John P. Davies

Sac1, a putative regulator that is critical for survival of Chlamydomonas reinhardtii during sulfur deprivation

The sac1 mutant of Chlamydomonas reinhardtii is aberrant in most of the normal responses to sulfur limitation; it cannot synthesize arylsulfatase, does not take up sulfate as rapidly as wild‐type cells, and does not synthesize periplasmic proteins that normally accumulate during sulfur‐limited growth. Here, we show that the sac1 mutant dies much more rapidly than wild‐type cells during sulfur deprivation; this emphasizes the vital role of the acclimation process. The loss of viability of the sac1 mutant during sulfur deprivation is only observed in the light and is mostly inhibited by DCMU. During sulfur‐stress, wild‐type cells, but not the sac1 mutant, downregulate photosynthesis. Thus, death of the sac1 mutant during sulfur deprivation is probably a consequence of its inability to downregulate photosynthesis. Furthermore, since SAC1 is necessary for the downregulation of photosynthesis, the process must be highly controlled and not simply the result of a general decrease in protein synthesis due to sulfur limitation. Genomic and cDNA copies of the SAC1 gene have been cloned. The deduced amino acid sequence of Sac1 is similar to an Escherichia coli gene that may involved in the response of E.coli to nutrient deprivation.

Chlamydomonas reinhardtii Genome Project. A Guide to the Generation and Use of the cDNA Information

The National Science Foundation-funded Chlamydomonas reinhardtii genome project involves (a) construction and sequencing of cDNAs isolated from cells exposed to various environmental conditions, (b) construction of a high-density cDNA microarray, (c) generation of genomic contigs that are nucleated around specific physical and genetic markers, (d) generation of a complete chloroplast genome sequence and analyses of chloroplast gene expression, and (e) the creation of a Web-based resource that allows for easy access of the information in a format that can be readily queried. Phases of the project performed by the groups at the Carnegie Institution and Duke University involve the generation of normalized cDNA libraries, sequencing of cDNAs, analysis and assembly of these sequences to generate contigs and a set of predicted unique genes, and the use of this information to construct a high-density DNA microarray. In this paper, we discuss techniques involved in obtaining cDNA end-sequence information and the ways in which this information is assembled and analyzed. Descriptions of protocols for preparing cDNA libraries, assembling cDNA sequences and annotating the sequence information are provided (the reader is directed to Web sites for more detailed descriptions of these methods). We also discuss preliminary results in which the different cDNA libraries are used to identify genes that are potentially differentially expressed.

Mutations in an Auxin Receptor Homolog AFB5 and in SGT1b Confer Resistance to Synthetic Picolinate Auxins and Not to 2,4-Dichlorophenoxyacetic Acid or Indole-3-Acetic Acid in Arabidopsis

Although a wide range of structurally diverse small molecules can act as auxins, it is unclear whether all of these compounds act via the same mechanisms that have been characterized for 2,4-dichlorophenoxyacetic acid (2,4-D) and indole-3-acetic acid (IAA). To address this question, we used a novel member of the picolinate class of synthetic auxins that is structurally distinct from 2,4-D to screen for Arabidopsis (Arabidopsis thaliana) mutants that show chemically selective auxin resistance. We identified seven alleles at two distinct genetic loci that conferred significant resistance to picolinate auxins such as picloram, yet had minimal cross-resistance to 2,4-D or IAA. Double mutants had the same level and selectivity of resistance as single mutants. The sites of the mutations were identified by positional mapping as At4g11260 and At5g49980. At5g49980 is previously uncharacterized and encodes auxin signaling F-box protein 5, one of five homologs of TIR1 in the Arabidopsis genome. TIR1 is the recognition component of the Skp1-cullin-F-box complex associated with the ubiquitin-proteasome pathway involved in auxin signaling and has recently been shown to be a receptor for IAA and 2,4-D. At4g11260 encodes the tetratricopeptide protein SGT1b that has also been associated with Skp1-cullin-F-box-mediated ubiquitination in auxin signaling and other pathways. Complementation of mutant lines with their corresponding wild-type genes restored picolinate auxin sensitivity. These results show that chemical specificity in auxin signaling can be conferred by upstream components of the auxin response pathway. They also demonstrate the utility of genetic screens using structurally diverse chemistries to uncover novel pathway components.

Mutants of Chlamydomonas with Aberrant Responses to Sulfur Deprivation

In the absence of sulfur, Chlamydomonas reinhardtii, a unicellular green alga, increases its rate of sulfate import and synthesizes several periplasmic proteins, including an arylsulfatase (Ars). These changes appear to help cells acclimate to a sulfur-deficient environment. The elevated rate of sulfate import results from an increase in the capacity and affinity of the transport system for sulfate. The synthesis of Ars, a periplasmic enzyme that cleaves sulfate from aromatic compounds, enables cells to use these molecules as a source of sulfur when free sulfate is not available. To characterize the ways in which C. reinhardtii perceives changes in the sulfur status of the environment and regulates its responses to these changes, we mutagenized cells and isolated strains exhibiting aberrant accumulation of Ars activity. These mutants were characterized for Ars activity, ars mRNA accumulation, periplasmic protein accumulation, and sulfate transport activity when grown in both sulfur-sufficient and sulfur-deficient conditions. All of the mutants exhibited pleiotropic effects with respect to several of these responses. Strains harboring double mutant combinations were constructed and characterized for Ars activity and ars mRNA accumulation. From the mutant phenotypes, we inferred that both positive and negative regulatory elements were involved in the acclimation process. Both the epistatic relationships among the mutations and the effects of the lesions on the responses of C. reinhardtii to sulfur limitation distinguished these mutants from similar mutants in Neurospora crassa.

Characterization of Sulfate Transport in Chlamydomonas reinhardtii during Sulfur-Limited and Sulfur-Sufficient Growth

We have characterized sulfate transport in the unicellular green alga Chlamydomonas reinhardtii during growth under sulfur-sufficient and sulfur-deficient conditions. Both the Vmax and the substrate concentration at which sulfate transport is half of the maximum velocity of the sulfate transport (K1/2) for uptake were altered in starved cells: the Vmax increased approximately 10-fold, and the K1/2 decreased approximately 7-fold. This suggests that sulfur-deprived C. reinhardtii cells synthesize a new, high-affinity sulfate transport system. This system accumulated rapidly; it was detected in cells within 1 h of sulfur deprivation and reached a maximum by 6 h. A second response to sulfur-limited growth, the production of arylsulfatase, was apparent only after 3 h of growth in sulfur-free medium. The enhancement of sulfate transport upon sulfur starvation was prevented by cycloheximide, but not by chloramphenicol, demonstrating that protein synthesis on 80S ribosomes was required for the development of the new, high-affinity system. The transport of sulfate into the cells occurred in both the light and the dark. Inhibition of ATP formation by the antibiotics carbonylcyanide m-chlorophenylhydrazone and gramicidin-S and inhibition of either F- or P-type ATPases by N,N-dicyclohexylcarbodiimide and vanadate completely abolished sulfate uptake. Furthermore, nigericin, a carboxylate ionophore that exchanges H+ for K+, inhibited transport in both the light and the dark. Finally, uptake in the dark was strongly inhibited by valinomycin. These results suggest that sulfate transport in C. reinhardtii is an energy-dependent process and that it may be driven by a proton gradient generated by a plasma membrane ATPase.

Sac3, an Snf1-like Serine/Threonine Kinase That Positively and Negatively Regulates the Responses of Chlamydomonas to Sulfur Limitation

The Sac3 gene product of Chlamydomonas positively and negatively regulates the responses of the cell to sulfur limitation. In wild-type cells, arylsulfatase activity is detected only during sulfur limitation. The sac3 mutant expresses arylsulfatase activity even when grown in nutrient-replete medium, which suggests that the Sac3 protein has a negative effect on the induction of arylsulfatase activity. In contrast to its effect on arylsulfatase activity, Sac3 positively regulates the high-affinity sulfate transport system—the sac3 mutant is unable to fully induce high-affinity sulfate transport during sulfur limitation. We have complemented the sac3 mutant and cloned a cDNA copy of the Sac3 gene. The deduced amino acid sequence of the Sac3 gene product is similar to the catalytic domain of the yeast Snf1 family of serine/threonine kinases and is therefore classified as a Snf1-related kinase (SnRK). Specifically, Sac3 falls within the SnRK2 subfamily of kinases from vascular plants. In addition to the 11 subdomains common to Snf1-like serine/threonine kinases, Sac3 and the plant kinases have two additional subdomains and a highly acidic C-terminal region. The role of Sac3 in the signal transduction system that regulates the responses of Chlamydomonas to sulfur limitation is discussed.

Sequences controlling transcription of the Chlamydomonas reinhardtii beta 2-tubulin gene after deflagellation and during the cell cycle.

In Chlamydomonas reinhardtii, transcripts from the beta 2-tubulin gene (tubB2), as well as those from other tubulin-encoding genes, accumulate immediately after flagellar excision as well as at a specific time in the cell cycle. Control of tubB2 transcript accumulation following deflagellation is regulated, at least partially, at the transcriptional level. We have fused the tubB2 promoter to the arylsulfatase (ars) reporter gene, introduced this construct into C. reinhardtii, and compared expression of the chimeric gene with that of the endogenous tubB2 gene. After flagellar excision, transcripts from the tubB2/ars chimeric gene accumulate with kinetics similar to those of transcripts from the endogenous tubB2 gene. The tubB2/ars transcripts also accumulate in a cell cycle-specific manner; however, chimeric transcripts are more abundant earlier in the cell cycle than the endogenous tubB2 transcripts. To elucidate transcriptional control of tubB2, we have mutated or removed sequences in the tubB2 promoter and examined the effect on transcription. The tubB2 promoter shares features with the promoters of other tubulin-encoding genes; these include a GC-rich region between the TATA box and the transcription initiation site and multiple copies of a 10-bp sequence motif that we call the tub box. The tubB2 gene contains seven tub box motifs. Changing the GC-rich region to an AT-rich region or removing three of the seven tub box motifs did not significantly affect transcription of the chimeric gene. However, removing four or five tub box motifs prevented increased transcription following deflagellation and diminished cell cycle-regulated transcription from the tubB2 promoter.

The sac Mutants of Chlamydomonas reinhardtii Reveal Transcriptional and Posttranscriptional Control of Cysteine Biosynthesis

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. reinhardtiigenes encoding 5′-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. Thesac1 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 aSAC2-dependent process.

Sulfur availability and the SAC1 gene control adenosine triphosphate sulfurylase gene expression in Chlamydomonas reinhardtii.

A Chalmydomonas reinhardtii adenosine triphosphate (ATP) sulfurylase cDNA clone (pATS1) was selected by complementing a mutation in the ATP sulfurylase gene (cysD) of Escherichia coli. E. coli cysD strains harboring pATS1 grow on medium containing sulfate as the sole sulfur source and exhibit ATP sulfurylase activity. The amino acid sequence of the C. reinhardtii ATP sulfurylase, derived from the nucleotide sequence of the complementing gene (ATS1), is 25 to 40% identical to that of ATP sulfurylases in other eukaryotic organisms and has a putative transit peptide at its amino terminus. ATP sulfurylase mRNA was present when cells were grown in sulfur-replete medium, but accumulated to higher levels when the cells were exposed to sulfur-limiting conditions. Furthermore, sulfur-stress-induced accumulation of the ATS1 transcript was reduced in a strain defective in SAC1, a gene that is critical for acclimation to sulfur-limited growth.

Responses to Deficiencies in Macronutrients

All organisms are able to acclimate to less than optimal environmental conditions. In this chapter we discuss how Chlamydomonas reinhardtii acclimates to deficiencies in macronutrients. The physiological changes that occur during macronutrient starvation can be classified as specific and common responses. Specific responses occur during limitation for a particular nutrient, while common responses occur during limitationfor any nutrient.Specific responses include changes in nutrient transport characteristics and induction of nutrient scavenging systems. General responses include decreases in cell division, photosynthesis and respiration. The decrease in photosynthesis has been shown to be essential for surviving severe sulfur-stress as a mutant unable to decrease photosynthesis cannot survive these conditions. We propose a model of how specific and general responses to nutrient limitation are regulated and discuss how mutants deficient in these responses fit in to this model.