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.

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.

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.

Mechanisms of Plant Tolerance to RNA Viruses Induced by Plant-Growth-Promoting Microorganisms.

Plant viruses are globally responsible for the significant crop losses of economically important plants. All common approaches are not able to eradicate viral infection. Many non-conventional strategies are currently used to control viral infection, but unfortunately, they are not always effective. Therefore, it is necessary to search for efficient and eco-friendly measures to prevent viral diseases. Since the genomic material of 90% higher plant viruses consists of single-stranded RNA, the best way to target the viral genome is to use ribonucleases (RNase), which can be effective against any viral disease of plants. Here, we show the importance of the search for endophytes with protease and RNase activity combined with the capacity to prime antiviral plant defense responses for their protection against viruses. This review discusses the possible mechanisms used to suppress a viral attack as well as the use of local endophytic bacteria for antiviral control in crops.

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.

Expression profiles and hormonal regulation of tobacco expansin genes and their involvement in abiotic stress response.

Changes in the expression levels of tobacco expansin genes NtEXPA1, NtEXPA4, NtEXPA5, and NtEXPA6 were studied in different organs of tobacco (Nicotiana tabacum L.) as well as in response to phytohormone and stress treatments. It was shown that NtEXPA1, NtEXPA4 and NtEXPA5 transcripts were predominantly expressed in the shoot apices and young leaves, but almost absent in mature leaves and roots. The NtEXPA6 mRNA was found at high levels in calluses containing a large number of undifferentiated cells, but hardly detectable in the leaves of different ages and roots. In young leaves, expression levels of NtEXPA1, NtEXPA4 and NtEXPA5 genes were induced by cytokinins, auxins and gibberellins. Cytokinins and auxins were also found to increase NtEXPA6 transcripts in young leaves but to the much lower levels than the other expansin mRNAs. Expression analysis demonstrated that brassinosteroid phytohormones were able either to up-regulate or to down-regulate expression of different expansins in leaves of different ages. Furthermore, transcript levels of NtEXPA1, NtEXPA4, and NtEXPA5 genes were increased in response to NaCl, drought, cold, heat, and 10µM abscisic acid (ABA) treatments but reduced in response to more severe stresses, i.e. cadmium, freezing, and 100µM ABA. In contrast, no substantial changes were found in NtEXPA6 transcript level after all stress treatments. In addition, we examined the involvement of tobacco expansins in the regulation of abiotic stress tolerance by transgenic approaches. Transgenic tobacco plants with constitutive expression of NtEXPA1 and NtEXPA5 exhibited improved tolerance to salt stress: these plants showed higher growth indices after NaCl treatment and minimized water loss by reducing stomatal density. In contrast, NtEXPA4-silenced plants were characterized by a considerable growth reduction under salinity and enhanced water loss. Our findings indicate that expression levels of all studied tobacco expansins genes are modulated by plant hormones whereas NtEXPA1, NtEXPA4, and NtEXPA5 expansins may be involved in the regulation of stress tolerance in tobacco plants.

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.

Role of AINTEGUMENTA-like gene NtANTL in the regulation of tobacco organ growth.

The Nicotiana tabacum AINTEGUMENTA-like gene (NtANTL), encoding one of AP2/ERF transcription factors, is a putative ortholog of the AtANT gene from Arabidopsis thaliana. In wild-type tobacco plants, the NtANTL gene was expressed in the actively dividing young flowers, shoot apices, and calluses, while the level of its mRNA increased considerably after treatment with exogenous 6-benzylaminopurine, indoleacetic acid and 24-epibrassinolide. We found a positive correlation among the expression levels of NtANTL, cyclin NtCYCD3;1 and cyclin-dependent kinase NtCDKB1-1 genes, suggesting possible molecular links between AINTEGUMENTA and cell cycle regulators in tobacco plants. However, no correlation was observed between NtANTL, NtCYCD3;1 and NtCDKB1-1 expression levels in response to NaCl and ABA. These observations indicate that the transcription factor NtANTL was not involved in the regulation of the cellular response to salinity nor did it affect the expression of NtCYCD3;1 and NtCDKB1-1 when tobacco plants were exposed to salt stress and ABA. In addition, we generated transgenic tobacco plants with both up-regulated and down-regulated expression of the NtANTL gene. Constitutive expression of the NtANTL gene contributed to an increase in the size of leaves and corolla of transgenic plants. Transgenic plants with reduced expression of the NtANTL gene had smaller leaves, flowers and stems, but showed a compensatory increase in the cell size of leaves and flowers. The results show the significance of the NtANTL gene for the control of organ growth by both cell division and expansion in tobacco 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.

Cytokinin stimulates chloroplast transcription in detached barley leaves.

Chloroplasts are among the main targets of cytokinin action in the plant cell. We report here on the activation of transcription by cytokinin as detected by run-on assays with chloroplasts isolated from apical parts of first leaves detached from 9-d-old barley (Hordeum vulgare) seedlings and incubated for 3 h on a 2.2 x 10(-5) m solution of benzyladenine (BA). Northern-blot analysis also detected a BA-induced increase in the accumulation of chloroplast mRNAs. A prerequisite for BA activation of chloroplast transcription was preincubation of leaves for 24 h on water in the light, resulting in a decreased chloroplast transcription and a drastic accumulation of abscisic acid. Cytokinin enhanced the transcription of several chloroplast genes above the initial level measured before BA treatment, and in the case of rrn16 and petD even before preincubation. Cytokinin effects on basal (youngest), middle, and apical (oldest) segments of primary leaves detached from plants of different ages revealed an age dependence of chloroplast gene response to BA. BA-induced stimulation of transcription of rrn16, rrn23, rps4, rps16, rbcL, atpB, and ndhC required light during the period of preincubation and was further enhanced by light during the incubation on BA, whereas activation of transcription of trnEY, rps14, rpl16, matK, petD, and petLG depended on light during both periods. Our data reveal positive and differential effects of cytokinin on the transcription of chloroplast genes that were dependent on light and on the age (developmental stage) of cells and leaves.