Light-Induced Basic/Helix-Loop-Helix64 Enhances Anthocyanin Biosynthesis and Undergoes CONSTITUTIVELY PHOTOMORPHOGENIC1-Mediated Degradation in Pear

Abstract

Light is indispensable for the anthocyanin accumulation of red pear (Pyrus pyrifolia). Anthocyanin biosynthesis is catalyzed by a series of enzymes encoded by structural genes, which are regulated by a MYB-basic/helix-loop-helix-WD repeat (MYB-bHLH-WDR [MBW]) complex. The bHLH proteins of subgroup (SG) IIIf are believed to be involved in the regulation of anthocyanin accumulation. In this study, we revealed that pear PpbHLH64, which belongs to SGIIIb, positively regulates anthocyanin biosynthesis and is regulated by light at the transcriptional and posttranslational levels. Specifically, an exposure to light induced PpbHLH64 expression and anthocyanin accumulation in pear fruit and calli. Under light conditions, pear calli overexpressing PpbHLH64 exhibited enhanced red coloration, whereas the anthocyanin accumulation decreased in the PpbHLH64-RNA interference calli. Additionally, the transient overexpression of PpbHLH64 in pear fruit peel increased anthocyanin accumulation, whereas the virus-induced gene silencing of PpbHLH64 had the opposite effect. Further analyses indicated that PpbHLH64 is a transcriptional activator that directly binds to the promoter of UDP-GLUCOSE:FLAVONOID 3-O-GLYCOSYLTRANFERASE to upregulate expression. Moreover, PpbHLH64 interacted with PpMYB10, but not with PpMYB114, to form an MBW complex that significantly induces the accumulation of anthocyanins. Furthermore, PpbHLH64 was targeted by CONSTITUTIVE PHOTOMORPHOGENIC1 in darkness for subsequent degradation by the 26S proteasome. A genetic analysis indicated that PpbHLH64 functions downstream of B-BOX18, a component of the light signal transduction pathway. However, we were unable to detect the direct interaction between PpbHLH64 and PpBBX18. The characterization of PpbHLH64 in this study highlights the importance of SGIIIb bHLH proteins for light-induced anthocyanin accumulation.

Figure 1.

Phylogenetic analysis of some of the bHLH family proteins from Arabidopsis and pear. Accession numbers in green and orange refer to Arabidopsis and pear proteins, respectively. PpbHLH64 and AtICE1 are highlighted with a yellow background.

Figure 2.

Light-responsive PpbHLH64 expression. A, Anthocyanin accumulation in the cv Hongzaosu fruit peel after the light treatment. B, Changes in the fruit peel anthocyanin contents during the light treatment. C, PpbHLH64 expression pattern during the treatment. D to G, PpMYB10 (D), PpMYB114 (E), PpUFGT (F), and PpbHLH3 (G) expression patterns. H, Color changes in the pear calli due to the light treatment. I, Anthocyanin accumulation in pear calli after the light treatment. J, PcbHLH64 expression pattern during the light treatment. K, GUS staining results for transgenic pear calli harboring proPpbHLH64-GUS after the light treatment. L, GUS activities of the transgenic pear calli presented in K. WT, Wild type. Error bars represent the sd of three biological replicates. Asterisks indicate significant differences by two-tailed Student’s t test (**P < 0.01). Bars = 1 cm.

Figure 3.

Functional analysis of PpbHLH64 in pear calli and fruit. A, Anthocyanin accumulation in pear calli. B, Relative PpbHLH64 transcript abundance in the pear calli presented in A. C, Anthocyanin contents in the pear calli presented in A. D, Transient overexpression of PpbHLH64 induced anthocyanin biosynthesis in the ‘Zaosu’ pear fruit peel after a 3-d blue light treatment. E, Relative PpbHLH64 transcript abundance in the transiently overexpressing fruit peel. F, Anthocyanin contents near the injection sites of the transiently overexpressing pears. G, Transient silencing of PpbHLH64 decreased anthocyanin biosynthesis in ‘Hongzaosu’ pear fruit after a 5-d blue light treatment. H, Relative PpbHLH64 transcript abundance in the transiently silenced fruit. I, Anthocyanin contents near the injection sites of the transiently silenced pear fruit. WT, Wild type. Error bars represent the sd of three biological replicates. Asterisks indicate significant differences by two-tailed Student’s t test (*P < 0.05 and **P < 0.01). Bars = 1 cm.

Figure 4.

PpbHLH64 subcellular localization and transcriptional activation. A, Subcellular localization of PpbHLH64 in N. benthamiana leaf cells. Bars = 25 μm. B, Transcriptional activation associated with PpbHLH64 in yeast cells. The β-galactosidase activities reflect the transcriptional activities. DDO, SD/−Trp/−Leu medium; TDO, SD/−Trp/−Leu/−His medium. C, Analysis of the cis-elements in the PpUFGT promoter region. D, PpbHLH64 induced PpUFGT transcription in dual-luciferase assays. E, Yeast one-hybrid analysis of the interaction between PpbHLH64 and the PpUFGT promoter. F, Results of the ChIP-qPCR analysis of the direct binding of PpbHLH64 to the G-box-containing fragments of the PpUFGT promoter. G, Direct binding of PpbHLH64 to the G-box of the PpUFGT promoter under in vitro conditions. H, A mutation to the G-box prevents the binding of PpbHLH64 to the PpUFGT promoter. Error bars for dual-luciferase assays represent the sd of three independent experiments involving six replicates each. Error bars for GUS staining assays and ChIP-qPCR assays represent the sd of three independent experiments involving three replicates each. Asterisks indicate significant differences by two-tailed Student’s t test (*P < 0.05 and **P < 0.01).

Figure 5.

PpbHLH64 interacts with PpMYB10 and activates PpUFGT expression. A, Interactions of PpMYB10, PpMYB114, and PpbHLH64 in a yeast two-hybrid assay, with pGADT7-T (T) and pGBKT7-53 (53) as positive controls. DDO, SD/−Trp/−Leu medium; QDO, SD/−Trp/−Leu/−Ade/−His medium. B, Interactions of PpMYB10, PpMYB114, and PpbHLH64 in N. benthamiana leaves based on luciferase complementation imaging assays. Bars = 1 cm. C and D, Physical association between PpbHLH64 and PpMYB10 confirmed by a pull-down assay (C) and a BiFC assay (D). Bars = 25 μm (D). E, Dual-luciferase assays for the activation of the PpUFGT promoter by PpMYB10 or PpMYB114 and PpbHLH64. Error bars represent the sd of four biological replicates. Different lowercase letters above the error bars indicate significant differences according to a one-way ANOVA with Tukey’s test. F, The mutation to the G-box or MBS decreased the activation of the PpUFGT promoter by PpMYB10-PpbHLH64. Asterisks indicate significant differences by two-tailed Student’s t test (*P < 0.05 and **P < 0.01). G, EMSA results for the PpMYB10-GST fusion protein and the PpUFGT promoter probe after PpbHLH64-HIS fusion protein supplementation. + represents the addition of a protein; + and ++ represent fivefold and 10-fold increases in the amounts of the PpbHLH64-HIS fusion protein, respectively.

Figure 6.

Combining PpbHLH64 and PpMYB10 enhanced the anthocyanin biosynthesis in pear fruit. A, Transient expression of PpbHLH64, PpMYB10, and PpMYB10-PpbHLH64 in ‘Hongzaosu’ pear fruit after a 3-d blue light treatment. Bars = 1 cm. B, Anthocyanin contents in the fruit presented in A. C and D, Relative PpMYB10 (C) and PpbHLH64 (D) transcript levels in different combinations. E, Relative PpUFGT transcript levels in different combinations. Error bars represent the sd of three biological replicates. Lowercase letters above the error bars indicate significant differences determined by one-way ANOVA followed by multiple comparisons with Tukey’s test (P < 0.05).

Figure 7.

PpbHLH64 is targeted by PpCOP1 and degraded by the 26S proteasome. A, Changes in the PpbHLH64 abundance in transgenic pear calli under a light-dark cycle. The PpbHLH64 protein was detected by immunoblotting with the anti-GFP monoclonal antibody. Calli were treated with or without MG132 (75 µm) under light and dark conditions. B, Ubiquitination of the PpbHLH64 protein in darkness. The PpbHLH64-GFP fusion protein was immunoprecipitated from the transgenic calli with the anti-GFP antibody and then analyzed with the anti-GFP (left) and anti-ubiquitin (right) antibodies. IB, Immunoblot; IP, immunoprecipitation; Ub(n), ubiquitin. C to E, PpCOP1 interacted with PpbHLH64 in BiFC assays (C), firefly luciferase complementation imaging assays (D), and yeast two-hybrid assays (E). Bars = 1 cm. F, In vitro ubiquitination assays involving PpCOP1 and PpbHLH64. Proteins were analyzed in gel blots with anti-ubiquitin (left) and anti-His (right) antibodies. G, Cell-free degradation assays of purified PpbHLH64-HIS proteins in the protein extract of PpCOP1-GFP transgenic pear calli as labeled. Samples were incubated in the degradation buffer with MG132 (75 µm) or dimethyl sulfoxide (DMSO). The abundance of PpbHLH64 was visualized by immunoblotting with the anti-His antibody. Actin was used as the loading control.

Figure 8.

PpbHLH64 functions downstream of PpBBX18. A, Anthocyanin accumulation in pear calli with different constructs. Bar = 1 cm. B and C, PpBBX18 (B) and PpbHLH64 (C) expression patterns in transgenic pear calli. D, Anthocyanin contents in the transgenic pear calli after a 3-d light treatment. E and F, Expression levels of anthocyanin biosynthesis-related genes (PcCHS, PcCHI, PcDFR, PcANS, PcUFGT, PcMYB10, and PcbHLH3) in transgenic pear calli in darkness (E) and under light (F). Error bars represent the sd of three biological replicates. Asterisks indicate significant differences by two-tailed Student’s t test (*P < 0.05 and **P < 0.01).

Figure 9.

Working model for PpbHLH64-mediated light-induced fruit coloration in red pears. In darkness, activated PpCOP1 targets PpbHLH64 and PpMYB10 for ubiquitination, which subsequently triggers their proteolysis via the 26S proteasome. Under light, PpMYB10 and PpbHLH64 function downstream of PpBBX18. Simultaneously, PpCOP1 activity is inhibited, resulting in the accumulation of both PpbHLH64 and PpMYB10. Accumulated PpMYB10 and PpbHLH64 bind to the MBS and the G-box motif, respectively, in the promoter of anthocyanin biosynthesis-related structural genes to activate transcription. Additionally, PpWDR interacts with PpbHLH64 and enhances the transcriptional activation by the PpMYB10-PpbHLH64 complex. U, Ubiquitin.