Analysis of solutes and metabolites in single plant cell vacuoles by capillary electrophoresis

Abstract

Capillary electrophoresis (CE) is demonstrated as a useful technique for the determination of a large number of solutes and metabolites such as inorganic anions, carboxylic acids, sugars and amino acids in single plant cell vacuoles. Sample volumes in the picoliter range can be extracted from single vacuoles and divided into subsamples for subsequent analyses. The challenges involved with such low volume samples are the low analyte amounts present, which require CE systems with sensitive detection approaches for the different compounds, and the danger of contamination and evaporation, which makes minimized sample handling under an inert protective medium essential. Three different separation and detection approaches were used in this investigation. For the determination of inorganic anions and carboxylic acids in vacuolar samples, indirect UV detection using salicylic acid as background electrolyte provides sufficient sensitivity. Amino acids were derivatised on-column with o-phthaldialdehyde-2-mercaptoethanol, separated by micellar electrokinetic chromatography and detected with fluorescence. For sugars a separation and direct UV detection based on chelation with Cu(II) was employed. The determination of these compounds in single vacuoles of epidermal or mesophyll cells of wheat leaves is shown.