Genome-wide identification of UDP-glycosyltransferase gene family in Dendrobium huoshanense and functional characterization of DhUGT44 mediating flavonoid 7-O-glycosylation and cold tolerance

Abstract

UDP-glycosyltransferases (UGTs) play essential roles in glycosylating plant specialized metabolites, thereby modulating their stability, solubility, and bioactivity to regulate growth, development, and environmental adaptation. Dendrobium huoshanense , a rare and endangered medicinal orchid endemic to China, is renowned for its rich bioactive glycosides, including polysaccharides, flavonoids, and alkaloids, which underpin its high medicinal value. However, systematic genome-wide identification and evolutionary analysis of the UGT gene family in Dendrobium remain limited, hindering their application in stress tolerance and quality improvement. In this study, we performed the first comprehensive genome-wide identification and functional characterization of the UGT family in D. huoshanense by integrating bioinformatics analyses with molecular experiments. A total of 71 DhUGT genes were identified and phylogenetically classified into 12 subfamilies. These genes are unevenly distributed across chromosomes, with tandem duplications as the primary driver of family expansion. Cis-regulatory element analysis showed that most DhUGT promoters are enriched with phytohormone- and abiotic stress-responsive motifs. By integrating multi-omics transcriptomic data, we identified DhUGT44 (subfamily L) as a high priority candidate, exhibiting consistently high expression across tissues, cultivation conditions, and growth stages. Its promoter contains cold-responsive elements, and RT-qPCR confirmed significant induction under low temperature stress in leaves. Subcellular localization assays demonstrated dual localization of DhUGT44 to the plasma membrane and chloroplasts. In vitro enzymatic assays and transient expression in Nicotiana benthamiana revealed that DhUGT44 specifically catalyzes 7- O -glycosylation of flavonoid substrates. Furthermore, heterologous overexpression of DhUGT44 in Arabidopsis thaliana significantly enhanced primary root growth under cold stress compared to wild-type plants, indicating its positive role in cold tolerance. This study provides the first systematic insight into the evolutionary dynamics and transcriptional regulation of the UGT family in D. huoshanense , while elucidating the flavonoid glycosylation function and cold tolerance regulatory role of DhUGT44 . These findings offer valuable genetic resources and a theoretical basis for molecular breeding and metabolic engineering of stress tolerance and bioactive compounds in Dendrobium .