title: Enhanced Ascorbate Regeneration Via Dehydroascorbate Reductase Confers Tolerance to Photo-Oxidative Stress in Chlamydomonas reinhardtii.
abstract: The role of ascorbate (AsA) recycling via dehydroascorbate reductase (DHAR) in the tolerance of Chlamydomonas reinhardtii to photo-oxidative stress was examined. The activity of DHAR and the abundance of the CrDHAR1 (Cre10.g456750) transcript increased after moderate light (ML; 750 µmol m-2s-1) or high light (HL; 1,800 µmol m-2s-1) illumination, accompanied by dehydroascorbate (DHA) accumulation, decreased AsA redox state, photo-inhibition, lipid peroxidation, H2O2overaccumulation, growth inhibition and cell death. It suggests that DHAR and AsA recycling is limiting under high-intensity light stress. The CrDHAR1 gene was cloned and its recombinant CrDHAR1 protein was a monomer (25 kDa) detected by Western blot that exhibits an enzymatic activity of 965 µmol min-1mg-1protein. CrDHAR1 was overexpressed driven by a HSP70A:RBCS2 fusion promoter or down-regulated by artificial microRNA (amiRNA) to examine whether DHAR-mediated AsA recycling is critical for the tolerance of C. reinahartii cells to photo-oxidative stress. The overexpression of CrDHAR1 increased DHAR protein abundance and enzyme activity, AsA pool size, AsA:DHA ratio and the tolerance to ML-, HL-, methyl viologen- or H2O2-induced oxidative stress. The CrDHAR1-knockdown amiRNA lines that have lower DHAR expression and AsA recycling ability were sensitive to high-intensity illumination and oxidative stress. The glutathione pool size, glutathione:oxidized glutathione ratio and glutathione reductase and ascorbate peroxidase activities were increased in CrDHAR1-overexpressing cells and showed a further increase after high-intensity illumination but decreased in wild-type cells after light stress. The present results suggest that increasing AsA regeneration via enhanced DHAR activity modulates the ascorbate-glutathione cycle activity in C. reinhardtii against photo-oxidative stress.
authors.affiliation: Department of Marine Biotechnology and Resources, National Sun Yat-sen University, Kaohsiung 804, Taiwan.|These authors contributed equally to this work.
authors.forename: Shu-Tseng
authors.initials: ST
authors.lastname: Lin
authors.affiliation: Department of Marine Biotechnology and Resources, National Sun Yat-sen University, Kaohsiung 804, Taiwan.|These authors contributed equally to this work.
authors.forename: Chih-Wen
authors.initials: CW
authors.lastname: Chiou
authors.affiliation: Department of Biological Sciences, National Sun Yat-sen University, Kaohsiung 804, Taiwan.
authors.forename: Yen-Lin
authors.initials: YL
authors.lastname: Chu
authors.affiliation: Department of Marine Biotechnology and Resources, National Sun Yat-sen University, Kaohsiung 804, Taiwan.
authors.forename: Yu
authors.initials: Y
authors.lastname: Hsiao
authors.affiliation: Department of Marine Biotechnology and Resources, National Sun Yat-sen University, Kaohsiung 804, Taiwan.
authors.forename: Yu-Fei
authors.initials: YF
authors.lastname: Tseng
authors.affiliation: Department of Marine Biotechnology and Resources, National Sun Yat-sen University, Kaohsiung 804, Taiwan.
authors.forename: Yi-Chun
authors.initials: YC
authors.lastname: Chen
authors.affiliation: Department of Biological Sciences, National Sun Yat-sen University, Kaohsiung 804, Taiwan.
authors.forename: Hsien-Jung
authors.initials: HJ
authors.lastname: Chen
authors.affiliation: Department of Marine Biotechnology and Resources, National Sun Yat-sen University, Kaohsiung 804, Taiwan.|The Asia-Pacific Ocean Research Center, National Sun Yat-sen University, Kaohsiung 804, Taiwan.
authors.forename: Hsin-Yang
authors.initials: HY
authors.lastname: Chang
authors.affiliation: Department of Marine Biotechnology and Resources, National Sun Yat-sen University, Kaohsiung 804, Taiwan tmlee@mail.nsysu.edu.tw.|The Asia-Pacific Ocean Research Center, National Sun Yat-sen University, Kaohsiung 804, Taiwan.|Doctoral Degree Program in Marine Biotechnology, National Sun Yat-sen University, Kaohsiung 804, Taiwan.
authors.forename: Tse-Min
authors.initials: TM
authors.lastname: Lee
mesh_terms: D000222:Adaptation, Physiological / Q000187:drug effects / Q000528:radiation effects*; D001205:Ascorbic Acid / Q000378:metabolism*; D001483:Base Sequence; D016825:Chlamydomonas reinhardtii / Q000201:enzymology* / Q000528:radiation effects*; D002734:Chlorophyll / Q000378:metabolism; D000077194:Chlorophyll A; D015536:Down-Regulation / Q000235:genetics; D005453:Fluorescence; D018506:Gene Expression Regulation, Plant / Q000187:drug effects / Q000528:radiation effects; D005978:Glutathione / Q000378:metabolism; D006861:Hydrogen Peroxide / Q000633:toxicity; D008027:Light; D018384:Oxidative Stress / Q000528:radiation effects*; D010088:Oxidoreductases / Q000378:metabolism*; D010269:Paraquat / Q000633:toxicity; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D014170:Transformation, Genetic / Q000187:drug effects / Q000528:radiation effects
chemical_list: D012333:RNA, Messenger; D002734:Chlorophyll; D006861:Hydrogen Peroxide; D010088:Oxidoreductases; C020666:glutathione dehydrogenase (ascorbate); D005978:Glutathione; D010269:Paraquat; D001205:Ascorbic Acid; D000077194:Chlorophyll A
keywords: 
  Chlamydomonas
  ; Ascorbate; Ascorbate–glutathione cycle; Dehydroascorbate reductase; High-intensity light stress