Nitrogen-Regulated Theanine and Flavonoid Biosynthesis in Tea Plant Roots: Protein-Level Regulation Revealed by Multiomics Analyses

Theanine and flavonoids (especially
proanthocyanidins) are the
most important and abundant secondary metabolites synthesized in the
roots of tea plants. Nitrogen promotes theanine and represses flavonoid
biosynthesis in tea plant roots, but the underlying mechanism is still
elusive. Here, we analyzed theanine and flavonoid metabolism in tea
plant roots under nitrogen deficiency and explored the regulatory
mechanism using proteome and ubiquitylome profiling together with
transcriptome data. Differentially expressed proteins responsive to
nitrogen deficiency were identified and found to be enriched in flavonoid,
nitrogen, and amino acid metabolism pathways. The proteins responding
to nitrogen deficiency at the transcriptional level, translational
level, and both transcriptional and translational levels were classified.
Nitrogen-deficiency-responsive and ubiquitinated proteins were further
identified. Our results showed that most genes encoding enzymes in
the theanine synthesis pathway, such as CsAlaDC, CsGDH, and CsGOGATs, were repressed by
nitrogen deficiency at transcriptional and/or protein level(s). While
a large number of enzymes in flavonoid metabolism were upregulated
at the transcriptional and/or translational level(s). Importantly,
the ubiquitylomic analysis identified important proteins, especially
the hub enzymes in theanine and flavonoid biosynthesis, such as CsAlaDC,
CsTSI, CsGS, CsPAL, and CsCHS, modified by ubiquitination. This study
provided novel insights into the regulation of theanine and flavonoid
biosynthesis and will contribute to future studies on the post-translational
regulation of secondary metabolism in tea plants.