Cyclic mononucleotides modulate potassium and calcium flux responses to H2O2 in Arabidopsis roots.

Cyclic mononucleotides are messengers in plant stress responses. Here we show that hydrogen peroxide (H2O2) induces rapid net K(+)-efflux and Ca(2+)-influx in Arabidopsis roots. Pre-treatment with either 10µM cAMP or cGMP for 1 or 24h does significantly reduce net K(+)-leakage and Ca(2+)-influx, and in the case of the K(+)-fluxes, the cell permeant cyclic mononucleotides are more effective. We also examined the effect of 10µM of the cell permeant 8-Br-cGMP on the Arabidopsis microsomal proteome and noted a specific increase in proteins with a role in stress responses and ion transport, suggesting that cGMP is sufficient to directly and/or indirectly induce complex adaptive changes to cellular stresses induced by H2O2.

Noninvasive microelectrode ion flux estimation technique (MIFE) for the study of the regulation of root membrane transport by cyclic nucleotides.

Changes in ion permeability and subsequently intracellular ion concentrations play a crucial role in intracellular and intercellular communication and, as such, confer a broad array of developmental and adaptive responses in plants. These changes are mediated by the activity of plasma-membrane based transport proteins many of which are controlled by cyclic nucleotides and/or other signaling molecules. The MIFE technique for noninvasive microelectrode ion flux measuring allows concurrent quantification of net fluxes of several ions with high spatial (µm range) and temporal (ca. 5 s) resolution, making it a powerful tool to study various aspects of downstream signaling events in plant cells. This chapter details basic protocols enabling the application of the MIFE technique to study regulation of root membrane transport in general and cyclic nucleotide mediated transport in particular.