C3 Carbon Reduction Cycle

Secondary article
Article Contents

Christine A Raines, University of Essex, Colchester, Essex, UK

Julie C Lloyd, University of Essex, Colchester, Essex, UK

. Introduction
. Enzymes and Reactions of the Cycle

The C3 carbon reduction cycle is the primary pathway of carbon fixation in all
photosynthetic organisms, reducing CO2 from the atmosphere to form carbohydrates; in
higher plants it takes place in the chloroplast stroma.

Introduction
The C3 photosynthetic carbon reduction cycle was rst
elucidated by Calvin, Bassham and Benson in the 1950s in a
series of experiments using the green alga Chlorella and
radiolabelled carbon. This cycle, frequently referred to as
the Calvin cycle, uses the products of the light reactions of
photosynthesis, ATP and NADPH, to x atmospheric
CO2 into carbon skeletons that are used directly for starch
and sucrose biosynthesis (Figure 1). The cycle can be divided
into three stages. The rst of these is carboxylation, in
which the enzyme ribulose-1,5-bisphosphate carboxylase/
oxygenase (Rubisco) (EC 4.1.1.39) catalyses the xation of
CO2 to the acceptor molecule ribulose 1,5-bisphosphate.
This reaction results in the formation of the rst stable
compound in the cycle, 3-phosphoglycerate, a threecarbon sugar that gives the cycle its name. Carboxylation
is followed by the reduction phase producing triose
phosphates and the nal, regenerative, phase resulting in
the production of the CO2 acceptor molecule, ribulose 1,5bisphosphate. The catalytic activities of a number of
enzymes within the cycle are highly regulated, ensuring
that a balance is maintained between the carbon leaving the
cycle and that remaining for synthesis of the CO2 acceptor
molecule.

Enzymes and Reactions of the Cycle

Carboxylation of ribulose 1,5-bisphosphate
The rst reaction of the C3 photosynthetic carbon
reduction cycle is the binding of CO2 to the acceptor
molecule, ribulose l,5-bisphosphate, to form two molecules
of 3-phosphoglycerate. This carboxylation reaction, catalysed by Rubisco, is unique to photosynthetic organisms.
In higher plants the functional 550 kDa Rubisco holoenzyme comprises eight identical large and eight identical
small subunits, with one catalytic site located on each of the
large subunits. This enzyme is easily the most abundant
protein on earth, constituting up to 50% of leaf soluble
protein in C3 plants. The reason for the high concentrations of Rubisco is that it has a poor anity for its
substrate, CO2 (Km  10 mmol L 2 1) and it also has an

. Regulation of Enzymes
. Control of Gene Expression

extremely low turnover number (3.3 s 2 1), so that large

amounts of the enzyme are needed to catalyse the uxes
required for photosynthesis. Rubisco is a bifunctional
enzyme and in addition to catalysing the carboxylation
reaction it can also catalyse the oxygenation of ribulose
1,5-bisphosphate at the same active site. This oxygenation
reaction diverts carbon from the Calvin cycle to the
photorespiratory pathway, releasing CO2 and NH3. The
carboxylation and oxygenation reactions of Rubisco are
competitive and the ratio of these reactions is determined
by the relative concentrations of CO2 and O2. As a
consequence of this feature of the enzyme, present-day
high atmospheric concentrations of oxygen relative to CO2
favour photorespiration and are estimated to result in the
loss of up to 40% of xed carbon.

Reduction of 3-phosphoglycerate
The second phase of the cycle is the production of triose
phosphates from the carboxylation product, 3-phosphoglycerate. First, 3-phosphoglycerate is phosphorylated by
the enzyme phosphoglycerate kinase (EC 2.7.2.3), forming
1,3-bisphosphoglycerate, which is then reduced by glyceraldehyde 3-phosphate dehydrogenase (EC 1.2.1.13) to
glyceraldehyde 3-phosphate, consuming ATP and
NADPH. Triose-phosphate isomerase catalyses the reversible isomerization of glyceraldehyde 3-phosphate to
dihydroxyacetone phosphate, although the equilibrium is
strongly biased towards dihydroxyacetone phosphate.
Five of every six molecules of triose phosphate produced
are required for regeneration, but one molecule represents
net product and can leave the cycle either for export from
the chloroplast or for use in starch biosynthesis.

Regeneration of ribulose 1,5-bisphosphate

In the third phase of the cycle, the CO2 acceptor molecule
ribulose 1,5-bisphosphate is regenerated from triose
phosphates through a series of sugar condensation and
carbon rearrangement reactions. Condensation of the
triose phosphates (glyceraldehyde 3-phosphate and dihydroxyacetone phosphate) by aldolase (EC 4.1.2.13) yields
fructose 1,6-bisphosphate. This six-carbon sugar is then
irreversibly hydrolysed to the monophosphate form,

ENCYCLOPEDIA OF LIFE SCIENCES / & 2001 Nature Publishing Group / www.els.net

C3 Carbon Reduction Cycle

CO2

ADP

ATP
3-Phosphoglycerate

NADPH

Glycerate
1,3-bisphosphate

NADP+
Glyceraldehyde
3-phosphate

Pi
4

Ribulose
1,5,-bisphosphate
Sedoheptulose
1,7-bisphosphate

ADP
11

Dihydroxy acetone phosphate

ATP
Ribulose
5-phosphate

Export to cytosol
(sucrose
biosynthesis)

Pi
Erythrose
4-phosphate

Sedoheptulose
7-phosphate

Fructose
1,6-bisphosphate

10
9

Ribose
5-phosphate

Pi
2-carbon
transfer

Fructose
6-phosphate

Starch
biosynthesis
(chloroplast)

Xylulose
5-phosphate
Figure l Reactions of the C3 carbon reduction cycle. The enzymes catalysing each step are: (1) ribulose-1,5-bisphosphate carboxylase/oxygenase
(Rubisco); (2) phosphoglycerate kinase; (3) glyceraldehyde-3-phosphate dehydrogenase; (4) triose-phosphate isomerase; (5) aldolase; (6) fructose-1,6bisphosphatase; (7) transketolase; (8) sedoheptulose-1,7-bisphosphatase; (9) ribulose-phosphate epimerase; (10) ribose-5-phosphate isomerase; (11)
phosphoribulokinase. ADP, adenosine diphosphate; ATP, adenosine triphosphate; NADP, nicotineadenine dinucleotide phosphate (NADP 1 , oxidized
form; NADPH, reduced form).

fructose 6-phosphate, by fructose-1,6-bisphosphatase (EC

3.1.3.37). The enzyme transketolase (EC 2.2.1.1) then
performs a two-carbon transfer from fructose 6-phosphate
to glyceraldehyde 3-phosphate, forming xylulose 5-phosphate and erythrose 4-phosphate. Transketolase uses
thiamin pyrophosphate as a prosthetic group to mediate
the two-carbon transfer. The resulting erythrose 4phosphate is combined with dihydroxyacetone phosphate,
in a reaction again catalysed by aldolase, to form
sedoheptulose 1,7-bisphosphate. This seven-carbon product is hydrolysed by sedoheptulose-1,7-bisphosphatase
(EC 3.1.3.37), yielding sedoheptulose 7-phosphate. Transfer of two carbons from sedoheptulose 7-phosphate to
glyceraldehyde 3-phosphate by transketolase produces
ribose 5-phosphate and xylulose 5-phosphate. Ribose 5phosphate and xylulose 5-phosphate are converted to
ribulose 5-phosphate by ribose-5-phosphate isomerase and
ribulose-phosphate epimerase, respectively. The nal step
converts ribulose 5-phosphate to the CO2 acceptor
molecule ribulose 1,5-bisphosphate by the action of
phosphoribulokinase (EC 2.7.1.19) in an irreversible
reaction utilizing ATP.

Regulation of Enzymes
The activity of a number of enzymes in the cycle, including
Rubisco, phosphoglycerate kinase, transketolase, fructose-1,6-bisphosphatase and sedoheptulose-1,7-bisphosphatase, is regulated by large increases in the pH and
Mg2 1 concentrations in the chloroplast stroma that occur
on illumination of a previously darkened leaf. In addition
to this general activation by light, the activity of a number
of enzymes is regulated by further specic mechanisms
described below.

Rubisco regulation
The activity of the rst enzyme in the C3 carbon reduction
cycle, Rubisco, is highly regulated at a number of dierent
levels. First, it must be converted from an inactive to an
active form before it is able to catalyse the xation of CO2.
This process of carbamoylation involves the binding of
CO2 and Mg2 1 to a lysine residue adjacent to the catalytic
site. Another protein, Rubisco activase, is involved in
mediating light activation and is itself subject to light
regulation via the thioredoxin system described below
(Zhang and Portis, 1999). Rubisco activase functions by
removing inhibitors bound to the Rubisco active site on

ENCYCLOPEDIA OF LIFE SCIENCES / & 2001 Nature Publishing Group / www.els.net

C3 Carbon Reduction Cycle

transition from darkness to light. These inhibitors prevent

activation (carbamoylation) of the enzyme and may
include the substrate ribulose 1,5-bisphosphate, the
products of alternative reactions of Rubisco, and the
potent inhibitor 2-carboxyarabinitol phosphate (although
this compound is not found in all species). Rubisco activase
requires ATP to function, linking activation to the
production of ATP and further coordinating carbon
xation with the light reactions of photosynthesis.

involve a conserved sequence motif, suggesting that this

mechanism has evolved independently on more than one
occasion.
In the leaf, light regulation of Calvin cycle enzyme
activity acts as an important on/o switch to prevent futile
cycling of carbon in the dark. In addition, it is now thought
that thiol regulation of the Calvin cycle also acts to
modulate enzyme activity in response to transient alterations in the light environment, such as shading and
sunecks (Scheibe, 1991; Ruelland and Migniac-Maslow,
1999).

Thioredoxin regulation
The activity of a number of Calvin cycle enzymes,
glyceraldehyde-3-phosphate dehydrogenase, fructose-1,6bisphosphatase, sedoheptulose-1,7-bisphosphatase and
phosphoribulokinase, is modulated by light. Although
the dark activity of these enzymes varies, the cycle is
essentially inactive until illumination takes place, when
there is a rapid increase in carbon ux through the cycle.
Light activation utilizes the reducing power produced by
the photosynthetic light reactions, which is transferred
from ferredoxin to thioredoxin f in a reaction catalysed by
the enzyme ferredoxin/thioredoxin reductase (Figure 2).
Thioredoxin then binds to the inactive target enzyme and
reduces the regulatory disulde bond. The enzyme is
activated by the associated change in conformation, and
oxidized thioredoxin is released.
The cysteine residues involved in thiol activation of
fructose-1,6-bisphosphatase, sedoheptulose-l,7-bisphosphatase and phosphoribulokinase have been identied
using site-directed mutagenesis (Jaquot et al., 1995;
Brandes et al., 1996; Dunford et al., 1998). It appears that
thiol-mediated activation of these enzymes does not

Another level of regulation of the activity of the Calvin

cycle may operate via the formation of transient multienzyme complexes between enzymes of the cycle. For
example, glyceraldehyde 3-phosphate and phosphoribulokinase, have been shown to interact with a small nuclear
encoded chloroplast protein, CP12, forming a complex
that is reversibly dissociated by NADPH (Wedel et al.,
1997). This may be an additional mechanism for light
regulation of phosphoribulokinase activity in vivo. Further
complexes involving dierent combinations of enzymes
have also been proposed and these may have a role in
channelling intermediates between enzymes, improving the
eciency of the cycle (Suss et al., 1993).

Control of carbon fixation

In recent years it has become possible to address the
relative importance of individual enzymes in controlling
the ux of carbon xation. Studies of transgenic plants

Reduced

Oxidized

Ferredoxin

Thioredoxin

e
Photosynthetic
electron transport

LIGHT

Multiprotein complexes

ACTIVE
Reduced
Target
enzyme

SH

SH

Ferredoxin/thioredoxin
reductase
Ferredoxin

Oxidized

Target
enzyme

Thioredoxin

SH
SH
Reduced

S
Oxidized
INACTIVE

Figure 2 Ferredoxin/thioredoxin-mediated light regulation of enzyme activity. Ferredoxin is reduced by electrons from the photosynthetic electron
transport chain and in turn the enzyme ferredoxin/thioredoxin reductase brings about the reduction of thioredoxin f. Finally, reduced thioredoxin f
activates the target enzyme by reducing a disulfide bridge between two cysteine residues, forming two thiol groups.

ENCYCLOPEDIA OF LIFE SCIENCES / & 2001 Nature Publishing Group / www.els.net

C3 Carbon Reduction Cycle

with altered levels of individual enzymes in the C3 cycle

have revealed that no single enzyme has complete control
of carbon xation. In fact, control is shared among a
number of enzymes with Rubisco, sedoheptulose-1,7bisphosphatase and aldolase having prominent roles (Stitt
and Schultz, 1994; Haake et al., 1998; Harrison et al.,
1998). Interestingly, enzymes with highly regulated activity, such as phosphoribulokinase, do not necessarily have
any control on carbon xation, whereas aldolase, which
catalyses a reversible reaction, does partly determine the
rate at which carbon is xed.

Control of Gene Expression

The genes encoding the enzymes of the Calvin cycle are,
with the exception of the large subunit of Rubisco, located
in the nuclear genome. The large subunit of Rubisco is
chloroplast encoded. As a result, the proteins are
synthesized in the cytosol with N-terminal extensions
termed transit sequences, which direct their import into the
chloroplast stroma (Chen and Schnell, 1999). The transit
sequences are subsequently cleaved from the mature
protein. In recent years the genes encoding the enzymes
of the cycle have been cloned and this has allowed studies of
the control of their expression to be carried out. Not
surprisingly there is coordination of both mRNA synthesis
and protein accumulation during leaf development and
chloroplast maturation (Raines et al. 1991). Much of the
control has so far been found to occur at the level of gene
transcription.
Light has a central role in regulating the biogenesis of the
photosynthetic apparatus during chloroplast development, including switching on the expression of genes
encoding enzymes of the C3 photosynthetic carbon
reduction cycle. Plants grown in darkness have very low
levels of C3 cycle mRNAs, but on exposure to light there is
a rapid induction of mRNA synthesis within 1 h with
maximum levels nally attained after 12 days of a normal
lightdark growth regime. If the light is again withdrawn,
these mRNA levels gradually decline, although there may
be some detectable even after 48 h of darkness. Induction
on re-illumination of such plants is more rapid than in
plants that have never been exposed to light, suggesting
that the leaf has been primed to respond by previous
completion of light-induced chloroplast development. The
light response of C3 cycle genes appears to be mediated via
a number of photoreceptors, including phytochrome and
the blue light receptor, cryptochrome. Understanding the
light regulation of gene expression is a very active area of
research, but as yet no complete picture has emerged. The
processes involved appear to be complex, involving many
regulatory proteins and crosstalk between the dierent
photoreceptor signalling pathways (Terzaghi and Cashmore, 1995).
4

In mature leaves, C3 expression of photosynthetic

carbon reduction cycle genes is sensitive to environmental
and metabolic signals, providing long-term mechanisms
for the plant to regulate primary carbon xation. High
levels of glucose and sucrose have been shown to be
associated with reduced levels of a number of Calvin cycle
mRNAs, including those encoding the small subunit of
Rubisco, sedoheptulose-1,7-bisphosphatase and fructose1,6-bisphosphatase. This feedback mechanism, which
again appears to act at the level of transcription, might
be important for sourcesink regulation in the plant
(Krapp et al., 1993). The signalling pathway involved in
glucose repression of gene expression is not known,
although it has been suggested that the enzyme hexokinase
might be involved. Recently it has been shown that
photosynthesis genes can respond at the transcript level
to nutrient status, namely N and P levels, and that this is
modulated by carbohydrate status. These results suggest
that this interaction between carbohydrate and nutrient
status may function as a long-term strategy in the control
of primary carbon metabolism.

References
Brandes HK, Larimer FW and Hartman FC (1996) The molecular
pathway for the regulation of phosphoribulokinase by thioredoxin f.
Journal of Biological Chemistry 271: 33333335.
Chen XJ and Schnell DJ (1999) Protein import into chloroplasts. Trends
in Cell Biology 9: 222227.
Dunford RP, Durrant MC, Catley MA and Dyer TA (1998) Location of
the redox-active cysteines in chloroplast sedoheptulose-1,7-bisphosphatase indicates that it is allosteric regulation is similar but not
identical to that of fructose-1,6-bisphosphatase. Photosynthesis
Research 58: 221230.
Haake V, Zrenner R, Sonnewald U and Stitt M (1998) A moderate
decrease of plastid aldolase activity inhibits photosynthesis, alters the
levels of sugars and starch and inhibits growth of potato plants. The
Plant Journal 14: 147145.
Harrison EP, Willingham NM, Lloyd JC and Raines CA (1998) Reduced
sedoheptulose-1,7-bisphosphatase levels in transgenic tobacco lead to
decreased photosynthetic capacity and altered carbohydrate accumulation. Planta 204: 2736.
Jacquot J-P, Lopez-Jaramillo J, Chueca A et al. (1995) High-level
expression of recombinant pea chloroplast fructose-1,6-bisphosphatase and mutagenesis of its regulatory site. European Journal of
Biochemistry 229: 675681.
Krapp A, Hofman B, Schafer C and Stitt M (1993) Regulation of the
expression of rbcS and other photosynthetic genes by carbohydrates: a
mechanism for the sink regulation of photosynthesis? The Plant
Journal 3: 817828.
Raines CA, Lloyd JC and Dyer TA (1991) Molecular biology of the C3
photosynthetic carbon reduction cycle. Photosynthesis Research 27: 1
14.
Ruelland E and Migniac-Maslow M (1999) Regulation of chloroplast
enzyme activities by thioredoxins: activation or relief from inhibition?
Trends in Plant Science 4: 136141.
Scheibe R (1991) Redox modulation of chloroplast enzymes. Plant
Physiology 96: 13.

ENCYCLOPEDIA OF LIFE SCIENCES / & 2001 Nature Publishing Group / www.els.net

C3 Carbon Reduction Cycle

Stitt M and Schulze D (1994) Does Rubisco control the rate of

photosynthesis and plant growth? An exercise in molecular ecophysiology. Plant, Cell and Environment 17: 465487.
Suss K-H, Arkona C, Manteell R and Adler K (1993) Calvin cycle
multi-enzyme complexes are bound to chloroplast thylakoid membranes of higher plants in situ. Proceedings of the National Academy of
Sciences of the USA 90: 55145518.
Terzaghi WB and Cashmore AR (1995) Light-regulated transcription.
Annual Review of Plant Physiology and Plant Molecular Biology 46:
445474.
Wedel N, Soll J and Paap BK (1997) CP12 provides a new mode of light
regulation of Calvin cycle activity in higher plants. Proceedings of the
National Academy of Sciences of the USA 94: 1047910484.
Zhang N and Portis AR (1999) Mechanism of light regulation of
Rubisco: a specic role for the larger Rubisco activase isoform
involving reductive activation by thioredoxin-f. Proceedings of the
National Academy of Sciences of the USA 96: 94389443.

Geiger DR and Servaites JC (1995) Diurnal regulation of photosynthetic

carbon metabolism in C3 plants. Annual Review of Plant Physiology
and Plant Molecular Biology 45: 253256.
Hartman FC and Harpel MR (1994) Structure, function, regulation and
assembly of d-ribulose-1,5-bisphosphate carboxylase/oxygenase. Annual Review of Biochemistry 63: 197234.
Heldt H-W (1997) Photosynthetic CO2 assimilation by the Calvin cycle.
In: Plant Biochemistry and Molecular Biology, pp. l48165. Oxford:
Oxford University Press.
Portis AR (1992) Regulation of Rubisco activity. Annual Review of Plant
Physiology and Plant Molecular Biology 43: 415437.
Quick WP and Neuhaus HE (1997) The regulation and control of
photosynthetic carbon assimilation. In: Foyer CH and Quick WP (eds)
A Molecular Approach to Primary Metabolism in Higher Plants, pp.
4162. London: Taylor and Francis.
Taiz L and Zeiger E (1998) Photosynthesis: carbon reactions. In: Plant
Physiology, 2nd edn, pp. 195225. Sunderland, MA: Sinauer
Associates Inc.

Further Reading
Buchanan BB (1980) Role of light in the regulation of chloroplast
enzymes. Annual Review of Plant Physiology 31: 341374.

ENCYCLOPEDIA OF LIFE SCIENCES / & 2001 Nature Publishing Group / www.els.net