C₄ and CAM

The Tragedy of Rubisco: An Enzyme That Makes Mistakes

Imagine the most abundant enzyme on Earth. Its total mass on the planet is about 10 million tonnes—roughly 2 kilograms for every inhabitant of the Earth (Medvedev, 2012). It is the enzyme that feeds the entire biosphere. And at the same time, it is one of the slowest and most imperfect enzymes in nature.

Rubisco catalyses the reaction between ribulose‑1,5‑bisphosphate (RuBP) and carbon dioxide, yielding two molecules of 3‑phosphoglycerate—the very C₃ compound that gave its name to all C₃ plants (Taiz et al., 2023). A seemingly simple and reliable reaction. But, as we mentioned in the lecture on respiration, Rubisco has another function: oxygenase activity.

This feature of Rubisco was discovered in the 1970s, when researchers using the isotope ¹⁸O showed that Rubisco can bind not only CO₂ but also molecular oxygen (Morot‑Gaudry et al., 2012). CO₂ and O₂ are competitive substrates. And the competition is unequal.

Why Is the Competition Unequal?

In the modern atmosphere, which formed about 350 million years ago, oxygen content is _{21%, while carbon dioxide is only} 0.04% (420 ppm). This means that oxygen around Rubisco is about 500 times more abundant than carbon dioxide (Schopfer & Brennicke, 2016). Imagine a worker on a factory floor who must pick the right part, but the wrong parts outnumber the correct ones by 500 times. How long will he work without errors?

For C₃ plants, photorespiration can account for 15 to 50% of the photosynthetic rate (Kuznetsov, 2006). In hot conditions, when stomata close to conserve water, the internal CO₂ concentration drops, and the percentage of oxygen‑dependent "errors" by Rubisco can exceed 50% (Morot‑Gaudry et al., 2012).

Here is what happens at the molecular level. Rubisco binds O₂ to RuBP, and instead of two molecules of 3‑phosphoglycerate, it produces one molecule of 3‑phosphoglycerate and one molecule of 2‑phosphoglycolate (Taiz et al., 2023). 2‑Phosphoglycolate is a toxic compound that must be rapidly detoxified.

The Glycolate Cycle: An Expensive "Error Correction"

To process phosphoglycolate, the plant launches an entire metabolic pathway, called the glycolate cycle or photorespiration (Taiz et al., 2023). This is a true metabolic "conveyor" that operates at the junction of three organelles: chloroplast, peroxisome, and mitochondrion (Schopfer & Brennicke, 2016).

Let us trace this pathway (Taiz et al., 2023; Morot‑Gaudry et al., 2012):

1. In the chloroplast, phosphoglycolate is dephosphorylated to glycolate, which exits the chloroplast into the cytosol.

2. In the peroxisome, glycolate is oxidised by glycolate oxidase to glyoxylate. This reaction produces toxic hydrogen peroxide (H₂O₂), which is immediately broken down by catalase.

3. Glyoxylate undergoes transamination to become the amino acid glycine.

4. In the mitochondrion, the key step occurs: two molecules of glycine combine to form one molecule of serine, releasing one molecule of CO₂ and ammonia.

5. Serine returns to the peroxisome, then is converted to glycerate, which re‑enters the chloroplast and is phosphorylated to 3‑phosphoglycerate, returning to the Calvin cycle.

What is the net result (Taiz et al., 2023; Morot‑Gaudry et al., 2012)? From two molecules of phosphoglycolate (4 carbon atoms) we obtain one molecule of 3‑phosphoglycerate (3 carbon atoms). One carbon atom is irreversibly lost as CO₂. Moreover, the process consumes ATP and reducing equivalents that could have been used for carbohydrate synthesis. Pure losses!

An Evolutionary Paradox

A legitimate question arises: why hasn't nature gotten rid of this defective enzyme? Why does the most abundant carboxylase on Earth continue to make mistakes?

The answer lies in evolutionary history (Schopfer & Brennicke, 2016). Photosynthesis arose about 2.5–3 billion years ago, when the Earth's atmosphere was oxygen‑free and contained much more CO₂. Under such conditions, Rubisco was an almost ideal carboxylase, working at full capacity. Then, about 2.4 billion years ago, an event occurred that forever changed the biosphere—the Great Oxidation Event. Bacteria performing oxygenic photosynthesis saturated the atmosphere with oxygen. The concentration of CO₂ began to fall. Rubisco found itself in a completely new, hostile atmosphere. But evolution cannot simply "rewrite" the genetic code of this key enzyme—it is too integrated into cellular metabolism (Medvedev, 2012).

The specificity of Rubisco for CO₂ relative to O₂, expressed as the specificity factor S_c/o, is 82–90 for C₃ plants, 70–82 for C₄ plants, and only about 40 for cyanobacteria (Taiz et al., 2023). The higher the S_c/o, the better the enzyme selects CO₂, but the slower it works. This is a fundamental trade‑off (Lambers & Oliveira, 2019).

The Solution: Raise the CO₂ Concentration

Modern C₃ plants have three ways to minimise photorespiration (Taiz et al., 2023; Connor et al., 2011):

1. Lower O₂ concentration—impossible under normal respiration.

2. Raise CO₂ concentration in the atmosphere—impossible globally, but possible locally, inside the leaf.

3. Lower temperature—impossible in the tropics.

It is the second pathway—creating a zone of elevated carbon dioxide within the leaf—that became the key evolutionary solution. Nature took two different routes:

• Spatial separation—C₄ photosynthesis.
• Temporal separation—CAM photosynthesis.

Both strategies use the same molecular mechanism—double carboxylation. The first carboxylation is fast, energy‑efficient, and insensitive to oxygen—it acts as a "pump" that collects CO₂. The second carboxylation is slow and highly CO₂‑specific (classical Rubisco)—it occurs in a zone where the CO₂ concentration is much higher than in the atmosphere (Taiz et al., 2023; Schopfer & Brennicke, 2016).

Key Takeaway of This Section

The main reason for the emergence of C₄ and CAM pathways is an evolutionary response to the imperfection of Rubisco. In an atmosphere with high O₂ and low CO₂, plants faced a dilemma: either tolerate enormous carbon losses through photorespiration (up to 50%!) or develop mechanisms to concentrate CO₂ around Rubisco. This "evolutionary race" led to the emergence of two alternative strategies.

Neither C₄ nor CAM create a new enzyme—they simply reorganise already existing molecular machinery (Schopfer & Brennicke, 2016):

• Phosphoenolpyruvate carboxylase (PEPCase, PEP carboxylase)—an enzyme that in C₃ plants participates in anaplerotic reactions and pH regulation.
• Calvin cycle enzymes—the same as in C₃ plants.
• The difference lies only in where and when these reactions occur.

That is why studying C₄ and CAM photosynthesis is not merely an introduction to botanical exotica. It is an understanding of fundamental physiological adaptations that allow plants to survive and be productive in a wide range of environments—from arid deserts to tropical savannas and hot fields planted with maize and sugarcane.

In the next part of our lecture, we will examine in detail how C₄ photosynthesis is organised and why it allows plants to win the struggle for survival in hot climates. Then we will meet CAM plants, which took a different path—they did not rearrange leaf anatomy but simply changed their daily rhythm.

The Anatomical Solution: Kranz Anatomy

The most obvious feature when looking at a C₄ plant leaf is its anatomy (Schopfer & Brennicke, 2016; Medvedev, 2012).

Recall the leaf structure of a C₃ plant: palisade parenchyma (columnar tissue) and spongy parenchyma—all mesophyll where photosynthesis occurs. In C₄ plants, it is different:

• The vascular bundles (veins) are surrounded by a double ring of cells.
• The inner ring—bundle sheath cells. They are large, with thick walls and large chloroplasts that often contain much starch.
• The outer ring—mesophyll cells. They are small and tightly packed.

This structure is called Kranz anatomy (from the German Kranz—"wreath" or "crown") (Schopfer & Brennicke, 2016). This anatomy is not mere decoration. It provides a diffusion barrier that prevents the CO₂ accumulated in the inner cells from escaping back into the atmosphere. It is this barrier that makes C₄ photosynthesis so efficient (Taiz et al., 2023).

It is important to note that Kranz anatomy is not the only possible option. In some plants of the family Chenopodiaceae (e.g., Bienertia and Suaeda aralocaspica), C₄ photosynthesis occurs within a single cell (Taiz et al., 2023; Schopfer & Brennicke, 2016). In that case, specialised regions of the cell perform the functions of mesophyll and sheath. This shows that the essential requirement for C₄ photosynthesis is not the presence of two cell types, but the creation of a zone of elevated CO₂ concentration, surrounded by a diffusion barrier.

Two Carboxylases: A Game‑Changing Tandem

The key idea of C₄ photosynthesis is two carboxylases working sequentially in two different cellular compartments (Taiz et al., 2023; Morot‑Gaudry et al., 2012).

First carboxylase: PEP carboxylase (PEPCase)

In mesophyll cells, an enzyme that plays a minor role in C₃ plants works—phosphoenolpyruvate carboxylase (PEPCase). It does not bind CO₂ itself but bicarbonate (HCO₃⁻), which is formed from CO₂ by the enzyme carbonic anhydrase (Taiz et al., 2023; Morot‑Gaudry et al., 2012).

PEP carboxylase has three key properties (Taiz et al., 2023):

1. It is not inhibited by oxygen—it does not suffer from photorespiration.

2. It has a high affinity for its substrate—it works efficiently even at low CO₂ concentrations.

3. It works fast—the reaction rate is significantly higher than that of Rubisco.

The substrate for PEP carboxylase is phosphoenolpyruvate (PEP)—a three‑carbon compound. The reaction produces oxaloacetate—a four‑carbon acid (hence the name C₄ photosynthesis).

Oxaloacetate is reduced to malate (or converted to aspartate)—these C₄ acids become the transport form of CO₂ (Taiz et al., 2023; Morot‑Gaudry et al., 2012). This is a true "molecular container" for CO₂, which prevents it from leaking out and allows safe transport of carbon deeper into the leaf.

Second carboxylase: Rubisco (in bundle sheath cells)

The C₄ acid (malate or aspartate) passes through plasmodesmata from the mesophyll into the bundle sheath cells. There, decarboxylation (removal of CO₂) occurs (Schopfer & Brennicke, 2016; Taiz et al., 2023):

The released CO₂ creates in the bundle sheath cells a concentration 10–20 times higher than atmospheric (Schopfer & Brennicke, 2016). Under these conditions, Rubisco shows practically no oxygenase activity—photorespiration is suppressed. It is here, in the bundle sheath cells, that the classical Calvin cycle with normal Rubisco operates (Taiz et al., 2023; Kuznetsov, 2006).

At the same time, the pyruvate formed during decarboxylation returns to the mesophyll, where it is regenerated into PEP (with the expenditure of ATP)—the CO₂ acceptor. The cycle closes.

Three Subtypes of C₄ Plants

Nature did not stop at one solution. Depending on which enzyme is involved in the decarboxylation of C₄ acids in the bundle sheath cells, C₄ plants are divided into three subtypes (Taiz et al., 2023; Morot‑Gaudry et al., 2012; Medvedev, 2012):

These differences are not just academic interest. They reflect the evolutionary diversity of C₄ plants, which arose independently in different lineages—by various estimates, from 45 to 66 times in the history of angiosperms (Taiz et al., 2023; Schopfer & Brennicke, 2016). This is a striking example of convergent evolution: similar adaptations arise independently in unrelated groups under similar ecological pressures.

The Cost: Energy Expenditure

It is important to understand that the C₄ pump does not work for free. It consumes additional energy (Taiz et al., 2023; Morot‑Gaudry et al., 2012):

• Regenerating PEP from pyruvate requires 2 molecules of ATP for each molecule of CO₂ "pumped".
• In the Calvin cycle, fixing CO₂ requires another 3 ATP and 2 NADPH.

Thus, for fixing one CO₂ molecule, a C₄ plant uses 5 ATP and 2 NADPH, whereas a C₃ plant uses only 3 ATP and 2 NADPH.

At low temperatures, when photorespiration in C₃ plants is minimal, the C₄ pathway is energetically less advantageous (Connor et al., 2011). This is why C₄ plants dominate in warm climates (above 25–30 °C) but are practically absent from cold regions.

Key Takeaway of This Section

C₄ photosynthesis is a "molecular pump" that creates a zone of elevated CO₂ concentration around Rubisco. This pump works through:

1. Cellular separation (or, in some species, intracellular)—mesophyll captures CO₂, bundle sheath cells fix it.

2. Two carboxylases—fast PEP carboxylase is not afraid of oxygen; slow Rubisco works in an environment where its errors are minimised.

3. Additional energy costs—the price for efficiency.

As a result, the plant can almost completely suppress photorespiration and conserve water, which is especially important in hot and dry climates.

Connection to Previous Sections

Recall what we discussed in the lecture on respiration: photorespiration is not just a "mistake" of Rubisco, but also an important metabolic process linked to nitrogen metabolism and protection against photooxidation. However, in C₄ plants, although this process does not disappear entirely, it becomes so insignificant that it is hard to detect by standard methods.

C₄ plants are characterised by:

• Absence of measurable Warburg effect (inhibition of photosynthesis by oxygen) (Schopfer & Brennicke, 2016).
• Very low CO₂ compensation point—around 2–10 ppm (compared to 40–60 ppm for C₃ at 25 °C) (Morot‑Gaudry et al., 2012).
• High water‑use efficiency—250–350 g of water per 1 g of dry matter produced, versus 450–950 g for C₃ plants (Tretyakov, 2000).

Energy Balance: Where Is It Advantageous and Where Not?

At first glance, the C₄ pathway seems unequivocally better: it suppresses photorespiration and improves water‑use efficiency. However, this comes at the cost of extra energy. As we recall, fixing one CO₂ molecule in a C₄ plant costs 5 ATP, whereas in C₃ it costs only 3 ATP (Taiz et al., 2023; Morot‑Gaudry et al., 2012).

But at low temperatures (below ~20–25 °C), photorespiration in C₃ plants is minimal. Under these conditions, the extra two ATP spent on the C₄ pump become unjustified—C₃ plants are energetically more efficient (Connor et al., 2011; Schopfer & Brennicke, 2016).

Key difference:

• C₃ plants win in cool and moist climates where photorespiration is low.
• C₄ plants become competitive at temperatures above 25–30 °C, when photorespiration in C₃ rises sharply, and the extra energy cost of the C₄ pathway is offset by the suppression of photorespiration (Taiz et al., 2023).

That is why in temperate zones C₃ plants predominate, while in the tropics and subtropics C₄ plants dominate. For example, in North America the boundary between C₃ and C₄ grasses lies roughly at 40°N latitude, correlating with the temperature of the growing season (Taiz et al., 2023; Schopfer & Brennicke, 2016). In Australia, the distribution of C₄ species is determined by the temperature of the warmest month (Lambers & Oliveira, 2019).

Water‑Use Efficiency (WUE): The Main Weapon of C₄

One of the most famous advantages of C₄ plants is high water‑use efficiency. This is related to how the C₄ pump works and how it affects stomatal conductance.

Recall that in C₄ plants, the intercellular CO₂ concentration (Cᵢ) is significantly lower than in C₃—about 100–150 ppm versus 250–280 ppm (Taiz et al., 2023; Connor et al., 2011). This means that to maintain the same CO₂ flux into the leaf, C₄ plants require lower stomatal conductance—stomata can be more closed.

Since water loss through transpiration is directly proportional to stomatal conductance, C₄ plants lose less water per unit of CO₂ fixed (Taiz et al., 2023; Medvedev, 2012).

The numbers speak for themselves (Tretyakov, 2000):

• C₃ plants use 450–950 g of water to produce 1 g of dry matter.
• C₄ plants use only 250–350 g of water.

That is almost a twofold advantage! This is why C₄ plants dominate in arid regions, savannas, and steppes. During droughts, when stomata close, C₄ plants maintain photosynthetic activity longer than C₃ plants (Pessarakli, 2020).

Note that the difference in WUE is not due to any special "economical" transpiration mechanism. It is a direct consequence of the C₄ pump, which allows stomata to be more closed at the same photosynthetic rate (Connor et al., 2011; Schopfer & Brennicke, 2016).

Nitrogen‑Use Efficiency (NUE): Saving on Protein

The second important advantage of C₄ plants is more efficient use of nitrogen (Brown, 1978; Lambers & Oliveira, 2019).

Rubisco is the most abundant protein on Earth, and it contains a lot of nitrogen. In C₃ plants, Rubisco can account for up to 20–30% of total soluble leaf protein, and together with other photosynthetic proteins, up to 50% of total leaf nitrogen (Lambers & Oliveira, 2019; Taiz et al., 2023).

In C₄ plants, the situation is different:

• Rubisco works in an environment with high CO₂ concentration, so its catalytic efficiency can be utilised almost 100%.
• C₄ plants need significantly less Rubisco—about 3–6 times less than C₃ plants per unit of photosynthetic activity (Schopfer & Brennicke, 2016; Taiz et al., 2023).

Result: C₄ plants have higher photosynthetic activity per unit of nitrogen (PNUE—photosynthetic nitrogen‑use efficiency). This gives them an advantage on nitrogen‑poor soils, where competition for this resource is particularly intense (Brown, 1978; Lambers & Oliveira, 2019).

It is important to note that C₄ plants do not necessarily have a lower total nitrogen requirement—their high productivity may even increase absolute nitrogen uptake. But per unit of fixed carbon, less nitrogen is used (Connor et al., 2011). This is especially noticeable in C₄ grasses compared to C₃ legumes (Sadras & Calderini, 2015).

Temperature Optimum: Why C₄ Plants Love Heat

The third key advantage of C₄ plants is their tolerance to high temperatures (Schopfer & Brennicke, 2016; Taiz et al., 2023).

In C₃ plants, rising temperatures cause two unfavourable effects:

1. The solubility of CO₂ in water decreases faster than that of O₂, increasing the O₂/CO₂ ratio in the cell.

2. Rubisco's specificity for CO₂ decreases—oxygenase activity rises faster than carboxylase activity.

As a result, at temperatures above 30 °C, photorespiration in C₃ plants becomes enormous. In C₄ plants, where Rubisco is protected by high CO₂ concentration, this does not happen. Therefore, the temperature optimum for photosynthesis in C₄ plants is higher than in C₃: 30–45 °C versus 15–25 °C (Schopfer & Brennicke, 2016; Taiz et al., 2023).

A classic example is the desert plant Tidestromia oblongifolia, whose photosynthesis is maximal at 47 °C (Schopfer & Brennicke, 2016; Lambers & Oliveira, 2019). In such extreme conditions, C₃ plants simply cannot survive.

Evolutionary Victory: Why C₄ Plants Conquered the Planet

C₄ photosynthesis did not appear yesterday. The first C₄ plants emerged about 30 million years ago, but their widespread expansion began about 6–8 million years ago (Taiz et al., 2023; Schopfer & Brennicke, 2016).

What triggered this "explosion"? The decline in atmospheric CO₂ concentration during the late Miocene. When CO₂ fell below a critical threshold (~500–600 ppm), C₃ plants faced a sharp increase in photorespiration. C₄ plants, able to concentrate CO₂, gained the upper hand and began to outcompete C₃ plants in warm regions (Taiz et al., 2023; Connor et al., 2011).

Today, C₄ plants make up about 5% of all higher plant species, but they account for roughly 20–25% of global terrestrial primary production (Taiz et al., 2023; Lambers & Oliveira, 2019). Among the most important agricultural crops, the C₄ pathway is used by maize, sorghum, sugarcane, and millet.

When Does C₄ Not Win?

The advantages of C₄ photosynthesis do not always manifest. Here are conditions in which C₄ plants lose out:

1. Shade: under low light, the extra energy‑consuming C₄ pump becomes a burden (Connor et al., 2011; Lambers & Oliveira, 2019). That is why C₄ plants are practically absent from forest understories.

2. Cold climate: at temperatures below 15–20 °C, C₄ plants lose their advantage due to the low efficiency of enzymes, especially pyruvate phosphate dikinase, which is very sensitive to cold (Lambers & Oliveira, 2019; Schopfer & Brennicke, 2016).

3. High CO₂: if atmospheric CO₂ rises to 600–800 ppm, photorespiration in C₃ plants will decrease, and the C₄ advantage may disappear (Schopfer & Brennicke, 2016; Taiz et al., 2023).

Summary Comparison of C₃ and C₄

Key Takeaway of This Section

C₄ photosynthesis is an evolutionary adaptation to hot, dry, and arid climates where C₃ plants suffer from photorespiration and water shortage. The advantages of C₄ are expressed in:

1. High WUE—up to twice as efficient in water use.

2. High NUE—less nitrogen spent on the photosynthetic apparatus.

3. Heat tolerance—maintains high photosynthesis at 30–45 °C.

But these advantages come at a price: extra energy (2 ATP per CO₂), complex anatomy, low efficiency in shade and cold. Therefore, C₄ plants are not an "improved version" of photosynthesis but a specialised strategy that works brilliantly in its ecological niches.

Connection to Previous Sections

Earlier we discussed that photorespiration is not just a carbon loss but also an important process linked to nitrogen metabolism, protection against photooxidation, and amino acid synthesis. In C₄ plants, this process does not disappear completely but becomes so minor that it is difficult to detect by standard methods. C₄ plants retain all photorespiratory enzymes—they are simply not needed in the same amount (Taiz et al., 2023).

In the next part, we will meet CAM photosynthesis—another alternative strategy that differs from C₄ fundamentally: not spatial but temporal separation of the two carboxylases.

Night Phase: Storing Carbon Dioxide

Unlike C₄ plants, CAM plants lack Kranz anatomy and two cell types. Everything happens in the same cells, but at different times of day (Taiz et al., 2023; Morot‑Gaudry et al., 2012).

At night, the stomata of CAM plants are wide open. This seems odd—since there is no light for photosynthesis at night. But for CAM plants, night is a time of active work (Schopfer & Brennicke, 2016; Taiz et al., 2023).

During the night:

1. CO₂ from the atmosphere enters the leaf cells through open stomata.

2. In the cytosol, PEP carboxylase (the same as in C₄ plants!) binds HCO₃⁻ (formed from CO₂ by carbonic anhydrase) with phosphoenolpyruvate, producing oxaloacetate.

3. Oxaloacetate is reduced to malate using NADH (not NADPH, as in C₄ plants).

4. Malate is actively transported into the vacuole and accumulates there as malate or malic acid. By morning, the vacuolar concentration of malate can reach 100 mM, acidifying the cell sap (pH drops to 3.5–4.0) (Taiz et al., 2023; Morot‑Gaudry et al., 2012; Schopfer & Brennicke, 2016).

Thus, during the night, the plant stores CO₂ in the form of organic acid. The vacuole acts as a reservoir, allowing a significant amount of carbon to be accumulated without immediate fixation in the Calvin cycle.

The source of phosphoenolpyruvate is stored starch, which is hydrolysed to glucose at night and then through glycolysis yields PEP (Morot‑Gaudry et al., 2012; Schopfer & Brennicke, 2016).

Day Phase: Using the Stored Carbon

During the day, stomata are closed (to prevent water loss). Photosynthesis proceeds using the malate stored overnight (Taiz et al., 2023; Lambers & Oliveira, 2019):

1. Malate leaves the vacuole into the cytosol.

2. In the cytosol or chloroplasts, decarboxylation of malate occurs, catalysed by malic enzyme (NAD⁺‑ or NADP⁺‑dependent, depending on the CAM subtype).

3. The released CO₂ enters the chloroplasts and is fixed by Rubisco in the Calvin cycle—just as in C₃ plants.

4. At this time, the CO₂ concentration in the leaf is very high (may reach 10,000 ppm or more), so photorespiration is fully suppressed (Taiz et al., 2023).

The product of decarboxylation—pyruvate—regenerates PEP (with ATP expenditure) and can be used for starch synthesis, which will again serve as a source of PEP the next night.

Key difference from C₄: in C₄ plants, CO₂ is "pumped" from mesophyll to bundle sheath cells; in CAM plants, CO₂ is "stored" at night in the vacuole and released during the day. But the molecular machinery—the same enzymes: PEP carboxylase, malate dehydrogenase, malic enzyme, Rubisco (Taiz et al., 2023; Morot‑Gaudry et al., 2012).

Four Phases of the CAM Cycle

For convenience, the daily rhythm of CAM plants is divided into four phases (Taiz et al., 2023; Schopfer & Brennicke, 2016):

In nature, the prominence of phases depends on species, light conditions, and humidity. In many CAM plants, phases II and IV can be very short or absent altogether (Taiz et al., 2023; Lambers & Oliveira, 2019).

Energetics and Productivity of CAM

The CAM pathway, like C₄, requires additional energy expenditure. Extra ATP is used for malate transport into the vacuole and for regeneration of PEP from pyruvate (Taiz et al., 2023; Lambers & Oliveira, 2019).

However, the main limitation of CAM is vacuolar capacity. Only a limited amount of malate can be stored at night, so daytime CO₂ fixation is constrained by vacuole volume. This explains why most CAM plants have succulent leaves or stems—large vacuoles provide greater storage capacity for malate (Schopfer & Brennicke, 2016; Lambers & Oliveira, 2019).

Overall, CAM plant productivity is lower than that of C₄ and even many C₃ plants. However, there are exceptions: with good irrigation and nutrition, pineapple and agaves can yield as much as C₄ crops (up to 20–30 t/ha dry matter per year) (Taiz et al., 2023; Lambers & Oliveira, 2019).

CAM‑Idling and Facultative CAM

CAM photosynthesis is not rigid. Many plants can switch between C₃ and CAM modes depending on conditions (Taiz et al., 2023; Lambers & Oliveira, 2019).

Facultative CAM (or inducible CAM) appears under drought, salinity, or high temperature. A classic example is Mesembryanthemum crystallinum (ice plant). Under normal water supply, it behaves as a C₃ plant. Under salinity or drought, stomata close during the day and the CAM cycle is activated (Lambers & Oliveira, 2019; Schopfer & Brennicke, 2016).

CAM‑idling is an extreme mode where the plant keeps stomata closed both day and night during severe drought. In this case, CO₂ released by respiration is refixed through the CAM cycle (recycling). Photosynthesis nearly stops, but the plant survives conditions in which others die (Taiz et al., 2023; Lambers & Oliveira, 2019).

CAM‑cycling is another variant where malate accumulates at night, but stomata are closed and external CO₂ is not taken up. All carbon for malate comes from respiration. This reduces CO₂ loss from respiration and saves water (Lambers & Oliveira, 2019).

The Main Advantage of CAM: Incredible WUE

CAM plants are absolute record holders for water‑use efficiency. Recall the values (Tretyakov, 2000; Lambers & Oliveira, 2019):

What is the secret? CAM stomata open at night, when temperatures are low, humidity is high, and the water vapour gradient between leaf and atmosphere is minimal. Thus, transpirational water loss is minimal. During the day, when the gradient is steep, stomata are closed, and water is conserved (Taiz et al., 2023; Schopfer & Brennicke, 2016).

This is why CAM plants dominate in extremely arid habitats: deserts, semi‑deserts, rocky outcrops, and epiphytic conditions (on tree trunks in tropical forests where water is unavailable for long periods) (Taiz et al., 2023; Lambers & Oliveira, 2019).

C₄ and CAM: Two Strategies on the Same Foundation

Now that we know both mechanisms, we can clearly see their similarities and differences:

Similarities:

• Both use double carboxylation (PEP carboxylase + Rubisco).
• Both raise CO₂ concentration around Rubisco, suppressing photorespiration.
• Both use additional energy (ATP) for the "pump".

Differences:

• C₄: spatial separation (mesophyll and bundle sheath cells).
• CAM: temporal separation (night and day).

Ecological niches:

• C₄—hot and relatively moist or seasonally dry climates (savannas, steppes, tropical pastures, agricultural fields).
• CAM—extremely arid or periodically dry climates (deserts, semi‑deserts, epiphytic habitats, rocky outcrops).

Key Takeaway of This Section

CAM photosynthesis is a survival strategy in extreme drought based on temporal separation of the two carboxylases. At night, stomata open and the plant stores CO₂ as malate in vacuoles. During the day, stomata close and the stored CO₂ is used for photosynthesis.

The main advantage of CAM is extremely high water‑use efficiency (WUE), achieved through nocturnal stomatal opening. The price is limited productivity due to small vacuolar capacity and extra energy costs.

Connection to Previous Sections

Recall that we discussed photorespiration as a "release" of excess photochemical energy. In CAM plants, this release also occurs, but it is minimised because Rubisco works under high CO₂ concentration (Taiz et al., 2023). However, unlike C₄, CAM plants lack Kranz anatomy, and during the transition from night to day, brief photorespiration may occur, especially in phases II and IV.

Now that we have covered all three strategies (C₃, C₄, and CAM), it is time to compare them and understand under which conditions each is evolutionarily best. This is the subject of the final section of our lecture.

Comparative Table: C₃, C₄, and CAM

Let us gather everything we have learned into a single comparative table. It will help us see the logic of the distribution of the three strategies in nature (Taiz et al., 2023; Connor et al., 2011; Lambers & Oliveira, 2019; Schopfer & Brennicke, 2016; Tretyakov, 2000; Kuznetsov, 2006).

When Does C₃ Win?

The C₃ strategy is the original and universal one. It dominates in temperate latitudes, where temperatures rarely exceed 25–30 °C and humidity is high enough for stomata to remain open most of the day (Taiz et al., 2023; Connor et al., 2011).

Key advantages of C₃ (Lambers & Oliveira, 2019; Schopfer & Brennicke, 2016):

• Low energy cost—no extra ATP for a pump.
• Efficiency at low temperatures—enzymes work well, photorespiration is minimal.
• Broad range of adaptations—enormous diversity of C₃ species adapted to a wide variety of conditions.
• High efficiency in shade—under low light, the C₄ pump becomes a burden.

Where C₃ loses:

• In hot climates—photorespiration increases sharply.
• Under drought—low WUE leads to rapid dehydration.
• On nitrogen‑poor soils—high nitrogen costs for Rubisco.

Examples: Wheat and rice—two of the world's most important food crops—are C₃ plants. That is why breeders have long dreamed of "installing" a C₄ mechanism in them. But, as we will see shortly, this task is extremely challenging (Connor et al., 2011; Taiz et al., 2023).

When Does C₄ Win?

The C₄ strategy is an adaptation to heat and drought. It dominates in the tropics and subtropics, where C₃ plants suffer from photorespiration and water shortage (Taiz et al., 2023; Schopfer & Brennicke, 2016).

Key advantages of C₄ (Taiz et al., 2023; Lambers & Oliveira, 2019; Connor et al., 2011):

• Suppression of photorespiration—almost complete, even at high temperatures.
• High WUE—up to twice as efficient in water use compared to C₃.
• High NUE—more CO₂ fixed per unit of nitrogen.
• High productivity—under optimal conditions, they give the highest biomass increments.

Where C₄ loses (Taiz et al., 2023; Schopfer & Brennicke, 2016):

• In cold climates—enzymes of the C₄ pathway (especially pyruvate phosphate dikinase) are cold‑sensitive.
• Under low light—extra energy costs are not recovered.
• On very poor soils—high productivity requires adequate nutrition.

Examples: Maize and sugarcane are among the most productive crops on the planet. It is the C₄ pathway that underpins their phenomenal yields, especially in warm climates. Sorghum and millet are crucial crops in the arid regions of Africa and Asia.

When Does CAM Win?

The CAM strategy is an adaptation to extreme drought. It is a survival pathway where water is the critical limiting factor (Taiz et al., 2023; Lambers & Oliveira, 2019).

Key advantages of CAM (Taiz et al., 2023; Lambers & Oliveira, 2019; Schopfer & Brennicke, 2016):

• Incredibly high WUE—absolute record among all plants (up to 3–5 times higher than C₄).
• Ability to survive without water for months—CAM‑idling allows enduring prolonged droughts.
• Flexibility—many CAM plants can switch between C₃ and CAM depending on conditions.

Where CAM loses (Taiz et al., 2023; Lambers & Oliveira, 2019):

• Low productivity—limited vacuolar capacity prevents storing much CO₂.
• Slow growth—compared to C₄ and even many C₃.
• Specialised anatomy—succulence requires high investment in structural tissues.

Examples: Pineapple and agave are CAM crops that produce yields in conditions where other crops cannot survive. Cacti and euphorbias are dominants of desert ecosystems. Epiphytic orchids, which in tropical forests often experience water shortage, also use CAM.

Where Are C₄ and CAM Found in Nature?

The ecological distribution of the three strategies is one of the clearest examples of the link between plant physiology and climate (Taiz et al., 2023; Connor et al., 2011; Schopfer & Brennicke, 2016).

C₄ plants:

• Dominate in savannas, tropical and subtropical steppes.
• In Australia, C₄ species make up over 60% of the flora in regions with warm and humid summers.
• In North America, the boundary between C₃ and C₄ grasses lies roughly at 40°N latitude, correlating with the temperature of the warmest month (Taiz et al., 2023).
• In mountains, C₄ plants occur only up to a certain altitude—above the line where temperature falls below critical, they are replaced by C₃ species.

CAM plants:

• Dominate in deserts and semi‑deserts on all continents.
• Occupy niches with periodic drought—epiphytes in tropical forests, rocky outcrops, sandy soils with low water‑holding capacity.
• In families where CAM species occur (Cactaceae, Crassulaceae, Euphorbiaceae, Bromeliaceae, Orchidaceae), one can trace an evolutionary transition from C₃ to CAM as an adaptation to decreasing humidity (Taiz et al., 2023; Lambers & Oliveira, 2019).

Interesting fact: In a single family (e.g., Euphorbiaceae or Chenopodiaceae), one can find C₃, C₄, and CAM species! This shows that transitions between strategies occurred many times during evolution under environmental pressures (Schopfer & Brennicke, 2016; Lambers & Oliveira, 2019).

Trends: How Climate Is Changing the Distribution of Strategies

The modern world is changing, and the distribution of C₃, C₄, and CAM plants is also not static (Taiz et al., 2023; Connor et al., 2011).

Rising atmospheric CO₂:

• Since C₃ plants respond more strongly to elevated CO₂ (their photosynthesis is CO₂‑limited), they may gain an advantage.
• At CO₂ concentrations of ~600–800 ppm, the C₄ advantage could be minimised (Schopfer & Brennicke, 2016; Taiz et al., 2023).

Global warming:

• Rising temperatures may expand the ranges of C₄ plants at the expense of C₃.
• However, increased drought may favour CAM plants.

Agriculture:

• Breeders are actively looking for ways to "introduce" the C₄ mechanism into C₃ crops (rice is a major target, as it feeds half of humanity) (Connor et al., 2011; Taiz et al., 2023).
• This task is extremely challenging, as it requires not only changes in the enzymatic apparatus but also a restructuring of leaf anatomy. Early successes in creating C₄ rice have not yet led to practical results.

Final Summary: Evolutionary Logic of the Three Strategies

To conclude our lecture, let us summarise the main points:

1. C₃ photosynthesis—the original strategy, efficient in cool and moist climates. Its main drawback is photorespiration caused by Rubisco's imperfection.

2. C₄ photosynthesis—an evolutionary response to heat and drought. Spatial separation of the two carboxylases creates a zone of high CO₂ concentration around Rubisco, suppressing photorespiration and improving WUE and NUE. It is a high‑productivity strategy in warm climates.

3. CAM photosynthesis—an evolutionary response to extreme drought. Temporal separation of the two carboxylases allows CO₂ to be stored at night (when stomata are open) and used during the day (when stomata are closed). It is a survival strategy in conditions where water is the main limiting factor.

Each strategy is not "better" or "worse"; they are specialised adaptations to different ecological conditions. Understanding these adaptations is the key to successful management of agricultural crops and predicting changes in the planet's vegetation cover.