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Environmental and Experimental Botany 61 (2007) 152–158

Interaction of carbon and nitrogen assimilation estimated by l3C and 15Ntracing in detached leaves of spinach

Tae-Hwan Kim a,∗, Tadakatsu Yoneyama b

a Department of Animal Science, Institute of Agricultural Science & Technology, College of Agriculture & Life Science, Chonnam National University,Buk-Gwangju, P.O. Box 205, 500-600 Gwangju, Korea

b Department of Applied Biological Chemistry, Graduate School of Agricultural and Life Sciences, The University of Tokyo,Bunkyo-ku, Tokyo 113-8657, Japan

Received 12 October 2006; received in revised form 27 April 2007; accepted 13 May 2007

bstract

To investigate short term interaction between recently fixed carbon and assimilated nitrogen, the fates of pulse-fed 13C and continuously-applied5N in sugar, nitrate, organic acid, amino acid and insoluble compound fractions from detached leaves of 5-weeks old spinach (Spinacia oleracea. cv. Lead) were followed under four light/NO3

− combined treatments. At the termination of a 75 min pulse 13CO2 feeding (defined as 0 h), theotal amounts of the 13C recently fixed by detached leaves in the light were slightly higher for N-free and 1 mM NO3

− medium than for 4 mMO3

−. In shaded leaves, 13C fixation and 15NO3− uptake, respectively, decreased to 11.2% and 71.3% of the fully illuminated leaves exposed to the

ame 4 mM NO3− concentration. In the light at 0 h, the incorporation of 13C was mostly into sugars, followed by undefined 13C (total 13C – sum of

nalyzed sub-fractions). In shaded leaves, the largest incorporation was found in the undefined C, followed by insoluble C and sugars. The uptakef 15NO3

− from 0 to 3 h was 128, 665 and 620 �g g−1 DW and the percentages of 15N present as reduced forms were 80, 77 and 41, respectively,

n the leaves exposed to, in mM, 1 NO3

−, 4 NO3− in the light and 4 NO3

− in the shaded condition; thus, shading did a small decrease in uptake butgreat decrease in reduction of 15NO3

−. Under light treatment, the synthesis of reduced 15N was negatively related to the reduction of 13C sugarsrom 0 to 3 h, but positively related to amounts of organic acid 13C at 3 h.

2007 Elsevier B.V. All rights reserved.

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eywords: Detached leaves; C and N labeling; C and N assimilation; Spina

. Introduction

In green tissues the reduction of CO2 with production ofucrose is not an isolated process. It interacts through, fornstance, competition for reducing power with other biosyntheticathways. One of the most important ones is the assimilation ofO3

− through reduction to NH4+ and subsequent incorporation

nto carbon chains leading to the production of amino acids.ptake of nitrate into root symplasm against electrochemicalradient (Clarkson et al., 1992) and nitrate transport derived

rom root symplasm into the xylem (Jackson et al., 1980) requireetabolic energy. In chloroplasts, photogenerated ATP, NADPH

nd reduced ferredoxin are required for assimilation of CO2 intoriose phosphates, reduction of NO2

− to NH4+ and a further

∗ Corresponding author. Tel.: +82 62 530 2126; fax: +82 62 530 2129.E-mail address: grassl@chonnam.ac.kr (T.-H. Kim).

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098-8472/$ – see front matter © 2007 Elsevier B.V. All rights reserved.oi:10.1016/j.envexpbot.2007.05.003

ssimilation into amino acids (Lea and Miflin, 1973; Losada etl., 1981). In addition, a continual supply of reducing equiva-ents, ATP and C skeletons is required for sustained enzymaticssimilation of nitrate into protein and nucleic acids (Beeversnd Hageman, 1980; Miflin and Lea, 1980). The overall reduc-ion of nitrate to the level of ammonia is an energy-dependentrocess (eight electrons per mM NH4

+ generated), just as CO2eduction.

Extensive research has been devoted to determine the effectsf carbohydrate availability on assimilation of NO3

−. Natu-al fluctuations in carbohydrate (energy) status occur duringhe daily light/dark cycle, and carbohydrates reserves becomeeverely depleted with extended darkness (Kerr et al., 1985).imitations in carbohydrate supply during darkness are likely to

e largely responsible for the decreased rates of NO3

− uptakeHansen, 1980; Ourry et al., 1996), and reduction (Reed et al.,983). Rufty et al. (1989) reported that a capacity for 15NO3

−eduction and synthesis of 15N-insoluble reduced was clearly

and Experimental Botany 61 (2007) 152–158 153

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T.-H. Kim, T. Yoneyama / Environmental

etained even when uptake was severely restricted and min-mal carbohydrate reserves remained in tissues. Although aunctional dependence of N uptake on photosynthetic activ-ty and carbohydrate availability has been inferred in manyxperimental studies (Clement et al., 1978; Rufty et al., 1989),ost evidence for carbohydrate limitation hypothesis, invoking

ausal relationships between photosynthetic activity, carbohy-rate translocation, root respiration and NO3

− assimilation,emains obscure.

Of particular interest in the previous experiment with intactpinach plants was the interaction of metabolism of the currentlyxed 13C and the newly assimilated 15N during 6 h of continuous5NO3

− absorption and chase feeding of non-labeled CO2 (Kimt al., 2002). It was noteworthy that the decreased amount of3C-labeled sugars was paralleled by an increased 15N-labelingn reduced N (total 15N – nitrate 15N) in roots, stems and leaves.he topic of this study was the interaction between nitrogenssimilation and the allocation of photoassimilated carbon. Itocused on the quantification of metabolites originating fromurrently fixed C and newly absorbed N during the applicationf different forms and levels of N. The availability of reduced

was influenced by conducting experiments in the shade asell as in the light. The changes in 13C and 15N amounts in theiochemical compounds were discussed in terms of interactionetween C and N assimilation in detached leaves.

. Materials and methods

.1. Plant culture

Spinach (Spinacia oleracea L. cv. Lead) seeds were gem-nated on wet filter paper, and then transferred to wetouble sheets of filter paper (15 cm × 30 cm) in an incuba-or at 15–25 ◦C, to allow the elongation of roots and shoots.wo-weeks old seedlings were transplanted to 3 L pots andydroponically grown on a complete nutrient solution contain-ng, in mM, 4.0 NO3

−, 1.0 P, 4.0 K, 2.0 Ca, 2.0 Mg andicronutrients (Fe, B, Mn, Zn, Cu, Mo) with the adjusted pH

.4. The nutrient solution was continuously aerated and renewedvery 5 days. Natural light was supplemented with 150 �molhotons m−2 s−1 by metal halide lamps for 18 h per day. After 5eeks of hydroponic culture (the early flowering stage), selected

eaves of uniform size were cut with a razor blade directly abovehe stubble level.

.2. 13CO2 and 15N labeling and harvest

The experiment was designed with five treatments consistingf different light/nitrogen combinations. Three N-treatments (N-ree, 1 or 4 mM NO3

−) with three replications were applied toetached leaves placed in 100 ml flasks (two leaves per flask) inhe light. An additional set of 12 flasks with 4 mM NO3

− wereovered with shading for blocking about 75% of the light. All

asks were placed in a 13CO2 feeding chamber at 25 ◦C. Lightas supplied at approximately 850 �mol photons m−2 s−1 at thelant top level by 400 W metal halide lamps. A water-flow filteretween the lamps and plants removed the heat radiation. The

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ig. 1. Experimental design with the periods of 15NO3− and 13CO2 feeding. At

he time indicated by ⇓, plants were transferred to the growth chamber. At theimes indicated by ↓, plants were harvested.

3CO2 feeding and 15N labeling schedule is depicted in Fig. 1.Fifteen minutes before feeding 13CO2, the labeling with 15N

as carried out by placing the detached leaves in 50 ml nutri-nt solution either without N (N-free) or containing 1 or 4 mM5NO3

− (10.3 15N atom% excess). The 15N was continuouslyed until each sampling time corresponding the hours after thend of 13CO2 feeding (0 h). The pulse-chase 13CO2 labelingas adapted. Feeding of 13CO2/12CO2 (51.0% 13C atom excess;

3CO2 = 270.7 ppm and 12CO2 = 355.1 ppm) was started 15 minfter initiation of the 15N treatment. A constant l3C excessas maintained in the chamber by continuously mixing and

ntroducing 12CO2 and 13CO2 by an operation of mass-flow con-roller (Ohkura Electric Co. Ltd., Tokyo, Japan) in response tohe outputs of differential-type infrared 13CO2 analyzer (Japanpectroscopic Co. Ltd., Tokyo, Japan). Industrial CO2 gas withatural l3C abundance was used as the source of 12CO2. The 13C-nriched CO2 gas was generated from a reaction of Ba13CO290.1 atom%) with 1 M HCI and collected under vacuum at liq-id nitrogen temperature. After 75 min of 13CO2 labeling (0 h),he assimilation chamber was opened and quickly purged withmbient air. Sampling was then carried out at 0, 1, 2 and 3 h afterhe end of 13CO2 feeding. Two detached leaves per flask wereombined to one sample. The samples were immediately frozennd stored at −70 ◦C until further analysis.

.3. Chemical fractionation and isotope analysis

About 200 mg of finely ground freeze-dried sample wasxtracted with 25 ml of four volumes ethanol:1 volume waterhile heated on a hot plate for 5 min. The 80% ethanol-soluble

raction was filtered, centrifuged, and passed through H+ columnDowex 50 W X 8). The pH of one-fifth of the solution collectedrom the H+ column was adjusted to 7.0 and this solution was

oncentrated to a final volume of 0.5 ml (nitrate fraction). Theemaining four-fifths were passed through a column in the for-ate form (Dowex 1). The collected solution was concentrated

o 2.0 ml (soluble sugars fraction). Amino acids were eluted with

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54 T.-H. Kim, T. Yoneyama / Environmental

5 ml of 0.5N HCl from the Dowex 50W column and concen-rated to 1.0 ml. Organic acids were eluted with 25 ml of 4N HClrom the Dowex 1 column and concentrated to 2.0 ml. Concen-ration was achieved by drying the collected solutions by rotaryacuum evaporation at 30 ◦C and dissolving the residues withistilled water to obtain the final volume of each fraction. Theesidues of ethanol extraction and filtration were dried for 24 h tobtain dry weight. The resulting dried samples were designateds the insoluble fractions for C and N.

Preliminary experiments, using single mode analysis with anNCA-SL mass spectrometer, showed that above 25 �g of N and0 �g of C per sample were required (Kim et al., 2002). The solidamples (total C, total N and residues) were precisely measurednto tin capsules. For the solution samples, an appropriate sampleolume, usually 0.1 ml, was dropped into tin capsules to giveore than minimum sample size as described above. The tin

apsules containing solution were quickly cooled with liquiditrogen, and then dried in a freeze-dryer. The residues in tinapsules were employed for their N or C content and 15N or 13Cbundance. 13C-enriched glycine (1.88 13C atom%) and 15N-nriched l-glutamic acid (0.362 15N atom%) were used as theeferences for C and N analysis, respectively.

These obtained 15N and 13C abundance were converted toelative specific activity (RSA, i.e. % of recently incorporatedtoms relative to total number of atoms in the sample) usingqs. (1) and (3). The amounts of newly absorbed 15N (NAN)r recently fixed 13C (RFC) incorporated into the organic com-ounds were calculated per gram dry weight using Eqs. (2) and4),

For nitrogen,

SAN =15N atom% measured − natural 15N atom%

15N atom% administered 15NO3 − natural15N atom%

×100 (1)

n which the natural 15N atom% was adopted from the 15N atomf non-15N-fed plants.

AN = RSAN × N content measured in a fraction

100(2)

For carbon,

SAC =13C atom% measured − natural13C atom%

13C atom% 13CO2supplied − natural13C atom%

×100 (3)

n which the natural 13C atom% was adopted from the 13Ctom% of non-13C-fed plants.

FC = RSAC × C content measured in a compound

100(4)

n the obtained data, considerable quantitative differences could

e estimated between the sum of C (13C) and N (15N) contentsn analyzed biochemical sub-fractions and total C (13C) and N15N) measured. The difference will be referred to either as thendefined C and N fraction.

t(de

xperimental Botany 61 (2007) 152–158

. Results

.1. Total C and N partitioning

The C content in biochemical fractions of detached leavesontinued to increase from 0 to 3 h only for sugars (data nothown). On average, sugars increased from about 9.3% to 15.4%f total carbon in the full light, while they stayed unchanged atbout 6.8% in the shaded leaves. The C attribution to amino acidsas the lowest (1.1–1.6% of total C) for all five light/nitrogen

ombined treatments. Little change occurred in C content ofmino acids and insoluble fractions. The C contents in the unde-ned fraction in the light treatments greatly decreased whereas

hat in the shaded remained relatively constant. A clear changen the amounts of nitrogen in biochemical fractions was foundnly for the nitrate fraction in the shaded leaves treated withmM NO3

− (data not shown).

.2. Relative specific activity of 13C and 15N in biochemicalractions

The changes in the percentages of 13C assimilated into var-ous biochemical compounds are shown in Fig. 2. The highestSAC values in the light were observed in sugars and the sec-nd highest in amino acids, which became close to the RSAC ofndefined C after 2 h in the chase period. The RSAC values ofugars and amino acids sharply decreased immediately after ter-ination of 13CO2 for the N-free and 1 mM NO3

− treatments.he decrease of the RSAC of sugars for 4 mM NO3

− started ath in both light and shaded conditions. In the light, the RSACalues of organic acids and insoluble C remained at the lowestevel. It was noteworthy that in the shaded leaves the RSAC val-es of amino acids were higher than those of sugars throughouthe whole experimental period.

The changes in the percentage of 15N assimilated into variousitrogenous compounds during the continuous 15N feeding arehown in Fig. 3. In the light, the RSAN values of all N compoundsxamined highly increased as NO3

− supply level was increasedrom 1 to 4 mM (note the y-axis with different scales). Aminocids showed a vast increase in RSAN values from 0 to 3 h inoth light and shade conditions, particularly prominent undermM NO3

− in the light. When compared to two plots of 4 mMO3

−, shading decreased the RSAN-amino acid in half, butontinuously increased up to 11.8% at 3 h.

.3. Partitioning of 13C and 15N

The partitioning of recently fixed 13C (RFC) in the biochem-cal compounds from 0 to 3 h is summarized in Table 1. After5-min of 13CO2 feeding (0 h), total amount of RFC in N-freeedium was relatively higher than in other N treatments in the

ight.Shading greatly decreased 13C fixation, presenting 8.7% of

he mean value obtained from three N treatments in the light38.1 mg g−1 DW at 3 h). During the 3 h chase period, the largestecrease in RFC occurred in N-free medium, while the small-st change took place in 4 mM NO3

− medium. At 0 h, in the

T.-H. Kim, T. Yoneyama / Environmental and Experimental Botany 61 (2007) 152–158 155

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ig. 2. Percentages of recently fixed C in analyzed carbon compounds, expressight/NO3

− combinations. Each value is the mean ± S.E. for three or four replcids; (�) organic acids; (�) undefined C; (♦) insoluble compounds.

ight, RFC was largely assimilated into sugars (on average 47%f the total amount of RFC) and into undefined C (on aver-ge 25%), but under shaded condition mainly to undefined

(45%) and much smaller to sugars (20%) and insoluble C18%). In the light, the largest decrease in RFC-sugars (−34.2%)as observed in N-free medium during 3 h of chase, but theecrease was much smaller as NO3

− supply level increased.he RFC-organic acids remained the smallest and most con-

−

tant (in particular 4 mM NO3 medium) of all C compoundsxamined during the 3 h chase period, suggesting active trans-er of 13C-organic acids to the non-metabolic compartmentlikely the vacuole). RFC-amino acids also decreased, and at

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ig. 3. Percentages of newly absorbed N in analyzed nitrogenous compounds, expreifferent light/NO3

− combinations. Each value is the mean ± S.E. for three or four repmino acids; (�) undefined N; (♦) insoluble compounds.

the relative specific activity (RSA), in the spinach leaves treated with different. The bold line indicates the period of 13CO2 feeding: (©) sugars; (�) amino

otably higher rates than those of other C compounds for allour treatments, suggesting that newly formed 13C-amino acidsn the metabolic compartment (likely the cytosol and chloro-last) were further metabolized. The RFC-amino acids fractionst 3 h decreased by 83%, 61%, 56% and 50% of the initialmount (0 h) in the N-free, 1 mM, 4 mM NO3

− in the light andmM NO3

− in the shade, respectively. It is noteworthy thathe RFC-insoluble C significantly increased in 4 mM NO3

−

n the light despite a general trend of decrease with advanc-ng 13C chase time. The RFC-undefined C also significantlyecreased during 3 h of chase (except for 4 mM NO3

− in theight).

ssed as the relative specific activity (RSA), in the spinach leaves treated withlicates. The bold line indicates the period of 15NO3

− feeding: (©) nitrate; (�)

156 T.-H. Kim, T. Yoneyama / Environmental and Experimental Botany 61 (2007) 152–158

Table 1Changes in the amounts (mg g−1DW) of recently fixed 13C in biochemical fractions of spinach leaves treated with different light/NO3

− combinations

Light/nitrogen treatment Biochemical fraction Hours after termination of 13CO2 feeding

0 1 2 3

Light/N-free Sugars 26.3 ± 0.47 19.6 ± 1.09 24.1 ± 0.56 17.3 ± 1.07Organic acids 1.6 ± 0.04 1.1 ± 0.05 1.0 ± 0.03 0.9 ± 0.01Amino acids 1.8 ± 0.19 0.8 ± 0.10 0.5 ± 0.09 0.3 ± 0.05Insoluble C 12.5 ± 1.98 10.8 ± 0.17 11.4 ± 1.14 8.5 ± 0.57Undefined C 20.7 ± 1.70 12.8 ± 1.01 9.3 ± 0.81 7.6 ± 0.53Total C 62.9 ± 3.35 45.1 ± 2.21 46.3 ± 1.85 34.6 ± 4.06

Light/1 mM NO3− Sugars 23.8 ± 0.54 19.8 ± 2.31 19.3 ± 1.37 17.6 ± 0.46

Organic acids 1.7 ± 0.07 1.1 ± 0.15 1.0 ± 0.10 1.0 ± 0.03Amino acids 1.8 ± 0.20 1.1 ± 0.04 0.8 ± 0.10 0.7 ± 0.04Insoluble C 12.9 ± 1.38 11.1 ± 1.03 9.1 ± 0.11 12.6 ± 0.81Undefined C 11.1 ± 0.89 8.6 ± 0.59 8.4 ± 0.71 8.7 ± 0.58Total C 51.3 ± 1.61 41.7 ± 6.09 38.6 ± 1.53 40.6 ± 0.50

Light/4 mM NO3− Sugars 18.7 ± 0.10 17.0 ± 0.32 16.9 ± 0.29 15.1 ± 0.41

Organic acids 1.3 ± 0.04 1.2 ± 0.13 1.1 ± 0.01 1.2 ± 0.09Amino acids 1.8 ± 0.11 0.9 ± 0.07 0.8 ± 0.05 0.8 ± 0.02Insoluble C 7.0 ± 0.68 10.4 ± 0.80 10.7 ± 0.90 12.8 ± 1.24Undefined C 7.2 ± 0.76 6.9 ± 1.23 8.9 ± 0.56 9.1 ± 0.61Total C 36.0 ± 0.30 36.4 ± 0.20 38.4 ± 1.72 39.0 ± 2.08

Shade/4 mM NO3− Sugars 0.8 ± 0.08 0.6 ± 0.09 0.6 ± 0.06 0.5 ± 0.08

Organic acids 0.3 ± 0.05 0.3 ± 0.03 0.4 ± 0.07 0.4 ± 0.05Amino acids 0.4 ± 0.06 0.3 ± 0.05 0.2 ± 0.03 0.2 ± 0.02Insoluble C 0.7 ± 0.07 0.7 ± 0.06 0.6 ± 0.05 0.9 ± 0.08

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The assimilation of newly absorbed 15N (NAN) to differ-nt N fractions is summarized in Table 2. After 90-min of 15Needing (0 h), total amounts of NAN in detached leaves were

ignificantly increased by increasing the external NO3

− supplyrom 1 to 4 mM in the light. Shading reduced total amounts ofAN by 89% at 0 h. During 3 h continuous 15N feeding after ter-

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able 2hanges in the amount (�g g−1DW) of the newly absorbed 15N in biochemical frach continuous 15NO3

− absorption after an initial 90 min of 15NO3− absorption (0 h)

ight/nitrogen treatment Biochemical fraction Hours after ter

0

ight/1 mM NO3− Nitrate 18.6 ± 0.1

Amino acids 12.6 ± 3.3Insoluble N 13.2 ± 1.9Undefined N 19.8 ± 6.2Total N 64.2 ± 4.9

ight/4 mM NO3− Nitrate 54.0 ± 2.7

Amino acids 78.8 ± 3.0Insoluble N 29.5 ± 2.4Undefined N 27.3 ± 5.8Total N 189.5 ± 5.4

hade/4 mM NO3− Nitrate 33.7 ± 4.5

Amino acids 29.5 ± 3.6Insoluble N 11.1 ± 1.5Undefined N 26.0 ± 8.9Total N 100.4 ± 2.8

ata are mean ± S.E. for n = 3.

1.7 ± 0.05 0.9 ± 0.04 1.3 ± 0.093.6 ± 0.03 2.7 ± 0.25 3.3 ± 0.18

ination of 13C labeling, NAN-nitrate increased in response toO3

− concentration and shading treatment. When compared tohe NAN-nitrate under 1 mM NO3

− in the light at 3 h, 6.1-fold

ncrease by increasing N supplying level to 4 mM and 14.5-old increase by increasing N supplying level + shading wasbserved. The NAN-amino acids, undefined N and insoluble N

tions of spinach leaves treated with different light/NO3− combinations during

mination of 13CO2 feeding

1 2 3

32.4 ± 5.4 29.8 ± 3.8 25.3 ± 3.345.8 ± 4.4 47.8 ± 3.3 51.0 ± 2.722.2 ± 2.1 44.3 ± 12.4 63.2 ± 9.234.7 ± 9.8 50.8 ± 12.4 52.8 ± 3.8

135.2 ± 15.7 172.7 ± 26.7 192.6 ± 13.1

67.0 ± 3.6 123.6 ± 5.2 153.6 ± 23.084.9 ± 27.9 197.2 ± 22.6 273.1 ± 29.059.3 ± 7.8 127.2 ± 16.6 266.6 ± 48.6

130.0 ± 8.8 145.4 ± 16.0 161.1 ± 24.2341.2 ± 41.9 593.3 ± 48.1 854.4 ± 40.2

148.1 ± 17.2 228.9 ± 10.6 367.8 ± 49.945.1 ± 1.6 92.6 ± 4.0 134.4 ± 14.317.1 ± 2.4 26.5 ± 4.8 73.6 ± 23.644.7 ± 13.2 85.7 ± 22.7 144.2 ± 42.6

254.9 ± 5.7 433.7 ± 17.6 720.2 ± 44.4

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esponded to light/nitrogen treatments. Comparing two mediaupplied with 4 mM NO3

−, shading decreased the amountsf 15N in three reduced N fractions (amino acids + undefined+ insoluble N) by 49.5%.

. Discussion

The uptake of external N by detached leaves (Table 2) wasuch lower than that of intact plants (Kim et al., 2002). This is

n contrast to observations for tea plants (Morita et al., 1998), inhich detached leaves absorbed more nitrate than intact plantid. The amount of newly absorbed 15N (NAN) in nitrate fractiont 3 h was increased six times by increasing NO3

− supply-ng level from 1 to 4 mM in the light (Table 2). Gojon et al.1991) reported that increasing NO3

− concentration from 1.5 to0 mM resulted in a 45% increase of net NO3

− uptake, and thatO3

− reduction was markedly stimulated by the increase of netO3

− uptake. Van Guy et al. (1991) also confirmed for detachedheat leaves that the rates of uptake, assimilation and accumu-

ation of NO3− were positively related to the concentration of

O3− in the external solution. In the present experiment, at 3 h,

7% and 82% of the total newly absorbed 15N were found ineduced N compounds in 1 and 4 mM NO3

− in the light, respec-ively, while only 49% in 4 mM in the shade (Table 2). Theselearly indicate that light acts as a strong stimulating factor foritrate reduction in plant tissues. Rufty et al. (1989) reported thatxtended darkness and associated carbohydrates/energy stressonsistently affected in proportional terms NO3

− reduction > Nranslocation > NO3

− uptake.At the end of 75 min of 13CO2 feeding (0 h), spinach leaves

upplied with 4 mM NO3− in the shade fixed only 11% 13CO2

Table 1), and absorbed 53% 15NO3− (Table 2) of those in the

ight. The accumulation of 15N-nitrate throughout the experi-ental period was prominent in the leaves supplied with 4 mMO3

− under shaded condition. These suggest that shadingould restrict the assimilation of newly absorbed NO3

−. Under

haded condition, energy for 15NO3

− uptake and assimilationay partly come from stored carbohydrates like starch (Huppe

nd Turpin, 1994). Several authors have reported that the limi-ation in carbohydrate supply was responsible for the decreased

bs

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ig. 4. The relation between the synthesis of 15N-labeled reduced N and decrease omounts of 13C and 15N between 0 and 6 h for leaf blades and between 1 and 6 h forO3

−; (�) light/4 mM NO3−; (�) shade/4 mM NO3

−.

xperimental Botany 61 (2007) 152–158 157

itrogen uptake (Ta and Ohira, 1981; Morgan and Jackson,988). 13CO2 fixation in N-free medium was the highest at 0 h26.3 mg g−1 DW) and then sharply decreased with the highestate of all treatments investigated during 3 h of chase (55% ofhe initial 13C amount). After 3 h of chase in the light, 20.1% ofotal fixed 13C at 0 h was lost in 1 mM NO3

− but no significanthange in 4 mM NO3

− (Table 1).During the 3 h 13C chase period, the sizes of the sugar pools

arkedly increased in the light whereas these remained rel-tively constant in the shaded leaves (data not shown). Thisncrease of sugars in the light was not observed in the leaf bladesf intact plants, and about 79% of total sugars derived from theewly fixed C was exported from the leaf blades of intact spinachlants during a 6 h 13C chase period (Kim et al., 2002). How-ver, in this study, maximum decrease (−32.4%) of the newlyxed 13C in sugars fraction (13C-sugars) was recorded in N-ree medium, suggesting that the export of sugars was highlyestricted in detached leaves. The absence of sink organs for sug-rs in detached leaves is possibly the reason for lower loss of 13Coss and for high accumulation of sugars. The amounts of 13C inugars in detached leaves supplied with 4 mM NO3

− in the lightnd shaded conditions were slightly decreased during the 3 h 13Chase period (Table 1), when the synthesis of reduced 15N fromhe newly absorbed nitrate occurred (Table 2). The processesf NO3

− assimilation and sucrose synthesis compete for pho-osynthetic energy (reductant) and carbon skeletons (Gerhardtt al., 1987). The decrease in incorporation of fixed 13C intougars at high concentrations of external NO3

− was not due ton enhanced respiratory catabolism of sugars but was mainlyhe result of a repressive effect of NO3

− on sugar synthesis. Noncrease in 13C amounts in other C compounds, which could beetabolized from the sugars, was observed (except for 4 mM in

he light). These results indicate that further turnover into other Compounds of recently synthesized sugars was greatly repressedn detached leaves, which had no sink organs for photosyntheticssimilates. In intact plants, we found a positive correlation

etween the synthesis of reduced 15N and the degradation ofugars-l3C within three organs (Kim et al., 2002).

The data obtained also showed that 13C incorporation intondefined C and insoluble C was very rapid in all treatments, but

f 13C-labelled sugars (A) and 13C-organic acids at 3 h (B). The differences inpetioles or roots were used for calculation: (�) light/N-free; (©) light/1 mM

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58 T.-H. Kim, T. Yoneyama / Environmental

epressed by NO3− (more at 4 mM than at 1 mM) (Table 1). The

ndefined C fraction showed the behavior of an active metabo-ite of C assimilation, represented by a rapid incorporation of3C and a high decrease from 0 to 3 h. The highest C/N ratios,uggesting the presence of low N compounds, were observedn this fraction in all treatments (data not shown). Therefore,t is assumed that the undefined C fraction possibly includesigh quantities of carbohydrates such as sugars-P and sugarucleotides (Kim et al., 2002), and these may be distributedredominantly in the chloroplast and cytosol (Winter et al.,994).

In the light, the amount of 13C fixed during 75 min pulse waseduced with increasing NO3

− concentrations from 0 to 1 ormM (Table 1). Van Guy et al. (1991) estimated that the ratef CO2 fixation by detached wheat leaves decreased by 10% inesponse to an increase in NO3

− concentration from 0 to 40 mM.he decrease in 13CO2 fixation was significantly related to the

ncreased 15NO3− absorption (r = 0.944, p ≤ 0.001, data not

hown) and to increased 15NO3− assimilation during the initial

0 min feeding (r = 0.936, p ≤ 0.001, data not shown). Exceptor the shaded conditions, the decrease in 13C-labeled sugarsuring the 3 h chase was negatively related to the increased 15N-abeled reduced N (r = 0.783, p ≤ 0.01, Fig. 4A). This suggestshat the supply of reductants by photo-reactions other than fromugars may increase the apparent efficiency of nitrate reduc-ion in green tissues (Noctor and Foyer, 1998), although sugarsere available for nitrate reduction. In addition, the amounts of

3C-organic acids after 3 h chase were positively related to thencreased 15NO3

− reduction from 0 to 3 h (r = 0.985, p ≤ 0.001,ig. 4B). The function of a relation between the formation ofeduced 15N from nitrate and organic acid formation may beo neutralize OH−, produced during nitrate reduction (Ravennd Smith, 1976), and thus formed organic acids may be trans-erred to the non-metabolic compartment (the vacuole) (Wintert al., 1994) as indicated by no metabolism of 13C-organic acidsTable 1).

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