Arabidopsis S-sulfocysteine synthase activity is essential for chloroplast function and long-day light-dependent redox control

Abstract

In bacteria, the biosynthesis of Cys is accomplished by two enzymes that are encoded by the cysK and cysM genes. CysM is also able to use thiosulfate as a substrate to produce S-sulfocysteine. In plant cells, the biosynthesis of Cys occurs in the cytosol, mitochondria, and chloroplasts. Chloroplasts contain two O-acetylserine(thiol)lyase homologs, which are encoded by the OAS-B and CS26 genes in Arabidopsis thaliana. An in vitro enzymatic analysis of the recombinant CS26 protein demonstrated that this isoform possesses S-sulfocysteine synthase activity and lacks O-acetylserine(thiol)lyase activity. In vivo functional analysis of this enzyme in knockout mutants demonstrated that mutation of CS26 suppressed the S-sulfocysteine synthase activity that was detected in the wild type; furthermore, the cs26 mutants exhibited a reduction in size and showed paleness, but penetrance of the growth phenotype depended on the light regime. The cs26 mutant plants also had reductions in chlorophyll content and photosynthetic activity (neither of which were observed in oas-b mutants) as well as elevated glutathione levels. However, cs26 leaves were not able to properly detoxify reactive oxygen species, which accumulated to high levels under long-day growth conditions. The transcriptional profile of the cs26 mutant revealed that the mutation had a pleiotropic effect on many cellular and metabolic processes. Our findings reveal that S-sulfocysteine and the activity of S-sulfocysteine synthase play important roles in chloroplast function and are essential for light-dependent redox regulation within the chloroplast.

Figure 1.

Cys Biosynthesis Network and Subcellular Localization of SAT and OASTL Enzymes in Arabidopsis Cells. The biosynthesis of Cys can occur in any of three different cellular compartments: the cytosol, the chloroplast, and the mitochondria. The biosynthetic enzymes are represented by five different SAT isoforms and eight functional OASTL isoforms.

Figure 2.

Electrospray Ionization Mass Spectra of S-Cys in Positive Ionization Mode. (A) The theoretical mass spectrum and the molecular structure of S-Cys. (B) The mass spectrum of a standard solution of S-Cys. (C) The mass spectrum of the enzymatic reaction products produced by recombinant His-CS26 using thiosulfate and OAS as substrates

Figure 3.

Fragmentation Pattern of S-Cys Samples by Electrospray Ionization/MS in Positive Ionization Mode. (A) Mass spectrum of the S-Cys ion [M+H]⁺ isolated in the ion trap. (B) MS² fragmentation pattern of the parental ion at m/z 201.9. (C) MS³ fragmentation pattern of the parental ion at m/z 120.0. The arrow indicates the fragmented ion. (D) Details of the ionization and fragmentation of S-Cys samples and identification of the detected ions.

Figure 4.

Partial Alignment of the Chloroplastic OAS-B and CS26 Deduced Amino Acid Sequences. The alignment was created with the Vector NTI software to compare the proteins encoded by the cysK and cysM genes from E. coli and S. typhimurium. The blue arrow indicates the β-sheet domains deduced from the crystal structure of the cytosolic OAS-A1 from Arabidopsis (Bonner et al., 2005).

Figure 5.

Phenotypes of the Null Mutants cs26 and oas-b and the Complemented Line cs26:P35S-CS26. (A) Plants grown on soil under long-day (LD) or short-day (SD) conditions for 3 and 4 weeks, respectively. (B) Plants grown on soil under continuous light for 4 weeks.

Figure 6.

Pigment Contents and Photosynthetic Activity in Leaves of the Null Mutants cs26 and oas-b. (A) Chlorophylls a and b and carotenoid contents were measured in acetone extracts of leaves of the wild type and mutant plants grown under long-day conditions for 4 weeks. Values are means ± sd of three independent determinations. Asterisks indicate statistical significance of P < 0.01. (B) Photosynthetic oxygen evolution measured with an oxygen electrode from disks excised from leaves of wild-type and mutant plants grown under long-day conditions for 4 weeks. A representative experiment is shown. (C) Photosynthetic oxygen evolution measured with an oxygen electrode from disks excised from leaves of wild-type and mutant plants grown under short-day conditions for 5 weeks. A representative experiment is shown.

Figure 7.

Detection of Accumulation of ROS in the Leaves of the Null Mutant cs26. (A) and (B) Histochemical detection of the superoxide radical anion by staining with NBT chloride in the wild-type (A) and cs26 mutant (B) in leaves from 3-week-old plants grown in long-day conditions. (C) and (D) Histochemical detection of H₂O₂ by staining with DAB in the wild-type (C) and cs26 mutant (D) leaves from 3-week-old plants growing in long-day conditions. (E) and (F) Detection of the superoxide radical anion in the wild-type (E) and cs26 mutant (F) leaves from 5-week-old plants grown in short-day conditions. (G) and (H) Detection of H₂O₂ in the wild-type (G) and cs26 mutant (H) leaves from 5-week-old plants grown in short-day conditions. The experiments were repeated at least five times with similar results.

Figure 8.

SSCS Activities in Whole-Leaf and Chloroplast Extracts. SSCS activity was measured in whole-leaf (white columns) and chloroplast extracts (black columns) from the wild-type and the oas-b and cs26 mutant lines. Values are means ± sd of three independent determinations.

Figure 9.

Transcriptional Profile of the cs26 Mutant Grown under Long- and Short-Day Photoperiod. (A) Phenotypes of cs26 and wild-type plants grown with long-day (LD) and short-day (SD) conditions. (B) Venn diagram showing the number of genes that changed in expression in the cs26 mutant compared with wild-type plants in long-day (yellow circle) and short-day (blue circle) conditions. The intersection (green area) shows the number of genes that are common in both profiles. Blue arrows show the number of genes that are upregulated. Red arrows show the number of genes that are downregulated. (C) Gene Ontology categorization of biological processes of the genes that have modified expression in long-day (yellow bars) and short-day (blue bars) growth conditions.