Identification and validation of reference genes with stable expression under elicitor treatments of the medicinal plant Arnica montana L.

Abstract

Background: In view of enhancing secondary metabolites biosynthesis in Arnica montana through elicitation, comprehensive studies are needed to fully understand the molecular background of biosynthetic pathways in this species. Analysis of transcriptional changes via RT-qPCR technique might shed light on the molecular mechanisms underlying plant reaction to elicitors. This study aimed to identify reference genes which are stably expressed in Arnica under methyl jasmonate, salicylic acid, and yeast extract treatment to provide the basis for current and future gene expression studies in this important medicinal plant. Results: The expression stability of nine candidate reference genes was evaluated using four widely used algorithms (geNorm, NormFinder, BestKeeper, and ΔCt method). A comprehensive analysis of the obtained results showed that the most stably expressed pair of genes under elicitation conditions was ATP-synthase and ACT. The PP2A and TUBb were the pair of least stable candidates as they presented substantial variation in transcript levels in response to elicitor agents. For validation purposes, the transcriptional profile of PAL, 4CL and HQT genes was analyzed. Substantial induction of two of these biosynthetic genes was confirmed after methyl jasmonate treatment. Conclusions: The ATP-synthase in combination with ACT were identified as the best endogenous controls for RT-qPCR data normalization in elicitation studies of A. montana. The research outcomes shed light on transcriptional changes associated with arnica’s response to elicitation and contribute to the understanding of secondary metabolism regulation in medicinal plants.