1. Iuliana-Cristina Simeoni, Ciprian Chis, Oana Sicora, Dalton Carmel and Cosmin Ionel Sicora Email: cosmin.sicora@gmail.com Changes In Photosystem II (PSII) Function During Dark and Light Cycles In Cyanobacterium Cyanothece sp. ATCC 51142 Summary: Cyanobacteria, green algae and higher plants have highly conserved photosynthetic machinery. The D1 protein, one of the core proteins of Photosystem II, is an indispensable component of oxygenic photosynthesis. In cyanobacteria, the D1 protein is encoded by a psbA gene family, whose members are differentially expressed according to the different environmental conditions. Cyanothece sp. ATCC 51142 is an unicellular, aerobic, diazotrophic cyanobacterium which fixes N 2 in the dark. In Cyanothece the psbA gene family is composed of 5 members, encoding different isoforms of the D1 protein. According to Murray (2012, DOI: 10.1007/s11120-011-9714-5), a new type of D1 protein is postulated, D1 rogue, which blocks the PSII during the night and allows fixation of nitrogen. In this study we show changes in PSII function in cells grown under 12 hours light/12 hours dark cycles, in Cyanothece sp. ATCC 51142 which has a hypothetical D1rogue (D1r) protein, and in Synechocystis sp. PCC 6803, a cyanobacterium which lack a gene that encodes D1r protein (used as a control). We also show variation in the number active centers number of PSII during the Light/Dark cycles, recorded as a reversible decrease of fluorescence amplitude. This results are very important to understand the functionality of PSII in the larger context of adaptation to environmental cues. Ministry of National Education, Biological Research Center Jibou, Romania Introduction: The psbA gene family in Cyanothece sp. ATCC 51142 contains 5 psbA genes differentially expressed according to environmental conditions. Previous studies have shown 1 gene to be induced during the dark phase of a light/dark cycle. This gene has been reported earlier by J. Murray to encode a divergent D1 protein called D1 rogue. Based on its sequence, this protein is proposed to have a modified donor and acceptor side that would inhibit the normal function of PSII electron transport chain. We investigated the Q A - reoxidation using flash fluorescence on samples from dark and light regimes, to detect the possible functional traces of D1r protein. Materials and Methods: Cyanothece sp. ATCC 51142 was obtained from Biological Research Institute from Cluj Napoca, Romania. Synechocystis sp. PCC 6803 was obtained from Pasteur Culture Collection. Both strains were grown at 30ºC and 50μmol photons m -2 s -1 light, until at a chlorophyll concentration of 6 μg chl ml -1. In order to investigate the changes in PSII function during 12 h light/12 h dark cycles, we designed the following experiments: We performed flash fluorescence measurements at Cyanothece sp. ATCC 51142, every 60 minutes during 26 h, both in the absence and presence of DCMU (3-(3,4-Dichlorophenyl)-1,1-dimethylurea). We also measured chlorophyll fluorescence in presence of Lincomycin (300µg/ml), an inhibitor that blocks the synthesis of new proteins. As control, similar measurements were also performed on Synechocystis sp. PCC 6803. Samples were also take for RNA, DNA, protein and flash fluorescence measurements. The fluorescence measurements were made with an FL3500 fluorometer from Photon Systems Instruments, we used a Q A recombination protocol, than the results were processed in the Origin.v8.0 program and for each sample was applied a Joliot Correction before plotting the graphs. Results Conclusions During the dark period the acceptor side of PSII is modified by a slowdown of Q A to Q B transfers, that is most evident in centers that have an empty Q B pocket at the time of the flash. This change is reversed in about one hour after the light is turned on. The ON-set of a fast phase on the flash fluorescence decay curve in presence of DCMU during dark period suggests an inhibition of Water Oxidation Complex that is rapidly reversed during the light phase. During the dark period, the amplitude of the flash fluorescence curve, (F M - F 0 ), proportional with number of PS II active centers is decreased to about 40%. This suggests a temporary inhibition, during the night, of the active PSII centers. The Lincomycin treatment, applied 80 minutes before the light was turned off, reduced the change on acceptor side and donor side as well, but did not change significantly the evolution of the active PS II centers. This suggests that at least in part, the changes observed during the dark period require de novo protein synthesis. Using a general D1 antibody we have recorded on Western Blot significant changes in total D1 protein amount during the light/dark cycle consistent with the changes seen on the amplitude of flash fluorescence. As a control strain we used Synechocystis sp. PCC 6803 (no presence of D1r protein). In this case we did not see the changes of the same amplitude on the donor and acceptor side of PSII as in the case of Cyanothece sp. ATCC 51142. We conclude that D1r protein from Cyanothece sp. ATCC 51142 may have a role in the photosystemII function changes seen during the light/dark cycles. Figure 2: Changes of acceptor side of PSII function during a 12h Light/12h Dark cycle in Cyanothece sp. ATCC 51142 Figure 3: Changes of donor side of PSII function during a 12h Light/12h Dark cycle in Cyanothece sp. ATCC 51142 Figure 4: Changes in the number of PSII active centers during a 12h Light/12h Dark cycle in Cyanothece sp. ATCC 51142 in absence (a) and presence (b) of DCMU Figure 5: Changes of acceptor side of PSII function during a 12h Light/12h Dark cycle in Cyanothece sp. ATCC 51142, in absence (a) and presence of Lincomycin (b). Figure 6: Changes of donor side of PSII function during a 12h Light/12h Dark cycle in Cyanothece sp. ATCC 51142, in absence (a) and presence of Lincomycin (b). Figure 7: T2 parameter evolution on the acceptor side of PSII function during a 12h Light/12h Dark cycle in Cyanothece sp. ATCC 51142, in absence and presence of Lincomycin Figure 8: Changes of acceptor side (a) and donor side (b) of PSII function during a 12h Light/12h Dark cycle in Synechocystis sp. PCC 6803. Changes in the PSII number of active centers in absence (a) and presence (b) of DCMU Figure 9: Changes in total D1 protein amount during a 12 h Light/12 h Dark Cycle in Cyanothece sp. ATCC 51142 Figure 1: Diagram representing the Light/Dark protocols used during our experiments Acknowledgement: This work was supported by a grant of the Romanian National Authority for Scientific Research, CNCS-UEFISCDI, project number PN-II-ID-PCE-2011-3-0765