Transcriptomic analysis of tea plant (Camellia sinensis) revealed the co-expression network of 4111 paralogous genes and biosynthesis of quality-related key metabolites under multiple stresses.

The tea plant is an essential economic plant in many countries. However, its growing season renders them vulnerable to stresses. To understand the transcriptomic influences of these stresses on tea plants, we sequenced and analyzed the transcriptomes under drought, high-temperature, and pest. Paralogs were identified by comparing 14 evolutionarily close genomes. The differentially expressed paralog (DEPs) genes were analyzed regarding single or multiple stresses, and 1075 of the 4111 DEPs were commonly found in all the stresses. The co-expression network of the DEPs and TFs indicated that genes of catechin biosynthesis were associated with most transcription factors specific to each stress. The genes playing a significant role in the late response to drought and pest stress mainly functioned in the early response to high-temperature. This study revealed the relationship between stress and regulation of QRM synthesis and the role of QRMs in response to these (a)biotic stresses.

Integrated transcriptomics and metabolomics analysis of catechins, caffeine and theanine biosynthesis in tea plant (Camellia sinensis) over the course of seasons.

BACKGROUND: Catechins, caffeine, and theanine as three important metabolites in the tea leaves play essential roles in the formation of specific taste and shows potential health benefits to humans. However, the knowledge on the dynamic changes of these metabolites content over seasons, as well as the candidate regulatory factors, remains largely undetermined. RESULTS: An integrated transcriptomic and metabolomic approach was used to analyze the dynamic changes of three mainly metabolites including catechins, caffeine, and theanine, and to explore the potential influencing factors associated with these dynamic changes over the course of seasons. We found that the catechins abundance was higher in Summer than that in Spring and Autumn, and the theanine abundance was significantly higher in Spring than that in Summer and Autumn, whereas caffeine exhibited no significant changes over three seasons. Transcriptomics analysis suggested that genes in photosynthesis pathway were significantly down-regulated which might in linkage to the formation of different phenotypes and metabolites content in the tea leaves of varied seasons. Fifty-six copies of nine genes in catechins biosynthesis, 30 copies of 10 genes in caffeine biosynthesis, and 12 copies of six genes in theanine biosynthesis were detected. The correlative analysis further presented that eight genes can be regulated by transcription factors, and highly correlated with the changes of metabolites abundance in tea-leaves. CONCLUSION: Sunshine intensity as a key factor can affect photosynthesis of tea plants, further affect the expression of major Transcription factors (TFs) and structural genes in, and finally resulted in the various amounts of catechins, caffeine and theaine in tea-leaves over three seasons. These findings provide new insights into abundance and influencing factors of metabolites of tea in different seasons, and further our understanding in the formation of flavor, nutrition and medicinal function.

Exploring plant metabolic genomics: chemical diversity, metabolic complexity in the biosynthesis and transport of specialized metabolites with the tea plant as a model.

The diversity and complexity of secondary metabolites in tea plants contribute substantially to the popularity of tea, by determining tea flavors and their numerous health benefits. The most significant characteristics of tea plants are that they concentrate the complex plant secondary metabolites into one leaf: flavonoids, alkaloids, theanine, volatiles, and saponins. Many fundamental questions regarding tea plant secondary metabolism remain unanswered. This includes how tea plants accumulate high levels of monomeric galloylated catechins, unlike the polymerized flavan-3-ols in most other plants, as well as how they are evolved to selectively synthesize theanine and caffeine, and how tea plants properly transport and store these cytotoxic products and then reuse them in defense. Tea plants coordinate many metabolic pathways that simultaneously take place in young tea leaves in response to both developmental and environmental cues. With the available genome sequences of tea plants and high-throughput metabolomic tools as great platforms, it is of particular interest to launch metabolic genomics studies using tea plants as a model system. Plant metabolic genomics are to investigate all aspects of plant secondary metabolism at the genetic, genome, and molecular levels. This includes plant domestication and adaptation, divergence and convergence of secondary metaboloic pathways. The biosynthesis, transport, storage, and transcriptional regulation mechanisms of all metabolites are of core interest in the plant as a whole. This review highlights relevant contexts of metabolic genomics, outstanding questions, and strategies for answering them, with aim to guide future research for genetic improvement of nutrition quality for healthier plant foods.

The Tea Tree Genome Provides Insights into Tea Flavor and Independent Evolution of Caffeine Biosynthesis.

Tea is the world's oldest and most popular caffeine-containing beverage with immense economic, medicinal, and cultural importance. Here, we present the first high-quality nucleotide sequence of the repeat-rich (80.9%), 3.02-Gb genome of the cultivated tea tree Camellia sinensis. We show that an extraordinarily large genome size of tea tree is resulted from the slow, steady, and long-term amplification of a few LTR retrotransposon families. In addition to a recent whole-genome duplication event, lineage-specific expansions of genes associated with flavonoid metabolic biosynthesis were discovered, which enhance catechin production, terpene enzyme activation, and stress tolerance, important features for tea flavor and adaptation. We demonstrate an independent and rapid evolution of the tea caffeine synthesis pathway relative to cacao and coffee. A comparative study among 25 Camellia species revealed that higher expression levels of most flavonoid- and caffeine- but not theanine-related genes contribute to the increased production of catechins and caffeine and thus enhance tea-processing suitability and tea quality. These novel findings pave the way for further metabolomic and functional genomic refinement of characteristic biosynthesis pathways and will help develop a more diversified set of tea flavors that would eventually satisfy and attract more tea drinkers worldwide.

Association mapping of caffeine content with TCS1 in tea plant and its related species.

Caffeine is the most abundant purine alkaloid in majority of tea plant and its related species. This purine alkaloid contributes to the important flavor and health attributes of tea. Tea caffeine synthase 1 (TCS1, EC 2.1.1.159/2.1.1.160) gene plays a crucial role in caffeine biosynthesis. The objective of this study was to investigate the genetic relationship between the TCS1 and caffeine content of tea plant and its related species using association mapping. We identified 87 single-nucleotide polymorphisms (SNPs, π = 0.00447) by resequencing the TCS1 locus of 44 tea accessions. Linkage disequilibrium (LD) analysis showed that LD did not extend over the entire gene (r(2) < 0.1, within 1000 bp). Two cleaved amplified polymorphism sequence (CAPS) markers were developed from sequence variations (SNP4318 and SNP6252). By association mapping, we identified SNP4318 associated with caffeine content in four environments, explaining 4.0%-7.7% of the phenotypic variance. We also validated the significant marker-trait associations in site-directed mutagenesis experiments. Examination of allelic variation and linkage disequilibrium by a candidate-gene-based approach can help to decipher the genetic basis of caffeine biosynthesis. Moreover, the SNP marker identified in this study can potentially be applied for future marker-assisted selection to improve tea quality.

Natural allelic variations of TCS1 play a crucial role in caffeine biosynthesis of tea plant and its related species.

Tea caffeine synthase 1 (TCS1) is an enzyme that catalyzes the methylation of N-3 and N-1 and considered to be the most critical enzyme in the caffeine biosynthetic pathway of tea plant. This study shows that TCS1 has six types of allelic variations, namely, TCS1a, TCS1b, TCS1c, TCS1d, TCS1e, and TCS1f, with a 252 bp insertion/deletion mutation in the 5'-untranslated region. Among tea plant and its related species, TCS1a is the predominant allele, and TCS1b-f are the rare alleles that mainly appear in few wild germplasms. The full-length cDNA sequences of three new alleles, namely, TCS1d, TCS1e, and TCS1f, were isolated from specific germplasms, and all of recombinant proteins have higher caffeine synthase (CS, EC 2.1.1.160) activity than theobromine synthase (TS, EC 2.1.1.159). Amino acid residue 269 is responsible for the difference in TCS activity and substrate recognition, which was demonstrated by using site-directed mutagenesis experiments. Furthermore, natural variations in TCS1 change the transcription levels. There are two molecular mechanisms controlling the caffeine biosynthesis in low-caffeine-accumulating tea germplasms, i.e., TCS1 allele with low transcription level or its encoded protein with only TS activity. Allelic variations of TCS1 play a crucial role in caffeine biosynthesis. Taken together, our work provides valuable foundation for a comprehensive understanding of the mechanism of caffeine biosynthesis in section Thea plants and useful guidance for effective breeding.

Global transcriptome and gene regulation network for secondary metabolite biosynthesis of tea plant (Camellia sinensis).

BACKGROUND: Major secondary metabolites, including flavonoids, caffeine, and theanine, are important components of tea products and are closely related to the taste, flavor, and health benefits of tea. Secondary metabolite biosynthesis in Camellia sinensis is differentially regulated in different tissues during growth and development. Until now, little was known about the expression patterns of genes involved in secondary metabolic pathways or their regulatory mechanisms. This study aimed to generate expression profiles for C. sinensis tissues and to build a gene regulation model of the secondary metabolic pathways. RESULTS: RNA sequencing was performed on 13 different tissue samples from various organs and developmental stages of tea plants, including buds and leaves of different ages, stems, flowers, seeds, and roots. A total of 43.7 Gbp of raw sequencing data were generated, from which 347,827 unigenes were assembled and annotated. There were 46,693, 8446, 3814, 10,206, and 4948 unigenes specifically expressed in the buds and leaves, stems, flowers, seeds, and roots, respectively. In total, 1719 unigenes were identified as being involved in the secondary metabolic pathways in C. sinensis, and the expression patterns of the genes involved in flavonoid, caffeine, and theanine biosynthesis were characterized, revealing the dynamic nature of their regulation during plant growth and development. The possible transcription factor regulation network for the biosynthesis of flavonoid, caffeine, and theanine was built, encompassing 339 transcription factors from 35 families, namely bHLH, MYB, and NAC, among others. Remarkably, not only did the data reveal the possible critical check points in the flavonoid, caffeine, and theanine biosynthesis pathways, but also implicated the key transcription factors and related mechanisms in the regulation of secondary metabolite biosynthesis. CONCLUSIONS: Our study generated gene expression profiles for different tissues at different developmental stages in tea plants. The gene network responsible for the regulation of the secondary metabolic pathways was analyzed. Our work elucidated the possible cross talk in gene regulation between the secondary metabolite biosynthetic pathways in C. sinensis. The results increase our understanding of how secondary metabolic pathways are regulated during plant development and growth cycles, and help pave the way for genetic selection and engineering for germplasm improvement.

Occurrence and de novo biosynthesis of caffeine and theanine in seedlings of tea (Camellia sinensis).

Caffeine (1,3,7-trimethyl xanthine) and theanine (γ-glutamyl-L-ethylamide) are the major nitrogen-containing secondary metabolites in tea leaves. The aim of the present study was to elucidate the relative concentration and amounts of these compounds and the de novo biosynthetic activity in different parts of tea seedlings grown for 27-, 106- and 205 days. The results indicated that caffeine and its biosynthetic activity occur only in leaves and stems, while theanine is distributed in all organs, including roots. The concentration of caffeine and theanine in leaves ranged from 0.3-1.1 mg N/g and 0.1-0.5 mg N/g fresh weight, respectively. A higher concentration of theanine was found in roots (0.5-1.1 mg N). The total amounts of theanine expressed as g N/seedling were 1.1-1.5 times higher than that of caffeine. The high biosynthetic activity of caffeine from NH4+ was found in young leaves during the first 106 days after germination. Theanine biosynthetic activity probably occurs in roots, since higher 15N atom% excess was observed in roots during the first 27 days. Theanine may be synthesized mainly in roots and translocated to leaves. The de novo biosynthesis of caffeine and theanine in tea seedlings and their accumulation and translocation are discussed.

Revisiting caffeine biosynthesis--speculations about the proximate source of its purine ring.

The prevailing hypothesis of caffeine biosynthesis starting from xanthosine was combined with Kremers' speculation on NAD as a biochemical precursor of caffeine and trigonelline in coffee. This bold sketch together with a few free-spirited ideas may channel future caffeine biosynthesis studies into novel directions.

Biosynthesis of caffeine underlying the diversity of motif B' methyltransferase.

Caffeine (1,3,7-trimethylxanthine) and theobromine (3,7-dimethylxanthine) are well-known purine alkaloids in Camellia, Coffea, Cola, Paullinia, Ilex, and Theobroma spp. The caffeine biosynthetic pathway depends on the substrate specificity of N-methyltransferases, which are members of the motif B' methyl-transferase family. The caffeine biosynthetic pathways in purine alkaloid-containing plants might have evolved in parallel with one another, consistent with different catalytic properties of the enzymes involved in these pathways.

Transcriptomic analysis of Camellia ptilophylla and identification of genes associated with flavonoid and caffeine biosynthesis.

Camellia ptilophylla, or cocoa tea, is naturally decaffeinated and its predominant catechins and purine alkaloids are trans-catechins and theobromine Regular tea [Camellia sinensis (L.) O. Ktze.] is evolutionarily close to cocoa tea and produces cis-catechins and caffeine. Here, the transcriptome of C. ptilophylla was sequenced using the 101-bp paired-end technique. The quality of the raw data was assessed to yield 70,227,953 cleaned reads totaling 7.09 Gbp, which were assembled de novo into 56,695 unique transcripts and then clustered into 44,749 unigenes. In catechin biosynthesis, leucoanthocyanidin reductase (LAR) catalyzes the transition of leucoanthocyanidin to trans-catechins, while anthocyanidin synthase (ANS) and anthocyanidin reductase (ANR) catalyze cis-catechin production. Our data demonstrate that two LAR genes (CpLAR1 and CpLAR2) by C. ptilophylla may be advantageous due to the combined effects of this quantitative trait, permitting increased leucoanthocyanidin consumption for the synthesis of trans-catechins. In contrast, the only ANS gene observed in C. sinensis (CsANS) shared high identity (99.2%) to one homolog from C. ptilophylla (CpANS1), but lower identity (~80%) to another (CpANS2). We hypothesized that the diverged CpANS2 might have lost its ability to synthesize cis-catechins. C. ptilophylla and C. sinensis each contain two copies of ANR, which share high identity and may share the same function. Transcriptomic sequencing captured two N-methyl nucleosidase genes named NMT1 and NMT2. NMT2 was highly identical to three orthologous genes TCS2, PCS2, and ICS2, which did not undergo methylation in vitro; in contrast, NMT1 was less identical to TCS, PCS and ICS, indicating that NMT1 may undergo neofunctionalization.

Producing low-caffeine tea through post-transcriptional silencing of caffeine synthase mRNA.

In this study, attempt has been made to produce a selected cultivar of tea with low-caffeine content using RNAi technology. The caffeine biosynthetic pathway in tea has been proposed to involve three N-methyltransferases such as xanthosine methyltransferase, 7-N-methylxanthine methyltransferase and 3, 7-dimethylxanthine methyltransferase. Last two steps of caffeine biosynthesis in tea have been known to be catalyzed by a bifunctional enzyme known as caffeine synthase. To suppress the caffeine synthesis in the selected tea [Camellia sinensis (L.) O. Kuntze] cv. Kangra jat, we isolated a partial fragment of caffeine synthase (CS) from the same cultivar and used to design RNAi construct (pFGC1008-CS). Somatic embryos were transformed with the developed construct using biolistic method. Transformed somatic embryos showed reduction in the levels of CS transcript expression as well as in caffeine content. Plants were regenerated from the transformed somatic embryos. Transgenic plants showed a significant suppression of CS transcript expression and also showed a reduction of 44-61% in caffeine and 46-67% in theobromine contents as compared to the controls. These results suggest that the RNAi construct developed here using a single partial fragment of CS gene reduced the expression of the targeted endogenous gene significantly. However, the reduction in theobromine content in addition to caffeine documented the involvement of this single CS in the catalysis of last two methyl transfer steps in caffeine biosynthesis of tea.

Agrobacterium-mediated silencing of caffeine synthesis through root transformation in Camellia sinensis L.

Tea [Camellia sinensis (L.) O. Kuntze] is a perennial and most popular non-alcoholic caffeine-containing beverage crop. Tea has several constraints for its genetic improvement such as its high polyphenolic content and woody perennial nature. The development of transgenic tea is very difficult, laborious, and time taking process. In tea, regeneration requires minimum 8-12 months. In view of this, attempt has been made in this article to develop a rapid, efficient, and quite economical Agrobacterium-mediated root transformation system for tea. The feasibility of the developed protocol has been documented through silencing caffeine biosynthesis. For this, one-month-old tea seedlings were exposed to fresh wounding at the elongation zone of roots and were inoculated with Agrobacterium tumefaciens cultures carrying a RNAi construct (pFGC1008-CS). The pFGC1008-CS contained 376 bp of caffeine synthase (CS) cDNA fragment in sense and antisense direction with an intron in between. This has made the RNAi construct to produce a hairpin RNA (ihpRNA). The suppressed expression of CS gene and a marked reduction in caffeine and theobromine contents in young shoots of tea seedlings were obtained after root transformation through Agrobacterium infiltration. Such transformation system could be useful for functional analysis of genes in tea like woody and perennial plants.

Essential region for 3-N methylation in N-methyltransferases involved in caffeine biosynthesis.

The caffeine biosynthetic pathway is composed of three methylation steps, and N-methyltransferase catalyzing each step has high substrate specificity. Since the amino acid sequences among coffee 7-methylxanthosine synthase (CmXRS1), theobromine synthase, and caffeine synthase are highly homologous to each other, these substrate specificities seem to be determined in a very restricted region. The analysis of site-directed mutants for CmXRS1 that naturally acts at the initial step, i.e., 7-N methylation of xanthosine, revealed that the activity of 3-N methylation needs a histidine residue at corresponding position 161 in the CmXRS1 sequence. We succeeded in producing the mutant enzyme which can catalyze the first and second methylation steps in caffeine biosynthesis.

Expression for caffeine biosynthesis and related enzymes in Camellia sinensis.

Caffeine (1,3,7-trimethylxanthine) is a purine alkaloid that is present in high concentrations in the tea plant Camellia sinensis. Caffeine synthase (CS, EC 2.1.1.160) catalyzes the S-adenosyl-L-methionine-dependent N-3- and N-1-methylation of the purine base to form caffeine, the last step in the purine alkaloid biosynthetic pathway. We studied the expression profile of the tea caffeine synthase (TCS) gene in developing leaves and flowers by means of northern blot analysis, and compared it with those of phenylalanine ammonia lyase (PAL, EC 4.3.1.5), chalcone synthase (CHS, EC 2.3.1.74), and S-adenosyl-L-methionine synthase (SAMS, EC 2.5.1.6). The amount of TCS transcripts was highest in young leaves and declined markedly during leaf development, whereas it remained constant throughout the development of the flower. Environmental stresses other than heavy metal stress and plant hormone treatments had no effect on the expression of TCS genes, unlike the other three genes. Drought stress suppressed TCS gene expression in leaves, and the expression pattern mirrored that of the dehydrin gene. The amounts of TCS transcripts increased slightly on supply of a nitrogen source. We discuss the regulation of TCS gene expression.

Caffeine biosynthesis and degradation in tea [Camellia sinensis (L.) O. Kuntze] is under developmental and seasonal regulation.

To study caffeine biosynthesis and degradation, here we monitored caffeine synthase gene expression and caffeine and allantoin content in various tissues of four Camellia sinensis (L.) O. Kuntze cultivars during non-dormant (ND) and dormant (D) growth phases. Caffeine synthase expression as well as caffeine content was found to be higher in commercially utilized tissues like apical bud, 1st leaf, 2nd leaf, young stem, and was lower in old leaf during ND compared to D growth phase. Among fruit parts, fruit coats have higher caffeine synthase expression, caffeine content, and allantoin content. On contrary, allantoin content was found lower in the commercially utilized tissues and higher in old leaf. Results suggested that caffeine synthesis and degradation in tea appears to be under developmental and seasonal regulation.

Expression of caffeine biosynthesis genes in tea (Camellia sinensis).

Using semi-quantitative reverse transcription-PCR, we studied the expression of genes encoding caffeine synthase (TCS1), inosine-5'-monophosphate dehydrogenase (TIDH), S-adenosyl-L-methionine synthase (sAMS), phenylalanine ammonia-lyase (PAL) and alpha-tubulin (Tua1) in young and mature leaves, stems and roots of 4-month-old tea seedlings and young and old tea tissue cultures. The amounts of transcripts of TCS1 were much higher in young leaves than in other parts of the plant. Expression of TIDH was greater in leaves than in other parts. Little difference in the amounts of transcripts of PAL, sAMS and Tua1 was found between various organs of tea seedlings. Larger amounts of transcripts of TCS1 and PAL were found in young callus tissues than in old tissues. These results support our conclusion deriving from previous enzymatic and metabolic studies that caffeine is synthesized mainly in young leaf tissues. We propose that marked caffeine biosynthesis in young leaves is dependent on a greater expression of the TCS1 gene in the organ.

Towards generating caffeine-free tea by metabolic engineering.

Tea is a rich source of antioxidants which are contributing substantially to the promotion of health and the prevention of various chronic diseases. Despite the fact that tea has various important compounds, it also contains a purine alkaloid, caffeine. High intake of tea leads to an increase in level of caffeine in addition to its important antioxidant constituents. Increased level of caffeine causes several health related problems. Therefore, tea can become a most useful source of beneficial compounds, if only its caffeine level is either decreased or eliminated all together from the plant itself. This could be achieved through either of the techniques; overexpressing caffeine degradative pathway genes or silencing caffeine biosynthesis pathway gene. The identification and cloning of caffeine biosynthesis in tea and degradative genes in microorganisms opens up the possibility of using genetic engineering to produce naturally decaffeinated tea. Here we review these different strategies which can be employed to make caffeine-free tea, a human health beneficial drink.