A novel orphan gene <i>CsOG3</i> drives CsMYB44-dependent cold tolerance in tea plant

Tea plant (Camellia sinensis) is an important thermophilic crop in China. Understanding the cold response mechanisms will be helpful to improve the yield and tea quality against cold stress. Orphan genes, which lack homologs in other lineages, play critical contributions in plant environmental adaptability, yet the characteristics and roles of orphan genes in tea plants, particularly regarding cold tolerance, remain largely unexplored. In the current study, we systematically identified 2,793 orphan genes of tea plant using both genomic and transcriptomic datasets. These orphan genes exhibited simpler gene structures, shorter lengths, fewer introns, higher isoelectric points and lower expression abundance compared with the evolutionarily-conserved genes. We further characterized an orphan gene named CsOG3 that may play roles in tea plant cold resistance. Silencing of CsOG3 reduced the cold tolerance level of tea plant seedlings, while overexpression of CsOG3 significantly enhanced the cold resistance of tobacco and tea plants. By regulatory elements and expression correlation analysis, we identified a cold-induced MYB transcription factor-CsMYB44, which is involved in regulating CsOG3. Functional validation using dual-luciferase reporter and yeast one-hybrid assays reveal that CsMYB44 could bind to the promoter and directly activate the expression of CsOG3. In vivo repression of CsMYB44 also significantly reduced the cold tolerance of tea plants. This report comprehensively presented the architecture of tea plant orphan genes and highlighted the contribution of CsOG3 modulated by CsMYB44 against the cold stress in tea plants, broadening our understanding of plant orphan genes and the contribution in environmental adaptation.