Proline induces calcium-mediated oxidative burst and salicylic acid signaling

Although free proline accumulation is a well-documented phenomenon in many plants in response to a variety of environmental stresses, and is proposed to play protective roles, high intracellular proline content, by either exogenous application or endogenous over-production, in the absence of stresses, is found to be inhibitory to plant growth. We have shown here that exogenous application of proline significantly induced intracellular Ca²⁺ accumulation in tobacco and calcium-dependent ROS production in Arabidopsis seedlings, which subsequently enhanced salicylic acid (SA) synthesis and PR genes expression. This suggested that proline can promote a reaction similar to hypersensitive response during pathogen infection. Other amino acids, such as glutamate, but not arginine and phenylalanine, were also found to be capable of inducing PR gene expression. In addition, proline at concentration as low as 0.5 mM could induce PR gene expression. However, proline could not induce the expression of PDF1.2 gene, the marker gene for jasmonic acid signaling pathway. Furthermore, proline-induced SA production is mediated by NDR1-dependent signaling pathway, but not that mediated by PAD4. Our data provide evidences that exogenous proline, and probably some other amino acids can specifically induce SA signaling and defense response.     

SLC36A4 (hPAT4) is a high affinity amino acid transporter when expressed in Xenopus laevis oocytes

The SLC36 family of transporters consists of four genes, two of which, SLC36A1 and SLC36A2, have been demonstrated to code for human proton-coupled amino acid transporters or hPATs. Here we report the characterization of the fourth member of the family, SLC36A4 or hPAT4, which when expressed in Xenopus laevis oocytes also encodes a plasma membrane amino acid transporter, but one that is not proton-coupled and has a very high substrate affinity for the amino acids proline and tryptophan. hPAT4 in Xenopus oocytes mediated sodium-independent, electroneutral uptake of [(3)H]proline, with the highest rate of uptake when the uptake medium pH was 7.4 and an affinity of 3.13 μM. Tryptophan was also an excellently transported substrate with a similarly high affinity (1.72 μM). Other amino acids that inhibited [(3)H]proline were isoleucine (K(i) 0.23 mM), glutamine (0.43 mM), methionine (0.44 mM), and alanine (1.48 mM), and with lower affinity, glycine, threonine, and cysteine (K(i) >5 mM for all). Of the amino acids directly tested for transport, only proline, tryptophan, and alanine showed significant uptake, whereas glycine and cysteine did not. Of the non-proteogenic amino acids and drugs tested, only sarcosine produced inhibition (K(i) 1.09 mM), whereas γ-aminobutyric acid (GABA), β-alanine, L-Dopa, D-serine, and δ-aminolevulinic acid were without effect on [(3)H]proline uptake. This characterization of hPAT4 as a very high affinity/low capacity non-proton-coupled amino acid transporter raises questions about its physiological role, especially as the transport characteristics of hPAT4 are very similar to the Drosophila orthologue PATH, an amino acid "transceptor" that plays a role in nutrient sensing.     

Amino acid uptake by Ricinus communis roots: characterization and physiological significance

Abstract Roots of sterile-grown, intact 6-day-old seedlings of Ricinus communis possess at least two independent active amino acid uptake systems, one for neutral and one for basic amino acids. The kinetics of uptake of L-proline and L-arginine, which were taken as representative substrates for the two systems, are biphasic. At low concentrations (0.01–0.5 mol m^?3) Michaelis -Menten kinetics prevail, changing to a linear concentration dependence at higher substrate concentrations (1–50 mol m^?3). L-glutamate uptake velocity is linear over the whole substrate concentration range. For comparison the uptake kinetics of nitrate and ammonium were determined as well as interactions among the different nitrogen sources. The K_m value for nitrate uptake was 0.4 mol m^?3, and for ammonium 0.1 mol m^?3. The uptake capacity for nitrate or ammonium was approximately the same as for amino acids. The interaction between the uptake systems for organic and inorganic nitrogen is small. Two hypotheses for the physiological significance of amino acid uptake by roots were considered: (i) Uptake of amino acids from the soil-determination of amino acids in soil and in soil water indicates that they might contribute 15–25% to the nitrogen nutrition of the plant. (ii) Amino acid uptake systems of root cells serve primarily as retrieval of amino acids delivered from the phloem- it was found that ¹⁴C L-glutamine, which was delivered to the cotyledon and transported to the root via the phloem, was not lost by the roots, whereas it appeared in the bathing medium if L-glutamine was applied externally to the root to compete for the uptake sites; this suggests that an apoplastic pool of amino acids in the root exists due to their efflux from the phloem.     

Uptake of L-proline by Histoplasma capsulatum.

The uptake and incorporation of L-proline by yeast cells of the dimorphic zoopathogen Histoplasma capsulatum were studied. The amino acid was assimilated in at least two ways: by an active transport system with a Km of 1.7 X 10(-5) M and by simple diffusion. The active transport system was sterospecific and severely restricted to neutral aliphatic side-chain amino acids. Certain analogues inhibited L-proline uptake and prevented incorporation of the amino acid into cellular constituents. The inhibition of L-proline uptake by L-leucine was competitive. Since L-leucine and L-proline are seemingly transported by a system with similar characteristics, must be concluded, as originally postulated, that the buckled ring of L-proline, in solution, acts as an aliphatic side chain and that this cyclic amino acid is transported by a system more or less specific for amino acids with neutral aliphatic side chains.     

Influence of some exogenous amino acids on the production of maize embryogenic callus and on endogenous amino acid content

The effects of four exogenous amino acids (proline, glycine, asparagine and serine) on the production of maize embryogenic callus and on its endogenous amino acid content have been investigated. For this purpose, an established embryogenic line of Type 1 callus from the inbred W64Ao2 has been used. From the results it may be concluded that a concentration of proline exceeding 6 mM is negative for the production of embryogenic callus. When proline is eliminated from the medium, other amino acids tested in certain concentrations yield a percentage of embryogenic callus production that exceeds or equals that of proline. The endogenous free proline content in embryogenic callus is significantly higher than that in non-embryogenic callus regardless of proline presence in the medium. The only exception are the glycine-containing media, in which endogenous free alanine of embryogenic callus increases at the expense of endogenous free proline. This study suggest a positive role of endogenous free proline or alanine accumulation in the embryogenic callus production which might be related to an adaptation to the metabolic changes produced by in vitro culture and embryogenesis induction. Furthermore, these results indicate that treatments with amino acids that are different from proline can be used to improve the efficiency of embryogenic callus production from well established maize callus cultures.Abbreviations Ala alanine - Asn asparagine - 2,4-d 2,4-dichlorophenoxyacetic acid - EC embryogenic callus - nEC non-embryogenic callus - Gaba gamma-aminobutyric acid - Glu glutamic acid - Gly glycine - Pro proline - Ser serine     

How many Na+-dependent carriers for L-alanine and L-proline in the eel intestine? Studies with brush-border membrane vesicles

Using brush-border membrane (BBM) vesicles prepared from the intestine of the European eel, the specificity of L-alanine and L-proline Na+-dependent transport was investigated by measuring the uptake of isotopically labelled substrates. In the presence of Na+ ions, cross-inhibition between alanine and proline transports was observed; in addition alpha-(methylamino)isobutyric acid (MeAIB) inhibited proline but had no effect on alanine uptake. These results can be explained by the presence, in eel intestinal BBM vesicles, of at least two distinct agencies for Na+-dependent proline and alanine translocation. The first system is specific for alanine and short-chain neutral amino acids; the second system, specific for imino acids and the N-methylated analogues, is regulated by alanine concentration.     

Effect of cysteamine on amino acid pools of cultured fibroblasts and the uptake of L-proline

Cysteamine when incubated with human normal and cystinotic fibroblasts produces significant alterations in cellular amino acid pools with a decrease in proline, glycine, histidine, and methionine as well as branch chain and dibasic amino acids. Serine content was increased while aspartate, glutamate and glutamine were unchanged. The uptake of L-proline by cysteamine treated cells was impaired suggesting that altered uptake of amino acids may be a factor in the cysteamine effect.     

Regulation of proline and ethylene levels in rape seedlings for freezing tolerance

This study was aimed to investigate the possibility of regulating free proline content and ethylene production in the resistant to abiotic stress cv. ‘Hornet H’ and the tolerant to stress cv. ‘Sunday’ of winter rapeseed seedlings by pretreatment with exogenous L-proline and L-glutamine in non-acclimated and cold-acclimated seedlings in relation to freezing tolerance. The ratio of proline content in acclimated (at 4°C) versus non-acclimated (18°C) ‘Hornet H’ seedlings increased 2.12-fold and in ‘Sunday’ seedlings 1.95-fold. Exogenously applied, proline and glutamine produced a positive effect on free proline content in both cold-acclimated and non-acclimated seedlings. At a temperature of -1°C the proline content significantly increased in non-acclimated and especially in cold-acclimated seedlings. At an intensified freezing temperature (?3°C, ?5°C, ?7°C), the proline content decreased in comparison with that at ?1°C, but glutamine, especially proline, in cold-acclimated seedlings takes part in free proline level increase and in seedlings’ resistance to freezing. Ethylene production increased in cold-acclimated conditions and under the effect of exogenous proline and glutamine. In freezing conditions, ethylene production decreased, but in cold-acclimated seedlings and under pretreatment of proline and glutamine the ethylene synthesis was intensive. Thus, free proline content and ethylene production increase in cold-acclimated winter rapeseed seedlings and under pretreatment with glutamine and especially with proline. Free proline is involved in the response to cold stress, and its level may be an indicator of cold-hardening and freezing tolerance, but the role of ethylene in the regulation of cold tolerance remains not quite clear.     

Chloroquine,a novel inhibitor of amino acid transport by rat renal brush border membrane vesicles

Summary Chloroquine is an antimalarial and antirheumatic lysosomotropic drug which inhibits taurine uptake into and increases efflux from cultured human lymphoblastoid cells. It inhibits taurine uptake by rat lung slices and affects the uptake and release of cystine from cystinotic fibroblasts. Speculations on its mode of action include a proton gradient effect, a non-specific alteration in membrane integrity, and membrane stabilization. In this study, the effect of chloroquine on the uptake of several amino acids by rat renal brush border membrane vesicles (BBMV) was examined. Chloroquine significantly inhibited the secondary active, NaCl-dependent component of 10µM taurine uptake at all concentrations tested, but did not change equilibrium values. Analysis of these data indicated that the inhibition was non-competitive. Taurine uptake was reduced at all osmolarities tested, but inhibition was greatest at the lowest osmolarity. Taurine efflux was not affected by chloroquine, nor was the NaCl-independent diffusional component of taurine transport. Chloroquine (1 mM) inhibited uptake of the imino acids L-proline and glycine, and the dibasic amino acid L-lysine. It inhibited the uptake of D-glucose, but not the neutral-amino acids L-alanine or L-methionine. Uptake of the dicarboxylic amino acids, L-glutamic acid and L-aspartic acid, was slightly enhanced. With regard to amino acid uptake by BBMV, these findings may support some of the currently proposed mechanisms of the action of chloroquine but further studies are indicated to determine why it affects the initial rate of active amino acid transport.     

Absence of derepression of amino acids transport in Candida

The transport of glycine, L-alanine, L-proline, L-leucine, L-lysine, L-phenylalanine and L-glutamic acid did not enhance in various strains of Candida cells, when they were grown in proline containing medium or preincubated with proline. However, under similar conditions, a significant enhancement in the level of accumulation of amino acids (derepression) was observed in Saccharomyces cerevisiae X-2180-A2 (GAP+) cells, which was sensitive to ammonium ions (NH4+). As expected, the derepression was absent in GAP- cells of S. cerevisiae X-2180 (GAP- mutant). In contrast to S. cerevisiae (GAP+) cells, the increase in few amino acids uptake in different Candida strains, grown in proline or preincubated in proline, could not be inhibited by cycloheximide, NH4+ or their D-stereoisomers. It appears that derepression of amino acids transport, a well known phenomenon in S. cerevisiae, may not exist in Candida species.     

UPTAKE SPECIFICITY FOR ORGANIC SUBSTRATES BY THE MARINE DIATOM MELOSIRA NUMMULOIDES1

Melosira nummuloides, clone Mel-3, shows a very high specificity with regard to its ability to take up organic substrates. Amino acids supplied in the medium at 1 X 10^-4 M are taken up at initial rates of the same order of magnitude as that of photoassirnilation of COj. However, sugars, sugar alcohols, or organic acids supplied at the same concentration are not taken up. The mechanism for uptake of amino acids appears to require energy, since tlie uptake of the amino acid analog α-aminoisobutyric acid is strongly inhibited by 2 f-dinitrophenul. The uptake mechanism does not appear to be inducible. The ability of M. numinuloides to utilize amino acids as a nitrogen source is quite restricted. Arginine, ghttamine, asparagine, proline, and glutamic acid were good nitrogen sources. Seventeen other amino acids, including α-aminoisobutyric acid, were unsatisfactory for growth, although they were rapidly taken up from the medium.     

Different mechanisms of energy coupling for transport of various amino acids in cells of Mycobacterium phlei.

Whole cells of Mycobacterium phlei were shown to actively accumulate proline, leucine, lysine, tryptophan, histidine, glutamine, and glutamic acid to different steady state levels. The transport of proline, in contrast to that of other amino acids, was found to be insensitive to various respiratory inhibitors, e.g. cyanide, arsenate, azide, and sulfhydryl reagents. However, oxygen was an obligatory requirement for the uptake of proline, as well as for the other amino acids. The results indicate that the energy requirements for proline uptake are different from those of other amino acids. In contrast to the system from Escherichia coli, the mode of energy transduction for the uptake of proline, glutamine, and glutamic acid is different even though these amino acids are shock resistant in the M. phlei system.     

Regulation of nitrate uptake by amino acids in maize cell suspension culture and intact roots

The effect of amino acids on nitrate transport was studied in Zea mays cell suspension cultures and in Zea mays excised roots. The inclusion of aspartic acid, arginine, glutamine and glycine (15mM total amino acids) in a complete cell-culture media containing 1.0 mM NO₃ ^- strongly inhibited nitrate uptake and the induction of accelerated uptake rates. The nitrate uptake rate increased sharply once solution amino acid levels fell below detection limits. Glutamine alone inhibited induction in the cell suspension culture. Maize seedlings germinated and grown for 7 days in a 15 mM mixture of amino acids also had lower nitrate uptake rates than seedlings grown in 0.5 mM Ca(NO₃)₂ or 1 mM CaCl₂. As amino acids are the end product of nitrate assimilation, the results suggest an end-product feed-back mechanism for the regulation of nitrate uptake.     

Cationic and anionic amino acid transport studies in rat red blood cells

The transport of L-proline, L-lysine and L-glutamate in rat red blood cells has been studied. L-proline and L-lysine uptake were Na⁺-independent. When the concentration dependence was studied both showed a non-saturable uptake assimilable to a difussion-like process, with high Kd values (0.718 and 0.191 min^–1 for L-proline and L-lysine respectively). Rat red blood cells showed high impermeability to L-glutamate. No sodium dependence was observed and the Kd value was low (0.067 min^–1). Our results show firstly, that rat red blood cells do not have amino acid transport systems for anionic and cationic amino acids and secondly that erythrocytes show no sodium-dependent L-proline transport, and that these cells are very permeable to this amino acid.Abbreviations MeAIB methyl aminoisobutyric acid     

Effects of Free Fatty Acids on Synaptosomal Amino Acid Uptake Systems

Abstract: The Na⁺-dependent synaptosomal uptakes of proline, aspartic acid, glutamic acid, and γ-aminobutyric acid were strongly inhibited by monounsaturated fatty acids. With oleic acid, half-maximal inhibition was observed at about 15 μM. The Na⁺-independent uptakes of leucine, phenylalanine, histidine, and valine were less sensitive to inhibition by the unsaturated fatty acids. In contrast, the uptakes of all of these amino acids were unaffected by saturated fatty acids. The inhibition of proline uptake (and that of the other Na⁺-dependent amino acids) by oleic acid was overcome by the addition of serum albumin and the data presented further indicate that the previously reported stimulation of proline uptake by albumin could be related to its fatty acid binding properties.     

Proline excretion by Escherichia coli K12

Proline excretion from proline overproducing strains of E. coli K12 has been studied as a model chemical production system. We have isolated proline overproducing mutants of E. coli and have shown that uncontrolled synthesis is not sufficient to cause excretion of this amino acid. An episomal mutation causing proline over production has been introduced into a series of otherwise isogenic strains that bear well defined, chromosomal lesions affecting the active uptake and catabolism of L-proline. A syntropism test reveals that L-proline is excreted by overproducing strains only if transport and/or catabolism are impaired. Dansyl derivatization and chromatographic analysis of culture supernatants shows that proline is the only amino acid excreted. Batch cultures of an excreting strain in an amino acid production medium yield culture supernatants containing 1 g proline/L, whereas no proline is detectable in supernatants derived from cultures of an overproducing strain with normal transport and catabolic activities. These data reveal that genetic lesions eliminating active uptake can be used to specifically enhance metabolite excretion.     

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.