Chloroplast avoidance movement: a novel paradigm of ROS signalling.

The damaging effects of supra-optimal irradiance on plants, often turning to be lethal, may be circumvented by chloroplast avoidance movement which realigns chloroplasts to the anticlinal surfaces of cells (parallel to the incident light), essentially minimizing photon absorption. In angiosperms and many other groups of plants, chloroplast avoidance movement has been identified to be a strong blue light (BL)-dependent process being mediated by actin filaments wherein phototropins are identified as the photoreceptor involved. Studies through the last few decades have identified key molecular mechanisms involving Chloroplast Unusual Positioning 1 (CHUP1) protein and specific chloroplast-actin (cp-actin) filaments. However, the signal transduction pathway from strong BL absorption down to directional re-localization of chloroplasts by actin filaments is complex and ambiguous. Being the immediate cellular products of high irradiance absorption and having properties of remodelling actin as well as phototropin, reactive oxygen species (ROS) deemed to be more able and prompt than any other signalling agent in mediating chloroplast avoidance movement. Although ROS are presently being identified as fundamental component for regulating different plant processes ranging from growth, development and immunity, its role in avoidance movement have hardly been explored in depth. However, few recent reports have demonstrated the direct stimulatory involvement of ROS, especially H2O2, in chloroplast avoidance movement with Ca2+ playing a pivotal role. With this perspective, the present review discusses the mechanisms of ROS-mediated chloroplast avoidance movement involving ROS-Ca2+-actin communication system and NADPH oxidase (NOX)-plasma membrane (PM) H+-ATPase positive feed-forward loop. A possible working model is proposed.

Orchestration of Cu-Zn SOD and class III peroxidase with upstream interplay between NADPH oxidase and PM H+-ATPase mediates root growth in Vigna radiata (L.) Wilczek.

Post-germination plant growth depends on the regulation of reactive oxygen species (ROS) metabolism, spatiotemporal pH changes and Ca+2 homeostasis, whose potential integration has been studied during Vigna radiata (L.) Wilczek root growth. The dissipation of proton (H+) gradients across plasma membrane (PM) by CCCP (protonophore) and the inhibition of PM H+-ATPase by sodium orthovanadate repressed SOD (superoxide dismutase; EC 1.15.1.1) activity as revealed by spectrophotometric and native PAGE assay results. Similar results derived from treatment with DPI (NADPH oxidase inhibitor) and Tiron (O2- scavenger) denote a functional synchronization of SOD, PM H+-ATPase and NOX, as the latter two enzymes are substrate sources for SOD (H+ and O2-, respectively) and are involved in a feed-forward loop. After SOD inactivation, a decline in apoplastic H2O2 content was observed in each treatment group, emerging as a possible cause of the diminution of class III peroxidase (Prx; EC 1.11.1.7), which utilizes H2O2 as a substrate. In agreement with the pivotal role of Ca+2 in PM H+-ATPase and NOX activation, Ca+2 homeostasis antagonists, i.e., LaCl3 (Ca+2 channel inhibitor), EGTA (Ca+2 chelator) and LiCl (endosomal Ca+2 release blocker), inhibited both SOD and Prx. Finally, a drastic reduction in apoplastic OH (hydroxyl radical) concentrations (induced by each treatment, leading to Prx inhibition) was observed via fluorometric analysis. A consequential inhibition of root growth observed under each treatment denotes the importance of the orchestrated functioning of PM H+-ATPase, NOX, Cu-Zn SOD and Prx during root growth. A working model demonstrating postulated enzymatic synchronization with an intervening role of Ca+2 is proposed.

Congruence between PM H+-ATPase and NADPH oxidase during root growth: a necessary probability.

Plasma membrane (PM) H+-ATPase and NADPH oxidase (NOX) are two key enzymes responsible for cell wall relaxation during elongation growth through apoplastic acidification and production of ˙OH radical via O2˙-, respectively. Our experiments revealed a putative feed-forward loop between these enzymes in growing roots of Vigna radiata (L.) Wilczek seedlings. Thus, NOX activity was found to be dependent on proton gradient generated across PM by H+-ATPase as evident from pharmacological experiments using carbonyl cyanide m-chlorophenylhydrazone (CCCP; protonophore) and sodium ortho-vanadate (PM H+-ATPase inhibitor). Conversely, H+-ATPase activity retarded in response to different ROS scavengers [CuCl2, N, N' -dimethylthiourea (DMTU) and catalase] and NOX inhibitors [ZnCl2 and diphenyleneiodonium (DPI)], while H2O2 promoted PM H+-ATPase activity at lower concentrations. Repressing effects of Ca+2 antagonists (La+3 and EGTA) on the activity of both the enzymes indicate its possible mediation. Since, unlike animal NOX, the plant versions do not possess proton channel activity, harmonized functioning of PM H+-ATPase and NOX appears to be justified. Plasma membrane NADPH oxidase and H+-ATPase are functionally synchronized and they work cooperatively to maintain the membrane electrical balance while mediating plant cell growth through wall relaxation.

Integrated role of ROS and Ca+2 in blue light-induced chloroplast avoidance movement in leaves of Hydrilla verticillata (L.f.) Royle.

Directional chloroplast photorelocation is a major physio-biochemical mechanism that allows these organelles to realign themselves intracellularly in response to the intensity of the incident light as an adaptive response. Signaling processes involved in blue light (BL)-dependent chloroplast movements were investigated in Hydrilla verticillata (L.f.) Royle leaves. Treatments with antagonists of actin filaments [2,3,5-triiodobenzoic acid (TIBA)] and microtubules (oryzalin) revealed that actin filaments, but not microtubules, play a pivotal role in chloroplast movement. Involvement of reactive oxygen species (ROS) in controlling chloroplast avoidance movement has been demonstrated, as exogenous H2O2 not only accelerated chloroplast avoidance but also could induce chloroplast avoidance even in weak blue light (WBL). Further support came from experiments with different ROS scavengers, i.e., dimethylthiourea (DMTU), KI, and CuCl2, which inhibited chloroplast avoidance, and from ROS localization using specific stains. Such avoidance was also partially inhibited by ZnCl2, an inhibitor of NADPH oxidase (NOX) as well as 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU), a photosynthetic electron transport chain (ETC) inhibitor at PS II. However, methyl viologen (MV), a PS I ETC inhibitor, rather accelerated avoidance response. Exogenous calcium (Ca+2) induced avoidance even in WBL while inhibited chloroplast accumulation partially. On the other hand, chloroplast movements (both accumulation and avoidance) were blocked by Ca+2 antagonists, La3+ (inhibitor of plasma membrane Ca+2 channel) and ethylene glycol-bis(2-aminoethylether)-N,N,N',N'-tetraacetic acid (EGTA, Ca+2 chelator) while LiCl that affects Ca+2 release from endosomal compartments did not show any effect. A model on integrated role of ROS and Ca+2 (influx from apolastic space) in actin-mediated chloroplast avoidance has been proposed.