8.1 Introduction

Functional genomics play important role in plant improvement (Mondal 2013; Mukhopadhaya et al. 2013). In tea, functional genomics work was initiated with the isolation of chalcone synthase gene from a Japanese green tea cultivar ‘Yabukita’ (Takeuchi et al. 1994a). Since then a significant amount of genomic work has been done in tea and its wild relatives targeting isolation and characterization of trait-specific genes and their expression under varying conditions (Table 8.1). It has been evident that two types of efforts have been made: (i) cloning of individual gene associated with particular trait and (ii) differential gene expression which leads to identification of genes that are associated to a trait.

8.2 Cloning and Characterization of Individual Genes

Interestingly, majority of the genes cloned so far belongs to quality followed by yield as these two parameters that have major demand in the commercial tea cultivations.

8.2.1 Quality Related Genes

Tea is valued for its cup quality. Therefore, research efforts of tea are biased towards the improvement of quality. Similarly, in the area of genomics, several individual genes of various biochemical pathways were targeted, cloned and characterized which are discussed below (Fig. 8.1).

Fig. 8.1
figure 1

Summary of major biochemical pathways involved in quality of made tea and genes of enzyme that are cloned (partially or fully) from tea, denoted by italics. : anthocyanidin reductase (ANR), anthocyanidin synthase (ANS), cinnamate 4-hydroxylase (C4H), 4-coumaroyl-CoA ligase (4CL), chalcone synthase (CHS), chalcone isomerase (CHI), Caffeoy-CoA 3-O-methyl transferase (CCoAOMT), 3-deoxy-D-arabinoheptulosonate 7-phosphate synthase (DAHPS), dihydroflavonol 4-reductase (DFR), 3-dehydroquinate synthase (DHQS), 3-dehydroquinate dehydratase (DHD), epicatechins 1-O-galloyl-β-Dglucose O-galloyltransferase (ECGT), flavanone 3-hydroxylase (F3H), flavonoid 39-hydroxylase (F3′H), flavonoid 39, 59-hydroxylase (F3′5′H), flavonol synthase (FLS), flavone synthase II (FSII), galloylated catechins hydrolase (GCH), inosine-5-monophosphate (IMP), leucoanthocyanidin reductase (LAR), 7 methyl xanthosine synthase (7-MXS), phenylalanine ammonia lyase (PAL), shikimate dehydrogenase (SDH), Theobromine synthase (TBS), caffeine synthase (TCS1), inosine-5-monophosphate dehydrogenase (TIDH), UDP-glucose:flavonoid 3-O-glucosyltransferase (UFGT), UDP glucose galloyl-1-O-β-D-glucosyltransferase (UGGT), uridine diphosphate Glycosyltransferase (UGT), xanthosine-5-monophosphate (XMP), Sg4, Sg7, WD40, bHLH24 are the transcription factors as reported by Zhao et al. (2012)

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8.2.1.1 Theanine Biosynthesis Related Genes

Theanine, a glutamate derivative is the most dominant amino acid in tea plant, which although is found in all the organs and tissues, yet with maximum amount in tender leaves (Sugiyama and Sadzuka 2004). Theanine is one of the constituents which provide the briskness of tea, a parameter which is used to determine the quality of black tea. It had been documented that theanine, makes up approximately 50 % of total the amino acids of tea. It is synthesized in the roots and is transported to the tender parts of the plant for storage. In the presence of sunlight, it is converted to polyphenols. Apart from its contribution in the taste of tea, it has some medicinal values as well (Sugiyama and Sadzuka 2003). Glutamine synthase, an important enzyme of theanine biosynthesis pathway is involved in assimilation of ammonia generated by various biochemical processes, had been cloned from tea (Rana et al. 2008a, b). Based on their cellular locations of isozymes, they were classified into two groups- GS3A (cytosolic GS) and GS2 (chloroplast GS). The distinct, cell-specific patterns of expression conferred by the promoters for GS2 and GS3A suggesting the different roles in metabolism (Edwards et al. 1990).

8.2.1.2 Flavonoid Biosynthesis Related Genes

Polyphenols content in tea leaf account for almost 15–35 % of the total dry weight of the plants. Polyphenols are chemical compounds such as flavonoids and tannins found naturally in tea. Among the polyphenols, catechins are the most abundant, account for 80 % of the total polyphenols. Owing to its importance, several genes of this pathway such as PAL, chalcone synthase (CHS), anthocyanidin synthase (ANS), anthocyanidin reductase (ANR), chalcone isomerase (CHI), flavanone-3-hydroxylase (F3′H), flavanone-3-hydroxylase (F3′5′H), leucoanthocyantin reductase (LAR), dihydroflavonol 4-reductase (DFR) had been isolated, cloned and characterized from tea plants (Table 8.1). Among them, PAL catalyses the initial step for catechins biosynthesis, via phenyl-propanoid pathway, by deamination of phenylalanine evolving ammonium ion and trans-cinnamic acid. On the other hand, CHS and CHI (chalcone isomerase) catalyse the formation of flavonoids from flavanone (2S)-naringenin by sequential reactions of 4-coumaroyl-CoA and three molecules of malonyl-CoA (Matsumoto et al. 1994a).

Table 8.1 Genes cloned from Camellia species
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In a pioneer work, young leaves of the widely cultivated Japanese cultivar ‘Yabukita’ was used by Matsumoto et. al. (1994b) to construct the cDNA library and hybridized with rice PAL cDNA probe to clone the tea PAL gene. Alignments of amino acid sequence of tea PAL protein revealed higher homology with dicots than monocots confirming its evolutionary proximity in relation with dicot PAL genes. Later, a 280 bp from 3′ UTR of this gene was used as probe to test the segregation pattern among the mapping population. It showed that PAL in tea was multi-allelic and inherited as per the Mendelian monogenic ratio. Similarly, using heterologus probe, CHS gene of tea was cloned by screening the cDNA library which showed 85 % similarity between parsley CHS and CsCHS(Takeuchi et al. 1994a). Later several genes related to biosynthesis of flavonoids including LAR (Ma et al. 2010) had been cloned in tea (Table 8.1).

Further DFR, from tea (CsDFR) was also cloned. In vivo expression analysis through Q-PCR indicated younger leaves had higher level of expression than older leaves under controlled condition. However, it was down-regulated under dehydration stress and ABA treatment, but up-regulated upon wounding. Interestingly, all the regulation of this gene was modulated with catechins content in the respective tissue or stress (Singh et al. 2009a). Anthocyanidin reductase, a key enzyme of epicatechins pathway of tea (CsANR) had been cloned (Singh et al. 2009b). The functional protein, upon its expression in E. coli, catalysed the conversion of cyanidin to epicatechins in the presence of NADPH. PAL and C4H are two important enzymes in catechin biosynthesis. Fragment of C4H gene was isolated from tea by differential display and later full length gene was cloned using RACE technology. It had been found that catechin contents decreased in response to drought, ABA and GA3 treatments but increased in response to wounding. Interestingly, the expression of CsPAL and CsC4H transcripts were in similar trend along with catechin contents under the above treatments. Therefore, a positive correlation between catechin contents and gene expression was established during catechin biosynthesis (Singh et al. 2009c). Flavanone 3-hydroxylase (F3′H), an important enzyme of catechin biosynthesis pathway catalyses the stereospecific hydroxylation of (2S)-naringenin to form (2R, 3R)-dihydrokaempferol. The CsF3′H was cloned from tea and expressed in E. coli which yielded a functional protein. Furthermore, a positive correlation was detected between the concentration of catechins and CsF3′H expression at different developmental stages of tea plants (Singh et al. 2008).

Polyphenol oxidase is an important enzyme which plays a vital role during fermentation of black tea processing. Using degenerate primers, Zhao et al. 2001 were first to clone this gene from young tea leaf. Phylogenetic analysis indicated a close proximity of CsPPO to other woody plants. Later, in order to understand the allelic variation, CsPPO was cloned from five different genotypes of tea and potential genic-SNPs were detected. Wu et al. 2010 also cloned PPO from tea. It had a signal peptide targeting the chloroplast and three Cu binding domains. The mature PPO showed high expression and enzyme activity in E. coli strain BL21 under pET30c expression vector. 4-Coumaroyl-CoA (4CL) is another gene of catechin biosynthesis pathway. The full-length cDNA cloning of Cs4CL and its association with catechin accumulation in tissue was established. Altering catechin content through drought stress, ABA, GA3 treatments and wounding established a strong positive correlation coefficient between catechins content and the expression of Cs4CL (Rani et al. 2009).

To understand the molecular mechanism, Pang et al. (2013) characterized the gene of key enzymes involved in polyphenolic proanthocyanidins pathway (PA) of tea. Recombinant protein of tea leucoanthocyanidin reductase (CsLAR) expressed in E. coliwas found to be functional and with leucocyanidin as substrate, could produce the product 2R, 3S-trans-flavan-ol (+)-catechin under in vitro conditions. Two genes encoding anthocyanidin reductase, CsANR1and CsANR2, were also expressed in E. coli,and the recombinant proteins exhibited similar kinetic properties. Both converted cyanidin to a mixture of (+)-epicatechin and (-)-catechin, although in different proportions, indicating that both enzymes possess epimerase activity and hence functional in nature (Pang et al. 2013).

8.2.1.3 Purine Biosynthesis Related Genes

Caffeine, theobromine and theophylline are considered as purine alkaloids and flavour forming chemicals present in some species of Camellia. Caffeine and theobromine are purine alkaloids that are present in high concentrations in tea. However, most members of the genus Camellia contain no purine alkaloids. The major steps of caffeine biosynthetic pathway are: xanthosine → 7-methylxanthosine → 7-methylxanthine → theobromine → caffeine (Suzuki 1972; Ashihara and Kubota 1987; Ashihara et al. 1996, 1997; Kato et al. 1996; Deng et al. 2008).

Among the pivotal enzymes, S-adenosylmethionine synthase, convert methionine to S-adenosylmethionine. It serves as the methyl donor in transmethylation reactions and also acts as the precursor in ethylene and polyamine biosynthesis. So, isolation and cloning of SAMwas might pave the way for further study and manipulation of caffeine biosynthesis, stress and senescence physiology. Accordingly a CsSAM gene was cloned and ORF was found to be 1303 bp in length with 349 amino acids. This is an essential step to manipulate the caffeine biosynthesis in tea plant (Feng and Liang 2001).

In tea, among the three genes namely 7-N-methyltransferase (7-NMT), 3-N-methyltransferase (3-NMT) and theobromine N-methyltransferase (1-NMT), two (3-NMT and 1-NMT) are highly homologous due to similarity of many properties and are considered them as single enzyme, caffeine synthase (CS). Initially using degenerate primer, partially CsCSgene and later full length was cloned from young leaf of tea using RACE-PCR (Kato et al. 1999). The substrate specificity of the recombinant enzyme, after expression of cDNA in E. coli, remained similar to that of the original tea enzyme. The cloning of CS gene led to the development of transgenic caffeine deficient tea plants by antisense technology (Kato et al. 2000).

Tracer experiments using [8-14C] adenine and [8-14C] theobromine showed that the purine alkaloid pathway is not fully functional in leaves of purine alkaloid-free species. Interestingly, in purine alkaloid-free Camellia species, sufficient evidence was obtained to show the occurrence of genes that are homologous to caffeine synthase. Recombinant enzymes derived from purine alkaloid-free species showed only theobromine synthase activity. Unlike the caffeine synthase gene, these genes were expressed more strongly in mature tissue than in young tissue (Isida et al. 2009).

Mohanpuria et al. (2009) suggested that caffeine metabolism in tea appears to be dependent on developmental and seasonal factors. Expression of caffeine synthase as well as caffeine content were found to be higher in harvestable plant parts such as apical bud, 1st leaf, 2nd leaf, young stem, and was lower in older leaf during non-dormant growth periods compared to dormant growth phase.

8.2.1.4 Floral Aroma Formation Related Genes

Aroma is an important yardstick to determine quality of black tea and primarily the aroma forming compounds are produced by endogenous enzymes such as β-glucosidases and β-primeverosides. In order to increase quality of made tea more, it is obvious to pursue ways that ensure an increment in tea aroma forming compounds. The β-glucosidases a hydrolases was found to be involved in the formation of floral aroma (Takeo 1981). Later on many aroma forming compounds had been isolated and identified from green, as well as black tea leaves. Off late, the concept of hydrolyzation of polyglycoside by β-glucosidase vis-a-vis β-primeverosides and subsequent removal of floral aroma were established. It is a disaccharide specific glycosidase which hydrolyzes aroma precursors of β-primeverosides to liberate aroma compounds. Using degenerate primer, full length cDNA sequence of β-primeverosidase genes were cloned and characterized (Mizutani et al. 2002). It had 50–60 % identical protein sequence with β-glucosidases of other plants and classified this as family 1 glycosyl hydrolase. The enzyme expressed in E. coli hydrolyzed β-primeverosides to liberate a primeverose unit and aglycons under in vitro condition. It was confirmed that the β-primeverosidase selectively hydrolyzed the β-glycosidic bond of β-primeverosides between the disaccharide and the aglycons (Mizutani et al. 2002). Later, the β-glucosidase gene of tea was also cloned (Li et al. 2004b). It shared 40–60 % similarity in nucleotide sequence with other plants. Upon expressing in heterologus system in E. coli, it possessed normal bioactivity by breaking down the glycosidic bonds. Subsequently, the spatio-temporal expression pattern of the aforesaid genes in different leaves of young shoot was determined in tea (Zhao et al. 2006a). Also, it revealed the variation of expression pattern according to leaf position.

8.2.2 Abiotic Stress Related Genes

Differential expression of a cytosolic glutamine synthetase (CsGS) from tea during developmental stages and light or dark conditions on the utilization of nitrate as well as ammonia had been reported (Rana et al. 2010). The CsGS transcript expression level was highest in apical bud, gradually reduced towards the older leaf and was found to be lowest in the fourth leaf. Exposure to both nitrate and ammonia N-sources showed enhancing effect on CsGS enzyme activity during light but under dark, ammonium increased CsGS activity and nitrate had inhibitory effect (Rana et al. 2010).

Ammonium transporters (AMT) are involved in nitrogen absorption of plant roots, therefore important for utilization in nitrogenous fertilizer. This gene had also been cloned from tea root (Taniguchi and Tanaka 2004). Cold is an important abiotic stress for tea cultivation as it hampers the productivity of tea significantly. However not much cold responsive genes had been cloned from tea, except, CsCOR1, which had been cloned recently from tea leaves (Li et al. 2010). The deduced amino acid sequence contained hydrophobic N-terminus as a signal peptide and a chareteristic glycine, arginine and proline rich C-terminal domain. The gene was found to be localized in the cell-wall of transgenic tobacco by CsCOR1::GPF fusion approach. Its (CsCOR1) expression was up-regulated under cold and dehydration stress.

Sulphate assimilation is an important metabolic pathway for plants and ATP sulfurylase, the first enzyme of this pathway, converts ATP and sulphate to adenosine phosphosulphate. Two cDNAs encoding ATP sulfurylase (APS1 and APS2) were cloned from tea (Zhu et al. 2008a). While, ORF of APS1 was 1,415 bp in length with 360 amino acids, yet APS2 ORF was found to be 1706 bp in length which encoded 465 amino acids. They shared 59.6 % identity with each other. The predicted amino acid sequences exhibited 86 % and 84 % similarity to ATP sulfurylases of Medicago truncatula and Solanum tuberosum respectively.

A higher level of selenium is toxic to plants. Enzyme selenocysteine methyltransferase (SMT) methylates selenocysteine to Se-methyl selenocysteine and thus play vital role in toxicity removal. Full length cDNA of CsSMT had been cloned from tea (Zhu et al. 2008b). This sequence of amino acids shared 74 % and 69 % identity with Astragalus bisulcatus SMT and Brassica. oleracea SMT respectively. Expression of CsSMT correlated with the presence of SMT enzyme activity in cell extracts and bacterial recombinant CsSMT had higher tolerance to selenate and selenite.

8.2.3 Biotic Stress Related Genes

Pests and pathogens are harmful agents that act upon the bush health and productivity. Although both pest and diseases are important for tea cultivation, yet not much works have been done, primarily due to lack of tolerant genotype in tea. Wang et al. (2005) cloned the cystain gene with the help of degenerate primers from tea. The deduced amino acid sequence contained the motif QXVXG conserved among most members of cystatin superfamily. In an another study, eight wound/pathogen inducible cDNAs were cloned and the structure was analysised through in silico (Yoshida and Homma 2005). It had been found with high sequence homology with other wound/ pathogen inducible genes of herbaceous plants.

8.2.4 Energy Metabolism Related Genes

Green plants, by virtue of chlorophyll, absorb solar energy to produce organic substance and release oxygen through the process of photosynthesis. Though solar energy is the basis for photosynthesis, excessive energy may result in photo-oxidative damage to photosynthetic apparatus and other cell components. There exists some mechanism to protect the plants from such adverse effects. In tea plants two photosynthesis related genes had been cloned, which were photosystem II protein D1 and violaxanthin de-epoxidase genes (VDE). Among them, VDE had been studied extensively. Under excess light intensity, violaxanthin converted to antheraxanthin and ultimately to zeaxanthin by VDE, whereas under low light intensity zeaxanthin epoxidase (ZE) catalyse the reverse reaction. Zeaxanthin can protect photosynthetic apparatus from excessive irradiation. Thus VDE can be considered as a vital enzyme in the xanthophyll cycle-dependent photo protective mechanism. Using degenerate primers, Wei et al. (2003) cloned full length cDNA of VDE gene. It was demonstrated that the gene contained lipocalin signature which considered to be most active domains in VDE (Wei et al. 2004).

RUBISCO (Ribulose 1, 5-bisphosphate carboxylase) is a key enzyme in energy metabolism of the Calvin cycle pathway of photosynthesis. The full length RUBISCO small subunit (RbcS) was cloned from tea. The amino acids sequence had a high identity to those of other plant RbcSgenes. It had three conserved domains as well as a protein kinase C phosphorylation site, one tyrosine kinase phosphorylation site and two N-myristoylation sites. The Q-PCR analysis showed that the expression of RbcSwas the highest in youngest tea leaf (Ye et al. 2009).

8.2.5 Developmentally Regulated Genes

Microtubules composed of α-tubulin and β-tubulin dimers are one of the most important constituents of eukaryotic cytoskeleton. It is an important structural element involved in mitosis, cytokinesis and vesicular transport. Sediment directions of microfilaments on cell-walls, which control the growth of cells, are also regulated by microtubules. The β-tubulin gene had been cloned from tea (Takeuchi et al. 1994b). The putative amino acid sequence had a high similarity to β-tubulin genes from other plants.

The eukaryotic nucleosome octamer core consists of the four histones, H3, H2A, H2B and H4. Using differential display and RACE-PCR, a full-length histone H3.1 cDNA (CsH3) was cloned from tea leaves. During active growth, higher expression was observed in apical buds and decreased gradually with maturity of the leaf. During dormancy, drought stress and ABA treatments, the expression of CsH3 was severely down-regulated but up-regulated by GA3 treatment. Positive co-relations between CsH3 and active cellular growth suggested its role in plant growth and development (Singh et al. 2009d).

On the other hand, lipoxygenase (LOXs) is a large gene family which catalyzes the hydroperoxidation of free polyunsaturated fatty acids into different biologically active compounds and had been isolated from tea plant (CsLOX1). Heterologous expression in yeast showed that CsLOX1 protein conferred a dual positional specificity and hence it was named 9/13-CsLOX1. Analysis of the isolation and expression of the LOX gene in tea plant indicated that the acidic CsLOX1 together with its primary and final products played an important role in regulating cell death related to flower senescence and the JA-related defensive reaction of the plant to phloem-feeders (Liu and Han 2010). Pollen tube growth in tea was promoted by Tua 1 gene. The cDNA-AFLP technique was used by Fang et al. (2006) to isolate transcript-derived fragments corresponding to these genes. The complete cDNA sequence of this gene had been cloned which had two N-glycosylation sites and four protein kinase C phosphorylation sites.

8.2.6 Other Genes

Ribosomes play an important role in plant evolution with 5.8S, 16S and 26S rRNA being the prime components of ribosome. The QM like protein gene encodes for ribosomal protein which has a function of ribosome stability. Full length QM cDNA from tea (CsQM) had been cloned (Singh et al. 2009d) which shared 71–87 % and 85–91 % identity at nucleotide and amino acid sequences, respectively with QM genes isolated from other woody plants.

8.3 Differentially Expressed Genes

Understanding the global transcript changes under a stress, cell types, tissue or developmental stage is a robust approach. Although several techniques are available (Mondal and Sutoh 2013; Das et al. 2013), suppression subtractive hybridization (SSH), cDNA AFLP have been widely used to study the differential gene expression in tea. For an example, SSH had been used to understand the molecular regulation of secondary metabolism pathways, in the young leaves (Park et al. 2004; Chen et al. 2005), to identify the cold responsive genes (Wang et al. 2009), to identify dormancy-associated genes (Wang et al. 2010; Krishnaraj et al. 2011; Paul and Kumar 2011; Yang et al. 2012a; Phukon et al. 2012 ;Thirugnanasambantham et al. 2013) and to identify the drought responsive genes (Sharma and Kumar 2005; Das et al. 2012; Gupta et al. 2012; Muoki et al. 2012; Gupta et al. 2013). A forward SSH library under drought stress was constructed from TV-23 (a drought tolerant cultivar) roots, to ascertain 123 drought responsive genes (Das et al. 2012). Recently 7 cDNA libraries from various organs of tea plants were used to generate 17,458 ESTs of tea (Taniguchi et al. 2012). Interestingly, to investigate the molecular mechanism by which light regulates phenylpropanoid metabolism, a light-induced SSH library of tea calli was constructed. Several genes in lignin biosynthesis pathways were identified in the light-induced library (Wang et al. 2012). To understand molecular mechanism of enhanced aroma under mild infestation of green leaf hopper (Empoasca vitis Göthe) in tea leaf, a SSH library was constructed and several genes were identified (Yang et al. 2011) and interestingly it was found that insect infection triggered the several genes that were responsible for quality. Furthermore, through cDNA-AFLP approach, they identified 108 transcript-derived fragments that were expressed differentially in the leaf of drought tolerant tea cultivars (Gupta et al. 2013). Eungwanichayapant and Popluechai (2009) reported that the expression of phenylalanine ammonia-lyase 1, chalcone synthase, dihydroflavonol 4-reductase, leucoanthocyanidin reductase, and flavanone 3-hydroxylase genes were higher in the young shoots than in the mature leaves which were obvious as most of the quality compounds were produced by these genes. These genes are normally found to be more expressive in young leaves. Nineteen genes were found to be up-regulated with Ectropic obliqua infection as detected by SSH, differential screening, and Q-PCR analysis. These genes were involved in JA synthesis, cell-wall modification, metabolism of secondary metabolites as well as carbohydrate, pathogen defensive response and oxidative stress protection. The results showed that Ectropic obliqua infections activated defense related genes and induced tea defense responses (Qiao et al. 2011).

In Taiwan, ‘oriental beauty’, a high flavoured made tea from leaf hopper infestation is very popular. In order to know the genes responsible for the same, gene expression profile and biochemical profile were done by DNA microbead array, a fluorescence labeled based technique where different fluorescence DNAs are sorted out by cell sorter machine. Interestingly several stress responsive genes were found to be up-regulated in infested tea leaves (Choi et al. 2007). Darjeeling teas of India are the highest grown teas in the world and best -known for its flavour, aroma and quality. Apart from the genetic makeup of the plant, reports suggested that insect infestation, particularly jassids and thrips triggered the aroma and flavour formation in Darjeeling tea. Several genes and transcription factors were identified through the SSH library of leaf that were highly infested by these insects (Gohain et al. 2012).

Expression of genes encoding enzymes involved in flavan-3-ol biosynthesis pathway such as, CHS, CHI, F3H, F3′-5′H, DFR, ANS, ANR and LARwas investigated. Transcripts of all genes, except LAR, were more abundant in leaves and stems than in roots and cotyledons. No significant difference of expression was found for the transcript of LAR. In tea, flavan-3-ols are produced by a naringenin-chalcone to naringenin to dihydrokaempferol pathway. Dihydrokaempferol is a branch point in the synthesis of (-)-epigallocatechin-3-O-gallate and other flavan-3-ols which can be formed by routes beginning with either a flavonoid 30-hydroxylase mediated conversion of the flavonol to dihydroquercetin or a flavonoid 30,50-hydroxylase-catalysed conversion to dihydromyricetin with subsequent steps involving sequential reactions catalysed by dihydroflavanol 4-reductase, anthocyanidin synthase, anthocyanidin reductase and flavan-3-ol gallate synthase (Ashihara et al. 2010). Expression of PAL, CHS, DFR were differentially expressed under red and blue light, however, their expression were lower in mature leaf than the younger leaf (Takechi and Matsumoto 2003).

R2R3-MYB, bHLH, and WD40 transcription factors are well-known to control multiple enzymatic steps in the biosynthetic pathway responsible for the production of flavonoids, important secondary metabolites of tea. The presence of 73 R2R3-MYB, 49 bHLH, and 134 WD40 transcripts were identified through in silico analysis of 127,094 unigenes of tea. In silico analysis of protein sequences of CsMYB4-1, CsMYB4-2, CsMYB4-3, CsMYB4-4, CsMYB5-1, and CsMYB5-2 indicated presence of [DE]Lx2[RK]x3 Lx6 Lx3R motif, potentially contributing to the specificity of the bHLH partner in the stable MYB-bHLH complex. Q-PCR analysis validated selected genes and their expression profiles under various developmental stages and treatment conditions, including hormones (Zhao et al. 2012).

To investigate the molecular mechanisms by which light regulates phenylpropanoid metabolism, light-induced SSH cDNA libraries from tea calli was prepared. Nine diverse ESTs involved in phenylpropanoid biosynthesis were detected in the library. Among them, CsDFR gene (CsDFR2) was found to be up-regulated by light but had sequence difference with previously reported CsDFR gene (CsDFR1) of tea. The key phenylpropanoid compounds and representative genes expression analysis implied that light could be effective for activation of the biosynthesis of phenylpropanoids. Compared to the dark treatment as control, levels of lignins, catechins, and PAs were increased 3.46, 3.00, and 1.21-fold, in light-induced calli, respectively. Lignin biosynthesis genes, involved in CCoAOMT, HCT and CCR, were identified in the light-induced SSH library (Wang et al. 2012a). The plant hormone, auxin plays a key role in adventitious rooting. To increase our understanding of genes involved in adventitious root formation, Wei et al. (2013) identified differentially expressed transcripts in single nodal cuttings of tea treated with or without IBA. A total of 77 differentially expressed transcripts, including 70 up-regulated and 7 down-regulated sequences, were identified that were expressed under IBA treatment. Influences of shade on flavonoid biosynthesis in relation to expression of the flavonoid pathway genes in tea leaves were analyzed. It was found that shade had notable effects on both flavonoids (including catechins, O-glycosylated flavonols) and proanthocyanins (PAs) and lignin biosynthesis, but had no significant effect on anthocyanin accumulation. Among all the compounds, the concentration of PAs and O-glycosylated flavonols under shaded leaves reduced more than other compounds, compared to the sunlight-exposed leaves. Expressions of PAL, flavanone 3-hydroxylase, flavonoid 3-hydroxylase, DFR and ANR1 were notably correlated with the concentration of PAs in leaves, and expression of CHS and flavonoid 3,5-hydroxylase (F3′-′5H) were correlated with the concentration of O-glycosylated flavonols. It was suggested that polymerization of catechins and glycosylation of flavonols might be key pathways of flavonoid metabolism in tea leaves affected by shade treatment. On the other hand, phenolic acids were increased under shade and a negative correlation with lignin accumulation suggested that phenolic acids might compete for the same substrate with lignins and flavonoids in tea leaves under different illumination conditions (Wang et al. 2012c).

In order to identify the differentially expressed genes related to morphological variation of callus, Yang et al. (2012a) analyzed two contrasting callus lines (Yunjing 63Y and Yunjing 63X) that showed different morphological characteristics and catechin contents. Yunjing 63Y callus was yellow and compact, while yunjing 63X callus was white and loose. Using cDNA AFLP, they identified 68 genes that were differentially expressed between the two lines. Of the 68 differentially expressed ESTs, 40 showed higher expressions in Yunjing 63Y and remaining 28 showed higher expressions in Yunjing 63X. Gray blight disease (Pestalotiopsis theae) is an economically important diseases of tea which account enormous loss to the tea industry. Senthilkumar et al. (2012) used SSH technique to identify the differential gene expression pattern during gray blight disease infection in tolerant cultivar UPASI-10. Majority of the up-regulated genes were related to hypersensitive response and reactive oxygen species production.

Three tea clones with varying degree of polyphenol content were selected to identify genes that were related to polyphenol biosynthetic pathway. Totally, 1,680 gene tags were obtained from the cDNA library to develop the first cDNA microarray of tea plant. Each gene probe in the microarray was duplicated in the slides. Finally, two genes that were closely related to tea aroma were selected for Q-PCR analysis to validate the cDNA microarray. The newly developed cDNA microarray could be applied in various tea research fields to make the high-throughput detection in the gene expression profiling (Zhao et al. 2006b). Cross species hybridization were also attempted to understand the gene expression of tea. Using Arabidopsis cDNA array, Venkatesh et al. (2006) found that highest level of induction of CHS gene in the tea leaf during the second flush (a period where flavonoids content of tea was found to be highest in tea planataion of Assam, India). On the other hand, expressions of cinnamyl alcohol dehydrogenase (CAD), cinnamyl CoA reductase (CCR) and POD involved in the synthesis of lignin were reduced during the second flush. These observations suggested that CHS may be a key enzyme involved in catechin biogenesis in tea leaf. Similarly, to identify, the Al uptake responsive genes in tea, Murayama et al. (2007) used cross-species microarray analysis. For the target gene of microarray analysis, approximately 8,500 cDNA fragments were identified from Arabidopsis thaliana. According to the signal intensity of cDNA detected by microarray analysis, putative genes including auxin-induced protein, zinc finger family protein, blue copper binding protein, were found to be expressed differentially under Al treatment in tea. Therefore, it was suggested that antioxidant enzymes genes and synthesis of auxin as well as cell-wall genes might be induced by Al treatment in tea root tips.

To understand the molecular mechanism of albinism, tea cultivar ‘Anji Baicha’, was selected which was a special green-revertible albino mutant, widely cultivated in China for producing high quality green tea. A total of 671 differentially expressed genes at different albescent stages were identified using cDNA microarray analysis. The corresponding genes were involved in energy metabolism, carbon fixation, cell expansion, secondary metabolism, plant growth and defence, and other physiological processes including protein, nucleotide synthesis, etc. Particularly, some differentially expressed genes encoding important catalyzing enzymes or regulatory proteins which took part in chlorophyll biosynthesis or chloroplast development were identified. Some candidate genes possibly related to the albino process were further analyzed by Q-PCR. The present study gave some useful clues for genes worthy of further understanding the albino phenotype of ‘Anji Baicha’ and also provided a model for utilization of the microarray technology in the tea plant (Ma et al. 2012).

8.4 Proteomics and Metabolomics

Proteomics study correlates the potential protein modifications to particular phenotypes through the techniques such as HPLC, mass spectrometry, SDS-PAGE, two-dimensional gel electrophoresis, in silico protein modeling, matrix-assisted laser desorption/ionization (MALDI) etc.

Li et al. (2008) did the proteomics analysis to identify the differentially expressed proteins of pollen that were associated with cold stress. Further, changes in protein expression, in the embryo of tea in response to desiccation, had been investigated and differentially expressed proteins were identified. Around twenty-three different proteins related to defense response, metabolism and redox status were up-regulated under desiccation. This finding is particularly important as tea seeds being recalcitrant, maintain high rate of metabolism and water content during the seed maturation stage (Chen et al. 2011). In China, an albino mutant of tea were identified which had typical white leaf phenotype. In order to understand the protein involve in albino leaf, proteomics analysis were done which identified 61 proteins during the three developmental stages of an albino cultivar of tea. Important proteins that played crucial roles in the periodic albinism were identified (Li et al. 2011). Altogether these studies basically identified the differentially expressed proteins with compared to their control counterparts which at this stage mere indicate the various pathways that are involved in that particular situation.

Metabolomics is also powerful technique and in tea particularly very relevant as quality is a complex trait, mixture of several metabolites of various nature. Metabolic characteristics associated with climatic variables, were investigated by 1H NMR spectroscopy on tea grown in different areas of South Korea, thereby allowing for the assessment of quality strategy in green tea production (Lee et al. 2010). Differential metabolites expressed under shade grown tea plants had been detected by LC-MS and GC-MS spectrometry coupled with a multivariate data set which helped to understand the effect of shade on tea growth (Ku et al. 2010). Further, the dependence of global green tea metabolome on plucking positions was investigated through NMR analysis coupled with multivariate statistical data set which correlated the age of plucking shoots with quality of made tea (Lee et al. 2011).

8.5 System Biology

Systems biology is a biology-based inter-disciplinary field of study that focuses on complex interactions within biological systems, using a more holistic perspective. Tea quality is a highly complex trait and tea manufacture induces a variety of stresses that affect quality. Using cross species Affymetrix Arabidopsisgenome arrays, Marshall et al. (2007) attempted to track transcriptional changes occurring during wounding and withering of the leaves to identify metabolic pathways that could influence tea aroma and flavour. Arabidopsismetabolic SBML (Hucka et al. 2003) network data from AraCyc (Mueller et al. 2003), KEGG and Reactome were collated and merged, then subsequently overlaid with the tea expression data. Sub networks were constructed by connecting the shortest paths between the differentially expressed genes and the downstream aroma-related compounds, therefore identifying the pathways involved in aroma.

8.6 Bioinformatics

In silico analysis is an integral part of the functional genomics today. With the accumulation of large scale ESTs in the public domain, researchers started to use for various studies. The emerging computational approach provides a better alternative process of development of SSRs from the ESTs than the conventional methods. In the present study, 12,851 ESTs of tea, were mined for the development of microsatellites. Finally, 6,148 (4,779 singletons and 1,369 contigs) non-redundant ESTs were found using various computational tools. Out of total 3822.68 kb sequence examined, 1,636 (26.61 %) ESTs containing 2,371 SSRs, were detected with a density of 1 SSR/1.61 kb leading to development of 245 primer pairs. These mined EST-SSR markers will help further in the study of variability, mapping, evolutionary relationship in tea. In addition, these developed SSRs can also be applied for various studies across species (Sahu et al. 2012). Using similar approaches, miRNAs of tea were identified along with their stem-loop structure from the ESTs (Das and Mondal 2010; Prabu and Mandal et al. 2010).

Structural analysis of a cytosolic glutamine synthetase from tea (CsGS) had been conducted employing computational techniques. This was conducted to compare its structural aspects with other known structures of CsGS. The disordered residues and their distribution in CsGS were in close comparison to earlier reported GS. The 3-D structure of CsGS protein also showed high degree of similarity with the only known crystal structure of GS from maize (Yadav 2009). Similarly, coding sequence of the ICE1in tea was analyzed with CodonW, CHIPS (codon heterozygosity in a protein coding sequence) and CUSP (create a codon usage table) programs, while compared with the genome of tea plant and ICE1from seven plant species. The results showed that ICE1of tea plant was bias toward the synonymous codons with A and T. (Shi et al. 2012).

The stearoyl-acylcarrier protein desaturase (SAD) gene is widely present in all kinds of plants. Tea SAD gene (CsSAD) sequence was analyzed by various bioinformatics tools such as CodonW, CHIPS, and CUSP programs, and compared with publicly available tea sequences, other SAD genes from 11 plant species. It had been found that CsSAD gene had similar codon usage bias. The CsSAD gene had a bias toward the synonymous codons with A and T at the third codon position. Compared with monocotyledons such as Triticum aestivum and Zea mays, the differences in codon usage frequency between the CsSAD gene and dicotyledons such as Arabidopsis thaliana and Nicotiana tobacum were less. Therefore, A. thaliana and N. tobacum expression systems may be more suitable for the expression of the CsSAD gene. The analysis result of SAD genes from the 12 plant species also showed that most of the SAD genes were biased toward the synonymous codons with G and C at the third codon position (Pan et al. 2013).

8.7 High Throughput Sequencing

Recently, the next generation sequencing technology, specially RNA-seq is widely used for identification of differentially expressed genes (Mondal and Rana 2013; Mukhopadhayay et al. 2013). For the first time, an extensive transcriptome dataset had been generated by RNA-Seq from the young leaf of tea plants. The coverage of the transcriptome was comprehensive enough to discover all known genes of several major metabolic pathways. This transcriptome dataset can serve as an important public information platform for gene expression, genomics, and functional genomics studies in tea (Shi et al. 2011). In the same year, Jiang et al. (2011) did deep transcriptome sequencing of C. oleifera, C. chekiangoleosa and C. brevistyla using 454 GS FLX platform. After removal of the adaptors as well as low complexity sequences, finally 182,766, 190,545 and 132,147 reads of C. oleifera, C. chekiangoleosa and C. brevistyla, were generated respectively. These reads were assembled into 49,909 contigs which were annotated using BLAST searches. Based on the information, they could estimated the similarity of gene expression in different species, detected and validated SNPs.

In order to know the genes involved secondary metabolism, high-throughput sequencing of tea leaf transcriptoms was done. A total of 437,908 reads were generated using 454 GS FLX platform. De novo assembly of those reads yielded 25,637 unigenes, 22,872 of which were annotated by BLAST searches against public databases. Some abundant transcripts were found to be related to developmentally regulated in plants, including ubiquitin/26S proteasome, lipid transfer protein, PPR containing protein, small GTPase, expansin, transport inhibitor response 1 and thioredoxin. Additionally, 3,767 EST-SSRs were identified as potential molecular markers in our unigenes. A total of 100 PCR primer pairs used in initial screening test among the 20 tea genotypes successfully identified 36 polymorphic loci (Wu et al. 2013).

Changes that occur at the molecular level in response to low temperature are poorly understood in tea plants. To elucidate the molecular mechanisms of cold acclimation, Wang et al. (2013) employed RNA-Seq and digital gene expression (DGE) technologies to the study of genome-wide expression profiles during cold acclimation in tea plants. In total, 1,770 differentially expressed transcripts were identified, of which 1,168 were up-regulated and 602 down-regulated. These included a group of cold sensor or signal transduction genes, cold-responsive transcription factor genes, plasma membrane stabilization related genes, osmosensing-responsive genes, and detoxification enzyme genes. DGE and Q-PCR analysis further confirmed the results from RNA-SEq. Pathway analysis indicated that the ‘carbohydrate metabolism pathway’ and the ‘calcium signaling pathway’ might play a vital role in tea plants’ responses to cold stress (Wang et al. 2013).

8.8 Conclusion

Functional and structural genomics both are new development of biotechnology. These ‘omics’ approaches along with other advance techniques of microscopy as well as DNA sequencing are very potential for deciphering several fundamental questions of gene expression. However, large-scale application of ‘omics’ has not been done perhaps primarily due to the reason of non-availability of complete genome sequence of tea. Although in several aspects of tea molecular biology work can be intended for, yet priority should be given: (i) to undertake a de novo whole genome sequencing for development of reference genome, (ii) to re-sequence popular trait-specific cultivars or extreme genotypes to generate large scale SNPs markers and their utilizations for association mapping, gene introgression, high density linakge map constructions etc., (iii) some other aspects which need an urgent priority in tea are genome-wide discovery of small RNAs, high throughput allele-mining, sequence information-based conservation and utilization of tea genetic resources etc.