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. 2024 Jun 23;119(3):1433–1448. doi: 10.1111/tpj.16866

The R2R3 MYB Ruby1 is activated by two cold responsive ethylene response factors, via the retrotransposon in its promoter, to positively regulate anthocyanin biosynthesis in citrus

Yuxin Wang 1,#, Shaojia Li 1,2,3,#, Yanna Shi 1,2,3,#, Shouzheng Lv 1, Changqing Zhu 1,2,3, Changjie Xu 1,2,3, Bo Zhang 1,2,3, Andrew C Allan 4,5, Donald Grierson 6, Kunsong Chen 1,2,3,✉
PMCID: PMC13087489  PMID: 38922743

SUMMARY

Anthocyanins are natural pigments and dietary antioxidants that play multiple biological roles in plants and are important in animal and human nutrition. Low temperature (LT) promotes anthocyanin biosynthesis in many species including blood orange. A retrotransposon in the promoter of Ruby1, which encodes an R2R3 MYB transcription factor, controls cold‐induced anthocyanin accumulation in blood orange flesh. However, the specific mechanism remains unclear. In this study, we characterized two LT‐induced ETHYLENE RESPONSE FACTORS (CsERF054 and CsERF061). Both CsERF054 and CsERF061 can activate the expression of CsRuby1 by directly binding to a DRE/CRT cis‐element within the retrotransposon in the promoter of CsRuby1, thereby positively regulating anthocyanin biosynthesis. Further investigation indicated that CsERF061 also forms a protein complex with CsRuby1 to co‐activate the expression of anthocyanin biosynthetic genes, providing a dual mechanism for the upregulation of the anthocyanin pathway. These results provide insights into how LT mediates anthocyanin biosynthesis and increase the understanding of the regulatory network of anthocyanin biosynthesis in blood orange.

Keywords: blood orange, anthocyanin accumulation, low temperature, ethylene response factors

Significance Statement

Anthocyanins are natural pigments and dietary antioxidants that play multiple biological roles in plants and are important in human nutrition. Low temperature is a necessary condition for anthocyanin accumulation in blood orange and the retrotransposon promoter of Ruby1 controls this process. Our results provide upstream insights into its cold response and enrich the understanding of the regulatory network of anthocyanin biosynthesis in blood orange. These results are of fundamental importance and great practical significance.

INTRODUCTION

Anthocyanins are important secondary metabolites in higher plants that belong to a branch of the flavonoids family of compounds (Landi et al., 2015; Naing & Kim, 2021; Santos‐Buelga et al., 2014). They serve as water‐soluble pigments to facilitate pollination and seed dispersal and also function as antioxidants to protect plants from biotic and abiotic stresses (Landi et al., 2015; Naing & Kim, 2021; Santos‐Buelga et al., 2014). The accumulation of anthocyanins in edible organs, especially fruits, enhances their esthetic value and provides versatile health properties such as obesity reduction, diabetes reversal, anti‐inflammatory activity, and disease intervention, which may be associated with their effects on gut microbiota (Alipour et al., 2016; de Pascual‐Teresa et al., 2010; Khan et al., 2020; Middleton et al., 2000). Blood orange is a special variety of citrus that accumulates significant amounts of anthocyanins in the flesh and has attracted keen attention from consumers. There are at least two types of blood orange originating from Europe and China, each with a transposon insertion that confers cold induction of the transcriptional activator Ruby1 (Butelli et al., 2012). The consumption of raw fruits as well as processed products such as juice and supplements made from blood orange shows huge potential (Bonina et al., 2002; Fabroni et al., 2016; Riso et al., 2005). However, there is a requirement for cold to stimulate anthocyanin accumulation in blood orange, and this is usually variable and insufficient due to the environmental conditions, especially in warm regions (Butelli et al., 2012; Carmona et al., 2017). Elucidating the underlying regulatory mechanisms of temperature‐mediated anthocyanin accumulation in blood orange is not only of fundamental importance for understanding plant responses to the environment but also has great practical significance.

The biosynthesis pathway of anthocyanin involves a series of enzymes encoded by chalcone synthase (CHS), chalcone isomerase (CHI), flavanone‐3‐hydroxylase (F3H), flavonoid 3‐hydroxylase (F3′H)/flavonoid 3′,5′‐hydroxylase (F3′5′H), dihydroflavonol 4‐reductase (DFR), anthocyanidin synthase (ANS), and UDP‐glucose: flavonoid 3 glucosyltransferase (UFGT) (Berardi et al., 2021; Koes et al., 2005; Tohge et al., 2015; Winkel‐Shirley, 2001). The transportation of anthocyanin is dependent on the transporters such as glutathione S‐transferase (GST) and MATE‐transporters, which are necessary for the accumulation of anthocyanin in vacuole (Marinova et al., 2007; Xue et al., 2022). The transcription initiation of these genes is primarily governed by a conserved MYB‐bHLH‐WD40 ternary complex (Hichri et al., 2011; Jaakola, 2013; Xu et al., 2015), in which the R2R3 MYB transcription factor (TF) acts as a core component by specifying DNA binding site in the target gene promoters (Allan et al., 2008; Heppel et al., 2013; Karppinen et al., 2021). The key anthocyanin‐activating MYB has been characterized in various fruit species such as tomato, apple, pear, kiwifruit, and strawberry (Hu et al., 2016; Jiang et al., 2023; Luo et al., 2022; Menconi et al., 2023; Wang, Li, et al., 2022; Wang, Moss, et al., 2022; Wang, Wang, et al., 2022; Yao et al., 2017). In citrus, through comparison of multiple germplasm resources that have distinct color characteristics, the MYB gene Ruby1, which exhibits diversity in terms of gene structure and promoter constitution, has been demonstrated to be crucial for anthocyanin biosynthesis (Butelli et al., 2012, 2017; Huang et al., 2018). Analysis of the complete sequence of Ruby1 revealed that an insertion of a transposable element in its promoter region upregulates transcriptional activity leading to the accumulation of functional Ruby1 protein and the subsequent biosynthesis of anthocyanins in blood orange (Butelli et al., 2012, 2017; Huang et al., 2018).

In a diverse range of plants, the transcript abundance of the activating MYB determines the extent of anthocyanin accumulation. Recently, researchers have focused on identifying the upstream regulators of these key MYBs. In pear, PpBBX16 and PpWRKY44 were characterized as upstream activators of PpMYB10 by overexpression or silencing experiments, leading to either anthocyanin accumulation or inhibition (Alabd et al., 2022; Bai, Tao, Tang, et al., 2019; Bai, Tao, Yin, et al., 2019). In strawberry, the up‐regulation of the ERF TF FaRAV1 accompanies anthocyanin accumulation during the ripening stage, and further investigation revealed that FaRAV1 positively regulates anthocyanin biosynthesis via activation of FaMYB10 (Zhang et al., 2020). In peach, the key gene for the blood‐flesh trait is PpBL (encoding a NAC TF), which activates PpMYB10.1 in maturing fruit (Zhou et al., 2015). Moreover, various other TFs have also been reported to participate in the regulation of anthocyanin biosynthesis by interacting separately with MYB (An et al., 2018, 2020, 2021). For example, GLK1 was reported to affect the transcriptional activation activities of MYB75, MYB90, and MYB73 through protein–protein interactions, thereby mediating the spatial distribution pattern of anthocyanins in Arabidopsis (Li et al., 2023).

A range of environmental factors such as light, temperature, and nutrients can effectively trigger or impede the accumulation of anthocyanins. Relevant studies have been carried out and indicate that there is a sophisticated and hierarchical regulatory network of anthocyanin biosynthesis in tandem with environmental factors. For example, in apple, pear, and sweet cherry, the accumulation of anthocyanin is normally limited to the peel, with a tendency to produce more anthocyanin on the side of the fruit exposed to the sun, indicating a strict dependency on light (Ma et al., 2021; Tao et al., 2020; Wang et al., 2023; Wang, Li, et al., 2022; Wang, Moss, et al., 2022; Wang, Wang, et al., 2022). HY5 and BBX‐involved regulatory pathways have been reported to play an important part in light‐mediated anthocyanin biosynthesis in a range of species (Alabd et al., 2022; Bai, Tao, Tang, et al., 2019; Bai, Tao, Yin, et al., 2019; Fang et al., 2019). In blood orange, by contrast, temperature is the vital factor that influences anthocyanin accumulation and prevailing cold conditions during ripening is required for intense color formation (Butelli et al., 2012). Postharvest cold temperatures also enhance pigmentation in blood orange, providing a practical way to ensure stable pigmentation (Butelli et al., 2012; Carmona et al., 2017). Unlike light‐mediated anthocyanin biosynthesis, the mechanism of temperature‐mediated anthocyanin biosynthesis still lacks clarity. In peach, DNA demethylation of anthocyanin biosynthetic genes as well as PpbHLH3 appears to be key to postharvest temperature‐dependent anthocyanin accumulation (Zhu et al., 2020). In apple, MdbHLH3 promotes anthocyanin accumulation in response to low temperature (LT) (Xie et al., 2012). In citrus, previous studies have revealed that a transposable element inserted into the promoter of CsRuby1 is the key to low‐temperature‐induced anthocyanin biosynthesis in blood orange (Butelli et al., 2012; Huang et al., 2019). However, the upstream and cooperative regulators of CsRuby1 remain to be uncovered.

The APETALA2/ethylene (AP2/ERF) TFs belong to a plant‐specific gene family that is divided into five categories: AP2, ERF, DREB, RAV, and Soloist (Licausi et al., 2013; Mizoi et al., 2012). Members of this family participate in the regulation of multiple biological processes by binding to the promoter cis‐elements such as the GCC‐box and dehydration responses element/C‐repeat element (DRE/CRT) in the promoters of target genes (Sakuma et al., 2002; Shoji et al., 2013). In addition to ripening, the response to cold signals is considered as one of the most important roles that ERFs play and has been extensively reported in both model plants and fruit crops. For example, DREB/CBF TFs regulate responsiveness to LT signals in plants. It is recognized that the ICE (Inducers of CBF expression)–CBF (C‐repeat binding factor)–COR (cold‐responsive genes) regulatory pathway plays a central role in the regulation of the cold stress response (Liu et al., 2019; Song et al., 2021). Two cold‐responsive TFs PtrERF9 and PtrERF108 were identified in trifoliate orange, which contribute to cold tolerance (Khan et al., 2021; Zhang et al., 2022). In addition, it has been shown that ERFs participate in the regulation of anthocyanin biosynthesis, mainly in pear (ERF3, ERF24, ERF96, ERF4.1/4.2, and ERF9) (Jiang et al., 2023; Ni et al., 2019, 2023; Sun et al., 2023) and in apple (MdERF1B and MdERF38) (An et al., 2020; Zhang, Xu, et al., 2018; Zhang, Yin, et al., 2018). Most recently, PsERF1B was found to enhance anthocyanin biosynthesis during plum flesh‐reddening in response to cold storage (Chang et al., 2023). Taken together, it is reasonable to suggest that ERFs play a role in low‐temperature‐induced anthocyanin biosynthesis in citrus.

In this study, a DRE/CRT cis‐element was found in the promoter of CsRuby1. CsERF054 and CsERF061 were then identified as important regulators of anthocyanin biosynthesis in response to LT in blood orange, which significantly activated the expression of CsRuby1 via directly binding to the DRE/CRT cis‐element in the retrotransposon within the promoter. In addition, CsERF061 can also strongly regulate the expression of anthocyanin biosynthetic genes via protein–protein interaction with CsRuby1. These findings enrich the regulatory mechanism of anthocyanin biosynthesis in citrus and build an additional link between ERFs and low‐temperature‐induced anthocyanin biosynthesis.

RESULTS

10°C promotes anthocyanin accumulation, whereas 20°C inhibits anthocyanin accumulation during postharvest in blood orange

Anthocyanin accumulation in blood orange flesh, consisting mainly of cyanidin 3‐glucoside (C3G) and cyanidin 3‐(6′′‐malonyl) glucoside (C3G6M) (Huang et al., 2019), is dependent on LTs (Butelli et al., 2012; Huang et al., 2019). To better evaluate how environmental factors, mainly including light and temperature, influence anthocyanin accumulation in blood orange and whether it is effective to use temperature treatment postharvest, we separated blood orange fruits into five groups and treated each group with different light and temperature conditions. In the preharvest groups, both naturally illuminated and shaded fruits showed continuous pigmentation and no visible differences were observed between the two groups (Figure S1), indicating that light is not a factor for anthocyanin accumulation in blood orange flesh. In the postharvest groups, 5 and 10°C differentially promoted anthocyanin accumulation: Fruits stored at 5°C showed similar pigmentation patterns as the preharvest groups and fruits stored at 10°C accumulated dramatically more anthocyanins (Figure 1a; Figure S1). This contrasted, however, with fruits stored at 20°C, which lacked anthocyanins (Figure 1a), indicating that LT is a necessary inducer of anthocyanin accumulation and 10°C is likely to be a good temperature for enhancing fruit quality. We measured the total anthocyanin contents of each group, and the results were consistent with the results of visible inspection (Figure 1b; Figure S2). We further tested the contents of the predominant anthocyanin components, cyanidin 3‐glucoside and cyanidin 3‐(6′′‐malonyl) glucoside using high‐performance liquid chromatography (HPLC) analysis, and the results showed that they both accumulated in line with the total anthocyanin contents (Figure S3). 10 and 20°C were then chosen to investigate the mechanism by which LT promotes anthocyanin accumulation in blood orange.

Figure 1.

Figure 1

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The effect of different temperatures on anthocyanin accumulation and relative expression of related genes during postharvest storage of blood orange.

(a) Phenotypes of blood orange flesh during postharvest storage at different temperatures.

(b) Anthocyanin contents in blood orange flesh during postharvest storage.

(c) Relative expressions of anthocyanin biosynthetic genes CsCHS1, CsCHS2, CsCHI, CsF3H, CsDFR, CsANS, CsDFR and CsUFGT; anthocyanin transportation gene: CsGST. The results in (b, c) are from three biological replicates and presented as means ± SD. Statistically significant differences were assessed using Student's t‐test (***P < 0.001; ns, no significance).

The results of reverse transcription quantitative PCR (RT‐qPCR) showed that the transcript levels of anthocyanin‐related structural genes (CsCHS1, CsCHS2, CsCHI, CsF3H, CsDFR, CsANS, CsUFGT, and CsGST) were all strongly induced after 10 days at 10°C and most continued to increase further thereafter (Figure 1c), indicating that higher anthocyanin content is a result of the overall up‐regulation of genes related to anthocyanin biosynthesis and transportation. Furthermore, the expression levels of CsRuby1 (a key MYB TF gene required for anthocyanin biosynthesis in citrus, and a homolog of MYB AtPAP from Arabidopsis) (Butelli et al., 2012) and CsNoemi (an identified bHLH TF gene required for anthocyanin biosynthesis in citrus) (Butelli et al., 2019) also increased in line with the expression of anthocyanin biosynthetic genes (Figure 2a), indicating that LT might be involved in the regulation of anthocyanin biosynthesis via the upregulation of CsRuby1 and CsNoemi expressions.

Figure 2.

Figure 2

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Relative expression of CsRuby1, CsNoemi, CsERF054, CsERF061 and CsRAP2.1 and the transcriptional activity of the CsRuby1 promoter.

(a) Relative expressions of two regulators required for anthocyanin biosynthesis in citrus: CsRuby1 (an R2R3 MYB transcription factor) and CsNoemi (a bHLH transcription factor).

(b) The transcriptional activity of the CsRuby1 promoter at 20 and 10°C and the transcriptional activity of the CsRuby1 promoter with mutant DRE/CRT cis‐element at 10°C.

(c) Relative expression levels of CsERF054, CsERF061 and CsRAP2.1. The results in (a–c) are from three biological replicates and presented as means ± SD. Statistically significant differences were assessed using Student's t‐test (*P < 0.05, **P < 0.01, ***P < 0.001).

Screening of potential cis‐elements responsible for the cold‐response of CsRuby1

To better understand how CsRuby1 responds to the cold signal, we screened for potential cis‐elements in the CsRuby1 promoter and detected a dehydration response element/C‐repeat element (DRE/CRT) (i.e., the binding sites of DREB TFs) (Figure S4a), suggesting that LT might regulate CsRuby1 expression through DREB TFs. A previous study also speculated that this element is a crucial switch of the LT responsiveness of the retrotransposon‐containing promoter of CsRuby1 through comparing it with the CsRuby1 promoter without retrotransposon insertion in purple pummelo (Huang et al., 2018). To examine whether the transcriptional activity of CsRuby1 promoter was induced by LT through the DRE/CRT cis‐element (CCGAC), a LUC reporter gene was fused downstream from the original promoter and a mutant promoter with CCGAC altered to AAAAA, respectively (Figure S4b). A REN reporter gene driven by the 35S‐promoter was used as an internal reference. The resulting construct was transiently transformed into tobacco leaves and treated with neither 20 or 10°C. The ratio of LUC and REN was used to measure the promoter activity. The activity of CsRuby1 promoter was significantly higher at 10°C and decreased when mutated (Figure 2b), indicating that CsRuby1 is transcriptionally up‐regulated by activators present at higher concentrations at 10°C compared with 20°C and that the CCGAC sequence plays an indispensable role in this process.

Identification of candidate regulators of CsRuby1

RNA‐seq analysis was then performed on fruits stored at 10 and 20°C at three different time points (0, 10, and 30 days), and the expression pattern of 32 DREBs was analyzed (Figure S4b). Of these, Cs_ont_2g021580.1, Cs_ont_5g049390.1 and Cs_ont_3g005380.2 were found to be induced by LT. The expression patterns of these DREBs are similar to the accumulation of CsRuby1 transcripts (Figure S4b), which was confirmed using RT‐qPCR (Figure 2c). Additionally, the expression pattern of these three ERFs were tested using fruit still developing on‐tree. Results showed that these three ERFs are not induced during natural ripening, suggesting they are not involved in developmentally‐induced anthocyanin biosynthesis (Figure S5). A phylogenetic tree was constructed using the amino acid sequence of the three screened ERFs, all ERFs from Arabidopsis thaliana, anthocyanin‐related ERFs from other plants and two cold‐responsive ERFs identified in trifoliate orange, which showed that Cs_ont_2g021580.1, Cs_ont_5g049390.1 and Cs_ont_3g005380.2 are most closely related to AtERF054, AtERF061, and AtRAP2.1 and show little homology with previously reported anthocyanin‐related ERF genes (Figure S6). These were renamed as CsERF054, CsERF061, and CsRAP2.1. Notably, CsERF061 has been identified previously as a positive regulator of carotenoid biosynthesis (Zhu et al., 2021). Amino acid sequence alignment showed the presence of the AP2 domain in all these proteins, and an intact EAR motif (DLNxxP) in the C‐terminal region of CsRAP2.1, indicating that it is likely to be a transcriptional repressor (Figure S7). The results of the dual‐luciferase assay showed that CsRAP2.1 represses the activity of the CsRuby1 promoter (Figure S8a), while a yeast one‐hybrid assay indicated that there is no direct interaction between them (Figure S8b). CsRAP2.1 may regulate CsRuby1 indirectly and play a negative role in balancing LT‐induced anthocyanin biosynthesis. As candidates induce of anthocyanin biosynthesis during LT, CsERF054 and CsERF061 were selected for further study.

CsERF054 and CsERF061 directly bind to the CsRuby1 promoter via the DRE/CRT cis‐element

Yeast one‐hybrid assays were carried out to confirm the interaction between CsERF054, CsERF061, and the full‐length CsRuby1 promoter (Figure 3a). The results showed that the transformed yeast cells containing pGADT7‐CsERF054/CsERF061 and the CsRuby1 promoter could grow on SD/−Leu medium with 125 ng ml−1 aureobasidin A (AbA), whereas yeast cells containing empty pGADT7 and the CsRuby1 promoter could not, indicating that CsERF054 and CsERF061 directly bind to the CsRuby1 promoter in the yeast one‐hybrid system. The specificity of CsERF054 and CsERF061 binding to the CsRuby1 promoter was confirmed by electrophoretic mobility shift assay (EMSA) (Figure 3b). A biotin‐labeled probe was designed from the region −750 to −714 bp containing the sequence CCGAC and a mutant probe was designed with CCGAC mutated to AAAAA. Recombinant CsERF054/CsERF061 protein with a His‐tag at the N terminus was able to bind the biotinylated probe but did not bind the mutant probe. The intensities of the shifted bands were reduced by the addition of increasing concentrations of cold probe, indicating that CsERF054 and CsERF061 could recognize and directly bind to the promoter of CsRuby1 via the DRE/CRT cis‐element, which is inside the transposable element in blood orange but not in other oranges.

Figure 3.

Figure 3

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CsERF054 and CsERF061 interact with the promoter of CsRuby1 through DRE/CRT cis‐element.

(a) Yeast‐one‐hybrid analysis. Autoactivation of the CsRuby1 promoter was tested on SD/−Ura medium with 125 ng ml−1 aureobasidin A (AbA). The interaction between CsERF054, CsERF061 and the CsRuby1 promoter was analyzed on SD/−Leu medium in the presence of 125 ng/mL AbA. The empty pGADT7 vector was used as a negative control.

(b) Electrophoretic mobility shift assay confirmed that CsERF054 and CsERF061 bind directly to the DRE/CRT cis‐element in the CsRuby1 promoter. The wildtype probe was a biotin‐labeled fragment of the CsRuby1 promoter containing the DRE/CRT cis‐element, whereas the competitor probe was an unlabelled probe. The mutant cold probe was the same as the labeled wild type probe but with ACCGAC mutated to TTTTTT. The purified His‐tagged CsERF054 and CsERF061 proteins were used.

CsERF054 and CsERF061 positively regulate anthocyanin biosynthesis via activation of CsRuby1 gene

The ability of CsERF054 and CsERF061 to trans‐activate the CsRuby1 promoter was investigated using a dual‐luciferase assay. The results showed that both CsERF054 and CsERF061 significantly trans‐activated the promoter of CsRuby1, approximately 14.7‐ and 6.8‐fold, respectively (Figure 4a). When transiently expressed as a 35S:CsERF054/CsERF061‐GFP fusion in tobacco leaves, GFP and mCherry fluorescent signals were both located in nuclei (Figure 4b), indicating that CsERF054 and CsERF061 are nuclear localized activators of CsRuby1.

Figure 4.

Figure 4

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CsERF054 and CsERF061 are positive regulators of anthocyanin biosynthesis functioning by activation of CsRuby1 gene.

(a) Dual‐luciferase assays were used to measure regulatory effect of the CsERF054 and CsERF061 on the CsRuby1 promoter. The LUC/REN ratio of the empty vector (SK) plus promoter was used as calibrator (set at a value of 1).

(b) Subcellular localization of CsERF054 and CsERF061 proteins shows they were located in the nucleus.

(c, e) Relative expression levels of CsERF054/CsERF061, CsRuby1 and CsNoemi in tobacco leaves.

(d, f) Relative expression levels of anthocyanin biosynthetic genes in tobacco leaves. The results in (a, c–f) are from three biological replicates and are presented as means ± SD. Statistically significant differences were assessed using Student's t‐test (***P < 0.001; ns, no significance).

There is currently no available transgenic system to verify gene function in blood orange. Attempts were made to generate anthocyanin‐accumulated callus, using expression of CsRuby1 and CsNoemi, but growth was inhibited (data not shown). N. benthamiana transient gene expression/color assays were performed to test evidence for CsERF054/CsERF061‐mediated regulation of genes encoding anthocyanin biosynthesis enzymes. ProCsRuby1:CsRuby1‐35S:CsNoemi (the promoter of CsRuby1 driving CsRuby1 and the 35SCaMV promoter driving CsNoemi) were transformed into tobacco leaves together with either CsERF054/CsERF061 or empty vector (Figure S9a). After 7 days of transformation, detectable anthocyanins were accumulated (Figure S9b). When CsERF054/CsERF061 was co‐expressed with ProCsRuby1:CsRuby1‐35S:CsNoemi in tobacco leaves, significantly higher anthocyanin accumulation was observed (Figure S9b), and the expression of CsRuby1 was strongly up‐regulated (Figure 4c,e), indicating that the overexpression of both CsERF054 and CsERF061 activated the expression of CsRuby1. Correspondingly, seven important anthocyanin biosynthetic genes in tobacco were all up‐regulated (Figure 4d,f). These results confirmed that CsERF054 and CsERF061 are positive regulators of anthocyanin biosynthesis, functioning by activating the expression of CsRuby1.

CsERF061 interacts with CsRuby1 to transactivate the promoters of anthocyanin biosynthetic genes

Dual‐luciferase assays were used to further explore the transactivation effects of CsERF054/CsERF061 on the promoters of anthocyanin biosynthetic genes, including CsCHS1, CsCHS2, CsCHI, CsF3H, CsDFR, CsANS, and CsUFGT. However, it appeared that neither CsERF054 nor CsERF061 alone could activate the expression of these genes (Figure 5a; Figure S10).

Figure 5.

Figure 5

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Interactions between CsERF061 and CsRuby1 synergistically activates the promoters of seven anthocyanin biosynthetic genes.

(a) Effect of the CsERF061 and CsRuby1 individually and in combination on the activation of the promoter of anthocyanin biosynthetic genes. The LUC/REN ratio of the empty vector (SK) plus promoter was used as calibrator (set at a value of 1). The results are from three biological replicates and presented as means ± SD. Statistically significant differences were assessed using one‐way anova testing. Different letters indicate significant differences at P < 0.05. Red coloured star indicates there is combinatorial effect between CsERF061 and CsRuby1.

(b, c) Physical interaction between CsERF061 and CsRuby1 determined by bimolecular fluorescence complementation assay and luciferase complementation imaging assay. The pairs of fusion proteins tested were CsERF061‐YFPN/C + CsRuby1‐YFPC/N and CsERF061‐nLUC/cLUC + CsRuby1‐cLUC/nLUC. The other combinations were negative controls.

Several studies have shown that TFs act to regulate anthocyanin biosynthetic genes via an interaction with an activating MYB (Alabd et al., 2022; Bai, Tao, Tang, et al., 2019; Bai, Tao, Yin, et al., 2019; Zhang et al., 2020; Zhou et al., 2015). Therefore, the combinatorial effects of CsERF054/CsERF061 and CsRuby1 were investigated. The dual‐luciferase results showed that CsERF061 significantly enhanced the degree of activation by CsRuby1 on the promoters of CsCHS1 (19.6‐ to 94.9‐fold), CsCHI (0.8‐ to 2.1‐fold), CsF3H (7.2‐ to 17.5‐fold), and CsDFR (94.5‐ to 214.0‐fold) (Figure 5a). However, CsERF054 showed no such combinatorial effects with CsRuby1 (Figure S10). These results suggest that a fully functional CsERF061 requires the participation of CsRuby1 and that there might be physical interactions between them. Bimolecular fluorescence complementation (BiFC) assays were conducted to validate the potential interactions between CsERF061 and CsRuby1 (Figure 5b). Co‐expression of CsERF061‐YFPC/YFPN and CsRuby1‐YFPN/YFPC in tobacco leaves produced obvious YFP fluorescent signals, while other negative control combinations showed no YFP signals, indicating the existence of a protein–protein interaction between CsERF061 and CsRuby1. Luciferase complementation imaging (LCI) assays were conducted and the results supported the conclusion that there is a specific interaction between CsERF061 and CsRuby1 (Figure 5c).

CsERF061 promotes CsRuby1‐mediated anthocyanin biosynthesis

In order to explore the function of CsERF061 in regulating anthocyanin biosynthesis, transient overexpression of CsERF061, CsRuby1, and CsNoemi individually or in combination was performed in tobacco (Nicotiana tabacum) leaves and verified by qRT‐PCR amplification. Although neither CsRuby1 nor CsNoemi overexpression alone caused anthocyanin accumulation in tobacco leaves after infiltration for 7 days, the co‐transfection of CsRuby1 and CsNoemi led to faint reddening in tobacco leaves (Figure 6a). Furthermore, the total anthocyanin content was greatly increased by the co‐transfection of CsERF061 with the combination of CsRuby1 and CsNoemi compared to that without CsERF061 (Figure 6a,b). The expression of CsERF061 was greatly up‐regulated in the area co‐injected with CsERF061‐CsRuby1‐CsNoemi (Figure 6c), indicating that the gene was successfully transferred and overexpressed. Compared with the result of co‐transfection of CsRuby1 and CsNoemi, the co‐transfection of CsERF061‐CsRuby1‐CsNoemi in tobacco leaves activated the expression of the important structural genes directly catalyzing anthocyanin production (Figure 6c), indicating that CsERF061 enhanced the activation activity of CsRuby1 on anthocyanin biosynthetic pathway gene promoters, thereby upregulating anthocyanin biosynthesis and resulting in more intense leaf reddening.

Figure 6.

Figure 6

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CsERF061 promotes anthocyanin biosynthesis via interaction with CsRuby1.

(a) Overexpression of CsERF061 and CsRuby1‐CsNoemi together further promotes the accumulation of anthocyanin in tobacco leaves. The experiment was repeated six times independently, with similar results.

(b) Relative expression levels of CsRuby1, CsNoemi and CsERF061 in tobacco leaves.

(c) Relative expression levels of anthocyanin biosynthetic genes in tobacco leaves. The results in (b, c) are from three biological replicates and presented as means ± SD. Statistically significant differences were assessed using Student's t‐test (*P < 0.05, **P < 0.01, ***P < 0.001; ns, no significance).

DISCUSSION

Temperature plays an important role in anthocyanin accumulation in blood orange

High anthocyanin content is an important factor when considering the consumption of fruits and vegetables. The biosynthesis of anthocyanin in plants is greatly affected by multiple internal and external factors. Investigating the underlying molecular mechanism can provide a theoretical basis for improving this aspect of fruit quality. The effects of temperature on anthocyanin accumulation appear to vary considerably in positively regulate anthocya various species. LT is normally considered an inducer of anthocyanin biosynthesis and an optimal LT can lead to the greatest level of anthocyanin accumulation, as was shown in peach and plum (Xu et al., 2023; Zhu et al., 2020). In strawberry, on the contrary, LT inhibits anthocyanin accumulation (Mao et al., 2022). In citrus, many species have lost the ability to accumulate anthocyanins in their fruits compared with the ancestral types (Butelli et al., 2012; Carmona et al., 2017). There are two exceptions, blood orange and purple pummelo (Butelli et al., 2017; Huang et al., 2018, 2019). In purple pummelo, the accumulation of anthocyanin is limited to the peel and is induced by light (Huang et al., 2019). In blood orange, anthocyanin accumulation occurs in both flesh and peel, showing independence of light but is strongly induced by LT (Butelli et al., 2017; Huang et al., 2018, 2019) (Figure 7).

Figure 7.

Figure 7

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A working model showing the molecular mechanism regulating low‐temperature induced anthocyanin biosynthesis in blood orange.

Low‐temperature induced CsERF054 and CsERF061 directly bind to the CsRuby1 promoter through the DRE/CRT element to promote its expression; CsRuby1 then transactivates the target anthocyanin biosynthetic genes. In addition, CsERF061 interacts directly with CsRuby1 to synergistically trans‐activate the promoters of seven anthocyanin biosynthetic genes.

In the present study, we observed that blood orange fruits stored at 10°C accumulate large amounts of anthocyanins, whereas fruits stored at 20°C accumulated virtually none (Figure 1a,b), which is consistent with a previous study (Carmona et al., 2017). The increased accumulation of anthocyanins at 10°C is explained by the overall up‐regulation of anthocyanin‐related structural genes and TFs CsRuby1 and CsNoemi (Figures 1c and 2a). MYB TFs are known to be the most direct regulators of anthocyanin biosynthesis and are responsible for the transcriptional initiation of a series of anthocyanin biosynthetic genes (Allan et al., 2008; Heppel et al., 2013; Jaakola, 2013; Karppinen et al., 2021; Xu et al., 2015). These results indicate that CsRuby1 acts as an important regulator in LT‐induced anthocyanin biosynthesis.

The activation of CsRuby1 is related to the DRE/CRT cis‐element and upstream regulators CsERF054 and CsERF061

Although the anthocyanin biosynthetic pathway and key activating MYBs are highly conserved in different species, distinct regulatory mechanisms upstream of these MYBs have been found, such as BBX in pear (Alabd et al., 2022), NAC in peach (Zhou et al., 2015) and RAV in strawberry (Zhang et al., 2020). TFs normally function through binding to specific cis‐elements in the promoters of target genes (Sakuma et al., 2002; Shoji et al., 2013). In citrus, the promoter of Ruby1 exhibits diversity with three main types in anthocyanin‐accumulating varieties: Tcs1‐insertion types (in derivatives of European blood oranges), Tcs2‐insertion types (in derivatives of Chinese blood orange), and no insertion types (in purple pummelo) (Butelli et al., 2012; Huang et al., 2019). The Tcs1‐insertion and Tcs2‐insertion type promoters respond to LT and different blood orange varieties display the same pattern of cold‐induced and fruit‐specific anthocyanin accumulation (Butelli et al., 2012). However, no‐insertion type promoter only responds to light via the cis‐acting GCC box, explaining why purple pummelo only accumulates anthocyanin in the peel (Huang et al., 2019). In the present study, we used Tarocco blood orange, which is the main Chinese cultivar of blood orange and harbors the Tcs1‐insertion type promoter. The effect of LT on the activity of the Tcs1‐insertion type CsRuby1 promoter was manifested in tobacco leaves by lowering the temperature to 10°C (Figure 2b). This suggests that there is a general cold response involving TFs that can activate gene expression. Subsequently, two TFs belonging to the DREB sub‐clade, CsERF054 and CsERF061, were found to be induced by LT (Figure 2c; Figure S4b) and to trans‐activate the CsRuby1 promoter by directly binding to the DRE/CRT cis‐element (Figures 3a,b and 4a), indicating that CsERF054 and CsERF061 might be crucial cold‐responsive upstream regulators of CsRuby1 in Tarocco blood orange. Citrus is a perennial woody plant, and in previous studies, callus, fruits, and seedlings were normally used to verify gene functions (Fang et al., 2023; Hu et al., 2021; Liu et al., 2022; Zhao et al., 2021). However, there is a lack of tools to verify gene functions in citrus; the anthocyanin accumulation occurs in the very late stages of maturity, when the fruits are not suitable for transient transfection; and blood orange itself does not have seeds therefore seedling populations can not be obtained. Therefore, tobacco transient gene expression assays were performed to acquire evidence for the in vivo function of CsERF054/061. When we transiently expressed CsERF054 and CsERF061 in tobacco leaves together with Pro:CsRuby1‐CsRuby1, there were increased expression of CsRuby1 and biosynthesis of anthocyanin (Figure 4c,e; Figure S9). This shows that CsERF054 and CsERF061 positively regulate anthocyanin biosynthesis by transactivation of CsRuby1 (Figure 7).

We analyzed the sequences of the LTR region in the Tcs1 and Tcs2 transposons and found that the Tcs2‐LTR does not possess the same DRE‐CRT element as the Tcs1‐LTR (Figure S11), which supports the previous suggestion that different members of the same retrotransposons family may alter the expression of nearby genes through parallel but distinct mechanisms (Butelli et al., 2012). Thus, it remains to be established whether the ERF054 and ERF061‐Ruby1 regulatory mechanism found in the present study exists in other varieties of blood orange, since the different transposon‐insertions may have arisen independently during domestication (Butelli et al., 2012).

Multiple ERF‐type TFs have been reported to play a role in the regulation of anthocyanin biosynthesis through the regulation of MYBs, including FaRAV1 in strawberry (Zhang et al., 2020), MdERF1B in apple (Wang, Li, et al., 2022; Wang, Moss, et al., 2022; Wang, Wang, et al., 2022) and PpERF105 in pear (Ni et al., 2021). According to the phylogenetic analysis, these ERFs are distributed among various clades (Figure S6), showing the diversity and complexity of the regulators upstream of MYBs. In addition, we also found a potential repressive TF CsRAP2.1 containing an EAR motif, which repressed the activity of the CsRuby1 promoter but could not directly bind to it (Figure S7). Research in pears has identified a repressive TF PpERF4.1/4.2 with an EAR motif that negatively regulates anthocyanin biosynthesis by diminishing the function of PpMYB114 (Sun et al., 2023). In contrast, another transcription activator, PpERF105, inhibits anthocyanin biosynthesis by inducing the expression of the repressor‐type R2R3‐MYB gene PpMYB140 (Ni et al., 2021). These results revealed multiple ways in which ERFs play negative role in anthocyanin biosynthesis. In this study, it was found that CsRAP2.1 could not directly bind to, or repress the promoters of CsERF054/061, indicating that the CsRAP2.1 does not repress the activity of CsRuby1 via CsERF054/061 (Figure S12). However, protein–protein interactions between CsRAP2.1 and CsERF054/CsERF061 were observed by BiFC and LCI assays (Figure S13), suggesting that CsRAP2.1 may have repression effects on CsRuby1 by sequestering its activators. These results indicate a fine‐tuning regulatory mechanism involving multiple activator and repressor ERFs regulating anthocyanin biosynthesis in blood orange, which deserves future investigation.

CsERF061 upregulates anthocyanin biosynthesis by multiple mechanisms

In addition to activating the expression of MYBs in other fruits, ERFs have been reported to directly activate the expression of structural genes of anthocyanin biosynthesis. In apple, MdERF109 positively regulates early‐stage light‐induced anthocyanin accumulation by binding to the promoters of MdCHS, MdUFGT, and MdbHLH3 (Ma et al., 2021). In strawberry, FaRAV1 positively regulates anthocyanin biosynthesis via activation of FaMYB10 as well as anthocyanin biosynthetic genes (Zhang et al., 2020). To further investigate the mechanism by which CsERF054 and CsERF061 regulate the biosynthesis of anthocyanin, we studied their effects on the promoters of anthocyanin biosynthetic genes. However, according to the results of dual‐luciferase assay, CsERF054 or CsERF061 alone showed no activation effects on the promoters of structural genes (Figure 6a; Figure S10).

There are extensive synergistic effects of multiple TFs operating in regulation of fruit quality (Fang et al., 2023; Liu et al., 2022; Shi et al., 2022; Zhao et al., 2021). Recent research has provided evidence for interactions between AP2/ERF and MYB TFs in regulating multiple aspects of fruit quality such as aroma (Zhang, Xu, et al., 2018; Zhang, Yin, et al., 2018) and texture (Zeng et al., 2015). In terms of anthocyanin synthesis, MYBs usually function together with a bHLH and WD40 (Chen et al., 2022; Tao et al., 2020; Zhou et al., 2022). Previous studies identified some cases where ERFs regulate anthocyanin accumulation through interaction with MYB. In apple, MdERF38 is involved in drought‐induced anthocyanin accumulation via interaction with MdMYB1 (An et al., 2020); MdERF1B positively regulates anthocyanin biosynthesis both by interacting with MdMYB9 and MdMYB11 and binding to their promoters to activate their expression (Wang, Li, et al., 2022; Wang, Moss, et al., 2022; Wang, Wang, et al., 2022). In pear, ERF3, ERF24, and ERF96 are positive regulators of anthocyanin biosynthesis that function through interactions with MYB114 and bHLH3 (Jiang et al., 2023; Ni et al., 2019). In the present results, we showed that, in addition to transactivation of the CsRuby1 promoter by CsERF061 and CsERF054, CsERF061 also transactivates the promoters of anthocyanin biosynthetic genes by interacting with CsRuby1, with CsCHS1, CsCHI, CsF3H and CsDFR showing the most significant upregulation (Figure 5a). Further investigation indicated the importance of a protein–protein interaction between CsERF061 and CsRuby1 (Figure 5b,c). Furthermore, the total anthocyanin content was greatly increased by the co‐transfection of CsERF061 with the combination of CsRuby1 and CsNoemi compared to that without CsERF061, providing further evidence for the in vivo function of CsERF061. Although it has been found that ERFs participate in anthocyanin biosynthesis by regulation or interaction with MYB, to our best knowledge, it has not been reported that the same ERF regulates anthocyanin biosynthesis through these two mechanisms simultaneously. Although CsERF054 and CsERF061 belong to the same sub‐family of ERFs and share similar mechanisms in terms of regulating the expression of CsRuby1, there are still differences in their functions (Figure 7), providing an intriguing topic for future study.

CONCLUSION

The cold‐inducible TF genes CsERF054 and CsERF061 were identified and found to positively regulate anthocyanin accumulation by two distinct mechanisms. CsERF054 and CsERF061 activate the expression of CsRuby1 through directly binding to its promoter, while CsERF061 interacts with CsRuby1 to synergistically transactivate the promoters of anthocyanin biosynthetic genes. These results build a link between ERF TFs and LT‐induced anthocyanin biosynthesis and provide new insights into the regulatory mechanisms of cold‐induced anthocyanin biosynthesis in blood orange.

MATERIALS AND METHODS

Plant materials and treatment

Blood orange (Citrus sinensis cv Tarocco) was grown in a commercial orchard in Wenzhou (Zhejiang, China) and the trees used for sample collection were cultivated with standard horticultural practices. At the initial stage of pigmentation, uniform fruits without signs of disease or mechanical damage were selected and randomly divided into five groups. Two groups of fruit were maintained on the tree (preharvest groups), one of which was naturally illuminated while the second group of fruit was individually shaded using aluminum foil. Three groups were picked from the tree (postharvest groups) and transported promptly to the laboratory. Subsequently, the three groups were stored at 5, 10, and 20°C respectively, with 85% humidity in constant darkness. Pulps were sampled and frozen in liquid nitrogen rapidly at 0, 10, 20, 30, and 40 days during the treatment period and then stored at −80°C for further use.

Tobacco plants (N. benthamiana and N. tabacum) used for dual‐luciferase assays, subcellular localization, bimolecular fluorescence complementation, firefly LCI, and transient overexpression assays were grown in a greenhouse with a standard control (25°C, 75% humidity, 16‐h light and 8‐h dark).

Measurement of the anthocyanin content

The total anthocyanin was extracted as described previously with some modifications (Zhang et al., 2020). Approximately 1 g of sample powder was weighed accurately and used for extraction with 4 ml of 0.1% formic acid‐methanol and then incubated at 4°C in the dark for 12 h. The supernatant was collected by centrifugation. The extraction procedure was repeated again, and the obtained supernatants were combined for further measurement. The total anthocyanin content was determined by the pH difference method. Then, the absorbance of the obtained solution from extraction was measured in a UV‐2550 spectrophotometer at 510 and 700 nm.

The anthocyanin components and contents were tested by HPLC analysis using a Waters Alliance 2695 system (Waters Corp., Milford, MA, USA) equipped with a reverse‐phase C18 column (4.6 × 250 mm, 5 μm; Waters Corp.). The combined supernatants were evaporated at 30°C in an evaporator, resuspended in 1 ml 0.1% formic acid‐methanol and filtered through a 0.22‐μm Millipore membrane before testing. The detection procedure was set as described previously (Zhang et al., 2020). C3G and C3G6M were used as standards.

RNA extraction and RT‐qPCR

Total RNA was extracted from fruit flesh using the TRIzol Reagent Kit (Ambion, Hopkinton, MA, USA). HiScript® II Q Select RT SuperMix (Vazyme, Nanjing, China) was used to remove genomic DNA contamination from total RNA and synthesize the first‐strand cDNA. RT‐qPCR analysis was conducted using ChamQ Universal SYBR qPCR Master Mix (Vazyme) with a CFX96 instrument (Bio‐Rad, Hercules, CA, USA) according to the procedure recommended by the manufacture. Citrus actin (XM_006464503) was used as the housekeeping gene (Li et al., 2017). Relative gene expression was calculated using the 2−ΔCT method. Primers for RT‐qPCR analysis are listed in Table S1.

Dual‐luciferase assay

Dual‐luciferase assays were performed as described in a previous study (Li et al., 2017). The coding sequences of TFs were recombined into the pGreen II 0029 62‐SK vector. The promoters of the CsRuby1 and anthocyanin biosynthetic genes were recombined into the pGreen II 0800‐LUC vector. All recombinant constructs were electroporated into Agrobacterium tumefaciens strain GV3101 and then were used for infiltration into tobacco (N. benthamiana) leaves with a combination ratio of TF:promoter = 10:1. Empty pGreenII 0029 62‐SK vector (SK) served as control and at least three biological replicates were set. After 3 days of infiltration, the LUC and REN activities were examined using dual‐luciferase reagents (Promega, Madison, WI, USA). The TF‐promoter interaction was evaluated by LUC/REN. The primers used are listed in Table S1.

Subcellular localization analysis

The full‐length CsERF054 and CsERF061 coding sequences without the stop codon were fused to the C terminus of the pCAMBIA1300‐sGFP vector. The fusion constructs were electroporated into A. tumefaciens GV3101 and infiltrated into N. benthamiana (with nucleus‐located mCherry) leaves. The fluorescence of GFP and RFP was imaged with a Nikon A1‐SHS confocal laser scanning microscope (Tokyo, Japan) after 48 h. The excitation wavelength for GFP and RFP fluorescence was 488 nm, and fluorescence was detected at 490–520 nm (Hu et al., 2021). The primers used are listed in Table S1.

Yeast one‐hybrid assay

Yeast one‐hybrid assays were performed using the Matchmaker Gold Yeast One‐Hybrid System kit (Takara, Beijing, Japan). The promoter sequence of CsRuby1 was inserted into the pAbAi vector, and the coding sequences of CsERF054, CsERF061, and CsRAP2.1 were inserted into the pGADT7 vector. Promoter auto‐activation was detected on SD medium lacking Ura in the presence of AbA. The protein‐DNA interaction was verified on SD medium lacking Leu in the presence of AbA. The primers used are listed in Table S1.

Recombinant protein purification

The full length of CsERF054 and CsERF061 without the stop codon were inserted into pET‐32a (Clontech, Kusatsu, Japan) (Hu et al., 2021). The recombinant vector was transformed into Escherichia coli strain BL21 and then incubated with LB medium at 37°C until OD600 = 0.6. Then, 0.5 mm isopropyl β‐d‐1‐thiogalactopyranoside was added to the medium and incubated at 16°C for 20 h. The E. coli cells were collected with 1 × PBS, and ultrasonicated on ice at 200 W with on/off at 3 sec/2 sec cycle for 15 min, and centrifuged at 9000  g for 20 min at 4°C. Subsequently, the Ni‐NTA resin (Transgene, Beijing, China) was added to the supernatant to combine His‐tagged proteins at 4°C for 1 h (Liu et al., 2022), which were eluted with elution buffers containing increasing concentrations of imidazole (10, 30, 50, 75, and 100 mm). The primers used are listed in Table S1.

Electrophoretic mobility shift assay

Electrophoretic mobility shift assay detection was performed using the LightShift Chemiluminescent EMSA kit (Thermo Fisher Scientific, Waltham, MA, USA) according to the manufacturer's instructions. The probes were labeled from the 3′ biotin terminal by HuaGene (Shanghai, China), annealed with complementary oligonucleotide, heated at 95°C for 5 min, and then gradually reduced to 25°C at a rate of 0.1 sec−1, and transformed into a double‐stranded DNA probe. The probes used for EMSA are listed in Table S1. The binding specificity was tested by mutant probe and competition with unlabelled probes (10, 50, 100 and 300‐fold unlabelled oligonucleotides).

BiFC assay

Bimolecular fluorescence assays were performed as described previously (Li et al., 2017). The coding sequences of CsERF061 and CsRuby1 without the stop codon were cloned and inserted into vectors containing C‐ and N‐terminal fragments of YFP. The recombinant constructs were electroporated into A. tumefaciens GV3101 and infiltrated into N. benthamiana (with nucleus‐located mCherry) leaves, with a set of negative controls. The YFP fluorescence was imaged 2 days after infiltration using a Nikon A1‐SHS confocal laser scanning microscope. The excitation wavelength for YFP was 488 nm, and fluorescence was detected at 520–560 nm. The primers used are listed in Table S1.

Firefly LCI assay

The full coding sequence of CsERF061 and CsRuby1 were cloned and inserted into pCAMBIA1300‐cLUC and pCAMBIA1300‐nLUC (Wei et al., 2022). The recombinant plasmids were transformed into A. tumefaciens strain EHA105 and infiltrated into N. benthamiana leaves. Two days after infiltration, 0.2 mm luciferin was infiltrated into the same positions at which A. tumefaciens was infiltrated, the leaves were kept in a dark room for 20 min, and the luciferase activity was detected using the NightSHADE LB 985 system (Berthold, Bad Wildbad, Germany). The primers used are listed in Table S1.

Transient overexpression in tobacco leaves

The coding sequences of CsERF054 and CsMYB061 were cloned and inserted into pGreenII0029 62‐SK vectors (Hellens et al., 2005) and then transformed separately into A. tumefaciens EHA105. The CsRuby1 promoter, CsRuby1 coding sequence, 35S promoter, CsNoemi were amplified and fused together by fusion PCR to construct a CsRuby1 promoter:CsRuby‐35S:CsNoemi fragment. The fragment was then inserted into pGreenII0029 62‐SK vectors and transformed into A. tumefaciens EHA105. CsERF061‐SK/CsMYB305‐SK/SK and CsRuby1 promoter:CsRuby1‐35S promoter:CsNoemi‐SK at a 1:1 ratio were then transiently expressed using the same method as described above for the dual‐luciferase assay. After 7 days of infiltration, the color phenotype was observed and photographed. The primers used are listed in Table S1.

CONFLICT OF INTEREST

The authors declare no conflicts of interest.

OPEN RESEARCH BADGES

This article has earned an Open Data badge for making publicly available the digitally‐shareable data necessary to reproduce the reported results. The data is available at https://www.ncbi.nlm.nih.gov/bioproject/PRJNA1098784/ (NCBI accession number: PRJNA1098784).

Supporting information

Figure S1. Phenotypes of blood orange flesh in different treatments.

Figure S2. Anthocyanin contents in blood orange flesh during treatment.

Figure S3. Content of predominant anthocyanin components cyanidin 3‐glucoside and cyanidin 3‐(6′′‐malonyl) glucoside in blood orange flesh at five time points.

Figure S4. Analysis of the CsRuby1 promoter and screening of candidate DREB genes.

Figure S5. Relative expression of CsERF054, CsERF061 and CsRAP2.1 in fruit on the tree and fruit stored at 10°C after harvest.

Figure S6. Phylogenetic tree constructed using the amino acid sequence of three screened ERFs (red), all ERFs of Arabidopsis thaliana (gray), anthocyanin related ERFs in other species (black) and cold‐responsive transcription factors PtrERF9 and PtrERF108 (blue) identified in trifoliate orange.

Figure S7. The amino acid sequence alignment analysis of CsERF054, CsERF061, CsRAP2.1 and AtCsERF054, AtCsERF061 and AtRAP2.1.

Figure S8. The relationship between CsRAP2.1 and the promoter of CsRuby1.

Figure S9. CsERF054 and CsERF061 activate anthocyanin biosynthesis in Nicotiana benthamiana leaves.

Figure S10. Effect of the CsERF061 and CsRuby1 individually and in combination on the activation of the promoter of anthocyanin biosynthetic genes.

Figure S11. Alignment of LTR region in the Tcs1‐insertion and Tcs2‐insertion types Ruby1 promoter.

Figure S12. The CsRAP2.1 does not bind to or repress the promoter of CsERF054/061.

Figure S13. The interaction between CsRAP2.1.

TPJ-119-1433-s002.pdf (1.5MB, pdf)

Table S1. Primers used in this study.

TPJ-119-1433-s001.xlsx (14.2KB, xlsx)

ACKNOWLEDGEMENTS

We would like to thank Dr. Wenli Liu for data analysis, Mr. Xufeng Shen for sample collections, Rong Jin for the help with tobacco plant care. This work was supported by the National Key Research and Development Program of China (2022YFD2100100), the National Natural Science Foundation of China (31801591), the 111 Project (B17039), and Zhejiang Provincial Cooperative Extension Project of Agricultural Key Technology (2022XTTGGP01).

DATA AVAILABILITY STATEMENT

All data generated or analyzed during this study are included in this published article. (and its supplementary information files).

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Figure S1. Phenotypes of blood orange flesh in different treatments.

Figure S2. Anthocyanin contents in blood orange flesh during treatment.

Figure S3. Content of predominant anthocyanin components cyanidin 3‐glucoside and cyanidin 3‐(6′′‐malonyl) glucoside in blood orange flesh at five time points.

Figure S4. Analysis of the CsRuby1 promoter and screening of candidate DREB genes.

Figure S5. Relative expression of CsERF054, CsERF061 and CsRAP2.1 in fruit on the tree and fruit stored at 10°C after harvest.

Figure S6. Phylogenetic tree constructed using the amino acid sequence of three screened ERFs (red), all ERFs of Arabidopsis thaliana (gray), anthocyanin related ERFs in other species (black) and cold‐responsive transcription factors PtrERF9 and PtrERF108 (blue) identified in trifoliate orange.

Figure S7. The amino acid sequence alignment analysis of CsERF054, CsERF061, CsRAP2.1 and AtCsERF054, AtCsERF061 and AtRAP2.1.

Figure S8. The relationship between CsRAP2.1 and the promoter of CsRuby1.

Figure S9. CsERF054 and CsERF061 activate anthocyanin biosynthesis in Nicotiana benthamiana leaves.

Figure S10. Effect of the CsERF061 and CsRuby1 individually and in combination on the activation of the promoter of anthocyanin biosynthetic genes.

Figure S11. Alignment of LTR region in the Tcs1‐insertion and Tcs2‐insertion types Ruby1 promoter.

Figure S12. The CsRAP2.1 does not bind to or repress the promoter of CsERF054/061.

Figure S13. The interaction between CsRAP2.1.

TPJ-119-1433-s002.pdf (1.5MB, pdf)

Table S1. Primers used in this study.

TPJ-119-1433-s001.xlsx (14.2KB, xlsx)

Data Availability Statement

All data generated or analyzed during this study are included in this published article. (and its supplementary information files).

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