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.     

Inhibition of mitochondrial electron transport is the prime cause behind proline accumulation during mineral deficiency in Oryza sativa

The concentration of proline in shoots of rice (Oryza sativa) seedlings raised in distilled water was about 3.3 times higher than in the seedlings raised in modified B₅ medium. The shoots of seedlings raised in B₅ medium which was depleted of calcium, iron, magnesium or potassium had a higher concentration of proline than those grown in standard B₅ medium. The shoots of seedlings raised in distilled water with iron had a lower level of proline than those in distilled water. These results suggest that iron deficiency leads to high proline accumulation.The electron transport activity of mitochondria from shoots of etiolated seedlings raised in distilled water or iron-depleted B₅ medium was significantly lower than those from equivalent seedlings raised with an iron source. As suppression in mitochondrial electron transport leads to an increase in NADH/NAD⁺ ratio, we propose that the prime cause of the proline accumulation during iron deficiency is to readily maintain NADH/NAD⁺ ratio.     

Counteraction of Salinity Stress on Wheat Plants by Grain Soaking in Ascorbic Acid, Thiamin or Sodium Salicylate

The interactive effects of salinity stress (40, 80, 120 and 160 mM NaCl) and ascorbic acid (0.6 mM), thiamin (0.3 mM) or sodium salicylate (0.6 mM) were studied in wheat (Triticum aestivum L.). The contents of cellulose, lignin of either shoots or roots, pectin of root and soluble sugars of shoots were lowered with the rise of NaCl concentration. On the other hand, the contents of hemicellulose and soluble sugars of roots, starch and soluble proteins of shoots, proline of either shoots or roots, and amino acids of roots were raised. Also, increasing NaCl concentration in the culture media increased Na⁺ and Ca²⁺ accumulation and gradually lowered K⁺ and Mg²⁺ concentration in different organs of wheat plant. Grain soaking in ascorbic acid, thiamin or sodium salicylate could counteract the adverse effects of NaCl salinity on the seedlings of wheat plant by suppression of salt stress induced accumulation of proline.     

Implication of ABA and proline on cell membrane injury of water deficit stressed barley seedlings

The aim of this work was to examine the ability of ABA and proline to counteract the deleterious effect of water deficit stress on cell membrane injuries. Six-day-old seedlings of two barley genotypes (cv. Aramir, line R567) were treated with ABA (2·10⁻⁴ M) or proline (0.1 M) for 24 h, and then subjected to osmotic stress for 24h, by immersing their roots in polyethylene glycol (PEG 6000) solution of osmotic potential of −1.0 MPa and −1.5 MPa or by submerging the leaf pieces in PEG solution of osmotic potential of −1.6 MPa. Pretreatment of plants with ABA and proline caused an increase of free proline level in the leaves. Plants treated with ABA exhibited a lower membrane injury index under water stress conditions than those untreated even when no effect of this hormone on RWC in the leaves of stressed plants was observed. Pretreatment of plants with proline prevented to some extent membrane damage in leaves of the stressed seedlings, but only in the case when stress was imposed to roots. Improvement in water status of leaves was also observed in seedlings pretreatment with proline. The protective effect of both ABA and proline was more pronounced in line R567 that exhibited higher membrane injury under water deficit stress conditions.     

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.     

Accumulation of some nitrogen compounds in response to salt stress and their relationships with salt tolerance in rice (Oryza sativa L.) seedlings

The salt-induced accumulation of some nitrogen compounds (free amino acids, ammonium and urea) in shoots of eight rice cultivars differing in salt tolerance was investigated. Salt treatment (100 mM, 6 days) significantly increased the proline content of shoots but this appeared to be a reaction to stress damage and not associated with salt tolerance, because proline contents were higher in the more sensitive cultivars. Besides proline, some other free amino acids also accumulated leading to a significant increase in the total amino acid content of the stressed seedlings. High levels of free ammonium also accumulated under conditions of stress; this was highly correlated with the accumulation of Na⁺ in the shoots and negatively correlated with salt tolerance. The accumulation of ammonium was positively correlated with the accumulation of many free amino acids, and also associated with the production of urea in the stressed seedlings. Results from the present investigations suggest that an increase in the concentration of some free amino acids including proline, may be a result of the reassimilation of the stress-induced ammonium. A high capacity to assimilate ammonium may be an important factor in alleviating the consequence of stress because ammonium can be toxic at high concentrations.     

Effects of NaCl stress on germination, antioxidant responses, and proline content in two rice cultivars

We investigated the physiological and biochemical bases for salt tolerance in two rice (Oryza sativa L.) cultivars — relatively salt-tolerant ‘Dongjin’ and salt-sensitive ‘Kumnam’. Salinized hydroponic cultures were studied at the germination and seedling stages. NaCI inhibited germination more severely in ‘Kumnam’ than in ‘Dongjin’. Increasing the salt concentration also deterred growth to a larger extent in the former. Moreover, the leaves of ‘Kumnam’ exhibited greater increases in lipid peroxidation and Na⁺ accumulation than those of ‘Dongjin’ under stress. The activities of constitutive and salt-induced superoxide dismutase (SOD, EC 1.15.1.1) and ascorbate peroxidase (AP, EC 1.11.1.11) were also higher in ‘Kumnam’, while only catalase (CAT, EC 1.11.1.6) activity was slightly higher in stressed plants of ‘Dongjin’. The positive correlation between leaf proline levels and NaCI concentration was more evident in ‘Kumnam’. However, ‘Dongjin’ seeds, which had higher germinability in the presence of NaCI, also contained more proline. These results suggest that the higher salt tolerance in ‘Dongjin’ seedlings could be ascribed to their lower NaCI accumulations in the leaves. This presumably is due to reductions in the uptake or transport rates of saline ions to the shoots from the roots. Finally, we believe that the higher germination rate by ‘Dongjin’ is caused by its higher seed proline content.     

Role of Ca2+ in Drought Stress Signaling in Wheat Seedlings

Plants use complex signal transduction pathways to perceive and react to various biotic and/or abiotic stresses. As a consequence of this signaling, plants can modify their metabolism to adapt themselves to new conditions. One such change is the accumulation of proline in response to drought and salinity stresses. We have studied drought and salinity induced proline accumulation and the roles of Ca²⁺ (10 mM) and indoleacetic acid (IAA, 0.3 mM) in this response. Subjecting seedlings to both drought (6% polyethylene glycol, PEG) and salinity (150 mM NaCl) stress resulted in a dramatic increase in proline accumulation (7-fold higher than control level). However, the application of Ca²⁺ along with these stress factors had different effects. Unlike the salinity stress, Ca²⁺ prevented the drought induced proline accumulation indicating that these stress factors use distinct signaling pathways to induce similar responses. Experiments with IAA and EGTA (10 mM) supported this interpretation and suggested that Ca²⁺ and auxin participate in signaling mechanisms of drought-induced proline accumulation. Drought and salt stress-induced proline accumulation was compared on salt resistant (cv. Gerek 79) and salt sensitive (cv. Bezostaya) wheat varieties. Although proline level of the first was twofold lower than that of the second in control, relative proline accumulation was dramatically higher in the case of the salt resistant wheat variety under stress conditions.     

Polyamine, carbohydrate, and proline content changes during salt stress exposure of aspen (Populus tremula L.): involvement of oxidation and osmoregulation metabolism

Excess salt in the soil solution affects the plant either through osmotic or ionic effects. Poplar trees, as fast growing pioneer tree species, are thought to be potential suitable candidates for afforestation on saline soils. Osmotic and oxidative stress induced by salinity could be reduced by the production and accumulation of compatible solutes and osmoprotectants in the plant. In this respect, metabolites of this type could be interesting markers for the improvement of salt stress tolerance in breeding programmes. Results have shown that Populus tremula was able to cope with up to 150 mM NaCl without any effect on plant survival. During stress application, the endogenous level of malondialdehyde did not vary significantly between the treatments, indicating that the level of lipid peroxidation was similar in the control and in the stressed plants. However, proline, spermine, sucrose, mannitol, and raffinose levels increased temporarily or throughout the salt treatment. All these molecules are more or less closely related to antioxidant or osmoprotectant mechanisms during stress, suggesting a key role for these compatible solutes, osmoprotectants, and their metabolism for salt stress resistance. The accumulation of free proline, sucrose and mannitol, and the transitory increase in spermine level observable during low and high NaCl application must be considered as general salt stress reaction markers.     

Salinity-induced changes in two cultivars of Vigna radiata: responses of antioxidative and proline metabolism

The influence of increasing salinity stress on plant growth, antioxidant enzymes and proline metabolism in two cultivars of Vigna radiata L. (cv. Pusa Bold and cv. CO 4) was investigated. Salt stress was imposed on 30-days-old cultivars with four different concentrations of NaCl (0, 100, 200 and 300 mM). The roots and shoots of CO 4 showed greater reduction in fresh weight, dry weight and water content when compared to Pusa Bold with increasing salt stress. Under salinity stress, the roots and shoots of CO 4 exhibited higher Na⁺: K⁺ ratio than Pusa Bold. The activities of reactive oxygen species (ROS) scavenging enzymes and reduced glutathione (GSH) concentration were found to be higher in the leaves of Pusa Bold than in CO 4, whereas oxidized glutathione (GSSG) concentration was found to be higher in the leaves of CO 4 compared to those in Pusa Bold. Our studies on oxidative damage in two Vigna cultivars showed lower levels of lipid peroxidation and H₂O₂ concentration in Pusa Bold than in CO 4 under salt stress conditions. High accumulation of proline and glycine betaine under salt stress was also observed in Pusa Bold when compared to CO 4. The activities of proline biosynthetic enzymes were significantly high in Pusa Bold. However, under salinity stress, Pusa Bold showed a greater decline in proline dehydrogenase (ProDH) activity compared to CO 4. Our data in this investigation demonstrate that oxidative stress plays a major role in salt-stressed Vigna cultivars and Pusa Bold has efficient antioxidative characteristics which could provide better protection against oxidative damage in leaves under salt-stressed conditions.     

Growth, proline accumulation and ion content in sodium chloride-stressed callus of date palm

Summary Growth and physiological responses of date palm. Phoenix dactylifera L. cv. Barhee, callus to salinity stress were examined. Callus induced from shoot tips of offshoots was cultured on Murashige and Skoog medium supplemented with NaCl at concentrations ranging from 0 to 225 mM, in consective increments of 25 mM. Data obtained after 6 wk of exposure to salt have shown a significant increase in callus proliferation in response to 25 mM NaCl the lowest level tested, beyond which callus weight decreased. At 125 mM NaCl and higher, callus growth was nearly completely inhibited. Physiological studies on callus exposed to salt stress have shown an increase in proline accumulation in response to increased salinity. Proline accumulation was correlated to callus growth inhibition. Furthermore, increasing the concentration of NaCl in the culture medium generally resulted in a steady increase in Na⁺ and reduction in K⁺ concentrations. However, at 25 mM NaCl, the only level at which callus growth was significantly enhanced, an increase in K⁺ content was noted, in comparison to the NaCl free control. In response to increasing external NaCl level, the Na⁺/K⁺ ratio increased The Na⁺/K⁺ ratio was positively correlated to proline accumulation and hence callus growth inhibition. This study provides, an understanding of the response of date palm callus to salinity, which is important for future studies aimed at developing strategies for selecting and characterizing somaclonal variants tolerant to salt stress.     

Steady state proline levels in salt-shocked barley leaves

Excised barley (Hordeum vulgare var Larker) leaves were treated with salt solutions or wilted. After the treatment period, the leaves were allowed to recover in a 50 millimolar sucrose and 1 millimolar glutamate solution, and proline, Na⁺, and K⁺ were measured at intervals. Na⁺ and K⁺ concentrations stayed at a constant high level after the salt treatments, and proline increased to a steady state concentration in response. The relationship between the maximum rate of proline accumulation and the Na⁺ concentration reached in each experiment was linear. The final steady state proline concentration reached was also directly proportional to the Na⁺ concentration. For a given Na⁺ concentration in the leaves, the steady state proline level was greater when 410 millimolar NaCl was added to the leaves than when 205 millimolar NaCl was added. These results are consistent with proline acting as a compatible cytoplasmic solute, balancing an accumulation of salts outside of the cytoplasm.

In contrast to the proline levels in salt-shocked leaves, the concentrations in wilted leaves decreased to near control levels within 24 hours of relief of stress.

     

Stress‐induced changes in polyamine and tyramine levels can regulate proline accumulation in tomato leaf discs treated with sodium chloride

The effect of salt stress on proline (Pro) accumulation and its relationship with the changes occurring at the level of polyamine (PA) metabolism and tyramine were investigated in leaf discs of tomato (Lycopersicon esculentum). The rate of accumulation of Pro, PA and tyramine was higher in the salt-sensitive than in the salt-tolerant cultivar. In the salt-sensitive cultivar, Pro started to accumulate 4 h after the onset of the NaCl treatment, its maximum level being reached 27 h later. The lag phase was associated with a rapid decrease in putrescine (Put) and spermidine (Spd) and some increase in 1,3-diaminopropane (Dap), a product of Spd and/or spermine (Spm) oxidation. This was followed by an increase in agmatine (Agm), cadaverine (Cad), Spm and tyramine. α-DL-difluoromethylarginine (DFMA), an inhibitor of arginine decarboxylase (ADC, EC 4.1.1.19), induced a decrease in the Put level in both control and stressed discs, while α-DL-difluoromethylomithine (DFMO), an inhibitor of ornithine decarboxylase (ODC, EC 4.1.1.17), caused a decrease in Spd and Spm levels only in salinized discs. These data suggest that ADC is operating under both control and stress conditions, whereas ODC activity is promoted only in response to salt stress. DFMA also depressed the salt-induced Pro accumulation while DFMO did not inhibit this response. In salt-stressed leaf discs, the decrease in Spd level in response to methylglyoxal-bis-(guanylhydrazone) (MGBG) or cyclohexylammonium (CHA) treatment suggests that salt stress did not block SAM decarboxylase or Spd synthase activities. However, the increased level of Dap reflected a salt stress-promoted oxidation of PA. CHA and MGBG had no effect on Pro accumulation. Putrescine, Dap and especially tyramine supplied at low concentrations stimulated the Pro response which was, however, suppressed by application of Spm. Treatment with aminoguanidine, an inhibitor of diamine oxidases, also strongly inhibited Pro accumulation. These data suggest that salt-induced Pro accumulation in tomato leaf discs is closely related to changes in their PA metabolism, either via substrate-product relationships or regulatory effects at target(s) which remain to be characterized.     

Differential biochemical responses of wheat shoots and roots to nickel stress: antioxidative reactions and proline accumulation

Wheat (Triticum aestivum L. cv. ‘Zyta’) seedlings were treated with 10, 100 and 200 μM Ni. Tissue Ni accumulation, length, relative water content (RWC), proline and H₂O₂ concentrations as well as the activities of superoxide dismutase (SOD), catalase (CAT), ascorbate peroxidase (APX), guaiacol peroxidase (POD) and glutathione S-transferase (GST) were studied in the shoots and roots after 6 days of Ni exposure. Treatment with Ni, except for its lowest concentration, resulted in a significant reduction in wheat growth. In comparison to the shoots, the roots showed greater inhibition of elongation, which corresponded with higher accumulation of Ni in these organs. Both shoots and roots responded to Ni application with a decrease in RWC and enhancement in proline concentration. Greater dehydration of the shoot tissue was accompanied by more intense accumulation of proline. Treatment of the wheat seedlings with the highest concentration of Ni led to about 60% increase in H₂O₂ concentration in both studied organs. Apart from CAT, constitutive activities of antioxidative enzymes were much higher in the roots than in the shoots. Exposure of the seedlings to Ni resulted in SOD activity decline, which was more marked in the roots. While the shoots showed a substantial decrease (up to 30%) in CAT activity, in the roots the activity of this enzyme remained unchanged. After Ni application APX, POD and GST activities increased several-fold in the shoots, whereas in the roots they were not significantly altered. The results suggest that differential antioxidative responses of the shoots and roots of wheat seedlings to Ni stress might be related to diverse constitutive levels of antioxidant enzyme activities in both organs.     

Medicago truncatula stress associated protein 1 gene (MtSAP1) overexpression confers tolerance to abiotic stress and impacts proline accumulation in transgenic tobacco

Stress associated proteins (SAP) have been already reported to play a role in tolerance acquisition of some abiotic stresses. In the present study, the role of MtSAP1 (Medicago truncatula) in tolerance to temperature, osmotic and salt stresses has been studied in tobacco transgenic seedlings. Compared to wild type, MtSAP1 overexpressors were less affected in their growth and development under all tested stress conditions. These results confirm that MtSAP1 is involved in the response processes to various abiotic constraints. In parallel, we have performed studies on an eventual link between MtSAP1 overexpression and proline, a major player in stress response. In an interesting way, the results for the transgenic lines did not show any increase of proline content under osmotic and salt stress, contrary to the WT which usually accumulated proline in response to stress. These data strongly suggest that MtSAP1 is not involved in signaling pathway responsible for the proline accumulation in stress conditions. This could be due to the fact that the overexpression of MtSAP1 provides sufficient tolerance to seedlings to cope with stress without requiring the free proline action. Beyond that, the processes by which the MtSAP1 overexpression lead to the suppression of proline accumulation will be discussed in relation with data from our previous study involving nitric oxide.     

Hydrogen sulfide donor sodium hydrosulfide-improved heat tolerance in maize and involvement of proline

Hydrogen sulfide (H₂S) has long been considered as a phytotoxin, but nowadays as a cell signal molecule involved in growth, development, and the acquisition of stress tolerance in higher plants. In the present study, hydrogen sulfide donor, sodium hydrosulfide (NaHS), pretreatment markedly improved germination percentage of seeds and survival percentage of seedlings of maize under heat stress, and alleviated an increase in electrolyte leakage of roots, a decrease in tissue vitality and an accumulation of malondialdehyde (MDA) in coleoptiles of maize seedlings. In addition, pretreatment of NaHS could improve the activity of Δ¹-pyrroline-5-carboxylate synthetase (P5CS) and lower proline dehydrogenase (ProDH) activity, which in turn induced accumulation of endogenous proline in maize seedlings. Also, application of proline could enhance endogenous proline content, followed by mitigated accumulation of MDA and increased survival percentage of maize seedlings under heat stress. These results suggest that sodium hydrosulfide pretreatment could improve heat tolerance of maize and the acquisition of this heat tolerance may be involved in proline.