Thylakoid protein FPB1 synergistically cooperates with PAM68 to promote CP47 biogenesis and Photosystem II assembly.

In chloroplasts, insertion of proteins with multiple transmembrane domains (TMDs) into thylakoid membranes usually occurs in a co-translational manner. Here, we have characterized a thylakoid protein designated FPB1 (Facilitator of PsbB biogenesis1) which together with a previously reported factor PAM68 (Photosynthesis Affected Mutant68) is involved in assisting the biogenesis of CP47, a subunit of the Photosystem II (PSII) core. Analysis by ribosome profiling reveals increased ribosome stalling when the last TMD segment of CP47 emerges from the ribosomal tunnel in fpb1 and pam68. FPB1 interacts with PAM68 and both proteins coimmunoprecipitate with SecY/E and Alb3 as well as with some ribosomal components. Thus, our data indicate that, in coordination with the SecY/E translocon and the Alb3 integrase, FPB1 synergistically cooperates with PAM68 to facilitate the co-translational integration of the last two CP47 TMDs and the large loop between them into thylakoids and the PSII core complex.

CBSX2 is required for the efficient oxidation of chloroplast redox-regulated enzymes in darkness.

Thiol/disulfide-based redox regulation in plant chloroplasts is essential for controlling the activity of target proteins in response to light signals. One of the examples of such a role in chloroplasts is the activity of the chloroplast ATP synthase (CFoCF1), which is regulated by the redox state of the CF1γ subunit and involves two cysteines in its central domain. To investigate the mechanism underlying the oxidation of CF1γ and other chloroplast redox-regulated enzymes in the dark, we characterized the Arabidopsis cbsx2 mutant, which was isolated based on its altered NPQ (non-photochemical quenching) induction upon illumination. Whereas in dark-adapted WT plants CF1γ was completely oxidized, a small amount of CF1γ remained in the reduced state in cbsx2 under the same conditions. In this mutant, reduction of CF1γ was not affected in the light, but its oxidation was less efficient during a transition from light to darkness. The redox states of the Calvin cycle enzymes FBPase and SBPase in cbsx2 were similar to those of CF1γ during light/dark transitions. Affinity purification and subsequent analysis by mass spectrometry showed that the components of the ferredoxin-thioredoxin reductase/thioredoxin (FTR-Trx) and NADPH-dependent thioredoxin reductase (NTRC) systems as well as several 2-Cys peroxiredoxins (Prxs) can be co-purified with CBSX2. In addition to the thioredoxins, yeast two-hybrid analysis showed that CBSX2 also interacts with NTRC. Taken together, our results suggest that CBSX2 participates in the oxidation of the chloroplast redox-regulated enzymes in darkness, probably through regulation of the activity of chloroplast redox systems in vivo.

NDH-1L with a truncated NdhM subunit is unstable under stress conditions in cyanobacteria.

Cyanobacterial NdhM, an oxygenic photosynthesis-specific NDH-1 subunit, has been found to be essential for the formation of a large complex of NDH-1 (NDH-1L). The cryo-electron microscopic (cryo-EM) structure of NdhM from Thermosynechococcus elongatus showed that the N-terminus of NdhM contains three ß-sheets, while two α-helixes are present in the middle and C-terminal part of NdhM. Here, we obtained a mutant of the unicellular cyanobacterium Synechocystis 6803 expressing a C-terminal truncated NdhM subunit designated NdhMΔC. Accumulation and activity of NDH-1 were not affected in NdhMΔC under normal growth conditions. However, the NDH-1 complex with truncated NdhM is unstable under stress. Immunoblot analyses showed that the assembly process of the cyanobacterial NDH-1L hydrophilic arm was not affected in the NdhMΔC mutant even under high temperature. Thus, our results indicate that NdhM can bind to the NDH-1 complex without its C-terminal α-helix, but the interaction is weakened. NDH-1L with truncated NdhM is more prone to dissociation, and this is particularly evident under stress conditions.

Reduction in chloroplastic ribulose-5-phosphate-3-epimerase decreases photosynthetic capacity in Arabidopsis.

Chloroplast ribulose-5-phosphate-3-epimerase (RPE) is a critical enzyme involved in the Calvin-Benson cycle and oxidative pentose phosphate pathways in higher plants. Three Arabidopsis rpe mutants with reduced level of RPE were identified through their high NPQ (nonphotochemical quenching) phenotype upon illumination, and no significant difference of plant size was found between these rpe mutants and WT (wild type) plants under growth chamber conditions. A decrease in RPE expression to a certain extent leads to a decrease in CO2 fixation, V cmax and J max. Photosynthetic linear electron transport was partially inhibited and activity of ATP synthase was also decreased in the rpe mutants, but the levels of thylakoid protein complexes and other Calvin-Benson cycle enzymes in rpe mutants were not affected. These results demonstrate that some degree of reduction in RPE expression decreases carbon fixation in chloroplasts, which in turn feedback inhibits photosynthetic electron transport and ATP synthase activity due to the photosynthetic control. Taken together, this work provides evidence that RPE plays an important role in the Calvin-Benson cycle and influences the photosynthetic capacity of chloroplasts.

Rubredoxin 1 Is Required for Formation of the Functional Photosystem II Core Complex in Arabidopsis thaliana.

Chloroplast thylakoid protein rubredoxin 1 (RBD1) in Chlamydomonas and its cyanobacterial homolog RubA contain a rubredoxin domain. These proteins have been proposed to participate in the assembly of photosystem II (PSII) at early stages. However, the effects of inactivation of RBD1 on PSII assembly in higher plants are largely unclear. Here, we characterized an Arabidopsis rbd1 mutant in detail. A drastic reduction of intact PSII complex but relatively higher levels of assembly intermediates including PSII RC, pre-CP47, and pre-CP43 were found in rbd1. Polysome association and ribosome profiling revealed that ribosome recruitment of psbA mRNA is specifically reduced. Consistently, in vivo protein pulse-chase labeling showed that the rate of D1/pD1 synthesis is significantly reduced in rbd1 compared with WT. Moreover, newly synthesized mature D1 and pD1/D2 can assemble into the PSII reaction center (RC) complex but further formation of larger PSII complexes is nearly totally blocked in rbd1. Our data imply that RBD1 is not only required for the formation of a functional PSII core complex during the early stages of PSII assembly but may also be involved in the translation of D1 in higher plants.

Stromal Protein Chloroplast Development and Biogenesis1 Is Essential for Chloroplast Development and Biogenesis in Arabidopsis thaliana.

Although numerous studies have been carried out on chloroplast development and biogenesis, the underlying regulatory mechanisms are still largely elusive. Here, we characterized a chloroplast stromal protein Chloroplast Development and Biogenesis1 (CDB1). The knockout cdb1 mutant exhibits a seedling-lethal and ivory leaf phenotype. Immunoblot and RNA blot analyses show that accumulation of chloroplast ribosomes is compromised in cdb1, resulting in an almost complete loss of plastid-encoded proteins including the core subunits of the plastid-encoded RNA polymerase (PEP) RpoB and RpoC2, and therefore in impaired PEP activity. Orthologs of CDB1 are found in green algae and land plants. Moreover, a protein shows high similarity with CDB1, designated as CDB1-Like (CDB1L), is present in angiosperms. Absence of CDB1L results in impaired embryo development. While CDB1 is specifically located in the chloroplast stroma, CDB1L is localized in both chloroplasts and mitochondria in Arabidopsis. Thus, our results demonstrate that CDB1 is indispensable for chloroplast development and biogenesis through its involvement in chloroplast ribosome assembly whereas CDB1L may fulfill a similar function in both mitochondria and chloroplasts.

F-Type ATP Synthase Assembly Factors Atp11 and Atp12 in Arabidopsis.

Atp11p and Atp12p are members of two chaperone families essential for assembly of the mitochondrial ATP synthase in Saccharomyces cerevisiae and Homo sapiens. However, the role of their homologs in higher plants is unclear with regard to the assembly of both chloroplast ATP synthase (cpATPase) and mitochondrial ATP synthase (mtATPase). Here, we show that loss of either Atp11 or Atp12 is lethal in Arabidopsis. While Atp12 is only localized in mitochondria, Atp11 is present both in chloroplasts and mitochondria. Yeast two-hybrid analyses showed that, as their homologs in yeast, Atp11 specifically interacts with the ß subunit of the mtATPase and cpATPase, and Atp12 interacts with the α subunit of the mtATPase, implying that Atp11 and Atp12 fulfill a conserved task during assembly of ATP synthase. However, the binding sites for Atp11 in the ß subunit of mtATPase and cpATPase are slightly different, suggesting that the mechanisms of action may have evolved in different ways. Although Atp11 interacts with cpATPase ß subunit as the two assembly factors BFA3 and BFA1, they bind to different sites of the ß subunit. These results indicate that Atp11 is involved in the assembly of both cpATPase and mtATPase but Atp12 is specifically required for the assembly of mtATPase in higher plants.

Na2CO3-responsive Photosynthetic and ROS Scavenging Mechanisms in Chloroplasts of Alkaligrass Revealed by Phosphoproteomics.

Alkali-salinity exerts severe osmotic, ionic, and high-pH stresses to plants. To understand the alkali-salinity responsive mechanisms underlying photosynthetic modulation and reactive oxygen species (ROS) homeostasis, physiological and diverse quantitative proteomics analyses of alkaligrass (Puccinellia tenuiflora) under Na2CO3 stress were conducted. In addition, Western blot, real-time PCR, and transgenic techniques were applied to validate the proteomic results and test the functions of the Na2CO3-responsive proteins. A total of 104 and 102 Na2CO3-responsive proteins were identified in leaves and chloroplasts, respectively. In addition, 84 Na2CO3-responsive phosphoproteins were identified, including 56 new phosphorylation sites in 56 phosphoproteins from chloroplasts, which are crucial for the regulation of photosynthesis, ion transport, signal transduction, and energy homeostasis. A full-length PtFBA encoding an alkaligrass chloroplastic fructose-bisphosphate aldolase (FBA) was overexpressed in wild-type cells of cyanobacterium Synechocystis sp. Strain PCC 6803, leading to enhanced Na2CO3 tolerance. All these results indicate that thermal dissipation, state transition, cyclic electron transport, photorespiration, repair of photosystem (PS) II, PSI activity, and ROS homeostasis were altered in response to Na2CO3 stress, which help to improve our understanding of the Na2CO3-responsive mechanisms in halophytes.

PPR Protein BFA2 Is Essential for the Accumulation of the atpH/F Transcript in Chloroplasts.

As a fascinating and complicated nanomotor, chloroplast ATP synthase comprises nine subunits encoded by both the nuclear and plastid genomes. Because of its uneven subunit stoichiometry, biogenesis of ATP synthase and expression of plastid-encoded ATP synthase genes requires assistance by nucleus-encoded factors involved in transcriptional, post-transcriptional, and translational steps. In this study, we report a P-class pentatricopeptide repeat (PPR) protein BFA2 (Biogenesis Factor required for ATP synthase 2) that is essential for accumulation of the dicistronic atpH/F transcript in Arabidopsis chloroplasts. A loss-of-function mutation in BFA2 results in a specific reduction of more than 3/4 of chloroplast ATP synthase, which is likely due to the absence of dicistronic atpH/F transcript. BFA2 protein contains 22 putative PPR motifs and exclusively localizes in the chloroplast. Bioinformatics and Electrophoretic Mobility Shift Assays (EMSA) analysis showed that BFA2 binds to the consensus sequence of the atpF-atpA intergenic region in a sequence-specific manner. However, translation initiation of the atpA was not affected in the bfa2 mutant. Thus, we propose that the chloroplast PPR protein BFA2 mainly acts as barrier to prevent the atpH/F transcript degradation by exoribonucleases by binding to the consensus sequence of the atpF-atpA intergenic region.

Comparative Study Reveals Insights of Sheepgrass (Leymus chinensis) Coping With Phosphate-Deprived Stress Condition.

Sheepgrass [Leymus chinensis (Trin.) Tzvel] is a valuable forage plant highly significant to the grassland productivity of Euro-Asia steppes. Growth of above-ground tissues of L. chinensis is the major component contributing to the grass yield. Although it is generally known that this species is sensitive to ecosystem disturbance and adverse environments, detailed information of how L. chinensis coping with various nutrient deficiency especially phosphate deprivation (-Pi) is still limited. Here, we investigated impact of Pi-deprivation on shoot growth and biomass accumulation as well as photosynthetic properties of L. chinensis. Growth inhibition of Pi-deprived seedlings was most obvious and reduction of biomass accumulation and net photosynthetic rate (Pn) was 55.3 and 63.3%, respectively, compared to the control plants grown under Pi-repleted condition. Also, we compared these characters with seedlings subjected to low-Pi stress condition. Pi-deprivation caused 18.5 and 12.3% more reduction of biomass and Pn relative to low-Pi-stressed seedlings, respectively. Further analysis of in vivo chlorophyll fluorescence and thylakoid membrane protein complexes using 2D-BN/SDS-PAGE combined with immunoblot detection demonstrated that among the measured photosynthetic parameters, decrease of ATP synthase activity was most pronounced in Pi-deprived plants. Together with less extent of lipid peroxidation of the thylakoid membranes and increased ROS scavenger enzyme activities in the leaves of Pi-deprived seedlings, we suggest that the decreased activity of ATP synthase in their thylakoids is the major cause of the greater reduction of photosynthetic efficiency than that of low-Pi stressed plants, leading to the least shoot growth and biomass production in L. chinensis.

OHP1, OHP2, and HCF244 Form a Transient Functional Complex with the Photosystem II Reaction Center.

The reaction center (RC) of photosystem II (PSII), which is composed of D1, D2, PsbI, and cytochrome b559 subunits, forms at an early stage of PSII biogenesis. However, it is largely unclear how these components assemble to form a functional unit. In this work, we show that synthesis of the PSII core proteins D1/D2 and formation of the PSII RC is blocked specifically in the absence of ONE-HELIX PROTEIN1 (OHP1) and OHP2 proteins in Arabidopsis (Arabidopsis thaliana), indicating that OHP1 and OHP2 are essential for the formation of the PSII RC. Mutagenesis of the chlorophyll-binding residues in OHP proteins impairs their function and/or stability, suggesting that they may function in the binding of chlorophyll in vivo. We further show that OHP1, OHP2, and HIGH CHLOROPHYLL FLUORESCENCE244 (HCF244), together with D1, D2, PsbI, and cytochrome b559, form a complex. We designated this complex the PSII RC-like complex to distinguish it from the RC subcomplex in the intact PSII complex. Our data imply that OHP1, OHP2, and HCF244 are present in this PSII RC-like complex for a limited time at an early stage of PSII de novo assembly and of PSII repair under high-light conditions. In a subsequent stage of PSII biogenesis, OHP1, OHP2, and HCF244 are released from the PSII RC-like complex and replaced by the other PSII subunits. Together with previous reports on the cyanobacterium Synechocystis, our results demonstrate that the process of PSII RC assembly is highly conserved among photosynthetic species.

Nucleus-Encoded Protein BFA1 Promotes Efficient Assembly of the Chloroplast ATP Synthase Coupling Factor 1.

F-type ATP synthases produce nearly all of the ATP found in cells. The catalytic module F1 commonly comprises an α3ß3 hexamer surrounding a γ/ε stalk. However, it is unclear how these subunits assemble to form a catalytic motor. In this work, we identified and characterized a chloroplast protein that interacts with the CF1ß, γ, and ε subunits of the chloroplast ATP synthase and is required for assembly of its F1 module. We named this protein BIOGENESIS FACTOR REQUIRED FOR ATP SYNTHASE1 (BFA1) and determined its crystal structure at 2.8-Å resolution. BFA1 is comprised primarily of two interacting ß-barrels that are oriented nearly perpendicularly to each other. The contact region between BFA1 and the CF1ß and γ subunits was further mapped by yeast two-hybrid assays. An in silico molecular docking analysis was performed and revealed close fitting contact sites without steric conflicts between BFA1 and CF1ß/γ. We propose that BFA1 acts mainly as a scaffold protein promoting the association of a CF1α/ß heterodimer with CF1γ. The subsequent assembly of other CF1α/ß heterodimers may shift the position of the CF1γ subunit to complete assembly of the CF1 module. This CF1 assembly process is likely to be valid for other F-type ATP synthases, as their structures are highly conserved.

LOW PHOTOSYNTHETIC EFFICIENCY 1 is required for light-regulated photosystem II biogenesis in Arabidopsis.

Photosystem II (PSII), a multisubunit protein complex of the photosynthetic electron transport chain, functions as a water-plastoquinone oxidoreductase, which is vital to the initiation of photosynthesis and electron transport. Although the structure, composition, and function of PSII are well understood, the mechanism of PSII biogenesis remains largely elusive. Here, we identified a nuclear-encoded pentatricopeptide repeat (PPR) protein LOW PHOTOSYNTHETIC EFFICIENCY 1 (LPE1; encoded by At3g46610) in Arabidopsis, which plays a crucial role in PSII biogenesis. LPE1 is exclusively targeted to chloroplasts and directly binds to the 5' UTR of psbA mRNA which encodes the PSII reaction center protein D1. The loss of LPE1 results in less efficient loading of ribosome on the psbA mRNA and great synthesis defects in D1 protein. We further found that LPE1 interacts with a known regulator of psbA mRNA translation HIGH CHLOROPHYLL FLUORESCENCE 173 (HCF173) and facilitates the association of HCF173 with psbA mRNA. More interestingly, our results indicate that LPE1 associates with psbA mRNA in a light-dependent manner through a redox-based mechanism. This study enhances our understanding of the mechanism of light-regulated D1 synthesis, providing important insight into PSII biogenesis and the functional maintenance of efficient photosynthesis in higher plants.

[Progress in improving photosynthetic efficiency by synthetic biology].

The contradiction between the increasing population and the decrease of tillable land areas is becoming more and more serious in our country. Food security is an important guarantee for sustainable development of our national economy. Photosynthesis is the basis for crop yield. Improving crop photosynthetic efficiency is one of the important approaches to increase crop yield. In this review, we summarized the recent advances in engineering photosynthetic performance by synthetic biology from three key aspects including absorption, transduction and conversion of light energy, light utilization efficiency and carbon assimilation. We also addressed the prospects of its application in increasing photosynthetic efficiency through synthetic biology principles, which may provide important theoretical support and key biotechnology to increase grain production.

A Light Harvesting Complex-Like Protein in Maintenance of Photosynthetic Components in Chlamydomonas.

Using a genetic approach, we have identified and characterized a novel protein, named Msf1 (Maintenance factor for photosystem I), that is required for the maintenance of specific components of the photosynthetic apparatus in the green alga Chlamydomonas reinhardtii Msf1 belongs to the superfamily of light-harvesting complex proteins with three transmembrane domains and consensus chlorophyll-binding sites. Loss of Msf1 leads to reduced accumulation of photosystem I and chlorophyll-binding proteins/complexes. Msf1is a component of a thylakoid complex containing key enzymes of the tetrapyrrole biosynthetic pathway, thus revealing a possible link between Msf1 and chlorophyll biosynthesis. Protein interaction assays and greening experiments demonstrate that Msf1 interacts with Copper target homolog1 (CHL27B) and accumulates concomitantly with chlorophyll in Chlamydomonas, implying that chlorophyll stabilizes Msf1. Contrary to other light-harvesting complex-like genes, the expression of Msf1 is not stimulated by high-light stress, but its protein level increases significantly under heat shock, iron and copper limitation, as well as in stationary cells. Based on these results, we propose that Msf1 is required for the maintenance of photosystem I and specific protein-chlorophyll complexes especially under certain stress conditions.

Loss of algal Proton Gradient Regulation 5 increases reactive oxygen species scavenging and H2 evolution.

We have identified hpm91, a Chlamydomonas mutant lacking Proton Gradient Regulation5 (PGR5) capable of producing hydrogen (H2 ) for 25 days with more than 30-fold yield increase compared to wild type. Thus, hpm91 displays a higher capacity of H2 production than a previously characterized pgr5 mutant. Physiological and biochemical characterization of hpm91 reveal that the prolonged H2 production is due to enhanced stability of PSII, which correlates with increased reactive oxygen species (ROS) scavenging capacity during sulfur deprivation. This anti-ROS response appears to protect the photosynthetic electron transport chain from photo-oxidative damage and thereby ensures electron supply to the hydrogenase.

CHLORORESPIRATORY REDUCTION 9 is a Novel Factor Required for Formation of Subcomplex A of the Chloroplast NADH Dehydrogenase-Like Complex.

In vascular plants, the chloroplast NADH dehydrogenase-like (NDH) complex, a homolog of respiratory NADH:quinone oxidoreductase (Complex I), mediates plastoquinone reduction using ferredoxin as an electron donor in cyclic electron transport around PSI in the thylakoid membrane. In angiosperms, chloroplast NDH is composed of five subcomplexes and forms a supercomplex with PSI. The modular assembly of stroma-protruded subcomplex A, which corresponds to the Q module of Complex I, was recently reported. However, the factors involved in the specific assembly steps have not been completely identified. Here, we isolated an Arabidopsis mutant, chlororespiratory reduction 9 (crr9), defective in NDH activity. The CRR9 gene encodes a novel stromal protein without any known functional domains or motifs. CRR9 is highly conserved in cyanobacteria and land plants but not in green algae, which do not have chloroplast NDH. Blue native-PAGE and immunoblot analyses of thylakoid proteins indicated that formation of subcomplex A was impaired in crr9 CRR9 was specifically required for the accumulation of NdhK, a subcomplex A subunit, in NDH assembly intermediates in the stroma. Furthermore, two-dimensional clear native/SDS-PAGE analysis of the stroma fraction indicated that incorporation of NdhM into NDH assembly intermediate complex 400 was impaired in crr9 These results suggest that CRR9 is a novel factor required for the formation of NDH subcomplex A.