A novel calcium binding site in the slow vacuolar cation channel TPC1 senses luminal calcium levels.

Cytosolic calcium homeostasis is pivotal for intracellular signaling and requires sensing of calcium concentrations in the cytosol and accessible stores. Numerous Ca²âº binding sites have been characterized in cytosolic proteins. However, little is known about Ca²âº binding inside organelles, like the vacuole. The slow vacuolar (SV) channel, encoded by Arabidopsis thaliana TPC1, is regulated by luminal Ca²âº. However, the D454/fou2 mutation in TPC1 eliminates vacuolar calcium sensitivity and increases store calcium content. In a search for the luminal calcium binding site, structure modeling indicated a possible coordination site formed by residues Glu-450, Asp-454, Glu-456, and Glu-457 on the luminal side of TPC1. Each Glu residue was replaced by Gln, the modified genes were transiently expressed in loss-of-TPC1-function protoplasts, and SV channel responses to luminal calcium were recorded by patch clamp. SV channels lacking any of the four negatively charged residues appeared altered in calcium sensitivity of channel gating. Our results indicate that Glu-450 and Asp-454 are directly involved in Ca²âº binding, whereas Glu-456 and Glu-457 are probably involved in connecting the luminal Ca²âº binding site to the channel gate. This novel vacuolar calcium binding site represents a potential tool to address calcium storage in plants.

Stomatal guard cells co-opted an ancient ABA-dependent desiccation survival system to regulate stomatal closure.

During the transition from water to land, plants had to cope with the loss of water through transpiration, the inevitable result of photosynthetic CO2 fixation on land [1, 2]. Control of transpiration became possible through the development of a new cell type: guard cells, which form stomata. In vascular plants, stomatal regulation is mediated by the stress hormone ABA, which triggers the opening of the SnR kinase OST1-activated anion channel SLAC1 [3, 4]. To understand the evolution of this regulatory circuit, we cloned both ABA-signaling elements, SLAC1 and OST1, from a charophyte alga, a liverwort, and a moss, and functionally analyzed the channel-kinase interactions. We were able to show that the emergence of stomata in the last common ancestor of mosses and vascular plants coincided with the origin of SLAC1-type channels capable of using the ancient ABA drought signaling kinase OST1 for regulation of stomatal closure.