Plant Physiol. (1981) 67, 37-420032-0889/81/67/0037/06/$00.50/0

Synthesis, Storage, and Utilization of Amino Compounds in WhiteLupin (Lupinus albus L.)'

Received for publication May 23, 1980

JOHN S. PATE, CRAIG A. ATKINS, DAVID F. HERRIDGE2, AND DAVID B. LAYZELL3Department of Botany, University of Western Australia, Nedlands, Western Australia, 6009

ABSTRACT

Changes in total N and in free amino compounds were foliowed duringgrowth of nodulated white lupin. Leaflets contained the greatest fractionof plant N but had lower proportions (1 to 4%) of their N in soluble aminoform than stem + petioles (10 to 27%) and reproductive parts (15 to 33%).Mobilization offree amino compounds from plant parts to fruits contnrbutedat most only 7% of the total N intake of fruits, compared with 50% inmobilization of other forms of N and 43% from fixation during fruiting.Asparagine was usually the most abundant free amino compound in plantparts, followed by glutamine and alanine. Valine, glycine, isoleucine, as-partic acid and -aminobutyric acid comprised the bulk of the remainingsoluble amino N. Composition of tissue pools of amino-N closely resembledthat of xylem and phloem exudates. Data on N flow and utilization werecombined with information on composition of transport fluids to quantifysyntheses, exchanges, and consumptions of asparagine, glutamine, asparticacid, and valine by organs of the 51- to 58-day plant. These aminocompounds carried 56, 29, 5, and 2%, respectively, of the N exported fromnodules and contributed in roughly commensurate proportions to transportexchanges and N increments of plant parts. There were, however, morethan expected involvements of glutamine and valine in mobilization of Nfrom lower leaves, of asparagine in xylem to phloem transfer, and ofaspartic acid in cycling of N through the root, and there was a less thanexpected participation of aspartic acid in xylem to phloem transfer and inphloem translocation to the shoot apex. The significance of these differ-ences is discussed.

In many plants, metabolism ofN is heavily biased towards thesynthesis, transport, and storage of specific solutes, such as amides,ureides, or non-protein amino acids (14, 22), and this poseschallenging questions as to why compounds of this kind predom-inate and how their synthesis and turnover are regulated relativeto the demands of growing plant parts for N in protein synthesis.

Before attempting to answer these questions at the subcellularlevel, knowledge is required on the sources and sinks within theplant for the solutes in question, on the transport exchanges of thesolutes between plant parts in xylem and phloem, and on how thefilling and emptying of storage pools ofN interact quantitativelywith the loading and unloading of transport channels with specificsets of solutes. The study presented here attempts to develop such

'This work was supported by funds from the Australian ResearchGrants Committee and the Wheat Industry Research Council.

2Recipient ofa scholarship from the Australian Meat Industry ResearchCouncil.

3Recipient of a scholarship from the Natural Science and EngineeringResearch Council of Canada. Present address: Boyce Thompson Institute,Cornell University, Ithaca, NY 14853.

37

an overview for the principal amino compounds of nodulatedwhite lupin (Lupinus albus L.), using data from a companion studyon the partitioning of C, N, and H20 in the species (5) andadditional information on the composition of transport fluids andpools of soluble amino-N. The study benefits extensively fromearlier work on the species (11, 15, 16).

MATERIALS AND METHODS

Plant Material. Effectively nodulated (Rhizobium WU425)plants of white lupin (L. albus L. cv Ultra) were grown in sandculture in a naturally lit glasshouse and supplied throughoutgrowth with N-free mineral nutrients. The culture period was Julyto November, the normal growing season for the species in West-ern Australia.

Life Cycle Study of Free Amino Compounds in Plant Parts andTransport Fluids. This involved harvesting of samples of 20 plantsat 10-day intervals through the growth cycle of 135 days. Rootbleeding (xylem) sap and stem base phloem sap were collectedfrom the plants at the time of harvest, using techniques describedelsewhere (15, 18).

Plants were separated into leaflets, stem + petioles, nodulatedroot, and reproductive parts (inflorescences, flowers, and fruits).Suitably sized aliquots of fresh plant material from each plantfraction were extracted with 80%o (v/v) ethanol and the extractswere evaporated to dryness and partitioned between petroleumether and water. The resulting aqueous fractions were assayed foramino compounds using a Beckman 118 amino acid analyzer,operated in a "physiological fluids-low temperature" mode usinga lithium-based buffer system to separate amides and non-proteinamino acids. Plant parts were assayed for total N by Kjeldhalanalysis, and the samples of xylem and phloem sap were assayedfor amino compounds using the amino acid analyzer as above.The data enabled the distribution of N among amino com-

pounds to be studied for both the soluble amino fraction of plantparts and the samples of xylem sap and phloem sap. Estimatesthen were made of the proportions of the total N of each plantpart present as free amino compounds.

Construction of Models Depicting Transport and Utilization ofAmino Compounds over a Specific Interval of Growth. The aminocompounds selected were Asn, Gln, Asp, and Val. These fourcompounds accounted for over 95% of the fixed N exported fromnodules in xylem, so that study of their utilization provided analmost complete inventory of the fate of recently assimilated Nwithin the plant.The study period selected was 51 to 58 days after sowing, when

the plants had commenced to flower on their primary shoots andwere forming lateral shoots at the three top nodes of their mainstems. A companion study (5) describing the partitioning of C, N,and H20 in plants of this age provided the data for fluxes of totalN between plant parts. This information was combined withanalyses of transport fluids and certain information from isotopelabeling studies to construct models of flow and utilization of

Plant Physiol. Vol. 67, 1981

amino compounds.The rationale for collecting transport fluids (see Fig. 3A) was

essentially as in earlier studies (5, 15). Phloem sap of petioles wasused to study translocate exported from leaves, phloem sap fromspecific sites on the stem assayed the upward- and downward-moving streams of assimilates supplying shoot apex and root,respectively, and root xylem bleeding provided information on thenitrogenous compounds exported from nodulated roots in xylem.Phloem sap samples were collected with reference to three nutri-tional strata of the main shoot (Fig. 3A): (a) a lower zone of eightleaves on the main stem (Li), shown in ["4Clurea-feeding experi-ments (see ref. 5) to be translocating exclusively to roots and lowerregions of the stem; (b) a midzone of four main stem leaves (L2),found to be feeding photosynthate to both root and apical regionsof the shoot; and (c) an upper zone of four or five main stemleaves (L3) immediately under the primary inflorescence andsupplying assimilates to this inflorescence and lateral shoots sub-tended at the top three or four nodes of the stem. Phloem sapfrom petioles was collected and bulked according to region oforigin (Fig. 3A, L1, L2, and La) and phloem sap from stem tissuewas obtained at the base and the top of the main stem and also atthe points of demarcation between zones SP,, SP2, and SP3 of thestem (Fig. 3A).

Additional information on transport of amino compounds inxylem was obtained by recovering tracheal sap by vacuum extrac-tion of segments of main stem SP1 and SP2 (Fig. 3A). Thesecollections were designed to test whether the proportions of Asn,Gln, Val, and Asp in xylem changed appreciably as transpirationalfluid ascended the stem. Xylem bleeding sap was also collectedfrom lower regions of the primary root. The Rhizobium strain usedpromoted strong "crown" nodulation of roots, so that in manyplants it was possible to collect xylem exudate from root tissue cutdistal to the lowest nodule (Fig. 3A). Where subsequent inspectionof the excavated root proved this to have been the case, the sapsample was retained for analysis and judged to contain N com-pounds resulting from N catabolism in the root or N cyclingthrough roots from the shoot system. Root bleeding sap collectedat ground level (Fig. 3A) was assumed also to contain exportproducts from N-fixing nodules so that any differences in com-position between it and the xylem sap of lower non-nodulatedportions of the root were assumed to provide information on therelative amounts of Asn, Gin, Val, and Asp contributed to xylemfrom root nodules. As mentioned in a companion study (5), allsites of collection of xylem and phloem sap were monitored duringeach 24-h interval of the study period, including three night-timesamples and eight day-time samples. The pooled samples from asite were considered to be representative of the average sapcomposition at that site over the study period. Earlier studies (15,16) defined the extent of diurnal fluctuations in sap compositionand the likely effect of such variations on the precision of modelsderived from the data.

RESULTS AND DISCUSSION

POOLS OF TOTAL N AND SOLUBLE AMINO-N IN PLANT PARTS

These quantities varied during growth of leaflets, stem + pe-tioles, nodulated roots, and reproductive parts (Fig. 1). MaximumN content of nonreproductive parts occurred during midfruiting(100 days), at which time leaflets contained 47% of plant total N,nodulated root contained 26%, stem + petioles contained 18%,and reproductive parts contained 9%. Reproductive parts con-tained from 15 to 33% of their N as soluble amino-N over thegrowth period; stem + petioles, 10 to 27%; nodulated roots, 3 to7%; and leaflets, only 1 to 4%. As in other legumes (3), stems wereprincipal sites for storage of soluble amino-N, but this form ofNrepresented only a small proportion of the total N content. Poolsof free amino compounds in L. albus built up especially over the

period from flowering (51 days) to midfruiting (91 days) and therewas evidence (Fig. 1) that these soluble N fractions were utilizedfor seed filling. Judging from the decline in soluble amino-N overthe period 90 to 130 days, mobilization from the pools of freeamino compounds in vegetative parts of the shoot and the nodu-lated root would have provided, at most, only 7% of the totalintake of N by fruits. Net losses of other N fractions fromnonreproductive parts would have fulfilled a further 50%o of thefruits' N intake, whereas N2 fixation during fruiting appeared tohave met the remaining 43% (see also ref. 17).

COMPOSITION OF SOLUBLE AMINO FRACTIONS OF PLANT ORGANSAND SAMPLES OF PHLOEM AND XYLEM SAP (FIG. 2)

Asn was at all times the major soluble amino compound ofplant parts, except in old nodulated roots in which Gin predomi-nated. Asn also comprised 55 to 70%o of the N of xylem sap andslightly less (40 to 60%) of the N of stem base phloem sap. Leafletswere generally less rich in Asn than other plant parts. Gin andAla were usually the most abundant compounds after Asn,whereas Val, Glu, Ile, Asp, and y-Abu comprised most of the restof the amino-N recovered in assays of the ethanolic extracts. Proaccumulated in significant amount in flowers and young fruits,and Gln tended to be at a proportionately higher level in xylemand phloem sap than in tissue pools of amino-N. Transport fluidswere relatively less rich than tissue pools in Ala and y-Abu.Organs with high proportions of their total N as free aminocompounds had very high proportions (up to 80o) of their solubleamino-N as Asn.

Despite the above differences in composition, there were strongresemblances in composition between tissue pools and transportfluids, indicating that similar sets and proportions of amino com-pounds were used for storage and translocation of N.

MODELING FLOW AND UTILIZATION OF AMINO COMPOUNDS DURING51- TO 58-DAY INTERVAL OF GROWTH

Models were constructed around the flow profile for N (Fig.3B) derived in a companion study (5) on C, N, and H20 utilization

300

200

100

z01E

LEAFLETS

10

10 50 90 130

STEM +

PET IOLES

10 50 90 130

10 50 90 130 10 50 90 130

days after sowingFIG. 1. Changes during growth in total N and ethanol-soluble amino-

N in plant parts of L. albus relying on root nodules for N supply. InsolubleN + soluble forms of N other than amino compounds are shown as thedifference between total N and free amino-N.

38 PATE ET AL.

se

(o

50

Plant Physiol. Vol. 67, 1981 AMINO COMPOUNDS IN WHITE LUPIN

60 as-o~~~~~~~~~g

,q0 60 so709

. 400

E20 asn

LEAFLETS

E30 50 70 90

c ~~~~~other

0060

LA 40

asn

20

NODULATED ROOTS

30 50 70 90 110

30 50 70 90 1)0

otherNys

hr

v abuilegluasp

agin

FRUITSa

70 90 110 130

other

asn

STEM BASE-PHLOEM SAP

other 40 60 8 100

thr

ilea

ser 9vala

asn

XYLEM SAP

40 60 80 loo

days after sowing *

FIG. 2. Changes during growth in percentage composition of the poolsof amino compounds in ethanol-soluble fractions of plant parts and inxylem sap and stem base phloem sap of L. albus relying on root nodulesfor N supply.

Table I. Proportions of Total N as Specific Amino Compounds inTransport Fluids Collectedfrom Nodulated Plants of L. albus L. over

Period 51 to 58 Days after SowingSap samples were taken from sites shown in Fig. 3A.

Percentage of Total N' of sapClass of Sap and Site of Collection on as

PlantAsn Gln Asp Val

Root bleeding (xylem) sapbBelow nodulated zone of root 23.5 10.7 19.6 3.9Top of root system 56.0 28.9 7.8 2.8

Stem tracheal (xylem) sapbLower part of stem (SP,) 51.3 25.1 5.4 3.9Mid half of stem (SP2) 48.5 22.6 6.2 4.7

Phloem bleeding sapbPetioles, leaves 1 to 8 (L1) 40.7 27.5 4.9 6.2Petioles, leaves 9 to 12 (L2) 44.7 27.4 4.4 4.2Petioles, leaves 13 to 15 or 16 (L3) 40.9 25.8 6.4 6.0Base of stem (below SP,) 39.9 28.4 4.9 6.2Stem between SP, and SP2 42.7 28.7 4.4 5.8Stem between SP2 and SP3 49.9 27.9 2.6 5.1Top of stem (above SP3) 58.2 20.8 1.9 3.5

More than 96% of the N of sap samples was recovered as aminocompounds.

b Pooled sample from 11 collections (three at night, eight in the day)made during the study period.

of white lupin. Combining this information with data on percent-age composition of transport fluids (Table I), estimates were madeof the amounts of Asn, Gln, Asp, and Val moving in xylem andphloem of the various segments of the plant's transport systems.From these data, the net intake or export of an amino acid byeach part of the plant was determined. The following assumptionswere considered to apply to the models.

(a) Mass flow in xylem and phloem represented the only formsof long distance transport, and amino compounds were carried inthese flow streams in the proportions shown in Table I.

(b) N cycling through the root (Fig. 3B) was carried as Asn,Gin, Asp, and Val in the proportions indicated from analysis of

Table II. Estimates of Contributions of Diferent Amino Compounds inXylem to Phloem Transfer ofN in Upper Stems of 51- to 58-day

Nodulated L. albus L.

N Concentration

Average

Amino Com- In Stem in Petiolepounds Top Phloem Difference Differ-

Philoem P in Phloemb encecSapa Upper (L2

Leaves'

yg/ml %Asn 1605 543 1062 71Gln 574 337 237 16Asp 51 67 (-16) 0Val 97 64 33 2Totald 2759 1263 1496 100

a From amino acid analyses of a pooled sample of sap representingseven separate collection times during the period.

b Concentration difference in stem-top phloem sap - average in petiolephloem sap in upper leaves.

c Concentration difference of amino compound (footnote b) as percent-age of concentration difference (footnote b) in total N of sap.

d More than 96% of the N of phloem sap was recovered as aminocompounds.

xylem sap collected below the nodulated zone of the root (TableI).

(c) Total amounts of Asn, Gln, Val, and Asp leaving the root inxylem were as indicated from composition of xylem bleeding sapcollected from the top of the root. Amounts of these amino acidsexported from nodules as fixed N then were computed as thedifference between total amounts exported from the root and theamounts of the compounds estimated to be cycling through theroot, as computed above (assumption b).

(d) Transfer ofN from xylem to phloem in upper regions of theshoot (Fig. 3B) involved the four amino compounds in the pro-portions indicated by comparisons of their respective concentra-tions in petiole phloem sap of upper (L2 + L3) leaves and phloemsap collected from the top of the stem. These companrsons (TableII) indicated that 71% of the N in xylem to phloem transfer wasas Asn, 16% as Gln, and 2% as Val. There appeared to be no netflux of Asp to phloem from stem tissue since the concentration ofthis compound in upper stem (above SP3) phloem sap was notsignificantly different from that in the parent phloem streamsfrom L2 and L3 leaflets. The data (Table II) agreed closely with anearlier study (15) of xylem to phloem transfer in the species.

(e) Flow profiles for the three strata of leaves included data fornet amounts of amino acid-N entering in xylem and leaving inphloem, as estimated respectively from xylem (tracheal) sap com-position for that part of the shoot and petiole phloem sap com-position for the relevant leaves. Estimates were also made of theamounts of entering amino acid utilized by the leaf or cycledthrough the leaf by xylem to phloem transfer in unmetabolizedform, using data from studies (1, 2) in which 14C- and '5N-labeledamino compounds were supplied to shoots of L. albus through thetranspiration stream. From these studies it was concluded that92% of the Asn, 77% of the Gln, 35% of the Val, and 19% of theAsp translocated from mature leaves in phloem were likely tohave come from direct xylem to phloem transfer in the leaf.Applying these values to all three strata of leaves in the studyhere, the likely proportions of each amino acid cycling throughleaves, in unmetabolized form, were determined.

39

I

PATE ET AL. Plant Physiol. Vol. 67, 1981

AA A

iO**15~ ,,1 ASPARAGINE

S+P3

S+2

S+P

B

L S P3 3

L3 S+P

1iS+P

NR

A

GLUTAMINE

NR

Phicem Trnpout

DonaedX rom Orgemns

mo ~- Xylem to Phineun Trea

Phkwen to Xylem Transfer

FIG. 3. A, diagrammatic representation of 51- to 58-day L. albus plantshowing sites of collection of xylem and phloem fluids and directions offlow of photosynthate from leaves as indicated in [t4Clurea-feeding studies(see ref. 5). B, flow profile for total N in the 51- to 58-day plant as

determined in a companion study (5). The partitioning and utilization byplant parts of 1000 units by weight of fixed N are depicted. L,, leaflets ofleaves at lowest 8 nodes; L2, leaflets of leaves 9 to 12; L3, leaflets of leaves13 to 16 or 17; SPI to SP3 (representing strata similar to those of leaves),stem + petiole segments; NR, nodulated root; A, apical inflorescence +lateral shoots developing at the top three nodes of the stem.

PROFILES FOR TRANSPORT AND INCORPORATION OF TOTAL N, ASN,GLN, VAL, AND ASP IN 51 TO 58-DAY PLANT

Comparisons were made between the transport profiles for totalN (Fig. 3B) and for each of the four major amino compounds(Fig. 4) using flow diagrams in which lines of differing thicknessdepicted the relative amounts of N flowing in different segmentsof the xylem and phloem transport systems. The numbers given inthe figures refer to amounts oftotal N or amino acid-N transportedto, incorporated into, or exported from plant parts relative to a

net intake by the plant of 1,000 units of fixed N.In the flow diagram for total N (Fig. 3B), all exchanges between

plant parts in xylem and phloem balanced the observed changesin the total N content of plant parts over the study period (see ref.5 for a description of the modeling technique used). The flowpatterns for amino acids, by contrast, utilized data for amino acidcomposition of transport fluids at specific exchange sites (Table I)and the predictions from labeling studies of amino acid cyclingthrough leaves mentioned above. The flow diagrams consequentlyprovided quantitative,data for specific fluxes of compounds trans-located to plant parts in xylem and phloem. On the basis of theseflux values, amounts of amino acid consumed or produced bydifferent parts of the system were estimated. Constructed in thismanner, inputs and outputs of amino acid by different segmentsof the system did not necessarily balance arithmetically, incon-sistencies being particularly evident for the minor xylem constit-uents Asp and Val. The conclusions drawn for each amino com-

pound were as follows.Asn and Gln (Fig. 4, A and B). The amides showed similar

patterns of flow and resembled closely the profile for transportand utilization of total N (Fig. 3B). Of the fixed N exported fromnodules, 56% was as Asn and 29% as Gln, and, were there no

discrimination in loading of the amides onto transport channels

S+P3

S+P2

S+P

A A

D ,<

L3

L S+P3 3

L2 S+P2

L, S+p L

NR NR

FIG. 4. Profile of synthesis, transport, and consumption of asparagine(A), glutamine (B), aspartate (C), and valine (D) in 51- to 58-day L. albusrelying on root nodules for N supply. Amounts of amino acid transportedand utilized are expressed on a N (by weight) basis relative to a net intakeby the whole plant of 1000 units of fixed N. See Fig. 3 for description ofplant parts, sites of collection of transport fluids, and flow profile for totalN.

or metabolic transformations en route to plant parts, their respec-

tive involvements in the net N increment of organs would havebeen expected to be in these same proportions. However, becauseof (a) differences in ratios of Asn to Gln in different transportpathways (Table I), (b) a disproportionately high involvement ofAsn in transfer from xylem to phloem (Table II), and (c) a

disproportionately high involvement of Gln in net loss ofN fromsenescing leaves, the extents to which the amides contributed N toplant parts varied appreciably from the proportions present in theincoming xylem stream (Table III). Asn, for example, contributeda more than expected amount ofN to stem + petioles (67% versus

56% expected from xylem sap composition); Gln contributed amore than expected amount (42% versus 29%) to the nodulatedroot and a less than expected amount (22% versus 29%) to theinflorescence plus lateral axes. Despite these differences, amidescomprised the bulk of the N intake of all plant parts, indicatingthe importance of these compounds as sources of N for aminoacid and protein synthesis.

Several possible metabolic routes for the utilization of Asn havebeen suggested (6) and the mechanisms involved in legumes haveyet to be clearly defined. The presence of an active asparaginase

40

A B

S+P3

S+P2

S+P

A

NR

Xylem Trpout

A Xylem Bleding Sap

Phboem Bleeding Sap

IV Extracted Trechee Sap

PS-

AMINO COMPOUNDS IN WHITE LUPIN

Table III. Contributions of Transported Amino Compounds to NetIncrements ofN in Plant Parts ofNodulated L. albus L. over Period 51 to

58 Days after Sowing

Percentage Contributionato N Increment of Plant

Receptor Organ (Mode of Intake) Pan through Net Import

Asn Gln Asp Val

Nodulated root (phloem import) 52.2 41.6 _b 8.0Inflorescence and lateral apices (xylem andphloem import) 54.4 21.8 3.6 4.0

Stem and petioles (lateral uptake from xy-lem and phloem) 67.0 23.2 1.6 4.4

Leaflets of leaves 9 to 16 (L2 and L3) (xylemimport) 53.3 24.7 8.1 5.7a Derived from models of flow and utilization of total N and amino

compounds shown in Fig. 4.b , root showed greater export of Asp in xylem than was imported in

phloem (Fig. 4).

in developing legume seeds (2, 8, 20) and the accumulation of'5NH3 in endosperm of lupin seeds supplied 16N(amide)-Asn inphloem (2) have indicated that deamidation followed by assimi-lation ofNH3 is a likely route ofAsn catabolism in seeds, althougha similar enzyme system has not been demonstrated in vegetativetissues (21) (C. A. Atkins and A. Sim, unpublished data). Enzymicand labeling studies of leaves of pea (10) and soybean (21) haveindicated that Asn may first transfer its amino group with subse-quent deamidation of the succinamic acids formed. Whatever themechanism of Asn utilization, a rapid and effective mechanism isrequired for assimilation of the resulting NH3. In photosynthetictissues, this is likely to be the glutamine synthetase/glutamatesynthase system of chloroplasts (12).

Since there is no evidence to date to support transfer of theamide-N ofAsn to an acceptor keto acid, such a route seems likelyfor Gln utilization (9, 12), although deamidation of Gln byglutaminase could well represent an alternative mechanism.The flow diagrams for the two amides differed significantly for

the lower (L1) leaflets (Fig. 4) which were in negative balance forN. They exported somewhat more Gln in phloem than theyimported in xylem, while importing and exporting almost balanc-ing amounts of Asn. The predominance of Gln, in this case, mightreflect differing specificities of the respective synthetases for Ndonors since Gln synthetases utilize NH3 most readily (12),whereas Asn synthetases show greatest activity when utilizing Glnrather than NH3 as a substrate (7, 19). Thus, continued Gln exportwould be likely to preclude a high rate of synthesis of Asn.

Similanty in behavior of the two amides extended to relativeextents of cycling of fixed N through the root (6.4% of N cycledas Asn, 5.5% as Gln) and to the relative extents to which xylemand phloem contributed each compound to the apical region ofthe shoot (Table IV).Asp (Fig. 4C). A major feature of the profile for this compound

was its involvement in export ofN from root tissue in the xylem.Some of this Asp (58%) was depicted as coming from phloem toxylem transfer, largely as Asp which had cycled through lowerleaves, whereas the remainder was presumed to have arisen fromstorage pools or N catabolism in the root. There might also besignificant export from roots of Asp derived from deamidation ofAsn, especially when translocation from the shoot provided theroot with an excess of Asn.Asp was exceptional in being supplied in greater amount to the

apical region of the shoot in xylem (69%) than in phloem (31%)(Table IV). Low phloem translocation of Asp to the shoot apexwas due to the apparent absence of xylem to phloem transfer of

Table IV. Proportional Contributions of Xylem and Phloem to Transfer ofAmino Compounds to Young Apical Regions of Shoot of 51- to 58-day-

Nodulated L. albus L.

Proportion (%) of Intake ofAmino Compound by Api-cal Shoot Partsa Provided

Amino Compounds by

Xylem Phloem

Asn 36.2 63.8Gln 42.9 57.1Asp 68.6 31.4Val 48.1 51.9Total amino compoundsb 40.4 59.6

a Inflorescence of main shoot and young lateral shoots developing attop three nodes of stem.

b Assumed that amino compounds were the only forms ofN translocatedin xylem and phloem (Table I).

the compound in the upper stem (Table II and see ref. 15) and tothe relatively low level of translocation of Asp from upper leaves(Fig. 4C). 14C- and '5N-labeling experiments have shown that Aspis not readily exchanged between xylem and phloem in leaflets ofL. albus but is extensively metabolized to yield a variety of soluteswhich may be retained by the leaf or exported in phloem as aminoacids other than aspartate (1). Thus, the N and C of xylem-borneAsp would be donated to the shoot apex largely as compoundsother than Asp.

Val (Fig. 4D). Flow in xylem to the upper leaves (L2 and L3)was particularly prominent for Val, and the model suggested thatthis involved release of Val to xylem from stem tissue, productionof Val in nodules, and the cycling of Val through roots. A secondmajor feature was the large translocation of Val from lower leaves(L3), most of this coming from mobilization ofN in the senescingleaf rather than from xylem to phloem transfer of currentlytransported Val. Most phloem-borne Val was donated to the root,which proved to be a major sink for this amino acid, as it was forAsn and Gln, but not Asp.

In other respects the pattern of transport and utilization of Valwas in proportion to its relative abundance (5.4% of xylem N) inthe transpiration stream. Thus, 3.9% of the N involved in xylemto phloem transfer in the top of the stem was as Val (Table II),and intake of Val by the apical region of the shoot through xylemand phloem, and incorporation of Val into leaflets and stem +petiole dry matter were roughly as expected from the flow profilefor total N (Table III; cf. Figs. 3B and 4D).

CONCLUSION

The study presented here showed strong resemblances in com-position between soluble reserves and translocated forms of N inthe white lupin, implying a ready interchange of these two classesof N during growth of the species. The small size of pools of freeamino compounds in plant parts relative to the fluxes and rates ofincorporation ofN in the plant body indicated that the growth ofeach organ was closely geared to its ability to metabolize effec-tively the compounds which it received through xylem andphloem. The models of amino acid flow predicted that Asn, Gln,Val, and Asp were all readily accepted as sources of N by plantorgans, but sufficient quantitative differences existed in the bal-ance of these four compounds in the various transport channels tosuggest a measure of selectivity among organs in loading of xylemand phloem with the solutes under study.The study highlighted the dominant role of the amides, and

especially Asn, in the nitrogen nutrition ofall organs and indicatedthe need for a clearer understanding of the biochemistry of thesynthesis and utilization ofamides, especially in photosynthesizing

Plant Physiol. Vol. 67, 1981 41

42 PATE ET AL.

leaves. This and other studies using '4C labeling (1, 11, 13)demonstrated the direct transfer of solutes, particularly Asn, be-tween xylem and phloem in leaves and stems. This direct transfermechanism showed a specificity for amino acids which suggestedthat specific transport sites were responsible for phloem loadingof the compounds (see also ref. 4).

Acknowledgments-The assistance of L. Watson and E. Rasins is gratefullyacknowledged.

LITERATURE CITED

1. ATKINS CA, JS PATE, DL McNEIL 1981 Phloem loading and metabolism ofxylem-borne amino compounds in fruiting shoots of a legume. J. Exp Bot Inpress

2. ATKINS CA, JS PATE, PJ SHARKEY 1975 Asparagine metabolism: key to thenitrogen nutrition of developing legume seeds. Plant Physiol 56: 807-812

3. HERRIDGE DF, CA ATKINS, JS PATE, RM RAINBIRD 1978 Allantoin and allantoicacid in the nitrogen economy of the cowpea ( Vigna unguiculata (L.) Walp.).Plant Physiol. 62: 495-498

4. HOUSLEY TL, LE SCHRADER, M MILLER, TL SETrER 1979 Partitioning of [14C]-photosynthate and long-distance translocation of amino acids in prefloweringand flowering, nodulated, and non-nodulated soybeans. Plant Physiol 64: 94-98

5. LAYZELL DB, JS PATE, CA ATKINS, DT CANVIN 1980 Partitioning ofcarbon andnitrogen and the nutrition of root and shoot apex in a nodulated legume. PlantPhysiol 67: 30-36

6. LEA PJ, L FOWDEN 1975 Asparagine metabolism in higher plants. BiochemPhysiol Pflanzen 168 (suppl): 3-14

7. LEA PJ, L FOWDEN 1975 The purification and properties ofglutamine-dependentasparagine synthetase extracted from Lupinus albus. Proc R Soc Lond B BiolSci 192: 13-26

8. LEA PJ, L FOWDEN, BJ MIFLIN 1978 The purification and properties of asparag-inase from Lupinus species. Phytochemistry 17: 217-222

Plant Physiol. Vol. 67, 1981

9. LEWIS OAM, TA PROBYN 1978 15N incorporation and glutamine synthetaseinhibition studies of nitrogen assimilation in leaves of the nitrophile Daturastramonium L. New Phytol 81: 519-526

10. LLOYD NDH, KW Joy 1978 2-Hydroxy-succinamic acid: a product ofasparaginemetabolism in plants. Biochem Biophys Res Commun 81: 186-192

11. McNEIL DL, CA ATKINS, JS PATE 1979 Uptake and utilization of xylem-borneamino compounds by shoot organs of a legume. Plant Physiol. 63: '1076-1081

12. MIFLIN BJ, PJ LEA 1977 Amino acid metabolism. Annu Rev Plant Physiol 28:299-329

13. PATE JS 1976 Nutrient mobilization and cycling: case studies for carbon andnitrogen in organs of a legume. In: IF Wardlaw, JB Passioura, eds, Transportand Transfer Processes in Plants. CSIRO Conf, Canberra, Australia, pp 253-345

14. PATE JS 1980 Transport and partioning of nitrogenous solutes. Annu Rev PlantPhysiol 31: 313-340

15. PATE JS, CA ATKINS, K HAMEL, DL McNEIL, DB LAYZELL 1979 Transport oforganic solutes in phloem and xylem of a nodulated legume. Plant Physiol 63:1082-1088

16. PATE JS, DB LAYZELL, DL McNEIL 1979 Modeling the transport and utilizationof carbon and nitrogen in a nodulated legume. Plant Physiol 63: 730-737

17. PATE JS, FR MINCHIN 1980 Comparative studies ofcarbon and nitrogen nutritionof selected grain legumes. In: R. J. Summerfield and A. H. Bunting, eds.,Advances in Legume Science Vol. I Royal Botanic Gardens, Kew, England,pp. 105-114

18. PATE JS, PJ SHARxEY, OAM LEWIS 1974 Phloem bleeding from legume fruits. Atechnique for study of fruit nutrition. Planta 120: 229-243

19. RoGNEs SE 1970 A glutamine-dependent asparagine synthetase from yellowlupine seedlings. FEBS Lett 10: 62-66

20. SODEK L, PJ LEA, BJ MIFLIN 1980 Distribution and properties of a potassium-dependent asparaginase isolated from developing seeds of Pisum sativum andother plants. Plant Physiol. 65: 22-26

21. STREETER JG 1977 Asparaginase and asparagine transaminase in soybean leavesand root nodules. Plant Physiol 60: 235-239

22. ZIEGLER H 1975 Nature of transported substances. In MH Zimmermann, JAMilburn, eds, Encylopedia Plant Physiol, New Series Vol 1. Springer-Verlag,Heidelberg, pp 59-100