Complementation Cloning and Characterization of the Pyrroline 5-Carboxylate Reductase Gene from Drosophila melanogaster

The first insect cDNA and genomic sequences encoding pyrroline 5-carboxylate reductase (EC 1.5.1.2) have been isolated from Drosophila melanogaster. The cDNA sequence was identified by interspecies complementation of an E. coli proline auxotroph and encodes a protein 280 amino acids in length with 25–41% identity to pyrroline 5-carboxylate reductases isolated from other organisms. The corresponding gene is single copy and is located at cytological position 91E-F, and in one of the P1 clones in that region. With a single 61-bp intron, and an impressively small 135- to 200-bp region that presumably acts as a bidirectional promoter, the gene itself shows remarkable economy. The calculated molecular weight of 29,700 predicts that the native enzyme is likely an octomer. Sequencing of the promoter region and expression studies, as well as the known function of the enzyme in redox regulation and the high levels of free proline in insects, suggest that this housekeeping gene encodes an enzyme with a crucial role in intermediary metabolism.     

Extraordinarily high density of unrelated genes showing overlapping and intraintronic transcription units

The cloning of pyrroline 5-carboxylate reductase from Drosophila melanogaster was accomplished by cDNA complementation of an Escherichia coli proline auxotroph. The corresponding P5cr gene is tightly clustered with three other expressed coding regions. A bidirectional promoter, an overlapping 3'UTR and an intraintronic sequence may all be found in only 4.3 kb, making this the most densely clustered region of unrelated genes in any eukaryote.     

Stimulation of phosphoribosyl pyrophosphate and purine nucleotide production by pyrroline 5-carboxylate in human erythrocytes

Recent studies have shown that pyrroline 5-carboxylate, the intermediate in the interconversions of proline, ornithine, and glutamate, can regulate the metabolism of erythrocytes. We now report that the formation of 5-phosphoribosyl 1-pyrophosphate (PP-Rib-P) was markedly stimulated by pyrroline 5-carboxylate in intact red cells. The production of PP-Rib-P is an important point of regulation in nucleotide metabolism. We found that pyrroline 5-carboxylate increased glucose metabolism through the oxidative arm of the pentose shunt, ribose 5-phosphate formation, and PP-Rib-P production and subsequently augmented purine nucleotide production through the salvage pathway in erythrocytes. We now report that pyrroline 5-carboxylate markedly stimulated the net synthesis of inosine monophosphate from hypoxanthine in intact human red cells so that the pool of inosine monophosphate became 20-30% of the total pool of purine nucleotides. Inosine monophosphate has been considered to be a "mobile pool" of purines, i.e. a reservoir from which peripheral tissues can be supplied; the effect of pyrroline 5-carboxylate on the inosine monophosphate pool may be a mechanism for regulating the function of erythrocytes in purine delivery.     

Enzymes metabolizing delta1-pyrroline-5-carboxylate in rat tissues.

The direction and capacity for the metabolism of delta1-pyrroline-5-carboxylate in a number of rat tissues ere investigated by measuring the activities of delta1-pyrroline-5-carboxylate reductase, delta1-pyrroline-5-carboxylate dehydrogenase and proline oxidase. Each of these enzymes catalyzed unidirectional reactions in which delta1-pyrroline-5-carboxylate was either the substrate or product. Delta1-Pyrroline-5-carboxylate reductase activities that were much higher than any previously reported were obtained by avoiding its inactivation in the cold. delta1-Pyrroline-5-carboxylate dehydrogenase, previously said to act on both D- and L-isomers of delta1-pyrroline-5-carboxylate, acted only on the L-isomer. Proline oxidase could not be measured in two adult tissues, in which an inhibitor appeared after birth. The activity of delta1-pyrroline-5-carboxylate reductase significantly paralleled that of ornithine aminotransferase in 23 tissues, showing a widespread potential for proline synthesis from ornithine. An independently distributed potential in fewer tissues for proline degradation to alpha-oxoglutarate was shown by the significantly similar tissue distributions of proline oxidase. Delta1-pyrroline-5-carboxylate dehydrogenase and glutamate dehydrogenase. Reverse metabolism of glutamate or proline to ornithine would be atypical in rat tissues with these distributions of unidirectional enzyme reactions.     

Transgenic Medicago truncatula plants that accumulate proline display nitrogen-fixing activity with enhanced tolerance to osmotic stress

Legume root nodule nitrogen-fixing activity is severely affected by osmotic stress. Proline accumulation has been shown to induce tolerance to salt stress, and transgenic plants over-expressing Delta(1)-pyrroline-5-carboxylate synthetase (P5CS), which accumulates high levels of proline, display enhanced osmotolerance. Here, we transformed the model legume Medicago truncatula with the P5CS gene from Vigna aconitifolia, and nodule activity was evaluated under osmotic stress in transgenic plants that showed high proline accumulation levels. Nitrogen fixation was significantly less affected by salt treatment compared to wild-type (WT) plants. To our knowledge, this is the first time that transgenic legumes have been produced that display nitrogen-fixing activity with enhanced tolerance to osmotic stress. We studied the expression of M. truncatula proline-related endogenous genes M. truncatulaDelta(1)-pyrroline-5-carboxylate synthetase 1 (MtP5CS1), M. truncatulaDelta(1)-pyrroline-5-carboxylate synthetase 2 (MtP5CS2), M. truncatula ornithine delta-aminotransferase (MtOAT), M. truncatula proline dehydrogenase (MtProDH) and a proline transporter gene in both WT and transgenic plants. Our results indicate that proline metabolism is finely regulated in response to osmotic stress in an organ-specific manner. The transgenic model allowed us to analyse some of the biochemical and molecular mechanisms that are activated in the nodule in response to high salt conditions, and to ascertain the essential role of proline in the maintenance of nitrogen-fixing activity under osmotic stress.     

Structural analogues of pyrroline 5-carboxylate specifically inhibit its uptake into cells

Summary Pyrroline 5-carboxylate, a naturally occurring intermediate, is a potent activator of redox-dependent metabolic pathways. This effect of pyrroline 5-carboxylate is due, at least in part, to the special mechanism mediating its entry into cells. Using Chinese hamster ovary cells we recently characterized the cellular uptake of pyrroline 5-carboxylate as a process transferring oxidizing potentialpari passu with cell entry, a process consistent with group translocation. We sought to identify specific inhibitors to probe this unique uptake mechanism, to blockade the metabolic effects of pyrroline 5-carboxylate, and to provide strategies to identify the putative carrier protein. Because pyrroline 5-carboxylate, a ring structure with a tertiary nitrogen, is in spontaneous equilibrium with glutamic--semialdehyde, an openchain structure, we tested analogues of both. Most open-chain aldehydes at 10mm had little effect on the uptake of pyrroline 5-carboxylate. Although succinic semialdehyde did inhibit, its effect was nonspecific in that the uptake of (methylamino) isobutyric acid was inhibited as much as the uptake of pyrroline 5-carboxylate. In contrast, pyrroline 2-carboxylate and other cyclic compounds with teriary nitrogens, e.g., pyridines, were specific inhibitors of pyrroline 5-carboxylate uptake. Respective potencies of pyridine derivatives depended on the nature and location of constituent groups. Kinetics studies showed that these inhibitors were competitive with pyrroline 5-carboxylate and the most potent inhibitor, 2,6-pyridinedicarboxaldehyde, exhibited aK ₁₂ of 0.27±0.05mm. In the face of their effect on P5C uptake, the most potent of thse analogues, 2-pyridinecarboxaldehyde and 2,6-pyridinedicarboxaldehyde, did not inhibit the activity of pyrroline 5-carboxylate to proline. Nevertheless, the analogues markedly inhibited the stimulatory effect of P5C on the pentose phosphate shunt. Importantly, not only did 2-pyridinecarboxaldehyde protect the pyrroline 5-carboxylate uptake mechanism from the inhibitory effects of a sulfhydryl-reactive agent, but also its inhibitory effect became irreversible in the presence of sodium cyanoborohydride. These inhibitors may help discriminate events mediated by the transport carrier from those mediated by intracellular metabolism and may provide a method for identifying and characterizing the putative carrier for P5C.     

Alterations in the activities of the enzymes of proline metabolism in Ragi (Eleusine coracana) leaves during water stress

Free proline content in Ragi (Eleusine coracana) leaves increased markedly (6 to 85 fold) as the degree of water stress, created by polyethylene gylcol treatment, was prolonged There was also a marginal increase in soluble proteins in the stressed leaves as compared to that in the controls. Water stress stimulated the activities of ornithine aminotransferase and pyrroline-5-carboxylate reductase, the enzymes of proline biosynthesis and markedly inhibited the enzymes involved in proline degradation viz., proline oxidase and pyrroline-5-carboxylate dehydrogenase. These results suggest that increase in free proline content of Ragi leaves could be due to enhanced activities of the enzymes synthesizing proline but more importantly due to severe inhibition of the enzymes degrading proline. These observations establish for the first time, the pathway of proline metabolism in plants by way of detection of the activities of all the enzymes involved and also highlight the role of these enzymes in proline accumulation during water stress.     

Metabolic implications of stress-induced proline accumulation in plants

In many plants, free proline accumulates in response to the imposition of a wide range of biotic and abiotic stresses. Controversy has surrounded the extent to which this shift in nitrogen metabolism benefits plants under adverse environmental conditions. Most attempts to account for the phenomenon have focused on the ability of proline to mediate osmotic adjustment, stabilise subcellular structures and scavenge free radicals. However, often the cytoplasmic pool of free proline even after the imposition of stress is insufficient size to account for pronounced biophysical effects.Alternatively, selective preservation of this stress-induced response may relate to endpoints other than simply augmenting the cellular pool of free proline. Proline accumulation may reduce stress-induced cellular acidification or prime oxidative respiration to provide energy needed for recovery. High levels of proline synthesis during stress may maintain NAD(P)+/NAD(P)H ratios at values compatible with metabolism under normal conditions. Consideration of the cofactor preference of plant 1-pyrroline-5-carboxylate (P5C) reductase as well as the in vivo concentrations of the two pyridine nucleotide cofactors and their respective redox ratios suggests that even a small increase in proline biosynthesis might have a large impact on the level of reduction of the cellular NADP pool. The increased NADP+/NADPH ratio mediated by proline biosynthesis is likely to enhance activity of the oxidative pentose phosphate pathway. This would provide precursors to support the demand for increased secondary metabolite production during stress as well as nucleotide synthesis accompanying the accelerated rate of cell division upon relief from stress, when oxidation of proline is likely to provide an important energy source for ADP phosphorylation. Thus, the extreme sensitivity of the metabolic processes of proline synthesis and degradation themselves may be of benefit by regulating metabolic processes adversely affected by stress. This viewpoint is supported by consideration of other physiological phenomena not directly related to stress responses, but in which proline metabolism may also play a regulatory role.A mechanism is proposed whereby the interconversions of proline and P5C in different cell types and the associated transfer of redox potential between tissues may constitute a form of metabolic signalling within higher plants. Stress-related alterations in proline metabolism may impinge on systems of redox control of plant gene expression.     

Transfer of 1-pyrroline-5-carboxylate as oxidizing potential from hepatocytes to erythrocytes.

The interconversions of proline and 1-pyrroline-5-carboxylate form an intercellular cycle that is the basis of a metabolic interaction between hepatocytes and erythrocytes. The cycle transfers oxidizing potential from hepatocytes to erythrocytes, which stimulates pentose phosphate pathway in erythrocytes. This interaction depends on the differential metabolism of proline and 1-pyrroline-5-carboxylate in erythrocytes and hepatocytes and consists of the following: in hepatocytes proline oxidase converts proline into 1-pyrroline-5-carboxylate, which is released into the medium and taken up by erythrocytes; erythrocyte 1-pyrroline-5-carboxylate reductase converts 1-pyrroline-5-carboxylate into proline and concomitantly generates NADP+; the generated oxidizing potential drives glucose metabolism through the pentose phosphate pathway in erythrocytes; finally, erythrocytes release proline into the medium, enabling it to re-enter hepatocytes and repeat the cycle. The increased activity of the pentose phosphate pathway in erythrocytes may enhance the production of 5-phosphoribosyl pyrophosphate, a necessary moiety for the processing of purines.     

Enzymes of orithine metabolism in adult developing rat intestine

The levels of 11 enzymes, most of them involved in the metabolism of orithine, were measured in whole upper intestine, or in duodenum, small intestine and colon of adult rats. The developmental formations in small intestine of arginase, orithine aminotransferase, and orithine transcarbamylase were compared with those in liver. Changes with age (late gestation to adult) of the intestinal activities of pyrroline-5-carboxylate reductase, proline oxidase and glutamyl transpeptidase are also described.The results suggests that the proximal part of the intestine is well endowed with enzymes involved in the conversion of ornithine to proline as well as to citrulline. Fetal intestine is rich in proline oxidase and pyrroline-5-carboxylate reductase. The peak levels of ornithine aminotraferase found in intestine in the first 3 postnatal weeks were higher than seen in any other rat tissue.Some of the properties of arginase, ornithine aminotransferase and pyrroline-5-carboxylate reductase in small intestine were compared with those in liver. Isozymes of arginase in small intestine differed from those in liver; the kinetic properties of ornithine aminotransferase were similar in the two tissues. In intestine of 14-day-old rats, the orithine aminotransferase reaction was reversible, forming ornithine from pyrroline-5-carboxylate. The intestinal pyrroline-5-carboxylate reductase was cold-labile as was the hepatic enzyme in rat.     

Proline in the Osmoregulation of Brevibacterium lactofermentum

Osmoregulation in Brevibacterium lactofermentum was studied. Proline was accumulated up to approximately 35mg/g dry cell weight in the cells of a wild strain of the bacterium grown under osmotic stress. The osmotic tolerance of a proline auxotroph mutant obtained from the bacterium was lower than that in the wild strain. The activity of pyrroline-5-carboxylate reductase, one of the enzymes in the proline biosynthetic pathway, increased about 3-fold when the cells of B. lactofermentum were grown under osmotic stress. These data indicated that proline is important in osmoregulation in the bacterium.     

Proline and Proline Metabolising Enzymes in in-vitro Selected NaCl-tolerant Brassica juncea L. under Salt Stress

The effect of salt stress was studied on proline accumulationand the activities of proline metabolic pathway enzymes in seedlingand leaf tissue of two genetically stable lines (SR2P1-2 andSR3P6-2) of in vitro selected NaCl-tolerant plants and parentcultivar Prakash of Brassica juncea L. Salt stress caused differentialenhancement in proline level in both seedlings and leaf tissueof plants at different developmental stages. The magnitude ofincrease in proline content was higher in SR3P6-2 line in seedlings(34 fold at 140 meq^-1 NaCl) as well as leaves (16 fold at 40d after sowing at 100 meq^-1 NaCl) compared to the parent cv.Prakash (29 fold in seedlings and five fold in leaves) and SR2P1-2(21 fold in seedlings and five fold in leaves) at similar stresslevels. Salt stress also resulted in changes in the activitiesof enzymes of proline metabolism. The activities of prolinebiosynthetic enzymes, pyrroline-5-carboxylate reductase andornithine aminotransferase, increased under salt stress bothin the seedlings and leaves. The range of increase in the activitiesof the two enzymes was relatively higher in SR3P6-2 (3·3-3·9fold) compared to the SR2P1-2 (1·8-2·8 fold) andparent cv. Prakash (1·5-2·8 fold). The activityof proline degrading enzyme, proline oxidase, decreased undersalt stress in both the tissues of all the lines; the reductionin activity was relatively greater in SR3P6-2 compared to SR2P1-2or cv. Prakash. The trend of changes in the enzyme activitieswas in tune with the increase in proline level, the magnitudeof change did not match the extent of increase in proline level.Copyright1995, 1999 Academic Press Brassica juncea L., NaCl-tolerant somaclones, proline content, ornithine aminotransferase, proline oxidase, pyrroline 5-carboxylate reductase     

Enzymes of ornithine metabolism in adult and developing rat intestine.

The levels of 11 enzymes, most of them involved in the metabolism of ornithine, were measured in whole upper intestine, or in duodenum, small intestine and colon of adult rats. The developmental formations in small intestine of arginase, ornithine aminotransferase, and ornithine transcarbamylase were compared with those in liver. Changes with age (late gestation of adult) of the intestinal activities of pyrroline-5-carboxylate reductase, proline oxidase and glutamyl transpeptidase are also described. The results suggest that the proximal part of the intestine is well endowed with enzymes involved in the conversion of ornithine to proline as well as to citrulline. Fetal intestine is rich in proline oxidase and pyrroline-5-carboxylate reductase. The peak levels of ornithine aminotransferase found in intestine in the first 3 postnatal weeks were higher than seen in any other rat tissue. Some of the properties of arginase, ornithine aminotransferase and pyrroline-5-carboxylate reductase in small intestine were compared with those in liver. Isozymes of arginase in small intestine differed from those in liver; the kinetic properties of ornithine aminotransferase were similar in the two tissues. In intestine of 14-day-old rats, the ornithine aminotransferase reaction was reversible, forming ornithine from pyrroline-5-carboxylate. The intestinal pyrroline-5-carboxylate reductase was cold-labile as was the hepatic enzyme in rat.     

Water and salt stress-induced alterations in proline metabolism of Triticum durum seedlings

Many plants accumulate proline as a non-toxic and protective osmolyte under saline or dry conditions. Its accumulation is caused by both the activation of its biosynthesis and inactivation of its degradation. We report here on the alterations induced by water and salt stress in the proline metabolism and amino acid content of 5-day-old seedlings of Triticum durum cv. Simeto. Most of the amino acids showed an increase with the induction of either stress, but proline increased more markedly than did other amino acids. We also measured the activities of two enzymes, Δ¹-pyrroline-5-carboxylate (P5C) reductase (EC 1.5.1.2) and proline dehydrogenase (EC 1.5.1.2), which are involved in proline biosynthesis and catabolism, respectively. The activity of P5C reductase was enhanced during both water and salt stress, while proline dehydrogenase was inhibited only during salt stress. The results indicate that synthesis de novo is the predominant mechanism in proline accumulation in durum wheat. Use of a cDNA clone that encodes P5C-reductase from Arabidopsis thaliana , showed no differences in the gene expression between controls and stressed plants, implying that the increase in enzyme activity is unrelated to the expression of this gene.     

Overproduction of proline in transgenic hybrid larch (Larix x leptoeuropaea (Dengler)) cultures renders them tolerant to cold,salt and frost

Cold, salt and frost are important environmental stresses in forest trees and may significantly reduce productivity. Elevated levels of proline are associated with these stresses and may help alleviate their effects. Transgenic hybrid larch Larix leptoeuropaea has been produced expressing a Vigna aconitifolia gene for pyrroline 5-carboxylate synthase, the rate-limiting step in proline synthesis. Embryogenic masses of hybrid larch were co-cultivated with Agrobacterium tumefaciens harbouring a binary vector expressing the gene. The integration of the gene into the plant genome was confirmed by Southern blot and by proline content analysis. There was an approximately 30-fold increase in proline level in transgenic tissue compared to non-transformed controls. The transgenic tissue lines were significantly more resistant to cold, salt, and freezing stresses and grew under conditions (200mM NaCl or 4 °C) that completely inhibited the growth of control cell lines. Our results indicated that introduction of proline over-accumulation into forest trees might be an effective strategy for ameliorating the effects of environmental stresses.