Method to improve drought tolerance in plants

Details Method to improve drought tolerance in plants Method to improve drought tolerance in plants

2000-05-10

2003-10-21

McElwain, Elizabeth F. (Department: 1638)

Multicellular living organisms and unmodified parts thereof and

Method of introducing a polynucleotide molecule into or...

C435S320100, C435S468000, C800S298000

Reexamination Certificate

active

06635803

ABSTRACT:

BACKGROUND OF THE INVENTION
Unpredictable rainfall, increases in soil salinity, and low temperature at the beginning or end of the growing season often result in decreased plant growth and crop productivity. These three environmental factors share at least one element of stress and that is water deficit or dehydration.
Drought is a significant problem in agriculture today. Over the last 40 years, for example, drought accounted for 74% of the total U.S. crop losses of corn (U.S. Department of Agriculture, 1990. Agricultural Statistics. U.S. Government Printing Office, Washington, D.C.). To sustain productivity under adverse environmental conditions, it is important to provide crops with a genetic basis for coping with water deficit, for example, by breeding water retention and/or drought tolerance mechanisms into crops so that they can grow and yield under these adverse conditions.
When the rate of transpiration exceeds that of water uptake or supply, water deficit occurs and wilting symptoms appear. The responses of plants to water deficits include leaf rolling and shedding, stomata closure, leaf temperature increases, and wilting. Metabolism is also profoundly affected. General protein synthesis is inhibited and significant increases in certain amino acid pools, such as proline, become apparent (Barnett et al., 1966). During these water deficit periods, the photosynthetic rate decreases with the ultimate result of loss in yield (Boyer, 1976). If carried to an extreme, severe water deficits result in death of the plant.
Moreover, fresh water is increasingly becoming a scarce and threatened resource in large part due to agricultural production (Serageldin, 1995). Some studies have suggested that partial reduction in stomatal apertures could optimize CO
2
and H
2
O exchange, particularly in light of rising atmospheric CO
2
levels (Morison et al., 1987) and thus optimize CO
2
flow into leaves for photosynthesis and water loss through transpiration. Classical studies showed that light-induced stomatal opening is mediated by K
+
and anion accumulation in guard cells (Imamura, 1943; Humble et al., 1971). Biophysical, second messenger regulation and physiological studies have suggested inward-rectifying K
+
(K
+
in
) channels provide a major pathway for K
+
uptake into guard cells during stomatal opening (Schroeder et al., 1994; Müller-Röber et al., 1998).
In addition to its role in stomatal movements (Schroeder et al., 1984; Schroeder et al., 1987; Schroeder et al., 1994; MacRobbie, 1998; Müller-Röber et al., 1998), K
+
in
channels have been proposed to function in K
+
uptake in roots (Findlay et al., 1994; Gassmann et al., 1994; Maathuis et al., 1995; Roberts et al., 1995; Hirsch et al., 1998), leaf movements (Kim et al., 1993), and nutrient transport in vascular tissues (Wegner et al., 1994; Gaymard et al., 1998). Molecular analyses of insertional disruption mutants in the Arabidopsis K
+
channel genes AKT1 and SKOR1 provide further evidence for roles of K
+
channels in K
+
uptake in roots (Hirsch et al., 1998) and in K
+
release into the xylem sap (Gaymard et al., 1998). However, molecular physiological analyses of the functions of other plant K
+
channels have not yet been reported.
KAT1, a K
+
in
channel protein, is expressed predominately in guard cells (Müller-Röber et al., 1995; Nakamura et al., 1995). Transgenic Arabidopsis that express a mutant of the Arabidopsis KAT1, which has a reduced sensitivity to Cs
+
block, were demonstrated to exhibit partial light-induced stomatal opening in the presence of Cs
+
concentrations that ordinarily inhibit stomatal opening in wild-type (Ichida et al., 1997). While Ichida et al. (1997) provide molecular evidence that transgenic K
+
in
channels in guard cells play a role in light-induced stomatal opening in the presence of Cs
+
, and that KAT1 functions as a plasma membrane K
+
in
channel in planta, these results were obtained with agents, i.e., pharmacological blockers, which can potentially affect multiple mechanisms in plant cells. For example, Ba
2+
blocks K
+
in
and outward-rectifying K
+
channels (Schroeder et al., 1987). Moreover, although 10 mM Ba
2+
blocks 90% of K
+
in
channel currents in faba bean guard cells (Schroeder et al., 1987), Ba
2+
does not affect the final stomatal apertures but affects only the rate of stomatal opening (Kelly et al., 1995). Further, several genetic loci in Arabidopsis affect Cs
+
sensitivity (Sheahan et al., 1993). Thus, the interpretation of pharmacological data relating to K
+
in
channels can be problematic.
Further, electrophysiological studies on guard cell K
+
channels have not been accompanied thus far by measurements of K
+
contents in guard cells to verify the proposed function of K
+
in
channels in K
+
uptake.
Thus, what is needed is a method to determine whether the specific inhibition of K
+
in
channel activity, e.g., in the absence of pharmacological blockers, is effective to inhibit light-induced stomatal guard cell opening in plants. What is also needed is a method which inhibits light-induced stomatal opening in a plant so as to increase drought tolerance in the plant.
SUMMARY OF THE INVENTION
The invention provides a method for increasing drought tolerance in a plant. The method comprises inhibiting or disabling K
+
in
channel activity in the stomatal guard cells of the plant, e.g., in a dicot or a monocot. K
+
in
channel activity may be measured by methods well known to the art including, but not limited to, K
+
uptake, e.g., into stomatal guard cells, K
+
currents, light-induced stomatal opening, final stomatal aperture diameter, or transpirational water loss from, for example, leaves of the plant. In one embodiment of the invention, the K
+
in
channel activity in the plant is inhibited or disabled by the expression of a DNA segment in the plant, e.g., a DNA segment which encodes a gene product that inhibits or reduces K
+
in
channel activity in the stomatal guard cells of the plant, for example, a gene product which interacts with a K
+
in
channel protein, e.g., a calcium-dependent protein kinase (Li et al., 1998) or protein phosphatase 2A (Li et al., 1994), and/or a gene product which encodes a K
+
channel protein. Thus, the plant may be a transgenic plant, the genome of which is augmented by a DNA segment such as one encoding an &agr; or &bgr; subunit of a K
+
channel. Preferably, the DNA segment encodes a K
+
in
channel protein, e.g., KAT1 (Nakamura et al., 1995), KST1 (Müller-Röber et al., 1995), AKT1 or AKT2. Also preferably, the DNA segment comprises a gene, e.g., the KAT1 gene, with at least one dominant negative mutation.
As described hereinbelow, transgenic Arabidopsis plants were generated that expressed dominant negative point mutations in KAT1. Patch-clamp analyses with transgenic guard cells from two independent lines showed that K
+
in
peak currents were reduced by about 75% compared to controls at −180 mV, which resulted in significant inhibition of light-induced stomatal opening. Analysis of intracellular K
+
content with sodium hexanitrocobaltate (III) showed that K
+
uptake was also significantly reduced in guard cells of two strong suppressor lines during light-induced stomatal opening. Moreover, transpirational water loss from leaves was reduced. Interestingly, plant growth improved under limited watering conditions in these K
+
in
channel suppressor lines. Further, comparisons of guard cell K
+
in
current magnitudes among 4 different transgenic lines with different levels of expression of the dominant negative kat1 gene provided quantitative data on the range of activities of K
+
in
channels required for guard cell K
+
uptake during light-induced stomatal opening. These data provide molecular evidence that K
+
in
channels function as a major pathway for K
+
uptake

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