Variation of theanine, methylxanthines and flavanoids compositions in leaves and shoots of tea plant cultivar Kolkhida in the conditions of humid subtropics of Russia: seasonal dynamics, stages of leaf maturity, and provision with biogenic elements

Biological specifics of the synthesis of specialized metabolites by tea plants, as well as the influence of many environmental factors on these processes, underlie the variation of their compositions in different varieties and regions of cultivation. From this point of view, it is important to determine the initial natural metabolic profile of domestic tea varieties grown in the natural conditions of the humid subtropics of Russia, as well as its transformation under the influence of a set of factors. In this work, for the first time we have established the basic metabolic profile of the tea plant (Camellia sinensis L.) cv. Kolkhida, including several groups of specialized organic components (flavan-3-ols, flavanols, methylxanthines, theanine), which are accumulated in the environmental conditions of the Sochi Black Sea region. The aim of the work was to establish the content and variation ranges of theanine and complex secondary metabolites (catechins, methylxanthines, flavanols) in shoots and leaves of different maturity of cv. Kolkhida, a standard cultivar for the Black Sea coast of the Russian Federation, in seasonal dynamics with application of fertilizers. The studies were conducted in the humid subtropics of Russia (the Black Sea coast of the Krasnodar Territory, Sochi, Lazarevsky District, Uch-Dere) in 2022-2023 on a vegetatively propagated full-aged plantation of cv. Kolkhida (established in 1983). The brown forest acidic soil with low-humus and thick medium-clayey was formed on eluvium-diluvium of clay shales (argillites) in the experimental plot. To assess the seasonal dynamics of formation and accumulation of metabolites, samples of 3-leaf flush (young shoot consisting of an apical bud with three leaves of different maturity) were collected during the periods of the most intensive flush formation of the 1st (May, June) and 2nd (July, August) growth waves on the optimal (N240P70K90) and deficient (N0P0K0) nutrient conditions. For a comparative assessment of the metabolic profile of leaves of different maturity stages, as well as to establish the effect of zinc in the summer period (July), young tea shoots were selected in two treatments of the field experiment, contrasting in the soils zinc provision: N240P70K90 (control), N240P70K90 + Zn4.3 (after annual 15-year application of zinc sulfate to the surface soil layer). The parts of the shoot (bud, 1st, 2nd, 3rd leaf) were grouped. The contents of specialized components in plant samples - caffeine, theobromine, theanine, (+)-catechin (K), (-)-epicatechin (EC), (+)-gallocatechin (GC), (-)-epigallocatechin (EGC), (-)-catechin-3-gallate (CG), (-)-epicatechin-3-gallate (ECG), (-)-gallocatechin-3-gallate (GCG), (-)-epigallocatechin-3-gallate (EGCG), rutin, flavanol glycosides (quercetin and kaempferol) - were assessed by high-performance liquid chromatography on a chromatograph from Agilent Technologies (model 1100, USA). In the seasonal dynamics of young tea shoots (3-leaf flush), significant fluctuations in theanine (3-12 mg/g) and total catechins (170-250 mg/g) were observed along with a relatively stable content of caffeine (25-35 mg/g) and theobromine (2.0-3.5 mg/g). The content of quercetin (1.6-3.2 mg/g) and kaempferol (0.4-3.0 mg/g) decreased from May to August, and the amount of quercetin glycoside (rutin) was within 0.95-1.76 mg/g. Increase of drought and hyperthermia from spring to summer lead to decrease in the content of theanine and flavanol O-glycosides by 2-3 folds along with increase the proportion of catechins by 1.2-1.5 folds. Significant difference in the amount of the studied metabolites was observed among the apical bud and the 3rd shoot leaf. Thus, the youngest part of the shoot (apical bud) was characterized by increased accumulation of theanine (5.0±0.1 mg/g), caffeine (42.0±2.0 mg/g) and catechin gallates (228.0-245.0 mg/g). While in third leaf, the content of these components decreased on average by 1.5 times, but the proportion of simple catechins increased on average by 2.5 folds. Different supply of plants with biogenic elements changed the metabolic profile of tea leaves to a lesser extent in comparison with seasonal variability. Nevertheless, increased supply of tea plants with NPK and Zn elements, led to increase in theanine content by 2.0 and 1.2 folds, respectively, while the amount of simple catechins was decreased by 1.3 folds. The following revealed patterns are promising for use in agricultural technology: the regulatory role of NPK elements in the induction of theanine synthesis and inhibiting of phenolic metabolism; a change in the initial balance of theanine and caffeine acting in different directions in the tea leaf under the influence of zinc, which determine the calming or stimulating effect of the tea beverage.