Endomicrobiome of in vitro and natural plants deciphering the endophytes-associated secondary metabolite biosynthesis in Picrorhiza kurrooa, a Himalayan medicinal herb

Picrorhiza kurrooa an endangered high-altitude Himalayan medicinal herb, is used as a potent hepatoprotective due to the presence of various secondary metabolites, with picrosides being the most bioactive. In-vitro propagation is used as a sustainable strategy for its conservation and cultivation. The in-vitro propagation produces P. kurrooa plants (Tc) in mass, but these plants have reduced secondary metabolites (especially picrosides) compared to plants growing in their natural habitats (Wt). Therefore, considering the well-established role of microbes in secondary metabolite biosynthesis, in this study, endomicrobiome of P. kurroa plants (Tc and Wt) was explored. Using high-throughput DNA-sequencing, the endophytic communities associated with leaves, roots, and rhizomes of Wt and Tc plants were characterized. Diversity analysis revealed a loss of diversity during in-vitro propagation, and the abundant phyla were Proteobacteria, Bacteroidetes, Parcubacteria (OD1), Firmicutes, and Verrucomicrobia. Besides, the presence of distinct genera specific to different parts of Wt plants was also revealed. Quantification of secondary metabolites demonstrated the reduced accumulation of picrosides and intermediates of picroside biosynthesis in the Tc plants compared to Wt plants. Host-secondary metabolite production was positively correlated to microbial community abundance, suggesting a dynamic interplay of host-endomicrobiota interaction. Predictive functional analysis revealed the abundance of enzymes of secondary metabolite biosynthesis (especially MVA/MEP and phenylpropanoid/shikimate pathway involved in picrosides biosynthesis) in the associated-endophytic community with predominance in roots and rhizomes of Wt plants. This investigation provides novel insight into the change in the endomicrobiome of Wt and Tc plants and their correlation to the biosynthesis of secondary metabolites, and that needs to be considered for cultivation practices.IMPORTANCE Picrorhiza kurrooa is a major source of picrosides, potent hepatoprotective molecules. Due to the ever-increasing demands, overexploitation has caused an extensive decline in its population in the wild and placed it in the endangered plants' category. At present plant in-vitro systems are widely used for the sustainable generation of P. kurrooa plants, and also for the conservation of other commercially important, rare, endangered, and threatened plant species. Furthermore, the in-vitro-generated plants had reduced content of therapeutic secondary metabolites compared to their wild counterparts, and the reason behind, not well-explored. Here, we revealed the loss of plant-associated endophytic communities during in-vitro propagation of P. kurrooa plants which also correlated to in-planta secondary metabolite biosynthesis. Therefore, this study emphasized to consider the essential role of plant-associated endophytic communities in in-vitro practices which may be the possible reason for reduced secondary metabolites in in-vitro plants.