Integrated Transcriptomic and Metabolomic Analysis Reveals the Mechanism Underlying Trichome Formation on the Stem Epidermis of A. mongholicus and Its Impact on Bioactive Compound Accumulation

doi:10.13560/j.cnki.biotech.bull.1985.2026-0172

Abstract

Abstract:

Objective This study aimed to identify key genes regulating stem trichome formation in Astragalus mongholicus and to investigate how pilose and glabrous stems affect the accumulation of flavonoids and saponins. Methods Phenotypic characteristics of glabrous and pilose stems were examined using scanning electron microscopy (SEM). An integrated approach combining transcriptome sequencing (RNA-seq) and untargeted metabolomics (UPLC-MS/MS) was employed to elucidate the regulatory mechanism of trichome formation and associated metabolic changes. Furthermore, high-performance liquid chromatography (HPLC) was employed to analyze the effects of these two phenotypes on the accumulation of major bioactive compounds in both stems and roots. Results SEM revealed that the stem epidermal trichomes of A. mongholicus are non-glandular trichomes. RNA-seq analysis identified 15 917 differentially expressed genes (DEGs), which were significantly enriched in the pathways related to trichome development, plant hormone signal transduction, and flavonoid biosynthesis. Among them, the trichome development-related genes GIS, GIS3-like, GIS3, HAT1 and ETC1 may be highly involved in the formation of non-glandular trichomes. Metabolomics profiling revealed 670 differentially accumulated metabolites (DAMs) in the epidermis of glabrous stems using the pilose stem epidermis as a control, among which 443 metabolites were up-regulated and 227 were down-regulated. The DAMs are mainly enriched in the biosynthetic pathways of phenylalanine, tyrosine and tryptophan, as well as nucleotide metabolism pathways. HPLC quantification confirmed a significant decrease (P<0.01) in the content of calycosin-7-O-β-D-glucoside in glabrous stems. In the roots of glabrous plants, the contents of calycosin and total content of the four saponin components were significantly reduced (P<0.05). Conclusions Several candidate genes regulating stem trichome formation in A. mongholicus were screened out. The stem trichomes of A. mongholicus are closely associated with the accumulation of bioactive ingredients including isoflavonoids and saponins. The pilose stem phenotype exhibits better agronomic performance than the glabrous stem phenotype.

Key words: Astragalus mongholicus, metabolomics, transcriptomics, non-glandular trichomes, flavonoids, saponins

LEI Zi-ying, WU Yan-hui, ZHAO Sheng-ting, ZHANG Yu, WANG Jia-lei, HE Run-li, LIU Jian-xia, WANG Rui-jun. Integrated Transcriptomic and Metabolomic Analysis Reveals the Mechanism Underlying Trichome Formation on the Stem Epidermis of A. mongholicus and Its Impact on Bioactive Compound Accumulation[J]. Biotechnology Bulletin, doi: 10.13560/j.cnki.biotech.bull.1985.2026-0172.

Figures/Tables 12

Fig. 1 PCA scoring plot (A) and volcano plot of DEGs (B)

Fig. 2 GO enrichment analysis plot of DEGsThe first circle represents the enriched terms and their corresponding categories, namely biological process (blue), molecular function (gray), and cellular component (red). The second circle shows the number of background genes in each term and the statistical significance value -log10 (q value) for the category; a longer bar indicates more genes enriched in the GO term, and a darker color denotes a smaller q value, meaning more significant enrichment. The third circle indicates the total number of foreground genes, with a longer bar standing for a larger number of genes. The fourth circle with bars of different heights represents the enrichment factor, where a higher bar corresponds to a larger enrichment factor

Fig. 3 KEGG enrichment analysis plot of DEGs

Table 1 Linear regression equations for flavonoids and saponins in A. mongholicus

化合物 Compound	线性方程 Linear regression equation	R²	线性范围 Linear range （μg）
毛蕊异黄酮苷	Y=39 109 157 C+180 233	0.999 5	0.050-4.970
芒柄花苷	Y=27 793 749 C-48 296	0.999 8	0.128-7.680
毛蕊异黄酮	Y=6 355 527 C-7 323	0.999 9	0.038-2.280
芒柄花素	Y=5 436 799 C-20 586	0.999 8	0.031-1.860
黄芪甲苷	Y=1.660 2 C+5.335 3	0.999 2	0.180-3.300
黄芪皂苷Ⅰ	Y=1.543 2 C+5.947 6	0.999 9	0.588-9.420
黄芪皂苷Ⅱ	Y=1.585 7 C+5.442	0.999 5	0.300-4.800
黄芪皂苷Ⅲ	Y=1.323 2 C+6.408	0.999 8	0.242-3.880

Fig. 4 Heat map of hormone-related DEGs (A) and differentially expressed transcription factors (B)The data in the heatmap are row-normalized relative expression (TPM) values. Red indicates upregulated relative expression within each row and blue indicates downregulation. The same below

Table 2 Determination results of flavonoid and saponin contents in A. mongholicus (x¯±s, n=10)

组别（mg/g）	PHS	GHS	PHR	GHR
毛蕊异黄酮苷	0.11±0.04	0.04±0.01**	1.48±0.50	1.76±0.35
芒柄花苷	0.04±0.01	0.04±0.01	0.53±0.37	0.44±0.17
毛蕊异黄酮	0.15±0.08	0.18±0.06	0.25±0.12	0.13±0.07^#
芒柄花素	0.01±0.01	0.02±0.01*	0.04±0.03	0.02±0.03
4种异黄酮总和	0.32±0.08	0.28±0.05	2.30±0.52	2.35±0.50
黄芪甲苷	-	-	0.25±0.07	0.26±0.08
黄芪皂苷Ⅰ	-	-	1.05±0.24	0.78±0.35
黄芪皂苷Ⅱ	-	-	0.61±0.18	0.48±0.16
黄芪皂苷Ⅲ	-	-	0.08±0.01	0.13±0.07
4种皂苷成分总和	-		1.98±0.36	1.65±0.36^#

Fig. 5 Heat map of flavonoid-related DEGs

Fig. 6 Validation results of RT-qPCRThe histograms represent the relative expression levels of each target gene measured by RT-qPCR (2-ΔΔCt), and the red lines indicate the corresponding RNA-seq (TPM values), x¯±s, n=3

Fig. 7 PCA (A) and OPLS-DA (B) score plots of metabolitesPC1 represents the first principal component and PC2 the second principal component, with the percentages indicating their respective explanation rates of the dataset; each dot in the figure denotes a sample, and samples from the same group are colored the same

Fig. 8 Data plot of DAMsA: volcano plot of DAMs; B: classification chart of DAMs; C: heat map of differentially accumulated flavonoid metabolites

Fig. 9 KEGG enrichment analysis plot of DAMs

Fig. 10 Diagram of DEGs and DAMs in the flavonoid pathway of stemsMetabolites are marked in boxed labels: red indicates significantly increased metabolite content, while green indicates decreased metabolite content. Related genes are annotated beside the arrows: red represents upregulated genes, green represents downregulated genes, and blue indicates genes with both upregulated and downregulated expression patterns. Rectangular boxes of different colors in the diagram correspond to distinct metabolic branches: the yellow box denotes the phenylpropanoid pathway, the pink box represents the isoflavonoid pathway, and the blue box refers to the flavone and flavonol biosynthesis pathway

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