Reversibly glycosylated polypeptides (RGPs) have been implicated in polysaccharide biosynthesis. In plants, these proteins may function, for example, in cell wall synthesis and/or in synthesis of starch. We have isolated wheat (Triticum aestivum) and rice (Oryza sativa) Rgp cDNA clones to study the function of RGPs. Sequence comparisons showed the existence of two classes of RGP proteins, designated RGP1 and RGP2. Glucosylation activity of RGP1 and RGP2 from wheat and rice was studied. After separate expression of Rgp1 and Rgp2 in Escherichia coli or yeast (Saccharomyces cerevisiae), only RGP1 showed self-glucosylation. In Superose 12 fractions from wheat endosperm extract, a polypeptide with a molecular mass of about 40 kD is glucosylated by UDP-glucose. Transgenic tobacco (Nicotiana tabacum) plants, overexpressing either wheat Rgp1 or Rgp2, were generated. Subsequent glucosylation assays revealed that in RGP1-containing tobacco extracts as well as in RGP2-containing tobacco extracts UDP-glucose is incorporated, indicating that an RGP2-containing complex is active. Gel filtration experiments with wheat endosperm extracts and extracts from transgenic tobacco plants, overexpressing either wheat Rgp1 or Rgp2, showed the presence of RGP1 and RGP2 in high-molecular mass complexes. Yeast two-hybrid studies indicated that RGP1 and RGP2 form homo-and heterodimers. Screening of a cDNA library using the yeast two-hybrid system and purification of the complex by an antibody affinity column did not reveal the presence of other proteins in the RGP complexes. Taken together, these results suggest the presence of active RGP1 and RGP2 homo-and heteromultimers in wheat endosperm.Reversibly glycosylated polypeptides (RGPs) are thought to be involved in polysaccharide metabolism. Polysaccharides are the main components of the plant cell wall. In dicots, 20% of the primary cell wall consists of the polysaccharide xyloglucan, whereas in monocots, this hemicellulose makes up 2% of the primary cell wall (Darvill et al., 1980). Xyloglucan consists of a ␤-1,4-glucan backbone with xylosyl and xylosyl-galactosyl-fucosyl side chains (Hayashi, 1989). The transferases responsible for the addition of these side chains are localized to the Golgi apparatus (Brummell et al., 1990;Driouich et al., 1993;Staehelin and Moore, 1995). RGP1 has been implicated in xyloglucan biosynthesis in pea (Pisum sativum; Dhugga et al., 1997). This protein, a 40-kD doublet, which could be glycosylated with radiolabeled UDP-Glc in the presence of Mn 2ϩ or Mg 2ϩ , is associated with Golgi membranes as shown by density gradient centrifugation (Dhugga et al., 1991). Immunolocalization experiments showed RGP1 to be present in transGolgi dictyosomal cisternae (Dhugga et al., 1997). An Arabidopsis homologue was found to be mostly soluble, but also to be associated with membranes (Delgado et al., 1998). Glycosylation of the pea protein with UDP-Glc, UDP-Xyl, and UDP-Gal in a ratio similar to that of the typical sugar composition of xyloglucan (UDP-Glc:UDP-Xyl:UDP-Gal ϭ 10...

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SummaryStarch granule size is an important parameter for starch applications in industry. Starch granules are formed in amyloplasts, which are, like chloroplasts, derived from proplastids.

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