Springer 2005

Photosynthesis Research (2005) 86: 475–489 DOI: 10.1007/s11120-005-4048-9

Review

Insights into the acclimation of Chlamydomonas reinhardtii to sulfur deprivation Steve V. Pollock1,*, Wirulda Pootakham1,2, Nakako Shibagaki1, Jeffrey L. Moseley1 & Arthur R. Grossman1 1

Department of Plant Biology, The Carnegie Institution, 260 Panama Street, Stanford, CA 94305, USA; Department of Biological Sciences, Stanford University, Stanford, CA 95305, USA; *Author for correspondence (e-mail: [email protected]) 2

Received 20 December 2004; accepted in revised form 17 March 2005

Key words: arylsulfatase, cross-talk, nutrient deprivation, nutrient sensing, protein kinase, random insertional mutagenesis, regulation of transport, SAC1, SAC3, signal transduction, sulfate

Abstract During sulfur deprivation, the photosynthetic green alga Chlamydomonas reinhardtii develops a highaffinity 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.

Introduction Sulfur (S) is an essential element for all organisms and is present in proteins, lipids, carbohydrates, and several metabolites. The sulfate anion (SO42)) is the preferred source of S for most organisms. It is transported into cells, and because it is a relatively inert molecule, it must be activated by the enzyme ATP sulfurylase prior to assimilation. Adenosine phosphosulfate (APS), the activated form of SO42), can serve as a substrate for SO42) reduction or can be phosphorylated by APS kinase to yield 3¢-phosphoadenosine phosphosulfate (PAPS). PAPS can be used by sulfotransferases to catalyze the sulfation of various cellular metabolites including choline, glucosides and proteins. In the pathway leading to the reduction of SO42), the S of APS is reduced to sulfite by APS

reductase, and sulfite is further reduced to sulfide and incorporated into the amino acids cysteine and methionine, the methyl donor S-adenosylmethionine (AdoMet), and the antioxidant glutathione (Leustek and Saito 1999). S can be limiting in the environment and strongly influence ecosystem composition. Depriving plants of S can result in stunted plant growth and reduced seed quality and yields, and limit plant productivity in certain agricultural settings (Mahler and Maples 1986, 1987; Warman and Sampson 1994). In recent history, many soils have accumulated high S levels as a result of either the administration of fertilizers with excess SO42) salts or exposure to pollutants present in acid rain (Cole and Johnson 1977; Johnson et al. 1982; David et al. 1988; MacDonald et al. 1991). However, with increased fertilizer purity and decreased

476 occurrence of acid rains, low levels of available S in diverse ecosystems may limit the growth and development of vascular plants (Marschner 1995). Furthermore, much of the SO42) in the soil may not be readily available to plants or microbes. The SO42) anion can be adsorbed onto the surface of soil particles or covalently bonded to organic molecules in the form of SO42) esters and sulfonates (reviewed in Grossman and Takahashi 2001); organic forms of sulfate cannot be directly used by plants (generally, sulfate is cleaved from organic compounds, making it available for uptake and assimilation). Many prokaryotic and eukaryotic organisms respond to S deficiency by increasing their capacity for scavenging S from both internal and external resources. An increased SO42) transport capacity and the synthesis of an extracellular arylsulfatase (ARS) accompany the acclimation of specific fungi and bacteria to S deprivation (Scott and Metzenberg 1970; Adachi et al. 1973). Extracellular ARS hydrolyzes aromatic SO42) esters in the environment of the organism, making free SO42) available for uptake and assimilation (de Hostos et al. 1988). S-limited C. reinhardtii cells also synthesize an extracellular ARS and exhibit an increased capacity for taking up free SO42). This review emphasizes potential mechanisms that control S-deprivation responses in C. reinhardtii.

Responses of C. reinhardtii to S limitation C. reinhardtii exhibits a suite of responses when challenged with extreme environmental conditions that help efficiently capture the limiting nutrient, ameliorate the stress conditions and slow or halt cell growth and division. These responses have been classified into those that are general and may occur under a number of different stress conditions and those that are specific and are solely associated with a particular stress condition such as S deprivation. Temperature, light quality and quantity, nutrient status, and osmotic conditions are all perceived by cellular sensors that transduce signals to the nucleus to modify the rate and specificity of transcription; these sensors may also influence the rate of RNA degradation and the activation state of various polypeptides as a consequence of posttranslational modification.

Some of the general and specific responses of C. reinhardtii to S deprivation have been characterized and at least two regulatory elements involved in this control have been identified. In contrast, there is still almost no information concerning elements that regulate the responses of vascular plants to S starvation. Interestingly, there does seem to be some commonality between the regulation of phosphorus-deprivation responses of Arabidopsis thaliana and C. reinhardtii (Wykoff et al. 1999; Rubio et al. 2001). This raises the possibility (but more information is needed) that other pathways involved in regulating nutrientdeprivation responses may be similar among the eukaryotic algae, and between eukaryotic algae and vascular plants. General responses to nutrient limitation The general responses to nutrient limitation are those associated with deprivation of the organism for any of the essential nutrients. These responses include the cessation of DNA replication and cell division, and a decrease in many aspects of cellular metabolism. Of all of the metabolic processes analyzed during nutrient deprivation, photosynthesis has been most extensively studied. A decrease in photosynthetic oxygen evolution during growth of C. reinhardtii cells in the light is critical for survival of the cells during S deprivation (Davies et al. 1996). However, there appear to be many different mechanisms for modifying the activity of the photosynthetic apparatus. During both S and phosphorus (P) deprivation, there is a dramatic loss of photosynthetic electron transport activity at the level of Photosystem (PS) II (Wykoff et al. 1998). The decline in PS II activity correlates with a decrease in the maximal quantum efficiency of PS II and the accumulation of QB (secondary quinone electron acceptor of PS II) nonreducing centers; these centers can perform a charge separation but are unable to reduce the plastoquinone pool. Furthermore, both S and P deprivation result in a reduced efficiency of excitation energy transfer to PS II reaction centers, which correlates with increased dissipation of absorbed light energy as heat and a transition of the photosynthetic apparatus from state 1 to state 2. In state 2, a significant proportion of the photosynthetic light harvesting complex of PS II becomes associated with PS I where the excitation energy can be

477 quenched by P700, the PS I reaction center trap (Wykoff et al. 1998). Specific responses to S limitation Specific responses to S deficiency are different than those associated with P or nitrogen limitation. They include increased capacity of the cell to transport and/or assimilate exogenous SO42) and the restructuring of cellular features to conserve S resources. Also, levels of transcripts encoding enzymes associated with SO42) assimilation generally increase in response to S deprivation (Davies and Grossman 1998; Grossman and Takahashi 2001; Zhang et al. 2004). Figure 1 depicts the C. reinhardtii cell and the pathways associated with S assimilation and the regulation of the cellular responses to S deprivation.

ARS and extracellular polypeptides A number of organisms can access S that is covalently associated with organic molecules in the environment through the synthesis and secretion of sulfatases and sulfonatases. Several classes of sulfatases from a variety of different organisms have been isolated and characterized. These enzymes hydrolyze esterified SO42) from organic molecules, promoting S cycling in the environment. The synthesis of some sulfatases is regulated by SO42) availability; there is an inverse correlation between the level of free SO42) in the soil solution and soil sulfatase activity. Soils containing elevated arylsulfatase (ARS) activity are likely to be limiting for free SO42). C. reinhardtii synthesizes a prominent extracellular ARS in response to S limitation (Lien and Schreiner 1975; de Hostos et al. 1988, 1989). This ARS is associated with the proteinaceous

Figure 1. A simplified depiction of the subcellular localization and expression of polypeptides involved in the assimilation of SO42) in C. reinhardtii during S deprivation. Polypeptides known to play a role in the acclimation to S deprivation, SAC1 and SAC3, are also shown. The dashed arrows represent signal transduction cascades that have not yet been characterized. The values in parentheses are the fold-induction of transcript abundance in cells deprived of S as measured by microarray analysis (Zhang et al. 2004). The abbreviations are: APS, adenosine 5¢-phosphosulfate; CrSultr, C. reinhardtii SO42) transporter; Ser, serine; Cys, cysteine; OAS, O–acetyl–L–serine; PAPS, 3¢-phosphoadenosine 5¢-phosphosulfate; the remainder of the abbreviations are defined in the text. Cysteine synthesis may also occur in the chloroplast.

478 cell wall and has been purified and characterized from mutant strains that are unable to assemble their cell wall (de Hostos et al. 1989); thus, ARS is secreted into the medium making it relatively easy to isolate and characterize. ARS of C. reinhardtii is a glycoprotein, with at least 3 sugar attachment sites. It has a molecular mass of approximately 70 kDa and can hydrolyze esterified SO42) associated with various organic compounds, releasing free SO42) for assimilation by the cells. There appear to be nine ARS genes on the C. reinhardtii as predicted from the version 2.0 genome. However, since the genome sequence is only 90% complete, and some of the sequence is not of high quality, this number cannot be firmly established. A number of extracellular polypeptides, in addition to ARS, are synthesized by C. reinhardtii in response to S deprivation. Although the enzymatic activities of these proteins have not been established, they may be alkylsulfatases, sulfonatases, sulfoquinovose degrading enzymes or choline sulfatases. Two prominent extracellular proteins of apparent molecular masses of 76 kDa (designated ExtraCellular Protein 76; ECP76) and 84 kDa (ECP84) are synthesized in response to S deprivation (Takahashi et al. 2001). These proteins were isolated from the medium of a mutant strain unable to assemble the cell wall (the extracellular proteins remain free in the medium). The N-termini of both ECP76 and ECP84 were sequenced and the limited sequence information was used to identify the genes encoding these proteins in C. reinhardtii EST and genomic libraries. The identified cDNA clones were sequenced and the deduced amino acid sequences were shown to be significantly similar to a number of different cell wall proteins. However, the unique feature of these deduced protein sequences, is that between them they contain a single S-containing amino acid (in the mature polypeptides); other cell wall polypeptides contain many S amino acids. The RNAs encoding these proteins rapidly accumulate during S deprivation, and are also very rapidly degraded when S is added back to the depleted cultures (the half life of these mRNAs is approximately 10 min) (Takahashi et al. 2001). SO42) transport activity The characterization of SO42) transport in C. reinhardtii during S-limited and S-sufficient

growth has been reported (Yildiz et al. 1994). Both the maximum velocity (Vmax) and the substrate concentration at which SO42) transport is at half-maximum velocity (K1/2) for uptake were altered in S-starved cells; the Vmax for SO42) increased approximately 10-fold while the K1/2 decreased approximately 7-fold. This suggests that S-deprived C. reinhardtii cells make a highaffinity SO42) transport system that can be detected within an hour of S deprivation. The development of enhanced SO42) transport activity upon S starvation is blocked by cycloheximide, but not by chloramphenicol, demonstrating that protein synthesis on 80S ribosomes is required for the induced transport activity. Moreover, SO42) transport in C. reinhardtii is an energy-dependent process and it may be driven by a proton gradient generated by a plasma membrane ATPase (Yildiz et al. 1994). Analysis of the C. reinhardtii genome sequence has led to the identification of seven genes that encode proteins with strong sequence similarity to known SO42) transporters. Of these seven genes, the deduced amino acid sequences of three, designated SULTR1, SULTR2, and SULTR3, are similar to the H+/SO42) co-transporters from vascular plants, including A. thaliana and Stylosanthes hamata. SO42) transporters are comprised of 12 transmembrane domains followed by a linking region that connects to a C-terminal STAS (Sulfate Transporter and Anti-Sigma antagonist) domain, which is found in different anion transporters and extends into the cytoplasm of the cell. There are at least three other predicted proteins, designated SAC proteins, that have significant similarity to Na+/SO42) co-transporters, although they also resemble the regulatory element SAC1 (see below). A gene encoding a subunit of a bacterial-type SO42) transporter (Laudenbach and Grossman 1991) in C. reinhardtii, designated SULP, has been cloned (Chen et al. 2003), and its deduced amino acid sequence shares a significant level of similarity with chloroplast SO42) permeases from Marchantia polymorpha and Nephroselmis olivacea. This type of SO42) permease is of prokaryotic origin as evident from its high similarity with the SO42) permease of the cyanobacterium Synechococcus sp. PCC7942. Recently, SULP has been shown to localize to the chloroplast envelope, and analyses of antisense strains suggest that it plays an

479 important role in the transport of SO42) into chloroplasts (Chen and Melis 2004). The STAS domains of C. reinhardtii SO42) transporters Members of the SLC26 family of anion transporters have a carboxy terminal STAS domain following an amino terminal catalytic region (Mount and Romero 2004), while the potential Na+/SO42) transporters contain no obvious STAS domain. Like most of the A. thaliana SO42) transporters, the C. reinhardtii SULTR1, SULTR2, and SULTR3 polypeptides all possess a C-terminal STAS domain (Figure 2a). The STAS domain present on the SULTR/SLC26 transporters share significant similarity with bacterial anti-sigmafactor antagonists such as SpoIIAA of Bacillus subtilis (Aravind and Koonin 2000). SpoIIAA positively regulates RNA polymerase activity by interacting with the anti-sigma factor SpoIIAB, which in turn allows for the initiation of sporulation (Ho et al. 2003). The phosphorylation of SpoIIAA at Ser-58 is thought to be involved in the

regulatory mechanism (Najafi et al. 1995); an analogous residue (Ser/Thr) with the potential to be phosphorylated is present in the STAS domain of the SO42) transporters (Figure 2a). Mutations within the STAS domains of various members of the SO42) transporter family are known to alter their transporter activity and can result in serious diseases in humans, including diastrophic dysplasia, Pendred syndrome and congenital chloride diarrhea (Everett et al. 1997; Makela et al. 2002; Chernova et al. 2003). These findings suggest that the STAS domain contributes to the catalytic, biosynthetic or regulatory aspects of anion transporters. In studies using the hDRA gene (SLC26A3) of humans and the A. thaliana SO42) transporter SULTR1;2, the removal of the STAS domain abolished anion transport activity. Furthermore, STAS domains from different SO42) transporters can markedly affect the kinetic characteristics of transporter activity, suggesting that this domain has a role in modulating transport activity either directly or indirectly (Chernova et al. 2003; Shibagaki and Grossman 2004).

Figure 2. (a) Amino acid sequence alignments among STAS domains, and with the antisigma factor antagonist 1H4Z from B. subtilis. The sequences of the C. reinhardtii transporters are denoted CrSultr1 and CrSultr2, while the A. thaliana transporters are denoted AtSultr1;1 to AtSultr4;1. Black shading indicates amino acids that are identical in at least 40% of the sequences. Grey shading represents amino acids that are similar in at least 20% of the sequences. S/T indicates the serine/threonine residue thought to be phosphorylated. (b) A diagram of the predicted structure of the SAC1 polypeptide showing the transmembrane domains as solid rectangles, and the two TrkA–C domains as open rectangles. The analysis was performed with the SMART prediction program.

480 Among the three putative H+/SO42) transporters in C. reinhardtii, SULTR1 has a C-terminal domain with features that are most typical of STAS domains that have been previously analyzed (Aravind and Koonin 2000) (Figure 2a). The STAS domain of the deduced SULTR2 polypeptide has additional amino acid residues relative to most other STAS domains. However, like SULTR1, SULTR2 retains the putative phosphorylation site at the N-terminal end of the a2 region, although this residue is a Thr rather than a Ser. It is not known whether phosphorylation of the STAS domain of either the C. reinhardtii or plant transporters takes place or has any role in SO42) transport. However, site-directed mutagenesis of the putative phosphorylation site was found to disturb A. thaliana SO42) transport activity (unpublished results, Shibagaki and Grossman). The most distant member of the C. reinhardtii putative SO42) transporter family, SULTR3, also has a putative STAS domain that lacks a predicted phosphorylation site, and the core of the enzymatic domain of the protein is disrupted by long loops. Some sequence differences among the transport proteins may be a consequence of incorrect protein predictions associated with the current version of the C. reinhardtii genome sequence. For these reasons, the SULTR3 STAS domain was not included in the alignment presented in Figure 2a. S deprivation, anaerobiosis, and hydrogen production As previously discussed, photosynthetic oxygen evolution in C. reinhardtii markedly decreases following removal of S from the growth medium (Wykoff et al. 1998). Respiration continues during the decline in photosynthetic activity and the cultures become anaerobic when maintained in a closed system since oxygen is being consumed faster than it is evolved (Ghirardi et al. 2000; Melis et al. 2000). The anaerobic conditions elicit expression of two iron hydrogenases that can catalyze the production of H2 in the light (Forestier et al. 2003). Hence, a better understanding of S-deprivation responses may provide insights into the features of cellular metabolism that facilitate the generation of H2. Detailed studies of the biochemical and morphological changes that occur during the induction of hydrogen production

have been previously reported (Zhang et al. 2002; Kosourov et al. 2003). For a comprehensive review of H2 evolution by C. reinhardtii see Melis et al. (2004).

Regulation of S-deprivation responses SAC1 Specific polypeptides necessary for the proper regulation of S deprivation responses in C. reinhardtii have been identified. SAC1 (Sulfur ACclimation protein 1) appears to be required for many responses associated with S limitation (Davies et al. 1996). Strains with mutations in the SAC1 gene exhibit lower SO42) transport activity, are unable to synthesize extracellular ARS, and show no significant increase in many transcripts associated with S deprivation responses (Zhang et al. 2004); these include transcripts encoding ARS, ATP sulfurylase (ATS1 and ATS2), serine acetyltransferase (SAT1), and the ferredoxin-dependent sulfite reductase (SIR1). Furthermore, the sac1 mutant cannot down-regulate photosynthetic electron transport activity and dies quickly when it is placed in S-deficient medium in the light. The SAC1 gene encodes a polypeptide predicted to have 10 transmembrane domains. This protein is similar to ion transporters from a number of different organisms, with the greatest degree of similarity to a Na+/SO42) transporter from rat kidneys. Although it is possible that SAC1 can function in the uptake of SO42), the phenotype of the sac1 mutant strongly suggests that it plays an important role in regulating cellular responses to S deprivation (Davies et al. 1996). Similarly, Snf3p of S. cerevisiae has strong sequence similarity to a glucose transporter, but appears to function in the acclimation of the cells to the glucose status of the medium (Bisson et al. 1987). While the signaling mechanism used by SAC1 has not been established, SAC1 is predicted to have a large intracellular loop, located between transmembrane helices 4 and 5, with two TrkA domains that have been suggested to bind NAD+ or another unidentified ligand (Schlosser et al. 1993; Anantharaman et al. 2001) (Figure 2b). Thus, this region of the protein has the potential to function in signaling as a consequence of interactions with an intermediate metabolite and/or specific proteins.

481 SAC1-like genes In addition to SAC1, there are three genes on the C. reinhardtii genome (ID numbers: C_150155, C_150148, and C_370034) predicted to encode proteins with strong sequence similarity to Na+/ SO42) transporters. Partial cDNA sequences of these three genes are available, confirming that they are expressed. Microarray analyses have shown that at least one of the SAC1-like mRNAs increases in abundance by approximately 10 fold when the algal cells experience S deprivation (Zhang et al. 2004). Recent work has shown that the transcripts for at least two of the three genes encoding potential Na+/SO42) transporters increase substantially during S deprivation (Pootakham and Grossman, unpublished). Use of a targeted silencing method such as RNA interference (Rohr et al. 2004) may help establish a role for these polypeptides in the acclimation of cells to S deprivation. SAC3 Another polypeptide involved in the acclimation of C. reinhardtii to S deprivation is SAC3. The deduced amino acid sequence of SAC3 places it into a large family of Ser/Thr protein kinases that are related to the Snf1p kinase of S. cerevisiae which, like Snf3p, is part of the regulatory circuitry that allows the organism to acclimate to glucose levels in the environment. Unlike the sac1 mutant, the sac3 mutant exhibits constitutive, lowlevel synthesis of ARS under S-replete condition. Interestingly, this mutant also does not exhibit an increased maximal level of SO42) uptake during S deprivation (Davies et al. 1994, 1999). We have identified 10 genes on the C. reinhardtii genome that encode proteins with similarity to SAC3. An alignment of these polypeptides is shown in Figure 3. Interestingly, another member of this SAC3-like gene family was identified in a screen for strains that exhibited aberrant expression of ARS activity (Pollock and Grossman, unpublished results). Two mutants, both disrupted in the gene described by model C_50170 (http:// genome.jgi-psf.org/chlre2/chlre2.home.html), had low levels of ARS activity following exposure of the cells to S deprivation. These strains are designated ars11 and ars44 in Table 1. Interestingly, two A. thaliana SNF1-related protein kinases (AKIN10 and SRK2C) were recently shown to be involved in

controlling stress-responsive gene expression (Farras et al. 2001; Umezawa et al. 2004). These results raise the possibility that at least some of the remaining 8 proteins that constitute the SAC3-Ser/ Thr protein kinase family in C. reinhardtii play a role in the acclimation of this alga to S deprivation or to other stress conditions. Several genes are regulated at the level of mRNA accumulation by S availability Recently, microarray analyses of gene expression during S starvation were reported by Zhang et al. (2004). Most transcripts for polypeptides involved in the accumulation and assimilation of SO42) increase when wild-type cells are deprived of SO42), with the exceptions of SIR3 (sulfite reductase) and AKN2 (APS kinase). The transcripts for the two latter polypeptides change very little following exposure of the cells to S deprivation. Transcripts encoding ARS1, ATP sulfurylase (ATS1), and ECP76 were previously shown to increase in response to S deprivation, confirming the microarray results (de Hostos et al. 1989; Yildiz et al. 1996; Takahashi et al. 2001). Interestingly, a rise in the level of the transcript encoding a putative selenobinding protein (SBDP) was also detected. SBDP may be important for sequestering selenate for detoxification since the transport machinery of the cells would have a high affinity for SO42) and possibly an elevated capacity for selenate uptake during S deprivation. The SDBP may also be important for efficient uptake and utilization of trace amounts of selenate from SO42) depleted medium since selenate is required for certain C. reinhardtii enzymes, including methionine-S-sulfoxide reductase (Novoselov et al. 2002), and most of the selenate used by C. reinhardtii in culture is derived from trace contamination of the SO42) salts used to make the growth medium. Transcripts encoding proteins involved in PS I, PS II, the light harvesting complex, and photosynthetic electron transport decreased in abundance to between 25% and 50% of the initial level following 24 h of exposure of C. reinhardtii cells to growth in medium lacking SO42). As mentioned previously, transcripts encoding SAC1-like, potential Na+/SO42) transporters increased during S deprivation. However, there appears to be little increase in transcripts encoding the SULTR1 and SULTR3 transport polypeptides (Pootakham

482

Figure 3. Alignment of the SAC3 and predicted SAC3-like protein kinases. The first sequence is of SAC3 (GI:406389), and the nine others are of predicted proteins that show similarity to SAC3. Black shading indicates amino acids that are identical in at least 40% of the sequences. Grey shading represents amino acids that are similar in at least 20% of the sequences. The designation to the left of each sequence is the gene model number, as given by the gene model track on the C. reinhardtii genome browser (http://genome.jgi-psf.org/ chlre2/chlre2.home.html). ClustalW (Thompson et al. 1994) was used for the alignments.

483 Table 1. Sulfur acclimation mutants (ars). The gene model numbers and predicted protein functions are reported on the JGI genome browser (http://genome.jgi-psf.org/chlre2/chlre2.home.html). Complementation of the mutant phenotype and further characterization of the mutants need to be completed prior to drawing strong conclusions concerning the roles of the tagged genes in acclimation and SO42) assimilation Mutant Gene model ars1 ars5 ars9 ars10 ars11 ars18 ars24 ars32 ars44 ars53 ars66 ars73a ars73b ars75 ars76 ars83b ars122 ars401

C_220146 C_4170002 C_550005 C_200039 C_50170 C_1010021 C_760042 C_60149 C_50170 C_120027 C_1440003 C_190070 C_350122 C_110116/C_110014 C_490030 C_180128 C_160132 C_690036

Protein prediction Unknown SPX domain putatively involved in sensing. Predicted Na+-dependent transporter Clathrin adaptor complexes medium subunit family protein Similar to SAC3: Possible dual-specificity Ser/Thr/Tyr kinase. Endonuclease Phosphoinositide polyphosphatase-like Cyclic nucleotide dependent protein kinase Similar to SAC3: dual-specificity Ser/Thr/Tyr kinase. HydA1- iron hydrogenase precursor Unknown Unknown Receptor, 9 transmembrane domains Single insertion probably affects two genes: SAC1, and 7 ankyrin repeat containing protein kinase APPLE domain: mediates dimer formation. Two transmembrane domains. 2 DOMAINS: NAD-dependent glycerol–3–phosphate dehydrogenase / Phosphoserine phosphatase 3 DOMAINS: 3·TPR; CS; and SGS (implicated to play a role in SCF-mediated ubiquitination) Guanylate or adenylate cyclase

and Grossman, unpublished), with a modest increase in the transcript for SULTR2 during S deprivation. These findings raise a number of speculative possibilities. The Na+/SO42) transporters may be the dominant transporters that function during S deprivation. Alternatively, the activity of the H+/SO42) transporters may not be controlled at the level of transcription, but may be modulated by post-translational modifications that occur as the cells become limited for S. An example of the control of transport processes by posttranslational modification was reported for the nitrate transporter of A. thaliana, which is converted from a low affinity to a high affinity transporter by phosphorylation (Liu and Tsay 2003). Cross-talk between nutritional deficiency response pathways A number of studies using fungal and plant model organisms have demonstrated that different nutritional deficiency-specific regulatory pathways can have target genes in common (reviewed by Gasch and Werner-Washburne 2002). Many genes involved in the environmental stress responses of S. cerevisiae are regulated similarly during P and S

deprivation (Saldanha et al. 2004). Similarly, microarray expression experiments have demonstrated that the transcripts encoding PO43), potassium and iron transporters in tomato were coordinately elevated by deficiencies for any one of these nutrients (Wang et al. 2002). More specific cross-regulation of P and S metabolism in S. cerevisiae was indicated by the finding that the P-deprivation-responsive transcription factor Pho4p can functionally substitute for the sequencespecific DNA-binding protein Cp1p, which is required for activation of methionine biosynthetic genes. Activation of Pho4p by P-deprivation, Pho4p overexpression or use of constitutive alleles leads to suppression of methionine auxotrophy of the cep1 mutant (O’Connell and Baker 1992). Preliminary evidence points to some degree of cross-regulation of the P- and S-deficiency response pathways in C. reinhardtii. Comparison of the results of microarray analysis of gene expression from P- and S-deprived cells reveals that a number of stress genes, including some encoding chaperones and proteases are regulated similarly during either nutrient deficiency (Zhang et al. 2004, and Moseley and Grossman, unpublished). However, since these genes do not appear

484 to be controlled by either SAC1 or PSR1 (regulator that controls many of the P-starvation responses) regulatory factors, it seems likely that elevated transcripts from these genes reflect secondary effects of both nutrient stress conditions. On the other hand, the transcript abundance pattern for psr1, a mutant unable to acclimate properly to P-deprivation, reveals that a subset of the transcripts encoding proteins involved in S-assimilation increase in cultures that are P-deprived (but are replete with respect to S). A significant level of ARS activity is detectable in P-deprived psr1 cells but not in wild-type cells (Moseley and Grossman, unpublished data). One explanation is that P-deprived psr1 mutant might suffer from S limitation because of decreased SO42) uptake or assimilation. Since the first step in SO42) assimilation involves the generation of APS (an ATPdependent reaction), severe P-starvation of psr1 may limit substrate availability. If this is the case, however, it is unclear why only a subset rather than all of the S-assimilation genes would be activated. An alternative hypothesis is that the expression of ARS and of a subset of the S-assimilation genes in P-deprived psr1 is a result of aberrant signaling through the S-deprivation response pathway; this is currently being examined more carefully. We have recently discovered that cells grown in PO42)-replete, SO42)-depleted medium, with an organic S source, synthesizes a secreted phosphatase that is not the same as any of the phosphatases expressed during P-starvation. Expression of this phosphatase activity is SAC1-dependent, but we have not yet ruled out the possibility that the activity arises from the broad substrate specificity of ARS (Pootakham and Grossman, unpublished). Nevertheless, this result suggests that S-deprivation may change the cell’s metabolic requirements for P. Such observations may open a window into the complex interactions among regulatory pathways that control nutrient stress responses.

extracellular ARS. When cells are grown on solid medium with limiting S, they form colonies and ARS is secreted into the agar medium. The chromogenic substrate, 5–bromo–4–chloro–3–indolyl sulfate (X- SO42)), can be sprayed onto the colonies and upon hydrolysis by ARS, a blue precipitate (X) forms a halo around the colony (Figure 4). This colorimetric assay provides a simple screen for identifying mutant strains that either cannot synthesize active ARS following S starvation of the cells, or that produce ARS even under nutrient-replete conditions. Random insertional mutagenesis has been used to successfully generate both types of SAC pathway mutants in C. reinhardtii. SAC1 and SAC3, discussed above, were insertional mutants isolated from a screen based on the hydrolysis of X-SO42) (Davies et al. 1996, 1999). Unfortunately, most of the remaining mutants from that screen did not show segregation of the insertion (the ARG7 gene) with the sac phenotype. For those mutants, physically mapping the phenotype to a specific nuclear gene (Kathir et al. 2003) would identify the lesion responsible for the sac phenotype. We are currently using a modified mutagenesis procedure (Pollock and Grossman, unpublished) in which the paramomycin resistance gene (AphVIII) is randomly inserted into the C. reinhardtii genome (Sizova 2001) to generate novel mutants in the SAC response pathway. Figure 5 shows ARS activity for 24 mutants, and Table 1 lists the disrupted genes in 18 of the recently identified insertional mutants. Of the 18 mutants generated,

Screening for mutants with aberrant acclimation responses ars) mutants The acclimation of C. reinhardtii to S deprivation can be monitored by assaying for the activity of

Figure 4. Screening paromomycin resistant colonies for ARS activity (based on cleavage of 5–bromo–4–chloro–3–indolyl SO42)) on solid medium with no added SO42). The mutant ars73b is unable to synthesize active ARS when deprived of S. D66 is the paramomycin-sensitive parental strain.

485 500 450

PNP production (% of WT)

400 140 130 120 110 100 90 80 70 60 50 40 30 20 10 0 D6

6

4 b 5 6 b 7 8 0 2 4 3 5 7 3 6 1 a 6 9 1 8 2 1 5 ars ars ars ars ars1 ars1 ars3 ars4 ars5 ars5 ars5 ars6 ars6 ars7ars73 ars73 ars7 ars7ars83ars10ars10ars12ars12ars12

Figure 5. ARS activity associated with potential mutants synthesizing aberrant levels of active ARS. The assay was performed in liquid medium following 18–20 h of S deprivation. The substrate used for the assays was q-nitrophenylsulfate and the product of the reaction q-nitrophenol (PNP) was assayed spectrophotometrically by measuring the absorbance at 410 nm.

only two appear to be in the same gene (ars11 and ars44 have an insertions at different locations in the same SAC3-like gene), and one mutant is likely a novel sac1 allele (ars75). While complementation of the mutant phenotype and further characterization of the mutants must be completed prior to drawing strong conclusions based on these data, these data suggest that there are a number of different loci in C. reinhardtii that influence acclimation of the cells to S deprivation. selR mutants An alternative approach to generating mutants in the SO42)signaling pathway is to select for selenate resistance in the absence of SO42). Selenate is thought to be taken up and incorporated into selenocysteine and selenomethionine by the SO42) uptake and assimilation pathways (Fu et al. 2002; Novoselov et al. 2002). The exact mechanism underlying selenate toxicity is not known, but selenate is likely reduced to selenite in the chloroplast and the selenite may elicit the formation of toxic levels of superoxides (Bebien et al. 2002). Selenate resistance selections have been performed with S. cerevisiae and A. thaliana to generate mutants in SO42) transporters and regulatory ele-

ments (Cherest et al. 1997; Shibagaki et al. 2002). A proof of concept for the selection was established with the sac mutants. The sac1 mutant (see above) is unable to induce high capacity SO42) uptake or activate genes associated with the reduction and assimilation of SO42). This mutant most probably does not accumulate high intracellular levels of selenate/selenite during S deprivation, and it was shown to be considerably more resistant to selenate than the parental strain. The sac3 mutant was also more resistant to selenate than wild-type cells (although not as resistant as sac1). Using the information concerning the sensitivity of wild-type cells and the sac mutants to selenate, we performed a selection that identified many strains that are selenate resistant (selR mutants). Table 2 lists the disrupted genes in the selR mutants. Interestingly, all of the mutants so far generated synthesize wild-type levels of ARS during S deprivation, raising the possibility that the SO42) transporters and or SO42) assimilation in C. reinhardtii may be regulated, at least in part, by an ARS-independent pathway. Further analysis will demonstrate whether or not these strains are reduced in their ability to take up or assimilate SO42). Similar to the ARS screen above, complementation of the mutant phenotype and further characterization of the mutants needs to be com-

486 Table 2. Selenate resistant mutants (sel). The selenate resistant mutants presented in this table were obtained from approximately 12,000 primary, paromomycin-resistant transformants. Complementation of the mutant phenotype and further characterization of the mutants need to be completed prior to drawing strong conclusions concerning the roles of the tagged genes in acclimation and SO42) assimilation Mutant

Gene model

Protein prediction

sel1 sel11

C_660040 C_510055 C_190006 C_560008 C_720054 C_60070 C_60184 ? C_1120033

Ubiquitin carboxyl-terminal hydrolase Protein tyrosine kinase Protein kinase ARIADNE-like; E2 dependent ubiquitin-protein ligase Elongation factor or GTP binding protein Unknowns

sel13 sel14 sel15 sel16 sel20

pleted prior to drawing strong conclusions with respect to these data.

Progress toward elucidating the regulation of S deprivation responses We are currently attempting to identify all of the components of the SAC pathway by performing a saturating, random, insertional mutagenesis. Tables 1 and 2 list the mutants and the disrupted genes in the mutant strains that have been obtained so far. A speculative model, integrating the existing SAC proteins with the identification of some of the novel mutants described above is shown in Figure 6. It is likely that the mutated gene in ars11 and ars44 is linked to the mutant phenotype since the lesion in these two strains are allelic, containing insertions in the same Ser/Thr protein kinase. Furthermore, the putative guanylate cyclase mutant (ars401) can be complemented with a fragment of DNA containing the guanylate cyclase gene. We are not yet confident that insertion of the AphVIII gene, encoding paramomycin resistance (the marker in the transformation vector used for the insertional mutagenesis), into the genome in the other mutants is linked to the observed phenotype; generally, the observed phenotype cosegregates with the introduced marker gene approximately 50% of the time. As depicted in the speculative model of Figure 6, and discussed by Davies et al. (1996), SAC1 is likely the SO42) sensor of the cell. It may function as a transporter (although this has not been established), and at the same time monitor the SO42) concentration in the environment

Protein serine/threonine phosphatase

surrounding the cell. In S-replete conditions (Figure 6a), signaling from SAC1 may occur through ARS11, a protein kinase homologous to the SAC3 polypeptide (see amino acid alignment in Figure 3; C_50170). ARS11 may interact with SAC3 to facilitate repression of the SO42)responsive promoters in the nucleus via the action of the repressor designated in this diagram as Y (not yet identified), although it is also possible that SAC3 causes repression through a pathway independent of ARS11. Furthermore, under these conditions the transcription factor X (not yet identified) is maintained in an inactive state, either because it cannot be phosphorylated by ARS11 and/or a phosphatase is actively removing the phosphate group from this putative regulator. When the cells experience S deprivation (Figure 6b), SAC1 signals, with the binding of a cyclic nucleotide monophosphate, through ARS11 to de-repress the SO42)-responsive promoters, and through SAC3 to activate the SO42) transporters, possibly by causing phosphorylation of a Thr residue in the STAS domain at the C-terminal end of the transporters. ARS11 may directly or indirectly phosphorylate the transcription factor designated in the diagram as X, which positively regulates many of the S-stress associated genes. Phosphatases may help maintain SAC3 and ARS11 in a dephosphorylated state. We have isolated mutants that have disruptions in three loci predicted to encode for protein phosphatases (Tables 1 and 2: C_760042, C_180128, and C_1120033) that may operate in this system. In addition, protein degradation may also be involved in controlling the acclimation process

487 (a)

High SO4 2SO 4 2- transporter

SAC1

PPaseA

ARS11 kinase SAC3 kinase

X

P

Y

transporters

P

Conclusions and future directions ARS

(b)

Low SO 42SO42transporter

SAC1 cNMP

ARS11 kinase

SAC3 kinase

P

PPaseB

Y

In sum, our results suggest that a SAC1 complex initiates a S-status-dependent phosphorelay that controls the acclimation of the cells to S deprivation through the regulation of both transcriptional and post-transcriptional processes. The characterization of the mutants and the model presented in Figure 6 provide a tentative framework for using both biochemical and genetic tools to precisely define regulatory events that control the physiology of C. reinhardtii cells when they are limited for S.

X

P

Our knowledge concerning the mechanism of the S-deprivation responses in photosynthetic organisms is limited. Acclimation of C. reinhardtii to nutrient limitation may be akin to analogous processes in vascular plants, although this conclusion is still tentative and will require further corroboration. Future work will be directed at elucidating the S response mechanism of C. reinhardtii by saturating for mutants of the SAC pathway, complementing the mutant strains, targeting the interference of mRNAs that are putative components of the SAC pathway, examining protein–protein and protein– DNA interactions, and characterizing the SO42) transporters and their regulation. Acknowledgements

transporters

ARS

Figure 6. Speculative model of S-dependent signal transduction operating in C. reinhardtii. The model depicts the activity of the signal transduction cascade in S-replete (a) and S-depleted (b) environments. Under replete conditions, SAC3 freely phosphorylates Y, an unidentified peptide, to repress the transcription of S-responsive promoters. This phosphorylation may not depend on SAC1 or ARS11. When cells are exposed to a S-deficient environment, the putative SAC1 S-sensing complex, composed of SAC1, ARS11, SAC3, and a nucleotide cyclase, prevents SAC3 phosphorylation of Y, de-repressing the transcription of SO42) responsive promoters. The same complex further enhances transcription by activation of X, another unidentified regulatory component. SAC3 is also required for the induction of the high affinity state of the SO42) transporters. Currently, no experimental evidence has shown that protein– protein interactions occur between these regulators. cNMP, cyclic nucleotide monophosphate. PPase, protein phosphatase.

since a number of genes encoding components of the ubiquitin pathway were isolated in the selR selection; these results need to be extended with additional characterization of the mutant strains.

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