Plant Physiol. (1975) 56, 148-156 Nitrate Translocation by Detopped Corn Seedlings' Received for publication November 19, 1974 and in revised form March 25, 1975 FERNANDO N. EZETA AND WILLIAM A. JACKSON2 Department of Soil Science, North Carolina State University, Raleigh, North Carolina 27607 ABSTRACT Five-day-old seedlings of corn (Zea mays L.) grown without nitrate were decapitated and exposed to 0.5 mM KNOs or 0.5 mM KCI in aerated solutions at 30 C. Uptake of nitrate, chlo- ride, and potassium was determined by replacing solutions hourly and measuring their depletion. Translocation of these ions and of organic nitrogen was determined by hourly analy- sis of the vascular exudate. Nitrate reduetion was estimated by the difference between nitrate uptake and nitrate recovered in the tissue and exudate. Nitrate uptake exhibited its usual pat- tern of apparent induction resulting in the development of an accelerated uptake phase. Chloride uptake remained fairly constant throughout the experimental period. Translocation of nitrate increased progressively for at least 7 hours whereas chlo- ride translocation reached a maximum about the 3d hour and then declined to a lower rate than nitrate translocation. Nitrate uptake and translocation were restricted by anaerobiosis, by 20 and 40 C relative to 30 C, and by 0.05 mM 6-methylpurine, an RNA-synthesis inhibitor. Accumulation, reduction and trans- location of nitrate had different sensitivities to all these factors. The effect of 0.05 mM 6-methylpurine was more detrimental to nitrate translocation and nitrate reduction than to nitrate up- take. Ambient nitrate, relative to chloride, enhanced the exuda- tion volume and the translocation of organic nitrogen within 4 hours from initiation of the experiments. Translocation of ni- trate and organic nitrogen decreased shortly after removal of external nitrate. The higher rates of organic nitrogen transloca- tion which occurred during nitrate uptake indicates either (a) rapid translocation of amino acids synthesized from the enter- ing nitrate, or (b) an accelerated rate of protein turnover and a resulting enhancement in translocation of endogenous amino acids. Following its absorption by root tissue, nitrate may be re- duced and assimilated, stored in vacuoles, or translocated to the shoots. That deposited in the vacuoles of root cells, how- ever, may be only slowly available for translocation (17), for maintenance of net nitrate reductase synthesis (17), and for nitrate reduction (9). Relatively high activities of nitrate reductase have been determined in the apical region of corn 'Paper No. 4491 of the Journal Series of the North Carolina Agricultural Experiment Station, Raleigh, N.C. These investigations were supported in part by the United States Atomic Energy Com- mission, Grant AT-(40-1)-2410. 'Present address: Centro Internaciona de la papa, Apartado 5969, Lima, Peru. roots (10, 29, 32), and experiments with 'N indicate that sub- stantial nitrate reduction and translocation of the reduced nitrogen can occur in corn roots (16). The capacity of the root system to reduce nitrate affects the proportion of entering nitrate which is released to the translocation stream (34). The relative rate of nitrate translocation may therefore be affected by the rate of uptake, the induction and activity of nitrate reductase, and the rate of deposition in the vacuoles of the root cells. The purpose of the present investigations was to examine some of the factors which regulate nitrate translocation to the shoots. Detopped, dark-grown corn seedlings were employed in which an accelerated rate of nitrate uptake develops after an initially slow uptake rate (17, 18). Data are presented to sup- port the concept that continuous nitrate uptake is required to maintain significant translocation of nitrate. In addition, the translocation of organic nitrogen was strongly dependent upon continuous nitrate uptake. MATERIALS AND METHODS The procedures were, in general, similar to those described by Jackson et al. (17). All experiments were conducted with corn (Zea mays L.) hybrid DeKalb XL45. Four days after germination in darkness with 0.1 mm CaSO, the secondary roots were excised and five seedlings were assembled in a Plexiglas holder especially designed to fit the top of a 75-ml Taylor tube. Seedlings were then placed in a pretreatment solution for 24 to 36 hr in darkness prior to initiating the ex- periment. The pretreatment solutions contained per liter 0.25 mM (NH)2SO,, 0.6 mmY K2SO4, 0.4 mM KH2PO4, 1.6 mM MgSO4, 0.8 mm CaSO4, and 250 mg CaCO,. Iron was supplied as Fe-EDTA at 1.8 mg per liter; the remaining five micro- nutrients were present at one-fifth the concentration of Hoag- land's solution (13). The standard uptake solution contained 0.5 mM KNO or 0.5 mM KCl plus 0.25 mm CaSO4, pH 6. For the experiments involving temperature, 6-methylpurine, or anaerobiosis vari- ables, the uptake solutions also contained 1 mm Na 2-[N- morpholino]ethanesulfonate as a buffer and the bactericide chloramphenicol at 20 jtg mP. Except where indicated, solu- tions were continuously aerated and the temperature was maintained at 30 C. Solutions were changed hourly and nitrate, chloride, and potassium uptake determined from depletion of the solutions. Five plants per 70 ml were employed for each replication. Four of these five-seedling units were used as replicates for each treatment and the data presented are the averages of the four replicates. Immediately after placing the plants in the uptake solutions, the mesocotyls were excised below the first node 3 cm above the seed. Exuding vascular sap was collected continuously with 100-,ul glass capillaries. Estimates of the amounts col- lected hourly were obtained by weight. The exudate collected from each set of five plants were dispensed into vials contain- 148 https://plantphysiol.org Downloaded on May 26, 2021. - Published by Copyright (c) 2020 American Society of Plant Biologists. All rights reserved.
Transcript
Plant Physiol. (1975) 56, 148-156
Nitrate Translocation by Detopped Corn Seedlings'Received for publication November 19, 1974 and in revised form March 25, 1975
FERNANDO N. EZETA AND WILLIAM A. JACKSON2Department of Soil Science, North Carolina State University, Raleigh, North Carolina 27607
ABSTRACT
Five-day-old seedlings of corn (Zea mays L.) grown withoutnitrate were decapitated and exposed to 0.5 mM KNOs or 0.5mM KCI in aerated solutions at 30 C. Uptake of nitrate, chlo-ride, and potassium was determined by replacing solutionshourly and measuring their depletion. Translocation of theseions and of organic nitrogen was determined by hourly analy-sis of the vascular exudate. Nitrate reduetion was estimated bythe difference between nitrate uptake and nitrate recovered inthe tissue and exudate. Nitrate uptake exhibited its usual pat-tern of apparent induction resulting in the development of anaccelerated uptake phase. Chloride uptake remained fairlyconstant throughout the experimental period. Translocation ofnitrate increased progressively for at least 7 hours whereas chlo-ride translocation reached a maximum about the 3d hour andthen declined to a lower rate than nitrate translocation. Nitrateuptake and translocation were restricted by anaerobiosis, by 20and 40 C relative to 30 C, and by 0.05 mM 6-methylpurine, anRNA-synthesis inhibitor. Accumulation, reduction and trans-location of nitrate had different sensitivities to all these factors.The effect of 0.05 mM 6-methylpurine was more detrimental tonitrate translocation and nitrate reduction than to nitrate up-take.
Ambient nitrate, relative to chloride, enhanced the exuda-tion volume and the translocation of organic nitrogen within 4hours from initiation of the experiments. Translocation of ni-trate and organic nitrogen decreased shortly after removal ofexternal nitrate. The higher rates of organic nitrogen transloca-tion which occurred during nitrate uptake indicates either (a)rapid translocation of amino acids synthesized from the enter-ing nitrate, or (b) an accelerated rate of protein turnover anda resulting enhancement in translocation of endogenous aminoacids.
Following its absorption by root tissue, nitrate may be re-duced and assimilated, stored in vacuoles, or translocated tothe shoots. That deposited in the vacuoles of root cells, how-ever, may be only slowly available for translocation (17), formaintenance of net nitrate reductase synthesis (17), and fornitrate reduction (9). Relatively high activities of nitratereductase have been determined in the apical region of corn
'Paper No. 4491 of the Journal Series of the North CarolinaAgricultural Experiment Station, Raleigh, N.C. These investigationswere supported in part by the United States Atomic Energy Com-mission, Grant AT-(40-1)-2410.
'Present address: Centro Internaciona de la papa, Apartado5969, Lima, Peru.
roots (10, 29, 32), and experiments with 'N indicate that sub-stantial nitrate reduction and translocation of the reducednitrogen can occur in corn roots (16). The capacity of the rootsystem to reduce nitrate affects the proportion of enteringnitrate which is released to the translocation stream (34). Therelative rate of nitrate translocation may therefore be affectedby the rate of uptake, the induction and activity of nitratereductase, and the rate of deposition in the vacuoles of theroot cells.The purpose of the present investigations was to examine
some of the factors which regulate nitrate translocation to theshoots. Detopped, dark-grown corn seedlings were employedin which an accelerated rate of nitrate uptake develops after aninitially slow uptake rate (17, 18). Data are presented to sup-port the concept that continuous nitrate uptake is required tomaintain significant translocation of nitrate. In addition, thetranslocation of organic nitrogen was strongly dependent uponcontinuous nitrate uptake.
MATERIALS AND METHODS
The procedures were, in general, similar to those describedby Jackson et al. (17). All experiments were conducted withcorn (Zea mays L.) hybrid DeKalb XL45. Four days aftergermination in darkness with 0.1 mm CaSO, the secondaryroots were excised and five seedlings were assembled in aPlexiglas holder especially designed to fit the top of a 75-mlTaylor tube. Seedlings were then placed in a pretreatmentsolution for 24 to 36 hr in darkness prior to initiating the ex-periment. The pretreatment solutions contained per liter 0.25mM (NH)2SO,, 0.6 mmYK2SO4, 0.4 mM KH2PO4, 1.6 mMMgSO4, 0.8 mm CaSO4, and 250 mg CaCO,. Iron was suppliedas Fe-EDTA at 1.8 mg per liter; the remaining five micro-nutrients were present at one-fifth the concentration of Hoag-land's solution (13).The standard uptake solution contained 0.5 mM KNO or
0.5 mM KCl plus 0.25 mm CaSO4, pH 6. For the experimentsinvolving temperature, 6-methylpurine, or anaerobiosis vari-ables, the uptake solutions also contained 1 mm Na 2-[N-morpholino]ethanesulfonate as a buffer and the bactericidechloramphenicol at 20 jtg mP. Except where indicated, solu-tions were continuously aerated and the temperature wasmaintained at 30 C. Solutions were changed hourly and nitrate,chloride, and potassium uptake determined from depletion ofthe solutions. Five plants per 70 ml were employed for eachreplication. Four of these five-seedling units were used asreplicates for each treatment and the data presented are theaverages of the four replicates.
Immediately after placing the plants in the uptake solutions,the mesocotyls were excised below the first node 3 cm abovethe seed. Exuding vascular sap was collected continuouslywith 100-,ul glass capillaries. Estimates of the amounts col-lected hourly were obtained by weight. The exudate collectedfrom each set of five plants were dispensed into vials contain-
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ing 5 ml of distilled H.0 and frozen until analysis could beperformed. Unless otherwise indicated, the term "transloca-tion" is used herein to refer to the material recovered in thevascular exudate. For nitrate, this is an underestimation of thequantity transported out of the root system because some
nitrate accumulated in the scutella and the 3-cm mesocotylsegment. In a number of experiments under standard con-ditions (0.5 mm KNO., 30 C), the exudate contained 72 to 82%of the total nitrate in the seed, mesocotyl, and exudate. Theamount of chloride, potassium, and organic nitrogen in theexudate includes that derived from the seed as well as thattransported out of the root system. Chloride and potassiumappearance in the exudate was strongly dependent upon thesolution bathing the roots, suggesting that direct translocationout of the root system was largely controlling the rate. As willbe shown below, organic nitrogen appearance in the exudatewas also strongly dependent on the composition of the ambientsolution.
After the appropriate exposure period, the tissue was rinsed,blotted, weighed, and frozen. It was then homogenized withmethanol-chloroform-water (13:4:3). In some instances, whenonly nitrate, chloride, and potassium were to be determined,the tissue was extracted with hot water. There was no differencebetween the two procedures for these three constituents.Nitrate in the nutrient solutions, exudate and root, seed, andmesocotyl extracts was analyzed by a nonautomated modifica-tion of the method of Lowe and Hamilton (26). Chloride was
determined by titration with a Cotlove chloridometer. Potas-sium was determined flame photometrically using lithium as
an internal standard. Soluble organic nitrogen was measuredin the exudate and in tissue extracts by the ninhydrin reaction,following the procedure of Yemm and Cocking (35), usingfreshly made ammonium sulfate solutions as standards.The quantity of nitrate reduced during the experiments was
calculated as the difference between the amount taken up andthe amounts accumulated in the tissue plus that in the exudate.All data presented, including that in other plant parts and inexudate, are based on a unit weight of fresh root tissue and are
the means of four replicates. Smoothed curves were fitted tothe cumulative uptake data by eye. The rates, shown in theinserts of the figures, are means of the four replications foreach measurement period and are plotted at the midpoints ofthese periods.
RESULTS
Uptake and Translocation of Nitrate and Chloride and Ef-fect of Anaerobiosis. Cumulative nitrate uptake (Fig. 1) showedits characteristic increase in rate leading to an acceleratedphase (cf., 17). Cumulative chloride uptake (Fig. 1) was es-
sentially linear, and the uptake of both ions was greatly re-
stricted by anaerobiosis. During the initial period of exposure
to the solutions, nitrate uptake rates were slightly less thanchloride uptake rates (Fig. 1, cf. Fig. 11), but the rates of ni-trate uptake were higher than those observed for chloride up-
take after the second hr. The rates of nitrate translocation in-creased steadily while chloride translocation rates increasedat first and then declined slightly after 2 hr (Fig. 2). In the ab-
sence of 02, little nitrate was translocated and the rate of chlo-ride translocation decreased progressively.A decrease in organic nitrogen translocation was observed
after the 3rd hr in KCl while the plants in KNO. maintainedhigher rates of organic nitrogen translocation (Fig. 3). In bothtreatments, lack of 02 resulted in a marked decline in organic
nitrogen translocation. The relative proportions of absorbed
nitrate that were translocated, accumulated, or reduced by the
149
roots was also strongly affected by the N, treatment. Trans-location (nitrate in seed + mesocotyl + exudate), as a percent-age of the amount absorbed (33.92 and 6.75 umoles g' for the
aerated and N2 treatments, respectively) decreased from 32%in air to 19% in N2. Reduction decreased from 50% to 42%.In contrast, accumulation of nitrate in the root tissue increasedfrom 18% to 39%.
la
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FIG. 1. Nitrate and chloride uptake by detopped seedlings from0.5 mM KNOs or KCI, and the effect of anaerobiosis. Solutions were
changed hourly and uptake determined by measuring loss fromsolution.
0
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FIG. 2. Nitrate and chloride translocation to the vascularexudate of detopped seedlings exposed to 0.5 mMKNO0 or KCI, and
the effect of anaerobiosis.
Plant Physiol. Vol. 56, 1975
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Flo. 3. Organic nitrogen translocation to the vascular exudateof detopped seedlings exposed to 0.5 mM KNO3 or KCI, and theeffect of anaerobiosis.
Tranlocation of Previously Accumulated Niate. Seedlingswere exposed to three concentrations (0.5, 2.5, and 15 mM) ofKNO. for 5 hr. At the end of this initial period a group ofseedlings was harvested. The remainder were transferred to 0.5mM KCI for an additional 5 hr. During the pretreatment, in-creasing the solution concentration from 0.5 mm to 2.5 mmKNO. resulted in an enhancement of nitrate translocation (Fig.4). Only a slight further increase occurred as the ambient con-centration increased from 2.5 mm to 15 mM KNO,. Nitratetranslocation sharply decreased during the first hr in 0.5 mMKCl with all three KNO. pretreatments (Fig. 4). Very lowtranslocation rates occurred by the end of the 5-hr chase pe-riod (Fig. 4) although significant amounts of nitrate were stillpresent in the roots at this time (Table I). No nitrate was re-covered in the ambient KCI during the chase period. For the0.5 and 2.5 mm KNO, pretreatments, the quantity of nitratelost from the root tissue during the chase period was greaterthan the quanity recovered in the exudate (Table I). For the15 mM KNOs pretreatment, the nitrate lost from the root tis-sue was less than that recovered in the exudate (Table I), indi-
cating movement out of the seed or mesocotyl cells into theexudate. Since nitrate in the mesocotyl and seed was not de-termined in this experiment the amount of nitrate reductionduring the 51 to 10-hr period is not known exactly. With cer-tain assumptions, calculations can be made to show that rela-tively little nitrate was reduced during this time. For the 0.5mM KNOS pretreatment, the assumptions are (a) nitrate accu-mulated in seed + mesocotyl during the 0- to 5-hr pretreat-ment was 33% of that accumulated in the exudate (cf. "Mate-rials and Methods"), and (b) all of the previously accumulatedseed + mesocotyl nitrate was subsequently reduced during the5- to 10-hr chase period. With these assumptions, the nitratereduced during the chase period was 2.9 pmoles g'. This esti-mate is a maximal value and is small compared with reductionvalues around 15 /Amoles g1 obtained in a number of other ex-periments over similar time periods with nitrate present in theambient solution. Similar calculations for the 2.5 and 15 mmKNO3 pretreatments (for which the relative nitrate distributionbetween mesocotyl + seed versus exudate is not known) re-
0 1 2 3 4 5 6 7 8 9 10HOURS
FIo. 4. Nitrate translocation to the vascular exudate duringexposure for the first 5 hr to three KNOs concentrations and forthe second 5 hr to 0.5 mm KCI.
Table I. Accumulation and Translocation of NitrateThe data represent nitrate accumulated in root tissue and nitrate translocated to the exudate of 5-day-old detopped corn plants
during exposure for 5 hr to three concentrations of ambient KNO3 and during a subsequent 5-hr period in 0.5 mm KCl.
T Root Nitrate Nitrate in Exudate Treatment Root Nitrate Nitrate in Exudate ofROOtS5 tote Deposited inContent at 5 Hr 0 to 5 Hr 5 to 10 Hr Content at 10 Hr 5 to 10 Hr 10 Hr Mesocotyl or Seed'
FIG. 5. Organic nitrogen translocation to the vascular exudateduring exposure for the first 5 hr to three KNO3 concentrations andfor a second 5 hr to 0.5 mim KCI.
veal values of less than 5 pmoles g-1 for maximal nitrate reduc-tion during the chase period. The evidence therefore indicatesthat nitrate reduction, as well as nitrate translocation, was sig-nificantly curtailed in the absence of ambient nitrate.Toward the end of the 5-hr pretreatment, translocation of
organic nitrogen was increased slightly by the two higher KNO,concentrations (Fig. 5). After transfer to KCI, the organic ni-trogen translocation rates declined steadily, although not asrapidly as those of nitrate (cf. Fig. 4).
Temperature Effects on Nitrate Uptake and Translocation.Larger total amounts of nitrate (Fig. 6) and potassium (Fig. 7)were taken up at 30 C than at 20 C or 40 C. Uptake of bothions was restricted more at 20 C than at 40 C but at both ad-verse temperatures the tendency was for the nitrate uptakerate to increase with time (Fig. 6). Translocation of both ni-trate and potassium was strongly restricted at 20 C and 40 C(Figs. 8 and 9), and potassium translocation rates exceedednitrate translocation rates at all three temperatures throughoutthe experiment. Uptake and translocation of nitrate were af-fected more adversely by the two temperature extremes thanwere uptake and translocation of potassium. Translocation oforganic nitrogen was restricted more at 20 C than at 40 C (Fig.10) as was translocation of endogenous chloride (data notshown).
Striking decreases in exudate volume and in amount andpercentage of nitrate accumulated in the exudate and seed +mesocotyl were observed at the two temperature extremes (Ta-ble II). In spite of a marked decrease in uptake, nitrate accu-mulation in the roots was unaffected at 40 C. At 20 C, onlyhalf as much nitrate accumulated in the roots as at 30 C butthis constituted a greater percentage of that taken up. Reduc-tion was restricted at 20 C and 40 C, but the relative propor-tion reduced at both extremes was greater than at 30 C. Valuesof Qlo, calculated for the 20 to 30 C interval, were significantlyhigher for nitrate translocation and exudate volume (4.6) thanfor nitrate uptake, accumulation in the roots, or reduction
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Effect of temperature on nitrate uptake from 0.5 mm
1 4HOURS
FIG. 7. Effect of temperature on potassium uptake from 0.5 amiKNOs.
(Table II). The Q1o values for translocation of endogenouschloride and organic nitrogen were 2.1 and 2.4, respectively,whereas potassium uptake and translocation had Q.o valuesof 1.9 and 2.9, respectively.
Effect of 6-Methylpurine on Uptake and Translocation ofNitrate, Chloride, and Potassium. Previous investigations (17)
P-lant Physiol. Vol. 56, 1975 151
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FIG. 8. Effect of temperature on nitrate translocation to thevascular exudate of detopped seedlings exposed to 0.5 mm KNO3.
-30 - 6-z. 5-
41~~4
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purne an 0niio fRAsnhss(0.A .5m,ti
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FIG. 9. Effect of temperature on potassium translocation to thevascular exudate of detopped seedlings exposed to 0.5 nmm KNOs.
revealed that nitrate uptake was restricted by 0.5 mm 6-methyl-purine, an inhibitor of RNA synthesis (20). At 0.05 mm, thiscompound largely eliminated development of the acceleratedrate of nitrate uptake (Fig. 1 1). It also restricted chloride (Fig.11) and potassium (Fig. 12) uptake, but significant uptake ofall three ions was still evident after 6 hr exposure to the in-hibitor. Translocation of these ions, on the other hand, was
Plant Physiol. Vol. 56, 1975
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=L 0~40500 |- 9 °z
0: 0O 2 3 4 56
o1 HOURSci, 400z
TEMPERATURE
7 20 C300 0 30 C
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FiG. 10. Effect of temperature on translocation of organicnitrogen to the vascular exudate of detopped seedlings exposed to0.5 mm KNO3.
Table II. Nitrate Metabolism in Tissue and ExudateThe data represent exudation volume, nitrate uptake, nitrate
accumulated in tissue and exudate, and nitrate reduced by 5-day-old detopped corn plants during exposure for 6 hr to 0.5 mm KNO3at three temperatures. Numbers in parentheses refer to percentageof total nitrate uptake at each temperature.
AccumulatedTemp. Exudate Uptake in Exudate + Accumulated ReducedVolume Utk Seed+ in Roots
curtailed completely by this time (Figs. 13 and 14). The effectof the inhibitor on nitrate translocation was evident during the1st hr while chloride translocation appeared to be affectedslightly later (Fig. 13). Potassium translocation from KCI wasrestricted earlier by 6-methylpurine than was potassium trans-location from KNO3 (Fig. 14). How 6-methylpurine exertsthese complex effects on uptake and transport of individualions is not clear. It is clear, however, that nitrate translocationwas more restricted than nitrate uptake and this difference insensitivity of the two processes to the inhibitor was also evi-dent with chloride and potassium.
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Neither nitrate nor chloride accumulation in the root tissuewas depressed by 6-methylpurine; instead, a slight increase intheir nitrate and chloride content was observed. At the end ofthe experiment, nitrate accumulation in the roots relative to
nitrate uptake, was increased by 6-methylpurine while its rela-tive reduction was strongly decreased (Table III).
Cumulative exudation patterns as affected by nitrate orchloride and by the presence of 6-methylpurine are shown in
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presenc(closedym)0o05mM 6MP
0
NITRATE 0 0
CHLORIDE
00 2 3 4 5 6 7
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FiG. 11. Nitrate (circles) and chloride (squares) uptake from
0.5 mm KNO& or KCI as affected by absence (open symbols) or
presence (closed symbols) of 0.05 mm 6-methylpurine.
HOURS
Fio. 12. Potassium uptake from 0.5 mm KNO. (circles) or 0.5mM KC1 (squares) as affected by absence (open symbols) or presence(closed symbols) of 0.05 mm 6-methylpurine.
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0s.5mM 6MP0-J - +10 -
sm s NITRATE 0 @CHLORIDE0
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Fie. 13. Nitrate (circles) and chloride (squares) translocationto the vascular exudate of detopped seedlings exposed to 0.5 mmiKN0a or KCI in the absence (open symbols) or presence (closedsymbols) of 0.05 mm 6-methylpurine.
40w .8
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2 ;=
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0Q05mM 6MP
20-NITRATE 0 0CHLORIDE0
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Ficy. 14. Potassium translocation to the vascular exudate ofdetopped seedlings exposed to 0.5 mm KNOa (circles) or 0.5 mmKCl (squares) in the absence (open symbols) or presence (closedsymbols) of 0.05 fmm 6-methylpurine.
Plant Physiol. Vol. 56, 1975 153
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Table III. Nitrate Metabolisni in Tissue anid ExYudateThe data represent nitrate uptake, nitrate accumulated in tissue
and exudate, and nitrate reduced by 5-day-old detopped cornplants during exposure for 7 hr to 0.5 mm KNO3 in the presenceand absence of 0.05 mm 6-methylpurine. Numbers in parenthesisrefer to percentage of total nitrate uptake for each treatment.
AccumulatedTreatment Uptake in Exudate + Accumulated ReducedMesocotyl + in Roots Reud
Seed
.sinole g-l
0.5 mM KNO 57.6 17.1 16.5 24.0(29.7) (28.5) (41.7)
FIG. 15. Exudation volume of detopped seedlings exposed to0.5 mM KNO3 (circles) or 0.5 mM KCI (squares) in the absence(open symbols) or presence (closed symbols) of 0.05 mm 6-methyl-purine.
Figure 15. Exudation rates clearly were enhanced with nitraterelative to chloride after the 3rd hr. The exudation rate in thepresence of nitrate was depressed by 6-methylpurine after the3rd hr whereas the rate was depressed after the 2nd hr withKCI.
DISCUSSION
Nitrate Uptake and Translocation. Increasing rates of ni-trate uptake were observed upon first exposure of the roots tonitrate (Figs. 1, 6, and 11). Potassium uptake rates were ini-tially higher than nitrate uptake and either remained relativelyconstant (Fig. 7) or increased slightly (Fig. 12). Once the ac-celerated phase of nitrate uptake had developed, its hourly
rate was nearly equal to that of potassium (Figs. 6 versus 7;11 versus 12). During the time period of these experiments, noconsistent difference in potassium uptake was evident betweenthe nitrate and chloride treatments (Fig. 12) although differ-ences in favor of KNO3 do develop after 8 to 10 hr (unpub-lished data).The percentage of absorbed nitrate which was recovered in
the xylem exudate increased steadily, reaching 25 to 30% ofthe hourly uptake rate after 6 to 7 hr. The concentration ofnitrate in the exudate reached 15 to 20 mm. Even higher con-centrations of nitrate in xylem exudate have been reported forother species in longer term experiments (7, 25). Some nitratewas detected in the mesocotyl and attached seed of nitrate-exposed plants. Movement of ions out of the xylem vesselsinto the adjacent tissue in the stem or shoots or in the upperparts of roots has been indicated previously (22, 23).
Nitrate translocation rapidly decreased when nitrate uptakewas abolished by removal of ambient nitrate (Fig. 4; cf. 17).Whether preloading the root tissue to concentrations substan-tially greater than 18 tsmoles g-' (Table I) would prevent thisimmediate decline cannot be ascertained from the presentdata. Under the experimental conditions reported here, itseems clear that much of the nitrate in the root tissue was in astorage pool and that the rate of removal from this pool wasnot rapid enough to sustain sizeable nitrate translocation rates.
Nitrate transport from the solution into the xylem is againstan electrochemical gradient (12), although the location of thepumping mechanism is not precisely known. Nitrate transloca-tion exhibited dependency on aerobic conditions (Fig. 2) andtemperature (Fig. 8). Moreover, it was inhibited by the pres-ence of 0.05 mim 6-methylpurine (Fig. 13). In each instance,the inhibiting treatments resulted in a decrease in quantities ofnitrate translocated as a percentage of that taken up (TablesII and III). In contrast, the quantities accumulated by the rootsas a percentage of uptake were increased. The data indicate agreater sensitivity of the nitrate translocation process to al-terations in aerobic metabolism, and possibly to protein syn-thesis (24), than either nitrate uptake or nitrate accumulationin the root cells.When plants were exposed to nitrate, the exudation volume
was invariably higher than when exposed to chloride (Fig. 15).This has been a consistent observation in a number of otherexperiments not reported here, the difference always beingnoted by the 3rd or 4th hr. An enhancing effect of nitrate overchloride on exudation volumes has been previously reported (3,31). Nitrate also enhanced the exudation volumes relative toother forms of nitrogen (27, 28). Xylem exudation is a func-tion of the osmotic pressure difference between the xylem sapand the root-bathing solution and it also depends on the hy-draulic conductivity of the root tissue (15, 22). For the ob-served difference in exudation (Fig. 15) to occur, exposure ofthe roots to nitrate as opposed to chloride must have inducedthe deposition of larger amounts of osmoticum in the xylem,or it must have enhanced the hydraulic conductivity. It is pos-sible that both effects occur at the same time. In the event,such changes had to occur fairly soon after exposure to theuptake solutions because we have consistently detected thedifferences in exudation rates within 4 hr.
After the first few hr, more inorganic nitrogen was recov-ered in the xylem exudate of seedlings exposed to KNO3 solu-tion than those exposed to KCl (Fig. 3); and transferring theplants from KNOS to KCI resulted in a decreased rate of or-ganic nitrogen translocation (Fig. 5). Hence, continuous nitrateuptake was required for sustained organic nitrogen depositionin the exudate. Organic nitrogen translocation, similar to ni-
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trate translocation, was dependent upon solution areration(Fig. 3); it was restricted at 20 C and 40 C (relative to 30 C,Fig. 10), and it was strongly inhibited by 0.05 mm 6-methyl-purine (data not shown). It is not yet known whether the en-hancement in organic nitrogen translocation in the presence ofambient nitrate resulted from translocation of organic nitrogensythesized from the entering nitrate or from the nitrate caus-ing an increased translocation from endogenous organic nitro-gen sources. Enhanced translocation of cations under nitratenutrition has been frequently reported (1, 3, 5, 8, 19, 21). Withthe detopped corn seedlings used in the present investigations,the effect of nitrate on potassium translocation was not ob-served until 4 or 5 hr elapsed (Fig. 14) and in some experi-ments it took longer. The effect of nitrate on organic nitrogentranslocation has been consistently observed prior to the 4thhr.By applying suction tension to the cut end of the stump of
detopped plants it has been shown that an increased flux ofwater across the root results in enhanced rates of translocationof nitrate and potassium (2). It is possible that removal ofthese ions from the xylem by a constant flushing of the vesselsprevents theeir backward leakage into the cells of the stelarparenchyma (6). Alternatively, enhanced removal of ions up-ward in the xylem with the increased water flux could main-tain a more efficient active translocation mechanism whichotherwise might be inhibited by high ion concentrations (14).If either of these postulates is valid, it follows that the nitrate-stimulated water flux (the mechanism of which is unknown atpresent) would serve to enhance the translocation of mosttransportable ions and organic substances.
Nitrate Reduction. About 40% of the absorbed nitrate wasreduced after 6 or 7 hr under the standard conditions of 0.5mM KNOS at 30 C (Tables II and III). Nitrate reductase is in-duced in corn roots shortly after exposure to nitrate, most ofit in the apical portions (17, 29, 32). A nitrate reductase couldhave been induced in the scutella (32) even though only verylittle nitrate accumulated there in the present experiments; itis not possible to say with certainty where the bulk of the ni-trate reduction took place. Nitrate reduction, as a percentageof nitrate taken up, was severely decreased by 6-methylpurine(Table III), less so by anaerobiosis (cf. "Results"), and wasslightly increased by 20 C and 40 C relative to 30 C (Table II).It is possible that the two potential sites of nitrate reduction(roots and scutella) were not equally sensitive to the imposedadverse conditions.
Basal regions of roots are able to absorb substantial quanti-ties of nitrate (10; Volk and Jackson, unpublished data), andan inhibitor of nitrate reductase is present in the region 2 to 3cm above the root apex (33). It follows that the apical cm maybe active in nitrate reduction whereas the basal and major por-tion may be more active in accumulation and translocation.Nitrate accumulation, translocation, and reduction have beenvisualized as competitive processes (4, 30, 34); the present re-sults further suggest that the degree of dominance of any oneof these processes is not uniformly distributed along the roots.
In the absence of external nitrate, the processes of nitratetranslocation and reduction will depend on the supply of ni-trate from a storage pool. Removal of the external nitrate re-sulted in only small amounts being reduced in a subsequent5-hr period in KCl (Table I). The concept of a short life ni-trate reductase-inducing pool, dependent on continuous nitratesupply (11, 9), appears applicable to corn roots because thereis a net decay of the enzyme soon after removal of nitrate fromthe medium (17, 29) even though the tissue still contains ap-preciable nitrate. Similarly, nitrate translocation rates decreased
immediately after removal from the nitrate solutions (Fig. 4).The data illustrate that storage nitrate was effectively isolatedfrom both the translocation pathway and the reduction path-way. The implication is that export of nitrate from root cellvacuoles was quite restricted under these experimental condi-tions.
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