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

Abstract

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.

HPLC and high-throughput sequencing revealed higher tea-leaves quality, soil fertility and microbial community diversity in ancient tea plantations: compared with modern tea plantations

“…Except the TK with a relatively higher loss that was translated into AK under the effect of related microbial communities, the natural cultivation way was conducive to a growth over 100 years in ancient tea plantations because of the higher soil fertility. The higher AN, AP and AK improved theanine and flavonoid biosynthesis activities in tea plant physiology through glutamine synthetase, phenylpropanoid, flavonoid pathways [ 35 , 36 ], which might contribute to the accumulations of free amino acids, gallic acid, caffeine and EGC in tea-leaves. Additionally, the photosynthetic capacity potentially impacted catechins biosynthesis (such as EGC) in tea plant [ 37 ], which might cause the significant ( P < 0.05) differences of EGC, EGCG and GCG between modern and ancient tea plantations.…”

Flavonoids Are Intra- and Inter-Kingdom Modulator Signals

“…Often, essential macro-and micro-nutrients, such as N, P, Fe and Mn, are poorly bioavailable for the plant that, among other strategies, secretes flavonoids to enhance their mobilization and solubility [101]. In fact, aside from their well-known role as chemoattractors and nod genes inducers in Rhizobium-legume symbiosis [102,103], flavonoid biosynthesis was reported to be upregulated under nitrogen deficiency in tea and tomato plants as a stress response [104,105]. Flavonoid exudation also increased in apple trees subjected to phosphorus depletion, in order to cope with the nutritional stress by prompting either Fe reduction or chelation and consequently P solubilization [106].…”

Theanine, a tea-plant-specific non-proteinogenic amino acid, is involved in the regulation of lateral root development in response to nitrogen status

“…Exogenous auxin increases LR number and promotes the anticlinal division of pericycle cells [ 22 ]. In Arabidopsis , auxin transporter-like protein 3 (LAX3) induces the expression of cell-wall-remodeling genes, resulting in cell wall breakdown and therefore boosting LR emergence [ 17 , 23 ]. Auxin signaling mediated by Auxin/Indole-3-Acetic Acid (Aux/IAA) and auxin response factors (ARFs) regulates LR development by inducing the expression of LATARAL ROOT PRIMORDIUM ( LRP1 ) in LR meristem [ 24 ].…”