Elicitor-mediated enhanced accumulation of ursolic acid in apple cell cultures

Plant cell cultures (PCCs) have been used for years as bio factories for the production of secondary metabolites (SMs). The reason of such interest is that plants deliver a broad spectrum of natural substances. Nevertheless, these molecules are often present at low abundance and only occur in specific plant organs. To overcome this issue, PCCs may represent a tool for in-depth studies of specific SM pathways and therefore serve as alternative platforms to enhance the production of highvalue compounds [1]. In particular, triterpenic acid have long been investigated for their anticancer properties. Ursolic acid belongs to this category, and it is recognized for its activity against several types of cancer and its low toxicity after administration [2]. Previous studies demonstrated that apple, among its several SMs, also contains high concentrations of UA. According to this, our work first aimed to the development of leaf-derived cell cultures from an apple landrace native to southern Italy. Thereafter, we analyzed the SMs content within the apple leaf calli, demonstrating the presence of a good amount of UA, although lower compared to the one found within apple leaf. Given this, the use of salycilic acid (SA) as elicitor was assessed, as it is known for its several functions, such as inducing seed germination and flowering, controlling photosynthesis and enzyme activities, therefore stimulating the SM production [3]. We tested the addition of SA 50 and 100 mg/L to the medium for the cultivation of leaf-derived cell cultures. After 3 weeks of treatment with SA 50 mg/L we found that the amount of UA increased and was comparable to the raw leaf material. The results of our analysis also revealed an increase in the growth of cell cultures with the addition of SA 50 mg/L compared with nontreated samples (Figure 1.). Considering this, the expression of the genes oxidosqualene cyclase 1 (OSC1) and betaamyrin 28-monooxygenase (CYP716A175), related to the production of UA, was examined by real-time PCR. Notably, data revealed the higher expression of these two genes after the addition of SA 50 mg/L to the medium of apple cell cultures (Figure 2.) showing the transcriptomic analysis to be in line with the metabolomic analysis. Since CYP716A175 gene has not been fully studied yet, further investigations are needed to characterize the structure of this enzyme and explain its role in apple triterpenic acid pathway.
[1] T. Wu, S.M. Kerbler, A.R. Fernie, Y. Zhang Plant Commun (2021) 100235
[2] S. Zafar, K. Khan, A. Hafeez et al. Cancer Cell Int (2022) 399
[3] B. Ali Biocatal Agric Biotechnol (2021) 101884