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Plant Physiol. (1982) 70, 476-482
Metabolism and Translocation of Allantoin in Ureide-Producing Grain Legumes' Received for publication February 8, 1982 and in revised form April 21, 1982
CRAIG A. ATKINS, JOHN S. PATE, ANNE RITCHIE, AND MARK B. PEOPLES Botany Department, University of Western Australia, Nedlands 6009, Western Australia, Australia ABSTRACT Transfer of the nitrogen and carbon of allantoin to amino acids and protein of leaflets, stems and petioles, apices, peduncles, pods, and seeds of detached shoots of nodulated cowpea ( Vigna unguiculata L. Walp. cv. Caloona) plants was demonstrated folowing supply of 12-14C1, 11,3"Niallantoin in the transpiration stream. Throughout vegetative and reproductive growth all plant organs showed significant ureolytic activity and readily metabolized 12-'4Clallantoin to 4CO2. A metabolic pathway for ureide nitrogen utilization via aliantoic acid, urea, and ammonia was indicated. Levels of ureolytic activity in extracts from leaves and roots of nodulated cowpea were consistently maintained at higher levels than in non-nodulated, N03- grown plants. I"4ClUreides were recovered in extracts of aphids (Aphis craccivora and Macrosiphum euphorbieae) feeding at different sites on cowpea plants supplied with 12-'4Clallantoin through the transpiration stream or to the upper surface of single leaflets. The data indicated that the ureides were effectively transferred from xylem or leaf mesophyll to phloem, and then translocated in phloem to fruits, apices, and roots.
The ureides, allantoin, and allantoic acid are major nitrogenous products of nitrogen fixation in nodulated cowpea (10) and soybean (14, 18) and apparently provide much of the nitrogen required for protein synthesis and growth during plant development (10, 19). Although extracts of tissues from cowpea and other 'ureide-producing' grain legumes (10, 20) show allantoinase (EC 188.8.131.52) activity, the metabolic pathway utilizing allantoic acid and releasing nitrogen for amino acid synthesis has not been defined. Allantoicase (EC 184.108.40.206) activity was not detectable in extracts of soybean seedlings (9), and even though urease (EC 220.127.116.11) is apparently a constitutive enzyme in many plants, including soybean (see 17), the exact nature of the relationship between ureolytic activity (12) and ureide metabolism has yet to be resolved. Transport of ureides in xylem from nodules and nodulated root systems (10, 14, 18) has been well documented, but subsequent transfer from the xylem to phloem streams supplying fruits, apices, roots, and developing leaves has not been established. The present investigation uses 14C, '5N-labeled allantoin to investigate transfer of the nitrogen and carbon of ureides to amino acids and protein, to elucidate sites and metabolic pathways involved in ureide utilization, and, using aphids, to demonstrate involvement of phloem in ureide transport to sites of utilization in cowpea shoots. ' Supported by grants from the Australian Research Grants Committee and the United Nations Development Program through a cooperative project with the International Institute of Tropical Agriculture, Ibadan,
MATERIALS AND METHODS Plant Material. Effectively nodulated legumes were grown in nitrogen-free sand culture (11) in a naturally lighted glasshouse. Cowpea ( Vigna unguiculata L. Walp. cv. Caloona) and mung bean (Vigna radiata L. Wilczek) were inoculated with Rhizobium strain CB756, soybean (Glycine max L. Merr. cv. Bragg) with a commercial peat Rhizobium inoculum (Root Nodule Pty. Ltd., Nitrogerm Group H), and white lupin (Lupinus albus L. cv. Ultra) with Rhizobium strain WU425 at sowing. Cowpea, soybean, and mung bean were grown with a day/night temperature regimen of 35/ 25°C and lupin with a regimen of 30/18°C. Non-nodulated cowpeas were grown in the same way as nodulated plants except that seed was not inoculated and the nutrient solution contained 10 mm KNO3 (2). Preparation of "C- and "N-Labeled Allantoin. Samples of [2'4C]uric acid (I mm, 50 liCi; Amersham (Australia)) and [1,3'5N]uric acid (3 mm, 96.4 atom % excess 15N; Merck Sharpe and Dohme, Canada) were dissolved in 5 ml of 20 mm Tricine and adjusted to pH 10.0 with NaOH. After adding 0.5 to 1.0 mg of purified urate oxidase (EC 18.104.22.168; Sigma type IV) the mixture was incubated at 30'C for 36 h with constant shaking to ensure aeration. Ion exchange chromatography (10) of aliquots of the incubation mixture showed a high yield of allantoin with usually less than 10% uric acid remaining, and insignificant hydrolysis of allantoin to allantoic acid (Fig. 1). Purity of the double-labeled allantoin fraction (Fig. 1) was further demonstrated by quantitative yield of [14C]allantoic acid following alkaline hydrolysis, and contamination with urea was disproved by absence of 14C02 evolution after treatment with urease. Following incubation, the bulk reaction mixture was passed through a 3 x 0.5 cm Dowex 50, H+-form column followed by a 3 x 0.5 cm Dowex 1, formateform column, and the labeled allantoin eluted with water. The treatment removed the Tricine buffer, uricase protein, allantoic acid, uric acid, and an unknown acidic reaction product (Fig. 1). The final allantoin preparations were then neutralized and stored at -20°C. Metabolism of Xylem-Borne ["CI, [15N]Allantoin. Vegetative and reproductive fruiting shoots of nodulated cowpea were cut beneath water and allowed to transpire for 30 min in xylem sap diluted 6-fold with water. The xylem sap had been previously collected as exudate from the root system following decapitation of nodulated cowpea plants identical with those used in the labeling study. Groups of three shoots were transferred to 30 ml of diluted xylem sap containing 2 mm allantoin labeled with 5.4 ,uCi of 14C and 0.45 mg of "5N. After 24 h shoots were harvested and component organs homogenized in cold 80% (v/v) ethanol. The ethanol extracts were dried, dissolved in water, and washed with petroleum ether to remove pigments. The resulting water extracts were then fractionated into acid and neutral and basic solutes using a Dowex 50 H+ resin column, and the labeling in allantoin and allantoic acid determined as described previously (1, 10). Basic fractions were separated into component amino acids
PETIOLE 150 50 100 ELUTION VOLUME (ml) FIG. 1. Ion exchange chromatographic separation of products of [2"4Cluric acid metabolism by urate oxidase. Note that allantoin peak is shown on a scale one-tenth that of following peaks.
using an amino acid analyzer (4). The ethanol insoluble residue dried and finely milled. "C in the ethanol soluble fractions, in isolated compounds, or in ethanol insoluble fractions solubilized in hyamine 10 x hydroxide was determined bRr liquid scintillation, with appropriate corrections for quenching. N in extracts, insoluble residues, or isolated compounds was measured by optical emission spectrometry (16) following steam distillation of ammonia from Kjeldahl digests. Metabolism of 12-'4CIAlIantoin by Tissue Slices. Freshly harvested, chilled plant material was finely sliced (0.5-1.0 mm strips) and samples (0.1 g fresh weight) added to serum vials containing 2.7 ml of either 50 mm K-phosphate buffer (pH 6.3) or 59 mm Tris-HCl (pH 7.5). The vials were placed under vacuum so that the tissue pieces were visibly 'wetted' and sank to the bottom of the buffer solution. A small tube containing 0.5 ml of 0.1 M NaOH was inserted and after the vials were closed, 0.3 ml of 10 M [2'4C]allantoin (2.2 x 104 dpm/,umol) was added through the serum cap. The vials were then incubated in the dark in a shaking bath at 30°C and, after varying periods (30-120 min), the reactions were terminated by adding 0.5 ml of 4 M HC104. The closed vials were held on ice for a further 2 h to ensure complete absorption of C02, opened, and the NaOH removed for 14C measurement by liquid scintillation spectrometry. Assay of Ureolytic Activity. Extracts of freshly harvested plant tissues were prepared using two volumes of 50 mm Hepes-NaOH (pH 8) containing 5 mM DTT. The homogenate was filtered through 100 ,um mesh and the filtrate used as source of enzyme. The reaction mixture comprised 50 mm Hepes-NaOH (pH 8), 1 mM DTT, 5 mm ["'C]urea (1.4 x 103 dpm/,umol), and 0.5 ml of plant extract in a total volume of 3 ml, contained in a closed, 34-
C Z 40
STEMn+ STEM+ PETIOLE
LEAF PEDUN - CLE
STEM+ LEAF PEDUN -CLE PETIOLE
z0 3 20
:1 AtF14IArn STEM+ LEAF PETIOLE
STEM+ LEAF PEOUN rIU
FIG. 2. Fresh weight (A) and distribution of 14C (B) and '5N (C) between organs of cut vegetative or reproductive shoots of nodulated cowpea plants supplied 2 mM [2-'4C], [1,3-15N]allantoin (6 X 105 dpm of '4C/ml; 48.2 atom % excess '5N) for 24 h in transpiration stream. Soluble and insoluble fractions were separated following extraction in 80% (v/v) ethanol at harvest.
ml serum vial with a small tube containing 0.5 ml of 0.1 M NaOH inserted in the vial to trap released '4CO2. After 15 min at 30°C in a shaking bath in darkness, the reactions were terminated and 14C in the trap measured as indicated above. Labeling Studies using Aphids. Actively growing colonies of the black cowpea aphid (Aphis craccivora) and the green potato aphid (Macrosiphum euphorbiae) were cultured on cowpea and soybean plants during mid-vegetative and reproductive growth. In a first experiment, aphids were collected from leaflets and fruits 90 or 180 min after placing cut, transpiring reproductive shoots of
cowpea in a feeding solution (diluted xylem sap) containing I mM [2-1 C]allantoin (4 ,uCi). The labeled solution was completely taken
up by the shoots in 20 min and was replaced with diluted, unlabeled xylem sap for the remainder of the feeding period. In a second experiment, the upper surface of a leaflet of the top three trifoliolate leaves of a cowpea plant was wetted with 0.01% (w/v) Triton X-100 followed by 20,il of 1 mm [2-'4C]allantoin (3 14C). The plant was held in sunlight and 90 to 110 min after administration of labeled allantoin, groups of feeding aphids (29-
ATKINS ET AL. Plant Physiol. Vol. 70, 1982 Table 1. Distribution of "C among Compounds of the Soluble Fraction from Different Organs of Vegetative and Reproductive Shoots of Cowpea The organs were supplied 2 mm [2-14Clallantoin (6 x 105 dpm/ml) in the transpiration stream for 24 h. % 14C of Soluble Fraction as Plant Part 14C of Soluble Fraction Acidic and . Allantoic Basic. neta8 Allantom ai neutral' acid dpm X 1o-4 Vegetative shoot Stem and petioles 296 2.5 55.7 Leaflets 53 8.2 75.7 Apex 8 4.8 24.5 Reproductive shoot Stem and petioles 162 2.3 50.9 Leaflets 66 4.3 59.6 Peduncles 45 2.2 55.3 Pods 19 4.2 64.3 Seeds 11 2.1 37.5 a Acidic and neutral compounds other than allantoin and allantoic acid.
38.1 12.3 57.7
3.7 3.8 12.9
43.3 32.1 36.3 29.7 56.3
3.5 3.9 6.2 1.8
Table II. 5N Labeling of the Basic Fraction of the Soluble Extractfrom Different Organs of Cut Vegetative and Reproductive Shoots of Cowpea The organs were supplied '5N as 2 mm [l1,3-'5Njallantoin (48.2 atom % excess 15N) for 24 h in the transpiration stream. Plant Part Total N "5N Excess mg jg Vegetative.shoot Stem and petioles 3.57 17.4 Leaflets 3.19 8.4 Apex 1.11 3.2 Reproductive shoot Stem and petioles 3.27 2.7 Leaflets 4.84 11.4 Peduncle 1.36 0.7 Fruits 1.93 1.9
ALLANTOIN METABOLISM AND TRANSLOCATION Table III. Distribution of '4C and '5N among Amino Compounds from the Basic Fraction of the Soluble Extract
of the Stem and Petioles and the Leaflets of Cut Vegetative Shoots of Cowpea The tissues were supplied 2 mm 12-'4C1, 1[,3-'5Nlallantoin (6 x I05 dpm/ml, 48.2 atom % excess '5N) for 24 h in the transpiration stream. Stem and Petioles Leaflets Compound 14c 5N 14C 15N
Urea Aspartate Threonine and serine Asparagine and glutamatec Glutamine Proline Glycine Alanine
dpm x 10-3
14.4 2.2 5.3 15.3
6.0 12.5 20.8 9.3 5.1 13.4 0.3 0.8 0.8 7.4 4.7 3.6 NDb a-Aminobutyrate 1.6 1.9 Arginine a Not assayed as the small amounts of nitrogen precluded b Less than 0.1 x I 03dpm or 0.01 15N.
content ,g a 0.64 4.82 0.92 ND 1.80 _
dpm x 10-3
2.2 ND 5.5 8.7
0.5 12.0 35.7 0.8 0.9 1.3 14.3 11.5 0.2
content Ag -
0.59 4.40 0.01 0.02
Table IV. Rate of Formation of '4CO2 from the Metabolism of [2-'4CJAllantoin by Intact Tissue Slices of the Vegetative and Reproductive Parts from Four Legumes Mature Stems and Mature Root Young Species Stage Pod Seed Leaflets Petioles Leaflets Roots Tips nmol/h.gfresh wt Vegetative plant parts Reproductive plant parts 93 196 62 33 31 Vigna unguiculata Vigna unguiculata Youngb 116 69 30 187 14 2 9 Gycine max MaturingC 134 36 -a 238 139 Matured Vigna radiata 38 5 4 18 199 Lupinus albus Glycine max Young 61 Maturing 36 Mature 14 Vigna radiata 10 Young 10 Maturing 110 18 a Not assayed. h Early embryo expansion. c Midway through cotyledon filling. d Dry seed at final harvest. shoot, only a small proportion of incoming allantoin was stored general the labeling reflected the abundance of each of the com(Table I). Peduncles also apparently functioned in the storage of pounds analyzed except for proline, which in stems and petioles ureides, whereas in pod tissue there was extensive metabolism to occurred at a high level but which contained little 14C or '5N. form other solutes. The distinct difference in the proportion of '4C Small amounts of 14C-labeled urea were recovered from certain recovered as ureides shown by pods compared with seeds (Table tissue extracts (Table III) but the levels of nitrogen in urea were IB) suggests that one function of pods may be to metabolize low and precluded assay for "5N. ureides and transfer the nitrogen to seeds as other compounds. Application of [2-'4CJallantoin to tissue slices of different organs
Labeling of the basic fractions of plant organs with 4C (Table I) indicated that a small amount of the carbon of allantoin had been utilized for amino acid synthesis, and recovery of "5N in these fractions was consistent with the transfer of ureide nitrogen to amino nitrogen (Table II). Organs differed in the amount of '5N found in the basic fraction of each, with the highest values being recorded for petioles of the vegetative shoot and for leaflets
of vegetative and reproductive nodulated plants of cowpea, soybean, and mung bean resulted in release of 14CO2 (Table IV). Of the vegetative tissues tested, mature leaflets and young developing fruit tissues were the most active in ureide metabolism. In contrast to the above three ureide-producing species, the leaves and stems of L. albus, a species transporting amides but not ureides as nitrogenous solutes of xylem and phloem (4), showed virtually no
of the reproductive shoot (Table II). These basic fractions were further analyzed by separating the component amino acids and measuring both '4C and '5N in the major compounds (Table III). Most amino acids showed a lower '4C/'6N ratio (average - 4.8 X 103 dpm of '4C/,ug '5N) than the supplied allantoin (13.2) consistent with a more ready incorporation of the ureide N than of the urea C of allantoin in their synthesis. Asparagine was the predominant amino acid of the soluble pools of both organs and in each case was the most heavily labeled, especially with '5N. In
activity in releasing "4CO2 from the labeled allantoin (Table IVA). Tissue extracts from all organs of nodulated cowpea plants exhibited considerable ureolytic activity during vegetative and early reproductive growth (Fig. 3A). Similar plants cultured on nitrate and not nodulated also showed some ureolytic activity, but enzyme levels were generally lower than in nodulated plants (Fig. 3B). This difference was especially marked for roots and leaves. Translocation of Allantoin within Shoots. Administration of xylem-borne [2-'4Clallantoin as a 20-min pulse in the transpiration
the lower surface of an attached leaf following application of [2"Clallantoin to the outer margin of the upper surface of the same leaf. In cowpea and soybean leaves after I h, ''C ureide constituted 39% to 40% of label recovered from aphids (Table VI). Aphids collected from soybean leaves after a further 30 min contained considerably more 14C, but the proportion as ureide was less, presumably due to more extensive metabolism in the plant or the aphids. Aphids used in these experiments secreted a copious, sugar-rich, honeydew consistent with their feeding from phloem. Serial sections cut of fixed material embedded in glycol methacrylate indicated that the stylets of the feeding aphids terminated in the phloem of the vascular bundles of fruit or leaf.
DISCUSSION Based on studies of ureide flow in xylem and ureide and total s N accumulation in plant tissues, Herridge et al. (10) concluded m that allantoin and allantoic acid provide the bulk of the nitrogen required for protein synthesis throughout shoot development of E nodulated cowpea. The present study provides direct evidence for the utilization of ureide nitrogen in the synthesis of soluble amino _acids and insoluble nitrogen-containing compounds. The demonstration of differential enrichment of amino acid and insoluble fractions of plant organs with the '5N as opposed to the ''C of fed , 0.6 ailantoin, indicates a preferential utilization of ureide nitrogen in pathways of nitrogen assimilation in the shoot. Previous studies found that allantoinase activity is widely dis0 tributed in vegetative and reproductive tissues of cowpea (10) and 0.4 soybean (20). Demonstration in this study of labeled allantoic acid a: in all organs of cowpea shoots following xylem uptake of [2"Clallantoin is consistent with such activity. Similarly, detection of [''Clurea in tissue extracts of the same fed shoots suggests an involvement of allantoicase (EC 22.214.171.124) in the cleavage of ["C] allantoic acid. However, urea would also be formed were allantoic \ o / Ptleaf ~j40 acid to be degraded by a mechanism involving allantoic acid Z FIG. 3. Ditiuto furoyiatvti xrct fcstemp+ amidohydrolase (EC 126.96.36.199) and ureidoglycolase (EC 188.8.131.52), x s a petiole with attendant formation of ammonia, ureidoglycine, and ureidoglycolate (19, 21). Intact tissues of cowpea, especially mature 0 A leaflets and stems, were shown here to readily metabolize [2-'4C] allantoin to form "CO2, and this, coupled with the capacity of extracts from all tissues of cowpea to hydrolyze urea, indicates FI.3. Distribution of ureolytic activity in of component that allantoin is metabolized via allantoic acid and urea to amnon-nodulated (B) cowpea plants during organs of nodulated (A) monia and CO2 (see also 10, 19, 20). Although the 15N labeling of determined by development. Onset of ureide export from nodules component amino acids of the soluble pool of both stem and in tissue xylem sap analysis previous study (5). In (A), samples of petioles and leaflets (Table III) does not indicate the major route extracted for assay excluded nodules. for ammonia reassimilation, the wide range of compounds labeled stream to cut, fruiting cowpea shoots resulted in labeling of the by ['5N]allantoin indicates that ureide nitrogen is readily utilized. ethanol soluble fraction of aphids feeding on the leaves and As with seeds of many other legumes (6), the cotyledons of subtended fruits (Table V). A significant proportion of the "C cowpea contain considerable ureolytic activity (Fig. 3). Although recovered in aphids at both feeding sites at 90 or 180 min was ureides constitute a small proportion of total seed nitrogen of identified as '4C-labeled ureide and at each site the proportion of cowpea (e.g. 4% ), the intense hydrolysis of nucleic acids ''C as ureide increased with time. [''Clurea was not detected or known to occur following the onset of germination of legume was barely detectable (leaflets 180 min) in the aphids. The identity seeds (15) might well be a source of purines and ureides (7), of labeled solutes other than ureides and urea was not determined. whereas the transient increases in ureolytic activity shown here Labeled allantoin applied to the upper surface of photosynthe- for seedling root and shoot are consistent with these organs sizing leaflets of intact plants resulted in transfer of "C to aphids engaging in the metabolism of ureides and/or urea translocated feeding on the petioles of the fed leaf and on the peduncles, fruits, from the cotyledons. Thereafter, as the cotyledonary source of or flowers subtended by the fed leaves. Labeled carbon was also nitrogen is progressively utilized, the specific activity of the seedpresent in aphids feeding on reproductive organs subtended by a ling tissues for urea hydrolysis declines. Although all tissues of leaf which was not labeled, or on lower parts of the main stem cowpea show ureolytic capacity throughout development, activisituated some 15 cm below the nearest fed leaflets (Fig. 4). In all ties are particularly high in the root and leaf tissues of nodulated cases, extracts of aphids collected 90 min after applying [2- plants following the onset of ureide export to the shoot (see also "4Clallantoin contained 14C ureide. The total amount of 14C re- 5). The high rates of activity in these organs presumably reflect covered and the proportion of this as ureide (1.5-58.5% of total the high requirement for urea metabolism in nodulated plants due to the predominance of ureides as a nitrogen source from the root. '4C) varied markedly between sites. Translocation of allantoin over shorter distances was demon- In non-nodulated plants grown with nitrate, where ureides have strated by collecting aphids feeding on veins of the central part of been shown to be present at much lower levels than in effectively 0.2
Table V. Recovery of '4C in Ethanol Extracts from Aphids Feeding on the Leaflets and Fruits of Cut Shoots of Cowpea Supplied 4 pCi (2-'4C)Allantoin in the Transpiration Stream in the Light The labeled solution was taken up by the shoots within 20 min and was replaced by distilled H20 for a further 70 or 160 min before the aphids were harvested. Shoots had two mature leaves each subtending two well developed fruits. FeigTime FeedingTime
Fraction of Extract Aphid
Site of Aphid Collection
Ureideb Total Urea
ND, not detectable. h Allantoin plus allantoic acid. c Values in parentheses are percentage of total
1.40 (39.7) 9.03
Ureide Total Urea Ureide Total Urea Ureide
dpm x 10-3/loo mg aphid 2.79 NDa 0.38 (13.7)c 3.53
2.47 (27.4) 12.36 0.04 (0.3) 5.88 (47.8)
"4C as allantoin or urea. Table VI. Recovery of 14C in Ethanol Extractsfrom Aphids Feeding on the Lower Epidermis of Attached Cowpea and Soybean Leaflets on Which 4 ,uCi (2-'4C)Allantoin Was Applied to the Periphery of the Upper Epidermis Plants were kept in the light and aphids collected 60 or 90 min after applying the labeled solution. All collected aphids were feeding from the midregion of the leaflet at least 1.5 cm proximal to the upper labeled leaf margin. Feeding Fraction of Species 14C Content Time Aphid Extract min dpm x 10-J/O00 mg aphid Total 60 13.17 Cowpea NDa Urea 5.08 (39.0)c Ureide" 6.29 Total 60 Soybean ND Urea
677.62 (58.5) 9.48 (3.2)
90 r. . L- ' *
sites of aphid
collection 3uCi( 2-14C)
allantoin applied to upper surface
FIG. 4. Recovery of 14C and proportion as 14C ureide (allantoin and allantoic acid) in extracts of aphids collected from feeding sites on an intact, nodulated cowpea plant supplied 3 uCi [2-'4C]allantoin to each three leaflets of uppermost trifoliolates. Aphids were collected 90 to 110 min after application of labeled substrate to upper leaflet surfaces.
nodulated plants (2), ureolytic activity proved to be lower in root and leaves. Similar variations in urease activity in response to the presence or absence of added urea have been described for a number of plants (see 17). This study demonstrates the recovery of 14C ureide as a signifi-
Ureide Total Urea Ureide
2.57 (40.4) 89.39 0.15 (0.2) 4.58 (5.1)
ND, not detectable.
hAllantoin plus allantoic acid. c Values in parentheses are the percentage of total
14C as allantoin or
cant proportion of the total "C in ethanolic extracts of aphids feeding on fruits, flowers, leaflets, peduncles, and stems, following application of [2-14C]allantoin to the upper surface of leaflets (Table VI, Fig. 4). It is concluded that ureide, as allantoin or allantoic acid, is effectively loaded onto phloem streams moving upward to fruits or downward to the root system. The parallel observations on the fate of transpirationally fed allantoin indicate that ureides are also exchanged freely from xylem to phloem probably using similar mechanisms and routings to those suggested for xylem-to-phloem transfer of amides in L. albus (3, 4,
13). Due to metabolism and utilization of ureides within the aphid, the proportion of 14C recovered as allantoin and allantoic acid at any site is likely to be a minimum value and is likely to underes-
timate seriously the ureide content of the translocation stream. Clearly, too, ureides are readily metabolized by stems and petioles and leaflets. Thus, depending on the relationship between the source organ and the collection site and on the residence time of the labeled phloem sap within the aphids prior to collection, the wide variation in the 4C ureide content of aphids found in the feeding experiments could be expected. The high values (58.5%, Fig. 4) indicate, however, that not only are ureides mobile in phloem, but that they may constitute a significant source of translocated nitrogen for protein synthesis in phloem-fed organs. Acknowledgments-Skilled technical assistance of E. Raisins, M. Sartori, D. Waldie, and L. Owen is gratefully acknowledged.
8. 9. 10. 11. 12.
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