Redox control of ntcA gene expression in Synechocystis sp. PCC 6803. Nitrogen availability and electron transport regulate the levels of the NtcA protein.

In this work we have studied the influence of the cellular redox status in the expression of the Synechocystis sp. PCC 6803 ntcA gene. Two different ntcA transcripts with different 5' ends were detected, depending on the different dark/light or nitrogen availability conditions. Accumulation of a 0.8-kb ntcA message was light and nitrogen dependent, whereas a longer 1.2-kb ntcA transcript was neither light nor nitrogen regulated. NtcA protein levels increased concomitantly with the accumulation of the 0.8-kb ntcA transcript. The light-dependent accumulation of the ntcA gene and the NtcA protein was sensitive to electron transport inhibitors. In addition, Glc-grown Synechocystis sp. cells showed a similar ntcA expression pattern in darkness to that observed under illumination. These data suggested that electron transport, and not light per se may regulate ntcA gene expression. Primer extension analysis, together with gel mobility-shift assays, demonstrated that in vitro, the Synechocystis sp. NtcA protein specifically bound to the putative promoter region from the light/nitrogen-dependent ntcA transcript but not to that from the constitutive 1.2-kb ntcA mRNA. Band-shift experiments carried out in the presence of thiol oxidizing/modifiying agents and different reducing/oxidizing conditions suggested that NtcA binding to its own promoter was under a thiol-dependent redox mechanism. Our results suggest that the cellular redox status plays a central role in the autoregulatory mechanism of the NtcA protein.

UV-B radiation induced exchange of the D1 reaction centre subunits produced from the psbA2 and psbA3 genes in the Cyanobacterium synechocystis sp. PCC 6803.

UV-B irradiation of Synechocystis 6803 cells inhibits photosystem II activity, which can be restored via de novo synthesis of the D1 (and D2) reaction center subunits. Recently we have shown that of the two psbA genes that encode identical D1 proteins in Synechocystis 6803, UV-B preferentially enhances the transcription of psbA3 compared to that of psbA2 [Máté, Z., Sass, L., Szekeres, M., Vass, I. and Nagy, F. (1998) J. Biol. Chem. 273, 17439-17444]. Here we studied the effect of UV-B on the synthesis of the D1 protein from the psbA2 and psbA3 genes in the P7 mutant of Synechocystis 6803. In this mutant, psbA2 carries the Ala251-->Val point mutation, which confers resistance to the photosystem II electron transport inhibitor metribuzin, but psbA3 is the same as in the wild-type. By applying variable chlorophyll fluorescence measurements to distinguish between metribuzin-sensitive and metribuzin-resistant photosystem II centers we quantified the amount of the D1 protein produced from each of the psbA3 and psbA2 genes. When the cells were exposed to UV-B light, the fraction of D1 protein produced from the psbA3 gene was increased from 15-20 to 32-40% of the total D1. This effect was reversible by transferring the cells to visible light. The rate of D1 production from psbA3 increased with increasing UV-B intensities, and was a transient phenomenon at low UV-B levels (0.1 microE x m-2 x s-1). It is concluded that the enhancement of psbA3 gene transcription by UV-B light leads to enhanced D1 protein synthesis from this gene. Our findings demonstrate that the main role of psbA3 transcription activated by UV-B is to increase the size of the psbA mRNA pool available for translation when a rapid repair of the D1 protein is needed under UV-B stress.

Redox control of psbA gene expression in the cyanobacterium Synechocystis PCC 6803. Involvement of the cytochrome b(6)/f complex.

We investigated the role of the redox state of the photosynthetic and respiratory electron transport chains on the regulation of psbA expression in Synechocystis PCC 6803. Different means to modify the redox state of the electron carriers were used: (a) dark to oxidize the whole electron transport chain; (b) a shift from dark to light to induce its reduction; (c) the chemical interruption of the electron flow at different points to change the redox state of specific electron carriers; and (d) the presence of glucose to maintain a high reducing power in darkness. We show that changes in the redox state of the intersystem electron transport chain induce modifications of psbA transcript production and psbA mRNA stability. Reduction of the intersystem electron carriers activates psbA transcription and destabilizes the mRNA, while their oxidation induces a decrease in transcription and a stabilization of the transcript. Furthermore, our data suggest that the redox state of one of the electron carriers between the plastoquinone pool and photosystem I influences not only the expression of the psbA gene, but also that of other two photosynthetic genes, psaE and cpcBA. As a working hypothesis, we propose that the occupancy of the Q(0) site in the cytochrome b(6)/f complex may be involved in this regulation.

Expression of the psbA gene during photoinhibition and recovery in Synechocystis PCC 6714: inhibition and damage of transcriptional and translational machinery prevent the restoration of photosystem II activity.

The D1 reaction center protein of the photosystem II complex is very sensitive to light. It is continuously being damaged, degraded and resynthesized. Under high light, photosystem II inactivation is observed. This is because the rate of D1 damage is faster than that of its replacement. This process can be reversed if exposure to high light is not too long. In this work we study the changes that occur in the transcriptional and translational machinery that could lead to irreversible photoinhibition in Synechocystis PCC 6714. In the first minutes of photoinhibition, high light induced an accumulation of psbA mRNA due to an increase in psbA transcription initiation. Although the transcription rate of other photosynthetic genes (e.g. psaE and cpcB-cpcA) declined, the high turnover of the psbA transcript was maintained for a long time. When the light stress was too long, the stability of psbA mRNA increased and the psbA transcription rate appeared to decrease. A high level of psbA mRNA was maintained even though translation no longer occurred and the cells were unable to recover. Experiments to measure newly synthesized D1 incorporation into the thylakoid membranes during recovery in the presence of rifampicin showed that the initiation of transcription was not required for translation of psbA mRNA when photoinhibition was still reversible. Since psbA translation did not depend on the level of psbA transcript or on the initiation of psbA transcription, we propose that damage to the translational machinery also occurred during light stress, leading to the inhibition of D1 synthesis and to irreversible photoinhibition.

S1 destabilization and higher sensitivity to light in metribuzin-resistant mutants.

Mutations in the secondary quinone electron acceptor (QB) pocket of the D1 protein conferring a modification on the donor side of photosystem II (PSII) have been characterized by gene cloning and sequencing in two metribuzin-resistant mutants of Synechocystis PCC 6714. The mutations induce different herbicide resistances: in M30, a point mutation at the codon 248, isoleucine to threonine, results in resistance only to metribuzin; in M35, a single mutation, Ala251Val, confers metribuzin, atrazine, and ioxynil resistance. As with other herbicide-resistant mutants, M30 and M35 present modifications in the electron transfer between the primary quinone electron acceptor (QA) and QB. In addition, they have a modified oscillatory pattern of oxygen emission: after dark adaptation, the maximum oscillation is shifted by one flash. Both mutants have a higher concentration of the redox state in the dark-adapted state than the wild type. The mutations render the oxygen-evolving system more accessible to cell reductants. The mutation Ala251Val also confers to PSII an increased sensitivity to high light. We have already demonstrated that under light stress a double mutant, AzV (Ala251Val, Phe211Ser), lost the ability to recover the PSII activity sooner than the wild type. Here, we confirm that the modification of the alanine-251 is responsible for this specific sensitivity to high light. We conclude that specific mutations of the QB pocket modify the behavior of the cells under light stress and have an effect on the structure of the D1 protein in the other side of the membrane.