Determination of chlorophyll fluorescence parameters in plants
Determination of plant chlorophyll fluorescence parameters is to master the basic principles and methods of portable chlorophyll fluorescence meter to determine chlorophyll fluorescence; to understand the physiological significance of chlorophyll fluorescence parameters and its application in the study of plant photosynthetic physiology, adversity physiology and so on.
Principle
The basic principle of the determination of chlorophyll fluorescence parameters in plants is that the energy conversion of photosynthesis is mainly a charge separation process in the reaction centers (photosystems I and II), i.e. the transfer of electrons from a specific chlorophyll molecule (P700 or P680) to an electron acceptor. It has been shown that chlorophyll fluorescence in living organisms is mainly related to photosystem II (Fig. 19-1). The light energy absorbed by plants is divided into three main components: photochemistry (P), chlorophyll fluorescence (F) and heat dissipation (D), which are related as follows:
P + F + D = 1
The chlorophyll fluorescence analysis technique accurately obtains the amount of photosynthesis and related plant growth potential data by measuring the amount of chlorophyll fluorescence. Chlorophyll fluorescence kinetics has a unique role in determining the absorption, transfer, dissipation, and distribution of light energy by the photosystem during leaf photosynthesis. Compared with the "apparent" indicators of gas exchange, chlorophyll fluorescence parameters have a more important role to play in the determination of photosynthesis. Chlorophyll fluorescence parameters are more reflective of the "intrinsic" characteristics. In this experiment, the main parameters of chlorophyll fluorescence were measured by modulated chlorophyll fluorometer PAM (WALZ).
As the photochemical light intensity increases, the photochemical quantum yield P gradually decreases, while the dissipated quantum yield D gradually increases. This gives rise to complex changes in the fluorescence yield F. An important breakthrough in fluorescence research was the temporary saturation of photosystem II with a saturation pulse (P = 0). The values of chlorophyll fluorescence parameters vary depending on the growth condition of the plant leaf, its location, and the light, so that there are some differences in chlorophyll fluorescence yields between leaves.
Operation method
Determination of chlorophyll fluorescence parameters in plants
Principle
The basic principle of the determination of chlorophyll fluorescence parameters in plants is that the energy conversion of photosynthesis is mainly a charge separation process in the reaction centers (photosystems I and II), i.e. the transfer of electrons from a specific chlorophyll molecule (P700 or P680) to an electron acceptor. It has been shown that chlorophyll fluorescence in living organisms is mainly related to photosystem II (Fig. 19-1). The light energy absorbed by plants is divided into three parts: photochemistry (P), chlorophyll fluorescence (F) and heat dissipation (D), and the relationship between them is as follows: P + F + D = 1 Chlorophyll fluorescence analysis technology accurately obtains the amount of photosynthesis and the related growth of the plant by measuring the amount of chlorophyll fluorescence. photosynthesis and related plant growth potential data by measuring chlorophyll fluorescence. Chlorophyll fluorescence kinetics has a unique role in determining the absorption, transfer, dissipation and distribution of light energy by the photosystem during leaf photosynthesis. Compared with the "apparent" indicators of gas exchange, chlorophyll fluorescence parameters have a more important role to play in the determination of photosynthesis. Chlorophyll fluorescence parameters are more reflective of the "intrinsic" characteristics. In this experiment, the main parameters of chlorophyll fluorescence were measured by modulated chlorophyll fluorometer PAM (WALZ). As the photochemical light intensity increases, the photochemical quantum yield P gradually decreases, while the dissipated quantum yield D gradually increases. This gives rise to complex changes in the fluorescence yield F. An important breakthrough in fluorescence research was the temporary saturation of photosystem II with a saturation pulse (P = 0). The values of chlorophyll fluorescence parameters vary depending on the growth condition of the plant leaf, its location, and the light, so that there are some differences in chlorophyll fluorescence yields between leaves.
Materials and Instruments
Material: plant leaves. Move The basic procedure for the determination of plant chlorophyll fluorescence parameters can be divided into the following steps: 1. Installation and connection of the instrument: connect the optical fiber to the main control unit and leaf clamp. One end of the fiber optic must be connected to the master unit via a three-hole fiber optic connector located on the front panel. The other end of the fiber optic must be fixed to the leaf clamp B. The leaf clamp B should also be connected to the master unit via a LASER. The other end of the fiber optic must be secured to the Leaf Clamp B. The Leaf Clamp B should also be connected to the Master Unit through the LEAF CLIP connector. 2. Power on: Press the "POWER ON" button to turn on the built-in computer and the green indicator light starts to flash to indicate that the instrument is working normally. The PAM will then be displayed in the display of the main control unit. 3. 3. PAM main control unit: There are keys on the PAM main control unit, the functions of the main keys are briefly described. Esc: exits the menu or report file; Edit: opens the report file; Pulse: open to stop the saturation pulse at a fixed time interval; Fm: opens the saturation pulse measurement Fo, Fm and Fv/Fm after dark adaptation of the blade; Menu: opens the main menu of the kinetics window; Shift: this key only works in combination with other keys; +: increases the numerical (parameter) settings for the selected zone; -: decreases the numerical (parameter) setting of the selected zone; Store: stores the recorded kinetic curve; Com: opens the command menu; ◀: the pointer moves left; ▶: shift the pointer right; ▲: the pointer moves up; ▼: pointer moves down; ▼: pointer moves up; ▼: pointer moves down; Act: turn on photochemical light; Yield: turn on a saturation pulse to determine the photosystem H effective quantum yield F/F 'm of the illuminated state. 4. measurement: (1) Adjust the F by choosing the appropriate measurement light intensity, gain and distance between the sample and the optical fiber. 200~400 mV. Meanwhile, in order to avoid human error, it is recommended to set a reasonable saturation pulse intensity and duration by checking the fluorescence kinetic change curve obtained during the saturation pulse. This is achieved by pressing the Pulse kinetics function in the Com menu. (2) Acquisition of Fo, Fm and Fv/Fm : Fo can be determined by pressing "shift return" to bring up the menu and execute Fo determina-tion , Fo can also be measured by pressing the key of the external keyboard, and Fo can be measured by pressing "Fm" or pressing the key of the external keyboard. Fm can be measured by pressing the "Fm" key or the "M" key of the external keyboard, and Fv/Fm will also be obtained automatically. (3) To obtain the quantum yield Yield: Simply press the "Yield" key. Or move the pointer to "RUN" to activate it. 5. Data output: (1) Connect the RS data cable with the PAM master unit. (2) Enter the Dynamics window and press the "Menu" key to enter the Data sub-menu and select TransferFiles and press the Enter key. (3) A window opens to select the ComPort of the RS data line. After selecting and activating the ComPort, another window appears showing the data file stored in the PAM. Double-click on the file to transfer it. 6. Turning off the instrument: Press "Com" to bring up a command selection menu, press "V" to select "Quitprogram" and press enter to turn off the instrument. Remove the optical fiber and leaf clip B and put them into the special case for fluorescence instrument.
Equipment: PAM2100 chlorophyll fluorometer.
Fo: original fluorescence yield, also called basic fluorescence, is the initial fluorescence yield when the PSII reaction center (after sufficient dark adaptation) is in a completely open state.
Fm: maximal fluorescence yield, is the fluorescence yield when the PSII reaction center is completely closed. It is usually measured after the leaves have been dark-adapted for 20 min.
Fv = Fm-Fo: variable fluorescence, reflecting the maximum potential for electron transfer in PSH. Measured after dark adaptation.
Fv/Fm: the maximum photochemical efficiency when the PSII reaction center is fully open under dark adaptation, reflecting the maximum light energy conversion efficiency of PSII reaction center.
Fv/Fo: represents the potential photochemical activity of PSⅡ, which is proportional to the number of active reaction centers.
Fo ': initial fluorescence under photoacclimation.
Fm ': maximum fluorescence under photoacclimation.
Fv ' = Fm ' -Fo ': variable fluorescence under light adaptation.
Fv ' /Fm ': maximum photochemical efficiency of PSII under photoacclimation, which reflects the photochemical efficiency when the PSII reaction center is fully open in the presence of heat dissipation, also known as maximum antenna conversion efficiency.
Ft (or Fs ): steady-state fluorescence yield.
φPSⅡ = (Fm' -Fs ) /Fm ': the actual photochemical efficiency of PSⅡ, which reflects the actual photochemical efficiency in the case of partial closure of the PSⅡ reaction center under illumination. qP = (Fm'-Fs) /Fm ': the actual photochemical efficiency of PSⅡ when the PSⅡ reaction center is completely open.
qP = ( Fm ' -Fs )/( Fm ' -Fo '): photochemical quenching coefficient, which reflects the degree of opening of the PSII reaction center.
1-qP: used to indicate the degree of closure of PSII reaction center.
(iii) Experimental Measurements1. Determination of leaf Fo, Fm and Fv/Fm: Mature leaves of two tree species were selected from the campus to determine Fo, Fm and Fv/Fm, and to compare the similarities and differences between different plants. 2.
2. Determination of quantum yield of leaf blade: Determination of Yield of mature leaves of experimental plants selected in the campus, and compare the similarities and differences between different plants.
Caveat
1. It is prohibited to connect an external power supply while the power is on.
2. Excessive bending of the optical fiber is prohibited.
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