Method for monitoring real-time dynamic change of swelling pressure of plant cells

Abstract

The invention discloses a real-time dynamic monitoring method for plant cell swelling pressure. The method comprises the following steps: (a) monitoring AT and BT cutting crystal frequency parameter changes delta fAT and delta fBT caused by cell mechanical property changes of plant cells under different osmotic pressures or other stress conditions in real time on the basis of double-resonance piezoelectric cytometry; (b) calculating the integral delta S of the surface stress generated by the cells in the thickness direction according to the frequency parameter change delta f on the basis of a stress equation; and (c) calculating the swelling pressure P according to the surface stress based on the stress relationship between the thin-wall theory and the Young-Laplacian equation. The method for measuring the plant cell swelling pressure has the advantages of being lossless, real-time and continuous.

Description

Technical Field

[0001] The invention belongs to the technical fields of plant physiology, agriculture, biological science and technology and crop information detection, and particularly relates to a method for monitoring the dynamic changes of plant cell turgor pressure in real time. Background Art

[0002] The plant cell wall-plasma membrane-cytoskeleton continuum is a highly dynamic structure, and the dynamic changes in interfacial forces (including turgor pressure) at the plant cell wall-plasma membrane-cytoskeleton interface play a crucial role in plant growth, development, and responses to environmental stress. For example, during organ development, turgor-driven hydraulics and cell growth, as well as cell wall biomechanics, are as crucial as gene regulation, protein interactions, and subcellular hormone levels. However, currently, general botany and plant physiology laboratories lack instrumentation for measuring cell wall mechanics and turgor pressure. Pressure chamber methods for measuring turgor pressure are only suitable for plant organs. Turgor pressure is defined at the cellular level as the pressure exerted on the cell wall by the volume expansion of plant cells due to water absorption. Therefore, accurate quantification of turgor pressure must be performed at the cellular level. To date, the main methods used for measuring turgor pressure include: the initial plasmolysis method, the pressure probe method and its subsequent improvements (picoliter manometer), the ball pressure method, and the recently developed atomic force microscopy (AFM) method. However, these methods have several drawbacks, such as the risk of cell damage or the requirement for applying a certain amount of force, which prevents real-time, continuous monitoring. This is also why cell turgor pressure measurement technology has not been widely promoted. Global climate change has led to an increase in the intensity and frequency of drought events, and soil salinization is becoming increasingly serious. Drought and salt stress are the most important abiotic stresses, and their common mechanism is water deficit. Similarly, in plants sensing water deficit and non-water stress, little is known about the role of the plant cell wall-plasma membrane-cytoskeleton continuum in stress sensitivity and signal transduction, as well as the key molecules involved. Plant cell turgor pressure is a key physiological indicator of water deficit and growth status. Monitoring plant turgor pressure in real time can provide important scientific guidance for crop growth and stable yield and increased income. Under abiotic stresses such as drought and salt, plant cells lose water and produce negative turgor pressure. However, current turgor pressure methods are not suitable for measuring negative turgor pressure, and therefore have limited application in abiotic stress research and screening of stress-resistant crop varieties. Conventional testing of stress-resistant varieties mainly relies on field experiments, which are time-consuming and labor-intensive. Currently, crop stress-resistant breeding mainly combines molecular breeding with crop phenotyping, lacking a platform for continuous cellular phenotyping and functional testing of both. Summary of the Invention

[0003] The present invention aims to overcome the deficiencies of the prior art and provide a method for monitoring the dynamic changes in plant cell turgor pressure in real time, which does not damage the test cells and can continuously monitor the real-time changes in plant cell turgor pressure under different conditions for a long time.

[0004] In order to achieve the above-mentioned purpose of the present invention, the following technical solutions are adopted:

[0005] The method for monitoring the dynamic changes of plant cell turgor pressure in real time comprises the following steps:

[0006] (1) Modifying molecules or materials that can electrostatically or chemically interact with cell walls or protoplasts on AT-cut and BT-cut double-resonance quartz crystal white pieces or conductive electrode surfaces or insulating films on electrode surfaces, wherein the conductive electrode materials are metal materials compatible with cell biotechnology and ITO materials that are both conductive and transparent and allow optical microscopy observation; the insulating film material is SiO2; the molecules or materials that can electrostatically or chemically interact with cell walls or protoplasts are materials that promote the adhesion of plant cells or protoplasts; the materials that promote the adhesion of plant cells or protoplasts are positively charged poly-L-lysine, chitosan or PDADMAC, including the following plant cell transmembrane force-sensitive proteins or small molecules: a) RGD and Fibronectin that can interact with clusterin-like receptor kinase (LecRLK) and AT14A; b) pectin that interacts with cell wall-attached receptor-like kinase (WAK); c) polyclonal antibodies for the extracellular domain that interact with FEKONIA in the Catharanthus roseus receptor-like kinase family; d) Formin in the Formin family 1. A polypeptide fragment with specific interaction; the AT-cut quartz crystal and the BT-cut quartz crystal have the same frequency, surface morphology and are modified with the same surface adhesion molecules;

[0007] (2) The modified AT-cut and BT-cut quartz crystals of step (1) are placed in a detection cell, and the walled plant cells or wall-removed protoplasts to be measured are added to the detection cell, and the frequency change Δf of the AT-cut and BT-cut chips caused by the adhesion, growth or adverse stress of the plant cells on the AT-cut and BT-cut chips is monitored in real time based on dual-resonance piezoelectric cytometry. AT and △f BT ; Based on the stress equation, the frequency parameter changes Δf AT and △f BT Calculate the integral ΔS of the stress exerted by cells on the chip surface in the thickness direction; calculate the turgor pressure P from the surface stress based on the stress relationship of thin shell theory and the Young-Laplace equation;

[0008] The calculation formula of the integral ΔS of the cell surface stress in the thickness direction is:

[0009] △S t = f r -1 (K AT -K BT ) -1 (△f t AT tq AT -△f t BT t q BT ) (1)

[0010] where K AT =2.75×10 -l2 cm 2 dyn -1 , K BT =-2.65×10 -l2 cm 2 dyn -1 are the stress coefficients of AT and BT cut quartz crystals respectively; tq AT with tq BT are the thickness of AT and BT cut quartz crystals, respectively, which are related to the crystal frequency; fr AT =fr BT =fr is the resonant frequency of AT and BT cut quartz crystals, △f t AT and △f t BT are the frequency shift of the quartz crystal relative to its reference point at any time t;

[0011] The real-time turgor pressure P is calculated as follows:

[0012] P t =2△S t ·h t / (R t ·δ t ) (2)

[0013] Where h is the cell wall thickness, R is the cell radius, and δ is the cell thickness.

[0014] These parameters are also dynamic parameters that change over time. Cell radius and thickness can be obtained in real time by optical microscopy, and cell wall thickness can be measured by electron microscopy or other methods or selected based on values ​​reported in the literature.

[0015] For cells that maintain a spherical shape, the calculation formula for turgor pressure P is simplified to:

[0016] P t =△S t ·h t / R t 2 (3).

[0017] Preferably, step (1) includes at least one AT-cut and one BT-cut quartz crystal, which are simultaneously used for measuring cell mechanical properties based on the piezoelectric effect.

[0018] Preferably, the plant cells include cells at different stages of cell growth and all mature plant cells.

[0019] Preferably, the plant cells to be measured are highly viable, dispersed, and synchronized single cell populations.

[0020] Preferably, the plant cells include suspension cells cultured from plant seeds and callus tissue, cells directly isolated and extracted from different plant organs (roots, stems, leaves) and growth cycles (such as rice seedling stage and ear stage); also include all other walled cells, including fungi, algae, and bacteria, and can also be used for the determination of hydrostatic pressure of de-walled protoplasts.

[0021] Preferably, the plant cell is approximately spherical, and turgor pressure can be directly calculated using formula (3). For protoplasts, h is the thickness of the protoplast cortex. For other plant cell shapes, including ellipsoids, polyhedrons, spindles, columns, and elongated tubes, turgor pressure can be calculated by converting the cell into a sphere of the same volume, or by model calibration.

[0022] Preferably, the temperature of the environment in which the cells are located during measurement is 22-27° C., and the pH is 5.5-6.0.

[0023] More preferably, the temperature of the environment in which the cells are located during measurement is 25° C. and the pH is 5.8.

[0024] The testing method of the present invention does not damage plant cells and can continuously monitor the changes in the real-time turgor pressure of plants under different conditions for a long time. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 The dynamic changes of the relationship between rice cell turgor pressure and extracellular osmotic pressure.

[0026] Figure 2 It shows the dynamic changes of the relationship between rice protoplast turgor pressure (hydrostatic pressure) and extracellular osmotic pressure.

[0027] Figure 3 Fitting the Sigmoidal equation for the relationship between rice cell turgor pressure and water potential.

[0028] Figure 4 Fitting the Sigmoidal equation for the relationship between rice protoplast turgor (hydrostatic pressure) and water potential.

[0029] Figure 5 :9MHz dual-resonance piezoelectric cytometry was used to monitor the changes in turgor pressure of 50,000 rice cells under different concentrations of PEG6000 simulated drought stress.

[0030] Figure 6:5MHz dual-resonance piezoelectric cytometry was used to monitor the changes in turgor pressure of 60,000 rice protoplasts under different concentrations of PEG6000 simulated drought stress.

[0031] Figure 7 : Real-time turgor response curves of drought-tolerant variety LH-1 (LH-1) and conventional variety 6527 under 5%-25% PEG6000 simulated drought stress were monitored by 9MHz dual-resonance piezoelectric cytometry.

[0032] Figure 8 : Real-time turgor response curves of drought-tolerant variety Lühan 639 (LH-639) and conventional variety Huanghuazhan (Hhz) under 5%-25% PEG6000 simulated drought stress were monitored by 9MHz dual-resonance piezoelectric cytometry.

[0033] Figure 9 : A 9MHz ITO chip monitors the changes in turgor pressure of 90,000 tobacco cells under 50-150mM NaCl simulated salt stress.

[0034] Figure 10 : A 9MHz ITO chip monitors the changes in turgor pressure of 90,000 Arabidopsis cells under 50-150mM NaCl simulated salt stress.

[0035] Figure 11 :5MHz dual-resonance piezoelectric cytometry was used to monitor the changes in turgor pressure of 10,000 rice cells under the action of different concentrations (1-8 mg / L) of the auxin IAA. DETAILED DESCRIPTION

[0036] In order to more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the specific embodiments or the description of the prior art. Obviously, the drawings described below are some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0037] Example 1

[0038] The method for monitoring the dynamic changes of turgor pressure of rice cells under different osmotic pressures comprises the following steps:

[0039] (a) Real-time monitoring of the frequency parameter changes Δf of two-cut chips in plant cells under different osmotic stresses based on dual-resonance piezoelectric cytometry t AT and △f t BTFirst, the polycationic PDADMAC was used to electrostatically modify the surface electrode of an 8MHz quartz crystal in a cell pool. Second, culture medium was added and the frequency change of the crystal was monitored using dual-resonance piezoelectric cytometry. Then, a rice callus cell suspension (approximately 20,000 cells) was added, and the cells adhered to the electrode surface under the action of electrostatics. The osmotic pressure of the solution in the cell pool was then gradually changed. Finally, frequency monitoring data of the culture medium, cell adhesion, and different osmotic pressures were obtained.

[0040] (b) Based on the stress equation, the integral of the cell surface stress in the thickness direction, ΔS, is calculated from the frequency parameter change Δf: First, the monitoring data is subtracted from the culture medium data to obtain the frequency change Δf caused by the cell and osmotic pressure changes; then, according to the stress calculation equation, the cell surface stress integral ΔS is calculated from the frequency change Δf of the two different cut types, AT and BT. ΔS = f r -1 (K AT -K BT ) -1 (△f AT t q AT -△f BT t q BT ).

[0041] (c) Calculation of turgor pressure P from surface stress based on the thin-wall theory and the stress relationship of the Young-Laplace equation: The ratio of the cell wall to the cell radius of rice callus cells is less than 0.2, which meets the thin-wall model conditions, and the cells are spherical. Therefore, the turgor pressure P can be calculated from the cell surface stress integral ΔS, cell wall thickness h, and cell radius R according to the optimized turgor pressure equation: P = ΔS·h / R 2 .

[0042] Example 2

[0043] A method for monitoring the dynamic changes of real-time turgor pressure (hydrostatic pressure) of rice protoplasts under different osmotic pressures comprises the following steps:

[0044] (a) Real-time monitoring of frequency parameter changes Δf caused by plant cells using dual-resonance piezoelectric cytometry: First, the surface electrodes of a 9 MHz quartz crystal in a cell pool were electrostatically modified using polycationic PDADMAC. Second, culture medium was added, and the frequency changes of the crystal were monitored using dual-resonance piezoelectric cytometry. Next, a rice protoplast suspension (approximately 50,000 protoplasts) was added, and the cells adhered to the electrode surface under the action of electrostatic force. The osmotic pressure of the solution in the cell pool was then gradually changed. Finally, frequency monitoring data under different osmotic pressures, culture medium, protoplast adhesion, and different protoplast conditions were obtained.

[0045] (b) Based on the stress equation, the integral ΔS of the protoplast surface stress in the thickness direction is calculated from the frequency parameter change Δf: First, the monitoring data is subtracted from the culture medium data to obtain the frequency change Δf caused by the protoplast and osmotic pressure changes; then, according to the stress calculation equation, the integral ΔS of the protoplast surface stress is calculated from the frequency change Δf of the two different cut types AT and BT, ΔS = f r -1 (K AT -K BT ) -1 (△f AT t q AT -△f BT t q BT ).

[0046] (c) Calculation of turgor pressure P from surface stress based on the thin-wall theory and the stress relationship of the Young-Laplace equation: The ratio of the plasma membrane to the protoplast radius of rice protoplasts is less than 0.2, which meets the thin-wall model conditions. The protoplasts are spherical. Therefore, the turgor pressure (hydrostatic pressure) P can be calculated from the surface stress integral ΔS, the plasma membrane thickness h, and the protoplast radius R according to the optimized turgor pressure equation: P = ΔS·h / R 2 .

[0047] Dynamic changes in the relationship between turgor pressure / hydrostatic pressure and extracellular osmotic pressure Figures 1 to 2 As shown. Among them, Figure 1 is the relationship between rice cell turgor pressure and the corresponding extracellular osmotic pressure, Figure 2 The relationship between the hydrostatic pressure of rice protoplasts and the corresponding extracellular osmotic pressure is shown in Figure 2. The turgor pressure and water potential of rice cells, as well as the hydrostatic pressure and water potential of rice protoplasts, were measured and then nonlinearly fitted. When the intracellular and extracellular water are in equilibrium, the intracellular and extracellular water potentials are equal, and the extracellular water potential is equal to the inverse of the extracellular osmotic pressure. The relationship between the turgor pressure and water potential of rice cells was fitted using the Sigmoidal equation:

[0048] P=2.139-2.8589 / (1+e (Ψ+0.334) / 0.2776 ), (R 2 =0.93381, P < 0.05), see Figure 2 The relationship between hydrostatic pressure and water potential of rice protoplasts was fitted by Sigmoidal equation: P = 0.32135-0.33495 / (1+e (Ψ-0.61733) / 0.61472 ),

[0049] (R 2 =0.99069, P < 0.05), see Figure 4It can be seen that the fitting accuracy of the established relationship model between turgor pressure P and water potential Ψ is higher than 0.93, indicating that this relationship model can be widely used in the real-time prediction of turgor pressure / hydrostatic pressure and water potential of plant cells.

[0050] Example 3 Some application scenarios of the method of the present invention (results see Figures 5 to 11 )

[0051] like Figures 5 to 11 The following are some application scenarios of the method of the present invention:

[0052] Figure 5 : 9MHz dual-resonance piezoelectric cytometry was used to monitor changes in turgor pressure in 50,000 rice cells under simulated drought stress with varying concentrations of PEG6000. Before PEG6000 stress, rice cell turgor pressure remained near zero, a state of equilibrium. Under 5%-15% PEG6000 stress, rice cells experienced transient negative turgor pressure, which increased with increasing PEG6000 concentration before rebounding to positive values. At 25% PEG6000, the cells experienced the maximum negative turgor pressure, which then recovered slightly but never returned to positive values, potentially exceeding the maximum PEG6000 concentration for rice drought tolerance.

[0053] Figure 6 : 5MHz dual-resonance piezoelectric cytometry was used to monitor changes in turgor pressure in 60,000 rice protoplasts under simulated drought stress with different concentrations of PEG6000. Under 5%-25% PEG6000 stress, protoplasts also produced negative turgor pressures followed by a rebound phenomenon, and the extreme negative turgor pressure increased with increasing PEG6000 concentration. However, the decline to the negative extreme turgor pressure and the rebound rate were slower than those of parietal cells, and the rebound values ​​remained negative or near 0. Under 25% PEG6000 treatment, protoplasts only slightly recovered after reaching the negative extreme turgor pressure.

[0054] Figure 5 and Figure 6 The results showed that the responses of cells with and without cell walls to drought turgor pressure were different.

[0055] Figure 7 :9MHz dual-resonance piezoelectric cytometry monitoring of the real-time turgor response curves of the drought-tolerant variety Green Han No. 1 (LH-1) and the conventional variety 6527 under 5%-25% PEG6000 simulated drought stress. PEG6000 concentration: (A): 5%,

[0056] (B): 10%, (C): 15%, (D): 20%, (E): 25%). The results showed that at the same PEG6000 concentration, the negative turgor pressure value produced by Luhan No. 1 was much smaller than that of the control variety 6527.

[0057] Figure 8: Real-time turgor response curves of drought-tolerant variety LH-639 (LH-639) and conventional variety Huanghuazhan (Hhz) under 5%-25% PEG6000 simulated drought stress monitored by 9MHz dual-resonance piezoelectric cytometry. PEG6000 concentration:

[0058] (A): 5%, (B): 10%, (C): 15%, (D): 20%, (E): 25%). The results showed that at the same PEG6000 concentration, the negative turgor pressure value produced by Lvhan 639 was much smaller than that of the control variety Huanghuazhan.

[0059] Figure 7 and Figure 8 The results showed that the drought-tolerant rice varieties had a smaller decrease (change) in turgor pressure under the same drought stress than the control conventional rice varieties, and had stronger drought resistance, which was consistent with the known field test results.

[0060] Figure 9 A 9MHz ITO chip monitored changes in turgor pressure in 90,000 tobacco cells under simulated salt stress of 50-150mM NaCl. Under 50-75mM NaCl, cells contracted, generating negative turgor. At 100-125mM NaCl, positive turgor pressure began to develop, and plasmolysis began to be observed in the corresponding cells. At the moment of adding 150mM NaCl, turgor pressure plummeted, and plasmolysis was completely observed in the corresponding cells. This indicates that saltwort cells can tolerate NaCl concentrations of 100-125mM.

[0061] Figure 10 A 9MHz ITO chip monitored the turgor pressure changes of 90,000 Arabidopsis cells under simulated salt stress of 50-150mM NaCl. Within the tested NaCl concentration range, no drastic drop in turgor pressure or obvious plasmolysis, as seen in saltwort cells, was observed. Furthermore, Arabidopsis cells are smaller than tobacco cells, suggesting a stronger tolerance to salt stress.

[0062] Figure 11 A 5MHz dual-resonance piezoelectric cytometry technique was used to monitor the turgor pressure changes in 10,000 rice cells exposed to different concentrations of the auxin IAA (1-8 mg / L). At 1 mg / L IAA, turgor pressure decreased, favoring cell growth. At concentrations greater than 2 mg / L, turgor pressure increased, hindering cell growth. Low and high concentrations exhibit opposite effects, demonstrating the dual nature of auxin.

Claims

1. A method for monitoring the dynamic changes of plant cell turgor pressure in real time, characterized in that: The method comprises the following steps: (1) Modifying molecules or materials that can electrostatically or chemically interact with cell walls or protoplasts on AT-cut and BT-cut double-resonance quartz crystal white pieces or conductive electrode surfaces or insulating films on electrode surfaces, wherein the conductive electrode materials are metal materials compatible with cell biotechnology and ITO materials that are both conductive and transparent and allow optical microscopy observation; the insulating film material is SiO2; the molecules or materials that can electrostatically or chemically interact with cell walls or protoplasts are materials that promote the adhesion of plant cells or protoplasts; the materials that promote the adhesion of plant cells or protoplasts are positively charged poly-L-lysine, chitosan or PDADMAC, including the following plant cell transmembrane force-sensitive proteins or small molecules: a) RGD and Fibronectin that can interact with clusterin-like receptor kinases and AT14A; b) pectin that interacts with cell wall-bound receptor kinases; c) polyclonal antibodies for the extracellular domain that interact with FEKONIA in the Catharanthus roseus receptor kinase family; d) polyclonal antibodies for the extracellular domain that interact with Formin in the Formin family.

1. A polypeptide fragment with specific interaction; the AT-cut quartz crystal and the BT-cut quartz crystal have the same frequency, surface morphology and are modified with the same surface adhesion molecules; (2) The modified AT-cut and BT-cut quartz crystals of step (1) are placed in a detection cell, and the walled plant cells or wall-removed protoplasts to be measured are added to the detection cell, and the frequency change Δf of the AT-cut and BT-cut chips caused by the adhesion, growth or adverse stress of the plant cells on the AT-cut and BT-cut chips is monitored in real time based on dual-resonance piezoelectric cytometry. AT With Δf BT ; Based on the stress equation, the frequency parameter changes Δf AT With Δf BT Calculate the integral ΔS of the stress exerted by cells on the chip surface in the thickness direction; calculate the turgor pressure P from the surface stress based on the stress relationship of thin shell theory and the Young-Laplace equation; The calculation formula of the integral ΔS of the cell surface stress in the thickness direction is: ΔS t = f r -1 (K AT -K BT ) -1 (Δf t AT t q AT -Δf t BT t q BT ) (1) where K AT =2.75×10 -l2 cm 2 dyn -1 , K BT =-2.65×10 -l2 cm 2 dyn -1 are the stress coefficients of AT and BT cut quartz crystals respectively; tq AT with tq BT are the thickness of AT and BT cut quartz crystals, respectively, which are related to the crystal frequency; f r AT =f r BT =f r is the resonant frequency of AT and BT cut quartz crystals, Δf t AT With Δf t BT are the frequency shift of the quartz crystal relative to its reference point at any time t; The real-time turgor pressure P is calculated as follows: P t =2ΔS t ·h t / (R t ·d t ) (2) Where h is the cell wall thickness, R is the cell radius, and δ is the cell thickness; For cells that maintain a spherical shape, the calculation formula for turgor pressure P is simplified to: P t =ΔS t ·h t / R t 2 (3)。 2. The method for monitoring the dynamic changes of plant cell turgor pressure in real time according to claim 1, characterized in that: The step (1) includes at least one AT-cut quartz crystal and one BT-cut quartz crystal, which are simultaneously used for measuring the mechanical properties of cells based on the piezoelectric effect.

3. The method for monitoring the dynamic changes of plant cell turgor pressure in real time according to claim 1, wherein: The plant cells include cells at different stages of cell growth and all mature plant cells.

4. The method for monitoring the dynamic changes of plant cell turgor pressure in real time according to claim 3, wherein: The plant cells measured were highly active, dispersed, and synchronized single cell populations.

5. The method for monitoring the dynamic changes of plant cell turgor pressure in real time according to claim 3, wherein: The plant cells include suspension cells cultured from plant seeds and callus tissue, cells directly separated and extracted from different organs and growth cycles of plants; they also include all other walled cells, including fungi, algae, and bacteria, and can also be used to measure the hydrostatic pressure of wall-removed protoplasts.

6. The method for monitoring the dynamic changes of plant cell turgor pressure in real time according to claim 1, wherein: The plant cell is approximately spherical, and turgor pressure can be directly calculated using formula (3). For protoplasts, h is the thickness of the protoplast cortex. For plant cells of other shapes, including ellipsoids, polyhedrons, spindles, columns, and elongated tubes, turgor pressure can be calculated by converting the cell into a sphere of the same volume, or by using model calibration.

7. The method for monitoring the dynamic changes of plant cell turgor pressure in real time as claimed in claim 1, characterized in that: During the measurement, the cells were placed in an environment with a temperature of 22-27°C and a pH of 5.5-6.

0.

8. The method for monitoring the dynamic changes of plant cell turgor pressure in real time as claimed in claim 7, characterized in that: During the measurement, the cells were placed in an environment with a temperature of 25°C and a pH of 5.8.

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