Starch Branching Enzyme cDNA from Solanum tuberosum

Starch the main storage carbohydrate in higher plants Ta& 1. Characterjstjcs of the SBE c-NA from p a t o and is composed of the two polysaccharides, amylopectin (approximately 75%) and amylose (approximately 25%). Amylopectin is a highly branched a-1,4 glucan containing a1,6 branch points, whereas amylose is composed of long, linear a-1,4 glucans, some of which have very few a-1,6 branch points. Starch is synthesized in chloroplasts and amyloplasts by the action of three enzymic activities: ADP-Glc pyrophosphorylase (EC 2.7.7.27), starch synthase (EC Source: 2.4.1.21), and SBE (EC 2.4.1.18) (reviewed in Preiss, 1991). ADP-Glc pyrophosphorylase catalyzes the formation of ADP-Glc, which is the Glc donor for the synthesis of amylose carried out by starch synthase, whereas SBE catalyzes the conversion of amylose to amylopectin by adding linear chains of a-1,4 glucans to amylose through a-1,6 branch points. Here we present the isolation of a branching enzyme cDNA from potato (Solanum tuberosum), which is an important prerequisite for further detailed studies of the mechanisms of starch biosynthesis at the enzyme and gene levels. A full-length cDNA clone encoding SBE was isolated from a potato sprout cDNA library using a partial potato SBE cDNA clone that was originally isolated with a heterologous cDNA encoding a pea SBE (Table I). The 3114-bp DNA sequence revealed an ORF that begins with an ATG initiation codon at coordinate 121 bp and ends with a TGA codon at coordinate 2704 bp. The ORF includes 861 amino acids, which has significant similarity to SBE I from maize and rice (Baba et al., 1991; Nakamura and Yamanouchi, 1992), indicating that the 3.1-kb cDNA encodes potato SBE. The protein encoded by the ORF has a calculated M, of 99,083, whereas the purified branching enzyme from potato tubers has been reported to have M, values in the range from 79,000 to 103,000 (Vos-Scheperkeuter et al., 1989; Blennow and Johansson, 199 1). The understanding of these discrepancies must await further analysis of the purified enzyme. SBE has been located in the amyloplast-stroma (Kram et al., 1993), suggesting that the deduced amino-terminal sequence of SBE contains a transit peptide that targets SBE to the amyloplast. In accordance with this prediction, the SBE amino terminus has some features in common with chloroplast transit peptides (Gavel and von Heine, 1990), i.e. a high content of Ser and Thr residues and a central, positively charged domain. Moreover, the hydropathicity profiles of the amyloplast transit peptide from the potato granule-bound