Laezza, Carmen (2024) Annurca apple callus culture: an avant-garde food source of high-value metabolites. [Tesi di dottorato]

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Abstract

The Annurca apple (Malus pumila Miller) is a cultivar native to Southern Italy and listed as Protected Geographical Indication (PGI) product. This apple is enriched in polyphenols (hydroxycinnamic acids, dihydrochalcones, catechin, epicatechin and procyanidins) and triterpenoids, in particular triterpenic acids, with health-promoting properties. Although these natural compounds are in high demand in the pharmaceutical, cosmetic and food markets, their production depends on the seasonality of plants and implies excessive use of water and land. Plant cell cultures (PCCs) are a “ready-to use” technology that allows specialized metabolites to be synthetized year-round and consistently without impacting the environment. This thesis aimed to explore the main natural compounds accumulating in callus cultures from the Annurca apple variety to propose their potential use also as a future novel food. Three different callus cultures were developed from peel, pulp and leaf of this variety. A UHPLC method was set up to decode the metabolomic profiles of the apple-derived calli. The results indicated the callus cultures from the peel and the pulp of the Annurca apple as the most enriched in polyphenols and triterpenoids. In particular, chlorogenic acid, procyanidin B2, epicatechin, catechin, phloridzin and ursolic acid were found to be the most abundant. To ameliorate this accumulation, elicitation was employed as a method to stimulate cell reprogramming and a major synthesis of natural compounds within calli. Different elicitors were tested, and yeast extract (YE) elicitation was able to generate a defence-related response within apple calli thus leading to higher production of the metabolites. Furthermore, the antioxidant and antibacterial activities of the extracts from the calli were tested, indicating the one from peel-derived calli as a good candidate to reduce the growth of foodborne pathogens such as Staphylococcus aureus and Bacillus cereus (Chapter 2 and 3). A comprehensive metabolomic analysis showed that peel-derived calli resulted remarkably affected by YE treatment and in particular, an increase in the biosynthesis of phloridzin and ursolic acid was detected after elicitation. These results were corroborated by RNA sequencing that demonstrated an upregulation of genes involved in dihydrochalcone synthesis, such as MdCHS and MdUGT88F1 genes encoding for the chalcone synthase and the UDP-glucose:phloretin 2’-O-glucosyltransferase, respectively. In contrast, the expression level of the genes directly related to ursolic acid synthesis (MdOSC1 and MdCYP716A175 encoding for the oxydosqualene cyclase 1 and the triterpene monooxygenase, respectively) were only slightly influenced by YE treatment. The investigations also demonstrated that the elicitor did not induce significant alteration of polyphenol and triterpenic acid biosynthesis in pulp-derived calli compared to peel-derived ones (Chapter 4). Considering these results, a different elicitation method, near ultraviolet radiation (NUV), was employed to boost triterpenic acid (TA) accumulation in leaf-derived calli, as they were faster to produce and analyse. Moreover, a single-cell method was developed to better investigate the TA pathway in these calli. Single-cell transcriptomic and metabolomic analyses demonstrated that a high heterogeneity in the synthesis of TAs was clearly detectable across the cells composing the callus mass, highlighting the challenge in establishing a stable cell line that consistently produces bioactive compounds. Furthermore, the NUV elicitation induced a more homogeneous production of TAs across the cell population of the callus (Chapter 5). Altogether, the results obtained in this thesis provide more detailed insights on the functionality of callus cultures for the synthesis of natural compounds thanks to the use of the newest biotechnological approaches.

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