Two banana fruit ripening-related C2H2 zinc finger proteins are transcriptional repressors of ethylene biosynthetic genes

• Two C2H2-ZFPs genes, named as MaC2H2-1/2, were identified and characterized from banana fruit.
• MaC2H2-1/2 localized preferentially to the nucleus.
• MaC2H2-1/2 were transcriptional repressors.
• Ripening-induced MaC2H2-1/2 bound to the promoters of the key ethylene biosynthetic genes MaACS1 and MaACO1, and repressed their activities.

C2H2 zinc finger proteins (C2H2-ZFPs), as one of the largest TF families in eukaryotes, play an essential role in plant development and stress responses, but their involvement in the ripening of economically important fruits, as well as their transcriptional regulatory mechanisms, remain largely unclear. In this study, two C2H2-ZFPs, named as MaC2H2-1/2, were identified and characterized from banana fruit. MaC2H2-1/2 contain a typical ERF-associated amphiphilic repression (EAR) motif at their C-terminus. Subcellular localization demonstrated that MaC2H2-1/2 proteins were both localized to the nucleus. Dual-luciferase reporter assay (DLR) showed that MaC2H2-1/2 were transcriptional repressors in vivo. Gene expression analysis revealed a significant induction of MaC2H2-1/2 transcripts during the ripening of banana fruit with three different ripening characteristics caused by natural, ethylene-induced, and 1-methylcyclopropene (1-MCP)-delayed treatments, which correlated well with ethylene production. More importantly, electrophoretic mobility shift assay (EMSA) and transient expression showed that MaC2H2-1/2 bound to the promoters of the key ethylene biosynthetic genes MaACS1 and MaACO1, and repressed their activities. Collectively, these results suggest that MaC2H2-1/2 are transcriptional repressors and may mediate a finely tuned regulation of ethylene production during banana fruit ripening, possibly via transcriptional repression of ethylene biosynthetic genes, expanding the knowledgement of the involvement of C2H2 zinc finger proteins in fruit ripening.