Spatial Genomic Resource Reveals Molecular Insights into Key Bioactive-Metabolite Biosynthesis in Endangered Angelica glauca Edgew.

Angelica glauca Edgew, which is an endangered medicinal and aromatic herb, is a rich source of numerous industrially important bioactive metabolites, including terpenoids, phenolics, and phthalides. Nevertheless, genomic interventions for the sustainable utilization and restoration of its genetic resources are greatly offset due to the scarcity of the genomic resources and key regulators of the underlying specialized metabolism. To unravel the global atlas of the specialized metabolism, the first spatial transcriptome sequencing of the leaf, stem, and root generated 109 million high-quality paired-end reads, assembled de novo into 81,162 unigenes, which exhibit a 61.53% significant homology with the six public protein databases. The organ-specific clustering grouped 1136 differentially expressed unigenes into four subclusters differentially enriched in the leaf, stem, and root tissues. The prediction of the transcriptional-interactome network by integrating enriched gene ontology (GO) and the KEGG metabolic pathways identified the key regulatory unigenes that correspond to terpenoid, flavonoid, and carotenoid biosynthesis in the leaf tissue, followed by the stem and root tissues. Furthermore, the stem and root-specific significant enrichments of phenylalanine ammonia lyase (PAL), cinnamate-4-hydroxylase (C4H), and caffeic acid 3-O-methyltransferase (COMT) indicate that phenylalanine mediated the ferulic acid biosynthesis in the stem and root. However, the root-specific expressions of NADPH-dependent alkenal/one oxidoreductase (NADPH-AOR), S-adenosyl-L-methionine-dependent methyltransferases (SDMs), polyketide cyclase (PKC), and CYP72A15 suggest the "root" as the primary site of phthalide biosynthesis. Additionally, the GC-MS and UPLC analyses corresponded to the organ-specific gene expressions, with higher contents of limonene and phthalide compounds in the roots, while there was a higher accumulation of ferulic acid in the stem, followed by in the root and leaf tissues. The first comprehensive genomic resource with an array of candidate genes of the key metabolic pathways can be potentially utilized for the targeted upscaling of aromatic and pharmaceutically important bioactive metabolites. This will also expedite genomic-assisted conservation and breeding strategies for the revival of the endangered A. glauca.

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

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.

Genome-wide transcriptional analysis unveils the molecular basis of organ-specific expression of isosteroidal alkaloids biosynthesis in critically endangered Fritillaria roylei Hook.

Fritillaria roylei Hook. is a critically endangered high altitude Himalayan medicinal plant species with rich source of pharmaceutically active structurally diverse steroidal alkaloids. Nevertheless, except few marker compounds, the chemistry of the plant remains unexplored. Therefore, in the current study, transcriptome sequencing efforts were made to elucidate isosteroidal alkaloids biosynthesis by creating first organ-specific genomic resource using bulb, stem, and leaf tissues derived from natural populations of Indian Himalayan region. Overall, 349.9 million high quality paired-end reads obtained using NovaSeq 6000 platform were assembled (de novo) into 82,848 unigenes and 31,061 isoforms. Functional annotation and organ specific differential expression (DE) analysis identified 2488 significant DE transcripts, grouped into three potential sub-clusters (sub-cluster I: 728 transcripts; sub-cluster II: 446 transcripts and Sub-cluster III: 1314 transcripts). Subsequently, pathway enrichment (GO, KEGG) and protein-protein network analysis revealed significantly higher enrichment of phenyl-propanoid and steroid backbone including terpenoid, sesquiterpenoid and triterpenoid biosynthesis in bulb. Additionally, upregulated expression of cytochrome P450, UDP-dependent Glucuronosyltransferase families and key transcription factor families (FAR1, bHLH, GRAS, C2H2, TCP and MYB) suggests 'bulb' as a primary site of MVA mediated isosteroidal alkaloids biosynthesis. The comprehensive elucidation of molecular insights in this study is a first step towards the understanding of isosteroidal alkaloid biosynthesis pathway in F. roylei. Furthermore, key genes and regulators identified here can facilitate metabolic engineering of potential bioactive compounds at industrial scale.