psaC Gene Research Articles

Effects and mechanisms of proanthocyanidins-derived carbon dots on alleviating salt stress in rice by muti-omics analysis

Carbon dots (CDs) with different structures were prepared by electrolysis (PE-CDs) and hydrothermal (PH-CDs) methods using proanthocyanidins as precursors. The smaller size and lower zeta potential enabled the PE-CDs treated rice seedlings to exhibit greater resistance to salt stress. The fresh weight of rice seedlings under salt stress was significantly increased by spraying CDs every other day for two weeks. PE-CDs treated group exhibited a faster electron transport rate, and the SOD activity and flavonoid content were 2.5-fold and 0.23-fold higher than those of the salt stress-treated group. Furthermore, the metabolomics and transcriptomics analysis revealed that the PsaC gene of photosystem I was significantly up-regulated under PE-CDs treatment, which accelerated electron transfer in photosystem I. The up-regulation of BX1 and IGL genes encoding indole synthesis allowed rice to enhance stress tolerance through tryptophan and benzoxazine biosynthesis pathways. These findings offer help in purposefully synthesizing CDs and boosting food production.

Chloroplast-related host proteins interact with NIb and NIa-Pro of soybeans mosaic virus and induce resistance in the susceptible cultivar

To gain a deeper understanding of the molecular mechanisms involved in viral infection and the corresponding plant resistance responses, it is essential to investigate the interactions between viral and host proteins. In the case of viral infections in plants, a significant portion of the affected gene products are closely associated with chloroplasts and photosynthesis. However, the molecular mechanisms underlying the interplay between the virus and host chloroplast proteins during replication remain poorly understood. In our previous study, we made an interesting discovery regarding soybean mosaic virus (SMV) infection in resistant and susceptible soybean cultivars. We found that the photosystem I (PSI) subunit (PSaC) and ATP synthase subunit α (ATPsyn-α) genes were up-regulated in the resistant cultivar following SMV-G7H and SMV-G5H infections compared to the susceptible cultivar. Overexpression of these two genes within the SMV-G7H genome in the susceptible cultivar Lee74 (rsv3-null) reduced SMV accumulation, whereas silencing of the PSaC and ATPsyn-α genes promoted SMV accumulation. We have also found that the PSaC and ATPsyn-α proteins are present in the chloroplast envelope, nucleus, and cytoplasm. Building on these findings, we now characterized protein-protein interactions between PSaC and ATPsyn-α with two viral proteins, NIb and NIa-Pro, respectively, of SMV. Through co-immunoprecipitation (Co-IP) experiments, we confirmed the interactions between these proteins. Moreover, when the C-terminal region of either PSaC or ATPsyn-α was overexpressed in the SMV-G7H genome, we observed a reduction in viral accumulation and systemic infection in the susceptible cultivar. Based on these results, we propose that the PSaC and ATPsyn-α genes play a modulatory role in conferring resistance to SMV infection by influencing the function of NIb and NIa-Pro-in SMV replication and movement. The identification of these photosynthesis-related genes as key players in the interplay between the virus and the host provides valuable insights for developing more targeted control strategies against SMV. Additionally, by utilizing these genes, it may be possible to genetically engineer plants with improved photosynthetic efficiency and enhanced resistance to SMV infection.

Physcomitrium patens pentatricopeptide repeat protein PpPPR_32 is involved in the accumulation of psaC mRNA encoding the iron sulfur protein of photosystem I.

Pentatricopeptide repeat (PPR) proteins are involved in RNA metabolism and also play a role in posttranscriptional regulation during plant organellar gene expression. Although a hundred of PPR proteins exist in the moss Physcomitrium patens, their functions are not fully understood. Here, we report the function of P-class PPR protein PpPPR_32 in P. patens. A transient expression assay using green fluorescent protein demonstrated that the N-terminal region of PpPPR_32 functions as a chloroplast-targeting transit peptide, indicating that PpPPR_32 is localized in chloroplasts. PpPPR_32 knockout mutants grew autotrophically but with reduced protonema growth and the poor formation of photosystem I (PSI) complexes. Quantitative real-time reverse transcription-polymerase chain reaction and RNA gel blot hybridization analyses revealed a significant reduction in the transcript level of the psaC gene encoding the iron sulfur protein of PSI but no alteration to the transcript levels of other PSI genes. This suggests that PpPPR_32 is specifically involved in the expression level of the psaC gene. Our results indicate that PpPPR_32 is essential for the accumulation of psaC transcript and PSI complexes.

Photosynthesis-related genes induce resistance against soybean mosaic virus: Evidence for involvement of the RNA silencing pathway.

Increasing lines of evidence indicate that chloroplast‐related genes are involved in plant–virus interactions. However, the involvement of photosynthesis‐related genes in plant immunity is largely unexplored. Analysis of RNA‐Seq data from the soybean cultivar L29, which carries the Rsv3 resistance gene, showed that several chloroplast‐related genes were strongly induced in response to infection with an avirulent strain of soybean mosaic virus (SMV), G5H, but were weakly induced in response to a virulent strain, G7H. For further analysis, we selected the PSaC gene from the photosystem I and the ATP‐synthase α‐subunit (ATPsyn‐α) gene whose encoded protein is part of the ATP‐synthase complex. Overexpression of either gene within the G7H genome reduced virus levels in the susceptible cultivar Lee74 (rsv3‐null). This result was confirmed by transiently expressing both genes in Nicotiana benthamiana followed by G7H infection. Both proteins localized in the chloroplast envelope as well as in the nucleus and cytoplasm. Because the chloroplast is the initial biosynthesis site of defence‐related hormones, we determined whether hormone‐related genes are involved in the ATPsyn‐α‐ and PSaC‐mediated defence. Interestingly, genes involved in the biosynthesis of several hormones were up‐regulated in plants infected with SMV‐G7H expressing ATPsyn‐α. However, only jasmonic and salicylic acid biosynthesis genes were up‐regulated following infection with the SMV‐G7H expressing PSaC. Both chimeras induced the expression of several antiviral RNA silencing genes, which indicate that such resistance may be partially achieved through the RNA silencing pathway. These findings highlight the role of photosynthesis‐related genes in regulating resistance to viruses.

Severe Plastid Genome Size Reduction in a Mycoheterotrophic Orchid, Danxiaorchis singchiana, Reveals Heavy Gene Loss and Gene Relocations.

Danxiaorchis singchiana (Orchidaceae) is a leafless mycoheterotrophic orchid in the subfamily Epidendroideae. We sequenced the complete plastome of D. singchiana. The plastome has a reduced size of 87,931 bp, which includes a pair of inverted repeat (IR) regions of 13,762 bp each that are separated by a large single copy (LSC) region of 42,575 bp and a small single copy (SSC) region of 17,831 bp. When compared to its sister taxa, Cremastra appendiculata and Corallorhiza striata var. involuta, D. singchiana showed an inverted gene block in the LSC and SSC regions. A total of 61 genes were predicted, including 21 tRNA, 4 rRNA, and 36 protein-coding genes. While most of the housekeeping genes were still intact and seem to be protein-coding, only four photosynthesis-related genes appeared presumably intact. The majority of the presumably intact protein-coding genes seem to have undergone purifying selection (dN/dS < 1), and only the psaC gene was positively selected (dN/dS > 1) when compared to that in Cr. appendiculata. Phylogenetic analysis of 26 complete plastome sequences from 24 species of the tribe Epidendreae had revealed that D. singchiana diverged after Cr. appendiculata and is sister to the genus Corallorhiza with strong bootstrap support (100%).

Differential expression of wheat transcriptomes in response to varying cadmium concentrations

This study aims to understand the changes in the transcriptome of durum wheat (Tricitum durum cv. Balcali-85) upon exposure to varying Cd concentrations using mRNA differential display (mRNA DD) technique. Sequence analyses of the two heavily induced genes upon exposure to Cd showed high homology to NADH dehydrogenase subunit 1 (EC907725) and PsaC gene encoding a photosystem 1 (PS 1) 9 kDa subunit protein (EC907731). Additionally, three differentially expressed genes (EC907726, EC907729 and EC907730) were identified. Their sequence analyses revealed no significant homologies to known genes. The expressions of NADH dehydrogenase subunit 1 and PsaC genes were confirmed by Northern blot analysis and quantified by real time PCR. This is the first report for the induction of NADH dehydrogenase subunit 1 gene during Cd stress in wheat.

Post-transcriptional Control of Chloroplast Gene Expression: ACCUMULATION OF STABLE psaC mRNA IS DUE TO DOWNSTREAM RNA CLEAVAGES IN THE ndhD GENE

Intergenic cleavages, intron splicing, and editing of primary transcripts of the plastid ndhH-D operon produce multiple overlapping RNAs, of which the most abundant by far is the monocistronic 400-nucleotide mRNA of psaC (encoding the PsaC protein of photosystem I), in contrast with the low level of transcripts of the six ndh genes. Like other plastid operons containing genes for functionally unrelated proteins, the contrasting accumulation of ndh and psaC transcripts provides a model to investigate the mechanisms of the post-transcriptional control of gene expression, a feature of chloroplast genetic machinery, with a minimum of interference by transcriptional control. In leek (Allium porrum L), the ndhD transcript (which follows the psaC gene and ends the ndhH-D operon) requires C --> U editing to restore its start codon and may be used as a marker for the processing of psaC and ndhD transcripts. By determining the editing state and 5' end sequences of specific transcripts, we demonstrated that stable monocistronic psaC mRNA results from downstream cleavages in the ndhD sequence, which renders non-functional ndhD transcripts as by-products. Alternative psaC-ndhD intergenic cleavages produce complete mRNAs for both genes, but only take place in precursors containing editing-restored ndhD start codons. Hence, post-transcriptional control acts by promoting the ndhD cleavage alternative, which allows the accumulation of psaC mRNA at the expense of ndhD mRNA levels.

The chloroplast ycf7 (petL) open reading frame of Chlamydomonas reinhardtii encodes a small functionally important subunit of the cytochrome b6f complex.

The small chloroplast open reading frame ORF43 (ycf7) of the green unicellular alga Chlamydomonas reinhardtii is cotranscribed with the psaC gene and ORF58. While ORF58 has been found only in the chloroplast genome of C.reinhardtii, ycf7 has been conserved in land plants and its sequence suggests that its product is a hydrophobic protein with a single transmembrane alpha helix. We have disrupted ORF58 and ycf7 with the aadA expression cassette by particle-gun mediated chloroplast transformation. While the ORF58::aadA transformants are indistinguishable from wild type, photoautotrophic growth of the ycf7::aadA transformants is considerably impaired. In these mutant cells, the amount of cytochrome b6f complex is reduced to 25-50% of wild-type level in mid-exponential phase, and the rate of transmembrane electron transfer per b6f complex measured in vivo under saturating light is three to four times slower than in wild type. Under subsaturating light conditions, the rate of the electron transfer reactions within the b6f complex is reduced more strongly in the mutant than in the wild type by the proton electrochemical gradient. The ycf7 product (Ycf7) is absent in mutants deficient in cytochrome b6f complex and present in highly purified b6f complex from the wild-type strain. Ycf7-less complexes appear more fragile than wild-type complexes and selectively lose the Rieske iron-sulfur protein during purification. These observations indicate that Ycf7 is an authentic subunit of the cytochrome b6f complex, which is required for its stability, accumulation and optimal efficiency. We therefore propose to rename the ycf7 gene petL.

Absence of PsaC subunit allows assembly of photosystem I core but prevents the binding of PsaD and PsaE in Synechocystis sp. PCC6803

In photosystem I (PSI) of oxygenic photosynthetic organisms the psaC polypeptide, encoded by the psaC gene, provides the ligands for two [4Fe-4S] clusters, FA and FB. Unlike other cyanobacteria, two different psaC genes have been reported in the cyanobacterium Synechocystis 6803, one (copy 1) with a deduced amino acid sequence identical to that of tobacco and another (copy 2) with a deduced amino acid sequence similar to those reported for other cyanobacteria. Insertion of a gene encoding kanamycin resistance into copy 2 resulted in a photosynthesis-deficient strain, CDK25, lacking the PsaC, PsaD and PsaE polypeptides in isolated thylakoid membranes, while the PsaA/PsaB and PsaF subunits were found. Growth of the mutant cells was indistinguishable from that of wild-type cells under light-activated heterotrophic growth (LAHG). A reversible P700+ signal was detected by EPR spectroscopy in the isolated thylakoids during illumination at low temperature. Under these conditions, the EPR signals attributed to FA and FB were absent in the mutant strain, but a reversible Fx signal was present with broad resonances at g = 2.079, 1.903, and 1.784. Addition of PsaC and PsaD proteins to the thylakoids gave rise to resonances at g = 2.046, 1.936, 1.922, and 1.880; these values are characteristic of an interaction-type spectrum of FA- and FB-. In room-temperature optical spectroscopic analysis, addition of PsaC and PsaD to the thylakoids also restored a 30 ms kinetic transient which is characteristic of the P700+ [FA/FB]- backreaction. Expression of copy 1 was not detected in cells grown under LAHG and under mixotrophic conditions. These results demonstrate that copy 2 encodes the PsaC polypeptide in PSI in Synechocystis 6803, while copy 1 is not involved in PSI; that the PsaC polypeptide is necessary for stable assembly of PsaD and PsaE into PSI complex in vivo; and that PsaC, PsaD and PsaE are not needed for assembly of PsaA-PsaB dimer and electron transport from P700 to Fx.

The PsaC Protein Is Necessary for the Stable Association of the PsaD, PsaE, and PsaL Proteins in the Photosystem I Complex: Analysis of a Cyanobacterial Mutant Strain

The PsaC protein binds two 4Fe-4S centers, FA and FB, in the photosystem I (PSI) protein complex. In the T398 strain of Anabaena variabilis ATCC 29413, the psaC gene encoding this protein has been insertionally inactivated by the introduction of a neomycin resistance gene cartridge in the coding region. Photosystem I complex was purified through native gel electrophoresis of β-dodecyl maltoside solubilized thylakoid membranes from wild-type and T398 strains of Anabaena 29413. The PSI complex from T398 strain retained functionally active P700, the reaction center chlorophylls. Interestingly, purified PSI complex from T398 cells lacked the PsaD, PsaE, and PsaL polypeptides. Western analysis with polyclonal antibodies raised against these proteins indicated that the two stromal exposed polypeptides, PsaD and PsaE, are absent in isolated thylakoid membranes from T398 cells. The PsaL polypeptide could be detected at a level comparable to that in wild-type thylakoid membranes, although it is absent in the PSI preparation from the mutant. These observations suggest that the PsaC protein is essential for the stable association of PsaD and PsaE, two hydrophilic, extrinsic polypeptides. Moreover, PsaL, a hydrophobic protein is loosely associated with PSI and is lost during the isolation of the PSI complex.

Sequence of a psaC Gene from the Cyanobacterium Synechococcus sp. PCC 6301

The psaC gene encodes PsaC, the apoprotein for the terminal iron-sulfur clusters, FA and FB, in the PSI reaction center of cyanobacteria, algae, and higher plants. PsaC functions as a membrane-bound oxidoreductase, accepting electrons from the Fx iron-sulfur cluster located on the PsaA/ PsaB heterodimer and donating them to the soluble electron transfer proteins Fd and flavodoxin (reviewed in Bryant, 1992). The objective of our work is to clarify the role of PsaC in linear and cyclic electron transfer. The experimental organism is Synechococcus sp. PCC 6301, a unicellular, freshwater cyanobacterium that is used extensively for physiological, biochemical, and spectroscopic studies of photosynthesis. The PSI complex of Synechococcus sp. PCC 6301 is widely used in structural and functional studies of the FA and FB ironsulfur clusters (Parrett et al., 1990). We became interested in the psaC sequence of Synechococcus sp. PCC 6301 because there are subtle differences in the electron paramagnetic resonance spectra of the FA and FB iron-sulfur clusters between cyanobacteria and higher plants. This suggests that there may be a correlation with structure: in all organisms studied thus far, the higher-plant PsaC protein differs from the cyanobacterial PsaC protein in two regions: at position 37, higher plants contain a Lys whereas cyanobacteria contain a neutral amino acid (Gly or Ala), and at positions 70 and 71, dicots contain a Trp-His pair, monocots contain a Gly-Pro pair, and cyanobacteria contain a GlyAla pair. No sequence for the PsaC protein of Synechococcus sp. PCC 6301 is available, even though entire sequences are available for PsaD and PsaE and partial sequences are available for PsaL and PsaF. This information is relevant because

The psaC genes of Synechococcus sp. PCC7002 and Cyanophora paradoxa: cloning and sequence analysis

The psaC genes of the cyanobacterium, Synechococcus sp. PCC7002, and of the cyanelle genome of the phylogenetically ambiguous biflagellate, Cyanophora paradoxa, were cloned, mapped and sequenced. The PsaC proteins of both species exhibit high degrees (approx. 95%) of sequence similarity to the PsaC proteins of other cyanobacteria as well as the chloroplast-encoded proteins of green algae and higher plants. The Synechococcus sp. PCC7002 psaC gene is transcribed as a monocistronic mRNA of approx. 350–400 nt, and transcription is initiated 51 nt upstream from the translational start codon. As found for the chloroplasts of higher plants, the C. paradoxa psaC gene is encoded within the small single-copy region of the cyanelle genome. In contrast to results obtained for chloroplasts and for the cyanobacterium Synechocystis sp. PCC6803, neither psaC gene is flanked by genes encoding components of the NAD(P)H dehydrogenase complex.

Nucleotide sequence and expression of the gene for the 9 kDa FA/FB component of photosystem I from the cyanobacterium Anabaena sp. PCC7120.

The nucleotide sequence of an 813 bp Ssp I-Hinc II fragment containing the psaC gene from Anabaena sp. PCC 7120 is reported. The gene encodes a polypeptide of 81 amino acids, has a single transcript of size approximately 480 nucleotides and a single startpoint of transcription. It is flanked by a ribosome binding site on the 5' end and a potential transcription terminator on the 3' end. Comparison of the amino acid sequences of the psaC gene product from sixteen organisms shows that cyanobacteria group as a cluster distinct from monocotyledonous and dicotyledonous plants and that individual amino acids are diagnostic of each cluster.

Directed mutagenesis of an iron-sulfur protein of the photosystem I complex in the filamentous cyanobacterium Anabaena variabilis ATCC 29413.

In oxygenic photosynthetic organisms the PSI-C polypeptide, encoded by the psaC gene, provides the ligands for two [4Fe-4S] centers, FA and FB, the terminal electron acceptors in the photosystem I (PSI) complex. An insertion mutation introduced in the psaC locus of the filamentous cyanobacterium Anabaena variabilis ATCC 29413 resulted in the creation of a mutant strain, T398-1, that lacks the PSI-C polypeptide. In medium supplemented with 5 mM fructose, the mutant cells grew well in the dark. However, when grown in the same medium under light, the doubling rate of T398-1 cells was significantly decreased. In intact cells of T398-1, bicarbonate-dependent whole-chain electron transport (PSII and PSI) could not be detected, although partial electron transport reactions involving either one of the two photosystems could be measured at significant rates. The low-temperature EPR signals attributed to the [4Fe-4S] centers FA and FB were absent in the mutant cells. Chemical titration measurements indicated that the ratios of chlorophyll to the primary donor P700 were virtually identical in membranes from the wild-type and mutant cells. Moreover, room-temperature optical spectroscopic analysis of the thylakoid membranes isolated from T398-1 showed flash-induced P700 oxidation followed by dark rereduction, indicating primary photochemistry in PSI. Thus stable assembly of the reaction center of PSI can occur in the absence of the Fe-S cluster cofactors FA and FB. These studies demonstrate that Anabaena 29413 offers a useful genetic system for targeted mutagenesis of the PSI complex.

Directed chloroplast transformation in Chlamydomonas reinhardtii: insertional inactivation of the psaC gene encoding the iron sulfur protein destabilizes photosystem I.

The chloroplast gene psaC encoding the iron sulfur protein of photosystem I (PSI) from the green alga Chlamydomonas reinhardtii has been cloned and characterized. The deduced amino acid sequence is highly related to that of higher plants and cyanobacteria. Using a particle gun, wild type C. reinhardtii cells have been transformed with a plasmid carrying the psaC gene disrupted by an aadA gene cassette designed to express spectinomycin/streptomycin resistance in the chloroplast. Transformants selected on plates containing acetate as a reduced carbon source and spectinomycin are unable to grow on minimal medium lacking acetate and are deficient in PSI activity. Southern blot analysis of total cell DNA of the transformants shows that the wild type psaC gene has been replaced by the interrupted psaC gene through homologous recombination. While authentic transcripts of the psaC gene are no longer detected, aadA gives rise to a few transcripts in the transformants. Biochemical analysis indicates that neither PSI reaction center subunits nor the seven small subunits belonging to PSI accumulate stably in the thylakoid membranes of the transformants. Pulse-chase labeling of cell proteins shows that the PSI reaction center subunits are synthesized normally but turn over rapidly in the transformants. We conclude that the iron sulfur binding protein encoded by the psaC gene is an essential component, both for photochemical activity and for stable assembly of PSI. The present study suggests that any chloroplast gene encoding a component of the photosynthetic apparatus can be disrupted in C. reinhardtii using the strategy described.

PsaD is required for the stable binding of PsaC to the photosystem I core protein of Synechococcus sp. PCC 6301.

The psaC gene product from Synechococcus sp. PCC 7002 and the psaD gene product from Nostoc sp. PCC 8009 were synthesized in Escherichia coli and purified to homogeneity. Incubation of the PsaC apoprotein with the Synechoccus sp. PCC 6301 photosystem I core protein in the presence of FeCl3, Na2S, and beta-mercaptoethanol resulted in a time-dependent transition in the flash-induced absorption change from a 1.2-ms, P700+ FX- back-reaction to a long-lived, P700+ [FA/FB]- back-reaction. ESR studies showed that FB and FA were photoreduced about equally at 19 K, and while the resonances were shifted upfield, they remained as broad as in the free PsaC holoprotein. When the reconstituted complex was purified in a sucrose gradient containing 0.1% Triton X-100, most of the optical absorption transient reverted to that characteristic of the P700+ FX- back-reaction. Addition of purified PsaD to the incubation mixture led to a greater extent of recovery of electron flow to FA/FB for any given concentration of PsaC. ESR studies showed that FA, rather than FB, became the preferred electron acceptor at 19 K; moreover, the resonances moved upfield and sharpened to become nearly identical with those of a control photosystem I complex. When the sample was purified in a sucrose gradient containing 0.1% Triton X-100, the long-lived P700+ [FA/FB]- optical transient remained stable. Analysis by denaturing polyacrylamide gel electrophoresis showed that the PsaC and PsaD proteins had rebound to the photosystem I core. The data indicate that although PsaC can bind loosely, the presence of PsaD leads to a stable, isolatable photosystem I complex which is spectroscopically indistinguishable from the native complex. Since a PsaC1 fusion protein which contains an amino-terminal extension of five amino acids (MEHSM...) does not bind in the absence of PsaD [Zhao, J., et al. (1990) FEBS Lett. 276, 175-180], the N-terminus of the PsaC protein could provide a site of interaction with the photosystem I core. We propose that the binding of PsaC to the PsaA/PsaB heterodimer is potentiated by insertion of the FA/FB clusters into PsaC, and stabilized by the presence of PsaD.

Partial conservation of the 5? ndhE-psaC-ndhD 3? gene arrangement of chloroplasts in the cyanobacterium Synechocystis sp. PCC 6803: implications for NDH-D function in cyanobacteria and chloroplasts

The psaC gene, which encodes the 8.9 kDa iron-sulfur containing subunit of Photosystem I, has been sequenced from Synechocystis sp. PCC 6803 and shows greater similarity to reported plant sequences than other cyanobacterial psaC sequences. The deduced amino acid sequence of the protein encoded by the Synechocystis psaC gene is identical to the tobacco PSA-C sequence. In plants psaC is located in the small single-copy region of the chloroplast genome between two genes (designated ndhE and ndhD) with similarity to genes encoding subunits of the mitochondrial NADH Dehydrogenase Complex I. The 5' ndhE-psaC-ndhD3' gene arrangement of higher plants is only partially conserved in Synechocystis. An open reading frame (ORF) upstream of the Synechocystis psaC gene has 85% identity to the tobacco ndhE gene. Downstream of psaC there is a 273 bp ORF with 48% identity to the 5' portion of the tobacco ndhD gene (1527 bp). psaC, ndhE and the region of similarity to ndhD are present in a single copy in the Synechocystis genome. Part of the wheat ndhD gene was sequenced and used as a probe for the presence of the 3' portion of the ndhD gene. The wheat ndhD probe did not hybridize to Synechocystis or Anabaena sp. PCC 7120 genomic DNA, but did hybridize to Oenothera chloroplast DNA. These results indicate the complete ndhD gene is absent in two cyanobacteria, and raises the question of what role, if any, the ndhD gene product plays in the facultative heterotroph Synechocystis sp. PCC 6803.

Reconstitution of the iron-sulfur clusters in the isolated F A/F B protein: EPR spectral characterization of same-species and cross-species Photosystem I complexes

The ESR spectra of the F A /F B iron-sulfur clusters in spinach and Synechococcus sp. PCC 6301 Photosystem I complexes differ slightly, but characteristically. In the fully reduced state, the spinach F A /F B resonances appear at g = 2.051, 1.941, 1.923, 1.887, while the Synechococcus sp. PCC 6301 F A /F B resonances appear at g = 2.047, 1.937, 1.917 and 1.879; both are independent of the details of Photosystem I isolation or the method of reduction. When the spinach or Synechococcus sp. PCC 6301 F A /F B holoprotein is removed from the thylakoid membrane by solvent extraction, the ESR resonances broaden and the iron-sulfur clusters become labile, degrading rapidly to the level of zero-valence sulfur. We show that the clusters can be reinserted in vitro by incubating the isolated F A /F B apoprotein for 12 h with FeCl 3 and Na 2 S in the presence of β-mercaptoethanol under strictly anaerobic conditions. Upon chemical reduction with Na 2 S 2 O 4 at pH 10, the rebuilt spinach F A /F B protein shows the broadened resonances of F A and F B , but when rebound to a Synechococcus sp. PCC 6301 Photosystem I core protein, the hybrid spinach- Synechococcus Photosystem I complex shows sharpened resonances with g -values of 2.052, 1.941, 1.922 and 1.886. These g -values are similar to those of the native spinach Photosystem I complex. In contrast, when a spinach or Synechococcus sp. PCC 6301 F A /F B holoprotein is rebound to a Photosystem I core protein from the same species, the F A /F B resonances appear identical to their respective control Photosystem I complexes. These results indicate that the altered ESR spectrum reported earlier on reconstitution of a solvent-extracted spinach F A /F B holoprotein with a Synechococcus sp. PCC 6301 Photosystem I core protein (Golbeck et al. (1988) FEBS Lett. 240, 9–14) is a consequence of the cross-species reconstitution and does not result from damage to the F A /F B holoprotein incurred during isolation. This in vitro reconstitution protocol circumvents the need to isolate the labile F A /F B holoprotein, and makes possible reinsertion of the iron-sulfur clusters following modification of the F A /F B apoprotein or the psaC gene.

Reconstitution of electron transport in photosystem I with PsaC and PsaD proteins expressed in Escherichia coli

A fusion protein, denoted PsaCl, which contains an amino-terminal extension of five amino acids (MEHSM...) and is derived from an in vitro modified form of the psaC gene of Synechococcus sp. PCC 7002, has been over-expressed in Escherichia coli. The product of the psaD gene of Nostoc sp. PCC 8009 has similarly been over-expressed. The PsaCl and PsaD proteins can be combined with the photosystem I core protein of Synechococcus sp. PCC 6301 to reconstitute electron transport from P700 to the terminal F A/F B acceptors. Reconstitution was found to be absolutely dependent on reinsertion of the iron-sulfur clusters in the PsaCI apoprotein and on the presence of the PsaD protein. This implies that the PsaCl holoprotein does not bind solely to the PsaA/PsaB heterodimer but rather that its interaction with these proteins is mediated through the PsaD protein.

Structural and functional properties of the cyanobacterial photosystem I complex

Photosystem I (PSI) complexes have been isolated from two cyanobacterial strains, Synechococcus sp. PCC 7002 and 6301. These complexes contain six to seven low molecular mass subunits in addition to the two high molecular mass subunits previously shown to bind the primary reaction center components. Chemical cross-linking of ferredoxin to the complex identified a 17.5-kDa subunit as the ferredoxin-binding protein in the Synechococcus sp. PCC 6301-PSI complex. The amino acid sequence of this subunit, deduced from the DNA sequence of the gene, confirmed its identity as the psaD gene product. A 17-kDa subunit cross-links to the electron donor, cytochrome c-553, in a manner analogous to the cross-linking of plastocyanin to the higher plant PSI complex. Using antibodies raised against the spinach psaC gene product (a 9-kDa subunit which binds Fe-S centers A and B), we identified an analogous protein in the cyanobacterial PSI complex.