2. Metabolic pathways in higher plants and their
determination
Brief study of basic metabolic pathways and formation of different
secondary metabolites through these pathways- Shikimic acid pathway,
Acetate pathways and Amino acid pathway.
•The sum total of all the enzymatic mediated reactions occurring in the
cell is collectively called metabolism.
•The reaction sequences occurring within organisms in an orderly and
regulated way are known as metabolic pathways and the compounds
formed during metabolism are called metabolites.
•During the process of metabolism many intermediate metabolites are
formed which is used to denote the chemical pathways of metabolism.
3.
4. As a result of metabolic process in plants, plant synthesizes
primary plant metabolites and secondary plant metabolites.
Primary plant metabolites- These are considered as basic plant
constituents like sugars, amino acids, coenzyme-A, mevalonic acid
and lipids. They are widely distributed in plants in large quantities; are
directly involved in growth, development and reproduction.
They do not possess biological or pharmacological action.
The primary metabolite are the starting material for biosynthesis of
Secondary Metabolite.
Secondary plant metabolites- These are biosynthesized from the
primary metabolites e.g- alkaloids, glycosides, tannins, flavonoids,
terpenoids and volatile oils etc.
5. They are more limited in distribution, not directly involved in the
growth and development, they have possess biological or
pharmacological action on humans and animals.
Basic Metabolic Pathway: The building blocks for secondary
metabolites are derived from primary metabolism. How metabolites from
photosynthesis, glycolysis, and the Krebs cycle are tapped off from
energy-generating processes to provide biosynthetic intermediates.
The most important building blocks used in the biosynthesis of
secondary metabolites are derived from the intermediates acetyl
coenzyme A (acetyl-CoA), shikimic acid, mevalonic acid, and 1-
deoxyxylulose 5-phosphate.
6.
7. Shikimic Acid Pathway: The shikimate pathway provides an
alternative route to aromatic compounds, particularly the aromatic
amino acids (Phenylalanine, Tyrosine, and Tryptophan).
This pathway is employed by microorganisms and plants, but not by
animals, and accordingly the aromatic amino acids feature among
those essential amino acids for human beings, to be obtained from
the diet.
Shikimic acid is named after the highly toxic Japanese shikimi
(Illicium anisatum) flower from which it was first isolated.
The shikimate pathway (shikimic acid pathway) is a seven-step
metabolic pathway used by bacteria, archaea, fungi, algae, some
protozoans, and plants for the biosynthesis of aromatic amino acids.
8. The seven enzymes involved in the shikimate pathway are-
1- DAHP synthase (3-deoxy-D-arabinoheptulosonate 7-Phosphate)
2- 3-dehydroquinate synthase
3- 3-dehydroquinate dehydratase
4- Shikimate dehydrogenase
5- Shikimate kinase
6- EPSP synthase (5-enolpyruvylshikimate-3-Phosphate)
7- Chorismate synthase.
The pathway starts with two substrates, phosphoenol pyruvate and
erythrose-4-phosphate, and ends with chorismate (chrorismic acid), a
substrate for the
9.
10.
11. Acetate-Mevalonate Pathway/Isoprenoid Pathways
The mevalonate pathway, also known as the isoprenoid pathway or
HMG-CoA reductase pathway is an essential metabolic pathway present
in eukaryotes, archaea, and some bacteria.
It is one of the important pathways for the synthesis of different type of
secondary metabolite of plant cells.
Different straight chain compound and aromatic compounds get
biosynthesized from this acetate by different metabolic pathways.
This pathways are mainly useful for formation of fatty acid and sterols.
12.
13.
14.
15. Geranylgeranyl pyrophosphate- It is an intermediate in the
biosynthesis of diterpenes and diterpenoids.
It is also the precursor to carotenoids, gibberellins, tocopherols, and
chlorophylls.
Prenylated Protein- It is a process for a Post- translation modification of
proteins by the attachment of an isoprenoid to the C-terminal of Cysteine
residues.
Dolichols- Dolichols play a role in the co-translational modification of
proteins known as N-glycosylation in the form of dolichol phosphate.
Dolichols are the major lipid component.
16. Sterols- Phytosterols, more commonly known as plant sterols, have
been shown in clinical trials to block cholesterol absorption sites in the
human intestine, thus helping to reduce cholesterol absorption in
humans. Phytosterols as a nutritional supplement.
Heme- It is a precursor to hemoglobin, which is necessary to bind
oxygen in the bloodstream.
Ubiquinone- It is lipophilic metabolite that functions in the electron
transport chain in the plasma membranes of prokaryotes, and the inner
mitochondrial membranes of eukaryotes, apart from its roles as an
antioxidant and in the regeneration of tocopherols.
Cholesterol- It is a fat-like, waxy substance that helps your body make
cell membranes, many hormones, and vitamin D.
17. Pharmacology
A number of drugs target the mevalonate pathway:
1. Statins (used to decrease cholesterol levels);
2. Bisphosphonates (used to treat various bone-degenerative diseases)
A number of diseases affect the mevalonate pathway:
Mevalonate Kinase Deficiency
1. Mevalonic Aciduria
2. Hyperimmunoglobulin D Syndrome (HIDS).
22. Utilization of radioactive isotopes in the
investigation of biogenetic studies
Isotopes = Iso (same) + topes (place) Occupy Same place in Periodic
table. Elements with same atomic number, but different atomic weight.
(Same number of proton but differ in neutron).
Example - 12C, 13C, 14C (Isotopes). and 1H (Hydrogen), 2H
(Deuterium), 3H (Tritium).
Radioisotopes are radioactive isotopes of an element. They can also be
defined as atoms that contain an unstable combination of neutrons and
protons, or excess energy in their nucleus.
Radioactive Isotopes: 1H, 14C, 24Na, 42K, 35S, 35P, and 131I are the
examples of isotopes.
23. The following two types of isotopes are used for labelling-
1) Radioactive isotopes- 1H, 14C, 24Na, 42K, 35S, 35P, and 131I.
2) Stable Isotopes- 2H, 13C, 15N, 18O
Before selecting an isotope, the following points should be considered:
1) Its initial concentration should be sufficient enough to hold up dilution
during the metabolic processes.
2) Its physical and chemical properties should be well-known for proper
labeling.
3) It should have a higher half-life.
4) It should be actively involved in biosynthesis.
5) It should not destroy the reaction system.
24. Utilization of radioactive isotopes
Radioactive tracers are radioactive isotopes used in biogenetic studies.
These are organic compounds whose one or more atoms have been
replaced with a radionuclide. Due to their property of radioactive decay,
the radioactive tracers are used to investigate the mechanism of chemical
reactions by tracing the path followed by the radioisotope from reactants
to products.
Examples of radioisotope tracers used frequently to trace the path of
biochemical reactions are hydrogen, carbon, phosphorus, sulphur, and
iodine.
25. BIOGENETIC STUDIES
Introduction Living plants are the biosynthetic laboratory as many
primary and secondary metabolites are synthesized in them, through
various biosynthetic pathways.
These pathways involve different intermediate steps that can be identified
by using different scientific techniques.
Biogenetic investigation in plants can be performed by the following
methods:
1) Using isolated tissue
2) Grafting methods
3) Use of Mutant strains
4) Tracer technique
26. Tracer Technique
These radioactive isotopes or tracers are used in tracer techniques for
investigating the biosynthetic pathways.
In this technique, a labeled compound is used to investigate the different
intermediates and various steps in biosynthetic pathways in plants by
tracing them at a given rate and time.
This technique also uses a labeled compound which is introduced into
the plant system.
After which the compound becomes a part of the general metabolic pool,
and gets involved in the related reaction of the particular plant system.
27. For this technique, the radioactive carbon (14C), Hydrogen (3H), and
Sulphur (35S), and Phosphorus (32P) are used for analyzing the various
biosynthesis pathways.
Significance of Tracer Technique
The tracer technique has the following significances:
1) It is highly sensitive.
2) It is used in the living system.
3) A number of isotopes are available that can be used in this technique.
4) It is a more reliable and easy process of administration and isolation.
5) It provides accurate results if used with proper metabolic time and
technique.
28. The following techniques are used for separation and
detection of the plant metabolites-
1- GM Counter (Geiger Muller Counter)
2- Liquid scintillation chamber
3- Auto Radiography
4- Mass Spectrophotometer
5- NMR Spectrophotometer
6- Gas ionization chamber
29. Application of Tracer Technique
Tracer technique has the following applications:
1) It is used to trace the biosynthetic pathway of prunasine
(cyanogenetic glycoside) by the incorporation of 14C into
Phenylaniline.
2) It utilizes 14C acetate to analyze the relationship between 4-methyl
sterols and 4,4 dimethyl sterols.
3) It utilizes 14C, 3H labeled mevalonic acid for studying squalene
cyclisation.
4) It utilizes 2-C mevalonate to investigate terpenoid biosynthesis by
chloroplast isolated in organic solvent.
30. 5) It is used to study cinnamic acid formation from labelled coumarin in
pathway of coumarin.
6) It utilises 14C or 15N labelled precursor to determine the origin of
carbon and nitrogen atoms of purine ring system.
7) It utilises labelled phenylalanine to study scopoletin formation.
8) It utilises 45Ca as a tracer to analyse the uptake of calcium by plants
from soil (CaO and CaCO2).
