Mechanism Analysis Of NAC Transcription Factors In Regulation Of Chilling Tolerance And Ripening In Banana Fruit

Banana is a typical climacteric fruit, characterized by a peak ethylene production that orchestrates ripening-associated processes, which results in a short post-harvest life at ambient temperature. Low temperature storage is the most effective method to reduce respiration and extend storage period of banana.However, banana fruits are very sensitive to low temperature and will develop chilling injury when temperature is below 13 ℃, resulting in reduced commercial quality.Therefore, understanding the mechanism of chilling tolerance and ripening in banana fruit is important for maintaining quality and extending shelf-life of the fruit.The plant-specific NAC(NAM, ATAF1/2 and CUC2) transcription factors(TFs) play important roles in plant growth, development and stress responses. Our previous research has shown that two NAC TFs, Ma NAC1 and Ma NAC2 are ethylene-inducible, and associated with banana fruit ripening. However, the regulatory network of Ma NAC1/2 ordinating propylene-induced cold tolerance andfruit ripening, andthe mechanisms of action of the other NAC TFs involved in fruit ripeningare largely unknown.In this study, we further analysis the mechanism of NAC TFs in regulation of chilling tolerance and ripening in banana fruit. The results are as follows.1. Ma NAC1 may be involved in propylene-induced cold tolerance of banana fruits through its interaction with ICE1-CBF cold signalling pathway. RT-q PCR showed that Ma NAC1 was noticeably induced by cold stress or following propylene treatment during cold storage. Transient protoplast assays showed that Ma NAC1 promoter was activated by cold stress and ethylene treatment. These data indicate that Ma NAC1 is cold inducible and might be related to propylene-induced cold tolerance of banana fruits. In addition, yeast one-hybrid(Y1H), electrophoretic mobility shift assay(EMSA) and transient expression assays demonstrated Ma NAC1 as a novel direct target of Ma ICE1, and that the ability of Ma ICE1 binding to Ma NAC1 promoter might be enhanced by Ma ICE1 phosphorylation and cold stress.2. Ma NAC1/Ma NAC2 and Ma XB3 may be antagonistically involved in ethylene biosynthesis and signaling during banana fruit ripening.Transcriptional activity assay showed that Ma NAC1 and Ma NAC2 are transcriptional repressors. EMSA, chromatin immunoprecipitation(Ch IP) and transient expression assays revealed that Ma NAC1 and Ma NAC2 repressed the expression of Ma RTH1 and Ma ERF11, which might be negative regulators of ethylene signaling and biosynthesis and fruit ripening, by direct binding to their promoters. Protein-protein interaction, ubiquitination and degradation assays showed the RING E3 Ligase Ma XB3 interacted with Ma NAC2, Ma ACS1 and Ma ACO1 and mediated the ubiquitinations of Ma NAC2, Ma ACS1 and Ma ACO1 for proteasome degradation,and therefore suppressed the transcriptional repression ability of Ma NAC2, and ethylene biosynthesis.Intriguingly, Ma NAC1 and Ma NAC2 antagonized Ma XB3 via directly binding to its promoter, and repressing its expression, suggesting a feedback regulatory mechanism of maintaining the stability of Ma NAC2, Ma ACS1 and Ma ACO1.3.Ma SPL1, Ma MADS1 and Ma NAC10-12 may be involved in banana fruit ripening through constituting regulatory cascades mediatingethylene biosynthesis.In order to identify banana NAC TF members whose mechanisms are different to Ma NAC1 and Ma NAC2 in regulation of fruit ripening, eleven NAC genes, termed Ma NAC7-Ma NAC17, were isolated from banana fruits. Based on expression patterns and transcriptional regulation activity, ethylene- and ripening-induced transcriptional activators Ma NAC10 and-11, and ethylene- and ripening-downregulated transcriptional repressors Ma NAC12,-13 and-14, were selected among these 11 NAC TFs. Ma NAC10 and Ma NAC11 cooperatively activated the expression of Ma ACS1 and Ma ACO1, whereas Ma NAC12 repressed Ma ACS1 and Ma ACO1.To further identify the upstream components of Ma NAC10-12, it was found that Ma SPL1 activated the expression of Ma NAC10 and Ma NAC11 by direct binding to their promoters, while Ma MADS1 might act as a transcription repressor of Ma NAC12 by suppressing its promoter activity.In addition, Ma XB3 mediated the ubiquitinations of Ma NAC10 and Ma NAC11 for proteasome degradation, and therefore suppressed the transcriptional activation ability of Ma NAC10/11 to Ma ACS1 or Ma ACO1.