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Apple NAC transcription factor MdNAC52 regulates biosynthesis of anthocyanin and proanthocyanidin through MdMYB9 and MdMYB11
Plant Science 289 (2019) 110286
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Apple NAC transcription factor MdNAC52 regulates biosynthesis of anthocyanin and proanthocyanidin through MdMYB9 and MdMYB11
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Qingguo Sun, Shenghui Jiang, Tianliang Zhang, Haifeng Xu, Hongcheng Fang, Jing Zhang, ⁎ Mengyu Su, Yicheng Wang, Zongying Zhang, Nan Wang, Xuesen Chen National Key Laboratory of Crop Biology, College of Horticulture Science and Engineering, Shandong Agricultural University, Tai-An, Shandong, 271018, China
A R T I C LE I N FO
A B S T R A C T
Keywords: Apple NAC transcription factor Light Anthocyanin Proanthocyanidin MYB transcription factors
Anthocyanin and proanthocyanidin (PA) play important roles in plant growth and development. Although previous studies have identified many of the transcription factors involved in the anthocyanin and PA pathway, the regulation mechanisms of these pathways remain poorly understood. In this study, we identified a NAC transcription factor, MdNAC52, whose gene transcript levels increased during apple coloration. Apple calli overexpressing MdNAC52 accumulated anthocyanin. Yeast one-hybrid, electrophoretic mobility shift, chromatin immunoprecipitation, and luciferase reporter assays showed that MdNAC52 could interact with the promoters of MdMYB9 and MdMYB11 to regulate anthocyanin biosynthesis. MdNAC52 was targeted by MdHY5 in response to light. Interestingly, MdNAC52 participated in the regulation of PA biosynthesis through controlling the expression of MdMYB9 and MdMYB11. MdNAC52 could also bind to the LAR promoter to regulate its expression and promote PA synthesis. Overall, these findings establish that MdNAC52 binds to the promoters of MdMYB9 and MdMYB11 to promote anthocyanin and PA biosynthesis and directly regulates LAR to modulate PA metabolism. Our study provides new insights into the roles of a NAC transcription factor in regulating anthocyanin and PA accumulation in apple.
1. Introduction Flavonoids are plant secondary metabolites that play important roles in growth and development. Flavonoids include anthocyanins, flavonols, and proanthocyanidins (PAs) [1], which are present in the form of glycosides and methylated derivatives [2]. Flavonoids are diverse in structure and have a wide range of biological functions. For example, the accumulation of anthocyanins in the vacuoles of plant epidermal cells can protect plants from damage caused by ultraviolet radiation [3]. The accumulation of anthocyanins can confer red, blue, and purple colors to organs and tissues, which can help plants to attract pollinators and seed dispersers [4]. Proanthocyanins are generally colorless in plant cells, but are easily oxidized to form brownish substances that bind to each other within the cell [5]. They also have antioxidant activity and are associated with cardiovascular health, so their dietary intake is beneficial for human health [6–9]. In recent years, the flavonoid pathway has been widely studied in various plant species. The gene encoding chalcone synthase, CHS, was
the first flavonoid biosynthesis gene to be isolated [10]. This gene was isolated from parsley in the 1980s. Subsequently, flavonoid biosynthesis pathways have been studied in Zea mays, petunia, snapdragon, Arabidopsis, grape, and other species [11–15]. Key enzymes in the pathway have gradually been identified. The late stage of anthocyanin biosynthesis depends mainly on structural genes such as DFR encoding dihydroflavonol 4-reductase, ANS encoding anthocyanin synthase, and UFGT encoding glycosyltransferase (UFGT) [16]. The synthesis of PA mainly depends on leucoanthocyanidin reductase (LAR) and anthocyanidin reductase (ANR) [16]. Plant growth and development not only depends on a series of structural genes, but also on a large number of transcription factors (TFs). In the flavonoid pathway, the MYB–bHLH–WD40 (MBW) complex regulates the accumulation of anthocyanin and PA synergistically by controlling the expression of structural genes [17]. Previous studies have shown that MdbHLH3, a low-temperature-induced bHLH TF in apple, interacts with MdMYB1 through two regions (amino acids 1–23 and 186–228) [18]. MdEIL1 functions upstream of MdMYB1 and
Abbreviations: PA, proanthocyanidin; TF, transcription factor; DAFB, days after full bloom; DMACA, 4-dimethylaminocinnamaldehyde; SD/−T/−L/−H, the Murashige and Skoog medium lacking Trp Leu, and His; 3-AT, 3-amino-12,4-triazole; EMSA, electrophoretic mobility shift assay; ChIP, chromatin immunoprecipitation-qPCR; LUC, luciferase reporter assays ⁎ Corresponding author. E-mail address: [email protected] (X. Chen). https://doi.org/10.1016/j.plantsci.2019.110286 Received 14 June 2019; Received in revised form 26 August 2019; Accepted 25 September 2019 Available online 27 September 2019 0168-9452/ © 2019 Elsevier B.V. All rights reserved.
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37°52′, E121°39′), Shandong, China. Fruits were bagged at around 30 days after full bloom (DAFB). Samples for these experiments were collected at 0, 2, 4, 6, 8 and 12 days after the fruits were de-bagged. All samples were stored at −80 °C. ‘Orin’ apple calli were grown on Murashige and Skoog medium containing 0.5 mg L−1 2,4-dichlorophenoxyacetic acid and 1 mg L−1 6benzyl-aminopurine for genetic transformation. The calli were cultured at 25 °C in darkness.
activates its expression, resulting in anthocyanin biosynthesis, MdMYB1 binds to MdERF3, a key regulator in the ethylene pathway [19]. An [20] found that both MdMYB9 and MdMYB11 directly bind to the promoters of LAR, ANR, and ANS, whereas MdbHLH3 is recruited upstream of MdMYB9 and MdMYB11 and regulates their transcription. MdMYBPA1 not only promotes the accumulation of anthocyanin in response to low temperature via its interaction with MdbHLH33, but also promotes the PA biosynthesis by forming multimers with MdMYB9/11 [21]. It is noteworthy that not only MYB TFs but also other TFs are involved in the flavonoid pathway. Previous studies have shown that TTG2, a WRKY TF, interferes with the production of tannins in the seed coat [22]. The E3 ubiquitin ligase COP1/SPA interacts with PAP1 and PAP2, and these two proteins are required for the production of anthocyanin [23]. MdHY5 can bind to FLS (encoding flavonol synthase) to promote and regulate flavonoid metabolism in apple [24]. A previous study showed that, under light conditions, genes associated with flavonoid synthesis were up-regulated in transgenic lines overexpressing AtNAC78 [25]. The NAC TFs make up one of the largest gene families. In Arabidopsis, nearly 90 NAC proteins have been identified [26]. All NAC TFs contain a plant-specific highly conserved N-terminal domain known as the NAC domain [27]. The NAC TFs are involved in plant growth and development. For example, NACs participate in signaling of auxins, abscisic acid, and ethylene [28]. Genetic and abiotic factors such as drought, temperature, and light affect the biosynthesis of anthocyanin and PA [29]. Among these factors, light is an important environmental stimulus promoting anthocyanin accumulation. The mechanism of light-induced regulation of anthocyanin biosynthetic pathways has been determined. In Arabidopsis, the expression of MYB (PAP1 and PAP2) and bHLH (TT8) promote anthocyanin accumulation in response to light [30]. In apple, MdCOP1 promotes the expression of MdMYB1 in the nucleus to regulate anthocyanin biosynthesis in the light, but MdCOP1 degrades MdMYB1 in the nucleus under dark conditions [31]. Ultraviolet light induces the expression of MdBBX20, which encodes a B-box zinc finger protein that promotes anthocyanin accumulation synergistically with MdHY5 [32]. HY5 also functions upstream of light-induced MYB transcription to promote anthocyanin biosynthesis [33]. In light-grown leaf tissues, cryptochrome 1 (cy1) is involved in controlling the expression of CHS to increase anthocyanin biosynthesis in response to ultraviolet-B radiation [30]. Transcriptome sequencing was performed on Yanfu 3 and Yanfu 8, showed that some TF families such as MYB, WRKY and NAC have different transcript levels in the two materials, we found a TF MdNAC52 which the transcript level was higher in the apple line ‘YanFu 8′ than in ‘YanFu 3′, and the ‘YanFu 8′ peel is deeper and full red, while the ‘YanFu 3′ peel is lighter than ‘YanFu 8′, those indicating that MdNAC52 may participate in anthocyanin biosynthesis [34]. In this study, we cloned MdNAC52 and found that its encoded protein was located in the nucleus. During apple coloration, the expression of MdNAC52 increased. ‘Orin’ apple calli overexpressing MdNAC52 (OE-MdNAC52) showed enhanced anthocyanin accumulation and PA biosynthesis. Further analyses showed that MdNAC52 could regulate the expression of the target genes MdMYB9 and MdMYB11 to promote anthocyanin and PA biosynthesis, and that MdHY5 could bind to the promoter of MdNAC52 to promote its expression. In addition, MdNAC52 directly regulated LAR to modulate PA metabolism. Our study reveals the detailed mechanism of the regulation of anthocyanin and PA accumulation, and provides a theoretical basis to enrich genetic resources related to flavonoid biosynthesis.
2.2. Transformation of apple calli with MdNAC52 MdNAC52 was inserted into the pRI101-AN vector with a GFP tag to generate the recombinant plasmid 35S:MdNAC52-GFP. The recombinant plasmid was inserted into Agrobacterium tumefaciens LBA4404, which was then incubated with 2-week-old apple calli for 30 min at room temperature. The calli were transferred to MS solid medium with 250 μg mL−1 carboxybenzyl (Solarbio, Beijing, China; http://solarbio.bioon.com.cn/) and 50 μg mL-1 kanamycin (Solarbio) to select for transformants. 2.3. Measurement of anthocyanin content Each sample (0.5 g) was ground to a powder in liquid nitrogen and then incubated in 5 mL 1% (v/v) HCl-methanol for 24 h at 4 °C in darkness. After centrifugation, KCl and NaAc buffers were added to aliquots of the supernatant, which were then mixed and incubated for 20 min at 4 °C in darkness. The solutions were centrifuged at 8000 rcf (×g) for 15 min. The absorbance of the supernatant was measured using a UV-2450 spectrophotometer (Shimadzu, Kyoto, Japan) at 510 nm and 700 nm (i.e., OD510 and OD700). The anthocyanin content was calculated using the following formula: ΔA × 5 × 0.005 × 1000 × 449.2/(26,900 × 0.5), where ΔA = (OD510 − OD700 at pH = 1.0) − (OD510 − OD700 at pH = 4.5). 2.4. 4-Dimethylaminocinnamaldehyde staining and determination of PA content The presence of PA was detected by 4-dimethylaminocinnamaldehyde (DMACA) staining, and the PA content was determined as described by Li [35]. The sample was ground in liquid nitrogen, 0.45 g of the powder was suspended in a mixture of 1 mL of 70% acetone solution (v/v) and 0.1% (w/v) ascorbic acid, and the mixture was incubated in the dark at 4 °C for 30 min. This was repeated three times, then the mixture was centrifuged and the supernatant was collected. The supernatant (2.5 mL) was mixed with 3 mL ether at −20 °C. The soluble liquid PA separated into the lower liquid phase. Then, 2 mL soluble PA, 0.5 mL 2% (w/v) DMACA, and 1 mL methanol were mixed and incubated at room temperature for 20 min. The absorbance was measured at 643 nm using a UV–vis spectrophotometer (UV-2450; Shimadzu). The concentration of PA was determined by comparison with a standard curve prepared using a catechin reference standard (Sigma, St Louis, MO, USA). 2.5. RNA extraction and RT-PCR analyses Total RNA was extracted using an RNAprep Pure Plant Kit (Tiangen, Beijing, China; http://www.tiangen.com/en/). The RNA was reversetranscribed into to cDNA using the RevertAidTM First Strand cDNA Synthesis Kit (TransGen, Beijing, China; http://www.transbionovo. com/) according to the manufacturer’s instructions. The 20-μL reaction mixture was prepared according to the instructions of the SYBR Green PCR Master Mix Kit, and contained 10 μL 2.5 × ReaL MasterMix/ 20 × SYBR solution, 7 μL ddH2O, 1 μL cDNA (50 ng·μL−1), and 1 μL each of upstream and downstream primers (5 μmol L·L−1). The analyses were conducted using a CFX96 Real-Time PCR Detection system (BioRad, Hercules, CA, USA). Three replicates were prepared for each
2. Materials and methods 2.1. Plant materials Trees of the apple cultivar ‘YanFu 3′ were grown in Yan Tai (N 2
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Fig. 1. Phylogenetic tree, sequence analyses, and subcellular localization of MdNAC52. (A) Phylogenetic analysis of NAC proteins from different species. Genes involved in the construction of phylogenetic tree are listed in Supplementary Table S3. (B) Amino acid sequence alignment of NAC transcription factors containing NAC domain. (C) Subcellular localization of 35S::MdNAC52-GFP fusion construct and 35S::GFP construct.
2.6. Cloning of MdNAC52 and bioinformatics analysis
sample, and the transcript level of MdActin was determined as the internal control. The data were analyzed using the (Ct) 2 –ΔΔCt method [36]. The primers used for RT-PCR are listed in Supplementary Table S2.
MdNAC52 (MD01G1094000) was amplified in vitro using phusion polymerase. The primers are listed in Supplementary Table S1. The 3
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Fig. 2. Anthocyanin accumulation and transcript levels of anthocyanin-related genes in ‘YanFu 3′ apples. (A) Mature fruits harvested on days 0, 2, 4, 6, 8, and 12 after de-bagging. (B) Anthocyanin contents in apple skins of ‘Yanfu 3′. (C) Transcript levels of MdNAC52 and related genes in anthocyanin biosynthesis pathway.
MMG solution (100 mL 0.8 mol·L−1 mannitol; 3 mL 1 mol·L−1 MgCl2; 2.667 mL 0.3 mol·L−1 MES). The GFP fluorescence in protoplasts was detected under a fluorescence microscope.
software MEGA5.1 and DNAMAN were used to construct the phylogenetic tree and align amino acid sequences. 2.7. Extraction of protoplasts and subcellular localization analyses
2.8. Yeast one-hybrid assay Calli harboring OE-MdNAC52 fused to a GFP tag were added to 10 ml cell hydrolysis solution, vacuum infiltrated for 30 min, kept in the dark at room temperature for 12 h, and gently shaken to release protoplasts. To this mixture was added an equal volume of W5 solution (1.333 mL 0.3 mol·L−1 MES; 1 mL 1 mol·L−1 KCl; 6.16 mL 5 mol·L−1 NaCl; 25 mL 1 mol·L−1 CaCl2), and then the protoplasts were filtered through a nylon cloth (75 μm pore diameter). The obtained filtrate was centrifuged at 500 rpm for 10 min, the supernatant was discarded, and the precipitate was added to 10 mL W5 solution. After 30 min, the supernatant was discarded and the protoplasts were suspended in 2 mL
For the yeast one-hybrid (Y1H) assays, each of the MdNAC52 and MdHY5 sequences was inserted into the pGADT7 vector to generate the MdNAC52-AD and MdHY5-AD constructs, respectively. Additionally, each of the MdNAC52, MdMYB9, MdMYB11, and MdLAR promoter sequences was inserted into the pHIS2 vector. The recombinant plasmids with the MdNAC52 or MdHY5 sequence and promoter fragments were co-transformed into the Y187 strain, then the interactions were examined on the medium lacking Trp, Leu, and His (SD/−T/−L/−H) with 3-amino-1,2,4-triazole (3-AT) at the optimal screening 4
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Fig. 3. Functional characterization of MdNAC52 overexpressed in ‘Orin’ apple calli. (A) ‘Orin’ calli overexpressing MdNAC52 (OE1–3) and ‘Orin’ calli containing empty GFP vector (GFP). (B) Anthocyanin contents in MdNAC52-OE calli and GFP calli. (C) PCR analysis confirming presence of transgene in OE-MdNAC52 calli. (D) Western blotting analysis of OE-MdNAC52 calli. (E) Transcript levels of MdNAC52 and genes encoding anthocyanin biosynthesis-related enzymes and TFs in MdNAC52-OE calli and GFP calli.
2.10. LUC reporter assay
concentration.
The sequences of MdNAC52 and MdHY5 were each recombined into the pHBT-AvrRpm1 effector, and the promoter sequences of MdNAC52, MdMYB9, MdMYB11, and MdLAR were inserted into the pFRK1-LUCnos reporter. For the analyses, 1 μL UBQ-GUS, 3 μL pFRK1-LUC-nos recombinant reporter, and 6 μL recombinant effector were co-transformed into ‘Orin’ apple callus protoplasts in the presence of 40% polyethylene glycol for transient expression. After incubation for 6 h at 24 °C, the fluorescence levels of LUC and GUS were tested using a multimode plate reader (Victor X4, PerkinElmer, Norwalk, CT, USA; http://www.perkinelmer.com/). Relative LUC activity was determined based on the ratio of LUC to GUS values.
2.9. Electrophoretic mobility shift assay The coding sequences of MdNAC52 and MdHY5 were each recombined into the vector pET-32a(+) (His-Tag), and each recombinant plasmid was transformed into Escherichia coli BL21 (DE3). The fusion protein was purified using a His-tagged protein purification kit (CWBIO Inc., Beijing, China). Electrophoretic mobility shift assays (EMSAs) were conducted using a LightShiftTM EMSA Optimization and Control Kit (Thermo Scientific, Waltham, MA, USA; https://www.thermofisher. com). The reaction mixture contained 2 μL 1 × binding buffer (2.5% glycerol, 5 mM MgCl3, 10 mM EDTA, 50 mM KCl,), 10 μL protein, and 1 μL unlabeled probe. The mixture was reacted at room temperature for 15 min, and then 1 μL labeled probe was added to initiate the 15-min competition reaction. 5
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Fig. 4. Interaction between MdNAC52 and promoters of MdMYB9 and MdMYB11. (A and B) Yeast one-hybrid assays showing interaction between MdNAC52 and promoters of MdMYB9 and MdMYB11. (C and D) Electrophoretic mobility shift assay showing direct binding of MdNAC52-HIS fusion protein to ACACGT and ACGTGT motifs in promoters of MdMYB9 and MdMYB11 in vitro. Red letters indicate NAC binding site. Labeled probe was incubated with MdNAC52-HIS protein; unlabeled probe served as competitor. (E) Binding of MdNAC52 to MdMYB9 and MdMYB11 promoters in vivo in ChIP-PCR assay. MdNAC52-GFP bound to ACACGT and ACGTGT motifs in MdMYB9 and MdMYB11 promoters. (F) Luciferase assays verifying that MdNAC52 activates promoters of MdMYB9 and MdMYB11 (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.).
and MdHY5 fused to a GFP tag were analyzed in the ChIP-qPCR assay. The ChIP experiment was carried out using a ChIP kit (Upstate Biotech, Lake Placid, NY, USA) following the manufacturer’s instructions. The amount of immunoprecipitated chromatin was detected by PCR. The
2.11. Chromatin immunoprecipitation-qPCR analysis The chromatin immunoprecipitation (ChIP) experiment was conducted as described by He [37]. Transgenic calli carrying MdNAC52 6
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Fig. 5. MdHY5 and MdNAC52 transcript levels in ‘Orin’ apple calli exposed to light as determined by RT-PCR. (A) Transcript levels of MdNAC52 and MdHY5. (B) Correlation analysis between transcript levels of MdNAC52 and MdHY5.
experiment was repeated three times.
3.2. Anthocyanin, proanthocyanidin content and TF transcript levels during apple coloration
3. Results
After the fruits were de-bagged, the peel gradually turned red and the anthocyanin content increased, meanwhile, the proanthocyanidin content was reduced. (Fig. 2A, B). To study the expression pattern of MdNAC52, we monitored its transcript levels in ‘YanFu 3′ during apple coloration, and found that its transcript levels increased from 0 d to 8 d. We also analyzed the transcript levels of anthocyanin-related structural genes and TFs. The transcript levels of key genes for anthocyanin synthesis (MdDFR, MdANS, MdUFGT) peaked at 8 d. The MdNAC52 transcript level was consistent with the expression levels of these genes and those of the related TF MdMYB1, which also peaked at 8 d after debagging, while MdMYB9, MdMYB11 have showed different expression pattern (Fig. 2C). And the transcript levels of proanthocyanidin-related structural genes MdLAR, MdANR were consistent with the proanthocyanidin content, minimum at 8 d (Fig. 2C). These observations suggested that MdNAC52 may act as an anthocyanin regulator in apple.
3.1. Phylogenetic tree, sequence analyses, and subcellular localization of MdNAC52 The NAC domains of predicted and known NAC family proteins were classified into subgroups I–XVI according to sequence similarity [38]. To analyze the relationship between MdNAC52 and other NACs, we constructed a phylogenetic tree. The results showed that MdNAC52 belongs to subgroup Ⅳ(ONAC003). Many studies have shown that NAC proteins are involved in plant growth and environmental adaptation [39,40]. AtNAC19, AtNAC72 are in subgroup Ⅱ and are important for tolerance to drought [41]. The NAC proteins in the subgroup Ⅳ might be participated in the diversity of developmental stages and tissuespecific [42]. The NAC domain is composed of approximately 100 amino acid residues [43]. Amino acid sequence alignment analyses confirmed that MdNAC52 contains a NAC domain (Fig. 1B). To test whether MdNAC52 was localized in the nucleus like other TFs, we constructed the 35S::MdNAC52-GFP vector and transformed it into ‘Orin’ calli, with 35S::GFP as the control. Protoplasts were isolated from the transgenic calli, and the subcellular localization of the fusion protein was detected by fluorescence microscopy. 35S::MdNAC52-GFP was localized in the nucleus, while 35S::GFP was distributed throughout the protoplasts (Fig. 1C). These findings indicated that MdNAC52 was localized in the nucleus.
3.3. MdNAC52 promotes anthocyanin accumulation in apple calli To explore the role of MdNAC52 in regulating anthocyanin accumulation, we generated ‘Orin’ calli overexpressing MdNAC52 (OENAC52), and verified the presence of the transgene by PCR and western blot assays (Fig. 3C, D). Under light conditions, OE-NAC52 calli gradually became red, while the calli transformed with the GFP vector did not (Fig. 3A). Spectrophotometric analyses confirmed that the anthocyanin content was significantly higher in OE-NAC52 calli than in the 7
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Fig. 6. Binding of MdHY5 to promoter of MdNAC52. (A and B) Yeast one-hybrid assays showing interaction between MdHY5 and MdNAC52 promoter. (C) Binding of MdHY5 to MdNAC52 promoter in vivo in ChIP-PCR assay. MdHY5-GFP bound to G-box motif in MdNAC52 promoter. (D) Electrophoretic mobility shift assay confirming direct binding of MdHY5-HIS fusion protein to G-box in promoter of MdNAC52 in vitro. Red letters indicate HY5 binding site. Labeled probe was incubated with MdHY5-HIS protein; unlabeled probe served as competitor. (E) Effects of MdHY5 on promoter activity of MdNAC52 as determined by luciferase reporter assay (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.).
we created Y187 yeast strains containing ProMdMYB9-PHIS2 and ProMdMYB11-PHIS2 vectors. These yeast strains could not grow on SD/ −T/−H screening medium with 3-AT (120 mM) (Fig. 4A). Next, combinations of MdNAC52-AD with ProMdMYB9-PHIS2 or ProMdMYB11-PHIS2 were co-transformed into Y187 yeast strains. The transformed strains were able to grow on SD/−T/−L/−H selection medium containing 120 mM 3-AT. These findings confirmed that MdNAC52 could interact with the promoters of MdMYB9 and MdMYB11 (Fig. 4B). The target gene promoters contained NAC-binding motifs (ACACGT, ACGTGT). The results of the EMSA and ChIP analyses confirmed that MdNAC52 could bind to these motifs in the promoters of its target genes (Fig. 4C–E). To demonstrate the effect of MdNAC52 on the promoters of MdMYB9 and MdMYB11, we performed a luciferase reporter assay. For
GFP calli (Fig. 3B). Next, RT-PCR analyses confirmed that the transcript levels of genes encoding structural enzymes and TFs related to anthocyanin synthesis were higher in OE-NAC52 calli than in control GFP calli (Fig. 3E). This result indicated that overexpression of MdNAC52 resulted in anthocyanin accumulation. 3.4. MdNAC52 binds to promoters of MdMYB9 and MdMYB11 to increase anthocyanin accumulation The transcript levels of MdMYB9 and MdMYB11 were increased in OE-NAC52 calli, but the transcript level of MdMYB1 was not up-regulated. Therefore, we speculated that MdNAC52 may act upstream of MdMYB9 and MdMYB11. To explore the relationship between MdNAC52 and MdMYB9/MdMYB11, we conducted Y1H assays. First, 8
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Fig. 7. Increased accumulation of proanthocyanin (PA) in ‘Orin’ calli overexpressing MdNAC52. (A) DMACA staining of MdNAC52-OE ‘Orin’ calli (OE1–3) and ‘Orin’ calli containing GFP vector (GFP). (B) PA extracts from MdNAC52-OE calli and GFP calli. (C) PA contents in MdNAC52-OE calli and GFP calli. (D) Transcript levels of PA pathway-related genes in MdNAC52-OE calli and GFP calli.
3.6. Overexpression of MdNAC52 promotes soluble PA accumulation in ‘Orin’ calli
the assay, we constructed pMdMYB9-LUC and pMdMYB11-LUC reporters and the effector 35S:NAC52. MdNAC52 had certain activation effects on MdMYB9 and MdMYB11 in these LUC reporter assays (Fig. 4F).
MdMTB9 and MdMYB11 could promote PA accumulation [20], and our results confirmed that they functioned downstream of MdNAC52. The OE-NAC52 calli showed increased soluble PA contents. After staining with DMACA, the OE-NAC52 calli were deep blue, while GFP calli were lighter (Fig. 7A, B). A spectrophotometric analysis confirmed that the soluble PA content of OE-NAC52 calli was 36 times that of GFP calli (Fig. 7C). Subsequently, we used RT-PCR analyses to detect differences in gene expression between OE-NAC52 and GFP calli. The results showed that the transcript levels of MdMYB9, MdMYB11, LAR and ANR were higher in OE-NAC52 calli than in GFP calli (Fig. 7D). The transcript level of LAR was significantly up-regulated in OENAC52 calli. We speculated that MdNAC52 can directly bind to MdLAR to affect PA biosynthesis. To test this idea, we performed a Y1H assay, found that MdNAC52 interacted with the MdLAR promoter (Fig. 8A, B). In EMSA, ChIP, and luciferase reporter assays, MdNAC52 bound to the ACACGT cis-element in the MdLAR promoter and activated its expression (Fig. 8C, D, E). These results showed that MdNAC52 acts upstream of MdMYB9 and MdMYB11 to promote the PA biosynthesis and directly regulates MdLAR to modulate PA metabolism.
3.5. MdNAC52 is induced by light and acts downstream of MdHY5 The transcript level of MdNAC52 increased during apple coloration. To explore the effect of light on MdNAC52, we exposed ‘Orin’ calli to light and monitored the transcript level of MdNAC52 by RT-PCR. Its transcript level increased upon exposure to light and peaked at 24 h. We also monitored the transcript level of MdHY5, which encodes a lightregulator, and found that its transcript levels were consistent with those of MdNAC52 (Fig. 5A). A correlation analysis confirmed that the transcript level of MdHY5 was positively correlated with that of MdNAC52 (Fig. 5B). At the same time, we also examined the expression levels of MdMYB9, MdMYB11 and MdLAR, they induced by light was not obvious, the transcript levels increased at the later stage of light treatment (Fig. 5A). To investigate the relationship between MdNAC52 and MdHY5, Y1H assays were conducted. In these assays, MdHY5 interacted with the MdNAC52 promoter (Fig. 6A, B). The MdNAC52 promoter contains a Gbox, which has been verified to bind to the HY5 TF. In EMSA and ChIP assays, MdHY5 bound to the G-box cis-element in the MdNAC52 promoter (Fig. 6C, D). We also found that MdHY5 positively affected the promoter activity of MdNAC52 in a LUC reporter assay (Fig. 6E).
4. Discussion 4.1. MdNAC52 participates in anthocyanin accumulation Anthocyanin biosynthesis is regulated transcriptionally by wellstudied MYB, basic bHLH, and WD40 classes of TFs [44]. These 9
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Fig. 8. Interaction between MdNAC52 with MdLAR promoter. (A and B) Yeast one-hybrid assays confirming interaction between MdNAC52 and MdLAR promoter. (C) Binding of MdNAC52 to MdLAR promoter in vivo in ChIP-PCR assay. MdNAC52-GFP bound to ACACGT motif in MdLAR promoter. (D) Electrophoretic mobility shift assay showing binding of MdNAC52-HIS fusion protein to ACACGT in promoter of MdLAR in vitro. Red letters indicate NAC binding site. Labeled probe was incubated with MdNAC52-HIS protein; unlabeled probe served as competitor. (E) Luciferase assays verifying that MdNAC52 activates promoter of MdLAR (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.).
biosynthesis in response to sucrose treatment [52]. In our study, overexpression of MdNAC52 in ‘Orin’ calli increased anthocyanin accumulation. Structural genes in the anthocyanin pathway were also upregulated in OE-MdNAC52 calli. Two MYB TF genes, MdMYB9 and MdMYB11, also showed increased transcript levels in the OE-MdNAC52 calli. Further experiments demonstrated that MdNAC52 could bind to the promoters of MdMYB9 and MdMYB11 to promote anthocyanin accumulation. These results indicated that MdNAC52 acts upstream of MdMYB9 and MdMYB11 to regulate anthocyanin biosynthesis. Those results are similar to those reported for BL in blood-fleshed peach [53]. Unexpectedly, the expression of MdMYB1 was not up-regulated in OEMdNAC52 calli, which plays a key role during apple coloration [31]. The results indicated that MdNAC52 may function independently from MdMYB1 and the MdNAC52 is not such a key TF in apple coloration like MdMYB1, but it is involved in anthocyanin pathway and its expression
regulators form a MBW complex that binds to promoters of genes in the phenylpropanoid and anthocyanin biosynthetic pathway [17]. In apple, the MBW complex is critical for the anthocyanin synthesis pathway [45]. It has been reported that anthocyanin synthesis is affected by MYB TFs such as MdMYB1, MdMYBA, MdMYB10, and MdMYB16 [46–48]. Other TFs are also involved in the anthocyanin synthesis pathway. In apple, the zinc finger TF HY5 regulates itself and promotes anthocyanin accumulation by binding to the G-box in the MdMYB1 promoter [33,49]. Zhang et al. (2018) found that the ethylene-responsive TF MdERF1B regulates the accumulation of anthocyanin in apple [50]. The B-box zinc finger protein MdBBX20 can promote anthocyanin accumulation in response to low temperature [32]. In previous studies, overexpression of MdWRKY11 up-regulated the expression of F3H, DFR, ANS, and UFGT and promoted anthocyanin accumulation in apple calli [51]. In Arabidopsis, AtNAC32 was found to repress anthocyanin 10
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Fig. 9. Proposed model for MdNAC52-regulated anthocyanin and PA biosynthesis.
in the GFP control calli, and the transcript levels of MdMYB9, MdMYB11, and MdLAR were significantly up-regulated in OEMdNAC52 calli. The EMSA and other assays indicated that MdNAC52 could interact with the promoter of MdLAR and activate its expression. These results showed that MdNAC52 mediates the expression of MdMYB9 and MdMBY11 to regulate PA production, and also directly regulates MdLAR to increase PA accumulation. In summary, we have identified a NAC transcription factor MdNAC52, which promotes the biosynthesis of anthocyanin and PA in apple. The transcript levels of MdNAC52 increase during apple coloration. MdNAC52 interacts with the promoters of MdMYB9 and MdMYB11 to promote anthocyanin and PA accumulation, and binds to the promoter of the structural gene MdLAR in the PA pathway to regulate PA synthesis (Fig. 9). Our study provides novel insights into the role of a NAC transcription factor in regulating anthocyanin and PA accumulation in apple.
contribute to accumulating the anthocyanin. Together, these results show that the MdNAC52 TF controls the anthocyanin pathway through binding to the promoters of genes encoding anthocyanin-related MYB TFs. 4.2. Light-induced MdNAC52 acts downstream of MdHY5 Light affects anthocyanin synthesis by affecting the expression of genes encoding key enzymes in anthocyanin synthesis [54]. MdMYB1 and MdMYBA are anthocyacnin regulators that were first isolated from apple peel, and may function as photosensitizers [46]. In petunia, activation of the anthocyanin pathway requires complex interactions between developmental signals and environmental signals such as light [55]. In Arabidopsis, the transcript level of AtPAP1 associated with anthocyanin synthesis is up-regulated by light [30]. AtHY5 plays a role in the anthocyanin pathway by regulating the expression of MYB genes such as AtMYBL2, AtMYB111, and AtMYB12 [56]. In this study, we monitored the transcript levels of MdNAC52 using cDNA isolated from ‘Orin’ calli exposed to light. The transcript level of MdNAC52 gradually increased from 0 h to 24 h under light conditions, and the transcript level of MdHY5, which encodes a light-induced factor, exhibited the same pattern. A correlation analysis showed that the transcript level of MdHY5 was positively correlated with that of MdNAC52. Binding analyses confirmed that MdHY5 could bind to the promoter of MdNAC52 and activate gene transcription. Overall, these results showed that light induces HY5 expression, and then HY5 regulates the downstream TFs that control the anthocyanin pathway.
Funding This work was supported by the National Natural Science Foundation of China (31572091 and 31730080), National Key Research and Development Project of China (2016YFC0501505), and Agroscientific Research in the Public Interest (201303093). Acknowledgments We appreciate the help of Shenghui Jiang, Nan Wang, and Zongying Zhang in setting up the experiments. We thank Jennifer Smith, PhD, from Liwen Bianji, Edanz Group China (www.liwenbianji.cn/ac), for editing the English text of a draft of this manuscript.
4.3. MdNAC52 participates in PA accumulation As well as anthocyanins, PAs are also important flavonoid compounds. In plants, PAs participate in growth and development. Some TFs regulate both anthocyanin biosynthesis and PA biosynthesis. For example, Wang [21] found that the TF MdMYBPA1 could promote the synthesis of anthocyanin and PA. In apple, the ethylene pathway TF MdERF1B was shown to interact with a MYB TF to regulate anthocyanin and PA biosynthesis [50]. A previous study showed overexpression of MdMYB9 and MdMYB11 promoted not only anthocyanin accumulation but also PA accumulation [20]. In this study, we found that MdNAC52 acted upstream of MdMYB9 and MdMYB11 to regulate anthocyanin accumulation. Interestingly, the accumulation of PA was significantly higher in OE-MdNAC52 calli than
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Apple NAC transcription factor MdNAC52 regulates biosynthesis of anthocyanin and proanthocyanidin through MdMYB9 and MdMYB11
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Qingguo Sun, Shenghui Jiang, Tianliang Zhang, Haifeng Xu, Hongcheng Fang, Jing Zhang, ⁎ Mengyu Su, Yicheng Wang, Zongying Zhang, Nan Wang, Xuesen Chen National Key Laboratory of Crop Biology, College of Horticulture Science and Engineering, Shandong Agricultural University, Tai-An, Shandong, 271018, China
A R T I C LE I N FO
A B S T R A C T
Keywords: Apple NAC transcription factor Light Anthocyanin Proanthocyanidin MYB transcription factors
Anthocyanin and proanthocyanidin (PA) play important roles in plant growth and development. Although previous studies have identified many of the transcription factors involved in the anthocyanin and PA pathway, the regulation mechanisms of these pathways remain poorly understood. In this study, we identified a NAC transcription factor, MdNAC52, whose gene transcript levels increased during apple coloration. Apple calli overexpressing MdNAC52 accumulated anthocyanin. Yeast one-hybrid, electrophoretic mobility shift, chromatin immunoprecipitation, and luciferase reporter assays showed that MdNAC52 could interact with the promoters of MdMYB9 and MdMYB11 to regulate anthocyanin biosynthesis. MdNAC52 was targeted by MdHY5 in response to light. Interestingly, MdNAC52 participated in the regulation of PA biosynthesis through controlling the expression of MdMYB9 and MdMYB11. MdNAC52 could also bind to the LAR promoter to regulate its expression and promote PA synthesis. Overall, these findings establish that MdNAC52 binds to the promoters of MdMYB9 and MdMYB11 to promote anthocyanin and PA biosynthesis and directly regulates LAR to modulate PA metabolism. Our study provides new insights into the roles of a NAC transcription factor in regulating anthocyanin and PA accumulation in apple.
1. Introduction Flavonoids are plant secondary metabolites that play important roles in growth and development. Flavonoids include anthocyanins, flavonols, and proanthocyanidins (PAs) [1], which are present in the form of glycosides and methylated derivatives [2]. Flavonoids are diverse in structure and have a wide range of biological functions. For example, the accumulation of anthocyanins in the vacuoles of plant epidermal cells can protect plants from damage caused by ultraviolet radiation [3]. The accumulation of anthocyanins can confer red, blue, and purple colors to organs and tissues, which can help plants to attract pollinators and seed dispersers [4]. Proanthocyanins are generally colorless in plant cells, but are easily oxidized to form brownish substances that bind to each other within the cell [5]. They also have antioxidant activity and are associated with cardiovascular health, so their dietary intake is beneficial for human health [6–9]. In recent years, the flavonoid pathway has been widely studied in various plant species. The gene encoding chalcone synthase, CHS, was
the first flavonoid biosynthesis gene to be isolated [10]. This gene was isolated from parsley in the 1980s. Subsequently, flavonoid biosynthesis pathways have been studied in Zea mays, petunia, snapdragon, Arabidopsis, grape, and other species [11–15]. Key enzymes in the pathway have gradually been identified. The late stage of anthocyanin biosynthesis depends mainly on structural genes such as DFR encoding dihydroflavonol 4-reductase, ANS encoding anthocyanin synthase, and UFGT encoding glycosyltransferase (UFGT) [16]. The synthesis of PA mainly depends on leucoanthocyanidin reductase (LAR) and anthocyanidin reductase (ANR) [16]. Plant growth and development not only depends on a series of structural genes, but also on a large number of transcription factors (TFs). In the flavonoid pathway, the MYB–bHLH–WD40 (MBW) complex regulates the accumulation of anthocyanin and PA synergistically by controlling the expression of structural genes [17]. Previous studies have shown that MdbHLH3, a low-temperature-induced bHLH TF in apple, interacts with MdMYB1 through two regions (amino acids 1–23 and 186–228) [18]. MdEIL1 functions upstream of MdMYB1 and
Abbreviations: PA, proanthocyanidin; TF, transcription factor; DAFB, days after full bloom; DMACA, 4-dimethylaminocinnamaldehyde; SD/−T/−L/−H, the Murashige and Skoog medium lacking Trp Leu, and His; 3-AT, 3-amino-12,4-triazole; EMSA, electrophoretic mobility shift assay; ChIP, chromatin immunoprecipitation-qPCR; LUC, luciferase reporter assays ⁎ Corresponding author. E-mail address: [email protected] (X. Chen). https://doi.org/10.1016/j.plantsci.2019.110286 Received 14 June 2019; Received in revised form 26 August 2019; Accepted 25 September 2019 Available online 27 September 2019 0168-9452/ © 2019 Elsevier B.V. All rights reserved.
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37°52′, E121°39′), Shandong, China. Fruits were bagged at around 30 days after full bloom (DAFB). Samples for these experiments were collected at 0, 2, 4, 6, 8 and 12 days after the fruits were de-bagged. All samples were stored at −80 °C. ‘Orin’ apple calli were grown on Murashige and Skoog medium containing 0.5 mg L−1 2,4-dichlorophenoxyacetic acid and 1 mg L−1 6benzyl-aminopurine for genetic transformation. The calli were cultured at 25 °C in darkness.
activates its expression, resulting in anthocyanin biosynthesis, MdMYB1 binds to MdERF3, a key regulator in the ethylene pathway [19]. An [20] found that both MdMYB9 and MdMYB11 directly bind to the promoters of LAR, ANR, and ANS, whereas MdbHLH3 is recruited upstream of MdMYB9 and MdMYB11 and regulates their transcription. MdMYBPA1 not only promotes the accumulation of anthocyanin in response to low temperature via its interaction with MdbHLH33, but also promotes the PA biosynthesis by forming multimers with MdMYB9/11 [21]. It is noteworthy that not only MYB TFs but also other TFs are involved in the flavonoid pathway. Previous studies have shown that TTG2, a WRKY TF, interferes with the production of tannins in the seed coat [22]. The E3 ubiquitin ligase COP1/SPA interacts with PAP1 and PAP2, and these two proteins are required for the production of anthocyanin [23]. MdHY5 can bind to FLS (encoding flavonol synthase) to promote and regulate flavonoid metabolism in apple [24]. A previous study showed that, under light conditions, genes associated with flavonoid synthesis were up-regulated in transgenic lines overexpressing AtNAC78 [25]. The NAC TFs make up one of the largest gene families. In Arabidopsis, nearly 90 NAC proteins have been identified [26]. All NAC TFs contain a plant-specific highly conserved N-terminal domain known as the NAC domain [27]. The NAC TFs are involved in plant growth and development. For example, NACs participate in signaling of auxins, abscisic acid, and ethylene [28]. Genetic and abiotic factors such as drought, temperature, and light affect the biosynthesis of anthocyanin and PA [29]. Among these factors, light is an important environmental stimulus promoting anthocyanin accumulation. The mechanism of light-induced regulation of anthocyanin biosynthetic pathways has been determined. In Arabidopsis, the expression of MYB (PAP1 and PAP2) and bHLH (TT8) promote anthocyanin accumulation in response to light [30]. In apple, MdCOP1 promotes the expression of MdMYB1 in the nucleus to regulate anthocyanin biosynthesis in the light, but MdCOP1 degrades MdMYB1 in the nucleus under dark conditions [31]. Ultraviolet light induces the expression of MdBBX20, which encodes a B-box zinc finger protein that promotes anthocyanin accumulation synergistically with MdHY5 [32]. HY5 also functions upstream of light-induced MYB transcription to promote anthocyanin biosynthesis [33]. In light-grown leaf tissues, cryptochrome 1 (cy1) is involved in controlling the expression of CHS to increase anthocyanin biosynthesis in response to ultraviolet-B radiation [30]. Transcriptome sequencing was performed on Yanfu 3 and Yanfu 8, showed that some TF families such as MYB, WRKY and NAC have different transcript levels in the two materials, we found a TF MdNAC52 which the transcript level was higher in the apple line ‘YanFu 8′ than in ‘YanFu 3′, and the ‘YanFu 8′ peel is deeper and full red, while the ‘YanFu 3′ peel is lighter than ‘YanFu 8′, those indicating that MdNAC52 may participate in anthocyanin biosynthesis [34]. In this study, we cloned MdNAC52 and found that its encoded protein was located in the nucleus. During apple coloration, the expression of MdNAC52 increased. ‘Orin’ apple calli overexpressing MdNAC52 (OE-MdNAC52) showed enhanced anthocyanin accumulation and PA biosynthesis. Further analyses showed that MdNAC52 could regulate the expression of the target genes MdMYB9 and MdMYB11 to promote anthocyanin and PA biosynthesis, and that MdHY5 could bind to the promoter of MdNAC52 to promote its expression. In addition, MdNAC52 directly regulated LAR to modulate PA metabolism. Our study reveals the detailed mechanism of the regulation of anthocyanin and PA accumulation, and provides a theoretical basis to enrich genetic resources related to flavonoid biosynthesis.
2.2. Transformation of apple calli with MdNAC52 MdNAC52 was inserted into the pRI101-AN vector with a GFP tag to generate the recombinant plasmid 35S:MdNAC52-GFP. The recombinant plasmid was inserted into Agrobacterium tumefaciens LBA4404, which was then incubated with 2-week-old apple calli for 30 min at room temperature. The calli were transferred to MS solid medium with 250 μg mL−1 carboxybenzyl (Solarbio, Beijing, China; http://solarbio.bioon.com.cn/) and 50 μg mL-1 kanamycin (Solarbio) to select for transformants. 2.3. Measurement of anthocyanin content Each sample (0.5 g) was ground to a powder in liquid nitrogen and then incubated in 5 mL 1% (v/v) HCl-methanol for 24 h at 4 °C in darkness. After centrifugation, KCl and NaAc buffers were added to aliquots of the supernatant, which were then mixed and incubated for 20 min at 4 °C in darkness. The solutions were centrifuged at 8000 rcf (×g) for 15 min. The absorbance of the supernatant was measured using a UV-2450 spectrophotometer (Shimadzu, Kyoto, Japan) at 510 nm and 700 nm (i.e., OD510 and OD700). The anthocyanin content was calculated using the following formula: ΔA × 5 × 0.005 × 1000 × 449.2/(26,900 × 0.5), where ΔA = (OD510 − OD700 at pH = 1.0) − (OD510 − OD700 at pH = 4.5). 2.4. 4-Dimethylaminocinnamaldehyde staining and determination of PA content The presence of PA was detected by 4-dimethylaminocinnamaldehyde (DMACA) staining, and the PA content was determined as described by Li [35]. The sample was ground in liquid nitrogen, 0.45 g of the powder was suspended in a mixture of 1 mL of 70% acetone solution (v/v) and 0.1% (w/v) ascorbic acid, and the mixture was incubated in the dark at 4 °C for 30 min. This was repeated three times, then the mixture was centrifuged and the supernatant was collected. The supernatant (2.5 mL) was mixed with 3 mL ether at −20 °C. The soluble liquid PA separated into the lower liquid phase. Then, 2 mL soluble PA, 0.5 mL 2% (w/v) DMACA, and 1 mL methanol were mixed and incubated at room temperature for 20 min. The absorbance was measured at 643 nm using a UV–vis spectrophotometer (UV-2450; Shimadzu). The concentration of PA was determined by comparison with a standard curve prepared using a catechin reference standard (Sigma, St Louis, MO, USA). 2.5. RNA extraction and RT-PCR analyses Total RNA was extracted using an RNAprep Pure Plant Kit (Tiangen, Beijing, China; http://www.tiangen.com/en/). The RNA was reversetranscribed into to cDNA using the RevertAidTM First Strand cDNA Synthesis Kit (TransGen, Beijing, China; http://www.transbionovo. com/) according to the manufacturer’s instructions. The 20-μL reaction mixture was prepared according to the instructions of the SYBR Green PCR Master Mix Kit, and contained 10 μL 2.5 × ReaL MasterMix/ 20 × SYBR solution, 7 μL ddH2O, 1 μL cDNA (50 ng·μL−1), and 1 μL each of upstream and downstream primers (5 μmol L·L−1). The analyses were conducted using a CFX96 Real-Time PCR Detection system (BioRad, Hercules, CA, USA). Three replicates were prepared for each
2. Materials and methods 2.1. Plant materials Trees of the apple cultivar ‘YanFu 3′ were grown in Yan Tai (N 2
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Fig. 1. Phylogenetic tree, sequence analyses, and subcellular localization of MdNAC52. (A) Phylogenetic analysis of NAC proteins from different species. Genes involved in the construction of phylogenetic tree are listed in Supplementary Table S3. (B) Amino acid sequence alignment of NAC transcription factors containing NAC domain. (C) Subcellular localization of 35S::MdNAC52-GFP fusion construct and 35S::GFP construct.
2.6. Cloning of MdNAC52 and bioinformatics analysis
sample, and the transcript level of MdActin was determined as the internal control. The data were analyzed using the (Ct) 2 –ΔΔCt method [36]. The primers used for RT-PCR are listed in Supplementary Table S2.
MdNAC52 (MD01G1094000) was amplified in vitro using phusion polymerase. The primers are listed in Supplementary Table S1. The 3
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Fig. 2. Anthocyanin accumulation and transcript levels of anthocyanin-related genes in ‘YanFu 3′ apples. (A) Mature fruits harvested on days 0, 2, 4, 6, 8, and 12 after de-bagging. (B) Anthocyanin contents in apple skins of ‘Yanfu 3′. (C) Transcript levels of MdNAC52 and related genes in anthocyanin biosynthesis pathway.
MMG solution (100 mL 0.8 mol·L−1 mannitol; 3 mL 1 mol·L−1 MgCl2; 2.667 mL 0.3 mol·L−1 MES). The GFP fluorescence in protoplasts was detected under a fluorescence microscope.
software MEGA5.1 and DNAMAN were used to construct the phylogenetic tree and align amino acid sequences. 2.7. Extraction of protoplasts and subcellular localization analyses
2.8. Yeast one-hybrid assay Calli harboring OE-MdNAC52 fused to a GFP tag were added to 10 ml cell hydrolysis solution, vacuum infiltrated for 30 min, kept in the dark at room temperature for 12 h, and gently shaken to release protoplasts. To this mixture was added an equal volume of W5 solution (1.333 mL 0.3 mol·L−1 MES; 1 mL 1 mol·L−1 KCl; 6.16 mL 5 mol·L−1 NaCl; 25 mL 1 mol·L−1 CaCl2), and then the protoplasts were filtered through a nylon cloth (75 μm pore diameter). The obtained filtrate was centrifuged at 500 rpm for 10 min, the supernatant was discarded, and the precipitate was added to 10 mL W5 solution. After 30 min, the supernatant was discarded and the protoplasts were suspended in 2 mL
For the yeast one-hybrid (Y1H) assays, each of the MdNAC52 and MdHY5 sequences was inserted into the pGADT7 vector to generate the MdNAC52-AD and MdHY5-AD constructs, respectively. Additionally, each of the MdNAC52, MdMYB9, MdMYB11, and MdLAR promoter sequences was inserted into the pHIS2 vector. The recombinant plasmids with the MdNAC52 or MdHY5 sequence and promoter fragments were co-transformed into the Y187 strain, then the interactions were examined on the medium lacking Trp, Leu, and His (SD/−T/−L/−H) with 3-amino-1,2,4-triazole (3-AT) at the optimal screening 4
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Fig. 3. Functional characterization of MdNAC52 overexpressed in ‘Orin’ apple calli. (A) ‘Orin’ calli overexpressing MdNAC52 (OE1–3) and ‘Orin’ calli containing empty GFP vector (GFP). (B) Anthocyanin contents in MdNAC52-OE calli and GFP calli. (C) PCR analysis confirming presence of transgene in OE-MdNAC52 calli. (D) Western blotting analysis of OE-MdNAC52 calli. (E) Transcript levels of MdNAC52 and genes encoding anthocyanin biosynthesis-related enzymes and TFs in MdNAC52-OE calli and GFP calli.
2.10. LUC reporter assay
concentration.
The sequences of MdNAC52 and MdHY5 were each recombined into the pHBT-AvrRpm1 effector, and the promoter sequences of MdNAC52, MdMYB9, MdMYB11, and MdLAR were inserted into the pFRK1-LUCnos reporter. For the analyses, 1 μL UBQ-GUS, 3 μL pFRK1-LUC-nos recombinant reporter, and 6 μL recombinant effector were co-transformed into ‘Orin’ apple callus protoplasts in the presence of 40% polyethylene glycol for transient expression. After incubation for 6 h at 24 °C, the fluorescence levels of LUC and GUS were tested using a multimode plate reader (Victor X4, PerkinElmer, Norwalk, CT, USA; http://www.perkinelmer.com/). Relative LUC activity was determined based on the ratio of LUC to GUS values.
2.9. Electrophoretic mobility shift assay The coding sequences of MdNAC52 and MdHY5 were each recombined into the vector pET-32a(+) (His-Tag), and each recombinant plasmid was transformed into Escherichia coli BL21 (DE3). The fusion protein was purified using a His-tagged protein purification kit (CWBIO Inc., Beijing, China). Electrophoretic mobility shift assays (EMSAs) were conducted using a LightShiftTM EMSA Optimization and Control Kit (Thermo Scientific, Waltham, MA, USA; https://www.thermofisher. com). The reaction mixture contained 2 μL 1 × binding buffer (2.5% glycerol, 5 mM MgCl3, 10 mM EDTA, 50 mM KCl,), 10 μL protein, and 1 μL unlabeled probe. The mixture was reacted at room temperature for 15 min, and then 1 μL labeled probe was added to initiate the 15-min competition reaction. 5
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Fig. 4. Interaction between MdNAC52 and promoters of MdMYB9 and MdMYB11. (A and B) Yeast one-hybrid assays showing interaction between MdNAC52 and promoters of MdMYB9 and MdMYB11. (C and D) Electrophoretic mobility shift assay showing direct binding of MdNAC52-HIS fusion protein to ACACGT and ACGTGT motifs in promoters of MdMYB9 and MdMYB11 in vitro. Red letters indicate NAC binding site. Labeled probe was incubated with MdNAC52-HIS protein; unlabeled probe served as competitor. (E) Binding of MdNAC52 to MdMYB9 and MdMYB11 promoters in vivo in ChIP-PCR assay. MdNAC52-GFP bound to ACACGT and ACGTGT motifs in MdMYB9 and MdMYB11 promoters. (F) Luciferase assays verifying that MdNAC52 activates promoters of MdMYB9 and MdMYB11 (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.).
and MdHY5 fused to a GFP tag were analyzed in the ChIP-qPCR assay. The ChIP experiment was carried out using a ChIP kit (Upstate Biotech, Lake Placid, NY, USA) following the manufacturer’s instructions. The amount of immunoprecipitated chromatin was detected by PCR. The
2.11. Chromatin immunoprecipitation-qPCR analysis The chromatin immunoprecipitation (ChIP) experiment was conducted as described by He [37]. Transgenic calli carrying MdNAC52 6
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Fig. 5. MdHY5 and MdNAC52 transcript levels in ‘Orin’ apple calli exposed to light as determined by RT-PCR. (A) Transcript levels of MdNAC52 and MdHY5. (B) Correlation analysis between transcript levels of MdNAC52 and MdHY5.
experiment was repeated three times.
3.2. Anthocyanin, proanthocyanidin content and TF transcript levels during apple coloration
3. Results
After the fruits were de-bagged, the peel gradually turned red and the anthocyanin content increased, meanwhile, the proanthocyanidin content was reduced. (Fig. 2A, B). To study the expression pattern of MdNAC52, we monitored its transcript levels in ‘YanFu 3′ during apple coloration, and found that its transcript levels increased from 0 d to 8 d. We also analyzed the transcript levels of anthocyanin-related structural genes and TFs. The transcript levels of key genes for anthocyanin synthesis (MdDFR, MdANS, MdUFGT) peaked at 8 d. The MdNAC52 transcript level was consistent with the expression levels of these genes and those of the related TF MdMYB1, which also peaked at 8 d after debagging, while MdMYB9, MdMYB11 have showed different expression pattern (Fig. 2C). And the transcript levels of proanthocyanidin-related structural genes MdLAR, MdANR were consistent with the proanthocyanidin content, minimum at 8 d (Fig. 2C). These observations suggested that MdNAC52 may act as an anthocyanin regulator in apple.
3.1. Phylogenetic tree, sequence analyses, and subcellular localization of MdNAC52 The NAC domains of predicted and known NAC family proteins were classified into subgroups I–XVI according to sequence similarity [38]. To analyze the relationship between MdNAC52 and other NACs, we constructed a phylogenetic tree. The results showed that MdNAC52 belongs to subgroup Ⅳ(ONAC003). Many studies have shown that NAC proteins are involved in plant growth and environmental adaptation [39,40]. AtNAC19, AtNAC72 are in subgroup Ⅱ and are important for tolerance to drought [41]. The NAC proteins in the subgroup Ⅳ might be participated in the diversity of developmental stages and tissuespecific [42]. The NAC domain is composed of approximately 100 amino acid residues [43]. Amino acid sequence alignment analyses confirmed that MdNAC52 contains a NAC domain (Fig. 1B). To test whether MdNAC52 was localized in the nucleus like other TFs, we constructed the 35S::MdNAC52-GFP vector and transformed it into ‘Orin’ calli, with 35S::GFP as the control. Protoplasts were isolated from the transgenic calli, and the subcellular localization of the fusion protein was detected by fluorescence microscopy. 35S::MdNAC52-GFP was localized in the nucleus, while 35S::GFP was distributed throughout the protoplasts (Fig. 1C). These findings indicated that MdNAC52 was localized in the nucleus.
3.3. MdNAC52 promotes anthocyanin accumulation in apple calli To explore the role of MdNAC52 in regulating anthocyanin accumulation, we generated ‘Orin’ calli overexpressing MdNAC52 (OENAC52), and verified the presence of the transgene by PCR and western blot assays (Fig. 3C, D). Under light conditions, OE-NAC52 calli gradually became red, while the calli transformed with the GFP vector did not (Fig. 3A). Spectrophotometric analyses confirmed that the anthocyanin content was significantly higher in OE-NAC52 calli than in the 7
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Fig. 6. Binding of MdHY5 to promoter of MdNAC52. (A and B) Yeast one-hybrid assays showing interaction between MdHY5 and MdNAC52 promoter. (C) Binding of MdHY5 to MdNAC52 promoter in vivo in ChIP-PCR assay. MdHY5-GFP bound to G-box motif in MdNAC52 promoter. (D) Electrophoretic mobility shift assay confirming direct binding of MdHY5-HIS fusion protein to G-box in promoter of MdNAC52 in vitro. Red letters indicate HY5 binding site. Labeled probe was incubated with MdHY5-HIS protein; unlabeled probe served as competitor. (E) Effects of MdHY5 on promoter activity of MdNAC52 as determined by luciferase reporter assay (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.).
we created Y187 yeast strains containing ProMdMYB9-PHIS2 and ProMdMYB11-PHIS2 vectors. These yeast strains could not grow on SD/ −T/−H screening medium with 3-AT (120 mM) (Fig. 4A). Next, combinations of MdNAC52-AD with ProMdMYB9-PHIS2 or ProMdMYB11-PHIS2 were co-transformed into Y187 yeast strains. The transformed strains were able to grow on SD/−T/−L/−H selection medium containing 120 mM 3-AT. These findings confirmed that MdNAC52 could interact with the promoters of MdMYB9 and MdMYB11 (Fig. 4B). The target gene promoters contained NAC-binding motifs (ACACGT, ACGTGT). The results of the EMSA and ChIP analyses confirmed that MdNAC52 could bind to these motifs in the promoters of its target genes (Fig. 4C–E). To demonstrate the effect of MdNAC52 on the promoters of MdMYB9 and MdMYB11, we performed a luciferase reporter assay. For
GFP calli (Fig. 3B). Next, RT-PCR analyses confirmed that the transcript levels of genes encoding structural enzymes and TFs related to anthocyanin synthesis were higher in OE-NAC52 calli than in control GFP calli (Fig. 3E). This result indicated that overexpression of MdNAC52 resulted in anthocyanin accumulation. 3.4. MdNAC52 binds to promoters of MdMYB9 and MdMYB11 to increase anthocyanin accumulation The transcript levels of MdMYB9 and MdMYB11 were increased in OE-NAC52 calli, but the transcript level of MdMYB1 was not up-regulated. Therefore, we speculated that MdNAC52 may act upstream of MdMYB9 and MdMYB11. To explore the relationship between MdNAC52 and MdMYB9/MdMYB11, we conducted Y1H assays. First, 8
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Fig. 7. Increased accumulation of proanthocyanin (PA) in ‘Orin’ calli overexpressing MdNAC52. (A) DMACA staining of MdNAC52-OE ‘Orin’ calli (OE1–3) and ‘Orin’ calli containing GFP vector (GFP). (B) PA extracts from MdNAC52-OE calli and GFP calli. (C) PA contents in MdNAC52-OE calli and GFP calli. (D) Transcript levels of PA pathway-related genes in MdNAC52-OE calli and GFP calli.
3.6. Overexpression of MdNAC52 promotes soluble PA accumulation in ‘Orin’ calli
the assay, we constructed pMdMYB9-LUC and pMdMYB11-LUC reporters and the effector 35S:NAC52. MdNAC52 had certain activation effects on MdMYB9 and MdMYB11 in these LUC reporter assays (Fig. 4F).
MdMTB9 and MdMYB11 could promote PA accumulation [20], and our results confirmed that they functioned downstream of MdNAC52. The OE-NAC52 calli showed increased soluble PA contents. After staining with DMACA, the OE-NAC52 calli were deep blue, while GFP calli were lighter (Fig. 7A, B). A spectrophotometric analysis confirmed that the soluble PA content of OE-NAC52 calli was 36 times that of GFP calli (Fig. 7C). Subsequently, we used RT-PCR analyses to detect differences in gene expression between OE-NAC52 and GFP calli. The results showed that the transcript levels of MdMYB9, MdMYB11, LAR and ANR were higher in OE-NAC52 calli than in GFP calli (Fig. 7D). The transcript level of LAR was significantly up-regulated in OENAC52 calli. We speculated that MdNAC52 can directly bind to MdLAR to affect PA biosynthesis. To test this idea, we performed a Y1H assay, found that MdNAC52 interacted with the MdLAR promoter (Fig. 8A, B). In EMSA, ChIP, and luciferase reporter assays, MdNAC52 bound to the ACACGT cis-element in the MdLAR promoter and activated its expression (Fig. 8C, D, E). These results showed that MdNAC52 acts upstream of MdMYB9 and MdMYB11 to promote the PA biosynthesis and directly regulates MdLAR to modulate PA metabolism.
3.5. MdNAC52 is induced by light and acts downstream of MdHY5 The transcript level of MdNAC52 increased during apple coloration. To explore the effect of light on MdNAC52, we exposed ‘Orin’ calli to light and monitored the transcript level of MdNAC52 by RT-PCR. Its transcript level increased upon exposure to light and peaked at 24 h. We also monitored the transcript level of MdHY5, which encodes a lightregulator, and found that its transcript levels were consistent with those of MdNAC52 (Fig. 5A). A correlation analysis confirmed that the transcript level of MdHY5 was positively correlated with that of MdNAC52 (Fig. 5B). At the same time, we also examined the expression levels of MdMYB9, MdMYB11 and MdLAR, they induced by light was not obvious, the transcript levels increased at the later stage of light treatment (Fig. 5A). To investigate the relationship between MdNAC52 and MdHY5, Y1H assays were conducted. In these assays, MdHY5 interacted with the MdNAC52 promoter (Fig. 6A, B). The MdNAC52 promoter contains a Gbox, which has been verified to bind to the HY5 TF. In EMSA and ChIP assays, MdHY5 bound to the G-box cis-element in the MdNAC52 promoter (Fig. 6C, D). We also found that MdHY5 positively affected the promoter activity of MdNAC52 in a LUC reporter assay (Fig. 6E).
4. Discussion 4.1. MdNAC52 participates in anthocyanin accumulation Anthocyanin biosynthesis is regulated transcriptionally by wellstudied MYB, basic bHLH, and WD40 classes of TFs [44]. These 9
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Fig. 8. Interaction between MdNAC52 with MdLAR promoter. (A and B) Yeast one-hybrid assays confirming interaction between MdNAC52 and MdLAR promoter. (C) Binding of MdNAC52 to MdLAR promoter in vivo in ChIP-PCR assay. MdNAC52-GFP bound to ACACGT motif in MdLAR promoter. (D) Electrophoretic mobility shift assay showing binding of MdNAC52-HIS fusion protein to ACACGT in promoter of MdLAR in vitro. Red letters indicate NAC binding site. Labeled probe was incubated with MdNAC52-HIS protein; unlabeled probe served as competitor. (E) Luciferase assays verifying that MdNAC52 activates promoter of MdLAR (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.).
biosynthesis in response to sucrose treatment [52]. In our study, overexpression of MdNAC52 in ‘Orin’ calli increased anthocyanin accumulation. Structural genes in the anthocyanin pathway were also upregulated in OE-MdNAC52 calli. Two MYB TF genes, MdMYB9 and MdMYB11, also showed increased transcript levels in the OE-MdNAC52 calli. Further experiments demonstrated that MdNAC52 could bind to the promoters of MdMYB9 and MdMYB11 to promote anthocyanin accumulation. These results indicated that MdNAC52 acts upstream of MdMYB9 and MdMYB11 to regulate anthocyanin biosynthesis. Those results are similar to those reported for BL in blood-fleshed peach [53]. Unexpectedly, the expression of MdMYB1 was not up-regulated in OEMdNAC52 calli, which plays a key role during apple coloration [31]. The results indicated that MdNAC52 may function independently from MdMYB1 and the MdNAC52 is not such a key TF in apple coloration like MdMYB1, but it is involved in anthocyanin pathway and its expression
regulators form a MBW complex that binds to promoters of genes in the phenylpropanoid and anthocyanin biosynthetic pathway [17]. In apple, the MBW complex is critical for the anthocyanin synthesis pathway [45]. It has been reported that anthocyanin synthesis is affected by MYB TFs such as MdMYB1, MdMYBA, MdMYB10, and MdMYB16 [46–48]. Other TFs are also involved in the anthocyanin synthesis pathway. In apple, the zinc finger TF HY5 regulates itself and promotes anthocyanin accumulation by binding to the G-box in the MdMYB1 promoter [33,49]. Zhang et al. (2018) found that the ethylene-responsive TF MdERF1B regulates the accumulation of anthocyanin in apple [50]. The B-box zinc finger protein MdBBX20 can promote anthocyanin accumulation in response to low temperature [32]. In previous studies, overexpression of MdWRKY11 up-regulated the expression of F3H, DFR, ANS, and UFGT and promoted anthocyanin accumulation in apple calli [51]. In Arabidopsis, AtNAC32 was found to repress anthocyanin 10
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Fig. 9. Proposed model for MdNAC52-regulated anthocyanin and PA biosynthesis.
in the GFP control calli, and the transcript levels of MdMYB9, MdMYB11, and MdLAR were significantly up-regulated in OEMdNAC52 calli. The EMSA and other assays indicated that MdNAC52 could interact with the promoter of MdLAR and activate its expression. These results showed that MdNAC52 mediates the expression of MdMYB9 and MdMBY11 to regulate PA production, and also directly regulates MdLAR to increase PA accumulation. In summary, we have identified a NAC transcription factor MdNAC52, which promotes the biosynthesis of anthocyanin and PA in apple. The transcript levels of MdNAC52 increase during apple coloration. MdNAC52 interacts with the promoters of MdMYB9 and MdMYB11 to promote anthocyanin and PA accumulation, and binds to the promoter of the structural gene MdLAR in the PA pathway to regulate PA synthesis (Fig. 9). Our study provides novel insights into the role of a NAC transcription factor in regulating anthocyanin and PA accumulation in apple.
contribute to accumulating the anthocyanin. Together, these results show that the MdNAC52 TF controls the anthocyanin pathway through binding to the promoters of genes encoding anthocyanin-related MYB TFs. 4.2. Light-induced MdNAC52 acts downstream of MdHY5 Light affects anthocyanin synthesis by affecting the expression of genes encoding key enzymes in anthocyanin synthesis [54]. MdMYB1 and MdMYBA are anthocyacnin regulators that were first isolated from apple peel, and may function as photosensitizers [46]. In petunia, activation of the anthocyanin pathway requires complex interactions between developmental signals and environmental signals such as light [55]. In Arabidopsis, the transcript level of AtPAP1 associated with anthocyanin synthesis is up-regulated by light [30]. AtHY5 plays a role in the anthocyanin pathway by regulating the expression of MYB genes such as AtMYBL2, AtMYB111, and AtMYB12 [56]. In this study, we monitored the transcript levels of MdNAC52 using cDNA isolated from ‘Orin’ calli exposed to light. The transcript level of MdNAC52 gradually increased from 0 h to 24 h under light conditions, and the transcript level of MdHY5, which encodes a light-induced factor, exhibited the same pattern. A correlation analysis showed that the transcript level of MdHY5 was positively correlated with that of MdNAC52. Binding analyses confirmed that MdHY5 could bind to the promoter of MdNAC52 and activate gene transcription. Overall, these results showed that light induces HY5 expression, and then HY5 regulates the downstream TFs that control the anthocyanin pathway.
Funding This work was supported by the National Natural Science Foundation of China (31572091 and 31730080), National Key Research and Development Project of China (2016YFC0501505), and Agroscientific Research in the Public Interest (201303093). Acknowledgments We appreciate the help of Shenghui Jiang, Nan Wang, and Zongying Zhang in setting up the experiments. We thank Jennifer Smith, PhD, from Liwen Bianji, Edanz Group China (www.liwenbianji.cn/ac), for editing the English text of a draft of this manuscript.
4.3. MdNAC52 participates in PA accumulation As well as anthocyanins, PAs are also important flavonoid compounds. In plants, PAs participate in growth and development. Some TFs regulate both anthocyanin biosynthesis and PA biosynthesis. For example, Wang [21] found that the TF MdMYBPA1 could promote the synthesis of anthocyanin and PA. In apple, the ethylene pathway TF MdERF1B was shown to interact with a MYB TF to regulate anthocyanin and PA biosynthesis [50]. A previous study showed overexpression of MdMYB9 and MdMYB11 promoted not only anthocyanin accumulation but also PA accumulation [20]. In this study, we found that MdNAC52 acted upstream of MdMYB9 and MdMYB11 to regulate anthocyanin accumulation. Interestingly, the accumulation of PA was significantly higher in OE-MdNAC52 calli than
Appendix A. Supplementary data Supplementary material related to this article can be found, in the online version, at doi:https://doi.org/10.1016/j.plantsci.2019.110286. References [1] C.A. Williamsa, R.J. Grayer, Anthocyanins and other flavonoids, Nat. Prod. Rep. 21 (2004) 539–573. [2] M. Škerget, P. Kotnik, M. Hadolin, A.R. Hraš, M. Simonič, Ž. Knez, Phenols, proanthocyanidins, flavones and flavonols in some plant materials and their antioxidant activities, Food Chem. 89 (2005) 191–198.
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