Metabolic pathways in plants and their determination

Metabolic pathways or Biogenetic pathways

Metabolic pathways or Biogenetic pathways are the pathways that lead to the formation of a chemical compound in plants. These chemical compounds are classified into two types: a) Primary metabolites and b) Secondary metabolites

a) Primary metabolites: are the metabolites/ chemical compounds which are essential for the growth, development, and reproduction in all organisms. Carbohydrates, fats, proteins, and nucleic acids are examples of primary metabolites.

b) Secondary metabolites: are the metabolites/ chemical compounds that are not essential OR not directly involved in the normal growth, development, and reproduction of living organisms. They are biosynthetically derived from primary metabolites. Thus, they are known as secondary metabolites. Alkaloids, tannins, terpenes, volatile oil, steroids, lignin, phenolic compounds, flavonoids are examples of secondary metabolites. Protection of the plants from UV rays, microbes, and pest infestation are the significant functions of secondary metabolites.

Building blocks: are required for the formation of secondary metabolites that are derived from primary metabolites. Surprisingly very few building blocks are required for secondary metabolite production. Acetyl coenzyme A (acetyl-CoA), Shikimic acid, Mevalonic acid, 1-deoxyxylulose 5-phosphate, and Amino acids are the most important building blocks involved in the biosynthesis of secondary metabolites are derived from.

The various Metabolic pathways which lead to the production of primary and secondary metabolites are listed below:

A) TCA Cycle

The TCA cycle is the central pathway in which various metabolites are formed. It mainly occurs in the matrix of mitochondria³. It is mainly responsible for the production of primary metabolites.

Significance of the TCA cycle:

1) The primary function of the TCA cycle is to provide energy in the form of ATP.

2) It is a significant pathway for the oxidation of carbohydrates, fats, lipids, and proteins³.

The steps involved in the TCA cycle are discussed below:

1) Two molecules of acetyl CoA and one molecule of oxaloacetate undergoes condensation to form a six-carbon compound known as citrate.

2) Citrate undergoes isomerization to form isocitrate.

3) Isocitrate undergoes oxidation in the presence of enzyme isocitrate dehydrogenase which results in the formation of α-ketoglutarate. During this step, CO₂ is released and one molecule of NADH is formed.

4) α-ketoglutarate formed is further oxidized to form succinyl CoA. The CoA part comes from the acetyl CoA. Also, one NADH molecule is released in this step.

5) Succinyl CoA is converted to succinate. One molecule of GTP is released.

6) Further, succinate in the presence of succinate dehydrogenase is converted to fumarate, and simultaneously one molecule of FADH₂ is formed.

7) Fumarate is converted to malate. This step is catalyzed by the enzyme fumarase.

8) Malate in presence of malate dehydrogenase is converted to oxaloacetate. Further oxaloacetate in presence of citrate synthase enzyme gets converted to citrate³.

B) Calvin Cycle

The Calvin cycle is the cycle of chemical reactions which helps the plants in the process of carbon fixation. Through this cycle, the plants fix carbon from atmospheric CO2 into the form of three-carbon sugars².

Steps in the Calvin cycle:

Step 1 Carbon Fixation: In this step CO₂ from the atmosphere binds to the ribulose-1,5-bisphosphate (a five-carbon receptor molecule) which resulted in the formation of six-carbon compound. This step is catalyzed by the enzyme RuBP carboxylase/oxygenase. The six-carbon compound formed is further split into a three-carbon compound known as phosphoglyceric acid.

Step 2 Reduction phase: The phosphoglyceric acid compound formed in the first step is converted to glyceraldehyde-3 phosphate. The energy required for this step comes from ATP and NADPH. NADPH donates electrons to the phosphoglyceric acid to form glyceraldehyde-3 phosphate, hence this step is known as reduction step.

Step 3 Carbohydrates formation/ regeneration phase: The glyceraldehyde-3 phosphate formed is converted back to sugar molecule such as glucose.

Step 4 Regeneration phase: Some of the glyceraldehyde-3 phosphate is converted to a five-carbon ribulose-1,5-bisphosphate compound that accepts the new carbon molecules.

Significance of the Calvin cycle:

Calvin cycle helps in the formation of three-carbon sugars in plants. These three-carbon sugars are used by the plants to build nucleotides, amino-acids, other sugars, and complex long-chain carbohydrates like glucose, starch, and cellulose².

C) Embden Meyerhof Pathway

abbreviated as EMP pathway was discovered by Gustav Embden, Otto Meyerhof, and Jakub Karol Parnas. In this pathway, glucose undergoes breakdown to generate energy in the form of ATP. Hence this pathway is also known as glycolysis (glyco: glucose; lysis: breakdown) pathway. The main site where glycolysis takes place is in the cytoplasm of all the living cells¹.

The steps involved in the glycolysis are mentioned listed below:

Step 1: Glucose in presence of the enzyme glucokinase is converted to glucose-6-PO₄. The latter undergoes isomerization in presence of the enzyme of isomerase to form fructose-6-PO₄.

Step 2: Fructose-6-PO₄ is converted to fructose-1,6-diphosphate in presence of the enzyme Phosphofructokinase.

Step 3: fructose-1,6-diphosphate in presence of the enzyme aldolase is converted to glyceraldehyde-3-PO₄. The latter undergoes phosphorylation to form glyceraldehyde-1,3-biPO_4.

Step 4: The glyceraldehyde-1,3-biPO₄ undergoes dephosphorylation to form 3-phosphoglycerate.

Step 5: The 3-phosphoglycerate undergoes isomerization to form 2-phosphoglycerate. The 2-phosphoglycerate in presence of enolase enzyme gets converted to phosphoenolpyruvate.

Step 6: The phosphoenolpyruvate in presence of pyruvate kinase is converted to pyruvate.

Step 7: Pyruvate can undergo reactions to form either lactate or acetaldehyde or formate.

Step 8: If acetaldehyde is formed it further undergoes fermentation in presence of alcohol dehydrogenase enzyme to form ethanol.

Biosynthesis of carbohydrates in plants: The various Metabolic pathways for the production of carbohydrates in plants are mentioned below.

D) Gluconeogenesis

Gluconeogenesis is the pathway responsible for synthesis on new glucose molecules. The pathway works in the opposite direction of the glycolysis pathway. The gluconeogenesis pathway starts in the mitochondria and ends in the endoplasmic reticulum. The glyceraldehyde-3-PO₄is the product of photosynthesis in plants. This product is stored inside chloroplasts in the form of starch or stored in other plant tissues in the form of glucose or sucrose. Similarly, stored fats are also converted to glucose and sucrose when seed germination occurs⁴.

E) Pentose Phosphate Pathway

It is a pathway that occurs in parallel to the glycolysis pathway. In this pathway energy in the form of ATP is not utilized. It is also known as hexose monophosphate shunt. The glucose pentose pathway consists of two important phases 1) oxidative phase and 2) non-oxidative pathway

1) The oxidative phase (irreversible reaction) is the phase in which NADPH is generated. The steps involved in the oxidative phase are mentioned below:

Step 1: Glucose-6-PO₄ (intermediate product of glycolysis pathway) in presence of the enzyme glucose-6-PO₄ dehydrogenase is converted to 6-phosphogluconolactone.

Step 2: 6-phosphogluconolactone is converted to 6-phosphogluconate. Gluconolactonase enzyme catalyzes this reaction.

Step 3: 6-phosphogluconate is converted to ribulose-5-PO₄. The reaction is catalyzed by enzyme 6-phosphogluconate dehydrogenase.

Significance of the oxidative phase:

Results in the formation of 2 NADPH molecules which are utilized by the plant cells in various Metabolic pathways or biogenetic pathways.

2) Non-oxidative phase (reversible reactions): is the phase in which 5 carbon sugars moiety is formed. The steps are mentioned below:

Step 1: Ribulose-5-PO₄ is converted into two different 5-carbon molecules (i.e. ribose-5-PO₄ and xylulose-5-PO₄

Step 2: Further depending upon the needs of the plant cell, 2 molecules of ribose-5-PO₄ undergoes conjugation to form a C₁₀ sugar moiety. The C₁₀sugar moiety formed can further undergo chemical interconversion to form C₇ and C₃ sugar molecules.

Significance of the non-oxidative phase: ribose-5-PO₄ obtained from this phase is used for the building of DNA/RNA molecules¹².

F) Glycogen Synthesis Pathway

The excess of glucose produced by plants is stored in the form of glycogen. The various steps involved in the glycogen synthesis are mentioned below:

1) Glucose undergoes phosphorylation to form glucose-6-PO₄ in presence of the enzyme hexokinase/glucokinase.

2) Glucose-6-PO₄ is converted to glucose-1-PO₄ in presence of the enzyme phosphoglucomutase. The latter is converted to UDP-glucose in presence of the enzyme glucose-1-PO₄ uridylyltransferase.

3) UDP-glucose is converted to glycogen in presence of the enzyme glycogen synthase⁵.

G) Shikimic Acid Pathway

The shikimic acid pathway is responsible for the metabolism of carbohydrates and amino acids. A combination of phosphoenolpyruvate, a glycolytic pathway intermediate, and erythrose 4-phosphate from the pentose phosphate pathway leads to the formation of Shikimic acid. The Shikimic acid formed is responsible for the production of a variety of secondary metabolites such as aromatic amino acids, phenols, cinnamic acid derivatives, lignans, and alkaloids. Shikimic acid is an important intermediate that leads to the formation of Chorismic acid. Chorismic acid acts as a substrate for the formation of anthranilic acid and tryptophan.

The steps involved in the Shikimic acid pathway are discussed in brief:

Step 1: The phosphoenolpyruvic acid and erythrose-4-phosphate undergo condensation to form 2-keto-3-deoxy-7-phospho-D-glucoheptonic acid. The latter undergoes cyclization to form 3-Dehydroquinic acid.

Step 2: 3-Dehydroquinic acid undergoes dehydration to form 3-Dehydroshikimic acid. The latter undergoes keto-enol tautomerism to form Shikimic acid.

Step 3 Shikimic acid undergoes phosphorylation to form Shikimic acid 3-phosphate.

Step 4: Shikimic acid 3-phosphate undergoes dehydration and condensation to form chorismic acid.

Step 5: Chorismic acid acts as a substrate for the formation of anthranilic acid and tryptophan. Also, chorismic acid undergoes isomerization to form prephenic acid (a substrate for the formation of phenylalanine and tyrosine)¹¹.

H) The acetate hypothesis

As per the acetate hypothesis, acetate occupies the central position concerning the general metabolism/ production of secondary metabolites in plants. The acetate pathway is an important pathway for the production of various secondary metabolites such as straight-chain and aromatic compounds. In the acetate pathway, acetate is the starting material. The acetate is utilized is in the form of acetyl CoA¹¹.

I) Acetate mevalonate pathway/ Isoprenoid pathway

Acetyl CoA is the starting material for acetate mevalonate pathway. After a series of steps, it leads to the formation of mevalonate or mevalonic acid. Through this pathway, various isoprenoid compounds such as terpenes and steroids are obtained. Acetate mevalonate pathway contributes to the synthesis of 1/3^rd of the total known secondary metabolites. This pathway occurs in eukaryotic cells such as plants, animals, and some bacterial cells.

The acetate mevalonate pathway leads to the biosynthesis of active isoprene units. The isoprene units obtained are considered as important active building blocks/ universal precursors. Examples of isoprene units are Isopentyl pyrophosphate and Dimethyl allyl pyrophosphate. These isoprene units contribute to the biosynthesis of many other secondary metabolites such as Anthraquinoes, naphthoquinones, terpenoids, glycosides, alkaloids.

The steps involved in the acetate mevalonate pathway are listed below:

2 molecules of Acetyl CoA (the end product of glycolysis) undergo condensation to form Acetoacetyl-CoA.
Acetoacetyl-CoA undergoes condensation with 1 acetyl CoA to form HMG-CoA in presence of HMG CoA synthase.
HMG CoA further undergoes a reduction in the presence of HMG-CoA reductase and NADPH to form Mevalonate (important intermediate).
Mevalonate undergoes phosphorylation (twice) to form 5-pyrophosphomevalonate. The latter undergoes dehydration and decarboxylation and leads to the formation of Isopentyl pyrophosphate and Dimethyl allyl pyrophosphate.
Isopentyl pyrophosphate and Dimethyl allyl pyrophosphate undergoes condensation to form geranyl pyrophosphate (a C₁₀ monoterpene).
Geranyl pyrophosphate undergoes condensation with isopentyl pyrophosphate to form farnesyl pyrophosphate (a C₁₅ sesquiterpene)
If farnesyl pyrophosphate undergoes condensation with isopentyl pyrophosphate, geranylgeranyl pyrophosphate (a C20).
Squalene (C₄₀ compound, important in cholesterol synthesis) is formed if farnesyl pyrophosphate undergoes condensation with farnesyl pyrophosphate¹¹.

J) Biosynthesis of lipids in plants

The de novo synthesis is the main pathway in plants responsible for the synthesis of lipids or fatty acids. The most common fatty acids synthesized by the de novo pathway are palmitic, oleic, linoleic, and linolenic acids. The main site for de novo fatty acid biosynthesis is the plastid compartment in the plant cell. Acetyl CoA is the substrate molecule for fatty acid synthesis.

There is two main enzyme system responsible for fatty acid synthesis. 1) acetyl CoA carboxylase and 2) fatty acid synthases.

The acetyl CoA obtained from TCA cycle has two fates:

1) Acetyl CoA is utilized as the starting material in plant cells for the synthesis of steroidal compounds (secondary metabolites) by the acetate mevalonate pathway.

2) Acetyl CoA can be utilized as a substrate molecule for the synthesis of complex fatty acids. Malonyl CoA, palmitate is some of the intermediates formed during the reaction. Fatty acid synthase is the class of enzyme utilized in fatty acid synthesis¹³.

K) Biosynthesis of volatile oils

Mevalonic acid pathway and 2-methylerythritol-4-phosphate (MEP) pathway are the major pathways by which plants biosynthesize the volatile oils. Various classes of terpenes (such as Monoterpenes, sesquiterpenes, diterpenes, triterpenes, tetraterpenes, and polyterpenes) are produced through this pathways⁵.

Notations

Terpenes: are hydrocarbon compounds that are the most important constituents present in essential oils. Terpenes are made up of isoprene units (5 carbon atoms)⁶.
Monoterpenes: belongs to the class of terpenes containing 2 isoprene units (10 carbon atoms)⁶.
Sesquiterpenes: belongs to the class of terpenes containing 3 isoprene units (15 carbon atoms)⁶.
Diterpenes: belongs to the class of terpenes containing 4 isoprene units (20 carbon atoms)⁶.
Triterpenes: belongs to the class of terpenes containing 6 isoprene units (30 carbon atoms)⁶.
Tetraterpenes: belongs to the class of terpenes containing 6 isoprene units (40 carbon atoms)⁶.
Photosynthesis: is a process by which plants produce organic compounds such as carbohydrates using H₂O, CO₂, minerals, and light energy⁷.
NADH: also known as Nicotinamide adenine dinucleotide which participates in various electron transfer chain reactions in the body⁸.
NADPH: also known as nicotinamide adenine dinucleotide phosphate is produced from the various biochemical process such as photosynthesis. It is also used in the biosynthesis of various compounds such as lipids, nucleotides, and amino acids⁹.
FADH₂: known as Flavin adenine dinucleotide. It is an important energy molecule that is utilized by the plant cells in the process of cellular respiration.
ATP: is known as Adenosine triphosphate is an energy-carrying molecule present in the cells of all living things¹⁰.
ADP: Adenosine diphosphate also known as adenosine pyrophosphate (APP) is the metabolite of ATP. ADP can be converted to ATP or adenosine monophosphate (AMP)¹⁰.