Solubilization of an Arabinan Arabinosyltransferase Activity from Mung Bean Hypocotyls

Abstract

The biosynthesis of polysaccharides destined for the plant cell wall and the subsequent assembly of the cell wall are poorly understood processes that are currently the focus of much research. Arabinan, a component of the pectic polysaccharide rhamnogalacturonan I, is composed of arabinosyl residues connected via various glycosidic linkages, and therefore, the biosynthesis of arabinan is likely to involve more than one arabinosyltransferase. We have studied the transfer of [ 14 C]arabinose (Ara) from UDP-l-arabinopyranose onto polysaccharides using microsomal membranes isolated from mung bean (Vigna radiata) hypocotyls. [ 14 C]arabinosyl and [ 14 C]xylosyl residues were incorporated into endogenous products due to the presence of UDP-Xyl-4-epimerase activity. Enzymatic digestion of endogenous products with endo-arabinanase released very little radiolabeled sugars, whereas digestion with arabinofuranosidase released some [ 14 C]Ara. Microsomal membranes solubilized with the detergent octyl glucoside were able to add a single [ 14 C]Ara residue onto (135)-linked ␣-l-arabino-oligosaccharide acceptors. The reaction had a pH optimum of 6.5 and a requirement for manganese ions. However, enzymatic digestion of the radiolabeled oligosaccharides with endo-arabinanase and arabinofuranosidases could not fully release the radiolabeled Ara residue, indicating that the [ 14 C]Ara residue was not a (132)-, (133)-, or (135)-linked ␣-l-arabinofuranosyl residue. Rather, mild acid treatment of the product suggested that the radiolabeled Ara residue was in a pyranose conformation, and this result was confirmed by thin-layer chromatography of radiolabeled partially methylated sugars. Using microsomal membranes separated on a discontinuous sucrose gradient, the arabinosyltransferase activity appears to be mainly localized to Golgi membranes.Pectic polysaccharides are major components of primary plant cell walls and middle lamella. Three main types of pectic polysaccharides have been identified, homogalacturonan, rhamnogalacturonan I (RG I), and rhamnogalacturonan II (RG II). RG I is composed of a backbone of alternating [34-␣-d-GalA-(132)-␣-l-Rha- (13] in which some of the rhamnose residues are substituted with complex side chains containing Ara and Gal residues. The composition and size of these side chains varies depending on the plant species, tissue type, and stage of development (O'Neill et al., 1990;Carpita and Gibeaut, 1993). In general, the arabinan side chains consist of a linear chain of (135)-linked ␣-l-arabinofuranosyl residues that may be substituted at the O-3 and/or O-2 with additional arabinofuranosyl residues (Carpita and Gibeaut, 1993;Schols and Voragen, 2002).The study of the biosynthesis of plant cell wall polysaccharides in plants is a growing field, but to date, very few genes encoding glycosyltransferases have been characterized (Edwards et al., 1999;Perrin et al., 1999;Faik et al., 2002). Notably, many different transferases must be involved in pectin biosynthesis, but until very recently, none had been isola...

ARABINAN DEFICIENT 1 Is a Putative Arabinosyltransferase Involved in Biosynthesis of Pectic Arabinan in Arabidopsis

“…In microsomal or Golgi membranes, we have detected incorporation of arabinofuranose from UDP-arabinopyranose in both wheat (Triticum aestivum; Porchia et al, 2002) and mung bean (Vigna radiata; Nunan and Scheller, 2003). However, after solubilization we could never detect any incorporation of arabinofuranose but only of arabinopyranose and the activity was very low (Nunan and Scheller, 2003). We interpret these findings as evidence that the membranes contain a mutase activity that is closely coupled with the transferase activity.…”

“…This would suggest a possible mechanism for incorporation of arabinofuranose into plant polymers, but unfortunately no protein in Arabidopsis appears to be even remotely similar to bacterial UDP-galactopyranose mutase. In microsomal or Golgi membranes, we have detected incorporation of arabinofuranose from UDP-arabinopyranose in both wheat (Triticum aestivum; Porchia et al, 2002) and mung bean (Vigna radiata; Nunan and Scheller, 2003). However, after solubilization we could never detect any incorporation of arabinofuranose but only of arabinopyranose and the activity was very low (Nunan and Scheller, 2003).…”

“…Very few of the glycosyltransferases have been identified (for a recent review, see Scheible and Pauly, 2004). Assays for the measurement of some of the glycosyltransferase activities involved in pectin biosynthesis have been described, including homogalacturonan galacturonosyltransferase (Villemez et al, 1965;Doong et al, 1995), galactan galactosyltransferase (Geshi et al, 2002), and arabinan arabinosyltransferase (Nunan and Scheller, 2003). However, none of the pectin synthesizing glycosyltransferases have been isolated.…”

The Interconversion of UDP-Arabinopyranose and UDP-Arabinofuranose Is Indispensable for Plant Development inArabidopsis

“…The mutase could be expected to be associated, for instance, with enzymes involved in arabinan biosynthesis, but if such direct interactions exist, they are not very strong. It has been observed that microsomes from potato and mung bean can incorporate arabinofuranose into polysaccharides with UDP-Arap as substrate, whereas isolated Golgi from these dicot species do not have this ability, consistent with a very loose association between RGP and Golgi in dicots (Nunan and Scheller, 2003;Konishi et al, 2006).…”

Manganese Efficiency in Barley: Identification and Characterization of the Metal Ion Transporter HvIRT1

“…These include (1) oxalate oxidase, which catalyzes the conversion of oxalate and oxygen into hydrogen peroxide and carbon dioxide (Requena and Bornemann, 1999); (2) the Mn-containing superoxide dismutase, located in the mitochondria, which protects the tissue from oxidative stress (Bowler et al, 1991;Alscher et al, 2002); and (3) the oxygen-evolving complex of PSII (Britt, 1996;Clemens et al, 2002;Rutherford and Boussac, 2004). In addition, Mn is an unspecific activator of a number of different enzymes, such as decarboxylases and dehydrogenases in the tricarboxylic acid cycle, Phe ammonia-lyase in the shikimic acid pathway, and in several glycosyltransferases in the Golgi apparatus (Marschner, 1995;Nunan and Scheller, 2003). Mn deficiency causes interveinal chlorosis and reduction in the content of fructans and structural carbohydrates, resulting in slack leaves (Pearson and Rengel, 1997).…”