Sean M. Hird

Nucleotide sequence of a wheat chloroplast gene encoding the proteolytic subunit of an ATP-dependent protease

ATP-dependent proteolysis of newly synthesised proteins has been detected in isolated pea chloroplasts [ 1, 2]. Liu and Jagendorf [3] demonstrated that an ATP-dependent protease degraded incomplete polypeptides and non-assembled chloroplast polypeptides accumulated in chloroplasts isolated from pea shoots that had been treated with cycloheximide. Several ATP-dependent proteolysis systems have been described from prokaryotic and eukaryotic organisms [4, 5]. In eukaryotic organisms a major system involves ubiquitin conjugation to target proteins and their subsequent degradation by an ATP-dependent protease [4]. In Escherichia coli, two distinct ATP-dependent proteases, Lon and Clp, have been described [5-9]. The Clp protease has recently been characterised and shown to consist of an 83 kDa regulatory subunit (CIpA) with ATPase activity and a 23 kDa subunit (CIpP) with proteolytic activity [6-10]. Comparisons of the gene sequences for these subunits reveal that they have homologues in a wide range of prokaryotic and eukaryotic organisms [ 11, 12]. The clpP gene is homologous to an open reading frame in the chloroplast genomes of tobacco, Marchantia and rice [12], whereas the clpA gene is homologous to two nuclear genes in tomato [ 11]. These nuclear genes encode proteins which are very similar to each other and which are predicted to contain N-terminal presequences for plastid targeting [11 ]. These results suggest that chloroplasts contain an ATP-dependent protease homologous to the Clp protease of E. coli. We have determined the nucleotide sequence of a region of wheat (Triticum aestivum cv. Mardler) chloroplast DNA upstream of the psbB gene for the 47 kDa chlorophyll a-binding protein of photosystem II and have detected the presence of an open reading frame, orf216, encoding a protein homologous to the ClpP subunit. The gene is contained within the 3.3 kb BamHI fragment B 15 in the plasmid pTac2.33 [ 13 ]. The nucleotide sequence is presented in Fig. 1. Orf216 is located 493 bp upstream ofpsbB and is transcribed divergently. No recognisable ribosome-binding site is located just upstream from the translation initiation codon of orf216. The open reading frame contains a short region (nucleotides 587-637) which is very similar (identical nucleotides at 48 out of 51 positions) to part of a variable copy number nuclear DNA sequence from rice [14]. Another variable copy number nuclear DNA se-

The gene for the 10 kDa phosphoprotein of photosystem II is located in chloroplast DNA

Nucleotide sequencing of a region of wheat chloroplast DNA between the genes for the 47 kDa chlorophyll a‐binding protein of photosystem II (psbB) and cytochrome b‐563 (petB) has revealed an open reading frame of 73 codons. This open reading frame has been identified as the gene (psbH) for the 10 kDa phosphoprotein of photosystem II by comparison with the published N‐terminal amino acid sequence and amino acid composition of the purified spinach protein. The predicted sequence of the protein shows some homology with the N‐terminal region of the light‐harvesting chlorophyll a/b‐binding protein of photosystem II (LHCII).

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.

Differential expression of the psbB and psbH genes encoding the 47 kDa chlorophyll a-protein and the 10 kDa phosphoprotein of photosystem II during chloroplast development in wheat.

SummaryThe nucleotide sequence of a region of wheat chloroplast DNA containing the psbB gene for the 47 kDa chlorophyll a-binding protein of photosystem II has been determined. The gene encodes a polypeptide of 508 amino acid residues which is predicted to contain six hydrophobic membrane-spanning regions. The psbB gene is located 562 bp upstream of the psbH gene for the 10 kDa phosphoprotein of photosystem II. A small open reading frame of 38 codons is located between psbB and psbH, and on the opposite strand the psbN gene, encoding a photosystem II polypeptide of 43 amino acid residues, is located between orf38 and psbH. S1 nuclease mapping indicated that the 5′ ends of transcripts were located 371 and 183 bp upstream of the psbB translation initiation codon. Predominant transcripts of 2.1 kb and 1.8 kb for psbB and 0.4 kb for psbH were present in RNA isolated from etiolated and greening wheat seedlings. Immunodecoration of Western blots indicated that the 47 kDa polypeptide was absent, or present in very low amounts, in dark-grown tissue and accumulated on greening, whereas the 10 kDa polypeptide was present in similar amounts in both dark-grown and greening seedlings. The 10 kDa polypeptide was phosphorylated in vitro by incubating wheat etioplast membranes with [γ3 2P] ATP.

Location and nucleotide sequence of the gene for cytochrome b-559 in wheat chloroplast DNA

SummaryThe gene for the apoprotein of cytochrome b-559 has been located in wheat chloroplast DNA by coupled transcription-translation of fragments of chloroplast DNA in a cell-free system from Escherichia coli and by nucleotide sequence analysis. The gene is located 1.5 kbp downstream from the gene for cytochrome f and is transcribed in the opposite direction. Nucleotide sequence determination revealed an open reading frame coding for a hydrophobic protein of 83 amino acid residues (Mr 9434). The deduced amino acid sequence of the first 27 codons corresponds exactly to the determined N-terminal amino acid sequence of spinach cytochrome b-559. Another open reading frame of 39 codons is located 10 bp downstream from the gene for cytochrome b-559. A putative ribosome-binding site, AGGAGG, for this open reading frame overlaps the translation stop codon, UAG, of the cytochrome b-559 gene. The deduced amino acid sequence from this open reading frame shows similarities to the N-terminal sequence of cytochrome b-559. Both sequences contain a histidine residue located in a putative membrane-spanning region of the polypeptide.

Genes and Polypeptides of Photosystem II

Photosystem II catalyses the light-driven transfer of electrons from water to plastoquinone, producing oxygen and generating a proton gradient across the thylakoid membrane. The complex may be regarded as made up of three assemblies of polypeptides: a light-harvesting complex (LHCH), a core complex containing the reaction centre and two antenna chlorophyll proteins, and an extrinsic complex concerned with oxygen evolution. Photosystem II is structually the most complex of the supramolecular assemblies of the thylakoid membrane and is currently recognised to be composed of at least 20 different polypeptides.1

Chloroplast Genes for Photosynthetic Membrane Components

The light reactions of photosynthesis take place in chloroplast thylakoid membranes, whose components are specially arranged to allow efficient light-harvesting, electron transfer and proton translocation. These photosynthetic components are assembled into four supramolecular membrane complexes which make up the bulk of the thylakoid membrane system. The four complexes are photosystems I and II, the cytochrome b — f complex and ATP synthase. Our aim is to understand the synthesis and assembly of the components of these complexes. This requires the localisation and characterisation of the genes for these components. For the past few years we have been studying the contribution of the chloroplast genetic system to the synthesis of thylakoid membrane proteins in pea (Pisum sativum) and wheat (Triticum aestivum). We have located and characterised chloroplast genes for 18 identified thylakoid proteins, as well as several open reading frames for putative membrane proteins. The genes for identified components are listed in Table 1, together with the sizes of the gene products deduced from nucleotide sequences. The location of these genes on the physical maps of chloroplast DNA from pea and wheat is shown in Fig. 1.

Genes for Photosystem II Polypeptides

Photosystem II catalyses the light-driven transfer of electrons from water to plastoquinone, producing oxygen and generating a proton gradient across the thylakoid membrane. The complex may be regarded as made up of three assemblies of polypeptides: a light-harvesting complex (LHCII), an intrinsic membrane complex containing the reaction centre and two antenna chlorophyll proteins, and an extrinsic complex concerned with oxygen evolution. Photosystem II is structurally the most complex of the supramolecular assemblies of the thylakoid membrane and is currently recognised to be composed of at least 20 different polypeptides1.

Characterisation of Genes for Components of Photosystem II in Wheat Chloroplast DNA

The light-driven reactions of photosynthesis are performed by a set of highly specialised protein complexes intercalated into the thylakoid membrane. The multisubunit photosystem II is responsible for the conversion of light energy into an electron motive force across the membrane resulting in the reduction of the plastoquinone pool and the evolution of oxygen.