Inhibition of hexokinase and expression of asparagine synthetase and beta-galactosidase genes during sugar feeding and starvation of asparagus (Asparagus officinalis) callus cultures

Hexokinase has an important role in hexose metabolism and in signalling sug ar status in plants. The aim of this study was to inhibit hexose phosphoryl ation using a hexokinase inhibitor (glucosamine), and to determine the effe cts on expression of asparagine synthetase (AS) and beta-galactosidase duri ng glucose feeding and starvation of asparagus (Asparagus officinalis L.) c allus cultures. After 48 h without glucose and a further 24-h incubation wi th a range of hexoses and analogues, expression of both AS and beta-galacto sidase was repressed by D-glucose, D-galactose, and 2-deoxyglucose, but the genes were not repressed when glucose was absent, or when 3-O-methylglucos e or L-glucose were supplied. Glucose-and fructose-phosphorylating activity was determined in extracts from callus cultures which had been exposed to glucose, 2-deoxyglucose, 2-deoxyglucose with glucosamine or mannitol (as an osmotic control), or glucose-free media. After 48 h on glucose-free media and 48-h incubation with 2-deoxyglucose and glucosamine, glucose- and fruct ose-phosphorylating activities were reduced by 68 and 83%, respectively. Wh en glucose was present in the cultures, there was no expression of AS trans cripts, but when glucose was absent, AS was highly expressed. AS expression was reduced when 2-deoxyglucose was present for 48 h, even when glucosamin e or mannitol was also present in the culture media. beta-galactosidase was highly expressed when glucose was absent, but expression was very low in a ll of the treatments which contained 2-deoxyglucose (including the glucosam ine and mannitol treatments). The results suggest AS and beta-galactosidase are sugar regulated, but inconsistencies, particularly reduced AS expressi on in the presence of glucosamine, are discussed in relation to the possibi lities that multiple forms of hexokinases exist which might be differential ly affected by glucosamine, and that uptake and distribution of 2-deoxygluc ose and glucosamine are limited.