Channelrhodopsin-mediated optogenetics highlights a central role of depolarization-dependent plant proton pumps.

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.

Insect haptoelectrical stimulation of Venus flytrap triggers exocytosis in gland cells.

The Venus flytrap Dionaea muscipula captures insects and consumes their flesh. Prey contacting touch-sensitive hairs trigger traveling electrical waves. These action potentials (APs) cause rapid closure of the trap and activate secretory functions of glands, which cover its inner surface. Such prey-induced haptoelectric stimulation activates the touch hormone jasmonate (JA) signaling pathway, which initiates secretion of an acidic hydrolase mixture to decompose the victim and acquire the animal nutrients. Although postulated since Darwin's pioneering studies, these secretory events have not been recorded so far. Using advanced analytical and imaging techniques, such as vibrating ion-selective electrodes, carbon fiber amperometry, and magnetic resonance imaging, we monitored stimulus-coupled glandular secretion into the flytrap. Trigger-hair bending or direct application of JA caused a quantal release of oxidizable material from gland cells monitored as distinct amperometric spikes. Spikes reminiscent of exocytotic events in secretory animal cells progressively increased in frequency, reaching steady state 1 d after stimulation. Our data indicate that trigger-hair mechanical stimulation evokes APs. Gland cells translate APs into touch-inducible JA signaling that promotes the formation of secretory vesicles. Early vesicles loaded with H+ and Cl- fuse with the plasma membrane, hyperacidifying the "green stomach"-like digestive organ, whereas subsequent ones carry hydrolases and nutrient transporters, together with a glutathione redox moiety, which is likely to act as the major detected compound in amperometry. Hence, when glands perceive the haptoelectrical stimulation, secretory vesicles are tailored to be released in a sequence that optimizes digestion of the captured animal.