Anthocyanins constitute a colorful family of plant specialized compounds derived from Lphenylalanine, with functions in plants such as pigmentation of flowers and fruits, UV-protection, among others. Although the enzymatic steps leading to production of anthocyanins are well known in petunia, there is little evidence on how the enzymes are spatially organized to channel the pathway towards the biosynthesis of specific compounds on-demand, how is their regulation at the protein level and how these compounds are transported from biosynthesis to storage. The existence of enzyme complexes (metabolons) with different configurations provides a plausible explanation for the metabolic control and flexibility, nevertheless the experimental proofs are still elusive and the dynamic regulation remains largely unknown.

The aim of this PhD thesis was to develop untargeted multi-omics approaches to unveil the enzymatic networks governing biosynthesis of anthocyanins in petunia during flower development.

Integration of temporal multi-omics dataset of Petunia inflata, encompassing global proteomics and metabolomics of flowers at different developmental stages, allowed to establish patterns of coexpressed proteins in relation to the profile of anthocyanin accumulation. The robust weighted gene co-expression network analysis WGCNA analysis led to the discovery of a new 4CL paralog dedicated to the biosynthesis of anthocyanins in petunia.

The cutting-edge TurboID-mediated proximity labeling was established in petunia and exploited to study dynamic protein networks in the anthocyanin pathway in vivo. For this, the endoplasmic reticulum (ER) membrane anchored cinnamic acid 4-hydroxylase (C4H) and the soluble glutathione transferase (AN9) from Petunia inflata were used as baits, coupled to TurboID and expressed transiently in petunia tissues. In the presence of biotin, TurboID releases a cloud of reactive biotin that binds covalently as a molecular tag to all proteins near the bait. In these proximity labeling experiments, the biotinylated proteins were isolated using streptavidin pull down and quantified by mass spectrometry. Among the enriched proteins in C4H-TurboID and AN9-TurboID systems, we identified multiple soluble enzymes from the anthocyanin pathway as well as additional ER membrane anchored cytochrome P450s, suggesting that the pathway is spatially organized and in association with the ER membrane mediated by cytochrome P450s.

The results of this thesis provides the basis to further investigate metabolon formation in the anthocyanin pathway, using petunia as a model plant. Combining different untargeted strategies will allow us to better understand how plants can modulate the biosynthesis of a large array of chemical compounds, such as anthocyanins, in a tailored manner in addition to set the foundation knowledge for future production of bioactive molecules in heterologous systems.