The Light Independent Reaction

Question 1

Describe the Calvin cycle

Answer 1

1. CO2 enters the leaf through the stomata and diffuses into the stroma of the chloroplast
2. Here CO2 is combined with RuBP (ribulose biphosphate) catalysed by the enzyme rubisco
3. This gives an unstable 6C compound which then breaks down into 2 molecules of a 3C compound which is called GP
4. ATP from the light dependent reaction provides energy to turn the 3C - GP, into a different 3C compound called TP (triose phosphate). The reaction also requires H+ ions Which come from reduced nadp, so turns reduced nadp into nadp (H+ ions used from light dependent reaction)
5. Some TP is used into useful organic compounds e.g. glucose, and some continues to regenerate RuBP
6. 5/6 molecule of TP produced in the cycle aren’t used to make hexose sugars, but to regenerate RuBP
7. Regenerating RuBP uses the rest of ATP from the light dependent reaction

Question 2

Where does the light independent reaction take place

Answer 2

In the stroma of the chloroplasts

Question 3

How are the chloroplasts adapted to carrying out the light independent reaction of photosynthesis

Answer 3

Fluid of stroma contains all enzymes needed for light independent reaction

Stroma fluid surrounds the grana and so the products of the light dependent reaction in the grana can readily diffuse into the stroma

It contains both DNA and ribosomes so it can quickly and easily manufacture some of the proteins involved in the light independent reaction

Question 4

What are the limiting factors of photosynthesis

Answer 4

Light intensity- provide energy for light dependent reaction, higher intensity, more energy it provides. At the saturation point ( where it levels off) increasing intensity after this point makes no difference, because something else has become the limiting factor

Temp- around 25 degrees. If below 10 enzymes become inactive. More then 45 they start to denature - stomata close at high temp to avoid loosing water, this means CO2 can’t get in the leaf slowing rate of photosynthesis

Co2 concentration- no higher then 0.4%, any higher stomata will close

Question 5

Describe the Use of chromatography to investigate the pigments isolated from leaves of different plants

Answer 5

1. Cut a TLC plate into small strips e.g.1.25 x 6.7 cm, so that they fit your tubes. Do not touch the surface of the plates.
2. Place 2 - 3 wheat or grass leaves on a slide. Use a second slide to scrape the juice out.
3. Add 6 drops of propanone to the green mush and mix. Transfer the dark green liquid to a small watch glass.
4. Repeat steps 2 and 3 until you have about 20 drops of extract. Use a hair dryer to remove all the water from the extract.
5. When the extract is completely dry, add 3 - 4 drops of propanone and mix the extract with a fine paint brush.
6. Use the brush to transfer tiny drops of extract to the TLC strip. Keep the spot diameter to less than 2 mm. Dry the spot thoroughly between each addition. Repeat until the spot is a very dark green.
7. Slot the TLC strip into a slit in the cork and put it into an empty tube. Mark the tube below the level of the spot. Remove the TLC strip.
8. Add running solvent to the mark. Put the TLC strip back in the tube. Make sure that it does not touch the sides of the tube. Watch the chromatogram develop.
9. After about 4 minutes remove the strip and immediately use a pencil to mark the solvent front.
10. Measure the distance run by the solvent front and by each of the pigments. All measurements should be made from the centre of the original spot to the front of each pigment spot.
11. Calculate how far the pigment has gone relative to the solvent front. This is the Rf value. (Rf = the distance run by the pigment divided by the distance run by the solvent.)

Question 6

Describe the Investigation into the effect of a named factor on the rate of dehydrogenase activity in extracts of chloroplasts

Answer 6

1. Cut a few leaves into pieces
2. Grind up leaf pieces wish some chilled isolation solution
3. Transfer the liquid to centrifuge tubes and centrifuge them at high speed for 10 mins. This will make the chloroplasts gather at the bottom of each tube forming a pellet
4. Get rid of the liquid from the top of the tubes, leaving pellet at bottom
5. Resuspend the pellets in fresh, chilled isolation. This is your chloroplast extract. Store it on ice for the rest of the experiment
6. Set up a colorimeter with a red filter and zero if using a curvette containing the chloroplast extract and distilled water
7. Set up test tube rack at a set distance from the bench lamp. Switch the lamp on
8. Our a test tube in the rack, add a set volume of chloroplasts extract to the tube and set of volume of DCPIP, mix the contents in the tube together
9. Immediately take a sample of the mixture from the tube and add it to a clean curvette. Then place the curvette in your colorimeter and record absorbance. Do this every 2 mins for 10 mins
10. Repeat steps 7 and 9 for each distance

If dehydrogenase activity is taking place, the absorbance will decrease as the DCPIP gets reduced and looses its blue colour