Propidium iodide competes with Ca(2+) to label pectin in pollen tubes and Arabidopsis root hairs.

Peter K. Hepler,Lawrence J. Winship,Eric Lubeck,Caleb M. Rounds

doi:10.1104/pp.111.182196

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.

Highlights

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
Competition studies indicate that PI and Ca2+ bind to the same sites in cell walls. Supporting these studies, we demonstrate that pectin methyl esterase (PME) creates more sites for PI binding, presumably by demethoxylating HGs as they are secreted, and that pectinase reduces PI fluorescence dramatically
Because PI vitally labels lily and tobacco pollen cell walls and predicts growth rate, we asked whether a similar phenomenon existed in two other well-studied tip-growing plant cells: the Arabidopsis root hair and pollen tube

Summary

Introduction

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. PI at low yet useful concentration is vital and does not alter the tip-focused Ca2+ gradient or growth oscillations. A positively charged phenanthridine derivative, the propidium ion stains cell walls but does not pass through the intact cell membranes of living cells It readily diffuses into dead cells and forms highly fluorescent complexes by intercalation between base pairs of double-stranded nucleic acids, acting as an excellent indicator for cell vitality (Hudson et al, 1969). We test two hypotheses: first, that PI stains other tip-growing cells with pectin-containing cell walls; and second, that PI and Ca2+ bind to the same sites in these walls

Methods

Results

Conclusion