Extraction of apoplastic sap from plant roots by centrifugation

A centrifugal method for extracting apoplastic sap from roots of lupin (Lupinus angustifolius) and pea (Pisum sativum) plants, and a method to analyse malic dehydrogenase in the sap using capillary electrophoresis, are described. Osmolality of apoplastic sap was relatively constant at relative centrifugal forces (RCFs) between 600 and 3000 g for lupin, and between 600 and 4000 g for pea. Electropherograms of a marker enzyme (malic dehydrogenase) and other components in apoplastic and symplastic saps revealed that contamination occurred at 7000 g. It is suggested that apoplastic sap expelled from plant roots at RCF between 600 and 3000 g is free from symplastic contamination, and is regarded as being of apoplastic origin. The proposed method was used to measure apoplastic pH changes in the plant roots in response to external pH, ammonium, nitrate and vanadate.