The impact of serine acetyltransferases and their product O-acetylserine on the sulfur deficiency response in Arabidopsis thaliana

Plants assimilate sulfur by the assimilatory sulfate reduction pathway. The final step of this pathway is the incorporation of sulfide into the carbon backbone O-acetylserine (OAS) by O-acetylserine(thiol)lyases (OAS-TL), resulting in cysteine. OAS production is performed by three major isoforms of the serine acetyltransferases (SERAT), which form cysteine synthase complexes (CSC) with the OAS-TL. During sulfur deficiency, sulfide levels decrease, OAS levels increase, and the CSCs dissociate. Furthermore, the genes encoding two additional isoforms of SERAT subgroup three are upregulated with unknown functions. This work investigates the potential involvement of these factors in the sulfur deficiency response. Previous studies suggested that the cytosolic CSC (cCSC), composed of SERAT1;1 and OAS-TLA, senses sulfur deficiency and controls the transcriptional response toward this stress in the model plant Arabidopsis thaliana. Although transcriptional delays observed in loss-of-cCSC mutants during earlier studies were not confirmed under the here applied experimental conditions, transcriptome analyses with newly generated oastlA and serat1;1 mutants using CRISPR/Cas9 indicate a possible involvement of the cCSC in balancing primary energy metabolism with immune responses under control condition. A previous study suggested the interaction of SERAT3s with SERAT1;1 in planta, especially in response to sulfur deficiency. The interaction of recombinant purified SERAT3;2 with SERAT1;1 was verified in vitro and resulted in the formation of hetero-hexamers with reduced OAS-TL A binding capacity. The in vivo analysis of a SERAT3;2-mCherry fusion protein verified its predominantly cytosolic localization and uncovered an unexpected localization in the nucleus, indicating independent functions of SERAT3;2, since SERAT1;1 is exclusively localized in the cytosol. The SERAT3;2 minimal native promoter composed of 460 bps upstream of the start codon was found to contain a SURE core motive in the 5’ UTR of the mRNA and was shown to induce the SERAT3;2-mCherry expression under sulfur deficiency and in a cell type specific manner. While the double mutant serat3;1s3;2 (serat3;1 x serat3;2) did not display any visible phenotype, mRNA sequencing of leaves from serat3;1s3;2 uncovered activation of the OAS gene cluster, mimicking a pre-induced sulfur deficiency response in control condition and over-induced response when cultured with limited sulfur supply. My findings demonstrate a generally repressive function of SERAT group three in regulating sulfur deficiency-related genes. Furthermore, I analyzed the impact of CSCs in different subcellular compartments on Arabidopsis developmental plasticity under sulfate limitation. OAS was identified as a key signal for long-term adaptation to sulfur deficiency, promoting an increased root-to-shoot FW ratio. OAS functioned independently of CSCs or its function as cysteine precursor because cysteine or glutathione could not complement the OAS-limiting phenotype in the triple knockout mutant serat tko, lacking all three major SERAT isoforms. Glutathione was shown to be crucial for the maintenance of primary root length and root FW in sulfur deficiency. The sulfur-specific transcription factor SLIM1 was found to be involved in the signaling response, probably working downstream of OAS. In conclusion, these findings highlight the diverse functions of the SERAT protein family in primary sulfur metabolism. While SERAT isoforms of groups one and two generate OAS as a cysteine precursor in non-stress conditions, OAS accumulation in sulfur deficiency is a central signal initiating long-term physiological adaptation. SERATs of group three play a modulating role as transcriptional repressors on sulfur deficiency-induced genes, fine-tuning the response.