Abstract

Phytochrome photoreceptors regulate vital adaptations of plant development, growth, and physiology depending on the ratio of red and far-red light. The light-triggered Z/E isomerization of a covalently bound bilin chromophore underlies phytochrome photoconversion between the red-absorbing Pr and far-red-absorbing Pfr states. Compared to bacterial phytochromes, the molecular mechanisms of signal propagation to the C-terminal module and its regulation are little understood in plant phytochromes, not least owing to a dearth of structural information. To address this deficit, we studied the Arabidopsis thaliana phytochrome A (AtphyA) at full length by cryo-electron microscopy (cryo-EM). Following heterologous expression in Escherichia coli, we optimized the solvent conditions to overcome protein aggregation and thus obtained photochemically active, near-homogenous AtphyA. We prepared grids for cryo-EM analysis of AtphyA in its Pr state and conducted single-particle analysis. The resulting two-dimensional class averages and the three-dimensional electron density map at 17 Å showed a homodimeric head-to-head assembly of AtphyA. Docking of domain structures into the electron density revealed a separation of the AtphyA homodimer at the junction of its photosensor and effector modules, as reflected in a large void in the middle of map. The overall architecture of AtphyA resembled that of bacterial phytochromes, thus hinting at commonalities in signal transduction and mechanism between these receptors. Our work paves the way toward future studies of the structure, light response, and interactions of full-length phytochromes by cryo-EM.

Highlights

  • Especially structural investigation, we set out to establish an efficient protocol for the heterologous expression in E. coli of Arabidopsis thaliana phytochrome A (AtphyA) at full length

  • To improve chromophore supply during expression, 0.5 mM δ-aminolevulinic acid was added as a precursor in the biosynthesis of porphyrin (Shemin and Russell, 1953), from which in turn biliverdin and other bilins derive

  • The N-terminal extension (NTE) has been implicated in light-dependent conformational changes and interactions with the phytochrome-interacting factors, the molecular underpinnings of these processes are unclear

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Summary

Introduction

Identified first among the plant sensory photoreceptors (Butler et al, 1959), phytochromes (phy) serve as ratiometric sensors of red and far-red (i.e., near-infrared) light and coordinate a wide range of vital adaptations of physiology, for instance shade avoidance, morphogenesis, development, and the timing of germination and flowering (Casal, 2013; Hughes, 2013; Analysis of A. thaliana phyA by Cryo-Electron MicroscopyPham et al, 2018; Legris et al, 2019; Rockwell and Lagarias, 2020). Land plants express a varying number of phys, with the model organism Arabidopsis thaliana possessing five, denoted AtphyA through AtphyE. Owing to their abundance and predominant role in photomorphogenesis, AtphyA and AtphyB have been studied more extensively than AtphyC-AtphyE (Legris et al, 2019). Phys of land plants generally adopt their red-absorbing Pr state which is characterized by the bilin chromophore in the Z configuration of its C15〓C16 double bond (see Figure 1b of Golonka et al, 2019; Legris et al, 2019). The Pfr state reverts to the Pr state thermally or upon illumination with far-red light

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