Is the infiltration-centrifugation technique appropriate for the isolation of 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 species. Large differences with respect to infiltrability of the leaves, which correlated inversely with stomatal and mesophyll resistance, became apparent. Osmolality of infiltration medium (deionised water, 0.2 mM CaCl2, 10 mM KCl, 180 mM 2-[N-morpholino]ethane-sulphonic acid) and incubation time, time passed between onset of infiltration and end of centrifugation, revealed relatively little influence on the composition of the apoplastic washing fluid (AWF). In contrast, the pH of the infiltrated solution highly influenced 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 time, Ca2+ concentration in the AWF increased even more. Since Ca2+ cannot originate from the cytoplasm, the suitability of HPI as marker for cytoplasmic contamination is questioned. From the composition of the AWF, it is concluded that, if centrifugation force does not exceed 1 000 g, cytoplasmic contamination is negligible and that the infiltration-centrifugation technique reveals an easy and inexpensive way to study apoplastic solutes. The infiltration-centrifugation method was also suitable to determine apoplastic air volume (Vair) and apoplastic water volume (Vwater), which are necessary for the calculation of the ion concentration in the leaf apoplast. It could be shown that the leaves of different species and the apical and basal leaves of single plants differ in Vair and Vwater.