Method for improving salt tolerance of soybeans under salt stress

Abstract

The invention belongs to the technical field of saline-alkali soil improvement, and particularly relates to a method for improving salt tolerance of soybeans under salt stress, boric acid is applied to the soybeans under salt stress, soybean seedlings are used as materials, a nutrient solution culture method is adopted, the boric acid is applied to the soybean seedlings under salt stress simulated by 100 mM NaCl, and the final concentration of the boric acid is larger than 25 [mu] M; preferably, the final concentration of the boric acid is 150 [mu] M. According to the method, boric acid is used for improving the salt tolerance of soybeans for the first time, under salt stress, boric acid is applied, the growth vigor and photosynthesis of the soybeans can be improved, oxidative damage of plants is relieved, the content of sodium ions on the overground part is reduced, and phenylpropane metabolism of roots is specifically started; the method is scientific, reliable and good in repeatability, has important scientific value, provides important reference for improving the salt tolerance of soybeans, improves the yield of soybean crops, and provides a new scheme for effective utilization of saline-alkali soil.

Description

Technical field:

[0001] The invention belongs to the technical field of saline-alkali land improvement and crops, and particularly relates to a method for improving the salt tolerance of soybeans under salt stress. Boron application under salt stress can improve the salt tolerance of soybeans under salt stress. Background technology:

[0002] More than 833 million hectares of land are affected by salinization, accounting for 20% of the world's total cultivated land. As global climate conditions continue to deteriorate and humans use incorrect irrigation methods, soil salinization will cause the global irrigated land to decrease by 1% to 2% each year, seriously affecting the environment and ecology of more than 80 countries and regions around the world, and threatening the food security of more than 1.5 billion people worldwide.

[0003] Salinized soil contains a lot of Na + , Cl - , which not only reduces the surrounding water potential and thus the water absorption rate of the soil, inducing osmotic stress in the plants, resulting in the inhibition of plant growth and stomata closure; but also absorbs Na + , Cl - Large amounts of ROS enter the plant body, destroying the plant cell structure, causing ion stress, inhibiting plant growth, and even causing death. Both osmotic stress and ion stress can cause the accumulation of ROS, induce oxidative stress in plants, lead to plant membrane peroxidation, and increase membrane permeability.

[0004] Reactive oxygen species (ROS) are signal molecules that control cell apoptosis in animals and plants, mainly including O2. - 、H2O2、OH - and other substances, which have strong reducing properties. Excessive ROS can destroy components such as cell membranes and proteins, and have a strong toxic effect on plant cells. In the long evolution, plants have formed a complete reactive oxygen scavenging system that can control reactive oxygen below the toxic level to maintain normal physiological metabolic activities. Plants mainly remove excess reactive oxygen in plants through antioxidant enzyme systems and antioxidant systems. Among them, the antioxidant enzyme system mainly includes superoxide dismutase (SOD), catalase (CAT), and peroxidase (POD). Adversity stress can cause changes in antioxidant enzyme activity. At the same time, studies have shown that phenylpropanoid metabolism is involved in plant response to adverse stress (salt stress, heavy metals, drought). Under salt stress, highland barley activated the phenylpropanoid metabolic pathway and increased the content of stress-resistant substances such as polyphenols, flavonoids, and lignin. Exogenous NO was applied under aluminum stress to remove excess reactive oxygen in watermelon by activating the phenylpropanoid metabolic pathway and antioxidant enzyme system. Under drought stress, maize activated four metabolic pathways, including plant hormone signal transduction, phenylpropanoid biosynthesis, glutathione metabolism, and starch and sucrose metabolism, to respond to drought stress.

[0005] Boron (B) is one of the essential trace elements for the growth and development of higher plants. It directly or indirectly affects the integrity of cell walls and cell membranes, cell elongation and division, plant reproductive growth, carbohydrate synthesis, and hormone production and synthesis. Studies have shown that boron plays an important role in improving plant resistance to stress (drought, heavy metals). In addition, there is a certain relationship between boron and salt. AR et al. found that salt can induce a decrease in boron concentration in plants; Letey J et al. found that increased soil salinity can alleviate boron toxicity in broccoli.

[0006] Soybeans are rich in protein and lipids. They can be used as oil crops and bean products, and are also important feed for animal husbandry and poultry farming. They are also one of the most widely planted crops in the world. Soybeans are moderately salt-tolerant crops. High salt stress will not only seriously affect the agronomic traits of soybeans, including height, leaf size, biomass, internode bundles, number of branches, number of pods, single plant weight and 100-grain weight, but also affect the protein content of soybeans. Under 7.0ds / m salt stress, soybean yield reduction is as high as 42%. Therefore, it is necessary to study how to improve the salt tolerance of soybeans. Summary of the invention:

[0007] The purpose of the present invention is to break the existing technical limitations and provide a new idea of ​​applying boron to improve the salt tolerance of soybean under salt stress (improve the growth potential and photosynthesis of crops, reduce the oxidative damage of plants, reduce the content of sodium ions in the aboveground parts, and specifically activate the phenylpropanoid metabolism of roots).

[0008] In order to achieve the above object, the present invention provides a method for improving the salt tolerance of soybean under salt stress, wherein boron is applied to the soybean under salt stress, and the boron is derived from boric acid (H3BO3).

[0009] The present invention uses soybean seedlings as materials and adopts a nutrient solution culture method to apply boron to the soybean seedlings under salt stress simulated by 100mM NaCl, and the final concentration of boron is greater than 25μM; preferably, the final concentration of boron is 150μM.

[0010] Preferably, boric acid is applied to soybean seedlings under salt stress simulated by 100 mM NaCl, and the final concentration of the boric acid is greater than 25 μM; preferably, the final concentration of the boric acid is 150 μM.

[0011] The present invention explores whether boron can improve the salt tolerance of soybean by applying 150 μM boric acid under salt stress simulated by 100 mM NaCl. Since the optimal boron concentration of soybean growth nutrient solution is 25 μM under normal plant growth, 0 and 100 mM salt concentrations and two boron concentrations (25, 150 μM) are used to conduct the experiment, and 4 groups are set, (1) control (CK): 25 μM H3BO3+0 mM NaCl; (2) +B: 150 μM H3BO3+0 mM NaCl; (3) +NaCl: 100 mM NaCl+25 μM H3BO3; (4) +NaCl+B, 100 mM NaCl+150 μM H3BO3. By analyzing the growth, photosynthesis, oxidative damage, etc. of each group of soybeans, it was found that when normally growing soybeans are in a salt stress environment, applying additional boric acid can improve the growth potential of soybean plants, reduce oxidative damage to plants, reduce the sodium ion content in the aboveground parts, and specifically activate phenylpropanoid metabolism in the roots. Therefore, boric acid can improve the salt tolerance of soybean plants under salt stress and reduce the impact of salt stress on plants.

[0012] Compared with the prior art, the present invention utilizes boric acid for the first time to improve the salt tolerance of soybeans. Under salt stress, the application of boric acid can improve the growth potential and photosynthesis of soybeans, reduce the oxidative damage of plants, reduce the content of sodium ions in the aboveground parts, and specifically activate the phenylpropanoid metabolism of roots. The method of the present invention is scientific and reliable, has good repeatability, has important scientific value, provides an important reference for improving the salt tolerance of soybeans, increases the yield of soybean crops, and provides a new solution for the effective utilization of saline-alkali land. Description of the drawings:

[0013] Figure 1 This is a schematic diagram of the principle of applying boron to alleviate soybean salt stress according to the present invention.

[0014] Figure 2 A picture of soybean germination.

[0015] Figure 3 This is a picture of soybeans in nutrient solution.

[0016] Figure 4 The results of the effect of boron on the growth of soybean under salt stress, where A is a growth photo; B is a root length picture; C is a leaf picture; D is a plant height statistic; E is a root length statistic; F is a ground fresh weight statistic; G is a root fresh weight statistic; H is a soybean seedling dry weight statistic. Different letters (a, b, c) indicate significant differences at the (P<0.05) level.

[0017] Figure 5The results of the effect of boron on photosynthesis of soybean under salt stress, where A is the net photosynthetic rate; B is the stomatal conductance; C is the transpiration rate; D is the intercellular carbon dioxide concentration; E is chlorophyll a; F is chlorophyll b; G is the total chlorophyll content; H is carotenoids. Different letters (a, b, c) indicate significant differences at the (P<0.05) level. (Independent sample t test, n=3, * is P<0.05).

[0018] Figure 6 The results of the effect of boron on oxidative damage of soybean under salt stress, where A is the H2O2 content in leaves; B is the H2O2 content in roots; C is the MDA content in leaves; D is the MDA content in roots; E is the relative electrical conductivity of leaves; and F is the relative electrical conductivity of roots. Different letters (a, b, c) indicate significant differences at the (P<0.05) level.

[0019] Figure 7 The results of the effect of boron on the antioxidant system of soybean under salt stress, where A and B are SOD in leaves and roots, C and D are POD in leaves and roots, E and F are CAT in leaves and roots. Different letters (a, b, c) indicate significant differences at the (P<0.05) level.

[0020] Figure 8 The results of the effect of boron on the content of soybean osmotic substances under salt stress, where A and B are the proline content in leaves and roots, respectively; C and D are the soluble protein content in leaves and roots, respectively. Different letters (a, b, c) indicate significant differences at the (P<0.05) level.

[0021] Fig. 9 The results of the effects of boron on the ion content of sodium, potassium and calcium in soybean under salt stress, where A is the sodium ion content in soybean leaves, stems and roots; B is the potassium ion content in soybean leaves, stems and roots; C is the calcium ion content in soybean leaves, stems and roots; D, E, and F are the sodium ion, potassium ion and calcium ion contents in the root cell wall, respectively. (Independent sample t test, n=3, * is P<0.05). Different letters (a, b, c) indicate significant differences at the (P<0.05) level.

[0022] Fig.10 Effect of boron on total sodium in soybean under salt stress + (A), K + (B) Ca 2+ (C) and Na + Effect of transport coefficient (D). The values ​​in the figure are the mean ± SD of three replicates. Different lowercase letters indicate differences among treatments at the P < 0.05 level.

[0023] Fig.11 Effect of boron application on sodium in soybean leaves (A), stems (B) and roots (C)+ / K + The effect bar graph is the mean ± SD of three replicates. Different letters (a, b, c) indicate significant differences at the (P < 0.05) level.

[0024] Fig.12 These are pictures of the heat map, variation and principal component analysis (PCA) of the effects of boron on soybean root metabolism under salt stress, where A is the total metabolism heat map; B is the Venn diagram; C is the principal component analysis diagram; and D is the total metabolites and significantly up-regulated / down-regulated metabolites.

[0025] Fig.13 The results of the KEGG pathway for the enrichment of differential metabolites in roots under salt stress induced by boron, where A is +NaCllvs.CK; B is +NaCl+B vs.+NaCl.

[0026] Fig.14 Figure 2 shows the effect of boron on the phenylpropanoid metabolic pathway of roots under salt stress. A is a circle-shaped heat map of differential metabolites in the phenylpropanoid metabolic pathway; B is a principal component analysis of differential metabolites in the phenylpropanoid metabolic pathway; and C is a loading matrix of differential metabolites in the phenylpropanoid metabolic pathway.

[0027] Fig.15 The results of the analysis of metabolite content in the phenylpropanoid metabolic pathway of soybean roots under +NaCl vs. CK. Red / green indicates a significant increase / decrease in metabolites, respectively. Black / blue indicates undetected / unchanged metabolites, respectively. (Independent sample t-test, n=3, * is P<0.05).

[0028] Fig.16 The results of the analysis of metabolite content in the phenylpropanoid metabolic pathway of soybean roots under +NaCl+B vs. +NaCl, red / green indicates a significant increase / decrease in metabolites, respectively. Black / blue indicates undetected / unchanged metabolites, respectively. (Independent sample t-test, n=3, * is P<0.05).

[0029] Specific implementation:

[0030] The technical solution of the present invention is further described in detail below through specific embodiments and in conjunction with the accompanying drawings.

[0031] Embodiment 1:

[0032] The present invention relates to a method for improving the salt tolerance of soybean under salt stress, which specifically comprises the following steps:

[0033] 1. Cultivating soybean seedlings:

[0034] (1) Select healthy, full-grained, smooth-skinned, disease-free soybean (Qihuang 34) seeds for cultivation. Wash the soybeans and soak them in a 10 wt% sodium hypochlorite (NaClO) aqueous solution for 10 minutes for disinfection. After disinfection, rinse them with tap water and then wash them with purified water three times;

[0035] (2) Soak the cleaned soybean seeds in pure water for 6-8 hours, and then place them in a humid and dark environment. The soybean seeds will germinate in about 48 hours;

[0036] (3) The germinated soybeans were transferred to a moist, bright place and given sufficient water every day. After one week, when the cotyledons turned green, they were transferred to a black plastic bucket (4 L) filled with purified water.

[0037] (4) When the first true leaf unfolds, transfer it to the modified Hoagland nutrient solution.

[0038] The formula of the improved Hoagland nutrient solution is: macroelements: calcium nitrate (Ca(NO3)2) 5mmol / L, potassium nitrate (KNO3) 5mmol / L, magnesium sulfate (MgSO4) 2mmol / L, ammonium phosphate (NH4H2PO4) 1mmol / L, trace elements: ammonium molybdate ((NH4)2MoO4) 0.5μmol / L, manganese chloride (MnCl2) 9.14μmol / L, copper sulfate (CuSO4) 0.32μmol / L, zinc sulfate (ZnSO4) 0.77μmol / L, and sodium iron ethylenediaminetetraacetate (Fe-EDTA) 50μmol / L.

[0039] 2. Experimental Grouping:

[0040] The soybean seedlings in the modified Hoagland nutrient solution were randomly divided into 4 groups. Since the boron concentration required for normal growth of soybean is 25 μM, (1) the control group (CK) was set up, which was a normal growth group and was given boric acid (final concentration was 25 μM); (2) the boron application group (+B), on the basis of normal growth of soybean, the amount of boric acid applied was increased to make the final boric acid concentration of 150 μM; (3) the salt stress group (+NaCl), under the normal growth conditions of soybean, sodium chloride stress was applied, that is, sodium chloride (final concentration was 100 mM) and boric acid (final concentration was 25 μM); (4) the salt stress boron application treatment group (+NaCl+B), which was given sodium chloride (final concentration was 100 mM) and boric acid (final concentration was 150 μM).

[0041] After 18 days of treatment, the growth of soybeans in the four groups was observed, and the fresh weight, dry weight, plant height and root length of the plants were measured and photographed. Figure 4 As shown. Figure 4AC shows that compared with the control group, the soybeans in the salt stress group grew significantly worse, with reduced leaf area and severe yellowing; compared with the salt stress group, the soybeans in the salt stress boron treatment group grew significantly better, with increased leaf area and restored leaf color, but still yellow ( Figure 4 C) From Figure 4 It can be seen that the fresh weight of soybean in the salt stress group ( Figure 4 F; 4G), dry weight ( Figure 4 H), plant height ( Figure 4 D) Root length ( Figure 4 E) The lowest; increasing the application of boron can significantly enhance the growth potential of soybean. Therefore, additional application of boron helps to improve the salt tolerance of soybean seedlings.

[0042] 3. Index Testing Process and Results

[0043] The photosynthesis, oxidative damage (hydrogen peroxide, malondialdehyde, relative conductivity), antioxidant enzyme system, osmotic substances (proline, soluble protein), sodium, potassium and calcium ion content and root metabolites of the above four groups of soybean seedlings were tested to verify the effect of boron on soybean under salt stress. The specific process is as follows:

[0044] 1. Effect of Boron on Photosynthesis of Soybean Seedlings under Salt Stress

[0045] The photosynthetic parameters of soybean leaves were measured using a photosynthesis meter from 9:00 am to 12:00 pm, mainly including net photosynthetic rate, transpiration rate, stomatal conductance and intercellular carbon dioxide concentration of leaves; in order to evaluate the chlorophyll content, the leaves were immersed in 95% alcohol for quantitative analysis. The maximum absorption of chlorophyll a, chlorophyll b and carotenoids was measured at three wavelengths (665, 649 and 470 nm) using a spectrophotometer. The results are shown in Figure 2. Figure 5 shown.

[0046] from Figure 5 AD shows that under salt stress, application of boric acid increased the stomatal conductance, transpiration rate, and photosynthetic rate of soybean, and reduced the intercellular carbon dioxide concentration of soybean leaves. Figure 5 EH showed that salt stress caused a decrease in chlorophyll a, b, and carotenoid contents. However, under salt stress, boric acid application increased chlorophyll a, b, and carotenoid contents and increased the level of photosynthetic pigments, thereby alleviating the harmful effects of salt stress.

[0047] 2. Effect of boron on oxidative damage in soybean seedlings under salt stress

[0048] Determination of MDA content: Take 0.15g of sample and add phosphate buffer (pH=7.8, 0.05mM) to grind and extract, centrifuge at 4℃ and take the supernatant for use. Add 0.5mL of 0.5% thiobarbituric acid solution to the supernatant after centrifugation at 4℃, heat at 100℃ for 20min and centrifuge, take the supernatant and measure the absorbance at 450nm, 532nm and 600nm respectively.

[0049] Determination of H2O2 content: Take 0.15g sample, add 1.5mL acetone and homogenize in an ice bath, centrifuge at 4℃, take the supernatant, add the required reagents according to the instructions of the kit manufacturer (Beijing Solebow Technology Co., Ltd.), let stand at room temperature for 5min, and measure the absorbance at 415nm.

[0050] The relative conductivity was determined by the immersion method. 0.1 g of fresh roots of equivalent size were selected, washed with ultrapure water and then dried, cut into appropriate strips, placed in a graduated test tube containing 10 ml of deionized water, covered and immersed at room temperature for 12 h. The conductivity of the extract (R1) was determined using a conductivity meter (DDS-307A), then heated in a boiling water bath for 30 min, cooled to room temperature, shaken and measured again for conductivity (R2). Relative conductivity = R1 / R2×100%.

[0051] The results of the test are as follows Figure 6 As shown. Salt stress leads to an increase in H2O2 and MDA content ( Figure 6 AD), the relative conductivity of leaves and roots increased ( Figure 6 EF). However, compared with the +NaCl treatment, the +NaCl+B treatment significantly reduced the H2O2, MDA contents and relative conductivity of the seedlings.

[0052] 3. Effect of boron on the antioxidant system of soybean seedlings under salt stress

[0053] The SOD enzyme activity was determined by the nitro blue tetrazolium reduction method. 0.05M PBS (pH=7.8) (1.5mL), 130mM L-methionine solution (0.3mL), 750μM nitro blue tetrazolium solution (0.3mL), 100μM EDTA-Na2 solution (0.3mL), 20μM riboflavin solution (0.3mL) and distilled water (250μL) were added to 50μL of extract (supernatant after centrifugation at 4°C). After mixing, the control tube was placed in a dark place, and the other tubes were reacted in daylight for 20min, and then the absorbance was measured at 560nm.

[0054] The method for determining CAT enzyme activity is as follows: 0.3% H2O2 (1 mL), H2O (1.9 mL) and supernatant (100 μL) were added to the reaction system, and the absorbance was read at 240 nm every half minute for a total of 210 seconds.

[0055] The POD enzyme activity was determined by the guaiacol method. 1 mL of 0.05 M PBS (pH = 7.0) and 0.95 mL of 0.2% guaiacol were added to the test tube. After mixing, the mixture was preheated in a 34°C water bath. 0.3% H2O2 (1 mL) and 0.05 mL of the extract were added to each sample. The rate of change of the absorbance within 210 seconds was recorded at 470 nm, and the data was recorded every half minute.

[0056] The results of the test are as follows Figure 7 As shown in Figure 2, the activities of SOD, POD and CAT in soybean leaves were significantly reduced under salt stress. In contrast, the application of boric acid under salt stress significantly enhanced their activities ( Figure 7 A, C, and E in the middle). Under salt stress, the root SOD activity decreased, while the POD and CAT activities increased; compared with the +NaCl treatment, the SOD activity increased but not significantly under the +NaCl+B treatment, while the POD and CAT activities decreased significantly ( Figure 7 (B, D, F).

[0057] 4. Effect of Boron on Osmotic Substances of Soybean Seedlings under Salt Stress

[0058] The proline content was determined by the ninhydrin colorimetric principle. First, a series of proline solutions were prepared. Proline solution (2 mL), glacial acetic acid (2 mL) and acidic ninhydrin solution (2 mL) were taken respectively. After boiling in a water bath for 30 min, 4 mL of toluene was added after cooling. After standing, colorimetry was performed at 520 nm and a standard curve was drawn. Another 0.15 g sample was extracted with 1.5 mL of 3% sulfosalicylic acid at 100 ° C for 10 min. After obtaining the supernatant, the absorbance was determined in the same way, and the proline content was obtained from the standard curve.

[0059] Soluble protein was determined by Coomassie Brilliant Blue G-250 staining. Bovine serum albumin was weighed to prepare gradient concentration standard solution, and the absorbance was measured at 595 nm to draw a standard curve. In addition, the supernatant was obtained by referring to the MDA extraction method, and the extract (0.05 mL) and Coomassie Brilliant Blue G-250 solution (5 mL) were added to a 10 mL centrifuge tube, mixed thoroughly and allowed to stand for 2 minutes, and the absorbance at 595 nm was measured, and the protein content of the sample was calculated using the standard curve.

[0060] The results of the test are as follows Figure 8 shown. Figure 8 AB showed that proline levels in soybean seedlings were significantly increased under salt stress, especially in leaves. However, the application of boron under salt stress led to a significant decrease in these elevated proline concentrations. In addition, a similar trend was found in soluble protein. Salt stress led to an increase in soluble protein content in leaves and roots, while boron treatment led to a subsequent decrease in its content ( Figure 8 CD).

[0061] 5. Effects of Boron on Sodium, Potassium and Calcium Contents in Soybean Seedlings under Salt Stress

[0062] The root cell walls were extracted in sequence using a mixture of ice water, ethanol, methanol, chloroform, and acetone: roots and leaves were homogenized in ice water at a mass volume ratio of 1 g:10 mL, and then centrifuged for 10 minutes; after repeating twice, the precipitate was washed three times with 80% ethanol to remove chlorophyll, sugars, and other soluble compounds; it was then rinsed once with a methanol-chloroform mixture (1:1, v / v) to remove certain sugars and lipids, and then rinsed a final time with acetone to remove proteins, phenols, and other residual substances; the resulting precipitate was dried and designated as cell wall material. 0.2 g of leaf, stem, root, and root cell walls were carbonized in an electric furnace, and incinerated at 500°C for 4 hours; after cooling, 10 ml of 0.1 M HCl solution was added; the boron content was determined by curcumin colorimetry; the resulting filtrate was then analyzed using a flame photometer to determine the Na in the root, stem, leaf, and root cell walls. + , K + and Ca 2+ The results are as follows Fig. 9 Statistical analysis of total Na in soybeans + , K + and Ca 2+ The content of Na + The transport coefficient is Fig.10 The sodium-potassium ratio of roots, stems and leaves was calculated, and the results are as follows Fig.11 shown.

[0063] from Fig. 9 AC and Fig.10 AC shows that compared with the control group, the Na content of plants in the salt stress group was + The content of K + and Ca 2+ Compared with the salt stress group, the Na content of plants in the salt stress boron treatment group was significantly reduced. + The content of K + The content of Ca increased (leaves, stems and roots), 2+ The content increased (stem). This indicates that salt stress reduces the K content in plants. + and Ca 2+ content, but will significantly increase Na + The content of Na + The content of K + The content of Ca 2+ Although there was an increase, it did not reach a significant difference. + The transport coefficient ( Fig.10D) increased sharply under salt stress, and boron application under salt stress reduced Na + Transport coefficient.

[0064] from Fig.11 It can be seen that compared with the control group, the sodium-potassium ratio of the roots, stems and leaves of the salt stress group was significantly increased; compared with the salt stress group, the sodium-potassium ratio of the leaves and stems of the salt stress treatment group was significantly reduced, indicating that boron application under salt stress will reduce the sodium-potassium ratio of the aboveground part ( Fig.11 AB).

[0065] from Fig. 9 DF shows that compared with the salt stress group, the Na + , K + and Ca 2+ The levels increased significantly, indicating that with the application of boron under salt stress, Na + , K + and Ca 2+ The levels increased significantly.

[0066] Na + The transport coefficient of aboveground Na + Content / underground Na + Content = (stem + leaf) Na + Content / root Na + content

[0067] 6. Effects of Boron on Metabolic System of Soybean Seedlings under Salt Stress

[0068] A root sample weighing 0.1 g was taken, crushed in liquid nitrogen, and then suspended in 500 μL of 80% methanol. After centrifugation, the supernatant was diluted with LC-MS grade water to a methanol concentration of 53%. Subsequently, the mixture was centrifuged again to obtain the final supernatant. UHPLC-MS / MS analysis was performed at "Novogene Co., Ltd" using "UHPLC, ThermoFisher" combined with "Orbitracp QExactiveTM HF-X mass spectrometer, ThermoFisher", and the raw data was processed by Compound Discoverer 3.3 "CD3.3, ThermoFisher" to identify and quantify metabolites. The identified metabolites were annotated using the KEGG database "https: / / www.genome.jp / kegg / pathway.html" and the HMDB database "https: / / hmdb". ca / metabolites” and lipid map database “http: / / www.lipidmaps.org”. To identify differential metabolites, the following selection criteria were used: VIP score greater than 1, p value less than 0.05, fold change (FC) ≥ 2 or FC ≤ 0.5.) The results are shown in Figure 12-15 shown.

[0069] Fig.12 This is the soybean root metabolism map. To further explain the soybean root response mechanism after boron addition under salt stress, this study analyzed the response mechanism of boron-salt interaction on root metabolites and metabolic pathways based on metabolic analysis. The heat map analysis of the total metabolome showed that +NaCl+B treatment had a significant effect on the content of most metabolites, and some metabolites were significantly upregulated after boron addition under salt stress ( Fig.12 A). The principal component analysis (PCA) score plot can reflect the degree of variation between boron salt treatments. Two principal components were selected based on their contribution rates: the first principal component (PC1) and the second principal component (PC2), which accounted for 24.42% and 14.43% of the total variance, respectively. It can be clearly observed that each individual treatment group had good repeatability and good discreteness between different treatment groups ( Fig.12 C), which well illustrates that the overall metabolic characteristics detected are stable and the data are reliable.

[0070] Differential metabolites were collected by pairwise comparison (+B vs.CK, +NaCl vs.CK, +NaCl+B vs.+NaCl). A total of 1,899 metabolites were detected. Compared with the control group, 94 metabolites in the boron group were significantly upregulated and 173 metabolites were significantly downregulated; in the salt stress group, 608 metabolites were seriously affected, of which 278 metabolites were upregulated and 330 metabolites were downregulated. Under salt stress, 242 differential metabolites were affected in the boron treatment group compared with the salt stress group, of which 171 metabolites were upregulated and 71 metabolites were downregulated ( Fig.12 D).

[0071] KEGG pathway enrichment analysis was performed on the differential metabolites after salt stress and salt stress with boron treatment to determine the main physiological metabolism and signal transduction pathways. The 20 most significantly enriched KEGG pathways were selected in +NaCl vs.CK and +NaCl+B vs.+NaCl, respectively. The results are as follows Fig.13 Among them, pyrimidine metabolism, unsaturated fatty acid metabolism, diterpene biosynthesis, phosphatidylinositol signaling system, inositol phosphate signaling system and other pathways were enriched in +NaCl vs.CK, +NaCl+B vs.+NaCl ( Fig.13 ).

[0072] Fig.14 The present invention detects 12 substances in the phenylpropanoid and flavonoid synthesis pathways and draws a heat map, which intuitively provides a global view of the changes in metabolites in the phenylpropanoid and flavonoid synthesis pathways under salt and boron treatment. Fig.14 A). HCA classified metabolites into two categories, namely cluster 1 (1a and 1b) and cluster 2 (2a and 2b). Compared with the control group, salt stress upregulated the content of 2a (phenylalanine) and downregulated the content of metabolites in cluster 1, especially cluster 1a (daidzein, daidzein, naringenin, and trifoliate safflower glycosides). Compared with salt stress, the boron treatment group under salt stress increased the content of each metabolite in cluster 1a and cluster 2b ( Fig.14 A).

[0073] PCA analyzed the above 22 differential metabolites. The scores of the two principal components PC1 and PC2 were 39.96% and 18.51%, respectively. The +NaCl and +NaCl+B treatments in PC1 and PC2 were clearly separated ( Fig.14 B). The loading matrix of PC1 shows that coumarin, daidzein, daidzein, daidzein, licorice, formononetin and other ordinates have large absolute values, which indicates that they have a high contribution rate to the separation between the treatments in PC1. Similarly, coniferin, eleutheroside B, daidzein, hydroxycinnamic acid, morinol and other substances have a great contribution to the separation between +NaCl and +NaCl+B treatments on PC2 ( Fig.14 C).

[0074] Fig.15 and Fig.16 The results of the analysis of metabolites in the phenylpropanoid metabolic pathway of soybean roots under +NaCl vs. CK, +NaCl+B vs. +NaCl. In the soybean root system, compared with the control group, the salt stress group significantly increased the content of daidzein in the phenylpropanoid metabolic pathway and reduced the content of flavonoids such as lignin, naringenin, and daidzein ( Fig.15 ). Compared with the salt stress group, the salt stress boron treatment group not only increased the content of antioxidant active substances such as cinnamic acid, 5-O-caffeoylshikimic acid, coumarin, sinapyl alcohol, eleutheroside B, and coniferin, but also enhanced the content of flavonoids such as biochanin A, trifoliate bean glycoside, and daidzein ( Fig.16 ).

Claims

1. A method for improving the salt tolerance of soybean under salt stress, characterized in that: Application of boron to soybean under salt stress.

2. The method for improving salt tolerance of soybean under salt stress according to claim 1, characterized in that: The boron is derived from boric acid.

3. The method for improving the salt tolerance of soybean under salt stress according to claim 1, characterized in that: Soybean seedlings were used as materials and cultured in nutrient solution. Boron was applied to the soybean seedlings under salt stress simulated by 100 mM NaCl, and the final concentration of boron was greater than 25 μM.

4. The method for improving salt tolerance of soybean under salt stress according to claim 3, characterized in that: The final concentration of boron was 150 μM.

Images