Starch degradation by alpha-amylase in tobacco leaves during the curing process

Tobacco (Nicotiana tabacum L.) leaf starch is degraded to sugars through curing (42 degrees C/82.3% relative humidity/72h). Total carbohydrate content remained almost constant, starch content decreased markedly, and soluble sugar content (mostly glucose) increased. -Amylase and starch phosphorylase activities increased sixfold and threefold, respectively, whereas -amylase activity was unaltered and isoamylase activity decreased. Increased -amylase activity was accompanied by increased -amylase protein levels. Although tobacco has four -amylase gene members, only NtAMY1 mRNA levels increased. For other starch degradation genes, such as NtBAM1 and NtBMY2 (-amylase), NtISO1 and NtISO2 (isoamylase) and NtGWD1 and NtGWD3 (glucan water dikinase), the mRNA levels remained unaltered during the first 48h of curing. NtAMY1 expression was induced by osmotic stress but was unaffected by high temperature and/or injury stresses. Similarly, soluble sugar contents were largely increased by osmotic stress. This suggests that starch is degraded by -amylase during curing and that -amylase is coded by NtAMY1, induced by osmotic stress.