Abstract
Auxin signaling, which is crucial for normal plant growth and development, mainly depends on ARF–Aux/IAA interactions. However, little is known regarding the regulatory effects of auxin signaling on anthocyanin metabolism in apple (Malus domestica). We investigated the functions of MdARF13, which contains a repression domain and is localized to the nucleus. This protein was observed to interact with the Aux/IAA repressor, MdIAA121, through its C-terminal dimerization domain. Protein degradation experiments proved that MdIAA121 is an unstable protein that is degraded by the 26S proteasome. Additionally, MdIAA121 stability is affected by the application of exogenous auxin. Furthermore, the overexpression of MdIAA121 and MdARF13 in transgenic red-fleshed apple calli weakened the inhibitory effect of MdARF13 on anthocyanin biosynthesis. These results indicate that the degradation of MdIAA121 induced by auxin treatment can release MdARF13, which acts as a negative regulator of the anthocyanin metabolic pathway. Additionally, yeast two-hybrid, bimolecular fluorescence complementation, and pull-down assays confirmed that MdMYB10 interacts with MdARF13. A subsequent electrophoretic mobility shift assay and yeast one-hybrid assay demonstrated that MdARF13 directly binds to the promoter of MdDFR, which is an anthocyanin pathway structural gene. Interestingly, chromatin immunoprecipitation–quantitative real-time PCR results indicated that the overexpression of MdIAA121 clearly inhibits the recruitment of MdARF13 to the MdDFR promoter. Our findings further characterized the mechanism underlying the regulation of anthocyanin biosynthesis via Aux/IAA–ARF signaling.
Highlights
Color is an important fruit quality index that influences the purchasing behavior of consumers to some degree[1]
In protoplasts expressing 35S::MdARF13-green fluorescent protein (GFP), the GFP signal was observed only in the nucleus, while the GFP signal was detected throughout the control protoplasts (Fig. 2c), implying that MdARF13 is localized to the nucleus
The MdARF13 expression levels in calli treated with 40 μmol/L NAA were 2–5-fold higher than in calli exposed to the other NAA concentrations
Summary
Color is an important fruit quality index that influences the purchasing behavior of consumers to some degree[1]. As a type of secondary metabolite, anthocyanins constitute one of the main pigments responsible for fruit color[2,3]. Their positive effects on human health include enhancing vascular elasticity, preventing cardiovascular disease, and protecting the liver from damage[4,5,6,7]. Two MYB TF genes (MdMYB1 and MdMYBA), which were first isolated from fruit skin, have been confirmed to be responsible for the accumulation of anthocyanin[15,16]. MdMYB10 consists of a characteristic R6 rearrangement in its promoter that is
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