Quantification of Apoplastic Potassium Content by Elution Analysis of Leaf Lamina Tissue from Pea (Pisum sativum L. cv Argenteum)

Abstract

K+ content and concentration within the apoplast of mesophyll tissue of pea (Pisum sativum L., cv Argenteum) leaflets were determined using an elution procedure. Following removal of the epidermis, a 1 centimeter (inside diameter) glass cylinder was attached to the exposed mesophyll tissue and filled with 5 millimolar CaCI2 solution (10C). From time-course curves of cumulative K+ diffusion from the tissue, the amount of K+ of extracellular origin was estimated. Apoplastic K+ contents for leaves from plants cultured in nutrient solution containing 2 or 10 millimolar K+ were found to range from 1 to 4.5 micromoles per gram fresh weight, comprising less than 3% of the total K+ content within the lamina tissue. Assuming an apoplastic solution volume of 0.04 to 0.1 milliliters per gram fresh weight and a Donnan cation exchange capacity of 2.63 micromoles per gram fresh weight (experimentally determined), the K+ concentration within apoplastic solution was estimated at 2.4 to 11.8 millimolar. Net movement of Rb+ label from the extracellular compartment within mesophyll tissue into the symplast was demonstrated by pulse-chase experiments. It was concluded that the mesophyll apoplast in pea has a relatively low capacitance as an ion reservoir. Apoplastic K+ content was found to be highly sensitive to changes in xylem solution concentration.The apoplast within leaf tissue constitutes a potentially important pathway for K+ transport between vascular and mesophyll tissues (6,22,23) accumulate extracellulary within the vascular bundles, whereas lower concentrations would exist within the cell wall of mesophyll tissue more distant from the minor veins.The specific role(s) ofthe apoplast as related to K+ transport and accumulation within leaf lamina tissue cannot be fully elucidated without quantitative analysis of extracellular K+ content and concentration (20). Several indirect approaches have been reported for collection of apoplastic sap and estimation of ionic concentrations including analysis of xylem exudates, vacuum perfusion of leaf lamina discs (1), and pressure dehydration of leaves (14). X-ray microanalysis (7, 9, 12) and ion-sensitive microelectrodes have been used to measure directly extracellular ion concentrations ofepidermal guard cells (2) and the extensor and flexor cells within the pulvinus of legume leaves (26). The primary limitation of these procedures is the inability to estimate the ionic content within the apoplastic reservoir of the mesophyll.Apoplastic ion content is a function of the amount of ion bound to fixed, negatively charged sites within the cell wall, the Donnan free space, and that in solution within the cell wall. Although the cation exchange capacity of leaf cell walls has been investigated for a variety of plant species (19, 25), the 'functional' volume of apoplastic solution in situ is more difficult to estimate. The volume within a unit fresh weight of tissue in theory is influenced by the thickness of the cell walls and the mean size of mesophyll cells. In addition, the vo...

Time dependent K+ currents through plasmalemma of laticifer protoplasts from Hevea brasiliensis

“…Figure 3 summarizes the current-voltage relationships for the increasing external KCl concentrations from 0.1 to 100 mW. Figure 3A shows ohmic current-voltage relationships, the magnitude of this current being sigmficantly increased beyond 10 xaM, suggesting the control of this current for physiological K" concentrations (Long and Widders 1990). Figure 3B shows that varying KCl from 0.1 to 100 mM reduced the steady-state current resulting from the time-dependent activation of the channels.…”

Protein extraction for proteome analysis from cacao leaves and meristems, organs infected by Moniliophthora perniciosa, the causal agent of the witches' broom disease

“…Several methods of isolation of apoplastic fluid have been developed such as application of pressure using the Scholander bomb [35], the vacuum perfusion method [36], the elution method [37] and the infiltration-centrifugation method. The latter has been used in different variations: centrifugation technique without infiltration process [25], infiltration technique without a centrifugation process [38] or a combination of both, the infiltration-centrifugation technique [39][40][41].…”

Cloning of Arabidopsis and barley cDNAs encoding HAK potassium transporters in root and shoot cells

“…These characteristics fulfil the requirements of the 'active' K + uptake that roots need for a competent K + uptake at low K + (Schroeder and Fang 1991, Kochian and Lucas 1993, Maathuis and Sanders 1993, Walker et al 1996, Maathuis et al 1997b), but are apparently unnecessary or even inappropriate, because they give rise to energy wasting, for cells facing millimolar K + concentrations. Although this consideration may lead to the conclusion that transporters of the HAK family (from now on we will use the generic name of HAK for the eukaryotic members of the bacterial and eukaryotic Kup-HAK type of transporters) cannot be universal plant K + transporters because in many plant cells the concentration of K + in the apoplast is millimolar (Lowen and Satter 1989, Long and Widders 1990, Mü hling and Sattelmacher 1997, other facts must also be taken into account. The first is, obviously, that not all the transporters in the Kup-HAK family must be high-affinity K + -H + symporters, but more importantly, in some cases non-root plant cells do in fact take up K + from micromolar concentrations (Reed andBonner 1974, Blatt 1985), following kinetics very similar to those exhibited by root cortical and epidermal cells (Smith andEpstein 1964, Osmond andLaties 1968).…”