PbMYB120 Negatively Regulates Anthocyanin Accumulation in Pear

Abstract

Subgroup 4 R2R3 MYBs play vital roles in the regulation of anthocyanin biosynthesis. However, there is limited knowledge regarding the functions of MYB repressors in pear (Pyrus × bretschneideri). Here, PbMYB120 was identified as a potential regulator of anthocyanin biosynthesis. A phylogenetic analysis revealed that PbMYB120 was clustered into the FaMYB1-like clade of the subgroup 4 R2R3 MYBs. PbMYB120 was expressed higher in red peels than in green peels in five pear cultivars. PbMYB120 expression was positively correlated with anthocyanin accumulation. However, the transient overexpression of PbMYB120 led to the inhibition of anthocyanin accumulation and PbUFGT1 expression. Promoter binding and activation assays indicated that PbMYB120 binds to the promoter of PbUFGT1 and represses the promoter's activity. Thus, the inhibition of anthocyanin accumulation by PbMYB120 may be correlated with the repression of PbUFGT1. Furthermore, during anthocyanin induction, the expression levels of anthocyanin activators and PbMYB120 were upregulated. This study demonstrated that PbMYB120 was highly expressed in pear tissues having higher anthocyanin accumulations but acted as a repressor in the regulation of anthocyanin accumulation. PbMYB120 may work coordinately with anthocyanin activators and serve as a balancer of anthocyanin accumulation.

Keywords: PbMYB120; anthocyanin; pear; repressor; subgroup 4 MYBs.

Figure 1

Characterization of PbMYB120. (A) Phylogenetic analyses of the subgroup 4 R2R3 MYBs. The subgroup 4 R2R3 MYBs were classified into two subclades: Clade Ⅰ (AtMYB4-like) and Clade Ⅱ (FaMYB1-like). R3 MYBs were added as the out-group. PbMYB3 and PbMYB120 are marked with solid circles. Protein sequences of the subgroup 4 R2R3 MYBs and R3 MYBs used were obtained from the NCBI database. Protein accessions are listed in the “Materials and Methods”. (B) Amino acid sequence alignment of typical subgroup 4 R2R3s and the identification of conserved domain motifs. The identified conserved domain motifs are marked with text boxes and wireframes. N1 domain (bHLH-binding domain): [D/E]Lx2[R/K]x3Lx6Lx3R; N2 domain: Characteristic sequence signatures of different clades, with DNEI in the AtMYB4-like repressors and DNEV in the FaMYB1-like repressors; C1 domain: LIsrGIDPxT/SHRxI/L; C2 domain (the EAR repression motif): pdLNLD/ELxiG/S; the C1 and C2 domains are characteristic features of the subgroup 4 R2R3 MYB repressors; C3 domain (zinc finger-like motif): CX2CX9CXC; C4 domain (GY/FDFLGL motif): contributes to the interaction with the Sensitive to ABA and Drought 2 (SAD2); C5 domain: TLLLFR-type repressor motif, which was identified in AtMYBL2 and some FaMYB1-like repressors.

Figure 2

Expression analyses of PbMYB120 in fruit of five pear cultivars having uneven coloration. (A) Fruit coloration of five pear cultivars, ‘Bartlett’, ‘Clap’s Favorite’, ‘Conference’, ‘Red silk’ and ‘5 Hao’. (B) Analyses of anthocyanin contents and expression levels of PbMYB120 and anthocyanin biosynthesis-related genes in the red and green peels of the five tested pear cultivars. Data are the means ± standard errors (SEs) of three biological replicates. Asterisks indicate significant differences as assessed by Student’s t test: * p < 0.05, ** p < 0.01, *** p < 0.001. (C) Correlation analyses between the expression of PbMYB120 and both anthocyanin accumulation and expressions of anthocyanin biosynthesis-related genes. Data represent Pearson’s correlation coefficients that were calculated using SPSS 13.0. * p < 0.05, ** p < 0.01.

Figure 3

Transient overexpression and protein interaction analysis of PbMYB120. (A) Phenotypes of ‘Zaosu’ and ‘Red Bartlett’ fruit transiently overexpressing PbMYB120 and the determination of the transfection efficiency as assessed by GUS staining. (B) Analysis of anthocyanin biosynthesis and PbMYB120 expression in ‘Zaosu’ and ‘Red Bartlett’ fruit transiently overexpressing PbMYB120. (C) Promoter-binding analyses of anthocyanin late biosynthetic genes (LBGs) and regulatory genes as assessed by Y1H assays, and a PbUFGT1 promoter activation analysis as assessed by the dual-luciferase reporter assay. Data are the means ± SEs of three biological replicates. Asterisks indicate significant differences as assessed by Student’s t test: * p < 0.05, ** p < 0.01.

Figure 4

Role of PbMYB120 in increase and decrease of anthocyanin accumulation. (A) Analysis of anthocyanin accumulation and PbMYB120 expression during light-induced fruit coloration of ‘Red Zaosu’. Young fruit of ‘Red Zaosu’ were bagged and faded completely after 30 days of shading. Days 1, 4, and 11 represent 1, 4, and 11 days after removing the fruit bags, respectively. (B) Analyses of anthocyanin accumulation and PbMYB120 expression during the natural fading of leaf coloration of ‘Zaosu’ and ‘Red Zaosu’. Young leaves with red coloration and mature leaves with green coloration were analyzed. Leaves were light- or deep-red colored at the young stages. The red coloration decreased during the leaf developmental process and disappeared eventually. Leaves were green colored at the mature stages. (C) Expression analysis of genes involved in anthocyanin pathway in light-induced fruit coloration of ‘Red Zaosu’. Data are the means ± SEs of three biological replicates. Differences between two samples were assessed using Student’s t test at a 95% confidence level and are indicated by asterisks (* p < 0.05, ** p < 0.01). Differences among the three samples were assessed using one-way ANOVA with Tukey’s honestly significant difference test at a 95% confidence level and are indicated by lowercase letters above the error bar.

Figure 5

Predicted mechanism of PbMYB120 in the regulation of anthocyanin accumulation. PbMYB120 inhibited anthocyanin biosynthesis by the direct repression of PbUFGT1. PbMYB120 was expressed in an anthocyanin-dependent manner. PbMYB120′s expression may be induced by light, anthocyanin activators, or increased anthocyanin accumulation. It formed a negative feedback loop regulating anthocyanin accumulation by the direct repression of PbUFGT1 and possible indirect interference with the MYB-bHLH-WD40 (MBW) complex.