Secondary Metabolites & Plant DefencePankaj Kumar Pant Lecturer Dept. Of Biotech. & Allied Sciences

Secondary Metabolites Secondary metabolites are generally those chemicals that have no significant role in plant s growth and development. Plant secondary metabolites differs from primary metabolites in being their specified occurrence.

Secondary metabolites are also known as the Natural products or Secondary Products.

Role of Secondary metabolites Protection against the different organisms. Serves as the attractants for the insects pollinators. Functions as the plant-plant competition and plant -microbe interection.

Classes of Secondary metabolites There are generally three major classes of secondary metabolites1.Terpenes 2.Phenolics 3.Nitrogen containing compounds

Terpenes or Terpenoids Terpenes are derived from the five carbon compounds known as the Isoprene units. The Isoprene is the basic structural unit of the terpenes. Thus, the terpenoids are occasionally known as the is Isoprenoides.

Classification of terpenoides There are different classes of terpenoides based on to the number of carbon units, these are 1. Monoterpenoids( with 10 carbons) 2.Sesqueterpenoides (with 15 carbon units) 3.Diterpenes(with 20 carbon atoms) 4.Triterpenoides(with 30 carbon atoms) 5.Tetraterpenoides(with 40 carbon atoms) 6.Polyterpenoides( {C5}n)

Terpene Biosyenthesis Terpenes are generally synthesized from primary metabolites by - Mevalonic acid pathway - Methylerythretol phosphate pathway(MEP)

Mevalonic Acid Pathway Acetyl Co-A is the main precursor. Three molecules of the Acetyl-CoA are utilized in stepwise manner to yeild the Mevalonic acid. The mevalonic acid is then phosphorylated, decarboxylated and dehydrated to form Isopentenyl diphosphate (IPP).

Methylerythretol Phosphate pathway MEP is yet another pathway leading towards the synthesis of the Isopentenyl diphosphate IPP. -The pathway generally involves the use of glycolysis intermediates. - This pathway is operated in the chloroplast and plastids.

IPP LEADS TO TERPNOIDS IPP generally present along with it s isomeric form called Dimethylallyl diphosphate (DPP). Both the IPP and DPP are the active constituents of the terpene biosyenthesis. - IPP and DPP reacts to form the Geranyl diphosphate (GPP)

GPP joins another molecule of the IPP to form the Farnesyl diphosphate(FPP). Farnesyl diphosphate is the main precursor of the all the sequeterpenes. Addition of yet another IPP lead to the formation Of Geranylgeranyldiphosphate(GGPP)

Terpenes And Their Role Terpenes are generally toxic in nature and are generally feeding deterrent to many plant feeding insects. Pyrethroides, Saponins,Phytoecdysomes essentialoils, are some of the important terpenoides.

Pyrethroides- A monoterpenoid ester. Found among the members of chrysanthemum species and are generally good sources of the insecticides commercially available. Essential oils- Essential oils are generally mixture of volatile monoterpenes and sesqueterpenes, lend a specific smell to the foliage.

- Essential oils has the insect repellant acitivities Azadirachitin is a non volatile limonoide { a group of triterpenoide}. Phytoecdysomes- isolated for the first time from Podophyllum vulgare. -It has the resemebelence with the molting hormone.

In the insect it thus erupts the molting and other necessary developmental processes. Cardiniloides- Generally the group of glycosides that taste better and are extremely toxic to the higher animals. -They generally have the dramatic effect on to the heart rate by influencing Na/K ion channel.

- Digitalis have been a good source of the cardinolides for the treatment of the cardiac disease. Saponin - a steroid and triterpenoide glycoside. - Posses soap like properties. - Posses the lipid soluble and water soluble elements

- Saponin works by disturbing the sterol uptake from the digestive system. - Disturbs the membranes after entering the blood stream. - Forms the complex with the sterols.

Phenolic Compounds Phenolics are characterised by the presence of the 6 membered benzene ring along with the alcohol ( OH). Phenolics provides -Mechanical support for the pollination. -Protection against the herbivores and pathogens. -Protection against the harmful radiations.

Biosyenthesis of Phenolics There are basically two general pathways are involved 1. Shikimik acid pathway. 2. Melonic acid pathway.Melonic acid a pathway is found in fungi and bacteria and is less common in higher plants.

Shikimik Acid Pathway Shikmik acid pathway generally involves the conversion of the simple carbohydrate precursor to Aromatic amino acid Phenylalanine generally. Animals generally posses no Shikmik acid pathway thus, the aromatic amino acid are essential nutrients in the diet.

From Phenylalanine to Phenolics The most generalised way to form the phenolics generally involves the removal of the NH3 to form the Cinnamic acid. The above reaction generally involves the use of the enzyme Phenlyalanine ammonia Lyase i.e (PAL). PAL Is the most studied enzyme in plant secondary metabolite production.

PAL generally occupies the position in between the primary and secondary metabolism. PAL activity generally increases during the environmentel,nutrient,light and fungal infections. There are cases in he plant where there are several copies of the PAL are present.

The reactions further involves the addition of the hydroxl groups and other substituents. The Trans-Cinnamic acid,p-Coumaric acid, and their derivative so formed are simple phenolic compounds called phenyl-propanoids. These simple phenolics have property to get activated in presence of light.

Furanocoumarins- Posses the property to resist the phototoxicity. - Under the influence of sunlight i.e UV-A(320400nm) the furanocoumarin gets activated. - Activated furanocoumarin attach it self to the double helix of the DNA and bind to the cytosine and thymine thus blocks the transcription and repair pathways.

AllelopathyPhenolic compounds like Caffeic acid and Ferulic acid generally lead to the inhibition of seed germination in nearby species a phenomenon known as the Allelopathy.

Lignin- Highly branched phenyl-propanoid compound. - The lignin is generally bound covalently to the polysaccharide and the cellulose. - Thus difficult to isolate & so structure not been correctly elucidated. - Made up of three phenyl-propanoid phenols

Coniferyl Coumaryl Sinapyl These components are added enzymatically that involves the use of the free radicals. Structural analysis generally showed the C-O-C, C-C bonding in the three dimensional orientation.

Lignin works as a protective agent in plant by1. Providing the physical strength that repels the herb feeder. 2. By binding to the protein and cellulose and so reducing their digestibility in their feeders. 3. By accumulating at the site of wounding and infection.

Flavinoids The largest class of the phenolics. The structure generallly consists of the 15 carbons. Out of which 12 present as the two benzene ring and the rest three are present as the bridge carbons.

FLAVONOIDE GROUPS Generally there are four major classes of the flavnoides1.Anthocynin 2.Flavones 3.Flavonols 4.Isoflavones

Anthocynins There are generally two types of the plant pigments1.Carotenoides- Yellow,red,orange colored 2.Flavonoides- With a range of color -Anthocynins are glycosides with sugar at the position 3. -Anthocynins without the sugar are known as the Proanthocynidines.

Flavones and Flavonols as Phtoprotectants Flavones and Flavonols functions as to have protection against the UV-B(280-320 nm). These pigments generally accumulates at the epidermal cells and so absorb the harmful radiation and allows only the photosyenthetic radiations.

Isoflavones Isoflavnoides generally posses the ring B is shifted. Isoflavones are generally the antimicrobial agents. Found mostly in the legumes. They generally posses the anti-cancer and anti-estrogenic activity.

Tannins Tannins is a category of plant phenolic that generally been utilized for the leather syenthesis. Tannin generally bind to the collagen protein increasing their resistance to the heat, water and microbes.

Classes of Tannins Tannins are generally been classified in two categories1. Condensed tannins. 2.Hydrolyzable tannins. - Condensed tannins- Made from the polymerization of the flavonoid units. - Found in woody plants, they could be hydrolyzed to their anthocynidines, thus are known as pro-anthocyanidins.

Hydrolyzable Tannin- They are generally heterogeneous polymers containing the Phenolic acids specially the Gallic acid and the simple sugar. They are generally much more easily hydrolyzed; needs dilute acid.

The Tannin have the property to bind the proteins nomspecifically thereby inhabiting their proper biological action. In the gut of many herbivores the tannin is hydrolyzed to their respective quinoid form. Quinoid forms are generally strong electrophiles that reacts with the nucleophiles NH2 and SH of the protein.

The Tannin also inactivates the herbivore digestion by forming the complex aggregates that are difficult to digest. However some of the Rodents and Rabbits are much resistant to the Tannin toxicity as they posses the proteins with a high Proline content.

Alkaloids; N containing compounds Alkaloids are group of compounds that are alkaline at the. Contains the N atom that is generally protonated. Protonated N lead to a characterstic positive charge to the alkaloids. Alkaloids are generally derived from some of amino acids i.e Lysine ,Tyrosine and Tryptophan.

Stychrine, Conine, Atropine are the classical examples of the Alkaloids. Alkaloids generally effects the nervous system specially chemical transmitters, ,membrane transport,protein syenthesis and many other enzyme activities.

Cynogenic glycosides Cynogenic glycosides are the one of the compounds of Alkaloid category. Found commonly in the Legumes and Grasses. Cynogenic glycosides are not posinous in themselves but they breaks down into the components that are poisonous. Cynogenic glycosides generally releases the poisonous gas HCN.

Steps involved in Cynogenic glycoside Breaking The Cynogenic glycosides are generally break down into toxic components in two steps1.The sugar is cleaved by glycosidase produces the -Hydroxynitrile or cynohydrin. 2.The cynohydrin is cleaved by Hydroxynitrile lyase to liberate the HCN.THE HCN GENERALLY BINDS TO THE METALLOPROTEIN LIKE CYTOCHROME OXIDASE,A KEY ENZYME IN RESPIRATION.

Plant are generally resistant to their own Cynogenic glycosides because the enzyme are separated in different compartments. Cynogenic glycosides are generally feeding detterents.

GLUCOSINOLATES Glucosinolates is another category of the Alkaloid Glycosides. Found commonly in the Brassicaceae family. The sugar moiety is generally attached to the Sulphur group Generally the compounds are responsible for the typical smell of some vegetables like Cabbage, Broccoli and Redishes.

Glucosinolate breakdown Glucosinolate breakdown is a two step process1. The first step involves the breaking of SugarSulphur bond to form a Aglycone,the step is catalyzed by enzyme Thioglucosidase or Myrosinase. 2. Depending on to the condition the resulting aglycone is rearranged and loses the sulphate and gives the Isothiocynates and nitriles.

Isothiocynates and nitriles are generally the defensive compounds due to their toxicity and feeding repellent property.

Like cynogenic glycosides the enzymes are seprated in different compartments.

Non protein amino acids as defence components There are many amino acid in addition to the standard 20 amino acids these amino acids are called Unusual amino acids. These amino acids are generally not incorporated into the protein and present in free form. Canavanine- A unusual amino acid and a close analogue of the Arginine.

- Canavanine sometime mistakenly incorporated into the protein. - This canavanine is identified by the herbivore enzyme that usually bind to the Arginine t-RNA. - The unusual appearance of the Canavanine in place of Arginine lead to inactivation of protein.

Defence responses in plants There are generally two types of plant responses to the pathogenic attacks1. Constitutive Defence response . 2. Induced defence response. Constitutive plant response-Are the mechanism that are always present. -Responsible elements are generally stored compounds or their precursors.

Induced plant defence response-Are initiated only after the actual damage occurs. -Thus, is a kind of rehabilitation action.

Elicitors;Induction of series of action The insect derived compounds that are responsible for the wound response by plant. A generalized group have been identified acts as the elicitors, these compounds known commonly as Fatty acid-Amino acid. The biosynthesis of these compounds generally depends upon the plant as source of fatty acid and insect as amino acid source.

The fatty acid component generally includes the Linoleic acid (18:2) and Linolenic acid (18:3). Plant part when ingested by insect then, enzyme in the gut generally conjugated the insect derived amino acid commonly Glutamine with plant derived fatty acid. Further processing of this conjugate is found in some insect species.

Elicitors Induces; Jasmonic acid Octadecanoid pathway- when a elicitor is introduced then it results in a signal transduction network The major signal transduction network is generally Ocatdecanoid pathway (Chloroplast and peroxisome). In the plants a major response to the insect attack is formation of jasmonic acid formation through the well known Octadecanoid signaling pathway.

Role of Jasmonic acid Jasmonic acid generally plays it s part by Increasing the transcription of the genes involved in the defence.A CLOSE OBSERVATION OF ARABIDOPSIS MUTANT LACKING THE ACTIVE GENE SHOWED A PRONOUNCED ATTACK BY INSECT THAT DON T ATTACK ARABIDOPSIS GENERALLY.

Plant proteins and their role In defence There are some plant proteins that have some role in defenceAmylase inhabitors- Generally blocks the action of the starch digesting enzyme amylase. Lectins- Lectins are generally carbohydrate binders, they binds to the carbohydrate associated with the membrane and inhabit the nutrient uptake.

Proteinase inhabitors- Proteinsase inhabitors are generally best known anti-digestive proteins. - Found in case of the legumes tomatoes and other plants. - After entering the herbivore digestive system they binds to the active site of the protein hydrolyzing enzymes, thereby inhabiting them.

Systemic Defence In case of tomatoes the rapid accumulation of the