Isolation of intact vacuoles from Arabidopsis rosette leaf–derived protoplasts

Abstract

Vacuoles are very prominent compartments within plant cells, and understanding of their function relies on knowledge of their content. Here, we present a simple vacuole purification protocol that was successfully used for large-scale isolation of vacuoles, free of significant contamination from other endomembrane compartments. This method is based on osmotic and thermal disruption of mesophyl-derived Arabidopsis protoplasts, followed by a density gradient fractionation of the cellular content. The whole procedure, including protoplast isolation, takes approximately 6 h. INTRODUCTIONLarge-scale isolation and characterization of cellular organelles offers excellent opportunities in plant systems biology 1 . Detailed qualitative and quantitative knowledge of the composition of a particular organelle allows for investigation of subcellular pathways through the identification of novel protein components and the analysis of cellular sinks. This also facilitates detailed comparative studies between species or between mutant and wild-type plants. In contrast to some organelles, such as mitochondria and chloroplasts, that are relatively easy to obtain in a pure form 2,3 , plant vacuoles are extremely fragile, and their isolation using conventional tissue homogenization and fractionation schemes can be remarkably challenging and often results in a subcellular fraction without the same properties as intact mature vacuoles 4 . The first successful technique to overcome these difficulties was based on protoplast isolation and subsequent gentle release of vacuoles by osmotic shock 5,6 . Later large-scale isolation protocols employed gradient fractionation of osmotically released organelles 7,8 , and the method was further optimized by application of both osmotic and thermal shocks to the isolated protoplasts 9,10 . As an alternative to the protoplast methods, which are applicable only to relatively soft tissues, vacuoles can be isolated by slicing the plant material and releasing contents into a medium with a perfectly adjusted osmoticum 11 . In subsequent studies, numerous vacuolar constituents have been determined, including sugars and amino acids 12 , hydrolases 13 and ions 14 .

Nitrogen Use Efficiency Is Mediated by Vacuolar Nitrate Sequestration Capacity in Roots of Brassica napus

“…Similar to the above assays, root tissues (1.0 g) were collected from B. napus at seedling and flowering stages and from Arabidopsis (0.5 g) seedlings and used to isolate intact protoplasts and vacuoles as described by Robert et al (2007), with minor modifications as outlined in Huang et al (2012) and Han et al (2015b). The purified protoplasts were divided into two equal aliquots, with one of them used for isolation of vacuoles.…”

“…Roots of both species were collected similarly as that used for V-ATPase and V-PPase activities, then used for vacuole isolation and measurement of NO 3 2 flux in vacuoles as described by Robert et al (2007), with minor modification (Han et al, 2015b). Net fluxes of NO 3 2 in vacuoles were measured noninvasively using SIET (scanning ion-selective electrode technique, SIET system BIO-003A; Younger USA Science and Technology Corporation).…”

Evidence for autophagy-dependent pathways of rRNA turnover inArabidopsis

“…Vacuoles were purified from Arabidopsis rosette leaves grown under LD conditions during early bolting 92 and frozen in liquid nitrogen. Vacuole purity was assessed by immunoblotting using antibodies against the endoplasmic reticulum resident protein BiP (Enzo Life Sciences, AD1-SPA-818, 1:1000 dilution) and the vacuolar protein aleurain (1:500 dilution).…”

Mobilization of Rubisco and Stroma-Localized Fluorescent Proteins of Chloroplasts to the Vacuole by anATGGene-Dependent Autophagic Process

“…Protoplasts were prepared from the concanamycin A-treated leaves by the procedure of Asai et al (2000), except that 1 mM concanamycin A was added to each preparation medium. Vacuoles were prepared from the protoplasts by the procedure of Robert et al (2007).…”