Identification, cDNA Cloning, and Gene Expression of Soluble Starch Synthase in Rice (Oryza sativa L.) Immature Seeds

Abstract

Three forms of soluble starch synthase were resolved by anionexchange chromatography of soluble extracts from immature rice (Oryza safiva 1.) seeds, and each of these forms was further purified by affinity chromatography. The 55-, 57-, and 57-kD proteins in the three preparations were identified as candidates for soluble starch synthase by western blot analysis using an antiserum against rice granule-bound starch synthase. It is interesting that the aminoterminal amino acid sequence was identical among the three proteins, except that the 55-kD protein lacked eight amino acids at the amino terminus. Thus, these three proteins are products of the same gene. lhe cDNA clones coding for this protein have been isolated from an immature rice seed library in Agtll using synthetic oligonucleotides as probes. The deduced amino acid sequence of this protein contains a lysine-X-glycine-glycine consensus sequence for the ADP-glucose-binding site of starch and glycogen synthases. Therefore, we conclude that this protein corresponds to a form of soluble starch synthase in immature rice seeds. The precursor of the enzyme contains 626 amino acids, including a 113-residue transit peptide at the amino terminus. lhe mature form of soluble starch synthase shares a significant but low sequence identity with rice granule-bound starch synthase and Escherichia coli glycogen synthase. However, several regions, including the substrate-binding site, are highly conserved among these three enzymes. Blot hybridization analysis demonstrates that the gene encoding soluble starch synthase is a single-copy gene in the rice genome and is expressed in both leaves and immature seeds. These results suggest that soluble and granule-bound starch synthases play distinct roles in starch biosynthesis of plant.

Function and Characterization of Starch Synthase I Using Mutants in Rice

“…3B). The predicted molecular mass of rice SSI calculated from the DNA sequence of the gene coding for the SSI mature protein is 65 kD (Baba et al, 1993;Jiang et al, 2004), which is smaller than the apparent molecular mass (71 kD) estimated on SDS-PAGE. Similar differences in molecular mass were also reported for SSI proteins from maize and potato tubers (Edwards et al, 1995).…”

“…Prior to this event, the expression of the OsSSI gene starts at DAF 1 (Hirose and Terao, 2004;Ohdan et al, 2005) and is maintained at high levels until DAF 15 (Baba et al, 1993;Hirose and Terao, 2004;Ohdan et al, 2005). The differences in the amylopectin chainlength distribution pattern between e72/2 and the wild type were analyzed during endosperm development (DAF 7-25; Fig.…”

“…The complete purification of SS isozymes from plants is difficult because of the inherent instability of the enzymes. Nevertheless, some biochemical studies of SS isozymes expressed in Escherichia coli (Imparl-Radosevich et al, 1998Senoura et al, 2004) and analysis of partially purified SS isozymes from plant tissues (Baba et al, 1993;MuForster et al, 1996;Cao et al, 2000) have been reported. Two SS activity peaks, designated SSI and SSII, have been fractionated by anion-exchange chromatography from soluble extracts of maize (Zea mays; Ozbun et al, 1971;Boyer and Preiss, 1981) and rice (Oryza sativa; Tanaka and Akazawa, 1971) developing endosperm.…”

Interaction with amylopectin influences the ability of granule-bound starch synthase I to elongate malto-oligosaccharides

“…The effect of amylopectin on the properties of GBSSI in a soluble state suggests that interactions between GBSSI and amylopectin within the matrix of the granule may be essential to allow processive elongation of malto-oligosaccharides by GBSSI, and thus to allow amylose biosynthesis. The processive mode of elongation may also explain the contribution of GBSSI to the synthesis of very long chains in the amylopectin fraction of starch granules isolated from higher plants [3,12] and Chlamydomonas [14]. Overall, we suggest that the ability to interact with the amylopectin matrix and thus attain a processive mode of action may be the primary feature that distinguishes GBSSI from isoforms of starch synthase which cannot synthesize amylose.…”

“…First, the amylopectin-synthesizing isoforms have Nterminal domains which GBSSI lacks (SSI [3,4] ; SSII [5,6] ; SSIII [7][8][9]). The amino acid sequences of the N-terminal domains are not highly conserved between orthologues from different plant species, and their function is unknown.…”