MYC2, a basic helix‐loop‐helix (bHLH) transcription factor, is a key regulator in the activation of jasmonate (JA) response. However, the molecular details of MYC2 involving in methyl jasmonate (MeJA)‐induced chilling tolerance of fruit remain largely unclear. In the present work, two MYC2 genes, MaMYC2a and MaMYC2b, and one homolog of the inducer of the C‐repeat‐binding factor (CBF) gene, MaICE1 were isolated and characterized from banana fruit. MaMYC2s and MaICE1 were found to be all localized in the nucleus. In addition, the proline‐rich domain (PRD) and the acidic domain (AD) in the N‐terminus were important for the transcriptional activation of MaMYC2 in yeast cells. Unlike MaICE1's constitutive expression, MaMYC2a and MaMYC2b were induced rapidly following MeJA treatment during cold storage. Moreover, protein–protein interaction analysis confirmed that MaMYC2s interacted with MaICE1. The expression of ICE‐CBF cold‐responsive pathway genes including MaCBF1, MaCBF2, MaCOR1, MaKIN2, MaRD2 and MaRD5 was also significantly induced by MeJA. Taken together, our work provides strong evidence that MaMYC2 is involved in MeJA‐induced chilling tolerance in banana fruit through physically interacting and likely functionally coordinating with MaICE1, revealing a novel mechanism for ICE1 in response to cold stress as well as during development of induced chilling tolerance.

The involvement of ethylene response factor (ERF) transcription factor (TF) in the transcriptional regulation of ethylene biosynthesis genes during fruit ripening remains largely unclear. In this study, 15 ERF genes, designated as MaERF1–MaERF15, were isolated and characterized from banana fruit. These MaERFs were classified into seven of the 12 known ERF families. Subcellular localization showed that MaERF proteins of five different subfamilies preferentially localized to the nucleus. The 15 MaERF genes displayed differential expression patterns and levels in peel and pulp of banana fruit, in association with four different ripening treatments caused by natural, ethylene-induced, 1-methylcyclopropene (1-MCP)-delayed, and combined 1-MCP and ethylene treatments. MaERF9 was upregulated while MaERF11 was downregulated in peel and pulp of banana fruit during ripening or after treatment with ethylene. Furthermore, yeast-one hybrid (Y1H) and transient expression assays showed that the potential repressor MaERF11 bound to MaACS1 and MaACO1 promoters to suppress their activities and that MaERF9 activated MaACO1 promoter activity. Interestingly, protein–protein interaction analysis revealed that MaERF9 and -11 physically interacted with MaACO1. Taken together, these results suggest that MaERFs are involved in banana fruit ripening via transcriptional regulation of or interaction with ethylene biosynthesis genes.

The plant-specific NAC (NAM, ATAF1/2, and CUC2) transcription factors (TFs) play important roles in plant growth, development, and stress responses. However, the precise role of NAC TFs in relation to fruit ripening is poorly understood. In this study, six NAC genes, designated MaNAC1–MaNAC6, were isolated and characterized from banana fruit. Subcellular localization showed that MaNAC1–MaNAC5 proteins localized preferentially to the nucleus, while MaNAC6 was distributed throughout the entire cell. A transactivation assay in yeast demonstrated that MaNAC4 and MaNAC6, as well as their C-terminal regions, possessed trans-activation activity. Gene expression profiles in fruit with four different ripening characteristics, including natural, ethylene-induced, 1-methylcyclopropene (1-MCP)-delayed, and a combination of 1-MCP with ethylene treatment, revealed that the MaNAC genes were differentially expressed in peel and pulp during post-harvest ripening. MaNAC1 and MaNAC2 were apparently upregulated by ethylene in peel and pulp, consistent with the increase in ethylene production. In contrast, MaNAC3 in peel and pulp and MaNAC5 in peel were constitutively expressed, and transcripts of MaNAC4 in peel and pulp and MaNAC6 in peel decreased, while MaNAC5 or MaNAC6 in pulp increased slightly during fruit ripening. Furthermore, the MaNAC2 promoter was activated after ethylene application, further enhancing the involvement of MaNAC2 in fruit ripening. More importantly, yeast two-hybrid and bimolecular fluorescence complementation analyses confirmed that MaNAC1/2 physically interacted with a downstream component of ethylene signalling, ethylene insensitive 3 (EIN3)-like protein, termed MaEIL5, which was downregulated during ripening. Taken together, these results suggest that MaNACs such as MaNAC1/MaNAC2, may be involved in banana fruit ripening via interaction with ethylene signalling components.

Real-time reverse transcription PCR (RT-qPCR) is a preferred method for rapid and accurate quantification of gene expression studies. Appropriate application of RT-qPCR requires accurate normalization though the use of reference genes. As no single reference gene is universally suitable for all experiments, thus reference gene(s) validation under different experimental conditions is crucial for RT-qPCR analysis. To date, only a few studies on reference genes have been done in other plants but none in papaya. In the present work, we selected 21 candidate reference genes, and evaluated their expression stability in 246 papaya fruit samples using three algorithms, geNorm, NormFinder and RefFinder. The samples consisted of 13 sets collected under different experimental conditions, including various tissues, different storage temperatures, different cultivars, developmental stages, postharvest ripening, modified atmosphere packaging, 1-methylcyclopropene (1-MCP) treatment, hot water treatment, biotic stress and hormone treatment. Our results demonstrated that expression stability varied greatly between reference genes and that different suitable reference gene(s) or combination of reference genes for normalization should be validated according to the experimental conditions. In general, the internal reference genes EIF (Eukaryotic initiation factor 4A), TBP1 (TATA binding protein 1) and TBP2 (TATA binding protein 2) genes had a good performance under most experimental conditions, whereas the most widely present used reference genes, ACTIN (Actin 2), 18S rRNA (18S ribosomal RNA) and GAPDH (Glyceraldehyde-3-phosphate dehydrogenase) were not suitable in many experimental conditions. In addition, two commonly used programs, geNorm and Normfinder, were proved sufficient for the validation. This work provides the first systematic analysis for the selection of superior reference genes for accurate transcript normalization in papaya under different experimental conditions.

Summary: Although starch degradation has been well studied in model systems such as Arabidopsis leaves and cereal seeds, this process in starchy fruits during ripening, especially in bananas, is largely unknown. In this study, 38 genes encoding starch degradation‐related proteins were identified and characterized from banana fruit. Expression analysis revealed that 27 candidate genes were significantly induced during banana fruit ripening, with concomitant conversion of starch‐to‐sugars. Furthermore, iTRAQ‐based proteomics experiments identified 18 starch degradation‐associated enzymes bound to the surface of starch granules, of which 10 were markedly up‐regulated during ripening. More importantly, a novel bHLH transcription factor, MabHLH6, was identified based on a yeast one‐hybrid screening using MaGWD1 promoter as a bait. Transcript and protein levels of MabHLH6 were also increased during fruit ripening. Electrophoretic mobility shift assays, chromatin immunoprecipitation and transient expression experiments confirmed that MabHLH6 activates the promoters of 11 starch degradation‐related genes, including MaGWD1, MaLSF2, MaBAM1, MaBAM2, MaBAM8, MaBAM10, MaAMY3, MaAMY3C, MaISA2, MaISA3 and MapGlcT2‐2 by recognizing their E‐box (CANNTG) motifs present in the promoters. Collectively, these findings suggest that starch degradation during banana fruit ripening may be attributed to the complex actions of numerous enzymes related to starch breakdown at transcriptional and translational levels, and that MabHLH6 may act as a positive regulator of this process via direct activation of a series of starch degradation‐related genes.