PGR5-Dependent Cyclic Electron Flow Protects Photosystem I under Fluctuating Light at Donor and Acceptor Sides

Abstract

In response to a sudden increase in light intensity, plants must cope with absorbed excess photon energy to protect photosystems from photodamage. Under fluctuating light, PSI is severely photodamaged in the Arabidopsis (Arabidopsis thaliana) proton gradient regulation5 (pgr5) mutant defective in the main pathway of PSI cyclic electron transport (CET). Here, we aimed to determine how PSI is protected by two proposed regulatory roles of CET via transthylakoid ΔpH formation: (1) reservation of electron sink capacity by adjusting the ATP/NADPH production ratio (acceptor-side regulation) and (2) down-regulation of the cytochrome b ₆ f complex activity called photosynthetic control for slowing down the electron flow toward PSI (donor-side regulation). We artificially enhanced donor- and acceptor-side regulation in the wild-type and pgr5 backgrounds by introducing the pgr1 mutation conferring the hypersensitivity of the cytochrome b ₆ f complex to luminal acidification and moss Physcomitrella patens flavodiiron protein genes, respectively. Enhanced photosynthetic control partially alleviated PSI photodamage in the pgr5 mutant background but restricted linear electron transport under constant high light, suggesting that the strength of photosynthetic control should be optimized. Flavodiiron protein-dependent oxygen photoreduction formed a large electron sink and alleviated PSI photoinhibition, accompanied by the induction of photosynthetic control. Thus, donor-side regulation is essential for PSI photoprotection but acceptor-side regulation also is important to rapidly induce donor-side regulation. In angiosperms, PGR5-dependent CET is required for both functions.

Figure 1.

Proposed mechanisms for the regulation of photosynthetic electron transport under high light by PGR5/PGRL1-dependent CET. LET and CET are indicated by blue and red arrows, respectively. In angiosperms, CET consists of two pathways: the PGR5/PGRL1- and NDH-dependent pathways. In response to a sudden increase in light intensity, PGR5/PGRL1-dependent CET backflows electrons from PSI to the PQ pool without net NADPH production and generates ΔpH across the thylakoid membrane via the Q cycle in the Cyt b₆f complex. In the PSI acceptor-side regulation, pmf composed of ΔpH and ΔΨ drives ATP synthesis via ATP synthase and adjusts the ATP/NADPH production ratio, which is required for operating the Calvin-Benson cycle and photorespiration. This mechanism alleviates the PSI acceptor-side limitation of electron transport by increasing electron sink capacity downstream of PSI. In the PSI donor-side regulation, luminal acidification slows plastoquinol oxidation at the Cyt b₆f complex to prevent excess electron flow toward PSI. This mechanism is called photosynthetic control. In addition, the luminal acidification induces qE quenching in the PSII antenna to discard excess photon energy as heat. FNR, Fd:NADP⁺ oxidoreductase; PC, plastocyanin; PGA, 3-phosphoglycerate; RuBP, ribulose 1,5-bisphosphate.

Figure 2.

Growth and thylakoid protein accumulation in wild-type (WT), pgr1, pgr5, pgr1 pgr5-1, and pgr1 pgr5-2 plants. A, Plants were grown in a growth chamber at 50 to 60 μmol photons m⁻² s⁻¹ under short-day conditions (9 h of light/15 h of dark) for 40 d. B, BN-PAGE analysis of thylakoid protein complexes. The gel was stained with Bio-Safe Coomassie stain. The positions of photosynthetic complexes are indicated on the right. C, Immunoblot analysis of photosynthetic proteins in the chloroplast membrane fractions. Antibodies used are indicated on the right.

Figure 3.

Impact of the enhanced photosynthetic control response by the pgr1 mutation on electron transport during steady-state photosynthesis. The light intensity dependence of PSII and PSI photosynthetic parameters was monitored in the wild type (WT) and pgr1, pgr5, and pgr1 pgr5 mutant alleles. A to C, PSI parameters donor-side limitation of PSI [Y(ND)], acceptor-side limitation of PSI [Y(NA)], and quantum yield of PSI [Y(I)], respectively. D to F, PSII parameters relative ETR(II), NPQ, and quantum yield of PSII [Y(II)], respectively (biological replicates n = 8–9 ± sd).

Figure 4.

Enhanced photosynthetic control response by the pgr1 mutation protects PSI from photodamage under fluctuating light. The effect of fluctuating light on photosynthetic parameters was monitored in the wild type (WT) and pgr1, pgr5, and pgr1 pgr5 mutant alleles. A to D, Y(I), Y(ND), Y(NA), and NPQ, respectively (biological replicates n = 7–9 ± sd). Leaf discs from plants dark adapted for 20 min were exposed to fluctuating light: black in the top bar, darkness; white, low light (47 µmol photons m⁻² s⁻¹); yellow, 1 min of high light (1,529 µmol photons m⁻² s⁻¹). E and F, PSII and PSI photoinhibition, respectively, in the wild type and pgr1, pgr5, and pgr1 pgr5 mutant alleles under fluctuating light. After the fluctuating light treatment, the leaf discs were sandwiched between wet tissue paper and incubated in the dark for 25 min, and then F_v/F_m and Pm were measured. Their relative values are shown against the values before the treatment (biological replicates n = 7–9 ± sd). Columns with the same letters are not significantly different between genotypes (Tukey-Kramer test, P < 0.05).

Figure 5.

Enhanced electron sink capacity downstream of PSI by PpFlv protects PSI from photodamage under fluctuating light. A and B, PSII and PSI photoinhibition, respectively, in the wild type (WT), pgr5-1, WT+35S;PpFlv no.13, and pgr5-1+35S;PpFlv no.13 under fluctuating light (1 min of high light of 1,529 µmol photons m⁻² s⁻¹ and 5 min of low light of 47 µmol photons m⁻² s⁻¹; biological replicates n = 8–9 ± sd). C and D, PSII and PSI photoinhibition, respectively, in the wild type, pgr5-1, WT+35S;PpFlv no.13, and pgr5-1+35S;PpFlv no.13 under fluctuating light (1 min of high light of 1,886 µmol photons m⁻² s⁻¹ and 5 min of low light of 47 µmol photons m⁻² s⁻¹; biological replicates n = 9–10 ± sd). Leaf discs from plants dark adapted for 20 min were exposed to fluctuating light (1 min of high light of 1,529 or 1,886 µmol photons m⁻² s⁻¹ and 5 min of low light of 47 µmol photons m⁻² s⁻¹). After the fluctuating light treatment, the leaf discs were sandwiched between wet tissue paper and incubated in the dark for 25 min, and then F_v/F_m and Pm were measured. Their relative values are shown against the values before the treatment. Columns with the same letters are not significantly different between genotypes (Tukey-Kramer test, P < 0.05).