Nucleic acid encoding GAI gene of Arabidopsis thaliana

Details Nucleic acid encoding GAI gene of Arabidopsis thaliana Nucleic acid encoding GAI gene of Arabidopsis thaliana

2001-07-25

2004-09-21

Mehta, Ashwin (Department: 1638)

Multicellular living organisms and unmodified parts thereof and

Method of introducing a polynucleotide molecule into or...

The polynucleotide alters plant part growth

C435S320100, C435S419000, C435S468000, C435S471000, C536S023600, C800S260000

Reexamination Certificate

active

06794560

ABSTRACT:

BACKGROUND OF THE INVENTION
This invention relates to the genetic control of growth and/or development of plants and the cloning and expression of genes involved therein. More particularly, the invention relates to the cloning and expression of the GAI gene of
Arabidopsis thaliana
, and homologues from other species, and use of the genes in plants.
An understanding of the genetic mechanisms which influence growth and development of plants, including flowering, provides a means for altering the characteristics of a target plant. Species for which manipulation of growth and/or development characteristics may be advantageous includes all crops, with important examples being the cereals, rice and maize, probably the most agronomically important in warmer climatic zones, and wheat, barley, oats and rye in more temperate climates. Important crops for seed products are oil seed rape and canola, sugar beet, maize, sunflower, soyabean and sorghum. Many crops which are harvested for their roots are, of course, grown annually from seed and the production of seed of any kind is very dependent upon the ability of the plant to flower, to be pollinated and to set seed. In horticulture, control of the timing of growth and development, including flowering, is important. Horticultural plants whose flowering may be controlled include lettuce, endive and vegetable brassicas including cabbage, broccoli and cauliflower, and carnations and geraniums. Dwarf plants on the one hand and over-size, taller plants on the other may be advantageous and/or desirable in various horticultural and agricultural contexts.
Arabidopsis thaliana
is a favourite of plant geneticists as a model organism. Because it has a small, well-characterized genome, is relatively easily transformed and regenerated and has a rapid growing cycle, Arabidopsis is an ideal model plant in which to study growth and development and its control.
Many plant growth and developmental processes are regulated by specific members of a family of tetracyclic diterpenoid growth factors known as gibberellins (GA)
1
. The gai mutation of Arabidopsis confers a dwarf phenotype and a dramatic reduction in GA-responsiveness
2-9
. Here we report the molecular cloning of gai via Ds transposon mutagenesis.
The phenotype conferred by the Ds insertion allele confirms that gai is a gain-of-function mutation, and that the wild-type allele (GAI) is dispensable
5,6
. GAI encodes a novel polypeptide (GAI) of 532 amino acid residues, of which a 17 amino acid domain is missing in the gai mutant polypeptide. This result is consistent with GAI acting as a plant growth repressor whose activity is antagonized by GA. Though we are not to be bound by any particular theory, gai may repress growth constitutively because it lacks the domain that interacts with the GA signal. Thus according to this model GA regulates plant growth by de-repression.
gai is a dominant, gain-of-function mutation, which confers a dark-green, dwarf phenotype, and interferes with GA reception or subsequent signal-transduction
2-9
. Dominant mutations conferring similar phenotypes are known in other plant species, including maize
10-12
and wheat
13
. The latter are especially important because they are the basis of the high-yielding, semi-dwarf wheat varieties of the ‘green revolution’
14
. The increased yield of these varieties is due to an increased grain production per ear, and superior straw strength. The shorter, stronger straw greatly reduces the losses resulting from lodging, that is flattening of standing wheat plants by rain/wind. We set out to clone gai from Arabidopsis because of its importance to the understanding of GA signal-transduction, and because of the potential for use of GA-insensitivity in the development of wheat and other crops such as oil-seed rape and rice which may show improvement as great as that already seen in wheat.
SUMMARY OF THE INVENTION
According to a first aspect of the present invention there is provided a nucleic acid molecule comprising a nucleotide sequence encoding a polypeptide with GAI function. The term “GAI function” indicates ability to influence the phenotype of a plant like the GAI gene of
Arabidopsis thaliana
. “GAI function” may be observed phenotypically in a plant as inhibition, suppression, repression or reduction of plant growth which inhibition, suppression, repression or reduction is antagonised by GA. GAI expression tends to confer a dwarf phenotype on a plant which is antagonised by GA. Overexpression in a plant from a nucleotide sequence encoding a polypeptide with GAI function may be used to confer a dwarf phenotype on a plant which is correctable by treatment with GA.
Also according to an aspect of the present invention there is provided a nucleic acid molecule comprising a nucleotide sequence encoding a polypeptide with ability to confer a gai mutant phenotype upon expression. gai mutant plants are dwarfed compared with wild-type, the dwarfing being GA-insensitive.
By gibberellin or GA is meant a diterpenoid molecule with the basic carbon-ring structure shown in FIG.
1
and possessing biological activity, i.e. we refer to biologically active gibberellins.
Biological activity may be defined by one or more of stimulation of cell elongation, leaf senescence or elicitation of the cereal aleurone &agr;-amylase response. There are many standard assays available in the art, a positive result in any one or more of which signals a test gibberellin as biologically active
28,29,30
.
Assays available in the art include the lettuce hypocotyl assay, cucumber hypocotyl assay, and oat first leaf assay, all of which determine biological activity on the basis of ability of an applied gibberellin to cause elongation of the respective tissue. Preferred assays are those in which the test composition is applied to a gibberellin-deficient plant. Such preferred assays include treatment of dwarf GA-deficient Arabidopsis to determine growth, the dwarf pea assay, in which internode elongation is determined, the Tan-ginbozu dwarf rice assay, in which elongation of leaf sheath is determined, and the d5-maize assay, also in which elongation of leaf sheath is determined. The elongation-bioassays measure the effects of general cell elongation in the respective organs and are not restricted to particular cell types.
Further available assays include the dock (Rumex) leaf senescence assay and the cereal aleurone &agr;-amylase assay. Aleurone cells which surround the endosperm in grain secrete &agr;-amylase on germination, which digests starch to produce sugars then used by the growing plant. The enzyme production is controlled by GA. Isolated aleurone cells given biologically active GA secrete &agr;-amylase whose activity can then be assayed, for example by measurement of degradation of starch.
Structural features important for high biological activity (exhibited by GA
1
, GA
2
, GA
4
and GA
7
) are a carboxyl group on C-6 of B-ring; C-19, C-10 lactone; and &bgr;-hydroxylation at C-3. &bgr;-hydroxylation at C-2 causes inactivity (exhibited by GA
8
, GA
29
, GA
34
and GA
51
). gai mutants do not respond to GA treatment, e.g. treatment with GA
1
, GA
3
or GA
4
.
Treatment with GA is preferably by spraying with aqueous solution, for example spraying with 10
−4
M GA
3
or GA
4
in aqueous solution, perhaps weekly or more frequently, and may be by placing droplets on plants rather than spraying. GA may be applied dissolved in an organic solvent such as ethanol or acetone, because it is more soluble in these than in water, but this is not preferred because these solvents have a tendency to damage plants. If an organic solvent is to be used, suitable formulations include 24 &eegr;l of 0.6, 4.0 or 300 mM GA
3
or GA
4
dissolved in 80% ethanol. Plants, e.g. Arabidopsis, may be grown on a medium containing GA, such as tissue culture medium (GM) solidified with agar and containing supplementary GA.
Nucleic acid according to the present invention may have the sequence of a wild-type GAI gene of
Arabidopsis thaliana
, or be a mutant, derivative, variant or allele of the

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