Article Figures & Data
Figures
- Figure 1.
Identification of two ERF17 genes in the apple genome. A, Phylogenetic analysis of homologous ERF17 transcription factors. MDP0000704216 and MDP0000649022 locate on apple chromosome 2 (Chr2) and chromosome 15 (Chr15), respectively. The numbers on the tree represent nucleotide substitutions. B, Relative expression of MDP0000704216 (left) and MDP0000649022 (right) in peels of cv Red Fuji (gray) and cv Zisai Pearl (black) various days after flower blossom (DAFB). An actin fragment was amplified as an internal control. Values are means ± sd (n = 3). Asterisks indicate statistically significant differences by Student’s t test (*, P < 0.05 and **, P < 0.01) compared with cv Red Fuji peel. ND, Not detected.
- Figure 2.
Chl degradation of fruit peel in cv Red Fuji and Zisai Pearl during development and variation in ERF17 gene and ERF17 protein sequences. A, Appearance of fruits and Chl content during development. Error bars indicate sd of three biological replicates. DAFB, Days after flower blossom; FW, fresh weight. Bar = 1 cm. B, Variations in the ERF17 cDNA fragment between the ERF17-3S, ERF17-6S, and ERF17-8S alleles (for full sequences, see Supplemental Fig. S1). C, Variations in the ERF17 amino acid sequences between the ERF17-3S, ERF17-6S, and ERF17-8S alleles.
- Figure 3.
Analyzing progeny from a cross between cv Red Fuji and Zisai Pearl. A, Appearance of ERF17-3S/ERF17-8S genotype fruits during development. Bar = 1 cm. B, Appearance of ERF17-3S/ERF17-6S genotype fruits during development. C, Chl degradation during development of the ERF17-3S/ERF17-8S genotype. D, Chl degradation during development of the ERF17-3S/ERF17-6S genotype. DAFB, Days after flower blossom; FW, fresh weight. Error bars in C and D indicate sd of three biological replicates.
- Figure 4.
Transcriptional regulation activity of ERF17 allele proteins. A dual-luciferase reporter assay of ERF17 allele proteins using an Arabidopsis protoplast system is shown. The plasmid ratio used (pTRL:reporter:effector) was 1:6:6. Values are means ± sd (n = 3). Asterisks indicate statistically significant differences by Student’s t test (*, P < 0.05 and **, P < 0.01) compared with the binding domain (BD).
- Figure 5.
ERF17 and Chl degradation-related gene expression in fruit peels during development. A, Gene expression in fruit peels of cv Red Fuji and Zisai Pearl during development. B, Gene expression in fruit peels from a cross between cv Red Fuji and Zisai Pearl: 07-130, 11-132, and 16-122 represent the ERF17-3S/ERF17-8S genotype, and 04-153, 06-174, and 09-129 represent the ERF17-3S/ERF17-6S genotype. Error bars indicate sd of three biological replicates. DAFB, Days after flower blossom.
- Figure 6.
ERF17 regulates Chl degradation-related genes. A, Yeast one-hybrid assay showing the activity of LacZ reporters driven by the PPH and NYC promoters and activated by activation domain (AD) fusion with ERF17-8S and ERF17-3S (blue color). The empty vectors pB42AD and pLacZi were used as negative controls (white color). BD, Binding domain. B, Gel-shift analysis of ERF17-8S and ERF17-3S binding to the promoters of PPH and NYC. EMSAs of a GST-labeled probe with ERF17-8S and ERF17-3S proteins are shown. DNA probes represent the promoters of PPH and NYC (probe sequences are shown in Supplemental Table S3). Protein/DNA complexes are indicated by arrows. C, SPR binding profiles of ERF17-8S and ERF17-3S proteins to the promoters of PPH and NYC. Protein concentrations were 275 nm in all lines but the bottom line, for which it was 17.2 nm. RU, Response units.
- Figure 7.
ERF17 is essential for Chl degradation in apple fruit. A, Apple fruit peel coloration. Three ERF17 genotypes were overexpressed in apple fruits by two A. tumefaciens-mediated transient transformation methods (injection and infiltration). Apples injected and infiltrated with the empty pBI1300 vector were used as controls. B, Chl content around the infiltration sites of apple peels in mg g−1 fresh weight (FW). C, Relative expression levels of MdERF17, MdNYC, and MdPPH in apple fruit peels around the infiltration sites determined using reverse transcription real-time quantitative PCR (RT-qPCR). An actin fragment was amplified as an internal control. Values are means ± sd (n = 3) in B and C. Asterisks indicate statistically significant differences by Student’s t test (**, P < 0.01) compared with the control. ND, Not detected.
- Figure 8.
Schematic model of the regulation of the apple peel degreening regulatory module. The increased Ser repeat number in the coding region of ERF17 enhances its transcriptional regulatory activity and its binding ability to the promoter of Chl degradation-related genes. NYC and PPH are required for Chl degradation, which can contribute to apple fruit pigment. An elevated expression level of NYC and PPH improves apple fruit peel degradation and promotes pigment accumulation.
Tables
Protein DNA Association Rate Constant Dissociation Rate Constant Equilibrium Dissociation Constant m−1 s−1 s−1 m ERF17-8S PPH 1.480 × 104 4.521 × 10−3 3.055 × 10−7 ERF17-3S PPH 0.969 × 104 8.352 × 10−3 8.623 × 10−7 ERF17-8S NYC 5.686 × 104 5.208 × 10−3 9.159 × 10−8 ERF17-3S NYC 6.638 × 104 8.648 × 10−3 1.303 × 10−7 - Table III. Nucleotide polymorphisms in ERF17 for Malus spp.
m = number of sequences, S = number of segregating sites, ps = S/m, Θ = ps/a1, π = nucleotide diversity, and D is the Tajima test statistic.
Species m ps Θ π D Neutral Mutation Range (Tajima’s D) M. asiatica 7 0.025054 0.010226 0.010271 0.024563 −1.498 to 1.728 M. baccata 10 0.021361 0.007551 0.005696 −1.17371 −1.559 to 1.719 M. prunifolia 13 0.047918 0.015442 0.017332 0.551679 −1.580 to 1.708 M. domestica 29 0.062706 0.015967 0.008435 −1.77898 −1.581 to 1.714 M. sieversii 13 0.030438 0.010079 0.008785 −0.58541 −1.580 to 1.708
Supplemental Data
Supplemental Figures and Tables
Files in this Data Supplement:
- Supplemental Data - Supplemental Figures 1-7 and Supplemental Tables 1-5
