Proline dehydrogenase and pyrroline-5-carboxylate reductase from pumpkin cotyledons

Activity of proline dehydrogenase and pyrroline-5-carboxylate reductase was greatest after 5 and 7 days germination in green and etiolated cotyledons respectively of pumpkin (Cucurbita moschata Poir. cv. Dickinson Field). The ratio of pyrroline-5-carboxylate reductase to proline dehydrogenase activity was constant throughout germination. Both enzymes were purified 30-fold but the ratio pyrroline-5-carboxylate reductase—proline dehydrogenase activity was constant throughout purification. However, this ratio decreased with storage, especially in purified preparations. Both enzymes were stable at high temperature and the ratio pyrroline-5-carboxylate reductase—proline dehydrogenase remained unchanged on heating. Proline dehydrogenase and pyrroline-5-carboxylate reductase were inhibited by sodium bisulfite and cysteine. ATP, ADP and NADP caused inhibition of both enzymes. Proline dehydrogenase utilized NAD but not NADP. Pyrroline-5-carboxylate reductase had a 2.5-fold greater activity with NADH than NADPH. Most of the data presented suggest that proline dehydrogenase and pyrroline-5-carboxylate reductase activities occur on the same protein molecule.     

Leaf position-dependent changes in proline,pyrroline-5-carboxylate reductase activity and water relations under salt-stress in genetically stable salt-tolerant somaclones ofBrassica juncea L.

The magnitude of the effect of salt stress on proline content, pyrroline-5-carboxylate (P5C) reductase activity and water relations was found to be leaf position dependent in an advance generation (R4) of twoBrassica juncea L. somaclones (SR-2 and SR-3) selected in vitro for NaCl-tolerance and the parent cv. Prakash. Free proline content and P5C reductase activity increased with increase in salt stress in all the lines but at different rates; the maximum increase being in the SR-3 derived somaclonal line. At 100 mM NaCl, SR-3 showed a nearly 19 fold increase in proline content compared to a 4–5 fold increase in the other two genotypes. The proline level and P5C reductase activity of the first (youngest) leaf was higher than in the other leaves and decreased linearly with increase in age of the leaf in all the lines. The relationship between relative water content and osmotic potential of the leaves at different positions also varied. The results indicate that a significant effect of salt may appear non-significant if the position of the leaves is not taken into account while sampling.     

Differential responses of the enzymes involved in proline biosynthesis and degradation in drought tolerant and sensitive cotton genotypes during drought stress and recovery

The relative water content (RWC), free proline levels and the activities of enzymes involved in proline metabolism were studied in drought tolerant (Ca/H 680) and drought sensitive (Ca/H 148) genotypes of cotton (Gossypium hirsutum L.) during induction of water stress and posterior recovery. Water stress caused a significant increase in proline levels and P5CS activity in leaves of both tolerant and sensitive genotypes, whereas the activity of P5CR increased minimally and the activity of OAT remains unchanged. The activity of PDH decreased under drought stress in both the genotypes. The leaf of tolerant genotype maintained higher RWC, photosynthetic activity and proline levels, as well as higher P5CS and P5CR activities under water stress than that of drought sensitive genotype. The drought induced proline levels and activities of P5CS and P5CR declined and tend to be equal to their respective controls, during recovery, whereas the PDH activity tends to increase. These results indicate that induction of proline levels by up regulation of P5CS and down regulation of PDH may be involved in the development of drought tolerance in cotton.     

Solute levels and osmoregulatory enzyme activities in reed plants adapted to drought and saline habitats

The pattern of solute accumulation and the activities of key enzymes involved in metabolism of proline and betaine were investigated in three ecotypes of reed from different habitats: swamp reed (SR), dune reed (DR), and heavy salt meadow reed (HSR). The two terrestrial reed ecotypes, DR and HSR, exhibited a higher capacity for osmotic adjustment; they accumulated higher contents of K⁺ and Ca²⁺ in the leaves in comparison with SR. DR also had the highest soluble sugar content in its leaves. HSR has higher levels of Na⁺ in its root environment and this was reflected by considerable accumulation of Na⁺ in the HSR rhizome. However, the different zones of its leaf lamina (upper, middle and lower) did not exhibit increased levels of Na⁺, suggesting that HSR has the ability to accumulate Na⁺ in the rhizome to protect the shoots from excessive Na⁺ toxicity. DR and HSR had higher levels of proline and betaine in the leaves than did SR. This difference was consistent with the activities of the various biosynthetic enzymes: betaine aldehyde dehydrogenase (BADH), pyrroline-5-carboxylate reductase (P5CR) and ornithine--aminotransferase (OAT) were enhanced in DR and HSR as compared to SR, whereas proline oxidase (PO) activities were inhibited. These findings suggest that changes in the activities of enzymes involved in osmotregulation might play important roles in the adaptation of reed, a hydrophilic plant, to more extreme dune and saline habitats. The relative contributions of the various proline synthetic pathways are also discussed.     

A one-step assay for pyrroline-5-carboxylate synthase using a short AG1-X8 column

A rapid and simple method for assay of pyrroline-5-carboxylate synthase is presented. In this method, the incubation is terminated by raising the pH of incubation mixture to 10, and [14C]pyrroline 5-carboxylate produced from the substrate, [14C]glutamate, is first converted quantitatively to [14C]proline by reduction with NaBH4 at pH 10 and then the proline is allowed to pass through column of AG1-X8 anion exchanger under the conditions where the glutamate is completely retained by the column. Radioactive counting of the eluate gives the synthase activity. The entire procedure takes only one hour.     

Biochemical responses to drought stress in mulberry (<Emphasis Type="Italic">Morus alba</Emphasis> L.): evaluation of proline,glycine betaine and abscisic acid accumulation in five cultivars

Five popularly grown mulberry cultivars (K-2, MR-2, TR-10, BC2-59 and S-13) were subjected to drought stress by withholding irrigation, to obtain leaf water potentials (Ψ_w) ranging from −0.75, −1.50 and −2.25 MPa. Accumulation of proline, glycine betaine and abscisic acid (ABA) were quantified in control and water stressed mulberry leaves. The activities of enzymes involved in proline accumulation including glutamate dehydrogenase (EC1.4.1.2-4), pyrroline-5-carboxylate synthetase (EC 1.2.1.41), pyrroline-5-carboxylate reductase (EC1.5.1.2), ornithine transaminase (EC 2.6.1.13) were significantly enhanced in the leaves of all the cultivars with decreasing leaf water potentials, while the activities of proline dehydrogenase (EC 1.5.1.2) were reduced with progressive increase in water stress. Accumulation of proline, glycine betaine and abscisic acid was relatively higher in S-13 and BC2-59 compared to K-2, MR-2 and TR-10 under water deficit conditions. Our results demonstrate that S-13 and BC2-59 have superior osmoprotectant mechanisms under water-limited growth regimes.     

Water stress-induced alterations in the proline metabolism of drought-susceptible and -tolerant cassava (Manihot esculenta) cultivars

The free proline levels and activities of ornithine aminotransferase (EC 2.6.1.13) and proline oxidase (EC 1.5.2.2), two of the enzymes involved in proline metabolism were studied during the induction of water stress in a drought susceptible (M-4) and a drought tolerant (S-1315) cultivar of cassava ( Manihot esculenta Crantz). Water stress induced by polyethylene glycol (MW 6000, osmotic potential — 1.65 MPa) caused a ca 25-fold increase in proline in young excised leaves of the susceptible cultivar (M-4) while the increase was about 9-fold in the tolerant cultivar (S-1315). The activity of ornithine aminotransferase (OAT), a key enzyme involved in the biosynthesis of proline, was found to increase 3-fold in water stressed leaves of M-4 and about 2-fold in those of S-1315. The activity of proline oxidase, which is involved in the degradation of proline to pyrroline-5-carboxylate, was reduced by 50% in M-4 and nearly 25% in S-1315 on water stress. Comparison of the kinetic properties of OAT showed that the enzyme from water-stressed leaves is more stable to heat inactivation compared to that of control. These results indicate that during water stress there are alterations in the metabolism of proline in cassava, and the extent of alteration varies between drought-susceptible and -tolerant cultivars.     

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.     

Mutant cell lines resistant to azetidine carboxylic acid: Quantitative and qualitative differences in pyrroline-5-carboxylate synthase activity

Mutant Chinese hamster lung fibroblasts were selected that are resistant to the proline analog L-azetidine-2-carboxylic acid. Resistance in the two mutant cell lines is associated with two distinct alterations in pyrroline-5-carboxylate synthase, the enzyme that catalyzes the proline biosynthetic step leading from glutamic acid to pyrroline-5-carboxylate. In one mutant cell line, pyrroline-5-carboxylate synthase specific activity is increased 30-fold over the level in control cells. In the other mutant line, pyrroline-5-carboxylate synthase activity is not increased, but the enzyme has become insensitive to inhibition by ornithine and proline.     

Proline accumulation induced by excess nickel in detached rice leaves

The regulation of proline accumulation in detached rice leaves exposed to excess NiSO₄ was investigated. NiSO₄ treatment increased proline and Ni contents but had no effect on relative water content, indicating that proline accumulation in Ni-exposed detached rice leaves was due to Ni uptake per se, rather than to water stress. Proline accumulation caused by NiSO₄ was related to protein hydrolysis, a decrease in proline dehydrogenase activity, and a decrease in proline utilization. It seems that an increase in the content of ammonia and an increase in the activities of Δ¹-pyrroline-5-carboxylate reductase and ornithine-δ-aminotransferase play minor if any role in Ni-induced proline accumulation in detached rice leaves.     

盐胁迫下小麦苗叶片吡咯-5-羧酸还原酶活性和游离脯氨酸积累

受NaCl、KCl或MsCl_2胁迫的小麦幼苗,当外部溶液的渗透势由—160 kPa下降到—900 kPa时,叶片吡咯—5—羧酸还原酶(PSC)活性增高;渗透势由—900 kPa降低到—1500kPa,酶活性下降;胁迫 1d和 2d的幼苗,还原酶活性显著增加;3～6d酶活性无大变化。游离脯氨酸含量随溶液渗透势下降和培养时间的延长而提高。胁迫解除后酶活性和脯氨酸含量均降低。受NcCl胁迫义在ABA影响下的幼苗,P5C还原酶活性和脯氨酸含量高于仅受NaCl胁迫的幼苗。     

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.     

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.     

Metabolism of arginine in lactating rat mammary gland.

Significant activities of the four enzymes needed to convert arginine into proline and glutamate (arginase, ornithine aminotransferase, pyrroline-5-carboxylate reductase and pyrroline-5-carboxylate dehydrogenase) develop co-ordinately in lactating rat mammary glands in proportion to the increased production of milk. No enzymes were detected to carry out the reactions of proline oxidation or reduction of glutamate to pyrroline-5-carboxylate. Minces of the gland converted ornithine into proline and into glutamate plus glutamine. These conversions increased during the cycle of lactation in proportion to the increased milk production and to the content of the necessary enzymes. The minced gland did not convert labelled ornithine into citrulline, confirming the absence from the gland of a functioning urea cycle, and did not convert labelled proline or glutamate into ornithine. A metabolic flow of labelled arginine to proline and glutamate in mammary gland was confirmed in intact animals with experiments during which the specific radioactivity of proline in plasma remained below that of the proline being formed from labelled arginine within the gland. It was concluded that arginase in this tissue had a metabolic role in the biosynthesis of extra proline and glutamate needed for synthesis of milk proteins.     

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     

The effect of NaCl on proline accumulation in potato seedlings and calli

The effects of salt stress were studied on the accumulation and metabolism of proline and its correlation with Na⁺ and K⁺ content in shoots and callus tissue of four potato cultivars, viz., Agria, Kennebec (relatively salt tolerant), Diamant and Ajax (relatively salt sensitive). Na⁺ and proline contents increased in all cultivars under salt stress. However, K⁺ and protein contents decreased in response to NaCl treatments. The activities of enzymes involved in proline metabolism, Δ¹-pyrroline-5-carboxylate synthetase (P5CS) and proline dehydrogenase (ProDH) increased and decreased, respectively, in response to elevated NaCl concentrations. The changes of P5CS and ProDH activities in more salt sensitive cultivars (Diamant, Ajax) were more than those in the tolerant ones. Then the stimulation of synthesis in combination with a partially increase of protein proteolysis, a decrease in proline utilization and inhibition of oxidation resulted in high proline contents in seedlings and calli under salt stress. In callus tissue, reduced growth and cell size may be partially responsible for high proline accumulation in response to high NaCl levels. However, although the basic proline contents in the seedlings of more salt tolerant cultivars were higher than the sensitive ones, a clear relationship was not generally observed between accumulation of proline and salt tolerance in potato.     

Exogenous ornithine is an effective precursor and the δ-ornithine amino transferase pathway contributes to proline accumulation under high N recycling in salt-stressed cashew leaves

The role of the δ-ornithine amino transferase (OAT) pathway in proline synthesis is still controversial and was assessed in leaves of cashew plants subjected to salinity. The activities of enzymes and the concentrations of metabolites involved in proline synthesis were examined in parallel with the capacity of exogenous ornithine and glutamate to induce proline accumulation. Proline accumulation was best correlated with OAT activity, which increased 4-fold and was paralleled by NADH oxidation coupled to the activities of OAT and Δ¹-pyrroline-5-carboxylate reductase (P5CR), demonstrating the potential of proline synthesis via OAT/P5C. Overall, the activities of GS, GOGAT and aminating GDH remained practically unchanged under salinity. The activity of P5CR did not respond to NaCl whereas Δ¹-pyrroline-5-carboxylate dehydrogenase was sharply repressed by salinity. We suggest that if the export of P5C from the mitochondria to the cytosol is possible, its subsequent conversion to proline by P5CR may be important. In a time-course experiment, proline accumulation was associated with disturbances in amino acid metabolism as indicated by large increases in the concentrations of ammonia, free amino acids, glutamine, arginine and ornithine. Conversely, glutamate concentrations increased moderately and only within the first 24 h. Exogenous feeding of ornithine as a precursor was very effective in inducing proline accumulation in intact plants and leaf discs, in which proline concentrations were several times higher than glutamate-fed or salt-treated plants. Our data suggest that proline accumulation might be a consequence of salt-induced increase in N recycling, resulting in increased levels of ornithine and other metabolites involved with proline synthesis and OAT activity. Under these metabolic circumstances the OAT pathway might contribute significantly to proline accumulation in salt-stressed cashew leaves.     

Proline biosynthesizing enzymes (glutamate 5-kinase and pyrroline-5-carboxylate reductase) from a model cyanobacterium for desiccation tolerance

Drought is the most important abiotic stress, challenging sustainable agriculture globally. For desiccation being the multigenic trait, a combination of identified genes from the appropriate organism may render crop tolerant to the water stress. Among the compatible solutes, proline plays multifaceted role in counteracting such stress. The genes encoding proline biosynthesizing enzymes, glutamate 5-kinase (G5K), and pyrroline-5-carboxylate reductase (P5CR) from the low-desiccation-tolerant cyanobacterium Anabaena sp. PCC 7120, were cloned and overexpressed in Escherichia coli BL21(DE3) individually. The recombinant E. coli cells harboring G5K, failed to exhibit enhanced desiccation tolerance relative to those with P5CR that showed increased growth/survival over the wild type. This may be ascribed to the overexpression of the reductase gene. Multiple sequence alignment showed P5CR to be conserved in all the organisms. We hypothesize that P5CR gene from high-desiccation-tolerant cyanobacteria may be adopted as the candidate for making transgenic N₂-fixing cyanobacterium for paddy fields and/or crop development in future.     

Proline accumulation and the expression of Δ1-pyrroline-5-carboxylate synthetase in two safflower cultivars

Proline (Pro) accumulation under water stress was measured in safflower (Carthamus tinctorius L.) drought tolerant cv. A1 and sensitive cv. Nira. Activities of pyrroline-5-carboxylate reductase (P5C reductase) and pyrroline-5-carboxylate synthetase (P5C synthetase), two enzymes involved in the Pro biosynthetic pathway were also estimated. Water stress resulted in a reduction in the leaf dry mass and chlorophyll content along with a gradual accumulation of Pro. RT-PCR results show higher expression of Δ¹-pyrroline-5-carboxylate synthetase (p5cs) gene in correlation with up-regulated Pro accumulation in cv. A1. P5C reductase was found to be the Pro synthesis rate limiting whereas P5C synthetase did not show any specific response to the drought stress in both cultivars.     

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.