The MdBBX22-miR858-MdMYB9/11/12 module regulates proanthocyanidin biosynthesis in apple peel

Abstract

Proanthocyanidins (PAs) have antioxidant properties and are beneficial to human health. The fruit of apple (Malus × domestica Borkh.), especially the peel, is rich in various flavonoids, such as PAs, and thus is an important source of dietary antioxidants. Previous research on the regulation of PAs in apple has mainly focussed on the transcription level, whereas studies conducted at the post-transcriptional level are relatively rare. In this study, we investigated the function of mdm-miR858, a miRNA with multiple functions in plant development, in the peel of apple fruit. We showed that mdm-miR858 negatively regulated PA accumulation by targeting MdMYB9/11/12 in the peel. During fruit development, mdm-miR858 expression was negatively correlated with MdMYB9/11/12 expression and PA accumulation. A 5'-RACE experiment, GUS staining assays and transient luminescent assays indicated that mdm-miR858 cleaved and inhibited the expression of MdMYB9/11/12. Overexpression of mdm-miR858 in apple calli, tobacco and Arabidopsis reduced the accumulation of PAs induced by overexpression of MdMYB9/11/12. Furthermore, we found that MdBBX22 bound to the mdm-miR858 promoter and induced its expression. Overexpression of MdBBX22 induced the expression of mdm-miR858 to inhibit the accumulation of PAs in apple calli overexpressing MdMYB9/11/12. Under light stress, MdBBX22 induced mdm-miR858 expression to inhibit PA accumulation and thereby indirectly enhanced anthocyanin synthesis in the peel. The present results revealed that the MdBBX22-miR858-MdMYB9/11/12 module regulates PA accumulation in apple. The findings provide a reference for further studies of the regulatory mechanism of PA accumulation and the relationship between PAs and anthocyanins.

Keywords: MdMYB9/11/12; MdBBX22; apple; light stress; mdm-miR858; proanthocyanidins.

Figure 1

Expression of mdm‐miR858 is negatively correlated with PA accumulation during apple peel development. (a) Apple ‘Starkrimson Delicious’ fruit developmental series. Fruit were harvested at 30, 60, 90, 120 and 140 DAFB. (b) The contents of PAs in the peel of apple ‘Starkrimson Delicious’ fruit at five time points in (a). (c) Correlation analysis of the expressions of known and conservative miRNAs (r ≤ −0.8) based on normalized deep‐sequencing counts with contents of PAs. (d) mdm‐miR858 normalized counts in the peel of apple ‘Starkrimson Delicious’ fruit at five time points in (a). The counts represent the averages of three biological replicates. (e) and (f) Expression pattern of mdm‐miR858 and MdMYB9/11/12 during peel development in apple ‘Starkrimson Delicious’ fruit. Error bars represent the standard deviation of three biological replicates.

Figure 2

mdm‐miR858 cleaves and inhibits the expression of MdMYB9/11/12. (a) The binding positions of mdm‐miR858 in the target genes MdMYB9/11/12. Orange numbers in parentheses indicate the proportion of 5′ RACE clones at the sites where the target genes are cleaved by mdm‐miR858 in peel of apple ‘Starkrimson Delicious’ fruit. (b) GUS phenotype observed by histochemical staining in tobacco leaves co‐transformed with mdm‐miR858 and MdMYB9/11/12. (c) Quantitative detection of GUS activity in tobacco leaves of experiments in (b). Error bars represent the standard deviation of three biological replicates. Different letters above the bars indicate a significant difference (P < 0.05, one‐way ANOVA and LSD test).

Figure 3

mdm‐miR858 targets MdMYB9/11/12 to regulate the expression of downstream structural genes. (a) Transient expression assays showing that mdm‐miR858 targets MdMYB9/11/12 to represses the expression of MdLAR and MdANR. Representative images of N. benthamiana leaves 72 h after infiltration are shown. (b) Relative LUC/REN ratio in (a). REN—Renilla luciferase activity; LUC—firefly luciferase activity. The value for pGreenII 62‐SK+ MdANRpro:LUC and pGreenII 62‐SK+ MdLARpro:LUC were set to 1. Error bars represent the standard deviation of three biological replicates.(** indicates P < 0.01(Student’s t‐test))

Figure 4

mdm‐miR858 targets MdMYB9/11/12 to inhibit PA accumulation in apple calli. (a) DMACA staining of ‘Orin’ apple calli (WT: wild‐type apple calli; MdMYB9‐OX: MdMYB9 overexpression apple calli; MdMYB11‐OX: MdMYB11 overexpression apple calli; MdMYB12‐OX: MdMYB12 overexpression apple calli; mdm‐miR858‐OX/MdMYB9‐OX: overexpression of mdm‐miR858 in the background of MdMYB9 overexpression apple calli; mdm‐miR858‐OX/MdMYB11‐OX: overexpression of mdm‐miR858 in the background of MdMYB11 overexpression apple calli; mdm‐miR858‐OX/MdMYB12‐OX: overexpression of mdm‐miR858 in the background of MdMYB12 overexpression apple calli). (b) Detection of the expression levels of mdm‐miR858 and MdMYB9/11/12 of apple calli from WT and transgenic lines in (a). (c) The contents of PAs in apple calli from WT and transgenic lines in (a). Error bars represent the standard deviation of three biological replicates. **P < 0.01 (Student’s t‐test).

Figure 5

mdm‐miR858 targets MdMYB9/11/12 to inhibit PA accumulation in Arabidopsis. (a) Unstained (upper row) and DMACA‐stained (lower row) seeds of tt2 mutant and transgenic lines (tt2 mutant: tt2 mutant Arabidopsis; MdMYB9‐OX: MdMYB9 overexpression Arabidopsis; MdMYB11‐OX: MdMYB11 overexpression Arabidopsis; MdMYB12‐OX: MdMYB12 overexpression Arabidopsis; mdm‐miR858‐OX/MdMYB9‐OX: overexpression of mdm‐miR858 in the background of MdMYB9 overexpression Arabidopsis; mdm‐miR858‐OX/MdMYB11‐OX: overexpression of mdm‐miR858 in the background of MdMYB11 overexpression Arabidopsis; mdm‐miR858‐OX/MdMYB12‐OX: overexpression of mdm‐miR858 in the background of MdMYB12 overexpression Arabidopsis). (b) The content of total PAs in seeds of tt2 mutant and transgenic lines in (a). (c) Detection of the expression levels of mdm‐MIR858 and MdMYB9/11/12 in seeds of tt2 mutant and transgenic lines in (a) by semiquantitative RT‐PCR. (d) Detection of the expression levels of flavonoid biosynthesis‐related genes in seeds of tt2 mutant and transgenic lines in (a) using RT‐qPCR. Error bars represent the standard deviation of three biological replicates. ** P < 0.01 (Student’s t‐test).

Figure 6

MdBBX22 binds the promoter of mdm‐miR858 and activates its expression. (a) Transient expression assay showing that MdBBX22 activates the expression of mdm‐miR858. Representative images of N. benthamiana leaves 72 h after infiltration are shown. (b) Relative LUC/REN ratio in (a). The value for empty vector pGreenII 62‐SK+ mdm‐miR858pro:LUC was set to 1. Error bars represent the standard deviation of three biological replicates. **P < 0.01 (Student’s t‐test). (c) Yeast one‐hybrid assay showing direct binding of MdBBX22 to the G‐box motif in mdm‐miR858 promoter. The mdm‐miR858/mdm‐miR858m promoter fragments inserted into the pHIS2 vector are shown in Figure S11. (d) EMSA showing that the MdBBX22‐HIS protein binds to G‐box motif in mdm‐miR858 promoter. The ‘+’ and ‘−’ indicate presence and absence, respectively. (e) ChIP‐PCR showing the in vivo binding of MdBBX22 to the mdm‐miR858 promoter. Three regions (S1–S3) of mdm‐miR858 promoter were investigated. Apple calli overexpressing the GFP sequence were used as negative controls. Error bars represent the standard deviation of three biological replicates. **P < 0.01 (Student’s t‐test).

Figure 7

MdBBX22 inhibits MdMYB9/11/12‐induced PA accumulation by promoting the expression of mdm‐miR858 in apple calli. (a) DMACA staining of ‘Orin’ apple calli (MdMYB9‐OX: MdMYB9 overexpression apple calli; MdMYB11‐OX: MdMYB11 overexpression apple calli; MdMYB12‐OX: MdMYB12 overexpression apple calli; MdMYB12m‐OX: MdMYB12m overexpression apple calli; MdBBX22‐OX/MdMYB9‐OX: overexpression of MdBBX22 in the background of MdMYB9 overexpression apple calli; MdBBX22‐OX/MdMYB11‐OX: overexpression of MdBBX22 in the background of MdMYB11 overexpression apple calli; MdBBX22‐OX/MdMYB12‐OX: overexpression of MdBBX22 in the background of MdMYB12 overexpression apple calli; MdBBX22‐OX/MdMYB12m‐OX: overexpression of MdBBX22 in the background of MdMYB12m overexpression apple calli). (b) Detection of the expression levels of MdBBX22, mdm‐miR858 and MdMYB9/11/12 of apple calli from transgenic lines in (a). (c) The contents of PAs in apple calli from transgenic lines in (a). Error bars represent the standard deviation of three biological replicates. **P < 0.01 (Student’s t‐test).

Figure 8

MdBBX22 induces the expression of mdm‐miR858 to inhibit PA accumulation and alters metabolism to enhance anthocyanin synthesis in apple peel under light stress. (a) The contents of total PAs and anthocyanin in the peel under light stress. (b) Expression patterns of MdBBX22, mdm‐miR858 and MdMYB11/12 in the peel under light stress. (c) Transient transformation assays in apple peel. MdBBX22‐OX: MdBBX22 transient overexpression; mdm‐miR858‐OX: mdm‐miR858 transient overexpression; STTM858‐OX: STTM858 transient overexpression; MdMYB12m‐OX: MdMYB12m transient overexpression; pTRV2‐MdBBX22: MdBBX22 transient suppression and pTRV2‐MdMYB11/12: MdMYB11/12 transient suppression. The black arrows represent the injection sites. (d) Anthocyanin content of apple peel around the injection sites in (c). (e) The content of total PAs in apple peel around the injection sites in (c). (f) Detection of the expression levels of MdBBX22, mdm‐miR858 and MdMYB11/12 of apple peel around the injection sites in (c) (** indicates P < 0.01(Student’s t‐test)). Error bars represent the standard deviation of three biological replicates. Different letters above the bars indicate a significant difference (P < 0.05, one?way ANOVA and LSD test) in (d) and (e). **P < 0.01 (Student's t‐test) in (f).