Sulfur is an essential element in proteins, lipids, and some critical metabolites. The work proposed in this grant continues an exploration of the regulatory aspects of sulfur deprivation responses in the alga Chlamydomonas. Regulatory mutants aberrant for sulfur-deprivation responses and suppressor strains will be characterized to help identify interactions among signaling factors. Wild-type and various mutant strains will be queried with genome-wide methods for examining the transcriptome under nutrient-replete and sulfur-starvation conditions, using both microarray and Solexa sequencing technologies. Focused intellectual and technical efforts will be directed toward establishing more biochemical links between identified regulators. A diversity of protein-protein interaction assays, including the yeast two-hybrid system, the split ubiquitin and split GFP systems and the classical co-immunoprecipitation assays will facilitate these studies, providing strong insights into the regulatory circuits that control nutrient limitation responses in photosynthetic organisms. Finally, analyses will be performed to help understand the two tiers of the sulfur-deprivation response that have recently been discovered; one is protein synthesis-independent while the other is protein synthesis-dependent.
In a broader sense, the proposed work will help untangle some of the complexities associated with the regulatory machinery that is required for photosynthetic cells to cope with deprivation conditions and ultimately help us understand the impact of sulfur compounds in our diets and on global weather patterns; some sulfur compounds strongly influence the quality and nutritional value of food while others have profound effects on the climate of the Earth. The project will train a post doctoral student and will involve collaboration with Dr. Charles Hauser and his undergraduate students at St. Edward's University, who will participate in summer exchange and in the analysis of microarray results. St. Edward's University is a minority serving institution.
Sulfur, most frequently found in the environment as the sulfate anion, is a macronutrient that can be limiting to the growth of plants in the natural environment as well as in agricultural settings. Sulfur is required for the synthesis of lipids, polysaccharides, regulatory molecules, the ubiquitous antioxidant glutathione and the heavy metal-chelating compound phytochelatin (which binds to and minimizes the toxicity of heavy metals). Furthermore, a number of sulfur metabolites are volatile and are released into the environment; some of these volatile compounds impact cloud formation while others may be important for the resistance of plants to insects and pathogens. Therefore, it is critical to understand the ways in which sulfur is metabolized, how this metabolism is controlled by environmental factors and the strategies used by organisms to ameliorate the effects of sulfur-deprivation conditions. In a larger sense, this work has helped elucidate a poorly understood aspects of metabolism that is critical for survival of both single cells and more complex organisms. The complexity of the regulatory machinery associated with sulfur deprivation responses has been surprising. We have been able to identify and elucidate the functions of many regulatory elements associated with sulfur acclimation responses, including components of a phosphorelay control system (e.g. specific serine threonine kinases and a putative sensor molecule located in the cytoplasmic membrane that resembles a sulfate transporter). We have begun to understand how sulfur deprivation impacts photosynthetic function, transport processes, sulfate assimilation pathways and the mechanisms for redistributing sulfur among the different compounds in the cell. We identified all of the sulfate transporters and demonstrated which of the transporters become more abundant during sulfur deprivation and how the individual transporters impact the uptake process under conditions of sulfur limitation. The work has also revealed a more global aspect of the acclimation response in that there appears to be a restructuring of the cell that minimizes the use of sulfur in proteins and lipids. In addition, we have demonstrated that the acclimation response is tiered, with the first tier associated with transport of sulfate into the cell and the second tier (which is temporally distinct and requires protein synthesis) associated with the ways in which the cell redistributes and allocates its sulfur resources.. However, there is still much more to learn about the ways in which cells deal with sulfur in the environment and the layers of regulation that modulate cellular metabolism to accomodate the availability of this element.
