Nitro‐blue tetrazolium staining of starch‐synthesizing enzymes in developing rice grains

An in situ nitro‐blue tetrazolium (NBT) staining method was used to locate the activities of enzymes involved in starch synthesis in developing grains of field grown rice ( Oryza sativa L.) cultivar Tainung 67 and its early ripening mutant SA 419. The results indicated that all the tested enzymes, including sucrose synthase, invertase, hexokinase, ADP‐glucose pyrophosphorylase, UDP‐glucose pyrophosphorylase and starch synthase, were detectable in developing rice endosperms by using NBT staining. The activity of these enzymes was also measurable by using chemical assays. The expression of enzyme activity, as indicated by the formation of blue formazan precipitate, in growing grain of Tainung 67 were visualized in entire endosperm at 11 days after anthesis (DAA). However, NBT staining shifted from central to peripheral region of endosperm after 18 DAA, and the staining disappeared at 25 DAA. Similar staining patterns were also observed in the growing grains of mutant SA 419, but the shift of staining from central to peripheral endosperm occurred at 11 DAA. Electron microscopy examinations showed that the growing patterns of starch granules, sampled from central and peripheral regions of endosperm, varied between two cultivars, with SA 419 growing faster than Tainung 67. Both cultivars showed that the shifts of NBT staining pattern coincided with the changes in the growing pattern of starch granules located in central and peripheral regions of endosperms. The NBT staining results showed that the activities of sucrose to starch conversion enzymes in mutant SA 419 declined and disappeared earlier than its wild‐type Tainung 67 and therefore it ripened earlier than cultivar Tainung 67. Copyright © 2005 Society of Chemical Industry