## 2 Results and Discussion
### Transcriptome Sequencing  
To achieve a global overview of the gene expression profile in _C. sinensis_, cDNA libraries of five tissues were constructed and sequenced. Pyrosequencing of five different tissues generated 148075495 reads, including 36293915 from the bud, 25938680 from FL, 28732060 from ML, 28438538 from SL and 28672302 from the stem (Supplemental Table S1). All sequences were assembled to obtain 1447580 contigs.  
According to the functional annotation, 82 genes involved in terpen pathway have been identified, annotated as 28 enzymes in two known terpene pathways MVA and MEP [15; 16] (Figure 1). Heatmaps constructed using 82 genes (Figure 3) showed that bud, SL and FL were clustered together but separate from ML, indicating the patterns of sequencing were changed according to the different tissues and position in the plant. To validate the accuracy of differential gene expressions, 12 genes in the terpene pathways were randomly selected and their expression levels were checked by qRT-PCR using specific primers as shown in Supplemental Table S2. As shown in, the expression patterns of the 12 genes were in accordance with those of RNA-Seq data.  
Figure 2: A heatmap coupled with clustering analysis for 41 terpen detected in five tissues of tea plants. ML, mature leaf; SL, the second leaf; FL, the first leaf.  
In plants, two biosynthetic pathways are responsible for the synthesis of IPP and DMAPP, the universal precursors of all terpenoids. Although the subcellular compartmentalization of the two pathways allows them to operate independently, metabolic cross-talk between the two pathways has been reported [22; 23]. For the MVA pathway, 33 genes were identified and annotated as AACT, HMGS, HMGR, MVK, MPDC, PMK, IDI and FDS. IPP derived from the MVA pathway in the cytosol is further acted upon by IDI, a divalent, metal ion-requiring enzyme, to form dimethylallyl diphosphate (DMAPP). Then condensation of one DMAPP and two IPP molecules catalyzed by FDS leads to the formation of FPP(C\({}_{15}\)), the precursor of sesquiterpenes [24].  
There are 20 genes in the MEP pathway, including DXS, DXR, MCT, CMK, MDS, HDS and HDR. This pathway leads to the formation of GPP (C\({}_{10}\)), the unique precursor for monoterpenes [24]. The MEP pathway was first elucidated in _Escherichia coli_, and plant homologs have been characterized using a combination of biochemical and genomic approaches [25]. Geranyl diphosphate synthase catalyzes the condensation of one molecule IPP and DMAPP to produce GPP (C\({}_{10}\)), the universal precursor of monoterpenes [24]. Then, an array of TPS catalyzes FPP and GPP to generate kinds of terpenoids. Taken all together, 29 genes were annotated as TPSs, including CIN/LIC, MYS/OCS, LIS/NES, NUDX, FAS, CPS/HUS, SQS and BAS, and among them, nerolidol synthase (CsNES) [17], \(\beta\)-ocime  
Figure 3: A heatmap coupled with clustering analysis of 82 terpen related genes in five tissues of tea plants. ML, mature leaf; SL, the second leaf; FL, the first leaf.  
synthesize (CsOCS) [18] and linalool synthase (CsLIS) [19] were recently functionally characterized in _C. sinensis_ plants.  
Six enzymes were identified to generate monoterpenes, and 11 enzymes catalyze FPP to generate sesquiterpenes. Two farnesene synthase (FAS) was identified; the highest reads per kilobase per million (RPKM) value is for the bud, followed by FL and stem.