The Vacuolar Proton-Cation Exchanger EcNHX1 Generates pH Signals for the Expression of Secondary Metabolism in Eschscholzia californica

Abstract

Cell cultures of Eschscholzia californica react to a fungal elicitor by the overproduction of antimicrobial benzophenanthridine alkaloids. The signal cascade toward the expression of biosynthetic enzymes includes (1) the activation of phospholipase A2 at the plasma membrane, resulting in a peak of lysophosphatidylcholine, and (2) a subsequent, transient efflux of vacuolar protons, resulting in a peak of cytosolic H + . This study demonstrates that one of the Na + /H + antiporters acting at the tonoplast of E. californica cells mediates this proton flux. Four antiporter-encoding genes were isolated and cloned from complementary DNA (EcNHX1-EcNHX4). RNA interference-based, simultaneous silencing of EcNHX1, EcNHX3, and EcNHX4 resulted in stable cell lines with largely diminished capacities of (1) sodium-dependent efflux of vacuolar protons and (2) elicitor-triggered overproduction of alkaloids. Each of the four EcNHX genes of E. californica reconstituted the lack of Na + -dependent H + efflux in a Dnhx null mutant of Saccharomyces cerevisiae. Only the yeast strain transformed with and expressing the EcNHX1 gene displayed Na + -dependent proton fluxes that were stimulated by lysophosphatidylcholine, thus giving rise to a net efflux of vacuolar H + . This finding was supported by three-dimensional protein homology models that predict a plausible recognition site for lysophosphatidylcholine only in EcNHX1. We conclude that the EcNHX1 antiporter functions in the elicitor-initiated expression of alkaloid biosynthetic genes by recruiting the vacuolar proton pool for the signaling process.

Beyond the patch‐clamp resolution: functional activity of nonelectrogenic vacuolar NHX proton/potassium antiporters and inhibition by phosphoinositides

“…It would be interesting to test whether PI(4,5)P 2 specifically regulates the plasma membrane protein NHX7/SOS1. Of note is that phosphatidic acid produced by phospholipase PLDα in response to salt stress activates MPK6, which in turn phosphorylates and likely activates NHX7/SOS1 (Yu et al ., 2010), whereas ScNHX1 of Saccharomyces cerevisiae and EcNHX1 from the plant Eschscholzia californica (California golden poppy) are stimulated by lysophosphatidylcholine, another signalling lipid that is produced by plasma membrane‐localized phospholipase A2 (Weigl et al ., 2016). These findings suggest that regulation of NHX exchangers by bioactive lipids, either directly or indirectly, is a common and physiologically relevant feature.…”