Respiratory burst oxidase homologs (Rbohs) are critical enzymes involved in the generation of reactive oxygen species (ROS) that play an important role in plant growth and development as well as various biotic and abiotic stresses in plants. Thus far, there have been few reports on the characterization of the Rboh gene family in Citrus. In this study, seven Rboh genes (CsRbohA~CsRbohG) were identified in the Citrus sinensis genome. The CsRboh proteins were predicted to localize to the cell membrane. Most CsRbohs contained four conserved domains, an EF-hand domain, and a transmembrane region. Phylogenetic analysis demonstrated that the CsRbohs were divided into five groups, suggesting potential distinct functions and evolution. The expression profiles revealed that these seven CsRboh genes displayed tissue-specific expression patterns, and five CsRboh genes were responsive to cold stress. Fourteen putative cis-acting elements related to stress response, hormone response, and development regulation were present within the promoters of CsRboh genes. The in-silico microRNA target transcript analyses indicated that CsRbohE might be targeted by csi-miR164. Further functional and physiological analyses showed that the knockdown of CsRbohD in trifoliate orange impaired resistance to cold stress. As a whole, our results provide valuable information for further functional studies of the CsRboh genes in response to cold stress.

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