L-theanine, or called as γ-glutamyl-L-ethylamide or γ-ethylamino-L-glutamic acid, is a unique non-protein amino acid in tea plant (Camellia sinensis (L.) O. Kuntze) (Deng et al., 2008). All theanine that naturally exists in tea plant belongs to L-theanine, D-theanine can only be produced industrially. L-theanine is an important indicator in the quality evaluation of green tea because of the particular umami taste that largely influences the flavor of tea (Yamaguchi and Ninomiya, 2000). The functions of theanine related to food science and human nutrition have been extensively studied since theanine was first discovered in tea plant leaves. Previous studies have shown that theanine can improve memory and learning ability by activating relative central neurotransmitters (Haskell et al., 2008). To a certain extent, theanine can reduce blood-pressure, maintain stability, and promote relaxation and concentration by inhibiting the negative effects of caffeine (Yokogoshi et al., 1995; Kakuda et al., 2000). In addition, theanine performs positive functions in anti-diseases action, including enhancing anti-tumor activity, preventing vascular diseases, and neuroprotection (Rogers et al., 2008; Liu et al., 2009; Zhao and Zhao, 2014). Numerous physiological functions make L-theanine as one of the hot spots in the development and utilization of functional components in tea.

As an important and highly abundant free amino acid in tea plants, L-theanine, almost can be detected in all tissues (Deng et al., 2009). However, L-theanine content in tea plants considerably varies among different cultivars and tissues. For example, the tea plant cultivars with small leaf contain higher amino acid contents than the cultivars with large leaf (Jiang, 2013). The small-leaf tea cultivars are suitable for producing the green tea, and the large-leaf cultivars are used to process the black tea (Wu et al., 2014). In general, the theanine content of the young leaves is the highest, and followed by the old leaves, root, and stem (Selvendran and Selvendran, 1973). Within a year, the theanine content is the highest in spring, and then decreases gradually, after which it rises during autumn to a certain degree. In winter (dormancy period), the theanine in tea plant is accumulated and stored in the root. Until to the March and April of the next year (germination period), theanine shifts to shoots and remains at a relative low level in roots until to August (Takeo, 1979). Moreover, L-theanine content is influenced by natural environmental conditions, such as light intensity and concentration of ammonia (Ashihara, 2015).

The theanine metabolism pathway, including synthesis, transportation, and hydrolysis, has been studied for more than sixty years since theanine was discovered by Sakato (Deng et al., 2010). Numerous pivotal enzymes participate in the metabolism pathway of theanine. The gene expression levels of theanine metabolic enzymes largely influence the content, distribution, and metabolism rules of theanine in tea plants (Shi et al., 2011). In theanine biosynthesis, numerous structural genes encode enzymes that directly catalyze reaction steps that lead to the formation of theanine. The genes encoding TS and GS have been identified and cloned from tea plant, and the sequences of TS and GS genes present high consistency (Okada et al., 2006; Rana et al., 2008a). The partial coding sequences of GOGAT and GDH have been logged in GenBank. The genes in tea plant encoding to ALT and two tea-specific enzymes, AIDA, and ThYD, are not identified gene sequences (Tsushida and Takeo, 1985; Shi et al., 2011). However, SAMDC and ADC, which are both pivotal enzymes in polyamine metabolism, play similar roles with AIDA.

Ethylamine, one of the hydrolyzates of theanine, is further oxidized to acetaldehyde by AO, which is another precursor of catechin phloroglucinol that participates in catechin biosynthesis (Kito et al., 1968). In addition, NiR, which provides NH₄⁺ for GS/ GOGAT cycle and α-ketoglutarate reductive amination reaction, performs important functions in theanine synthesis. Interestingly, similar to GS in bacteria, γ-GGT can also catalyze theanine synthesis with glutamine and ethylamine as substrate (Mu et al., 2015).

To obtain novel insight into L-theanine metabolism in tea plant, the genes encoding the enzymes involved in the theanine metabolism pathway are identified based on GenBank and our tea plant transcriptome (Wu et al., 2014). The expression patterns of seventeen metabolic-related genes and L-theanine content in the bud and 1st leaf, 2nd leaf, 3rd leaf, old leaf, stem, and root of the three selected tea cultivars were detected and analyzed by RP-HPLC method. The three C. sinensis cultivars, ‘Huangjinya’, ‘Anjibaicha’, and ‘Yingshuang’, were used in this experiment (Figure 1). Based on the obvious difference of geographic and climate characteristics in these three tea production areas, the morphology and physiology of the three tea plants were different. The theanine contents and the ability of adaptation to a single or multiple stresses were different among the three tea plant cultivars. The mainly characteristics of the three tea plant cultivars were listed in Table 1. This work is aimed to predict the relationship between structural genes and L-theanine content and the distribution at transcript level, and to provide reference for the further study of regulation mechanisms of theanine metabolism.

The L-theanine metabolism pathway involves in the unique accumulation and translocation pathway of nitrogen in tea plant, which belongs to one of perennial economic crops with ammonium resistance (Konishi et al., 1969). At present, a few studies about the enzymes involved in the theanine metabolism pathway at gene level have been reported (Shi et al., 2011; Li et al., 2015b). To better understand the theanine metabolic mechanism at molecular level and provide references for identification and breeding of high theanine tea plant resources, it is essential to explore the correlations between theanine metabolic related genes and contents.

In this study, different tissues of three tea plant cultivars ‘Huangjinya’, ‘Anjibaicha’, and ‘Yingshuang’ were selected, including buds and 1st leaf, 2nd leaf, 3rd leaf, old leaf, stems, and roots. Among different tissues, the stem shows the lowest theanine content, thereby indicating that stems may only act as the transport part of theanine (Oh et al., 2008). In the ‘Huangjinya’ and ‘Anjibaicha’, root theanine content was close to the content of bud and 1st leaf, and higher than previously reported. This finding may be due to variety specificity and weakened transportation of theanine from root to leaves and bud at the re-greening phase of albino tea (Hahlbrock et al., 1971). Among the different cultivars, they presented highly similar distribution tendencies of theanine contents from bud to root. The theanine content in ‘Huangjinya’, ‘Anjibaicha’, and ‘Yingshuang’ decreased by degrees among the tissues except stems, thereby it confirmed that albino tea plants contained higher theanine concentration than non-albino varieties (Li et al., 2011; Feng et al., 2014). Particularly, the high theanine content of ‘Huangjinya’ explained why it is provided with high quality and high popularity.

In qRT-PCR analysis, the high expression levels of most theanine metabolic genes in ‘Huangjinya’ with high theanine concentration. It was basically consistent with the prediction of transcriptome by RPKM values, that is the higher transcripts levels of related genes in small-leaf tea cultivars (‘Chawansanhao’ and ‘Anjibaicha’, higher theanine content) than that in large-leaf cultivar (‘Ruchengmaoyecha’, lower theanine content). In ‘Huangjinya’, the expression levels of CsNR and CsNiR reached to several hundreds of times higher than the other two cultivars. While the NR and NiR play key role in providing the source of ammonium in theanine synthesis (Yang et al., 2013). In addition, the high expression levels of CsTS1, CsTS2, CsGS2, CsADC, and CsSAMDC in ‘Huangjinya’ also offered the possibility for synthesis of abundant theanine.

However, the expression patterns of the selected genes among tissues exist difference at different degrees, even though the genes belong to a family. The CsTS1 and CsTS2, CsGS1, and CsGS2 showed almost opposite correlations with the theanine content in leaves. The transcript levels of CsTS1 and CsTS2 in root were lower than in leaves, but in shoot were less than and more than in mature leaves, respectively. Those comparison results among shoot, mature leaves, and root were identical with the results of Deng (Deng et al., 2008). The negative correlation between mRNA levels of CsGS1 and theanine concentration in tea plant was comparable to that, which was previously reported for rice (Oryza sativa L.) and tobacco (Nicotiana tabacum L.) transfected CsGS gene. In rice and tobacco, the overexpression of transfected CsGS gene caused the decrease in free amino acids, including glutamic acid and glutamine (Migge et al., 2000; Cai et al., 2009). The positive correlation between mRNA level of CsGDH2 and theanine content, possibly because GDH is strongly activated by abundant amide compounds, such as glutamine and theanine, to assist GS/GOGAT (Rana et al., 2008b). In addition, most genes showed lower expression levels in root than that in other tissues. However, the expression level of CsALT was up-regulated in root and much higher than that in stem and leaves. The CsALT was the key gene in the process from ammonia converted to ethylamine. The ethylamine showed high conversion rate to synthesize theanine in root (Deng et al., 2009). Then the theanine might act as an easily transported nitrogenous compound were transported to other tea plant tissues (Deng et al., 2012). The high expression level of CsALT in root was consistent with the high synthesis rate of theanine, and promoted indirectly the transportation of theanine.

In this study, some genes expression level showed positive correlations with theanine contents among different tea plant cultivars, which explained the potential reason of the tea plant cultivar contains high amount of theanine at molecular level. However, some other theanine metabolic related genes exhibited negative correlations among different tissues. Ashihara mentioned that the root of tea plant played important roles in theanine synthesis. The synthetic theanine is preferentially transported and accumulated in the tender tissues (Ashihara, 2015). In addition, the theanine in mature leaves were probably degraded more quickly than in young leaves. Tsushida and Takeo also demonstrated that the degradation products of theanine as substrates were used for the synthesis of other polyphenols and other amino acids (Tsushida and Takeo, 1985).

In addition, we can see the high similarity of expression profiles of CsGOGAT-Fe and CsGOGAT-NAD(P)H, CsADC and CsSAMDC, CsNR and CsNiR, CsPAO and CsCuAO, CsGGT1 and CsGGT3 among different parts. It may be deduced that they play synergistic roles in regulation of theanine metabolism. Therefore, the theanine metabolism is co-regulated through many related genes, which may apply synergistic or antagonistic effect. The tissue, cultivar, developmental stages, and growth environment also can influence the expression profiles of theanine metabolic related genes, the genes regulation of theanine metabolism became complicated. This study revealed the correlation between theanine content and genes expression to a certain extent, and provide support for the high theanine tea plant germplasm at gene level, as well as molecular theory foundation for quality improvement of tea plants.

JZ and ZL initiated and designed the research. ZL and JZ performed the experiments. ZL, ZW, HL, YW, and JZ analyzed the data. JZ contributed reagents, materials, analysis tools. ZL wrote the paper. JZ and ZL revised the paper.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.