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Soluble sugars are an important determinant of fruit taste, but their accumulation mechanisms remain elusive. In this study, we report two vacuolar invertase inhibitor genes involved in sugar accumulation in peach, PpINHa and PpINH3. Transient overexpression of PpINH3 in peach fruits resulted in an increase in sugar content, while the opposite trend was detected for PpINHa. Unexpectedly, PpINH3 and PpINHa both had no physical interaction with vacuolar invertase (VIN). Moreover, the PpVIN genes had no or extremely low expression in fruits at the ripening stage. These results suggested that the regulatory role of PpINHa and PpINH3 in sugar accumulation is unlikely due to their interaction with PpVINs. Additionally, overexpression of PpINHa and PpINH3 had an impact on transcription of genes related to fruit sugar metabolism and transport, which is likely responsible for their regulatory role in fruit sugar accumulation. Altogether, these results indicated an important role of PpINHs in fruit accumulation in peach. Our study provides new insights into molecular mechanisms underlying sugar accumulation, which could be useful for genetic improvement of fruit taste in breeding programs of peach and other fruit crops. [ABSTRACT FROM AUTHOR]

SUMMARY: Fruit ripening is a complex developmental process, which is modulated by both transcriptional and post‐translational events. Control of fruit ripening is important in maintaining moderate quality traits and minimizing postharvest deterioration. In this study, we discovered that the transcription factor MaMYB4 acts as a negative regulator of fruit ripening in banana. The protein levels of MaMYB4 decreased gradually with banana fruit ripening, paralleling ethylene production, and decline in firmness. DNA affinity purification sequencing combined with RNA‐sequencing analyses showed that MaMYB4 preferentially binds to the promoters of various ripening‐associated genes including ethylene biosynthetic and cell wall modifying genes. Furthermore, ectopic expression of MaMYB4 in tomato delayed tomato fruit ripening, which was accompanied by downregulation of ethylene biosynthetic and cell wall modifying genes. Importantly, two RING finger E3 ligases MaBRG2/3, whose protein accumulation increased progressively with fruit ripening, were found to interact with and ubiquitinate MaMYB4, contributing to decreased accumulation of MaMYB4 during fruit ripening. Transient overexpression of MaMYB4 and MaBRG2/3 in banana fruit ripening delayed or promoted fruit ripening by inhibiting or stimulating ethylene biosynthesis, respectively. Taken together, we demonstrate that MaMYB4 negatively modulates banana fruit ripening, and that MaMYB4 abundance could be regulated by protein ubiquitination, thus providing insights into the role of MaMYB4 in controlling fruit ripening at both transcriptional and post‐translational levels. [ABSTRACT FROM AUTHOR]

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Summary: Fruit ripening is a critical phase in the production and marketing of fruits. Previous studies have indicated that fruit ripening is a highly coordinated process, mainly regulated at the transcriptional level, in which transcription factors play essential roles. Thus, identifying key transcription factors regulating fruit ripening as well as their associated regulatory networks promises to contribute to a better understanding of fruit ripening. In this study, temporal gene expression analyses were performed to investigate banana fruit ripening with the aim to discern the global architecture of gene regulatory networks underlying fruit ripening. Eight time points were profiled covering dynamic changes of phenotypes, the associated physiology and levels of known ripening marker genes. Combining results from a weighted gene co‐expression network analysis (WGCNA) as well as cis‐motif analysis and supported by EMSA, Y1H, tobacco‐, banana‐transactivation experimental results, the regulatory network of banana fruit ripening was constructed, from which 25 transcription factors were identified as prime candidates to regulate the ripening process by modulating different ripening‐related pathways. Our study presents the first global view of the gene regulatory network involved in banana fruit ripening, which may provide the basis for a targeted manipulation of fruit ripening to attain higher banana and loss‐reduced banana commercialization. [ABSTRACT FROM AUTHOR]

Key message: Banana MaBZR1/2 interact with MaMPK14 to enhance the transcriptional inhibition of cell wall modifying genes including MaEXP2, MaPL2 and MaXET5. Fruit ripening and softening, the major attributes to perishability in fleshy fruits, are modulated by various plant hormones and gene expression. Banana MaBZR1/2, the central transcription factors of brassinosteroid (BR) signaling, mediate fruit ripening through regulation of ethylene biosynthesis, but their possible roles in fruit softening as well as the underlying mechanisms remain to be determined. In this work, we found that MaBZR1/2 directly bound to and repressed the promoters of several cell wall modifying genes such as MaEXP2, MaPL2 and MaXET5, whose transcripts were elevated concomitant with fruit ripening. Moreover, yeast two-hybrid (Y2H) and bimolecular fluorescence complementation (BiFC) assays indicated that MaBZR1/2 physically interacted with a mitogen-activated protein kinase MaMPK14, and this interaction strengthened the MaBZR1/2-mediated transcriptional inhibitory abilities. Collectively, our study provides insight into the mechanism of MaBZR1/2 contributing to fruit ripening and softening, which may have potential for banana molecular improvement. [ABSTRACT FROM AUTHOR]

Banana fruit (Musa acuminate L.) ripening is a complex genetical process affected by multiple phytohormones and expression of various genes. However, whether plant hormone brassinosteroid (BR) is involved in this process remains obscure. In this work, three genes that encode BR core signaling components brassinazole resistant (BZR) proteins, namely MaBZR1 to MaBZR3, were characterized from banana fruit. MaBZR1‐MaBZR3 exhibited both nuclear and cytoplasmic localization and behaved as transcription inhibitors. Expression analysis showed that MaBZR1/2/3 were continuously decreased as fruit ripening proceeded, indicating their negative roles in banana ripening. Moreover, gel shift and transient expression assays demonstrated that MaBZR1/2 could suppress the transcription of ethylene biosynthetic genes, including MaACS1, MaACO13 and MaACO14, which increased gradually during the banana ripening, via specifically binding to CGTGT/CG sequence in their promoters. Importantly, exogenous application of BRs promotes banana ripening, which is presumably due to the accelerated expression of MaACS1 and MaACO13/14, and consequently the ethylene production. Our study indicates that MaBZR1/2 act as transcriptional repressors of ethylene biosynthetic genes during banana fruit ripening. [ABSTRACT FROM AUTHOR]

Summary: Starch degradation is a necessary process determining banana fruit quality during ripening. Many starch degradation‐related genes are well studied. However, the transcriptional regulation of starch degradation during banana fruit ripening remains poorly understood. In this study, we identified a MYB transcription factor (TF) termed MaMYB3, as a putative protein binding the promoter of MaGWD1, a member of glucan water dikinase (GWD) family which has been demonstrated as an important enzyme of starch degradation. MaMYB3 was ripening‐ and ethylene‐repressible, and its expression was negatively correlated with starch degradation. Acting as a nucleus‐localized transcriptional repressor, MaMYB3 repressed the transcription of 10 starch degradation‐related genes, including MaGWD1, MaSEX4, MaBAM7‐MaBAM8, MaAMY2B, MaAMY3, MaAMY3A, MaAMY3C, MaMEX1, and MapGlcT2‐1, by directly binding to their promoters. Interestingly, a previously identified activator of starch degradation‐related genes, MabHLH6, was also suppressed by MaMYB3. The ectopic overexpression of MaMYB3 in tomato down‐regulated the expression of starch degradation‐related genes, inhibited starch degradation and delayed fruit ripening. Based on these findings, we conclude that MaMYB3 negatively impacts starch degradation by directly repressing starch degradation‐related genes and MabHLH6, and thereby delays banana fruit ripening. Collectively, our study expands our understanding of the complex transcriptional regulatory hierarchy modulating starch degradation during fruit ripening. Significance Statement: MaMYB3 negatively impacts starch degradation by the direct repression of MabHLH6 and starch degradation‐related genes, and thus delays banana fruit ripening. Our findings illustrate the complex transcriptional regulatory hierarchy modulating starch degradation during fruit ripening. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Fruit ripening is a complex, genetically programmed process involving the action of critical transcription factors (TFs). Despite the established significance of dehydration-responsive element binding (DREB) TFs in plant abiotic stress responses, the involvement of DREBs in fruit ripening is yet to be determined., Here, we identified four genes encoding ripening-regulated DREB TFs in banana ( Musa acuminata), MaDREB1, MaDREB2, MaDREB3, and MaDREB4, and demonstrated that they play regulatory roles in fruit ripening., We showed that MaDREB1-MaDREB4 are nucleus-localized, induced by ethylene and encompass transcriptional activation activities. We performed a genome-wide chromatin immunoprecipitation and high-throughput sequencing (ChIP-Seq) experiment for MaDREB2 and identified 697 genomic regions as potential targets of MaDREB2. MaDREB2 binds to hundreds of loci with diverse functions and its binding sites are distributed in the promoter regions proximal to the transcriptional start site (TSS). Most of the MaDREB2-binding targets contain the conserved (A/G)CC(G/C)AC motif and MaDREB2 appears to directly regulate the expression of a number of genes involved in fruit ripening. In combination with transcriptome profiling (RNA sequencing) data, our results indicate that MaDREB2 may serve as both transcriptional activator and repressor during banana fruit ripening., In conclusion, our study suggests a hierarchical regulatory model of fruit ripening in banana and that the MaDREB TFs may act as transcriptional regulators in the regulatory network. [ABSTRACT FROM AUTHOR]

Our previous studies have indicated that the banana ripening-induced MaNAC1, a NAC ( NAM, ATAF1/2 and CUC2) transcription factor ( TF) gene, is regulated by ethylene during fruit ripening, and propylene, a functional ethylene analogue, induces cold tolerance of banana fruits. However, the involvement of MaNAC1 in propylene-induced cold tolerance of banana fruits is not understood. In the present work, the possible involvement of MaNAC 1 in cold tolerance of banana fruits was investigated. MaNAC1 was noticeably induced by cold stress or following propylene treatment during cold storage. Transient protoplast assays showed that MaNAC1 promoter was activated by cold stress and ethylene treatment. Yeast one-hybrid ( Y1 H), electrophoretic mobility shift assay ( EMSA) and transient expression assays demonstrated MaNAC1 as a novel direct target of MaICE1, and that the ability of MaICE1 binding to MaNAC1 promoter might be enhanced by MaICE1 phosphorylation and cold stress. Moreover, yeast two-hybrid ( Y2 H) and bimolecular fluorescence complementation ( BiFC) analyses revealed physical interaction between MaNAC1 and MaCBF1, a downstream component of inducer of C-repeat binding factor (CBF) expression 1 ( ICE1) in cold signalling. Taken together, these results suggest that the cold-responsive MaNAC1 may be involved in cold tolerance of banana fruits through its interaction with ICE1- CBF cold signalling pathway, providing new insights into the regulatory activity of NAC TF. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Harvested banana fruit ripened under warm temperatures above 24 °C remain green peel, leading to severe economic loss. E3 ubiquitin-ligases, as the major components in the ubiquitination pathway, have been implicated to play important roles in temperature-stress responses. However, the molecular mechanism underlying high temperature-triggered stay-green ripening bananas in association with E3 ubiquitin-ligases, remains largely unknown. In this study, a RING-type E3 ubiquitin ligase termed MaLUL2, was isolated and characterized from banana fruit. The MaLUL2 gene contains 1095 nucleotides and encodes a protein with 365 amino acids. The MaLUL2 protein contains a domain associated with RING2 (DAR2) and a RING domain, which are the typical characteristics of RING-type E3 ligases. MaLUL2 expression was up-regulated during high temperature-induced green ripening. Subcellular localization showed that MaLUL2 localized in the nucleus, cytoplasm, and plasma membrane. MaLUL2 displayed E3 ubiquitin ligase activity in vitro. More importantly, transient overexpression of MaLUL2 in banana fruit peel increased the level of ubiquitination in vivo and led to a stay-green phenotype, accompanying with decreased expression of chlorophyll catabolic genes. Collectively, these findings suggest that MaLUL2 might act as a negative regulator of chlorophyll degradation and provide novel insights into the regulatory mechanism of high temperature-induced green ripening bananas. [ABSTRACT FROM AUTHOR]

Graphical model for the role of MaXB3-MaNAC029 module in linking ethylene biosynthesis and quality formation during banana fruit ripening. [Display omitted] • MaNAC029 trans -activates ethylene biosynthetic genes. • MaNAC029 enhances genes involved in metabolic processes related to fruit quality formation. • MaXB3 attenuates MaNAC029-mediated enhancements by proteasomal degradation of MaNAC029. • MaXB3-MaNAC029 connects ethylene biosynthesis with fruit quality formation during ripening. Ethylene regulates ripening by activating various metabolic pathways that controlcolor, aroma, flavor, texture, and consequently, the quality of fruits. However, the modulation of ethylene biosynthesis and quality formation during banana fruit ripening remains unclear. The present study aimed to identify the regulatory module that regulates ethylene and fruit quality-related metabolisms during banana fruit ripening. We used RNA-seq to compare unripe and ripe banana fruits and identified a ripening-induced NAC transcription factor, MaNAC029. We further performed DNA affinity purification sequencing to identify the MaNAC029′s target genes involved in ethylene biosynthesis and fruit quality formation, and electrophoretic mobility shift assay, chromatin immunoprecipitation with real-time polymerase chain reaction and dual luciferase assays to explore the underlying regulatory mechanisms. Immunoprecipitation combined with mass spectrometry, yeast two-hybrid assay, and bimolecular fluorescence complementation assay were used to screen and verify the proteins interacting with MaNAC029. Finally, the function of MaNAC029 and its interacting protein associated with ethylene biosynthesis and quality formation was verified through transient overexpression experiments in banana fruits. The study identified a nucleus-localized, ripening-induced NAC transcription factor MaNAC029. It transcriptionally activated genes associated with ethylene biosynthesis and a variety of cellular metabolisms related to fruit quality formation (cell wall degradation, starch degradation, aroma compound synthesis, and chlorophyll catabolism) by directly modulating their promoter activity during ripening. Overexpression of MaNAC029 in banana fruits activated ethylene biosynthesis and accelerated fruit ripening and quality formation. Notably, the E3 ligase MaXB3 interacted with and ubiquitinated MaNAC029 protein, facilitating MaNAC029 proteasomal degradation. Consistent with this finding, MaXB3 overexpression attenuated MaNAC029-enhanced ethylene biosynthesis and quality formation. Our findings demonstrate that a MaXB3-MaNAC029 module regulates ethylene biosynthesis and a series of cellular metabolisms related to fruit quality formation during banana ripening. These results expand the understanding of the transcriptional and post-translational mechanisms of fruit ripening and quality formation. [ABSTRACT FROM AUTHOR]

Banana (Musa acuminata) fruits ripening at 30 °C or above fail to develop yellow peels; this phenomenon, called green ripening, greatly reduces their marketability. The regulatory mechanism underpinning high temperature-induced green ripening remains unknown. Here we decoded a transcriptional and post-translational regulatory module that causes green ripening in banana. Banana fruits ripening at 30 °C showed greatly reduced expression of 5 chlorophyll catabolic genes (CCGs), MaNYC1 (NONYELLOW COLORING 1), MaPPH (PHEOPHYTINASE), MaTIC55 (TRANSLOCON AT THE INNER ENVELOPE MEMBRANE OF CHLOROPLASTS 55), MaSGR1 (STAY-GREEN 1), and MaSGR2 (STAY-GREEN 2), compared to those ripening at 20 °C. We identified a MYB transcription factor, MaMYB60, that activated the expression of all 5 CCGs by directly binding to their promoters during banana ripening at 20 °C, while showing a weaker activation at 30 °C. At high temperatures, MaMYB60 was degraded. We discovered a RING-type E3 ligase MaBAH1 (benzoic acid hypersensitive 1) that ubiquitinated MaMYB60 during green ripening and targeted it for proteasomal degradation. MaBAH1 thus facilitated MaMYB60 degradation and attenuated MaMYB60-induced transactivation of CCGs and chlorophyll degradation. By contrast, MaMYB60 upregulation increased CCG expression, accelerated chlorophyll degradation, and mitigated green ripening. Collectively, our findings unravel a dynamic, temperature-responsive MaBAH1–MaMYB60–CCG module that regulates chlorophyll catabolism, and the molecular mechanism underpinning green ripening in banana. This study also advances our understanding of plant responses to high-temperature stress. [ABSTRACT FROM AUTHOR]

Starch is an essential natural carbohydrate source for human beings and the main storage carbohydrate for bananas during growth and development. Starch degradation is an indispensable cause of banana fruit softening and flavor formation. Although AP2/ERF transcription factors (TFs) contribute to texture, color, and flavor changes during fruit ripening, the mechanism of AP2/ERF TFs regulating starch degradation in bananas is not fully understood. Here, we identified an EDLL motif-containing AP2/ERF member MaERF96L in banana fruit, and found its expression was upregulated by ethylene during ripening. Meanwhile, MaERF96L was a nuclear-localized protein and acted as a transcriptional activator. Importantly, further in vitro and vivo assays showed that MaERF96L directly bound to the promoter of one starch degradation enzyme gene MaGWD1 and enhanced its transcription. Moreover, MaERF96L transient overexpression in banana fruit up-regulated MaGWD1 expression and promoted starch degradation, which in turn contributed to advance ripening. In summary, these findings demonstrate that MaERF96L could be involved in starch degradation pathway during banana fruit ripening, partly through the positive transcriptional regulation of MaGWD1 , thereby expanding our understanding towards the regulatory network of fruit starch degradation. • A transcriptional activator MaERF96L is identified from banana fruit. • MaERF96L directly targets MaGWD1 and activates its transcription. • MaERF96L transient overexpression in banana fruit promotes starch degradation during ripening. [ABSTRACT FROM AUTHOR]

Fleshy fruit undergoes a complex ripening process accompanied with changes in aroma, color, flavor, and texture, which is regulated by hormonal, genetic, and environmental factors. However, the transcriptional cascade mechanism underlying fruit ripening regulation remains obscure. Here, we characterized a transcriptional repressor, MaMADS68, screened from transcriptomic data related to low-temperature-affected ripening. In vitro and in vivo assays revealed that MaMADS68 binds directly to the promoters of a subset of fruit ripening-related genes, including MaAAT1 , MaEXP2 , MaEXP15L , MaXTH23L , MaXTH25L , MaACS1 , along with a MaWRKY49 that activates MaACS1 expression, to repress their transcription. Notably, yeast one-hybrid screening assay revealed that MaWRKY147, another ripening-induced transcriptional repressor, directly binds to and represses the promoter of MaMADS68 , weakening MaMADS68′s repressive activity on ripening-related target genes owing to their expression and thus resulting in fruit ripening delay. Collectively, the results of this study illustrate the molecular basis of the transcriptional cascade MaWRKY147-MaMADS68 in modulating low-temperature-affected banana fruit ripening, advancing our understanding of the transcriptional regulatory mechanisms underlying fruit ripening. • Pre-storaged at low temperature (7 °C) hinders banana fruit ripening. • A transcriptional repressor MaMADS68 was identified from banana fruit. • MaMADS68 represses the transcription of fruit ripening-related target genes. • MaWRKY147 directly represses the transcription of MaMADS68. [ABSTRACT FROM AUTHOR]

Sucrose, a predominant sweetener in banana (Musa acuminata) fruit, determines sweetness and consumer preferences. Although sucrose phosphate synthase (SPS) is known to catalyze starch conversion into sucrose in banana fruit during the ripening process, the SPS regulatory mechanism during ripening still demands investigation. Hence, this study discovered that the MaSPS1 expression was promoted during ethylene-mediated ripening in banana fruit. MaNAC19, recognized as the MaSPS1 putative binding protein using yeast one-hybrid screening, directly binds to the MaSPS1 promoter, thereby transcriptionally activating its expression, which was verified by transient overexpression experiments, where the sucrose synthesis was accelerated through MaNAC19-induced transcription of MaSPS1. Interestingly, MaXB3, an ethylene-inhibited E3 ligase, was found to ubiquitinate MaNAC19, making it prone to proteasomal degradation, inhibiting transactivation of MaNAC19 to MaSPS1 , thereby attenuating MaNAC19-promoted sucrose accumulation. This study's findings collectively illustrated the mechanistic basis of a MaXB3-MaNAC19- MaSPS1 regulatory module controlling sucrose synthesis during banana fruit ripening. These outcomes have broadened our understanding of the regulation mechanisms that contributed to sucrose metabolism occurring in transcriptional and post-transcriptional stages, which might help develop molecular approaches for controlling ripening and improving fruit quality. [ABSTRACT FROM AUTHOR]

• CT inhibits green ripening in bananas under high temperature storage. • CT upregulates expression of chlorophyll catabolic genes and MaCBF1. • MaCBF1 directly activates the transcription of chlorophyll catabolic genes. • Transient expression of MaCBF1 alleviates high temperature-caused green ripening. Inhibition of peel de-greening in postharvest bananas under high temperature storage, resulting in green ripening, causes significant deterioration in fruit quality. Herein, we reported that cold treatment accelerated chlorophyll degradation of postharvest banana fruit at 30 °C, which was associated with the upregulated expression of MaCBR (Chlorophyll b reductase) and MaSGR1 (Stay-green 1). Moreover, cold treatment increased the expression of C-repeat binding factor MaCBF1. MaCBF1 bound directly to the promoters of MaCBR and MaSGR1 and activated their expressions. More importantly, transient expression of MaCBF1 in bananas enhanced chlorophyll degradation and weakened the repression of de-greening caused by high temperature. In summary, the cold treatment promotes chlorophyll catabolism by activating MaCBF1-induced transcriptional activation of MaCBR and MaSGR1 , and attenuates high temperature-caused green ripening in bananas. These results study expand the understanding of the molecular events of high temperature-inhibited chlorophyll degradation and provide a feasible strategy to alleviate green ripening of banana fruit. [ABSTRACT FROM AUTHOR]

Deacetylation of core histone proteins mediated by histone decetylases (HDACs) is an important machinery in regulating gene repression in multiple plant developmental processes including fruit ripening and senescence. The ripening of banana fruit is a highly coordinated and developmentally programmed event which involves the genetic and epigenetic regulation of ripening-associated genes. Despite the biological functions of HDACs in model plants have been extensively studied, knowledge about HDAC gene family in banana is very limited. In this work, a total of 17 HDACs were identified in the genome of banana, among which 12, 2 and 3 members are grouped into RPD3/HDA1, SIR2 and HD2 sub-families respectively, based on the sequence similarity and phylogeny. Expression profiling revealed that these MaHDAC s were differentially expressed during banana fruit ripening. Of these MaHDACs , MaHDA6 showed the highest inducement in fruit ripening and thus was selected for further study. MaHDA6 was localized within the nucleus and contained histone deacetylase activity in plant cells. Furthermore, chromatin immunoprecipitation (ChIP) assays indicated that MaHDA6 was capable of binding to the promoters of MaERF11/15 , the genes in ethylene signaling pathway. Particularly, the levels of acetylation of histones H3 and H4 in MaERF11/15 ’s regulatory regions were obviously decreased at ripening stage, which was in accordance with their decreased expression levels. Taken together, our findings suggest that various MaHDACs have distinct expression patterns during banana ripening and that MaHDA6 might be involved in the ripening presumably via regulating the transcription of MaERF11/15 by histone deacetylation. [ABSTRACT FROM AUTHOR]

Fruit ripening and softening is a complex physiological process that is governed by the expression of a wide range of genes, and histone modification is a critical mechanism for precise gene expression. Although previous studies have indicated that NAC transcription factor (TF) and histone deacetylase individually play important roles in fruit ripening, the molecular connection between these two proteins within the regulatory system underlying banana fruit ripening is poorly understood. Here, we characterized a banana NAC MaNAC154 that was a negative regulator of banana fruit ripening. MaNAC154 was shown to localize in the nucleus, and it could target the promoters of MaEXP1/2 , MaPL2 , MaPG1/X3 and MaXTH5/23/28 to repress their transcription. Importantly, MaNAC154 was found to interact with a histone deacetylase MaHDA6, and their interaction significantly enhanced MaNAC154-mediated transcriptional repression capacity. Moreover, the acetylation levels of histones H3 and H4 of MaEXP1/2 , MaPL2 , MaPG1/X3 and MaXTH5/23/28 were elevated in the ripening stage. Overall, our data establish a coordinated mechanism underpinning histone deacetylation and TF-mediated gene repression for banana fruit ripening, providing a novel molecular basis for controlling mechanism of fruit ripening and softening. [Display omitted] • A transcriptional repressor MaNAC154 was identified from banana fruit. • MaNAC154 represses the transcription of cell wall modifying genes. • The first report of a regulatory model for the interaction between NAC and HDAC. • MaHDA6 enhances MaNAC154 transcriptional repression via histone deacetylation. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Highlights: [•] Two EBF genes designated as MaEBF1 and MaEBF2 were isolated and characterized from banana fruit. [•] MaEBF1 and MaEBF2 showed differential expression patterns during fruit ripening. [•] Promoter activity of MaEBF2 was activated by ethylene application. [•] MaEBF2 was able to physically interact with MaEIL5. [Copyright &y& Elsevier]

• MaRTH1 was down-regulated in the banana fruit ripening process. • Transient over-expression of MaRTH1 delayed banana fruits ripening. • MaNAC2 directly repressed MaRTH1 expression. • MaXB3 suppresses the ability of MaNAC2 in repressing the transcription of MaRTH1. RTE1 (Reversion to Ethylene Sensitivity 1) and its homolog RTH play important role in ethylene response through mediating the receptor signaling output, but their role in climacteric fruit ripening is not clear. In this study, we found two RTE1/RTH genes MaRTH1 and MaRTH2 in banana genome. MaRTH1 expression was inhibited by ethylene and down-regulated during ripening, whereas MaRTH2 expression showed no significant changes during the whole ripening process. Transient over-expression of MaRTH1 in bananas inhibited the effect of exogenous ethylene on endogenous ethylene production, thereby delayed fruit ripening. Using MaRTH1 promoter as a target, we performed yeast one-hybrid screening and isolated the NAC transcription factor MaNAC2, a banana ripening-associated transcriptional repressor degraded by an E3 ligase MaXB3 through ubiquitination. DNA-protein binding assays further confirmed that MaNAC2 repressed the expression of MaRTH1 by directly targeting its promoter. Moreover, the MaNAC2 transcriptional repression to MaRTH1 was inhibited by MaXB3, and this inhibitory action was attenuated by the proteasome inhibitor MG132. Collectively, these findings reveal that MaRTH1 , which is modulated by MaXB3-MaNAC2 regulatory module, functions as a negative regulator of ethylene response during banana ripening. [ABSTRACT FROM AUTHOR]

• Sizes of starch granules and crystallinity were reduced during ripening. • Activities and transcripts of amylase and isoamylase were enhanced in fruit ripening. • MaEIL2 trans-activates the transcription of MaAMY3 , MaISA2 and MaISA3. Starch is the major reserve carbohydrate in nature, which possesses nutritional property and industrial applications. Starch degradation is an important contributor to softening and sweetening of banana fruit, but more details in this process are not fully understood. In this study, the contents of total starch, amylose and amylopectin were gradually decreased during banana fruit ripening, which is in parallel with the increased levels of total soluble sugars. Particularly, reduced sizes and elongated shapes of starch granules, as well as decreased crystallinity were observed as ripening proceeds, which is largely due to the digestion of starch degradation enzymes such as amylase and isoamylase. Importantly, an ethylene signaling component MaEIL2 bound to the promoters of amylase and isoamylase encoding genes MaAMY3 , MaISA2 and MaISA3 , and stimulated their transcription. Overall, these findings reveal that starch-sugar transformation during banana ripening is mediated by enzymatic hydrolysis, and that ethylene signaling component MaEIL2 positively modulates starch breakdown via trans-activation of MaAMY3 , MaISA2 and MaISA3. [ABSTRACT FROM AUTHOR]

• MabZIP74 is gradually decreased during banana ripening. • MabZIP74 is a nuclear protein with transcriptional repression ability. • MabZIP74 binds to and represses the promoters of MaACO1/4. • MaMAPK11-3 interacts with and phosphorylates MabZIP74. Fruit ripening is an economically important process of many edible fruits, which is controlled by a multi-level regulatory network coordinating the spatio-temporal expression of a large array of genes and/or proteins. In banana, ethylene orchestrates the ripening process, but the regulatory mechanism(s) controlling the expression of genes encoding ethylene biosynthesis-related enzymes is not fully understood. Here, we report the identification and characterization of a basic leucine zipper (bZIP) transcription factor MabZIP74 from banana fruit. Its gradually decreased with the progression of banana fruit ripening, implying a negative role for MabZIP74 in banana fruit ripening. MabZIP74 is localized in the nucleus and displays transcriptional inhibitory activity. More specifically, MabZIP74 was found to be a transcriptional repressor of ethylene biosynthetic genes MaACO1 and MaACO4 , the accumulation of which paralleled ethylene production during banana ripening. Importantly, a protein kinase MaMAPK11-3 interacted with and phosphorylated MabZIP74, which attenuated MabZIP74-mediated transcriptional repression of MaACO1 and MaACO4 during ripening. Taken together, our findings reveal a novel MabZIP74-mediated regulatory network involved in ethylene biosynthesis and fruit ripening in banana. These results advance our knowledge on fruit ripening and provide additional opportunities for genetic improvement of banana fruit ripening. [ABSTRACT FROM AUTHOR]

• MabHLH7 was ethylene-inducible and nuclear-localized, and its transcript level was increased during banana fruit ripening. • MabHLH7 can directly bind to and activate the promoters of several cell wall-modifying-related genes. • Our findings establish a transcriptional regulatory network during banana fruit ripening. The basic helix-loop-helix (bHLH) family proteins, a group of functionally diverse transcription factors (TFs), control a series of plant biological processes. However, the involvement of bHLH TFs in fruit ripening still rarely reported. In this study, a banana fruit bHLH TF, named as MabHLH7, was identified and characterized. MabHLH7 was ethylene-inducible and nuclear-localized, and its transcript level was increased during banana fruit ripening. More importantly, MabHLH7 can directly bind to and activate the promoters of several cell wall-modifying-related genes, including MaXTH12 , MaEXP2/21 , MaPME4/5 , MaPG4, and MaPL1/2, which were significantly enhanced in the ripening stage. Overall, our findings establish a transcriptional regulatory network during banana fruit ripening, in which MabHLH7 activates a small subset of cell wall-modifying-related genes through directly binding to their promoters. [ABSTRACT FROM AUTHOR]