Guanidine production by plant homoarginine-6-hydroxylases.

Metabolism and biological functions of the nitrogen-rich compound guanidine have long been neglected. The discovery of four classes of guanidine-sensing riboswitches and two pathways for guanidine degradation in bacteria hint at widespread sources of unconjugated guanidine in nature. So far, only three enzymes from a narrow range of bacteria and fungi have been shown to produce guanidine, with the ethylene-forming enzyme (EFE) as the most prominent example. Here, we show that a related class of Fe2+- and 2-oxoglutarate-dependent dioxygenases (2-ODD-C23) highly conserved among plants and algae catalyze the hydroxylation of homoarginine at the C6-position. Spontaneous decay of 6-hydroxyhomoarginine yields guanidine and 2-aminoadipate-6-semialdehyde. The latter can be reduced to pipecolate by pyrroline-5-carboxylate reductase but more likely is oxidized to aminoadipate by aldehyde dehydrogenase ALDH7B in vivo. Arabidopsis has three 2-ODD-C23 isoforms, among which Din11 is unusual because it also accepted arginine as substrate, which was not the case for the other 2-ODD-C23 isoforms from Arabidopsis or other plants. In contrast to EFE, none of the three Arabidopsis enzymes produced ethylene. Guanidine contents were typically between 10 and 20 nmol*(g fresh weight)-1 in Arabidopsis but increased to 100 or 300 nmol*(g fresh weight)-1 after homoarginine feeding or treatment with Din11-inducing methyljasmonate, respectively. In 2-ODD-C23 triple mutants, the guanidine content was strongly reduced, whereas it increased in overexpression plants. We discuss the implications of the finding of widespread guanidine-producing enzymes in photosynthetic eukaryotes as a so far underestimated branch of the bio-geochemical nitrogen cycle and propose possible functions of natural guanidine production.

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.

The first and committed step in proline synthesis from glutamate is catalyzed by δ1 -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.

A standalone editing protein deacylates mischarged canavanyl-tRNAArg to prevent canavanine incorporation into proteins.

Error-free translation of the genetic code into proteins is vitally important for all organisms. Therefore, it is crucial that the correct amino acids are loaded onto their corresponding tRNAs. This process is highly challenging when aminoacyl-tRNA-synthetases encounter structural analogues to the native substrate like the arginine antimetabolite canavanine. To circumvent deleterious incorporation due to tRNA mischarging, editing mechanisms have evolved. However, only for half of the tRNA synthetases, editing activity is known and only few specific standalone editing proteins have been described. Understanding the diverse mechanisms resulting in error-free protein synthesis is of great importance. Here, we report the discovery of a protein that is upregulated upon canavanine stimulation in bacteria that live associated with canavanine-producing plants. We demonstrate that it acts as standalone editing protein specifically deacylating canavanylated tRNAArg. We therefore propose canavanyl-tRNAArgdeacylase (CtdA) as systematic name. Knockout strains show severe growth defects in canavanine-containing media and incorporate high amounts of canavanine into the proteome. CtdA is frequently found under control of guanidine riboswitches, revealing a functional connection of canavanine and guanidine metabolisms. Our results are the first to show editing activity towards mischarged tRNAArg and add to the puzzle of how faithful translation is ensured in nature.

Error-free translation is one of the most vital processes in all living organisms, but can be substantially challenged by compounds that mimic amino acids. Canavanine, or 5-oxa-arginine, is used as an antimetabolite by higher plants that is toxic due to its incorporation into proteins. We report the discovery of a standalone editing protein specifically deacylating canavanylated tRNAArg that enables the legume rhizosphere inhabitant Pseudomonas canavaninivorans to prevent canavanine mis-incorporation into its proteome. Our results are the first to show editing activity towards mischarged tRNAArg and add to the puzzle of how faithful translation is ensured in nature.

Guanidino acid hydrolysis by the human enzyme annotated as agmatinase.

Guanidino acids such as taurocyamine, guanidinobutyrate, guanidinopropionate, and guanidinoacetate have been detected in humans. However, except for guanidionacetate, which is a precursor of creatine, their metabolism and potential functions remain poorly understood. Agmatine has received considerable attention as a potential neurotransmitter and the human enzyme so far annotated as agmatinase (AGMAT) has been proposed as an important modulator of agmatine levels. However, conclusive evidence for the assigned enzymatic activity is lacking. Here we show that AGMAT hydrolyzed a range of linear guanidino acids but was virtually inactive with agmatine. Structural modelling and direct biochemical assays indicated that two naturally occurring variants differ in their substrate preferences. A negatively charged group in the substrate at the end opposing the guanidine moiety was essential for efficient catalysis, explaining why agmatine was not hydrolyzed. We suggest to rename AGMAT as guanidino acid hydrolase (GDAH). Additionally, we demonstrate that the GDAH substrates taurocyamine, guanidinobutyrate and guanidinopropionate were produced by human glycine amidinotransferase (GATM). The presented findings show for the first time an enzymatic activity for GDAH/AGMAT. Since agmatine has frequently been proposed as an endogenous neurotransmitter, the current findings clarify important aspects of the metabolism of agmatine and guanidino acid derivatives in humans.

Interplay between Proline Metabolism and ROS in the Fine Tuning of Root-Meristem Size in Arabidopsis.

We previously reported that proline modulates root meristem size in Arabidopsis by controlling the ratio between cell division and cell differentiation. Here, we show that proline metabolism affects the levels of superoxide anion (O2•-) and hydrogen peroxide (H2O2), which, in turn, modulate root meristem size and root elongation. We found that hydrogen peroxide plays a major role in proline-mediated root elongation, and its effects largely overlap those induced by proline, influencing root meristem size, root elongation, and cell cycle. Though a combination of genetic and pharmacological evidence, we showed that the short-root phenotype of the proline-deficient p5cs1 p5cs2/P5CS2, an Arabidopsis mutant homozygous for p5cs1 and heterozygous for p5cs2, is caused by H2O2 accumulation and is fully rescued by an effective H2O2 scavenger. Furthermore, by studying Arabidopsis mutants devoid of ProDH activity, we disclosed the essential role of this enzyme in the modulation of root meristem size as the main enzyme responsible for H2O2 production during proline degradation. Proline itself, on the contrary, may not be able to directly control the levels of H2O2, although it seems able to enhance the enzymatic activity of catalase (CAT) and ascorbate peroxidase (APX), the two most effective scavengers of H2O2 in plant cells. We propose a model in which proline metabolism participates in a delicate antioxidant network to balance H2O2 formation and degradation and fine-tune root meristem size in Arabidopsis.

Cytosolic proline is required for basal freezing tolerance in Arabidopsis.

The amino acid proline accumulates in many plant species under abiotic stress conditions, and various protective functions have been proposed. During cold stress, however, proline content in Arabidopsis thaliana does not correlate with freezing tolerance. Freezing sensitivity of a starchless plastidic phosphoglucomutase mutant (pgm) indicated that localization of proline in the cytosol might stabilize the plasma membrane during freeze-thaw events. Here, we show that re-allocation of proline from cytosol to vacuole was similar in the pyrroline-5-carboxylate synthase 2-1 (p5cs2-1) mutant and the pgm mutant and caused similar reduction of basal freezing tolerance. In contrast, the starch excess 1-1 mutant (sex1-1) had even lower freezing tolerance than pgm but did not affect sub-cellular localization of proline. Freezing sensitivity of sex1-1 mutants affected primarily the photosynthetic electron transport and was enhanced in a sex1-1::p5cs2-1 double mutant. These findings indicate that several independent factors determine basal freezing tolerance. In a pgm::p5cs2-1 double mutant, freezing sensitivity and proline allocation to the vacuole were the same as in the parental lines, indicating that the lack of cytosolic proline was the common cause of reduced basal freezing tolerance in both mutants. We conclude that cytosolic proline is an important factor in freezing tolerance of non-acclimated plants.

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.

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.

Proline Accumulation in Pollen Grains as Potential Target for Improved Yield Stability Under Salt Stress.

Seed yield, a major determinant for the commercial success of grain crops, critically depends on pollen viability, which is dramatically reduced by environmental stresses, such as drought, salinity, and extreme temperatures. Salinity, in particular, is a major problem for crop yield known to affect about 20% of all arable land and cause huge economic losses worldwide. Flowering plants are particularly sensitive to environmental stress during sexual reproduction, and even a short exposure to stressing conditions can severely hamper reproductive success, and thus reduce crop yield. Since proline is required for pollen fertility and accumulates in plant tissues in response to different abiotic stresses, a role of proline in pollen protection under salt stress conditions can be envisaged. In this perspective, we analyze old and new data to evaluate the importance of pollen development under saline conditions, and discuss the possibility of raising proline levels in pollen grains as a biotechnological strategy to stabilize seed yield in the presence of salt stress. The overall data confirm that proline is necessary to preserve pollen fertility and limit seed loss under stressful conditions. However, at present, we have not enough data to conclude whether or not raising proline over wildtype levels in pollen grains can effectively ameliorate seed yield under saline conditions, and further work is still required.

Differential Contribution of P5CS Isoforms to Stress Tolerance in Arabidopsis.

Proline accumulation is a widespread response of plants to salt stress as well as drought and cold stress. In most plant species, two isoforms of pyrroline-5-carboxylate synthetase (P5CS) catalyze the first step in proline biosynthesis from glutamate. In Arabidopsis, these isoforms differ in their spatial and temporal expression patterns, suggesting sub-functionalization. P5CS1 has been identified as the major contributor to stress-induced proline accumulation, whereas P5CS2 has been considered important for embryo development and growth. In contrast to previous results, our analysis of P5CS1- and P5CS2-GFP fusion proteins indicates that both enzymes were exclusively localized in the cytosol. The comparison of the susceptibility of p5cs1 and p5cs2 mutants to infection with Pseudomonas syringae and salt stress provided novel information on the contribution of the two P5CS isoforms to proline accumulation and stress tolerance. In agreement with previous studies, salt-stressed p5cs1 mutants accumulated very little proline, indicating that P5CS1 contributed more to stress-induced proline accumulation, whereas its impact on stress tolerance was rather weak. Germination and establishment of p5cs2 mutants were impaired under ambient conditions, further supporting that P5CS2 is most important for growth and development, whereas its contribution to stress-induced proline accumulation was smaller than that of P5CS1. In contrast to p5cs1 mutants or wildtype plants, p5cs2 mutants were only weakly affected by sudden exposure to a high NaCl concentration. These findings show that proline content, which was intermediate in leaves of p5cs2 mutants, was not directly correlated with stress tolerance in our experiments. In rosettes of NaCl-exposed p5cs2 mutants, nearly no accumulation of Na+ was observed, and the plants showed neither chlorosis nor reduction of photosynthesis. Based on these data, we suggest a function of P5CS2 or P5CS2-mediated proline synthesis in regulating Na+ accumulation in leaves and thereby salt stress tolerance.

A Theophylline-Responsive Riboswitch Regulates Expression of Nuclear-Encoded Genes.

Riboswitches are small cis-regulatory RNA elements that regulate gene expression by conformational changes in response to ligand binding. Synthetic riboswitches have been engineered as versatile and innovative tools for gene regulation by external application of their ligand in prokaryotes and eukaryotes. In plants, synthetic riboswitches were used to regulate gene expression in plastids, but the application of synthetic riboswitches for the regulation of nuclear-encoded genes in planta remains to be explored. Here, we characterize the properties of a theophylline-responsive synthetic aptazyme for control of nuclear-encoded transgenes in Arabidopsis (Arabidopsis thaliana). Activation of the aptazyme, inserted in the 3' UTR of the target gene, resulted in rapid self-cleavage and subsequent decay of the mRNA. This riboswitch allowed reversible, theophylline-dependent down-regulation of the GFP reporter gene in a dose- and time-dependent manner. Insertion of the riboswitch into the ONE HELIX PROTEIN1 gene allowed complementation of ohp1 mutants and induction of the mutant phenotype by theophylline. GFP and ONE HELIX PROTEIN1 transcript levels were downregulated by up to 90%, and GFP protein levels by 95%. These results establish artificial riboswitches as tools for externally controlled gene expression in synthetic biology in plants or functional crop design.

Appropriate Activity Assays Are Crucial for the Specific Determination of Proline Dehydrogenase and Pyrroline-5-Carboxylate Reductase Activities.

Accumulation of proline is a widespread plant response to a broad range of environmental stress conditions including salt and osmotic stress. Proline accumulation is achieved mainly by upregulation of proline biosynthesis in the cytosol and by inhibition of proline degradation in mitochondria. Changes in gene expression or activity levels of the two enzymes catalyzing the first reactions in these two pathways, namely pyrroline-5-carboxylate (P5C) synthetase and proline dehydrogenase (ProDH), are often used to assess the stress response of plants. The difficulty to isolate ProDH in active form has led several researchers to erroneously report proline-dependent NAD+ reduction at pH 10 as ProDH activity. We demonstrate that this activity is due to P5C reductase (P5CR), the second and last enzyme in proline biosynthesis, which works in the reverse direction at unphysiologically high pH. ProDH does not use NAD+ as electron acceptor but can be assayed with the artificial electron acceptor 2,6-dichlorophenolindophenol (DCPIP) after detergent-mediated solubilization or enrichment of mitochondria. Seemingly counter-intuitive results from previous publications can be explained in this way and our data highlight the importance of appropriate and specific assays for the detection of ProDH and P5CR activities in crude plant extracts.

DEG10 contributes to mitochondrial proteostasis, root growth, and seed yield in Arabidopsis.

Maintaining mitochondrial proteome integrity is especially important under stress conditions to ensure a continued ATP supply for protection and adaptation responses in plants. Deg/HtrA proteases are important factors in the cellular protein quality control system, but little is known about their function in mitochondria. Here we analyzed the expression pattern and physiological function of Arabidopsis thaliana DEG10, which has homologs in all photosynthetic eukaryotes. Both expression of DEG10:GFP fusion proteins and immunoblotting after cell fractionation showed an unambiguous subcellular localization exclusively in mitochondria. DEG10 promoter:GUS fusion constructs showed that DEG10 is expressed in trichomes but also in the vascular tissue of roots and aboveground organs. DEG10 loss-of-function mutants were impaired in root elongation, especially at elevated temperature. Quantitative proteome analysis revealed concomitant changes in the abundance of mitochondrial respiratory chain components and assembly factors, which partially appeared to depend on altered mitochondrial retrograde signaling. Under field conditions, lack of DEG10 caused a decrease in seed production. Taken together, our findings demonstrate that DEG10 affects mitochondrial proteostasis, is required for optimal root development and seed set under challenging environmental conditions, and thus contributes to stress tolerance of plants.

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 ß-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.

Small One-Helix Proteins Are Essential for Photosynthesis in Arabidopsis.

The extended superfamily of chlorophyll a/b binding proteins comprises the Light-Harvesting Complex Proteins (LHCs), the Early Light-Induced Proteins (ELIPs) and the Photosystem II Subunit S (PSBS). The proteins of the ELIP family were proposed to function in photoprotection or assembly of thylakoid pigment-protein complexes and are further divided into subgroups with one to three transmembrane helices. Two small One-Helix Proteins (OHPs) are expressed constitutively in green plant tissues and their levels increase in response to light stress. In this study, we show that OHP1 and OHP2 are highly conserved in photosynthetic eukaryotes, but have probably evolved independently and have distinct functions in Arabidopsis. Mutations in OHP1 or OHP2 caused severe growth deficits, reduced pigmentation and disturbed thylakoid architecture. Surprisingly, the expression of OHP2 was severely reduced in ohp1 T-DNA insertion mutants and vice versa. In both ohp1 and ohp2 mutants, the levels of numerous photosystem components were strongly reduced and photosynthetic electron transport was almost undetectable. Accordingly, ohp1 and ohp2 mutants were dependent on external organic carbon sources for growth and did not produce seeds. Interestingly, the induction of ELIP1 expression and Cu/Zn superoxide dismutase activity in low light conditions indicated that ohp1 mutants constantly suffer from photo-oxidative stress. Based on these data, we propose that OHP1 and OHP2 play an essential role in the assembly or stabilization of photosynthetic pigment-protein complexes, especially photosystem reaction centers, in the thylakoid membrane.

Role of proline and GABA in sexual reproduction of angiosperms.

Two glutamate derivatives, proline and γ-aminobutyric acid (GABA), appear to play pivotal roles in different aspects of sexual reproduction in angiosperms, although their precise function in plant reproduction and the molecular basis of their action are not yet fully understood. Proline and GABA have long been regarded as pivotal amino acids in pollen vitality and fertility. Proline may constitute up to 70% of the free amino acid pool in pollen grains and it has been recently shown that Arabidopsis mutants affected in the first and rate-limiting step in proline synthesis produce aberrant and infertile pollen grains, indicating that proline synthesis is required for pollen development and fertility. Concerning GABA, a large body of evidence points to this glutamate derivative as a key determinant of post-pollination fertilization. Intriguingly, proline has also been associated with pollination, another aspect of sexual reproduction, since honeybees were reported to show a strong preference for proline-enriched nectars. In this review, we survey current knowledge on the roles of proline and GABA in plant fertility, and discuss future perspectives potentially capable to improve our understanding on the functions of these amino acids in pollen development, pollination, and pollen tube guidance.

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.

Functional properties and structural characterization of rice δ(1)-pyrroline-5-carboxylate reductase.

The majority of plant species accumulate high intracellular levels of proline to cope with hyperosmotic stress conditions. Proline synthesis from glutamate is tightly regulated at both the transcriptional and the translational levels, yet little is known about the mechanisms for post-translational regulation of the enzymatic activities involved. The gene coding in rice (Oryza sativa L.) for δ(1)-pyrroline-5-carboxylate (P5C) reductase, the enzyme that catalyzes the second and final step in this pathway, was isolated and expressed in Escherichia coli. The structural and functional properties of the affinity-purified protein were characterized. As for most species, rice P5C reductase was able to use in vitro either NADH or NADPH as the electron donor. However, strikingly different effects of cations and anions were found depending on the pyridine nucleotide used, namely inhibition of NADH-dependent activity and stimulation of NADPH-dependent activity. Moreover, physiological concentrations of proline and NADP(+) were strongly inhibitory for the NADH-dependent reaction, whereas the NADPH-dependent activity was mildly affected. Our results suggest that only NADPH may be used in vivo and that stress-dependent variations in ion homeostasis and NADPH/NADP(+) ratio could modulate enzyme activity, being functional in promoting proline accumulation and potentially also adjusting NADPH consumption during the defense against hyperosmotic stress. The apparent molecular weight of the native protein observed in size exclusion chromatography indicated a high oligomerization state. We also report the first crystal structure of a plant P5C reductase at 3.40-Šresolution, showing a decameric quaternary assembly. Based on the structure, it was possible to identify dynamic structural differences among rice, human, and bacterial enzymes.