Extending the Anion Channelrhodopsin-Based Toolbox for Plant Optogenetics

Abstract

Optogenetics was developed in the field of neuroscience and is most commonly using light-sensitive rhodopsins to control the neural activities. Lately, we have expanded this technique into plant science by co-expression of a chloroplast-targeted β-carotene dioxygenase and an improved anion channelrhodopsin <i>Gt</i>ACR1 from the green alga <i>Guillardia theta</i>. The growth of <i>Nicotiana tabacum</i> pollen tube can then be manipulated by localized green light illumination. To extend the application of analogous optogenetic tools in the pollen tube system, we engineered another two ACRs, <i>Gt</i>ACR2, and ZipACR, which have different action spectra, light sensitivity and kinetic features, and characterized them in <i>Xenopus laevis</i> oocytes, <i>Nicotiana benthamiana</i> leaves and <i>N. tabacum</i> pollen tubes. We found that the similar molecular engineering method used to improve <i>Gt</i>ACR1 also enhanced <i>Gt</i>ACR2 and ZipACR performance in <i>Xenopus laevis</i> oocytes. The ZipACR1 performed in <i>N. benthamiana</i> mesophyll cells and <i>N. tabacum</i> pollen tubes with faster kinetics and reduced light sensitivity, allowing for optogenetic control of anion fluxes with better temporal resolution. The reduced light sensitivity would potentially facilitate future application in plants, grown under low ambient white light, combined with an optogenetic manipulation triggered by stronger green light.