The fate of proline in the African fruit beetle Pachnoda sinuata

Metabolic pathways of proline consumption in working flight muscles and its resynthesis were investigated in the African fruit beetle, Pachnoda sinuata.Mitochondria isolated from flight muscles oxidise proline, pyruvate and α-glycerophosphate, but not palmitoyl-carnitine. At low proline concentrations, the respiration rate during co-oxidation of proline and pyruvate is additive, while at high proline concentrations it is equal to the respiration rates of proline oxidation.Flight muscles have high activities of alanine aminotransferase and NAD⁺-dependent malic enzyme which are involved in proline metabolism. Glycogen phosphorylase and glyceraldehyde-3-phosphate dehydrogenase (carbohydrate breakdown) also display high activities, whilst 3-hydroxyacyl-CoA dehydrogenase (fatty acid oxidation) showed low activity.During the oxidation of proline, mitochondria isolated from flight muscles produce equimolar amounts of alanine. The rates of oxygen consumption by the mitochondria during this process lead to the conclusion that proline is partially oxidised. This is confirmed by the incorporation of radiolabel from pre-injected [U-¹⁴C] proline into alanine during a flight experiment with P. sinuata.Proline is resynthesised, in vitro, from alanine and acetyl-CoA in the fat body. High activities of enzymes catalysing such pathways (alanine aminotransferase, 3-hydroxyacyl-CoA dehydrogenase and NADP⁺-dependent malic enzyme) were found. The in vitro production of proline from alanine is equimolar suggesting that resynthesis of one proline molecule is accomplished from one alanine molecule and one acetyl-CoA molecule. One source of the acetyl-CoA for the in vitro synthesis of proline is the lipid stores of the fat body.Proline synthesis by fat body tissue is controlled by feedback. Alanine aminotransferase is competitively inhibited by high proline concentrations.     

Mineral composition of and proline accumulation by zostera marina L. in response to environmental salinity

Plants of Zostera marina L. collected in the field were grown for several weeks at different salinities under controlled conditions. After this period plants were harvested and the plant tissue water was analysed for proline, Na, K and Cl, which increased in response to seawater concentration as did osmolality. Proline accumulated up to 22 mmoll⁻¹ tissue water at the highest seawater concentration. Proline synthesis or breakdwon was found to be slow in response to changes in environmental salinity. Nitrogen levels in Zostera were high (5% dry weight); a possible drain of proline on plant nitrogen is discussed. In vitro activity of malate dehydrogenase (MDH) was inhibited by NaCl; proline did not affect MDH activity, not even at 1 mol proline l⁻¹. The role of proline in osmotic adaptation of Zostera marina is discussed.     

Effects of NaCl on Proline Synthesis and Utilization in Excised Barley Leaves

Proline accumulation in NaCl-treated excised barley (Hordeum vulgare var Larker) leaves was studied. Leaves were treated by placing the cut end in NaCl solutions and allowing the salt to enter the leaf via the transpiration stream. Leaves treated this way maintained turgor while the sodium content increased and the osmotic potential decreased. Proline began accumulating after 12 hours and continued accumulating over the subsequent 12-hour period at an average rate of 0.6 micromoles per hour per gram fresh weight.

During the time proline was accumulating, [¹⁴C]glutamate was added to measure the effects of salt on proline synthesis from glutamate and [¹⁴C] proline was added in separate experiments to determine the effect of salt on proline utilization. Salt treatment dramatically increased proline synthesis from glutamate. Proline utilization by oxidation and for protein synthesis was decreased by 50 and 60%, respectively, by the salt treatment.

These effects are similar to the effects of drought and abscisic acid in barley leaves. The results indicate that common mechanisms cause proline to accumulate under these different stresses.

     

Increased proline transport resulting from growth of normal and Kirsten sarcoma virus-transformed BALB 3T3 cells in the presence of N(6), O(2')-dibutyryl cyclic adenosine 3',5'-monophosphate.

Proline transport in Kirsten sarcoma virus-transformed BALB 3T3 (Ki-3T3) cells was increased approximately twofold by 0.5 mm dibutyryl cAMP (dbcAMP), and the increase was observed whether transport was assayed in the presence or absence of cycloheximide. Two days of exposure to the analog was required for maximum stimulation. Increased proline transport contributed almost entirely to the increased incorporation of [¹⁴C]proline into noncollagen protein but for only 13% of the increased incorporation into collagen of dbcAMP-treated Ki-3T3 cells. Proline transport was further characterized using an assay system containing 0.1 mm cycloheximide, which did not affect transport over a 30-min period. The K_m for proline was decreased from 6.5 to 3.4 mm by dbcAMP treatment of Ki-3T3. Proline transport in Ki-3T3 proceeds almost entirely via the A system, and the effect of dbcAMP appears to be on this system specifically since glycine and glutamine transport, which are heterogeneous, were not affected but transport of N-methylaminoisobutyrate, a specific A system substrate, was increased by dbcAMP treatment. Although 0.5 mm butyrate increased proline transport in Ki-3T3 cells to a similar degree as dbcAMP, the effect of the latter appeared related to its action as a cAMP analog since N⁶-monobutyryl cAMP, having a stable butyryl group, and 8-bromo-cAMP also increased proline transport while dbcGMP did not. The rate of proline transport in normal BALB 3T3 cells was only 30–40% lower than that of Ki-3T3 cells at various growth stages, and dbcAMP and 8-bromo-cAMP treatment also increased proline transport in the normal cells. The results of these studies suggest that dbcAMP and other cAMP analogs induce the synthesis of an altered component of the A system for amino acid transport and that the effect of these compounds is unrelated to the effect of transformation on proline transport.     

Effect of sodium and calcium chlorides,abscisic acid and proline on callus cultures ofArachis hypogaea L.

Callus cultures ofArachis hypogaea L. cv. JL-24 adapted to 200 mM NaCl (otherwise lethal to cells) were used for the study. Calli grew slowly when transferred to 250 mM NaCl, but the growth was enhanced when ABA was included in the medium. ABA induced increase in growth of callus was not accompanied by corresponding increase in internal free proline levels. 0.5 mM of CaCl₂ ameliorated the negative effect of NaCl indicating that cells require a specific Ca²⁺/Na⁺ ratio for their growth. Proline content also increased at this ratio thereby suggesting that increase in growth at 0.5 mM Ca²⁺ may be due to an increase in proline content. However, exogenous proline did not increase the growth of callus (adapted to 200 mM), and higher concentrations even inhibited the growth. This shows that proline is not required for growth or adaptation of cells to salt stress, but is produced as a consequence of stress.     

Effects of proline and carbohydrates on the metabolism of exogenous proline by excised bean leaves in the dark

Proline was metabolized when vacuum infiltrated into starved bean (Phaseolus vulgaris L.) leaves from plants previously in the dark for 48 hours, but an equivalent increase in protein proline was not observed. When ¹⁴C-proline was infiltrated into starved leaves, a large percentage of the ¹⁴C was recovered in other amino acids, organic acids, and CO₂, in addition to that recovered as protein proline. However, extensive oxidation of proline was observed only if enough proline was added to increase substantially the endogenous concentration of proline. Increasing the endogenous concentration did not affect the amount of proline that was incorporated into protein.     

Solubilization of a functionally active proline carrier from membranes of Escherichia coli with an organic solvent.

A proline transport carrier was extracted from the membranes of Escherichia coli with acidic n-butanol. Vesicles reconstituted from the butanol extract and E. coli phospholipids and preloaded with K⁺ showed rapid uphill uptake of proline when energy was supplied as a membrane potential introduced by K⁺-diffusion via valinomycin. Proline uptake by the reconstituted vesicles, like that of intact cells and isolated membrane vesicles, was inhibited by 3,4-dehydroproline, SH reagents, and a proton conducting uncoupler. Reconstituted vesicles of mutants defective in proline transport showed little or no proline uptake. The proline carrier was partially purified from the extract and separated from the bulk of phospholipids on Sephadex LH-20.     

Metabolism of Proline, Glutamate, and Ornithine in Proline Mutant Root Tips of Zea mays (L.)

In excised pro_1-1 mutant and corresponding normal type roots of Zea mays L. the uptake and interconversion of [¹⁴C]proline, [¹⁴C]glutamic acid, [¹⁴C]glutamine, and [¹⁴C]ornithine and their utilization for protein synthesis was measured with the intention of finding an explanation for the proline requirement of the mutant. Uptake of these four amino acids, with the exception of proline, was the same in mutant and normal roots, but utilization differed. Higher than normal utilization rates for proline and glutamic acid were noted in mutant roots leading to increased CO₂ production, free amino acid interconversion, and protein synthesis. Proline was synthesized from either glutamic acid (or glutamine) or ornithine in both mutant and normal roots; it did not accumulate but rather was used for protein synthesis. Ornithine was not a good precursor for proline in either system, but was preferentially converted to arginine and glutamine, particularly in mutant roots. The pro_1-1 mutant was thus not deficient in its ability to make proline. Based on these findings, and on the fact that ornithine, arginine, glutamic acid and aspartic acid are elevated as free amino acids in mutant roots, it is suggested that in the pro_1-1 mutant proline catabolism prevails over proline synthesis.     

Regulation of pyruvate oxidation in blowfly flight muscle mitochondria: Requirement for ADP

Blowfly (Phormia regina) flight muscle mitochondria oxidized pyruvate (+ proline) in the presence of either ADP (coupled respiration) or carbonylcyanide-p-trifluoromethoxyphenylhydrazone (FCCP-uncoupled respiration). There was an absolute requirement for ADP (K_m = 8.0 μm) when pyruvate oxidation was stimulated by FCCP in the presence of oligomycin. This requirement for ADP was limited to the oxidation of pyruvate; uncoupled α-glycerolphosphate oxidation proceeded maximally even in the absence of added ADP. Atractylate inhibited uncoupled pyruvate oxidation whether added before (>99%) or after (95%) initiation of respiration with FCCP. In the presence of FCCP, oligomycin, and limiting concentrations of ADP (less than 110 μm), there was a shutoff in the uptake of oxygen. This inhibition of respiration was completely reversed by the addition of more ADP. Plots of net oxygen uptake as a function of the limiting ADP concentration were linear; the observed ADP/O ratio was 0.22 ± 0.025. An ADP/O ratio of 0.2 was predicted if phosphorylation occurred only at the succinyl-CoA synthetase step of the tricarboxylate cycle. Experiments performed in the presence of limiting concentrations of ADP, and designed to monitor changes in the mitochondrial content of ADP and ATP, demonstrated that the shutoff in oxygen uptake was not due to the presence of a high intramitochondrial concentration of ATP. Indeed, ATP, added to the medium prior to the addition of FCCP, inhibited uncoupled pyruvate oxidation; the apparent K_I was 0.8 mm. These results are consistent with the hypothesis that it is the intramitochondrial ATP/ADP ratio that is one of the controlling factors in determining the rate of flux through the tricarboxylate cycle. Changes in the mitochondrial content of citrate, isocitrate, α-ketoglutarate, and malate during uncoupled pyruvate oxidation in the presence of a limiting concentration of ADP were consistent with the hypothesis that the mitochondrial NAD⁺-linked isocitric dehydrogenase is a major site for such control through the tricarboxylate cycle.     

Abscisic Acid accumulation is not required for proline accumulation in wilted leaves

Leaves from dark-grown barley (Hordeum vulgare L. var Larker) seedlings grown in the presence and absence of fluridone were used to determine whether or not abscisic acid (ABA) accumulation was necessary for proline to accumulate in wilted tissue. Wilted tissue (polyethylene glycol-treated) leaves from fluridone-grown seedlings did not accumulate ABA but did accumulate proline at a rate that was not different from the non-fluridone-treated leaves. Thus ABA accumulation is not required for wilting-induced proline accumulation in barley leaves. Proline accumulation in wilted leaves from the wilty tomato (Lycopersicon esculentum) mutant, flacca, was compared to that in the wild type, Rheinlands Ruhm. Proline accumulated in wilted leaves from flacca. The rate of accumulation was faster in flacca compared to the rate in the wild type because the wilty mutant wilted faster. ABA accumulated in wilted leaves from the wild type but not in the wilty mutant. This result is a further confirmation that ABA accumulation is not required for wilting-induced proline accumulation. These results are significant in that proline accumulation in barley leaves can be induced independently by any one of three treatments: wilting, ABA, or salt.     

Proline Biosynthesis Is Required for Endoplasmic Reticulum Stress Tolerance in Saccharomyces cerevisiae

The amino acid proline is uniquely involved in cellular processes that underlie stress response in a variety of organisms. Proline is known to minimize protein aggregation, but a detailed study of how proline impacts cell survival during accumulation of misfolded proteins in the endoplasmic reticulum (ER) has not been performed. To address this we examined in Saccharomyces cerevisiae the effect of knocking out the PRO1, PRO2, and PRO3 genes responsible for proline biosynthesis. The null mutants pro1, pro2, and pro3 were shown to have increased sensitivity to ER stress relative to wild-type cells, which could be restored by proline or the corresponding genetic complementation. Of these mutants, pro3 was the most sensitive to tunicamycin and was rescued by anaerobic growth conditions or reduced thiol reagents. The pro3 mutant cells have higher intracellular reactive oxygen species, total glutathione, and a NADP⁺/NADPH ratio than wild-type cells under limiting proline conditions. Depletion of proline biosynthesis also inhibits the unfolded protein response (UPR) indicating proline protection involves the UPR. To more broadly test the role of proline in ER stress, increased proline biosynthesis was shown to partially rescue the ER stress sensitivity of a hog1 null mutant in which the high osmolality pathway is disrupted.     

Role of Δ1-Pyrroline-5-Carboxylate Dehydrogenase Supports Mitochondrial Metabolism and Host-Cell Invasion of Trypanosoma cruzi

Proline is crucial for energizing critical events throughout the life cycle of Trypanosoma cruzi, the etiological agent of Chagas disease. The proline breakdown pathway consists of two oxidation steps, both of which produce reducing equivalents as follows: the conversion of proline to Δ¹-pyrroline-5-carboxylate (P5C), and the subsequent conversion of P5C to glutamate. We have identified and characterized the Δ¹-pyrroline-5-carboxylate dehydrogenase from T. cruzi (TcP5CDH) and report here on how this enzyme contributes to a central metabolic pathway in this parasite. Size-exclusion chromatography, two-dimensional gel electrophoresis, and small angle x-ray scattering analysis of TcP5CDH revealed an oligomeric state composed of two subunits of six protomers. TcP5CDH was found to complement a yeast strain deficient in PUT2 activity, confirming the enzyme''s functional role; and the biochemical parameters (K_m, k_cat, and k_cat/K_m) of the recombinant TcP5CDH were determined, exhibiting values comparable with those from T. cruzi lysates. In addition, TcP5CDH exhibited mitochondrial staining during the main stages of the T. cruzi life cycle. mRNA and enzymatic activity levels indicated the up-regulation (6-fold change) of TcP5CDH during the infective stages of the parasite. The participation of P5C as an energy source was also demonstrated. Overall, we propose that this enzymatic step is crucial for the viability of both replicative and infective forms of T. cruzi.     

The role of Pro-P5C Cycle in chs mutants of Arabidopsis under cold stress

Proline metabolism is implicated in plant responses to abiotic stresses, including the chilling stress. During proline catabolism, the two-step oxidation of proline is performed by the continuous actions of proline dehydrogenase (ProDH), which produces Δ¹-pyrroline-5-carboxylate (P5C), and P5C dehydrogenase (P5CDH), which oxidizes P5C to glutamate. The Arabidopsis thaliana chilling mutants chs1 and chs2 are sensitive to chilling temperatures of 13–18°C. For a better understanding of Arabidopsis responses to chilling stress, 4-week-old wild-type (WT) and chs1 and chs2 lines, with three plants in each group, were subjected to chilling stress (13°C), cold stress (4°C), or remained under normal conditions (23°C); and several factors including the expression of ProDH2 and P5CDH genes, POX (peroxidase) and SOD (superoxide dismutase) activities, as well as MDA and proline contents were examined. Our results showed an increase in the proline content in all lines under chilling conditions. In addition, a greater expression of ProDH2 and a lower expression of P5CDH were observed, leading us to speculate a greater breakdown of proline into P5C and a consequent overproduction of ROS in the ETC cycle. The higher POX and SOD activities and a higher MDA content in chs mutants at 13°C are in line with this speculation. Finally, cold-treated plants (4°C) only showed an increase in proline levels.     

Citrate transport in corn mitochondria

Citrate uptake by corn mitochondria (Zea mays L. B73 × Mol9) was investigated by osmotic swelling and [¹⁴C]citrate accumulation. Uptake driven by passive influx, ammonium gradients, and respiration was followed. There was no requirement for phosphate and/or malate to secure citrate uptake, although under some conditions these additives were promotive. Inhibition of the phosphate and dicarboxylate carriers did not eliminate citrate uptake. Citrate_in/malate_out exchange occurs, but at a rate too slow to account for observed citrate uptake, and depletion of endogenous malate only reduced citrate uptake by 38%. It was concluded that citrate can be rapidly accumulated by a mechanism other than by exchange for dicarboxylates. The effect of uncoupler on respiration-driven [¹⁴C]citrate accumulation, and studies of passive swelling using ionophores and uncouplers indicated that the major avenue of citrate uptake is by H⁺/citrate co-transport with a pH optimum near 4.5. The in vivo role of this mechanism is not yet understood.     

Comparison between a Stable NaCl-Selected Nicotiana Cell Line and the Wild Type : K, Na, and Proline Pools as a Function of Salinity

An NaCl-resistant line has been developed from suspension-cultured tobacco cells (Nicotiana tabacum/gossii) by stepwise increases in the NaCl concentration in the medium. Resistance showed stability through at least 24 generations in the absence of added NaCl.

Above an external NaCl concentration of 35 millimolar, proline concentration in the selected cells rose steeply with external NaCl, particularly so above 100 millimolar NaCl. Proline accumulation in the wild type was far slighter. Selected cells which had been grown for 24 generations in the absence of added NaCl accumulated proline strongly on re-exposure to NaCl medium, indicating stability of this character. Proline accumulation was fully reversible with a half-time of about 6 hours. When selected cells were transferred sequentially to lower and lower NaCl concentrations, their proline content fell to the level corresponding to the new NaCl concentration. The NaCl-selected cells responded to water stress (i.e. added mannitol) by accumulating markedly more proline than did the wild type.

The addition of Ca²⁺ to the growing and rinsing media minimized Na⁺ and K⁺ binding in the Donnan free space of cell walls and thus allowed assessment of intracellular Na⁺ and K⁺. In both cell types, internal Na⁺ content rose steadily as a function of external NaCl concentration. In the course of 7 days in NaCl media, the wild type cells lost a considerable part of their K⁺ content, the extent of the loss increasing with rise in external NaCl concentration. The selected cells, by contrast, lost no K⁺ at external NaCl concentrations below 50 millimolar external NaCl, and at higher concentrations lost less than the wild type.

     

Leishmania tarentolae: proline anabolism in promastigotes.

Using ¹⁴C-labeled substrates in conjunction with thin-layer Chromatographic radioautography, studies were done to determine the precursors for proline synthesis utilized by promastigotes of Leishmania tarentolae. The only substances in Trager's Defined Medium capable of acting as proline precursors under the conditions studied were l-arginine and l-ornithine. None of the other substrates tested (16 amino acids, five purines and pyrimidines, and glucose) were converted to proline. l-glutamate, l-aspartate, and d-glucose did not act as precursors for the imino acid although they stimulated oxygen uptake better than did l-arginine and l-ornithine. Proline synthesis was unaffected by the presence of preformed proline indicating the absence of end product feedback inhibition.     

Proline Metabolism Increases katG Expression and Oxidative Stress Resistance in Escherichia coli

The oxidation of l-proline to glutamate in Gram-negative bacteria is catalyzed by the proline utilization A (PutA) flavoenzyme, which contains proline dehydrogenase (PRODH) and Δ¹-pyrroline-5-carboxylate (P5C) dehydrogenase domains in a single polypeptide. Previous studies have suggested that aside from providing energy, proline metabolism influences oxidative stress resistance in different organisms. To explore this potential role and the mechanism, we characterized the oxidative stress resistance of wild-type and putA mutant strains of Escherichia coli. Initial stress assays revealed that the putA mutant strain was significantly more sensitive to oxidative stress than the parental wild-type strain. Expression of PutA in the putA mutant strain restored oxidative stress resistance, confirming that depletion of PutA was responsible for the oxidative stress phenotype. Treatment of wild-type cells with proline significantly increased hydroperoxidase I (encoded by katG) expression and activity. Furthermore, the ΔkatG strain failed to respond to proline, indicating a critical role for hydroperoxidase I in the mechanism of proline protection. The global regulator OxyR activates the expression of katG along with several other genes involved in oxidative stress defense. In addition to katG, proline increased the expression of grxA (glutaredoxin 1) and trxC (thioredoxin 2) of the OxyR regulon, implicating OxyR in proline protection. Proline oxidative metabolism was shown to generate hydrogen peroxide, indicating that proline increases oxidative stress tolerance in E. coli via a preadaptive effect involving endogenous hydrogen peroxide production and enhanced catalase-peroxidase activity.     

Response of two tomato cultivars to field-applied proline under irrigation with saline water: Growth,chlorophyll fluorescence and nutritional aspects

The response of tomato (Solanum lycopersicum L.) to abiotic stress has been widely investigated. Recent physiological studies focus on the use of osmoprotectants to ameliorate stress damage, but experiments at a field level are scarce. Two tomato cultivars were used for an experiment with saline water (6.57 dS m^?1) and subsurface drip irrigation (SDI) in a silty clay soil. Rio Grande is a salinity-tolerant cultivar, while Heinz-2274 is the salt-sensitive cultivar. Exogenous application of proline was done by foliar spray at two concentrations (10 and 20 mg L^?1) during the flowering stage. Control plants were treated with saline water without proline. Proline at the lower concentration (10 mg L^?1) increased dry mass of different plant organs (leaves, stems, and roots) and it improved various chlorophyll a fluorescence parameters compared with controls. Regarding mineral nutrition, K⁺ and P were higher in different organs, while low accumulation of Na⁺ occurred. However, Mg²⁺ was very high in all tissues of Rio Grande at the higher concentration of proline applied. Thus, the foliar spray of proline at 10 mg L^?1 increased the tolerance of both cultivars. The growth of aboveground biomass of Heinz-2274 was enhanced by 63.5%, while Rio Grande improved only by 38.9%.     

Effects of hydroxyproline on the growth of excised root segments of Pisum sativum under aseptic conditions

Summary The cis and trans isomers of 4-hydroxy-l-proline stimulated the extension growth of excised 2–4 mm pea root segments during culture. Increase in the uptake and subsequent incorporation of [¹⁴c]leucine into proteins was inhibited by both l-isomers, and so also were changes in chloride uptake capacity and in protein metabolism measured in terms of invertase and peroxidase activities. Changes in [¹⁴C]proline uptake and incorporation, and in respiration, were unaffected. Proline had no effect on changes in extension growth or protein metabolism but did prevent the effects of both hydroxyproline isomers. Azetidine-2-carboxylic acid inhibited extension growth and all the aspects of protein metabolism studied, the effects again being all prevented by proline. It is suggested that hydroxyproline enhances growth by interfering with protein synthesis in the cell walls.