Restoration of rapidly reversible photoprotective energy dissipation in the absence of PsbS protein by enhanced DeltapH

Abstract

Variations in the light environment require higher plants to regulate the light harvesting process. Under high light a mechanism known as non-photochemical quenching (NPQ) is triggered to dissipate excess absorbed light energy within the photosystem II (PSII) antenna as heat, preventing photodamage to the reaction center. The major component of NPQ, known as qE, is rapidly reversible in the dark and dependent upon the transmembrane proton gradient (ΔpH), formed as a result of photosynthetic electron transport. Using diaminodurene and phenazine metasulfate, mediators of cyclic electron flow around photosystem I, to enhance ΔpH, it is demonstrated that rapidly reversible qE-type quenching can be observed in intact chloroplasts from Arabidopsis plants lacking the PsbS protein, previously believed to be indispensible for the process. The qE in chloroplasts lacking PsbS significantly quenched the level of fluorescence when all PSII reaction centers were in the open state (F(o) state), protected PSII reaction centers from photoinhibition, was modulated by zeaxanthin and was accompanied by the qE-typical absorption spectral changes, known as ΔA(535). Titrations of the ΔpH dependence of qE in the absence of PsbS reveal that this protein affects the cooperativity and sensitivity of the photoprotective process to protons. The roles of PsbS and zeaxanthin are discussed in light of their involvement in the control of the proton-antenna association constant, pK, via regulation of the interconnected phenomena of PSII antenna reorganization/aggregation and hydrophobicity.

FIGURE 1.

A, comparison of 9-AA fluorescence quenching and chlorophyll fluorescence quenching in control and DAD-treated intact Arabidopsis chloroplasts. Chlorophyll fluorescence quenching and 9-AA fluorescence quenching (9-aa) during a 5-min light/5-min dark cycle in control and DAD-treated WT(z) and npq4(z) chloroplasts. Light intensity was 350 μmol of photons m⁻² s⁻¹. B, NPQ during a 5-min light/5-min dark cycle in control WT(z) (white triangles), control npq4(z) (black triangles), DAD-treated WT(z) (white squares), and DAD-treated npq4(z) (black squares) chloroplasts. Light intensity was 350 μmol of photons m⁻² s⁻¹, data are average of three independent experiments ± S.E.

FIGURE 2.

Photochemical quenching (qP) during a 5-min light/5-min dark cycle in control WT(z) (white triangles), control npq4(z) (black triangles), DAD-treated WT(z) (white squares), and DAD-treated npq4(z) (black squares) chloroplasts. Light intensity was 350 μmol of photons m⁻² s⁻¹, data are average of three independent experiments ± S.E.

FIGURE 3.

A, NPQ during a 5-min light/5-min dark cycle in PMS-treated WT(z) (white circles), PMS-treated npq4(z) (black circles), DAD/DCMU-treated WT(z) (white squares), DAD/DCMU-treated npq4(z) (black squares), DAD/nigericin-treated WT(z) (white triangles) and DAD/nigericin-treated npq4(z) (white triangles) chloroplasts. Light intensity was 350 μmol of photons m⁻² s⁻¹, data are average of three independent experiments ± S.E. B, effect of actinic (635 nm) and far-red (710 nm) illumination intensity on the level of 9-AA fluorescence quenching in npq4(z) chloroplasts in the presence and absence of DAD. Data are average of three independent experiments ± S.E.

FIGURE 4.

Effect of DAD on the qE-related absorption changes in the Soret region. A, kinetics of 535 nm absorption change induced during a 5-min light/5-min dark cycle in control and DAD-treated WT(z) and npq4(z) chloroplasts, the reference wavelength used was 565 nm. Light-minus-dark recovery absorption difference spectra in control and DAD-treated (B) WT(z) chloroplasts (C) npq4(z) chloroplasts. Light intensity was 350 μmol of photons m⁻² s⁻¹.

FIGURE 5.

Titrations of qE versus ΔpH in control WT(z) (red triangles), DAD-treated WT(z) (red circles), control WT(v) (blue triangles), DAD-treated WT(v) (blue circles), control npq4(z) (white triangles), DAD treated npq4(z) (white circles), control npq4(v) (black triangles), and DAD-treated npq4(v) (black circles) chloroplasts. Light intensity was varied between 0 and 350 μmol of photons m⁻² s⁻¹ to achieve different values of ΔpH. Right pointing arrow indicates the enhancement in ΔpH observed above the physiological maximum (dashed line) in the presence of DAD. Data are average of three independent experiments ± S.E. Potential systematic error in estimation of absolute values of ΔpH based on 1000% uncertainty (or 10 times variation) in the total volume of the lumen (1 pH unit) is indicated by the bidirectional arrow above the plots.

FIGURE 6.

Effect of lowering the pH (at time point shown by arrow marked +HCL) of the bulk medium on qE relaxation in the dark following 5 min illumination to induce maximum qE in DAD-treated (A) WT(z) and (B) npq4(z) chloroplasts. Light intensity in all cases was 350 μmol of photons m⁻² s⁻¹. Data are average of three independent experiments ± S.E.

FIGURE 7.

A, titrations of qE maintained in the dark versus bulk pH in DAD-treated WT(z) (red circles), DAD-treated WT(v) (blue circles), DAD-treated npq4(z) (white circles), and DAD-treated npq4(v) (black circles) chloroplasts. Solid lines are fits of the data presented in Fig. 5 to show close agreement between the two sets of data. B, effect of lowering the pH of the bulk medium on qP levels in the dark following 5 min illumination to induce maximum qE in DAD-treated WT(z) (white circles) and npq4(z) (black circles) chloroplasts. Shown for comparison is the qP level in the dark following 5 min illumination where only the lumen pH level fell below 8.0 (due to ΔpH formation) during illumination, DAD-treated WT(z) (white squares) and npq4(z) (black squares) chloroplasts.