Integrated Analysis of Transcriptome and Metabolome and Evaluation of Antioxidant Activities in Lavandulapubescens

Abstract

Lavandula pubescens, belonging to the Labiatae family, is a newly discovered strongly aromatic species of lavender that is potentially beneficial for human health. Given the economic importance of lavender species, we sought in this study to characterize the terpenoid biosynthesis of L. pubescens by obtaining transcriptomic and metabolic datasets. Transcriptome analysis of L. pubescens grown aseptically in tissue culture medium yielded 124,233 unigenes with an average length of 470 bp and N50 value of 522 bp from 9,476,122,928 raw reads. In order to provide relevant biological information, the unigenes were annotated using the following public databases: National Center for Biotechnology Information (NCBI) nucleotide (NT) and non-redundant protein (NR), Brassica (BRAD), Arabidopsis Information Resource (TAIR), Clusters of Orthologous Groups (COG), and Gene Ontology (GO). NR annotation results revealed that L. pubescens is genetically closely related to Sesamum indicum. On the basis of the transcriptome data, a total of 14 cDNA clones encoding the terpene biosynthetic genes LpDXS, LpMCT, LpMCS, LpHDR, LpIDI, LpAACT, LpHMGS, LpHMGR, LpMVK, LpPMK, LpMVD, LpGPPS, LpSQS, and LpGGPPS were identified in L. pubescens. These were quantified in the roots, stems, and leaves of L. pubescens using quantitative real-time polymerase chain reaction (qRT-PCR), which revealed that the gene expression levels were higher in the leaves and stems than in the roots, which was found to be consistent with the levels of ursolic and oleanolic acids in the different organs using high-performance liquid chromatography (HPLC). A total of 48 hydrophilic metabolites were identified and quantified in the organs using gas chromatography time-of-flight mass spectrometry (GC-TOFMS). Furthermore, the antioxidant activity of an ethyl acetate extract of L. pubescens leaves was examined using different methods to determine the potential therapeutic properties. A reducing power assay revealed that the absorbance values increased in a concentration-dependent manner, whereas a 2,2-diphenyl-1-picrylhydrazyl radical scavenging assay indicated the strong activity (60.4 ± 0.9%) of the ethyl acetate extract at a concentration of 100 µg/mL, which also showed strong hydrogen peroxide (57.4 ± 2.7%), superoxide radical (62.1 ± 0.7%), and hydroxyl radical (58.6 ± 0.4%) scavenging activities.

Keywords: Lavandula pubescens; antioxidant capacity; metabolome; transcriptome.

Figure 1

The phenotype of L. pubescens grown in a greenhouse.

Figure 2

The proposed pathway for the biosynthesis of oleanolic acid and ursolic acid. The terpenoid biosynthetic genes identified in this study are indicated in red: MVA, mevalonic acid; MEP, 2-C-methyl-D-erythritol 4-phosphate; IPP, isopentenyl diphosphate; DMAPP, isomer dimethylallyl diphosphate; AACT, acetyl-CoA acetyltransferase; HMGS, hydroxymethylglutaryl-CoA synthase; HMGR, 3-hydroxy-3-methylglutaryl-CoA reductase; MVK, mevalonate kinase; PMK, phosphomevalonate kinase; MVD, mevalonate-5-pyrophosphate decarboxylase; DXS, 1-deoxy-D-xylulose-5-phosphate synthase; DXR, 1-deoxy-D-xylulose 5-phosphate reductoisomerase; MCT, 2-C-methyl-D-erythritol 4-phosphate cytidylyltransferase; CMK, 4-diphosphocytidyl-2-C-methyl-D-erythritol kinase; MCS, 2-C-methyl-D-erythritol 2,4-cyclodiphosphate synthase; HDS, 4-hydroxy-3-methylbut-2-enyl-diphosphate synthase; HDR, 4-hydroxy-3-methylbut-2-enyl diphosphate reductase; IDI, isopentenyl diphosphate isomerase; GPPS, geranyl diphosphate synthase; GGPPS, geranylgeranyl diphosphate synthase; FPPS, farnesyl pyrophosphate synthase; SQS, squalene synthase; and SQE, squalene epoxidase.

Figure 3

e-value distribution (A), percentage similarity (B), and species distribution (C) of the nonredundant (NR) annotation results for Lavandula pubescens unigenes.

Figure 4

Expression of terpene biosynthetic genes in different organs of Lavandula pubescens.

Figure 5

(A) scores and loading plots of PCA model and (B) scores and loading plots of PLDA model obtained from metabolites detected in different organs of L. pubescens using HPLC and GC-TOFMS.

Figure 6

Correlation matrix of metabolites detected in different organs of L. pubescens. Each square indicates the Pearson’s correlation coefficient of a pair of compounds, and the value of the correlation coefficient is represented by the intensity of blue or red colors, as indicated on the color scale.

Figure 7

The antioxidant activity of an ethyl acetate extract of Lavandula pubescens leaves. (A) Reducing power activity; (B) 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging activity; (C) Hydrogen peroxide scavenging activity; (D) Superoxide radical scavenging activity; (E) Hydroxyl radical scavenging activity. The blue bars represent values obtained using an ethyl acetate extract of L. pubescens leaves and the red bars are values obtained using ascorbic acid.