Tea Caffeine: Metabolism, Functions, and Reduction ... - Springer Link

Food Sci. Biotechnol. 19(2): 275-287 (2010) DOI 10.1007/s10068-010-0041-y

RESEARCH REVIEW

Tea Caffeine: Metabolism, Functions, and Reduction Strategies Prashant Mohanpuria, Vinay Kumar, and Sudesh Kumar Yadav

Received: 27 October 2009 / Revised: 30 December 2009 / Accepted: 7 January 2010 / Published Online: 30 April 2010 © KoSFoST and Springer 2010

Abstract Tea is a product made up from topmost part of in various soft drinks. Along with other methylxanthines the plant Camellia sinensis. This part includes bud, first leaf (next to bud), second leaf, and stem (spanning from bud to second leaf). Tea is the second most consumed beverage in the world, well ahead of coffee, beer, wine, and carbonated soft drinks. Clinical studies have demonstrated that one of the harmful effects of tea over consumption, at least in sensitive peoples is due to its caffeine content. In view of this, major points discussed in this article are the following: i) a brief overview on tea and its biochemical composition, ii) health effects of tea drink, iii) caffeine metabolism and its functions, iv) possible strategies for caffeine reduction in tea, and v) feasibility of tea improvement through biotechnological approaches.

Keywords: tea, caffeine, caffeine metabolism, tea improvement, gene silencing

1. Introduction Tea [Camellia sinensis (L.) O. Kuntze] is the oldest, cheapest, and most popular non-alcoholic caffeinecontaining beverage in the world. It is commercially utilized for its tender shoots i.e., bud (topmost leaf), first leaf (next to bud), second leaf, and stem (spanning from bud to second leaf) is used for tea manufacturing. In Asian countries like China and India, tea drinking is a ritual and a life style. India occupies first rank in consumption and second rank in production of tea after China (1). Caffeine (1,3,7-trimethylxanthine) is one of the few plant products with which general public is readily familiar, because of its occurrence in beverages such as coffee and tea as well as Prashant Mohanpuria, Vinay Kumar, Sudesh Kumar Yadav ( ) Biotechnology Division, Institute of Himalayan Bioresource Technology, CSIR, Palampur, HP 176061, India Tel: +91-1894-233339; Fax: +91-1894-230425 E-mail: [email protected]; [email protected]

including theobromine (3,7-dimethylxanthine), paraxanthine (1,7-dimethylxanthine), and methyluric acids, caffeine is a member of a group of compounds known as purine alkaloids and is naturally found in coffee, tea, mate, guarana, cola, and cocoa. Tea is grown in about 30 countries and is most widely consumed beverage next to water worldwide. This is consumed by over 2/3 of the world population (1,2). The genus Camellia as a whole is endemic to south-east Asia and it comes under Theaceae family which includes 8 other genera. Among these genera Camellia is the largest one. The modern generic name Camellia is dedicated to Moravian Jesuit Kamel, a German missionary stationed in Philippines. According to this, tea was found in Asia during the later half of the 17 century. Tea is essentially an out-breeding crop and its 3 races (Chinary, Assamica, and Cambod) are highly interfertile. The enormous natural hybridization between tea species and large-scale dispersal of tea seeds over centuries, true-to-type tea plants are now rare, and are very difficult to find today. Now a day, the commercial tea is produced from these hybrids possessing mixed taxonomic characters and occupies the largest tea area under cultivation through seed propagation (3). Wight (4) proposed tea classification as C. sinensis for the China tea plants, Camellia assamica (Masters) for the Assam tea plants, and C. assamica sub sp. lasiocalyx (Planch. MS) or Cambodiensis for the Southern form (Indo-China) of tea plants. Of the total tea produced and consumed worldwide, 78% is in the form of black tea primarily consumed in Western countries. While 20% is consumed as green tea mainly in China, Japan, Korea, India, and a few countries in North Africa and the Middle East. Only 2% is produced as oolong tea and is the main tea beverage in southern China and Taiwan (5). In recent years, tea quality has gained considerable significance as consumers have become more health conscious. However, the quality is a composite character th

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determined by various constituents of tea such as polyphenols, catechins, proteins, enzymes, caffeine, carbohydrates, and inorganics (1). The presence of flavonoids in green and black tea provides antioxidative, anticarcinogenic, and antiarteriosclerotic like various health beneficial properties (6). In addition to various beneficial properties, high intake of tea can cause some adverse effects associated with caffeine such as palpitations, gastrointestinal disturbances, anxiety, tremor, increased blood pressure, and insomnia (7). The reported anticancerous as well as stroke preventive effects of drinking tea, especially green tea can be amplified by an absence of caffeine (8,9). Tea is a woody perennial having an average life of more than 60 years. Its long life cycle, self-incompatibility, and high inbreeding depression usually limit the genetic improvements by conventional breeding methods. While chemical based industrial decaffeination process is expensive and produces low-flavored products (2). Only after the year 2000, several caffeine biosynthesis pathway gene(s) from tea, coffee, and cocoa have been successfully reported (10,11). Thus, possibility of obtaining transgenic with low caffeine using genetic engineering has increased. Tea can become a most useful source of beneficial compounds upon reduction or elimination of caffeine from the plant. This can be achieved by silencing caffeine biosynthesis pathway gene(s) (12). Under these circumstances, genetic engineering seems to be promising approach to improve its genetic characters in a short time with the above limitations. Till date, only transgenic coffee plants have been produced with reduced caffeine content using RNA interference (RNAi) approach (13). The cloning of the various caffeine biosynthesis pathway genes from tea and coffee has now facilitated the use of genetic engineering towards the production of low-caffeine/caffeine-deficient beverages like tea.

2. Biochemical Composition of Tea Tea is composed of more than 700 chemical constituents (14). Major chemical constituents of tea are presented with their composition in Table 1. The beneficial effects of tea have been mainly attributed to the antioxidant properties of polyphenolic compounds particularly the catechins, which make up 30% of the dry weight of green tea leaves. The major catechins present in fresh tea leaves are (-)epigallocatechin gallate (EGCG), (-)-epigallocatechin (EGC), (-)-epicatechin gallate (ECG), and (-)-epicatechin (EC). EGCG is the major catechin in tea accounting for more than 10% on a dry weight basis, while other catechins are present in smaller amounts (6). Due to the differences in processing of tea after harvest, the catechins are present in higher quantity in green tea than in black or

P. Mohanpuria et al.

Table 1. Major chemical constituents and their composition in tea Constituent Polyphenols Amino acids Proteins Polysaccharides Lipids Organic acids Methylxznthines Minerals

Content (% dry wt) 30-40 4-5 14-17 14-25 2 1.5 7-9 5-6

oolong tea. However, the amount of caffeine in all 3

varieties of tea is unaffected by the different processing methods (15). In addition to the polyphenols, tea leaves are an important source of methylxanthines, amino acid, minerals, and trace elements such as potassium, magnesium, calcium, nickel, and zinc which are essential to human health (16). On dry weight basis out of 7-9% of the total methylxanthines, caffeine (3-6%), theobromine (0.1%), and theophylline (0.02%) have major physiological and pharmacological effects on some body systems such as central nervous, cardiovascular, gastrointestinal, respiratory, and renal systems (17,18). The amino acids play an important role in the development of tea aroma during the processing of black tea. Among these, aspartic acid, glutamic acid, serine, glutamine, tyrosine, valine, phenylalanine, leucine, isoleucine, and Ltheanine are found to be the principal amino acids present in the tea leaves while asparagine is formed during withering of tea. L-Theanine (γ-ethylamino-L-glutamic acid) is a unique, neurologically-active amino acid naturally found in the tea. It is a free (non-protein) amino acid found almost exclusively in tea plants (Camellia sp.), constituting 1-2% of the dry weight of tea leaves and accounting for about 50% of the total free amino acids (19). The ascorbic acid is detected only in green tea extracts and is absent in black tea extracts. The highest ascorbic acid content of green tea was recorded as 367 mg/100 g and 64.2% of this amount was extracted by a normal infusion process (20). Besides water-soluble components, tea leaves possess several lipid-soluble chemicals such as carotenoids and tocopherols. Similar to ascorbic acid, these are also detected in green tea and are not present in black tea. Several carotenoids including β-carotene, lutein, neoxanthin, and violaxanthin were found in fresh tea leaves (21). All these carotenoids are oxidatively degraded to several volatile and non-volatile products during the fermentation stage of black tea manufacture (22). The amounts of tocopherols are also higher in green tea leaves than black tea. α-Tocopherol is present in the highest

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amount among 4 tocopherol isomers. Carotenoids (23) and tocopherols (24) showed antioxidative and anticarcinogenic activities. Therefore, green tea containing these constituents may play a different role in the physiological effects compared to black tea. 3. Tea as a Health Drink

There is a very long history of association of tea with health. It has evolved from domain of medicine in ancient China to its current global status of beverage having the largest consumption after water (25). Chinese were the first to use tea as medicinal drink and later as beverage. They have been doing so for the past 3000 years. Tea is consumed in the form of black tea (fermented tea), green tea (non-fermented tea), and oolong tea (semi-fermented tea). Since tea is made using boiling sterile water, it is one of the safest beverages. Due to health beneficial properties of tea, the world’s most popular beverage has gained prominence during the last 2 decades (26). Tea contains various polyphenolic compounds such as catechins and flavonoids. Tea is the best dietary source of catechins. Tea flavonoids have various beneficial effects on human health upon their consumption as a tea drink such as effective antioxidant because of their free radical scavenging and metal chelating ability. Thus, tea provides protection against inflammation, clastogenesis, and several types of cancer. It reduces DNA damage and mutagenesis due to oxidative stress or the presence of pro-mutagens through antioxidant function. Tea flavonoids also involve in blocking activation pathways of mutagens and in suppressing the transcription of enzymes involved in DNA damage and mutagenesis (27). ‘Tea drinking’ is associated with various health promoting properties including reduction of cholesterol, immunity against intestinal disorders, depression of hypertension, antimicrobial effects, protection against cardiovascular diseases by inhibiting low-density lipoprotein peroxidation, suppression of fatty acid synthase (27), arthritis (28), osteoporosis (29), and dental caries (30). The EGCG, a major polyphenol found in tea is a widely studied chemo-preventive agent with potential anticancer activity. It inhibits angiogenesis and metastasis, and induces growth arrest and apoptosis through regulation of multiple signaling pathways (31). Recently, it has been suggested that daily consumption of tea reduced type 2 diabetes by 42% (32). The health benefits of green tea include antibacterial (33), anti-HIV (34), anti-aging (35), and anti-inflammatory activities (36). It also provides protection against Parkinson’s disease (37), Alzheimer’s disease, and ischemic damage (38). While the health benefits of black tea include antiviral due to its enzyme-inhibiting and receptor-blocking properties. Black tea intake improves bone mineral density. Black tea

also affects the motility and absorption of microflora present in gastrointestinal tract by influencing the hormonal balance and antioxidant function (27). L-Theanine, present in the tea serves as anti-stress and mood modulator through an inhibition of cortical neuron excitation (39). Several reports demonstrate the neuroprotective effect of L-theanine in cortical neurons via the antagonistic role which suggest its functional role in brain dynamics (40). L-Theanine is being used in candies, herb tea, cocoa drinks, beverages, chocolates, puddings, jellies, chewing gums, and other confectioneries for its relaxation effect. Intake of L-theanine by drinking green tea promotes the generation of alpha-waves. Such waves are known to produce an awake, alert, and relaxed physical and mental conditions. L-Theanine is responsible for giving characteristic specific ‘umami’ taste and flavor to green tea infusion. Intake of L-theanine also improves the learning ability (19). Administration of L-theanine in animals has resulted in reduced blood pressure and inhibits the excitatory effects of caffeine (40). L-Theanine has been studied extensively for its effects on tumor cells and the sensitivity of these cells to chemotherapeutic agents. In tumor cells, L-theanine competitively inhibits the glutamate transport and decreased intracellular glutathione levels. L-Theanine also inhibits the efflux of chemotherapeutic agents such as doxorubicin, idarubicin, cisplatin, and irinotecan causing them to accumulate in tumor cells. Thus L-theanine provides protection to the normal cells from damage by these drugs. This protective action of L-theanine is attributed to its antioxidant activity, specifically by maintaining cellular glutathione levels (41,42). 4. Caffeine

Out of about 50,000 known secondary metabolites produced by plants, more than 12,000 are alkaloids. Most common secondary metabolites are anthocyanins, flavonoids, quinines, lignin, steroids, and terpenoids. Caffeine is a typical purine alkaloid and found in more than 60 different plant species including coffee, tea, kola nuts, guarana berries, Yerba mate, and cacao beans (43). 4.1. Biological roles of caffeine

Broadly, two hypotheses have been made regarding the role of accumulated high concentrations of caffeine in tea, coffee, and few other plants such as in allelopathy and in chemical defense (2,43). Till date, its physiological functions have not been completely understood. However, chemical defense against pathogens and herbivores, and allelopathic effects against competing plant species have been proposed (44). Also, the role of caffeine synthesizing enzymes has been indicated during growth and ripening of

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Coffea arabica and Coffea canephora fruits (45). Along

with other compounds present in tea leaves, caffeine is involved in plant defense by acting against invading pathogens such as insects, bacteria, fungi, and viruses (46).

4.1.1. Role of caffeine in allelopathy The ‘allelopathic

or autotoxic function theory’ proposes that caffeine in seed coats and falling plant parts is released into the soil and inhibits the germination of seeds of other competing plant species (47). Caffeine has been shown to completely suppress germination of Amaranthus spinosu at a concentration of 1,200 ppm (48). It has also been reported that caffeine reduces access to nutrients and water by inhibiting mitosis in the roots of many plants (49,50). Its powerful allelochemical properties can be assessed by providing caffeine treatment to model plants from seed germination to further plant growth and development.


inhibiting growth of rice seedlings as compared to dimethylxanthines such as theobromine and theophylline. Treatment of plants with 2.5 mM caffeine inhibited the growth of roots to a greater extent than shoots and it had suggested that shoots could have more effective mechanism than roots for maintaining growth in the presence of caffeine. Retardation in seedling size including root and shoot size, yellowing, and decrease in chlorophyll content of tobacco and Arabidopsis seedlings upon 1 and 5 mM caffeine treatment indicated that caffeine exposure induced early senescence in plants. Also, the caffeine exposure was found to decrease the expression and activity of Rubisco in both the plants. But still it is unknown whether caffeine induces senescence that leads to down-regulation of Rubisco or if it is the caffeine down-regulated Rubisco that leads to senescence (61).

4.3. Influence of caffeine on human health 4.1.2. Role of caffeine in plant defense The ‘chemical More than 80% of the world’s population ingests caffeine

daily, making it the most widely consumed drug in history. It is well known that caffeine acts as a stimulant in humans. This is due to the chemical similarity of caffeine with adenine that blocks adenosine receptors in nerve cells. Generally, human beings consume 2-3 cups of tea daily. One cup of tea contains about 60 mg of caffeine. High intake of tea leads to an increase in caffeine level in addition to its important antioxidant constituents. Higher levels of caffeine can produce adverse effects on human health, at least in sensitive peoples. This leads to various problems such as palpitations, gastrointestinal disturbances, anxiety, tremor, increased blood pressure, and insomnia (7). It may also leads to higher risk of developing bone problems, including osteoporosis as well as problems in metal absorption, excretion, and reabsorption processes in intestine and kidney and iron deficiency anemia (62,63). Caffeine also appears to increase the risk of coronary heart 4.2. Influence of caffeine on plant growth and development disease (64). Further, tea taken up by women during Adverse effect of caffeine consumption has been well pregnancy has proven to be more harmful as this leads to documented in animals and in human beings. However, a the birth defects in babies and produces infertility (65). few studies have been conducted to see the effect of Thus reproductive-aged and pregnant women are ‘at risk’ caffeine exposure on plant growth and developments. subgroups of the population and require specific advice on Methylxanthines and their analogues have been found to moderating their daily caffeine intake (66). It is believed that habitual daily use of 500-600 mg be inhibiting the seed germination in Cicer arietinum (54). Earlier, Stallwood and Davidson (55) reported the inhibitory caffeine (4 to 7 cup of coffee or 7 to 9 cup of tea) leads to influence of methylxanthine on proliferating cells, however, a significant health risk. If such intake is sustained, it may that was reversed on colchicine application. A mitotic results in ‘caffeinism’. Caffeinism refers to a syndrome delay and a potentiation of the chromosome damage by characterized by a range of adverse reactions such as caffeine post-treatment have been shown in proliferating restlessness, anxiety, irritability, agitation, muscle tremor, plant cells (56). Several studies have reported that caffeine insomnia, headache, diuresis, sensory disturbances (e.g., treatment affected the chromosome damage at mitotic tinnitus), cardiovascular symptoms (e.g., tachycardia, stage of plant cell division (57-59). Smyth (60) reported arrhythmia), and gastrointestinal disturbances like nausea, that caffeine, a trimethylxanthine is more effective in vomiting, diarrhea, etc (7,12,17,26). defense theory’ proposes that caffeine in young leaves, fruits, and flower buds provides protection to soft tissues of plant against pathogenic insect larvae, beetles, and herbivores (51,52). Caffeine acts as a repellent and pesticide against tobacco hornworms (52). It has been shown that by spraying 1% caffeine solution on tomato leaves deters feeding by tobacco hornworms, while 0.010.1% solutions of caffeine on cabbage leaves and orchids significantly affect the slugs and snails feeding (53). Recently, anti-herbivore trait against tobacco cut worms has been developed in transgenic tobacco by accumulating caffeine up to 5 µg/g fresh weight (44). It has been assumed that caffeine produces direct interference with the pest metabolic pathways and acts as one of signaling molecules to activate plant defense responses in plants (44).

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4.4. Caffeine metabolism in plants 4.4.1. Caffeine biosynthesis Caffeine was discovered

from tea and coffee in the early 1820s. However, its biosynthetic pathway was established only after 2000. The major caffeine biosynthesis pathway involves conversion of xanthosine to caffeine via 7-methyl-xanthosine, 7methylxanthine, and theobromine (Fig. 1). Pathway is essentially the same in other purine producing plants such as mate (Ilex paraguariensis), cacao (Theobroma cacao), and coffee (67,68). The 1st, 3rd, and 4th steps in the major pathway are catalyzed by N-methyl-transferases that use Sadenosyl-L-methionine (SAM) as methyl donor (69). The 2 final steps in tea are catalyzed by caffeine synthase (CS), a bifunctional enzyme comprising 2 SAM-dependent Nmethyltransferase (NMT) activities. The 2nd step of caffeine biosynthesis is catalyzed by an N-methylnucleosidase. It has been suggested that the methyl transfer in the 1st step and nucleoside cleavage in the 2nd step of caffeine biosynthesis in coffee might be coupled and catalyzed by a single enzyme (70). In addition, conversion of 7methylxanthine to caffeine via paraxanthine has been reported as one of the number of minor pathways operating in young leaves of tea. The paraxanthine N-methyltransferase catalyzes the conversion of paraxanthine to caffeine has been found in the chloroplasts of tea (71). While in coffee, green-fluorescence protein fusion method could localize this enzyme in the cytoplasm (72,73). The purification and characterization of CS from young tea leaves has suggested it as a stroma enzyme. Thus, caffeine appears to be synthesized in the cytoplasm and translocated to vacuole. But the mechanism of translocation is not yet known. Gene encoding caffeine synthase has been cloned from young tea leaves (74). Three types of complementary DNAs (cDNAs) i.e., CaXMT1, CaMXMT2, and CaDXMT1 encoding N-methyltransferases that catalyze the formation of 7-methylxanthosine from xanthosine, theobromine, and caffeine respectively, have been cloned from immature fruits of coffee (75). Xanthosine, an initial substrate of purine alkaloid synthesis is derived from adenine and guanine nucleotide pools which are produced by de novo and salvage pathways. The conversion of xanthosine via adenosine monophosphate (AMP), inosine monophosphate (IMP), and xanthosine monophosphate (XMP) is likely predominating. SAM cycle also produces adenosine which is converted to AMP directly or indirectly via adenine (76). In addition to this, purine nucleotides are also synthesized de novo from non-purine precursors such as CO2, 10formyltetrahydrofolate, 5-phosphoribosyl-1-pyrophosphate, and the amino acids, glycine, glutamine, and aspartate (77). In tea, single NMT has dual function in caffeine synthesis (74). High expression of gene encoding this NMT could be responsible for the production of high caffeine content in

Fig. 1. Schematic representation of caffeine biosynthesis and degradation. The major biosynthetic pathway of caffeine starts

from xanthosine which is supplied by , AMP/GMP and adenosyl-L-methionine (SAM) routes. The major steps in caffeine biosynthesis are catalyzed by following enzymes: (1) Xanthosine -methyltransferase (XMT); (2) -methylnucleosidase; (3) Monomethylxanthine -methyltransferase (MXMT); (4) Caffeine synthase or dimethylxanthine -methyltransferase (DXMT). The 1, 3, and 4 step catalyzing enzymes are dependent on SAM. In tea, 3 and 4 steps are found be catalyzed by a dual functional caffeine synthase. Caffeine degraded to xanthine via theophylline in both coffee and tea. Xanthine is then catabolized to urea, CO2, and NH3 through conventional purine catabolism pathway. However, the exact mechanism of caffeine degradation is still not known. demethylases (NDM) are expected in caffeine degradation pathway. de novo

N

S

N

N

N

N

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tea young leaves. While coffee plants are equipped with multiple sets of enzymes, specifically for the final 2 steps of major caffeine biosynthesis pathway which are necessary for constitutive production of caffeine in relevant tissues (75).

4.4.2. Caffeine degradation A caffeine degradation

pathway has been documented in coffee. Caffeine is catabolized to xanthine via theophylline and 3-methylxanthine, which then degrade further by the conventional purine catabolism pathway to CO2, NH3, and urea via uric acid, allantoin, and allantoic acid (78). The caffeine degradation pathway is shown along with its synthesis (Fig. 1). Caffeine degradation pathways have also been well studied in microorganisms like bacteria, fungi, and yeast (79-81). Although, demethylase (s) activity has been detected in these microorganisms, but it has not been elucidated so far in tea or coffee. However, based on tracer experiments several catabolites of caffeine degradation have been identified in C. arabica leaves (82) and also in tea. Tea and coffee are very close to each other in terms of caffeine containing plants. Therefore, tea might possess more or less similar catabolic pathway for caffeine as been predicted in coffee. Caffeine is mainly produced in young leaves of tea plants and continuously accumulates during its maturation. However, it is slowly catabolized with the age of the tissue. It is expected that demethylase(s) is present in tea for the removal of methyl groups from caffeine to convert it into xanthine. But still no such enzyme activity has been isolated from the higher plants (2). Allantoin content measurement in different tissues can be an important aspect in measuring caffeine degradation as it has been reported as one of the important products of caffeine catabolism in plants (83). Based on allantoin content in various tissues of 4 tea cultivars during nondormant and dormant growth phases, caffeine degradation has been found to be inversely correlated with the caffeine synthesis. Our study has documented that synthesis and degradation of caffeine in tea appears to be under developmental and seasonal regulation (84). It has also been documented that the ratio of biosynthetic and degradation enzymes in fruit and leaves determine the caffeine content. Degradation of caffeine in fruits was rapid as compared to leaves (85,86). There could be 2 possibilities, either production or degradation of caffeine is faster in fruit tissues of tea depending upon the ratio of caffeine biosynthesis and degradation enzymes or caffeine accumulates in fruit tissues for a short period of interval.

4.4.3. Characteristics of caffeine biosynthesis pathway enzymes and genes Caffeine biosynthesis pathway

enzymes are basically group of methyltransferases. Enzymatic methylation is a ubiquitous reaction that occurs in diverse


group of organisms, including bacteria, fungi, plants, and mammals. This results in the modification of both small and macromolecules for different functional and regulatory purposes. Plant N-methyltransferases are placed in a subclass of enzymes transferase that catalyze the transfer of a methyl group from one compound to another and act on proteins, DNA, phosphoethanolamine, and secondary metabolites. A new family of methyltransferases (Type 3) has been identified which includes the SAM dependant enzymes such as SAMT (SAM:salicylic acid carboxyl methyltransferase), BAMT (SAM:benzoic acid carboxyl methyltransferase), and JMT (SAM:jasmonic acid carboxyl methyltransferase) in addition to NMTs of caffeine biosynthesis pathway. Among these, only SAMT has been structurally characterized (87). The first gene encoding NMT of caffeine biosynthesis pathway was cloned and characterized from tea leaves and was named as tea caffeine synthase1 (TCS1) (74). Various NMTs from different tissues of coffee plant were biochemically characterized and their encoding genes were cloned. This include 7-methylxanthine methyltransferase1 (MXMT1) (72), 7-methylxanthosine synthase1 (XRS1) (88), coffee caffeine synthase1 (CCS1), and coffee theobromine synthase (CTS1 and CTS2) (11) characterized from leaf and endosperm of coffee. Three SAM-dependent NMTs named as xanthosine methyltransferase (XMT), 7-methylxanthosine methyltransferase (MXMT2) or theobromine synthase, and 3,7-dimethylxanthine methyltransferase (DXMT) or caffeine synthase were biochemically characterized from immature coffee fruit (75). Recently, genes encoding theobromine synthase from theobromine accumulating Camellia plants were isolated and characterized (89). In coffee, NMTs are present in isoforms with different properties. This suggests that caffeine is synthesized through multiple pathways in coffee depending on availability and concentration of the substrates (11,72,75,88). Using bimolecular fluorescence complementation study, it has been shown that all 3 enzymes, XMT, MXMT, and DXMT form homodimers as well as hetero-dimers in planta. However, hetero-dimer formation has no effect on the individual enzymatic activities (73). The XMT has been found to exhibit coupled activities for methyltransferase and nucleosidase cleavage (70). The NMTs have appeared to evolve from Omethyl transferases based on their conserved sequence regions. However, slight variations in their coding regions alter substrate specificity for methylation (70). Although, NMTs have high protein sequence homology (>80% identity) but they exhibit remarkable substrate selectivity (90). The amino acid sequences of MXMT and CTS proteins have revealed that substrate specificity is determined by a small number of key amino acids (72). Using crystallography analyses, histidine-160 was considered as critical for substrate selectivity or binding (70). While in XMT, it was

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exemplified by serine-316 being central to the recognition of xanthosine. Crystallographic data also elucidated the structure of XMT and DXMT. Both consist of 2 domains, methyltransferase domain and α-helical cap domain (70). The characterization of different caffeine biosynthesis pathway genes or cDNA sequences has been done from coffee and tea. A tea TCS1 has 40% homology to CCS1 of coffee at nucleotide level. At the protein levels, they share close homology only for 4 highly conserved regions motif A, motif B’, motif C, and YFFF. These regions are responsible for the N-methylating catalytic reactions, where motif A, B’, and C are proposed to be the SAMbinding motifs for the caffeine production in plants (11,88). 4.4.4.

Tissue

specific

distribution

and

expression

of

Caffeine is accumulated in seeds, cotyledons, and young leaves. Based on transcript accumulation analyzed by semiquantitative-PCR, constitutive production of caffeine in relevant tissues has been reported in coffee. The transcript for caffeine synthase gene was accumulated predominantly in immature fruits, a major site of caffeine synthesis. While transcripts for other Nmethyltransferase genes of caffeine biosynthesis pathway were accumulated in leaves, floral buds, and immature fruits (75). Caffeine biosynthesis activity has been found in various other tissues such as stamens and petals of tea prior to the opening of flowers, pericarp, and seeds of tea fruits (91). A seasonal variation of caffeine biosynthesis has also been investigated through high performance liquid chromato-graphy (HPLC) in C. sinensis. This indicated that caffeine biosynthesis is restricted to young leaves of new shoots during spring season (92). Using semiquantitative-PCR, expression of genes encoding TCS1, inosine-5'-mono-phosphate dehydrogenase (TIDH), Sadenosyl-L-methionine synthase (sAMS), phenylalanine ammonia-lyase (PAL), and α-tubulin (Tua1) were studied in young and mature leaves, stems, and roots of tea seedlings. The amount of TCS1 transcript was found to be much higher in young leaves than in other parts of the plant which suggested high caffeine biosynthesis and caffeine content (93). Recently, we analyzed the caffeine biosynthesis and degradation by monitoring the caffeine synthase gene expression and caffeine and allantoin content in various tissues of 4 tea cultivars during non-dormant and dormant growth phases (84). During winter, tea undergoes dormancy phase and its apical bud is either not available or available with very low level of important biochemical constituents. Therefore, this period for tea is known as dormant growth phase. Caffeine synthase gene expression as well as caffeine content was found to be higher in commercially utilized tissues like apical bud, first leaf, second leaf, young stem, and was lower in old leaf during non-dormant caffeine

synthase

genes

compared to dormant growth phase. Among fruit parts, fruit coats showed higher caffeine synthase gene expression, caffeine content, and allantoin content as compared to cotyledons. On contrary, allantoin content was found lower in the commercially utilized tissues and higher in old leaf. Thus, caffeine synthesis and degradation in tea appeared to be under developmental and seasonal regulation (84). 4.5. Strategies for caffeine reduction

Caffeine content of tea can be reduced by 4 different approaches. One is conventional breeding approach. This is a very difficult approach because of woody and perennial nature of tea crop. Tea may take 25 or more years to stabilize the desired traits. Tea has long life cycle of 100 years, self-incompatibility and high inbreeding depression which limit its genetic improvement through the selection of elites from existing natural populations or by the development of biclonal hybrids. In addition, a low fruit set and the difficulty in rooting of vegetatively propagated cuttings in a nursery provide further constraints at every stage of the selection, hybridization, and propagation in multi-location trials. Also, biotic and/or abiotic stressresistant mutants are unavailable in tea (94). Second is chemical based approach. This has been attempted to produce low caffeine teas. Chloroform or methylene chloride has been used as an effective solvent for extracting caffeine from tea leaf. However, it is not widely accepted by consumers because of toxicity of the solvent residue left (95). Decaffeination using supercritical carbon dioxide is effective and leaves no solvent residues (96), but it needs expensive equipment. Sawdust lignocellulose columns can be used to separate caffeine from tea extracts (95), but they are difficult to use for decaffeination of tea leaves. Still search is going on to find inexpensive and safe decaffeination chemicals which leave no solvent residue. This is an important area of research for the tea processing field. Later on, hot water treatment was considered as a safe and inexpensive method for decaffeinating the green tea. However, when the rolled leaf and dry tea were decaffeinated by this method, a large percentage of tea catechins were lost and the process was considered to be not suitable for processing black tea (97). The third approach is microbial degradation of caffeine. The first report on caffeine degradation by microorganisms was in early 1970’s. The bacteria like Pseudomonas species and fungi such as Aspergillus and Penicillium species were found to be efficient in degradation of caffeine. Thus, these microorganisms can be utilized for reducing caffeine content in caffeine bearing plants (98). Using this approach, in situ lowering of caffeine in tea leaves was documented without affecting the quality of other tea components (99). The fourth approach is genetic engineering, where

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Fig. 2. Representative model of RNA interference (RNAi) mechanism operating in plants. RNAi mechanism is initiated by 21-26nt

small interfering RNAs (siRNAs) which are processed from long double stranded RNAs (dsRNAs) through RNase III (DCL; Dicer like) and 21nt micro RNAs (miRNAs) which are transcribed from miRNA gene in the genome. The siRNAs can also be used as primers for the generation of new dsRNA by RNA-dependent RNA polymerase (RdRp). The new dsRNA can subsequently serve as target for the DCL and are processed into secondary siRNAs. In this manner silencing signal is amplified. In plants, nuclear-localized DCL1 appears to be responsible for processing of both pri (primary) - and pre (precursor) -miRNAs to generate mature miRNAs. The mature single stranded miRNAs and siRNAs are loaded into RNA induced silencing complex (RISC) to direct mRNA cleavage, translational inhibition, chromatin modification, or transcriptional gene silencing (TGS).

different strategies can potentially be used to reduce the caffeine content. As caffeine degradation pathway mediated by demethylases is known in bacteria, fungi, and yeast (100,101), such gene(s) from either a microorganism or plants can be overexpressed in tea to enhance the caffeine degradation. However, the caffeine degradation pathway and its enzymatic steps have not been elucidated in tea so far. Only the caffeine catabolites were observed in C. arabica leaves (60) and thus there would be possibility of demethylases. Caffeine biosynthesis pathway gene(s) from tea and coffee have been isolated and even expressed in prokaryotic as well as eukaryotic system such as tobacco. Thus, a possibility to use this gene sequence from tea for the purpose of caffeine reduction using antisense and RNA interference (RNAi) technology has produced (12). In antisense technology, a cDNA sequence of the gene of interest is cloned in antisense orientation with respect to the promoter into a suitable plant transformation vector and mobilized in plant tissue via Agrobacterium or biolistic method of gene delivery. In the transgenic plants, antisense mRNA transcribed from antisense DNA would be complementary to the sense mRNA of the gene of interest and thus both would hybridize. This hybrid would be inhibited for protein synthesis or broken down quickly by RNases (104). In this manner, caffeine biosynthesis pathway can be halted and low or no caffeine tea can be produced. In RNAi, the double stranded RNA rather than the single

stranded RNA as in antisense is the interfering agent. The RNAi technique has high potential and high degree of specificity for target gene silencing by cleavage of mRNA. Its systemic nature, high degree of amplification of effects and persistence through generations has greatly attracted the attention of researchers globally for functional analysis of genes (103).

4.5.1. Caffeine reduction through RNAi approach

Before explaining caffeine reduction through RNAi approach, it is very important to understand about this technique and its mechanism exists in plants (Fig. 2). RNAi is a post-transcriptional, RNA dependent, sequencespecific gene silencing mechanism. This mechanism is conserved in a wide range of eukaryotes including plants, fungi, nematodes, protozoa, insects, and vertebrates which reflects its ancient evolutionary origin to protect the genome of a species (104). It is triggered by exogenously supplied double-stranded RNA (dsRNA) that produces small interfering RNAs (siRNAs) into cells or endogenously produced small non-coding RNAs known as micro RNAs (miRNAs) (105). The siRNAs are produced from replication of plant RNA viruses, transgenic inverted repeats, and amplify through RNA-dependent RNA polymerases (RdRps) in plants. The phenomenon of RNAi was first identified as ‘co-suppression’ by Napoli et al. (106) in transgenic petunia flowers where the introduction

Tea Caffeine

of a chalcone synthase gene of flavonoid biosynthesis pathway resulted in white petunias instead of the expected deep blue coloration. Plant produces 2 distinct classes of siRNAs with 2nucleotide 3' overhangs i.e., short (21-23 nucleotides) and long (24-26 nucleotides) siRNAs. However, in case of plants, both siRNA and miRNA are produced by RNase III- like enzymes called DICER-LIKE (DCL) (107). The miRNAs are chemically and functionally similar to siRNAs but are produced endogenously from miRNA genes present in the genome. They are produced through transcription by RNA polymerase II into long primary transcripts (pri-miRNAs) which are further processed into precursor miRNAs (pre-miRNAs). In plants nuclearlocalized DCL1 appears to be responsible for processing of both pri- and pre-miRNAs to generate mature miRNAs. Dicer family members are large, multidomain proteins that contain a putative RNA helicase domain (at N-terminal), 1 PAZ (Piwi/Argonaute/Zwille) protein-protein interaction domain, 2 tandem ribonuclease III (RNase III) which are believed to mediate ATP-dependent endonucleolytic cleavage of dsRNA into siRNAs and 1 or 2 dsRNAbinding domains (at C-terminal). In plants and worms, amplification of the silencing signal and cell-to-cell RNAi spreading has been observed (108). The siRNAs can also be used as primers for the generation of new dsRNA by RdRp. The new dsRNAs subsequently serve as target for the DCL and are processed into secondary siRNAs (109). The movement of dsRNA or siRNA between plant cells can take place through plasmodesmata and/or via the vascular system. The mature single stranded miRNAs or siRNAs are loaded into RNA induced silencing complex (RISC). RISC also contains Argonaute proteins and directs mRNA cleavage or translational inhibition of target genes (110,111). They can also involve in transcriptional silencing through RNAdirected DNA methylation or post-transcriptional silencing through histones modification (112). The current evidences have shown that miRNAs are important regulatory factor which play multiple roles in biological processes, including development, cell proliferation, apoptosis, and stress responses. RNAi mechanism in plants has numerous distinctive features, including signal amplification, shortand long-range signaling and transitivity, and transcriptional gene silencing responses. Still the whole mechanism of RNAi in plants is yet to be known. For genetic improvement of crop plants, RNAi has more advantageous in terms of its efficiency and stability in its ability to suppress transgene expression in multigene families in a regulated manner (113). A few efforts have been made towards producing lowcaffeine or caffeine-deficient beverage. The blocking of IMP dehydrogenase, which converts IMP to XMP has

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been assumed to be a key step to produce caffeine-deficient tea. Thus, ribavirin (1-L-D-ribofuranosyl- 1,2,4-triazole-3carboxamide), a potent inhibitor of IMP dehydrogenase was used to inhibit caffeine biosynthesis. The IMP dehydrogenase-deficient plants were suggested as a potential source of good quality caffeine-deficient tea and coffee plants. In addition to this, caffeine-deficient fully flavored coffee plants were produced using RNAi approach. The 3'-untranslated region and the coding region of CaMXMT cDNA were used to design 2 RNAi constructs, RNAi-S having a short insert of 150 bp and RNAi-L having a long insert of 360 bp. These constructs were used for Agrobacterium-mediated transformation of C. arabica and C. canephora plants. The resulted transgenic lines had suppressed the transcript of CaXMT and CaDXMT in addition to CaMXMT and a marked reduction of 30-80 and 50-70% in their theobromine and caffeine contents, respectively, was obtained as compared with control (13). These plants retained the same morphology as that of untransformed coffee plants and had the same profile of cellular metabolites as wild type plants except the reduced caffeine content. As the caffeine biosynthesis emerges from purine nucleotide biosynthesis, the transgenic plants so generated were examined for the production of any abnormal compound(s) through the purine metabolism. Caffeine biosynthesis from [8-14C]adenine, a most efficient exogenously supplied precursor for caffeine synthesis was investigated together with the overall metabolism. It was found that metabolism of [8-14C]adenine was shifted from purine alkaloid synthesis to degradation into CO2 via purine conventional catabolism. While no accumulation of any unusual compounds specific to the transgenic plants were found (114). The development of decaffeinated coffee through RNAi has opened up the possibility of reducing caffeine in tea through the same approach. Hence, fully-flavored low caffeine or caffeine free tea plants can be generated. The demand for decaffeinated beverages has increased in last few years. Such products can certainly contribute to the share of world market (2). Till now there is no report on silencing caffeine synthase gene in tea through RNAi. However, the down regulation of caffeine synthase transcript expression and reduction of caffeine content in somatic embryos has been observed in our laboratory documented the feasibility of using RNAi for caffeine reduction in tea.

4.5.2. Feasibility of tea improvement through genetic modifications As the world market has critical standards for tea produced from different parts of the globe to attain a significant commercial value and thus, crop improvement combining both yield and quality are important (115). Tea has several constraints for its improvement which we have already discussed earlier in this review. Transgenic

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technology has immense potential for genetic improvement of tea. Several transgenic plants have been produced in a wide range of genera but the development of transgenic tea plants remained difficult. Due to lack of suitable tea regeneration as well as transformation protocols, there were no reports on tea transgenesis before 2000. Tea has been shown low competence for transformation as well as regeneration because of the presence of high levels of polyphenols with germicidal property (116,117). So, optimization of transformation efficiency, reproducibility, and regeneration is very critical. Genetic transformation of tea through Agrobacterium tumefaciens has been attempted by several workers using different explants such as in vitro leaves, somatic embryos and embryogenic tissues (118, 119). However, somatic embryogenesis was considered one of the most worked out regeneration system in tea. It was used successfully in the production of first healthy transgenic tea plants using GUS reporter and NPT-II marker genes via Agrobacterium-mediated transformation (94). The transgenic microshoots were multiplied in vitro. However, hardening of tissue culture raised tea founds difficulty. For this, microshoots were grafted on to the seedling-derived root-stocks of the same cultivar. Grafting is an alternative propagation technique where fresh single leaf internode cutting of both root-stock and scion is used. In this technique, scion from a quality cultivar is grafted on to the root-stock from a drought tolerant or high yielding cultivar. Upon grafting, the scion and stock influence each other for the characters. Thus, composite plant combines both yield and quality characters resulting in significant increase of yield with better quality than either of the nongrafted cultivars (120). Biolistic-mediated genetic transformation is another method used for producing transgenic tea plants. Recently, tea plants expressing stress tolerance genes were developed (121). The osmotin gene was introduced into tea somatic embryos at the globular stage of development via microprojectile bombardment. Transgene effects were studied on the accumulation of storage reserves and desiccation tolerance during embryo maturation. This has resulted in several fold increase in storage reserves, normal maturation and 57-60% germination of tea somatic embryos. This has also provided a solution to the developmental problems associated with recalcitrant seeds of tea (122). In addition, first time successful implication of transient RNAi in tea somatic embryos through glutathione synthetase, an important enzyme of glutathione biosynthesis has opened up the way of using tea for gene silencing studies (103).

Acknowledgments Authors are thankful to Dr. P.S.

Ahuja, Director, IHBT for his valuable suggestions during writing of this article. The research work in our laboratory is supported by the research grants from Council for


Scientific and Industrial Research (CSIR) and Department of Science and Technology (DST; Grant No GAP095), Govt. of India, New Delhi. Prashant Mohanpuria is also thankful to CSIR for providing research fellowship in the form of SRF. The IHBT number for this article is 2040.

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