Low environmental temperatures promote anthocyanin accumulation and fruit colouration by up-regulating the expression of genes involved in anthocyanin biosynthesis and regulation in many fruit trees. However, the molecular mechanism by which fruit trees regulate this process in response to low temperature (LT) remains largely unknown. In this study, the cold-induced bHLH transcription factor gene MdbHLH3 was isolated from an apple tree and was found to interact physically and specifically through two regions (amino acids 1–23 and 186–228) at the N terminus with the MYB partner MdMYB1 (allelic to MdMYB10). Subsequently, MdbHLH3 bound to the promoters of the anthocyanin biosynthesis genes MdDFR and MdUFGT and the regulatory gene MdMYB1 to activate their expression. Furthermore, the MdbHLH3 protein was post-translationally modified, possibly involving phosphorylation following exposure to LTs, which enhanced its promoter-binding capacity and transcription activity. Our results demonstrate the molecular mechanism by which MdbHLH3 regulates LT-induced anthocyanin accumulation and fruit colouration in apple.
Anthocyanins are biosynthesized through the flavonoid pathway, in which biosynthetic genes are coordinately modulated by a conserved MYB-bHLH-WD40/WDR (MBW) regulatory complex. The members of this regulatory complex, such as bHLH and MYB transcription factors (TFs), have been intensively studied for two decades in herbaceous model plants, including Arabidopsis, Petunia, maize, snapdragon and tomato (Koes et al. 2005; Ramsay & Glover 2005; Ballester et al. 2010; Albert et al. 2011). The crucial role of this complex in the regulation of anthocyanin biosynthesis in higher plants has been well documented across many species (Koes et al. 2005; Ramsay & Glover 2005; Allan et al. 2008). Furthermore, locus mutation and genetic manipulation of bHLH and MYB genes have produced an increasing number of red or purple staple, fruit and ornamental crops that are rich in anthocyanins, which provide enhanced health benefits for consumers and unprecedented aesthetics (Escribano-Bailón, Santos-Buelga & Rivas-Gonzalo 2004; Allan et al. 2008; Butelli et al. 2008; Tanaka & Ohmiya 2008; Kovinich et al. 2011). Therefore, interest in discovering new regulatory genes in economically important plants has been growing. Recently, an increasing number of MYB TFs have been characterized from woody fruit trees (Allan et al. 2008; Deluc et al. 2008; Lin-Wang et al. 2010), suggesting that the MBW model operates in perennial deciduous trees. However, their bHLH interaction partners have not been functionally characterized in most plants, especially in deciduous ornamental and fresh fruit trees.
The fact that red organ colouration is more prominent in cooler, temperate regions and seasons indicates that there is a temperature-specific effect on anthocyanin biosynthesis in plants (Ubi et al. 2006; Lin-Wang et al. 2011), which increases concerns about the effect of ongoing climate warming on red organ colouration. In fact, leaf colouration in Japanese maple has been markedly delayed as a consequence of global warming in the past half century (Ibáñez et al. 2010). Similarly, climate warming appears to profoundly influence the suitability of regions for apple and citrus production (Sugiura & Yokozawa 2004). Thus, it is proposed that the global atmospheric warming trend will affect red organ colouration, thereby resulting in a world with fewer bright colours. Elucidation of the molecular mechanism by which environmental temperatures affect anthocyanin synthesis and organ colouration in plants is important for the maintenance of ornamental plants and fresh fruit production.
It has been well documented that low temperature (LT) stimulates anthocyanin accumulation by up-regulating the expression of biosynthetic genes (Shvarts, Borochov & Weiss 1997; Ubi et al. 2006; Yamane et al. 2006; Steyn et al. 2009; Crifòet al. 2011). Furthermore, increasing evidence indicates that the expression of the members of the MBW complex, specifically bHLHs and MYBs, is modulated by environmental temperatures and other stimuli (Ban et al. 2007; Rowan et al. 2009; Lin-Wang et al. 2011), suggesting a novel role for the MBW regulatory complex in response to environmental effects on anthocyanin synthesis in plants. However, the molecular mechanism by which MBW components regulate anthocyanin biosynthesis and organ colouration in response to environmental stimuli is largely unknown (Gonzalez 2009).
In the anthocyanin-associated MBW regulatory complex, MYB TFs specifically regulate anthocyanin biosynthesis (Allan et al. 2008). However, bHLH TFs and WD proteins function pleiotropically in multiple processes (Ramsay & Glover 2005). In Arabidopsis, the bHLH TFs EGL3 and GL3 function in both the biosynthesis of anthocyanins and the formation of trichomes and root hairs (Ramsay & Glover 2005). In addition, bHLH TFs such as ICE1 are associated with functions in cold signalling pathways (Chinnusamy et al. 2003;Chinnusamy, Zhu & Zhu 2007). Taking into consideration that LT induces anthocyanin accumulation in apple led us to the hypothesis that certain bHLH TFs are involved in both the regulation of anthocyanin biosynthesis and cold responses. In apple, bHLH TF genes, such as MdbHLH3 and MdbHLH33, have been characterized in relation to anthocyanin biosynthesis. The combination of MdbHLH3 with MdMYB10 (allelic to MdMYB1) confers visible anthocyanin phenotypes in tobacco leaves in transient co-expression experiments (Espley et al. 2007). AtbHLH2 plus either PdmMYB10 or PprMYB10 confers the same phenotype (Lin-wang et al. 2010). In addition, the expression of MdbHLH300 is inhibited in apple fruits grown under hot climatic conditions (Lin-Wang et al. 2011). However, it is still largely unknown whether and how apple MdbHLHs interact physically with MYB TFs to regulate anthocyanin biosynthesis genes and how they promote anthocyanin accumulation and fruit colouration in response to low environmental temperatures.
In this study, the MdbHLH3 gene was isolated based on its differential expression in apple fruits showing poor or good colouration which were exposed to high and LTs, respectively. The function of this gene in anthocyanin biosynthesis and fruit colouration was characterized, particularly under LT. Finally, the role of MdbHLH3 in the genetic manipulation of anthocyanin enrichment in plants was discussed.
MATERIALS AND METHODS
Apple fruits were collected from adult trees of the ‘Red Delicious’ cultivar (Malus domestica Borkh.). Bagged fruits were harvested 140 days after full bloom (DAFB). The skin of the fruits was peeled, obtaining less than 1 mm of cortical tissue, for anthocyanin measurements and other analyses.
Calli of the ‘Orin’ cultivar and in vitro cultures of the ‘Gala’ cultivar were used for genetic transformations and other analyses. The calli were grown on Murashige and Skoog (MS) medium supplemented with 0.5 mg L−1 indole-3-acetic acid (IAA) and 1.5 mg L−1 6-benzylaminopurine (6-BA) at 25 °C in the dark. The ‘Gala’ cultures were grown on MS subculture medium containing 0.6 mg L−1 6-BA and 0.2 mg L−1 IAA, rooted in MS rooting medium containing mg L−1 IAA at 25 °C under a 16 h photoperiod (30 µmol m−2 s−1, cool white fluorescent lamps).
Tobacco (Nicotiana benthamiana) was cultivated in a growth room at 25 °C using natural light with a daylight extension of 14 h until at least six leaves were available for infiltration with Agrobacterium.
Determination of total anthocyanins
Anthocyanin content was measured using 0.2 g of the cultures or 0.3 g of apple skin in 1 mL 1% (v/v) HCl-methanol for 24 h at room temperature in the dark. After centrifugation for 5 min at 13 000 g, the upper aqueous phase was subjected to spectrophotometric quantification at 530, 620 and 650 nm using a UV–vis spectrophotometer (SHIMADZU UV-2450, Kyoto, Japan). The relative anthocyanin content was determined with the following formula: OD = (A530 – A620) – 0.1(A650 – A620) (Lee & Wicker 1991). One unit of anthocyanin content was expressed as a change of 0.1 OD (unit × 103 g−1 FW).
Gene cloning and expression analysis
Total RNA was isolated from plant material for gene cloning and expression analyses using TRIzol reagent (Invitrogen, Carlsbad, CA, USA). Genomic DNA was extracted from 0.2 g of young apple leaves using a DNeasy plant mini kit (QIAGEN, Shanghai, China). First-strand cDNA was synthesized using Oligo dT with a RevertAidTM First Strand cDNA Synthesis Kit (Fermentas, Hanover, MD, USA).
For real-time RT-PCR analysis, cDNA was diluted 1:20 with water, and the reactions were performed using SYBR Green MasterMix (SYBR Premix EX Taq TM, Dalian, China), as described by the manufacturer. The primers employed for flavonoid structural genes and the thermocycling conditions are described in Takos et al. (2006). Real-time qPCR was performed with iQ SYBR Green Supermix and an iCycler iQ5 system (Bio-Rad, Hercules, CA, USA). Relative quantification of specific mRNA levels was performed using the cycle threshold (Ct) 2–ΔΔCt method (Software IQ5 2.0; Livak & Schmittgen 2001). The qRT-PCR efficiency (E) for each gene was obtained by calculating the kinetic curve (Liu & Saint 2002). For all analyses, the signal obtained for a gene of interest was normalized against the signal obtained for the MdACTIN gene. All samples were tested in three to four biological replicates.
For the semi-quantitative RT-PCR, the reactions were carried out according to the manufacturer's instructions (Transgene, Beijing, China) using the following thermal profile: pre-incubation at 95 °C for 5 min followed by 30 cycles of 95 °C (30 s), 58 °C (30 s) and 72 °C (30 s), with a final extension at 72 °C for 5 min. All of the primers used in this study are listed in Supporting Information Appendix S1.
Yeast two-hybrid assays
Yeast transformants were exhaustively screened on synthetic defined (SD) media (-Leu/-Trp/-His/-Ade) according to the manufacturer's instructions (Clontech, Palo Alto, CA, USA). The MdbHLH3 coding sequence was excised using either BamHI single digestion or BamHI-SalI double digestion (for some regions containing deletions) and subcloned into pGAD424 to generate an in-frame fusion with the GAL4 activation domain (GAD). The domain-deleted form (1–201) of MdMYB1 was excised using BamHI-SalI double digestion and subcloned into pGBT9 to generate an in-frame fusion with the GAL4 DNA-binding domain (GBD) because full-length MdMYB1 is self-activated in β-galactosidase assays in yeast. MdbHLH3 full-length (1) or domain-deletion constructs [2-(1–346), 3-(207–710), 4-(1–228), 5-(207–464), 6-(400–710), 7-(24–228), 8-(57–228), 9-(107–228), 10-(1–90), 11-(1–129), 12 -(1–133) and 13-(1–186)] were cloned into the pGAD424 vector and fused with BD DNA. The two plasmids were co-transformed into the yeast (Saccharomyces cerevisiae) AH109 strain using the lithium acetate method and cultured at 30 °C. The resultant yeast transformants were exhaustively screened on SD (-Leu/-Trp/-His/-Ade) medium according to the manufacturer's instructions (Clontech, Mountain View, CA, USA). Cells were plated onto selective medium lacking Trp and Leu (-Trp/-Leu), and putative transformants were subsequently transferred to medium lacking Trp, Leu, His and adenine (-Leu/-Trp/-His/-Ade). After colonies were transferred to filter paper, β-galactosidase filter assays were conducted using the substrate 5-bromo-4-chloro-β-D-galactoside (X-Gal) as described by Halfter, Ishitani & Zhu (2000). Another apple bHLH protein, MdbHLH33, was used as a negative control.
Constructs for investigating in planta interactions using BiFC assays were produced in the pSPYNE-35S and pSPYCE-35S vectors and the co-transformation vector 35S:P19. Firstly, the coding regions of MdbHLH3, MdMYB1, MdbHLH33 and MdMYB31 were cloned into pSPYNE-35S and pSPYCE-35S, which contain DNA encoding the N or C-terminal regions of YFP (YFPN or YFPC), respectively, to generate the plasmids MdbHLH3/MdMYB1/MdbHLH33-YFPN and MdbHLH3/MdMYB1/MdMYB31-YFPC according to previous protocols (Walter et al. 2004). The primers used for plasmid construction are presented in the online Supporting Information Appendix S1.
These constructs were transformed into the Agrobacterium tumefaciens GV3101 strain through electroporation. The A. tumefaciens strains containing different constructs were incubated, harvested and resuspended in infiltration buffer (10 mm MES, 0.2 mm acetosyringone and 10 mm MgCl2) at a final concentration of OD600 = 0.5. Equal volumes of different combinations of Agrobacterium strains were mixed and co-infiltrated into onion epidermis cells through Agrobacterium infection. The Petri dishes were incubated at 24 °C for 48 h prior to detection of YFP fluorescence.
The YFP expression in the onion epidermis cells was examined using a confocal microscope (Zeiss LSM 510 Meta, Jena, Germany), with excitation at 488 nm and detection with a 500–530 nm band-path filter for YFP.
Electrophoretic mobility shift assays (EMSA)
EMSAs were performed using biotin-labelled probes and the Lightshift Chemiluminescent EMSA kit (Pierce, Rockford, IL, USA) according to the manufacturer's instructions. A volume containing 50 ng of recombinant His-MdbHLH3 was purified for the binding reactions. Synthetic oligonucleotides for the MdDFR and MdUFGT promoters were biotinylated and heated at 100 °C for 5 min, then allowed to anneal to the double-stranded oligonucleotides for 12 h at room temperature. The binding reactions were carried out in a total volume of 20 µL of a solution containing 25 mm HEPES-KOH (pH 7.5), 100 mm KCI, 0.1 mm ethylenediaminetetraacetic acid (EDTA), 17% glycerol, 1 mm DTT, 4 µg of poly (dI-dC), 50 ng of purified protein, ∼1 pmol of labelled probe and competitor DNA (25, 100 or 400 pmol). The assay mixtures were incubated for 20 min at room temperature. Then, the reaction mixtures were layered on 6% acrylamide gels containing 0.5% TBE buffer and 3.6% glycerol. After a pre-run in the 0.5% TBE buffer for 1 h at 100 V at room temperature, the samples were electrophoresed for an additional 2 h under the same conditions.
Following native polyacrylamide gel electrophoresis, the DNA was transferred to positively charged nylon membranes (Hybond N+; Amersham, Little Chalfont, Buckinghamshire, UK), and the signal was detected using the chemiluminescent nucleic acid detection method (Pierce).
Chromatin immunoprecipitation (ChIP)-PCR analysis
The ChIP experiment was performed as described by He et al. (2005), with some modifications. The immunoprecipitation, elution and reverse cross-linking of chromatin were performed using a ChIP Kit (Upstate, Lake Placid, NY, USA, no. 17–295). An anti-GFP antibody (Abcam, Cambridge, UK) was used for ChIP. The amount of immunoprecipitated chromatin was determined via PCR. The experiment was repeated three times.
Transient dual luciferase assay
Transient expression assays were performed in N. benthamiana leaves. The MdUFGT promoter was cloned into pGreen 0800 at the KpnI and PstI sites to fuse it with the luciferase reporter gene (ProMdUFGT-LUC). MdbHLH3, MdMYB1 and MdbHLH33 were cloned into the pGreenII 62-SK vector (Hellens et al. 2000). The transient expression assays were performed in tobacco (N. benthamiana) leaves. The Agrobacterium strains containing different constructs were incubated, harvested and resuspended in infiltration buffer (10 mm MES, 0.15 mm acetosyringone and 10 mm MgCl2) at a final concentration of OD600 = 0.2 and then incubated at room temperature without shaking for 2 h prior to infiltration. The infiltrations were performed using a needleless syringe in young leaves of N. benthamiana. Transient expression was assayed 3 d after inoculation.
Firefly and Renilla(Ren) luciferase were assayed using dual luciferase assay reagents (Beyotime, Nanjing, Jiangsu, China). Three days after inoculation, 2 cm leaf discs (six technical replicates from each plant) were removed and ground in 500 µL of passive lysis buffer (PLB). Subsequently, 10 µL of a 1/100 dilution of this crude extract was assayed in 90 µL of luciferase assay buffer, and the resultant chemiluminescence was measured. Ren luciferase test buffer (100 µL) was added, and a second chemiluminescence measurement was recorded. Absolute relative luminescence units (RLU) were measured using a GloMax20/20 Luminometer (Turner Biosystems, Sunnyvale, CA, USA) according to the manufacturer's instructions, with a 5 s delay and 15 s integrated measurements.
Agrobacterium-mediated genetic transformation of MdbHLH3 into apple materials
The A. tumefaciens LBA4404 strain was grown in LB media supplemented with 50 µg mL−1 of kanamycin and 50 µg mL−1 of rifampicin. The MdbHLH3-GFP construct consists of the coding sequence under the control of a 35S promoter cloned into the pBIN m-gfp5-ER vector. The control construct contained the 35S:GFP construct in the same vector. The binary vector pBI121-MdbHLH3 contained MdbHLH3 cDNA driven by a 35S promoter. For transformation of ‘Orin’ calli, 7-day-old calli grown in liquid medium were co-cultured with A. tumefaciens LBA4404 carrying MdbHLH3-GFP, pBI121-MdbHLH3 or other vectors. The calli were co-cultured on MS medium containing 0.5 mg L−1 IAA, 1.5 mg L−1 6-BA and 8 g L−1agar for 2 d at 25 °C. Subsequently, the calli were washed three times with sterile water and transferred to MS medium supplemented with 250 mg·L−1 of carbenicillin and 30 mg L−1 of kanamycin or 20 mg L−1 of hygromycin for transgene selection.
Transgenic ‘Gala’ plants were generated from leaf fragments through Agrobacterium-mediated transformation, as previously reported by Yao et al. (1995).
Histochemical staining to detect GUS activity in transgenic calli was performed as previously described by Jefferson, Kavanagh & Bevan (1987). Two-week-old calli were immersed in GUS staining buffer (1 mm 5-bromo-4-chloro-3-indolyl-β-glucuronic acid solution in 100 mm sodium phosphate, pH 7.0, 0.1 mm EDTA, 0.5 mm ferricyanide, 0.5 mm ferrocyanide and 0.1% Triton X-100), and after applying vacuum for 5 min, they were incubated at 37 °C overnight. After staining, the calli were photographed to record the deposition of the transgene product.
For quantitative analysis of GUS, 0.5 g of calli was extracted with 1 mL of GUS extraction buffer. The concentration of total protein in the crude extract was quantified using the RC DC Protein Assay kit (Bio-Rad). The extract (50 µL) was then added to 950 µL of GUS extraction buffer containing 1 mm 4-MUG and incubated at 37 °C. An aliquot of the reaction mixture (100 µL) was added to 900 µL of stop solution (1 M sodium carbonate) every 10 min. The resultant fluorescence was measured using a Hitachi F-4500 spectrofluorometer (Hitachi, Tokyo, Japan) at an excitation wavelength of 365 nm and an emission wavelength of 450 nm. The analysis was repeated three times, and the mean values for each construct were compared.
Construction of viral vectors and agro-inoculation of apple fruit
The viral vector pIR (Peretz et al. 2007) was used for agro-inoculation. To construct the MdbHLH3 silencing vector, the 5′-UTR of MdbHLH3 (bases 112–342) was amplified from apple cDNA. The resultant PCR product was confirmed by sequencing and inserted at both ends of MdbHLH3 (between the KpnI and PstI sites upstream of MdbHLH3 and between the XbaI and SalI sites downstream of it). The resultant construct was designated pIR-antiMdbHLH3. To construct the overexpression vector, the ORF of MdbHLH3 was inserted into the pIR vector between the AvrII and Eco52I sites, while MdMYB1 was inserted between the HindIII and SalI sites. The resultant constructs were designated pIR-MdbHLH3 and pIR-MdMYB1, respectively. The IL-60-BS vector was used as a helper plasmid.
Bagged apples that had been freshly harvested from trees were used for infiltration. The recipient apples were punctured with a hypodermic needle. The infiltrations were performed using a needleless syringe, and approximately 200 ng of DNA (in 100 µL) was pipetted into the tube until it was partially absorbed by the apples. For colouration, the debagged apples were infiltrated with pIR-antiMdbHLH3 and kept in the dark at 4 °C for 10 d. Subsequently, these apples were infiltrated and maintained for an additional 4 d under 24 h of continuous white light (200 µmol m−2 s−1) with supplemental UVB (280 to 320 nm) at 17 °C in a growth chamber. The debagged apples infiltrated with either pIR-MdbHLH3 or pIR-MdMYB1 were kept overnight in the dark at room temperature and were subsequently exposed to white light and UVB at 17 or 27 °C in a growth chamber for another 2 or 5 d for colouration. The helper plasmid IL-60-BS was used in all infiltrations.
Immunoblot analysis for protein accumulation and phosphorylation
An MdbHLH3 affinity purified rabbit polyclonal antibody against the specific synthetic MdbHLH3 peptide NH2-CQRSRSSSGEMQRSNS-COOH was obtained from GenScript (Nanjing, China). The antibody was resuspended in phosphate-buffered saline (PBS) at a final concentration of 14.4 mg mL−1. To detect the MdbHLH3-GFP protein in transgenic plants, 2-week-old, dark-grown calli were either kept in darkness or treated at LT, as indicated. The treated calli were dried with a paper towel and immediately extracted in denaturing extraction buffer (100 mm Tris-HCl, pH 7.8, 1 mm EDTA-Na2, 1% PVP, 200 mm sucrose, 10 mmβ-MeOH) at a ratio of 2:1 (v/w). A protease inhibitor cocktail (1X; Roche, Indianapolis, IN, USA) and 2 mm phenylmethylsulfonyl fluoride (PMSF) were also added during the extraction. The plant extract was boiled for 4 min and cleared via centrifugation at 12 000 g for 15 min. Total protein supernatants were separated on 10% sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS–PAGE) gels, blotted onto polyvinylidene difluoride (PVDF) membranes, and probed with anti-GFP or anti-MdbHLH3 antibodies. Protein analysis was conducted by immunoblotting using a previously described method (Shen et al. 2007) employing a 1:2000 dilution of anti-GFP or a 1:1000 dilution of an anti-MdbHLH3 antibodies. A peroxidase-labelled goat anti-rabbit antibody (ZSGB-BIO, Beijing, China) at a 1:20 000 dilution was used as a secondary antibody.
For phosphorylation immunoblot analyses, the total protein supernatants were separated on 6.5% SDS–PAGE gels and probed with anti-GFP antibodies. For Western blot band detection, Chemiluminescent HRP Substrate (Millipore, Billerica, MA, USA) was used to obtain enhanced chemiluminescence (ECL), which was subsequently visualized on X-ray film.
LT-induced MdbHLH3 is a positive regulator of fruit colouration in apple
Compared with high temperatures (HTs) of 27 °C, LTs of 17 °C noticeably promoted UVB-induced anthocyanin accumulation and red colouration in apples from the ‘Red-Delicious’ cultivar (Fig. 1a; Supporting Information Fig. S1a,b). A bHLH gene was isolated based on its differential expression in apple skins of bright and pale red fruits, as shown in Fig. 1a. The predicted apple bHLH protein from the ‘Red-Delicious’ cultivar shares a high identity with the known MdbHLH3 protein (Espley et al. 2007; Supporting Information Fig. S2). BLAST searches through the apple genome database found that they were allelic to MDP0000225680 on Chromosome11. The predicted apple bHLH protein was designated MdbHLH3. Compared with bHLH proteins in other plant species, MdbHLH3 exhibited high similarity to Arabidopsis TT8/HLH042 and other bHLH protein-associated anthocyanin biosynthesis, especially at the N terminus and in the bHLH domain (Supporting Information Figs S3 & S4). Expression analyses demonstrated that both the transcript and protein levels of MdbHLH3 increased with the bright red colouration under LT but decreased with the pale red colouration in the debagged apples under HT (Fig. 1b,c), suggesting its involvement in LT-induced fruit colouration in apples.
To characterize the function of MdbHLH3 in red fruit colouration, viral vector-mediated suppression was carried out using bagged fruits immediately following their detachment from the apple tree. The pIR-anti MdbHLH3 construct containing the antisense 5′-UTR of MdbHLH3 was used for infiltration. The results showed that MdbHLH3 suppression inhibited red colouration in the skin around the infiltration site, while the empty vector control did not influence the red colouration (Fig. 1d). In addition, MdbHLH3 suppression noticeably down-regulated the expression levels of the anthocyanin synthesis genes MdDFR and MdUFGT in the skin around the injection sites (Fig. 1e and Supporting Information Fig. S5). Therefore, MdbHLH3 is a positive regulator of anthocyanin biosynthesis and fruit colouration in apple. Moreover, the expression of MdMYB1 was slightly decreased, suggesting that MdbHLH3 may regulate MdMYB1.
The N terminus of MdbHLH3 interacts with MdMYB1 physically and specifically
MdMYB1 is a master regulator of anthocyanin biosynthesis and fruit colouration in apples (Takos et al. 2006). To verify whether MdbHLH3 interacts with MdMYB1, yeast two-hybrid (Y2H) assays were performed. Positive β-gal activity was observed in yeast containing pGBT9-MdMYB1 plus pGAD424-MdbHLH3 grown on -T/-L/-H/-A screening medium, but not in those containing pGBT9-MdMYB1 plus the empty pGAD424 vector or pGBT9-MdMYB1 plus pGAD424-MdbHLH33 (Fig. 2b), indicating that MdbHLH3 interacted with MdMYB1 physically and specifically.
In addition, serially truncated MdbHLH3 mutants were used in Y2H assays to determine the region that is critical for the specific interaction of MdbHLH3 with MdMYB1 (Fig. 2a). The results showed that two regions (amino acids 1–228 and 1–336) at the N terminus of MdbHLH3 interacted strongly with MdMYB1, while regions at the C terminus (207–710 and 400–710) did not. Furthermore, serial truncations of the N terminus of MdbHLH3 resulted in weak (amino acids 24–228) or no interaction (amino acids 1–90, 1–129, 1–133 and 1–186) with MdMYB1, indicating that two regions (amino acids 1–23 and 186–228) at the N terminus of MdbHLH3 are crucial for the interaction between MdbHLH3 and MdMYB1 (Fig. 2b).
To further characterize the in vivo interaction between MdbHLH3 and MdMYB1, bimolecular fluorescence complementation (BiFC) assays were performed. Two-construct combinations, that is, MdbHLH3-YN plus MdMYB1-YC or MdMYB1-YN plus MdbHLH3-YC, were co-transformed into onion epidermal cells. As a result, a strong yellow fluorescent signal was observed in the nuclei of cells transformed with either of the two combinations (Fig. 2c). In contrast, there was no fluorescent signal observed in cells co-transformed with MdbHLH3-YN plus the empty vector pSPYCE-35S (YC) or pSPYNE-35S (YN) plus MdMYB1-YC. Therefore, MdbHLH3 interacted strongly with MdMYB1. In contrast, no fluorescence was detected in the cells containing combinations of MdbHLH3-YN plus MdMYB31-YC and MdbHLH33-YN plus MdMYB1-YC (Fig. 2c), indicating that MdbHLH3 interacted specifically with MdMYB1 in plant cells in vivo.
MdbHLH3 binds specifically to the MdDFR and MdUFGT promoters
The results presented in Fig. 1e suggested that MdbHLH3 positively regulated the expression of the MdDFR and MdUFGT genes in apple skin. It is well known that bHLH proteins recognize MYC cis-elements with the consensus sequence CANNTG, also referred to as the E-box (Fisher & Goding 1992). The MdDFR promoter contains 10 putative MYCs, while the MdUFGT promoter has 17, among which three MYCs share the same consensus sequence, CATTTG. In addition, there are two low-temperature-responsive (LTR) cis-elements in the MdUFGT promoter with the core sequence CCGAAA.
To determine whether MdbHLH3 binds to the MYC-recognition site CATTTG in the MdDFR and MdUFGT promoters, we expressed and purified recombinant His- MdbHLH3 fusion proteins. DNA fragments containing the E-box sequence CATTTG were used as probes to examine possible interactions with His- MdbHLH3 in an electrophoresis mobility shift assay (EMSA). As a result, specific DNA-MdbHLH3 protein complexes were detected when the CATTTG-containing sequence was used as a labelled probe (Fig. 3a). The formation of these complexes was reduced when increasing amounts of the unlabelled MYC competitor probe with the same sequence were added. This competition was not observed when using the mutated version (MdDFRm, AATTTC; Fig. 3b). This specificity of competition confirms the hypothesis that the interaction between DNA and MdbHLH3 requires the MYC recognition sequence.
In addition, an EMSA was used to test the possible interaction between the MdbHLH3 protein and the LTR element in the MdUFGT promoter. A labelled probe containing the LTR element CCGAAA detected the formation of specific DNA- MdbHLH3 protein complexes, which was suppressed gradually by the addition of increasing amounts of an unlabelled LTR competitor, but not by the addition of a mutated LTR (MdUFGTm, CCCTAAA) competitor (Fig. 3b). These results demonstrate that MdbHLH3 binds specifically to the MYC and LTR cis-elements in the MdDFR and MdUFGT promoters in vitro.
To verify the specific in vivo binding of MdbHLH3 to the MdDFR and MdUFGT promoters, we generated a transgenic apple calli containing MdbHLH3-GFP using the vector pBIN-GFP as a control. The MdDFR and MdUFGT promoters were used to test in vivo binding using a ChIP-PCR assay. The results showed that the MdDRF and MdUFGT promoter regions containing E-box and LTR cis-elements were all enriched by ChIP in the MdbHLH3-GFP transgenic calli compared with those containing the pBIN-GFP control, while other regions were not (Fig. 3c). These results provide in vivo evidence of the binding of MdbHLH3 to the MdDFR and MdUFGT promoters.
MdbHLH3 promotes anthocyanin accumulation by activating the promoter of biosynthesis genes in apple calli
To verify whether MdbHLH3 activates the MdUFGT promoter, a transient dual luciferase assay was performed. Constructs were tested for the capacity to activate their encoded proteins using a co-transfected MdUFGT promoter fused to the luciferase (LUC) reporter gene in Nicotiana benthamiana leaves. The results showed that each of the MdbHLH3 and MdMYB1 constructs showed noticeable activation of the MdUFGT promoter based on LUC gene expression, indicating that they immediately activate the MdUFGT promoter. In addition, the combination of the MdbHLH3 and MdMYB1 constructs resulted in enhanced activation of the MdUFGT promoter based on LUC expression relative to either gene alone, while the MdbHLH3 and MdMYB31 combination did not (Fig. 3d), indicating that the specific interaction between the MdbHLH3 and MdMYB1 proteins enhances activation through the MdUFGT promoter.
Moreover, a stable transformation was carried out to confirm the activation of MdbHLH3 through the MdUFGT promoter using the GUS reporter gene. Firstly, the constructs PMdUFGT:GUS and 35S:MdbHLH3 were genetically transformed into apple calli (Fig. 3e). The result showed that the transgenic calli containing PMdUFGT:GUS plus 35S:MdbHLH3 exhibited much higher GUS activity than PMdUFGT:GUS transgenic calli under normal conditions at 25 °C, indicating that MdbHLH3 activates GUS transcription driven by the MdUFGT promoter (Fig. 3f).
MdbHLH3-GFP transgenic apple calli were used to observe the anthocyanin phenotype at 25 °C under UVB light or in the dark. The results showed that under UVB light, the MdbHLH3-GFP transgenic calli accumulated more anthocyanins and therefore had a redder appearance than the pBIN-GFP control (Fig. 4a). Thus, MdbHLH3 promotes anthocyanin accumulation by activating the promoters of biosynthesis genes.
Post-transcriptional modification of the MdbHLH3 protein is associated with regulation of anthocyanin accumulation in response to LT in apple calli
In the GUS assay, PMdUFGT:GUS transgenic calli exhibited 1.5-fold higher GUS activity at 17 °C than at 25 °C. In transgenic calli co-transformed with PMdUFGT:GUS plus 35S:MdbHLH3, the LT of 17 °C induced approximately 2.5-fold higher GUS activity than the HT of 25 °C (Fig. 3e,f). Similarly, the MdbHLH3-GFP transgenic calli generated fivefold more anthocyanins at 17 °C than at 25 °C (Fig. 4b). Therefore, lower temperatures are more conducive to the MdbHLH3-mediated activation of the transcription of downstream genes and the promotion of subsequent anthocyanin accumulation in apple calli.
In addition, expression analysis showed that the expression of MdMYB1 increased in both pBIN-GFP control and MdbHLH3 transgenic calli after treatment with the LT of 17 °C. However, MdbHLH3 is induced only by the LT of 17 °C in pBIN-GFP control and is not induced in MdbHLH3 transgenic calli, compared with the 25 °C treatment (Fig. 4c). Considering that LT induced remarkably high GUS activity and anthocyanin accumulation, it is proposed that post-translational modification of the MdbHLH3 protein may occur under LT. To verify this hypothesis, an immunoblot assay was performed in MdbHLH3-GFP transgenic calli using an anti-GFP antibody. The MdbHLH3-GFP protein was examined to detect rapid changes in molecular behaviour immediately following LT treatment. Using PAGE conditions optimized to detect changes in apparent molecular mass, a small shift in electrophoretic mobility was observed within 15 min of calli exposure to a temperature of 17 °C (Fig. 4e), indicating that MdbHLH3 was post-translationally modified. Furthermore, no shift in electrophoretic mobility was detected in transgenic calli that were treated with a temperature of 17 °C plus staurosporine(stau), a protein kinase C inhibitor (Fig. 4e), providing evidence of post-translational modification of the MdbHLH3 protein, which may involve phosphorylation.
It has been reported that post-translational modification affects the DNA-binding activity of many transcription factors (Yang, Sharrocks & Whitmarsh 2003). A ChIP-PCR assay was conducted to examine whether LT affects the in vivo binding activity of MdbHLH3 to the MdDFR and MdUFGT promoters. The results showed that the MdDFR and MdUFGT promoter regions were enriched in transgenic calli grown at 17 °C compared with being grown at 25 °C, indicating that the LT enhanced the in vivo binding activity of the MdbHLH3 protein to the MdDFR and MdUFGT promoters (Fig. 4d) and subsequently promoted anthocyanin accumulation in apple calli (Fig. 4a).
MdbHLH3 promotes LT-induced anthocyanin accumulation in transgenic apple plantlets
To identify its in planta function, MdbHLH3 was introduced into ‘Gala’ apple cultivars through Agrobacterium-mediated transformation. A total of six transgenic lines were obtained, as indicated by increased MdbHLH3 expression (Supporting Information Fig. S6). Three of these lines, L1, L2 and L3, were chosen for further investigation. Under normal conditions at 25 °C, transgenic plantlets produced more anthocyanins in their shoots and roots than wild-type (WT) plantlets (Fig. 5a,b). The transgenic plantlets also generated more MdDFR and MdUFGT transcripts compared with WT (Fig. 5c), demonstrating that MdbHLH3 enhanced anthocyanin biosynthesis by up-regulating downstream genes.
When cultured under an LT of 17 °C, both WT and transgenic in vitro plantlets showed markedly increased accumulation of anthocyanins and expression of MdDFR and MdUFGT compared with the results obtained at 25 °C. However, the transgenic lines produced much greater amounts of anthocyanins and therefore appeared redder in colour than the WT control, further indicating that MdbHLH3 is involved in LT-induced anthocyanin biosynthesis in apple.
Viral vector-mediated MdbHLH3 overexpression promotes LT-induced red colouration in apple fruits
The viral construct pIR-MdbHLH3 containing the MdbHLH3 ORF was used for infiltration, while the vector pIR-MdMYB1 served as a positive control. The results showed that MdMYB1 overexpression improved the red colouration around the infiltration site compared with the empty vector control, indicating that this methodology was feasible for use in apples. Following MdbHLH3 injection, red colouration was observed in the skin around the infiltration site 2 days after infiltration (DAI) at 17 °C, whereas reddish colouration around the infiltration site did not appear until 5 DAI at 27 °C. In contrast, no red colouration was observed in apples infiltrated with the empty vector (Fig. 6a). These data indicated that MdbHLH3 overexpression promoted red fruit colouration, and this colouration was enhanced by LT.
Furthermore, infiltration with pIR-MdMYB1 and pIR-MdbHLH3 induced the overexpression of MdMYB1 and MdbHLH3, respectively, and subsequently increased the expression of MdUFGT and MdDFR in the skin around the infiltration sites (Fig. 6b). Therefore, MdbHLH3 overexpression promoted fruit colouration by up-regulating the expression of anthocyanin synthesis genes and subsequent anthocyanin accumulation.
Interestingly, MdbHLH3 overexpression also enhanced the expression of MdMYB1 (Fig. 6b), whereas its suppression slightly inhibited the expression of MdMYB1 (Fig. 1e). To determine whether MdbHLH3 regulates MdMYB1, the MdMYB1 promoter was analysed for possible MdbHLH3-binding cis-elements. The results showed that E-box elements were present in the MdMYB1 promoter sequence. Using 35S:MdbHLH3-GFP transgenic apple calli and the anti-GFP antibody, ChIP-PCR assays showed that MdbHLH3 bound directly to three regions containing E-box elements within the MdMYB1 promoter (Fig. 6c). Therefore, MdbHLH3 regulates the expression of MdMBY1 by directly binding to its promoter.
In the ternary MBW complex which is necessary for anthocyanin regulation, pleiotropic bHLH TFs interact with specific MYB partners to modulate the expression of downstream biosynthesis genes. Two decades of research on this complex have shown that several bHLH TFs, including AmDelila, AtTT8, AtGL3, AtEGL3, PhAN1, PhJAF13 and GhMYC1, interact with their respective MYB partners AmRosea1, AtPAP1, AtTT2, PhAN2 and GhMYB10 to regulate anthocyanin or proanthocyanidin biosynthesis in herbaceous model plants, such as snapdragon, Arabidopsis, Petunia and Gerbera hybrida (Elomaa et al. 2003; Koes et al. 2005; Ramsay & Glover 2005).
Recently, an increasing number of MYB TFs necessary for anthocyanin biosynthesis have been identified from deciduous fruit trees (Allan et al. 2008; Lin-Wang et al. 2010). However, only a few bHLH TF genes, such as VvMYC1, MdbHLH3 and MdbHLH33, have been characterized as being associated with anthocyanin biosynthesis in fruit trees (Espley et al. 2007; Hichri et al. 2010). To our knowledge, several combinations of bHLH and MYB TFs, for example, MdMYB1 and AtEGL3 in cultured grape cells as well as MdMYB10 and MdbHLH3, AtbHLH2 and PdmMYB10 (or PprMYB10) in tobacco leaves, confer visible anthocyanin phenotypes in transient coexpression experiments (Takos et al. 2006; Espley et al. 2007; Lin-Wang et al. 2010). However, there is no direct evidence to support any physical interaction between them. In this study, Y2H and BiFC assays provided strong evidence that MdbHLH3 interacted physically and specifically with MdMYB1 (an allele of MdMYB10) in apples, further confirming that the MBW regulatory complex functions in woody plants.
The N terminus of bHLH proteins is generally indispensible for the interaction with MYB and contains the MYB interaction region (MIR, Pattanaik, Xie & Yuan 2008). However, the specific amino acids in the MIR of bHLH factors that are responsible for the interaction with MYB TFs have not yet been identified in anthocyanin-related bHLH regulators in plants (Feller et al. 2011). Our data narrowed the MIR site to two regions in MdbHLH3, that is, amino acids 1–24 and 168–228, as the deletion of either of these regions abolished the interaction with MdMYB1 (Fig. 2b). This is the first time that such a narrow map of the MIR of an anthocyanin-associated bHLH protein has been reported in plants.
Not all E-box elements served as MdbHLH3 binding sites, indicating that, in addition to the hexamer per se, flanking DNA sequences also contribute to DNA-binding specificity (Grove et al. 2009). Apart from the canonical E-box, MdbHLH3 also bound to an LTR element within the MdUFGT promoter, indicating its potential involvement in LT-induced anthocyanin biosynthesis. Furthermore, in Arabidopsis, MYB TF PAP1 regulates bHLH TF TT8, and TT8 automatically regulates itself (Baudry, Caboche & Lepiniec 2006). In this study, it was found that MdbHLH3 also bound to the promoter of MdMYB1 and regulated its expression (Fig. 6c), providing solid molecular evidence of direct regulation of the expression of MYB genes by a bHLH TF. In addition, MdMYB10, which is found in red-flesh apple cultivars, is an allele of MdMYB1. Its constitutive expression results from self-activating feedback associated with the R6 promoter (Espley et al. 2009). Thus, it is proposed that MdbHLH3-mediated MdMYB1 up-regulation magnifies the regulatory signal of the anthocyanin biosynthesis pathway as part of the autoregulatory loop proposed by Espley et al. (2009).
It is well known that the MBW complex plays a crucial role in the regulation of anthocyanin biosynthesis (Ramsay & Glover 2005). Increasing evidence is now supporting the hypothesis that the members of the MBW complex, especially bHLH, are involved in environmental responsiveness. For example, in Petunia and Asiatic lilies, high light induces anthocyanin accumulation in vegetative tissues by up-regulating the expression of AN1-clade bHLH genes, but not JAF13-clade genes, which are constitutively active and independent of light stress (Nakatsuka et al. 2009; Albert et al. 2011). In Arabidopsis, bHLH members of the MBW complex such as the EGL3 and TT8 genes are down-regulated by HT and low light (Gonzalez 2009), while GL3 is involved in the response to nutrient depletion (Lillo, Lea & Ruoff 2008). In contrast, the expression of GL3 and EGL3 is induced by LT (Olsen et al. 2009).
Among the MYB genes, PAP1 expression is inhibited by HT in Arabidopsis (Rowan et al. 2009). In apple, the expression of MYB activator genes, especially MdMYB10, is down-regulated in fruits grown under natural or artificially hot conditions compared with temperate conditions (Lin-Wang et al. 2011). Therefore, it has been proposed that temperature affects anthosynthesis accumulation via MYB activator genes, especially MdMYB10. However, the effect of temperature on the expression of MdbHLH genes is disputable. It appears that that MdbHLH3 and MbHLH33 are expressed at higher levels in the temperate season than the hot season (see fig. 9 in Espley et al. 2007), whereas MdbHLH300, but not MdbHLH3 and MdbHLH33, is down-regulated in fruits grown in a hot climate compared with a temperate climate (Lin-Wang et al. 2011). Our data showed that MdbHLH3 expression is also induced at both the transcriptional and translational levels by LT, providing additional evidence related to bHLH TFs in support of the involvement of the MBW complex at the transcriptional level in plant responses to environmental factors (Gonzalez 2009; Lin-Wang et al. 2011). The fact that LT induces anthocyanin accumulation suggests that there is crosstalk between the anthocyanin biosynthesis and cold signalling pathways. In Arabidopsis, freezing-sensitive mutants, such as sfr3, sfr4, sfr6 and sfr7, do not accumulate anthocyanin following cold acclimation (McKown, Kuroki & Warren 1996).
Most recently, it was reported that the JAZ proteins interact with MBW components to repress anthocyanin accumulation, and jasmonate-induced JAZ degradation rescues this repression (Qi et al. 2011). Considering that the MBW components are regulated by various environmental stimuli (Gonzalez 2009), it is reasonable to conclude that the MBW complex is a central regulatory module with respect to anthocyanin biosynthesis in response to environmental stimuli, which provides new information regarding the role of the MBW complex in plants.
Post-translational modification is one of the common ways of regulating gene expression in organisms. For example, phosphorylation plays a central role in the regulation of signal transduction within various secondary metabolic pathways, such as the carotenoid, shikimate/phenylpropanoid and terpenoid pathways (Pawson & Scott 2005; Xie, Kapteyn & Gang 2008). Phosphorylation can alter protein activity or subcellular localization and target proteins to effect dynamic changes in protein complexes (Peck 2006). The binding activity of transcription factors to gene promoters is regulated either positively or negatively by phosphorylation (Yang et al. 2003). Among bHLH TFs, AtICE1 is phosphorylated in response to a cold signal and, thus, activates the expression of AtCBFs only under cold stress, despite the fact that it is constitutively expressed. AtICE1 is also modified by ubiquitination and Sumoylation under cold stress (as reviewed by Chinnusamy et al. 2007). In the present study, it was found that the MdbHLH3 protein was post-translationally modified in response to LT, which may explain the enhanced binding of MdbHLH3 to E-box cis-elements in the MdUFTG and MdDFR promoters under LT. Therefore, it can be hypothesized that plants recruit the MBW complex to regulate anthocyanin biosynthesis in response to environmental temperatures not only at the transcriptional and translational levels but also at the post-translational level.
A model summarizing our findings regarding the genetic interactions of MdbHLH3 and its role in the regulation of anthocyanin accumulation and fruit colouration in response to LTs is presented in Fig. 7. Cereal, ornamental and fruit crops and trees not only produce economically important products with aesthetically pleasing colours, but they also provide bioactive and healthful nutrition value for consumers. Our findings regarding the regulatory mechanism involved in the anthocyanin biosynthesis pathway are likely to favour the development of novel biotechnological tools for the generation of enhanced plants with an optimized anthocyanin content or improved organ colouration, especially for deciduous ornamental and fruit trees against the background of global warming.
The authors would like to thank Dr Andrew C. Allan and Rogers Hellens for the pGreen 0800-LUC, pGreen 62-sk binary vectors and providing sequence information of MdbHLH3; Dr Bao-Xiu Qi for BiFC vectors; Dr Ilan Sela for IL-60-BS and pIR binary vectors. This work was supported by National Basic Research Program of China (2011CB100600), National High Technology Research and Development Program of China (2011AA100204), and Fok Ying Tung Education Foundation (111024).