MaNAC029 modulates ethylene biosynthesis and fruit quality and undergoes MaXB3-mediated proteasomal degradation during banana ripening

Abstract

Introductions: Ethylene regulates ripening by activating various metabolic pathways that controlcolor, aroma, flavor, texture, and consequently, the quality of fruits. However, the modulation of ethylene biosynthesis and quality formation during banana fruit ripening remains unclear.

Objectives: The present study aimed to identify the regulatory module that regulates ethylene and fruit quality-related metabolisms during banana fruit ripening.

Methods: We used RNA-seq to compare unripe and ripe banana fruits and identified a ripening-induced NAC transcription factor, MaNAC029. We further performed DNA affinity purification sequencing to identify the MaNAC029's target genes involved in ethylene biosynthesis and fruit quality formation, and electrophoretic mobility shift assay, chromatin immunoprecipitation with real-time polymerase chain reaction and dual luciferase assays to explore the underlying regulatory mechanisms. Immunoprecipitation combined with mass spectrometry, yeast two-hybrid assay, and bimolecular fluorescence complementation assay were used to screen and verify the proteins interacting with MaNAC029. Finally, the function of MaNAC029 and its interacting protein associated with ethylene biosynthesis and quality formation was verified through transient overexpression experiments in banana fruits.

Results: The study identified a nucleus-localized, ripening-induced NAC transcription factor MaNAC029. It transcriptionally activated genes associated with ethylene biosynthesis and a variety of cellular metabolisms related to fruit quality formation (cell wall degradation, starch degradation, aroma compound synthesis, and chlorophyll catabolism) by directly modulating their promoter activity during ripening. Overexpression of MaNAC029 in banana fruits activated ethylene biosynthesis and accelerated fruit ripening and quality formation. Notably, the E3 ligase MaXB3 interacted with and ubiquitinated MaNAC029 protein, facilitating MaNAC029 proteasomal degradation. Consistent with this finding, MaXB3 overexpression attenuated MaNAC029-enhanced ethylene biosynthesis and quality formation.

Conclusion: Our findings demonstrate that a MaXB3-MaNAC029 module regulates ethylene biosynthesis and a series of cellular metabolisms related to fruit quality formation during banana ripening. These results expand the understanding of the transcriptional and post-translational mechanisms of fruit ripening and quality formation.

Keywords: Banana fruit; E3 ligase; Ethylene; Fruit quality formation; NAC; Ripening.

Graphical abstract

Fig. 1

Molecular characterization of MaNAC029. (A) Expression of MaNAC029 in banana fruit with three different ripening characteristics: natural, ethylene-induced and 1-MCP-delayed ripening. The open triangle indicates the beginning of ethylene production for each treatment, while the closed triangle indicates the peak of ethylene production. (B)MaNAC029 promoter activity in response to ethylene. Data shown are the mean ± SE of six replicates (**, P < 0.01). (C) Sub-cellular localization of MaNAC029 in tobacco BY-2 protoplasts. NLS-mCherry was used as a nuclear marker. Bars, 25 μm. (D) Transcriptional activation of MaNAC029 in yeast cells. The left panel shows the schematic representation of the domain structure of MaNAC029. NaNAC029 contains a highly conserved NAC domain at the N-terminal, further divided into five conserved subdomains (A–E, shown in yellow). The full-length proteins (MaNAC029), N-terminal fragment (MaNAC029-N) and C-terminal fragment (MaNAC029-C) were fused with the GAL4 DNA-binding domain. The pGBKT7 vector (BD) was used as a negative control and P53-BD + T-antigen-AD as a positive control. (E) trans-activation of MaNAC029 in tobacco leaves. The LUC/REN ratio of the empty pBD vector (negative control) was considered the calibrator (set as 1). VP16 (a potent transcriptional activator) was used as a positive control. Data shown are the mean ± SE of six replicates (**, P < 0.01).

Fig. 2

Genome-wide identification of MaNAC029-targeted genes by DAP-seq. (A) Distribution of the enriched DAP-seq fragments in different regions of the annotated genes. The peaks in the promoter region (<1000 bp and 1000–2000 bp), downstream of the promoter, 5′ UTR, 3′ UTR, or exon are shown. (B) De novo motifs enriched near the MaNAC029-binding sites. (C) GO biological process functional classes of the targets associated with MaNAC029 DAP-seq peaks. (D) KEGG pathway enrichment analysis of MaNAC029-binding genes (top 20 enriched pathways are presented.). (E) Venn diagram showing the overlap of genes targeted by MaNAC029 from DAP-seq and the genes differentially expressed between the ripe and unripe fruits identified based on RNA-seq. (F) Heatmap of differentially expressed MaNAC029 target genes between unripe and ripe fruits involved in ripening and quality formation. The green–red color and the white–purple scale represents log₁₀ transformed FPKM value and log2 fold change in expression, respectively.

Fig. 3

MaNAC029 directly binds to and activates the promoters of genes for ethylene biosynthesis and fruit quality formation. (A) Electrophoretic mobility shift assay showing in vitro binding of MaNAC029 to the promoters of genes associated with ethylene biosynthesis and a variety of cellular metabolisms related to fruit quality formation. The probe sequences corresponding to the target gene promoters are shown at the top, with red letters indicating the MaNAC029-binding motif. Plus sign indicate increasing amounts of unlabeled wild type for competition. (B) Chromatin immunoprecipitation-quantitative PCR analysis showing in vivo binding of MaNAC029 to its target gene promoters. The promoter structure of the target genes is shown on the left. The probes used for the assays are underlined in black. Data shown are the mean ± SE of three replicates (** P < 0.01). (C) MaNAC029-mediated trans-activation of target promoters. The LUC/REN value of the empty vector plus promoter reporter served as a calibrator (set as 1). Data shown are the mean ± SE of six replicates (** P < 0.01).

Fig. 4

Transient overexpression of MaNAC029 in banana fruit accelerates ethylene biosynthesis and quality formation during ripening. (A) Appearance of banana fruit transiently overexpressing MaNAC029 and empty vector during ripening. (B) Western blot shows MaNAC029 protein levels in bananas fruits transiently overexpressing the empty vector and MaNAC029. Western blot was repeated three times (Fig. S10). (C) Color index, ethylene production, firmness, starch content, and total chlorophyll content of MaNAC029-overexpressing and control banana fruits. Error bars indicate the SE of the mean of six replicates (**, P < 0.01). (D) Relative abundance of MaACS1, MaACO1, MaACO13, MaEXP2, MaEXP15, MaXTH28, MaXTH30, MaGWD1, MaAAT1, MaSGR1 and MaPPH mRNA in MaNAC029-overexpressing and control banana fruit. Data shown are the mean ± SE of six replicates (** P < 0.01).

Fig. 5

MaXB3 interacts with and ubiquitinates MaNAC029.(A) Y2H assay shows the interaction between MaXB3 and MaNAC029. Yeast cells cotransformed with DBD-p53 and AD-T were used as the positive control, and those with DBD-Lam and AD-T as the negative control. (B) BiFC analysis shows the interaction between MaNAC029 and MaXB3 in tobacco BY-2 protoplasts. NLS-mCherry was used as a nuclear marker. Bar, 25 μm. (C) Co-immunoprecipitation assay of unripe and ripe banana fruits shows the interaction between MaXB3 and MaNAC029, and the role of MaXB3 in MaNAC029 ubiquitination. anti-ubiquitin and anti-MaNAC029 antibodies were used to detect the ubiquitinated MaNAC029 protein. The presence of MaXB3 in the immunoprecipitated complex was analyzed using the anti-MaXB3 antibody. (D) MaXB3 directly ubiquitinates MaNAC029 in vitro. The activity of the MaXB3 in MaNAC029 ubiquitination was tested in the presence and absence of ubiquitin, E1, E2, MBP-MaXB3 and GST-MaNAC029-N. (E) In vivo ubiquitination of MaNAC029 by MaXB3. MaNAC029-GFP and MaXB3-His, MaNAC029-GFP and Empty-His, Empty-GFP and MaXB3-His, or Empty-GFP and Empty-His, were transiently expressed in tobacco leaves and immunoprecipitated with anti-GFP antibody. The interaction between MaXB3 and MaNAC029 was detected using an anti-GFP or anti-His antibody. The ubiquitin levels of MaNAC029 were detected using the anti-ubiquitin antibody. Western blot was repeated three times for C, D, and E (Fig. S11, S12 and S13).

Fig. 6

MaXB3 degrades MaNAC029 and attenuates MaNAC029-mediated trans-activation of target genes.(A) Proteasome-mediated degradation of MaNAC029. MaNAC029 fused with GFP and LUC was co-expressed with MaXB3 or empty vector in tobacco leaves in the presence or absence of proteasome inhibitor (MG132). Western blot was performed using an anti-GFP antibody to analyze the abundance of MaNAC029 protein (top panel). The MaNAC029 protein level was quantified by measuring luciferase activity (bottom panel). Data shown are the mean ± SE of six replicates (** P < 0.01). (B) MaXB3 attenuates the MaNAC029-mediated trans-activation of targets. The LUC reporter driven by the promoters of target genes was co-expressed in tobacco leaves with either MaNAC029- or MaXB3-expressing effector plasmid alone or together in the presence or absence of MG132. Data shown are the mean ± S. of six biological replicates (**, P < 0.01). (C–I) Transient overexpression of MaXB3 in banana fruit delays ripening and quality formation. (C) The appearance of banana fruits transiently overexpressing empty vector (control) or MaXB3 during ripening. (D) Western blot of fruits transiently overexpressing empty vector and MaXB3. (E–F) Changes in the color index, ethylene production, firmness, starch content, and total chlorophyll content in MaXB3-overexpressing and control banana fruits. Error bars indicate the SE from six replicates (**, P < 0.01). (G) Relative mRNA abundance of MaNAC029 downstream genes in MaXB3-overexpressing and control banana fruits. Data shown are the mean ± SE of three replicates. (H) Western blot of MaNAC029 protein in MaXB3-overexpressing and control banana fruit. (I) Chromatin immunoprecipitation-quantitative PCR shows MaNAC029 binding to its target promoters in MaXB3-overexpressing and control fruit. Data shown are the mean ± SE of three replicates (** P < 0.01). Western blot was repeated thrice for A, D, and H (Fig. S14, S15 and S16).

Fig. 7

Proposed model demonstrating MaXB3-MaNAC029 module regulates ethylene biosynthesis and a series of cellular metabolisms related to quality formation during fruit ripening. Ethylene-induced MaNAC029 trans-activates MaACS1, MaACO1 and MaACO13, forming a positive feedback loop to regulate ethylene biosynthesis. Meanwhile, ethylene down-regulated MaXB3 ubiquitinates and degrades MaNAC029, MaACS1 and MaACO1, creating a negative feedback loop. MaNAC029 enhances the transcription of various genes involved in chlorophyll, starch and cell wall degradation, and aroma formation, leading to the formation of banana fruit quality, while MaXB3 attenuates these MaNAC029-mediated enhancements. Our findings represent a novel regulatory module, MaXB3-MaNAC029, regulating ethylene biosynthesis and a variety of cellular metabolisms associated with quality formation during banana fruit ripening. Arrows indicate positive effect, and bar indicates a negative regulation.