Cooperative regulation of light-harvesting complex II phosphorylation via the plastoquinol and ferredoxin-thioredoxin system in chloroplasts

Abstract

Light induces phosphorylation of photosystem II (PSII) proteins in chloroplasts by activating the protein kinase(s) via reduction of plastoquinone and the cytochrome b(6)f complex. The recent finding of high-light-induced inactivation of the phosphorylation of chlorophyll a/b-binding proteins (LHCII) of the PSII antenna in floated leaf discs, but not in vitro, disclosed a second regulatory mechanism for LHCII phosphorylation. Here we show that this regulation of LHCII phosphorylation is likely to be mediated by the chloroplast ferredoxin-thioredoxin system. We present a cooperative model for the function of the two regulation mechanisms that determine the phosphorylation level of the LHCII proteins in vivo, based on the following results: (i) Chloroplast thioredoxins f and m efficiently inhibit LHCII phosphorylation. (ii) A disulfide bond in the LHCII kinase, rather than in its substrate, may be a target component regulated by thioredoxin. (iii) The target disulfide bond in inactive LHCII kinase from dark-adapted leaves is exposed and easily reduced by external thiol mediators, whereas in the activated LHCII kinase the regulatory disulfide bond is hidden. This finding suggests that the activation of the kinase induces a conformational change in the enzyme. The active state of LHCII kinase prevails in chloroplasts under low-light conditions, inducing maximal phosphorylation of LHCII proteins in vivo. (iv) Upon high-light illumination of leaves, the target disulfide bond becomes exposed and thus is made available for reduction by thioredoxin, resulting in a stable inactivation of LHCII kinase.

Figure 1

Modulation of PSII protein phosphorylation by electron transfer inhibitors, reducing agents, and quantity of light. (A and B) Thylakoids isolated from dark-adapted leaves were phosphorylated in vitro for 15 or 30 min in the presence of the following reagents: 20 μM 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) or 10 μM 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone (DBMIB) (A) or 2 mM trans-4,5-dihydroxy-1,2-dithiane (DTTox), 2 mM DTT, or 10 mM ascorbate (B). Immunoblots present the phosphorylation level of the D1 and LHCII proteins. (C) Steady-state phosphorylation level of PSII proteins in vivo. Leaf discs were incubated in darkness (D) or illuminated for 2 h at a photon flux density of 30 (LL), 200 (GL), or 1000 (HL) μmol·m⁻²·s⁻¹. Phosphorylated PSII proteins were detected by immunoblotting with phosphothreonine antibody. P-CP43, P-D2, P-D1, and P-LHCII, phosphorylated forms of CP43, D2, D1, and LHCII proteins, respectively.

Figure 2

Effect of chloroplast thioredoxins on the phosphorylation of LHCII and D1 proteins. Thylakoids isolated from dark-adapted leaves were incubated in darkness in the presence of thioredoxin f or m at the concentrations indicated. DTT (0.2 mM) was included in the incubation medium to keep thioredoxins in their reduced form. A minor effect of DTT on thylakoid protein phosphorylation (see Fig. 3) was always subtracted from the inhibition observed with thioredoxins. After incubation for 10 min in darkness, protein phosphorylation was initiated by the addition of ATP and turning the light on. Phosphorylation levels of proteins were detected as described in Fig. 1, and the blots were quantified by scanning. Results are means ± SD, n = 2.

Figure 3

Inhibition of LHCII and D1 protein phosphorylation by thiol reagents. Thylakoids isolated from dark-adapted leaves were incubated in darkness without (white bar) and with DTT (dark gray bars) or NEM (light gray bars) for 10 min. Thereafter, in vitro protein phosphorylation was initiated. In experiments indicated by hatched bars, the thylakoid membranes were first incubated with 2 mM DTT, which was subsequently washed out by pelleting the thylakoids and resuspending them in the same buffer either without thiol reagents (hatched white bars) or with various concentrations of NEM (hatched gray bars), and incubated in darkness for 10 min before the phosphorylation assay. The concentrations of DTT and NEM are indicated under (or above) the bars. The phosphorylation levels of the D1 and LHCII proteins were analyzed as described in Fig. 2. Results are means ± SD, n = 2–4.

Figure 4

Activation of LHCII kinase decreases the sensitivity of LHCII phosphorylation to thiol reagents. Thylakoids isolated from dark-adapted leaves were incubated for 5 min either in darkness (gray bars) or in light (striped bars). Thiol reagents were subsequently added, and incubation continued for 5 more min before initiation of the phosphorylation assay. Activation of the kinase and phosphorylation of thylakoid proteins were also conducted in total darkness in the presence of NADPH and ferredoxin (hatched bars). The sequential order of the addition of thiol reagents and NADPH + ferredoxin is indicated in the figure. In all cases, the thylakoid protein phosphorylation was initiated by the addition of ATP. Phosphorylation levels of LHCII and D1 proteins were identical in the control assays without thiol reagents, regardless of the activation of the kinase in darkness or in light before the initiation of phosphorylation. DTT (2 mM) and thioredoxins (2 μM) were used in the assay medium. The phosphorylation levels of the D1 and LHCII proteins were analyzed as described in Fig. 2. Results are means ± SD, n = 2–4.

Figure 5

Model for the regulation of LHCII phosphorylation in vivo. LHCII kinase is inactive in darkness because of the oxidized state of the Q_o site in the cytochrome b₆/f complex. Light initiates the electron transport in thylakoid membranes, resulting in activation of the LHCII kinase via binding of plastoquinol to the cytochrome b₆/f complex. This activation induces a conformational change in the LHCII kinase with a concomitant burial and protection of the target regulatory disulfide bond against reduction. Such an active state prevails in chloroplasts under low light conditions. In high light, the regulatory disulfide bond becomes exposed and reduced via the ferredoxin–thioredoxin system, resulting in an inactive state of LHCII kinase despite the reduced state of the Q_o site in cytochrome b₆/f complex. The thicknesses of the black arrows indicate the amount of reduced thioredoxins under various light conditions.