Integrated metabolomic and transcriptomic analyses reveal anthocyanin biosynthesis mechanisms and the regulatory role of LjAN2 in Lonicera japonica

Lonicera japonica flowers are a very commonly used traditional Chinese herb. Anthocyanins are the source of flower pigments, and also are renowned for their therapeutic activities. However, the specific anthocyanin composition and regulatory mechanisms governing their accumulation in L. japonica varieties remain unclear. Here, we first investigated the changes in flower color and anthocyanin content during development in the green flower (GFLJ) and the purple flower (PFLJ) cultivars of L. japonica. Results show GFLJ has green flowers and PFLJ has purple flowers, which are especially remarkable during S3-S4 stages. Accordingly, PFLJ had much higher (>10 times) anthocyanins contents at all the six flower stages than those of GFLJ. Further metabolomic analysis in S3 stage flowers found that most anthocyanins showed increased accumulation, whereas flavones and flavonols showed decreased accumulation in PFLJ compared to GFLJ. Transcriptome analysis identified 21 (85.7 % upregulated) anthocyanin synthase gene DEGs, and 23 MYB transcription factor (TF) DEGs (play essential roles in regulating anthocyanin biosynthesis). In addition, 19 GST and 14 MATE DEGs (play key roles in anthocyanins accumulation) were identified. Further, we found a novel MYB TF (LjAN2) that showed much higher expression in PFLJ. LjAN2 overexpression in tobacco led to purple leaves, and the upregulation of anthocyanin synthase genes (NtCHS and NtANS), as well as increased anthocyanin accumulation. This research offers a comprehensive understanding of the molecular basis of anthocyanin biosynthesis in L. japonica, highlighting its potential applications in the pharmaceutical industry.