psbK Gene Research Articles

Plastid genome and composition analysis of two medical ferns: Dryopteris crassirhizoma Nakai and Osmunda japonica Thunb.

BackgroundDryopteris crassirhizoma Nakai and Osmunda japonica Thunb. are ferns that are popularly used for medicine, as recorded by the Chinese pharmacopoeia, and are distributed in different regions of China. However, O. japonica is not record in the Standards of Chinese Herbal Medicines in Hong Kong. Research on identification methods of D. crassirhizoma and O. japonica is necessary and the phylogenetic position of the two species should be identified. The plastid genome is structurally highly conserved, providing valuable sources of genetic markers for phylogenetic analyses and development of molecule makers for identification.MethodsThe plastid genome DNA was extracted from both fern species and then sequenced on the Illumina Hiseq 4000. Sequences were assembled into contigs by SOAPdenovo2.04, aligned to the reference genome using BLAST, and then manually corrected. Genome annotation was performed by the online DOGMA tool. General characteristics of the plastid genomes of the two species were analyzed and compared with closely related species. Additionally, phylogenetical trees were reconstructed by maximum likelihood methods. The content of dryocrassin of the two species were determined according to the Standards of Chinese Herbal Medicines in Hong Kong.ResultsThe genome structures of D. crassirhizoma and O. japonica have different characteristics including the genome size, the size of each area, gene location, and types. Moreover, the (simple sequence repeats) SSRs of the plastid genomes were more similar to other species in the same genera. Compared with D. fragrans, D. crassirhizoma shows an inversion (approximately 1.6 kb), and O. japonica shows two inversions (1.9 kb and 216 bp). The nucleotide diversity (polymorphism information, Pi) analysis showed that the psbK gene and rpl14-rpl16 region have the highest Pi value in Dryopteris, and the ycf2-CDS3 and rpl14-rpl16 regions show the highest Pi vale in O. japonica. Phylogenetic analyses showed that the two species were grouped in two separate clades from each other, with both individually located with other members of their genus. The marker content of dryocrassin is not found in O. japonica.ConclusionsThe study is the first to identify plastid genome features of D. crassirhizoma and O. japonica. The results may provide a theoretical basis for the identification and the application of the two medically important fern species.

Evidence That the PsbK Polypeptide Is Associated with the Photosystem II Core Antenna Complex CP43

PsbK is encoded by the chloroplast psbK gene and is one of the small polypeptides of photosystem II (PSII). This polypeptide is required for accumulation of the PSII complex. In the present study, we generated an antibody against recombinant mature PsbK of Chlamydomonas and used it in Western blots to localize PsbK in the PSII core complex. PsbK was found in the thylakoid membranes, and purification of the PSII core complex from detergent-solubilized thylakoid membranes showed that PsbK is tightly associated with the PSII core complex. We used potassium thiocyanate to separate PSII into subcore complexes, including the D1/D2/cytochrome b559 reaction center complex, CP47, and CP43, and we found that PsbK co-purifies with one of the core antenna complexes, CP43, during ion exchange chromatography. Subsequent gel filtration chromatography of the purified CP43 confirmed that PsbK is tightly associated with CP43. Steady-state levels of PsbK were also determined in Chlamydomonas mutants expressing various levels of PSII. Quantitative Western blotting revealed that the levels of PsbK in these mutants are approximately equal to those of CP43, suggesting that PsbK is stable only when associated with CP43 in the chloroplast. Together, our results indicate that PsbK is an integral part of the PSII complex and may participate in the assembly and stability of the PSII complex.

Directed disruption of the Chlamydomonas chloroplast psbK gene destabilizes the photosystem II reaction center complex.

Using particle gun-mediated chloroplast transformation we have disrupted the psbK gene of Chlamydomonas reinhardtii with an aadA expression cassette that confers resistance to spectinomycin. The transformants are unable to grow photoautotrophically, but they grow normally in acetate-containing medium. They are deficient in photosystem II activity as measured by fluorescence transients and O2 evolution and they accumulate less than 10% of wild-type levels of photosystem II as measured by immunochemical means. Pulse-labeling experiments indicate that the photosystem II complex is synthesized normally in the transformants. These results differ from those obtained previously with similar cyanobacterial psbK mutants that were still capable of photoautotrophic growth (Ikeuchi et al., J. Biol. Chem. 266 (1991) 1111-1115). In C. reinhardtii the psbK product is required for the stable assembly and/or stability of the photosystem II complex and essential for photoautotrophic growth. The data also suggest that the stability requirements of the photosynthetic complexes differ considerably between C. reinhardtii and cyanobacteria.

Subunit III of the chloroplast ATP-synthase can form a Ca 2+-binding site on the lumenal side of the thylakoid membrane

Subunit III, the 8 kDa component of the chloroplast CF 0 H + channel, was isolated and purified from pea thylakoids for the purpose of studying its Ca 2+-binding properties. After n-butanol extraction and ether precipitation, HPLC purification was accomplished using a poly(styrenedivinylbenzene) column which removes lipid and protein contaminations. The main components of protein contamination were two hydrophobic proteins of near 4 kDa molecular mass, psaI and psbK gene products associated with PSI and PSII reaction centers, respectively. Purified subunit III as well as the unfractionated organic-solvent soluble preparation were used in a 45Ca 2+-ligand blot assay known to detect high affinity Ca 2+-binding sites in proteins. Polypeptides were separated with SDS-PAGE and were transferred onto PVDF membranes. Treatment of the membrane with 45CaCl 2 in the presence of 10-fold excess of MgCl 2 and 200-fold excess KCl led to the labeling of only the 8 kDa polypeptide. The Ca 2+ binding was inhibited after derivatizing aqueously exposed carboxyl groups with a water soluble carbodiimide plus a nucleophile, after de-formylation of the N-terminal methionine, or with a subsequent treatment with La 3+. Ca 2+ binding was maximum at pH 7.5–8.5 and was greatly decreased at acidic pH. Dicyclohexylcarbodiimide treatment (no nucleophile was added) of thylakoid membranes, which derivativizes the hydrophobically located Glu-61, decreased the electrophoretical mobility of isolated subunit III but did not inhibit the Ca 2+ binding. The data indicate that the carbonyl group of the formylated N-terminal Met-1 and probably the carboxyl group of the subunit III C-terminal Val-81 provide some of seven essential oxygen ligands normally required for defining a Ca 2+-binding site in proteins. It is probable, but not yet established that an oligomeric form of subunit III polypeptides is essential for forming the Ca 2+-binding site. Based on the accepted models for the hairpin conformation of the subunit III, it does seem clear that the Ca 2+-binding site can form on the lumenal side of the membrane in the functional CF 0 structure.

Characterization of the psbK locus of Synechocystis sp. PCC 6803 in terms of Photosystem II function

The psbK gene encodes a small protein of Photosystem II. The gene has previously been cloned and sequenced in Synechocystis sp. PCC 6803. Our new results, presented here, confirm the conclusions of Ikeuchi et al. Based on Northern hybridization and primer extension analyses, we show that psbK is transcribed as a monocistronic message in this cyanobacterium. Analysis of DNA sequence immediately upstream of the transcription start site revealed an E. coli-like-10 consensus sequence. A deletion mutant was constructed where the psbK gene was replaced by a kanamycin resistant cartridge. In situ complementation experiments, as well as Southern and Northern hybridization analyses, confirmed that the mutant strain contains a lesion in psbK. The psbK-less mutant could grow photoautotrophically as well as photoheterotrophically both in liquid culture and on agar plates. The rate of growth was slightly less compared with the wild-type as clearly observed by in situ complementation experiments. Although the mutant showed correspondingly lower rates of electron transport, thermoluminescence, oxygen flash yield and chlorophyll a fluorescence measurements did not detect any significant modification of the reactions of PS II. Moreover, the mutant was no more susceptible to excess light than the wild-type. It is, therefore, concluded that the product of the psbK gene is not crucial for PS II activity and possibly plays some other role in the metabolism of Synechocystis.

Cloning of the psbK gene from Synechocystis sp. PCC 6803 and characterization of photosystem II in mutants lacking PSII-K

We cloned and sequenced the psbK gene, coding for a small photosystem II component (PSII-K), from the transformable cyanobacterium, Synechocystis sp. PCC 6803, and determined the N-terminal sequence of mature PSII-K. The psbK gene product is processed by cleaving off eight amino acid residues from the N terminus. A mutant lacking psbK was constructed; this mutant grew photoautotrophically, but its growth rate was reduced. The number of photosystem II reaction centers on a chlorophyll basis was decreased by less than a factor of 2 in the psbK-deletion mutant. In Synechocystis sp. PCC 6803, the psbK gene is transcribed as a single gene and is not part of an operon. Single-site mutations were introduced into psbK leading to early termination or deletion of the presequence. The phenotype of these mutants strongly resembles that of the psbK deletion mutant, indicating that indeed the change in phenotype in the deletion mutant is directly correlated with PSII-K. PSII-K is not essential for photosystem II assembly or activity but is needed for optimal photosystem II function.

Low-molecular-mass proteins in cyanobacterial photosystem II: Identification of psbH and psbK gene products by N-terminal sequencing

The O 2-evolving photosystem II core complex was isolated from a thermophilic cyanobacterium, Synechococcus vulcanus Copeland. Analysis by SDS-polyacrylamide gel electrophoresis revealed that the complex contained at least seven low-molecular-mass proteins in addition to the well characterized CP47 apoprotein, CP43 apoprotein, 33 kDa extrinsic protein, D1 protein, D2 protein and large subunit of cytochrome b-559. The separation profiles of these low-molecular-mass proteins were very similar between cyanobacterial and higher plant PS II. N-terminal sequences of the 6.5 kDa and 3.9 kDa proteins of cyanobacterial core complex were determined after blotting to a polyvinylidene difluoride membrane. The sequence of the 6.5 kDa protein showed high homology with an internal sequence of plant psbH gene product, so-called 10 kDa phosphoprotein, but did not conserve the Thr residue which is specifically phosphorylated in plants. The sequence of the 3.9 kDa protein corresponded to the K protein of higher plants (mature form of psbK gene product). These results indicate that the products of both psbH and psbK genes are present in cyanobacterial PS II as well as being associated with the O 2-evolving core complex.

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.

A new photosystem II reaction center component (4.8 kDa protein) encoded by chloroplast genome

The photosystem II reaction center complex, so-called D1-D2-cytochrome b-559 complex, isolated from higher plants contains a new component of about 4.8 kDa [(1988) Plant Cell Physiol. 29, 1233–1239]. The partial amino acid sequence of this component from spinach was determined after release of N-terminal blockage. The determined sequence matched an open reading frame (ORF36) of the chloroplast genome from tobacco and liverwort, which is located downstream from the psbK gene and forms an operon with psbK. The predicted product consists of 36 amino acid residues and has a single membrane-spanning segment. High homology between the tobacco and liverwort genes, and its presence in the reaction center complex suggest an important role for this component in the photosystem II complex. Since this gene corresponds to a part of the formerly designated psbI gene, we propose to revise the definition of psbI as the gene encoding the 4.8 kDa reaction center component.