Ascorbate peroxidase 4 plays a role in the tolerance of Chlamydomonas reinhardtii to photo-oxidative stress

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.

Figure 1

Changes in cell growth (A), lipid peroxidation (TBARS) (B), and ROS (H₂DCFDA) (C) in Chlamydomonas reinhardtii CC-400 under 50 (NL) and 1,400 (HL) μE·m⁻²·s⁻¹ conditions. Data are expressed as the mean ± SD (n = 3) and analyzed by the t-test.

Figure 2

Changes in APX activity (A) and transcript levels of CrAPX1 (B), CrAPX2 (B), and CrAPX4 (B) in Chlamydomonas reinhardtii under 50 (NL) and 1,400 (HL) μE·m⁻²·s⁻¹ conditions. Data are expressed as the mean ± SD (n = 3). The asterisk indicates a significant difference between NL and HL treatments at the same time point by the t-test (*P < 0.05; **P < 0.01; ***P < 0.001).

Figure 3

APX activity (A) and transcript levels of CrAPX4 (B), CrAPX1 (C), CrAPX2 (D), CrMDAR1 (E), CrDHAR1 (F), CrGSHR1 (G), and CrGSHR2 (H) in Chlamydomonas reinhardtii wild-type CC-400, vector-only control (V15), and CrAPX4 knockdown lines (APX4-ami 9, APX4-ami 53, APX4-ami 56, APX4-ami 59, and APX4-ami 65) under 50 μE·m⁻²·s⁻¹ condition. The data are expressed as the mean ± SD (n = 3) and different symbols indicate significant difference analyzed by Duncan’s new multiple range test (P < 0.05).

Figure 4

The transcript levels of CrAPX1 (A), CrAPX2 (B), and CrAPX4 (C) and the activity of APX (D) in Chlamydomonas reinhardtii wild-type CC-400, vector-only control (V15), and CrAPX4 knockdown lines (APX4-ami 56, APX4-ami 59, and APX4-ami 65) under 50 μE·m⁻²·s⁻¹ condition. The data are expressed as the mean ± SD (n = 3) and different symbols indicate significant difference analyzed by Duncan’s new multiple range test (P < 0.05).

Figure 5

The changes in the expression of genes and activity of enzymes in the ascorbate–glutathione cycle under HL condition. The transcript levels of CrVTC2 (A), CrMDAR1 (B), CrDHAR1 (C), CrGSHR1 (D) and CrGSHR2 (E) and the activity of MDAR (F), DHAR (G), and GR (H) in Chlamydomonas reinhardtii wild-type CC-400, vector-only control (V15), and CrAPX4 knockdown lines (APX4-ami 56, APX4-ami 59, and APX4-ami 65) 1 h after exposure to 50 (NL) and 1,400 (HL) μE·m⁻²·s⁻¹. The data are expressed as the mean ± SD (n = 3) and different symbols indicate significant difference analyzed by Duncan’s new multiple range test (P < 0.05).

Figure 6

The viability assay of Chlamydomonas reinhardtii wild-type CC-400, vector-only control (V15), and CrAPX4 knockdown lines (APX4-ami 56, APX4-ami 59, and APX4-ami 65) 1 h (A) and 9 h (B) after exposure to 50 (NL) and 1,400 (HL) μE·m⁻²·s⁻¹. Three biological replicates have been shown and the cell colony in the CrAPX4 knockdown lines was normal 1 h after HL treatment but absent after prolonged HL treatment (9 h).

Figure 7

Lipid peroxidation (TBARS) (A, B) and ROS (H₂DCFDA fluorescence) (C, D) of Chlamydomonas reinhardtii CrAPX4 knockdown lines (APX4-ami 56, APX4-ami 59, and APX4-ami 65) under 50 (NL) and 1,400 (HL) μmol·m⁻²·s⁻¹ conditions for 1 h (A, C) and 6 h (B, D). The controls are the wild-type CC-400 and vector-only line (V15). The data are expressed as the mean ± SD (n = 3) and different symbols indicate significant difference analyzed by Duncan’s new multiple range test (P < 0.05).

Figure 8

Changes in total AsA (A, E), AsA (B, F), DHA (C, G), and AsA/DHA ratio (D, H) of the Chlamydomonas reinhardtii CrAPX4 knockdown lines (APX4-ami 56, APX4-ami 59, and APX4-ami 65) under 50 (NL) and 1,400 (HL) μE·m⁻²·s⁻¹ conditions for 1 h and 6 h. The controls are the wild-type CC-400 and vector-only line (V15). Data are expressed as the mean ± SD (n = 3) and different symbols indicate significant difference by Duncan’s new multiple range teat (P < 0.05).