Involvement of CsERF2 in leaf variegation of Cymbidium sinense ‘Dharma’

Main conclusion CsERF2, an ethylene response factor, plays a role in leaf variegation. Leaf variegation is a main ornamental characteristic in Cymbidium sinense ( C . sinense ). However, the mechanisms of leaf color variegation remain largely unclear. In the present study, we analyzed the cytological and physiological features, as well as molecular analyses of leaves from wild-type (WT) and leaf variegation mutants of Cymbidium sinense ‘ Dharma ’. Chloroplasts with typical and functional structures were discovered in WT and green sectors of the mutants leaves (MG), but not in yellow sectors of the mutant leaves (MY). The activities of key enzymes involved in chlorophyll (Chl) degradation and their substrate contents were significantly increased in MY. Genes related to Chl degradation also showed a significant up-regulation in MY. Transcriptomic analysis showed that the expression of all identified ethylene response factors (ERFs) was significantly up-regulated, and the 1-aminocyclopropane-1-carboxylic acid (ACC) content in MY was significantly higher compared with MG. QRT-PCR analysis validated that the expression levels of genes related to Chl degradation could be positively affected by ethylene (ETH) treatment. Stable overexpression of CsERF2 in Nicotiana tabacum ( N. tabacum ) led to a decrease in Chl content and abnormal chloroplast. Transcriptomic analysis and qRT-PCR results showed that the KEGG pathway related to chloroplast development and function showed significant change in transgenic N. tabacum . Therefore, the leaf color formation of C . sinense was greatly affected by chloroplast development and Chl metabolism. CsERF2 played an important role in leaf variegation of C . sinense .