Identification of methyl jasmonate-induced cardiac glycosides and related biosynthetic transcripts from callus culture of Calotropis gigantea using transcriptome and metabolite profiling

Abstract

Cardiac glycosides (CGs) are well known for treating congestive heart failure, and several CGs like digoxin, digitoxin, and ouabain are marketed as drugs. In the present study, we have biosynthesized two CGs (CGCL520/227 and CGCL534/209) and elicited them up to 537- and 357-fold respectively in response to methyl jasmonate (MJ) treatment. For identification of the key enzyme involved in its biosynthesis, a comparative transcriptome sequencing of control and MJ elicited (75.0 mg L⁻¹ for 3 d) callus culture was done. A total of 17,898 transcripts were expressed across all samples. Annotated unigenes were functionally categorized based on gene ontology. A total of 7625 unigenes were significantly matched in the KEGG database involved in 151 different plant metabolism pathways. Upon digital expression analysis, 2924 MJ-responsive transcripts were identified, and among them 166 were unique for MJ-treated samples. A majority of upregulated transcripts were categorized under hydrolase activity, oxido-reductase activity, metabolic processes, and carbohydrate metabolic process. Based on their role in terpenoid, steroid, and cardenolide pathways, 295 putative unigenes representing 24 gene families involved in CG biosynthesis were identified. Expression analysis revealed that 12 transcripts involved in steroid and cardenolide biosynthetic pathways were upregulated in response to MJ. The highest expression was recorded for squalene monooxygenase (SMO) with 43-fold upregulation, followed by sterol delta7 reductase (DWF5) with 22.2-fold. C-5 sterol desaturase (STE1), 4-diphosphocytidyl-2-C-methyl-D-erythritolkinase/4diphosphocytidyl-2C-methyl-D-erythritol synthase (CMK), 4-hydroxy-3-methylbut-2-enyl diphosphate reductase (HDR), acetyl-CoA C-acetyltransferase (AACT), mono-oxygenases (MO), and progesterone 5β-reductase (PBR) showed high and significant expressions of 16.4-, 16.1-, 14.8-, 14.7-, 13.4-, and 11.3-fold, respectively. This study not only identifies MJ-responsive CGs and related transcripts involved in CG biosynthesis, but also provides scope for the development of biotechnological process for biosynthesis and enrichment of targeted CGs using identified rate-limiting key enzymes.