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Abstract

In plants, the synthesis of cysteine occurs via a two-step process catalyzed by serine acetyltransferase (SERAT) and O-acetylserine(thiol)lase (OAS-TL) enzymes. The two enzymes can interact reversibly, thereby forming the cysteine synthase (CS) complex, which is present in the cytosol, mitochondria, and plastids. This research aimed to investigate the cytosolic and mitochondrial CS complexes in Arabidopsis thaliana to determine whether there are any compartment-specific differences in their regulation. Moreover, the significance of SERAT in the regulatory function of the CS complex and cysteine synthesis was explored. Therefore, a specific mutation in a conserved serine was implemented, localized in S102 and S210 in cytosolic AtOAS-TL A and mitochondrial AtOAS-TL C, respectively. The mutation had been previously studied in rice and represented a stable CS complex, which O-acetylserine (OAS) could not dissociate. Earlier findings have demonstrated that substituting Ser102 with Asn in the OAS-TL A protein results in complete inactivation of the corresponding mutant OAS-TL AS102N protein. The findings of this study also indicated a significant reduction in enzymatic activity of the OAS-TL CS210N compared to the wild-type OAS-TL C. Comparative in vitro analysis indicated that the CS complex composed of cytosolic AtOAS-TL AS102N and either cytosolic AtSERAT1;1 or mitochondrial AtSERAT2;2 displayed a notably higher level of resistance to OAS dissociation. Nonetheless, even though the interaction between mitochondrial AtOAS-TL CS210N and mitochondrial AtSERAT2;2 seemed inseparable, it was observed that mitochondrial AtOAS-TL CS210N was unable to establish a non-dissociable complex with cytosolic AtSERAT1;1. Upon subsequent in vivo analysis, it was observed that both oastla and wild-type (WT) plants, which were complemented with cytosolic AtOAS-TL AS102N , exhibited increased levels of OAS, cysteine, and glutathione. This finding confirms the formation of a stable CS complex, which results in constant stimulation of the SERAT within the complex. However, upon complementing the serat1;1 or a1;1 mutants with AtOAS-TL AS102N , no noticeable increase in the levels of OAS or thiols was observed in comparison to the control condition. The findings in the absence of SERAT1;1 confirm that it is indeed the CS complex formation through mutated AtOAS-TL AS102N within the cytosolic environment leads to the observed synthesis of OAS and thiols in the wildtype background. Moreover, upon investigation of the metabolite content in transformants that were complemented with mutated mitochondrial AtOAS-TL CS210N , it was discovered that both WT (AtOAS-TL CS210N ) and oastlc (AtOAS-TL CS210N ) transformants exhibited an increase in OAS steady-state level. Additionally, the cysteine content in the WT (AtOAS-TL CS210N ) and the glutathione content in oastlc (AtOAS-TL CS210N ) indicated a notable increase. In conclusion, the findings suggest that the modified version of mitochondrial AtOAS-TL CS210N is capable of generating a stable CS complex in Arabidopsis, leading to an increase in OAS production as it consistently activates SERAT2;2. Finally, the effects of cadmium stress on the root growth of the transformants were assessed as an indirect approach to evaluate the levels of flux of OAS and thiols compared to their steady-state levels. Under cadmium treatment, the oastla (AtOAS-TL A) transformants exhibited a comparable root length to that of the control condition. This finding suggests that the transformants resist high concentrations of cadmium, which is attributed to the higher quantity of phytochelatins. Correspondingly, both serat1;1 (AtOAS-TL AS102N ) and a1;1 (AtOAS-TL AS102N ) displayed a significant reduction in root length under cadmium treatment, implying the critical role of functional endogenous SERAT to uninterrupted synthesis of OAS and thiols. Taken together, the findings in this study suggest that the CS complex within the chloroplast of the monocotyledonous rice system demonstrates comparable functionality to various cellular compartments in the dicotyledonous Arabidopsis model. The identified mutation in AtOAS-TL consistently yields a stabilized CS complex within the cell. Nevertheless, this process is contingent upon an active and functional SERAT.