Effect of N source on concentration of Rubisco in Eucalyptus diversicolor, as measured by capillary electrophoresis

Proteins in plant tissues have been extensively characterised by conventional methods such as liquid chromatography and polyacrylamide gel electrophoresis - methods that are tedious and time-consuming. Capillary electrophoresis is potentially a more simple and cost-effective method (with respect to time and consumables) but needs substantial development, especially for native plants which are frequently poor in protein and rich in interfering substances (oils, tannins, phenols). We report here the development of capillary electrophoresis (CE) for the separation of SDS-protein complexes (by molecular mass) and their quantification in plant tissues, In Leaf extracts, two peaks dominated the electropherograms, these peaks had migration times corresponding to the small and large subunits of Rubisco (ribulose-1,5-bisphosphate carboxylase/oxygenase; EC 4.1.1.39) and co-migrated with added purified Rubisco. Linearity of peak area, reproducibility of migration time and peak areas for the small and large subunit were excellent, suggesting Rubisco could be quantified with a high degree of accuracy. We determined how the concentration (0.5 or 4 mM) and form of N applied (nitrate versus ammonium) affects partitioning of N to Rubisco in seedlings of Eucalyptus diversicolor. Analysis of extracts from leaves of E. diversicolor was only possible after precipitation of proteins with trichloroacetic acid (TCA). Precipitation with TCA was highly reproducible and recovery of added Rubisco through procedures of extraction, precipitation and analysis were close to 100% for both subunits. An 8-fold difference in the concentration of N applied did not affect total N, the concentration of Rubisco or the fraction of N present as Rubisco. The similarity of total N may well reflect faster rates of growth in those plants receiving 4 mM N, and a subsequent 'dilution' of tissue N. The N source did not affect total N, the concentration of Rubisco or the fraction of N present as Rubisco. Despite similar Rubisco concentrations, the total concentration of soluble proteins was greater in ammonium-grown plants.