Development and Application of a Fast Gas Chromatographic Method Offer New Insights into l‑theanine Production Regulation in Camellia sinensis L

Tea is the most consumed beverage worldwide, and l-theanine in tea leaves significantly affects their flavor and market quality. We have developed and validated a fast and reliable gas chromatographic method with flame ionization detection (GC-FID) to quantify l-theanine after its extraction from Camellia sinensis (tea plant) and derivatization. The procedure was completed in 40 min, from extraction to chromatographic analysis, with a recovery rate of more than 93% and allowing a high sample throughput. The GC-FID intraday precision was within 0.57–2.28%, while the interday precision ranged from 1.57 to 13.48%. The intraday accuracy ranged from −6.84 to 5.26%, while the interday accuracy ranged from −1.08 to 3.12%. The limit of detection was 2.28 μg/mL, and the limit of quantification was 6.47 μg/mL. The GC-FID method was validated by high-performance liquid chromatography with UV detection (HPLC–UV) and was used to investigate the biosynthesis and regulation of l-theanine in tea plants. We found that plants fed with ethylamine significantly increased l-theanine concentrations in roots, while exogenous supplementation of glutamic acid, carbamide, and glutamine did not significantly affect the l-theanine level in roots. Our results also indicated that roots were not indispensable for the biosynthesis of l-theanine, which was detected in undifferentiated embryonic calluses in concentrations (g/100 g dry weight) as high as in leaves of whole plants (1.67 and 1.57%, respectively) and without any exogenous theanine precursor supplementation.