A method for improving drought resistance and antioxidant capacity of poplar

Abstract

The application discloses a method for improving drought resistance and oxidation resistance of poplar by improving physiological indexes of the poplar, and the method is irrigating poplar plants with L-glutamic acid solution to improve the physiological indexes of the poplar, including: increasing the relative water content of leaves; reducing the leaf water loss rate; maintaining the stability of the leaf electrolyte permeability; increasing the endogenous proline content of leaves; reducing the expression amount of endogenous proline dehydrogenase, increasing the expression amount of endogenous pyrroline-5-carboxylic acid reductase, reducing the expression amount of endogenous pyrroline-5-carboxylic acid dehydrogenase, and increasing the expression amount of endogenous pyrroline-5-carboxylic acid synthetase; improving the rehydration capacity of drought stress leaves; increasing the content or activity of endogenous catalase; increasing the content or activity of endogenous peroxidase; and increasing the content or activity of endogenous superoxide dismutase.

Description

Technical Field

[0001] The invention belongs to the field of forestry and relates to a method for improving the drought resistance and antioxidant capacity of poplars. Background Art

[0002] Drought is a complex hydrological and climatic disaster characterized by long duration and destructive power. Globally, drought is becoming increasingly severe, causing changes in the distribution, structure, community composition, and biodiversity of forests, severely impacting forestry development worldwide. The area of ​​arid and semi-arid regions in my country is increasing, and the survival rate of artificial afforestation is only 4% to 30%. Poplar (Populus L.), a traditional fast-growing windbreak tree species in northern my country and a renewable bioenergy plant, plays a vital role in windbreak and sand fixation, water conservation, and timber supply.

[0003] Poplar is a fast-growing artificial tree species in my country. Its growth and development has long been restricted by various stresses. Therefore, methods to improve poplar's resistance to drought stress are of great significance.

[0004] Glutamate (also known as α-aminoglutarate) is a ubiquitous amino acid that plays a crucial role in amino acid metabolism. Glutamate also serves as a metabolite, a substrate for energy production, a nutrient, a determinant of protein structure, and even as a signaling molecule involved in various biological reactions. Summary of the Invention

[0005] In order to solve the problems existing in the prior art, the present invention provides a method for improving the stress resistance of poplars by improving their physiological indicators, wherein the method comprises watering the poplar plants with a glutamic acid solution;

[0006] Improving the physiological indicators of poplars is improving the first group of physiological indicators of poplars and / or improving the second group of physiological indicators of poplars;

[0007] The improvement of the first group of poplar physiological indicators includes:

[0008] Increase the relative water content of leaves;

[0009] Reduce leaf water loss rate;

[0010] Maintaining the stability of leaf electrolyte permeability;

[0011] Increase the endogenous proline content in leaves;

[0012] reducing the expression of endogenous proline dehydrogenase, increasing the expression of endogenous pyrroline-5-carboxylic acid reductase, reducing the expression of endogenous pyrroline-5-carboxylic acid dehydrogenase, and increasing the expression of endogenous pyrroline-5-carboxylic acid synthetase; and

[0013] Improve the rehydration capacity of drought-stressed leaves;

[0014] The second group of improved poplar physiological indicators includes:

[0015] Increase the content or activity of endogenous catalase;

[0016] increasing the level or activity of endogenous peroxidase; and

[0017] Increase the content or activity of endogenous superoxide dismutase.

[0018] In some embodiments, the glutamate is L-glutamate.

[0019] In some embodiments, the glutamate solution is a 5-15 mmol / L glutamate aqueous solution.

[0020] In some embodiments, the glutamate solution is a 10 mmol / L glutamate aqueous solution.

[0021] In some embodiments, the soil moisture content of the poplar plant is maintained at 70-80%.

[0022] In some embodiments, the soil moisture content of the poplar plant is maintained at 75%.

[0023] In some embodiments, the growth conditions of the poplar are: 14-18 hours of sunlight per day, a temperature of 20-28°C, and a light intensity of 40-60 μmol·m -2 s -1 .

[0024] In some embodiments, the growth conditions of the poplar are: 16 hours of sunlight per day, a temperature of 23-25°C, and a light intensity of 50 μmol·m -2 s -1 .

[0025] In some embodiments, the poplar is 84K poplar.

[0026] In some embodiments, the stress resistance is drought resistance and / or antioxidant capacity. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 Schematic diagram of the experimental design for glutamate treatment.

[0028] Figure 2 Shown are the effects of glutamate treatment on 84K poplar under drought stress.

[0029] Figure 3 Shown are the physiological indices of leaves after exogenous glutamate treatment.

[0030] Figure 4Shown are the effects of glutamate treatment on proline metabolism.

[0031] Figure 5 Shown are the determination of hydrogen peroxide content and analysis of antioxidant enzyme activities after exogenous glutamate treatment.

[0032] Figure 6 Shown are the analysis of leaf physiological indicators after rehydration.

[0033] Figure 7 Shown are the effects of exogenous glutamate on reactive oxygen species content and antioxidant enzyme activities under drought stress. DETAILED DESCRIPTION

[0034] To make the objectives, technical solutions and advantages of the present invention more clear, the embodiments of the present invention will be described in further detail below with reference to the accompanying drawings.

[0035] Test materials

[0036] Populus alba × P. glandulosa '84K' (abbreviated as "84K Poplar") was cultured in 380 mL tissue culture flasks. The stem tips (approximately 3 cm long) of the tissue culture seedlings were transferred to rooting medium and grown in a light culture room for one month, reaching a plant height of approximately 10 cm. The culture conditions were: 16 h light / 8 h dark, temperature 25°C, and light intensity 12,000 Lux. Tissue culture seedlings that had grown well for one month were transferred to seedling pots (10 cm × 10 cm × 8 cm) filled with nutrient soil and cultured in conventional soil for one month, reaching a plant height of approximately 35 cm. The culture conditions were: 16 h light / 8 h dark, temperature 23-25°C, and light intensity 50 μmol·m -2 s -1 .

[0037] Experimental design

[0038] 1. Drought test after applying exogenous glutamate

[0039] In order to determine the optimal time for exogenous glutamate application, 84K poplars grown in seedling pots for two months were used as materials. The 84K poplars were treated with exogenous glutamate (10 mmol·L) on the 0th, 1st, 2nd and 3rd day before drought stress (n days before drought stress in this invention refers to the interval of n days between watering with glutamate solution and drought stress). -1 Root irrigation was performed with an L-glutamic acid aqueous solution (50 mL per seedling pot). Without L-glutamic acid irrigation, 50 mL of clean water was added daily. A control group with normal watering was also set up (50 mL of clean water was added daily). The treatments were designated: G0, G1, G2, G3, and control. All procedures maintained soil moisture at approximately 75%.

[0040] On this basis, each group of G0, G1, G2, G3 and the control was divided into two groups: one group continued to water normally on the basis of the above operations to maintain the soil moisture content at about 75%; the other group was subjected to drought treatment after applying glutamate, namely, drought after applying glutamate (G0+drought), drought after applying glutamate 1 day (G1+drought), drought after applying glutamate 2 days (G2+drought), drought after applying glutamate 3 days (G3+drought) and drought after normal watering (drought).

[0041] The soil was naturally drought-dried for 6 days (the soil moisture content dropped to about 25%). Each treatment was repeated 6 times and the experimental results were repeated 3 times. The leaf phenotypes were observed during the treatment period to determine the optimal time for glutamate application (see Experimental Design for details). Figure 1 a).

[0042] The above experimental analysis determined that the optimal application time of glutamate was 0 days. Subsequently, glutamate was irrigated on the 84K poplar 0 days before drought stress and physiological indicators were measured. The experiment included four treatments: normal watering (control), normal watering followed by drought (drought), normal watering after glutamate application (glutamate), and drought after glutamate application (glutamate + drought). Each treatment had 8 biological replicates and the experiment was repeated 3 times. Samples were taken at three time points: no treatment (start of the experiment: D0), drought after glutamate treatment (4th day of the experiment: D4), and 6 days of drought stress after glutamate treatment (10th day of the experiment: D10). Different indicators were tested. For details of the specific experimental treatments, see Figure 1 a.

[0043] 2. Rehydration Test

[0044] Two-month-old 84K poplars were used as materials in seedling pots. After 6 days of natural drought (soil moisture content dropped to 25%), they were watered with 50 mL of water (H2O), 10 mmol·L -1 L-glutamic acid aqueous solution (Glu) and 10mmol·L -1 L-aspartic acid aqueous solution (Asp) was used to observe the rehydration. Samples were taken at three time points: untreated (start of the experiment: D0), 6 days after drought stress (6th day of the experiment: D6), and 3 hours after rehydration and recovery (D6+3h), and the corresponding physiological indicators were measured. For details of the specific experimental treatments, see Figure 1 b.

[0045] exist Figure 1In the figure, a indicates the drought test after exogenous glutamic acid treatment. D0: untreated; D4: drought after glutamic acid treatment; D10: drought stress for 6 days; control: normal watering; drought: drought after normal watering; G0: normal watering after applying glutamic acid; G0+drought: drought after applying glutamic acid; G1: normal watering after applying glutamic acid for 1 day; G1+drought: drought after applying glutamic acid for 1 day; G2: normal watering after applying glutamic acid for 2 days; G2+drought: drought after applying glutamic acid for 2 days; G3: normal watering after applying glutamic acid for 3 days; G3+drought: drought after applying glutamic acid for 3 days. B indicates the rehydration test. D0: untreated; D6: drought stress for 6 days; D6+3h: rehydration recovery culture for 3 hours; H2O: irrigation of 50 mL water after drought stress; Glu: irrigation of 50 mL 10 mmol·L -1 L-glutamic acid solution; Asp: irrigation of 50 mL 10 mmol·L -1 L-aspartic acid solution.

[0046] Test method

[0047] I. Real-time fluorescent quantitative PCR analysis

[0048] Real-time fluorescent quantitative PCR analysis was performed on the 84K poplar proline synthesis and degradation related genes at different times of glutamic acid treatment. The 5th and 6th leaf blades of the 84K poplar under normal growth and after different treatments were taken, RNA was extracted for reverse transcription, and Vazyme ChamQ SYBR qPCR Master Mix (Nanjing Novozyme Biotech Co., Ltd., Catalog No.: Q311-02) was used according to the instructions, 2 -ΔΔCT The expression amount was calculated, the reference gene was Actin, 4 biological replicates and 3 test replicates were set for each treatment.

[0049] II. Measurement of leaf relative water content

[0050] The 8th leaf blades of the 84K poplar under normal growth and after different treatments were taken to measure the relative water content. The fresh weight of the taken leaf blades was immediately weighed and recorded as FW, the leaf blades were completely immersed in deionized water at room temperature for 24 h, the surface water was absorbed with filter paper and the saturated fresh weight of the leaf blades was weighed and recorded as TW. The leaf blades were then placed in an envelope and dried in a 75℃ oven until the weight was constant, and the weight was weighed and recorded as DW. The relative water content of the leaf blades = (FW-DW) / (TW-DW) x 100%.

[0051] III. Measurement of leaf angle

[0052] The leaf inclination angle of the fifth functional leaf (L5) of 84K poplar trees, both grown normally and after various treatments, was measured. The plant was held vertically above the ground. The angle at which the L5 leaf area was minimized was selected. A time-lapse camera (ATLI, T100 TS) was used to take photos every half hour at a fixed position 60 cm in front of the plant. Using Image J software, the angle between the leaf base and tip was measured with a protractor relative to the main stem. Three biological replicates and three experimental replicates were used for each treatment.

[0053] 4. Electrolyte Permeability Measurement

[0054] Electrolyte permeability was determined by taking the seventh leaf of a normally grown 84K poplar and one of the various treatments. The leaves were rinsed with deionized water, dried with filter paper, and five small discs punched at each end of the main vein using a 6mm diameter punch. These discs were placed with tweezers in a 10mL centrifuge tube containing 2mL of deionized water. The tubes were completely immersed in a vacuum for 20 minutes. The pressure was then reduced to 0.08 MPa. 6mL of deionized water was added to the tubes, and the tubes were shaken at 25°C and 200 rpm for 1 hour. The conductivity was measured with a conductivity meter and recorded as S1. The tubes were sealed and placed in a boiling water bath for 15 minutes. After the solution cooled to room temperature, the conductivity was measured and recorded as S2. Electrolyte permeability was calculated as (S1 / S2) × 100%.

[0055] 5. Leaf water loss rate measurement

[0056] Leaf water loss (LW) was measured from the fourth leaf of a normal-growing 84K poplar and from various treatments. The fresh weight (FW) of the treated leaves was immediately weighed and then left at room temperature for 4 hours, with the desiccated weight (DW) recorded every 0.5 hours. Finally, the leaves were oven-dried at 75°C until constant weight was achieved, and the dry weight (DW) was calculated. The leaf water loss rate (LW) of each sample was calculated at each measurement time point. The calculation formula is: Leaf water loss rate = (FW - desiccated weight) / (FW - DW) × 100%.

[0057] 6. Glutamate Content Measurement

[0058] The 5th and 6th leaves of 84K poplars with normal growth and different treatments were taken to measure the content of glutamate. A glutamate content detection kit (Beijing Solebaugh Technology Co., Ltd., Cat. No. BC1580) was used. Glutamate dehydrogenase (GDH) catalyzes glutamate and nicotinamide adenine dinucleotide (NAD) to produce α-ketoglutarate, NADH and NH4 + , will cause the absorbance at 340nm to rise. By measuring the change of absorbance at 340nm within 5min, the glutamate content is calculated. Glutamate content / (μmol·mL -1) is the x-axis, the absorbance value ΔA of the sample increased within 5 minutes is the y-axis, and the standard curve y = kx + b is drawn. According to the sample mass, the following is calculated: glutamic acid content / (μmol·g -1 )=x / W, W: sample mass.

[0059] 7. Proline Content Measurement

[0060] The 5th and 6th leaves of 84K poplar with normal growth and different treatments were taken to determine the proline content. A proline content detection kit (Beijing Solebaugh Technology Co., Ltd., Cat. No. BC0290) was used. The proline content was determined based on the absorption peak at 520nm when proline reacts with acidic ninhydrin solution to generate red after heating treatment. About 0.1g of leaf tissue (W) was weighed, 1mL of extract was added, and the extract was shaken in boiling water for 10min. Centrifuged at room temperature for 10min, the supernatant was taken, cooled, and then added to the acidic ninhydrin solution in a boiling water bath for 30min. The concentration of the proline standard solution was taken as the horizontal axis x, and ΔA was taken as the vertical axis. 标准 =A 标准管 -A 空白管 Draw a standard curve for the vertical axis, and substitute the absorbance changes (ΔA) of different samples at a wavelength of 520 nm into the standard curve to obtain x / (μg·mL -1 ) Calculated according to sample mass: Proline content / (μg·g -1 )=x / W.

[0061] 8. Reactive oxygen content measurement

[0062] The 5th and 6th leaves of 84K poplar with normal growth and different treatments were taken to measure the concentrations of hydrogen peroxide (H2O2) and superoxide anion (O2· - ) content, using the hydrogen peroxide content detection kit and superoxide anion content detection kit (Beijing Solebow Technology Co., Ltd., catalog number BC3595, BC1295), the sample was thoroughly ground with liquid nitrogen, 0.1g (W) was taken, 1mL of the extract was added and vortexed, centrifuged at 4°C, and the supernatant was used as the test solution. The reagents were added to the centrifuge tube according to the steps and mixed, and the absorbance values ​​were measured at wavelengths of 415nm and 530nm, respectively, to calculate ΔA 标准 =A 标准管 -A 空白管 , ΔA 样本 =A 测定管 -A 空白管 Calculate H2O2 content according to sample mass, H2O2 content / (μmol·g -1 )=2×(ΔA 样本 / ΔA 标准 ) / W.

[0063] O2· - The content is determined by ΔA 标准As the y-axis, different standard solution concentrations as the x-axis, draw the standard curve y = kx + b, and change ΔA 样本 Substitute the standard curve to obtain the x value, O2· - Content / (μmol·mg -1 )=2x / W.

[0064] IX. Measurement of Antioxidant Scavenging Enzyme Activity

[0065] The activities of catalase (CAT) (Beijing Solebaugh Technology Co., Ltd., Catalog No. BC0200), peroxidase (POD) (Beijing Solebaugh Technology Co., Ltd., Catalog No. BC0090), and superoxide dismutase (SOD) (Beijing Solebaugh Technology Co., Ltd., Catalog No. BC0170) were measured from the fifth and sixth leaves of normally grown and treated 84K poplars. After thorough grinding with liquid nitrogen, 0.1 g (W) of the sample was added to 1 mL of the extract and vortexed. The supernatant was then centrifuged and used as the test solution. After adding the assay working solution as described, the mixture was immediately mixed for 5 seconds and the absorbance at different wavelengths was measured. CAT activity was defined as the degradation of 1 μmol H2O2 per gram of tissue in the reaction system at 240 nm; POD activity was defined as the change in absorbance of 0.01 per gram of tissue per minute per milliliter of reaction system at 470 nm; SOD activity was defined as the SOD activity in the reaction system when the inhibition percentage in the xanthine oxidase coupled reaction system was 50% at 560 nm. The activities of different antioxidant enzymes were calculated according to the sample mass: CAT / (U·g -1 )=764.5×ΔA / W,POD / (U·g -1 )=7133×ΔA / W,SOD / (U·g -1 ) = 11.11 × [inhibition percentage / (1-inhibition percentage)] / W, ΔA = A 测定 -A 对照 , inhibition percentage = (A 空白 -A 对照 ) / A 测定 ×100%.

[0066] 10. Data Analysis

[0067] Excel and SPSS statistical software were used for data processing and analysis. SPSS 17.0 data processing software was used to perform independent sample t-test on sample data, and the significance of differences was tested at the 0.05 level. Excel was used for data analysis and chart drawing.

[0068] Example 1: Effect of exogenous glutamate treatment before drought stress on drought resistance of 84K poplar

[0069] Glutamate was applied to the plants 0, 1, 2, and 3 days before drought stress, and the phenotypes were observed. The relative water content of the leaves was measured 6 days after drought stress. It was found that the drought symptoms of the plants irrigated with glutamate 0 day before drought stress appeared more slowly. The degree of drought stress damage from strong to weak was drought, G3+drought, G2+drought, G1+drought, G0+drought (see Figure 2 a, c). During drought, leaves of 84K poplars wilt and droop, demonstrating sensitivity to drought stress. However, plants treated with glutamate showed reduced sensitivity to drought. The relative water content of leaves in G0+ drought-treated plants was 1.60 times higher than that in drought-treated plants, demonstrating strong drought tolerance.

[0070] When all treatments showed wilting and drooping phenotypes, rewatering (50 mL) was performed and photos were taken every half hour. The functional leaf inclination angle was measured and it was found that the leaves of the plants treated with glutamic acid recovered first, and the recovery speed was from fast to slow in the order of G0+drought, G1+drought, G2+drought, G3+drought, drought (see Figure 2 b, d) indicate that 84K plants irrigated with glutamate 0 day before drought stress had a stronger rehydration response. In summary, glutamate treatment reduced the sensitivity of plants to drought, and 0 day was selected as the optimal application time for subsequent physiological index measurements.

[0071] Figure 2 (a) Phenotype of drought-stressed plants after exogenous glutamate treatment; (b) Phenotype of plants rewatered after drought stress; (c) Relative water content of L8 leaves after 6 days of drought stress; (d) Inclination angle of L5 leaves after rewatering. G3+drought: drought after 3 days of glutamate application; G2+drought: drought after 2 days of glutamate application; G1+drought: drought after 1 day of glutamate application; G0+drought: drought after glutamate application; drought: drought after normal watering. Arrows in Figure a indicate the beginning of leaf wilting after drought stress; arrows in Figure b indicate the beginning of leaf recovery after rewatering. Scale bar = 5 cm.

[0072] Example 2: Effect of exogenous glutamate on cold resistance of 84K poplar

[0073] 1. Physiological changes in 84K poplar leaves under drought stress induced by exogenous glutamate treatment

[0074] Further testing Figure 1 a shows the changes in physiological indicators of 84K poplars under four modes of drought stress: control, G0 (glutamate), drought, and G0 + drought (glutamate + drought) at 0d (D0), 4d, and 10d. Compared with the control, after the application of glutamate (D4), there was no significant change in the relative water content and electrolyte permeability of the leaves of the plants. Compared with the control, after 6d of drought stress (D10), the relative water content of the leaves of 84K poplars treated with drought decreased by 34.2%, while the relative water content of the plants treated with glutamate + drought decreased by only 14.2% ( Figure 3 a) As can be seen, irrigation of glutamic acid can effectively reduce the impact of drought on the decrease of leaf relative water content. After 6 days of drought stress, the electrolyte permeability of drought-treated plants increased significantly, 2.73 times that of the control 84K poplar; while the electrolyte permeability of glutamic acid+drought-treated plants changed less, and was not significantly different from that of the control 84K poplar Figure 3 b) As can be seen, irrigation of glutamic acid can effectively maintain the stability of leaf electrolyte permeability and reduce the damage of drought stress on poplar. Before drought, compared with the control plants, the leaf water loss rate of glutamic acid-treated plants was significantly reduced; after 6 days of drought, the leaf water loss rate of the drought plants was lower than that of the non-drought plants Figure 3 c, d) Compared with the drought plants, the leaf water loss rate of the glutamic acid+drought plants was significantly reduced (Figure d). The above results show that the application of exogenous glutamic acid can reduce the leaf water loss rate, enhance the water retention capacity, reduce the damage of drought stress on the cell membrane permeability, and enhance the drought tolerance of 84K poplar.

[0075] II. The effect of exogenous glutamic acid treatment on the accumulation of osmotic regulation substances in 84K poplar under drought stress

[0076] When plants are water-deficient, plant cells will synthesize some osmotic regulation substances to reduce osmotic potential, maintain cell osmotic balance, and prevent cell water loss. After the application of exogenous glutamic acid, the glutamic acid content of plant leaves was 1.85 times that of the control treatment Figure 3 e) Therefore, according to the difference in absorbance at different standard concentrations, a standard curve was drawn to determine the glutamic acid and proline content of different treatment plants. After the application of glutamic acid, the proline content of the four treatments showed no significant change Figure 3 f) Compared with the glutamic acid treatment (D4), the glutamic acid content increased less after 6 days of drought (D10), but the glutamic acid+drought treatment had a significant increase, 1.28 times that of the control. The proline content of D10 increased except for the control, among which the proline content of the glutamic acid+drought treatment was the highest, 2.41 times that of the control and 1.21 times that of the drought treatment Figure 3 e, f).

[0077] After the application of glutamic acid, compared with the control, the expression of proline synthesis-related genes pyrroline-5-carboxylate synthetase (P5CS), pyrroline-5-carboxylate reductase (P5CR), and pyrroline-5-carboxylate dehydrogenase (P5CDH) increased significantly, 2.47 times, 2.57 times, and 8.89 times that of the control, respectively, and the expression of proline reductase (PDH) decreased by 48.8% Figure 4 ) After 6 days of drought stress, compared with the drought treatment, the expression of proline synthesis genes P5CS and P5CR in the glutamic acid+drought treatment increased by 23.0% and 32.5%, respectively Figure 4 b, d), the expression of proline degradation gene PDH decreased by 20.4%, and the expression of P5CDH decreased slightly ( Figure 4 c) The above results indicate that glutamate treatment increases glutamate content in plant leaves, but this is metabolized as the plants grow. When plants are subjected to drought stress, glutamate acts as a precursor for proline synthesis, inducing the synthesis and accumulation of proline, improving osmotic regulation and reducing the plant's sensitivity to drought.

[0078] Figure 3 (a): Relative leaf water content; (b): Electrolyte permeability; (c): Leaf water loss after glutamate treatment (D4); (d): Leaf water loss after drought stress (D10); (e): Glutamate content; (f): Proline content. D0: Untreated; D4: Drought onset after glutamate treatment; D10: Six days of drought stress. Values ​​are expressed as mean ± standard error (n = 3). Different letters indicate significant differences at P < 0.05 according to Duncan's multiple range test. Same below.

[0079] Figure 4 In the figure, a: relative expression level of pyrroline-5-carboxylate dehydrogenase (P5CDH); b: relative expression level of pyrroline-5-carboxylate synthase (P5CS); c: relative expression level of proline dehydrogenase (PDH); d: relative expression level of pyrroline-5-carboxylate reductase (P5CR). D0: untreated; D4: drought stress followed by glutamate treatment; D10: 6 days of drought stress. The four adjacent data bars represent the control, G0 (glutamate), drought, and G0 + drought (glutamate + drought) treatment modes, respectively.

[0080] summary:

[0081] (1) D4, compared with the control, the glutamate content in the G0 (glutamate) group increased significantly; D10, compared with the control, the glutamate content in the G0 (glutamate) group increased slightly, which indicates that in the absence of drought stress, the glutamate content brought about by glutamate irrigation will gradually metabolize and stabilize.

[0082] (2) D4, compared with the drought group, the proline content in the G0+drought (glutamic acid + drought) group was slightly increased. D10, compared with the drought group, the proline content in the G0+drought (glutamic acid + drought) group was significantly increased.

[0083] (3) Regardless of D4 or D10, compared with the control, the expression of PDH in the G0 (glutamate) group was downregulated, while the expression of P5CR was upregulated. Compared with the drought group, the expression of PDH in the G0+drought (glutamate+drought) group was downregulated, while the expression of P5CR was upregulated. Both were beneficial to increasing the proline content in the tissues, indicating that glutamate was involved in the proline synthesis pathway.

[0084] Both P5CDH and P5CS were upregulated in the G0 (glutamate) group compared to the control on both Day 4 and Day 10. P5CS was upregulated in the G0+drought (glutamate+drought) group compared to the drought group. On Day 10, P5CDH was downregulated in the G0+drought (glutamate+drought) group compared to the drought group, indicating that glutamate affects the proline degradation pathway. In the absence of drought stress, both P5CDH and P5CS were upregulated, minimizing the physiological balance of glutamate concentrations and preventing exogenous glutamate from disrupting homeostasis. However, in the presence of drought stress on Day 10, P5CDH was downregulated, indirectly reducing proline consumption, while P5CS was upregulated, indirectly promoting proline synthesis. PDH was downregulated, directly reducing proline consumption, while P5CR was upregulated, directly promoting proline synthesis. This suggests that in the absence of drought stress, glutamate has little effect on glutamate and proline, but after drought stress, exogenous glutamate can significantly increase proline content. Based on the unknown metabolic pathways and regulatory mechanisms, we have achieved a good effect of regulating the endogenous proline content through exogenous glutamate to improve the drought resistance of poplar trees.

[0085] Effects of exogenous glutamate treatment on hydrogen peroxide content and antioxidant scavenging enzyme activity in 84K poplar under drought stress

[0086] Reactive oxygen, as a byproduct of plant cell metabolism, increases significantly after plants are stressed and can cause oxidative damage to cells. To investigate the oxidative damage of plants after glutamate treatment with drought stress, the present invention measured Figure 1 a shows the H2O2 content of 84K poplar leaves under glutamate treatment and drought conditions in the four models of control, G0 (glutamate), drought, and G0 + drought (glutamate + drought). Compared with the control, the H2O2 accumulation in glutamate-treated plants was significantly reduced after glutamate application. After 6 days of drought stress (D10), the H2O2 content in drought-treated 84K poplars increased by 46.1%, while the H2O2 content in glutamate + drought plants increased by only 17.9%, which was significantly lower than that in drought-treated plants ( Figure 5 a).

[0087] The activities of active oxygen scavenging enzymes CAT, POD, and SOD showed the same trend during the drought test. Compared with the control, glutamate treatment significantly increased the activities of CAT, POD, and SOD in leaves by 34.5%, 32.5%, and 9.7%, respectively. After drought stress, glutamate-treated 84K poplar plants maintained higher levels of antioxidant enzyme activity, with CAT, POD, and SOD activities being 1.67 times, 1.35 times, and 1.17 times higher than those in drought-treated plants, respectively. Figure 5b, c, d). Higher CAT and POD activities accelerated the decomposition of hydrogen peroxide, resulting in a 19.3% decrease in hydrogen peroxide content compared to drought-treated plants. Therefore, glutamate treatment alleviated oxidative damage caused by the large amount of reactive oxygen species produced by drought stress, thereby synergistically enhancing drought tolerance in 84K poplar.

[0088] Figure 5 In the figure, a: hydrogen peroxide content; b: catalase activity; c: peroxidase activity; d: superoxide dismutase activity. D0: untreated; D4: drought stress after glutamate treatment; D10: 6 days of drought stress.

[0089] Example 3: Exogenous glutamate affects the growth recovery of 84K poplar after drought stress

[0090] 1. Physiological changes in leaves treated with exogenous glutamate after drought stress

[0091] To investigate the effect of exogenous glutamate (Glu) on the recovery of 84K poplar after drought stress, a rehydration test was conducted on drought-stressed plants. Aspartic acid (Asp), an acidic amino acid similar to glutamate, was added as a control to verify that glutamate plays a specific role in the rehydration process. Phenotypic observations showed that after rehydration of wilted plants, the fifth and sixth leaves responded first. Compared with the rehydration treatment, the plants treated with glutamate recovered first ( Figure 6 a).

[0092] Compared with 84K poplar leaves treated with drought stress for 6 days (D6), the relative water content of leaves treated with rehydration, glutamic acid and aspartic acid increased by 22.4%, 36.9% and 24.6%, respectively; the electrolyte permeability decreased by 18.6%, 28.8% and 18.0%, respectively (D6+3h). Figure 6 These physiological indicators showed that glutamate treatment alleviated cell membrane damage caused by drought stress, increased relative water content in leaves, and enhanced plant recovery ability.

[0093] Figure 6 In the middle, a: plant phenotype. H2O: 50 mL water was poured after drought stress; Glu: 50 mL L-glutamic acid aqueous solution (10 mmol·L) was poured after drought stress. -1 ); Asp: After drought stress, 50 mL of L-aspartic acid aqueous solution (10 mmol·L -1 b: Leaf relative water content; c: Electrolyte permeability; d: Glutamate content; e: Proline content. D0: Untreated; D6: 6 days of drought stress; D6+3h: 3 hours of rehydration and recovery. Scale bar = 5 cm.

[0094] 2. Effects of exogenous glutamate on glutamate and proline content in poplars under drought stress

[0095] There was no significant change in glutamate content in the three treatments without treatment (D0) and after 6 days of drought stress, but the glutamate content increased by 41.8% ( Figure 6 d). After 3 hours of rehydration, the proline content increased in both the rehydration treatment and the glutamic acid treatment, but the increase in the glutamic acid treatment was more significant, 1.48 times that of the rehydration treatment, while there was no significant change in the aspartic acid treatment ( Figure 6 e). This indicates that glutamate treatment increases the glutamate content in plants, induces the synthesis and accumulation of proline, maintains cell osmotic balance, and prevents water deficit.

[0096] Effects of exogenous glutamate on reactive oxygen species content and antioxidant scavenging enzyme activity under drought stress

[0097] When plants are subjected to drought stress, the dynamic balance of active oxygen in the plant body is broken, the active oxygen content increases, and the accumulation of hydrogen peroxide and superoxide anions increases. After 3 hours of rehydration, the content of hydrogen peroxide and superoxide anions in the rehydrated plants decreased significantly, while the reduction in hydrogen peroxide in the glutamic acid-treated plants was significantly greater than that in the rehydrated 84K poplar. The hydrogen peroxide content was ranked from high to low in the order of H2O>Asp>Glu( Figure 7 a, b). After rehydration, the activities of CAT and POD increased by 19.0% and 62.7% respectively after glutamate treatment compared with those after rehydration treatment ( Figure 7 c, d). The results showed that glutamate treatment enhanced the activity of antioxidant enzymes, alleviated the oxidative damage caused by increased reactive oxygen species, and accelerated the recovery ability of 84K poplar.

[0098] Figure 7 a: hydrogen peroxide content; b: superoxide anion content; c: catalase activity; d: peroxidase activity. H2O: rehydration after drought stress; Glu: 50 mL L-glutamic acid solution (10 mmol·L) was irrigated after drought stress. -1 ); Asp: After drought stress, 50 mL of L-aspartic acid solution (10 mmol·L -1 ); D0: before treatment; D6: 6 days of drought stress; D6+3h: rehydration and recovery for 3 hours.

[0099] discuss

[0100] Through the experiment of applying glutamate at different times before drought, it was found that the shorter the interval between glutamate application and drought treatment, the stronger the drought tolerance of the plant; the longer the interval between glutamate application and drought treatment, the weaker the drought tolerance of the plant ( Figure 2 After 6 days of drought stress, the proline content in the glutamic acid + drought treatment plants was the highest, 1.21 times that of the drought treatment plants ( Figure 3 f), and the expression of proline synthase (P5CS) and pyrroline-5-carboxylate reductase (P5CR) increased after glutamate administration ( Figure 4 b, d), indicating that exogenous glutamate enhances the osmotic regulation ability of 84K poplar by accumulating proline under drought stress, thereby alleviating the stress of drought on 84K poplar to a certain extent. In the present invention, glutamate treatment increases the expression of proline synthesis genes (P5CS, P5CR) in 84K poplar, decreases the expression of degradation genes (PDH), increases the content of glutamate and proline, increases the concentration of osmotic substances in the leaf cytoplasm, and decreases the osmotic potential, thereby alleviating the damage caused by water stress. Therefore, the water loss rate of leaves of glutamate-treated plants under drought stress is significantly lower than that of control plants ( Figure 3 d). In contrast, the relative water content of leaves in plants treated with glutamate + drought was significantly higher than that in drought-treated plants. This indicates that the increase in glutamate and proline content can maintain cellular osmotic balance, hinder the further occurrence and development of water deficit, enhance plant drought tolerance, and facilitate rapid recovery of plants after rehydration. In addition to glutamate being used as an amino acid precursor to synthesize proline, glutamate itself also acts as an osmotic regulating substance, accumulating in plant cells in a free form, reducing water loss by lowering cell osmotic potential. The accumulation of reactive oxygen species in plants treated with glutamate was significantly reduced compared to the control ( Figure 5 a), the antioxidant enzyme activity increased significantly, which also proved that glutamate alleviated the oxidative stress damage of drought to plants to a certain extent. Compared with the drought treatment, the proline content of 84K poplar treated with glutamate increased significantly after drought ( Figure 3 f), glutamate can serve as a precursor for proline synthesis. Therefore, we believe that glutamate induces the synthesis of proline, regulates the permeability of plants under drought stress, enhances the activity of antioxidant enzymes, eliminates excessive reactive oxygen species, and thus enhances the tolerance of plants to drought stress.

[0101] When plants are stressed, the balance between reactive oxygen free radicals and antioxidant enzymes is disrupted. The accumulation of reactive oxygen species damages the cell membrane system, causing electrolyte extravasation in the cells and increasing conductivity. In the rehydration experiment, glutamate treatment reduced cell membrane damage in 84K poplars, significantly reducing electrolyte permeability and contributing to the rapid recovery of the plants after rehydration ( Figure 6 a, c). To avoid damage from reactive oxygen species, plants develop an effective defense mechanism, in which the main function is the reactive oxygen species scavenging enzyme system, such as POD, SOD, and CAT, which can effectively remove H2O2 and O2 in plants. - , inhibiting the accumulation of high concentrations of reactive oxygen species, preventing membrane lipid peroxidation and cell death. In the present invention, whether glutamate is applied before drought or rewatered after drought, the addition of glutamate can rapidly increase the enzyme activities of POD and CAT in the plant body ( Figure 5 b, c; Figure 7 c, d), reducing the accumulation of active oxygen. The present invention shows that exogenous glutamic acid can improve the drought tolerance of the woody plant 84K poplar and enhance its resistance to environmental stress.

[0102] Applying glutamate before drought treatment increased glutamate content in plant leaves, induced the expression of proline biosynthesis genes, increased proline accumulation, reduced osmotic potential, reduced leaf water loss, and enhanced leaf water retention. It also increased the activity of antioxidant enzymes (CAT, POD, and SOD) and reduced H₂O₂ accumulation, thereby alleviating the negative symptoms of drought stress and enhancing plant drought tolerance. Applying glutamate after drought stress enhanced the scavenging activity of the antioxidant enzymes CAT and POD, reducing cell membrane damage caused by lipid peroxidation and accelerating plant recovery from drought stress. In summary, glutamate can act as both a drought stress alleviator and a restorative agent in woody plants, modulating plant physiological metabolism to mitigate the damage caused by drought stress. This provides important evidence for the role of glutamate in drought tolerance in forest trees.

[0103] It is understood from common technical knowledge that the present invention may be implemented by other embodiments that do not depart from its spirit or essential features. Therefore, the embodiments disclosed above are, in all respects, merely illustrative and not exclusive. All modifications within the scope of the present invention or equivalent to the scope of the present invention are intended to be encompassed by the present invention.

Claims

1. A method for improving the stress resistance of poplars by improving their physiological indicators, the method comprising watering the poplars with a glutamic acid solution; The poplar is 84K poplar; The improvement of poplar physiological indicators includes: Increase the relative water content of leaves; Reduce leaf water loss rate; Maintaining the stability of leaf electrolyte permeability; Increase the endogenous proline content in leaves; Reduce the expression of endogenous proline dehydrogenase, increase the expression of endogenous pyrroline-5-carboxylic acid reductase, reduce the expression of endogenous pyrroline-5-carboxylic acid dehydrogenase, and increase the expression of endogenous pyrroline-5-carboxylic acid synthetase; Improve the rehydration capacity of drought-stressed leaves; Increase the content or activity of endogenous catalase; increasing the level or activity of endogenous peroxidase; and Increase the content or activity of endogenous superoxide dismutase; The glutamic acid is L-glutamic acid; The glutamate solution is a 10 mmol / L glutamate aqueous solution; Maintain the soil moisture content of the poplar plants at 75%; The stress resistance includes drought resistance and antioxidant capacity.

2. The method according to claim 1, wherein The growth conditions of the poplar trees were as follows: 14-18 h of sunlight per day, a temperature of 20-28 °C, and a light intensity of 40-60 μmol·m -2 s -1 .

3. The method according to claim 2, wherein The growth conditions of the poplar trees were as follows: 16 h of sunlight per day, temperature 23-25 ​​°C, and light intensity 50 μmol·m -2 s -1 .

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