Activities of sucrose metabolism enzymes in glycerol-grown suspension cultures of sweet orange ( Citrus sinensis L. Osbeck)

Abstract

Activities of the enzymes involved in sucrose metabolism were investigated in embryogenic nucellus-derived calli of ‘Hamlin’ sweet orange, Citrus sinensis L. Osbeck, cultured in suspension medium containing glycerol or sucrose as the only carbon sources. Activities of sucrose phosphate synthase (SPS) and sucrose synthase (SS), determined after 14 days of culture, were about 2.5-fold higher in the 2% glycerol-grown tissues, as compared to the 5% sucrose-grown tissues. Furthermore, activity of SPS was about two-fold and three-fold higher in the glycerol- and sucrose-grown tissues, respectively, when 40 mM glucose 6-phosphate (G6P) was added to the assay medium containing uridine 5′-diphosphoglucose (UDPG) and fructose 6-phosphate. SPS activity increased 43% for the glycerol-grown tissues and 130% for the sucrose-grown tissues when only UDPG and G6P were present in the assay reaction. Gel electrophoretic analysis and catalytic determination confirmed the presence of phosphoglucose isomerase in the tissues of both glycerol- and sucrose-containing medium. Arbutin, an inhibitor of SS in plants, depressed most activity of SS in both glycerol- and sucrose-grown tissues when 5 mM was added to the assay medium. High activities of SPS, SS and invertase and appreciable accumulation of sucrose, reducing sugars and starch in the glycerol-grown tissues indicate the ability of ‘Hamlin’ orange tissue cultures to efficiently use glycerol as a source of carbon for growth and differentiation.