The FIBRILLIN multigene family in tomato, their roles in plastoglobuli structure and metabolism.

Plastoglobuli (PG) are plant lipoprotein compartments, present in plastid organelles. They are involved in the formation and/or storage of lipophilic metabolites. FIBRILLINs (FBNs) are one of the main PG-associated proteins and are particularly abundant in carotenoid-enriched chromoplasts found in ripe fruits and flowers. To address the contribution of different FBNs, independently and in combination, to isoprenoid formation and sequestration, a multiplex gene editing approach was undertaken in tomato. This approach generated a suite of single and high-order fbn mutants that were shown to lack transcripts and respective protein products. The major PG-related FBNs in tomato chosen for this study are SlFBN1, SlFBN2a, SlFBN4 and SlFBN7a. When knocked out independently, functional redundancy was revealed. However, paralog-specific roles were detected regulating specific isoprenoids (e.g. plastochromanol 8) or plastidial esterification capability. In addition, high-order fbn mutants displayed altered isoprenoid chromoplast sequestration patterns, notably with a significant reduction in carotenes (phytoene and phytofluene) in the PG fraction. Proteomic analysis confirmed the absence of PG-core associated proteins, including NAD(P)H-ubiquinone oxidoreductase C1, tocopherol cyclase (VTE1) and phytol esterase (PES1/PYP). Perturbations to the ultrastructure of the plastid were revealed, with aberrant PG formation and morphology predominating in high-order mutants. Global lipidome profiles also highlighted broader changes directly affecting storage and plastid membrane lipids, for example, tri- and diacylglycerides and galactolipid species. Collectively, these results support both structural and metabolic roles of SlFBNs in PGs. The findings expose fundamental aspects of metabolic compartmentalisation in plant cells and the importance of lipoprotein particles for plastid integrity and functionality.