Generation of gene-activating pH signals in plant cells

细胞内pH的局部峰值是植物几种胁迫信号转导和细胞分化过程中必不可少的中间产物。尽管它们具有核心功能，但pH峰产生及其基因激活效应背后的机制在很大程度上尚不清楚。在模式植物加州棘球藻中，我们的实验室发现，激发子挑战的细胞产生细胞质pH变化，进而诱导生物碱的生物合成。信号转导始于质膜，激发子触发溶血磷脂酰胆碱(LPC)的产生，激活液泡质子钠离子依赖的瞬时外流。该项目旨在为不同的液泡膜Na+/H+逆向转运体(NHx)参与细胞质pH漂移的产生提供分子证据，并表征它们在二次生物合成表达中的信号作用。缺乏NHx蛋白的细胞系将通过RNAi介导的沉默所有或部分NHx家族成员在埃希尔齐斯获得。NHx缺乏的影响将在细胞质和液泡pH的动态平衡、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。ΔpH信号下游上调和/或磷酸化的基因控制蛋白将通过对特定EST文库的高通量测序、磷酸蛋白质组分析和DNA结合分析来鉴定。

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.