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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.

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.

Ripening of fleshy fruits involves both diverse post-translational modifications (PTMs) and dynamic transcriptional reprogramming, but the interconnection between PTMs, such as protein phosphorylation and transcriptional regulation, in fruit ripening remains to be deciphered. Here, we conducted a phosphoproteomic analysis during banana (Musa acuminata) ripening and identified 63 unique phosphopeptides corresponding to 49 proteins. Among them, a Musa acuminata basic leucine zipper transcription factor21 (MabZIP21) displayed elevated phosphorylation level in the ripening stage. MabZIP21 transcript and phosphorylation abundance increased during banana ripening. Genome-wide MabZIP21 DNA binding assays revealed MabZIP21-regulated functional genes contributing to banana ripening, and electrophoretic mobility shift assay, chromatin immunoprecipitation coupled with quantitative polymerase chain reaction, and dual-luciferase reporter analyses demonstrated that MabZIP21 stimulates the transcription of a subset of ripening-related genes via directly binding to their promoters. Moreover, MabZIP21 can be phosphorylated by MaMPK6-3, which plays a role in banana ripening, and T318 and S436 are important phosphorylation sites. Protein phosphorylation enhanced MabZIP21-mediated transcriptional activation ability, and transient overexpression of the phosphomimetic form of MabZIP21 accelerated banana fruit ripening. Additionally, MabZIP21 enlarges its role in transcriptional regulation by activating the transcription of both MaMPK6-3 and itself. Taken together, this study reveals an important machinery of protein phosphorylation in banana fruit ripening in which MabZIP21 is a component of the complex phosphorylation pathway linking the upstream signal mediated by MaMPK6-3 with transcriptional controlling of a subset of ripening-associated genes.

In flowering plants, repression of the seed maturation program is essential for the transition from the seed to the vegetative phase, but the underlying mechanisms remain poorly understood. The B3-domain protein VIVIPAROUS1/ABSCISIC ACID-INSENSITIVE3-LIKE 1 (VAL1) is involved in repressing the seed maturation program. Here we uncovered a molecular network triggered by the plant hormone brassinosteroid (BR) that inhibits the seed maturation program during the seed-to-seedling transition in Arabidopsis (Arabidopsis thaliana). val1-2 mutant seedlings treated with a BR biosynthesis inhibitor form embryonic structures, whereas BR signaling gain-of-function mutations rescue the embryonic structure trait. Furthermore, the BR-activated transcription factors BRI1-EMS-SUPPRESSOR 1 and BRASSINAZOLE-RESISTANT 1 bind directly to the promoter of AGAMOUS-LIKE15 (AGL15), which encodes a transcription factor involved in activating the seed maturation program, and suppress its expression. Genetic analysis indicated that BR signaling is epistatic to AGL15 and represses the seed maturation program by downregulating AGL15. Finally, we showed that the BR-mediated pathway functions synergistically with the VAL1/2-mediated pathway to ensure the full repression of the seed maturation program. Together, our work uncovered a mechanism underlying the suppression of the seed maturation program, shedding light on how BR promotes seedling growth.

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.

Banana fruit is prone to chilling injury (CI) during cold storage, resulting in quality deterioration and commodity reduction. The hot water treatment (HWT), dipping banana fruit in hot water (52 °C) for 3 min, reduced CI symptom at 7 °C storage. The purpose of this study was to investigate the potential molecular mechanism of HWT on the alleviation of CI of postharvest banana fruit. It was found that HWT treatment obviously inhibited the increases in CI index, relative electrolytic leakage, and the contents of malonaldehyde (MDA) and O

The regulatory role of cytokinins (CTKs) in leaf senescence has been documented in different species, including Chinese flowering cabbage. However, its physiological and molecular basis relatively remains unknown. In this study, exogenous application of a CTK analogue 6-benzylaminopurine (6-BA) significantly retarded leaf senescence of Chinese flowering cabbage, with less chlorophyll degradation and lower accumulation of reactive oxygen species (ROS) and malondialdehyde compared with the control group. Meanwhile, higher levels of soluble sugars and proteins, flavonoids, cellulose, amino acids, total phenols, procanthocyanins, and vitamin C were retained in 6-BA-treated leaves. 6-BA treatment also prevented the decline in endogenous CTK content and the increase in ethylene, abscisic acid, and jasmonic acid contents. Moreover, the comparative transcriptome analysis revealed that a total of 21,895 differently expressed genes (DEGs) were identified from four comparisons of 6-BA treatment versus the control during senescence. Further analysis showed that most of the identified DEGs were enriched in ROS, respiratory metabolism, and phytohormone pathways, and a total of 50 classes of transcription factors that were possibly involved in modulating these DEGs were obtained. The transcriptional levels of 18 DEGs were verified by Quantitative real-time PCR (qRT-PCR), which confirmed the accuracy of the transcriptomic data. Overall, these findings and data provide a comprehensive view of physiological and molecular events concerning with the CTK-mediated leaf senescence and -maintained quality in economical leafy vegetables.

Ethylene plays a critical regulatory role in climacteric fruit ripening, and its biosynthesis is fine-tuned at the transcriptional and posttranslational levels. Nevertheless, the mechanistic link between transcriptional and posttranslational regulation of ethylene biosynthesis during fruit ripening is largely unknown. This study uncovers a coordinated transcriptional and posttranslational mechanism of controlling ethylene biosynthesis during banana (Musa acuminata) fruit ripening. NAC (NAM, ATAF, and CUC) proteins MaNAC1 and MaNAC2 repress the expression of MaERF11, a protein previously known to negatively regulate ethylene biosynthesis genes MaACS1 and MaACO1. A RING E3 ligase MaXB3 interacts with MaNAC2 to promote its ubiquitination and degradation, leading to the inhibition of MaNAC2-mediated transcriptional repression. In addition, MaXB3 also targets MaACS1 and MaACO1 for proteasome degradation. Further evidence supporting the role of MaXB3 is provided by its transient and ectopic overexpression in banana fruit and tomato (Solanum lycopersicum), respectively, which delays fruit ripening via repressing ethylene biosynthesis and thus ethylene response. Strikingly, MaNAC1 and MaNAC2 directly repress MaXB3 expression, suggesting a feedback regulatory mechanism that maintains a balance of MaNAC2, MaACS1, and MaACO1 levels. Collectively, our findings establish a multilayered regulatory cascade involving MaXB3, MaNACs, MaERF11, and MaACS1/MaACO1 that controls ethylene biosynthesis during climacteric ripening.

Horticultural commodities suffer chilling injury following exposure to extremely low temperatures, which results in visible symptoms and considerable quality loss. Therefore, it is of significance to understand the mechanism of this physiological disorder and to develop effective strategies to control it. Chilling stress causes alteration in structure and function of the plasma membrane, which is assumed to be the primary event in response to cold stress. During this process, the membrane lipid metabolism plays a pivotal role in membrane fluidity and stability. In this review, we summarized the possible roles of membrane lipid metabolism in the development of chilling injury, having the potential for developing effective strategies to alleviate chilling injury in horticultural products under refrigerated storage in practice.

Carotenoids are a class of bioactive compounds that exhibit health-promoting properties for humans, but their regulation in bananas during fruit ripening remains largely unclear. Here, we found that the total carotenoid content continued to be elevated along the course of banana ripening and peaked at the ripening stage followed by a decrease, which is presumably caused by the transcript abundances of carotenoid biosynthetic genes MaLCYB1.1 and MaLCYB1.2. Moreover, a ripening-inducible transcription factor MaSPL16 was characterized, which was a nuclear protein with transactivation activity. Transient transformation of MaSPL16 in banana fruits led to enhanced transcript levels of MaLCYB1.1 and MaLCYB1.2 and hence the total carotenoid accumulation. Importantly, MaSPL16 stimulated the transcription of MaLCYB1.1 and MaLCYB1.2 through directly binding to their promoters. Collectively, our findings indicate that MaSPL16 behaves as an activator to modulate banana carotenoid biosynthesis, which may provide a new target for molecular improvement of the nutritional and bioactive qualities of agricultural crops that accumulate carotenoids.

The performance of p-Anisaldehyde (PAA) for preserving pitaya fruit quality and the underpinning regulatory mechanism were investigated in this study. Results showed that PAA treatment significantly reduced fruit decay, weight loss and loss of firmness, and maintained higher content of total soluble solids, betacyanins, betaxanthins, total phenolics and flavonoids in postharvest pitaya fruits. Compared with control, the increase in hydrogen peroxide (H2O2) content and superoxide anion (O2•−) production was inhibited in fruit treated with PAA. Meanwhile, PAA significantly improved the activity of antioxidant enzymes superoxide dismutase (SOD), peroxidase (POD) and catalase (CAT). Moreover, PAA-treated pitaya fruit maintained higher ascorbic acid (AsA) and reduced-glutathione (GSH) content but lower dehydroascorbate (DHA) and oxidized glutathione (GSSG) content, thus sustaining higher ratio of AsA/DHA and GSH/GSSG. In addition, activities of ascorbate peroxidase (APX), glutathione reductase (GR), monodehydroascorbate reductase (MDHAR) and dehydrogenation ascorbic acid reductase (DHAR), as well as the expression of HpSOD, HpPOD, HpCAT, HpAPX, HpGR, HpDHAR and HpMDHAR, were enhanced after PAA treatment. The findings suggest that postharvest application of PAA may be a reliable method to control postharvest decay and preserve quality of harvested pitaya fruit by enhancing the antioxidant potential of the AsA-GSH cycle and activating an antioxidant defense system to alleviate reactive oxygen species (ROS) accumulation.

Pitaya (Hylocereus) is the most economically important fleshy-fruited tree of the Cactaceae family that is grown worldwide, and it has attracted significant attention because of its betalain-abundant fruits. Nonetheless, the lack of a pitaya reference genome significantly hinders studies focused on its evolution, as well as the potential for genetic improvement of this crop. Herein, we employed various sequencing approaches, namely, PacBio-SMRT, Illumina HiSeq paired-end, 10× Genomics, and Hi-C (high-throughput chromosome conformation capture) to provide a chromosome-level genomic assembly of ‘GHB’ pitaya (H. undatus, 2n = 2x = 22 chromosomes). The size of the assembled pitaya genome was 1.41 Gb, with a scaffold N50 of ~127.15 Mb. In total, 27,753 protein-coding genes and 896.31 Mb of repetitive sequences in the H. undatus genome were annotated. Pitaya has undergone a WGT (whole-genome triplication), and a recent WGD (whole-genome duplication) occurred after the gamma event, which is common to the other species in Cactaceae. A total of 29,328 intact LTR-RTs (~696.45 Mb) were obtained in H. undatus, of which two significantly expanded lineages, Ty1/copia and Ty3/gypsy, were the main drivers of the expanded genome. A high-density genetic map of F1 hybrid populations of ‘GHB’ × ‘Dahong’ pitayas (H. monacanthus) and their parents were constructed, and a total of 20,872 bin markers were identified (56,380 SNPs) for 11 linkage groups. More importantly, through transcriptomic and WGCNA (weighted gene coexpression network analysis), a global view of the gene regulatory network, including structural genes and the transcription factors involved in pitaya fruit betalain biosynthesis, was presented. Our data present a valuable resource for facilitating molecular breeding programs of pitaya and shed novel light on its genomic evolution, as well as the modulation of betalain biosynthesis in edible fruits.

Low temperature leads to abnormal ripening and poor quality of the harvested banana fruit, which is an urgent problem limiting the development of industry in China. To comprehensively understand the mechanism underlying low-temperature-affected ripening, we performed comparative RNA-Seq analysis of ethylene-induced ripening of banana fruit after 3 days of pre-storage at 7 °C and 22 °C. A total of 986 differentially expressed genes (DEGs) were identified in both RT-0 d versus RT-3 d, LT-0 d versus LT-3 d, RT-0 d versus LT-0 d and RT-3 d versus LT-3 d, and the RNA-Seq outputs of 15 randomly selective DEGs were verified using qRT-PCR. Among the 986 DEGs obtained in the four groups, 9 MYB genes (MaMYB75/281/219/4/151/156/3/37 and MaMYB3R1) and 32 genes related to carotenoid biosynthesis (MaPSY1/2a), flavor formation (MaLOX6, MaADH7, MaAAT1), sucrose transport (MaSUS2/4), ethylene production (MaSAM1, MaACO9/10/12, MaACS1/12), starch degradation (MaAMY1A/1B, MaPHS1/2, MaMEX2, MapGlcT1) and cell wall degradation (MaPG3/X1, MaPME25/41, MaXTH5/7/22/23/25, MaEXP2/20/A1/A15) were characterized. Combining transcription factor binding site (TFBS) analysis as well as cis-acting element analysis, the regulatory network of low-temperature-affected ripening mediated by MYBs were constructed. The data generated in this study may unravel the transcriptional regulatory network of MYBs associated with low-temperature-affected ripening and provide a solid foundation for future studies.

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.

Linoleic acid (LA; C18:2) and α-linolenic acid (ALA; C18:3) are two essential unsaturated fatty acids that play indispensable roles in maintaining membrane integrity in cold stress, and ω-3 fatty acid desaturases (FADs) are responsible for the transformation of LA into ALA. However, how this process is regulated at transcriptional and posttranscriptional levels remains largely unknown. In this study, an MYB transcription factor, MaMYB4, of a banana fruit was identified and found to target several ω-3 MaFADs, including MaFAD3-1, MaFAD3-3, MaFAD3-4 and MaFAD3-7, and repress their transcription. Intriguingly, the acetylation levels of histones H3 and H4 in the promoters of ω-3 MaFADs were elevated in response to cold stress, which was correlated with the enhancement in the transcription levels of ω-3 MaFADs and the ratio of ALA/LA. Moreover, a histone deacetylase MaHDA2 physically interacted with MaMYB4, thereby leading to the enhanced MaMYB4-mediated transcriptional repression of ω-3 MaFADs. Collectively, these data demonstrate that MaMYB4 might recruit MaHDA2 to repress the transcription of ω-3 MaFADs by affecting their acetylation levels, thus modulating fatty acid biosynthesis. Our findings provided new molecular insights into the regulatory mechanisms of fatty acid biosynthesis in cold stress in fruits.

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.

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.

Autophagy is a critical intracellular solute trafficking and degradation pathway associated with leaf senescence. Autophagy-related genes (ATGs), are indispensable for autophagosome formation and autophagic process, but the underlying molecular functions of ATGs during leaf senescence in harvested leafy vegetables such as Chinese flowering cabbage remain largely unknown. In this study, 53 ATG genes from Chinese flowering cabbage were identified and studied. Notably, the expression of three ATG genes, BrATG5b, BrATG8e-1 and BrATG8h-1, was strongly up-regulated during postharvest leaf senescence. These genes were induced by senescence-promoting phytohormones methyl jasmonate (MeJA) and abscisic acid (ABA), and repressed by senescence-delaying hormones cytokinin 6-benzyladenine (6-BA) and gibberellic acid (GA3). We identified a MYB transcriptional factor, BrMYB108, which showed a similar expression pattern as that of BrATG5b, BrATG8e-1 and BrATG8h-1. This was localized in the nucleus and possessed trans-activation activity. Further, DNA-protein interaction assays showed that BrMYB108 directly binds to BrATG5b, BrATG8e-1 and BrATG8h-1 promoters and enhances their expression. In summary, this study unravels a novel transcription factor that activates the expression of senescence-associated ATGs, thereby acting as a potential positive regulator of postharvest leaf senescence in Chinese flowering cabbage.

Chilling injury (CI) of banana represents a serious problem constraining postharvest preservation in the industry. Application of ethylene was found to efficiently attenuate CI of banana, however, the mechanism of which remains to be investigated. Here, we found that application of ethrel could ameliorate the CI of banana, as evidenced by lower CI index, electrolytic leakage and malondialdehyde content. Meanwhile, expression of phosphatidic acid (PA) formation genes MaPLDδ1, MaPLDδ5 and MaDGK2, as well as two transcriptional regulators MabHLH060 and MabHLH183 was significantly increased with the progression of CI, but decreased under ethrel treatment. MabHLH060 and MabHLH183 were shown to locate in nucleus and have transactivation ability. Importantly, these two transcriptional regulators MabHLH060/183 recognized the E-box elements in the promoters of MaPLDδ1, MaPLDδ5 and MaDGK2, and promoted their transcription. Together, these findings suggest that ethylene-alleviated CI of banana may involve the MabHLH060/183 module in modulating the expression of PA formation genes.

SlARF10 plays an important role in the chlorophyll accumulation and photosynthesis of tomato plants, and regulation of its expression affects the starch, fructose, and sucrose content of fruit., The photosynthesis of green tomatoes contributes to fruit growth and carbon economy. The tomato auxin response factor 10 (SlARF10) belongs to the ARF family and is located in nucleus. In this study, we found that SlARF10 was highly expressed in green fruit. Overexpression of SlARF10 in fruit produced a dark-green phenotype whilst knock-down by RNAi produced a light-green phenotype. Autofluorescence and chlorophyll content analyses confirmed the phenotypes, which indicated that SlARF10 plays an important role in chlorophyll accumulation. Overexpression of SlARF10 positively affected photosynthesis in both leaves and fruit. Furthermore, SlARF10-overexpression lines displayed improved accumulation of starch, fructose, and sucrose in fruit, whilst SlARF10-RNAi lines showed decreased accumulation of starch and sucrose. Regulation of SlARF10 expression altered the expression of AGPase starch biosynthesis genes. SlARF10 positively regulated the expression of SlGLK1, POR, CBP1, and CBP2, which are related to chlorophyll metabolism and regulation. Electrophoretic mobility shift assays confirmed that SlARF10 directly targets to the SlGLK1 promoter. Our results thus indicate that SlARF10 is involved in chlorophyll accumulation by transcriptional activation of SlGLK1 expression in tomato fruit, and provide insights into the link between auxin signaling, chloroplast activity, and sugar metabolism during tomato fruit development.

Both NAC transcription factors (TFs) and reactive oxygen species (ROS) are known to be involved in leaf senescence. However, how NAC TFs modulate ROS metabolism associated with leaf senescence remains largely uncharacterized, especially during leaf senescence of postharvest economically leafy vegetables such as Chinese flowering cabbage. Here, we found that expression levels of two genes BrRbohB and BrRbohC-like encoding ROS-producing enzymes respiratory burst oxidase homologues (RBOHs) were increased consistently with the progression of postharvest leaf senescence, exhibiting a good correlation with ROS accumulation and chlorophyll degradation, as well as expressions of two chlorophyll catabolic genes ( CCGs), BrNYC1 and BrNYE1. Significantly, a novel, nuclear-localized transcriptional activator BrNAC055 was identified, and observed to show a similar expression pattern with BrRbohB, BrRbohC-like, BrNYC1 and BrNYE1. Further gel mobility shift and dual luciferase reporter assays confirmed that BrNAC055 bound directly to the NAC binding sequence (NACBS) in BrRbohB, BrRbohC-like, BrNYC1, and BrNYE1 promoters, and activated their activities. Moreover, transient overexpression of BrNAC055 in tobacco leaves made an increased ROS level and accelerated chlorophyll degradation via the up-regulation of NbRbohA and NbSGR1, resulting in the promoted leaf senescence. On the basis of these findings, we conclude that BrNAC055 acts as a transcriptional activator of ROS production and chlorophyll degradation by activating the transcriptions of RBOHs and CCGs and thereby accelerates leaf senescence in Chinese flowering cabbage.

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 MaHDACs 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.

The transcriptional regulation of aroma formation genes remains poorly understood in the banana. In this work, we found that the expressions of a subset of aroma biosynthetic genes including MaOMT1, MaMT1, MaGT1, MaBCAT1, MaACY1, MaAGT1, and BanAAT, as well as two bZIP genes, MabZIP4 and MabZIP5, were down-regulated when prestored at 7 °C compared to those prestored at 22 °C during the ripening process of banana. Furthermore, MabZIP4 and MabZIP5 were shown to be able to activate the transcription of these aroma biosynthetic genes. Importantly, MabZIP4 directly binds to BanAAT promoter, while MabZIP5 binds to the promoters of MaMT1, MaACY1, MaAGT1, and BanAAT via the G-box motif, implicating the diverse functional significances of MabZIPs in controlling aroma biosynthesis in banana. Overall, this work sheds new insights on the understanding of transcriptional regulatory mechanisms associated with aroma formation during banana ripening.

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.

Protein degradation is an integral process of leaf senescence, and this catabolism is strongly associated with proteases in diverse plant species. However, the characterization of proteases as well as their upstream regulators during postharvest senescence, especially in economically important leafy vegetables such as Chinese flowering cabbage, remains to be determined. In this study, temporal and spatial gene expression patterns of eight proteases showed that three of them, namely, BrAPM1, BrASPG2 and BrSAG12, were most up-regulated during postharvest leaf senescence. Their expressions were also significantly induced by senescence-accelerating phytohormones abscisic acid (ABA) and methyl jasmonate (MeJA), but were inhibited by senescence-delaying hormones gibberellin (GA3) and cytokinin (6-BA). Importantly, using yeast one-hybrid screening, we identified a Dof (DNA-binding with one finger) transcription factor, BrDof2.4, as the putative binding protein of BrAPM1, BrASPG2 and BrSAG12 promoters. Nucleus-localized BrDof2.4 was senescence-, ABA- and MeJA-repressible, but was GA3- and 6-BA-inducible. Furthermore, gel mobility shift and transient dual luciferase reporter assays revealed that BrDof2.4 suppressed the transcription of BrAPM1, BrASPG2 and BrSAG12, through binding to their promoters. Taken together, our findings demonstrate that BrDof2.4 acts as a potential repressor of postharvest leaf senescence in Chinese flowering cabbage by the direct suppression of protease expression. Our work contributes towards improving the technology for increasing the shelf-life and commercial value of this important leafy vegetable.

Phytohormone gibberellins (GAs) regulate leaf senescence. A global view of the regulatory pathways associated with GA-mediated inhibition of leaf senescence, especially in economically important leafy vegetables such as Chinese flowering cabbage, remains unclear. In this study, exogenous supply of gibberellin (GA3) delayed postharvest Chinese flowering cabbage leaf senescence. By comparative transcriptome analysis of leaf tissue, a total of 4682, 5059, and 2362 differentially expressed genes (DEGs) were identified between the comparison of 0d (cabbages at the harvest day) and CK3d (control cabbages at day 3 post-harvest), 0d and GA3d (GA3-treated cabbages at day 3 post-harvest), and CK3d and GA3d, respectively. Further analysis of identified DEGs involved in chlorophyll and GA degradation during leaf senescence, and the activity of chlorophyll catabolic genes (CCGs) BrPPH and BrRCCR, and bioactive GA degradation gene BrGA2ox1, was greatly reversed with GA3 application during leaf senescence. More importantly, a NAC transcription factor (TF), BrNAC087, was found to be associated with leaf senescence. BrNAC087 is a nuclear localized gene with transcriptional activity, highly expressed during senescence but downregulated by GA3. Significantly, BrNAC087 positively regulated BrPPH, BrRCCR and BrGA2ox1 expression by binding to NAC-binding sequences in their promoters in vitro and in vivo. These findings collectively provide evidence for a new molecular regulatory pathway explaining, at least in part, a mechanistic basis for gibberellin-mediated leaf senescence inhibition in postharvest Chinese flowering cabbage.

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.

Chilling injury (CI) represents a physiological disorder caused by improper low temperature management, which affects the postharvest quality and marketing potential of banana fruit. In this study, postharvest application of glycine betaine (GB) can significantly reduce the CI incidence of banana fruit during cold storage, as observed by lower CI index, electrolyte leakage, malondialdehyde (MDA) contents, and higher values of lightness, chlorophyll, soluble sugar contents. Moreover, GB not only increased the antioxidant substances such as total phenolics, glutathione and ascorbic acids (AsA), but also elevated the enzyme activities and gene expression of the antioxidant enzymes including ascorbate peroxidase (APX), catalase (CAT), superoxide dismutase (SOD) and peroxidase (POD). In addition, GB could obviously enhance the total antioxidant capacity of banana fruit under refrigerated storage. Collectively, these findings suggest that GB-attenuated CI incidence in banana fruit during cold storage might be, at least partially, ascribed to the enhancement of antioxidant system involving antioxidant substances, as well as enzyme activities and gene expression of antioxidant enzymes.

The efficacy of melatonin on postharvest quality of Chinese flowering cabbage was investigated in this study. Treatment of Chinese cabbage with melatonin significantly delayed leaf yellowing and maintained higher contents of nutrients such as soluble sugars, protein, vitamin C, total flavonoids and phenols during a 7-day storage. Also, melatonin enhanced the activities and expression level of glucose-6-phosphate dehydrogenase (G6PDH) and 6-phosphogluconate dehydrogenase (6-PGDH). Melatonin treatment decreased tissue weight loss, respiration rate, activities of phosphohexose isomerase (PHI), succinate dehydrogenase (SDH), cytochrome C oxidase (CCO), and ascorbic acid oxidase (AAO) and the expression of their corresponding genes. Additionally, melatonin-treated leaves maintained high energy status, as evidenced by higher total ATPase and nicotinamide adenine dinucleotide kinase (NADK) activities, higher energy charge level, adenosine triphosphate (ATP) and adenosine diphosphate (ADP) contents, lower level of adenosine monophosphate (AMP) and transcript abundance of respiratory enzymes. These findings collectively suggest that melatonin maintains leaf quality of postharvest Chinese flowering cabbage might be, at least in part, due to the reduced ratio of Embden-Meyerhof-Parnas pathway (EMP), tricarboxylic acid (TCA) cycle, and cytochrome pathway (CCP), a relatively higher level of pentose phosphate pathway (PPP) and energy status.

Cell wall polysaccharides in fruits act a pivotal role in their resistance to fungal invasion. Lasiodiplodia theobromae (Pat.) Griff. & Maubl. is a primary pathogenic fungus causing the spoilage of fresh longan fruit. In this study, the influences of L. theobromae inoculation on the disassembly of cell wall polysaccharides in pericarp of fresh longans and its association with L. theobromae-induced disease and softening development were investigated. In contrast to the control, samples with L. theobromae infection showed more severe disease development, lower firmness, lower amounts of cell wall materials, covalent-soluble pectin, ionic-soluble pectin, cellulose and hemicellulose, whereas higher value of water-soluble pectin, higher activities of cell wall polysaccharide-disassembling enzymes (cellulase, β-galactosidase, polygalacturonase and pectinesterase). These findings revealed that cell wall polysaccharides disassembly induced by enzymatic manipulation was an essential pathway for L. theobromae to infect harvested longans, and thus led to the disease occurrence and fruit softening.

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 Ma GWD 1, Ma LSF 2, Ma BAM 1, Ma BAM 2, Ma BAM 8, Ma BAM 10, Ma AMY 3, Ma AMY3 C, Ma ISA 2, Ma ISA 3 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.