Is the infiltration-centrifugation technique appropriate for the isolationof apoplastic fluid? A critical evaluation with different plant species

The suitability of the infiltration-centrifugation method for collection of apoplastic fluid from intact leaves was evaluated for different plant spec ies. Large differences with respect to infiltrability of the leaves, which correlated inversely with stomatal and mesophyll resistance, became apparen t, Osmolality of infiltration medium (deionised water, 0.2 mM CaCl2, 10 mM KCI, 180 mM 2-[N-morpholino]ethane-sulphonic acid) and incubation time, tim e passed between onset of infiltration and end of centrifugation, revealed relatively little influence on the composition of the apoplastic washing fl uid (AWF), In contrast, the pH of the infiltrated solution highly influence d the concentration of sucrose and hexoses, With increasing centrifugation force, hexosephosphate isomerase (HPI) activity in the AWF, which was taken as an indication for cytoplasmic contamination, increased. At the same tim e, Ca2+ concentration in the AWF increased even more. Since Ca2+ cannot ori ginate from the cytoplasm, the suitability of HPI as marker for cytoplasmic contamination is questioned. From the composition of the AWF, it is conclu ded that, if centrifugation force does not exceed 1000 g, cytoplasmic conta mination is negligible and that the infiltration-centrifugation technique r eveals an easy and inexpensive way to study apoplastic solutes. The infiltr ation-centrifugation method was also suitable to determine apoplastic air v olume (V-air) and apoplastic water volume (V-water), which are necessary fo r the calculation of the ion concentration in the leaf apoplast, It could b e shown that the leaves of different species and the apical and basal leave s of single plants differ in V-air and V-water.