Light at the end of the tunnel: FRAP assay reveals that plant vacuoles start as a tubular network

Abstract

Plant vacuoles play key roles in cellular homeostasis performing catabolic and storage functions, regulating pH and ion balance 1,2. The essential role of vacuoles for plant cell viability makes them a notoriously difficult subject to study. As a consequence, there is still no consensus on the mechanism of vacuolar establishment and the source of membrane material for it. Our previous suggestion of endoplasmic reticulum (ER) being the main contributor of membrane for growing young vacuoles3 was recently challenged in a study proposing that plant vacuoles are formed de novo via homotypic fusion of multivesicular bodies (MVBs)4. Authors of this work pointed out issues that might explain our seemingly contradictory observations and we have thus carefully revaluated our hypothesis. Using the Arabidopsis thaliana root as a model, we provide a systematic overview of successive vacuolar biogenesis stages, starting from the youngest cells proximate to the quiescent center. We validate our previous conclusions by demonstrating that the vacuolar dye BCECF is fully suitable for studying the organelle morphology and provide 3D models from vacuoles of all developmental stages. We established a customized FRAP assay and proved that even at the earliest stages of biogenesis, vacuoles comprise a connected network. Together, this adds to a growing body of evidence indicating that vacuolar structures cannot originate solely from MVBs.

Networked proteins redundantly interact with VAP27 and RABG3 to regulate membrane tethering at the vacuole and beyond

“…Together with the partial ER-localization of NET4 and its ability to interact with the ER-PM contact site protein VAP27 (Wang et al, 2014), this establishes a direct connection between the ER and the vacuole. This could represent an efficient way to provide ER-derived lipids to the expanding vacuole or to deliver distinct sets of proteins to developing (pro-) vacuoles (Viotti et al, 2013; Minina et al, 2021). NET3- and NET4-mediated bridging of the ER and the vacuole to establish a membrane contact site would be novel in plants.…”

Recent advances in plant endomembrane research and new microscopical techniques

“…The classic model as demonstrated by 2D‐TEM indicates that vacuoles stem from Golgi and post‐Golgi‐derived vesicles, which fuse with each other to form the central vacuole (Marty, 1999). Research using 3D confocal imaging of living cells with 200‐nm resolution together with fluorescence recovery after photobleaching (FRAP) assay indicated that vacuoles originated from tubular network (Minina et al ., 2021). Immunogold‐TEM of high‐pressure frozen/freeze‐substituted, and serial block‐face scanning electron microscope (SBF‐SEM) of chemically fixed samples have revealed a single interconnected vacuole inside the cell which originates from the ER, together with connection of ER‐vacuole and ER‐to‐vacuole transfer (Viotti et al ., 2013; Kolb et al ., 2015; Lofke et al ., 2015; Scheuring et al ., 2016).…”