1,720,972 research outputs found
A Specific and Sensitive Enzymatic Assay for the Quantitation of L-Proline
No full textBecause proline accumulates rapidly in response to several stress conditions such as drought and excess salt, increased intracellular levels of free proline are considered a hallmark of adaptive reactions in plants, particularly in response to water stress. Proline quantitation is easily achievable by reaction with ninhydrin, since under acidic conditions peculiar red or yellow reaction products form with this unique cyclic amino acid. However, little attention has been paid to date to cross-reaction of ninhydrin with other amino acids at high levels, or with structurally related compounds that may also be present at significant concentrations in plant tissues, possibly leading to proline overestimation. In vitro at high pH values, δ1-pyrroline-5-carboxylate reductase, the enzyme catalyzing the second and last step in proline synthesis from glutamate, was early found to catalyze the reverse oxidation of proline with the concomitant reduction of NAD(P)+ to NAD(P)H. Here we characterized this reverse reaction using recombinant enzymes from Arabidopsis thaliana and Oryza sativa, and demonstrated its utility for the specific quantification of L-proline. By optimizing the reaction conditions, fast, easy, and reproducible measurement of L-proline concentration was achieved, with similar sensitivity but higher specificity than the commonly used ninhydrin methods
Enzymology and Regulation of δ1-Pyrroline-5-Carboxylate Synthetase 2 From Rice
No full textUnder several stress conditions, such as excess salt and drought, many plants accumulate proline inside the cell, which is believed to help counteracting the adverse effects of low water potential. This increase mainly relies upon transcriptional induction of δ1-pyrroline-5-carboxylate synthetase (P5CS), the enzyme that catalyzes the first two steps in proline biosynthesis from glutamate. P5CS mediates both the phosphorylation of glutamate and the reduction of γ-glutamylphosphate to glutamate-5-semialdehyde, which spontaneously cyclizes to δ1-pyrroline-5-carboxylate (P5C). In most higher plants, two isoforms of P5CS have been found, one constitutively expressed to satisfy proline demand for protein synthesis, the other stress-induced. Despite the number of papers to investigate the regulation of P5CS at the transcriptional level, to date, the properties of the enzyme have been only poorly studied. As a consequence, the descriptions of post-translational regulatory mechanisms have largely been limited to feedback-inhibition by proline. Here, we report cloning and heterologous expression of P5CS2 from Oryza sativa. The protein has been fully characterized from a functional point of view, using an assay method that allows following the physiological reaction of the enzyme. Kinetic analyses show that the activity is subjected to a wide array of regulatory mechanisms, ranging from product inhibition to feedback inhibition by proline and other amino acids. These findings confirm long-hypothesized influences of both, the redox status of the cell and nitrogen availability, on proline biosynthesis
Editorial: Amino Acids in plants: regulation and functions in development and stress defense
Full text linkFor quite a long time, research on amino acid metabolism received only limited attention in the areas of plant physiology and biochemistry. Owing to the essential function of amino acids in protein synthesis, it was tempting to assume that plants use and metabolize amino acids in the same manner as microorganism or humans do. However, as fully autotrophic organisms, plants face fundamentally different challenges compared to organisms that make a living on plant- (or algae-) produced biomass. Furthermore, plants produce literally hundreds of non-proteinogenic amino acids or amino acid-derived secondary metabolites and thus require a very distinct regulatory system to coordinate the needs of primary and secondary metabolism. The distribution of amino acid metabolism between roots and aboveground organs or among the different subcellular compartments adds another level of complexity further distinguishing plants from prokaryotes or animals.
From the perspective of human nutrition, the amino acids that we cannot synthesize by ourselves are the most interesting. Accordingly, the efforts to understand the routes and regulation of Lys, Met, and Trp biosynthesis has boosted amino acid research in plants, since these three amino acids are often contained in limiting amounts in staple crops. From an agricultural perspective, amino acid biosynthetic pathways that are exclusively found in plants were analyzed and used for the development of herbicides.
At present, the threats of climate change and groundwater contamination by excess nitrogen fertilization call for a re-focusing of our efforts. Optimal productivity is currently achieved by massive application of nitrogen-containing fertilizers and is yet limited by the efficiency of the plants in taking up, distributing, storing and assimilating the nitrogen, with all these processes being governed by amino acid metabolism and transport. Similarly, many defense responses of plants against biotic or abiotic stress involve metabolic adjustments in amino acid metabolism to counteract detrimental environmental impacts directly or to provide precursors for defense compounds. Several amino acids emerge as important signaling molecules that orchestrate plant growth and development by integrating the metabolic status of the plant with environmental signals, especially in stressful conditions. However, our knowledge about amino acid metabolism, the mechanisms that regulate the levels of free amino acids and the diverse functions of amino acids in plants is far from complete.
This Research Topic aims to collect contributions from different facets of amino acid metabolism, transport or signaling to bring together and integrate into a comprehensive view the latest advances in our understanding of the multiple functions of amino acids in plants
Requirement of Proline Synthesis during Arabidopsis reproductive development
No full textBackground
Gamete and embryo development are crucial for successful reproduction and seed set in plants, which is often the determining factor for crop yield. Proline accumulation was largely viewed as a specific reaction to overcome stress conditions, while recent studies suggested important functions of proline metabolism also in reproductive development. Both the level of free proline and proline metabolism were proposed to influence the transition to flowering, as well as pollen and embryo development.
Results
In this study, we performed a detailed analysis of the contribution of individual proline biosynthetic enzymes to vegetative development and reproductive success in Arabidopsis. In contrast to previous reports, we found that pyrroline-5-carboxylate (P5C) synthetase 2 (P5CS2) is not essential for sexual reproduction although p5cs2 mutant plants were retarded in vegetative development and displayed reduced fertility under long-day conditions. Single mutant plants devoid of P5CS1 did not show any developmental defects. Simultaneous absence of both P5CS isoforms resulted in pollen sterility, while fertile egg cells could still be produced. Expression of P5C reductase (P5CR) was indispensable for embryo development but surprisingly not needed for pollen or egg cell fertility. The latter observation could be explained by an extreme stability of P5CR activity, which had a half-life time of greater than 3 weeks in vitro. Expression of P5CR-GFP under the control of the endogenous P5CR promoter was able to restore growth of homozygous p5cr mutant embryos. The analysis of P5CR-GFP-fluorescence in planta supported an exclusively cytoplasmatic localisation of P5CR.
Conclusions
Our results demonstrate that potential alternative pathways for proline synthesis or inter-generation transfer of proline are not sufficient to overcome a defect in proline biosynthesis from glutamate during pollen development. Proline biosynthesis through P5CS2 and P5CR is limiting for vegetative and reproductive development in Arabidopsis, whereas disruption of P5CS1 alone does not affect development of non-stressed plants
A complex array of factors regulate the activity of Arabidopsis thaliana δ1-pyrroline-5-carboxylate synthetase isoenzymes to ensure their specific role in plant cell metabolism
No full textThe first and committed step in proline synthesis from glutamate is catalyzed by delta1-pyrroline-5-carboxylate synthetase (P5CS). Two P5CS genes have been found in most angiosperms, one constitutively expressed to satisfy proline demand for protein synthesis, the other stress-induced. Despite the number of papers to investigate regulation at the transcriptional level, to date, the properties of the enzymes have been subjected to limited study. The isolation of Arabidopsis thaliana P5CS isoenzymes was achieved through heterologous expression and affinity purification. The two proteins were characterized with respect to kinetic and biochemical properties. AtP5CS2 showed KM values in the micro- to millimolar range, and its activity was inhibited by NADP+, ADP and proline, and by glutamine and arginine at high levels. Mg2+ ions were required for activity, which was further stimulated by K+ and other cations. AtP5CS1 displayed positive cooperativity with glutamate and was almost insensitive to inhibition by proline. In the presence of physiological, nonsaturating concentrations of glutamate, proline was slightly stimulatory, and glutamine strongly increased the catalytic rate. Data suggest that the activity of AtP5CS isoenzymes is differentially regulated by a complex array of factors including the concentrations of proline, glutamate, glutamine, monovalent cations and pyridine dinucleotides
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
Going Beyond Counting First Authors in Author Co-citation Analysis
Full text linkThe present study examines one of the fundamental aspects of author co-citation analysis (ACA) - the way co-citation
counts are defined. Co-citation counting provides the data on which all subsequent statistical analyses and mappings
are based, and we compare ACA results based on two different types of co-citation counting - the traditional type that
only counts the first one among a cited work's authors on the one hand and a non-traditional type that takes into
account the first 5 authors of a cited work on the other hand. Results indicate that the picture produced through this non-traditional author co-citation counting contains more coherent author groups and is therefore considerably clearer. However, this picture represents fewer specialties in the research field being studied than that produced through the traditional first-author co-citation counting when the same number of top-ranked authors is selected and analyzed. Reasons for these effects are discussed
Variations on the Author
Full text link“Variations on the Author” discusses two of Eduardo Coutinho’s recent films (Um Dia na Vida, from 2010, and Últimas Conversas, posthumously released in 2015) and their contribution to the general question of documentary authorship. The director’s filmography is characterized by a consistent yet self-effacing form of authorial self-inscription: Coutinho often features as an interviewer that rather than express opinions propels discourses; an interviewer that is good at listening. This mode of self-inscription characterizes him as an author who is not expressive but who is nonetheless markedly present on the screen. In Um Dia na Vida, however, Coutinho is completely absent form the image, while Últimas Conversas, on the contrary, includes a confessional prologue that moves the director from the margins to the center of his films. This article examines the ways in which these works stand out in the filmography of a director who offers new insights into the notion of cinematic authorship
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