix) enhanced grain yield; (x) enhanced ploidy of the seed;
(xi)
enhanced endosperm size; (xii) reduced apical dominance; (xiii) increased
bushiness;
(xiv) enhanced lateral root formation; (xv) enhanced rate of lateral root
production; (xvi)
enhanced nitrogen-fixing capability; (xvii) enhanced nodulation or nodule
size; (xviii)
reduced or delayed leaf chlorosis; (xix) reduced or delayed leaf necrosis;
(xx) partial
or complete inhibition of the arrest of DNA replication in a plant cell under
growth-
limiting conditions; (xxi) enhanced endoreplication and/or enhanced
endoreduplication;
and (xxii) enhanced cell expansion.
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In a particularly preferred embodiment of the invention, a genetic sequence
encoding
a cyclin protein is expressed ectopically in a plant , or a plant part, cell,
tissue or organ,
to: (i) shorten the G2 phase of the cell cycle; and/or (ii) to shorten the
G2/M phase
transition of a cell; and/or (iii) to inhibit root regeneration from calli;
and/or (iv) to
stimulate shoot formation from calli; and/or (v) to promote, stimulate or
enhance
bushiness of a plant; and/or (vi) to inhibit, delay, or reduce apical
dominance in a plant;
and/or (vii) to modify source/sink relationships in the plant, and
particularly in the seed;
and/or (viii) to inhibit, delay, or reduce leaf senescence (chlorosis and/or
necrosis);
and/or (ix) to enhance endoreplication, endoreduplication, or otherwise modify
DNA
synthesis by overriding the DNA synthesis checkpoint in a cell.
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.
The term "cell cycle" as used herein shall be taken to include the cyclic
biochemical
and structural events associated with growth and with division of cells, and
in particular
with the regulation of the replication of DNA and mitosis. Cell cycle includes
phases
called: GO (gap 0), G1 (gap 1), DNA replication (S), G2 (gap 2), and mitosis
including
cytokinesis (M). Normally these four phases occur sequentially. However, the
cell
cycle also includes modified cycles such as endomitosis, acytokinesis,
polyploidy,
polyteny, endopolyploidisation and endoreduplication or endoreplication.
The term "cell cycle interacting protein", "cell cycle protein", or "cell
cycle control
protein" as denoted herein means a protein which exerts control on or
regulates or is
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required for the cell cycle or part thereof of a cell, tissue, organ or whole
organism
and/or DNA replication. It may also be capable of binding to, regulating or
being
regulated by cyclin dependent kinases or their subunits. The term also
includes
peptides, polypeptides, fragments, variant, homologues, alleles or precursors
(eg
preproproteins or preproteins) thereof.
Cell cycle 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
(Norbury and Nurse 1992; Nurse 1990; Ormond and Francis 1993) and the
contributing papers therein (Doerner et al. 1996; Elledge 1996; Francis and
Halford
1995; Francis et al. 1998; Hirt et al. 1991; Mironov et al. 1999) which are
incorporated
herein by way of reference.
The term "cell cycle control gene" refers to any gene or mutant thereof which
exerts
positive or negative control on, or is required for, chromosomal DNA
synthesis, mitosis
(preprophase band, nuclear envelope, spindle formation, chromosome
condensation,
chromosome segregation, formation of new nuclei, formation of phragmoplast,
etc)
meiosis, cytokinesis, cell growth, or endoreduplication. The term "cell cycle
control
gene" also includes any and all genes that exert control on a cell cycle
protein as
hereinbefore defined, including any homologues of CDKs, cyclins, E2Fs, Rb,
CKI, Cks,
cyclin D, cdc25, Wee1, Nim1, MAP kinases, etc. Preferably, a cell cycle
control gene
will exert such regulatory control at the post-translation level, via
interactions involving
the polypeptide product expressed therefrom.
More specifically, cell cycle control genes are all genes involved in the
control of entry
and progression through S phase. They include, not exclusively, genes
expressing
"cell cycle control proteins" such as cyclin dependent kinases (CDK), cycline
dependent kinase inhibitors (CKI), D, E and a cyclins, E2F and DP
transcription
factors, pocket proteins, CDC7/DBF4 kinase, CDC6, MCM2-7, Orc proteins, cdc45,
components of SCF ubiquitin ligase, PCNA, and DNA-polymerase, amongst others.
The term " cell cycle control protein" includes cyclins a, B, C, D and E,
including
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CYCA1;1, CYCA2;1, CYCA3;1, CYCB1;1, CYCB1;2, CYCB2;2, CYCD1;1, CYCD2;1.
CYCD3;1, and CYCD4;1 (Evans et al. 1983; Francis et al. 1998; Labbe et al.
1989;
Murray and Kirschner 1989; Renaudin et al 1996; Soni et al 1995; Sorrell et al
1999;
Swenson et al 1986); cyclin dependent kinase inhibitor (CKI) proteins such as
ICK1
(Wang et al 1997), FL39, FL66, FL67 (PCT/EP98/05895), Sic1, Far1, Rum1, p21,
p27,
p57, p16, p15, p18, p19 (Elledge 1996; Pines 1995), p14 and p14ARF; p13suc1 or
CKS1At (De Veylder et al 1997; Hayles and Nurse 1986) and nim-1 (Russell and
Nurse 1987a; Russell and Nurse 1987b; Fantes 1989; Russell and Nurse 1986;
Russell and Nurse 1987a; Russell and Nurse 1987b) homologues of Cdc2 such as
Cdc2MsB (Hirt et al 1993) CdcMs kinase (Bogre et al 1997) cdc2 T14Y15
phosphatases such as Cdc25 protein phosphatase or p80cdc25 (Bell et al 1993;
Elledge 1996; Kumaghi and Dunphy 1991; Russell and Nurse 1986) and Pyp3
(Elledge
1996) cdc2 protein kinase or p34cdc2 (Colasanti et al 1991; Feiler and Jacobs
1990;
Hirt et al 1991; John et al 1989; Lee and Nurse 1987; Nurse and Bissett 1981;
Ormond
and Francis 1993) cdc2a protein kinase (Hemerly et al 1993) cdc2 T14Y15
kinases
such as wee1 or p107wee1 (Elledge 1996; Russell and Nurse 1986; Russell and
Nurse 1987a; Russell and Nurse 1987a; Sun et al 1999) 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
p34cdc28 (Nasmyth 1993; Reed et al. 1985) p40M015 (Fesquet et al 1993; Poon et
al. 1993) chk1 kinase (Zeng et al 1998) cds1 kinase (Zeng et al 1998) growth
associated H1 kinase (Labbe et al 1989; Lake and Salzman 1972; Langan 1978;
Zeng
et al 1998) MAP kinases described by (Binarova et al 1998; Bogre et al 1999;
Calderini
et al 1998; Wilson et al 1999).
Other cell cycle control proteins are involved in cyclin D-mediated entry of
cells into G1
from GO include pRb (Xie et al., 1996; Huntley et al., 1998), E2F, RIP, MCM7,
and the
pRb-like proteins p107 and p130.
Other cell cycle control proteins 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
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complex, such as, but not limited to, the CDC7, DBF4 and MBF proteins.
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
any other
cell cycle control protein, or in regulating the half-life of said other cell
cycle control
protein. The term "protein turnover" is to include all biochemical
modifications of a
protein leading to the physical or functional removal of said protein.
Although not
limited to these, examples of such modifications are phosphorylation,
ubiquitination
and proteolysis. Particularly preferred proteins which are involved in the
proteolysis
of one or more of any other 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 et al 1998) and Plesse
et al
in (Francis et al 1998)).
For the present purpose, the term "cell cycle control genes" shall further be
taken to
include any one or more of those gene that are involved in the transcriptional
regulation of cell cycle control gene expression such as transcription factors
and
upstream signal proteins. Additional cell cycle control genes are not
excluded.
For the present purpose, the term "cell cycle control genes" shall further be
taken to
include any cell cycle control gene or mutant thereof, which is affected by
environmental signals such as for instance stress, nutrients, pathogens, or by
intrinsic
signals such as an animal mitogen or plant hormone (auxin, cytokinin,
ethylene,
gibberellic acid, abscisic acid and brassinosteroid).
In a preferred embodiment, the cell cycle control protein of the present
invention is
involved in controlling or regulating the length of the G2 phase of the cell
cycle and/or
the transition from the G2 phase to the M phase. As will be apparent from the
description contained herein, a cell cycle control protein that is capable of
regulating
the length of the G2 phase and/or the G2/M transition will be capable of
modifying the
duration of the cell cycle and the time taken by a cell to exit the cell cycle
and
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commence cell division.
Even more preferably, the cell cycle control protein of the present invention,
when
expressed or over-expressed in a plant cell, tissue or organ, is capable of
shortening
the length of the G2 phase of the cell cycle and/or reducing the transition
from the G2
phase to the M phase and, as a consequence, can be used to advance cell
division.
Still more preferably, the cell cycle control protein is a cyclin protein or a
homologue,
analogue or derivative thereof, and still more preferably a mitogenic cyclin
protein,
such as, for example, a cyclin B protein or a cyclin B-like protein.
In a particularly preferred embodiment of the invention, the cell cycle
control protein
is a cyclin B protein comprising the alfalfa CycMs2 protein or a biologically-
active
homologue, analogue or derivative thereof and, in particular, a plant-derived
homologue of the alfalfa CycMs2 protein. The present invention clearly
contemplates
the use of functional homologues of the alfalfa CycMs2 protein and is not to
be limited
in application to the use of nucleotide sequences encoding the alfalfa CycMs2
protein.
"Homologues" of a cyclin protein, such as cyclin B, in particular homologues
of
CycMs2, are those peptides, oligopeptides, polypeptides, proteins and enzymes
which
contain amino acid substitutions, deletions and/or additions relative to the
cyclin
polypeptide with respect to which they are a homologue, without altering one
or more
of its cell cycle control properties, in particular without reducing the
cyclin B or cyclin
B-like activity of the resulting polypeptide with respect to its ability to
induce one or
more cytokinin-mediated and/or gibberellin-mediated effects in a plant cell,
tissue,
organ or whole organism. For example, a homologue of the alfalfa CycMs2
polypeptide
will consist of a bioactive amino acid sequence variant of said polypeptide.
To produce such homologues, amino acids present in the cyclin polypeptide 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 ~i-sheet structures, and so on.
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Substitutional variants are those in which at least one residue in the cyclin
protein
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 cyclin 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 amino acid sequence of the cyclin protein.
Amino acid variants of the cyclin protein 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
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 cyclin protein, such as cyclin B, in particular analogues of
CycMs2,
are defined as those peptides, oligopeptides, polypeptides, proteins and
enzymes
which are functionally equivalent to the cyclin with respect to which they are
analogous.
Analogues of a cyclin protein will preferably exhibit like bioactivity in
inducing one or
more cytokinin-mediated and/or gibberellin-mediated effects in plant cells,
tissues,
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organs or whole organisms,
Preferably, an analogue of a cyclin protein is a cell cycle control protein,
or a modified
cell cycle control protein, which produces the same modifications to cell
fate, plant
morphology, biochemistry or physiology when ectopically-expressed in a plant,
as
observed for the CycMs2 protein. More preferably, an analogue of a cyclin
protein, in
particular cyclin B, is a cell cycle control protein other than a Cdc25
protein, or a Cdc2
protein.
"Derivatives" of a cyclin protein, such as cyclin B, in particular derivatives
of CycMs2,
are those peptides, oligopeptides, polypeptides, proteins and enzymes which
comprise
at least about five contiguous amino acid residues of a naturally-occurring
cyclin
polypeptide, in particular the alfalfa CycMs2 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
cyclin 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 cyclin 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 a cyclin
protein,
such as, for example, a cyclin B protein, and, in particular the alfalfa
CycMs2
polypeptide, include those molecules incorporating single or multiple
substitutions,
deletions and/or additions therein, such as carbohydrates, lipids andlor
proteins or
polypeptides. Naturally-occurring or altered glycosylated or acylated forms of
cyclin
B are also contemplated by the present invention. Additionally, homopolymers
or
heteropolymers comprising one or more copies of the cyclin 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-
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mediated effects in plant cells, tissues, organs or whole organisms.
Particularly preferred homologues, analogues and derivatives of a cyclin
protein which
are contemplated for use in performing the present invention are derived from
plants
or capable of being expressed therein.
To effect expression of the cyclin 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,
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-
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 cyclin 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
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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.
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, dexamethasone-
responsive
or tetracycline-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, dexamethasone-inducible, or
tetracycline-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.
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The terms "plant-operable" and "operable in a plant" when used herein, in
respect of
a promoter sequence, shall be taken to be equivalent to a plant-expressible
promoter
sequence.
S In the present context, a "regulatable promoter sequence" is a promoter that
is capable
of conferring expression on a structural gene in a particular cell, tissue, or
organ or
group of cells, tissues or organs of a plant, optionally under specific
conditions,
however does generally not confer expression 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
within the scope of such promoters are cell-specific promoter sequences,
tissue-
specific promoter sequences, inducible promoter sequences, organ-specific
promoter
sequences, cell cycle specific gene 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 an
excisable genetic element.
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.
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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.
Those skilled in the art will be aware that an "inducible promoter" is a
promoter the
transcriptional activity of which is increased or induced in response to a
developmental,
chemical, environmental, or physical stimulus.
Similarly, the term "cell cycle specific" shall be taken to indicate that
expression is
predominantly cyclic and occurring in one or more, not necessarily consecutive
phases
of the cell cycle albeit not necessarily exclusively in cycling cells.
As will be apparent from the preceding description, the present invention does
not
require the exclusive expression of the cyclin 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 cyclin 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 cyclin
protein
in the plant cell, tissue or organ, will confer expression in a limited 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
cyclin
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 cyclin protein
from
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publicly-available or readily-available sources, without undue
experimentation.
In this regard, constitutive promoters can be made tissue-specific, cell-
specific, cell-
cycle specific or organ-specific, by adding regulatory elements from regulated
tissue-
s specific, cell-specific, cell-cycle specific or organ-specific promoters to
the constitutive
promoter sequence. Alternatively, the otherwise constitutive expression
activity of the
promoter sequence may be regulated by integrating the promoter sequence and
cell
cycle control gene in one or more excisable genetic elements.
As used herein, the term "an excisable genetic element" shall be taken to
refer to any
nucleic acid which comprises a nucleotide sequence which is capable of
integrating
into the nuclear, mitochondrial, or plastid genome of a plant, and
subsequently being
autonomously mobilised, or induced to mobilise, such that it is excised from
the
original integration site in said genome. By "autonomously mobilised" is meant
that the
genetic element is excised from the host genome randomly, or without the
application
of an external stimulus to excise. In performing the present invention, the
genetic
element is preferably induced to mobilise, such as, for example, by the
expression of
a recombinase protein in the cell which contacts the integration site of the
genetic
element and facilitates a recombination event therein, excising the genetic
element
completely, or alternatively, leaving a "footprint", generally of about 20
nucleotides in
length or greater , at the original integration site.
Preferably, the excisable genetic element comprises a transposable genetic
element,
such as, for example, Ac, Ds, Spm, or En, or alternatively, on or more loci
for
interaction with a site-specific recombinase protein, such as, for example,
one or more
lox or frt nucleotide sequences.
Known site-specific recombination systems, for example the cre/lox system and
the
flplfrt system which comprise a loci for DNA recombination flanking a selected
gene,
specifically lox or frt genetic sequences, combination with a recombinase, cre
or flp,
which specifically contacts said loci, producing site-specific recombination
and deletion
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of the selected gene. In particular, European Patent No. 0228009 (E.I. Du Pont
de
Nemours and Company) published 29 April, 1987 discloses a method for producing
site-specific recombination of DNA in yeast utilising the crellox system,
wherein yeast
is transformed with a first DNA sequence comprising a regulatory nucleotide
sequence
and a cre gene and a second DNA sequence comprising a pre-selected DNA segment
flanked by two lox sites such that, upon activation of the regulatory
nucleotide
sequence, expression of the cre gene is effected thereby producing site-
specific
recombination of DNA and deletion of the pre-selected DNA segment. United
States
Patent No. 4,959,317 (E.I. Du Pont de Nemours and Company) filed 29 April 1987
and
International Patent Application No. PCT/US90/07295 (E.I. Du Pont de Nemours
and
Company) filed 19 December, 1990 also disclose the use of the cre/lox system
in
eukaryotic cells.
A requirement for the operation of site-specific recombination systems is that
the loci
for DNA recombination and the recombinase enzyme contact each other in vivo,
which
means that they must both be present in the same cell. The prior art means for
excising unwanted transgenes from genetically-transformed cells all involve
either
multiple transformation events or sexual crossing to produce a single cell
comprising
both the loci for DNA recombination and the site-specific recombinase.
A "site-specific recombinase" is understood by those skilled in the relevant
art to mean
an enzyme or polypeptide molecule which is capable of binding to a specific
nucleotide
sequence, in a nucleic acid molecule preferably a DNA sequence, hereinafter
referred
to as a "recombination locus" and induce a cross-over event in the nucleic
acid
molecule in the vicinity of said recombination locus. Preferably, a site-
specific
recombinase will induce excision of intervening DNA located between two such
recombination loci.
The terms "recombination locus" and "recombination loci" shall be taken to
refer to any
sequence of nucleotides which is recognized and/or bound by a site-specific
recombinase as hereinbefore defined.
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A number of different site specific recombinase systems can be used, including
but not
limited to the Cre/lox system of bacteriophage P1, the FLP/FRT system of
yeast, the
Gin recombinase of phase Mu, the Pin recombinase of E.coli, the Ping, PinD and
PinF
from Shigella, and the R/RS system of the psR1 plasmid. Some of these systems
have
already been used with high efficiency in plants, such as tobacco, and A.
thaliana.
Preferred site-specific recombinase systems contemplated for use in the gene
constructs of the invention, and in corijunction with the inventive method,
are the
bacteriophage P1 Cre/lox system, and the yeast FLP/FRT system. The site
specific
recombination loci for each of these two systems are relatively short, only 34
by for the
lox loci, and 47 by for the frt loci.
In a most particularly preferred embodiment, however, the recombination loci
are lox
sites, such as lox P, lox 8, Lox L or lox R or functionally-equivalent
homologues,
analogues or derivatives thereof. Lox sites may be isolated from bacteriophage
or
bacteria by methods known in the art (Hoess et al., 1982). It will also be
known to
those skilled in the relevant art that lox sites may be produced by synthetic
means,
optionally comprising one or more nucleotide substitutions, deletions or
additions
thereto.
The relative orientation of two recombination loci in a nucleic acid molecule
or gene
construct may influence whether the intervening genetic sequences are deleted
or
excised or, alternatively, inverted when a site-specific recombinase acts
thereupon. In
a particularly preferred embodiment of the present invention, the
recombination loci are
oriented in a configuration relative to each other such as to promote the
deletion or
excision of intervening genetic sequences by the action of a site-specific
recombinase
upon, or in the vicinity of said recombination loci.
The present invention clearly encompasses the use of gene constructs which
facilitate
the expression of a site-specific recombinase protein which is capable of
specifically
contacting the excisable genetic element, in conjunction with the gene
constructs
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containing the cell cycle control protein-encoding gene. A single gene
construct may
be used to express both the site-specific recombinase protein and the cell
cycle control
protein, or alternatively, these may be introduced to plant cells on separate
gene
constructs.
For example, the recombinase gene could already be present in the plant genome
prior to transformation with the gene construct of the invention, or
alternatively, it may
be introduced to the cell subsequent to transformation with the gene construct
of the
invention, such as, for example, by a separate transformation event, or by
standard
plant breeding involving hybridisation or cross-pollination. In one embodiment
of the
current invention, the recombinase gene is supplied to the transgenic plants
containing
a vector backbone sequence flanked by recombination sites by sexual crossing
with
a plant containing the recombinase gene in it's genome. Said recombinase can
be
operably linked to either a constitutive or an inducible promoter. The
recombinase
gene can alternatively be under the control of single subunit bacteriophage
RNA
polymerase specific promoters, such as a T7 or a T3 specific promoter,
provided that
the host cells also comprise the corresponding RNA polymerase in an active
form. Yet
another alternative method for expression of the recombinase consists of
operably
linking the recombinase open reading frame with an upstream activating
sequence
fired by a transactivating transcription factor such as GAL4 or derivatives
(US5801027,
W097/30164, W098/59062) or the Lac repressor (EP0823480), provided that the
host
cell is supplied in an appropriate way with the transcription factor.
Alternatively, a substantially purified recombinase protein could be
introduced directly
into the eukaryotic cell, eg., by micro-injection or particle bombardment.
Typically, the
site-specific recombinase coding region will be operably linked to regulatory
sequences
enabling expression of the site-specific recombinase in the eukaryotic cell.
In a
preferred embodiment of the present invention, the site-specific recombinase
sequences is operably linked to an inducible promoter.
Dual-specific recombinase systems can also be employed, which may employ a
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recombinase enzyme in conjunction with direct or indirect repeats of two
different site-
specific recombination loci corresponding to the dual-specific recombinase,
such as
that described in International Patent Publication No. W099/25840.
As will be known to those skilled in the art, for recombination mediated by a
transposon
to occur, a pair of DNA sequences comprising inverted repeat transposon border
sequences, flanking the excisable genetic element sequence, and a specific
transposase enzyme, are required. The transposase catalyzes a recombination
reaction only between two transposon border sequences.
A number of different plant-operable transposon/transposase systems can be
used
including but not limited to the Ac/Ds system, the Spm system and the Mu
system. All
of these systems are operable in Zea mays, and at least the AclDs and the Spm
system function in other plants.
Preferred transposon sequences for use in the gene constructs of the invention
are the
Ds-type and the Spm-type transposons, which are delineated by border sequences
of
only 11 by and 13 by in length, respectively.
As with the use of site-specific recombinase systems, the present invention
clearly
encompasses the use of gene constructs which facilitate the expression of a
transposase enzyme which is capable of specifically contacting the transposon
border
sequence, in conjunction with the gene constructs containing thecell cycle
control
protein-encoding gene. A single gene construct may be used to express both the
transposase and the cell cycle control protein, or alternatively, these may be
introduced to plant cells on separate gene constructs.
For example, the transposase-encoding gene could already be present in the
plant
genome prior to transformation with the gene construct of the invention, or
alternatively, it may be introduced to the cell subsequent to transformation
with the
gene construct of the invention, such as, for example, by a separate
transformation
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event, or by standard plant breeding involving hybridisation or cross-
pollination.
Alternatively, a substantially purified transposase protein could be
introduced directly
into the eukaryotic cell, eg., by micro-injection or particle bombardment.
Typically, the
transposase coding region will be operably linked to regulatory sequences
enabling
expression of the transposase in the eukaryotic cell. In a preferred
embodiment of the
present invention, the transposase-encoding sequence is operably linked to an
inducible promoter.
In the present context, transposon border sequences are organized as inverted
repeats flanking the excisable genetic element. As transposons often re-
integrate at
another locus of the host's genome, segregation of the progeny of the hosts in
which
the transposase was allowed to act might be necessary to separate transformed
hosts
containing only the genes) of interest and transformed hosts containing only
the cell
cycle control protein-encoding gene.
Likewise, the site-specific recombinase gene or transposase gene present in
the host's
genome can be removed by segregation of the progeny of the hosts to separate
transformed hosts containing only the genes) of interest and transformed hosts
containing only the site-specific recombinase gene or transposase gene.
Alternatively,
said site-specific recombinase gene or transposase gene are included in the
same or
in a different excisable genetic element as thecell cycle control protein-
encoding gene.
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
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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 gene constructs of the present
invention
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-~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.
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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,
such as,
for example, the SAUR gene promoter, the parAs and parAt gene promoters(van
der
Zaal et al., 1991; Gil et al., 1994; Niwa et al., 1994); or a gibberellin-
inducible promoter
such as the Amy32b gene promoter (Lanahan et al. 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 Adh1 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.
In yet another alternative embodiment, the promoter is a cell cycle specific
gene
promoter, such as, for example, the Cdc2a promoter sequence (Chung and Parish
1995) or the PCNA promoter sequence (Kosugi et al, 1991, Kosugi and Ohashi
1997).
Preferred embodiments of the invention relate to the effects) of cytokinins on
the
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3
determination of cell fate and plant development and cellular processes
therefor.
However, the present invention clearly contemplates the broad application of
the
inventive method to the modification of a range of cellular processes involved
in
determining cell fate and plant cell development, including but not limited to
modifying
the length of the cell cycle, and in particular, modifying the length of the
G2 phase;
modifying the duration of the G2/M phase transition; the advancement of cell
division;
determination of cell fate and in particular root development and/or seed
development; the modification of source/sink relationships, and/or the
inhibition of root
growth and/or inhibition of root apical dominance and/or the delay of
senescence
and/or modifying shoot apical dominance.
Preferred embodiments of the invention also relate to specific the effects) of
hormones such as cytokinins and/or gibberellins on plant metabolism. With
respect
to such hormone-related effects, the present invention clearly contemplates
the broad
application of the inventive method to the modification of a range of cellular
processes
that are mediated by cytokinins and/or gibberellins, including but not limited
to cellular
development and/or cell fate; the advancement of cell division;the
modification of
source/sink relationships, and/or the inhibition of root growth and/or the
inhibition of
root apical dominance and/or the delay of senescence andlor the initiation,
promotion,
stimulation or enhancement of seed development and/or tuber formation and/or
shoot
initiation and/or dwarfism and/or pigment synthesis and/or the modifcation of
shoot
apical dominance (i.e. bushiness) of the plant.
Accordingly, in a particularly preferred embodiment of the present invention,
there is
provided a method of shortening the duration of the G2 phase of the cell cycle
and/or
shortening the G2/M phase transition of a cell comprising expressing the
alfalfa
CycMs2 protein or a homologue, analogue or derivative thereof, or a modified
substrate of cyclin B that mimics the effect of cyclin B operably under the
control of a
regulatable promoter sequence as described supra.
CA 02364566 2001-08-24
WO 00/52169 PCT/AU00/00137
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CA 02364566 2001-08-24
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In the present context, the term "substrate of cyclin B" shall be taken to
refer to any
protein that interacts with cyclin B or cyclin B/CDK complex 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.
The term "modified substrate of cyclin B" refers to a homologue, analogue or
derivative
of a substrate of cyclin B that mimics the effect of alfalfa CycMs2 activity
described
herein. For example, substitution of amino acids in a cyclin-dependent kinase
(CDK)
can produce one or more cytokinin-like effects in a plant that are similar to
those
observed following constitutive cyclin B expression in the plant.
Accordingly, similar effects to the cyclin B-induced effects obtained by
expressing
alfalfa CycMs2 under control of the regulatable promoter, can be obtained by
expressing the cyclin B 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 cyclin B, in
particular
alfalfa CycMs2, and one or more cyclin B substrates and/or one or more
modified
cyclin B substrates, operably under the control of a regulatable promoter that
is
selected for a particular application as described herein. The present
invention also
extends to the co-expression of cyclin B with another synergistic or non-
antagonistic
cell cycle control protein, such as Cdc25, amongst others.
In another particularly preferred embodiment of the present invention, there
is provided
a method of advancing cell division in a plant cell, tissue or organ
comprising
expressing the alfalfa CycMs2 protein or a homologue, analogue or derivative
thereof,
or a modified substrate of cyclin B that mimics the effect of cyclin B
operably under the
control of a regulatable promoter sequence.
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By "advancing cell division" is meant that the duration between the
commencement of
the cell cycle and entry of the cell into mitosis or alternatively or in
addition, the
duration between the commencement of the G2 phase of the cell cycle and entry
of
the cell into mitosis is shortened. In the present context this is achieved by
reducing
7the duration of the cell in the G2 phase of the cell cycle and/or by reducing
the
duration of the G2/M phase transition.
In yet another particularly preferred embodiment of the present invention,
there is
provided a method of altering cell fate or development in a plant cell,
tissue, organ or
whole plant comprising expressing the alfalfa CycMs2 protein or a homologue,
analogue or derivative thereof, or a modified substrate of cyclin B that
mimics the
effect of cyclin B operably under the control of a regulatable promoter
sequence.
Preferably, the cell fate or development that is altered or modified by the
performance
of the present invention comprises a process that is regulated by the G2 phase
of the
cell cycle and/or by the duration of G2 and/or by the duration of the G2/M
phase
transition. In a more preferred embodiment, the cell fate or development
comprises
root development; and/or seed development, in particular grain production and
yield;
and/or one or more sink/source relationships of the plant, such as carbon
partitioning;
and/or tissue sensecence. Other processes are not excluded.
In another particularly preferred embodiment of the present invention, there
is provided
a method of modifying sink/source relationships of a plant tissue, organ or
whole plant
comprising expressing the alfalfa CycMs2 protein or a homologue, analogue or
derivative thereof, or a modified substrate of cyclin B that mimics the effect
of cyclin
B operably under the control of a regulatable promoter sequence.
As will be known to those skilled in the art, the term "sink/source
relationship" refers
to the flux of carbon, in particular, in the form of sucrose or triose or
triose phosphate,
or other carbon-containing compound, from a source organelle, cell, tissue or
organ,
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such as but not necessarily limited to photosynthetic tissues (eg. flag
leaves), to a
particular storage organelle, cell, tissue or organ (the "sink") and to the
regulation of
carbon flux and/or storage in the sink by such a process. Accordingly,
sink/source
relationships include such processes as carbon partitioning between sucrose
and
starch within a particular cell, and the regulation of seed starch deposition
by
photosynthesis and/or photosynthate transport to the endosperm (i.e. source
limitation), and by sink-limiting processes such as endosperm or storage
cotyledon
ATP/Pi ratio, and the level of starch-metabolising enzymes (eg. ADP-glucose
pyrophosphorylase; starch synthase) within the endosperm or storage cotyledon,
amongst others. Those skilled in the art will be aware that sink strength is
possibly the
most important yield component.
Cytokinins are known to promote phloem unloading of metabolites, and in
immature
seeds cell division activity is correlated with a high endogenous cytokinin
level,
particularly in maize and legumes. Likewise, hexoses in very young Vicia faba
seeds
stimulate cell division while sucrose stimulates starch formation at a later
stage of seed
development. Sucrose-to-hexose conversion is controlled by invertase and
indeed
invertase activity is high in very young seeds. Hexose-to-sucrose conversion
is
controlled by sucrose phosphate synthase, leading eventually to starch
synthesis.
Invertase converts sucrose in a sink (meristem, storage organ, seed, etc.)
into glucose
while glucose stimulates cell division. Cell division produces cell mass in
the sink.
Cytokinins are part of the signal transduction chain linking the incoming
sucrose to the
activation of cell divisions. Cytokinins act downstream of the sucrose-to-
hexose
conversion regulated by invertase, but may have a pleiotropic effect on
invertase and
the G2/M transition (and possibly also on G1/S transition). At some point in
seed
development, invertase activity goes down, or glucose is activately converted
into
starch by activation of sucrose phosphate synthase. Cell division activity is
stopped,
to be followed by cell expansion (including endoreduplication) and starch
biosynthesis.
In seeds, grain filling occurs, whilst in leaf initials on meristems, leaf
expansion and
development occurs. Whilst not being bound by any theory or mode of action,
ectopic
overexpression of a mitotic cyclin, such as CycMs2, has the same effect as
cytokinin
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in that it drives the cell precociously into mitosis, resulting in a shortened
G2 phase.
A short G2 phase (and/or a short G1 phase) are defining features of a cell
that is
actively-dividing. Thus, in a mitotic cyclin-overexpressing cell, the G2/M
transition is
uncoupled from its normal external signal, glucose and cytokinin.
S
In one application of this phenomenon, such an uncoupling of sink/source
relationships
is useful in regulating the production of seeds. For example, a transgenically-
controlled
mitotic cyclin level in young seeds can render these cells more or less
responsive to
the incoming glucose/cytokinin signal, resulting in more or fewer cells in the
endosperm of the seed until glucose-to-sucrose conversion occurs.
In a further application, the production of potato tubers may be regulated. In
potato
tubers, sucrose induces stolon tips to develop into tubers. However, cytokinin
is
required to initiate cell divisions in the stolon. A role for glucose in
activation of cell
division is not known. Ectopic expression of mitotic cyclins, such as, for
example, a
cyclin B protein and, in particular, the alfalfa CycMs2 protein, can
substitute for the
cytokinin effect and stimulate cell division independently from the incoming
tuberization
signal.
Accordingly, it is possible to increase or decrease sink strength of the seed
or tuber,
and, by extrapolation, of any other sink. Being the work horses of the cell
cycle and
the endpoints of the signal transduction chain originating from the incoming
sucrose.
CDKs might be the final and crucial determinants of sink strength.
The promoter selected for use according to this embodiment may be any promoter
sequence operable in the tissue or tissues in which carbon flux is to be
modified.
Accordingly, in a related embodiment of the present invention, there is
provided a
method of increasing seed set and/or seed size and/or seed production and/or
grain
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yield in a plant comprising expressing the alfalfa CycMs2 protein or a
homologue,
analogue or derivative thereof, or a modified substrate of cyclin B that
mimics the
effect of cyclin B, and in particular that mimics the effect of alfalfa
CycMs2, 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 ~i-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. By virtue of being linked to cell expansion and metabolic activity,
endoreplication and endoreduplication are generally considered as an important
factor
for increasing yield (Traas et al 1998). As grain endosperm development
initially
includes extensive endoreplication (Olsen et al 1999), enhancing, promoting or
stimulating this process is likely to result in increased grain yield.
Enhancing,
promoting or stimulating cell division during seed development is an
alternative way
to increase grain yield. As shown herein, cyclin B cooperates with Cdc25 to
override
the DNA synthesis checkpoint in cells. This can produce endoreplication and
endoreduplication, and stimulate cell division.
Accordingly, in a preferred embodiment, the cyclin protein-encoding gene,
preferably
the CycMs2 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
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protein and endosperm-expressible promoter provides the additional advantage
of
increasing the grain size and grain yield of the plant.
Endosperm-specific promoters that can be used to drive cyclin protein
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 be used, including the barley blz2 gene promoter, the rice
prolamin
NRP33 promoter, the rice REB promoter, 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 cyclin protein expression.
Promoters
derived from those genes that are expressed in the endosperm at the stage when
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 cyclin-encoding 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 cyclin protein
activity in
the endosperm, by the ectopic expression of the cyclin-encoding gene, in
particular the
CycMs2 gene, therein. In cases where exogenous cytokinin is used to increase
grain
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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 (Chatfield and Armstrong 1987; reviewed by Morris
et al
1993).
In another related 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 alfalfa CycMs2 protein or a homologue, analogue or derivative
thereof,
or a modified substrate of cyclin B that mimics the effect of cyclin B, in
particular that
mimics the effect of alfalfa CycMs2,operably under the 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 particularly preferred embodiment of the present invention, there
is provided
a method of inhibiting root development and/or root growth from plant cell,
meristem
or other tissue, organ or whole plant comprising expressing the alfalfa CycMs2
protein
or a homologue, analogue or derivative thereof, or a modified substrate of
cyclin B that
mimics the effect of cyclin B operably under the control of a regulatable
promoter
sequence.
By inhibiting "root development" is meant that the formation of a root
structure or root-
like structure from a meristem is prevented or delayed or repressed,
irrespective of
whether or not the meristem is developmentally committed to forming a root
structure
(i.e. irrespective of whether or not the meristem is a root meristem).
By inhibiting "root growth" is meant that the continued growth of a committed
root
structure or root-like structure from an existing root or root meristem is
prevented,
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delayed, or represseds.
Accordingly, this embodiment of the present invention relates to the
prevention of
visible root structures or root-like structures from appearing in plants. As
exemplified
herein, the present inventors have demonstrated that the ectoptic expression
of alfalfa
CycMs2 in transgenic tobacco plants inhibits the auxin-mediated development of
roots
from cultured leaf disc tissue. Accordingly, the ectopic expression of cyclin
B proteins
in plant cells is capable of antagonising auxin-mediated processes in plants,
compatible with the concept that to initiate such processes, in particular
root
development, the cell must remain in the G2 phase of the cell cycle for a
longer period
than would otherwise be the case. Whilst not being bound by any theory or mode
of
action, since ectopic expression of CycMs2 shortens G2 and/or the G2/M phase
transition, cells do not remain in G2 for a sufficient time to initiate auxin-
mediated
processes such as root development.
In the performance of this embodiment of the invention, it is preferred that
the
promoter selected for regulating cyciin B expression is a root-expressible or
meristem-
expressible promoter sequence such as those listed in Table 1 and in
particular, the
meristem-expressible PCNA promoter sequence. However, since this embodiment of
the invention is also applicable to the prevention of root regeneration in
cultured cells
and tissues, it will also be apparent that any promoter that is operable in
the cell or
tissue where inhibition of root regeneration is desired will be useful. For
example, a
leaf-operable promoter is preferred for use in preventing root regeneration
from leaf
disc tissue. Alternatively, any regulatable constitutive promoter may also be
useful in
performing this embodiment of the invention.
In another particularly preferred embodiment of the present invention, there
is provided
a method of delaying senescence of a plant tissue, organ or whole plant
comprising
expressing the alfalfa CycMs2 protein or a homologue, analogue or derivative
thereof,
or a modified substrate of cycfin B that mimics the effect of cyclin B
operably under the
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control of a regulatable promoter sequence. This embodiment of the present
invention
also relates to the prevention, delay or reduction of leaf chlorosis and/or
leaf necrosis
in plants.
Preferably, the promoter selected for use in performing this embodiment of the
invention is operable in the green tissues of the plant and in particular, in
the leaves.
Accordingly, the use of a strong promoter such as one of the known Cab
promoters,
the SAM22 promoter, or the rbcs-1A 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 another preferred embodiment of the present invention, there is provided a
method
of modifying shoot apical dominance or bushiness of a plant, comprising
expressing
the alfalfa CycMs2 protein or a homologue, analogue or derivative thereof, or
a
modified substrate of cyclin B that mimics the effect of cyclin B, in
particular that
mimics the effect of alfalfa CycMs2, 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, the shortened G2
transition
modifies cellular metabolism at the level of carbon partitioning to modify th
degree of
branch formation in the plant, thereby modfying auxin-induced apical dominance
in the
plant.
In another 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 alfalfa CycMs2 protein or a
homologue,
analogue or derivative thereof, or a modified substrate of cyclin B that
mimics the
effect of cyclin B operably under the control of a stem-expressible promoter
sequence.
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Preferably, the stem-expressible promoter sequence is derived from the rbcs-7A
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 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 alfalfa
CycMs2 protein or a homologue, analogue or derivative thereof, or a modified
substrate of cyclin B that mimics the effect of cyclin B, and in particular
that mimics the
effect of alfalfa CycMs2, 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
economic/ 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
cyclin
B under control of a promoter that is operable in vascular tissue and
preferably, in
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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 auxins and are
therefore the
preferential tissue for cyclin B overproduction.
In another preferred embodiment of the present invention, there is provided a
method
of modifying lateral root production in a plant comprising expressing the
alfalfa CycMs2
protein or a homologue, analogue or derivative thereof, or a modified
substrate of
cyclin B that mimics the effect of cyclin B, in particular that mimics the
effect of alfalfa
CycMs2, 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 modifying the nitrogen-fixing capability of a plant comprising
expressing the
alfalfa CycMs2 protein or a homologue, analogue or derivative thereof, or a
modified
substrate of cyclin B that mimics the effect of cyclin B, in particular that
mimics the
effect of alfalfa CycMs2, operably under the control of a nodule-specific
promoter
sequence.
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.
thaliana
or other plants.
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In a further preferred embodiment of the present invention, the alfalfa CycMs2
protein
or a homologue, analogue or derivative thereof, or a modified substrate of
cyclin B that
mimics the effect of cyclin B, in particular that mimics the effect of alfalfa
CycMs2, is
expressed in one of the specialised minority of plant tissues in which the
activation of
S 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 advancing
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 alfalfa CycMs2 protein or a homologue,
analogue
or derivative thereof, or a substrate or modified substrate of cyclin B that
mimics the
effect of cyclin B operably under the control of a meristem specific promoter
sequence.
Without being bound by any theory or mode of action, the shortened G2 phase
and/or
shortened G2/M phase transition in the intercalary meristem of the youngest
stem
internode as a consequence of increased cyclin B activity therein results in
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 cyclin
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
gene
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construct or vector into plant cells by transformation or transfection means.
The
nucleic acid molecule or a gene 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 gene 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 gene construct or vector or an active
fragment thereof
comprising a cyclin B gene, in particular the CycMs2 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 gene construct or vector or an active
fragment thereof
comprising a cyclin B gene, in particular the CycMs2 gene, operably under the
control
of the regulatable promoter sequence is stably integrated into the genome of
the cell.
Accordingly, in a further preferred embodiment, the present invention provides
a
method of modifying one or more plant morphological andlor biochemical and/or
physiological characteristics comprising
(i) introducing to a plant cell, tissue or organ a gene construct or vector
comprising a nucleotide sequence that encodes a cyclin protein, such as, for
example, a cyclin B protein, and in particular, the CycMs2Cdc25 protein, or a
homologue, analogue or derivative thereof, operably in connection with a
regulatable promoter sequence selected from the list comprising cell-specific
promoter sequences, tissue-specific promoter sequences, inducible promoter
sequences, organ-specific promoter sequences and cell cycle gene promoter
sequences to produce a transformed or transfected cell; and
(ii) expressing said cyclin protein in one or more of said cells, tissues or
organs
of the plant.
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In an alternative embodiment, the inventive method comprises regenerating a
whole
plant from the transformed cell.
Means for introducing recombinant DNA into plant tissue or cells include, but
are not
limited to, transformation using CaClz 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
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
gene 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 ~m
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 gene construct of the present invention and a whole plant regenerated
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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
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.
Preferably, the transformed plants are produced by a method that does not
require the
application of exogenous cytokinin and/or gibberellin during the tissue
culture phase,
such as, for example, an in planfa transformation method. In a particularly
preferred
embodiment, plants are transformed by an in planta method using Agrobacterium
fumefaciens such as that described by Bechtold et al., (1993) or Clough et al
(1998),
wherein A. tumefaciens is applied to the outside of the developing flower bud
and the
binary vector DNA is then introduced to the developing microspore and/or
macrospore
and/or the developing seed, so as to produce a transformed seed without the
exogenous application of cytokinin and/or gibberellin. Those skilled in the
art will be
aware that the selection of tissue for use in such a procedure may vary,
however it is
preferable generally to use plant material at the zygote formation stage for
in plants
transformation procedures.
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
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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
S 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 gene constructs
and
vectors designed to facilitate the introduction and/or expression and/or
maintenance
of the cyclin protein-encoding sequence and regulatable promoter into a plant
cell,
tissue or organ.
In addition to the cyclin protein-encoding sequence and regulatable promoter
sequence, the gene 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
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 gene constructs
of the
present invention include the Agrobacterium tumefaciens nopaline synthase
(NOS)
gene terminator, the Agrobacterium tumefaciens octopine synthase (OCS) gene
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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-1A 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 gene 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 gene 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 f1-on
and colE1
origins of replication.
The gene construct may further comprise a selectable marker gene or genes that
are
functional in a cell into which said gene 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
selection of cells which are transfected or transformed with a gene 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, ~i-glucuronidase (GUS) gene, chloramphenicol
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acetyltransferase (CAT) gene, green fluorescent protein (gfp) gene (Haseloff
et al,
1997), and luciferase gene, amongst others.
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 cyclin 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, a plant-
expressible
cell cycle specific gene promoter, or alternatively, a plant-expressible
constitutive
promoter sequence such that said plant-expressible constitutive promoter
sequence
and said nucleotide sequence encoding a cyclin 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,
ornamental
plant, food crop, tree, or shrub selected from the list comprising Acacia
spp., Acerspp.,
Acfinidia spp.,Aesculus spp., Agathis australis, Albizia amara, Alsophila
tricolor,
Andropogon spp., Arachis spp, Areca catechu, Astelia fragrans, Astragalus
cicer,
Baikiaea plurijuga, Betula spp., Brassica spp., Bruguiera gymnorrhiza, Burkea
africana,
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 dealbafa,
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., Eucalypfus spp., Euclea schimperi, Eulalia villosa, Fagopyrum spp.,
Feijoa
sellovviana, Fragaria spp., Flemingia spp, Freycinetia banksii, Geranium
thunbergii,
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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 incarnata, 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., Ornithopus
spp., Oryza spp., Peltophorum africanum, Pennisetum spp., Persea gratissima,
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, Pseudotsuga
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, Sfiburus alopecuroides,
Stylosanthos
humilis, Tadehagi spp, Taxodium distichum, Themeda triandra, Trifolium spp.,
Triticum
spp., Tsuga heterophylla, Vaccinium spp., Vicia spp. Vitis vinifera, Watsonia
pyramidata, Zantedeschia aethiopica, Zea mays, amaranth, artichoke, asparagus,
broccoli, brussel sprout, cabbage, canola, carrot, cauliflower, celery,
collard greens,
flax, kale, lentil, oilseed rape, okra, onion, potato, rice, soybean, straw,
sugarbeet,
sugar cane, sunflower, tomato, squash, 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.
Accordingly, the present invention clearly extends to any plant produced by
the
inventive method described herein, and any and all plant parts and propagules
thereof.
The present invention extends further to encompass the progeny derived from a
primary transformed or transfected cell, tissue, organ or whole plant that has
been
produced by the inventive method, the only requirement being that said progeny
exhibits the same genotypic and/or phenotypic characteristics) as that (those)
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characteristics) that has (have) been produced in the parent by the
performance of
the inventive method.
By "genotypic characteristic" is meant the composition of the genome and, more
particularly, the introduced gene encoding the cyclin protein.
By "phenotypic characteristic" is meant one or more plant morphological
characteristics
and/or plant biochemical characteristics and/or plant physiological
characteristics that
are produced by ectopic expression of a cyclin protein in a plant.
Preferably, the plant is produced according to the inventive method is
transfected or
transformed with a genetic sequence, or amenable to the introduction of a
protein, by
any art-recognised means, such as microprojectile bombardment, microinjection,
Agrobacterium-mediated transformation (including in planta transformation),
protoplast
fusion, or electroporation, amongst others.
The present invention is further described with reference to the following non-
limiting
Examples and to the drawings.
EXAMPLE 1
Regulatable ectopic expression of alfalfa CycMs2 mitotic cyclin in tobacco
The cycMs2 alfalfa mitotic cyclin was expressed as a fusion protein with
haemaglutinin
(HA) in tobacco plants under the control of tetracycline-regulatable promoter
construct.
To facilitate the detection of the transgene, the haemaglutinin epitope tag
(HA) was
fused to the C-terminus of the CycMs2 coding region, to produce the fusion
designated
as "CycMs2-HA".
After Agrobacterium-mediated transformation, the expression of CycMs2-HA was
determined in leaves from 5 independent transgenic lines treated with 1 mg/l
CI-
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tetracyclin for 24 h (Figure 1-1). In all of these transgenic lines, the mRNA
of cycMs2-
HA transgene could be detected by RNA-blot hybridisation. In 4 lines, the
expression
was strictly dependent on the addition of tetracycline.
A suspension culture was initiated on 2,4-D-containing medium, using the line
2 plant
(Figure 1-1) as starting material. The expression of cycMs2-HA was still
strictly
dependent on tetracycline in cultured cells and the accumulation of cycMs2-HA
mRNA
could already be detected after 10 minutes of incubation with 1 mg/I CI-
tetracycline
(Figure 1-2).
To determine the optimal tetracycline concentration for expression of the
CycMs2-HA
transgene, cultured cells were treated with a range of CI-tetracycline
concentrations
for 24 h, and the expression of the CycMs2-HA protein was detected by protein
blotting
with the HA antibody (Figure 1-3, upper panel). A sharp increase in the
production of
CycMs2-HA protein was found at 0.01 mg/I CI-tetracycline concentration.
To determine whether the ectopically-expressed CycMs2-HA protein forms an
active
complex with cyclin-dependent kinase (CDK), we immunopurified the complex from
cell
extract treated with different tetracycline concentrations and measured the
protein
kinase activity of the CDK. The increase in activity of CycMs2-HA complexed
with
CDK was correlated with the amount of expressed cycMs2-HA cyclin protein in
these
extracts, indicating that the amount of cyclin is rate-limiting to produce
active CDK
complexes (Figure 1-3, middle panel). When different CDK complexes were
isolated
by the binding to the p13s~°,_protein, CDK activities were comparable
in cells
expressing or not expressing the CycMs2-HA protein, indicating that the CycMs2-
HA
associated CDK activity is only a minor portion of the total CDK activity in
these
extracts (Figure 1-3, lower panel).
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EXAMPLE 2
The CycMs2-mitotic cyclin protein is in the nucleus
By indirect immunofluorescence microscopy using anti-HA antibody as a probe,
the
localisation of the ectopically-expressed CycMs2-HA protein was determined
(Figure
2). In the presence of tetracycline, the CycMs2-HA protein was detected in the
nucleus of around 60% of cells (Figures 2-1 and 2-2). No signal was detectable
without incubation of cells in tetracycline (Figures 2-3 and 2-4).
To confirm the nuclear localisation by other means, cells were fractionated
into
cytoplasm and nucleus and the activity of the CycMs2-HA associated CDK was
determined by measuring the protein kinase activity of the complex
immunoprecipitated from nuclear and cytoplasmic extracts. The majority of
CycMs2-
HA associated kinase activity was present in the nucleus (Figure 3).
The intracellular localisation of the CycMs2 in tobacco cells was also
determined using
GFP and GFP-protein fusion genes placed in a regulated plant expression vector
pBIN-HygTX. In the binary vector pBIN-HygTX the expression is directed by a
modified cauliflower mosaic virus (CaMV) 35S promoter combined with the
regulatable
tetracyclin expression system (Weinmann et al, Plant J. 1994 Apr;S(4):559-69;
Gatz
et al at 1992, Plant J 2(3), 397-404). A modified version of GFP with s65T
mutation
and altered codon usage was used (Sheen et al 1995, Plant J 8(5) 777-784).
Figure
10 indicates that the CycMs2-GFP fusion is constitutively localised to the
nucleus and
absent in cells in telophase (arrow). Epifluorescence microphotograph of GFP
fluorescence (A); and DIC phase contrast image of the cell (B).
EXAMPLE 3
The length of the G2 phase is shortened in cells that ectopically express
the CycMs2-HA mitotic cyclin
Having determined that the amount of cyclin is rate limiting to form an active
CDK
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complex in plant cells, we asked if the ectopic expression of CycMs2 mitotic
cyclin in
G2 cells is sufficient to enter mitosis.
Cell divisions were synchronised by releasing cells from a block by the
inhibition of
DNA synthesis with aphidicolin in cultured cells. The expression of CycMs2-HA
protein
was induced by adding 0.1 mg/I CI-tetracycline, after removing aphidicolin,
and the
progression of cell cycle was followed by measuring the DNA content of cells
with flow
cytometry (Pfosser et al., 1995), counting the number of mitotic cells and
counting the
mitosis-specific microtubule structures (Figure 4).
In tetracycline-treated cells, the number of mitotic divisions started to
increase 8 h after
aphidicolin release and the maximal number of mitotic divisions were found at
10 h
(Figure 5A). In contrast, cells incubated without tetracycline entered mitosis
2 hours
later, and the highest number of mitotic divisions was achieved at 12 h. A
similar 2
hour advance in the formation of mitotic microtubule structures were observed
in cells
ectopically expressing the mitotic CycMs2-HA cyclin protein in the presence of
tetracyclin.
The measurement of DNA content by flow cytometry further confirmed these
results
(Figure 4-3).
While at 8 h a similar distribution of nuclei with G2 and G1 DNA contents was
found
in tetracycline-treated and control cells, at 10 h in the presence of
tetracycline a high
proportion of cells already had nuclei with G1 DNA content, indicating that
they passed
through mitosis.
From these experiments we can conclude that the ectopic expression of cycMs2
mitotic cyclin in G2 cells does not induce mitosis directly but advances cells
to enter
mitosis 2 hours earlier (Figure 5-4).
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As independent molecular proof for the advancement of cell divisions in the
cells
ectopically expressing the CycMs2-HA protein in G2 phase, mRNA was prepared
from
these cells and hybridised with an HA fragment to detect the transgene (Figure
5).
Additionally, the expression of S phase and mitosis-specific marker genes were
also
monitored, in particular the histone H4 and an endogenous mitotic tobacco
cyclin
(cycM) as shown in Figure 5.
Data shown in Figure 5 indicate that histone H4 mRNA was similarity present in
samples with aphidicolin and at 3 h both with and without tetracycline,
however at 20
h in the presence of tetracycline a higher level of histone H4 expression was
observed
than that in the control samples, indicating that some cells reached the
second S
phase at 20 h when the mitotic cyclin is ectopically expressed. The expression
of the
tobacco cycM mitotic cyclin followed the number of mitotic cells during the
synchronous cell division and was similarity advanced by about 2 hours in the
presence of tetracycline.
EXAMPLE 4
Ectopic expression of CycMs2 mitotic cyclin inhibits root regeneration
in culture from tobbaco leaves
Regeneration of shoots and roots from tobacco leaves is dependent on the
auxin/cytokinin ratio: high auxin to cytokinin concentration favours root
regeneration,
while high cytokinin to auxin concentration favours shoot regeneration.
In in vitro regeneration experiments, with leaf disks of the transgenic line
containing the
tetracycline inducible cycMs2 gene, the regeneration of shoots still required
cytokinin
even in the presence of ectopic mitotic cyclin expression, but root formation
was
inhibited by ectopic cyclin expression (Figure 6).
We measured the DNA content of nuclei from leaves treated for 5 days with
different
NAA and BAP concentrations in the presence or absence of tetracyclin (Figure
7). A
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higher percentage of cells with G2 DNA content was found in leaf cells from
wild type
plants treated with high auxin to cytokinin ratio favouring root formation
than in leaves
treated with high cytokinin to auxin ratio, favouring shoot regeneration. This
observation is compatible with the notion that cells spend more time in G2
when
cultured on medium favouring root formation compared to the treatment
favouring
shoot formation. In leaf disks from transgenic tobacco plants ectopically
expressing
cycMs2 the number of cells in G2 was reduced indicating a shortened G2 phase.
EXAMPLE 5
DISCUSSION
Plants are mutlicellular organisms with defined shapes and sizes. A
developmentally
determined body plan is elaborated by the controlled timing and orientation of
cell
divisions in restricted zones called meristems. Conserved regulators of cell
division
in eukaryotes are the cyclin-dependent protein kinases (CDKs). The expression
of
cyclin genes are tightly regulated in plants, e.g. B-type cyclins were only
found in
mitotic cells. We used a tetracycline regulatable promoter construct to study
if cyclin
expression is limiting in the timing of cell divisions. The expression of a
mitotic cyclin,
the alfalfa cycMs2, results in an elevated CDK activity, and cells with
increased cyclin
amount entered mitosis earlier. We tested the consequences of a shortened G2
phase on the auxin- and cytokinin-dependent regeneration of shoots and roots
from
leaf disks. A premature passage through mitosis inhibits root formation, thus
having
a cytokinin-like effect.
We propose a model, shown in Figure 8, that in which shoot versus root
regeneration
is regulated by the G1 and G2 exit points from the cell cycle. According to
our model,
regeneration of roots and root development and/or initiation depends upon
whether a
cell exits the cell cycle in the G1 or G2 phase. Roots are formed when cells
exit the cell
cycle in G2 phase, while shoot formation is dependent on an exit from G1 phase
or
following a shortened G2 phase.
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EXAMPLE 5
Expression of CycMs2 under the control of the patatin gene promoter
increases tuber size and number in potato plants
The CycMs2 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 Agrobacterium tumefaciens, and the introduced into
potato
plants.
The CycMs2 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 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 CycMs2 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-
CycMs2 transgenes is seen on tuber initiation. However, the Class I patatin
promoter
drives CycMs2 expression very early after tuber initiation onwards, allowing a
maximal
impact of CycMs2 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 - CycMs2 transgene
increases both
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the size and number of tubers.
EXAMPLE 6
Expression of CycMs2 under the control of endosperm-specific promoters
increases grain size and yield of grain crop plants
The alfalfa CycMs2 coding sequence is placed operably in connection with the
endosperm-specific Itr1 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 CycMs2 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 gene 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.
EXAMPLE 7
Expression of CycMs2 under the control of the cab-6 or ubi7 promoters
reduces leaf necrosis and chlorosis in lettuce plants
The alfalfa CycMs2 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
CycMs2 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-
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transformed control plants.
EXAMPLE 8
Ectopic expression of the CycMs2 (Medsa CycB2;2) mitotic cyclin mimics
cytokinin effects in dark-grown seedlings
Tobacco seedlings of CycMs2 T2 transformants (TM100 2/5) and transformants
with
a control plasmid (pain-HygTX) (see Example 1 for construct and transformation
details) were germinated and grown in the dark for 14 days and then placed on
light.
Photographs were taken 18 and 24 days after germination (see Figure 9). The
TM100
2/5 seedlings show a retardation in growth (fresh weight) of approximately 20-
30 % in
comparison to the controls. The roots of the TM100 2/5 seedlings show more
branching, there is reduced root length and hypocotyl elongation is strongly
retarded
in comparison to controls.
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