Proteasomal degradation of MaMYB60 mediated by the E3 ligase MaBAH1 causes high temperature-induced repression of chlorophyll catabolism and green ripening in banana.

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.

Banana MaNAC1 activates secondary cell wall cellulose biosynthesis to enhance chilling resistance in fruit.

Chilling injury has a negative impact on the quantity and quality of crops, especially subtropical and tropical plants. The plant cell wall is not only the main source of biomass production, but also the first barrier to various stresses. Therefore, improving the understanding of the alterations in cell wall architecture is of great significance for both biomass production and stress adaptation. Herein, we demonstrated that the cell wall principal component cellulose accumulated during chilling stress, which was caused by the activation of MaCESA proteins. The sequence-multiple comparisons show that a cold-inducible NAC transcriptional factor MaNAC1, a homologue of Secondary Wall NAC transcription factors, has high sequence similarity with Arabidopsis SND3. An increase in cell wall thickness and cellulosic glucan content was observed in MaNAC1-overexpressing Arabidopsis lines, indicating that MaNAC1 participates in cellulose biosynthesis. Over-expression of MaNAC1 in Arabidopsis mutant snd3 restored the defective secondary growth of thinner cell walls and increased cellulosic glucan content. Furthermore, the activation of MaCESA7 and MaCESA6B cellulose biosynthesis genes can be directly induced by MaNAC1 through binding to SNBE motifs within their promoters, leading to enhanced cellulose content during low-temperature stress. Ultimately, tomato fruit showed greater cold resistance in MaNAC1 overexpression lines with thickened cell walls and increased cellulosic glucan content. Our findings revealed that MaNAC1 performs a vital role as a positive modulator in modulating cell wall cellulose metabolism within banana fruit under chilling stress.

MaHsf24, a novel negative modulator, regulates cold tolerance in banana fruits by repressing the expression of HSPs and antioxidant enzyme genes.

Transcriptional regulation mechanisms underlying chilling injury (CI) development have been widely investigated in model plants and cold-sensitive fruits, such as banana (Musa acuminata). However, unlike the well-known NAC and WRKY transcription factors (TFs), the function and deciphering mechanism of heat shock factors (HSFs) involving in cold response are still fragmented. Here, we showed that hot water treatment (HWT) alleviated CI in harvested banana fruits accomplishing with reduced reactive oxygen species (ROS) accumulation and increased antioxidant enzyme activities. A cold-inducible but HWT-inhibited HSF, MaHsf24, was identified. Using DNA affinity purification sequencing (DAP-seq) combined with RNA-seq analyses, we found three heat shock protein (HSP) genes (MaHSP23.6, MaHSP70-1.1 and MaHSP70-1.2) and three antioxidant enzyme genes (MaAPX1, MaMDAR4 and MaGSTZ1) were the potential targets of MaHsf24. Subsequent electrophoretic mobility shift assay (EMSA), chromatin immunoprecipitation coupled with quantitative PCR (ChIP-qPCR) and dual-luciferase reporter (DLR) analyses demonstrated that MaHsf24 repressed the transcription of these six targets via directly binding to their promoters. Moreover, stably overexpressing MaHsf24 in tomatoes increased cold sensitivity by suppressing the expressions of HSPs and antioxidant enzyme genes, while HWT could recover cold tolerance, maintaining higher levels of HSPs and antioxidant enzyme genes, and activities of antioxidant enzymes. In contrast, transiently silencing MaHsf24 by virus-induced gene silencing (VIGS) in banana peels conferred cold resistance with the upregulation of MaHSPs and antioxidant enzyme genes. Collectively, our findings support the negative role of MaHsf24 in cold tolerance, and unravel a novel regulatory network controlling bananas CI occurrence, concerning MaHsf24-exerted inhibition of MaHSPs and antioxidant enzyme genes.

E3 ligase MaNIP1 degradation of NON-YELLOW COLORING1 at high temperature inhibits banana degreening.

Banana (Musa acuminata) fruit ripening under high temperatures (>24 °C) undergoes green ripening due to failure of chlorophyll degradation, which greatly reduces marketability. However, the mechanism underlying high temperature-repressed chlorophyll catabolism in banana fruit is not yet well understood. Here, using quantitative proteomic analysis, 375 differentially expressed proteins were identified in normal yellow and green ripening in banana. Among these, one of the key enzymes involved in chlorophyll degradation, NON-YELLOW COLORING 1 (MaNYC1), exhibited reduced protein levels when banana fruit ripened under high temperature. Transient overexpression of MaNYC1 in banana peels resulted in chlorophyll degradation under high temperature, which weakens the green ripening phenotype. Importantly, high temperature induced MaNYC1 protein degradation via the proteasome pathway. A banana RING E3 ligase, NYC1-interacting protein 1 (MaNIP1), was found to interact with and ubiquitinate MaNYC1, leading to its proteasomal degradation. Furthermore, transient overexpression of MaNIP1 attenuated MaNYC1-induced chlorophyll degradation in banana fruits, indicating that MaNIP1 negatively regulates chlorophyll catabolism by affecting MaNYC1 degradation. Taken together, the findings establish a post-translational regulatory module of MaNIP1-MaNYC1 that mediates high temperature-induced green ripening in bananas.

E3 ubiquitin ligase MaRZF1 modulates high temperature-induced green ripening of banana by degrading MaSGR1.

High temperatures (>24°C) prevent the development of a yellow peel on bananas called green ripening, owing to the inhibition of chlorophyll degradation. This phenomenon greatly reduces the marketability of banana fruit, but the mechanisms underlining high temperature-repressed chlorophyll catabolism need to be elucidated. Herein, we found that the protein accumulation of chlorophyll catabolic enzyme MaSGR1 (STAY-GREEN 1) was reduced when bananas ripened at high temperature. Transiently expressing MaSGR1 in banana peel showed its positive involvement in promoting chlorophyll degradation under high temperature, thereby weakening green ripening phenotype. Using yeast two-hybrid screening, we identified a RING-type E3 ubiquitin ligase, MaRZF1 (RING Zinc Finger 1), as a putative MaSGR1-interacting protein. MaRZF1 interacts with and targets MaSGR1 for ubiquitination and degradation via the proteasome pathway. Moreover, upregulating MaRZF1 inhibited chlorophyll degradation, and attenuated MaSGR1-promoted chlorophyll degradation in bananas during green ripening, indicating that MaRZF1 negatively regulates chlorophyll catabolism via the degradation of MaSGR1. Taken together, MaRZF1 and MaSGR1 form a regulatory module to mediate chlorophyll degradation associated with high temperature-induced green ripening in bananas. Therefore, our findings expand the understanding of posttranslational regulatory mechanisms of temperature stress-caused fruit quality deterioration.

MaMYB4 is a negative regulator and a substrate of RING-type E3 ligases MaBRG2/3 in controlling banana fruit ripening.

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.

Phosphorylation of transcription factor bZIP21 by MAP kinase MPK6-3 enhances banana fruit ripening.

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.

A tomato NAC transcription factor, SlNAM1, positively regulates ethylene biosynthesis and the onset of tomato fruit ripening.

Fruit ripening in tomato (Solanum lycopersicum) is the result of selective expression of ripening-related genes, which are regulated by transcription factors (TFs). The NAC (NAM, ATAF1/2, and CUC2) TF family is one of the largest families of plant-specific TFs and members are involved in a variety of plant physiological activities, including fruit ripening. Fruit ripening-associated NAC TFs studied in tomato to date include NAC-NOR (non-ripening), SlNOR-like1 (non-ripening like1), SlNAC1, and SlNAC4. Considering the large number of NAC genes in the tomato genome, there is little information about the possible roles of other NAC members in fruit ripening, and research on their target genes is lacking. In this study, we characterize SlNAM1, a NAC TF, which positively regulates the initiation of tomato fruit ripening via its regulation of ethylene biosynthesis. The onset of fruit ripening in slnam1-deficient mutants created by CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats and CRISPR-associated protein 9) technology was delayed, whereas fruit ripening in OE-SlNAM1 lines was accelerated compared with the wild type. The results of RNA-sequencing (RNA-seq) and promoter analysis suggested that SlNAM1 directly binds to the promoters of two key ethylene biosynthesis genes (1-aminocyclopropane-1-carboxylate synthase: SlACS2 and SlACS4) and activates their expression. This hypothesis was confirmed by electrophoretic mobility shift assays and dual-luciferase reporter assay. Our findings provide insights into the mechanisms of ethylene production and enrich understanding of the tomato fruit ripening regulatory network.

Deciphering transcriptional regulators of banana fruit ripening by regulatory network analysis.

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.

MaXB3 Modulates MaNAC2, MaACS1, and MaACO1 Stability to Repress Ethylene Biosynthesis during Banana Fruit Ripening.

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.

Four HD-ZIPs are involved in banana fruit ripening by activating the transcription of ethylene biosynthetic and cell wall-modifying genes.

KEY MESSAGE: Four MaHDZs are possibly involved in banana fruit ripening by activating the transcription of genes related to ethylene biosynthesis and cell wall degradation, such as MaACO5, MaEXP2, MaEXPA10, MaPG4 and MaPL4. The homeodomain-leucine zipper (HD-ZIP) proteins represent plant-specific transcription factors, which contribute to various plant physiological processes. However, little information is available regarding the association of HD-ZIPs with banana fruit ripening. In this study, we identified a total of 96 HD-ZIP genes in banana genome, which were divided into four different groups consisting of 35, 31, 9 and 21 members in the I, II, III and IV subfamilies, respectively. The expression patterns of MaHDZ genes during fruit ripening showed that MaHDZI.19, MaHDZI.26, MaHDZII.4 and MaHDZII.7 were significantly up-regulated in the ripening stage and thus suggested to be potential regulators of banana fruit ripening. Furthermore, MaHDZI.19, MaHDZI.26, MaHDZII.4 and MaHDZII.7 were found to localize exclusively in the nucleus and exhibit transcriptional activation capacities. Importantly, MaHDZI.19, MaHDZI.26, MaHDZII.4 and MaHDZII.7 stimulated the transcription of several ripening-related genes including MaACO5 related to ethylene biosynthesis, MaEXP2, MaEXPA10, MaPG4 and MaPL4 were associated with cell wall degradation, through directly binding to their promoters. Taken together, our findings expand the functions of HD-ZIP transcription factors and identify four MaHDZs likely involved in regulating banana fruit ripening by activating the expression of genes related to ethylene biosynthesis and cell wall modification, which may have potential application in banana molecular breeding.

Banana MaBZR1/2 associate with MaMPK14 to modulate cell wall modifying genes during fruit ripening.

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.

The Ubiquitin E3 Ligase MaLUL2 Is Involved in High Temperature-Induced Green Ripening in Banana Fruit.

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.

A banana R2R3-MYB transcription factor MaMYB3 is involved in fruit ripening through modulation of starch degradation by repressing starch degradation-related genes and MabHLH6.

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.

Melatonin delays leaf senescence of Chinese flowering cabbage by suppressing ABFs-mediated abscisic acid biosynthesis and chlorophyll degradation.

Melatonin and abscisic acid (ABA) play contrasting roles in regulating leaf senescence in plants. The molecular mechanism underlying the interaction between melatonin and ABA involved in leaf senescence, however, remains poorly defined. Herein, we found that exogenous application of melatonin delayed the senescence of Chinese flowering cabbage, accompanied by reduced expression of chlorophyll catabolic and ABA biosynthetic genes, and a lower endogenous ABA level. Significantly, three nucleus-localized transcriptional activators BrABF1, BrABF4, and BrABI5 were identified, and their expressions were repressed by melatonin. In vitro and in vivo binding experiments revealed that BrABF1, BrABF4, and BrABI5 activated the transcription of a series of ABA biosynthetic and chlorophyll catabolic genes by physically binding to their promoters. Moreover, transient over-expression of BrABF1, BrABF4, and BrABI5 in tobacco leaves induced ABA accumulation and promoted chlorophyll degradation by upregulating tobacco ABA biosynthetic and chlorophyll catabolic genes, resulting in the accelerated leaf senescence. These effects were significantly attenuated by melatonin treatment. Our findings suggest that melatonin-mediated inhibition of leaf senescence involves suppression of ABFs-mediated ABA biosynthesis and chlorophyll degradation. Unraveling of the molecular regulatory mechanism of leaf senescence controlled by ABA and melatonin expands our understanding of the regulation of this phenomenon and offers potentially more effective molecular breeding strategies for extending the shelf-life of Chinese flowering cabbage.

MaBZR1/2 act as transcriptional repressors of ethylene biosynthetic genes in banana fruit.

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.

Activation of the Transcription of BrGA20ox3 by a BrTCP21 Transcription Factor Is Associated with Gibberellin-Delayed Leaf Senescence in Chinese Flowering Cabbage during Storage.

Several lines of evidence have implicated the involvement of the phytohormone gibberellin (GA) in modulating leaf senescence in plants. However, upstream transcription factors (TFs) that regulate GA biosynthesis in association with GA-mediated leaf senescence remain elusive. In the current study, we report the possible involvement of a TEOSINTE BRANCHED1/CYCLOIDEA/PCF (TCP) TF BrTCP21 in GA-delayed leaf senescence in Chinese flowering cabbage. Exogenous GA3 treatment maintained a higher value of maximum PSII quantum yield (Fv/Fm) and total chlorophyll content, accompanied by the repression of the expression of senescence-associated genes and chlorophyll catabolic genes, which led to the delay of leaf senescence. A class I member of TCP TFs BrTCP21, was further isolated and characterized. The transcript level of BrTCP21 was low in senescing leaves, and decreased following leaf senescence, while GA3 could keep a higher expression level of BrTCP21. BrTCP21 was further found to be a nuclear protein and exhibit trans-activation ability through transient-expression analysis in tobacco leaves. Intriguingly, the electrophoretic mobility shift assay (EMSA) and transient expression assay illustrated that BrTCP21 bound to the promoter region of a GA biosynthetic gene BrGA20ox3, and activated its transcription. Collectively, these observations reveal that BrTCP21 is associated with GA-delayed leaf senescence, at least partly through the activation of the GA biosynthetic pathway. These findings expand our knowledge on the transcriptional mechanism of GA-mediated leaf senescence.

BrTCP7 Transcription Factor Is Associated with MeJA-Promoted Leaf Senescence by Activating the Expression of BrOPR3 and BrRCCR.

The plant hormone jasmonic acid (JA) has been recognized as an important promoter of leaf senescence in plants. However, upstream transcription factors (TFs) that control JA biosynthesis during JA-promoted leaf senescence remain unknown. In this study, we report the possible involvement of a TEOSINTE BRANCHED1/CYCLOIDEA/PCF (TCP) TF BrTCP7 in methyl jasmonate (MeJA)-promoted leaf senescence in Chinese flowering cabbage. Exogenous MeJA treatment reduced maximum quantum yield (Fv/Fm) and total chlorophyll content, accompanied by the increased expression of senescence marker and chlorophyll catabolic genes, and accelerated leaf senescence. To further understand the transcriptional regulation of MeJA-promoted leaf senescence, a class I member of TCP TFs BrTCP7 was examined. BrTCP7 is a nuclear protein and possesses trans-activation ability through subcellular localization and transcriptional activity assays. A higher level of BrTCP7 transcript was detected in senescing leaves, and its expression was up-regulated by MeJA. The electrophoretic mobility shift assay and transient expression assay showed that BrTCP7 binds to the promoter regions of a JA biosynthetic gene BrOPR3 encoding OPDA reductase3 (OPR3) and a chlorophyll catabolic gene BrRCCR encoding red chlorophyll catabolite reductase (RCCR), activating their transcriptions. Taken together, these findings reveal that BrTCP7 is associated with MeJA-promoted leaf senescence at least partly by activating JA biosynthesis and chlorophyll catabolism, thus expanding our knowledge of the transcriptional mechanism of JA-mediated leaf senescence.

Pitaya HpWRKY3 Is Associated with Fruit Sugar Accumulation by Transcriptionally Modulating Sucrose Metabolic Genes HpINV2 and HpSuSy1.

Sugar level is an important determinant of fruit taste and consumer preferences. However, upstream regulators that control sugar accumulation during fruit maturation are poorly understood. In the present work, we found that glucose is the main sugar in mature pitaya (Hylocereus) fruit, followed by fructose and sucrose. Expression levels of two sucrose-hydrolyzing enzyme genes HpINV2 and HpSuSy1 obviously increased during fruit maturation, which were correlated well with the elevated accumulation of glucose and fructose. A WRKY transcription factor HpWRKY3 was further identified as the putative binding protein of the HpINV2 and HpSuSy1 promoters by yeast one-hybrid and gel mobility shift assays. HpWRKY3 was localized exclusively in the nucleus and possessed trans-activation ability. HpWRKY3 exhibited the similar expression pattern with HpINV2 and HpSuSy1. Finally, transient expression assays in tobacco leaves showed that HpWRKY3 activated the expressions of HpINV2 and HpSuSy1. Taken together, we propose that HpWRKY3 is associated with pitaya fruit sugar accumulation by activating the transcriptions of sucrose metabolic genes. Our findings thus shed light on the transcriptional mechanism that regulates the sugar accumulation during pitaya fruit quality formation.

A comprehensive investigation of starch degradation process and identification of a transcriptional activator MabHLH6 during banana fruit ripening.

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.