EPSIN1 and MTV1 define functionally overlapping but molecularly distinct trans-Golgi network subdomains in Arabidopsis.

The plant trans-Golgi network (TGN) is a central trafficking hub where secretory, vacuolar, recycling, and endocytic pathways merge. Among currently known molecular players involved in TGN transport, three different adaptor protein (AP) complexes promote vesicle generation at the TGN with different cargo specificity and destination. Yet, it remains unresolved how sorting into diverging vesicular routes is spatially organized. Here, we study the family of Arabidopsis thaliana Epsin-like proteins, which are accessory proteins to APs facilitating vesicle biogenesis. By comprehensive molecular, cellular, and genetic analysis of the EPSIN gene family, we identify EPSIN1 and MODIFIED TRANSPORT TO THE VACUOLE1 (MTV1) as its only TGN-associated members. Despite their large phylogenetic distance, they perform overlapping functions in vacuolar and secretory transport. By probing their relationship with AP complexes, we find that they define two molecularly independent pathways: While EPSIN1 associates with AP-1, MTV1 interacts with AP-4, whose function is required for MTV1 recruitment. Although both EPSIN1/AP-1 and MTV1/AP-4 pairs reside at the TGN, high-resolution microscopy reveals them as spatially separate entities. Our results strongly support the hypothesis of molecularly, functionally, and spatially distinct subdomains of the plant TGN and suggest that functional redundancy can be achieved through parallelization of molecularly distinct but functionally overlapping pathways.

Pectin methylesterase activity is required for RALF1 peptide signalling output.

The extracellular matrix plays an integrative role in cellular responses in plants, but its contribution to the signalling of extracellular ligands largely remains to be explored. Rapid alkalinisation factors (RALFs) are extracellular peptide hormones that play pivotal roles in various physiological processes. Here, we address a crucial connection between the de-methylesterification machinery of the cell wall component pectin and RALF1 activity. Pectin is a polysaccharide, contributing to the structural integrity of the cell wall. Our data illustrate that the pharmacological and genetic interference with pectin methyl esterases (PMEs) abolishes RALF1-induced root growth repression. Our data suggest that positively charged RALF1 peptides bind negatively charged, de-methylesterified pectin with high avidity. We illustrate that the RALF1 association with de-methylesterified pectin is required for its FERONIA-dependent perception, contributing to the control of the extracellular matrix and the regulation of plasma membrane dynamics. Notably, this mode of action is independent of the FER-dependent extracellular matrix sensing mechanism provided by FER interaction with the leucine-rich repeat extensin (LRX) proteins. We propose that the methylation status of pectin acts as a contextualizing signalling scaffold for RALF peptides, linking extracellular matrix dynamics to peptide hormone-mediated responses.

Endocytic trafficking promotes vacuolar enlargements for fast cell expansion rates in plants.

The vacuole has a space-filling function, allowing a particularly rapid plant cell expansion with very little increase in cytosolic content (Löfke et al., 2015; Scheuring et al., 2016; Dünser et al., 2019). Despite its importance for cell size determination in plants, very little is known about the mechanisms that define vacuolar size. Here, we show that the cellular and vacuolar size expansions are coordinated. By developing a pharmacological tool, we enabled the investigation of membrane delivery to the vacuole during cellular expansion. Our data reveal that endocytic membrane sorting from the plasma membrane to the vacuole is enhanced in the course of rapid root cell expansion. While this 'compromise' mechanism may theoretically at first decelerate cell surface enlargements, it fuels vacuolar expansion and, thereby, ensures the coordinated augmentation of vacuolar occupancy in dynamically expanding plant cells.