Photosynthesis

The carbon reactions (Dark Reactions)

Overall Perspective• Light reactions:

– Harvest light energy– Convert light energy

to chemical energy

• Dark Reactions:– Expend chemical energy– Fix Carbon [convert CO2

to organic form]

At the end of the light reactions

• The reaction of the light reaction is:– CO2 +H2O (CH2O) + O2

• Recent estimates indicate that about 200 billion tones of CO2 (Mr = 44) are converted to biomass each year– 40 % of this is from marine

phytoplankton– The bulk of the carbon is incorporated

into organic compounds by the carbon reducing reactions (dark reactions) of photosynthesis

At the end of the light reactions• The reactions catalyzing

the reduction of CO2 to carbohydrates are coupled to the consumption of NADPH and ATP by enzymes found in the stroma– fluid environment

• These reactions were thought to be independent of the light reactions– So the name “dark

reactions” stuck

• However, these chemical reactions are regulated by light– So are called the “carbon

reactions” of photosynthesis

Overview of the carbon reactions

• The Calvin cycle:• Stage 1:

– CO2 accepted by Ribulose-1,5-bisphosphate.

– This undergoes carboxylation• Has a carboxyl group

(-COOH) attached to it

– At the end of stage 1, CO2 covalently linked to a carbon skeleton forming two 3-phosphycerate molecules.

Carboxylation: The first step is the most important

• Step 1: The enzyme RUBISCO (Ribulose bis-phosphate carboxylase oxygenase) carries out this conversion

• Rubisco accounts for 40% of the protein content of chloroplasts – is likely the most abundant protein on Earth

• Rubisco is, in fact, very inefficient, and that a mechanism has evolved to deal with this handicap

Overview of the carbon reactions• The Calvin cycle:

• Stage 2:– Each of the two 3-

phosphycerate molecules are altered.

– First phosphorylated through the use of the 3 ATPs generated during the light reaction.

– Then reduced through the use of the 2 NADPHs generated during the light reaction.

– Forms a carbohydrate• glyceraldehyde-3-

phosphate

3-phosphycerate molecules are altered

• First phosphorylated through the use of the 3 ATP molecules generated during the light reaction– Forms 1,3-bisphosphoglycerate

• Then reduced through the use of the 2 NADPH molecules generated during the light reaction– Forms glyceraldehyde-3-phosphate

• Note the formation of triose phosphate

Overview of the carbon reactions

• The Calvin cycle:• Stage 3:

– Regeneration of Ribulose-1,5-bisphosphate.

– This requires the coordinated action of eight reaction steps

• And thus eight specific enzymes

– Three molecules of Ribulose-1,5-bisphosphate are formed from the reshuffling of carbon atoms from triose phosphate.

Regeneration of Ribulose-1,5-bisphosphate

• The Calvin cycle reactions regenerate the biochemical intermediates needed for operation

• More importantly, the cycle is Autocatalytic– Rate of operation can be enhanced by increasing

the concentration of the intermediates in the cycle

• So, Calvin cycle has the metabolically desirable of producing more substrate than is consumed– Works as long as the produced triose phosphate is

NOT diverted elsewhere (as in times of stress or disease)

Overview of the carbon reactions

• The Calvin cycle:• The cycle runs six

times:– Each time incorporating a

new carbon . Those six carbon dioxides are reduced to glucose:

– Glucose can now serve as a building block to make:• polysaccharides • other monosaccharides • fats • amino acids • nucleotides

Only one-sixth of the triose phosphate is used for

polysaccharide production• Synthesis of polysaccharides, such as starch

and sucrose, provide a sink – Ensures an adequate flow of carbon atoms

through the cycle IF CO2 is constantly available

• During a steady rate of photosynthesis 5/6 of the triose phosphates are used for the regeneration of Ribulose-1,5-bisphosphate

• 1/6 is transported to the cytosol for the synthesis of sucrose or other metabolites that are converted to starch in the chloroplast

Regulation of the Calvin cycle

• The high energy efficiency of the Calvin cycle indicates that some form of regulation ensures that all intermediates in cycle:– Are present at adequate concentrations– The cycle is turned off when it is not

needed in the dark

• Remember:– These are the “carbon reactions”, NOT the “dark

reactions”

• Many factors regulate the Calvin cycleMany factors regulate the Calvin cycle

Regulation of the Calvin cycle

• 1: The pH of the stroma increases as protons are pumped out of it through the membrane assembly of the light reactions.– The enzymes of the Calvin Cycle function

better at this higher pH.

• 2: The reactions of the Calvin cycle have to stop when they run out of substrate– as photosynthesis stops, there is no more

ATP or NADPH in the stroma for the dark reactions to take place.

Regulation of the Calvin cycle

• 3: The light reactions increase the permeability of the stromal membrane to required cofactors – Mg ions are required for the Calvin Cycle.

• 4: Several enzymes of the Calvin Cycle are activated by the breaking of disulphide bridges of enzymes involved in the working of the cycle. – the activity of the light reactions is

communicated to the dark reactions by an enzyme intermediate

When conditions are not optimum

Photorespiration • Occurs when the CO2 levels inside a leaf

become low – This happens on hot dry days when a plant is

forced to close its stomata to prevent excess water loss

• If the plant continues to attempt to fix CO2 when its stomata are closed– CO2 will get used up and the O2 ratio in the leaf

will increase relative to CO2 concentrations

• When the CO2 levels inside the leaf drop to around 50 ppm, – Rubisco starts to combine O2 with Ribulose-1,5-

bisphosphate instead of CO2

Photorespiration• Instead of producing 2 3C

PGA molecules, only one molecule of PGA is produced and a toxic 2C molecule called phosphoglycolate is produced

• The plant must get rid of the phosphoglycolate

• The plant immediately gets rid of the phosphate group – converting the

molecule to glycolic acid

Photorespiration• The glycolic acid is then

transported to the peroxisome and there converted to glycine– Peroxisomes are

ubiquitous organelles that function to rid cells of toxic substances

• The glycine (4 carbons) is then transported into a mitochondria where it is converted into serine (3 carbons)– Releases CO2

Photorespiration• The serine is then used to

make other organic molecules

• All these conversions cost the plant energy and results in the net lost of CO2 from the plant

• 75% of the carbon lost during the oxygenation of Rubisco is recovered during photorespiration and is returned to the Calvin cycle

The C4 Carbon cycle

The C4 carbon Cycle• The C4 carbon Cycle occurs in 16 families

of both monocots and dicots. – Corn– Millet– Sugarcane– Maize

• There are three variations of the basic C4 carbon Cycle – Due to the different four carbon molecule

used

The C4 carbon Cycle• This is a biochemical

pathway that prevents photorespiration

• C4 leaves have TWO chloroplast containing cells– Mesophyll cells– Bundle sheath (deep in the leaf so

atmospheric oxygen cannot diffuse easily to them)

• C3 plants only have Mesophyll cells

• Operation of the C4 cycle requires the coordinated effort of both cell types– No mesophyll cells is more than

three cells away from a bundle sheath cells

• Many plasmodesmata for communication

The C4 carbon Cycle• Four stages:• Stage 1:• In Mesophyll cell

– Fixation of CO2 by the carboxylation of phosphenol-pyruvate (primary acceptor molecule)

– forms a C4 acid molecule– Malic acid and/or aspartate

• Stage 2:– Transport of the C4 acid

molecule to the bundle sheath cell

The C4 carbon Cycle• Stage 3:

– Decarboxylation of the C4 acid molecule (in bundle sheath)

– Makes a C3 acid molecule – This generates CO2 – This CO2 is reduced to

carbohydrate by the Calvin cycle

• Stage 4:– The C3 acid molecule

(pryuvate) is transported back to mesophyll cells

– Regeneration of phosphenol-pyruvate

The C4 carbon Cycle• Regeneration of phosphenol-pyruvate consumes two high

energy bonds from ATP• Movement between cells is by diffusion via plasmodesmata • Movement within cells is regulated by concentration

gradients • This system generates a higher CO2 conc in bundle sheath

cells than would occur by equilibrium with the atmosphere– Prevents photorespiration!!!!!!!!!!

The C4 carbon Cycle• The net effect of the C4 carbon Cycle is to

convert a dilute CO2 solution in the mesophyll into a concentrated solution in the bundle sheath cells– This requires more energy than C3 carbon

plants

• BUT – This energy requirement is constant no matter what the environmental conditions

• Allows more efficient photosynthesis in hotter conditions

Crassulacean Acid Metabolism

(CAM Plants)

CAM Plants• The CAM mechanism enables plants to

improve water efficiency– CAM plant

• Loses 50 – 100 g water for every gram of CO2 gained

– C4 plant• Loses 250 – 300 g water for every gram of CO2 gained

– C3 plant• Loses 400 – 500 g water for every gram of CO2 gained

• Similar to C4 cycle– In CAM plants formation of the C4 acid is both

temporally and spatially separated

CAM Plants• At night:• Stomata only open at night

when it is cool

• CO2 is captured by phosphenol-pyruvate carboxylase in the cytosol – leaves become acidic

• The malic acid formed is stored in the vacuole– Amount of malic acid

formed is equal to the amount of CO2 taken in

CAM Plants• During the day:• Stomata close, preventing water

loss, and further uptake of CO2

• Malic acid is transported to the chloroplast and decarboxylated to release CO2

• This enters the Calvin cycle as it can not escape the leaf– Pyruvate is converted to starch in

the chloroplast – regenerates carbon acceptor

Phosphorylation regulates phosphenol-pyruvate (PEP)

carboxylase• CAM and C4 plants require a separation of the initial carboxylation from the following de-carboxylation

• Diuranal regulation is used

• IN CAM PLANTS:-• Phosphorylation of the serine

residue of phosphenol-pyruvate (PEP) carboxylase (Ser-OP) yields a form of the enzyme which is active at night– This is relatively

insensitive to malic acid

Photophorylation regulates phosphenol-pyruvate

(PEP)carboxylase• During the day:

• De-Phosphorylation of the serine (ser-OH) gives a form of the enzyme which is inhibited by malic acid

• THIS IS THE OPPOSITE WAY AROUND FOR C4 PLANTS!

Summary• The reduction of CO2 to carbohydrate via

photosynthesis is coupled to the consumption of ATP and NADPH

• CO2 is reduced via the Calvin cycle – Takes place in the stroma (soluble phase)

• CO2 and water combine with Ribulose-1,5-bisphosphate in the following reaction – CO2 +H2O (CH2O) + O2

• Regeneration of the carrier is required for the cycle to continue

Summary• The Calvin cycle requires the joint action

of several light-dependant systems– Changes in ions (Mg+ and H+)– Changes in effector metabolites

(enzyme substrates)– Changes in protein-mediated systems

(rubisco activase)

• Rubisco can also act as an oxygenase– The carboxylation & oxygenation

reactions take place at the active sites of rubisco.

Summary• C4 and CAM plants Prevent

photorespiration!!!!!• C4 leaves have TWO chloroplast containing

cells– Mesophyll cells– Bundle sheath

• CAM Plants drastically reduce water lass– CAM plant

• Loses 50 – 100 g water for every gram of CO2 gained

– C4 plant• Loses 250 – 300 g water for every gram of CO2 gained

– C3 plant• Loses 400 – 500 g water for every gram of CO2 gained

Any Questions?