Dark-adapted spinach thylakoid protein heterogeneity offers insights into the photosystem II repair cycle

In higher plants, thylakoid membrane protein complexes show lateral heterogeneity in their distribution: photosystem (PS) II complexes are mostly located in grana stacks, whereas PSI and adenosine triphosphate (ATP) synthase are mostly found in the stroma-exposed thylakoids. However, recent research has revealed strong dynamics in distribution of photosystems and their light harvesting antenna along the thylakoid membrane. Here, the dark-adapted spinach (Spinacia oleracea L.) thylakoid network was mechanically fragmented and the composition of distinct PSII-related proteins in various thylakoid subdomains was analyzed in order to get more insights into the composition and localization of various PSII subcomplexes and auxiliary proteins during the PSII repair cycle. Most of the PSII subunits followed rather equal distribution with roughly 70% of the proteins located collectively in the grana thylakoids and grana margins; however, the low molecular mass subunits PsbW and PsbX as well as the PsbS proteins were found to be more exclusively located in grana thylakoids. The auxiliary proteins assisting in repair cycle of PSII were mostly located in stroma-exposed thylakoids, with the exception of THYLAKOID LUMEN PROTEIN OF 18.3 (TLP18.3), which was more evenly distributed between the grana and stroma thylakoids. The TL29 protein was present exclusively in grana thylakoids. Intriguingly, PROTON GRADIENT REGULATION5 (PGR5) was found to be distributed quite evenly between grana and stroma thylakoids, whereas PGR5-LIKE PHOTOSYNTHETIC PHENOTYPE1 (PGRL1) was highly enriched in the stroma thylakoids and practically missing from the grana cores. This article is part of a Special Issue entitled: Photosynthesis Research for Sustainability: Keys to Produce Clean Energy.     

Photoprotection in an ecological context: the remarkable complexity of thermal energy dissipation

This review places photoprotection into the context of ecology and species diversity. The focus is on photoprotection via the safe removal - as thermal energy - of excess solar energy absorbed by the light collecting system, which counteracts the formation of reactive oxygen species. An update on the surprisingly complex, multiple variations of thermal energy dissipation is presented, placing these different forms into ecological and genetic contexts. Zeaxanthin-facilitated, flexible thermal dissipation associated with the PsbS protein and controlled by the trans-thylakoid pH gradient apparently occurs ubiquitously in plants, and can become sustained (and thus less flexible) at low temperatures. Long-lived, slow-growing plants with low intrinsic capacities for photosynthesis have greater capacities for this flexible dissipation than short-lived, fast-growing species. Furthermore, potent, but inflexible (zeaxanthin-facilitated) thermal dissipation, prominent in evergreen species under prolonged environmental stress, is characterized with respect to the involvement of photosystem II core rearrangement and/or degradation as well as the absence of control by trans-thylakoid pH and, possibly, PsbS. A role of PsbS-related proteins in photoprotection is discussed.     

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

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.     

Winter down-regulation of intrinsic photosynthetic capacity coupled with up-regulation of Elip-like proteins and persistent energy dissipation in a subalpine forest

Overwintering, sun-exposed and photosynthetically inactive evergreens require powerful photoprotection. The goal of this study was to seasonally characterize photosynthesis and key proteins/components involved in electron transport and photoprotection. Maximal photosystem II (PSII) efficiency and photosynthetic capacity, amounts of zeaxanthin (Z), antheraxanthin (A), pheophytin and proteins (oxygen-evolving 33 kDa protein (OEC), PSII core protein D1 and subunit S (PsbS) protein, and members of the early light-inducible protein (Elip) family) were assessed in five conifer species at high altitude and in ponderosa pine (Pinus ponderosa) at moderate altitude during summer and winter. Relative to summer, winter down-regulation of photosynthetic capacity and loss of PSII efficiency at the high-altitude sites were paralleled by decreases in OEC, D1, and pheophytin; massive nocturnal retention of (Z + A) and up-regulation of two to four proteins cross-reactive with anti-Elip antibodies; and no change in PsbS amount. By contrast, ponderosa pine at moderate altitude exhibited no down-regulation of photosynthetic capacity, smaller depressions in PSII efficiency, and less up-regulation of Elip family members. These results support a function for members of the Elip family in the acclimation of sun-exposed needles that down-regulate photosynthesis during winter. A possible role in sustained photoprotection is considered.     

Corrections to current approaches used to calculate energy partitioning in photosystem 2

We analyzed several approaches dealing with the components of non-photochemical energy dissipation and introduced improved versions of the equations used to calculate this parameter. The usage of these formulae depends on the conditions of the sample (acclimation to dark or irradiation, presence or absence of the “actinic light”). The parameter known as “excess” cannot be used as a component of energy partitioning. In reality, this parameter reflects the differences between potential and actual quantum yields of photochemistry.     

Light-harvesting antenna composition controls the macrostructure and dynamics of thylakoid membranes in Arabidopsis

We characterized a set of Arabidopsis mutants deficient in specific light-harvesting proteins, using freeze-fracture electron microscopy to probe the organization of complexes in the membrane and confocal fluorescence recovery after photobleaching to probe the dynamics of thylakoid membranes within intact chloroplasts. The same methods were used to characterize mutants lacking or over-expressing PsbS, a protein related to light-harvesting complexes that appears to play a role in regulation of photosynthetic light harvesting. We found that changes in the complement of light-harvesting complexes and PsbS have striking effects on the photosystem II macrostructure, and that these effects correlate with changes in the mobility of chlorophyll proteins within the thylakoid membrane. The mobility of chlorophyll proteins was found to correlate with the extent of photoprotective non-photochemical quenching, consistent with the idea that non-photochemical quenching involves extensive re-organization of complexes in the membrane. We suggest that a key feature of the physiological function of PsbS is to decrease the formation of ordered semi-crystalline arrays of photosystem II in the low-light state. Thus the presence of PsbS leads to an increase in the fluidity of the membrane, accelerating the re-organization of the photosystem II macrostructure that is necessary for induction of non-photochemical quenching.     

Mechanisms shaping the synergism of zeaxanthin and PsbS in photoprotective energy dissipation in the photosynthetic apparatus of plants

Safe operation of photosynthesis is vital to plants and is ensured by the activity of processes protecting chloroplasts against photo-damage. The harmless dissipation of excess excitation energy is considered to be the primary photoprotective mechanism and is most effective in the combined presence of PsbS protein and zeaxanthin, a xanthophyll accumulated in strong light as a result of the xanthophyll cycle. Here we address the problem of specific molecular mechanisms underlying the synergistic effect of zeaxanthin and PsbS. The experiments were conducted with Arabidopsis thaliana, using wild-type plants, mutants lacking PsbS (npq4), and mutants affected in the xanthophyll cycle (npq1), with the application of molecular spectroscopy and imaging techniques. The results lead to the conclusion that PsbS interferes with the formation of densely packed aggregates of thylakoid membrane proteins, thus allowing easy exchange and incorporation of xanthophyll cycle pigments into such structures. It was found that xanthophylls trapped within supramolecular structures, most likely in the interfacial protein region, determine their photophysical properties. The structures formed in the presence of violaxanthin are characterized by minimized dissipation of excitation energy. In contrast, the structures formed in the presence of zeaxanthin show enhanced excitation quenching, thus protecting the system against photo-damage.     

Winter acclimation of PsbS and related proteins in the evergreen Arctostaphylos uva-ursi as influenced by altitude and light environment

The evergreen groundcover bearberry (Arctostaphylos uva-ursi [L.] Sprengel) was characterized over two successive years (2002-2004) from both sun-exposed and shaded sites at a montane ponderosa pine and subalpine forest community of 1900- and 2800-m-high altitudes, respectively. During summer, photosynthetic capacities and pre-dawn photosystem II (PSII) efficiency were similarly high in all four populations, and in winter, only the sun-exposed and shaded populations at 2800 m exhibited complete down-regulation of photosynthetic oxygen evolution capacity and consistent sustained down-regulation of PSII efficiency. This photosynthetic down-regulation at high altitude involved a substantial decrease in PSII components [pheophytin, D1 protein, oxygen evolving complex ([OEC)], a strong up-regulation of several anti-early-light-inducible protein (Elip)- and anti-high-light-inducible protein (Hlip)-reactive bands and a warm-sustained retention of zeaxanthin and antheraxanthin (Z + A). PsbS, the protein modulating the rapid engagement and disengagement of Z +A in energy dissipation, exhibited its most pronounced winter increases in the shade at 1900 m, and thus apparently assumes a greater role in providing rapidly reversible zeaxanthin-dependent photoprotection during winter when light becomes excessive in the shaded population, which remains photosynthetically active. It is attractive to hypothesize that PsbS relatives (Elips/Hlips) may be involved in sustained zeaxanthin-dependent photoprotection under the more extreme winter conditions at 2800 m.