Publications - Cell and Metabolic Biology

The serine carboxypeptidase-like protein 1-O-sinapoylglucose:malate sinapoyltransferase (SMT) catalyzes the transfer of the sinapoyl moiety of 1-O-sinapoylglucose to malate in the formation of sinapoylmalate in some members of the Brassicaceae. Rabbit polyclonal monospecific antibodies were raised against the recombinant SMT produced in Escherichia coli from the corresponding Arabidopsis thaliana (L.) Heynh. cDNA. Immunoblot analysis of protein from different Arabidopsis tissues showed that the SMT is produced in all plant organs, except in the seeds and young seedlings. The enzyme was most abundant in older seedlings as well as in rosette leaves and the flowering stem of the plant. Minor amounts were found in the cauline leaves, flower buds and siliques. Traces were detected in the root and flowers. Arabidopsis and transgenic tobacco (Nicotiana tabacum L.) plants expressing the full-length Arabidopsis SMT containing an N-terminal signal peptide showed apparent molecular masses of the protein of 52–55 kDa. The difference of ca. 8 kDa compared to the recombinant protein produced in E. coli was shown to be due to post-translational N-glycosylation of SMT in plants. Immunofluorescent labeling of Arabidopsis leaf sections localized SMT to the central vacuoles of mesophyll and epidermal cells. Comparable leaf sections of an SMT deletion mutant showed no vacuolar immunofluorescent labeling. We conclude that Arabidopsis SMT is synthesized as a precursor protein that is targeted to the endoplasmic reticulum where the signal peptide is removed. The correct N-terminus of the recombinantly produced SMT protein lacking the signal peptide was confirmed by Edman degradation. The protein is probably glycosylated in the Golgi apparatus from where it is subsequently routed to the vacuole.

Sinapoylmalate is one of the major phenylpropanoid metabolites that is accumulated in the vegetative tissue of Arabidopsis thaliana. A thin-layer chromatography-based mutant screen identified two allelic mutant lines that accumulated sinapoylglucose in their leaves in place of sinapoylmalate. Both mutations were found to be recessive and segregated as single Mendelian genes. These mutants define a new locus called SNG1 for sinapoylglucose accumulator. Plants that are homozygous for the sng1 mutation accumulate normal levels of malate in their leaves but lack detectable levels of the final enzyme in sinapate ester biosynthesis, sinapoylglucose:malate sinapoyltransferase. A study of wild-type and sng1 seedlings found that sinapic acid ester biosynthesis in Arabidopsis is developmentally regulated and that the accumulation of sinapate esters is delayed in sng1 mutant seedlings.