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

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.     

Two amino acid changes in the R3 repeat cause functional divergence of two clustered <Emphasis Type="Italic">MYB10</Emphasis> genes in peach

R2R3-MYB genes play a pivotal role in regulating anthocyanin accumulation. Here, we report two tandemly duplicated R2R3-MYB genes in peach, PpMYB10.1 and PpMYB10.2, with the latter showing lower ability to induce anthocyanin accumulation than the former. Site-directed mutation assay revealed two amino acid changes in the R3 repeat, Arg/Lys⁶⁶ and Gly/Arg⁹³, responsible for functional divergence between these two PpMYB10 genes. Anthocyanin-promoting activity of PpMYB10.2 was significantly increased by a single amino acid replacement of Arg⁹³ with Gly⁹³. However, either the Gly⁹³ → Arg⁹³ or Arg⁶⁶ → Lys⁶⁶ substitutions alone showed little impact on anthocyanin-promoting activity of PpMYB10.1, but simultaneous substitutions caused a significant decrease. Reciprocal substitution of Arg/Gly⁹³ could significantly alter binding affinity to PpbHLH3, while the Arg⁶⁶ → Lys⁶⁶ substitution is predicted to affect the folding of the MYB DNA-binding domain, instead of PpbHLH3-binding affinity. Overall, the change of anthocyanin-promoting activity was accompanied with that of bHLH-binding affinity, suggesting that DNA-binding affinity of R2R3-MYBs depends on their bHLH partners. Our study is helpful for understanding of functional evolution of R2R3-MYBs and their interaction with DNA targets.     

Genome-wide analysis of ACO and ACS genes in pear (Pyrus ussuriensis)

The “Nanguo” pear is a typically climacteric fruit and ethylene is the main factor controlling the ripening process of climacteric fruit. Ethylene biosynthesis has been studied clearly and ACC synthase (ACS) is the rate-limited enzyme. ACO (ACC oxidase) is another important enzyme in ethylene biosynthesis. By exploring the pear genome, we identified 13 ACS genes and 11 ACO genes, respectively, and their expression patterns in fruit and other organs were investigated. Among these genes, 11 ACS and 8ACO genes were expressed in pear fruits. What’s more, 4 ACS and 3ACO genes could be induced by Ethephon and inhibited by 1-MCP treatment. This study is the first time to explore ACS and ACO genes at genome-wide level and will provide new data for research on pear fruit ripening.