Molecular genetics of sulphate assimilation in 'Arabidopsis thaliana'
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The steps involved in plant reductive assimilation of sulphate to sulphide for incorporation into cysteine are not clear. The aim of the project described in this thesis was the isolation of genes encoding sulphate assimilation enzymes which will provide molecular tools for unravelling this key metabolic pathway. Functional complementation of the Escherichia coli cysteine auxotrophic strain JM15 (serine acetyltransferase deficient) using an Arabidopsis thaliana cDNA library in the expression vector YES resulted in the isolation of at least three members of an A. thaliana multigene family encoding serine acetyltransferase. Characterisation of one clone, Sat-1, showed that it conferred serine acetyltransferase activity (with apparent KM for substrates acetyl CoA and L-serine of 0.043 and 3.47 mM, respectively) on strain JM15. The 1515 bp full-length cDNA encodes a deduced protein of 391 amino acids, SAT-1, that has significant identity with bacterial and plant serine acetyltransferases, and that contains a putative N-terminal organellar targeting peptide. Southern hybridisation indicated that Sat-1 is present as a single copy in A. thaliana, while northern analysis revealed a single message of 1.5 kb. Using the A. thaliana cDNA library in the expression vector YES, cDNAs encoding a novel putative "APS reductase" were obtained by functional complementation of E. coli cysteine auxotrophic strains JM81A (adenosine 5'-phosphosulphate [APS] kinase deficient) and JM96 (3'-phosphoadenosine-5'-phosphosulphate [PAPS] reductase deficient). Retransformation of three clones (Papsr-19, Papsr-26 and Papsr-43) encoding different putative APS kinase isoforms into strain JM96 conferred low PAPS reductase activity on the mutant, although this activity was thioredoxin-independent unlike wild-type bacterial activity. The putative APS reductase has a PAPS reductase-like C-terminal domain, but further analysis demonstrated that the enzyme accepts APS in preference to PAPS as substrate and has a thioredoxin-like C-terminal domain. Isolation and characterisation of these genes invites a new hypothesis for plant reductive sulphate assimilation and provides direction for future research.
Thesis, PhD Doctor of Philosophy
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