Plant metabolism ALL

Question 1

Plant organisation/structure

Answer 1

• Vacuole = 95-98% of cell volume. separates from cytosol by tonoplast
• Adjacent cells are connected w/ plasmodesmata. Allows free diffusion of small compounds up to 8kDa btw cells
• Apoplasm = space outside plasma membrane where material can diffuse freely

Question 2

Carbohydrate flexibility/oxidation

Answer 2

• Plants use ↑ range of carbohydrates as respiratory substrates
• Sucrose, metabolic flexibility
• Starch in plastid or fructans in vacuole
• Other C sources e.g. raffinose

Question 3

Experiment

Varying response to developmental requirements

Answer 3

• 14C supplied to exiled maize root tips + different sections of root tip separated + fractionated
• Co2 contains ↓ proportion of 14C, acidic compounds the most
• Different components synthesised in different proportion in each section

Question 4

Experiment

Alternative metabolic pathways

Answer 4

• 14C experiments
• DHAP + G3P = mirrors (1-3) (6-4)
• C3+4 = COOH in pyruvate → CO2 in TCA
• Rest = acetyl coA
• Hypothetically, Cs are release 3/4, 2/5, 1/6
• C3/4 = labels in aa, so not just CO2 (must be another route for making organic aa → PEP carboxylase)
• C1+6 should be equivalent. C1 actually > C6 (alternative route for decarboxylation of glucose → PPP)
• C2 should > Co2 from C6 but x (alternative route for release of CO2 from C6 → UDP glucose)

Question 5

Experiment

Plants contain multiple isoforms

Answer 5

• Ion exchange chromatography column showed 2 peaks for PFK

Question 6

Carbohydrate oxidation isoforms

Answer 6

• Plastids + cytosol
• Encoded for by different genes
• Duplication occurs as compromise btw requirements in pathway (C oxidation needed for E source vs source of intermediates that can act as precursors (plastid))
• 2 roles could conflict, need pathway to be regulated in response to demand for both
• Translators that catalyser controlled exchange of specific intermediates

Question 7

Analysis of plant metabolism

Answer 7

Difficulties

1. Measuring E activity
   - Tissue disruption = breaking tough cell wall
   - Enzyme inactivation - proteases + polyphenols in vacuole
   - Assay of enzyme - contains isozymes so have to compromise conditions
2. Measuring metabolites (metabolite levels)
   - Low amounts - cytosol could only occupy 2-5% of cell. Need ↑ tissue and have ↑ background noise
   - Breakdown extraction - phosphatases/hydrolases in vacuole = released. Mixes + distorts levels
3. Metabolic flux measurements
   - Low metabolic rates, difficult to measure substrate utilisation through depletion
   - Diversity of substrates/ complexity of pathways = ↑ end-products + pathways

Question 8

Plant transfer

Answer 8

1. Transfer w/o vector
   - Direct uptake into naked protoplast e.g. w/ electroporation (24,000V)
   - Biolistic gun, most popular, small gold particle coated w/ solution of DNA + fired into plant tissue
2. Virus mediated transfer
   - Either DNA or RNA viruses
   - Advantage = can simply infect plant cells + virus can replicate within infected tissue
   - Disadvantage = most viruses are RNA-based, handling difficult and ↑ levels of unpredictable recombinatino
3. Agrobacterium-mediated transfer
   - Technically simple, ↑ capacity for gene insertion

Question 9

Agrobacterium natural

Answer 9

• Moves to plant w/ cellular damage in response to phenolic compounds e.g. acetosyringone
• Then transfers tDNA into host
• Genes encoded in tDNA:
  1. E for growth regulators e.g. auxins + cytokines then → synthesis of auxins → de-deifferentiation + multiplication of infected cells → crown gall tumor
  2. Genes responsible for synthesis of opines. Used as source of N + respiration substrate in bacteria
  3. Vir genes (8 transcriptional units VirA-H e.g. VirA/6 encodes 2-component system responsible for detecting phenolic compounds)

Question 10

Agrobacterium technical

Answer 10

• tDNA region x have to be in same plasmid. Often vectors modified → binary vector system
• Vir genes maintained in separate large plasmid + tDNA region is excised to small plasmid (easier to manipulate)
• tDNA plasmid could tDNA region w/ genes for opines removed + replaced w/ kanamycin resistance + GOI
• Can select, transfer to different medium + grow to a good size

Question 11

Agrobacterium floral dip procedure

Answer 11

• Take immature Arabidopsis seedlings + submerge in medium w/ Agrobacterium
• Flowers fertilised + seeds spread on soil + germinated. Treat w/ kanamycin + select what grows

Question 12

How can Agrobacterium be used to manipulate enzymes of metabolism

Answer 12

1. PFP
   - Antisense inhibition
   - Coding region (PFP) inserted in reverse btw promoter + terminator that allows expression in cells
   - Catalytic capacity of PFP ↓ by 80-90% WT + x effect respiration
2. PFK
   - Coding region in sense orientation
   - 20x normal PFK in selected transformed lines
   - PFK = thought to be rate-limiting stage. But even though ↑ ↑ expression, x impact on rate

Question 13

Insertional mutagenesis

Answer 13

• Ablate expression of individual genes in plants
• Exploits agrobacterium + ability to insert v large pieces of foreign DNA into host
• E.G. used tDNA to ablate expression of glycolytic E like glycerate mutase (plastidic)
• → ↑ normal lipids (conversion of 3PGA-2PGA x essential)
• If ablate genes encoding plastid enzymes (2-PG→PEP), → normal lipid production (cytosolic pathway can supply PEP needed)
• x necessarily pathway used by WT but just plastid x essential

Question 14

Light vs Dark reaction

Answer 14

• Dark reaction = CO2 fixation, E located in stroma

- Light reaction = energy harvesting, in ↑ structured thylakoids

Question 15

Electron transport chain

Answer 15

• PSI absorbs photos that excite e-, e- donated to e- carriers, hole in PSI is filled by PSII and hole in PSII is filled by oxidation in water
• PSII starts with strong oxidant, generates weak reductant
• PSI starts with weak oxidant, generates strong reductant

Question 16

LHCII structure

Answer 16

• Has core complex D1 + D2
• Surrounding core complex = peripheral antennas encoded by 6 genes Lhcb1-6
• Lhcb1-3 = variable numbers, Lhcb4-6 usually = single q

Question 17

Evidence for coordination btw PSI + PSII

Answer 17

• Illuminate plant w/ PSII light
• Initial ↑ chlorophyll fluorescence
• Turn on PSI light, ↑ in photosynthesis + small ↓ in fluorescence (PSI pulls e-s from PSII)
• Turn off PSI ↓ photosynthesis, ↑ in fluorescence
• Inverse relationship btw ↑ in PSI photosynthesis + ↓ of fluorescence in PSII = redistribution

Question 18

PsaH, L + O

Answer 18

• Structure to 3.3A
• PsaH,L+O form interface btw LHCI+LHCII
• Particularly, PsaL interacts w/ phosphate on residue 3 threonine
• For lHCII to share excitation E w/ PSI, need ↑ ordered chlorophyll.
• PsaO = associated w/ 2 chlorophyll molecules positioned in orientations + at distances that allow transfer of E

Question 19

Transcriptional redox control of PSI + PSII

Answer 19

• CSK responds to amount of oxidised PQ through identification of BS for PQ on CSK
• Autophosphorylated CSK → activation.
• Facilitates phosphorylation of sigma factor associated w/ plastid-encoded RNA pol
• In phosph. form, RNA pol can transcribe PSII, PSI transcribed weakly
• Reduced PQ = PSI + II genes both transcribe

Question 20

4 Factors required for net CO2 assimilation Calvin cycle

Answer 20

1. Irreversible. Keq for Ru1,5BP → 3PG = 10^6
   - Means ↑ photosynthesis products even when ↓ precursor
2. 1st step has ↑ affinity for CO2. Km = 9um (E close to saturation in physiological conditions)
3. Regenerates initial CO2 acceptor
4. Cycle adjusts levels of RuBP to ensure con. of CO2 acceptor = maximum

Question 21

Rubisco effector activation

Answer 21

• Activator = carbamylated
• Inhibitor = decarbamylated form
• Inhibitor to decarbamylated shifts eq. to decarbamylated form so ↑ proportion of E = inactive
• Tight-binding inhibitors, released slowly
• E.G = RuBP itself. Binds to inactive decarbam. form + released v slowly

Question 22

Reasons for regulation of the Calvin Cycle

Answer 22

• Adjustment to changes in light intensity/CO2 availability
• Switch off in dark to avoid depleting NADH/ATP
• Intermediates are shared w/ pathway of carbohydrate oxidation

Question 23

SBPase, FBPase, PRK

Answer 23

• Alkaline pH optimum + magnesium dependent (↑ [MG2+] ↑ activity] + redox state
• So activity goes from ↑ in light to ↓ in dark by 95%
• Thioredoxin reduce disulphide bridges in target e.g. PRK, 2 lys = oxidised w/ BS for ATP at AS. Reduced so open bs so ATP can bind. Changes conformation of the E
• Plants w/ 1/2 SBPase WT activity = significant but non-proportional inhibition (Cj = 0.1-0.2) due to ↓ regenerative capacity of TCA
• Transformants w/ 15% PRK activity had Cj = 0.06, even smaller than FBPase
• FBPase x effect photosynthesis until 60% removed
• Enzymes that thought to have ↑ control x but are ↑ regulatable

Question 24

Explanation of SBPase,FBPase,PRK activity

Answer 24

• PRK as an example
• PRK activity depends on relative conc. of reactants (+ve), products (-ve) + modulators (e.g. 3PGA -ve)
• If remove PRK, R5P→R1,5BP slows down
• But, R5P made at previous rate + R1,5BP removed at previous rate. R1,5BP levels ↓ + R5P levels ↑
• ↑ in R5P (product) activate E + ↓ in product (R1,5BP) + 3PGA also help activate

Question 25

Explanation of aldolase activity

/ Reversible E

Answer 25

• WATCH AGAIN PLEASE!!!!
• Previously thought inappropriate site that flux controlled through
• Reduction of aldolase below 30% of WT → significant ↓ in rate
• ↓ expression of aldolase inhibits photosynthesis in low light due to RuBP efficiently catalysing regenerative part of cycle → inhibition
• ↓ in high light by restricting regeneration of ATP. In high light Cj = 0.5 (more than 1/2 control in aldolase)

Question 26

Metabolic integration btw bacteriod + host plant cell

Answer 26

• E + reducing power for bacterial N fixation is provided by plant in form of dicarboxylates
• To release ammonia to host cell, need mandatory supply of aa to bacteroid by host

Question 27

GS assimilatory role

Answer 27

• GS has ↑ Km for ammonium binding, (more likely to be involved in assimilation)
• Labelling experiments = label is 1st incorporated into glutamine amide group. Then GOGAT transfers the N from the amide to amino group in glutamate

Question 28

Compartmentalisation of GOGAT/GS cycle

Answer 28

In roots:

• GS is mainly cytosolic, some evidence for plastid
• GOGAT is plastidic, needs reducing power that comes from plastidic oxidative PPP

In leaves:

• GS = cyt (GS1), mit (GS2) + plastidic (GS3) forms
• GS1 = in phloem cells, makes glutamine
• GS2 = in mesophyll cells, recovers photorespiratory ammonia
• GOGAT is also plastidic

Question 29

Regulation of GS1 expression

Answer 29

• Regulation occurs at multiple levels

- GS = target for trying to improve N use efficiency, if overexpress GS, ↑ capture of ammonium + ↑ efficiency

Question 30

Carbon skeletons for N assimilation
(Citrate)
(Asparagine)

CHECKKKKKKKKKKK!!!!!!!!!!!!!!!!!!

Answer 30

• C skeletons drawn from variety of int. from TCA, glycolytic + PPP.
• At some point in each pathway, have aminotransferase that removes NH3 from Glu/Gln + transfers to C skeleton
• Citrate → a-ketoglut + glutamate
• Need to replace citrate
• Depends on PEPC to make OAA
• Although mostly cytosolic, some evidence of PEPC plastidic isoforms in rice

CHECK THIS!

• In illuminated leaves, C skeletons for N assimilation are derived from citrate
• Stored + synthesised during dark period
• Using Jnc + 15N/13C labelling data
• Looked at Glu-C2 + barely any molecules so glutamate is made from 2-a ketogenic x 13C
• Look at Jcn, Glu labelled w/ 13C + 15J
• Conclude most 2-aketo made overnight in dark before could incorporate any 13C into system
• Asparagine export from legume root nodules requires OAA as a C source for asp synthesis

Question 31

Glutamate dehydrogenase

Answer 31

• Reversible enzyme
• x in favour of reductive amination
• ↑ Km so ↓ affinity for ammonium
• Catabolic role e.g. detection of glutamate ox by isolated mitochondria in absence of aminotransferase activity
• ↑ GDH activity in senescence + in C starved cell suspension cultures. Both systems are running out of respiratory substrates, re-mobilise aa for other purposes

Question 32

Sources of ammonia

Answer 32

1. Ammonium uptake from the soil
   - NH4+ = main form of inorganic N in acidic soil
   - Low and high affinity ammonium transporters occur in the plasma membrane - AMTI transporters
2. Symbiotic N fixation
   - Nitrogenase
3. Photorespiration
   - Cxygenase activity of Rubisco
   - Prevents accumulation of toxic 2-phosphoglycerate
   - Ammonia release = glycine decarboxylase + serine hydroxymethyl transferase
   - Ammonium production = 10x assimilation. Re-assimilation = important
   - Evidence: photo respiratory mutants lacking GS/GOGAT die in air but grow normally in 1% O2. Shows NH3 re-assimilation x essential for growth of C3 + photoresp. x essential
4. Recycling of protein + aa
   - Oxidative de-am of glutamate is followed by re-assim. of NH4+ into transport when protein hydrolysis occurs e.g. during germination
5. Nitrate upatke + reduction
   - NO3- = main arouce of inorganic N in soil
   - Uptake is carrier mediated

Question 33

Nitrate uptake

Answer 33

• Some are constitutively expressed (low/high) + some are inducible by nitrate (high)
• 2 families
  1. NRT1/NPF = encodes low + dual affinity transporters
  2. NRT2 = high affinity transporters
• Nitrate uptake is driven by H+ cotransport
• Experiment: NRT1 expressed in Xenopus. Electrogenic NO3- transporter w/ an activity that ↑ as the external medium was acidified. Conclude NRT1 catalyses uptake w/ at least 2H+ in cotransport

Question 34

Nitrate reductase regulation

CHECKKKKKKKKKKK!!!!!!!!!!!!!!!!!!

Answer 34

• NIA = sensitive to conditions plant is in
• Reversible activation occurs when leaves = exposed to light or high CO2
• Phosphorylated/dephosh. forms of NIA are both active; Ca2+ dependent phosphorylation of a Ser permits binding of inhibitory 14-3-3 protein
• In light, leads to production of reduced ferredoxin. Reduced ferredoxin also reduces thioredoxin + involved in activation of ↑ E e.g. Calvin cycle, favours reduction of nitrate → nitrite → ammonium
• Signals prevent phosphorylation of NIA + inactivation by 14-3-3
• In the dark, photosystems x active, thioredoxin is oxidised. Activates OPP + produces NADPH which reduces ferredoxin which supplies reductants to Nitrite reductase +GS
• Inhibitory signals from chloroplast lead to phosph. of nitrate reductase + inhibition by 14-3-3
• Rate of flux nitrate → nitrate is reduced, slower assimilation of nitrate
• Store carbon skeletons for use in light

Question 35

Nitrate availability + role in regulation

Answer 35

• ↑ changes in gene expression depending on N-containing metabolites
• Availability of NO3- → signals from hormones, aa, nitrate
• Evidence that nitrate itself plays a role in regulating gene expression:
• Transcripts that ↑ rapidly in response to NO3- (sensing nitrate itself)
• NiA mutants. Compare WT plants grown on low NO3- (N deficient) w/ NIA mutants grown on high NO3- (N deficient too).
• Find both nitrate + product of nitrate influence plant responses

Analysis of 2o metabolism in tobacco using NIA mutants

• Nitrate availability regulates shift from N-containing alkaloids to C-rich phenylpropanoids during N deficiency
• N-replete WT + nitrate-grown NIA mutants have low levels of C-rich 2o metabolism → nitrate inhibits phenypropanoid synthesis
• Genes for phenylpropanoid metabolism are induced in N-deficient WT (↓ nitrate) x nitrate-grown NIA mutants (↑ nitrate) → low nitrate is signal for switch to C-rich compounds during N-deficiency

Demonstrate that nitrate has direct effect on N metabolism:

• Get induction of NRT1/2, NIA, NII< GS/GOGAT
• Also ↑ rate of NO3- uptake, activity of nitrate reductase
• Also affects synthesis of organic acids needed for NO3- assimilation. Induces PEPC, pyruvate kinase, citrate synthase genes (↑ activity of E + levels of malate + 2-oxoglut)
• Effects = in both roots and leaves (nitrate sensing = in both tissue)
• Rapid change in gene expression = primary nitrate response

Question 36

NO3- signalling

Answer 36

• Metabolism of NO3- also generates signals
• NO3- uptake + assimilation are inhibited by Gln, Asparagine + other aa
• WT plants show marked diol changes in transcript levels + E activities but in NIA-deificeint mutants, have ↑ levels of mRNA + E activity but no variation (nitrate alone x only signal but powerful signal)
• Sugars often complement the effect of NO3- on gene expression
• Sugars induce NRT1/2, NIA, genes for GS, PEPC

Question 37

NO3- sensing

Also note plant PII homologue acts as a glutamine sensor. Different from 2 component regulatory system of bacteria

Answer 37

• NO3- sensor likely senses extracellular NO3- pool as cytosolic pool is generally unresponsive to changes in extracellular NO3- (excess NO3- can be stored in vacuole)

Evidence:

1. NO3- transporter is involved in NO3- signalling
   - Split root experiment. Single root Arabidopsis has access to ↑ NO3-
   - Mutant NRT1.1 shows ↓ lateral root formation in response to external NO3-
   - Either nitrate sensor or facilitator for nitrate uptake into nitrate-sensing cells (less likely)
   - Mutant NRT2.1 shows lateral root formation w/o external NO3-
   - Consistent of NRT2.1 as a low NO3- sensor or signal transducer

NRT1.1 as Nitrate sensor

• NRT1 mutant chl1-9 = defective in NO3- transport but still triggers intracellular response to NO3-
• At ↓ NO3-, phosphate of NRT1 at Thr101 converts transporter from low to high affinity + intracellular response to NO3- = down regulated (or change in Vmax?)
• Nrt1.1 = transporter of nitrate + receptor

Question 38

Long range signalling mechanism

Answer 38

• In addition to uptake of NO3- at root, also need long range signalling to communicate N+C status of the root
• NRT2.1 = ↑ affinity transporter is repressed by signals from shoots (signals could be ammonium + glutamine but x established)
• NRT1.1 x sense shoot N status

Evidence
- Status of shoot reported to shoot
- Loss of function hy5-526 = in screen for impaired shoot-illumination-promoted root growth
- HY5 = TF that is a shoot-to-root mobile signal. NRT2.1 expression + N uptake depends on + maintains balance of C + N (shoot to root)
ALSO
(root to shoot)
- Thought mobile C-terminally encoded peptide (CEP) reports root N status to shoot
- CEP = transported through xylem to leaves
- x made in N-rich roots on N deficient
- When reaches leaf, CEP binds to receptor + leads to synthesis of glutaredoxin (phloem-mobile + go back to roots)
- In N-rich patch, glutaredoxin further activates NRT2.1 expression + promotes uptake of N2

Question 39

Local signalling to coordinate N + C signalling

Answer 39

• Localised supply of nitrate stimulates elongation of lateral roots in Arabidopsis
• 1mM nitrate lateral root grows due to accumulation of auxin at apical meristem.
• Glutamine x nitrate = less root growth. Flow of auxin back through Nrt1.1 back to origin of IAA
• Thought return of IAA is impaired in nitrate x glutamine (nitrate competes for transport w/ auxin)
• Evidence = chl1 mutant. x transport either nitrate or auxin. Elongate roots irrespective of nitrate of glutamine
• Nrt1.1 has 3 functions: transports nitrate, auxin + receptor that detects external nitrate

Question 40

How does levels of F2,6BP change

Answer 40

• 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 41

Carbon partitioning during photosynthesis

Answer 41

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 42

Evidence of impact of F2,6BP on flux

Answer 42

• 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 43

Sucrose P synthase (SPS)

Answer 43

• 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 44

Which SPS gene is responsible for sucrose

Answer 44

• 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 45

Sucrose synthesis + trehalose 6P control

Answer 45

• 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

Question 46

Overview of assimilation in dark

Answer 46

• Starch accumulates in the light + degraded in the dark
• In dark, mobilisation occurs at same rate so decline is linear
• Ensures starch reserves are almost completely gone by end of night

Question 47

How do enzymes access insoluble starch granules

Answer 47

• Glucosyl units within glucan chains are phosphorylated by glucan water dikinase (GWD) using ATP
• GWD phosphorylates around 1 in every 30 glucosyl units in a-glycan polymer C6
• Steric effects + -ve charge of phosphorylation disrupts crystalline structure of granule + allows more E to enter
• Phosphoglucan water dikinase (PWD) adds additional P onto C3. Further disrupts structure
• Allows access to hydrolytic E that cleave a1,4 + a1,6 that link glucosyl units
• Phosphate removed by phosphatases + oligosaccharides are further cleaved by B-amalases that release maltose

Question 48

Pathway of C export at night

Answer 48

• Maltose itself is exported directly from cytoplasm → cytosol
• Maltose is condensed to longer chain glucans
• Accumulate + = substrate for a-glucan phosphorylase.
• Cleaves a1,4 glucan bonds + releases glucose1-P
• Glucose1P → other hexose-P ultimately forming sucrose that can be exported from the leaf even at night
• Thought maltose degradation involving resynthesis of cytosolic oligosaccharides could help buffer against provision of substrates

Evidence

• Characterised critical mutants defective in starch degradation
• Mex1 mutant = defective in maltose transport. Low state of starch mobilisation in dark

Question 49

Experiments to show control of starch degradation

Answer 49

Experiment 1

• Genetically identical Arabidopsis plants are grown in 2 different conditions
  1. = 12h light, 12h dark (normal). Starch accumulates in day + is degraded linearly + completely in dark
  2. = 6h light, 18h dark. Starch accumulates in day + completely degraded at night
• Rate of starch degradation is slower than 12h dark plants to ensure starch synthesis supplies x exhausted
• Amount of starch accumulates in 6h light is > than 1st 6h of 12light/12 dark plants
• Recognise growing in limited photoperiod + adjust rate of starch synthesis

Experiment 2

• Test if plants can test length of time passed since ‘dawn’ at start of day
• Introduce skeleton photoperiod of where plants grown in normal 12h dark/light cycle + are subjected to start of the day to few hrs of illumination
• Light switched off for photoperiod + switched on for last few hours for ‘normal’ photoperiod then off
• Accumulates less starch compared to normal
• Know don’t count time since light was last switched on as would only recognise few short hours + would degrade slowly
• So, plants determine passage of time based on time since dawn

Experiment 3

• Plants grown in 14hr light + 14hr dark (28 hr total)
• Compromises growth as rather than recognising 28hr day, think 24hr
• After 14hr light, think night =10hr so adjust starch degradation so used by end of 24th hour
• Starvation = last 4 hrs

Question 50

Phloem unloading

Answer 50

• 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 51

Methods of sucrose breakdown

Answer 51

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 52

Evidence for methods of sucrose breakdown

Answer 52

• 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 53

Starch synthesis in storage tissue

Answer 53

• 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 54

What crosses amyloplast membrane?

Answer 54

• 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 55

Endosperm AGPase

Answer 55

• 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 56

Regulation of starch synthesis

Answer 56

• 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 57

Evidence for ATPase modification

Answer 57

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 58

How is redox state of AGPase affected

Answer 58

• 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 59

Summary of regulation for AGPase

Answer 59

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

Question 60

Potential substrates for the TCA

Answer 60

• 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 61

Experimental evidence for TCA substrates

Answer 61

• 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 62

Non-cyclic flux modes in TCA

Answer 62

• 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 63

ETC in plant mitochondria

Answer 63

• 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 64

Respiratory chain supercomplexes

Answer 64

• 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 65

Regulation + role of alternative oxidase / dehydrogenase

Answer 65

• 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 66

Alternative oxidase (AOX) properties

Answer 66

• 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 67

Regulation of AOX

Answer 67

• 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 68

Transcriptional regulation of AOX

Answer 68

• 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 69

Role of AOX

Answer 69

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 70

Uncoupling protein

Answer 70

• 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 71

Mitochondrial activity in illuminated leaves

Answer 71

• 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 72

Respiration is essential in illuminated leaves

Answer 72

• 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 73

Reducing power from the chloroplasts

Answer 73

• 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 74

Other sources reducing power of ETC

Answer 74

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

Question 75

Nitrogen fixation

Nitrogenase

Answer 75

• Dinitrogenase reductase = Fe protein, supplies reducing power, also MoFe protein that reduces N2
• Symbiotic N fixing = when N fixing bacteria associate with a plant
• E.g. rhizobia + legumes forming root nodules

Question 76

Nodules development in legumes 1

Answer 76

• Flavonoids released by roots of host plant → change in rhizobia gene expression (nod genes)
• Nod genes allow synthesis of nod factors (lipochitin oligosaccharides) that initiate change in host gene expression (ENOD genes encoding for early nodulins)
• In addition to change gene expression, Nod factors also trigger influxes, membrane depolarisation

Question 77

Nod factors

Answer 77

• Typically lipochitin oligosaccharide w/ NAG backbone
• Chemical modifications on terminal residue are important for interaction with host plant + encodes specificity of interaction
• Plant releases compounds into rhizosphere, some detected by Rhizobia which produces a particular Nod factor that if recognised by plant starts formation of nodule

Question 78

Nod factor signalling

Answer 78

• DS signalling component identified by genetic analysis of Lotus japonius
• Signal received by receptor kinase. Causes Ca2+ influx which impacts TF NSP1/2
• NSP1/2 can also be activated by DMI1/2 (ligand gated cation channel/ receptor kinase) which triggers ca2+ spiking around nucleus + activates DMI3 (calcium/calmodulin-dependent protein kinase)
• If deregulate DM13 by removing autoinhibition domain, causes nodulation w/o rhizobia (rhizobia critical in cascade)

Ubiquination

• LYK3 receptor kinase = nod factor protein that assists binding of nod factor to kinase
• W/o nod factor, PUBI = E3 ubiquitin ligase that is active. Ubiquinates a protein needed for infection + infection x
• When nod factor binds LYK3, LYK3 phosphorylates PUB1 + inactivates

Bacterial exopolysaccharide (EPS)

• Important for host invasion by symbiotic bacteria
• In Lotus japonicus, nod factor signal transduction induces transcription of the host gene Epr3
• This encodes a receptor protein similar to NRF1 family which can recognise EPS

Question 79

Nodule development in legumes 2

During infection

Answer 79

1. Bacteria attach to root harms promote growth + curling
2. Modification of cell wall followed by invagination of the plasma membrane → infection thread
3. Invagination propagates into root cortex + nodule primordial is formed in cortex
4. LOF + GOF mutations in cytokinin receptor show activation of LHK1 is sufficient to trigger nodule formation

• Signalling coordinates activity at surface vs centre of root cortex (cortex is ready for invading bacteria)

Question 80

Role of nodule inception protein in nodulation (NIN)

Answer 80

• NIN - bifunctional TF suppressing ENOD11 expression in epidermis + promoting transcription of cytokinin receptor CRE1 in root cortex
• Nod factors arrive at root hair + trigger expression of NIN
• NIN activates the cortical program leading to organogenesis
• NIN activates NPL that breaks down cell wall + allows invag. of plasma membrane to create infection thread
• Some signal triggers cytokinin production that → expression of NIN in cortex
• Sets up +ve feedback loop: production of NIN ↑ expression of CRE1, which ↑ zone within root sensitive to cytokinin. Eventually NIN inhibited
• NIN controls a diversity of functions inc. cell wall modification via NPL, GA biosynthesis by genes like CPS1, nutrient uptake + DNA synthesis

Question 81

Nodule development in legumes - 3

Nodule formation

Answer 81

1. The infection thread extends into the nodule primordium, allowing rhizobia to enter the plant.
2. Bacteria are released into the cytoplasm, forming symbiosomes that occupy up to 80% of the cell volume and contain up to 20 bacteria surrounded by the plant-derived peribacteroid membrane.
3. The bacteria differentiate into endosymbiotic bacteroids + the nodule primordium develops into a mature nodule.

Question 82

Symbiotic bacterial gene expression

Answer 82

• Nod, nol and noe genes are required for the synthesis of the nod factors that initiate nodule development:
  1. The nodD gene product activates the expression of the other nod genes after forming a complex with secondary metabolites in the root exudate
  2. NodA-C gene products make lipochitin oligosaccharide backbone
  3. Other nod gene products determine specificity by controlling the chemical modification of terminal residues
  4. Nif genes = both symbiotic bacteria + N2 fixing. Gene products Include Fe protein + MoFe, regulatory proteins for bif gene expression, NifA/L to name a few
  5. Fix genes - only occur in symbiotic N2 fixing bacteria. Gene products include components of O2 sensing system (FixJ,L,K) + structural proteins of high affinity bacterial terminal oxidase. Protect nitrogenase from denaturation by O2

Question 83

Oxygen sensing

Answer 83

• In K pneumoniae, NifA-induced transcription of nifHDK is inhibited by
  NifL-NifA interactions in the presence of O2
• In symbiotic diazotrophs, further control = FixL (haem protein) acts as O2 sensor in bacterial membrane, deoxy-FixL is a protein kinase, oxy-FixL is a protein phosphatase
• When ↓ O2 in periplasm, FixL = deoxygenated + = kinase making FixJ-P
• FixJ activates FixK which triggers formation of structural proteins for terminal oxidase. Also activates NifA which promotes expression of nifHDK

Question 84

Maintaining an anerobic environment

Answer 84

3 factors help ↓ O2 conc.

1. Restricted diffusion, based on a variable permeability barrier controlling O2 exchange at the nodule periphery and the reduction of intercellular air spaces.
2. Binding to leghaemoglobin reduces the free O2 concentration in the host cytosol to 10-25 nM. RNAi-induced abolition of leghaemoglobin synthesis prevents symbiotic N fixation.
   - Bacterial respiration, aided by the ↑ affinity terminal oxidase (Km ~ 7 nM), acts as a major oxygen sink.
   - Low O2 limits nodule respiration + nitrogenase. Effect greater in bacteroid than host cytosol as host mit. localise at cell surface near intracellular air spaces

Question 85

Nitrogen sensing

Answer 85

• Free living diazotrophs use 2 component N control system to regulate Nif gene expression
• NtrB/C. Less common in symbiotic diazotrophs, reflects role of symbiotic diazotroph as source of fixed N for host + importance of O2 sensing
• NtrC-P activates NifA/L

Question 86

Symbiotic plant gene expression

Answer 86

1. Early nodulins
   - Tissue specific expression = essential for successful infection + nodule development
   - Epidermal Nod factor-induced ENOD expression occurs in the vicinity of actively growing root hairs
   - Pectate lyase induced in the nodule primordium degrades the plant cell wall around the infection thread, facilitating the formation of symbiosomes
2. Late nodulins
   - Establishes metabolic conditions for N2 fixation in the host cytosol:
   - leghaemoglobin for controlling oxygen availability
   - glutamine synthetase for ammonium assimilation
   - sucrose synthase for sucrose breakdown

Question 87

Problems with Rubisco

Answer 87

• Slow turnover 3s-1
• ↑ Kc so typically -50% saturated w/ CO2 in C3 plants
• Rather poor selectivity for CO2 via O2 under physiological conditions

Question 88

Experimental evidence for photorespiration

Answer 88

1. O2 inhibition of photosynthesis
   - Soybean plants maintained at 2 different CO2 conc. (275ppm + 73ppm)
   - As [O2] ↑, rate of CO2 uptake ↓
   - For 73ppm, CO2 uptake became -ve
2. Post-illumination ‘burs’ of CO2
   - Measured gas exchange characteristics of a leaf
   - Leaf initially exposed to v ↑ light intensity + exposed to successfully lower intensities
   - W/ each transition, photosynthesis rapidly ↓ + ‘overshoots’. Balanced back to new steady state- post illumination burst
   - Thought light-dependent transport processes immediately change but takes longer for other metabolic processes to adjust

Question 89

Properties of Rubisco + Structure

Answer 89

• Carboxylase: RuBP + CO2 → glycerate 3P + O2
• Oxygenase: RuBP + O2 → phosphoglycolate
• Balance btw photoresp: photosynthesis ↑ w/ ↑ temperature
• At ↑ temp, V(O2) ↑ more than V(CO2) + solubility of CO2 ↓ more than O2

Structure

• 550kDa hexadecameric structure
• 8 large subunits (LSU), 55kDa
• 8 small subunits (SSU), 13-15kDa
• Tetramer of LSU capped by pair of SSU tetramers

Question 90

Photorespiration

Answer 90

• In chloroplast:
• RuBP + O2 0 → phosphoglycolate (toxic)
• Immediately dephosph. → glycolate (phosphoglycolate phosphatase)
• Moves to peroxisome
• In peroxisome:
• Glycolate ox to glyoxylate which is converted by transaminase to glycine
• Glycine moves into mit.
• In mit.
• Glycine decarboxylation releases CO2
• Serine formed passes back into peroxisome.
• Transaminated to hydroxypyruvate which is reduced to glycerate (hydroxypyruvate reductase)
• Glycerate moves back into chloroplast in exchange for glycolate
• Glycolate → glycerate 3P + returns to cytoplasm

Question 91

2 key E of photoresp.

Answer 91

1. Glutamine synthase
   - Chloroplast
   - Assimilates NH3 into organic form
   Glutamate + NH3 + ATP → Glutamine + ADP + Pi
2. Glycine decarboxylase
   - Mitochondria
   - 4 protein system, ↓ Glycolic = ↑ GDC activity, ↓ levels of CO2 acceptor RubP so important feedback signal
   - Releases CO2 + NH3 (toxic if accumulate)
   2 Glycine + H2O + NAD+ → 1 Serine + CO2 + NH3 + NADH

Question 92

Purpose + cost of photorespiration

Answer 92

• Detoxifies phosphoglycolate
• In glycine decarboxylase reaction, 1 CO2 released, 3CO2 recycled
• Can salvage 75% of C that would be lost w/o complicated mechanisms (25% CO2 lost directly tho)
• Some benefits e.g. produces Ser + dissipates excess excitation E from chloroplast to mit.

But metabolic cost

• ATP:glycerate kinase in final step + glutamine synthetase reaction = essential for re-assimilation of NH3
• Reduced ferredoxin needed for GOGAT reaction
• H202 in peroxisome, NH3 need to be detoxified

Question 93

Why does photorespiration exist

Answer 93

• If so disadvantageous, would be lost w/ evolution
• Kozaki + Takeba experiment 1996:
• Hypothesised E cost of photoresp. in certain times could be adv. if plant has excess photon E or reducing equiv.
• Transgenic tobacco plants either ↑ or ↓ of GS + control
• Antisense GS show ↓ post illumination bursts of CO2 when lights switched off
• Control = normal
• GS overexpression = enhanced post illumination bursts of CO2 output
• Examined different responses to photoinhib. damage in CO2-free air→ GS over expressing = less reductions in e- transport rate/ETR, control = progressive decline, GS antisense = greater reduction in ETR in high light
• Consistent w/ photoresp. playing role in ↓ photoinhibitroy damage to e- transport system under high light

Question 94

Is Rubisco lazy

+

Evidence Rubisco specificity = evolutionary selection

Answer 94

• Early relic as early atmosphere had ↓ O2 so not influenced by ability to distinguish btw
• 1000x ↑ selectivity for CO2 than o2. But O2 conc. = 500x that of CO2 in air
• Specificity factor = Kcat/Km
• Anaerobic Bacteria = ↑ CO2, ↓ O2. Sc/o = 6-41
• C4 higher plants w/ CCM, Sc/o = 70-82
• Given evolutionary span, rather limited range of values, thought Rubisco operates under restraint. But range shows can respond to selection pressures
• Variation = compromise: a TS for CO2 aids discrimination btw CO2 + O2 (↑ for CO2) but resemblance to 6C carboxyketone intermediate causes the int. to bind so tightly, catalytic max = restricted
• Inverse relationship btw Sc/o + Kcat (as make TS ↑ specific to CO2, ↓ opportunity for tighter binding/ ↓ ROR)

Question 95

Rate limiting step (RLS) Rubisco photorespiration

Answer 95

• Step where E-substrate complex is resolved to release 2 3PGA molecules = kinetic RLS
• Trade-off to max. selectivity of CO2 - make resolving step difficult

Question 96

Nitrate assimilation + photorespiration

Answer 96

• Inhibition of photosynthesis inhibits nitrate assimilation + absorption (Bloom et al 2010)
• Most plants get N from environment in form of inorganic nitrate or ammonium + legumes can assimilate N2
• Used 3 conditions: normal O2+ CO2 (1), ↑ CO2 + normal O2 (2), normal CO2, ↓ O2 (3)
• 2+3 suppress photoresp + N assimilation
• Possible as in cytosol photorespiration produces reducing equiv. needed for N assimilation
• Change in assimilatory quotient which reflects shoot NO3- assimilation is ↓ at ↑ CO2
• Photoresp. = embedded in N assimilation

Question 97

C4 photosynthesis discovery

Answer 97

• 3 research groups simultaneously found in 1960, one = Hatch + Slack in Australia
• In certain tropical grasses, 1st formed products after 14CO2 feeding are labelled 4C compounds x 3C
• 14C label quickly moves into usual 3C compounds

Question 98

Anatomy of C4 plants

Answer 98

• Kranz anatomy
• Large bundle sheath cells (BS) surrounding vascular bundles contain prominent chloroplasts arranged around their outer walls
• Vascular bundles are separated by 2 layers of intervening mesophyll cells
• Explains difficulty in finding Rubisco (Rubisco present but entered contained in bundle sheath cells)

Question 99

C4 pathway

Answer 99

1st step = carboxylation phase in mesophyll cells. Co2→HCO3- which combines w/ phosphorylation-pyruvate → oxaloacetate

2. = decarboxylation phase inside thick-walled bundle-sheath cells, malate→pyruvate w. release of CO2
   - Shuttling of CO2 from mesophyll to bundle sheet. PEPC = v efficient E + draws down CO2 in mesophyll to 150ppm (2000 in bundle sheath)
   - Bundle sheath cells have chloroplasts w/ thylakoid lamellae deficient in PSII so x O2 evolution in BS cell. Good for rubisco (suppresses photorespiration) but lacks NADPH so Calvin cycle x have enough reducing power
   - Means activity of Calvin cycle is split as only 1/2 NADPH is supplies to drive cycle, 1/2 = supplied inside the mesophyll cell

Question 100

C4 advantages

Answer 100

• Provides Rubisco w/ ↑ CO2 for carboxylase reaction

- Suppresses oxygenase activity, overall v efficient C fixation

Question 101

Key E in the C4 pathway

Answer 101

1. Carbonic anhydrase
   CO2 + H2O → H2Co3 → HCO3- + H+
   - Rate of spontaneous hydration of CO2 = 10,000 x too slow to provide bicarbonate at rate needed
   - So present at ↑ quantity + ensures CO2 hydration = good rate
2. Phosphoenol pyruvate
3. Decarboxylase e.g. malic E
   malate + NADP+ → pyruvate + CO2 + NADPH
   - CO2 released builds up locally to ↑ conc. inside bundle sheath cell, helps Rubisco bet at max efficiency
4. PEP regeneration e.g. PPDK
   - Extra E requirement for pathway (2 ATP per CO2 fixed)

Question 102

3 subtypes of C4 plant

Answer 102

• Variation lies mainly in localisation of decarboxylating E in bundle sheet
  1. NADP-malic E (chloroplast)
• ↑ common form, malate enters bundle sheath + is ox. decarboxyl. to pyruvate + frees CO2
  2. NAD malic E (mitochondria)
• Aspartate into bundle sheath. Transaminated to oxaloacetate
• Malic E converts malate → pyruvate + CO2
  3. PEP carboxykinase (cytosol)
• more complex
• Asp coming in → oxaloacetate → PEP, uses ATP + releases CO2
• To support ATP requirement, ↑ activity of malic E in mit.

Question 103

Tissue specific gene expression

Answer 103

• C4 photosynthesis involves partitioning of photosynthetic E btw 2 cell types (mesophyll + bs)
• Hypothesis for control at molecular level = PPDK or PEP carboxykinase = expressed in mesophyll x bs cells
  Model A = C4-specific sequence elements are present in upstream promoter region of gene although both C3 + C4 have needed TF. Thought correct
  Model B = PPDK has upstream promoter sequence in C3+4 plants but gene is only transcribed in C4 as TF present in C4 cells

Experiment

• Upstream promoter sequence from PEP carboxykinase of C4 is ligated to Gus reporter gene
• When gene is expressed in a tissue if sugar substrate is produced, blue dye is expressed. Blue = mesophyll cells
• If use C3 promoter, blue = mesophyll + BS
• Conclude = specific sequence elements present in promoter of C4 gene that can confer specificities of expression. Supports A

Question 104

Key properties of PEPC

Answer 104

• ↑ specific activity in C4
• Effective Km for CO2, lower than Rubisco in C3 plants
• Regulated by reversible phosphorylation
• Allosterically activated by G6P, Gly, Ala
• Allosterically inhibited by malate, OAA, Asp

C3 vs C4

• C4 = distinct Ser774 in region 5. C4 determinant
• C3 = Alanine774
• Phosphorylation site of PEPC = at N terminus. One phosphorylated, C4 has ↑ affinity for substrate + ↓ affinity for PEP
• Also phosphorylated C4 has ↓ sensitivity for inhibition by malate

Question 105

CO2 diffusion gradients in C3 vs C4

Answer 105

• C4 plants confer ↑ efficient H20 use so can grow in hot + semi-arid environment
• Greater draw down of CO2 in C4 plants = direct function of kinetic properties of PEPC vs Rubisco + directly leads to ↓ transpiration rates

Question 106

Ecological characteristics of C4 plants

Answer 106

• Total 7500 species (around 3% of all flowering plants)
• Have high max rate of photosynthesis and growth
• More H20 efficient than C3 plants
• 2 main ecological groups = tropical + subtropical grasses vs highly stress-tolerant plants

Question 107

C4 advantages / disadvantages

Answer 107

Advantages
- C4 photosynthesis effectively eliminates photorespiration, more water/nitrogen use efficient + permits growth under ↑ temperature conditions

Disadvantages
- ↑ energetic cost + intrinsic cold sensitivity

Question 108

Crassulacean acid metabolism (CAM) phases

Answer 108

• 2nd form of CO2-conc. mechanism
• Phases
• All occurs in 1 cell
  1. Acidification, net CO2 fixation, PEPC, malic acid ↑ (Dark period)
  2. PEPC → Rubisco
  3. Deacidification, Co2 refixation + RUBISCO
  4. Net Co2 fixation, RUBISCO/PEPC
  (Light period for 2-4, malic E ↓
• Take up most CO2 during night (accumulates malic acid + degrades glucan)
• Transient burst of CO2 fixation during start of day

Question 109

Carbon flow in CAM

Answer 109

Dark

• All C flow is in just mesophyll (C4 = mesophyll + BS)
• CO2 taken up from atmosphere by open stomata at night, hydrated to form carbonic anhydrase bicarbonate. Binds to PEP → OAA (PEPC) → Malate (MDH)
• Can’t use photosynthesis as night
• Some malate equilibrates w/ pool in mit + enters TCA (not lots)
• Want to accumulate malate as a C source for light period. If accumulated in cytosol, would feedback + inhibit PEPC, so needs to be removed to vacuole.
• Accumulates as malic acid + ↓ pH6 to pH3 in vacuole by end of dark period
• ATP needed to drive proton pump to bring H+ into vacuole to ↓ pH
• 1 ATP used to drive proton pump per malate accumulated in vacuole + 0.5 ATP per malate produced in glycolysis = 0.5 per malate

Light
- Efflux of malic E out of vacuole + charge-balancing H+ out
- In cytosol, decarb. by malic E, releases CO2 which is liberated in cell cytoplasm at ↑ conc.
- Then processed in Calvin cycle
- Stomata closed so CO2 builds up to 1% in phase 3 (10x ↑ than C4)
- ↑ CO2 conc. maximises carboxylation of Rubisco + almost completely suppresses oxygenase. TCA v efficient
2 ATP per malate for PPDK, 1 per malate for PGK + 0.5 per malate for AGPase = 3.5ATP

Overall = 4ATP needed + E requirements of Calvin cycle
- More complicated in reality, around 641 reactants

Question 110

C4 vs CAM

Answer 110

• Both ↑ carboxylation efficiency of Rubisco by repressing photorespiration
• Both have evolved many times (>60) independently, strong evidence for functional significance in improving efficiency of photosynthesis
  C4
• Characteristic of warm, high light but not necessarily H20 limited environment
• NADP Mc subtype = found in ↓ arid environment than PEPCK + NAD-Me
• Similar C flow to CAM but PEPC active during light so malate formed + immediately transported from mesophyll to BS where decarboxylated + CO2 conc. around Rubisco
  CAM
• Warm, high light. Almost always assoc. w/ H20-limited environments as closure of stomata in day + confining CO2 to dark period = associated w/ conserving H20
• CO2 fixed by PEPC→OAA→malate, stored in vacuole then releases CO2 in light period + is assimilated through Calvin cycle

Question 111

E.G. of Cam plant environment

Answer 111

• Economic importance e.g. Vanilla planifolia - cultivation in Madagascar

Question 112

Developmental + environmental induction of CAM

Answer 112

• CAM may be facultatively expressed in response to stress e.g. exposed to hot dry conditions, has epidermal storage cells that can take up NaCl
• Can be used to investigate regulation

Question 113

Regulation of PEPC

Could add circadian clocks if want!!!!

Answer 113

• Ideally, PEPC is active in dark + off in light
• Max catalytic capacity actually x change that much day-night
• Instead, malate sensitivity of PEPC (Ki malate) is ↓ at night than day. Dramatic change, 5% activity in 2mM malate in day vs 80% at night
• Substrate affinity of PEPC is higher at night than during day (0.2mm in night vs 0.8mm in day for 1/2 max activity)
• Night time E = PEPC, malic-acid accumulation
• Day time = malic-acid release, decarboxylation, Rubisco

Question 114

Evolution of oxygenic photosynthesis + Rubisco

Answer 114

• Early atmosphere of earth = high CO2 conc. but negligible O2
• Atmospheric O2 ↑ steeply during great oxidation event
• All organisms that do oxygenic photosynthesis use Rubisco for the primary CO2 assimilation reaction, despite its inefficiencies
• Originally thought 2 types of ancestral Rubisco: 1A + 1B
• Interestingly, Higher plants have 2x specificity compared to 1A, 1B + bacteria. Indicates constraints under Rubisco operates, only 2x ↑ over ↑ ↑ time
• Cyanobacterium ↑ CO2 conc. of around 250um, have different CO2 conc. mechanism based on pumping HCO3- out into chloroplast. Highest turnover number + photosynthesis (then CC4 then C4 then C3)

Question 115

Evolution of C3 to C4

Answer 115

• BS cells enlarge and mitochondria (with GDC) become localized to inner wall of BS cells
• In M cells, GDC lost from mitochondria + concentrated in BS cells, where chloroplasts become appressed to mitochondria
• In M cells, Rubisco lost from chloroplasts and isoform of PEPC becomes highly expressed; BS cells acquire chloroplast C4-decarboxylase (NADP-ME) and lose PSII
• Any photorespiratory metabolism now restricted to BS cells and resulting CO2 efficiently refixed by Rubisco

Question 116

Single celled C4

Answer 116

• Only few examples e.g. large-celled halophyte Bienertia cycloptera
• Immunoblotting showed Rubisco = localised to basal part of cell + malic E = restricted to basal part
• PPDK = restricted to upper cell + PEPC is restricted round cell periphery
• Thought once C4 acid formed, decarboxylated by malic E + pyruvate formed in base of cell. Intermediates diffuse up through cytosol to top part of cell where PPDK converts pyruvate → PEP

Question 117

C4 evolution

Answer 117

• Reconstruction of CO2 conc. during Cenozoic era
• Showed significant ↓ in CO2 during Eocene + Oligocene epochs
• Thought where C4/CAM emerged
• Different E discriminate against 13C to different degrees (PEPC discriminates less than Rubisco). Through teeth of fossil equids, found towards end of Miocene become more like C4
• Thought due to aridification of earth’s climate
• Through molecular evolutionary sequence analysis, thought CAM evolved at same time of aridification

Question 118

Mechanism of Rubisco carboxylation + activation

Answer 118

• lys201 in AS is covalently modified by carboxylation to be active
• Forms a carbamyl group that binds mg2+ that interacts w/ substrate at AS + allows Rubisco to bend
• R1,5BP binds forms an enediol
• Mobile loop of TIM barrel forms a multilayered lid that closes the AS and generates physical environment for electrophilic attack of RuBP by CO2 for O2. CO2 directly attacks enediol, not AS
• Carbamylated form = binds RuBP tightly + Co2 can bind
• Decarbamylated form = RuBP binds across AS, CO2 x bind
• Enedoil intermediate reacts specifically w/ O2/CO2
• Even though 25% = O2, CO2 is favoured as is 25xO2 in solution and 500xO2 in air

Question 119

Rubisco activase

Answer 119

• In plants + some algae, needed to allow rapid formation of critical carbamate in AS of Rubisco
• Activase produces 2 protein products which activate
• Ru1,5BP binds stronger to AS when the carbamate is present + ↑ slows down the ‘activation’
• In the light, activase promotes release of RuBP from catalytic site as changes site
• Has ATPase activity to induce structural changes to rubisco
• Also needed as in darkness Rubisco is inhibited by competitive inhibitor CAIP which binds tightly to the AS of carbamylated Rubisco + inhibits activity. Activase promotes release

Question 120

PSI/PSII Overview

Answer 120

• Light reaction found in thylakoid in chloroplast
• Non cyclic photosynthesis = uses both PSI + PSII. Generates proton motive force + NADPH
• Cyclic = uses just PSI, Fd e- are donated back to ETC btw PSII + PSI e.g. PQ . Makes proton motive force so ATP
• PSI chlorophyll centre P700 absorbs best at 700nm
• PSII “ “ P680 absorbs best at 680nm

Question 121

PSI/PSII structure

Answer 121

PSI
- Located in stroma lamella of thylakoid

PSII
- Stacked in grana domain

• Both have core complex + peripheral antenna system, light harvesting complex 1/2
• E.g. in PSII supercomplex, PsbA-D make up catalytic centre. PsbA/D make up photochemical RC. Also have 12 membrane spanning subunits in core complex e.g. in spinach = PsbE-X
• LHCII = 30% of total protein in chloroplast membrane, ↑ abundant. Acts as a heterotrimer constituted by Lhcb1-2

Question 122

Reasons for coordinating activities of PSI+2

Answer 122

1. Maximise efficiency of light utilisation
   - PSII absorbs best at 680nm, PSI at 700nm. Natural light can result in imbalance of E distribution. As PSI + II are connected in series, can be issue for noncyclic e- transport. Unequal rates of light E conversion = photosynthesis limited by photosystem that receives less E
2. Avoid photo-inhibition due to over excitation of PSII . However, stn7 mutants argue against this + there are more effective mechanisms for photoprotection that exist

Question 123

State transitions (short term adaptation)

Answer 123

• When PSII>PSI activity (state 2). Occurs in PSII light
• PSI>PSII = PSI light. Redistributes E from light saturated PSI

1. Plastoquinone pool sensing
   - PII preferentially excited, PQ pool is reduced (over-reduction of ETC btw PSI + PSII as PSII gives ↑ e- than PSI)
   - PI preferentially excited, PQ is oxidised by faster transfer of e-s in PSI
   - State 1 to state 2 = LHCII from PSI to PSII
2. Binding of reduced PQ to cytb6f
   - Qo pocket in cytb6f = formed by cytb6, subunit IV + Rieske protein. PQH2 binds here + causes conf. change in downstream region of Gly-rich hinge. Allows Rieske protei to transfer e-s from PQH2 to cytochrome (research paper written by Shapiguzor et al)
   - x clear how signal from Qo side is transmitted to catalytic domain on stromal site
   - State1-2 transition, part of cytb6f is displaced from grana to cytosol
3. Kinase activation
   - When reduced PQ binds cytb6f, specific kinase is activated
   - Possible large-scale protein domain movement (Gly-rich region = due to change in protein kinase state transition 7 (Stn7), activating it
4. LHCII phosphorylation
   - LHCII trimers are linked to PSII core w/ LHCII proteins CP26/CP29
   - LhcbM1/2 are specifically phosphorylated. Non-phosph. LHCII has 3 membrane spanning helices + unstructured N. Upon Phosphoenolpyruvate. of Thr, amino terminal forms a helix that intercalates btw 2 membrane spanning helices + changes orientation
   - CP26/29 dissociate upon phosphorylation
   - LHCII proteins associated w/ PSII are forced to discociate when minor LHCIIs are unlocked
   - PsaH+L form docking site for LHCII (mutant PsaH x have state transitions)

Question 124

State transition function hypotheses

Answer 124

1. Surface charge hypothesis
   - Components of PSII are conc. in regions of the membrane that are closely appressed, like in thylakoids
   - Unappressed regions have components of PSI e.g. stroma
   - Phosphorylation ↑ -ve charge on cytoplasmic surface of appressed domain of thylakoid membrane
   - This change in charge is enough to overcome attractive forces which hold together LHCIIs on adjacent domains
   - Complexes migrate to unappressed regions where ↑ distance + cations e.g. Mg2+ ↓ repulsive forces
   - Issues: if protein phospho. change electrostatic potential throughout membrane domain, how can it avoid changes in interactions btw each protein + all others
2. Molecular recognition hypothesis
   - Electrostatic forces exerted initially by phosphorylation are v intramolecular + lead to ↑ structural changes that change interaction of membrane proteins by effects on respective docking surfaces
   - Phosphorylation of membrane proteins ↑ -ve charge at phosphorylation site
   - changes electrostatic interactions btw sc of phosphorylated aa + other aa nearby
   - Large compensations allow change in 2o structure of polypeptide segment containing phosphorylated site. In LCHII, forms a helix + phosphate group neutralises int. of -ve charges
   - Local 2o structure can cause change to 3o structure, which could change shape of a surface phosphoprotein, ↓ complementarity w/ neighbouring complex
   - ↓ sum of interactions holding 2 proteins means separate + diffuse freely

Question 125

Long term response to light imbalance

Answer 125

• Can change stoichiometry of PSII/PSI to being subjected to stable light quality gradient over a long period of time
• Chloroplast sensor kinase (CSK) = sensor histidine kinase that communicates the redox state of PQ transcriptional apparatus’s. Initiates change in stoichiometry
• In PSI light, Sig-1 is phosphorylated
• CSK is autophosph. + activated using both SIG-1 + PTK as substrates
• Phospho-sig1 represses transcription at the psa promoter, allowing transcription of psb genes
• Phospho-PTK (inactive) usually keeps chloroplast transcription low by phosphorylating PEP, now x suppress chloroplast transcription non-specifically as it is inactive
• ↑ stoichiometry of PSII relative to PSI

Question 126

Calvin cycle overview

Answer 126

• Takes place in stroma
  1. C fixation
• Inorganic CO2 molecule combines w/ organic 5C acceptor (RuBP) → 6C (Rubisco). Splits into 2x3PGA

2. Reduction
   - ATP + NADPH used to covert 3PGA into molecules of 3C sugar G3p
3. Regeneration
   - Some G3P molecules go to making glucose, others are recycle to generate RuBP. Requires ATP

Question 127

Contribution of Rubisco to control of Calvin cycle

Answer 127

• Small no of reactions are removed from thermodynamic eq.
• Net flux here depends on current rate of catalysis so plausible these E regulate flux through the pathway
• Subject to high regulatability
• Contribution of Rubisco to control photosynthesis depends on past + present conditions e.g.
• Use antisense tobacco plant w/ ↓ expression of Rubisco
• When Rubisco ↓ to 60% of WT + grown in ambient light, photosynthesis only slightly inhibited (c=0.05-0.15)
• When grown in low light + ↑ light intensity, near proportional relation btw amount of Rubisco + rate (C>0.9)
• Proposed 1-sided limitation of photosynthesis by Rubisco would hinder use of resources so disadvantage. Response could be for Rubisco to change amount of itself + other proteins

Question 128

Other ways of controlling Calvin cycle

Coordinating a balance btw starch + sucrose synthesis

Answer 128

• SBPase (15% of levels cause drop in starch synthesis), aldolase, Rubisco + PRKase also have selective inhibition to starch synthesis
• Transketolase causes preferential ↓ in sugars, but starch synthesis remained high until photosynthesis strongly inhibited
• Exact mechanism for partitioning unclear.
• ↓ levels of E inhibit photosynthesis
• Inhibition of starch synthesis causes ↑ of phosphorylated int. + ↓ of free organic phosph. when 30% fo aldolase
• Shows integrative nature…

Question 129

Bypass 1

Answer 129

• Plant glycolate oxidase uses molecular O2 + needs to be contained in peroxisomes to avoid H202 release into metabolically active compounds
• Glycolate dehydrogenase from E coli uses NAD+ instead of O2 as an e- acceptor to oxidise glycolate
• The 3 subunits of glycolate dehydrogenase are introduced into the plant, as well as glyoxylate carboligase + tartronic semialdehyde reductase (TSR)
• Here, 2C2 compounds (glyoxylate) are converted to 1 C3 compound w/ release of CO2
• Pros/Cons
• CO2 released into chloroplast stroma not mit., ↑ chloroplastic CO2 conc. ↓ probability of further oxygenation + ↑ CO2 fixation
• Ammonia release is abolished so no refixation
• Using glycolate dehydrogenase ↓ consumption of reducing equivalents
• Transmembrane transport is avoided
• Has ↑ biomass by 50% according to Kebeish et al

Question 130

Bypass 2 / Carvalho bypass

Answer 130

• Similar to bypass 1, phosphoglycolate → glycolate catalysed by PGLP
• Clycolate is converted to glyoxylate which is converted to hydroxypyruvate directly in the peroxisome using glyoxylate carboligase to tartronic semialdehyde + CO2
• Hydroxypyruvate isomerase converts tartronic semialdehyde → hydroxypyruvate

Pros

• E are directed to peroxisome, make use of glyoxylate formed
• ↓ no, of transport steps, theoretically ↓ E consumption
• Like bypass 1, abolished ammonia release, 25% C from glycolate is release as CO2 + 3/4C from gylcolate converted back to PGA

Cons

• Glyoxylate is diverted away from Gly in a deleterious short-circuit of photorespiration metabolism
• Experimental evidence shows x enhance photosynthesis + E consumption is only slightly lower than photorespiration

Question 131

Bypass 3/ Maier

Answer 131

• Characterised by complete oxidation of glycolate in chloroplasts
• Glycolate is ox. by glyoxylate by glycolate oxidase + H2O2 detoxified through expression of a plastid-targeted catalase
• Glyoxylate is condensed w/ acetyl coA to give malate, which is oxidised to regenerate acetyl coA using activities of NADP-dependent malic E + pyruvate dehydrogenase

Pros

• CO2 released is shifted to chloroplast
• NAD(P)H is made in both malic E reaction + pyruvate dehydrogenase reaction so bonus 2 additional reducing equiv/ per glycolate
• 3PG x produced so x cost for re-reduction of 3-PGA into Calvin cycle

Cons

• Depletes Calvin cycle of intermediates as 2CO2 released that have to be refixed (costs 4.5ATP + 3 reducing equivalents)
• Conflicting experimental results: Maier et al found biomass ↑, Xin et al simulation ↓ rate by 31% in Arabidopsis

Question 132

Other bypasses

Answer 132

• Weber et al aimed to abolish release of CO2 altogether through recycling glyoxylate into central metabolism through ds cycle of prokaryotic 3-hydroxypropionate bicycle
• Pyruvate = trick by-product as E needed to re-assimilate into CBC

Question 133

General photorespiratory bypasses

Answer 133

• Circumventing photorespiration restricts regeneration of int. but not critical as have excess C
• Levels of photoresp. int. like Ser or glycerate are likely to be affected due to diversion of C away from native photoresp. pathway or ↑ E costs due to transport of int.
• But, symptomatic of any changes to plant metabolism
• Potential feasibility of bypasses may depend on whether or not release of CO2 into chloroplast improves CO2 fixation or if it ↓ probability of O2 fixation
• Particularly w/ bypass 3
• E.g. if photoresp Co2 release in mit. causes significant ↑ flux of CO2 escaping from atmosphere, photoresp. CO2 release in chloroplast could ↑ photosynthesis by ↑ CO2 fixation
• But, if CO2 release outside chloroplast is re-refixed efficiently, relocation of CO2 should make x different to photosynthetic efficiency + could event be wasteful
• Exact desirability could depend on type of plant + level of CO2 fixation

Question 134

Products of photosynthesis

Starch + Sucrose

Answer 134

Sucrose

• 3-PGA exported from chloroplast in exchange for Pi to the cytosol
• Converted to F6P then sucrose
• Accumulates at the start of the day faster then used but then synthesis slows down + is used up
• Rate of sucrose synthesis has to be coordinated w/ photosynthesis (if sucrose made faster than photosynthesis, less C3P, less RU1,5Bp, less photosynthesis) (if slower, inorganic Pi ↓, so less ATP so x make triose P)

Starch

• In chloroplast
• F6P converted to ADPGlc then starch
• Slowly ↑ then declines as degraded as respiratory substrate

3-PGA can be retained in chloroplast to make starch or exported to sucrose

Question 135

Sucrose regulation

Look at feedworward flashcard + F2,6BP

Answer 135

• At end of day, high sucrose. Have mechanism for restricting further synthesis
• Means ↑ F2,6Bp which inhibits FBPase
• Triose-P exported from chloroplast accumulate
• Have ↓ inorganic Pi in cytosol, needed to be imported to chloroplast in exchange for triose-P. Limits export + can limit max rate of CO2 assimilation
• Need alternative regeneration. AGPase activated: - ↑ triose-P + ↓ Pi in chloroplastlast ↑ in 3PGA/Pi which activates AGPase
• Leads to ↑ starch synthesis, makes inorganic Pi which allows resynthesis of ATP