Expression of a-amylase genes during cereal grain germination and seedling growth is regulated negatively by sugar in embryos and positively by gibberellin (GA) in endosperm through the sugar response complex (SRC) and the GA response complex (GARC), respectively. We analyzed two a-amylase promoters, aAmy3 containing only SRC and aAmy8 containing overlapped SRC and GARC. aAmy3 was sugar-sensitive but GA-nonresponsive in both rice (Oryza sativa) embryos and endosperms, whereas aAmy8 was sugar-sensitive in embryos and GA-responsive in endosperms. Mutation of the GA response element (GARE) in the aAmy8 promoter impaired its GA response but enhanced sugar sensitivity, and insertion of GARE in the aAmy3 promoter rendered it GA-responsive but sugar-insensitive in endosperms. Expression of the GAREinteracting transcription factor MYBGA was induced by GA in endosperms, correlating with the endosperm-specific aAmy8 GA response. aAmy8 became sugar-sensitive in MYBGA knockout mutant endosperms, suggesting that the MYBGA-GARE interaction overrides the sugar sensitivity of aAmy8. In embryos overexpressing MYBGA, aAmy8 became sugar-insensitive, indicating that MYBGA affects sugar repression. a-Amylase promoters active in endosperms contain GARE, whereas those active in embryos may or may not contain GARE, confirming that the GARE and GA-induced MYBGA interaction prevents sugar feedback repression of endosperm a-amylase genes. We demonstrate that the MYBGA-GARE interaction affects sugar feedback control in balanced energy production during seedling growth and provide insight into the control mechanisms of tissue-specific regulation of a-amylase expression by sugar and GA signaling interference.
Overexpression of bacterial-derived starch metabolic enzymes in plant starch storage organs represents a valuable strategy for improving starch quality, bioprocessing and nutritional value. Transgenic rice seeds producing a thermostable and bifunctional starch hydrolase, amylopullulanase (APU) from Thermoanaerobacter ethanolicus 39E, were generated. Starch in these seeds could be hydrolyzed with optimal temperatures between 85 and 95 °C, which resulted in complete conversion of starch into soluble sugars and production of protein-enriched flour within a few hours. By expressing various levels of APU, rice seeds containing reduced amounts of amylose, which is an important factor affecting starch quality, were obtained without a significant impact on grain yield. Elevation in granule-bound pullulanase activity correlates with the reduction of amylose in developing APU-containing rice seeds. APU was found to be localized within amyloplasts and in cell walls, which could be the result of overexpression of APU with a signal peptide. This study establishes novel approaches to alter starch properties, accelerate bioprocessing of starch and production of protein-enriched flour from rice seeds, and could significantly impact the industrial and food uses of cereals.
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