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.

Endophytic Plant Growth-Promoting Bacterium Bacillus subtilis Reduces the Toxic Effect of Cadmium on Wheat Plants.

Heavy metal ions, in particular cadmium (Cd), have a negative impact on the growth and productivity of major crops, including wheat. The use of environmentally friendly approaches, in particular, bacteria that have a growth-stimulating and protective effect, can increase the resistance of plants. The effects of the pre-sowing seed treatment with the plant growth-promoting endophyte Bacillus subtilis 10-4 (BS) on cadmium acetate (Cd)-stressed Triticum aestivum L. (wheat) growth, photosynthetic pigments, oxidative stress parameters, roots' lignin content, and Cd ions accumulation in plants were analyzed. The results showed that the tested Cd-tolerant BS improved the ability of wheat seeds to germinate in the presence of different Cd concentrations (0, 0.1, 0.5, and 1 mM). In addition, the bacterial treatment significantly decreased the damaging effects of Cd stress (1 mM) on seedlings' linear dimensions (lengths of roots and shoots), biomass, as well as on the integrity and permeability of the cell walls (i.e., lipid peroxidation and electrolyte leakage) and resulted in reduced H2O2 generation. The pretreatment with BS prevented the Cd-induced degradation of the leaf photosynthetic pigments chlorophyll (Chl) a, Chl b, and carotenoids. Moreover, the bacterial treatment intensified the lignin deposition in the roots under normal and, especially, Cd stress conditions, thereby enhancing the barrier properties of the cell wall. This manifested in a reduced Cd ions accumulation in the roots and in the restriction of its translocation to the aboveground parts (shoots) of the bacterized plants under Cd stress in comparison with non-bacterized controls. Thus, the pre-sowing seed treatment with the endophyte BS may serve as an eco-friendly approach to improve wheat production in Cd-contaminated areas.

Effect of Seed Priming with Endophytic Bacillus subtilis on Some Physio-Biochemical Parameters of Two Wheat Varieties Exposed to Drought after Selective Herbicide Application.

Wheat plants are frequently exposed to combined herbicide and drought stress (HDS) which induces complex responses negatively, affects productivity, and is becoming more exacerbated with current climate change. In this work, we studied the influence of seed priming with endophytic bacteria Bacillus subtilis (strains 104 and 26D) on growth and tolerance of two wheat (Triticum aestivum L.) varieties (E70-drought tolerant; SY-drought susceptible) exposed to soil drought after application of selective herbicide Sekator® Turbo in pot experiments under controlled conditions; 17-day-old plants sprayed with herbicide and after 3 days were subjected to soil drought by stopping irrigating the plants for 7 days with subsequent resumption of normal irrigation (recovery). Additionally, the growth of tested strains (104, 26D) in the presence of different concentrations of herbicide Sekator® Turbo and drought (PEG-6000) were evaluated. It was established that both strains are herbicide and drought tolerant and capable to improve seed germination and early seedlings' growth under different herbicide and drought stress degrees. The results of pot experiments showed that HDS exposure declined growth (plant length, biomass), photosynthetic pigments (chlorophyll a and b), leaf area, and increased lipid peroxidation (LPO) and proline accumulation in plants, demonstrating higher damaging effects for SY variety. Strains 104 and 26D mitigated (in different levels) such negative impacts of HDS on growth of both varieties by increasing length of roots and shoots, biomass, photosynthetic pigments (chlorophyll a and b), and leaf area, reducing stress-caused LPO (i.e., malondialdehyde), and regulating proline biosynthesis, as well as contributing to a faster recovery of growth, photosynthetic pigments, and redox-status of plants in post-stress period in comparison with non-primed plants. These ultimately manifested in forming a better grain yield of both varieties primed with 104, 26D, and exposed to HDS. Thus, both strains 104 and 26D (which are herbicide and drought tolerant) may be used as seed priming agents to improve wheat HDS tolerance and grain yield; however, strain 104 more effectively protected plants of E70, while strain 26D-plants of SY. Further research should be focused on understanding the mechanisms that determine the strain and variety-specificity of endophytic symbiosis and the role of bacteria in the modulation of physiological states of primed plants under stress conditions, including HDS.

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 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.

The Effect of Endophytic Bacteria Bacillus subtilis and Salicylic Acid on Some Resistance and Quality Traits of Stored Solanum tuberosum L. Tubers Infected with Fusarium Dry Rot.

The effect of endophytic Bacillus subtilis (strains 10-4, 26D) and their compositions withsalicylic acid (SA) on some resistance and quality traits of stored potatoes infected with Fusariumdry rot were studied. The experiments were carried out on hydroponically grown Solanumtuberosum L. tubers that were infected before storage with Fusarium oxysporum and coated with B.subtilis 10-4, 26D with and without exogenous SA, and then stored for six months. It has been shownthat 10-4, 26D, 10-4 + SA, and 26D + SA reduced in different levels (up to 30-50%) the incidence ofF. oxysporum-caused dry rot (with the highest effect for 10-4 + SA). SA notably enhanced the positiveeffect of 10-4, while for 26D, such an effect was not observed. All of the tested treatments increasedamylase (AMY) and AMY inhibitors activity in infected tubers, while decreased Fusarium-inducedprotease activity (except in the case of 10-4 + SA, which promoted a slight increase) was revealed.10-4, 26D, and their compositions with SA decreased (in different degrees) the pathogen-causedlipid peroxidation, proline, and reducing sugars accumulation in potatoes after long-term storage.It was also discovered 10-4 and 26D, regardless of SA presence, decrease pathogen-inducedglycoalkaloids α-Solanine and α-Chaconine accumulation and preserved increased levels of starchand total dry matter in infected stored potatoes. The findings indicate endophytic B. subtilis and itscompositions with SA is a promising eco-friendly and bio-safe approach to cope with postharvestdecays of potato during long-term storage; however, when developing preparations-compositionsit should take into account the strain-dependent manner of B. subtilis action together with SA.

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.

Effect of constitutive expression of Arabidopsis CLAVATA3 on cell growth and possible role of cytokinins in leaf size control in transgenic tobacco plants.

We generated transgenic tobacco plants (Nicotiana tabacum L.) with overexpression of the Arabidopsis thaliana CLAVATA3 (CLV3) gene which is known to be a negative regulator of cell division. Surprisingly, most of the 35S::CLV3 transgenic plants showed no phenotypic differences with the wild type plants. However, there were considerable changes in the morphological parameters between 35S::CLV3 overexpressors and wild type plants. As expected, the number of meristematic cells in the shoot apical meristem was reduced in 35S::CLV3 plants as compared to the wild type plants. Moreover, overexpression of CLV3 exerted morphological changes not only to shoot apical meristem but also to leaves and flowers. Thus, transgenic plants were characterized by reduced number of epidermal and mesophyll cells as well as stomatal pores in mature leaves. However, there was a compensatory increase in leaf cell size of 35S::CLV3 plants that contributed to maintenance of organ size within the normal range. We observed that expression of cell expansion-promoted genes, expansin NtEXPA4 and endo-xyloglucan transferase NtEXGT, were elevated in mature leaves. In contrast, there was a decrease in the transcript level of the cell division-related AINTEGUMENTA-like (NtANTL) gene in 35S::CLV3 transgenic plants. In addition, we detected an increase in cytokinin level without any changes in the contents of IAA and ABA in 35S::CLV3 overexpressors. Interestingly, cytokinin treatment was shown to stimulate the expression of NtEXPA4 and NtEXGT genes in 35S::CLV3 transgenic plants. We propose that observed compensatory cell expansion in leaves of 35S::CLV3 transgenic plants may be due, at least in part, to a possible link between cytokinin signalling and cell expansion-related genes.

Effects of Bacillus subtilis on some physiological and biochemical parameters of Triticum aestivum L. (wheat) under salinity.

Endophytic strain Bacillus subtilis (B. subtilis) 10-4, producing indole-3-acetic acid (IAA) and siderofores but not active in phosphate solubilization, exerted a protective effect on Triticum aestivum L. (wheat) plant grown under salinity (2% NaCl) stress. Exposure to salt stress resulted in an essential increase of proline (Pro) and malondialdehyde (MDA) level in the seedlings. At the same time the seedlings inoculated with B. subtilis 10-4 were characterized by decreased level of stress-induced Pro and MDA accumulation. It was revealed that both B. subtilis 10-4 and salinity caused increase in the content of endogenous salicylic acid (SA) in wheat seedlings as compared to SA content in the control, while B. subtilis 10-4 suppressed stress-induced SA accumulation. Water storage capacity (WSC) in leaf tissues was increased and stress-induced hydrolysis of statolite starch in root cap cells of the germinal roots was reduced by B. subtilis 10-4. The obtained data indicated that the activation of the defense reactions induced by B. subtilis 10-4 induced defense reactions may be connected with their ability to decrease the level of stress-induced oxidative and osmotic stress in seedlings and with the increase of endogenous SA level that can make a significant contribution to the implementation of the protective effect of B. subtilis 10-4 and is manifested in the improvement of plant growth, WSC of leaves and slowing down of the process of statolite starch hydrolysis under salinity.

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.

Cytokinin oxidase is involved in the regulation of cytokinin content by 24-epibrassinolide in wheat seedlings.

Fast and stable 2-fold accumulation of cytokinins (CKs) was detected initially in roots and then in shoots of 4-day-old wheat (Triticum aestivum L.) seedlings in the course of their treatment with 0.4µM 24-epibrassinolide (EBR). Elevated cytokinin level has been maintained only in the presence of EBR, while the hormone removal has led to return of cytokinin concentration to the control level initially in the roots and then in the shoots. EBR-induced accumulation of cytokinins was accompanied by inhibition of both cytokinin oxidase (CKX) (cytokinin oxidase/dehydrogenase, EC 1.5.99.12) activity and expression of the gene coding for this enzyme, and on the contrary the decline in CKs level resulted in increase in these characteristics up to the control level in roots and then in shoots. Sharp accumulation of cytokinin O-glucosides has been discovered in response to EBR-treatment suggesting fast EBR-induced activation of production of cytokinins, which excessive amounts were transferred into the storage forms. The obtained data provide evidence for the involvement of EBR in regulation of cytokinin level in wheat seedlings.