An Integrative Transcriptional Network Revealed Spatial Molecular Interplay Underlying Alantolactone and Inulin Biosynthesis in Inula racemosa Hook f

Abstract

Inula racemosa Hook. f. (Pushkarmula), a perennial Himalayan herb known for its aromatic and phytopharmaceutical attributes, is not yet explored at genomic/transcriptomic scale. In this study, efforts were made to unveil the global transcriptional atlas underlying organ-specific specialized metabolite biosynthesis by integrating RNA-Seq analysis of 433 million sequenced reads with the phytochemical analysis of leaf, stem, and root tissues. Overall, 7242 of 83,772 assembled nonredundant unigenes were identified exhibiting spatial expression in leaf (3761), root (2748), and stem (733). Subsequently, integration of the predicted transcriptional interactome network of 2541 unigenes (71,841 edges) with gene ontology and KEGG pathway enrichment analysis revealed isoprenoid, terpenoid, diterpenoid, and gibberellin biosynthesis with antimicrobial activities in root tissue. Interestingly, the root-specific expression of germacrene-mediated alantolactone biosynthesis (GAS, GAO, G8H, IPP, DMAP, and KAO) and antimicrobial activities (BZR1, DEFL, LTP) well-supported with both quantitative expression profiling and phytochemical accumulation of alantolactones (726.08 μg/10 mg) and isoalantolactones (988.59 μg/10 mg), which suggests "roots" as the site of alantolactone biosynthesis. A significant interaction of leaf-specific carbohydrate metabolism with root-specific inulin biosynthesis indicates source (leaf) to sink (root) regulation of inulin. Our findings comprehensively demonstrate the source-sink transcriptional regulation of alantolactone and inulin biosynthesis, which can be further extended for upscaling the targeted specialized metabolites. Nevertheless, the genomic resource created in this study can also be utilized for development of genome-wide functionally relevant molecular markers to expedite the breeding strategies for genetic improvement of I. racemosa.

Keywords: Germacrene A; Inula racemosa; alantolactones; interactome; inulin; sesquiterpene lactones; transcriptome.

Figure 1

Phytochemical screening of leaf, stem, and root tissue of Inula racemosa. (A) Salkowski’s test of leaf, stem, and root tissue recorded higher accumulation of terpenoids with appearance of reddish-brown color; (B) alkaline reagent test recorded presence of flavonoids with appearance of yellow color in the leaf and root tissue. (C) Graph representing percent of total flavonoid content (TFC) with significant appearance in the leaf followed by root tissues. (D) Chromatogram of leaf, stem, and root extracts. The peak labelled as 1 represents costunolides; 2: dehydrocostuslactone; 3: isoalantolactone; and 4: alantolactones in the respective three tissue extracts. (E) Organ-specific quantification of dehydrocostuslactone, isoalantolactone; and alantolactones in leaf, root and stem extract.

Figure 2

Global details of total sequenced reads of I. racemosa generated from illumina NovaSeq 6000 platform. (A) Quality filtering of sequenced reads. (B) Assembly statistics. (C) BUSCO score of assembled unigenes. (D) Open reading frame prediction in de novo assembled sequence representing 58% of complete coding region. (E) Venn diagram illustrating annotation of unigenes with five public protein databases.

Figure 3

Organ-specific expression analysis of three tissues, viz., leaf, root, and stem of I. racemosa. (A) Pearson’s correlation among leaf, stem, and root tissue. (B) Bubble plot representing normalized tissue-specific gene expression. (C–E) Gene expression sub-clustering of significantly expressed unigenes based on median FPKM values.

Figure 4

Spatially enriched metabolic pathways in leaf, stem, and root tissue of I. racemosa. (A) Significantly enriched photosynthesis and oxidative phosphorylation metabolic pathways. (B) Flavonoid biosynthesis; (C) starch and sucrose metabolism; (D–F) terpenoid metabolic biosynthesis pathways including (D) sesqui- and triterpenoids, (E) terpenoid backbone biosynthesis & diterpenoid biosynthesis; (G) Carotenoid biosynthesis in the leaf, stem, and root tissue of I. racemosa. The organ specific spatial enrichment representing (green) enrichment specifically in the leaf tissue; pink in the stem; and brown in the root tissue.

Figure 5

(A) Predicted interrologous transcriptional interactome network, (B) table representing network statistics, (C–E) significantly enriched unigenes corresponding to (C) terpenoids, (D) diterpenoids, and (E) the CYP450 family. Predicted significant interactions of unigenes involved in starch and sucrose metabolism regulating (F) inulin biosynthesis and (G) transporters. Enriched interaction representing unigenes involved in (H) lipid transfer proteins and (I) antiviral pathogenesis.

Figure 6

Integrated predicted network with spatially enriched sesquiterpenoid, monoterpenoid, and diterpenoid biosynthesis pathways, comprising unigenes corresponding to (A) terpenoid biosynthesis, (B) CYP450 family corresponding to monoterpenoid biosynthesis and (C) diterpenoid, (D) sesquiterpenoid, and triterpenoid biosynthesis in Inula racemsa.

Figure 7

(A) Predicted network representing enriched unigenes corresponding to starch and sucrose metabolic biosynthesis complementing the (B) enriched KEGG pathway; (C) inulin synthesis involving hydrolysis of sucrose followed by conversion of beta-D fructose to inulin via FFT gene.

Figure 8

Predicted network representing (A) antiviral pathogenesis-related and (B) phytohormones. (C) KEGG based spatial enrichment of auxin, brassinosteroid and ethylene related pathways involved in antimicrobial activities in I. racemosa.

Figure 9

qRT-PCR based validation of RNA-Seq data using 13 unigenes involved in terpenoid biosynthesis (IPP: DMAP, KAO, GAS, G8H), photosynthesis (PSI, Chl-a-b), starch and sucrose metabolism regulating inulin biosynthesis (STP, SST1, FFT, cellulose syn), and antiviral pathogenesis (BZR1, DEFL, LTP) in (A) Leaf vs. Stem; (B) Root vs. Stem and (C) Root vs Leaf tissue. (D) Correlation plot representing strong correlation between RNA-Seq and qRT-PCR data.

Figure 10

Illustration representing spatial expression of terpenoid and inulin biosynthesis in I. racemosa.