Transport of organic solutes in Phloem and xylem of a nodulated legume

Collections of xylem exudate of root stumps or detached nodules, and of phloem bleeding sap from stems, petioles, and fruits were made from variously aged plants of Lupinus albus L. relying on nodules for their N supply. Sucrose was the major organic solute of phloem, asparagine, glutamine, serine, aspartic acid, valine, lysine, isoleucine, and leucine, the principal N solutes of both xylem and phloem. Xylem sap exhibited higher relative proportions of asparagine, glutamine and aspartic acid than phloem sap, but lower proportions of other amino acids. Phloem sap of petioles was less concentrated in asparagine and glutamine but richer in sucrose than was phloem sap of stem and fruit, suggesting that sucrose was unloaded from phloem and amides added to phloem as translocate passed through stems to sinks of the plant. Evidence was obtained of loading of histidine, lysine, threonine, serine, leucine and valine onto phloem of stems but the amounts involved were small compared with amides. Analyses of petiole phloem sap from different age groups of leaves indicated ontogenetic changes and effects of position on a shoot on relative rates of export of sucrose and N solutes. Diurnal fluctuations were demonstrated in relative rates of loading of sucrose and N solutes onto phloem of leaves. Daily variations in the ability of stem tissue to load N onto phloem streams were of lesser amplitude than, or out of phase with fluctuations in translocation of N from leaves. Data were related to recent information on C and N transport in the species.     

Amino Acid Release from the Seed Coat of Developing Seeds of Vicia faba and Pisum sativum

After removal of the embryo from developing ovules of Viciafaba L. and Pisum sativum L., seed-coat exudates were collectedand the amino acid fraction of the exudate was analyzed. InV. faba, alanine was the most important compound of the aminoacid fraction. In P. sativum, alanine and glutamine were thetwo most important components, whereas only small amounts ofasparagine were present. Comparison with published data suggeststhat seed-coat exudates may differ from phloem sap in the relativeimportance of these amino acids. Pisum sativum, pea, Vicia faba, broad bean, amino acid transport, amino acid unloading, seed-coat exudate, seed development     

Characteristics of Sugar, Amino Acid and Phosphate Release from the Seed Coat of Developing Seeds of Vicia faba and Pisum sativum

After removal of the embryo from developing seeds of Vicia fabaL. and Pisum sativum L., the ‘empty’ ovules werefilled with a standard solution (pH 5.5). Seed coat exudatesof both species were collected during relatively long experiments(up to about 12 h) and the concentration of sugar (mainly sucrose),amino acids and phosphate in the exudate measured. A discussionis presented on the amino acid/sugar ratio and the phosphate/sugarratio in the seed coat exudate. A pretreatment (15 min) withp-chloromercuribenzenesulphonic acid (PCMBS) reduced the releaseof sugar, amino acids and phosphate from broad bean seed coats.After excision of ‘empty’ ovules of Vicia faba andPisum sativum from the maternal plant, 2–4 h after thistreatment a strong difference became visible between sucroserelease from excised seed coats and sucrose release from attachedseed coats. Similarly, when the rate of phloem transport ofsucrose into an ‘empty’ ovule of Vicia faba or Pisumsativum was reduced by a sub-optimal mannitol concentrationin the solution, a reduced rate of sugar release from the seedcoat could be observed. Excision and treatment with a sub-optimalmannitol concentration reduced the release of amino acids toa lesser extent than for sucrose. These treatments did not reducethe rate of phosphate release from the seed coat. Key words: Seed development, Seed coat exudate, Phloem transport     

Nitrogen nutrition and metabolic interconversions of nitrogenous solutes in developing cowpea fruits

Budgets for import and utilization of ureide, amides, and a range of amino acids were constructed for the developing first-formed fruit of symbiotically dependent cowpea (Vigna unguiculata [L.] Walp. cv Vita 3). Data on fruit total N economy, and analyses of the xylem and phloem streams serving the fruit, were used to predict the input of various solutes while the compositions of the soluble and protein pools of pod, seed coat, and embryo were used to estimate the net consumption of compounds. Ureides and amides provided virtually all of the fruit's N requirements for net synthesis of amino compounds supplied inadequately from the parent plant. Xylem was the principal source of ureide to the pod, while phloem was the major source of amides to pod and seed. All fruit parts showed in vitro activity of urease (EC 3.5.1.5), allantoinase (EC 3.5.2.5), asparaginase (EC 3.5.11), ammonia-assimilating enzymes and aspartate and alanine aminotransferases (EC 2.61.1 and EC 2.6.1.1.2). Asparagine:pyruvate aminotransferase (EC 2.6.1.14) was recovered only from the pod. The pod was initially the major site for processing and incorporating N; later seed coats and finally embryos became predominant. Ureides were broken down mainly in the pod and seed coat. Amide metabolism occurred in all fruit organs, but principally in the embryo during much of seed growth. Seed coats released N to embryos mainly as histidine, arginine, glutamine, and asparagine, hardly at all as ureide. Amino compounds delivered in noticeably deficient amounts to the fruit were arginine, histidine, glycine, glutamate, and aspartate, while seeds received insufficient arginine, histidine, serine, glycine, and alanine. Quantitatively based schemes are proposed depicting the principal metabolic transformation accompanying N-flow between seed compartments during development.     

Effect of Potassium on Sucrose and Amino Acid Release from the Seed Coat of Developing Seeds of Pisum sativum

After removal of the embryo from developing seeds of Pisum sativum,the ‘empty’ ovules (seed coats without enclosedembryo) were filled with a solution (pH 5.5) containing mannitol(usually 400 mM) to which various salts were added. A solutioncontaining two isotopes ((a) [²H]-sucrose/[–¹⁴C]aminoisobutyricacid (AIB) or (b) [³H]valine/[₁₄C]asparagine mixture) was administeredto the plant via the petiole subtending the fruiting node, and[²H]solute and [¹⁴C]solute unloading from the seed coat wasmeasured, in pulse-labelling experiments of about 5 h. The presenceof 25 or 50 mM K⁺ in the ‘empty’ ovule enhancedthe release of sucrose from the seed coat particularly duringthe first hours of the experiment, but the stimulating effectof K⁺ on the release of labelled solutes derived from aminoacids was much smaller. The presence of 25 mM CaCl₂ did notaffect the release of sucrose or amino acids from the seed coat.The effect of K⁺ on sucrose and amino acid release is explainedas an inhibition of sucrose and amino acid resorption from theseed coat apoplast into seed coat cells, after unloading fromthe seed coat unloading sites. It is suggested that amino acidrelease is much less affected by K⁺ than sucrose release, becausefar less resorption of amino acids by seed coat parenchyma cellstakes place during amino acid transport into the seed coat cavity. Pisum sativum, pea, assimilate transport, assimilate unloading, seed-coat exudate, seed development, sucrose resorption, surgical treatment     

Transport of Photoassimilate in Pea Ovules

Interpretation of tracer washout from an attached empty seedcoat depends on whether photoassimilate within the apoplastof the seed coat is absorbed by the seed coat tissues. Usingsucrose trapping procedures, we were unable to see any evidencefor sucrose uptake from the seed coat apoplast which would beneeded to provide the seed coat with its carbohydrate requirementsif phloem unloading were into the apoplast. Once released intothe apoplast photoassimilate is unavailable to the seed coattissue. Changes between equimolar solutions of sorbitol andsorbitol/sucrose mixes induced small transient responses inseed coat unloading which suggest that sorbitol and sucrosehad different reflection coefficients and gave water relationresponses with rapid, and fatiguable, osmoregulation withinthe seed coat. Immediate inhibition of seed coat unloading with PCMBS is reported,followed by inhibition of import into the entire pod. PCMBSappears to be xylem mobile, thereby quickly being dispersedthroughout the entire experimental pod. A complex CCCP responseis reported, which is consistent with immediate inhibition ofsymplastic transport followed by membrane disruption. AlthoughCCCP inhibited seed coat unloading, there was no effect on ovuleimport. This has been interpreted as evidence that the seedcoat has an active role in control of photoassimilate importinto ovules. Key words: Pisum sativum, phloem unloading, seed coat unloading     

Allantoinase and asparaginase activities in maturing fruits of nodulated and non -nodulated soybeans

Immature fruits of soybean ( Glycine max L. Merr. cv. Santa Rosa) were found to contain high ureide/amino acid ratios for plants dependent on atmospheric nitrogen (nodulated), but low ratios for plants cultivated on NO⁻₃ (non-nodulated). The pod tissue was responsible for almost all this difference, which reflects the N metabolism of these plants (nodulated:urcide-based; NO⁻₃ dependent: asparagine based). The capacity of fruit tissues to utilize ureides and asparagine via allantoinase (EC 3.5.2.5) and asparaginase (EC 3.5.1.1) was investigated during fruit development. Both enzymes were present in crude desalted extracts of all parts of the fruit analysed (pod, cotyledon and seed coat). Asparaginase was detected in pod tissue only at early stages and with very low activities, whereas high activities of allantoinase (up to 20 [imol pod⁻¹ h⁻¹) were present after this organ reached full expansion. The cotyledons contained most of the allantoinase and asparaginase activities of the seed, the highest activities being recorded during the period of rapid protein accumulation. There was little difference in the activity patterns for nodulated and NO⁻₃-grown plants, despite the large difference in nitrogen nutrition of the fruits.     

Concentrations of sucrose and nitrogenous compounds in the apoplast of developing soybean seed coats and embryos

The apoplast of developing soybean (Glycine max cv Hodgson) embryos and seed coats was analyzed for sucrose, amino acids, ureides, nitrate, and ammonia. The apoplast concentration of amino acids and nitrate peaked during the most rapid stage of seed filling and declined sharply as the seed attained its maximum dry weight. Amino acids and nitrate accounted for 80 to 95% of the total nitrogen, with allantoin and allantoic acid either absent or present in only very small amounts. Aspartate, asparagine, glutamate, glutamine, serine, alanine, and γ-aminobutyric acid were the major amino acids, accounting for over 70% of the total amino acids present. There was a nearly quantitative conversion of glutamine to glutamate between the seed coat and embryo, most likely resulting from the activity of glutamate synthase found to be present in the seed coat tissue. This processing of glutamine suggests a partly symplastic route for solutes moving from the site of phloem unloading in the seed coat to the embryo.     

Mobilization of Minerals to Developing Seeds of Legumes

The mineral nutrition of fruiting plants of Pisum sativum L.,Lupinus albus L. and Lupinus angustifolius L. is examined insand cultures supplying adequate and balanced amounts of essentialnutrients. Changes in content of specific minerals in leaves,pods, seed coat, and embryo are described. P, N and Zn tendto increase precociously in an organ relative to dry matteraccumulation, other elements more or less parallel with (K,Mn, Cu, Mg and Fe) or significantly behind (Ca and Na) dry weightincrease. Some 60–90 per cent of the N, P and K is lostfrom the leaf, pod and seed coat during senescence, versus 20–60per cent of the Mg, Zn, Mn, Fe and Cu and less than 20 per centof the Na and Ca. Mobilization returns from pods are estimatedto provide 4–39 per cent of the seeds' accumulations ofspecific minerals, compared with 4–27 per cent for testatransfer to the embryo. Endosperm minerals are of only minorsignificance in embryo nutrition. Comparisons of the mineral balance of plant parts of Lupinusspp. with that of stem xylem sap and fruit tip phloem sap supportthe view that leaves and pod are principal recipients of xylem-borneminerals and that export from these organs via phloem is themajor source of minerals to the seeds. Endosperm and embryodiffer substantially in mineral compostition from phloem sap,suggesting that selective uptake occurs from the translocationstream during seed development. Considerable differences are observed between species in mineralcomposition of plant organs and in the effectiveness of transferof specific minerals to the seeds Differences between speciesrelate principally to Ca, Na and certain trace elements.     

Phloem Loading and Metabolism of Xylem-Borne Amino Compounds in Fruiting Shoots of a Legume

Cut, fruiting shoots of Lupinus albus L. supplied with ¹⁴C-and ¹⁵N-labelled L-asparagine, L-glutamine, L-aspartic acid,or L-glutamic acid through the transpiration stream readilytransferred the labelled carbon and nitrogen of each compoundto pods and seeds of fruits. A time course of labelling of phloemsap collected from petioles and fruit tips following feedingof labelled asparagine indicated that xylem to phloem exchangein leaflets was an immediate and effective route of transferof the amide to fruits and that this and the loading onto phloemof additional asparagine from unlabelled pools of the amidein stems furnished a major source of the nitrogen for fruitfilling. Xylem to phloem exchange of nitrogen was accomplishedin different ways for each amino acid. The amide nitrogen ofasparagine was transferred mainly in the unmetabolized compound,the nitrogen of aspartate and glutamate largely in a wide rangeof amino acids synthesized in the leaf, and the amide nitrogenof glutamine was transferred in a manner intermediate betweenthese extremes. Glutamine and asparagine were the principalphloem solutes labelled with nitrogen from any of the suppliedcompounds, but the photosynthetically produced amino acids,glutamate, aspartate, serine, alanine, and valine also became¹⁵N-labelled in phloem. The main pathway for glutamine synthesisin vegetative parts of the shoot appeared to be by amidationof glutamate, but asparagine was not considered to be derivedsimilarly from aspartate. Leaflets metabolized glutamine morereadily than asparagine, but in each case the amide nitrogenwas used for synthesis of a variety of amino acids and the carbonwas recovered largely in non-amino compounds.     

Phloem Unloading within the Seed coat of Pisum sativum Observed using Surgically Modified Seeds

Phloem unloading in pea seed coats was observed by removingthe embryos from developing seeds and washing the attached coatswith a weakly buffered solution. The quantity of labelled photosynthateappearing in the washing solution varied immediately when thesolute concentration was changed, and is shown to be an osmoticresponse. This response is predicted by the Münch theoryof phloem transport with concentration dependent unloading.Respiratory inhibitors and the sulphydryl modifying reagentPCMBS had a slow effect upon the washout of tracer, which arrivedwithin the seed coat prior to inhibitor application, but completelystopped any washout of tracer arriving after its application.This time-course suggests that the inhibitors were not directlyinhibiting unloading, but preventing further tracer from enteringthe region of unloading within the seed coat. Phloem unloadingwithin the seed coats of Pisum appears to be passive and notdependent upon a PCMBS-sensitive carrier. Key words: Pisum sativum, seeds, phloem unloading     

Synthesis and Interconversion of Amino Acids in Developing Cotyledons of Pea (Pisum sativum L.)

Freshly isolated cotyledons from 10-day developing pea (Pisum sativum) seeds were fed radiolabeled precursors for 5 hours, and the specific radioactivity of the free and total protein amino acids was determined using a dansylation procedure. When the seven most abundant amino acids in phloem exudate of pea fruits (asparagine, serine, glutamine, homoserine, alanine, aspartate, glycine) were fed singly, their carbon was distributed widely among the aliphatic amino acids, proline and tryptophan; sporadic labeling of tyrosine and histidine also occurred. Feeding of glucose led to relatively greater labeling of aromatic amino acids including phenylalanine. The data support the involvement of known plant pathways in these interconversions. Labeling patterns were consistent with participation of the cyanoalanine pathway in the conversion of serine to homoserine, and with the synthesis of histidine from adenosine. All of the labeled amino acids were incorporated into protein.     

The Effect of Fruit Shading on Yield in Pisum sativum L.

Fruits of Pisum sativum L. cv. Feltham First which initiatesonly one flower per flowering node, were selectively shadedunder varying levels of defoliation. The purpose of the experimentswas to ascertain whether the foliage could compensate for lossof the fruit's contribution to its own growth. There was evidenceof this, but fruit and seed weight per fruit and per plant werereduced by fruit shading at all levels of defoliation. The lossin yield due to shading suggested that the contribution fromthe fruit was at least 12 per cent. The number of seeds whichdeveloped to maturity was the yield component most affectedby treatment. There was no evidence to suggest that shadinghad a different quantitative effect on final weight at differentnodes, but it did increase flower abscission at the first foweringnode in an experiment done at low radiant exposure. In an experimentat higher radiant exposure, very few flowers abscised at theearlier nodes, but leaflet removal reduced final fruit yieldat the first flowering node to a greater degree than at thesecond. These differential responses could contribute to variabilityof seed size in a crop of vining peas. Pisum sativum, pea, fruit, pod, light, shading, photosynthesis, yield     

Translocation of Nitrogenous Compounds in Symbiotic and Nitrate-Fed Amide-Exporting Legumes

Peoples, M. B., Sudin, M. N. and Herridge, D. F. 1987. Translocationof nitrogenous compounds insymbiotic and nitrate-fed amide-exportinglegumes.–J. exp. Bot. 38: 567–579. The transport of nitrogen from the roots and nodules of chickpea(Cicer anetinum L.), lentil (Lens culinaris Medic), faba bean(Vicia faba L.) and pea (Pisum sativum L.) was examined in glasshouse-grownplants supplied either with nitrate-free nutrients or with nutrientssupplemented with 1,2,4 or 8 mol m^-3153N-nitrate. A sixth treatmentcomprised uninoculated plants supplied with 8–0 mol m^-31513N-nitrate. For each species, more than 75% of the nitrogenwas exported from the nodules as the amides, asparagine andglutamine. In fully symbiotic plants, the amides also dominatednitrogen transport to the shoot When N2 fixation activity wasdecreased by the addition of nitrate to the rooting medium,the N-composition of xylem exudate and stem solutes changedconsiderably. The relative concentrations of asparagine tendedto increase in the xylem whilst those of glutamine were reduced;the levels of nitrate increased in both xylem exudate and thesoluble nitrogen pool of the stem with a rise in nitrate supply.The changes in relative nitrate contents reflected generallythe contributions of root and shoot to overall nitrate reductaseactivity at the different levels of nitrate used. The relationshipsbetween the relative contents of xylary or stem nitrate andamino nitrogen and the plants' reliance on N2 fixation (determinedby the ¹⁵N isotope dilution procedure) were examined. Data suggestthat compositional relationships based on nitrate may be reasonableindicators of symbiotic dependence for all species under studyexcept faba bean when greater than 25% of plant nitrogen wasderived from N2 fixation. Key words: Nitrogen, translocation, legumes     

Allantoin and Allantoic Acid in Tissues and Stem Exudate from Field-grown Soybean Plants

Samples of stem exudate and plant tissue collected from field-grown soybean (Glycine max [L.] Merr.) plants were analyzed for allantoin and allantoic acid. Nitrogen in nitrate plus amino acids exceeded ureide N concentration in stem exudate prior to flowering. During all of reproductive development (from about 40 days after planting until maturity), ureide N concentration was two to six times greater than amino acid plus nitrate N concentration. Allantoin and allantoic acid, not asparagine, are the principal forms of nitrogen transported from nodulated roots to shoots of the soybean plant. During pod and seed development ureide N comprised as high as 2.3, 37.7, and 15.8% of total N in leaf blades, stems + petioles, and fruits, respectively. The concentration of ureide in stems and fruits declined to nearly zero at maturity.     

Nitrogen redistribution during grain growth in wheat (Triticum aestivum L.)

The technique of EDTA-enhanced phloem exudation (King and Zeevaart, 1974: Plant Physiol. 53, 96–103) was evaluated with respect to the collection and identification of amino acids exported from senescing wheat leaves. Whilst the characteristics of the exudate collected conform with many of the accepted properties of phloem exudate, unexpectedly high molar proportions of phenylalanine and tyrosine were observed. By comparing exudation into a range chelator solutions with exudation into water, the increased exudation of phenylalanine and tyrosine relative to the other amino acids occurring when ethylene-diaminetetracetic acid was used, was considered to an artefact.In plants thought to be relying heavily on mobilisation of protein reserves to satisfy the nitrogen requirements of the grain, the major amino acids present in flag-leaf phloem exudate were glutamate, aspartate, serine, alanine and glycine. Only small proportions of amides were present until late in senescence when glutamine became the major amino acid in phloem exudate (25 molar-%). Glutamine was always the major amino acid in xylem sap (50 molar-%).The activities of glutamine synthetase (EC 6.3.1.2), glutamate synthase (EC 1.4.7.1), glutamate dehydrogenase (EC 1.4.1.3) and asparagine synthetase (EC 5.3.5.4) were measured in the flag leaf throughout the grain-filling period. Glutamine synthetase and glutamate-synthase activities declined during this period. Glutamate-dehydrogenase activity was markedly unchanged despite variation in the number of multiple forms visualised after gel electrophoresis. The activity of the enzyme reached a peak only very late in the course of senescence of the flag leaf. No asparagine-synthetase activity could be detected in the flag leaf during the grain-filling period.II. Peoples et al. (1980)     

Unusual Network of Internal Phloem in the Pod Mesocarp of Cowpea [Vigna unguiculata (L.) Walp. (Fabaceae)]

A mycelium-like network of internal phloem was observed in theinner mesocarp of the lateral pod walls of the fruit of certaingenotypes of cowpea [Vigna unguiculata (L.) Walp.] In the cultivarVita 3, the network consists of single, or rarely double, strandsof sieve elements and associated phloem parenchyma, orientedmainly parallel with the fibres of the adjacent endocarp, andstretching marginally beyond the sheets of fibres to connectabove and below with the outermost phloem of the longitudinalstrands of the dorsal and ventral sutures of the fruit. Theinternal phloem network does not relate conformationally to,or interconnect with the conventional (xylem+phloem) vasculatureof the mid mesocarp of the pod wall. In Vita 3, sieve elementsdifferentiate in the internal phloem after those in the majorveins of the pod, but before the presumptive endocarp fibrescommence wall thickening. The pod walls of twenty-one otherspecies of legumes proved negative for internal phloem, whileof nine varied genotypes of cowpea examined, six proved positive,three negative for the trait. Presence of internal phloem incowpea is not always associated with presence of endocarp fibresor necessarily with large fruits with large seeds. Possiblefunctions suggested for the phloem network are to provide assimilatesfor fibre wall thickening or to transport solutes to or fromsites of temporary storage in the fleshy inner layers of thepod wall. Internal phloem, legume fruit, translocation, mesocarp, pod wall, Vigna unguiculata, cowpea     

Modeling C and N transport to developing soybean fruits

Xylem sap and phloem exudates from detached leaves and fruit tips were collected and analyzed during early pod-fill in nodulated soybeans (Glycine max (L.) Merr. cv Wilkin) grown without (−N) and with (+N) NH₄NO₃. Ureides were the predominant from (91%) of N transported in the xylem of −N plants, while amides (45%) and nitrate (23%) accounted for most of the N in the xylem of +N plants. Amino acids (44%) and ureides (36%) were the major N forms exported in phloem from leaves in −N plants, but amides (63%) were most important in +N plants. Based on the composition of fruit tip phloem, ureides (55% and 33%) and amides (26% and 47%) accounted for the majority of N imported by fruits of −N and +N plants, respectively.

C:N weight ratios were lowest in xylem exudate (1.37 and 1.32), highest in petiole phloem (24.5 and 26.0), and intermediate in fruit tip exudate (12.6 and 12.1) for the −N and +N treatments, respectively. The ratios were combined with data on fruit growth and respiration to construct a model of C and N transport to developing fruits. The model indicates xylem to phloem transfer provides 35% to 52% of fruit N. Results suggest the phloem entering fruits oversupplies their N requirement so that 13% of the N imported is exported from fruit in the xylem.

     

Metal Complexation in Xylem Fluid : I. CHEMICAL COMPOSITION OF TOMATO AND SOYBEAN STEM EXUDATE

Xylem fluid was analyzed for numerous solutes to characterize chemically the sap as a medium for forming and transporting metal complexes. The stem exudate was collected hourly for 8 hours from topped 31-day-old soybean (Glycine max L. Merr.) and 46-day-old tomato (Lycopersicon esculentum Mill.) plants grown in normal (0.5 micromolar) and Za-phytotoxic nutrient solutions. Soybean plants were grown in the normal and high-Zn solutions for 24 days; tomato plants were grown for 32 days. The exudate was analyzed for seven organic acids, 22 amino acids, eight inorganic solutes, apparent ionic strength, and pH. Significant changes in many solutes occurred over the 8-hour sampling period. These fluctuations depended on plant species, individual solute, and Zn treatment, and demonstrated that extrapolation of xylem-fluid analyses to whole-plant xylem sap is valid only for sap samples collected shortly after topping a plant. Exudate pH decreased over the 8-hour period for both species; exudate ionic strength increased for tomato and decreased for soybean. At the normal-Zn treatment (0 to 1 hour), the highest acid micromolar concentrations in soybean exudate were: asparagine, 2,583; citric, 1,706; malic, 890; and malonic, 264. Under the same conditions, the highest acid micromolar concentrations in tomato exudate were: maleic, 1,206; malic, 628; glutamine, 522; citric, 301; and asparagine, 242. Cysteine and methionine were above detection limits only in soybean exudate. Zinc phytotoxicity caused significant changes in many solutes. The analyses reported here provide a comprehensive data base for further studies on metal-complex equilibria in xylem fluid.     

Spontaneous Phloem bleeding from cryopunctured fruits of a ureide-producing legume

The vasculature of the dorsal suture of cowpea (Vigna unguiculata [L.] Walp) fruits bled a sugar-rich exudate when punctured with a fine needle previously cooled in liquid N₂. Bleeding continued for many days at rates equivalent to 10% of the estimated current sugar intake of the fruit. A phloem origin for the exudate was suggested from its high levels (0.4-0.8 millimoles per milliliter) of sugar (98% of this as sucrose) and its high K⁺ content and high ratio of Mg²⁺ to Ca²⁺. Fruit cryopuncture sap became labeled with ¹⁴C following feeding of [¹⁴C]urea to leaves or adjacent walls of the fruit, of ¹⁴CO₂ to the pod gas space, and of [¹⁴C] asparagine or [¹⁴C]allantoin to leaflets or cut shoots through the xylem. Rates of translocation of ¹⁴C-assimilates from a fed leaf to the puncture site on a subtended fruit were 21 to 38 centimeters per hour. Analysis of ¹⁴C distribution in phloem sap suggested that [¹⁴C]allantoin was metabolized to a greater extent in its passage to the fruit than was [¹⁴C] asparagine. Amino acid:ureide:nitrate ratios (nitrogen weight basis) of NO₃-fed, non-nodulated plants were 20:2:78 in root bleeding xylem sap versus 90:10:0.1 for fruit phloem sap, suggesting that the shoot utilized NO₃-nitrogen to synthesize amino acids prior to phloem transfer of nitrogen to the fruit. Feeding of ¹⁵NO₃ to roots substantiated this conclusion. The amino acid:ureide ratio (nitrogen weight basis) of root xylem sap of symbiotic plants was 23:77 versus 89:11 for corresponding fruit phloem sap indicating intense metabolic transfer of ureide-nitrogen to amino acids by vegetative parts of the plant.