Plant lecture 8 - C metabolism in heterotrophic tissue

Question 1

Phloem unloading

Answer 1

• Sucrose is delivered to sink tissues via specialised cells - phloem sieve element cells
• Sucrose = is actively loaded into sieve elements at source and moves to sink
• Moves into cell wall space/apoplast via transporter
• Sucrose either taken directly up by parenchyma cells via sucrose co-transporter or hydrolysed within apoplast to hexose which can then be translocated to parenchyma
• Can also move to parenchyma via plasmodesmata
• Different tissues rely on different methods e.g. developing seeds x have plasmodesmata btw maternal + offspring so taken up via apoplastic route

Question 2

Methods of sucrose breakdown

Answer 2

1. Invertase
   - Catalyses hydrolysis of sucrose into glucose + fructose
   - Irreversible, found mainly in cytosol, vacuole + axoplasm
   - Neutral pH optimum (cytosolic isoform), or acidic pH optimum (vacuole + apoplasm isoform)
   - Makes glucose that can be phosphorylated to G6P
2. Sucrose synthase
   - Near-eq reaction found in cytosol
   - Neutal pH optimum
   - Makes UDPGlc which can → Glc1P through action of UGPase

• Catalytic capabilities of invertase + sucrose synthase x reveal route of sucrose breakdown, pea root INV =1/5SUSY but in pea embryo, INV = 5SUSY. In reality E rarely operates in vivo at max rate and either alone are sufficient
• In some tissues SUSY activity dominates, in others invertase

Question 3

Evidence for methods of sucrose breakdown

Answer 3

• Rug4 mutants = wrinkled (have ↓ starch than WT, ↓ structural integrity)
• Have x detectable SusY activity + invertase only slightly ↑
• Rug4 gene isolated + compared to WT. Found mutant allele in coding region for SUSY (clear example of absence of SUSY alone compromises ability to take up sucrose)
  BUT
• Arabidopsis has 6 SUS gene, use genetic crosses to ablate individual genes. Plant happy. Only double mutant Sus1/4 have an effect for growth
• More limited impact

Invertase
- Of 22 iNV genes, inv1/2 appear to be essential for growth in arabidopsis. Obvious disruption

Question 4

Starch synthesis in storage tissue

Answer 4

• In heterotrophic cells like starch production in chloroplast, starch synthesis precursor = ADPGlc
• Provides Glc needed for amylose (straight chain polymer of glucose, glucosyl units linked a1,4) + amylopectin (also a1,4 + branched at a1,6)
• ADPGlc is made from Glc1P + catalysed by ADP glucose pyrophosphorylase (AGPase)
• AGPase = usually in plastid. In developing endosperm = both plastid + cytosol

Question 5

What crosses amyloplast membrane?

Answer 5

• Final stage of starch synthesis must occur in amyloplast but know initial steps in degradation of sucrose = in cytosol, so what crosses membrane
• Thought could be like chloroplast where sucrose → hexoseP → triose P (DHAP/G3P), cross membrane via translator → F6P → starch

Evidence against

• x find F1,6Bpase in amyloplast so couldn’t convert triose P to hexose P
• Also x find translocator TPT, instead - GPT (catalyses 1:1 counter exchange of G6P for inorganic phosphate)
• Labelling experiment. If sequence of events above, would expect label of starch glucosyl units to be the same for C1+6 of glucosyl chains
• In fact, label remains in original position, so hexose P x convert to triose P and vice versa

Question 6

Endosperm AGPase

Answer 6

• AGPase found in both plastid + cytosol
• ADPGlc can be imported to amyloplast w/ adenylate translocator
• Analysis of mutants defective in starch accumulation in seeds. 1st mutant = brittle1, has mutation in locus encoding translocator, 2nd = shrunken 1, mutation in gene for cytosolic AGPase
• Abblation of either prevents normal accumulation of starch so cytosolic formation of ADPGlc = important

Question 7

Regulation of starch synthesis

Answer 7

• Needed to avoid depletion of pools of intermediates needed for supply precursors for metabolism
• In photosynthetic leaves, AGPase regulates starch synthesis. Modulated by 3PGA/Pi. Same as non-photosynthetic leaves but different:
  1. 3C metabolite x an intermediate in starch synthesis in non-photosynthetic cells so not obvious how works
  2. Starch synthesis is not correlated w/ 3PGA content of non-photosynthetic tissues
• Suggests other factor

Question 8

Evidence for ATPase modification

Answer 8

Transcription

• If prevent supply of sucrose to tuber, ↓ in starch synthesis. ↓ rate of transcription of subunits encoding AGPase
• But x change amount of protein or catalytic activity of AGPase, so starch synthesis changes happen more rapidly than transcription

Covalent modification

• Specific disulphide bonds btw 2 Cys separate subunits of S polypeptide that make up heterotetrameric AGPase is ox/reduced
• With DTT, disulphide bridge = fully reduced. In both attached + detached for 1 day tuber, subunit = reduced 50kDa monomer
• W/o DTT, at partly attached, partly dimer and partly monomer. 1 day after, all oxidised/dimer

Correlates with kinetic properties:
Substrate affinity is ↓ in dimer / ↑ in monomer
Sensitivity to 3PGA is ↓ in dimer / ↑ in monomer

Question 9

How is redox state of AGPase affected

Answer 9

• Similar to covalent modification of Calvin cycle enzymes
• In non photosynthetic plants, there is no reduced Fd as no light reaction
• NADPH is used as source e- for reduction

Experiment

• NADPH reduction of AGPBase using protein NTrC which has both NTR and TRX/thioredoxin domain
• Showed increasing AGPase activity due to ↑ in NADPH + ↑ in conc of. NTRC
• Sucrose content in root depends on photosynthetic activity in leaves: in light, ↑ supply of sucrose, roots have ↑ sucrose so AGPase = reduced + active
• In dark, ↓ sucrose form shoots + leaves, ↓ sucrose for AGPase, inactive

Question 10

Summary of regulation for AGPase

Answer 10

1. Transcriptional control
   Modulation of rate of expression of genes encoding both subunits of ATPase
2. PTM
   - Redox regulation that determines the relative proportions of ↑ active monomeric/ ↓ dimeric form of the E
3. Allosteric regulation
   - Activation by 3PGA/Pi ratio