Abstract
In higher plants cysteine biosynthesis is catalyzed by O-acetylserine(thiol)lyase (OASTL) and represents the last step of the assimilatory sulfate reduction pathway. It is mainly regulated by provision of O-acetylserine (OAS), the nitrogen/carbon containing backbone for fixation of reduced sulfur. OAS is synthesized by Serine acetyltransferase (SERAT), which reversibly interacts with OASTL in the cysteine synthase complex (CSC). In this study we identify and characterize the SERAT gene family of the crop plant Vitis vinifera. The identified four members of the VvSERAT protein family are assigned to three distinct groups upon their sequence similarities to Arabidopsis SERATs. Expression of fluorescently labeled VvSERAT proteins uncover that the sub-cellular localization of VvSERAT1;1 and VvSERAT3;1 is the cytosol and that VvSERAT2;1 and VvSERAT2;2 localize in addition in plastids and mitochondria, respectively. The purified VvSERATs of group 1 and 2 have higher enzymatic activity than VvSERAT3;1, which display a characteristic C-terminal extension also present in AtSERAT3;1. VvSERAT1;1 and VvSERAT2;2 are evidenced to form the CSC. CSC formation activates VvSERAT2;2, by releasing CSC-associated VvSERAT2;2 from cysteine inhibition. Thus, subcellular distribution of SERAT isoforms and CSC formation in cytosol and mitochondria is conserved between Arabidopsis and grapevine. Surprisingly, VvSERAT2;1 lack the canonical C-terminal tail of plant SERATs, does not form the CSC and is almost insensitive to cysteine inhibition (IC50 = 1.9 mM cysteine). Upon sulfate depletion VvSERAT2;1 is strongly induced at the transcriptional level, while transcription of other VvSERATs is almost unaffected in sulfate deprived grapevine cell suspension cultures. Application of abiotic stresses to soil grown grapevine plants revealed isoform-specific induction of VvSERAT2;1 in leaves upon drought, whereas high light- or temperature- stress hardly trigger VvSERAT2;1 transcription.
Highlights
Cysteine biosynthesis is the exclusive entry point of reduced sulfur into primary metabolism of enterobacteria and phototrophic organism
In this study we addressed for the first time the importance of cysteine biosynthesis in different sub-cellular compartments of vascular land plants by characterization of the Serine acetyltransferase (SERAT) protein family of grapevine
Degenerate primers were designed to a consensus plant SERAT sequence obtained by aligning all nucleotide sequences from verified SERAT genes deposited in NCBI
Summary
Cysteine biosynthesis is the exclusive entry point of reduced sulfur into primary metabolism of enterobacteria and phototrophic organism. In these organisms, Cys is synthesized from free sulfide and O-acetylserine (OAS). Sulfur is taken up by all organisms as sulfate, an oxidized form of sulfur that is exclusively reduced to sulfide in plastids of all so far analyzed phototrophic organisms, including green algae, mosses and vascular plants (Shibagaki and Grossman, 2008; Khan et al, 2010). It gives rise to presumably hundreds of down-stream metabolites that contain reduced sulfur as functional constituent e.g. iron-sulfur clusters in diverse proteins, vitamins, glucosinolates
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