laboratory

PART 3. STRUCTURE OF THE PLANT LEAF AND CHLOROPLASTS

While stems and some flower parts are green, the plant organ designed for optimal photosynthesis is the leaf. In a typical flowering plant, the leaf is broad to provide surface area for light absorption, but very thin since light cannot penetrate deeply into living tissue.

A diagram of a typical leaf is shown below. It is cut in both longitudinal and cross section to show the cellular structure within. Note the different cell types which carry out different functions in the leaf.

leaf Most of the chloroplasts in the leaf are located within the mesophyll cells. The mesophyll region contains spaces where water and gases can diffuse through the leaf. The epidermis forms a protective layer on both sides of the leaf. It has openings called stomata that allow gas exchange; usually carbon dioxide enters, whereas oxygen leaves the leaf. The vein contains tubes that bring water into the leaf from the roots and collect sugars for distribution throughout the plant.

Leaf peel

The openings within the epidermis are called stomata (singular is stoma). On either side of a stoma lies a guard cell, a unique cell type that can change shape to open or close the stoma. Water vapor escapes from the leaf through open stomata on a hot day. If there is insufficient water in the soil under these conditions, the stomata will close to prevent water loss. Most stoma are located on the underside of the leaf.

1. Peel the epidermis from the underside of a zebrina leaf as described by your TA. Place a small piece of tissue on a slide with the underside up. Add 2 or 3 drops of water to cover the tissue, add a coverslip, and observe under your microscope. Look for stomata and identify the guard cells surrounding them.

Due to lack of space, use only one microscope per table, but be sure that everyone at your table views the slide. Have the TA check your preparation before continuing.

2. When you have a good preparation, count the number of stomata in your field of view with the 40X objective and answer question 4.

Leaf cross section

View this stained leaf section which is displayed as if using a "virtual light microscope". Magnify the image and move to a region that contains the clearest view of internal structure, including a stoma. Perform a screen capture of the magnified leaf section, then label:
1) guard cells surrounding a closed stoma, 2) an open stoma, and 3) a mesophyll cell containing chloroplasts. Submit your labeled image to WebAssign for question 5.

Chloroplast structure

As you know by now, photosynthesis occurs within the chloroplasts of eukaryotic cells. Chloroplasts are larger than mitochondria and can be seen more easily by light microscopy. Since they contain chlorophyll, which is green, chloroplasts can be seen without staining and are clearly visible within living plant cells. However, viewing the internal structure of a chloroplast requires the magnification of an electron microscope.

plant cells These living plant cells are viewed by light microscopy. Note the many green chloroplasts within each cell.
mitochondrion Here is a diagram showing the internal structure of a chloroplast. By now, you should be familiar with its parts and know the role
that each structure plays in photosynthesis. Note that the grana are stacks of individual units called thylakoids. Chlorophyll molecules are embedded within the thylakoid membranes.

Now, it is time to view a real chloroplast by transmission electron microscopy. Study this transmission electron micrograph of a spinach leaf cell, locate a chloroplast and capture the image for labeling. The micrograph is displayed as if using a "virtual electron microscope", so you will need to magnify the image and move to a region that contains the clearest view of chloroplast internal structures. Perform a screen capture of the chloroplast, then label:
1) a thylakoid, 2) a granum, 3) the stroma, and 4) the outer chloroplast membrane. Submit your labeled image to WebAssign for question 6.

PART 4. MEASURING EFFECTS OF LIGHT ON THE RATE OF PHOTOSYNTHESIS

As the pigments that we just examined absorb light, water is split and the photosystems energize electrons to a higher energy level. These high-energy electrons are essential to ATP production and reduction of NADP+ to NADPH.  Both ATP and NADPH are necessary for carbon fixation in the Calvin cycle.

If thylakoid structure is disrupted, the electron transport chain is broken and there is nothing to accept electrons. However, the photosystems within the thylakoids may still be able to absorb light and excite electrons. With no electron acceptor available, these energized electrons give off energy as heat and light (at a longer wavelength than was absorbed) and fall back to a low energy state. This will be demonstrated by the TA, using a solution of thylakoids in alcohol.

You will now perform a series of assays using the Hill reaction, which measures the generation of excited electrons in the first step of the "light reactions" of photosynthesis.

DPIP
In the Hill reaction, chloroplasts are isolated and their membranes disrupted. In place of NADP+, which is no longer available to the electron transport chain,
2,6-dichlorophenol- indophenol (DPIP) is added to the solution and acts as a final acceptor for the energized electrons. When DPIP is reduced, it changes from blue to colorless. Thus, by using DPIP in an assay, we can subject the isolated thylakoids to different conditions and use the color changes we observe as an indicator of relative photosynthetic rate .   
Changes in the color of DPIP as it is reduced (accepts electrons)