Uptake Assimilation N03 NH4' Nitrogen- DeficientPerennial ...NH4' uptake amounted to 65 mgN, while NO3- uptake was reducedto 34mgNculture-'. Nitrateuptakeratesweredecreas-ingconsiderablyby8to

Plant Physiol. (1988) 88, 1303-13090032-0889/88/88/1303/07/$01.00/0

Uptake and Assimilation of N03 and NH4' by Nitrogen-Deficient Perennial Ryegrass Turf

Received for publication January 12, 1988 and in revised form July 15, 1988

DANIEL C. BowMAN*1 AND JACK L. PAULDepartment ofEnvironmental Horticulture, University ofCalifornia, Davis, California 95616

ABSTRACT

Assimilation ofNO3- and NHI' by perennial ryegrass (Lolium perenneL.) turf, previously deprived of N for 7 days, was examined. Nitrogenuptake rate was increased up to four- to five-fold for both forms of N byN-deprivation as compared to N-sufficient controls, with the deficiency-enhanced N absorption persisting through a 48 hour uptake period.Nitrate, but not NH4R, accumulated in the roots and to a lesser degree inshoots. By 48 hours, 53% of the absorbed NO3- had been reduced,whereas 97% of the NH4 had been assimilated. During the early stages(O to 8 hours) of N03 uptake by N-deficient turf, reduction occurredprimarily in the roots. Between 8 and 16 hours, however, the site ofreduction shifted to the shoots. Nitrogen form did not affect partitio ingof the absorbed N between roots (40%) and shoots (60%) but did affectgrowth. Compared to N03-, NH.g uptake inhibited root, but not shoot,growth. Total soluble carbohydrates decreased in both roots and shootsduring the uptake period, principally the result of fructan metabolism.Ammonium uptake resulted in greater total depletion of soluble carbo-hydrates in the root compared to N03- uptake. The data indicate that Nassimilation by ryegrass turf utilizes stored sugars but is also dependenton current photosynthate.

Nitrogen deficiency enhances N uptake by turfgrasses (3, 4) aswell as other crop plants (1,20). Periods ofN deprivation, rangingin duration from 1 to 4 weeks (3) to as short as 4 h (4), increasedthe uptake of both N03 -N and NH4+-N by mature perennialryegrass turf. Nitrogen equivalent to a normal 50 kg N ha-'application (approximately 1 lb N/1000 ft2) was absorbed in 2 dor less (3), and it was suggested that such rapid N absorptionmight be the principal cause of the short-lived growth responsetypically observed with turf following application of inorganic Nfertilizers. Implicit in this suggestion is that the absorbed N isalso rapidly assimilated. The literature contains numerous re-ports on NO3- assimilation by N-deficient graminaceous plants(7, 8, 12, 17, 23), which describe a pattern of accumulation,reduction, and translocation. Other studies (21, 22) have revealedthe dominant role of shoots in the assimilation of NO3- androots in assimilation ofNH4'. However, there are no comparabledata for mowed turf systems recovering from N deficiency.

In addition to increasingN uptake, nitrogen deprivation resultsin accumulation ofsoluble carbohydrates in roots, both in peren-nial ryegrass (4, 29) and other species (7, 17, 28). High levels ofcarbohydrates may be involved both in the uptake and assimi-lation ofN (1, 13, 17, 28), although there is controversy regardingthe role of increased root carbohydrates in deficiency-enhanced

' Present address: Department of Plant Science, University ofNevada,Reno, NV 89557.

uptake (4, 9, 18, 19). The importance of carbohydrates in Nassimilation is, by comparison, much clearer. Supplying both anenergy source and carbon skeletons, total soluble sugars decreaserapidly in N-deficient plants upon resupply ofN (13, 17, 28). Itis apparently not known which of the various sugars in rootsystems are specifically involved in assimilation, although glucosepreferentially stimulated both uptake and reduction of NO3- bydwarf bean (13).

This paper presents results of an investigation into the assim-ilation ofboth NO3- and NH4' by N-deficient perennial ryegrassturf. Whereas N assimilation has typically been studied withseedlings or excised plant tissues (12, 16, 23, 28), the presentinvestigation has used a mature, mowed turf system as theexperimental unit. Nitrogen assimilation was followed over timeby tissue analysis and, in the case of NO3;, by exudate analysis.Additionally, changes in individual soluble carbohydrates, in-cluding fructans, were measured.

MATERIALS AND METHODSPlant Culture. A detailed description of the turf culture system

used in these experiments has been presented (3). Briefly, peren-nial ryegrass (Lolium perenne L.) 'Manhattan II' turf was grownin solution culture in round plastic containers, 167 cm2 in area,containing 2.3 L of aerated nutrient solution. The standardnutrient solution used for establishment contained 1.25 mmKNO3, 1.25 mM Ca(NO3)2, 0.5 mM MgSO4, and 0.25 mmKH2PO4 plus micronutrients at full concentration of Hoaglandsolution (14). Iron was supplied at 1 mg Fe L' as Fe-EDDHA.A minus-N solution in which the NO3- salts were replaced byS042- salts was substituted to produce N-deficient turf. The initialpH of both solutions was 6.0. Supplemental Fe as FeSO4 7H20was periodically added at a rate of 0.4 mg Fe L-'. Solutions werechanged and the turf was mowed at 4 cm every 4 to 7 d asdetermined by growth.

Experiments were conducted in a controlled environmentgrowth chamber operated for the 16 h light period (0600-2200h) at 24C and for the 8 h dark period at 18C. RH wasapproximately 80%. The PPFD2 at plant height was 400 Amolm-2 s ', supplied by incandescent and cool-white fluorescentlamps. Turf cultures, previously grown for 9 weeks in the green-house, were acclimated to the growth chamber for 1 week priorto the experimental treatments.

Experiment 1. This experiment examined changes in tissue Nand soluble sugars during the rapid uptake of either NO3- orNH4' by N-deficient turf. The turf cultures were grown on minus-N solution for 7 d and mowed 3 d before initiating uptake. Toovercome the initial period of low NO3- uptake by N-deprivedroots, NO3- uptake was induced starting 6 h prior to the experi-ment with additions of 1 mg N03 -N culture-' every 2 h. At

2Abbreviations: PPFD, photosynthetic photon flux density; NRA,nitrate reductase activity.

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Plant Physiol. Vol. 88, 1988

1200 h, cultures were transferred to 2.3 L of the uptake solutionwhich consisted ofminus-N solution supplemented with 1.5 mMNO from KNO3 or 1.5 mM NH4 from (NH4)2SO4.Net uptake was determined over a 48 h period by measuring

the depletion ofN from solution at 3 to 8 h intervals, correctedfor loss of solution by evapotranspiration and sampling. Knownamounts ofN stock solutions were added to the uptake solutionsas required to maintain the concentration above 1 mM N. ThepH was also adjusted frequently to maintain it at 6.0 ± 0.5.Uptake solutions were replaced at 24 h. Cumulative uptake (mgN culture-') is shown in Figure 1 fit to a cubic spline curve.Uptake rates were estimated from the first derivative ofthe curveevaluated at the sampling time points and are presented as mgN culture-' h-'.

Individual cultures (four per sampling per N form) were har-vested for tissue analysis at 0, 6, 12, 24 and 48 h and separatedinto root and shoot tissue. Fresh samples were quickly dried ina microwave oven to approximately 90% of oven dryness andthen oven dried at 70°C for at least 24 h. The dried tissue wasground to pass through a 40 mesh screen. Plus-N controls wereharvested at 0 and 48 h.Experiment 2. This experiment differed from experiment 1 in

that the assimilation of NO3- only was investigated over theshorter period of 16 h to determine the relative role of shoot androot in NO3- reduction. This was accomplished by measuringthe quantity ofNO3- transported from roots to shoots with time.Xylem flux of NO3- was estimated by periodically collectingexudate from four separate cultures. Fifteen min before eachharvest at 4, 8, 12, and 16 h, shoots of these separate cultureswere excised approximately 0.5 cm above the crown and blotteddry. Exudate which collected at the cut surface in 15 min wasabsorbed with preweighed filter paper disks. The disks werereweighed and extracted with 5 mL of deionized water, and the

z J_cs

-IE 1,a)0

10c

>0-

EC-)

z

Ea)

a)c)0

4-,

FIG. 1. Cumuby perennial ryedeprived ofN forOpen symbols, D~means of four sai

NO3- content of the extract was determined. Transpiration wasmeasured gravimetrically on a separate group ofcultures. Nitrateflux to the shoot was then estimated as the product of transpi-ration and NO3- concentration in the exudate. This method issimilar to that used by Shaner and Boyer (26) and assumes thatthe discrete values for NO3- flux obtained at 4 h intervals arerepresentative of each preceding interval. Supporting this isconsiderable evidence that NO3 translocation is closely associ-ated with current uptake (10, 25). It should be noted that fresh,intact shoots were excised at each sampling, since prolongedexcision may disrupt N uptake and metabolism (5, 23, 25). Inaddition to the changes described above, the photoperiod wasextended through the 16 h duration of this experiment. Solublecarbohydrates were determined for root tissue only.

Tissue and Solution Analyses. Nitrate and NH4I were deter-mined both in the uptake solution and in aqueous tissue extractsby the Carlson method (6). KjeldahlN (reduced N) was measuredby a method modified to exclude NO3- from the analysis (2).

Soluble carbohydrates in the tissue were analyzed by a modi-fication of the method of Steen and Larsson (27). One hundredmg of ground tissue was extracted for 45 min in 5 mL of 0.1 Mammonium acetate (pH 5.0) in a shaking waterbath at 60°C. Theextract was centrifuged and the supernatant used for subsequentassays. Glucose in the supematant was determined directly by acolorimetric assay (Sigma serum glucose procedure No. 115);fructose was measured in a duplicate assay after addition ofphosphoglucose isomerase (D-glucose-6-phosphate ketol-isomer-ase, EC 5.3.1.9, obtained from Sigma), based on the method ofBernt and Bergmeyer (1). Sucrose and fructans in the remainingextract were acid hydrolyzed by mixing equal volumes of theextract and 0.15 N H2SO4 and heating the mixture for 60 min at90°C. This solution was cooled and neutralized with KOH, andglucose and fructose were determined as above. Sucrose contentwas calculated as twice the increase in glucose following acidhydrolysis and corrected for that present initially. The calculationassumes that the contribution of the terminal glucosyl residuefrom the fructans is insignificant. Similarly, the content of fruc-tans in the tissue was calculated from the increase in fructosefollowing acid hydrolysis. In a preliminary experiment, starchwas determined for both N-deficient and N-supplied tissue bythe method of Steen and Larsson (27) and found to be presentat very low levels. Consequently, starch was excluded from theseanalyses. Results are expressed as mg glucose equivalents pergram dry weight and are the means of four replicate samples.

RESULTS),-*̂*.. Experiment 1. N deprivation for 7 d resulted in a 24% increase

D in the root dry weight and a 24% decrease in shoot dry weight_L) compared to +N controls (Table I). Cultures were very uniform

\°0 at the start of this experiment, containing a total of 151 ± 6 and331 ± 12 mg N culture-' in the -N and +N tissues, respectively.40\ \ | Nitrate was very low in the -N tissues, amounting to only 1.4

%\;o .mg N culture-', while there was 74 mg N03--N in the controls.\ Consequently, N absorbed by -N cultures during the 48 h uptake0o period represented a substantial addition to the extant N pool

.,-'.\ and was easily quantified by chemical analysis.I.._.-^_^~^- ^ ^ ==^ Although +N and -N cultures differed by a total of 180 mg~A~A.A N, this value does not take into account differences in dry weight.

Adjusting for these differences, it is estimated that relative to the0 12 24 36 48 +N controls, N-deprived turf had a total N deficit at time 0 of

Hours 138 mg N, with 63% and 37% of the total deficit in the shootsHou-rs and roots, respectively.

Alative uptake (A) and uptake rate (B) ofNO3- and NH4' Nitrogen Uptake. N deficiency increased uptake of NO3- and,grass turf as a function of N deprivation. Turf was NH4' by perennial ryegrass four- to fivefold over +N controls7 d (circles) or supplied continuously with N (triangles). (Fig. 1). Due to the 6 h preinduction period, no lag phase of403- uptake; closed symbols, NH4 uptake. Values are NO3- uptake was noted. Cumulative uptake by -N turf wasmples with SE bars all smaller than the symbols. essentially identical for the two N forms over the first 24 h, and

1304 BOWMAN AND PAUL

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NITROGEN ASSIMILATION BY RYEGRASS TURF

Table I. Initial Dry Weight andN Content ofboth Minus-N and Plus-N Perennial Ryegrass Turf

Reduced-N NO3--N Total-NDry weight' S/R Content Content Content N DefiCitb

Shoot Root Total Shoot Root Shoot Root Shoot Root Total Shoot Root Total

g culture-' mgN culture-'Minus N turf 5.43 3.67 9.10 1.48 103 47 1.1 0.3 104 47 151 87 51 138

(0.26) (0.24) (0.38) (6) (2) (0.2) (0.04) (6) (2) (6)Plus N turf 7.16 2.95 10.11 2.43 199 58 53 21 252 79 331

(0.19) (0.08) (0.20) (8) (4) (4) (2) (8) (4) (12)a Values are means of four samples ± SD, in parentheses. b N deficit is calculated as the difference in percent total N between plus-N and

minus-N shoot or root multiplied by the dry wt of the minus-N shoot or root.

amounted to 87 mg N culture-'. During the 24 to 48 h period,NH4' uptake amounted to 65 mg N, while NO3- uptake wasreduced to 34 mg N culture-'. Nitrate uptake rates were decreas-ing considerably by 8 to 12 h and leveled off near the +N controlvalues of 1 mg N culture-' h-' by 32 h. By contrast, NH4' uptakeleveled off between 12 and 24 h at a rate nearly twice the +Ncontrol and remained so through 48 h. Plus-N controls absorbedapproximately 40% more NH4+-N than N03--N during the 48h uptake period.Growth During N Uptake. There was no difference in the

pattern of shoot growth between the two N forms (Fig. 2), withapproximately equal increases of 0.6 g dry weight culture-'measured during each 24 h period. Root growth, however, wasaffected by N form during the uptake period. Ammonium-treated turf had little or no root growth during the 48 h experi-ment, whereas N03 -treated roots gained 0.4 g dry weight duringthe first 24 h, followed by no increase in dry weight during the24 to 48 h interval.

Nitrogen Assimilation. The fate of absorbed N was followedby chemical analysis oftissue. Uptake estimated by tissue analysis

6.8

6.0 -*NH4 _ v6.4-

6.0- T/0

5.656

t4.1

0~~~~~~~~~~~~

D3 37.b>9 ,00 3.5 B

0 12 24 36 48Hours

FIG. 2. Dry weight ofshoots (A) and roots (B) ofN-deprived perennialryegrass turf during 48 h N uptake period. Open circles, NO3- uptake;closed circles, NH4' uptake. Values are means of four samples + SEwhere larger than the symbols.

correlated extremely well (r2 = 0.996) with that measured bysolution depletion for both forms of N.Accumulation of nitrate occurred mainly in the roots and

accounted for 61 to 67% of the absorbed NO3- during the first6 to 12 h (Fig. 3). By the end of the 48 h uptake period, 53% ofthe N absorbed had been reduced. Nitrate concentration in-creased from 203 and 74 yg N g-' dry weight in the shoot androot, respectively, to 2881 and 9696 ,ug N g-1 during the experi-ment (data not presented). Uptake was nearly equal to N03-reduction between 24 and 48 h, with accumulation decreased to19% of the uptake during that period. Reduced N increasedequally in the roots and shoots during the first 12 h. After 12 h,reduced N continued to increase in the shoots but remainedessentially constant in the roots through 48 h.Ammonium assimilation followed a much different pattern

than that for NO3- (Fig. 4). Accumulation ofNH4+ (Fig. 4, inset)was principally in the roots and was considerably less than theNO3- accumulated by the NO3--treated turf. In root tissue, NH4+increased from 40 ,ug N g-' at h 0 to a maximum of 1330 ,ug Ng-'in the roots at 12 h, after which it decreased to approximately

z 80 A-ATotalcl) 0E 80 -

60-Z n40 ___ __ A

20

20

120 B

100z o)-o 0 80 -0) ~~~~~~~~~~A

2C 60 -

CDE 40 -

20 ,/..-::

00 12 24 36 48

HoursFIG. 3. Nitrate uptake and accumulation (A) and reduced N accu-

mulation (B) in shoots and roots of N-deprived perennial ryegrass turf.Values are presented as the increase in N content (mg per culture) as afunction of time. Values are the means of four samples with SE bars allsmaller than the symbols.

1305



Plant Physiol. Vol. 88, 1988

-o

0

a)

0~

z

+1

z

--, 150I125

3 100

E 75z 50u)

*25

0 14, '- a I--- . I

0 1 2 24 36 48Hours

FIG. 4. Ammonium uptake (open circles) and accumulation of re-

duced N in the shoots (closed circles) and roots (open triangles) of N-deprived perennial ryegrass turf. Inset, the increase of NH4' (mg N perculture) in root, shoot, and whole plant tissue. Values are means of foursamples ± SE where larger than the symbols.

700 ,ug N g-' (data not presented). Ammonium in the shootnever exceeded 350 jig N g-'. On a whole plant basis, NH4'accumulation amounted to 19% of that absorbed by 6 h but wasreduced to 4.7% and 3.2% at 24 and 48 h, respectively. Parti-tioning of the N absorbed between roots and shoots was approx-imately equal through 12 h; thereafter the shoots were thedominant sink.A summary of absorbed N partitioning between tissues for

both NO3- and NH4' (Table II) indicates the increasingly dom-inant role of the shoot as N sink during the 48 h uptake period.Relative partitioning ofthe absorbed N was very similar betweenN forms by 48 h, with approximately 60% measured in theshoots and 40% in the roots. This ratio is identical to the initialshoot to root ratio ofthe -N cultures (Table I) and is very similarto the relative N deficit in -N shoots and roots, based ondifferences in the percent N in the tissue as compared to +Ncontrols.

Soluble Carbohydrates. Total soluble carbohydrate levels wereincreased threefold and twofold in the shoots and roots, respec-tively, of turf deprived of N for 7 d (Table III), with the largestincrease occurring in the fructans. Glucose and fructose eachrepresented less than 5% of the total soluble carbohydrates, withsucrose and fructans being the predominant sugars.

Nitrogen uptake decreased total sugar levels in both shootsand roots (Figs. 5 and 6), and in general the pattern of loss wassimilar between N forms. Glucose and fructose concentrations

changed very little with either N form over time (data notpresented). The decrease in total soluble carbohydrates in theshoot was caused primarily by the depletion of fructans. Sucroseconcentration in the shoot was relatively stable at 3.0 to 4.4%with both forms of N; fructans were decreased from 5.8% to1.7% and 2.6% with N03 and NH4,, respectively.Ammonium uptake caused a greater decrease in root carbo-

hydrates than did NO3 uptake (Figs. 5, bottom; 6, bottom), dueto the loss of nearly 70% of the sucrose in addition to completedepletion of the fructans. By comparison, while fructans weresimilarly depleted in NO3-treated roots, sucrose concentrationwas fairly stable, decreasing only 14% by 48 h. Consequently, bythe end of 48 h, total soluble sugars in the roots had droppedfrom 2.8 to 1.4% with NO3- and to 0.6% with NH4+.

Experiment 2. Uptake and accumulation of NO3- over 16 h(Table IV) were very similar to experiment 1, although the uptakemechanism was apparently not fully induced at the start of theexperiment (14.8 versus 22.8 mg N absorbed during the 0-4 and4-8 h periods). Nitrate reduction was relatively constant duringthe four 4-h periods, ranging from 7.1 to 11.5 mg N/period withtotal reduction for the entire 16 h amounting to 50% ofthe NO3-absorbed. Nitrate translocation to the shoot, estimated by xylemflux, increased from 19% of the current uptake rate at 4 h to63% at 12 h (Table IV). Nitrate reduction in the shoot was quitelow during 0 to 8 h but increased approximately sixfold duringthe 8 to 16 h period. The pattern of NO3- reduction in the rootwas reversed, with a relatively high average rate during 0 to 8 hof 1.68 mg N culture-' h-', followed by a decrease of nearly 80%in the rate during the 8 to 16 h period to 0.32 mg N culture-'h-'. Ofthe NO3- reduced, the greater percentage was reduced bythe roots during 0 to 8 h (81%) and by the shoots during 8 to 16h (87%).The initial concentration of soluble sugars in the roots was

similar to that in experiment 1 as was the decrease in sugarsconcomitant with NO3- uptake (data not presented). The loss ofcarbohydrates amounted to an average of 56 mg glucose equiv-alents per root system per culture with fructans being primarilyresponsible for the decrease.

DISCUSSION

The absorption of NO3- and NH4+ was similar over 48 h,exhibiting comparable repression of the initially rapid uptakethrough 12 to 18 h. Root carbohydrate levels remained highduring the decrease in uptake rates and indicate that the decreasewas either unrelated to energy supply or that the carbohydrateswere insufficiently metabolized to meet the demand (23). Thedecrease in uptake could also be due to NO3- filling of the rootvacuolar compartment, as suggested by Glass et al. (11). Netuptake would then be regulated primarily by xylem loading.The two N forms differed in their assimilation; 53% of the

Table II. Nitrogen Partitioning in N-Deficient Perennial Ryegrass Turfduring the Absorption ofEither N03-or NH4,

Partitioning of Absorbed N

Nitrate AmmoniumTime

Shoot Root Shoot Root

N03--N Red. Na Total N03- Red. N Total Red. Nb Red. N.

h % ofN increase6 23C 13 36 48 16 64 51 4912 15 25 40 42 18 60 52 4824 16 26 42 41 17 58 55 4548 14 45 59 30 1 1 41 61 39

a Reduced N. b Reduced N is equivalent to total N since no increase in nitrate was detected. c Allvalues are cumulative and are based on the increase in N over time 0 samples, determined from tissue analysis.

1306 BOWMAN AND PAUL




Table III. Initial and Final Concentrations ofSoluble Carbohydrates in Shoot and Root Tissue ofMinus-N and Plus-N Perennial Ryegrass Turf,Experiment I

Values are means offour samples ± SD, in parentheses.

0 Hours 48 Hours

Sugar -N plants +N plants -N plants +N plants

Shoot Root Shoot Root +NO3 shoot Root +NH4+ shoot Root +NO3 shoot Root +NH4+ shoot Root

mg/g dry wtGlucose 3.3 0.1 1.1 0.4 1.5 0 2.2 0.1 0.9 0 1.4 0.3

(0.6) (0.1) (0.2) (0.2) (0.2) (0.2) (0.2) (0.4) (0.2) (0.4)Fructose 3.9 0.9 2.2 0.6 2.9 0.5 2.5 0.6 1.7 0.3 2.2 0.7

(0.6) (0.2) (0.2) (0.4) (0.4) (0.2) (0.2) (0.4) (0.8) (0.2) (0.4) (0.2)Sucrose 44 15 27 10 36 13 34 5 27 8 27 3.9

(2) (4) (2) (4) (6) (2) (4) (1) (2) (2) (2) (1.6)Fructans 58 12 4 2.6 17 0.3 26 0.1 0.6 0.1 0.6 0.7

(6) (2) (2) (0.6) (4) (0.3) (4) (0.1) (0.6) (0.1) (0.6) (0.7)Total 109 28 34 14 57 14 64 5.7 30 8.4 3 1 5.6

(6) (4) (2) (2) (6) (4) (4) (0.8) (2) (0.6) (4) (1.2)

100

80

~0

a)

v-0)

U)

E

60

401

20 1

0

30

25

20

154

10

5

OL1 2 24 36 48

HoursFIG. 5. Changes in the concentration of sucrose, fructans, and total

soluble carbohydrates in the shoot and root during N03- uptake by N-deficient perennial ryegrass turf. Values are means of four samples ± SE

where larger than the symbols.

NO3- was assimilated compared to 97% assimilation of theabsorbed NH,' by 48 h. This compares to values in the literaturefor NO3- reduction (as percent of N absorbed) by N-deficientplants over similar time periods ranging from lows of 20 to 30%in barley (12) and dwarf bean (5) to highs of 64 to 80% in wheat(7, 17, 23) and corn (16). Very little free NH4+-N accumulatedin the tissues during NH4+-N uptake, consistent with resultsusing +N barley plants (21, 22).Accumulation of both NO3- and reduced N in NO3-treated

roots (experiment 1) approached maximum levels by 12 h, afterwhich shoot accumulation of reduced N became the predomi-nant fate of the absorbed NO3-. This shift from root to shoot

100

80

-o

40

a)a)

C,)

0

:3

E

20

0

30

25

20

15

10

5

0 12 24 36 48

HoursFIG. 6. Changes in the concentration of sucrose, fructans, and total

soluble carbohydrates in the shoot and root during NH4' uptake by N-deficient perennial ryegrass turf. Values are means of four samples ± SE.Symbols as in Figure 5.

accumulation is roughly coincident with the shift in NO3- re-duction from root to shoot noted in experiment 2 and suggeststhat following resupply of NO3-, reduction in the root functionsmainly to provide the root system with reduced N. Upon satis-fying the rootArequirement, root NRA may be repressed, possiblyby assimilation products or a decrease in cytoplasmic [NO3-]due to decreased uptake or, as noted in experiment 2, increasedtranslocation. Consequently, reduction would be shifted to theshoot. Gojon et al. (12) have reported a similar shift of N03-assimilation from root to shoot in N-deficient barley and cornseedlings. This pattern also agrees with reports that crop plants

NT A-A sucrose

O A-A fructans1\-0OTNC°

A~~~

Shoot A

1 1

RoT

Root\

\.-

Root A a5

. . . .

a I 2 -A a I - A

1307

w)



Table IV. Uptake, Accumulation, Translocation, and Reduction ofNO3- by Minus-N Perennial Ryegrass Turf, Experiment 2Values are means of four samples ± SD, in parentheses.

Time Accumula- Transloca- Shoot Shoot Root ReductionInterval Uptake tion tiona Reduction Accumula- Reductionb Reductionc Shoot Root

h mg N culture-' %

0-4 14.8 7.8d 2.8 7.0 0.9 1.9 5.1 27 73(0.8) (1.2) (1.2) (0.2)

4-8 22.8 13.1 6.4 9.7 5.2 1.2 8.5 12 88(0.8) (2.4) (1.2) (0.6)

8-12 19.3 7.8 12.2 11.5 1.4 10.8 0.7 94 6(1.2) (1.4) (3.0) (0.2)

12-16 17.2 8.4 8.2 8.8 1.2 7.0 1.8 80 20(0.8) (0.8) (1.2) (0.2)

a Translocation is based on the N03--N flux determination derived from measurements of xylem exudate and is calculated for the preceding 4 hperiod. b Shoot reduction is calculated as the difference between N03--N translocation and shoot accumulation for each time interval. c Rootreduction is calculated as the difference between whole plant reduction and calculated shoot reduction. d This value based on three samples.

grown under steady state, +N conditions reduce most NO3- inthe shoot (21-23). Nitrate uptake by the N-stressed turf nearedsteady state by 24 to 48 h; NO3- reduction during this periodamounted to 81% of absorption compared to the steady statevalue of 79 to 80%. Reduced N in the tissue of N03A-treatedcultures failed to reach the levels in the +N controls. By com-parison, NH4' uptake by minus-N turf continued at elevatedrates through 48 h. Reduced N in the NH4+-treated minus-Nplants nearly equaled (shoots) and exceeded (roots) the reducedN levels in NH4+-treated plus-N controls (Table II). The turfthus appears to quickly reestablish conditions found in +Ncontrols following resupply of NO3- but not NH4+.The failure of-N roots to increase in dry weight during NH4+

absorption, as compared to those absorbing NO3- (Fig. 2) indi-cates that either NH4+ inhibits photosynthate translocation tothe root or that the requirements for carbon chains by NH4+assimilation in the root takes precedence over growth. The latteris supported by the role of roots as the primary tissue of NH4+assimilation (21, 22). Considerable growth occurred in the N-stressed roots during the first but not the second 24 h period ofNO3- uptake. This is possibly the result of photosynthate diver-sion to NO3- reduction and renewed growth in the shoot duringthe second period.

Similar amounts of total carbohydrates were depleted duringN uptake (262 versus 246 mg glucose equivalents per culture forNO3- and NH4', respectively). This loss was mainly due tomobilization of fructan reserves. Fructans accumulate in thetissue of numerous species, often in lieu of starch, during thedevelopment of N deficiency (4, 24). Their direct involvementin N metabolism has apparently not been reported previously,while data with wheat (28) suggest fructans do not participate inNO3- assimilation. Sucrose appears to be either not involved inN assimilation by perennial ryegrass or buffered against changesin pool size. Humphries (15) reached a similar conclusion forbarley, reporting that reducing sugars rather than sucrose wereinvolved in N assimilation. The exception to this may be duringNH4+ assimilation in roots (Fig. 6, bottom), where the demandfor carbon chains is extremely high, resulting in decreased sucroseconcentrations and nearly depleted total carbohydrates.The depletion of soluble carbohydrates during N absorption

must be considered with some caution, since the values do notmeasure turnover of potentially dynamic pools. If the pools arerelatively static (i.e. little turnover) and represent the primarysource of carbohydrates for N assimilation, then there should bea predictable stoichiometry between carbohydrates depleted andN assimilated. Assuming glutamine as the product ofassimilation

and ignoring other energy costs, five glucose equivalents will fixeight NH4,. The decrease in whole-plant carbohydrates duringuptake of both NO3- and NH4' amounted to 1.4 mmol perculture and could thus account for the assimilation of 2.2 mmolN. By comparison, 4 mmol NO3- and 10.5 mmol NH4' perculture were actually assimilated. This indicates that currentphotosynthate, in addition to stored carbohydrates, is involvedto a large extent in the assimilation ofNH4' and to a lesser extentin NO3- assimilation. A similar conclusion with regard to N03assimilation may be made from the data ofChampigny et al. (7,their Table 2); over a 7 h NO3- uptake period, N-deficient wheatseedlings reduced 20.4 zmol NO3- but lost only 0.05 ,umolglucose equivalents per seedling. Our data are also consistentwith the decreased growth of roots absorbing NH4' being due todiversion of photosynthate from growth to N assimilation.These results indicate that the rapid absorption ofN by peren-

nial ryegrass, resulting from N deprivation, is matched by equallyrapid assimilation of NH4'. Nitrate is both reduced and accu-mulated, with overall assimilation being less than with NH4+.Roots are initially responsible for most NO3- reduction, but afterapproximately 12 h reduction occurs primarily in the shoots.Due to the rapid uptake and assimilation of both NO3- andNH4+, fertilizing turf with soluble inorganic N sources will likelyalleviate N-stress for only very brief periods.

LITERATURE CITED

1. BERNT E, HU BERGMEYER 1974 D-Fructose. In HV Bergmeyer, ed, Methodsof Enzymatic Analysis III. Academic Press, NY, pp 1304-1307

2. BOWMAN DC, JL PAUL, RM CARLSON 1988 A method to exclude nitrate fromKjeldahl digestion of plant tissues. Common Soil Sci Plant Anal 19: 205-213

3. BOWMAN DC, JL PAUL, WB DAVIS 1988 Characterization of NO3- and NH4'uptake by N-deficient perennial ryegrass 'Manhattan II' J Am Soc Hort Sci(in press)

4. BOWMAN DC 1987 Uptake and assimilation of nitrate and ammonium bynitrogen-deficient perennial ryegrass turf. PhD dissertation, University ofCalifornia, Davis

5. BRETELER H, CH HANISCH TEN CATE 1980 Fate of nitrate during initial nitrateutilization by nitrogen-depleted dwarf bean. Physiol Plant 48: 292-296

6. CARLSON RM 1986 Continuous flow reduction of nitrate to ammonia withgranular zinc. Anal Chem 58: 1590-1591

7. CHAMPIGNY ML, E BISMUTH, A TALOUIZTE, G GUIRAUD 1984 The role oftheroots in nitrate reduction and mobilization of the carbohydrate product ofphotosynthesis for amino acid synthesis in Triticum aestivum seedlings. InC Sybesma, ed, Advances in Photosynthesis Research, Vol III. MartinusNijhoff/Dr W Junk, The Hague, pp 875-878

8. CLEMENT CR, MJ HOPPER, LHP JONES, EL LEAFE 1978 The uptake of nitrateby Lolium perenne from flowing nutrient solution. II. Effect of light, defol-iation, and relationship to CO2 flux. J Expt Bot 29: 1173-1183

9. DODDEMA H, H OTrEN 1979 Uptake of nitrate by mutants of Arabidopsis

1308 BOWMAN AND PAUL Plant Physiol. Vol. 88, 1988




thaliana, disturbed in uptake or reduction of nitrate. Physiol Plant 45: 339-346

10. EZETA FN, WA JACKSON 1975 Nitrate translocation by detopped corn seed-lings. Plant Physiol 56: 148-156

11. GLASS ADM, RG THOMPSON, L BORDELEAU 1985 Regulation of NO3 influx

in barley. Plant Physiol 77: 379-38112. GOJON A, JF SOUSSANA, L PASSAMA, P ROBIN 1986 Nitrate reduction in roots

and shoots of barley (Hordeum vulgare L.) and corn (Zea mays L.) seedlingsI. '5N study. Plant Physiol 82: 254-260

13. HANISCH TEN CATE CH, H BRETELER 1981 Role of sugars in nitrate utilizationby roots of dwarf bean. Physiol Plant 52: 129-135

14. HOAGLAND DR, DI ARNON 1950 The water culture method for growing plantswithout soil. Calif Agri Exp Stn Bull 347, pp 31-32

15. HUMPHRIES EC 1956 The relation between the rate of nutrient uptake byexcised barley roots and their content of sucrose and reducing sugars. AnnBot 20: 411-417

16. IVANKO S, J INGVERSEN 1971 Investigation on the assimilation of nitrogen bymaize roots and the transport of some major nitrogen compounds by xylemsap. Physiol Plant 24: 59-65

17. JACKSON WA, KD KwIK, RJ VOLK 1976 Nitrate uptake during recovery fromnitrogen deficiency. Physiol Plant 36: 174-181

18. JACKSON WA, KD KWIK, RJ VOLK, RG BUTZ 1976 Nitrate influx and effluxby intact wheat seedlings: Effects of prior nitrate nutrition. Planta 132: 149-156

19. LEE RB 1982 Selectivity and kinetics of ion uptake by barley plants followingnutrient deficiency. Ann Bot 50: 429-449

20. LEE RB, KA RUDGE 1986 Effects of nitrogen deficiency on the absorption ofnitrate and ammonium by barley plants. Ann Bot 57: 471-486

21. LEWIS OAM, DM JAMES, EJ HEWIrr 1982 Nitrogen assimilation in barley(Hordeum vulgare L. cv. Mazurka) in response to nitrate and ammoniumnutrition. Ann Bot 49: 39-49

22. LEWIS OAM, S CHADWICK 1983 An '5N investigation into nitrogen assimila-tion in hydroponically-grown barley (Hordeum vulgare L. cv. Clipper) inresponse to nitrate, ammonium and mixed nitrate and ammonium nutrition.New Phytol 95: 635-646

23. MINOrrI PL, WA JACKSON 1970 Nitrate reduction in the roots and shoots ofwheat seedlings. Planta 95: 36-44

24. POLLOCK CJ 1986 Fructans and the metabolism of sucrose in vascular plants.New Phytol 104: 1-24

25. RuFrY TW, RJ VOLK, PR MCCLURE, DW ISRAEL, CD RAPER 1982 Relativecontent of NO3- and reduced N in xylem exudate as an indicator of rootreduction of concurrently absorbed '5NO3 . Plant Physiol 69: 166-170

26. SHANER DL, JS BOYER 1976 Nitrate reductase activity in maize (Zea mays L.)leaves. I. Regulation by nitrate flux. Plant Physiol 58: 499-504

27. STEEN E, K LARSSON 1986 Carbohydrates in roots and rhizomes of perennialgrasses. New Phytol 104: 339-346

28. TALOUIZTE A, ML CHAMPIGNY, E BISMUTH, A MOYSE 1984 Root carbohydratemetabolism associated with nitrate assimilation in wheat previously deprivedof nitrogen. Physiol Veg 22: 19-27

29. VOSE PB, EL BREESE 1964 Genetic variation in the utilization of nitrogen byryegrass species Lolium perenne and L. mulliflorum. Ann Bot 28: 251-270

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Uptake Assimilation N03 NH4' Nitrogen- DeficientPerennial ...NH4' uptake amounted to 65 mgN, while NO3- uptake was reducedto 34mgNculture-'. Nitrateuptakeratesweredecreas-ingconsiderablyby8to

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