Red peel regulator 1 links ethylene response factor 25 and β-citraurin biosynthetic genes to regulate ethylene-induced peel reddening in citrus

Abstract

The reddish apocarotenoid β-citraurin, produced by CAROTENOID CLEAVAGE DIOXYGENASE 4b (CsCCD4b), is responsible for peel reddening in citrus (Citrus spp.). Ethylene induces the characteristic red color of citrus peel, but the underlying molecular mechanism remains largely unclear. Here, we identified red peel regulator 1 (CsRP1), a trihelix transcriptional activator that regulates ethylene-induced peel reddening by directly binding to a key V-myb avian myeloblastosis viral oncogene homolog (MYB)-binding site in the CsCCD4b promoter, thus activating its transcription. Furthermore, 2 drought-responsive cis-elements in the CsRP1 promoter are bound by the ethylene response factor ethylene response factor 25 (CsERF25). We reconstructed the CsERF25-CsRP1-CsCCD4b transcriptional regulatory cascade through transient expression of CsERF25 and CsRP1 in citrus peel and via stable transformation of citrus calli. In this cascade, CsERF25 expression was induced by ethylene to activate CsRP1 expression, and then, CsRP1 directly induced CsCCD4b transcription to catalyze β-citraurin biosynthesis. CsRP1 and CsERF25 also bound to the promoters of other carotenogenic genes and induced their transcription, thereby promoting β-citraurin accumulation. Collectively, our findings reveal a complex regulatory network modulating ethylene-induced citrus peel reddening and provide innovative strategies for improving the nutritional and esthetic values of citrus and other fruit crops.

Figure 1.

Ethylene promotes β-citraurin accumulation and fruit coloration. A) Phenotype of treated fruit under various treatment. Bars = 2 cm. Images were digitally extracted for comparison. 1-MCP, 1-methylcyclopropene. B) Contents of β-citraurin and C) expression levels of CsCCD4b in peel of treated fruit. “Newhall” navel orange fruit was harvested at 180 DAF. DW, dry weight. D) The carotenoid metabolic pathway of “Newhall” navel orange fruit under ethylene treatment. Labeled genes indicate that the carotenogenic genes are induced by ethylene treatment. Numbers represent the maximum multiple of carotenogenic genes activation by ethylene treatment during the whole storage. B, C) Data are expressed as means ± SD of 3 biological replicates. Asterisks indicate statistically significant differences by Student's t-test (*P < 0.05; **P < 0.01; n.s., no significant difference).

Figure 2.

Ethylene-induced CsRP1 directly activates CsCCD4b expression. A) Expression of CsRP1 under various treatments. 1-MCP, 1-methylcyclopropene. B) Interactions of CsRP1 with CsCCD4b promoter by Y1H assay. Empty PGADT7 + pAbAi-ProCsCCD4b and PGADT7-Rec-p53 + p53-AbAi were used as the negative control (N. Control) and positive control (P. Control), respectively. SD/-Leu/AbA²⁰⁰ indicates SD/-Leu medium supplemented with 200 ng ml⁻¹ aureobasidin A (AbA) which was used as a yeast growth inhibitor. C) Interactions between CsRP1 and biotin-labeled CsCCD4b promoter fragment (hot probe) containing SNP mutation identified by EMSA. Labeled letters indicate the binding motifs and blue letters denote their corresponding mutant motifs. The red line indicates the position of the SNP mutation. “+” and “−” present the presence and absence of the indicated probe or protein, respectively. Increased amounts (50- and 100-folds) of the unlabeled DNA fragments (cold probe) were added as competitors. Purified MBP-tagged CsRP1 protein was used in EMSA, and purified MBP protein was used as a N. Control. Red arrows point to the positions of protein–DNA complexes or free probes. D) ChIP-PCR assay showed the interaction of CsRP1 and the region containing SNP mutation on the CsCCD4b promoter, and this interaction was enhanced by ethylene. The gray line represents putative MBS motif, and the red line indicates the position of SNP mutation. Cross-linked chromatin was precipitated by GFP antibody, and the resulting DNA fragment was amplified by qPCR. E) Schematic diagram of reporter and effector constructs used in dual-LUC assay. PK7-Ev indicates PK empty vector (control). Dual-LUC assay showed that CsRP1 activated the CsCCD4b promoter and this activation was intensified by ethylene treatment. ACC represents the materials treated with ethylene. A, D, and E) Data are expressed as means ± SD of 3 biological replicates. Asterisks indicate statistically significant differences by Student's t-test (**P < 0.01; n.s., no significant difference).

Figure 3.

CsRP1 is essential for ethylene-induced CsCCD4b expression and peel reddening. A to D) Stable transformation of CsRP1 in citrus calli. A) Phenotypes. NH and RM calli were derived from “Newhall” navel orange (C. sinensis L. Osbeck., “NH”) and “Marsh” grapefruit (C. paradise Macf., “RM”). The G allele of the G-to-A SNP was detected in the CsCCD4b promoter in genomic DNA NH calli, whereas the CCD4b promoter from RM genomic DNA harbored the A allele. WT, wild type. OE-CsRP1 and RNAi-CsRP1 indicate overexpressing and interfering CsRP1 in citrus calli, respectively. B) Expression levels of CsRP1. C) Total carotenoid content. DW, dry weight. D) Expression levels of CsCCD4b. E to H) Transient expression of CsRP1 in “Newhall” navel orange fruit. E) Phenotypes. PK7-Ev and RNAi were used as control, respectively. PK7-CsRP1 and RNAi-CsRP1 indicate overexpressing and interfering CsRP1, respectively. Ethylene treatment was performed at 3 d after infiltration. Bars = 2 cm. F) Total β-citraurin content. DW, dry weight. Expression levels of CsCCD4b (G) and CsRP1 (H). B to D, F to H) Data are expressed as means ± SD of 3 biological replicates. Asterisks indicate statistically significant differences by Student's t-test (*P < 0.05; **P < 0.01; n.s., no significant difference).

Figure 4.

Ethylene-induced CsRP1 directly activates the expression of β-citraurin biosynthesis genes. A) Y1H assay identified interactions between CsRP1 and target gene promoters. Empty PGADT7 + pAbAi-ProCBG and PGADT7-Rec-p53 + p53-AbAi were used as the N. Control and P. Control, respectively. CBG, β-citraurin biosynthesis genes. AbA was used as a yeast cell growth inhibitor. SD/-Leu/AbA¹⁵⁰ indicates SD/-Leu medium supplemented with 150 ng ml⁻¹ AbA. B to D) ChIP-PCR assay showed the interaction between CsRP1 and promoter regions of CsPSY, CsHYD, and CsNCED2, and this interaction was intensified by ethylene. The gray lines represent putative MBSI motifs in these promoters. Cross-linked chromatin was precipitated by GFP antibody, and the resulting DNA fragment was amplified by qPCR. E) Schematic diagram of reporter and effector constructs used in dual-LUC assay. CBG, β-citraurin biosynthesis genes. PK7-Ev indicates PK7 empty vector (control). F) Dual-LUC assay showed that CsRP1 activated the promoters and this activation was enhanced by ethylene treatment. ACC represents the materials treated by ethylene. B, C, D, and F) Data are expressed as means ± SD of 3 biological replicates. Asterisks indicate statistically significant differences by Student's t-test (*P < 0.05; **P < 0.01; n.s., no significant difference).

Figure 5.

Ethylene-activated CsERF25 directly activates CsRP1 expression. A) Expression of CsERF25 under various treatments. 1-MCP, 1-methylcyclopropene. B) Y1H assay identified interactions between CsERF25 and CsRP1 promoter. Empty PGADT7 + pAbAi-ProCsRP1 and PGADT7-Rec-p53 + p53-AbAi were used as the N. Control and P. Control, respectively. SD/-Leu/AbA²⁰⁰ indicates SD/-Leu medium supplemented with 200 ng ml⁻¹ AbA which was used as a yeast growth inhibitor. C) EMSA identified interactions between CsERF25 and biotin-labeled CsRP1 promoter fragment (hot probe) containing the DRE elements. Labeled letters represent the bind motifs and their corresponding mutant motifs. The gray line indicates the position of the SNP mutation. “+” and “−” indicate the presence and absence of the indicated probe or protein, respectively. Increased amounts (100-folds) of the unlabeled DNA fragments (cold probe) were added as competitors. Purified MBP-tagged CsERF25 protein was used in EMSAs, and purified MBP protein was used as a negative control. Red arrows point to the positions of protein–DNA complexes or free probes. D) ChIP-PCR assay showed the interaction between CsERF25 and CsRP1 promoter regions and this interaction was intensified by ethylene. These gray lines represent putative DRE elements. Cross-linked chromatin was precipitated by GFP antibody, and the resulting DNA fragment was amplified by qPCR. E) Schematic diagram of reporter and effector constructs used in dual-LUC assay. PK7-Ev indicates PK7 empty vector as (control). Dual-LUC assay showed that CsERF25 activated the CsRP1 promoter and this activation was intensified by ethylene. ACC represents the materials treated by ethylene. A, D, and E) Data are expressed as means ± SD of 3 biological replicates. Asterisks indicate statistically significant differences by Student's t-test (*P < 0.05; **P < 0.01; n.s., no significant difference).

Figure 6.

CsERF25 is essential for ethylene-induced peel reddening and CsCCD4b and CsRP1 expression. A to E) Stable transformation of CsERF25 in citrus calli. A) Phenotypes. NH and RM calli were derived from “Newhall” navel orange (C. sinensis L. Osbeck., “NH”) and “Marsh” grapefruit (C. paradise Macf., “RM”). The G allele of the G-to-A SNP was detected in the CsCCD4b promoter in genomic DNA NH calli, whereas the CCD4b promoter from RM genomic DNA harbored the A allele. WT, wild type. OE-CsERF25 and RNAi-CsERF25 indicate overexpressing and interfering CsERF25 in citrus calli, respectively. The expression levels of CsERF25 (B), CsRP1 (D), and CsCCD4b (E). C) Total carotenoid content. DW, dry weight. F to J) Transient expression of CsERF25 in “Newhall” navel orange fruit. F) Phenotypes. PK7-Ev and RNAi were used as control, respectively. PK7-CsERF25 and RNAi-CsERF25 indicate CsERF25 overexpressing and interfering in citrus fruit, respectively. Ethylene treatment was performed at 3 d after infiltration. Bars = 2 cm. The expression levels of CsERF25 (G), CsRP1 (I), and CsCCD4b (J). H) The β-citraurin content. DW, dry weight. B to E, G to J) Data are expressed as means ± SD of 3 biological replicates. Asterisks indicate statistically significant differences by Student's t-test (*P < 0.05; **P < 0.01; n.s., no significant difference).

Figure 7.

Ethylene-promoted CsERF25 directly activates the expression of β-citraurin biosynthesis genes. A) Y1H assay identified interactions between CsERF25 and target gene promoters. Empty PGADT7 + pAbAi-ProCBG and PGADT7-Rec-p53 + p53-AbAi were used as the N. Control and P. Control, respectively. CBG, β-citraurin biosynthesis genes. AbA was used as a yeast cell growth inhibitor. SD/-Leu/AbA^x indicates SD/-Leu medium supplemented with AbA at various concentrations (200 ng ml⁻¹ as the basal concentration of ProCsPDS and ProCsLCYB). SD/-Leu/AbA^x denotes SD/-Leu medium supplemented with AbA at various concentrations (150 ng ml⁻¹ as the basal concentration of ProCsHYD, ProCsZEP, and ProCsNCED2). B to G) ChIP-PCR assay showed the interaction between CsERF25 and the promoter regions of CsPSY, CsPDS, CsLCYB, CsHYD, CsZEP, and CsNCED2, respectively, and this interaction was enhanced by ethylene. The gray lines represent putative ERF TF binding motifs on the promoters. The sequences of potential binding motifs for ERF25 on target gene promoters are shown below: ProCsPSY (P2: 5′-AAGTTTGAATTTAT-3′), ProCsPDS (P2: 5′-TATATTTCAAACTT-3′; P3: 5′-AGCGTCGGCTT-3′), ProCsLCYB (P2: 5′-GCAGCCGCCAAT-3′), ProCsHYD (P1: 5′-GCTGTCGGTTT—TTCGGCGGCCGA-3′; P3: 5′-CACAATTCAAAAAA-3′), ProZEP (P1: 5′-CTACCGACCCA—ATTGTCGGACG-3′; P3: 5′-GAAGCCGTCGGAGA-3′), and ProNCED2 (P1: 5′-AAGGTCGGTGC-3′). Cross-linked chromatin was precipitated by GFP antibody and the resulting DNA fragment was amplified by qPCR. H) Schematic diagram of reporter and effector constructs used in dual-LUC assay. CBG, β-citraurin biosynthesis genes. PK7-Ev indicates PK7 empty vector (control). I) Dual-LUC assay showed that CsERF25 activated these promoters and this activation was intensified by ethylene. ACC represents the materials treated with ethylene. B to G, I) Data are expressed as means ± SD of 3 biological replicates. Asterisks indicate statistically significant differences by Student's t-test (*P < 0.05; **P < 0.01. n.s., no significant difference).

Figure 8.

CsERF25 regulates ethylene-induced peel reddening in CsRP1-dependent manner. A to C) Interference of CsERF25 and CsRP1 in the corresponding overexpressing “Newhall” navel orange fruit. A) Phenotypes. Bars = 2 cm. B) Expression levels of CsRP1, CsERF25, and CsCCD4b. C) β-citraurin content. DW, dry weight. PK7-Ev and RNAi were used as control, respectively. PK7-CsERF25/CsRP1 and RNAi-CsERF25/CsRP1 indicate overexpressing and interfering CsERF25/CsRP1 in citrus fruit, respectively. PK7-CsERF25 + RNAi-CsRP1 and PK7-CsRP1 + RNAi-CsERF25 indicate interfering CsRP1 in CsERF25-overexpressing fruit and interfering CsERF25 in CsRP1-overexpressing fruit, respectively. D to F) Interference of CsERF25 and CsRP1 in the corresponding overexpressing NH calli. D) Phenotypes. E) Expression levels of CsRP1, CsERF25, and CsCCD4b. F) Total carotenoid content. DW, dry weight. WT, wild type. OE and RNAi indicate overexpressing and interfering in citrus calli, respectively. OE-CsERF25 + RNAi-CsRP1 and OE-CsRP1 + RNAi-CsERF25 indicate interfering CsRP1 in CsERF25-overexpressing fruit and interfering CsERF25 in CsRP1-overexpressing fruit, respectively. B, C, E, and F) Data are expressed as means ± SD of 3 biological replicates. Asterisks indicate statistically significant differences by Student's t-test (*P < 0.05; **P < 0.01; n.s., no significant difference).

Figure 9.

Model in which CsERF25–CsRP1–CsCCD4b transcriptional regulatory cascade regulates ethylene-induced fruit coloration and positive feedback regulatory loop between ethylene and ABA in the coloration and carotenoid metabolism of “Newhall” navel orange fruit. Ethylene-induced CsERF25 activates CsRP1 expression through sequence-specific interactions between CsERF25 and DRE elements on the CsRP1 promoter. CsRP1 enhances CsCCD4b expression by specifically binding the MBSI motif on the CsCCD4b promoter, and then, more CsCCD4b proteins catalyze the biosynthesis of β-citraurin, thus promoting the peel reddening of “Newhall” navel orange fruit. Moreover, CsERF25 and CsRP1 also directly bind the promoters of other β-citraurin biosynthetic genes (including CsPSY, CsPDS, CsLCYB, CsHYD, CsZEP, and CsNCED2) and induce their expressions to provide substantial precursors for β-citraurin biosynthesis. In addition, ethylene significantly promotes ABA biosynthesis, meanwhile promoting fruit coloration and carotenoid accumulation. In return, ABA also induces ethylene production, fruit coloration, and carotenoid metabolism in a feedback regulation manner. “+” represents promotion. Solid lines represent direct regulation. Dashed lines represent multistep syntheses. GGPP, geranylgeranyl diphosphate; CsPSY, phytoene synthase; CsPDS, phytoene desaturase; CsLCYB, lycopene β-cyclase; CsHYD, β-carotene hydroxylase; CsZEP, zeaxanthin epoxidase; CsNCED2, 9-cis-epoxycarotenoid dioxygenase 2; CsCCD4b, carotenoid cleavage dioxygenase 4. CsACS1/9, ACC synthase 1/9; CsACO1/4, ACC oxidase 1/4.