# Triterpennoids Biosynthesis Regulation for Leaf Coloring of Wheel Wingnut (_Cyclocarya paliurus_)
## 3 Results
### Transcriptomic and Metabolomic Association Analysis  
According to the transcriptome and metabolome analysis, we constructed the triterpenoid biosynthesis pathway to uncover the mechanism regulating triterpenoids accumulation during leaf development of LC1 [11; 12; 13]. In Figure 3, structure genes showed significant expression differences; most detected structure genes showed a random regulating trend except HMGS (hydroxymethylglutural-CoA synthase), HMGR (hydroxymethylglutural-CoA reductase), and FPPS (farnesyl-diphosphate synthase). HMGS and HMGR belong to the MVA pathway of terpenoid biosynthesis pathway, promoting the transformation from acetyl CoA to MVA. On the contrary, the MEP pathway was not active compared to the MVA pathway and DXS (1-deoxy-D-xylulose-5-phosphate synthase) was relatively silent during leaf development. In addition to the triterpenoids backbones biosynthesis pathway, FPPS (farnesyl-diphosphate synthase) exhibited an active expression level while other structure genes were relatively silent including GPPS (geranyl-diphosphate synthase) and SM (squalene monooxygenase). In the lupane-type triterpenoid saponins biosynthesis pathway, LS (lupeol synthase) DEGs were detected with a relatively random expression level. Metabolome analysis results showed that total triterpenoids increased during leaf growth, and most triterpenoids including madasiatic acid and 3-O-(2-O-Acetyl-glucosyl) oleanolic acid exhibited significant increases. It could be inferred that up-regulation of the MVA pathway promoted an increase in total triterpenoids. Figure 2: Differential expressed genes (DEG) in growing leaves of LC1. (**A**) PCA plots displaying DEG of LC1 leaves in A, B and C stage with 3 replicates, (**B**) Volcano plots displaying the up-regulated, down-regulated, and no-regulated genes between green-colored and red-colored leave samples, (**C**) Venn diagram showing the shared and unique DEGs between the 3 compared groups of leave samples. AG = genes expression in old green leave period; BG = gene expression in semi-red leave period; CG = gene expression in new red leave period; (**D**) DEGs clusters according to the expression level in different leave coloring period. (**E**) Top KEGG terms contributed by DEGs of CG vs. AG. Figure 3: Modulation of triterpenoids biosynthesis pathway genes during growing process of LC1 leaves. The histogram displays the expression levels of the genes (FPKM value). The charts in blue and red represent the down and upregulated structural genes. AACT: acetyl-CoA C-acetyltransferase, HMGR: hydroxymethylglutaryl-CoA reductase, HMGS: hydroxymethylglutaryl-CoA synthase, PMD: phosphomevalonate decarboxylase, IPK: isopentenyl phosphate kinase, MK: mexulonate kinase, PMK: phosphomevalonate kinase, MDC: dipphosphorvalonate decarboxylase, GPPS: geranyl-diphosphate synthase, DXR: 1-deoxy-D-xylulose-5-phosphate reducto isomerase, DXS: 1-deoxy-D-xylulose-5-phosphate synthase, MCT: 2-C-methyl-D-eryththird 4-phosphate cytidyltransferase, MDS: 2-C-methyl-D-eryththird 2,4-cyclodiphosphate synthase, CMK: 4-diphosphocytidyl-2-C-methyl-D-eryththird 1-phosphate cytidylyltransferase. HDR: 4-hydroxy-3-methylbut-2-en-1-yl diphosphate reductase, HDS: (E)-4-hydroxy-3-methylbut-2-enyl-diphosphate synthase, IDI: isopentenyl-diphosphate Delta-isomerase, IPP: Isopentenyl pyrophosphate, GPPS: geranyl-diphosphate synthase, FPPS: farnesyl-diphosphate synthase, SM: squalene monooxygenase, SS: squalene synthase, LS: lupeol synthase, LSS: lunosterol synthase, CAS: cycloartenol synthase, \(\alpha\)-AS: \(\alpha\)-amyrin synthase, \(\beta\)-AS: \(\beta\)-amyrin synthase, DDS: dammareediol II synthase. According to previous research, CYP450 s and glycosyltransferase (UGTs) have been considered to be involved in the biosynthesis of triterpene saponins [9]. CYP450 s catalyzes the structural modification of the triterpene backbones for hydroxylation, carboxylation, alkylation, kekylation, and dehydrodehydration to form the intermediate of triterpene saponins.