In vitro biosynthesis of 1,4-β-galactan attached to rhamnogalacturonan I

Abstract

The biosynthesis of galactan was investigated using microsomal membranes isolated from suspension-cultured cells of potato (Solanum tuberosum L. var. AZY). Incubation of the microsomal membranes in the presence of UDP-[14C]galactose resulted in a radioactive product insoluble in 70% methanol. The product released only [14C]galactose upon acid hydrolysis. Treatment of the product with Aspergillus niger endo-1,4-beta-galactanase released 65-70% of the radioactivity to a 70%-methanol-soluble fraction. To a minor extent, [14C]galactose was also incorporated into proteins, however these galactoproteins were not a substrate for Aspergillus niger endo-1,4-beta-galactanase. Thus, the majority of the 14C-labelled product was 1,4-beta-galactan. Compounds released by the endo-1,4-beta-galactanase treatment were mainly [14C]galactose and [14C]galactobiose, indicating that the synthesized 1,4-beta-galactan was longer than a trimer. In vitro synthesis of 1,4-beta-galactan was most active with 6-d-old cells, which are in the middle of the linear growth phase. The optimal synthesis occurred at pH 6.0 in the presence of 7.5 mM Mn2+. Aspergillus aculeatus rhamnogalacturonase A digested at least 50% of the labelled product to smaller fragments of approx. 14 kDa, suggesting that the synthesized [14C]galactan was attached to the endogenous rhamnogalacturonan I. When rhamnogalacturonase A digests of the labelled product were subsequently treated with endo-1,4-beta-galactanase, radioactivity was not only found as [14C]galactose or [14C]galactobiose but also as larger fragments. The larger fragments were likely the [14C]galactose or [14C]galactobiose still attached to the rhamnogalacturonan backbone since treatment with beta-galactosidase together with endo-1,4-beta-galactanase digested all radioactivity to the fraction eluting as [14C]galactose. The data indicate that the majority of the [14C]galactan was attached directly to the rhamnose residues in rhamnogalacturonan I. Thus, isolated microsomal membranes contain enzyme activities to both initiate and elongate 1,4-beta-galactan sidechains in the endogenous pectic rhamnogalacturonan I.

Accumulation of antioxidants in rice callus (Oryza sativa L.) induced by β-glucan and salt stress

“…Gunter and Ovodov, (2002) observed that polysaccharides in Silene vulgaris callus is the primary metabolite (polysaccharide) cell wall component permanently biosynthesized through cells. Therefore, the results presented here are consistent with those of Geshi et al, (2000); Goubet and Morvan, (1994). These studies reported that polysaccharides have important roles in the expansion phase and cell division.…”

Pectin Biosynthesis: GALS1 in Arabidopsis thaliana Is a β-1,4-Galactan β-1,4-Galactosyltransferase

“…Most likely, one or two as yet unidentified galactosyltransferases are required to initiate the b-1,4-galactan side chains. This activity has been described in an extract from potato that could initiate but not elongate b-1,4-galactan side chains in vitro (Geshi et al, 2000). Future studies should clarify this but will require a set of putative oligosaccharide substrates that are not easily obtained.…”

Biosynthesis of Pectin

“…The assays have been further developed with solubilization of the membranes and the use of exogenous acceptors (Geshi et al, 2000(Geshi et al, , 2002Peugnet et al, 2001;Ishii et al, 2004). Activities were found in flax (Linum usitatissimum; Peugnet et al, 2001) and potato (Geshi et al, 2002) that could transfer Gal from UDP-Gal onto RGI acceptors, resulting in products that could be digested with galactosidase but not with endo-b-1,4-galactanase.…”