Nitrate Absorption and Assimilation in Ryegrass as
Influenced by Calcium and Magnesium'Received for publication March 9, 1972
M. A. MORGAN,2 W. A. JACKSON, AND R. J. VOLKDepartment of Soil Science, North Carolina State University, Raleigh, North Carolina 27607
ABSTRACT
The absorption and assimilation patterns of 35NO0- suppliedas the Ca2+ and Mg2+ salts to intact ryegrass (Lolium perenne)seedlings were compared. No statistically significant effect ofambient cation on the amounts of 35NO3- absorbed was observedin the initial six hours, but during the subsequent six hours,absorption from Ca ('5NO:) 2 exceeded that from Mg ('lNO3)2Lower rates of '5NO,- assimilation were found in roots ex-
posed to Mg('-NO3)2 than in those exposed to Ca('5NO3)2. Itwas proposed that Mg2+ initiated a restriction in 15NO0- reduc-tion in roots, probably as a consequence of a Mg2+-inducedphysiological Ca2" deficiency. Lower '5N translocation rates werealso observed from Mg('`NO3)2. These effects of Mg2+ in de-pressing 5NO0- assimilation and translocation occurred priorto an effect on '5NO0- uptake.
In shoots, larger amounts of reduced '5N products occurredwith Ca(CN03)2 than with Mg('5NO3)2. It was concluded thatthis was due to enhanced translocation of 5NO3- (and possiblyits reduced products) in presence of Ca22 rather than to spe-cific cation effects on 15N03- assimilation in the shoots.
Beneficial effects of ambient Ca2+, relative to Mg-+, on NO:-absorption by nitrogen-depleted wheat seedlings have been re-ported (18), but whether this effect invariably occurs is notknown. Similarly, the suggestion (17) that ambient Ca2- en-hances NO3- translocation also requires further documentation.
Relatively little direct information is available regarding theextent to which absorbed NO3- is reduced in root tissue of in-tact plants. Nitrate reductase activities generally are lower inroots than in leaves (2) although sizable activities have beenobserved in the former (14-16, 27). Moreover, the presence ofamides, amino acids, and ureides in the bleeding xylem sap ofdecapitated plants exposed to NO:- indicates a significant ca-pacity for NO3- reduction in the intact root tissue of somespecies (3. 22, 23, 29, 30). Whether Ca" and Mg2- are directlyinvolved in NO:- assimilation by roots has not been examined,although the accumulation of NO2- in shoots (7, 24), and lackof carbohydrates in roots of Ca"+-deficient plants (I1, 20) sug-gest such an involvement for Ca22.The paucity of information on NO:- absorption, reduction,
' Paper No. 3685 of the Journal Series of the North CarolinaState University Agricultural Experiment Station, Raleigh, NorthCarolina. These investigations were supported by the United StatesAtomic Energy Agency, Grant AT(40-1)-2410.
2 Present address: Department of Soil Science, Faculty of Agri-culture, University College, Glasnevin, Dublin 9, Ireland.
and assimilation by roots, and the possibility of Ca22 modifyingthese processes, prompted the present investigation. We rea-soned that Ca'', or Mg2, or both, might affect NO3- reductionby roots by: (a) influencing the rate of NO.- absorption, therebyinfluencing both the amount of NO3- available for reductionand the amount of nitrate reductase (a substrate-inducible en-zyme); (b) directly altering the in vivo activities of NO3- reduc-tion and assimilatory enzymes; and (c) modifying the transloca-tion of NO:- to the shoots, thereby indirectly altering theamount of NO3- available for reduction in the roots. We reporthere results of two experiments with perennial ryegrass (Loliumperenne). The first was conducted to determine the generalpattern of "'N uptake from Ca(QNO3)2 and its relative distribu-tion between roots and shoots. The more detailed second ex-periment compared the effects of ambient Ca2+ and Mg-2 on the'N absorption, assimilation, and translocation patterns ofplants supplied with highly enriched '"NO.;-. The data indicatethat absence of Ca2- in the ambient medium resulted in altera-tions in the products of '3NO;- assimilation prior to the timethat net "'NO:- uptake was affected significantly.
MATERIALS AND METHODS
Experiment I, 30-Day-Old Plants. Seeds of perennial rye-grass (L. perenne) were surface-sterilized with 5% H202 andgerminated in opaque polyethylene cups with bottoms ofstainless steel screen. Developing roots thus grew through thescreen into aerated nutrient solution. Each cup (one culture)contained 25 seedlings after thinning. Forty cultures wereplaced in 13 liters of aerated nutrient solution and the plantswere grown in a chamber maintained at 24 to 25 C during thelight period (16 hr, 75.6 hectolux at leaf surface) and at 18 to19 C during the dark period. The nutrient solution contained50 uM K .SO4, 50 jM MgSO4. 0.1 mm Ca(H2PO4)2, 0.25 mMNaNO:,, 1 mg iron per liter as FeEDTA, and one-fifth the traceelements supplied by Hoagland's solution (8). Solutions werechanged every other day. At 28 days after sowing, NaNO3 wasdeleted from the solution and 2 days later the experiment wasinitiated.
Immediately prior to application of the treatment solutionroots were rinsed by dipping 10 times in each of two 15-litertanks of distilled water and extraneous water allowed to drainoff. Roots were then exposed to an aerated solution containing0.25 mm Ca(QNO2.)2 enriched to 46.7 atom % "2N. Light in-tensity was 54.0 hectolux and temperature 28 ± I C. Exceptfor the initial 30 min when the acidity rose to pH 5.9, solutionacidity was maintained at pH 6.5 + 0.3 by periodic addition ofdilute H..SO, or NaOH. Duplicate cultures were harvested at0, 0.5, 1, 2, 4, 8, 12. and 16 hr after exposure to '`N; a singleculture was harvested after 24 hr. After rinsing the roots inredistilled water and blotting dry, roots and shoots wereweighed separately, freeze-dried, reweighed, and ground in aWiley mill to pass a 40-mesh screen. The tissue was then
485 www.plantphysiol.orgon February 9, 2020 - Published by Downloaded from
FIG. 1. Deposition of 15N in roots, shoots, and intact seedlings(open symbols) and percentage of total absorbed '5N03- recoveredin the shoots (closed symbols) of 30-day-old seedlings exposed toCaC5NO3)2.
analyzed for total nitrogen (21) using Devarda's alloy to reduceNO3- and for `5N enrichment by mass spectrometric methods(26).
Experiment II, 21-Day-Old Plants. Cultures contained 45 to50 seedlings per cup after thinning. Plants were grown in achamber at 22 + 1 C during the light period (16 hr, 238 hecto-lux) and at 18 1 C during the dark period. Four cultureswere placed in 1-liter plastic beakers containing 820 ml ofaerated nutrient solution, containing 0.1 mm KH2PO4, 0.25 mMCa(N03)2, and 0.25 mm MgSO4. Iron and other trace elementswere supplied as in experiment I. These solutions were replacedevery other day, and 2 days prior to the experiment theCa(NO3)2 concentration was doubled.Two experimental treatments were applied 1 hr after the
beginning of the light period on the 21st day after sowing.Following thorough rinsing and draining of the roots as
previously described, plants were placed in the aerated singlesalt solutions of CaC5NO3)2 or Mg(5NO3)2, both applied at0.75 mm and 97.5 atom % '5N enrichment. During the 12-hrabsorption period, illumination was maintained at 238 hectoluxand air temperature at 24 + 1 C. Initial solution acidity was
pH 5.0, but it rose rapidly to 6.8. Four replicates of two cul-tures each were harvested after 0-, 3-, 6-, and 12-hr exposureto each treatment solution. Root and shoot samples were thentreated as outlined for experiment I.
Tissue nitrogen was separated into four fractions. Weighedsamples were extracted with cold, 70% ethanol in an Omni-mixer and the insoluble residue was separated by Buchner fil-tration. The ethanol-soluble portion was evaporated to drynessby means of a warm air stream and partitioned into chloro-form-soluble and water-soluble constituents. Following acidifi-cation of the latter to pH 2.7, Dowex 50W-X8 resin in hydro-gen form was used to separate NO:- from reduced nitrogenouscompounds, which were eluted with 1.2 N K2SO4.The NO:- content of the treatment solutions was determined
by ultraviolet absorption at 207 nm (1). Tissue NO- was de-termined by the method of Johnson and Ulrich (12). Totalnitrogen in the other three fractions was determined by Cope's
Plant Physiol. Vol. 50, 1972
procedure (5). The '5N enrichment of all fractions was de-termined by mass spectrometry (26).
All data were calculated on the basis of a unit weight of dryroot tissue and analyzed statistically by the analysis of variancetechnique. Appropriate LSD values are indicated in the figuresby the ordinate scale values of vertical lines.
RESULTS
The patterns of 5N0,- absorption and 15N translocation bythe 30- and 21-day-old plants exposed to Ca(15NO)2 weresurprisingly similar in view of their different ages, differentgrowing conditions, and the different experimental conditionsduring the absorption period. In both instances, a decline in therate of absorption occurred after the first few hours (Figs. 1and 2A) as a consequence of decreasing accumulation of 15N inroot tissue. During this period the deposition of 15N in shootsoccurred at nearly constant rates (~420 and 490 ,ug "5N per gdry roots per hr for the 30- and 21-day-old plants, respectively).With 21-day-old plants, 5NO3- absorption during the first 6hr was unaffected by the accompanying cation (Fig. 2A). By12 hr, however, significantly less (p <s 0.01) 15NO3- had beenabsorbed from Mg('5NO3)2, due primarily to a diminished ac-cumulation of 15N in the roots (Fig. 2B).
Accumulation of '5NO;- in the roots was significantly greater(p < 0.01) during the 3- to 6-hr period when Mge5NO3)2, ratherthan CaC5NO,)2, was applied (Fig. 3). In the shoots, however,the Mg('5N03)2 treatment resulted in less accumulation of`5NO-, particularly between 6 and 12 hr (p < 0.01). In con-trast to 15NO,-, amounts of 15N-labeled water-soluble reducedconstituents (amino acids, amides) in the shoots of both treat-ments exceeded those in the roots (Fig. 4) after the 3rd hr. Inboth roots and shoots, Ca(`5NO3)2 rather than Mg(Q5NO3)2, re-sulted in greater accumulation of '5N in the water-soluble re-duced N fraction (Fig. 4). The difference was evident in theroots by the 3rd hr (p < 0.05) and became highly significant(p < 0.01) by the 6th hr. In spite of sizable variation in the '5Nof this fraction in the shoots, the advantage for the Ca(C5NO3)2treatments was evident (p < 0.05) by the 6th hr. On theother hand, accumulation of "5N in the ethanol-insoluble frac-tion of both roots and shoots was not significantly affectedduring the first 6 hr (Fig. 5). Between 6 and 12 hrs, however, sig-nificantly greater (p < 0.01) `5N accumulation in the ethanol-insoluble fraction occurred in both tissues of the Ca('5NO3)2treatment compared to Mg('5NO3)2. Chloroform-soluble "5Nconstituted a relatively small proportion of the total 15N ab-sorbed (data not presented). No significant influence of theambient cation on this fraction was detectable in the root tissue;in the shoots the advantage for Ca2+ was statistically significant(p < 0.01) only by the 12th hr.
DISCUSSION
Influence of Ca2` versus Mg` on '5NO3- Absorption. It hasbeen suggested that ambient Ca2+ enhances anion uptake byneutralizing negative charges in the boundary region of absorb-ing cells (10, 25), while Mg2, is less effective (9). In the presentstudies, however, no significant difference between the twocations was observed in net '5NO0;- uptake until after 6 hr (Fig.2A). Since 1 hr was sufficient for Mg(NO)2 solutions to displacethe peripheral Ca2+ from roots of ryegrass similar to those usedhere (19). the initial displacement of Ca2+ from the roots didnot alter the net '5NO- absorption rates. The decrease in up-take from the Mg('5N03)2 treatment after 6 hr was associatedwith a smaller amount of '5N reaching the shoots (Fig. 2B).Significant ambient cation effects on the magnitude of the'5NO,- (Fig. 3) and water-soluble reduced '5N (Fig. 4) poolsoccurred prior to the effect on net '-NO,- absorption (Fig. 2A).
FIG. 2. Deposition of 15N in intact seedlings (A) and total 'IN accumulation in roots (B) and shoots (B) of 21-day-old plants exposed to
Ca('5NO3)2 or Mg('5N03)2.
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FIG. 3. Accumulation of "NO3- in roots and shoots of 21-day-old seedlings exposed to Ca(QNO3)2 or Mg("5NO,3)2.
The data therefore indicate an influence of the ambient cationon nitrogen assimilation reactions prior to an influence on net"NO.- uptake.
Influence of Ca'+ versus Mg2+ on "N Translocation. After thefirst 2 or 3 hr, translocation of absorbed "'N to the shoots oc-
0 3 6 9 12
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FIG. 4. Accumulation of water-soluble reduced "N in roots andshoots of 21-day-old seedlings exposed to Ca("NO3)2 or Mg(15N03)2.
curred at nearly constant rates when 30-day-old or 21-day-oldplants were exposed to Ca(C'NO,)2. The constant rate occurredin spite of a decreasing rate of accumulation of "N in the roottissue (Figs. 1 and 2B). Even during the first 3 hr the amount of"N reaching the shoots constituted nearly 30% of the amount
FIG. 5. Accumulation of ethanol-insoluble '5N in roots (A) and shoots (B) of 21-day-old seedlings exposed to Ca('5NO:)2 or Mg(15N03)2.
absorbed. It therefore appears that the rate of 'N translocationwas not closely dependent upon the rate of '1N accumulationin root tissue, and that an efficient translocation system wasoperating in concert with the 'l NO.- accumulation and assimila-tion systems in the root tissue.
Exposure to Mg('5NO3)2, rather than Ca(' NO;)2, decreasedthe translocation of '5N, particularly after 6 hr (Fig. 2B).Calculations of the proportion of total absorbed N which wasrecovered in the shoots (percentage translocation) reveal a sig-nificantly (p . 0.01) higher value (39.7%) for Ca(5'NO)2 thanfor Mg('NO)2 (34.7%) by the 6th hr although the total amountof l'NO:- absorbed (Fig. 2A) was nearly identical for the twotreatments at this time.
Concentrations of NOs- and water-soluble reduced N com-pounds in the xylem translocation stream can vary considerably(22, 23, 29, 30). It is therefore not possible to decide forcertain whether the lower `5N translocation in the Mg('`N`O)treatment was due entirely to lower translocation of '-NO0-, en-tirely to lower translocation of water-soluble reduced 1"N. or acombination of the two effects. The data do indicate, however,that 1'NO- translocation was depressed. A minimal value for"NO:- translocated during a specific time is given by the in-crease in 'NOs- in the shoots during that time. For the 6- to12-hr period, the increase in '5NO- was considerably greaterfor the Ca('5NO3)2 treatment (698 ag "N per g dry roots) thanfor the Mg(Q5NOs)2 treatment (392 tig '5NO-N per g dry roots:cf. Fig. 3). The total reduced '-N in the shoots during thisperiod increased by a greater amount with Ca(Q NO;)2 thanwith Mg(l'NO3)2 (2250 versus 1603 gug "3N per g dry roots, re-spectively, Fig. 6) which indicates that l'NO;- reduction in theshoots was not impaired by Ca2+ relative to Mg2+. Hence, theincrease in '5NO- in the shoots indicates an advantage for Ca"relative to Mg2+ in the process of l-NO:. translocation. A bene-
ficial influence of ambient Ca2 on NO.- translocation was sug-gested earlier by Minotti et al. (17).
Influence of Ca2 versus Mg2- on '5NO:- Assimilation inRoots. Reduced N continued to accumulate in the rootsthroughout the 12-hr absorption period with a maximal ratein the 3- to 6-hr period (Fig. 6A). The decline after 6 hr wasassociated with decreased rates of accumulation of 'lN in thewater-soluble reduced fraction (Fig. 4). Accumulation of theethanol-insoluble fraction continued nearly linearly to 1 2 hr(Fig. 5). We assume that translocation of water-soluble reducedN from roots to shoots occurred at a greater rate than the re-
verse process (23). The quantities of all reduced 15N in theroots at each harvest (Fig. 6A) therefore represent only mini-mal values of the actual amounts reduced there. For theCa('`NO3)2 treatment, these minimal rates of reduction in theroot tissue during the 0- to 3-, 3- to 6-, and 6- to 12-hr time pe-riods were 163, 251, and 181 Mug 15N per hr per g dry roots, re-spectively. The corresponding values for the Mg(l'NO)2 treat-ment were 143, 216, and 138 Mlg '5N per hr per g dry roots, whichindicates less efficiency in reduction of the absorbed l-NO:-. Dur-ing the 3- to 6-hr period, when the minimal rate of l NO- reduc-tion clearly became greater with Ca(' NO)2, the accumulationrates of 'NO:s- were 279 and 493 Ag '5N per hr per g dry roots forthe Ca(5NO)2 and Mg('5NO)2 treatments, respectively (Fig. 3).The greater reduction rate with Ca2+ was therefore not due to ahigher substrate concentration. The advantage for Cal in totalamount of reduced '5N present in the roots was highly signifi-cant (p < 0.01) by the 6th hr (Fig. 6A). Similarly. the propor-tion of total ':N in the roots present in reduced forms was alsosignificantly (p < 0.01) greater for Ca('NO:)2 by the 6th hr(Fig. 7). Since the Mg('NOs)2. did not increase the amount ofreduced l N in the shoots (Fig. 6B) or increase the relative pro-portion of N present there in reduced form (Fig. 7), this treat-
HOURS HOURSFIG. 6. Accumulation of reduced 15N in roots (A) and shoots (B) in 21-day-old seedlings exposed to Ca(15N03)2 and Mg('5N03)2.
2
L.S.D.'C
P90.10 PEO.05 PiO.0IROOTSI I
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LL 80 INTACT SEEDUNGS I I
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FIG. 7. Percentage of total absorbed '5N03--N in roots, shoots, and intact seedlings recovered in the reduced N fraction of 21 -day-old seedlingsexposed to Ca('5NO3)2 and Mg(Q5NO3)2.
ment resulted in a restriction in the rate of "'NO;- reduction inthe roots. Furthermore, this restriction apparently occurredduring the 3- to 6-hr period, i.e., prior to a restriction in `NO:-absorption (cf. Fig. 2A).The consistency of Ca` versus Mg'- in promoting higher
amounts of reduced "N in the roots indicates that during ex-posure to Mg(`NO3)2 there was a restriction in the reduction ot"5NO;- to ammonium, or a restriction in the amination reactionsleading to the formation of amino acids, or both. Failure ottransport of the intermediates in these reactions to the ap-propriate reaction centers, as well as decreased concentrationsor activities of the appropriate enzymes, could account for thedepressed rates. The onset of the restriction in "NO,- reductionof the roots exposed to Mg("'NO,)2 must have been fairly rapidbecause statistically significant (p s, 0.05) cation effects wereexerted on the water-soluble reduced "N fraction of the rootsby the 3rd hr (Fig. 4). We propose that the restriction resultedfrom suboptimal cell surface Ca''+ in the Mg(1'NO,), treatment.The roots contained sizeable Mg" concentrations prior to ex-posure to the "NO;- solutions and were not depleted of Mg"+appreciably during exposure to Ca("oNO,)2 (19). Upon exposureto Mg("'NO,)2, however, two distinct Ca2+ fractions in the rootswere clearly delineated, the largest of which was rapidly dis-placed within the 1st hr (19). The second fraction was onlyvery slowly displaced for at least 12 hr. Thus, ryegrass rootsexposed to Mg("NO3)2 for more than 1 hr were probably de-prived of physiologically sufficient amounts of Ca2+. AlthoughCa`5 deficiency did not influence the efficiency of nitrate reduc-tion in wheat roots (4, 17), a physiological deficiency of Ca2+ inour ryegrass roots seems the best explanation for the restrictionin reduction of 15NO,- when Mg("NO,)2, rather than Ca("4NO)2,was applied.
Increased vacuolization (28) is one of the changes (13) inroot cells resulting from low ambient Ca+'. Accordingly, greatercompartmentalization of "NO;- away from reactions centerscould have occurred with Mg("'NO,)2 compared to Ca("5NO,)2,the result being an increased "'NO;- accumulation (Fig. 3) anddecreased "NO3- reduction (Fig. 7). Since membrane turnoverrates are rapid, possibly approaching 5 min (6), there appearsto have been ample opportunity for the cation treatments tohave exerted differential influences on membrane synthesis andreconstruction in the present experiments.
Influence of Ca2+ versus Mg2+ on "NO.- Assimilation inShoots. By the 6th hr, the total quantities of reduced "'N inthe shoots exceeded that in the roots (Fig. 6) and as early as the3rd hr the proportion of total "N in the shoots occurring in re-duced form was substantially greater than in the roots (65 and39%, respectively; Fig. 7). The water-soluble reduced "N frac-tion of the roots tended to become saturated by the 6th hr but"N continued to accumulate at sizable rates in this fraction ofthe shoots (Fig. 4). It is possible that the upward translocationprocess was partially responsible for the continued accumula-tion of "N in the water-soluble reduced "N fraction of theshoots (Fig. 4) while removal from this fraction into ethanol-insoluble components (Fig. 5) was proceeding vigorously in theshoot tissue. Because of uncertainty in the magnitude of theupward translocation of water-soluble reduced "N, the presentdata do not permit an accurate estimate of the relative effi-ciencies of the roots and shoots in "'NO,- reduction and as-similation.
Incorporation of "5NO,- into the reduced components of theshoots was consistently greater with Ca("'NO,), than withMgC'NO,)2 (Figs. 4 and 5). The larger quantities of "5N presentin the shoots with Ca("NO,)2 (Fig. 2B) were due. in part atleast, to the greater rate of "'NO:- translocation discussed previ-ously. Calculations of the proportion of total "5N present which
was recovered in reduced components of the shoots indicates,in contrast to the roots, no statistically significant differencebetween the cation treatments (Fig. 7). hence, the enhanced"N assimilation in the shoots with Ca(" NO,), was probablydue to the greater translocation rates of '"NO,- (and possiblywater-soluble reduced "N) than to any direct cation effect onthe assimilation processes in shoots.
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