Final Activity and Management Report Summary - APOSD (The acclimation of photosynthetic organisms to sulphur deprivation) In the absence of sulphur (S), Chlamydomonas reinhardtii increases the abundance of several transcripts encoding proteins associated with S acquisition and assimilation, conserves S amino acids, and acclimates to suboptimal growth conditions. A positive regulator, SAC1 (for sulphur acclimation protein 1), and a negative regulator, SAC3, were shown to participate in the control of these processes. During the research project supported by the Marie Curie fellowship we identified, isolated and investigated a mutant affected in a gene encoding a SNRK2 (for SNF1-related protein kinase 2) kinase designated SNRK2.1. We probed that SNRK2.1 is a major positive regulator with a crucial role in the signalling cascade for both the S assimilation pathway and S deprivation responses. Like the sac1 mutant, the snrk2.1 mutant was deficient in the expression of S-responsive genes although the effects of SNRK2.1 mutation on specific transcripts expressions and general adaptative responses to S deficiency were more severe than those observed for the sac1 mutant. Moreover, SNRK2.1 is epistatic over SAC3. These results have been published (Plant Physiology, Gonzalez-Ballester et al., 2008). We have also identified three sulphate transporters (SLT1, SLT2 and SULTR2), examined their regulation, and established their biogenesis and subcellular locations. Upon S starvation of wild-type Chlamydomonas cells, their transcripts markedly accumulated. This transcript accumulation is SNRK2.1-dependent. All three transporters were localised to the plasma membrane, and their turnover rates were significantly impacted by S availability. We employed a reverse genetics approach to identify mutants for each of the sulphate transporters. These mutants were used to establish the critical role and functionality of these three sulphate transporters in the transport of sulphate into S-deprived cells. These results were published in Plant Physiology (Pootakham et al., 2010). Moreover, the Chlamydomonas transcriptome was characterized from nutrient-replete and S-depleted wild-type and snrk2.1 mutant cells. The transcriptome analyses used microarray hybridization and RNA-seq technology. RNA-seq is a novel accurate technology that allows a deep coverage of the transcriptome; we were the first group that reported the use of this technology in Chlamydomonas and one of the first among plants. Transcripts responsive to S deprivation included those encoding proteins involved in S acquisition and assimilation, synthesis of S-containing metabolites, Cys degradation, and S recycling. Furthermore, we noted potential modifications of cellular structures during S deprivation, including the cell wall and complexes associated with the photosynthetic apparatus. Moreover, the data suggest that S-deprived cells accumulate proteins with few S-containing amino acids. Most of the S deprivation responses are controlled by the SNRK2.1 protein kinase. The snrk2.1 mutant exhibits a set of unique responses during both S-replete and S depleted conditions that are not observed in wild-type cells; among them are the expression of genes related with hydrogen production and amelioration of oxidative stress. The transcriptome results for wild-type and mutant cells strongly suggest the occurrence of massive changes in cellular physiology and metabolism as cells become depleted for S, and they also reveal acclimation aspects that are likely critical for cell survival. These results were published in Plant Cell (González-Ballester et al., 2010). We have also studied the interactions of the S- and phosphorous (P)-deficiency responses in Chlamydomonas. The Chlamydomonas PSR1 gene is required for proper acclimation of the cells to P deficiency. P-starved psr1 mutants show signs of secondary S starvation, exemplified by the accumulation of transcripts encoding proteins involved in S scavenging and assimilation.
