Effects of Nod Factors on Alfalfa Root Hair Ca++ and H+ Currents and on Cytoskeletal Behavior

Abstract

In order to understand more about the early responses of alfalfa root hairs to nodulation factors, we used the nonintrusive ion-specific vibrating microelectrode [1,2] to determine the time course of calcium and proton currents around mature and growing root hairs before and after nod factor stimulation. Fluorescence video microscopy was used to study the actin cytoskeleton in normally growing root hairs as well as changes in the actin staining patterns after application of Nod factors over a period of 12 hours. In growing root hairs, the calcium gradient indicates a net influx of calcium ions, while the direction of the proton gradient indicates a net efflux of protons from the root hair tip. No significant gradient was observed in non-growing root hairs. Root hairs perfused with NodRm-IV (S) had a consistent and dramatic rise in proton efflux within five to seven minutes. The response of the Ca++ flux to Nod factors was more complicated with an oscillating pattern of Ca++ influx and efflux recorded at the root tip. The network of actin present in normally growing root hairs is described. After Nod factor exposure, the dynamic changes of the actin filament network over time were found to involve breakdown and rearrangements. The early ionic changes in response to nodulation factors are temporally related to the early membrane depolarization reported by Ehrhardtet al.[3] and the observed changes in the actin cytoskeleton as well as other previously observed rearrangements [4] of the cytoplasmic structure.