SummaryThe two-electron reduction of sulfate to sulfite in plants is mediated by 5 0 -adenylylsulfate (APS) reductase, an enzyme theorized to be a control point for cysteine synthesis. The hypothesis was tested by expression in Arabidopsis thaliana under transcriptional control of the CaMV 35S promoter of the APS reductase from Pseudomonas aeruginosa (PaAPR) fused with the rbcS transit peptide for localization of the protein to plastids. PaAPR was chosen for the experiment because it is a highly stable enzyme compared with the endogenous APS reductase of A. thaliana, and because PaAPR is catalytically active in combination with the plant thioredoxins m and f indicating that it would likely be catalytically active in plastids. The results indicate that sulfate reduction and O-acetylserine (OAS) production together limit cysteine synthesis. Transgenic A. thaliana lines expressing PaAPR accumulated sulfite, thiosulfate, cysteine, c-glutamylcysteine, and glutathione. Sulfite and thiosulfate increased more than did cysteine, c-glutamylcysteine and glutathione. Thiosulfate accumulation was most pronounced in flowers. Feeding of OAS to the PaAPRexpressing plants caused cysteine and glutathione to increase more rapidly than in comparably treated wild type. Both wild-type and transgenic plants accumulated sulfite and thiosulfate in response to OAS feeding. The PaAPR-expressing plants were slightly chlorotic and stunted compared with wild type. An attempt to uncover the source of thiosulfate, which is not thought to be an intermediate of sulfate reduction, revealed that purified b-mercaptopyruvate sulfurtransferase is able to form thiosulfate from sulfite and b-mercaptopyruvate, suggesting that this class of enzymes could form thiosulfate in vivo in the presence of excess sulfite.

Algae and vascular plants are cysteine (Cys) prototrophs. They are able to import, reduce, and assimilate sulfate into Cys, methionine, and other organic sulfur-containing compounds. Characterization of genes encoding the enzymes required for Cys biosynthesis from the unicellular green alga Chlamydomonas reinhardtii reveals that transcriptional and posttranscriptional mechanisms regulate the pathway. The derived amino acid sequences of the C. reinhardtii genes encoding 5Ј-adenylylsulfate (APS) reductase and serine (Ser) acetyltransferase are orthologous to sequences from vascular plants. The Cys biosynthetic pathway of C. reinhardtii is regulated by sulfate availability. The steady-state level of transcripts and activity of ATP sulfurylase, APS reductase, Ser acetyltransferase, and O-acetyl-Ser (thiol) lyase increase when cells are deprived of sulfate. The sac1 mutation, which impairs C. reinhardtii ability to acclimate to sulfur-deficient conditions, prevents the increase in accumulation of the transcripts encoding these enzymes and also prevents the increase in activity of all the enzymes except APS reductase. The sac2 mutation, which does not affect accumulation of APS reductase transcripts, blocks the increase in APS reductase activity. These results suggest that APS reductase activity is regulated posttranscriptionally in a SAC2-dependent process.Plants and algae are primary producers. They absorb sunlight and through photosynthesis, convert it into chemical energy stored as carbohydrate. They also import inorganic nutrients from their environment and convert them into biologically active compounds. Photosynthesis and nutrient acquisition are coordinated with growth.Photosynthetic organisms acclimate to nutrientdeficient conditions through a suite of physiological changes that can be classified as general and specific (Harder and Dijkhuizen, 1983;Davies and Grossman, 1998). General responses, which occur when an organism is deprived of any essential nutrient, include a decrease in the rate of photosynthesis, a decrease in or cessation of cell division, and an accumulation of starch or glycogen. Specific changes occur in response to loss of a specific nutrient and are different for each nutrient. Specific responses are those that enable the organism to scavenge the limiting nutrient from internal or external sources, and those that increase the efficiency of nutrient assimilation.The ability to sense and respond to a nutrientlimiting environments is necessary for an organism to successfully compete in natural environments where nutrients are often limiting. However, the mechanisms used to sense nutrient availability and control physiological changes in response to nutrient limitation are largely unknown. We are using the unicellular green alga Chlamydomonas reinhardtii as a model system to study sulfur metabolism and the response to sulfur-deficient conditions. Sulfur is considered a macronutrient because it is required at relatively high levels. Sulfur is a constituent of proteins, lipids, carbohydrates, el...