Analysis method of water physiological status of wheat leaves under combined atmospheric and soil drought stress

Abstract

The present invention discloses a method for analyzing the physiological state of wheat leaf water under the combined drought stress of the atmosphere and soil, including cultivating wheat seeds, and after germination and seedling determination, subjecting the cultivated wheat plants to drought stress, setting three levels of soil moisture and two levels of saturated water vapor pressure difference, and simulating the experimental method of soil and atmospheric dual drought stress by controlling the upper and lower limits of soil moisture and different levels of saturated water vapor pressure difference; starting relevant measurements at the heading stage, keeping different soil moisture levels within the set upper and lower limits during the measurement, measuring flag leaf gas exchange parameters, leaf water potential, measuring wheat leaf hydraulic conductivity, and measuring leaf hydraulic conductivity vulnerability curve, and deeply analyzing the experimental data. The present invention deeply reveals the physiological adaptation mechanism and water utilization strategy of wheat under soil drought, atmospheric drought and their combined stress, and finds that wheat can regulate stomatal behavior according to different drought types to change the path of drought affecting photosynthetic rate.

Description

Technical Field

[0001] The invention relates to the technical field of agricultural planting, and in particular to a method for analyzing the physiological state of water in wheat leaves under combined drought stress of the atmosphere and soil. Background Art

[0002] The saturated vapor pressure deficit (VPD) is the difference between the atmospheric saturated vapor pressure and the actual vapor pressure. It reflects the atmospheric moisture status and is a key climate factor affecting plant stomatal conductance. It is also a key driving force for regulating water transport in plants. The intensification of global climate change has led to the frequent occurrence of combined stress of VPD and soil water (SM) deficit. Under drought stress, plants control gas exchange and water absorption, conduction and loss by regulating water transport and stomatal opening and closing to maintain water balance, thereby ensuring their survival and growth. The response of crops to the combined stress of VPD and SM is far more complex than that of a single stress. The response to this combined stress is not a simple superposition of the effects of the two stresses, but may produce synergistic or antagonistic effects, and involves complex physiological feedback and multiple adaptive mechanisms. Clarifying these response mechanisms is of vital significance for predicting and adapting to future climate change. Summary of the invention

[0003] The present invention provides a method for analyzing the water physiological state of wheat leaves under combined atmospheric and soil drought stress, aiming to understand how soil drought, high VPD and the dual stress of the two affect the water relationship and physiological and biochemical factors of wheat leaves; clarify the stomatal-hydraulic synergistic relationship of wheat leaves, and illustrate the water utilization strategy of crops in response to dual atmospheric and soil drought; and determine the regulation path and indicator effect of the hydraulic-stomatal-photosynthetic synergistic relationship under different VPDs.

[0004] To achieve the above object, the present invention provides the following technical solutions:

[0005] The method for analyzing the physiological state of water in wheat leaves under combined atmospheric and soil drought stress provided by the present invention comprises the following steps:

[0006] S1. Cultivated wheat seeds,

[0007] Cultivation method: Before sowing, apply ammonium sulfate and potassium dihydrogen phosphate to supplement nitrogen, phosphorus and potassium fertilizers for crops, and apply 152 mg nitrogen, 120 mg phosphorus and 120 mg potassium to each pot with water;

[0008] After sowing, Hoagland nutrient solution (5 mM KNO 3 , 5 mM Ca(NO 3 ) 2 ·4H 2 O, 1 mM KH 2 PO 4 and 1 mM MgSO 47H 2 O, 1ml·l -1 trace elements, pH = 6.0) for irrigation;

[0009] S2. After germination and seedling determination (about 2 weeks), the cultivated wheat plants were subjected to drought stress. Three levels of soil moisture and two levels of saturated water vapor pressure difference were set. The experimental method of simulating soil and atmosphere dual drought stress was achieved by controlling the upper and lower limits of soil moisture and different levels of saturated water vapor pressure difference.

[0010] S3, maintaining drought treatment until the beginning of heading stage, and using Li-COR6800 photosynthesis measurement system to measure flag leaf gas exchange parameters under different conditions;

[0011] S4, measuring leaf water potential before dawn and at noon using a portable plant water potential pressure chamber;

[0012] S5. The transpiration-driven method was used to measure the hydraulic conductivity of wheat leaves and the leaf hydraulic conductivity vulnerability curve was drawn.

[0013] S6. After the determination of leaf gas exchange parameters, select leaves adjacent to the flag leaf with the same treatment and immediately freeze them with liquid nitrogen and store them in a -40°C refrigerator to determine the auxin and abscisic acid content in the leaves;

[0014] S7. Use a SPAD-502 chlorophyll meter to measure the chlorophyll value of the same part near the photosynthetic leaf; collect other leaves from the same part, dry and grind them, and then boil them, and use a Kjeldahl nitrogen analyzer to measure the nitrogen content per unit mass of the leaves;

[0015] S8. Conduct an in-depth analysis of the experimental data to reveal the physiological adaptation mechanism and water utilization strategy of wheat under dual drought stress, and analyze the synergistic relationship between stomatal conductance and leaf hydraulic conductance and net photosynthetic rate through the structural equation model.

[0016] Furthermore, in step S1, the wheat variety material is "Jinchun No. 6", and during the cultivation process, the soil used is nutrient soil with a field water holding capacity of 0.33 cm 3 cm -3 , bulk density is 1.4 g cm -3 .

[0017] Further, in step S2, the three levels of soil moisture are high soil moisture, medium soil moisture, and low soil moisture, and the corresponding upper and lower limits of soil moisture are 85%-95% field water holding capacity, 65%-75% field water holding capacity, and 50%-60% field water holding capacity, respectively; the two levels of saturated vapor pressure difference are conventional VPD and VPD stress, respectively, the conventional VPD is set to 0.79 kPa, and the corresponding environmental conditions are normal temperature and high humidity, consisting of 25°C and 75% relative humidity, and the VPD stress is set to 1.43 kPa, and the corresponding environmental conditions are normal temperature and low humidity, consisting of 25°C and 55% relative humidity.

[0018] Furthermore, in step S3, the gas exchange parameters include the net photosynthetic rate, transpiration rate and instantaneous water use efficiency of the wheat flag leaf. During the measurement, the leaf chamber temperature is set at 26-28°C and the light intensity is set at 1200 μmol / m -2 s -1 , CO 2 The concentration was set at 400 ppm.

[0019] Furthermore, the method for measuring leaf water potential is: measuring the pre-dawn leaf water potential and the noon leaf water potential through a portable plant water potential pressure chamber; selecting a flag leaf with consistent growth in each treatment, cutting it with scissors and immediately moving it into the portable plant water potential pressure chamber, and extending the leaf incision at the air chamber cover; after the air chamber is tightened, slowly pressurizing the air chamber, and observing the incision with the help of a magnifying glass; when small water droplets just appear at the incision, immediately stop pressurizing and read the pressure value.

[0020] Furthermore, in step S5, the method for determining the blade hydraulic conductivity comprises the following steps:

[0021] S511, selecting flag leaf wheat plants after the gas exchange measurement, cutting them with scissors and immediately placing them in water, and performing secondary shearing under water to remove bubbles in the leaves;

[0022] S512, placing the cut plants in a dark environment for rehydration, placing the leaves under an irradiation lamp to induce the opening of the leaf stomata during measurement, and after balancing for 30 minutes, cutting off the new fully expanded leaves with leaf sheaths underwater, and quickly connecting the cut leaves underwater to a pre-filled water pipe, and the other end of the water pipe is connected to a water source placed on a balance;

[0023] S513, after the blade is connected to the water pipe, record the weight change on the balance and calculate the instantaneous water flow velocity, and stop recording after the water flow velocity is stable for at least 15 minutes;

[0024] S514. After the balance is turned off for counting, the blade is quickly removed from the water pipe and stored in a ziplock bag. After the blade is balanced, it is used to determine the final leaf water potential and leaf area. The leaf hydraulic conductivity is calculated as follows:

[0025] K leaf =F / (LA×Ψ final ) (1);

[0026] In the formula, K leaf is the leaf hydraulic conductivity, F is the instantaneous water velocity, LA is the leaf area, Ψ final It is the final leaf water potential of the leaf.

[0027] Furthermore, in step S5, the blade hydraulic conductivity vulnerability curve is calculated as follows:

[0028] S521, Measure transpiration rate using the LI-COR 6800 photosynthesis measurement system;

[0029] S522. Take out the leaves, quickly put them into a sealed bag with a wet paper towel, and then store them in a dark environment for about 30 minutes to reach equilibrium. Then measure the leaf water potential, and calculate the maximum hydraulic conductivity of the leaves as follows:

[0030] K max = E / (0-Ψ leaf ) (2);

[0031] In the formula, K max is the maximum hydraulic conductivity of the leaf, E is the transpiration rate, Ψ leaf is the leaf water potential;

[0032] S523, placing the remaining wheat plants after the above rehydration on the laboratory table for different lengths of time and dehydrating them to different degrees, then wrapping the plants with different dehydration degrees with black plastic bags and balancing for at least 30 minutes; measuring the initial water potential of the top and bottom leaves of the plants at different dehydration times, and using the middle leaves to measure the leaf hydraulic conductivity of wheat at different dehydration degrees according to the above method for measuring hydraulic conductivity. The leaf hydraulic conductivity loss percentage PLC is calculated using the following formula:

[0033] PLC=100×(1-K leaf / K max ) (3);

[0034] S524. The fragility curves of various species were fitted using the least squares method based on empirical functions to determine the xylem water potential and the slope of the fragility curve at the inflection point when the hydraulic conductivity was lost by 50%:

[0035] PLC=a / (1+exp(S(Ψ leaf -c)) (4);

[0036] Where Ψ leaf is the leaf water potential, a is the maximum PLC, S is the slope of the fragility curve at the inflection point, and c is K leaf Reduces water potential by 50%.

[0037] Based on the above technical solution, the embodiments of the present invention can at least produce the following technical effects:

[0038] (1) The method for analyzing the water physiological state of wheat leaves under combined atmospheric and soil drought stress provided by the present invention clarifies the response characteristics of water physiology of wheat leaves under combined atmospheric and soil drought. leaf ), photosynthetic rate (A n ) and stomatal conductance (g s ) were significantly reduced, and the reduction under dual stress was greater than that under any single stress. PD , ABA, IAA, SPAD value and N mass There were significant interactive effects. Under the dual drought of atmosphere and soil, ABA and IAA increased significantly, while Ψ PD 、N mass These changes reflect the physiological responses of plants to drought conditions, including the decline of gas exchange parameters and the adjustment of other related factors.

[0039] (2) The method for analyzing the water physiological state of wheat leaves under combined atmospheric and soil drought stress provided by the present invention discovered the changes in the water use strategy of wheat leaves under dual atmospheric and soil drought. s The downward trend is higher than K leaf The loss is slower, indicating that wheat adopts non-isohydric stomatal regulation to maintain photosynthetic rate. s and K leaf These results indicate that under low VPD, wheat prefers to maintain carbon assimilation at the expense of hydraulic imbalance through isohydric stomatal regulation, while under high VPD, wheat prefers to avoid hydraulic imbalance by sacrificing carbon assimilation through stomatal closure through non-isohydric regulation.

[0040] (3) The method for analyzing the water physiological state of wheat leaves under combined atmospheric and soil drought stress provided by the present invention reveals the main pathways for regulating photosynthetic rate of wheat under different VPDs. n With g s Strong coupling, K leaf The changes are mainly caused by MD caused by the change of; while at high VPD, due to Ψ MD A nThe direct impact of A n With g s The coupling relationship between them, K leaf The changes are mainly caused by changes in ABA. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on the structures shown in these drawings without paying creative work.

[0042] Figure 1 is the net photosynthetic rate of the leaves in the embodiment of the present invention (A n ), stomatal conductance (g s ), hydraulic conductivity (K leaf ) between them; (a) is the relationship between stomatal conductance and net photosynthetic rate, (b) is the relationship between hydraulic conductance and net photosynthetic rate, and (c) is the relationship between hydraulic conductance and stomatal conductance;

[0043] Figure 2 is the net photosynthetic rate of the leaves in the embodiment of the present invention (A n ), stomatal conductance (g s ), hydraulic conductivity (K leaf ) and leaf noon water potential (Ψ MD ), (a) is the relationship between leaf hydraulic conductance and leaf noon water potential, (b) is the relationship between stomatal conductance and leaf noon water potential, (c) is the relationship between leaf net photosynthetic rate and leaf noon water potential;

[0044] Figure 3 is the fragility curve of leaves treated with different VPDs in the embodiment of the present invention;

[0045] Figure 4 is the leaf hydraulic conductivity (K) under VPD and SM treatments in the embodiment of the present invention leaf ), net photosynthetic rate (A n ), stomatal conductance (g s ) and P50 regression analysis.

[0046] Figure 5 is the correlation analysis between the physiological and biochemical traits of leaves under low VPD and high VPD in the embodiment of the present invention;

[0047] Figure 6 The structural equation model (SEM) of the present invention is used to test the net photosynthetic rate (A) under low VPD (a) and high VPD (b). n) and their standardized total effect (c).

[0048] In the figure: H-SM is high soil moisture content; M-SM is medium soil moisture content; L-SM is low soil moisture content; low VPD is normal VPD; high VPD is VPD stress. DETAILED DESCRIPTION

[0049] The technical solutions in the embodiments of the present invention will be described clearly and completely below. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments in the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention. In addition, the technical solutions between the various embodiments can be combined with each other, but they must be based on the ability of ordinary technicians in this field to implement them. When the combination of technical solutions is contradictory or cannot be implemented, it should be considered that such a combination of technical solutions does not exist and is not within the scope of protection required by the present invention.

[0050] The objective of the present invention is achieved through the following technical solutions:

[0051] The experiment was conducted in an artificial climate chamber at the China Institute of Water Resources and Hydropower Research's test base in Daxing District, Beijing. The climate chamber is equipped with an automatic ventilation system, a temperature and humidity control system, and an LED plant growth lamp. The rated light intensity of the LED is 1000 μmol / m -2 ·s -1 . The daily light hours are 7 am and 7 pm, a total of 12 hours. Two VPD levels were set in this experiment: conventional VPD, 0.79 kPa (low VPD, normal temperature and high humidity, consisting of 25°C and 75% relative humidity) and VPD stress, 1.43 kPa (high VPD, normal temperature and low humidity, consisting of 25°C and 55% relative humidity). The night temperature and humidity settings were the same for the two VPD environments, 18°C ​​and 80% respectively. The irrigation water volume was controlled according to the upper and lower limits of soil moisture content (SM). Three levels of high (H-SM), medium (M-SM), and low (L-SM) were set, and the corresponding upper and lower limits of SM were 85%-95% field water holding capacity (θ FC ), 65%-75%θ FC 、50%-60%θ FC Each treatment had 9 replicates.

[0052] The spring wheat variety used for the test is "Jinchun No. 6", the soil used in the test is matrix soil, the volume of the test pot used is 5.6L (top diameter 24cm, bottom diameter 20cm, depth 25cm, pot weight 0.2kg), and the potted matrix soil is 2.3kg (including pot weight). FC 33%cm 3 cm-3 , dry bulk density is 1.4 g cm -3 Before seedling emergence and treatment, irrigate to θ FC The amount of irrigation was determined by weighing every day. Ammonium sulfate and potassium dihydrogen phosphate were applied to supplement nitrogen, phosphorus and potassium fertilizers for crops before sowing. 152 mg nitrogen, 120 mg phosphorus and 120 mg potassium were applied to each pot with water. 25 wheat seeds were sown in each pot at a sowing depth of 2-3 cm. After sowing, each pot was irrigated with Hoagland nutrient solution (5 mM KNO 3 , 5 mM Ca(NO 3 ) 2 ·4H 2 O, 1 mM KH 2 PO 4 and 1 mM MgSO 4 7H 2 O, 1ml·l -1 Trace elements, pH = 6.0) to θ FC . After emergence, thin out the seedlings until 10 plants with similar growth are retained in each pot, and start water deficit treatment 2 weeks after sowing. During the deficit treatment, all pots are irrigated with the same volume of Hoagland solution. The water applied with the nutrient solution is included in the irrigation volume. The experimental water is tap water, and the concentration of mineral ions carried in the water is ignored. There was no water leakage during the experiment. The dynamic change data of the temperature, relative humidity and saturated water vapor pressure deficit in the climate chamber during the experimental treatment period were recorded by the automatic detection equipment in the climate chamber. For the H-SM, M-SM, and L-SM treatments, the weighing method was used to determine the change process of SM. The barrels were weighed at 8:00 every day, and the weight change of each barrel was recorded. SM was always controlled at around 2.58-3kg, 2.02-2.3kg, and 1.6-1.88kg (including the pot weight).

[0053] 1. After all drought stress treatments were completed, the leaves of the test samples were uniformly measured. The measurement indexes and the measurement methods were as follows:

[0054] Example 1: Determination of gas exchange parameters

[0055] The photosynthetic instantaneous value was measured using the Li-COR6800 photosynthetic measurement system from 10:00 to 14:00. The leaf chamber parameters were set according to the light intensity and temperature and humidity at the time of measurement. The leaf chamber temperature was set at 26-28°C and the light intensity was set at 1200 μmol / m -2 s -1 , CO 2 The concentration was set at 400 ppm. Five replicates were measured for each treatment. n ) and transpiration rate (T r ) is the instantaneous water use efficiency (WUE ins ).

[0056] VPD affects photosynthetic rate (A n ), intercellular carbon dioxide concentration (C i ) and stomatal guide (g s ) was significantly affected. Under the H-SM treatment, compared with the low VPD treatment, the A n , C i , g s Significantly reduced WUE by 8.97%, 10.50% and 35.09% ins The change was not significant. Under the M-SM treatment, compared with the low VPD treatment, the A n , C i , g s Significantly reduced WUE by 15.78%, 16.87% and 52.83% ins Under L-SM treatment, since the values ​​of various parameters in low VPD treatment were also lower, the gas exchange parameters and WUE in high VPD treatment were ins The values ​​did not change significantly.

[0057] SM vs A n , C i , g s and WUE ins The impact is significant. Under low VPD, the A of M-SM is lower than that of H-SM. n , C i , g s Reduced by 5.68%, 0.62% and 5.35%, WUE ins Increased by 1.70%; L-SM's A n , C i , g s Significantly lower by 31.14%, 22.44% and 66.07%, WUE ins Compared with H-SM, the A of M-SM increased significantly by 50.27% under high VPD. n , C i , g s Significantly reduced WUE by 12.73%, 7.68% and 32.43% ins The A of L-SM increased significantly by 15.69%. n , C i , g s Significantly reduced WUE by 21.21%, 11.21% and 43.24% ins Significantly increased by 24.02%;

[0058] VPD and SM to A n , g s and WUE insThere was a significant interaction effect. Compared with the treatment without water stress (low VPD H-SM treatment), the A n , C i and g s The WUE values ​​were significantly reduced by 20.6%-28.2%, 17.39%-20.54% and 54.8%-61.7%, respectively. ins Significantly increased by 31.27%-40.58%.

[0059] Example 2: Determination of leaf water potential

[0060] Determination of predawn leaf water potential (Ψ PD ) and noon leaf water potential (Ψ MD ) were taken before turning on the lights (before 7:00) and at noon (11:30-13:30). In each treatment, the flag leaves with the same growth were selected, cut with scissors and immediately moved into the portable plant water potential pressure chamber, and the leaf cut was extended at the air chamber cover. After the air chamber was tightened, the air chamber was slowly pressurized while observing the cut with the help of a magnifying glass. When small water droplets just appeared at the cut, the pressure was stopped and the pressure value was read. Five replicates were measured for each treatment.

[0061] Example 3: Determination of blade hydraulic conductivity

[0062] The transpiration driven method (EFM) was used to measure the leaf hydraulic conductivity (K leaf ), select the flag leaf wheat plants after the gas exchange measurement (leaving a leaf sheath length of about 5 cm), cut them with scissors and immediately put them in water, and perform secondary shearing underwater to remove bubbles in the leaves. After that, place the cut plants in a dark environment for rehydration. During the measurement, place the leaves under an irradiation lamp to induce the opening of the leaf stomata. After balancing for 30 minutes, cut off the new fully expanded leaves with leaf sheaths underwater, and quickly connect the cut leaves to a pre-filled water pipe underwater, and the other end of the water pipe is connected to a water source placed on a balance. After the leaves are connected to the water pipe, start recording the weight change on the balance every 10 seconds and calculate the instantaneous water flow rate (F), until the water flow rate is stable for at least 15 minutes and then stop recording. After turning off the balance counting, quickly remove the leaves from the water pipe and store them in a ziplock bag. After the leaves are balanced, they are used to measure the final leaf water potential (Ψ final ) and leaf area (LA). Each treatment was repeated 3 times, K leaf The calculation is as follows:

[0063] K leaf =F / (LA×Ψ final ) (1).

[0064] VPD affects blade hydraulic conductivity (Kleaf ) has a significant impact on MD and PD The effect was not significant. Under the H-SM treatment, compared with the low VPD treatment, the K leaf The K of the high VPD treatment was significantly reduced by 29.18% under the M-SM treatment compared with that of the low VPD treatment. leaf The K of the high VPD treatment was significantly reduced by 35.04% under L-SM treatment compared with that of the low VPD treatment. leaf Significantly reduced by 38.91%

[0065] SM vs K leaf and MD The effect was significant. Under low VPD, K in L-SM treatment was lower than that in H-SM treatment. leaf and MD significantly decreased by 50.50% and 35.56%; under high VPD, K in L-SM treatment was significantly lower than that in H-SM treatment. leaf and MD A significant decrease of 57.30% and 43.07%.

[0066] VPD and SM are only for Ψ PD There was a significant interaction effect, and the SM under high VPD was significantly smaller than that under low VPD. leaf ,Ψ MD and PD The reductions were significant at 55.11%-69.75%, 18.52%-45.19% and 63.34%-93.34%, respectively.

[0067] Example 4: Determination of leaf hydraulic conductivity vulnerability curve

[0068] During the flowering period, the sampling time was before turning on the lights. In each treatment, 20 tillering plants with three fully expanded leaves (leaving a leaf sheath length of about 5 cm) were selected, all plants were cut with scissors and immediately placed in water, and pruned twice underwater, and then placed in a dark environment for rehydration. Wheat K leaf The measurement method is as follows: Select wheat plants with fully developed flag leaves (leaf sheath length is about 5 cm), cut them with scissors, and immediately immerse them in water. Prune them twice to eliminate any remaining bubbles. Then, take out the test leaves and illuminate them with a lamp to induce the opening of the stomata. After reaching equilibrium for 3 minutes, use the LI-COR 6800 photosynthesis measurement system to measure the transpiration rate (E). When the leaves reach a stable transpiration state, record E. Then, immediately take out the leaves, quickly put them in a sealed bag with a wet paper towel, and then store them in a dark environment for about 30 minutes to reach equilibrium, and then measure Ψ leafEach treatment was repeated 5 times. leaf The calculation is as follows:

[0069] K max = E / (0-Ψ leaf ) (2);

[0070] This measurement value is the maximum hydraulic conductivity of the blade (K max The remaining wheat plants after rehydration were placed on the laboratory table for different lengths of time and dehydrated to different degrees. The plants with different dehydration degrees were then wrapped in black plastic bags and allowed to balance for at least 30 minutes. The initial water potential values ​​(Ψ) of the top and bottom leaves of the plants at different dehydration times were measured. 0 ), the value should be less than 0.1MPa (for severely dehydrated plants, the value can be less than 0.3MPa). Use the middle leaf to measure the hydraulic conductivity of wheat at different dehydration levels according to the above method. leaf ). leaf The percentage loss (PLC) is calculated using the following formula:

[0071] PLC=100×(1-K leaf / K max ) (3);

[0072] The least square method based on empirical functions was used to fit the vulnerability curves of each species to determine the xylem water potential (P 50 ) and the fragility curve at the inflection point (P 50 ) at the slope (S).

[0073] PLC=a / (1+exp(S(Ψ leaf -c)) (4);

[0074] Where Ψ leaf is the leaf water potential, a is the maximum PLC, S is the slope of the fragility curve at the inflection point, and c is P 50 . Where S is the slope of the fragility curve at the inflection point, expressed as the percentage loss of hydraulic conductivity per megapascal (MPa) change (% / MPa). S reflects the sensitivity of embolism to propagate in the xylem network. The larger S is, the faster the embolism propagates. A fragility curve was measured for each treatment, and the R package “fitplc” was selected to fit the fragility curve.

[0075] Example 5: Determination of endogenous plant hormones

[0076] After the determination of leaf gas exchange parameters, leaves adjacent to the flag leaf with the same treatment were immediately frozen with liquid nitrogen and stored in a -40°C refrigerator. Endogenous plant hormones were determined by enzyme-linked immunosorbent assay, and the contents of auxin (IAA) and abscisic acid (ABA) in leaves were determined using a kit (BNTY) produced by Beijing Beinong Tianyi Biotechnology Co., Ltd.

[0077] Example 6: Determination of chlorophyll and leaf nitrogen content

[0078] The chlorophyll (SPAD) value of the same part near the photosynthetic leaf was measured using a SPAD-502 chlorophyll meter; other leaves from the same part were collected, dried, ground, and boiled, and the nitrogen content per unit mass of the leaves (N mass ), with 3 replicates for each treatment.

[0079] VPD significantly affected the values ​​of abscisic acid (ABA), auxin (IAA) and chlorophyll (SPAD), and had a significant effect on the nitrogen content of leaves (N mass ) had no significant effect. Under H-SM treatment, compared with low VPD, ABA did not change significantly under high VPD treatment, IAA increased significantly by 4.79% under high VPD treatment, and SPAD decreased significantly by 7.69%; under M-SM treatment, compared with low VPD, ABA and IAA increased significantly by 77.29% and 13.15% under high VPD treatment, and SPAD decreased significantly by 6.44%; under L treatment, compared with low VPD, ABA and IAA increased significantly by 48.18% and 11.42% under high VPD treatment, and SPAD did not change significantly.

[0080] SM had a significant effect on ABA, IAA and SPAD, and had a significant effect on N mass The effect was not significant. Under low VPD, compared with the H-SM treatment, ABA and IAA in the L-SM treatment increased significantly by 25.53% and 6.50%, and SPAD decreased significantly by 9.06%. Under high VPD, compared with the H-SM treatment, ABA and IAA in the L treatment increased significantly by 104.05% and 7.32%, and SPAD did not change significantly.

[0081] VPD and SM had significant interactive effects on ABA, IAA, SPAD and leaf Nmass. Compared with the treatment without water stress, ABA and IAA under the dual drought of air and soil increased significantly by 63.57%-84.98% and 17.68-22.89%, respectively. mass and SPAD decreased significantly by 6.50% and 9.25%-11.10%.

[0082] 2. Correlation between various physiological and ecological parameters

[0083] 1. Gas exchange parameters and leaf hydraulic conductivity

[0084] The regression fitting results of gas exchange parameters and hydraulic traits under low VPD and high VPD showed that A n With g s and K leaf With g s There was a significant positive correlation under both VPD conditions, and the linear regression relationship was not affected by SM and VPD treatments. Figure 1 (a) and Figure 1 (b) g s With K leaf There is a piecewise linear relationship between them, g s Follow K leaf There is an obvious threshold effect when K leaf At more than 10.60mmolm -2 s -1 MPa -1 (Threshold) stage change g s Basically remain unchanged, only K leaf When it is below the threshold, g s It starts to decrease linearly, see Figure 1 (c).

[0085] 2. Gas exchange parameters, leaf water conductance and leaf water potential

[0086] K leaf With MD The linear regression relationship between them was affected by VPD, and the slope of the regression line in the low VPD treatment was significantly smaller than that in the high VPD treatment. s Rapid drop in K leaf The threshold value corresponds to MD Displayed as low VPD greater than high VPD, refer to Figure 2 (a) g s With MD The linear regression relationship between the two was affected by VPD, and the slope of the regression line in the high VPD treatment was significantly smaller than that in the low VPD treatment. 50 The mean value is less than the above g s The noon leaf water potential point corresponding to the rapid decline threshold is shown in Figure 2 (b) g s The fast falling threshold corresponds to Ψ MD The low VPD is shown to be greater than the high VPD, which is consistent with Figure 2 a The results are consistent, that is, when facing SM stress, under the same SM level, high VPD and low VPD g s First reach the rapid decline threshold point. n With MD There is a significant linear regression between A n WithMD There was no significant difference in the slope and intercept of the regression line between the two VPD treatments. Figure 2 (c).

[0087] 3. Trade-off between hydraulic safety and efficiency

[0088] SM deficiency P 50 Significantly reduced. At low VPD, P 50 The decrease from -0.44MPa in H-SM treatment to -0.74MPa and -0.82MPa in M-SM and L-SM treatments was 68.18% and 86.36%, respectively. Under high VPD, the decreases in M-SM and L-SM treatments were 5.37% and 24.73%, respectively. Figure 3 ). Compared with low VPD, P 50 Significantly reduced, H-SM, P 50 The P value under M-SM and L-SM decreased significantly from -0.44 MPa under low VPD to -0.93 MPa under high VPD, a decrease of 111%. 50 The decrease from low VPD to high VPD is 32.43% and 41.46% respectively.

[0089] Each treatment P 50 Its P 50 The results of the slope S value corresponding to the treatment showed that with the decrease of SM, the S value decreased, and the decrease of the S value under low VPD was greater than that under high VPD treatment. leaf The decline in leaf The sensitivity of the decrease is low, that is, P 50 When it is more negative, K leaf It began to decline, showing higher hydraulic security and lower hydraulic transmission efficiency. leaf , A n , g s With P 50 There is a significant positive correlation, see Figure 4 .

[0090] 4. Pathways of crop stress response

[0091] like Figure 5 As shown in the figure, the correlation analysis (CA) between the various indicators of different SM treatments under high and low VPD showed that under low VPD, An and gs (R 2 =0.82, p<0.01) and K leaf (R 2 =0.73, p<0.05) showed a significant positive correlation, A n With MD There is no significant correlation between ABA and A under high VPD.n With g s (R 2 =0.85, p<0.01), K leaf (R 2 =0.85, p<0.01) and Ψ MD (R 2 =0.92, p<0.001) and was significantly positively correlated with ABA.

[0092] Structural equation modeling (SEM) was used to elucidate the relationship between A n Different response mechanisms, SEM explained the A n The chi-square (χ2) test and the root mean square error of approximation (RMSEA) showed that the model fit was good. Figure 6 (a) and Figure 6 (b).

[0093] At low VPD, Ψ MD To K leaf The significant positive effect increased g s A n The contribution of ABA to K leaf Significant negative effect increases g s A n The path analysis showed that due to the leaf A n The direct impact of A n With g s The coupling relationship between them, K leaf The changes in ABA were mainly caused by the changes in the SEM. The standardized net effect analysis showed that the Figure 6 (c), A n Mainly affected by MD and ABA, followed by g s and K leaf , ABA and g at low VPD s A n The total effect of the influence tends to be neutralized. The standardized total effect results show that A n Mainly affected by MD and ABA, followed by g s and K leaf In addition, under low VPD treatment, MD and ABA vs. A n The total effect of g was smaller than that of high VPD. s A n The total effect of the impact is greater than that of high VPD, Kleaf A n The total effect of the impact is similar at high and low VPD, see Figure 6 (c).

[0094] The above shows and describes the basic principles, main features and advantages of the present invention. It should be understood by those skilled in the art that the present invention is not limited by the above embodiments, and the above embodiments and descriptions are only for explaining the principles of the present invention. Without departing from the spirit and scope of the present invention, the present invention may have various changes and improvements, which fall within the scope of the present invention to be protected. The scope of the present invention to be protected is defined by the attached claims and their equivalents.

Claims

1. A method for analyzing the physiological state of water in wheat leaves under combined atmospheric and soil drought stress, characterized in that: The steps include: S1, cultivated wheat, Cultivation method: Before sowing, ammonium sulfate and potassium dihydrogen phosphate were applied to supplement nitrogen, phosphorus and potassium fertilizers for crops, and 152 mg nitrogen, 120 mg phosphorus and 120 mg potassium were applied to each pot with water; after sowing, Hoagland nutrient solution was used for irrigation; S2. After germination and seedling determination, the cultivated wheat plants were subjected to drought stress. Three levels of soil moisture and two levels of saturated water vapor pressure difference were set. The experimental method of simulating soil and atmosphere dual drought stress was implemented by controlling the upper and lower limits of soil moisture and different levels of saturated water vapor pressure difference. S3, maintaining drought treatment until the beginning of heading stage, and using Li-COR6800 photosynthesis measurement system to measure flag leaf gas exchange parameters under different conditions; S4, measuring leaf water potential before dawn and at noon using a portable plant water potential pressure chamber; S5. The transpiration-driven method was used to measure the hydraulic conductivity of wheat leaves and the leaf hydraulic conductivity vulnerability curve was drawn. The method for measuring blade hydraulic conductivity comprises the following steps: S511, selecting flag leaf wheat plants after the gas exchange measurement, cutting them with scissors and immediately placing them in water, and performing secondary shearing under water to remove bubbles in the stems; S512, placing the cut plants in a dark environment for rehydration, placing the leaves under an irradiation lamp to induce the opening of the leaf stomata during measurement, and after balancing for 30 minutes, cutting off the new fully expanded leaves with leaf sheaths underwater, and quickly connecting the cut leaves underwater to a pre-filled water pipe, and the other end of the water pipe is connected to a water source placed on a balance; S513, after the blade is connected to the water pipe, record the weight change on the balance and calculate the instantaneous water flow velocity, and stop recording after the water flow velocity is stable for at least 15 minutes; S514. After the balance is turned off for counting, the blade is quickly removed from the water pipe and stored in a ziplock bag. After the blade is balanced, it is used to determine the final leaf water potential and leaf area. The leaf hydraulic conductivity is calculated as follows: K leaf = F / (LA× Ψ final )(1); In the formula, K leaf is the hydraulic conductivity of the blade, F is the instantaneous water velocity, LA is the leaf area, Ψ final is the final leaf water potential of the leaf; The calculation method of the blade hydraulic conductivity vulnerability curve is as follows: S521, Measure transpiration rate using the LI-COR 6800 photosynthesis measurement system; S522. Take out the leaves, quickly put them into a sealed bag with a wet paper towel, and then store them in a dark environment for about 30 minutes to reach equilibrium. Then measure the leaf water potential, and calculate the maximum hydraulic conductivity of the leaves as follows: K max = E / (0− Ψ leaf )（2）； In the formula, K max is the maximum hydraulic conductivity of the blade, E is the transpiration rate, Ψ leaf is the leaf water potential; S523, placing the remaining wheat plants after the above rehydration on a laboratory table and performing dehydration treatments of different degrees for different lengths of time, then wrapping the plants of different dehydration degrees with black plastic bags and balancing for at least 30 minutes; measuring the initial water potential of the two leaves at the top and bottom of the plant at different dehydration times, using the middle leaf to measure the leaf hydraulic conductivity of wheat at different dehydration degrees according to the method of measuring leaf hydraulic conductivity and the method of measuring leaf maximum hydraulic conductivity, and calculating the leaf hydraulic conductivity loss percentage PLC using the following formula: PLC=100×(1- K leaf / K max )(3); S524. The fragility curves of each species were fitted using the least squares method based on empirical functions to determine the xylem water potential and the slope of the fragility curve at the inflection point when the hydraulic conductivity was lost by 50%: PLC=a / (1+exp(S( Ψ leaf -c))(4); In the formula Ψ leaf is the leaf water potential, a is the maximum PLC, S is the slope of the fragility curve at the inflection point, and c is K leaf Reduce water potential by 50%; S6. After the determination of leaf gas exchange parameters, select leaves adjacent to the flag leaf with the same treatment and immediately freeze them with liquid nitrogen and store them in a −40°C refrigerator to determine the auxin and abscisic acid content in the leaves; S7, using a chlorophyll meter to measure the chlorophyll value of the same part near the photosynthetic leaf; collecting other leaves in the same part, drying and grinding them, and then boiling them, and using a nitrogen analyzer to measure the nitrogen content per unit mass of the leaves; S8. Conduct an in-depth analysis of the experimental data to reveal the physiological adaptation mechanism and water utilization strategy of wheat under dual drought stress, and analyze the synergistic relationship between stomatal conductance and leaf hydraulic conductance and net photosynthetic rate through the structural equation model.

2. The method for analyzing the physiological state of water in wheat leaves under combined atmospheric and soil drought stress according to claim 1, characterized in that: In step S1, the wheat variety is "Jinchun No. 6". During the cultivation process, the soil used is nutrient soil with a field water holding capacity of 0.33 and a bulk density of 1.4 g·cm -3 .

3. The method for analyzing the physiological state of water in wheat leaves under combined atmospheric and soil drought stress according to claim 1, characterized in that: In step S2, the three levels of soil moisture are high soil moisture, medium soil moisture, and low soil moisture, and the corresponding upper and lower limits of soil moisture are 85%-95% field water holding capacity, 65%-75% field water holding capacity, and 50%-60% field water holding capacity, respectively; the two levels of saturated vapor pressure difference are conventional VPD and high VPD stress, respectively, the conventional VPD is set to 0.79 kPa, and the corresponding environmental conditions are normal temperature and high humidity, consisting of 25°C and 75% relative humidity, and the VPD stress is set to 1.43 kPa, and the corresponding environmental conditions are normal temperature and low humidity, consisting of 25°C and 55% relative humidity.

4. The method for analyzing the physiological state of water in wheat leaves under combined atmospheric and soil drought stress according to claim 1, characterized in that: In step S3, the gas exchange parameters include the net photosynthetic rate, transpiration rate and instantaneous water use efficiency of wheat leaves. During the measurement, the leaf chamber temperature was set at 26-28°C and the light intensity was set at 1200 μmol / m -2 s -1 , CO2 concentration is set to 400ppm.

5. The method for analyzing the physiological state of water in wheat leaves under combined atmospheric and soil drought stress according to claim 1, characterized in that: The method for measuring leaf water potential is as follows: measuring the leaf water potential before dawn and at noon by a portable plant water potential pressure chamber; selecting a flag leaf with consistent growth in each treatment, cutting it with scissors and immediately moving it into the portable plant water potential pressure chamber, and extending the leaf cutout at the air chamber cover; after the air chamber is tightened, slowly pressurizing the air chamber, and observing the cutout with the help of a magnifying glass; and stopping pressurizing and reading the pressure value immediately when small water droplets just appear at the cutout.

Images