Relations between intracellular pH and gamma-aminobutyric acid levels in tea (Camellia sinensis) leaves, after two types of anaerobic treatment

Sammanfattning: Tea (Camellia sinensis) is one of the three major non-alcoholic beverages in the world. In Gabaron tea, the levels of γ-aminobutyric acid (GABA) are more than 1.3 mg/g. In the human body, GABA lowers blood pressure and cholesterol levels. GABA in plants is mainly produced by two pathways: the glutamate (Glu) decarboxylation pathway (dominated by Glu decarboxylase; GAD), and the polyamine oxidation pathway (dominated by polyamine oxidase; PAO). In previous studies, it was demonstrated that GABA in tea tissue originates mainly from Glu decarboxylation. Intracellular pH is an important regulator of cell physiological activities. Intracellular pH influences enzyme activity and important metabolic processes, including DNA replication, RNA and protein synthesis, as well as cell growth. The purpose of this project was to develop a detection method for intracellular pH of tea leaves, use the method to analyze the relationship between GABA enrichment and pH change during the production of Gabaron tea. To this end, fresh tea leaves (Longjin 43) were picked and exposed to two kinds of anaerobic treatment (N2, CO2). The intracellular pH of the upper and lower mesophyll cells of the two kinds of anaerobically treated tea leaves was measured by fluorescent dye (BCECF/AM). Combined with the changes of pH and the levels of two amino acids (Glu, GABA), the relationship between N2 and CO2 and intracellular pH during GABA enrichment was followed. The results showed that after different anaerobic treatments (N2 or CO2), the pH value of cells in the upper and lower mesophyll of tea leaves varied depending on treatment. For tea leaves treated with CO2, the pH in the upper and lower mesophyll cells increased after 1 h, and returned to normal after 6 h. Combined with the levels changes of the two amino acids, it is speculated that under high concentration of CO2, the stomata of tea leaves were still open, and anaerobic respiration was carried out at this time. Excessive CO2 causes the internal environment of cells to become acidic. At this time, GAD is activated. GAD consumes a lot of protons and produces GABA, which reduces damage of the cell membrane under acidic conditions. For tea leaves treated with N2, the intracellular pH of the upper mesophyll first increased and then decreased, while the intracellular pH of the lower mesophyll first decreased and then increased. It is speculated that in the high concentration of N2, the stomata of the lower mesophyll are closed, the tricarboxylic acid cycle is inhibited, and protons cannot be consumed, resulting in the acidic cytoplasm. After 6 h of treatment, GAD is activated, consuming protons and producing GABA. Future work should focus on pH determination after short-term anaerobic treatment. The GAD activity during anaerobic treatment and the detection of stomatal opening and closing of upper and lower epidermis during different anaerobic treatments are also necessary experiments to verify the hypothesis.