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Abstract Background Plants have evolved a panoply of specialized metabolites that increase their environmental fitness. Two examples are caffeine, a purine psychotropic alkaloid, and crocins, a group of glycosylated apocarotenoid pigments. Both classes of compounds are found in a handful of distantly related plant genera (Coffea, Camellia, Paullinia, and Ilex for caffeine; Crocus, Buddleja, and Gardenia for crocins) wherein they presumably evolved through convergent evolution. The closely related Coffea and Gardenia genera belong to the Rubiaceae family and synthesize, respectively, caffeine and crocins in their fruits. Results Here, we report a chromosomal-level genome assembly of Gardenia jasminoides, a crocin-producing species, obtained using Oxford Nanopore sequencing and Hi-C technology. Through genomic and functional assays, we completely deciphered for the first time in any plant the dedicated pathway of crocin biosynthesis. Through comparative analyses with Coffea canephora and other eudicot genomes, we show that Coffea caffeine synthases and the first dedicated gene in the Gardenia crocin pathway, GjCCD4a, evolved through recent tandem gene duplications in the two different genera, respectively. In contrast, genes encoding later steps of the Gardenia crocin pathway, ALDH and UGT, evolved through more ancient gene duplications and were presumably recruited into the crocin biosynthetic pathway only after the evolution of the GjCCD4a gene. Conclusions This study shows duplication-based divergent evolution within the coffee family (Rubiaceae) of two characteristic secondary metabolic pathways, caffeine and crocin biosynthesis, from a common ancestor that possessed neither complete pathway. These findings provide significant insights on the role of tandem duplications in the evolution of plant specialized metabolism.

While caffeine biosynthetic pathways have been extensively studied in tea ( I Camellia sinensis i L) and coffee plants, the regulation of caffeine biosynthesis is not understood (Zhao I et al i ., 2020). Keywords: caffeine biosynthesis; MYB factor; regulation; Thea plants EN caffeine biosynthesis MYB factor regulation Thea plants 1012 1014 3 05/26/22 20220601 NES 220601 As the most well-known and globally consumed central nervous system stimulant, caffeine is a purine alkaloid natural product usually derived from tea and coffee. [Extracted from the article]

Background: Plants have evolved a panoply of specialized metabolites that increase their environmental fitness. Two examples are caffeine, a purine psychotropic alkaloid, and crocins, a group of glycosylated apocarotenoid pigments. Both classes of compounds are found in a handful of distantly related plant genera (Coffea, Camellia, Paullinia, and Ilex for caffeine; Crocus, Buddleja, and Gardenia for crocins) wherein they presumably evolved through convergent evolution. The closely related Coffea and Gardenia genera belong to the Rubiaceae family and synthesize, respectively, caffeine and crocins in their fruits. Results: Here, we report a chromosomal-level genome assembly of Gardenia jasminoides, a crocin-producing species, obtained using Oxford Nanopore sequencing and Hi-C technology. Through genomic and functional assays, we completely deciphered for the first time in any plant the dedicated pathway of crocin biosynthesis. Through comparative analyses with Coffea canephora and other eudicot genomes, we show that Coffea caffeine synthases and the first dedicated gene in the Gardenia crocin pathway, GjCCD4a, evolved through recent tandem gene duplications in the two different genera, respectively. In contrast, genes encoding later steps of the Gardenia crocin pathway, ALDH and UGT, evolved through more ancient gene duplications and were presumably recruited into the crocin biosynthetic pathway only after the evolution of the GjCCD4a gene. Conclusions: This study shows duplication-based divergent evolution within the coffee family (Rubiaceae) of two characteristic secondary metabolic pathways, caffeine and crocin biosynthesis, from a common ancestor that possessed neither complete pathway. These findings provide significant insights on the role of tandem duplications in the evolution of plant specialized metabolism. [ABSTRACT FROM AUTHOR]

Main conclusion: The roles of microRNA-mediated epigenetic regulation were highlighted in the bud dormancy–activity cycle, implying that certain differentially expressed miRNAs play crucial roles in apical bud burst, such as csn-miR319c/TCP2. microRNAs (miRNAs) are a class of small non-coding RNAs that regulate gene expression by targeting mRNA transcripts for cleavage or directing translational inhibition. To investigate whether miRNAs regulate bud dormancy–activation transition in tea plant, which largely affects the yield and price of tea products and adaptability of tea trees, we constructed small RNA libraries from three different periods of bud dormancy–burst transition. Through sequencing analysis, 262 conserved and 83 novel miRNAs were identified, including 118 differentially expressed miRNAs. Quantitative RT-PCR results for randomly selected miRNAs exhibited that our comprehensive analysis is highly reliable and accurate. The content of caffeine increased continuously from the endodormancy bud to flushing bud, and differentially expressed miRNAs coupling with their targets associated with bud burst were identified. Remarkably, csn-miR319c was downregulated significantly from the quiescent bud to burst bud, while its target gene CsnTCP2 (TEOSINTE BRANCHED/CYCLOIDEA/PROLIFERATING CELL FACTOR 2) displayed opposite expression patterns. Co-transformation experiment in tobacco demonstrated that csn-miR319c can significantly suppress the functions of CsnTCP2. This study on miRNAs and the recognition of target genes could provide new insights into the molecular mechanism of the bud dormancy–activation transition in tea plant. [ABSTRACT FROM AUTHOR]

Purine bases and nucleosides are produced by turnover of nucleotides and nucleic acids as well as from some cellular metabolic pathways. Adenosine released from the S -adenosyl-L-methionine cycle is linked to many methyltransferase reactions, such as the biosynthesis of caffeine and glycine betaine. Adenine is produced by the methionine cycles, which is related to other biosynthesis pathways, such those for the production of ethylene, nicotianamine and polyamines. These purine compounds are recycled for nucleotide biosynthesis by so-called “salvage pathways”. However, the salvage pathways are not merely supplementary routes for nucleotide biosynthesis, but have essential functions in many plant processes. In plants, the major salvage enzymes are adenine phosphoribosyltransferase (EC 2.4.2.7) and adenosine kinase (EC 2.7.1.20). AMP produced by these enzymes is converted to ATP and utilised as an energy source as well as for nucleic acid synthesis. Hypoxanthine, guanine, inosine and guanosine are salvaged to IMP and GMP by hypoxanthine/guanine phosphoribosyltransferase (EC 2.4.2.8) and inosine/guanosine kinase (EC 2.7.1.73). In contrast to de novo purine nucleotide biosynthesis, synthesis by the salvage pathways is extremely favourable, energetically, for cells. In addition, operation of the salvage pathway reduces the intracellular levels of purine bases and nucleosides which inhibit other metabolic reactions. The purine salvage enzymes also catalyse the respective formation of cytokinin ribotides, from cytokinin bases, and cytokinin ribosides. Since cytokinin bases are the active form of cytokinin hormones, these enzymes act to maintain homeostasis of cellular cytokinin bioactivity. This article summarises current knowledge of purine salvage pathways and their possible function in plants and purine salvage activities associated with various physiological phenomena are reviewed. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Caffeine biosynthesis involves sequential methylation of the purine ring by the transfer of methyl group from the methyl donor S-adenosyl methionine. Genes that code for the individual methyl transferase have a high degree of sequence homology. To achieve decaffeination by genetic engineering of coffee, the effect and efficiency of different post-transcriptional gene silencing (PTGS) constructs were evaluated. Sense, antisense and invert repeat constructs were developed using a 339 bp fragment from the conserved region of the coffee N-methyltransferase genes involved in caffeine biosynthesis. The silencing constructs were mobilized into Agrobacterium tumefaciens (EHA101). Sonication assisted Agrobacterium-mediated transformation of the somatic embryos of C.canephora was performed, followed by regeneration of the transformants. Molecular analysis and identification of flanking regions confirmed the stable integration of the transgenes in the transformants and their integration in non coding regions of the genome, respectively. Effectiveness of the silencing constructs was evaluated by transcript analysis of the targeted genes by RT-PCR and northern blotting. The silencing efficiency was further evaluated by estimation of the purine alkaloid content in the transgenic lines using HPLC. The three constructs differed in their silencing efficiencies and specificity. Though the constructs were not specific to a single N-methyltransferase, transformants obtained were mainly affected in one of the N-methyltransferases. Results show that the use of homologous coding sequence in the PTGS constructs results in a much higher efficiency in silencing the caffeine biosynthetic pathway. Further, the lowering of caffeine content should be the preferred over complete decaffeination due to extremely low survival rate of transgenic plants with extremely low levels of caffeine. [ABSTRACT FROM AUTHOR]

Abstract: There are numerous reports on intronic miRNAs from plants, most of which are involved in the regulation of unrelated genes. Some of the target genes are antagonistic to the host genes. Intronic miRNAs in animal systems, however, are known to have synergistic effects. This article is the first to report a similar regulatory effect of a miRNA originating from an intron in plants. NMT genes involved in caffeine biosynthesis were silenced to obtain transformants with reduced caffeine. Transcript analysis revealed the accumulation of transcripts for a related NMT gene (CaMTL1) in transformants bearing either antisense or RNAi constructs. The altered expression was assumed to relate to the silencing of the NMT genes. Bioinformatics analysis of the genes involved in biosynthesis revealed the presence of an intronic miRNA originating from the intron of the theobromine synthase gene targeting CaMTL1. The putative miRNA was cloned and sequenced. Modified 5′-RLM-RACE mapping of the cleavage site and subsequent Northern blotting experimentally demonstrated the presence and activity of such a miRNA in Coffea canephora. This novel regulatory mechanism previously unreported in plants will shed more light onto the evolution of multigene families and the role of introns in this process. [Copyright &y& Elsevier]

The GCAs are new tetraploid interspecific hybrids developed in Madagascar from Coffea eugenioides, C. canephora and C. arabica. Selected GCA having genotype UF1023 contained 0.37% DW caffeine and no detectable theobromine in green beans. Low caffeine accumulation in GCA plants is due mainly to the low biosynthetic activity of purine alkaloids, possibly the extremely weak N-methyltransferase reactions in caffeine biosynthesis. No significant catabolic activity of caffeine was found in GCA-UF1023, in common with almost all coffee plants including C. arabica. [ABSTRACT FROM AUTHOR]

Abstract: Caffeine is synthesized from the precursor xanthosine through three methylation and one nucleoside removal steps. Methylation is catalyzed by N-methyltransferases, designated as CaXMT1, CaMXMT1 and CaDXMT1, which, respectively, convert xanthosine into 7-methylxanthosine, 7-methylxanthine into 3,7-dimethylxanthine, and 3,7-dimethylxanthine into 1,3,7-trimethylxanthine (caffeine). In the present study, we examined their cytological and biochemical properties using fusion proteins with fluorescent proteins. All three enzymes were found to localize in cytosol as visualized by green fluorescence protein fusions. The possibility of dimer formation among these enzyme proteins was examined in vivo by transient expression of bimolecular fluorescence complementation of yellow fluorescent protein (YFP) using onion epidermal cell layers. Results showed that each enzyme protein formed a homo-dimer in cytosol as seen by a clear reconstituted YFP fluorescence. In addition, each enzyme also formed a hetero-dimer with each of the other two enzymes in cytosol. The biological significance of dimerization among structurally resembling methyltransferases involved in caffeine biosynthesis is discussed. [Copyright &y& Elsevier]

Abstract: In anti-sense and RNA interference transgenic plants of Coffea canephora in which the expression of CaMXMT1 was suppressed, caffeine biosynthesis from [8-14C]adenine was investigated, together with the overall metabolism of [8-14C]adenine. Compared with wild type control plants, total purine alkaloid biosynthesis from adenine and conversion of theobromine to caffeine were both reduced in the transgenic plants. As found previously, [8-14C]adenine was metabolised to salvage products (nucleotides and RNA), to degradation products (ureides and CO2) and to purine alkaloids (theobromine and caffeine). In the transgenic plants, metabolism of [8-14C]adenine shifted from purine alkaloid synthesis to purine catabolism or salvage for nucleotides. HPLC analysis revealed a significantly reduced caffeine content in the transgenic plants. A small quantity (less than 20nmolg−1 fresh weight) of xanthosine had accumulated in at least one of the transgenic plants. [Copyright &y& Elsevier]

The aim of this study was to investigate the S-adenosylmethionine dependent N-methyltransferase(s) (NMT) associated with the three methylation steps in the caffeine biosynthesis pathway in tea (Camellia sinensis L.). NMT activity in cell-free preparations from young leaves was purified by anion-exchange and gel-filtration column chromatography. In both systems, a single zone of NMT activity, with broad substrate specificity was detected. The N-3 position of dimethylxanthine and monomethylxanthines was methylated more readily than N-1 while comparatively little substitution occurred at the N-7 locus. When xanthosine was used as a substrate only the N-7 position was methylated. These results indicate that a single NMT may participate in the conversion of xanthosine to caffeine. The apparent Mr of the NMT, estimated by gel filtration chromatography, was 61 000. The substrate specificity of the NMT is compatible with the operation of a xanthosine → 7-methylxanthosine → 7-methylxanthine → theobromine → caffeine pathway as the main biosynthetic route to caffeine in young tea leaves. The data also indicate that the conversion of 7-methylxanthine → paraxanthine → caffeine may function as one of a number of minor pathways that also contribute, to the production of caffeine. [ABSTRACT FROM AUTHOR]

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., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Caffeine (Cf) is one of the important components of plant-derived drinks, such as tea, coffee, and cola. It can protect soft tissues from being infected by pathogens and is also medically beneficial for human health. In this review, we first introduced the Cf biosynthesis pathways in plants and the related N -methyltransferases (NMTs), with a focus on the current research status of the substrate specificity, structural basis for substrate recognition, and catalytic mechanism in members of the caffeine synthase gene family. In addition, we addressed the expression characteristics and potential regulatory mechanisms of NMTs and also projected the future research directions. The goal was to summarize the Cf biosynthetic pathway and related NMTs in plants and to provide the molecular basis for regulating the caffeine biosynthesis, so as to effectively guide future tea and coffee breeding.

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.

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.

Engineered microbial biosynthesis of plant natural products can support manufacturing of complex bioactive molecules and enable discovery of non-naturally occurring derivatives. Purine alkaloids, including caffeine (coffee), theophylline (antiasthma drug), theobromine (chocolate), and other methylxanthines, play a significant role in pharmacology and food chemistry. Here, we engineered the eukaryotic microbial host Saccharomyces cerevisiae for the de novo biosynthesis of methylxanthines. We constructed a xanthine-to-xanthosine conversion pathway in native yeast central metabolism to increase endogenous purine flux for the production of 7-methylxanthine, a key intermediate in caffeine biosynthesis. Yeast strains were further engineered to produce caffeine through expression of several enzymes from the coffee plant. By expressing combinations of different N-methyltransferases, we were able to demonstrate re-direction of flux to an alternate pathway and develop strains that support the production of diverse methylxanthines. We achieved production of 270μg/L, 61μg/L, and 3700μg/L of caffeine, theophylline, and 3-methylxanthine, respectively, in 0.3-L bench-scale batch fermentations. The constructed strains provide an early platform for de novo production of methylxanthines and with further development will advance the discovery and synthesis of xanthine derivatives., Competing Interests: statement The authors declare competing financial interests in the form of a pending patent application., (Copyright © 2016 International Metabolic Engineering Society. Published by Elsevier Inc. All rights reserved.)

S-Adenosyl-l-methionine (SAM) dependent xanthosine methyltransferase (XMT) is the key enzyme that catalyzes the first methyl transfer in the caffeine biosynthesis pathway to produce the intermediate 7-methylxanthosine (7mXR). Although XMT has been a subject of extensive discussions, the catalytic mechanism and nature of the substrate involved in the catalysis are still unclear. In this paper, quantum mechanical/molecular mechanical (QM/MM) molecular dynamics (MD) and free energy (potential of mean force or PMF) simulations are undertaken to determine the catalytic mechanism of the XMT-catalyzed reaction. Both xanthosine and its monoanionic form with N3 deprotonated are used as the substrates for the methylation. It is found that while the methyl group can be transferred to the monoanionic form of xanthosine with a reasonable free energy barrier (about 17 kcal/mol), that is not the case for the neutral xanthosine. The results suggest that the substrate for the first methylation step in the caffeine biosynthesis pathway is likely to be the monoanionic form of xanthosine rather than the neutral form as widely adopted. This conclusion is supported by the pKa value on N3 of xanthosine both measured in aqueous phase and calculated in the enzymatic environment. The structural and dynamics information from both the X-ray structure and MD simulations is also consistent with the monoanionic xanthosine scenario. The implications of this conclusion for caffeine biosynthesis are discussed.

Convergent evolution is a process that has occurred throughout the tree of life, but the historical genetic and biochemical context promoting the repeated independent origins of a trait is rarely understood. The well-known stimulant caffeine, and its xanthine alkaloid precursors, has evolved multiple times in flowering plant history for various roles in plant defense and pollination. We have shown that convergent caffeine production, surprisingly, has evolved by two previously unknown biochemical pathways in chocolate, citrus, and guaraná plants using either caffeine synthase- or xanthine methyltransferase-like enzymes. However, the pathway and enzyme lineage used by any given plant species is not predictable from phylogenetic relatedness alone. Ancestral sequence resurrection reveals that this convergence was facilitated by co-option of genes maintained over 100 million y for alternative biochemical roles. The ancient enzymes of the Citrus lineage were exapted for reactions currently used for various steps of caffeine biosynthesis and required very few mutations to acquire modern-day enzymatic characteristics, allowing for the evolution of a complete pathway. Future studies aimed at manipulating caffeine content of plants will require the use of different approaches given the metabolic and genetic diversity revealed by this study., Competing Interests: The authors declare no conflict of interest.

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., (Copyright © 2016 Elsevier Masson SAS. All rights reserved.)

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., (Copyright © 2016 Elsevier Masson SAS. All rights reserved.)

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.

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.

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.

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.

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.

Coffee is a valuable beverage crop due to its characteristic flavor, aroma, and the stimulating effects of caffeine. We generated a high-quality draft genome of the species Coffea canephora, which displays a conserved chromosomal gene order among asterid angiosperms. Although it shows no sign of the whole-genome triplication identified in Solanaceae species such as tomato, the genome includes several species-specific gene family expansions, among them N-methyltransferases (NMTs) involved in caffeine production, defense-related genes, and alkaloid and flavonoid enzymes involved in secondary compound synthesis. Comparative analyses of caffeine NMTs demonstrate that these genes expanded through sequential tandem duplications independently of genes from cacao and tea, suggesting that caffeine in eudicots is of polyphyletic origin., (Copyright © 2014, American Association for the Advancement of Science.)

The caffeine biosynthetic pathway is of considerable importance for the beverage and pharmaceutical industries which produces two blockbuster products: theobromine and caffeine. The major biochemistry in caffeine biosynthesis starts from the initial substrate of xanthosine and ends with the final product caffeine, with theobromine serving as an intermediate. The key enzyme, S-adenosyl-l-methionine (SAM) dependent 3,7-dimethyl-xanthine methyltransferase (DXMT), catalyzes two important methyl transfer steps in caffeine biosynthesis: (1) methylation of N3 of 7-methylxanthine (7mX) to form theobromine (Tb); (2) methylation of N1 of theobromine to form caffeine (Cf). Although DXMT has been structurally characterized recently, our understanding of the detailed catalytic mechanism and role of key catalytic residues is still lacking. In this work, the quantum mechanical/molecular mechanical (QM/MM) MD and free energy simulations are performed to elucidate the catalytic mechanism of the enzyme-catalyzed reactions and to explain experimental observations concerning the activity of this enzyme. The roles of certain active-site residues are studied, and the results of computer simulation seem to suggest that a histidine residue (His160) at the active site of DXMT may act as a general base/acid catalyst during the methyl transfer process.

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.

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.

Caffeine functions in the chemical defense against biotic attackers in a few plant species, including coffee and tea. Transgenic tobacco plants that endogenously produced caffeine by expressing three N-methyltransferases involved in the caffeine biosynthesis pathway exhibited a strong resistance to pathogens and herbivores. Here we report that transgenic Chrysanthemum, which produced an equivalent level of caffeine as the tobacco plants at approximately 3 μg g(-1) fresh tissues, also exhibited a resistance against grey mold fungal attack. Transcripts of PR-2 gene, a marker for pathogen response, were constitutively accumulated in mature leaves without pathogen attack. The levels of salicylic acid and its glucoside conjugate in mature leaves of the transgenic lines were found to be 2.5-fold higher than in the wild type control. It is suggested that endogenous caffeine stimulated production and/or deposition of salicylates, which possibly activated a series of defense reactions even under non-stressed conditions.

Plants produce up to 100,000 secondary metabolites. One of their biological functions is self-denfese, and it is referred as chemical defense, directly and/or indirectly counteracting biotic and abiotic stresses. Alkaloids constitute 12% of total secondary metabolites, and some of them exhibit detrimental effects on living organisms. Caffeine (1,3,7-trimethylxanthine) is a member of purine alkaloids, and its exogenous application to plants at relatively high concentrations (0.01-0.1%) effectively repelled herbivores and pathogenic microbes. This allowed the construction of transgenic crops that endogenously produce caffeine to tolerate stresses. Experimentally, tobacco and chrysanthemum were successfully transformed with three distinct N-methyltranferases involved in the caffeine biosynthesis pathway. They produced 0.4-5 mug caffeine/g tissue (5 x 10(-4)%), this being three magnitudes lower than values found in caffeine-producing plants and in vitro experiments. Nevertheless, they exhibited strong repellence against pest insects, and high resistance to virus and bacterial infection. They also exhibited accelerated self-defense, as estimated by constitutive expression of defense-related genes, and by elevated production of salicylic acid, a critical signaling molecule for defense response. Since caffeine content was low in transgenic lines, observed effects might not be direct, but rather indirect. We presume that, as endogenously produced caffeine could be toxic, the host plants activated its own self-defense system, which commonly occurs regarding other stresses. Eventually the host became on standby to cope with a broad range of biotic stresses. The procedure resembles mammalian vaccination, in which antigen-antibody system is critical. We propose that plants can also be vaccinated as far as proper "antigenic" chemicals are expressed in planta.

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.

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.

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.

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.

Details of the recently elucidated biosynthetic pathways of caffeine and related purine alkaloids are reviewed. The main caffeine biosynthetic pathway is a sequence consisting of xanthosine-->7-methylxanthosine-->7-methylxanthine-->theobromine-->caffeine. Genes encoding N-methyltransferases involved in three of these four reactions have been isolated and the molecular structure of N-methyltransferases investigated. Pathways for the catabolism of caffeine have also been studied, although there are currently no reports of enzymatic and genetic studies having been successfully carried out. Metabolism of purine alkaloids in species including Camellia, Coffea, Theobroma and Ilex plants is summarised, and evidence for the involvement of caffeine in chemical defense and allelopathy is discussed. Finally, information is presented on metabolic engineering that has produced coffee seedlings with reduced caffeine content, and transgenic caffeine-producing tobacco plants with enhanced disease resistance.

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.

Caffeine (1,3,7-trimethylxanthine) is a secondary metabolite produced by certain plant species and an important component of coffee (Coffea arabica and Coffea canephora) and tea (Camellia sinensis). Here we describe the structures of two S-adenosyl-l-methionine-dependent N-methyltransferases that mediate caffeine biosynthesis in C. canephora 'robusta', xanthosine (XR) methyltransferase (XMT), and 1,7-dimethylxanthine methyltransferase (DXMT). Both were cocrystallized with the demethylated cofactor, S-adenosyl-L-cysteine, and substrate, either xanthosine or theobromine. Our structures reveal several elements that appear critical for substrate selectivity. Serine-316 in XMT appears central to the recognition of XR. Likewise, a change from glutamine-161 in XMT to histidine-160 in DXMT is likely to have catalytic consequences. A phenylalanine-266 to isoleucine-266 change in DXMT is also likely to be crucial for the discrimination between mono and dimethyl transferases in coffee. These key residues are probably functionally important and will guide future studies with implications for the biosynthesis of caffeine and its derivatives in plants.

The new generic and species name Amorosia littoralis gen. sp. nov. is introduced for the conidial dematiaceous hyphomycete isolated from the littoral zone in The Bahamas and reported in 2001 to produce the novel aza-anthraquinone scorpinone, and also caffeine. No satisfactory generic placement was found at the time, but subsequent morphological and molecular investigations reveal that a new generic name is required. The new genus has some similarity to several fungi described in Trichocladium, but differs substantially from the type species of that genus in the form of the conidia and the lack of ornamentation. BLAST studies using the 18S and 28S rDNA gene sequences place the new genus in the Sporormiaceae. In addition to the morphological studies, an ultrastructural examination of the conspicuous porate septa of hyphae showed that they do not belong to a basidiomycete.

Caffeine (1,3,7-trimethylxanthine) is one of the most widely used plant secondary metabolites, primarily as a stimulant and an ingredient in drugs. In nature, caffeine is believed to function in chemical defense, acting as an antiherbivory and allelopathic agent, and therefore it might be employed to protect agriculturally important crop plants. In coffee plants, caffeine is synthesized from the precursor xanthosine in four steps, three N-methylations and removal of ribose. We had previously isolated genes encoding three distinct N-methyltransferases, and we demonstrated production of recombinant enzymes that yielded caffeine in in vitro reconstitution experiments. When these caffeine biosynthetic pathway genes were simultaneously expressed in tobacco plants (Nicotiana tabacum), caffeine was successfully produced up to 5 microg/g fresh weight in leaves. The leaves were unpalatable to tobacco cutworms (Spodoptera litura). This repellent action appeared to be more widely applicable to lepidopteran caterpillars as observed with small white (Pieris rapae) fed on Chinese cabbages that had been top-treated with caffeine. Our recent results suggest a novel approach to strengthen anti-herbivore traits by producing caffeine in crop plants.

In this review, the biosynthesis, catabolism, ecological significance, and modes of action of purine alkaloids particularly, caffeine, theobromine and theophylline in plants are discussed. In the biosynthesis of caffeine, progress has been made in enzymology, the amino acid sequence of the enzymes, and in the genes encoding N-methyltransferases. In addition, caffeine-deficient plants have been produced. The ecology of purine alkaloids has not proved to be particularly promising. However, advances have been made in insecticidal and allelopathic fields, and in the role of microorganisms play in the changes that these compounds undergo in the soil. Caffeine inhibits cell plate formation during telophase throughout the development of coffee plants and other species.

Caffeine (1,3,7-trimethylxanthine) and theobromine (3,7-dimethylxanthine) are the major purine alkaloids in plants. To investigate the diversity of N-methyltransferases involved in purine alkaloid biosynthesis, we isolated the genes homologous for caffeine synthase from theobromine-accumulating plants. The predicted amino acid sequences of N-methyltransferases in theobromine-accumulating species in Camellia were more than 80% identical to caffeine synthase in C. sinensis. However, there was a little homology among the N-methyltransferases between Camellia and Theobroma. The recombinant enzymes derived from theobromine-accumulating plants had only 3-N-methyltransferase activity. The accumulation of purine alkaloids was, therefore, dependent on the substrate specificity of N-methyltransferase determined by one amino acid residue in the central part of the protein.

N-Methyltransferases (NMTs) catalyze the three SAM dependent sequential methylation of xanthosine, producing caffeine in Coffea species. In the present work, a PCR based genome walking method was adopted to isolate and clone the promoter for the NMT gene. Inspection of the promoter sequence revealed the presence of several motifs important for the regulation of the gene expression. The whole fragment was fused to the beta-glucuronidase (gus) reporter gene and used in Agrobacterium tumefaciens mediated transformation of Nicotiana tabacum. GUS assays proved that the isolated promoter was able to direct the expression of the reporter gene in transgenic tobacco. Based on the promoter sequence, primer was designed and the genomic fragment comprising the promoter and its corresponding gene was amplified and cloned. Sequencing of one of the genomic clones revealed the presence of four exons and three introns in NMT gene. The differences in the restriction pattern among the genomic clones were studied using PCR-RFLP. This is the first report of cloning of the promoter for a gene involved in caffeine biosynthetic pathway and it opens up the possibility of studying the molecular mechanisms that regulate the production of caffeine.

Caffeine (1,3,7-trimethylxanthine) is derived from xanthosine through three successive transfers of methyl groups and a single ribose removal in coffee plants. The methyl group transfer is catalyzed by N-zmethyltransferases, xanthosine methyltransferase (XMT), 7-methylxanthine methyltransferase (MXMT) and 3,7-dimethylxanthine methyltransferase (DXMT). We previously cloned three genes encoding each of these N-methyltransferases from coffee plants, and reconstituted the final sequence of the caffeine synthetic pathway in vitro. In the present study, we simultaneously expressed these coffee genes in tobacco plants (Nicotiana tabacum), using a multiple-gene transfer method, and confirmed successful caffeine production up to 5 microg g(-1) fresh weight in leaves of the resulting transgenic plants. Their effects on feeding behavior of tobacco cutworms (Spodoptera litura), which damage a wide range of crops, were then examined. Leaf disc choice test showed that caterpillars selectively fed on the wild-type control materials, or positively avoided the transgenic materials. The results suggest a novel approach to confer self-defense by producing caffeine in planta. A second generation of transgenic crops containing caffeine may save labor and agricultural costs and also mitigate the environmental load of pesticides in future.

The adverse side effects of caffeine have increased the market for decaffeinated coffee to about 10% of coffee consumption worldwide (http://www.ncausa.org), despite the loss of key flavour compounds in the industrial decaffeinating process. We have discovered a naturally decaffeinated Coffea arabica plant from Ethiopia, a species normally recognized for the high quality of its beans. It should be possible to transfer this trait to commercial varieties of arabica coffee plants by intraspecific hybridization--a process likely to be simpler than an interspecific hybridization strategy, which could require more than 30 years of breeding to fix the decaffeinated trait and would probably result in an inferior cup of coffee.