Xyloglucan metabolizing enzyme activities in suspension cultured tobacco cells

Xyloglucan was isolated from cells and medium of suspension cultures of tobacco, Nicotiana tabacum L. cv. Petit Havana SR1. Cellulase, 1,4-beta-D-glucan 4-glucanohydrolase (EC 3.2.1.4), purified from the insoluble fraction of tobacco callus, degraded the polydisperse tobacco xyloglucan (tobXG) with abundant molecular weights around 150 kDa to products focusing around 60 kDa. No oligosaccharides were found in the hydrolysate, and no xyloglucan endotransglycosylase (XET) activity could be ascribed to the cellulase when using tobXG together with [H-3] alditols of the low molecular weight fraction (6-12 kDa) of the cellulase digest as substrates for the enzyme in an XET test. However, the presence of XET activity in the tobacco cellulase was clearly demonstrated with tamarind xyloglucan (tamXG) together with oligosaccharidyl-[H-3] alditols of tamXG as substrates. A soluble extract from tobacco callus possessed XET activity with the two substrates from tobXG as well as with those from tamXG. Glc, Xyl and Ara constituted between 95% and 99% of the sugars found in acid hydrolysates of tobXG from cells and medium of suspension cultures. Gal and Man were the remaining components. TobXG moved as one spot on electrophoresis at pH 6.6, while an additional weak and faster moving spot was observed in some of the preparations when using a berate buffer at pH 9.4. The results are discussed in terms of the regulation of growth and morphogenesis in tobacco tissue.