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Differential Regulation of Nitrate Reductase Induction in Roots and Shoots of Cotton Plants1

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Differential Regulation of Nitrate Reductase Induction in Roots and Shoots of Cotton Plants1 Plant Physiol. (1975) 55, 178-182 Differential Regulation of Nitrate Reductase Induction in Roots and Shoots of Cotton Plants1 Received for publication June 14, 1974 and in revised form September 19, 1974 JOHN W. RADIN Western1 Cottotn Researcch Laboratory, Agriclultlural Research Service, Uniited States Department of Agriciultulre, Phoeniix, Ar-izo,ia 85040 ABSTRACT by amino acids in higher plants, the conclusions are again contradictory. Filner (7) and Heimer and Filner (8, 9), work- The induction of nitrate reductase activity in root tips of ing with tobacco cell cultures, carefully separated and analyzed cotton (Gossypium hirsutum L.) was regulated by several the regulation by amino acids of both NR activity and nitrate amino acids and by ammonium. Glycine, glutamine, and as- uptake. They found a complex pattern of control of both paragine strongly inhibited induction of activity by nitrate and activities, including repression and derepression of enzyme also decreased growth of sterile-cultured roots on a nitrate synthesis. Stewart (26), working with Lemnna minzor, similarly medium. Methionine, serine, and alanine weakly inhibited in- found amino acid-induced decreases in NR activity that were duction, and 11 other amino acids had little or no effect. Am- independent of nitrate uptake. Sims et al. (24), also with L. moniuni also decreased induction in root tips, but was most m?iinor, showed limited evidence for a slight inhibition of NR, effective only at pH 7 or higher. The optimum conditions for presumably allosteric, by certain amino acids. In contrast, ammonium regulation of induction were identical to those for Ingle et al. (11). and Schrader and Hageman (23) found no growth of sterile-cultured roots on ammonium as the sole amino acid effects on NR activity in radish cotyledons and nitrogen source. Aspartate and glutamate strongly stimulated corn leaves, respectively. The present paper reports evidence induction, but several lines of evidence indicated that the for the regulation of NR by ammonium and amino acids in mechanism of this response was different from that elicited by roots, but not in shoots, of cotton plants. the other amino acids. The effects of amino acids on induction appeared to be independent of nitrate uptake. MATERIALS AND METHODS In green shoot tissues, all attempts to (lenionstrate regula- tion of induction by amino acids failed. The great difference in Chemicals. L-Amino acids and chloramphenicol were pur- observed responses of root and shoot to amino acids suggests chased from Calbiochem. that their nitrate reductase activities are regulated differently. Induction of NR Activity in Roots. Procedures were similar Differential regulation of this enzyvme is consistent with the to those described earlier (19). Seeds of cotton (Gossypiumn responses of root and shoot nitrate reductase activity to nitrate. Ihirsuttumii L. cv. 'Deltapine 16') were germinated for 2 to 3 days at 30 C in vermiculite moistened with tap water. Root tips 15 to 20 mm long were excised and transferred to an induction medium on moist filter paper in Petri dishes. The medium contained 100 mm KNOI, 20 mm glucose. and 10 1tg/ml chloramphenicol to retard bacterial growth. In addition, it contained the compounds to be tested and was usually The enzyme nitrate reductase occupies a control point in buffered at pH 5.4 with 10 mm phosphate, although the effects the pathway of nitrate assimilation (1). Activity of the enzyme of amino acids were little changed by omission of the buffer. fluctuates widely in response to many environmental or physio- Normally, the root tips were induced at room temperature logical factors, such as the presence of reduced N in the (24 C) for 4 to 5 hr, at which time they contained maximum growth medium. The various ways by which enzyme activity activity (19). can be affected include de novo synthesis and turnover (12, Sterile Culture of Roots. Seeds were germinated on filter 30), allosteric modifications (24, 28), and activation and paper in Petri dishes using standard aseptic techniques. After inactivation (16, 27). In addition, cofactor availability can 2 days at 30 C, 10-mm root tips were excised and transferred limit in vivo rates of nitrate reduction (5, 13, 18). to 50-ml culture flasks containing 10 ml of sterile liquid Many reports have appeared on the effects of ammonium medium. The medium contained, in mmoles/l: CaCl2, 1.03; salts on nitrate reduction. Observations range from a promo- MgSO4, 3.16; KCl, 1.66; KH2PO4, 0.15; MnSO4, 0.03; H3B,3, tion of activity, both with and without nitrate present (11, 0.024; ZnSO4. 0.0094; KI, 0.0045; Fe ethylenediaminedi-(o- 23), to inhibition, either by repression or by inactivation (16, hydroxyphenyl) acetate, 0.0027; Na2MoO4, 0.0010; and in 25. 26). In studies of the possible regulation of NR2 activity mg/l: nicotinic acid, 0.5; thiamin-HCl, 0.1; pyridoxine HCl, 0.1, and sucrose, 20,000. In addition, the medium contained additions of 3 mm KNO3, 2 mM KHCO3, 5 mM amino acids, 1 Contribution of the Agricultural Research Service, United and (NH4)2SO as necessary. The pH was adjusted to the States Department of Agriculture, in cooperation with the Arizona Agricultural Experiment Station. Journal Paper 2290 of the Arizona Agricultural Experiment Station. I Mention of a proprietory product does not constitute endorse- 2 Abbreviation: NR: nitrate reductase. ment by the United States Department of Agriculture. 178 Plant Physiol. Vol. 55, 1975 CONTROL OF NITRATE REDUCTASE ACTIVITY 179 proper value with 0.1 M HCl or 0.1 M KOH before auto- Table I. Effects of Aminzo Acids anid Ammonium at 2 mM onz the claving. Each flask contained four or five roots, and was Iniduictiont of NR Activity in Several Experimentts incubated at room temperature on a reciprocal shaker. Root tips were incubated for 4 hr in 100 mm KNO3 plus 20 mm Incubation of Leaf Discs. Discs were cut from mature glucose, pH 5.4, in darkness. Leaf discs were incubated for 4 hr leaves of greenhouse-grown plants as previously described in 100 mm KNO3 in the light. All amino acids were not included (18). The discs were incubated 4 hr at room temperature in in every experiment, but each amino acid was tested at least three Petri dishes, either in darkness or under fluorescent lamps times in each tissue. Values shown are means of the several trials, (6400 lux). The induction medium contained 100 mM KNO3, expressed as percentage of controls. Control activities varied from 10 mm phosphate buffer, pH 5.4, and the compounds to be 1.23 to 7.06 nmoles root-' hr-' in roots and from 7.21 to 13.26 tested. After incubation, discs remained turgid and apparently ,umoles (g fresh weight)-' hr-' in leaf discs. healthy. Induction of NR Activity in Nitrate-free Plants. Seeds were NR Activity germinated in a greenhouse for 5 to 7 days in vermiculite Treatment moistened with tap water. The green cotyledons were excised Root tips Leaf discs just below the node and placed in aerated induction solutions described for incubation of leaf discs. Induction was carried % of control out in darkness at room temperature. After 4 hr, cotyledons Gln 42 94 were removed from the flasks, blotted, weighed, and assayed. Gly 43 99 Assay for NR Activity. Procedures for the in vivo assay were Asn 50 115 described previously (18, 19). In roots, generally only the Met 61 108 apical 2-mm sections were assayed. All root-tip assays were Ser 66 112 run in duplicate or triplicate. Ala 75 103 Nitrate Accumulation in Roots. Net accumulation of nitrate Leu 85 101 in 20-mm root tips was determined after incubation in the Ammonium 89 98 standard induction medium either with or without 1 mM Thr 91 104 Na2WO4 present. Duplicate samples of 20 roots were washed Trp j 93 96 with water, weighed, and placed in test tubes containing 10 Lys 94 95 ml of water. tubes were His 94 100 The capped loosely and heated in a Val 94 100 boiling water bath for 30 min, then cooled to room tempera- Ile 96 105 ture for nitrate analysis. This method, similar to that of Ferrari et Arg 100 103 al. (6), gave better results than homogenization and centrifu- Pro 101 104 gation. Tyr 104 102 Nitrate was determined with a nitrate ion electrode (Orion Phe 107 115 Research Inc.) and with a Leeds and Northrup model 7413 Glu 167 99 pH/ specific ion meter. Duplicate samples agreed well. Asp 179 114 RESULTS Effects of Amino Acids on NR Induction. Table I sum- of NR induction in root tips and in green tissue was investi- marizes the results of many experiments carried out with gated further, with emphasis on the inhibitory amino acids excised root tips or leaf discs. Although the NR activity of glycine, glutamine, and asparagine; the stimulatory amino acids the controls varied among trials, the effects of the amino glutamate and aspartate; and ammonium. acids, expressed as percentages, were reproducible. In root Induction in root tips seemed to be more sensitive to glycine tips, several amino acids affected induction of NR. At 2 mM, than to other amino acids, showing some response to a con- glutamine was the most effective inhibitor of induction, fol- centration of 0.1 mm (Fig. 1). Glutamine and asparagine were lowed in order by glycine, asparagine, methionine, serine, and less effective at low concentration, with threshold levels of alanine. Eleven amino acids, classified here as neutral, showed 0.3 and 1 mm, respectively (Fig. 1). At these concentrations, mild inhibition or promotion of activity (limits set arbitrarily which can be considered substrate levels, the possibility can- at 15% deviation from the controls).
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