In vitro demetalation of central magnesium in various chlorophyll derivatives using Mg-dechelatase homolog from the chloroflexi Anaerolineae.

In the metabolic pathway of chlorophylls (Chls), an enzyme called STAY-GREEN or SGR catalyzes the removal of the central magnesium ion of Chls and their derivatives to their corresponding free bases, including pheophytins. The substrate specificity of SGR has been investigated through in vitro reactions using Chl-related molecules. However, information about the biochemical properties and reaction mechanisms of SGR and its substrate specificity remains elusive. In this study, we synthesized various Chl derivatives and investigated their in vitro dechelations using an SGR enzyme. Chl-a derivatives with the C3-vinyl group on the A-ring, which is commonly found as a substituent in natural substrates, and their analogs with ethyl, hydroxymethyl, formyl, and styryl groups at the C3-position were prepared as substrates. In vitro dechelatase reactions of these substrates were performed using an SGR enzyme derived from an Anaerolineae bacterium, allowing us to investigate their specificity. Reactivity was reduced for substrates with an electron-withdrawing formyl or sterically demanding styryl group at the C3-position. Furthermore, the Chl derivative with the C8-styryl group on the B-ring was less reactive for SGR dechelation than the C3-styryl substrate. These results indicate that the SGR enzyme recognizes substituents on the B-ring of substrates more than those on the A-ring.

Anthocyanins act as a sugar-buffer and an alternative electron sink in response to starch depletion during leaf senescence: a case study on a typical anthocyanic tree species, Acer japonicum.

We hypothesized that anthocyanins act as a sugar-buffer and an alternative electron sink during leaf senescence to prevent sugar-mediated early senescence and photoinhibition. To elucidate the anthocyanin role, we monitored seasonal changes in photosynthetic traits, sugar, starch and N contents, pigment composition, and gene expression profiles in leaves exposed to substantially different light conditions within a canopy of an adult tree of fullmoon maple (Acer japonicum). Enhancement of starch amylolysis accompanied with cessation of starch synthesis occurred in the same manner independent of light conditions. Leaf sugar contents increased, but reached upper limits in the late stage of leaf senescence, even though leaf anthocyanins further increased after complete depletion of starch. Sun-exposed leaves maintained higher energy consumption via electron flow than shade-grown leaves during leaf N resorption. Thus, anthocyanins accumulated in sun-exposed leaves might have a regulative role as a sugar buffer, retarding leaf senescence, and an indirect photoprotective role as an alternative sink for electron consumption to compensate declines in other metabolic processes such as starch and protein synthesis. In this context, anthocyanins might be key substrates protecting both outer-canopy leaves (against photoinhibition) and inner-canopy leaves (via shading by outer-canopy leaves) from high light stress during N resorption.

Structural Characterization of the Chlorophyllide a Oxygenase (CAO) Enzyme Through an In Silico Approach.

Chlorophyllide a oxygenase (CAO) is responsible for converting chlorophyll a to chlorophyll b in a two-step oxygenation reaction. CAO belongs to the family of Rieske-mononuclear iron oxygenases. Although the structure and reaction mechanism of other Rieske monooxygenases have been described, a member of plant Rieske non-heme iron-dependent monooxygenase has not been structurally characterized. The enzymes in this family usually form a trimeric structure and electrons are transferred between the non-heme iron site and the Rieske center of the adjoining subunits. CAO is supposed to form a similar structural arrangement. However, in Mamiellales such as Micromonas and Ostreococcus, CAO is encoded by two genes where non-heme iron site and Rieske cluster localize on the distinct polypeptides. It is not clear if they can form a similar structural organization to achieve the enzymatic activity. In this study, the tertiary structures of CAO from the model plant Arabidopsis thaliana and the Prasinophyte Micromonas pusilla were predicted by deep learning-based methods, followed by energy minimization and subsequent stereochemical quality assessment of the predicted models. Furthermore, the chlorophyll a binding cavity and the interaction of ferredoxin, which is the electron donor, on the surface of Micromonas CAO were predicted. The electron transfer pathway was predicted in Micromonas CAO and the overall structure of the CAO active site was conserved even though it forms a heterodimeric complex. The structures presented in this study will serve as a basis for understanding the reaction mechanism and regulation of the plant monooxygenase family to which CAO belongs.

Reversible down-regulation of photosystems I and II leads to fast photosynthesis recovery after long-term drought in Jatropha curcas.

Jatropha curcas is a drought-tolerant plant that maintains its photosynthetic pigments under prolonged drought, and quickly regains its photosynthetic capacity when water is available. It has been reported that drought stress leads to increased thermal dissipation in PSII, but that of PSI has been barely investigated, perhaps due to technical limitations in measuring the PSI absolute quantum yield. In this study, we combined biochemical analysis and spectroscopic measurements using an integrating sphere, and verified that the quantum yields of both photosystems are temporarily down-regulated under drought. We found that the decrease in the quantum yield of PSII was accompanied by a decrease in the core complexes of PSII while light-harvesting complexes are maintained under drought. In addition, in drought-treated plants, we observed a decrease in the absolute quantum yield of PSI as compared with the well-watered control, while the amount of PSI did not change, indicating that non-photochemical quenching occurs in PSI. The down-regulation of both photosystems was quickly lifted in a few days upon re-watering. Our results indicate, that in J. curcas under drought, the down-regulation of both PSII and PSI quantum yield protects the photosynthetic machinery from uncontrolled photodamage.

Dynamic seasonal changes in photosynthesis systems in leaves of Asarum tamaense, an evergreen understorey herbaceous species.

BACKGROUND AND AIMS: Evergreen herbaceous species in the deciduous forest understorey maintain their photosystems in long-lived leaves under dynamic seasonal changes in light and temperature. However, in evergreen understorey herbs, it is unknown how photosynthetic electron transport acclimates to seasonal changes in forest understorey environments, and what photoprotection systems function in excess energy dissipation under high-light and low-temperature environments in winter. METHODS: Here, we used Asarum tamaense, an evergreen herbaceous species in the deciduous forest understorey with a single-flush and long-lived leaves, and measured photosynthetic CO2 assimilation and electron transport in leaves throughout the year. The contents of photosynthetic proteins, pigments and primary metabolites were determined from regularly collected leaves. KEY RESULTS: Both the rates of CO2 assimilation and electron transport under saturated light were kept low in summer, but increased in autumn and winter in A. tamaense leaves. Although the contents of photosynthetic proteins including Rubisco did not increase in autumn and winter, the proton motive force and ΔpH across the thylakoid membrane were high in summer and decreased from summer to winter to a great extent. These decreases alleviated the suppression by lumen acidification and increased the electron transport rate in winter. The content and composition of carotenoids changed seasonally, which may affect changes in non-photochemical quenching from summer to winter. Winter leaves accumulated proline and malate, which may support cold acclimation. CONCLUSIONS: In A. tamaense leaves, the increase in photosynthetic electron transport rates in winter was not due to an increase in photosynthetic enzyme contents, but due to the activation of photosynthetic enzymes and/or release of limitation of photosynthetic electron flow. These seasonal changes in the regulation of electron transport and also the changes in several photoprotection systems should support the acclimation of photosynthetic C gain under dynamic environmental changes throughout the year.

Heterologous complementation systems verify the mosaic distribution of three distinct protoporphyrinogen IX oxidase in the cyanobacterial phylum.

The pathways for synthesizing tetrapyrroles, including heme and chlorophyll, are well-conserved among organisms, despite the divergence of several enzymes in these pathways. Protoporphyrinogen IX oxidase (PPOX), which catalyzes the last common step of the heme and chlorophyll biosynthesis pathways, is encoded by three phylogenetically-unrelated genes, hemY, hemG and hemJ. All three types of homologues are present in the cyanobacterial phylum, showing a mosaic phylogenetic distribution. Moreover, a few cyanobacteria appear to contain two types of PPOX homologues. Among the three types of cyanobacterial PPOX homologues, only a hemJ homologue has been experimentally verified for its functionality. An objective of this study is to provide experimental evidence for the functionality of the cyanobacterial PPOX homologues by using two heterologous complementation systems. First, we introduced hemY and hemJ homologues from Gloeobacter violaceus PCC7421, hemY homologue from Trichodesmium erythraeum, and hemG homologue from Prochlorococcus marinus MIT9515 into a ΔhemG strain of E. coli. hemY homologues from G. violaceus and T. erythraeum, and the hemG homologue of P. marinus complimented the E. coli strain. Subsequently, we attempted to replace the endogenous hemJ gene of the cyanobacterium Synechocystis sp. PCC6803 with the four PPOX homologues mentioned above. Except for hemG from P. marinus, the other PPOX homologues substituted the function of hemJ in Synechocystis. These results show that all four homologues encode functional PPOX. The transformation of Synechocystis with G. violaceus hemY homologue rendered the cells sensitive to an inhibitor of the HemY-type PPOX, acifluorfen, indicating that the hemY homologue is sensitive to this inhibitor, while the wild-type G. violaceus was tolerant to it, most likely due to the presence of HemJ protein. These results provide an additional level of evidence that G. violaceus contains two types of functional PPOX.

Exposure to strong irradiance exacerbates photoinhibition and suppresses N resorption during leaf senescence in shade-grown seedlings of fullmoon maple (Acer japonicum).

Leaves of fullmoon maple (Acer japonicum) turn brilliant red with anthocyanins synthesis in autumn. Based on field observations, autumn coloring mainly occurs in outer-canopy leaves exposed to sun, whereas inner-canopy leaves remain green for a certain longer period before finally turn yellowish red with a smaller amount of anthocyanins. Here, we hypothesized that outer-canopy leaves protect themselves against photooxidative stress via anthocyanins while simultaneously shading inner canopy leaves and protecting them from strong light (holocanopy hypothesis). To test this hypothesis, we investigated photoinhibition and leaf N content during autumn senescence in leaves of pot-grown seedlings of fullmoon maple either raised under shade (L0, ≈13% relative irradiance to open) or transferred to full sunlight conditions on 5th (LH1), 12th (LH2), or 18th (LH3) Oct, 2021. Dry mass-based leaf N (Nmass) in green leaves in shade-grown seedlings was ≈ 30 mg N g-1 in summer. Nmass in shed leaves (25th Oct to 1st Nov) was 11.1, 12.0, 14.6, and 10.1 mg N g-1 in L0, LH1, LH2, and LH3 conditions, respectively. Higher Nmass was observed in shed leaves in LH2, compared to other experimental conditions, suggesting an incomplete N resorption in LH2. Fv/Fm after an overnight dark-adaptation, measured on 19th Oct when leaf N was actively resorbed, ranked L0: 0.72 > LH3: 0.56 > LH1: 0.45 > LH2: 0.25. As decreased Fv/Fm indicates photoinhibition, leaves in LH2 condition suffered the most severe photoinhibition. Leaf soluble sugar content decreased, but protein carbonylation increased with decreasing Fv/Fm across shade-grown seedlings (L0, LH1, LH2, and LH3) on 19th Oct, suggesting impaired photosynthetic carbon gain and possible membrane peroxidation induced by photooxidative stress, especially in LH2 condition with less N resorption efficiency. Although the impairment of N resorption seems to depend on the timing and intensity of strong light exposure, air temperature, and consequently the degree of photoinhibition, the photoprotective role of anthocyanins in outer-canopy leaves of fullmoon maple might also contribute to allow a safe N resorption in inner-canopy leaves by prolonged shading.

Crystal structure and reaction mechanism of a bacterial Mg-dechelatase homolog from the Chloroflexi Anaerolineae.

Chlorophyll degradation plays a myriad of physiological roles in photosynthetic organisms, including acclimation to light environment and nutrient remobilization during senescence. Mg extraction from chlorophyll a is the first and committed step of the chlorophyll degradation pathway. This reaction is catalyzed by the Mg-dechelatase enzyme encoded by Stay-Green (SGR). The reaction mechanism of SGR protein remains elusive since metal ion extraction from organic molecules is not a common enzymatic reaction. Additionally, experimentally derived structural information about SGR or its homologs has not yet been reported. In this study, the crystal structure of the SGR homolog from Anaerolineae bacterium was determined using the molecular replacement method at 1.85 Å resolution. Our previous study showed that three residues-H32, D34, and D62 are essential for the catalytic activity of the enzyme. Biochemical analysis involving mutants of D34 residue further strengthened its importance in the functioning of the dechelatase. Docking simulation also revealed the interaction between the D34 side chain and central Mg ion of chlorophyll a. Structural analysis showed the arrangement of D34/H32/D62 in the form of a catalytic triad that is generally found in hydrolases. The probable reaction mechanism suggests that deprotonated D34 side chain coordinates and destabilizes Mg, resulting in Mg extraction. Besides, H32 possibly acts as a general base catalyst and D62 facilitates H32 to be a better proton acceptor. Taken together, the reaction mechanism of SGR partially mirrors the one observed in hydrolases.

BRZ-INSENSITIVE-PALE GREEN 1 is encoded by chlorophyll biosynthesis enzyme gene that functions in the downstream of brassinosteroid signaling.

Brassinosteroids (BRs), a kind of phytohormone, have various biological activities such as promoting plant growth, increasing stress resistance, and chloroplast development. Though BRs have been known to have physiological effects on chloroplast, the detailed mechanism of chloroplast development and chlorophyll biosynthesis in BR signaling remains unknown. Here we identified a recessive pale green Arabidopsis mutant, Brz-insensitive-pale green1 (bpg1), which was insensitive to promoting of greening by BR biosynthesis-specific inhibitor Brz in the light. BPG1 gene encoded chlorophyll biosynthesis enzyme, 3, 8-divinyl protochlorophyllide a 8-vinyl reductase (DVR), and bpg1 accumulated divinyl chlorophylls. Chloroplast development was suppressed in bpg1. Brz dramatically increased the expression of chlorophyll biosynthesis enzyme genes, including BPG1. These results suggest that chlorophyll biosynthesis enzymes are regulated by BR signaling in the aspect of gene expression and BPG1 plays an important role in regulating chloroplast development.

Characterization of photosystem II assembly complexes containing ONE-HELIX PROTEIN1 in Arabidopsis thaliana.

The assembly process of photosystem II (PSII) requires several auxiliary proteins to form assembly intermediates. In plants, early assembly intermediates comprise D1 and D2 subunits of PSII together with a few auxiliary proteins including at least ONE-HELIX PROTEIN1 (OHP1), OHP2, and HIGH-CHLOROPHYLL FLUORESCENCE 244 (HCF244) proteins. Herein, we report the basic characterization of the assembling intermediates, which we purified from Arabidopsis transgenic plants overexpressing a tagged OHP1 protein and named the OHP1 complexes. We analyzed two major forms of OHP1 complexes by mass spectrometry, which revealed that the complexes consist of OHP1, OHP2, and HCF244 in addition to the PSII subunits D1, D2, and cytochrome b559. Analysis of chlorophyll fluorescence showed that a major form of the complex binds chlorophyll a and carotenoids and performs quenching with a time constant of 420 ps. To identify the localization of the auxiliary proteins, we solubilized thylakoid membranes using a digitonin derivative, glycodiosgenin, and separated them into three fractions by ultracentrifugation, and detected these proteins in the loose pellet containing the stroma lamellae and the grana margins together with two chlorophyll biosynthesis enzymes. The results indicated that chlorophyll biosynthesis and assembly may take place in the same compartments of thylakoid membranes. Inducible suppression of the OHP2 mRNA substantially decreased the OHP2 protein in mature Arabidopsis leaves without a significant reduction in the maximum quantum yield of PSII under low-light conditions, but it compromised the yields under high-light conditions. This implies that the auxiliary protein is required for acclimation to high-light conditions.

Insights into the structure and function of the rate-limiting enzyme of chlorophyll degradation through analysis of a bacterial Mg-dechelatase homolog.

The Mg-dechelatase enzyme encoded by the Stay-Green (SGR) gene catalyzes Mg2+ dechelation from chlorophyll a. This reaction is the first committed step of chlorophyll degradation pathway in plants and is thus indispensable for the process of leaf senescence. There is no structural information available for this or its related enzymes. This study aims to provide insights into the structure and reaction mechanism of the enzyme through biochemical and computational analysis of an SGR homolog from the Chloroflexi Anaerolineae (AbSGR-h). Recombinant AbSGR-h with its intact sequence and those with mutations were overexpressed in Escherichia coli and their Mg-dechelatase activity were compared. Two aspartates - D34 and D62 were found to be essential for catalysis, while R26, Y28, T29 and D114 were responsible for structural maintenance. Gel filtration analysis of the recombinant AbSGR-h indicates that it forms a homo-oligomer. The three-dimensional structure of AbSGR-h was predicted by a deep learning-based method, which was evaluated by protein structure quality evaluation programs while structural stability of wild-type and mutant forms were investigated through molecular dynamics simulations. Furthermore, in concordance with the results of enzyme assay, molecular docking concluded the significance of D34 in ligand interaction. By combining biochemical analysis and computational prediction, this study unveils the detailed structural characteristics of the enzyme, including the probable pocket of interaction and the residues of structural and functional importance. It also serves as a basis for further studies on Mg-dechelatase such as elucidation of its reaction mechanism or inhibitor screening.

Enrichment of chlorophyll catabolic enzymes in grana margins and their cooperation in catabolic reactions.

During leaf senescence, chlorophyll a and b are degraded through several enzymatic reactions, including chlorophyll b reductase, 7-hydroxymethyl chlorophyll a reductase, and Mg-dechelatase. Considering that the intermediates of the chlorophyll breakdown pathway are highly photoreactive, cooperative and efficient reactions of chlorophyll metabolic enzymes may protect chloroplasts from potential photo-oxidative damage. Here, we investigated the sub-organellar localization and cooperative reactions of the enzymes involved in the chlorophyll breakdown pathway by the fractionation of thylakoid membranes and enzymatic assays using recombinant proteins. We found that these enzymes were enriched in the grana margin fraction. Furthermore, we found that chlorophyll b reductase and Mg-dechelatase efficiently catabolized chlorophylls bound to the chlorophyll-protein complexes when these two enzymes were mixed. These results suggest that the co-localization of chlorophyll catabolic enzymes enables efficient chlorophyll breakdown. The results from this study highlight a key step forward in the investigation of the photosystem breakdown process.

Distribution and functional analysis of the two types of 8-vinyl reductase involved in chlorophyll biosynthesis in marine cyanobacteria.

In the chlorophyll biosynthesis pathway, the 8-vinyl group of the chlorophyll precursor is reduced to an ethyl group by 8-vinyl reductase. Two isozymes of 8-vinyl reductase have been described in oxygenic photosynthetic organisms: one encoded by BciA and another by BciB. Only BciB contains an [Fe-S] cluster and most cyanobacteria harbor this form; whereas a few contain BciA. Given this disparity in distribution, cyanobacterial BciA has remained largely overlooked, which has limited understanding of chlorophyll biosynthesis in these microorganisms. Here, we reveal that cyanobacterial BciA encodes a functional 8-vinyl reductase, as evidenced by measuring the in vitro activity of recombinant Synechococcus and Acaryochloris BciA. Genomic comparison revealed that BciB had been replaced by BciA during evolution of the marine cyanobacterium Synechococcus, and coincided with replacement of Fe-superoxide dismutase (SOD) with Ni-SOD. These findings imply that the acquisition of BciA confers an adaptive advantage to cyanobacteria living in low-iron oceanic environments.

Degradation of the photosystem II core complex is independent of chlorophyll degradation mediated by Stay-Green Mg2+ dechelatase in Arabidopsis.

During leaf senescence, the degradation of photosystems and photosynthetic pigments proceeds in a coordinated manner, which would minimize the potential photodamage to cells. Both photosystem I and II are composed of core complexes and peripheral antenna complexes, with the former binding chlorophyll a and the latter binding chlorophyll a and b. Although the degradation of peripheral antenna complexes is initiated by chlorophyll degradation, it remains unclear whether the degradation of core complexes and chlorophyll is coordinated. In this study, we examined the degradation of peripheral antenna and core complexes in the Arabidopsis sgr1/sgr2/sgrl triple mutant, lacking all the isoforms of chlorophyll a:Mg2+ dechelatase. In this mutant, the degradation of peripheral antenna complexes and photosystem I core complexes was substantially retarded, but the core complexes of photosystem II were rapidly degraded during leaf senescence. On the contrary, the photosynthetic activity declined at a similar rate as in the wild type plants. These results suggest that the degradation of photosystem II core complexes is regulated independently of the major chlorophyll degradation pathway mediated by the dechelatase. The study should contribute to the understanding of the complex molecular mechanisms underlying the degradation of photosystems, which is an essential step during leaf senescence.

Unique Peripheral Antennas in the Photosystems of the Streptophyte Alga Mesostigma viride.

Land plants evolved from a single group of streptophyte algae. One of the key factors needed for adaptation to a land environment is the modification in the peripheral antenna systems of photosystems (PSs). Here, the PSs of Mesostigma viride, one of the earliest-branching streptophyte algae, were analyzed to gain insight into their evolution. Isoform sequencing and phylogenetic analyses of light-harvesting complexes (LHCs) revealed that M. viride possesses three algae-specific LHCs, including algae-type LHCA2, LHCA9 and LHCP, while the streptophyte-specific LHCB6 was not identified. These data suggest that the acquisition of LHCB6 and the loss of algae-type LHCs occurred after the M. viride lineage branched off from other streptophytes. Clear-native (CN)-polyacrylamide gel electrophoresis (PAGE) resolved the photosynthetic complexes, including the PSI-PSII megacomplex, PSII-LHCII, two PSI-LHCI-LHCIIs, PSI-LHCI and the LHCII trimer. Results indicated that the higher-molecular weight PSI-LHCI-LHCII likely had more LHCII than the lower-molecular weight one, a unique feature of M. viride PSs. CN-PAGE coupled with mass spectrometry strongly suggested that the LHCP was bound to PSII-LHCII, while the algae-type LHCA2 and LHCA9 were bound to PSI-LHCI, both of which are different from those in land plants. Results of the present study strongly suggest that M. viride PSs possess unique features that were inherited from a common ancestor of streptophyte and chlorophyte algae.

Substitution of Deoxycholate with the Amphiphilic Polymer Amphipol A8-35 Improves the Stability of Large Protein Complexes during Native Electrophoresis.

Native polyacrylamide gel electrophoresis (PAGE) is a powerful technique for protein complex separation that retains both their activity and structure. In photosynthetic research, native-PAGE is particularly useful given that photosynthetic complexes are generally large in size, ranging from 200 kD to 1 MD or more. Recently, it has been reported that the addition of amphipol A8-35 to solubilized protein samples improved protein complex stability. In a previous study, we found that amphipol A8-35 could substitute sodium deoxycholate (DOC), a conventional electrophoretic carrier, in clear-native (CN)-PAGE. In this study, we present the optimization of amphipol-based CN-PAGE. We found that the ratio of amphipol A8-35 to α-dodecyl maltoside, a detergent commonly used to solubilize photosynthetic complexes, was critical for resolving photosynthetic machinery in CN-PAGE. In addition, LHCII dissociation from PSII-LHCII was effectively prevented by amphipol-based CN-PAGE compared with that of DOC-based CN-PAGE. Our data strongly suggest that majority of the PSII-LHCII in vivo forms C2S2M2 at least in Arabidopsis and Physcomitrella. The other forms might appear owing to the dissociation of LHCII from PSII during sample preparation and electrophoresis, which could be prevented by the addition of amphipol A8-35 after solubilization from thylakoid membranes. These results suggest that amphipol-based CN-PAGE may be a better alternative to DOC-based CN-PAGE for the study of labile protein complexes.

Subcellular localization of chlorophyllase2 reveals it is not involved in chlorophyll degradation during senescence in Arabidopsis thaliana.

Chlorophyllase (CLH), which catalyzes the release of the phytol chain from chlorophyll (Chl), has been long considered to catalyze the first step of Chl degradation. Arabidopsis contains two isoforms of CLH (CLH1 and CLH2), and CLH1 was previously demonstrated to be localized in tonoplast and endoplasmic reticulum, and not be involved in Chl degradation. In contrast, CLH2 possesses a predicted signal-peptide for chloroplast localization, and phylogenetic analysis of CLHs in Arabidopsis and other species also indicate that CLH2 forms a different clade than CLH1. Therefore, the possibility remains that CLH2 is involved in the breakdown of Chl. In the current study, clh mutants lacking CLH2 or both CLH isoforms were analyzed after the induction of senescence. Results indicated that the clh knockout lines were still able to degrade Chl at the same rate as wild-type plants. Transgenic Arabidopsis plants were generated that constitutively expressed either CLH2 or CLH2 fused to a yellow fluorescent protein (YFP). Observations made using confocal microscopy indicated that CLH2-YFP was located external to chloroplasts. Additionally, in overexpression plants, CLH2 was enriched in tonoplast and endoplasmic reticulum fractions following membrane fractionation. Based on the collective data, we conclude that CLH2 is not involved in Chl breakdown during senescence in Arabidopsis.

Formation of a PSI-PSII megacomplex containing LHCSR and PsbS in the moss Physcomitrella patens.

Mosses are one of the earliest land plants that diverged from fresh-water green algae. They are considered to have acquired a higher capacity for thermal energy dissipation to cope with dynamically changing solar irradiance by utilizing both the "algal-type" light-harvesting complex stress-related (LHCSR)-dependent and the "plant-type" PsbS-dependent mechanisms. It is hypothesized that the formation of photosystem (PS) I and II megacomplex is another mechanism to protect photosynthetic machinery from strong irradiance. Herein, we describe the analysis of the PSI-PSII megacomplex from the model moss, Physcomitrella patens, which was resolved using large-pore clear-native polyacrylamide gel electrophoresis (lpCN-PAGE). The similarity in the migration distance of the Physcomitrella PSI-PSII megacomplex to the Arabidopsis megacomplex shown during lpCN-PAGE suggested that the Physcomitrella PSI-PSII and Arabidopsis megacomplexes have similar structures. Time-resolved chlorophyll fluorescence measurements show that excitation energy was rapidly and efficiently transferred from PSII to PSI, providing evidence of an ordered association of the two photosystems. We also found that LHCSR and PsbS co-migrated with the Physcomitrella PSI-PSII megacomplex. The megacomplex showed pH-dependent chlorophyll fluorescence quenching, which may have been induced by LHCSR and/or PsbS proteins with the collaboration of zeaxanthin. We discuss the mechanism that regulates the energy distribution balance between two photosystems in Physcomitrella.