Coumarin enhances nitrate uptake in maize roots through modulation of plasma membrane H+ -ATPase activity.

Coumarin is one of the simplest plant secondary metabolites, widely distributed in the plant kingdom, affecting root form and function, including anatomy, morphology and nutrient uptake. Although, some plant responses to coumarin have been described, comprehensive knowledge of the physiological and molecular mechanisms is lacking. Maize seedlings exposed to different coumarin concentrations, alone or in combination with 200 µm nitrate (NO3- ), were analysed, through a physiological and molecular approach, to elucidate action of coumarin on net NO3- uptake rate (NNUR). In detail, the time course of NNUR, plasma membrane (PM) H+ -ATPase activity, proton pumping and related gene expression (ZmNPF6.3, ZmNRT2.1, ZmNAR2.1, ZmHA3 and ZmHA4) were evaluated. Coumarin alone did not affect nitrate uptake, PM H+ -ATPase activity or transcript levels of ZmNRT2.1 and ZmHA3. In contrast, coumarin alone increased ZmNPF6.3, ZmNAR2.1 and ZmHA4 expression in response to abiotic stress. When coumarin and NO3- were concurrently added to the nutrient solution, a significant increase in the NNUR, PM H+ -ATPase activity, together with ZmNAR2.1:ZmNRT2.1 and ZmHA4 expression was observed, suggesting that coumarin affected the inducible component of the high affinity transport system (iHATS), and this effect appeared to be mediated by nitrate. Moreover, results with vanadate, an inhibitor of the PM H+ -ATPase, suggested that this enzyme could be the main target of coumarin. Surprisingly, coumarin did not affect PM H+ -ATPase activity by direct contact with plasma membrane vesicles isolated from maize roots, indicating its possible elicitor role in gene transcription.

Development of Fe-deficiency responses in cucumber (Cucumis sativus L.) roots: involvement of plasma membrane H(+)-ATPase activity.

One of the mechanisms through which some strategy I plants respond to Fe-deficiency is an enhanced acidification of the rhizosphere due to proton extrusion. It was previously demonstrated that under Fe-deficiency, a strong increase in the H(+)-ATPase activity of plasma membrane (PM) vesicles isolated from cucumber roots occurred. This result was confirmed in the present work and supported by measurement of ATP-dependent proton pumping in inside-out plasma membrane vesicles. There was also an attempt to clarify the regulatory mechanism(s) which lead to the activation of the H(+)-ATPase under Fe-deficiency conditions. Plasma membrane proteins from Fe-deficient roots submitted to immunoblotting using polyclonal antibodies showed an increased level in the 100 kDa polypeptide. When the plasma membrane proteins were treated with trypsin a 90 kDa band appeared. This effect was accompanied by an increase in the enzyme activity, both in the Fe-deficient and in the Fe-sufficient extracts. These results suggest that the increase in the plasma membrane H(+)-ATPase activity seen under Fe-deficiency is due, at least in part, to an increased steady-state level of the 100 kDa polypeptide.

Plasma Membrane H+-ATPase in Maize Roots Induced for NO3- Uptake.

Plasma membrane H+-ATPase was studied in maize (Zea mays L.) roots induced for NO3- uptake. Membrane vesicles were isolated by means of Suc density gradient from roots exposed for 24 h either to 1.5 mM NO3- or 1.5 mM SO4-. The two populations of vesicles had similar composition as shown by diagnostic inhibitors of membrane-associated ATPases. However, both ATP-dependent intravesicular H+ accumulation and ATP hydrolysis were considerably enhanced (60-100%) in vesicles isolated from NO3--induced roots. Km for Mg:ATP and pH dependency were not influenced by NO3- treatment of the roots. ATP hydrolysis in plasma membrane vesicles for both control and NO3--induced roots was not affected by 10 to 150 mM NO3- or Cl-. On the other hand, kinetics of NO3-- or Cl--stimulated ATP-dependent intravesicular H+ accumulation were modified in plasma membrane vesicles isolated from NO3-- induced roots. Immunoassays carried out with polyclonal antibodies against plasma membrane H+-ATPase revealed an increased steady-state level of the enzyme in plasma membrane vesicles isolated from NO3--induced roots. Results are consistent with the idea of an involvement of plasma membrane H+-ATPase in the overall response of roots to NO3-.

Evidence for two different nitrate-reducing activities at the plasma membrane in roots of Zea mays L.

Plasma-membrane (PM) vesicles isolated from 6-d-old corn roots by sucrose gradient centrifugation or two-phase partitioning showed an NADH-dependent nitrate reductase (NR) activity averaging at 40 nmol per milligram PM protein per hour. This membrane-associated NR activity could not be removed from two-phase partitioned PM vesicles by salt washing, osmotic shock treatment, sonication, or freeze-thawing to reverse vesicle sidedness. Therefore, it could not be attributed to contamination of membrane vesicles by the soluble, cytosolic NR. Plasma-membrane vesicles reduced NO~ in the presence of the electron donors NADH or NADPH at an activity ratio of 2.2. The NADH- and NADPH-dependent NR activities of outside-out oriented PM vesicles differed in their sensitivity toward the detergent Brij 58,leading to a latency of 65% or 29% using NADH or NADPH as electron donor, respectively. The activities of NO 3 reduction in the presence of saturating concentrations of NADH and NADPH were additive. Furthermore,both activities were characterized by a different pH dependence with a pH optimum of 7.5 for the NADH-dependent activity and of 6.8 for the NADPH-dependent activity. The membrane-associated NAD(P)H-dependent NR activities responded to different nitrogen nutrition of plants in a manner different from the soluble forms of the enzyme. The data confirm the existence of a corn PM NR and suggest that there may be two different NO3-reducing enzymes located at the PM of corn roots.