Plant lecture extra

Question 1

Rubisco activase

Answer 1

• 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 2

PSI/PSII Overview

Answer 2

• 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 3

PSI/PSII structure

Answer 3

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 4

Reasons for coordinating activities of PSI+2

Answer 4

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 5

State transitions (short term adaptation)

Answer 5

• 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 6

State transition function hypotheses

Answer 6

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 7

Long term response to light imbalance

Answer 7

• 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 8

Calvin cycle overview

Answer 8

• 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 9

Contribution of Rubisco to control of Calvin cycle

Answer 9

• 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 10

Other ways of controlling Calvin cycle

Coordinating a balance btw starch + sucrose synthesis

Answer 10

• 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 11

Bypass 1

Answer 11

• 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 12

Bypass 2 / Carvalho bypass

Answer 12

• 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 13

Bypass 3/ Maier

Answer 13

• 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 14

Other bypasses

Answer 14

• 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 15

General photorespiratory bypasses

Answer 15

• 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