Distribution of the mevalonate and glyceraldehyde phosphate/pyruvate pathways for isoprenoid biosynthesis in unicellular algae and the cyanobacterium Synechocystis PCC 6714

Isopentenyl diphosphate, the universal isoprenoid precursor, can be produced by two different biosynthetic routes: either via the acetate/mevalonate (MVA) pathway, or via the more recently identified MVA-independent glyceraldehyde phosphate/pyruvate pathway. These two pathways are easily differentiated by incorporation of [1-13C]glucose and analysis of the resulting labelling patterns found in the isoprenoids. This method was successfully applied to several unicellular algae raised under heterotrophic growth conditions and allowed for the identification of the pathways that were utilized for isoprenoid biosynthesis. All isoprenoids examined (sterols, phytol, carotenoids) of the green algae Chlorella fusca and Chlamydomonas reinhardtii were synthesized via the GAP/pyruvate pathway, as in another previously investigated green alga, Scenedesmus obliquus, which was also shown in this study to synthesize ubiquinone by the same MVA-independent route. In the red alga Cyanidium caldarium and in the Chrysophyte Ochromonas danica a clear dichotomy was observed: as in higher plants, sterols were formed via the MVA route, whereas chloroplast isoprenoids (phytol in Cy. caldariumand O. danica and β-carotene in O. danica) were synthesized via the GAP/pyruvate route. In contrast, the Euglenophyte Euglena gracilis synthesized ergosterol, as well as phytol, via the acetate/MVA route. Similar feeding experiments were performed with the cyanobacterium SynechocystisPCC 6714 using [1-13C]- and [6-13C]-glucose. The two isoprenoids examined, phytol and β-carotene, were shown to have the typical labelling pattern derived from the GAP/pyruvate route.

Structural Analysis of the QB Pocket of the D 1 Subunit of Photosystem II in Synechocystis PCC 6714 and 6803

Various herbicides inhibit photosynthesis by displacing the second electron acceptor QB from its binding site at the D 1 protein. Different amino acid substitutions within this binding site have been found to reduce herbicide affinities, thereby conferring herbicide resistance. In Synechocystis PCC 6714 we have selected 7 single mutants and 6 double mutants resistant to various herbicides due to amino acid substitutions at different positions in the QB pocket. Characterization of these mutants consists in molecular determination of the mutations in the psbAl genes and in transformation of Synechocystis PCC 6803 by the cloned mutated genes to analyze the role of the mutations in the mutant phenotypes. Comparison with equivalent Chlamydomonas mutants is presented. These studies allow us to specify the interactions of several amino acid residues with herbicides and QB and with each other. Furthermore some Synechocystis mutants present additional characteristics such as an increased sensitivity to photoinhibition, or resistance to formate or modification of the oscillatory pattern of oxygen evolution. Among the 6 point-mutations giving herbicide resistance, only those located at the limit of the loop and the parallel helix produced additional effects on photosystem II function.

Mutations in the Subunit of Photosystem II and Resistance to the Phenol Type Herbicide Ioxynil in Synechocystis PCC 6714 and 6803

Several herbicides block the photosystem II electron transfer because they compete with QB, the second stable electron acceptor of photosystem II for binding to the D1 subunit. We have previously isolated a mutant of Synechocystis 6714 in which Asn is replaced by Thr at position 266 of D1 (G. Ajlani, I. Meyer, C. Vernotte, and Astier, FEBS Lett. 246, 207-210 (1989)) and presenting resistance to ioxynil but not to DCMU . In this report we present selection, from this mutant, of a double mutant with an additional substitution at position 264 (Ser by Ala). The sensitivity of this mutant toward several herbicides is given and compared to those of the mutants having only one substitution at 266 and one substitution at 264. It was also compared to a mutant of Chlamydomonas having the same substitutions. This allows us to discuss the interaction of various herbicides with the D1 protein and to compare the herbicide binding niches of Chlamydomonas and Synechocystis.