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 The international C4 rice consortium aims to introduce into rice a high capacity photosynthetic mechanism, the C4 pathway, to increase yield. The C4 pathway is characterised by a complex combination of biochemical and anatomical specialisation that ensures high CO2 partial pressure at RuBisCO sites in bundle sheath (BS) cells. Here we report an update of the progress of the C4 rice project. Since its inception in 2008 there has been an exponential growth in synthetic biology and molecular tools. Golden Gate cloning and synthetic promoter systems have facilitated gene building block approaches allowing multiple enzymes and metabolite transporters to be assembled and expressed from single gene constructs. Photosynthetic functionalisation of the BS in rice remains an important step and there has been some success overexpressing transcription factors in the cytokinin signalling network which influence chloroplast volume. The C4 rice project has rejuvenated the research interest in C4 photosynthesis. Comparative anatomical studies now point to critical features essential for the design. So far little attention has been paid to the energetics. C4 photosynthesis has a greater ATP requirement, which is met by increased cyclic electron transport in BS cells. We hypothesise that changes in energy statues may drive this increased capacity for cyclic electron flow without the need for further modification. Although increasing vein density will ultimately be necessary for high efficiency C4 rice, our modelling shows that small amounts of C4 photosynthesis introduced around existing veins could already provide benefits of increased photosynthesis on the road to C4 rice.

18 O 2 labeling was used to discriminate between O 2 evolution in photosynthetic water splitting and O 2 consumption. In wild type, ϳ20% of electrons originated from water was targeted to O 2 under air level CO 2 conditions but increased up to 60% in severe limitation of C i . Gas exchange experiments with ⌬flv1, ⌬flv3, and ⌬flv1/⌬flv3 mutants demonstrated that a considerable amount of electrons in these mutants is directed to photorespiration under C i deprivation. This assumption is in line with increased transcript abundance of photorespiratory genes and accumulation of photorespiratory intermediates in the WT and to a higher extent in mutant cells under C i deprivation.

C 4 photosynthesis is characterised by a CO 2 concentrating mechanism that operates between mesophyll and bundle sheath cells increasing CO 2 partial pressure at the site of Rubisco and photosynthetic efficiency. Electron transport chains in both cell types supply ATP and NADPH for C 4 photosynthesis. Cytochrome b 6 f is a key control point of electron transport in C 3 plants. To study whether C 4 photosynthesis is limited by electron transport we constitutively overexpressed the Rieske FeS subunit in Setaria viridis . This resulted in a higher Cytochrome b 6 f content in mesophyll and bundle sheath cells without marked changes in the abundances of other photosynthetic proteins. Rieske overexpression plants showed better light conversion efficiency in both Photosystems and could generate higher proton-motive force across the thylakoid membrane underpinning an increase in CO 2 assimilation rate at ambient and saturating CO 2 and high light. Our results demonstrate that removing electron transport limitations can increase C 4 photosynthesis.

Flavodiiron proteins are known to have crucial and specific roles in photoprotection of photosystems I and II in cyanobacteria. The filamentous, heterocyst-forming cyanobacterium Anabaena sp. strain PCC 7120 contains, besides the four flavodiiron proteins Flv1A, Flv2, Flv3A, and Flv4 present in vegetative cells, two heterocyst-specific flavodiiron proteins, Flv1B and Flv3B. Here, we demonstrate that Flv3B is responsible for light-induced O 2 uptake in heterocysts, and that the absence of the Flv3B protein severely compromises the growth of filaments in oxic, but not in microoxic, conditions. It is further demonstrated that Flv3B-mediated photosynthetic O 2 uptake has a distinct role in heterocysts which cannot be substituted by respiratory O 2 uptake in the protection of nitrogenase from oxidative damage and, thus, in an efficient provision of nitrogen to filaments. In line with this conclusion, the Δflv3B strain has reduced amounts of nitrogenase NifHDK subunits and shows multiple symptoms of nitrogen deficiency in the filaments. The apparent imbalance of cytosolic redox state in Δflv3B heterocysts also has a pronounced influence on the amounts of different transcripts and proteins. Therefore, an O 2 -related mechanism for control of gene expression is suggested to take place in heterocysts.nitrogen fixation | oxygen protection | photosynthesis

a b s t r a c tFlavodiiron proteins present in many prokaryotic and some eukaryotic organisms have a capacity to protect cells against nitrosative or oxidative stress. In Anabaena sp. PCC 7120, Flv1 and Flv3 proteins are encoded by families of two genes. We demonstrate here that flv1A and flv3A genes are up-regulated in vegetative cells in low CO 2 and high light conditions. In contrast, flv1B and flv3B genes are expressed in N 2 -fixing conditions and corresponding proteins are located exclusively in heterocysts. It is suggested that Flv1B and Flv3B protect enzymes of N 2 -fixation in heterocysts of Anabaena 7120 by reducing molecular oxygen directly to water.

Summary Introduction of a C4 photosynthetic mechanism into C3 crops offers an opportunity to improve photosynthetic efficiency, biomass and yield in addition to potentially improving nitrogen and water use efficiency. To create a two‐cell metabolic prototype for an NADP‐malic enzyme type C4 rice, we transformed Oryza sativa spp. japonica cultivar Kitaake with a single construct containing the coding regions of carbonic anhydrase, phosphoenolpyruvate (PEP) carboxylase, NADP‐malate dehydrogenase, pyruvate orthophosphate dikinase and NADP‐malic enzyme from Zea mays, driven by cell‐preferential promoters. Gene expression, protein accumulation and enzyme activity were confirmed for all five transgenes, and intercellular localization of proteins was analysed. 13CO2 labelling demonstrated a 10‐fold increase in flux though PEP carboxylase, exceeding the increase in measured in vitro enzyme activity, and estimated to be about 2% of the maize photosynthetic flux. Flux from malate via pyruvate to PEP remained low, commensurate with the low NADP‐malic enzyme activity observed in the transgenic lines. Physiological perturbations were minor and RNA sequencing revealed no substantive effects of transgene expression on other endogenous rice transcripts associated with photosynthesis. These results provide promise that, with enhanced levels of the C4 proteins introduced thus far, a functional C4 pathway is achievable in rice.

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