PsbS-dependent enhancement of feedback de-excitation protects photosystem II from photoinhibition

Abstract

Feedback de-excitation (qE) regulates light harvesting in plants to prevent inhibition of photosynthesis when light absorption exceeds photosynthetic capacity. Although the mechanism of qE is not completely understood, it is known to require a low thylakoid lumen pH, de-epoxidized xanthophylls, and the photosystem II protein PsbS. During a short-term 4-h exposure to excess light, three PsbS- and qE-deficient Arabidopsis thaliana mutants that differed in xanthophyll composition were more photoinhibited than the wild type. The extent of photoinhibition was the same in all of the mutants, suggesting that qE capacity rather than xanthophyll composition is critical for photoprotection in short-term high light, in contrast to longer-term high light conditions (days) when additional antioxidant roles of specific xanthophylls are evident. Plants with a 2-fold increase in qE capacity were generated by overexpression of PsbS, demonstrating that the level of PsbS limits the qE capacity in wild-type Arabidopsis. These results are consistent with the idea that variations in PsbS expression are responsible for species-specific and environmentally induced differences in qE capacity observed in nature. Furthermore, plants with higher qE capacity were more resistant to photoinhibition than the wild type. Increased qE was associated with decreased photosystem II excitation pressure and changes in the fractional areas of chlorophyll a fluorescence lifetime distributions, but not the lifetime centers, suggesting that qE protects from photoinhibition by preventing overreduction of photosystem II electron acceptors. Engineering of qE capacity by PsbS overexpression could potentially yield crop plants that are more resistant to environmental stress.

Fig 1.

Characteristics of LL (150 μmol photons m⁻²⋅s⁻¹)-grown wild-type and mutant Arabidopsis plants before, during, and after treatment with HL (1,700 μmol photons m⁻²⋅s⁻¹). (A) qE. F_m′ was measured at the indicated time points in LL or HL, and F_v and F_m were measured after 15 min in the dark. qE was calculated as (F_m − F_m′)/F_m′. Data are shown as the means ± SE (n ≥ 12). (B) Xanthophyll cycle de-epoxidation state ([A+Z]/[V+A+Z]). Data are shown as the means ± SE (n = 4). (C) PS II photochemical efficiency (F_v/F_m). Data are shown as the means ± SE (n ≥ 12).

Fig 2.

Apparent quantum yield of oxygen evolution [Φ(O₂)] before, during, and after HL treatment of wild type and npq4. Data are shown as the means ± SE (n = 4). At the 1- and 4-h HL time points, the wild type and npq4 differ significantly (P < 0.05, t test).

Fig 3.

DNA, RNA, and protein analyses in wild-type plants (wt), psbS-overexpressing lines (L5 and L17), and the npq4 mutant. (A) DNA gel blot analysis. Genomic DNA (8 μg) was digested with XhoI and hybridized with a psbS probe. (B) RNA gel blot analysis. Total RNA (10 μg) was hybridized with a psbS probe. rRNA was visualized by staining of the blot with methylene blue. (C) Immunoblot analysis of PsbS and D1.

Fig 4.

NPQ induction curves in wild type, L5, L17, and npq4. Plants were dark-adapted overnight before exposure to actinic light of 2,000 μmol photons m⁻²⋅s⁻¹. Data are shown as the means ± SE (n = 3).

Fig 5.

Characteristics of LL-grown wild type, L5, L17, and npq4 plants before, during, and after HL treatment. Data are shown as the means ± SE (n = 15). (A) qE. F_m′ was measured at the indicated time points in LL or HL, and F_v and F_m were measured after 15 min in the dark. qE was calculated as (F_m − F_m′)/F_m′. F_m′ could not be measured after the overnight dark periods, so no qE values were calculated for the O/N D and R O/N time points. (B) Xanthophyll cycle de-epoxidation state ([A+Z]/[V+A+Z]). (C) PS II photochemical efficiency (F_v/F_m). O/N D, overnight dark; R, recovery in LL; R O/N, recovery overnight in the dark.

Fig 6.

Light response curves for NPQ and PS II excitation pressure. Chlorophyll fluorescence was measured during exposure of attached rosette leaves to an increasing series of PFDs for 5 min each. Data are shown as the means ± SE (n = 3). (A) NPQ. (B) PS II excitation pressure (1-qP).