chlorophyll-a and Chromosome-Deletion

The light-independent pathway of chlorophyll synthesis which occurs in some lower plants and algae is still largely unknown. We have characterized a chloroplast mutant, H13, of Chlamydomonas reinhardtii which is unable to synthesize chlorophyll in the dark and is also photosystem I deficient. The mutant has a 2.8 kb deletion as well as other rearrangements of its chloroplast genome. By performing particle gun mediated chloroplast transformation of H13 with defined wild-type chloroplast DNA fragments, we have identified a new chloroplast gene, chlN, coding for a 545 amino acid protein which is involved in the light-independent accumulation of chlorophyll, probably at the step of reduction of protochlorophyllide to chlorophyllide. The chlN gene is also found in the chloroplast genomes of liverwort and pine, but is absent from the chloroplast genomes of tobacco and rice.

Topics: Algal Proteins; Amino Acid Sequence; Animals; Base Sequence; Chlamydomonas reinhardtii; Chlorophyll; Chloroplasts; Chromosome Deletion; DNA; Light; Molecular Sequence Data; Mutation; Plant Proteins; Restriction Mapping; RNA; Sequence Homology, Nucleic Acid

The photosystem II (PSII) reaction center complex coordinates a cluster of Mn atoms that are involved in the accumulation of oxidizing equivalents generated by light-induced charge separations within the intrinsic portion of the PSII complex. A 33-kDa extrinsic protein, termed the Mn-stabilizing protein (MSP), has been implicated in the stabilization of two of the four Mn atoms of the cluster, yet the precise role of this protein in O2 evolution remains to be elucidated. Here we describe the construction of a mutant of the cyanobacterium Synechocystis sp. PCC6803 in which the entire gene encoding MSP has been deleted. Northern and immunoblot analyses indicate that other PSII proteins are expressed and accumulated, despite the absence of MSP. Fluorescence emission spectra at 77 K indicate PSII assembles in the mutant, but that the binding of MSP is required for the normal fluorescence characteristics of the PSII complex, and suggest a specific interaction between MSP and CP47. Fluorescence induction measurements indicate a reduced rate of forward electron transport to the primary electron donor, P680, in the mutant. It is concluded that in contrast to previous reports, MSP is not required for the assembly of active PSII complexes nor is it essential for H2O-splitting activity in vivo.

Topics: Blotting, Northern; Chlorophyll; Chromosome Deletion; Cloning, Molecular; Cyanobacteria; Light-Harvesting Protein Complexes; Manganese; Mutation; Oxygen; Photosynthesis; Photosynthetic Reaction Center Complex Proteins; Photosystem II Protein Complex; Polymerase Chain Reaction; Restriction Mapping; RNA, Messenger; Spectrometry, Fluorescence

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.

Topics: Base Sequence; Chlorophyll; Chromosome Deletion; Cyanobacteria; Cytochrome b Group; Light-Harvesting Protein Complexes; Membrane Proteins; Molecular Sequence Data; Molecular Weight; Mutation; Oligonucleotide Probes; Photosynthetic Reaction Center Complex Proteins; Photosystem II Protein Complex; Phycobilisomes; Plant Proteins; Polymerase Chain Reaction; Spectrometry, Fluorescence

Photosynthesis is the hallmark of plant life and is the only plastid metabolic process known to be controlled by plastid genes. The complete loss of photosynthetic ability, however, has occurred on several independent occasions in parasitic flowering plants. Some of these plants are known to lack chlorophyll and certain photosynthetic enzymes, but it is not known to what extent changes have occurred in the genes encoding the photosynthetic apparatus or whether the plants even maintain a plastid genome. Here we report that the nonphotosynthetic root parasite Epifagus virginiana has a plastid chromosome only 71 kilobases in size, far smaller than any previously characterized land plant plastid genome. The Epifagus plastid genome has lost most, if not all, of the 30 or more chloroplast genes for photosynthesis and most of a large family of plastid genes, the ndh genes, whose products may be involved in a plastid respiratory chain. The extensive changes in Epifagus plastid gene content must have occurred in a relatively short time (5-50 x 10(6) yr), because Striga asiatica, a related photosynthetic parasite, has a typical complement of chloroplast genes for photosynthesis and chlororespiration. The plastid genome of Epifagus has retained transcribed ribosomal RNA and ribosomal protein genes, suggesting that it expresses one or more gene products for plastid functions not related to photosynthesis.

Topics: Blotting, Northern; Chlorophyll; Chromosome Deletion; DNA Probes; Organelles; Photosynthesis; Plants; RNA, Ribosomal

In order to identify specific cis-acting elements which regulate the expression of the divergent Cab22R and Cab22L genes of Petunia, we conducted systematic mutational studies of the 1 kb intergenic promoter region. Sequence analysis revealed three GATA box sequence repeats positioned between the TATA and CAAT box elements. These GATA elements are conserved in corresponding promoter regions of all LHCII Type I Cab genes in Petunia and other dicotyledonous plants we have examined. Site-specific mutations in the CAAT box and the GATA box elements of the Cab22R promoter resulted in 8-fold and 5-fold reductions in Cab22R transcript levels respectively. A deletion of 52 bp, adjacent and upstream from the CAAT box (-92 to -145) in the Cab22R promoter reduced transcript levels 20-fold. This deletion contains a region of 13 bp which is conserved between many Petunia Cab genes. These results indicate that the quantitative expression of the Cab22 promoters is regulated by multiple cis-acting elements including CAAT and GATA box elements as well as sequences located between -92 and -145. The deletion of the region between -92 and -145 is partially compensated by homologous sequences present in the adjacent divergent promoter Cab22L.

Topics: Base Sequence; Chlorophyll; Chromosome Deletion; Genes; Introns; Light-Harvesting Protein Complexes; Molecular Sequence Data; Mutation; Photosynthetic Reaction Center Complex Proteins; Plant Proteins; Plants; Promoter Regions, Genetic

We studied cis regulatory elements controlling the light-dependent organ-specific expression of Arabidopsis thaliana chlorophyll a/b binding protein gene (cab1) by stably transforming tobacco plants using a tumor-inducing (Ti) plasmid vector system. The results from the 5' and internal deletion analyses indicate that there are at least three cis-acting elements that are involved in the light-dependent developmental expression of cab1 gene. Two such elements are located at the immediate upstream regulatory region and the other element is located at the further upstream region. The 1120-base-pair (bp) DNA fragment containing the immediate and far upstream region can confer light-inducible organ specificity on the truncated nos promoter. However, deletion of the 39-bp DNA fragment at the immediate upstream regulatory region from this hybrid promoter resulted in a nonfunctional promoter, revealing that the 39-bp region is important for the cab promoter specificity. Further analyses of this region suggest that a potential Z-DNA-forming sequence (ATACGTGT) is involved in light-dependent developmental expression of the cab1 gene. Two additional Z-DNA-forming sequences (ACACATAT) that are inverted repeats of this sequence are also found in the upstream region where the additional regulatory elements are expected.

Topics: Base Sequence; Chlorophyll; Chromosome Deletion; DNA; Gene Expression Regulation; Light-Harvesting Protein Complexes; Molecular Sequence Data; Mutation; Photosynthetic Reaction Center Complex Proteins; Plant Proteins; Plants; Promoter Regions, Genetic