Fig 1.
Heat map of the expression levels of ripening-associated durian ERF family genes at three different stages (unripe, midripe, and ripe) during fruit post-harvest ripening.
The cluster was generated using the Euclidian distance method according to gene expression values (RPKM) from the transcriptome data. For each row, blue and red correspond, respectively, to low and high expression values. For a given gene and stage, the expression value corresponds to the mean of the RPKM value.
Table 1.
Members of the ERF transcription factor family identified in durian (Durio zibethinus L.) pulps.
Fig 2.
Motif organization of ripening-associated durian ERFs (DzERFs).
A schematic distribution of 10 conserved motifs identified by MEME suite version 5.1.0. is presented. Motifs 1 and 2 correspond to the DNA binding domain (AP2/ERF domain). The functions of other eight motifs are still unknown and must be further investigated.
Fig 3.
Phylogenetic tree of the amino acid sequences of the ripening-associated durian ERFs (DzERFs).
The deduced full-length amino acid sequences of DzERFs were aligned with protein sequences of ERFs from tomato (Solanum lycopersicum; SlERFs), banana (Musa acuminata; MaERFs), and previously characterized ERFs from climacteric fruit crops (apple: MdERFs; pear: PpERFs; papaya: CpERF; kiwi: AdERF; peach: PpeERF; persimmon: DkERFs) to construct the phylogenetic tree using MEGA X software and the neighbor-joining method (with 1000 bootstrap replicates, a JTT model, and pairwise gap deletion using a bootstrap test of phylogeny with the minimum evolution test and default parameters). The previously characterized ERFs are highlighted with a frame.
Fig 4.
Tissue-specific expression profile of ripening-associated durian ERFs (DzERFs) in the Musang King cultivar at the ripe stage.
We used the publicly available Illumina RNA-seq data to analyze the expression levels of ripening-associated DzERFs in root, stem, leaf, and fruit pulp tissues. For each DzERF, higher expression is presented in red; otherwise, blue is used. The heatmap was generated using MetaboAnalyst 4.0, an open source R-based program. Data were sum-normalized, log transformed, and autoscaled.
Fig 5.
Gene expression correlation of ripening-associated durian ERFs (DzERFs).
(A) Heatmap of hierarchical clustering of Pearson’s correlations (R) for 34 ripening-associated DzERFs and previously identified ripening-related genes. Genes with a normalized expression level (RPKM) > 1 were log2 transformed before analysis and were designated as expressed. The DzERFs for which the expression decreased are highlighted with a red frame. The DzERFs for which the expression increased and ripening-related genes are highlighted with a blue frame. (B) Correlation network analysis of 34 ripening-associated DzERFs, previously identified ripening-related genes, and a previously characterized member of the ARF TF family (DzARF2A). The thickness of the line corresponds to the correlation strength. Red lines represent positive correlations, whereas blue lines indicate negative correlations.
Fig 6.
Fold changes in expression levels of candidate ripening-associated durian ERFs (DzERFs) at three different stages (unripe, midripe, and ripe) during the post-harvest ripening of durian fruit (Monthong cultivar) and under three different ripening conditions.
(A and B) The relative gene expression levels of DzERF6 and DzERF9 were calculated using the 2−ΔΔCt method, and levels were normalized by the geometric mean of reference genes and the unripe stage as the control. Three independent biological replicates were used. An asterisk above the bars indicates a significant difference at P < 0.05 (*). (C and D) The relative expression levels of DzERF6 and DzERF9 were also quantified under three different ripening conditions, natural (control), ethylene-induced, and 1-MCP-delayed ripening by using the 2−ΔΔCt method, and levels were normalized by the geometric mean of reference genes and the natural ripening condition as the control. Three independent biological replicates were used. An asterisk above the bars indicates a significant difference at P < 0.05 (*).
Fig 7.
Auxin-responsiveness of candidate ripening-associated durian ERFs (DzERFs).
Fold changes in expression levels of DzERF6 (A) and DzERF9 (B) in durian leaves of the Monthong cultivar treated with 0 (control), 10, 20, and 40 μM indole-3-acetic acid (IAA) for 2 h were calculated using the 2−ΔΔCt method, and levels were normalized by the geometric mean of reference genes and the control samples (0 μM IAA). Three independent biological replicates were used. An asterisk above the bars indicates a significant difference at P < 0.05 (*).
Fig 8.
A general scheme depicting the role of ripening-associated DzERF6 and DzERF9 in the regulatory network mediating durian fruit ripening.
Data from this study and our previous one [33] are integrated and presented. DzERF9 might exert its effect via the ethylene-dependent ripening pathway by positively regulating the transcription of ethylene biosynthetic genes (ACC synthase; ACS and ACC oxidase; ACO). It appears that the expression of DzERF9 is positively regulated by both auxin and ethylene. DzARF2A is a positive regulator of fruit ripening that functions by trans-activating the ethylene biosynthetic genes. DzARF2A might interact with DzERF9, and together they act as a positive regulator of durian fruit ripening. As a negative regulator of fruit ripening, DzERF6 represses the transcription of ethylene biosynthetic genes. Arrows indicate positive regulation (activation) whereas blunt-ended lines indicate negative regulation (repression). The dashed lines denote our proposed regulatory role during ripening.