Analysis of Cytosolic and Plastidic Serine Acetyltransferase Mutants and Subcellular Metabolite Distributions Suggests Interplay of the Cellular Compartments for Cysteine Biosynthesis in Arabidopsis

Abstract

In plants, the enzymes for cysteine synthesis serine acetyltransferase (SAT) and O-acetylserine-(thiol)-lyase (OASTL) are present in the cytosol, plastids and mitochondria. However, it is still not clearly resolved to what extent the different compartments are involved in cysteine biosynthesis and how compartmentation influences the regulation of this biosynthetic pathway. To address these questions, we analysed Arabidopsis thaliana T-DNA insertion mutants for cytosolic and plastidic SAT isoforms. In addition, the subcellular distribution of enzyme activities and metabolite concentrations implicated in cysteine and glutathione biosynthesis were revealed by non-aqueous fractionation (NAF). We demonstrate that cytosolic SERAT1.1 and plastidic SERAT2.1 do not contribute to cysteine biosynthesis to a major extent, but may function to overcome transport limitations of O-acetylserine (OAS) from mitochondria. Substantiated by predominantly cytosolic cysteine pools, considerable amounts of sulphide and presence of OAS in the cytosol, our results suggest that the cytosol is the principal site for cysteine biosynthesis. Subcellular metabolite analysis further indicated efficient transport of cysteine, gamma-glutamylcysteine and glutathione between the compartments. With respect to regulation of cysteine biosynthesis, estimation of subcellular OAS and sulphide concentrations established that OAS is limiting for cysteine biosynthesis and that SAT is mainly present bound in the cysteine-synthase complex.

Cysteine-Generated Sulfide in the Cytosol Negatively Regulates Autophagy and Modulates the Transcriptional Profile in Arabidopsis

“…Subcellular sulfide concentrations have been determined in plastids (125 mM) and in the cytosol (55 mM) (Krueger et al, 2009). In plants, sulfate reduction takes place in plastids (Takahashi et al, 2011), which is consistent with the large amount of sulfide in plastids.…”

Signaling in the plant cytosol: cysteine or sulfide?

“…The observation of the cytosol as the major site of Cys synthesis correlates well with the estimated Cys concentrations >300 and <10 µM in other cell compartments (Krueger et al 2009 ). However, Cys is a toxic molecule when it is present above a certain concentration threshold, due to the high reactivity of its thiol.…”

“…In Arabidopsis, the analysis of specific mutants of members of the SAT and OASTL family has demonstrated that mitochondria are the predominant source of OAS in vivo and that cytosol is the compartment where the major bulk of Cys synthesis takes place, while chloroplasts provide the sulfide via the assimilatory sulfate reduction (Haas et al 2008 ;Heeg et al 2008 ;Krueger et al 2009 ;Watanabe et al 2008a , b ). However, when a compartment is affected by mutations in any SAT or OASTL isoform, the other compartments are able to provide Cys for plant viability.…”

Sulfite Reductase Defines a Newly Discovered Bottleneck for Assimilatory Sulfate Reduction and Is Essential for Growth and Development in Arabidopsis thaliana

“…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 Watanabe et al, 2008aWatanabe et al, , 2008bKrueger 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 Kopriva, 2006).…”