MYB transcription factors are one of the largest TF families involved in plant growth and development as well as biotic and abiotic stresses. In this study, we report the identification and functional characterization of a stress-responsive MYB gene (GhMYB3) from drought stress related transcriptome of upland cotton. GhMYB3, belonging to the R2R3-type, has high sequence similarity with AtMYB3 and was localized in the nucleus. Silence of GhMYB3 enhanced the drought tolerance of cotton seedlings and plants, reduced the water loss rate, and enhanced stomatal closure. In addition, GhMYB3i lines exhibited less ROS accumulation, as well as higher antioxidant enzyme activity and increased content of anthocyanins and proanthocyanidins than WT plants after drought stress. The expression level of flavonoid biosynthesis- and stress-related genes were up-regulated in GhMYB3i lines under drought stress condition. These results demonstrated that GhMYB3 acted as a negative regulator in upland cotton response to drought stress by regulating stomatal closure and ROS accumulation.
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