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Cyanobacteria are the most ancient organisms performing oxygenic photosynthesis, and they are the ancestors of plant plastids. All plastids contain the plastid terminal oxidase (PTOX), while only certain cyanobacteria contain PTOX. Many putative functions have been discussed for PTOX in higher plants including a photoprotective role during abiotic stresses like high light, salinity and extreme temperatures. Since PTOX oxidizes PQH 2 and reduces oxygen to water, it is thought to protect against photo-oxidative damage by removing excess electrons from the plastoquinone (PQ) pool. To investigate the role of PTOX we overexpressed rice PTOX fused to the maltose-binding protein (MBP-OsPTOX) in Synechocystis sp. PCC 6803, a model cyanobacterium that does not encode PTOX. The fusion was highly expressed and OsPTOX was active, as shown by chlorophyll fluorescence and P 700 absorption measurements. The presence of PTOX led to a highly oxidized state of the NAD(P)H/NAD(P) + pool, as detected by NAD(P)H fluorescence. Moreover, in the PTOX overexpressor the electron transport capacity of PSI relative to PSII was higher, indicating an alteration of the photosystem I (PSI) to photosystem II (PSII) stoichiometry. We suggest that PTOX controls the expression of responsive genes of the photosynthetic apparatus in a different way from the PQ/PQH 2 ratio. This article is part of the themed issue ‘Enhancing photosynthesis in crop plants: targets for improvement’.

Cyanobacterial flavodiiron proteins (FDPs; A-type flavoprotein, Flv) comprise, besides the β-lactamase–like and flavodoxin domains typical for all FDPs, an extra NAD(P)H:flavin oxidoreductase module and thus differ from FDPs in other Bacteria and Archaea. Synechocystis sp. PCC 6803 has four genes encoding the FDPs. Flv1 and Flv3 function as an NAD(P)H:oxygen oxidoreductase, donating electrons directly to O 2 without production of reactive oxygen species. Here we show that the Flv1 and Flv3 proteins are crucial for cyanobacteria under fluctuating light, a typical light condition in aquatic environments. Under constant-light conditions, regardless of light intensity, the Flv1 and Flv3 proteins are dispensable. In contrast, under fluctuating light conditions, the growth and photosynthesis of the Δ flv1(A) and/or Δ flv3(A) mutants of Synechocystis sp. PCC 6803 and Anabaena sp. PCC 7120 become arrested, resulting in cell death in the most severe cases. This reaction is mainly caused by malfunction of photosystem I and oxidative damage induced by reactive oxygen species generated during abrupt short-term increases in light intensity. Unlike higher plants that lack the FDPs and use the Proton Gradient Regulation 5 to safeguard photosystem I, the cyanobacterial homolog of Proton Gradient Regulation 5 is shown not to be crucial for growth under fluctuating light. Instead, the unique Flv1/Flv3 heterodimer maintains the redox balance of the electron transfer chain in cyanobacteria and provides protection for photosystem I under fluctuating growth light. Evolution of unique cyanobacterial FDPs is discussed as a prerequisite for the development of oxygenic photosynthesis.

Summary Ferredoxin:NADP oxidoreductases (FNRs) constitute a family of flavoenzymes that catalyse the exchange of electrons between ferredoxin and NADP(H). In cyanobacteria FNR provides NADPH for photoautotrophic metabolism, but the enzyme is also capable of oxidizing NADPH providing reduced ferredoxin. In the cyanobacterium Synechocystis sp. strain PCC6803, the unique petH gene has two translation products depending on growth conditions. As a consequence two isoforms of the FNR accumulate – FNRL and FNRS. In the present work, analysis of petH expression reveals that different transcriptional start points (tsp) are responsible for this differential translation initiation. Under standard conditions (where FNRL accumulates), two tsps were found at −52 and −34 relative to the first translation start site. Under nitrogen-starvation conditions (where FNRS accumulates) a tsp was mapped at −126 relative to the first translation start site. Therefore, the transcript responsible for FNRS translation is longer than that producing FNRL. In addition, expression of the short or long transcript in E. coli resulted in the accumulation of FNRL or FNRS respectively. This result demonstrates that translation can initiate at two different sites, 336-bases apart (ATG-1 to ATG-113), depending only on the 5′UTR structure.

Synechocystis strain PCC 6803 exhibits similar levels of cyclic AMP (cAMP) and cyclic GMP (cGMP). A thorough analysis of its genome showed that Cya2 (Sll0646) has all the sequence determinants required in terms of activity and purine specificity for being a guanylyl cyclase. Insertional mutagenesis of cya2 caused a marked reduction in cGMP content without altering the cAMP content. Thus, Cya2 represents the first example of a prokaryotic guanylyl cyclase.

In several cyanobacteria, petH, the gene encoding ferredoxin:NADP oxidoreductase (FNR), is transcribed from at least two promoters depending on growth conditions. Two transcripts (short and long) are translated from two different translation initiation sites, resulting in two isoforms (large and small, respectively). Here, we show that in Synechocystis PCC6803 the global transcriptional regulator NtcA activates transcription from the distal petH promoter. Modification of the NtcA-binding site prevents NtcA binding to the promoter in vitro and abolishes accumulation of the small isoform of FNR in vivo. We also demonstrate that a similar petH transcription and translation regime occurs in other cyanobacteria. The conditions under which this system operates provide hints for the function of each FNR isoform.

In photosynthetic organisms, ferredoxin:NADP(+) oxidoreductase (FNR) is known to provide NADPH for CO(2) assimilation, but it also utilizes NADPH to provide reduced ferredoxin. The cyanobacterium Synechocystis sp. strain PCC6803 produces two FNR isoforms, a small one (FNR(S)) similar to the one found in plant plastids and a large one (FNR(L)) that is associated with the phycobilisome, a light-harvesting complex. Here we show that a mutant lacking FNR(L) exhibits a higher NADP(+)/NADPH ratio. We also purified to homogeneity a phycobilisome subcomplex comprising FNR(L,) named FNR(L)-PC. The enzymatic activities of FNR(L)-PC were compared with those of FNR(S). During NADPH oxidation, FNR(L)-PC exhibits a 30% decrease in the Michaelis constant K(m)((NADPH)), and a 70% increase in K(m)((ferredoxin)), which is in agreement with its predicted lower activity of ferredoxin reduction. During NADP(+) reduction, the FNR(L)-PC shows a 29/43% decrease in the rate of single electron transfer from reduced ferredoxin in the presence/absence of NADP(+). The increase in K(m)((ferredoxin)) and the rate decrease of single reduction are attributed to steric hindrance by the phycocyanin moiety of FNR(L)-PC. Both isoforms are capable of catalyzing the NADP(+) reduction under multiple turnover conditions. Furthermore, we obtained evidence that, under high ionic strength conditions, electron transfer from reduced ferredoxin is rate limiting during this process. The differences that we observe might not fully explain the in vivo properties of the Synechocystis mutants expressing only one of the isoforms. Therefore, we advocate that FNR localization and/or substrates availability are essential in vivo.

In cyanobacteria, light energy is mainly harvested for photosynthesis by the phycobilisome (PBS): a large pigment-protein complex. This complex is composed of heterodimeric phycobiliproteins that are assembled with the aid of linker polypeptides in order to optimize light-energy absorbance and transfer to photosystem II. The core membrane linker subunit (L(CM)) is a fascinating multifunctional polypeptide that participates in the PBS structure, function and anchoring to the photosynthetic membrane. Sequence analysis has defined several domains within the L(CM) polypeptide. The C-terminal portion contains two to four repeated domains that are similar to the conserved domains of linker polypeptides and are believed to play the same role. The N-terminal portion is similar to phycobiliproteins (PB-domain) and carries, like phycobiliproteins, a covalently linked phycobilin chromophore. This domain is interrupted by a so-called PB-loop insertion. The PB-domain of the L(CM) is thus regarded as one of the core subunits, with its PB-loop protruding towards the photosynthetic membrane. The PB-loop was thought to be involved in the attachment of the PBS to the photosynthetic membrane. We generated an apcE gene (encoding L(CM)), in which we deleted the sequence encoding 54 amino acids of the PB-loop domain. The modified gene was expressed in a Synechocystis PCC6714 strain in which the apcE gene had been inactivated. The truncated polypeptide was functionally equivalent to the wild-type L(CM); PBSs were assembled and functioned as in the wild-type. The PB-loop of the L(CM) seems thus dispensable for the PBS biogenesis and function.

Different classes of herbicides block the electron transfer at the level of photosystem II. The herbicides and QB bind in the same region of the D1 protein which belongs to the PSII core complex (1). The resistance to an herbicide is due to a decrease in the affinity constant of the herbicide for its site.

A Synechocystis 6714 mutant resistant to the phenol-type herbicide ioxynil was isolated and characterized. Sensitivity to DCMU and atrazine was measured in whole cells and isolated thylakoids. The mutant presents the same sensitivity to atrazine as the wild type and a slightly increased sensitivity to DCMU. A point mutation has been found at codon 266 in the psbAI coding locus (AAC to ACC) resulting in an amino acid change from asparagine to threonine in the D1 protein.

Mutations conferring herbicide resistance in 3 mutant strains of the cyanobacterium Synechocystis 6714 have been characterized by gene cloning and sequencing. The mutants display very different phenotypes: DCMU-IIA is DCMU-resistant and atrazine-resistant, DCMU-IIB is DCMU-resistant and atrazine-sensitive, and Az-V is DCMU-sensitive, atrazine-resistant and presents particular photoinhibition properties. These mutants were originally obtained either by one-step selection (DCMU-IIA) or by two-step selection (DCMU-IIB and Az-V). psbA copies carrying herbicide resistance have been identified by transformation experiments as psb AI in all cases. Sequences of the psb AI copy of each mutant have been compared to the wild-type sequence. In the single mutant DCMU-IIA, a point mutation at codon 264 (Ser----Ala) results in resistance to both DCMU and atrazine. In the double mutants DCMU-IIB and Az-V, two point mutations were found. DCMU-IIB was derived from DCMU-IIA and had acquired a second mutation at codon 255 (Phe----Leu) resulting in a slight increase in DCMU resistance and complete abolition of atrazine resistance. Az-V contains two changes at codons 211 (Phe----Ser) and 251 (Ala----Val) resulting in high atrazine resistance but only slight DCMU resistance.