Abstract
Ascorbate peroxidase (APX; EC 1.11.1.11) activity and transcript levels of CrAPX1, CrAPX2, and CrAPX4 of Chlamydomonas reinhardtii increased under 1,400 μE·m−2·s−1 condition (HL). CrAPX4 expression was the most significant. So, CrAPX4 was downregulated using amiRNA technology to examine the role of APX for HL acclimation. The CrAPX4 knockdown amiRNA lines showed low APX activity and CrAPX4 transcript level without a change in CrAPX1 and CrAPX2 transcript levels, and monodehydroascorbate reductase (MDAR), dehydroascorbate reductase (DHAR), and glutathione reductase (GR) activities and transcript levels. Upon exposure to HL, CrAPX4 knockdown amiRNA lines appeared a modification in the expression of genes encoding the enzymes in the ascorbate–glutathione cycle, including an increase in transcript level of CrVTC2, a key enzyme for ascorbate (AsA) biosynthesis but a decrease in MDAR and DHAR transcription and activity after 1 h, followed by increases in reactive oxygen species production and lipid peroxidation after 6 h and exhibited cell death after 9 h. Besides, AsA content and AsA/DHA (dehydroascorbate) ratio decreased in CrAPX4 knockdown amiRNA lines after prolonged HL treatment. Thus, CrAPX4 induction together with its association with the modulation of MDAR and DHAR expression for AsA regeneration is critical for Chlamydomonas to cope with photo-oxidative stress.
Highlights
Ascorbate peroxidase (APX; EC 1.11.1.11) activity and transcript levels of CrAPX1, CrAPX2, and CrAPX4 of Chlamydomonas reinhardtii increased under 1,400 μE·m−2·s−1 condition (HL)
Using the subcellular localization prediction programs, ChloroP 1.1 and TargetP 1.1, we found that CrAPX1 and CrAPX2 show dual localization in chloroplast and mitochondrium and CrAPX4 is a chloroplastic enzyme
Whether the downregulation of CrAPX4 expression affected the expression of other APX isoforms and the genes encoding the enzymes in the ascorbate–glutathione cycle was elucidated
Summary
Ascorbate peroxidase (APX; EC 1.11.1.11) activity and transcript levels of CrAPX1, CrAPX2, and CrAPX4 of Chlamydomonas reinhardtii increased under 1,400 μE·m−2·s−1 condition (HL). The evolution analysis of APX isoforms in the chloroplast by Maruta, Sawa, Shigeoka and Ishikawa (2016)[25] shows a similar result in Arabidopsis but APX4 and APX6 are excluded because they do not have APX activity due to the substitution of nucleotides or amino acids essential for APX activity They find that only one chloroplastic APX protein, that is, CrAPX1, exists in Chlamydomonas and no APX isoforms can be detected in cytosolc and peroxisome. The expression of MDAR, DHAR, and GR in CrAPX4 knockdown amiRNA lines as well as ascorbate hemostasis was examined to see whether the ascorbate–glutathione cycle was affected by the knockdown of CrAPX4 expression
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