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

Abstract

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.

Protein phosphorylation modification of a transcription factor hastens the ripening of banana fruit.

Introduction

Fruit ripening is a genetically programmed series of events requiring the up- and downregulation of a variety of genes designed to alter the postharvest quality attributes, which are closely linked with the consumer preference and acceptance, such as color, texture, aroma, flavor, and nutritional compounds (Li et al., 2019; Chen et al., 2020). Meanwhile, fruit ripening has a negative effect on shelf-life due to softening primarily caused by disassembly of cell wall, and this causes considerable losses throughout the food distribution chain. Factors influencing fruit ripening are complex, which involve developmental, hormonal, epigenetic regulation, as well as environmental stimuli (Tang et al., 2020). Hence, a better understanding of fruit ripening may provide guidance not only for the maintenance of quality traits, but also for the proper application of postharvest practices and strategies in manipulating fruit ripening and extending shelf-life.

Studies on the transcription regulation of fruit ripening have established the regulatory models for ripening, due to the characterization of three spontaneous ripening mutants, namely ripening-inhibitor (rin), nonripening (nor), and Colorless nonripening (Cnr) that encode transcription factors (TFs) belonging to the MCM1-agamous-deficiens-serum response factor (MADS)-box, NAM-ATAF1/2-CUC2 (NAC), and SQUAMOSA promoter-binding protein (SBP)-like families, respectively (Wang et al., 2020a). Besides these TFs, other TFs have been identified to control fruit ripening, including ethylene response factor, ethylene-insensitive 3/ethylene-insensitive 3 like, homeo-box, basic helix–loop–helix (bHLH), MYB, and so on (Karlova et al., 2014). Exploring the roles of these ripening-associated TFs is an effective tool for understanding the mechanisms underlying fruit ripening. The basic leucine zipper (bZIP) TF constitutes one of the largest and the most diverse TF families in plants and features a highly conserved DNA-binding domain with approximate 60–80 amino acids (aa) in length consisting of a conserved basic region and a leucine zipper (Jakoby et al., 2002). Numerous studies have revealed that bZIP TFs are involved in a series of biological processes, such as plant growth and development, and response to abiotic/biotic stresses (Dröge-Laser et al., 2018; Yu et al., 2020). In addition, some bZIP TFs are involved in the formation of quality attributes during fruit ripening. For example, over-expression of SlbZIP1 and SlbZIP2 in tomato (Solanum lycopersicum) fruits resulted in higher sugar and amino acid contents (Sagor et al., 2016). Banana (Musa acuminata) MabZIP4/5 are involved in aroma production during fruit ripening by controlling the expression of aroma biosynthetic genes (Guo et al., 2018). It should be noted that bZIP proteins function as either transcriptional activators or repressors of various signaling pathways by specifically targeting the cis-elements containing a core ACGT, such as the G-box (CACGTG), C-box (GACGTC), and A-box (TACGTA) motifs, in the promoters of their target genes (Jakoby et al., 2002).Banana MabZIP93 trans-activates the expression of cell wall-modifying genes including pectate lyase2 (MaPL2), pectinesterase1 (MaPE1), xyloglucan endo-transglycosylase/hydrolase23 (MaXTH23), and xyloglucan galactosyltransferase1 (MaXGT1; Wu et al., 2019), while MabZIP74 acts as a transcriptional repressor of ethylene biosynthetic genes 1-amino-cyclopropane-1-carboxylic acid oxidase1/4 (MaACO1/4) during fruit ripening (Liang et al., 2020). Interestingly, these two MabZIP proteins were found to be phosphorylated by mitogen-activated protein kinases (MAPKs; Wu et al., 2019; Liang et al., 2020), implying that protein phosphorylation may have an effect on the transcriptional activity and stability of MabZIPs.

Protein phosphorylation is one of the most important and ubiquitous post-translational modifications (PTMs). In plants, ∼47% of expressed proteins display phosphorylation in at least one instance (Mergner et al., 2020). Protein phosphorylation modulates a variety of cellular processes, including protein activity, subcellular location, protein–protein interaction, and stability (Olsen et al., 2006), and it is catalyzed by protein kinases such as MAPKs, receptor-like kinases, sucrose nonfermenting1-related protein kinases, and calcium-dependent protein kinases (CDPKs). MAPKs are a family of protein kinases that constitute the MAPK cascade. A typical MAPK signaling cascade is composed of three protein kinases sequentially activated via phosphorylation by an upstream kinase: MAP kinase kinase kinase (MAPKKK), MAP kinase kinase (MAPKK), and MAPK (Rodriguez et al., 2010). As the terminal player of the signaling cascades, the active MAPKs recognize and phosphorylate-specific substrate proteins to function in various signaling networks. These substrates incorporate enzymes, structural proteins, and TFs. Recently, phosphorylation modification of TFs has received much attention, as TFs function as key regulators in the signaling pathways. For example, phosphorylation of GsERF7 modified its translation from cytoplasm to nuclei and elevated its transcriptional activation ability in adaptation of soybean (Glycine max) to environmental stresses (Feng et al., 2021). In rice (Oryza sativa), WRKY53 phosphorylation by GSK2 promoted its degradation (Tian et al., 2021), but its phosphorylation by MAPK6 elevated its transcriptional activity (Tian et al., 2017). The positive or negative effects of phosphorylation on the function of TFs might depend on substrate and protein kinase combination. However, only a few target proteins of MAPKs have been identified to date, and a comprehensive interaction map between many MAPKs and their substrates remains largely unknown, requiring further investigation to clarify.

Banana is an important fruit crop grown throughout the year in the tropical and subtropical regions of the world with an annual global production of about 114 million tons from an area of 5.6 million ha (Food and Agriculture Organization [FAO], 2018). Being climacteric, bananas are usually harvested at mature-green stage, and postharvest ripening is artificially carried out by treatment with exogenous ethylene or ethylene-generated chemicals under controlled conditions. Temperature, packaging, mechanical injury, concentration, and exposure time of ethylene are the major factors affecting the ripening process as well as quality attributes such as texture, color, flavor, and desirable bioactive components (Duan et al., 2007). Therefore, studying the mechanisms underlying banana ripening would be a reasonable way to ensure the quality and extend the shelf-life of this fresh fruit. TFs as well as their transcriptional networks related to fruit ripening of banana have been identified and involve MaERF11, MaDREB2, MaMADS1/2, MaNAC1/2, MaTCP5/19/20, MaMYB3, MabHLH6/7, and MabZIP5/6/74/93 (Shan et al., 2012; Xiao et al., 2013, 2018; Elitzur et al., 2016; Han et al., 2016; Kuang et al., 2017; Guo et al., 2018; Song et al., 2018, 2020; Wu et al., 2019; Liang et al., 2020). Interestingly, based on the fruitENCODE database, the ripening regulatory circuit of banana has two positive feedback loops, in which the first one is NAC-type positive loop, and the second one maintains the ethylene biosynthesis when the first loop is hindered, and this could explain the fact that 1-methylcyclopropene (1-MCP) cannot prohibit banana ethylene generation once ripening is initiated (Lü et al., 2018). More recently, using the weighted gene co-expression network analysis and cis-motif analysis, 25 TFs have been characterized as important ripening regulators, which participate in different ripening-related pathways (Kuang et al., 2021). However, despite the abundant evidence for the involvement of transcriptional regulators in banana ripening, details of the PTMs in modulating the transcription activity of TFs remain largely unknown. Deciphering the functions of TFs in the regulation of banana ripening holds great potential for further understanding fruit ripening and may provide tools for increasing agricultural productivity.

In this study, we performed a comparative phosphoproteomic analysis and identified many proteins that display diverse phosphorylation patterns during banana fruit ripening. We found that a bZIP-type protein MabZIP21 showed elevated phosphorylation content during fruit ripening. Genome-wide binding site analysis revealed that MabZIP21 targets a subset of fruit ripening-related genes. Furthermore, we showed that a protein kinase MaMPK6-3 directly interacts with and phosphorylates MabZIP21, and that phosphorylation of MabZIP21 increases its transcriptional activation activity on the ripening-related genes. Our data provide insights into phosphorylation modification of bZIP TF in banana fruit ripening.

Results

Phosphoproteomic analysis reveals an involvement of MabZIP21 phosphorylation in banana fruit ripening

To obtain a global view of the phosphoproteome of banana during the ripening process, banana fruit ripening was induced by exogenous ethylene treatment (Supplemental Figure S1A). As expected, banana fruit ripening occurred, as evidenced by the appearance of ethylene production, showing a maximal level at 3 d after treatment (Supplemental Figure S1B). Meanwhile, we detected the accumulation of phosphoproteins during banana ripening using anti-phospho-Ser/Thr/Tyr antibody. We found that the accumulation of phosphoproteins gradually increased as ripening proceeded, and peaked at Day 3 followed by a decline (Supplemental Figure S1C), suggesting that banana fruit ripening accompanies protein phosphorylation. Based on these data, we chose the samples of 0 day (unripening) and 3 d after ethylene treatment (ripening) to perform phosphoproteomic assay, and each sample contains three biological replicates. Analysis of the data by principal component analysis showed that the population of phosphopeptides in ripe banana clustered far from that of the unripe fruit (Figure 1A), indicating the change of phosphoproteins in banana ripening. Moreover, we identified a total of 63 unique phosphopeptides corresponding to 49 proteins from banana pulp during fruit ripening based on the phosphoproteomics analysis using unripe and ripe bananas (Figure 1B;Supplemental Data Set S1). Among the phosphopeptides, 95.24% were due to single phosphorylation, and 4.76% were due to double phosphorylation (Figure 1C). The distribution of phosphorylation events was Ser (90.48%) and Thr (9.52%; Figure 1C).

Figure 1.

Analysis of phosphoproteins in ripening bananas. A, Principal component analysis of three biological replicates in bananas at 0 and 3 d after ethylene treatment. B, Number of peptides and unique proteins identified from phosphoproteomic assay. C, Frequency distribution of all the identified phosphopeptides based on the number of phosphorylation sites and phosphorylated amino acids. D, Sequence motif analysis of phosphorylation sites. The numbers in brackets indicate the percentage of phosphorylation sites.

To identify which individual or classes of kinases were involved in protein phosphorylation during banana fruit ripening, we analyzed the phosphorylation motifs from the differentially phosphorylated proteins using the MEME MOMO online software. Three phosphorylation motifs including motifs [SP], [SD], and [RxxS] were identified, which correspond to the potential substrates of MAPK, Acidic kinase, and CDPK, occupying 28.6%, 20.6%, and 15.9%, respectively (Figure 1D). Notably, 49 phosphoproteins displayed elevated levels in phosphorylation in ripe fruit (Supplemental Figure S2 and Supplemental Data Set S1), suggesting that banana fruit ripening accompanies the phosphorylation of a variety of proteins. Among these differential phosphoproteins, a total of three TFs, which display elevated phosphorylation levels during banana fruit ripening were identified, including two bZIPs (Ma04_p35070 and Ma10_p15920) and one HD-ZIP (Ma08_p23500). Among these TFs identified, MabZIP21 (Ma04_p35070) showed substantially high levels of protein phosphorylation (23.26-fold) in ripe banana, and we thus focused on it for further study.

To investigate the possible role of MabZIP21 in banana fruit ripening, we first monitored its expression profiles during banana fruit ripening using reverse transcription quantitative polymerase chain reaction (RT-qPCR). Supplemental Figure S3 indicates the change of ripening parameters such as ethylene production, firmness, and chromaticity in bananas during natural, ethylene-promoted, and 1-MCP-alleviated ripening. Evaluation of ethylene production at different types of fruit ripening illustrated that the climacteric ethylene peaks appeared at 17, 3, and 27 d of natural, ethylene-promoted, and 1-MCP-alleviated ripening, with maximum production of 2.4, 2.6, and 2.5 μL ethylene kg⁻¹ h⁻¹, respectively. Fruit firmness dropped rapidly as fruit ripening progressed in all treatments, which explains the occurrence of fruit softening during ripening (Supplemental Figure S3B). Similarly, the hue values declined continuously in all ripening patterns, consistent with the pigment development from green to yellow color. Meanwhile, the transcript of MabZIP21 substantially increased in the pulp of bananas during fruit ripening in all treatments, and maintained its maximum level at the ripening stage (Figure 2A).

Figure 2.

Change of MabZIP21 transcript and phosphorylation abundance during banana fruit ripening. A, Expression profile of MabZIP21 in bananas with natural, ethylene-induced, and 1-MCP-delayed ripening. The relative expression level is shown as a ratio relative to 0 days of natural ripening, which was set at 1. The data represent mean ±sd (n = 3 biological replicates). B, Subcellular localization of MabZIP21 expressed in tobacco leaf cells. Green fluorescence was captured by fluorescence microscopy 3 d after infiltration. Scale bar = 25 μm. C, Transcriptional activation capacity of MabZIP21 in plant cells. Compared with the pBD control, pBD-MabZIP21 significantly stimulated the expression of the LUC reporter. The ratio of LUC to REN of the pBD vector was used as a calibrator (value set as 1). The data represent mean ±sd (n = 6 biological replicates). The different letters denote significant differences (one-way ANOVA-multiple comparisons, P < 0.05). D, Phosphorylation abundance of MabZIP21 in unripe and ripe banana fruit at 0 and 3 d after ethylene treatment, respectively. The MabZIP21 proteins were immunoprecipitated using anti-MabZIP21 antibody from the proteins extracted in unripe and ripe banana, and then subjected to immunoblot analysis using anti-phospho-Ser/Thr/Tyr and anti-MabZIP21 antibodies, respectively. E, Quantification of immunoblot bands using Image Lab 5.1 software. Value shows the phosphorylated MabZIP21 in 3 d relative to that in 0 days (set at 1). The data represent mean ±sd (n = 3 biological replicates).

To confirm the subcellular localization of MabZIP21, the MabZIP21-GFP and GFP control in GV3101 were injected into tobacco (Nicotiana benthamiana) leaf cells. Our results showed that green fluorescent signals of the MabZIP21-GFP fusion protein was observed throughout the entire cell, which is similar to that of the GFP control (Figure 2B). To further examine the transcriptional activity of MabZIP21, a GAL4 binding domain of BD-MabZIP21 fusion protein was produced as effector, which can target the GAL4 DNA-binding site in front of the luciferase (LUC) reporter, according to Han et al. (2016). Co-infiltration of the effector and reporter plasmids into tobacco leaf cells showed that MabZIP21 has transcriptional activity in vivo, as MabZIP21 could activate the transcription of LUC reporter gene (Figure 2C).

To detect the phosphorylation abundance of MabZIP21 in banana fruit ripening, we firstly generated polyclonal anti-MabZIP21 antibody, which picked up the MabZIP21 recombinant protein only (Supplemental Figure S4). Then MabZIP21 was immunoprecipitated from proteins in unripe and ripe banana fruit using anti-MabZIP21 antibody, and then followed by phosphorylation detection using anti-phospho-Ser/Thr/Tyr antibody. The phosphorylation level of MabZIP21 was much stronger in ripe banana than in unripe fruit (Figure 2, D and E), which is in line with the findings of phosphoproteomics.

MabZIP21 controls a subset of ripening-associated genes

In order to gain insight into the mechanism by which MabZIP21 controls banana ripening, we assessed MabZIP21 DNA-binding sites using DNA-affinity purification sequencing (DAP-Seq) technology, which has been recently used for high-throughput identifying TF binding sites in vitro (O’Malley et al., 2016). Two biological replicates of the DAP-Seq analysis revealed 849 overlapping peaks (Figure 3A;Supplemental Data Set S2), which were regarded as the high-confidence MabZIP21 binding regions and were used for further study. Furthermore, the MabZIP21 binding regions were significantly enriched mostly (73.26%) in 3-kb upstream of the transcription start site (TSS; Figure 3B), primarily concentrating on the region of −400-bp upstream of TSS (Figure 3C), suggesting that MabZIP21 may bind to these regions to regulate gene transcription. The distribution of MabZIP21 binding sites on each chromosome was proportional to chromosomal sizes, which suggests that MabZIP21 has no preference for a specific chromosome (Figure 3D). MabZIP21 binding motifs were predicted using MEME analysis (Machanick and Bailey, 2011). Results showed that MabZIP21 recognizes a cis-acting element with the core sequence of “ACGT” (Figure 3E). In addition, among the overlapping set of the target genes, the most significantly enriched kyoto encyclopedia of genes and genomes (KEGG) categories (false discovery rate [FDR] <0.05) belong to “metabolic pathways”, “biosynthesis of secondary metabolites”, and “plant hormone signal transduction” (Figure 3F).

Figure 3.

Genome-wide binding study of MabZIP21 using DAP-Seq analysis. A, DAP-Seq using two biological replicates revealed 849 high-confidence MabZIP21 binding peaks. B, Distribution of MabZIP21 association sites within genic regions, including 5′-UTR (untranslated region), promoter, 3′UTR, exon, and intron. C, MabZIP21 association sites are highly enriched in proximal to the transcriptional start sites. D, Distribution of MabZIP21-binding sites across the eleven Musa chromosomes. DAP-Seq peaks indicate the MabZIP21-binding sites. E, The potential DNA-binding motif of MabZIP21 analyzed according to DAP-Seq results. F, Pathway enrichment analysis of MabZIP21-binding genes. Each circle represents an enriched category compared with the whole genome after FDR correction (FDR < 0.05). The size of each circle is proportional to the number of genes annotated to the node.

It is well accepted that fruit ripening is concomitant with ethylene production and change in color, texture, aroma, and flavor, thus genes involved in ethylene biosynthesis, color formation, cell wall hydrolysis, volatile production, sugar accumulation, and transportation are regarded as ripening-associated genes (Li et al., 2021). To study the potential MabZIP21’s target genes that are involved in fruit ripening, we selected several ripening-associated genes such as MaACO1, MaPG3, MaPL5, MaPL15, MaPE42, MaPE51, and MaEXPA15 from the DAP-Seq data, and validated the binding of MabZIP21 with these promoters by conducting electrophoretic mobility shift assay (EMSA). We first expressed and purified GST-MabZIP21 fusion protein from Escherichia coli. The probes derived from the bound peaks of these genes in the dataset were biotin-labeled. As expected, MabZIP21 bound to the regulatory regions of MaACO1, MaPG3, MaPL5, MaPL15, MaPE42, MaPE51, and MaEXPA15 by recognizing the predicted binding motif, but the glutathione S-transferase (GST) protein alone did not (Figure 4A). In competition assays, with excess of cold probe, the intensity of mobility shift signal decreased, while with same concentration of mutated probes, the mobility shift was still clearly observed, suggesting that these binding were specific. To determine whether MabZIP21 directly associates with the promoters of these ripening-associated genes in vivo, chromatin immunoprecipitation (ChIP)-polymerase chain reaction (PCR) assays were performed. The predicted cis-element-containing promoter regions of MaACO1, MaPG3, MaPL5, MaPL15, MaPE42, MaPE51, and MaEXPA15 were enriched by anti-MabZIP21 antibody compared with IgG control (Figure 4B). Together, these findings indicate that MabZIP21 is likely involved in banana ripening at least in part by targeting a subset of ripening-associated genes.

Figure 4.

Direct binding of MabZIP21 to the promoters of ripening-associated genes in banana. A, EMSA assays showing that the GST-MabZIP21 fusion protein binds to the promoters of MaPG3, MaPL15, MaPL5, MaPE42, MaPE51, MaEXPA15, and MaACO1. – or + represents absence or presence, respectively. ++ indicates increasing amounts of unlabeled WT or mutated probes for competition. B, ChIP-qPCR assay showing the binding of MabZIP21 to the MaPG3, MaPL15, MaPL5, MaPE42, MaPE51, MaEXPA15, and MaACO1 promoters in vivo. The black square indicates MabZIP21 binding site identified by DAP-Seq analysis, and the black triangle represents the TSS. The data represent mean ± sd (n = 3 biological replicates). The different letters denote significant differences (one-way ANOVA-multiple comparisons, P < 0.05).

Identification of interacting proteins of MabZIP21

Based on the above findings that MabZIP21 displays high level of phosphorylation in the ripening of banana fruit (Supplemental Data Set S1; Figure 2, D and E), and that MAPK-mediated phosphorylation site occupies the largest portion during the ripening (Figure 1D), it seems plausible that MabZIP21 might have a relationship with protein kinases such as MAPK. To detect whether MabZIP21 interacts with MAPK, a yeast two-hybrid (Y2H) screening was employed. Each coding sequence of the 10 MAPK candidates was inserted into the pGADT7 vector fused with activation domain, while MabZIP21 was inserted into the pGBKT7 vector fused with BD. Since MabZIP21 has self-activation activity when it was subcloned into pGBKT7, we added 60-mM 3-AT (3-aminotriazole; a competitive inhibitor of HIS3 reporter gene) for excluding its self-activation before Y2H test. Preliminary experiments revealed that MabZIP21 was able to interact with MaMPK6-3, but not with the other nine candidates (Supplemental Figure S5). The expression of MaMPK6-3 gradually increased during banana fruit ripening (Supplemental Figure S6A). The interaction between MabZIP21 and MaMPK6-3 was further validated by Y2H (Figure 5A). Interestingly, we also found that MabZIP21 could interact with MabZIP21 itself (Figure 5A), indicating that MabZIP21 might form homodimer in gene regulation. In vitro pull-down assays illustrated that maltose binding protein (MBP)-MaMPK6-3 and MBP-MabZIP21 bound to GST-MabZIP21, respectively, but not to the GST alone protein (Figure 5B). Interestingly, bimolecular fluorescence complementation (BiFC) assays using N. benthamiana leaf epidermal cells indicated that MabZIP21-YNE and MaMPK6-3-YCE, MaMPK6-3-YNE and MabZIP21-YCE can reconstitute the YFP signals in the nuclear compartment of transformed cells, corresponding to the nuclear marker NLS-mCherry (Figure 5C), although the GFP fluorescence of both MaMPK6 and MabZIP21 was detected in the nucleus and cytoplasm (Figure 2B;Supplemental Figure S6B). Likewise, MabZIP21-YNE and MabZIP21-YCE form YFP signals in both the nucleus and cytoplasm (Figure 5C). Consistently, co-immunoprecipitation (Co-IP) assays using GFP-MabZIP21 and His-MaMPK6-3 or His-MabZIP21 co-infiltrated into N. benthamiana leaf cells indicated that His-MaMPK6-3 or His-MabZIP21 was detected in the immunoprecipitated proteins (Figure 5D). These results indicate that MabZIP21 can interact with MaMPK6-3 and MabZIP21 itself in vitro and in vivo.

Figure 5.

MabZIP21 physically interacts with MaMPK6-3 and itself in vitro and in vivo. A, Y2H assay showing that MabZIP21 could interact with MaMPK6-3 and MabZIP21. Transformed yeast cells were grown on the control medium DDO (SD/–Trp/–Leu) and selective medium QDO (SD/–Trp/–Leu/–His/–Ade). AD, activation domain; DBD, DNA-binding domain. B, In vitro pull-down assay confirming the interactions of MabZIP21 with MaMPK6-3 and MabZIP21. Recombinant MBP-MaMPK6-3 or MBP-MabZIP21 was used as prey and pulled down using protein crude extract of GST or GST-MabZIP21 as bait. C, BiFC assay verifying the interaction between MabZIP21 and MaMPK6-3, MabZIP21. NLS-mCherry was used as an indicator for nucleus. The right gels indicate the expression of fusion protein detected by immunoblot using anti-MYC and anti-HA antibodies, respectively. D, Co-IP assay illustrating that MabZIP21 associates with MaMPK6-3 and MabZIP21 in N. benthamiana leaves in vivo. E, DLR assay showing that the interaction between MabZIP21 and MaMPK6-3 enhances MabZIP21-mediated transcriptional activation ability. MabZIP20-2 was used as a negative control. The relative LUC activities normalized to the REN activity are shown (LUC/REN). The data represent mean ± sd (n = 6 biological replicates). The different letters denote significant differences (one-way ANOVA-multiple comparisons, P < 0.05).

To determine whether MaMPK6-3 affects MabZIP21-mediated transcriptional activity, we conducted transient LUC expression assays in tobacco leaf cells. As shown in Figure 5E, the expression of MabZIP21 alone could activate the transcription of its target genes, as shown by the higher relative LUC/REN ration than that of the control. Overexpression of MaMPK6-3 alone did not affect LUC activity of MaPG3 and MaPE42, but enhanced that of MaPL15, MaPL5, MaPE51, MaEXPA15, MaACO1, possibly via the uncharacterized mechanisms. More importantly, however, a synergistic effect was observed when MaMPK6-3 was combined with MabZIP21, observed with the promoters of MaACO1, MaPG3, MaPL5, MaPL15, MaPE42, MaPE51, and MaEXPA15, pointing to an additive action of these two proteins in the regulation of ripening-associated genes. These observations suggest that the interaction with MaMPK6-3 enhances the transcriptional activation ability of MabZIP21.

Transient overexpression of MaMPK6-3 promotes banana ripening and protein phosphorylation

To determine the possible role of MaMPK6-3 in banana fruit ripening, we transiently overexpressed MaMPK6-3 into banana fruit, due to the difficulty in stable transformation. First, we infiltrated red inks from the top of banana fruit, and found that the pulp tissue was pervaded by red ink (Figure 6A), suggesting that the infiltration of Agrobacterium carrying a given plasmid in banana fruit could be an alternative strategy in analyzing gene function. After carefully examining the protein contents of HA-tag in infiltrated bananas, the MaMPK6-3-overexpressed fruit displayed faster ripening than the fruit expressing empty vector (Figure 6, B and C). We then detected the abundance of phosphoproteins in these two groups of fruits using anti-phospho-Ser/Thr/Tyr antibody. Immunoblot analysis indicated that banana overexpressing MaMPK6-3 contained more phosphoproteins than the control fruit (Figure 6, D and E). These findings suggest that MaMPK6-3 plays a role in banana ripening and protein phosphorylation.

Figure 6.

Transient overexpression of MaMPK6-3 in banana fruit. A, Schematic diagram for banana infiltration with red inks. B, Ripening phenotypes of banana fruits transiently expressing MaMPK6-3-HA or empty vector. C, Immunoblot analysis of HA-tag in infiltrated bananas using anti-HA antibody. Actin was used as a loading control. D, Abundance of phosphoproteins in bananas transiently expressing MaMPK6-3-HA or empty vector. Immunoblot was analyzed using anti-phospho-Ser/Thr/Tyr antibody. Actin was used as the loading control. E, Quantitative analysis of immunoblot bands using the Image Lab 5.1 software. The data represent mean ± sd (n = 3 biological replicates).

MaMPK6-3 phosphorylates MabZIP21 at S436 and T318 sites

The finding that MaMPK6-3 interacts with MabZIP21 raised the question of whether MaMPK6-3 is able to directly phosphorylate MabZIP21. To address this question, we employed an in vitro phosphorylation assays using the phos-tag sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS–PAGE) technologies. GST-MabZIP21 and MBP-MaMPK6-3 recombinant proteins were successfully expressed and purified, respectively. As shown in Supplemental Figure S7, a mobility shift corresponding to phospho-GST-MabZIP21 by MBP-MaMPK6-3 in the phosphate-affinity SDS–PAGE, while treatment of alkaline phosphatase (AP) abolished the phosphorylated band of GST-MabZIP21, supporting the notion that MaMPK6-3 phosphorylates MabZIP21.

To map the MabZIP21 site(s) phosphorylated by MaMPK6-3, we cut out bands from gels containing GST-MabZIP21 and phosphorylatable GST-MabZIP21 and performed liquid chromatography tandem mass spectrometry (LC–MS/MS) analyses. Two phosphorylation sites (Ser-436 and Thr-318) were found in phosphorylatable GST-MabZIP21 (Figure 7, A and B), but no phosphorylation residues were found in GST protein. It should be noted that the identified phosphorylated sites (Ser-436 and Thr-318) were different with the site (Ser-422) found in phosphoproteomic assays (Supplemental Data Set S1).

Figure 7.

Identification of phosphorylation sites of MabZIP21. A and B, Mass spectrum showing two phosphorylated peptides (MFGGPTpSPLQR, ADVEpTLR) of MabZIP21 by MaMPK6-3. C, In vitro phosphorylation assay of recombinant MaMPK6-3, MabZIP21, and the phosphomutant forms of MabZIP21 in which Thr-318 and Ser-436 were each or both replaced with Ala. Proteins were separated by Phos-Tag SDS–PAGE and analyzed by western blot. As for phosphatase treatment, protein extracts were incubated with alkaline phosphatase (37°C, 10 min) before Phos-Tag SDS–PAGE separation and immunoblot analysis. D, In vivo phosphorylation assay of MabZIP21 by MaMPK6-3. The GFP-MabZIP21 proteins were immunoprecipitated using anti-GFP antibody from tobacco leaf transiently expressing GFP-MabZIP21 or GFP-MabZIP21 phosphomutants with or without the co-expression of His-MaMPK6-3, and then they were subjected to immunoblot assays using anti-phospho-Ser/Thr/Tyr, anti-GFP, and anti-His antibodies, respectively. E, MabZIP21^S436A/T318A affects its interaction with MaMPK6-3 in Y2H assay. MabZIP21 interacts with MaMPK6-3, while mutation of Thr-318 and Ser-436 to Ala-318 and Ala-436 in MabZIP21 inhibited the interaction with MaMPK6-3. F, MabZIP21^S436A/T318A inhibits the interaction with MaMPK6-3 in BiFC assay. Co-expression of MabZIP21 and MaMPK6-3 induces strong YFP signals, whereas mutation of Thr-318 and Ser-436 to Ala-318 and Ala-436 in MabZIP21 abolished the interaction between MabZIP21 and MaMPK6-3 in tobacco leaf cells. The right panels indicate the expression of fusion protein detected by immunoblot using anti-MYC and anti-HA antibodies, respectively. Scale bar = 25 μm. G, Mutation of Thr-318 and Ser-436 to Ala-318 and Ala-436 or Glu-318 and Glu-436 has no effect on the subcellular localization of MabZIP21. Green fluorescence was captured by fluorescence microscopy 3 d after infiltration. Scale bar = 25 μm.

Phosphorylation occurs generally in serine (Ser) or threonine (Thr) residues, resulting in acidic phosphoserine or phosphothreonine. In phosphomimetic mutants the phosphorylation sites are replaced with negatively charged amino acids such as glutamic acid (Glu) or aspartic acid (Asp) by site-directed mutagenesis, while in the nonphosphorylatable mutants they are substituted with positively charged alanine (Ala; Dóczi and Bögre, 2018). To verify these phosphorylation sites, on one hand Ser-422 was mutated to Ala (MabZIP21^S422A) and Glu (MabZIP21^S422E) to mimic the nonphosphorylation and phosphorylation, respectively; on the other hand, Ser-436 and Thr-318 were substituted with Ala (MabZIP21^S436A/T318A) and Glu (MabZIP21^S436E/T318E), respectively. Results showed that replacing S422 with either Ala or Glu had no effect on MabZIP21’s trans-activation ability, subcellular localization, or interaction with MaMPK6-3 in Y2H and BiFC assays (Supplemental Figure S8). Additionally, MaMPK6-3 was able to phosphorylate MabZIP21, but not MabZIP21^S436A/T318A in vitro and in vivo assays (Figure 7, C and D). Y2H and BiFC analyses revealed that the nonphosphorylatable mutant MabZIP21^S436A/T318A abolished the interaction with MaMPK6-3 completely (Figure 7, E and F). Interestingly, both MabZIP21^S436A/T318A and MabZIP21^S436E/T318E were exclusively targeted in cytoplasm and nucleus (Figure 7G), suggesting that phosphomimetic or nonphosphorylatable version has no effect on MabZIP21 localization. Moreover, compared with the wild-type (WT) of MabZIP21, phosphomimetic form MabZIP21^S436E/T318E possessed strong transcriptional activation activity, while phosphonull form MabZIP21^S436A/T318A had relatively low activation capacity in plant cells (Figure 8A). Additionally, the co-expression of MabZIP21 with MaMPK6-3 could elevate the transcription of a subset of ripening-associated genes, but no significant increment of target gene transcription was detected when MabZIP21^S436A/T318A and MaMPK6-3 were co-expressed (Figure 8B). Furthermore, the binding of phosphorylated MabZIP21 on target gene promoter was investigated using EMSA assays. Compared to MabZIP21 alone, the binding strength of MabZIP21 to target promoter was enhanced by adding purified MaMPK6-3 to the reaction. Similarly, the phosphomimetic version MabZIP21^S436E/T318E also exhibited higher DNA-binding ability. In contrast, the nonphosphorylatable mutant MabZIP21^S436A/T318A displayed less binding capacity regardless of the presence or absence of MaMPK6-3 (Figure 8, C and D). Overall, these results establish that the Ser-436 and Thr-318 but not Ser-422 are the major phosphorylation sites of MabZIP21 by MaMPK6-3, and that MaMPK6-3-mediated phosphorylation could enhance MabZIP21 transcriptional activation ability.

Figure 8.

Impact of Thr-318 and Ser-436 phosphorylation on MabZIP21 activity. A, Phosphomimetic form of MabZIP21 increased its transcriptional activity in plant cells, whereas the dephosphorylation version decreased its trans-activation capacity. The data represent mean ±sd (n = 6 biological replicates). The different letters denote significant differences (one-way ANOVA-multiple comparisons, P < 0.05). B, Mutation of Thr-318 and Ser-436 to Ala-318 and Ala-436 did not show additive effect of MabZIP21 and MaMPK6-3 on the target gene activation. The data represent mean ± sd (n = 6 biological replicates). The different letters denote significant differences (one-way ANOVA-multiple comparisons, P < 0.05). C, EMSA showing the binding of MabZIP21 incubated with MaMPK6-3 or MabZIP21 phosphomutants to the target promoter. Phosphomimetic form of MabZIP21 elevated its DNA-binding ability, while the dephosphorylation version decreased its DNA-binding capacity. D, Quantitative analysis of shift bands using the Image Lab 5.1 software. The data represent mean ± sd (n = 3 biological replicates).

The S436 and T318 phosphorylation of MabZIP21 accelerates banana fruit ripening

To examine whether S436 and T318 phosphorylation in MabZIP21 affects banana fruit ripening, banana fruit at pre-climacteric stage were transiently transformed with Agrobacterium carrying the 35S::MabZIP21^S436A/T318A-HA or 35S::MabZIP21^S436E/T318E-HA constructs, and then the fruits were treated by ethylene treatment (Figure 9A). Immunoblot assays using anti-HA antibody indicated higher levels of HA tag in bananas transiently expressing MabZIP21^S436A/T318A-HA and MabZIP21^S436E/T318E-HA, indicating the efficiency of transient overexpression (Figure 9B). As shown in Figure 9, C–E, the introduction of the 35S::MabZIP21^S436E/T318E-HA vector resulted in an accelerated-ripening phenotype, which corresponded to the faster decline of fruit firmness, peel color as well as earlier appearance of climacteric ethylene peak, while fruit expressing 35S::MabZIP21^S436A/T318A-HA displayed similar symptoms as the control. Additionally, the expression of MabZIP21’s target genes including MaACO1, MaPG3, MaPL5, MaPL15, MaPE42, MaPE51, and MaEXPA15 was significantly enhanced in the early stage of MabZIP21^S436E/T318E-overexpressed fruit (Figure 9F). Overall, these observations demonstrate that the S436 and T318 phospho-mimicking form of MabZIP21 is able to hasten banana ripening by significantly enhancing the expression of a subset of ripening-associated genes.

Figure 9.

Functional analysis of phosphomutant forms of MabZIP21 in banana fruit using transient overexpression system. A, Ripening phenotypes of banana fruits transiently expressing MabZIP21^S436A/T318A-HA, MabZIP21^S436E/T318E-HA, or empty vector. B, Immunoblot analysis of HA-tag in infiltrated bananas using anti-HA antibody. Actin was used as a loading control. C, Fruit firmness, (D) ethylene production, and (E) color index in infiltrated banana fruits during ripening. The data represent mean ±sd (n = 3 biological replicates). F, Expression of MabZIP21, MaPG3, MaPL15, MaPL5, MaPE42, MaPE51, MaEXPA15, and MaACO1 transcripts in infiltrated banana fruits during ripening. The data represent mean ±sd (n = 3 biological replicates). The different letters denote significant differences (one-way ANOVA-multiple comparisons, P < 0.05).

MabZIP21 activates the transcription of MaMPK6-3 and itself

TFs generally regulate their transcription to form the feedback regulation (Mao et al., 2011; Schmidt et al., 2013). To investigate whether MabZIP21 could regulate the transcription of itself and MaMPK6-3, we first performed EMSAs. Strong binding of MabZIP21 on the promoters of MabZIP21 and MaMPK6-3 was observed, and addition of excess unlabeled probes but not the mutated probes could decrease the binding (Figure 10A). The binding of MabZIP21 on the promoters of MabZIP21 and MaMPK6-3 was further verified by ChIP-qPCR assays (Figure 10B). In addition, a transcriptional regulation activity assay indicated that the expression of MabZIP21-pro::LUC and MaMPK6-3-pro::LUC was significantly enhanced by transient overexpression of MabZIP21 (Figure 10C). These findings indicate that MabZIP21 positively regulates MaMPK6-3 and its transcription.

Figure 10.

MabZIP21 could stimulate the transcription of MaMPK6-3 and MabZIP21. A, EMSA showing that MabZIP21 binds to the promoters of MaMPK6-3 and MabZIP21. The DNA sequences that contained the WT or mutated forms were used as probes for DNA binding competition. – or + represents absence or presence, respectively. ++ indicates increasing amounts of unlabeled WT or mutated probes for competition. B, ChIP-qPCR assay indicating MabZIP21 enriched on MaMPK6-3 and MabZIP21 promoters. Banana fruit tissues were used to prepare chromatin and ChIP was performed with anti-MabZIP21 or IgG control. The data represent mean ±sd (n = 3 biological replicates). **P < 0.01 (Student’s t test). C, Transient transcriptional activity assay showing the activation of the MaMPK6-3 and MabZIP21 promoters by MabZIP21. The relative LUC activities normalized to the REN activity are shown (LUC/REN). The data represent mean ±sd (n = 6 biological replicates). **P < 0.01 (Student’s t test).

Discussion

In this study, we explored whether phosphorylation of TFs is involved in banana fruit ripening. We performed a quantitative phosphoproteomic analysis during banana fruit ripening and identified 49 phosphoproteins (representing 63 phosphopeptides) that were related to banana fruit ripening (Figure 1;Supplemental Data Set S1). Previously, a total of 352 and 144 phosphoproteins corresponding to 438 and 167 phosphopeptides were quantified from the seedlings of Cavendish Banana and Dajiao, respectively, which reveals the genetic diversity in cold response between these two banana crops (Yang et al., 2012). In contrast to the situation in other plants such as Arabidopsis (Arabidopsis thaliana) and maize (Zea mays), where a large number of phosphoproteins have been identified, we showed that relatively small number of phosphoproteins were found in banana fruit. Perhaps, the low abundance of phosphoproteins is too low to be detected using these strategies, or perhaps the phosphoproteins are difficult to isolate in fruits because of the abundant secondary metabolites, especially in banana fruit. Additionally, it is worth pointing out that most phosphoproteins showed increased phosphorylation levels in the ripening stage of banana fruit (Supplemental Figures S1C and S2), implying that fruit ripening is a biochemical and physiological process involving the action of protein phosphorylation. Therefore, studying the expression pattern of these phosphoproteins could help understand the molecular mechanism underlying banana fruit ripening.

We identified a putative bZIP TF (MabZIP21) as the highest phosphorylation level within TFs detected during banana ripening (Supplemental Data Set S1). It has been reported that bZIP TFs play a critical role in fruit ripening. For example, MdbZIP44 is involved in anthocyanin accumulation during apple ripening via targeting anthocyanin biosynthetic genes and interacting with MdMYB1 (An et al., 2018). In our previous study, MabZIP4/5 modulate aroma formation through controlling the transcription of a subset of aroma-producing genes (Guo et al., 2018). It should be noted that there are 121 bZIPs in banana genome (Hu et al., 2016), only a few of bZIP members have been implicated or demonstrated to have a role in fruit ripening. In this context, accumulation of MabZIP21 transcript was enhanced in the banana fruit ripening (Figure 2A), implying a positive role of MabZIP21 in fruit ripening. Moreover, MabZIP21 has transcriptional activation activity (Figure 2C). Using the DAP-Seq technology, we characterized the DNA-binding feature of MabZIP21 (Figure 3;Supplemental Data Set S2). We found that the binding regions of MabZIP21 were close to the transcription start site of the target genes, indicating that MabZIP21 has a role in gene regulation. More importantly, we identified a conserved binding motif for MabZIP21 via de novo motif discovery in sequences under DAP-Seq peaks, and found the core DNA cis-element (ACGT) that could be recognized by other bZIP proteins as well (Jakoby et al., 2002). Several ripening-associated genes including MaACO1, MaPG3, MaPL5, MaPL15, MaPE42, MaPE51, and MaEXPA15 harbor this MabZIP21-binding site in their promoters, and EMSA and ChIP-qPCR assays confirmed the binding of MabZIP21 to these promoters (Figure 4). It should be pointed out that we used the raw type of MabZIP21 in DAP-Seq analysis in the current study, and it would be interesting to use the phospho-mimicking form of MabZIP21 in DAP-Seq experiment and identify the difference between these two types of proteins in target gene regulation. Additionally, MabZIP21 can feedback regulate its transcription (Figure 10), which corroborates with that of WRKY, SERF1, and MaDREB2 (Mao et al., 2011; Schmidt et al., 2013; Kuang et al., 2017). Future research will focus on details of the action of MabZIP21 in regulating banana fruit ripening using the stable transgenic strategy.

The enhanced phosphorylation contents of MabZIP21 in banana ripening (Figure 2, D and E;Supplemental Data Set S1) led us to identify its potential interaction partner MaMPK6-3. Interaction between MabZIP21 and MaMPK6-3 was further validated by Y2H, BiFC, GST pull-down, and Co-IP methods (Figure 5, A–D), and this interaction could enhance MabZIP21-mediated transcriptional activation (Figure 5E). The expression of MaMPK6-3 was gradually increased following banana fruit ripening (Supplemental Figure S6A). Furthermore, transient overexpression of MaMPK6-3 in banana fruit could not only accelerate fruit ripening to some extent, but also enhance the phosphorylation levels of total proteins (Figure 6). These findings together suggest that MaMPK6-3 may have a role in banana fruit ripening.

MAPK-mediated phosphorylation of TFs affects their DNA-binding capacity, subcellular localization, protein stability, transcriptional activation ability, and protein–protein interaction (Zhang et al., 2016). For example, Arabidopsis MPK3/6 interact with and phosphorylate SPL protein, thus contributing to SPL stability and anther development (Zhao et al., 2017). Intriguingly, in banana fruit, MaMPK2 enhanced the transcriptional activation activity of MabZIP93 (Wu et al., 2019), whereas MaMAPK11-3 attenuated MabZIP74-mediated transcriptional inhibition via MAPK-mediated protein phosphorylation (Liang et al., 2020). In the current study, we found that MaMPK6-3 phosphorylated MabZIP21. Interestingly, the phosphorylation sites (Ser-436 and Thr-318) identified in MabZIP21 by LC-MS/MS are different with the site (Ser-422) identified by phosphoproteomic analysis (Supplemental Data Set S1). This may be attributed to the following reasons. First, relatively fewer phosphoproteins and phosphorylated sites were found by phosphoproteomic analysis in the present work, and the immunoprecipated peptides containing phosphorylated Ser-436 and Thr-318 sites may be too low to be quantified. Further improvement of the protein quality and immunoprecipitation efficiency may help to identify more phosphorylated sites. Secondly, different materials were used for analyses in two assays. Ser-422 site identified by phosphoproteomics used banana fruit for analysis, whereas the other two sites identified by LC–MS/MS analysis were from in vitro expressed GST-MabZIP21 proteins.

The nonphosphomutant form MabZIP21^S436A/T318A displayed neither interaction with MaMPK6-3 nor phosphorylation by MaMPK6-3 (Figure 7, E and F). Moreover, phosphomimetic form MabZIP21^S436E/T318E displayed stronger transcriptional activation ability on the target genes, while MabZIP21^S436A/T318A did not because of the prevention of S436/T318 phosphorylation (Figure 8). Importantly, transient overexpression of MabZIP21^S436E/T318E in banana fruit could upregulate the expression of MaACO1, MaPG3, MaPL5, MaPL15, MaPE42, MaPE51, and MaEXPA15 and thus accelerate fruit ripening (Figure 9). These results were consistent with the findings of Zhou et al. (2020), who showed that Thr-229 residue is essential for the full activity of WRKY33 in transcriptionally activating the expression of camalexin biosynthetic genes. Interestingly, phosphomutant variants in these two residues (S436E/A and T318E/A) of MabZIP21 were transiently expressed in N. benthamiana, but no substantial difference in subcellular localization was observed relative to WT MabZIP21 regardless of phosphomimetic or phosphonull versions (Figure 7G). Similarly, the interaction between MabZIP21 and itself also occurred in both cytoplasm and nucleus (Figure 5C). In general, most of the interaction between two TFs happens in the nucleus. Similar with our findings, WRKY40 translocates from the nuclear compartment to cytoplasm because of its interaction with an ABA receptor (Shang et al., 2010). This suggests that the subcellular localization of MabZIP21 might be regulated via an uncharacterized mechanism. Given that MaMPK6-3 is a component in MAPK cascade, it would be interesting to investigate whether other MAPK cascade components such as MAPKK or MAPKKK are involved in the regulation of MaMPK6-3 or MabZIP21 activity during banana fruit ripening. In contrast, our data displayed that neither of the phosphomutant forms (S422A and S422E) affects MabZIP21’s transcriptional activity, localization or interaction with MaMPK6-3 (Supplemental Figure S8). We assumed that Ser-422 in MabZIP21 may be phosphorylated by other kinases. Further investigation will be performed to study the role of Ser-422 phosphorylation in regulating MabZIP21 protein function in banana fruit.

Based on the current data, we proposed a working model for the role of MabZIP21 in controlling banana fruit ripening (Supplemental Figure S9). Ethylene induces the transcription of MabZIP21 and MaMPK6-3, and then MaMPK6-3 interacts with and phosphorylates MabZIP21 at both S436 and T318 sites. The phosphorylated form of MabZIP21 transactivates the transcription of ripening-associated genes such as MaACO1, MaPG3, MaPL5, MaPL15, MaPE42, MaPE51, and MaEXPA15, as well as MabZIP21 and MaMPK6-3 to feedback regulate the MaMPK6-3-MabZIP21 module, thus ultimately resulting in banana fruit ripening. Findings reported in this study provide insight into phosphorylation of MabZIP21 by MAPK in regulating fruit ripening.

Materials and methods

Plant material and treatments

Mature green banana fruits (Musa acuminata AAA group, Cavendish subgroup) were obtained from a commercial farm in Guangdong, China. Three postharvest treatments, including an untreated control (natural ripening), ethylene-induced ripening (100 μL L⁻¹ ethylene, 18 h), 1-MCP-delayed ripening (0.5 μL L⁻¹ 1-MCP, 18 h), were performed as described previously (Xiao et al., 2018). Each treatment contained three biological replicates, and each replicate consists of approximately 120 fruit. After each treatment, fruit were held at 22°C and 90% relative humidity until fully ripe. At each sampling point, three replicates of five fruits each were collected from each treatment. All samples were frozen in liquid nitrogen and stored at −80°C for further use.

Fruit-ripening measurement

Ethylene production, firmness, and peel color are ripening indexes that are used to evaluate the ripening procedure of banana, according to Shan et al. (2012). Ethylene production was evaluated from three biological replicates each consisting of three fruits at the given sampling times. The three fruits were held in a plastic box capped with a rubber stopper at 22 ± 1°C for 3 h. A 1-mL sample of headspace gas was taken and quantified for ethylene by means of gas chromatography (Model GC-17A; Shimadzu Co., Kyoto, Japan) fitted with an activated alumina column (200 cm × 0.3 cm) and a flame ionization detector, with the injector temperature, column temperature, and detector temperature of 120°C, 60°C, and 60°C, respectively.

Fruit firmness was detected by a penetrometer (Model Instron 5542; Instron Co., USA) equipped with a cylindrical flat-surfaced plunger (6-mm diameter). A small slice of banana fruit skin was peeled off and then firmness was recorded in three different fruit, with three different points per fruit. The results were expressed as Newton (N).

Peel color was assessed by a chroma meter (chroma meter CR-300 series; MINOLTA, Japan). Peel color was recorded from three different fruit, with three different points per fruit and results were expressed as h°. The value of h° is associated with the change of peel color from green to yellow.

Phosphoproteomic quantification

To analyze the phosphoproteomic change during banana fruit ripening, three biological replicates were collected from the pulp in banana fruit at 0 and 3 d after ethylene treatment, respectively. Samples of 0 days denote unripe stage, and those of 3 d represent ripe stage. Each biological repeat was pooled by three technical replicates. In each technical replicate, five bananas were used. The phosphoproteomic analysis was conducted by APTBIO Co. Ltd (Shanghai, China). Total proteins were extracted from banana fruit tissues using a 10% (w:v) trichloroacetic acid/acetone solution based on the previous methods (Wang et al., 2006). Protein concentrations were quantified using the Bradford methods (Bradford, 1976). Two hundred micrograms of proteins for each sample were incorporated into 30-μL SDS/DTT/Tris (SDT) buffer (4% [w:v] SDS, 100-mM DTT, 150-mM Tris–HCl, pH 8.0). The detergent, DTT and other low-molecular weight components were removed using UA buffer (8-M urea, 150-mM Tris–HCl, pH 8.0) by repeated ultrafiltration (Microcon units, 10 kDa). Then 100-μL iodoacetamide (50-mM IAA in UA buffer) was added to block reduced cysteine residues and the samples were incubated for 30 min in darkness. The filters were washed with 100-μL UA buffer three times and then 100-μL NH₄HCO₃ buffer twice. Finally, the protein suspensions were digested with 4-μg trypsin in 40-μL NH₄HCO₃ buffer overnight at 37°C, and the resulting peptides were collected as a filtrate. Peptides in the filtrate were desalted using a C18 SPE cartridge. Peptides were labeled using the iTRAQ Reagent-8plex Multiplex Kit (AB SCIEX) following the manufacturer’s instructions. The labeled phosphopeptides were enriched using TiO₂ beads. After centrifugation, the beads were washed with 50 μL of washing buffer I (30% [v:v] acetonitrile, 1% [v:v] trifluoroacetic acid) three times and then 50 μL of washing buffer II (80% [v:v] acetonitrile, 0.1% [v:v] trifluoroacetic acid) three times to remove the remaining nonadsorbed material. Finally, the phosphopeptides were eluted with 50 μL of elution buffer (40% [v:v] acetonitrile, 15% [v:v] NH₃·H₂O) three times. The enriched phosphopeptide fractions were analyzed with EASY-nLC 1000 Liquid Chromatography System (Thermo Scientific). Samples were injected into an EASY-Column (20-mm length × 100-μm diameter; particle size: 5-μm diameter; Thermo Fisher Scientific) with a flow rate of 250 nL min⁻¹. The mobile phases consisted of 0.1% (v/v) formic acid (A), 0.1% (v/v) formic acid, and 84% (v/v) acetonitrile (B) in a two-step linear gradient of 0%–55% B for 220 min, 55%–100% B for 8 min, and 100% B for 12 min. LC/MS–MS analysis is performed in Q-Exactive Mass Spectrometer (Thermo Fisher) according to the previous method (Wang et al., 2020b). MS spectra were analyzed using Mascot software (version 2.2) against the banana genome hub (http://banana-genome-hub.southgreen.fr/). The ripening-responsive phosphopeptides were identified while satisfying an FDR of ≤0.05. The FDR was calculated with Benjamini–Hochberg procedure. The phosphorylation sites with localization probability >0.75 were selected. Sequence motif analysis of phosphorylation sites of the phosphopetides was performed using Multiple Em for Motif Elicitation Modification Motifs (MEME MOMO) online software (https://meme-suite.org/meme/tools/momo). The significance was set at 0.00001, the width was set at 15, and the minimum number of occurrences was set at 5.

Antibodies and immunoblot analysis

Anti-MabZIP21, -GST, -MBP, -His, -GFP, -HA, -Myc, and -phospho-Ser/Thr/Tyr antibodies were used in this study. The anti-MabZIP21 (186–198 aa, VKGNASTVQHKAG) antibody was produced by HUABIO Co., Ltd (Hangzhou, Zhejiang, China). Anti-GST, -MBP, -His, -GFP, -Myc, and -phospho-Ser/Thr/Tyr were purchased from Abcam Company (Cat. No. ab9085, ab9084, ab9108, ab290, ab32, and ab15556). Anti-HA was purchased from Sigma–Aldrich (Cat. No. H6908). Proteins were resolved by SDS–PAGE and transferred to NC membrane for immunoblot assays. Detection was carried out using the chemiluminescent substrate SuperSignal West Pico (Thermo) for horse-radish peroxidase and imaged on a ChemiDocTM MP Imaging System (Bio-Rad).

Gene expression analysis

Total RNA was extracted from the pulp tissues of each sample using the hot borate method (Wan and Wilkins, 1994). The first-strand cDNA was generated by PrimeScript RT Reagent Kit with gDNA Eraser (TaKaRa). RT-qPCR was carried out on a Bio-Rad CFX96 Real-Time PCR System using the SYBR Green PCR Supermix kit (Bio-Rad Laboratories). MaRPS2 was used as the reference gene to normalize the gene expression levels, based on our previous report on the selection of dependable reference genes for gene expression in banana fruit (Chen et al., 2011). The primers used in this study are listed in Supplemental Table S1.

Subcellular localization

The complete open reading frame (ORF) of MabZIP21 was amplified and inserted into the pEAQ-GFP vector. The MabZIP21-GFP plasmid was electroporated into the Agrobacterium tumefaciens strain EHA105, and injected into the leaves of 4- to 6-week-old N. benthamiana plants, using a needleless 1-mL syringe. NLS-mCherry was used as the nuclear marker. After 36 h of infiltration, GFP and mCherry fluorescences were observed under a fluorescence microscope (Zeiss Axioskop 2 Plus). The experiment was repeated three times with similar results. The excitation wave lengths for GFP and mCherry detection were 488 and 561 nm, respectively. The experiment was repeated three times with similar results.

DAP-Seq and data analysis

DAP-Seq was performed according to the method as described previously (O'Malley et al., 2016) with minor adjustments. The gDNA was isolated from banana fruit, then sheared and purified using AMPure XP beads (Beckman). Libraries were constructed using the NEXTflex Rapid DNA-Seq Kit (Bioo Scientific) as per the manufacturer’s recommendations. The coding sequence of MabZIP21 was inserted into a pFN19K HaloTag T7 SP6 Flexi expression vector. Halo–MabZIP21 fusion protein was produced using the TNT SP6-Coupled Wheat Germ Extract System (Promega) according to the protocols provided by the manufacturer. Produced proteins were directly captured using Magne Halo Tag Beads (Promega). The protein-bound beads were incubated with adaptor-ligated gDNA fragments, washed and eluted. Eluted DNA fragments were PCR amplified and sequenced on an Illumina NavoSeq. Reads were mapped to the banana genome. DAP-Seq peaks were called using GEM (Genome Wide Binding Event Finding and Motif Discovery). Association of DAP-Seq peaks located upstream or downstream of the TSS within 3 kb were analyzed using Homer (Heinz et al., 2010), based on the General Feature Format files. Motif discovery was performed using MEME-ChIP (Machanick and Bailey, 2011) to identify the motifs to which MabZIP21 bound. The genes of banana were annotated with KEGG by KOBAS 3.0 (KEGG Orthology Based Annotation System), and further KEGG pathway enrichment was also carried out with the KOBAS, with FDR used in the multiple comparisons (FDR < 0.05).

Transient overexpression analysis in banana fruit

The full-length sequences of MabZIP21^T318A/S436A, MabZIP21^T318E/S436E, and MaMPK6-3 were cloned into the pCXUN-HA vector, respectively. The MabZIP21^T318A/S436A-HA, MabZIP21^T318E/S436E-HA, MaMPK6-3-HA plasmids and the vector control pCXUN-HA were transformed into the A. tumefaciens strain EHA105, then separately injected into the mature-green banana fruits through the distal end (Xiao et al., 2018; Zhang et al., 2018; Shan et al., 2020). Transformed fruits were treated with 100-μL L⁻¹ ethylene and held at 22°C and 90% relative humidity for 5 d. Samples were collected on Day 0, 1, 2, 3, 4, and 5 for the measurement of color index, fruit firmness, ethylene production, gene expression, and protein accumulation (Shan et al., 2020). Immunoblot assay was performed to examine the efficiency of the transient expression of the target genes in banana fruit.

Dual-LUC reporter assay

For transcriptional activity assay of MabZIP21, the GAL4 binding domain (BD)-MabZIP21 fusion protein that binds to the GAL4 DNA-binding sites of the firefly LUC reporter was produced. The LUC reporter includes four upstream GAL4 DNA-binding sites and the 35S::renilla luciferase (REN) internal control. For the assay of the binding activity of MabZIP21 and MaMPK6-3 to the MaACO1, MaPG3, MaPL5, MaPL15, MaPE42, MaPE51, and MaEXPA15 promoters, these promoters were cloned into pGreenII 0800-LUC double-reporter vector (Hellens et al., 2005), while ORFs of MabZIP21 and MaMPK6-3 were cloned into the pEAQ vector (Han et al., 2016) as effectors. The constructed effector and reporter plasmids were co-transformed into tobacco leaves by A. tumefaciens strain EHA105 as described previously (Hellens et al., 2005). After 2 d, LUC and REN activities were measured using the dual-luciferase assay reagents (Promega). The analysis was executed using the Luminoskan Ascent Microplate Luminometer (Thermo Fisher Scientific). The results were indicated by the ratio of LUC to REN. At least six transient assay measurements were included for each pair.

EMSA

The ORF of MabZIP21 was cloned into GST tag and expressed in BM Rosetta (DE3) by induction with 0.5 mM of IPTG (isopropyl-β-d-thiogalactopyr-amoside) for 6 h at 28°C. The recombinant protein was purified with Glutathione Sepharose 4B (GE Healthcare) according to the manufacturer’s instructions. The EMSA was performed using the EMSA kit (Thermo Scientific) according to the instructions. The GST-MabZIP21 protein and biotin-labeled probes were incubated together, while unlabeled cold and mutated probes were used as competitors, and the GST protein alone served as negative control. Biotin-labeled DNA was detected by the chemiluminescence method according to the manufacture’s protocol on a ChemiDoc MP Imaging System (Bio-Rad).

As for the supershift assays, the GST-MabZIP21 and GST-MabZIP21^T318A/S436A proteins were incubated with MBP-MaMPK6-3 in kinase assay buffer (20-mM Tris-HCl buffer, 100-mM NaCl, 20-mM MgCl₂, 2-mM DTT, 10 mM of ATP) at 30°C for 30 min. After reactions, the mixtures, as well as GST-MabZIP21, GST-MabZIP21^T318A/S436A, or GST-MabZIP21^T318E/S436E protein were added with biotin-labeled probes for EMSA assays. The Image Lab 5.1 software was used to quantify the gel band intensities.

Y2H screening

The Y2H screening was performed using the Matchmaker GoldYeast Two-Hybrid Systems (Clontech) according to the manufacturer’s recommendations. The full-length MabZIP21 was cloned into pGBKT7 vector (used as the bait plasmid). The ORFs of the candidate kinases were individually cloned into the pGADT7 vector (used as the prey plasmid). The bait and prey constructs were co-transformed into yeast strain Y2H Gold by the lithium acetate method and grown on the control medium DDO (SD/–Leu/–Trp) for 3 d following the manufacturer’s protocol. Transformed colonies were plated onto the selective medium QDO (SD/–Leu/–Trp/–Ade/–His) with 60-mM 3-AT (3-aminotriazole), and the possible interaction was evaluated according to their growth status and the activity of α-galactosidase.

BiFC analysis

To yield constructs for BiFC assay, full-length coding sequences of MabZIP21 and MaMPK6-3 (without their stop codons) were subcloned into pSPYNE or pSPYCE vectors (Walter et al., 2004), respectively. Expressions of target genes alone were used as negative controls. The plasmids were introduced into N. benthamiana leaf cells by infiltration as reported (Walter et al., 2004). Infected tissues were analyzed at 48 h after infiltration, and YFP fluorescence was captured using a florescence microscope (Zeiss Axioskop 2 plus). Expression of fusion proteins was detected by immunoblot analysis with anti-HA and anti-MYC antibodies, respectively.

In vitro GST pull-down assay

The full-length sequences of MabZIP21 and MaMPK6-3 were cloned into MBP tag, and recombinant proteins were expressed and purified with amylose (NEB Company). The in vitro protein–protein interaction assays were carried out according to the GST Protein Interaction Pull-Down Kit (Thermo). The eluted proteins were separated on an sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS–PAGE) gel and detected by immunoblot using anti-MBP (Abcam) or anti-GST (Abcam) antibodies, respectively. Detection was carried out using the chemiluminescent substrate SuperSignal West Pico (Thermo) for horse-radish peroxidase and imaged on a ChemiDocTM MP Imaging System (Bio-Rad).

In vivo Co-IP assay

To give rise to MabZIP21-GFP, MabZIP21-His, and MaMPK6-3-His constructs, full-length MabZIP21 or MaMPK6-3 was inserted into pEAQ-HT-His and pEAQ-HT-GFP vectors, respectively (Sainsbury et al., 2009), and the three resulting constructs were then introduced into A. tumefaciens strain EHA105, following co-infiltrated into 4- to 6-week-old tobacco leaves using a 1-mL needleless syringe. In vivo Co-IP assays were performed as described by Han et al. (2016). Thirty-six hours after infiltration, tobacco leaves were ground in liquid nitrogen. Proteins were extracted using an extraction buffer (50-mM Tris-HCl [pH 7.4], 150-mM NaCl, 2-mM MgCl₂, 20% [v/v] glycerol, 5-mM DTT, and 0.1% [v/v] Nonidet P-40) including protease inhibitor cocktail. Tobacco cell debris was pelleted and the supernatant was incubated with 10 μL of anti-GFP antibody (Abcam) at 4°C overnight to capture the epitope-tagged protein. The second day, 50 μL of protein A agarose beads (Roche) was added. After 4 h of incubation at 4°C, the beads were centrifuged and washed three times using a washing buffer (50-mM Tris-HCl [pH 7.4], 150-mM NaCl, 2-mM MgCl₂, 10% [v/v] glycerol, 5-mM DTT, and 0.1% [v/v] Nonidet P-40). Proteins were eluted with 40 mL of 2.5 SDS sample buffer, separated by SDS–PAGE and then subjected to immunoblotting analysis using anti-His antibody (Abcam) and anti-GFP antibody (Abcam) for Co-IP assay. Detection was carried out using the chemiluminescent substrate SuperSignal West Pico (Thermo) for horse-radish peroxidase and imaged on a ChemiDoc MP Imaging System (Bio-Rad).

ChIP-qPCR analysis

ChIP was performed as described by Han et al. (2016). The banana fruit pulp was submerged in 1% (v/v) formaldehyde to cross-link genomic DNA and protein. The chromatin was sheared to an average length of 500 bp by sonication and immunoprecipitated with the specific anti-MabZIP21 antibody. ChIP assays were repeated with three biological replicates. The DNA immunoprecipitated by anti-MabZIP21 antibody was analyzed using real-time qPCR in triplicate. IgG was used as an internal negative control to the ChIP-qPCR enrichment signal.

In vitro phosphorylation assay

To analyze the phosphorylation of MabZIP21 by MaMPK6-3, 3 μg of the GST-MabZIP21 protein and 1 μg of the MBP-MaMPK6-3 protein were simultaneously added to the kinase assay buffer to a final volume of 25 μL (20-mM Tris–HCl buffer, 100-mM NaCl, 20-mM MgCl₂, 2-mM DTT, 10-mM of ATP). For the AP treatment, 1 μL of fast alkaline phosphatase (Thermo Fisher) was added into the 25-μL kinase assay buffer. Samples were mixed gently and kept at 30°C for 30 min. Samples were subsequently separated using 10% (w/v) SDS–PAGE, with 0.1 mM of MnCl₂ and 0.1 mM of Phos-tag (Wako), and the proteins were detected using anti-GST antibody (Sigma).

In vivo phosphorylation assay

The ORFs of MabZIP21 and MabZIP21^T318A/S436A were inserted into pEAQ-HT-GFP vector to construct MabZIP21-GFP and MabZIP21^T318A/S436A-GFP plasmids, while the ORFs of MabZIP21 and MaMPK6-3 were inserted into pEAQ-HT-His vector to give rise to MabZIP21-His and MaMPK6-3-His constructs (Sainsbury et al., 2009). The resulting constructs were introduced into A. tumefaciens strain EHA105, and then infiltrated into 4- to 6-week-old tobacco leaves using a 1-mL needleless syringe. Thirty-six hours after infiltration, tobacco leaves were ground in liquid nitrogen, and proteins were extracted using an extraction buffer (50-mM Tris–HCl [pH 7.4], 150-mM NaCl, 2-mM MgCl₂, 20% [v/v] glycerol, 5-mM DTT, and 0.1% [v/v] Nonidet P-40) including protease inhibitor cocktail. Tobacco cell debris was pelleted, and the supernatant was incubated with 10 μL of anti-GFP antibody (Abcam) at 4°C overnight to capture the epitope-tagged protein. The second day, 50 μL of protein A agarose beads (Roche) was added. After 4 h of incubation at 4°C, the beads were centrifuged and washed three times using a washing buffer (50-mM Tris–HCl [pH 7.4], 150-mM NaCl, 2-mM MgCl₂, 10% [v/v] glycerol, 5-mM DTT, and 0.1% [v/v] Nonidet P-40). The immunoprecipitated proteins were released from the beads by boiling in SDS loading buffer and analyzed by immunoblot using anti-His antibody (Abcam), anti-GFP antibody (Abcam), and anti-phospho-Ser/Thr/Tyr antibody (Abcam), respectively. Detection was carried out using the chemiluminescent substrate SuperSignal West Pico (Thermo) for horse-radish peroxidase and imaged on a ChemiDoc MP Imaging System (Bio-Rad).

As for studying in vivo phosphorylation of MabZIP21 in banana fruit, total proteins were extracted from banana pulp tissue, and anti-MabZIP21 antibody was added to immunoprecipitate MabZIP21 protein. The immunoprecipitated proteins were detected using anti-phospho-Ser/Thr/Tyr and anti-MabZIP21 antibodies, respectively.

LC–MS/MS analysis

Phosphorylated MabZIP21 proteins were obtained by in vitro phosphorylation with MaMPK6-3, and then were resolved in a 10% (w/v) SDS–PAGE gel. The protein band corresponding to MabZIP21 was visualized by Coomassie brilliant blue staining, and the gel slice was subjected to in-gel tryptic digestion. The tryptic peptides were dissolved in 0.1% formic acid (solvent A), directly loaded onto a home-made reversed-phase analytical column (15-cm length, 75 μm i.d.). The gradient comprised an increase from 6% to 23% solvent B (0.1% [v/v] formic acid in 98% [v/v] acetonitrile) over 16 min, 23% to 35% in 8 min and climbing to 80% in 3 min then holding at 80% for the last 3 min, all at a constant flow rate of 400 nL·min⁻¹ on an EASY-nLC 1000 UPLC system (Thermo Scientific).

The peptides were subjected to NSI source followed by MS/MS in Q Exactive Plus (Thermo) coupled online to the UPLC. The electrospray voltage applied was 2.0 kV. The m/z scan range was 350–1,800 for full scan, and intact peptides were detected in the Orbitrap at a resolution of 70,000. Peptides were then selected for MS/MS using NCE setting as 28 and the fragments were detected in the Orbitrap at a resolution of 17,500. A data-dependent procedure that alternated between one MS scan followed by 20 MS/MS scans with 15.0 s dynamic exclusion. Automatic gain control was set at 50,000.

MabZIP21 mutagenesis was performed using Mut Express II Fast Mutagenesis Kit V2 (C214-01; Vazyme Biotech). The phosphomutant versions of MabZIP21 were named MabZIP21^S436A/T318A and MabZIP21^S436E/T318E.

Statistical analysis

Statistical differences between samples were analyzed by Student’s t test (P < 0.05 or P < 0.01), and statistical comparisons among the mean values were performed using one-way analysis of variance (ANOVA), followed by Duncan’s multiple range test at the P < 0.05 level.

Accession numbers

Sequence data of this study can be found in banana genome under the following accession numbers: MabZIP21 (Ma04_g35070), MabZIP6 (Ma02_g14450), MabZIP93 (Ma11_g04880), MabZIP116 (Ma06_g01870), MaMPK6-3 (Ma02_g21170), MaMPK4 (Ma11_g02790), MaMPK6-2 (Ma05_g06410), MaMPK7 (Ma04_g11230), MaMPK9-2 (Ma09_g00730), MaMPK9-3 (Ma04_g03870), MaMPK9-4 (Ma04_g11200), MaMPK20-1 (Ma09_g06560), MaMPK20-2 (Ma06_g28840), MaMPK20-3 (Ma00_g00010), MaMPK20-4 (Ma01_g05530), MaPG3 (Ma02_g04450), MaPL15 (Ma03_g13710), MaPL5 (Ma06_g30000), MaPE42 (Ma06_g30950), MaEXPA15 (Ma06_g12190), MaPE51 (Ma10_g04350), and MaACO1 (Ma07_g19730).

Supplemental data

The following materials are available in the online version of this article.

Supplemental Figure S1. Change of phosphorylation proteins during banana fruit ripening.

Supplemental Figure S2. Heat map showing the phophopeptides with different abundance in unripe and ripe bananas.

Supplemental Figure S3. Change of bananas with natural, ethylene-induced, and 1-MCP-delayed ripening.

Supplemental Figure S4. Specificities of anti-MabZIP21 antibody.

Supplemental Figure S5. Y2H analysis of possible interaction between MabZIP21 and MaMAPKs.

Supplemental Figure S6. Characterization of MaMPK6-3.

Supplemental Figure S7. In vitro phos-tag-based phosphorylation assay showing that MaMPK6-3 phosphorylates MabZIP21.

Supplemental Figure S8. Mutation of Ser-422 of MabZIP21 to Ala or Glu has no effect on transcriptional activation, subcellular localization, or interaction with MaMPK6-3.

Supplemental Figure S9. A postulated model showing that MabZIP21 associates with MaMPK6-3 in controlling banana ripening.

Supplemental Table S1. Summary of primers used in this study.

Supplemental Data Set S1. Phosphopeptides in banana ripening.

Supplemental Data Set S2. Binding regions of MabZIP21 revealed by DAP-Seq.

 

J.F.K. conceived the project, supervised the work, and wrote the manuscript; C.J.W. performed most of the experiments, analyzed the data; W.S. analyzed the data; L.S.Z., W.W., Y.Y.Y., and Y.F.G. performed some of the experiments; X.C.L., M.B., J.Y.C., and W.J.L. gave advices and revised the manuscript, and all the authors approved the final manuscript.

The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (https://academic.oup.com/plphys/pages/general-instructions) is: Jianfei Kuang (jfkuang@scau.edu.cn).