Extending the Anion Channelrhodopsin-Based Toolbox for Plant Optogenetics.

Yang Zhou,Shiqiang Gao,Meiqi Ding,Georg Nagel,Kai R Konrad,Xiaodong Duan

doi:10.3390/membranes11040287

Yang Zhou, Shiqiang Gao + Show 4 more

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https://doi.org/10.3390/membranes11040287

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Journal: Membranes	Publication Date: Apr 14, 2021
Citations: 10	License type: CC BY 4.0

Affiliation: University of Würzburg, Leibniz Association

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 GtACR1 from the green alga Guillardia theta. The growth of Nicotiana tabacum 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, GtACR2, and ZipACR, which have different action spectra, light sensitivity and kinetic features, and characterized them in Xenopus laevis oocytes, Nicotiana benthamiana leaves and N. tabacum pollen tubes. We found that the similar molecular engineering method used to improve GtACR1 also enhanced GtACR2 and ZipACR performance in Xenopus laevis oocytes. The ZipACR1 performed in N. benthamiana mesophyll cells and N. tabacum 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.

Highlights

Optogenetics employs rhodopsins or other photoreceptors to manipulate defined events in biological systems with high spatial and temporal resolution [1,2]
Anion channelrhodopsins GtACR1, GtACR2, and ZipACR were synthesized by Invitrogen GeneArt Gene Synthesis according to the published sequence
In comparison to ACR2 and ZipACR, both engineered ACR2 2.0 and ZipACR 2.0 exhibited higher expression level in the plasma membrane of oocytes and resulted in improved photocurrents (Figure 1c,d), which is similar to ACR1 2.0 (Figure 1b–d)

Summary

Introduction

Optogenetics employs rhodopsins or other photoreceptors to manipulate defined events in biological systems with high spatial and temporal resolution [1,2]. The most commonly used optogenetic tools are the microbial rhodopsins with light-gated ion channel or pump functions [3]. The discovery of Channelrhodopsin-2 (ChR2), a light-sensitive cation channel from the unicellular green alga Chlamydomonas reinhardtii, paved the way for optogenetic applications [4]. The light-sensitive anion channelrhodopsin GtACR1 from the green algae Guillardia theta was discovered later [10] and enabled electric silencing in certain neurons via hyperpolarizing the plasma membrane [11,12,13,14]. With the unique advantages of fine spatial and temporal resolution, microbial opsin-based optogenetic tools advanced neuroscience research greatly [1,15]

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