Transgenic plants expressing a MAPKKK protein kinase domain

Details Transgenic plants expressing a MAPKKK protein kinase domain Transgenic plants expressing a MAPKKK protein kinase domain

1999-08-10

2003-09-02

Bui, Phuong T. (Department: 1638)

Multicellular living organisms and unmodified parts thereof and

Method of introducing a polynucleotide molecule into or...

C800S279000, C800S287000, C800S298000, C800S289000, C800S295000, C800S306000, C800S317000, C435S069100, C435S418000, C435S419000, C435S468000, C435S320100, C536S023100, C536S023200, C536S023600, C536S024100

Reexamination Certificate

active

06613959

ABSTRACT:

BACKGROUND OF THE INVENTION
This invention relates to the manipulation of plant gene expression and the production of transgenic plants.
Auxin is an essential plant hormone that regulates diverse processes, such as cell division and elongation, root and leaf development, apical dominance, tropism, and reproduction (Davies, P. J., In: Plant hormones, ed., Davies, P. J., pp. 1-12, Kluwer, Dordrecht, Netherlands, 1995). The auxin response is regulated by a complex signaling network, and reflects a balance between auxin and other synergistical or antagonistical signaling pathways in plant cells (Bellincampi et al.,
Plant Cell
8: 477-487, 1996; Coenen et al.,
Trends Plant Sci.
2: 351-356, 1997). A primary event of auxin action is the activation of many early response genes. Extensive studies of the early response gene promoters have identified several auxin responsive cis-elements and trans-acting factors (Abel et al.,
Plant Physiol.
111: 9-17, 1996; Ulmasov et al.,
Science
276: 1865-1868, 1997). Although genetic approaches have significantly advanced our understanding of auxin action (Walden et al.,
Trends Plant Sci.
1: 335-339, 1996; Leyser,
Curr. Biol.
8: R305-R307, 1998; Guilfoyle,
Trends Plant Sci.
3: 205-207, 1998), the molecular mechanisms underlying signal transduction pathways that control auxin responsive transcription remain largely unknown.
In yeast, worms, insects, and mammals, the primary responses to hormone, growth, and stress signals are mediated by a conserved signaling cascade consisting of three protein kinases, the mitogen-activated protein kinase (MAPK), mitogen-activated protein kinase kinase (MAPKK), and mitogen-activated protein kinase kinase kinase (MAPKKK). MAPKKK phosphorylates and activates MAPKK that, in turn, phosphorylates and activates MAPK. The activated MAPK can be translocated into the nucleus where it phosphorylates transcription factors that control gene expression (Herskowitz,
Cell
80: 187-197, 1995; Kyriakis et al.,
J. Biol. Chem.
271: 24313-24316, 1996). Although many plant MAPK, MAPKK, and MAPKKK homologues have been identified based on sequence conservation and functional complementation in yeast, their precise physiological functions in plants are largely unknown (Hirt,
Trends Biol. Sci.
2: 11-15, 1997). It also remains unclear whether and how these homologues constitute specific MAPK kinase cascades (Mizoguchi et al.,
Trends Biotech.
15: 15-19, 1997).
Plants are constantly exposed to environmental stimuli that influence their growth and development. Adverse environmental conditions, including heat, salinity, freezing, and drought, greatly compromise plant productivity and reduce crop yield. Genetic approaches have been taken to enhance plant tolerance to stresses through alteration of osmolytes, osmoprotectants, membrane fatty acids, channels, transcription factors, and enzymes that scavenge active oxygen species by transferring or mutating individual stress target genes. A need in the art therefore exists for developing molecular strategies that enable plants to have resistance or tolerance to adverse environmental conditions.
SUMMARY OF THE INVENTION
The invention is based on applicants' discovery that a mitogen-activated protein kinase kinase kinase (MAPKKK) polypeptide, such as NPK1 of tobacco and the ANPs of Arabidopsis, is involved in signaling the activation of stress protective gene transcription, repression of early auxin response gene transcription, and the alteration of seed development. Accordingly, the invention involves methods of genetically engineering plants to produce altered, agronomic, physiological, or developmental changes in plants by expressing a transgene including DNA encoding a kinase domain of a MAPKKK within the tissues of the plants. In particular, it has been found that it is possible to engineer plants that express a recombinant MAPKKK that are resistant to a broad spectrum of stresses (e.g., drought, increased salinity, heat shock, and freezing temperature), that have repressed early auxin gene expression, or that have altered seed development.
In one aspect, the invention therefore features a method for increasing stress resistance or tolerance in a plant. The method, in general, includes the steps of: (a) introducing into plant cells a transgene including DNA encoding a kinase domain of a MAPKKK operably linked to a promoter functional in plant cells to yield transformed plant cells; and (b) regenerating a transgenic plant from the transformed cells, wherein the kinase domain of the MAPKKK is expressed in the cells of the transgenic plant, thereby increasing the level of stress resistance or tolerance in the transgenic plant. In preferred embodiments, the expression of the DNA encoding the kinase domain activates the expression of a stress-inducible gene (e.g., a gene encoding a glutathione S-transferase, an asparagine synthetase, or a heat shock protein). In particular applications, the method is especially useful for providing to a plant resistance or tolerance to an environmental stress. Exemplary environmental stresses include, without limitation, those which occur upon exposure of the transgenic plant to limited or inadequate water availability (e.g., drought conditions), excess salt or osmotic conditions, excess temperature conditions (e.g., heat, cold, or frost), excess light, a pathogen, a chemical (e.g. a metal, herbicides, and pollutants), an oxidative stress, UV light, and wounding. In preferred embodiments, the plant is protected against multiple stress conditions.
In another aspect, the invention features a method for reducing the action of an auxin in a plant. The method includes the steps of: (a) introducing into plant cells a transgene including DNA encoding a kinase domain of a MAPKKK operably linked to a promoter functional in plant cells to yield transformed plant cells; and (b) regenerating a transgenic plant from the transformed cells, wherein the kinase domain of the MAPKKK is expressed in the cells of the transgenic plant, thereby reducing the action of the auxin in the transgenic plant. In preferred embodiments, the expression of the DNA encoding the kinase domain represses the expression of an early-auxin gene (e.g., those which are under the control of a promoter which is substantially identical to the GH3 promoter or a promoter which includes the ER7 element).
In still another aspect, the invention features a method for altering seed development. In particular, the method includes the steps of: (a) introducing into plant cells a transgene including DNA encoding a kinase domain of a MAPKKK operably linked to a promoter functional in plant cells to yield transformed plant cells; and (b) regenerating a transgenic plant from the transformed cells, wherein the kinase domain of the MAPKKK is expressed in the cells of the transgenic plant, thereby altering the development of a seed in the transgenic plant. In preferred embodiments, the expression of the DNA encoding the kinase domain enriches endosperm development, enriches embryo development, or attenuates seed development. In yet other preferred embodiments, the attenuation of the seed development results in a seedless plant (e.g., a seedless fruit or vegetable).
In yet another aspect, the invention features a method for increasing the yield or productivity of a transgenic plant. The method generally includes the steps of: (a) introducing into plant cells a transgene including DNA encoding a kinase domain of a MAPKKK operably linked to a promoter functional in plant cells to yield transformed plant cells; and (b) regenerating a transgenic plant from the transformed cells, wherein the kinase domain of the MAPKKK is expressed in the cells of the transgenic plant, thereby increasing the yield of the transgenic plant.
In related aspects of the invention, the invention features a plant (or plant cell, plant tissue, plant organ, or plant component) including a recombinant transgene capable of expressing a kinase domain of a MAPKKK, wherein the transgene is expressed in the transgenic plant under the control of

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