Comparative transcriptome and tissue-specific expression analysis of genes reveal tissue-cultured plants as an alternative source for phenylethanoids and phenylpropanoids in Rhodiola imbricata (Edgew.).

Rhodiola imbricata (Crassulaceae) is a traditional trans-Himalayan endangered medicinal herb with immense therapeutic applications. Over the years, over-exploitation, un-managed harvesting, and lack of captive cultivation procedures persuaded threat to its wild habitat. Plant tissue culture and RNA-Seq-based molecular bioprospection of key regulatory genes aid the understanding of molecular dynamics involved in specialized metabolites (phenylethanoids and phenylpropanoids) biosynthesis and its sustainable production. Hence, comparative transcriptomic analysis was performed using leaf and root tissues from the wild and tissue-cultured plants, revealing tissue-specific production of salidroside and rosavin. The transcriptome profiling resulted in 345 million high-quality reads yielding 92,380 unique transcripts with an N50 of 1260 bp. Tissue-specific gene expression analysis revealed that both phenylethanoids and phenylpropanoids biosynthesis are predominantly associated with the shikimate pathway. In addition to RNA-Seq data, the downstream biosynthesis pathways genes viz., phospho-2-dehydro-3-deoxyheptonate aldolase (DAHPS), 3-dehydroquinate synthase (DHQS), shikimate kinase (SK), chorismate mutase (CM), arogenate dehydrogenase (TYRAAT), aromatic-L-amino-acid decarboxylase (TDC), phenylalanine ammonia-lyase (PAL), 4-coumarate-CoA ligase (4-CL), cinnamoyl-CoA reductase (CCR), and cinnamyl alcohol dehydrogenase (CAD) showed higher expression pattern in wild plant tissues compared to tissue-cultured plants. The transcript fold expression determined by RT-qPCR results followed similar patterns as those observed in RNA-seq and targeted metabolite profiling data. Salidroside and rosavin content in wild plants exhibited 2.40 fold and 1.77 fold increase accumulation compared to the tissue-cultured plant. The present investigation explained the tissue and condition-specific significant differences between the expression of proposed biosynthetic pathway genes and salidroside and rosavin content. Additionally, NAC, bHLH, and ARF were the most abundant transcription factor families found in the transcriptomic analysis of R. imbricata. The generated transcriptome dataset provides a valuable gene(s)/transcription factors hub that can be used for the sustainable production of salidroside and rosavin in R. imbricata under tissue culture conditions.

Comparative transcriptome analysis infers bulb derived in vitro cultures as a promising source for sipeimine biosynthesis in Fritillaria cirrhosa D. Don (Liliaceae, syn. Fritillaria roylei Hook.) - High value Himalayan medicinal herb.

Fritillaria cirrhosa D. Don (Liliaceae, syn. Fritillaria roylei Hook.) is a critically endangered medicinal herb of immense importance due to its pharmaceutical bioactive compound, especially sipeimine, used for the treatment of chronic respiratory disorders. However, the industrial demand for sipeimine solely depends on its endangered natural habitat. Therefore; there is an utmost need for its biodiversity conservation as well as for the sustainable utilization of phytochemicals. Plant cell culture and transcriptomics-based molecular bioprospection of key regulatory genes involved in sipeimine biosynthesis as such will play a crucial role in exploring the unexplored traits, that are in supply crisis or nearly in extinction stage. De novo comparative transcriptome sequencing of the bulb (in vivo), callus, and regenerated plantlets (in vitro) resulted in more than 150 million high-quality paired-end clean reads that assembled into final 31,428 transcripts. Functional annotation and unigenes classification with multiple public databases such as KEGG, Refseq, Uniprot, TAIR, GO, and COG, etc. along with chemical structures and functional biocatalytic activity analysis of different steroidal alkaloids facilitated the identification of 30 unigenes specific to sipeimine biosynthesis. Additionally, ABC transporters and TFs like bHLH, MYC, MYB, and WRKY suggests their possible role in metabolite translocation and regulation in vivo as well as in vitro tissues. Differential gene expression and quantitative analysis revealed that the MVA pathway probably the predominant route for 5C intermediate (IPP & DMAPP) biosynthesis. Further, the genes involved in the downstream biosynthesis pathway viz. SQLE, CAS1, SMT1, SMO1, SMO2, SC5DL, DHCR7, DHCR24, CYP710A, 3ß-HSD, CYP90D2, and CYP374A6 shown similar expression pattern with RNA-Seq and qRT-PCR findings. The positive correlation between higher expression of proposed biosynthetic pathway genes and relatively higher accumulation of sipeimine in differentiated naturally grown bulb tissues (in vivo), undifferentiated cells (callus), and de-differentiated tissues i.e. regenerated plantlets (in vitro) has been evident from the present study. Comprehensive genomic resources created in F. cirrhosa will provide strong evidence of bulb derived in vitro culture as an alternative promising source for steroidal alkaloids biosynthesis and metabolite upscaling through genetic and metabolic engineering.

Effect of Elicitors on Morpho-Physiological Performance and Metabolites Enrichment in Valeriana jatamansi Cultivated Under Aeroponic Conditions.

The use of new agricultural technologies such as soilless and aeroponic cultivation systems is a valuable approach to medicinal plant production. The present study investigated the prospects of enhancing yield and secondary metabolite production in Valeriana jatamansi under aeroponic cultivation using elicitors, such as yeast extract and methyl jasmonate. Plants were evaluated by measuring growth parameters, photosynthetic rate, and secondary metabolites contents (on a dry weight basis). Maximum plant height (36.83 cm), leaf number (17.67), rootlet number (37.33), and rootlet length (6.90 cm) were observed at 0.5 mg/L yeast extract treatment; whereas treatment levels of 1.5 mg/L yeast extract and 150 µM methyl jasmonate resulted in maximum leaf length (6.95 cm) and leaf width (5.43 cm), respectively. Maximum photosynthetic rate (5.4053 µmol m-2s-1) and stomatal conductance (0.0656 mmol m-2s-1) were recorded at treatment levels of 0.5 mg/L and 1.5 mg/L yeast extract respectively, whereas at 150 µM methyl jasmonate treatment, transpiration rate was 0.9046 mmol m-2s-1. In aeroponic cultivation, the maximum content of valerenic acid and hydroxy valerenic acid was detected in leaf (2.47 and 8.37 mg/g) and root (1.78 and 7.89 mg/g) at treatment levels of 100 µM and 150 µM methyl jasmonate, respectively. Acetoxy valerenic acid was highest in leaf (1.02 mg/g) at 1.5 mg/L yeast extract, and in the root (2.38 mg/g) at 150 µM methyl jasmonate. Gas chromatography-mass spectrometry analysis identified twenty-eight volatile compounds in roots, of which three-isovaleric acid (6.72-50.81%), patchouli alcohol (13.48-25.31%) and baldrinal (0.74-25.26%)-were the major constituents. The results revealed that, besides roots, leaves could also be utilized as a prominent alternative source for targeted secondary metabolites. In conclusion, aeroponic cultivation offers year-round quality biomass production and ease to access subsequent roots harvest in V. jatamansi, to meet the demand of the pharmaceutical industries.

Effect of salicylic acid on the activity of PAL and PHB geranyltransferase and shikonin derivatives production in cell suspension cultures of Arnebia euchroma (Royle) Johnst--a medicinally important plant species.

Cell suspension cultures of Arnebia euchroma were established from the friable callus on liquid Murashige and Skoog medium supplemented with 6-benzylaminopurine (10.0 µM) and indole-3-butyric acid (5.0 µM). Salicylic acid was used to study its effect on the enzymes which participate in shikonin biosynthesis with respect to metabolite (shikonin) content in the cell suspension culture of A. euchroma. In our study, phenylalanine ammonia lyase and PHB geranyltransferase were selected from the entire biosynthetic pathway. Results showed that phenylalanine ammonia lyase is responsible for growth and PHB geranyltransferase for metabolite production. Salicylic acid exhibited an inverse relationship with the metabolite content (shikonin); salicylic acid (100 µM) completely inhibited shikonin biosynthesis. The results presented in the current study can be successfully employed for the metabolic engineering of its biosynthetic pathway for the enhancement of shikonin, which will not only help in meeting its industrial demand but also lead to the conservation of species in its natural habitat.