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Forward genetics was used to isolate Chlamydomonas reinhardtii mutants with altered abilities to acclimate to sulfur (S) deficiency. The ars76 mutant has a deletion that eliminates several genes, including VACUOLAR TRANSPORTER CHAPERONE1 (VTC1), which encodes a component of a polyphosphate polymerase complex. The ars76 mutant cannot accumulate arylsulfatase protein or mRNA and shows marked alterations in levels of many transcripts encoded by genes induced during S deprivation. The mutant also shows little acidocalcisome formation compared with wild-type, S-deprived cells and dies more rapidly than wild-type cells following exposure to S-, phosphorus-, or nitrogen (N)-deficient conditions. Furthermore, the mutant does not accumulate periplasmic L-amino acid oxidase during N deprivation. Introduction of the VTC1 gene specifically complements the ars76 phenotypes, suggesting that normal acidocalcisome formation in cells deprived of S requires VTC1. Our data also indicate that a deficiency in acidocalcisome function impacts trafficking of periplasmic proteins, which can then feed back on the transcription of the genes encoding these proteins. These results and the reported function of vacuoles in degradation processes suggest a major role of the acidocalcisome in reshaping the cell during acclimation to changing environmental conditions., (© 2014 American Society of Plant Biologists. All rights reserved.)

During sulfur (S) deprivation, the unicellular alga Chlamydomonas reinhardtii exhibits increased expression of numerous genes. These genes encode proteins associated with sulfate (SO4(2-)) acquisition and assimilation, alterations in cellular metabolism, and internal S recycling. Administration of the cytoplasmic translational inhibitor cycloheximide prevents S deprivation-triggered accumulation of transcripts encoding arylsulfatases (ARS), an extracellular polypeptide that may be important for cell wall biosynthesis (ECP76), a light-harvesting protein (LHCBM9), the selenium-binding protein, and the haloperoxidase (HAP2). In contrast, the rapid accumulation of transcripts encoding high-affinity SO4(2-) transporters is not affected. These results suggest that there are two tiers of transcriptional regulation associated with S deprivation responses: the first is protein synthesis independent, while the second requires de novo protein synthesis. A mutant designated ars73a exhibited low ARS activity and failed to show increases in ECP76, LHCBM9, and HAP2 transcripts (among others) in response to S deprivation; increases in transcripts encoding the SO4(2-) transporters were not affected. These results suggest that the ARS73a protein, which has no known activity but might be a transcriptional regulator, is required for the expression of genes associated with the second tier of transcriptional regulation. Analysis of the ars73a strain has helped us generate a model that incorporates a number of complexities associated with S deprivation responses in C. reinhardtii.

Chlamydomonas (Chlamydomonas reinhardtii) exhibits several responses following exposure to sulfur (S)-deprivation conditions, including an increased efficiency of import and assimilation of the sulfate anion (SO(4)(2-)). Aspects of SO(4)(2-) transport during S-replete and S-depleted conditions were previously studied, although the transporters had not been functionally identified. We employed a reverse genetics approach to identify putative SO(4)(2-) transporters, examine their regulation, establish their biogenesis and subcellular locations, and explore their functionality. Upon S starvation of wild-type Chlamydomonas cells, the accumulation of transcripts encoding the putative SO(4)(2-) transporters SLT1 (for SAC1-like transporter 1), SLT2, and SULTR2 markedly increased, suggesting that these proteins function in high-affinity SO(4)(2-) transport. The Chlamydomonas sac1 and snrk2.1 mutants (defective for acclimation to S deprivation) exhibited much less of an increase in the levels of SLT1, SLT2, and SULTR2 transcripts and their encoded proteins in response to S deprivation compared with wild-type cells. All three transporters were localized to the plasma membrane, and their rates of turnover were significantly impacted by S availability; the turnover of SLT1 and SLT2 was proteasome dependent, while that of SULTR2 was proteasome independent. Finally, mutants identified for each of the S-deprivation-responsive transporters were used to establish their critical role in the transport of SO(4)(2-) into S-deprived cells.

In the absence of sulfur (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 sulfur acclimation protein 1), and a negative regulator, SAC3, were shown to participate in the control of these processes. In this study, we investigated two allelic mutants (ars11 and ars44) affected in a gene encoding a SNRK2 (for SNF1-related protein kinase 2) kinase designated SNRK2.1. Like the sac1 mutant, both snrk2.1 mutants were deficient in the expression of S-responsive genes. Furthermore, the mutant cells bleached more rapidly than wild-type cells during S deprivation, although the phenotypes of ars11 and ars44 were not identical: ars11 exhibited a more severe phenotype than either ars44 or sac1. The phenotypic differences between the ars11 and ars44 mutants reflected distinct alterations of SNRK2.1 mRNA splicing caused by insertion of the marker gene. The ars11 phenotype could be rescued by complementation with SNRK2.1 cDNA. In contrast to the nonepistatic relationship between SAC3 and SAC1, characterization of the sac3 ars11 double mutant showed that SNRK2.1 is epistatic to SAC3. These data reveal the crucial regulatory role of SNRK2.1 in the signaling cascade critical for eliciting S deprivation responses in Chlamydomonas. The phylogenetic relationships and structures of the eight members of the SNRK2 family in Chlamydomonas are discussed.

Chlamydomonas reinhardtii is a unicellular green alga whose lineage diverged from land plants over 1 billion years ago. It is a model system for studying chloroplast-based photosynthesis, as well as the structure, assembly, and function of eukaryotic flagella (cilia), which were inherited from the common ancestor of plants and animals, but lost in land plants. We sequenced the approximately 120-megabase nuclear genome of Chlamydomonas and performed comparative phylogenomic analyses, identifying genes encoding uncharacterized proteins that are likely associated with the function and biogenesis of chloroplasts or eukaryotic flagella. Analyses of the Chlamydomonas genome advance our understanding of the ancestral eukaryotic cell, reveal previously unknown genes associated with photosynthetic and flagellar functions, and establish links between ciliopathy and the composition and function of flagella.

During sulfur deprivation, the photosynthetic green alga Chlamydomonas reinhardtii develops a high-affinity sulfate uptake system and increases the expression of genes encoding proteins involved in sulfur assimilation. Although two regulatory elements, SAC1 and SAC3, have been shown to be required for normal acclimation of C. reinhardtii to sulfur deprivation, a number of other regulatory elements appear to also be involved. The molecular mechanisms by which these regulatory elements function are largely unknown. This manuscript presents our current knowledge of sulfur deprivation responses and the regulation of these responses in C. reinhardtii. In addition, we present preliminary results of a sub-saturation screen for novel sulfur acclimation mutants of C. reinhardtii. A speculative model, incorporating the activities of established regulatory elements with putative novel components of the signal transduction pathway(s) is discussed.