Theaceae species are dicotyledonous angiosperms with extremely high ornamental and economic value. The chloroplast genome is traditionally used to study species evolution, expression of chloroplast genes and chloroplast transformation. Codon usage bias (CUB) analysis is beneficial for investigations of evolutionary relationships and can be used to improve gene expression efficiency in genetic transformation research. However, there are relatively few systematic studies of the CUB in the chloroplast genomes of Theaceae species. In this study, CUB and nucleotide compositions parameters were determined by the scripts written in the Perl language, CodonW 1.4.2, CU.Win2000, RStudio and SPSS 23.0. The chloroplast genome data of 40 Theaceae species were obtained to analyse the codon usage (CU) characteristics of the coding regions and the influence of the source of variation on CUB. To explore the relationship between the CUB and gene expression levels in these 40 Theaceae plastomes, the synonymous codon usage order (SCUO) and measure independent of length and composition (MILC) values were determined. Finally, phylogenetic analysis revealed the genetic evolutionary relationships among these Theaceae species. Our results showed that based on the chloroplast genomes of these 40 Theaceae species, the CUB was for codons containing A/T bases and those that ended with A/T bases. Moreover, there was great commonality in the CUB of the Theaceae species according to comparative analysis of relative synonymous codon usage (RSCU) and relative frequency of synonymous codon (RFSC): these species had 29 identical codons with bias (RSCU > 1), and there were 19 identical high-frequency codons. The CUB of Theaceae species is mainly affected by natural selection. The SCUO value of the 40 Theaceae species was 0.23 or 0.24, and the chloroplast gene expression level was moderate, according to MILC values. Additionally, we observed a positive correlation between the SCUO and MILC values, which indicated that CUB might affect gene expression. Furthermore, the phylogenetic analysis showed that the evolutionary relationships in these 40 Theaceae species were relatively conserved. A systematic study on the CUB and expression of Theaceae species provides further evidence for their evolution and phylogeny.

Theanine, a tea plant-specific non-proteinogenic amino acid, is the most abundant free amino acid in tea leaves. It is also one of the most important quality components of tea because it endows the “umami” taste, relaxation-promoting, and many other health benefits of tea infusion. Its content in tea leaves is directly correlated with the quality and price of green tea. Theanine biosynthesis primarily occurs in roots and is transported to new shoots in tea plants. Recently, great advances have been made in theanine metabolism and transport in tea plants. Along with the deciphering of the genomic sequences of tea plants, new genes in theanine metabolic pathway were discovered and functionally characterized. Theanine transporters were identified and were characterized on the affinity for: theanine, substrate specificity, spatiotemporal expression, and the role in theanine root-to-shoot transport. The mechanisms underlying the regulation of theanine accumulation by: cultivars, seasons, nutrients, and environmental factors are also being rapidly uncovered. Transcription factors were identified to be critical regulators of theanine biosynthesis. In this review, we summarize the progresses in theanine: biosynthesis, catabolism, and transport processes. We also discuss the future studies on theanine in tea plants, and application of the knowledge to crops to synthesize theanine to improve the health-promoting quality of non-tea crops.

Free amino acids, including theanine, glutamine and glutamate, contribute greatly to the pleasant taste and multiple health benefits of tea. Amino acids in tea plants are mainly synthesized in roots and transported to new shoots, which are significantly affected by nitrogen (N) level and forms. However, the regulatory amino acid metabolism genes have not been systemically identified in tea plants. Here, we investigated the dynamic changes of free amino acid contents in response to N deficiency and forms in tea plant roots, and systemically identified the genes associated amino acid contents in individual metabolism pathways. Our results showed that glutamate-derived amino acids are the most dynamic in response to various forms of N and N deficiency. We then performed transcriptomic analyses of roots treated with N deficiency and various forms of N, and differentially expressed amino acid metabolic genes in each pathway were identified. The analyses on expression patterns and transcriptional responses of metabolic genes to N treatments provided novel insights for the molecular basis of high accumulation of theanine in tea plant root. These analyses also identified potential regulatory genes in dynamic amino acid metabolism in tea plant root. Furthermore, our findings indicated that the dynamic expression levels of CsGDH, CsAlaDC, CsAspAT, CsSDH, CsPAL, CsSHMT were highly correlated with changes of amino acid contents in their corresponding pathways. Herein, this study provides comprehensive insights into transcriptional regulation of amino acid metabolism in response to nitrogen deficiency and nitrogen forms in tea plant root.

Theanine, one of the most important components of teas, confers the umami taste and relaxation effect of tea infusion. As a non-proteinogenic amino acid, theanine solely accumulates to high levels in tea plants (Camellia sinensis L.); however, the underlying mechanism remains largely unknown. Theanine is biosynthesized from ethylamine and glutamate by CsTSI (Theanine Synthetase I), with ethylamine being synthesized from alanine, by alanine decarboxylase. Although CsAlaDC exhibited alanine decarboxylase activity, in vitro, the in vivo role in tea plants has not been characterized. In this study, we found ethylamine content in tea roots to be highly correlated with theanine content (r=0.883, p <0.01), and expression levels of CsAlaDC were similarly highly correlated with the accumulation of ethylamine (r=0.903, p <0.01) and theanine (r=0.881, p <0.01). CsAlaDC-expressing tobacco leaves produced high levels of ethylamine, but did not produce detectable theanine. However, high levels of theanine were produced in leaves co-expressing CsAlaDC and CsTSI. These results indicate CsAlaDC and CsTSI act coordinately in determining the high level of theanine biosynthesis. This is also the first report to synthesize theanine in a non-tea plant, which offers the potential to generate relaxation-promoting foods by synthesizing theanine in crops.