Ornithine-δ-aminotransferase is essential for Arginine Catabolism but not for Proline Biosynthesis

<p>Abstract</p> <p>Background</p> <p>Like many other plant species, Arabidopsis uses arginine (Arg) as a storage and transport form of nitrogen, and proline (Pro) as a compatible solute in the defence against abiotic stresses causing water deprivation. Arg catabolism produces ornithine (Orn) inside mitochondria, which was discussed controversially as a precursor for Pro biosynthesis, alternative to glutamate (Glu).</p> <p>Results</p> <p>We show here that ornithine-δ-aminotransferase (δOAT, At5g46180), the enzyme converting Orn to pyrroline-5-carboxylate (P5C), is localised in mitochondria and is essential for Arg catabolism. Wildtype plants could readily catabolise supplied Arg and Orn and were able to use these amino acids as the only nitrogen source. Deletion mutants of δ<it>OAT</it>, however, accumulated urea cycle intermediates when fed with Arg or Orn and were not able to utilize nitrogen provided as Arg or Orn. Utilisation of urea and stress induced Pro accumulation were not affected in T-DNA insertion mutants with a complete loss of δ<it>OAT </it>expression.</p> <p>Conclusion</p> <p>Our findings indicate that δOAT feeds P5C exclusively into the catabolic branch of Pro metabolism, which yields Glu as an end product. Conversion of Orn to Glu is an essential route for recovery of nitrogen stored or transported as Arg. Pro biosynthesis occurs predominantly or exclusively via the Glu pathway in Arabidopsis and does not depend on Glu produced by Arg and Orn catabolism.</p

Non-redundant functions of two proline dehydrogenase isoforms in Arabidopsis

<p>Abstract</p> <p>Background</p> <p>Proline (Pro) accumulation is a widespread response of prokaryotic and eukaryotic cells subjected to osmotic stress or dehydration. When the cells are released from stress, Pro is degraded to glutamate by Pro-dehydrogenase (ProDH) and Pyrroline-5-carboxylate dehydrogenase (P5CDH), which are both mitochondrial enzymes in eukaryotes. While <it>P5CDH </it>is a single copy gene in Arabidopsis, two <it>ProDH </it>genes have been identified in the genome. Until now, only <it>ProDH1 </it>(At3g30775) had been functionally characterised.</p> <p>Results</p> <p>We demonstrate vasculature specific expression of the Arabidopsis <it>ProDH2 </it>gene (At5g38710) as well as enzymatic activity and mitochondrial localisation of the encoded protein. Expression levels of <it>ProDH2 </it>are generally low, but increased in senescent leaves and in the abscission zone of floral organs. While sucrose represses <it>ProDH2 </it>expression, Pro and NaCl were identified as inducers. Endogenous <it>ProDH2 </it>expression was not able to overcome Pro sensitivity of <it>ProDH1 </it>mutants, but overexpression of a GFP-tagged form of ProDH2 enabled the utilisation of Pro as single nitrogen source for growth. Amongst two intronic insertion mutants, one was identified as a null allele, whereas the other still produced native <it>ProDH2 </it>transcripts.</p> <p>Conclusions</p> <p>Arabidopsis possesses two functional ProDHs, which have non-redundant, although partially overlapping physiological functions. The two ProDH isoforms differ with respect to spatial, developmental and environmental regulation of expression. While <it>ProDH1 </it>appears to be the dominant isoform under most conditions and in most tissues, <it>ProDH2 </it>was specifically upregulated during salt stress, when <it>ProDH1 </it>was repressed. The characterisation of <it>ProDH2 </it>as a functional gene requires a careful re-analysis of mutants with a deletion of <it>ProDH1</it>, which were so far considered to be devoid of ProDH activity. We hypothesise that ProDH2 plays an important role in Pro homeostasis in the vasculature, especially under stress conditions that promote Pro accumulation.</p

Proline metabolism and transport in plant development

Proline fulfils diverse functions in plants. As amino acid it is a structural component of proteins, but it also plays a role as compatible solute under environmental stress conditions. Proline metabolism involves several subcellular compartments and contributes to the redox balance of the cell. Proline synthesis has been associated with tissues undergoing rapid cell divisions, such as shoot apical meristems, and appears to be involved in floral transition and embryo development. High levels of proline can be found in pollen and seeds, where it serves as compatible solute, protecting cellular structures during dehydration. The proline concentrations of cells, tissues and plant organs are regulated by the interplay of biosynthesis, degradation and intra- as well as intercellular transport processes. Among the proline transport proteins characterized so far, both general amino acid permeases and selective compatible solute transporters were identified, reflecting the versatile role of proline under stress and non-stress situations. The review summarizes our current knowledge on proline metabolism and transport in view of plant development, discussing regulatory aspects such as the influence of metabolites and hormones. Additional information from animals, fungi and bacteria is included, showing similarities and differences to proline metabolism and transport in plant

Proline synthesis in developing microspores is required for pollen development and fertility

Background: In many plants, the amino acid proline is strongly accumulated in pollen and disruption of proline synthesis caused abortion of microspore development in Arabidopsis. So far, it was unclear whether local biosynthesis or transport of proline determines the success of fertile pollen development. Results: We analyzed the expression pattern of the proline biosynthetic genes PYRROLINE-5-CARBOXYLATE SYNTHETASE 1 & 2 (P5CS1 & 2) in Arabidopsis anthers and both isoforms were strongly expressed in developing microspores and pollen grains but only inconsistently in surrounding sporophytic tissues. We introduced in a p5cs1/p5cs1 p5cs2/P5CS2 mutant background an additional copy of P5CS2 under the control of the Cauliflower Mosaic Virus (CaMV) 35S promoter, the tapetum-specific LIPID TRANSFER PROTEIN 12 (Ltp12) promoter or the pollen-specific At5g17340 promoter to determine in which site proline biosynthesis can restore the fertility of proline-deficient microspores. The specificity of these promoters was confirmed by β-glucuronidase (GUS) analysis, and by direct proline measurement in pollen grains and stage-9/10 anthers. Expression of P5CS2 under control of the At5g17340 promoter fully rescued proline content and normal morphology and fertility of mutant pollen. In contrast, expression of P5CS2 driven by either the Ltp12 or CaMV35S promoter caused only partial restoration of pollen development with little effect on pollen fertility. Conclusions: Overall, our results indicate that proline transport is not able to fulfill the demand of the cells of the male germ line. Pollen development and fertility depend on local proline biosynthesis during late stages of microspore development and in mature pollen grains

A Specific and Sensitive Enzymatic Assay for the Quantitation of L-Proline

Because 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.publishe

Physiological implications of arginine metabolism in plants

Nitrogen is a limiting resource for plant growth in most terrestrial habitats since large amounts of nitrogen are needed to synthesize nucleic acids and proteins. Among the 21 proteinogenic amino acids, arginine has the highest nitrogen to carbon ratio, which makes it especially suitable as a storage form of organic nitrogen. Synthesis in chloroplasts via ornithine is apparently the only operational pathway to provide arginine in plants, and the rate of arginine synthesis is tightly regulated by various feedback mechanisms in accordance with the overall nutritional status. While several steps of arginine biosynthesis still remain poorly characterized in plants, much wider attention has been paid to inter- and intracellular arginine transport as well as arginine-derived metabolites. A role of arginine as alternative source besides glutamate for proline biosynthesis is still discussed controversially and may be prevented by differential subcellular localization of enzymes. Apparently, arginine is a precursor for nitric oxide (NO), although the molecular mechanism of NO production from arginine remains unclear in higher plants. In contrast, conversion of arginine to polyamines is well documented, and in several plant species also ornithine can serve as a precursor for polyamines. Both NO and polyamines play crucial roles in regulating developmental processes as well as responses to biotic and abiotic stress. It is thus conceivable that arginine catabolism serves on the one hand to mobilize nitrogen storages, while on the other hand it may be used to fine-tune development and defense mechanisms against stress. This review summarizes the recent advances in our knowledge about arginine metabolism, with a special focus on the model plant Arabidopsis thaliana, and pinpoints still unresolved critical questions

Enzymology and Regulation of δ¹-Pyrroline-5-Carboxylate Synthetase 2 From Rice

Under 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.publishe

Proline Metabolism and its Functions in Development and Stress Tolerance.

Most green plants are fully autotrophic organisms and can produce their entire biomass from inorganic molecules with the help of light energy captured by photosynthesis. Energy from photosynthesis is thereby not only needed to reduce CO2 to carbohydrates but also to assimilate nitrogen, phosphorus, and sulfur from inorganic salts for the biosynthesis of proteins and nucleic acids). In contrast, most non-photosynthetic organisms, including animals and humans, depend on the uptake of organic material both as energy source and as building material. This fundamental difference exists since the development of oxygenic photosynthesis by cyanobacteria, which were later converted to endosymbiotic chloroplasts in eukaryotic algae and plants. It is therefore not surprising that despite the use of identical building blocks in all living organisms, i.e., nucleotides, amino acids, and carbohydrates, the pathways to acquire or synthesize these building blocks are not identical in distantly related groups of organisms. Knowledge about human metabolism can therefore only serve to guide investigations of regulatory and metabolic pathways in plant primary metabolism but not as a direct template

Functional characterization and expression analysis of rice δ¹-pyrroline-5-carboxylate dehydrogenase provide new insight into the regulation of proline and arginine catabolism

While intracellular proline accumulation in response to various stress conditions has been investigated in great detail, the biochemistry and physiological relevance of proline degradation in plants is much less understood. Moreover, the second and last step in proline catabolism, the oxidation of δ(1)-pyrroline-5-carboxylic acid (P5C) to glutamate, is shared with arginine catabolism. Little information is available to date concerning the regulatory mechanisms coordinating these two pathways. Expression of the gene coding for P5C dehydrogenase was analyzed in rice by real-time PCR either following the exogenous supply of amino acids of the glutamate family, or under hyperosmotic stress conditions. The rice enzyme was heterologously expressed in E. coli, and the affinity-purified protein was thoroughly characterized with respect to structural and functional properties. A tetrameric oligomerization state was observed in size exclusion chromatography, which suggests a structure of the plant enzyme different from that shown for the bacterial P5C dehydrogenases structurally characterized to date. Kinetic analysis accounted for a preferential use of NAD(+) as the electron acceptor. Cations were found to modulate enzyme activity, whereas anion effects were negligible. Several metal ions were inhibitory in the micromolar range. Interestingly, arginine also inhibited the enzyme at higher concentrations, with a mechanism of uncompetitive type with respect to P5C. This implies that millimolar levels of arginine would increase the affinity of P5C dehydrogenase toward its specific substrate. Results are discussed in view of the involvement of the enzyme in either proline or arginine catabolism.publishe