Root Respiration Associated with Ammonium and Nitrate Absorption and Assimilation by Barley ' Arnold

We examined nitrate assimilation and root gas fluxes in a wildtype barley (Hordeum vulgare L. cv Steptoe), a mutant (narla) deficient in NADH nitrate reductase, and a mutant (narla;nar7w) deficient in both NADH and NAD(P)H nitrate reductases. Estimates of in vivo nitrate assimilation from excised roots and whole plants indicated that the narla mutation influences assimilation only in the shoot and that exposure to N03induced shoot nitrate reduction more slowly than root nitrate reduction in all three genotypes. When plants that had been deprived of nitrogen for several days were exposed to ammonium, root carbon dioxide evolution and oxygen consumption increased markedly, but respiratory quotient-the ratio of carbon dioxide evolved to oxygen consumed did not change. A shift from ammonium to nitrate nutrition stimulated root carbon dioxide evolution slightly and inhibited oxygen consumption in the wild type and narla mutant, but had negligible effects on root gas fluxes in the narla;nar7w mutant. These results indicate that, under NH4' nutrition, 14% of root carbon catabolism is coupled to NH4' absorption and assimilation and that, under N03nutrition, 5% of root carbon catabolism is coupled to N03absorption, 15% to N03assimilation, and 3% to NH4+ assimilation. The additional energy requirements of N03assimilation appear to diminish root mitochondrial electron transport. Thus, the energy requirements of NH4' and N03absorption and assimilation constitute a significant portion of root respiration. Nitrogen assimilation is among the most energy-intensive processes in plants, requiring the transfer of two electrons per N03 converted to NO2, six electrons per N02 converted to NH4', and two electrons and one ATP per NH4' converted to glutamate. To provide sufficient electrons for these reactions, plants may divert reductant from mitochondrial electron transport. During dark N03assimilation, shoots of a higher plant (8) and algae (18, 29) evolved CO2 significantly faster than they consumed O2, presumably because the TCA cycle or the oxidative pentose phosphate pathway catabolized substrates and transferred some electrons to N03and N02rather than to O2. These results indicate that, in the dark, shoots expend up to 25% of their respiratory energy on nitrogen assimilation (8). Plants assimilate in the root virtually all of the NH4' and ' Supported in part by U.S. Department of Agriculture CSRS grant 88-37264-3857 and National Science Foundation grants BSR8416893, BSR-8806585, and DCB-8916637 to A.J.B. from 5 to 95% of the N03absorbed from the rhizosphere (1, 20). Estimates of root nitrogen acquisition and the associated energy transfers have been limited (2, 4, 5, 10, 12, 1416, 23, 26, 27), and these could not distinguish among expenditures for tissue maintenance, root growth, NH4' and NO3absorption, and NH4' and N03assimilation. Root respiration is usually determined from net 02 uptake, yet N03-, N02-, and NH4' can substitute for 02 as electron acceptors during nitrogen assimilation. Root carbon catabolism might be a more pertinent measure, but analysis of dissolved CO2 has required discontinuous sampling (24, 29) or elevated CO2 concentrations (17). The present study employed an instrumentation system that simultaneously monitors root fluxes of C02, 02, NH4', N03-, and H+ at normal physiological concentrations (6). With this system, we evaluated the relationships among NH4' and NO3absorption, assimilation, and root respiration in barley genotypes having normal or deficient levels of root NO3reductase activity. MATERIALS AND METHODS Barley Genotypes and Growth Conditions We examined three genotypes of the barley cultivar, Hordeum vulgare L. cv Steptoe: a wild type, a narla mutant (line Az12) with defects in the structural gene for NADH-specific NR,2 and a narla;nar7w mutant (line Azl2;Az7O) with defects in the structural genes for both NADH-specific and NAD(P)H-bispecific NR (28). The in vitro activities of the NR isozymes are well characterized in these genotypes: for the wild type, only NADH-specific NR is active in the shoots, whereas both NR isozymes are active in the roots; activity of NAD(P)H-bispecific NR is higher in the root and shoot of the narla mutant than in the wild type, whereas activity of NADH-specific NR is very low in this mutant; the narla;nar7w mutant has negligible activities of either isozyme (28). Seeds of the genotypes were sprouted on wet germination paper. After 3 to 4 d, the seedlings were suspended by foam plugs around the stem above light-tight root boxes. These boxes held 4 L of a one-tenth strength modified Hoagland nutrient solution containing only 100 $M NH4+ for the N03assimilation experiments and both 100 Mm NH4' and 700 Mm NO3for the RQ experiments. This solution was replaced daily except for plants that were nitrogen-deprived, which 2 Abbreviations: NR, nitrate reductase; RQ, respiratory quotient.