Sulfur metabolism and chromium tolerance in the unicellular green alga Scenedesmus acutus: role of Serine Acetyl Transferase (SAT).

The sulfur metabolic pathway is essential for the maintenance of cellular redox balance and for the detoxi7ication of heavy metals through a process known as sulfur-enhanced defense (SED). Previous studies conducted in our laboratory have indicated a correlation between sulfate metabolism and chromium tolerance in two strains of the unicellular green alga Scenedesmus acutus, with different metal sensitivity: a wild type (wt) and a chromium tolerant strain (Cr-t). Both strains show an enhanced chromium tolerance after sulfur starvation, and the Cr-t shows higher sulfur content and consequently higher cysteine levels than the wt strain (Sardella et al. 2019). At the core of sulfur metabolism is the cysteine synthase complex (CSC), which is composed of a hexamer of Serine Acetyltransferase (SAT) and two dimers of O-acetyl-Serine (Thiol) Lyase (OAS-TL), and represents an enzymatic and regulatory hub present in different cell compartments (plastids, mitochondria and cytosol), coordinating sulfate assimilation and cysteine biosynthesis. Within the complex, SAT is active and produces O-acetylserine (OAS) while OASTL plays a regulatory role, becoming active as a free dimer in cysteine synthesis when the complex disaggregates. The CSC is stabilized by sul7ide and destabilized by its 7inal product, OAS, which acts as a positive feedback regulator on sulfate uptake/assimilation pathway (Wirtz et al. 2006). OAS production is thus crucial in two ways as OAS acts as a positive regulator and represents the substrate for cysteine production, which, conversely, has a role in negative feedback. SAT and OAS-TL activities differ in the three cell compartments in which CSC is present and while cysteine synthesis mainly occurs in the cytosol, OAS production is mainly located within mitochondria. Moreover, the isoforms involved in CSC formation in different cell compartments show different cysteine feedback sensitivity (Noji et al. 1998). To assess if SAT plays a role in determining the different Cr(VI) tolerance of the two strains, we analyzed the two SAT isoforms present in the S. acutus genome (SaSAT1 and SaSAT2). Our analysis revealed differences in the SaSAT C-terminal domain, thought to be responsible for binding to OASTL. Albeit no information could be found regarding their cell compartmentalization in algae, by inferring homology with the Arabidopsis thaliana 5 SAT isoforms, SaSAT1 likely represents the chloroplastic/mitochondrial isoform, and SaSAT2 the cytosolic one. The analysis of SaSAT1 and SaSAT2 expression after Cr(VI) exposure following a 7-day pre-culture in standard or sulfur-deprived medium indicates that both isoforms are inducible in response to sulfur deprivation and metal exposure. The expression of the two genes is, however, differently regulated in the two strains, which show different basal levels of SaSAT1 and different stress responsiveness for both SaSAT isoforms. In the wt, SaSAT1 seems insensitive to chromium stress and strongly activated by sulfur deprivation. In contrast, SaSAT2 transcription is enhanced by Cr(VI) in S-suf7icient condition, but sulfur deprivation overwhelms the effects of Cr(VI) exposure in S-replete condition. In the Cr-t strain, however, both genes are less responsive to sulfur deprivation than in wt and are induced by Cr(VI) exposure in both nutrient conditions. The higher Cr-t strain SaSAT1 expression levels in S-suf7icient condition suggests a higher OAS production in this strain, which may induce a precocious positive feedback regulation of sulfate assimilation pathway as sul7ide levels start to decrease and OAS begins to accumulate, thus allowing a greater sulfur uptake, a higher cysteine production and a prompter defense toward Cr(VI) toxicity.