Robert L. Travis

Comparative Induction of Nitrate and Nitrite Uptake and Reduction Systems by Ambient Nitrate and Nitrite in Intact Roots of Barley (Hordeum vulgare L.) Seedlings

The induction by ambient NO3- and NO2- of the NO3- and NO2- uptake and reduction systems in roots of 8-d-old intact barley (Hordeum vulgare L.) seedlings was studied. Seedlings were induced with concentrations of NaNO3 or NaNO2 ranging from 0.25 to 1000 [mu]M. Uptake was determined by measuring the depletion of either NO3- or NO2- from uptake solutions. Enzyme activities were assayed in vitro using cell-free extracts. Uptake and reduction systems for both NO3- and NO2- were induced by either ion. The Km values for NO3- and NO2- uptake induced by NO2- were similar to those for uptake induced by NO3-. Induction of both the uptake and reduction systems was detected well before any NO3- or NO2- was found in the roots. At lower substrate concentrations of both NO3- and NO2- (5–)10 [mu]M), the durations of the lag periods preceding induction were similar. Induction of uptake, as a function of concentration, proceeded linearly and similarly for both ions up to about 10 [mu]M. Then, while induction by NO3- continued to increase more slowly, induction by NO2- sharply decreased between 10 and 1000 [mu]M, apparently due to NO2- toxicity. In contrast, induction of NO3- reductase (NR) and NO2- reductase (NiR) by NO2- did not decrease above 10 [mu]M but rather continued to increase up to a substrate concentration of 1000 [mu]M. NO3- was a more effective inducer of NR than was NO2-; however, both ions equally induced NiR. Cycloheximide inhibited the induction of both uptake systems as well as NR and NiR activities whether induced by NO3- or NO2-. The results indicate that in situ NO3- and NO2- induce both uptake and reduction systems, and the accumulation of the substrates per se is not obligatory.

Differential effect of amino acids on nitrate uptake and reduction systems in barley roots.

This study was conducted to determine whether the inhibition of nitrate reductase activity (NRA; EC 1.6.6.1) in barley (Hordeum vulgare L. var. CM-72) roots by the amino acids (glutamic, aspartic, glutamine and asparagine) is a direct effect or indirect due to inhibition of the NO(3)(-) uptake system. Roots of 8-day-old intact seedlings were supplied with the amino acids (I mM) individually either with NO(3)(-) (0.1 or 10 mM) or roots were pretreated with the amino acids and then supplied with NO(3)(-) only. Nitrate uptake was determined by following NO(3)(-) depletion from the uptake solution containing 0.1 mM NO(3)(-). All the amino acids inhibited the increase in NO(3)(-) uptake similarly (50-60%) when the roots were supplied with 0.1 mM NO(3)(-). Pretreatment with glutamic and aspartic acids was more inhibitory (70-80%) than with glutamine and asparagine (30%). The amino acids partially inhibited (35%) the induction of NRA in roots supplied with 0.1 mM NO(3)(-); however, no inhibition occurred at 10 mM NO(3)(-). Likewise, pretreatment with glutamic or aspartic acid inhibited the induction of NRA at 0.1 mM NO(3)(-) but not at 10 mM NO(3)(-). In contrast, pretreatment with glutamine or asparagine had no effect on the subsequent induction of NRA, even at 0.1 mM NO(3)(-). The results suggest that, at low NO(3)(-) supply, the inhibition of induction of NRA by the amino acids is a result of the lack of substrate availability due to inhibition of the NO(3)(-) uptake system.

Evidence for Substrate Induction of a Nitrate Efflux System in Barley Roots.

Induction of an NO3- efflux system in intact barley (Hordeum vulgare L.) roots was demonstrated. Since the measurement of NO3- efflux is dependent on its accumulation, experiments were devised to facilitate accumulation under noninducing conditions. This was accomplished by incubating seedlings in 10 mM NO3- in the presence of RNA and protein synthesis inhibitors. Under these conditions NO3- uptake is mediated by constitutive high- and low-affinity transport systems. Control roots were incubated with 1.0 mM NO3-. This resulted in the accumulation of similar levels of NO3- in both treated and control roots; however, cytoplasmic NO3- efflux from inhibitor-treated roots was much lower than from control roots. Following a brief lag period, efflux rates increased rapidly in the presence of NO3- for 8 to 12 h. The NO3- efflux system was also induced by ambient NO2-. After induction the efflux system was relatively stable in the presence of RNA and protein synthesis inhibitors as long as NO3- or NO2- was present. These results suggest that NO3- efflux may be an inducible system requiring both RNA and protein synthesis, as does induction of the uptake system. The efflux system, however, has a much slower turnover rate than the uptake system.

Stimulation of Nitrate and Nitrite Efflux by Ammonium in Barley (Hordeum vulgare L.) Seedlings

The inhibitory effect of NH4+ on net NO3- uptake has been attributed to an enhancement of efflux and, recently, to an inhibition of influx. To study this controversy, we devised treatments to distinguish the effects of NH4+ on these two processes. Roots of intact barley (Hordeum vulgare L.) seedlings, uninduced or induced with NO3- or NO2-, were used. Net uptake and efflux, respectively, were determined by following the depletion and accumulation in the external solutions. In roots of both uninduced and NO2- -induced seedlings, NO3- efflux was negligible; hence, the initial uptake rates were equivalent to influx. Under these conditions, NH4+ had little effect on NO3- uptake (influx) rates by either the low- or high-Km uptake systems. In contrast, in plants preloaded with NO3-, NH4+ and its analog CH3NH3+ decreased net uptake, presumably by enhancing NO3- efflux. The stimulatory effect of NH4+ on NO3- efflux was a function of external NH4+ and internal NO3- concentration. These results were corroborated by the absence of any effect of NH4+ on NO2- uptake unless the roots were preloaded with NO2-. In this case NH4+ increased efflux and decreased net uptake. Hence, the main effect of NH4+ on net NO3- and NO2- uptake appears to be due to enhancement of efflux and not to inhibition of influx.

Effect of ammonium on the regulation of nitrate and nitrite transport systems in roots of intact barley (Hordeum vulgare L.) seedlings

The effect of NH4+on the regulation of NO3−and NO2−transport systems in roots of intact barley (Hordeum vulgareL.) seedlings grown in NO3−or NO2−was studied. Ammonium partially inhibited “induction” of both transport systems. The inhibition was less severe in NO2−-fed than in NO3−-fed seedlings, presumably due to lower uptake of NH4+in the presence of NO2−. In seedlings pretreated with NH4+subsequent “induction” was inhibited only when NH4+was also present during “induction”, even though pretreated roots accumulated high levels of NH4+. This indicates that inhibition may be regulated by NH4+concentration in the cytoplasm rather than its total accumulation in roots. L-Methionine sulfoximine did not relieve the inhibition by NH4+, suggesting that inhibition is caused by NH4+itself rather than by its assimilation product(s). Ammonium inhibited subsequent expression of NO3−transport activity similarly in roots grown in 0.1, 1.0, or 10 mM NO3−for 24 h (steady-state phase) or 4 d (decline phase), indicating that it has a direct, rather than general feedback effect. “Induction” of the NO3−transport system was about twice as sensitive to NH4+as compared to the NO2−transport system. This may relate to higher turnover rates of membraneassociated NO3−-transport proteins.

THE EFFECT OF ULTRAVIOLET RADIATION ON WHEAT ROOT VESICLES ENRICHED IN PLASMA MEMBRANE

Abstract— The irradiation of plant cells with UV radiation (254nm) causes various solutes to leak from the cells. Vesicles enriched in plasma membranes were prepared from wheat roots. These were used to determine whether UV radiation alters membrane function by direct action on the membranes and to distinguish between the chemical effects produced by high and low fluences of UV. The plasma membrane‐associated K+‐stimulated ATPase was very sensitive to UV radiation (100% inhibition with 1.35kJ/m2). ATPase activity measured in the absence of K+ and K+‐stimulated ATPase activity measured in the presence of diethylstilbestrol were much less sensitive. Lipid breakdown, as measured by malondialdehyde production, occurred only at UV fluences greater than 1.8 kJ/m2.

Effect of pH and Calcium on Short-Term NO3- Fluxes in Roots of Barley Seedlings

The effect of pH and Ca2+ on net NO3- uptake, influx, and efflux by intact roots of barley (Hordeum vulgare L.) seedlings was studied. Seedlings were induced with NO3- or NO2-. Net NO3- uptake and efflux, respectively, were determined by following its depletion from, and accumulation in, the external solution. Since roots of both uninduced and NO2--induced seedlings contain little internal NO3-, initial net uptake rates are equivalent to influx (M. Aslam, R.L. Travis, R.C. Huffaker [1994] Plant Physiol 106: 1293–1301). NO3- uptake (influx) by these roots was little affected at acidic pH. In contrast, in NO3--induced roots, which accumulate NO3-, net uptake rates decreased in response to acidic pH. Under these conditions, NO3- efflux was stimulated and was a function of root NO3- concentration. Conversely, at basic pH, NO3- uptake by NO3-- and NO2--induced and uninduced roots decreased, apparently because of the inhibition of influx. Calcium had little effect on NO3- uptake (influx) by NO2-- induced roots at either pH 3 or 6. However, in NO3--induced roots, lack of Ca2+ at pH 3 significantly decreased net NO3- uptake and stimulated efflux. The results indicate that at acidic pH the decrease in net NO3- uptake is due to the stimulation of efflux, whereas at basic pH, it is due to the inhibition of influx.

Enhancement of nitrate reductase activity and metabolic nitrate concentration by methionine sulfoximine in barley roots

Abstract The enhancement of nitrate reductase activity (NRA) and metabolic NO 3 − concentration by methionine sulfoximine (MSO) in roots of 8-day-old intact barley ( Hordeum vulgare L.) seedlings was studied. NRA was induced with 0.05–100 mM NO 3 − with or without 0.25 mM MSO and assayed in vitro using NADH as the electron donor. Metabolic NO 3 − concentration was estimated by the anaerobic in vivo NRA assay method. In the absence of MSO, induction of NRA reached a plateau at 0.1 mM external NO 3 − , even though root NO 3 − accumulation continued at higher external NO 3 − concentrations. MSO enhanced the induction of NRA in roots exposed to NO 3 − concentrations greater than 0.1 mM, although total NO 3 − accumulation in MSO-treated roots decreased. In the absence of MSO, induction of NRA reached a plateau after 6 h exposure to 100 mM NO 3 − , whereas in the presence of MSO the enzyme activity increased gradually up to 24 h. Metabolic NO 3 − concentrations were similar in roots induced with 0.1 and 100 mM NO 3 − . MSO had no effect on the metabolic NO 3 − concentration when roots were induced with 0.1 mM NO 3 − , but it increased the metabolic NO 3 − concentration in roots induced with 100 mM NO 3 − by 2-fold between 6 and 24 h. The results indicate that in the absence of MSO the metabolic NO 3 − pool is saturated at 0.1 mM exogenous NO 3 − and that enhancement of NRA by MSO at higher NO 3 − levels may be due to increased accumulation of NO 3 − in that pool.

Comparisons of plasma membrane polypeptides from soybean and alfalfa

Abstract Polypeptides were solubilized with sodium dodecyl sulfate from plasma membrane vesicles of eight varieties of soybean roots [ Glycine max (L.) Merr.] and of cultured alfalfa cells ( Medicago sativa L.). The solubilized polypeptides were analysed by 2D-polyacrylamide gel electrophoresis. Apparent isoelectric point and MW values were obtained for 80 soybean plasma membrane polypeptides and 44 alfalfa plasma membrane polypeptides. From these data composite distribution patterns were constructed, which are representative of the soybean or alfalfa 2D-gels, respectively. The results showed that the general polypeptide staining patterns were similar for all the soybean varieties, but some minor differences were evident. The alfalfa electrophoretograms differed markedly from the soybean electrophoretograms in specific details, though some general pattern similarities were noted. The data are discussed in terms of a physiological role for the integral plasma membrane polypeptides and in terms of the potential for distinguishing among soybean varieties and between species at the plasma membrane polypeptide level.

Response of Acala Cotton to Nitrogen Rates in the San Joaquin Valley of California

The responses of Acala cotton (Gossypium hirsutum L.) in California to a range of applied nitrogen (N) treatments were investigated in a 5-year, multisite experiment. The experiment’s goals were to identify crop growth and yield responses to applied N and provide information to better assess the utility of soil residual N estimates in improving fertilizer management. Baseline fertilizer application rates for the lowest applied N treatments were based on residual soil nitrate-N (NO3-N) levels determined on soil samples from the upper 0.6 m of the soil collected prior to spring N fertilization and within 1 week postplanting each year. Results have shown positive cotton lint yield responses to increases in applied N across the 56 to 224 kg N/ha range in only 41% (16 out of 39) of test sites. Soil NO3-N monitoring to a depth of 2.4 m in the spring (after planting) and fall (postharvest) indicate most changes in soil NO3 occur within the upper 1.2 m of soil. However, some sites (those most prone to leaching losses of soluble nutrients) also exhibited net increases in soil NO3-N in the 1.2- to 2.4-m depth zone when comparing planting time vs. postharvest data. The lack of yield responses and soil NO3-N accumulations at some sites indicate that more efforts should be put into identifying the amount of plant N requirements that can be met from residual soil N, rather than solely from fertilizer N applications.