Propidium iodide competes with Ca(2+) 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.

Figure 1.

PI fluorescence and growth rate oscillate in lily pollen tubes (A and B), Arabidopsis root hairs (C–E), and Arabidopsis pollen tubes (F and G). A, The top panel shows a DIC image of a lily pollen tube, and the bottom panel shows PI fluorescence of the same tube. The PI fluorescence is pseudocolored, with white representing high signal and blue representing low signal. Bar = 10 μm. B, Growth rate (blue) and PI fluorescence (red) are plotted on a line graph. Both oscillate with the same period but with different phases. C, DIC image (top panel) and PI fluorescence image (bottom panel) of an Arabidopsis root hair. Bar = 10 μm. D, Two PI fluorescence images of the same root hair focused on the apex representing peak (top) and trough (bottom) PI signals. Bar = 5 μm. E, A line graph showing the growth rate (blue) and peak PI fluorescence at the apex (red) for the same root hair shown in C and D. F, The top panel shows a DIC image of an Arabidopsis pollen tube, and the bottom panel shows PI fluorescence of the same tube. The PI fluorescence is pseudocolored, with white representing high signal and blue representing low signal. Bar = 5 μm. G, Growth rate (blue) and PI fluorescence (red) are plotted on a line graph. Both oscillate with the same period but with different phases. The growth rate between individual 3-s frames was smaller than the pixel size for our optics in both Arabidopsis cell types; to remove the noise this generated, a four-image (pollen) or five-image (root hair) running average is shown. A.U., Arbitrary units.

Figure 2.

PI fluorescence decreases with increasing Ca²⁺ concentration. A, Arabidopsis roots incubated in 6.4 μm PI in water with increasing amounts of Ca²⁺. B and C, Fluorescence of PI in different concentrations of PI and Ca²⁺ (B) or PI, Mg²⁺, and Ca²⁺ (C). In B, squares represent 100 μm Ca²⁺ (dashed line), triangles represent 10 μm Ca²⁺ (dotted and dashed line), and circles represent 1 mm Ca²⁺ (solid line). In B and C, regression lines represent Hill plots. In B, fluorescence depends on the concentration of Ca²⁺. Decreasing Ca²⁺ results in more fluorescence at a given PI concentration. Each point represents the average of at least 20 pollen tubes, and error bars represent se. In C, triangles represent 10 μm Ca²⁺ (dotted and dashed line), squares represent 100 μm Ca²⁺ (dashed line), diamonds represent 10 μm Ca²⁺ + 90 μm Mg²⁺ (dotted line), and circles represent 10 μm Ca²⁺ plus 1 mm Mg²⁺ (solid line). A.U., Arbitrary units.

Figure 3.

PI growth inhibition depends on Ca²⁺ concentration (A) but not Mg²⁺ (B). Increasing concentrations of PI inhibit pollen tube growth. This can be mitigated by increasing the concentration of Ca²⁺ or exacerbated by decreasing the concentration. In A, squares represent 100 μm Ca²⁺ (dashed line), triangles represent 10 μm Ca²⁺ (dotted and dashed line), and circles represent 1 mm Ca²⁺ (solid line). Regression lines in A represent sigmoidal plots. Each point represents the average of at least 40 pollen tubes, and error bars represent se. B shows a similar experiment performed with Mg²⁺. An increase in Mg²⁺ concentration does not reverse PI inhibition, although a dramatic decrease in growth rate can be seen at the higher Mg²⁺ concentration.

Figure 4.

Increasing PI does not alter growth oscillations. Pollen tubes were grown in LPGM and then plated, and PI was added. Time series from growing tubes with appreciable clear zones were collected, and the growth rates and periods were collected. A, The entire data set at all concentrations shows no significant correlation between growth rate and period. Plus signs represent no PI, squares represent 10 μm PI, diamonds represent 20 μm PI, triangles represent 40 μm PI, and white circles represent 80 μm PI. r² = 0.16. B, The average period is plotted against the increasing PI concentrations. Error bars represent se. n > 8 for each concentration. No linear correlation is seen, as the r² value is 0.07.

Figure 5.

PME causes a dramatic increase in PI fluorescence at the pollen tube apex. A, DIC (top) and PI fluorescence (bottom) images of a pollen tube before (0 min) and after (20 min) treatment with PME. At 20 min, the tip is clearly thicker and the fluorescence has increased dramatically. Bar = 10 μm. B, A line graph representing the time course for the same pollen tube shown in A. Blue represents the growth rate, and red represents the PI fluorescence at the pollen tube apex. Note that both growth rate and PI apex fluorescence continue to oscillate until the pollen tube stops, and then growth rate plummets and PI fluorescence surges in tandem. The arrow in B shows where PME was added to the slide. A.U., Arbitrary units.

Figure 6.

Pectinase decreases PI fluorescence and stops growth. A, Pectinase was added to growing lily pollen tubes. The top panels show DIC images before and after pectinase addition. The bottom panels show the PI fluorescence of the same pollen tube at the same time points with the same look-up table. B, Mean intensity of a line scan down the middle of six pollen tubes before treatment with pectinase on the left and after treatment on the right. The gray lines represent se.

Figure 7.

PI does not interfere with tip-focused cytosolic Ca²⁺ gradient ([Ca²⁺]_i). A, DIC (top), PI fluorescence (middle), and [Ca²⁺]_i (bottom) images of a growing pollen tube that has been injected with fura-2 dextran and labeled with PI. Bar = 10 μm, and the Ca²⁺ gradient scale is in nanomolar. B, Line graphs showing the growth rate (top), apical PI signal (middle), and [Ca²⁺]_i (bottom) at the apex of the pollen tube shown in A. Growth rate, PI, and [Ca²⁺]_i continue to oscillate in the same manner as each of the phenomena measured individually. A.U., Arbitrary units.

Figure 8.

Model for binding of Ca²⁺ and PI to HGs during tip growth. Disorganized chains of highly methoxylated HG leave the growing cell by exocytosis. In the cell wall, PME demethoxylates the chains, creating linear regions of negatively charged carboxyl groups. In the presence of Ca²⁺, cooperative binding occurs between HG chains, creating a stiff gel. In the presence of Ca²⁺ and PI, both ions bind to carboxyls, although PI cannot cross-link.