The metabolism of theanine — a unique non-proteinogenic amino acid — is a necessary biological process during the planting and production of tea (Camellia sinensis) that determines tea quality.
A proposed model for CsMYB73 negatively regulating the accumulation of theanine mediated by CsGGT2 and CsGGT4 in tea shoots of ‘Huangkui’ returning green from yellow. The blue arrows indicate theanine metabolism involved in CsGGT2. The orange arrows indicate the theanine metabolism involved in CsGGT4. The black arrows represent upregulated and downregulated gene expression and theanine content. The gray arrows represent a positive effect, and the gray vertical lines represent an inhibitory effect. Glu – glutamine; CsGGT2 and CsGGT4 – γ-glutamyl-transpeptidase from tea plants; CsMYB73 – transcription factor from tea plants. Ellipses embedded with question marks represent unverified regulatory factors that may participate in this model. During the yellowing period of ‘Huangkui’ leaves, a significant suppression of CsMYB73 further inhibited the theanine hydrolase CsGGT2 activity, and activated the theanine synthase CsGGT4 activity, resulting in a higher level of theanine accumulated in tea shoots. As tea shoots transition from yellow to green, the upregulation of CsMYB73 leads to an enhancement in the activity of theanine hydrolase CsGGT2, while simultaneously suppressing the synthesis of theanine by the theanine synthase CsGGT4. Consequently, this process culminates in a persistent reduction in the accumulation level of theanine in ‘Huangkui’ shoots. Image credit: Chang et al., doi: 10.1093/hr/uhae012.
The accumulation of theanine in tea leaves is a dynamic process influenced by environmental factors and plant development stages.
While past studies have identified key enzymes involved in its biosynthesis and breakdown, the precise transcriptional regulation governing these processes has remained elusive.
“As an economically important crop, tea leaves are generally selected for making tea, with good economic value and health effects,” said Anhui Agricultural University plant biologist Manman Chang and colleagues.
“As a unique non-proteinogenic amino acid, theanine is dynamically regulated by environmental conditions and developmental cues.”
“Similar to glutamine, theanine acts as a form of nitrogen storage and transport and has vital physiological functions in plant growth.”
“Generally, theanine is mainly synthesized in the roots, which is transported to young leaves and other tissues of tea plant for use,” they added.
“During leaf maturation, theanine levels decrease progressively from young leaves to older leaves.”
“It is well documented that theanine accumulation is strongly influenced by factors such as seasons, development, and environmental influences.”
“Molecular mechanisms responsible for theanine biosynthesis and degradation, however, are yet to be elucidated.”
In their study, the authors investigated the roles of specific genes and transcription factors in determining theanine accumulation, offering novel insights into the molecular basis of tea plant biology.
They focused on the functional divergence between two key enzymes, CsGGT2 and CsGGT4, and their transcriptional regulator, CsMYB73.
Through biochemical assays, they established that CsGGT4 exhibits significantly higher catalytic efficiency for theanine synthesis, whereas CsGGT2 primarily functions as a theanine hydrolase, breaking down theanine.
Advanced homology modeling and molecular docking analyses further revealed structural differences between these enzymes, explaining their distinct roles.
Notably, CsGGT4 showed a stronger affinity for ethylamine and glutamine, key substrates in theanine biosynthesis.
Further molecular investigations demonstrated that CsMYB73, a nucleus-localized transcription factor, directly binds to the promoters of CsGGT2 and CsGGT4, exerting opposite regulatory effects: it activates CsGGT2 while repressing CsGGT4, ultimately reducing theanine accumulation in tea shoots.
The researchers also observed that sustained high levels of CsMYB73 expression correlated with increased CsGGT2 activity and diminished CsGGT4 function, leading to lower theanine content.
These discoveries reveal a sophisticated genetic control mechanism governing theanine metabolism and open up new possibilities for precision breeding in tea cultivation.
“Our study provides a comprehensive understanding of the transcriptional regulation of theanine metabolism in tea plants,” said Dr. Jun Sun, also from Anhui Agricultural University.
“By uncovering the roles of CsMYB73, CsGGT2, and CsGGT4, we are paving the way for genetic interventions that could enhance tea quality, a factor of great economic and cultural importance.”
The findings appear in the journal Horticulture Research.
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Manman Chang et al. 2024. CsMYB73 negatively regulates theanine accumulation mediated by CsGGT2 and CsGGT4 in tea shoots (Camellia sinensis). Horticulture Research 11 (3); doi: 10.1093/hr/uhae012
