A sucrose binding protein homologue from soybean affects sucrose uptake insuspension-cultured transgenic tobacco cells

We have obtained Nicotiana tabacum transgenic cell lines expressing a sucro se binding protein (sbp) homologue gene from soybean (Glycine max L.), desi gnated s-64, either in the sense or antisense orientation. Sense cell lines over-accumulated the S-64 protein, whereas the antisense cell lines had re duced levels of the endogenous homologue protein. Sucrose uptake experiment s were conducted by incubating suspension-cultured tobacco cells with radio labeled sucrose at pH 4.5 or 7.0. Raising the extracellular pH to 7.0 cause d an inhibition of radiolabeled carbon uptake efficiency, which was attribu ted to the pH-sensitivity of cell-wall invertase (EC 3.2.1.26), H+/hexose t ransporter and/or H+/sucrose symporter activities. Because SEP-mediated suc rose uptake has been shown to be insensitive to extracellular pH in yeast, we performed the sucrose uptake experiments in sense and antisense cultured cells at pH 7.0. Under this condition, the level of SEP homologue correlat ed with the efficiency of radiolabeled uptake by the transgenic tobacco cel ls. Furthermore, manipulation of S-64 levels altered sucrose-cleaving activ ities in a metabolic compensatory manner. Enhanced accumulation of S-64 cau sed an increase in intracellular sucrose synthase (cleavage, EC 2.4.1.13) a ctivity with a concomitant decline in cell-wall invertase activity. This re sult may reflect a metabolic adjustment of the sense cell lines caused by i ts high efficiency of direct sucrose uptake as disaccharide; In contrast, t he level of cell-wall invertase activity was remarkably increased in antise nse cells, favoring the invertase-dependent sugar uptake system. Collective ly, these results may establish a functional link between radiolabeled infl ux and S-64 accumulation, suggesting that SEP affects sucrose uptake in sus pension-cultured cells. (C) 2000 Editions scientifiques et medicales Elsevi er SAS.