1,720,969 research outputs found
Differential accumulation of γ–aminobutyric acid in elicited cells of two rice cultivars showing contrasting sensitivity to the blast pathogen.
No full textIntracellular free amino acid pools were quantified in suspension cultured cells of a blast-sensitive and a blast-resistant rice genotype at increasing time after the treatment with Magnaporthe oryzae cell wall hydrolysates. Besides some expected variations in free phenylalanine, a remarkable early increase of γ–aminobutyric acid (GABA) levels was evident in both cultivars. Glutamate decarboxylase activity and protein levels were unaffected. GABA homeostasis was recovered in the sensitive cultivar 48 h after the treatment. On the contrary, a further GABA accumulation and a general increase of most amino acids were found at this later stage in the resistant genotype, which showed a higher decrease of cell viability as a consequence of elicitor addition. Data support a recently-hypothesized role of GABA metabolism in the plant response to fungal pathogens
Plant P5C reductase as a new target for aminomethylenebisphosphonates.
No full textA series of N-substituted derivatives of aminomethylenebisphosphonic acid were evaluated as potential inhibitors of -pyrroline-5-carboxylate reductase (EC 1.5.1.2), the enzyme that catalyzes the last step in proline biosynthesis, partially purified from Arabidopsis thaliana suspension cultured cells. At millimolar concentrations, three compounds out of 26 were found to interfere with the catalytic mechanism. One of them, namely, 3,5-dichloropyridyl-aminomethylenebisphosphonic acid, retained such inhibitory activity in the micromolar range. Kinetic analyses ruled out the possibility that the inhibition could simply rely upon the chelating properties of bisphosphonates and showed mechanisms of a noncompetitive type against NADH and an uncompetitive type against pyrroline-5-carboxylic acid, with K
I values of 199 +/- 6 and 10.3 +/- 1.5 uM, respectively. A computer-aided docking analysis, performed on the basis of the crystal structure of the enzyme from Streptococcus pyogenes, suggested that this phosphonate may interact with amino acid residues near the binding site of pyrroline-5-carboxylic acid, thus blocking the substrate in a pocket and preventing its interaction with NADH. Because in higher plants the step catalyzed by pyrroline-5-carboxylate reductase is shared by all pathways leading to proline synthesis, such a compound may represent a lead structure to be exploited for the design of new substances endowed with herbicidal activity
acts as co‐substrate
No full text• Δ1-pyrroline-5-carboxylate (P5C) reductase catalyses the final step of proline synthesis in plants. In Arabidopsis thaliana, protein levels are correlated neither to the corresponding mRNA copy numbers, nor to the intracellular proline concentrations. The occurrence of post-translational regulative mechanisms was therefore hypothesized, but never assessed.
• The purification of A. thaliana P5C reductase was achieved through either a six-step protocol from cultured cells, or heterologous expression of AtP5CR in E. coli. The protein was characterized with respect to structural, kinetic, and biochemical properties.
• P5C reductase was able to use either NADPH or NADH as the electron donor, with contrasting affinities and maximum reaction rates. The presence of equimolar levels of NADP+ completely suppressed the NADH-dependent activity, whereas the NADPH-dependent reaction was mildly affected. Proline inhibited only the NADH-dependent reaction. At physiological levels, increasing concentrations of salt steadily inhibited the NADH-dependent activity, but were stimulatory of the NADPH-dependent reaction.
• The biochemical properties of A. thaliana P5C reductase suggest a complex regulation of enzyme activity by the redox status of the pyridine nucleotide pools, and the levels of proline and chloride in the cytosol. Data support a so far underestimated role of P5C reductase in controlling stress-induced proline accumulation
Carotenoid cleavage in chromoplasts of white and yellow-fleshed peach varieties
No full textBackground: In peach fruit, carotenoid accumulation in the mesocarp causes the difference between yellow and white genotypes. The latter are generally characterized by a peculiar and more intense aroma, because of higher release of volatiles deriving from dioxygenase-catalysed breakdown of the tetraterpene skeleton. The rate of carotenoid oxidation was investigated in peach (Prunus persica L.) fruits harvested at various stages of development. Two couples of white and yellow-fleshed isogenic varieties and an ancestral white-fleshed genotype were analysed, which had previously shown to differ in Carotenoid Cleavage Dioxygenase 4 allelic composition resulting in various combinations of putatively active/inactive proteins. Results: Carotenoid bleaching activity was localized in the insoluble fraction of fruit flesh chromoplasts. Higher rates of trans-β-apo-8′-carotenal than β-carotene bleaching suggest that the first cleavage reaction is the rate-limiting step. Consistently, HPLC analysis did not show the appearance of coloured intermediates in reaction mixtures. High levels of substrate breakdown were found during the initial phases of fruit development in all genotypes examined, whereas significant differences were evident during the second exponential growth phase and ripening onset. Also, the ratio of carotene versus carotenale utilization varied significantly. Conclusion: Pattern comparison among activity levels measured in vitro on chromoplast enriched fractions suggests that cleavage enzyme(s) other than Carotenoid Cleavage Dioxygenase 4 play a significant role in carotenoid breakdown during fruit development and ripening
Synthesis and evaluation of effective inhibitors of plant δ1-pyrroline-5-carboxylate reductase
No full textAnalogues of previously studied phenyl-substituted aminomethylene-bisphosphonic acids were synthesized and
evaluated as inhibitors of Arabidopsis thaliana δ1-pyrroline-5-carboxylate reductase. With the aim of improving their effectiveness, two main modifications were introduced into the inhibitory scaffold: the aminomethylenebisphosphonic moiety was replaced with a hydroxymethylenebisphosphonic group, and the length of the molecule was increased by replacing the methylene linker with an ethylidene chain. In addition, chlorine atoms in the phenyl ring were replaced with various other substituents. Most of the studied derivatives showed activity in the micromolar to millimolar range, with two of them being more effective than the lead compound, with concentrations inhibiting 50% of enzyme activity as low as 50 μM. Experimental evidence supporting the ability of these inhibitors to interfere with proline synthesis in vivo is also shown
Phytotoxicity of aminobisphosphonates targeting both δ1-pyrroline-5-carboxylate reductase and glutamine synthetase
No full textBACKGROUND: Dual-target inhibitors may contribute to the management of herbicide-resistant weeds and avoid or delay the selection of resistant biotypes. Some aminobisphosphonates inhibit the activity of both glutamine synthetase and δ1-pyrroline-5-carboxylate (P5C) reductase in vitro, but the relevance of the latter in vivo has yet to be proven. This study aimed at demonstrating that these compounds can also block proline synthesis in planta. RESULTS: Two aminophosphonates, namely 3,5-dichlorophenylamino-methylenebisphosphonic acid and 3,5-dibromophenylaminomethylenebis phosphonic acid (Br2PAMBPA), showed inverse effectiveness against the two partially purified target enzymes from rapeseed. The compounds showed equipotency in inhibiting the growth of rapeseed seedlings and cultured cells. The analysis of amino acid content in treated cells showed a strong reduction in glutamate and glutamate-related amino acid pools, but a milder effect on free proline. In the case of Br2PAMBPA, toxic P5C levels accumulated in treated seedlings, proving that the inhibition of P5C reductase takes place in situ. CONCLUSIONS: Phenyl-substituted aminobisphosphonates may be regarded as true dual-target inhibitors. Their use to develop new active principles for crop protection could consequently represent a tool to address the problem of target-site resistance among weeds
Sublethal detergent concentrations increase metabolization of recalcitrant polyphosphonates by the cyanobacterium Spirulina platensis
No full textAs a consequence of increasing industrial applications, thousand tons of polyphosphonates are introduced every year into the environment. The inherent stability of the C–P bond results in a prolonged half-life. Moreover, low uptake rates limit further their microbial metabolization. To assess whether low detergent concentrations were able to increase polyphosphonate utilization by the cyanobacterium Spirulina platensis, tolerance limits to the exposure to various detergents were determined by measuring the growth rate in the presence of graded levels below the critical micellar concentration. Then, the amount of hexamethylenediamine-N,N,N′,N′-tetrakis(methylphosphonic acid) that is metabolized in the absence or in the presence of sublethal detergent concentrations was quantified by 31P NMR analysis on either P-starved or P-fed cyanobacterial cultures. The strain tolerated the presence of detergents in the order: nonionic > anionic > cationic. When added to the culture medium at the highest concentrations showing no detrimental effects upon cell viability, detergents either improved or decreased polyphosphonate
utilization, the anionic sodium dodecyl sulfate being the most
beneficial. Metabolization was not lower in P-fed cells—a
result that strengthens the possibility of using, in the future, this strain for bioremediation purposes
Effectiveness and mode of action of phosphonate inhibitors of plant glutamine synthetase
No full textBACKGROUND: Aiming at the rationale design of new herbicides, the availability of the three-dimensional structure of the target enzyme greatly enhances the optimization of lead compounds and the design of derivatives with increased activity. Among the most widely exploited herbicide targets is glutamine synthetase. Recently, the structure of a cytosolic form of the maize enzyme has been described, allowing verifying whether steric, electronic and hydrophobic features of a compound are in agreement with inhibitor-protein interaction geometry.
RESULTS: Three series of compounds (aminophosphonates, hydroxyphosphonates and aminomethylenebisphosphonates) were evaluated as possible inhibitors of maize glutamine synthetase. Aminomethylenebisphosphonate derivatives substituted in the phenyl ring retained the inhibitory potential, whereas variations in the scaffold, i.e. the replacement of the second phosphonate moiety with a hydroxyl or an amino residue, resulted in a significant loss of activity. A kinetic characterization showed a mechanism of non competitive type against glutamate and uncompetitive against ATP. A docking analysis suggested the mode of bisphosphonate binding to the active site.
CONCLUSION: Results allowed defining the features required to maintain or enhance the biological activity of these compounds, which represent lead structures to be further exploited for the design of new substances endowed with herbicidal activity
Synthetic analogues of the natural compound cryphonectric acid interfere with the photosynthetic electron transport chain through two different mechanisms.
No full textA series of isobenzofuran-1(3H)-ones (phthalides), analogues of the naturally occurring phytotoxin cryphonectric acid, were designed, synthesized, and fully characterized by NMR, IR, and MS analyses. Their synthesis was achieved via condensation, aromatization, and acetylation reactions. The measurement of the electron transport chain in spinach chloroplasts showed that several derivatives are capable of interfering with the photosynthetic apparatus. Few of them were found to inhibit the basal rate, but a significant inhibition was brought about only at concentrations exceeding 50 μM. Some other analogues acted as uncouplers or energy transfer inhibitors, with a remarkably higher effectiveness. Isobenzofuranone addition to the culture medium inhibited the growth of the cyanobacterium Synechococcus elongatus, with patterns consistent with the effects measured in vitro upon isolated chloroplasts. The most active derivatives, being able to completely suppress algal growth at 20 μM, may represent structures to be exploited for the design of new active ingredients for weed control
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