A voltage-dependent chloride channel fine-tunes photosynthesis in plants

Abstract

In natural habitats, plants frequently experience rapid changes in the intensity of sunlight. To cope with these changes and maximize growth, plants adjust photosynthetic light utilization in electron transport and photoprotective mechanisms. This involves a proton motive force (PMF) across the thylakoid membrane, postulated to be affected by unknown anion (Cl(-)) channels. Here we report that a bestrophin-like protein from Arabidopsis thaliana functions as a voltage-dependent Cl(-) channel in electrophysiological experiments. AtVCCN1 localizes to the thylakoid membrane, and fine-tunes PMF by anion influx into the lumen during illumination, adjusting electron transport and the photoprotective mechanisms. The activity of AtVCCN1 accelerates the activation of photoprotective mechanisms on sudden shifts to high light. Our results reveal that AtVCCN1, a member of a conserved anion channel family, acts as an early component in the rapid adjustment of photosynthesis in variable light environments.

Figure 1. Chloroplast localization of VCCN1 and VCCN2 in Arabidopsis.

(a) GFP and chlorophyll fluorescence imaged in leaf protoplasts from Arabidopsis wild-type plants (control) and vccn mutants transformed with AtVCCN-GFP fusions. Scale bars, 20 μm. (b,c) Localization of AtVCCN1 and AtVCCN2 in chloroplast and thylakoid subfractions by immunoblotting with an anti-GFP antibody. Chloroplasts (Clp), envelope (Env), stroma (Str), thylakoids (Thl), grana (Gr) and stroma lamellae (Str.L) were purified from leaves of plants transformed with AtVCCN1-GFP or AtVCCN2-GFP. Purity of the fractions was validated using antibodies against marker proteins for the respective compartments: chloroplast outer envelope membrane translocon complex Toc34 protein, ribulose bisphosphate carboxylase large subunit RbcL, photosystem I subunit PsaB and photosystem II subunit CP43.

Figure 2. Recombinant AtVCCN1 displays a voltage-sensitive anion channel activity in planar lipid bilayers.

(a) Current traces recorded in symmetric KCl (representative of 15 experiments) or KNO₃ (representative of 26 experiments) solutions (cis side: 100 mM, trans side: 100 mM) at indicated potentials (cis side) are shown (c, closed state; o1, o2, o3, open states). The arrow in the upper right panel indicates a subconductance state. (b) Current traces recorded before and after addition of 20 μM DIDS (left: 100 mM KCl, n=4; right: 100 mM KNO_3, n=6; see also Supplementary Fig. 6c; for amplitude histograms, see Supplementary Figs 7a,8e). The general anion channel blocker DIDS caused inhibition of the activity in accordance with AtVCCN1 being an anion channel.

Figure 3. Kinetics of proton motive force and photosynthetic acclimation on transition from dark to light.

(a) Kinetics of ECS_t (total electrochromic shift, a measure of total proton motive force size, PMF) in wild-type plants (wt), vccn1-1 knockout mutant and mutant overexpressing AtVCCN1 (oeVCCN1) were recorded at 650 μmol photons per m² per s after 30 min dark adaptation. (b) Fraction of PMF contributed by transmembrane electric-potential gradient (ΔΨ) and H⁺ concentration gradient (ΔpH) at two time points in the light. (c) H⁺ conductivity through ATP synthase (g_H⁺) was determined from ECS decay kinetics. (d) Kinetics for induction of non-photochemical quenching (NPQ) as a measure of photoprotective mechanisms were recorded during 10 min of illumination at 650 μmol photons per m² per s. (e) Photosystem II quantum yield (Φ_II) as a measure of linear electron transport was calculated from the same experiment as NPQ. Data are the means ±s.e.m. (n=5). Where not visible, error bars are smaller than the symbols. Asterisks denote a statistically significant difference between wt and mutants (Student's t-test, P<0.05).

Figure 4. Dynamics of photosynthesis and photoprotection during transitions from low to high light.

(a) Induction of non-photochemical quenching (NPQ) is slowed down in vccn1-1 but approaches wild-type (wt) levels with each transition from low light (70 μmol photons per m² per s) to high light (2,000 μmol photons per m² per s). NPQ is induced faster in oeVCCN1 and decays slower than in wt. (b) Photosystem II quantum yield (Φ_II) as a measure of electron transport is not changed in vccn1-1, but is reduced in oeVCCN1 when compared with the wt. Data are the means ±s.e.m. (n=5). Where not visible, error bars are smaller than the symbols. Asterisks denote a statistically significant difference between wt and mutants (Student's t-test, P<0.05). (c) Simplified model for sequence of events in the regulation of photosynthesis by AtVCCN1. At the onset of illumination (grey bar), electron transport-coupled H⁺ pumping into the thylakoid lumen results in the formation of proton motive force (PMF), consisting of a major transmembrane electric-potential gradient (ΔΨ) and a minor pH gradient (ΔpH). AtVCCN1 is activated by ΔΨ and partially dissipates it by anion influx. This phenomenon allows a rapid increase in ΔpH/ΔΨ ratio, which is maintained during the remaining illumination. AtVCCN1 also fine-tunes the size of total PMF (not illustrated in this model), which in turn regulates H⁺ efflux from the thylakoid lumen through ATP synthase. Electron transport and activation of NPQ are modulated by the ΔpH/ΔΨ ratio. After sudden shift from low light to high light (white bar), the ΔpH/ΔΨ ratio is changed by an increase in ΔΨ, but AtVCCN1 re-establishes a steady-state, resulting in rapid adjustment of photosynthesis.

Figure 5. Thylakoid ultrastructure.

Representative transmission electron micrographs are shown for leaf chloroplasts from 8-week-old wild-type (wt) plants and vccn1-1 mutant fixed after 16 h of darkness or 3 h after the onset of illumination (150 μmol photons per m² per s). The mutant shows consistently longer thylakoid stacks (grana) than wt in the dark state. In light conditions, the grana of vccn1-1 are also longer, but often display a curved ‘banana-like' shape (observed in 20–50% of the studied plastid sections). Scale bars, 1 μm (upper panels) and 200 nm (lower panels).