Global organization of phenylpropanoid and anthocyanin pathways revealed by proximity labeling of trans-cinnamic acid 4-hydroxylase (CYP73A412) in Petunia inflata petal protoplasts

The phenylpropanoid pathway is one of the main carbon sinks in plants, channeling phenylalanine towards thousands of products including monolignols, stilbenes, flavonoids and volatile compounds. The enzymatic steps involved in many of these pathways are well characterized, however the physical organization of these enzymes within the plant cell remains unknown Proximity-dependent labeling allows untargeted determination of protein interactions in vivo, and therefore stands as an attractive alternative to targeted binary assays for determining global protein-protein interaction networks. In this study, we show a TurboID-based proximity labeling system developed to study protein interaction networks of the core phenylpropanoid pathway in petunia. Here, the endoplasmic reticulum (ER) membrane anchored cytochrome P450 cinnamic acid 4-hydroxylase (C4H, CYP73A412) from Petunia inflata was coupled to TurboID and expressed in protoplasts derived from anthocyanin-rich petunia petals. Potential interactors were isolated using streptavidin beads, digested and quantified by mass spectrometry. Among the enriched proteins, we identified multiple soluble enzymes from the late anthocyanin pathway, other CYP73 isoforms, as well as additional ER membrane anchored CYPs including p-coumaric acid 3-hydroxylase (C3H, CYP98A2). Our results suggest that CYP73A412 co-localizes with enzymes from the phenylpropanoid- and downstream anthocyanin pathways, supporting the idea that CYP73s may serve as ER anchoring points for these metabolic pathways. Moreover, this study demonstrates the feasibility of using protoplasts to perform global mapping of protein network for enzymes in their native cellular environment.