Plant breathing

3.1. Glycolysis — Producing Pyruvate and the First “Release” of Energy

Glycolysis (from Greek glykys — sweet, lysis — splitting) is a universal, evolutionarily ancient pathway of sugar oxidation that occurs in the cytosol of all living cells. In plants, it has several important peculiarities.

The main function of glycolysis is to convert hexoses (primarily sucrose, glucose, and fructose) into organic acids — pyruvate and malate. Concurrently, the cell obtains:

• a small amount of ATP (substrate‑level phosphorylation, without oxygen),
• the reductant NADH,
• valuable intermediate products (phosphoenolpyruvate, 3‑phosphoglycerate, glucose‑6‑phosphate) that can be used for the synthesis of amino acids, lipids, nucleotides (Taiz et al., 2023; Hopkins & Hüner, 2009).

Peculiarities of plant glycolysis:

1. Substrate — not only glucose, as in animals, but primarily sucrose (the main transport form of carbohydrates in plants) (Schopfer & Brennicke, 2016).

2. There are two ways of sucrose cleavage: via invertase (hydrolysis to glucose and fructose) or via sucrose synthase (yielding UDP‑glucose and fructose). The second pathway is energetically more favourable because it immediately gives glucose‑1‑phosphate without ATP expenditure (Taiz et al., 2023; Третьяков и др., 2000).

3. Phosphorylation of fructose‑6‑phosphate can occur not only via ATP (ATP‑dependent phosphofructokinase) but also via pyrophosphate (PPᵢ) through PPᵢ‑dependent phosphofructokinase. This is a unique feature of plants, allowing ATP economy under energy‑deficient conditions (Taiz et al., 2023).

4. End product — not only pyruvate but, in some cases, malate (via sequential action of PEP carboxylase and malate dehydrogenase). Malate can be stored in the vacuole or enter mitochondria for oxidation (Lambers & Oliveira, 2019; Morot‑Gaudry et al., 2012).

Thus, glycolysis is a bridge between carbohydrate “fuel” and further oxidative pathways. It works under both aerobic and anaerobic conditions, giving the cell ATP even when oxygen is scarce (in that case, pyruvate enters fermentation, but more on that later).

3.2. Oxidative Pentose Phosphate Pathway — Supplier of NADPH and Building Blocks

The oxidative pentose phosphate pathway (OPPP), sometimes called the apotomic pathway, runs parallel to glycolysis and also starts with glucose‑6‑phosphate. But its role is entirely different.

In the oxidative phase of the OPPP, the following occurs:

• oxidation of glucose‑6‑phosphate with the production of NADPH,
• and subsequent release of CO₂ (this is the only sugar oxidation pathway that gives CO₂ without mitochondrial involvement).

In the non‑oxidative phase, from pentoses (ribulose‑5‑phosphate) through a series of reversible reactions, various sugar phosphates are synthesized: ribose‑5‑phosphate, erythrose‑4‑phosphate, fructose‑6‑phosphate, and triose phosphates (Schopfer & Brennicke, 2016; Taiz et al., 2023).

The main function of the OPPP is to supply the cell with the reductant NADPH (not ATP!). NADPH is used in biosynthetic processes: fatty acid synthesis, reduction of glutathione and ascorbate (antioxidant defence), nitrate reduction in plastids (Lambers & Oliveira, 2019; Marschner, 2012).

In addition, the OPPP provides:

• ribose‑5‑phosphate — the backbone for nucleotide and nucleic acid synthesis,
• erythrose‑4‑phosphate — together with phosphoenolpyruvate (from glycolysis) serves as the starting point for the shikimate pathway, which leads to the synthesis of aromatic amino acids, lignin, flavonoids, coumarins (Morot‑Gaudry et al., 2012; Третьяков и др., 2000).

The OPPP is especially active in tissues with high rates of synthesis (meristems, developing seeds, roots) and under oxidative stress, when large amounts of NADPH are needed for antioxidant systems (Lambers & Oliveira, 2019).

3.3. Tricarboxylic Acid Cycle (TCA Cycle, Krebs Cycle) — Supplier of Reductants

After glycolysis, pyruvate (and often malate in plants) enters the mitochondria. Inside the matrix, pyruvate is oxidized to acetyl‑CoA (this reaction gives NADH and CO₂), and then acetyl‑CoA enters the tricarboxylic acid cycle (TCA cycle).

What happens in the TCA cycle?

The acetyl group (C₂) condenses with oxaloacetate (C₄) to form citrate (C₆).

Then follows a series of oxidative decarboxylations, as a result of which:

• three molecules of CO₂ are released (per pyruvate),
• four NADH and one FADH₂ are formed,
• one molecule of ATP is synthesized (substrate‑level phosphorylation at the level of succinyl‑CoA) (Taiz et al., 2023; Hopkins & Hüner, 2009).

The main task of the TCA cycle is not ATP synthesis, but mass production of reduced coenzymes (NADH and FADH₂). They will then go to the electron transport chain to generate ATP. The TCA cycle also supplies intermediates for biosynthesis: α‑ketoglutarate and oxaloacetate — for amino acid synthesis (glutamate, aspartate), succinyl‑CoA — for porphyrin synthesis (including chlorophyll) (Morot‑Gaudry et al., 2012).

In plants, the TCA cycle has an important feature: in the mitochondrial matrix, there is malic enzyme, which can oxidatively decarboxylate malate to pyruvate (right inside the mitochondrion). This allows mitochondria to use malate (e.g., accumulated in vacuoles or formed during glycolysis) as a full respiratory substrate, bypassing pyruvate dehydrogenase. In addition, this pathway supplies malate to the citrate cycle even when pyruvate is scarce (Lambers & Oliveira, 2019; Taiz et al., 2023).

3.4. Electron Transport Chain and Oxidative Phosphorylation — ATP Synthesis

The final stage of respiration takes place on the inner mitochondrial membrane. Here, the electron transport chain (ETC) operates, consisting of four multi‑protein complexes (I–IV) and two mobile carriers — ubiquinone (ubiquinol) and cytochrome c.

NADH and FADH₂ formed in the TCA cycle (and partly in glycolysis) donate their electrons to the ETC. Electrons move from more negative potential (NADH, −320 mV) to more positive (oxygen, +820 mV). During this, three proton pumps (complexes I, III, and IV) translocate protons from the matrix into the intermembrane space. An electrochemical proton gradient (proton‑motive force) is created, which is used by ATP synthase (complex V) to synthesize ATP from ADP and inorganic phosphate (Schopfer & Brennicke, 2016; Taiz et al., 2023).

The theoretical yield per NADH is about 2.5 ATP (in experiments 2.4–2.7), and per FADH₂ about 1.5 ATP. Thus, upon complete oxidation of one glucose molecule (via glycolysis, TCA cycle, and ETC), up to 38 molecules of ATP can be obtained, most of which (~34) come from oxidative phosphorylation (Медведев, 2012; Третьяков и др., 2000). It is the ETC that is the main ATP generator under aerobic conditions. Without oxygen, it stops, and the cell must make do with only two ATP from substrate‑level phosphorylation in glycolysis.

Summarizing

• Glycolysis — provides pyruvate, a little ATP and NADH, as well as intermediates for synthesis.
• OPPP — provides NADPH and pentoses, erythrose‑4‑phosphate — for nucleotide and aromatic compound biosynthesis.
• TCA cycle — provides NADH, FADH₂, and carbon skeletons for amino acid and other metabolite synthesis.
• ETC and oxidative phosphorylation — use NADH and FADH₂ to synthesize ATP with the participation of oxygen.

All four pathways are tightly linked and regulated to maintain the current balance of ATP, reductants, and building blocks. This is not just a “conveyor belt” — it is a flexible system that reconfigures depending on tissue age, light conditions, temperature, and nutrient availability.

In the next chapter, we will discuss how plant respiration differs from that of animals, and why plants possess the so‑called “alternative pathway” — cyanide‑resistant respiration.

4.1. Alternative Oxidase (AOX) — “Cyanide‑Resistant Respiration”

The classic way to test whether the ETC is working is to add cyanide (CN⁻). Cyanide blocks complex IV (cytochrome oxidase), and in animals, respiration stops completely. In most plants, this does not happen — respiration continues, albeit at a lower rate. This phenomenon is called cyanide‑resistant respiration, and it is due to the presence of alternative oxidase (AOX) (Schopfer & Brennicke, 2016; Taiz et al., 2023).

AOX is a small integral protein embedded in the inner mitochondrial membrane on the matrix side. It accepts electrons directly from ubiquinol (reduced ubiquinone) and transfers them to oxygen, reducing it to water. In doing so, complexes III and IV are bypassed entirely, and therefore two of the three proton pumps do not work. Electrons that pass through AOX yield not 2.5 ATP per NADH, but only about 1 ATP (or even 0 if electrons come from succinate) (Lambers & Oliveira, 2019).

Importantly: AOX is not merely an “inefficient” pathway. It is activated in response to certain conditions. Its activity is regulated at several levels:

• Redox state of the ubiquinone pool: AOX is turned on when ubiquinol accumulates, i.e., when the ETC is “overloaded” with electrons.
• Activation by pyruvate: in the presence of pyruvate, AOX affinity for ubiquinol increases sharply, and it begins to work even when the main chain is not fully loaded.
• Transcriptional regulation: AOX genes are actively expressed under stress (cold, drought, salt, pathogens), under phosphorus deficiency, and also in response to high light intensity (Lambers & Oliveira, 2019; Taiz et al., 2023).

Why would a plant need such a “wasteful” pathway? There are several reasons, all with profound physiological meaning.

The first function — protection against oxidative stress. When the ETC is overloaded with electrons and ADP is low (the cell does not consume ATP), electrons may be transferred directly to oxygen at early stages of the chain (e.g., from ubiquinol or complex I), forming superoxide radical (O₂⁻) and other reactive oxygen species (ROS). ROS damage membranes, proteins, and DNA. By diverting excess electrons from ubiquinol, AOX reduces the likelihood of ROS formation, acting as a “safety valve” (Taiz et al., 2023; Медведев, 2012).

The second function — thermogenesis (heat production). In some plant organs — for example, in the inflorescences of aroids (spadices of Arum, Sauromatum, Symplocarpus) — AOX activity increases to such an extent that the tissue temperature rises 10–15 °C above ambient. This occurs at strictly defined developmental stages and serves to evaporate volatile aromatic compounds that attract pollinating insects (Lambers & Oliveira, 2019; Schopfer & Brennicke, 2016). In Nelumbo nucifera (lotus), the flowers even maintain a constant temperature of about 30 °C while external temperature fluctuates from 10 to 30 °C — a unique case of thermoregulation in plants (Taiz et al., 2023).

The third function — “dissipation” of excess reductants. During periods when carbohydrates are abundant but the demand for ATP and biosynthesis is low (e.g., in roots with excess sugar supply from leaves), AOX allows the “burning” of excess NADH without accumulating it and without blocking TCA cycle and glycolysis. This maintains metabolic flexibility and allows the cell to switch quickly between modes (Lambers & Oliveira, 2019; Третьяков и др., 2000).

The fourth function — sustaining respiration when the main chain is inhibited. Some secondary metabolites (e.g., cyanogenic glycosides, hydrogen sulfide, high CO₂ concentrations) may partially block the cytochrome pathway. Under such conditions, AOX becomes a life‑saving backup pathway, allowing at least some energy to be obtained and substrate oxidation to continue (Taiz et al., 2023).

4.2. Additional NAD(P)H Dehydrogenases — Bypassing Complex I

In animals, NADH formed in the matrix is oxidized only through complex I (NADH dehydrogenase), which is coupled to proton pumping. Plants have at least four additional dehydrogenases that work “bypassing” complex I and do not generate a proton gradient (Taiz et al., 2023; Lambers & Oliveira, 2019).

• Internal, rotenone‑insensitive NADH dehydrogenase (NDin) — located on the inner side of the inner membrane, oxidizes matrix NADH but does not pump protons. It is activated when the matrix is highly reduced.
• External NADH dehydrogenase (NDex) — oxidizes cytosolic NADH, transferring electrons to ubiquinone. This allows mitochondria to directly use NADH formed in glycolysis, without the need for shuttling mechanisms (as in animals).
• External and internal NADPH dehydrogenases — oxidize NADPH, linking respiration to the pentose phosphate pathway and antioxidant systems.

All these dehydrogenases yield about 1 ATP less per electron pair compared to complex I. But they provide flexibility: the cell can oxidize different reductants (NADH, NADPH) from different sources, independently of ADP levels and without tight coupling to ATP synthesis.

4.3. Uncoupling Protein (UCP) — Controlled Proton “Leak”

In animals, there is thermogenin (UCP1) in brown adipose tissue, which creates a proton “shunt” across the membrane, uncoupling oxidation and phosphorylation to generate heat. In plants, homologues of this protein — UCPs (uncoupling proteins) — have been found, which are activated by fatty acids and reactive oxygen species. Their role is also to lower the proton gradient and prevent overloading of the ETC with electrons, reducing ROS formation (Taiz et al., 2023; Lambers & Oliveira, 2019).

4.4. Summary: Why Does the Plant Need All This Diversity?

In animals, the mitochondrion is a “power station” operating at maximum efficiency. In plants, the mitochondrion is a universal regulatory centre that must simultaneously:

• supply the cell with ATP,
• provide reductants for biosynthesis and stress defence,
• utilize excess carbohydrates without allowing toxic accumulation,
• prevent the formation of reactive oxygen species,
• function under conditions where the main respiratory pathway is limited (high ATP/ADP, low temperature, inhibitors).

All the “additional” pathways — AOX, bypass dehydrogenases, UCP — do not reduce efficiency “for nothing.” They give the plant metabolic plasticity and resilience in a fluctuating environment, where photosynthesis, growth, and energy demands may not coincide in time or space (Lambers & Oliveira, 2019; Taiz et al., 2023).

Moreover, we now know that AOX and bypass pathways are not “backup options” but constantly operating components of normal metabolism (Del‑Saz et al., 2018). For example, in many species, even under optimal conditions, up to 40–50% of respiration may proceed through AOX (measured by oxygen isotope fractionation) (Lambers & Oliveira, 2019). This means that the plant “plans” to use incomplete coupling as part of its everyday strategy.

5.1. Energy for Cytosolic Processes

Photosynthesis generates ATP and NADPH in chloroplasts, but these compounds cannot freely leave the chloroplast. The chloroplast envelope is impermeable to ATP and NADPH; they are used mainly inside the chloroplast — for CO₂ fixation in the Calvin cycle and for nitrite reduction (Hopkins & Hüner, 2009; Taiz et al., 2023). Meanwhile, many energy‑consuming processes occur in the cytosol:

• sucrose synthesis from triose phosphates,
• phloem loading (active transport of sucrose into sieve tubes),
• nitrate reduction (in roots and sometimes in leaves),
• maintenance of ion gradients across the plasma membrane,
• biosynthesis of cellulose and other cell‑wall components,
• operation of proton pumps that drive secondary transport.

All this “cytosolic” demand for ATP is covered mainly by oxidative phosphorylation in mitochondria, even in the light (Lambers & Oliveira, 2019; Morot‑Gaudry et al., 2012). In other words, the chloroplast provides “raw materials” (sugars and reductants), and the mitochondrion converts them into usable energy for the rest of the cell. Without respiration in the light, export of assimilates from the leaf and whole‑plant growth would be impossible (Connor et al., 2011; Третьяков и др., 2000).

5.2. Carbon Skeletons for Nitrogen Metabolism

One of the most important roles of respiration in the light is supplying the cell with organic acids for amino acid synthesis. When nitrate (NO₃⁻) is actively taken up and reduced to ammonium (NH₄⁺) in leaves, there is a need for rapid incorporation of ammonium into amino acids, primarily glutamine and glutamate. This requires α‑ketoglutarate (from the TCA cycle) and oxaloacetate (also from the TCA cycle).

During daylight hours, the rate of nitrogen metabolism increases sharply because reducing power (NADPH and ferredoxin) comes from the photosynthetic electron transport chain. However, the carbon skeletons themselves — α‑ketoglutarate and oxaloacetate — can only be formed in the TCA cycle, i.e., through respiration (Schopfer & Brennicke, 2016; Taiz et al., 2023). If the TCA cycle stops, nitrogen assimilation becomes impossible even with abundant light and nitrates. This is one reason why respiration does not cease during the day: it provides substrates for glutamine synthetase and other enzymes of the nitrogen assimilation pathway (Lambers & Oliveira, 2019; Morot‑Gaudry et al., 2012).

In addition, intermediates are withdrawn from the TCA cycle for the synthesis of many amino acids (glutamate, aspartate, alanine, and aromatic amino acids via the shikimate pathway), as well as for porphyrins (chlorophyll, haems) and purine nucleotides. Respiration in the light is not “waste” of carbon but its redistribution toward building blocks (Taiz et al., 2023).

5.3. Photorespiration — “Work” of Mitochondria in the Light

A particularly close connection between chloroplasts and mitochondria is evident in the process of photorespiration (or the C₂ cycle). Photorespiration occurs when RuBisCO (ribulose‑bisphosphate carboxylase/oxygenase) incorporates O₂ instead of CO₂, forming glycolate. Glycolate leaves the chloroplast, passes through peroxisomes, and enters mitochondria, where two molecules of glycine (derived from glycolate) are oxidized to one molecule of serine. During this:

• CO₂ is released (this is the “light‑dependent” CO₂ emission),
• NH₄⁺ is formed (which is then re‑assimilated in chloroplasts),
• and, most importantly for us, large amounts of NADH are generated in mitochondria, which immediately enter the electron transport chain and yield ATP (Schopfer & Brennicke, 2016; Taiz et al., 2023).

Interestingly, during the day, photorespiration is the main supplier of NADH for mitochondria in leaves. Estimates show that the electron flux from the glycine dehydrogenase complex can be several times higher than that from the TCA cycle (Lambers & Oliveira, 2019). Thus, the respiratory chain during the day uses “fuel” that comes directly from photorespiratory metabolism, not only from the classical TCA cycle. This shows how deep the integration of the two processes — photosynthesis and respiration — is in a working leaf.

5.4. Light Regulation of Respiration: Switching On and Off

How does the cell “decide” that during the day the mitochondrion should work, but not as it does at night? Plants have several mechanisms of light‑dependent regulation of respiration.

• Pyruvate dehydrogenase (PDH), the enzyme that channels pyruvate into the TCA cycle, is inhibited by phosphorylation (by a specific kinase) in the light. This reduces the flow of pyruvate into the TCA cycle during the day (Schopfer & Brennicke, 2016; Taiz et al., 2023). However, PDH activity does not drop to zero — a small basal flux is maintained to supply the TCA cycle with intermediates for biosynthesis (see section 5.2).
• At the same time, alternative oxidase (AOX) can be activated in the light, especially at high intensity, to dissipate excess electrons if the cytochrome pathway becomes overloaded (Lambers & Oliveira, 2019). This prevents ROS formation and protects chloroplasts from photoinhibition.
• Substrate availability also changes: in the light, there is abundant malate (from photosynthesis and from the vacuole), which can be oxidized in mitochondria via malic enzyme, providing NADH and replenishing the TCA cycle (Morot‑Gaudry et al., 2012).

Overall, respiration during the day “switches”: the share of classical TCA‑dependent pyruvate oxidation decreases, but the roles of photorespiration and malate oxidation increase. This supplies both ATP and carbon skeletons, but in different proportions than at night.

5.5. Interaction Between Chloroplasts and Mitochondria: Metabolite Exchange

Modern research shows that there is intensive metabolite exchange between chloroplasts and mitochondria. For example, the malate shuttle: malate from the chloroplast leaves into the cytosol, then enters the mitochondrion, where it is oxidized to oxaloacetate with the formation of NADH (and hence ATP), while oxaloacetate is converted to aspartate and returns to the chloroplast, where it serves as an amino group acceptor (Taiz et al., 2023; Lambers & Oliveira, 2019). This exchange allows the chloroplast to dispose of excess reductants, while the mitochondrion obtains “fuel” for ATP production even when pyruvate input is low.

In addition, exchange of TCA cycle intermediates such as 2‑oxoglutarate (for glutamine synthesis in the chloroplast) and glycolate and glycine (photorespiration) occurs through the cytosol. The mitochondrion, therefore, is not merely an energy “battery” but an active participant in the metabolic network of the leaf, integrating light and respiratory processes (Schopfer & Brennicke, 2016; Morot‑Gaudry et al., 2012).

5.6. Summary: Photosynthesis and Respiration — Not Competitors but Partners

So, why is respiration not switched off during the day?

• Because the chloroplast cannot supply ATP for the cytosol and other organelles — it uses ATP mainly for its own needs.
• Because nitrogen metabolism requires a constant supply of carbon skeletons from the TCA cycle, especially at high rates of nitrate reduction.
• Because photorespiration, which supplies CO₂ and NH₄⁺, provides the mitochondrion with NADH and keeps the respiratory chain working even when the TCA cycle is partially inhibited.
• Because the mitochondrial electron transport chain acts as a protective mechanism, preventing over‑reduction of chloroplast redox systems and ROS formation.

Thus, photosynthesis and respiration operate synchronously, forming a single functional ensemble. The former generates organic substances and reducing equivalents, the latter converts them into energy, reductants, and building blocks, distributing them among growth, transport, synthesis, and defence. Without respiration, photosynthesis could not realize its potential for biomass production.

This concludes our overview of plant respiration. In subsequent lectures, we will delve deeper into specific aspects: the physiological role of alternative pathways, the effects of stress on respiration, and the relationship between respiration and crop productivity. But most importantly, you now have a systemic understanding that respiration is not “wastefulness” or “losses,” but a necessary and highly organized part of plant life that enables its existence and adaptation in a constantly changing environment.