Mitogen-activated protein kinase 14-mediated phosphorylation of MaMYB4 negatively regulates banana fruit ripening

Abstract

Mitogen-activated protein kinase (MAPK/MPK) cascades play crucial parts in plant growth, development processes, immune ability, and stress responses; however, the regulatory mechanism by which MAPK affects fruit ripening remains largely unexplored. Here, we reported that MaMPK14 cooperated with MaMYB4 to mediate postharvest banana fruit ripening. Transient overexpression of individual MaMPK14 and MaMYB4 in banana fruit delayed fruit ripening, confirming the negative roles in the ripening. The ripening negative regulator MaMYB4 could repress the transcription of genes associated with ethylene biosynthesis and fruit softening, such as MaACS1, MaXTH5, MaPG3, and MaEXPA15. Furthermore, MaMPK14 phosphorylated MaMYB4 at Ser160 via a direct interaction. Mutation at Ser160 of MaMYB4 reduced its interaction with MaMPK14 but did not affect its subcellular localization. Importantly, phosphorylation of MaMYB4 by MaMPK14 enhanced the MaMYB4-mediated transcriptional inhibition, binding strength, protein stability, and the repression of fruit ripening. Taken together, our results delineated the regulation pathway of MAPK module during banana fruit ripening, which involved the phosphorylation modification of MaMYB4 mediated by MaMPK14.

Figure 1

MaMPK14 transient overexpression delays banana fruit ripening. a Illustration of successful injection of Agrobacterium solution into banana fruit. b The appearance of banana fruit during ripening was recorded for MaMPK14 and control (empty vector) transiently overexpressing. c qRT-PCR and d Western blot determination of MaMPK14 overexpression in banana fruit. e Patterns of variation in physiological indicators of infiltrated banana fruits.

Figure 2

MaMYB4 is a substrate for MaMPK14. a Y2H assay for MaMPK14 and MaMYB4 interaction. The CDS of MaMPK14 and MaMYB4 were constructed into BD and AD vectors, respectively, then transferred into the yeast (Gold Y2H) strain. A positive result was recorded if the plates could be grown on SD/−Leu-Trp-Ade-His (containing 30 mM 3-AT) plates as well as turning blue in the presence of the chromogenic substrate X-α-Gal. b BiFC showing the interaction of MaMPK14 and MaMYB4. MaMPK14 and MaMYB4 was constructed onto YNE and YCE vector, respectively. The fusion constructs were introduced to tobacco leaves by agro-infiltration. Fluorescent signal was observed with fluorescence microscopy. Bars = 25 μm. c Co-localization of MaMPK14-GFP and MaMYB4-mCherry in N. benthamiana. Fluorescent signals of GFP and mCherry empty plasmids were applied as negative controls and NLS-mCherry was used as an indicator of nuclear localization. Bars = 25 μm. d MaMPK14 and MaMYB4 in vivo interaction was detected by CoIP. Tobacco leaves co-expressing His-MaMPK14 and MaMYB4-GFP or GFP, were used for immunoprecipitation with GFP antibody and performed western blot with His and GFP antibodies. e GST pull-down to investigate MaMPK14-MaMYB4 interactions. MBP-tag MaMPK14 protein was incubated with GST-tag MaMYB4 or GST protein, and then the bound protein was detected via western blotting with MBP and GST antibodies, respectively. f MaMPK14 phosphorylates MaMYB4 in vitro. GST-MaMYB4 protein was separated in Phos-tag SDS-PAGE gels and subjected to western blot with GST antibody. The upper band is indicated as phosphorylated MaMYB4 and the lower as unphosphorylated.

Figure 3

Relative expression of MaMYB4 and MaBRA1/2 target genes during transient overexpression of MaMPK14 banana fruit ripening. Each gene’s expression level is expressed as a ratio relative to the 0 d of control group, it was set as 1. Each value represents the means ± SE of three replicates. The ^** and ^* denote significant differences between treatments (Student’s t-test, P < 0.05 or P < 0.01), respectively.

Figure 4

Substitution of Ser160 with Ala160 weakened the phosphorylation and transcriptional repression activity of MaMYB4. a Mass spectrometry analysis showed that Ser-160 in MaMYB4 was phosphorylated by MaMPK14 in vivo. b The interaction of MaMYB4^S160A with MaMPK14 was investigated by Y2H assay. The CDS of MaMPK14 and MaMYB4^S160A were constructed into BD and AD vectors, respectively, then transferred into the yeast (Gold Y2H) strain. Positive result was recorded if the plates could be grown on SD/−Leu-Trp-Ade-His plates as well as turning blue in the presence of the chromogenic substrate X-α-Gal. c BiFC assay of MaMPK14 and MaMYB4^S160A interaction. MaMYB4^S160A was constructed onto YNE vector and MaMPK14 constructed onto YCE vector. They were infiltrated to tobacco leaves and observed with fluorescence microscopy. Bar = 25 μm. d Co-localization of MaMPK14-GFP and MaMYB4^S160A-mCherry in N. benthamiana. Fluorescent signals of GFP and mCherry empty plasmids were applied as negative controls, and NLS-mCherry served as an indicator for nuclear localization. Bar = 25 μm. e Firefly luciferase complementation imaging assay was performed to compare the strength of interaction between MaMYB4 or MaMYB4^S160A and MaMPK14 in N. benthamiana leaves. Then Luc/MaMYB4-cLuc, MaMPK14-nLuc/MaMYB4-cLuc and nLuc/MaMYB4^S160A-cLuc, MaMPK14-nLuc/MaMYB4^S160A-cLuc were co-transformed into tobacco leaves and examined. Each value represents the average SE of six biological replicates. Statistical comparison of means was performed via one-way ANOVA at the P < 0.05 level. fIn vitro kinase assay comparing MaMYB4 and MaMYB4^S160A phosphorylation by MaMPK14. MBP-MaMPK14 protein was incubated with GST-MaMYB4 or GST-MaMYB4^S160A of ALP and ATP in the presence (+) or absence (−) in buffer. GST-MaMYB4 or GST-MaMYB4^S160A proteins were separated in Phos-tag SDS-PAGE gels and performed western blot with GST antibody. g Schematic diagram of vector construction. h Effects of MaMYB4^S160A on the transcription of MaACS1, MaXTH5, MaPG3, and MaEXPA15. Substitution of Ser160 with Ala attenuated MaMYB4 transcriptional repression activity. Each value represents the average SE of six biological replicates. Statistical comparison of means was performed via one-way ANOVA at the P < 0.05 level.

Figure 5

MaMPK14-mediated phosphorylation of MaMYB4 enhances its DNA-binding ability and stability. a Phosphorylation of MaMYB4 by MaMPK14 enhances its DNA-binding ability. The phosphorylated form of MaMYB4 enhanced ability to bind DNA, whereas the dephosphorylated version reduced its DNA-binding capacity. Labelled probe was incubated with phosphorylated MaMYB4 or MaMYB4^S160A by MaMPK14 and unphosphorylated MaMYB4 or MaMYB4^S160A in a control reaction without MaMPK14. Protein inputs were detected using an GST or MBP antibody. b GFP-tagged MaMYB4 with or without S/A substitution at S160 was transiently co-expressed with His-tagged MaMPK14 and His-tagged MaBRG2/3 (as E3, interacting with and ubiquitinating MaMYB4) in N. benthamiana leaves by agroinfiltration. Protein accumulation was examined by western blotting at 3 d after infiltration. MG132 was applied 12 h before sample harvesting, DMSO was used as a mock control, and Actin served as a loading control.

Figure 6

Phosphorylation of MaMYB4 represses fruit ripening. a Phosphorylation abundance of MaMYB4 in unripe and ripe banana fruit, respectively. The MaMYB4 proteins were immunoprecipitated using anti-MaMYB4 antibody from the proteins extracted in unripe and ripe banana at 0 and 3 d after ethylene treatment, and then subjected to immunoblot analysis using anti-phospho-Ser/Thr/Tyr and anti-MaMYB4 served as a loading control. b Photograph of banana fruit during ripening was recorded for MaMYB4, MaMYB4^S160A, and control (empty vector) transiently overexpressing. c qRT-PCR and d Western blot to determine overexpression of MaMYB4 or MaMYB4^S160A overexpression in banana fruit. e Changes in physiological indicators of infiltrated banana fruits at each storage time. f Relative mRNA abundance of MaACS1, MaXTH5, MaPG3, and MaEXPA15 in MaMYB4 or MaMYB4^S160A overexpressed and control fruits during fruit ripening. Each gene’s expression level is expressed as a ratio relative to the 0 d of control group, it was set as 1. Each value represents the means ± SE of three to six biological replicates. The ** and * denote significant differences between treatments (Student’s t-test, P < 0.05 or P < 0.01), respectively.

Figure 7

Proposed model of MaMPK14 in controlling banana ripening. As for the MaMPK14-MaMYB4 module, in unripe banana, highly expressed MaMPK14 phosphorylates a transcriptional repressor MaMYB4 mainly at Ser160 site to form a repressor complex that synergistically represses the transcription of ripening-associated genes MaACS1, MaPG3, MaXTH5, and MaEXPA15, which represses their transcriptional expression. During fruit ripening, accumulation of MaMPK14 and MaMYB4 is inhibited by ethylene and accumulation of MaBRG2/3 is induced. MaBRG2/3 ubiquitinate MaMYB4 for proteasome-dependent degradation, which weakens the repressive effect on the ripening-associated genes, resulting in gene activation and fruit ripening. As for the MaMPK14-MaBZR1/2 module, a similar regulatory mechanism may exist in which MaMPK14 negatively regulates banana fruit ripening by interacting with MaBZR1/2 and affecting their transcriptional activity on the target genes (MaACS1, MaACO13, MaACO14, MaPL2, MaXTH5, and MaEXP2). A solid line indicates the confirmed findings, whereas a dotted line implies the predicted results. Arrows denote positive regulation, while a T bar represents negative regulation.