MESA COLLEGE, SAN DIEGO
SCHOOL OF MATHEMATICS & NATURAL SCIENCE
General Biology (BIO107); Instructor: Elmar Schmid, Ph.D.
C
Chhaapptteerr 77:: TThhee D
Daarrkk R
Reeaaccttiioonn ooff P
Phhoottoossyynntthheessiiss
- Part II The C
Caallvviinn--B
Beennssoonn ccyyccllee

In this section we will look at the second part of the photosynthesis process – The dark
reaction – in more detail. Since the events of the dark reactions lead to the production of
sugars, primarily in form of glucose and fructose, plants are the primary producers of food
materials of all major food chains on planet Earth

The key event of the dark reaction is a cyclical chemical reaction pathway called the
Calvin-Benson cycle.

The Calvin cycle regenerates its starting material after molecules enter and leave the
cycle. In a nutshell: The atmospheric gas carbon dioxide (CO2) enters the cycle and
leaves as sugar. The cycle spends the energy of ATP and the reducing power of
electrons carried by NADPH to make the sugar. The actual sugar product of the Calvin
cycle is not glucose, but a three-carbon sugar, glyceraldehyde-3-phosphate (G3P).

Due to the importance of plants as the primary suppliers of raw materials, most
importantly in form of glucose, fructose and starch, to most forms of life, the CalvinBenson cycle (or short: Calvin cycle) has to be considered as the single most
important biological process on planet Earth

The Calvin cycle which is a cyclical series of chemical reactions takes place in the
stroma of the chloroplast.

The Calvin cycle builds-up (= synthesizes) the energy-rich 3 carbon molecule
glyceraldehyde-3-phosphate (= G3P) from CO2, ATP and NADPH + H+
 CO2 is extracted from air, which diffuses freely into the
chloroplast via special leaf openings, called stomata
 ATP and NADPH + H+ is supplied via the two light reactions in PS I and PS II
(see part I)

G3P the end product of the Calvin cycle is the key precursor molecule of all important 6
carbon sugar molecules, most importantly of glucose and fructose.
TThhee tthhrreeee kkeeyy sstteeppss ooff tthhee C
Caallvviinn ccyyccllee
11.. C
Caarrbboonn ffiixxaattiioonn

3C
CO
O222 molecules are combined with 3 molecules of the 5 carbon- molecule R
Riibbuulloossee-bbiisspphhoosspphhaattee ((R
i
b
P
2
)
Rib-P2) to receive 6 molecules of the 3 carbon compound
33--P
Phhoosspphhooggllyycceerriicc aacciidd ((== 33--P
PG
G))
 this reaction is catalyzed by Rubisco, the key enzyme of the Calvin cycle
 of C:
3 xC
C +
3 +
3 xC
C55
15


6 xC
C33
18
1
MESA COLLEGE, SAN DIEGO
SCHOOL OF MATHEMATICS & NATURAL SCIENCE
General Biology (BIO107); Instructor: Elmar Schmid, Ph.D.
C
Caarrbboonn ffiixxaattiioonn rreeaaccttiioonn ooff tthhee R
Ruubbiissccoo eennzzyym
mee
2
MESA COLLEGE, SAN DIEGO
SCHOOL OF MATHEMATICS & NATURAL SCIENCE
General Biology (BIO107); Instructor: Elmar Schmid, Ph.D.

Despite its central role in PS, the Rubisco
enzyme is remarkably inefficient and
slow
 typical enzymes can process
more than 1000 molecules per
second
 Rubisco fixes only about three
CO2 molecules per second

Plant cells compensate for this slow rate
by building lots of the enzyme

Since chloroplasts are literally packed with
Rubisco, it makes it
the most plentiful single enzyme on
planet Earth!
M
Mooddeell ooff tthhee ccaarrbboonn--ffiixxiinngg
eennzzyym
mee R
Ruubbiissccoo
 based on X-ray crystallography
Plants & Algae:
 8 copies of a large protein chain
 8 copies of a smaller protein chain
 contains Mg2+ as co-factor
22.. E
Enneerrggyy ccoonnssuum
mppttiioonn aanndd rreeddooxx rreeaaccttiioonnss

During reduction, each 3-phosphoglycerate receives a second phosphate group from ATP
to form 1,3 bisphosphoglycerate.

Then a pair of electrons from NADPH reduces each 1,3 bisphosphoglycerate to G3P. The
electrons reduce a carboxyl group to a carbonyl group.

These 2 chemical reactions of the Calvin cycle consume energy from 6 ATP and oxidize
6 NADPH + H+ molecules to reduce 6 molecules of 3-PG to the energy-rich G3P
molecule
3
MESA COLLEGE, SAN DIEGO
SCHOOL OF MATHEMATICS & NATURAL SCIENCE
General Biology (BIO107); Instructor: Elmar Schmid, Ph.D.
33.. R
Reelleeaassee ooff oonnee m
moolleeccuullee ooff G
G33P
P ppeerr ccoom
mpplleettee ccyyccllee &
&R
Reeggeenneerraattiioonn ooff R
RuuB
BP
P

5 of the synthesized 6 G3P (C3) molecules remain in the Calvin cycle to recover 3
molecules of Rib-P2 (C5), which can re-enter the cycle

Only one molecule G3P (C3) leaves the Calvin cycle and is available for the
subsequent glucose (C6) synthesis

In the last phase, the five G3P molecules which remained within the cycle are rearranged
to regenerate the initial CO2 acceptor molecule RuBP. 3 RuBP molecules are formed
from the 5 G3P molecules. To achieve this, the cycle spends three more molecules of
ATP (one per RuBP) to complete the cycle and prepare for the next.
4
MESA COLLEGE, SAN DIEGO
SCHOOL OF MATHEMATICS & NATURAL SCIENCE
General Biology (BIO107); Instructor: Elmar Schmid, Ph.D.

Since G3P is only a 3 carbon compound (C3) and glucose is a six carbon molecule, it
takes two rounds of the Calvin cycle to finally make one glucose molecule (C6).

Since plants and other photosynthesizing life forms, such as algae, usually produce more
sugar than they actually need for maintaining their vital biological functions, many sugars
are stock-piled within the cells as storage sugars in form of starch in roots, tubers and
fruits.

The photosynthesis reaction of plants produces billions of tons of organic matter or biomass each year. This bio-production is unmatched by any other chemical process on
Earth! (see Graphic below). Most of the organic matter produced by plants via
photosynthesis is the ultimate source of food for virtually all organisms on our planet.
5
MESA COLLEGE, SAN DIEGO
SCHOOL OF MATHEMATICS & NATURAL SCIENCE
General Biology (BIO107); Instructor: Elmar Schmid, Ph.D.

Plants and other photosynthesizing life forms, such as algae and diatoms, are the
foundation of our Earth’s diverse food chains. Life on Earth as we know it is not possible
without this unique and truly fascinating biological process called photosynthesis!
S
Suum
mm
maarryy:: TThhee ccrruucciiaall cchheem
miiccaall sstteeppss ooff tthhee C
Caallvviinn ccyyccllee
(molecule numbers shown for two complete rounds within the cycle
to make one molecule of glucose )

The Calvin cycle is the plants core chemical reaction and is the ‘turbine’ of its sugar
manufactory process

The sugar produced during the Calvin cycle is used by the plants as:
1.
fuel molecule for cellular respiration
2.
as nectar for insect attraction or
3.
as starting material for the biosynthesis of structural molecules, e.g. cellulose or
storage molecules, e.g. starch
6
MESA COLLEGE, SAN DIEGO
SCHOOL OF MATHEMATICS & NATURAL SCIENCE
General Biology (BIO107); Instructor: Elmar Schmid, Ph.D.
Carbon fixation and release in numbers
• Carbon fixation rate:
5g CO2 / m2 x day
(temperate forest)
• Global carbon fixation
100 Giga t C / y
(due to photosynthesis)
• Global carbon fixation
92 Giga t C / y
(due to Ocean uptake)
• Global atmospheric carbon
• Carbon (CO2) release
750 Giga t C
5.4 Giga t C / y
(anthropogenic, fossil fuels)
• Plant and soil respiration
50 + 50 Giga t C / y
Global atmospheric CO2 concentration
7
MESA COLLEGE, SAN DIEGO
SCHOOL OF MATHEMATICS & NATURAL SCIENCE
General Biology (BIO107); Instructor: Elmar Schmid, Ph.D.
TThhee ddiiffffeerreenntt ccaarrbboonn ffiixxaattiioonn ssttrraatteeggiieess ooff ppllaannttss

One of the reasons for the great evolutionary success of plants on Earth lays in their
adaptive flexibility and in their different ways of taking up and fixing atmospheric CO2
as well as saving water during photosynthesis.

The different carbon fixation strategies we observe in modern plants today is the
adaptive consequence to one of the major problems facing all terrestrial plants dehydration. Plants cannot live without water, which – as we learned earlier – is their
ultimate electron donating resource.

At times, solutions to this “water problem” conflict with other metabolic processes,
especially photosynthesis. For example, the stomata are not only the major route for gas
exchange (CO2 in and O2 out), but also for the evaporative loss of water.

On hot, dry days plants close the stomata to conserve water, but this causes problems for
the ongoing photosynthesis process, especially in C3 plants.

Depending on their way of fixing the air’s trace gas CO2 (only 0.03% of air is CO2!), plants
are classified into three groups:
11..
C
C33--ppllaannttss

In C3 plants initial fixation of CO2 occurs via the Rubisco enzyme and results in a
three-carbon compound, 3-phosphoglycerate.

C3 plants are draught-sensitive and close their stomata (= leaf openings) on hot, dry days
to prevent loss of water.

When their stomata are closed on a hot, dry day, CO2 levels drop as CO2 is consumed in
the fully running Calvin cycle. At the same time, O2 levels rise as the light reaction
converts light to chemical energy.

While Rubisco normally favors CO2 over O2 as substrate, when the O2/CO2 ratio
increases (on a hot, dry day with closed stomata), Rubisco tends to add O2 instead of
CO2 to RuBP.

As a result of this, the Rubisco enzyme of the Calvin cycle incorporates O2 into
Ribulose-Bisphosphate (RibP2) instead of CO2. As a consequence Rubisco enzyme
cleaves and retrieves only one 3-PG (= C3) molecule (instead of two) and one C2
compound (= Phospho-glycolate) and triggers a process called photorespiration
 since Rubisco is an extraordinarily ancient enzyme that evolved when the
planet's atmosphere lacked oxygen, it “never learned” how to distinguish
CO2 from oxygen

In photorespiration the uptake of O2 instead of carbon dioxide by Rubisco leads to one
C3 molecule and one C2 molecule:
8
MESA COLLEGE, SAN DIEGO
SCHOOL OF MATHEMATICS & NATURAL SCIENCE
General Biology (BIO107); Instructor: Elmar Schmid, Ph.D.
1. 3-phosphoglyceric acid (3-PG) (= C3)
 just as in the Calvin cycle under “normal” temperature conditions
22.. P
Phhoosspphhoo--ggllyyccoollaattee ((== C
C22))
 this molecule enters the peroxisome where it forms an intermediate molecule
(= glycoxylate) under consumption of oxygen (see Graphic below)
 a further derivative of the intermediate C2 molecule (= glycine) enters the
mitochondrion where it is cleaved to CO2 and water
The major chemical events during photorespiration in C3-Plants
- Happens at high light intensities
- Happens at high daylight temperatures
Plant Cell
2-Oxoglutarate
P O – CH2 – CH – COOH
3-PG
Calvin
Cycle
P O – CH2 – COOH
L- Glycine
Phosphoglycolate
O2
NH4+
H2O2
Glu
Pi
Rubisco
HOCH2 – COOH
Glycolate
Rib-P2
Peroxisome
CO2 Mitochondrion
NADH + H+
O2
CO2
Glycolate
Chloroplast
Oxygenase
act.
NAD+
H2N – CH2 – COOH
H2N – CH2 – COOH
Serine
L- Glycine (2x)
Hydroxymethyl
H2N – CH2 – COOH
L- Serine
NH4+
Stomata
closed
Glycoxylate
Transferase
Graphics©E.Schmid/SWC2003

Photorespiration, similar to cellular respiration, consumes oxygen and releases carbon
dioxide, but unlike photosynthesis yields no sugar and does nnoott produce ATP
molecules. This outcome is very disadvantageous for the many C3 agricultural plants
(and especially the farmers who agriculture them for achieving high yields and profits).

Photorespiration decreases photosynthetic output by siphoning organic material from
the Calvin cycle.

One favored hypothesis for the existence of photorespiraton found in C3 plants is that it is
evolutionary baggage. When Rubisco first evolved in the first photosynthesizing life forms
on planet Earth, the atmosphere had far less O2 (than the 21% today) and more CO2 than
it does today. The inability of the active site of Rubisco enzyme to effectively discriminate
9
MESA COLLEGE, SAN DIEGO
SCHOOL OF MATHEMATICS & NATURAL SCIENCE
General Biology (BIO107); Instructor: Elmar Schmid, Ph.D.
between O2 and CO2 would have made little difference then.

But today in a high oxygen atmosphere of more than 20% it does make a difference.
Photorespiration can drain away as much as 50% of the carbon fixed by the Calvin cycle
on a hot, dry day, leading to much lower biomass yield.

Since many important agricultural plants, e.g. soybean, wheat and rice, are C3 plants,
with photorespiration significantly lowering the agricultural yield, scientists fear that rising
temperatures due to global warming might have a negative impact on human food supply
in the future.

Therefore, scientists try to genetically replace the inefficient plant Rubisco enzyme within
agriculturally relevant C3 plants and replace its gene with the gene of an enzyme from
green algae that captures CO2 more quickly and more efficiently.
22..
C
C44--ppllaannttss

They evolved special adaptations to save water and also to prevent photorespiration

C4-plants also close their stomata on dry, hot days, but they have a special enzyme
which fixes CO2 into a four carbon (= C4) compound instead of incorporating it into 3PGA.

In C4 plants, mesophyll cells incorporate CO2 into organic molecules. In these cells, the
key enzyme, phosphoenolpyruvate carboxylase, adds CO2 to phosphoenolpyruvate
(PEP) to form oxaloacetetate. PEP carboxylase has a very high affinity for CO2 and can
fix CO2 efficiently when rubisco cannot - on hot, dry days with the stomata closed. The
PEP carboxylase enzyme does not switch to O2 incorporation under dry conditions.

The mesophyll cells pump these four-carbon compounds into bundle-sheath cells.

The bundle sheath cells strip a carbon, as CO2, from the four-carbon compound and
return the three-carbon remainder to the mesophyll cells. The bundle sheath cells then
uses Rubisco enzyme to start the Calvin cycle with an abundant supply of CO2, without
going into photorespiration.

Therefore, on hot, dry days C4 plants can continue to efficiently fix carbon in the
Calvin cycle even though the CO2 concentration within the leaf is low due to the stomata
closure.

Important agricultural plants e.g. corn and sugar cane, are C4 plants. The C4 plants
evolved in the tropics as an adaptive response to the permanently hot-dry climate in these
regions.
10
MESA COLLEGE, SAN DIEGO
SCHOOL OF MATHEMATICS & NATURAL SCIENCE
General Biology (BIO107); Instructor: Elmar Schmid, Ph.D.
33..

C
CA
AM
M ((== ccrraassssuullaacceeaann aacciidd m
meettaabboolliissm
m)) ppllaannttss
A second strategy to minimize photorespiration is found in succulent plants, cacti,
pineapples, and several other plant families. These plants, known as CAM plants for
crassulacean acid metabolism (CAM), open stomata during the night and close them
during the day.
11
MESA COLLEGE, SAN DIEGO
SCHOOL OF MATHEMATICS & NATURAL SCIENCE
General Biology (BIO107); Instructor: Elmar Schmid, Ph.D.

These plants, which usually grow in very arid parts on planet Earth, conserve water by
allowing opening of its stomata and influx of CO2 only at night. Temperatures are
typically lower at night and humidity is higher.

During the night, CAM plants fix CO2 into a variety of organic acids, mostly maleic acid, in
mesophyll cells.

During the day, the light reactions supply ATP and NADPH to the Calvin cycle and CO2 is
released from the organic acids.

These unique plants have evolved in form of e.g. cacti, pineapple and succulents (ice
plant!) as a response to persistently dry and hot climates.
12
MESA COLLEGE, SAN DIEGO
SCHOOL OF MATHEMATICS & NATURAL SCIENCE
General Biology (BIO107); Instructor: Elmar Schmid, Ph.D.
P
Phhoottoossyynntthheessiiss,, TTooxxiinnss &
&E
Ennvviirroonnm
meennttaall P
Poolllluuttaannttss

Photosynthesis is a highly complex biological process, enabled and driven by many
enzymes and enzyme systems, which work in well-defined structures in well ordered
and integrated sequences (= enzyme cascades)

These enzymes and structures, playing a crucial role in photosynthesis, are the point of
attack of many molecules released by bacteria, fungi or herbivorous insects
 e.g. Fusarium solani, a fungal pathogen of many plants, produces
Naphthazarian toxins which destroy the chloroplast membranes
 as a consequence the chlorophyll of the affected leaves bleaches and the
plant starts to wilt

The “photosynthesis bioapparatus” is also highly vulnerable to many environmental
pollutants released by human activity and is purposely attacked by commonly used
herbicidal agents

Critical plant-harming environmental pollutants are sulfur dioxide (= SO2) and ozone (=
O3), which are common air pollutants in today’s urban and inner-city areas
 Sulfur dioxide results from the burning of fossil fuels such as oil, gasoline
and coal (especially brown coal combustion)
 Ozone is a highly aggressive by-product of automobile exhaust and may
accumulate in urban areas on sunny days
 it is estimated that air pollution may reduce yields of some farm crops by
as much as 20 percent!

Herbicides which are commonly used in agricultural weed control and known to target
the enzymatic reactions, structures or processes in the chloroplasts are:
1. Triazines (e.g. Evik, Bladec) and Phenylureas (e.g. Lorox, Spike)
 site of action in the chloroplast is the D-1 quinone-binding protein of the
photosynthetic electron transport chain
2. Diphenylether and Bipyridylium herbicides
 these contact herbicides destroy plant cell membranes after activation by
exposure to sunlight and formation of aggressive oxygen compounds such as
hydrogen peroxide
3. Bipyridiliums (e.g. Paraquat)
 the non-selective weed controlling herbicide Paraquat (Gramoxone Extra) is
activated by the photosystem I (PSI) and destroys the chloroplast by generating
free radicals
4. Diphenylethers (e.g. Blazer, Cobra)
 these herbicides inhibit the protoporphyrinogen oxidase enzyme of the electron
transport chain
13
MESA COLLEGE, SAN DIEGO
SCHOOL OF MATHEMATICS & NATURAL SCIENCE
General Biology (BIO107); Instructor: Elmar Schmid, Ph.D.

Certain herbicides work as so-called un-couplers, e.g. DNP or CCCP and prevent the
synthesis of ATP in chloroplasts by shuttling protons across the thylacoid membrane
 they do not interfere with the passage of electrons down the electron
transport chain to NADP, but destroy the proton gradient essential for ATP
synthesis
14