Skip to main page content
Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
, 67 (8), 2263-75

The Banana Fruit Dof Transcription Factor MaDof23 Acts as a Repressor and Interacts With MaERF9 in Regulating Ripening-Related Genes

Affiliations

The Banana Fruit Dof Transcription Factor MaDof23 Acts as a Repressor and Interacts With MaERF9 in Regulating Ripening-Related Genes

Bi-hong Feng et al. J Exp Bot.

Abstract

The DNA binding with one finger (Dof) proteins, a family of plant-specific transcription factors, are involved in a variety of plant biological processes. However, little information is available on their involvement in fruit ripening. We have characterized 25 MaDof genes from banana fruit (Musa acuminata), designated as MaDof1-MaDof25 Gene expression analysis in fruit subjected to different ripening conditions revealed that MaDofs were differentially expressed during different stages of ripening. MaDof10, 23, 24, and 25 were ethylene-inducible and nuclear-localized, and their transcript levels increased during fruit ripening. Moreover, yeast two-hybrid and bimolecular fluorescence complementation analyses demonstrated a physical interaction between MaDof23 and MaERF9, a potential regulator of fruit ripening reported in a previous study. We determined that MaDof23 is a transcriptional repressor, whereas MaERF9 is a transcriptional activator. We suggest that they might act antagonistically in regulating 10 ripening-related genes, including MaEXP1/2/3/5, MaXET7, MaPG1, MaPME3, MaPL2, MaCAT, and MaPDC, which are associated with cell wall degradation and aroma formation. Taken together, our findings provide new insight into the transcriptional regulation network controlling banana fruit ripening.

Keywords: Banana; Dof; ERF; Musa acuminate; fruit ripening; protein interaction; transcriptional regulation..

Figures

Fig. 1.
Fig. 1.
(A) Multiple sequence alignment of the conserved Dof domain of 25 MaDof proteins. Identical and similar amino acids are indicated by black and grey shading, respectively. Gaps were introduced to optimize alignment. The NLS motif, and conserved cysteine residues (C1–C5) within the Dof domain are indicated in the alignment. (B) Phylogenetic tree of Dofs. Banana MaDofs were aligned with the Arabidopsis, rice, and tomato Dof family. The multiple alignment was made using ClustalW, and the phylogenetic tree was constructed with MEGA 5.0 using a bootstrap test of phylogeny with minimum evolution test and default parameters.
Fig. 2.
Fig. 2.
Expressions of MaDof10, 23, 24, and 25 in pulp during three ripening behaviours: natural (control), ethylene-induced, and 1-MCP-delayed ripening. The expression levels of each gene are represented as a ratio relative to the harvest time (0 d of control), which was set at 1. Each value represents the mean ± SE of three biological replicates. The physiological data related to fruit ripening and softening, including changes in fruit firmness and ethylene production in banana fruit subjected to three different ripening conditions, have been described in Shan et al. (2012).
Fig. 3.
Fig. 3.
Subcellular localization of MaDof10, 23, 24, and 25 in tobacco leaves. MaDof10, 23, 24, and 25 fused with the GFP or GFP positive control were infiltrated into tobacco leaves via A. tumefaciens strain GV3101. After 48h of infiltration, GFP fluorescence signals were visualized using a fluorescence microscope. Merge indicates a digital merge of bright field and fluorescent images. Images were taken in a dark field for green fluorescence, while the outline of the cell and the Merged images were photographed in a bright field. Scale bars, 25 μm.
Fig. 4.
Fig. 4.
Transcriptional activation of MaDof10, 23, 24, and 25 in yeast. The coding regions of MaDof10, 23, 24, and 25, as well as C- and N-terminal derivatives of MaDof25, were cloned into the pGBKT7 (GAL4BD) vector to create the pGBKT7-MaDof10, 23, 24, 25, 25-N, and 25-C constructs. All of the constructs mentioned above, together with the positive control (pGBKT7-53 + pGADT7-T) and negative control (pGBKT7) were transformed into yeast strain AH109. Yeast clones transformed with different constructs were grown on SD plates without tryptophan (SD/−Trp) or without tryptophan, histidine, and adenine but containing 125 μM aureobasidin A (SD/−Trp−His−Ade) for 3 d at 30°C. Transcription activation was monitored by the detection of yeast growth and an α-Gal assay.
Fig. 5.
Fig. 5.
In vitro and in vivo interaction between MaDof23 and MaERF9. (A) A Y2H assay for the interaction between MaDof23 and MaERF9. The coding regions of MaDof10, 23, 24, and 25-N were fused with pGBKT7 (BD) and the coding region of MaERF9 with pGADT7 (AD) vectors as indicated, and co-transformed into the yeast strain Gold Y2H. The ability of yeast cells to grow on QDO medium (SD/−Leu−Trp−Ade−His but containing 125 μm aureobasidin A), and to turn blue in QDO medium containing 4mg mL−1 X-α-Gal, was scored as a positive interaction. (B) BiFC in tobacco leaf epidermal cells showing the interaction between MaDof23 and MaERF9 in living cells. MaDof23 and MaERF9 were fused with the N-terminus of YFP (YNE) or the C-terminus of YFP (YCE), as indicated, and co-transfected into N. benthamiana leaves by A. tumefaciens infiltration. Expressions of MaDof23 or MaERF9 alone were used as negative controls. YFP indicates fluorescence of YFP; Merge indicates a digital merge of bright field and fluorescent images. Scale bar, 30 μm.
Fig. 6.
Fig. 6.
Transcriptional repression or activation ability of MaDof23 or MaERF9 in tobacco leaves. (A and C) Diagram of the various constructs used in this assay. The double-reporter plasmids contained LUC luciferase fused with 5 × GAL4 and CaMV35S or 5 × GAL4 and the minimal TATA region of CaMV35S, and REN luciferase driven by CaMV35S. The effector plasmids contained the MaDof23 or MaERF9 genes fused to GAL4BD driven by the CaMV35S. pBD was used as a negative control, while the GAL4BD fused with VP16 activation domain was used as positive control. (B and D) The dual REN/LUC reporter and effectors were co-transformed into tobacco leaves using A. tumefaciens strain GV3101. After 48h of infiltration, LUC and REN luciferase activities were assayed, and the transcription repression or activation ability of MaDof23 or MaERF9 is indicated by the ratio of LUC to REN. The ratio of LUC to REN of the pBD vector was used as a calibrator (set as 1). Each value represents the means of six biological replicates, and vertical bars represent the SE. Asterisks indicate a statistically significant difference compared with pBD by one-way ANOVA; *P < 0.05, **P < 0.01. This figure is available in colour at JXB online.
Fig. 7.
Fig. 7.
Antagonistic action of MaDof23 and MaERF9 in transcriptional regulation of 10 ripening-related genes including MaEXP1/2/3/5, MaXET7, MaPG1, MaPME3, and MaPL2 associated with cell wall degradation; and MaCAT and MaPDC associated with aroma formation. (A) Diagram of the various constructs used in this assay. The double-reporter plasmids contained the promoter of the 11 ripening-related genes fused to LUC luciferase and REN luciferase driven by CaMV35S. The effector plasmids contained MaDof23 or MaERF9 driven by CaMV35S. (B) The reporter and effector vectors were co-introduced into tobacco leaves using A. tumefaciens strain GV3101. After 48h of infiltration, LUC and REN luciferase activities were assayed, and the repression or activation of MaDof23 or MaERF9 to the promoter was showed by the ratio of LUC to REN. The ratio of LUC to REN of the empty vector plus promoter vector was used as a calibrator (set as 1). Each value represents the mean ± SE of six biological replicates. Asterisks indicate a statistically significant difference by one-way ANOVA; *P < 0.05, **P < 0.01. + or – indicates that the effector was present or absent in the combinations. This figure is available in colour at JXB online.

Similar articles

See all similar articles

Cited by 17 articles

See all "Cited by" articles

References

    1. Asif MH, Lakhwani D, Pathak S, Gupta P, Bag SK, Nath P, Ttivedi PK. 2014. Transcriptome analysis of ripe and unripe fruit tissue of banana identifies major metabolic networks involved in fruit ripening process. BMC Plant Biology 14, 316. - PMC - PubMed
    1. Ba LJ, Shan W, Kuang JF, Feng BH, Xiao YY, Lu WJ, Chen JY. 2014a. The banana MaLBD (LATERAL ORGAN BOUNDARIES DOMAIN) transcription factors regulate EXPANSIN expression and are involved in fruit ripening. Plant Molecular Biology Reporter 32, 1103–1113.
    1. Ba LJ, Shan W, Xiao YY, Chen JY, Lu WJ, Kuang JF. 2014b. A ripening-induced transcription factor MaBSD1 interacts with promoters of MaEXP1/2 from banana fruit. Plant Cell Reports 33, 1913–1920. - PubMed
    1. Bapat VA, Trivedi PK, Ghosh A, Sane VA, Ganapathi TR, Nath P. 2010. Ripening of fleshy fruit: molecular insight and the role of ethylene. Biotechnology Advances 28, 94–107. - PubMed
    1. Cai XF, Zhang YY, Zhang CJ, Zhang TY, Hu TX, Ye J, Zhang JH, Wang TT, Li HX, Ye ZB. 2013. Genome-wide analysis of plant-specific Dof transcription factor family in tomato. Journal of Integrative Plant Biology 55, 552–566. - PubMed

Publication types

MeSH terms

Feedback