Reactive oxygen species function as signaling molecules in controlling plant development and hormonal responses

Abstract

Reactive oxygen species (ROS) serve as second messengers in plant signaling pathways to remodel plant growth and development. New insights into how enzymatic ROS-producing machinery is regulated by hormones or localized during development have provided a framework for understanding the mechanisms that control ROS accumulation patterns. Signaling-mediated increases in ROS can then modulate the activity of proteins through reversible oxidative modification of specific cysteine residues. Plants also control the synthesis of antioxidants, including plant-specialized metabolites, to further define when, where, and how much ROS accumulate. The availability of sophisticated imaging capabilities, combined with a growing tool kit of ROS detection technologies, particularly genetically encoded biosensors, sets the stage for improved understanding of ROS as signaling molecules.

Keywords: Abscisic acid; Arabidopsis; Auxin; Class III peroxidases; Ethylene; Guard cells; HyPer7; Hydrogen peroxide; Pollen; Reactive oxygen species; Respiratory burst oxidase homologs; Root hairs; Tomato; roGFP2-Orp1.

Figure 1. ROS are produced during development and in response to hormone perturbation.

(a) Confocal images of wild-type (WT) Arabidopsis roots treated with IAA (adapted from Ref. [33]) or the ethylene precursor, ACC (adapted from Ref. [31], and stained with the hydrogen peroxide selective probe, peroxy orange 1 (PO1), revealing elevated ROS in root hair forming cells. Scale bars = 200 μm (b) Confocal images of WT tomato guard cells treated with ABA and stained with the general ROS sensor, dichlorofluorescein (DCF) (in green), visualized across a 45 min time course, with chlorophyll autofluorescence in magenta. ROS increases are observed in multiple subcellular locations with significant increases in the number and intensity of cytosolic puncta. Scale bar = 5 μm. Adapted from Ref. [82]. Copyright 2017 American Society of Plant Biologists. (c) Confocal images of WT tomato pollen grains stained with PO1 and the general ROS sensor DCF revealing differences in localized ROS accumulation around the pore from which pollen tubes emerge. Scale bars = 10 μm. Adapted from Ref. [51]. (d) Confocal images of Arabidopsis Col-0 and tt7-2, a mutant with a defect in quercetin synthesis, stained with the superoxide selective stain, dihydroethidium (DHE), which is visualized as a heat map. The tt7 mutant has increased lateral root formation and elevated DHE signal over the lateral root primordia (indicated with caret symbol). Scale bar = 50 μm. Adapted from Ref. [85].

Figure 2. ROS-generating enzymes and their location within a cell.

In the apoplastic space, ROS are produced by RBOHs [19], Cu/Zn superoxide dismutases (SOD) in cotton [41,128], the newly characterized Mn-SOD, MSD2 in Arabidopsis, as well as class III peroxidase (PRX) [37]. The best established signaling circuit is RBOH produced superoxide, (using NADPH as an electron donor), which is then converted to H₂O₂ by SOD. H₂O₂ then enters cells via aquaporins [21]. PRX enzymes have complex biochemistry but have been reported to scavenge hydrogen peroxide when in the resting state, but also the resting state enzyme can be reduced by superoxide and generate hydroxyl radicals when in the presence of hydrogen peroxide. PRX also have the ability to indirectly produce superoxide when oxidized, which can also be converted to H₂O₂ via SOD [37]. All three isoforms of SOD are also present within the cell found in the cytosol, chloroplasts (a), mitochondria, (b) and peroxisomes (c) [41,129]. ROS has been reported to travel from metabolic organelles to the nucleus and calcium treatment of isolated tobacco nuclei can generate ROS although the mechanism for this ROS synthesis is unknown [130]. Created with Biorender.com.

Figure 3. roGFP2-Orp1 is a genetically encoded ratiometric bioreporter for H₂O_2.

(a) Schematic detailing the biochemical mechanism of roGFP2-Orp1 oxidation by H₂O₂ and resulting changes in fluorescence [10] Created with Biorender.com.(b) Representative micrographs of guard cells from individual excitation channels as well as ratiometric images following reduction with DTT or oxidation with H₂O₂ to illustrate the properties of the sensor. Fully reduced roGFP2 displays high signal intensity after excitation with the 488 nm laser line (magenta), while low-signal intensity excitation with the 405 nm laser lines yields low signal intensity (cyan). As roGFP2-Orp1 becomes fully oxidized, excitation at 405 nm results in increased emission intensity, while excitation with the 488 nm laser results in decreased emission intensity than when it is fully reduced. Ratiometric images were generated using the Redox Ratio Analysis software [131] to display fluorescence ratios calculated from the GFP fluorescence images excited at 488 nm and 405 nm.