WITHDRAWN: Effective symbiosis and activation of protective antioxidant systems for increasing soybean tolerance to drought

doi:10.21203/rs.3.rs-2942450/v1

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WITHDRAWN: Effective symbiosis and activation of protective antioxidant systems for increasing soybean tolerance to drought

https://doi.org/10.21203/rs.3.rs-2942450/v1

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Background and Aims

In the face of increasing drought associated with a warming climate, encouraging rhizobial symbioses represents a promising Nature-Based Solution for providing legume crops with ecological nitrogen and increasing drought resistance. In addition, to effectively resist drought, plants must adapt under stress and activate key systems for antioxidant protection.

The aim of the present study is to explore the role of protective antioxidant systems in the drought resistance of soybean, depending on the effectiveness of forming a symbiotic interaction with the nodula bacteria Bradyrhizobium japonicum.

Methods

The study uses microbiological, biochemical, physiological approaches and employs various symbiotic soybean systems based on Bradyrhizobiumstrains and Tn5 mutants, differing in activity and virulence.

Results

The effective symbiotic systems are able to activate the key antioxidant enzymes superoxide dismutase and catalase under prolonged drought, thus maintaining the prooxidant-antioxidant balance of plants and the functioning of the symbiotic relationship under stress conditions. The less effective and ineffective symbiotic systems are unable to provide the soybean plants with antioxidant protection due to the significant development of drought-related oxidative processes, as evidenced by excessive production of hydrogen peroxide and intensification of lipid peroxidation.

Conclusion

The formation of a tolerant soybean-rhizobial symbiosis is the result of the combined ability of both partners, i.e. the macro- and microsymbiont, to realize their adaptive potential and regulate redox homeostasis under effects of drought. This is achieved by activating key antioxidant enzyme systems, thus maintaining the prooxidant-antioxidant status of the symbiotic system.

oxidative processes

antioxidant enzymes

nodulation potential

nitrogen-fixing activity

drought

symbiosis

Drought is a major abiotic stressor that significantly affects the yield and quality of crops in many regions of the world, and can pose a serious threat to food security (FAO 2021). The situation has been exacerbated by global climate changes, accompanied by aridization of the air, frequent droughts and a lack of soil moisture (IPCC 2022). Therefore, the study of stress tolerance of cultivated plants has become a priority area of research around the world, with the aim of creating optimal solutions for reducing crop losses and maintaining the sustainable development of agriculture in the face of climate change. Research into sustainable and integrated water resources management is particularly important, especially in agriculture and plant food production, which are the largest consumers of water. Freshwater can be seen as the thread that ties all aspects of society together. It supports life on Earth and drives sustainable economic development. Healthy rivers, lakes, and wetlands not only provide drinking water and maintain valuable ecosystems, but also support agriculture and drought prevention, among others (UNESCO IHP IX, 2022).

Consequently, the most important challenge for both agricultural management and water management is how to increase the quantity and quality of water (W) and food production resources, while increasing biodiversity (B), ecosystem services (S) for society, and resilience (R) to the impact of prolonged drought periods for plants (benefits of WBSR) (Zalewski et al. 2020; Kiedrzyńska et al. 2021). Therefore, a priority area of research concerns the stress tolerance of cultivated plants; more specifically, the creation of optimal solutions for reducing crop losses and maintaining the sustainable development of agriculture in the face of climate change.

One of the most widely-grown and used oilseeds is soybean. Its uses range from human foods to precursor materials, through animal foods, industrial products and ingredients (Gaonkar and Rosentrater, 2019). Presently, soybean production occupies approximately 119 million ha worldwide, with a total annual production of 319 MMT (FAOSTAT 2013–17). Global production of soybean increased from 155.1 million metric tons in 1999 to 284 million metric tons in 2013 (www.soystats.com). Currently, the share of soybean in global oilseed production is around 55%, and over the last 10 years, its production has expanded at a rate of over 5% per year. Soybean has the maximum global production share of all oilseed crops (53%), with rapeseed, cotton, and peanut contributing 15%, 10% and 9%, respectively.

The legumes (Fabaceae) are capable of forming symbiotic relationships with nodule bacteria (Rhizobiaceae) that fix atmospheric nitrogen in the form of organic compounds. This fixation takes place during vital periods of plant growth and development, supporting the development of stable and environmentally-sustained crops (Wang et al. 2018; Khatun et al. 2021). Such biological fixation represents a natural source of nitrogen for plants that does not threaten the state of ecological processes in the environment (Raza et al. 2019; Sousa et al. 2022). Therefore, the development of effective leguminous-rhizobial symbiotic systems with active strains of nodule bacteria can play an important role in increasing the efficiency of environmental nitrogen fixation by plants while also building their tolerance to the effects of climate change.

The self-regulation processes aimed at eliminating the water imbalance in plants are of particular importance as they may help maintain the concentration gradient of water and water vapor through the soil – plant – atmosphere system (Sun et al. 2020). Water imbalance can arise due to an uneven distribution of precipitation and increase in air temperature during atmospheric and soil drought (Mamenko 2019). The reactions of plants to these factors are complex and depend on the depth and duration of stress, the stage of plant growth and the phase of development (Bandurska 2022). The loss of water initiates regulatory processes that influence gene expression and plant metabolism, thus shaping the adaptive potential of the plant in arid conditions and increasing drought resistance (Hura et al. 2022).

Biochemical protection systems play an important role in the adaptation of plants to adverse environmental factors, including drought. Among these, considerable attention has been paid to clarifying the role of pro-oxidant-antioxidant systems in the metabolism and formation of plant resistance under the action of stress factors (Laxa et al. 2019; Mishra 2021). Antioxidant systems are involved in the neutralization of reactive oxygen species (ROS), which can cause oxidative damage to membrane structures under conditions of high stress (Hasanuzzaman et al. 2020). It has also been demonstrated that ROS, especially hydrogen peroxide, can also act as signaling molecules that participate in the activation of defense systems under the effects of stress, such as the synthesis of antioxidant enzymes (Singh et al. 2019; Nyzhnyk et al. 2022). Such signaling influences plant growth and development processes under both optimal plant growing conditions and during biotic and abiotic stresses (Mhamdi and Van Breusegem 2019; Mamenko 2021). Hydrogen peroxide has also been found to indirectly influence the cascade of reactions regarding the movement of calcium ions and the activation of mitogen-activated protein kinases (MAPKs) (Slesak 2007; Mittler et al. 2011; Kots et al. 2019).

Many ROS receptors are found in cell membranes, where most of the starting enzyme systems are located; these have been found to be involved in the initiation of calcium, lipoxygenase, phosphatide-oxalate signaling systems (Mittler 2017). Therefore, there is no doubt that lipid peroxidation processes participate in the regulation of plant cell homeostasis, including inter alia the establishment of microbial-plant interactions (Mamenko and Kots 2020). The activation of lipid peroxidation is one of the first non-specific links in the general stress response and can initiate other defense mechanisms; the products of the lipid peroxidation process are considered as indicators and primary mediators of stress (Ayala et al. 2014). It has been demonstrated that the most informative such indicator is a shift in the balance towards pro-oxidants (Barrera et al. 2018).

The main initiator of free radical oxidation of membrane lipids is superoxide, which is generated in many spontaneous and enzymatic oxidation reactions, and the products of its secondary transformation can be singlet oxygen, hydroxyl radical, hydrogen peroxide, organic peroxides and their radicals (Mittler 2017). Therefore, superoxide dismutase (SOD), which catalyzes the formation of hydrogen peroxide and oxygen from superoxide anion radicals, is considered a key enzyme in the protection of living organisms from oxidative destruction (Raychauhuri and Deng 2000; Morgun et al. 2021). The effective functioning of SOD is largely determined by the functioning of other components of the defense system, in particular those that dispose of excessive amounts of hydrogen peroxide (Mamenko et al. 2018; Bossolani et al. 2022). An entire complex of enzymes and substrates is involved in the elimination of hydrogen peroxide. One of the key antioxidant enzymes that protects the aerobic cell from the toxic effect of hydrogen peroxide (H₂O₂) is catalase (CAT) (Gudeva et al. 2019). Research has shown that CAT activity is related to the activity of symbiotic nitrogen fixation in legume root nodules and depends on the efficiency of the legume-rhizobial symbiosis (Kots et al. 2019).

In legumes, rhizobial infection significantly modifies the metabolism of the host plant, thus favoring more optimal conditions for infection and nodulation, and this may be a prerequisite for the further effective functioning of the legume-rhizobial symbiosis (Hawkins and Oresnik 2021). The effectiveness of this symbiosis can be increased by selecting modern legumes varieties and using more effective new nodule bacteria strains, which will be capable of active nodulation and nitrogen fixation during the formation of symbiosis (Datta et al. 2015; Vorobey and Kots 2018).

Therefore, it is important to understand the role played by key components of drought-related antioxidant defense systems to clarify the adaptive responses demonstrated by symbioses of soybean with the nodula bacteria Bradyrhizobium japonicum with different activities and levels of virulence. Such information will be valuable for assessing the tolerance of the formed symbioses, as well as establishing the specificity of their adaptation potential in the face of climate change, while optimizing the efficiency of atmospheric molecular nitrogen fixation.

The aim of the research is to explore the role of protective antioxidant systems in the drought resistance of soybean, depending on the effectiveness of forming a symbiotic interaction with nodula bacteria (Bradyrhizobium japonicum).

The study tested the following hypotheses: the formation of protective reactions of soybean plants in symbiosis with B. japonicum under the influence of drought is associated with changes in the activity of the key antioxidant enzymes superoxide dismutase and catalase, which leads to the regulation of the content of hydrogen peroxide and the intensity of lipoperoxidation processes.

Research objects and the design of experiment

Soybean plants (Glycine max (L.) Merr.) of the Vasylkivska variety ‒ mid-early variety (Steppe, Forest Steppe, Polissya) were selected for the study, originators: Breeding and Genetic Institute – National Center for Seed Science and Varietal Research; National Scientific Center - Institute of Agriculture of the National Academy of Sciences; Institute of Plant Physiology and Genetics of the National Academy of Sciences of Ukraine, 2003.

Before sowing, soybean seeds were inoculated for an hour with a suspension of nodule bacteria Bradyrhizobium japonicum, and then sown in the substrate. A culture of slow-growing nodule bacteria was cultivated on a mannitol-yeast nutrient medium for nine days at 26–28°С (bacterial titer 10⁸ cells in 1 mL). The inoculation titer was 200–300 thousand rhizobia cells per seed.

Soybean-rhizobial symbioses (inoculation types) of different efficiency were created with the participation of nodule bacteria, which differ in activity and virulence. These were taken from the museum collection of microorganisms kept at the Institute of Plant Physiology and Genetics of the National Academy of Sciences of Ukraine (Table 1).

The soybean plants were planted in pots. Each of the five inoculation types (strains) (Fig. 1) was planted as six repetitions: three for the Control variant and three for the Dry-Restoration variant. All seeds were planted in a sterile substrate (sand) with the addition of Herligel nutrient mixture (0.25 standard nitrogen) and grown under natural light.

In the Control variant, optimal water conditions, i.e. 60% of the total moisture capacity (TMC), was maintained throughout the 19 days of the experiment. In the Dry-Restoration variant, water stress was induced by decreasing TMC to 30% for five days during the third true leaf stage and for seven days during the budding stage; following this, the water conditions were restored to the optimal level (60% TMC) in the flowering stage and maintained for seven days.

Soybean plant material and root nodules were sampled in all stages of experiments: the third true leaf (day 5), budding (day 12), and flowering (day 19).

Measurement of the intensity of lipid peroxidation (LPO)

The level of lipid peroxidation (LPO) was determined by reaction with thiobarbituric acid; the content of the final product, malondialdehyde (MDA) was determined using a UV-1900 scanning two-beam spectrophotometer (Shimadzu, Japan). To obtain the plant extract, samples of plant material (0.5 g) were homogenized with 3 mL of distilled water. Following which, 3 ml of 0.1% trichloroacetic acid (TCA) solution was added to the homogenate and homogenized a second time. Two samples of 2 mL each were taken from the obtained homogenate. One (control) was treated with 2 mL of 20% TCA, while the other (test) was treated with 2 mL of 0.5% thiobarbituric acid in a 20% solution of TCA. The samples were incubated for 30 min in a water bath, cooled and centrifuged for 10 min at 3000 rpm. The supernatant liquid was carefully taken with a syringe into test tubes and measured on a spectrophotometer at λ = 532 nm. The degree of non-specific absorption was calculated at λ = 600 nm. The results are presented in terms of the number of nmol of MDA per gram of dry weight, using the molar extinction coefficient of 1.55 × 105 cm^-1 · M^-1 (Heath and Paker 1968).

The mass of dry matter (DM) was determined by drying samples of above-ground and underground parts of plants to a constant value at a temperature of +105ºС.

Determination of the content of hydrogen peroxide (H₂O₂)

The H₂O₂ content was determined by the Sagisaka ferrothiocyanite method (Sagisaka 1976). Briefly, plant material was extraction (ratio 1˸3) with a cooled solution of 5% TCA. The supernatant was obtained by centrifugation at 14,000 rpm for 5 minutes (4ºС). The concentration of H₂O₂ was determined by a color reaction with potassium thiocyanate at a wavelength of 480 nm and calculated using a calibration curve constructed with known concentrations of H₂O₂. The results are presented in μmol per g of DM.

Extraction and determination of superoxide dismutase (SOD) activity

To obtain the enzyme extract, a weight of plant material (0.2 g) was ground in a mortar with 4 ml of 50 mM phosphate buffer (pH 7.5), which contained 2 mM EDTA, 1 mM PMSF, 5 mM β-mercaptoethanol and 1% polyvinylpyrrolidone. The homogenate was centrifuged at 10,000 rpm for 20 minutes at 4ºС. The activity of SOD in the supernatant (EC 1.15.1.1) was determined by its ability to inhibit the photochemical reduction of nitroblue tetrazolium (Raychauhuri and Deng 2000). The reaction mixture contained 50 mM phosphate buffer (pH 7.8), 13 mM methionine, 2 μM riboflavin, 63 μM p-nitroblue tetrazolium, 0.1 mM EDTA, and 100 μL of enzyme extract. The reaction proceeded for 15 min at a light intensity of 70 μmoL μmoL quanta/(m2 · s) illumination by fluorescent lamps with a power of 15 W. The optical density was measured at 560 nm. The results are presented in units of enzyme activity (act unit) per mg of protein in the supernatant.

The content of total soluble protein in the enzyme extract was determined according to Bradford (Bradford, 1976).

Extraction and determination of catalase activity (CAT)

To obtain the enzyme extract, a portion of the plant material was triturated (ratio 1˸ 2) with a cooled 0.5 M Tris-HCl buffer (pH 7.8) containing 5 mM β-mercaptoethanol and 0.1% polyvinylpyrrolidone. The homogenate was centrifuged at 10,000 rpm (4ºС) for 20 minutes. The supernatant was taken and assayed for CAT activity (EC 1.11.1.6.) by the development of a color reaction with ammonium molybdate; the concentration was measured at a wavelength of 410 nm according to Korolyuk (1998). The results are presented in units of enzyme activity (act unit) per mg of protein in the supernatant.

Evaluation of the effectiveness of soybean-rhizobial symbiosis

The effectiveness of each soybean-rhizobial symbiosis was evaluated by diagnosing the nodulation ability of rhizobia. Twenty typical plants of each experimental variant were taken and the roots were washed; the nodules were then separated and their mean number and mass per plant was counted, thereby determining the nodulation potential of the studied rhizobia.

In addition, the nitrogen-fixing activity of the soybean nodules was measured on an Agilent GC system 6850 gas chromatograph (USA) with a flame ionization detector (Hardy 1968). Gas separation was carried out on a column (Supelco Porapak N) at a thermostat temperature of 55°C and a detector temperature of 150°C. The carrier gas was helium (20 mL per one minute). The volume of the analyzed gas mixture sample was 1 cm³. Pure ethylene (Sigma-Aldrich, No. 536164, USA) was used as a standard. Total nitrogen fixation activity (TNFA) was expressed in molar units of ethylene formed (μmoL С₂Н₄) per plant in hour. The value of the specific nitrogen fixation activity (SNFA) was calculated per unit mass of nodules and expressed in molar units of ethylene formed per hour (μmoL С₂Н₄/g nodules·h).

Statistical analysis

The analysis was performed based on the inoculation type of soybean roots by Bradyrhizobium japonicum, as well as on the variant and day of the experiment. Descriptive statistics were calculated, including the activity of antioxidant enzymatic complexes, the number and mass of nodules on soybean roots and nitrogen fixation activity. Statistical analysis and box plot generation were performed using the STATISTICA ver. 13.3 software package (StatSoft 2011). The basic statistics are provided in the Supplementary Materials.

An unconstrained ordination using Principal Component Analysis (PCA) was performed to visualize the direction and magnitude of changes during experiment on a multidimensional space. The analysis for the two sets of variables considered: 1) antioxidant enzymatic complexes, expressed by SOD, MDA, CAT; and 2) effectiveness of soybean-rhizobial symbiosis, expressed by the number and mass of nodules on soybean roots. Multidimensional analysis and ordination plots were performed using the CANOCO 5 package (ter Braak and Šmilauer 2012). The points in the presented PCA ordination plots correspond to the centroids calculated from three repetitions performed in the experiment, for each studied case.

Prooxidant-antioxidant state of soybean-rhizobial symbiosis

H₂O₂ content

Higher levels of H₂O₂ were recorded during the twelve days of drought compared to controls in all studied cases (Fig. 2); however, only a slight difference between stressed and control plants was observed for strain PC-08 (24.0 μmol/g DW for stress and 27.3 μmol/g DW for control). After twelve days of dry conditions, the highest H₂O₂ content (41.7 μmol/g DW) was recorded for strain 604k, and the lowest (15.4 μmol/g DW) for Tn5 mutant 113.

H₂O₂content decreased in the post-stress period in all cases, but for Tn5 mutant 113, such a decrease was only weak. In the case of PC-08 and Tn5 mutant B1-20, H₂O₂ content returned to the level scored for controls in the post-stress period (Fig. 2). However, in the case of the other variants, H₂O₂ content was still much higher than recorded for control plants. Besides strain 634b, changes in H₂O₂ concentration after the restoration of water supply were also observed in control plants. However, the direction of such changes varied. An increase in H₂O₂ content was observed in two mutants (Tn5 mutant B1-20 and Tn5 mutant 113), but a decrease was scored in strains PC-08 and 604k.

MDA concentration

Two patterns of changes in MDA concentrations during the experiment were observed. The first pattern, observed in strains 634b, PC-08, and Tn5 mutant B1-20, is characterized by a similar level of MDA after five days of drought in stress plants compared to control (both variants did not exceed 150 nmoL/g DW), a significant increase in MDA after twelve days of drought (up to 200 nmoL/g DW), and a return to control levels during restoration (Fig. 3). The second pattern, observed for Tn5 mutant 113 and strain 604k, is characterized by significant differences in MDA concentration between stress and control plants throughout the experiment. In this pattern, the MDA content in the stressed plants was above 400 nmoL/g DW after five days of the experiment and increased even further after twelve days. The decrease in MDA during the restoration phase did not reach the levels scored for control variants (Fig. 3).

SOD activity

After five days of the experiment, no significant differences in SOD activity were observed between control plants and stressed plants in the strains 634b, PC-08, and Tn5 mutant B1-20 (Fig. 4). In these cases, SOD activity did not exceed 150 U/mg of protein·min. After twelve days of drought, SOD activity increased significantly for these inoculants, especially in the case of Tn5 mutant B1-20, which rose to a level of 255.4 U/mg protein·min. In these cases, the restoration of water supply returned the SOD activity to the level observed in control plants.

Different scenarios were observed for Tn5 mutant 113 and strain 604k. There were evident differences in SOD activity between control and stressed plants, even after five days of drought. Stressed plants scored two to three times higher results. Surprisingly, after the next seven days of drought, the level of SOD activity decreased, especially in the case of strain 604k (Fig. 4). In the restoration phase, such a trend continued for Tn5 mutant 113, but in 604k, there was a significant increase in SOD activity to 358.5 U/mg protein·min. Therefore, in these two cases, the level of SOD activity did not return to the level presented by control plants. During the entire experiment, an increase in SOD activity was only observed in control plants of strain 604k.

CAT activity

In all cases studied, either no significant differences in CAT activity were noted between stressed plants and control plants after five days, or only slight differences (Fig. 5). However, in two cases (Tn5 mutant B1-20 and strain 604k), the differences were more noticeable, with the results for the stressed variant being about 30% higher than those for the control plants. After twelve days of exposure to drought, a significant increase in CAT activity was noted in all cases, with particularly high scores (above 6 and 8 U/mg protein) in soybean plants inoculated with PC-08 (60% higher than controls), Tn5 mutant 113 (120%), and 604k (112%). In the other cases, the differences in CAT activity in favor of the stressed conditions were 35% for strain 634b and 45% for Tn5 mutant B1-20.

After water stress was removed, CAT activity for strains 634b, PC-08, and Tn5 mutant B1-20 returned to control levels (Fig. 5). However, for the other two variants (Tn5 mutant 113 and 604k), CAT activity did not return to the levels scored for control plants. At the end of the experiment, CAT activity was 50% higher than the control in Tn5 mutant 113, and 63% higher in strain 604k.

Combination of MDA, SOD and CAT results

In the ordination plots based on MDA, SOD, and CAT results, the first PCA axis corresponded to increasing values of all three parameters (Fig. 6). Strains 634b, PC-08, and Tn5 mutant B1-20 demonstrated effective restoration of metabolic processes: the plants subjected to twelve days of drought and then returned to optimal water conditions, exhibited similar positions on the PCA ordination diagram as control plants, along the first PCA axis (Fig. 6). Conversely, for the Tn5 mutant 113 and 604k symbiotic systems, metabolic processes were not restored to the control level, as revealed by the PCA analysis. In those cases, the direction of changes after the restoration phase was associated with the second PCA axis (Fig. 6).

Most of the analyzed symbiotic systems undergoing water stress exhibited a relatively more significant increase in CAT activity (consistent with the direction of the CAT vector) than SOD content during the five and twelve days of drought (Fig. 6).

Formation and functioning of soybean-rhizobial symbiosis

The number and the mass of nodules

In control variants, the number of nodules in strains 634b, PC-08, and mutants Tn5 B1-20 and 113 ranged from 10 to 20 throughout the experiment (Fig. 7). In these cases, stress variants (5 and 12 days of drought) showed values lower by 25-66%, and regeneration did not lead to a significant increase in the number of nodules. After restoring water conditions, the stress variants still showed lower nodule evaluations compared to the control. The lowest differences between control and stress-subjected plants were observed in the Tn5 B1-20 mutant. In contrast, in the case of strain 604k, a significant increase in the number of nodules was observed, especially in control plants, which had over 100 nodules at the end of the experiment. After five and twelve days of drought, the plants achieved a 31.2% and 40.4% increase in the number of nodules, respectively. Restoring water supply resulted in an increase in the number of nodules in the stress variant, but it still did not reach the level scored for control (it was lower by 51.3%).

An increasing mass of nodules was observed in all cases and in all variants of the experiment (Fig. 8). The smallest mass of nodules was recorded for the Tn5 113 mutant and the 604k strain, which did not exceed 0.3 g at the end of the experiment. Differences between stress and control variants were observed in practically all cases. Only slight differences or no differences between stressed and control plants were noted for the PC-08 strain and the 604k strain after five days of drought. After twelve days of drought in all cases, significant differences between stress and control variants were visible. The most effective growth of nodules after restoring water supply was observed in the PC-08 strain, where a small difference was observed in relation to control during the post-stress period.

Combination of the number and the mass of nodules

The PCA ordination revealed that nodulation patterns, based on a combination of the number and mass of nodules, were mainly explained by the first PCA axis, accounting for a large part of the variability (76%-96%); both parameters were found to increase in value (Fig. 9).

At the end of the experiment, all soybean-rhizobial symbioses in the control group exhibited higher values on PCA1 compared to the stressed variants (Fig. 9). Only strain 634b and Tn-5 mutant B1-20 sustained increases along the vector accounting for the mass of nodules in the control variants (Fig. 9), while other inoculation types showed different behavior in changing the position of control variants on the PCA diagram.

Among the stressed variants, only Tn-5 mutant B1-20 demonstrated a relatively higher importance of the number of nodules after twelve days of drought. After the restoration of water supply, changes according to PCA1 were observed in all cases (Fig. 9). In Tn-5 mutant B1-20, a reversal of change was observed in the importance of the mass of nodules.

Nitrogen fixation activity

Data indicates a significant inhibition of the average activity of the nitrogenase enzyme complex, as measured by TNFA in nodules, for stress variants compared to the control (Fig. 10). Strain 604k showed no total nitrogen fixation, while Tn5 mutant 113 showed an insignificant level of total nitrogen fixation (Fig. 10).

Different patterns were observed in SNFA for two cases: strain 634b and Tn5 mutant B1-20, where the average values for stressed variants were higher. In Tn5 mutant 113, a much lower level of nitrogen fixation was observed for SNFA (Fig. 10), and no specific nitrogen fixation was recorded for strain 604k.

Prooxidant-antioxidant state of soybean-rhizobial symbiosis

H₂O₂ content

Following exposure to external factors, including drought, plants undergo significant metabolic changes aimed at realizing their adaptive potential (Raza et al. 2019). However, these result in stress characterized by variation in genome expression, and the activation of a cascade of biochemical reactions resulting in further significant alterations in metabolic processes (Fernandez-Göbel et al. 2019). This is manifested by the level of LPO in cell membranes being altered, caused by excessive production of ROS (Guo et al. 2018); thus, disturbing the prooxidant-antioxidant balance in the plant cells.

H₂O₂ is known to be produced in plant reactions in response to environmental factors. It can act as a signaling molecule and induce the expression of "protective" genes, apparently by activating protein kinases and protein phosphorylation; they play important roles in the adaptation of the plant to stress (Slesak et al. 2007). In particular, the exposure of Arabidopsis cells in the presence of H₂O₂ was found to lead to changes in the expression of about 175 genes; among these, 113 encoded proteins with antioxidant functions or were associated with protective responses to stress, and the remainder encoded proteins with signaling functions (Davison et al. 2002).

The analysis showed that prolonged drought induces an increase in H₂O₂content compared to the control in all studied symbiotic soybean systems (Fig. 2). Among the effective symbiotic systems, a slight difference in H₂O₂production between control and stressed plants was recorded in soybeans with active РС 08 rhizobia. After resuming watering of the plants, there is a trend towards slowing down of H₂O₂ production and its level approaching the control level in symbiotic systems of soybean with the strain РС 08 and Tn5 mutant B1-20 (Fig. 2).

In the inefficient symbiotic system with the participation of strain 604k, a significant increase in H₂O₂ content was recorded under prolonged drought. After the stress was removed, this level only partially returned to control values. In contrast, no recovery of H₂O₂ content was recorded in the post-stress period for the less active Tn5 mutant 113.

Hence, less-effective and ineffective symbiotic systems were distinguished by the increased development of oxidizing processes during prolonged drought, and an inability to slow their development after stress. However, like in the effective symbiotic systems, the development of drought-induced oxidative processes decreased as optimal conditions for plant growth and development returned.

MDA concentration

ROS can exert an influence on plant tissues via the activation of LPO, which can cause damage to cell membranes (Mittler, 2017). In addition, ROS and lipid peroxidation products are known to have signaling functions (Noctor et al., 2018). Furthermore, the intensity of oxidative processes occurring under stress can influence the ability of plants to realize their adaptation potential (Singh et al., 2019; Mishra, 2021).

LPO is the primary reaction in the destruction of membrane lipoprotein complexes and the disruption of their transport functions; it also suppresses energy generation processes, which ultimately reduces cell vitality (Alché 2019). These processes also play key roles in the renewal and repair of functioning structures and lipoprotein membranes and increase the power and buffer capacity of the redox system, and thus the efficiency of enzymatic and non-enzymatic antioxidant protection. In addition, lipid peroxides and catalysts of peroxidase reactions can precisely regulate LPO reactions in membrane structures (Laxa et al. 2019).

Two patterns of change in MDA concentration were noted during the experiment. In effective symbiotic systems (strain 634b and РС 08, Tn5 mutant B1-20), similar dynamics of MDA were noted: a slight increase in its content in stress variants under moderate dehydration (day 5) compared to the control, followed by a significant increase during prolonged drought exposure (day 12 ). In the post-stress period, the content of MDA in effective symbiotic systems was restored to control levels (Fig. 3). This indicates a slowdown in the development of the lipid peroxidation process in these symbioses with the return of optimal growing conditions.

In the less-effective (Tn5 mutant 113) and ineffective (strain 604k) symbiotic systems, significant differences in MDA concentration between stressed and control plants were recorded during the experiment. This is evidenced by the significant accumulation of the product of lipid peroxidation in soybean nodules during the drought. During the recovery phase, the MDA content of soybean nodules in these symbioses did not reach the levels estimated for the control variants. This indicates a significant development of lipid peroxidation processes of cell membranes, induced by drought, in soybean with low-active and inactive rhizobia, which led to obvious metabolic disorders and the inability to restore its functioning to an optimal level in the post-stress period.

SOD activity

Under the influence of adverse factors, SOD activation is intended to protect plant cells and tissues from oxidative damage due to increased ROS production. However, SOD activity can increase or decrease in response to stress, depending on the intensity of the action of the stress factor (intensity and duration of the impact of stress), as well as on the adaptive capacity of the plant (Gill et al. 2015). Although the activation of latent forms of SOD or the synthesis of new enzyme molecules can occur under stress, resulting in increased activity, this activity decreases when a certain level of oxidative stress is reached (Saed-Moucheshi et al. 2021).

It was found that moderate drought did not induce significant changes in SOD activity in soybean nodules in effective symbiotic systems. This is evidenced by the absence of significant differences between control plants and plants that experienced stress. Prolonged drought leads to an increase in enzyme activity, which was especially evident in an effective symbiotic system involving the Tn5 mutant B1-20. Restoration of irrigation returned SOD activity to the level of control plants in all effective soybean-rhizobial symbioses (Fig. 4). As such, in the effective symbiotic systems of soybeans, the activation of SOD was recorded as a protective reaction in response to the long-term effect of drought, which ensured the neutralization of excessive ROS formation and contributed to the recovery of plant metabolism after stress.

In less-effective and ineffective symbiotic systems, visible differences in SOD activity between control and stressed plants were observed, even under moderate effects of drought on day 5. Surprisingly, with the strengthening of the effect of drought on day 12, a decrease in the dynamics of SOD activity was found, especially in the inactive symbiosis (strain 604k) (Fig. 4). This trend continued after the resumption of irrigation in the ineffective symbiosis with Tn5 mutant 113; however, a rapid increase in enzyme activity was observed after stress in the ineffective symbiosis with strain 604k (Fig. 4). In both symbiotic systems (Tn5 mutant 113 and strain 604k), nodule SOD activity did not return to the level of control plants in the post-stress period. This indicates that these symbiotic systems have a weak ability to neutralize the consequences of drought-induced oxidative stress.

Activity of CAT

In the cell, CAT is a SOD synergist that prevents the accumulation of hydrogen peroxide, which is a SOD inhibitor. In addition, in nodules, CAT activity reflects metabolic changes that occur in the cells of the root cortex when they are infected with rhizobia (Troitska et al. 2000). CAT is compartmentalized in special microbodies called peroxisomes. They have an oxidative type of metabolism, which is characterized by plasticity, i.e., the enzyme composition of peroxisomes can vary depending on organism, cell type, tissue type and external conditions (Rio et al. 2006). The synthesis of CAT in peroxisomes is induced by its substrate, and enough produced hydrogen peroxide is required for the manifestation of enzyme activity (Rio et al. 2006).

In all studied symbiotic systems under moderate drought, either no significant differences were observed, or only minor differences in CAT activity between stressed and control plants (Fig. 5). During long-term stress, a significant increase in CAT activity was recorded in all soybean symbiotic systems.

For effective symbiotic systems of soybean, an increase in the activity of CAT under prolonged exposure to drought and a restoration of the activity level to the control after exposure to stress was shown (Fig. 5). This testifies to the efficient operation of the catalase enzymatic complex for the disposal of excessive production of H₂O₂ under the effects of stress, which led to adaptive changes in metabolism and contributed to the rapid restoration of its functioning to an optimal level after the effects of stress.

In low-effective and ineffective soybean-rhizobial symbioses, CAT activity increased rapidly under prolonged exposure to drought and did not return to the levels estimated for control plants (Fig. 5). This proves a significant disruption of the CAT enzyme complex in these symbioses induced by drought, as well as their inability to mobilize their antioxidant properties after exposure to stress.

Combination of results of MDA, SOD and CAT

Analysis of the results shows that water deficit induced a relatively more significant increase in the activity of CAT than SOD during the drought. Efficient systems symbiotic systems demonstrated efficient recovery of metabolic processes: plants subjected to a 12 days of drought and then returned to optimal water conditions showed the same positions on the PCA ordination diagram. In less-effective and ineffective symbiotic systems, the level of development of prooxidant-antioxidant processes was not restored to the optimal level, as in the control, and the direction of changes after the recovery phase was related to the second axis of PCA (Fig. 6).

Formation and functioning of soybean-rhizobial symbiosis

The symbiosis of legumes with nodule bacteria is an effective biological nitrogen-fixing system which has great ecological significance for preserving and reproducing the quality of the environment. The symbiotic relationships between the plants and bacteria occur in root nodules, in which bacteria are isolated from the host cell by a symbiosome, a membrane that regulates the exchange between the symbionts (Andrews and Andrews 2017). The nitrogenase activity of the bacteroids, and therefore the availability of nitrogen in plants, is facilitated by the supply of carbon (Larrainzar et al. 2009). Therefore, there is a close relationship between carbon and nitrogen metabolism, and thus between bacteroid substances and those produced by the plant. The host plant provides the root nodules access to photoassimilates, which are a source of energy and carbon and are necessary for nitrogen fixation (Cerezini 2016; McCormick 2018).

Number and mass of nodules

During the growing season of plants from the stages of three true leaves to flowering, we recorded an increase in the mass of nodules on soybean roots in all symbiotic systems and variants of the experiment. However, according to the indicator of nodule mass, differences between the stressed and control variants were observed in almost all soybean-rhizobial symbioses, which was especially noted in long-term drought conditions.

Among effective symbiotic systems, the smallest differences in the number of nodules between control and stressed plants were observed in soybeans in symbiosis with Tn5 mutant B1-20. While the most efficient growth of nodule mass after restoration of water supply was observed in strain PC-08, where there was little difference in relation to the control in the post-stress period.

In the ineffective symbiotic system with the participation of strain 604k, a significant increase in the number of nodules is observed during the growing season, especially in control plants. However, in this symbiotic system, drought induces a significant decrease in the number of nodules on the roots, while restoration of water supply does not lead to an increase in their number to the control level. The smallest mass of nodules was recorded for soybean in symbiosis with the less-active Tn5 mutant 113 and the inactive strain 604k under drought conditions. This indicates that soybean in symbiosis with these rhizobia is not capable of fully realizing its nodulation potential in drought conditions.

Combination of number and mass of nodules

At the end of the experiment, all soybean-rhizobial symbioses of the control group showed higher PCA1 values compared to the stressed variants (Fig. 9). After the restoration of water supply, changes according to PCA1 were observed in all cases (Fig. 9). Among the investigated soybean-rhizobial symbioses, only soybeans in symbiosis with strain 634b and Tn5 mutant B1-20 showed an increase along the vector, taking into account the mass of nodules in the control variants (Fig. 9).

Nitrogen fixation activity

In effective symbiotic systems of soybean, suppression of nitrogen-fixing activity by soybean nodules during drought and partial restoration of its activity after stress was noted. However, the effective symbiotic systems (strain 634b and Tn5 mutant B1-20) demonstrated an increase in specific nitrogen fixation under the effects of drought, and a return to control values in post-stress period. These findings confirm the efficiency of nitrogen fixation per unit mass of nodules and prove that in the effective symbioses, the symbiotic apparatus was maintained under the influence of drought.

While nitrogen fixation was not recorded in soybean inoculated with the inactive strain (604k), significant inhibition was detected while in those infected with Tn5 mutant 113 under the effects of drought and after watering was resumed.

The formation of effective drought tolerance in symbiotic soybean systems is accompanied by the preservation of the optimal prooxidant-antioxidant balance by activating key antioxidant enzymes, viz. superoxide dismutase and catalase, and regulating the development of oxidative processes. This helps maintain redox homeostasis and improve the effectiveness of the soybean ‒ Bradyrhizobium japonicum symbiosis in drought conditions. Ineffective and less-effective soybean symbiotic systems are unable to fully implement antioxidant protection systems during water stress due to the excessive production of hydrogen peroxide and intensification of lipoperoxidation processes. Therefore, in soybean-rhizobial symbioses, a key role in the formation of drought tolerance is played by the coordinated activity of the macro- and microsymbionts, which allows them to realize their adaptive potential and regulate redox homeostasis during water stress by activating key antioxidant enzyme systems. The presented approach demonstrates that the use of active virulent Bradyrhizobium strains can play a key role in increasing the implementation of protective and nitrogen-fixing properties in soybean under drought conditions.

Bradyrhizobium japonicum – B. japonicum; reactive oxygen species – ROS; lipid peroxidation – LPO; malondialdehyde – MDA; hydrogen peroxide – H₂O₂; trichloroacetic acid – TCA; superoxide dismutase – SOD; catalase – CAT; Total nitrogen fixation activity – TNFA; specific nitrogen fixation activity – SNFA; total moisture capacity – TMC; dry matter – DM

Acknowledgments

The research was carried out within the framework of tasks under the target program of fundamental and applied research of the National Academy of Sciences of Ukraine and as part of an internship funded by the Polish Phytopathological Society, as well as under the Program in support of Displaced Ukrainian Scholars organized jointly by the Polish Academy of Sciences and the National Academy of Sciences of the United States, and also under the statute research of the European Regional Centre for Ecohydrology of the Polish Academy of Sciences under the auspices of UNESCO.

Funding

The authors declare that no funds, grants, or other support were received during the preparation of this manuscript.

Competing interests

The authors have no relevant financial or non-financial interests to disclose.

Author Contributions

All authors contributed to the study conception and design. Material preparation, data collection and analysis were performed by [Tetiana Nyzhnyk], [Marcin Kiedrzyński] and [Edyta Kiedrzyńska]. The first draft of the manuscript was written by [Tetiana Nyzhnyk] and all authors commented on previous versions of the manuscript. Supervision [Sergii Kots] and [Maciej Zalewski]. All authors read and approved the final manuscript.

Data availability

The datasets generated during and/or analyzed during the current study are available from the corresponding author on reason able request.

Conflict of interest

Authors declare that they have no known conflict of interest.

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Table 1

Symbiotic characteristics of the Bradyrhizobium japonicum used to create soybean-rhizobial symbioses with different degrees of effectiveness
Bradyrhizobium japonicum	Symbiotic characteristic	Selection	Reference
Effective symbiotic systems Glycine max ‒ Bradyrhizobium japonicum
Strain 634b	Active, virulent	Isolated from soybean nodules by analytical selection	Novikova et al. (1984)
Strain РС-08	Active, virulent	Isolated from soybean nodules by analytical selection	Vorobey et al. (2013)
Tn5-mutant В1–20	Active, virulent	Created by the method of transposon mutagenesis with the participation of Esherichia coli S17-1 with the plasmid pSUP5011, which contains the Tn5 transposon (pSUP5011::Tn5mob)	Kots et al. (2018)
Less-effective symbiotic systems Glycine max ‒ Bradyrhizobium japonicum
Tn5-mutant 113	Less-active, virulent	Created from the original strain 634b by transposon mutagenesis at the Institute of Plant Physiology and Genetics of the National Academy of Sciences of Ukraine	Kots (2019)
Ineffective symbiotic systems Glycine max ‒ Bradyrhizobium japonicum
Strain 604k	Inactive, highly virulent	A mutant that has lost the ability for effective symbiosis; it was isolated from the nodule when strain 604k was passed through soy plants in a high radiation zone - the "cesium spot"	Tolkachov et al. (1995)

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WITHDRAWN: Effective symbiosis and activation of protective antioxidant systems for increasing soybean tolerance to drought

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