ELUCIDATION OF

BIOSYNTHETIC PATHWAYS
 Biosynthesis of Primary Metabolites
Living plants are solar-powered biochemical and
biosynthetic laboratory which manufactures both
primary and secondary metabolites from air, water,
minerals and sunlight.

The primary metabolites like sugars, amino acids & fatty acids
that are needed for general growth & physiological development of
plant which distributed in nature & also utilized as food by man.

The secondary metabolites such as alkaloids, glycosides,

Flavonoids, volatile oils etc are biosynthetically derived
from primary
metabolites.

Biosynthetic reactions are replica of common organic reactions like

catalytic reactions, phosphorylation, hydride transfer, oxidation,
 Metabolism & Metabolic Pathways
Metabolic Pathway: A complete set of chemical
reactions that occur in living cells, allowing cells to grow
and reproduce, maintain their structures, and respond to
their environments.
Living cell require energy for biosynthesis, transport of
nutrient, motility and maintenance.
Energy is obtained from the catabolism of carbon
compounds (carbohydrate)
Carbohydrates are synthesized from CO2 and H2O in the
present of light by photosynthesis.
difference between Primary and secondary
metabolites
 ~ produce energy to the cell

~ requires energy

glucose to glycogen
 Introduction

• Living plants considered as huge biosynthetic

laboratory primary as well as secondary
metabolites.
• Different biosynthetic pathway:-
-Shikmic acid pathway
-Mevalonic acid pathway
-Amino acids pathway
-HMP Shunt
-Glycolysis
• Various intermediate steps are involved in biosynthetic
pathway in plants can be investigated by means of
following techniques: -

• Tracer technique
• Use of isolated organ
• Grafting methods
• Use of mutant strain

In this technique different isotope, mainly the

radioactive isotopes are incorporated into
presumed precursor of plant metabolites and
are used as marker in biogenic experiments.
 Radioisotopes
• 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.
• The term isotope is formed from the Greek roots isos
("equal") and topos ("place"), meaning "the same
place“.
• Isotope, one of two or more species of atoms of a
chemical element with the same atomic number and
position in the periodic table and nearly identical
chemical behaviour but with different atomic masses
and physical properties.
• All isotopes of a given element have the same
number of protons but different numbers of
neutrons in each atom.
• Every chemical element has one or more
isotopes.
• Examples: Carbon-12, Carbon-13, and Carbon-
14 are three isotopes of the element Carbon
with mass numbers 12, 13, and 14, respectively.
The atomic number of carbon is 6, which means
that every carbon atom has 6 protons, so that
the neutron numbers of these isotopes are 6, 7,
and 8 respectively.
 Radioactive Decay

• An unstable atomic nucleus spontaneously

loses energy by emittingionizing
particles
radiation. and
Radioactive isotope Parent Nuclide Daughter
Nuclide
• When an unstable nucleus decays, it may give:
1) Alpha or helium Radiation.
2) Beta or Electron Radiation.
3) Gamma Radiation.
More examples
Penetration power of the Radiations
 Half-life
• The half life of radioisotope is the time for the radiation level to decrease (decay) to
one half of the original value.
• Naturally occurring tend to have longer half lives.
• Used in nuclear medicine have short half lives.
 Tracer
technique
 It can be defined as techniques which utilizes a
labelled compound to find or to trace the different
intermediates and various steps in biosynthetic
pathways in plants, at a given rate and time.
OR
In this technique different isotope, mainly the
radioactive isotopes are incorporated into presumed
precursor of plant metabolites and are
used as marker in biogenic experiments.
• The labelled compound can be prepared by use of
two types of isotopes.
i) Unstable Radioactive isotopes.
ii) Stable isotopes.

i) Unstable Radioactive isotopes: - [e.g. 1H, 14C,

24Na, 42K, 35S, 35P, 131I decay with emission
of radiation] For biological investigation –
carbon & hydrogen.
• For metabolic studies – S, P, and alkali and
alkaline earth metals are used.
• For studies on protein, alkaloids, and amino
acid–labelled nitrogen atom give more specific
information.
• 3H compound is commercially available.

ii) Stable isotopes: - [e.g. 2H, 13C, 15N, 18O] Used

for labelling compounds as possible intermediates
in biosynthetic pathways.
• Usual method of detection are: –
• MASS spectroscopy [15N, 18O]
• NMR spectroscopy [2H, 13C]
• Scintillation counter
• Geiger-Mueller Counter
• Autoradiography
Requirement for tracer technique

1. Preparation of labelled compound.

2. Introduction of labelled compound
into a biological system.
3. Separation & determination of
labelled compound in various
biochemical fractions at later time.
 1.Preparation of Labelled Compound:-
 The labelled compound can be produced by growing chlorella in
atmosphere of 14CO2 All carbon compounds get
14
C labelled.
 The 3H (tritium) labelled compound are commercially available.
Tritium labelling is effected by catalytic exchange in aqueous
media by hydrogenation of unsaturated compound with tritium
gas. Tritium is pure β emitter of low intensity &
its radiation energy is lower
 2. Introduction of labelled compound:-
PRECAUTION:-
• The precursorshould react at necessarysite of synthesisin
plant.
• Plant at the experiment time should
synthesize the compound under investigation.
• The dose given is for short period.
Methods of Radioactive Labelling
 Root feeding (Hydroponic culture): From roots, roots
are the main site for biosynthesis.(Rauwolfia)
 Direct injection:Administered through injection,
for hollowstem plants (Umbeliferious, opium, etc)
 Infiltration: Infiltrations can be made into rigid stems by using a
wick consisting of a thread drawn through the stem and dipping
into the labelled solution; alternatively, a flap can be cut in the
stem and this dipped into the solution to be infiltrated.
3. Separation and detection of compound:-

• Geiger–Muller counter.
• Liquid Scintillation counter.
• Gas ionization chamber.
• Bernstein–Bellentine counter.
• Mass spectroscopy.
• NMR electrode meter.
• Autoradiography.
• Radio paper chromatography.
 Methods
1. PRECURSOR PRODUCT SEQUENCE:
• In this technique, the presumed precursor of the constituent under
investigation on a labelled form is fed into the plant and after a
suitable time the constituent is isolated, purified and radioactivity is
determined.
Disadvantage:
• The radioactivity of isolated compound alone is not usually
sufficient evidence that the particular compound fed is direct
precursor, because substance may enter the general metabolic
pathway and from there may become randomly distributed through
a whole range of product.
Application:
• Stopping of hordenine production in barley seedling after 15–20
days of germination.
• Restricted synthesis of hyoscine, distinct from hyoscyamine in
Datura stramonium.
• This method is applied to the biogenesis of morphine & ergot
alkaloids.
 2. DOUBLE & MULTIPLE LABELLING:-
• This method give the evidence for nature of
biochemical incorporation of precursor arises double & triple
labelling.
• In this method specifically labelled precursor and their
subsequent degradation of recover product are more
employed.
• Application: -
• This method is extensively applied to study the biogenesis of plant
secondary metabolite.
• Used for study of morphine alkaloid. E.g. Leete, use Doubly
labelled lysine used to determine which hydrogen of lysine
molecule was involved in formation of piperidine ring of
anabasine in Nicotina glauca.
2. Competitive feeding experiments can be of value
in determining which of two possible intermediates is
normally used by the plant. In its simplest form,
without taking into account a number of other factors,
competitive feeding could distinguish whether B or B
´ was the normal intermediate in the formation of C
from A.
Inactive B and B´ are fed with labelled A to separate
groups of plants and a control is performed by feeding
labelled A only to another group. If the incorporation
of radioactivity into C is inhibited in the plants
receiving B, but is unaffected in the group receiving B
´, then we may conclude that the pathway from A to
C probably proceeds via B.
 COMPETITIVE FEEDING:
• If incorporation is obtained it is necessary to consider whether this infact, the
normal route of synthesis in plant not the subsidiary pathway.
• Competitive feeding can distinguish whether B & B’ is normal intermediate
in the formation of C from A.

Application: -
• This method is used for elucidation of biogenesis of Tropane alkaloids.
• Biosynthesis of hemlock alkaloids (coniine, conhydrine etc) e.g. biosynthesis
of alkaloids of Conium maculactum (hemlock) using 14C labelled
compounds.
3. SEQUENTIAL ANALYSIS:
• The principle of this method of investigation is to grow
Chlorella in atmosphere of 14CO2 & then analyze the at
given time interval to obtain the sequence in which various
correlated compound become labelled.
Application:-
• 14CO2 & sequential analysis has been very
successfully used in elucidation of carbon in
photosynthesis.
• Determination of sequential formation of opium,
hemlock and tobacco alkaloids.
• Exposure as less as 5 min. 14CO2, is used in
detecting biosynthetic sequence as-
• Piperitone --------- (-) Menthone ---------- (-) Menthol in
Mentha piperita.
 AUTORADIOGRAPHY
An autoradiograph is an image on an x-ray film or
nuclear emulsion produced by the pattern of decay
emissions (e.g., beta particles or gamma rays) from a
distribution of a radioactive substance. Alternatively,
the autoradiograph is also available as a digital image
(digital autoradiography)

General principle of autoradiography. A silver halide (AgX) is

ionized by the radiation emitted from radioisotopes, forming
Ag+ ions. Ag+ is then reduced and converted to metallic Ag by a
developer reagent (usually containing AgNO3), which
precipitates within the gelatin emulsion of the X-ray film. The
reduction and precipitation are stopped by emerging the film in
a fixative solution forming the final image.
Radiography is the visualisation of the pattern of
distribution of radiation. In general, the radiation
consists of X-rays, gamma (g ) or beta (b ) rays,
and the recording medium is a photographic film.
For classical X-rays, the specimen to be
examined is placed between the source of
radiation and the film, and the absorption and
scattering of radiation by the specimen produces
its image on the film. In contrast,
in autoradiography the specimen itself is the
source of the radiation, which originates from
radioactive material incorporated into it.
 Application of tracer technique

 Study of squalene cyclization by use of 14C, 3H

labelled mevalonic acid.

 Interrelationship among 4 – methyl sterols & 4, 4

dimethyl sterols, by use of 14C acetate.
 Terpenoid biosynthesis by chloroplast isolated in
organic solvent, by use of 2- 14C mevalonate.
 Study the formation of cinnamic acid in pathway
of coumarin from labelled coumarin.
 Origin of carbon & nitrogen atoms of purine
ring system by use of 14C or 15N labelled
precursor.
 Study of formation of scopoletin by use of
labelled phenylalanine.
 By use of 45Ca as tracer, - find out the uptake of
calcium by plants from the soil. (CaO & CaCo2).
 By adding ammonium phosphate labelled with
32P of known specific activity the uptake of
phosphorus is followed by measuring the
radioactivity as label reaches first in lower part of
plant, than the upper part i.e. branches, leaves etc.
 Advantages

 High sensitivity.
 Applicable to all living organism.
 Wide ranges of isotopes are available.
 More reliable, easily administration &
isolation procedure.
 Gives accurate result, if proper metabolic time &
technique applied.
 Isolated Organs, Tissues and Cells
• The cultivation of isolated organs and tissues of
plants eliminates interference from other parts of the
plant which may produce secondary changes in the
metabolites. It can be used for feeding experiments in
conjunction with labelled compounds and is also
useful for the determination of the site of synthesis of
particular compounds.
• Isolated shoots of plants, when placed in a suitable
solution or in water, will usually remain turgid for
some days and during this time presumably have a
normal metabolism; soon, however, a pathological
metabolism commences.
• The technique can be refined by aseptically
connecting the cut end of the shoot to a reservoir
of suitable sterile nutrient, when the shoots will
remain normal for much longer periods. Such
shoots often develop roots at the cut ends—a
factor which could invalidate the results of an
experiment.
• Isolated leaves can be similarly maintained. Rooted
leaves have been used in studies on Nicotiana and
Datura. By this method a large quantity of root is
obtained with a relatively small amount of aerial
parts. It has the advantage that the nutrient solution
requires no sugar, as sufficient starch is synthesized
in the leaf and consequently bacterial and fungal
growth in the nutrient solution is minimized.
 Grafts

• Grafting techniques have considerable use in

biosynthetic studies, particularly for the
determination of the sites of primary and secondary
metabolism of some secondary plant products.
Alkaloid formation by grafted plants has been
extensively studied in Nicotiana and the tropane
alkaloid-producing Solanaceae. Thus, tomato scions
grafted on to Datura stocks accumulate tropane
alkaloids, whereas Datura scions on tomato stocks
contain only a small amount of tropane alkaloids.
This suggests the main site of alkaloid synthesis to
be the Datura roots.
• However, interspecific grafts involving D.
ferox and D. stramonium demonstrate that
secondary modifications of alkaloids do
occur in the aerial parts of these plants. This
has been demonstrated conclusively by
feeding alkaloid-free scions of D. ferox on
Cyphomandra betacea stocks with
hyoscyamine; on subsequent analysis,
hyoscine was isolated from the leaves.
• Large numbers of mutant strains of microorganisms have
been produced which lack a particular enzyme, which
results in their metabolism being blocked at a particular
stage. Such an organism may accumulate the
intermediate compound immediately before the block
and for its survival may require an artificial supply of
another intermediate which arises after the block. Such
organisms are obviously useful materials in biosynthetic
studies and have proved of major importance in some
investigations.
• A mutant of Lactobacillus acidophilus, by its ability to
utilize a constituent of ‘brewer’s solubles’ but not acetate,
led to the isolation of mevalonic acid, an important
intermediate of the isoprenoid compound pathway.
• Ultraviolet-induced auxotrophic mutants of
ergot with respect to a number of amino
acids have been produced; cultures of these
have been used to inoculate rye and the
resulting alkaloid contents of the sclerotia
have been investigated.