1. Characterization of the response of Synechococcus sp. WH7803 to high-light and UV radiations Ludovic Joubin, Christophe Six, Frédéric Partensky, and Laurence Garczarek Station Biologique, CNRS, F-29682 Roscoff, France. Introduction Albeit sunlight is essential for phototrophs, high-light and especially associated UV-radiations (UV-A and B) can severely injure organisms with effects ranging from molecular to physiological levels (Häder et al.). Impact of these radiations is particularly important in oligotrophic and mesotrophic waters where Synechococcus constitutes one of the most abundant photosynthetic prokaryotes (Partensky et al. 1999) In order to better understand which mechanisms are involved in the Synechococcus sp.WH7803 response to these stresses, we used a transcriptomic approach coupled with biophysical measurements (PAM fluorimetry) in cultures previously acclimated to different light irradiances. Transcriptional variations Figure 2 shows that both stresses induced a similar response of the photosynthetic apparatus and photoprotection system. Indeed, upregulation of photosystem II genes (psbA and psbD) indicates an increased turnover of the reaction center II, allowing its repair following photoinhibition, as well as enhanced dissipation of excess light energy. Moreover, both stresses seemed to induce a reduction of carbon fixation as indicated by the repression of Rubisco (rbcL). Thus, both light and dark photosynthetic reactions were affected. The crtR gene, likely involved in photoprotection, is also upregulated under HL and UV stresses, suggesting an activation of zeaxanthin-binding proteins. In contrast, psaA mRNA levels strongly decreased, possibly reflecting a modulation of the PSI : PSII ratio. Decrease of mpeD trancript levels (and all other phycobilisome components; not shown) pointed out a global down-regulation of light-harvesting capacities in response to high energy radiations. Surprisingly, genes encoding proteins involved in protection against reactive oxygen species (sodB and katG) were not significantly activated and the catalase gene expression even seemed to be slightly repressed during both stresses. Concerning the DNA repair genes, while none of them were differentially regulated under HL stress, the recombinase A gene (recA) was strongly induced upon UV stress. This clearly points out that DNA damages are specifically induced by UV radiations and not HL stress alone. Figure 3 : Transcriptional variations of recA, psbA and psaA genes under UV stress conditions after acclimation to different growth irradiances psbA recA psaA Effect of light acclimation on the transcriptional response to UV stress Figure 2 : Relative expression of selected genes under a 24 h UV- (left column) or HL- stress (right column) for ML- acclimated Synechococcus cultures. Genes are classified according to the function of their encoding proteins. Only the variations of gene expression higher than 2 or lower than 0.5 have been considered significant. Synechococcus cultures acclimated to different light irradiances showed a similar transcriptional response to UV stress (Fig. 3), characterized by an increased level of psbA and recA transcripts and a repression of the psaA gene. However, the response was faster and stronger in the LL- than in the ML- and HL- acclimated cultures. In agreement with these results, basal expression levels for most genes but recA were much higher in ML- and HL- acclimated cultures (not shown). Thus, prior acclimation to higher light photon fluxes appears to better prepare the cells to undergo UV light stress and favours a quick acclimation to new light conditions. UV stress HL stress Photosynthesis DNA repair and oxidative stress Others 15µE 75 µE400 µE Shift HL Shift HL Shift ML Shift UV Shift UV Shift UV LL ML HL Experimental approaches Figure 1 : Schematic representation of the experimental protocol.. LL = Low Light, ML = Medium Light, HL = High Light. To compare the effect of UV- and high light- stresses, Synechococcus WH7803 cultures were submitted to either UV (UV-A: 5 W.m -2, UV-B: 0.5 W.m -2 ) or high light (HL) stresses (Fig. 1). These stresses have been performed by shifting cells to a 5-times higher irradiance compared to initial irradiance conditions. The influence of light acclimation has been investigated by comparing the response to these stresses of Synechococcus cultures acclimated to three different light intensities: Low Light (LL; 15 µE.m -2.s -1 ), Medium Light (ML; 75 µ.m -2.s -1 ) and High Light (HL; 400 µE.m -2.s -1 ). To obtain an overview of the response of Synechococcus sp. WH7803, real time RT-PCR has been optimized to measure the expression of about 15 relevant genes which could be involved in the response to UV and light stresses (Table 1). This work was done in the framework of the EU program MARGENES, the European Network of Excellence Marine Genomics and the National Program UVECO. Gene Product Description Photosynthetic genes psbA psbD psaA mpeD Photoprotection crtR DNA repair recA ORF0264 Detoxification in Reactive Oxygen Species (ROS) sodB katG Cellular membrane rlpA Carbon Fixation rbcL General stress response groes Table 1 : Genes selected to study the Synechococcus sp. WH7803 response to high light- and UV- stresses by quantitative real time RT-PCR Effect of HL- and UV- stresses on Synechococcus cultures acclimated to ML. Although this work is still in a preliminary stage, it allowed us to start apprehending Synechococcus sp. WH7803 response to HL and UV stresses and the effect of the light history of cells on their resistance to these stresses. Indeed, both stresses seemed to induce a large reorganization of the photosynthetic apparatus as shown by the acceleration of the turnover of PSII transcripts while the PSI and phycobilisome transcripts were strongly repressed. Furthermore, induction of the gene encoding the β-carotene hydroxylase (crtR), which is likely involved in zeaxanthin synthesis, seems to indicate the occurrence of photoprotection mechanisms of the cells by carotenoid compounds. This will be further studied by HPLC measurements of evolution of cell pigment content during the course of these stresses. Whereas the photosynthetic apparatus seemed to react similarly to both stresses, Synechococcus cultures recovered much better after HL- than after UV-stress (not shown). This phenomenon suggests that specific damages occurred in response to UV stress compared to HL stress. Surprisingly, genes encoding proteins involved in protection against reactive oxygen species (sodB, katG) and pyrimidine dimers (Photolyase) were not activated in response to these stresses. However, strong activation of the gene encoding the recombinase A (recA) suggests the occurrence of DNA damages specifically under UV stress. Complementary analyses to better understand the Synechococcus strategies to respond to reactive oxygen species (ROS) and pyrimidine dimers are currently in progress. Microarray analyses supported by the Marine Genomics Europe network (Synchips project, cf poster by Gregor Gierga) are currently initiated to further characterize the response of this strain to light and UV stresses. References Partensky F, Blanchot J, Vaulot D: Differential distribution and ecology of Prochlorococcus and Synechococcus in oceanic waters: a review. Edited by: Charpy L, Larkum AWD. Monaco: Musée Océanographique; 1999:457-475. Häder D.-P., Kumar H.D., Smith R.C. and. Worrest R.C : Effects on aquatic ecosystems. Journal of Photochemistry and Photobiology B: Biology, Volume 46, Issues 1-3, October 1998, Pages 53-68 Sinha R. P., Singh N., Kumar A., Kumar H. D., Häder M. and Häder D. -P. : Effects of UV irradiation on certain physiological and biochemical processes in cyanobacteria. Journal of Photochemistry and Photobiology B: Biology, Volume 32, Issues 1-2, January 1996, Pages 107-113 Ralph P. J. and Gademan R.: Rapid light curves: A powerful tool to assess photosynthetic activity. Aquatic Botany, Volume 82, Issue 3, July 2005, Pages 222-237 Hihara Y, Kamei A, Kanehisa M, Kaplan A and Ikeuchi M. : DNA microarray analysis of cyanobacterial gene expression during acclimation to high light. Plant Cell. 2001 Apr;13(4):793-806. Measurements of Photosystem II quantum yield variations were performed using a PAM fluorimeter in order to characterize cultures acclimated to different light regimes (Ralph et al. 2005). Over an irradiance gradient (Fig. 4), maximal yield was attained at a lower irradiance for LL-acclimated cultures than for HL ones. Under stress conditions (Fig. 5), the quantum yield of PSII was affected by both HL and UV stresses, although more strongly by the latter. This reflected photosystem II photoinhibition. Moreover, as noted at the transcriptional level, UV stress seemed to affect more strongly LL-acclimated cultures than HL, particularly at the beginning of the stress. At last, PSII quantum yield increases following the shift of an HL-acclimated cultures to ML conditions, suggesting the mobilization of short-term acclimation processes to a lower level of light. Figure 4 : Comparison of photosynthetic yield with regards to the light acclimation intensities performed by PAM fluorimeter. Figure 5 : Quantum yield of the photosystem II under a 24h UV stress for LL- and HL-acclimated cultures and after a shift to HL condition for the LL and a shift to ML for the HL cells. Photosynthetic activity Conclusions and future work D1 D2 psaA MpeD CrtR RecA ORF0264 SodB KatG RlpA RbcL GroES Reaction Center Photosystem II Reaction Center Photosystem I Phycoerythrin-associated linker β-carotene hydroxylase Recombinase A Photolyase Superoxide dismutase Catalase Lipoprotein A Rubisco large subunit Chaperon protein