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. The essential role of vacuoles for plant cell viability makes them a notoriously difficult subject to study impeding reaching the consensus on the mechanism of vacuolar establishment and the source of membrane material for it. Our previous suggestion of endoplasmic reticulum being the main membrane contributor for the tubular network of young vacuoles was recently challenged in a study proposing that young plant vacuoles comprise a set of individual vesicles that are formed de novo via homotypic fusion of multivesicular bodies (MVBs). To resolve these seemingly contradictory observations we have carefully revaluated both hypotheses. Here we provide a systematic overview of successive vacuolar biogenesis stages in Arabidopsis root, 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 organelles morphology and provide 3D models of vacuoles at all developmental stages. Furthermore, we established a customized FRAP assay and proved that even at the earliest stages of biogenesis, vacuoles comprise a connected network. Finally, we summarized the new and pre-existing evidence substantiating that vacuolar structures cannot originate solely from MVBs.