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

Abstract

Hexokinase has an important role in hexose metabolism and in signalling sugar status in plants. The aim of this study was to inhibit hexose phosphorylation using a hexokinase inhibitor (glucosamine), and to determine the effects on expression of asparagine synthetase (AS) and (3‐galactosidase during glucose feeding and starvation of asparagus (Asparagus officinalis L.) callus cultures. After 48 h without glucose and a further 24‐h incubation with a range of hexoses and analogues, expression of both AS and β‐galactosidase was repressed by d‐glucose, d‐galactose, and 2‐deoxyglucose, but the genes were not repressed when glucose was absent, or when 3‐O‐methylglucose 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 fructose‐phosphorylating activities were reduced by 68 and 83%, respectively. When glucose was present in the cultures, there was no expression of AS transcripts, but when glucose was absent, AS was highly expressed. AS expression was reduced when 2‐deoxyglucose was present for 48 h, even when glucosamine or mannitol was also present in the culture media. P‐galactosidase was highly expressed when glucose was absent, but expression was very low in all of the treatments which contained 2‐deoxyglucose (including the glucosamine and mannitol treatments). The results suggest AS and P‐galactosidase are sugar regulated, but inconsistencies, particularly reduced AS expression in the presence of glucosamine, are discussed in relation to the possibilities that multiple forms of hexokinases exist which might be differentially affected by glucosamine, and that uptake and distribution of 2‐deoxyglucose and glucosamine are limited.