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Sea buckthorn ( Hippophae rhamnoides L.) is a plant with economic and ecological value. It is uniquely capable of growing well under salt and drought stress. WRKY transcription factors play important roles in the ability of plants to resist stress. In this study, 48 HrWRKY genes were identified based on RNA sequencing of H. rhamnoides . Evaluation of expression pattern of HrWRKY1 , HrWRKY17 , HrWRKY18 , HrWRKY21 , HrWRKY33-2 , HrWRKY40-2 , HrWRKY41 , and HrWRKY71 suggested that they were involved in abiotic stress. Interestingly, HrWRKY21 , one of eight HrWRKY genes, was a positive regulator of abiotic stress tolerance in H. rhamnoides . In addition, most morphological attributes of roots in transgenic Nicotiana tabacum lines (overexpressing HrWRKY21 ) were also markedly increased compared with the wild-type (WT), including total lengths, specific root lengths, and surface areas. Stress tolerance of transgenic lines was also correlated with higher antioxidant activity (SOD and POD), lower percentage of relative conductivity (REC), and lower activity of malondialdehyde (MDA) under stress conditions. These findings represent a foundation of knowledge about the molecular mechanisms driving resistance to adverse conditions in plants; they are a promising step towards development of tree cultivars with improved tolerance to abiotic stress.