Ethylene enhances MdMAPK3-mediated phosphorylation of MdNAC72 to promote apple fruit softening

Abstract

The phytohormone ethylene plays an important role in promoting the softening of climacteric fruits, such as apples (Malus domestica); however, important aspects of the underlying regulatory mechanisms are not well understood. In this study, we identified apple MITOGEN-ACTIVATED PROTEIN KINASE 3 (MdMAPK3) as an important positive regulator of ethylene-induced apple fruit softening during storage. Specifically, we show that MdMAPK3 interacts with and phosphorylates the transcription factor NAM-ATAF1/2-CUC2 72 (MdNAC72), which functions as a transcriptional repressor of the cell wall degradation-related gene POLYGALACTURONASE1 (MdPG1). The increase in MdMAPK3 kinase activity was induced by ethylene, which promoted the phosphorylation of MdNAC72 by MdMAPK3. Additionally, MdPUB24 functions as an E3 ubiquitin ligase to ubiquitinate MdNAC72, resulting in its degradation via the 26S proteasome pathway, which was enhanced by ethylene-induced phosphorylation of MdNAC72 by MdMAPK3. The degradation of MdNAC72 increased the expression of MdPG1, which in turn promoted apple fruit softening. Notably, using variants of MdNAC72 that were mutated at specific phosphorylation sites, we observed that the phosphorylation state of MdNAC72 affected apple fruit softening during storage. This study thus reveals that the ethylene-MdMAPK3-MdNAC72-MdPUB24 module is involved in ethylene-induced apple fruit softening, providing insights into climacteric fruit softening.

Figure 1.

Ethylene promotes apple fruit softening and MdMAPK3 activity during storage. A) Apple fruits were collected 140 d after full bloom (DAFB, the commercial harvest day). No treatment, apple fruit not receiving treatment; Eth treatment, apple fruit treated with 0.1% [v/v] ethephon; 1-MCP treatment, apple fruit treated with 1 μL L^–1 of the ethylene inhibitor 1-MCP. Scale bar, 2 cm. B) Ethylene production and (C) fruit firmness in fruits receiving the indicated treatments. Fruits sampled at each sampling point were divided into 3 subgroups (10 fruit per subgroup) and each subgroup was considered 1 biological replicate. Three biological replicates were analyzed. Values are means ± standard error (Se). Statistical significance was determined using Student's t-test (**P < 0.01). n.s., no significant difference. D) RT-qPCR analysis of relative MdPG1 expression during apple fruit storage. Storage after harvest at RT is shown in days. For RT-qPCR, fruits sampled at each sampling point were divided into 3 subgroups (5 fruits per subgroup). Fruit flesh from each subgroup was evenly mixed for RNA isolation. RNA isolation from each group was considered 1 biological replicate. Three biological replicates were analyzed. Values are means ± Se. Statistical significance was determined using Student's t-test (**P < 0.01). n.s., no significant difference. E) Immunoblot analysis of MdMAPK activity levels during fruit storage using an anti-phospho-p44/42 antibody. Tubulin was used as a loading control.

Figure 2.

MdMAPK3 promotes apple fruit softening during storage. A) Apple fruit transiently overexpressing MdMAPK3 (MdMAPK3-OE) or empty vector (control) during storage. MdMAPK3 was overexpressed in apple fruit using Agrobacterium-mediated injection. MdMAPK3-OE fruits were harvested 7 d after injection and stored at RT for 30 d. DAI, days after infiltration; DAS, days after storage. Scale bar, 2 cm. B) Immunoblot analysis of proteins from apple fruit injected with empty vector or MdMAPK3-Myc and detected using an anti-Myc antibody. Fruit tissue at the injection region was used for protein extraction. Tubulin was used as a loading control. C) Relative MdMAPK3 expression in the fruits shown in (A) by RT-qPCR analysis. D) Apple fruit firmness during storage. Fruits sampled at each sampling point were divided into 3 subgroups (5 fruits per subgroup) and each subgroup was considered 1 biological replicate. Three biological replicates were analyzed. Values are means ± Se. Statistical significance was determined using Student's t-test (**P < 0.01). n.s., no significant difference. E) Relative MdPG1 expression by RT-qPCR analysis. For RT-qPCR, 3 biological replicates were used as described in Fig. 1. Values represent means ± Se. Statistical significance was determined using Student's t-test (**P < 0.01). n.s., no significant difference.

Figure 3.

MdMAPK3/MdNAC72 interaction and MdNAC72 protein abundance. A) MdMAPK3 and MdNAC72 interact in a Y2H assay. DDO, synthetic defined (SD) medium lacking Trp and Leu; QDO, SD medium lacking Trp, Leu, His, and Ade; QDO/X/A, QDO medium containing X-α-gal and aureobasidin A. BD and AD vectors were used as negative controls. SV40/P53 was used as the positive control. B) Pull-down assay showing that MdMAPK3 interacts with MdNAC72 in vitro. Recombinant MdNAC72-MBP was incubated with immobilized MdMAPK3-GST and detected for immunoblot analysis using an anti-MBP antibody (upper panel) or an anti-GST antibody (bottom panel). The asterisk represents the target protein. C) Confirmation of the interaction between MdMAPK3 and MdNAC72 by a co-IP assay. MdMAPK3-GFP and MdNAC72-FLAG were overexpressed in apple fruit calli; an anti-FLAG antibody was used for immunoprecipitation. Anti-GFP and anti-FLAG antibodies were used for immunoblotting. D) A firefly LCI assay confirms the interaction between MdMAPK3 and MdNAC72 in N. benthamiana leaves. E) Immunoblot analysis of MdNAC72 abundance during fruit storage using a specific anti-MdNAC72 antibody. Tubulin was used as a loading control.

Figure 4.

MdNAC72 represses MdPG1 transcription by binding to its promoter. A) Y1H assay showing that MdNAC72 directly binds to the MdPG1 promoter. The basal concentration of AbA (aureobasidin A) used was 250 ng mL^–1. The empty vector and the MdPG1 promoter were used as negative controls. The Rec-P53+P53-promoter was used as a positive control. B) Electrophoretic mobility shift assay (EMSA) to assess the binding of MdNAC72 to the CACG or CGTG binding sites in the MdPG1 promoter. The hot probes (probe 1 and probe 2, Supplemental Data Set 4) were biotin-labeled fragments of the MdPG1 promoter containing the NAC binding site. The cold probes were unlabeled and used as competitive probes (100×). The mutant cold probe consisted of an unlabeled hot probe with 2 nucleotides mutated. Recombinant MdNAC72-MBP was purified from E. coli and used for DNA-binding assays. C) ChIP-qPCR assay showing that MdNAC72 binds to the MdPG1 promoter in vivo. The various crosslinked chromatin samples were extracted from MdNAC72-FLAG apple fruit calli and immunoprecipitated with an anti-FLAG antibody. The eluted DNA was used to amplify the sequences neighboring the NAC binding site using qPCR. Four different regions (S1–S4) were investigated. Fruit calli injected with the empty vector (35S:FLAG) were used as a negative control. The ChIP-qPCR assay was performed 3 times, and the enriched DNA fragments in each ChIP were considered 1 biological replicate for qPCR. Values are means ± Se. Statistical significance was determined using Student's t-test (**P < 0.01). n.s., no significant difference. D) β-glucuronidase activity assay showing that MdNAC72 represses MdPG1 promoter activity. The 35S:MdNAC72 effector vector together with the proMdPG1:GUS promoter, or a mutated promoter (m1proMdPG1, m2proMdPG1, and m1m2proMdPG1), were infiltrated into N. benthamiana leaves to analyze GUS activity. The 35S:LUC was included as an internal control for normalization of transformation efficiency. Three independent infiltration experiments were performed; values are means ± Se. Statistical significance was determined using Student's t-test (**P < 0.01). n.s., no significant difference.

Figure 5.

MdMAPK3 phosphorylates MdNAC72 to suppress its transcriptional repression activity. A) Phos-tag mobility shift assay showing that MdMAPK3 phosphorylates MdNAC72, but not MdNAC72AA, in vitro. CA-MdMAPK3, constitutively active form. MdNAC72 variants harboring alanine substitutions at S74 and/or S220 (MdNAC72^S74A; MdNAC72^S220A; MdNAC72AA, MdNAC72^S74A,S220A). Proteins were separated on a phos-tag gel and an immunoblot analysis was performed using an anti-His antibody to detect MdNAC72. The slowly migrating band on the phos-tag gel represents phosphorylated MdNAC72-His (upper panel). Proteins were also separated on a normal SDS-PAGE gel to verify equal loading (bottom panel). B) MdMAPK3 phosphorylates MdNAC72 in vivo. Proteins were immunoprecipitated from transgenic apple calli (35S:FLAG-MdNAC72, 35S:FLAG-MdNAC72+35S:MdMAPK3, or 35S:FLAG-MdNAC72AA) using an anti-FLAG antibody and separated using SDS-PAGE for immunoblot analysis. An anti-phosphoSer/Thr antibody was used to detect MdNAC72 phosphorylation. Eth, apple calli treated with ethylene. C) In vitro cell-free degradation assay showing that MdNAC72 degradation rate is slower in protein extracts from MdMAPK3-silenced (MdMAPK3-AS) transgenic apple calli compared with a control calli (transformed with the empty vector). Recombinant MdNAC72-His was added to total protein extracts, incubated for the indicated times, and subjected to immunoblot analysis using an anti-His antibody. Transgenic apple calli transformed with the empty vector were treated with 50 μm MG132. Tubulin was used as a loading control. D) ChIP-qPCR assay showing that ethylene treatment decreases the binding of MdNAC72 to the MdPG1 promoter, but not of MdNAC72AA. Crosslinked chromatin samples were extracted from 35S:FLAG, 35S:FLAG-MdNAC72, and 35S:FLAG-MdNAC72AA fruit calli (no treatment) or treated with ethylene and precipitated using an anti-FLAG antibody. ProPG1-S3 and ProPG1-S4 refer to the MdPG1 promoter regions from Fig. 4C. For ChIP-qPCR, 3 biological replicates were analyzed as described in Fig. 4. Values are means ± Se. Statistical significance was determined using Student's t-test (**P < 0.01). n.s., no significant difference. E) GUS activity assay showing that MdNAC72 is phosphorylated by MdMAPK3 to upregulate MdPG1 transcript levels. The proMdPG1:GUS reporter, together with the effector constructs 35S:MdNAC72 or 35S:MdNAC72 and 35S:MdMAPK3, was separately infiltrated into N. benthamiana leaves to assess GUS activity. Three independent infiltrations were performed as described in Fig. 4. MG132, N. benthamiana leaves treated with 50 μm MG132. F) Dual-luciferase (LUC) reporter assay showing that MdNAC72 phosphorylation by MdMAPK3 upregulates MdPG1 transcription. The reporter proMdPG1:LUC, together with the effectors 35S:MdNAC72 or 35S:MdNAC72 and 35S:MdMAPK3, was separately infiltrated into N. benthamiana leaves to assess LUC activity. MG132, N. benthamiana leaves treated with 50 μm MG132.

Figure 6.

MdPUB24 interacts with MdNAC72. A) Y2H assay showing that MdPUB24 interacts with MdNAC72 in yeast; the assay was performed as described in Fig. 3. B) A pull-down assay showing that MdPUB24 interacts with MdNAC72 in vitro. Recombinant MdNAC72-MBP was incubated with immobilized MdPUB24-His and detected for immunoblot analysis using an anti-MBP antibody (upper panel) or an anti-His antibody (bottom panel). The asterisk represents the target protein. C) Co-IP assay showing that MdPUB24 interacts with MdNAC72 in vivo. MdPUB24-GFP and MdNAC72-FLAG were overexpressed in apple calli. An anti-FLAG antibody was used for immunoprecipitation analysis. Anti-GFP and anti-FLAG antibodies were used for immunoblotting. D) Firefly LCI assay verifying that MdPUB24 and MdNAC72 interact in N. benthamiana leaves.

Figure 7.

MdPUB24 ubiquitinates MdNAC72 and promotes apple fruit softening. A) MdPUB24 ubiquitinates MdNAC72 in vitro. MdNAC72-His was used to detect potential E3 ubiquitin ligase activity, in the presence of ATP, ubiquitin, E1, E2, and recombinant MdPUB24-MBP. An immunoblot analysis was used to detect the ubiquitination of MdNAC72 using an anti-ubiquitin (Ub) antibody. B) Ubiquitination of MdNAC72 in MdNAC72-FLAG and MdPUB24-OE+MdNAC72-FLAG pretreated (50 μm MG132) calli. MdNAC72 was immunoprecipitated using an anti-FLAG antibody and the anti-FLAG antibody was used to detect ubiquitinated MdNAC72-FLAG. C) Cell-free degradation assay for recombinant MdNAC72-His in protein extracts from MdPUB24-silenced (MdPUB24-AS) transgenic apple calli. MdNAC72-His abundance was determined by immunoblot analysis using an anti-His antibody. Empty vector or MdPUB24-AS transgenic apple calli were separately treated with 50 μm MG132, and tubulin was used as the loading control. D) Apple fruit transiently overexpressing MdPUB24 (MdPUB24-OE) or empty vector (control) during storage. MdPUB24 was overexpressed in apple fruit using Agrobacterium-mediated transient injection. MdPUB24-OE fruits were harvested 7 d after injection and stored at RT for 30 d. DAI, days after infiltration; DAS, days after storage. Scale bar, 2 cm. E) Immunoblot analysis using an anti-GFP antibody of proteins from apple fruit injected with an empty vector or MdPUB24-GFP. Fruit tissues at the injection region were used for protein detection. Tubulin was used as the loading control. F) Relative MdPUB24 expression by RT-qPCR. G) Firmness of injected apple fruit during storage. Three biological replicates were performed as described in Fig. 2D. H) Relative MdPG1 expression in MdPUB24-OE apple fruit during storage as detected by RT-qPCR. Fruits injected with empty vectors were used as control. For RT-qPCR, 3 biological replicates were performed as described in Fig. 1. I and J) The ubiquitination level of MdNAC72 increases and MdNAC72 abundance decreases in MdPUB24-OE apple fruit during storage. Tubulin was used as the loading control.

Figure 8.

MdMAPK3-mediated phosphorylation of MdNAC72 enhances its ubiquitination by MdPUB24, which upregulates MdPG1 transcription. A) In vitro pull-down assay showing that the interaction between MdPUB24 and MdNAC72 is enhanced by MdMAPK3. Recombinant MdNAC72-His and MdMAPK3-GST were incubated with immobilized MdPUB24-MBP. The pulled-down fractions were visualized using an anti-His antibody. B) Co-IP assay showing that MdMAPK3 promotes the interaction between MdPUB24 and MdNAC72 in vivo. An anti-GFP antibody was used for immunoprecipitation from protein extracts of transgenic apple calli (MdPUB24-GFP MdMAPK3-Myc and MdPUB24-GFP). Anti-MdNAC72 and anti-Myc antibodies were used for immunoblotting. C) In vivo ubiquitination assay showing that MdMAPK3 promotes the ubiquitination of MdNAC72 by MdPUB24. Proteins were extracted from N. benthamiana leaves transiently expressing MdNAC72-FLAG and MdPUB24-GFP or MdNAC72-FLAG and MdPUB24-GFP and MdMAPK3 pretreated with 50 μm MG132. MdNAC72 was immunoprecipitated using an anti-FLAG antibody. Immunoblot analysis was used to detect the ubiquitination of MdNAC72 by MdPUB24 using an anti-Ub antibody. D) In vitro ubiquitination assay showing that phosphorylation of MdNAC72 is a prerequisite for its ubiquitination by MdPUB24. Recombinant MdNAC72-His and MdNAC72AA-His were analyzed for ubiquitination in the presence of ATP, ubiquitin, E1, E2, and recombinant MdPUB24-MBP. An immunoblot analysis was used to detect the ubiquitination of MdNAC72 or MdNAC72AA using an anti-Ub antibody. E) In vivo ubiquitination assay showing that MdMAPK3 phosphorylates MdNAC72, which enhances the ubiquitination of MdNAC72 by MdPUB24. Proteins were extracted from N. benthamiana leaves transiently expressing MdNAC72-FLAG and MdPUB24-GFP or MdNAC72-FLAG and MdPUB24-GFP and CA-MdMAPK3 pretreated with 50 μm MG132. MdNAC72 protein immunoprecipitated using an anti-FLAG antibody was incubated with λPP at 30°C for 30 or 60 min. Immunoblot analysis was used to detect the ubiquitination of MdNAC72 by MdPUB24 using an anti-Ub antibody. Immunoblot analysis for MdPUB24-GFP or CA-MdMAPK3-Myc proteins using anti-GFP or anti-Myc antibodies, respectively, indicated equal loading. F and G) Ethylene promotes the phosphorylation and ubiquitination of MdNAC72, which is suppressed in MdMAPK3-silenced (MdMAPK3-AS) apples. MdMAPK3-AS fruits were harvested 7 d after injection and stored at RT for 30 d. The fruits were treated with ethylene. Empty vector-infected fruits were used as controls. DAI, days after infiltration; DAS, days after storage. Total protein was extracted from each sample to analyze MdNAC72 phosphorylation and ubiquitination. MdNAC72 was immunoprecipitated using an anti-MdNAC72 antibody. Anti-phosphoSer/Thr and anti-Ub antibodies were used to detect the phosphorylation (F) and ubiquitination (G) of MdNAC72. H) GUS activity assay showing that MdMAPK3 phosphorylates MdNAC72 to promote its ubiquitination by MdPUB24, which suppresses the transcriptional repression of the MdPG1 promoter by MdNAC72. The proMdPG1:GUS reporter, together with the effectors 35S:MdNAC72, 35S:MdPUB24, and 35S:MdMAPK3 or 35S:MdNAC72AA, 35S:MdPUB24, and 35S:MdMAPK3, was separately infiltrated into N. benthamiana leaves to assess GUS activity. The reporter vector proMdPG1:GUS, together with the effectors 35S:MdNAC72 and 35S:MdMAPK3, was infiltrated into N. benthamiana leaves as a control. Eth, N. benthamiana leaves treated with ethylene. Three independent infiltration experiments were performed, as described in Fig. 4. I) Dual-LUC reporter assay showing that MdMAPK3 phosphorylates MdNAC72 to promote its ubiquitination by MdPUB24, thereby suppressing the transcriptional repression of the MdPG1 promoter by MdNAC72. The reporter proMdPG1:LUC, together with the effectors 35S:MdNAC72, 35S:MdPUB24, and 35S:MdMAPK3 or 35S:MdNAC72AA, 35S:MdPUB24, and 35S:MdMAPK3, was separately infiltrated into N. benthamiana leaves to assess LUC activity. Eth, N. benthamiana leaves treated with ethephon.

Figure 9.

Effect of the MdNAC72 phosphorylation state on apple fruit softening. A) Apple fruit transiently overexpressing various MdNAC72 variants (MdNAC72 or MdNAC72AA) or an empty vector (control) during storage. Transient overexpression in apple fruit was accomplished using Agrobacterium-mediated injection. Overexpressing fruits (MdNAC72-OE or MdNAC72AA-OE) were harvested 7 d after injection and stored at RT for 30 d. DAI, days after infiltration; DAS, days after storage. B) Immunoblot analysis of proteins from apple fruit transformed with empty vector, MdNAC72-FLAG, or MdNAC72AA-FLAG using an anti-FLAG antibody. The fruit tissues at the injection region were used for protein detection. Tubulin was used as the loading control. C) Relative MdNAC72 expression as detected by RT-qPCR in the fruits shown in (A). D) Firmness of injected apple fruit during storage. Three biological replicates were performed as described in Fig. 2D. E) Relative MdPG1 expression as determined by RT-qPCR. For RT-qPCR, 3 biological replicates were used, as described in Fig. 1. Values are means ± Se. Statistical significance was determined using Student's t-test (**P < 0.01). n.s., no significant difference.

Figure 10.

A model showing the molecular mechanism of MdMAPK3-mediated phosphorylation of MdNAC72 in ethylene-induced fruit softening. When a ripe apple fruit with low ethylene levels enters storage conditions, MdNAC72 abundance is maintained at high levels, which inhibits MdPG1 transcription, and the apple fruit is firm. Following storage, high levels of ethylene production promote MdMAPK3 phosphorylation of MdNAC72, leading to its ubiquitination and degradation by ethylene-induced MdPUB24. Consequently, MdNAC72 protein abundance declines and MdPG1 transcript levels increase, leading to pectin degradation and fruit softening.