Glutamine synthetase and glutamate dehydrogenase isoforms in maize leaves:localization, relative proportion and their role in ammonium assimilation or nitrogen transport

Mesophyll cells (MCs) and bundle-sheath cells (BSCs) of leaves of the C-4 p lant maize (Zen mays L.) were separated by cellulase digestion to determine the relative proportion of the glutamine synthetase (GS; EC 6.3.1.2) or th e NADH-glutamate dehydrogenase (GDH; EC 1.4.1.2) isoforms in each cell type . The degree of cross-contamination between our MC and BSC preparations was checked by the analysis of marker proteins in each fraction. Nitrate reduc tase (EC 1.6.6.1) proteins (110 kDa) were found only in the MC fraction. In contrast, ferredoxin-dependent glutamate synthase (Fd-GOGAT: EC 1.4.7.1) p roteins (160 kDa) were almost exclusively present in the BSC fraction. Thes e results are consistent with the known intercellular distribution of nitra te reductase and Fd-GOGAT proteins in maize leaves and show that the cross- contamination between our MC and BSC fractions was very low. Proteins corre sponding to cytosolic GS (GS-1) or plastidic GS (GS-2) were found in both t he MC and BSC fractions. While equal levels of GS-I (40 kDa) and GS-2 (44 k Da) polypeptides were present in the BSC fraction, the GS-I protein level i n the MC fraction was 1.8-fold higher than the GS-2 protein pool. Following separation of the GS isoforms by anion-exchange chromatography of IMC or B SC soluble protein extracts, the relative GS-I activity in the MC fraction was found to be higher than the relative GS-2 activity. In the BSC fraction , the relative GS-I activity was very similar to the relative GS-2 activity . Two isoforms of GDH with apparent molecular weights of 41 kDa and 42 kDa, respectively. were detected in the BSC fraction of maize leaves. Both GDH isoenzymes appear to be absent from the MC fraction. In the BSCs, the level of the 42-kDa GDH isoform was 1.7-fold higher than the level of the 41-kDa GDH isoform. A possible role for GS-I and GDH coacting in the synthesis of glutamine for the transport of nitrogen is discussed.