Soybean MAPK, GMK1 Is dually regulated by phosphatidic acid and hydrogen peroxide and translocated to nucleus during salt stress

Mitogen-activated protein kinase (MAPK) is activated by various biotic and abiotic stresses. Salt stress induces two well-characterized MAPK activating signaling molecules, phosphatidic acid (PA) via phospholipase D and phospholipase C, and reactive oxygen species (ROS) via nicotinamide adenine dinucleotide phosphate (NADPH)-oxidase. In our previous study, the activity of soybean MAPK, GMK1 was strongly induced within 5 min of 300 mM NaCl treatment and this early activity was regulated by PA. In this study, we focused on the regulation of GMK1 at the later stage of the salt stress, because its activity was strongly persistent for up to 30 min. H₂O₂ activated GMK1 even in the presence of PA generation inhibitors, but GMK1 activity was greatly decreased in the presence of diphenyleneiodonium, an inhibitor of NADPH-oxidase after 5 min of the treatment. On the contrary, the n-butanol and neomycin reduced GMK1 activity within 5 min of the treatment. Thus, GMK1 activity may be sustained by H₂O₂ 10 min after the treatment. Further, GMK1 was translocated into the nucleus 60 min after NaCl treatment. In the relationship between GMK1 and ROS generation, ROS generation was reduced by SB202190, a MAPK inhibitor, but was increased in protoplast overexpressing TESD-GMKK1. However, these effects were occurred at prolonged time of NaCl treatment. These data suggest that GMK1 indirectly regulates ROS generation. Taken together, we propose that soybean GMK1 is dually regulated by PA and H₂O₂ at a time dependant manner and translocated to the nucleus by the salt stress signal.     

Higher accumulation of gamma-aminobutyric acid induced by salt stress through stimulating the activity of diamine oxidases in Glycine max (L.) Merr. roots.

Polyamines (PAs) are assumed to perform their functions through their oxidative product such as gamma-aminobutyric acid (GABA) formation. However, there is only limited information on the interrelation between PA degradation and GABA accumulation under salt stress. In order to reveal a quantitative correlation between PA oxidation and GABA accumulation, the effects of treatments with different NaCl concentrations, along with aminoguanidine (AG, a specific inhibitor of diamine oxidases (DAO; EC: 1.4.3.6)) and a recovery test from salt stress on endogenous free PAs, gamma-aminobutyric acid (GABA) accumulation and DAO activity were determined in roots of soybean [Glycine max (L.) Merr.] cultivar Suxie-1. The results showed that the levels of putrescine (Put), cadaverine (Cad), and spermidine (Spd) decreased significantly with increasing salt concentrations. This occurred because salt stress strongly promoted DAO activity to stimulate PA degradation. GABA accumulation increased with growing NaCl concentrations, about an 11- to 17-fold increase as compared with the control plants. AG treatment increased the accumulation of endogenous free PAs as a result of a strong retardation of DAO activity, but decreased GABA accumulation. The recovery for 6 days in 1/2 Hoagland solution from 100mM NaCl stress resulted in a decrease in DAO activity, a rebound of PA levels and a simultaneous reduction of GABA content. A close correlation was observed between the changes in DAO activity and GABA accumulation. The results indicated that higher GABA accumulation (about 39%) induced by salt stress could come from PA degradation, suggesting that PAs might perform their functions through GABA formation under salt stress.     

Substrate preference of stress-activated phospholipase D in Chlamydomonas and its contribution to PA formation

In response to various environmental stress conditions, plants rapidly form the intracellular lipid second messenger phosphatidic acid (PA). It can be generated by two independent signalling pathways via phospholipase D (PLD) and via phospholipase C (PLC) in combination with diacylglycerol kinase (DGK). In the green alga Chlamydomonas, the phospholipid substrates for these pathways are characterized by specific fatty acid compositions. This allowed us to establish: (i) PLD's in vivo substrate preference; and (ii) PLD's contribution to PA formation during stress signalling. Accordingly, G-protein activation (1 micro m mastoparan), hyperosmotic stress (150 mm NaCl) and membrane depolarization (50 mm KCl) were used to stimulate PLD, as monitored by the accumulation in 5 min of its unique transphosphatidylation product phosphatidylbutanol (PBut). In each case, PBut's fatty acid composition specifically matched that of phosphatidylethanolamine (PE), identifying this lipid as PLD's favoured substrate. This conclusion was substantiated by analysing the molecular species by electrospray ionization-mass spectrometry (ESI-MS/MS), which revealed that PE and NaCl-induced PBut share a unique (18 : 1)2-structure. The fatty acid composition of PA was much more complex, reflecting the different contributions from the PLC/DGK and PLD pathways. During KCl-induced stress, the PA rise was largely accounted for by PLD activity. In contrast, PLD's contribution to hyperosmotic stress-induced PA was less, being approximately 63% of the total increase. This was because the PLC/DGK pathway was activated as well, resulting in phosphoinositide-specific fatty acids and molecular species in PA.     

Effect of ABA on the contents of proline, polyamines, and cytokinins in the common ice plants under salt stress

The effects of ABA treatment on the contents of proline, polyamines (PA), and cytokinins (CK) in the facultative halophyte the common ice plant (Mesembryanthemum crystallinum L.) subjected to salt stress were studied. Plants grown in the phytotron chamber on Jonson nutrient medium for 6 weeks were subjected to 6-day-long salinity by a single NaCl adding to medium. During first three days of salinity, half plants of each treatment were placed for 30 min on nutrient medium containing 0, 100, or 300 mM NaCl plus ABA in the final concentration of 1 μM. Salinity reduced biomass accumulation and water and chlorophyll contents in plants. This was accompanied by the increase in the levels of MDA, proline, and sodium ions. ABA treatment of salt-stressed plants favored biomass accumulation and photosynthetic pigment protection, reduced the intensity of oxidative stress and the level of NaCl-induced proline accumulation. ABA treatment increased the contents of putrescine (Put) and spermidine (Spd) in the leaves and roots of control plants (not subjected to salt stress), reduced the losses of Put in the leaves and roots and Spd in the roots in the presence of 100 mM NaCl, and suppressed cadaverine (Cad) accumulation in the roots in the presence of 300 mM NaCl. In the presence of NaCl, ABA reduced the contents of zeatin and zeatin riboside and increased the level of zeatin-O-glucoside in the roots and isopentenyladenosine and isopentenyladenine in the leaves. Thus, ABA protective action under salinity can be realized through the weakening of oxidative stress (a decrease in MDA content) and the regulation of PA, proline, and CK metabolism, which has a great significance in plant adaptation to injurious factors.     

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 abscisic acid on the contents of polyamines and proline in common bean plants under salt stress

The effects of ABA treatment on the contents of polyamines (PAs) and proline (Pro) in the glycophyte Phaseolus vulgaris L. during plant adaptation to salt stress were studied. Two-week-old common bean seedlings grown in the phytotron chamber on the Jonson nutrient medium were subjected to salinity for 6 days by one-time NaCl addition to medium up to final concentrations of 50 and 100 mM. During first three days of salinity, the root system was daily treated with ABA (1, 5, 10, or 50 μM) for 30 min. Salt stress (100 mM NaCl) elevated the level of endogenous ABA, increased the content of Pro 14-fold, reduced sharply the content of free PAs (putrescine, spermidine, spermine, and cadaverine), and the accumulation of 1,3-diaminopropan, a product of oxidation of high-molecular PAs. Common bean plant treatment with 1 μM ABA weakened the adverse effects of salt stress (100 mM NaCl), which was manifested in the maintenance of plant growth, stimulation of chlorophyll (a and b) and carotenoid accumulation, a stabilization of water and Na⁺ balance. Seedling treatment with ABA suppressed NaCl-induced Pro and intracellular ABA accumulation and restored the levels of putrescine and spermidine. The content of spermine in the leaves of plants subjected to salt stress and treated with ABA was approximately threefold higher than in control plants, whereas the content of cadaverine increased under similar conditions more than fivefold. Simultaneously, the contents of 1,3-diaminopropan and malondialdehyde as well as activity of superoxide dismutase were reduced, which indicates a weakening of oxidative stress, one of the possible causes of defensive ABA effects against salt stress. In addition, the suppression by exogenous ABA of Pro accumulation and stimulation of PA content under salt stress confirm indirectly our hypothesis that ABA is involved in the coordinated regulation of two biosynthetic pathways, Pro and PA formation, which use a common precursor, glutamate, and play an important protective role during stress in plants.     

Ameliorative role of salicylic acid and spermidine in the presence of excess salt in <Emphasis Type="Italic">Lycopersicon esculentum</Emphasis>

Salicylic acid (SA) and polyamines (PA) are widely used to overcome various abiotic stresses including salt (NaCl) stress in plants. In the present investigation, co-application efficacies of SA and PA on the salt stress (200 mM NaCl) were evaluated in Lycopersicon esculentum. After transplantation, at 10-d stage, seedlings were exposed to NaCl through soil and then allowed to grow till 30-d stage. At 31-d stage of growth, plants were sprayed with double distilled water (control) or spermidine (1.0 mM) and/or SA (10^–5 M). The salt stress significantly reduced the growth, gas-exchange parameters, but increased antioxidant enzymes and proline content in the leaves. Moreover, the loss caused by salt stress was successfully restored by the following treatment of spermidine and SA.     

Contribution of putrescine degradation to proline accumulation in soybean leaves under salinity

Proline accumulation was studied in the leaves of Glycine max (L.) Merr. subjected to salt stress in the presence of aminoguanidine (AG, a specific inhibitor of diamine oxidase, DAO) and exogenous putrescine (Put). Both DAO activity and proline content were increased while endogenous Put content was decreased in soybean leaves under 50 to 150 mM NaCl. There was a negative correlation between proline accumulation and endogenous Put content. The addition of AG during NaCl stress inhibited DAO activity, caused Put accumulation and a 15 to 20 % decrease in proline content. Application of 1 mM Put to NaCl solution markedly increased proline content. The promotive effect of Put application could be alleviated by the treatment with Put plus AG. Moreover an application of AG had no effect on proline accumulation in soybean seedlings grown under normal condition. These results indicate that the quantitative contribution of Put degradation to proline formation is 15 to 20 %.     

Influence of exogenous application of glycinebetaine on antioxidative system and growth of salt-stressed soybean seedlings (Glycine max L.)

Glycinebetaine is one of the most competitive compounds which play an important role in salt stress in plants. In this study, the enhanced salt tolerance in soybean (Glycine max L.) by exogenous application of glycinebetaine was evaluated. To improve salt tolerance at the seedling stage, GB was applied in four different concentrations (0, 5, 25 and 50 mM) as a pre-sowing seed treatment. Salinity stress in the form of a final concentration of 150 mM sodium chloride (NaCl) over a 15 day period drastically affected the plants as indicated by increased proline, MDA and Na⁺ content of soybean plants. In contrast, supplementation with 50 mM GB improved growth of soybean plants under NaCl as evidenced by a decrease in proline, MDA and Na⁺ content of soybean plants. Further analysis showed that treatments with GB, resulted in increasing of CAT and SOD activity of soybean seedlings in salt stress. We propose that the role of GB in increasing tolerance to salinity stress in soybean may result from either its antioxidant capacity by direct scavenging of H₂O₂ or its role in activating CAT activity which is mandatory in scavenging H₂O₂.     

Heme oxygenase up-regulation under salt stress protects nitrogen metabolism in nodules of soybean plants

The behaviour of enzymes involved in nitrogen metabolism, as well as oxidative stress generation and heme oxygenase gene and protein expression and activity, were analysed in soybean (Glycine max L.) nodules exposed to 50, 100 and 200 mM NaCl concentrations. A significant increase in lipid peroxidation was found with 100 and 200 mM salt treatments. Moreover, superoxide dismutase, catalase and peroxidase activities were decreased under 100 and 200 mM salt. Nitrogenase activity and leghemeoglobin content were diminished and ammonium content increased only under 200 mM NaCl. At 100 mM NaCl, glutamine synthetase (GS) and NADH-glutamate dehydrogenase (GDH) activities were similar to controls, whereas a significant increase (64%) in NADH-glutamate synthase (GOGAT) activity was observed. GS activity did not change at 200 mM salt treatment, but GOGAT and GDH significantly decreased (40 and 50%, respectively). When gene and protein expression of GS and GOGAT were analysed, it was found that they were positively correlated with enzyme activities. In addition, heme oxygenase (HO) activity, protein synthesis and gene expression were significantly increased under 100 mM salt treatment. Our data demonstrated that the up-regulation of HO, as part of antioxidant defence system, could be protecting the soybean nodule nitrogen fixation and assimilation under saline stress conditions.     

Identification and biochemical characterization of 20S proteasome in wheat roots under salt stress

In order to gain a better understanding of proteases state in wheat roots under salt stress, the roots of wheat (Triticum aestivum L., H6756) exposed to 300 mM NaCl were investigated at different days after treatment (DAT). The results showed that the content of soluble proteins decreased continuously, but the activity of total proteases increased gradually, and five root endopeptidase isoenzymes (RE1-5) were detected by natural gradient polyacrylamide gel electrophoresis (GPAGE) with supplement of gelatin as a substrate. Among five REs, RE1 was not only detected in salt-treated roots, but also obviously possessed the higher activity, whereas its protein amount decreased gradually during the salt treatment process. The biochemical characters of RE1 were examined afterwards. The results showed that its molecular mass was about 740 kDa and its optimal temperature was about 40°C. But the optimal pH was 5 to substrate gelatin or 7–8 to substrate casein. Its activity was obviously inhibited by 10 mM PMSF and 20 μM MG115, and RE1 was further confirmed as 20S proteasome by Western blotting. The results in this study suggested that 20S proteasome of wheat roots might be an important protease accumulated in roots in response to salt stress.     

Proline and its metabolism enzymes in cucumber cell cultures during acclimation to salinity

Proline is an important osmolyte appearing as the result of salt stress response of plants. In the present study, we measured the proline concentration, activities of pyrroline-5-carboxylate synthetase (P5CS), pyrroline-5-carboxylate reductase (P5CR), and proline dehydrogenase (PDH) key regulatory enzymes in the biosynthesis and degradation of proline in the acclimated (AC20) and the non-acclimated (NAC) cucumber cell suspension cultures subjected to moderate (150 mM NaCl; AC20–150, NAC-150, respectively) and severe (200 mM NaCl; AC20–200, NAC-200, respectively) salt stress. The data showed that salt stress brought about a linear increase in proline content in both types of cultures. However, in the acclimated culture proline accumulation was observed earlier, in third hour after stress. Only in the acclimated culture moderate and severe stresses up-regulated P5CS activity throughout the experiment, whereas the activity of P5CR grew in response to both NaCl concentrations only in 24th and 48th hour. The severe salt stress resulted in decrease in P5CR in NAC-200 cultures. In response to salt stress, both types of cell suspension cultures reacted with decline in PDH activity below the spectrophotometrically detected level. Cell cultures vigor correlated with salt concentration and time of exposure to the stress factor. Both NaCl concentrations caused linear decline in vigor of the non-acclimated culture up to 80–90 % at the end of the experiment, whereas in the acclimated culture significant decrease by about 30–40 % was reached in 24th hour after stress. The presented data suggest that acclimation to salt stress up-regulated proline synthesis enzyme activity and caused intensive accumulations of proline by inhibiting its oxidation.     

Sea fennel (<Emphasis Type="Italic">Crithmum maritimum</Emphasis> L.) under salinity conditions: a comparison of leaf and root antioxidant responses

The present study was carried out to compare the effect of NaCl on growth, cell membrane damage, and antioxidant defences in the halophyte Crithmum maritimum L. (sea fennel). Physiological and biochemical changes were investigated under control (0 mM NaCl) and saline conditions (100 and 300 mM NaCl). Biomass and growth of roots were more sensitive to NaCl than leaves. Roots were distinguished from leaves by increased electrolyte leakage and high malondialdehyde (MDA) concentration. Superoxide dismutase (SOD), catalase (CAT) and ascorbate peroxidase (APX) activities, ascorbic acid (AA) and glutathione (GSH) concentrations were lower in the roots than in the leaves of control plants. The different activity patterns of antioxidant enzymes in response to 100 and 300 mM NaCl indicated that leaves and roots reacted differently to salt stress. Leaf CAT, APX and glutathione reductase (GR) activities were lowest at 300 mM NaCl, but they were unaffected by 100 mM NaCl. Only SOD activity was reduced in the latter treatment. Root SOD activity was significantly decreased in response to 300 mM NaCl and root APX activity was significantly higher in plants treated with 100 and 300 mM compared to the controls. The other activities in roots were insensitive to salt. The concentration of AA decreased in leaves at 100 and 300 mM NaCl, and in roots at 300 mM NaCl, when compared to control plants. The concentrations of GSH in NaCl-treated leaves and roots were not significantly different from the controls. In both organs, AA and GSH were predominating in the total pool in ascorbic acid and glutathione, under control or saline conditions.     

Proteome analysis of soybean hypocotyl and root under salt stress

To evaluate the response of soybean to salt stress, the related changes in protein expression were investigated using the proteomic approach. Soybean plants were exposed to 0, 50, 100, and 200 mM NaCl. Especially at 200 mM, the length and fresh weight of the hypocotyl and root reduced under salt stress, while the proline content increased. Proteins from the hypocotyl and root treated with 100 mM NaCl were extracted and separated by two-dimensional polyacrylamide gel electrophoresis; 321 protein spots were detected. In response to salt stress, seven proteins were reproducibly found to be up- or down-regulated by two to sevenfold: late embryogenesis-abundant protein, beta-conglycinin, elicitor peptide three precursor, and basic/helix-loop-helix protein were up-regulated, while protease inhibitor, lectin, and stem 31-kDa glycoprotein precursor were down-regulated. These results indicate that salinity can change the expression level of some special proteins in the hypocotyl and root of soybean that may in turn play a role in the adaptation to saline conditions.     

野生大豆P5CS基因的克隆及对盐胁迫反应

逆境下植物大量积累脯氨酸是减轻胁迫伤害的一种自我保护机制。本研究应用同源克隆方法从NaCl处理的野生大豆中克隆获得一个脯氨酸合成酶(P5CS)基因,命名为GsP5CS。该基因核苷酸序列全长2.232 kb,含一个2148bp开放阅读框,编码715个氨基酸,包含有高等植物P5CS蛋白质的5个主要功能域,与菜豆PvP5CS1基因核苷酸序列相似性高达98.79%。Real Time PCR分析显示该基因受轻度盐胁迫诱导上调表达,根中表达高峰出现在200 mmol/L NaCl处理下,相对表达量为对照的5.83倍;叶片中表达高峰出现在300 mmol/L NaCl处理条件下,相对表达量为对照的12.78倍。并且该基因在根和叶片中的表达模式和脯氨酸含量的变化模式相同。上述结果说明,GsP5CS可能参与野生大豆脯氨酸合成。     

Role of phospholipase D in metabolic reactions of transgenic tobacco cax1 cells under the influence of salt stress

This work was aimed at investigating the primary reactions of plant cell metabolism in response to salt stress. It was found that the phospholipase D regulatory enzyme is activated in wild-type and transgenic cax1 tobacco plants during the early stages of the influence of salt stress. We have shown that a disturbance in the intracellular homeostasis of calcium ions and oppression of phospholipase D activity decrease the resistance of tobacco plants under the influence of salinity and also indicate the involvement of such systems in signaling during stress adaptation of plants.