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.

N-Methyltransferases of Caffeine Biosynthetic Pathway in Plants.

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.

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.

Tandem gene duplications drive divergent evolution of caffeine and crocin biosynthetic pathways in plants.

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.

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.

Understanding the Catalytic Mechanism of Xanthosine Methyltransferase in Caffeine Biosynthesis from QM/MM Molecular Dynamics and Free Energy Simulations.

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.

Engineering a microbial platform for de novo biosynthesis of diverse methylxanthines.

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.

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.

The coffee genome provides insight into the convergent evolution of caffeine biosynthesis.

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.

Quantum mechanical/molecular mechanical study of catalytic mechanism and role of key residues in methylation reactions catalyzed by dimethylxanthine methyltransferase in caffeine biosynthesis.

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.

Involvement of a novel intronic microRNA in cross regulation of N-methyltransferase genes involved in caffeine biosynthesis in Coffea canephora.

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.

In vitro effects of caffeine in growth cartilage of rats / Efeitos in vitro da cafeína na cartilagem de crescimento de ratos

OBJETIVO: Avaliar os efeitos in vitro da cafeína na proliferação, apoptose e expressão de transcriptos gênicos de diferenciação condrogênica na cartilagem de crescimento.MÉTODO: As epífises cartilaginosas de fêmures de ratos neonatos foram divididas em dois subgrupos: os tratados com cafeína e o grupo controle, ambos observados nos tempos de 0, 7, 14 e 21 dias. As epífises cartilaginosas de fêmures de cada subgrupo e de cada tempo foram submetidas à histomorfometria, análise imunoistoquímica, técnica de túnel e RT-PCR em tempo real.RESULTADO: A diminuição da atividade proliferativa e o aumento de condroblastos em apoptose aos 21 dias foram encontrados em ambos os subgrupos. Entretanto a diminuição da proliferação celular causada pela cafeína foi menor quando comparada ao grupo controle e aumentou significativamente a expressão de transcriptos gênicos para diferenciação condrogênica, representada pelo SOX-9 e pelo RUNX-2. Entretanto o cultivo in vitro com cafeína demostrou efeitos antagônicos: apesar dos efeitos positivos na proliferação e diferenciação de condroblatos, cafeína aumentou a apoptose, caracterizada pelo aumento da expressão de caspase-3 e do numero de células em apoptose (p< 0.05).CONCLUSÃO: A cafeína apresenta efeitos antagônicos in vitro na cartilagem em crescimento, aumentando a proliferação, diferenciação e apoptose celular. Estudo experimental.

OBJECTIVE: To evaluate the in vitro effetcs of caffeine on proliferation, apoptosis and gene transcripts expression of chondrogenic differentiation in growth cartilage.METHODS: The cartilaginous epiphyses of femurs of newborn rats, which were divided into two subgroups: treated with caffeine and control group, both observed over the time periods of 0, 7, 14 and 21 days. The cartilaginous epiphyses of femurs of each subgroup and each time span were subjected to histomorphometric, immunohistochemical analysis, Tunel technique and RT-PCR in real time.RESULTS: The decrease in proliferative activity and the increase of apoptotic chondroblasts at 21 days were found regardless of the subgroup. However, the decrease in cell proliferation caused by caffeine was lower than in the control group and significantly increased the expression of gene transcripts for chondrogenic differentiation, represented by SOX-9 and RUNX-2. However, the in vitro culture with caffeine revealed antagonistic effects: despite the positive effect on chondroblasts proliferation and differentiation, caffeine increased apoptosis, characterized by increased expression of caspase 3 and of the number of cells undergoing apoptosis (p<0.05).CONCLUSION: Caffeine presents antagonistic effects in vitro on growth cartilage, increasing the proliferation, differentiation and cell apoptosis. Experimental Study.