Combined metabolomic and transcriptomic analysis reveals key candidate genes involved in the regulation of flavonoid accumulation in Anoectochilus roxburghii

doi:10.1016/j.procbio.2020.01.004

Process Biochemistry

Volume 91, April 2020, Pages 339-351

https://doi.org/10.1016/j.procbio.2020.01.004 Get rights and content

Highlights

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A. roxburghii was first characterized using high-throughput sequencing technologies and metabolomics.
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Correlation analysis between metabolites and regulatory genes revealed flavonoid accumulation-related regulatory genes.
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This study screened and obtained key genes potentially involved in the accumulation of flavonoids in A. roxburghii.
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This study provides an in-depth understanding of the molecular mechanisms underlying flavonoid accumulation in A. roxburghii.

Abstract

Anoectochilus roxburghii, a species used in Chinese herbal medicine, has unique characteristics. This plant has important medicinal and ornamental value and is distributed primarily in China, including Fujian, Zhejiang, Jiangxi, and Guizhou provinces, and in Taiwan. Flavonoids are involved in leaf pigment formation and are major pharmacodynamic substances. However, the molecular mechanisms that regulate accumulation of flavonoids remain unclear, which has significantly limited their application. To elucidate the molecular mechanisms underlying the regulation of flavonoid accumulation in A. roxburghii, root, stem and leaf samples were collected for constructing transcriptomic and metabolomic datasets using RNA sequencing and liquid chromatography-mass spectrometry (LC–MS) techniques. Sequencing of the transcriptomes of the different organs generated 60.2 Gb of data, which were assembled into 186,865 unigenes. Metabolomic analysis resulted in the identification of 10,690 metabolites. Based on the ESI⁺ mode, 4,010, 4,008 and 4,013 metabolites were annotated in roots, stems, and leaves, respectively; based on the ESI– mode, 1,530, 1,530, and 1,531 metabolites were annotated, respectively. Differential analyses of the transcriptome and flavonoid metabolism of the different organs revealed the greatest significant differences between roots and leaves, followed by differences between stems and leaves; differences between roots and stems were the smallest. According to analysis of differentially expressed genes (DEGs), the secondary metabolism-related DEGs between roots and stems, between roots and leaves, and between leaves and stems were classified into 16, 21, and 19 secondary metabolic Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways, respectively. Among these pathways, flavonoid biosynthesis, flavone and flavonol biosynthesis, and isoflavonoid biosynthesis significantly differed in terms of both gene expression and secondary metabolism. Moreover, the key genes involved in flavonoid accumulation were comprehensively analyzed using metabolic and transcriptomic data, and ten transcription factor-encoding genes and fourteen flavonoid biosynthesis genes were identified. Specifically, four of the ten transcription factor-encoding genes appear to activate CHS, CHI, CYP75A and ANR gene expression. The combined approach used in this study provides an in-depth understanding of the molecular regulatory mechanisms underlying flavonoid accumulation in A. roxburghii and is a powerful tool that can uncover valuable information for plant breeders.

Graphical abstract

Introduction

Anoectochilus roxburghii (Wall.) Lindl is a perennial evergreen plant species of the Orchidaceae family. A. roxburghii has unique characteristics and value for use in Chinese herbal medicine; indeed, it is often used as an important medicinal and health care product in China and other Asian countries [1]. This plant is known as “the king of medicine” because of its multiple pharmacological effects and is applied for treating liver diseases, cardiovascular diseases, diabetes, cancer, and nephritis [[1], [2], [3], [4]]. Because it is a small, exquisite plant with gracefully shaped leaves and networks of golden yellow veins, A. roxburghii also has high ornamental value and is prized for its indoor foliage [2]. Owing to its important medicinal and ornamental value, A. roxburghii is receiving increased attention.

The secondary metabolites of A. roxburghii, especially flavonoids, are involved in leaf pigment formation and are a major source of the pharmacological activities of this species [5,6]. Therefore, it is important to study the molecular regulatory mechanisms underlying flavonoid accumulation in A. roxburghii. Furthermore, the contents of flavonols, such as quercetin, kaempferol and isorhamnetin, are often used as indicators to measure the quality of A. roxburghii [1,6]. Additionally, A. roxburghii is rich in flavonoid compounds, including dihydroquercetin, quercetin, kaempferol, and myricetin [2,6], and differences in the contents of quercetin, kaempferol, myricetin, and total flavonoids can affect pigment formation [6,7].

In recent years, long-term harvesting of A. roxburghii has led to a scarcity of wild resources of this species, pushing it to the verge of extinction. Although wild A. roxburghii was once the only source, the success of artificial cultivation techniques has revolutionized the procurement of the plant [8,9]. Recent studies regarding the medicinal value of A. roxburghii have focused primarily on resource collection of varieties, quality evaluation, active ingredient analysis, and pharmacological effects. Conversely, few studies have investigated the molecular genetics of A. roxburghii, including molecular identification and genetic diversity analyses. Genomic information concerning A. roxburghii is also not available, which has been the primary bottleneck for molecular genetics research in this species [2,8,9]. However, there has been marked progress in plant functional genomics in recent years. In general, transcriptomics is widely used to elucidate the biosynthesis pathways and regulatory mechanisms of key drug-related metabolites in different medicinal plant species [10,11]. Additionally, metabolomic analyses of medicinal plants have greatly facilitated the identification of the metabolic pathways of active pharmaceutical compounds [12]. Hence, integration of transcriptomics and metabolomics has been widely applied to identify the accumulation mechanisms underlying key metabolic pathways, especially the biosynthetic mechanisms of nonmodel plant species [11,13].

In this study, a transcriptomic database of A. roxburghii organs was constructed using a sequencing platform and bioinformatic analysis. Analysis of the expression profiles of different organs utilizing this database will aid in the identification of functional genes in A. roxburghii, laying the foundation for molecular genetics investigations in this species. To date, the molecular regulatory mechanisms of flavonoid accumulation in A. roxburghii affecting leaf pigment production and drug efficacy remain unclear, yet the metabolic pathways and metabolites in different organs, including roots, stems, and leaves, can be analyzed via metabolomics to identify differences in flavonoid accumulation. The data obtained in this study provide important technical support for future research on the accumulation and metabolic regulation of flavonoids in different organs of A. roxburghii and will assist in the identification of other active drug compounds.

Section snippets

Sample collection

Sample materials were collected from Nanjing, Zhangzhou, Fujian Province, China, and identified as A. roxburghii. In March 2016, A. roxburghii was cultivated in Nanjing according to the underwood planting method. The planting site is located at 24′ 26″ north, 117′ 36″ east, at approximately 300 m above sea level, and is characterized as a southern Fujian hilly landform. The annual average temperature is 20.4 °C, and the annual rainfall is 2,001.3 mm. The forest is dominated by evergreen

Construction and functional analysis of the transcriptome libraries of A. roxburghii roots, stems and leaves

The transcriptomes of the root, stem, and leaf libraries of A. roxburghii were sequenced, resulting in 60.2 Gb of data. After raw data were filtered and removed, the average sizes of clean reads of the transcriptomic data were 44.60, 44.58, and 44.61 Mb for roots, stems, and leaves, accounting for 78.87 %, 78.68 %, and 77.24 % of the total reads, respectively. The Q20 (%) clean reads accounted for 97.99 %, 97.70 %, and 97.69 % of the clean reads from the root, stem, and leaf libraries,

Discussion

Transcription factors are involved in many biological processes, and their gene expression is likely related to different traits of different organs; however, flavonoid accumulation is also involved in many biological processes. Therefore, genes related to the different traits of different organs might also be linked to flavonoid accumulation processes [24,[1], [2], [3], [4], [5], [6], [7], [8], [9], [10], [11], [12], [13], [14], [15], [16], [17], [18], [19], [20], [21], [22], [23], [24], [25],

Conclusion

In this study, A. roxburghii was for the first time characterized by high-throughput sequencing and metabolomic techniques. In total, 186,865 unigenes and 10,690 metabolites were identified by transcriptomic and metabolomic approaches. By comprehensively analyzing metabolomic and transcriptomic data, we investigated the regulatory network underlying flavonoid accumulation in different A. roxburghii organs. Correlation analysis between the metabolites and genes revealed regulatory genes related

Author contributions statement

SL obtained the funding and designed and supervised all the experiments. YC performed most of the experiments, analyzed the results, and wrote the article. JS performed some of the experiments. WP critically reviewed the manuscript. All authors read and approved the final manuscript.

Funding

This work was financially supported by grants from the Department of Technology and Science of Fujian Provincial Government (Grant No. 2017J01614), the Fujian Department of Finance Scientific Research Project (Grant No. k81139231) and the Education Department Project of Fujian Province (Grant No. JAT160169).

Declaration of Competing Interest

The authors declare that they have no competing interests.

Acknowledgments

The authors would like to acknowledge the staff of the State Forestry Administration Engineering Research Center of Chinese Fir for their excellent technical support.

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Light quality affects plant leaf growth and its metabolism of amino acids and polyphenols. Anoectochilus roxburghii has been used for food, medicine, and ornamental purposes. To obtain the optimal light quality for improved accumulation of metabolites in A. roxburghii in vitro. The seedings were cultured under the white light (W), blue light (B), red light (R), red: blue light = 1:2 (R1B2), red: blue light = 1:1 (R1B1), red: blue light = 2:1 (R2B1). Twenty compounds in raw leaves were in situ rapidly identified within 20 s by neutral desorption-extractive electrospray ionization mass spectrometry (ND-EESI-MS) without any sample pretreatment, including eight amino acids, eleven polyphenols, and kinsenoside. Meanwhile, leaf area and the activities of superoxide dismutase (SOD), peroxidase (POD), and catalase (CAT) were evaluated. Our results showed that leaf area and antioxidant enzyme activities of the R2B1 were significantly higher than B or R group. The red-blue light increased the content of isoleucine, histidine, serine, arginine, and promoted the accumulation of caffeic acid, ferulic acid, quercetin, myricetin, kaempferol, and rutin in phenylpropane metabolism, especially in the R2B1, which could be used as the optimal light quality. ND-EESI-MS could enhance our understanding of the effect of light quality on the accumulation of amino acids and polyphenols in A. roxburghii.
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Water stress is an important factor affecting herb quality and yield and exogenous spermidine (Spd) addition can enhance dehydration tolerance of plants. Anoectochilus roxburghii (Wall.) Lindl is a perennial herb widely used in medicine, health care, and beauty industry, yet, its dehydration resistance remains largely unknown. In this view, the current research aimed at elucidating the drought resistance mechanism of A. roxburghii under PEG-induced water stress at different levels (10%, 20%, and 30% PEG6000). In particular, the morphological, physiological, and biochemical changes occurring in leaves treated with (0.5 mM, 5 mM) or without Spd were discussed under moderate water stress (20% PEG6000). Results showed that water stress significantly inhibited the growth of A. roxburghii by decreasing the activities of superoxide dismutase (SOD), peroxidase (POD), catalase (CAT), arginine decarboxylase (ADC), S-adenosylmethionine decarboxylase (SAMDC), and polyamine oxidase (PAO). The contents of soluble protein, soluble proline, hydrogen peroxide (H₂O₂), malondialdehyde (MDA), and the relative conductivity (REC) of leaves increased by 27.32%, 31.37%, 68.84%, 103.61%, and 63.64%, respectively. Under dehydration condition, foliar spraying Spd effectively improved plant growth with the contents of soluble protein and soluble proline increased, while the H₂O₂, REC, and MDA contents decreased. The SOD activity of 0.5 mM and 5 mM treatments were 1.13 and 1.01 times higher than only 20% PEG treatment, respectively. Similarly, the POD activity of 0.5 mM and 5 mM treatments increased by 52.82% and 34.11%, while the CAT activity of 0.5 mM and 5 mM treatments increased by 34.39 and 33.15 U/mgprot, respectively. In addition, exogenous Spd application significantly increased the key enzyme activities of polyamine metabolism (ADC, SAMDC, and PAO) under moderate water stress. In conclusion, exogenous Spd addition with low concentration (0.5 mM) improved the resistance of A. roxburghii against water stress by increasing the antioxidant enzymes activities and endogenous polyamines contents. This study helps to the understanding of dehydration resistance of A. roxburghii and provide guidelines for its cultivation under water deficit condition.
Flavonoid profile of Anoectochilus roxburghii (Wall.) Lindl. Under short-term heat stress revealed by integrated metabolome, transcriptome, and biochemical analyses
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Global warming severely threatens plant growth, and could lead to yield reduction. Although findings suggest that flavonoids play important roles in biological process in plants, their response to heat stress in Anoectochilus roxburghii (Wall.) Lindl. remains unclear. Here, we aimed to examine the flavonoid profile of A. roxburghii under heat stress and assess the effect of exogenous application of quercetin on heat stress tolerance. Metabolome analysis showed that quercetin, tricetin, isorhamnetin, scutellarein, and 4′,7-Isoflavandiol were the main upregulated flavonoids in A. roxburghii, based on variable importance in the projection >1 and with fold change >2. Determination of the concentrations of the flavonoids using a standard curve revealed that quercetin, kaempferol, and isorhamnetin contents increased by 8.24-, 7.55-, and 5.01-fold, respectively, during heat stress, whereas rutin concentration decreased from 83.04 to 80.89 mg/kg (dry weight). Additionally, transcriptome analysis indicated increased expression of several genes in flavonoid biosynthesis pathways, including phenylalanine ammonia-lyase and chalcone synthase. Moreover, exogenous application of quercetin improved the antioxidant capacity and physiological parameters, including photosynthetic rate and chlorophyll content, of A. roxburghii under heat stress. Overall, the flavonoid profile of A. roxburghii under short-term heat stress was characterized based on integrated metabolomic, transcriptomic, and biochemical analyses, providing new insights for improving the biological value of A. roxburghii.
Combined analysis of transcriptomics and metabolomics revealed complex metabolic genes for diterpenoids biosynthesis in different organs of Anoectochilus roxburghii
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Citation Excerpt :
Therefore, the research on the diterpenoids biosynthesis of A. roxburghii is beneficial to further explore its formation mechanism of medicinal value. In recent years, with the development of biological technology in the application of medicinal plants, the combination of transcriptomics and metabolomics analysis method has been widely used in the research of biosynthesis and regulatory mechanisms of active ingredients in medicinal plants (Chen, Yao, et al., 2020; Chen, Pan, Jin, & Lin, 2020; Gong et al., 2020; Lin et al., 2019; Nett, Lau, & Sattely, 2020; Zhang, Li, Chen, Meng, & Guo, 2020). Nett et al. established a metabolic route of tropolone-containing colchicine alkaloids by transcriptomics, metabolomics analysis and pathway reconstruction, which almost completely elucidated the biosynthetic pathway of colchicine (Nett, Lau, & Sattely, 2020).
Diterpenoids with a wide variety of biological activities from Anoectochilus roxburghii, a precious medicinal plant, are important active components. However, due to the lack of genetic information on the metabolic process of diterpenoids in A. roxburghii, the genes involved in the molecular regulation mechanism of diterpenoid metabolism are still unclear. This study revealed the complex metabolic genes for diterpenoids biosynthesis in different organs of A. roxburghii by combining analysis of transcriptomics and metabolomics.
The differences in diterpenoid accumulation in roots, stems and leaves of A. roxburghii were analyzed by metabonomic analysis, and its metabolic gene information was obtained by transcriptome sequencing. Then, the molecular mechanism of differential diterpenoid accumulation in different organs of A. roxburghii was analyzed from the perspective of gene expression patterns.
A total of 296 terpenoid metabolites were identified in the five terpenoid metabolic pathways in A. roxburghii. There were 38, 34, and 18 diterpenoids with different contents between roots and leaves, between leaves and stems, and between roots and stems, respectively. Twenty-nine metabolic enzyme genes with 883 unigenes in the diterpenoid synthesis process were identified, and the DXS and FDPS in the terpenoid backbone biosynthesis stage and CPA, GA20ox, GA3ox, GA2ox, and MAS in the diterpenoid biosynthesis stage were predicted to be the key metabolic enzymes for the accumulation of diterpenoids. In addition, 14 key transcription factor coding genes were predicted to be involved in the regulation of the diterpenoid biosynthesis. The expression of genes such as GA2ox, MAS, CPA, GA20ox and GA3ox might be activated by some of the 14 transcription factors. The transcription factor NTF-Y and PRE6 were predicted to be the most important transcription factors.
This study determined 29 metabolic enzyme genes and predicted 14 transcription factors involved in the molecular regulation mechanism of diterpenoid metabolism in A. roxburghii, which provided a reference for the further study of the molecular regulation mechanism of the accumulation of diterpenoids in different organs of A. roxburghii.
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Combined metabolomic and transcriptomic analysis reveals key candidate genes involved in the regulation of flavonoid accumulation in Anoectochilus roxburghii

Highlights

Abstract

Graphical abstract

Introduction

Section snippets

Sample collection

Construction and functional analysis of the transcriptome libraries of A. roxburghii roots, stems and leaves

Discussion

Conclusion

Author contributions statement

Funding

Declaration of Competing Interest

Acknowledgments

J. Ethnopharmacol.

Carbohydr. Polym.

J. Ethnopharmacol.

Prog. Biophys. Mol. Biol.

Toxicon

J. Chromatogr. B Analyt. Technol. Biomed. Life Sci.

Trends Plant Sci.

J. Photochem. Photobiol. B

Plant Physiol. Biochem.

Am. J. Clin. Nutr.

Gene

Curr. Opin. Biotechnol.

Plant Sci.

Simultaneous Structural Identification of Natural Products in Fractions of Crude Extract of the Rare Endangered Plant Anoectochilus roxburghii Using 1H NMR/RRLC-MS Parallel Dynamic Spectroscopy

Int. J. Mol. Sci.

Effects of supplemental lighting with different light qualities on growth and secondary metabolite content of Anoectochilus roxburghii

PeerJ

Effects of Light Quality on Morphology, Enzyme Activities, and Bioactive Compound Contents in Anoectochilus roxburghii

Front. Plant Sci.

The complete chloroplast genome sequence of Anoectochilus roxburghii

Mitochondrial DNA A DNA Mapp. Seq. Anal.

DNA barcodes for the identification of Anoectochilus roxburghii and its adulterants

Planta

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Plant Cell Rep.

Combining metabolomics and transcriptomics to characterize tanshinone biosynthesis in Salvia miltiorrhiza

BMC Genomics

Current advances in the metabolomics study on Lotus seeds

Front. Plant Sci.

Network analysis of the metabolome and transcriptome reveals novel regulation of potato pigmentation

J. Exp. Bot.

HLAscan: genotyping of the HLA region using next-generation sequencing data

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Sci. Data

De novo assembly and comparative analysis of the transcriptome of embryogenic callus formation in bread wheat (Triticum aestivum L.)

BMC Plant Biol.

DEGseq: an R package for identifying differentially expressed genes from RNA-seq data

Bioinformatics

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PLoS One

Simultaneous untargeted and targeted metabolomics profiling of underivatized primary metabolites in sulfur-deficient barley by ultra-high performance liquid chromatography-quadrupole/time-of-flight mass spectrometry

Plant Methods