Carbon assimilation

2.1. Why Is Rubisco the Most Important Enzyme?

Rubisco catalyses the very first reaction on the path from inorganic to organic carbon: the addition of CO₂ to a five‑carbon sugar — ribulose‑1,5‑bisphosphate (RuBP). This is the primary reaction of carbon fixation. Without it, no organic matter would exist at all.

But here we encounter our first surprise. Rubisco is a very slow enzyme. Its catalytic number (turnover number), i.e., the number of CO₂ molecules that one enzyme molecule can convert per second, is only 1–6 for higher plants (Taiz et al., 2023). For comparison, many other enzymes work thousands of times faster. How, then, can plants grow at all with such a “lazy” enzyme?

The answer is paradoxical: the plant compensates for slowness with an enormous quantity of Rubisco. In the leaves of C₃ plants, this enzyme accounts for up to 50 % of all soluble proteins! This is a colossal investment of nitrogen. By some estimates, the total amount of Rubisco on the planet is about 10 million tonnes (Medvedev, 2012). This is why Rubisco is called the most abundant enzyme in the biosphere. Plants literally “overwhelm” the problem of slowness with quantity.

Important to understand: Rubisco’s slowness is not a flaw but an evolutionarily developed compromise between speed and accuracy. We will return to this.

2.2. Structure of Rubisco: A Complex Ensemble

Rubisco is a large protein complex consisting of eight large subunits (L) and eight small subunits (S), forming an L₈S₈ structure. The molecular mass of the whole complex reaches 540–550 kDa (Medvedev, 2012; Morot‑Gaudry et al., 2012). It is important to note that the catalytic centre is located on the large subunits. The small subunits have an auxiliary function, stabilising the structure, but their exact role is not fully understood.

From the perspective of plant physiology, it is fundamentally important that the genes for the large subunits are located in the chloroplast genome, while the genes for the small subunits are in the nuclear genome. This means that synthesis of the active enzyme requires coordinated work of two genetic systems: the chloroplast and the nucleus. The small subunit proteins are synthesised in the cytosol, transported into the chloroplast, where they assemble with the large subunits synthesised on site. This assembly process requires chaperones (helper proteins) and ATP energy (Medvedev, 2012).

2.3. The Dual Nature of Rubisco: Carboxylase and Oxygenase

Now we come to the most important point — the main compromise built into Rubisco’s structure. This enzyme can catalyse not one but two competing reactions:

1. Carboxylase reaction (main):

Rubisco + CO₂ + RuBP → two molecules of 3‑phosphoglycerate (PGA). This is the path to carbohydrates.

2. Oxygenase reaction (side but inevitable):

Rubisco + O₂ + RuBP → one molecule of PGA + one molecule of 2‑phosphoglycolate.

Both reactions take place in the same active centre. CO₂ and O₂ compete for binding to the same substrate — RuBP. In essence, Rubisco does not perfectly distinguish between these two gases, and this is its “imperfection” (Hopkins and Hüner, 2009; Morot‑Gaudry et al., 2012).

Question: why did evolution not create a more selective enzyme?

The answer lies in Earth’s history. Rubisco originated about 3 billion years ago, when the atmosphere was rich in CO₂ and contained virtually no O₂. Under those conditions, oxygen was not a problem. However, with the advent of oxygenic photosynthesis, the atmosphere became saturated with O₂. Rubisco “did not have time” to adapt, and its oxygenase activity became a physiological reality that plants are forced to cope with (Hopkins and Hüner, 2009).

Moreover, there is an evolutionary trade‑off between speed and specificity: enzymes that bind CO₂ rapidly (high turnover) generally have lower specificity for CO₂ over O₂, and vice versa. Rubisco from C₃ plants has high specificity (S_c/o around 80–90) but low speed. Rubisco from C₄ plants and algae, which have CO₂‑concentrating mechanisms, is faster but less specific (Lambers & Oliveira, 2019; Taiz et al., 2023). This is a classic trade‑off.

2.4. Activation of Rubisco: An Enzyme That Needs to Be “Switched On”

We often think of enzymes as constantly working molecules. With Rubisco, this is not the case. In darkness, it is in an inactive or low‑activity form. For it to work, it must be activated. This is a physiologically important point, because it links Rubisco activity to the presence of light — not directly, but through conditions in the chloroplast stroma.

Rubisco activation occurs in several steps (Taiz et al., 2023; Medvedev, 2012):

1. Carbamylation. A lysine residue is located at the active centre of Rubisco, which is protonated in the dark. In the light, when the stromal pH rises (due to proton transfer into the thylakoids), this lysine becomes deprotonated and capable of binding CO₂. One molecule of CO₂ (not substrate, but activator) attaches to this lysine, forming a carbamate group (–NH‑COO⁻).

2. Mg²⁺ binding. The formed carbamate electrostatically attracts an Mg²⁺ ion, which stabilises the active conformation of the enzyme. Only after this does Rubisco become capable of binding substrate CO₂ and RuBP.

It is important that both conditions — the rise in pH and the increase in Mg²⁺ concentration in the stroma — are direct consequences of light reactions: proton transport into the lumen leads to alkalinisation of the stroma, and as charge compensation, Mg²⁺ ions leave the lumen into the stroma (Taiz et al., 2023). Thus, Rubisco activation depends indirectly on light.

However, even in the active form, Rubisco can be blocked by inhibitors — for example, the substrate RuBP, which binds to the non‑carbamylated form of the enzyme and blocks activation, or 2‑carboxy‑D‑arabinitol‑1‑phosphate (CA1P), which binds to the active form. These inhibitors accumulate in the dark and must be removed.

2.5. Rubisco Activase: The Enzyme That Services Enzymes

To free the active centre from inhibitors, plants use a special protein — Rubisco activase. This is an ATP‑dependent enzyme that physically interacts with Rubisco, changes its conformation, and promotes the release of inhibitors (Farquhar & Sharkey, 1994; Taiz et al., 2023).

Why is this physiologically important? Because Rubisco activase is sensitive to temperature and the energy status of the cell. Under heat stress, Rubisco activase activity declines, leading to Rubisco deactivation and a drop in photosynthesis (Lambers & Oliveira, 2019). This is one reason why photosynthesis may weaken on a hot midday even under sufficient light.

Thus, Rubisco activity in the leaf is a dynamic balance between activation (carbamylation) and inhibition. Light regulates this balance via pH and Mg²⁺, as well as through the redox state affecting Rubisco activase.

2.6. Physiological Meaning of Rubisco’s Dual Function

Now we can answer the key question: why is Rubisco imperfect?

Because its oxygenase activity is not a mistake but a price for universality and evolutionary heritage. In modern plant physiology, photorespiration (the process that starts with the oxygenase reaction) is no longer seen as “useless waste.” On the contrary, it performs important protective functions under excess light and when stomata are closed, when internal CO₂ falls and O₂ rises (Hopkins and Hüner, 2009). In addition, photorespiration provides a flow of metabolites, linking photosynthesis with nitrogen metabolism and redox regulation (Taiz et al., 2023). We will discuss this in detail in the section on photorespiration.

Remember the main point: Rubisco is not a perfect tool, but it is precisely because of this “imperfection” that all modern regulatory mechanisms of photosynthesis, including the C₄ pathway, CAM metabolism, and photorespiration, have evolved.

So, let us summarise Rubisco:

• It is the most abundant enzyme on Earth.
• It catalyses the first step of CO₂ fixation, but does so slowly.
• It has dual function: carboxylase (useful) and oxygenase (side).
• This duality is an evolutionary compromise linked to the history of the atmosphere.
• Rubisco requires activation (carbamylation + Mg²⁺) and maintenance (Rubisco activase), which connects its operation to light reactions.
• Its oxygenase activity triggers photorespiration — a process now regarded as an important physiological mechanism, not an error.

Now that we have met the key enzyme, let us move on to how its product — 3‑phosphoglycerate — is converted into carbohydrates. This occurs in the Calvin cycle, where ATP and NADPH find their ultimate application.

3.1. Three Stages — Three Steps of Logic

The entire Calvin cycle can be divided into three major stages. They are sequential but closely interconnected. Let us examine each from a physiological perspective.

Stage 1. Carboxylation (or CO₂ fixation)

This is the very reaction we already discussed in the Rubisco section. One molecule of CO₂ attaches to the five‑carbon ribulose‑1,5‑bisphosphate (RuBP). Upon hydrolysis of the unstable six‑carbon intermediate, two molecules of 3‑phosphoglycerate (PGA) are formed (Taiz et al., 2023; Schopfer & Brennicke, 2016). It is important to emphasise: at this stage, energy is not yet expended. The reaction is exergonic, proceeding toward PGA formation. Rubisco does its job, but the product — PGA — is not yet a carbohydrate in the full sense. It is an oxidised form (a carboxylic acid), and it needs to be reduced.

Stage 2. Reduction of PGA to triose phosphate

This is where the actual “construction” begins. The two PGA molecules formed in the first stage undergo sequential transformations:

• First, PGA is phosphorylated by ATP. The enzyme phosphoglycerate kinase transfers a phosphate group from ATP to the carboxyl group of PGA, forming 1,3‑bisphosphoglycerate. This is a high‑energy compound.
• Then 1,3‑bisphosphoglycerate is reduced by NADP‑dependent glyceraldehyde‑3‑phosphate dehydrogenase using NADPH. This yields glyceraldehyde‑3‑phosphate (GAP) — a three‑carbon sugar that is already a true carbohydrate (triose) (Farquhar & Sharkey, 1994; Morot‑Gaudry et al., 2012).

Thus, for one CO₂ molecule (which gives two PGA molecules), 2 ATP and 2 NADPH are required to obtain two molecules of triose phosphate. However, as we will see later, this is not yet the final balance.

Stage 3. Regeneration of RuBP

Now we have six molecules of triose phosphate (GAP) — exactly the number formed when three CO₂ molecules are fixed (since each CO₂ gives two PGA, and then two GAP). Of these six trioses, only one is a net product of CO₂ assimilation. The remaining five must go toward regenerating the original acceptor — RuBP — so that the cycle can continue (Taiz et al., 2023; Lambers & Oliveira, 2019).

This stage is a series of enzymatic rearrangements in which carbon skeletons are rebuilt: trioses (C₃) are converted into tetroses (C₄), pentoses (C₅), hexoses (C₆), and sedoheptulose (C₇), and then back to ribulose‑5‑phosphate, which is subsequently phosphorylated by ATP to RuBP. These reactions are catalysed by enzymes such as aldolase, transketolase, phosphatases, and isomerases. To regenerate three RuBP molecules from five GAP molecules, another 3 molecules of ATP are needed (one for each RuBP formed). This phosphorylation is catalysed by phosphoribulokinase (Schopfer & Brennicke, 2016; Kuznetsov & Dmitrieva, 2006).

3.2. Final Balance: Inputs and Outputs

Now we can summarise the quantitative result of the Calvin cycle. Consider three turns of the cycle to obtain neat whole numbers:

Inputs:

• 3 molecules of CO₂ (fixed)
• 3 molecules of RuBP (accept CO₂, but then regenerated)
• 6 molecules of NADPH (used in the reduction stage)
• 9 molecules of ATP (6 for PGA phosphorylation, 3 for RuBP regeneration)

Outputs:

• 1 molecule of glyceraldehyde‑3‑phosphate (GAP) — net product, which can be directed to starch or sucrose synthesis.
• 3 molecules of RuBP — regenerated and ready for a new cycle.

Per fixed CO₂ molecule:

Or, if counting to glucose (6 CO₂ → 1 hexose), 12 NADPH and 18 ATP are required (Medvedev, 2012). This ratio — 2 NADPH and 3 ATP per carbon — is a key constant of C₃ photosynthesis. It determines how much light energy must be converted into chemical energy to ensure continuous cycle operation.

Remember: The Calvin cycle is not just a “dark phase.” It is a sophisticated enzymatic machine that requires not only reducing power (NADPH) but also energy (ATP) at two fundamentally different steps: the actual reduction of carbon and the maintenance of the acceptor capacity of the system.

3.3. Where Does the Triose Phosphate — the Cycle’s Product — Go?

One molecule of GAP leaving the cycle is not an end product in terms of storage. It serves as a “building block” that can be used in several ways (Farquhar & Sharkey, 1994; Taiz et al., 2023):

• In the chloroplast, GAP is used for the synthesis of transitory starch. This is a temporary depot that accumulates during the day and is consumed at night.
• GAP can be exported from the chloroplast via the triose phosphate translocator in exchange for inorganic phosphate. In the cytosol, triose phosphates are used to synthesise sucrose — the main transport form of carbohydrates, which enters the phloem and is distributed throughout the plant.
• Some GAP goes into the synthesis of amino acids, fatty acids, and other metabolites — this already links photosynthesis to nitrogen and sulfur metabolism.

Thus, the product of the Calvin cycle serves as a kind of “distribution centre” from which carbon flows diverge to various plant needs.

3.4. Why Is the Calvin Cycle Often Called the “Slow” Part of Photosynthesis?

Despite the complexity of the cycle, its rate is limited not by any single reaction but by a combination of several key steps. These primarily include:

1. Rubisco activity, which, as we recall, is low and requires constant activation.

2. The rate of RuBP regeneration, which depends on the activity of enzymes in the second half of the cycle and on ATP availability.

3. The availability of NADPH and ATP, determined by the rate of light reactions.

It is important to understand: although the Calvin cycle is called “dark reactions,” it cannot operate in the dark (Hopkins & Hüner, 2009). Why? Not only because there is no supply of ATP and NADPH in the dark. Moreover, many enzymes of the cycle (including Rubisco, phosphoribulokinase, fructose‑1,6‑bisphosphatase) require light activation via the ferredoxin–thioredoxin system, which we will discuss in the next section. Without light, they are simply inactive. So the name “dark reactions” is long outdated — we now call them carbon reactions or carbon assimilation reactions, emphasising their dependence on products and regulation of the light phase (Taiz et al., 2023).

3.5. The Calvin Cycle and Photorespiration — Two Sides of the Same Coin

We already know that Rubisco can act as an oxygenase. When this occurs, instead of two PGA molecules, one PGA and one molecule of 2‑phosphoglycolate are formed. This product cannot be used in the Calvin cycle — it is toxic and inhibits some enzymes. It must be “disposed of” through a complex pathway involving chloroplasts, peroxisomes, and mitochondria. This pathway is called photorespiration or the C₂ cycle.

It is important to note that the Calvin cycle and photorespiration are functionally linked — they compete for the same substrate (RuBP) and determine the net efficiency of photosynthesis. The more oxygenase reaction, the more carbon is lost as CO₂ and the more energy is spent on the photorespiratory pathway. Therefore, in C₃ plants under hot and dry conditions, when stomata are closed and internal CO₂ concentration falls, photorespiration can “consume” up to 50 % of assimilated carbon (Kuznetsov & Dmitrieva, 2006). This is one of the most important physiological constraints, which evolution overcame by creating C₄ and CAM mechanisms for concentrating CO₂.

Summary

The Calvin cycle is not just a biochemical diagram. It is:

• The central pathway of carbon reduction, where the products of light reactions are used.
• Three stages: carboxylation (CO₂ capture), reduction (conversion of PGA to triose), and regeneration (restoration of the RuBP acceptor).
• Strict stoichiometry: 3 CO₂ + 6 NADPH + 9 ATP → 1 triose phosphate + regenerated 3 RuBP.
• The cycle’s product (GAP) is distributed between starch, sucrose, and other metabolite synthesis.
• The cycle does not operate without light — not only because of the absence of ATP and NADPH but also because of the light‑dependent activation of its key enzymes.
• It is closely coupled with photorespiration, and their balance determines the net efficiency of photosynthesis.

Now that we understand the logic of the cycle, we must answer the logical question: “How does light control the operation of this cycle, besides supplying ATP and NADPH?” This is the subject of the next section of our lecture.

4.1. Light Changes the Internal Environment of the Chloroplast

Let us start with the simplest but important point. When light strikes the thylakoid membranes, the electron transport chain is activated. One consequence of this is proton transport from the stroma into the thylakoid lumen. As a result, the stromal pH rises from about 7.0 in the dark to 8.0 in the light (Taiz et al., 2023; Schopfer & Brennicke, 2016). At the same time, as charge compensation, magnesium ions (Mg²⁺) leave the thylakoids into the stroma, and their concentration increases from 2–3 mM to 5 mM.

Why is this important for the Calvin cycle? Because several key enzymes of this cycle require an alkaline environment and the presence of Mg²⁺ for their activity:

• Rubisco (activation via carbamylation, as we recall, requires Mg²⁺) (Farquhar & Sharkey, 1994).
• Fructose‑1,6‑bisphosphatase (FBPase) — an enzyme that removes a phosphate group from fructose‑1,6‑bisphosphate, converting it to fructose‑6‑phosphate. This enzyme has a pH optimum around 8.0.
• Sedoheptulose‑1,7‑bisphosphatase (SBPase) — a similar enzyme working in the RuBP regeneration stage.
• Phosphoribulokinase (PRK) — the enzyme that phosphorylates ribulose‑5‑phosphate to RuBP using ATP.

In the dark, at pH 7.0 and low Mg²⁺, these enzymes are virtually inactive. When light is switched on, the environment changes, and they “switch on.” This is the first level of regulation, rapid and reversible (Hopkins & Hüner, 2009).

4.2. The Ferredoxin–Thioredoxin System — The Master Regulator

However, the change in pH and Mg²⁺ concentration is only part of the picture. A much more elegant and specific mechanism was discovered later — the ferredoxin–thioredoxin system. It directly couples the electron flow from photosystem I with the activation of Calvin‑cycle enzymes.

How does it work?

1. In the light, photosystem I reduces ferredoxin — a small water‑soluble protein containing an iron‑sulfur cluster.

2. Reduced ferredoxin transfers electrons to the enzyme ferredoxin‑thioredoxin reductase.

3. This enzyme reduces thioredoxin — a small regulatory protein (about 12 kDa) that contains two cysteine residues capable of forming a disulfide bond (S–S) or, in the reduced state, two sulfhydryl groups (–SH HS–).

4. Reduced thioredoxin (with –SH groups) interacts with target enzymes, reducing disulfide bonds in their structure. This leads to conformational changes and activation of the enzyme. In the dark, when electron flow ceases, thioredoxin becomes oxidised, and the enzymes return to their inactive form (Taiz et al., 2023; Schopfer & Brennicke, 2016; Medvedev, 2012).

This mechanism was first described for fructose‑1,6‑bisphosphatase and later found for other key enzymes (Lambers & Oliveira, 2019).

Which Calvin‑cycle enzymes are regulated via thioredoxin?

• Fructose‑1,6‑bisphosphatase (FBPase) — activated by reduction.
• Sedoheptulose‑1,7‑bisphosphatase (SBPase) — activated by reduction.
• Phosphoribulokinase (PRK) — activated by reduction.
• NADP‑dependent glyceraldehyde‑3‑phosphate dehydrogenase (GAPDH) — activated by reduction (Schopfer & Brennicke, 2016).

Note that all these enzymes are located at two key points of the cycle — at the reduction stage (GAPDH) and at the RuBP regeneration stage (FBPase, SBPase, PRK). By activating them, light “turns on” the entire cycle, precisely at the points where energy is expended.

4.3. Is Rubisco Regulated via Thioredoxin?

Rubisco itself, as we recall, is activated through carbamylation and Mg²⁺ binding. But here, too, there is a connection to light via thioredoxin, though not direct. The activity of Rubisco activase — the enzyme that removes inhibitors from Rubisco — also depends on the redox state. In some plant species, Rubisco activase contains regulatory cysteines, and its activity is modulated via thioredoxin (Taiz et al., 2023). Thus, even Rubisco activation is indirectly tied to light regulation.

4.4. Assembly of Supramolecular Complexes — Another Layer of Regulation

But that is not all. In recent years, it has been discovered that some Calvin‑cycle enzymes assemble into inactive supramolecular complexes in the dark. For example, glyceraldehyde‑3‑phosphate dehydrogenase (GAPDH) and phosphoribulokinase (PRK) in the dark bind to a small regulatory protein CP12, forming a ternary complex in which both enzymes are inactive (Taiz et al., 2023; Lambers & Oliveira, 2019). CP12 contains four cysteines capable of forming two disulfide bonds.

In the light, reduced thioredoxin reduces the disulfide bonds in CP12 and in PRK itself. This causes the complex to dissociate, and the enzymes are released in active form. Such a mechanism ensures a very rapid transition from the “off” state to the “on” state — literally within seconds after illumination. In the dark, the enzymes reassemble into complexes and are inactivated.

Important: All these regulatory mechanisms (pH, Mg²⁺, thioredoxin, complex assembly) act in concert and rapidly. Thanks to them, when switching from dark to light, the Calvin cycle can be activated within minutes, even though the enzymatic reactions themselves are relatively slow (Farquhar & Sharkey, 1994).

4.5. Physiological Meaning: Why Is This Important?

Why does the plant need such a complex multi‑level regulation? The answer is obvious: saving resources and preventing futile cycles.

Imagine if the Calvin cycle were active in the dark. In the dark, there is no photosynthetic electron transport, no supply of NADPH and ATP from light reactions. But if the cycle enzymes were active, they could attempt to reduce CO₂ using ATP and NADPH obtained from respiration (mitochondrial). This would lead to waste of energy — essentially, running idle. Moreover, without a constant supply of CO₂ (stomata are often closed at night) and without acceptor regeneration (which requires ATP), the cycle would quickly stall, but with useless resource consumption.

In addition, there is a risk of futile cycles. In the chloroplast stroma, besides the Calvin cycle, there is the oxidative pentose phosphate pathway, which can operate in the reverse direction. If both pathways were active simultaneously, carbon could be cyclically reduced and oxidised, wasting ATP with no useful result. Light regulation prevents this: in the dark, Calvin‑cycle enzymes are inactive, while enzymes of the oxidative pentose phosphate pathway are, on the contrary, activated (via the same thioredoxin but with opposite effects) (Hopkins & Hüner, 2009).

Thus, light regulation of the Calvin cycle is not just “on” and “off.” It is a system that:

• Saves ATP and reducing equivalents in the dark.
• Prevents futile cycles between assimilation and dissimilation.
• Ensures instant startup of photosynthesis when light appears.
• Coordinates chloroplast function with the overall energy state of the cell.

Summary

So, why does the Calvin cycle only work in the light? Because:

1. Light changes the pH and Mg²⁺ concentration in the stroma, which is necessary for the activity of several key enzymes.

2. Light via the ferredoxin–thioredoxin system directly reduces disulfide bonds in enzymes, activating them.

3. Light causes dissociation of inactive supramolecular complexes, releasing enzymes in active form.

4. This regulation prevents useless energy expenditure in the dark and rapidly activates assimilation when illumination resumes.

Thus, the light regulation of the Calvin cycle is one of the most elegant examples of integration of photophysical and biochemical processes in plant physiology. Light does not merely provide energy; it is a signal that reorganises the entire enzymatic apparatus of the chloroplast.

5.1. Where Does Photorespiration Come From? The Oxygenase Reaction of Rubisco

We already know that Rubisco is a dual‑function enzyme. Under normal atmospheric conditions (21 % O₂, 0.04 % CO₂), about 20–25 % of the reactions catalysed by Rubisco are not carboxylation but oxygenation of ribulose‑1,5‑bisphosphate (Farquhar & Sharkey, 1994; Hopkins & Hüner, 2009). As a result, instead of two molecules of 3‑phosphoglycerate (PGA), one molecule of PGA and one molecule of 2‑phosphoglycolate are formed (Taiz et al., 2023).

Why so much? Because in the atmosphere, O₂ is about 500 times more abundant than CO₂. Although Rubisco “prefers” CO₂ by about 80–100 times (its specificity), the huge excess of O₂ makes the oxygenase reaction inevitable (Lambers & Oliveira, 2019). As temperature rises, Rubisco’s specificity for CO₂ decreases, and the proportion of oxygenase reaction increases — which is why photorespiration intensifies in heat (Morot‑Gaudry et al., 2012).

2‑Phosphoglycolate is a toxic compound. It cannot be used in the Calvin cycle and inhibits some enzymes. Therefore, the plant is forced to “dispose” of it through a complex metabolic pathway involving three organelles: chloroplast, peroxisome, and mitochondrion.

5.2. The Metabolic Pathway of Photorespiration: Cooperation of Three Organelles

Photorespiration is not just a side reaction but a full‑fledged metabolic cycle, often called the C₂ cycle or glycolate pathway (after the researcher Tolbert who described it). Let us follow the carbon path (Taiz et al., 2023; Medvedev, 2012).

1. In the chloroplast: 2‑Phosphoglycolate formed by the oxygenase reaction is dephosphorylated by the enzyme phosphoglycolate phosphatase to glycolate. Glycolate leaves the chloroplast through specific transporters in the chloroplast membrane.

2. In the peroxisome: Glycolate is oxidised by glycolate oxidase to glyoxylate. This consumes O₂ and produces H₂O₂. The toxic hydrogen peroxide is immediately decomposed by catalase into water and oxygen. Then glyoxylate undergoes transamination with glutamate to form glycine. Glycine leaves the peroxisome and goes to the mitochondrion.

3. In the mitochondrion: Two molecules of glycine (each with 2 carbon atoms) undergo a reaction catalysed by the glycine decarboxylase complex (GDC) and serine hydroxymethyltransferase (SHMT). The products are: one molecule of serine (3 carbons), one molecule of CO₂ (released), one molecule of NH₃ (ammonia), and NAD⁺ is reduced to NADH.

4. Return to the peroxisome: Serine returns to the peroxisome, where through a series of reactions (deamination, reduction) it is converted to glycerate.

5. Return to the chloroplast: Glycerate enters the chloroplast, is phosphorylated by glycerate kinase using ATP, and becomes 3‑phosphoglycerate (PGA) — the same product that can be fed into the Calvin cycle (Hopkins & Hüner, 2009; Taiz et al., 2023).

Thus, from two molecules of glycolate (4 carbon atoms), one CO₂ molecule (1 carbon) is lost, while three carbon atoms are returned to the Calvin cycle as PGA. This means that 75 % of the carbon that could have been lost is salvaged. If counted from the original two RuBP molecules (10 carbon atoms), the final result is three PGA molecules (9 carbon atoms) — 90 % of the carbon is recovered (Morot‑Gaudry et al., 2012).

5.3. The Energy Cost of Photorespiration

Photorespiration is an expensive process. For “disposal” of two molecules of phosphoglycolate, the following is required:

• An extra molecule of ATP (for glycerate phosphorylation).
• Reducing equivalents (NADH produced in mitochondria during GDC operation, but then used for hydroxypyruvate reduction in peroxisomes).
• Reassimilation of ammonia (NH₃), released in mitochondria, requires another molecule of ATP and reduced ferredoxin (via glutamine synthetase and glutamate synthase) (Taiz et al., 2023).

In total, each photorespiratory cycle “consumes” additional energy and reducing equivalents that could have been used for CO₂ fixation. Moreover, the released CO₂ is partially lost, although some may be refixed if active Rubisco is nearby.

Under hot, dry conditions, when stomata are closed and internal CO₂ concentration drops, the proportion of oxygenase reaction increases. Under such conditions, losses to photorespiration can reach 30–50 % of potentially fixed carbon (Kuznetsov & Dmitrieva, 2006). This is why in the tropics and subtropics evolution took the path of creating the C₄ pathway, which minimises photorespiration.

5.4. Why Do Plants Need Photorespiration? (Physiological Significance)

If photorespiration is so costly, why has it been retained in evolution? Why did natural selection not eliminate plants with “imperfect” Rubisco? The answer is multifaceted, and today researchers identify at least four important functions of photorespiration.

5.5. Why Do C₄ and CAM Plants Have Negligible Photorespiration?

Now it becomes clear why C₄ and CAM plants have virtually no photorespiration. They have mechanisms for concentrating CO₂ around Rubisco:

• In C₄ plants, this is achieved by spatial separation: primary CO₂ fixation (via PEP carboxylase) occurs in mesophyll cells, and then CO₂ is released in bundle‑sheath cells, where Rubisco is located. The CO₂ concentration there can be 10–20 times higher than atmospheric, which almost completely suppresses the oxygenase reaction (Morot‑Gaudry et al., 2012; Taiz et al., 2023).
• In CAM plants (Crassulacean acid metabolism), the same is achieved by temporal separation: at night, CO₂ is fixed by PEP carboxylase into malate, and during the day, malate is decarboxylated, creating a high CO₂ concentration around Rubisco while stomata are closed (Lambers & Oliveira, 2019).

This allows C₄ and CAM plants to avoid the energy costs of photorespiration and to compete successfully in hot, dry, and water‑limited environments.

5.6. Photorespiration as an Evolutionary Compromise: A Final View

So, how should we view photorespiration in plant physiology today?

1. It is an inevitable consequence of Rubisco’s structure, which originated in an oxygen‑free atmosphere and was not “designed” for the modern O₂ level. Changing Rubisco without losing its catalytic properties is extremely difficult (Farquhar & Sharkey, 1994).

2. It is not a mistake but a compromise. Plants pay for photorespiration with part of their assimilated carbon and energy, but in return they gain protection from photoinhibition, integration with nitrogen metabolism, and the ability to survive under CO₂ deficiency (Hopkins & Hüner, 2009).

3. It is an important regulatory element of photosynthesis, helping to balance electron, carbon, and nitrogen fluxes in the cell (Taiz et al., 2023).

4. It is an evolutionary “bridge” to more advanced strategies. It is precisely the “imperfection” of Rubisco that stimulated the emergence of C₄ and CAM pathways, which became important adaptive strategies in the tropics and arid zones (Lambers & Oliveira, 2019).

Summary of the “Photorespiration” Section

Photorespiration begins with the oxygenase reaction of Rubisco, producing 2‑phosphoglycolate.

2‑Phosphoglycolate is disposed of via the C₂ cycle, involving chloroplast, peroxisome, and mitochondrion.

From two molecules of glycolate, one CO₂ is lost, and three return to the Calvin cycle (75 % recycling).

Photorespiration requires additional ATP and reducing equivalents.

Physiological functions of photorespiration:

• Salvages carbon from complete loss.
• Protects against photoinhibition by acting as an electron sink under excess light.
• Integrates carbon and nitrogen metabolism (NH₃ reassimilation, serine/glycine synthesis).
• Participates in redox signalling (via H₂O₂).

C₄ and CAM plants “bypass” photorespiration through CO₂ concentration, making them more efficient in hot and dry climates.

Today, photorespiration is regarded as an evolutionary compromise, not a mistake of nature — it is the price for Rubisco’s versatility and, at the same time, an important adaptive mechanism.

Lecture Conclusion

Dear listeners! We have travelled from the question “How does CO₂ become carbohydrate?” to the understanding that carbon assimilation is a complex, multi‑step, and highly regulated process. We have seen:

• That Rubisco is a slow but ubiquitous enzyme that guards the entry of carbon into the organic world.
• That the Calvin cycle is not just a set of reactions but a logically constructed machine with three functional stages, where each molecule of ATP and NADPH has its strict place.
• That light regulates this cycle not only through energy supply but also via the ferredoxin–thioredoxin system, which “switches on” key enzymes.
• That photorespiration is not a “breakdown” but an inevitable and even beneficial companion of C₃ photosynthesis, helping the plant survive under stress, but at the cost of energy and some carbon.

All these elements together constitute the physiological portrait of carbon assimilation in plants. Understanding this portrait is necessary not only for fundamental science but also for applied tasks — increasing crop yields, breeding for photosynthetic efficiency, and adapting plants to climate change.