Distribution of Nitrate-assimilating Enzymes between Mesophyll Protoplasts and Bundle Sheath Cells in Leaves of Three Groups of C₄ Plants

Abstract

Intercellular distribution of enzymes involved in amino nitrogen synthesis was studied in leaves of species representing three C4 groups, i.e. Sorghum bicolor, Zea mays, Digitaria sanguinalis (NADP malic enzyme type); Panicum miliaceum (NAD techniques for separation of mesophyll and bundle sheath cells and also because of the recent progress in understanding the route of amino acid biosynthesis in leaves of C3 plants. The suggestion that glutamate dehydrogenases are responsible for the incorporation of nitrogen into the a-amino group of amino acids through the reductive amination of a-ketoglutarate in presence of ammonia (3) is now regarded by Lea and Miflin (16) as a minor or insignificant pathway. The newly discovered enzymes, glutamine synthetase and glutamate synthase, in leaves of C3 plants has led to the proposal of an alternate pathway for nitrate assimilation (16). The enzyme glutamine synthetase catalyzes the ATP-driven addition of ammonia to glutamate (10,21,22). The amide group of glutamine is then transferred to aketoglutarate by glutamate synthase (16).In leaves, the biological reduction of nitrate to ammonia and the subsequent assimilation of ammonia to organic form utilize photosynthetic energy (3). Since C4 plants have two photosynthetic cell types, mesophyll and bundle sheath, it is of interest to consider the mechanism and localization of nitrate-assimilating enzymes in these species. We utilized species from the three groups of C4 plants in the present work for studying nitrateassimilating enzyme distribution, and the results are discussed in relation to the glutamine synthetase-glutamate synthase pathway of nitrogen assimilation.In C4 plants, the amino acid aspartate is an early labeled product of photosynthesis apparently associated with the C4 pathway (6, 9, 24) rather than being formed from an intermediate of the carbon reduction pathway as occurs in C3 plants (29). Aspartate synthesis in C4 plants takes place in their mesophyll cells (12), and a continual shuttle of aspartate and alanine is proposed between mesophyll and bundle sheath cells which would maintain a nitrogen and carbon balance between the cell types (13,25).In view of this, there has been recent interest in elucidating the mechanism of a-amino nitrogen synthesis in leaves of C4 plants (19,23 MATERIALS AND METHODS Plant Material. The plants used were Zea mays L., Sorghum bicolor (L.) Moench., Digitaria sanguinalis (L.) Scop., Panicum miliaceum L., and Panicum maximum Jacq. The plants were grown in a growth chamber under a daily regime of 16 hr light and 8 hr dark with a day temperature of 30 C and a night temperature of 20 C. Light was provided by a combination of incandescent and fluorescent lamps giving a quantum flux density of 50 to 70 nanoeinsteins/cm2* sec between wavelengths of 400 and 700 nm. The plants were watered daily with nutrient solution described earlier by Beevers et al. (4). Mature leaves from plants 10 to 20 days old were excised after an initial 1 to 2 hr of light exposure following the 8-hr dark...

Effects of Supplemental Nitrate Application on the Activity of Some Nitrogen Assimilation Enzymes and Leaf Tissue Productivity in Maize Seedlings

“…It was confirmed by light micros- copy observations that the mesophyll protoplast fraction is free of contamination by bundle sheath cells after enzymatic digestion and subsequent purification. The chlorophyll afb ratios and the activity of PEP carboxylase, as a marker enzyme of mesophyll cells (13), in both fractions were comparable with the values reported previously in mesophyll protoplasts and bundle sheath strands of maize leaves (15,16). These results altogether indicate no substantial cross-contamination between the two fractions in these preparations.…”

“…Maize leaf glutamine synthetase has been known to be present in both the mesophyll and bundle sheath cells (15,16). We thus attempted to investigate the effect of ambient nitrate on the levels of its components.…”

Functional Differentiation of Bundle Sheath and Mesophyll Maize Chloroplasts Determined by Comparative Proteomics

“…Several groups analyzed the localization of nitrogen assimilation in C4 plants and designated M cells as the compartment where primary N assimilation takes place Edwards, 1975, 1976;Harel et al, 1977;Becker et al, 1993). However, conflicting conclusions were presented for the localization of GS and Fd-GOGAT involved in secondary N assimilation (Rathnam and Edwards, 1976;Harel et al, 1977;Becker et al, 1993Becker et al, , 2000. This might well have been the result of the lengthy preparations of M and BC cells using enzymatic digestion at 37C.…”

Pathway for Nitrate Assimilation in Corn (Zea mays L.) Leaves

“…It was shown that malate could be coupled to nitrate reduction by corn leaves by using higher concentrations (27). Investigations with C-4 plants have shown that nitrate assimilation occurs primarily in the mesophyll cells (I 1, 24,29); however, these reports did not consider the possibility that the C4 photosynthetic system could provide malate as an energy source reduction. In corn, malate is a primary product of CO2 fixation in mesophyll chloroplasts, while hexose and starch production is confined primarily to the bundle sheath cells (7,12).…”