Glucose Sensor MdHXK1 Phosphorylates and Stabilizes MdbHLH3 to Promote Anthocyanin Biosynthesis in Apple

Glucose induces anthocyanin accumulation in many plant species; however, the molecular mechanism involved in this process remains largely unknown. Here, we found that apple hexokinase MdHXK1, a glucose sensor, was involved in sensing exogenous glucose and regulating anthocyanin biosynthesis. In vitro and in vivo assays suggested that MdHXK1 interacted directly with and phosphorylated an anthocyanin-associated bHLH transcription factor (TF) MdbHLH3 at its Ser361 site in response to glucose. Furthermore, both the hexokinase_2 domain and signal peptide are crucial for the MdHXK1-mediated phosphorylation of MdbHLH3. Moreover, phosphorylation modification stabilized MdbHLH3 protein and enhanced its transcription of the anthocyanin biosynthesis genes, thereby increasing anthocyanin biosynthesis. Finally, a series of transgenic analyses in apple calli and fruits demonstrated that MdHXK1 controlled glucose-induced anthocyanin accumulation at least partially, if not completely, via regulating MdbHLH3. Overall, our findings provide new insights into the mechanism of the glucose sensor HXK1 modulation of anthocyanin accumulation, which occur by directly regulating the anthocyanin-related bHLH TFs in response to a glucose signal in plants.


Author Summary
Glucose is considered as a major regulatory molecule in addition to being essential metabolic nutrients and structural components in higher plants. As is well known, hexokinase1 (HXK1) is a glucose sensor that integrates diverse signals to govern gene expression and plant growth in response to environmental cues. Previously, it is reported that the nuclear HXK1 forms a glucose signaling complex core with the vacuolar H + -ATPase B1 (VHA-B1) and the 19S regulatory particle of proteasome subunit (RPT5B), which influences the transcription of target genes. However, it is yet unknown if and how HXK1 directly targets TFs to modulate their function in the nucleus in plants. Our results reveal the important roles of MdHXK1 protein kinase in phosphorylating MdbHLH3 TF to modulate anthocyanins accumulation in response to glucose in apple.
Introduction biosynthesis by directly binding to the promoters of not only anthocyanin structural genes, such as DFR and UFGT, but also anthocyanin-associated MYB TF genes to activate their expression [31][32][33][34]. Interestingly, MdbHLH3 protein promotes anthocyanin accumulation partially through a putative phosphorylation modification in response to low temperature in apple [32]. However, the protein kinase that mediates the phosphorylation of MdbHLH3 protein is unknown.
Sugars induce anthocyanin biosynthesis in various plant species [35][36][37]. First, they provide carbon sources, skeletons and glucosides for anthocyanin biosynthesis [38,39]. Second, they increase the expression levels of biosynthetic structural genes and regulatory MYB genes [37,40]; however, the precise mechanism by which sugars modulate these genes remains unknown. The present study found that a protein kinase, MdHXK1, is involved in the regulation of anthocyanin biosynthesis in response to glucose by interacting with the phosphorylating and stabilizing MdbHLH3 protein. Subsequently, the function of MdHXK1 in the modulation of anthocyanin accumulation was characterized in apple calli and fruits. Finally, the potential application of HXK1-mediated glucose signaling in the genetic improvement of horticultural traits is discussed.

Results
MdHXK1 modulates anthocyanin accumulation mainly through glucose signaling, but not through the catalytic pathway, under the high-glucose condition Previous studies have verified that glucose significantly induces anthocyanin biosynthesis in Arabidopsis seedlings [36]. Similarly, the effect of different concentrations of glucose (0-6%, w/ v) and the HXK inhibitor glucosamine on anthocyanins accumulation was tested in in vitro shoot cultures of the 'Gala' apple cultivar. The results showed that glucose promotes anthocyanin accumulation in an HXK-dependent manner in apple (S1 Fig; S5 Text).
Because glucose controls anthocyanin accumulation in an HXK-dependent pathway in apple, it is reasonable to propose that this process is regulated by the catalytic or signaling function of HXK. We isolated the MdHXK1 gene from apple to investigate this possibility. The predicted MdHXK1 protein is highly homologous with AtHXK1, which functions as not only a catalytically active kinase but also a glucose sensor in Arabidopsis [11,41]. Two catalytically inactive HXK1 mutants have been identified in Arabidopsis, namely, HXK1 S177A and HXK1 G104D ; these mutants retain signaling functions but not catalytic activities [11]. To investigate whether Ser and Gly at positions 177 and 104 of HXK1, respectively, are conserved between apple and Arabidopsis, an alignment of the amino acid sequence of MdHXK1 with AtHXK1 was performed. The result showed that apple MdHXK1 protein exhibited a high sequence similarity (77.31% identity) to Arabidopsis AtHXK1 (S2 Fig). The positions of 177 and 104 of apple MdHXK1 are the Ser and Gly residues, respectively, which were the same as those of Arabidopsis AtHXK1 (Fig 1A). These results suggest that the catalytically inactive apple MdHXK1 proteins MdHXK1 S177A and MdHXK1 G104D also exercised their functions in signaling but not catalytic activities, similar to Arabidopsis.
To rapidly determine whether the catalytically important G104 and S177 is involved in glucose-induced anthocyanin accumulation, two point mutations, i.e., MdHXK1 G104D and MdHXK1 S177A , were made to the MdHXK1 protein. A total of three types of 35S-driven vectors of 35S::MdHXK1, 35S::MdHXK1 S177A and 35S::MdHXK1 G104D were generated and genetically transformed into apple calli of the 'Orin' cultivar ( Fig 1B). Subsequently, these three transgenic apple calli and the wild-type (WT) control were used for immunoblotting assays with an anti-MdHXK1 antibody. The result demonstrated that the protein abundance of MdHXK1 was  G104D and 35S::MdHXK1 S177A transgenic apple calli treated with 6% glucose. Note: Before being treated with 6% exogenous glucose, these apple calli were suffered from a increased by 4.2-, 3.9-and 4.0-fold in the 35S::MdHXK1, 35S::MdHXK1 G104D and 35S:: MdHXK1 S177A transgenic apple calli, respectively, compared with the WT control (Fig 1C), indicating that the target genes were successfully transformed into and expressed in the transgenic apple calli. In addition, qPCR assays showed that MdHXK1 repressed two classes of photosynthesis genes including MdSBP and MdCAA in 35S::MdHXK1, 35S::MdHXK1 G104D and 35S::MdHXK1 S177A transgenic apple calli but not WT apple calli (Fig 1D), suggesting that Gly104 and Ser177 mutations have similar effect on MdHXK1 as Arabidopsis AtHXK1.
As a result, the three transgenic apple calli produced nearly the same levels of anthocyanins to each other but at a considerably higher level than in the WT control under 6% glucose conditions ( Fig 1B and 1E), indicating that MdHXK1 and two point mutants successfully function to promote anthocyanin accumulation in these transgenic apple calli. In addition, the glucose phosphorylation activities were determined for the WT and these transgenic apple calli. The results showed that the 35S::MdHXK1 transgenic calli exhibited higher glucose phosphorylation activity than the 35S::MdHXK1 S177A and 35S::MdHXK1 G104D calli and the WT control ( Fig  1F). However, there was no significant difference in glucose phosphorylation activities among the 35S::MdHXK1 S177A and 35S::MdHXK1 G104D transgenic calli and the WT control ( Fig 1F). Collectively, these results suggest that MdHXK1 modulates anthocyanin accumulation mainly through glucose signaling, but not the catalytic pathway, under the high-glucose (6%) conditions. Furthermore, the WT and aforementioned three transgenic calli were also treated with a low glucose concentration (1%) to induce anthocyanin accumulation. The result demonstrated that the 35S::MdHXK1 S177A and 35S::MdHXK1 G104D transgenic apple calli produced more anthocyanins than the WT controls but less anthocyanins than the 35S::MdHXK1 transgenic calli (S3A and S3B Fig). However, the glucose phosphorylation activities of the 35S:: MdHXK1 G104D and 35S::MdHXK1 S177A transgenic apple calli showed no significant difference compared with the WT control but were considerably lower than the activities for the 35S:: Taken together, these results indicate that MdHXK1 induces anthocyanin accumulation depending on both the catalytic activity and signaling under low-glucose conditions but mainly depending on signaling under high-glucose conditions.

MdHXK1 interacts with MdbHLH3 via the conserved hexokinase_2 domain
To screen the target protein of MdHXK1 in its signal pathway, the 35S::MdHXK1-Myc vector was constructed and genetically transformed into apple calli (S4A Fig). The 35S::MdHXK1-Myc transgenic calli were used for co-immunoprecipitation (Co-IP) against the monoclonal anti-Myc antibody (S4B Fig). Subsequently, the Co-IPed proteins were analyzed with LC/MS to identify the potential proteins that interact with the MdHXK1 protein. The results showed that the anthocyanin-associated bHLH TF MdbHLH3 is a candidate (S1 Text).
To determine whether MdHXK1 interacts with MdbHLH3 protein, yeast two-hybrid (Y2H) assays were performed. MdHXK1 protein contains two conserved hexokinase domains, i.e., hexokinase_1 and hexokinase_2 (S4C Fig). Therefore, the full-length cDNA of MdHXK1 gene dark (24 hours dark)-induced glucose starvation to deplete endogenous glucose. (C) Western blotting analysis of MdHXK1 protein abundance in the WT and transgenic apple calli. (D) MdHXK1-mediated glucose-dependent gene repression. MdSBP, sedoheptulose-biphosphatase (accession no. XM_008384867); MdCAA, carbonic anhydrase (accession no. XM_008387117). (E) and (F) Anthocyanin content (E) and glucose phosphorylation activity (F) in the WT and transgenic apple calli. The data are shown as the mean ± SE, which were analyzed based on more than 9 replicates. Statistical significance was determined using Student's t-test in different apple calli lines. n.s., P > 0.01; *P < 0.01. doi:10.1371/journal.pgen.1006273.g001 was divided into two fragments, i.e., MdHXK1 1-245aa and MdHXK1 245-498aa . Subsequently, the full-length cDNA and two truncated mutants of the MdHXK1 gene were inserted into the pGBT9 vector, independently, as the bait vectors. Moreover, the full-length MdbHLH3 cDNA and its serially truncated mutants, as previously reported by Xie et al. [32], were inserted into the pGAD424 vector as the prey vectors. The different combinations of bait and prey vectors were transformed into yeast for Y2H assays. The results indicated that the full-length MdHXK1 strongly interacted with the full-length MdbHLH3 proteins. Furthermore, the truncated peptide MdbHLH3 346-709aa , i.e., the C-terminus of MdbHLH3, interacted with MdHXK1 proteins at the hexokinase_2 domain MdHXK1 245-498aa but not at the hexokinase_1 domain MdHXK1 1-245aa (Fig 2A).
To further verify the interaction between MdHXK1 and MdbHLH3, an in vivo Co-IP assay using 35S::MdbHLH3-GFP transgenic apple calli was conducted. The result indicated that the MdbHLH3-GFP fusion protein, but not the GFP negative control, interacted with MdHXK1 in apple calli (Fig 2B). In addition, a GST pull-down assay showed that a GST-tagged MdbHLH3 physically interacted with a His-tagged MdHXK1 in vitro (Fig 2C). These results indicate that the hexokinase_2 domain of MdHXK1 physically interacts with the C-terminus of the MdbHLH3 protein.

Glucose induces phosphorylation of the MdbHLH3 protein at the Ser 361 site
To examine how MdbHLH3 protein responds to glucose, an expression vector 35S:: MdbHLH3-Myc was constructed and genetically transformed into apple calli (S5A Fig). After treatment with or without 6% glucose, the 35S::MdbHLH3-Myc overexpressing calli were used for Western blotting with the anti-Myc antibody. The results showed that the position of the MdbHLH3 proteins shifted from a faster-to a slower-migrating band in the transgenic apple calli treated with 6% glucose compared to those without glucose (Fig 3A), indicating that the MdbHLH3 protein was post-translationally modified in response to glucose. Furthermore, treatment with calf intestine alkaline phosphatase (CIP), which cleaves exposed phosphate residues from ribonucleotides and deoxyribonucleotides, converted the slower-migrating form of MdbHLH3 to the faster-migrating form (Fig 3A), indicating that the glucose-induced posttranslational modification for the MdbHLH3 protein in apple calli is predominantly a phosphorylation.
To examine the potential phosphorylation sites of the MdbHLH3 protein, the glucoseinduced phosphorylated MdbHLH3 protein in the 35S::MdbHLH3-Myc overexpressing calli was captured with anti-Myc antibody-conjugated agarose beads and separated in an SDS-PAGE gel. After proteolytic digestion and purification, the protein sample was subjected to liquid chromatography-tandem mass spectrometry (LC-MS/MS) to detect the phosphorylation sites. The serine at residue 361 (Ser 361 ) of the MdbHLH3 protein exhibited a high phosphopeptide signal intensity (Fig 3B; S2 and S4 Texts), suggesting that it is a potential phosphorylation site.
Subsequently, a monoclonal antibody specifically against the MdbHLH3 phosphorylation site at residue 361 was prepared and named as the anti-MdbHLH3 S361 antibody (S5B Fig). This antibody specifically recognized the glucose-induced phosphorylation of MdbHLH3 protein in the WT apple calli (Fig 3C), which was consistent with the results shown in Fig 3A. These results indicate that glucose induces the phosphorylation of the MdbHLH3 protein at the Ser 361 site in apple calli.
To examine whether the glucose concentration influences the phosphorylation of the MdbHLH3 protein, the WT apple calli were treated for 30 min with glucose concentrations of 0%, 1%, 3% and 6% and then used for immunoblotting with the anti-MdbHLH3 S361 antibody. The results showed that the MdbHLH3 protein was not phosphorylated when the calli grew in absence of glucose, whereas the phosphorylation intensity of the MdbHLH3 protein increased gradually with glucose concentration (Fig 3D). Moreover, apple calli were treated with 6% glucose for different times (0, 10, 20, 30 and 60 min) to examine whether treatment time affects the  phosphorylation of the MdbHLH3 protein. The results showed that the phosphorylation intensity of MdbHLH3 proteins in the calli gradually increased with the treatment duration ( Fig 3E). These results indicate that the MdbHLH3 protein is phosphorylated in response to glucose and that this modification is positively associated with glucose concentration and treatment time.
In addition, glucose-induced phosphorylation of the MdbHLH3 protein could be observed in apple leaves (S5C Fig), indicating that the glucose-induced phosphorylation of the MdbHLH3 protein occurred in different apple tissues and organs.

Glucose-induced MdbHLH3 phosphorylation is required for MdHXK1
Considering the interaction between MdHXK1 and MdbHLH3 proteins, it is reasonable to hypothesize that the MdHXK1 protein kinase mediates the phosphorylation of MdbHLH3 protein in apple calli. To verify this hypothesis, new transgenic apple calli, 35S::antiMdHXK1, were obtained, which contained an antisense fragment specific to MdHXK1 cDNA and exhibited considerably lower transcript and protein levels of MdHXK1 than the WT control (S6A and S6B  Fig). Subsequently, immunoblotting assays with the anti-MdbHLH3 S361 antibody were performed using the WT control and the 35S::MdHXK1 and 35S::antiMdHXK1 transgenic apple calli after treatment with or without glucose. The result showed that the 35S::MdHXK1 overexpressing calli exhibited a considerably higher phosphorylation level of the MdbHLH3 protein, whereas that of the 35S::antiMdHXK1-suppressing calli were lower than the WT control in response to glucose treatment ( Fig 4A). This result suggests that the MdHXK1 protein kinase is necessary, if not sufficient, for the glucose-induced phosphorylation of the MdbHLH3 protein in apple calli.
To further verify that MdHXK1 directly phosphorylates the MdbHLH3 protein, in-gel assays were conducted using prokaryon-expressed and purified MdHXK1-GST and  35-242aa (hexokinase_1), MdHXK1 245-491aa (hexokinase_2), MdHXK1 1-21aa+35-242aa (Signal peptide + hexokinase_1), MdHXK1 1-21aa+245-491aa (Signal peptide + hexokinase_2) and MdHXK1 1-499aa (Signal peptide + hexokinase_1 + hexokinase_2) were transformed into the WT apple calli. Protein amounts were normalized based on the protein folds of the WT control. (E) Co-localization analysis of the full-length or truncated mutants of MdHXK1-GFP and MdbHLH3-RFP in vivo. The full-length and truncated mutants of MdHXK1 as mentioned in (D) were fused to the green fluorescent protein (GFP) tag. The full-length MdbHLH3 was fused to the red fluorescent protein (RFP) tag. For each image, two constructs, as indicated, were transferred into protoplasts of apple calli cells and then analyzed using confocal microscopy. Yellow colors in the merged images indicate the co-localization of the two signals. Bars = 20 μm. Note: In (C) and (D), protein bands were quantified by scanning densitometry using a Hewlett Packard Scanjet scanner and Scanplot software. All of the protein amounts were normalized based on the protein folds of band 1.
MdbHLH3-His fusion proteins. As a result, MdbHLH3 protein was phosphorylated by the recombined MdHXK1 ( Fig 4B). Furthermore, this in vitro phosphorylation assays were performed with anti-MdbHLH3 S361 antibody. The result showed that MdbHLH3-His proteins were phosphorylated by MdHXK1, while MdbHLH3 mutation MdbHLH3 S361A -His were not (S7 Fig). These results demonstrated that the MdbHLH3 protein is a direct substrate of the MdHXK1 protein kinase.
In addition, the phosphorylation status of the MdbHLH3 protein was determined in the glucose-treated 35S::MdHXK1-, 35S::MdHXK1 G104D -and 35S::MdHXK1 S177A -overexpressing apple calli lines. Interestingly, there was no visible difference in the phosphorylation levels of these three transgenic calli (Fig 4C), indicating that the abolishment of MdHXK1 catalytic function as indicated by the phosphorylation activity is unable to affect the phosphorylation level of the MdbHLH3 protein.
In addition to the hexokinase_1 and hexokinase_2 domains, MdHXK1 also contains a signal peptide ranging from 1 to 22 amino acid residues at the N-terminus (S4C Fig). Given that signal peptides are polypeptide chains that are used as 'address labels' for sorting proteins to their correct subcellular destinations, it was hypothesized that the signal peptide of MdHXK1 is involved in the MdbHLH3 phosphorylation process. To verify this hypothesis, three vectors of MdHXK1 cDNA including the signal peptide domain, i.e., 35S::MdHXK1 1-21aa+35-242aa -Myc, 35S::MdHXK1 1-21aa+245-491aa -Myc and 35S::MdHXK1 1-499aa -Myc, were constructed and successfully transformed into the WT apple calli (Fig 4D). The resulting transgenic calli were used for immunoblotting assays with anti-Myc and anti-MdbHLH3 S361 antibodies. The results showed that the phosphorylation intensities of MdbHLH3 proteins were considerably higher in the 35S::MdHXK1 1-21aa+245-491aa -Myc and 35S::MdHXK1 1-499aa -Myc transgenic calli than in the WT control. However, the level of MdbHLH3 phosphorylation was highly similar in the 35S::MdHXK1 1-21aa+35-242aa -Myc transgenic calli as in the WT control ( Fig 4D). Therefore, the signal peptide and hexokinase_2 domain are crucial for MdHXK1-mediated phosphorylation of the MdbHLH3 protein.
Taken together, the signal peptide and hexokinase_2 domain of the MdHXK1 protein are essential for its nuclear co-localization together with the MdbHLH3 protein, which is crucial for MdHXK1-mediated phosphorylation of the MdbHLH3 protein.

Phosphorylation modification stabilizes the MdbHLH3 protein and enhances its transcriptional activation of downstream genes
To examine whether MdHXK1 influences the stability of MdbHLH3 proteins, the prokaryonexpressed and purified MdbHLH3-GST fusion proteins were incubated with plant total proteins that were extracted from the WT control and the 35S::MdHXK1 and 35S::antiMdHXK1 transgenic apple calli. Subsequently, protein degradation assays were performed. The results showed that MdbHLH3-GST proteins were more stable in the protein extracts of the 35S:: MdHXK1 transgenic calli than in those of the WT control ( Fig 5A, 5B and 5E), whereas they were degraded at a more rapid speed in the protein extracts of 35S::antiMdHXK1 transgenic calli compared to those of the WT control (Fig 5C and 5E). These results suggest that MdHXK1-mediated phosphorylation of the MdbHLH3 protein may increase its stability.
To further verify that phosphorylation influences the stability of the MdbHLH3 protein, a sitedirected S361A mutation was introduced into the MdbHLH3 protein. The mutated cDNA MdbHLH3 S361A was inserted into the expression vector for prokaryon-expression and purification of MdbHLH3 S361A -GST fusion proteins, which were then incubated with the total proteins extracted from the WT calli. The protein sample was used for Western blotting with the anti-GST antibody. The results showed that the MdbHLH3 S361A -GST proteins degraded at a rapid speed compared with the wild-type MdbHLH3-GST proteins (Figs 4B, 5D and 5E), indicating that the inhibition of phosphorylation promoted the degradation of MdbHLH3 proteins. In addition, MdHXK1 also enhanced the stability of the endogenous MdbHLH3 proteins (S8A-S8D Fig).
To examine whether phosphorylation of the MdbHLH3 protein influences its binding capacity to the downstream genes, such as MdMYB1, MdANS and MdUFGT, the 35S:: MdbHLH3-Myc and 35S::MdbHLH3 S361A -Myc transgenic apple calli were used for ChIP-PCR analysis (Fig 5F; S9 Fig). The results showed that the phosphorylated MdbHLH3-Myc protein exhibited a higher enrichment in the promoters of MdMYB1 and anthocyanins biosynthetic structural genes than the non-phosphorylated MdbHLH3 S361A -Myc (Fig 5G). As a result, those genes showed higher expression levels in the 35S::MdbHLH3-Myc transgenic apple calli than the MdbHLH3 S361A -Myc apple calli (Fig 5H). Furthermore, the abundance of the endogenous MdbHLH3 and MdMYB1 proteins were higher in 35S::MdHXK1 overexpressing calli but lower in 35S::antiMdHXK1 suppressing calli than in the WT control ( Fig 5I).
Therefore, phosphorylation modification stabilizes the MdbHLH3 protein and enhances its transcriptional activation of downstream genes.

MdHXK1 promotes anthocyanin accumulation in an MdbHLH3-dependent manner
To examine whether and how MdHXK1 influences anthocyanin accumulation, the full-length sense ORFs and antisense cDNA fragments of MdHXK1 and MdbHLH3 (or MdbHLH3 S361A ) were inserted into the expression vectors downstream of 35S promoters independently. The  (H), the data are shown as the mean ± SE, which were analyzed based on more than 9 replicates. Statistical significance was determined using Student's t-test in different apple calli lines. n.s., P > 0.01; *P < 0.01; **P < 0 .001.  (Fig 6A). The MdHXK1 and MdbHLH3 genes were successfully overexpressed or suppressed in the corresponding calli compared with the WT control (Fig 6B), indicating that the genetic transformation was successful in apple calli. As downstream genes, the transcript levels of MdANS and MdUFGT genes were positively correlated with that of the MdbHLH3 gene; however, MdANS and MdUFGT were considerably lower in the 35S::MdbHLH3 S361A transgenic calli than in the 35S::MdbHLH3 calli (Fig 6B). In addition, the transcription activity of the MdANS promoter was positively associated with the transcript level of MdHXK1 genes (S10 Fig). These transgenic apple calli were used to determine the anthocyanin content. The results showed that overexpression of MdHXK1 and MdbHLH3, either alone or together, noticeably enhanced the anthocyanin content in the corresponding transgenic calli compared with the WT control ( Fig 6C). Moreover, the 35S::MdbHLH3 S361A transgenic calli produced less anthocyanins than the 35S::MdbHLH3 calli (Fig 6C), indicating that the phosphorylation site Ser361 is crucial for MdbHLH3 to regulate the biosynthesis of anthocyanins.
Furthermore, the 35S::MdHXK1 transgenic calli produced more anthocyanins, but the 35S:: MdHXK1+35S::antiMdbHLH3 calli produced less than the WT control (Fig 6A and 6C), indicating that the suppression of the MdbHLH3 gene inhibited the MdHXK1-mediated increase of anthocyanin biosynthesis. Therefore, MdHXK1 regulates anthocyanin accumulation at least partially, if not completely, depending on the presence of MdbHLH3.

MdHXK1 works together with MdbHLH3 to modulate anthocyanin accumulation in apple fruits
To investigate whether MdHXK1 and MdbHLH3 regulate anthocyanin accumulation in apple fruits in a similar manner as in calli, a viral vector-based method was applied to alter their expression using vector pRI for overexpression and vector TRV for suppression. Four viral constructs, including pRI-MdHXK1, TRV-MdHXK1, pRI-MdbHLH3 and TRV-MdbHLH3, were obtained. Each construct and two combinations, i.e., TRV-MdHXK1+pRI-MdbHLH3 and pRI-MdHXK1+TRV-MdbHLH3, were used for fruit infiltration, with the empty vectors as controls (Fig 7A). The results showed that the transcript levels of MdHXK1 and MdbHLH3 genes were enhanced after being infiltrated with pRI-MdHXK1 and pRI-MdbHLH3 but decreased with TRV-MdHXK1 and TRV-MdbHLH3, respectively ( Fig 7B).
Subsequently, anthocyanin levels were measured in apple peel tissues around the sites infiltrated with the different viral constructs. The results showed that both MdHXK1 and MdbHLH3 positively regulate anthocyanin accumulation and that the MdHXK1-mediated anthocyanin accumulation required MdbHLH3 in apple fruits (Fig 7C), similar to the apple calli ( Fig 6C).

Discussion
Sugar-induced anthocyanin accumulation is important for not only proper cell function [23,24] but also the quality formation of ornamental crops and fresh fruits [37,40,42]. Therefore, it is critical to elucidate the molecular mechanism underlying sugar-induced anthocyanin accumulation. The present study found that the glucose sensor MdHXK1, a hexokinase protein, stabilized the bHLH TF MdbHLH3 by phosphorylation modification, leading to an enhanced anthocyanin accumulation in apple.  In (B) and (C), the data are shown as the mean ± SE, which were analyzed based on more than 9 replicates. Statistical significance was determined using Student's t-test in different apple calli lines. n.s., P > 0.01; *P < 0.01; **P < 0 .001. doi:10.1371/journal.pgen.1006273.g006 , the data are shown as the mean ± SE, which were analyzed based on more than 9 replicates. Statistical significance was determined using Student's t-test in different apple calli lines. n.s., P > 0.01; *P < 0.01; **P < 0 .001. Glucose-induced anthocyanin accumulation results from MdHXK1-dependent glucose signaling and metabolic functions Sugars are the major sources of carbon and energy metabolites and play key roles in plant growth and development. Sugars also act as effective signaling molecules throughout plant life [43,44]. In Arabidopsis, HXK1 is a crucial enzyme in glucose catabolism; HXK1 senses glucose and initiates its signaling pathway [11]. Glucose-promoted aliphatic glucosinolate biosynthesis is regulated by HXK1-mediated signaling via the MYB TFs MYB28 and MYB29 [45]. Most recently, it was reported that glucose treatment greatly enhances anthocyanin content and induces the expression of PsWD40-2, PsMYB2, PsCHS1, PsCHI1 and PsF3'H1 through glucose signaling in Paeonia suffruticosa cut flowers [37]. Among the WBM genes, MYB and WD40 genes, but not bHLH genes, are induced at the transcriptional level by glucose. The present study found that a glucose-dependent signaling pathway is involved in the regulation of anthocyanin accumulation in apple. This process depended on functional MdHXK1, which directly phosphorylated and stabilized the WBM component MdbHLH3 protein at the post-translational level (Figs 6A-6C and 7A-7C). MdbHLH3 modulates both anthocyanin biosynthetic structural genes and the regulatory MdMYB1 gene, thereby promoting anthocyanin accumulation [32].
Furthermore, anthocyanin accumulation is induced by glucose, which is not due to the osmotic effects of glucose (S1C and S1D Fig) [46]. MdHXK1 promoted anthocyanins accumulation mainly via the glucose signaling pathway under the high-glucose condition (Fig 1B-1F) and via both glucose metabolism and the signaling pathway under the low-glucose condition (S3 Fig). Given that sugars are the major sources of carbon and energy, higher plants require sugars for normal metabolism [9]. Glucose provides carbon skeletons for anthocyanin biosynthesis via its HXK1-dependent catalytic metabolism pathway, especially under low-glucose conditions (S3 Fig) [47]. Moreover, the MdHXK1-dependent glucose signaling pathway also plays a vital role in anthocyanin biosynthesis (Fig 1B-1E; S3 Fig). Therefore, glucose promotes anthocyanin biosynthesis depending on both signaling and metabolism under low-glucose conditions in apple. However, when carbohydrates are derived from glucose to meet the needs of anthocyanin synthesis under high-glucose conditions (e.g., 6% glucose), the glucose mainly served as signaling molecules to initiate anthocyanin biosynthesis (Fig 1B-1E). Taken together, glucose-induced anthocyanin accumulation is the result of MdHXK1-dependent glucose signaling together with catalytic metabolism pathways.
In the present study, the catalytic and signaling functions of MdHXK1 were characterized using its two catalytically inactive mutants, MdHXK1 S177A and MdHXK1 G104D , in apple (Fig 1), both of which retain signaling functions but not catalytic activities, similar to their activities in Arabidopsis [11]. Most recently, Feng et al. [48] successfully resolved the crystal structures of two AtHXK1 inactive forms, AtHXK1 S177A and AtHXK1 G104D , and analyzed the biochemical properties of AtHXK1 in Arabidopsis. These findings provide biochemical and structural insights into how HXK1 functions at the atomic level, thereby providing a structural explanation for the dual functions of HXK1 in plants.

The nuclear HXK1 complex regulates glucose-mediated transcription activation
As the most important glucose sensor, HXK1 is involved in diverse signaling functions, particularly in the regulation of gene expression. In plants, HXK1 is mainly localized in the cytosol; however, a minor degree of HXK1 is also present in the nucleus [49]. In the nucleus, this minor portion of HXK1 interacts with the B1 subunit of the V-ATPase (VHA-B1) and with a 19S regulatory particle of the proteasome subunit (RPTB5), leading to the formation of unexpected nuclear HXK1 complexes [49]. A large number of putative TFs identified in the nuclear HXK1 complexes interact directly with VHA-B1 and/or RPT5B but not directly with HXK1 [49]. In addition, nuclear-localized HXK1 has also been implicated in the control of the transcriptional activity and proteasome-mediated degradation of EIN3 (ethylene-insensitive3), a key transcriptional regulator in ethylene signaling [50]. The present study found that HXK1 directly interacted with MdbHLH3 (Fig 2B-2D), a key bHLH transcriptional regulator in anthocyanin biosynthesis [32]. However, it is unclear whether the HXK1/VHA-B1/RPT5B nuclear complex is also involved in these processes.
Furthermore, a R2R3 MYB regulator MdMYB1 interacts with the N-terminus of MdbHLH3 to regulate anthocyanin biosynthesis [32]. The present study found that the hexokinase_2 domain of MdHXK1 strongly interacted with the C-terminus of MdbHLH3 to modulate anthocyanin accumulation (Fig 2B). Therefore, there is no competition for the interaction of the MdbHLH3 protein with MdMYB1 and MdHXK1 in the regulation of anthocyanin biosynthesis.

MdbHLH3 phosphorylation may be an MdHXK1-mediated single-site phosphorylation event
In apples, a putative phosphorylation modification is involved in the MdbHLH3-mediated anthocyanin accumulation in response to low temperature [32]. However, the protein kinase that mediates the phosphorylation of MdbHLH3 protein is not yet identified. The present study found that the MdHXK1 protein kinase is directly involved in the glucose-induced phosphorylation of MdbHLH3 protein, thereby modulating anthocyanin biosynthesis (Fig 4A and  4B). In addition, the hexokinase_2 domain of MdHXK1, which may be required for signal peptide cleavage based on its functions in protein secretion and subcellular localization [51,52], plays a key role in the phosphorylation of the MdbHLH3 protein (Fig 4D and 4E).
Additionally, several bHLH TFs are phosphorylated by external environmental stimuli. For example, multiple light-induced Ser/Thr phosphorylation sites are found in the phyB-interacting bHLH TF PIF3 in Arabidopsis [53]. Multisite light-induced phosphorylation of the bHLH TFs PIF1 and PIF5 has been confirmed using photobiological and genetic approaches [54,55]. In addition to PIFs, another bHLH TF, TWIST1, is phosphorylated at Thr125 and Ser127 to control pro-metastatic functions in prostate cancer cells [56]. In contrast to the aforementioned bHLH TFs, the bHLH TF speechless is phosphorylated to promote stomatal development at a single serine 186 site in Arabidopsis [57]. Similarly to the bHLH TF speechless, only a single phosphorylation site in the bHLH TF MdbHLH3 protein was detected in apple (Fig 3B  and 3C; S2 Text), suggesting that MdbHLH3 phosphorylation may be a single-site phosphorylation event in apple or at least that its Serine 361 plays a crucial role in anthocyanin biosynthesis (Figs 5D-5H, 6 and 7).

MdHXK1 protein kinase stabilizes MdbHLH3 to regulate the expression of anthocyanin biosynthesis genes
As is well known, the MYB-bHLH-WDR (MBW) complex plays an important role in regulating anthocyanin and proanthocyanidin biosynthesis. In apple, MdbHLH3 physically interacts with MdMYB1 and specifically binds to the promoters of anthocyanin structural genes, such as MdDFR and MdUFGT, to promote anthocyanin accumulation [32]. Moreover, MdbHLH3 interacts with MdMYB9 and MdMYB11 to regulate the JA-induced biosynthesis of anthocyanin and proanthocyanidin [58]. In the present study, MdbHLH3 promoted anthocyanin accumulation in apple calli and apple fruits (Figs 6 and 7). In addition, MdbHLH3 also promotes malate accumulation in the vacuole by indirectly regulating the vacuolar transport system in apple [59]. Similarly, the increase of malate content in 35S::MdHXK1-overexpressing apple calli accumulated more malate than the WT control (S11 Fig), possibly due to the MdHXK1-mediated stabilization of MdbHLH3.
The glucose supply promotes anthocyanin biosynthesis and organ coloration in different plant species, such as Arabidopsis, grape, and Paeonia suffruticosa [36,37,40]. However, the mechanism underlying the glucose signaling-mediated regulation of MYB TFs, WDR and anthocyanin structural genes remains unclear [37,40]. Here, a working model is proposed to illuminate how glucose regulates anthocyanin accumulation in apple (Fig 8). Under glucose deprivation conditions, the kinase activity of MdHXK1 is inactivated and fails to phosphorylate MdbHLH3 protein (Fig 3A and 3C). As a result, a small amount of MdbHLH3 protein binds to the promoters of anthocyanin structural genes, leading to reduced anthocyanin accumulation (Figs 6 and 7). When exposed to glucose, the kinase activity of MdHXK1 is activated, and then, MdHXK1 phosphorylates and stabilizes the MdbHLH3 protein, which further regulates the expression of the anthocyanin biosynthetic genes and the regulatory MYB genes (Figs 4A, 4B, 5 and 6B), ultimately enhancing anthocyanin biosynthesis in apple (Figs 6A, 6C, 7A and 7C). In addition, ectopic expression of the apple MdHXK1 gene also increased anthocyanin accumulation in the transgenic Arabidopsis (S12 Fig), suggesting that the mechanism by which HXK1 controls anthocyanin accumulation in response to glucose is conserved in different species.
In summary, the current study provides new insights into the molecular mechanism of MdHXK1 stabilization of the MdbHLH3 protein, which occurs via phosphorylation, thereby promoting the accumulation of anthocyanins in plant cells in response to glucose signals. Because color is one of the most eye-catching traits for fresh fruits and ornamental plants [60,61], there is considerable interest for the organ coloration in the breeding programs for these economically important plants. Taken together, the regulatory mechanism uncovered in the present study is also useful for the development of novel biotechnological strategies for improving the quality of apple fruit and other horticultural crops.

Plant materials and growth conditions
The in vitro shoot cultures of apple were obtained from detoxified buds of 'Gala' apples. They were maintained at 25°C under long-day conditions (16 h light/8 h dark) on Murashige and Skoog (MS) medium supplemented with 0.8 mg L -1 6-BA and 0.2 mg L -1 IAA and subcultured at a 4-week interval before being used for further studies. The apple calli used in this study were induced from the young embryos of the 'Orin' apple (Malus domestica Borkh.). The calli were grown on MS medium containing 0.5 mg of L-1 indole-3-acetic acid (IAA) and 1.5 mg of L-1 6-benzylaminopurine (6-BA) at 25°C in the dark. The apple calli were subcultured three times at 15-day intervals before being used for genetic transformation and in other assays. Additionally, all the apple calli were suffered from a dark (24 hours dark)-induced glucose starvation before being treated with exogenous glucose in this study, unless noted otherwise.
The apple fruits used for the injection of viral vectors were collected from mature trees of the cultivar 'Red Delicious' that were grown in a commercial orchard near Tai-An City. Fruits were bagged at 35 DAB (days after blooming); the bagged fruits were harvested at 140 DAB and de-bagged before injection.
The present study used the Arabidopsis (Arabidopsis thaliana) ecotype 'Columbia,' the MdHXK1 overexpression line MdHXK1-OVX1, the glucose-insensitive mutant gin2, and the function-complementary line MdHXK1-R1 (overexpression of MdHXK1 in a gin2 mutant background). Seeds were surface sterilized with 70% (v/v) ethanol and sown on 0.8% (w/v) agar plates containing half-strength MS medium and different glucose concentrations. The seeds were stratified for three days at 4°C and transferred into constant light (100 μmol m 2 s -1 ) at 20°C for 2 weeks of growth. Before being used for exogenous glucose treatment, 2-weeks-old Arabidopsis plants were suffered from a dark (24 hours dark)induced glucose starvation.

Construction of the expression vectors and genetic transformation
To construct MdHXK1 and MdbHLH3 sense overexpressing and antisense suppressing vectors, the full-length cDNA of MdHXK1 and MdbHLH3, a specific fragment of MdHXK1 and a conserved fragment of MdbHLH3 were isolated from 'Gala' apple using RT-PCR. Furthermore, truncated sense overexpression vectors, including MdHXK1 35-242aa , MdHXK1 245-491aa , MdHXK1 1-21aa+35-242aa and MdHXK1 1-21aa+245-491aa , were also isolated from 'Gala' apple using RT-PCR. All of the cDNA were digested with EcoRI/BamHI and cloned into the pRI plant transformation vector downstream of the CaMV 35S promoter. All of the primers used in this study are listed in S3 Text.
In addition, two point mutants of MdHXK1, namely, G104D (altering Glycine to Aspartate at position 104) and S177A (altering Serine to Alanine at position 177), and the MdbHLH3 point mutant Ser 361A (mutation of Serine to Alanine at position 361), were obtained using sitedirected mutagenesis methods. The resulting cDNA were digested with EcoRI/BamHI and cloned into the pRI plant transformation vector downstream of the CaMV 35S promoter. The primers used in this study are listed in S3 Text.
In addition, the full-length cDNA of MdHXK1 and MdbHLH3 were also cloned into the PRI plant transformation vector with a Myc tag downstream of the CaMV 35S promoter, and subsequently, the recombined expression vectors MdHXK1-Myc and MdbHLH3-Myc were used for genetic transformation.
For Arabidopsis transformation, the 35S::MdHXK1 vector plasmid was introduced into WT (Col-0) and the glucose-insensitive mutant gin2 via the Agrobacterium strain GV3101 using a floral dip method [59]. The seeds of the transgenic plants were individually harvested and subsequently selfed. Homozygous transgenic lines were used for further investigation.

RNA extraction and quantitative RT-PCR assays
RNA extraction and quantitative RT-PCR (qRT-PCR) assays were performed with the methods described by Hu et al. [59]. All of the primers used for qRT-PCR are listed in S3 Text.

Protein extraction and Western blotting
Protein extraction and Western blotting assays were conducted as described by Hu et al. [59]. The monoclonal antibody of anti-MdHXK1, anti-MdbHLH3 S361 (specifically against the MdbHLH3 phosphorylation site at residue 361) and anti-GST antibody were prepared by the Abmart Company (Shanghai, China).

Yeast two-hybrid assays
Yeast two-hybrid assays were performed using the Matchmaker GAL4-based two-hybrid system (Clontech, Palo Alto, CA, USA). Full-length cDNA and truncated mutants of MdHXK1, including MdHXK1 1-245 aa and MdHXK1 245-498aa , were inserted into the pGBT9 vector. The associated yeast two-hybrid vectors of MdbHLH3, which were inserted into vector pGAD424, are detailed in Xie et al. [32]. All of the constructs were transformed into yeast strain AH109 using a lithium acetate method. Yeast cells were cultured on minimal medium -Leu/-Trp according to the manufacturer's instructions. Transformed colonies were plated onto minimal medium -Leu/-Trp/-His/-Ade with or without β-galactosidase to test for possible interactions.

Co-immunoprecipitation (Co-IP) procedures
The WT and 35S::MdbHLH3-GFP transgenic apple calli were treated with 50 μM MG132 for 16 h to stabilize the MdbHLH3-GFP and MdHXK1 proteins. The Co-IP was carried out as described by Oh et al. [62]. The eluted samples were immunoblotted using anti-GFP and anti-MdHXK1 antibodies.

GST pull-down assays
For the GST pull-down assays, full-length cDNA of MdbHLH3 were inserted into the pGEX-4T-1 vector, whereas that of MdHXK1 was inserted into pET-32a. All of the recombinant proteins were used to perform GST pull-down assays as described by Oh et al. [62].

Identification of phosphorylation sites using LC-MS/MS
MdbHLH3 proteins were immunoprecipitated with anti-MdbHLH3 antibody-conjugated agarose beads and then separated on an SDS-PAGE gel and stained with Coomassie brilliant blue (CBB). The band containing phosphorylated MdbHLH3 protein was cut from the stained SDS-PAGE gel. The protein digestion, phosphopeptide enrichment, mass spectrometry data acquisition, data analysis, and label-free quantitation were carried out as described by Wang et al. [63].

Detection of phosphoproteins
The MdbHLH3-Myc transgenic apple calli were pre-incubated in MS medium plus 6% glucose with or without 5 U of calf intestine alkaline phosphatase (CIP) for 1 and 3 h. Subsequently, proteins extraction was performed for Western blotting assays with an anti-Myc antibody. Actin served as a protein-loading control.

In vitro kinase assay
A total of 0.2 μg of recombinant His-tagged protein kinase MdHXK1 and 1 μg of MdbHLH3 S361A -GST and normal MdbHLH3-GST proteins were incubated in 25 μL of reaction buffer [20 mM Tris (pH 7.5), 5 mM MgCl 2 , 10 mM NaCl and 2 mM DTT] with 100 μM ATP and [λ-32 P]ATP (0.2 mCi per reaction) at room temperature for 30 min. Recombinant MdbHLH3 S361A -GST was served as a negative control in the in vitro kinase assay. The phosphorylated proteins were visualized using autoradiography after separation on a 12% SDS-PAGE gel.

Construction of the viral vectors and transient expression in apple fruits
To construct antisense expression viral vectors, a specific fragment of MdHXK1 and a conserved fragment of MdbHLH3 were amplified with RT-PCR using apple fruit cDNA as the template. The resulting products were cloned into the tobacco rattle virus (TRV) vector in the antisense orientation under the control of the dual 35S promoter. The vectors were named TRV-MdHXK1 and TRV-MdbHLH3. To generate overexpression viral vectors, full-length cDNA of MdHXK1 and MdbHLH3 were inserted into the IL-60 vector under the control of the 35S promoter. The resulting vectors were named MdHXK1-IL and MdbHLH3-IL.
The antisense expression viral vectors were transformed into Agrobacterium tumefaciens strain GV3101 for inoculations. Fruit infiltrations were performed as previously described [59]. The injected apple fruits were kept in the dark at room temperature for two days and subsequently placed in the highlight at 10°C for one week. The peel of the injected part was then harvested for gene expression analysis and anthocyanin content determination.

Analysis of glucose phosphorylation activity
Glucose phosphorylation activity was measured using an enzyme-linked assay according to Schaffer and Petreikov [64]. The assays contained a total volume of 1 mL of 30 mM HEPES--NaOH, pH 7.5, 2 mM MgCl 2 , 0.6 mM EDTA, 9 mM KCl, 1 mM NAD, 1 mM ATP, and 1 unit of NAD-dependent glucose-6-phosphate dehydrogenase (G6PDH). To assay glucose phosphorylation, 25 μL of the desalted extract was added to start the reaction under 25°C. Reduction of NAD within 5 min was monitored by the increase in absorption at 340 nm. Activity was calculated in terms of μmol of NAD reduced per minute.

Transient expression in protoplasts of apple calli cells and fluorescence detection
Protoplasts isolated apple calli cells were prepared and transformed as described by Sheen [64]. The fluorescence in transformed cells was detected with a confocal laser scanning microscope (Zeiss LSM 510 META), with excitation wavelengths of 488 nm and 543 nm using an argon laser and an emission wavelength of 505-530 nm and over 560 nm using a BP filter or excitation wavelengths of 458 nm and 514 nm using an argon laser, and an emission wavelength of 475-525 nm and 530-600 nm using a BP filter. A total of 20-30 cells were imaged for each experiment. Co-expressed proteins in the same protoplasts of apple calli cells were detected in the same Pinhole.

Determination of the total anthocyanin content
The total anthocyanins were extracted using a methanol-HCl method and detected as described by Hu et al. [59].

Statistical analysis
Samples were analyzed in triplicates, and the data are expressed as the mean ± standard deviation unless noted otherwise. Statistical significance was determined using Student's t-test. A difference at P 0.01 was considered significant, and P 0.001 was considered extremely significant. The phenotype as indicated by a red color for anthocyanin accumulation in in vitro shoot cultures of the 'Gala' apple cultivar treated with 6% glucose and 6% mannitol or 6% glucose plus glucosamine. (D) The anthocyanin content of apple shoot cultures in (C). The data are shown as the mean ± SE, which were analyzed based on more than 9 replicates. Statistical significance was determined using Student's t-test in apple shoot cultures. n.s., p > 0.01; ÃÃ p < 0.001. (TIF)