Multicellular living organisms and unmodified parts thereof and – Method of introducing a polynucleotide molecule into or... – The polynucleotide alters plant part growth
Reexamination Certificate
2000-07-05
2004-08-10
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, C800S286000, C800S287000, C800S298000
Reexamination Certificate
active
06774284
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to polynucleotides which encode plant polypeptides and which exhibit senescence-induced expression, transgenic plants containing the polynucleotides in antisense orientation and methods for controlling senescence in plants. More particularly, the present invention relates to plant lipase genes whose expression is induced by the onset of senescence and the use of the lipase gene to control senescence in plants.
DESCRIPTION OF THE PRIOR ART
Senescence is the terminal phase of biological development in the life of a plant. It presages death and occurs at various levels of biological organization including the whole plant, organs, flowers and fruit, tissues and individual cells.
Cell membrane deterioration is an early and fundamental feature of senescence. Metabolism of lipids, in particular membrane lipids, is one of several biochemical manifestations of cellular senescence. Rose petals, for example, sustain an increase in acyl hydrolase activity as senescence progresses that is accompanied by a loss of membrane function (Borochov, et al., Plant Physiol., 1982, 69, 296-299). Cell membrane deterioration is an early and characteristic feature of senescence engendering increased permeability, loss of ionic gradients and decreased function of key membrane proteins such as ion pumps (Brown, et al., Plant Physiol.: A Treatise, Vol. X. Academic Press, 1991, pp.227-275). Much of this decline in membrane structural and functional integrity can be attributed to lipase-mediated phospholipid metabolism. Loss of lipid phosphate has been demonstrated for senescing flower petals, leaves, cotyledons and ripening fruit (Thompson, J. E., Senescence and Aging in Plants, Academic Press, San Diego, 1988, pp. 51-83), and this appears to give rise to major alterations in the molecular organization of the membrane bilayer with advancing senescence that lead to impairment of cell function. In particular, studies with a number of senescing plant tissues have provided evidence for lipid phase separations in membranes that appear to be attributable to an accumulation of lipid metabolites in the membrane bilayer (McKersie and Thompson, 1979, Biochim. Biophys. Acta, 508: 197-212; Chia, et al., 1981, Plant Physiol., 67:415-420). There is growing evidence that much of the metabolism of lipids in senescing tissue is achieved through senescence-specific changes in gene expression (Buchanan-Wollaston, V., J. Exp. Bot., 1997, 307:181-199).
The onset of senescence can be induced by different factors both internal and external. For example, ethylene plays a role in many plants in a variety of plant processes such as seed germination, seedling development, fruit ripening and flower senescence. Ethylene production in plants can also be associated with trauma induced by mechanical wounding, chemicals, stress (such as produced by temperature and water amount variations), and by disease. Ethylene has been implicated in the regulation of leaf senescence in many plants, but evidence obtained with transgenic plants and ethylene response mutants has indicated that, although ethylene has an effect on senescence, it is not an essential regulator of the process. In many plants ethylene seems to have no role in fruit ripening or senescence. For example in the ripening of fruits of non-climacteric plants such as strawberry, in senescence of some flowers such as day lilies and in leaf senescence in some plants, such as Arabidopsis, and in particular, in the monocots there is no requirement for ethylene signaling (Smart, C. M., 1994, New Phytology, 126:419-448; Valpuesta, et al., 1995, Plant Mol. Biol., 28:575-582).
External factors that induce premature initiation of senescence include environmental stresses such as temperature, drought, poor light or nutrient supply, as well as pathogen attack. As in the case of natural (age-related) senescence, environmental stress-induced senescence is characterized by a loss of cellular membrane integrity. Specifically, exposure to environmental stress induces electrolyte leakage reflecting membrane damage (Sharom, et al., 1994, Plant Physiol., 105:305-308; Wright and Simon, 1973, J. Exp. Botany, 24:400-411; Wright, M., 1974, Planta,120:63-69; and Eze et al., 1986, Physiologia Plantarum, 68:323-328), a decline in membrane phospholipid levels (Wright, M., 1974, Planta,120:63-69) and lipid phase transitions (Sharom, et al., 1994, Plant Physiol., 105:305-308), all of which can be attributed to the action of lipase. Plant tissues exposed to environmental stress also produce ethylene, commonly known as stress ethylene (Buchanan-Wollaston, V., 1997, J. Exp. Botany, 48.181-199; Wright, M., 1974, Planta,120:63-69). As noted above, ethylene is known to cause senescence in some plants. Membrane deterioration leading to leakage is also a seminal feature of seed aging, and there is evidence that this too reflects deesterification of fatty acids from membrane phospholipids (McKersie, B. D., Senarata, T., Walker, M. A., Kendall, E. J. and Hetherington, P. R. In: Senescence and Aging in Plants, Ed. L. D. Nooden and A. C. Leoopold, academic Press, 1988. PP 441-464).
Presently, there is no widely applicable method for controlling onset of senescence caused by either internal or external, e.g., environmental stress, factors. At present, the technology for controlling senescence and increasing the shelf-life of fresh, perishable plant produce, such as fruits, flowers and vegetables relies primarily upon reducing ethylene biosynthesis. For example, U.S. Pat. No. 5,824,875 discloses transgenic geranium plants which exhibit prolonged shelf-life due to reduction in levels of ethylene resulting from the expression of one of three 1-amino-cyclopropane-1-carboxylate (ACC) synthase genes in antisense orientation. Consequently, this technology is applicable to only a limited range of plants that are ethylene-sensitive.
The shelf-life of some fruits is also extended by reducing ethylene biosynthesis, which causes ripening to occur more slowly. Since senescence of these fruits is induced after ripening, the effect of reduced ethylene biosynthesis on shelf-life is indirect. Another approach used to delay fruit ripening is by altering cellular levels of polygalacturonase, a cell-wall softening enzyme that is synthesized during the early stages of ripening. This approach is similar to controlling ethylene biosynthesis in that it, too, only indirectly affects senescence and again, is only applicable to a narrow range of plants.
Thus, there is a need for a method of controlling senescence in plants which is applicable to a wide variety of plants. It is therefore of interest to develop senescence modulating, technologies that are applicable to all types of plants, regardless of ethylene sensitivity.
SUMMARY OF THE INVENTION
This invention is based on the discovery and cloning of a full length cDNA clone encoding a carnation senescence-induced lipase and a full-length cDNA clone encoding
Arabidopsis thaliana
senescence-induced lipase. The nucleotide sequences and corresponding amino acid sequences for the senescence-induced lipase genes are disclosed herein. The nucleotide sequence of the carnation senescence-induced lipase gene has been successfully used as a heterologous probe to detect corresponding genes or RNA transcripts in several plants that are similarly regulated.
The invention provides a method for genetic modification of plants to control the onset of senescence, either age-related senescence or environmental stress-induced senescence. The senescence-induced lipase nucleotide sequences of the invention, fragments thereof, or combinations of such fragments, are introduced into a plant cell in reverse orientation to inhibit expression of the endogenous senescence-induced lipase gene, thereby reducing the level of endogenous senescence-induced lipase and altering senescence in the transformed plant.
Using the methods of the invention, transgenic plants are generated and monitored for growth and development. Plants or detached parts of plants (e.g., cuttings, flowers,
Hong Yuwen
Hudak Katalin
Thompson John E.
Wang Tzann-Wei
Kenyon & Kenyon
Mehta Ashwin
Senesco, Inc.
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