Evidence for a Role of Calcium in Nitrate Assimilation in Wheat Seedlings

Abstract

Severely Ca-deficient Triticum aesfivum L. seedlings accumulated high levels of nitrite and moderate levels of nitrate and organic nitrogen, but oontained unaltered levels of hydroxy)lamine. Nitrite accumulation was not related to molybdenum deficiency or altered cellular pH. Nitrate reductase was decreased by Ca defioiency, apparently by repression of enzyme synthesis from accumulated nitrite and not by inhibition of enzyme aotivity. Nitrite reductase and NADP diaphora,se activities were noat affected by Ca de,ficency, and Ca did not restore activity to nitrite reductase inactivated by cyanide. The results indicated that the role of Ca is in intracelilular transport of nitrite and not in induction or aotivity of enzymes. Magnesium, manganese, copper, and iron have been reported as cofactors or constituents of nitrite reductase enzyme (15, 16, 21, 26). Calcium has not been shown to play a specific role in nitrogen metabolism, althotugh an association between calcium and nitrogen contents of plants has long been known (19) and calcium has been implicated in nitrogen assimilation (18) and symbiotic fixation (2). Intermediates of nitrogen assimillation between nitrate and ammonia seldom reach appreciable levels in plants' tissues. Nitrite accumulation has been found in algae cufltured in media of low pH or under other-specific conditions (13). Nitrite accumulation in higher plants is rare (7), but has been demonstrated to occur briefly in tomato plants given molybdenum after undergoing deficiency of the element (24) and in forage crops degraded by bacteria after harvest (5). In a study on effect of planit nuttrition on nitrogen assimillation in wheat (9), it was observed that crude ni,trate reductase enzyme extracts of calcium-deficient plants contained substantiall quantities of nitrite. This report considers nitrite aoctcmulation associalted with cal,cium deficiency in relation to contents of other constituents and activities of enzymes involved in nitrate and nitrite metabolism. Materials and Methods 'Pawnee' wheat seedlings (Triticum aestivum L.) were germinated in vermiculite, and 1 week after being planted, were transplanted to nuttrient solutions in 2-liter styrene containers. Macronu-trients were prepared from reagent quality chem-2.5 mm K,S04, 2 mM MgSO4, and 0, 0.05, 0.5, or 5.0 mM CaCl2. Micronutrients were supplied as recommended by Johnson et al. (11). Every 4 days, iron was added to al'l containers at the rate of 1 ml of 0.6 % FeSO4-0.4 % tartaric acid solution per liter of nultrient solution, and the pH of the nutrient solutions was adjusted to 5.0 with HCI. All treatments were Treplicated 4 times. The plants were grown in environmental chambers at 200 day and 100 night temperatures with a 16-hour light period. Lighting was provided by 16 160-watt fluorescent anid 6 300-watt incandescent lanmps. Relative humidity in ithe charnbers was maintained at about 40 %. Leaf blades were sampled after 3 weeks of growth and assayed for nitrate and nitrite by the method of Wooliley, Hicks, and Hageman (27); hydroxylamine by the method of Yasphe, Halpern, and Grossowicz (28); and ammonia 'by the method of Thompson anld Morrison (25). Interference of nitrite with the hydroxylamine assay was compensated by subtracting the color formed when oxidation of hydroxylamine was prevented with thiosull-fate. The pH of the tissne was determined on a 10-fold diluted homogenate of blade tissue, and calcium and molybdenum contents were determined by atomic absorption spectrophotometry. Nitrate redu1ctase was extracted from leaf blades and assayed by the method of Hageman and Flesher (8). Enzyme blanks of all samples were prepared by omitting NADH from the assay mixtuires. Nitrite reductase was extracted with 50 mM tris (pH 7.5) containing 1 mim cyste.ine-HGl and partially purified by the acetone fractionation procedure of Joy and Hageman (12). Coild (-200) acetone was added-to the extract to 33 % (v/v) concentration and the resulting debris were removed by centrifugation. The stupernatant was diluted to 75 % (v/v) acetone and the precipitate 775 PLANT PHYSIOLOGY' obtained after centri fugation was restuspended anid dialysed 12 hoturs against a 50-fold volume of extraction media. The dithionite a,ssay mixture used for nitrite reductase (12) wa,s modified to contain 20 tmo'les K,HPO4 (,pH 7.5), 0.9 jkmo'le KNOO, 2.0 kmoles benzyl vio,logen, 7.5 Kmoles Na2S2O4, and 0.5 ml enzyme preparation in a total volume of 3 ml. NADP diaphorase activity of the extracts was determined by the method of Avron anid Jagendorf (1) by measuiring the change ill absorban,cy at 650 m/i for 2 minuites. Sodium 2,6-dichiloroubenzenioieindopheinol was used as the electron acceptor. An enzyme preparation from plants growni 3 weeks in complete nutrient soluttion contain.ing 5 mM calcium was extracte,d as described above and dia,lysed 6 hours against extraction media containing 1 mm KCN, and 12 houirs against the same solution withotut KCN. The ability of 0.1 to 5.0 prmoles of CaCI].. to restore activity to nitrite redutctase in the dithionite assay was determined and compared witlh activity of a isimilar enzyme preparation dialysed 18 hours againist extractioni media only. AIll enzyme assays were ruin at 300 and, except for diaphora,se, for 20 minuites. Protein content of-the enzyme preparations was determine,d by the Folin phenol me,thod of Mildler (14). Reagents foir the assays were obtained from S;gma Chemical Company (St. Louis, Missouri) and Mann Research Laboratories (New York, New,r York). Results The appearaince of the plan'ts at sampling is shown in figure 1. Plants grow,n wit,h,o.ut calcium l)roduced little growtlh after transplanting and were generally chlorotic when sampled. Plan,ts grown in 0.05 mm calcium, produced very distorted growth, especialily of the newer leaves formed after transplanting. There was little dlifference between the 2 higher calcium treatments an,d Ino symptoms of calcitum deficiency were noted. Substituting am-monium for nitrate in the nuitrient media did not alter the calcitum deficiency symptoms, as was noted by Skok (23), and dlid no,t cau,se accumullatioon of any compouind giving a reaction for nitrite. Nitrate accumulated in calc:tium-deficient plants, buit mtuch less than nitrite relative to the respective quanitities presen;t in plants grown with stufficient calcium (table I). Lea,f blades of plantb grown under the 2 dower calcitum devels contained suib-stantial quantities of ni,trite compared withl sliglht traces detected under the 2 higher calcitum treatments. No difference in hydroxylami11 content among the calcium treatments wa's noted. Ammonia s,ligh,tly increased in tihe cailcium-deficient plants, probably as a restult o'f secondary rathlel-than l)ri-mary effects of calcitum dleficiency. Nitrite accumulation was not associated with any apparent effect of callciuim deficiency on the pH of the plant tissuie. Low pH has been shown, however, to cautse accumulation of ni'tri'te in green algae b)y enhancinig non-enzymic reduct.ion of nitrate w.bile inhibiting nitrite reductase enzyme (13). Likewi,se, molybdenutm determination,s in.dicated the eilement was not deficient at sampling or presuimably anytime during plant growtth. Similar experiments involving deficiencies of other niutrients, including magnesium, manganese, iron, and copper, have been conducted (9) and no accumulation of nitrite 'has been observed. Absence of nitrite accumul.,ation uinder deficiencies of these nutrien,ts 'likely is not good evidence of their possible ro,le in nitrite reduction. It i's noteworthy, however, that the effect of calcitum deficiency on nitrite accumullation was mulch greater thani the effects of deficiencies of the othe;r nuttrients. Nitrate reductase activity was mulch lower in calcitum-deficient seedlings than in n1ormal seedli!lgs (table II). Suibstantial amouints of endogenoius nitrite were present in crui(le extracts from deficient plants, but probably did not cau1se significant inhi-hition of enzyme activity (10, 22). Efforts to remove the nitrite by dialysis, while successful, caused complete 'loss of nitrate redutctase activity in alil samples. Thus, it was impossible to determine whether results obtained with the crude uindiallysed extracts accurately reflected the situation in vivo. Nitrite reducta-se and NADP diaphorase activities were !similar in deficient and normal plants (table II). The slight reduction of activitiei in deficient plants was not sufficient to account for the marked accumullation of ii:trite, btut instead wvas Table I. Growth, Conttents of Nitrate, Nitrite, Hydroxylamine, Ammnoniia, Calcium and Molybdenum, and pH of Diluted Homiiogeniate of WVheat Seedlings after Three Weeks Assays were condticted as described in Materials and Methods. All values except calcium and molybdenum contenits are on a fresh weight basis.