Theanine is synthesized mainly in tea roots from glutamate and ethylamine. Although glutamate is widespread in plants, ethylamine accumulates strongly in Camellia species, making its supply a major factor limiting theanine production. Earlier work established that alanine decarboxylase converts alanine into ethylamine and that the tea enzyme CsAlaDC is closely related in sequence to serine decarboxylases, despite showing a different substrate preference. What remained unresolved was how this specialized activity arose, how gene duplication shaped the pathway, and which residues separated the two catalytic functions. Because of these challenges, deeper investigation is needed into how the CsAlaDC gene evolved from CsSDC and which molecular changes gave the enzyme its alanine-specific activity.
Researchers from the Tea Research Institute of the Chinese Academy of Agricultural Sciences and the Tea Research Institute of Hangzhou Academy of Agricultural Science reported the study in Horticulture Research, published (DOI 10.1093/hr/uhag153) online on April 17, 2026. The work reconstructs the evolutionary route from the conserved serine decarboxylase gene CsSDC to the specialized alanine decarboxylase gene CsAlaDC and experimentally tests the molecular changes underlying that transition. By linking gene-family evolution to enzyme function, the study provides a mechanistic explanation for a key step that helped establish efficient theanine biosynthesis in tea.
The team first surveyed alanine decarboxylase and serine decarboxylase homologs across 48 plant species and analyzed related genes in 19 Camellia species, revealing a lineage-specific expansion that produced four conserved family members in the examined Camellia genomes. Two additional genes, CsASDC1 and CsASDC2, occupied intermediate evolutionary positions between CsSDC and CsAlaDC, while alanine decarboxylase homologs showed predominantly root-based expression in the species where they were detected. Functional assays showed that the intermediate proteins retained serine decarboxylase activity. The researchers then compared CsSDC and CsAlaDC sequences, identified 21 consistently divergent amino-acid positions, and exchanged those residues between the two enzymes. The engineered proteins underwent an almost complete switch in substrate preference, showing that these positions collectively encode functional specialization. Structure-guided single-residue tests narrowed the key determinants further: phenylalanine at position 106 (Phe106) and glycine at position 168 (Gly168) were indispensable for CsAlaDC activity, while corresponding substitutions in CsSDC generated alanine decarboxylase activity. The codon changes TAC to TTT and TGT to GGT therefore emerged as pivotal evolutionary events. In planta tests reinforced the result. Overexpressing the CsAlaDC gene in Arabidopsis thaliana increased ethylamine and theanine, while transient expression of mutant forms in Nicotiana benthamiana confirmed the importance of the two residues.
The authors said the study shows how a familiar enzyme framework can be repurposed during evolution to create a new metabolic function. Rather than treating tea’s theanine pathway as a fixed biochemical feature, they said the results reveal a traceable sequence of gene duplications and catalytic changes that helped establish efficient ethylamine production. They emphasized that identifying Phe106 and Gly168 moves the story from broad evolutionary inference to specific molecular mechanisms, making it possible to test how individual sequence changes reshaped substrate recognition and ultimately contributed to a distinctive chemical trait of tea.
The findings could inform future efforts to improve theanine production through molecular breeding, metabolic engineering, or structure-guided enzyme design. Because the CsAlaDC enzyme controls ethylamine formation, tuning CsAlaDC expression or CsAlaDC catalytic efficiency may offer a route to increase precursor supply in tea or other biological production systems. The study also provides candidate residues for engineering alanine decarboxylase performance more precisely, rather than relying only on whole-gene overexpression. However, the reduced growth observed in CsAlaDC-overexpressing Arabidopsis thaliana suggests that metabolic gains may carry physiological costs if pathway activity is pushed too far. Future work will therefore need to balance theanine enhancement with plant growth, metabolic homeostasis, crop performance, and the stability of desirable traits under field conditions.
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References
DOI
10.1093/hr/uhag153
Original Source URL
https://doi.org/10.1093/hr/uhag153
Funding information
This work was supported by the Zhejiang Science and Technology Major Program on Agricultural New Variety Breeding-Tea Plant (2021C02067-7-1)；and the China Agriculture Research System of MOF and MARA (CARS-19).
About Horticulture Research
Horticulture Research is an open access journal of Nanjing Agricultural University and ranked number one in the Horticulture category of the Journal Citation Reports ™ from Clarivate, 2023. The journal is committed to publishing original research articles, reviews, perspectives, comments, correspondence articles and letters to the editor related to all major horticultural plants and disciplines, including biotechnology, breeding, cellular and molecular biology, evolution, genetics, inter-species interactions, physiology, and the origination and domestication of crops.