Optimising carbon fixation 1 LECTURE 3

Once CO2 has been delivered to the chloroplast stroma

• reacts with the primary caboxylase→ Rubisco

When did Rubisco evolve

• 3.6-3.8 billion years ago

• before the atmosphere became oxygenated

  • 2.4 bya

Rubisco features

• most abundant protein on Earth

• accounts for up to 50% of the total soluble protein in a leaf

• corresponds to 10kg of protein per person

• L8S8 version→ 550kiloDaltons

  • one of the largest most complex enzymes known

Variety of Rubisco strucutres

• some variation in structure and function

But what version do plants and cyanobacteria use

L8S8 version:

• 4 pairs of large subunits

• 4 small subunits

  • on top and below to hold it together

• each pair of subunits sits top to tail

  • enclose two active sites

Biosynthesis of Rubisco invovles:

Multiple steps of

1. transcription

2. translation

3. assembly of subunits

Function of Rubisco: even though pressure to select CO2 over O2 for the past 2 billion years

Rubisco still only poorly distinguishes one from the other!

• compete for the activated sites on Rubisco

  → carboxylase : oxygenase events

@ 20 degrees and atmospheric oxygen levels (21%)

carboxylase: oxygenase

4:1

Value of Km for Co2

9 microMol but [CO2] is 0.04% in present day

→ means that Rubisco can only ever work at half maximum velocity

How Carboxylase part of Rubisco works

1. combines CO2 with a 5 carbon compound

   • RuBP→ Ribulose 1,5-biphosphate

2. Creates 3-PGA

   • phosphoglyceric acid

How Oxygenase Rubisco works

No carbon assimilation

1. Fixes O2 to RuBP

2. Makes 2-phosphoglycolate (PG)

But phosphoglycolate is inhibits some enzymes in the Calvin cycle

Must be removed and made into molecules that are compatible with plant metabolism→ Photorespiration

Photorespiration: What organelles are involved

1. peroxisomes

2. mitochondria

3. many others

This is so the PG can be removed so it doesn’t effect the calvin cycle enzymes

Photorespiration: What happens?

1. PG is converted into PGA

   • releases CO2

   • Uses ATP and NADPH

2. Converted back to RuBP

   • Uses ATP and NADPH

overall: recycles the toxic waste→ releases CO2 in the light

Why is this allowed to happen?

1. Cannot distinguish between CO2 and O2 very well

2. Maybe it is advantageous?→ linked to

   • Sources of Serine

   • Role in nitrogen assimilation

3. Too difficult to change a highly conserved and massive protein

   • one mutation/change could result in failure

     → no longer viable

     • important role in photosynthesis

       • very costly if wrong change was made

Why is photorespiration bad for plant!

1. Energetically expensive

2. Product inhibits Calvin cycle enzymes

3. Stop CO2 taken up

4. Also releases CO2

Overall really decreases overall CO2 fixation and Calvin cycle

• photosynthesis is down→ cannot grow as much etc…

Blackman’s limiting factor analysis can be used to compare the sensitivity and operating efficiency of Rubisco

Plotting Carbon assimilation rates as a function of CO2 concentration

In order to remove the effects of stomatal limitation

Plot

y→photosynthetic CO2 assimilation rate

• A, micro.mol CO2 m-2s-1

against

x→ mesophyll gas phase CO2 concentration

• Ci, ppm or micromol mol-1

Analysis of this plot

Initial slope

• revealed the carboxylation efficiency of Rubisco

X-axis Intercept

• give CO2 compensation point

  • where A=0 and Co2 uptake is balanced by (photo)-respiration release)

  • →is a measure of oxygenase activity!

Because Rubisco catalysis is sensitive to O2

• When decrease O2, there is a reduction in oxygenase activity!

  → leads to more CO2 assimilation

At low O2 concentration→ graph @ 1.9% vs 20%

1. Reduced oxygenase activity

2. Carboxylase Efficiency (Rubisco activity) Increased

3. CO2 Compensation point reduced: from 40-10

   • Meaning that less CO2 concentration is needed to get CO2 assmilation to meet CO2 loss

     → Less CO2 lost because photorespiration is lower!

4. Max assmilation is higher

5. A Decrease Assilimation at high CO2 (at the end of the graph)

Why is there decreased assimilation at high CO2?

• Due to PO43- limitations in the stroma

  • i.e something else becomes the limiting factor!

Therefore to limit oxygenase activity and photorespiration:

• Need to lower O2

• Increase CO2 vs O2

  → concentrate CO2

→ This is the strategy used by C4 and CAM plants to icrease the efficiency of Rubisco

So does increasesing CO2 in the atmosphere help photosynthesis then?

Last 200 years, 280 ppm → 400ppm

BUT→ also comes with

• increased temp

• drought

• atmospheric pollution

OVERALL: outweighs the benefits for the productivity of natural vegetation and crops

Balancing the benefits and negatives

Depends on the

• species

• region

Issue with increased temperatures and drought

Warmer and water is limiting:

• Photorespiration is more costly

Due to this

There has been convergent evolution to tackle this:

All using carbon concentrating mechanisms to increase the efficiency of carboxylation and water use

1. Cyanobacteria

   • Concentrate CO@ in carboxysomes

2. Algae

   • Concentrate CO2 in pyrenoids within their chloroplasts

3. Plants

   • C4 and CAM plants

The C3 pathway→ usual photosynthesis

1. Rubisco fix CO2 to Ribuloe Biphosphate

2. Makes 9-carbon intermediate

3. Cleaved into two 3 carbon molecules (phospholyceric acid) PGA

   • This is the 3 carbon major product (C3)

4. Reduced to glyceraldehyde 3-phosphate (G3P)

   • useing ATP and NADPH

5. Regenerated into RuBP with ATP

C3 plants

1. Tomatoes

2. Barley

3. Wheat

4. Potatoes

5. Soybean

Many staple diets worldwide

Where are C3 plants found

• temperate biomes

• tropical forest biomes

C4 Plant pathway (general)

1. CO2 converts into carbonic acid

   • with carboninc anhydrase

2. This is fixed to phosphoenolpyruvate (PEP) into Oxaloaxetate (OAA) using PEP carboxylase (PEPC)

3. OAA rapidly converted int malate or aspartate

   • This is the C4 primary product

4. This C4 is then moved, decarboxylated and the CO2 off of this is used by Rubisco for the normal Calvin cycle process

How C4 mechanism concentrates CO2

Spatial Separation

1. C4 intermediates made in the mesophyll

2. Transported to bundle sheath cells

3. Decaboxylated here

4. So the Rubisco in the bundle sheath cells are surrounded by a higher concentration of CO2

→ Leads to concentrations of 1-2%

Overall: means that the rubsico has less O2, so less oxygenase reaction, less photorespiration and more photosynthesis!

Types of C4 plants

Found in 18 families

1. Grasses:

2. Corn (Maize, Zea mays)

3. Sugar cane

4. Sorgum

5. Pearl Millet

6. Foxtail millet

7. Tef

3% of angiosperms are C4

→ 25% of terrestrial productivity!

C4 pathway (in depth)

1. Fixation

2. Transport

3. Decarboxylation

4. Transport and Regeneration

1. Fixation

Mesophyll cells

1. Carbonic anhydrase CO2→ HCO3-

2. PEPC (found in cytoplasm) activated in the light via phosphorylation of specific kinase

3. Makes C4 malate and aspartate

   • PEP +HCO3 → OAA

   • OAA→ malate and aspartate

Why is PEPC used?

• Much higher affinity (low Km) for inorganic carbon

• Compared to Rubisco

→ maintains a steep diffusion gradient for CO2 into the leaf!

• Lots of CO2 can get in→ great for concentrating the CO2 inside

2. Transport

1. Malate and aspartate moved to bundle sheath

2. Via symplastic connections→ plasmodesmata

3. Decarboxylation

1. Regenerates CO2 from C4 organic acids

2. Forms 1-2% CO2 concentration in the bundle sheath

3. Rubisco is therefore much more efficient now

   • photorespiration completely suppressed

4. initiates the Calvin cycle

4. Transport and regeneration

1. C3 residue diffused back to mesophyll cells

Why?

• Needs to be regenerated back into PEP so the mesophyll cells have something PEPC can convert again

• Uses:

  • pyruvate phosphate dikinase (PPDK)

  • ATP

To achieve this spatial separation

• Have kranz “wreath” anatomy

Kranz Wreath anatomy

1. Bundle sheath surrounding vascular tissue

   • Thick walled

   • suberized→ impregnate with suberin in cell wall

     → Increase space for photosynthesis

2. Many plasmodesmata

   • connecting mesophyll → bundle sheath

3. Higher Vein density to C3 plants

   → coz more products made??

4. More chloroplasts in bundle sheath

   • Than C3 plants

     → coz more photosynthesis happening?

     → less in mesophyll so more PEPC than rubisco so can concentrate CO2 more?

What this enables

• Efficient transport of metabolise between cells

• Efficient carbon fixation by Rubisco in the bundle sheath

Why called a wreath?

Advantages of suppressing photorespiration

1. Energetics→ cheaper at higher temp

2. CO2 compensation point→ lower

3. Efficiency of light use (quantum yield)→higher

4. Water Use→ higher efficiency

1. Energetics→ How costly is C4?

• Regeneration in C4 costs 2 ATP per CO2 used

THEREFORE: more expensive than C3 pathway (if photorespiration did not occur)

1. Enegetics→ C3 vs C4 at 20 degrees

• similar energetic ATP costs

1. Energetics→ As temp increases from C3 to C4

• Carboxylase rate doubles

But

• Oxygenase rate trebles

In C3 plants

1. Energetics→ when is C4 more energetically beneficial?

• Photorespiration increases with temperature

• But C4 costs do not increase with temperature

• So at higher temperatures, C4 becomes less costly than C3, per CO2 assimilated

2. CO2 compensation point

• C3→ 50-70ppm

  • and increases with temperature

• C4→ 0-10 ppm

  • lower compensation point

  • Coz less CO2 is being lost

  • Do not need to compensate at much!

3. Efficiency of light use→ Initial slope of graph

Quantum yield:

• Higher for C4 plants

  → fix more CO2 per photon absorbed

4. Water use

• PEPC higher affinity for CO2

• Stomata do not need to be as wide to get enough CO2 in

• So Stomata have lower conductance

  • not easy for gases to move in and out

  • Because stomata closed

• For the same CO2 assimilation rate

• Means:

  • Higher water use (assimilation/evaporation)

  • Less water lost!

4. Water use→ ecological advantage

E.g Grasses:

• Found in fire-dominated Savannah regions

  • tropics

• Tall grass in

  • high latitude

  • North America

Where C4 normally found

• hot summers

• tropical regions

C4 plants in the UK?

Probably not the best climate for it:

• typically found in association with hot summers or tropical regions

→ Only 4 species are native

1. Salsola kali

2. Spartina X

3. Cyperus longus

4. Atriplex lacinata

Growing C4 crops in the UK?

1. Maize:

   • often sickly

   • chlorotic

   → Not great

2. Miscanthus

   • Offers the prospect of biofuels for the future

When C4 pathway evolved?

• 20 mya

• When CO2 levels fell to around 280ppm

  • When huge climate change due to uplift of the himalayan plateau

How many times has it evolved?

• 60 times independently in angiosperms

What this suggests?

• Might be quite an easy step to make?

Is evolving from C3 to C4 easy?

• Found correlation between bundle sheath physiology in C4

to

• prescence of high activity of enzymes used for decarboxylation in cells around vascular system in C3 (in tobacco)

• showed the products decarboxylated in the regions around the stem and then fixed carbon exported to the growing apices

→ Showed there are similar biochemistry found in the bundle sheath and the pathway just had to be rewired

Future of C4 plants

• Want to make Rice a C4 plant

• Needed for food source

• Feeds the most people

  → Make C4 to increase the yield