Respiration in Plants

All living organisms need energy for carrying out daily life activities — absorption, transport, movement, reproduction, or even breathing. Where does all this energy come from? We know we eat food for energy, but how is this energy extracted from food? How is it utilised? Do all foods give the same amount of energy? Do plants "eat"? And micro-organisms — do they eat "food"?

The process of breathing is intimately connected to the process of release of energy from food. All the energy required for life processes is obtained by the oxidation of some macromolecules that we call food. Green plants and cyanobacteria can prepare their own food by the process of photosynthesis, trapping light energy and converting it into chemical energy stored in the bonds of carbohydrates like glucose, sucrose, and starch.

We must remember that in green plants, not all cells, tissues, and organs photosynthesise. Only cells containing chloroplasts — most often located in the superficial layers — carry out photosynthesis. Hence, even in green plants, all other organs, tissues, and cells that are non-green need food for oxidation. Food has to be translocated to all non-green parts.

Animals are heterotrophic, obtaining food from plants directly (herbivores) or indirectly (carnivores). Saprophytes like fungi are dependent on dead and decaying matter. Ultimately, all food that is respired for life processes comes from photosynthesis.

12.1 Do Plants Breathe?

The answer to this question is not quite so direct. Yes, plants require O₂ for respiration to occur and they also give out CO₂. Hence, plants have systems in place that ensure the availability of O₂. Plants, unlike animals, have no specialised organs for gaseous exchange but they have stomata and lenticels for this purpose.

There are several reasons why plants can get along without respiratory organs:

• Each plant part takes care of its own gas-exchange needs. There is very little transport of gases from one plant part to another.
• Plants do not present great demands for gas exchange. Roots, stems, and leaves respire at rates far lower than animals do. Only during photosynthesis are large volumes of gases exchanged, and each leaf is well adapted to take care of its own needs during these periods.
• The distance that gases must diffuse even in large, bulky plants is not great. Each living cell in a plant is located quite close to the surface of the plant.

In stems, the "living" cells are organised in thin layers inside and beneath the bark. They also have openings called lenticels. The cells in the interior are dead and provide only mechanical support. Thus, most cells of a plant have at least a part of their surface in contact with air. This is also facilitated by the loose packing of parenchyma cells in leaves, stems, and roots, which provide an interconnected network of air spaces.

Photosynthesis, of course, takes place within the chloroplasts (in eukaryotes), whereas the breakdown of complex molecules to yield energy takes place in the cytoplasm and in the mitochondria (also only in eukaryotes). The breaking of the C–C bonds of complex compounds through oxidation within the cells, leading to release of considerable amount of energy, is called respiration.

Usually carbohydrates are oxidised to release energy, but proteins, fats, and even organic acids can be used as respiratory substances in some plants, under certain conditions. During oxidation within a cell, all the energy contained in respiratory substrates is not released free into the cell, or in a single step. It is released in a series of slow, step-wise reactions controlled by enzymes, and it is trapped as chemical energy in the form of ATP.

The energy released by oxidation in respiration cannot be used directly but is used to synthesise ATP, which is broken down whenever and wherever energy needs to be utilised. Hence, ATP acts as the energy currency of the cell. This energy trapped in ATP is utilised in various energy-requiring processes of the organisms, and the carbon skeleton produced during respiration is used as precursors for biosynthesis of other molecules in the cell.

If this energy is to be useful to the cell, it should be able to utilise it to synthesise other molecules that the cell requires. The strategy that the plant cell uses is to catabolise the glucose molecule in such a way that not all the liberated energy goes out as heat. The key is to oxidise glucose not in one step but in several small steps, enabling some steps to be just large enough such that the energy released can be coupled to ATP synthesis.

During the process of respiration, oxygen is utilised, and carbon dioxide, water, and energy are released as products. The combustion reaction requires oxygen. But some cells live where oxygen may or may not be available. There are sufficient reasons to believe that the first cells on this planet lived in an atmosphere that lacked oxygen. Even among present-day living organisms, we know of several that are adapted to anaerobic conditions. Some of these organisms are facultative anaerobes, while in others the requirement for anaerobic condition is obligate. In any case, all living organisms retain the enzymatic machinery to partially oxidise glucose without the help of oxygen. This breakdown of glucose to pyruvic acid is called glycolysis.

12.2 Glycolysis

The term glycolysis has originated from the Greek words, glycos for sugar, and lysis for splitting. The scheme of glycolysis was given by Gustav Embden, Otto Meyerhof, and J. Parnas, and is often referred to as the EMP pathway. In anaerobic organisms, it is the only process in respiration.

Glycolysis occurs in the cytoplasm of the cell and is present in all living organisms. In this process, glucose undergoes partial oxidation to form two molecules of pyruvic acid. In plants, this glucose is derived from sucrose, which is the end product of photosynthesis, or from storage carbohydrates. Sucrose is converted into glucose and fructose by the enzyme invertase, and these two monosaccharides readily enter the glycolytic pathway.

While studying the steps of glycolysis, note the steps at which utilisation or synthesis of ATP or NADH + H⁺ take place:

• ATP is utilised at two steps: first in the conversion of glucose into glucose-6-phosphate, and second in the conversion of fructose-6-phosphate to fructose-1,6-bisphosphate.
• Fructose-1,6-bisphosphate is split into dihydroxyacetone phosphate and 3-phosphoglyceraldehyde (PGAL).
• There is one step where NADH + H⁺ is formed from NAD⁺ — when PGAL is converted to 1,3-bisphosphoglycerate (BPGA). Two redox-equivalents are removed (in the form of two hydrogen atoms) from PGAL and transferred to a molecule of NAD⁺.
• The conversion of BPGA to 3-phosphoglyceric acid (PGA) is an energy-yielding process; this energy is trapped by the formation of ATP.
• Another ATP is synthesised during the conversion of PEP to pyruvic acid.

Figure 12.1 — Steps of glycolysis (EMP pathway): Glucose is converted to 2 molecules of pyruvic acid through 10 enzymatic steps in the cytoplasm

Pyruvic acid is the key product of glycolysis. What is the metabolic fate of pyruvate? This depends on the cellular need. There are three major ways in which different cells handle pyruvic acid produced by glycolysis. These are lactic acid fermentation, alcoholic fermentation, and aerobic respiration.

12.3 Fermentation

In fermentation, say by yeast, the incomplete oxidation of glucose is achieved under anaerobic conditions by sets of reactions where pyruvic acid is converted to CO₂ and ethanol. The enzymes pyruvic acid decarboxylase and alcohol dehydrogenase catalyse these reactions.

In both lactic acid and alcohol fermentation, not much energy is released — less than seven per cent of the energy in glucose is released, and not all of it is trapped as high-energy bonds of ATP. Also, the processes are hazardous — either acid or alcohol is produced.

What then is the process by which organisms can carry out complete oxidation of glucose and extract the energy stored to synthesise a larger number of ATP molecules needed for cellular metabolism? In eukaryotes, these steps take place within the mitochondria and this requires O₂. Aerobic respiration is the process that leads to a complete oxidation of organic substances in the presence of oxygen, and releases CO₂, water, and a large amount of energy present in the substrate. This type of respiration is most common in higher organisms.

12.4 Aerobic Respiration

For aerobic respiration to take place within the mitochondria, the final product of glycolysis — pyruvate — is transported from the cytoplasm into the mitochondria. The crucial events in aerobic respiration are:

• The complete oxidation of pyruvate by the stepwise removal of all the hydrogen atoms, leaving three molecules of CO₂.
• The passing on of the electrons removed as part of the hydrogen atoms to molecular O₂ with simultaneous synthesis of ATP.

The first process takes place in the matrix of the mitochondria while the second process is located on the inner membrane of the mitochondria.

Pyruvate, which is formed by the glycolytic catabolism of carbohydrates in the cytosol, after it enters mitochondrial matrix undergoes oxidative decarboxylation by a complex set of reactions catalysed by pyruvic dehydrogenase. The reactions catalysed by pyruvic dehydrogenase require the participation of several coenzymes, including NAD⁺ and Coenzyme A.

During this process, two molecules of NADH are produced from the metabolism of two molecules of pyruvic acid (produced from one glucose molecule during glycolysis). The acetyl CoA then enters a cyclic pathway, Tricarboxylic Acid (TCA) cycle, more commonly called Krebs' cycle after the scientist Hans Krebs who first elucidated it.

12.5 Tricarboxylic Acid Cycle

The TCA cycle starts with the condensation of acetyl group with oxaloacetic acid (OAA) and water to yield citric acid. The reaction is catalysed by the enzyme citrate synthase and a molecule of CoA is released.

• Citrate is then isomerised to isocitrate.
• It is followed by two successive steps of decarboxylation, leading to the formation of α-ketoglutaric acid and then succinyl-CoA.
• In the remaining steps of the citric acid cycle, succinyl-CoA is oxidised to OAA allowing the cycle to continue.
• During the conversion of succinyl-CoA to succinic acid, a molecule of GTP is synthesised. This is a substrate-level phosphorylation.
• In a coupled reaction, GTP is converted to GDP with the simultaneous synthesis of ATP from ADP.

Figure 12.3 — The Citric Acid (Krebs') Cycle: Acetyl CoA enters the cyclic pathway in the mitochondrial matrix, producing 3 NADH, 1 FADH₂, and 1 GTP per turn

There are three points in the cycle where NAD⁺ is reduced to NADH + H⁺, and one point where FAD⁺ is reduced to FADH₂. The continued oxidation of acetyl CoA via the TCA cycle requires the continued replenishment of oxaloacetic acid, the first member of the cycle. In addition, it also requires regeneration of NAD⁺ and FAD⁺ from NADH and FADH₂ respectively.

We have till now seen that glucose has been broken down to release CO₂, and eight molecules of NADH + H⁺ and two of FADH₂ have been synthesised, besides just two molecules of ATP in the TCA cycle. You may be wondering why we have been discussing respiration at all — neither O₂ has come into the picture, nor the promised large number of ATP has yet been synthesised. Also, what is the role of the NADH + H⁺ and FADH₂ that is synthesised?

12.6 Electron Transport System & Oxidative Phosphorylation

The following steps in the respiratory process are to release and utilise the energy stored in NADH + H⁺ and FADH₂. This is accomplished when they are oxidised through the electron transport system (ETS) and the electrons are passed on to O₂ resulting in the formation of H₂O. The metabolic pathway through which the electron passes from one carrier to another is called the ETS and it is present in the inner mitochondrial membrane.

Electrons from NADH produced in the mitochondrial matrix during the citric acid cycle are oxidised by an NADH dehydrogenase (Complex I), and electrons are then transferred to ubiquinone located within the inner membrane. Ubiquinone also receives reducing equivalents via FADH₂ (Complex II) that is generated during oxidation of succinate in the citric acid cycle.

The reduced ubiquinone (ubiquinol) is then oxidised with the transfer of electrons to cytochrome c via cytochrome bc₁ complex (Complex III). Cytochrome c is a small protein attached to the outer surface of the inner membrane and acts as a mobile carrier for transfer of electrons between Complex III and IV.

Complex IV refers to cytochrome c oxidase complex containing cytochromes a and a₃, and two copper centres.

Although the aerobic process of respiration takes place only in the presence of oxygen, the role of oxygen is limited to the terminal stage of the process. Yet, the presence of oxygen is vital, since it drives the whole process by removing hydrogen from the system. Oxygen acts as the final hydrogen acceptor.

Unlike photophosphorylation where it is the light energy that is utilised for the production of the proton gradient required for phosphorylation, in respiration it is the energy of oxidation-reduction utilised for the same process. It is for this reason that the process is called oxidative phosphorylation.

ATP Synthesis by ATP Synthase (Complex V)

The energy released during the electron transport system is utilised in synthesising ATP with the help of ATP synthase (Complex V). This complex consists of two major components:

Figure 12.5 — Electron Transport Chain & Oxidative Phosphorylation: Electrons from NADH and FADH₂ pass through Complexes I–IV, pumping H⁺ into the intermembrane space. ATP synthase (Complex V) uses the proton gradient to synthesise ATP via chemiosmosis

12.7 The Respiratory Balance Sheet

It is possible to make calculations of the net gain of ATP for every glucose molecule oxidised, but in reality this can remain only a theoretical exercise. These calculations can be made only on certain assumptions:

• There is a sequential, orderly pathway functioning, with one substrate forming the next, and with glycolysis, TCA cycle, and ETS pathway following one after another.
• The NADH synthesised in glycolysis is transferred into the mitochondria and undergoes oxidative phosphorylation.
• None of the intermediates in the pathway are utilised to synthesise any other compound.
• Only glucose is being respired — no other alternative substrates are entering in the pathway at any of the intermediary stages.

Yet, it is useful to do this exercise to appreciate the beauty and efficiency of the living system in extraction and storing energy. Hence, there can be a net gain of 38 ATP molecules during aerobic respiration of one molecule of glucose.

ATP Yield Summary — One Glucose Molecule

Comparison: Fermentation vs. Aerobic Respiration

12.8 Amphibolic Pathway

Glucose is the favoured substrate for respiration. All carbohydrates are usually first converted into glucose before they are used for respiration. Other substrates can also be respired, but they do not enter the respiratory pathway at the first step.

Since respiration involves breakdown of substrates, the respiratory process has traditionally been considered a catabolic process and the respiratory pathway as a catabolic pathway. But is this understanding correct?

What is important to recognise is that the very compounds that would be withdrawn from the respiratory pathway for the synthesis of said substrates are the same ones involved in their breakdown. Fatty acids would be broken down to acetyl CoA before entering the respiratory pathway when used as a substrate. But when the organism needs to synthesise fatty acids, acetyl CoA would be withdrawn from the respiratory pathway for it.

Similarly, during breakdown and synthesis of protein, respiratory intermediates form the link. Breaking down processes within the living organism is catabolism, and synthesis is anabolism.

12.9 Respiratory Quotient

Let us now look at another aspect of respiration. As you know, during aerobic respiration, O₂ is consumed and CO₂ is released. The ratio of the volume of CO₂ evolved to the volume of O₂ consumed in respiration is called the respiratory quotient (RQ) or respiratory ratio.

The respiratory quotient depends upon the type of respiratory substrate used during respiration.

Respiratory Quotient Values

12.10 Summary

12.11 Exercises