Regulation of nitrate uptake by amino acids in maize cell suspension culture and intact roots

The effect of amino acids on nitrate transport was studied in Zea mays cell suspension cultures and in Zea mays excised roots. The inclusion of aspartic acid, arginine, glutamine and glycine (15mM total amino acids) in a complete cell-culture media containing 1.0 mM NO₃ ^- strongly inhibited nitrate uptake and the induction of accelerated uptake rates. The nitrate uptake rate increased sharply once solution amino acid levels fell below detection limits. Glutamine alone inhibited induction in the cell suspension culture. Maize seedlings germinated and grown for 7 days in a 15 mM mixture of amino acids also had lower nitrate uptake rates than seedlings grown in 0.5 mM Ca(NO₃)₂ or 1 mM CaCl₂. As amino acids are the end product of nitrate assimilation, the results suggest an end-product feed-back mechanism for the regulation of nitrate uptake.     

Inorganic carbon fixation and metabolism in maize roots as affected by nitrate and ammonium nutrition

The effects of NO^?₃ and NH⁺₄ nutrition on the rates of dark incorporation of inorganic carbon by roots of hydroponically grown Zea mays L. cv. 712 and on the metabolic products of this incorporation, were determined in plants supplied with NaH¹⁴CO₃ in the nutrient solution. The shoots and roots of the plants supplied with NaH¹⁴CO₃ in the root medium for 30 min were extracted with 80%; (v/v) ethanol and fractionated into soluble and insoluble fractions. The soluble fraction was further separated into the neutral, organic acid, amino acid and non-polar fractions. The amino acid fraction was then analyzed to determine quantities and the ¹⁴C content of its individual components. The rates of dark incorporation of inorganic carbon calculated from H¹⁴CO^?₃ fixation and attributable to the activity of phosphoenolpyuvate carboxylase (EC 4.1.1.31), were 5-fold higher in ammonium-fed plants than in nitrate-fed plants after a 30-min pulse of ¹⁴C. This activity forms a small, but significant component of the carbon budget of the root. The proportion of ¹⁴C located in the shoots was also significantly higher in ammonium-fed plants than in nitrate-fed plants, indicating more rapid translocation of the products of dark fixation to the shoots in plants receiving NH⁺/sp₄ nutrition. Ammonium-fed plants favoured incorporation of ¹⁴C into amino acids, while nitrate-fed plants allocated relatively more ¹⁴C into organic acids. The amino acid composition was also dependent on the type of nitrogen supplied, and asparagine was found to accumulate in ammonium-fed plants. The ¹⁴C labelling of the amino acids was consistent with the diversion of ¹⁴C-oxaloacetate derived from carboxlyation of phosphoenolpyruvate into the formation of both asparatate and glutamate. The results support the conclusion that inorganic carbon fixation in the roots of maize plants provides an important anaplerotic source of carbon for NH⁺₄ assimilation.     

Nitrate reductase activity and uptake of nitrate and oxygen as affected by nitrate supply to detached maize organs

Supply of 1, 2, 5, 10 or 20 mM nitrate to detached roots, scutella or shoots from 5- to 6-d-old Zea mays L. seedlings increased in vitro nitrate reductase (NR) activity in all the organs and NADPH specific NR (NADPH:NR) activity in roots and scutella but not in the shoots. Usually 2 to 5 mM nitrate supported maximum enzyme activity, the higher concentration did not increase it further. The protein content in the roots, scutella and shoots increased up to 5, 2 and 20 mM medium nitrate, respectively. Nitrate uptake also increased with increasing nitrate concentration in roots and shoots, but it increased only slightly in the scutella. In both roots and scutella, methionine sulfoximine had no effect, while cycloheximide and tungstate abolished nitrate induced NADH:NR activity completely and NADPH:NR partially. Methionine sulfoximine increased nitrate uptake by roots and scutella slightly, but other inhibitors had no effect. The depletion of dissolved oxygen from the medium was lower in the presence of nitrate than in its absence or in the presence of ammonium, especially in the scutella. This revised version was published online in July 2006 with corrections to the Cover Date.     

Pattern of sulfate uptake during root elongation in maize: its correlation with productivity

The efficiency of sulfate uptake was evaluated in excised roots of 22 maize genotypes, 12 inbreds and 10 hybrids, in order to study the relationship between the kinetic characteristics of the uptake and the grain productivity. During root elongation, the uptake capacity showed a pulse which appeared when the root reached 1/3 to 1/2 of its final length. The size of the accumulated pool of sulfate was significantly correlated with the productivity. The kinetic parameters of the uptake, Vmax and Km, followed the same trend, showing pulses, whoxe maximum had the same position for Vmax and Km in each genotype. The variability with the genotype of the size and duration of the Vmax pulse was not strictly connected with that of Km. The main correlation between Vmax and Km patterns was the following; inbreds were generally characterized by low Vmax and low Km; hybrids by high Vmax and high Km. As a consequence, in most cases, the benefit of the heterotic stimulation of Vmax was contrasted by the loss of affinity of the transport system or the nutrients.     

Stress induction of abscisic acid in maize roots

Moderate water stresses in the range 0 to −0.6 MPa applied with PEG 6000 to excised roots of Zea mays L. var. LG 11 induced increases of up to four-fold in the amount of abscisic acid (ABA) determined in the tissue after a 12 h period of xylem exudation. The ABA concentration of xylem exudate collected after a 2 h water stress also increased by up to four-fold. Salt stresses, induced with NaCl solutions, resulted in similar increases in the ABA concentrations. ABA concentrations in both root tissue and xylem exudate were highest 4 h after removal of the stress and then declined over a subsequent 8 h period. These results are interpreted in support of the concept that root-produced ABA may have a role in the fine control of the plant's water balance.     

The effect of a day at low irradiance of a maize crop

During the growth of maize ( Zea mays L.cv. INRA F₇× F₂) under constant climatic conditions, the effects of reductions in irradiance simulating a cloudy day were studied. Hourly and daily measurements made in an assimilation chamber (C₂ 3A) showed important and lasting effects in root activity. After reduction of photosynthesis, it took approximately 2 hours to start a lowering of the uptake of NO₃⁻ and NH₄⁺, three hours for K⁺ and four hours for phosphate. Root respiration started to fall after 3 hours. The level to which these activities were reduced also varied. Phosphate uptake was reduced by a mean of 27%, nitrate uptake by 47%, and K⁺ uptake by 55% while the root respiration was reduced by 55%. After return to the initial irradiance, root activities took 3 days to recover their initial rates. Shoot respiration was re-established after one day, while the effects on photosynthesis and transpiration were immediate. The delay of the effect of a change of photosynthesis on the activities of the root, indicates the existence of considerable metabolic reserves. Over longer periods, root metabolism depends on photosynthetic assimilates, but in the short term it is much more dependent on the level of metabolic reserves than on the direct flow of photosynthetic translocates.     

Interaction between electron transport at the plasma membrane and nitrate uptake by maize (Zea mays L.) roots

Summary In the present study nitrate uptake by maize (Zea mays L.) roots was investigated in the presence or absence of ferricyanide (hexacyanoferrate III) or dicumarol. Nitrate uptake caused an alkalization of the medium. Nitrate uptake of intact maize seedlings was inhibited by ferricyanide while the effect of dicumarol was not very pronounced. Nitrite was not detected in the incubation medium, neither with dicumarol-treated nor with control plants after application of 100 M nitrate to the incubation solution. In a second set of experiments interactions between nitrate and ferricyanide were investigated in vivo and in vitro. Nitrate (1 or 3 mM) did neither influence ferricyanide reductase activity of intact maize roots nor NADH-ferricyanide oxidoreductase activity of isolated plasma membranes. Nitrate reductase activity of plasma-membrane-enriched fractions was slightly stimulated by 25 M dicumarol but was not altered by 100 M dicumarol, while NADH-ferricyanide oxidoreductase activity was inhibited in the presence of dicumarol. These data suggest that plasma-membrane-bound standard-ferricyanide reductase and nitrate reductase activities of maize roots may be different. A possible regulation of nitrate uptake by plasmalemma redox activity, as proposed by other groups, is discussed.Abbreviations ADH alcohol dehydrogenase - HCF III hexacyanoferrate III (ferricyanide) - ME NADP-dependent malic enzyme - NR nitrate reductase - PM plasma membrane - PM NR nitrate reductase copurifying with plasma membranes     

Inhibition of nitrate uptake by aluminium in maize

Experiments with two maize (Zea mays L.) hybrids were conducted to determine (a) if the inhibition of nitrate uptake by aluminium involved a restriction in the induction (synthesis/assemblage) of nitrate transporters, and (b) if the magnitude of the inhibition was affected by the concurrent presence of ambient ammonium. At pH 4.5, the rate of nitrate uptake from 240 μM NH₄NO₃ was maximally inhibited by 100 μM aluminium, but there was little measurable effect on the rate of ammonium uptake. Presence of ambient aluminium did not eliminate the characteristic induction pattern of nitrate uptake upon first exposure of nitrogen-depleted seedlings to that ion. Removal of ambient aluminium after six hours of induction resulted in recovery within 30 minutes to rates of nitrate uptake that were similar to those of plants induced in absence of aluminium. Addition of aluminium to plants that had been induced in absence of aluminium rapidly restricted the rate of nitrate uptake to the level of plants that had been induced in the presence of aluminium. The data are interpreted as indicating that aluminium inhibited the activity of nitrate transporters to a greater extent than the induction of those transporters. When aluminium was added at initiation of induction, the effect of ambient ammonium on development of the inhibition by aluminium differed between the two hybrids. The responses indicate a complex interaction between the aluminium and ammonium components of high acidity soils in their influence on nitrate uptake. ei]{gnA C}{fnBorstlap}     

Accumulation of potassium by differentiating metaxylem elements of maize roots

The previous demonstration that the large late metaxylem vessels of field-grown maize ( Zea mays L. cv. Rosella) roots do not lose their crosswalls until they are 20–30 cm from the tip, and that the presence of a soil sheath outside the root was indicative of immature vessels within, greatly strengthened the hypothesis that ion accumulation into these roots was by uptake into living xylem element vacuoles. Proposals that salt movement into the xylem was by leakage or secretion into dead vessels became much less plausible. Potassium concentration in the vacuoles of late metaxylem elements was measured by X-ray microanalysis in unetched fracture faces of bulk, frozen-hydrated pieces of sheathed roots, and found to be in the range 150–400 m M . Potassium concentration in open vessels of bare roots, measured both with the microprobe and by spectrophotometry of aspirated sap, was in the range of 5 to 25 m M . It is concluded that uptake of potassium (and possibly other ions) is into living xylem elements, and that its release to the transpiration stream occurs by the breakdown of their crosswalls and the addition of their vacuoles to the solution in the vessels above.     

Anomalous kinetics of sucrose uptake in maize scutellum slices1

Abstract The kinetics of sucrose uptake into maize scutellum slices showed that the uptake mechanism had a saturable component with a Km of l.5mol m^?3 sucrose. Nevertheless, uptake rate was constant (zero order) over extended periods of time until the bathing solution was nearly depleted of sucrose. It is concluded that these anomalous uptake kinetics reflect sucrose influx across the plasmalemma because of the following results: (a) Efflux of sucrose into buffer was negligible compared with uptake rate, (b) When slices were incubated in fructose, sucrose was synthesized and there was a net release of sucrose to the bathing solution until a steady-state was reached when influx and efflux were equal in magnitude. After the steady-state was reached, efflux of sucrose from the slices was nearly the same in magnitude as the estimated rate of uptake that would have occurred from bathing solutions initially containing the steady-state sucrose concentration, (c) Exchange of sucrose between bathing solution and slices was negligible compared with uptake rate, (d) Pretreatment of slices with uranyl nitrate abolished sucrose uptake, but uptake rate was re-established in these slices after treatment with HCl (pH 2). Uptake rate was set by the initial sucrose concentration of the bathing solution, and was not influenced by the level of endogenous sucrose or by the rate at which the sucrose concentration of the bathing solution declined. Abrupt increases in sucrose concentration during the uptake period increased the rate of uptake only if the concentration was increased above that at the start of the uptake period. Following abrupt decreases in sucrose concentration, there was a lag of about 30 min before uptake rate decreased greatly. If slices were washed and replaced in a fresh sucrose solution during the uptake period, a new uptake rate was set to correspond to the new initial sucrose concentration. It is suggested that the sucrose carrier has a transport site with a relatively low Km (much below 1.5mol m^?3) and that the measured Km (1.5mol m^?3) is that of a site that binds sucrose and thereby controls the rate of uptake. The low Km suggested for the transport site would explain the zero order kinetics but a model of the uptake mechanism that includes the control site cannot, as yet, be constructed from the data.     

Role of sugars in nitrate utilization by roots of dwarf bean

Nitrate uptake and in vivo, nitrate reductase activity (NRA) in roots of Phaseolus vulgaris, L. cv. Witte Krombek were measured in nitrogen-depleted plants of varying sugar status, Variation in sugar status was achieved at the start of nitrate nutrition by excision, ringing, darkness or administration of sugars to the root medium. The shape of the apparent induction pattern of nitrate uptake was not influenced by the sugar status of the absorbing tissue. When measured after 6 h of nitrate nutrition (0.1 mol m^?3), steady state nitrate uptake and root NRA were in the order intact>dark>ringed>excised. Exogenous sucrose restored NRA in excised roots to the level of intact plants. The nitrate uptake rate of excised roots, however, was not fully restored by sucrose (0.03–300 mol m^?3). When plants were decapitated after an 18 h NO₃^? pretreatment, the net uptake rate declined gradually to become negative after three hours. This decline was slowed down by exogenous fructose, whilst glucose rapidly (sometimes within 5 min) stimulated NG^?₃ uptake. Presumably due to a difference in NO₃^? due to a difference in NO₃^? uptake, the NRA of excised roots was also higher in the presence of glucose than in the presence of fructose after 6 h of nitrate nutrition. The sugar-stimulation of, oxygen consumption as well as the release of ¹⁴CO₂ from freshly absorbed (U-¹⁴C) sugar was the same for glucose and fructose. Therefore, we propose a glucose-specific effect on NO₃^? uptake that is due to the presence of glucose rather than to its utilization in root respiration. A differential glucose-fructose effect on nitrate reductase activity independent of the effect on NO₃^? uptake was not indicated. A constant level of NRA occurred in roots of NO₃^? induced plants. Removal of nutrient nitrate from these plants caused an exponential NRA decay with an approximate half-life of 12 h in intact plants and 5.5 h in excised roots. The latter value was also found in roots that were excised in the presence of nitrate, indicating that the sugar status primarily determines the apparent rate of nitrate reductase decay in excised roots.     

A chlorate-hypersensitive, high nitrate/chlorate uptake mutant of Arabidopsis thaliana

N-ethylmaleimide (NEM) Lit 10-100 μ M led to a strong inhibition of the auxin-induced elongation growth of colcoptile segments, while fusicoccin-enhanced growth was not affected. Growth inhibition occurred only if NEM and auxin were allowed to act simultaneously. Preincubation of plant segments with NEM in the absence of auxin caused no inhibition of a subsequent growth stimulation by auxin, whenever NEM was removed before the application of IAA. However, preincubation with NEM plus auxin led to a remaining growth inhibition, which could not be reversed by a second auxin incubation in the absence of NEM. Fusicoccin added to NEM- plus auxin-treated segments was able to restore growth. It is suggested that auxin causes the unmasking of essential SH-groups of a protein to which NEM links covalently. thus inhibiting the growth process. This assumption was further supported by labeling experiments wish [¹⁴C]-NEM using membranes of maize ( Zea mays L. cv. Inraplus) coleoptiles. Two membrane fractions (S₂= 480-1900 g; S₄= 4300-15000 g) revealed a significantly higher [¹⁴C]-NEM labeling in the presence of auxin (2,4-diehlorophe-noxyacctic acid compared to 2,6 dichlorophenoxyacetic acid). This effect disappeared when the membranes were previously washed with EGTA [ethyleneglycolbis-(β-aminoethylether)-N,N,Nr',N'-tetraacetic acid]. The auxin-induced sensitization of coleoptilc segments against thiol-reagents and the auxin-induced expression of SH-groups of proteins of isolated membranes from coleoptiles arc suggested to be events involved in the primary action of auxins.