Abstract
In higher plants, jasmonate ZIM-domain (JAZ) proteins negatively regulate the biosynthesis of anthocyanins by interacting with bHLH transcription factors. However, it is largely unknown if and how other regulators are involved in this process. In this study, the apple MdJAZ2 protein was characterized in regards to its function in the negative regulation of anthocyanin accumulation and peel coloration. MdJAZ2 was used as a bait to screen a cDNA library using the yeast two-hybrid method. The hypersensitive induced reaction (HIR) proteins, MdHIR2 and MdHIR4, were obtained from this yeast two-hybrid. The ZIM domain of MdJAZ2 and the PHB domain of the MdHIR proteins are necessary for their interactions. The interactions were further verified using an in vitro pull-down assay. Subsequently, immunoblotting assays demonstrated that MdHIR4 enhanced the stability of the MdJAZ2-GUS protein. Finally, a viral vector-based transformation method showed that MdHIR4 inhibited anthocyanin accumulation and fruit coloration in apple by modulating the expression of genes associated with anthocyanin biosynthesis.
Highlights
MYB TFs are the most intensively studied components of the MBW complex associated with anthocyanin biosynthesis in fruit trees[8,9,10]
Anthocyanin content in the apple peel injected with pIR-MdJAZ2 was much lower than in the apple peel injected with the empty control (Fig. 1B)
We previously reported that MdJAZ2 interacts with MdbHLH3 and inhibits the expression of MdMYB1, MdMYB9, MdMYB11 and their downstream anthocyanin structural target genes[15]
Summary
MYB TFs are the most intensively studied components of the MBW complex associated with anthocyanin biosynthesis in fruit trees[8,9,10]. In the classic Arabidopsis model of the JA signaling pathway, when the JA level is relatively low JAZ proteins accumulate and interact with the bHLH TF MYC2, attenuating the expression of the downstream JA-responsive genes[25]. The JAZ proteins play a principal role in various functions of JA They interact with the R2R3-MYB TFs, such as MYB21 and MYB24, to affect JA-regulated stamen development in Arabidopsis[31]. The JAZ proteins interact with the bHLHs, such as GL3, EGL3, and TT8, and with the MYB TFs, such as MYB75 and GL1, which regulate the expression of the MBW complexes associated with anthocyanin biosynthesis, attenuating their transcriptional function and, thereby, inhibiting anthocyanin biosynthesis[34]. MdJAZ2 interacts with MdbHLH3 and inhibits its transcriptional function, thereby, working as a negative regulator for anthocyanin biosynthesis[12,15]
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