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CARBON FIXATION. THE LIGHT INDEPENDENT REACTION. THE LIGHT INDEPENDENT REACTION. Enzyme controlled (Q 10  2) Located in the stroma of the chloroplast Occurs simultaneously with the light dependent reaction

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CARBON FIXATION


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carbon fixation

CARBON FIXATION

THE LIGHT INDEPENDENT REACTION

the light independent reaction
THE LIGHT INDEPENDENT REACTION
  • Enzyme controlled (Q10 2)
  • Located in the stroma of the chloroplast
  • Occurs simultaneously with the light dependent reaction
  • It can continue in the dark provided the necessary raw materials are available (CO2, NADPH + H+ and ATP)

© 2010 Paul Billiet ODWS

enzyme controlled reaction pathways
Enzyme controlled reaction pathways

A B C D E F

Ee

Ea

Ec

Ed

Ea

X = Precursor (raw material) added here

© 2010 Paul Billiet ODWS

enzyme controlled reaction pathways4
Enzyme controlled reaction pathways

To find out the sequence of the reactions and the point at which Xis added in, two approaches can be used:

  • Label and trace the products formed through time
  • Cut the supply of X and observe what happens to the intermediates in the pathway

e.g. in studying photosynthesis, cut the CO2 supply or switch off the lightso cutting the supply of ATP and NADPH+H+

© 2010 Paul Billiet ODWS

calvin and benson 1946 to 1953

Image Credit Scenedesmus

Calvin and Benson 1946 to 1953
  • Used 14C radioisotope for labelling
  • Unicellular algae: Chlorella and Scenedesmus
  • Simple plants which respond quickly to changes in the environment
  • So little time lag

© 2010 Paul Billiet ODWS

the lollipop vessel
The “Lollipop” vessel

A flat-sided, round flask containing the culture of algae

This shape:- provided even illumination of all the cells- permitted careful control of environmental conditions (e.g. pH, temperature)- permitted rapid mixing of contents- precise sampling time

© 2010 Paul Billiet ODWS

labelling and tracing carbon using 14 c
Labelling and tracing carbon using 14C
  • Add NaH14CO3 solution
  • At timed intervals the algae are sampled and killed by dropping in hot methanol
  • Two-way (2-dimensional) chromatography used to separate the compounds
  • Identify radioactively labelled compounds by autoradiography

© 2010 Paul Billiet ODWS

two way 2 dimensional chromatography

A. Mixture placed at the origin

C. Rotate the paper 90°

B.1st run

E. Autoradiograph reveals the compound/s which are labelled with 14C

D. 2nd run

Two-way (2-dimensional) chromatography

© 2010 Paul Billiet ODWS

results

2s

Results

Salisbury & Ross (1969) Plant Physiology Wadsworth

result
Result
  • The compound that became labelled first was Phosphoglyceric Acid (or Phosphoglycerate) PGA
  • PGA is a 3 carbon sugar
  • Therefore: Compound B + CO2 PGA

What is compound B?!!!

© 2010 Paul Billiet ODWS

light independent pathway
Light independent pathway

A B PGA D E F

Ee

Ea

Ec

Ed

Ea

CO2

What is compound B?!!!

© 2010 Paul Billiet ODWS

searching for the compound that combines with carbon dioxide
Searching for the compound that combines with carbon dioxide
  • Feed the algae NaH14CO3 for over 15 min
  • All the compounds involved in photosynthesis are labelled
  • Then reduce the level of NaH14CO3 rapidly

Prediction

  • PGA levels should fall rapidly
  • The compound that fixes CO2 should rise sharply

© 2010 Paul Billiet ODWS

result14
Result
  • This compound was found to be Ribulose Bisphosphate (RBP)
  • RBP is a 5 carbon sugar

Therefore

RBP + CO2 2PGA

5C + 1C = 2x3C

  • The enzyme controlling this important step is Ribulose Bisphosphate Carboxylase (Rubisco)

© 2010 Paul Billiet ODWS

light independent pathway15
Light independent pathway

A RBPPGA D E F

Ee

RUBISCO

Ec

Ed

Ea

CO2

© 2010 Paul Billiet ODWS

rubisco
RUBISCO

A large enzyme 500 000 Daltons

Inhibited by oxygen

A relatively slow enzyme

50% of chloroplast protein

Probably the most abundant protein on Earth

Consists of two subunits (1 large + 1 small)

The large subunit is coded for by a gene on chlDNA

Image Credit: Rubisco stick model

© 2010 Paul Billiet ODWS

carbon fixation and light
Carbon fixation and light
  • The light dependent pathways produce ATP and NADPH+H+ to be used in the light independent processes
  • Illuminate the algae for 15 min then cut the light

Result

  • PGA levels rise
  • Other compounds, after PGA in the pathway, fall

© 2010 Paul Billiet ODWS

carbon fixation and light18
Carbon fixation and light
  • The compound requiring the NADPH+H+ and ATP was found to be Glycerate 3-phosphate (GP)
  • GP is converted to various other three carbon sugars or Triose Phosphates (TP) some are used to produce hexoses such as fructose phosphate and glucose phosphate

© 2010 Paul Billiet ODWS

light independent pathway19
Light independent pathway

A RBPPGAGP E Hexoses

Ee

RUBISCO

Ec

Ed

Ea

12 ATP

12 NADPH + H+

CO2

© 2010 Paul Billiet ODWS

a curious turn

Image Credit: Salisbury & Ross (1969) Plant Physiology Wadsworth

A curious turn
  • When the light is cut interestingly RBP levels also fall
  • This is for two reasons
      • RBP requires ATP in its synthesis
      • RBP is formed from some of the GP
      • In other words the reaction pathway is a CYCLE

© 2010 Paul Billiet ODWS

squaring the circle the calvin benson cycle
Squaring the circle! The Calvin-Benson Cycle

6 RBP

(5C)

6CO2

(1C)

6 ATP

12 PGA

(3C)

Intermediates

12 TP

(3C)

12 GP

(3C)

1 Hexose

(6C)

12 ATP

12 NADPH + H+

photosynthesis overview

CHLOROPLAST

CYTOSOL

Light dependent reaction

O2

H2O

ATP

NADPH+H+

Light independent reaction

CO2

Sugars

Triose phospates

Fattyacids

Amino acids

Starch

PHOTOSYNTHESIS OVERVIEW

© 2010 Paul Billiet ODWS