Ellison A. Farmer, L. Wageningen UR. Pinghai Ding Use of nondestructive spectroscopy to assess chlorophyll and nitrogen in fresh leaves. Oregon State University. Dec, Marchand F. Borkowska B. Naidoo G. Atkinson C. Brown C. Zheng Y. Liu Y. Parelle J. Jamil M. Stagakis S. Van Heerden P. Biber P. Ekbladh G. Rutkowski K. Christensen B. Morris J. Rodo A. Depending on the number, distribution clumped or not and condition of thylakoids in your reaction tube, you may obtain varying reaction rates with a given light intensity.
However, that intensity may not be appropriate for all thylakoid preparations at all concentrations so you will have to experiment with both the light intensity and the concentration of your thylakoid suspension to find a rate that does not use up all of our substrate DCPIP too quickly before 90 sec.
The 15 s reading intervals consists of 10 s of thylakoid illumination and 5 s to read the absorbance in the spectrophotometer. Your goal is to establish conditions favorable for a 90 s experiment. This data collection at interval of 15 s should yield 7 data points i. This reaction is so slow it would not come to completion in 2 minutes or less. The trendline does not fit the data as nicely as the good example and there is a leveling off at the end suggesting that the DCPIP has been used up.
Measurement of Hill Reaction Rates: For each reaction tube, do the following in turn. Now you are ready to start your first trial reaction. The thylakoid suspension will settle over time so gently swirl to resuspend. Record absorbance readings and times in your lab notebook. Take care not to shield the tube from the light with your hand. Plot the data in excel as described below. If the majority of your data points are within the linear portion of the curve repeat steps Your goal is to complete 3 runs that give approximately the same rate slope of the line.
If your rate is too slow then increase the light intensity. If your rate is too fast then decrease the light intensity or decrease the amount of thylakoids. A description of simple linear regression and directions can also be found in Appendix E.
An Excel Workbook will open. If Microsoft Excel is already open, select New Workbook from the File menu to display a new spreadsheet. Assign a title to column A the x-axis of spreadsheet and enter time in seconds starting with zero. Assign a title to column B the y-axis and record the Anm values that correspond to each time point. To select data to be plotted, highlight both columns, including headers.
Click on the Charts tab below the toolbar. A gallery of chart types will appear below. Make sure that you choose the display option where the data points are NOT connected with a line. Examine the data points for linearity. If the curve begins to flatten, substrate may be depleted and those data points should be removed from the data in the scatter plot BEFORE going to the next step. Click on a data point to highlight all points that are linear make sure you have removed any that show evidence of limiting substrate.
Under the Chart menu, select Add Trendline. The Format Trendline window will open. Press Type in the left column to select linear regression. Gently squeeze the cheesecloth to express the liquid in both steps, then discard the cheesecloth and pulp in the garbage can.
To isolate chloroplasts, divide the extract into two equal portions and pour them into two 50mL plastic round-bottom, capless centrifuge tubes. After the spin, each pair of students continues with one of these tubes. From now on you are working in pairs. Carefully decant pour off the pale green supernatant and discard it. Save the green pellet chloroplast-enriched fraction.
Using a glass rod, gently resuspend the pellets by mixing 1—2mL of breaking medium into the pellet. Make sure the pellet is completely detached from the wall and mixed into the resuspension. Avoid air bubbles O2 that may oxidize enzymes and thereby reduce activity.
The breaking medium is intended to shock the chloroplasts osmotically, thereby breaking open the organelles' outer membranes and releasing the stroma while leaving the thylakoid membranes intact. Note the absence of sorbitol.
Bring the resuspended pellets to a volume of about 25mL with cold breaking medium 50mL centrifuge tube half-full , balance against a tube of water, and centrifuge at x g for 5min. Discard the supernatant. Resuspend the resulting pellet in 1.
This suspension is your stock preparation of thylakoids to be used in the Hill reaction. Keep it on ice. Table 1.A light meter and timer will be able. A gallery of community photosynthesises will appear below. Settle of thylakoid pour montage 1. Be paved to include appropriate units: time in films for the X axis and A nm for the Y infiltrate -no units. Certificate of Media Type of Medium.
Dec, Marchand F. The electrons extracted from water replace the electrons lost by the reaction center II chlorophyll. Repeat, starting with number 3, until you have a separate plot and trendline with equation and R-square value for each trial. Be sure to keep all beakers and buffers on ice. Pierre and Marie Curie Unversity. Assign a title to column A the x-axis of spreadsheet and enter time in seconds starting with zero.
You will test the effects of one environmental parameter on the Hill reaction rates. Rutkowski K. Your goal is to establish conditions favorable for a 90 s experiment. You will work with a partner and with another pair to obtain data at baseline and at conditions that test a variable that you hypothesize will show an increase or decrease from the baseline rate. P
Brown C. DCPIP accepts electrons between the electron chain components plastoquinone and cytochrome. Filter the suspension into ice-cold beakers first through two, then eight layers of cheesecloth the first filtration removes large debris, the second removes cell wall material and some nuclei. Rutkowski K.
The 15 s reading intervals consists of 10 s of thylakoid illumination and 5 s to read the absorbance in the spectrophotometer. However, that intensity may not be appropriate for all thylakoid preparations at all concentrations so you will have to experiment with both the light intensity and the concentration of your thylakoid suspension to find a rate that does not use up all of our substrate DCPIP too quickly before 90 sec. Be sure to keep all beakers and buffers on ice.
Gairola S. Christensen B. First, during the light reactions, the transport of electrons is coupled to the movement of protons from the stroma to the thylakoid lumen, forming a pH gradient across the thylakoid membrane. Quit out of all applications on your computer.
Please sign up today with your group to test the effects of one of the following parameters: temperature, light intensity, inhibitors or uncouplers. Borkowska B. Under Chart Options you can label the axes. Discard the supernatant. In a chilled blender, grind leaf tissue with 80mL ice-cold grinding medium for 5—10s at high speed.
Your goal is to establish conditions favorable for a 90 s experiment. You can also show or hide the gridlines as you choose.
Bushnell W. Variables Affecting the Rate of Photosynthesis: Temperature Consider the normal growing conditions which spinach usually tolerates. Brown C. Farmer, L.
Oecologia Francisco Javier Flores de Santiago Multiple approaches for assessing mangrove biophysical and biochemical variables using in situ and remote sensing techniques. You will practice these measurements until you get reproducible results. Ellison A.