Photosynthesis: the light-

independent reactions

Biol 3470

Plant Physiol Biotech

5.5 to 5.12

Lecture 9

Thurs. Feb. 2, 2006

From Rost et al., “Plant Biology”, 2nd edn

The process of carbon fixation in plants goes by many names

• Including:– The dark reactions– The enzymatic reactions of photosynthesis– Reductive pentose phosphate cycle– C3 cycle– The photosynthetic carbon reduction (PCR)

cycle (in the textbook)

The PCR cycle converts atmospheric carbon to organic molecules

• Convert CO2 to stable phosphorylated carbon intermediates (specifically- a three-carbon carbohydrate, 3-PGA)

• Uses the energy produced in the light-dependent reactions to reduce CO2

– Convert less complex → more complex molecules– Fight entropy

• Pathway elicited in late 1940s and early 50s by U.S. plant physiologist Melvin Calvin using labeled 14CO2 – feed plants 14CO2 – Allow metabolism– Kill, extract, examine small carbohydrates that contain 14C using

paper chromatography and autoradiography

The PCR cycle contains 3 distinct segmentsStep 1: Carboxylation fixes CO2

using the enzyme rubisco• 14CO2 fixed first into 3-

phosphoglycerate (3 Cs ≡ C3 cycle)– 3-PGA is the first organic product of

the PCR cycle

• Given this product and reactant, one would assume the plant substrate of the PCR cycle would have ___Cs

1

3 2

• Actually, the plant substrate for the PCR cycle is a five-carbon substrate, RuBP

Fig. 5.8

Fig. 5.9

Unstable intermediate is hydrolyzed

(2x )

Rubisco reaction

The key enzyme regulating carbon uptake by the PCR cycle is rubisco

Rubisco• Enzyme with a high affinity for CO2• Present in high amounts in the chloroplast stroma• Its activity maintains a CO2 gradient from the atmosphere • ΔGº′ = -35 kJ/mol (energetically favourable to occur

spontaneously)• But its activity requires ATP + NADPH made in the light

reactions elsewhere in PCR cycle

• e.g. in 2nd step: reduction of 3-PGA to G3P

• This is Step 2: Reduction

Fig. 5.10

Phosphorylate!

Reduce!

The final step in the PCR cycle regenerates the rubisco substrate

• This is accomplished via Step 3: Regeneration– Requires 1 ATP per CO2

• Note that the PCR cycle is autocatalytic– This means that it operates more quickly if

CO2 and/or RuBP pools are low (e.g. in the morning, when the RuBP supply is depleted)

PCR cycle activity must be integrated with plant carbon metabolism as a whole

• These include respiration (glycolysis) and macromolecule synthesis (for nucleic acids, lipids, carbohydrates, proteins)

• Thus, the PCR cycle activity must be regulated by a number of mechanisms• The plant wants to keep CO2 fixation rate high to make more organic carbon

Fig. 5.11

Rubisco

Auto

cata

lytic

RuB

P

rege

nera

tion

2. Reduction

3. Regener-ation

1. Carbox-ylation

• The carbon from 5 of every 6 molecules of G3P needs to be recycled to make RuBP and keep the cycle spinning

• Only around one-sixth of the carbon fixed is exported from the leaf and supports growth and metabolism

• To keep high CO2 fixation, the plant can prevent G3P export

The PCR cycle consumes the ATP and NADPH produced in the light-dependent reactions

Mechanisms of regulation of PCR cycle activity

• The activity of rubisco is regulated by light• Complex mechanism driven by uptake of protons by

thylakoid lumen between1. Mg2+→ moves lumen → stroma to compensate for H+ uptake by

thylakoids in light inactive active

(light)

Dark stroma pH = 5.0Light

Fig. 5.14

• Stromal pH ↑ activates rubisco

2. CO2 → binds to activating site on rubisco (not active site!) ≡ CARBAMYLATION

3. pH increase favours carbamylation (H+ sink in lumen)

Rubisco is now catalytically ready to fix atmospheric CO2!

Plant cells also respire: convert O2→CO21. Via mitochondiral respiration at night

– This is oxidative phosphorylation to generate ATP in the dark (R on diagram)

– This also happens in the light!

2. Via rubisco → can use O2 as a substrate in photorespiration (PR)

• Therefore, measuring NET gas exchange in photosynthetic organs is difficult!

• We can define an apparent photosynthesis rate

= CO2 fixation rate – CO2 evolution rate =gross p’syn – (mt R + PR)

• At a low atmospheric [CO2] these values (GP) and (R+PR) are equal

• this is the CO2 compensation point

Fig. 5.16

(mt)

(mt)

(Rubisco O2-ase)

(Rubisco CO2-ase)

(mt)

Photorespiration is due to rubisco’s oxygenase activity

• Makes 2-phospho-glycolate (2C) + 3-PGA from RuBP + O2

• This C in 2-P-glycolate not wasted but reassimilated by exchange of intermediates with 2 other organelles– Peroxisome– Mitochondria

Fig. 5.18

Exported or recycled to regenerate RuBP

The function of photorespiration is not immediately obvious

Energetically wasteful, so why do it? Thoughts and theories…

1. [O2] in atmosphere has been low during most of evolutionary history

• Therefore PR is an evolutionary relic?• No! PR mutants are lethal! → Therefore, PR is essential• No evolutionary pressure to get rid of O2-ase function

2. The salvage cycle does a good job of recovering photorespired C

• Each 2 turns of the 2-P-glycolate salvage cycle forms 4 3-PGA• 1 lost, 3 returned to the PCR cycle• Complex salvage pathway works well!

3. Metabolic safety valve?• PR protects against photoxidative damage by allowing P.E.T. to

continue at low [CO2]• e.g., under high light + low CO2 (photoinhibitory conditions, stomata

closed, water-stressed)

The chloroplast oxidative pentose phosphate cycle allows plants to make NADPH in the dark

• Shares intermediates with PCR• Both at once: FUTILE CYCLE!

– Use 3 ATP

– No CO2 fixation!

• Both pathways are light-regulated

PCR

a/k/a RPPC

OPPC

• Light induces changes the structure of the disulfide bonds of the pathways’ enzymes– PCR cycle enzymes active when reduced– OPPC cycle enzymes active when oxidized

Light Dark

PCR enzymes

√ X

OPPC enzymes

X √

Fig. 5.20

•Why have an OPPC?–Make NADPH in dark–Make ribose and deoxyribose for nucleic acid synthesis

1. Mesophyll• fewer chloroplasts

2. Bundle sheath cells• lots of chloroplasts• surround vascular tissue• thick cell walls prevent

diffusion of CO2 out of BS cells and traps photorespired CO2

• No mesophyll cells are more than 2-3 cells away from BS

• This ensures quick export of fixed CO2 as sucrose

• Many chloroplasts needed to fix high [CO2]

How can plants minimize PR and maximize GP?Plants are separated into 2 main groups based on their ability to do this:• Plants where 3-C 3-PGA is product of CO2 fix’n = C3• Plants where 4-C oxaloacetate is product of CO2 fix’n = C4• C4 plants have 2 distinct photosynthetic tissues

– Leaf anatomy differs from C3 leaf

Fig. 5.21: C4 leaf X-section (e.g., maize)

C4 plants are present in all 18 plant families• This includes flowering plants as wellC4 plants are:• Better at CO2 fixation (up to 3X more efficient)• Better at drought stress• Concentrate CO2 at rubisco active site and thus minimize

CO2 loss!• How do they do this?

– Fix CO2 into C4 organic acid in the mesophyll cell using PEP carboxylase (not rubisco!)

– Use a transporter to move the acid into the bundle sheath cell

– Release CO2 there

– Fix CO2 via PCR

– Recycle C3 acid released (pyruvate) back to mesophyll

PEP carboxylase malate malate

pyruvatepyruvate

Malic Enzyme

Fig. 5.22: the C4 carbon fixation pathway

• C4 metabolism pluses:– CO2 outcompetes O2 at

rubisco active site: Less PR! – Much lower compensation

point• maintain high CO2 fixation rates

when stomata are partially closed → conserves H2O

– Lower transpiration ratio = less H2O transported per CO2 assimilated

Using the C4 pathway to fix carbon is not always an advantage for the plant

• C4 metabolism minuses:– Need to “spend” 2 ATP per CO2 to recycle C3 acid back to the

mesophyll cells• C3 plants often have an ecological advantage

– Grow better in cooler climates and low irradiance– Higher CO2 assimilation rate in environments with lots of water

How do plants grow in the desert?• Use CAM metabolism: conserves

H2O• CAM plants have an inverted

stomatal cycle – Night: open– Day: closed

• Therefore CO2 uptake at night → accumulate malate in vacuole

• During day → convert malate to starch via PCR cycle

• Need PEPC as in C4 photosynthesis– Requires lots of PEP (PEPC

substrate), provided from glycolytic breakdown of starch

• CAM is similar to C4, but:– No specialized anatomy (specialized

cell types)– No closed cycle of carbon

intermediates

Night Day

PEPC

Large, watery

Decarbox-

ylation

Malic

enzyme

export

Fig. 5.26

CAM plants are evolutionarily adapted to live in low water environments

• CAM plants have even lower transpiration ratios than C4 plants BUT– Only fix <1/2 C of C3 and <1/3

of C4 plants → slow growers– But can

• continue CO2 uptake under H2O stress

• Reassimilate respired CO2

• Some plants can “switch on” CAM metabolism (facultative vs. obligatory)

From http://www.arizonensis.org/images/plantae/cereus_gigant.jpg