Propidium Iodide Competes with Ca2+ to Label Pectin in Pollen Tubes and Arabidopsis Root Hairs    

Abstract

We have used propidium iodide (PI) to investigate the dynamic properties of the primary cell wall at the apex of Arabidopsis (Arabidopsis thaliana) root hairs and pollen tubes and in lily (Lilium formosanum) pollen tubes. Our results show that in root hairs, as in pollen tubes, oscillatory peaks in PI fluorescence precede growth rate oscillations. Pectin forms the primary component of the cell wall at the tip of both root hairs and pollen tubes. Given the electronic structure of PI, we investigated whether PI binds to pectins in a manner analogous to Ca 2+ binding. We first show that Ca 2+ is able to abrogate PI growth inhibition in a dose-dependent manner. PI fluorescence itself also relies directly on the amount of Ca 2+ in the growth solution. Exogenous pectin methyl esterase treatment of pollen tubes, which demethoxylates pectins, freeing more Ca 2+ -binding sites, leads to a dramatic increase in PI fluorescence. Treatment with pectinase leads to a corresponding decrease in fluorescence. These results are consistent with the hypothesis that PI binds to demethoxylated pectins. Unlike other pectin stains, PI at low yet useful concentration is vital and specifically does not alter the tip-focused Ca 2+ gradient or growth oscillations. These data suggest that pectin secretion at the apex of tip-growing plant cells plays a critical role in regulating growth, and PI represents an excellent tool for examining the role of pectin and of Ca 2+ in tip growth.

Metabolic click-labeling with a fucose analog reveals pectin delivery, architecture, and dynamics in Arabidopsis cell walls

“…Calcium crosslinked pectin networks correlate with increased cell wall rigidity (43), and the presence of FucAl-containing fibrils in differentiated cells that have ceased expanding might reflect this rigidification process. In addition, recent data indicate that root-hair tips are enriched in pectins (44), and the bright FucAl-associated fluorescence we observed at root-hair primordia is in agreement with these results.…”

POLYGALACTURONASE INVOLVED IN EXPANSION3 Functions in Seedling Development, Rosette Growth, and Stomatal Dynamics in Arabidopsis thaliana

“…In cotyledons of seedlings, very young stomatal complexes that had not yet initiated a stomatal pore contained PGX3-GFP distributed evenly along the border between the two guard cells. Later, during pore formation, PGX3-GFP was enriched at the site of pore initiation along with propidium iodide (PI)-stained pectins (Rounds et al, 2011) (Figure 2C). Similar results were observed when ProPGX3:PGX3-GFP was expressed in a pgx3 mutant background (see below for descriptions of mutant and transgenic lines).…”

“…Using the cellulose dye Pontamine Fast Scarlet 4B (Anderson et al, 2010) to label stomata that had been induced to open or close by treatment with FC or ABA, we observed similar labeling patterns and changes in cellulose anisotropy (Rui and Anderson, 2016) in both genotypes (Supplemental Figures 8A and 8B), indicating that cellulose organization was more diffuse in guard cells surrounding open stomata but was more fibrillar in guard cells surrounding closed stomata in both Col controls and pgx3-1 mutants. To probe HG abundance and distribution in guard cells as a function of PGX3 expression, we applied either a chitosanoligosaccharide-Alexa 488 probe, COS 488 , which binds to negatively charged carboxyl groups on stretches of demethylesterified GalA residues in HG (Mravec et al, 2014), or propidium iodide, which binds to single demethylesterified GalA residues (Rounds et al, 2011) (Figure 6). These probes were used prior to immunolabeling because the latter method is not compatible with imaging of intact cells, whereas the cuticle permeability of these small molecular probes enabled labeling of intact guard cells without fixation or sectioning, allowing for quantitative analyses of labeling intensity in three dimensions.…”

Monitoring Polysaccharide Dynamics in the Plant Cell Wall

“…PI is a membrane-impermeable stain that competes with Ca 2+ ions to bind to demethylesterified pectins. When used at low concentrations, PI enables imaging in Arabidopsis root hairs and pollen tubes without altering cell growth (Rounds et al, 2011). Mutant pollen tubes with impaired exocytosis show repetitive bursts of growing tips at cell wall regions that accumulate PI-stained demethylesterified HG (Synek et al, 2017).…”