Sensing and signaling of oxidative stress in chloroplasts by inactivation of the SAL1 phosphoadenosine phosphatase

Abstract

Intracellular signaling during oxidative stress is complex, with organelle-to-nucleus retrograde communication pathways ill-defined or incomplete. Here we identify the 3'-phosphoadenosine 5'-phosphate (PAP) phosphatase SAL1 as a previously unidentified and conserved oxidative stress sensor in plant chloroplasts. Arabidopsis thaliana SAL1 (AtSAL1) senses changes in photosynthetic redox poise, hydrogen peroxide, and superoxide concentrations in chloroplasts via redox regulatory mechanisms. AtSAL1 phosphatase activity is suppressed by dimerization, intramolecular disulfide formation, and glutathionylation, allowing accumulation of its substrate, PAP, a chloroplast stress retrograde signal that regulates expression of plastid redox associated nuclear genes (PRANGs). This redox regulation of SAL1 for activation of chloroplast signaling is conserved in the plant kingdom, and the plant protein has evolved enhanced redox sensitivity compared with its yeast ortholog. Our results indicate that in addition to sulfur metabolism, SAL1 orthologs have evolved secondary functions in oxidative stress sensing in the plant kingdom.

Keywords: chloroplast; drought stress; redox regulation; retrograde signaling; stress sensing.

Fig. 1.

In vivo AtSAL1 activity is down-regulated by oxidative stress. (A) In vivo AtSAL1 activity is down-regulated by oxidative stress with negligible change in protein abundance (WW, well watered; MD, middrought; LD, late drought; HL, high light; MV, methyl viologen; H₂O₂, hydrogen peroxide). Activity was measured without any reducing agent, whereas protein electrophoresis and Western blotting were performed under reducing conditions for optimal protein transfer from gel to membrane. Similar results were obtained from two independent experiments. Means and SE for three to four biological replicates per treatment are shown. a, b, and c (P < 0.05) show significant differences between treatments. (B) In vivo AtSAL1 activity is sensitive to redox state. Disrupting redox homeostasis at photosystem I water–water cycle, ascorbate detoxification of ROS, cellular redox buffers, or regeneration of oxidized proteins (also see Table S1) results in significantly greater (*P < 0.05, **P < 0.01) down-regulation of AtSAL1 activity compared with WT under high-light stress. Some mutants show a trend of down-regulation in activity, but the differences were not significant. Means and SE for averaged relative activities of two biological replicates at three different concentrations of PAP per genotype are shown.

Fig. 2.

Regulation of AtSAL1 activity by redox state via its intramolecular Cys167–Cys190 disulfide. (A) Down-regulation of AtSAL1 activity by oxidation requires oxidation of cysteines because mutagenesis of cysteines to alanine in AtSAL1 abrogated redox sensitivity. The redox sensitivity correlates with a band directly beneath the full-length reduced protein (black arrow), which was determined to be a Cys167–Cys190 intramolecular disulfide (C). Vertical dashed lines indicate splicing and truncation of the gel shown in full in C. *P < 0.1. Activities of all proteins were assayed in the presence of 13.4 µM PAP. Means and SE of two independent experiments are shown. (B) Formation of disulfides in AtSAL1 by oxidation is rapidly reversed by returning the redox state to reducing conditions. Vertical dashed lines indicate splicing of the gel to show these three samples side by side; all samples were run on the same gel. (C) Determination of Cys–Cys disulfide pairs observed in WT AtSAL1 using cysteine to alanine substitution mutants of AtSAL1 under oxidation. Oxidized AtSAL1 proteins migrate at different rates to reduced AtSAL1 protein. The different Cys–Cys disulfide pairs were identified by cross-comparison with cysteine mutants: AtSAL1 containing a Cys167–Cys190 intramolecular disulfide (black triangles) migrates closest to reduced AtSAL1. The oxidized form is absent in all AtSAL1 mutants lacking either or both of Cys167 and Cys190. Other combinations such as the Cys21–Cys167 and Cys21–Cys190 disulfide did not correlate with the down-regulation of AtSAL1 activity by oxidation (A). These are likely nonspecifically formed during protein denaturation and SDS/PAGE. AtSAL1 containing the Cys167–Cys190 intramolecular disulfide is the only oxidized AtSAL1 species detected in endogenous plant protein samples pretreated with iodoacetamide to block reduced cysteines during protein extraction to prevent nonspecific disulfide formation (Fig. 6). Experiments were performed twice, with identical results.

Fig. 3.

Structural basis for redox regulation of AtSAL1 activity. (A) (Left) Structural elucidation of AtSAL1 reveals a dimerization interface and three potentially redox-sensitive cysteine residues. (Middle) A view of the 2mFo-dFc map (blue lines, contoured at 1.0 σ) centered on Cys119 which is located at the interface between chain A (orange sticks) and chain B (green sticks) or a view of the 2mFo-dFc map centered on Cys167 and Cys190. (Right) The disulfide bonds present in an energy minimized model of the oxidized AtSAL1 dimer. (B) Closure of loop 1 of AtSAL1, as predicted by normal mode analysis (NMA) (31). The lowest-frequency normal mode is shown. Positions of C_α atoms are shown as colored spheres, from the crystal structure (blue) to the most closed conformation (red). (C) Dimerization and disulfide formation reduces the mobility of key loops (loops 1, 3, 4, and 8) in AtSAL1. Energy minimized models of the oxidized AtSAL1 dimer (Left) or the reduced AtSAL1 monomer (Middle) are colored according to mobility (blue indicating least mobile and red indicating most mobile); for details of energy minimization and normal mode analysis, see Materials and Methods. (Right) Plot of NMA mobility by residue for the oxidized dimer and reduced monomer.

Fig. 4.

AtSAL1 is also regulated via dimerization involving Cys119. (A) AtSAL1 in monomer–dimer equilibrium detected in size exclusion chromatography of purified recombinant protein. Dimers were detected in at least three independent purification runs. (Inset) SDS/PAGE of monomeric and dimeric AtSAL1 indicating the proteins were of similar purity. Monomer and dimer masses were confirmed by SEC–MALLS (Fig. S1). (B) The monomer–dimer equilibrium can be shifted by an intermolecular disulfide under oxidizing conditions, thus increasing dimer abundance, or reduction of the disulfide by DTT dissociating the dimer. Whereas DTT is sufficient to achieve dimer separation, GSH is not. This is consistent with the relative redox potentials of these compounds: −264 mV at pH 7.4 for GSH compared with DTT (−360 mV) and the redox potential of disulfide bonds (ranging from −330 to −95 mV in thiol–disulfide oxidoreductases). Oxidation (DTT_ox, GSSG) increased dimer abundance to 100%, indicating formation of an intermolecular disulfide dimer under oxidation. Reversing the oxidation with reductant (DTT_ox + DTT and GSSG + DTT lanes) that breaks disulfide bonds shifts the equilibrium to monomer. The low resolution and fuzzy appearance of the higher MW bands are likely due to the type of gel (Tris-Glycine), lack of detergent (SDS), or reductant (DTT) that inhibits resolving native, folded proteins that are oxidized. Similar results were obtained in two independent experiments. (C) Under redox titration by DTT_ox in vitro, which induces formation of the Cys167–Cys190 disulfide, only dimeric AtSAL1 showed significant down-regulation of activity. Identical results were obtained from two independent experiments. (Inset) Dimerization is required for formation of the Cys167–Cys190 intramolecular disulfide that regulates AtSAL1 activity.

Fig. S1.

AtSAL1 exists in a monomer–dimer equilibrium. Purified AtSAL1 was exchanged into assay buffer and analyzed by MALLS following separation by size exclusion chromatography. Differential refractive index is shown as a blue line (right axis), and molecular mass derived from light scattering data is shown as red points (left axis). The expected masses of AtSAL1 monomer (37.5 kDa) and dimer (75 kDa) are indicated by dashed lines.

Fig. S2.

Prediction of coupled motions in AtSAL1. Covariance maps of C_α motions (74) calculated for the reduced AtSAL1 monomer and oxidized AtSAL1 dimer. Residues with coupled motions are shown in red, and residues with anticorrelated motions are shown in blue. The positions of the mobile loops described in Fig. 3C (loops 1, 3, and 8) are indicated.

Fig. 5.

AtSAL1 can be regulated by glutathionylation at redox-sensitive cysteines. (A) Glutathionylation of AtSAL1 with oxidized glutathione (GSSG; yellow arrows) results in formation of the intramolecular C167–C190 disulfide (black arrows), presumably via the thiol–disulfide exchange mechanism (34). Identical results were obtained in two independent experiments. (B) Observed shift in mass consistent with cysteine glutathionylation in AtSAL1 treated with GSSG compared with untreated AtSAL1. A representative m/z spectrum for Cys119 is shown. Charge is indicated in brackets. (C) Both monomeric and dimeric AtSAL1 are sensitive to glutathionylation, with decrease in activity in redox titration with GSH/GSSG (a less negative potential is more oxidizing). The redox midpoint potential (E_m) was close to physiological GSH/GSSG redox potential of Arabidopsis chloroplasts (35). Although dimeric AtSAL1 activity only decreased to 40% under fully oxidizing conditions compared with 10% for monomeric AtSAL1, the basal activity of dimeric AtSAL1 is already significantly lower than monomeric AtSAL1 under the same redox state (Table 1). Measurements were performed twice.

Fig. S3.

Formation of an intramolecular disulfide via a thiol–disulfide exchange initiated by glutathionylation of a cysteine residue. See ref. for review of this mechanism.

Fig. 6.

AtSAL1 is redox-regulated via intramolecular disulfide formation and dimerization in vivo, and it is sensitive to the chloroplast redox state. (A) Down-regulation of AtSAL1 activity and concomitant PAP accumulation correlates with formation of the Cys167–Cys190 intramolecular disulfide (black triangles) in endogenous AtSAL1 during drought stress. Means and SE are shown for n = 4 biological replicates for well-watered and n = 3 for drought. In contrast to Fig. 1A, leaf protein extracts were blocked with iodoacetamide, and then protein electrophoresis and Western blotting were performed under nonreducing conditions to visualize the Cys167–Cys190 disulfide. Loading control was Coomassie Blue staining. Similar results were obtained in two independent experiments. (B) The monomer–dimer equilibrium of AtSAL1 in vivo is shifted in favor of the dimer during oxidative stress, suggesting formation of the Cys119–Cys119 intermolecular disulfide to stabilize the dimer. Total leaf protein pooled from four biological replicates per treatment was resolved on Native-PAGE and immunoblotted, and the relative quantities of dimeric to monomeric AtSAL1 were estimated by image analysis on ImageJ. WW, well watered; MD, middrought; LD, late drought; HL, high light; MV, methyl viologen; H₂O₂, hydrogen peroxide.

Fig. S4.

Cys–Ala mutations negatively affect AtSAL1 protein stability and abundance. The yield of soluble AtSAL1 protein is drastically decreased when the redox-sensitive Cys residues were mutagenized to Ala. The SDS/PAGE gel shows semipurified recombinant WT or mutated AtSAL1 proteins after a soluble protein fraction from 7 mL of induced E. coli cells was incubated with Ni-NTA beads in a 1.5 mL Eppendorf tube, washed with 20 mM imidazole, and the bound AtSAL1+6X His–Ub fusion proteins (black triangles) eluted with 250 mM imidazole. The abundance of soluble AtSAL1 protein decreased with increasing number of Cys–Ala substitutions. The negative effect of the Cys–Ala mutations was reproducible in two independent transformed bacterial colonies.

Fig. S5.

Conservation of redox-sensitive cysteines in SAL1 orthologs in plants. The Cys119 involved in dimerization of AtSAL1 is moderately conserved in eudicots and non-Poaceae monocot plants but is absent in other lineages. The intramolecular disulfide pair Cys167–Cys190 is invariant across bryophytes, lycophytes, primitive angiosperm, eudicots, and non-Poaceae monocots. Cys190 is C-terminally shifted by seven amino acids in Poaceae monocot plants. Additionally, another cysteine is strongly conserved in the Poaceae family of monocots (Cys221 in OsSAL1; Fig. 7). In contrast to plant SAL1 orthologs (green), the fungal SAL1 ortholog in Saccharomyces cerevisiae HAL2 (blue) lacks Cys119 and Cys190.

Fig. 7.

Biochemical and structural evidence for conservation of redox sensitivity in a rice SAL1 ortholog. (A) Redox titration on OsSAL1 shows that the protein is redox sensitive and has a redox midpoint potential (E_m) in the physiologically relevant range. A less negative potential is more oxidizing. (B) Oxidation of AtSAL1 and OsSAL1 with GSSG similarly result in glutathionylation of the proteins, increasing their apparent molecular weight when resolved on nonreducing SDS/PAGE (yellow triangles). Vertical dashed lines indicate splicing and truncation of the gel to remove additional lanes not relevant to this result. (C) Comparison between redox-sensitive cysteine residues detected in structures of AtSAL1 and modeling of OsSAL1. Unlike AtSAL1 which contains both surface-exposed and intramolecular disulfide cysteines, OsSAL1 is predicted to contain surface exposed cysteines (marked in yellow). Both Cys203 and Cys221 of OsSAL1 are strongly conserved in Poaceae SAL1 orthologs (Fig. S5).

Fig. 8.

Enhancement of redox sensitivity in yeast ScHAL2 by introduction of the AtSAL1 intramolecular disulphide. (A) Structural alignment-guided introduction of the intramolecular disulfide from AtSAL1 (orange) into yeast ScHAL2 (gray) by the Tyr176Cys mutation. Thiol groups are indicated in yellow. (B) Introduction of additional disulfide in ScHAL2+3C results in increased redox sensitivity in vitro compared with WT ScHAL2. Means and SE from two independent experiments for specific activity at 3.35 µM PAP are shown. For full results, see Table S3. Asterisks indicate significant differences (P < 0.05). (C) Introduction of additional disulfide in ScHAL2+3C increased redox sensitivity and PAP accumulation in vivo when expressed in yeast Δhal2 cells under mild oxidative stress. Significant differences are indicated by a and b (P < 0.05). Error bars indicate SE; n = 3 independent cultures for all experiments. n.s., no significant difference.

Fig. S6.

WT and engineered ScHAL2. (A) Presence of a surface-exposed Cys349 in the ScHAL2 crystal structure (1KA1), which may explain the redox-sensitive activity observed in Fig. 8B. (B) The WT ScHAL2 and ScHAL2+3C proteins are equally active in vivo when expressed in yeast cells deficient in ScHAL2 (Δhal2) because both proteins complement PAP levels in Δhal2 to similar levels, albeit still about 10-fold higher than WT. Similar results were obtained in two independent experiments.

Fig. 9.

SAL1 as an oxidative stress sensor in the chloroplast for regulation of PAP-mediated retrograde signaling. Oxidative stresses (drought and HL) invoke physiological and biochemical changes in chloroplasts, such as ROS accumulation and altered redox poise. These are sensed by SAL1 via redox-mediated inhibition of SAL1 activity, enabling PAP accumulation. Stress responses, including transcription of stress-responsive genes, are activated. ROS sensors may also control other HL retrograde signals, such as β-cyclocitral or MEcPP, or use stromules; their identity and interaction with SAL1-PAP remain to be determined (gray arrows).