Molecular Regulation of Catalpol and Acteoside Accumulation in Radial Striation and non-Radial Striation of Rehmannia glutinosa Tuberous Root

Abstract

Rehmannia glutinosa L., a perennial plant of Scrophulariaceae, is one of the most commonly used herbs in traditional Chinese medicine (TCM) that have been widely cultivated in China. However, to date, the biosynthetic pathway of its two quality-control components, catalpol and acteoside, are only partially elucidated and the mechanism for their tissue-specific accumulation remains unknown. To facilitate the basic understanding of the key genes and transcriptional regulators involved in the biosynthesis of catalpol and acteoside, transcriptome sequencing of radial striation (RS) and non-radial striation (nRS) from four R. glutinosa cultivars was performed. A total of 715,158,202 (~107.27 Gb) high quality reads obtained using paired-end Illumina sequencing were de novo assembled into 150,405 transcripts. Functional annotation with multiple public databases identified 155 and 223 unigenes involved in catalpol and acteoside biosynthesis, together with 325 UGTs, and important transcription factor (TF) families. Comparative analysis of the transcriptomes identified 362 unigenes, found to be differentially expressed in all RS vs. nRS comparisons, with 143 upregulated unigenes, including those encoding enzymes of the catalpol and acteoside biosynthetic pathway, such as geranyl diphosphate synthase (RgGPPS), geraniol 8-hydroxylase (RgG10H), and phenylalanine ammonia-lyase (RgPAL). Other differentially expressed unigenes predicted to be related to catalpol and acteoside biosynthesis fall into UDP-dependent glycosyltransferases (UGTs), as well as transcription factors. In addition, 16 differentially expressed genes were selectively confirmed by real-time PCR. In conclusion, a large unigene dataset of R. glutinosa generated in the current study will serve as a resource for the identification of potential candidate genes for investigation of the tuberous root development and biosynthesis of active components.

Keywords: Rehmannia glutinosa L.; acteoside; biosynthetic pathway; catalpol; tuberous root.

Figure 1

Morphology and active compounds distribution of the R. glutinosa tuberous roots. (A) Cross sections of tuberous roots from 1706, BJ1, QH1, and W85; (B) HPLC chromatographic fierprints in radial striation and non-radial striation of tuberous roots, and monitored at 205 nm, the green arrow and red arrow point to peaks of catalpol and acteoside, respectively; (C) the contents of catalpol and total iridoid glycosides in radial striation and non-radial striation; (D) the contents of acteoside and total phenylethanoid glycosides in radial striation and non-radial striation. Data shows the average values ± SE of three independent experiments. * indicate significant difference at p < 0.05. Abbreviations: RS, radial striation; nRS, non-radial striation. The same as follows.

Figure 2

Characteristics of homology search of R. glutinosa unigenes. (A) Venn diagram of shared and unique unigenes in R. glutinosa. Annotation according to the nr, nt, Swiss-Prot, KEGG, COG, GO, and Interpro databases; (B) number and percentage of unigenes matching the 25 top species using BLASTx in the nr database.

Figure 3

Unigenes expressed number and level on different tissues. (A) The number of expressed genes in each sample; (B) Expressional levels of unigenes of all the samples. Up and down lines of colored squares indicate upper and lower quartiles, and middle lines indicate medians. Black balls indicate outliers of expression values.

Figure 4

Unigenes significantly differentially expressed between radial striation and non-radial striation in different R. glutinosa cultivars. (A), The up-regulated and down-regulated transcript numbers in radial striation and non-radial striation. X axis represents pairwise and Y axis means number of screened DETs. Green bar denotes down-regulated genes and red bar for the up-regulated. (B), Venn diagram analysis of the quantity of DETs identified in radial striation and non-radial striation.

Figure 5

Top 20 enriched KEGG pathways among the annotated DETs across four comparisons. (A) Significant enrichment pathways for genes in 1706_RS vs. 1706_nRS comparison. (B) Significant enrichment pathways for genes in BJ1_RS vs. BJ1_nRS comparison. (C) Significant enrichment pathways for genes in QH1_RS vs. QH1_nRS comparison. (D) Significant enrichment pathways for genes in W85_RS vs. W85_nRS comparison. The X-axis below represents KEGG pathways, and the Y-axis indicates the enrichment factor. Low q-values are shown in blue, and high q-values are depicted in white. Point size indicates DET number (more: big, less: small).

Figure 6

Expression patterns of catalpol biosynthetic unigenes between radial striations and non-radial striations of R. glutinosa. (A) Biosynthetic pathway of catalpol. (B) Heat map representing expression dynamics of unigenes involved in biosynthesis. The expression values (FPKM) for unigenes were log₁₀ transformed and scaled across each row, and the heatmap was generated by MultiExperiment Viewer (MeV). (C) Relative expression of candidate genes in involved in catalpol biosynthesis. These genes expression levels were all determined by Real-time PCR (qRT-PCR). Vertical bars indicate the standard deviation of three biological replicates. Asterisks (*) indicate a significant difference at the p < 0.05 level. Abbreviations: DXR, 1-deoxy-D-xylulose 5-phosphate reductoisomerase; GPPS, geranyl diphosphate synthase; IPI, isopentenyl-diphosphate Delta-isomerase; GES, geraniol synthase; G10H, Geraniol 10-hydroxylase; CPR, NADPH--cytochrome P450 reductase; 10HGO, 10-hydroxygeraniol dehydrogenase; IRS, iridoid synthase; CPM, Cytochrome P-450 monooxygenase; ALD, aldehyde dehydrogenase; UGT, Uridine diphosphate glycosyltransferase; F3D, flavanone 3-dioxygenase; UPD, uroporphyrinogen decarboxylase; UGD, UDP-glucuronic acid decarboxylase; SQM, squalene monooxygenase.

Figure 7

Expression patterns of acteoside biosynthetic unigenes between radial striations and non-radial striations of R. glutinosa. (A) Biosynthetic pathway of acteoside. The blue arrows indicate the tyrosine-derived pathway, and the purple arrows indicate the phenylalanine-derived pathway. The solid arrows denote known steps and the dashed arrows denote hypothetical steps. (B) Expression levels of the identified unigenes were annotated as enzyme coding genes from acteoside biosynthesis pathways. (C) Expression levels of unigenes involved in the biosynthesis of acteoside. Vertical bars indicate the standard deviation of three biological replicates. * indicate a significant difference at the p < 0.05 level. Abbreviations: PAL: phenylalanine ammonia-lyase; C4H, cinnamate-4-hydroxylase; C3H, coumarate-3-hydroxylase; TyDC, tyrosine decarboxylase; PPO, polyphenol oxidase; CuAO, copper-containing amine oxidase; ALDH, alcohol dehydrogenase; UGT, UDP-glucose glucosyltransferase; 4CL, 4-coumarate-CoA ligase; HCT, Shikimate O-hydroxycinnamoyltransferase.

Figure 8

Expression patterns of UGTs between radial striations and non-radial striations of R. glutinosa. (A) Heat map representing expression dynamics of unigenes belongs to UGT encoding genes. (B) Phylogenetic tree of UGT proteins was generated by the neighbor-joining method using software of MEGA 6.0. (C) Relative expression levels of four UGT genes in radial striation and non-radial striation from 1706, BJ1, QH1, and W85 by qRT-PCR. Vertical bars indicate the standard deviation of three biological replicates. * indicate a significant difference at the p < 0.05 level.

Figure 9

Expression patterns of TFs between non-radial striations than in radial striations of R. glutinosa. (A) Heat map representing expression dynamics of unigenes belongs to transcription factor genes. (B) Relative expression levels of four transcription factor genes in non-radial striations compared to radial striations from 1706, BJ1, QH1, and W85 by qRT-PCR. Vertical bars indicate the standard deviation of three biological replicates. * indicate a significant difference at the p < 0.05 level.