Exogenous Sodium Nitroprusside Affects the Redox System of Wheat Roots Differentially Regulating the Activity of Antioxidant Enzymes under Short-Time Osmotic Stress.

Nitric oxide (NO) is a multifunctional signalling molecule involved in the regulation of plant ontogenesis and adaptation to different adverse environmental factors, in particular to osmotic stress. Understanding NO-induced plant protection is important for the improvement of plant stress tolerance and crop productivity under global climate changes. The root system is crucial for plant survival in a changeable environment. Damages that it experiences under water deficit conditions during the initial developmental periods seriously affect the viability of the plants. This work was devoted to the comparative analysis of the pretreatment of wheat seedlings through the root system with NO donor sodium nitroprusside (SNP) for 24 h on various parameters of redox homeostasis under exposure to osmotic stress (PEG 6000, 12%) over 0.5-24 h. The active and exhausted solutions of SNP, termed as (SNP/+NO) and (SNP/-NO), respectively, were used in this work at a concentration of 2 × 10-4 M. Using biochemistry and light microscopy methods, it has been revealed that osmotic stress caused oxidative damages and the disruption of membrane cell structures in wheat roots. PEG exposure increased the production of superoxide (O2•-), hydrogen peroxide (H2O2), malondialdehyde (MDA), and the levels of electrolyte leakage (EL) and lipid peroxidation (LPO). Stress treatment enhanced the activities of superoxide dismutase (SOD), ascorbate peroxidase (APX), catalase (CAT), the excretion of proline, and the rate of cell death and inhibited their division. Pretreatment with (SNP/+NO) decreased PEG-induced root damages by differently regulating the antioxidant enzymes under stress conditions. Thus, (SNP/+NO) pretreatment led to SOD, APX, and CAT inhibition during the first 4 h of stress and stimulated their activity after 24 h of PEG exposure when compared to SNP-untreated or (SNP/-NO)-pretreated and stress-subjected plants. Osmotic stress triggered the intense excretion of proline by roots into the external medium. Pretreatment with (SNP/+NO) in contrast with (SNP/-NO) additionally increased stress-induced proline excretion. Our results indicate that NO is able to mitigate the destructive effects of osmotic stress on the roots of wheat seedlings. However, the mechanisms of NO protective action may be different at certain periods of stress exposure.

24-Epibrassinolide Reduces Drought-Induced Oxidative Stress by Modulating the Antioxidant System and Respiration in Wheat Seedlings.

Brassinosteroids (BRs) represent a group of plant signaling molecules with a steroidal skeleton that play an essential role in plant adaptation to different environmental stresses, including drought. In this work, the effect of pretreatment with 0.4 µM 24-epibrassinolide (EBR) on the oxidant/antioxidant system in 4-day-old wheat seedlings (Triticum aestivum L.) was studied under moderate drought stress simulated by 12% polyethylene glycol 6000 (PEG). It was revealed that EBR-pretreatment had a protective effect on wheat plants as evidenced by the maintenance of their growth rate, as well as the reduction in lipid peroxidation and electrolyte leakage from plant tissues under drought conditions. This effect was likely due to the ability of EBR to reduce the stress-induced accumulation of reactive oxygen species (ROS) and modulate the activity of antioxidant enzymes. Meanwhile, EBR pretreatment enhanced proline accumulation and increased the barrier properties of the cell walls in seedlings by accelerating the lignin deposition. Moreover, the ability of EBR to prevent a drought-caused increase in the intensity of the total dark respiration and the capacity of alternative respiration contributes significantly to the antistress action of this hormone.

Nitric Oxide (NO) Improves Wheat Growth under Dehydration Conditions by Regulating Phytohormone Levels and Induction of the Expression of the TADHN Dehydrin Gene.

Nitric oxide (NO) is a universal signaling molecule with important regulatory functions in the plant's life cycle and adaptation to a wide spectrum of environmental stresses including drought. The effect of pre-sowing seed treatment with the donor of NO sodium nitroprusside (SNP, 200 µM) on wheat Triticum aestivum L. plants subjected to dehydration (PEG-8000, 12%) was investigated. SNP pretreatment stimulated germination and seedling growth in normal conditions and protected them under dehydration. These effects were confirmed by percentage of seed germination, changes in fresh and dry weight of 5-6-day-old seedlings, as well as by seedlings' linear dimensions, visual appearance, and mitotic index of the root apical meristem. Assessment of the transpiration intensity (TI) and relative water content (RWC) showed that SNP pretreatment helped to maintain the water status of seedlings subjected to dehydration stress. The data obtained by enzyme-linked immunosorbent assay (ELISA) suggested that the positive effects of SNP may be due to its influence on the phytohormonal system. SNP pretreatment induced an increase in the level of indolylacetic acid (IAA) and especially cytokinins (CK), while essential changes in ABA content were not detected. Water deficiency caused a substantial increase in ABA content and a decrease in the levels of CK and IAA. Pre-sowing SNP treatment decreased stress-induced fluctuations in the content of all studied phytohormones. Using reverse-transcription PCR (RT-PCR), we obtained data on the increase in expression of the TADHN dehydrin gene in SNP-pretreated seedlings under normal and, especially, under dehydration conditions. These findings may indicate the participation of dehydrins in NO-induced defense reactions in wheat plants under water stress. Furthermore, exogenous NO had a stabilizing effect on membrane cellular structures, as evidenced by the reduction of electrolyte leakage (EL) levels and malondialdehyde (MDA) content in dehydrated wheat seedlings under the influence of pre-sowing SNP treatment.

The Contribution of Hormonal Changes to the Protective Effect of Endophytic Bacterium Bacillus subtilis on Two Wheat Genotypes with Contrasting Drought Sensitivities under Osmotic Stress.

A comparative analysis was conducted to evaluate the effects of seed priming with endophytic bacterium Bacillus subtilis 10-4 (BS) on the hormonal system and cell wall tolerance (lipid peroxidation (LPO), electrolyte leakage (EL), and root lignin deposition) of two Triticum aestivum L. (wheat) varieties with contrasting drought sensitivities (Ekada 70-drought-tolerant (DT); Salavat Yulaev-drought-sensitive (DS)) under normal conditions and 12% polyethylene glycol-6000 (PEG)-induced osmotic stress. The results showed that under normal conditions, the growth stimulation in wheat plants by BS was attributed to changes in the hormonal balance, particularly an increase in endogenous indole-3-acetic acid (IAA) accumulation. However, under stress, a significant hormonal imbalance was observed in wheat seedlings, characterized by a pronounced accumulation of abscisic acid (ABA) and a decrease in the levels of IAA and cytokinins (CK). These effects were reflected in the inhibition of plant growth. BS exhibited a protective effect on stressed plants, as evidenced by a significantly lower amplitude of stress-induced changes in the hormonal system: maintaining the content of IAA at a level close to the control, reducing stress-induced ABA accumulation, and preventing CK depletion. These effects were further reflected in the normalization of growth parameters in dehydrated seedlings, as well as a decrease in leaf chlorophyll degradation, LPO, and EL, along with an increase in lignin deposition in the basal part of the roots in both genotypes. Overall, the findings demonstrate that BS, producing phytohormones, specifically IAA and ABA, had a more pronounced protective effect on DT plants, as evidenced by a smaller amplitude of stress-induced hormonal changes, higher leaf chlorophyll content, root lignin deposition, and lower cell membrane damage (LPO) and permeability (EL) compared to DS plants.

The Effects of Seed Pretreatment with Endophytic Bacteria Bacillus subtilis on the Water Balance of Spring and Winter Wheat Seedlings under Short-Time Water Deficit.

We investigated the effect of pre-sowing seed treatment with endophytic Bacillus subtilis 10-4 (B. subtilis) on spring and winter wheat (Triticum aestivum L.; cultivars Ekada-70 (Ek) and Scepter (Sc), respectively) growth and tolerance under 1-24 h of drought stress, modulated by 12% polyethylene glycol 6000 (PEG). The results showed that drought decreased transpiration intensity (TI), root relative water content (RWC), osmotic potential (Ψπ) of cell sap, and induced proline accumulation and electrolyte leakage (EL) in both wheat cultivars. It was revealed that Sc was more responsive to PEG and B. subtilis treatments than Ek. Under drought, Ek did not significantly change root length, shoot height, or dry biomass. The pretreatment of wheat plants with B. subtilis performed significantly better under drought conditions through the enhanced TI, RWC, and Ψπ of the cell sap in comparison with the plants treated with 12% PEG alone. B. subtilis also reduced stress-caused EL, especially in the Sc cultivar. Under water deficit wheat seedlings, pretreated with B. subtilis, have a higher proline accumulation in comparison to untreated stressed plants. Taken together, our results demonstrate the crucial role of endophytic B. subtilis in ameliorating the adverse effects of water stress on the water balance of both winter and spring wheat cultivars.

Multiple Ways of Nitric Oxide Production in Plants and Its Functional Activity under Abiotic Stress Conditions.

Nitric oxide (NO) is an endogenous signaling molecule that plays an important role in plant ontogenesis and responses to different stresses. The most widespread abiotic stress factors limiting significantly plant growth and crop yield are drought, salinity, hypo-, hyperthermia, and an excess of heavy metal (HM) ions. Data on the accumulation of endogenous NO under stress factors and on the alleviation of their negative effects under exogenous NO treatments indicate the perspectives of its practical application to improve stress resistance and plant productivity. This requires fundamental knowledge of the NO metabolism and the mechanisms of its biological action in plants. NO generation occurs in plants by two main alternative mechanisms: oxidative or reductive, in spontaneous or enzymatic reactions. NO participates in plant development by controlling the processes of seed germination, vegetative growth, morphogenesis, flower transition, fruit ripening, and senescence. Under stressful conditions, NO contributes to antioxidant protection, osmotic adjustment, normalization of water balance, regulation of cellular ion homeostasis, maintenance of photosynthetic reactions, and growth processes of plants. NO can exert regulative action by inducing posttranslational modifications (PTMs) of proteins changing the activity of different enzymes or transcriptional factors, modulating the expression of huge amounts of genes, including those related to stress tolerance. This review summarizes the current data concerning molecular mechanisms of NO production and its activity in plants during regulation of their life cycle and adaptation to drought, salinity, temperature stress, and HM ions.

Role of Endogenous Salicylic Acid as a Hormonal Intermediate in the Bacterial Endophyte Bacillus subtilis-Induced Protection of Wheat Genotypes Contrasting in Drought Susceptibility under Dehydration.

Endophytic Bacillus subtilis is a non-pathogenic beneficial bacterium which promotes plant growth and tolerance to abiotic stresses, including drought. However, the underlying physiological mechanisms are not well understood. In this study, the potential role that endogenous salicylic acid (SA) plays in regulating endophytic B. subtilis-mediated drought tolerance in wheat (Triticum aestivum L.) was examined. The study was conducted on genotypes with contrasting levels of intrinsic drought tolerance (drought-tolerant (DT) cv. Ekada70; drought-susceptible (DS) cv. Salavat Yulaev). It was revealed that B. subtilis 10-4 promoted endogenous SA accumulation and increased the relative level of transcripts of the PR-1 gene, a marker of the SA-dependent defense pathway, but two wheat cultivars responded differently, with the highest levels exhibited in DT wheat seedlings. These had a positive correlation with the ability of strain 10-4 to effectively protect DT wheat seedlings against drought injury by decreasing osmotic and oxidative damages (i.e., proline, water holding capacity (WHC), and malondialdehyde (MDA)). However, the use of the SA biosynthesis inhibitor 1-aminobenzotriazole prevented endogenous SA accumulation under normal conditions and the maintenance of its increased level under stress as well as abolished the effects of B. subtilis treatment. Particularly, the suppression of strain 10-4-induced effects on proline and WHC, which are both contributing factors to dehydration tolerance, was found. Moreover, the prevention of strain 10-4-induced wheat tolerance to the adverse impacts of drought, as judged by the degree of membrane lipid peroxidation (MDA) and plant growth (length, biomass), was revealed. Thus, these data provide an argument in favor of a key role of endogenous SA as a hormone intermediate in triggering the defense responses by B. subtilis 10-4, which also afford the foundation for the development of the bacterial-induced tolerance of these two different wheat genotypes under dehydration.

Seed priming with endophytic Bacillus subtilis strain-specifically improves growth of Phaseolus vulgaris plants under normal and salinity conditions and exerts anti-stress effect through induced lignin deposition in roots and decreased oxidative and osmotic damages.

Bacillus subtilis is one of the non-pathogenic beneficial bacteria that promote plant growth and stress tolerance. In the present study, we revealed that seed priming with endophytic B. subtilis (strains 10-4, 26D) improved Phaseolus vulgaris L. (common bean) seed germination and plant growth under both saline and non-saline conditions. 10-4 and 26D decreased oxidative and osmotic damage to the plant cells since bacterial inoculations reduced lipid peroxidation and proline accumulation in plants under salinity. 26D and especially 10-4 preserved different elevated levels of chlorophyll (Chl) a and Chl b in bean leaves under salinity, while carotenoids (Car) increased only by 10-4 and slightly decreased by 26D. Under normal conditions, 10-4 and 26D did not affect Chl a and Car concentrations, while Chl b decreased in the same plants. Under non-saline and especially saline conditions, 10-4 and 26D significantly increased lignin accumulation in plant roots and the highest lignin content along with better growth and oxidative damages reduction was observed after 10-4 inoculation under salinity, indicating a major role of B. subtilis-induced strengthening the root cell walls in the implementation protective effect of studied bacteria on plants. Therefore, B. subtilis 10-4 and 26D exerts protective effects on the growth of common bean plants under salinity by regulating plant defense mechanisms and the major role in tolerance development may contribute through the activation by B. subtilis lignin deposition in roots. The obtained data also indicates a strain-dependent efficiency of endophytic B. subtilis since strains 10-4 and 26D differently improved growth attributes and modulates cellular response reactions of the same common bean plants both under normal and salinity conditions, that generates interest for further investigations in this direction.

Seed Priming with Endophytic Bacillus subtilis Modulates Physiological Responses of Two Different Triticum aestivum L. Cultivars under Drought Stress.

The protective effects against drought stress of the endophytic bacterium Bacillus subtilis 10-4 were measured by studying the priming response in two wheat (Triticum aestivum L.)-Ekada70 (E70) and Salavat Yulaev (SY)-lines, tolerant and susceptible to drought, respectively. B. subtilis 10-4 improved germination and growth parameters under normal conditions in both cultivars with the most pronounced effect observed in cv. E70. Under drought conditions, B. subtilis 10-4 significantly ameliorated the negative impact of stress on germination and growth of cv. E70, but had no protective effect on cv. SY. B. subtilis 10-4 induced an increase in the levels of photosynthetic chlorophyll (Chl) a, Chl b, and carotenoids (Car) in the leaves of cv. E70, both under normal and drought conditions. In cv. SY plants, bacterial inoculation decreased the contents of Chl a, Chl b, and Car under normal conditions, but pigment content were almost recovered under drought stress. B. subtilis 10-4 increased water holding capacity (WHC) of cv. E70 (but did not affect this parameter in cv. SY) and prevented the stress-induced decline in WHC in both cultivars. Notably, B. subtilis 10-4 increased endogenous salicylic acid (SA) concentration in both cultivars, especially in cv. E70. Moreover, B. subtilis 10-4 reduced drought-induced endogenous SA accumulation, which was correlated with the influence of endophyte on growth, indicating a possible involvement of endogenous SA in the implementation of B. subtilis-mediated effects in both cultivars. Overall, B. subtilis 10-4 inoculation was found to increase drought tolerance in seedlings of both cultivars, as evidenced by decreased lipid peroxidation, proline content, and electrolyte leakage from tissues of wheat seedlings primed with B. subtilis 10-4 under drought conditions.

Methyl jasmonate alleviates water stress-induced damages by promoting dehydrins accumulation in wheat plants.

The investigation of dehydrins participation in MeJA-induced protection of wheat plants (Triticum aestivum L.) from drought stress was performed. The dehydration was designed by the presence of mannitol in increasing concentration (3, 4, and 5%) in the growth medium of wheat seedlings. Pre-treatment of 3-days-old seedlings with 0.1 µM MeJA reduced the level of drought-induced growth retardation as well as membrane structures lesions. Exogenous MeJA enhanced accumulation of the TADHN dehydrin transcripts and dehydrin proteins with Mw 28 and 55 kDa in wheat seedlings under normal conditions and additionally increased their expression during dehydration. The obtained data may indicate the dehydrins involvement in MeJA protective effect on wheat plants from the damages caused by water deficit.

Wheat germ agglutinin is involved in the protective action of 24-epibrassinolide on the roots of wheat seedlings under drought conditions.

The involvement of wheat germ agglutinin (WGA) in the protective action of 24-epibrassinolide (EBR) against drought stress was studied in the seedling roots of two wheat cultivars differing in drought tolerance. Under dehydration conditions, the contents of ABA and WGA were shown to change significantly in the roots of either drought-tolerant cultivar Omskaya 35 or drought-sensitive cultivar Salavat Yulaev. Meanwhile, accumulation of either ABA or WGA started earlier and was two times greater in plants of drought-tolerant cultivar. Since WGA is an excreted protein, it is not surprising that the level of lectin in the roots gradually decreased by the 7th day of treatment due to its exudation into root environment. Pre-sowing treatment with EBR contributed to additional accumulation of lectin as compared to the control variants of either cultivar, while the hormone treatment did not change ABA content. Meanwhile, under conditions of drought, EBR-pretreated seedlings were characterized by lower level of accumulation of ABA and WGA in the roots. EBR application was found to prevent drought-induced inhibition of cell division in the root apical meristem, while WGA excreted into the root environment may contribute significantly to the effect.

Involvement of dehydrins in 24-epibrassinolide-induced protection of wheat plants against drought stress.

In this study, we performed a comparative analysis of the physiological and biochemical parameters of wheat cultivars with contrasting drought resistance, drought-resistant Omskaya 35 (O-35) and less drought-resistant Salavat Yulaev (SYu), during 7-day germination under drought stress simulated by 5% mannitol. In addition, we evaluated the effectiveness of pre-sowing seed treatment with 0.4 µM 24-epibrassinolide (EBR) used to increase the resistance of plants of both cultivars to drought stress. It was revealed that mannitol has caused significant changes in the hormonal balance of the plants of both cultivars, associated with abscisic acid (ABA) accumulation and decrease in the contents of indoleacetic acid (IAA) and cytokinins (CKs). It should be noted that more dramatic changes in the content of phytohormones were characteristic for seedlings of SYu cultivar, which was reflected in a stronger growth inhibition of these plants. Pretreatment with EBR mitigated the negative effect of drought on the hormonal status and growth of seedlings during their germination. Furthermore, we found that drought caused accumulation of dehydrin (DHN) proteins, especially of low molecular weight DHNs, whose abundance was 2.5 times greater in O-35 cultivar than in SYu plants. EBR-pretreated plants of both cultivars were characterized by the additional accumulation of DHNs, indicating their involvement in the development of the EBR-induced wheat drought resistance. The use of fluridone allowed us to demonstrate ABA-dependent and ABA-independent pathways of regulation of low molecular mass dehydrins accumulation by EBR in wheat plants of both cultivars under drought conditions.

Exogenous methyl jasmonate regulates cytokinin content by modulating cytokinin oxidase activity in wheat seedlings under salinity.

The treatment of 4-days-old wheat seedlings with methyl jasmonate (MeJA) in concentration optimal for their growth (0.1 µM) resulted in a rapid transient almost two-fold increase in the level of cytokinins (CKs). MeJA-induced accumulation of CKs was due to inhibition of both cytokinin oxidase (CKX) (cytokinin oxidase/dehydrogenase, EC 1.5.99.12) gene expression and activity of this enzyme. Pretreatment of wheat seedlings with MeJA decreased the growth-retarding effect of sodium chloride salinity and accelerated growth recovery after withdrawal of NaCl from the incubation medium. We speculate that this protective effect of the hormone might be due to MeJA's ability to prevent the salinity-induced decline in CK concentration that was caused by inhibition of gene expression and activity of CKX in wheat seedlings. The data might indicate an important role for endogenous cytokinins in the implementation of growth-promoting and protective effects of exogenous MeJA application on wheat plants.