Transcriptome and Metabolite analysis reveal candidate genes of the cardiac glycoside biosynthetic pathway from Calotropis procera.

Akansha Pandey,Vineeta Tripathi,Dipak Kumar Mishra,Sanjeev Kanojiya,Vishakha Swarnkar,Tushar Pandey,Piush Srivastava

doi:10.1038/srep34464

Akansha Pandey, Vineeta Tripathi + Show 5 more

Open Access

https://doi.org/10.1038/srep34464

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Abstract

Calotropis procera is a medicinal plant of immense importance due to its pharmaceutical active components, especially cardiac glycosides (CG). As genomic resources for this plant are limited, the genes involved in CG biosynthetic pathway remain largely unknown till date. Our study on stage and tissue specific metabolite accumulation showed that CG’s were maximally accumulated in stems of 3 month old seedlings. De novo transcriptome sequencing of same was done using high throughput Illumina HiSeq platform generating 44074 unigenes with average mean length of 1785 base pair. Around 66.6% of unigenes were annotated by using various public databases and 5324 unigenes showed significant match in the KEGG database involved in 133 different pathways of plant metabolism. Further KEGG analysis resulted in identification of 336 unigenes involved in cardenolide biosynthesis. Tissue specific expression analysis of 30 putative transcripts involved in terpenoid, steroid and cardenolide pathways showed a positive correlation between metabolite and transcript accumulation. Wound stress elevated CG levels as well the levels of the putative transcripts involved in its biosynthetic pathways. This result further validated the involvement of identified transcripts in CGs biosynthesis. The identified transcripts will lay a substantial foundation for further research on metabolic engineering and regulation of cardiac glycosides biosynthesis pathway genes.

Highlights

Calotropis procera is a medicinal plant of immense importance due to its pharmaceutical active components, especially cardiac glycosides (CG)
Based on CG profiling data, 3 month old whole seedlings were selected for transcriptome sequencing and analysis
With critical observation and the best of our knowledge, we found that 3 months old seedlings had maximum CGs accumulation which was validated through Cardiac Glycoside profiling at different developmental stages of C. procera

Summary

Introduction

Calotropis procera is a medicinal plant of immense importance due to its pharmaceutical active components, especially cardiac glycosides (CG). Many cardiac glycosides have been isolated from Calotropis procera like Calactin, Ascelpin, Calotropin, Uscharin, Calotoxin, Uscharidin, Frugoside, Voruscharin[26,27,28,29] These are known to inhibit the plasma membrane Na+/K+ ATPase[30] and have been used in congestive heart failure diseases for decades[31,32]. The functional groups and sugars attached to the genin unit impart structural diversity of these compounds[35] These compounds are present in different plant groups and have been well characterized for its chemical structures, but biosynthesis of these compounds have not been studied well till date[36,37]. Kreis et al suggested norcholanic acid pathway as precursor funnelling system at final stages of cardenolide biosynthesis47. 5β-POR and 3β-HSD which have a well-established role in pregnane pathway were able to catalyse norcholanic acid and their intermediates[48,49]

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