Dark Reaction

Calvin Cycle
C3, C4 and CAM Plants
Photorespiration
Factors Influencing
Photorespiration
 Calvin Cycle

Also called: the dark reaction, Carbon Fixation, or C3-
cycle.

It is a set of complex reactions that occurs in the stroma.

Uses ATP and NADPH from light reaction to add 1 CO2
to ribulose bisphosphate (RuBP)

To produce one glucose molecule, the cycle must turn 6
times: 6 CO2 1 glucose (C6H12O6)

This cost 18 ATP and 12 NADPH, all of which come
from light-dependent reaction.
CLAVIN CYCLE
Calvin Cycle
Summary of Calvin Cycle
 Three stages of Calvin Cycle
Calvin cycle can be divided into three stages:

1= Carbon fixation stage (CARBOXYLATION)

6 Ribulose bisphosphate (RuBP) + 6 CO2  yield twelve 3-

carbon phosphoglyceric acid (PGA) molecules

2- Synthesis of Glyceraldehyde 3-Phosphate (G3P)

(REDUCTION)

Phosphoglyceric acid (PGA) molecules are converted into
glyceraldehyde 3-Phophate (G3P) molecules

Energy is donated by ATP and NADPH
 Three stages of Calvin Cycle

3- Regeneration of Ribulose bis-phosphate (RuBP)

(REGENERATION)

10 of 12 G3P molecules converted into 6 RuBP molecules

2 of 12 G3P molecules used to synthesize 1 glucose
 First Stage: CO2-Fixation (CARBOXYLATION

The enzyme that catalyzes the first step in Calvin cycle is
Ribulose bisphosphate carboxylase

It is the most important enzyme in nature that carries “True” CO2
fixation reaction

The most abundant protein in the biosphere (~15- 50% of leaf
proteins)

Its overall reaction is believed to proceed
-
as follows:
O
H O
-
-
H P O-
H C O P O- H O CO H C OO
- 2 O - -
HC O PO O O O O
O C O - C C
O C O
H C OH Carboxylase HO C C - H C OH -
H C OH -
+ C OH O O O
- H O C H C O P O-
H C OH O H C OH - H C O P O-
O H C OH - H2O H
H C O P O- O O H O
H H C O P O- H C O P O-
O
H O H O
Ribulose-1,5- Enediolate β-Keto
bis-phosphate intermediate intermediate 3-Phosphoglycerate
 RuBP Carboxylase

RuBP Carboxylase in plants is a very
complex enzyme consisting of:

8 large catalytic subunits (477 residues,
each (blue &cyan)

Encoded by Chloroplast gene

8 small subunits (123 residues, each
(red)).

Encoded by nuclear genes

The large subunit has the catalytic
activity

The small subunit probably modulate the
activity by increasing Kcat. RuBisCO PDB 1RCX

Some bacteria contain only the large The enzyme fixes 10^11
subunit, with the smallest functional
unit being a homodimer, L2. tons of CO2 per year

The enzyme has low catalytic activity Crude oil consumption:
(Kcat=~ 3 S-1)
3*10^9 tons per year
 RuBisCO

RuBP Carboxylase can under certain condition work also as an oxygenase, thereby fixing
O2 instead of CO2, hence the name RuBisCO

The enzyme has much higher affinity for CO2 than O2  the oxygenase reaction is
significant only under conditions in which CO2 levels are low and O2 levels are high

Oxygenase reaction is responsible for Photorespiration (see later).

-
O
H P O- -
H C OO O - O O
O C
O C
CO2 H C OH - H C OH O
-
HO C C O
-
O H C O P O- H C O P O-
H O
-
-
yl a se O C
H H O
box
H C O P O- H O - H2O O
H C OH
O C O
H C O P O-
- Ca r O
H C O P O-
3-Phosphoglycerate
O C O
H C OH H O
C OH β-Keto
H C OH O
- H+ - intermediate
H C OH O
H C O P O-
H H C O P O- H O
-
O H O2 O -
O H C O P O- O
Ox C O O
-
yg O - C -
en HO C O O
- H C OH O O
Ribulose-1,5- Enediolate ase H C O P O- H C O P O-
bis-phosphate intermediate O C H O
H O
H C OH - H2O
O 2-Phosphoglycolate
3-Phosphoglycerate
H C O P O-
H O
 Second Stage: Synthesis of G3P (GAP)

The second stage of Calvin Cycle is like the Glycolysis running backward, except for

These reactions occur in the stroma of chloroplast  different enzymes

Chloroplast Dehydrogenase uses NADPH as e- donor, while the cytosolic Glycolysis enzyme
uses NAD+ as e- acceptor.

This is the most expensive stage in term of ATP and NADPH consumption

We know from the first stage that:

6 Ribulose bisphosphate (RuBP) + 6 CO2  yield twelve 3-carbon phosphoglyceric acid (PGA) molecules

These sets of reactions must run 12 times per glucose molecule synthesized

The sequence of events are proposed to proceed as follows:

Phosphoglycerate Dehydrogenase Triose Phosphate

Kinase Isomerase
- ADP O - H
O O ATP O PO3 2
C C O H
NADPH NADP+ H C OH
C
H C OH H C OH C O
-2 -2 H C OH
H C O PO3 H C O PO3 -2 -2
H C O PO3 H C O PO3
H H Pi
H
H
3-Phosphoglycerate 1,3 Bisphospho- D-Glyceraldehyde Dihydroxyacetone
Glycerate 3-Phosphate Phosphate
 Third stage of Calvin cycle

The second stage produces 12 G3P molecules

2 molecules are transported across the membrane to be used in the
synthesis of 1 glucose

The remaining 10 molecules are converted into 6 RuBP molecule
6 CO2
Inner chloroplast
Membrane
12
3-Phospho
6 RuBP Glycerate
Triose
2 Phosphate
Triose
Phosphate
10
 Third stage: Regeneration of (RuBP)

Complex series of reactions where five 3C-G3P molecules are
rearranged to make three 5C-RuBP molecules

An additional ATP molecule is consumed for generating RuBP (reaction is not shown)

Glyceraldehyde P Dihydroxyacetone P Carbons

C3 + C3 C6
Fructose 1,6-bisphosphate
C3 + C6 C4 + C5
P
Fructose 6-P Glyceraldehyde P C3 + C4 C7
Dihydroxyacetone P
Erythrose 4-P Xylulose 5-P C3 + C7 C5 + C5

Sedoheptulose 1,7-bisphosphate 5 C3 3 C5

Glyceraldehyde P Sedoheptulose 7-P

Ribose 5-P Xylulose 5-P

 Regeneration of (RuBP)
TI

These reactions are similar glyceraldehyde-3-P dihydroxyacetone-P
to Pentose Phosphate AL, FB
Pathway reactions, but fructose-6-P
running backward. TK

xyulose-5-P + erythrose-4-P
AL, SB

Overall: 5 C3  3 C5 sedoheptulose-7-P

Enzymes:

TI, Triosephosphate Isomerase TK

AL, Aldolase

FB, Fructose-1,6-bisphosphatase xylulose-5-P + ribose-5-P

SB, Sedoheptulose-Bisphosphatase
EP IS

TK, Transketolase

EP, Epimerase
(3) ribulose-5-P

IS, Isomerase PK

PK, Phospho-ribulokinase (3) ribulose-1,5-bis-P
 Dark Reactions

‘Calvin Cycle’

‘Carbon Reactions
Pathway’

Do not Require Light
Energy to Occur

Do Require Energy
Captured by Light
Reactions

http://www.ualr.edu/~botany/calvincycle.gif
 Dark Reactions

Occur at same Time as

Light Reactions

Cease Soon if Light
Energy Is not Available
to Make Light Reaction
Products

Exception: some
Xerophytes

http://www.ualr.edu/~botany/thylakoidmembrane.gif
 Dark Reactions

2 Main Steps

Carbon Dioxide Fixation

Sugar Formation

Via three stages

1) Carboxylation

2) Reduction

3) Regeneration

Occur in the Stroma

of the Chloroplasts
http://courses.cm.utexas.edu/jrobertus/ch339k/overheads-3/ch19_Dark-reactions.jpg
 Carbon Dioxide Fixation

‘Carbon Dioxide
Assimilation’

CO2 Is Incorporated
into a 3-Carbon or 4-
Carbon Chain

C3 Plants

C4 Plants

http://www.science.siu.edu/plant-biology/PLB117/JPEGs%20CD/0127.JPG
 Alternative mechanisms of C-fixation
Photorespiration may have drastic effect on the viability of plants:
In hot, dry weather: Stomata closed (preserve their H2O), O2 ↑ ,
CO2↓, Photorespiration ↑ photosynthesis ↓ (no glucose
synthesis)

Plants have special adaptations to limit the effect of photorespiration

C4-plants in Hot, moist environments.

15% of plants (e.g., corn, sugarcane , sorghum, millet, etc.)

They store CO2 temporarily as C4 molecule, giving them advantage
under high light, high temperature, low CO2 conditions.

CAM plants in hot arid climates

Many succulents plants (e.g., Cacti, Pineapple, agaves, etc.)

Stomata open during night, and close during the day

Use Crassulacean Acid Metabolism (CAM)
 Carbon Dioxide Fixation

C3 Plants

Most Plants Use an Enzyme Called RuBP
Carboxylase (RuBisCo) to Carry out the
CO2 Fixation

Enzymes Are Natural Proteins that Help
Catalyze/Carry out Reactions

Rubisco Is the most Abundant Enzyme
on Earth!

This Occurs in the Mesophyll Cells

Palisade or Spongy

Creates a 3-Carbon Product Ready for

Sugar Formation

Called C3 Plants because the 1st Stable
Carbon Chain Made from CO2 Has 3
Carbons

C3 Crops

Wheat, Soybeans, Cotton, Tobacco,
Small Grains, Legumes, Tomatoes,
Potatoes, Peppers, Cucurbits
http://www.uic.edu/classes/bios/bios100/lecturesf04am/rubisco01.jpg, http://www.palaeos.com/Eukarya/Lists/EuGlossary/Images/Rubisco.gif
 C4 Plants Carbon Dioxide Fixation

‘Hatch-Slack Pathway’

Process of CO2 Fixation for
many Plants of Dry or
Tropical Origins

Plants Use a Different Enzyme
Called PEP
(Phosphoenolpyruvate)
carbooxylase in the Mesophyll
Cells for CO2 Fixation

PEP Carboxylase Has a
much Higher Affinity for
CO2 than Does Rubisco

At Low CO2 Pressures,
Rubisco Doesn’t
Distinguish Well between
O2 and CO2 so Stomata
usually Have to Be Wide
Open for PS to Occur
http://www.ualr.edu/~botany/c4pathway.gif

Creates a 4-Carbon Product
 Carbon Dioxide Fixation

C4 Plants

This 4-Carbon Chain Is then
Transported into Bundle
Sheath Cells where the CO2
Is Released and then
Immediately Fixed by
Rubisco as Part of the C3
Cycle

Bundle Sheath Cells Are
Specialized Cells that
Surround the Vascular
Bundles in the Leaves

Same Fixation with Rubisco
as in C3 Plants but Occurs in
the Bundle Sheath Cells, not
Mesophyll Cells

http://gemini.oscs.montana.edu/~mlavin/b434/graphic/Leafc4a.jpg, http://www.ualr.edu/~botany/c4pathway.gif
 PEP Carboxylase vs. Rubisco

PEP Carboxylase Works Well at Warm Temperatures
but not Optimally at Cool Temps

This Is the Reason why C4 Grasses Are Referred to as
Warm Season Grasses, but Do not Compete Well with C3
Grasses at Cooler Temps

C4 Grasses Have an Edge in Dry Warm Sites or Open
Sunny Sites as They Can Keep Leaf Stomata Closed
during Mid-Day and Extract every Last CO2 Molecule in
the Leaf

In Contrast, C3 Grasses that Keep Stomata Closed in
Dry Sunny Sites Undergo High Amounts of Respiration
 C4-Plants

At least 19 plant families are C4 plants, e.g.
Sugarcane, corn, and millet.

The C-4 pathway is not an alternative to the Calvin or
even a net CO2 fixation

it is a mechanism for CO2 delivery system under condition
of O2 ↑ , CO2↓ to limit photorespiration

The C-4 plants has unique leaf anatomy to capture
CO2
 C4 Logistics

C4 plants: CO2 Fixation occurs
twice: First in mesophyll, then in
bundle-sheath cells

Mesophyll cells fix CO2 as C4
- -
O CO2 O O
PEP Carboxylase C C CH2 C
C C
CH2 -
O O
O O PO3 O
Pi
PEP Oxaloacetate

PEP Carboxylase has a very high affinity

for CO2  can fix CO2 when RuBP cannot

Mesophyll cells pump malate into
bundle sheath cells. There:

Malate  Pyruvate + CO2

CO2 is fixed by RuBISCO
 C3 & C4 plants

C4 Plants Can Produce 3 Times as much Dry Matter
per Unit of Water as C3 Plants

In hot environment:

C4 Plants have higher CO2 assimilation Rates (2-3 Times)
than that of C3 Plants  Efficient Plants

A Few C3 plants are as efficient as C4 Plants

In cooler Temperatures, C3 Plants have the

advantage:

PEP Carboxylase Needed to Incorporate CO2 into the 4-Carbon
Structure no Longer Functions

C4 Photosynthetic Rates Drop Dramatically or Stop
 Photorespiration

Plants that use the Calvin Cycles are called C3 Plants, because the 1st
Stable Carbon Chain Made from CO2 Has 3 Carbons

Nearly 80% of plants, e.g. Wheat, Soybeans, Cotton, Tobacco, Grains, Legumes,
Tomatoes, Potatoes, and Peppers

However, some plants close their stomata on hot, dry, & bright days to
save their H2O

As a result, CO2 in leaf is reduced

Rubisco can add O2 instead of CO2 to RuBP (Oxygenase Reaction)

Produces 2-C molecules instead of 3-C sugar molecules.

Produces no sugar molecules or no ATP.

Photorespiration occurs in light only

Occurs 1 out of 4 reactions under today’s atmospheric CO2

Rate increases with temperature
 Photorespiration

In the "normal" reaction, CO2 is joined with RUBP to form 2
molecules of 3PGA

In the process called photorespiration,

O2 replaces CO2 in a non-productive,
wasteful reaction

In C4-type plants photorespiration is suppressed

It has long been the dream of biologists to increase the

production of certain crop plants, such as wheat, that carry on
C3 photosynthesis by genetically re-engineer them to perform C4
photosynthesis

It seems unlikely that this goal will be accomplished in the near

future due to the complex anatomical and metabolic differences
that exist between C3- and C4-type plants http://www.marietta.edu/~spilatrs/biol103/photolab/photresp.html
 -
O
H P O- -
H C O O O - O O
O C
O C
CO2 H C OH - H C OH O
-
HO C C O
-
O H C O P O- H C O P O-
H O
-
-
yl a se O C
H H O
H C O P O- H O
b ox H C OH - H2O O
O C O
H C O P O-
- Ca r O
H C O P O-
3-Phosphoglycerate
O C O
H C OH H O
C OH β-Keto
H C OH O
- H+ - intermediate
H C OH O
H C O P O-
H H C O P O- H O
-
O H O2 O -
O H C O P O- O
Ox C O O
-
yg O - C -
en HO C O O
- H C OH O O
Ribulose-1,5- Enediolate ase H C O P O- H C O P O-
bis-phosphate intermediate O C H O
H O
H C OH - H2O
O 2-Phosphoglycolate
3-Phosphoglycerate
H C O P O-
H O
 Photorespiration

Respiration Driven by Light Energy

Occurs in Chloroplasts and other Structures in a

Photosynthetic Cell

Rubisco can React with Oxygen to Start a slightly

Different Series of Reactions

Result in a Loss or no Net Gain of Dry Matter for the Plant

Less ATP Is Produced from the Photorespiration

http://www.botany.hawaii.edu/faculty/webb/BOT311/BOT311-00/PSyn/Image81.gif
 Factors Influencing
Photorespiration

O2 : CO2 Ratio

If Cells Have Low O2 but Higher CO2, Normal

photosynthesis i.e. Calvin Cycle Dominates

C4 Plants Have Little Photorespiration because They

Carry the CO2 to the bundle Sheath Cells and can
Build up High [CO2]

Calvin Cycle Reactions always Favored over
Photorespiration

If Cells Have Higher O2 and Lower CO2,

Photorespiration Dominates

http://www.botany.hawaii.edu/faculty/webb/BOT311/BOT311-00/PSyn/Image81.gif
 Factors Influencing
Photorespiration

Light Intensity

Increasing Light Intensity will Increase Energy for
the Photorespiration Process and for photosynthesis

C3 Plants Light-Saturate at Lower Light Intensities

than C4 Plants

Reach Their ‘Break-Even Point’ at much Lower Light
Levels due to Increasing Photorespiration

http://www.botany.hawaii.edu/faculty/webb/BOT311/BOT311-00/PSyn/Image81.gif
 Factors Influencing Photorespiration

Temperature

Photorespiration
Increases with
Temperature

Plants Have
Optimum, Minimum
and Maximum Temp
Ranges

http://www.botany.hawaii.edu/faculty/webb/BOT311/BOT311-00/PSyn/Image81.gif
 Factors Influencing
Photorespiration

Net Photosynthesis or
Net Assimilation Rate

C4 Plants generally Have Net Assimilation Rates

about 2 to 3 Times that of C3 Plants

C4 Plants Are often Called Efficient Plants and C3

Plants Called Non-Efficient Plants

A Few C3 Plants Have Low Respiration and Similar

Assimilation Rates as C4 Plants

Sunflower

Peanut http://www.botany.hawaii.edu/faculty/webb/BOT311/BOT311-00/PSyn/Image81.gif
 Factors Influencing
Photorespiration

Net Photosynthesis or
Net Assimilation Rate

Cooler Temps Are the only Time when C3 Plants

Have Higher Net Assimilation Rates than C4 Plants

PEP Carboxylase Needed to Incorporate CO2 into the 4-
Carbon Structure no Longer Functions

C4 PS Rates Drop Dramatically or Stop

http://www.botany.hawaii.edu/faculty/webb/BOT311/BOT311-00/PSyn/Image81.gif
 Carbon Dioxide Fixation (C3 and C4
Pathways)

Both Types of Plants Use Energy from ATP and
NADPH2 to Carry out the Reactions

The Energy from ATP Is Given by ATP Giving up Its
3rd Phosphorus

ATP → ADP + P

The Energy from NADPH2
Giving up Its Hydrogens

NADPH2 → NADP + H2
 CAM Photosynthesis

Crassulacean Acid
Metabolism

Another Type of PS Carried
out only by Xerophytes

At Night

Stomata Are Open

Plants Fix CO2 into a 4-Carbon
Product

4-Carbon Product Stored
overnight in Vacuole

http://www.ualr.edu/~botany/c4andcam.jpg
 CAM Photosynthesis

During the Day

Stomata Are Closed

CO2 Is Released from the 4-
Carbon Produce

Normal Light and Dark
Reactions occur without
Stomata Opening

Allows the Plants to Conserve
Water during the Day

When Water Is Adequate,
these Plants usually Carry out
C3 PS

http://www.ualr.edu/~botany/c4andcam.jpg
 CAM Photosynthesis

CAM Plants

Cacti, Succulents

Crops include Pineapple,

Tequila Agave

http://www.ualr.edu/~botany/c4andcam.jpg
 CAM Photosynthesis

Alternative mechanisms of C-fixation is found in
Succulent plants of hot, arid environments: cacti,
pineapples, etc.

Plant family Crassulaceae  crassulacean acid
metabolism or CAM plants

These plants open their stomates during the night
and close them during the day

CAM plants partition carbon fixation by time

During the night: lower temps and higher humidity

CO2 is fixed into C4 molecules, and stored in large
vacuoles

During daylight: Higher Temps and lower humidity

Stomata closed for water conservation

NADPH and ATP are available

C4 molecules release CO2 to Calvin cycle
Overview of CAM
Comparison between C4 & CAM plants
 Regulation of Carbon Dioxide Fixation

Plant cells have chloroplasts that carry out

photosynthesis: CO2  glucose

Plant cells also have mitochondria and carry

out glycolysis, TCA, and oxidative phosphorylation:

Glucose  CO2

To prevent futile cycling of carbohydrate,

cells must regulate the activities of key Calvin
cycle enzymes

These enzymes respond indirectly to light activation.

light energy is available  the Calvin cycle proceeds.

If no light available, no fixation of CO2 occur

Among the key changes that regulate Calvin cycle versus respiration are:

Environment Factors: Light intensity, temperature, & availability of H2O,
CO2, O2

Cellular factors: cell state of key metabolites (NADPH, ATP, inhibitors,
reducing power etc.)
 Summary of Carbon fixation

Each method of photosynthesis has advantages and
disadvantages Depending on the climate (light, heat,
water, CO2, and O2)

C3 plants better adapted to:

Cold Temp (below 25C), moderate light, balanced CO2 & O2,
and High moisture

~ 80% of plants

C4 plants most adapted to:

high light intensities, high temperatures, Limited rainfall

CAM plants better adapted to extreme aridity (desert

conditions, low water)