psbE Genes Research Articles

Isolation of highly active photosystem II core complexes with a His-tagged Cyt b559 subunit from transplastomic tobacco plants

Photosystem II (PSII) is a huge multi-protein-complex consisting, in higher plants and green algae, of the PS II core and the adjacent light harvesting proteins. In the study reported here, N-terminal His-tags were added to the plastome-encoded α-subunit of cytochrome b 559 , PsbE, in tobacco plants, thus facilitating rapid, mild purification of higher plant PSII. Biolistic chloroplast transformation was used to replace the wildtype psbE gene by His-tagged counterparts. Transgenic plants did not exhibit an obvious phenotype. However, the oxygen evolution capacity of thylakoids prepared from the mutants compared to the wildtype was reduced by 10–30% depending on the length of the His-tag, although Fv/Fm values differed only slightly. Homoplasmic F1 plants were used to isolate PSII cores complexes. The cores contained no detectable traces of LHC or PsaA/B polypeptides, but the main core subunits of PSII could be identified using immunodetection and mass spectroscopy. In addition, Psb27 and PsbS were detected. The presence of the former was presumably due to the preparation method, since PSII complexes located in the stroma are also isolated. In contrast to previous reports, PsbS was solely found as a monomer on SDS-PAGE in the PSII core complexes of tobacco.

High Conservation of a 5′ Element Required for RNA Editing of a C Target in Chloroplast psbE Transcripts

C-to-U editing modifies 30-40 distinct nucleotides within higher-plant chloroplast transcripts. Many C targets are located at the same position in homologous genes from different plants; these either could have emerged independently or could share a common origin. The 5' sequence GCCGUU, required for editing of C214 in tobacco psbE in vitro, is one of the few identified editing cis-elements. We investigated psbE sequences from many plant species to determine in what lineage(s) editing of psbE C214 emerged and whether the cis-element identified in tobacco is conserved in plants with a C214. The GCCGUU sequence is present at a high frequency in plants that carry a C214 in psbE. However, Sciadopitys verticillata (Pinophyta) edits C214 despite the presence of nucleotide differences compared to the conserved cis-element. The C214 site in psbE genes is represented in members of four branches of spermatophytes but not in gnetophytes, resulting in the parsimonious prediction that editing of psbE C214 was present in the ancestor of spermatophytes. Extracts from chloroplasts from a species that has a difference in the motif and lacks the C target are incapable of editing tobacco psbE C214 substrates, implying that the critical trans-acting protein factors were not retained without a C target. Because noncoding sequences are less constrained than coding regions, we analyzed sequences 5' to two C editing targets located within coding regions to search for possible editing-related conserved elements. Putative editing cis-elements were uncovered in the 5' UTRs near editing sites psbL C2 and ndhD C2.

Knock-out of the plastid ribosomal protein L11 in Arabidopsis: effects on mRNA translation and photosynthesis.

The prpl11-1 mutant of Arabidopsis thaliana was identified among a collection of T-DNA tagged lines on the basis of a decrease in the effective quantum yield of photosystem II. The mutation responsible was localized to Prpl11, a single-copy nuclear gene that encodes PRPL11, a component of the large subunit of the plastid ribosome. The amino acid sequence of Arabidopsis PRPL11 is very similar to those of L11 proteins from spinach and prokaryotes. In the prpl11-1 mutant, photosensitivity and chlorophyll fluorescence parameters are significantly altered owing to changes in the levels of thylakoid protein complexes and stromal proteins. The abundance of most plastome transcripts examined, such as those of genes coding for the photosystem II core complex and RbcL, is not decreased. Plastid ribosomal RNA accumulates in wild-type amounts, and the assembly of plastid polysomes on the transcripts of the rbcL, psbA and psbE genes remains mainly unchanged in mutant plants, indicating that lack of PRPL11 affects neither the abundance of plastid ribosomes nor their assembly into polysomes. However, in vivo translation assays demonstrate that the rate of translation of the large subunit of Rubisco (RbcL) is significantly reduced in prpl11-1 plastids. Our data suggest a major role for PRPL11 in plastid ribosome activity per se, consistent with its location near the GTPase-binding centre of the chloroplast 50S ribosomal subunit. Additional effects of the mutation, including the pale green colour of the leaves and a drastic reduction in growth rate under greenhouse conditions, are compatible with reduced levels of protein synthesis in plastids.

The Chloroplast-encoded α Subunit of Cytochromeb-559 Is Required for Assembly of the Photosystem Two Complex in both the Light and the Dark in Chlamydomonas reinhardtii

The role of cytochrome b-559 in the photosystem two (PSII) complex has been investigated through the construction of a psbE null mutant by transformation of the chloroplast genome of the green alga Chlamydomonas reinhardtii. No PSII activity could be detected in this mutant either in oxygen evolution assays or by analysis of variable chlorophyll fluorescence. Immunoblotting experiments showed that the absence of PSII activity in the mutant was due to the loss of the PSII complex in both light-grown and dark-grown cultures. In contrast, the photosystem one reaction center polypeptide, PsaA, was present at wild-type levels in the mutant. RNA gel blot assays confirmed that the transcript levels for the psbA, psbD, and psbF genes were unaffected by disruption of the psbE gene, suggesting a post-transcriptional effect on their expression. Pulse-labeling experiments showed that either synthesis of PSII subunits was impaired in the psbE null mutant or there was extremely rapid degradation of newly synthesized subunits. Interestingly, the PsbE and PsbF subunits accumulated to wild-type levels in a psbA deletion mutant of C. reinhardtii, FuD7, which fails to synthesize D1 and assemble PSII. Our results provide evidence for a role for cytochrome b-559 in the early steps of assembly of the PSII complex, possibly as a redox-controlled nucleation factor that determines the level of PSII within the thylakoid membrane.

An unusual organization of the genes encoding cytochrome b559 in Chlamydomonas reinhardtii: psbE and psbF genes are separately transcribed from different regions of the plastid chromosome.

The psbE and psbF genes encode the apoproteins of cytochrome b559, an essential component of the pigment protein complex photosystem II. Together with psbL and psbJ, these genes constitute a single operon in all photosynthetic organisms examined thus far. We have cloned and sequenced the psbE and psbF genes of the Chlamydomonas reinhardtii plastid genome. The predicted amino-terminal domains of both polypeptides are more basic than those of other organisms, and the sequence of the psbE gene product indicates a departure from the 'positive-inside' rule for the insertion of proteins in the thylakoid membrane. Northern blot analysis demonstrated that psbE is transcribed into a 0.3 kb mRNA, while transcription of psbF and psbL genes results in a 0.9 kb transcript. The splitting of the psbEFLJ operon into separate transcription units suggests a unique mechanism of regulation of expression of these genes in C. reinhardtii.

Nucleotide sequence of the psbE, psbF and trnM genes from the chloroplast genome of Chlamydomonas reinhardtii

We have determined the nucleotide sequences of the psbE and psbF genes, which encode the α and β subunits, respectively, of cytochrome b-559, from the chloroplast genome of the green alga Chlamydomonas reinhardtii. In contrast to other organisms psbE is not co-transcribed with psbF. The primary structures of the gene products are very similar to the equivalent proteins in cyanobacteria and plants. Each subunit contains a single histidine residue that is thought to ligate haem. Upstream of the psbE gene, a trnM gene is located which encodes an elongator tRNA Met molecule.

The psaA operon pre‐mRNA of the Euglena gracilis chloroplast is processed into photosystem I and II mRNAs that accumulate differentially depending on the conditions of cell growth

The chloroplast genome of Euglena gracilis contains a psaA operon which encodes a lysine tRNA gene, trnK; psaA and psaB photosystem I genes, and psbE, psbF, psbL and psbJ photosystem II genes. The pre-mRNA of the psaA operon undergoes a complex processing pathway of 5' and 3' tRNA processing, splicing of 11 group II introns and one group II twintron, plus three intercistronic RNA cleavage events. The accumulated transcripts of the psaA operon have been characterized by Northern hybridization, S1 nuclease analysis and primer extension RNA sequencing. The mature 5' end of the psaA-psaB-psbE-psbF-psbL-psbJ hexacistronic transcript lies 8 nt downstream of the trnK gene, and is the result of intercistronic trnK-psaA cleavage. Other intercistronic processing events occur between the psaA and psaB genes and the psaB and psbE genes. Processing at the latter site produces a dicistronic mRNA of PSI genes and a tetracistronic mRNA of PSII genes. The PSI dicistronic transcript is further processed to monocistronic psaA and psaB mRNAs. Secondary structural motifs within the intercistronic regions may be recognition sites for processing. The steady-state levels of psaA operon mRNAs from Euglena grown under several different conditions have been determined. Accumulated transcripts from all growth conditions are spliced, and a proportion are also processed at the intercistronic sites. The products of intercistronic processing increase from heterotrophic dark- to heterotrophic light-grown Euglena, and from heterotrophic light- to photoautotrophic light-grown Euglena. The differential accumulation of psaA operon mRNAs may be a means of chloroplast gene regulation or, alternatively, a consequence of gene expression during chloroplast development.

Truncation of the COOH-terminal domain of the psbE gene product in Synechocystis sp. PCC 6803: requirements for photosystem II assembly and function.

The COOH-terminal domain of the 80-residue cytochrome b559 alpha-subunit (psbE gene product) in Synechocystis sp. PCC 6803 was sequentially truncated in order to determine the minimum polypeptide length needed for function and assembly. A stop codon was introduced into the Arg-50, Arg-59, or Tyr-69 codons of the psbE gene, generating mutants truncated by 31, 22, and 12 residues, respectively. Removal of 12 residues caused a decrease of 20% in PSII function. Truncation of 22 or 31 residues caused a large decrease (60-85%) in the photoautotrophic growth rate, the rate of O2 evolution, and the amplitude of the 77 K 696-nm fluorescence, and a concomitant increase in the constant yield fraction (F0/Fmax) of the chlorophyll fluorescence. The level of residual activity in the Arg50-stop mutant was 10-20% of the wild type, which was reflected in a similar low level of immunochemically detected D2 polypeptide. Quantitation of the PSII reaction center stoichiometry of the Arg50-stop mutant by analysis of [14C]DCMU binding also showed a 5-fold decrease (1:910 Chl in wild type and 1:5480 Chl in R50) in the PSII reaction center concentration. However, the KD value for DCMU in the residual 15% of the complexes to which it bound was approximately equal to that (25 nM) of the wild type. Northern blot analysis showed no decrease in the b559 psbE mRNA level. Chemical difference spectral analysis of heme content indicated that the level of native cytochrome b559 heme in the Arg50-stop mutant (1:640 Chl) was 80% that of wild type (1:510 Chl).(ABSTRACT TRUNCATED AT 250 WORDS)

The D1 protein of the photosystem II reaction‐centre complex accumulates in the absence of D2: analysis of a mutant of the cyanobacterium Synechocystis sp. PCC 6803 lacking cytochrome d559

The reaction center core of photosystem II, a multiprotein membrane bound complex, is composed of a heterodimer of two proteins, D1 and D2. A random mutagenesis technique was used to isolate a photosystem II deficient mutant, CP6t16, of the unicellular cyanobacterium, Synechocystis sp. PCC 6803. Nucleotide sequence analysis showed that the primary lesion in CP6t16 is an ochre mutation introducing a translational stop codon in the psbE gene, encoding the alpha-subunit of cytochrome b559, an integral component of the PSII complex. Analysis of the protein composition of CP6t16 thylakoid membranes isolated in the presence of serine protease inhibitors revealed that, in the absence of cytochrome b559, the D2 protein is also absent. However, the D1 protein is stably incorporated in these membranes, suggesting that the synthesis and integration of D1 are independent of those of D2 and cytochrome b559.

Thylakoid membrane protein topography: transmembrane orientation of the chloroplast cytochrome b-559 psbE gene product.

Protease accessibility and antibody to a COOH-terminal peptide were used as probes for the in situ topography of the Mr 10,000 psbE gene product (alpha subunit) of the chloroplast cytochrome b-559. Exposure of thylakoid membranes to trypsin or Staphylococcus aureus V8 protease cleaved the alpha subunit to a slightly smaller polypeptide (delta Mr approximately -1000) as detected on Western blots, without loss of reactivity to COOH-terminal antibody. The disappearance of the parent Mr 10,000 polypeptide from thylakoids in the presence of trypsin correlated with the appearance of the smaller polypeptide with delta Mr = -750, the conversion having a half-time of approximately 15 min. Exposure of inside-out vesicles to trypsin resulted in almost complete loss of reactivity to the antibody, showing that the COOH terminus is exposed on the lumenal side of the membrane. Removal of the extrinsic polypeptides of the oxygen-evolving complex resulted in an increase of the accessibility of the alpha subunit to trypsin. These data establish that the alpha subunit of cytochrome b-559 crosses the membrane once, as predicted from its single, 26-residue, hydrophobic domain. The NH2 terminus of the alpha polypeptide is on the stromal side of the membrane, where it is accessible, most likely at Arg-7 or Glu-6/Asp-11, to trypsin or V8 protease, respectively. As a consequence of this orientation, the single histidine residue in the alpha subunit is located on the stromal side of the hydrophobic domain.(ABSTRACT TRUNCATED AT 250 WORDS)

Targeted mutagenesis of the psbE and psbF genes blocks photosynthetic electron transport: evidence for a functional role of cytochrome b559 in photosystem II.

The genes encoding the two subunits (alpha and beta) of the cytochrome b559 (cyt b559) protein, psbE and psbF, were cloned from the unicellular, transformable cyanobacterium, Synechocystis 6803. Cyt b559, an intrinsic membrane protein, is a component of photosystem II, a membrane-protein complex that catalyzes photosynthetic oxygen evolution. However, the role of cyt b559 in photosynthetic electron transport is yet to be determined. A high degree of homology was found between the cyanobacterial and green plant chloroplastidic psbE and psbE genes and in the amino acid sequences of their corresponding protein products. Cartridge mutagenesis techniques were used to generate a deletion mutant of Synechocystis 6803 in which the psbE and psbF genes were replaced by a kanamycin-resistance gene cartridge. Physiological analyses indicated that the PSII complexes of the mutant were inactivated. We conclude that cyt b559 is an essential component of PSII.

Nucleotide sequence of the genes encoding cytochrome b-559 from the cyanelle genome of Cyanophora paradoxa

Cyanophora paradoxa is a flagellated protozoan which possesses unusual, chloroplast-like organelles referred to as cyanelles. The psbE and psbF genes, which encode the two apoprotein subunits of cytochrome b-559, have been cloned from the cyanelle genome of C. paradoxa. The complete nucleotide sequences of these genes and their flanking sequences were determined by the chain-termination, dideoxy method. The psbE gene is composed of 75 codons and predicts a polypeptide of 8462 Da that is seven to nine residues smaller than most other psbE gene products. The psbF gene consists of 43 codons and predicts a polypeptide of 4761 Da. Two open reading frames, whose sequences are highly conserved among cyanobacteria and numerous higher plants, were located in the nucleotide sequence downstream from the psbF gene. The first open reading frame, denoted psbI, is composed of 39 codons, while the second open reading frame, denoted psbJ, is composed of 41 codons. The predicted amino acid sequences of the psbI and psbJ gene products predict proteins of 5473 and 3973 Da respectively. These proteins are probably integral membrane proteins anchored in the membrane by a single, transmembrane alpha helix. The psbEFIJ genes are probably co-transcribed and constitute an operon as found for other organisms. Each of the four genes is preceded by a polypurine sequence which resembles the consensus ribsosome binding sequences for Escherichia coli.

Characterization and in vitro expression of the cytochrome b-559 genes of barley. II. In vitro transcription and translation.

The two cytochrome b-559 apoproteins of 9.4 kD and 4.5 kD molecular weight have been expressed in vitro using DNA templates containing either the two genes psbE and psbF in tandem or the individual genes. Transcription with E. coli RNA-polymerase or SP6 RNA-polymerase has been followed by translation in E. coli derived lysates. Simultaneous as well as independent synthesis of the apoproteins is possible. A 9.4 kD in vitro translation product has been identified as apoprotein I by immunoprecipitation with a monoclonal antibody specific for the C-terminal part of the 9.4 kD apoprotein of cytochrome b-559. The isolated psbF gene directs the synthesis of a translation product with a molecular weight of 4.5 kD corresponding to apoprotein II. Expression of the psbE gene requires the presence of endogenous regulatory sequences 5' upstream of psbE, while this is not the case for psbF. Additional in vitro translation products of 5.7 and 2.4 kD molecular weights are synthesized and probably translated from two reading frames starting with two different out-of-phase ATG codons in the nucleotide sequence of the psbE gene.

Stoichiometry and turnover of photosystem II polypeptides

Using Triton X-100 solubilization and sucrose gradient fractionation, a photosystem II core complex has been isolated from the green alga Dunaliella salina. The complex was similar to that previously isolated from spinach [(1985) FEBS Lett. 188, 68-72] being composed of five main polypeptides of apparent molecular masses, 47, 40, 32, 30 and 10 kDa. Studies using 14C indicate that with either dark or illuminated cells the Stoichiometry of these polypeptides is 1:1:1:1:1 with about 20 chlorophyll a molecules per monomeric unit. According to 35S labelling only the 32 kDa polypeptide, identified as the D1 or Q B binding protein by immunoblotting, is rapidly turning over at a rate greater than the growth rate. The level of labelled sulphur associated with each polypeptide is reasonably consistent with the amino acid content derived from gene sequences and identifies the 47, 40, 32, 30 and 10 kDa apoproteins as products of the psbB, psbC, psbA, psbD and psbE genes.