Detection of candidate proteins in the indican biosynthetic pathway of Persicaria tinctoria (Polygonum tinctorium) using protein–protein interactions and transcriptome analyses

Persicaria tinctoria (Polygonum tinctorium) synthesizes indican (indoxyl-β-D-glucoside) as a specialized metabolite. Indican is synthesized in the cytosol of leaf cells from indoxyl and UDP-glucose by the catalysis of indoxyl-β-D-glucoside synthase (PtIGS), then transported into vacuoles. As a portion of PtIGS is found on the microsomal membrane, we assume that it is present on the ER membrane as a large complex involving other indican metabolism-related proteins. Based on this hypothesis, the existence of such a complex was investigated using two separate approaches: a protein–protein interaction assay and transcriptome analysis. We first performed a co-immunoprecipitation using the anti-PtIGS antibody and a pull-down assay using recombinant PtIGS, then identified the candidate proteins through MS/MS analysis. Secondly, we performed a transcriptome analysis to examine the differential gene expression between the first and the second leaves. The expressions of candidate genes detected by protein–protein interaction analyses were collated with transcriptome data and validated by quantitative reverse transcription polymerase chain reaction, showing that the expression of sucrose synthase and cytochrome P450 genes decreased in the second leaves compared with the first leaves. Furthermore, we detected several additional proteins, such as heat shock and cytoskeletal proteins, suggesting that PtIGS may form a large complex, a metabolon.