Inhibition by Phenylglyoxal

Nitrate transport in excised corn (Zea mays L.) roots was inhibited by phenylglyoxal, but not by 4,4'-diisothiocyano-2,2'-stilbene disulfonic acid (DIDS) or fluorescein isothiocyanate (FITC). Inhibition of nitrate uptake by a 1-hour treatment with 1 millimolar phenylglyoxal was reversed after 3 hours, which was similar to the time needed for induction of nitrate uptake. If induction of nitrate uptake occurs by de novo synthesis of a nitrate carrier, then the resumption of nitrate uptake in the inhibitortreated roots may occur because of turnover of phenylglyoxal-inactivated nitrate carrier proteins. All three chemicals inhibited chloride uptake to varying degrees, with FITC being the strongest inhibitor. While inhibition due to DIDS was reversible within 30 minutes, both FITC and phenylglyoxal showed continued inhibition of chloride uptake for up to 3 hours after removal from the uptake solution. Assuming that the anion transporter polypeptide(s) carries a positive charge density at or near the transport site, the results indicate that the nitrate carrier does not carry any lysyl residues that are accessible to DIDS or FITC, whereas the chloride carrier does. Both chloride and nitrate carriers, however, seem to possess arginyl residues that are accessible to phenylglyoxal. With the increased cost of farm inputs such as nitrogen fertilizers, it is important to breed crop cultivars that absorb and use soil nitrogen more efficiently. The absorption of nitrate by plants is mainly determined by the root surface area and/or the capacity of nitrate uptake by root cells which, in turn, depends in part upon the number and/or efficiency of the nitrate carriers per unit area of the plasma membrane (1). Although the involvement of carriers in the transport of ions across the plant plasma membrane has long been recognized, it has been difficult to biochemically identify and characterize such carriers. A biochemical identification of a nitrate carrier, for example, is essential for effective manipulation of nitrate uptake by plants at the molecular level. This requires the development of certain probes that can be used to label and subsequently identify the polypeptide(s) involved in nitrate transport. Group-specific chemical reagents have been successfully used to identify the functionally essential amino acid residues in a number of enzymes (15, 27). Because of the high pKa (12.5) of their side chains, arginyl residues are positively charged at physiological pH. Based on this observation, it was suggested that enzymes which have anionic substrates will most likely have arI Supported in part by a Chancellors Patent Fund Award to K. S. D., and funds from the California Agricultural Experiment Station and the University of California, Riverside Botanic Gardens to J. G. W. 2 Present address: Department of Biological Sciences, Stanford University, Stanford, CA 94305. ginyl residue(s) at or near their active sites (27), and this hypothesis has received considerable support (4, 24, 26, 31). The other amino acid residues that can provide a positive charge at physiological pH are lysine (pKa 10.5) and histidine (pKa 6.0). Phenylglyoxal, a diketone that binds the guanidinium group of arginine (30), strongly inhibited chloride transport in mammalian red blood cells (3). Anion transport in red blood cells was also inhibited by the disulfonic acid stilbene and eosin derivatives, both of which bind the essential lysyl residue of the band 3 protein, which composes the anion transporter (17, 18, 22, 25). Likewise, DIDS,3 a disulfonic acid stilbene derivative, inhibited chloride uptake in corn roots and Chara (16, 19, 20). 14C-Phenylglyoxal, tritiated DIDS, and FITC were successfully used to specifically label and identify the anion transporter in red blood cells (3, 9, 29). Inhibition of chloride uptake by DIDS in corn roots and Chara was reversible, however, such that radiolabeled DIDS could not be used to label the chloride transporter protein of plant cells (16, 19). It is highly probable that the nitrate carrier of the plant cell plasma membrane also carries arginyl and/or lysyl residue(s) at or near the transport site. To our knowledge, no inhibitors, reversible or irreversible, for nitrate transport have so far been reported in plants. The availability of an irreversible inhibitor of nitrate transport could facilitate the identification of the plasma membrane-associated polypeptide(s) involved in nitrate transport. This study reports on the inhibition of nitrate transport by phenylglyoxal as it relates to the induction of nitrate uptake in excised corn roots. It further reports on the inhibition of chloride uptake by DIDS, FITC, and phenylglyoxal and demonstrates the involvement of different mechanisms in nitrate and chloride transport in corn roots. MATERIALS AND METHODS Plant Materials. Seeds of corn (Zea mays L.) hybrid WF 9 x MO 17 were grown in glass baking trays on a single layer of blotter paper saturated with 0.1 mM CaCl2 solution. The trays were covered with plastic wrap, which was perforated to allow gas exchange, and kept at 28°C in a growth chamber in the dark. Primary roots from 3.5-d-old seedlings were excised, cut into segments about 4 cm long, and placed in aerated, ice-cold water. Nitrate Uptake Assay. For studying the induction of nitrate uptake, 3 g of the fresh root segments were weighed into 50 ml capacity glass test tubes kept on ice. Uptake was started by adding 30 ml of uptake solution (0.5 mm KNO3, 0.25 mm Ca[NO3]2, 2 mm Mes, pH 6.0). Assay tubes were kept in a water bath maintained at 30°C and were aerated throughout the uptake period. Uptake was studied by monitoring the depletion of nitrate from the solution. Aliquots of 0.2 ml were taken at hourly intervals and nitrate was determined colorimetrically by the method 3Abbreviations: DIDS, 4,4'-diisothiocyano-2,2'-stilbene disulfonic acid; FITC, fluorescein isothiocyanate. 759 www.plantphysiol.org on December 30, 2017 Published by Downloaded from Copyright © 1988 American Society of Plant Biologists. All rights reserved. Plant Physiol. Vol. 86, 1988 of Cataldo et al. (7). Data are shown for representative experiments consisting of duplicate nitrate assays for duplicate root samples. Nitrate Pretreatment. It was determined that nitrate uptake in excised root segments from corn seedlings grown as described above, reached its maximum rate after about 3 h. Therefore, the root segments were routinely pretreated for 3 h in a solution containing 5 mm nitrate (2.5 mm KNO3 and 1.25 mm Ca[NO312) in 5 mm Mes (pH 6.0) unless specified otherwise. Inhibition of Nitrate Uptake. Inhibitors were added to the uptake assays to a final concentration ranging from 0.25 to 1.00 mM. The root segments were incubated with the inhibitor for 1 h before measurement of nitrate absorption. In one experiment (Fig. 4), the nitrate-pretreated roots were incubated in 1 mM inhibitor solution in 2 mm Mes (pH 6.0) for 1 h, and then uptake was studied either in the presence or absence of the inhibitor over a period of 6 h. Inhibition of Chloride Uptake. F6r measurement of chloride uptake, root segments were pretreated for 3 h in an aerated solution consisting of 0.1 mm CaSO4 in 2 mm Mes (pH 6.0). The pretreated root segments were then incubated in a solution containing 0.5 or 1.0 mm inhibitor, 0.1 mm CaSO4, 2 mm Mes (pH 6.0) for 1 h, at the end of which time they were transferred to uptake solution containing 0.5 or 1 mm inhibitor. The uptake solution consisted of 1 mm KCl, 0.1 mm CaSO4, 2 mm Mes (pH 6.0), and 36C1at a specific activity of 25 to 30 ,uCi per mmol chloride. After incubation for 10 min, the root segments were washed twice with ice-cold 0.1 mm CaSO4 solution for 5 min each and weighed into scintillation vials for determination of radioactivity in a liquid scintillation counter. Data are shown for representative experiments consisting of duplicate root samples. For the time-course experiment (Fig. 6), the pretreated root segments were incubated in a solution containing 1 mm inhibitor, 2mM Mes, 0.1 mm CaSO4 (pH 6.0) for 1 h, at the end of which time they were washed twice with water for 5 min each. The inhibitor-treated roots were kept in the aerated solution of the same composition as above minus the inhibitor. Chloride uptake was measured as a function of time as described above. RESULTS AND DISCUSSION Inhibition of Nitrate Uptake. Both DIDS and FITC carry amino reactive isothiocyanate group(s) that can react with the epsilon amino group of lysyl residues and the N-terminal a-amino group of polypeptide chains (12, 25). DIDS or FITS had little inhibitory effect on nitrate uptake in corn roots (Figs. 1 and 2). The slight inhibition by DIDS may simply result from general anionic competition at the transport site because DIDS has two well exposed negative charges on the sulfonate groups. In comparison to DIDS, FITC is relatively less negatively charged at physiological pH (32). It has one relatively hidden, negatively charged carboxyl group and a well exposed, but partially deprotonated hydroxyl group (pK = 6.5). A slight apparent stimulation in nitrate uptake, shown by 1 mm FITC, probably resulted from the simultaneous depletion of both FITC and nitrate from the uptake solution over the period of uptake. The absorption spectra of FITC and nitrate, in the solution used for the colorimetric nitrate assay, were found to slightly overlap each other (data not shown). This would result in inflated estimates of apparent nitrate loss from the uptake solution because at each time interval, both loss of nitrate and FITC were being assayed. " DIDS has long been known as a potent inhibitor of anion transport in mammalian red blood cells (6). The following observations demonstrated the specificity of DIDS for the anion transporter (band 3 protein) in red blood cells. One mole of DIDS per mole of the anion transporter completely inhibited sulfate flux into red blood cells (29). In the same study, almost 90% of the total radiolabeled DIDS bound to the red blood cells o 3.0 00.5mM DIDS