Starch branching enzymes in Solanum tuberosum and Arabidopsis thaliana

Starch is the major storage carbohydrate in higher plants and essentially consists of two major polysaccharide components, amylose and amylopectin. Amylopectin, which is the major component, is one of the largest natural macromolecules consisting of 103-1003 glucose units linked together by alfa(1,4)-bonds and branched by alfa(1,6)-linkages. Starch-branching enzyme (SBE) is the enzyme responsible for the branched chains of the amylopectin. Three multiple forms of SBE with apparent molecular masses of 103-, 97- and 80-kDa were isolated from potato tubers. Amino acid sequencing revealed that the enzymes belong to SBE-I isoform. Southern blot analysis and DNA sequencing suggested that the multiple forms of SBE-I are encoded from a single locus. It was found that a direct nucleotide repeat in the 3' end of the sbeI gene is responsible for the highly negatively charged C-terminal portion of the SBE-I. The C-terminal portion of the 103- kDa is easily removed in vitro by a number of proteases to result an 80-kDa enzyme form. The branching activity of the enzyme is preserved after this cleavage. It was concluded that the 80-kDa form is most likely an in vivo-processed form of the 103-kDa enzyme. A cDNA clone encoding the mature form of the SBE-I was isolated from potato tubers and expressed in Escherichia coli. A high specific enzyme activity of the recombinant protein was purified using a starch-affinity column. The recombinant SBE-I showed a 6-fold higher rate of branching amylose than of amylopectin. Two genes (Sbe2.1 and Sbe2.2) encoding two closely related SBE-II were isolated from Arabidopsis thaliana. DNA sequencing of the promotor region of the Sbe2.1 gene showed the existence of several putative sugar and light responsive cis-elements. Northern blot analysis showed two somewhat different expression patterns for the genes with respect to light and sugar. Plants incubated in light with exogenous supply of either glucose, fructose or sucrose responded to this condition with strong expression of both Sbe2 genes. The sugar-inducible expression was considerably repressed in the absence of light, indicating the importance of light for high gene expression.