Genome-wide Identification and Characterization of Wall-Associated Kinases, Molecular Docking and Polysaccharide Elicitation of Monoterpenoid Indole Alkaloids in Micro-propagated Catharanthus roseus

Abstract

Abstract Wall-associated kinases (WAKs) play a pivotal role in signal transduction to regulate growth and defense responses in plants. WAKs have been identified and characterized in various plant species, however, similar information for Catharanthus roseus is scarce. Genomic, CDS, and protein sequences of WAKs were obtained from medicinal plant genomics resource using homology modelling and were named CrWAKs. 3D structure of CrWAKs was predicted using AlphaFold and docked against tri-galacturonic acid and chitosan. Micropropagated C. roseus plants were elicited with chitosan and tri-galacturonic acid to produce monoterpenoid indole alkaloids (MIAs). Three MIAs, namely catharanthine, vindoline, and vinblastine, were isolated, purified, and quantified on LC-MS. Expression profiling of transcription factors and genes involved in MIA biosynthesis and identified CrWAKs was performed using qRT-PCR. Analyses showed 37 putative CrWAK genes present in the genome of C. roseus, the majority of them localized on the plasma membrane. Phylogenetic analysis unfolded six clusters of CrWAKs. Diverse cis-acting elements, including those involved in defense responses, were identified on the promotor regions of CrWAK genes. The highest binding affinity (-12.6 kcal/mol) was noted for CrWAK-22 against tri-galacturonic acid. Tri-galacturonic acid stimulated 2.5-fold higher production of vinblastine, 6-fold upregulation of the expression of ORCA3 transcription factor, and 6.14-fold upregulation of CrWAK-22 expression. Based on the current findings it was concluded that expression of CrWAKs induced by biotic elicitors may have an important role in the production of MIAs, which may serve as a basis for functional characterization and mechanistic explanation of their role in the biosynthesis of MIAs upon elicitation.