Plant lecture 6 - Partitioning of C

Question 1

How does levels of F2,6BP change

Answer 1

• PFK2 = kinase that makes F2,6BP, inhibited by triose-P, stimulated by hexose P
• F2,6BPase = phosphatase, triose P x have effect
• Kinase + phosphatase activities are found on 2 different AS within a single bifunctional peptide
• F2,6BP = signal metabolite that integrates concentrations of triose-P + hexose in the cytosol
• Overall F2,6BP depends on levels of inhibitory triose-P + stimulatory hexose-P

Question 2

Carbon partitioning during photosynthesis

Answer 2

Feed forward regulation

• Coordinates sucrose synthesis w/ the rate of photosynthetic C assimilation
• ↑ photosynthesis, ↑ 3PGA, ↑ triose-P in cytosol, ↑ F1,6BP
• This reduces inhibition in FBPase
• ↑ FBPase ↑ Glc6P so sucrose P synthase = activated + ↑ sucrose

Feedback regulation

• Coordination of photo assimilate C partitioning
• ↑ sucrose inhibits sucrose P synthase, leading to accumulation of hexoses (still made by FBPase)
• Build up of F2,6BP inhibits FBpase +
• Leads to ↑ 3-PGA, ↓ Pi in cytosol. Restricts export of C from chloroplast
• ↑ 3PGA/Pi in chloroplast, activates AGPase + ↑ STARCH

(F2n6BP + intermediate regulator), ↑ F26BP ↑ in flux to starch + ↓ in flux to sucrose (FBPase inhibited)

Evidence

• In vitro kinetics
• Correlative analysis used
• Change rate of CO2 assimilation by changing external conditions + study the impact on E + F2,6BP levels

Question 3

Evidence of impact of F2,6BP on flux

Answer 3

• Introduced 2 separate versions of construct that encoded bifunctional E to transgenic tobacco
• Used SDM to change AS of phosphatase domain.
• Changed to permanently on kinase (3x WT amount)
• Change ATP bs in kinase domain, fully switched on phosphatase
• As F2,6BP ↑, ↓ flux to sucrose + ↑ starch flux
• Response coefficient for starch synthesis = 0.7, sucrose = -0.5
• Definitive

Question 4

Sucrose P synthase (SPS)

Answer 4

• Modulated by G6P/Pi ratio
• 2 kinetically distinct forms: Ser158-P in spinach = inactive form, S158 = active
• Ser158-P/inactive = sensitive to Pi, Ser158/active = insensitive
• Ser158-P/inactive = sensitive + stimulated by F6P/G6P, Ser158/active = insensitive + ↑ affinity for F6P
• Ratio of active/inactive SPS = responsive to Glc6P/Pi levels: ↑ Glc6P/Pi, allosterically activates SPS + shifts relative proportion of SPS to ↑ active
• Complication = plants contain 3-4 SPS genes

Question 5

Which SPS gene is responsible for sucrose

Answer 5

• 3 separate gene families encode SPS
• Insertional mutagenesis used to ablate each gene separately
• For 1/4 genes, ↑ accumulation of starch turnover (deletion of this gene restricts sucrose production so to maintain CO2 assimilation, divert photo assimilation to starch) = SPSA1 gene

Question 6

Sucrose synthesis + trehalose 6P control

Answer 6

• Thought like F2,6BP, trehalose 6P could be signal metabolite + coordinate sucrose synthesis
• But, activity of sucrose itself can influence activity of trehalose P synthase + phosphatase
• ↑ sucrose activates + inhibits phosphatase so ↑ T6P
• Leads to phosphorylation/deactivation of SPS/ sucrose production
• In vivo change sucrose synthesis, x know exactly how works