Generation of gene-activating pH signals in plant cells

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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.