Imaging of the Yellow Cameleon 3.6 indicator reveals that elevations in cytosolic Ca2+ follow oscillating increases in growth in root hairs of Arabidopsis

Abstract

In tip-growing cells, the tip-high Ca(2+) gradient is thought to regulate the activity of components of the growth machinery, including the cytoskeleton, Ca(2+)-dependent regulatory proteins, and the secretory apparatus. In pollen tubes, both the Ca(2+) gradient and cell elongation show oscillatory behavior, reinforcing the link between the two. We report that in growing root hairs of Arabidopsis (Arabidopsis thaliana), an oscillating tip-focused Ca(2+) gradient can be resolved through imaging of a cytosolically expressed Yellow Cameleon 3.6 fluorescence resonance energy transfer-based Ca(2+) sensor. Both elongation of the root hairs and the associated tip-focused Ca(2+) gradient show a similar dynamic character, oscillating with a frequency of 2 to 4 min(-1). Cross-correlation analysis indicates that the Ca(2+) oscillations lag the growth oscillations by 5.3 +/- 0.3 s. However, growth never completely stops, even during the slow cycle of an oscillation, and the concomitant tip Ca(2+) level is always slightly elevated compared with the resting Ca(2+) concentration along the distal shaft, behind the growing tip. Artificially increasing Ca(2+) using the Ca(2+) ionophore A23187 leads to immediate cessation of elongation and thickening of the apical cell wall. In contrast, dissipating the Ca(2+) gradient using either the Ca(2+) channel blocker La(3+) or the Ca(2+) chelator EGTA is accompanied by an increase in the rate of cell expansion and eventual bursting of the root hair tip. These observations are consistent with a model in which the maximal oscillatory increase in cytosolic Ca(2+) is triggered by cell expansion associated with tip growth and plays a role in the subsequent restriction of growth.

Figure 1.

Arabidopsis root hairs show an oscillating tip-focused Ca²⁺ gradient that peaks after maximal growth. A, Root hairs undergoing tip growth in Arabidopsis plants expressing the Ca²⁺ sensor YC3.6 targeted to the cytosol were imaged every 3 s. Cytosolic Ca²⁺ levels were calibrated as described in “Materials and Methods” and pseudocolor coded according to the scale at right. Numbers represent time in seconds. Representative results of more than 40 measurements are shown. Bar = 10 μm. B, Quantitative analysis of cytosolic Ca²⁺ oscillations in a representative growing root hair. Ca²⁺ levels were measured in 5-μm² regions of interest (ROI) along the root hair length, as indicated in the inset. Increase in the FRET/CFP ratio reflects an increase in cytoplasmic Ca²⁺ level. C, Average Ca²⁺ levels during peaks and troughs of Ca²⁺ oscillations. Values selected for calculation of averages are depicted with the asterisk (peak) and arrowhead (trough) in B. D, Quantitative analysis of root hair growth rates and cytosolic Ca²⁺ levels at the root hair apex. Ca²⁺ was measured in the approximately 30-μm² ROI indicated in A. Representative results of more than 10 measurements are shown. E, Cross-correlation analysis of Ca²⁺ oscillations with growth oscillations indicates that the increases in cytosolic Ca²⁺ lag the increases in growth rate by approximately 5 s. Cross-correlation was performed on data from eight separate root hairs.

Figure 2.

Effects of La³⁺ on cytosolic Ca²⁺ and growth of Arabidopsis root hairs. A, Treatment with 200 μm La³⁺ triggers rapid dissipation of the tip-focused Ca²⁺ gradient in a growing root hair. Ca²⁺ levels were measured in the approximately 30-μm² ROI at the root hair apex indicated in Figure 1A. The rapid increase in Ca²⁺ at the end of this recording is due to Ca²⁺ entry during bursting of the root hair. The arrow denotes this Ca²⁺ increase due to bursting. B, Treatment with 200 μm La³⁺ causes acceleration of elongation and eventual bursting of growing root hairs. Representative results of seven (Ca²⁺) and 10 (growth) measurements are shown.

Figure 3.

Effects of the Ca²⁺ ionophore A23187 on cytosolic Ca²⁺ and growth of Arabidopsis root hairs. A, Treatment with 10 μm A23187 triggers a rapid increase of cytosolic Ca²⁺ in a growing root hair. Ca²⁺ levels were measured in the approximately 30-μm² ROI at the root hair apex indicated in Figure 1A. Representative results of 10 measurements are shown. B, Treatment with 10 μm A23187 arrests root hair tip growth. All detectable growth of the root hair had ceased by 14 s, when measurements resumed. The nongrowing root hair did not remain in the same plane as the root axis continued to shift after treatment, necessitating constant refocusing. At or below the limits of resolution for the tracker (0.2 μm min⁻¹), this combination of changes in focal plane and root expansion appear as very slow growth for this example. Representative results of 10 measurements are shown. C to F, Bright-field and fluorescence images of root hairs loaded with fluorescein diacetate and immersed in medium containing fluorescein dextran. C and D, Untreated growing control root hairs. Cell wall (arrowhead) thickness is indicated by the exclusion of cytosolic and extracellular fluorescein fluorescence. E and F, A23187-induced Ca²⁺ increase and growth inhibition are accompanied by a thickening of the apical root hair cell wall (arrowhead). Images were acquired 1 h after the start of ionophore treatment. Representative results of 11 measurements are shown. G to J, Bright-field and fluorescence images of root hairs expressing YFP-RabA4b. G and H, Untreated growing control root hairs. Note that YFP-RabA4b accumulates at the apex of the growing root hair. I and J, A23187-induced apical cell wall thickening is accompanied by an accumulation of YFP-RabA4b at the cell apex. The root hair was immersed in medium containing fluorescein dextran to help visualize cell wall thickness (arrowhead). Representative results of 10 measurements are shown. Bars = 10 μm.

Figure 4.

Temporal and spatial relationships between growth, the tip-focused Ca²⁺ gradient, surface (wall) pH, and surface (wall) ROS. Relative timings of growth, pH, and ROS were taken from Monshausen et al. (2007).