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

Eva Yuhua Kuo,Tse-Min Lee,Hsueh-Ling Chang,Shu-Tseng Lin

doi:10.3389/fpls.2020.00772

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 H2O2 and 0.1 mM NO donors enhanced autophagy induction and resulted in cell death after 24 h. The interaction of H2O2 and NO can be prevented by cPTIO treatment. This implies that NO is critical for the interaction of H2O2 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 H2O2 for HL-induced cell death. The relationships between autophagy and cell death are discussed.

Highlights

Whether the DAFFM fluorescence was attributable to Nitric oxide (NO) was confirmed by the treatment with an NO scavenger, cPTIO (400 μM). cPTIO was added twice, at 0 and 2.5 h, to remove the NO generated during high light (HL) illumination
CPTIO can cause an increase in the DAF-FM fluorescence under certain conditions due to oxidization of NO to the NO2 radical with the subsequent formation of N2O3 (NO2 + NO → N2O3), which can react with the DAF-FM dye to form the fluorescent DAF-2T (Mur et al, 2011; D’Alessandro et al, 2013), our current results showed that the presence of cPTIO effectively reduced the DAF-FM fluorescence in the HL-treated cells (Figures 1A,B)
It has been shown that C. reinhardtii can metabolize NO to N2O and this metabolism is dependent on photosynthetic electron transport

Summary

Introduction

Nitric oxide (NO), a short-lived, gaseous molecule that can be either enzymatically or non-enzymatically synthesized in plants (Besson-Bard et al, 2008; Palavan-Unsal and Arisan, 2009), has been recognized as a novel biological messenger in the regulation of various biochemical and physiological activities and stress responses (Anbar, 1995; Neill et al, 2008; Hasanuzzaman et al, 2010; Hayat et al, 2010; Siddiqui et al, 2011; Bajguz, 2014). Increasing importance has been attached to the role of NO in the regulation of high intensity light responses. Whether NO plays a protective or a harmful role in the response of plants to excessive light energy depends on the plant species. Illumination of the leaves of tall fescue (Festuca arundinacea Schreb.) at an intensity of 500 μmol m−2 s−1 triggers NO production against oxidative stress by increasing the activity of antioxidant enzymes and the content of antioxidants (Xu et al, 2010). Foresia et al (2010) has reported for a unicellular marine alga Ostreococcus tauri Gen et Sp-NOV that illumination at 400 μmol m−2 s−1 induces an NO burst, which is proposed to be a signal triggering a photoprotection mechanism against high light (HL)-induced oxidative damage. Dangeard that NO generated under very high intensity light (VHL; 3,000 μmol m−2 s−1) conditions is associated with VHLinduced cell death (Chang et al, 2013)

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