Potential Regulatory Differences of Endogenous Hormones on Flavonoids in Anoectochilus roxburghii at Different Growth Stages Based on Multi-omics Analysis

doi:10.13560/j.cnki.biotech.bull.1985.2025-0861

Abstract

Abstract:

Objective To investigate the influences of endogenous hormones at different growth stages of Anoectochilus roxburghii on flavonoid metabolism and to reveal the molecular basis for the differences in flavonoid accumulation. Method Using A. roxburghii as the experimental material, leaves from different growth stages (S3: 3 months, S6: 6 months, S10: 10 months) were selected. High-throughput RNA sequencing (RNA-Seq) and liquid chromatography-mass spectrometry (LC-MS/MS) were employed to obtain the transcriptomic and metabolomic data of A. roxburghii. Bioinformatics analysis was used to predict the differential enzyme genes related to flavonoids, metabolites, and differential hormone metabolites, and to explore the relationships among them. Result A total of 17 differential enzyme genes related to flavonoids, 16 differential metabolites, and 19 differential hormone metabolites were predicted. F3'H and FLS were identified as the key genes affecting flavonoid metabolism in the leaves of A. roxburghii, while cyanidin 5-O-β-D-glucoside 3-O-β-D-primeveroside and cyanidin 3-O-β-D-primeveroside might be the key metabolites causing the color differences in the leaves. Correlation analysis showed that 11 differential hormone metabolites were significantly related to 13 differential enzyme genes related to flavonoids, such as CHS, PGT1 and F3H. Moreover, 19 transcription factors were predicted to regulate eight differential enzyme genes, including F3H, F3'H, FLS, CHS, PGT1, and CCoAOMT. Among these transcription factors, 16 contained cis-elements responding to plant hormones (salicylic acid, abscisic acid, methyl jasmonate, gibberellin, ethylene, and auxin). The expressions of six genes (three transcription factors and three enzyme genes) were verified by qRT-PCR, and the results were consistent with the transcriptomic analysis. Conclusion At different growth stages, 16 transcription factors in A. roxburghii respond to five types of hormone elements, thereby regulating the expressions of flavonoid metabolism-related enzyme genes F3'H and FLS, and ultimately affecting the accumulation of flavonoid metabolites, delphinidin 5-O-β-D-glucoside 3-O-β-D-sambubioside and cyanidin 3-O-β-D-sambubioside.

Key words: Anoectochilus roxburghii, flavonoids, plant hormones, transcriptome, metabolome

ZENG Jing-jing, LUO Pan-lan, YAN Shu-jun, ZHENG Tao, YANG Jun-jie, CAI Kun-xiu, CAO Jia-yu, ZHANG Tian-xiang, LI Luan, CHEN Ying. Potential Regulatory Differences of Endogenous Hormones on Flavonoids in Anoectochilus roxburghii at Different Growth Stages Based on Multi-omics Analysis[J]. Biotechnology Bulletin, 2025, 41(12): 190-200.

Figures/Tables 8

Fig. 1 Anoectochilus roxburghii at different growth stagesFrom left to right, they are three-month-old, six-month-old, and ten-month-old, respectively

Fig. 2 Clustered heatmap of differential flavonoid and plant hormone metabolites in the leaves of A. roxburghii at various growth stagesA: Flavonoid metabolites; B: Plant hormone metabolites

Fig. 3 Hierarchical-clustering heatmap of flavonoid-synthesis-related differentially expressed genes in A. roxburghii leaves at different growth stages

Fig. 4 Integrated transcriptome-metabolome analysis of flavonoids in A. roxburghii leaves at different growth stages

Fig. 5 Correlation network between phytohormones and flavonoid-related genes in A. roxburghii leaves at different growth stagesPlant hormone metabolites are denoted by KEGG annotation numbers. Solid lines indicate positive regulation, and dashed lines indicate negative regulation

Fig. 6 Regulatory network between transcription factors and key enzyme genes of flavonoid synthesis in A. roxburghii leaves at different growth stages

Fig. 7 Cis-regulatory element profiling of flavonoid-related transcription factors in A. roxburghii leaves at different growth stages

Fig. 8 RT-qPCR analysis of differential enzymes and transcription factors in A. roxburghii leaves at different growth stages

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