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

这是一个POWRE项目，允许PI休假，在麦迪逊大学威斯康星州的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完全不同于PI的研究重点气孔保卫细胞; 2）这项研究将提供对植物基因组学和蛋白质组学新领域的关键接触，包括“动手”经验，这些技术随后将应用于PI自己的研究领域; 3）同样在POWRE奖的赞助下，PI将与埃德加斯伯丁教授合作（也在麦迪逊的威斯康星州大学）关于改进植物电生理学的本科实验室练习，Sussman博士的实验室正在鉴定所有35个基因中的T-DNA无效突变体，拟南芥基因组中编码钙调蛋白结构域激酶（CDPKs）的40个基因。来自Sussman博士实验室的初步数据表明，CDPKs 10和11可能参与植物对盐条件的反应，当植物在缺乏K+的情况下生长在高浓度NaCl上时，cdpk 10/11突变体植物表现出更好的存活率。 盐分是一个重要的农业问题，影响着全世界7%的耕地。 实验将测试两个假设CDPKs 10和11和植物对盐的反应：假设1众所周知，植物K+缺乏是盐胁迫的一个主要方面。因此，假设野生型CDPK 10和11（和/或其他CDPK）通过下调K+摄取转运蛋白降低盐耐受性;在cdpk 10/11突变体中，这种下调缺失，因此盐耐受性得到改善。该假设将通过以下方式进行检验：1）测量在限制与充足K+条件下生长的野生型与cdpk 10/11突变体植物中的生长、K+吸收和Na+吸收; 2）测量在cdpk 10/11与K+吸收通道akt 1和kat 1之间我将产生的三重突变体中的相同因子;和3）确定cdpk 10/11突变体与野生型CDPK 10/11基因的互补恢复野生型表型。 假设2 Ca ~（2+）的存在可以减轻NaCl对植物生长和K ~+状况的不利影响。最近显示，SOS 3蛋白与Ca 2+激活的磷酸酶的调节亚基钙调神经磷酸酶具有同源性，并且是这种Ca 2+效应所必需的（Liu和Zhu，1998，Science 280：1943）。sos 3突变体植物K+吸收不足。因此，假设SOS 3/钙调磷酸酶和CDPK 10/11靶向相同的K+转运蛋白（直接或通过信号级联），野生型SOS 3/钙调磷酸酶在盐水条件下上调该K+转运蛋白，野生型CDPK 10/11下调该K+转运蛋白。为了检验这一假设：1）在具有不同Ca 2+水平的盐条件下，在cdpk 10/11突变体与野生型植物中评价生长、K+和Na+吸收; 2）进行磷酸化测定以鉴定（作为2-D凝胶上的斑点）其磷酸化状态被野生型CDPK 10/11增强并被野生型SOS 3降低的根蛋白。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