L16 - The Synthesis of Carbohydrates from Photosynthates

Question 1

Give the three important molecules produced inside the stroma that are used in carbon export from the chloroplast.

Outline how they are generated

Answer 1

1) 3-PhosphoGlyceric Acid (3PGA)
- Generated by Rubisco

2) DiHydroxyAcetone Phosphate (DHAP)
-Triose Phosphate

3) Glyceraldehyde 3-Phosphate (Ga3P)
- Triose Phosphate

DHAP and Ga3P interconverted by Triose Phosphate Isomerase (TPI)

Triose phosphates (TPs) generated on cycle powered by ATP and NADPH to regenerate RuBP for Calvin Cycle (can also exit cycle as discussed in this lecture)

Question 2

Describe the evidence that show the 3 molecules listed previously can be exported from the chloroplast

Describe how this export occurs and what the main molecule exported is

Answer 2

• Difficult to measure export. Uptake experiments showed chloroplast envelope permeable to 3-PGA and TPs
• Triose phosphate translocator (TPT) uses ping pong mechanism
• TPs and 3-PGA exchanged for phosphate (Pi)
• TPT transports phosphate molecule, phosphate attached to 3C but not 2C or 4C
• More 3-PGA but transport of TPs favoured due to PH of stroma in light
• 20:1 ratio DHAP:Ga3P so mostly DHAP exported

Question 3

What are the final reactants required to form sucrose?

How are these reactants formed from the Triose Phosphates? Give the 5 steps

Answer 3

• UDP-glucose and fructose 6-phosphate (F6P)

1) TPs exported to cytosol
2) Converted to Fructose 1,6-BisPhosphate (FBP)
3) Converted to F6P (type of hexose-P)
4) F6P interchangeable with other hexose-Ps in “hexose-P pool” (F6P, G6P, G1P)
5) UDP-Glucose produced from Glucose-1-P (G1P) and UTP under UDP-G pyrophosphorylase catalyst

Question 4

Describe the importance of sucrose

Answer 4

• Major end-product of photosynthesis (over half of C fixed is in sucrose)
• Major translocated carbohydrate
• Can be important storage compound in some plants e.g. carrots, sugar beet

Question 5

Give the four enzymes in plants involved in sucrose metabolism. Also give the reactants each requires.

Sketch a diagram of all of these routes

Answer 5

1) Sucrose P Synthase (SPS)
- Sucrose P formed from UDP-Glucose and F6P

2) Sucrose P Phosphatase (SPPase)
- Sucrose formed from Sucrose P

3) Sucrose Synthase (SS)
- Reversible reaction between Sucrose and (Fructose and UDP-Glucose)

4) Invertase (INV)
- Breakdown of sucrose to (fructose + glucose)

Diagram seen on pg 4

Question 6

Which reaction pathway is used to form sucrose?

Give the 4 strands of evidence for this

Answer 6

• SPS and SPPase act in concert (not SS)

1) Thermodynamics favour SPS

2) Strong + correlation between SPS and in vivo sucrose synthesis
- SPS highest in leaves, lowest in roots where sucrose is degraded

3) 14 CO2 labels in order UDP-G, Sucrose P, Sucrose
- Sucrose made via Sucrose P

4) Fructosyl group of sucrose labelled w/ 14C prior to free fructose (i.e. free fructose not a reactant)

Question 7

How do the enzymes SPS and SPPase work together?

Give three points of evidence for this

Answer 7

• Form a complex to increase efficiency

1) Increased addition of SPPase to purified SPS increased the activity of SPS
2) Radio-labelled Sucrose P showed Sucrose P is channelled efficiently to SPPase
3) Bifluorescence complementation showed proteins physically interact in vivo

Question 8

How does the TPT relay information about the relative rates of sucrose synthesis?

Answer 8

• Pi released from FBPase and Sucrose Phophatase reactions returned to chloroplast vis TPT
• Slower sucrose production = less Pi = less available to return to chloroplast = reduced export of DHAP

Question 9

Explain briefly how sucrose synthesis is regulated.

Answer 9

• Fructose 1,6-bisphosphatase (FBPase) catalyses conversion of Fructose 1,6-bisphosphate to F6P
• FBPase inhibited by metabolite Fructose 2,6-bisPhosphate
• Sucrose levels and F2,6-bisPhosphate inversely correlated

Question 10

Give the two reactions and the corresponding enzymes that regulate Fructose 2,6-bisPhosphate

Answer 10

• Enzyme PhosphoFructoKinase II (PFKII) catalyses conversion from F6P to F2,6-bisPhosphate:

F6P + ATP = F2,6-bisPhosphate + ADP

• Enzyme F 2,6-BisPase catalyses conversion from F2,6-bisPhosphate to F6P:

F2,6-bisPhosphate + H20 = F6P + Pi

Question 11

How are the two reactions controlling Fructose 2,6-bisPhosphate activated and inhibited?

Sketch the relevant diagram

Answer 11

F6P to F2,6-bisPhosphate:
-Activated by sucrose metabolites Pi and F6P
-Inhibited by photosynthesis products 3-PGA and DHAP

F2,6-bisPhosphate to F6P:
- Inhibited by sucrose metabolites Pi and F6P

See diagram on pg 6

Question 12

Explain how the activation and inhibition of the two reactions controlling Fructose 2,6-bisPhosphate functions over a day

Answer 12

Night: F2,6-bisP high as Calvin-Benson cycle inactive = low cytosolic DHAP and PGA

Dawn: CO2 fixation begins, intermediates of Calvin-Benson cycle increase until threshold reached when TPs are exported to cytosol, inhibiting PFKII = less F2,6-bisPhosphate = sucrose synthesis starts

Later in Day: Sucrose synthesis starts to exceed demand in sink tissues and sucrose accumulates = increase in F6P and Pi = more F2,6-bisPhosphate = slowed sucrose synthesis