Title : Nitric oxide and reactive oxygen species regulation of NADPH oxidase for the high light induction of autophagy in Chlamydomonas reinhardtii
itric oxide (NO) is a signaling factors involved in the regulation of plant physiology and stress responses. The role of NO interaction with H2O2 in the regulation of NADPH oxidase (RBOL2) for the induction of autophagy and in Chlamydomonas reinhardtii was determined. 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, which can be 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 donor, S-nitroso-N-acetyl-penicillamine, 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 0.1 mM NO donor increased the abundance of ATG8 protein and CrATG transcripts, which can be suppressed by cPTIO, but did not influence cell viability. Treatment with 1 mM H2O2 and 0.1 mM NO donors enhanced autophagy induction and resulted in cell death after 24 h. This implies that NO is critical for the interaction of H2O2 and NO that induces cell death and autophagy. Furthermore, RBOL2 mutant showed enhanced autophagy expression and an exposure of RBOL2 mutant to 0.1 mM NO donors induced a higher autophagy expression. In conclusion, the present findings demonstrated that the NO interacts with H2O2 for HL-induced cell death and RBOL2 mediates the NO-induced autophagy in C. reinhardtii.