Plant lecture 13 - plant mitochondrial metabolism

Question 1

Potential substrates for the TCA

Answer 1

• Mainly carbohydrates
• Amino acids can be oxidised, but only in certain circumstances e.g. glucose during photorespiration
• B oxidation of lipids is largely peroxisomal
• Glycolysis produces PEP → pyruvate, pyruvate imported into mitochondria
• Another route = PEP →(PEPC) OAA → malate, malate then imported + converted to OAA or pyruvate w/ NAD malic E
• Needs transporter

Question 2

Experimental evidence for TCA substrates

Answer 2

• Butylmalonate inhibits malate. Pi + respiration ↓ in Aram maculatum as ↓ capacity to metabolise glucose + drive TCA
• Transgenic tobacco plants lacking cytosolic pyruvate kinase have WT phenotype (PEPC can provide route for pyruvate into mitochondria)
• Metabolic flux analysis shows flux through PEPC = 25-40% of pyruvate kinase route

Question 3

Non-cyclic flux modes in TCA

Answer 3

• TCA is embedded in broader metabolic network
• x necessarily support dominant cyclic flux where acetyl coA → citrate … → OAA etc.
• Non cyclic flux modes are common e.g. flux mode supporting N assimilation in Xanthium strumanium. Have fragments of TCA supporting flux from citrate to 2-oxoglutarate (clockwise) + OAA → fumarate (anticlockwise)

Question 4

ETC in plant mitochondria

Answer 4

• Also have complex 1-IV
• But, also have alternative dehydrogenases to access UQ pool that x involve complex 1 + alternative oxidases
• 2 are present on internal face of mitochondrial membrane (specific 1 NADH, 1 NADPH) + transporting e- to UQ pool
• Experiment : inhibit complex 4 w/ cyanide, still get respiration so alternative oxidase x involved in protein translocation

Question 5

Respiratory chain supercomplexes

Answer 5

• Through blue native gel polyacrylamide gel electrophoresis, thought existence of several complexes e.g. I + III2 in Arabidopsis + potato
• Proposed function = optimise e- transfer to match substrate availability + stabilise inner membrane to further optimise function

Question 6

Regulation + role of alternative oxidase / dehydrogenase

Answer 6

• Rotenome + cyanide insensitivity = presence of alternative dehydrogenases + oxidases, allows e-s to bypass blockages caused
• In vivo difficult to distinguish electron flow
• External dehydrogenases - activated by Ca2+, may be important in determining cytosolic redox balance under stress conditions, plausible
• Internal dehydrogenases - important in providing a sink for dissipating NADH made by GDL during respiration

Question 7

Alternative oxidase (AOX) properties

Answer 7

• Alternative oxidase = well-characterise E, 35kDa, di-iron carboxylate
• Thought alternative oxidase = only engaged when cytochrome pathway is fully saturated (evidence = detection of Fe(II)/Fe(III) EPR signals in the presence of O2)
• AOX + cytochrome oxidase discriminate btw 16O2 + 18O2 to different extents
• Showed starvation of cytochrome pathway x prerequisite for AOX activity

Question 8

Regulation of AOX

Answer 8

• Exists in inactive form w/ disulphide bridge. Can be reduced + activated
• Mechanism for sensing mitochondrial redox status via thioredoxin. Allows respond to ↑ active as NAD(P)+ ↓
• Activateable AOX can be activated by 2-oxo acids, mainly pyruvate. Likely when ↓ TCA flux, maybe due to ↓ e- flow through cytochrome pathway
• Both stages sense state of mid is reducing so can activate AOX

Question 9

Transcriptional regulation of AOX

Answer 9

• May be fully activated in vivo even under conditions when activity x required
• When AOX activity needs to be ↑ further, turn to transcriptional levels
• Levels ↑ in response to environmental stresses e.g. drought
• AOX levels also ↑ in tobacco cell suspense in response to various chemical treatments e.g. citrate
• AOX plays a role in modulating TCA flux and for preventing formation of ROS

Question 10

Role of AOX

Answer 10

1. Thermogenesis (minor role)
   - Well-established during flowering in water lilies
   - Heat generation promotes release of compounds that attract pollinators
   - If bypass H+ pumps, E that would be used to build H+ gradient = dissipate heat
2. Uncoupling TCA from proton pumping (major role)
   - Typically mit. → TCA → reducing power fed into ETC → H+ pump, make ATP
   - Need to uncouple TCA from proton pumping process
   - If x ↑ demand for process driven by H+ pump (↑ ATP) but still need to oxidise substrates in TCA
   - AOX can uncouple by taking surplus reducing power from TCA + directing it to O2 to make H2O
   - Experiment = overexpression of AOX when cytochrome pathway inhibited, promote cell growth, under expression inhibits growth, evidence that AOX allows uncoupling
3. Protection against oxidative stress
   - Activity of AOX ↑ under conditions that cause oxidative stress
   - AOX activity could reduce the formation of ROS produced by leakage of ETC by preventing over-reduction of UQ pool
   - Experiment = ↓ AOX levels ↑ ROS levels e.g. superoxide in transgenic tobacco

Question 11

Uncoupling protein

Answer 11

• Allows H+ to flow down gradient back into mit. matrix
• Helps regulate mitochondrial ROS production
• As it gets harder to pump H+ as H+ gradient ↑, UQ pool = over-reduced, ↑ chance of e= leaking out of ETC → ROS
• One ROS promotes oxidation of lipids in membrane which creates HNE int. that is an activator of an UCP
• Helps efficient photosynthesis
• Experiment = Insertional knockout of AtUCP1 in Arabidopsis ↑ ROS production, limited oxidative stress
• This ↓ photorespiration rates, ↓ C assimilation
• Conclude to get optimum metabolism, need UCP1 to modulate redox state

Question 12

Mitochondrial activity in illuminated leaves

Answer 12

• Illuminated leaf has active chloroplast that makes ATP, might be possible for mit. to have minor role

1. E- transport + uncoupling
   - Very flexible ET system, allows flexibility for coping w/ surges in reducing power in chloroplasts + mit.
   - Can synthesise ATP, useful for non-plastidic processes
2. TCA cycle
   - Proves C skeletons + reducing power
   - Interaction btw flux through TCA + photosynthesis
3. Other mit. pathways
   - Glycine oxidation in photorespiration in C3 leaves + in mit.
   - NAD-malic E activity in bundle sheet mit. of 2 tips of C4 plant

Question 13

Respiration is essential in illuminated leaves

Answer 13

• Oxidative phosphorylation is essential for photosynthesis
• Mit. FoF1-ATP synthase is ↑ sensitive to oligomycin than chloroplast FoF1 ATP synthase
• ↓ conc. of oligomycin (effects mit. x chloroplast) ↓ photosynthesis + ↓ cytosolic ATP/ADP
• Conclude = need E source outside of plastid for sucrose synthesis (outside plastid) + ATP x be exported in sufficient quantities to meet demand
• Chloroplast energy dissipation, such as nonphotochemical quenching, and the capacity of the ATP export shuttles limit the extent to which the chloroplast can meet the energy demands of the whole cell. Respiration = essential

Question 14

Reducing power from the chloroplasts

Answer 14

• Metabolic modelling shows that leaf energy balance requires mitochondrial respiration and export of chloroplast NADPH.
• Cytosolic ATP = mainly met by mit.
• Flow of reducing power from chloroplast largely goes to peroxisome, where hydroxypyruvate reductase (photoresp.) + some to ETC. Minimises photo damage

Experiment = Knockout plants w/o malate/oxaloacetate shuttle (takes reducing power from chloroplast to cytosol) have ↑ photo inhibition + ROS accumulation in ↑ light

• Flux through the shuttle is regulated by stromal NADP-dependent malate dehydrogenase (MDH) in the light by the ferredoxin- thioredoxin system.
• The triose-phosphate/3- phosphoglycerate shuttle = parallel route for exporting stromal reducing equivalents.
• Regulated via the light- dependent activation of NADP-glyceraldehyde-3- phosphate dehydrogenase (GAPDH).
• The shuttle can also export ATP, if ATP consumption in the chloroplast is restricted under stress conditions.

Question 15

Other sources reducing power of ETC

Answer 15

1. Photorespiration
   - Glycine oxidation in mit. provides reducing equivalents for oxidative phosphorylation in C3 leaves (exported through OAA/malate shuttle, directed to ETC)
   - Experiment = cytosolic mit. ATP/ADP ratios ↓ under non-photoresp. or w/ GDC inhibitor (GDC contributes to respiration)
2. TCA cycle
   - In illuminated leaves, partial inhibition of PDH restricts operation of TCA in the light
   - 13C experiments imply simultaneous flux through forwards and reverse
   - Labelling patterns for malate and citrate are inconsistent with a complete cyclic flux.
   - Antisense inhibition of TCA cycle effect of photosynthesis:
   - ↓ fumarate ↓ rate
   - ↓ malate dehydrogenase ↑ rate
   - ↓ isocitrate dehydrogenase x change rate
   - Implies TCA has to be analysed as a component of a wider metabolic pathway x discrete pathway