Stefano Cazzaniga

Light-induced dissociation of an antenna hetero-oligomer is needed for non-photochemical quenching induction.

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.

The Arabidopsis aba4-1 Mutant Reveals a Specific Function for Neoxanthin in Protection against Photooxidative Stress

The aba4-1 mutant completely lacks neoxanthin but retains all other xanthophyll species. The missing neoxanthin in light-harvesting 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 Mehlers reaction, whose rate is known to be enhanced in abiotic stress conditions.

Enhanced photoprotection by protein-bound vs free xanthophyll pools: a comparative analysis of chlorophyll b and xanthophyll biosynthesis mutants.

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.

Different Roles of α- and β-Branch Xanthophylls in Photosystem Assembly and 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 α-branch (chy1chy2lut5) or β-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 β-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 light-harvesting complexes, whereas carotenoid composition of photosystem II core complex was not influential. In depth analysis of the mutant phenotypes suggests that α-branch (lutein) and β-branch (zeaxanthin, violaxanthin, and neoxanthin) xanthophylls have distinct and complementary roles in antenna protein assembly and in the mechanisms of photoprotection.

The occurrence of the psbs gene product in chlamydomonas reinhardtii and in other photosynthetic organisms and its correlation with energy quenching

To avoid photodamage, photosynthetic organisms have developed mechanisms to evade or dissipate excess energy. Lumen overacidification caused by light‐induced electron transport triggers quenching of excited chlorophylls and dissipation of excess energy into heat. In higher plants participation of the PsbS protein as the sensor of low lumenal pH was clearly demonstrated. Although light‐dependent energy quenching is a property of all photosynthetic organisms, large differences in amplitude and kinetics can be observed thus raising the question whether a single common mechanism is in action. We performed a detailed study of PsbS expression/accumulation in Chlamydomonas reinhardtii and investigated its accumulation in other algae and plants. We showed that PsbS cannot be detected in Chlamydomonas under a wide range of growth conditions. Overexpression of the endogenous psbs gene showed that the corresponding protein could not be addressed to the thylakoid membranes. Survey of different unicellular green algae showed no accumulation of anti‐PsbS reactive proteins differently from multicellular species. Nevertheless, some unicellular species exhibit high energy quenching activity, suggesting that a PsbS‐independent mechanism is activated. By correlating growth habitat and PsbS accumulation in different species, we suggest that during the evolution the light environment has been a determinant factor for the conservation/loss of the PsbS function.

Arabidopsis Mutants Deleted in the Light-Harvesting Protein Lhcb4 Have a Disrupted Photosystem II Macrostructure and Are Defective in Photoprotection

Analysis of a series of mutants lacking each of the three isoforms of the Lhcb4 light-harvesting complex in Arabidopsis showed that this complex plays an important role in the macro-organization and photoprotection of photosystem II. The role of the light-harvesting complex Lhcb4 (CP29) in photosynthesis was investigated in Arabidopsis thaliana by characterizing knockout lines for each of the three Lhcb4 isoforms (Lhcb4.1/4.2/4.3). Plants lacking all isoforms (koLhcb4) showed a compensatory increase of Lhcb1 and a slightly reduced photosystem II/I ratio with respect to the wild type. The absence of Lhcb4 did not result in alteration in electron transport rates. However, the kinetic of state transition was faster in the mutant, and nonphotochemical quenching activity was lower in koLhcb4 plants with respect to either wild type or mutants retaining a single Lhcb4 isoform. KoLhcb4 plants were more sensitive to photoinhibition, while this effect was not observed in knockout lines for any other photosystem II antenna subunit. Ultrastructural analysis of thylakoid grana membranes showed a lower density of photosystem II complexes in koLhcb4. Moreover, analysis of isolated supercomplexes showed a different overall shape of the C2S2 particles due to a different binding mode of the S-trimer to the core complex. An empty space was observed within the photosystem II supercomplex at the Lhcb4 position, implying that the missing Lhcb4 was not replaced by other Lhc subunits. This suggests that Lhcb4 is unique among photosystem II antenna proteins and determinant for photosystem II macro-organization and photoprotection.

Zeaxanthin protects plant photosynthesis by modulating chlorophyll triplet yield in specific light-harvesting antenna subunits

Background: The plant carotenoid zeaxanthin is accumulated under excess light. Results: Zeaxanthin induces a red shift in the carotenoid triplet excited state spectrum and reveals a higher efficiency in controlling chlorophyll triplet formation. Conclusion: Binding of zeaxanthin to specific proteins modulates the yield of dangerous chlorophyll excited states and protects photosynthesis from over-excitation. Significance: Functional dissection of zeaxanthin-dependent photoprotective mechanisms is crucial for understanding how plants avoid photoinhibition. Plants are particularly prone to photo-oxidative damage caused by excess light. Photoprotection is essential for photosynthesis to proceed in oxygenic environments either by scavenging harmful reactive intermediates or preventing their accumulation to avoid photoinhibition. Carotenoids play a key role in protecting photosynthesis from the toxic effect of over-excitation; under excess light conditions, plants accumulate a specific carotenoid, zeaxanthin, that was shown to increase photoprotection. In this work we genetically dissected different components of zeaxanthin-dependent photoprotection. By using time-resolved differential spectroscopy in vivo, we identified a zeaxanthin-dependent optical signal characterized by a red shift in the carotenoid peak of the triplet-minus-singlet spectrum of leaves and pigment-binding proteins. By fractionating thylakoids into their component pigment binding complexes, the signal was found to originate from the monomeric Lhcb4–6 antenna components of Photosystem II and the Lhca1–4 subunits of Photosystem I. By analyzing mutants based on their sensitivity to excess light, the red-shifted triplet-minus-singlet signal was tightly correlated with photoprotection in the chloroplasts, suggesting the signal implies an increased efficiency of zeaxanthin in controlling chlorophyll triplet formation. Fluorescence-detected magnetic resonance analysis showed a decrease in the amplitude of signals assigned to chlorophyll triplets belonging to the monomeric antenna complexes of Photosystem II upon zeaxanthin binding; however, the amplitude of carotenoid triplet signal does not increase correspondingly. Results show that the high light-induced binding of zeaxanthin to specific proteins plays a major role in enhancing photoprotection by modulating the yield of potentially dangerous chlorophyll-excited states in vivo and preventing the production of singlet oxygen.

The Arabidopsis szl1 Mutant Reveals a Critical Role of β-Carotene in Photosystem I Photoprotection

Carotenes and their oxygenated derivatives, the xanthophylls, are structural determinants in both photosystems (PS) I and II. They bind and stabilize photosynthetic complexes, increase the light-harvesting capacity of chlorophyll-binding proteins, and have a major role in chloroplast photoprotection. Localization of carotenoid species within each PS is highly conserved: Core complexes bind carotenes, whereas peripheral light-harvesting systems bind xanthophylls. The specific functional role of each xanthophyll species has been recently described by genetic dissection, however the in vivo role of carotenes has not been similarly defined. Here, we have analyzed the function of carotenes in photosynthesis and photoprotection, distinct from that of xanthophylls, by characterizing the suppressor of zeaxanthin-less (szl) mutant of Arabidopsis (Arabidopsis thaliana) which, due to the decreased activity of the lycopene-β-cyclase, shows a lower carotene content than wild-type plants. When grown at room temperature, mutant plants showed a lower content in PSI light-harvesting complex I complex than the wild type, and a reduced capacity for chlorophyll fluorescence quenching, the rapidly reversible component of nonphotochemical quenching. When exposed to high light at chilling temperature, szl1 plants showed stronger photoxidation than wild-type plants. Both PSI and PSII from szl1 were similarly depleted in carotenes and yet PSI activity was more sensitive to light stress than PSII as shown by the stronger photoinhibition of PSI and increased rate of singlet oxygen release from isolated PSI light-harvesting complex I complexes of szl1 compared with the wild type. We conclude that carotene depletion in the core complexes impairs photoprotection of both PS under high light at chilling temperature, with PSI being far more affected than PSII.

Interaction between avoidance of photon absorption, excess energy dissipation and zeaxanthin synthesis against photooxidative stress in Arabidopsis

Plants evolved photoprotective mechanisms in order to counteract the damaging effects of excess light in oxygenic environments. Among them, chloroplast avoidance and non-photochemical quenching concur in reducing the concentration of chlorophyll excited states in the photosynthetic apparatus to avoid photooxidation. We evaluated their relative importance in regulating excitation pressure on photosystem II. To this aim, genotypes were constructed carrying mutations impairing the chloroplast avoidance response (phot2) as well as mutations affecting the biosynthesis of the photoprotective xanthophyll zeaxanthin (npq1) or the activation of non-photochemical quenching (npq4), followed by evaluation of their photosensitivity in vivo. Suppression of avoidance response resulted in oxidative stress under excess light at low temperature, while removing either zeaxanthin or PsbS had a milder effect. The double mutants phot2 npq1 and phot2 npq4 showed the highest sensitivity to photooxidative stress, indicating that xanthophyll cycle and qE have additive effects over the avoidance response. The interactions between non-photochemical quenching and avoidance responses were studied by analyzing the kinetics of fluorescence decay and recovery at different light intensities. phot2 fluorescence decay lacked a component, here named as qM. This kinetic component linearly correlated with the leaf transmittance changes due to chloroplast relocation induced by white light and was absent when red light was used as actinic source. On these basis we conclude that a decrease in leaf optical density affects the apparent non-photochemical quenching (NPQ) rise kinetic. Thus, excess light-induced fluorescence decrease is in part due to avoidance of photon absorption rather than to a genuine quenching process.

Domestication of the green alga Chlorella sorokiniana : reduction of antenna size improves light-use efficiency in a photobioreactor

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.