Theme : PHOTOSYNTHESIS, FLUORESCENCE AND WATER REGIME. Experiment 1: Determination of the rate of photosynthesis under different conditions

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Theme : PHOTOSYNTHESIS, FLUORESCENCE AND WATER REGIME Experiment 1: Determination of the rate of photosynthesis under different conditions Exp.

1 Theme : PHOTOSYNTHESIS, FLUORESCENCE AND WATER REGIME Experiment 1: Determination of the rate of photosynthesis under different conditions Exp.1a: Follow the changes in net photosynthesis rate depending on the irradiation of maize plants (Zea mays). Quantify the PN rate as the amount of CO2 (µmol) assimilated by leaf area unit (m 2 ) per 1 sec. Exp.1b: Follow the stomatal conductance, transpiration and net photosynthesis rate of sunflower leaf as influenced by wilting. For both experiments employ the TPS-1 instrument. Principle: Measurement of CO2 uptake is the most commonly used and easiest method for determining the photosynthesis rate of plants. Using infrared gas analysis techniques, we can readily determine CO2 concentrations to within 1 ppm (part per million) and instantaneous measurements are TPS-1 possible. The TPS-1 passes a measured flow of air over a leaf sealed into a chamber called the leaf cuvette. Using a valve, the TPS-1 first samples the CO2 and H2O in the air going to the cuvette and then in the air leaving the cuvette. From the flow rate and the change in the CO2 and H2O concentrations, the assimilation rate of CO2 and the transpiration rate of water can be determined. This is commonly referred to as the "open system method of measurement". This is the method used by the TPS-1 for CO2 measurements. The TPS-1 is a completely self contained unit with all the necessary pumps, flowmeters and valves for measurements in the laboratory, classroom or field. Measurements can be made under ambient or changing conditions (CO2, H2O and light) for response curves. Comparative spot measurements, both inter- and intraspecies, can readily be made. The transfer of the measured data to spreadsheet calculator (e.g. Excel) is at disposal. Prepare the TPS-1 instrument for measurement. Install a lamp in proximity of the instrument. Place a part of the leaf blade of the intact, well watered plant into the leaf cuvette and set all necessary parameters (light source, regime, length of measurement intervals (data acquisition intervals), leaf area etc.) and follow the changes in measured parameters. Set the measurement interval 1 min. First, cover the leaf chamber by a black cloth. After a few minutes (when the conditions in the chamber are stabilised) start to follow the measurement data. Do not forget to register the measurement number(s) to be able to identify later your own data! After a few minutes remove the black cloth and start to illuminate the leaf. Register the number of earliest measurement and let the instrument to measure the parameters for a few minutes. Then, cut the apical part of the plant (do not remove the leaf from the cuvette), register the measurement number and continue with the measurement until wilting of the plant is evident. A few minutes later stop the measurement. Export the data from the TPS-1 using TRANSFER application to EXCEL software and register the following parameters (date,

time, FAR (photosynthetic active radiation), transpiration, stomatal conductance, leaf temperature, net photosynthesis, internal leaf CO2 concentration.

2 time, FAR (photosynthetic active radiation), transpiration, stomatal conductance, leaf temperature, net photosynthesis, internal leaf CO2 concentration. Plot the rate of transpiration, net photosynthesis and stomatal conductance in a graph. Highlight the points when the conditions changed and specify the characters of the changes. Try to explain the reasons of the observed changes. Table of abbreviations abbrev parameter Plot Record Day Month Hour Minute CO2 Ref reference CO 2 concentration in ppm CO2 Diff difference in analysed and reference CO 2 concentrations in ppm PAR measured photosynthetic active radiation in μmol m -2 s -1 mb Ref reference water vapour pressure in mb (milibar) mb Diff Difference in analysed and reference water vapour pressures in mb Air Temp Leaf chamber air temperature in C Leaf Area Leaf area in the leaf cuvette in cm 2 Flow Air flow rate in the leaf cuvette in cm 3 min -1 Evap Transpiration rate in mmol H 2O m -2 leaf area s -1 GS Stomatal conductance in mmol H 2O m -2 stomatal area s -1 Leaf Temp Leaf temperature in C PN Net photosynthesis rate in μmol CO 2 m -2 s -1 C Int CO 2 concentration inside the leaf in ppm Experiment 2: Chlorophyll fluorescence induction kinetics in maize leaves. Compare the induction kinetics of chlorophyll (OJIP curves) of herbicide diurone (Exp. 2a) and heat (Exp. 2b) stressed plants with control variant to evaluate the status of photosynthetic apparatus. Use the pocket fluorimeter FluorPen 100 MAX-LM. Exp. 2a: Herbicide diurone DCMU (3-(3,4-dichlorophenyl)-1,1- dimethylurea) is a specific inhibitor of plastochinone-binding site and thus it blocks the electron transfer in photosystem II. Rapid phase of fluorescence induction is defined as a transition of chlorophylle fluorescence after short pulse of actinic light to darkness-adapted plants. The record of this type of fluorescence is called OJIP curve. After diurone treatment it usually does not exhibit the classical 4phase shape.

I P-peak Control Diurone-treated J O-origin The shape of OJIP curves Break transversally the maize leaf in two places (at the 2cm distance) to accelerate the herbicide action.

3 I P-peak Control Diurone-treated J O-origin The shape of OJIP curves Break transversally the maize leaf in two places (at the 2cm distance) to accelerate the herbicide action. Put the leaf for 30s into 10µM DCMU solution. Treate identically the control plant and put into water. Let plants adapt for min in the darkness to fully oxidize all reaction centra. Measure OJIP curves using fluorimeter FluorPen and transfer the data to PC. Compare the shape of individual OJIP curves and indicate the parameters in the table (Fv variable fluorescence Fv = Fp-Fo). FO FP FV FI FJ control herbicide diurone Exp. 2b: Incubate plants at 45 C for 10 min, then let 5min at rom temperature in light. Both, heatstressed and control plants let adapt for min in the darkness. Measure OJIP curves using fluorimeter FluorPen and transfer the data to PC. Compare the shape of individual OJIP curves and indicate the parameters in the table (Fv variable fluorescence Fv = Fp-Fo). FO FP FV FI FJ control high temperature Explain the differencies in OJIP curves obtained of control plants and plants under stress conditions. What is the effect of herbicide application and high temeprature?

4 FluorPen FP Operation Manual Operation Instructions In general: Use the MENU key to scroll through sequential menu options on the digital display. Use the SET key to select a menu option based on cursor (>) position.

6 Bluetooth Pairing Enabling Bluetooth in the FluorPen Switch on the FluorPen (press and hold the SET key). Scroll to the Setting menu (press the MENU key twice, then press the SET key once). Select BT_On to enable Bluetooth (press the SET key). * Keep in mind that the FluorPen turns off automatically after about 3 minutes of no action. Turning off the FluorPen always turns off Bluetooth. FluorPen Software Start the FluorPen program. Menu: Setup - Device ID Detects the Bluetooth connected device. Icon Explanation: Download Downloads data from the FluorPen to PC. Load Loads (opens) previously saved data files. Save Saves data to hard disc. Export Exports data in.txt format. Click the Download icon or select Device>Download. The Data table appears. Save the data. Switch off the Bluetooth. In the main menu of the Flourpen open DATA BROWSE AND ERASE. Erase measured data and switch it off, press for 5 seconds the MENU key. To visualize measurement in the graphic mode, click the Graph field in the bottom bar. Use the function printscreen to transfer the data to a file, which you can print afterwards. Ask the teacher for help if any problem emerges.

Experiment 3 : Experiment 3a: Simulate water transport in vascular tissues of sunflower using colour inks, follow time course and localisation of water (ink) transport.

7 Experiment 3 : Experiment 3a: Simulate water transport in vascular tissues of sunflower using colour inks, follow time course and localisation of water (ink) transport. Place 2 test tubes in the rack so that the necks will be in contact. Put in each test tube ink of different colour. Cut the plant with blade 5 cm above the substrate and transfer the cutting area as fast as possible into a vessel filled with water. Further manipulate with the plant material under water. Carry out 5 cm long longitudinal section in the middle of the stem. Each half of the stem place into one test tube and attach test tubes together. Put the plant under direct light (table lamp) and follow transport of the ink along the stem. Scheme of experiment Experiment 3b: Detect lignin in vascular tissues on transversal stem and petiole sections of sunflower. Prepare several thin transversal sections of another plant (node and internode stem and petiole sections) and put them into a drop of HCl-floroglucinol. After 5 min carry the sections into a drop of acid glycerol on a slide, cover with cover-glass and observe under microscope. Draw a schematic picture and explain where lignin was detected and why. Similarly prepare transversal sections of plants used in Experiment 2a and evaluate under microscope, do not use the dye. In which direction did the water movement occur? Why? What could happen if the longitudinal stem sections have not been carried out under water? Did the colour inks mix together in the plant tissues?

Transpiration. Evaluation copy

Transpiration. Evaluation copy Transpiration Computer 9 Water is transported in plants, from the roots to the leaves, following a decreasing water potential gradient. Transpiration, or loss of water from the leaves, helps to create