Sulfite Reductase Defines a Newly Discovered Bottleneck for Assimilatory Sulfate Reduction and Is Essential for Growth and Development in Arabidopsis thaliana[C][W]
Muhammad Sayyar Khan,a Florian Heinrich Haas,a Arman Allboje Samami,a Amin Moghaddas Gholami,b Andrea Bauer,b Kurt Fellenberg,c Michael Reichelt,d Robert Hänsch,e Ralf R. Mendel,e Andreas J. Meyer,a Markus Wirtz,a and Rüdiger Hella1
aHeidelberg Institute for Plant Sciences, University of Heidelberg, 69120 Heidelberg, Germany bGerman Cancer Research Center (DKFZ), 69120 Heidelberg, Germany
cTechnical University Munich, 85354 Freising, Germany
dMax Planck Institute for Chemical Ecology, 07745 Jena, Germany
eTechnical University Braunschweig, Institute of Plant Biology, 38106 Braunschweig, Germany 1Address correspondence to ; Email: firstname.lastname@example.org .
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.plantcell.org) is: Rüdiger Hell(email@example.com ).
[C]Some figures in this article are displayed in color online but in black and white in the print edition.
[W]Online version contains Web-only data.
Received January 18, 2010; Revised March 18, 2010; Accepted April 5, 2010.
The role of sulfite reductase (SiR) in assimilatory reduction of inorganic sulfate to sulfide has long been regarded as insignificant for control of flux in this pathway. Two independent Arabidopsis thaliana T-DNA insertion lines (sir1-1 and sir1-2), each with an insertion in the promoter region of SiR, were isolated. sir1-2 seedlings had 14% SiR transcript levels compared with the wild type and were early seedling lethal. sir1-1 seedlings had 44% SiR transcript levels and were viable but strongly retarded in growth. In mature leaves of sir1-1 plants, the levels of SiR transcript, protein, and enzymatic activity ranged between 17 and 28% compared with the wild type. The 28-fold decrease of incorporation of 35S label into Cys, glutathione, and protein in sir1-1 showed that the decreased activity of SiR generated a severe bottleneck in the assimilatory sulfate reduction pathway. Root sulfate uptake was strongly enhanced, and steady state levels of most of the sulfur-related metabolites, as well as the expression of many primary metabolism genes, were changed in leaves of sir1-1. Hexose and starch contents were decreased, while free amino acids increased. Inorganic carbon, nitrogen, and sulfur composition was also severely altered, demonstrating strong perturbations in metabolism that differed markedly from known sulfate deficiency responses. The results support that SiR is the only gene with this function in the Arabidopsis genome, that optimal activity of SiR is essential for normal growth, and that its
downregulation causes severe adaptive reactions of primary and secondary metabolism.
Plants take up the essential macronutrient sulfur from the soil in the form of sulfate. The uptake of sulfate and its subsequent assimilatory reduction into organic sulfur compounds proceed through a highly coordinated mechanism. First, uptake of sulfate is catalyzed by specific proton cotransporters in root epidermal cells. They belong to the group of high affinity sulfate transporters (SULTR group 1) and are inducible by external sulfate deprivation (Smith et al., 1997; Takahashi et al., 2000). Internal allocation of sulfate is catalyzed by members of the low affinity SULTR groups 2 and 3 (Hawkesford, 2008; Takahashi and Saito, 2008). Next, assimilatory reduction of sulfate is initiated by ATP-dependent activation of sulfate to adenosine 5′-phosphosulfate (APS) catalyzed by ATP sulfurylase (ATPS). Further activation with ATP is catalyzed by APS kinase and yields 3′-phosphoadenosyl-5′-phosphosulfate (PAPS). APS kinase is
present in plastids and the cytosol to provide PAPS for sulfation reactions by sulfotransferases (Mugford et al., 2009).
APS reductase (APR) in plastids from Arabidopsis thaliana and other plants strongly prefers APS instead of PAPS as a substrate, its expression responds to sulfate and nitrate availability, and a number of stress factors result in regulation of its activity (Leustek et al., 2000). In addition, flux analysis using 35S-labeled sulfate hinted that APR, after sulfate uptake, exerts the strongest control over flux through the sulfate reduction pathway in Arabidopsis (Vauclare et al., 2002) and is responsible for genetically determined variation in sulfate content in Arabidopsis ecotypes (Loudet et al., 2007).
In contrast with APR, the second enzyme of the free reduction pathway, sulfite reductase (SiR), has received little attention. Plant SiR is a plastid-localized soluble enzyme of two 65-kD subunits, contains a single siroheme and (4Fe-4S) cluster as prosthetic groups, and has a high affinity (Kmsulfite ~10 μM) for sulfite (Krüger and Siegel, 1982; Nakayama et al., 2000). Ferredoxin acts
as the physiological donor of the six electrons required for sulfite reduction, whereas bacterial SiR uses NADPH (Yonekura-Sakakibara et al., 2000). The structure, sequence, and ligands of SiR in bacteria, archea, and eukaryotes are similar to those of nitrite reductase, which catalyzes an equivalent reduction step in nitrate assimilation (i.e., a six-electron reduction of nitrite to ammonia) (Crane et al., 1995; Swamy et al., 2005). In Arabidopsis, SiR shows 19% identity with nitrite reductase (NiR) at the amino acid level. Phylogenetic analysis showed that both SiR and NiR arose from an ancient gene duplication in eubacteria, before the primary endosymbiosis that gave rise to plastids (Patron et al., 2008). SiR is able to reduce nitrite as well as sulfite, and substrate preferences can be converted by a single amino acid mutation (Nakayama et al., 2000). NiR has been suggested to operate similarly and to accept sulfite as substrate at a low rate (Schmidt and Jäger, 1992).
SiR is encoded by the only single-copy gene in primary sulfur metabolism in Arabidopsis, whereas the rice (Oryza sativa) and poplar (Populus spp) genomes each contain two copies (Bork et al., 1998; Kopriva, 2006). It is expressed in nearly all tissue types and shows the least transcriptional responses among sulfur-related genes in Arabidopsis in classical sulfate starvation experiments or under other stress conditions, according to a survey in microarray databases (Zimmermann et al., 2004). Expression changes were observed after treatment with SO2 (Brychkova et al., 2007), but these were not translated into significant changes of SiR enzyme activity under similar conditions (Lang et al., 2007). Activity of SiR is generally believed to be maintained in excess to scavenge potentially toxic sulfite (Leustek, 2002; Kopriva, 2006), based on flux control and APR overexpression experiments in Arabidopsis and maize (Zea mays; Tsakraklides et al., 2002; Vauclare et al., 2002; Martin et al., 2005). SiR potentially competes with two other enzymes for sulfite: UDP-sulfoquinosovyl synthase (SQD1) for sulfolipid synthesis in plastids (Benning, 2007) and sulfite oxidase (SO), a molybdoenzyme, in peroxisomes (Eilers et al., 2001; Hänsch and Mendel, 2005; Brychkova et al., 2007). However, the bulk of sulfite is normally channeled into the assimilatory reduction pathway for sulfur amino acid and protein biosynthesis. Assimilatory sulfate reduction is completed by the two-step process of Cys synthesis that is catalyzed by serine acetyltransferase (SAT) and O-acetylserine (thiol) lyase (OAS-TL) with O-acetylserine (OAS) as the intermediate. Both enzymes form a regulatory complex, the Cys synthase complex that controls SAT activity in reaction to intracellular changes of sulfide, OAS, and Cys concentrations (for review, see Hell and Wirtz, 2008). The Cys synthase complex is present in plastids, cytosol, and mitochondria with sulfide, OAS, and Cys being freely exchanged between the compartments in Arabidopsis (Heeg et al., 2008; Watanabe et al., 2008a, 2008b; Krueger et al., 2009). Metabolic repression by Cys and GSH and activation by OAS of genes encoding sulfate transporters, ATPS, and APR are major mechanisms discussed for regulation of primary sulfur metabolism (Leustek et al., 2000; Kopriva, 2006).
Here, we investigated two Arabidopsis lines with T-DNA insertions in the promoter region of SiR. Mutant line sir1-2 is early seedling lethal and unequivocally demonstrates that the free sulfate reduction pathway is essential for survival and cannot be compensated for by any other enzymatic process. In mature leaves, mutant sir1-1 has 28% of SiR activity and 3.6% of flux in the assimilatory reduction pathway in vivo compared with the wild type. sir1-1 has a strongly retarded growth phenotype, showing that in contrast with general assumptions, SiR can easily become limiting for growth. sir1-1 mutant plants are sensitive to cadmium due to lack of GSH for phytochelatin synthesis. Carbon, nitrogen, and sulfur composition are severely altered with a shift toward carbon-bound reduced nitrogen, indicating that lowered sulfite reduction leads to comprehensive reprogramming of primary metabolism.
Identification and Characterization of sir Mutants
Two independent Arabidopsis T-DNA insertion lines, further annotated as sir1-1 and sir1-2, were identified in the GABI-Kat collection center. In both lines, the T-DNA was inserted in the promoter region of SiR (Figure 1A). PCR-verified heterozygous sir1-2 plants segregated in two distinct classes of seedlings after germination in the absence of selection marker: (1) wild type-like seedlings (401/533, 75%; χ2 = 0.016) and (2) bleached seedlings that died at the two cotyledon stage after germination (132/533, 25%). Genotyping of these seedlings (Figure 1B) revealed that bleached sir1-2 seedlings were homozygous for the T-DNA insertion. On these plates, wild-type-like plants represented a pool of heterozygous sir1-2 (1/2) and segregating Columbia-0 (Col-0) plants (1/4), suggesting a single insertion of a recessive allele. Thus, the homozygous sir1-2 line is seedling lethal. Flanking sequences of the T-DNA were PCR amplified and sequenced to characterize the insertion sites. Alignment with the genomic sequence of SiR revealed the beginning of the left border of the T-DNA at position ?54 bp in sir1-2 relative to the
transcription start site in the promoter region of SiR (Figure 1A). Quantitative real-time PCR (qRT-PCR) detected successful transcription initiation corresponding to 14% of mature SiR transcript in the early seedling stage that may account for embryo development and germination of sir1-2 (Figure 1C). For genetic complementation, the heterozygous sir1-2 plants were transformed with a construct that expressed the SiR cDNA along with its plastid transit peptide under control of the constitutive 35S cauliflower mosaic virus promoter. The phenotype of sir1-2 was completely restored (Figure 1D), demonstrating that the loss of function of SiR was the cause of the early seedling lethality observed in the homozygous sir1-2 plants.
Molecular Identification of sir Mutants and Phenotype of sir1-2.
Like sir1-2, heterozygous sir1-1 plants also gave rise to two distinct classes of seedlings. Besides wild-type-like seedlings (352/471, 75%; χ2 = 0.018), severely growth-retarded and pale-looking
seedlings (119/471, 25%) could be identified after germination in the absence of selection marker. The growth-retarded sir1-1 seedlings were found to be homozygous for the T-DNA insertion by PCR analysis (Figure 2A), again pointing to a single insertion site. The insertion site of the T-DNA in sir1-1 was determined by PCR amplification of the flanking sequences and revealed the insertion of the T-DNA at position ?73 bp in the promoter region (Figure 1A). The viability of
homozygous sir1-1 seedlings was in contrast with the seedling lethal phenotype of the sir1-2 mutant. In the early seedling stage of sir1-1, the abundance of SiR mRNA was 44% (Figure 1C) compared with wild-type seedlings of the same developmental stage. Thus, the levels of 44 and
14% of mature SiR mRNA made the difference between survival and death of the early seedlings of sir1-1 and sir1-2, respectively. It is remarkable that both T-DNA insertions in the SiR promoter allowed transcription of intact mRNA and the 19 bp between the insertion sites were responsible for a twofold difference in steady state mRNA of SiR. Transcript levels of SiR in mature leaves of 7-week-old soil-grown homozygous sir1-1 plants were decreased to 17% compared with the wild type (Figure 2B). Accordingly, the amount of SiR protein (Figure 2C) was significantly reduced, and SiR activity also was lowered to 28% of the wild-type level (Figure 2B). This provides an explanation for the slower vegetative growth in comparison to the wild type that became more pronounced with time (Figure 2D). The homozygous sir1-1 line had the same number of leaves compared with the wild type of the same age, but they were clearly paler and smaller (Figure 3A). An approximately fivefold reduction in total biomass was observed between sir1-1 and Col-0 plants grown for 8 weeks on soil under short-day conditions (Figure 3B). Short-day conditions delayed flowering of soil-grown homozygous sir1-1 plants by ~4 weeks compared with Col-0. In spite of the retarded growth phenotype and delayed flowering, the homozygous sir1-1 plants were able to set seeds leading to viable offspring in the next generation. Thus, reduced expression of SiR causes a severe growth limitation. These results provide functional evidence that there is only a single annotated gene for SiR in the Arabidopsis genome and that its function is indispensible for survival. Nevertheless, the viability of sir1-1 allows investigation of the physiological role of SiR activity in plant metabolism. For that reason, all further experiments were performed with sir1-1.
Abundance and Activity of SiR in the T-DNA Insertion Line sir1-1.
Phenotype and Complementation of sir1-1 Plants.
Genetic and Chemical Complementation of sir1-1
Stable transformation of homozygous sir1-1 with the 35S:SiR overexpression construct completely restored the phenotype of sir1-1, including the pale low chlorophyll view (Figures 3A and 3B). A 2.5-fold higher enzymatic activity of SiR was observed as a result of the high expression rate of SiR driven by the 35S promoter (Figure 3C). This resulted in wild-type-like thiol levels (see Supplemental Figure 1 online) and normal growth of the complemented sir1-1 lines under growth chamber conditions (Figure 3B). The latter results indicate an adequate SiR activity in Col-0 for optimal growth. Chemical complementation by exogenous supply of the limiting product of the SiR reaction, sulfide, and GSH, the major low molecular weight thiol in plants, partially rescued the phenotype (Figure 3D). The total shoot biomass of homozygous sir1-1 plants was increased twofold by feeding of 0.1 mM Na2S and 2.7-fold by feeding of 1 mM GSH in comparison to untreated sir1-1. However, the differences between chemically complemented homozygous sir1-1 and Col-0 control plants were still significant (Figure 3D). Shifts of C, N, and S Metabolites in sir1-1
The consequences of reduced SiR activity for metabolic homeostasis were analyzed by determination of the steady state levels of primary and secondary metabolites in leaves of 7-week-old soil-grown wild-type and sir1-1 plants. Measurements of anion levels in homozygous sir1-1 plants revealed a ninefold accumulation of free sulfate, while the steady state level of nitrate was reduced threefold in comparison to the wild type, indicating a deregulation of nitrate and sulfate uptake and assimilation pathways (Figure 4A). Indeed, the accumulation of sulfate in leaves of sir1-1 was found to correlate with strongly increased uptake rates of sulfate in roots. Feeding of 35SO42? to roots of Arabidopsis wild-type and sir1-1 mutant plants revealed a 13-fold
increased rate of sulfate uptake in sir1-1 (2 ? 0.8 fmol 35S min?1 mg?1 fresh weight [FW] in
Col-0 compared with 26.6 ? 7.6 fmol 35S min?1 mg?1 FW in sir1-1).
Impact of Reduced SiR Activity on Metabolism in Leaves of sir1-1 Plants.
In agreement with reduced SiR activity, a 1.7-fold accumulation of sulfite levels was evident (Figure 4B). OAS, the activated amino acid for synthesis of Cys, was more than twofold accumulated due to decreased formation of sulfide (Figure 4C). Unexpectedly, the steady state level of Cys was 1.8-fold upregulated, while the level of GSH was not significantly affected (Figure 4D). Both thiols had been assumed to be downregulated in sir1-1, as a result of lower SiR activity. The analysis of glucosinolates, the major sulfur-containing secondary compounds in Arabidopsis revealed strong differences between sir1-1 and Col-0. The profiles of aliphatic glucosinolates, especially the major glucosinolate, glucoraphanin, were reduced (Figure 4E; see Supplemental Figure 2 online), causing a significant twofold reduction of the total glucosinolate content either by decreased biosynthesis or by enhanced turnover.
The analysis of total carbon (C) nitrogen (N) and sulfur (S) demonstrated significant reductions of total C (8%) and total N (11%) in sir1-1. A striking increase of total S of more than threefold was observed in the sir1-1 mutant (Figure 4F). This increase can be almost entirely attributed to the observed rise of free sulfate (Figure 4A) and not to organically bound sulfur (see Supplemental Figure 3A online). By contrast, the decrease in nitrate content accounted only partially for the lowered total nitrogen share in sir1-1 and strongly suggests that the organically bound nitrogen fraction was increased (see Supplemental Figure 3B online). These severe changes in the reduced carbon and nitrogen fractions prompted us to dissect the major C and N metabolites. In situ staining of leaves with iodine solution showed a clearly visible significant reduction in starch content 3 h after onset of light in source as well as sink leaves of rosettes of sir1-1 plants compared with the wild type (Figure 5A). On average, leaf starch content was reduced nearly threefold after extraction of starch from young leaves (Figure 5B). Sucrose contents were decreased by the same range and glucose and fructose were down by ~90% in sir1-1 (Figure 5B). While the levels of major free sugars were reduced, the opposite was observed for amino acids (Figure 5C). Contents of 14 out of 18 determined amino acids were higher in sir1-1 (see Supplemental Figure 4 online), including that of Met (Figure 5C). In total, the amount of free amino acids increased significantly by 55% in sir1-1 compared with the wild type. These findings suggest an enhanced fixation of reduced nitrogen into carbon, possibly on cost of sugars. Concomitantly, the mRNA levels of VEGETATIVE STORAGE PROTEIN1 (VSP1) and VSP2 in leaves of sir-1 plants were at least 10-fold enhanced according to expression analyses (Figure 5D). Taken together, these results indicate massive and far-reaching shifts between carbon, nitrogen, and sulfur fractions in response to the partial block in sulfite reduction.
Deregulation of Carbon- and Reduced Nitrogen-Containing Compounds in
S-Related Enzymatic Activities in sir1-1 Plants
The significantly higher sulfite contents in leaves of sir1-1 prompted us to test the activity of SO in these plants. The activity of SO in leaves of hydroponically grown sir1-1 plants was found to be 2.4-fold higher in comparison to Col-0 (Figure 6A). This observation points to the theoretical possibility of reoxidation of excess sulfite to sulfate in peroxisomes of sir1-1. The ninefold higher accumulation of foliar sulfate may therefore result from insufficient SiR activity but also reoxidation of toxic sulfite to sulfate by SO. The amount of SO protein was not increased in sir1-1 mutants according to immunoblotting using an SO-specific antiserum (see Supplemental Figure 5 online), suggesting a posttranslational upregulation of SO activity. SAT catalyzes the production of OAS that is converted by OAS-TL to Cys under consumption of sulfide. Despite twofold increases in OAS and Cys steady state-levels, the total activities of SAT and OAS-TL were not significantly affected in sir1-1 (Figure 6B). Accordingly, the amount of mitochondrial SAT3 protein, which functions as pacemaker of Cys synthesis in Arabidopsis by providing OAS (Haas et al., 2008), did not change, as shown by immunological detection using a SAT3 specific antiserum (Figure 6C). Mitochondrial OAS-TL C, which is supposed to function as a regulator of SAT3 activity in the Cys synthase complex (Wirtz and Hell, 2006), showed no changes in its abundance and neither did cytosolic OAS-TL A, which is responsible for the majority of Cys synthesis (Heeg et al., 2008). Furthermore, the amount of plastid OAS-TL B was not altered, despite presumably strongly decreased sulfide production in the plastids of sir1-1 plants (Figure 6D).
Abundance and Activity of SO, SAT, and OAS-TL in Leaves of sir1-1 Plants.
Incorporation of Radioactively Labeled Sulfate into Thiols and Protein
The higher steady state level of Cys in combination with unaffected SAT and OAS-TL activities
are in contrast with the retarded growth phenotype and the assumed reduced sulfide production in sir1-1 plants. To unravel the underlying fluxes of sulfur through the assimilatory sulfate reduction pathway of sir1-1 plants, the incorporation of radiolabeled sulfate (35SO42?) into Cys and GSH
was determined by feeding of leaf pieces according to Heeg et al. (2008). Time-resolved analysis of incorporation of the 35S label into Cys and GSH demonstrated a very strong reduction of flux through the sulfate assimilation pathways in sir1-1 plants compared with the wild type. Only 5.5% of incorporation of 35S label into Cys compared with the wild type was observed in leaves of sir1-1 after the 15-min feeding period. The incorporation was lower at the end of the 30-min chase period at time point 45 min, indicating the active turnover of Cys (Figure 7A). Most of the 35S-Cys went into the GSH pool (Figure 7B), and a smaller part was found in the protein fraction (Figure 7C), documenting active protein biosynthesis under these conditions. Thus, the total incorporation into organic sulfur compounds in sir1-1 after 15 min amounted to a 28-fold reduction compared with the wild type. The experiment was independently repeated using soil-grown plants instead of hydroponically grown plants to exclude influences of nutritional status of the plants. The distribution patterns were quite similar, albeit incorporation rates of the 35S-label were somewhat lower (see Supplemental Figure 6 online). The results from feeding experiments thus confirm a strongly reduced rate of sulfide formation and corroborate the unique function of SiR for the assimilatory sulfate reduction pathway.
Incorporation Rates of Sulfate in Wild-Type and sir1-1 Plants.
Response of sir1-1 Seedlings toward Cadmium Stress
Phytochelatins are enzymatically synthesized from GSH by cytosolic phytochelatin synthase for detoxification of heavy metal ions (Ha et al., 1999). For that reason, the reduced capacity of the sir1-1 plants to incorporate sulfur for synthesis of GSH was expected to result in a higher sensitivity toward cadmium. Wild-type Arabidopsis and sir1-1 seeds were germinated on solid medium containing increasing concentrations of Cd (0 to 100 μM CdCl2). After 14 d of treatment both Col-0 and sir1-1 showed a pale leaf phenotype and shortened primary roots (Figure 8A) as typical symptoms of Cd stress (Cobbett et al., 1998). The degree of root shortening was used for quantification of Cd tolerance. Since the root length of sir1-1 seedlings was already strongly reduced in comparison to Col-0 in absence of Cd, the reduction of root length was expressed as a percentage, whereby the root length of untreated plants of the respective genotype was set to
100% (Figure 8B). Upon exposure to 25, 50, and 100 μM CdCl2, reductions of 75, 87, and 92%, respectively, were observed in the root length of sir1-1 seedlings. By contrast, the magnitudes of reduction at the same CdCl2 concentrations for Col-0 were only 29, 53, and 67%, demonstrating that root growth of sir 1-1 was 2.6-fold more sensitive than Col-0 toward 25 μM CdCl2, for
instance. Taken together, the data demonstrate that reduced sulfur assimilation leads to higher Cd sensitivity in Arabidopsis.
Cadmium Sensitivity of sir1-1 Plants.
Response of Sulfur Metabolism at the Transcriptional Level
The impact of reduced SiR activity on the transcription of sulfur metabolism-related genes in leaves of 7-week-old soil-grown plants was investigated with a microarray carrying 920 genes related to primary metabolism and stress responses as described by Haas et al. (2008). Based on three biological repetitions of the wild type and sir1-1 with four technical replicates, each including dye swaps for each set, 67 genes were found to be significantly up- or downregulated in the leaves of hydroponically grown sir1-1 plants compared with Col-0 according to P values of <0.05. Most regulated genes were related to redox homeostasis (20), while genes of sulfur metabolism (11), pathogen resistance (11), glucosinolate synthesis (10), hormone synthesis and signaling (5), GSH transfer activity (4), sulfur-induced nonsulfur genes (3), and amino acid synthesis (3) were also found to be significantly changed in abundance (see Supplemental Table 1 online). The microarray analysis confirmed independently the downregulation of SiR transcript in mature leaves of sir1-1 (Figure 2C). Besides SiR, three genes of the primary sulfur assimilation pathway were significantly downregulated: ATPS4, APS REDUCTASE2 (APR2), and SULFATE TRANSPORTER 2.1 (SULTR2.1; Figure 9). SULTR2.1 is known to be specific for the vasculature and downregulated in leaves upon sulfur deficiency (Takahashi et al., 2000). ATPS4 catalyzes the activation of sulfate in plastids, which leads to formation of APS. APR2 is the key APR isoform in leaves for reduction of activated sulfate in APS to sulfite that is further reduced by SiR. Downregulation of APR2 was independently confirmed by real-time PCR and revealed 58% mRNA content in sir1-1 compared with wild-type plants (see Supplemental Figure 7 online). Most likely ATPS4 and APR2 are downregulated to avoid extensive accumulation of toxic sulfite, which cannot be incorporated into Cys as a result of reduced SiR activity in sir1-1. The stable total SAT and OAS-TL enzymatic activities and abundances of analyzed SAT and OAS-TL isoforms shown in leaves of sir1-1 (Figure 6) were further supported by unchanged SAT and OAS-TL transcript levels in sir1-1 (Figure 9). In accordance with unchanged SO protein contents, SO was not upregulated at the transcriptional level despite clearly increased enzymatic activity, leaving the possibility of posttranslational activation of SO in peroxisomes of sir1-1. Multiple genes involved