# Biosynthesis of Camphane Volatile terpenes in _Annomum_ _villosum_ Lour: Involved Genes and Enzymes
## 2 Results
### Functional Classification of the DEGs  
After a GO enrichment analysis of the DEGs, there were 76, 134, and 126 enriched GO terms, consisting of various biological processes in the three comparisons of G2 vs. G1, G3 vs. G2, and G3 vs. G1, respectively (Figure 3). There were 46, 14 and 16 terms in BP, CC, and MF functional ontologies in G2 vs. G1, respectively. Many of the enriched terms were found in the metabolism and cellular component, including'metabolic process' (GO:0008152), 'organic substance metabolic process' (GO:0071704), 'primary metabolic process' (GO:0044238), 'cell' (GO:0005623), 'cell part' (GO:0044464), and 'intracellular' (GO:0005622), which correspond to the results of the morphology changes. In contrast, a very high number of DEGs enriched GO terms for G3 vs. G2 and G3 vs. G1, which consist of'metabolic process' (GO:0008152), 'organic substance metabolic process' (GO:0071704), 'primary metabolic process' (GO:0044238), 'catalytic activity' (GO:0003824), and 'ion binding' (GO:0043167) (Table S1). This implied that secondary metabolites, including bornyl acetate, were mainly accumulated in the later stage, which agreed with the findings of the bornyl acetate content analysis of _A. villosum_ fruits at various growth stages.  
Figure 1: Changes in _Anomum villosum_ (_A. villosum_) Lour. fruit at different growing stages. (**A**) Development of _A. villosum_ 24 days after pollination. The figures below the picture correspond to the sampling time. Red scale bar, 1 cm. (**B**) Changes in fruit diameter of _A. villosum_. (**C**) The relative content percentage of bornyl acetate in _A. villosum_ at different growth stages. Values are shown as the mean \(\pm\) SD (\(n=3\)). Note: * indicates significant differences in the index values of various treatment durations (\(p<0.05\)).  
Figure 3: _Cont._  
Figure 2: Overview of transcriptome at different growing stages of fruit (**A**) Number of differentially expressed genes (DEGs) (log2 Foldchange \(\geq\) 1 and adjusted \(p\) < 0.05, \(p\)-value was adjusted using \(q\)-value). (**B**) Distribution of DEGs across the three developmental stages. (**C**) Heatmap showing the cluster analysis of all DEG expression profiling.  
The biological functions of various genes were coordinated in vivo, and pathway substantial enrichment allowed the identification of the key biochemical metabolic and signal transduction pathways involved in differentially expressed genes. Overall, KEGG pathway enrichment analysis revealed 40, 35 and 35 pathways in G2 vs. G1, G3 vs. G2, and G3 vs. G1, respectively (Table S2). The pathways in G2 vs. G1 were mainly involved in primary metabolite biosynthesis and metabolism, such as 'biosynthesis of amino acids' (ko01230), 'phenylalanine, tyrosine, and tryptophan biosynthesis' (ko00400),'starch and sucrose metabolism' (ko00500), and the signaling transduction pathway 'plant hormone signal transduction' (ko04075). In addition, we found several pathways associated with secondary metabolites both in G3 vs. G2 and G3 vs. G1. Besides the primary metabolite pathways, 'terpenoid backbone biosynthesis' (ko00900) and 'flavone and flavonoid biosynthesis' (ko00944) were found in both. After comparation of G3 vs. G1 and G3 vs. G2,'steroid biosynthesis' (ko00100) and 'brassinosteroid biosynthesis' (ko00905) were found in G3 vs. G1, but 'tropane, piperidine and pyridine alkaloid biosynthesis' (ko00960) and 'diterpenoid biosynthesis' (ko00904) were found in G3 vs. G2 (Figure 4). All these results suggest that the genes involved in camphane volatile terpene biosynthesis might show significant expression in later stages.  
Figure 4: _Cont._  
Figure 3: GO functional classification of DEGs. (**A**) G2 vs. G1. (**B**) G3 vs. G2. (**C**) G3 vs. G1.