Respiration Rates of Storage Tissue in Plants

Effect of "Aging" and Inhibitors on Respiration

Energy obtained from the oxidation of food is used by the cells of plants (and animals) to do work.  The synthesis of new cells and tissues for growth,  the transport of minerals across membranes and the opening of stomata for gas exchange are all obvious examples of cellular work requiring energy. Essentially all processes in living cells require energy.

Even though plants can make their own food through photosynthesis, they must break down some of that food to supply their own energy needs.  When all forms of food are metabolized in the plant, oxygen is consumed and energy is released, along with water and carbon dioxide, as seen in the equation below:

                        Food + O₂  CO₂  +  energy

If the starting material for respiratory breakdown of food is a sugar such as glucose a great deal of energy can be produced. The complete oxidation of a mole of glucose releases 673 kilocalories of energy:

                        C₆H₁₂O₆ (glucose)  +  6O₂ 6CO₂  + 6H₂O  +  673 kcal

Notice that the amount of O₂ consumed is equal to the amount of CO₂ produced.  Thus the rate of respiration of a plant oxidizing carbohydrate could be determined by measuring either the rate of O₂ consumption or the rate of CO₂ production.

Measuring Respiratory Rate of Potato Discs

You will be measuring respiration with an O₂ sensor that measures the amount of O₂ in the atmosphere of a chamber containing your plant material.  You will be able to determine the rate at which O₂ is being removed by determining the slope of the line recorded as the O₂ concentration changes. You must read the file “Measurement of Plant Respiration Using  an O₂ Sensor” on the Web at www.grinnell.edu/courses/bio/qubitmanual before proceeding with this experiment.

Materials

60 "aged" or fresh discs (8 mm in diameter by 2 mm thick) cut from potato tuber per treatment

Phosphate buffer, pH 5.0

CCCP, NaCN or KCN, NaN₃ (sodium azide), or potassium malonate inhibitor

Qubit O₂ sensor apparatus

Procedures

For this experiment potato tubers provide very suitable plant material.  For "aging," all tissues should be sliced into discs 8 mm in diameter by 2 mm thick and washed for about 24 hours in aerated tap water, with changes in water at least every 12 hours.  They may also be aged by covering with water and shaking continuously for about 24 hours, again with occasional changes of the water. 

Each student will be assigned the responsibility for one or more of the treatments in the table. Weigh the tissue and determine its volume before placing it in each treatment. Determine the volume of the tissue by placing it in a graduated cylinder containing a known volume of water and determining the volume of displacement of the tissue.  Subtract this volume from 47 ml, the total volume of the leaf chamber.  Use this difference as the volume of the leaf chamber in your calculations of respiratory rate.

Prepare the appropriate solutions  and allow tissues to soak in them for at least 15 min.  Remove the discs you are about to measure from the solution and blot with a paper towel.  Then place the discs in one or two layers in the leaf chamber of the O₂ sensor and measure respiration rates, as directed.  Begin your measurements with the control tissue, if that is one of your assigned treatments.

  Calculate average microliters of O₂ absorbed per gram of fresh weight per hour for each treatment following the instructions provided.  Pool your results with those of the other students so that everyone has all the class data.  List the results in the report sheet.

1.    Before starting the experiment adjust the time axis on both graphs to a maximum of 20 minutes by highlighting the maximum value present and then typing 20.  Hit return.  Set the maximum oxygen concentration on the upper graph to 22% and the minimum at 19% using the same technique.

2.    Remove the potato discs from one treatment solution and blot with a paper towel to remove excess liquid.  Don’t dry them excessively.  Distribute the discs evenly in the lower part of the leaf chamber, in one or two layers.  Ensure that a very thin coating of vacuum grease has been applied to the gaskets of the chamber.  If any grease is visible it will not be necessary to apply more. 

3.    Before attaching the lower part of the leaf chamber containing the potato discs, click on the Start button on the bottom left hand side of the computer screen.  The button will change to a Stop button and data will begin to appear on the two graphs on the screen, and as numerals on the bottom of the screen.  The initial O₂ concentration should be close to 20.7% O₂ and the light intensity readings should be close to zero since you will not be using the light source for this experiment.  

4.    Attach the lower chamber to the upper chamber with the thumb-screws, turning them finger tight only.  Seal the gas ports with the plugs provided.

5.    Follow the decline in O₂ concentration in the leaf chamber for the next 5-10 minutes or until you have a fairly linear rate of decline.  Click on the Stop button.

6.    Save your data by selecting Save As . . in the File menu.  Give your data an appropriate file name and save it to your data folder.  Remove the discs from the leaf chamber and, if you have not previously done so, measure their total volume by submerging them in a pre-measured amount of water in a graduated cylinder.  Record this volume in your data table along with the previously measured fresh weight of the discs.

7.    Repeat these measurements with the discs from the remaining treatments.

Data Analysis

The O₂ sensor measures only the partial pressure of O₂ present in the leaf chamber; it does not measure the rate at which this O₂ is produced or consumed.  To measure the rate of oxygen consumption in your experiment, you will need to measure the decrease in O₂ concentration within the leaf chamber as a function of time.  This is achieved by measuring the rate of the O₂ response which, when the x axis of your graphs is presented in minutes, will give a rate in %O₂ per min.  The procedure for analyzing your data is as follows:

1.      Open the file containing data from one of your experiments.  A command box will appear asking you whether or not you wish to load the calibration stored with your data file.  Answer Yes.  Your data will appear on the screen exactly as it appeared when you saved it at the end of the experiment.

2.       Select Examine from the Analyze menu at the top of the screen. A vertical line will appear on your graphs which can be moved along the data points on the graphs by moving the mouse.  Note that as you move the vertical line, the numerical display in the box on the screen will change to show you the exact O₂ concentration and time value at the point on the graph where the line is situated.

3.       Measure respiration rate during the linear part of the decrease in chamber O₂ concentration.  To do this, move the vertical line to the point on your O₂ data where you wish to start the measurement, click on the mouse button and hold it down.  Move the mouse over the part of the data you wish to analyze, and then release the mouse button capturing the portion to be analyzed inside the box that appears.

4.       Select Linear Fit from the Analyze menu. In the command box that is on the screen you will see the equation for a straight line, y = mx + b, along with values for m and b.  The value for m is the slope of the line, which is the rate of O₂ utilization.  Record this in your data table.  Close the box on the screen by clicking in the upper right hand corner.

5.       Measure respiration rate for each treatment by moving the vertical line to the linear part of the next set of data.  Select the next area of data to be analyzed by clicking and dragging with the mouse.  Again select Linear Fit from the Analyze menu. The equation on the screen will reflect the new m value for the range of data that you have selected. Record the new value of m in the Results section.

6.       Repeat this procedure for all of the treatments that you tested. 

Calculations

Each m value from each linear fit that you performed represents the rate of decrease of O₂ concentration in the chamber with time.  As such, each of these m values are rates of respiration expressed as %O₂ per min.  However, respiration is usually expressed in terms of ml O₂/ gfw/hr.  To make this conversion the following procedure is required:

Let us assume that the m value was x, i.e. the O₂ concentration of the chamber increased by x %O₂/min.  x %O₂ is equivalent to 10,000x parts per million (ppm) O₂ which, in turn, is equivalent to 10,000x ml of O₂ per liter of gas in the chamber.

         x %O₂/min = 10,000x ml O₂/min/liter of gas in the chamber

To obtain the respiratory rate we must now multiply by the volume of the chamber expressed in liters. The chamber is designed so that when closed it has a fixed internal volume of 0.047 liters. However the potato discs occupied some of this volume; thus the total volume of the chamber must be reduced by the volume of the potato discs

         mlitersO₂/min/liter X [0.047 liter - volume of leaf discs (liter)] = mliters O₂/min

However we would like to express the respiratory rate in ml O₂/gfw/hr.To do this we must perform the following calculations:

         mliters O₂/min X 60 min/hour = mliters O₂/hour

         mliters O₂/hour ÷ fresh weight (gm) = mliters O₂/gfw/hour

Results

Report Sheet

Effect of "Aging" and Inhibitors on Respiration

 Treatment                                               ml O₂ Absorbed/g fr wt/hr

Fresh tissue, control

"Aged tissue, control

Fresh + 0.02 mM CCCP

"Aged"+ 0.02 mM CCCP

Fresh + 1 mM CN^-

"Aged" + 1 mM CN^-

Fresh + 1 mM N₃-

"Aged" + 1 mM N_3-

Fresh + 50 mM malonate

"Aged" + 50 mM malonate

Show how you calculated these rates.

Questions:

1.     Did "aged" discs utilize oxygen at the same rate as fresh discs?  Discuss some of the reasons these rates may differ.

2.     Did CCCP inhibit or stimulate oxygen utilization, or both?  How could this substance increase rates?

3.     Did malonate influence the oxygen utilization of "aged" discs any differently than of fresh discs?  Explain.

4.    How do azide and cyanide inhibit respiration?  Did you see any differences between

aged and fresh tissue?  What might these be due to?