Extraction method of leaf apoplast solution

Abstract

The invention discloses a leaf apoplast solution extraction method, and relates to the technical field of plant leaves. The method for effectively extracting the AWF of the corn leaves is established by taking seedlings cultured by a nutrient solution as a test material, researching the extraction efficiency of the exosome with different centrifugal forces and different leaf curling angles in a centrifugal tube and optimizing the extraction centrifugal forces and the leaf curling angles, and can be applied to plant exosome research, so that the method for effectively extracting the AWF of the corn leaves is established. A foundation is laid for accuracy and reliability of research results such as proteomics and metabonomics, and reference is provided for optimization of AWF separation methods of other crops.

Description

Technical Field

[0001] The invention relates to the technical field of plant leaves, in particular to a method for extracting a leaf apoplast solution. Background Art

[0002] The first step to study the material composition, transcriptomics, proteomics and metabolomics of plant leaf apoplasts is to obtain a sufficient amount of apoplast wash fluid (AWF) that is not contaminated by cytoplasm.

[0003] Studying the composition of different cell apoplast solutions can provide a broader understanding of the metabolic dynamics of cells and organisms and their intercellular signal transduction regulatory mechanisms. The methods for extracting apoplast juice from various tissues include elution method, filter paper strip method, pressure method, microdialysis method, elution method and vacuum infiltration centrifugation method. The most mature AWF extraction method is vacuum infiltration centrifugation. Many publications have detailed methods for extracting apoplast fluid from plant leaves based on centrifugation. However, due to different plant species, organ structures and apoplast research contents, the parameters involved in obtaining AWF using vacuum infiltration centrifugation, such as vacuum infiltration, apoplast washing solution, centrifugation time, intensity, etc., are different.

[0004] However, the amount of AWF extracted by existing methods is very small, the operation technology requirements are high or it is easily contaminated and cannot meet most research needs. Although there are reports of vacuum infiltration centrifugation being used to separate leaf AWF from dicotyledonous crops such as Arabidopsis, cotton and tomato, there is still the problem of apoplast solution contamination, and the volume of apoplast solution extracted varies greatly among species or is relatively small.

[0005] Therefore, it is necessary to optimize the key parameters in the extraction process, further improve the extraction method, and propose a new solution that is simple to operate, efficient, and highly reproducible. Summary of the invention

[0006] The purpose of the present invention is to provide a method for extracting leaf apoplast solution to solve the technical problems raised in the background technology.

[0007] To achieve the above object, the present invention provides the following technical solution: a method for extracting leaf apoplast solution, comprising at least the following steps:

[0008] S1: Determine the experimental materials and process them to obtain materials for apoplast extraction, that is, obtain multiple samples of apoplast extraction;

[0009] S2: The first true leaf of each sample was collected, washed with pure water, dried with absorbent paper, and placed in a 50 mL centrifuge tube filled with pure water. The centrifuge tube lid was closed to prevent the leaves from floating on the water surface. The lid should not be tightened.

[0010] S3: Place the 50 mL centrifuge tube into a vacuum permeameter filled with crushed ice at the bottom for permeation treatment;

[0011] S4: After infiltration, the leaves were removed from the washing solution, gently wiped dry, and then weighed after infiltration;

[0012] S5: Place the leaf on the Parafilm membrane, roll up the leaf together with the Parafilm membrane using a 1 mL pipette tip, take another small piece of Parafilm membrane, fix the rolled Parafilm membrane together, and suspend it above the bottom of a 50 mL centrifuge tube;

[0013] S6: Place a 1 mL pipette in a 50 mL centrifuge tube with the tip facing downward and centrifuge at 2500 g, 5000 g and 8000 g for 10 min at 4°C. Transfer the supernatant to a fresh 0.5 mL test tube to obtain the leaf apoplast solution for subsequent analysis.

[0014] Furthermore, the leaf number and length parameters in S2 depend on the amount of apoplast solution that needs to be obtained.

[0015] Furthermore, the infiltration treatment in S3 at least includes the following steps:

[0016] Set the pressure to 0.5kg / cm 2 , firstly, infiltration was performed for 3 min;

[0017] After 3 min of infiltration, slowly release the air to remove the bubbles on the leaves in the centrifuge tube;

[0018] Repeat the above penetration steps 5 times until the leaves are completely penetrated and dark penetration areas are seen or the leaves no longer float.

[0019] Furthermore, the curling of the blades in S5 at least includes:

[0020] Apex90 (A90): The curling of the leaves starts from the tip of the leaves, and the curling angle is 90°, that is, the tip of the leaves curls at an angle of 90°;

[0021] Apex45 (A45): The curling of the leaves starts from the tip of the leaf, and the curling angle is 45°, that is, the tip of the leaf forms an angle of 45° toward the curling direction;

[0022] Base90 (B90): The curling of the leaves starts from the base of the leaves, and the curling angle is 90°, that is, the base of the leaves curls at a 90° angle;

[0023] Base45 (B45): The curling of the leaves starts from the leaf base, and the curling angle is 45°, that is, the leaf base forms an angle of 45° toward the curling direction;

[0024] 0apex (0a): The curl angle is 0°, and the tip of the leaf faces the bottom of the centrifuge tube, indicating that the leaf remains in a horizontal state with the tip facing downward;

[0025] 0base (0b): The curl angle is 0°, the leaf base is toward the bottom of the centrifuge tube, which means the leaf blade remains in a horizontal state with the leaf base facing downward;

[0026] Upper Surface (US): On the parafilm, the upper side of the leaf faces upwards;

[0027] Lower Surface (LS): On the parafilm membrane, the reverse side of the leaf faces upward.

[0028] Furthermore, the size of the Parafilm in S5 is 15*5 cm.

[0029] Compared with the prior art, the present invention has the following beneficial effects:

[0030] The present invention uses seedlings cultured in nutrient solution as test materials, studies the apoplast extraction efficiency at different centrifugal forces and different leaf curling angles in centrifuge tubes, and establishes a method for effectively extracting AWF from corn leaves by optimizing the extraction centrifugal force and leaf curling angle. The method can be applied to plant apoplast research, such as proteomics and metabolomics, to lay a foundation for the accuracy and reliability of research results, and provides a reference for the optimization of AWF separation methods for other crops.

[0031] Vacuum infiltration centrifugation is simple, efficient and reproducible, but it also has some problems, such as cell membrane rupture during centrifugation, which leads to contamination of the apoplast solution. To solve this problem, it is necessary to optimize some experimental parameters in the vacuum infiltration centrifugation process, such as vacuum infiltration time, centrifugation time, centrifugation intensity, etc., to reduce the degree of apoplast contamination as much as possible. The present invention optimizes the method for extracting apoplast solution from corn leaves by studying different centrifugal forces, leaf curling angles and initial placement directions, so as to obtain uncontaminated AWF. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings required for describing the embodiments will be briefly introduced below. Obviously, the accompanying drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other accompanying drawings can be obtained based on these accompanying drawings without paying creative work.

[0033] Figure 1 It is a schematic diagram of the process of the present invention;

[0034] Figure 2Schematic diagram of the volume of apoplast extracted from leaves with different curling angles of the present invention;

[0035] Figure 3 Schematic diagram of the effect of centrifugal force on the extraction of corn leaf apoplasts in the present invention;

[0036] Figure 4 Schematic diagram of calculation of apoplast hydration degree of the present invention, wherein Figure 4 A is the degree of apoplast hydration which can be determined by measuring the leaf weight during apoplast extraction; Figure 4 B is the hydration degree of apoplast extract under different centrifugation conditions;

[0037] Figure 5 is a schematic diagram of different centrifugal forces of the present invention, wherein Figure 5 A is the G6DPH activity of AWF under different centrifugal forces; Figure 5 B is the contamination rate of AWF under different centrifugal forces. DETAILED DESCRIPTION

[0038] The technical solutions in the embodiments of the present invention will be described clearly and completely below in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, rather than all the embodiments.

[0039] See also Figure 1 , a method for extracting leaf apoplast solution, comprising at least the following steps:

[0040] S1: Determine the experimental materials and process them to obtain materials for apoplast extraction, that is, obtain multiple samples of apoplast extraction;

[0041] In this embodiment, the corn variety Zhengdan 958 was used as the test material for apoplast extraction. Corn seeds of uniform size were selected and disinfected by soaking in 10% hydrogen peroxide solution for 20 minutes, washed with pure water to remove the hydrogen peroxide solution on the surface of the seeds, placed in a saturated calcium sulfate solution for ventilation and soaking for more than 6 hours at 25°C, and then raised by paper culture method, and hydroponically grown in a fully automatic artificial climate chamber at 27°C during the day / 20°C at night and a relative humidity of 75%.

[0042] The cells were cultured in a complete nutrient solution for 7 days, which consisted of: 1.0 mM K 2 HPO 4 ; 1.0 mM K 2 SO 4 ; 0.25 mM MgSO 4 ; 25 μM KCl; 2.0 mM Ca(NO 3 ) 2 ; 100μM EDTA-Fe; 12.5μMH 3 BO3 ; 1.0μM MnSO 4 ; 0.25μM CuSO 4 ; 1.0 μM ZnSO 4 ; 0.25 μM (NH 4 ) 6 Mo 7 O 24 The nutrient solution was replaced every two days and the pH of the nutrient solution was adjusted to 6. After 13 days, the first fresh true leaf was collected as the material for apoplast extraction.

[0043] S2: The first true leaf of each sample was collected, washed with pure water, dried with absorbent paper, and placed in a 50 mL centrifuge tube filled with pure water. The centrifuge tube lid was closed to prevent the leaves from floating on the water surface. The lid should not be tightened.

[0044] S3: Place the 50 mL centrifuge tube into a vacuum permeameter filled with crushed ice at the bottom for permeation treatment;

[0045] S4: After infiltration, remove the leaves from the washing solution, dry them gently and thoroughly, and then weigh them after infiltration;

[0046] S5: Place the leaf on the Parafilm membrane, roll up the leaf together with the Parafilm membrane using a 1 mL pipette tip, take another small piece of Parafilm membrane, fix the rolled Parafilm membrane together, and suspend it above the bottom of a 50 mL centrifuge tube;

[0047] S6: Place a 1 cm pipette in a 50 ml centrifuge tube with the tail end facing downward and centrifuge at 2500g, 5000g and 8000g for 10 min at 4°C. Then transfer the supernatant to a fresh 0.5 ml test tube to obtain the leaf apoplast solution for subsequent analysis.

[0048] The number and length of the leaves in S2 depend on the amount of apoplast solution that needs to be obtained. In this embodiment, the number of leaves is 10 and the length of the leaves is 4 cm-5 cm.

[0049] The infiltration process in S3 includes at least the following steps:

[0050] Set the pressure to 0.5kg / cm 2 , firstly, infiltration was performed for 3 min;

[0051] After 3 min of infiltration, slowly release the air to remove the bubbles on the leaves in the centrifuge tube;

[0052] Repeat the above penetration steps 5 times until the leaves are completely penetrated and dark penetration areas are seen or the leaves no longer float.

[0053] The leaf curling in S5 at least includes:

[0054] Apex90 (A90): The curling of the leaves starts from the tip of the leaves, and the curling angle is 90°, that is, the tip of the leaves curls at an angle of 90°;

[0055] Apex45 (A45): The curling of the leaves starts from the tip of the leaf, and the curling angle is 45°, that is, the tip of the leaf forms an angle of 45° toward the curling direction;

[0056] Base90 (B90): The curling of the leaves starts from the base of the leaves, and the curling angle is 90°, that is, the base of the leaves curls at a 90° angle;

[0057] Base45 (B45): The curling of the leaves starts from the leaf base, and the curling angle is 45°, that is, the leaf base forms an angle of 45° toward the curling direction;

[0058] 0apex (0a): The curl angle is 0°, and the tip of the leaf faces the bottom of the centrifuge tube, indicating that the leaf remains in a horizontal state with the tip facing downward;

[0059] 0base (0b): The curl angle is 0°, the leaf base is toward the bottom of the centrifuge tube, which means the leaf blade remains in a horizontal state with the leaf base facing downward;

[0060] Upper Surface (US): On the parafilm, the upper side of the leaf faces upwards;

[0061] Lower Surface (LS): On the parafilm membrane, the reverse side of the leaf faces upward.

[0062] The size of Parafilm in S5 is 15*5cm.

[0063] Based on the above steps, further data statistics are proposed:

[0064] The data were expressed as mean ± standard deviation (SD). FA test was used for comparison among the groups. P < 0.05 was considered to be statistically significant.

[0065] Results and Analysis:

[0066] Effect of curling angle on extraction of leaf apoplast

[0067] The apoplast is more likely to flow out of the wound site of the leaf, but the possibility of contamination by damaged cells will also increase. Therefore, the curling method of the leaf in the centrifuge tube may affect the apoplast extraction effect. For this, 12 types of leaf curling methods were confirmed, and their components are shown above. The apoplast of the leaves of corn seedlings was extracted by vacuum infiltration centrifugation, and the extraction efficiency of the extract was calculated based on the apoplast extraction volume (volume / mass, VWR). Figure 2It can be seen that the front and back sides of the leaves have a certain influence on the extraction efficiency of the apoplast. The apoplast extraction results of the leaves facing up (US) are higher. Under US conditions, the extraction results of 0a and 0b are higher, with the extraction results of 29.3 and 39.4 μL / g respectively. It can be seen that the extraction result of US0b is the highest.

[0068] Figure 2 Note: VWR stands for volume / mass; A90: the curling start point is the tip, and the curling angle is 90°; A45: the curling start point is the tip, and the curling angle is 45°; B90: the curling start point is the base, and the curling angle is 90°; B45: the curling start point is the base, and the curling angle is 45°; 0a: the curling angle is 0°, and the tip of the leaf faces the bottom of the centrifuge tube; 0b: the curling angle is 0°, and the base of the leaf faces the bottom of the centrifuge tube. US: the front side of the leaf faces up on the parafilm; LS: the back side of the leaf faces up on the parafilm; different lowercase letters in the same series indicate significant differences (P<0.05).

[0069] Effects of Centrifugal Force on the Extraction of Apoplast from Maize Leaves

[0070] The greatest influence on the efficiency of leaf apoplast extraction was the centrifugal force. Next, the effect of centrifugal force on the efficiency of leaf apoplast extraction was evaluated ( Figure 3 ). The purpose is to determine the centrifugation conditions to achieve the maximum extraction yield of exoplastids with a low contamination rate. At present, the centrifugal force for extracting corn leaf exoplastids with a large volume and a negligible contamination rate is 2500g, but the extraction volume is still low and does not meet the needs of most studies. Of course, experiments have also been conducted to extract AWF with a centrifugal force of 5000g, but the specific extraction efficiency and contamination rate are unclear. Therefore, the leaf exoplastids were extracted by centrifugation at 2500g, 5000g, and 8000g for 10 minutes to verify the optimal centrifugal force for a larger leaf exoplastid extraction volume. The permeation volume of each leaf mass before centrifugation under different centrifugal forces was similar. With the increase of centrifugal force, the extraction volume of the leaf volume increased as expected, and the extraction results were 47.4, 64.7, 154.2, and 222.8μL / g, respectively. The results showed that there was no difference in the exoplastid extraction results at 2500g and 3200g, and the extraction result at 8000g was the highest ( Figure 3 ).

[0071] To understand the extent to which the apoplast is filled with fluid after vacuum infiltration of maize leaves, we call this apoplast hydration. At the time of apoplast collection, the hydration level of the apoplast is unknown. Specifically, the apoplast hydration can be calculated based on the leaf weight measured before and after vacuum infiltration of the apoplast wash solution (water) and after centrifugation ( Figure 4 A) At the time of collection, it is assumed that the apoplasts are fully hydrated after vacuum infiltration with the apoplast wash solution and completely drained after centrifugation. Figure 4 As can be seen from B, the higher the centrifugal force, the higher the apoplast hydration, and the apoplast hydration at 8000g is close to 100%, indicating that the apoplast is almost completely excreted after hydration under 8000g conditions.

[0072] Evaluation and detection of contamination rate of leaf apoplast extract

[0073] After vacuum infiltration centrifugation, in order to evaluate the degree of cytoplasmic contamination of the apoplast extract to ensure the purity of the sample, the extract was tested for the activity of cellular enzymes, such as malate dehydrogenase (MDH) or G6PDH activity. A contamination rate of less than 3% is considered to be free of obvious contamination. G6PDH is an enzyme that is only commonly found in the cytoplasm and chloroplasts, so by detecting its activity, it can be determined whether the extracted apoplast is contaminated by the cytoplasm. The apoplast extracts obtained from 12 leaf curling angles had almost no detectable enzyme activity, and the enzyme activity was very low (data not shown).

[0074] See also Figure 4 and Figure 5 ,from Figure 4 As can be seen from A, the G6PDH activity of the apoplast extracted at 2500g was high, and there was no significant difference between the other three centrifugal forces. However, the apoplast contamination rate of the four centrifugal forces was less than 3% ( Figure 5 B). This indicates that the extracted apoplast is of high purity and is suitable for subsequent apoplast analysis.

[0075] The following discussion is further proposed:

[0076] Although the apoplast is an important physiological compartment in plant leaves, it is not often studied because it is difficult to detect its contents without destroying the surrounding tissue. One method that has facilitated the study of the apoplast is the vacuum infiltration centrifugation technique. Although AWF extraction may be limited by centrifugal force and the properties of the apoplast washing solution, this general method has been shown to be useful for the study of proteins, metabolites, and microorganisms localized in the apoplast. However, how to ensure that the integrity of the membrane is maintained during the extraction process while extracting as much apoplast as possible is still a difficulty in apoplast extraction.

[0077] The study was based on 2500g, and the effect of different curling angles of the leaves during centrifugation on the extraction was observed. The results showed that LU0b had the best extraction effect, and almost no contamination was detected. The different effects of curling angle on the efficiency of apoplast extraction may be that the wound of the leaf is facing the centrifugal direction, which is easy for the apoplast to flow out. But it is also easy to be contaminated by broken cells, but the final result shows that the contamination is low, indicating that its influence is small. Although the contamination effect of the cells damaged at the petiole incision on the apoplast extract may be extremely small and almost negligible, it is still necessary to clean the incision. The curling angle of the leaf has a certain effect on the apoplast extraction effect, but the contamination can be ignored, indicating that the cells damaged at the petiole incision have a very small effect on the contamination of the apoplast extract, almost no effect, which is consistent with the results of other experiments. On this basis, the effect of different centrifugal forces on AWF extraction was analyzed, and the results showed that 8000 had the best extraction effect, and the contamination rate was less than 1%. The centrifugal forces borne by leaves of different plants or different varieties of the same plant are different. It is reported that the centrifugal force taken for AWF extraction of broad bean leaves is 500-800g. The three centrifugal forces adopted by the present invention all have low AWF contamination rates, wherein the efficiency of 8000g is the highest, and the contamination rate is less than 1%. With the increase of centrifugal force, the cell enzyme activity of the apoplast extract of corn leaves also increases. There is a distortion phenomenon (possibly damage) in the appearance of the corn leaves after 8000g centrifugation, so further proof may be needed. Comprehensive comparison, 5000g as a centrifugal condition carries out AWF extraction on corn seedling leaves, and more AWF can be extracted without pollution, which will provide a good technical support for the research of apoplast proteomics, metabolomics, etc. in the future.

[0078] In summary:

[0079] The present invention uses indoor paper culture to cultivate seedlings for 13 days to optimize the method of extracting apoplasts from corn leaves:

[0080] (1) The effect of the curling angle of the blade during centrifugation on the AWF extraction efficiency was analyzed, and it was found that the AWF extraction efficiency was highest when the curling angle was downward at the wound flow. The higher the centrifugal force, the higher the AWF extraction efficiency, but the higher the degree of cell damage.

[0081] (2) 5000 g + front side up + cut side down are the optimal parameters for extracting corn leaf apoplast solution under the experimental conditions.

[0082] It will be apparent to those skilled in the art that the invention is not limited to the details of the exemplary embodiments described above and that the invention can be implemented in other specific forms without departing from the spirit or essential features of the invention. Therefore, the embodiments should be considered exemplary and non-limiting in all respects, and the scope of the invention is defined by the appended claims rather than the foregoing description, and it is intended that all variations falling within the meaning and scope of the equivalent elements of the claims be included in the invention. Any reference numeral in a claim should not be considered as limiting the claim to which it relates.

Claims

1. A method for extracting leaf apoplast solution, characterized in that: At least the following steps are included: S1: Determine the experimental materials and process them to obtain materials for apoplast extraction, that is, obtain multiple samples of apoplast extraction; S2: Leaves from the same leaf position were collected for each sample, washed with pure water, dried with absorbent paper, and placed in a 50 mL centrifuge tube filled with pure water. The centrifuge tube lid was closed to prevent the leaves from floating on the water surface. The lid should not be tightened. S3: Place the 50 mL centrifuge tube into a vacuum permeameter filled with crushed ice at the bottom for vacuum permeation treatment; S4: After infiltration, the leaves were removed from the washing solution, gently wiped dry, and then weighed after infiltration; S5: Place the leaf on the Parafilm membrane, roll up the leaf together with the Parafilm membrane using a 1 mL pipette tip, take another small piece of Parafilm membrane, fix the rolled Parafilm membrane together, and suspend it above the bottom of a 50 mL centrifuge tube; S6: Place a 1 mL pipette in a 50 mL centrifuge tube with the tip facing downward and centrifuge at 2500 g, 5000 g and 8000 g for 10 min at 4°C. Transfer the supernatant to a fresh 0.5 mL test tube to obtain the leaf apoplast solution for subsequent analysis.

2. A method for extracting leaf apoplast solution according to claim 1, characterized in that: The leaf number and length parameters in S2 depend on the amount of apoplast solution that needs to be obtained.

3. The method for extracting leaf apoplast solution according to claim 1, characterized in that: The infiltration treatment in S3 at least comprises the following steps: Set the pressure to 0.5kg / cm 2 , firstly, infiltration was performed for 3 min; After 3 min of infiltration, slowly release the air to remove the bubbles on the leaves in the centrifuge tube; Repeat the above penetration steps 5 times until the leaves are completely penetrated and dark penetration areas are seen or the leaves no longer float.

4. The method for extracting leaf apoplast solution according to claim 1, characterized in that: The curling of the blades in S5 at least includes: Apex90 (A90): The curling of the leaves starts from the tip of the leaves, and the curling angle is 90°, that is, the tip of the leaves curls at an angle of 90°; Apex45 (A45): The curling of the leaves starts from the tip of the leaf, and the curling angle is 45°, that is, the tip of the leaf forms an angle of 45° toward the curling direction; Base90 (B90): The curling of the leaves starts from the base of the leaves, and the curling angle is 90°, that is, the base of the leaves curls at a 90° angle; Base45 (B45): The curling of the leaves starts from the leaf base, and the curling angle is 45°, that is, the leaf base forms an angle of 45° toward the curling direction; 0apex (0a): The curl angle is 0°, and the tip of the leaf faces the bottom of the centrifuge tube, indicating that the leaf remains in a horizontal state with the tip facing downward; 0base (0b): The curl angle is 0°, the leaf base is toward the bottom of the centrifuge tube, which means the leaf blade remains in a horizontal state with the leaf base facing downward; Upper Surface (US): On the parafilm, the upper side of the leaf faces upwards; Lower Surface (LS): On the parafilm membrane, the reverse side of the leaf faces upward.

5. The method for extracting leaf apoplast solution according to claim 1, characterized in that: The size of the Parafilm in the S5 is 15*5cm.

Images