Knud W. Henningsen

Expression of the chlI, chlD, and chlH Genes from the Cyanobacterium Synechocystis PCC6803 in Escherichia coli and Demonstration That the Three Cognate Proteins Are Required for Magnesium-protoporphyrin Chelatase Activity

Magnesium-protoporphyrin chelatase catalyzes the first step unique to chlorophyll synthesis: the insertion of Mg2+ into protoporphyrin IX. Genes from Synechocystis sp. PCC6803 with homology to the bchI and bchD genes of Rhodobacter sp. were cloned using degenerate oligonucleotides. The function of these genes, putatively encoding subunits of magnesium chelatase, was established by overexpression in Escherichia coli, including the overexpression of Synechocystis chlH, previously cloned as a homolog of the Rhodobacter bchH gene. The combined cell-free extracts were able to catalyze the insertion of Mg2+ into protoporphyrin IX in an ATP-dependent manner and only when the products of all three genes were present. The ChlH, ChlI, and ChlD gene products are therefore assigned to the magnesium chelatase step in chlorophyll a biosynthesis in Synechocystis PCC6803. The primary structure of the Synechocystis ChlD protein reveals some interesting features; the N-terminal half of the protein shows 40-41% identity to Rhodobacter BchI and Synechocystis ChlI, whereas the C-terminal half displays 33% identity to Rhodobacter BchD. This suggests a functional as well as an evolutionary relationship between the “I” and “D” genes.

Structural genes for Mg-chelatase subunits in barley:Xantha-f, -g and-h

Barley mutants in the lociXantha-f, Xantha-g andXantha-h, when fed with 5-aminolevulinate in the dark, accumulate protoporphyrin IX. Mutant alleles at these loci that are completely blocked in protochlorophyllide synthesis are also blocked in development of prolamellar bodies in etioplasts. In contrast to wild type, thexan-f, -g and-h mutants had no detectable Mg-chelatase activity, whereas they all had methyltransferase activity for synthesis of Mg-protoporphyrin monomethyl ester. Antibodies recognising the CH42 protein ofArabidopsis thaliana and the OLIVE (OLI) protein ofAntirrhinum majus immunoreacted in wild-type barley with 42 and 150 kDa proteins, respectively. Thexan-h mutants lacked the protein reacting with antibodies raised against the CH42 protein. Twoxan-f mutants lacked the 150 kDa protein recognised by the anti-OLI antibody. Barley genes homologous to theA. majus olive and theA. thaliana Ch-42 genes were cloned using PCR and screening of cDNA and genomic libraries. Probes for these genes were applied to Northern blots of RNA from thexantha mutants and confirmed the results of the Western analysis. The mutantsxan-f27, -f40, -h56 and-h57 are defective in transcript accumulation while-h38 is defective in translation. Southern blot analysis established thath38 has a deletion of part of the gene. Mutantsxan-f10 and-f41 produce both transcript and protein and it is suggested that these mutations are in the catalytic sites of the protein. It is concluded thatXan-f and-h genes encode two subunits of the barley Mg-chelatase and thatXan-g is likely to encode a third subunit. The XAN-F protein displays 82% amino acid sequence identity to the OLI protein ofAntirrhinum, 66% to theSynechocystis homologue and 34% identity to theRhodobacter BchH subunit of Mg-chelatase. The XAN-H protein has 85% amino acid sequence identity to theArabidopsis CH42 protein, 69% identity to theEuglena CCS protein, 70% identity to theCryptomonas BchA andOlisthodiscus CssA proteins, as well as 49% identity to theRhodobacter BchI subunit of Mg-chelatase. Identification of the barleyXan-f andXan-h encoded proteins as subunits required for Mg-chelatase activity supports the notion that theAntirrhinum OLI protein and theArabidopsis CH42 protein are subunits of Mg-chelatase in these plants. The expression of both theXan-f and-h genes in wild-type barley is light induced in leaves of greening seedlings, and in green tissue the genes are under the control of a circadian clock.

Localization and nucleotide sequence of the gene for the membrane polypeptide D2 from pea chloroplast DNA

SummaryThe gene for the membrane polypeptide D2 has been mapped on the pea (Pisum sativum) chloroplast genome. The nucleotide sequence of the gene and its flanking regions is presented. The only large open reading frame in the sequence codes for a protein of MW 39.5 kD. A potential ribosome binding site is located 6 nucleotides upstream from the initiation codon and there are two sets of putative promotor sequences in the 5′ flanking region. The polypeptide has a high content of hydrophobic amino acids, predominatly grouped in clusters of 20 or more residues. The 3′ end of the D2 gene is overlapped by 50 nucleotides of a second open reading frame (UORF I) which is at least 369 nucleotides long. Based on current data we suggest the D2 polypeptide to be a constituent of photosystem II (PSII).

Sequence of two genes in pea chloroplast DNA coding for 84 and 82 kD polypeptides of the photosystem I complex

SummaryThe genes encoding the two P700 chlorophyll a-apoproteins of the photosystem I complex were localized on the pea (Pisum sativum) chloroplast genome. The nucleotide sequence of the genes and the flanking regions has been determined. The genes are separated by 25 bp and are probably cotranscribed. The 5′ terminal gene (psaA1) codes for a 761-residue protein (MW 84.1 kD) and the 3′ terminal gene (psaA2) for a 734-residue protein (MW 82.4 kD). Both proteins are highly hydrophobic and contain eleven putative membrane-spanning domains. The homology to the corresponding polypeptides from maize are 89% and 95% for psaA1 and psaA2, respectively. A putative promoter has been identified for the psaA1 gene, and potential ribosome binding sites are present before both genes.

A cDNA clone encoding a 10.8 kDa photosystem I polypeptide of barley

A cDNA clone encoding the barley photosystem I polypeptide which migrates with an apparent molecular mass of 16 kDa on SDS‐polyacrylamide gels has been isolated. The 634 bp sequence of this clone has been determined and contains one large open reading frame coding for a 15 457 Da precursor polypeptide. The molecular mass of the mature polypeptide is 10 821 Da. The amino acid sequence of the transit peptide indicates that the polypeptide is routed towards the stroma side of the thylakoid membrane. The hydropathy plot of the polypeptide shows no membrane‐spanning regions.

STRUCTURE AND EXPRESSION OF A NITRITE REDUCTASE GENE FROM BEAN (PHASEOLUS VULGARIS) AND PROMOTER ANALYSIS IN TRANSGENIC TOBACCO

A structural gene encoding nitrite reductase (NiR) in bean (Phaseolus vulgaris) has been cloned and sequenced. The NiR gene is present as a single copy encoding a protein of 582 amino acids. The bean NiR protein is synthezised as a precursor with an amino-terminal transit peptide (TP) consisting of 18 amino acid residues. The bean NiR transit peptide shows similarity to the TPs of other known plant NiRs.The NiR gene is expressed in trifoliate leaves and in roots of 20-day old bean plants where transcript accumulation is nitrate-inducible. Gene expression occurs in a circadian rhythm and induced by light in leaves of dark-adapted plants.A particular 100 bp sequence is present in the promoter and in the first intron of the NiR gene. Several copies of this 100 bp sequence are present in the bean genome. Comparisons between the promoter of the bean NiR gene and of two bean nitrate reductase genes (NR1 and NR2) show a limited number of conserved motifs, although the genes are presumed to be co-regulated. Comparisons are also made between the bean NiR promoter and the spinach NiR promoter.Transformation of tobacco plants with the bean NiR promoter fused to the GUS reporter gene (β-glucuronidase) shows that the bean NiR promoter is nitrate-regulated and that the presence of the 100 bp sequence influences the level of GUS activity. NiR-coding sequences are not required for nitrate regulation but have a quantitative effect on the measured GUS activity.

Structure of a 3.2 kb region of pea chloroplast DNA containing the gene for the 44 kD photosystem II polypeptide

SummaryThe gene for the 44 kD chlorophyll a-binding photosystem II polypeptide has been localized on the pea (Pisum sativum) chloroplast genome. The nucleotide sequence of the gene and its flanking regions has been analyzed. The gene codes for a polypeptide of 473 amino acid residues and is possibly cotranscribed with the gene for the D2 photosystem II polypeptide with which it has 50 bp in common. The amino acid sequences of the 44 kD polypeptides from pea, spinach and maize are approximately 95% homologous. Within the 1 kb fragment 3′ to the 44 kD gene a 93 bp tRNA-Ser (UGA) gene and an open reading frame of 62 codons (ORF 62) were identified. Both show high homology to corresponding genes 3′ to the 44 kD genes from spinach, maize and barley. The 44 kD gene and ORF 62 are encoded in the same strand, and have putative promoter sequences, ribosome binding sites and transcription termination signals.

Characterization of the fine structure and proteins from barley protein bodies

Abstract The fine structure and the membranes surrounding barley protein bodies have been separated from the proteins stored within the particles by sonication. The two fractions obtained have been characterized by electron microscopy and by immunoelectrophoretic analysis. Phytase activity was shown to be associated with the fine structure, and hordein was identified as the aleurin inside the protein bodies. The localization of phytic acid is discussed.

The influence of cations and methylamine on structure and function of thylakoid membranes from barley chloroplasts

The effects of cations on the structure and activity of isolated barley (Hordeum vulgare cv. Svalöfs Bonus) chloroplast lamellar systems were studied. Three separate effects of cations were recognizable. (1) Cations in high concentrations (in excess of 100–200 mM NaCl) are required to maintain granal stacks once the outer chloroplast envelope is broken. In 30 mM NaCl the majority of the lamellar systems exist as well-separated thylakoids, even in the presence of high concentrations of sucrose (330 and 660 mM). The lamellar systems photoreduce ferricyanide at coupled rates whether they are in the granal or non-granal configuration. (2) Cations are required for Hill reaction activity. This requirement becomes apparent following loss of thylakoid membrane integrity at very low cation concentrations. Maximum activation of the photoreduction of ferricyanide occurs at 30 mM NaCl, and divalent ions (Mg++, Ca++, Mn++) are 12 times more effective than monovalent ions (Na+, K+). (3) Cations are necessary to preserve the integrity of thylakoid membranes. Below about 8 mM NaCl, the thylakoids swell and this change is accompanied by progressive loss of Hill activity and the capacity to maintain a light-dependent proton gradient. Divalent cations are more than 200 times as effective as monovalent ions in preventing these changes. Hill reaction activity, but not proton pump activity, is regained by adding cations back to the swollen thylakoids (cation effect 2, above) and the new rate of Hill reaction activity is the same as in chloroplasts uncoupled with methylamine. The re-establishment of Hill reaction activity is accompanied by appression of the swollen thylakoids. The uncoupler methylamine causes stacking of thylakoids and collapsing of intrathylakoidal spaces.

Distribution of ATPase and ATP-to-ADP phosphate exchange activities in magnesium chelatase subunits of Chlorobium vibrioforme and Synechocystis PCC6803

Abstract Insertion of magnesium into protoporphyrin IX is a complex ATP-dependent reaction catalysed by the enzyme Mg-chelatase. Three separate proteins (Mg-chelatase subunits), designated as D, H and I, are involved in the chelation reaction. The genes encoding the Mg-chelatase subunits of the green sulfur bacterium Chlorobium vibrioforme and of the cyanobacterium Synechocystis strain PCC6803 were expressed in Escherichia coli. The recombinant proteins were purified, tested for ATPase and phosphate exchange activities, and compared with the activities of the corresponding subunits of Rhodobacter sphaeroides. The Synechocystis strain PCC6803 I subunit and the C. vibrioforme H and I subunits hydrolysed ATP at the rates of 2.0, 1.8 and 0.16 nmol (mg protein)–1 min–1, respectively. The ATPase activity of the C. vibrioforme H subunit was similar to that reported for the R. sphaeroides H subunit. The Synechocystis strain PCC6803 H subunit failed to hydrolyse ATP. The I subunit of Synechocystis strain PCC6803 and C. vibrioforme catalysed a transfer of PO4 from ATP to ADP (exchange activity) at the rate of 1.75 ± 0.15 nmol (mg protein)–1 min–1. This exchange rate was 300-fold lower than that reported for the R. sphaeroides I subunit. The PO4 exchange activities were correlated with the presence of the sequence GXRGTGKSTXVRALA in the primary structure of the three I subunits. Mg-chelatase activity was reconstituted by combining the three subunits of the same bacterium [rates of 41–89 pmol Mg-deuteroporphyrin (mg protein)–1 min–1]. Heterologous subunit combinations resulted in low or no Mg-chelatase activity.