Drought tolerant plants and methods of increasing drought...

Multicellular living organisms and unmodified parts thereof and – Method of introducing a polynucleotide molecule into or... – The polynucleotide alters plant part growth

Reexamination Certificate

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C800S278000, C800S286000

Reexamination Certificate

active

06756526

ABSTRACT:

SEQUENCE LISTING
A printed Sequence Listing accompanies this application, and has also been submitted with identical contents in the form of a computer-readable ASCII file on CDROM.
BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention generally relates to the field of plants. More particularly, the present invention involves plant responses to stress and methods of altering these responses. Still more particularly, the present invention involves transgenic plants which have altered expression of phospholipase D which thereby affects plant transpiration, respiration, and bioremediation. Finally, the present invention involves breeding and selecting such transgenic plants for growth in stress-prone areas.
2. Description of the Prior Art
Terrestrial plants lose water primarily via stomata, which are pores defined by pairs of guard cells. These guard cells and stomata are located throughout the epidermis of plant stems and leaves. When subjected to heat and light, each pair of guard cells separates, thereby forming the stomata therebetween wherein plant transpiration and respiration occur. During respiration, when the stomata are open, carbon dioxide and oxygen enter and exit the leaf. When carbon dioxide enters, it participates in photosynthesis and releases oxygen as a waste product. The oxygen then passes out of the leaf through the open stomata. Additionally, oxygen also enters the leaf and takes part in respiration, thereby forming carbon dioxide as a waste product. This carbon dioxide exits the leaf via the stomata.
During transpiration, water, in the form of vapor, exits the stomata. It has been determined that more than 90% of the water loss in terrestrial plants is through the stomata. Plants minimize water loss and evaporation through the stomata in a number of ways. For example, more stomata are located on the underside of a leaf (the side of the leaf which faces the ground) than on the upper side. Stomata also close at night in response to a decreased amount of light, thereby increasing water conservation. Stomata also close in response to decreasing amounts of available water. This stomatal closure is crucial for maintaining hydration status in leaves and therefore contributes to plant survival during times of drought.
Phospholipase D (PLD) hydrolyzes phospholipids, generating phosphatidic acid (PA) and free head groups. This enzyme has been implicated in various processes, including signal transduction, membrane trafficking, cytoskeletal rearrangement, and membrane degradation. Suppression of a PLD in Arabidopsis has been shown to decrease the rate of abscisic acid (ABA)-promoted senescence in detached leaves. Other experiments have shown that the addition of phosphatidic acid (PA), a potential PLD reaction product, to protoplasts of barley aleurone and
Vicia faba
guard cells partially mimicked the effect of ABA. Activity and gene expression of PLD also increased in tissues treated with ABA and in plants under water deficit. (Xu, et al.
Promoter Analysis and Expression of a Phospholipase D Gene from Castor Bean,
115 Plant Physiol 387-395 (1997); Jacob, et al.
Abscisic Acid Signal Transduction in Guard Cells is Mediated By Phospholipase D Activity,
96 PNAS 12192-12197 (1999); and Frank, et al.
Water Deficit Triggers Phospholipase D Activity in the Resurrection Plant Craterostigma plantagineum,
12 The Plant Cell 111-123 (2000)).
Because the physiological role of PLD in plants has not been established, the increases in PLD activities and gene expression shown in those studies provide no direct evidence for a role of PLD in plant response to ABA or water deficit. In addition, multiple forms of PLD in plants have been identified recently, and they exhibit different biochemical properties and patterns of expression. This raises a question of which PLD is involved in guard cell regulation. Moreover, the process which promotes stomatal closure during periods of drought stress has not heretofore been determined. Selective regulation and modification of stomatal closure would contribute to the development of drought resistant plants, plants with modified rates of respiration, transpiration, and bioremediation, and plants which react to drought stress in a quicker, more efficient manner.
SUMMARY OF THE INVENTION
The present invention overcomes the problems of the prior art and provides a distinct advance in the state of the art by providing methods of altering drought response in plants, genetically altered plants which have modified stomatal responses in comparison to wild-type plants, methods of selecting for plants having upregulated or down regulated stomatal closure, methods of testing plants for stomatal closure, and methods of differentiating between wild-type plants and plants which have been genetically altered according to the present invention.
It has now been determined that the hormone abscisic acid (ABA) promotes stomatal closure and that phospholipase D (PLD) participates in the regulation of stomatal closure induced by ABA and water stress. Three distinct PLDs, PLD&agr;, PLD&bgr; and PLD&ggr;, have been cloned from Arabidopsis (Dyer et al., 109 Plant Physiol 1497 (1995); Pappan et al., 272 J. Biol. Chem. 7048-7054 (1997); Qin et al., 272 J. Biol. Chem. 28267-28273 (1997)). PLD&agr; is expressed in Arabidopsis guard cells, and the introduction of a PLD&agr; antisense gene abrogated its expression. The sequence of the PLD&agr; antisense gene is provided herein as Sequence ID No. 1. Preferably, sequences having at least about 60% sequence similarity or 50% sequence identity with SEQ ID No. 1 are introduced into the PLD genome and suppress expression of PLD&agr;. More preferably, such sequences have at least about 70% sequence similarity or 65% sequence identity with SEQ ID No. 1. Most preferably, such sequences have at least about 90% sequence similarity or 85% sequence identity with SEQ ID No. 1. Plants expressing decreased amounts of PLD&agr; also exhibit a decreased sensitivity to ABA as well as impaired stomatal closure. PLD&agr;-depleted plants exhibited an accelerated rate of transpirational water loss and decreased ability to tolerate drought stress. Overexpression of PLD&agr; increased the leaf's sensitivity to ABA in promoting stomatal closure and decreased the rate of transpirational water loss. Thus, PLD plays a crucial role in controlling stomatal movement and the plant's tolerance to water deficit.
To investigate the function of PLD in plant-water relations, the presence of PLD&agr; in Arabidopsis guard cells was determined using immunolabeling with isoform-specific antibodies raised against PLD&agr;. To perform this testing, Arabidopsis plants were grown. After 4-5 weeks of growth, fully expanded Arabidopsis leaves were detached. Epidermal peels were collected from the abaxial side of Arabidopsis leaves immediately following detachments and incubated for 1 hour in a solution containing 5 mM MES-KOH (pH 6.1), 22 mM KCl, and 1 mM CaCl
2
. The peels were then fixed in 1.5% formaldehyde, 0.5% glutaraldehyde, 0.1 M PIPES, 5 mM EGTA, 2 mM MgCl
2
, and 0.05% Triton X-100, pH 6.9 for 35 minutes with gentle shaking. The fixed peels were washed in phosphate-buffered saline (PBS) for 30 minutes with three changes of solution. Then they were spread onto microscope slides, blotted to remove excess solution, and freeze-shattered using the methods of Wasteneys, et al.,
Freeze Shattering: A Simple and Effective Method for Permeabilizing Higher Plant Cell Walls,
188 Journal of Microscopy 51-61 (1997), the teachings of which are hereby incorporated by reference. Briefly, epidermal peels were collected from the abaxial side of Arabidopsis leaves immediately following their detachment and incubated for one hour in a solution containing 5 mM MES-KOH (pH 6.1), 22 mM KCl, and 1 mM CaCl
2
, Next, the peels were fixed and the fixed peels were washed in phosphate-buffered saline (PBS) for 30 minutes with three changes of solution. The peels were spread onto a microscope slide, blotted to remove excess solution, and then sandwiched wi

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