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First published online August 27, 2008; 10.1104/pp.108.125237 Plant Physiology 148:1055-1067 (2008) © 2008 American Society of Plant Biologists OPEN ACCESS ARTICLE
Mitochondrial Serine Acetyltransferase Functions as a Pacemaker of Cysteine Synthesis in Plant Cells1,[C],[W],[OA]Heidelberg Institute for Plant Sciences, Heidelberg University, 69120 Heidelberg, Germany (F.H.H., C.H., M.W., R.H.); and German Cancer Research Center, 69120 Heidelberg, Germany (R.Q., A.B.)
Cysteine (Cys) synthesis in plants is carried out by two sequential reactions catalyzed by the rate-limiting enzyme serine acetyltransferase (SAT) and excess amounts of O-acetylserine(thiol)lyase. Why these reactions occur in plastids, mitochondria, and cytosol of plants remained unclear. Expression of artificial microRNA (amiRNA) against Sat3 encoding mitochondrial SAT3 in transgenic Arabidopsis (Arabidopsis thaliana) plants demonstrates that mitochondria are the most important compartment for the synthesis of O-acetylserine (OAS), the precursor of Cys. Reduction of RNA levels, protein contents, SAT enzymatic activity, and phenotype strongly correlate in independent amiSAT3 lines and cause significantly retarded growth. The expression of the other four Sat genes in the Arabidopsis genome are not affected by amiRNA-SAT3 according to quantitative real-time polymerase chain reaction and microarray analyses. Application of radiolabeled serine to leaf pieces revealed severely reduced incorporation rates into Cys and even more so into glutathione. Accordingly, steady-state levels of OAS are 4-fold reduced. Decrease of sulfate reduction-related genes is accompanied by an accumulation of sulfate in amiSAT3 lines. These results unequivocally show that mitochondria provide the bulk of OAS in the plant cell and are the likely site of flux regulation. Together with recent data, the cytosol appears to be a major site of Cys synthesis, while plastids contribute reduced sulfur as sulfide. Thus, Cys synthesis in plants is significantly different from that in nonphotosynthetic eukaryotes at the cellular level.
Cys synthesis in plants is a fundamental process for protein biosynthesis and all anabolic pathways that require reduced sulfur. Bacteria and fungi are able to reduce sulfate by assimilatory sulfate reduction to sulfide and to integrate it into Cys in the cytosol. In contrast, mammals need to take up reduced sulfur as Met and can synthesize Cys via trans-sulfurylation in the cytosol. Protein biosynthesis in the mitochondria in animals and fungi receives Cys, therefore, from the cytosol. In plants, the assimilatory sulfate reduction pathway is localized to plastids, whereas Cys synthesis is found in the cytosol, plastids, and mitochondria. Why Cys synthesis in plants is localized in three compartments has long been speculated.
Serine acetyltransferase (SAT; also termed Serat; EC 2.3.1.30) catalyzes the activation of Ser by acetyl-CoA to O-acetylserine (OAS). In Arabidopsis (Arabidopsis thaliana), five genes encoding SAT1 to SAT5 are localized on the five chromosomes (Hell et al., 2002
The three major SATs, SAT5, SAT1, and SAT3, form the Cys synthase protein complex with the major OAS-TLs, A, B, and C, in the cytosol, plastids, and mitochondria, according to interaction studies of the Arabidopsis proteins (Bogdanova and Hell, 1997
The role of OAS-TL in the three compartments with independent protein biosynthesis was recently addressed using Arabidopsis T-DNA insertion lines. Knockout of cytosolic OAS-TL A apparently caused oxidative stress and lesions as well as sensitivity to heavy metals (Lopez-Martin et al., 2008
Expression of amiRNA against Sat3 Causes a Severe Growth Phenotype
According to in vitro analyses, the majority of SAT activity resides in mitochondria in pea leaves and heterotrophic Arabidopsis cell cultures (Ruffet et al., 1995
Several independent transformants (T1) were identified using kanamycin as a selectable marker. Nine plants of quite variable size survived (Fig. 1, A and B
), while numerous others failed to develop any further and died at the early rosette stage before production of flowers (data not shown). Determination of SAT3 protein levels in the surviving amiSAT3 lines by specific antibodies (Wirtz and Hell, 2003
Lines 4 and 5 showed intermediate growth retardation and were selected for further analyses. The T2 generation of amiSAT3 lines 4 and 5 confirmed the growth phenotypes of the T1 generation (Fig. 2 ). Growth rates of lines 4 and 5 differed significantly from those of the wild type from week 4 on after germination, and fresh weights of rosettes were 40% ± 4% and 33% ± 4%, respectively, of wild-type levels after 8 weeks.
Specific Down-Regulation of Mitochondrial SAT Causes Growth Retardation The amiSAT3 lines 4 and 5 were characterized in detail. First, the expression pattern of the Sat gene family was compared with the pattern in wild-type plants by quantitative real-time PCR. Lines 4 and 5 showed approximately 50% of wild-type Sat3 mRNA levels but no changes in abundance of Sat1, Sat2, Sat4, and Sat5 mRNA (Fig. 3A ). This pattern was independently confirmed by microarray analyses of amiSAT3 line 5 (see below). These findings confirmed the specificity of the amiRNA approach and demonstrated that no compensatory up-regulation of other Sat genes occurred in the transgenic lines. Second, SAT enzyme activities were determined from whole leaf protein extracts (Fig. 3B). The down-regulation of Sat3 mRNA was mirrored by decreases of SAT enzyme activity in the two lines. Finally, the same extent of decrease in lines 4 and 5 was observed with respect to protein levels using a SAT3-specific antiserum for immunoblotting and subsequent densitometric quantification (Fig. 3, C and D). Thus, the phenotype of growth retardation is caused by gene-specific down-regulation of the gene encoding mitochondrial SAT.
Flux of Ser into Cys and Glutathione Is Reduced in amiSAT3 Lines The causal relationship between reduced growth and specific down-regulation of the gene encoding mitochondrial SAT3 implies a reduced rate of OAS formation and ultimately of Cys synthesis, unless other SAT isoforms compensated for the decreased activity of the major SAT location in the cell in other ways than transcriptional increases. This assumption was tested by incubation of leaf discs in the light with radiolabeled [3H]Ser and quantification of label in Cys and glutathione (Fig. 4 ). Application of labeled Ser rather than sulfate was chosen to monitor flux via OAS but not of the assimilatory sulfate reduction pathway. [3H]Ser uptake of wild-type plants and amiSAT3 lines 4 and 5 was compared after 20 and 30 min, respectively. Radioactive labeling of Cys formed from [3H]OAS corresponded to half of the flux rate compared with the wild type at both time points due to down-regulation of mitochondrial SAT3 activity. An even stronger reduction of incorporation, down to 30% to 40%, was observed for glutathione. This difference suggests that, after uptake into the leaf cells, [3H]Ser was transported into the mitochondria to be used for the synthesis of [3H]OAS. This entered the cytosol to serve for the synthesis of Cys, which finally moved into the chloroplasts as substrate for the initial step of glutathione formation.
It is concluded that decreased mitochondrial SAT activity has a profound effect on flux into Cys and glutathione due to reduced overall OAS synthesis. As expected from the constant expression levels of the four unaffected SAT genes in Arabidopsis, no significant flux compensation could be observed, since the reductions in Sat3 mRNA and SAT3 protein levels were in the same range as the reductions of incorporation rates into Cys. Furthermore, these matching quantitative molecular and metabolic changes due to amiSAT3 expression imply that the contributions of the potentially important plastid SAT1 and cytosolic SAT5 to cellular OAS synthesis are rather small in vivo and by far exceeded by mitochondrial SAT3.
The overall cellular contents of major metabolites of primary sulfur metabolism were determined in leaves. OAS concentrations in lines 4 and 5 were decreased by 82% ± 2% and 83% ± 6% compared with the wild type and confirmed the down-regulation of the flux rate (Fig. 5A ). Remarkably, steady-state levels of Cys were doubled and those of glutathione increased up to 1.5-fold compared with wild-type levels (Fig. 5, B and C). Therefore, despite strongly reduced rates of synthesis, the free concentration of both thiols was maintained, at least at the whole cell level.
Also, the content of sulfate was significantly increased in the amiSAT3 lines (Fig. 6A ). It should be noted that phosphate contents increased 1.5- and 2-fold, respectively, in amiSAT lines 4 and 5, but not the levels of nitrate (Supplemental Fig. S2). Despite elevated sulfate levels, the total sulfur contents of these lines were not affected (Fig. 6B), as were the total carbon and nitrogen contents (Supplemental Fig. S3). The significant increase of sulfate in combination with unchanged levels of total sulfur levels point toward a deregulation of the ratio between oxidized and reduced sulfur in amiSAT3 lines.
Impact of Reduced OAS Synthesis on Expression of Sulfur Metabolism-Related Genes
The transcriptome response of amiSAT3 lines was investigated with an array containing 912 genes designed for the detection of even small changes of mRNA abundance, as is often observed with genes of primary metabolism. Sixty genes with different expression intensity by robust constitutive patterns according to Czechowski et al. (2005) Based on three biological repetitions of the wild type and amiSAT3 line 5 with four technical replicates, each including dye swaps for each set, 26 genes were found to be significantly up- or down-regulated according to P values lower than 0.05 (Table I ). Most of the significantly regulated genes (eight) belong to the category of sulfur metabolism, while a lower number of redox-related genes (six) and genes involved in hormone function (five), primary metabolism (four), and stress response (three) were also found to be significantly altered in abundance.
Microarray analyses of amiSAT3 line 5 confirmed the specificity and extent of down-regulation of Sat3 expression and the unchanged mRNA levels of the other four Sat genes that had been observed by real-time PCR (Fig. 3). Most interestingly, two genes upstream of SAT in assimilatory sulfate reduction were down-regulated: Aps4, encoding a ubiquitously expressed plastidic isoform of ATP-sulfurylase, which is responsible for the activation of sulfate prior to reduction (Hatzfeld et al., 2000a  ); and Apr2, encoding adenosine 5'-phosphosulfate reductase (APR), the major plastid isoform for the first reduction step. None of the other genes of primary sulfur metabolism was significantly affected (Fig. 7A
). The mRNA data of selected genes were complemented by immunoblotting of leaf proteins. Protein contents of sulfite reductase (SIR) and OAS-TL A, B, and C were unchanged, while APR protein was decreased by 40% in amiSAT3 line 5 (Fig. 7B). Thus, all three protein species precisely reflected the mRNA contents determined by microarray analysis, although it should be noted that the APR2 antiserum can probably cross-react with all three APR isoforms (Koprivova et al., 2000).
Only two genes out of the 152 candidate genes known for sulfur-dependent regulation (Hirai et al., 2003 ) were found to be significantly regulated in amiSAT3 line 5. Both genes, At1g33960 (avirulence-responsive protein) and At1g75300 (putative isoflavone reductase), are known to be transcriptionally regulated also by other environmental factors or hormone stimuli. Especially At1g75300 is regulated more strongly by methyl jasmonate (MJ) than by sulfur availability (www.genevestigator.ethz.ch). In this respect, it is noteworthy that the gene with the highest up-regulation (2.6-fold) is ST2a, a hydroxyjasmonate sulfotransferase (At5g0710), which is supposed to inactivate MJ by sulfonation (Gidda et al., 2003). Reponses to disease and pathogen attack have been known for a long time to be coordinated by MJ. All significantly regulated genes of the category stress response in this experiment (PR1, AIG1, and PRP1) are involved in responses to diseases or pathogens. In agreement with the idea of inactivation of MJ by induction of ST2a, all of them are down-regulated. Besides genes responding to MJ, two genes (GASA1 and GASA4) that were known as markers for GA stimulus are induced. Taken together, the expression data point toward a down-regulation of the sulfate assimilation pathway in amiSAT3 lines in order to coordinate plastid sulfate reduction with the reduced synthesis of OAS in mitochondria. Additionally, an inactivation of stress-responsive genes was observed, which could be a result of perturbed hormone metabolism.
The subcellular organization of Cys synthesis in plants differs strongly from that in other eukaryotes. Why plant SAT and OAS-TL form Cys in the cytosol, plastids, and mitochondria, the reasons for their strongly different activity ratios, and whether this distribution reflects redundancy or specific functions remained enigmatic (Fig. 8, A and B ). With respect to the second step of Cys synthesis, recent analyses of the OAS-TL protein family members in Arabidopsis using T-DNA insertion lines indicated redundancy as well as compartment-specific functions (Heeg et al., 2008 ; Lopez-Martin et al., 2008; Watanabe et al., 2008). In nonstressed plants, cytosolic OAS-TL turned out to be the most important isoform in terms of enzymatic activity (Heeg et al., 2008; Watanabe et al., 2008). However, a mitochondrial OAS-TL C mutant showed a mild growth phenotype despite a measurable OAS-TL C contribution of less than 5% of total activity. On the other hand, this little OAS-TL C activity alone was sufficient for comfortable growth under nonstress conditions in a double mutant without cytosolic OAS-TL A and plastid OAS-TL B (Heeg et al., 2008). At the same time, these mutants allowed us to conclude that Cys must be exchangeable between cytosol and organelles, at least in Arabidopsis, making an earlier hypothesis of a lack of such exchange as reason for the three sites of Cys very unlikely (Lunn et al., 1990). Feeding experiments with [35S]sulfate to oastl mutants furthermore confirmed the importance of the second step of Cys synthesis in the cytosol. The explanation for these findings was that the rate of formation of the reaction intermediate OAS in mitochondria dominates the overall rate of Cys synthesis in the cell (Fig. 8C; Heeg et al., 2008).
If this assumption was correct, one has to conclude that plastids and mitochondria provide most of the substrates sulfide and OAS, respectively, while the majority of Cys synthesis actually takes place in the cytosol. The experimental down-regulation of mitochondrial SAT activity in vivo, therefore, should result in reduced formation of OAS and Cys. Indeed, targeting of mitochondrial SAT3 by amiRNA resulted in reduced OAS contents, reduced rates of Cys synthesis, and reduced growth rates. Importantly, the degree of down-regulation of Sat3 mRNA, SAT3 protein levels, and overall SAT enzyme activity quantitatively matched the lowered OAS levels, Cys synthesis rates, and growth reduction. The amiRNA approach was shown by two independent methods to be specific with respect to the expression levels of the other four Sat genes. This demonstrates that mitochondrial SAT3 activity is indeed responsible for the bulk of OAS production under these conditions, and since SATs in the other compartments showed no compensation for the reduction, this isoform limited Cys production, as shown by retarded growth (Fig. 8D).
This strong dependence of total cellular Cys production on the pacemaking role of mitochondrial synthesis of OAS is unexpected, because until now cytosol and plastids were believed to be the major production sites of both OAS and Cys (Urano et al., 2000
The cytosol was assumed to be the major site for regulation of OAS synthesis as a consequence of the strong feedback inhibition of cytosolic SAT isoforms by Cys (50% inhibition of initial activity at 2–10 µM Cys; Noji et al., 1998; Saito, 2000
It is remarkable that radiolabel from [3H]Ser feeding appears in glutathione. This implies an uptake of Ser into mitochondria, formation of OAS, transport to the cytosol for Cys synthesis, and import into plastids to serve as substrate of
OAS contents were decreased in Arabidopsis amiSAT3 lines 4 and 5, but surprisingly, steady-state levels of Cys and glutathione were increased despite lowered flux into these thiols. Maintenance of Cys and glutathione contents was also observed in the Arabidopsis oastlC T-DNA mutant line, which lacked mitochondrial OAS-TL C and showed reduced growth (Heeg et al., 2008
The microarray analysis of leaves of the Arabidopsis amiSAT3 line 5 in comparison with wild-type plants revealed that none of the OAS-TL genes and none of the genes of sulfate uptake and primary sulfur metabolism were changed in expression, with the exception of one member of the Aps and Apr gene family each. Down-regulation of APR2 protein was confirmed by immunoblotting. Reduced activation and reduction of sulfate would explain the increased contents of sulfate in amiSAT3 lines. Especially the strong down-regulation of APR2 could be of particular importance, since lower APR2 activity in leaves was shown to be responsible for the higher sulfate content of the Arabidopsis ecotype Shahdara in comparison with ecotype Bay-0 (Loudet et al., 2007
Further putative signals for the down-regulation of APR2 are OAS and MJ. It has been repeatedly observed that OAS, possibly due to the dissociation of the CSC, acts as a signal for the entire sulfur assimilation pathway. Feeding of OAS regulates more than 650 and 850 genes in leaves and roots of Arabidopsis, respectively (Hirai et al., 2003
Specific down-regulation of mitochondrial SAT3 activity in Arabidopsis by amiRNA expression demonstrated that mitochondria, but not chloroplasts or the cytosol, are the dominant source of OAS in vivo. Despite strongly reduced flux into Cys in amiSAT3 plants, the steady-state levels of Cys and glutathione were elevated and presumably contributed to the repression of assimilatory sulfate reduction and accumulation of free sulfate. This, together with previous evidence, strongly suggests that OAS from mitochondria and sulfide from chloroplasts serve as substrates for the bulk of Cys synthesis in the cytosol.
General Cloning
Standard molecular biology technologies like growth of bacteria, plasmid isolation, and PCR were performed as described by Sambrook et al. (1989)
Transformation of Agrobacterium tumefaciens C58 with binary vectors and subsequent transformation and selection of Arabidopsis (Arabidopsis thaliana Col-0) were carried out as described by Clough and Bent (1998)
Hydrophilic metabolites were extracted from leaves of Arabidopsis plants according to Wirtz and Hell (2003)
Total soluble proteins were isolated with 0.5 mL of 50 mM HEPES, pH 7.4, 10 mM KCl, 1 mM EDTA, 10% glycerin, 30 mM dithiothreitol, and 0.5% phenylmethylsulfonyl fluoride from 0.2 g of leaf material that was ground to a fine powder in liquid nitrogen. Cell debris was removed by centrifugation at 16,000g and 4°C for 10 min. Proteins were quantified as described by Bradford (1976)
Total proteins from leaves were separated according to Laemmli (1970)
Twelve hours prior to the labeling experiment, soil-grown plants were enclosed in transparent plastic bags to ensure opening of the stomata. Leaf pieces (30 mg) were cut out from the interveinal fields of leaves and floated on the labeling solution (half-strength Hoagland medium containing 2.5 µM [3H]Ser [185–925 GBq mmol–1; Hartmann Analytic]) on a horizontal shaker with 60 rpm in the light (17 µE). Samples were taken after 20 and 30 min, washed twice with half-strength Hoagland medium [2.5 mM Ca(NO3)2, 2.5 mM KNO3, 0.5 mM MgSO4, 0.5 mM KH2PO4, 40 mM Fe-EDTA, 25 mMH3BO3, 2.25 mM MnCl2, 1.9 mM ZnSO4, 0.15 mM, CuSO4, and 0.05 mM (NH4)6MO7O24, pH 5.9], and frozen in liquid nitrogen. A negative control for contamination of the leaf surface with [3H]Ser was taken by dipping leaf pieces into the labeling solution for 1 s, prior to washing and harvesting like the samples. The samples were powdered using the Bio101 ThermoSavant Fast Prep system (Qbiogene) according to the manufacturer's instructions. The metabolites were extracted and separated prior to quantification of the incorporated 3H label as described by Heeg et al. (2008)
Design and Production of the Microarray
Sample Preparation, Hybridization, and Evaluation
Total RNA from leaf tissue was extracted with the RNeasy Plant Kit (Qiagen) according to the manufacturer's protocol. Total RNA was transcribed into cDNA and analyzed by quantitative real-time PCR as described by Talke et al. (2006)
Regression analyses of data sets were performed with SigmaPlot 8.0 that uses the Marquardt-Levenberg algorithm for determination of independent variables. Comparison of means from different sets of data was analyzed for statistical significance with the unpaired t test. Constant variance and normal distribution of data were carefully checked with SigmaStat 3.0 prior to statistical analysis.
The following materials are available in the online version of this article.
We are indebted to Dr. S. Kopriva (John Innes Institute, Norwich, UK) and Prof. Dr. D. Weigel (Max Planck Institute, Tübingen, Germany) for the kind gifts of the APR2 antiserum and the pRS300 vector, respectively. We thank S. Hassel (University of Heidelberg) for excellent technical assistance, Dr. Maria Bernal (University of Heidelberg) for support of quantitative real-time PCR analysis of the SAT gene family, and Dr. A. Meyer (University of Heidelberg) for critically reading the manuscript. Received June 24, 2008; accepted August 25, 2008; published August 27, 2008.
1 This work was supported by the Stiftung der deutschen Wirtschaft and the Schmeil-Foundation, Heidelberg (grants to F.F.H.), and by the Bioquant Landesgraduiertenkolleg Baden-Württemberg (grant to C.H.).  The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantphysiol.org) is: Rüdiger Hell (rhell{at}hip.uni-heidelberg.de).
[C] Some figures in this article are displayed in color online but in black and white in the print edition.
[W] The online version of this article contains Web-only data.
[OA] Open Access articles can be viewed online without a subscription. www.plantphysiol.org/cgi/doi/10.1104/pp.108.125237 * Corresponding author; e-mail rhell{at}hip.uni-heidelberg.de.
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-glutamylcysteine, the precursor of glutathione that is exclusively formed in plastids in Arabidopsis (Wachter et al., 2005
