Transcriptional and hormonal regulation of fruit flavour in apple and kiwifruit

Abstract

Transcriptional and hormonal regulation of fruit ripening was investigated in two climacteric fruit species, Malus x domestica Borkh. (apple) and Actinidia chinensis Planch. (kiwifruit). From apple, thirteen promoter fragments of ethylene biosynthesis genes (ACC synthase and ACC oxidase genes) and flavour and ripening genes such as MdAAT1 (alcohol acyl transferase), MdPG1 (polygalacturonase) and MdAFS1 (α-farnesene synthase) were cloned and screened against a panel of over eighty fruit expressed transcription factors (TFs). From kiwifruit, eleven ethylene biosynthesis and downstream flavour promoters were cloned and screened against over forty TFs to identify novel transcriptional regulators. In silico examination of cis-elements using PlantCARE identified a large selection of potential TF binding sites in all promoters cloned. Experiments using apple promoters confirmed that the SEPALLATA class transcription factor MdMADS9 activated the MdACS1 promoter in planta. Additionally, this promoter was activated by the related genes MdMADS6 and MdMADS8. All three TFs also activated the ethylene biosynthesis promoters MdACO1, MdACS3a and several downstream ripening genes such as MdAAT1, MdAFS1 and MdPG1. This shows the central importance of MADS box genes in activation of ethylene biosynthesis, as well as other ripening related genes. The screening of the apple promoters uncovered a large set of additional ripening activators including HMG (High Mobility Group) and NAC TFs, and many repressors from a number of different TF classes. The transcriptional regulation of apple ethylene biosynthesis and ripening is thus complex, and screening results suggest that apart from activation by MADS and ethylene, repression by auxin related TFs such as AUX/IAA and bZIPs may also be critical. In kiwifruit, ethylene biosynthesis during wounding and autocatalytic fruit ripening appears to be regulated by NAC TFs. Based on expression analysis, AcACS1 appears to be the most important ACC synthase gene in both processes, while AcACS2 is also induced. AcACS3 is only induced during the late autocatalytic ripening phase. Gel shift assays combined with promoter analysis shows that NAC TFs can bind directly to the AcACS1 promoter. NAC expression shows strong correlation with ACS and ACO expression, as well as ethylene release. Furthermore, promoter screening showed that these NAC TFs, as well as EIN3-like TFs (the downstream signalling component of the ethylene signalling pathway), can activate ripening genes such as AaTPS1 and AcDXS1 involved in terpene synthesis as well as the promoters of two ripening induced TFs, AcRIL and AcNAC3. Gel shift assays showed direct binding of NAC TFs to the AaTPS1 promoter. In kiwifruit there appears to be a central role for NAC TFs in controlling both wound and autocatalytic ethylene biosynthesis, as well as directly affecting ripening genes such as AaTPS1 and AcDXS1. Ripening genes also appear to be affected indirectly through ethylene signalling (EIN3-like TFs) during the autocatalytic ripening phase. A model is presented which compares and contrasts the transcriptional and hormonal network in apple and kiwifruit, occurring when the fruit ripens. This is compared to tomato, a model species for the study of ethylene regulated fruit ripening.