Expression of bacterial isochorismate synthase (EC 5.4.99.6) in transgenic root cultures ofRubia peregrina.

The gene coding for isochorismate synthase (EC 5.4.99.6) was amplified by the polymerase chain reaction fromEscherichia coli, cloned into a binary vector. and mobilised intoAgrobacterium rhizogenes LBA9402 which was used to transformRubia peregrina. Transgenic roots containing bacterial isochorismate synthase cDNA expressed twice as much isochorismate synthase activity (4.88 pkat/mg protein) as the control roots (2.45 pkat/mg protein) after 10 days in culture, and accumulatedca. 20% higher levels of total anthraquinones after 30 days in culture. Whilst the amount of total alizarin (free and bound) in the transgenic roots was 30% higher than in control roots, the level of free alizarin wasca. 85% higher.

Control of leaf and chloroplast development by the Arabidopsis gene pale cress.

Leaf plastids of the Arabidopsis pale cress (pac) mutant do not develop beyond the initial stages of differentiation from proplastids or etioplasts and contain only low levels of chlorophylls and carotenoids. Early in development, the epidermis and mesophyll of pac leaves resemble those of wild-type plants. In later stages, mutant leaves have enlarged intercellular spaces, and the palisade layer of the mesophyll can no longer be distinguished. To study the molecular basis of this phenotype, we cloned PAC and determined that this gene is regulated by light and has the capacity to encode an acidic, predominantly alpha-helical protein. The PAC gene appears to be a novel component of a light-induced regulatory network that controls the development of leaves and chloroplasts.

A putative anaerobic coproporphyrinogen III oxidase in Rhodobacter sphaeroides. I. Molecular cloning, transposon mutagenesis and sequence analysis of the gene.

A mutant of Rhodobacter sphaeroides, N1, has been isolated which is incapable of photosynthetic growth and, instead of synthesizing bacteriochlorophyll, N1 excretes coproporphyrin III into the growth medium. Using conjugative gene transfer, several clones were isolated from a R. sphaeroides gene library which restored normal pigment synthesis and photosynthetic growth to N1. Using transposon Tn5 mutagenesis, the gene was located to a 1.05 kb EcoRI fragment. Sequence and transcription analysis defined the position and expression of an open reading frame of approximately 920 bp, which is proposed as the anaerobic coproporphyrinogen III oxidase dedicated to bacteriochlorophyll biosynthesis.

Localized transposon Tn5 mutagenesis of the photosynthetic gene cluster of Rhodobacter sphaeroides.

Four genes essential for bacteriochlorophyll biosynthesis were known to be encoded within a 45 kb region of the Rhodobacter sphaeroides genome, the boundaries of which are defined by puh and puf genes for reaction-centre and light-harvesting LH1 complexes. The cluster is represented by eight overlapping inserts cloned in the mobilizable vector pSUP202. We have used localized transposon Tn5 mutagenesis to characterize this cluster further; a total of 87 independent insertions were generated which identify nine genes for bacteriochlorophyll biosynthesis, six for carotenoid biosynthesis, and puhA encoding the reaction-centre H subunit. This work provides an essential framework for a detailed study of the structure and expression of genes for photosynthesis in this bacterium.

psbG is not a photosystem two gene but may be an ndh gene.

A gene of the chloroplast genome has been designated the psbG gene on the basis that in maize the gene product is a 24-kDa polypeptide of photosystem two (PS2) (Steinmetz, A. A., Castroviejo, M., Sayre, R. T., and Bogorad, L. (1986) J. Biol. Chem. 261, 2485-2488). We have located and sequenced the equivalent gene in wheat (Triticum aestivum) and have raised specific antibodies to the gene product following its expression in Escherichia coli as a beta-galactosidase fusion protein. Using these antibodies, we have investigated the location of the gene product in various thylakoid membrane fractions of pea (Pisum sativum). The gene product of apparent molecular mass 27-28 kDa is severely depleted in PS2-enriched membrane preparations and its distribution between stromal and granal regions of the membrane is distinct to that of the psbC gene product which is known to be a core polypeptide of PS2. We therefore conclude that psbG does not code for a component of PS2 but instead suggest that it is present in a novel protein complex of the thylakoid membrane. On the basis of 1) the conserved overlap between psbG and ndhC, a chloroplast gene which shows significant homology to a mitochondrial gene that codes for a subunit of the NADH-ubiquinone oxidoreductase of mitochondria, and 2) sequence similarity between the psbG gene product and the ndh gene product of E. coli, which codes for a respiratory NADH dehydrogenase, we propose that this ill-defined complex functions as a NADH or NADPH-plastoquinone oxidoreductase.

Effect of oxygen on levels of mRNA coding for reaction-centre and light-harvesting polypeptides of Rhodobacter sphaeroides.

The relative levels of mRNA for the reaction-centre L and M subunits, B875 (LH1) alpha and beta polypeptides and B800-850 (LH2) alpha and beta polypeptides, have been measured during pigment induction of Rhodobacter sphaeroides. Over the 6 h of the experiment, bacteriochlorophyll levels increased by at least 100-fold. No transcripts for photosynthetic components were detectable at the start of induction; after 2 h the levels of transcripts from the puf operon (encoding reaction-centre and B875 subunits) had reached the maximum; these transcripts were 2.7 and 0.5 kb respectively. The transcript for the puc operon (B800-850 complex) was estimated to be 0.55 kb and reached a maximum level after 6 h. These results are consistent with the proposal that, during the assembly of the photosynthetic apparatus, the synthesis of B875 reaction-centre aggregates precedes that of the major antenna, B800-850.