High Light-Induced Nitric Oxide Production Induces Autophagy and Cell Death in Chlamydomonas reinhardtii

Abstract

Autophagy plays a role in regulating important cellular functions in response to stress conditions. The role of nitric oxide (NO) in the regulation of autophagy in Chlamydomonas reinhardtii has been not studied. Illumination of C. reinhardtii cells under a high light (HL, 1,600 µmol m −2 s −1) condition induced a NO burst through NO synthase-and nitrate reductase-independent routes, and cell death. The abundance of CrATG8 protein, an autophagy marker of C. reinhardtii, increased after HL illumination along with a linear increase in the transcript abundance of autophagy-associated genes (CrVPS34, CrATG1, CrATG3, CrATG4, CrATG6, CrATG7, CrATG8, and CrATG12), which were suppressed in the presence of an NO scavenger, 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (cPTIO). The cells were treated with NO donors, S-nitroso-N-acetyl-penicillamine, and S-nitrosoglutathione, under a normal light (50 µmol m −2 s −1) condition to elucidate the role of NO in autophagy activation and cell death. Treatment with 0.05 mM or 0.1 mM NO donors increased the abundance of ATG8 protein and CrATG transcripts, which were suppressed in the presence of cPTIO. Moreover, treatment with 0.05 mM NO donors did not affect cell viability, while 0.1 mM NO donors elicited a transient decrease in cell growth and death that recovered after 12 h. The transient effect could be prevented by the presence of cPTIO. However, treatment with 1 mM H 2 O 2 and 0.1 mM NO donors enhanced autophagy induction and resulted in cell death after 24 h. The interaction of H 2 O 2 and NO can be prevented by cPTIO treatment. This implies that NO is critical for the interaction of H 2 O 2 and NO that induces cell death and autophagy. Furthermore, exposure to 0.1 mM NO donors under a non-lethal HL condition (750 µmol m −2 s −1) evoked autophagy and cell death. In conclusion, the present findings demonstrated that the NO-mediated autophagy pathway is activated in C. reinhardtii under lethal high intensity illumination and may interact with H 2 O 2 for HL-induced cell death. The relationships between autophagy and cell death are discussed.

Structural and functional insights into nitrosoglutathione reductase from Chlamydomonas reinhardtii

“…NO metabolism is of particular importance in these organisms living in liquid micro-oxic environments, where the fermentative metabolism and the Hemoglobin-NO cycle are important players in cellular bioenergy [ 99 , 100 ]. In Chlamydomonas , the biological function of NO relates to responses to nitrogen and sulfur starvation, hypoxia/anoxia, high light and light to dark transitions [ 4 , 58 , 60 , 99 , 101 , 102 ]. In general, protein S-nitrosylation acts as the major mechanism propagating NO-dependent biological signaling and it can modulate protein function by altering enzymatic activity and/or protein structure [ [12] , [13] , [14] , 103 ].…”

“…Nitric oxide (•NO) is a relatively stable free radical widely recognized as a signaling molecule in oxygenic phototrophs where it controls multiple physiological processes ( e.g. development, stomatal closure, tolerance to metal toxicity, and adaptive response to abiotic and biotic stresses) [ [1] , [2] , [3] , [4] , [5] , [6] , [7] , [8] ]. The biological actions of •NO are mainly exerted by NO-derived reactive molecules through their ability to react with proteins and trigger the formation of post-translational modifications (PTMs) [ [9] , [10] , [11] , [12] ].…”

“…In green microalgae such as Chlamydomonas reinhardtii, NO signaling participates in the regulation of nutrients acquisition, photosynthetic efficiency, and other processes including autophagy and cell death [ 4 , [58] , [59] , [60] , [61] , [62] ], making its understanding of particular interest for biotechnological purposes. Recently, GSNO reducing activity has been measured in Chlamydomonas reinhardtii extracts following exposure to salt stress [ 63 ], but the underlying enzymes along with their functional features are yet to be uncovered.…”

Acclimation ofChlamydomonas reinhardtiito Nitric Oxide Stress Related to Respiratory Burst Oxidase-Like 2

“…Studying NO burst over a short-term period provided us a chance to elucidate the metabolic shift to a brief NO attack and the following acclimation processes in Chlamydomonas cells. We recently discovered that NO interacts with reactive oxygen species (ROS) to induce cell death in association with autophagy in Chlamydomonas cells under high intensity illumination (Kuo et al, 2020a). Fortunately, ROS over-production and oxidative damage were not found in the 0.3 mM SNAP treatment.…”

“…Nitric oxide (NO) is a crucial signaling molecule in diverse physiological processes in plants (Durner et al, 1999), including in the green alga Chlamydomonas , in which NO regulates many physiological processes and stress responses such as the remodeling of chloroplast proteins by the degradation of thylakoid cytochrome b 6 f complex and stroma ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) via FtsH and Clp chloroplast proteases under nitrogen (Wei et al, 2014) or sulfur starvation (de Mia et al, 2019). In Chlamydomonas , NO is also involved in cell death induced by ethylene and mastoparan (Yordanova et al, 2010), induction of oxidative stress under extreme high light (VHL, 3,000 μmol·m −2 ·s −1 ) (Chang et al, 2013), interaction of NO with hydrogen peroxide (H 2 O 2 ) for high light stress-induced autophagy and cell death (Kuo et al, 2020a), proline biosynthesis under copper stress (Zhang et al, 2008), and responses to salt stress (Chen et al, 2016). NO is responsible for the regulation of nitrogen assimilation by repressing the expression of nitrate reductase as well as nitrate and ammonium transporters (de Montaigu et al, 2010) and their enzyme activities (Sanz-Luque et al, 2013; Calatrava et al, 2017).…”