Biosynthesis of Camphane Volatile Terpenes in Amomum villosum Lour: Involved Genes and Enzymes

Abstract

Amomum villosum (A. villosum) Lour., a medicinal species of the Zingiberaceae family, is used for medical purposes. Bornyl acetate, camphor, and borneol are the main bioactive ingredients in A. villosum fruit, and the amount of bornyl acetate is regarded as a measure of the fruit's quality. In order to explore the major effective genes regulating the biosynthesis of camphane volatile terpenes in A. villosum, some DEGs involved in camphane volatile terpene biosynthesis and transcription factors were analyzed and summarized in this study. The result showed that the content of bornyl acetate was altered in the different growth stages. In particular, the significant change occurred from 7 to 30 DAP (days after pollination). The content of bornyl acetate at 30 DAP was 169.3% more than that at 7 DAP. In total, 4782 up-regulated and 5284 down-regulated unigenes were found in G2 vs. G1, as well as 3324 up-regulated and 5036 down-regulated unigenes in G3 vs. G1, and 3332 up-regulated and 4490 down-regulated unigenes in G3 vs. G2. A total of 323 up-regulated and 820 down-regulated unigenes were shared in three growth stage comparisons. We screened the genes that encode the enzymes most likely to inhibit bornyl diphosphate synthase, borneol dehydrogenase, and BAHD acyltransferases. Interestingly, we found that borneol dehydrogenase and bornyl diphosphate synthase displayed bi-substrate features, suggesting that a substrate of catalyzation is promiscuity in the biosynthesis downstream pathway, and the unknown bornyl pyrophosphate hydrolase may not be the specific enzyme for borneol formation. Additionally, the DXR, HDS, and IDS found in the PPI network would assist in the understanding of molecular regulation. The results of this study constructed DGE libraries and identified key genes related to camphane volatile terpenes, which laid a foundation for a deep investigation of the mechanism of volatile terpene biosynthesis, and provided a reference for mining other key genes in A. villosum fruits.

Keywords: biosynthesis pathway; camphane volatile terpenes; transcriptome.

Figure 1

Changes in Amomum 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 ± SD (n = 3). Note: * indicates significant differences in the index values of various treatment durations (p < 0.05).

Figure 2

Overview of transcriptome at different growing stages of fruit (A) Number of differentially expressed genes (DEGs) (log2 Foldchange ≥ 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.

Figure 3

GO functional classification of DEGs. (A) G2 vs. G1. (B) G3 vs. G2. (C) G3 vs. G1.

Figure 3

GO functional classification of DEGs. (A) G2 vs. G1. (B) G3 vs. G2. (C) G3 vs. G1.

Figure 4

KEGG pathway enrichment analysis of DEGs. (A) G2 vs. G1. (B) G3 vs. G2. (C) G3 vs. G1.

Figure 4

KEGG pathway enrichment analysis of DEGs. (A) G2 vs. G1. (B) G3 vs. G2. (C) G3 vs. G1.

Figure 4

KEGG pathway enrichment analysis of DEGs. (A) G2 vs. G1. (B) G3 vs. G2. (C) G3 vs. G1.

Figure 5

Network analysis of protein interaction based on the DEGs. (A) G2 vs. G1. (B) G3 vs. G1. (C) G3 vs. G2. Red nodes represent DXR, yellow IDS, and blue HDS.

Figure 6

Putative amphane volatile terpene biosynthesis pathway in Amomum villosum Lour. DXS: 1-deoxy-D-xylulose-5-phosphate synthase, EC: 2.2.1.7; DXR: 1-deoxy-D-xylulose-5-phosphate reductoisomerase, EC:1.1.1.267; MCT: 2-C-methyl-D-erythritol 4-phosphate cytidylyltransferase, EC:2.7.7.60; CMK: 4-(cytidine 5′-diphospho)-2-C-methyl-D-erythritol kinase, EC:2.7.1.148; MCS: 2-C-methyl-D-erythritol 2,4-cyclodiphosphate synthase, EC:4.6.1.12; HDS: 4-hydroxy-3-methylbut-2-en-1-yl diphosphate synthase, EC:1.17.7.1; IDS: 4-hydroxy-3-methylbut-2-en-1-yl diphosphate reductase, EC:1.17.1.2; AACT: acetyl-CoA acetyltransferase, EC:2.3.1.9; HMGS: 3-hydroxy-3-methylglutaryl coenzyme A synthase, EC:2.3.3.10; HMGR: 3-hydroxy-3-methylglutaryl coenzyme A reductase, EC:1.1.1.34; MK: mevalonate kinase, EC:2.7.1.36; PMK: phosphomevalonate kinase, EC:2.7.4.2; MVD: diphosphomevalonate decarboxylase, EC:4.1.1.33; IPI: isopentenyl-diphosphate Delta-isomerase, EC:5.3.3.2; GPPS: geranyl-diphosphate synthase, EC:2.5.1.1; BPPS: bornyl diphosphate synthase, EC: 5.5.1.8; BPPH: bornyl pyrophosphate hydrolase, EC: 3.1.7.3; BDH: borneol dehydrogenase, EC: 1.1.1.198; BAHD: BAHD acyltransferase, EC: 2.3.1.

Figure 7

Expression profile of DEGs in terpenoid and polyketide biosynthesis pathways and putative transcription factors. (A) Expressions of DEGs associated with biosynthesis pathways of terpenoids and polyketides. (B) Expressions of DEGs involved in transcription factors.

Figure 8

Validation of the RNA-Seq results with qRT-PCR experiments. Fifteen unigenes associated with the biosynthesis of camphane volatile terpenes were selected and subjected to qRT-PCR analysis. The expression levels between RNA-Seq and qRT-PCR experiments for these unigenes are shown in panels (a–o), respectively. The error bar for the qRT-PCR results corresponds to the variations among the three biological replicates and three technical replicates. The value of r is the Pearson’s correlation coefficients between the expression profiles obtained from the RNA-Seq and qRT-PCR experiments. Note: * indicates significant differences in the index values of various treatment durations (p < 0.05).