The Arabidopsis thylakoid chloride channel ClCe regulates ATP availability for light-harvesting complex II protein phosphorylation

Abstract

Coping with changes in light intensity is challenging for plants, but well-designed mechanisms allow them to acclimate to most unpredicted situations. The thylakoid K⁺/H⁺ antiporter KEA3 and the voltage-dependent Cl^- channel VCCN1 play important roles in light acclimation by fine-tuning electron transport and photoprotection. Good evidence exists that the thylakoid Cl^- channel ClCe is involved in the regulation of photosynthesis and state transitions in conditions of low light. However, a detailed mechanistic understanding of this effect is lacking. Here we report that the ClCe loss-of-function in Arabidopsis thaliana results in lower levels of phosphorylated light-harvesting complex II (LHCII) proteins as well as lower levels of the photosystem I-LHCII complexes relative to wild type (WT) in low light conditions. The phosphorylation of the photosystem II core D1/D2 proteins was less affected either in low or high light conditions. In low light conditions, the steady-state levels of ATP synthase conductivity and of the total proton flux available for ATP synthesis were lower in ClCe loss-of-function mutants, but comparable to WT at standard and high light intensity. As a long-term acclimation strategy, expression of the ClCe gene was upregulated in WT plants grown in light-limiting conditions, but not in WT plants grown in standard light even when exposed for up to 8 h to low light. Taken together, these results suggest a role of ClCe in the regulation of the ATP synthase activity which under low light conditions impacts LHCII protein phosphorylation and state transitions.

Keywords: ATP synthase; Arabidopsis thaliana; chloride channel (ClC); light-harvesting complex II (LHCII); low light acclimation; photosystem II; protein phosphorylation; proton motive force (PMF).

Figure 1

Phospho-Thr immunoblot of thylakoids from plants grown in short-day conditions. Wild-type plants (WT) and the clce mutant were grown using 8 h light (120 µmol photons m⁻² s⁻¹)/16 h dark cycles for 5 weeks. Overnight dark-acclimated plants were exposed to light at 15 µmol photons m^-2 s^-1 (LL1) for 2 h, followed by 1000 µmol photons m^-2 s^-1 (HL) for 2 h and back to 15 µmol photons m^-2 s^-1 (LL2) for 2 h. Thylakoids were isolated from leaves harvested at the given time points and light intensities. All thylakoids were loaded on equal chlorophyll basis of 0.5 µg. 100%, 50% and 25% WT sample after LL2 2h was used as loading control.

Figure 2

Levels of thylakoid protein complexes during long- and short-day conditions. Wild-type plants and mutants were grown using 16 h light (100 µmol photons m⁻² s⁻¹)/8 h dark cycles (long day) for 3 weeks (A, C, E) or 8 h light (120 µmol photons m⁻² s⁻¹)/16 h dark cycles (short day) for 5 weeks (B, D, F). (A, B) Large-pore blue-native gels (lpBN-PAGE) of thylakoids solubilized with 1% (w/v) n-dodecyl-β-D-maltoside (DM). (C, D) lpBN-PAGE of thylakoids solubilized with 1% (w/v) digitonin (DIG). (E, F) p-LHCB2 immunoblot from lpBN-PAGE of thylakoids solubilized with 1% (w/v) digitonin (DIG). Thylakoids were loaded on equal chlorophyll basis of 4 µg (A–D) and 1 µg (E, F). WT – Col-0, c – clce-2, k– kea3-1, v – vccn1-1, ck – clce-2kea3-1, cv – clce-2vccn1-1, kv – kea3-1vccn1-1, and kvc – kea3-1vccn1-1clce-2.

Figure 3

Thylakoid protein content and phosphorylation levels during long- and short-day conditions. Wild-type plants and mutants were grown using 16 h light (100 µmol photons m⁻² s⁻¹)/8 h dark cycles (long day) for 3 weeks or 8 h light (120 µmol photons m⁻² s⁻¹)/16 h dark cycles (short day) for 5 weeks. Immunoblots from thylakoids separated by SDS-PAGE and probed with LHCB1, p-LHCB1, LHCB2, p-LHCB2, p-Thr, STN7, CP47 (PSII) and PsaB (PSI) antibodies are shown. All thylakoids were loaded on equal chlorophyll basis of 0.5 µg, except 3 µg for STN7. 150%, 100%, 50% and 25% of WT samples were used as loading controls. WT – Col-0, c – clce-2, k– kea3-1, v – vccn1-1, ck – clce-2kea3-1, cv – clce-2vccn1-1, kv – kea3-1vccn1-1, and kvc – kea3-1vccn1-1clce-2.

Figure 4

Kinetics of proton motive force and H⁺ conductivity through ATP synthase. Electrochromic shift measurements (ECS) were performed on 30 min dark-acclimated wild-type and mutant plants grown in short-day conditions (120 µmol photons m⁻² s⁻¹) and illuminated at the indicated intensities. Total proton motive force (PMF) and ATP synthase H⁺ conductivity (g_H ⁺) were calculated from ECS decay kinetics as described in Methods. The plotted data are means ± S.E.M. (n = 6 plants). WT – Col-0, c – clce-2, k– kea3-1, v – vccn1-1, and kvc – kea3-1vccn1-1clce-2. Induction kinetics for the first 210 illumination are presented in Supplementary Figure 4 .

Figure 5

Steady-state proton motive force, H⁺ conductivity and H⁺ flux through the ATP synthase. Electrochromic shift measurements (ECS) were performed on 30 min dark-acclimated wild-type (WT) and mutant plants grown in short-day conditions (120 µmol photons m⁻² s⁻¹) and illuminated for 15 min at the indicated intensities. Total proton motive force (PMF), ATP synthase H⁺ conductivity (g_H ⁺) and H⁺ flux (v_H ⁺) were calculated from ECS decay kinetics as described in Methods. The plotted data were obtained from Figure 4 and represent means ± S.E.M. (n = 6 plants). WT – Col-0, c – clce-2, k– kea3-1, v – vccn1-1, and kvc – kea3-1vccn1-1clce-2. Different letters denote statistically significant differences among genotypes according to ANOVA (P< 0.05).

Figure 6

Fold change in the expression of CLCe, KEA3 and VCCN1 genes. (A) Wild type plants (WT, front bars) and clce mutants (bars behind, lighter color) were grown in short-day conditions with 8 h light (120 µmol photons m⁻² s⁻¹)/16 h dark for 6 weeks. Total RNA was isolated after 16-h dark and 3 h exposure to low light (LL, 15 µmol photons m⁻² s ⁻¹), growth light (GL, 120 µmol photons m⁻² s ⁻¹) or high light (HL, 650 µmol photons m⁻² s⁻¹), and changes in transcript abundance were determined by quantitative RT-PCR analysis. The expression of CLCe, KEA3 and VCCN1 genes was calculated relative to two reference genes and normalized to expression in samples collected after the 16-h of dark period. Data are the means ± S.E.M. (n = 4). Asterisks denote a statistically significant difference between each of the light treatments and the dark control according to Student’s t-test (P< 0.05). (B) WT and clce plants were grown in short-day LL condition (15 µmol photons m⁻² s⁻¹) for 8 months. Total RNA was isolated after 16-h dark and 3 h exposure to LL and changes in transcript abundance were determined by quantitative RT-PCR analysis. The relative expression of CLCe, KEA3 and VCCN1 genes was calculated as in (A). Data are the means ± S.E.M. (n = 4).