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.     

Xylem to phloem transfer of solutes in fruiting shoots of legumes,studied by a phloem bleeding technique

Summary Comparisons were made of the levels of various solutes in xylem (tracheal) sap and fruit tip phloem sap of Lupinus albus (L.) and Spartium junceum (L.). Sucrose was present at high concentration (up to 220 mg ml^-1) in phloem but was absent from xylem whereas nitrate was detected in xylem (up to 0.14 mg ml^-1) but not in phloem. Total amino acids reached 0.5–2.5 mg ml^-1 (in xylem) versus 16–40 mg ml^-1 in phloem. Phloem: xylem concentration ratios for mineral nutrients (K, Na, Mg, Ca, Fe, Zn, Mn, Cu) spanned the range 0.7 to 20, the ratios generally reflecting an element's phloem mobility and its availability to the xylem from the roots.The accessibility of nitrate to xylem and phloem was studied in Lupinus. Increasing the nitrate supply to roots from 100 to 1000 mg NO₃–Nl^-1 increased nitrate spill over into xylem, but nitrate always failed to appear in phloem. However, phloem loading of small amounts of nitrate was induced by feeding 750 or 1000 mg NO₃–Nl^-1 directly to cut shoots via the transpiration stream. Transfer of reduced nitrogen to phloem was demonstrated by feeding ¹⁵NO₃ to shoots and recovering ¹⁵N-enriched amides and amino acids in phloem sap. Increased nitrate supply to roots led to increased amino acid levels in xylem and phloem but did not alter markedly the balance between individual amino acids.The fate of xylem-fed ¹⁴C-labelled asparagine, glutamine and aspartic acid and of photosynthetically fed ¹⁴CO₂ was studied in Spartium, with reference to phloem transport to seeds. Substantial fractions of the ¹⁴C of all sources appeared in non-amino compounds. [¹⁴C]asparagine passed largely in unchanged form to the phloem whereas the ¹⁴C from aspartic acid or glutamine appeared in phloem attached to other amino acids (e.g. asparagine and glutamic acid). Serine, asparagine and glutamine were the main amino compounds labelled in phloem sap after feeding ¹⁴CO₂. The wide distribution of ¹⁴C amongst free and bound amino acids of seeds suggested that extensive metabolism of phloem-borne solutes occurred in the fruits.     

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.     

Distribution and metabolism of xylem-borne ureido and amino compounds in developing soybean shoots

Pulse-chase feeding (30-120 minutes) of ¹⁴C-labeled nitrogenous compounds to cut transpiring shoots was used to investigate the early fate of the major xylem-borne solutes in N₂-fixing soybean (Glycine max) plants at the V₄ growth stage. By comparison with the foliar distribution of [¹⁴C]inulin (a xylem marker), it was determined that the phloem supply of allantoin, allantoic acid, asparagine, glutamine, aspartate, and arginine, respectively, provided about 20, 10, three, two, five, and 20 times the ¹⁴C delivered to the developing trifoliolate in the xylem stream. Recovery of unmetabolized asparagine, aspartate, and arginine in this indicator trifoliolate, and significant declines in the percentage of ¹⁴C from allantoic acid and allantoin recovered in the first trifoliolate, provided some support for the direct xylem-to-phloem transfer of these compounds, but did not preclude the involvement of indirect transfer. Data on stem retention and foliar distribution, expressed as a function of the relative xylem sap composition, indicated that ureides provide the major sources of nitrogen to all plant parts. There was no consistent distinction in distribution patterns between pairs of similar anionic and neutral compounds. The extent of xylem-to-phloem transfer among the ureido or the amino compounds was inversely related to its prominence in xylem sap.     

The Kinetics of Phloem Loading of Valine in the Shoot of a Nodulated Legume (Lupinus albus L. cv. Ultra)

Phloem loading of several amino acids (D- and L-Val, Arg, Asn,Asp, Leu) was studied in shoots of L. albus using a phloem bleedingtechnique on both intact plants and detached shoots fed viathe transpiration stream. Val was singled out for intensivestudy due to the minimal amount of metabolism it underwent inthe shoot For the amino acids studied, the relationship between xylem,phloem, and leaflet concentrations was determined by the interactionof rates of xylem supply, metabolism, and export. At elevatedxylem fluid concentrations, low rates of loading of D-Val intothe phloem and little metabolism in the tissues resulted inhigh levels in the leaflets. For other amino acids (Arg, Asp,Leu) rapid metabolism in the leaflets prevented a build-up inconcentration in either phloem or leaflets. Asn was rapidlytransferred to the phloem, thus high levels in the xylem leadto high concentrations in the phloem without greatly affectingleaflet concentrations. L-Val responded in a manner intermediatebetween Asn and D-Val. A detailed study of L-Val showed it to be loaded into the phloemagainst a concentration gradient in both stem and leaflets.Some of this Val originated from the transpiration stream atboth locations but in the leaflets as much as 64% of the Valoriginated from other sources, e.g. recent photosynthesis. L-Valsupplied to the phloem in the stem was derived from a largestorage pool and did not come directly from the xylem fluid.As a consequence the rate of stem loading was independent ofshort-term fluctuations in the xylem fluid Val concentration.L-Val entering the leaflets in the xylem initially bypassedthe large storage pool and was loaded directly into the phloem.However, after 350 min the pools had reached an equilibriumand rate of phloem loading was dependent on total leaflet concentration.     

6(5)CARBOXYFLUORESCEIN AS A TRACER OF PHLOEM SAP TRANSLOCATION

6(5)carboxyfluorescein (6(5)CF), a polar fluorescein with an apparent pK of 6.3, was introduced, as a pH 6.3 solution, into the apoplast of lamina or petioles of mature soybean leaves. Freehand sections were prepared at various times and immediately observed with a fluorescence microscope. 6(5)CF-associated fluorescence appeared in all sink organs, from shoot apex to roots. It was strictly confined to the phloem regions, even after 4 days. Its transport into young leaves ceased at approximately the time they underwent sink-to-source transition. It was never transported between two leaflets of the same leaf. Its transport was interrupted by phloem destruction. All these transport characteristics were highly reproducible, and were paralleled by those of ¹⁴C transport after application of (¹⁴C)sucrose to leaf surfaces. In contrast with 6(5)CF, fluorescein was transported between mature leaves, and between leaflets of the same leaf. It was not restricted to phloem, and often appeared in the xylem region. These results indicate that 6(5)CF can be used to monitor phloem sap translocation in real time, in short- and long-term experiments.     

Synthesis, Storage, and Utilization of Amino Compounds in White Lupin (Lupinus albus L.)

Changes in total N and in free amino compounds were followed during growth of nodulated white lupin. Leaflets contained the greatest fraction of plant N but had lower proportions (1 to 4%) of their N in soluble amino form than stem + petioles (10 to 27%) and reproductive parts (15 to 33%). Mobilization of free amino compounds from plant parts to fruits contributed at most only 7% of the total N intake of fruits, compared with 50% in mobilization of other forms of N and 43% from fixation during fruiting. Asparagine was usually the most abundant free amino compound in plant parts, followed by glutamine and alanine. Valine, glycine, isoleucine, aspartic acid and γ-aminobutyric acid comprised the bulk of the remaining soluble amino N. Composition of tissue pools of amino-N closely resembled that of xylem and phloem exudates. Data on N flow and utilization were combined with information on composition of transport fluids to quantify syntheses, exchanges, and consumptions of asparagine, glutamine, aspartic acid, and valine by organs of the 51- to 58-day plant. These amino compounds carried 56, 29, 5, and 2%, respectively, of the N exported from nodules and contributed in roughly commensurate proportions to transport exchanges and N increments of plant parts. There were, however, more than expected involvements of glutamine and valine in mobilization of N from lower leaves, of asparagine in xylem to phloem transfer, and of aspartic acid in cycling of N through the root, and there was a less than expected participation of aspartic acid in xylem to phloem transfer and in phloem translocation to the shoot apex. The significance of these differences is discussed.     

The extrafloral nectaries of cowpea (Vigna unguiculata (L.) Walp.) II. Nectar composition,origin of nectar solutes,and nectary functioning

Nectar was collected from the extrafloral nectaries of leaf stipels and inflorescence stalks, and phloem sap from cryopunctured fruits of cowpea plants. Daily sugar losses as nectar were equivalent to only 0.1–2% of the plant's current net photosynthate, and were maximal in the fourth week after anthesis. Sucrose:glucose:fructose weight ratios of nectar varied from 1.5:1:1 to 0.5:1:1, whereas over 95% of phloem-sap sugar was sucrose. [¹⁴C]Sucrose fed to leaves was translocated as such to nectaries, where it was partly inverted to [¹⁴C]glucose and [¹⁴C]fructose prior to or during nectar secretion. Invertase (EC 3.2.1.26) activity was demonstrated for inflorescence-stalk nectar but not stipel nectar. The nectar invertase was largely associated with secretory cells that are extruded into the nectar during nectary functioning, and was active only after osmotic disruption of these cells upon dilution of the nectar. The nectar invertase functioned optimally (phloem-sap sucrose as substrate) at pH 5.5, with a starting sucrose concentration of 15% (w/v). Stipel nectar was much lower in amino compounds relative to sugars (0.08–0.17 mg g^-1 total sugar) than inflorescence nectar (22–30 mg g^-1) or phloem sap (81–162 mg g^-1). The two classes of nectar and phloem sap also differed noticeably in their complements of organic acids. Xylem feeding to leaves of a range of ¹⁴C-labelled nitrogenous solutes resulted in these substrates and their metabolic products appearing in fruit-phloem sap and adjacent inflorescence-stalk nectar. ¹⁴C-labelled asparagine, valine and histidine transferred freely into phloem and appeared still largely as such in nectar. ¹⁴C-labelled glycine, serine, arginine and aspartic acid showed limited direct access to phloem and nectar, although labelled metabolic products were transferred and secreted. The ureide allantoin was present in phloem, but absent from both types of nectar. Models of nectary functioning are proposed.     

Glutamine Transfer from Xylem to Phloem and Translocation to Developing Leaves of Populus deltoides

The distribution of ¹⁴C from xylem-borne [¹⁴C]glutamine, the major nitrogen compound moving in xylem sap of cottonwood (Populus deltoides Bartr. ex Marsh), was followed in rapidly growing shoots with a combination of autoradiographic, microautoradiographic, and radioassay techniques. Autoradiography and ¹⁴C analyses of tissues showed that xylem-borne glutamine did not move with the transpiration stream into mature leaves. Instead, most of it was transferred from xylem to phloem in the upper stem and then translocated to young developing tissues. Microautoradiography showed that metaxylem parenchyma, secondary xylem parenchyma, and rays were the major areas of uptake from xylem vessels in the stem. Accumulation in phloem (high ¹⁴C concentrations in sieve tubes) took place in internodes subtending recently mature leaves. Little ¹⁴C from xylem-borne glutamine was found in phloem of mature leaves, which indicates restricted retransport of glutamine that did enter the leaf. In the primary tissues of the upper stem, most ¹⁴C was found in the phloem. Cottonwood stems have an efficient uptake and transfer system that enhances glutamine movement to developing tissues of the upper stem.     

Nitrogen Nutrition and Xylem Transport of Nitrogen in Ureide-producing Grain Legumes

Xylem sap composition was examined in nodulated and nonnodulated cowpea (Vigna unguiculata [L.] Walp.) plants receiving a range of levels of NO₃ and in eight other ureide-forming legumes utilizing NO₃ or N₂ as sole source of nitrogen. A ¹⁵N dilution technique determined the proportions of plant nitrogen derived from N₂ in the nodulated cowpeas fed NO₃. Xylem sap composition of NO₃-fed, nodulated cowpea varied predictably with the relative extents to which N₂ and NO₃ were being utilized. The ratios of asparagine to glutamine (N/N) and of NO₃ to ureide (N/N) in xylem sap increased with increasing dependence on NO₃ whereas per cent of xylem nitrogen as ureide and the ratio of ureide plus glutamine to asparagine plus NO₃ (N/N) in xylem sap increased with increasing dependence on N₂ fixation. The amounts of NO₃ and ureides stored in leaflets, stems plus petioles, and roots of cowpea varied in a complex manner with level of NO₃ and the presence or absence of N₂ fixation. All species showed higher proportions of organic nitrogen as ureide and several-fold lower ratios of asparagine to glutamine in their xylem sap when relying on N₂ than when utilizing NO₃. In nodulated (minus nitrate) cowpea and mung bean (Vigna radiata [L.] Wilczek) the percentage of xylem nitrogen as ureide remained constant during growth but the ratio of asparagine to glutamine varied considerably. The biochemical significance of the above differences in xylem sap composition was discussed.     

Modeling the transport and utilization of carbon and nitrogen in a nodulated legume

An empirical modeling technique was developed for depicting quantitatively the transport and partitioning of photosynthetically fixed C and symbiotically fixed N during 10-day intervals of a 40-day period in the growth of nodulated plants of white lupin (Lupinus albus L. cv. Ultra). Model construction utilized data for C and N consumption of plant parts and C:N weight ratios of the xylem and phloem fluids serving specific plant organs. Formulas were derived from calculating the net transport of C and N between plant parts in xylem and phloem. The models provided quantitative information on the dependence of growing organs on xylem and phloem for their supply of C and N, the cycling of N through leaflets and of C through nodules, the extent of direct incorporation of fixed N into growing nodules, and the involvement of N from shoot translocate in the nutrition of the nodulated root. Stem plus petioles abstracted considerably more N from xylem than expected from their transpirational activity. Xylem to phloem transfer of recently fixed N in mature stem and petioles was substantiated by the models, being depicted as a device for dispensing N to growing parts of the shoot extra to that attracted transpirationally in xylem or received as translocate from leaflets.     

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.     

Assimilation and Transport of Nitrogen in Nonnodulated (NO(3)-grown) Lupinus albus L

The response of nonnodulated white lupin (Lupinus albus L. cv. Ultra) plants to a range of NO₃ levels in the rooting medium was studied by in vitro assays of extracts of plant parts for NO₃ reductase (EC 1.6.6.1) activity, measurements of NO₃-N in plant organs, and solute analyses of root bleeding (xylem) sap and phloem sap from stems and petioles. Plants were grown for 65 days with 5 millimolar NO₃ followed by 10 days with 1, 5, 15, or 30 millimolar NO₃. NO₃ reductase was substrate-induced in all tissues. Roots contained 76, 68, 62 and 31% of the total NO₃ reductase activity of plants fed with 1, 5, 15, and 30 millimolar NO₃, respectively. Stem, petioles, and leaflets contained virtually all of the NO₃ reductase activity of a shoot, the activity in extracts of fruits amounting to less than 0.3% of the total enzyme recovered from the plant. Xylem sap from NO₃-grown nonnodulated plants contained the same organic solutes as from nodulated plants grown in the absence of combined N. Asparagine accounted for 50 to 70% and glutamine 10 to 20% of the xylem-borne N. The level of NO₃ in xylem sap amounted to 4, 13, 12, and 17% of the total xylem N at 1, 5, 15, and 30 millimolar NO₃, respectively. Xylem to phloem transfer of N appeared to be quantitatively important in supplying fruits and vegetative apices with reduced N, especially at low levels of applied NO₃. NO₃ failed to transfer in any quantity from xylem to phloem, representing less than 0.3% of the phloem-borne N at all levels of applied NO₃. Shoot organs were ineffective in storing NO₃. Even when NO₃ was supplied in great excess (30 millimolar level) it accounted for only 8% of the total N of stem and petioles, and only 2 and 1% of the N of leaflets and fruits, respectively.     

Pathways of assimilation and transfer of fixed nitrogen in coralloid roots of cycad-Nostoc symbioses

Freshly detached coralloid roots of several cycad species were found to bleed spontaneously from xylem, permitting identification of products of nitrogen transfer from symbiotic organ to host. Structural features relevant to the export of fixed N were described for Macrozamia riedlei (Fisch. ex Gaud.) Gardn. the principal species studied. Citrulline (Cit), glutamine (Gln) and glutamic acid (Glu), the latter usually in a lesser amount, were the principal translocated solutes in Macrozamia (5 spp.), Encephalartos (4 spp.) and Lepidozamia (1 sp.), while Gln and a smaller amount of Glu, but no Cit were present in xylem sap of Bowenia (1 sp.),and Cycas (2 spp.). Time-course studies of ¹⁵N enrichment of the different tissue zones and the xylem sap of ¹⁵N₂-pulse-fed coralloid roots of M. riedlei showed earlier ¹⁵N incorporation into Gln than into Cit, and a subsequent net decline in the ¹⁵N of Gln of the coralloid-root tissues, whereas Cit labeling continued to increase in inner cortex and stele and in the xylem sap. Hydrolysis of the ¹⁵N-labeled Cit and Gln consistently demonstrated much more intense labeling of the respective carbamyl and amide groups than of the other N-atoms. Coralloid roots of M. riedlei pulse-fed ¹⁴CO₂ in darkness showed ¹⁴C labeling of aspartic acid (Asp) and Cit in all tissue zones and of Cit of xylem bleeding sap. Lateral roots and uninfected apogeotropic roots of M. riedlei and M. moorei also incorporated ¹⁴CO₂ into Cit. The ¹⁴C of Cit was restricted to the carbamyl-C. Comparable ¹⁵N₂ and CO₂-feeding studies on corallid roots of Cycas revoluta showed Gln to be the dominant product of N₂ fixation, with Asp and alanine as other major ¹⁴C-labeled amino compounds, but a total absence of Cit in labeled or unlabeled form.Abbreviations Ala alanine - Asp aspartic acid - Cit citrulline - Gln glutamine - Glu glutamic acid - Orn ornithine     

Photosynthetic formation of the aspartate family of amino acids in isolated chloroplasts

The metabolism of ¹⁴C-labeled aspartic acid, diaminopimelic acid, malic acid and threonine by isolated pea (Pisum sativum L.) chloroplasts was examined. Light enhanced the incorporation of [¹⁴C] aspartic acid into soluble homoserine, isoleucine, lysine, methionine and threonine and protein-bound aspartic acid plus asparagine, isoleucine, lysine, and threonine. Lysine (2 millimolar) inhibited its own formation as well as that of homoserine, isoleucine and threonine. Threonine (2 millimolar) inhibited its own synthesis and that of homoserine but had only a small effect on isoleucine and lysine formation. Lysine and threonine (2 millimolar each) in combination strongly inhibited their own synthesis as well as that of homoserine. Radioactive [1,7-¹⁴C]diaminopimelic acid was readily converted into [¹⁴C]threonine in the light and its labeling was reduced by exogenous isoleucine (2 millimolar) or a combination of leucine and valine (2 millimolar each). The strong light stimulation of amino acid formation illustrates the point that photosynthetic energy is used in situ for amino acid and protein biosynthesis, not solely for CO₂ fixation.     

Cytokinins in the Phloem Sap of White Lupin (Lupinus albus L.)

Cytokinin-like activity in samples of xylem and phloem sap collected from field-grown plants of white lupin (Lupinus albus L.) over a period of 9 to 24 weeks after sowing was measured using the soybean hypocotyl callus bioassay following paper chromatographic separation. The phloem sap was collected from shallow incisions made at the base of the stem, the base of the inflorescence (e.g. stem top), the petioles, and the base and tip of the fruit. Xylem sap was collected as root exudate from the stump of plants severed a few centimeters above ground level. Concentration of cytokinin-like substances was highest in phloem sap collected from the base of the inflorescence and showed an increase over the entire sampling period (from week 10 [61 nanogram zeatin equivalents] to week 24 [407 nanogram zeatin equivalents]). Concentrations in the xylem sap and in the other phloem saps were generally lower. Relatively high concentrations of cytokinin-like substances in petiole phloem sap (70 to 130 nanogram zeatin equivalents per milliliter) coincided in time with high concentrations in sap from the base of the inflorescence (see above). Concentrations in sap (phloem or xylem) from the base of the stem were very much lower. This finding is consistent with movement of cytokinins from leaves into the developing inflorescence and fruit, rather than direct input to the fruit from xylem sap. However, an earlier movement of cytokinins from roots into leaves via the xylem cannot be ruled out. Sap collected at an 18-week harvest was additionally separated by sequential C₁₈ reversed-phase high performance liquid chromatography → NH₂ normal phase high performance liquid chromatography, bioassayed, and then analyzed by electron impact gas chromatography-mass spectrometry. Identification of zeatin riboside and dihydrozeatin as two of the major cytokinins in combined sap samples was accomplished by gas chromatography-mass spectrometry-selected ion monitoring.     

The biosynthesis and translocation of 14C-IAA in Ricinus communis

The biosynthesis of ¹⁴C-IAA from ¹⁴C-tryptophan applied to abraded leaves of Ricinus communis and its subsequent export through the phloem were studied. Phloem sap was collected at intervals from incisions made in the stem below the IAA fed leaf. Any upward movement of label through the phloem or downward movement of phloem mobile compounds from leaves above the treated one were restricted by bark-ringing the plants.TLC and HPLC analyses of the collected sap indicate that some conversion of ¹⁴C-tryptophan to ¹⁴C-IAA had occurred. Subsequent GC-MS analysis of the HPLC purified samples of phloem sap revealed high levels of endogenous IAA transported from the fed leaf. The high ratio of unlabelled/labelled IAA in the phloem sap makes unequivocal confirmation by GC-MS of the predicted biosynthesis of ¹⁴C-IAA impossible. It is postulated that IAA is synthesised from tryptophan in mature leaves and exported to developing sink tissues with the flow of photoassimilates in the phloem.     

d-Glycerate Transport by the Pea Chloroplast Glycolate Carrier : Studies on [1-C]d-Glycerate Uptake and d-Glycerate Dependent O(2) Evolution

Rapid direct conversion of exogenously supplied [¹⁴C]aspartate to [¹⁴C] asparagine and to tricarboxylic cycle acids was observed in alfalfa (Medicago sativa L.) nodules. Aspartate aminotransferase activity readily converted carbon from exogenously applied [¹⁴C]aspartate into the tricarboxylic acid cycle with subsequent conversion to the organic acids malate, succinate, and fumarate. Aminooxyacetate, an inhibitor of aminotransferase activity, reduced the flow of carbon from [¹⁴C]aspartate into tricarboxylic cycle acids and decreased ¹⁴CO₂ evolution by 99%. Concurrently, maximum conversion of aspartate to asparagine was observed in aminooxyacetate treated nodules (30 nanomoles asparagine per gram fresh weight per hour. Metabolism of [¹⁴C]aspartate and distribution of nodulefixed ¹⁴CO₂ suggest that two pools of aspartate occur in alfalfa nodules: (a) one involved in asparagine biosynthesis, and (b) another supplying a malate/aspartate shuttle. Conversion of [¹⁴C]aspartate to [¹⁴C]asparagine was not inhibited by methionine sulfoximine, a glutamine synthetase inhibitor, or azaserine, a glutmate synthetase, inhibitor. The data did not indicate that asparagine biosynthesis in alfalfa nodules has an absolute requirement for glutamine. Radioactivity in the xylem sap, derived from nodule ¹⁴CO₂ fixation, was markedly decreased by treating nodulated roots with aminooxyacetate, methionine sulfoximine, and azaserine. Inhibitors decreased the [¹⁴C]aspartate and [¹⁴]asparagine content of xylem sap by greater than 80% and reduced the total amino nitrogen content of xylem sap (including nonradiolabeled amino acids) by 50 to 80%. Asparagine biosynthesis in alfalfa nodules and transport in xylem sap are dependent upon continued aminotransferase activity and an uninterrupted assimilation of ammonia via the glutamine synthetase/glutamate synthase pathway. Continued assimilation of ammonia apparently appears crucial to continued root nodule CO₂ fixation in alfalfa.     

Sucrose Transport and Phloem Unloading in Stem of Vicia faba: Possible Involvement of a Sucrose Carrier and Osmotic Regulation

Stems of Vicia faba plants were used to study phloem unloading because they are hollow and have a simple anatomical structure that facilitates access to the unloading site. After pulse labeling of a source leaf with ¹⁴CO₂, stem sections were cut and the efflux characteristics of ¹⁴C-labeled sugars into various buffered solutions were determined. Radiolabeled sucrose was shown to remain localized in the phloem and adjacent phloem parenchyma tissues after a 2-hour chase. Therefore, sucrose leakage from stem segments prepared following a 75-minute chase period was assumed to be characteristic of phloem unloading. The efflux of ¹⁴C assimilates from the phloem was enhanced by 1 millimolar p-chloromercuribenzene sulfonic acid (PCMBS) and by 5 micromolar carbonyl cyanide m-chlorophenly hydrazone (CCCP). However, PCMBS inhibited and CCCP enhanced general leakage of nonradioactive sugars from the stem segments. Sucrose at concentrations of 50 millimolar in the free space increased efflux of [¹⁴C]sucrose, presumably through an exchange mechanism. This exchange was inhibited by PCMBS and abolished by 0.2 molar mannitol. Increasing the osmotic concentration of the efflux medium with mannitol reduced [¹⁴C]sucrose efflux. However, this inhibition seems not to be specific to sucrose unloading since leakage of total sugars, nonlabeled sucrose, glucose, and amino acids from the bulk of the tissue was reduced in a similar manner. The data suggest that phloem unloading in cut stem segments is consistent with passive efflux of sucrose from the phloem to the apoplast and that sucrose exchange via a membrane carrier may be involved. This is consistent with the known conductive function of the stem tissues, and contrasts with the apparent nature and function of unloading in developing seeds.     

Xylem and Phloem transport and the functional economy of carbon and nitrogen of a legume leaf

Exchanges of CO₂ and changes in content of C and N were studied over the life of a leaf of Lupinus albus L. These data were combined with measurements of C:N weight ratios of xylem (upper stem tracheal) and phloem (petiole) sap to determine net fluxes of C and N between leaf and plant. Phase 1 of leaf development (first 11 days, leaf to one-third area) showed increasing net import of C and N, with phloem contributing 61% of the imported C and 18% of the N. ¹⁴C feeding studies suggested the potential for simultaneous import and export through phloem over the period 9 to 12 days. Phase 2 (11-20 days, leaf attaining maximum area and net photosynthesis rate) exhibited net import through xylem and increasing export through phloem. Eighty-two% of xylem-delivered N was consumed in leaf growth, the remainder exported in phloem. Phase 3 (20-38 days) showed high but declining rates of photosynthesis, translocation, and net export of N. Phase 4 (38-66 days) exhibited substantial losses of N and declining photosynthesis and translocation of C. C:N ratio of xylem sap remained constant (2.3-2.6) during leaf life; petiole phloem sap C:N ratio varied from 25 to 135 over leaf development. The relationships between net photosynthesis and N import in xylem were: phase 1, 4.8 milligrams C per milligram N; phase 2, 24.7 milligrams C per milligram N; phase 3, 91.9 milligrams C per milligram N; and phase 4, 47.7 milligrams C per milligram N.