The 1st International Electronic Conference on Plant Science (01/12/2020 - 15/12/2020): https://iecps2020.sciforum.net/

Section A. Plant Protection, Response to stress and Climate Change

The Acclimation Mechanisms of Chlamydomonas reinhardtii against Nitrosative Stress: A Role of NADPH Oxidase (RBOL2) in the Regulation of Nitric Oxide-Mediated ER Stress and Glutathione Redox State

Eva YuHua Kuo ¹ , Wen-Chyi Dai ¹ , Mu-Ting Lee ² , Zheng Yu Yun ² , Tse-Min Lee ^{* 3}

¹ Department of Marine Biotechnology and Resources, National Sun Yat-sen University, Kaohsiung, Taiwan

² Department of Marine Biotechnology and Resources, National Sun Yat-sen University, Kaohsiung, Taiwan.

³ Department of Marine Biotechnology and Resources, College of Marine Sciences, National Sun Yat-sen University, Kaohsiung 80424, Taiwan

Published: 30 November 2020 by MDPI in The 1st International Electronic Conference on Plant Science session Plant Nutrition, Plant Physiology and Metabolism

10.3390/IECPS2020-08609 (registering DOI)

Abstract:

Nitric oxide (NO) is a signal in the modulation of acclamatory responses to stress in plants. This study has investigated the metabolic shift in the acclimation of a green alga Chlamydomonas reinahrdii to NO stress. On exposure to 0.1 mM SNAP, a NO donor, causes a transient inhibition in photosysnthesis after 1 h, followed by a fast recovery. The RNA-seq results discover that NO enhances the catabolism of cysteine, threonine, aromatic amino acids, and branched-chain amino acids to other metabolites for the synthesis of functional compounds. NO also induces the endosomal protein trafficking system in association with the ubiquitinylation machinery (ubiquition, SNARE, and autophagy) for the degradation of sequestered damaged proteins to generate small molecules for re-synthesis of macromolecules, and triggers the protein modification system (heat proteins). The antioxidant defense system (VTC2, APX, MDAR, DHAR, GR, GPXH/GPX5, GSTS1, and GSTS2) and redox state (AsA/DHA and GSHH/GSSG) are activated by NO. Two PsbP-like proteins and LHCBM9 are induced for the acclimayion to NO stress. Besides, THB, FLVb, and CYP55B1 are induced to reduce NO level for NO detoxification, allowing the acclimation to NO stress. NO significantly induced NADPH oxidase, RBOL2, but slightly decreased RBOL1 expression. Unfolded protein response (UPR) in response to ER stress was inhibited by NO stress. Using RBOL2 mutant, the UPR was induced and the glutathione synthesis and redox state was inhibited. This finding demonstrates that NADPH oxidase (RBOL2)-mediated signaling pathway is involved in the NO-induced inhibition of ER stress and the induction of glutathione availability while other NO-induced metabolisms are under control by other signaling pathway. Our analyses provide a valuable tool for selecting genetic modification targets to understand acclamatory mechanisms to NO stress in Chlamydomonas.

Keywords: acclimation, antioxidant defense system, Chlamydomonas reinhardtii, ER stress, glutathione redox state, membrane trafficking system, nitrosative stress, photosynthesis, sulfur oemeostasis, transcriptome

11th Green Sustainable Biotechnology Technology International Symbosium, Kaohsiung, Taiwan

Optimization of docosahexaenoic acid (DHA) production of Aurantiochytrium strain BL1 using waste cooking oil as carbon source

Ze-Kuan Wu¹, Yuan Yu Chin¹, Mu-Ting Lee¹, Yi-Min Chen², Jo-Shu Chang^3,4, Tse-Min Lee^1,5

¹Department of Marine Biotechnology and Resource, National Sun Yat-sen University, Kaohsiung 804, Taiwan

²Research Center of Ocean Environment and Technology, National Cheng Kung University, Tainan 701, Taiwan

³Department of Chemical and Materials Engineering, Tunghai University, Taichung 407, Taiwan

⁴Department of Chemical Engineering, National Cheng Kung University, Tainan 701, Taiwan

⁵Doctoral Degree Program, National Sun Yat-sen University, Kaohsiung 804, Taiwan

Thraustochytrids that are heterokonts, which were first discovered in 1934, are a worldwide distributed decomposer in marine environments that occupies a distinct ecological niche in marine ecosystems. It utilizes dissolved organic matter (DOM) and particulate organic matter (POM). Several species synthesize high-value compounds, such as docosahexaenoic acid (DHA, C22: 6n−3). We have isolated indigenous Thraustochytrids from Taiwan with the potential for the high production of DHA. Among them, an Aurantiochytrium strain BL1 with high DHA production yield (12 mg/L) was isolated (Yang et al., 2010. Mar. Biotechnology. 12: 173–185).

The purpose is to develop a circular economy model to recycle materials from waste using Aurantiochytrium strain BL1 to synthesize DHA by utilization of waste cooking oil. The main discoveries are 1. It shows the ability in the utilization of waste cooking oil (120 g/L in the presence of 10% tween-80) for the production of DHA; 2. The optimal salinity for waste cooking oil as carbon source was 20 ppt and the optimal pH value was 6-8. The biomass production using waste cooking oil as carbon source can reach 17 g/L and DHA production is 366 mg/L; 3. When scales up using 5-L fermenter with 3 L culture medium containing 120 g/L waste cooking oil, we can obtain around 9 g/L biomass production with 7 days; 3. The boost of biomass during scale up process is a bottleneck for BL1 using waste cooking oil for the production of DHA; 4. The current finding reveals that Aurantiochytrium strain BL1 isolated from Taiwan is a potential organism for waste reuse in the production of high-value product (DHA), which is considered as the model of circular economy.

Plant Science Virtual 2020

September 10-11, 2020

Websute: https://plant-science-biology-conferences.magnusgroup.org/

Nitric oxide and reactive oxygen species regulation of NADPH oxidase for the high light induction of autophagy in Chlamydomonas reinhardtii

Eva YuHua Kuo¹, Yu Yuan Zheng² and Tse-Min Lee³

¹Department of Marine Biotechnology and Resources, National Sun Yat-sen University, Kaohsiung 80424, Taiwan, eyk1920@mail.nsysu.edu.tw

²Department of Marine Biotechnology and Resources, National Sun Yat-sen University, Kaohsiung 80424, Taiwan, ybndhs56@gmail.com

³Department of Marine Biotechnology and Resources, National Sun Yat-sen University, Kaohsiung 80424, Taiwan; Doctoral Degree Program in Marine Biotechnology, National Sun Yat-sen University, Kaohsiung 80424, Taiwan, tmee@mail.nsysu.edu.tw

Abstract: Nitric 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 (NADPH oxidase) mutant showed enhanced 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.

ASPB2021. July, 2021. (poster presentation)

NADPH oxidase associates with the modulation of antioxidant defense system in Chlamydomonas under high intensity illumination

Eva YuHua Kuo, Tsen-Hung Lin, and Tse-Min Lee

Department of Marine Biotechnology and Resources, National Sun Yat-sen University, Kaohsiung 80424, Taiwan

6th Edition of Global Conference on Plant Science and Molecular Biology

Sept 30-OCT 01, 2021

Website: https://plant-science-biology-conferences.magnusgroup.org/

NADPH oxidase-mediated signaling pathway mediates the high light activation of ascorbate-glutathione cycle (AGC) in Chlamydomonas reinhardtii against photo-oxidative stress

Eva YuHua Kuo¹ and Tse-Min Lee²

¹Department of Marine Biotechnology and Resources, National Sun Yat-sen University, Kaohsiung 80424, Taiwan, eyk1920@mail.nsysu.edu.tw

²Department of Marine Biotechnology and Resources, National Sun Yat-sen University, Kaohsiung 80424, Taiwan; Doctoral Degree Program in Marine Biotechnology, National Sun Yat-sen University, Kaohsiung 80424, Taiwan, tmee@mail.nsysu.edu.tw

The ascorbate-glutathione cycle (AGC) is essential for Chlamydomonas reinhardtii cells to cope with high light stress. NADPH oxidase acts as a key hub for the regulation of stress tolerance in plants. High-intensity illumination (HL, 1,200 mmol m^-2 s^-1) did not impact Chlamydomonas growth while the application of diphenyleneiodonium (DPI), an NADPH oxidase inhibitor, in the concentration of 5 or 10 mM inhibited cell growth along with an increase of reactive oxygen species (ROS, O₂^.-, H₂O₂, and ¹O₂) and lipid peroxidation. The RNA-seq analysis and enzyme activity assay reveal that HL triggered an increase in CAT, APX, DHAR and GR enzyme activity and FeSOD (FSD1), APX1, DHAR1, and GSHR1 transcript abundances. But, the increase in the transcript abundances of APX1 and GSHR1 by HL was enhanced in the presence of DPI. The induction in the activity of enzymes in AGC can be inhibited in the presence of 10 mM DPI, accompanied by increased ROS production. However, the increase in the transcript abundances of FSD1, APX1, DHAR1, and GSHR1 under HL conditions can be enhanced in the presence of DPI. It reflects that NADPH oxidase-mediated signaling pathway modulates the induction of AGC in ways other than mRNA level. In conclusion, the present findings demonstrate that NADPH oxidase modulates the upregulation of the ascorbate-glutathione cycle for the acclimation of Chlamydomonas cells to photo-oxidative stress.