Proline accumulation inside symbiosomes of faba bean nodules under salt stress

A two‐week salt treatment (NaCl, 100 m M ) induced a 50% inhibition of acetylene reduction activity (ARA) of faba bean ( Vicia faba L. var. minor cv. Soravi) nodules, associated with a large increase in the nodule pool of amino acids. The concentration of proline in the different nodule compartments was determined after calculating their respective volumes from their areas on electron micrographs. The proline concentration exhibited a large increase, especially in the cytosol where its amount was 8‐fold enhanced under salt stress, whereas the low proline content of bacteroids was less affected. Increase of proline concentration in faba bean nodules subjected to salt stress was correlated with an enhancement of the cytosolic Δ¹‐pyrroline‐5‐carboxylate synthetase (EC 2.7.2.11 + EC 1.2.1.41; P5CS) activity. Experiments with purified symbiosome preparations showed that the greatest proline content occurred in the peribacteroid space (PBS), where proline was the most abundant amino acid, with a concentration reaching 15.3 m M under salt stress. Proline accumulation in the PBS resulted both from a diffusive transport from the host cell to the symbiosomes through the peribacteroid membrane (PBM) and from the very low rate of uptake by faba bean bacteroids. This accumulation could be partly responsible for the 1.7‐fold enlargement of the symbiosome volume observed in salt‐stressed nodules. In incubations of bacteroids, isolated from salt‐stressed or unstressed plants and supplied with O₂ by purified oxyleghemoglobin, addition of proline stimulated neither O₂ consumption nor ARA. These results were consistent with proline playing a role as osmoticum, rather than energy source for bacteroid N₂ fixation in amide‐exporting legumes such as faba bean.     

Mistletoe viscotoxins induce membrane permeabilization and spore death in phytopathogenic fungi

Viscotoxins (Vts) are basic peptides expressed in mistletoe leaves, seeds and stems which have been shown to be cytotoxic to mammalian cells. The aim of this study was to analyse whether Vts were able to control and/or inhibit the growth of phytopathogenic fungi to obtain a clue to their biological function. Incubation of two Vt isoforms, VtA₃ and VtB, at a final concentration of 10 µ M resulted in a complete blockage of the germination of spores from three different pathogenic fungi. It was also shown that lower concentrations than 10 µ M of VtA₃ and VtB inhibit their mycelial growth in a dose-dependent manner. The protein dose required to inhibit the growth of Fusarium solani and Sclerotinia sclerotiorum to a 50% was between 1.5 and 3.75 µ M , which represents a potent activity. No significant differences in the antifungal potency for each Vt isoform, either VtA₃ and VtB, were observed, although they have been shown to exert differential cytotoxicity on mammalian cells. It was also demonstrated that Vts act as fungicidal compounds. To explore the basis of the antifungal activity the ability of VtA₃ to induce changes in membrane permeability and on the oxidative status of F. solani spores was analysed. By using a specific fluorescent probe on intact spores, it was demonstrated that VtA₃ produces rapid changes in fungal membrane permeability. It also induces H₂O₂ production verified by a histochemical staining. The data presented in this study support a direct role of Vts in the plant defence determined by their lethal effect on fungal pathogens.     

Triiodothyronine Is a High-Affinity Inhibitor of Amino Acid Transport System L1 in Cultured Astrocytes

Abstract: The relationship between the transport of thyroid hormones and that of amino acids was examined by measuring the uptake of amino acids that are characteristic substrates of systems L, A, and N, and the effect of 3,3',5-triiodo-L-thyronine (T₃) on this uptake, in cultured astrocytes. Tryptophan and leucine uptakes were rapid, Na⁺-independent, and efficiently inhibited by T₃ (half-inhibition at ∼ 2 μ M ). Two Na⁺-independent L-like systems (L₁ and L₂), common to leucine and aromatic amino acids, were characterized kinetically. System L₂ had a low affinity for leucine and tryptophan ( K _m= 0.3–0.9 m M ). The high-affinity system L₁ ( K _m∼ 10 μ M for both amino acids) was competitively inhibited by T₃ with a K _i of 2–3 μ M (close to the T₃ transport K _m). Several T₃ analogues inhibited system L₁ and the T₃ transport system similarly. Glutamine uptake and α-(methylamino)isobutyric acid uptake were, respectively, two and 200 times lower than tryptophan and leucine uptakes. T₃ had little effect on the uptakes of glutamine and α-(methylamino)isobutyric acid. The results indicate that the T₃ transport system and system L₁ are related.     

Kinetics of amino acid uptake by ectomycorrhizal roots

It is well established that ectomycorrhizal fungi can use amino acids as nitrogen and carbon sources, but data on the kinetic properties of amino acid uptake systems of ectomycorrhizal systems are scarce. Using ¹⁴C-labelled compounds we have determined the kinetics of uptake of amino acids by excised ectomycorrhizal roots for a range of distinct mycorrhizal types from three tree species, beech, spruce, and pine. All mycorrhizal types examined took up amino acids via high-affinity transport systems ( K _M values ranging from 19 to 233 mmol m^–3). A comparative analysis of kinetic parameters for uptake of amino acids and the ammonium analogue methylammonium showed that ectomycorrhizal roots have similar or even higher affinities (lower K _M values) for the amino acids, indicating that absorption of these organic forms of nitrogen (N) can contribute significantly to total N uptake by ectomycorrhizal plants. Analysis of amino acid uptake by ectomycorrhizal roots collected along a European north/south gradient of increasing mineral N pollution from northern Sweden to south Germany revealed no obvious trend in the uptake capabilities for amino acids by ectomycorrhizal roots in relation to the location of the sampling site on this gradient. Rather, the fungal species forming a particular morphotype was the factor determining uptake kinetics. It can therefore be deduced that the species composition of the fungal community will contribute significantly to the functional diversity of a population of mycorrhizal roots.     

Relationship between osmoprotection and the structure and intracellular accumulation of betaines by Escherichia coli

Abstract Naturally occuring betaines, especially glycine betaine and proline betaine, were accumulated by Escherichia coli from urine. In synthetic hyperosmotic medium, with an homologous series of added betaines, (CH₃)₃N⁺-(CH₂) _n -COO⁻, osmoprotective activity and intracellular accumulation decreased monotonically as n increased from 1 to 5. In contrast, α -substituted glycine betaines were accumulated in a similar manner to glycine betaine, but with different osmoprotective activities. Arsenobetaine, with a quaternary arsonium group, was also accumulated but amino acids which can become negatively charged in a chemically basic environment were not.     

Influence of nitrate and ammonium on the growth and 2,4-diaminobutyric acid composition of flatpea (Lathyrus sylvestris L.)

Abstract. Presence of 2.4-diaminobutyric acid (A₂bu), a neurotoxin, in tissues of flatpea ( Lathyrus sylvestris L.) necessitates a thorough understanding of the regulation of this nonprotein amino acid before the species can be recommended to livestock producers for forage applications. To determine how different concentrations and ratios of NO₃ and NH⁺₄ in growth media influence the levels of A₂bu and other free amino acids in the 'Lathco'flatpea cultivar, plants were grown hydroponically in controlled environments. The concentration of A₂bu was highest in tissues when the NO₃ to NH⁺₄ ratio in the nutrient solution was low. Responses of amides and other nonprotein amino acids, especially in the roots, followed a similar trend. Free protein amino acids in leaves and stems were generally unaffected by changes in NO₃ to NH⁺₄ ratios. In roots, protein amino acids increased as the NO₃ to NH⁺₄ ratio in the growth medium increased. Ammonium inhibited shoot and root growth; NO₃ alleviated the toxic effects of NH⁺₄. Soluble protein concentrations were higher in the shoots of NO₃-fed plants and in the roots of plants supplied with NH⁺₄. These results suggest that accumulation of A₂bu and other nonprotein amino acids, as well as asparagine and glutamine, plays a role in detoxification of NH⁺₄ and storage of N.     

The C Terminal Tail of the Histamine H2 Receptor Contains Positive and Negative Signals Important for Signal Transduction and Receptor Down-Regulation

Abstract: To examine the role of the C terminal tail in H₂ receptor regulation, three cDNAs, encoding truncated histamine H₂ receptor mutants (H₂T295, H₂T307, and H₂T341), were constructed and stably transfected in Chinese hamster ovary (CHO) cells. The amino acids before position 307 appear to be necessary for proper receptor transport or folding, as no detectable H₂ receptor binding of the H₂T295 was observed after transfection. Truncation of the C terminal tail by 51 amino acids (H₂T307) did not affect the binding properties of H₂ antagonists and histamine or histamine-induced signaling. Yet, removal of 17 amino acids generated a mutant receptor (H₂T341), which was able to form a ternary complex but was unable to fully activate the G_s protein on histamine exposure. Agonist-induced but not the cyclic AMP-dependent H₂ receptor down-regulation was more profound for the H₂T307 receptor, indicating that different structural elements of the H₂ receptor protein are involved in the cyclic AMP-dependent and independent pathways of H₂ receptor down-regulation. Taken together, in this study we identified regions in the C terminal tail of the H₂ receptor that act as positive and/or negative signals in H₂ receptor signaling and down-regulation.     

Differential Accumulation of Translatable mRNA Species During a Specific Host-pathogen Interaction Between Puccinia recondita tritici and Triticum aestivum

The time-course of mRNA induction in specific cultivar-race combinations was examined using a criss-cross interaction system consisting of nearly isogenic lines of wheat (Sinvalocho M.A. and Gamma 1R) and genetically related pathogenic races of leaf rust Puccinia recondita f. sp. tritici . Infection stimulated the differential accumulation of mRNA species, identified by in vitro translation, after only three days of inoculation with rust spores. Comparisons of different host-pathogen combinations showed polypeptide pattern changes likely to be associated with gene-for-gene relationships. At least two specific mRNAs which code for polypeptide bands of 34 and 24 kDa are associated with the compatible interaction mediated by genes A₁,/A₂ from Gamma 1R wheat line and virulence gene p_t/p₂ of rust race FO₁. Comparisons made using a mutant clone of rust, which elicits an inverse criss-cross relationship with the same wheat lines, are consistent with the proposed specificity of the detected changes.
In addition, two wheat mRNAs (coding for polypeptide bands of 20.5 and 32.5 kDa) were elicited by infection with rust race FO₁ regardless the plant genotype or reaction type. A cDNA clone involved in this kind of race specific induction has been isolated.     

Host plant effects on the performance of the aphid Aulacorthum solani (Kalt.) (Homoptera: Aphididae) at ambient and elevated CO2

In future elevated CO₂ environments, chewing insects are likely to perform less well than at present because of the effects of increased carbon fixation on their host plants. When the aphid, Aulacorthum solani was reared on bean ( Vicia faba ) and tansy ( Tanacetum vulgare ) plants under ambient and elevated CO_2, performance was enhanced on both hosts at elevated CO₂. The nature of the response was different on each plant species suggesting that feeding strategy may influence an insect's response to elevated CO₂. On bean, the daily rate of production of nymphs was increased by 16% but there was no difference in development time, whereas on tansy, development time was 10% shorter at elevated CO₂ but the rate of production of nymphs was not affected. The same aphid clone therefore responded differently to elevated CO₂ on different host plants. This increase in aphid performance could lead to larger populations of aphids in a future elevated CO₂ environment.     

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.     

Isolation and characterization of a general amino acid permease from the ectomycorrhizal fungus Amanita muscaria

A cDNA coding for a fungal amino acid transporter ( AmAAP1 ) was identified from Amanita muscaria ectomycorrhizas. The transporter gene was expressed at a basal level under all conditions investigated, but its expression was enhanced 10-fold in the absence of a N source utilized by the fungus. Nitrate was not a suitable N source for A. muscaria and resulted in maximal AmAAP1 expression. The expression of AmAAP1 in a yeast mutant revealed its function as a high-affinity amino acid transporter with a broad substrate spectrum. AmAAP1 takes up all investigated amino acids with K _m values between 22 μM for histidine and up to 100 μM for proline. Gene expression and amino acid uptake data together indicate two main functions for AmAAP1: uptake of amino acids from soil for fungal nutrition, and prevention of an amino acid loss by hyphal leakage in the absence of a suitable N source.     

Biosynthesis of aromatic amino acids by highly purified spinach chloroplasts – Compartmentation and regulation of the reactions

The ability of chloroplasts to synthesize aromatic amino acids from CO₂ was investigated using highly purified, intact spinach ( Spinacia oleracea L. cv. Viking II) chloroplasts and ¹⁴CO₂. Incorporation of ¹⁴C into aromatic amino acids was very low, however, and this was assumed to be due to lack of phosphoenolpyruvate (PEP), one of the substrates for the shikimate/arogenate pathway leading to aromatic amino acids in chloroplasts. Therefore, the glycolytic enzymes phosphoglycerate mutase (EC 2.7.5.3) and enolase (EC 4.2.1.11) were added to the ¹⁴CO₂ fixation medium in order to convert labelled 3-phosphoglycerate exported from the intact chloroplasts to 2-phosphoglycerate and PEP. In this way a part of the glycolytic pathway was reconstituted outside the chloroplasts to substitute for the cytoplasm lost on isolation. The presence of both enzymes in the medium increased incorporation of ¹⁴C into Tyr and Phe more than ten-fold and incorporation into Trp about two-fold, while total ¹³CO₂ fixation rates were not affected. Our results suggest that chloroplasts do not contain phosphoglycerate mutase or enolase, and that, in vivo, PEP is synthesized in the cytoplasm and imported to the chloroplast stroma for the biosynthesis of aromatic amino acids. The biosynthesis of all three aromatic amino acids was under feedback control. Using expected physiological concentrations (below 100 μ M ), each of the aromatic amino acids exerted a strict feedback inhibition of its own biosynthesis only.     

Transmission of pea bacterial blight (Pseudomonas syringae pv. pisi) from seed to seedling: effects of inoculum dose, inoculation method, temperature and soil moisture

Quantitative analyses of the utilization of amino acids by Lactococcus lactis subsp. cremoris FD1 in yeast extract medium (YE) and in casein peptone medium (CP) have been performed. Both free and peptide-bound amino acids were measured. In the CP most amino acids are peptide-bound and some amino acids are virtually only present in peptides. Thirty-six per cent of all peptide bonds in CP are hydrolysed during fermentation (6·3 mmol peptide bonds per gram biomass formed) and there is a transition of the growth rate related ATP consumption Y _xATP (mmol ATP g biomass^-1) from 25 mmol g^-1 to 71 mmol g^-1 coincident with a decrease of the peptide consumption. In YE most of the amino acids are on the free form and only 26% of the peptide bonds are hydrolysed during fermentation (1·5 mmol peptide bonds per gram biomass formed). A constant Y _xATP= 38 mmol g^-1 prevails throughout the fermentation in YE.     

The accumulation of proline in Prasiola crispa during winter in Antarctica

Samples of Prasiola crispa were collected in Antarctica throughout a 13-month period and analysed for free amino acids by HPLC. There was a marked increase in the levels of proline with the onset of winter, concurrent with a decrease in the other predominant amino acids. In January, proline constituted 1.2 ± 0.1 μ mol (g dry weight)⁻¹. whereas by mid-April it was the major component at 28.4 ± 2.9 μ mol (g dry weight)⁻¹. When winter samples were thawed in a growth cabinet, their proline content declined to 4.3 ± 0.5 μ mol (g dry weight)⁻¹ after 7 days. Measurements of photosynthetic quantum yield indicated that winter samples of P. crispa also recovered photosynthetic activity upon thawing. Amino acids and other solutes are involved in the preservation of photosynthetic activities during freezing and it seems probable that proline is involved in cryoprotection in this species. In summer samples, there was no evidence that proline levels in P. crispa increased with the conductivity of the water in which they were found growing.