Method for measuring size and connectivity of plant leaf light system II antenna

Abstract

The invention discloses a method for measuring the size and connectivity of a plant leaf light system II antenna, and relates to the field of plant leaf chlorophyll fluorescence determination.According to the method, an analytical solution of a QA reduction kinetics differential equation is provided, the uncertainty of parameter estimation is minimized, a PEA portable chlorophyll luminoscope is utilized, and the size and connectivity of a plant leaf light system II antenna are measured. A key parameter Fmj is obtained through a mathematical statistical method. According to the method for measuring the size and connectivity of the light system II antenna of the plant leaf, curve fitting and parameter calculation are carried out on the basis of solving the two technical problems, the method for reliably measuring and calculating the size and connectivity of the light system II antenna is provided, and then the method is used for related research of a photosynthesis efficiency regulation and control mechanism. The method provides a powerful tool for researching major scientific problems such as a crop high-light-effect adjusting mechanism and a forest carbon sink capacity adjusting mechanism, and has a wide application prospect.

Description

Technical Field

[0001] The invention relates to the field of plant leaf chlorophyll fluorescence measurement, in particular to a method for measuring the size and connectivity of a leaf photosystem II antenna. Background Art

[0002] Photosynthesis is one of the most important physiological processes of plants. The photosynthesis carried out by green plants is the energy basis for the survival of the biosphere and the main source of primary productivity in the biosphere. The regulatory mechanism of plant photosynthesis efficiency is an important research topic in terms of improving crop yields and estimating carbon sinks in ecosystems. Plant chlorophyll fluorescence technology is currently a commonly used and reliable method for detecting plant photosynthesis capacity efficiently and non-destructively. Based on the basic principles of chlorophyll fluorescence, researchers have invented various types of chlorophyll fluorescence meters, such as pulsed modulation fluorescence meters (such as PAM101-102-103), continuous plant efficiency analysis fluorescence meters (such as Plant Efficiency Analyser), etc., which have become fast and powerful tools in photosynthesis research. The application of these instruments also provides strong technical support for the study of rapid fluorescence induction dynamics.

[0003] During the transition from darkness to light, the chlorophyll fluorescence induction (FI) curve exhibits a complex rising morphology and can be divided into four transient stages, including OJIP ( Figure 1 ). In order to study the electron transfer and energy conversion of photosystem 2 (PSII) in plants and algae, scholars have conducted a lot of analytical research on FI. After the green organs of plants are fully dark-adapted, the electron acceptor of PSII: Q A (primary receptor quinone of PSII), Q B (PSII secondary receptor quinone) and PQ (plastoquinone) are all in an oxidized state (Krause and Weis 1991; Strasser et al. 1995). At this time, the ability of the PSII receptor side to accept electrons is the greatest, and the PSII reaction center can accept light quanta to the maximum extent, that is, it is in a "fully open" state. At this time, the fluorescence emitted by the sample after being exposed to light is the smallest, and it is in the initial phase "O" (20-50μs). When the sample is exposed to strong light, the electrons generated by the PSII reaction center after being excited are transferred to Q via Pheo (de-pheophytin). A , restore it to generate Q A - At this time, due to Q B Cannot get from Q in time A - Accepts electrons to oxidize it, creating Q A - The fluorescence rapidly rises to the J point (2ms). B Can be from Q A- Accepts electrons to form Q B - , Q B 2- , PQ - The fluorescence rises to point I (>20ms). Further strong light irradiation will cause the electron carriers in the entire PSII electron transport chain to enter a completely reduced state, at which point the PSII reaction center is completely closed, the excitation energy received cannot separate the original charge, the fluorescence output is the highest, and point P (>200ms) appears.

[0004] FI after point J is considered to be A The OJ stage is relatively simple, mainly reflecting the reduction of Q A There is experimental and theoretical evidence that the OJ phase is the "photochemical phase" related to the primary photochemical process. Therefore, researchers have used a variety of kinetic models to fit the OJ curve to derive PSII photochemical kinetics and structural parameters. Based on the "exciton / radical pair equilibrium" model, two important parameters, such as the PSII functional absorption cross-sectional area and its connectivity, can be obtained by fitting the OJ segment (Strasser et al. 2004; Schriber et al. 2012). These parameters are related to the estimation of the proportion of open PSII centers, S2Q A - It plays a very important indicative role in the research of charge recombination, photoprotection of non-photochemical quenching and photosynthetic efficiency related to crop yield, reflecting the changes in the ability of plant photosynthesis system to absorb, transmit and convert light energy.

[0005] At present, there is still uncertainty about whether there is connectivity between PSII antennas in terms of energy transfer. A mainstream view is that the photosystem 2 reaction center (PSII RCs) is embedded in a large light-harvesting pigment matrix, and energy may be transferred from a closed PSII antenna to the adjacent open PSII RCs, resulting in an S-shaped rise in fluorescence in the OJ segment. This model has been proven by OJ fitting techniques by Strasser, Schreiber et al., LIFT / RRP techniques by Kolber, Osmond et al., fluorescence lifetime analysis, exciton annihilation and time-resolved photoelectric experiments, and CCD spectral analysis. On the other hand, Laisk et al.'s improved single-flip flash-induced O2 analysis method does not support this model. They found that the PSII antenna has no antenna connectivity under steady-state illumination, which in turn affects the reliability of the PSII functional absorption cross section obtained by traditional methods.

[0006] Laisk et al. have two main doubts about the traditional OJ fitting method: First, the Q derived from the "exciton / radical pair equilibrium" model A The reduction dynamics differential equation currently has no analytical solution, and all existing fitting methods are based on numerical iteration processes, which makes the estimated parameter values ​​unreliable. mj The maximum chlorophyll fluorescence produced by a single-inversion saturation flash is always lower than that produced by multiple-inversion saturation pulses. m level (>200 ms). The maximum chlorophyll fluorescence yield obtained by the LIFT / FRR instrument is less than the yield obtained from the same leaf measured by the PAM instrument with saturation multiple reversal pulses. This quenching is due to the quenching of oxidized PQ. mj Reflects the fully reduced Q A state and participate in the OJ fitting process, so the appropriate F is determined in the OJ stage mj The single-flip flash function is very important (Schreiber 2012, 2023; Schreiber et al. 1995; Osmond et al. 2017; Keller et al. 2019). For commercial instruments like PEA, there is no single-flip flash function during the continuous recording of fluorescence from dark to bright, which leads to only empirical estimates of the PSII functional absorption cross section and its connectivity in studies using this instrument. In addition, the F obtained by a single-flip flash is not very accurate. mj Can it reflect the fully restored Q A Status is currently controversial. Summary of the invention

[0007] The purpose of the present invention is to use a traditional chlorophyll fluorescence meter (such as PEA) to provide a method for reliably measuring the antenna size and connectivity of the photosystem II of plant leaves on the basis of solving the two technical problems mentioned above, and then to use it in related research on the regulation mechanism of photosynthesis efficiency, and to serve major theoretical and practical topics such as high-yield and efficiency-enhancing crops and carbon sink regulation mechanisms of ecosystems.

[0008] To achieve the above purpose, we first obtain Q A Recover analytical solutions to the kinetic differential equations, thereby minimizing the uncertainty in parameter estimates.

[0009] The fluorescence yield before 2 ms (OJ, or O-I1) can be fitted by an equation (Schreiber et al., 2012) based on the “exciton-radical pair equilibrium model” (Lavergne and Trissl, 1995): (1) Where f(t) is the fluorescence yield at time t, Fo It's Q A The minimum fluorescence yield when fully oxidized, F mj It's Q A The maximum fluorescence yield at time point J when the reduction is complete, C(t) is the fraction of closed PSII reaction centers, and p is the connectivity parameter (Joliot).

[0010] In formula (1), when Q is not considered A - During reoxidation, the closing rate of the PSII reaction center can be fitted by a differential equation (Kolber, 1998): (2) in, s PSII is the functional absorption cross section of PSII, f is the proportion of light absorbed by PSII, and I is the intensity of the excitation light, in μmol photons m -2 s -1 In their study published in 1998, Kolber et al. only provided a numerical solution to differential equation (2). Here, we calculated the analytical solution of this equation using a self-made Matlab program code (Matlab, R2010b, the MathWorks, Natick, Massachusetts, USA): (3) where W(t) is the Lambert W function: (4) in lambertw yes Lambert-W The expression symbol for a function; it is the function f(W) = W*e W The inverse relation of W is an exponential function. When used in practice in this calculation, the W0 branch is applied because t≥0.

[0011] As mentioned above, Q is not considered in formula (3). A - Reoxidation process. When at time t, Q A - Reoxidation occurs, so the function of C(t) should be composed of three parts: (1) Q is not considered A - Reoxidation C(t) (Equation 3); (2) Oxidation rate constant ( k ox ); (3) time t: (5) ink ox It may contain several components (Kolber et al., 1998), and for the convenience of fitting, we consider the overall oxidation rate constant here.

[0012] For fitting purposes, F o The value is the minimum reliable fluorescence yield recorded at a time point of 50 μs using a PEA fluorimeter (Strasser 2004), and the value of f is the proportion of excitation energy allocated to PSII. In steady-state photosynthesis, f ranges between 0.47 and 0.50 in spinach, poplar, rice, and cotton leaves (Zhang et al., 2018), but its value in dark-adapted leaves has yet to be determined. Here, f is assumed to be 0.5. Therefore, if F mj It is known that there are three unknown variables in formula (5), namely s PSII , p and k ox To derive these three parameters, we use lsqcurvefit() Wrote a Matlab program code, lsqcurvefit() is a nonlinear curve fitting function based on the improved Gauss-Newton algorithm and the trust region method. It aims to find the minimum root mean square error (RMSE) among all potential fits. The RMSE is calculated as: (6) where Y i is the fluorescence measurement, Y i ¢ is the predicted value and n is the number of data.

[0013] F mj It is also one of the most important fluorescence parameters in the calculation. Its determination is important for the above model fitting. At the end of the OJ segment in the photochemical stage, Q A The conclusion of whether the oxidation is complete (Stirbet and Govindjee, 2012) is crucial. s PSII Will follow F mj For LIFT / FRR fluorometers (Kolber et al., 1998) and improved PAM fluorometers (Schreiber et al., 2011), a single flip flash or a series of single flip flashes is usually used at the J point to obtain the F mj The obtained data were fitted and basically conformed to the exciton-radical pair equilibrium model (Lavergne, Trissl, 1995). However, the PEA fluorometer cannot measure F at the J point. mj, in order to measure the F at the OJ stage without saturation pulses in the PEA fluorometer mj , we used mathematical statistics to calculate. According to the research of Kolber et al., s PSII It is independent of the excitation light intensity, so by analyzing s PSII The change of F mj is reliable, we set a series of light intensity gradients and mj Assuming a gradient of (ranging from the fluorescence yield at point J to a higher uncertainty value), each F mj Under the gradient, there are s PSII The relative root mean square error (RRMSE) of the predictions for different light intensities. We believe that by calculating the excitation light intensity, there is a parameter s PSII The lowest RRMSE is the F mj It is the F at point J. mj The lower the RRMSE value, the higher the fitting accuracy. The RRMSE equation is: (7) in Ȳ i yes Y i The average value of .

[0014] In order to verify the calculation, the present invention also actually measured k ox This parameter can be measured by using a PAM 101-102-103 fluorometer to measure Chl a The fluorescence yield of the flash-induced leaves was measured at room temperature using a pulse-modulated fluorometer (PAM101 and 103, Walz, Effeltrich, Germany). a Fluorescence yield decay. A single flip flash (full width at half maximum 6 μs) was emitted from an XE-STC xenon flash lamp unit (model XF-103, Walz). When the single flip flash was given, the weak monitoring light (450 nm) was switched to 100 kHz. Data acquisition (time 15 μs) was achieved using homemade equipment and a computer program (Chow and Hope 2004). Twenty flashes were emitted continuously every 5 s and the signals were averaged. Flash overload artifacts after the flash (the first 3-4 points) were deleted before kinetic fitting. The fitting followed a first-order reaction with k ox (Measured Q A Oxidation coefficient, ms -1) and amplitude (A) as unknown quantities. Fitting the flash-induced Chl a The decay equation of fluorescence yield is: (8) where y is the fluorescence yield and t is the time (ms). k ox and A were obtained by Origin 7 (Microcal Software Inc, Northhampton, MA, USA) based on the Levenberg-Marquardt method. k ox The initial values ​​of and a are assumed, and after multiple iterations, both tend to stable values. mj is reliable, then k ox The value of ¢ should be close to k ox The numerical value of .

[0015] A method for determining the size and connectivity of the antenna of the photosystem II of a plant leaf comprises the following steps: 1. Sample preparation: Take fresh spinach leaves, wipe off the dust on the surface, wrap them in moist gauze, and put the leaves directly into the leaf clip during measurement.

[0016] 2. Instrument preparation: Turn on the PEA instrument (Plant Efficiency Analyzer, Hansatech Instruments Ltd., King's Lynn, Norfolk, England) in advance and set the measurement intensity and measurement time to 3000, 3100, 3200, 3300, 3400, and 3500 (μmol m –2 s –1 The instrument records the fluorescence transient of the leaves from 10 μs to 1 s, with a data acquisition rate of 100,000 readings per second within 2 ms and 1,000 readings per second after 2 ms.

[0017] 3. Sample fixation and measurement: After dark adaptation for 30 minutes, place the material in the leaf clamp and start the measurement. Each leaf is measured six times at the same position. a Fluorescence transient measurements were performed six times using 3000, 3100, 3200, 3300, 3400, and 3500 μmol m –2 s –1 The cells were subjected to actinic light and dark adaptation was performed for 30 min between each measurement.

[0018] 4. Data processing: The fluorescence data is exported from the instrument, and the function analytical solution described in the present invention is used to fit the data using Matlab software, and finally the calculation results are exported to Excel for storage. s PSⅡ , Connectivity p , Oxidation coefficient k ox The parameter values ​​are shown in the figure ( Figure 2 ), the change of fitting time should have little effect on the three parameters, so it can be seen that the measured values ​​of antenna size and connectivity are more reliable before 500us. We speculate that since PEA provides continuous light, when the fitting time is greater than 500us, the parameters may be affected by the multi-flash effect.

[0019] The technical route of the present invention is shown in the figure ( Figure 3 ), due to the adoption of the above technology and calculation scheme, compared with the previous chlorophyll fluorescence parameter determination method, it has the following advantages: 1) Q A The analytical solution of the reduction kinetic differential equation minimizes the uncertainty of parameter estimation; 2) Using the PEA portable chlorophyll fluorescence instrument, the key parameter F is obtained by mathematical statistics. mj Through these two improvements, the PSII antenna size and connectivity of plants can be measured quickly and non-destructively in field surveys related to ecology, agronomy, forestry, and plant physiology, providing a powerful tool for studying major scientific issues such as the regulation mechanism of crop high light efficiency and the regulation mechanism of forest carbon sequestration capacity, and thus has broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 Schematic diagram of the OJIP curve of chlorophyll fluorescence induction dynamics Figure 2 The relationship between the photosynthetic parameters and the illumination time obtained by the method of the present invention is Figure 3 Technical route for determining the size and connectivity of antennas in plant leaf photosystem II DETAILED DESCRIPTION

[0021] The present invention is described in detail below in conjunction with the accompanying drawings and embodiments. However, it should be understood that the accompanying drawings are provided only for a better understanding of the present invention and should not be construed as limiting the present invention.

[0022] like Figure 2 As shown, the method for determining the antenna size and connectivity of the photosystem II of plant leaves provided by the present invention can obtain relatively reliable and regular results, and the instrument required for the experimental process is PEA (Plant Efficiency Analyser, continuous excitation plant efficiency analyzer).

[0023] In a preferred embodiment, in order to prevent the leaves from losing water during the measurement process, the leaf clip is covered with wet gauze and placed in a light-proof environment during the measurement process.

[0024] In a preferred embodiment, a statistical method is used to determine the appropriate F mj / F o , set 3500,3400,3300,3200,3100,3000 (μmol m -2 s -1 ) Six light intensity gradients.

[0025] In a preferred embodiment, to ensure the stability and accuracy of the measurement, each group of different light intensities is fixed at the same position on a leaf, dark adaptation treatment is performed for 30 minutes between each measurement, and movement of the leaf is minimized.

[0026] In a preferred embodiment, the sample to be tested is a fresh leaf.

[0027] The following describes in detail the operation and calculation method of measuring the antenna size and connectivity of the photosystem II of a plant leaf according to the present invention through a specific embodiment, comprising the following steps: 1. Collect fresh leaves to be tested, wipe off the dust on the surface, and wrap them with wet gauze for dark adaptation; 2. Turn on the PEA instrument (Plant Efficiency Analyzer, Hansatech Instruments Ltd., King's Lynn, Norfolk, England) and set the measurement time to 2 s. The PEA instrument records the fluorescence transient of the leaves from 10 μs to 1 s, with a data acquisition rate of 100,000 readings per second within 2 ms and 1,000 readings per second after 2 ms. 3. Place the leaf to be tested after dark adaptation for 30 minutes in the soaked leaf clip gasket, fix it in the leaf clip, with the upper surface of the leaf facing the light emission probe of the instrument, and cover the leaf clip with wet gauze; 4. Set the PEA instrument to measure the actinic light intensity to 3500 μmol m –2 s –1 , start measuring; 5. The leaves after the measurement were dark-adapted for another 30 min. After the adaptation, the PEA measurement light intensity was set to 3400 μmol m –2 s –1 , start measuring again; 6. The leaves after the measurement were dark-adapted for another 30 min. After the adaptation, the PEA measurement light intensity was set to 3300 μmol m –2 s –1 , start measuring again; 7. The leaves after the measurement were dark-adapted for another 30 min. After the adaptation, the PEA measurement light intensity was set to 3200 μmol m –2 s –1 , start measuring again; 8. The leaves after the measurement were dark-adapted for another 30 min. After the adaptation, the PEA measurement light intensity was set to 3100 μmol m –2 s –1 , start measuring again; 9. The leaves after the measurement were dark-adapted for another 30 min. After the adaptation, the PEA measurement light intensity was set to 3000 μmol m –2 s –1 , start measuring again; 10. After the measurement, set the instrument to "PC Mode" and connect it to the computer, and import the data into the computer for sorting; 11. Open Matlab software and run the self-written program to fit the OJ curve of each repetition and each light intensity, and obtain the antenna size and connectivity measured each time. Average the different light intensity data of each repetition, and use different fitting times to further organize and analyze the output parameters, and finally obtain the illumination time and the antenna size of the optical system II measured by the method of the present invention. s PSⅡ , Connectivity p , Oxidation coefficient k ox and F mj / F o The relationship between.

[0028] The correctness of the antenna size and connectivity of the blade optical system II provided by the present invention is verified, and the specific steps are as follows: 1. Collect 3 fresh spinach leaves, number them in sequence, and place them in wet gauze for dark adaptation for 30 minutes. 2. Turn on the PEA instrument (Plant Efficiency Analyzer, Hansatech Instruments Ltd., King's Lynn, Norfolk, England) and set the measurement light intensity to 3000, 3100, 3200, 3300, 3400, and 3500 (μmol m –2 s –1The instrument records the fluorescence transient of the leaves within a time range of 10 μs to 1 s, with a data acquisition rate of 100,000 readings per second within 2 ms and 1,000 readings per second after 2 ms.

[0029] 3. After dark adaptation for 30 min, the material was placed in a leaf clip and the measurement was started. The Chla fluorescence transient state was measured six times for each leaf, using 3000, 3100, 3200, 3300, 3400, and 3500 μmol m –2 s –1 The actinic light was used and a 30-min dark adaptation period was set between each measurement.

[0030] 4. The fluorescence data was exported from the instrument, and OJ curve fitting was performed using Matlab software according to the function analytical solution of the present invention, and finally the calculation results were exported to Excel table for storage.

[0031] 5. The above measurement results are shown in Figure 2. Considering p (≈0.5) unchanged before 500us ( Figure 2 ), we can infer the PSII functional absorption cross section to t = 0, when the PSII photochemical quantum efficiency should be maximum (= 0.84, Baker 2008), and other effects are minimal. Therefore, for spinach leaves, the optical PSII antenna size is about 8.0 / 0.84 = 9.52 nm 2 At 596 nm, Chl a and b The absorption cross section is 0.29A. Therefore, Chl a / Chl b Cross section (Chl a and Chl b has a similar absorption coefficient at this wavelength) is 3.6*0.29≈1.04. If we assume that 30% of the photosynthetic light utilization is due to accessory pigments, the remainder of the absorption cross section we measured will be composed of 640 Chl a +Chl b Molecules contribute and are shared by 3-5 PSIIs ( p ≈0.5, Joiliot 1973), corresponding to 134-223 Chl per PSII a +Chl bAccording to the results obtained by single-particle cryo-electron microscopy and biochemical assays (Wei et al., 2016), for spinach leaves, the size of PSII antenna should be between 128-213 chlorophyll molecules (Melis and Anderson, 1983), which proves that the measurement of leaf photosystem II antenna size and connectivity using the present invention is reliable.

[0032] The above embodiments are only used to illustrate the present invention, wherein the implementation steps of the methods can be varied, and any equivalent transformations and improvements based on the technical solution of the present invention should not be excluded from the protection scope of the present invention.

Claims

1. A method for determining the size and connectivity of the antenna of the photosystem II of a plant leaf, characterized in that: Got Q A The analytical solution of the reduced kinetic differential equations and the method of calculating the antenna size and connectivity of Photosystem II using the analytical solution are superior to the common iterative calculation method, minimizing the uncertainty of parameter estimation. In addition, the fluorescence parameter F, which is very important in model fitting and calculation, is mj This method uses mathematical statistics to analyze F mj To determine and actually measure k ox To verify the accuracy of this method.

2. The method for calculating antenna size and connectivity according to claim 1, characterized in that: Q A The analytical solution of the reduction kinetic differential equation is fitted and calculated based on the equation of the "exciton-radical pair equilibrium model", where f is assumed to be 0.

5. mj Under certain conditions, we used a nonlinear curve fitting function based on the improved Gauss-Newton algorithm and the trust region method lsqcurvefit() A Matlab program code was written to find the minimum root mean square error (RMSE) among all potential fits and derive the best σ PSII , p and k ox .

3. The method for calculating antenna size and connectivity according to claim 1, characterized in that: Determination of fluorescence parameter F using PEA fluorometer mj Since the PEA fluorescence instrument cannot measure F at point J, mj , in order to measure the F at the OJ stage without saturation pulses in the PEA fluorometer mj , mathematical statistics were used to calculate, by setting a series of light intensity gradients and mj Assuming a gradient of 1 / 2 (ranging from the fluorescence yield at point J to a higher uncertainty value), the F mj Under the gradient, σ PSII The relative root mean square error (RRMSE) of the predictions for different light intensities is obtained from the lowest RRMSE. mj best estimate of .

4. The method for calculating antenna size and connectivity according to claim 1, characterized in that: Comparison of PSII antenna size and connectivity estimates from this method between species or treatments is more reliable when other influences are minimized. For the PEA fluorometer, data points were fitted before 300 μs.

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