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Cluster bean-a ureide- or amide-producing legume?

Xylem sap of cluster bean (Cyamopsis tetragonoloba L. cv FS-277) and pigeonpea (Cajanus cajan cv UPAS-120) were analyzed for total nitrogen, amide nitrogen, and ureide nitrogen at flowering stage. Nitrogenase, uricase, and allantoinase were compared in nodules of cluster bean and pigeonpea. Xylem sap of cluster bean exhibited higher amounts of amides as compared to ureides, and the activities of uricase and allantoinase (ureide-producing enzymes) in nodules were also low, whereas the reverse was the case for pigeonpea. Based on these investigations, it has been concluded that cluster bean is an amide-producing legume rather than ureide-producing as had been reported earlier.

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Properties of peach flower buds which facilitate supercooling.

Water in dormant peach (Prunus persica [L.] Batsch. var. ;Harbrite') flower buds deep supercooled. Both supercooling and the freezing of water within the bud axis and primordium as distinct components depended on the viability of the bud axis tissue. The viability of the primordium was not critical. Supercooling was prevented by wounding buds with a dissecting needle, indicating that bud structural features were important. Bud morphological features appeared to prevent the propagation of ice through the vascular tissue and into the primordium. In dormant buds, procambial cells had not yet differentiated into xylem vessel elements. Xylem continuity between the bud primordium and adjacent tissues did not appear to be established until buds had deacclimated. It was concluded that structural, morphological, and physiological features of the bud facilitated supercooling.

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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.

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Nitrate Uptake and Partitioning by Corn Root Systems : Differential Effects of Ammonium among Genotypes and Stages of Root Development.

The relative effects of ammonium on nitrate uptake and partitioning during induction were compared among decapitated seedlings of three corn (Zea mays L.) genotypes at two developmental stages. This study tested the hypothesis that root systems efficient at translocating products of ammonium assimilation away from sites of nitrate uptake or reduction would exhibit less inhibition of nitrate uptake by ammonium compared to root systems with inefficient N translocation efficiency. Inhibition of nitrate uptake by ammonium was relatively slight at day 5 ranging from 0% to 20% among the three genotypes, as compared to greater inhibition, from 20% to 37%, at day 8. Five-day-old roots exhibited negligible xylem translocation capacity in comparison with those grown for 8 days. Thus, although the capability to translocate ammonium assimilates out of the root increased between days 5 and 8, inhibitory effects of ammonium also increased. In the absence of ammonium, nitrate uptake per unit root mass increased between days 5 and 8. This increased activity of the uptake system was proportionally more sensitive to ammonium.Partitioning of entering nitrate into the reduction process was positively correlated with lateral root development of the inbred root systems at 5 and 8 days. This is supportive of a localization of a major portion of nitrate reduction occurring in root apical regions. Nitrate reduction was the partitioning process most severely inhibited by ammonium in all cases, ranging from 39% to 55% inhibition. In contrast, ammonium-inhibition of nitrate accumulation in the root tissue and translocation via xylem vessels varied with genotype and root age.Two mechanisms of ammonium-inhibition of nitrate are implicated, one which directly affects nitrate reduction and the uptake system associated with it, and another which may involve potassium as an intermediate regulator of nitrate accumulation in the root tissue and nitrate translocation out of the root tissue.

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Sodium and potassium fluxes and compartmentation in roots of atriplex and oat.

K(+) and Na(+) fluxes and ion content have been studied in roots of Atriplex nummularia Lindl. and Avena sativa L. cv Goodfield grown in 3 millimolar K(+) with or without 3 or 50 millimolar NaCl. Compartmental analysis was carried out with entire root systems under steady-state conditions.Increasing ambient Na(+) concentrations from 0 to 50 millimolar altered K(+), in Atriplex, as follows: slightly decreased the cytoplasmic content (Q(c)), the vacuolar content (Q(v)), and the plasma membrane influx and efflux. Xylem transport for K(+) decreased by 63% in Atriplex. For oat roots, similar increases in Na(+) altered K(+) parameters as follows: plasma membrane influx and efflux decreased by about 80%. Q(c) decreased by 65%, and xylem transport decreased by 91%. No change, however, was observed in Q(v) for K(+). Increasing ambient Na(+) resulted in higher (3 to 5-fold) Na(+) fluxes across the plasma membrane and in Q(c) of both species. In Atriplex, Na(+) fluxes across the tonoplast and Q(v) increased as external Na(+) was increased. In oat, however, no significant change was observed in Na(+) flux across the tonoplast or in Q(v) as external Na(+) was increased. In oat roots, Na(+) reduced K(+) uptake markedly; in Atriplex, this was not as pronounced. However, even at high Na(+) levels, the influx transport system at the plasma membrane of both species preferred K(+) over Na(+).Based upon the Ussing-Teorell equation, it was concluded that active inward transport of K(+) occurred across the plasma membrane, and passive movement of K(+) occurred across the tonoplast in both species. Na(+), in oat roots, was actively pumped out of the cytoplasm to the exterior, whereas, in Atriplex, Na(+) was passively distributed between the free space, cytoplasm, and vacuole.

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Studies on Genetic Male-Sterile Soybeans : IV. Effect of Male Sterility and Source of Nitrogen Nutrition on Accumulation, Partitioning, and Transport of Nitrogen.

Soybean (Glycine max [L.] Merr.) germplasm, isogenic except for loci controlling male sterility (ms(1)) and nodulation (rj(1)), was used to investigate the effects of reproductive tissue development and source of nitrogen nutrition on accumulation, transport, and partitioning of nitrogen in a greenhouse experiment. Nodulated plants were supplied nitrogen-free nutrient solution, and nonnodulated plants were supplied nutrient solution containing 20 millimolar KNO(3). Plants were sampled from flowering until maturity (77 to 147 days after transplanting).Accumulation rates of nitrogen in whole plants during reproductive growth were not significantly different among the four plant types. Nitrogen accumulation in the sterile, nonnodulated plants, however, ceased 2 weeks earlier than in fertile, nonnodulated or fertile and sterile, nodulated plants. This early cessation in nitrogen accumulation resulted in sterile, nonnodulated plants accumulating significantly less whole plant nitrogen by 133 days after transplanting (DAT) than fertile, nonnodulated plants. Thus, changing the site of nitrogen assimilation from nodules (N(2)-fixing plants) to roots and leaves (NO(3)-fed plants) resulted in similar whole-plant nitrogen accumulation rates in fertile and sterile plants, despite the absence of seed in the latter.Leaflet and stem plus petiole tissues of both types of sterile plants had significantly higher nitrogen concentrations after 119 DAT than both types of fertile plants. Significantly higher concentrations and exudation rates of nonureide, reduced-nitrogen in xylem sap of sterile than of fertile plants after 105 DAT were observed. These latter results indicated possible cycling of nonureide, reduced-nitrogen from the downward phloem translocation stream to the upward xylem translocation stream in roots of sterile plants. Collectively, these results suggest a lack of sinks for nitrogen utilization in the shoots of sterile plants. Hence, comparison of nitrogen accumulation rates for sterile and fertile plants does not provide a definitive test of the hypothesis that reproductive tissue development limits photosynthate availability for support of N(2) fixation and nitrate assimilation in determinate soybeans.Nitrogen assimilation during reproductive growth met a larger proportion of the reproductive-tissue nitrogen requirement of nitrate-dependent plants (73%) than of N(2)-fixing plants (63%). Hence, vegetative-tissue nitrogen mobilization to reproductive tissue was a more prominent process in N(2)-fixing than in nitrate-dependent plants. N(2)-fixing plants partitioned nitrogen to reproductive tissue more efficiently than nitrate-dependent plants as the reproductive tissues of the former and latter contained 65 and 55%, respectively, of the whole-plant nitrogen at the time that nitrogen accumulation in reproductive parts had ceased (133 DAT).

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Characterization of water stress and low temperature effects on flower induction in citrus.

Experiments were conducted with containerized ;Tahiti' lime (Citrus latifolia Tan.) trees in order to define conditions needed to induce flowering. Cyclical or continuous water stress for 4 to 5 weeks induced flowering. Moderate (-2.25 megapascals, midday) or severe (-3.5 megapascals, midday) water stress as measured by leaf xylem pressure potential, for as little as 2 weeks induced flowering, but the response was more significant in severely stressed trees. Low temperature (18 degrees C day/10 degrees C night) induced a time dependent flowering response much like that of moderate water stress. Significantly negative leaf xylem pressure potentials as compared to controls were found only under water stress treatment, suggesting that a common stress-linked event, separate from low plant water potential is involved in floral induction. Leafless, immature cuttings from mature, field-grown trees were induced to flower by water stress treatment, suggesting that leaves are not essential for a flower inductive response.

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Nitrogen Enhancement of Phosphate Transport in Roots of Zea mays L. : I. Effects of Ammonium and Nitrate Pretreatment.

The effect of nitrogen status on phosphorous uptake and translocation was examined in 6-day-old dark-grown decapitated maize seedlings exposed to 25 micromolar phosphorous. Transfer to complete solutions containing 1 millimolar ammonium resulted in an increase in phosphorous uptake rate after 6 to 8 hours. The stimulus remained effective for at least 5.5 hours upon subsequent transfer to nitrogen-free solutions. Pretreatments for 16 hours with either nitrate or ammonium resulted in enhanced rates of subsequent phosphorous uptake and in enhanced translocation to the xylem of the exogenously supplied phosphorous. Both processes reached a plateau following pretreatment with 0.1 to 1.0 millimolar concentrations of either nitrogen ion. Further enhancement occurred with 10 millimolar nitrate, but not with 10 millimolar ammonium pretreatment. Although nitrogen pretreatments slightly increased the quantity of exogenous phosphorous retained in the root tissue, most of the extra phosphorous taken up by the nitrogen-pretreated seedlings was translocated to the xylem. The enhanced translocation, however, did not totally account for the increase in uptake implying a specific stimulation of the uptake process.

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Abscisic Acid is not the only stomatal inhibitor in the transpiration stream of wheat plants.

Xylem sap was collected from the transpiration stream of wheat (Triticum aestivum L.) plants and assayed for the presence of an inhibitor of transpiration using leaves detached from well-watered plants. Transpiration of detached leaves was reduced by nearly 60% by sap collected from plants in drying soil, and to a lesser extent (about 25%) by sap from plants in well-watered soil. As the soil dried the abscisic acid (ABA) concentration in the sap increased by about 50 times to 5 x 10(-8) molar. However, the ABA in the sap did not cause its inhibitory activity. Synthetic ABA of one hundred times this concentration was needed to reduce transpiration rates of detached leaves to the same extent. Furthermore, inhibitory activity of the sap was retained after its passage through an immunoaffinity column to remove ABA. Xylem sap was also collected by applying pressure to the roots of plants whose leaf water status was kept high as the soil dried. Sap collected from these plants reduced transpiration to a lesser extent than sap from nonpressurised plants. This suggests that the inhibitory activity was triggered partly by leaf water deficit and partly by root water deficit.

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Turgor and growth at low water potentials.

Turgor affects cell enlargement but has not been measured in enlarging tissue of intact plants when growth is inhibited by inadequate water. Mature or excised tissue can be problematic for these measurements because turgor may not be the same as in intact enlarging cells. Therefore, we measured the average turgor in the elongating region of intact stems of soybean (Glycine max [L.] Merr.) while the seedlings were exposed to low water potentials by transplanting to vermiculite of low water content. Stem growth was completely inhibited by the transplanting, and the average turgor decreased in the mature stem tissue. However, it did not decrease in the elongating region whether measured in intact or excised tissue (total of four methods). At the cellular level, turgor was uniform in the elongating tissue except at transplanting, when turgor decreased in a small number of cortical cells near the xylem. The reduced turgor in these cells, but constant turgor in most of the cells, confirmed that no general turgor loss had occurred but indicated that gradients in water potential extending from the xylem into the enlarging tissue were reduced, thus decreasing the movement of water into the tissue for cell enlargement. A modest growth recovery occurred after 2 days and was preceded by a recovery of the gradient. This suggests that under these conditions, growth initially was inhibited not by turgor loss but by a collapse of the water potential gradient necessary for the growth process.

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Root hypoxia reduces leaf growth : role of factors in the transpiration stream.

This study examined the potential role of restricted phloem export, or import of substances from the roots in the leaf growth response to root hypoxia. In addition, the effects of root hypoxia on abscisic acid (ABA) and zeatin riboside (ZR) levels were measured and their effects on in vitro growth determined. Imposition of root hypoxia in the dark when transpirational water flux was minimal delayed the reduction in leaf growth until the following light period. Restriction of phloem transport by stem girdling did not eliminate the hypoxia-induced reduction in leaf growth. In vitro growth of leaf discs was inhibited in the presence of xylem sap collected from hypoxic roots, and also by millimolar ABA. Disc growth was promoted by sap from aerated roots and by 0.1 micromolar ZR. The flux of both ABA and ZR was reduced in xylem sap from hypoxic roots. Leaf ABA transiently increased twofold after 24 hours of hypoxia exposure but there were no changes in leaf cytokinin levels.

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Evidence for Cytokinin Involvement in Rhizobium (IC3342)-Induced Leaf Curl Syndrome of Pigeonpea (Cajanus cajan Millsp.).

A uniquely abnormal shoot development (shoot tip-bending, leaf curling, release from apical dominance, and stunted growth) in pigeonpea (Cajanus cajan Millsp) induced by a nodulating Rhizobium strain, IC3342, is thought to be due to a hormonal imbalance. Amaranthus betacyanin bioassay indicated that xylem exudate and leaf extracts from pigeonpea plants with Rhizobium-induced leaf curl symptoms contained high concentrations of cytokinin relative to those in normal plants. Radioimmunoassay (RIA) of samples purified with high performance liquid chromatography revealed that zeatin riboside (ZR) and dihydrozeatin riboside (DZR) concentrations in xylem sap from plants with leaf curl symptoms were 7 to 9 times higher than those in the sap from symptomless, nodulated plants. The sap from symptomless plants nodulated by a Curl(-) mutant had ZR and DZR concentrations comparable to those in the normal plant sap. RIA indicated that the respective concentrations of zeatin and N(6)-isopenteny-ladenine in culture filtrates of the curl-inducing strain IC3342 were 26 and 8 times higher than those in filtrates of a related normal nodulating strain (ANU240). Gas chromatographic-mass spectrometric analyses revealed similar differences. Gene-specific hybridization and sequence comparisons failed to detect any homology of IC3342 DNA to Agrobacterium tumefaciens or Pseudomonas savastanoi genetic loci encoding enzymes involved in cytokinin biosynthesis.

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Temperature-dependent water and ion transport properties of barley and sorghum roots : I. Relationship to leaf growth.

Root temperature strongly affects shoot growth, possibly via "nonhydraulic messengers" from root to shoot. In short-term studies with barley (Hordeum vulgare L.) and sorghum (Sorghum bicolor L.) seedlings, the optimum root temperatures for leaf expansion were 25 degrees and 35 degrees C, respectively. Hydraulic conductance (L(p)) of both intact plants and detached exuding roots of barley increased with increasing root temperature to a high value at 25 degrees C, remaining high with further warming. In sorghum, the L(p) of intact plants and of detached roots peaked at 35 degrees C. In both species, root temperature did not affect water potentials of the expanded leaf blade or the growing region despite marked changes in L(p). Extreme temperatures greatly decreased ion flux, particularly K(+) and NO(3) (-), to the xylem of detached roots of both species. Removing external K(+) did not alter short-term K(+) flux to the xylem in sorghum but strongly inhibited flux at high temperature in barley, indicating differences in the sites of temperature effects. Leaf growth responses to root temperature, although apparently "uncoupled" from water transport properties, were correlated with ion fluxes. Studies of putative root messengers must take into account the possible role of ions.

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Nitrate-regulated growth and cytokinin responses in seminal roots of barley.

The influence of nitrate availability on growth of seminal roots, and root cytokinin levels, was studied in barley (Hordeum vulgare L. cv Golf). Nitrate was continuously supplied to initially N-starved seedlings at relative addition rates (RA) of 0.03 to 0.21 per day (standard cultures) or at RA 0.09 per day in split root cultures with the nitrate additions distributed in ratios of 100:0 or 80:20 to the two subroots. Data were collected both during a phase of acclimation (first 10 days of N additions) and in the acclimated stage (>10 days after onset of N additions). Limitation of whole-plant growth was observed at RA <0.15 per day. The lateral root frequency increased with RA in plants of equal chronological age. However, the lateral root frequency was related to root size rather than to RA; roots of uneven age but having comparable total root lengths also had comparable lateral root frequencies. Growth of individual subroots in split root systems during acclimation was proportional to the fraction of the total N addition that was fed to the root. All subroots had comparable relative growth rates in acclimated plants, and their lateral root frequency correlated with total root length in the same manner as in standard cultures. Onset of N additions in a 80:20 split root culture resulted in doublings of zeatin riboside (ZR) levels in shoots and in the "80" root, whereas the response of the "20" root was small. No effect of perturbed nitrate availability on xylem translocation of ZR was observed. The ZR levels remained higher in the "80" root during acclimation but returned to the level of the "20" root after acclimation. Root cytokinin levels and xylem translocation in acclimated standard cultures were unaffected by RA in the lower range but increased at high RA. Arguments for involvement of cytokinins in the nitrate-regulated growth response are discussed.

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Phenylalanine ammonia-lyase from loblolly pine : purification of the enzyme and isolation of complementary DNA clones.

Phenylalanine ammonia-lyase (EC 4.3.1.5) has been purified from differentiating secondary xylem of loblolly pine (Pinus taeda L.). Native molecular weight of the enzyme was estimated to be 280,000, with a subunit molecular weight of 74,000; isoelectric point, 5.8; and Michaelis constant for i-phenylalanine, 27 micromolar. No evidence was obtained for the existence of isoforms of the enzyme, nor for negative cooperativity of substrate binding. Polyclonal antibodies were raised against the phenylalanine ammonia-lyase subunit and used to identify a pal clone in an expression library of xylem complementary DNA (cDNA). Polymerase chain reaction, using oligonucleotide primers made from N-terminal amino acid sequence and from the 5' end of the clone isolated from the expression library, was also used to isolate cDNA clones. These methods yielded cDNA clones covering the protein coding region of the pal messenger RNA. Comparisons of nucleotide sequence of pal cDNAs from pine, bean, sweet potato, and rice showed 60 to 62% identity between the pine clone and the angiosperm clones.

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Structural Changes and Associated Reduction of Hydraulic Conductance in Roots of Sorghum bicolor L. following Exposure to Water Deficit.

The effects of a severe water deficit on total root (L(t)) and axial (L(x)) hydraulic conductances and on the development of the hypodermis, endodermis, and xylem were studied in sorghum (Sorghum bicolor L.). Water deficit was imposed in the upper rooting zone while the lower zones were kept moist. L(t) and L(x) were based on water flow rates obtained by applying suction to proximal xylem ends of excised roots. The development of the hypodermis, endodermis, and other tissues were examined by staining with fluorescent berberine hemisulfate and phloroglucinol-HCl. The L(t) value (x 10(-8) meters per second per megapascal) for unstressed control roots was 22.0 and only 5.9 for stressed roots. The low L(t) in stressed roots was attributed, in part, to accelerated deposition of lignin and suberin in the hypodermis and endodermis. Calcofluor, an apoplastic tracer that binds to cellulose, was blocked in stressed roots at the lignified and suberized outer tangential walls of the hypodermis but readily penetrated the cortical walls of similar root regions in controls where the casparian band was not developed. L(x) per unit root length was about 100 times lower in stressed roots than in controls because of the persistence of late metaxylem cross-walls and the smaller diameter and lower number of conductive protoxylem and early metaxylem vessels.

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Temperature-Dependent Water and Ion Transport Properties of Barley and Sorghum Roots : II. Effects of Abscisic Acid.

Water flux through excised roots (J(v)) is determined by root hydraulic conductance (L(p)) and the ion flux to the xylem (J(i)) that generates an osmotic gradient to drive water movement. These properties of roots are strongly temperature dependent. Abscisic acid (ABA) can influence J(v) by altering L(p), J(i), or both. The effects of root temperature on responses to ABA were determined in two species differing in their temperature tolerances. In excised barley (Hordeum vulgare L.) roots, J(v) was maximum at 25 degrees C; 10 micromolar ABA enhanced J(v), primarily by increasing L(p), at all temperatures tested (15-40 degrees C). In sorghum (Sorghum bicolor L.) roots, J(v) peaked at 35 degrees C; ABA reduced this optimum temperature for J(v) to 25 degrees C by increasing L(p) at low temperatures and severely inhibiting J(i) (dominated by fluxes of K(+) and NO(3) (-)) at warm temperatures. The inhibition of K(+) flux by ABA at high temperature was mostly independent of external K(+) availability, implying an effect of ABA on ion release into the xylem. In sorghum, ABA enhanced water flux through roots at nonchilling low temperatures but at the expense of tolerance of warm temperatures. These effects imply that ABA may shift the thermal tolerance range of roots of this heat-tolerant species toward cooler temperatures.

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The production and efflux of 4-aminobutyrate in isolated mesophyll cells.

The pathway of 4-aminobutyric acid (GABA) production and efflux was investigated in suspensions of mesophyll cells isolated from asparagus (Asparagus sprengeri Regel) cladophylls. Analysis of free amino acids demonstrated that, on a molar basis, GABA represented 11.4, 19, and 6.5% of the xylem sap, intact cladophyll tissue, and isolated mesophyll cells, respectively. l-Glu, a GABA precursor, was abundant in intact cladophylls and isolated cells but not in xylem sap. When cells were incubated with l-[U-(14)C]Glu, intracellular GABA contained less than 10% of the radioactivity found in intracellular Glu. However, GABA in the medium contained 78% of the radioactivity found in extracellular l-Glu metabolites. Incubation with l-[1-(14)C]Glu resulted in the appearance of unlabeled GABA, demonstrating its production through decarboxylation at carbon 1. GABA released to the medium from cells incubated with l-[U-(14)C]Glu had a specific activity of 18 nanocuries per nanomole, whereas GABA remaining in the cell had a specific activity of 2.25 x 10(-1) nanocuries per nanomole. In the presence of exogenous l-Glu, amino acid analysis and cell volume measurements indicated intracellular Ala and GABA concentrations of 4.2 and 1.4 millimolar, respectively. In the medium, however, the corresponding concentrations were 2 and 57 micromolar. The data indicate that l-Glu entering the cell is decarboxylated to GABA, and that specific and passive efflux is from this pool of recently synthesized GABA and not from a previously synthesized unlabeled pool of GABA.

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