Copy number variants in kiwifruit ETHYLENE RESPONSE FACTOR/APETALA2 (ERF/AP2)-like genes show divergence in fruit ripening associated cold and ethylene responses in C-REPEAT/DRE BINDING FACTOR-like genes

The ETYHLENE RESPONSE FACTOR/APETALA2 (ERF/AP2) transcription factors have been shown to control a wide range of developmental and environmental responses in plants. These include hormonal responses to ethylene and Abscisic Acid (ABA) as well as to cold and drought. In Actinidia chinensis (kiwifruit), ripening is unusual: although it is sometimes classed as a climacteric fruit (ethylene-associated ripening), much of fruit ripening occurs independently from autocatalytic ethylene production. Initiation of ripening appears to be strongly developmentally controlled and modulated by low temperature. In this study, fruit treated with different temperatures showed an increase in soluble sugar accumulation, and a corresponding increase in ß-AMYLASE (BAM) genes (predominantly BAM3.2 and BAM9) with lower temperatures. To investigate the potential role of the AP2/ERF gene family in the control of fruit ripening in kiwifruit this family was investigated further. Using the new genome annotation and further genome sequence analysis we identified 226 ERF-like genes, 10 AP2L/RAV-like genes and 32 AP2-like genes. An RNA-seq screen from kiwifruit of different maturities, and following treatment with ethylene and temperatures between 0 and 16°C, revealed 4%, 26% and 18% of the ERF-like genes were upregulated by maturation, ethylene and cold temperatures, respectively. Focusing on the C-REPEAT/DRE BINDING FACTOR (CBF) cold master regulators, nine potential genes were identified based on sequence similarity. Five of these CBF-like genes were found in a copy number variant (CNV) cluster of six genes on chromosome 14. Expression analysis showed that two homeologous genes (ERF41 and ERF180) increased in abundance with cold and ethylene, while the cluster of CNV CBF-like genes had lost the ability to respond to cold and increased only with ethylene, suggesting an evolutionary progression of function of these genes.