Plant metabolism ALL Flashcards

Question

Explanation of aldolase activity | / Reversible E

Answer 1

- 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)

Answer 2

- 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

Answer 3

- 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

Answer 4

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

Answer 5

- Regulation occurs at multiple levels | - GS = target for trying to improve N use efficiency, if overexpress GS, ↑ capture of ammonium + ↑ efficiency

Answer 6

- 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

Answer 7

- 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

Answer 8

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

Answer 9

- 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

Answer 10

- 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

Answer 11

- ↑ 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

Answer 12

- 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

Answer 13

- 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

Answer 14

- 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

Answer 15

- 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

Answer 16

- 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

Answer 17

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

Answer 18

- 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

Answer 19

- 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

Answer 20

- 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

Answer 21

- 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

Answer 22

- 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

Answer 23

- 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

Answer 24

- 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

Answer 25

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

Answer 26

- 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

Answer 27

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

Answer 28

- 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

Answer 29

- 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

Answer 30

- 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

Answer 31

- 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

Answer 32

- 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

Answer 33

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

Answer 34

- 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

Answer 35

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

Answer 36

- 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

Answer 37

- 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

Answer 38

- 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)

Answer 39

- 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

Answer 40

- 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

Answer 41

- 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

Answer 42

- 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

Answer 43

- 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

Answer 44

- 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

Answer 45

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

Answer 46

- 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

Answer 47

- 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

Answer 48

- 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

Answer 49

- 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.

Answer 50

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

Answer 51

- 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

Answer 52

- 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

Answer 53

- 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

Answer 54

- 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

Answer 55

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)

Answer 56

- 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

Answer 57

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.

Answer 58

- 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

Answer 59

- 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

Answer 60

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

Answer 61

- 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

Answer 62

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

Answer 63

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

Answer 64

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

Answer 65

- 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

Answer 66

- 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

Answer 67

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

Answer 68

- 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

Answer 69

- 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

Answer 70

- 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)

Answer 71

- 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

Answer 72

- 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

Answer 73

- 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

Answer 74

- 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)

Answer 75

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

Answer 76

- Provides Rubisco w/ ↑ CO2 for carboxylase reaction | - Suppresses oxygenase activity, overall v efficient C fixation

Answer 77

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)

Answer 78

- 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.

Answer 79

- 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

Answer 80

- ↑ 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

Answer 81

- 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

Answer 82

- 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

Answer 83

Advantages - C4 photosynthesis effectively eliminates photorespiration, more water/nitrogen use efficient + permits growth under ↑ temperature conditions Disadvantages - ↑ energetic cost + intrinsic cold sensitivity

Answer 84

- 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

Answer 85

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

Answer 86

- 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

Answer 87

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

Answer 88

- 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

Answer 89

- 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

Answer 90

- 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)

Answer 91

- 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

Answer 92

- 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

Answer 93

- 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

Answer 94

- 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

Answer 95

- 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

Answer 96

- 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

Answer 97

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

Answer 98

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

Answer 99

- 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)

Answer 100

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

Answer 101

- 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

Answer 102

- 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

Answer 103

- 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

Answer 104

- 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...

Answer 105

- 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

Answer 106

- 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

Answer 107

- 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

Answer 108

- 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

Answer 109

- 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

Answer 110

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

Answer 111

- 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

Brainscape's Knowledge GenomeTM

Plant metabolism ALL Flashcards

Brainscape's Knowledge Genome^TM