The oxidative stress and antioxidant systems in cucumber cells during acclimation to salinity

In the present study, we measured the markers of oxidative stress as well as activity of antioxidative enzymes and content of α-tocopherol in the acclimated and non-acclimated cucumber (Cucumis sativus L.) cell suspension cultures subjected to 150 and 200 mM NaCl. The content of carbonyl groups and lipid peroxidation were lower in the acclimated cultures than in the non-acclimated ones as well as their increases after NaCl treatments. Both NaCl concentrations enhanced activity of glutathione peroxidase in the examined cultures whereas activity of glutathione-S-transferase rose only in the acclimated ones. The increase in content of α-tocopherol induced by NaCl was more pronounced in the acclimated cultures. NaCl caused high decline in cell vigour in the non-acclimated cultures up to 80–90 % at the end of the experiment. The presented data suggest that the acclimated cultures coped with the salt stress better than the non-acclimated ones.     

Proline Metabolism and Cross-Tolerance to Salinity and Heat Stress in Germinating Wheat Seeds

Germination/growth of wheat (Triticum aestivum L., cv. Zimai 1) seeds and changes in the levels of proline and protein as well as in activities of key enzymes involved in proline metabolism in response to salinity-, heat-stresses and their cross-stress were studied. With decreasing water potential caused by increasing concentrations of NaCl, germination percentage, fresh weight of seedlings and protein amount markedly decreased, whereas proline amount slightly increased. The activities of pyrroline-5-carboxylate synthetase (P5CS), ornithine aminotransferase (OAT), and proline dehydrogenase (PDH) peaked at ?0.2 MPa water potential. Germination percentage and amounts of proline and protein increased as germination temperature elevated to 25°C from 15°C, and decreased above 25°C; fresh weight of seedlings increased to 30°C from 15°C, and decreased above 30°C. However, the activities of P5CS, OAT and PDH gradually decreased with elevaing temperature. Seeds pretreated at 33°C or in ?0.8 MPa NaCl solution for various time length increased tolerance to subsequent salt + water stress or heat stress, as measured by germination percentage and fresh weight of seedlings 5 days after beginning of experiment. The acquisition of cross-tolerance resulting in limitation of negative stress effects does not relate directly to proline level and activities of P5CS, OAT and PDH involved in proline metabolism. Proline amount as measured four days or later after stress imposition cannot be considered a symptom of salt-, water- and heat-stress injury or an indicator of the resistance.     

Production of secondary metabolites from cell and organ cultures: strategies and approaches for biomass improvement and metabolite accumulation

Pyrroline-5-carboxylate reductase (P5CR) lies at the converging point of the glutamate and ornithine pathways and is the last and critical enzyme in proline biosynthesis. In the present study, a P5CR gene, named IbP5CR, was isolated from salt-tolerant sweetpotato line ND98. Expression of IbP5CR was up-regulated in sweetpotato under salt stress. The IbP5CR-overexpressing sweetpotato (cv. Kokei No. 14) plants exhibited significantly higher salt tolerance compared with the wild-type. Proline content and superoxide dismutase and photosynthetic activities were significantly increased, whereas malonaldehyde content was significantly decreased in the transgenic plants. H₂O₂ was also found to be significantly less accumulated in the transgenic plants than in the wild-type. Overexpression of IbP5CR up-regulated pyrroline-5-carboxylate synthase gene and down-regulated proline dehydrogenase and P5C dehydrogenase genes under salt stress. The systemic up-regulation of reactive oxygen species (ROS) scavenging genes was found in the transgenic plants under salt stress. These findings suggest that overexpression of IbP5CR increases proline accumulation, which enhances salt tolerance of the transgenic sweetpotato plants by regulating osmotic balance, protecting membrane integrity and photosynthesis and activating ROS scavenging system. This study indicates that IbP5CR gene has the potential to be used for improving salt tolerance of plants.     

Glutamine synthetase and glutamate dehydrogenase contribute differentially to proline accumulation in leaves of wheat (Triticum aestivum) seedlings exposed to different salinity

To investigate the roles of ammonium-assimilating enzymes in proline synthesis under salinity stress, the activities of glutamine synthetase (GS; EC 6.3.1.2) and NADH-dependent glutamate dehydrogenase (NADH-GDH; EC 1.4.1.2) were determined in leaves of wheat (Triticum aestivum) seedlings exposed to salt stress at 150 and 300 mM NaCl for 5d. At the lower salinity, only GS activity increased markedly. At 300 mM NaCl, however, NADH-GDH activity increased while GS activity decreased. A significant accumulation of proline was found only at high-salinity exposure while glutamate, a proline precursor, increased dramatically under both low and high salinity. These data suggests that GS-catalysis might be the main glutamate synthesis pathway under low salinity. At 300 mM NaCl, glutamate seems to be preferentially produced through the process catalyzed by NADH-GDH. The increase of ammonium in salinity-stressed wheat seedlings might have resulted from increased photorespiration, which is responsible for the higher NADH-GDH activity. The activity of Delta(1)-pyrroline-5-carboxylate reductase (P5CR; EC 1.5.1.2) was significantly enhanced at 300 mM NaCl but remained unchanged at 150 mM. Delta(1)-Pyrroline-5-carboxylate synthetase (P5CS) activity did not show a specific response, indicating that P5CR might be the limiting step in proline synthesis from glutamate at high salinity.     

Ameliorative Effect by Calcium on NaCI Salinity Stress Related to Proline Metabolism in the Callus of Centella asiatica L

Soil salinity affects plant growth and development by way of osmotic stress. Compatible osmolytes are potent osmoprotectants that playa role in counteracting the effect of saline stress. Proline biosynthesis and catabolism were investigated in both the control and salt stressed calli. Proline content showed a steady increase in the calli of all NaCI treated media. Calli on CaCl₂ containing media did not show any increase in proline level compared to control calli. When the salinized media were supplemented with CaCl₂ the proline level drastically increased compared to the corresponding calli grown on salt alone. Similarly, the activity of proline biosynthetic enzyme, pyrroline-5-carboxylate synthetase (P5CS) under salt stress was higher in NaCl + CaCl₂ supplemented medium than the calli on the salinized medium alone. This suggested that the alleviation effect of calcium under saline condition was through modulation of the enzyme complexes that accelerate the rate of proline biosynthesis under salt stress. Similarly, the activity of proline degrading enzyme, proline oxidase was found to be lower in calli of all salt stressed media than control.     

Pyrroline-5-Carboxylate Reductase in Soybean Nodules : Comparison of the Enzymes in Host Cytosol, Bradyrhizobium japonicum Bacteroids, and Cultures

Characteristics of pyrroline-5-carboxylate reductase (P5CR) from Bradyrhizobium japonicum bacteroids and cultured rhizobia were compared with those of the enzyme in soybean nodule host cytosol. Reductase from host cytosol differed from that in bacteroids in: (a) the effect of pH on enzymic activity, (b) the capacity to catalyze both reduction of pyrroline-5-carboxylic acid and NAD⁺-dependent proline oxidation, (c) apparent affinities for pyrroline-5-carboxylic acid, and (d) sensitivities to inhibition by NADP⁺ and proline. The K₁ for proline inhibition of P5CR in bacteroid cytosol was 1.8 millimolar. The properties of P5CR in B. japonicum and bacteroid cytosol were similar. The specific activities of P5CR in the cytosolic fractions of the nodule host and the bacteroid compartment were also comparable.     

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.     

The effect of water deficit and excess copper on proline metabolism in Nicotiana benthamiana

Fluctuation in proline content is a widespread phenomenon among plants in response to heavy metal stress. To distinguish between the participation of water deficit and copper on changes in proline metabolism, potted plants and floating leaf discs of tobacco were subjected to CuSO₄ treatments. The application of copper increased the proline content in the leaves concomitantly with decreased leaf relative water content and increased abscisic acid (ABA) content in the potted plant. Excess copper increased the expression of two proline synthesis genes, pyrroline-5-carboxylate synthetase (P5CS) and ornithine aminotransferase (OAT) and suppressed proline catabolism gene, proline dehydrogenase (PDH). However, in the experiment with tobacco leaf discs floating on CuSO₄ solutions, the excess copper decreased proline content and suppressed the expression of the P5CS, OAT and PDH genes. Therefore, proline accumulation in the potted tobacco plants treated with excess Cu treatment might not be the consequence of the increased copper content in tobacco leaves but rather by the accompanied decrease in water content and/or increased ABA content.     

Subcellular compartmentation in control of converging pathways for proline and arginine metabolism in Saccharomyces cerevisiae.

Enzymes of proline biosynthesis and proline degradation which act on the same compound, delta 1-pyrroline-5-carboxylate, are physically separated in yeast cells. The enzyme responsible for the final step in proline biosynthesis, pyrroline-5-carboxylate reductase, converts pyrroline-5-carboxylate to proline and is located in the cytoplasm. The last enzyme in the proline degradative pathway, pyrroline-5-carboxylate dehydrogenase, converts pyrroline-5-carboxylate to glutamate and is found in the particulate fraction of the cell, presumably in the mitochondrion. By subcellular compartmentation, yeast cells avoid futile cycling between proline and pyrroline-5-carboxylate.     

Exogenous Spermidine Modulates Osmoregulatory Substances and Leaf Stomata to Alleviate the Damage to Lettuce Seedlings Caused by High Temperature Stress

Lettuce (Lactuca sativa L.) prefers cool environments, and high temperatures affect its yield and quality. Polyamines (PAs) have a mitigating effect on plant abiotic stresses. The effect of exogenous spermidine (Spd) on the osmoregulatory substances and stomata of seedlings of the non-heat-tolerant lettuce variety ‘Bei San 3’ under high temperature stress was investigated at 35 °C/30 °C (day/night) under spray treatment with Spd. The results showed that exogenous Spd increased the total fresh weight, root-to-shoot ratio, leaf length, leaf width, root volume, and root surface area of lettuce under high temperature stress and reduced levels of malondialdehyde. The endogenous polyamine content was changed, and endogenous spermidine (Spd) and putrescine (Put) were increased. The accumulation of six organic osmoregulatory substances was promoted, resulting in enhanced betaine aldehyde dehydrogenase (BADH), choline monooxygenase (CMO), proline catalase pyrroline-5-carboxylate synthase (P5CS), ornithine aminotransferase (OAT), and pyrroline-5-carboxylate reductase (P5CR) activity. The production and activity of the degrading enzymes proline dehydrogenase (PDH) and proline oxidase (POX) were inhibited, and the activity of glutamic acid decarboxylase (GAD), the key enzyme of γ-aminobutyric (GABA), was suppressed. In addition, exogenous Spd increased the contents of Ca, K, Fe, Mn, Zn, and NO₃^? ions in lettuce leaves under high temperature stress, promoted K⁺ efflux and Ca²⁺ influx, and reduced the relative stomatal aperture. In summary, exogenous Spd mitigates lettuce injury caused by high temperature stress by increasing the content of osmoregulatory substances and altering stomatal morphology.

     

Solubilization of a Proline Dehydrogenase from Maize (Zea mays L.) Mitochondria

L-Proline is oxidized to pyrroline-5-carboxylic acid in intact plant mitochondria by a proline dehydrogenase (EC 1.4.3) that is bound to the matrix side of the inner mitochondrial membrane (TE Elthon, CR Stewart [1981] Plant Physiol 67: 780-784). This investigation reports the first solubilization of the L-proline dehydrogenase (PDH) from plant mitochondria. The supernatant from NP-40-treated etiolated shoot mitochondria of maize, Zea mays L., reduced iodonitrotetrazolium violet in a proline dependent manner. The pH optimum for this activity was 8. The apparent K_m for proline was 6.6 millimolar. When supplied with proline, this solubilized PDH activity also synthesized pyrroline-5-carboxylic acid. The PDH activity was inhibited in vitro by 300 millimolar potassium chloride but not by 300 millimolar potassium acetate. The PDH activity had a molecular mass that was greater than 150 kilodaltons. Mitochondria were prepared from etiolated shoots grown in 100% water-saturated vermiculite (control) and 16% water-saturated vermiculite (stress). The specific activity of solubilized PDH from the stress treatment was 11% of the same activity from the control treatment. Oxygen uptake in the presence of proline and ADP (state 3 proline oxidation) by mitochondria from the stress treatment was 25% of the same rate by mitochondria from the control treatment. Mitochondria were also prepared 16 hours after rewatering the seedlings growing in the stress treatment. Both the solubilized PDH specific activity and state 3 proline oxidation returned to the control levels. The specific activities of the NAD⁺-dependent pyrroline-5-carboxylic acid dehydrogenase and cytochrome c oxidase in the solubilized preparations were unaffected by these stress and recovery treatments. Oxygen uptake rates by intact mitochondria in the presence of ADP and NADH, succinate or malate-pyruvate were also unaffected by these treatments.     

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     

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.     

The influence of NaCl on Δ1-pyrroline-5-carboxylate reductase in proline-accumulating cell suspension cultures of Mesembryanthemum nodiflorum and other halophytes

Pyrroline-5-carboxylate (P-5-C) reductase (EC 1.5.1.2) was extracted from cell suspension cultures, which proved to be very suitable for investigation of proline accumulation. Proline accumulation in cell suspensions of M. nodiflorum L., as well as P-5-C reductase activity and substrate affinity, increased with progressive adaptation to NaCl stress. In vitro NaCl treatment inhibited enzyme activity and decreased substrate affinity, independent of pretreatment of the cells. NaCl concentrations below 100 m M did not inhibit enzyme activity of adapted cells. High substrate concentrations counteracted in vitro NaCl inhibition (up to 200 m M ). Cycloheximide inhibited the increase of P-5-C reductase activity, as well as proline accumulation, after NaCl treatment, indicating stress-induced de novo synthesis of the enzyme. The different reactions of P-5-C reductase upon salt treatment are discussed with respect to its possible role in the regulation of proline accumulation.     

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.