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.     

Effects of salt stress on growth, mineral nutrition and proline accumulation in relation to osmotic adjustment in rice (Oryza sativa L.) cultivars differing in salinity resistance

Salt-resistant rice cultivars Nona Bokra and IR 4630 exposed at the seedling stage during one or two weeks to 0, 20, 30, 40 or 50 mM NaCl accumulated less Na, Cl, Zn and proline and more K at root and shoot levels than salt-sensitive I Kong Pao and IR 31785. Aiwu, a moderately resistant genotype, exhibited an intermediate behaviour. P transport from root to shoot was inhibited in the most sensitive cultivar IR 31785. Accumulation of Na and Cl and decrease in K content at the shoot level were restricted to the oldest leaves in salt-resistant genotypes while proline accumulated in the youngest leaves in all cultivars. In the presence of NaCl, the osmotic potentials of the roots and of the oldest and youngest leaves were lower in the salt-resistant than in the salt-sensitive genotypes, differences among genotypes increasing with stress intensity. Proline did not appear to be involved in osmotic adjustment in salt-stressed rice plants and the significance of its accumulation is discussed in relation to salinity resistance.Abbreviations cv(s). cultivar(s) - EC electrical conductivity - IKP I Kong Pao - J rate of ion transport - MCW methanol-chloroform-water - PAR photon flux density - Pc partitioning coefficient - RGR mean relative growth rate - RI resistance index - s osmotic potential     

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.     

不同耐盐性水稻幼苗根氨同化酶对盐胁迫的反应

在盐胁迫下,检测了耐盐性不同的水稻(Oryza sativa L.)品种根部氨同化酶及其相关参数的变化。结果表明,根的可溶性蛋白、谷氨酰胺合成酶(GS)及依赖于NADH的谷氨酸合酶(NADH-GOGAT)活性在高盐浓度下不同程度地降低,其影响大小依次为早花二号(盐敏感品种)、金珠一号(正常栽培品种)、津稻779(耐盐品种),与其耐盐性相一致。在盐胁迫条件下,在耐盐性较高的水稻品种中, GS和GOGAT活性比盐敏感品种高,NH4 浓度维持在较低的水平。Native-PAGE和活性染色结果表明,GSrb更容易受到外界环境的影响。在高浓度盐的胁迫下,早花二号、金珠一号的依赖于NADH的谷氨酸脱氢酶(NADH-GDH)活性都有较显著的升高,津稻779却无明显的变化,这和NH4 含量的变化相一致。盐不同程度地导致可溶性糖(TSS)在金珠一号和津稻779根部积累,而在早花2号的根部,可溶性糖的水平则随盐浓度的不同而表现出不同的变化。在所检测的品种中,脯氨酸的含量均有不同程度的升高,但在高盐浓度下,盐敏感品种的含量较低。这些结果提示,不同的水稻品种对盐胁迫的敏感程度与该品种GS以及GOGAT活性的高低有关。     

The response of Mo-hydroxylases and abscisic acid to salinity in wheat genotypes with differing salt tolerances

The differential responses of the wheat cultivars Shi4185 and Yumai47 to salinity were studied. The higher sensitivity of Yumai47 to salinity was linked to a greater growth reduction under salt stress, compared to more salt-tolerant Shi4185. Salinity increased the Na⁺, proline and superoxide anion radical (O₂ ^?) contents in both cultivars. Leaf Na⁺ content increased less in the more salt-tolerant cultivar Shi4185 than salt-sensitive Yumai47. The proline content increased more significantly in Shi4185 than Yumai47; on the contrary, superoxide anion radical content increased less in Shi4185 than Yumai47. This data indicated that wheat salinity tolerance can be increased by controlling Na⁺ transport from the root to shoot, associated with higher osmotic adjustment capability and antioxidant activity. Although salinity increased aldehyde oxidase (AO) activity and abscisic acid (ABA) content in the leaves and roots of both cultivars following the addition of NaCl to the growth medium, AO and ABA increased more in the salt-sensitive cultivar Yumai47 than the more salt-tolerant cultivar Shi4185. Xanthine dehydrogenase (XDH) activity in the leaves of both cultivars increased with increasing concentrations of NaCl; however, leaf XDH activity increased more significantly in Yumai47 than Shi4185. Root XDH activity in Shi4185 decreased with increasing NaCl concentrations, whereas salinity induced an increased root XDH activity in Yumai47. The involvement of AO and XDH enzymatic activities and altered ABA content in the response mechanisms of wheat to salinity are discussed herein.     

Cold acclimation of wheat (Triticum aestivum): Properties of enzymes involved in proline metabolism

Two cultivars of wheat ( Triticum aestivum L.), a winter wheat, Kharkov, and a spring wheat, Glenlea, were acclimated under controlled conditions at 2 temperatures, 5°C and 25°C with a 12-h photoperiod. Water content, protein and proline concentrations were determined. Enzymatic properties (activity and apparent energy of activation) were investigated for enzymatic systems involved in 2 pathways of proline metabolism, the glutamic acid and ornithine pathways. Four enzymes were studied, proline dehydrogenase (PDH, EC 1.5.1.2), glutamate dehydrogenase (GDH, EC 1.4.1.2-4), glutamine synthetase (GS, EC 6.3.1.2) and ornithine transaminase (OT, EC 2.6.1.13). Cold acclimation led to an accumulation of proline, a decrease in water content and an increase in soluble protein, especially in winter wheat. For both cultivars, cold acclimation modulated enzyme properties of PDH and GDH. Increased activities of GS and OT were observed as a result of cold acclimation in both cultivars, with the greatest increase in Kharkov. The apparent energy of activation of these 2 enzymes decreased, particularly for Kharkov, which accumulated proline in cold conditions.     

Signal regulation of proline metabolism in callus of the halophyte Nitraria tangutorum Bobr. grown under salinity stress

Callus of the halophyte Nitraria tangutorum Bobr. was used to investigate proline metabolism and its signal regulation under salinity stress. Enhanced levels of proline and hydrogen peroxide (H₂O₂) were observed in calli exposed to salinity stress, and elevated levels of calcium (Ca) were detected in early responses to 75?mM NaCl treatment. Additionally, NaCl treatment induced significant elevation of ornithine-??-aminotransferase (OAT) activity, but notable decreases occurred in the activities of glutamyl kinase (GK) and proline dehydrogenase (PDH). H₂O₂ scavenger dimethylthiourea and pyruvate inhibited the accumulation of proline and the stimulation of OAT in salinity-stressed calli. Moreover, the utilization of Ca chelator EGTA and Ca channel blocker verapamil abolished the enhancement of proline level induced by 75?mM NaCl treatment for 3?days. These results suggest that the accumulation of proline is correlated to the increase of OAT activity and the decrease of PDH activity in response to salinity, and that elevated Ca signal during the early stage of NaCl treatment and the excitation of OAT activity resulting from the increase of H₂O₂ generation are essential for proline accumulation in salinity-stressed calli.     

NaHSO3对盐胁迫下小麦幼苗氮同化酶及脯氨酸含量的影响

以普通小麦(Triticum aestivumL.)为材料,研究了NaHSO3对不同盐度胁迫下小麦幼苗氮素同化酶和脯氨酸含量的调节。结果表明,盐胁迫降低了叶片中硝酸还原酸(NR)的活性,加入NaHSO3之后,NR活性表现出进一步的降低。谷氨酰胺合成酶(GS)在低浓度盐胁迫下活性增加,在高浓度盐胁迫下活性降低;NaHSO3加入时,即便在低盐浓度下GS活性也降低。依赖于NADH的谷氨酸脱氢酶(NADH-GDH)和依赖于NADP的异柠檬酸脱氢酶(NADP-ICDH)的变化趋势一致,在盐胁迫下它们的活性都明显增加;NaHSO3加入促进了它们活性的进一步增加,尤其对NADH-GDH活性的促进更为明显。游离脯氨酸在高浓度盐胁迫下大量积累,在低浓度盐胁迫下含量增加不明显;NaHSO3促进了盐胁迫下脯氨酸的积累,提示了NaHSO3促进了盐胁迫下小麦幼苗碳氮营养元素的贮存。     

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.     

Kinetic Properties of Four Enzymes Involved in Proline Metabolism in Cold-acclimated Wheat

Two varieties of wheat (Triticum aestivum L.) a winter (Kharkov)and a spring (Glenlea), were acclimated under controlled conditionsat 5 °C and 25 °C (12 h photoperiod). Kinetic properties(K_m1 V_max/Km ratio and Q₁₀ as a function of reduction of substrateconcentration) were investigated for enzymatic systems involvedin two pathways of proline metabolism: the glutamic acid andthe ornithine pathways. Four enzymes were studied, namely prolinedehydrogenase (PDH, EC 1.5.1.2 [EC] ), glutamate dehydrogenase (GDH,EC 1.4.1.2 [EC] -4), glutamine synthetase (GS, EC 6.3.1.2 [EC] ) and ornithinetransaminase (OT, EC 2.6.1.13 [EC] ). Kinetic properties of thesefour enzymes proved to be modulated by cold acclimation, especiallyin Kharkov, the winter cultivar, which accumulates proline.Firstly, the synthesis of precursors of proline may be augmentedand the degradation of proline lessened by either decreasingthe K_m values of OT or increasing the K_m values of PDH. Secondly,the catalytic efficiency (V_max ratio) of GDH, GS, and OT isincreased. Thirdly, the lower values of Q₁₀ indicate a highcapacity of reaction of GS and OT.     

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.     

Peroxidase activities of two rice cultivars differing in salinity tolerance as affected by proline and NaCl

Proline content, ion accumulation, cell wall and soluble peroxidase activities were determined in control and salt-treated calli (150 nM NaCl) and whole plants (30 mM NaCl) of two rice cultivars (salt sensitive cv. IKP and salt tolerant cv. Aiwu). Under salinity, the highest accumulation of Na⁺, Cl^? and proline occurred in calli, roots and younger leaves of cv. IKP, coupled with the highest decrease in K⁺ content; accumulations of Na⁺ and Cl^? were restricted to older leaves in cv. Aiwu. Relative growth rates of calli and roots or shoots from both cultivars were not linked to peroxidase activities. High concentrations (1 M) of exogenously applied glycerol did not inhibitin vitro activities of soluble peroxidase extracted from control and salt-treated calli or plants. Conversely, 35–55% (in cv. IKP) or 60–80% (in cv. Aiwu) of soluble peroxidase activities were found in presence of isosmotic proline concentration. There were no differences between proline and glycerol effects onin vitro cell wall peroxidase activities.     

不同耐盐性水稻幼苗根氨同化酶对盐胁迫的反应

在盐胁迫下,检测了耐盐性不同的水稻(Oryza sativa L.)品种根部氨同化酶及其相关参数的变化.结果表明,根的可溶性蛋白、谷氨酰胺合成酶(GS)及依赖于NADH的谷氨酸合酶(NADH-GOGAT)活性在高盐浓度下不同程度地降低,其影响大小依次为早花二号(盐敏感品种)、金珠一号(正常栽培品种)、津稻779(耐盐品种),与其耐盐性相一致.在盐胁迫条件下,在耐盐性较高的水稻品种中,GS和GOGAT活性比盐敏感品种高,NH4 浓度维持在较低的水平.Native-PAGE和活性染色结果表明,GSrb更容易受到外界环境的影响.在高浓度盐的胁迫下,早花二号、金珠一号的依赖于NADH的谷氨酸脱氢酶(AADH-GDH)活性都有较显著的升高,津稻779却无明显的变化,这和NH4 含量的变化相一致.盐不同程度地导致可溶性糖(TSS)在金珠一号和津稻779根部积累,而在早花2号的根部,可溶性糖的水平则随盐浓度的不同而表现出不同的变化.在所检测的品种中,脯氨酸的含量均有不同程度的升高,但在高盐浓度下,盐敏感品种的含量较低.这些结果提示,不同的水稻品种对盐胁迫的敏感程度与该品种GS以及GOGAT活性的高低有关.     

Proline accumulation and glutamine synthetase activity are increased by salt-induced proteolysis in cashew leaves

In this study cashew (Anacardium occidentale) plants were exposed to a short- and long-term exposure to NaCl in order to establish the importance of the salt-induced proteolysis and the glutamine synthetase activity on the proline accumulation. The cashew leaf showed a prominent proline accumulation in response to salt stress. In contrast, the root tissue had no significant changes in proline content even after the drastic injury caused by salinity on the whole plant. The leaf proline accumulation was correlated to protease activity, accumulation of free amino acid and ammonia, and decrease of both total protein and chlorophyll contents. The leaf GS activity was increased by the salt stress whereas in the roots it was slightly lowered. Although the several amino acids in the soluble pool of leaf tissue have showed an intense increment in its concentrations in the salt-treated plants, proline was the unique to show a proportional increment from 50 to 100 mol m-3 NaCl exposure (16.37 to 34.35 mmol kg-1 DM, respectively). Although the leaf glutamate concentration increased in the leaves of the salt-stressed cashew plants, as compared to control, its relative contribution to the total amino acid decreased significantly in stressed leaves when compared to other amino acids. In addition, when the leaf discs were incubated with NaCl in the presence of exogenous precursors (Glu, Gln, Orn or Arg) involved in the proline synthesis pathways, the glutamate was unique in inducing a significant enhancement of the proline accumulation compared to those discs with precursor in the absence of NaCl. These results, together with the salt-induced increase in the GS activity, suggest an increase in the de novo synthesis of proline probably associated with the increase of the concentration of glutamate. Moreover, the prominent salt-induced proline accumulation in the leaves was associated with the higher salt-sensitivity in terms of proteolysis and salt-induced senescence as compared to the roots. In conclusion, the leaf-proline accumulation was due, at least in part, to the increase in the salt-induced proteolysis associated with the increments in the GS activity and hence the increase in the concentration of glutamate precursor in the soluble amino acid pool.     

Salt stress affects glutamine synthetase activity and mRNA accumulation on potato plants in an organ-dependent manner

Ammonium assimilation into glutamine and glutamate is vital for plant growth as these are precursors for almost all nitrogenous compounds. Ammonium can be assimilated onto nitrogenous organic compounds by the concerted action of two enzymes that compose the glutamine synthetase (GS, EC 6.3.1.2) – glutamate synthase (Fd-GOGAT, EC 1.4.7.1; NADH–GOGAT, EC 1.4.1.14) cycle. Ammonium may also be directly incorporated into glutamate by the glutamate dehydrogenase (GDH, EC 1.4.1.2) aminating reaction. However, as GDH reversibly deaminates glutamate, its physiological role in vivo remains controversial. Potato has been classified as moderately tolerant to salinity. Potato GS is encoded by a small multigene family which is differentially regulated in an organ and age-dependent way. In this study, the effect of increasing concentrations of salinity in the soil in GS activity and gene-specific mRNA accumulation levels were studied on potato leaves and roots, as well as the biochemical parameters protein, chlorophyll, lipid peroxidation and proline levels, in order to evaluate the severity of the imposed stress. The data obtained suggests that when potato plants are subjected to salt stress, increased ammonium assimilation occurs in roots, due to an increased GS accumulation, along with a decreased assimilation in leaves. Regarding GS gene-specific mRNA accumulation, an organ-dependent response was also observed that contributes for the detected alteration in the ammonium assimilatory metabolism. This response may be a key feature for future genetic manipulations in order to increase crop productivity in salty soils. The possible contribution of GDH for ammonia assimilation was also investigated.     

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.     

Effective salt criteria in callus-cultured tomato genotypes

Na+, Cl-, K+, Ca2+, and proline contents, the rate of lipid peroxidation level in terms of malondialdehyde (MDA) and chlorophyll content, and the changes in the activity of antioxidant enzymes, such as superoxide dismutase (SOD: EC 1.15.1.1), catalase (CAT: EC 1.11.1.6), ascorbate peroxidase (APX: EC 1.11.1.11), and glutathione reductase (GR: EC 1.6.4.2), in tissues of five tomato cultivars in salt tolerance were investigated in a callus culture. The selection of effective parameters used in these tomato genotypes and to find out the use of in vitro tests in place of in vivo salt tolerance tests were investigated. As a material, five different tomato genotypes during a 10-day time period were used, and 150 mM NaCl was applied at callus plant tissue. The exposure to NaCl induced a significant increase in MDA content in both salt-resistant and salt-sensitive cultivars. But the MDA content was higher in salt-sensitive cultivars. The chlorophyll content was more decreased in salt-sensitive than in salt-resistant ones. The proline amount was more increased in salt-sensitive than in salt-resistant ones. It has been reported that salt-tolerant plants, besides being able to regulate the ion and water movements, also exhibit a strong antioxidative enzyme system for effective removal of ROS. The degree of damage depends on the balance between the formation of ROS and its removal by the antioxidative scavenging system that protects against them. Exclusion or inclusion of Na+, Cl-, K+, and Ca2+, antioxidant enzymes and MDA concentration play a key protective role against stress, and this feature at the callus plant tissue used as an identifier for tolerance to salt proved to be an effective criterion.