Dynamic Contrast for Plant Phenotyping

Zsolt Kelemen,Vincent Croquette,Ruikang Zhang,Yannick Bellec,Lionel Gissot,Thomas Le Saux,Ludovic Jullien,Jean-Denis Faure,Raja Chouket

doi:10.1021/acsomega.0c00957

Abstract

Noninvasiveness, minimal handling, and immediate response are favorable features of fluorescence readout for high-throughput phenotyping of labeled plants.Yet, remote fluorescence imaging may suffer from an autofluorescent background and artificial or natural ambient light. In this work, the latter limitations are overcome by adopting reversibly photoswitchable fluorescent proteins (RSFPs) as labels and Speed OPIOM (out-of-phase imaging after optical modulation), a fluorescence imaging protocol exploiting dynamic contrast. Speed OPIOM can efficiently distinguish the RSFP signal from autofluorescence and other spectrally interfering fluorescent reporters like GFP. It can quantitatively assess gene expressions, even when they are weak. It is as quantitative, sensitive, and robust in dark and bright light conditions. Eventually, it can be used to nondestructively record abiotic stress responses like water or iron limitations in real time at the level of individual plants and even of specific organs. Such Speed OPIOM validation could find numerous applications to identify plant lines in selection programs, design plants as environmental sensors, or ecologically monitor transgenic plants in the environment.

Highlights

The rise of the human population combined with the requirement for a sustainable agriculture requires developing modern techniques for plant breeding, cultivation, and management of crops
Speed OPIOM has exploited reversibly photoswitchable fluorescent proteins (RSFPs), which can reversibly photoswitch between two states of distinct brightnesses by using two different wavelengths
The Speed OPIOM image of a targeted RSFP is obtained after matching the illumination parameters to its cross sections for fluorescence photoswitching at wavelengths λ1 and λ2 using the resonance conditions

Summary

Introduction

The rise of the human population combined with the requirement for a sustainable agriculture requires developing modern techniques for plant breeding, cultivation, and management of crops. High-throughput phenotyping tools providing quantitative analyses of plant structure and function are actively sought for developing highly productive strains or monitoring plants to determine their physiological state.[1−3] A fluorescence readout is advantageous: it is not invasive, requires minimal handling, and provides an immediate response. FP-mediated imaging can suffer from a significant autofluorescent background within a broad range of wavelengths (from UV to IR). The possibility to selectively record fluorescence emission with minimal contribution from an autofluorescence background and artificial or natural ambient light would definitively open new avenues for plant phenotyping

Methods

Results

Conclusion