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In plants, environmental stressors trigger plasma membrane depolarizations. Being electrically interconnected via plasmodesmata, proper functional dissection of electrical signaling by electrophysiology is basically impossible. The green alga Chlamydomonas reinhardtii evolved blue light-excited channelrhodopsins (ChR1, 2) to navigate. When expressed in excitable nerve and muscle cells, ChRs can be used to control the membrane potential via illumination. In Arabidopsis plants, we used the algal ChR2-light switches as tools to stimulate plasmodesmata-interconnected photosynthetic cell networks by blue light and monitor the subsequent plasma membrane electrical responses. Blue-dependent stimulations of ChR2 expressing mesophyll cells, resting around −160 to −180 mV, reproducibly depolarized the membrane potential by 95 mV on average. Following excitation, mesophyll cells recovered their prestimulus potential not without transiently passing a hyperpolarization state. By combining optogenetics with voltage-sensing microelectrodes, we demonstrate that plant plasma membrane AHA-type H+-ATPase governs the gross repolarization process. AHA2 protein biochemistry and functional expression analysis in Xenopus oocytes indicates that the capacity of this H+ pump to recharge the membrane potential is rooted in its voltage- and pH-dependent functional anatomy. Thus, ChR2 optogenetics appears well suited to noninvasively expose plant cells to signal specific depolarization signatures. From the responses we learn about the molecular processes, plants employ to channel stress-associated membrane excitations into physiological responses.

Summary During drought, abscisic acid (ABA) induces closure of stomata via a signaling pathway that involves the calcium (Ca2+)‐independent protein kinase OST1, as well as Ca2+‐dependent protein kinases. However, the interconnection between OST1 and Ca2+ signaling in ABA‐induced stomatal closure has not been fully resolved. ABA‐induced Ca2+ signals were monitored in intact Arabidopsis leaves, which express the ratiometric Ca2+ reporter R‐GECO1‐mTurquoise and the Ca2+‐dependent activation of S‐type anion channels was recorded with intracellular double‐barreled microelectrodes. ABA triggered Ca2+ signals that occurred during the initiation period, as well as in the acceleration phase of stomatal closure. However, a subset of stomata closed in the absence of Ca2+ signals. On average, stomata closed faster if Ca2+ signals were elicited during the ABA response. Loss of OST1 prevented ABA‐induced stomatal closure and repressed Ca2+ signals, whereas elevation of the cytosolic Ca2+ concentration caused a rapid activation of SLAC1 and SLAH3 anion channels. Our data show that the majority of Ca2+ signals are evoked during the acceleration phase of stomatal closure, which is initiated by OST1. These Ca2+ signals are likely to activate Ca2+‐dependent protein kinases, which enhance the activity of S‐type anion channels and boost stomatal closure.

In plants, antimicrobial immune responses involve the cellular release of anions and are responsible for the closure of stomatal pores. Detection of microbe-associated molecular patterns (MAMPs) by pattern recognition receptors (PRRs) induces currents mediated via slow-type (S-type) anion channels by a yet not understood mechanism. Here, we show that stomatal closure to fungal chitin is conferred by the major PRRs for chitin recognition, LYK5 and CERK1, the receptor-like cytoplasmic kinase PBL27, and the SLAH3 anion channel. PBL27 has the capacity to phosphorylate SLAH3, of which S127 and S189 are required to activate SLAH3. Full activation of the channel entails CERK1, depending on PBL27. Importantly, both S127 and S189 residues of SLAH3 are required for chitin-induced stomatal closure and anti-fungal immunity at the whole leaf level. Our results demonstrate a short signal transduction module from MAMP recognition to anion channel activation, and independent of ABA-induced SLAH3 activation.

Since a waxy cuticle covers outer leaf tissues, water vapor diffusion into the atmosphere 71 occurs mainly through the stomatal pores at the leaf surface. The size of the stomatal aperture is 72 tightly regulated to optimize gas exchanges between the leaf inner tissues and the atmosphere, 73 including CO 2 intake for photosynthesis and water loss by transpiration (Lawson and Blatt, 74 2014). This is achieved by fine tuning of the turgor pressure of the two guard cells which 75 surround the stomatal pore, and involves a complex coordinated activity of transport systems at 76 the guard cell plasma membrane and vacuolar membrane (Hedrich, 2012; Chen et al., 2012; Hills 77 et al., 2012;Kollist et al., 2014 4 nutrients from the roots, which take up these nutrients, to the aerial parts, to support plant growth 79 (Marschner et al., 1996). 80Potassium ion (K + ), as a major inorganic constituent of the plant cells and the most abundant 81 cation in the cytosol, is an essential macronutrient for growth and development. It is involved in 82 various functions including electrical neutralization of negative charges, control of cell 83 membrane polarization and osmoregulation (Clarkson and Hanson, 1980; Leigh and Wyn Jones, 84 1984). K + is thus the main cation absorbed by the roots and circulating within the plant at the 85 cellular or long distance levels. In guard cells, it is well known as a major contributor, with Cl -, 86 NO 3 -and malate, to the osmolarity (Raschke and Schnabl, 1978; Willner and Fricker, 1996). 87Stomatal opening is initiated by activation of plasma membrane proton pumps in guard cells, 88 which promotes K + influx through voltage-gated inward K + channels, as well as anion uptake 89 through H + -anion symporters (Blatt, 1987a; Schroeder et al., 1987;Roelfsema and Prins, 1997; 90 Talbott and Zeiger, 1998; Guo et al., 2003; Jezek and Blatt, 2017). Conversely, stomatal closure 91 requires inhibition of proton pumping at the guard cell membrane and activation of both anion 92 channels and voltage-gated outward K + channels. 93The molecular mechanisms responsible for inward and outward K + fluxes across the plasma 94 membrane have been extensively investigated in Arabidopsis. Shaker channel subunits, present as 95 a 9-member family in Arabidopsis, have been shown to form the major pathways for these fluxes 96 throughout the plant (Véry and Sentenac, 2003). In the Arabidopsis model species, four genes 97 encoding Shaker channel subunits have been identified as playing a major role in root to shoot K + 98 translocation and in stomatal movements. The SKOR subunit, which is expressed in root 99 pericycle and xylem parenchyma, forms outwardly-rectifying channels involved in K + secretion 100 into the xylem sap (Gaymard et al., 1998). In stomata, the inward Shaker channel subunits KAT1 101 and KAT2 are involved in guard cell K + uptake, and the outward Shaker channel GORK 102 mediates guard cell K + release (Ache et al., 2000;Pilot et al., 2001;Szyroki et al., 2001; Hosy et 103 al., 2003;Lebaudy...