Photoprotective qH in Arabidopsis occurs in the light-harvesting complex II trimer

Excess light can induce photodamage to the photosynthetic machinery, therefore plants have evolved photoprotective mechanisms such as non-photochemical quenching (NPQ). Different NPQ components have been identified and classified based on their relaxation kinetics and molecular players. The NPQ component qE is induced and relaxed rapidly (seconds to minutes), whereas the NPQ component qH is induced and relaxed slowly (hours or longer). Molecular players regulating qH have recently been uncovered, but the photophysical mechanism of qH and its location in the photosynthetic membrane have not been determined. Using time-correlated single-photon counting analysis of the Arabidopsis thaliana suppressor of quenching 1 mutant (soq1), which displays higher qH than the wild type, we observed shorter average lifetime of chlorophyll fluorescence in leaves and thylakoids relative to wild type. Comparison of isolated photosynthetic complexes from plants in which qH was turned ON or OFF revealed a chlorophyll fluorescence decrease specifically in the trimeric light-harvesting complex II (LHCII) fraction when qH was ON. LHCII trimers are composed of Lhcb1, 2 and 3 proteins, so CRISPR-Cas9 edited and T-DNA insertion lhcb1, lhcb2 and lhcb3 mutants were crossed with soq1. In soq1 lhcb1, soq1 lhcb2, and soq1 lhcb3, qH was not abolished, indicating that no single major Lhcb isoform is necessary for qH. Using transient absorption spectroscopy of isolated thylakoids, no spectral signatures for chlorophyll-carotenoid excitation energy quenching or charge transfer quenching were observed, suggesting that qH may occur through chlorophyll-chlorophyll excitonic interaction.

Photosynthetic Light-Harvesting (Antenna) Complexes—Structures and Functions

Chlorophylls and bacteriochlorophylls, together with carotenoids, serve, noncovalently bound to specific apoproteins, as principal light-harvesting and energy-transforming pigments in photosynthetic organisms. In recent years, enormous progress has been achieved in the elucidation of structures and functions of light-harvesting (antenna) complexes, photosynthetic reaction centers and even entire photosystems. It is becoming increasingly clear that light-harvesting complexes not only serve to enlarge the absorption cross sections of the respective reaction centers but are vitally important in short- and long-term adaptation of the photosynthetic apparatus and regulation of the energy-transforming processes in response to external and internal conditions. Thus, the wide variety of structural diversity in photosynthetic antenna “designs” becomes conceivable. It is, however, common for LHCs to form trimeric (or multiples thereof) structures. We propose a simple, tentative explanation of the trimer issue, based on the 2D world created by photosynthetic membrane systems.

Small-Angle X-Ray and Neutron Scattering on Photosynthetic Membranes

Ultrastructural membrane arrangements in living cells and their dynamic remodeling in response to environmental changes remain an area of active research but are also subject to large uncertainty. The use of noninvasive methods such as X-ray and neutron scattering provides an attractive complimentary source of information to direct imaging because in vivo systems can be probed in near-natural conditions. However, without solid underlying structural modeling to properly interpret the indirect information extracted, scattering provides at best qualitative information and at worst direct misinterpretations. Here we review the current state of small-angle scattering applied to photosynthetic membrane systems with particular focus on data interpretation and modeling.

The PsbS protein and low pH are necessary and sufficient to induce quenching in the light-harvesting complex of plants LHCII

Photosynthesis is tightly regulated in order to withstand dynamic light environments. Under high light intensities, a mechanism known as non-photochemical quenching (NPQ) dissipates excess excitation energy, protecting the photosynthetic machinery from damage. An obstacle that lies in the way of understanding the molecular mechanism of NPQ is the large gap between in vitro and in vivo studies. On the one hand, the complexity of the photosynthetic membrane makes it challenging to obtain molecular information from in vivo experiments. On the other hand, a suitable in vitro system for the study of quenching is not available. Here we have developed a minimal NPQ system using proteoliposomes. With this, we demonstrate that the combination of low pH and PsbS is both necessary and sufficient to induce quenching in LHCII, the main antenna complex of plants. This proteoliposome system can be further exploited to gain more insight into how PsbS and other factors (e.g. zeaxanthin) influence the quenching mechanism observed in LHCII.

A Lipid Bodies-Associated Galactosyl Hydrolase Is Involved in Triacylglycerol Biosynthesis and Galactolipid Turnover in the Unicellular Green Alga Chlamydomonas reinhardtii

Monogalactosyldiacylglycerol (MGDG) and digalactosyldiacylglycerol (DGDG) are the main constituent lipids of thylakoid and chloroplast envelop membranes. Many microalgae can accumulate large amounts of triacylglycerols (TAGs) under adverse environmental conditions, which is accompanied by degradation of the photosynthetic membrane lipids. However, the process mediating the conversion from galactolipids to TAG remains largely unknown. In this study, we performed genetic and biochemical analyses of galactosyl hydrolases (CrGH) identified in the proteome of lipid bodies of the green microalga Chlamydomonas reinhardtii. The recombinant CrGH was confirmed to possess galactosyl hydrolase activity by using o-nitrophenyl-β-D-galactoside as the substrate, and the Michaelis constant (Km) and Kcat of CrGH were 13.98 μM and 3.62 s−1, respectively. Comparative lipidomic analyses showed that the content of MGDG and DGDG increased by 14.42% and 24.88%, respectively, in the CrGH-deficient mutant as compared with that of the wild type cc4533 grown under high light stress conditions, and meanwhile, the TAG content decreased by 32.20%. Up-regulation of CrGH at both a gene expression and protein level was observed under high light stress (HL) conditions. In addition, CrGH was detected in multiple subcellular localizations, including the chloroplast envelope, mitochondria, and endoplasmic reticulum membranes. This study uncovered a new paradigm mediated by the multi-localized CrGH for the conversion of the photosynthetic membranes to TAGs.

A new function for the xanthophyll zeaxanthin: glueing chlorophyll biosynthesis to thylakoid protein assembly

Xanthophylls are coloured isoprenoid metabolites synthesized in many organisms with a variety of functions from the attraction of animals for impollination to absorption of light energy for photosynthesis to photoprotection against photooxidative stress. The finding by Proctor and co-workers makes a new addition to the last type of functions by showing that zeaxanthin is instrumental in coordinating chlorophyll biosynthesis with the insertion of pigment-binding proteins into the photosynthetic membrane by glueing the protein components catalyzing these functions into a supercomplex and regulating its activity.

A novel method produces native light-harvesting complex II aggregates from the photosynthetic membrane revealing their role in nonphotochemical quenching

Nonphotochemical quenching (NPQ) is a mechanism of regulating light harvesting that protects the photosynthetic apparatus from photodamage by dissipating excess absorbed excitation energy as heat. In higher plants, the major light-harvesting antenna complex (LHCII) of photosystem (PS) II is directly involved in NPQ. The aggregation of LHCII is proposed to be involved in quenching. However, the lack of success in isolating native LHCII aggregates has limited the direct interrogation of this process. The isolation of LHCII in its native state from thylakoid membranes has been problematic because of the use of detergent, which tends to dissociate loosely bound proteins, and the abundance of pigment–protein complexes (e.g. PSI and PSII) embedded in the photosynthetic membrane, which hinders the preparation of aggregated LHCII. Here, we used a novel purification method employing detergent and amphipols to entrap LHCII in its natural states. To enrich the photosynthetic membrane with the major LHCII, we used Arabidopsis thaliana plants lacking the PSII minor antenna complexes (NoM), treated with lincomycin to inhibit the synthesis of PSI and PSII core proteins. Using sucrose density gradients, we succeeded in isolating the trimeric and aggregated forms of LHCII antenna. Violaxanthin- and zeaxanthin-enriched complexes were investigated in dark-adapted, NPQ, and dark recovery states. Zeaxanthin-enriched antenna complexes showed the greatest amount of aggregated LHCII. Notably, the amount of aggregated LHCII decreased upon relaxation of NPQ. Employing this novel preparative method, we obtained a direct evidence for the role of in vivo LHCII aggregation in NPQ.

Photocatalytic plant LPOR forms helical lattices that shape membranes for chlorophyll synthesis

Chlorophyll (Chl) biosynthesis, crucial to life on Earth, is tightly regulated because its precursors are phototoxic1. In flowering plants, the enzyme Light-dependent Protochlorophyllide OxidoReductase (LPOR) captures photons to catalyze the penultimate reaction: the reduction of a double-bond within protochlorophyllide (Pchlide) to generate chlorophyllide (Chlide)2,3. In darkness, LPOR oligomerizes to facilitate photon energy transfer and catalysis4,5. However, the complete 3D structure of LPOR, the higher-order architecture of LPOR oligomers, and the implications of these self-assembled states for catalysis, including how LPOR positions Pchlide and the cofactor NADPH, remain unknown. Here we report the atomic structure of LPOR assemblies by electron cryo-microscopy (cryoEM). LPOR polymerizes with its substrates into helical filaments around constricted lipid bilayer tubes. Portions of LPOR and Pchlide insert into the outer membrane leaflet, targeting the product, Chlide, to the membrane for the final reaction site of chlorophyll biosynthesis. In addition to its crucial photocatalytic role, we show that in darkness LPOR filaments directly shape membranes into high-curvature tubules with the spectral properties of the prolammelar body, whose light-triggered disassembly provides lipids for thylakoid assembly. Our structure of the catalytic site, moreover, challenges previously proposed reaction mechanisms6. Together, our results reveal a new and unexpected synergy between photosynthetic membrane biogenesis and chlorophyll synthesis in plants orchestrated by LPOR.

Cortical photosynthesis as a physiological marker for grape breeding: methods and approaches

Photosynthesis occurring in chlorenchymal tissues of lignified branches of perennial plants (cortical photosynthesis) has a significant impact on their productivity and resistance to adverse environmental conditions, such as water deficiency and low temperatures. Cortical photosynthesis occurring under the outer bark of a lignified grape vine can become a convenient marker for breeding freeze-tolerant varieties. The following approaches can be undertaken to assess the functional state of the cortical photosynthetic apparatus: (1) analysis of the variable chlorophyll fluorescence parameters and (2) biochemical analysis of photosynthetic membrane preparations. To evaluate these approaches, in this work we have carried out the comparative analysis of characteristics of the cortical photosynthetic apparatus in grape varieties differing in freeze tolerance. This work was supported by grant №18-04-00079 from the Russian Foundation for Basic Research.