Spring.wmf (18300 bytes) Plant Physiology (Biology 327)  - Dr. Stephen G. Saupe;  College of St. Benedict/ St. John's University;  Biology Department; Collegeville, MN  56321; (320) 363 - 2782; (320) 363 - 3202, fax;    ssaupe@csbsju.edu

Autotrophism: Carbon Reactions (Calvin Cycle, C4, CAM)

I. The final frontier - Photosynthetic Carbon Reduction (step 3)

A. Carbon dioxide fixation
    Carbon dioxide is fixed (trapped, bound) to form an organic compound (phosphoglyceric acid, PGA)

  • carbon dioxide condenses with RuBP (ribulose bisphosphate; C5) to form 2 molecules of PGA (C3)
  • first product of carbon fixation is PGA (Calvin’s experiments)
  • catalyzed by the enzyme ribulose bisphosphate carboxylase (rubisco).
  • rubisco is the most abundant protein on earth; it makes up 50% of leaf protein
  • essentially, the carbon dioxide binds to the keto-carbon and then the resultant molecule splits

B. Reduction
    Step in which the temporary chemical (ATP) and reducing (NADPH) potentials that were generated in the light-dependent reactions are used to reduce the PGA (an acid) to a carbonyl (glyceraldehyde 3-phosphate; abbreviated G3P or GAP)

  • PGA is reduced to G3P
  • this is a two-step reaction sequence
  • first, PGA is phosphorylated with ATP to 1,3-bisphosophoglycerate which is subsequently reduced to G3P (note a phosphate is lost during this reaction). NADPH provides the electrons for the reduction
  • energy requirements - at this point in the cycle, for each carbon dioxide fixed, two ATP and two NADPH are required (one for each of the two PGA’s)

C. Rearrangement/Recharging/Release
    Complex series of reactions (rearrangment) that result in the net removal of a C3 carbohydrate from the cycle (release) and the production of the precursor to the starting material (recharging):

  • see overhead and diagram in text for details
  • the cycle must turn 3 times for the production of one net triose
  • the end product of the cycle is ribulose-5-P (RuP)
  • ATP converts ribulose-5-P to RuBP
  • ATP comes from the Z scheme

E. Summary
    The fixation of 1 carbon dioxide requires: 3 ATP and 2 NADPH.

II. Regulation of the Calvin Cycle
    We will not cover this in class except to say that regulation of the cycle is obviously important.  There are several regulatory controls:

    1. rubisco - light activated;
    2. allosteric regulation - rubisco has a binding site for CO2;
    3. rubisco activase - protein that "activates" rubisco; and
    4. Fd/thioredoxin - several enzymes require a reduction to become activated.

III. C3 Plants
    Plants that exhibit the type of photosynthetic carbon reduction that we described above are termed C3 plants. In other words, the first product of carbon dioxide fixation is a 3-carbon compound (PGA). Thus, when radioactively labeled carbon dioxide is fed to a plant, the first place that it shows up is PGA.

IV. Photorespiration
    Light stimulated production of carbon dioxide in the presence of oxygen

  • not associated with mitochondrial respiration
  • requires light
  • not accompanied by ATP synthesis
  • wastes energy (i.e., ATP, NADPH)

 A. Observations on photorespiration

  1. Not all plants photorespire - see plot of carbon dioxide production (= respiration rate) vs. time for tobacco and maize. Note the dark rates are the same, but the light rate is much greater in tobacco.
  2. Plants that photorespire typically show light saturation - points to note: (a) plot of carbon dioxide uptake (=Ps rate) vs. fluence for tobacco and maize in ambient oxygen (21%); (b) light saturation point - point at which increasing fluence yields a constant amount of photosynthesis; (c) light compensation point - fluence at which the amount of photosynthesis just equals the amount of respiration; and (d) note that plants that photorespire (like tobacco) have a higher light compensation point and light saturate.
  3. Plants that photorespire have a higher CO2 compensation point. In other words, it takes a greater amount of carbon dioxide to break even.
  4. Oxygen inhibits photosynthesis in plants that photorespire (called the Warburg effect) - plot carbon dioxide uptake vs. fluence for maize and tobacco at 1.5% oxygen
  5. Carbon dioxide is limiting to plants that photorespire - for evidence see: (a) plot of carbon dioxide fixation vs. carbon dioxide concentration for maize and red clover (photorespires); and (b) plot of carbon dioxide uptake vs. fluence for tobacco and maize at ambient oxygen at varying carbon dioxide levels.

B. Making sense of the data
    The data cited above suggest that carbon dioxide and oxygen have antagonistic (opposite) actions in photosynthesis and act in a competitive manner.

C. The problem - rubisco
    Unlike most enzymes, rubisco is not substrate specific - it also has an oxygenase function. In addition to its normal substrate (carbon dioxide) rubisco also binds oxygen to RuBP. Although rubisco has a higher affinity for binding carbon dioxide (Km = 9 �M), if enough oxygen is present, it acts as a competitive inhibitor (the Km for oxygen is 535 �M).

D. The reaction catalyzed by ribulose bisphosphate carboxylase/ oxygenase
    When rubisco binds oxygen to RuBP, the RuBP is essentially split in half to a 3 carbon piece and a 2 carbon fragment according to the following reaction:

RuBP + oxygen + rubisco PGA (C3)+ phosphoglycolate (C2)

Compare this to the normal reaction:

RuBP + oxygen + rubisco 2 PGA (C3)

Thus, rubisco has oxygenase activity as well as a carboxylase.

E. What determines which process will occur?  Oxygenase activity occurs when:

    1. carbon dioxide levels are low - during periods of active photosynthesis; and
    2. oxygen levels are high - due to the activity of PSII; high light intensity.

The ratio of [carbon dioxide]/[oxygen] ultimately determines the product of the rubisco reaction.

if [carbon dioxide/oxygen] = high; then it favors normal Calvin cycle
if [carbon dioxide/oxygen] = low; then it favors oxygenase activity

V. Photosynthetic carbon oxidation (PCO), or, Glycolate Cycle
    The purpose of this pathway is to metabolize and reclaim the carbon in phosphoglycolate

A. Overview of the major steps:

    1. The products of rubisco oxygenase activity are phosphoglycolate and PGA;
    2. PGA enters the Calvin cycle as normal;
    3. Phosphoglycolate is dephosphorylated to glycolate and is then shuttled out of the chloroplast into the peroxisome;
    4. Recall that peroxisomes are single membrane-bound organelles that contain catalase. They also have the marker enzyme glycolate oxidase;
    5. In the peroxisome, the glycolate is oxidized to glyoxylate by glycolate oxidase. This is a redox reaction. Oxygen gets reduced to hydrogen peroxide;
    6. Catalase converts the potentially destructive hydrogen peroxide to oxygen and water;
    7. Glyoxylate is converted to glycine (an amino acid) by a transamination reaction. Glycine is transported out of the peroxisome into the mitochondrion. Two glycine molecules condense to form serine releasing carbon dioxide. This process requires NADH;
    8. Serine is further metabolized in the peroxisome to glycerate;
    9. Glycerate enters the chloroplast, is phosphorylated and enters the Calvin cycle;

B. The Highlights - The glycolate cycle:

  • is oxidative;
  • occurs in three organelles;
  • reclaims some (75%), but not all, of the carbon from glycolate;
  • carbon dioxide is released in the mitochondria and is hence the reason this is a type of "respiration".

C. Why do plant photorespire?
    From a Darwinian perspective, we’d expect that this process would have been selected against. However, the fact that so many plants do it, suggests that it may have an unappreciated function. Possibilities include: (a) salvage the carbon lost during rubisco oxygenase action; (b) mechanism to help prevent destruction by excess light.

VI. C4 Photosynthesis, or, How maize avoids photorespiration
    Plants that avoid photorespiration have a unique modification of photosynthesis. They are called C4 plants because the first product of carbon dioxide fixation is a 4-carbon compound, not PGA as it is in C3 plants.

Examples: There are many plants that have this specialized modification. Found in many different and unrelated groups of plants which indicates that it apparently evolved independently several times. Even within a genus, some members can be C4 others C3.

    C4 photosynthesis is common in grasses like maize, sorghum, crabgrass and members of the Centrospermae (a closely related group of plants that includes Chenopodiaceae, Amaranthaceae, Aizoaceae, Nyctaginaceae, Portulaceae, Zygophyllaceae). Not all grasses are C4; for example, Kentucky blue grass (Poa pratensis; common lawn grass) is C3.

A. How do C4 plants avoid photorespiration?
    The answer is simple - C4 plants separate the site of oxygen production (PSII) from rubisco (Calvin cycle). But how? PSII and rubisco are placed in different:

    1. Cells. In typical C3 plants the chloroplasts are dispersed throughout the mesophyll. Usually there is a well-defined palisade and spongy layer. In contrast, C4's have a more or less uniform mesophyll layer with a well-developed bundle sheath around each vein. This is called Kranz anatomy, because the bundle sheaths appears like a wreath surrounding the vein. In C4 plants, the Calvin cycle activity occurs primarily in the bundle sheath cells, whereas PSII activity occurs in the mesophyll cells.
    2. Chloroplasts - The chloroplasts of C4 are dimorphic. Bundle sheath cell (BSC) chloroplasts are agranal. Recall that PSII occurs in the appressed regions of the chloroplasts. Thus, agranal chloroplasts have little PSII activity; but, they do have hi PSI activity. The mesophyll cell (MC) chloroplasts have typical granal stacking, but low rubisco activity. Thus, most carbon fixation (carbohydrate production) occurs in the bsc. Smart, eh?

B. Since C4 plants have separated the Calvin cycle PSII, there must be a mechanism to get carbon dioxide into the BSC since:

    1. there is relatively slow diffusion to deep, interior regions of the leaf, especially considering;
    2. the ambient level of carbon dioxide is low.

In order to solve this problem, plants required a mechanism to:

    1. fix carbon dioxide in regions of the leaf where it occurs in high concentration (i.e., MC). The enzyme that catalyzes this reaction is phosphoenolpyruvate carboxylase (PEPcase). This enzyme binds carbon dioxide (actually bicarbonate) to PEP to form oxaloacetate (reaction diagram). This reaction occurs in the cytoplasm. Note that OAA is a C4 compound. Hence these plants are called C4 - because the first product of carbon fixation is a four carbon compound.
    2. transport the fixed carbon dioxide (which is in the form of a C4 compound like malate or aspartate) from the MC to the BSC. OAA is converted to another C4 compound that, in turn, migrates to the BSC where it is decarboxylated and used in the Calvin cycle. The "leftover" C3 shuttles back to the MC to pick up another carbon dioxide and repeat the process.

C. General scheme - on overhead, covered in class

D. Details
    Note that there are at least three different types of C4 plants. They differ in specific form in which carbon dioxide is transported.

E. Advantages of C4 metabolism
    Plants that exhibit this type of photosynthesis are characteristic of hot, tropical environments that have a high light fluence. The advantage of C4 in these circumstances is that C4 metabolism:

    1. avoids the photorespiratory loss of carbon
    2. improves the water use efficiency of the plants
    3. results in higher rates of photosynthesis at high temperatures
    4. improves the efficiency of nitrogen utilization (because C3 require lots of rubisco)

VII. Crassulacean Acid Metabolism - CAM plants

A. Origin of the name
   Crassulacean refers to the Stonecrop family (Crassulaceae) and related succulents in which this process is common. To date, plants in more than 18 different families including Cactaceae (Cactus family) and Bromeliaceae (Pineapple family) have been shown to carry out CAM metabolism. Acid is derived from the observation that these plants accumulate large amounts of organic acids in the dark.

    Plants with CAM metabolism evolved in dry, hot, high light environments. This is largely a mechanism to conserve water. Recall the photosynthesis-transpiration compromise (paradox)? Plants in dry environments can’t afford to compromise - they loose too much water opening their stomates during the day. CAM plants solved this problem by opening up the stomates at night to obtain carbon dioxide. This strategy is just the reverse of "normal" plants. But, this presents another problem - ATP & NAPDH, which are products of the light dependent reactions, are not available when the carbon dioxide is fixed. The solution to this problem was to store the carbon dioxide during the night until ATP and NADPH were available the following day. Thus, there is a temporal separation of initial carbon fixation via PEPcase and the Calvin cycle (C4 plants have a spatial separation).

B. PEPcase
    This is the initial enzyme that fixes carbon dioxide. The product is ultimately malate which accumulates in the vacuole during the night (hence the "acid" term).

C. Sequence of events.
 stomates open  nocturnal transpiration (lower than diurnal) and carbon fixation by PEPcase  OAA produced reduced with NADPH to malate shuttled into vacuole acid content of vacuole increases starch depleted to provide PEP for carboxylation day stomates close transpiration decreased acid content decreases malate decarboxylated to provide carbon dioxide for Calvin cycle starch content increases

VIII. Comparison of C3, C4 and CAM Photosynthesis





Leaf anatomy

no distinct bundle sheath

Kranz anatomy

Usually no palisade cells, large vacuoles

Initial carboxylating enzyme




Product of CO2 fixation

PGA  (C3)

OAA (C4)

OAA (C4)


one type


one type

Theoretical energy requirements (CO2: ATP: NADPH)

1: 3: 2

1: 5 : 2

1: 6.5: 2

Transpiration ratio (g H2O/g dry wt)




Photosynthesis rate (mg CO2 fixed dm-2 h-1)

15 - 30

40 - 80


chl a/b ratio



2.5 - 3.0

Requirement for sodium as a micronutrient?




Carbon dioxide compensation point (ppm)

50 - 150 (Hi)

0-10 (low)

0-5 (in dark)

Response to light

Light saturation easily achieved

No light saturation


Photosynthesis inhibited by oxygen?




Photorespiration detectable?


Only in bundle sheath

Late afternoon

Temperature optimum for photosynthesis




Dry matter production (bushels/acre)

Low (26 � soybean; 30 � wheat)

High (87 � maize; 50 � sorghum)

low, variable

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Last updated:  03/23/2009     � Copyright  by SG Saupe