BBX16, a B-box protein, positively regulates light-induced anthocyanin accumulation by activating MYB10 in red pear

Abstract

The red coloration of pear (Pyrus pyrifolia) results from anthocyanin accumulation in the fruit peel. Light is required for anthocyanin biosynthesis in pear. A pear homolog of Arabidopsis thaliana BBX22, PpBBX16, was differentially expressed after fruits were removed from bags and may be involved in anthocyanin biosynthesis. Here, the expression and function of PpBBX16 were analysed. PpBBX16's expression was highly induced by white-light irradiation, as was anthocyanin accumulation. PpBBX16's ectopic expression in Arabidopsis increased anthocyanin biosynthesis in the hypocotyls and tops of flower stalks. PpBBX16 was localized in the nucleus and showed trans-activity in yeast cells. Although PpBBX16 could not directly bind to the promoter of PpMYB10 or PpCHS in yeast one-hybrid assays, the complex of PpBBX16/PpHY5 strongly trans-activated anthocyanin pathway genes in tobacco. PpBBX16's overexpression in pear calli enhanced the red coloration during light treatments. Additionally, PpBBX16's transient overexpression in pear peel increased anthocyanin accumulation, while virus-induced gene silencing of PpBBX16 decreased anthocyanin accumulation. The expression patterns of pear BBX family members were analysed, and six additional BBX genes, which were differentially expressed during light-induced anthocyanin biosynthesis, were identified. Thus, PpBBX16 is a positive regulator of light-induced anthocyanin accumulation, but it could not directly induce the expression of the anthocyanin biosynthesis-related genes by itself but needed PpHY5 to gain full function. Our work uncovered regulatory modes for PpBBX16 and suggested the potential functions of other pear BBX genes in the regulation of anthocyanin accumulation, thereby providing target genes for further studies on anthocyanin biosynthesis.

Keywords: BBX16; MYB10; anthocyanin accumulation; light; pear.

Figure 1

PpBBX16's expression pattern during light treatment in ‘Red Zaosu’ pear. The bagging treatment was performed at 15 DAFB in the orchard. The light treatment was performed using the bagged fruit at 150 DAFB. (a) Colour changes of ‘Red Zaosu’ during the treatment. (b, c) Changes in anthocyanin (b) and chlorophyll (c) contents during the treatment. (d) PpBBX16's expression pattern during the treatment. (e) Anthocyanin biosynthesis‐related genes’ expression profiles during the treatment. Error bars represent the standard deviations of three biological replicates.

Figure 2

Effects of PpBBX16's ectopic expression in Arabidopsis. (a) The phenotypes of wild‐type ‘Columbia 0’ and transgenic Arabidopsis. Three independent transgenic lines were used in the experiment. The anthocyanins overaccumulated in the seedlings (upper panel) and the tops of flower stalks (bottom panel) in the transgenic lines. (b) The PpBBX16's expression level in transgenic Arabidopsis lines. (c) The ectopic expression of PpBBX16 resulted in shortened hypocotyls. (d) The anthocyanin contents in the transgenic lines. (e) The expression levels of anthocyanin biosynthesis‐related genes (AtCHS, AtCHI, AtF3H, AtDFR, AtLDOX and AtPAP1) in transgenic lines. The error bars represent the standard deviations of three biological replicates. Asterisks indicate significant differences (two‐tailed Student's t‐test, * P < 0.05, ** P < 0.01).

Figure 3

PpBBX16 and PpHY5 jointly activated the anthocyanin biosynthesis. (a) Subcellular localization of PpBBX16 expressed in tobacco leaf cells. (b) Trans‐acting activity of PpBBX16 transformed into yeast cells. The β‐galactosidase activities reflected the trans‐acting activities. (c) Yeast one‐hybrid assays of PpBBX16 and the promoter of PpCHS or PpMYB10. PpBBX16 could not interact with either promoter region, even though they harboured G‐boxes and can be directly bound by PpHY5 (Tao et al., 2018). (d) PpBBX16 transcriptionally induced the activity of anthocyanin biosynthesis‐related genes (PpCHS, PpCHI, PpDFR and PpMYB10) in dual‐luciferase assay. (e, f) PpBBX16 interacting with PpHY5. The physical interaction of PpBB16 and PpHY5 was tested by yeast two‐hybrid assays (e) and BiFC assays (f). (g) PpBBX16 and PpHY5 jointly promoted the expression of PpMYB10 and PpCHS. Error bars for dual‐luciferase assays represent the standard deviation of three independent experiments each with six technical replicates. Lower case letters above bars indicate a significant difference determined by two‐way ANOVA followed by multiple comparisons with Tukey's test (P < 0.05). Asterisks indicate significant differences (two‐tailed Student's t‐test, * P < 0.05, ** P < 0.01).

Figure 4

The effects of PpBBX16 overexpression in pear calli. (a) PpBBX16's overexpression resulted in an anthocyanin accumulation after 2 days of the light treatment. (b) PpBBX16's expression level in transgenic pear calli. (c) Anthocyanin contents in transgenic pear calli after the light treatment. The corresponding anthocyanin extracts are shown above each bar. (d) The expression levels of anthocyanin biosynthesis‐related genes (PcCHS, PcCHI, PcDFR, PcANS, PcUFGT and PcMYB10) in pear calli. The error bars represent the standard deviations of three biological replicates. Asterisks indicate significant differences (two‐tailed Student's t‐test, * P < 0.05, ** P < 0.01).

Figure 5

RNA‐Seq analysis of PpBBX16 overexpression calli under light/dark condition. (a) The Venn graph of the numbers of differential expression genes between Empty and PpBBX16 overexpression calli under dark/light conditions. (b) The enrichment analysis of KEGG pathways in the differential expression genes between Empty and PpBBX16 overexpression calli under light condition.

Figure 6

Effects of the transient expression or silencing of PpBBX16 in pear fruit. (a) The transient expression of PpBBX16 in the fruit of ‘Korla’ pear induced anthocyanin biosynthesis, while the silencing of PpBBX16 suppressed anthocyanins’ accumulation. (b–d) The expression patterns of PpBBX16 (b), the anthocyanin contents (c) and the colour changes (d) of the fruit peels were analysed. (e) The expression levels of anthocyanin biosynthesis‐related genes were also affected by the transient expression and the silencing of PpBBX16 in fruit peels. The error bars represent the standard deviations of three biological replicates. Asterisks indicate significant differences (two‐tailed Student's t‐test, * P < 0.05, ** P < 0.01).

Figure 7

Proposed model of the mechanism of regulating anthocyanin accumulation through PpBBX16 in pear. (a) Red pear under dark conditions cannot accumulate anthocyanin. The dashed lines represent pathways already reported in Arabidopsis: Line 1, BBX proteins could be degraded through the 26S proteasome pathway by interactions with E3 ubiquitin ligases, such as COP1 (Datta et al., 2008). Line 2, The functions of BBX members can be suppressed by other BBX proteins (Holtan et al., 2011). (b) Red pear under light conditions accumulate anthocyanin through the PpBBX16‐involved pathway: PpBBX16 and PpHY5 jointly activate the expression of structural genes (Arrow 3) and PpMYB10 (Arrow 4), which further induces the expression levels of structural genes.