Research Interests:

My interests focus on understanding the mechanisms by which plants perceive and respond to environmental stresses such as high salinity and drought.  Detailed descriptions of specific research projects that emphasize different aspects of this general theme can be found by clicking on highlighted text.  Major research projects include: Molecular Genetics of Crassulacean Acid Metabolism (CAM) – a project to understand how CAM, an alternative type of photosynthetic carbon fixation that helps plants survive in arid climates, is controlled by environmental stress and circadian rhythms; Functional Genomics of Plant Stress Tolerance – a multi-institutional research program designed to identify and functionally analyze all genes that contribute to salinity and drought tolerance in a small collection of plant and non-plant model species; Engineering Environmental Stress Tolerance in Higher Plants – a practical project aimed at understanding how a unique set of unusual proteins called hydrophilins or dehydrins function to confer salinity, drought, and cold tolerance in higher plants; and Developing Paradigms for Functional Genomics of Protein Kinases and Phosphoproteins Using the calcium-dependent protein kinase (CDPK) Superfamily - a multi-institutional research program designed to identify CDPK substrates using substrate traps and interaction screens such as the yeast two-hybrid system, to determine the subcellular locations of membrane-associated or compartmentalized CDPKs, to use mass spectrometry to initiate identification of all CDPK phosphorylation sites in the plant proteome, and to develop lectures and on-line resources targeted for colleges without science research programs.

Students and postdoctoral research associates with broad interests in plant molecular genetics, genetic engineering of plants, and plant biochemistry are welcomed to pursue training opportunities in this laboratory. Our research is funded by the National Science Foundation (Integrative Plant Biology, Plant Genome, and 2010 Programs), the United States Department of Agriculture (CRGP-NRI Plant Responses to the Environment Program), and the Nevada Agricultural Experiment Station.

Recent Publications:

Cushman JC, Bohnert HJ (1999) Crassulacean acid metabolism: Molecular Genetics. Ann Rev Plant Physiol Plant Mol Biol 50: 305-332

Taybi T, Cushman JC (1999) Signaling Events Leading to Crassulacean Acid Metabolism (CAM) Induction in the Common Ice Plant, Mesembryanthemum crystallinum. Plant Physiol 121: 545-555

Cushman JC, Wulan T, Kuscuoglu N, Spatz MD (2000) Efficient plant regeneration of Mesembryanthemum crystallinum via somatic embryogenesis.  Plant Cell Rep 19: 459-463

Cushman JC, Bohnert HJ (2000) Genomic approaches to plant stress tolerance. Curr Opin Plant Biol 3: 117-124

Taybi T, Patil S, Chollet R, Cushman JC  (2000) A minimal Ser/Thr protein kinase circadianly regulates phosphoenolpyruvate carboxylase activity in CAM-induced leaves of Mesembryanthemum crystallinum. Plant Physiol 123: 1471-1482

Patharkar OR, Cushman JC (2000) A stress-induced calcium-dependent protein kinase from Mesembryanthemum crystallinum phosphorylates a two-component response regulator.  Plant J 24: 679-692

Bohnert HJ, CushmanJC (2001) The ice plant cometh: lessons in abiotic stress tolerance.  J Plant Growth Regulators 19: 334-346

Cushman JC, Taybi T, Bohnert HJ (2000) Induction of Crassulacean Acid Metabolism - Molecular Aspects. In: Advances in Photosynthesis: Physiology and Metabolism." Eds. R.C. Leegood, T.D. Sharkey, and S. von Caemmerer.  Kluwer Academic Publishers. vol. 9, pp. 551-582.

Cushman JC (2001) Crassulacean Acid metabolism (CAM): A plastic photosynthetic adaptation to arid environments. Plant Physiol. 127: 1439-1448.

Gong Z, Koiwa H, Cushman MA, Ray A, Bufford D, Kore-eda S, Matsumoto T, Zhu J, Cushman J, Bressan R, Hasegawa M (2001) Genes that are uniquely stress regulated in salt overly sensitive (sos) mutants. Plant Physiol 126: 363-375

Gehrig HH, Winter K, Cushman JC, Borland AM, Taybi T (2001) An improved RNA isolation method for succulent plant species rich in polyphenols and polysaccharides. Plant Molec Biol Rep 18: 369-376

Bohnert HJ, Ayoubi P, Borchert C, Bressan RA, Burnap RL, Cushman JC, Cushman MA, Deyholos M, Galbraith DW, Hasegawa PM, Jenks M, Kawasaki S, Koiwa H, Kore-eda S, Lee B-H, Michalowski,CB, Misawa E, Nomura M, Ozturk M, Postier B, Prade R, Song C-P, Tanaka Y, Wang H, Zhu J-K (2001) A genomics approach towards salt stress tolerance. Plant Physiol. Biochem 39: 1-17

Cushman JC, Borland AM (2002) Induction of Crassulacean acid metabolism by water limitation. Plant Cell Environ. in press.

Bohnert HJ, CushmanJC (2002) Plant and environmental stress adaptation strategies. In “Plant Biotechnology and transgenic plants”, Oksman-Caldentey KM, and Barz W, eds. Marcel Dekkar, New York, NY. in press.

Cushman JC, Bohnert HJ (2002) Induction of Crassulacean acid metabolism by salinity molecular aspects. In: “Salinity: Environment - Plants - Molecules." Eds. A. Läuchli and U. Lüttge. Kluwer Academic. Publishers, Inc. in press

Cushman JC (2002) Osmoregulation in plants: implications for agriculture. Amer Zool in press.

Soulages JL, Kim K, Walters C, Cushman JC (2002) Temperature-induced extended helix/random coil transitions in a Group 1 Late Embryogenesis Abundant Protein from Soybean. Plant Physiol in press

Molecular Genetics of Crassulacean Acid Metabolism (CAM)    

C₄ and Crassulacean acid metabolism (CAM) plants have evolved as alternative photosynthetic carbon fixation pathways that employ a "CO₂ pump" to concentrate CO₂ within plant tissues to improve their competitiveness under environmental stress conditions such as high light intensity, high temperatures, or low water availability (Cushman and Bohnert, 1999). Unlike C₃ and C₄ plants, CAM plants assimilate the bulk of atmospheric CO₂ into C4 acids predominantly at night and subsequently refix this CO₂ into carbohydrates during the following day. In facultative CAM plants, such as the halophytic ice plant, Mesembryanthemum crystallinum, environmental stress can induce the CAM pathway (Bohnert and Cushman, 2000). Little is known about the signal transduction events that lead to CAM induction. We have recently determined that Ca²⁺ plays a central role in the signaling events following stress perception (Taybi and Cushman, 1999). Calcium-dependent protein kinases (CDPKs) are major sensors of Ca²⁺ signatures in higher plants. We have begun to unravel the functional roles of stress-induced CDPKs (McCPK1) from M. crystallinum by defining proteins with which they interact such as two-component pseudo-response regulators (Patharkar and Cushman, 2000). Understanding the components of Ca²⁺ signaling pathways that mediate environmental stress responses will be essential to enhance future engineering strategies for more salt and drought tolerant crops. CAM is also controlled by a circadian rhythm. We have recently cloned and characterized a CDPK-related protein kinase responsible for phosphorylating phosphoenolpyruvate carboxylase (PEPc) kinase, a key component of the CO₂ pump (Taybi et. al. 2000). In CAM plants, nocturnal phosphorylation renders PEPC considerably less sensitive to inhibition by negative effectors, but both more active and more sensitive to activation by positive effectors. Expression of PEPc kinase is controlled by a circadian oscillator that largely restricts its own mRNA and protein expression to the night.  Circadian control of PEPC kinase provides one of the key regulatory steps in controlling the competing actions of PEPC and ribulose 1,5-bisphosphate carboxylase/oxygenase.

Compared to C₃ and C₄ plants, no genetic model exists for CAM plants.  To overcome this deficiency, our laboratory has initiated a large-scale genetic screen to isolate M. crystallinum mutants defective in CAM or that are salinity or drought stress tolerance. Since M. crystallinum is a facultative CAM plant, this pathway should not be essential for normal growth and development of the plant.  However, CAM may be essential for long-term survival and reproductive success of the plant under the prolonged conditions of salinity or drought stress encountered in its native habitat. Thus far, more than 25,000 plants have been screened and many putative CAM deficient mutants have been isolated and are being studied. The goal of this research is to identify and characterize key structural and regulatory components of this important photosynthetic adaptation. Large-scale EST sequencing efforts and expression profiling using cDNA microarrays are providing a rich source of sequence information for identifying novel genes or gene family members and expression patterns peculiar to CAM plants such as circadianly regulated genes.

Functional Genomics of Plant Stress Tolerance

Abiotic stress accounts for more crop productivity losses than any other factor. Yet our ability to improve plant stress tolerance remains limited due to our lack of understanding of the inherent complexity of stress signaling and adaptation processes. To overcome this problem, the University of Arizona, Oklahoma State University, University of Nevada, and Purdue University have formed the plant stress genomics consortium to isolate and functionally characterize the core set of stress-related genes that provide the basis for ionic or dehydration stress tolerance in plants (Cushman and Bohnert, 2000). Comparative analysis of a series of halophytic and glycophytic higher plant (Arabidopsis, Mesembryanthemum, and Oryza), and non-plant models (Aspergillus, Dunaliella, Synechocystis, and Saccharomyces) have identified evolutionarily conserved and unique stress defense mechanisms. Research strategies involve the functional identification and analysis of genes important for stress signaling and tolerance using large-scale EST sequencing, random and targeted mutagenesis, complementation and microarray-based expression screening, and promoter-trapping strategies. In addition, integrated bioinformatics resources for the collection, analysis, and distribution of materials and data generated by the consortium has been established. More detailed information about these projects are available at the following two websites: http://stress-genomics.org/ and http://osmid.org/.

Engineering Environmental Stress Tolerance in Higher Plants

Bacteria, algae, and plants have evolved several universal adaptation systems to desiccation, osmotic, and low temperature stresses.  In many prokaryotic and eukaryotic organisms, water deficit, high osmolarity, and low temperature stress results in the accumulation of a group of glycine-rich, hydrophilic proteins known as hydrophilins or late embryogenesis abundant (LEA) proteins. Such proteins may preserve protein structure and membrane integrity by binding water, preventing protein denaturation or renaturing unfolded proteins, and sequestering ions in stressed tissues.  Recent experimental evidence shows that constitutive expression of these distinct classes of biomolecules in transgenic plants can improve growth performance under salinity, drought, and low temperature stress conditions.  However, the exact function of LEA proteins remains uncertain.  Currently, our laboratory is focused on understanding the structure and function of a Group 1 (D-19) LEA protein (Soulages et al., 2002) and a Group 2 (D-11 or "dehydrin") LEA protein.  We hypothesize that such proteins will be important components of multi-gene strategies to genetically engineer improved environmental stress tolerance.

Developing Paradigms for Functional Genomics of Protein Kinases and Phosphoproteins Using the CDPK Superfamily

Protein phosphorylation is a major mode of regulation of metabolism, gene expression, and cellular architecture in eukaryotic cells, and defining phosphorylation-based regulatory networks is essential for understanding the function of the Arabidopsis genome. The goal of this project is to define phosphorylation networks that are related to the function of calcium-dependent protein kinases (CDPKs) and four closely related families; CDPK-related kinases (CRKs), phosphoenolpyruvate carboxylase kinases (PPCKs), PPCK-related kinases (PEPRKs), and SNF-1 related kinases (SnRKs). These 88 kinases, which represent about 9% of all the protein kinases in the Arabidopsis genome, are involved in all aspects of plant development and physiology and participate in the coupling of cellular responses to environmental and developmental signals. A list of these protein kinases including gene identification numbers is available at http://www.arabidopsis.org, and further information including links to database records is available at http://plantsp.sdsc.edu. This research will investigate the function of 64 members of these families through determination of the subcellular location of each kinase and identification of downstream targets and other proteins with which the kinases associate. This information will give insight into the physiological roles of each kinase by identifying signaling networks in which each participates. This research will also determine the target sequences in substrate proteins that are phosphorylated by each kinase, and these results will contribute to understanding the overlap in kinase function and cross-talk between signaling pathways. The results of this work will be made available on a yearly basis at the two URLs given above. This research is a collaborative effort among investigators at the University of Wisconsin, University of Florida, University of Nevada, Reno, and the Scripps Research Institute.

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