Plant Physiol. (1986) 82, 946-9510032-0889/86/82/0946/06/$0 1.00/0

Nitrogen Nutrition and Xylem Sap Composition of Peanut(Arachis hypogaea L. cv Virginia Bunch)'

Received for publication June 18, 1986 and in revised form August 21, 1986

MARK B. PEOPLES*, JOHN S. PATE, CRAIG A. ATKINS, AND FRASER J. BERGERSENCommonwealth Scientific and Industrial Research Organization, Division ofPlant Industry, GPO Box1600 Canberra, ACT, Australia, 2601, and Botany Department, University of Western Australia,Nedlands, WA, Australia, 6009

ABSTRACT

The principal forms of amino nitrogen transported in xylem werestudied in nodulated and non-nodulated peanut (Arachis hypogaea L.). Insymbiotic plants, asparagine and the nonprotein amino acid, 4-methyl-eneglutamine, were identified as the major components of xylem exudatecollected from root systems decapitated below the lowest nodule or abovethe nodulated zone. Sap bleeding from detached nodules carried 80% ofits nitrogen as asparagine and less than 1% as 4-methyleneglutamine.Pulse-feeding nodulated roots with `5N2 gas showed asparagine to be theprincipal nitorgen product exported from N2-fixing nodules. Maintainingroot systems in an N2-deficient (argon:oxygen, 80:20, v/v) atmospherefor 3 days greatly depleted asparagine levels in nodules. 4-Methyleneglu-tamine represented 73% of the total amino nitrogen in the xylem sap ofnon-nodulated plants grown on nitrogen-free nutrients, but relative levelsof this compound decreased and asparagine increased when nitrate wassupplied. The presence of 4-methyleneglutamine in xylem exudate didnot appear to be associated with either N2 fixation or nitrate assimilation,and an origin from cotyledon nitrogen was suggested from study ofchanges in amount of the compound in tissue amino acid pools and inroot bleeding xylem sap following germination. Changes in xylem sapcomposition were studied in nodulated plants receiving a range of levelsof 'IN-nitrate, and a 'IN dilution technique was used to determine theproportions of accumulated plant nitrogen derived from N2 or fed nitrate.The abundance of asparagine in xylem sap and the ratio of aspara-gine:nitrate fell, while the ratio of nitrate:total amino acid rose as plantsderived less of their organic nitrogen from N2. Assays based on xylemsap composition are suggested as a means of determining the relativeextents to which N2 and nitrate are being used in peanuts.

Despite the importance of peanut (Arachis hypogaea L.) as anoil or protein seed crop, relatively little research has been directedat the assimilation and metabolism of nitrogen in the species.The classic study ofFowden (7) identified the unusual substitutedamide, 4-methyleneglutamine, as the major form of nitrogentransported in xylem from roots to shoot and leaves of peanutseedlings, and a more recent study (23) confirmed this finding inshowing that both 4-Megln2 and the free acid 4-methyleneglu-tamic acid were synthesized rapidly from cotyledon nitrogenfollowing germination. Winter et al. (23) further showed that the

' Supported by the Australian Centre for International AgriculturalResearch (Project No. 8305).

2 Abbreviations: 4-Megln, 4-methyleneglutamine; 4-Meglu, 4-methy-leneglutamate; 4-Meglx, 4-methyleneglutamine + 4-methylenegluta-mate; NR, nitrate reductase.

levels of 4-Megln and 4-Meglu in tissue pools of soluble Ndeclined only gradually during plant growth, even under condi-tions of severe N deficiency, and prolonged 14CO2 feeding studiesprovided little or no evidence ofnet synthesis ofeithercompoundfollowing germination. This paper reassesses the nitrogen metab-olism of peanuts by identifying the principal forms of newlyassimilated nitrogen transported in xylem and determiningwhether analysis of xylem sap offers promise as a technique forevaluating the dependence of plants on symbiotically fixed orcombined forms of nitrogen. Use is made of nodule xylem sapcollection and '5N2 feeding to identify products of fixation, while'5N-nitrate feeding and NR assays are used to relate xylem sapcomposition to patterns of nitrate assimilation.

MATERIALS AND METHODSPLANT MATERIAL AND EXPERIMENTATION

Identification of the Reduced Forms of Nitrogen Exportedfrom the Root and Nodule (Water Cultured Plants). Seeds of thestudy species (Arachis hypogaea L. cv. Virginia Bunch) weregerminated in trays of sterilized sand (some uninoculated, othersinoculated at sowing with pure peat cultures ofRhizobium strainCB756; Group I; Agricultural Laboratories, Regents Park, NSW,Australia). Groups of four seedlings were transferred a few daysafter emergence to 3.5-L containers of aerated, N-free, one-tenthstrength Hoagland liquid culture solution and then grown in anaturally lit, temperature-controlled (27-30°C day, 20-22°Cnight) glasshouse. A week later 20 mm nitrate was introducedinto 5 of the 10 containers with uninoculated seedlings, and thisconcentration was maintained for the rest of the experiment.The remaining uninoculated plants and all nodulated plantsreceived no nitrogen during their growth. The level of the liquidin the culture pots containing inoculated plants was maintainedjustbelow the nodulated zone on the main root. Culture solutionswere replaced in all containers twice weekly until final harvest.No nodules developed on any uninoculated seedlings.Groups ofplants from each ofthe three treatments (symbiotic-

N, uninoculated-N, and +20 mm nitrate) were harvested at week6, and xylem exudates were collected as bleeding sap from plantsdecapitated at either the top of the root, at a site on the rootbelow the lowest nodule, or from detached nodules placed onmoistened filter paper in a Petri dish (13). All exudates wereimmediately frozen to prevent any potential breakdown ofamides or other amino acids and stored for analysis.

Water-cultured, nodulated plants (35-d-old) were used in a'5N2 feeding experiment, in which the lids of the culture con-tainers were sealed at the edge and around each stem withTerostat VII (Teroson G.m.b.H., Heidelberg, FRG), and theenclosed gas space above the nutrient liquid (approximately 250ml) was flushed for a few minutes with an Ar:02 gas mixture

946 www.plantphysiol.orgon April 7, 2019 - Published by Downloaded from

Copyright © 1986 American Society of Plant Biologists. All rights reserved.

XYLEM TRANSPORT OF NITROGEN IN PEANUT

(80:20, v/v) to remove atmospheric N2. The inlet and outletports were subsequently closed with rubber Suba Seals (WilliamFreeman, Barnsley, Yorkshire, UK) and 120 ml of gas wereremoved with a syringe. This was followed by the addition of 60ml '`N2 (99 atom % excess '5N) and a balance of air and O2 tomaintain 20% 02. The final Ar:N2 gas mixture of approximately1:1 was shown previously (16) not to interfere significantly witha nodulated root's capacity to fix N2. The root systems weremaintained in the '5N2-enriched atmosphere for 1.5 h beforebeing opened and flushed with air, with the exception of plantscollected at 20, 40, and 60 min after the commencement of '5N2-feeding. Plants were harvested at intervals up to 6 h (i.e. for 4.5h after opening). Xylem exudates were collected at 1.25, 2.75,and 5.75 h for a 15-min period prior to each harvest.

In a further experiment, sets of 20 (40-d-old) nodulated plantswere sealed in culture containers as above, and a moisturizedstream of either air or Ar:02 (80:20, v/v) was passed through theenclosed gas spaces of containers (1). The flushing of root systemswith an Ar.02 gas mixture does not affect the activity of thenodule's nitrogenase enzyme, as assessed by acetylene reductionassay, but suppresses ammonia production and the subsequentbiosynthesis of amino compounds (2). As a consequence there isa rapid run-down in the associated products of N2 fixation withtime (2, 16). The soluble-N pools of nodules from both control(air) and N2-deficient (Ar:02) peanut plants were extracted in80% (v/v) ethanol after 3 d of continuous Ar:02 treatment andtheir individual nitrogenous components separated and analyzed.

Study of Changes in the Composition of Translocated N andTissue-Soluble N Pools During Plant Development (Sand Cul-tured Plants). Seeds were sown in pots of sterilized sand in atemperature-controlled glasshouse and inoculated with the effec-tive Rhizobium strain CB756. Seedlings were watered daily andreceived N-free mineral nutrients throughout development (I15).Groups of 12 plants were harvested at intervals after sowing foridentification of the nitrogenous solutes in xylem exudate ortissue-soluble N pools. Root bleeding xylem saps were collectedfor up to 30 min following decapitation of the stems below thecotyledonary node. Plant tissues were separated into cotyledons,roots, and shoot, homogenized in cold 80% (v/v) ethanol, andtheir soluble components later partitioned between ether andwater prior to analysis ( 18).Study of the Effect of Nitrogen Nutrition on the Transport and

Assimilation of Nitrogen (Sand Cultured Plants). Nodulated sandcultured plants were maintained either on N-free nutrients, ortheir rooting medium flushed with '5N-nitrate (0.5-10 atom %excess '5N) culture solution (1, 2, 5, 10, 20, and 30 mm nitrate)replenished every 2 d from 14 d after sowing. The different levelsof nitrate were chosen to ensure that plants were grown over thecomplete range (0-100%) of symbiotic dependencies. Plantsfrom all treatments were harvested 8 weeks after sowing; rootxylem bleeding exudate was collected from 12 plants of eachnitrate regime. Plants were separated into nodules, roots, andshoots for dry weight measurements, Kjeldahl total N determi-nations, and analysis of the '5N enrichment by MS. The propor-tions of plant N coming from nitrate, N2, and cotyledonary Nwas estimated by an isotope dilution technique (14).A further group of peanut seedlings was fed with 2, 5, 10, or

20 mm nitrate, and the NR (EC 1.6.6.1) of roots or shoots wasextracted 6 weeks after sowing, by grinding (1 g: 3 ml and 1 g: 5ml, respectively) with ice-cold K-phosphate (50 mM, pH 8.5)containing 5 mM EDTA, 1% (w/v) casein, and 2.5% (w/v)insoluble PVP in a chilled mortar. Preliminary investigationsindicated that 20 mm cysteine or glutathione also had to beincluded in the extraction buffer to give the maximum yield ofstable NR activity. Enzyme activity of the supernatant wasdetermined immediately after centrifugation (20,000g for 10 minat 4°C) using the assay procedure described by Wallace (22).

Analysis of Nitrogenous Solutes. Concentrations of amidesand other amino acids in sap samples and tissue extracts weredetermined following their separation by an ion-exchange resinHPLC system based on lithium buffers and employing post-column ninhydrin detection (17). Ureides (allantoin and allan-toic acid) were either measured together as the phenylhydrazoneof glyoxylate (21), or separately on a Varian 5000 HPLC system(UV detector operating at 210 nm) employing an Aminex (Bio-Rad Chemical Division) HPX-87H column eluted at room tem-perature with 8 mM HSO4. Nitrate was determined by a salicylicacid method (6).

'5N Analysis of Plant Total N or Amino Compounds. Enrich-ment of '5N in labeled total N or in N of individual N soluteswas measured using a triple ion collector, dual inlet mass spec-trometer (VG SIRA 9; VG Isogas, Middlewich, Cheshire, UK),incorporating a direct inlet line from a hypobromite oxidationsystem (19). The nitrogenous solutes of the collected root xylembleeding saps, and the nodule-soluble N pools (extracted in 80%[v/v] ethanol [18]) were separated on a Beckman 118 aminoacid analyzer and the 'sN content of each compound assessedfollowing Kjeldahl digestion of relevant fractions of columneffluent and distillation of ammonia. Digestion and distillationprocedures in preparation for MS followed the techniques out-lined by Bergersen (4).

RESULTS

Principal Forms of Reduced Nitrogen in Xylem Saps Collectedfrom Peanut Roots and Nodules. Xylem exudates collected fromthree different locations in symbiotic plants (lower root belownodulated zone, detached nodules, and upper root above nodu-lated zone) and from the top of the root of non-nodulated plantsmaintained on N-free nutrients or 20 mM nitrate, showed distinctdifferences in the balance between principal nitrogenous solutes(Fig. 1). The concentration of total sap N varied between 70 4g/ml (lower root xylem sap) and 7 mg/ml (nodule bleeding sap).Asparagine and 4-Megln were the major components ofbleed-

ing (xylem) exudate collected from all parts of nodulated roots(Fig. 1), although the relative proportion ofthese two nitrogenoussolutes changed with the point of collection. Samples from thexylem of the lower root, presumed to contain only cycled N,recorded 44% N as asparagine and 20% as 4-Megln comparedwith 70% and 15%, respectively, for exudate from the top of thewhole nodulated root. The increased proportion of asparagine inthe upper root xylem was assumed to be due to an asparagine-dominated contribution of N from the nodules, and this wasborne out by the finding that 80% of nodule bleeding exudate Nwas in the form of this amide (<1% as 4-Megln). Total Nrecoverable by HPLC determinations of individual componentsaccounted for over 90% of the total sap N as determined byKjeldahl analysis, suggesting that there were no other major N-solutes in the xylem that were undetected by the analyticaltechniques used. Ureides (allantoin or allantoic acid) and 4-Meglu were not detected in any of the exudates.The root bleeding exudate of non-nodulated plants relying

solely on the N reserves ofcotyledons was dominated by 4-Megln(73% total N), with no other single amino compound represent-ing more than 10% of xylem N. When non-nodulated plantswere supplied with nitrate, however, there was a rise in therelative content of several amino acids, especially asparagine. 4-Megln accounted for only 32% of the amino-N of these sapsamples (Fig. 1).

Products of Symbiotic N2 Fixation as Identified by N2-Star-vation and '5N2-Feeding of Nodulated Root Systems. Whennodulated roots of peanut plants were continuously flushed withAr.02 (80:20) for 3 d, there was a substantial decline in thenodule concentrations ofasparagine (44% ofcontrol, maintained

947

www.plantphysiol.orgon April 7, 2019 - Published by Downloaded from Copyright © 1986 American Society of Plant Biologists. All rights reserved.

PEOPLES ET AL.

other

4-Megin

Asn

FIG. 1. The proportional composition ofamino compounds in xylemsaps collected from nodulated (no nitrate) and non-nodulated (zero or

20 mm nitrate) water-cultured Virginia Bunch peanut plants. Xylemexudates were collected as bleeding sap from plants decapitated at eitherthe top of the root, or at a site on the root below the lowest nodule, or

from detached nodules. Other compounds included Glu, Thr, Ser, Gly,Ala, Val, Cys, Met, Ile, Leu, Tyr, Phe, y-aminobutyric acid, His, Lys,and Arg. No 4-Meglu or ureide (allantoin and allantoic acid) was de-tected. Proportional compositions were determined from five replicatesamples; values varied with a SE of ±3%.

in air), glutamine (61% of control), and glutamate (32% ofcontrol). However, concentrations of other amino acids (partic-ularly 4-Megln, aspartate, and arginine), remained between 88and 100% of the air control plants. The fall in level ofasparaginefrom 40 to 17.5 ,mol/g fresh weight represented the largest singledrop for an amino acid in the nodule, suggesting a close associ-ation with symbiotic N2 fixation.

Figure 2 details the time-course of "N-enrichment and therelative abundance (percent of total N) of various nitrogenouscompounds in the nodule soluble-N pool and in root bleedingxylem exudate following labeling of intact nodulated plants with"5N2. Substantial '"N-enrichment was found in the asparagineand glutamine of nodule extracts (Fig. 2A) and in asparagine,aspartate, and glutamine in root xylem exudate collected justabove the nodulated zone (Fig. 2B). Considerable incorporationof "N also occurred into glutamate (from 2.6 to 12 atom %excess from 1.5 to 3 h after the commencement of "IN2-feeding)and alanine (between 3 and 4 atom % excess during the period1.5-6 h) in nodule extracts. Virtually no enrichment, however,was recorded for 4-Megln (Fig. 2), even up to 24 h after "N2-feeding (data not shown), despite the fact that this compoundrepresented a large proportion ofthe nodule soluble-N pool (Fig.2A). Asparagine represented between 76 and 96% of the total"N recovered in nodule extracts or xylem exudates, indicatingits predominant role in transport of currently fixed N to theshoot. Analysis of the distribution of '"N in the amide-N andamino-N of aspargine collected from nodules after 1 h of "N2-feeding showed a 4.7 atom % '"N in the amide group and a 2.7

10

5

z

E0

Plant Physiol. Vol. 82, 1986

- A Nodule ExtractU

0--o ~ sn

a a

/\ / Gin *-

/m,4-Megin

-A ____-A- A^~~~ ~I A,

Gin

4-

Mogin

Aen

0 2 4 6

Time after exposure to 1'N2 (hrs)FIG. 2. Time-course of incorporation of "N into various nitrogenous

components following a pulse feeding of "N2-gas to the nodulated rootsof water-cultured Virginia Bunch peanut plants. (A) the soluble N poolof nodule extracts, and (B) root bleeding xylem sap. Enclosed rootsystems were retained in a "N2 atmosphere (approximately 50 atom %excess "N) for 1.5 h before being flushed with air. The first subgroup ofplants were harvested 20 min after the commencement of "5N2 feedingand nodules were immediately extracted in 80% (v/v) ethanol. Xylemexudates were first collected at 1.25 h. Panels on the right-hand side referto the average amino acid composition (expressed on a % N basis) ofeither (A) nodule extracts, or (B) xylem exudate determined from fourreplicate samples. Values varied with a SE of ± 15 (A), and ±2% (B).

atom % '"N in the amino group, implying differences in the ratesof equilibration of "N with the precursor pools of amide andamino N supplying asparagine. Compartmentation with respectto the resident pools of soluble-N and the transport pool of thenodule was also apparent (Fig. 2) since "5N-enrichments of theamides in xylem sap were initially much higher, yet declined farmore rapidly than the soluble-N pool ofthe nodule. This impliedpreferential loading into xylem of metabolically active pools ofamino compounds in rapid equilibrium with the fixation process.Changes in the 4-Megin and 4-Meglu Content of Tissue Sol-

uble-N Pools and in the Composition of Xylem Translocated Nduring Plant Development. The data of Figure 3 depict changesin the levels of 4-Meglu and 4-Megln in cotyledons, roots andshoot during the first 72 d ofgrowth ofnodulated Virginia Bunchpeanut and relates the total plant content of 4-methylene substi-tuted compounds (denoted as 4-Meglx) to the total pool ofsoluble amino acids. Initially, levels of both 4-Meglu and 4-Megln were very high in cotyledons but declined thereafter ascotyledonary reserves were mobilized to other parts of the seed-ling. The 4-Meglu content of the shoot and 4-Megln in the rootdid not increase past d 20, although the level of 4-Megln contin-ued to increase in the shoot until around d 60. No 4-Meglu wasdetected in root (or nodule) extracts (Fig. 3). Total amount of

948

other Asp

Gin

4-

Megin

Asn

_ 100zat

la 80C

0

E 600U

1 40EaEj2 20

0

Gin IAsn

-other -

Gin -

Asp

Gln

other

VASP4-

Megin

Asn

I4-

Megin

Asn

IL Ii

etached -Cnodules

-1d

no N03nodulated plants

noN03 20mMN03non nodulated plants

II

r...)

www.plantphysiol.orgon April 7, 2019 - Published by Downloaded from Copyright © 1986 American Society of Plant Biologists. All rights reserved.

XYLEM TRANSPORT OF NITROGEN IN PEANUT

40

20

1-

4E

E

80

40

800

400 I

°'C.4-Meglx

20 40 60

Days after sowing

FIG. 3. Changes during growth and development of the VirginiaBunch cultivar of peanut plants in the tissue levels of (A) 4-Meglu, and(B) 4-Megln in cotyledons, roots and shoot, and (C) the total pool of 4-Meglx in relation to total plant amino acid content. Flowering com-menced 42 d after sowing and peg initiation began thereafter.

100

P

C,)cr

~0CL

00.

0U

.EE'5)x

801

60

40

20 _

20 40 60

Days after sowing

FIG. 4. Changes in the amino acid composition of root bleedingexudate during growth and development of nodulated Virginia Bunchpeanuts.

both compounds (4-Meglx) per plant remained fairly stablethroughout much of the experimental period, declining slightlyat the beginning of seed development (around 70 d after sowing).Pools of other free amino acids rose significantly during plantgrowth.Xylem sap analysis showed 4-Megln to be the principal amino

acid transported in xylem during the early seedling development(>80% of total sap N), but once an effective symbiosis had beenestablished, there was a progressive increase in the proportion ofxylem N as asparagine (Fig. 4). The relative contribution madeby aspartate and other protein amino acids to xylem translocated

N remained relatively unchanged between 16 and 76 d aftersowing (Fig. 4).Xylem Transport ofNitrogen in Nodulated Peanut Fed a Range

of Levels of '5N-Nitrate. Isotopic analysis of dry matter of plantsraised on different '5N-labeled nitrate regimes indicated an in-creasing dependence of the root, shoot and nodules upon nitrateas the level of applied nitrate was increased from 1 to 30 mm(Fig. 5A). At the highest level of nitrate (30 mM) the proportionof total N derived from fed nitrate was around 70% in the shootand root but only 46% for the nodules. Although the dependenceof the nodules upon nitrate was somewhat lower than the otherorgans, the shoot and root N content so dominated total plantN (nodule contribution fell from 3 to 0.6% of total plant N atthe highest nitrate level) that the proportion of plant N derivedfrom fed nitrate increased from 3.8% at 1 mm nitrate to 70.7%at 30 mm (Fig. 5B). Plants accumulated between 120.9 and 157.4mg N in the 8 weeks after sowing, and, assuming that the full Nreserves of the seed (42.4 mg N) had been available to the plant,all remaining plant N not attributable to the '5N-labeled nitratewas designated as originating from N2 fixation (Fig. SB).The xylem sap composition ofthe treatments (Fig. 6A) showed

a substantial decline in the relative asparagine content and acompensatory increase in sap nitrate as increased nitrate levelscaused plants to acquire less of their N from fixation. NRactivities of root and shoot extracts from peanuts fed a range ofnitrate levels (Table I) indicated that the nitrate reducing capacityof both organs increased significantly as root medium nitrateconcentrations were raised. Activities in the shoot, however, werehigher than those in roots at each nitrate dose, indicating adominant role of the shoot in nitrate assimilation. Changingrelative contributions of root and shoot nitrate reduction withincreasing nitrate levels were apparent in the compositionalrelationships depicted in Figure 6B. The relative abundance ofasparagine in xylem sap (asparagine-N: total amino acid N+nitrate-N, or asparagine-N:nitrate N) declined progressively asthe plant derived less of its organic N from N2, while the ratio of

0)._

0)

cCDL- zo- EC o

0

z

z

4-

P

1W,

80

60

401

20

U,

vv

80

60

40

20

1 0 20 30

N03 concentration in rootingmedium (mM)

FIG. 5. The (A) reliance ofvarious plant organs of nodulated VirginiaBunch peanuts on fed nitrate, and (B) the dependence of the whole planton N2 and nitrate as determined by a '5N isotope dilution technique.Plants were harvested after 6 weeks of growth on culture solutionscontaining "N-nitrate of known enrichment.

-_____________ other

4-Megin

Asn

/

AK shoot~~~~~~~~~~ I

root

- / nodulesin vA,;>'

1 0 20 30

\B\From added N03

From N2 fxation\

From cotyledonsI

1 ()( -

U' -.- -.- -.-

u -

949

www.plantphysiol.orgon April 7, 2019 - Published by Downloaded from Copyright © 1986 American Society of Plant Biologists. All rights reserved.

Plant Physiol. Vol. 82, 1986

(A

.0z

0

0)0.E0

0.

Coa.

n

E0

x

z

z

6 -

(Cl

Z0

r 7+

'0

c

Z Eo X

X4-0O oZr E

.(.

zz

0

zCo.

20 40 60 80 100

% plant N increment from N2

3020 10 5 2 1 0NO3 concentration in rooting medium

(mM)

FIG. 6. Changes in (A) the xylem sap composition of nodulatedVirginia Bunch peanuts fed a range of constantly maintained levels ofnitrate, and (B) the relationships between compositional features ofxylemsap and the dependence of plants on N2 fixation as opposed to nitratereduction. Other compounds depicted include Asp and those solutesindicated in the legend to Figure 1.

Table I. Influence ofNitrate Supply on the in Vitro Nitrate ReductaseActivities ofRoots and Shoots of42-d-Old Sand Cultured Peanuts

(Arachis hypogaea L. cv Virgina Bunch)NR Activity

Nitrate SuppliedRoot Shoot

mumol NO2 producedmm min' gI fresh wt2 3.7 7.55 6.2 9.810 8.7 19.920 11.8 30.0

nitrate-N to total amino acid N rose due to an increasing pro-portion of the fed nitrate escaping the reductase system of theroot.

DISCUSSION

The first stable product of N2 fixation in legume nodules isammonia (3), which is released from the bacteroids (5) to berapidly incorporated into the glutamine and glutamate pools of

the host cells of the nodule (10-12). Although the glutamine andglutamate components of peanut nodules were found to bereadily enriched with '5N following '5N2-gas feeding of nodulatedroots, little incorporation of '5N took place into the glutamine-derived 4-Megln (Fig. 2). With 4-Megln clearly not associatedwith the assimilation and transport of recently fixed N2, itspresence in xylem exudates of both symbiotic and nitrate-fedplants (Figs. 1 and 6), presumably represented the recirculationof a large pool of the compound synthesized at germination fromseed storage reserves ofN (23). A cotyledonary origin of4-Meglnwas substantiated in the present study by temporal changes in itslevels in cotyledons (Fig. 3B) and xylem sap (Fig. 4) duringgermination and by its dominance of xylem N in non-nodulatedplants maintained on N-free nutrients (Fig. 1). Following the netsynthesis of 4-Megln and 4-Meglu during early growth, the totalpool of these compounds within the plant remained relativelyconstant throughout much of the vegetative and early reproduc-tive phases of development (Fig. 3C) and declined only veryslowly during severe N-deficiency (23). It seems likely, therefore,that 4-Megln and 4-Meglu do not play a major role in the N-economy of peanuts, although 4-Megln may represent a largecomponent of transport N throughout much of early growth(Fig. 1). It is beyond the scope of the presept study to speculateupon the significance of the high levels of 4-Meglu and 4-Meglnfound; however, it has been suggested elsewhere that such non-protein amino acids may be involved with regulation of meta-bolic reactions or are important in protection of plants frompathogens or insect pests (8).

Asparagine was the principal carrier of newly assimilated '5Nin both nodule extracts and root bleeding xylem exudate (Fig. 2)following '5N2-feeding. It was also the major amino compound(80% of total N) in bleeding sap from detached nodules (Fig. 1)and was the main amino acid to disappear from the nodule-soluble N pool during a period of N2 starvation. It is difficult tototally reconcile the differences between the present investigationon effectively nodulated peanut plants and previous studies (7,23) which found very little asparagine in peanut xylem exudates.In the earlier study (7), 3-week-old plants were used, so it wasperhaps not surprising that asparagine assumed a minor role (seeFig. 4). The more recent study (23) reported that asparagineremained a small component ofxylem N up to 90 d after sowing.It is possible that cultivar or cultural differences may have favored4-Megln persistence; alternatively, treatments imposed by theprevious workers both prior to and during analysis might haveaffected amide recovery. The present paper and other unpub-lished results suggest that asparagine is invariably a major com-ponent of xylem exudates collected from a range of cultivarsgrown and analyzed in two different laboratories.

Asparagine appeared also to represent a transported productof nitrate reduction in non-nodulated plants (Fig. 1). However,an increased reliance ofnodulated plants on inorganic N resultedin a substantial fall in xylem levels of asparagine relative tonitrate (Fig. 6A). Although in vitro measurements ofNR activitymay not indicate the actual rate of nitrate assimilation in situ,NR is an inducible enzyme, the activity of which is related tothe flux of nitrate through metabolic pools (20). Assuming thatthe in vitro assays indicate the potential for nitrate reduction inplant tissue (22), the responses of the shoot and root reductasesystems to changing nitrate supply (Table I) appear to be con-sistent with the increased levels of nitrate detected in the xylemstream. Thus, the shoot seems to play a progressively moreimportant role in the reduction and assimilation of inorganic Nwith increasing nitrate levels in the rooting medium.

Since the bulk of the nitrogenous solutes of xylem sap arelikely to represent current products ofN uptake and assimilation,and since there are substantial differences in sap compositionbetween N2- and nitrate-fed peanut plants, it may be possible to

950 PEOPLES ET AL.

www.plantphysiol.orgon April 7, 2019 - Published by Downloaded from Copyright © 1986 American Society of Plant Biologists. All rights reserved.

XYLEM TRANSPORT OF NITROGEN IN PEANUT

devise some form of assay system based on xylem sap analyses,similar to those developed for ureide-exporting legumes (9, 14),to assess plant reliance on symbiosis in the presence of nitrate.Such an objective may not be as easily achieved with peanut asfor ureide-producers, however, because there is no N-solutespecific to either N2 fixation or nitrate assimilation. The use ofnitrate itself depends upon a correlation between the concentra-tion of this ion, its rate of uptake and the extent of storage,assimilation and reduction within the root. Nevertheless, it maybe feasible to use the relative amino acid and nitrate contents ofxylem exudates as indirect measures of symbiotic dependence.Three relationships which may be useful for peanut were exam-ined, namely, the proportion of sap N as asparagine, the ratio ofasparagine:nitrate, and the ratio of nitrate:total amino acid N.The first two parameters were shown to fall progressively andthe third to rise substantially with decreasing plant dependenceupon symbiosis (Fig. 6B). Of the three relationships selected, theproportion of asparagine in sap total N appeared to have themost linear response to changing reliance on N2 or nitrate. Asubsequent study (MB Peoples, JS Pate, unpublished data) com-paring xylem composition in 12-week-old Virginia Bunch peanutplants inoculated with the Rhizobium (CB756) used in this study,or with any one of a number of field isolated Rhizobium strainsfrom Thailand, showed xylem sap values for asparagine-N:totalsap N ranging from 0.85 to 0.88 for fully symbiotic plants to 0.3for nitrate-dependent plants, suggesting that this compositionalfeature might provide a reliable diagnostic tool for field meas-urement of symbiotic dependence. Other variables likely tocomplicate xylem sap assays such as plant genotype, plant age,site and mode of sap collection are currently under investigation.

Acknowledgments-The authors are greatly indebted to W. Wallace of WAITEAgricultural Research Institute of the University of Adelaide, South Australia, foridentifying the optimum conditions for extracting peanut nitrate reductase; G.O'Hara of Murdoch University, Western Australia, for provision of rhizobialcultures isolated from field-grown peanut plants in Thailand as part of ACIARProject No. 8329; and D. McNeil of the Western Australian Department ofAgriculture, Kununurra for provision ofseed stock. We also gratefully acknowledgethe skilled technical assistance of P. Sanford, E. Rasins and M. Unkovitch (Perth),and D. Hebb and D. Lilley (Canberra), and thank D. Waldie (Perth) and H. Tantala(Canberra) for the care and maintenance of plant material.

LITERATURE CITED

1. ATKINS CA, BJ SHELP, J Kuo, MB PEOPLES, JS PATE 1984 Nitrogen nutritionand the development and senescence of nodules of cowpea seedlings. Planta162: 3 16-326

2. ATKINS CA, JS PATE, BJ SHELP 1984 Effects of short-term N2 deficiency on N

metabolism in legume nodules. Plant Physiol 76: 705-7 103. BERGERSEN FJ 1965 Ammonia-an early stable product of nitrogen fixation

by soybean root nodules. Aust J Biol Sci 18: 1-94. BERGERSEN FJ 1980 Analysis of nitrogen fixation by direct means. In FJ

Bergersen, ed, Evaluating Biological Nitrogen Fixation. J Wiley & Sons,Chichester, UK, pp 65-110

5. BERGERSEN FJ, GL TURNER 1967 Nitrogen fixation by the bacteroid fractionof breis of soybean root nodules. Biochim Biophys Acta 141: 507-515

6. CATALDO DA, M HAROON, LE SCHRADER, VL YOUNGS 1975 Rapid colori-metric determination of nitrate in plant tissue by nitration of salicylic acid.Commun Soil Sci Plant Anal 6: 71-80

7. FOWDEN L 1954 The nitrogen metabolism of groundnut plants: the role of -y-methyleneglutamine and y-methyleneglutamic acid. Ann Bot 18: 417-440

8. FOWDEN L 1981 Non protein amino acids. In EE Conn, ed, The Biochemistryof Plants. A Comprehensive Treatise, Vol 7, Secondary Plant Products,Academic Press, New York, pp 215-247

9. HERRIDGE DF 1984 Effects of nitrate and plant development on the abundanceof nitrogenous solutes in root-bleeding and vacuum-extracted exudates ofsoybean. Crop Sci 25: 173-179

10. KENNEDY IR 1966 Primary products of symbiotic nitrogen fixation. I. Short-term exposures of serradella nodules to '5N2. Biochim Biophys Acta 130:285-294

11. KENNEDY IR 1966 Primary products of symbiotic nitrogen fixation II. Pulse-labelling of serradella nodules with '5N2. Biochim Biophys Acta 130: 295-303

12. OHYAMA T, K KUMAZAWA 1980 Nitrogen assimilation in soybean nodules. II.'5N2 assimilation in bacteroid and cytosol fractions of soybean nodules. SoilSci Plant Nutr 26: 205-213

13. PATE JS, BES GUNNING, LG BRIARTY 1969 Ultrastructure and functioning ofthe transport system of the leguminous root nodule. Planta 85: 11-34

14. PATE JS, CA ATKINS, ST WHITE, RM RAINBIRD, KC Woo 1980 Nitrogennutrition and xylem transport ofnitrogen in ureide-producinggrain legumes.Plant Physiol 65: 961-965

15. PATE JS, MB PEOPLES, CA ATKINS 1983 Post-anthesis economy of carbon ina cultivar of cowpea. J Exp Bot 34: 544-562

16. PATE JS, CA ATKINS, DB LAYZELL, BJ SHELP 1984 Effects of N2 deficiency ontransport and partitioning of C and N in a nodulated legume. Plant Physiol76: 59-64

17. PATE JS, MB PEOPLES, PJ STORER, CA ATKINS 1985 The extrafloral nectariesof cowpea ( Vigna unguiculata (L.) Walp.) II. Nectar composition, origin ofnectar solutes, and nectary functioning. Planta 166: 28-38

18. PEOPLES MB, JS PATE, CA ATKINS, DR MURRAY 1985 Economy of water,carbon, and nitrogen in the developing cowpea fruit. Plant Physiol 77: 142-147

19. PORTER LK, WA O'DEAN 1977 Apparatus for preparing nitrogen from am-monium chloride for nitrogen-15 determinations. Anal Chem 49: 514-516

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

21. TRIJBELS F, GD VOGELS 1966 Degradation of allantoin by Pseudomonasacidovorans. Biochim Biophys Acta 113: 292-301

22. WALLACE W 1986 The distribution of nitrate assimilation between the rootand shoot of legumes and a comparison with wheat. Physiol Plant 66: 630-636

23. WINTER HC, GK POWELL, EE DEKKER 1981 4-Methyleneglutamine in peanutplants: dynamics of formation, levels, and turnover in relation to other freeamino acids. Plant Physiol 68: 588-593

951

www.plantphysiol.orgon April 7, 2019 - Published by Downloaded from Copyright © 1986 American Society of Plant Biologists. All rights reserved.