Transport of amino acids (L-valine, L-lysine, L-glutamic acid) and sucrose into plasma membrane vesicles isolated from cotyledons of developing pea seeds

Transport of the amino acids L-valine, L-lysine, and L-glutamic acid and of sucrose was studied in plasma membrane vesicles isolated from developing cotyledons of pea (Pisum sativum L. cv. Marzia). The vesicles were obtained by aqueous polymer two-phase partitioning of a microsomal fraction and the uptake was determined after the imposition of a H(+)-gradient (DeltapH, inside alkaline) and/or an electrical gradient (Deltapsi, inside negative) across the vesicle membrane. In the absence of gradients, a distinct, time-dependent uptake of L-valine was measured, which could be enhanced about 2-fold by the imposition of DeltapH. The imposition of Deltapsi stimulated the influx of valine by 20%, both in the absence and in the presence of DeltapH. Uptake of L-lysine was more strongly stimulated by Deltapsi than by DeltapH, and its DeltapH-dependent uptake was enhanced about 6-fold by the simultaneous imposition of Deltapsi. In the absence of gradients the uptake of L-glutamic acid was about 2-fold higher than that of L-valine, but it was not detectably affected by DeltapH or Deltapsi. Although the transport of sucrose was very low, a stimulating effect of DeltapH could be clearly demonstrated. The results lend further support to the contention that during seed development cotyledonary cells employ H(+)-symporters for the active uptake of sucrose and amino acids.     

Proton-symport of L-valine in plasma membrane vesicles isolated from leaves of the wild-type and the Val(r)-2 mutant of Nicotiana tabacum L

Transport of amino acids across the plasma membranes of various cell types is a key process in controlling the nitrogen balance of leaves. We studied the transport of the neutral amino acid L-valine into plasma membrane vesicles obtained by aqueous polymer two-phase partitioning of a microsomal fraction isolated from leaves of the wild-type and the Val(r)-2 mutant of tobacco (Nicotiana tabacum L.). Initial influxes were determined after the imposition of a pH-gradient (DeltapH, inside alkaline) and/or an electrical gradient (Deltapsi, inside negative) across the vesicle membrane. The initial magnitudes of the imposed gradients were DeltapH=2 and Deltapsi=-68 mV. In vesicles from the wild-type, the DeltapH-dependent valine influx could be analysed into a high-affinity (Km approximately 20 microM) and a low-affinity (Km approximately 3 mM) component. The influx of valine by the low-affinity system was stimulated about twofold, and that by the high-affinity system more than sixfold by the imposition of Deltapsi. This strong stimulation of the high-affinity system may indicate that it transports 2H+/amino acid. In the Val(r)-2 mutant the high-affinity component appeared to be completely absent.     

The Mechanism of Amino Acid Efflux from Seed Coats of Developing Pea Seeds as Revealed by Uptake Experiments

The uptake of amino acids by excised seed coat halves of developing seeds of pea (Pisum sativum L.) was characterized. The influx of L-valine and L-glutamic acid was proportional to their external concentration, with coefficients of proportionality (k) of 11.0 and 7.1 [mu]mol g-1 fresh weight min-1 M-1, respectively. The influx of L-lysine could be analyzed into a component with linear kinetics (k = 8.1 [mu]mol g-1 fresh weight min-1 M-1) and one with saturation kinetics (Michaelis constant = 6.5 mM), but the latter may have resulted from the mutual interaction between the influx of the cationic lysine and the membrane potential. The influx of the amino acids was not affected by 10 [mu]M carbonylcyanide m-chlorophenylhydrazone, but was inhibited by about 50% in the presence of 2.5 mM p-chloromercuribenzene sulfonic acid. Conservative estimates of the permeability coefficients of the plasma membrane of seed coat parenchyma cells for lysine, glutamic acid, and several neutral amino acids were all in the range of 4 x 10-7 cm s-1 to 9 x 10-7 cm s-1, which is 4 to 5 orders of magnitude greater than those reported for artificial lipid bilayers. It is concluded that nonselective pores constitute a pathway in the plasma membrane for passive transport of amino acids. It is argued that this pathway is also used for the efflux of endogenous amino acids, the process by which nitrogen becomes available for the embryo.     

Differences in release of endogenous sugars and amino acids from attached and detached seed coats of developing pea seeds

The effect of p -chloromercuribenzenesulfonic acid (PCMBS), carbonylcyanide- m -chlorophenylhydrazone (CCCP) and a high apoplastic pH (pH 7.5 compared with pH 5.5) on the release of sugars (sucrose and glucose) and amino acids from attached and detached seed coats of Pisum sativum L. cv. Marzia into a bathing solution was measured by means of the 'empty seed coat technique'. PCMBS reduced the release of sugars and amino acids from attached as well as from detached seed coats, suggesting that carrier-mediated transport might be involved. CCCP reduced sugar release from attached seed coats while amino acid release was hardly affected. In experiments with detached seed coats CCCP had no effect on release of either sugar or amino acids, suggesting that it is not energy-dependent. Raising the pH of the bathing solution from pH 5.5 to pH 7.5 slightly increased sugar release from both attached and detached seed coats while amino acid release was not affected. This might indicate a role of the apoplastic pH in regulating sugar release from the seed coat via a retrieval mechanism. The presented data indicate that there are important differences between sugars and amino acids with respect to transport processes in the seed coat. This is supported by the observation that the rate of amino acid release from the seed coat was higher than the rate of sugar release. The release data of detached seed coats were subjected to compartmental analysis in order to calculate rate constants for release from cell compartments. In the case of sugars, the half-times for emptying the cytoplasmic and vacuolar compartment were 0.8 h and 12.5 h. respectively. For amino acids the half-times were 0.5 h for emptying the cytoplasmic and 3.8 h for emptying the vacuolar compartment.     

Kinetics and Specificity of Amino Acid Uptake by the Duckweed Spirodela polyrhiza (L.) Schleiden

Borstlap, A. G, Meenks, J. L. D., van Eck, W. F. and Bicker,J. T. E. 1986. Kinetics and specificity of amino acid uptakeby the duckweed Spirodela polyrhiza (L.) Schleiden.—J.exp. Bot. 37: 1020–1035. Uptake of ¹⁴C-labelled amino acids by intact, axenically grownplants of Spirodela polyrhiza (L.) Schleiden was investigated.Experiments in which uptake was measured from the decrease inthe amino acid concentration in the medium, indicated that saturableuptake conforms to the sum of two Michaelis-Menten terms, possiblycorresponding with a high-affinity and a low-affinity system.Further experiments with L-leucine, L-glutamic acid, and L-lysine,in which uptake was measured by assaying the amount of ¹⁴ inthe plants, showed the presence of a non-saturable componentin addition to the dual saturable uptake. Uptake of L-glutamic acid precipitously declined between pH4?0 and 6? and that of L-leucine between pH 4?0 and 8? whereasL-lysine uptake was optimal at pH 6?0. No evidence was foundthat the apparent high-affinity and low-affinity systems respondeddifferently to changes in external pH or to the addition ofCCCP. The non-saturable uptake component was not affected bychanges in external pH or by adding CCCP, and might have beendue to free space uptake. Mutual inhibition of uptake was found between acidic and neutralamino acids (L-leucine, L-methionine, L-glutamic acid) and betweenbasic amino acids (L-lysine, L-ornithine). The basic amino acidshad no effect on the uptake of L-leucine, L-methionine and L-glutamicacid, although the uptake of basic amino acids was inhibitedby glutaminc acid and several neutral amino acids. It is suggested that the duckweed has a high-affinity transportsystem for neutral and acidic amino acids, and a distinct high-affinitysystem for basic amino acids. It is argued that the first systemtransports zwitterionic amino acids (z-system), and that thesecond system transports cationic amino acids(y⁺-system). Thespecificity of the low-affinity system is less certain, butthere is some evidence that it is similar to that of their high-affinitycounterparts. Key words: Kinetics, membrane transport, pH-dependency, transport systems, uptake isotherms     

Alterations in fatty acyl composition can selectively affect amino acid transport in Saccharomyces cerevisiae

The fatty acid composition of yeast lipid was manipulated by using auxotrophic strain of S.cerevisiae, KD115, which requires unsaturated fatty acid (UFA) for its growth. It was possible to specifically enrich the yeast with different fatty acyl residues. As compared to wild type strain (S288C), the uptake of amino acids viz., L-alanine, glycine, L-glutamic acid, L-valine in KD115 was drastically reduced, however, the uptake of L-leucine and L-lysine was not affected by the change in lipid unsaturation. Kinetic studies revealed that KT and Jmax values for L-alanine were altered whereas for L-lysine they remained unaffected by UFA modification. Furthermore, unsaturation index for wild type cells was found to be fairly constant while it was variable in KD115 supplemented with different UFAs. It is observed that the variation in amino acid permeases activity which was affected by fluctuations in fatty acyl composition corresponds more to degree of unsaturation rather than growth stage of KD115.     

Decline in ribonucleic acid and protein synthesis with loss of viability during the early hours of imbibition of rye (Secale cereale L.) embryos.

The specificity of amino acid transport in normal (high-glutathione) sheep erythrocytes was investigated by studying the interaction of various neutral and dibasic amino acids in both competition and exchange experiments. Apparent Ki values were obtained for amino acids as inhibitors of L-alanine influx. Amino acids previously found to be transported by high-glutathione cells at fast rates (L-cysteine, L-alpha-amino-n-butyrate) were the most effective inhibitors. D-Alanine and D-alpha-amino-n-butyrate were without effect. Of the remaining amino acids studied, only L-norvaline, L-valine, L-norleucine, L-serine and L-2,4-diamino-n-butyrate significantly inhibited L-alanine uptake. L-Alanine efflux from pre-loaded cells was markedly stimulated by extracellular L-alanine. Those amino acids that inhibited L-alanine influx also stimulated L-alanine efflux. In addition, D-alanine, D-alpha-amino-n-biutyrate, L-threonine, L-asparagine, L-alpha, beta-diaminoproprionate, L-ornithine, L-lysine and S-2-aminoethyl-L-cysteine also significantly stimulated L-alanine efflux. L-Lysine uptake was inhibited by L-alanine but not by D-alanine, and the inhibitory potency of L-alanine was not influenced by the replacement of Na+ in the incubation medium with choline. L-Lysine efflux from pre-loaded cells was stimulated by L-alanine but not by D-alanine. It is concluded that these cells possess a highly selective stero-specific amino acid-transport system. Although the optimum substrates are small neutral amino acids, this system also has a significant affinity for dibasic amino acids.     

Postnatal amino acid uptake by the rat small intestine. Energetics of membrane transport systems for amino acids in the developing jejunum

The energetics of amino acid uptake by the developing small intestine was investigated in vitro. L-valine, L-leucine, L-phenylalanine, L-methionine, L-lysine and L-arginine were all actively transported by the newborn rat jejunum. Metabolic inhibitors (e.g. 2,4-dinitrophenol) significantly reduced uptake of all amino acids but uptake against a concentration gradient was not totally abolished. Uptake of all amino acids was reduced at low[Na+]. Inhibition of transport of neutral amino acids by reduced luminal [Na+] was greater than that of basic amino acids, and the tissue was barely able to concentrate the neutral amino acids. [Na+] affected the Michaelis constant (Km) of neutral transport systems for their substrates; for the basic amino acids Km values were unaffected by the presence or absence of Na+. Ouabain significantly inhibited neutral amino acid uptake but had no effect on L-lysine or L-arginine uptake. These results are discussed in terms of the Na+ gradient hypothesis for amino acid transport, and the site of energy input to active transport. The role of glycolysis in providing energy for intestinal transport in the neonatal rat and the efficiency of Na+ dependent and independent transport mechanisms are considered. It is concluded that the energetics of amino acid transport systems in neonatal and adult rats are essentially similar.     

Nitrate uptake in the halotolerant cyanobacterium Aphanothece halophytica is energy-dependent driven by DeltapH

The energetics of nitrate uptake by intact cells of the halotolerant cyanobacterium Aphanothece halophytica were investigated. Nitrate uptake was inhibited by various protonophores suggesting the coupling of nitrate uptake to the proton motive force. An artificially-generated pH gradient across the membrane (DeltapH) caused an increase of nitrate uptake. In contrast, the suppression of DeltapH resulted in a decrease of nitrate uptake. The increase of external pH also resulted in an enhancement of nitrate uptake. The generation of the electrical potential across the membrane (Deltapsi) resulted in no elevation of the rate of nitrate uptake. On the other hand, the valinomycin-mediated dissipation of Deltapsi caused no depression of the rate of nitrate uptake. Thus, it is unlikely that Deltapsi participated in the energization of the uptake of nitrate. However, Na(+)-gradient across the membrane was suggested to play a role in nitrate uptake since monensin which collapses Na(+)-gradient strongly inhibited nitrate uptake. Exogenously added glucose and lactate stimulated nitrate uptake in the starved cells. N, N'-dicyclohexylcarbodiimide, an inhibitor of ATPase, could alsoinhibit nitrate uptake suggesting that ATP hydrolysis was required for nitrate uptake. All these results indicate that nitrate uptake in A. halophytica is ATP-dependent, driven by DeltapH and Na(+)-gradient.     

Effect of the Osmotic Environment on the Balance between Uptake and Release of Sucrose and Amino Acids by the Seed Coat and Cotyledons of Developing Seeds of Pisum sativum

Excised seed-coat halves and cotyledons of developing seedsof Pisum sativum L. were incubated in a bathing medium (pH 5·5),in order to measure the release or uptake of sucrose and aminoacids. Net efflux of sucrose and amino acids was reduced bya 250 mol m ^–3 mannitol solution and a 400 mol m ^–3solution, in comparison with a 100 mol m^–3 control. Thiseffect could not be observed in the case of the amino acid analogue-aminoisobutyric acid (AIB). Net uptake of labelled sucroseor valine by cotyledons and seed coats was enhanced by a highosmolality of the bathing medium. The data on AIB and the datafrom uptake experiments support the view that net efflux ofassimilates is reduced by a high solute concentration in theapoplast (e.g. 400 mol m^–3 mannitol), via a stimulationof carrier-mediated sucrose and amino acid uptake into cotyledonaryand seed coat tissues. In experiments with attached empty ovulesof pea in a very early stage of development, sugar release fromthe seed coat was enhanced by a low osmolality of the apoplastsolution (e.g. 100 mol m^–3 mannitol, in comparison witha 400 mol m ^–3 control). This paradoxical effect may beobserved when the stimulatory effect on net assimilate effluxfrom seed coat tissues is exceeding the inhibitory effect onassimilate import into the seed coat. Key words: Seed development, turgor-sensitive transport, assimilate transport     

Some properties of thyroidal membrane adenosinetriphosphatase and iodide uptake: effects of basic polyamino acids.

Poly L-lysine, poly L-ornithine, and histone significantly inhibited the iodide uptake by the thyroid slices, as previously reported. These basic polymers diminshed Na, K-ATPase and concomitantly markedly elevated Mg-ATPase activity in the NaI-treated microsomal preparation and the plasma membrane fraction obtained from thyroid. Poly L-glutamic acid, which was noneffetive to the iodide uptake in vitro, did not show such phenomenon. K-dependent p-nitrophenylphosphatase activity which is considered to reflect the terminal step of the reaction sequence of Na, K-ATPase was also inhibited by poly L-lysine. The effects mentioned above of poly L-lysine and other basic polyamino acids on membrane ATPase system were only found in the preparations from thyroid. The inhibitory effect of these reagents on thyroidal iodide uptake was discussed in terms of the change in membrane ATPase activities.     

Proton-coupledl-lysine uptake by renal brush border membrane vesicles from mullet (Mugil cephalus)

The uptake of the basic amino acid, L-lysine, was studied in brush border membrane vesicles isolated from the kidney of the striped mullet (Mugil cephalus). The uptake of L-lysine was not significantly stimulated by a Na+ gradient and no overshoot was observed. However, when a proton gradient (pHo = 5.5; pHi = 8.3) was imposed across the membrane in the absence of Na+, uptake was transiently stimulated. When the proton gradient was short circuited by the proton ionophore, carbonylcyanide p-triflouromethoxyphenyl hydrazone, proton gradient-dependent uptake of lysine was inhibited. Kinetics of lysine uptake determined under equilibrium exchange conditions indicated that the Vmax increased as available protons increased (2.1 nmol/min/mg protein at pH 7.5 to 3.7 nmol/min/mg at pH 5.5), whereas the apparent Km (4.9 +/- 0.6 mM) was not altered appreciably. When membrane potential (inside negative) was imposed by K+ diffusion via valinomycin, a similar (but smaller) stimulation of lysine uptake was observed. When the membrane potential and the proton gradient were imposed simultaneously, a much higher stimulation in lysine uptake was shown, and the uptake of lysine was approximately the sum of the components measured separately. These results indicate that the uptake mechanism for basic amino acids is different from that of neutral or acidic amino acids and that the proton-motive force can provide the driving force for the uptake of L-lysine into the isolated brush border membrane vesicles.     

Influence of amino acids on gastric emptying in young pigs.

The effects of amino acids on gastric emptying and secretion were studied in four young conscious pigs provided with a chronic gastric cannula. A basal test meal of 500 ml containing 10 g citrus pectin, 17.5 g sucrose, 100 mg phenol red alone or with glycine, L- or DL-tryptophan, DL-methionine or L-glutamic acid was poured into the stomach and recovered 20 min later. Glycine at concentrations of 26.7--106.8 mM/l did significantly affect gastric emptying, although at the highest concentration gastric emptying appeared to slow down. L-lysine (41.0 mM/l), DL-methionine (40.3 mM/l or L-tryptophan (29.4 mM/l did not significantly affect the rate of stomach emptying; L-tryptophan increased the amount of Cl- and H+ secreted as compared with the basal diet alone. The effects of addition of the essential amino acids, L-lysine and DL-methionine, were compared with those of the nonessential glycine and L-glutamic acid. There was no significant difference in the rate of gastric emptying or secretion between them. The L and DL isomers of lysine and trypotphan were not found to differ significantly in their effects on gastric emptying and secretion.     

Quantitative Analysis of Photosynthate Unloading in Developing Seeds of Phaseolus vulgaris L. : II. Pathway and Turgor Sensitivity

Phloem import and unloading in perfused bean (Phaseolus vulgaris L.) seed coats were investigated using steady-state labeling. Though photosynthate import and unloading were significantly reduced by perfusion, measurements of photosynthate fluxes in perfused seed coats proved useful for the study of unloading mechanisms in vivo. Phloem import was stimulated by lowered seed coat cell turgor, as demonstrated by an increase in tracer and sucrose import to seed coats perfused with high concentrations of an osmoticum. The partitioning of photosynthates between retention in the seed coat and release to the perfusion solution also was turgor sensitive; increases in seed coat cell turgor stimulated photosynthate release to the apoplast at the expense of photosynthate retention within the seed coat. There was no evidence of a turgor-sensitive sucrose uptake mechanism in perfused seed coats. Thus, the turgor sensitivity of photosynthate partitioning within perfused seed coats was consistent with a turgor-sensitive efflux control mechanism. Measurements of tracer equilibration and sugar partitioning in perfused seed coats provided strong evidence for symplastic phloem unloading in seed coats.     

Postnatal amino acid uptake by the rat small intestine. Changes in membrane transport systems for amino acids associated with maturation of jejunal morphology.

The uptake of a number of amino acids by the developing small intestine of the rat was investigated in vitro. L-valine, L-leucine, L-methionine, L-phenylalanine, L-arginine and L-lysine were all taken up by active transport and concentrated within the jejunal mucosa. GABA was not actively transported by the jejunum. The kinetics of carrier transport of amino acids was determined from birth to maturity. The Michaelis constant (Km) of the L-leucine, L-methionine, L-arginine and l-lysine transport systems was found to be low postnatally and increased with age, particularly after the time of weaning. The rate of l-leucine, L-methionine, L-phenylalanine and L-lysine transport (Vmax) was high postnatally but decreased after weaning. Neutral amino acids were transported at higher rates than basic amino acids. l-arginine was poorly transported by the jejunum. The specificity of transport systems for amino acids was investigated in inhibition studies. Amino acid transport systems appeared to be polyfunctional in the postnatal period but were more specific in post-weaned animals. The changes in kinetics and specificity of amino acid transport in the small intestine are discussed with reference to their possible functional significance and to the maturational changes in the jejunum, particularly with the appearance of a functionally distinct absorptive cell lining the intestinal villi during the third postnatal week (the time of weaning).     

Improvement of pea biomass and seed productivity by simultaneous increase of phloem and embryo loading with amino acids

The development of sink organs such as fruits and seeds strongly depends on the amount of nitrogen that is moved within the phloem from photosynthetic‐active source leaves to the reproductive sinks. In many plant species nitrogen is transported as amino acids. In pea (Pisum sativum L.), source to sink partitioning of amino acids requires at least two active transport events mediated by plasma membrane‐localized proteins, and these are: (i) amino acid phloem loading; and (ii) import of amino acids into the seed cotyledons via epidermal transfer cells. As each of these transport steps might potentially be limiting to efficient nitrogen delivery to the pea embryo, we manipulated both simultaneously. Additional copies of the pea amino acid permease PsAAP1 were introduced into the pea genome and expression of the transporter was targeted to the sieve element‐companion cell complexes of the leaf phloem and to the epidermis of the seed cotyledons. The transgenic pea plants showed increased phloem loading and embryo loading of amino acids resulting in improved long distance transport of nitrogen, sink development and seed protein accumulation. Analyses of root and leaf tissues further revealed that genetic manipulation positively affected root nitrogen uptake, as well as primary source and sink metabolism. Overall, the results suggest that amino acid phloem loading exerts regulatory control over pea biomass production and seed yield, and that import of amino acids into the cotyledons limits seed protein levels.     

Abscisic Acid and its relationship to seed filling in soybeans

The effect of exogenous abscisic acid (ABA) on the rate of sucrose uptake by soybean (Glycine max L. Merr.) embryos was evaluated in an in vitro system. In addition, the concentrations of endogenous ABA in seeds of three soybean Plant Introduction (PI) lines, differing in seed size, were commpared to their seed growth rates. ABA (10⁻⁷ molar) stimulated in vitro sucrose uptake in soybean (cv `Clay') embryos removed from plants grown in a controlled environment chamber, but not in embryos removed from field-grown plants of the three PI lines. However, the concentration of ABA in seeds of the three field-grown PI lines correlated well with their in situ seed growth rates and in vitro [¹⁴C] sucrose uptake rates.

Across genotypes, the concentration of ABA in seeds peaked at 8.5 micrograms per gram fresh weight, corresponding to the time of most rapid seed growth rate, and declined to 1.2 micrograms per gram at physiological maturity. Seeds of the large-seeded genotype maintained an ABA concentration at least 50% greater than that of the small-seeded genotype throughout the latter half of seed filling. A higher concentration of ABA was found in seed coats and cotyledons than in embryonic axes. Seed coats of the large-seeded genotype always had a higher concentration of ABA than seed coats of the small-seeded line. It is suggested that this higher concentration of ABA in seed coats of the large-seeded genotype stimulates sucrose unloading into the seed coat apoplast and that ABA in cotyledons may enhance sucrose uptake by the cotyledons.

     

Energetics of alanine, lysine, and proline transport in cytoplasmic membranes of the polyphosphate-accumulating Acinetobacter johnsonii strain 210A.

Amino acid transport in right-side-out membrane vesicles of Acinetobacter johnsonii 210A was studied. L-Alanine, L-lysine, and L-proline were actively transported when a proton motive force of -76 mV was generated by the oxidation of glucose via the membrane-bound glucose dehydrogenase. Kinetic analysis of amino acid uptake at concentrations of up to 80 microM revealed the presence of a single transport system for each of these amino acids with a Kt of less than 4 microM. The mode of energy coupling to solute uptake was analyzed by imposition of artificial ion diffusion gradients. The uptake of alanine and lysine was driven by a membrane potential and a transmembrane pH gradient. In contrast, the uptake of proline was driven by a membrane potential and a transmembrane chemical gradient of sodium ions. The mechanistic stoichiometry for the solute and the coupling ion was close to unity for all three amino acids. The Na+ dependence of the proline carrier was studied in greater detail. Membrane potential-driven uptake of proline was stimulated by Na+, with a half-maximal Na+ concentration of 26 microM. At Na+ concentrations above 250 microM, proline uptake was strongly inhibited. Generation of a sodium motive force and maintenance of a low internal Na+ concentration are most likely mediated by a sodium/proton antiporter, the presence of which was suggested by the Na(+)-dependent alkalinization of the intravesicular pH in inside-out membrane vesicles. The results show that both H+ and Na+ can function as coupling ions in amino acid transport in Acinetobacter spp.     

Solute transport into healthy and powdery mildew-infected leaves of pea and uptake by powdery mildew mycelium

The transport of sugars and amino acids into the mycelium of Erysiphe pisi DC. was investigated using two different systems, intact leaf discs and mycelial suspensions. Of the sugars tested, glucose was preferentially taken up by both uninfected and mildew-infected leaf discs, whereas glutamine was taken up by both tissues at a higher rate than lysine or aspartic acid. Leaf discs from infected tissue had a greater uptake capacity than those from healthy tissue for both sugars and amino acids. The uptake of glucose was inhibited more markedly than that of sucrose and fructose by 10 μ m carbonyl cyanide m -chlorophenylhydrazone (CCCP), 1 m m N -ethylmaleimide (NEM), 1 m m diethyl pyrocarbonate (DEPC) and 1 m m phenylglyoxal, whereas 1 m m PCMBS ( p -chloro-mercuribenzenesulphonic acid) inhibited sucrose uptake to the greatest extent. Uptake of glutamine, lysine and aspartic acid was inhibited similarly by CCCP (80%), NEM (20%), DEPC (70%) and PCMBS (60%). Additionally, leaf discs were used to determine which solutes could be taken up from leaf tissue by the fungus. The uptake of sugars into the mycelium was greater than that of amino acids.
Suspensions of powdery mildew mycelium accumulated glucose at about three times the rate of sucrose or fructose, and the amino acid glutamine was taken up at three times the rate of lysine or aspartic acid. Spores separated from the suspension had a low uptake capacity.
When the reducing sugar concentration of leaf apoplastic fluid was estimated, leaves infected by powdery mildew had much higher amounts in the apoplast, whereas the activity of acid invertase also appeared to be higher in apoplastic fluids from infected leaves. When apoplastic fluid samples were run on SDS gels, an invertase antibody detected two bands in samples from infected tissues that were not found in the uninfected samples.