MdBBX22 regulates UV-B-induced anthocyanin biosynthesis through regulating the function of MdHY5 and is targeted by MdBT2 for 26S proteasome-mediated degradation.

Dear Editor,

Anthocyanin is a class of important secondary metabolite that contributes to the pigmentation of plant tissues (Jaakola, 2013). The biosynthesis of anthocyanin is through the phenylpropanoid pathway and is regulated by various environmental factors including ultraviolet‐B (UV‐B) light (Jaakola, 2013). UV‐B exposure contributes to anthocyanin biosynthesis, but the molecular mechanism is still not fully understood (Henry‐Kirk et al., 2018). ELONGATED HYPOCOYTL5 (HY5) is a basic leucine zipper (bZIP) transcription factor (TF) that plays a key role in light signalling as a positive regulator in light‐induced photomorphogenesis and anthocyanin biosynthesis (Gangappa and Botto, 2016). In this study, we verified that UV‐B treatment promoted anthocyanin accumulation in apple calli compared to controls (Figure 1a,b), and MdHY5 acted an essential role in this process as suppression of MdHY5 in apple calli resulted in an obvious decrease in UV‐B‐induced anthocyanin accumulation (Figure 1a,b).

Figure 1

MdBBX22 positively regulates UV‐B‐induced anthocyanin biosynthesis by enhancing the binding of MdHY5 to its target genes, and MdBT2 decreases MdBBX22‐promoted anthocyanin biosynthesis by degrading the MdBBX22 protein. (a) Phenotypes and (b) anthocyanin levels of apple calli in the dark (Dark) or in the light but with (+UV‐B) or without (‐UV‐B) UV‐B treatment. WT: wild‐type; MdHY5‐Anti: MdHY5 antisense suppression. ‐UV‐B: white fluorescent light; +UV‐B: white fluorescent light plus UV‐B. Apple calli of 15‐day‐old were treated with dark, ‐UV‐B, and +UV‐B for 10 days. Anthocyanin content of ‐UV‐B‐treated wild‐type apple calli was used as the reference and set to 1. Different letters above the bars indicated significant difference (P < 0.05) as obtained by one‐way ANOVA and LSD test. Asterisks denoted t‐test significance: *P < 0.05, **P < 0.01. (c) Detection of MdBBX22 transcription in response to UV‐B treatment using qRT‐PCR. ‘Fuji’ apple fruits were moved from white fluorescent light (‐UV‐B) to white fluorescent light plus UV‐B (+UV‐B). (d) Detection of the MdBBX22‐HIS fusion protein after UV‐B and MG132 treatments. ‘Fuji’ apple fruits were treated with white fluorescent light (‐UV‐B, Control) and white fluorescent light plus UV‐B (+UV‐B, UV‐B) for 5 days. Total proteins extracted from apple peel were incubated with the purified MdBBX22‐HIS protein. For MG132 treatment, total proteins of ‐UV‐B‐treated apple fruits were pre‐treated using 100 µM MG132 for 0.5 h. (e) Phenotypes and (f) anthocyanin levels of apple calli with (+UV‐B) or without (‐UV‐B) UV‐B treatments. WT: wild‐type; MdBBX22‐OX: MdBBX22‐overexpression; MdBBX22‐Anti: MdBBX22 antisense suppression. Apple calli of 15‐day‐old were treated with ‐UV‐B and +UV‐B for 3 days. (g) MdHY5 interacts with MdBBX22. MdHY5 (MDP0000586302) and MdBBX22 (MDP0000298804). (h) Phenotypes and (i) anthocyanin levels of apple calli with (+UV‐B) or without (‐UV‐B) UV‐B treatments. WT: wild‐type; MdBBX22‐OX: MdBBX22‐overexpression; MdHY5‐Anti: MdHY5 antisense suppression; MdBBX22‐OX/MdHY5‐Anti: antisense suppression of MdHY5 in the background of MdBBX22‐overexpression. Apple calli of 15‐day‐old were treated with ‐UV‐B and +UV‐B for 3 days. (j) and (k) EMSA showing that MdBBX22 enhances the transcriptional activity of MdHY5 on MdMYB10 and MdCHS. (l) MdHY5 interacts with MdBBX proteins. MdBBX10 (MDP0000733075), MdBBX20 (MDP0000177126), MdBBX22 (MDP0000298804), MdBBX23 (MDP0000222881), MdBBX24 (MDP0000800387), MdBBX25 (MDP0000901915), MdBBX33 (MDP0000697407), MdBBX37 (MDP0000157816), MdBBX43 (MDP0000140484), and MdBBX48 (MDP0000759984). (m) MdBBX22 interacts with MdBBX37 and MdBBX48 proteins. (n) MdBBX22 interacts with MdBT2. MdBT2 (MDP0000151000). (o) Expression of MdBT2 in response to UV‐B treatment using qRT‐PCR. (p) Detection of the MdBT2‐GST fusion protein after UV‐B and MG132 treatments. (q) Phenotypes and (r) anthocyanin contents of wild‐type (WT) and MdBT2 transgenic apple calli in response to UV‐B treatments. WT: wild‐type; MdBT2‐OX: MdBT2‐overexpression; MdBT2‐Anti: MdBT2 antisense suppressing apple calli. Apple calli of 15‐day‐old were treated with ‐UV‐B and +UV‐B for 4 days. (s) Phenotypes and (t) anthocyanin contents of wild‐type (WT) and transgenic apple calli. WT: wild‐type; MdBBX22‐OX: MdBBX22‐overexpression; MdBBX22‐OX/MdBT2‐OX: overexpression of MdBT2 in the background of MdBBX22‐overexpression; MdBBX22‐OX/MdBT2‐Anti: antisense suppression of MdBT2 in the background of MdBBX22‐overexpression. Apple calli of 15‐day‐old were treated with ‐UV‐B for 4 days. (u) MdBT2 promoted the degradation of the MdBBX22 protein in vitro. (v) MdBT2 promotes the ubiquitination of the MdBBX22 protein in vivo. (w) MdBT2 interacts with MdBBX proteins. (x) A working model of MdBBX22 functioning in UV‐B‐induced anthocyanin biosynthesis. The blue line represents post‐translational regulation; the red line represents transcriptional regulation, and the black line represents transcriptional and post‐translational regulations.

The B‐box (BBX) family proteins are a group of zinc finger TFs featuring one or two conserved B‐box motifs at their N‐termini and sometimes a CCT (CONSTANS, CO‐like, and TOC1) domain at the C‐termini (Gangappa and Botto, 2014). BBX proteins play an array of diverse physiological functions in plant growth and development, and in both biotic and abiotic stress responses (Gangappa and Botto, 2014). Previous reports have shown that BBX proteins are involved in regulating anthocyanin biosynthesis in pear and apple (Bai et al., 2019; Fang et al., 2019). Here, we identified the role of MdBBX22 in UV‐B‐induced anthocyanin biosynthesis. We found that UV‐B treatment stimulated the transcription of MdBBX22 (Figure 1c), and the stability of the MdBBX22 protein dramatically increased after UV‐B treatment (Figure 1d). These findings show that UV‐B affects the expression of MdBBX22 in both transcriptional and post‐transcriptional levels. Phenotype analysis further revealed that MdBBX22 was a positive regulator of the UV‐B‐induced anthocyanin biosynthesis (Figure 1e,f).

Since both MdBBX22 and MdHY5 are involved in the UV‐B‐induced anthocyanin biosynthesis in apple, we set to explore more of the relationship between these two proteins. Yeast two‐hybrid assays confirmed that MdBBX22 directly interacted with MdHY5 (Figure 1g). And suppression expression of MdHY5 in the MdBBX22‐overexpression background (MdBBX22‐OX/MdHY5‐Anti) significantly decreased MdBBX22‐promoted anthocyanin biosynthesis (Figure 1h,i), indicating that MdBBX22 promotes anthocyanin biosynthesis with partial dependency on MdHY5. We assessed whether MdBBX22 affected the binding of MdHY5 to its target genes. Therefore, we investigated the effect of MdBBX22 on the binding of MdHY5 to the promoters of MdMYB10 and MdCHS in a similar fashion. As expected, the MdBBX22‐MdHY5 interaction enhanced the binding of MdHY5 to its target genes (Figure 1j,k). These results provide evidence that MdBBX22 promotes UV‐B‐induced anthocyanin biosynthesis by MdBBX22‐MdHY5 interaction. Given that MdBBX22 has similar mechanisms to PpBBX16 (Bai et al., 2019) and MdBBX20 (Fang et al., 2019) in regulating anthocyanin biosynthesis, we suspect that they may play a redundant role in this process. In addition to MdBBX22, we also found that MdBBX24 and MdBBX33 interacted with MdHY5 (Figure 1l), indicating that MdHY5 might act a central role in the regulation of anthocyanin biosynthesis and BBXs‐HY5 interaction is a universal regulatory module (Gangappa and Botto, 2014, 2016). We also found that MdBBX22 interacted with MdBBX37 and MdBBX48 (Figure 1m), suggesting that MdBBX proteins might synergistically regulate anthocyanin biosynthesis.

Since UV‐B alleviated the degradation of the MdBBX22 protein (Figure 1d), we carried out a liquid chromatography/mass spectroscopy assay to identify the MdBBX22‐interacting partners with a potential role modulating the stability of the MdBBX22 protein. MdBT2 was identified to interact with MdBBX22 directly (Figure 1n). BT2 is a member of the Bric‐a‐Brack/Tramtrack/Broad (BTB) family of scaffold proteins that plays important roles in the activation of telomerase activity, in both male and female gametophyte development, in multiple hormone and stress responses, and in plant nitrogen use efficiency (Mandadi et al., 2009). BT2 protein possesses ubiquitination activity by forming the BT2‐CUL3‐RBX1 ubiquitin ligase complex (Mandadi et al., 2009). In apple, MdBT2 is characterized as a repressor in the regulation of iron homeostasis, anthocyanin biosynthesis and leaf senescence by degrading the MdbHLH104, MdMYB1 and MdbHLH93 proteins, respectively (An et al., 2019; Wang et al., 2018; Zhao et al., 2016). We noticed that UV‐B treatment suppressed the expression of MdBT2 in both transcriptional and post‐transcriptional levels (Figure 1o,p), which is the opposite to what was observed for MdBBX22 (Figure 1c,d). Moreover, MdBT2 was confirmed to be a negative regulator of the UV‐B‐induced anthocyanin biosynthesis by degrading the MdBBX22 protein through the 26S proteasome pathway (Figure 1q–v).

In addition to MdBBX22, we also found that several BBX proteins including MdBBX20, MdBBX23, MdBB24, MdBBX25, MdBBX33 and MdBBX43 were partners of MdBT2 (Figure 1w). Based on these data, it seems that the BBX protein family might be a universal ubiquitination substrate for MdBT2. Previous and current studies have revealed a series of MdBT2‐interacting proteins including MdbHLH104, MdMYB1, MdbHLH93, MdBBX20, MdBBX22, MdBBX23, MdBBX24, MdBBX25, MdBBX33 and MdBBX43 in apple (An et al., 2019; Wang et al., 2018; Zhao et al., 2016). MdBT2 interacts with these regulators and modulates their stability to be involved in multiple stress responses, indicating that MdBT2 might act as a fine‐tuning modulator to orchestrate a post‐transcriptional cascade in response to multiple stresses. Notably, the investigations that several BBXs act as ubiquitination target proteins of MdBT2 demonstrate that BT2‐BBX regulatory module of ubiquitination might be a universal regulatory mechanism in response to multiple stresses.

A possible working model is proposed here to summarize our findings (Figure 1x). MdBBX22 enhances the binding of MdHY5 to the promoters of MdMYB10 and MdCHS through directly interacting with MdHY5, thus to promote anthocyanin biosynthesis. In the absence of UV‐B, MdBT2 ubiquitinates and degrades the MdBBX22 protein, which decreases the MdBBX22‐promoted anthocyanin biosynthesis. In the presence of UV‐B, UV‐B promotes the expression of MdBBX22 in both transcriptional and post‐translational levels, which triggers UV‐B‐induced anthocyanin biosynthesis. The dynamic regulatory module of MdBT2‐MdBBX22‐MdHY5 plays a key role in UV‐B‐induced anthocyanin biosynthesis in apple.

Conflict of interest

The authors declare no conflict of interest.

Author contributions

Y.J.H. and J.P.A. conceived and designed the experiments. J.P.A. performed the research. J.P.A., X.X.W., X.W.Z., S.Q.B. and C.X.Y. analysed the data. J.P.A. and Y.J.H. wrote the paper.