Generation of gene-activating pH signals in plant cells

细胞内pH的局部峰值是植物细胞分化过程中几种胁迫信号转导的重要中间体。尽管它们具有重要的功能，但pH峰值产生及其基因激活效应背后的机制在很大程度上是未知的。在模式植物scholzia californica中，我们的实验室揭示了激发子激发的细胞产生细胞质pH值变化，进而诱导生物碱生物合成。信号转导起始于质膜，激发子触发溶血磷脂酰胆碱（LPC）的产生，LPC激活液泡质子的瞬时Na+依赖性流出。该项目旨在为不同的液泡膜Na+/H+反向转运蛋白（NHX）参与细胞质pH变化的产生提供分子证据，并表征其在次生生物合成表达中的信号转导作用。NHX蛋白缺陷的细胞系将通过RNAi介导的使Scholzia中的NHX家族的全部或一些成员沉默来产生。NHX缺乏的影响将在细胞质和液泡pH的稳态，激发子触发的pH峰的产生和基因激活信号的转导方面进行测试。将通过特定EST文库的高通量测序、磷酸化蛋白质组分析和DNA结合测定来鉴定Δ pH信号下游上调和/或磷酸化的基因控制蛋白。

Localized peaks of intracellular pH are essential intermediates in the transduction of several stress signals and in processes of cell differentiation in plants. Despite their central functions, the mechanisms behind the generation of pH peaks and their gene-activating effects are largely unknown. In the model plant Eschscholzia californica, our lab has revealed that elicitor-challenged cells generate cytoplasmic pH shifts that in turn induce alkaloid biosynthesis. Signaling starts at the plasma membrane with the elicitor-triggered production of lysophosphatidylcholine (LPC) which activates a transient, Na+-dependent efflux of vacuolar protons.The project aims to provide molecular evidence for the involvement of distinct tonoplast Na+/H+- antiporters (NHX) in the generation of cytoplasmic pH shifts, and to characterize their signaling effects at the expression of secondary biosynthesis. Cell lines deficient in NHX proteins will be generated by RNAi mediated silencing of all or some members of the NHX family in Eschscholzia. Effects of NHX deficiency will be tested with respect to the homeostasis of cytoplasmic and vacuolar pH, the generation of elicitor-triggered pH peaks and the transduction of gene-activating signals. Gene control proteins that are upregulated and/or phosphorylated downstream of the ΔpH-signal will be identified via high-throughput sequencing of specific EST libraries, phosphoproteome analysis and DNA-binding assays.