1. The carbon reactions (Dark Reactions)
Photosynthesis
The carbon reactions
(Dark Reactions)

2. Overall Perspective Light reactions: Dark Reactions:
Harvest light energy
Convert light energy to chemical energy
Dark Reactions:
Expend chemical energy
Fix Carbon [convert CO2 to organic form]

3. At the end of the light reactions
The reaction of the light reaction is:
CO2 +H2O (CH2O) + O2
Recent estimates indicate that about 200 billion tones of CO2 (Mr = 44) are converted to biomass each year
40 % of this is from marine phytoplankton
The bulk of the carbon is incorporated into organic compounds by the carbon reducing reactions (dark reactions) of photosynthesis

4. At the end of the light reactions
The reactions catalyzing the reduction of CO2 to carbohydrates are coupled to the consumption of NADPH and ATP by enzymes found in the stroma
fluid environment
These reactions were thought to be independent of the light reactions
So the name “dark reactions” stuck
However, these chemical reactions are regulated by light
So are called the “carbon reactions” of photosynthesis

5. Overview of the carbon reactions
The Calvin cycle:
Stage 1:
CO2 accepted by Ribulose-1,5-bisphosphate.
This undergoes carboxylation
Has a carboxyl group (-COOH) attached to it
At the end of stage 1, CO2 covalently linked to a carbon skeleton forming two phosphycerate molecules.

6. Carboxylation: The first step is the most important
Step 1: The enzyme RUBISCO (Ribulose bis-phosphate carboxylase oxygenase) carries out this conversion
Rubisco accounts for 40% of the protein content of chloroplasts
is likely the most abundant protein on Earth
Rubisco is, in fact, very inefficient, and that a mechanism has evolved to deal with this handicap

7. Overview of the carbon reactions
The Calvin cycle:
Stage 2:
Each of the two 3-phosphycerate molecules are altered.
First phosphorylated through the use of the 3 ATPs generated during the light reaction.
Then reduced through the use of the 2 NADPHs generated during the light reaction.
Forms a carbohydrate
glyceraldehyde-3-phosphate

8. 3-phosphycerate molecules are altered
First phosphorylated through the use of the 3 ATP molecules generated during the light reaction
Forms 1,3-bisphosphoglycerate
Then reduced through the use of the 2 NADPH molecules generated during the light reaction
Forms glyceraldehyde-3-phosphate
Note the formation of triose phosphate

9. Overview of the carbon reactions
The Calvin cycle:
Stage 3:
Regeneration of Ribulose-1,5-bisphosphate.
This requires the coordinated action of eight reaction steps
And thus eight specific enzymes
Three molecules of Ribulose-1,5-bisphosphate are formed from the reshuffling of carbon atoms from triose phosphate.

10. Regeneration of Ribulose-1,5-bisphosphate
The Calvin cycle reactions regenerate the biochemical intermediates needed for operation
More importantly, the cycle is Autocatalytic
Rate of operation can be enhanced by increasing the concentration of the intermediates in the cycle
So, Calvin cycle has the metabolically desirable of producing more substrate than is consumed
Works as long as the produced triose phosphate is NOT diverted elsewhere (as in times of stress or disease)

11. Overview of the carbon reactions
The Calvin cycle:
The cycle runs six times:
Each time incorporating a new carbon . Those six carbon dioxides are reduced to glucose:
Glucose can now serve as a building block to make:
polysaccharides
other monosaccharides
fats
amino acids
nucleotides

12. Only one-sixth of the triose phosphate is used for polysaccharide production
Synthesis of polysaccharides, such as starch and sucrose, provide a sink
Ensures an adequate flow of carbon atoms through the cycle IF CO2 is constantly available
During a steady rate of photosynthesis 5/6 of the triose phosphates are used for the regeneration of Ribulose-1,5-bisphosphate
1/6 is transported to the cytosol for the synthesis of sucrose or other metabolites that are converted to starch in the chloroplast

13. Regulation of the Calvin cycle
The high energy efficiency of the Calvin cycle indicates that some form of regulation ensures that all intermediates in cycle:
Are present at adequate concentrations
The cycle is turned off when it is not needed in the dark
Remember:
These are the “carbon reactions”, NOT the “dark reactions”
Many factors regulate the Calvin cycle

14. Regulation of the Calvin cycle
1: The pH of the stroma increases as protons are pumped out of it through the membrane assembly of the light reactions.
The enzymes of the Calvin Cycle function better at this higher pH.
2: The reactions of the Calvin cycle have to stop when they run out of substrate
as photosynthesis stops, there is no more ATP or NADPH in the stroma for the dark reactions to take place.

15. Regulation of the Calvin cycle
3: The light reactions increase the permeability of the stromal membrane to required cofactors
Mg ions are required for the Calvin Cycle.
4: Several enzymes of the Calvin Cycle are activated by the breaking of disulphide bridges of enzymes involved in the working of the cycle.
the activity of the light reactions is communicated to the dark reactions by an enzyme intermediate

16. When conditions are not optimum

17. Photorespiration Occurs when the CO2 levels inside a leaf become low
This happens on hot dry days when a plant is forced to close its stomata to prevent excess water loss
If the plant continues to attempt to fix CO2 when its stomata are closed
CO2 will get used up and the O2 ratio in the leaf will increase relative to CO2 concentrations
When the CO2 levels inside the leaf drop to around 50 ppm,
Rubisco starts to combine O2 with Ribulose-1,5-bisphosphate instead of CO2

18. Photorespiration
Instead of producing 2 3C PGA molecules, only one molecule of PGA is produced and a toxic 2C molecule called phosphoglycolate is produced
The plant must get rid of the phosphoglycolate
The plant immediately gets rid of the phosphate group
converting the molecule to glycolic acid

19. Photorespiration
The glycolic acid is then transported to the peroxisome and there converted to glycine
Peroxisomes are ubiquitous organelles that function to rid cells of toxic substances
The glycine (4 carbons) is then transported into a mitochondria where it is converted into serine (3 carbons)
Releases CO2

20. Photorespiration
The serine is then used to make other organic molecules
All these conversions cost the plant energy and results in the net lost of CO2 from the plant
75% of the carbon lost during the oxygenation of Rubisco is recovered during photorespiration and is returned to the Calvin cycle

21. The C4 Carbon cycle

22. The C4 carbon Cycle
The C4 carbon Cycle occurs in 16 families of both monocots and dicots.
Corn
Millet
Sugarcane
Maize
There are three variations of the basic C4 carbon Cycle
Due to the different four carbon molecule used

23. The C4 carbon Cycle
This is a biochemical pathway that prevents photorespiration
C4 leaves have TWO chloroplast containing cells
Mesophyll cells
Bundle sheath (deep in the leaf so atmospheric oxygen cannot diffuse easily to them)
C3 plants only have Mesophyll cells
Operation of the C4 cycle requires the coordinated effort of both cell types
No mesophyll cells is more than three cells away from a bundle sheath cells
Many plasmodesmata for communication

24. The C4 carbon Cycle Four stages: Stage 1: In Mesophyll cell Stage 2:
Fixation of CO2 by the carboxylation of phosphenol-pyruvate (primary acceptor molecule)
forms a C4 acid molecule
Malic acid and/or aspartate
Stage 2:
Transport of the C4 acid molecule to the bundle sheath cell

25. The C4 carbon Cycle Stage 3: Stage 4:
Decarboxylation of the C4 acid molecule (in bundle sheath)
Makes a C3 acid molecule
This generates CO2
This CO2 is reduced to carbohydrate by the Calvin cycle
Stage 4:
The C3 acid molecule (pryuvate) is transported back to mesophyll cells
Regeneration of phosphenol-pyruvate

26. The C4 carbon Cycle
Regeneration of phosphenol-pyruvate consumes two high energy bonds from ATP
Movement between cells is by diffusion via plasmodesmata
Movement within cells is regulated by concentration gradients
This system generates a higher CO2 conc in bundle sheath cells than would occur by equilibrium with the atmosphere
Prevents photorespiration!!!!!!!!!!

27. The C4 carbon Cycle
The net effect of the C4 carbon Cycle is to convert a dilute CO2 solution in the mesophyll into a concentrated solution in the bundle sheath cells
This requires more energy than C3 carbon plants
BUT – This energy requirement is constant no matter what the environmental conditions
Allows more efficient photosynthesis in hotter conditions

28. Crassulacean Acid Metabolism (CAM Plants)

29. CAM Plants
The CAM mechanism enables plants to improve water efficiency
CAM plant
Loses 50 – 100 g water for every gram of CO2 gained
C4 plant
Loses 250 – 300 g water for every gram of CO2 gained
C3 plant
Loses 400 – 500 g water for every gram of CO2 gained
Similar to C4 cycle
In CAM plants formation of the C4 acid is both temporally and spatially separated

30. CAM Plants At night: Stomata only open at night when it is cool
CO2 is captured by phosphenol-pyruvate carboxylase in the cytosol – leaves become acidic
The malic acid formed is stored in the vacuole
Amount of malic acid formed is equal to the amount of CO2 taken in

31. CAM Plants During the day:
Stomata close, preventing water loss, and further uptake of CO2
Malic acid is transported to the chloroplast and decarboxylated to release CO2
This enters the Calvin cycle as it can not escape the leaf
Pyruvate is converted to starch in the chloroplast – regenerates carbon acceptor

32. Phosphorylation regulates phosphenol-pyruvate (PEP) carboxylase
CAM and C4 plants require a separation of the initial carboxylation from the following de-carboxylation
Diuranal regulation is used
IN CAM PLANTS:-
Phosphorylation of the serine residue of phosphenol-pyruvate (PEP) carboxylase (Ser-OP) yields a form of the enzyme which is active at night
This is relatively insensitive to malic acid

33. Photophorylation regulates phosphenol-pyruvate (PEP)carboxylase
During the day:
De-Phosphorylation of the serine (ser-OH) gives a form of the enzyme which is inhibited by malic acid
THIS IS THE OPPOSITE WAY AROUND FOR C4 PLANTS!

34. Summary
The reduction of CO2 to carbohydrate via photosynthesis is coupled to the consumption of ATP and NADPH
CO2 is reduced via the Calvin cycle
Takes place in the stroma (soluble phase)
CO2 and water combine with Ribulose-1,5-bisphosphate in the following reaction
CO2 +H2O (CH2O) + O2
Regeneration of the carrier is required for the cycle to continue

35. Summary
The Calvin cycle requires the joint action of several light-dependant systems
Changes in ions (Mg+ and H+)
Changes in effector metabolites (enzyme substrates)
Changes in protein-mediated systems (rubisco activase)
Rubisco can also act as an oxygenase
The carboxylation & oxygenation reactions take place at the active sites of rubisco.

36. Summary C4 and CAM plants Prevent photorespiration!!!!!
C4 leaves have TWO chloroplast containing cells
Mesophyll cells
Bundle sheath
CAM Plants drastically reduce water lass
CAM plant
Loses 50 – 100 g water for every gram of CO2 gained
C4 plant
Loses 250 – 300 g water for every gram of CO2 gained
C3 plant
Loses 400 – 500 g water for every gram of CO2 gained

37. Any Questions?