Integrative Transcriptome and Metabolome Analysis to Reveal Red Leaf Coloration in Shiya Tea (Adinandra nitida)

Adinandra nitida, commonly known as Shiya tea, is a healthcare drink enriched in several phenolic acids and flavonoids, with a purple-red leaf variety possessing a unique flavor and a higher economic value. However, the mechanisms underlying leaf coloration and senescence discoloration remain unknown.

Here, we compared both varieties of A. nitida (purple-red leaf, RL, and green leaf, GL) at two stages of development. To make sure the difference in leaf color in these four groups, several indexes, leaf colorimetric differences, H2O2 content in leaf cells, and antioxidant enzymes activities (superoxide dismutase (SOD), catalase (CAT)) were measured. With the integration of metabolome and transcriptome becoming a trend, metabolites in four groups were detected using an Ultra performance liquid chromatography-tandem mass spectrometer (UPLC-MS/MS) system, and the transcriptome was performed after the extraction of RNA in samples. Afterward, the activities of laccase (LAC) and peroxidase (POD) were measured for further analysis.

The deeper or discoloration of leaf color was not caused by the reactive oxygen species (ROS) stress because the H2O2 content was similar for each group. And the SOD and CAT activities improved significantly in young leaves, especially RL_young. Metabolome data showed a large shift in four groups. By focusing on the variation of flavonoids and 1079 metabolites detected in both varieties, along with the accumulation of flavonoids and tannins, proanthocyanins (PAs) were mostly accumulated in young RL. Differential analysis of expressed genes (DEGs) revealed six genes associated with leaf discoloration as hub factors, of which ANRs (ANR1 and ANR2) were positively correlated with the accumulation of PA in RL.

Using integrate analysis of metabolome and transcriptome, our results revealed that six structural genes found in proanthocyanin biosynthesis, two reductases (ANR), two oxidative polymerases (POD64, LAC17) and two TFs (bHLH3 and MYB4) related to biosynthesis and polymerization of proanthocyanins were associated with not only the difference of GL and RL but also the faded coloration in two RL groups (RL_young and RL_old), which provided a foundation for further research on an understanding of the regulatory genes and the enzymes specific for proanthocyanidin biosynthesis, facilitating the genetic engineering of crops for beneficial metabolite accumulation.