The Nitrate-Responsive Protein MdBT2 Regulates ...ture, and nutrition, influence anthocyanin biosynthesis The Nitrate-Responsive Protein MdBT2 Regulates Anthocyanin Biosynthesis by

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Anthocyanins are a class of flavonoids and robust scavengers of reactive oxygen species, and they may act as critical regulators during various abiotic and bi-otic stresses (Chalker-Scott, 1999). In plants, they accu-mulate in the cell vacuole and cause various organs to develop bright colors that range from pink to purple, which are important for attracting pollinators and seed dispersal (Cipollini and Levey, 1997; Shang et al., 2011). Generally, their accumulation accompanies the devel-opment or ripening process in several organs, which in-clude flowers and fruits (Jimenez-Garcia et al., 2013). In addition, anthocyanin in the edible organs, especially

fruits, is recognized as a compound with potential health benefits for consumers due to its antioxidant properties (Pourcel et al., 2007).

The biosynthesis of anthocyanin represents a branch of the flavonoid pathway, and the catalytic enzymes and their encoding genes have been found in a variety of plant species (Koes et al., 2005; Hichri et al., 2011). Furthermore, the MYB/bHLH/WD40 (MBW) com-plex that contains MYB transcription factors (TFs), basic helix-loop-helix (bHLH) TFs and WD-repeat proteins acts as a core player that regulates anthocy-anin accumulation in plant species, including maize (Zea mays), antirrhinum (Antirrhinum majus), petunia (Petunia hybrida) and Arabidopsis (Arabidopsis thaliana; Ramsay and Glover, 2005; Gonzalez et al., 2008). In these plants, R2R3 MYB TFs, including ARABIDOPSIS THALIANA PRODUCTION OF ANTHOCYANIN PIGMENT1 (AtPAP1), AtPAP2, ZmC1, PhMYBAN2, and AmROSEA1/2, participate in regulating the expression of anthocyanin biosynthesis by binding directly to the promoters of the corresponding genes and then induc-ing anthocyanin accumulation (Zimmermann et al., 2004; Schwinn et al., 2006; Allan et al., 2008; Gonzalez et al., 2008). Increasing numbers of MYB TFs and other MBW regulators that modulate anthocyanin biosyn-thesis and fruit coloration have been isolated and char-acterized in fruit trees (Jaakola, 2013; Xu et al., 2015).

In higher plants, numerous developmental and envi-ronmental factors, including hormones, light, tempera-ture, and nutrition, influence anthocyanin biosynthesis

The Nitrate-Responsive Protein MdBT2 Regulates Anthocyanin Biosynthesis by Interacting with the MdMYB1 Transcription Factor1[OPEN]

Xiao-Fei Wang,a,b,c Jian-Ping An,a,b,c Xin Liu,a,b,c Ling Su,a,b,c Chun-Xiang You,a,b,c and Yu-Jin Haoa,b,c,2

aNational Key Laboratory of Crop Biology, Shandong Agricultural University, Tai-An 271018, Shandong, ChinabNational Research Center for Apple Engineering and Technology, Shandong Agricultural University, Tai-An 271018, Shandong, ChinacCollege of Horticulture Science and Engineering, Shandong Agricultural University, Tai-An 271018, Shandong, ChinaORCID ID: 0000-0001-7258-3792 (Y.-J.H.)

In addition to scavenging reactive oxygen species, anthocyanins are pigments that give organs their color. In apple (Malus do-mestica), R2R3-MYB transcription factor MdMYB1 is a master regulator of anthocyanin biosynthesis and fruit coloration. In this study, we found that MdMYB1 was degraded via a ubiquitin-dependent pathway in response to nitrate, an inhibitor of anthocy-anin synthesis. Using a yeast two-hybrid (Y2H) approach, we found that the BTB-TAZ protein encoded by the nitrate-responsive gene MdBT2 interacts with MdMYB1. Pull-down and coimmunoprecipitation assays supported this conclusion. In vivo and in vitro experiments revealed that MdBT2 promoted the ubiquitination and degradation of MdMYB1 through a cullin protein Md-CUL3-independent pathway. Expression analysis demonstrated that MdBT2 and MdMYB1 were inversely regulated by nitrate and other environmental signals. Furthermore, we used transgenic approaches in apple and Arabidopsis (Arabidopsis thaliana) to characterize the function of MdBT2 in regulating anthocyanin biosynthesis in response to nitrate. Our findings provide insight into a mechanism involving the MdBT2-MdMYB1 pathway that regulates anthocyanin accumulation in apple and possibly in other plant species.

1This work was supported by grants from the National Natural Science Foundation of China (31430074, 31601742 and U1706202), Ministry of Education of the People’s Republic of China (IRT15R42), MOA (CARS-27), and Shandong Province (SDAIT-06-03).

2Address correspondence to [email protected] author responsible for distribution of materials integral to

the findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantphysiol.org) is: Yu-Jin Hao ([email protected]).

Y.-J.H. conceived the original screening and research plans; Y.-J.H. and C.-X.Y. supervised the experiments; X.-F.W., J.-P.A., X.L., and L.S. performed most of the experiments; C.-X.Y. provided technical assistance; Y.-J.H. and X.-F.W. designed the experiments and ana-lyzed the data; X.-F.W. and Y.-J.H. conceived the project and wrote the article with contributions from all the authors; Y.-J.H. and C.-X.Y. supervised and complemented the writing.

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(Lancaster and Dougall, 1992; Jaakola, 2013). Recent re-search has improved understanding of the mechanisms by which these factors regulate anthocyanin biosynthesis through the MBW complex. These factors regulate MBW members at both the transcriptional and posttranscrip-tional levels (Jaakola, 2013; Xu et al., 2015). For example, the expression levels of ARABIDOPSIS THALIANA TRANSPARENT TESTA GLABRA1, ARABIDOPSIS THALIANA GLABRA3 (AtGL3), AtPAP1, and AtPAP2 are induced by low temperature and high light in Arabidopsis (Lea et al., 2007), and many anthocyanin- associated bHLH and MYB genes are regulated tran-scriptionally by light and temperature in fruit trees (Li et al., 2012; Xie et al., 2012). MBW members are modulated at the posttranslational level in response to various stimuli. ARABIDOPSIS THALIANA BRASS-INOSTEROID-INSENSITIVE2 inhibits MBW activity in response to brassinosteroid signaling by phosphor-ylating AtEGL3 and AtGL3, which are subsequently targeted for ubiquitin-proteasome system-dependent degradation through the 26S proteasome pathway (Pa-tra et al., 2013; Cheng et al., 2014). Both in Arabidopsis and in apple (Malus domestica), MYB TFs are ubiquiti-nated by ARABIDOPSIS THALIANA CONSTITU-TIVE PHOTOMORPHOGENIC1 (COP1) E3 ligase and subsequently degraded by the 26S ubiquitin-protea-some in response to photoperiod (Li et al., 2012; Maier et al., 2013). Recently, it was found that apple RING E3 ligase MYB30-INTERACTING E3 LIGASE1 inhib-its anthocyanin accumulation by ubiquitinating and degrading the MdMYB1 protein (An et al., 2017). In apple, MdbHLH3 is phosphorylated by hexokinase1 in response to exogenous Glc treatment (Hu et al., 2016).

Nitrogen is also a critical player that regulates antho-cyanin accumulation in plants. Generally, low nitrogen promotes, while nitrogen oversupply inhibits the accu-mulation of anthocyanin (Rubin et al., 2009; Zhou et al., 2012; Wang et al., 2015). Nitrate is not only an import-ant nutrient for plant growth and development, but it is also a crucial signal molecule for modulating the expression of numerous genes encoding transcription factors, protein kinases/phosphatases, and enzymes in response to various environments (Scheible et al., 2004; Ho et al., 2009). Nitrate represses anthocyanin accumulation mainly by decreasing the expression of AtPAP genes. Members of the LATERAL ORGAN BOUNDARIES DOMAIN gene family are induced by nitrate and act as negative regulators of the expres-sion of AtPAPs to modulate anthocyanin accumulation (Rubin et al., 2009). Therefore, the regulation of MYB genes at the transcriptional level is involved in the control of anthocyanin accumulation in response to ni-trate. Furthermore, the E3 ubiquitin ligase NITROGEN LIMITATION ADAPTATION participates in the reg-ulation of anthocyanin biosynthesis in response to limited nitrogen, suggesting that ubiquitination at the posttranslational level occurs in the regulation of an-thocyanin accumulation in response to nitrate (Peng et al., 2007, 2008). However, the molecular link between

ubiquitination modification and the MBW members in response to nitrate remains largely unknown.

In apple, the most important regulatory MYB TFs involved in regulating anthocyanin accumulation are MdMYBA, MdMYB1, and MdMYB10, which are ho-mologous to Arabidopsis AtPAP1 and allelic to each other (Espley et al., 2007, 2009; Allan et al., 2008). They are positive regulators of anthocyanin accumulation and fruit coloration (Espley et al., 2007, 2009). It is well known that MdMYB1 and its alleles are regulated at posttranslational levels in response to light (Li et al., 2012; Maier et al., 2013), and recent research demon-strates that MYB30-INTERACTING E3 LIGASE1 reg-ulates anthocyanin accumulation by modulating the degradation of MdMYB1 in response to abiotic stress (An et al., 2017). These findings demonstrate that post-translational modification is critical for environmen-tally regulated anthocyanin accumulation, which is mediated by MdMYB1.

In this study, the transcriptional and posttranslational modulation of MdMYB1 expression was examined in response to nitrate treatment. MdMYB1-interacting proteins were screened using an apple cDNA library with the Y2H method. Subsequently, their function in the ubiquitination and degradation of the MdMYB1 protein was characterized. Finally, the molecular mechanism by which the MdMYB1-interacting proteins regulate anthocyanin biosynthesis in response to ni-trate was elucidated and discussed.

RESULTS

MdMYB1 Is Degraded through the 26S Proteasome-Dependent Pathway in Response to Nitrate

Excessive nitrate noticeably inhibited anthocyanin biosynthesis in shoot cultures in vitro and in apple fruits (Supplemental Fig. S1; Ji et al., 2015). MdMYB1 is a positive regulator of anthocyanin biosynthesis and fruit coloration in apple (Li et al., 2012). The tran-script levels of MdMYB1 and anthocyanin biosynthetic genes, including CHALCONE SYNTHASE (MdCHS), DIHYDROFLAVONOL 4-REDUCTASE (MdDFR), and UDP-GLC:FLAVONOID 3-O-GLUCOSYLTRANSFERASE (MdUFGT), were negatively regulated by nitrate in ap-ple shoot cultures (Supplemental Fig. S2).

In addition, in vitro and in vivo assays of protein deg-radation were conducted to ascertain whether and how nitrate regulates the MdMYB1 protein at the posttrans-lational level. First, we performed a cell-free protein degradation assay. The tagged MdMYB1-HIS recombi-nant protein was expressed in and purified from Esch-erichia coli. Equal quantities of recombinant protein (100 ng) were then added to the total protein extract that was isolated from apple calli treated with KNO3 or KCl for different times. The immunoblot assays with an anti-HIS antibody showed that the MdMYB1- HIS protein was degraded faster in the protein extract

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MdBT2 Regulates Anthocyanin Biosynthesis


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that was isolated from the KNO3-treated calli than that from the KCl-treated calli (Fig. 1A).

To determine whether the degradation of nitrate-in-duced MdMYB1 depended on the 26S proteasome pathway, the effect of the proteasome inhibitor MG132 was tested using DMSO as a control. The protein sam-ples were extracted from apple calli that was pretreated with KNO3 and then treated with either DMSO or MG132 (50 µm) for 30 min. Subsequently, MdMYB1- HIS was added to the protein samples for the indicated times to detect the protein degradation of MdMYB1- HIS. Protein gel blots showed that MdMYB1 was de-graded in response to the KNO3 with DMSO treatment. However, MG132 treatment substantially inhibited the KNO3-induced recombinant MdMYB1 degradation compared with the DMSO treatment (Fig. 1B). These findings indicated that KNO3-induced MdMYB1 deg-radation was mediated at least partially through the 26S proteasome pathway.

To ascertain whether the nitrate-induced degradation of the MdMYB1 protein was mediated by the ubiqui-tin-dependent proteasome pathway, calli derived from the apple cultivar ‘Orin’ were used for genetic transfor-mation. 35S::MdMYB1-GFP and 35S::GFP (pBIN) con-structs were transformed genetically into these apple calli. The 35S::MdMYB1-GFP transgenic calli produced more MdMYB1 transcripts than the pBIN control (Fig. 2A), which was consistent with more anthocyanin ac-cumulation in the transgenic calli than in the control (Fig. 2, B and C). This indicated that MdMYB1 was overexpressed successfully and was functional in the transgenic calli. Then, MdMYB1-GFP transgenic calli were treated with the translational inhibitor cyclohex-imide and with KCl, KNO3, and MG132. Immunoblot analysis showed that the KNO3 treatment decreased

the abundance of MdMYB1-GFP protein remarkably compared with the KCl treatment (Fig. 2D), which sug-gested that the MdMYB1-GFP protein was degraded in response to nitrate. However, the MG132 treatment neutralized the KNO3-induced degradation of the MdMYB1-GFP protein (Fig. 2D), suggesting a possible ubiquitination modification for the MdMYB1 protein in response to nitrate. To verify this hypothesis, MdMYB1- GFP protein samples were immunoprecipitated from the transgenic calli with anti-GFP antibody and then used to examine ubiquitination with anti-ubiquitin and anti-GFP antibody. Polyubiquitinated Ubi(n)-MdMYB1-GFP protein was detected in the transgenic calli, and KNO3 induced a much higher level of ubiquitin than KCl (Fig. 2E). These data demonstrated that nitrate reg-ulated the abundance of MdMYB1 protein through the ubiquitin-mediated 26S proteasome pathway.

MdBT2 Interacts with MdMYB1 Protein

A Y2H screen was performed to identify the MdMYB1- interacting proteins that potentially mediate its ubiq-uitination and degradation. The truncated MdMYB1 peptide (amino acids 1–118), from which the C-terminal transcriptional activation domain had been deleted, was used as bait to screen a cDNA library that was ob-tained from the skin of a light-grown apple and fused to a synthetic activation domain in yeast. Among the positive colonies, a clone that contained a cDNA, which was part of a bric-à-brac, tramtrack, and broadcom-plex (BTB) domain family gene (MDP0000643281), was isolated. The gene was named MdBT2 because it was homologous to an Arabidopsis nitrate-responsive gene, AtBT2 (Supplemental Fig. S3A). Based on the phylo-genetic analysis and sequence alignment, the MdBT2 protein belonged to the BTB-transcriptional adapter zinc-finger (TAZ) family. There are five BTB-TAZ genes in the Arabidopsis and apple genomes, and these five apple MdBT proteins were named MdBT1, MdBT2, MdBT3.1, MdBT3.2, and MdBT4 based on their similarity to the Arabidopsis AtBTs (Supplmen-tal Fig. S3A). Online software CDD (Conserved Domain Database) at NCBI (National Center for Bio-technology Information) and InterPro at EMBL-EBI (European Bioinformatics Institute) were used to an-alyze conserved domains. The results showed that MdBT proteins also had a conserved BACK-like do-main, besides BTB and TAZ regions (Supplemental Fig. S3, B and C).

A Y2H assay was conducted to verify the interac-tion between MdBT2 and MdMYB1. Yeast cells that contained pGAD-MdMYB1 plus pGBD-MdBT2 grown well on -Trp/-Leu/-His/-Ade screening medium (Sup-plemental Fig. S4, A and B). The other four MdBT pro-teins were tested for interactions with the MdMYB1 protein, and the result showed thatMdMYB1 interacted weakly with MdBT1 but not with MdBT3.1, MdBT3.2, or MdBT4 (Supplemental Fig. S4, B and C). Subsequently, MdBT2 was chosen for further investigation.

Figure 1. Cell-free degradation assays of MdMYB1 in response to ni-trate treatment. A, MdMYB1 degradation in response to nitrate. Protein samples extracted from apple calli treated with 10 mm KNO3 or 10 mm KCl were incubated with MdMYB1-HIS recombinant protein for the indicated times. B, Proteasome inhibitor MG132 inhibited the nitrate- induced MdMYB1 degradation. The calli were treated with 10 mm KNO3 plus 0.5% DMSO or 50 µm MG132, and the protein extracts of treated calli were incubated with MdMYB1-HIS protein for the indicated times. For A and B, the anti-HIS antibody was used for immunoblotting. Protein levels at 0 h were set to 1.00, and MdACTIN was used as loading control.

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To examine the region necessary for the interaction between MdMYB1 and the MdBT2 protein, MdBT2 was divided to generate twelve vectors that contain different domains (Fig. 3A). A Y2H assay showed that all regions containing the BACK-lke domain (ami-no acids 129-195) interacted with MdMYB1 (Fig. 3B). However, the other regions without the BACK-like domain (BTB or TAZ domain) failed to interact with MdMYB1 (Fig. 3B), indicating that the BACK-like do-main was crucial for the interaction between MdBT2 and MdMYB1 (Fig. 3B).

To verify the interaction between MdBT2 and Md-MYB1, an in vitro pull-down analysis was performed. The recombinant MdBT2-HIS protein and the HIS control were incubated in vitro with the recombinant MdMYB1-GST protein. The protein was eluted with glu-tathione and immunoblotted with anti-GST and anti- HIS antibody. The results showed that MdBT2-HIS was pulled down, but HIS alone was not. Therefore, MdBT2-HIS interacted directly with the MdMYB1-GST protein (Fig. 3C). Furthermore, the interaction between MdMYB1 and MdBT2 was verified with a coimmuno-precipitation (co-IP) assay using 35S::MYC-MdBT2 + 35S::MdMYB1-GFP double-transgenic calli. The 35S::MYC-MdBT2 + 35S::GFP double-transgenic calli were used as the control. The protein samples extracted from two double-transgenic calli were used for

immunoprecipitation with an anti-MYC antibody. The eluted protein was then immunoblotted with anti- MYC and anti-GFP antibody. The result showed that the MdMYB1-GFP fusion protein, but not the GFP negative control, interacted with MdBT2 in apple cells in vivo.

To further confirm the interaction between MdBT2 and MdMYB1 proteins, a bimolecular fluorescence com-plementation (BiFC) assay was performed in Nicotiana benthamiana leaves. MdBT2 was fused to the N-terminal fragment of YFP to construct MdBT2-nYFP, and MdMYB1 was fused to the C-terminal fragment of YFP to construct MdMYB1-cYFP. A strong yellow fluores-cence signal was observed in the nucleus of cells trans-formed with MdBT2-nYFP and MdMYB1-cYFP (Fig. 3E). No fluorescent signal was observed in cells that were transformed with empty vector nYFP plus MdMYB1- cYFP or MdBT2-nYFP plus empty vector cYFP, which indicated that MdBT2 protein interacted physically with MdMYB1 in vivo.

BTB-TAZ proteins originated in Selaginella tamariscina and exist only in plants. They have similar numbers and structures in woody plants (Supplemental Table S1). AtBTs were identical to all five predicted MdBT proteins in that they all contained an N-terminal BTB domain, a BACK-like domian, a TAZ domain and a C-terminal calmodulin-binding domain (Supplemental

Figure 2. In vitro detection of MdMYB1 abundance in response to nitrate. A, Transcript levels of MdMYB1 in MdMYB1-GFP transgenic apple calli. MdACTIN was used as internal control. B, Phenotype of the 35S::MdMYB1-GFP transgenic apple calli. The transgenic calli (MdMYB1-GFP and pBIN-GFP) were treated with 10 mm KNO3 or 10 mm KCl medium for 3 d. C, Analysis of anthocyanin content in apple transgenic calli (MdMYB1-GFP and pBIN-GFP). Error bars represent the sd from three biological replicates with three parallel experiments. Statistical significance was determined using Student’s t test. **P < 0.01, and ***P < 0.001. D, Effect of MG132 on nitrate-regulated degradation of MdMYB1. The MdMYB1-GFP transgenic calli were treated with 250 µm cycloheximide plus 10 mm KCl, 10 mm KNO3, or 50 m MG132. An anti-GFP was used for immunoblotting, and MdACTIN was used as a loading control. E, Ubiquitination assay of MdMYB1 in samples used in D. The MdMYB1-GFP and the ubiquitinated MdMYB1-GFP were detected using anti-GFP (top) and anti-Ubi (bottom) antibodies, respectively. IP, Immuno-precipitate; IB, immunoblot; Ubi, ubiquitin.








Figure 3. MdBT2 interacts with MdMYB1. A, Y2H assay revealing an interaction between MdMYB1 with MdBT1 and MdBT2. The full-length coding sequences of MdMYB1 and MdBT1/2 were cloned into pGAD (pGAD-MdMYB1) and pGBD (pGBD-MdBT1/2), respectively. B, Relative β-gal activity in the ONPG assays. The activity of pGAD-MdMYB1 plus pGBD was set arbitrarily to 1. Error bars represent the sd from three biological replicates with three parallel experiments, and statistical sig-nificance was determined using Student’s t test. n.s., P > 0.05; *P < 0.05; **P < 0.01; ***P < 0.001. β-gal, β-galactosidase. C, Interaction of the N-terminal domain of MdMYB1 with MdBT2. Top, The schematic representation of the different MdMYB1 domains, and the indicated regions of MdMYB1 were cloned into the pGAD vector. D, Interaction of MdMYB1 with the BTB domain of MdBT2. Top, The schematic representation of the different MdBT2 domains, and the indicated regions of MdBT2 were cloned into the pGBD vector. E, Pull-down assay detection of the interaction of MdBT2 with MdMYB1. The recombinant MdBT2-HIS or HIS was incubated with MdMYB1-GST. Blots were first probed with anti-GST antibody and then detected with the anti-HIS antibody. F, Coimmunoprecipitation assay of MdBT2 and MdMYB1. MYC-MdBT2 plus MdMYB1-GFP proteins were immunoprecipitated with an anti-MYC antibody and immunoblotted with an anti-GFP antibody, while pBIN-GFP plus MYC-MdBT2 was used as a control. G, Bimolecular fluorescence complementation assay of interaction between MdBT2 and MdMYB1 in nuclei of epidermal cells of N. benthamiana. MdBT2-nYFP and MdMYB1-cYFP were used for the interaction assay, while nYFP plus MdMYB1-cYFP and MdBT2-nYFP plus cYFP were used as the controls. Scale bars, 10 μm. For A, C, and D, the interaction is indicated by yeast growth and X-α-Gal staining. Yeast grown in -T/-l (-Trp/-Leu) medium and -T/-l/-H/-A (-Trp/-Leu/-His/-Ade) medium are indicated. X-α-Gal represents 5-bromo-4-chloro-3-indolyl-α-d-galactopyranoside.


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Fig. S3, B and C, and Supplemental Fig. 5A). They also had similar protein and genome structures (Sup-plemental Fig. S5). MdBT1 and MdBT2 had the pre-dicted nuclear localization signals and nuclear export signals, but the other three MdBT proteins did not (Supplemental Fig. S5A). In addition, expression anal-ysis indicated that MdBT2 was a nitrogen-responsive gene (Supplemental Fig. S6, A and B) similar to AtBT2 (Mandadi et al., 2009). The nitrate-responsive analy-sis was supported further by the promoter analysis of MdBT2. The promoter of MdBT2 was fused to the GUS reporter gene. The resultant construct, pMdBT2::GUS, was transformed genetically into Arabidopsis. Subse-quently, the transgenic Arabidopsis was used to check the response to nitrate. KNO3 induced much higher GUS activity than KCl (Supplemental Fig. S6C), which demonstrated that the MdBT2 promoter was activated by nitrate.

MdBT2 Promotes Ubiquitination and Degradation of MdMYB1

The interaction analyses suggested that MdMYB1 is a potential substrate for MdBT2. To ascertain whether MdBT2 improved the ubiquitination and degradation of MdMYB1, protein degradation and ubiquitination assays were performed. First, a cell-free method was used to examine whether MdBT2 influenced the deg-radation of MdMYB1. The full-length cDNA in the sense direction and the cDNA fragments in the 5′ un-translated region (UTR) in the antisense direction of MdBT2 were transformed genetically into apple calli. Expression analysis demonstrated that MdBT2 tran-script levels increased in the MdBT2-overexpression (MdBT2-ovx) transgenic calli, but they decreased in the MdBT2-anti calli, compared with the wild-type control (Supplemental Fig. S7). The expression levels of the other four MdBT genes also changed (Supplemental Fig. S7), possibly due to the functional redundancy and mutual regulation among the MdBT genes.

Subsequently, total protein was extracted from the tested calli and incubated with the recombinant MdMYB1-HIS protein for different times. An immuno-blot assay was conducted with an anti-HIS antibody to check the abundance of the MdMYB1-HIS protein. The results showed that the MdMYB1-HIS fusion pro-tein was degraded faster in the protein extracts from MdBT2-ovx calli and slower in the protein extracts from MdBT2-anti transgenic calli than in the protein extracts from wild-type control (Fig. 4A), which indi-cated that MdBT2 promoted the degradation of the MdMYB1 protein. Furthermore, MG132 treatment in-hibited the MdBT2-mediated degradation of MdMYB1 (Fig. 4B), which suggested a possible involvement of the 26S proteasome pathway and ubiquitination modification.

To ascertain whether MdBT2 influenced the deg-radation of MdMYB1 protein in plant cells in vivo, three types of transgenic calli (i.e. 35S::MdMYB1-GFP, 35S::MdMYB1-GFP+35S::MdBT2-ovx, and 35S::MdMYB1-

GFP+35S::MdBT2-anti) were obtained. The protein ex-tracts from these calli were used for immunoblotting assays with an anti-GFP antibody. The results indicated that compared to the 35S::MdMYB1-GFP transgenic calli, the amount of MdMYB1-GFP protein was reduced in the 35S::MdMYB1-GFP+35S::MdBT2-ovx transgenic calli, but 35S::MdMYB1-GFP+35S::MdBT2-anti calli re-tained higher levels of the MdMYB1-GFP protein (Fig. 4C), which provided evidence that MdBT2 promoted the degradation of MdMYB1 in vivo.

The three transgenic calli were used to test the influ-ence of MdBT2 on the ubiquitination of the MdMYB1 protein. Total protein was extracted and used for im-munoprecipitation with an anti-GFP antibody. The immunoprecipitated proteins were immunoblotted with anti-Ubi antibody (Fig. 4D, top) and anti-GFP antibody (Fig. 4D, bottom). The quantity of high- molecular-weight polypeptides that corresponded to the polyubiquitinated forms of MdMYB1-GFP were higher in 35S::MdMYB1-GFP + 35S::MdBT2-ovx trans-genic calli, but they were lower in 35S::MdMYB1-GFP + 35S::MdBT2-anti calli than in the control (Fig. 4D), indi-cating that MdBT2 promotes the ubiquitination of Md-MYB1 protein. This indicated that MdBT2 promoted the ubiquitination of the MdMYB1 protein. There-fore, MdBT2 was involved in the ubiquitination and subsequent 26S proteasomal degradation of MdMYB1 protein.

MdBT2 Mediates the Degradation of MdMYB1 through an MdCUL3-Independent Pathway

Generally, BTB-TAZ proteins can interact with CUL3 proteins to form the Cullin-RING E3 ubiquitin ligases (CRL3) complex, which ubiquitinates and degrades target proteins (Gingerich et al., 2005; Hua and Vier-stra, 2011). In Arabidopsis, there are two CUL3 genes, AtCUL3A and AtCUL3B, and in apple, there are also two MdCUL3 genes, which are highly conserved. MdCUL3A (MDP0000458584) is similar to AtCUL3A, and it was cloned for further investigation. To test the interaction of MdBT2 with MdCUL3A, co-IP, pull-down, and Y2H assays were conducted. The constructs 35S::MdCUL3A-FLAG and 35S::FLAG were obtained and transformed genetically into 35S::MYC-MdBT2 transgenic calli. As a result, two double-transgenic calli, 35S::MYC-MdBT2+35S::MdCUL3A-FLAG and 35S::MYC- MdBT2+35S::FLAG, were obtained and used for im-munoprecipitation with an anti-MYC antibody. The immunoprecipitated proteins were immunoblotted with anti-MYC and anti-FLAG antibodies, respectively. The results showed that MdBT2 interacted with MdCUL3A (Fig. 5A). Subsequently, their interaction was verified by a pull-down assay (Fig. 5B). However, the Y2H assays failed to detect any interaction between MdBT2 and MdCUL3A (Supplemental Fig. S8A).

Subsequently, the sense 35S::MdCUL3A-FLAG con-struct was introduced genetically into apple calli. Ex-pression analysis showed that the MdCUL3A transcripts increased in the 35S::MdCUL3A-FLAG transgenic calli
















(Supplemental Fig. S8B). Anthocyanin accumulation was also detected in these calli. The 35S::MdCUL3A- FLAG calli generated anthocyanin at a level similar to the wild-type control, no matter in KNO3 or KCl treatment (Fig. 5, C and D). Subsequently, an anti- MdMYB1 antibody was used for immunoblotting as-says. The result showed that MdCUL3A transgenic apple calli and WT control exhibited a similar level of MdMYB1 proteins (Fig. 5E). Meanwhile, a cell-free assay was carried out using MdCUL3A transgenic calli, and the results showed that overexpression of MdCUL3A did not promote the degradation of MdMYB1 protein (Supplemental Fig. S8C), which indicated that MdBT2 promoted the degradation of MdMYB1 through the MdCUL3A-independent pathway.

To explain why MdCUL3A did not promote the degradation of the MdMYB1-HIS protein, a compat-ible pull-down assay was conducted to detect the ef-fect of MdCUL3A on the interaction between MdBT2 and MdMYB1. The interaction between MdCUL3A and MdBT2 repressed the interaction between MdBT2 and MdMYB1 (Fig. 5F), which demonstrated that MdCUL3A

had no effect on MdBT2-mediated degradation of the MdMYB1 protein.

Expression of MdBT2 and MdMYB1 Are Inversely Regulated

To gain further information on the action of MdBT2 on MdMYB1, besides posttranslational regulation, gene expression was analyzed to detect the response of MdBT2 and MdMYB1 to exogenous nitrogen/car-bon and light/dark. Nitrate noticeably induced the ex-pression of MdBT2, but Suc inhibited it (Fig. 6, A and B). Light repressed the expression of MdBT2, but dark-ness increased it (Fig. 6C). In contrast, the expression of MdMYB1 responded to the corresponding treatments in the opposite manner (Fig. 6, A and C). Considering the negative control that was exerted by MdBT2 on MdMYB1 protein stability, these data indicated that MdBT2 and MdMYB1 played key roles in the accumu-lation of plant anthocyanin at both transcriptional and posttranslational levels in response to nitrate and related external environmental factors.

Figure 4. MdBT2 regulates ubiquitination and degradation of MdMYB1. A, Cell-free degradation assays of MdMYB1-HIS pro-tein in the protein extract of transgenic apple calli. Protein extracts of transgenic calli (MdBT2-ovx and MdBT2-anti), and wild-type (WT) calli were incubated with MdMYB1-HIS protein for the indicated times. MdMYB1-HIS levels were visualized by immunoblotting using the anti-HIS antibody. B, Degradation of the recombinant MdMYB1-HIS proteins in the protein extracts of transgenic apple calli mediated by MG132. Apple calli were treated as indicated, and the protein extracts were incubated with MdMYB1-HIS protein for the indicated time points. MdMYB1-HIS levels were detected by the anti-HIS antibody. C, MdBT2-mediated protein stability of MdMYB1. The 35S::MdMYB1-GFP construct was transformed with or without expressing MdBT2, and the resultant 35S::MdMYB1-GFP + 35S::MdBT2 transgenic calli were analyzed by immunoblotting using the anti- GFP antibody. D, Detection of MdBT2-mediated ubiquitination of the MdMYB1 protein. MdMYB1-GFP was immunoprecipi-tated with anti-GFP antibody from the transgenic calli, and then the immunoprecipitated protein was examined using anti-Ubi antibody (top) and anti-GFP antibody (bottom). IP, Immunoprecipitate; IB, immunoblot; Ubi, ubiquitin. For A to C, MdACTIN was used as the loading control, and the protein levels at 0 h were set to 1.00.


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MdBT2 Negatively Regulates the Accumulation of MdMYB1-Mediated Anthocyanin

To verify the role of MdBT2 in regulating anthocyanin accumulation in response to nitrate, the transgenic calli and the wild-type control were treated with KCl and KNO3, and their anthocyanin contents were then de-tected. Less anthocyanin accumulated in KNO3-treated medium than that in KCl-treated medium for each type of apple calli (Fig. 7, A and C). Meanwhile, 35S::MdBT2-ovx transgenic calli produced less, and 35S::MdBT2-anti transgenic calli produced more anthocyanin than the wild-type control in the KCl medium (Figs. 7A, top, and

7C), which indicated that MdBT2 negatively regulates anthocyanin accumulation.

Because MdMYB1 is a master regulator of antho-cyanin biosynthesis, and MdBT2 promotes MdMYB1 degradation, it is reasonable to propose that MdBT2 negatively regulates accumulation of MdMYB1-medi-ated anthocyanin. To test this hypothesis, 35S::MdBT2-ovx and 35S::MdBT2-anti constructs were transformed genetically into 35S::MdMYB1-GFP transgenic calli, and they were treated with KNO3 and KCl. Anthocy-anin accumulation was induced in MdMYB1 trans-genic calli compared to the wild-type control. In addition, overexpression of MdBT2-ovx inhibited and

Figure 5. Effect of MdCUL3A on MdBT2-mediated stability of MdMYB1. A, Coimmunoprecipitation assays of MdCUL3A-FLAG and MYC-MdBT2. 35S::MdCUL3A-FLAG + 35S::MYC-MdBT2 or 35S::FLAG + 35S::MYC-MdBT2 transgenic apple calli were immunoprecipitated with an anti-MYC antibody and immunoblotted with an anti-FLAG antibody. B, Pull-down assay to ana-lyze the interaction of MdCUL3A with MdBT2. The recombinant MdCUL3A-HIS or HIS were incubated with MdBT2-GST. Blots were first probed with anti-GST antibody and then detected with the anti-HIS antibody. C, Phenotype of the MdCUL3A trans-genic apple calli. MdCUL3A-ovx transgenic apple calli and wild-type (WT) calli were treated with 10 mm KNO3 and 10 mm KCl medium for 3 d. D, Analysis of anthocyanin content in the MdCUL3A transgenic apple calli (MdCUL3A-FLAG transgenic apple calli). Error bars represent the sd from three biological replicates with three parallel experiments, and statistical significance was determined using Student’s t test. n.s., P > 0.05. E, Degradation assays of the recombinant MdMYB1-HIS protein regulated by MdCUL3A. Protein extracts of MdCUL3A-ovx transgenic calli and wild-type calli were incubated with MdMYB1-HIS protein for the indicated times. MdMYB1-HIS levels were visualized by immunoblotting using the anti-HIS antibody, MdACTIN was used as loading control, and the protein levels at 0 h were set to 1.00. F, Effect of MdCUL3A on the interaction between MdBT2 with MdMYB1. The recombinant MdBT2-GST plus MdMYB1-HIS were incubated with MdCUL3A-HIS or HIS, respectively. Blots were first probed with anti-GST and then detected with the anti-HIS antibody.





MdBT2-anti promoted accumulation of MdMYB1-im-proved anthocyanin (Fig. 7, A and C), which suggested that MdBT2 negatively regulated the accumulation of MdMYB1-mediated anthocyanin in apple calli.

To identify the function of MdBT2 in planta, the construct 35S::MdBT2-anti was introduced genetically into the ‘GL3’ apple genotype via Agrobacterium tume-faciens-mediated transformation. As a result, a total of six transgenic lines were obtained, as indicated by reduced MdBT2 expression (Supplemental Fig. S9A). Among them, L13, L15, and L23 were chosen to detect anthocyanin accumulation in response to nitrate. GL3 and 35S::MdBT2-anti transgenic plantlets generated less anthocyanin in KNO3-treated medium than in KCl-treated medium; however, 35S::MdBT2-anti trans-genic lines produced more anthocyanin than the wild-type control (Fig. 7, D and E).

Subsequently, an anti-MdMYB1 antibody was used to detect the level of MdMYB1 protein in the MdBT2- anti transgenic and WT plantlets. The result showed that MdBT2-anti transgenic plantlets produced more MdMYB1 proteins than the WT control (Fig. 7F). Mean-while, a cell-free method was used to examine whether MdBT2 influenced the degradation of the MdMYB1 protein. The result showed that MdMYB1-HIS fusion protein was degraded more slowly in the MdBT2-anti

plantlets than that in the GL3 control plantlets (Supple-mental Fig. S9B), which indicated that MdBT2-anti re-pressed the degradation of the MdMYB1 protein. This was further evidence that MdBT2 was necessary for the inhibition of anthocyanin biosynthesis in apples.

Both in transgenic apple calli and shoot cultures, ex-pression analysis demonstrated that MdBT2 exerted little influence on the expression of MdMYB1, but it significantly inhibited the expression of the anthocy-anin biosynthetic genes, including MdCHS, MdDFR, and MdUFGT (Supplemental Fig. S9C–D), which sup-ported the conclusion that MdBT2 regulated MdMYB1 mainly at the posttranslational, but not at the tran-scriptional level.

MdBT2 Functions in a Conserved Manner in Arabidopsis

Similar to our finding in this study, anthocyanin ac-cumulation was also inhibited by nitrate in Arabidop-sis (Rubin et al., 2009). As mentioned above, MdBT2 is a homolog of AtBT genes in Arabidopsis. To examine whether MdBT2 has a conserved function in Arabidop-sis, AtPAP1 was first isolated, and a Y2H assay was performed. The result showed that AtPAP1 interacted with MdBT2 (Supplemental Fig. S10A–B), which was then confirmed by a pull-down assay (Supplemental

Figure 6. Expression analysis of MdBT2 and MdMYB1 in response to environmental stimuli. A, Expression analysis of MdBT2 and MdMYB1 in response to nitrate. The apple plantlets were pretreated in nitrogen deprivation medium for 3 d and then treated with 10 mm KNO3 or KCl for the indicated times. The left and right y axes represent transcript levels of MdBT2 and MdMYB1, respectively. B, Expression analysis of MdBT2 and MdMYB1 in response to Suc. The apple plantlets were pretreated in exogenous carbon deprivation medium for 3 d and then treated with 100 mm Suc or mannitol (Man) for the indicated times. The left and right y axes represent transcript levels of MdMYB1 and MdBT2, respectively. C, Expression analysis of MdBT2 and MdMYB1 in response to light and darkness. For light treatment, apple plantlets were pretreated under continuous darkness for 5 d and then treated with light for the indicated times. For dark treatment, apple plantlets were pretreated under continuous light for 5 d and then treated with darkness for the indicated times. The left y axis represents transcript levels of MdMYB1 in response to light and transcript levels of MdBT2 in response to darkness, and the right y axis represents transcript levels of MdMYB1 in response to darkness and transcript levels of MdBT2 in response to light. For A to C, MdACTIN was used as internal control, and error bars represent the sd from three biological replicates with three parallel experiments.


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Fig. S10B). To further verify whether MdBT2 func-tioned in a conserved way in regulating anthocyanin biosynthesis in Arabidopsis, MdBT2 was transformed ectopically into the mutant bt2 (Supplemental Fig. S10C). The homozygous MdBT2-ovx/bt2 plants notice-ably inhibited anthocyanin accumulation, but bt2 ap-parently induced anthocyanin accumulation compared to wild type (Col-0; Supplemental Fig. S10, D and E). These findings were consistent with the gene expres-sion pattern of downstream genes in the anthocyanin biosynthesis pathway (Supplemental Fig. S10F), which indicated that MdBT2 recovered the functional defect in mutant bt2.

Because MdBT2 interacted with AtPAP1 and regu-lated anthocyanin accumulation in transgenic Arabi-dopsis, 35S::AtPAP1-GFP transgenic Arabidopsis lines (Hu et al., 2016) were used to confirm further whether the phenotype of anthocyanin biosynthesis was due to the regulation of AtPAP1 that was mediated by MdBT2. Three such independent lines were crossed with MdBT2-ovx to generate MdBT2-ovx/AtPAP1-GFP lines. The phenotypic analysis showed that compared

with the wild type (Col-0), AtPAP1-GFP lines notice-ably induced the accumulation of anthocyanin, but this effect was noticeably inhibited in the homozygous MdBT2-ovx/AtPAP1-GFP lines (Fig. 8, A and B). Expres-sion analysis showed that AtPAP1-GFP lines induced the transcripts of downstream genes in the anthocyan-in biosynthesis pathway, but this effect was noticeably inhibited in the homozygous MdBT2-ovx/AtPAP1-GFP lines (Fig. 8C). Therefore, it is speculated that the mech-anism by which BT2 protein regulates the anthocyanin accumulation is conserved in different plant species.

DISCUSSION

The MBW transcriptional complex plays a central role in regulating anthocyanin accumulation in plants (Gonzalez et al., 2008; Jaakola, 2013; Xu et al., 2015). The formation and activation of the MBW complex are controlled by a series of developmental and en-vironmental signals, including hormones, light, tem-perature, and nutrients, and they maintain a delicate

Figure 7. Effect of MdBT2 on MdMYB1-mediated anthocyanin accumulation in transgenic apple calli and plantlets. A, Phe-notype of MdBT2 transgenic apple calli. The transgenic apple calli and wild-type (WT) calli were treated as indicated. B, Phenotype of MdMYB1 and MdBT2 cotransformed apple calli. The apple calli were treated as indicated. C, Analysis of the anthocyanin contents in transgenic calli. Anthocyanin content of the apple calli (MdBT2-ovx, MdBT2-anti, wild-type, MdBT2-ovx/MdMYB1-GFP, MdBT2-anti/MdMYB1-GFP, and MdMYB1-GFP) was detected. D, Phenotype of the MdBT2-anti transgenic apple plantlets. The apple plantlets were treated as indicated, and the plantlets (top) and leaves (bottom) were photographed. E, Analysis of anthocyanin contents in the MdBT2-anti transgenic apple plantlets. F, Degradation assays of the recombinant MdMYB1-HIS protein regulated by MdBT2. Protein extracts of transgenic apple plantlets (MdBT2-anti) and GL3 control were incubated with MdMYB1-HIS protein for the indicated times. MdMYB1-HIS levels were visualized by immunoblotting using the anti-HIS antibody. MdACTIN was used as a loading control, and the protein level at 0 h was set to 1.00. For C and E, error bars represent the sd from three biological replicates with three parallel experiments, and statistical significance was deter-mined using Student’s t test. n.s., P > 0.05; *P < 0.05; **P < 0.01; ***P < 0.001.










balance of transcriptional activity, which is crucial for the regulation of anthocyanin biosynthesis.

Nitrate is a signaling molecule, and ARABIDOPSIS THALIANA NITRATE TRANSPORTER1 is a sensor that regulates the nitrate-mediated signal transduction pathway (Ho et al., 2009). For anthocyanin biosynthesis, MYB TFs are positive regulators, but nitrate is a negative regulator. However, it is largely unknown how these MYB TFs are regulated in response to nitrate, especially at a posttranslational level. In plants, the ubiquitin- proteasome pathway and the protein phosphorylation cascade are thought to mediate the responses to a vari-ety of internal hormonal and external environments. Re-cently, a Leu-rich-repeat receptor-like kinase regulated plant root architecture and anthocyanin accumula-tion in a systemic N-dependent manner (Bisseling and Scheres, 2014; Tabata et al., 2014), which indicated a pos-sible pathway involving a phosphorylation cascade of target proteins by which nitrate regulated anthocyanin

accumulation. In this study, a BTB-TAZ protein, MdBT2, was identified and characterized functionally. This pro-tein was induced by nitrate, and it was involved in the ubiquitination and degradation of the MdMYB1 pro-tein in response to nitrate. These results indicated that an ubiquitin-proteasome pathway played an important role in nitrate-mediated signal transduction and antho-cyanin biosynthesis.

BTB domain proteins are found primarily in Drosophila melanogaster. They contain a conserved BTB transcrip-tion regulation domain (Stogios et al., 2005). There are 149 and 81 BTB domain-containing proteins in rice (Oryza sativa) and Arabidopsis, respectively (Gingerich et al., 2007). In addition to the BTB domain, they have conserved protein-interacting domains, including MATH, NPH, ankyrin, TAZ, and TPR (Gingerich et al., 2005, 2007). Based on these protein-interacting do-mains, BTB proteins are divided into different subfam-ilies. Among them, MdBT2 belongs to the BTB-TAZ

Figure 8. Effect of MdBT2 on AtPAP1-mediated anthocyanin accumulation in Arabidopsis. A, Phenotype of transgenic Ara-bidopsis. AtPAP1-GFP transgenic Arabidopsis was crossed with MdBT2-ovx, and the phenotypes of MdBT2-ovx/AtPAP1-GFP, AtPAP1-GFP, and wild type (Col-0) was shown. B, Analysis of anthocyanin content in transgenic Arabidopsis (AtPAP1-GFP and MdBT2-ovx/AtPAP1-GFP) and wild type (Col-0). C, Expression analysis of anthocyanin regulatory (AtPAP1) and biosyn-thetic genes (AtCHS, AtDFR, and AtUFGT) in the Arabidopsis transgenic lines. For B and C, error bars represent the sd from three biological replicates with three parallel experiments, and statistical significance was determined using Student’s t test. *P < 0.05; **P < 0.01; ***P < 0.001.


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subfamily, which contains five BT proteins in Ara-bidopsis and apple, respectively (Du and Poovaiah, 2004). It features an N-terminal BTB domain, a cen-tral TAZ domain for protein-protein interactions and a C-terminal motif that interacts with calmodulin in a Ca2+-dependent manner (Du and Poovaiah, 2004).Here, a conserved BACK-like domain between BTB and TAZ domains was identified (Supplemental Fig. S3B). BACK-like domain is also found in BTB-BACK proteins in other eukaryotes, these BTB-BACK pro-teins usually contain a C-terminal KELCH domain fol-lowing the BACK region (Stogios and Privé, 2004). In Arabidopsis, 11 BTB-BACK proteins have been identi-fied. However, they do not contain KELCH-repeat fol-lowing BACK domain (Orosa et al., 2017). Here, it was found that BTB-TAZ proteins MdBTs have the BACK-like domain directly following a TAZ region (Supple-mental Fig. 3B and Supplemental Fig. 5A), suggesting that BTB-TAZ proteins may have biological function associated BACK-like domain.

Generally, BTB proteins are regarded as adaptors of the CRL3 complex that catalyzes E3 ubiquitin ligase activity (Hua and Vierstra, 2011). The BTB motif forms a complex with CUL3, but another protein interaction domain is responsible for recognizing the ubiquiti-nation targets (Petroski and Deshaies, 2005). In this study, we found that MdBT2 interacted with MdMYB1 through the BACK-like domain, but not through the BTB or TAZ protein interaction domain (Fig. 3A–B). In addition, MdCUL3A interacted with MdBT2 (Fig. 5, A and B), which indicated that MdBT2 recruited MdCUL3A to form the CRL3 complex. For BTB-BACK containing proteins SPOP (speckle-type POZ protein), however, the C-terminal BACK-like domain together with the adjacent BTB domian are essential for inter-action with CUL3 (Canning et al., 2013). In this study, both BTB and BACK-like domains may be required for the interaction between MdBT2 and MdCUL3A, which subsequently respressed the interaction between MdBT2 and MdMYB1 (Fig. 5F), therefore, MdBT2 reg-ulated anthocyanin biosynthesis in an MdCUL3A-in-dependent manner (Fig. 5C–F). In fact, multiple BTB proteins have been found to work through a non-E3 function and act by forming dimers or interacting with other non-CUL proteins (Luke-Glaser et al., 2005; Sto-gios et al., 2005; Leljak Levanić et al., 2012). On one hand, the BTB-MATH protein BTB-POZ AND MATH DOMAIN1 (BPM1) forms a complex with CUL3 to tar-get AtMYB56 for degradation, and it then modulates plant abscisic acid signaling and flowering time (Chen et al., 2015). On the other hand, colocalization analyses have shown a difference in the subcellular localization of AtBPM1 and CUL3 proteins, which imply that there were potentially unrelated functions of AtBPM1 when deprived of CUL3 (Leljak Levanić et al., 2012). In ap-ple, MdMYB1 interacted with the BACK-like domain of MdBT2 and degraded in an MdCUL3-independent manner (Fig. 3, A and B), while MdbHLH104 interacts with the TAZ domain of MdBT2 and degrades in an MdCUL3-dependent way (Zhao et al., 2016). Therefore,

it seems that MdBT2 may recruit MdCUL3 to form the E3 ligase complex to ubiquitinate and degrade the targeted proteins such as MdbHLH104 that interact with the TAZ domain of MdBT2. However, MdBT2 may also act as a scaffold protein that bridges an un-known ubiquitin E3 ligase and the target proteins such as MdMYB1, which interacts with the BACK-like do-main of MdBT2 protein, to mediate its ubiquitination and degradation. Meanwhile, transcripts of anthocy-anin biosynthetic genes were induced in the AtPAP1 transgenic Arabidopsis, which was noticeably inhibit-ed by MdBT2 (Fig. 8), indicating that MdBT2 repressed expression of anthocyanin biosynthetic genes through regulating protein stability of AtPAP1. A model con-sistent with these findings is presented in Figure 9. Furthermore, the interaction between MdBT2 and Md-CUL3A suggested that they formed a CRL3 complex to regulate the ubiquitination and degradation of tar-geted proteins that interact with the TAZ domain of MdBT2, but not with MdMYB1.

The antagonistic interconnection between the car-bon and nitrogen metabolic pathways regulates plant growth and development and anthocyanin accumu-lation (Coruzzi and Bush, 2001), and it is an import-ant research topic for fruit cultivation and breeding. SNF1-RELATED PROTEIN KINASE and Hexokinase (HXK) are crucial sensors and players in response to the energy supply (Sheen et al., 1999; Rolland et al., 2006; Ramon et al., 2008). BT2 was among the numerous po-tential target genes of the SNF1-RELATED PROTEIN KINASE and BASIC LEU-ZIPPER TFs based on mi-croarray data (Baena-González et al., 2007; Hanson et al., 2008).

HXK represses the expression of AtBT2 through a metabolic but not a signaling pathway (Kunz et al., 2015). In this study, nitrate and darkness induced, whereas Suc and light inhibited the expression of MdBT2 (Fig. 6; Supplemental Fig. S6). In addition, the expression pattern of MdMYB1 was opposite that of MdBT2 (Fig. 6). Most recently, through a supervised machine-learning algorithm, BT2 was predicted to be a central gene in nitrogen use, as demonstrated by func-tional identification in Arabidopsis and rice (Araus et al., 2016). In summary, BT2 may be a molecular switch at the transcriptional level that participates in carbon/ nitrogen nutrient-mediated anthocyanin accumula-tion. Meanwhile, previous research found that BT1 and BT2 were redundant in their efficiency of nitrogen use and gametophyte development in Arabidopsis (Robert et al., 2009; Araus et al., 2016). In this study, the expres-sion levels of the other four MdBT genes also changed (Supplemental Fig. S7), possibly due to the functional redundancy and mutual regulation among the MdBT genes.

In addition, a series of BTB proteins has been identi-fied and characterized with functions that are associated with multiple biological processes (Hepworth et al., 2005; Christians et al., 2009; Ni et al., 2014; Chen et al., 2015). Among the BTB proteins, BT2 belongs to the land- plant-specific BTB-TAZ subfamily, which is primarily












described as regulated by the zinc-finger protein TELOMERASE ACTIVATOR1 (Ren et al., 2007). BT2 is essential for female and male gametophyte develop-ment, which may occur through a PINOID-mediated auxin pathway in Arabidopsis (Ren et al., 2007; Robert et al., 2009). BT2 genes perceive and respond at the transcriptional level to carbon and nitrogen nutrients and to various hormonal and environmental signals (Mandadi et al., 2009), which was also shown in this study (Fig. 6). Interestingly, in addition to nitrogen, ex-ogenous carbon, photoperiod, and other environmen-tal signals also regulate expression of MdMYB1 and anthocyanin accumulation. This led us to speculate that the MdBT2-MdMYB1 complex played a critical role in environmental factors that modulated antho-cyanin biosynthesis. Together with previous research and our study, we propose a regulatory mechanism to explain how the MdBT2-MdMYB1 complex regulates anthocyanin biosynthesis in response to nitrogen and other environmental signals (Fig. 9).

Our hypothesis is that the transcripts of MdMYB1 are repressed directly by sufficient nitrogen and other environmental signals (darkness, exogenous carbon deficiency, and so on), whereas MdBT2 is up-regulated under these environmental factors; in this case, MdBT2 recruits and degrades the MdMYB1 protein through the 26S proteasome pathway, which prevents MdMYB1 from binding to the Myb binding sites of downstream anthocyanin biosynthetic genes and deactivates their expression. This ultimately leads to less anthocyanin accumulation (Fig. 9, top). In contrast, the transcripts

of MdMYB1 are induced under nitrogen deficiency and other environmental signals (light, exogenous carbon, and so on), and MdBT2 is down-regulated under these environmental factors. A decrease in the MdBT2 protein releases and enhances the abundance of the MdMYB1 protein, which promotes MdMYB1 to activate the expression of downstream anthocyanin biosynthetic genes. This ultimately leads to more an-thocyanin accumulation (Fig. 9, bottom). This study provides novel evidence on how the MdBT2-MdMYB1 complex modulates anthocyanin biosynthesis in re-sponse to nitrogen and other environmental cues.

MATERIALS AND METHODS

Plant Materials, Growth Conditions, and Treatments

Tissue cultures of apple (seedling of Malus × domestica cv ‘Royal Gala’) ‘GL3,’ which has a high adventitious regeneration capacity, were used in this study (Dai et al., 2013). The ‘GL3’ cultures and transgenic apple plantlets were grown on Murashige-Skoog (MS) medium containing 0.2 mg L−1 NAA, 0.6 mg L−1 6-BA, and 0.2 mg L−1 GA at a normal temperature under long-day conditions (25°C 16 h light/8 h dark). ‘Orin’ apple calli were cultured on MS medium with 1.5 mg L−1 2,4-D, and 0.4 mg L−1 6-BA at a normal temperature (25°C) in a continuous dark environment. Bagged fruits of the apple cultivar ‘Starkrimson apple’ that were grown for 100 d after blooming were harvested for in vitro apple fruit experiments. Arabidopsis (Arabidopsis thaliana) wild-type ecotype Columbia and the bt2-3 mutant were used for genetic transfor-mation and functional identification. The seedlings were treated for 3 d at 4°C before being grown on MS medium. The seedlings and plants were grown at 21°C under long-day conditions (16 h light/8 h dark).

Figure 9. A proposed model of the mechanism by which MdBT2 regulates anthocyanin biosynthesis. Top, High levels of nitrogen and other cues (darkness, exogenous carbon deficiency, etc.) induce the transcripts of MdBT2 while repressing the transcripts of MdMYB1. Enhanced abundance of MdBT2 recruits and degrades MdMYB1 protein, then prevents MdMYB1-in-duced expression of anthocyanin biosynthetic genes and anthocyanin accumulation. Bottom, Low nitrogen and other cues (light, exogenous carbon, etc.) repress the transcripts of MdBT2 and induce the transcripts of MdMYB1. Decreased MdBT2 levels promote MdMYB1 abundance and activate expression of downstream anthocyanin biosynthetic genes and anthocyanin accumulation.


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For phenotypic analyses of anthocyanin accumulation in ‘GL3’ plantlets, 1-month-old apple plantlets were treated on medium with the indicated amount of nitrate (KCl was added to maintain the same concentration) under long-day conditions (16 h light/8 h dark) for approximately 3 to 5 d (until there was an obvious difference in shoot colors), and then the samples were harvested to measure the anthocyanin content. Similar to plantlets, the tree branches carrying the bagged apple fruits were treated with the indicated amount of nitrate (0, 3, 15, 30, and 60 mm KNO3, with KCl added to maintain the same concentration) under continuous light for approximately 2 to 4 d (until there was an obvious difference in apple skin color) before the apple skins were harvested.

For the phenotypic analyses of the nitrate-deprivation response, apple calli and ‘GL3’ plants were transferred from nitrate-sufficient medium (MS medium with the indicated hormones, but without nitrogen, followed by the indicated amount of KNO3, 10 mm) to nitrate-deficient medium (MS medium with the indicated hormones but without nitrogen, followed by the indicated amount of KCl, 10 mm, as control) under continuous light for the indicated days.

One-month-old ‘GL3’ apple plantlets were used to evaluate expression of MdBT2 and MdMYB1. To determine the expression of MdBT2 in response to different kinds of nitrogen, the apple plantlets were treated with 10 mm of KNO3, NH4Cl, NH4NO3, Glu, Gln, and urea for the indicated times, respec-tively. For the nitrogen response, apple plantlets were pretreated with nitro-gen-deficient medium (MS medium with the indicated hormones but without nitrogen) for 3 d, then they were treated with 10 mm KNO3/KCl for the indicat-ed times. For the exogenous carbon response, apple plantlets were pretreated with exogenous carbon-deficient medium (MS medium with the indicated hormones but without Suc) for 3 d, then they were treated with 100 mm Suc/mannitol (Man) for the indicated times. For the light/dark response, apple plantlets were pretreated under continuous light/darkness for 5 d, and then they were treated with darkness/light for the indicated times.

Immunoblot analysis was performed as described previously (Zhao et al., 2016). For the ubiquitination and degradation of MdMYB1 protein, 35S::Md-MYB1-GFP calli were cotransformed with 35S::MdBT2-ovx and 35S::MdBT2-anti. The total protein was extracted from the transgenic calli and used for immu-noblot assays, and then ubiquitination was examined using anti-ubiquitin and anti-GFP antibody. Total protein extraction was performed as described below.

Plasmid Construction and Genetic Transformation

The PCR products were cloned into vector pEasy-T5 (Transgen; Supple-mental Table S2). All genes were cloned into an expression vector under the Cauliflower mosaic virus 35S promoter, which also contained protein tags.

The MdMYB1-GFP and AtPAP1-GFP vectors were generated using the modified pRI101 vector (Li et al., 2012; Hu et al., 2016). To construct the MdBT2-anti vectors, cDNA fragments of MdBT2 were inserted into the pCXSN vector (Chen et al., 2009). To construct the MdCUL3-ovx vector, the open reading frame of MdCUL3A was inserted into the pCXSN-FLAG (FLAG-MdCUL3) vec-tor. To construct the MdBT2-ovx (MYC-MdBT2) vector, the open reading frame of MdBT2 was inserted into the pCAMBIA1301 vector. To clone the MdBT2 promoter, a 2-kb-long genomic region, which was upstream of the MdBT2 start codon, was amplified and inserted into the pCAMBIA1391 vector (ABRC, http://www.arabidopsis.org; pMdBT2; Supplemental Table S3). pET32a and pGEX-4T-1 were used to construct the expression vector. pGAD424 and pGBT9 were used to construct the Y2H vector. The primers used for gene clon-ing and vector construction were listed on Supplemental Tables S2 and S3. Agrobacterium tumefaciens strain GV3101 was used to transform Arabidopsis, and A. tumefaciens strain LBA4404 was used to transform apple calli and apple plantlets. The transgenic lines were selected on medium that contained kana-mycin (30 mg/L for apple calli and 50 mg/L for Arabidopsis) or hygromycin B (15 mg/L for apple calli and 30 mg/L for Arabidopsis).

Identification and Sequence Analysis of BTB Proteins

To identify the BTB gene family of apples and other plant species, BTB pro-teins in Arabidopsis were first collected, which were then applied as query sequences to carry out searches against the proteome and genome files that were downloaded from the Phytozome database (http://www.phytozome.net/). Stand-alone versions of BLASTP and TBLASTN (http://blast.ncbi.nlm.nih.gov), which were available from NCBI, were used with an e-value cutoff of 1e-003. The screened BTB candidates were then determined using the domain analysis programs PFAM (http://pfam.xfam.org/), SMART (http://smart.

embl-heidelberg.de/), and CDD with the default cutoff parameters. Second, the domains of all plant BTB candidates were analyzed using a hidden Markov model (e-value ≤ 1e-20) and PFAM search. Then, we obtained the sequences by using PF00651, which contained a typical BTB domain.

Using similar methods, we obtained the sequences that included PF02135, PF03000, PF00917, PF13637, PF13424, PF00514, and PF00754, which contained a typical TAZ domain, NPH domain, MATH domain, ANK domain, TRP do-main, ARM domain, and F5/8 domain, respectively, from the plant genome sequences using a Perl-based script.

Online software CDD at NCBI (http://www.ncbi.nlm.nih.gov/) and Inter-Proat EMBL-EBI (http://www.ebi.ac.uk/interpro/) were used to identify the BACK-like domains of MdBT and AtBT proteins. DNAMAN was used to per-form multiple sequence alignments. The structures of the MdBT genes were obtained from the Phytozome database using a Perl-based program.

Gene Expression Analysis

Total RNA was extracted from ‘Orin’ calli, ‘GL3’ shoot cultures, ‘GL3’ trans-genic apple lines, and Arabidopsis using the RNA plant Plus reagent (Tiangen) and TRIzol reagent (Invitrogen) according to the manufacturers' instructions. First-strand cDNA was synthesized using a PrimeScript First Strand cDNA Synthesis Kit transcriptase (TaKaRa) following the manufacturer’s instructions.

The reverse transcription quantitative PCR (RT-qPCR) with solutions that contained SYBR Green PCR Master Mix was performed using the iCycler iQ5 system (Bio-Rad). Relative gene expression analyses were calculated with MdACTIN as the internal control. Three biological replicates were performed for each individual experiment. Supplemental Table S4 shows the primers used for RT-qPCR.

Y2H Analysis

Y2H studies were performed following the yeast maker Yeast Transforma-tion System 2 protocol (Clontech). The cDNA library was obtained from the apple skin and was constructed by the Oebiotech Company. The cDNAs of MdCUL3A and MdMYB1 were amplified (Supplemental Table S3) and insert-ed into pGBT9 and pGAD424, respectively. The MdBT cDNAs and truncated sequences (amino acids 1-335, 1-297, 1-247, 1-195, 1-128, 31-128, 129-335, 129-297, 129-247, 129-195, 196-335, 196-297) were amplified by PCR (Supplemental Table S3) and inserted into pGBT9. The plasmids of pGAD424-MdMYB1 and the pGBT9-MdBTs were cotransformed into Y2H Gold (Clontech). The yeasts were grown on selection medium supplemented with SD base/-L/-T (SD base/-Leu/-Trp) and then transferred onto selection medium supplemented with SD base/-L/-T/-H/-A (SD base/-Leu/-Trp/-His/-Ade), with or without 5-bromo-4-chloro-3-indolyl-α-d-galactopyranoside (X-α-Gal) to determine the interactions of the MdBTs with MdMYB1 and MdCUL3A. ONPG assays were used to measure the quantitative β-gal activity.

Pull-Down Analysis

Pull-down assays were conducted according to the Pierce GST Spin Pu-rification Kit protocol (Pierce). The full-length cDNAs of MdMYB1, MdBT2, and MdCUL3A were amplified using specific primers (MdMYB1-PD, MdBT2, and MdCUL3A; Supplemental Table S3) and inserted into pET32a to generate His-tagged recombinant protein and into pGEX-4T to generate GST-tagged recombinant protein. For protein expression, the plasmids were transformed into Escherichia coli BL21 (DE3; Transgene) and induced with 0.5 mm isopropyl β-d-1-thiogalactopyranoside for 4 h at 28°C. Then, the His-fused and GST-fused proteins were used for pull-down assays. The MdBT2-GST fused protein was first incubated with His-fused protein and then the mixed proteins were eluted with glutathione-agarose. The mixed proteins were incubated at least 1 h at 4°C with gentle shaking before centrifugation. The mixture was washed three times with wash buffer to remove nonspecific binding proteins. The pro-tein content of the eluted fractions was assessed by measuring the A280 to deter-mine whether the nonspecific protein had been removed. Finally, GST-tagged protein was eluted by adding elution buffer that contained glutathione, and the eluted proteins were analyzed by boiling and immunoblotting.

Co-IP Assays

IP assays were performed with a Pierce Classic Co-IP Kit (Thermo Fisher). Samples (500–1,000 µg) of the protein extracts were combined with 2 to 10 µg specific IP antibody per sample in a microcentrifuge tube and incubated for






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1 h to overnight at 4°C with gentle shaking. The reactions were then incubated with 20 µL protein A/G agarose for 1 to 2 h at 4°C to form the immune com-plex, and the resin was washed before elution. The eluted proteins were then boiled and analyzed as described above.

BiFC Assays

The full-length coding sequences of MdBT2 and MdMYB1 genes were inserted into the vectors 35S::pSPYNE-nYFP and 35S::pSPYCE-cYFP, respec-tively. The resultant constructs were transformed into A. tumefaciens LBA4404. Equal volumes of different combinations of A. tumefaciens strains were mixed and coinfiltrated into Nicotiana benthamiana leaves. Then the material was cul-tured at 23°C for 1 to 2 d, and YFP fluorescence was detected using a confocal laser-scanning microscope with excitation at 488 nm (Zeiss LSM 510 Meta).

Measurement of Anthocyanin

The methanol-HCl method was used to extract total anthocyanin from ap-ple calli and shoot cultures that had been pretreated with continuous light for 5 d (Lee and Wicker, 1991). Approximately 2 g of the samples were incubated in 5 mL of 1% (v/v) methanol-HCl overnight in the dark. The absorbance of each extract was measured at 530, 620, and 650 nm with a spectrophotometer (UV-1600; Shimadzu). The anthocyanin concentration was measured and cal-culated as described by Li et al. (2012).

Promoter-GUS Analysis

The pMdBT2::GUS vector was generated as described above. This ex-pression vector was transformed into Arabidopsis Col-0 plants (Bechtold and Pelletier, 1998). The transgenic Arabidopsis was grown on MS medium with 7% concentration of agarose (Sangon Biotech) for 7 d before treatment. For treatment, transgenic Arabidopsis seedlings grown on MS medium were transferred individually to medium that contained 10 mm KNO3 or 10 mm KCl for 12 h. After treatment, histochemical staining was performed as previously described (Jefferson et al., 1987). The promoter-GUS lines were selected by Hy-gromycin B at a concentration of 30 mg/L, and the homozygous pMdBT2::GUS (T2 generation) was used for GUS staining.

Protein Extraction and Immunoblotting

Proteins of transgenic apple calli were extracted with protein extraction buffer that contained 20 mm Tris (pH 7.4), 0.5 mm EDTA, 100 mm NaCl, 0.5 mm PMSF, 0.5% Nonidet P-40, and 0.5% protease inhibitor mixture (Sigma- Aldrich). Immunoblotting analysis was performed with 12% SDS-PAGE gels and PVDF membranes (Roche) using an electrotransfer apparatus (Bio-Rad). The PVDF membranes were combined with specific antibody (Sigma-Aldrich) before the proteins were visualized using ECL kits (Millipore). Protein abun-dance of MdMYB1 was detected by an anti-MdMYB1 monoclonal antibody (Abmart, Shanghai, China), MdACTIN protein was used as the loading control.

Ubiquitination Assay

To detect ubiquitination of MdMYB1 in vivo, 35S::MdMYB1-GFP, 35S::Md-MYB1-GFP, and 35S::MYC-MdBT2, or 35S::MdMYB1-GFP and 35S::MdBT2- anti cotransfected calli were obtained, the transgenic calli were treated on nitrate-sufficient medium (MS medium with the indicated hormones) under long-day conditions (16 h light/8 h dark) for approximately 3 to 5 d, and then the calli were treated with 50 µm MG132 for 10 h before extraction. The pro-tein extracts were immunoprecipitated using a Pierce Classic Protein A IP Kit as described (Thermo Fisher) with anti-GFP (Beyotime) and anti-Ubi (Sigma- Aldrich). The gel blot was probed with anti-Ubi (Sigma-Aldrich) or anti-GFP antibody, and the protein extract and immunoblotting were performed follow-ing the protocol as described above.

Cell-Free Degradation

MdMYB1-HIS protein was induced in cells (E. coli, BL21) as described above and eluted from His-nickel resin. Total proteins of transgenic MdBT2

and MdCUL3A apple calli and plantlets were extracted with degradation buf-fer (Wang et al., 2009). The protein concentration was then determined. Each reaction contained 500 µg of total transgenic apple calli protein and 100 ng of MdMYB1-HIS protein. For the proteasome inhibitor experiments, 50 µm MG132 (stock solution prepared with 0.5% DMSO) was added to the trans-genic apple calli protein 30 min prior to the cell-free degradation experiment. The reactions were incubated at 22°C. The quantified results were analyzed using Quantity One software (Bio-Rad).

Statistical Analysis

The data were expressed as the mean plus sd unless noted otherwise. Each experiment was repeated at least three times, and the results were based on the average of three parallel experiments. Statistical analysis was carried out using t tests, and differences were considered statistically significant when *P < 0.05, **P < 0.01, and ***P < 0.001.

Accession Numbers

Sequence data from this article can be found in the TAIR/GDR data libraries under accession numbers MdBT1 (MDP0000151000), MdBT2 (MDP0000643281), MdBT3.1 (MDP0000296225), MdBT3.2 (MDP0000187156), MdBT4 (MDP0000215415), MdMYB1 (MDP0000259614), MdCUL3A (MDP0000458584), AtBT2 (At3g48360), and AtPAP1 (At1g56650).

Supplemental Data

The following supplemental materials are available.

Supplemental Figure S1. Phenotype of apple shoot cultures and apple fruits in response to nitrate.

Supplemental Figure S2. The transcript levels of MdMYB1 and anthocy-anin biosynthetic genes MdCHS, MdDFR, and MdUFGT in response to nitrate in apple shoot cultures.

Supplemental Figure S3. Phylogenetic analysis and sequence alignment of the BTB-TAZ genes.

Supplemental Figure S4. Interaction of MdMYB1 with apple BTB-TAZ proteins.

Supplemental Figure S5. Protein and genome structure of the BTB-TAZ gene family.

Supplemental Figure S6. Expression analysis of MdBT2 in response to ni-trogen.

Supplemental Figure S7. Expression analysis of apple BTB-TAZ genes in transgenic apple calli.

Supplemental Figure S8. Interaction between MdBT2 and MdCUL3A, transcript levels of MdCUL3A in transgenic apple calli, and cell free degradation assays.

Supplemental Figure S9. Expression analysis of MdMYB1 and anthocyan-in biosynthetic genes, and cell free degradation assays in MdBT2 trans-genic apple calli and plantlets.

Supplemental Figure S10. Function of MdBT2 and its homolog AtBT2 in Arabidopsis.

Supplemental Table S1. The subfamily of BTB proteins in different species.

Supplemental Table S2. Primers used for gene and promoter cloning.

Supplemental Table S3. Primers used for yeast two-hybrid, pull-down, and BiFC assays.

Supplemental Table S4. Primers used for RT-qPCR analysis of different genes.


Wang et al.

















ACKNOWLEDGMENTS

We thank Professor Thomas Alan Gavin of Cornell University for language editing, Professor Zhi-Hong Zhang of Shenyang Agricultural University for providing apple ‘GL3’ shoot cultures, and Professor Remko Offringa of Leiden University, The Netherlands, for providing the Arabidopsis bt2-3 mutant.

Received February 27, 2018; accepted May 17, 2018; published May 28, 2018.

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