High-quality haplotype-resolved chromosome assembly provides evolutionary insights and targeted steviol glycosides (SGs) biosynthesis in Stevia rebaudiana Bertoni.

Stevia rebaudiana Bertoni is popular source of plant-derived low/no-calorie natural sweeteners (LNCSs), collectively known as steviol glycosides (SGs). Nevertheless, genetic predisposition for targeted biosynthesis of SGs is complex due to multi-substrate functionality of key uridine diphosphate glycosyltransferases (UGTs). Here, we created a high-quality monoploid assembly of 1.34 Gb with N50 value of 110 Mb, 55 551 predicted protein-coding genes, and ~80% repetitive regions in Rebaudioside-A (Reb-A) enriched cultivar of S. rebaudiana. Additionally, a haplotype-based chromosome assembly consisting of haplotype A and haplotype B with an overall genome size of 2.33Gb was resolved, harbouring 639 634 variants including single nucleotide polymorphisms (SNPs), indels and structural variants (SVs). Furthermore, a lineage-specific whole genome duplication analysis revealed that gene families encoding UGTs and Cytochrome-P450 (CYPs) were tandemly duplicated. Additionally, expression analysis revealed five tandemly duplicated gene copies of UGT76G1 having significant correlations with Reb-A content, and identified key residue (leu200val) in the glycosylation of Reb-A. Furthermore, missense variations identified in the acceptor region of UGT76G1 in haplotype resolve genome, transcriptional and molecular docking analysis were confirmed with resequencing of 10 diverse stevia genotypes (~25X). Gene regulatory network analysis identified key transcription factors (MYB, bHLH, bZIP and AP2-ERF) as potential regulators of SG biosynthesis. Overall, this study provides haplotype-resolved chromosome-level genome assembly for genome editing and enhancing breeding efforts for targeted biosynthesis of SGs in S. rebaudiana.

Genome-wide transcriptional profiling and physiological investigation elucidating the molecular mechanism of multiple abiotic stress response in Stevia rebaudiana Bertoni.

Considering the major source of plant-derived low/non-calorie steviol glycosides (SGs), comprehensive physiological, biochemical, and deep transcriptional investigations were conducted to explicit deeper insight into multiple abiotic stress responses in Stevia rebaudiana. The physiological indicators including photosynthesis, chlorophyll, relative water content, shoot growth, electrolyte leakage, and SG biosynthesis were negatively impacted under drought (DS), followed by salinity (SS) and waterlogging (WS). Global transcriptional analysis revealed significant upregulated expression of the genes encoding for ROS detoxification (GST, SOD, APX, glutathione peroxidase), osmotic adjustment (alpha-trehalose-phosphate and S-adenosylmethionine decarboxylase), ion transporters (CAX, NHX, CNGS, VPPase, VATPase), water channel (PIP1, TIP) and abiotic stress-responsive candidate genes (LEA, HSPs, and Dehydrins) regulating abiotic stress response in S. rebaudiana. These inferences were complemented with predicted interactome network that revealed regulation of energy metabolism by key stress-responsive genes (GST, HKT1, MAPKs, P5CSs, PIP), transcription factors (HSFA2, DREB1A, DREB2A), and abiotic stress responsive pathways (ABA, ethylene, ion stress). This is the first detailed study to comprehend the molecular regulation of stress response and their interplay under DS, SS, and WS. The key genes and regulators can be functionally validated, and will facilitate targeted gene editing for genetic improvement of crop sustainability under changing environmental conditions in S. rebaudiana.

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.

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.

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.

Transcriptional analysis reveals key insights into seasonal induced anthocyanin degradation and leaf color transition in purple tea (Camellia sinensis (L.) O. Kuntze).

Purple-tea, an anthocyanin rich cultivar has recently gained popularity due to its health benefits and captivating leaf appearance. However, the sustainability of purple pigmentation and anthocyanin content during production period is hampered by seasonal variation. To understand seasonal dependent anthocyanin pigmentation in purple tea, global transcriptional and anthocyanin profiling was carried out in tea shoots with two leaves and a bud harvested during in early (reddish purple: S1_RP), main (dark gray purple: S2_GP) and backend flush (moderately olive green: S3_G) seasons. Of the three seasons, maximum accumulation of total anthocyanin content was recorded in S2_GP, while least amount was recorded during S3_G. Reference based transcriptome assembly of 412 million quality reads resulted into 71,349 non-redundant transcripts with 6081 significant differentially expressed genes. Interestingly, key DEGs involved in anthocyanin biosynthesis [PAL, 4CL, F3H, DFR and UGT/UFGT], vacuolar trafficking [ABC, MATE and GST] transcriptional regulation [MYB, NAC, bHLH, WRKY and HMG] and Abscisic acid signaling pathway [PYL and PP2C] were significantly upregulated in S2_GP. Conversely, DEGs associated with anthocyanin degradation [Prx and lac], repressor TFs and key components of auxin and ethylene signaling pathways [ARF, AUX/IAA/SAUR, ETR, ERF, EBF1/2] exhibited significant upregulation in S3_G, correlating positively with reduced anthocyanin content and purple coloration. The present study for the first-time elucidated genome-wide transcriptional insights and hypothesized the involvement of anthocyanin biosynthesis activators/repressor and anthocyanin degrading genes via peroxidases and laccases during seasonal induced leaf color transition in purple tea. Futuristically, key candidate gene(s) identified here can be used for genetic engineering and molecular breeding of seasonal independent anthocyanin-rich tea cultivars.

Comparative transcriptome analysis revealed gamma-irradiation mediated disruption of floral integrator gene(s) leading to prolonged vegetative phase in Stevia rebaudiana Bertoni.

Stevia rebaudiana Bert. is getting global attention because of its ability to synthesize commercially important low/no calorie natural sweeteners (LNCSs) steviol glycosides (SGs). Considering, higher accumulation of SGs in vegetative phase followed by decrement during reproductive phase necessitate the understanding of different molecular components of floral transition to develop superior varieties/cultivars with prolonged vegetative phase in Stevia. Current comparative transcriptional analysis of low dose (5 kR) gamma-irradiated mutant genotype (SMG) with prolonged vegetative phase vis-à-vis background genotype (SBG) identified DGEs of major floral transition pathways, and expressed according to their physiological fate irrespective to SMG & SBG. Contrarily, reduced expression of floral integrator genes (FT and LEAFY) in mutant genotype suggests their involvement in prolonged vegetative phase phenotype. Likewise, GO and KEGG enrichment of photosynthesis and carbon assimilation efficiency might be associated with prolonged vegetative phase and higher accumulation of Stevioside content in mutant genotype. Furthermore, deviation of flowering related transcription factors (higher expressions except MIKS-type MADS-box SMG_PV compared to SBG_F) may possibly be correlated with low expression of floral integrator genes. Findings of current studies will facilitate the genetic manipulations and crop improvement efforts in Stevia through conventional breeding and genome editing approaches for increased SGs biosynthesis.