N. Delu-filho et al., A sucrose binding protein homologue from soybean affects sucrose uptake insuspension-cultured transgenic tobacco cells, PL PHYS BIO, 38(5), 2000, pp. 353-361
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.