PsbS plays a major role in activating the photoprotection mechanism known as "non-photochemical quenching," which dissipates chlorophyll excited states exceeding the capacity for photosynthetic electron transport. PsbS activity is known to be triggered by low lumenal pH. However, the molecular mechanism by which this subunit regulates light harvesting efficiency is still unknown. Here we show that PsbS controls the association/dissociation of a five-subunit membrane complex, composed of two monomeric Lhcb proteins (CP29 and CP24) and the trimeric LHCII-M. Dissociation of this supercomplex is indispensable for the onset of non-photochemical fluorescence quenching in high light, strongly suggesting that protein subunits catalyzing the reaction of heat dissipation are buried into the complex and thus not available for interaction with PsbS. Consistently, we showed that knock-out mutants on two subunits participating to the B4C complex were strongly affected in heat dissipation. Direct observation by electron microscopy and image analysis showed that B4C dissociation leads to the redistribution of PSII within grana membranes. We interpreted these results to mean that the dissociation of B4C makes quenching sites, possibly CP29 and CP24, available for the switch to an energy-quenching conformation. These changes are reversible and do not require protein synthesis/degradation, thus allowing for changes in PSII antenna size and adaptation to rapidly changing environmental conditions. Photosynthetic reaction centers exploit solar energy to drive electrons from water to NADP ϩ

When light absorbed by plants exceeds the capacity of photosynthesis, the xanthophyll violaxanthin is reversibly de-epoxidized to zeaxanthin in the so-called xanthophyll cycle. Zeaxanthin plays a key role in the protection of photosynthetic organisms against excess light, by promoting rapidly reversible (qE) and long-term (qI) quenching of excited chlorophylls, and preventing lipid oxidation. The photoprotective role of zeaxanthin, either free or bound to light-harvesting complexes (Lhcs), has been investigated by using mutants lacking Chl b (ch1) and/or specific xanthophyll species (npq, lut2). The ch1 mutation causes (1) the absence of Lhcb proteins; (2) strong reduction of the feedback de-excitation (qE); and (3) accumulation of xanthophylls as free pigments into thylakoids. Ch1 mutants showed extreme sensitivity to photo-oxidative stress in high light, due to higher singlet oxygen (¹O₂) release. The double mutant ch1npq1 was more sensitive to photo-oxidation than ch1, showing that zeaxanthin does protect lipids even when free in the membrane. Nevertheless, lack of zeaxanthin had a much stronger impact on the level of lipid peroxidation in Lhcs-containing plants (WT vs npq1) with respect to Lhc-less plants (ch1 vs ch1npq1), implying that its protective effect is enhanced by interaction with antenna proteins. It is proposed that the antioxidant capacity of zeaxanthin is empowered in the presence of PSII-LHCs-Zea complexes, while its effect on enhancement of qE only provides a minor contribution. Comparison of the sensitivity of WT vs npq1 plants to exogenous ¹O₂ suggests that besides the scavenging of ¹O₂, at least one additional mechanism is involved in chloroplast photoprotection.

Xanthophylls (oxygenated carotenoids) are essential components of the plant photosynthetic apparatus, where they act in photosystem assembly, light harvesting, and photoprotection. Nevertheless, the specific function of individual xanthophyll species awaits complete elucidation. In this work, we analyze the photosynthetic phenotypes of two newly isolated Arabidopsis mutants in carotenoid biosynthesis containing exclusively ␣-branch (chy1chy2lut5) or ␤-branch (chy1chy2lut2) xanthophylls. Both mutants show complete lack of qE, the rapidly reversible component of nonphotochemical quenching, and high levels of photoinhibition and lipid peroxidation under photooxidative stress. Both mutants are much more photosensitive than npq1lut2, which contains high levels of viola-and neoxanthin and a higher stoichiometry of light-harvesting proteins with respect to photosystem II core complexes, suggesting that the content in light-harvesting complexes plays an important role in photoprotection. In addition, chy1chy2lut5, which has lutein as the only xanthophyll, shows unprecedented photosensitivity even in low light conditions, reduced electron transport rate, enhanced photobleaching of isolated LHCII complexes, and a selective loss of CP26 with respect to chy1chy2lut2, highlighting a specific role of ␤-branch xanthophylls in photoprotection and in qE mechanism. The stronger photosystem II photoinhibition of both mutants correlates with the higher rate of singlet oxygen production from thylakoids and isolated lightharvesting complexes, whereas carotenoid composition of photosystem II core complex was not influential. In depth analysis of the mutant phenotypes suggests that ␣-branch (lutein) and ␤-branch (zeaxanthin, violaxanthin, and neoxanthin) xanthophylls have distinct and complementary roles in antenna protein assembly and in the mechanisms of photoprotection.Carotenoids are a group of C40 terpenoid compounds with a wide distribution in several biological taxa, ranging from archaea to bacteria, fungi, algae, and higher plants. Xanthophylls form a subgroup of oxygenated carotenoids, whose importance in the oxygenic photosynthesis is well known. Xanthophylls play essential roles in higher plant photosynthesis, as components of the photosynthetic apparatus of the chloroplast. In higher plants, ␤-carotene binds to reaction center subunits of both photosystem I (PSI) 4 and II (PSII), whereas xanthophylls are both accessory pigments and structural elements of light-harvesting complexes (Lhcs). Together with ␤-carotene, they act both as chromophores, absorbing light energy that is used in photosynthetic electron transport, and as photoprotectants of the photosynthetic apparatus from excess light and from the reactive oxygen species (ROS) that are generated during oxygenic photosynthesis. A remarkable characteristic of higher plant xanthophylls is that they show very similar spectral properties in the visible region. This evidence is apparently incoherent with the high conservation of their relative abundance across a range of pla...

The aba4-1 mutant completely lacks neoxanthin but retains all other xanthophyll species. The missing neoxanthin in lightharvesting complex (Lhc) proteins is compensated for by higher levels of violaxanthin, albeit with lower capacity for photoprotection compared with proteins with wild-type levels of neoxanthin. Detached leaves of aba4-1 were more sensitive to oxidative stress than the wild type when exposed to high light and incubated in a solution of photosensitizer agents. Both treatments caused more rapid pigment bleaching and lipid oxidation in aba4-1 than wild-type plants, suggesting that neoxanthin acts as an antioxidant within the photosystem II (PSII) supercomplex in thylakoids. While neoxanthin-depleted Lhc proteins and leaves had similar sensitivity as the wild type to hydrogen peroxide and singlet oxygen, they were more sensitive to superoxide anions. aba4-1 intact plants were not more sensitive than the wild type to high-light stress, indicating the existence of compensatory mechanisms of photoprotection involving the accumulation of zeaxanthin. However, the aba4-1 npq1 double mutant, lacking zeaxanthin and neoxanthin, underwent stronger PSII photoinhibition and more extensive oxidation of pigments than the npq1 mutant, which still contains neoxanthin. We conclude that neoxanthin preserves PSII from photoinactivation and protects membrane lipids from photooxidation by reactive oxygen species. Neoxanthin appears particularly active against superoxide anions produced by the Mehler's reaction, whose rate is known to be enhanced in abiotic stress conditions.

BackgroundThe utilization of biomass from microalgae for biofuel production is one of the key elements for the development of a sustainable and secure energy supply. Among the different microalgae, Chlorella species are of interest because of their high productivity, high lipid content, and resistance to the high light conditions typical of photobioreactors. However, the economic feasibility of growing algae at an industrial scale is yet to be realized, in part because of biological constraints that limit biomass yield. A key issue is the inefficient use of light due to uneven light distribution, and the dissipation of excess absorbed light as heat. The successful implementation of biofuel production facilities requires the development of algal strains with enhanced light use efficiency in photobioreactors. Such domestication strategies include decreasing the absorption cross section in order to enhance light penetration, increasing the size of metabolic sinks per chlorophyll and minimizing feedback energy dissipation.ResultsIn this work we applied random mutagenesis and phenotypic selection to the thermotolerant, fast-growing Chlorella species, C. sorokiniana. Truncated antenna mutants (TAMs) were selected that exhibited a lower fluorescence yield than the wild-type (WT) strain. Six putatively interesting mutants were selected by high throughput fluorescence video imaging, two of which, TAM-2 and TAM-4, were found to have approximately half the chlorophyll content per cell and LHCII complement per PSII with respect to the WT. In batch culture, TAM-2 showed an increased photon use efficiency, yielding a higher Pmax at saturating irradiances with respect to the WT. Cultivation of TAM-2 in both laboratory-scale and outdoor photobioreactors showed higher productivity than WT, with a 30% higher biomass yield in dense cell suspensions typical of industrial photobioreactors.ConclusionsThese results suggest that generation of mutants with low chlorophyll content can significantly improve the light-to-biomass conversion efficiency of C. sorokiniana under mass culture conditions. However, owing to the lack of sexual reproduction in this species, the presence of additional mutations might affect growth rate, suggesting that selection should include evaluation of multiple independent mutants for each desired phenotype.Electronic supplementary materialThe online version of this article (doi:10.1186/s13068-014-0157-z) contains supplementary material, which is available to authorized users.