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.

Iron-Sulfur Cluster Biogenesis and Iron Homeostasis in Cyanobacteria.

Iron-sulfur (Fe-S) clusters are ancient and ubiquitous cofactors and are involved in many important biological processes. Unlike the non-photosynthetic bacteria, cyanobacteria have developed the sulfur utilization factor (SUF) mechanism as their main assembly pathway for Fe-S clusters, supplemented by the iron-sulfur cluster and nitrogen-fixing mechanisms. The SUF system consists of cysteine desulfurase SufS, SufE that can enhance SufS activity, SufBC2D scaffold complex, carrier protein SufA, and regulatory repressor SufR. The S source for the Fe-S cluster assembly mainly originates from L-cysteine, but the Fe donor remains elusive. This minireview mainly focuses on the biogenesis pathway of the Fe-S clusters in cyanobacteria and its relationship with iron homeostasis. Future challenges of studying Fe-S clusters in cyanobacteria are also discussed.

Ssl3451 is Important for Accumulation of NDH-1 Assembly Intermediates in the Cytoplasm of Synechocystis sp. Strain PCC 6803.

Two mutants sensitive to high light for growth and impaired in NDH-1 activity were isolated from a transposon-tagged library of Synechocystis sp. strain PCC 6803. Both mutants were tagged in the ssl3451 gene encoding a hypothetical protein, which shares a significant homology with the Arabidopsis (Arabidopsis thaliana) CHLORORESPIRATORY REDUCTION 42 (CRR42). In Arabidopsis, CRR42 associates only with an NDH-1 hydrophilic arm assembly intermediate (NAI) of about 400 kDa (NAI400), one of total three NAIs (NAI800, NAI500 and NAI400), and its deletion has little, if any, effect on accumulation of any NAIs in the stroma. In comparison, the ssl3451 product was localized mainly in the cytoplasm and associates with two NAIs of about 300 kDa (NAI300) and 130 kDa (NAI130). Deletion of Ssl3451 reduced the abundance of the NAI300 complex to levels no longer visible on gels and of the NAI130 complex to a low level, thereby impeding the assembly process of NDH-1 hydrophilic arm. Further, Ssl3451 interacts with another assembly factor Ssl3829 and they have a similar effect on accumulation of NAIs and NdhI maturation factor Slr1097 in the cytoplasm. We thus propose that Ssl3451 plays an important role in accumulation of the NAI300 and NAI130 complexes in the cytoplasm via its interacting protein Ssl3829.

Light Intensity is Important for Hydrogen Production in NaHSO3-Treated Chlamydomonas reinhardtii.

Chlamydomonas reinhardtii is a unicellular green alga that can use light energy to produce H2 from H2O in the background of NaHSO3 treatment. However, the role of light intensity in such H2 production remains elusive. Here, light intensity significantly affected the yield of H2 production in NaHSO3-treated C. reinhardtii, which was consistent with its effects on the content of O2 and the expression and activity of hydrogenase. Further, NaHSO3 was found to be able to remove O2 via a reaction of bisulfite with superoxide anion produced at the acceptor side of PSI, and light intensity affected the reaction rate significantly. Accordingly, high light and strong light but not low light can create an anaerobic environment, which is important to activate hydrogenase and produce H2. Based on the above results, we conclude that light intensity plays an important role in removing O2 and consequently activating hydrogenase and producing H2 in NaHSO3-treated C. reinhardtii.

NDH-1 Is Important for Photosystem I Function of Synechocystis sp. Strain PCC 6803 under Environmental Stress Conditions.

Cyanobacterial NDH-1 interacts with photosystem I (PSI) to form an NDH-1-PSI supercomplex. Here, we observed that absence of NDH-1 had little, if any, effect on the functional fractions of PSI under growth conditions, but significantly reduced the functional fractions of PSI when cells of Synechocystis sp. strain PCC 6803 were moved to conditions of multiple stresses. The significant reduction in NDH-1-dependent functional fraction of PSI was initiated after PSII activity was impaired. This finding is consistent with our observation that the functional fraction of PSI under growth conditions was rapidly and significantly decreased with increasing concentrations of DCMU, which rapidly and significantly suppressed PSII activity by blocking the transfer of electrons from QA to QB in the PSII reaction center. Furthermore, absence of NDH-1 resulted in the PSI limitation at the functionality of PSI itself but not its donor-side and acceptor-side under conditions of multiple stresses. This was supported by the result of a significant destabilization of the PSI complex in the absence of NDH-1 but the presence of multiple stresses. Based on the above results, we propose that NDH-1 is important for PSI function of Synechocystis sp. strain PCC 6803 mainly via maintaining stabilization of PSI under conditions of environmental stresses.

The NDH-1L-PSI Supercomplex Is Important for Efficient Cyclic Electron Transport in Cyanobacteria.

Two mutants isolated from a tagging library of Synechocystis sp. strain PCC 6803 were sensitive to high light and had a tag in sll1471 encoding CpcG2, a linker protein for photosystem I (PSI)-specific antenna. Both mutants demonstrated strongly impaired NDH-1-dependent cyclic electron transport. Blue native-polyacrylamide gel electrophoresis followed by immunoblotting and mass spectrometry analyses of the wild type and a mutant containing CpcG2 fused with yellow fluorescent protein-histidine6 indicated the presence of a novel NDH-1L-CpcG2-PSI supercomplex, which was absent in the cpcG2 deletion mutant, the PSI-less mutant, and several other strains deficient in NDH-1L and/or NDH-1M. Coimmunoprecipitation and pull-down analyses on CpcG2-yellow fluorescent protein-histidine6, using antibody against green fluorescent protein and nickel column chromatography, confirmed the association of CpcG2 with the supercomplex. Conversely, the use of antibodies against NdhH or NdhK after blue native-polyacrylamide gel electrophoresis and in coimmunoprecipitation experiments verified the necessity of CpcG2 in stabilizing the supercomplex. Furthermore, deletion of CpcG2 destabilized NDH-1L as well as its degradation product NDH-1M and significantly decreased the number of functional PSI centers, consistent with the involvement of CpcG2 in NDH-1-dependent cyclic electron transport. The CpcG2 deletion, however, had no effect on respiration. Thus, we propose that the formation of an NDH-1L-CpcG2-PSI supercomplex in cyanobacteria facilitates PSI cyclic electron transport via NDH-1L.

A Cytoplasmic Protein Ssl3829 Is Important for NDH-1 Hydrophilic Arm Assembly in Synechocystis sp. Strain PCC 6803.

Despite significant progress in clarifying the subunit compositions and functions of the multiple NDH-1 complexes in cyanobacteria, the assembly factors and their roles in assembling these NDH-1 complexes remain elusive. Two mutants sensitive to high light for growth and impaired in NDH-1-dependent cyclic electron transport around photosystem I were isolated from Synechocystis sp. strain PCC 6803 transformed with a transposon-tagged library. Both mutants were tagged in the ssl3829 gene encoding an unknown protein, which shares significant similarity with Arabidopsis (Arabidopsis thaliana) CHLORORESPIRATORY REDUCTION7. The ssl3829 product was localized in the cytoplasm and associates with an NDH-1 hydrophilic arm assembly intermediate (NAI) of about 300 kD (NAI300) and an NdhI maturation factor, Slr1097. Upon deletion of Ssl3829, the NAI300 complex was no longer visible on gels, thereby impeding the assembly of the NDH-1 hydrophilic arm. The deletion also abolished Slr1097 and consequently reduced the amount of mature NdhI in the cytoplasm, which repressed the dynamic assembly process of the NDH-1 hydrophilic arm because mature NdhI was essential to stabilize all functional NAIs. Therefore, Ssl3829 plays an important role in the assembly of the NDH-1 hydrophilic arm by accumulating the NAI300 complex and Slr1097 protein in the cytoplasm.

NdhV Is a Subunit of NADPH Dehydrogenase Essential for Cyclic Electron Transport in Synechocystis sp. Strain PCC 6803.

Two mutants sensitive to heat stress for growth and impaired in NADPH dehydrogenase (NDH-1)-dependent cyclic electron transport around photosystem I (NDH-CET) were isolated from the cyanobacterium Synechocystis sp. strain PCC 6803 transformed with a transposon-bearing library. Both mutants had a tag in the same sll0272 gene, encoding a protein highly homologous to NdhV identified in Arabidopsis (Arabidopsis thaliana). Deletion of the sll0272 gene (ndhV) did not influence the assembly of NDH-1 complexes and the activities of CO2 uptake and respiration but reduced the activity of NDH-CET. NdhV interacted with NdhS, a ferredoxin-binding subunit of cyanobacterial NDH-1 complex. Deletion of NdhS completely abolished NdhV, but deletion of NdhV had no effect on the amount of NdhS. Reduction of NDH-CET activity was more significant in ΔndhS than in ΔndhV. We therefore propose that NdhV cooperates with NdhS to accept electrons from reduced ferredoxin.

Subunit Q Is Required to Stabilize the Large Complex of NADPH Dehydrogenase in Synechocystis sp. Strain PCC 6803.

Two major complexes of NADPH dehydrogenase (NDH-1) have been identified in cyanobacteria. A large complex (NDH-1L) contains NdhD1, NdhF1, and NdhP, which are absent in a medium size complex (NDH-1M). They play important roles in respiration, NDH-1-dependent cyclic electron transport around photosystem I, and CO2 uptake. Two mutants sensitive to high light for growth and impaired in cyclic electron transport around photosystem I were isolated from the cyanobacterium Synechocystis sp. strain PCC 6803 transformed with a transposon-bearing library. Both mutants had a tag in an open reading frame encoding a product highly homologous to NdhQ, a single-transmembrane small subunit of the NDH-1L complex, identified in Thermosynechococcus elongatus by proteomics strategy. Deletion of ndhQ disassembled about one-half of the NDH-1L to NDH-1M and consequently impaired respiration, but not CO2 uptake. During prolonged incubation of the thylakoid membrane with n-dodecyl-ß-D-maltoside at room temperature, the rest of the NDH-1L in ΔndhQ was disassembled completely to NDH-1M and was much faster than in the wild type. In the ndhP-deletion mutant (ΔndhP) background, absence of NdhQ almost completely disassembled the NDH-1L to NDH-1M, similar to the results observed in the ΔndhD1/ΔndhD2 mutant. We therefore conclude that both NdhQ and NdhP are essential to stabilize the NDH-1L complex.

NdhO, a subunit of NADPH dehydrogenase, destabilizes medium size complex of the enzyme in Synechocystis sp. strain PCC 6803.

Two mutants that grew faster than the wild-type (WT) strain under high light conditions were isolated from Synechocystis sp. strain PCC 6803 transformed with a transposon-bearing library. Both mutants had a tag in ssl1690 encoding NdhO. Deletion of ndhO increased the activity of NADPH dehydrogenase (NDH-1)-dependent cyclic electron transport around photosystem I (NDH-CET), while overexpression decreased the activity. Although deletion and overexpression of ndhO did not have significant effects on the amount of other subunits such as NdhH, NdhI, NdhK, and NdhM in the cells, the amount of these subunits in the medium size NDH-1 (NDH-1M) complex was higher in the ndhO-deletion mutant and much lower in the overexpression strain than in the WT. NdhO strongly interacts with NdhI and NdhK but not with other subunits. NdhI interacts with NdhK and the interaction was blocked by NdhO. The blocking may destabilize the NDH-1M complex and repress the NDH-CET activity. When cells were transferred from growth light to high light, the amounts of NdhI and NdhK increased without significant change in the amount of NdhO, thus decreasing the relative amount of NdhO. This might have decreased the blocking, thereby stabilizing the NDH-1M complex and increasing the NDH-CET activity under high light conditions.

Identification of biochemical association of phycobilisome with photosystems in cyanobacterial state transition.

State transition is a short-term balance mechanism of energy distribution between photosystem II (PSII) and PSI. Although light-induced state transition in cyanobacteria has been suggested to depend completely on the phycobilisome (PBS) movement between PSII and PSI, the biochemical evidence has not been clearly shown. In this study, we locked the association of PBS with PSII or PSI using glycinebetaine when cells attain State 1 or 2 by exposure to light of blue or green, respectively. Subsequently, the PBS-reaction centers were resolved by blue native polyacrylamide gel electrophoresis and two-dimensional electrophoresis, and then identified by western blot analysis. The results showed that in wild-type (WT) Synechocystis sp. strain PCC 6803, the PBS core always co-migrates with the PSII dimer during light-induced State 1-State 2 transition, but its rod leaves the PSII dimer in State 2 regardless of its co-migration in State 1. In the light-induced State 2, the co-migration of PBS rod with PSI trimer was observed in WT, but not in ΔndhB (M55), a State-2-transition-deficient mutant. This study first provided the biochemical evidence for the association of PBS with photosystems during cyanobacterial state transition.

NdhP is an exclusive subunit of large complex of NADPH dehydrogenase essential to stabilize the complex in Synechocystis sp. strain PCC 6803.

Two major complexes of NADPH dehydrogenase (NDH-1) have been identified in cyanobacteria. A large complex (NDH-1L) contains NdhD1 and NdhF1, which are absent in a medium size complex (NDH-1M). They play important roles in respiration, cyclic electron transport around photosystem I, and CO2 acquisition. Two mutants sensitive to high light for growth and impaired in NDH-1-mediated cyclic electron transfer were isolated from Synechocystis sp. strain PCC 6803 transformed with a transposon-bearing library. Both mutants had a tag in sml0013 encoding NdhP, a single transmembrane small subunit of the NDH-1 complex. During prolonged incubation of the wild type thylakoid membrane with n-dodecyl ß-d-maltoside (DM), about half of the NDH-1L was disassembled to NDH-1M and the rest decomposed completely without forming NDH-1M. In the ndhP deletion mutant (ΔndhP), disassembling of NDH-1L to NDH-1M occurred even on ice, and decomposition to a small piece occurred at room temperature much faster than in the wild type. Deletion of the C-terminal tail of NdhP gave the same result. The C terminus of NdhP was tagged by YFP-His6. Blue native gel electrophoresis of the DM-treated thylakoid membrane of this strain and Western analysis using the antibody against GFP revealed that NdhP-YFP-His6 was exclusively confined to NDH-1L. During prolonged incubation of the thylakoid membrane of the tagged strain with DM at room temperature, NDH-1L was partially disassembled to NDH-1M and the 160-kDa band containing NdhP-YFP-His6 and possibly NdhD1 and NdhF1. We therefore conclude that NdhP, especially its C-terminal tail, is essential to assemble NdhD1 and NdhF1 and stabilize the NDH-1L complex.