The aim is to elucidate the ionic components of the mechanism by which the growth hormone, indole acetic acid (IAA), causes loosening of cell walls to produce elongation growth of plant stems. There is a long-standing controversy over whether acidification of plant cell walls is a sufficient condition for growth to take place. Whether or not this 'acid-growth' hypothesis is accepted, the question remains, "What is the wall-loosening mechanism?" Is it pH-dependent wall enzyme activation, breakage of calcium bonds between wall components, or some other process? It is agreed that the mechanism certainly involves H+ and Ca2+, but there is lack of knowledge of what IAA-stimulated H+ and Ca2+ movements occur between plasma membrane, cell wall and external solution. This project is designed to provide that knowledge which underlies the mechanism of IAA action. The measurement of the fluxes of specific ions and their exchange in the cell wall under IAA treatment has not been done outside this laboratory.
Knowledge of the IAA-induced ion fluxes will link with and illuminate many other observations on the action of IAA on the cell wall, and models for it, but specific questions we seek to answer for oats and soybean are:
How do the time courses of all the ion fluxes relate to each other, to membrane potential changes and to growth, both for protoplasts and for tissue segments? The 10-minute 'lag' in IAA-induced growth and other responses (not found in response to fusicoccin) is particularly pertinent here.
Central to the project is the Microelectrode Ion Flux Estimation (MIFE) technique. In this technique the electrochemical potential gradient of an ion is measured by moving the tip of an ion selective microelectrode through a small distance in solution close to the tissue. The net flux is then calculated from that gradient, knowing the mobility and concentration of the ion (Newman et al., 1987; Ryan et al., 1990). A PC-based multi-channel electrometer data acquisition system has been developed. The system incorporates software so that up to 8 channels of data can be displayed, chart recorder-like, in real time on the screen. The new MIFE system allows more than 10-fold improvement in sensitivity over our previous system and time resolution down to 10 seconds for large fluxes. It allows a real-time estimation of fluxes and calculation of fluxes in non-planar geometries.
Fusicoccin causes an immediate H+ efflux from the plasmalemma. Fusicoccin therefore functions as a useful reference material for IAA which, by contrast, produces a delayed H+ efflux and may act primarily on the cell wall or a cytoplasmic component. We will therefore make measurements of the effects of each of IAA and fusicoccin on all plant material used. Application of auxin analogues, active and inactive, will allow further comparisons to be made.
Protoplasts, following IAA application to them, will show fluxes of H+ and Ca2+ very different from those from tissue containing cells with walls. A replicate MIFE system has been developed within this project, to allow measurement of ion fluxes from plant protoplasts prepared from the tissue types used.
We will also develop preparative and culture procedures for protoplasts taken from tissues used. The MIFE sensitivity is greatest at low concentrations of the ion being measured, whereas high millimolar concentrations of cations are usually required to maintain the health of protoplasts. We will particularly determine how low the Ca2+ concentration can be made, how much Ca2+ can be replaced by other divalents and how long protoplasts can remain healthy when transferred to a low-Ca2+ medium for IAA treatment and measurement. This will entail assessment of protoplast membrane integrity. Later in the project we will study protoplasts as they regenerate functional walls over a period of several days. If it appears that IAA may be having a direct effect on walls, we will then test the effect of IAA on wall preparations.
With the protoplasts we will measure ion fluxes caused by IAA or fusicoccin treatment. Studies of protoplast membrane potential, using standard microelectrode techniques while also measuring fluxes, will be made to allow comparison with cell membrane depolarisation and hyperpolarisation observed to occur with segments during IAA or fusicoccin action.
To elucidate further the roles of Ca2+ and H+ in auxin action, and the location of the action, we will modify their concentrations or functioning, for both tissue and protoplasts, by varying bathing Ca2+ or substituting Sr2+ for Ca2+ and by inhibiting the H+ pump. We will also measure cytoplasmic H+ and Ca2+ using ion selective microelectrodes and relate their changes to the flux changes. This will further test the hypothesis that IAA causes Ca2+ to enter the cytoplasm from outside.
During the above work on tissues, the Weak Acid Donnan-Manning model for fluxes (Arif & Newman, 1993) will be extended to deal more completely with all relevant ions.
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If you have comments or questions, please email me at Ian.Newman@utas.edu.au
© 1996 University of Tasmania