Relationship Between Primary Metabolism and Secondary Metabolite Accumulation

8/6/2019 Relationship Between Primary Metabolism and Secondary Metabolite Accumulation

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CIMAP WINTER TRAINING ON BIOTECHNOLOGYAND BIO-INFORMATICS-2006

RELATIONSHIP BETWEEN PRIMARY METABOLISM AND

SECONDARY METABOLITE ACCUMULATION. C-ASSIMILATION,METABOLITE UTILIZATION INTOPRODUCTION OF ESSENTIAL OILS AND ALKALOIDS IN SOMEIMPORTANT MEDICINAL AND AROMATIC PLANTS (MAPs).

N.K.SRIVASTAVA

DEPTT. OF PLANT PHYSIOLOGY

& BIOCHEMISTRY, CIMAP



PHYSIOLOGICAL BASIS OF MEDICINAL &

AROMATIC PLANTS (MAPs)²

Case histories reveal great diversity among cropplants (divided into groups based on metabolic end

products utilized) such as-sugar yielding

(sugarcane, sugar beet), starch yielding (cassava),

protein yielding (pea, legumes) oil yielding(mustard, sunflower) etc. Like wise MAPs for e.g..

(mints, poppy, turmeric, artemisia, catharanthus,

rose) occupy a unique position as being rich

repository of Secondary Metabolite yieldingcompounds (SM) prominently essential oils &

alkaloids. These SM are present in leaves, stems,

barks, roots, inflorescence & capsules



WHAT ARE SECONDARY METABOLITES-?

Secondary metabolites are chemicalcompounds not directly involved in the normal

growth, development or reproduction and in

this sense are "secondary". Yet, presence of

these compounds used as defense againstpredators, parasites, diseases, for interspecies

competition, and to facilitate the reproductive

processes (coloring agents, attractive smells,

etc). These include Alkaloids as Hyoscyamine,(Datura stramonium

), Atropine, ( Atropa

belladonna). Terpenoid as Azadirachtin (Neem

tree),Artemisinin,( Artemisiaannua

),Steroids and

Saponins



SECODARY METABOLITES (SM)

How are these metabolites (Essential oil/Alkaloids) accumulated?--Genetic, Protein,

Metabolite and Environmental effects.

SM accumulation is highly integrated with the physiological

processes of the whole plant as such it depends on the metabolic

state of the actual and adjacent synthesizing tissues. At the same

time SM production is an eco-physiological response/adaptation to a

particular set of environmental conditions. Physiological &

Biochemical basis of productivity encompasses- plant ontogeny,

sites of accumulation, C-metabolic efficiency of the tissues involved,

photoperiod, light quality, seasonal & climatic exposure/variations,elemental (nutritional) availability (Macro as N P K, and Micro as Fe,

Mn, B, Cu, Zn.) endogenous levels of PGRs, abiotic stress as water,

soil and microbial population and temperature.



SYSTEMS BIOLOGY APPROACH-AFUNCTIONAL APPROACH.



CONTENTS

1-Metabolic Pathways-Terminologies.

2-Linkages of Primary Metabolic Pathways.

3-Carbon Assimilation-Overview.

4-Components/Steps of Carbon Assimilation.5-Relation to Terpenoid metabolism.

6-Relation to Alkaloid metabolism.

7-Experimental procedures to measurephotosynthetic efficiency and carbon partitioning.

8-Literature



1-METABOLIC PATHWAYS-

TERMINOLOGIES.

METABOLISM.

Higher plants produce a huge number of organic

compounds which are essential to constructcell/tissue/organs and support metabolicprocesses during their growth. This is achievedby an integrated network of enzymes and

carefully controlled chemical reactionscollectively termed as METABOLICPATHWAYS.



1-METABOLIC PATHWAYS- PRIMARYMETABOLISM.

Some of the crucially important end productmolecules include CARBOHYDRATES, FATS,PROTEINS AND NUCLEIC ACIDS. Despite theextremely diverse characteristics of the plants,pathways leading to the production of thesemolecules, barring minor variations, are found tobe similar. The processes of these pathways aretermed as PRIMARY METABOLISM and theintermediary metabolites as PRIMARYMETABOLITES, such as sucrose in carbohydratepathways and amino acids in proteins/nucleic acid

pathways.



1-METABOLIC PATHWAYS-SECONDARYMETABOLIC PATHWAYS.

SECONDARY METABOLISM- along withprimary metabolic pathways, there also existsan area of metabolism concerned with

compounds which have limited distribution.Pathways leading to the production of suchmetabolites are termed as SECONDARYMETABOLIC PATHWAYS and compounds of

these pathways as SECONDARYMETABOLITES. Broad category of examplesinclude essential oils (terpenoid) and alkaloids.



1-UTILIZATION OF SM.

These secondary metabolic compounds are localized in sub-cellular, tissue, organ and also are plant specific. Presenceof these compounds at the same time impart a great eco-physiological advantages to the plant in the environment in

which they are present. Biomining of secondary metabolitesprovides majority of BIOLOGICALLY ACTIVE NATURALPRODUCTS. These find application in nutraceuticals, healthtonics, medicines, cosmetics etc. Studies on the area ofinter-relation ship between the primary & secondary

metabolism offers us mechanisms to understand how andwhy accumulation of secondary metabolites occur and howit can be regulated and manipulated to our advantage. Thisis the basis of emergence of new field ²METABOLICENGINEERING.



2-LINKAGES OF PRIMARY METABOLIC PATHWAYS,CASSIMILATES AS CENTRE STAGE MOLECULES.-



2-LINKAGES OF PATHWAYS-The major primary metabolic processes such as

PHOTOSYNTHESIS, GLYCOLYSIS AND KREB¶S CYCLE provide a bank of

metabolites which are utilized in pathways as:

1-Acetate pathway-By utilizing acetyl-CoA leading to theproduction of fatty acids and derivatives.

2-Shikimic acid pathway-leading to the production ofphenylalanine, tyrosine, tryptophan which act as precursors ofvariety of alkaloids.

3-Mevalonic acid pathway-leading to the production ofterpenoids and polymers.

4-Deoxyxylulose phosphate-together with mevalonic acid leadsto the production of isoprenoids ,terpenoids and steroids.





3-CARBON ASSIMILATION-Integrated process.



4-COMPONENTS/STEPS OF CARBON ASSIMILATION-

Leaves and its structure.

Nature of light-Photosynthetic Active Radiation(PAR)

Chlorophyll and accessory pigments.

Chloroplast.

Stages of photosynthesis-light and dark reaction.

Types of photosynthesis-C3 and C4 .Summary of chemical steps of carbon reductioncycle.



4-COMPONENTS OF CARBON ASSIMILATION-

Leaves and its structure.

Crosssection of

leaf

showing

anatomical

features

importantfor study of

photosynth

esis-stoma,

guard cells

,mesophyll

and veins.

OIL GLANDS




Stomata and stomatal apparatus.

Pea leaf stoma Stomatal apparatus of leaf

epidermal peel of corn.




NATURE OF LIGHT-PAR



4-COMPONENTS OF CARBON ASSIMILATION.

Chlorophyll and accessory pigments. A pigment is any substance whichabsorbs light. The colour of the pigment comes from the wavelength oflight reflected.eg. Chlorophyll absorbs all wavelength except green. Chl.a isthe major fraction and accessory pigments include Chl.b, (c, d, and e inalgae & protistans) xanthophylls & carotenoids.

Molecular structure.Absorption peaks

Mg



4- COMPONENTS OF CARBON ASSIMILATION-

CHLOROPLAST STRUCTURE-

Light

Lightindependent




Photo chemical reactions, Light and light independent (dark)

reactions.



4-COMPONENTS OF CARBON ASSIMILATION.Light reaction, PS-I & PS-II leading to production of O2 in presence ofMagnanin (water splitting protein).




Light independent steps of carboxylation,C3 cycle,3C molecule (PGA),

key enzyme- Rubisco.

R

U

BI

S

C

O



4-COMPONENTS OF CARBON ASSIMILATION.Initial steps of carboxylation in C4 cycle,4C molecule OAA, enzyme-PEP Case.




Photorespiration, loss of fixed CO2?.Protection against oxidative

damage.




Summary of chemical steps of C3 & C4 cycle.




End products of carbon assimilation with in the

plants may be presents as,Monosaccharide (as glucose, hexose's, pentose) diand tri saccharides.

Derivatives as alcohol, esters, and glycosides.

Polysaccharides as Glucan, fructans.

Structural components as cellulose, hemicelluloses.

Used as food reserves as starch.

Present as gums and mucilage.



5-Carbon assimilates in relation to terpenoid metabolism

Mevalonate pathway Deoxyxylulose pathway.





6-Intracellular C assimilate partitioning in relation toalkaloid production. Compartmentation of metabolitesin Catharanthus.

METABOLIC

POOLS OFSUGARS,AM

-INO ACIDS

ORGANIC

ACIDS



6-COMPONENTS OF CARBON ASSIMILATION

INTRINSIC, these include:

GENOTYPE

ONTOGENY

METABOLIC STATUS (YOUNG,MATURE,OLD)

EXTRINSIC, these include:NUTRIENT AVAILABILTY

ENVIRONMENTAL VARIABLES AS

LIGHT

TEMPERATURE

GASEOUS COMPOSITION (POLLUTION)



7-EXPERIMENTAL PROCEDURES TO MEASUREPHOTOSYNTHETIC CARBON EFFICIENCY

Parameters determining the photosynthetic capacity/efficiency ofthe leaves are ²incident Solar radiation,Total Chlorophyllcontent,CO2 uptake and its fixation.

1-SOLAR RADIATION:-This is the single most powerfulenvironmental factor.Plants respond best to the spectral region

between 400-800 nm which is called the Photosynthetic ActiveRadiations (PAR).

1a-MEASURMENT TERMINOLOGY-Solar radiations can bemeasured in three categories by different sensors:

A-Total energy is measured by a Radiometric sensor and the unit

of expression is w/m2/s.

B-Total intensity is measured Photometric sensor and the unit ofexpression is Lux/Klux.

C-Total radiation is measured by a Quantum sensor and the unit

of expression is Einstein(Mol)/m2/s.



7-Solar radiation (LIGHT) -A light meter is employed

for measurement.The overall system consists of a sensor and a

console either in analog or digital form with option for printout.

SENSOR ______________________

PAR/LUX LEAD CONSOLE

SYSTEM OVERVIEW

1c-Demonstration unit- LiCOR 188B Integrating

Quantum/Radiometer/Photometer.



7-FLOW SHEET FOR DETERMINATION OFCHLOROPHYLL CONTENT (leaf photochemical capacity)

Chop fresh leaves of known wt.& grind into fine paste in 80% acetone

|

pass through a filter paper and collect

filtrate

|

Make up to a known volume with 80 % acetone and measure absorbanceat 645 & 663 by a spectrometer. Note: This extraction should be

carried out in dim light,a pinch of CaCO3 is added to prevent

pheophytin formation.Calculation is based on the formula of Arnon1959.Variations in chlorophyll content will be demonstrated respect toleaf physiological capacity viz age..



7-DETERMINATION OF CARBON-DI-OXIDEEXCHANGE RATE (CER) of leaf-

Assimilation of CO2 is a multistep process startingfrom the uptake of CO2 into the leaf via thestomata.The rate at which this uptake occurs is calledCO2 Exchange Rate (CER).

Normal atmospheric CO2 concentration is 350 ppm.PRINCIPLE OF DETERMINATION

3a-Principle of measurement-CER measurement isbased on the principle of measuring decrease in CO2levels by an Infra-red Gas Analyzer (IRGA).

3b-Units of Expression-CER is expressed as -mgCO2/m2/s



7-Basically there are two approaches for determiningCER-

A-CLOSED SYSTEM-A leaf is enclosed in a leafchamber and inlet and outlet pipes are connected toIRGA.Closed systems are best suited for short termeffects & measurement of CER & also for thephotorespiration capacity.C4 plants can reduce

ambient CO2 to levels from 350 to 0-10 ppm where asC3 plants can reduce it up to 50 ppm.

B-OPEN SYSTEM-Commonly used to monitor steadystate CER.In this system inlet air tube is open and air

passes over the leaf As a result air leaving thechamber will have low CO2 and this is monitored byIRGA.

3d-Demonstration unit- Computerized Portable

Photosynthesis System Model CID-CI 310





7-PROCEDURE FOR FEEDING 14CO2 TO PLANTS ANDSUBSEQUENT EXTRACTION.

Extraction intooil in a mini

Clevengerapparatus orinto alkaloidsas perexperimental

needs.

Tracer fed plants.



GROWING PLANTS AND CULTURE CONDITIONS

1-SAND CULTURE

2-HYDROPONICS



SAND GROWN CHLOROPHYTUM ROOTS



VISUAL Fe DEFECIENCY RESPONSE LEFT-

MENTHA AND RIGHT-CITRONELLA



SAND CULTURE HYDROPONICS



7-SETUP FOR EXPOSURE OF PLANTS TOGESEOUS 14CO2



7-SETUP FOR EXPOSURE TO LIQUID 14C-

SOURCES AS SUCROSE, ACETATE, GLUCOSE



8-RECOMMENDED LITERATURE.Topic related

101 Reasons to learn more plant biology. The Plant Cell.1995.Light perception and signaling in higher plants. Current opinion in Plant Biology2003, 6, 446-452.

Sucrose to starch-A transition in plant molecular biology. TRENDS IN PLANT SCIENCE,7,2002,35-41.

Strategies for transgenic manipulation of monoterpene biosynthesis in plants.TRENDS IN PLANT SCIENCE,7, 2002,366-373.

Biosynthesis of Terpenoid Indole Alkaloids in Catharanthus. The Alkaloids. Ed.G.A.Cordell.

Sugar sensing in higher plants. TRENDS IN PLANT SCIENCE.1997,2, 208-

213.

Role of crop physiology in predicting gene-to-phenotype relationships. TRENDSIN PLANT SCIENCE.2004.9.1360-1368.

Systems biology: a brief overview. SCIENCE.2002.295.1662-1664.

8 S ifi l t l t d



8-Specific plant related.

Distribution of Photosynthetically fixed 14CO2 into essential oil inrelation to primary metabolites in developing peppermint Menthapiperita. leaves. Plant Science, 1991, 76, 153-157.

Interspecific variations in mints for photosynthetic efficiency and 14-Cprimary metabolic pool in relation to essential oil accumulation. PlantPhysiology,1991, 138, 650-654.

Distribution of Photosynthetically fixed 14CO2 into curcumin and

essential oil in relation to primary metabolites in developing turmeric(curcuma longa ) leaves. Plant Science, 2000,152, 165-171.

The substrate utilization and concentration of 14C photosynthate incitronella under Fe deficiency. Photosynthetica, 1998, 35, 391-398.

Utilization of Photosynthetically fixed 14CO2 into alkaloids in relationto primary metabolites in developing leaves of Catharanthus roseus.Photosynthetica, 2004, 42, 469-472.



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Relationship Between Primary Metabolism and Secondary Metabolite Accumulation