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. 2018 May 11;177(3):927–937. doi: 10.1104/pp.18.00421

Sulfur Partitioning between Glutathione and Protein Synthesis Determines Plant Growth1,[OPEN]

Anna Speiser a, Marleen Silbermann a, Yihan Dong a, Stefan Haberland a, Veli Vural Uslu a, Shanshan Wang a, Sajid AK Bangash b, Michael Reichelt c, Andreas J Meyer b, Markus Wirtz a, Ruediger Hell a,2
PMCID: PMC6053006  PMID: 29752309

Glutamate cysteine ligase activity determines flux of sulfur into protein synthesis via the Target of Rapamycin sensor kinase in Arabidopsis.

Abstract

Photoautotrophic organisms must efficiently allocate their resources between stress-response pathways and growth-promoting pathways to be successful in a constantly changing environment. In this study, we addressed the coordination of sulfur flux between the biosynthesis of the reactive oxygen species scavenger glutathione (GSH) and protein translation as one example of a central resource allocation switch. We crossed the Arabidopsis (Arabidopsis thaliana) GSH synthesis-depleted cadmium-sensitive cad2-1 mutant, which lacks glutamate cysteine (Cys) ligase, into the sulfite reductase sir1-1 mutant, which suffers from a significantly decreased flux of sulfur into Cys and, consequently, is retarded in growth. Surprisingly, depletion of GSH synthesis promoted the growth of the sir1-1 cad2-1 double mutant (s1c2) when compared with sir1-1. Determination of GSH levels and in vivo live-cell imaging of the reduction-oxidation-sensitive green fluorescent protein sensor demonstrated significant oxidation of the plastidic GSH redox potential in cad2-1 and s1c2. This oxidized GSH redox potential aligned with significant activation of plastid-localized sulfate reduction and a significantly higher flux of sulfur into proteins. The specific activation of the serine/threonine sensor kinase Target of Rapamycin (TOR) in cad2-1 and s1c2 was the trigger for reallocation of Cys from GSH biosynthesis into protein translation. Activation of TOR in s1c2 enhanced ribosome abundance and partially rescued the decreased meristematic activity observed in sir1-1 mutants. Therefore, we found that the coordination of sulfur flux between GSH biosynthesis and protein translation determines growth via the regulation of TOR.

One of the most delicate decisions for plants is balancing resources between growth and stress responses. A prime example is the partitioning of Cys for the synthesis of proteins to promote the growth and synthesis of glutathione (GSH), the universal scavenger of reactive oxygen species (ROS) in eukaryotes (Foyer and Noctor, 2011; Noctor et al., 2012).

Cys is the end product of the assimilatory sulfate reduction pathways and serves as the unique donor of reduced sulfur (S) for many S-containing compounds (e.g. Met, biotin, and glucosinolates). Its synthesis is catalyzed by O-acetylserine(thiol)lyase (OAS-TL), which replaces the activated acetyl moiety of O-acetylserine (OAS) with sulfide to form Cys. OAS production limits Cys synthesis and is catalyzed by serine acetyltransferase (SERAT). SERAT physically interacts with OAS-TL in the regulatory Cys synthase complex that is present in all subcellular compartments where protein synthesis occurs (Wirtz et al., 2010).

In contrast to OAS production, sulfide production by the assimilatory reduction of sulfate is restricted to plastids, since the two enzymes capable of transferring electrons to S are localized exclusively in this compartment. Adenosine-5-phosphosulfate reductase (APR) transfers two electrons from GSH to activated sulfate (adenosine-5-phosphosulfate) and releases sulfite. The sulfite is further reduced to sulfide by sulfite reductase (SiR), which accepts the required six electrons from ferredoxin (for review, see Takahashi et al., 2011). Notably, the condensation of Glu and Cys to γ-glutamylcysteine, which also is the committed step of GSH synthesis, is catalyzed exclusively in plastids by glutamate cysteine ligase (GCL, formerly known as γ-glutamylcysteine synthetase; Hell and Bergmann, 1990; Wachter et al., 2005).

Many studies have provided independent evidence for the importance of GSH synthesis during natural environmental stresses that impinge on redox homeostasis by the formation of ROS (Foyer and Noctor, 2011; Noctor et al., 2012). Consequently, decrease of GSH synthesis in the partial loss-of-function mutants of SiR (sir1-1) or GCL (cad2-1) results in sensitivity toward heavy metal-induced stress (Cobbett et al., 1998; Khan et al., 2010). The total loss-of-GCL-function mutant is embryo lethal, and this observation provides evidence for the essential relevance of this redox buffer in plants (Cairns et al., 2006). However, the remaining GCL activity in cad2-1 (40% of the wild-type level) is sufficient to allow wild-type-like growth under nonstressed conditions (Cobbett et al., 1998).

Protein synthesis is mandatory for cell growth and a relevant sink of energy and major nutrients, such as carbon (C), nitrogen (N), and S (for review, see Henriques et al., 2014). In all eukaryotes, protein synthesis and growth is controlled by the Target of Rapamycin (TOR) sensor kinase. Human TOR perceives signals from the hormone system, sugars, and amino acids and matches growth with environmental cues like starvation and infections (Demetriades et al., 2014; Yun et al., 2016). We recently demonstrated the regulation of TOR activity in response to the flux of the S-assimilation pathway. The sir1-1 mutant displayed decreased TOR activity, which explained the retarded growth phenotype and the substantially decreased translation in sir1-1 (Dong et al., 2017).

In this study, we addressed the importance of S partitioning in the primary metabolic network for adequate stress responses and growth by comprehensive analysis of the sir1-1 and cad2-1 mutants. Surprisingly, we found that the sir1-1 cad2-1 double mutant grew better than the sir1-1 single mutant. Determination of steady-state levels and feeding with radioactive tracers evidenced a significant induction of flux through the assimilatory sulfate reduction pathway in cad2-1. Furthermore, we demonstrated that the cad2-1 mutation results in a specific induction of TOR activity. Both the activation of assimilatory sulfate reduction and the induction of TOR activity caused a significantly higher de novo incorporation of S into proteins in the sir1-1 cad2-1 double mutant. The results provide molecular insights into the astonishing growth-promoting effect by the combination of two mutations that reduce the flux of S into the universal redox buffer GSH. In vivo determination of the cellular redox environment by noninvasive live-cell imaging of GCL-depleted mutants shed light on the importance of redox-induced flux control within the assimilatory sulfate reduction pathway.

RESULTS

Decreased GSH Synthesis Promotes the Growth of sir1-1

The importance of Cys partitioning between GSH synthesis and translation was addressed by crossing sir1-1, which has 18-fold lower flux of S into Cys than the wild type and strong growth retardation (Khan et al., 2010), with the cad2-1 mutant, which is affected in the GSH synthesis branch but has no growth phenotype under nonstress conditions (Supplemental Fig. S1). The identified homozygous sir1-1 cad2-1 (s1c2) double mutants were viable and displayed no developmental defects (Fig. 1A), although GSH affects the initiation and maintenance of cell division (Vernoux et al., 2000). Even more surprising, the s1c2 plants grew 5 times faster than sir1-1 under nonstressed conditions (Fig. 1B) and accumulated less anthocyanin (Fig. 1C). However, neither growth of s1c2 nor accumulation of the stress marker anthocyanin was completely restored to wild-type levels (Fig. 1, B and C). Furthermore, the introduction of the cad2-1 mutation in s1c2 partially rescued the delayed-flowering phenotype of sir1-1 (Fig. 1D).

Figure 1.

Figure 1.

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Crossing of sir1-1 with cad2-1 partially rescues the dwarf phenotype of sir1-1. A, Phenotypes of three representative rosettes of 7-week-old wild-type (WT), cad2-1, sir1-1, and s1c2 plants. Individual rosettes were extracted from separate images and are at the same scale. Bar = 2 cm. B, Fresh weight of whole rosettes of wild-type and mutant plants shown in A. Bars represent means ± se (n = 10). Different letters indicate statistically significant differences among the result groups (Holm-Sidak one-way ANOVA, P ≤ 0.05). C, Relative levels of anthocyanins in leaves of wild-type and mutant plants grown as described in A. The anthocyanin level of the wild type was set to 1. Bars represent means ± se (n = 4). Differences are indicated as in B. D, Time line of flower bud induction in wild-type and mutant plants under short-day conditions. Single blocks indicate the period when plants evolved flower buds. Data points were collected and plotted against time for five individuals of the wild type, cad2-1, and sir1-1 and for 18 individuals of s1c2. E, Impact of the pharmacological inhibition of GCL activity in the wild type and the sir1-1 mutant. Plants were treated with nontoxic (0–125 µm) and toxic (greater than 250 µm) concentrations of BSO, and root length was determined at day 7 after germination. Root growth in the absence of BSO was set to 100% for both genotypes. F, Phenotypes of wild-type and sir1-1 mutant seedlings grown on AT medium in the absence (control) or presence of 63 µm BSO. Bar = 1 cm.

Next, we inhibited GCL activity in the wild type and the sir1-1 mutant by external application of nontoxic (0–125 µm) and toxic (greater than 250 µm) concentrations of the well-established GCL inhibitor 1-buthionine-S-sulfoximine (BSO; Fig. 1E). Application of nontoxic BSO promoted the root growth of sir1-1 but did not affect the wild-type root (Fig. 1, E and F). Concentrations above 250 µm BSO inhibited the root growth of both genotypes (Fig. 1E). Taken together, these results demonstrate that moderate inhibition of GCL activity promotes the growth of sir1-1 but has no impact on the growth of the wild type under nonstressed conditions.

Impact of GCL on Steady-State Levels of S-Related Metabolites under Normal and Decreased Flux through the S Assimilation Pathway

The GSH steady-state level of s1c2 was indistinguishable from that of cad2-1 but was lower than in sir1-1 and the wild type, which strongly indicated that the flux of Cys into GSH was decreased in roots and shoots of the double mutant (Fig. 2, A and G). The lowered GCL activity of cad2-1 caused the accumulation of Cys, which is in full agreement with the initial characterization of cad2-1 (Cobbett et al., 1998) and the concept that the GSH pool represents a significant sink for de novo-synthesized Cys even in nonstressed plants. The accumulation of Cys was decreased significantly in s1c2 plants when compared with cad2-1 (Fig. 2B; P < 0.05), indicating an alternative use of Cys in s1c2 when compared with cad2-1.

Figure 2.

Figure 2.

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s1c2 exhibits intermediate levels of S-related metabolites and transcripts in leaves and roots, respectively. A to D, Steady-state levels of GSH (A), Cys (B), OAS (C), and sulfate (D) in rosette leaves of 7-week-old wild-type (WT), cad2-1, sir1-1, and s1c2 plants under short-day conditions. FW, Fresh weight. E and F, Relative transcript levels of sulfate transporters SULTR1;1 and SULTR1;2 in roots of wild-type and mutant plants. The wild-type transcript level was set to 1. For A to F, results represent means ± se (n = 3). Different letters indicate statistically significant differences among the result groups (Holm-Sidak one-way ANOVA, P ≤ 0.05). Asterisks indicate statistically significant differences between two single genotypes using Student’s t test (**, P ≤ 0.01). G, In situ staining of cytosolic GSH with monochlorobimane (green signal) in roots of wild-type, cad2-1, sir1-1, and s1c2 plants. Cells were stained with propidium iodide (red signal) to label cell walls and demonstrate cell viability. Bar = 100 µm.

As expected, Cys steady-state levels were not affected in nonstressed sir1-1 plants (Fig. 2B), despite the already known decrease of S flux into Cys (Khan et al., 2010). The substantial decrease of sulfide production in sir1-1 caused significant accumulation of OAS (Fig. 2C; P < 0.05). The OAS accumulation was lower in the double mutant when compared with sir1-1 (Fig. 2C), indicating the activation of sulfide production by the crossing of cad2-1 into the sir1-1 background. In agreement, the OAS steady-state level of cad2-1 also was lower when compared with the wild type (Fig. 2C). The decreased levels of OAS and the accumulation of Cys in cad2-1 and s1c2 are consistent with the efficient use of OAS for Cys synthesis when GCL activity is decreased in leaves.

Control of a High-Affinity Sulfate-Transport System by OAS

OAS is known to act as an S-starvation signal, which controls the transcription of high-affinity sulfate transporters located at the plasma membrane and, consequently, the incorporation of sulfate. We thus tested steady-state levels of sulfate and sulfate transporter SULTR1;1 and SULTR1;2 transcripts in the wild type and the mutants (Fig. 2, D–F). Transcription of SULTR1;1 and SULTR1;2 was associated with OAS steady-state levels in all analyzed plants (Fig. 2, C, E, and F). The strong induction of SULTR1;1 and SULTR1;2 in sir1-1 and s1c2 resulted in the significant accumulation of sulfate in leaves (P < 0.05). However, the s1c2 double mutant displayed significantly less induction of SULTR1;1 and less accumulation of leaf sulfate when compared with sir1-1 (Fig. 2, D and E; P < 0.05).

Impact of GCL and SiR on the Distribution of S and C between Sinks

The accumulation of sulfate causes a significant increase of total S content in sir1-1 and a substantial decrease of total C, but total N content is unaffected (Khan et al., 2010). This study independently confirms the previously observed alterations in the abundance of the major nutrients in sir1-1. Although the cad2-1 mutation alone had no impact on the distribution of total C and S, the s1c2 double mutant accumulated significantly less total S than sir1-1 (Fig. 3A; P < 0.05). Also, the decrease of total C from wild-type levels was less pronounced in s1c2 when compared with sir1-1 (Fig. 3B). These results reinforce the hypothesis that the accumulation of oxidized S drives the increase of total S in sir1-1. The total content of glucosinolates, a family of C/S-containing defense compounds, also was reduced significantly in s1c2 and sir1-1 but not in cad2-1 (Fig. 3C; P < 0.05; Supplemental Fig. S2). Remarkably, the amount of soluble protein-bound S was significantly higher in s1c2 when compared with sir1-1 (Fig. 3D; P < 0.001 using Student’s t test), strongly suggesting that decreased GCL activity allowed more free Cys to be translated into protein in the s1c2 plants.

Figure 3.

Figure 3.

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Total S, C, glucosinolate, and protein-bound S in leaves of wild-type (WT), cad2-1, sir1-1, and s1c2 plants. A to C, Total S (A), C (B), and glucosinolate (C) contents were determined from rosette leaves of 8-week-old plants. D, Quantification of total protein-bound S in soluble leaf protein. Bars represent means ± se (n = 5). Different letters indicate statistically significant differences among the result groups (Holm-Sidak one-way ANOVA, P ≤ 0.05). Asterisks indicate statistically significant differences between two single genotypes using Student’s t test (***, P ≤ 0.001). FW, Fresh weight.

GCL Activity Controls the Flux of Sulfate Reduction and Partitioning of Cys between Translation and GSH Synthesis

To provide direct evidence of the repartitioning of Cys from GSH synthesis to protein synthesis in the s1c2 mutant, we determined fluxes of S in all mutants by feeding with radioactive tracers.

Leaves from the wild type, cad2-1, sir1-1, and the s1c2 double mutant were treated with [35S]sulfate to determine the incorporation rates of S into GSH and proteins after the reduction of oxidized sulfate and the assimilation of S into Cys (Fig. 4). Incorporation rates of de novo-fixed S into protein or GSH were similar in the wild type under the applied experimental conditions (Fig. 4, B and C). As shown previously by Khan et al. (2010), the total incorporation of S is decreased dramatically in sir1-1 compared with the wild type (Fig. 4D), as a result of the lowered sulfate assimilation and incorporation into Cys (Fig. 4A). This limitation resulted in a 15-fold decrease of S incorporation into GSH in sir1-1, while S incorporation into proteins was less than 3-fold affected. We conclude that, under the limitation of sulfate assimilation, the recycling of the GSH pool is decreased sharply to favor protein biosynthesis.

Figure 4.

Figure 4.

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Incorporation of 35SO42− into low-molecular-weight thiols and proteins in wild-type (WT), cad2-1, sir1-1, and s1c2 leaves. Leaf discs of 7-week-old soil-grown wild-type and mutant plants were incubated with one-half-strength Hoagland solution containing 225 nm H235SO4 (1.5 mCi nmol−1; Hartmann Analytic) for 40 min. A and B, Incorporation of 35S into Cys (A) and GSH (B) was determined by scintillation counting after extraction of metabolites and separation by reverse-phase HPLC. C, Incorporation of 35S into protein was quantified after selective protein precipitation. D, Total sum of the incorporated 35S label in Cys, GSH, and proteins. Bars represent means ± se (n = 5). Different letters mark statistically significant differences between each genotype (Holm-Sidak one-way ANOVA, P ≤ 0.05). Asterisks indicate statistically significant differences between two single genotypes using Student’s t test (*, P ≤ 0.05; **, P ≤ 0,01; and ***, P ≤ 0.001). FW, Fresh weight.

The cad2-1 mutant displayed 3-fold less incorporation of 35S into GSH when compared with the wild type (Fig. 4B), which is in agreement with the strongly down-regulated GCL activity in cad2-1 leaves (Cobbett et al., 1998). Consequently, 35S-labeled Cys accumulated significantly and was more efficiently used for incorporation into proteins in cad2-1 compared with the wild type (Fig. 4, A and C; P < 0.05). Interestingly, the decrease of GCL activity in cad2-1 resulted in a minor increase of total 35S incorporation (P < 0.05), which must be due to the activation of sulfate reduction in leaves (Fig. 4D). The s1c2 mutant also displayed an increase of total 35S incorporation and a significant repartitioning of S from GSH synthesis to protein synthesis when compared with sir1-1 (Fig. 4, B–D; P < 0.05). Both factors contribute to the remarkable increase of protein-bound S in s1c2 and the lower accumulation of sulfate in s1c2 when compared with sir1-1 (Figs. 2D and 3D).

Decreased GCL Activity in the s1c2 Mutant Rescues TOR Inhibition in the sir1-1 Mutant

We recently showed that decreased protein translation in sir1-1 is due to specific down-regulation of the Glc-TOR axis causing decreased ribosome biogenesis (Dong et al., 2017). To explain the significant increase of 35S incorporation into proteins of s1c2 when compared with sir1-1 (P < 0.05), we determined the total amino acid level and the TOR activity by immunological detection of ribosomal protein S6 kinase (S6K) phosphorylation in the wild type and the mutants (Wullschleger et al., 2006; Xiong and Sheen, 2014). While none of the mutants displayed significantly altered levels of total amino acids (Supplemental Fig. S3; P > 0.05), the phosphorylation of S6K at Thr-449 was enhanced in s1c2 when compared with sir1-1 (Fig. 5A). This higher S6K phosphorylation status explains the higher abundance of total rRNAs in s1c2 when compared with sir1-1 (Fig. 5B; Supplemental Fig. S4), since S6K phosphorylation induces the transcription of the rDNA (Kim et al., 2014).

Figure 5.

Figure 5.

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Decrease of GCL activity causes the activation of TOR kinase. A, In vivo TOR kinase activity in leaves of 7-week-old wild-type, cad2-1, sir1-1, and s1c2 plants was determined by immunological detection of the phosphorylation status of the bona fide TOR substrate S6K with a phospho-specific antiserum. Detection of the total S6K level with a polyclonal antiserum and total protein by Coomassie Brilliant Blue staining served as a control. B, Ethidium bromide staining of total RNA extracted from plants analyzed in A. C, Visualization of meristematic activity by staining of roots from 2-week-old seedlings grown on one-half-strength Murashige and Skoog medium with 5-ethynyl-2′-deoxyuridine for 30 min. Bar = 75 µm. D, Phenotypes of wild-type, cad2-1, sir1-1, and s1c2 seedlings. Bar = 1 cm.

The enhanced TOR activity also might contribute to the faster growth of s1c2 when compared with sir1-1 (Fig. 1A), since TOR controls the apical meristematic activity of shoots (Pfeiffer et al., 2016) and roots (Xiong et al., 2013). We thus quantified cell division in the meristematic zone of wild-type and mutant roots by short-term staining with 5-ethynyl-2′-deoxyuridine (Fig. 5C). As shown previously, sir1-1 displays lower meristematic activity when compared with the wild type, which explains its slow growth phenotype (Dong et al., 2017). The root meristem activity of the cad2-1 mutant was indistinguishable from that of the wild type. However, when combined with sir1-1 in the s1c2 mutant, the cad2-1 mutation partially rescues the depleted meristematic activity of sir1-1. This TOR-induced activation of s1c2 root meristems provides a mechanistic explanation for the faster growth of s1c2 roots when compared with sir1-1 roots (Fig. 5D).

Decreased GCL Activity Causes Enhanced Oxidation of the Plastids and the Cytosol

The decrease of GCL activity in cad2-1 and the s1c2 double mutant resulted in repartitioning of S flux from GSH to protein synthesis when compared with the wild type and sir1-1, respectively. In both cases, the total incorporation of 35S also was enhanced by the lowered GCL activity. To provide a functional explanation for the significant increase of sulfate assimilation in both mutants with decreased GCL activity (P < 0.05), we immunologically determined the steady-state level of APR2, the key determinant of sulfate reduction rate in Arabidopsis (Arabidopsis thaliana; Loudet et al., 2007). No relevant alteration of APR2 content was detectable in any of the mutants (Fig. 6A). Since the activities of APR1, APR2, and APR3 are localized in plastids, and at least APR1 is known to be regulated by the redox environment (Bick et al., 2001), we tested the redox state of plastids in all mutants with the genetically encoded redox sensor redox-sensitive green fluorescent protein2 (roGFP2; Figs. 6, B–G, and 7). Despite the strong decrease of sulfate assimilation in sir1-1, the plastid GSH redox state of sir1-1 was unaffected in protoplasts derived from leaves and in intact roots of seedlings grown on one-half-strength Murashige and Skoog medium (Figs. 6G and 7). This finding is in agreement with the determined GSH levels of sir1-1 (Fig. 2A; Khan et al., 2010). In contrast, cad2-1 and s1c2 displayed a more oxidized redox environment, as expected from the significantly lower steady-state levels of GSH in leaves of both mutants (P < 0.05). The plastids of s1c2 were even more oxidized than cad2-1 plastids, due to the combination of decreased sulfate assimilation and decreased GCL activity in s1c2 (Fig. 6G). The oxidation of the GSH pool in plastids of cad2-1 and s1c2 was independently confirmed in intact roots after stable transformation of the roGFP2 probe (Fig. 7).

Figure 6.

Figure 6.

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The plastid GSH redox pool is more oxidized in protoplasts of s1c2. A, Immunological detection of APR2 using a polyclonal antibody against Arabidopsis APR2 in leaf protein extracts of wild-type (WT), cad2-1, sir1-1, and s1c2 plants. An APR2 knockout mutant (apr2-ko) served as a negative control. Equal loading was confirmed by staining the large subunit (LSU) of Rubisco protein in the same samples. B to F, Confocal images of representative leaf protoplasts from 6-week-old wild-type, cad2-1, sir1-1, and s1c2 plants that transiently express a plastid-targeted Grx1-roGFP2 sensor. B, Bright-field images. C, Chlorophyll autofluorescence. D, roGFP2 fluorescence excited at 488 nm. E, roGFP2 fluorescence excited at 405 nm. F, False-color images of the corresponding calculated 405/488-nm ratios (blue = reduced, red = oxidized). The ratio range has been adjusted to match the maximum change in roGFP2 fluorescence in the analyzed samples and does not represent the full dynamic range of the sensor. Bars = 20 µm. G, Quantification of the ratiometric roGFP2 fluorescence. Bars represent means ± se (n = 14–20). Different letters indicate statistically significant differences among the result groups (Holm-Sidak one-way ANOVA, P ≤ 0.05).

Figure 7.

Figure 7.

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The plastid GSH redox pool is more oxidized in roots of s1c2. A, False-color ratio images (405 nm/488 nm) calculated from confocal images of 1-week-old wild-type (WT), cad2-1, sir1-1, and s1c2 plants expressing a plastid-targeted Grx1-roGFP2 sensor (blue = reduced, red = oxidized). The ratio range has been adjusted to match the full dynamic range of the sensor. The wild type was treated with 10 mm DTT for full reduction and with 5 mm DPS (dipyridyl disulfide) for full oxidation. Bar = 20 µm. B, Quantification of the ratiometric roGFP2 fluorescence. Bars represent means ± se (n = 8–10). Different letters indicate statistically significant differences among the result groups (one-way ANOVA with Tukey’s multiple comparisons test, P ≤ 0.05).

Next, we tested the consequences of decreased GSH formation in plastids for the redox milieu of the cytosol and the nucleus in leaf epidermal and hypocotyl cells (Fig. 8; Supplemental Fig. S5). As expected, depletion of GCL activity caused significant oxidation of the nucleus and the cytosol in pavement cells of cad2-1 and s1c2 (P < 0.05). Similarly, the cytosol of hypocotyl cells suffering from depleted GCL activity was more oxidized. The sharp decrease of S flux into GSH in the sir1-1 mutant had only a minor impact on the nuclear and cytosolic redox milieu of pavement cell plants and no effect on the cytosolic redox status of hypocotyl cells (Supplemental Fig. S5).

Figure 8.

Figure 8.

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Inhibition of plastid-localized GCL and SiR causes oxidation of the cytosolic GSH redox pool. A to E, Confocal images of representative epidermal cells from 3-week-old wild-type (WT), cad2-1, sir1-1, and s1c2 plants expressing a cytosol-targeted Grx1-roGFP2 sensor. The ratio range has been adjusted to match the full dynamic range of the sensor. Bars = 30 µm. A, Bright-field images. B, Chlorophyll autofluorescence. C, roGFP2 fluorescence excited at 488 nm. D, roGFP2 fluorescence excited at 405 nm. E, False-color images of the corresponding calculated 405/488-nm ratios (blue = reduced, yellow = oxidized). F, Quantification of the ratiometric roGFP2 fluorescence. Bars represent means ± se (n = 20–50). Different letters indicate statistically significant differences among the result groups (one-way ANOVA with Tukey’s multiple comparisons test, P ≤ 0.05).

DISCUSSION

Repartitioning of Cys under Limited S Assimilation

Here, we show that decreased GCL activity in the cad2-1 mutant increases the flux of de novo-assimilated sulfate into proteins. At first glance, this finding appears obvious, since it is apparently explained by the lower requirement of Cys for GSH synthesis in cad2-1, which, in turn, can be used for protein translation. However, this intuitive statement is not the explanation. The cad2-1 mutation promotes de novo fixation of S in proteins by two independent mechanisms: (1) activation of sulfate assimilation, and (2) activation of translation.

Activation of S Assimilation by Decreased GCL Activity

The function of GSH as a ROS scavenger places it in the first line of defense against many environmental stresses. Consequently, GSH biosynthesis and its precursor supply are under the significant control of diverse stresses (Foyer and Noctor, 2011; Noctor et al., 2012). Under sulfate deprivation, a decreased GSH level was reported to trigger the transcription of plasmalemma-localized high-affinity sulfate transporters (Lappartient and Touraine, 1997). The importance of this demand-driven regulation is still controversial, since other signal molecules like OAS, sulfate, and sulfide also are affected by sulfate deficiency. Furthermore, an increase of GSH levels by the overexpression of GCL does not alter the transcription of S-assimilation genes (Buchner et al., 2004; Hartmann et al., 2004). Consequently, it is unclear if the low GSH level in cad2-1 and s1c2 serves as a demand signal and contributes to the activation of sulfate assimilation by enhancing sulfate uptake in roots. More likely is a direct activation of several enzymes in the S-assimilation pathway by oxidation of the plastid GSH pool in leaves of GCL-depleted plants.

Mechanistic insights into the regulation of S assimilation by the plastid redox milieu are provided at the atomic resolution by crystallization of plastid-localized ATP sulfurylase (ATPS) and adenosine-5′-phosphosulfate (APS) kinase (Ravilious et al., 2012; Herrmann et al., 2014). ATPS catalyzes the first step of the S-assimilation pathway and is activated under oxidative conditions to enhance the flux of S for efficient Cys and GSH synthesis (Herrmann et al., 2014; Jez et al., 2016). In contrast, oxidation activates APS kinase (Ravilious et al., 2012). Since APS kinase produces 3′-phosphoadenosine-5′-phosphosulfate (PAPS), the universal sulfate donor for diverse sulfation reactions in secondary S metabolism, its inactivation directs the flux of S into primary S metabolism, facilitating Cys biosynthesis (Mugford et al., 2009). APR competes with APS kinase for their common substrate APS at the first branch of the S-assimilation pathway. Remarkably, oxidizing conditions promote the activity of APR isoform 1 in vitro and total APR activity in vivo (Bick et al., 2001). These regulatory mechanisms define the first branching point of the assimilatory sulfate reduction as a hub for redox regulation in plastids.

Similar to sulfate assimilation, GSH synthesis is redox regulated by two thiol switches in GCL, which induce homodimerization and activate catalysis upon oxidation (Hothorn et al., 2006; Hicks et al., 2007). The above-described redox switch network will promote S assimilation for GSH synthesis under oxidizing conditions to maintain a suitable redox environment under diverse biotic and abiotic stresses that impinge on the GSH pool (Foyer and Noctor, 2011; Noctor et al., 2012; Chan et al., 2013). At the same time, it allows sufficient formation of PAPS under nonstressed conditions when GSH is in an almost entirely reduced state, due to the activity of GSH reductases and the NADPH-dependent thioredoxin system (Marty et al., 2009). The relevance of redox stress-induced resource allocation between primary and secondary S metabolism is reinforced by the finding that the by-product of sulfation reactions, 3′-phosphoadenosine 5′-phosphate (PAP), serves as a crucial retrograde signal in response to high-light and drought stress (Estavillo et al., 2011). Indeed, the PAP-degrading enzyme SAL1 (EC 3.1.3.31) also is redox regulated and controls, via retrograde signaling, the expression of nuclear genes controlling the plastid redox environment (Chan et al., 2016).

Despite the manifold and independent proofs for direct redox regulation of enzymes catalyzing the biosynthesis of Cys and GSH, the activation of transcription by decreased GSH level and loss-of-GSH feedback inhibition of GCL may contribute to the observed activation of GSH biosynthesis in s1c2 (Schnaubelt et al., 2015).

Activation of Translation by Decreased GCL Activity

S assimilation must be tightly controlled in plants due to the toxicity of sulfide (Birke et al., 2012, 2015b) and the essential function of sulfide as a reduced S donor for Cys synthesis in all subcellular compartments where protein synthesis occurs (Birke et al., 2013, 2015a). In this study, we provide direct evidence for the relevant coregulation of protein synthesis and GSH synthesis and uncover TOR as the mediator of decreased GCL activity to the translation machinery. TOR kinase is an essential integrator of diverse nutrient and stress signals and regulates ribosome biogenesis, translation reinitiation, stem cell activity, and lifespan in plants (Ren et al., 2012; Schepetilnikov et al., 2013; Xiong et al., 2013; Pfeiffer et al., 2016). TOR is regulated in response to osmotic stress, internal carbohydrate levels, or external nutrient supply (Mahfouz et al., 2006; Ren et al., 2012; Xiong et al., 2013). We recently showed that decreased flux through the S-assimilation pathway inactivates TOR (Dong et al., 2017), which is in full agreement with the observed activation of TOR in GCL-depleted plants displaying a high rate of S assimilation here. However, the mode of TOR activation in GCL-depleted plants remains unclear. Potential signals for the activation of TOR in GCL activity-depleted plants are enhanced plastid sulfide levels or the oxidized cytosol. Indeed, the cytosol is more oxidized in cad2-1 (Meyer et al., 2007), and oxidizing conditions stabilize the Saccharomyces cerevisiae TOR protein due to the formation of a disulfide bridge in its FATC domain (PFAM domain accession PFO02260). The disulfide bridge-forming Cys residues of yeast TOR are fully conserved in TOR kinases from human, Drosophila melanogaster, Caenorhabditis elegans, and Arabidopsis and can be oxidized by oxidized glutathione in vitro (Dames et al., 2005). The determined redox potential of the FATC domain of yeast TOR is −230 mV (Dames et al., 2005), suggesting a relevant oxidization of AtTOR by the cytosolic redox environment of GCL activity-depleted plants (Fig. 8; wild type, −310 mV, pad2-1, −275 mV; Meyer et al., 2007). The phytoalexin defective2-1 mutant is an allele of cad2-1 with a similar reduction of GCL activity and phenotype (Dubreuil-Maurizi et al., 2011). However, our data do not exclude the possibility of TOR activation by other mechanisms pleiotropically induced by the cad2-1 mutation. Potential mechanisms might be an alteration of auxin distribution or activation of TOR upstream kinases (Xiong et al., 2013). A mechanistic link between TOR and the levels of ROS/NADPH has been suggested previously due to the significant oxidation of the GSH pool after the inhibition of TOR activity by rapamycin treatment (Ren et al., 2012). Furthermore, TOR activity controls the transcription of genes encoding for GSH synthesis and sulfate assimilation (Xiong et al., 2013). Stabilization of TOR by oxidizing conditions would act as a negative feedback to maintain TOR activity and induce GSH synthesis genes to cope with the stress-induced oxidation of the cytosol. The mammalian TOR kinase also is activated upon the addition of Cys oxidants, but in this case, the TOR activation is dependent on the regulatory axis consisting of Tuberous Sclerosis Complex1 (TSC1)/TSC2 and the small GTPase Ras Homolog Enriched in Brain (Yoshida et al., 2011).

Furthermore, it is highly conceivable that cytosolic translation is regulated directly by ROS and reactive nitrogen species that may add specific feedback inputs under diverse environmental stresses. These direct feedback mechanism may include S-nitrosylation, S-glutathionylation, S-sulfenylation, S-sulfinylation, and S-sulfonylation of Cys residues in diverse proteins of the translation machinery (for review, see Moore et al., 2016). Consequently, the application of different stresses may result in particular adaptations of the cytosolic translation machinery and may differ from the response observed here of the translation machinery to the oxidized GSH redox potential in the absence of stress.

CONCLUSION

Our study provides mechanistic insights into the reallocation of S between GSH biosynthesis and protein translation. We identified the activation of the sensor kinase TOR as one trigger for the up-regulation of translation by the induction of rRNA transcription in plants with decreased GSH biosynthesis. The reactivation of TOR also reestablished meristem activity in the s1c2 double mutant, which offers a functional explanation for the observed cad2-1-induced growth-promoting effect in s1c2 when compared with sir1-1. The results provide further support for the substantial control of TOR on plant growth. Depletion of GCL activity was associated with significant oxidation of the plastid and the cytosolic GSH pools. The oxidation of the plastid GSH pool significantly induced sulfate assimilation and, consequently, the flux of S into Cys. This finding is mechanistically well explained by the numerous redox switches in critical enzymes of the primary and secondary S metabolism. It is conceivable but not proven yet that the oxidation of the cytosolic GSH pool in cad2-1 and s1c2 triggers the activity of plant TOR in a similar way to that shown for yeast TOR. Future work is required to address this hypothesis, which opens new avenues for TOR to control metabolism under diverse stress situations.

MATERIALS AND METHODS

Growth Conditions and Phenotype Determination

All experiments were performed using Arabidopsis (Arabidopsis thaliana) ecotype Columbia-0 as the wild-type control. Seeds for sir1-1 (GABI-Kat line 550A09; ∼30% remaining SiR activity) and cad2-1 plants (identified by Cobbett et al. [1998]; 40% remaining GCL activity) were obtained from the SALK collection (Salk Institute Genomic Analysis Laboratory). All seeds were verified for homozygosity of the corresponding T-DNA insertion with primers described by Khan et al. (2010) and Cobbett et al. (1998). Seeds were stratified on soil for 3 d at 4°C in the dark and transferred for germination to climate chambers. After 2 weeks, seedlings were transferred to individual pots and grown for another 6 weeks under short-day conditions (8.5-h light period) at 22°C during the light period and 18°C at night. The light intensity was set to 100 µE m−2 s−1, and the relative humidity was kept at 50%.

Determination of Metabolites

Leaf material was harvested 2 h after the onset of light and snap frozen in liquid nitrogen. The material was ground to a fine powder before extraction. Extraction, derivatization, separation, and quantification of thiols and OAS were performed as described by Heeg et al. (2008) in cooperation with the Metabolomics Core Technology Platform Heidelberg. Anions were extracted from around 0.1 g of leaf material with 300 µL of distilled, deionized water at 98°C for 30 min under constant shaking and determined according to Forieri et al. (2017). Glucosinolates were separated by HPLC and determined by UV light absorption as described by Burow et al. (2006) after extraction of 0.1 g of leaf material with 1 mL of 80% (v/v) methanol solution containing 0.05 mm intact 4-hydroxybenzylglucosinolate as an internal standard. Determination of anthocyanins followed the procedure described by Speiser et al. (2015).

Analysis of Total C, N, and S Contents

Total C, N, and S contents of 8-week-old Arabidopsis rosettes were determined using a Vario MAX CNS elemental analyzer (Elementar). After complete drying of the entire rosettes at 120°C for 16 h, the material was ground to fine powder. The elements were detected in the forms of CO2, N2, and SO2 by means of their thermal conductivity from 20 mg of material and quantified according to a standard calibration curve prepared with sulfadiazine.

Determination of Protein-Bound S

Total soluble proteins were extracted according to Heeg et al. (2008), with the exception that DTT and PMSF were omitted to avoid contamination of proteins with external S. The soluble proteins were separated from low-molecular-weight compounds by gel filtration with Ilustra NAP-10 columns (GE Healthcare Life Sciences) according to the manufacturer’s protocol. The resulting protein solution was freeze dried, and 10 mg of powder was used for analysis of total S content.

RNA Extraction and Analysis of Transcript Levels

Total RNA was extracted from 50 mg of frozen root material using the peqGOLD Total RNA Kit (peqGOLD) according to the manufacturer’s instructions. Reverse transcription quantitative PCR (RT-qPCR) analysis was performed using the qPCRBIOSyGreen Mix Lo-ROX (PCR Biosystems) in the Rotor-Gene Q cycler (Qiagen). Gene-specific primers used for RT-qPCR are listed in Supplemental Table S1.

Quantification of S Flux in Different Pools

Plants were grown on soil under short-day conditions for 7 weeks prior to analysis. Leaf discs were incubated for 40 min with one-half-strength Hoagland solution containing 225 nm 35S-labeled sulfate (specific activity, 1.5 mCi nmol−1). Quantification of incorporated 35S label in metabolites and proteins was performed as described by Heeg et al. (2008).

Determination of TOR Activity

For immunological detection of S6K phosphorylation, total soluble proteins were extracted from 50 mg of leaves with 0.25 mL of 2× Laemmli buffer supplemented with 1% phosphatase inhibitor cocktail 2 (Sigma). Proteins were denatured for 5 min at 95°C, separated by 10% SDS-PAGE, and subsequently transferred to nitrocellulose membranes. All antibody dilutions were prepared in 1× TBS-T buffer containing 5% BSA. The primary antibodies anti-S6k-p (Phospho-p70 S6 Kinase [p-Thr-389]; Cell Signaling, no. 9205; 1:5,000) and anti-S6K1/2 (Agrisera, no. AS1-1855; 1:5,000) were detected using the horseradish peroxidase-conjugated secondary antibody (Promega, no. W4011; 1:30,000). The signal was detected with the SuperSignal West Dura Extended Duration Substrate (Thermo Scientific, no. 34075). Signal intensities were quantified with Image Quant LAS 4000 software version 1.21 (GE Healthcare Life Sciences) after normalization to the loading control.

Staining of Meristematic Activity

Seven-day-old seedlings were grown on AT medium [2.5 mm Ca(NO3)2∙4H2O, 5 mm KNO3, 2 mm MgSO4∙6H2O, 2.5 mm KH2PO4, 50 μm Fe-EDTA, 70 μm H3BO3, 14 μm MnCl2∙4H2O, 1 μm ZnSO4∙7H2O, 0.5 μm CuSO4∙5H2O, 0.02 μm Na2MoO4∙2H2O, 0.01 µm CoCl2∙6H2O, pH 5.8 adjusted with KOH, and 0.6% agar] in short-day conditions. For staining of dividing cells, 5 µL of staining solution (1 μm 5-ethynyl-2′-deoxyuridine in liquid AT medium) was added to the root tips and incubated for 30 min. Next, the seedlings were fixed in 0.1 mL of 4% formaldehyde and 0.1% Triton X-100 in 1× PBS (137 mm NaCl, 2.7 mm KCl, 10 mm Na2HPO4, and 1.8 mm KH2PO4) for 30 min. After fixation, seedlings were incubated in the dark with 0.1 mL of Click-iT reaction cocktail (prepared according to the manufacturer’s protocol, Click-iT EdU Alexa Fluor 488 Imaging Kit; Invitrogen). The seedlings were washed with 1× PBS buffer two times and analyzed by a Leica DM IRB epifluorescence microscope with a FITC/GFP filter (AlexaFluor 488: excitation, 495 nm; emission, 519 nm). Images were recorded with a Leica DFC350 FX camera.

In Vivo Redox Imaging of Grx1-roGFP2

The isolation and transfection of mesophyll protoplasts were performed according to Yoo et al. (2007) with minor modifications. Instead of cutting the leaves in strips, the lower epidermis was peeled off with adhesive tape. The remaining leaves were incubated in enzyme solution for 1 h without vacuum infiltration. The cytosol-targeted and plastid-targeted Grx1-roGFP2 described by Schwarzländer et al. (2008) were used for Agrobacterium tumefaciens-mediated transformation of Arabidopsis by the floral dip method or by polyethylene glycol-mediated transfection of protoplasts. Confocal laser scanning microscopy and image processing of protoplasts and roots were performed according to Meyer et al. (2007).

Statistical Analysis

Statistical analysis was performed using the software SigmaPlot 12.5 (Systat). Different data sets were analyzed for statistical significance with ANOVA followed by the Student-Newman-Keuls or Keuls posthoc test. Different letters in the figures indicate significant differences (P < 0.05).

Accession Numbers

The double mutant s1c2 generated here is a cross of the accessions sir1-1 (GABI-Kat line 550A09) and cad2-1 (accession no. AF068299).

Supplemental Data

The following supplemental materials are available.

Dive Curated Terms

The following phenotypic, genotypic, and functional terms are of significance to the work described in this paper:

Acknowledgments

We thank the Metabolomics Core Technology Platform Heidelberg, funded by the DFG Excellence initiative, for excellent support during metabolite analysis.

Footnotes

1

Y.D. was supported by the Schmeil Foundation. S.A.K.B. received a scholarship from the Higher Education Commission of Pakistan and the Agricultural University Peshawar (grant number 360/SIBGE). Selected aspects of this work were supported by funds from the Deutsche Forschungsgemeinschaft: ME15679-1 to A.J.M.; HE1848/14-1, HE1848/15-1, and HE1848/16-1 to R.H.; and WI3560/1-1 and WI3560/2-1 to M.W.

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