Ethylene-Activated E3 Ubiquitin Ligase MdEAEL1 Promotes Apple Fruit Softening by Facilitating the Dissociation of Transcriptional Repressor Complexes

Abstract

Fruit of most apple varieties soften after harvest, and although the hormone ethylene is known to induce softening, the associated pathway is not well resolved. In this study, it is determined that MdEAEL1 (Ethylene-activated E3 ubiquitin Like 1) is specifically expressed during apple fruit postharvest storage, activated by ethylene, and interacts with the transcription factor MdZFP3 (zinc finger protein3). MdZFP3 is found to rely on an EAR (ethylene-responsive element binding factor-associated amphiphilic repression) motif to form a transcriptional repression complex with MdTPL4 (TOPLESS4)-MdHDA19 (histone deacetylase19), thereby downregulating the histone acetylation levels of the promoters of a range of cell wall degradation-related genes and inhibiting their transcription. MdEAEL1 ubiquitinates and degrades MdZFP3, leading to the disassembly of the MdZFP3-MdTPL4-MdHDA19 transcriptional repression complex. This process promotes the transcription of cell wall degradation-related genes, resulting in fruit softening during storage. Furthermore, the disassembly of the MdZFP3-MdTPL4-MdHDA19 transcriptional repression complex, mediated by MdEAEL1, upregulates the transcription of MdEAEL1 itself, creating a feedback loop that further promotes softening. This study elucidates the interplay between post-translational modifications of a transcription factor and its epigenetic modification to regulate fruit softening, and highlights the complexity of ethylene-induced softening.

Keywords: apple fruit; ethylene; fruit softening.

Figure 1

Ethylene promotes apple fruit softening and MdEAEL1 expression during storage. Apple fruits were harvested 140 d after full bloom and treated with ethylene (ethephon) and 1‐MCP (ethylene signaling inhibitor), or not treated. A) Changes in the appearance of apples during the 20 d storage period. B) Ethylene production, C) firmness, and D) water‐soluble pectin (WSP) were measured. FW, Fresh weight. The data are presented as means ± SE (n = 5 groups, 10 fruits per group). Statistical significance was assessed using Student's t‐test (**p < 0.01). Expression of E) MdPG1, F) MdPL5, G) Mdβ‐Gal9, H) Mdα‐AFase2, I) MdXET1, J) MdEXP8, and K) MdEAEL1 detected using reverse transcription‐quantitative PCR (RT‐qPCR). In (K), H is the harvest day (140 d after full bloom). The data are presented as means ± SE (n = 3 groups, 10 fruits per group). Statistical significance was assessed using Student's t‐test (**p < 0.01, *p < 0.05).

Figure 2

MdEAEL1 promotes apple fruit softening during storage. A) Apple fruit transiently overexpressing MdEAEL1 (MdEAEL1‐OE) or empty vector (pRI101‐Myc) during storage. MdEAEL1‐OE fruit were harvested 7 d after injection and stored at room temperature for 15 d. B) Proteins were extracted from apple fruit at the injection site. Immunoblot analysis was conducted using an anti‐Myc antibody for detection, with Coomassie brilliant blue (CBB) staining of protein extracts serving as a loading control. C) Reverse transcription‐quantitative PCR (RT‐qPCR) was used to detect the expression of MdEAEL1 in MdEAEL1‐OE fruit and the empty vector fruit. D) Fruit firmness and E) water‐soluble pectin (WSP) were measured. FW, Fresh weight. F) Fruit transiently silenced MdEAEL1 (MdEAEL1‐AS), with an empty vector as a control. MdEAEL1‐AS fruit were harvested 7 d after injection and stored at room temperature for 15 d. G) RT‐qPCR was used to detect the expression of MdEAEL1 in the fruit of MdEAEL1‐AS and Empty vector. H) Fruit firmness and I) water‐soluble pectin (WSP) were measured. FW, Fresh weight. DAI, days after infiltration; DAS, days after storage. For firmness and WSP determination, the data are presented as means ± SE (n = 5 groups, 5 fruits per group). For RT‐qPCR, the data are presented as means ± SE (n = 3 groups, 10 fruits per group). Statistical significance was assessed using Student's t‐test (**p < 0.01, *p < 0.05).

Figure 3

MdEAEL1 interaction with MdZFP3 and MdZFP3 expression. A) Interaction between MdEAEL1 and MdZFP3 in a yeast‐two‐hybrid (Y2H) assay. DDO, synthetic defined (SD) medium lacking Trp and Leu; QDO, SD medium lacking Trp, Leu, His, and Ade. Negative controls included BD (binding domain) and AD (activation domain) vectors, while the positive control used SV40/P53. B) Interaction between MdEAEL1 and MdZFP3 in N. benthamiana leaves via luciferase complementation imaging (LCI). C) Interaction between MdEAEL1 and MdZFP3 was verified using a co‐immunoprecipitation (co‐IP) assay. MdZFP3 tagged with Myc (MdZFP3‐Myc) and MdEAEL1 fused with green fluorescent protein (MdEAEL1‐GFP) were overexpressed in apple fruit calli, respectively. The immunoprecipitation analysis was conducted using an anti‐Myc antibody and immunoblotting was performed using anti‐GFP and anti‐Myc antibodies. D) Immunoblot analysis of MdZFP3 protein expression levels during apple fruit storage using an anti‐MdZFP3 antibody. Coomassie Brilliant Blue (CBB) staining of the protein extracts from apple fruit served as a control to ensure equal loading. DAS, days after storage.

Figure 4

MdZFP3 is a transcriptional repressor that negatively regulates apple fruit softening. A) Transcriptional repression assay of MdZFP3. pBD, empty vector, negative control. pBD‐VP16, VP16 transcriptional activator domain, positive control. B) MdZFP3 functions as a transcriptional repressor of promoters containing ZFP transcription factor 5 × binding element. For A and B, the dual LUC/REN reporter was co‐transfected into N. benthamiana leaves along with individual effector plasmids. MdZFP3mEAR, the amino acids LGLDL in the EAR motif of MdZFP3 were mutated to SDSDS. MdZFP3△EAR, the EAR motif deleted from MdZFP3. The data are presented as means ± SE (n = 3 independent transfected N. benthamiana leaves). Statistical significance was determined using Student's t‐test (**p < 0.01, *p < 0.05). C) Apple fruit transiently overexpressing MdZFP3 (MdZFP3‐OE) or empty vector (pRI101) during storage. MdZFP3‐OE fruit were harvested 7 d after injection and stored at room temperature for 15 d. D) Proteins were extracted from apple fruit (MdZFP3‐OE) at the injection site. Immunoblot analysis was conducted using an anti‐MdZFP3 antibody, with Coomassie brilliant blue (CBB) staining of protein extracts serving as a loading control. E) Reverse transcription‐quantitative PCR (RT‐qPCR) was used to detect the expression of MdZFP3 in the fruits of MdZFP3‐OE and Empty vector. F) Fruit firmness and G) water‐soluble pectin (WSP) were measured. FW, Fresh weight. H) Apple fruit with transiently silenced MdZFP3 expression (MdZFP3‐AS), with an empty vector as a control. MdZFP3‐AS fruit were harvested 7 d after injection and stored at room temperature for 15 d. I) Proteins were extracted from apple fruit (MdZFP3‐AS) at the injection site. Immunoblot analysis with an anti‐MdZFP3 antibody for detection, and Coomassie brilliant blue (CBB) staining of protein extracts serving as a loading control. J) RT‐qPCR analysis of the expression of MdZFP3 in the fruits of MdZFP3‐AS and Empty vector. K) Fruit firmness and L) water‐soluble pectin (WSP) were measured. FW, Fresh weight. DAI, days after infiltration; DAS, days after storage. The data statistical analysis was used as described in Figure 2.

Figure 5

MdZFP3 inhibits the expression of cell wall degradation‐associated genes by binding to their promoters. A) Yeast one‐hybrid (Y1H) assay showed that MdZFP3 directly binds to the promoters of MdPG1, MdPL5, Mdβ‐Gal9, Mdα‐AFase2, MdXET1, and MdEXP8. For ProMdPG1, ProMdPL5, ProMdβ‐Gal9, and ProMdEXP8, the basal concentration of AbA (aureobasidin A) was 200 ng mL⁻¹. For ProMdα‐AFase2, the concentration was 250 ng mL⁻¹. For ProMdXET1, the concentration was 150 ng mL⁻¹. B) Chromatin immunoprecipitation (ChIP)‐qPCR assay to assess the in vivo binding of MdZFP3 to the MdPG1, MdPL5, Mdβ‐Gal9, Mdα‐AFase2, MdXET1, and MdEXP8 promoters. Chromatin samples from 35S:MdZFP3‐GFP transgenic apple fruit calli were crosslinked and immunoprecipitated with an anti‐GFP antibody. The eluted DNA was used for qPCR amplification of sequences near the ZFP binding site, with different regions (S1–S4, S1–S3, or S1–S5) investigated. Negative control samples were obtained from 35S:GFP (empty vector) transgenic fruit calli. The data are presented as means ± SE (n = 3 independent transgenic calli). Statistical significance was assessed using a Student's t‐test (**p < 0.01, *p < 0.05). C) GUS reporter gene assays showing that MdZFP3 depends on EAR motif to inhibit the expression of cell wall degradation‐related genes. The GUS reporter plasmid was co‐transfected into N. benthamiana leaves along with individual effector plasmids. MdZFP3mEAR, the amino acids LGLDL in the EAR motif of MdZFP3 were mutated to SDSDS. MdZFP3△EAR, EAR motif deleted from MdZFP3. The data are presented as means ± SE (n = 3 independent transfected N. benthamiana leaves). Statistical significance was determined using Student's t‐test (**p < 0.01, *p < 0.05).

Figure 6

MdZFP3 interacts with MdTPL4 via the EAR motif to form the MdZFP3‐MdTPL4‐MdHDA19 complex. A) Yeast two‐hybrid (Y2H) assays showing that MdZFP3 interacts with MdTPL4, while MdZFP3mEAR, and MdZFP3△EAR do not interact with MdTPL4. B) The interaction relationship of MdTPL4 with MdZFP3, MdZFP3mEAR, or MdZFP3△EAR verified in co‐immunoprecipitation (co‐IP) assays. MdZFP3, MdZFP3mEAR, or MdZFP3△EAR tagged with Myc (MdZFP3‐Myc, MdZFP3mEAR‐Myc, or MdZFP3△EAR‐Myc) and MdTPL4 fused with FLAG (MdTPL4‐FLAG) were overexpressed in apple fruit calli. The immunoprecipitation analysis was conducted using an anti‐FLAG antibody and immunoblotting was performed using anti‐Myc and anti‐FLAG antibodies. C) The interaction relationship of MdTPL4 with MdZFP3, MdZFP3mEAR, or MdZFP3△EAR was confirmed in N. benthamiana leaves using luciferase complementation imaging (LCI). D) MdHDA19 interacts with MdTPL4 as shown in Y2H assays. E) The interaction of MdHDA19 with MdTPL4 was verified in a co‐IP assay. MdHDA19 tagged with GFP (MdHDA19‐GFP) and MdTPL4 fused with FLAG (MdTPL4‐FLAG) were overexpressed in apple fruit calli. The immunoprecipitation analysis was conducted using an anti‐FLAG antibody and immunoblotting was performed using anti‐GFP and anti‐FLAG antibodies. F) The interaction relationship MdHDA19 with MdTPL4 was confirmed in N. benthamiana leaves by LCI. G) MdHDA19 does not interact with MdTPL4, as shown in Y2H assays. The Y2H assay was performed as described in Figure 3. H) MdTPL4 mediates the interaction between MdZFP3 and MdHDA19 in a co‐IP assay. MdHDA19 tagged with GFP (MdHDA19‐GFP) and Myc‐fused MdZFP3 (MdZFP3‐Myc) were co‐expressed along with 35S:MdTPL4 in N. benthamiana leaves. As a control, only MdHDA19‐GFP and MdZFP3‐Myc were co‐expressed. The immunoprecipitation analysis was conducted using an anti‐GFP antibody and immunoblotting was performed using anti‐GFP and anti‐Myc antibodies. I) MdTPL4 mediates the interaction between MdZFP3 and MdHDA19 in Nicotiana benthamiana leaves via LCI assays. 35S:GUS, β‐glucuronidase protein is expressed in tobacco leaves as a negative control.

Figure 7

MdZFP3 recruits MdTPL4 and MdHDA19 to the promoter of its downstream genes, downregulating histone acetylation levels. A) DNA pull‐down assay showing that the MdPG1 promoter is bound by MdTPL4 and MdHDA19 through MdZFP3. In this assay, recombinant MdTPL4‐HIS and MdHDA19‐MBP were incubated with a biotin‐labeled 1000 bp DNA fragment of the MdPG1 promoter, along with MdZFP3‐GST or GST. The complexes were then pulled down using streptavidin agarose beads. Immunoblots were subsequently probed with anti‐His, anti‐MBP or anti‐GST antibodies. Chromatin immunoprecipitation (ChIP)‐qPCR analysis of B) H3K9Ac and C) H3K27Ac levels in the MdPG1, MdPL5, Mdβ‐Gal9, Mdα‐AFase2, MdXET1, and MdEXP8 promoters in MdZFP3‐OE and MdZFP3‐AS fruit 15 d after harvest. The empty vector transgenic fruits (Empty vector) were used as a control. The data are presented as means ± SE (n = 3 groups, 10 transgenic fruit per group). Statistical significance was assessed using Student's t‐test (**p < 0.01, *p < 0.05).

Figure 8

Ethylene promotes the ubiquitination of MdZFP3 by MdEAEL1, leading to MdZFP3 degradation. MdEAEL1 ubiquitination of MdZFP3 in vitro. The presence of ATP, ubiquitin, E1, E2, and recombinant MdEAEL1‐GST allowed the detection of potential E3 ubiquitin ligase activity using MdZFP3‐His as a substrate. Immunoblot analysis was performed to identify the ubiquitination of MdZFP3 using A) anti‐His or B) anti‐ubiquitin (Ub) antibodies. C) MdZFP3 ubiquitination in MdZFP3‐Myc and MdEAEL1‐OE + MdZFP3‐Myc apple calli (pretreated with 50 µm MG132). MdZFP3 was immunoprecipitated using an anti‐Myc antibody, and ubiquitinated MdZFP3‐Myc was detected using an anti‐Ub antibody. D) Cell‐free degradation assay performed using protein extracts from transgenic apple calli (MdEAEL1‐OE) and wild‐type (Wt) to assess the abundance of recombinant MdZFP3‐His. Immunoblot analysis was performed using an anti‐His antibody to determine MdZFP3‐His levels. MG132, wild‐type (Wt) and MdEAEL1‐OE transgenic apple calli were treated separately with 50 µm MG132. E) LUC reporter gene assay indicated that MdEAEL1 mediates the degradation of MdZFP3 through the 26S proteasome pathway. The reporter 35S:MdZFP3:LUC, alone or together with 35S:MdEAEL1, was infiltrated into N. benthamiana leaves to assess LUC activity. MG132, N. benthamiana leaves treated with 50 µm MG132. F) Ubiquitination assays of apple fruits silencing MdEAEL1 (MdEAEL1‐AS) or overexpressing MdEAEL1 (MdEAEL1‐OE) after 15 d of storage shows that MdEAEL1 can ubiquitinate MdZFP3. Proteins were extracted from apple pretreated with 50 µm MG132. MdZFP3 protein was immunoprecipitated using an anti‐MdZFP3 antibody. Immunoblotting was performed to detect the ubiquitination of MdZFP3 using an anti‐Ub. G) Abundance of the MdZFP3 protein in MdEAEL1‐OE or MdEAEL1‐AS apple fruit after 15 d of storage. H) The ubiquitination level of MdZFP3 gradually increased during the apple fruit storage and was enhanced by ethylene treatment. DAS, days after storage. I) The ethylene‐promoted ubiquitination of MdZFP3 was inhibited by silencing of MdEAEL1. “+” Ethylene treatment, “−” Non‐treatment. Ubiquitination assay was performed as described in F. In C, D, F, G, H, and I, Coomassie brilliant blue (CBB) staining of total protein extracts served as a control for equal sample loading.

Figure 9

MdEAEL1 mediates the disassembly of the MdZFP3‐MdTPL4‐MdHDA19 transcriptional repression complex, upregulating histone acetylation levels in the promoter region of MdZFP3 target genes. A) MdEAEL1 inhibits the interaction between MdZFP3 and MdTPL4 in a co‐IP assay. MdZFP3 tagged with Myc (MdZFP3‐Myc) and FLAG‐fused MdTPL4 (MdTPL4‐FLAG) were co‐expressed together with 35S:EAEL1 expressed in N. benthamiana leaves. As a control, only MdZFP3‐Myc and MdTPL4‐FLAG were co‐expressed. The immunoprecipitation analysis was conducted using an anti‐FLAG antibody and immunoblotting was performed using anti‐FLAG and anti‐Myc antibodies. Coomassie brilliant blue (CBB) staining of total protein extracts served as a control for equal sample loading. B) MdTPL4 inhibition of the interaction between MdZFP3 and MdTPL4 in N. benthamiana leaves assessed by luciferase complementation imaging (LCI). C) GUS reporter assays indicating that MdEAEL1 mediates the disassembly of the MdZFP3‐MdTPL4‐MdHDA19 complex, promoting the transcription of MdPG1. The GUS reporter plasmid was co‐transfected into N. benthamiana leaf together with individual effector plasmids. D) The LUC reporter was co‐transfected into N. benthamiana leaves together with individual effector plasmids. E) Chromatin immunoprecipitation (ChIP)‐qPCR analysis of H3K9Ac level at the MdPG1, MdPL5, Mdβ‐Gal9, Mdα‐AFase2, MdXET1, and MdEXP8 promoters in MdEAEL1‐OE and MdEAEL1‐AS fruit at 15 d after harvest. The empty vector transgenic fruit (Empty vector) were used as a control. The data statistical analysis was used as described in Figure 7B. In A–D, MG132 was used as a proteasome inhibitor.

Figure 10

MdEAEL1‐MdZFP3‐MdTPL4‐MdHDA19 module forms a feedback loop that suppressed the transcriptional repression activity of MdZFP3 on the MdEAEL1 promoter. Reverse transcription‐quantitative PCR (RT‐qPCR) analysis of the expression of MdEAEL1 in the fruit of A) MdZFP3‐AS and B) MdZFP3‐OE. The data statistical analysis was used as described in Figure 2. C) Yeast one‐hybrid (Y1H) assay showing that MdZFP3 directly binds to MdEAEL1 promoter. The basal concentration of AbA (aureobasidin A) used was 200 ng mL⁻¹. D) Chromatin immunoprecipitation (ChIP)‐qPCR assay demonstrating the in vivo binding of MdZFP3 to the MdEAEL1 promoter. The data statistical analysis was used as described in Figure 5B. E) GUS reporter assays showing that MdZFP3 depends on an EAR motif to inhibit the expression of MdEAEL1. The GUS reporter plasmid was co‐transfected into N. benthamiana leavestogether with individual effector plasmids. F) The LUC reporter was co‐transfected into N. benthamiana leaves together with individual effector plasmids. In E and F, MdZFP3mEAR, the amino acids LGLDL in the EAR motif of MdZFP3 were mutated to SDSDS. MdZFP3△EAR, MdZFP3 with a deleted EAR motif. G) GUS reporter assays indicating that MdEAEL1 mediates the disassembly of the MdZFP3‐MdTPL4‐MdHDA19 complex, promoting the transcription of MdEAEL1. The GUS reporter plasmid was co‐transfected into N. benthamiana leaves together with individual effector plasmids. MG132 was used as a proteasome inhibitor. In E and G, the data statistical analysis was used as described in Figure 5C. H) The LUC reporter was co‐transfected into N. benthamiana leaves together with individual effector plasmids. MG132 was used as a proteasome inhibitor. I) ChIP analysis of H3K9Ac level at the MdEAEL1 promoter in MdEAEL1‐OE and MdEAEL1‐AS fruit at 15 d after harvest. The empty vector transgenic fruit (Empty vector) were used as a control. The data statistical analysis was used as described in Figure 7B.

Figure 11

A model showing the ethylene‐activated E3 ubiquitin ligase MdEAEL1 mediating the disassembly of the MdZFP3‐MdTPL4‐MdHDA19 transcriptional repression complex, thereby forming a loop that promotes apple fruit softening. During storage, ripe apples with low ethylene levels maintain the stability of the MdZFP3‐TPL4‐HDA19 transcriptional repression complex, which prevents the transcription of cell wall degradation‐related genes. As ethylene production increases after storage, ethylene‐activated MdEAEL1 mediates the ubiquitination and degradation of MdZFP3, leading to the disassembly of the MdZFP3‐MdTPL4‐MdHDA19 transcriptional repression complex. This upregulates the acetylation levels of histones in the promoter regions of cell wall degradation‐related genes, resulting in elevated transcription levels of these genes and leading to fruit softening. The disassembly of the MdZFP3‐MdTPL4‐MdHDA19 complex triggers the transcription of MdEAEL1, forming a feedback loop that further promotes fruit softening.