Vacuolar Staining Methods in Plant Cells

Abstract

Commercially available fluorescent dyes enable the fast and specific visualization of plant vacuoles, allowing for investigation of membrane dynamics and vacuolar biogenesis in living cells. Here, we describe different approaches tinting the tonoplast or the vacuolar lumen with a range of dyes, and illustrate its utilization with established fluorescent-tagged marker lines.

Extracellular matrix sensing by FERONIA and Leucine‐Rich Repeat Extensins controls vacuolar expansion during cellular elongation in Arabidopsis thaliana

“…This allows us to assess the impact of cell wall acidification before the actual onset of elongation. Using the tonoplast stain MDY-64 or the tonoplast marker line pUBQ10::VAMP711-YFP (Geldner et al, 2009;Löfke et al, 2015;Scheuring et al, 2015), we revealed that cell wall acidification correlated with a dramatic alteration in vacuolar morphology (Fig 2A, C and E), leading to an increased vacuolar occupancy of the cell ( Fig 2B, D and F). Fusicoccin-induced cell wall acidification had an immediate (within 30 min) influence on the vacuolar lumen (Appendix Fig S2A and B), suggesting that cell wall A Representative images and quantification of vacuolar morphology of late meristematic cells.…”

“…We either directly acidified the rhizosphere (by lowering the pH of the media) or genetically (SAUR19 induction) as well as pharmacologically (fusicoccin treatment) induced the activity of the plasma membrane H + -ATPase (Marre, 1979;Spartz et al, 2014Spartz et al, , 2017. Using the tonoplast stain MDY-64 or the tonoplast marker line pUBQ10::VAMP711-YFP (Geldner et al, 2009;Löfke et al, 2015;Scheuring et al, 2015), we revealed that cell wall acidification correlated with a dramatic alteration in vacuolar morphology ( A Representative images and quantification of vacuolar morphology of late meristematic cells. We quantified the vacuolar morphology, depicting the dimension of the biggest luminal vacuolar structure Scheuring et al, 2016), in root epidermal cells of the late meristematic zone.…”

Flagellin peptide flg22 gains access to long-distance trafficking in Arabidopsis via its receptor, FLS2

“…Cellular trafficking of flg22 and the identity of fluorescently labeled compartments was evaluated by colocalization with dyes staining plasma membrane, endocytic vesicles and MVBs (FM4-64) (Bolte et al , 2004; van Gisbergen et al , 2008) or prevacuolar compartments/vacuoles (BCECF) (Brauer et al , 1995; Scheuring et al , 2015). FM4-64 has previously been used to confirm endocytosis of activated FLS2 (Beck et al , 2012).…”

Actin-dependent vacuolar occupancy of the cell determines auxin-induced growth repression

“…Whenever vacuolar morphology was analyzed, roots were mounted in PI solution (0.02 mg/mL) to counterstain cell walls. MDY-64, FM4-64, and BCECF staining was performed as described previously (32). Z-stacks were recorded with a step size of 420 nm, resulting in 25-35 Z-stack images per cell.…”