Improved drought tolerance in soybean by protein elicitor AMEP412 induced ROS accumulation and scavenging

Abstract Drought stress is a major limiting factor for soybean production. In this study, protein elicitor AMEP412 was applied on soybean seedlings to promote drought tolerance, and its correlation with alterations in the reactive oxygen species (ROS) in leaves was studied. The plants of soybean cv. Suinong 26 were sprayed with AMEP412 at the V1 stage and then treated with drought stress and rehydration. The alterations in phenotypes, physiological indices, ROS levels and antioxidant system as well as lipid peroxidation were monitored. The results showed that spraying AMEP412 significantly promoted the drought tolerance of soybean seedlings. Soybean seedlings pre-treated with AMEP412 performed better in phenotypes and physiological indexes, like leaf relative water content (RWC), leaf relative conductivity (RC) and osmolytes (free proline, soluble sugar and protein). In addition, the in situ and quantification detection of ROS revealed that AMEP412 pretreatment led to higher accumulation of ROS at the early stage and faster scavenging of ROS at the late stage. Moreover, AMEP412 pretreatment increased the activity of superoxide dismutase (SOD), guaiacol peroxidase (POD), catalase (CAT), ascorbate peroxidase (APX), monodehydroascorbate reductase (MDHAR), dehydroascorbate reductase (DHAR), glutathione reductase (GR), glutathione peroxidase (GPX), and the levels of ascorbate (AsA) and glutathione (GSH), but decreased the level of malondialdehyde (MDA) as compared with only drought treatment. Overall, the results indicated that AMEP412 was effective in alleviating the adverse effects of drought stress, which was partially attributable to the ROS accumulation and scavenging triggered by AMEP412.


Introduction
As a major agricultural crop, soybean (Glycine max) provides large amounts of vegetable protein and edible oil for humans and animals. However, the growth and yield of soybean are highly affected by drought stress [1]. Many references have reported the yield reduction caused by drought stress [2][3][4]. Thus there is a great need of applicable means to improve the drought tolerance of soybean.
As a kind of eco-friendly exogenous bio-stimulants, protein elicitors could induce plant biotic and abiotic stress resistance via signal recognition, signal transduction and defense gene regulation [5][6][7]. Many protein elicitors have been used to improve the drought resistance of plants [8][9][10][11]. AMEP412 is a newly identified protein elicitor from Bacillus subtilis, which could trigger plant defense response and promote plant resistance [12]. In our previous field application, AMEP412 treated soybean plants showed obvious drought tolerance, and resulted in less loss of the yield (unpublished data). However, the underlying mechanism still needed investigation.
Reactive oxygen species (ROS) are an unavoidable chemical entity of aerobic life [13]. Under normal conditions, ROS are continuously produced in plants at a relatively low level. When plants are exposed to environmental stress, the level of ROS increases significantly to activate stress related signaling pathways. However, further accumulation of ROS will result in oxidative damage and even programmed cell death [14][15][16][17]. Both drought stress and protein elicitors could trigger ROS production [18,19], indicating that the crosstalk between the two different external stimulants at ROS level might play a key role in the drought resistance induced by protein elicitors.
In this study, we performed AMEP412 treatment on soybean at the seedling stage, followed by the drought stress and rehydration. The effects of AMEP412 application on phenotypes, physiological indexes, ROS production and scavenging were monitored, and the influences of the altered ROS levels on antioxidant and lipid peroxidation system were also investigated. Our goal was to explore the soybean drought tolerance improved by AMEP412 at ROS level, and to provide experience for field application.

Plant materials and treatments
A pot experiment was conducted in the greenhouse at Heilongjiang Bayi Agricultural University, Daqing, Heilongjiang Province, China (45°50´N, 124°15´E) in the summer of 2021. The soybean cultivar Suinong 26 was used in the experiment. Soybean seeds were planted in plastic pots (7.5 cm in height and 9.3 cm in diameter) containing 0.4 kg soil, and six seeds were planted per pot. After one week, they were thinned to four seedlings of uniform size per pot. At the V1 stage, combinations of four different treatments were initiated: (i) water control (CK); (ii) drought stress (DS); (iii) water control treated with AMEP412 (ACK); (iv) drought stress pre-treated with AMEP412 (ADS). AMEP412 treatment was executed by spraying 100 μg/mL purified protein [12] on leaves (treatments iii and iv), whereas the others were sprayed with distilled water (treatments i and ii). The spray treatment was done only once, with 2 mL of protein solution or water per pot. On the same day, the drought treatments were set up by withholding water for 5 days and, subsequently, resupplying water at 10% (w/w) for another 5 days for the stressed plants to recover. In parallel, the control pots were watered daily at 10% (w/w) for 10 days. At the 1st, 3rd and 5th days of drought (DAD), and the 1st, 3rd and 5th days after resupplying water (DRW), leaf samples of each treatment were collected in triplicate for direct determination, or frozen in liquid nitrogen and stored at −80 °C for extraction.

Phenotype and physiological indexes
The phenotypes of the soybean plants in each treatment were recorded under control, drought stressed conditions and water resupplied conditions. The leaf relative water content (RWC) was calculated following Smart and Bingham's method [20]. Once the leaf fresh weight (LFW) was determined, the samples were soaked in distilled water for 24 h. Then the leaf turgid weight (LTW) of each sample was recorded. To determine the leaf dry weight (LDW), the leaf tissues were oven-dried at 70 °C for 72 h before weight was recorded. Finally, the RWC was calculated based on the following equation: RWC (%) = [(LFW-LDW)/ (LTW-LDW)] × 100.
The leaf relative conductivity (RC) was measured according to the method of Blum and Ebercon [21] with modifications. Leaves were excised and immersed in distilled water in test tubes overnight at room temperature. The conductivities of the water before boiling (R 1 ) and after boiling (R 2 ) were determined, and the electrolyte leakage in leaves was calculated based on the formula: RC (%) = (R 1 /R 2 )×100.
Proline content was measured according to a method previously published by Bates et al. [22]. Free proline was extracted from 0.5 g leaf sample with a 5 mL extraction solution of 3% aqueous sulfosalicylic acid. The homogenate was centrifuged at 12 000×g for 15 min. Then 2 mL of supernatant was incubated with 2 mL of glacial acetic acid and 2 mL acid ninhydrin at 100 °C for 1 h. After cooling the samples at room temperature, 5 mL toluene was added and mixed by vortexing. The absorbance was recorded at 520 nm.
The soluble sugar content was determined by spectrophotometer at 640 nm using an anthrone reagent [23], and the calibration curve was prepared using glucose as the standard. The soluble protein content was determined by the Brilliant Blue G-250 regent [24], using bovine serum albumin (BSA) as a standard.

In situ detection of ROS
Accumulation of hydrogen peroxide was detected by a peroxidase-dependent in situ histochemical staining procedure using 3,3-diaminobenzidine (DAB) [25] and superoxide ion using a superoxide-dependent reduction of nitro blue tetrazolium (NBT) [26]. Leaves were cut and then vacuum-infiltrated with 1 mg/mL DAB (pH 3.8) or 1 mg/mL NBT for 2 h. The treated leaves were incubated for more than 24 h in 70% ethanol and 5% glycerol to eliminate chlorophyll, observed for DAB and NBT deposits, and photographed.

Quantification of ROS production
The production of H 2 O 2 was determined using the method described by Tiedemann [27]. Leaf squares were washed using distilled water and incubated in 2 mL of a reagent mixture consisting of 50 mmol/L phosphate buffer pH 7.0, 0.05% guaiacol and 2500 U/ mL peroxidase (EC 1.11.1.7, Sigma Aldrich, P8375) for 2 h at room temperature in the dark. The release of H 2 O 2 was followed by measurement of absorbance at 450 nm.
The O 2 • generation was quantified based on NBT reduction activity, as described by El Hadrami et al. [28]. Leaf squares were washed using distilled water and immersed in 10 mmol/L potassium phosphate buffer, pH 7.8, containing 0.05% NBT and 10 mmol/L NaN 3 for 1 h. This mixture was heated at 85 °C for 15 min and then cooled quickly. The reduction activity of the soybean leaf was followed by measuring the absorbance at 580 nm.

Measurement of SOD, POD and CAT activities
Frozen leaves (0.5 g) were ground to a powder in liquid nitrogen and homogenized with 5 mL of 50 mmol/L sodium phosphate buffer (pH 7.8 for SOD and POD, and pH 7.0 for CAT) containing 0.1 mmol/L ethylene diamine tetraacetic acid (EDTA) and 1% (w/v) polyvinylpyrrolidone (PVP). The homogenate was centrifuged at 15 000×g for 30 min at 4 °C and the supernatant was used for measuring enzyme activities.
SOD activity was assayed by measuring the inhibition of NBT reduction according to the inhibition of nitro blue tetrazolium reduction at 560 nm [29]. POD activity was measured according to the changes in absorption at 470 nm due to guaiacol oxidation [30]. CAT activity was determined by measurement of the differential values between original and residual H 2 O 2 in the reaction solution at 240 nm [31]. All spectrophotometric analyses were performed on a UV-VIS spectrophotometer (ONLAB, EU-2000A).

Measurement of APX, MDHAR, DHAR, GR and GPX activities
Frozen leaves (0.5 g) were ground to a powder in liquid nitrogen and homogenized with 5 mL of 50 mmol/L sodium phosphate buffer (pH 7.5) containing 0.1 mmol/L Na 2 EDTA, 0.3% Triton X-100 and 4% (w/v) PVP. The homogenate was centrifuged at 15 000×g for 30 min at 4 °C. The supernatant were used for measurement of enzyme activities.
APX activity was assayed by monitoring the rate of reduced ascorbate acid (AsA) oxidation at 290 nm according to Nakano and Asada's method [32]. MDHAR activity was measured by recording the decrease in absorbance at 340 nm due to oxidation of reduced nicotinamide adenine dinucleotide phosphate (NADPH), as described by Murshed et al. [33]. The DHAR activity assay was carried out by measuring the increase in absorbance at 265 nm due to AsA formation, according to Nakano and Asada's method [32].
GR activity was measured by monitoring the oxidized glutathione (GSSG)-dependent oxidation of NADPH at 340 nm for 3 min, according to the method of Schaedle and Bassham [34]. GPX activity was assayed by measuring the rate of GSH-dependent oxidation of NADPH at 340 nm, as described by Navari-Izzo et al. [35].

Measurement of antioxidants (AsA and GSH)
Frozen leaves (0.5 g) were ground to a powder in liquid nitrogen and homogenized with 5 mL of 5% sulfosalicylic acid and centrifuged at 15 000×g for 30 min, and the supernatant was used for AsA and GSH assays. The AsA concentration was determined based on the reduction of ferric to ferrous ion with AsA in acid solution followed by the formation of the red chelate between ferrous ion and 2,2′-dipyridyl [36]. The total GSH was determined by the 5,5′-dithio-bis-nitrobenzoic acid (DTNB)-GR recycling procedure [37].

Measurement of MDA
Malondialdehyde (MDA) content was measured by the thiobarbituric acid (TBA) reaction as described by Heath and Packer [38], and was used to estimate lipid peroxidation. Frozen leaves (0.5 g) were ground to a powder in liquid nitrogen and homogenized with 5 mL of 5% trichloroacetic acid (TCA). The homogenate was centrifuged at 15 000×g for 10 min. The supernatants were mixed with the same volume of 20% TCA containing 0.5% TBA and kept at 95 °C for 30 min. The mixture was cooled rapidly and centrifuged again. The optical density of the supernatant was measured at 532 and 600 nm.

Phenotype and physiological indexes
Phenotype can directly reflect the drought resistance of soybean plants. As shown in Figure 1A, at 5DAD, the growth status of soybean plants (DS) was seriously suppressed, with most leaves wilted. In contrast, there were no visible wilting symptoms in AMEP412-pretreated soybean plants (ADS). At 5DRW, the soybean plants in DS and ADS treatments generally recovered from the drought stress ( Figure 1B). However, in DS treatment, permanent wilting was found on leaves at bottom layers. Compared with water controls, drought stress apparently affected the growth status of soybean plants, while AMEP412 pretreatment reduced the damage caused by drought stress. There was no significant difference in phenotypes between CK and ACK treatments.
Variations in RWC and RC were used as parameters to determine the stress status of plants [39]. The free proline, soluble sugar and soluble protein contents play important roles in osmoregulation and dehydration prevention under drought stress [40]. In the present study, drought stress significantly decreased the leaf RWC, but increased the leaf RC and osmolytes (free proline, soluble sugar and soluble protein) contents. Then all the indexes tended to recover gradually after rehydration ( Figure 2). Most of the peak and valley values appeared at 5DAD except for those of soluble sugar and soluble protein (peaked at 1DRW). Compared with DS treatment, ADS treatment significantly changed the values of RWC, RC, free proline, soluble sugar and soluble protein by 13.3%, 27.3%, 28.2%, 27.3%, and 7.6% at the peak and valley points, respectively (p < 0.05). In the non-drought stressed treatments (CK and ACK), AMEP412 also resulted in a similar but minor trend of the above indexes.

ROS determination
The in situ detection of ROS (H 2 O 2 and O 2 • ,) was executed by DAB and NBT staining, respectively. The straining degrees showed positive correlations with the ROS levels. As shown in Figure 3, in drought stress treatments (DS and ADS), significant ROS accumulations were observed at 5DAD and 5DRW. The difference probably lay in that the cleanup of ROS in AMEP412 pretreatment (ADS) was more effective, as compared with DS treatment. Certain amounts of staining spots were monitored in ACK treatment at 5DAD and 5DRW, indicating AMEP412 application could also trigger ROS accumulation throughout the experimental process.  had no significant differences between DS and ADS treatments. However, they showed a significant decline in ADS treatment at 5DRW, which decreased by 4.6 and 14.3%, as compared with the DS treatment, respectively (p < 0.05). In the non-drought stressed treatments (CK and ACK), AMEP412 led to a slight but non-significant increase in ROS levels.

Antioxidant enzyme activities
Plants employ many different types of antioxidant enzymes to work together to remove excessive ROS effectively [41]. SOD is a major scavenger of O 2 • , catalyzing its dismutation into H 2 O 2 and O 2 . Both CAT and POD scavenge the accumulated H 2 O 2 to non-toxic levels by converting it into H 2 O and O 2 . In the AsA-GSH cycle [42], APX utilizes AsA as a specific electron donor to reduce H 2 O 2 to water, and AsA is then regenerated by the action of MDHAR and DHAR. GPX catalyzes the reduction of H 2 O 2 to water by GSH, and GR catalyzes the NADPH-dependent regeneration of GSH.
In this experiment ( Figure 5), drought stress showed significant effect on the activities of the antioxidant enzymes, and peak values were obtained at 5DAD (GR and GPX) and 1DRW (SOD, POD, CAT, APX, MDHAR and DHAR). Then the recovered plants showed a gradual decline in enzyme activities. Notably, AMEP412 pretreatment further enhanced the alterations of antioxidant enzyme activities. ADS treatment significantly increased the SOD, POD, CAT, APX, MDHAR, DHAR, GR and GPX activities by 17.3%, 23.7%, 9.2%, 15.8%, 23.2%, 19.0%, 36.4% and 10.7% at the peak points, respectively, over DS treatment (p < 0.05). Furthermore, even at 5DRW, most of the enzyme activities (except for MDHAR and GPX) of the ADS treatment still kept at significantly higher levels, as compared with the DS treatment (p < 0.05). In water controls (CK and ACK), AMEP412 application also induced a significant but minor increase in some antioxidant enzyme activities (POD, APX and GPX).

Antioxidants
Antioxidants (AsA and GSH) play important roles in scavenging ROS through the AsA-GSH cycle [43]. Drought stress treatments (DS and ADS) significantly increased the AsA content ( Figure 6A) and GSH cotntent ( Figure 6B), which peaked at 1DRW and then decreased. In comparison with the drought-only treatment (DS), the AsA and GSH levels in the AMEP412 pretreatment (ADS) were generally elevated, increasing by 38.5% and 16.2% at 1DRW, respectively (p < 0.05). In water controls, AMEP412 treatment (ACK) also led to a significant increase in GSH content, when compared with CK treatment.

Lipid peroxidation
The degree of membrane lipid peroxidation in soybean leaves can be reflected by MDA content, which reflects the degree of cell membrane damage [44]. As shown in Figure 6C, marked enhancement in MDA content was found under drought stress conditions (DS and ADS), with a peak at 1DRW. ADS treatment continuously decreased the MDA content, which was 27.4% lower than that of DS treatment at 1DRW (p < 0.05).   Under water conditions (CK and ACK), the lipid peroxidation did not show significant changes.

Discussion
AMEP412, a protein elicitor produced by B. subtilis, is recently known as a biological agent for improving plant health by triggering plant immunity, inhibiting plant pathogens and killing harmful insects [12,45,46]. In this study, for the first time, the drought tolerance of soybean plants induced by AMEP412 was reported. During the process of drought stress and rehydration, no obvious wilting leaves were observed with the application of AMEP412, while the physiological parameters also agree with the symptoms. Compared with drought stress control, AMEP412 increased the ROS burst degree at the early stage, which induced higher production of antioxidant enzymes, antioxidants and osmolytes. Then the enhanced antioxidant system fastened the cleanup of ROS and alleviated the lipid peroxidation, which protected soybean plants from the damage caused by drought stress. This result enriched our knowledge of the function of AMEP412 and broadened its potential application scale.
Under drought stress, a rapid ROS accumulation would occur, leading to negative impact on antioxidant metabolism, and consequently cell peroxidation damage [47,48]. Plants have evolved enzymatic and non-enzymatic antioxidant systems to scavenge excessive ROS, which keep the cellular ROS level at a steady state. Once this balance is broken by continuous drought stress, oxidative damage and wilting symptoms appear. In this study, the soybean seedlings in DS treatment showed obvious wilting symptoms at 5DAD, indicating that the balance of ROS generation and scavenging was disturbed. The soybean seedlings in ADS treatment did not show wilting symptoms, implying the scavenging of ROS could still keep pace with the accumulation of ROS. Following rehydration, the production of ROS was restrained and the balance shifted to ROS scavenging, resulting in the recovery of the soybean plants from drought stress.
Apart from the destructive role of ROS, an early rise in ROS level could act as a signal for acclimation or defense response, which serves positive roles in plant growth and development [49]. By applying protein elicitors on plants, the signaling role of ROS has been understood well in the case of defense against pathogens [50], promotion in growth [51] and tolerance to drought stress [52]. In our previous study, we found that AMEP412 could trigger ROS burst and defensive enzymes expression, and then led to the defense response against disease [12]. In this study, the pretreatment with AMEP412 elevated the production of ROS at the early stage, and then led to an enhanced response of the ROS-scavenging system throughout the whole experimental process, resulting in the drought tolerance of soybean seedlings. This result suggested that the ROS accumulation trigged by AMEP412 activated certain downstream signaling cascades, and finally facilitated the ROS scavenging, which might partially be the mechanism underlying the drought tolerance induced by AMEP412.
It is now well established that both drought stress and protein elicitors could induce ROS to some degree [53]. In drought stress related references, the downstream signaling of ROS is likely to occur via calcium and reversible protein phosphorylation [54,55], which refer to Ca 2+ channels and mitogen-activated protein kinases (MAPKs), respectively. Furthermore, the levels of phytohormones like abscisic acid (ABA) and ethylene could also be modulated by ROS [56][57][58]. In plant immunity related reports, Ca 2+ signaling and MAPK cascades could also be activated by ROS burst [59][60][61], as well as downstream pathways mediated by hormones like salicylic acid (SA), jasmonic acid (JA) and ethylene [62,63]. Taken together, these observations suggest that ROS signaling induced by protein elicitors could evoke downstream responses that contribute to drought tolerance. The present study further indicated the existence of crosstalk between the two different external stimulants at ROS level.
In the present research, the drought tolerance of soybean improved by AMEP412 was only investigated at the seedling stage. However, drought stress at flowering and pod filling stages affects the soybean yield more directly [64]. So the influence of AMEP412 on soybean plants at these other stages will be studied in subsequent research. Furthermore, besides ROS, more aspects like photosynthesis, hormone balance and signaling transduction will be involved for better understanding of the underlying mechanism.

Conclusions
Drought stress remarkably affected the phenotypes and physiological indexes of soybean seedlings. As a response to drought stress, ROS were generated dramatically, followed by great alterations in the antioxidant and lipid peroxidation system. By application of AMEP412 before drought stress, the phenotypes of soybean plants gained notable improvements. Improvements were seen in the physiological indexes, like RWC, RC and osmolytes as well. Besides, higher accumulation of ROS was observed at the early stage, which led to enhanced antioxidative protection and resulted in accelerated ROS scavenging and decreased lipid peroxidation. Altogether the results suggested that AMEP412 obviously promoted the drought tolerance of soybean seedlings, which was at least partially attributed to the improvement at the ROS level.