psbF Genes Research Articles

The Chloroplast Genome of Endive (Cichorium endivia L.): Cultivar Structural Variants and Transcriptome Responses to Stress Due to Rain Extreme Events.

The chloroplast (cp) genome diversity has been used in phylogeny studies, breeding, and variety protection, and its expression has been shown to play a role in stress response. Smooth- and curly-leafed endives (Cichorium endivia var. latifolium and var. crispum) are of nutritional and economic importance and are the target of ever-changing breeding programmes. A reference cp genome sequence was assembled and annotated (cultivar 'Confiance'), which was 152,809 base pairs long, organized into the angiosperm-typical quadripartite structure, harboring two inverted repeats separated by the large- and short- single copy regions. The annotation included 136 genes, 90 protein-coding genes, 38 transfer, and 8 ribosomal RNAs and the sequence generated a distinct phyletic group within Asteraceae with the well-separated C. endivia and intybus species. SSR variants within the reference genome were mostly of tri-nucleotide type, and the cytosine to uracil (C/U) RNA editing recurred. The cp genome was nearly fully transcribed, hence sequence polymorphism was investigated by RNA-Seq of seven cultivars, and the SNP number was higher in smooth- than curly-leafed ones. All cultivars maintained C/U changes in identical positions, suggesting that RNA editing patterns were conserved; most cultivars shared SNPs of moderate impact on protein changes in the ndhD, ndhA, and psbF genes, suggesting that their variability may have a potential role in adaptive response. The cp transcriptome expression was investigated in leaves of plants affected by pre-harvest rainfall and rainfall excess plus waterlogging events characterized by production loss, compared to those of a cycle not affected by extreme rainfall. Overall, the analyses evidenced stress- and cultivar-specific responses, and further revealed that genes of the Cytochrome b6/f, and PSI-PSII systems were commonly affected and likely to be among major targets of extreme rain-related stress.

Whole-transcriptome RNA-seq, gene set enrichment pathway analysis, and exon coverage analysis of two plastid RNA editing mutants

ABSTRACTIn land plants, plastid and mitochondrial RNAs are subject to post-transcriptional C-to-U RNA editing. T-DNA insertions in the ORGANELLE RNA RECOGNITION MOTIF PROTEIN6 gene resulted in reduced photosystem II (PSII) activity and smaller plant and leaf sizes. Exon coverage analysis of the ORRM6 gene showed that orrm6–1 and orrm6–2 are loss-of-function mutants. Compared to other ORRM proteins, ORRM6 affects a relative small number of RNA editing sites. Sanger sequencing of reverse transcription-PCR products of plastid transcripts revealed 2 plastid RNA editing sites that are substantially affected in the orrm6 mutants: psbF-C77 and accD-C794. The psbF gene encodes the β subunit of cytochrome b559, an essential component of PSII. The accD gene encodes the β subunit of acetyl-CoA carboxylase, a protein required in plastid fatty acid biosynthesis. Whole-transcriptome RNA-seq demonstrated that editing at psbF-C77 is nearly absent and the editing extent at accD-C794 was significantly reduced. Gene set enrichment pathway analysis showed that expression of multiple gene sets involved in photosynthesis, especially photosynthetic electron transport, is significantly upregulated in both orrm6 mutants. The upregulation could be a mechanism to compensate for the reduced PSII electron transport rate in the orrm6 mutants. These results further demonstrated that Organelle RNA Recognition Motif protein ORRM6 is required in editing of specific RNAs in the Arabidopsis (Arabidopsis thaliana) plastid.

An Organelle RNA Recognition Motif Protein Is Required for Photosystem II Subunit psbF Transcript Editing.

Loss-of-function mutations in ORGANELLE RNA RECOGNITION MOTIF PROTEIN6 (ORRM6) result in the near absence of RNA editing of psbF-C77 and the reduction in accD-C794 editing in Arabidopsis (Arabidopsis thaliana). The orrm6 mutants have decreased levels of photosystem II (PSII) proteins, especially PsbF, lower PSII activity, pale green pigmentation, smaller leaf and plant sizes, and retarded growth. Stable expression of ORRM6 rescues the orrm6 editing defects and mutant phenotype. Unlike ORRM1, the other known ORRM plastid editing factor, ORRM6, does not contain RNA editing interacting protein/multiple organellar RNA editing factor (RIP/MORF) boxes, which are required for ORRM1 to interact with site-specific pentatricopeptide repeat protein editing factors. ORRM6 interacts with RIP1/MORF8, RIP2/MORF2, and RIP9/MORF9, known components of RNA editosomes. While some plastid RRM proteins are involved in other forms of RNA processing and translation, the primary function of ORRM6 is evidently to mediate psbF-C77 editing, like the essential site-specific pentatricopeptide repeat protein LOW PSII ACCUMULATION66. Stable expression in the orrm6 mutants of a nucleus-encoded, plastid-targeted PsbF protein from a psbF gene carrying a T at nucleotide 77 significantly increases leaf and plant sizes, chlorophyll content, and PSII activity. These transformants demonstrate that plastid RNA editing can be bypassed through the expression of nucleus-encoded, edited forms of plastid genes.

Spectroscopic and Functional Characterizations of Cyanobacterium Synechocystis PCC 6803 Mutants on and near the Heme Axial Ligand of Cytochrome b559 in Photosystem II

The functional role of cytochrome (cyt) b(559) in photosystem II (PSII) was investigated in H22K alpha and Y18S alpha cyt b(559) mutants of the cyanobacterium Synechocystis sp. PCC6803. H22K alpha and Y18S alpha cyt b(559) mutant carries one amino acid substitution on and near one of heme axial ligands of cyt b(559) in PSII, respectively. Both mutants grew photoautotrophically, assembled stable PSII, and exhibited the normal period-four oscillation in oxygen yield. However, both mutants showed several distinct chlorophyll a fluorescence properties and were more susceptible to photoinhibition than wild type. EPR results indicated the displacement of one of the two axial ligands to the heme of cyt b(559) in H22K alpha mutant reaction centers, at least in isolated reaction centers. The maximum absorption of cyt b(559) in Y18S alpha mutant PSII core complexes was shifted to 561 nm. Y18S alpha and H22K alpha mutant PSII core complexes contained predominately the low potential form of cyt b(559). The findings lend support to the concept that the redox properties of cyt b(559) are strongly influenced by the hydrophobicity and ligation environment of the heme. When the cyt b(559) mutations placed in a D1-D170A genetic background that prevents assembly of the manganese cluster, accumulation of PSII is almost completely abolished. Overall, our data support a functional role of cyt b(559) in protection of PSII under photoinhibition conditions in vivo.

Recombinant PsbF from Synechococcus sp. PCC 7002 forms b︰b homodimeric cytochrome b559

All organisms with oxygenic photosynthesis contain two photosystems: photosystem I (PS I) and photosystem II (PS II). The minimal photosystem II particles which are photochemically active contain three subunits: D1, D2 and cytochrome b559 (Cyt b559). The function of Cyt b559 remains unclear. We have successfully overexpressed the psbF gene, encoding the β subunit of Cyt b559, from a marine cyanobacterium Synechococcus sp. PCC 7002 as a fusion gene and obtained a redox-active form of Cyt b559. When the N-terminal GST protein of the fusion gene product was removed with thrombin, the PsbF protein was still redox-active, suggesting that the recombinant PsbF can form dimer in Escherichia coli. The absorption spectra of either the oxidized form or the reduced form of both GST fusion protein and the purified PsbF dimer and the difference spectra between the two forms are the same as that of the Cyt b559 isolated from the higher plants. Redox titration analysis of recombinant PsbF showed that the mid-point redox potential of the recombinant Cyt b559 was approximately 50 mV, which is close to the low potential of Cyt b559. The results are helpful to the understanding of localization and function of Cyt b559 on thylakoid membranes.

In vitro assembly of a β 2 cytochrome b559-like complex from the chemically synthesised β-subunit encoded by the Synechocystis sp. 6803 psbF gene

The α- and β-subunits of cytochrome b 559 encoded by the psbE and psbF gene, respectively, are essential components of photosystem II. The exact structure of this cytochrome is not yet known. The β-subunit of the Synechocystis sp. 6803 cytochrome b 559 complex was synthesised by means of solid-phase peptide synthesis. Under reducing conditions, two β-peptide molecules could be assembled specifically with one haem to form a β 2 cytochrome b 559-like complex. The spectral properties and the midpoint redox potential (48±5 mV) of the in vitro assembled β 2 cytochrome are nearly identical to those of the low potential form of the native cytochrome b 559.

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.

Introduction of a heterologous editing site into the tobacco plastid genome: the lack of RNA editing leads to a mutant phenotype.

The psbF mRNA is edited in spinach plastids by a C to U conversion, changing a serine to a conserved phenylalanine codon. In tobacco at this position a phenylalanine codon is present at the DNA level, and the psbF mRNA here is not edited. To test if the psbF editing capacity is evolutionarily conserved, the tobacco psbF gene was modified to match the corresponding spinach sequence. The endogenous tobacco gene was replaced with the modified copy using biolistic transformation. We report here that the heterologous editing site remains unmodified in transplastomic tobacco plants. The lack of editing is associated with slower growth, lowered chlorophyll content and high chlorophyll fluorescence, a phenotype characteristic of photosynthetic mutants. This finding confirms that the editing of the psbF mRNA is an essential processing step for protein function and thus provides direct proof for the biological significance of plant organellar RNA editing. Given that a mutant phenotype is associated with the lack of editing, it seems likely that the evolutionary loss of the site-specific capacity for psbF editing was preceded by the mutation that eliminated the editing requirement.

Site directed mutagenesis of the heme axial ligands of cytochrome b559 affects the stability of the photosystem II complex.

Cytochrome (cyt) b559, an integral membrane protein, is an essential component of the photosystem II (PSII) complex in the thylakoid membranes of oxygenic photosynthetic organisms. Cyt b559 has two subunits, alpha and beta, each with one predicted membrane spanning alpha-helical domain. The heme cofactor of this cytochrome is coordinated between two histidine residues. Each of the two subunit polypeptides of cyt b559 has one His residue. To investigate the influence of these His residues on the structure of cyt b559 and the PSII complex, we used a site directed mutagenesis approach to replace each His residue with a Leu residue. Introduction of these missense mutations in the transformable unicellular cyanobacterium, Synechocystis 6803, resulted in complete loss of PSII activity. Northern blot analysis showed that these mutations did not affect the stability of the polycistronic mRNA that encompasses both the psbE and the psbF genes, encoding the alpha and the beta subunits, respectively. Moreover, both of the single His mutants showed the presence of the alpha subunit which was 1.5 kd smaller than the same polypeptide in wild type cells. A secondary effect of such a structural change was that D1 and D2, two proteins that form the catalytic core (reaction center) of PSII, were also destabilized. Our results demonstrate that proper axial coordination of the heme cofactor in cyt b559 is important for the structural integrity of the reaction center of PSII.

Group II twintron: an intron within an intron in a chloroplast cytochrome b-559 gene.

The psbF gene of chloroplast DNAs encodes the beta-subunit of cytochrome b-559 of the photosystem II reaction center. The psbF locus of Euglena gracilis chloroplast DNA has an unusual 1042 nt group II intron that appears to be formed from the insertion of one group II intron into structural domain V of a second group II intron. Using both direct primer extension cDNA sequencing and cDNA cloning and sequencing, we have determined that a 618 nt internal intron is first excised from the 1042 nt intron of psbF pre-mRNA, resulting in a partially spliced pre-mRNA containing a 424 nt group II intron with a spliced domain V. The 424 nt intron is then removed to yield the mature psbF mRNA. Therefore, the 1042 nt intron of psbF is a group II intron within another group II intron. We use the term 'twintron' to define this new type of genetic element. Intermediates in the splicing pathway were detected by northern hybridization. Splicing of both the internal and external introns occurs via lariat intermediates. Twintron splicing was found to proceed by a sequential pathway, the internal intron being removed prior to the excision of the external intron. A possible mechanism for twintron formation by intron transposition is discussed.

Targeted Deletion Mutagenesis of the β Subunit of Cytochrome b559 Protein Destabilizes the Reaction Center of Photosystem II

Oligonucleotide-directed mutagenesis techniques were used to delete the psbF gene, encoding the beta subunit of the cytochrome b559 protein of the photosystem II complex in the cyanobacterium, Synechocystis 6803. Cyt b559 is an integral component of PS II complex. However, its precise functional role in PS II remains to be determined. Previously, we created a mutant in which the psbF gene as well as three of its neighbouring genes, psbE, psbL and psbJ were simultaneously deleted from the chromosome of Synechocystis 6803 (Pakrasi, Williams and Arntzen, EMBO J. 7, 325-332, 1988). This mutant had no PS II activity. However, the role of any one of the four individual gene products could not be determined by studying this mutant. The newly generated mutant, T256, had only one gene, psbF, deleted from the genome. This mutant was also impaired in its PS II activities. In addition, it had barely detectable levels of two other protein components, D1 (herbicide binding protein) and D2, of the reaction center of PS II, in its thylakoid membranes. In contrast, two other proteins of PS II, CP47 and CP43 were present in appreciable amounts. Fluorescence spectra (77 K) of the mutant showed the absence of a peak at 695 nm that was previously believed to originate from CP47. In addition, phycobilisomes, the light-harvesting antenna system of PS II, were found to be assembled normally in this mutant. We conclude that the presence of the beta subunit of Cyt b559 in the thylakoid membranes is critically important for the assembly of PS II reaction center.

A photosystem II polypeptide is encoded by an open reading frame co-transcribed with genes for cytochrome b-559 in wheat chloroplast DNA

The N-terminal amino acid sequence of a 3.2 kDa photosystem II polypeptide is shown to be identical to that of a polypeptide encoded by an open reading frame of 38 codons (orf38) in wheat chloroplast DNA. Orf38 is located just downstream of the psbE and psbF genes for the polypeptides of cytochrome b-559. Analysis of the transcription of this region of chloroplast DNA shows that psbE, psbF and orf38 are co-transcribed to give a 1.1 kb polycistronic transcript which also contains another open reading frame of 40 codons. The orf38 and orf40 products are hydrophobic polypeptides which are both predicted to span the thylakoid membrane once. Orf38 and orf40 are highly conserved, and map to similar locations adjacent to psbE and psbF, in all organisms from which this region of DNA has been sequenced. We propose that orf38 is named psbL.

A fifth chloroplast-encoded polypeptide is present in the photosystem II reaction centre complex

The small polypeptides present in the pea photosystem II reaction centre complex have been separated by high-resolution SDS-polyacrylamide gel electrophoresis and characterised by N-terminal amino acid sequencing. Two polypeptides are identified as the α- and β-subunits of cytochrome b559 which are the products of the pdbE and psbF genes. A third polypeptide, of 4.5 kDA, is identified as the product of a small open reading frame in chloroplast DNA. The gene for this polypeptide , psbI, is located just downstream of the psbK gene chloroplast DNA.

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.

Characterization and in vitro expression of the cytochrome b-559 genes of barley. I. Localization and sequence of the genes.

The psbE and psbF genes encoding the 9.4 and 4.4 kD apoproteins of cytochrome b-559 have been located in the chloroplast genome of barley. As in other plant species they are found adjacent to each other in the large single copy region of the chloroplast DNA. Both the nucleotide sequence and the deduced amino acid sequence for the two polypeptides are identical to that of wheat and more than 95% similar to those of spinach, tobacco and Oenothera. The region between the two genes spans 10 nucleotides (excluding the stopcodon) and contains a typical procaryotic ribosomal binding site. A dicistronic transcript is identified, but the presence of a ribosomal binding site between the two genes may allow independent translation.