POWRE: The Role of CDPKs in Plant Salt Tolerance: a T-DNA Mutagenesis Approach

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This is a POWRE project to allow the PI to take a sabbatical leave to work in thelaboratory of Dr. Michael Sussman at the University of Wisconsin, Madison. This fits the criteria of a POWRE award since: 1) this research concerns an area of plant science completely different from the PIs research focus on stomatal guard cells; 2) this research will provide a critical exposure to the new fields of plant genomics and proteinomics, including "hands-on" experience with a number of techniques which will subsequently be applied to the PI's own research area; 3) also under the auspices of the POWRE award, the PI will work with Prof. Edgar Spalding (also at the University of Wisconsin, Madison) on the improvement of an undergraduate laboratory exercise in plant electrophysiology, with the goal of incorporating this laboratory into her own teaching.Dr. Sussman's laboratory is in the process of identifying T-DNA null mutants in all of the 35 -40 genes in the Arabidopsis thaliana genome that encodes calmodulin-domain kinases (CDPKs). Preliminary data from Dr. Sussman's laboratory suggest that CDPKs 10 and 11 may be involved in plant responses to saline conditions, with cdpk10/11 mutant plants showing improved survival when plants are grown on elevated NaCl in the absence of K+. Salinity is an important agronomic problem, affecting 7% of arable land world-wide. The experiments will test two hypotheses regarding CDPKs 10 and 11 and plant response to salinity: Hypothesis 1 It is known that plant K+ deficiency is a major aspect of salt stress. It is therefore hypothesized that wild type CDPKs 10 and 11 (and/or other CDPKs) reduce salt tolerance by down-regulating a K+ uptake transporter; in the cdpk 10/11 mutants such down-regulation is missing, and so salt tolerance is improved. This hypothesis will be tested by: 1) measuring growth, K+ uptake, and Na+ uptake in wild type vs. cdpk 10/11 mutant plants grown under limiting vs. sufficient K+ conditions; 2) measuring the same factors in triple mutants that I will produce between cdpk 10/11 andthe K+ uptake channels akt1 and kat1; and 3) determining that complementation of the cdpk 10/11 mutant with the wild type CDPK 10/11 genes restores the wild type phenotype. Hypothesis 2 The presence of Ca2+ can reduce the deleterious effects of NaCl on plant growth and K+ status. It was recently shown that the SOS3 protein has homology to the regulatory subunit of the Ca2+-activated phosphatase, calcineurin, and is necessary for this Ca2+ effect (Liu and Zhu, 1998, Science 280: 1943). sos3 mutant plants are deficient in K+ uptake. It is therefore hypothesized that SOS3/calcineurin and CDPK 10/11 target the same K+ transporter (either directly or through a signaling cascade), with wild type SOS3/calcineurin upregulating that K+ transporter under saline conditions and wild type CDPK10/11 downregulating that K+ transporter. To test this hypothesis: 1) growth, K+ and Na+ uptake will be evaluated in cdpk 10/11 mutants vs wild type plants under saline conditions with different levels of Ca2+; 2) phosphorylation assays will be conducted to identify (as spots on 2-D gels) root proteins whose phosphorylation status is enhanced by wild type CDPKs 10/11 and reduced by wild type SOS3. Such proteins will be sequenced and identified using the technique of tandem mass spectrometry