pheophytin-a and gabaculine

To elucidate the mechanism of an irradiance-dependent adjustment in the chlorophyll (Chl) antenna size of Dunaliella salina, we investigated the regulation of expression of the Chl a oxygenase (CAO) and light-harvesting complex b (Lhcb) genes as a function of Chl availability in the photosynthetic apparatus. After a high-light to low-light shift of the cultures, levels of both CAO and Lhcb transcripts were rapidly induced by about 6-fold and reached a high steady-state level within 1.5 h of the shift. This was accompanied by repair of photodamaged photosystem II (PSII) reaction centers, accumulation of Chl a and Chl b (4:1 ratio), photosystem I (PSI), light-harvesting complex, and by enlargement of the Chl antenna size of both photosystems. In gabaculine-treated cells, induction of CAO and Lhcb transcripts was not affected despite substantial inhibition in de novo Chl biosynthesis. However, cells were able to synthesize and accumulate some Chl a and Chl b (1:1 ratio), resulting in a marked lowering of the Chl a to Chl b ratio in the presence of this inhibitor. Assembly incorporation of light-harvesting complex and a corresponding Chl antenna size increase, mostly for the existing photosystems, was noted in the presence of gabaculine. Repair of photodamaged PSII was not affected by gabaculine. However, assembly accumulation of new PSI was limited under such conditions. These results suggest a coordinate regulation of CAO and Lhcb gene transcription by irradiance, independent of Chl availability. The results are discussed in terms of different signal transduction pathways for the regulation of the photosynthetic apparatus organization by irradiance.

Topics: Apoproteins; Cells, Cultured; Chlorophyll; Chlorophyll A; Chlorophyta; Cyclohexanecarboxylic Acids; Light; Light-Harvesting Protein Complexes; Oxygenases; Photosynthesis; Photosynthetic Reaction Center Complex Proteins; Photosystem I Protein Complex; Photosystem II Protein Complex; Signal Transduction; Thylakoids

Gabaculine (2,3-dihydro 3-amino benzoic acid) is a potent inhibitor of tetrapyrrole biosynthesis in organisms that use the C5 pathway for the synthesis of delta-aminolaevulinic acid. Glutamate semialdehyde aminotransferase (GSA-AT), the enzyme catalysing the formation of this key precursor of tetrapyrroles, is normally inhibited by concentrations of gabaculine in the order of 5 microM. However, in Synechococcus 6301 strain GR6, a cyanobacterium that is resistant to 100 microM gabaculine, this enzyme has undergone two changes in structure: a deletion of three amino acids from positions 5 to 7 and the substitution of isoleucine for methionine at position 248. To establish the effect in vivo of these specific changes in the gene for GSA-AT (hemL), a suicide vector (pHS7) containing an antibiotic cassette was constructed to achieve the replacement, by homologous recombination, of the wild-type hemL gene in the chromosome by a modified form of the gene. Recombinant strains of Synechococcus 7942 obtained using pHS7-hemLGR6 were indistinguishable from Synechococcus 6301 GR6 in terms of the resistance of growth and of chlorophyll accumulation to high concentrations of gabaculine, while a wild-type recombinant produced using pHS7-hemLWT had retained its sensitivity. Southern hybridisation using gene probes for hemL, ampr and cmr confirmed that chromosomal integration of the plasmids had occurred in both WT and GR6 recombinants. Growth and chlorophyll accumulation in equivalent strains with the hemL gene containing either the deletion or the transition characteristic of Synechococcus 6301 GR6 were inhibited by 10 microM gabaculine. Consequently, resistance in vivo to high concentrations of this compound is dependent on both the changes in gene/enzyme structure. This investigation has established the effectiveness of the suicide vector pHS7 for studying the effect in vivo of specific changes in the hemL gene. It has also demonstrated that replacement of the wild-type gene by that from Synechococcus 6301 GR6 is sufficient to confer resistance in vivo to high concentrations of gabaculine.

Topics: Alleles; Chlorophyll; Chlorophyll A; Cyanobacteria; Cyclohexanecarboxylic Acids; Genes; Genetic Vectors; Intramolecular Transferases; Isomerases; Recombination, Genetic