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

这是一个power项目，允许PI休年假到威斯康星大学麦迪逊分校的Michael Sussman博士的实验室工作。这符合POWRE奖的标准，因为：1)这项研究涉及的植物科学领域与pi研究的气孔保护细胞完全不同；2)这项研究将为植物基因组学和蛋白质组学的新领域提供重要的接触机会，包括对许多技术的“动手”经验，这些技术随后将应用于PI自己的研究领域；同样在POWRE奖的赞助下，PI将与Edgar Spalding教授（同样在威斯康星大学麦迪逊分校）合作，改进本科生的植物电生理学实验室练习，目标是将该实验室纳入她自己的教学中。苏斯曼的实验室正在鉴定拟南芥基因组中编码钙调素结构域激酶（CDPKs）的35 -40个基因中的T-DNA无突变体。Sussman博士实验室的初步数据表明，cdpk10和11可能与植物对盐水条件的反应有关，当植物生长在没有K+的高NaCl环境中时，cdpk10/11突变体植物表现出更高的存活率。盐碱化是一个重要的农业问题，影响着全球7%的可耕地。实验将验证两个关于CDPKs 10和11与植物对盐的反应的假设：假设1已知植物K+缺乏是盐胁迫的一个主要方面。因此，假设野生型CDPKs 10和11（和/或其他CDPKs）通过下调K+摄取转运体来降低盐耐受性；在CDPK 10/11突变体中，这种下调缺失，因此耐盐性提高。这一假设将通过以下方法得到验证：1)测定野生型与cdpk 10/11突变型植物在限制与充足K+条件下的生长、K+吸收和Na+吸收；2)在cdpk 10/11和K+摄取通道akt1和kat1之间产生的三重突变体中测量相同的因子；3)确定cdpk 10/11突变体与野生型cdpk 10/11基因的互补恢复了野生型表型。假设2 Ca2+的存在可以降低NaCl对植物生长和K+状态的有害影响。最近的研究表明，SOS3蛋白与Ca2+活化磷酸酶钙调磷酸酶的调控亚基同源，并且是Ca2+作用所必需的（Liu和Zhu， 1998, Science 280: 1943）。sos3突变体植株缺乏K+吸收。因此，我们假设SOS3/calcineurin和CDPK10/11靶向相同的K+转运蛋白（直接或通过信号级联），野生型SOS3/calcineurin在盐水条件下上调K+转运蛋白，而野生型CDPK10/11下调K+转运蛋白。为了验证这一假设：1)在不同Ca2+水平的盐水条件下，将评估cdpk 10/11突变体与野生型植物的生长、K+和Na+吸收；2)磷酸化实验将用于鉴定被野生型CDPKs 10/11增强而被野生型SOS3降低磷酸化状态的根蛋白（作为2d凝胶上的斑点）。这些蛋白质将使用串联质谱技术进行测序和鉴定

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