Distinct contribution of two cyclic electron transport pathways to P700 oxidation

Abstract

Cyclic electron transport (CET) around Photosystem I (PSI) acidifies the thylakoid lumen and downregulates electron transport at the cytochrome b6f complex. This photosynthetic control is essential for oxidizing special pair chlorophylls (P700) of PSI for PSI photoprotection. In addition, CET depending on the PROTON GRADIENT REGULATION 5 (PGR5) protein oxidizes P700 by moving a pool of electrons from the acceptor side of PSI to the plastoquinone pool. This model of the acceptor-side regulation was proposed on the basis of the phenotype of the Arabidopsis (Arabidopsis thaliana) pgr5-1 mutant expressing Chlamydomonas (Chlamydomonas reinhardtii) plastid terminal oxidase (CrPTOX2). In this study, we extended the research including the Arabidopsis chlororespiratory reduction 2-2 (crr2-2) mutant defective in another CET pathway depending on the chloroplast NADH dehydrogenase-like (NDH) complex. Although the introduction of CrPTOX2 did not complement the defect in the acceptor-side regulation by PGR5, the function of the NDH complex was complemented except for its reverse reaction during the induction of photosynthesis. We evaluated the impact of CrPTOX2 under fluctuating light intensity in the wild-type, pgr5-1 and crr2-2 backgrounds. In the high-light period, both PGR5- and NDH-dependent CET were involved in the induction of photosynthetic control, whereas PGR5-dependent CET preferentially contributed to the acceptor-side regulation. On the contrary, the NDH complex probably contributed to the acceptor-side regulation in the low-light period but not in the high-light period. We evaluated the sensitivity of PSI to fluctuating light and clarified that acceptor-side regulation was necessary for PSI photoprotection by oxidizing P700 under high light.

Figure 1

Analysis of the CrPTOX2 level. Total leaf proteins were separated by SDS-PAGE and probed with the antibody raised against CrPTOX2. As loading controls, Cyt f was also immuno-detected and the gel was stained by Coomassie Brilliant Blue. Each fraction corresponding to 0.25 µg chlorophyll was loaded per lane.

Figure 2

Light-intensity dependence of chlorophyll fluorescence and P700 parameters during steady-state photosynthesis in the crr2-2 background. NPQ (A), Y(II) (B), 1-qL (C), Y(I) (D), Y(ND) (E), and Y(NA) (F) were analyzed in detached leaves from WT, crr2-2, and CrPTOX2 lines under the crr2-2 background (#4 and #5). Data represent mean ± SD (n = 5 biological replicates). Different letters indicate the statistical differences confirmed by the Tukey–Kramer test (P < 0.05). Asterisks indicate a statistically significant difference from crr2-2 (*P < 0.05, **P < 0.01), confirmed by the Dunnett test. Results of the statistical analyses are not shown when the difference is not significant between any genotypes. PPFD, photosynthetic photon flux density.

Figure 3

Chlorophyll fluorescence and P700 parameters during the induction of photosynthesis by actinic light (AL, 130 µmol photons m⁻² s⁻¹) in the crr2-2 background. NPQ (A), Y(II) (B), 1-qL (C), Y(I) (D), Y(ND) (E), and Y(NA) (F) were analyzed in detached leaves from WT, crr2-2, and CrPTOX2 lines under the crr2-2 background (#4 and #5). Dark and bright bars indicate dark and light period, respectively. Data represent mean ± SD (n = 4–5 biological replicates). Different letters indicate the statistical differences confirmed by the Tukey–Kramer test (P < 0.05). Asterisks indicate a statistically significant difference from crr2-2 (*P < 0.05, **P < 0.01), confirmed by the Dunnett test. Statistical analyses were performed every 10 s during the initial 60 s and then every 30 s.

Figure 4

Electrochromic shift (ECS) analysis during induction of photosynthesis. (A) The size of pmf formed in the light was calculated as ECSt/ECS_ST at each time point after the onset of actinic light. ECS_t and ECS_ST represent the light–dark difference of the ECS level and the ECS difference depending on a single turnover flash (see Materials and methods). (B) The proton conductivity of the thylakoid membrane (g_H⁺) was determined by monitoring the kinetics of ECS decay in the dark. Data represent mean ± SD (n = 5 biological replicates). Different letters indicate the statistical differences confirmed by the Tukey–Kramer test (P < 0.05). Asterisks indicate a statistically significant difference in the CrPTOX2 lines from the corresponding background plants (*P < 0.05, **P < 0.01), confirmed by the Dunnett test.

Figure 5

P700 parameters during the fluctuating light condition consisting of 5-min LL (white bars) of 40 µmol photons m⁻² s⁻¹ and 1-min HL (yellow bars) of 1,616 µmol photons m⁻² s⁻¹ in the WT background. A black bar represents the adaptation to room light. Y(I) (A), Y(NA) (B), and Y(ND) (C) were analyzed in detached leaves from WT and CrPTOX2 lines under the WT background (#1 and #2). Data represent mean ± SD (n = 5–6 biological replicates).

Figure 6

Chlorophyll fluorescence and P700 parameters during the fluctuating light condition consisting of 5-min LL (white bars) of 40 µmol photons m⁻² s⁻¹ and 1-min HL (yellow bars) of 1,616 µmol photons m⁻² s⁻¹ in the pgr5-1 background. A black bar represents the adaptation to room light. Y(II) (A), NPQ (B), 1-qL (C), Y(I) (D), Y(ND) (E), and Y(NA) (F) were analyzed in detached leaves from WT, pgr5-1, and CrPTOX2 lines under the pgr5-1 background (#1 and #2). Data represent mean ± SD (n = 5–6 biological replicates).

Figure 7

Evaluation of PSI photodamage during the fluctuating light condition consisting of 5-min LL of 40 µmol photons m⁻² s⁻¹ and 1-min HL of 1,616 µmol photons m⁻² s⁻¹. The Pm levels were determined before and after exposure to the fluctuating light. The ratio (after/before) is shown as a mean of four biological replicates in the WT and pgr5-1 background (A) and crr2-2 background (B). Error bars represent SD. Different letters indicate statistically significant differences by the Tukey–Kramer test (P < 0.05).

Figure 8

Chlorophyll fluorescence and P700 parameters during the fluctuating light condition consisting of 5-min LL (white bars) of 40 µmol photons m⁻² s⁻¹ and 1-min HL (yellow bars) of 1,616 µmol photons m⁻² s⁻¹ in the crr2-2 background. A black bar represents the adaptation to room light. NPQ (A), Y(II) (B), 1-qL (C), Y(I) (D), Y(ND) (E), and Y(NA) (F) were analyzed in detached leaves from WT, crr2-2, and CrPTOX2 lines under the crr2-2 background (#4 and #5). Data represent mean ± SD (n = 5–6 biological replicates).