Is the infiltration‐centrifugation technique appropriate for the isolation of apoplastic fluid? A critical evaluation with different plant species

Abstract

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.

Studying guard cells in the intact plant: modulation of stomatal movement by apoplastic factors

“…This value is in agreement with the free Ca 2+ concentration measured by Felle et al . (2000), who found an Felle et al (2000) 3 mM 10 mM Mühling and Läuchli (2000) 27 mM* 40 mM* Lohaus et al (2001) 13 mM Cl - Felle et al (2000) 1 mM 1 mM Lohaus et al (2001) 11 mM Ca 2+ Felle et al (2000) 64 µM Lohaus et al (2001) 700 µM pH Felle et al (2000) 5.1 5.5 Mühling and Läuchli (2000) 5.0 5.4…”

“…All cells in the leaf will alter the solute concentration depending on their transport properties. Information on the apoplastic solute composition of Vicia faba has been obtained by infiltration of intact leaves (Lohaus et al , 2001). For the dynamical changes in the ion concentration this method is less suitable, but rapid changes in ion concentration have been measured using fluorescent dyes (Mühling & Läuchli, 2000) or with ion selective electrodes brought in contact with the cell wall via an open stoma (Felle et al , 2000).…”

“…This may be due to the fact that most Ca 2+ in the apoplast is bound and will equilibrate with an infiltrated solution to get a uniform free Ca 2+ concentration. When these values are simply corrected for the dilution occurring during infiltration (Lohaus et al , 2001) an overestimation of the free Ca 2+ concentration will be made. The actual free Ca 2+ concentration is probably closer to the value not corrected for dilution, which is 0.08 mM.…”

“…Malate (Lohaus et al, 2001) 0.7 mM Amino acids (Lohaus et al, 2001) 9.6 mM Sucrose (Lohaus et al, 2001) 1.6 mM Hexoses (Lohaus et al, 2001) 0.7 mM…”

“…The values determined by Lohaus et al (2001) were obtained by infiltration of excised leaves, which were preincubated for 7 h in the light. The data of Mühling and Läuchli (2000) derive from measurements with fluorescent dyes fed to the leaf via the petiole or by infiltration of the leaf.…”