## 2 Results and Discussion
### Gene Function Annotation and Classification
rigescens_ through the KEGG database predicted a total of five categories (level 1, cellular processes, environmental information processing, genetic information processing, and metabolism and organismal systems), 31 sub-categories (level 2, Figure 6) and 238 pathways (level 3). Unigenes identified as related to the \"Translation\" (861, 10.77%), \"carbohydrate metabolism\" (852, 10.65%), \"Folding, sorting and degradation\" (699, 8.74%) and \"Signal transduction\" (685, 8.56%) were the top four representative pathways (Figure 6). Unigenes counts for \"Terpenoid backbone biosynthesis\", \"Monoterpenoid biosynthesis\", \"Diterpenoid biosynthesis\", \"Sesquiterpenoid and triterpen biosynthesis\", and \"Ubiquinone and other terpenoid-quinone biosynthesis\" were 55, 5, 22, 21, and 31, separately. These results indicated that the terpenoid pathways were active in flowering gentian, and the corresponding genes would be candidate genes for gentiopicroside biosynthesis. Gene expression was calculated using the RPKM method, which takes into account both sequencing depth and gene length effects on read count [22]. On the basis of the applied criteria _q_-value <0.005 and log2(foldchange) >1, 3306 genes (4.31% of all genes) were identified as significantly differentially expressed genes (DEGs) between these two tissues, which comprised 2204 up-regulated genes  
Figure 6: KEGG classification map. The ordinate is the name of the pathway, while the abscissa is the proportion of genes belonging to this pathway. These genes were divided into five branches: (A, Cellular Processes; B, Environmental Information Processing; C, Genetic Information Processing; D, Metabolism; E, Organismal Systems.) according to the metabolic pathway they participated in. (accounting for 67%) and 1102 down-regulated genes (33%) in leaves (Figure 7, Table S4). The log2(fold changes) ranged from one to 15. Not surprisingly, among these DEGs, most were related to photosynthesis, for example, ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO)-a, a key enzyme of the Calvin-Benson cycle of autotrophic CO2 assimilation [23], chloroplast chlorophyll a/b-binding protein, photosystem II 22 kDa protein gene, and chloroplastic ferredoxin genes, were all up-regulated over 10-fold in leaves compared to roots. The terpenoid biosynthesis related genes, such as geranyl diphosphate synthase (GPPS), geraniol synthase (GES), geraniol 10-hydroxylase (G10H), and iridoid oxidase (IO), four key enzymes involving monoterpene biosynthesis, were all up-regulated over 10-fold in leaves compared to roots. Of the down-regulated genes, a late embryogenesis abundant (LEA) protein, was 13-fold higher in roots than in leaves. Late Embryogenesis Abundant (LEA) proteins are a group of hydrophilic proteins with a high content of glycine, and are associated with stress tolerance in plants and animals through protecting enzymatic function and inhibition of aggregation in dehydration, heat, and salt stress [24, 25]. In _Arabidopsis thaliana_, overexpression of _LEA14_ enhances salt stress tolerance [26]. Ectopic expression of _ZmLEA5C_ in tobacco and yeast enhances their tolerance to osmotic and low temperature stresses [27]. A calcium-dependent protein kinase (CDPK) gene involved in plant defense responses [28] was nine-fold higher in roots than in leaves. Previous research suggests that CCaMK is an important  
Figure 7: Volcano plot of Leaf _ves_. Root in _G. rigescens_. The abscissa represents changes of gene expression (Leaf _vs._ Root). The ordinate represents the statistical significance of change of the amount of gene expression. The less _p_-value, the more -log10(_p_ value), and the more significance. The scattering dots represent genes, while the blue dots show genes without significant differences and _vice versa_ for red dots. component of the symbiosis signaling pathway [29; 30; 31; 32; 33; 34]. In _Zea mays_, calcium/calmodulin-dependent protein kinase (ZmCCaMK) is required for abscisic acid (ABA)-induced antioxidant defense systems [35].