ROLE OF SECONDARY METABOLITES

PLANT INNATE IMMUNITY

1. ROLE OF SECONDARY METABOLITES IN PLANT INNATE IMMUNITY PAT- 613 DISEASE RESISTANCE IN PLANTS (2+0) K.SIVAGNANAPAZHAM 2018601508

2. SECONDARY METABOLITES Organic compounds produced by Bacteria, Fungi or Plants which are not directly involved in the normal growth ,development or Reproduction of the organisms. Play an important role in plant defense mechanisms. Important functions such as protection, competition, and species interactions, but are not necessary for survival. Examples: Alkaloids,Antibiotics,Terpenoids,Quinolines,Phenazines , etc.

3. INNATE IMMUNITY Innate Immunity is an immunological subsystem that comprises the cells and mechanisms that provide the first line of defence from infection in a non specific manner. Play immediately within the hours of a pathogen enter in the body. Physical Barriers. Natural Immunity.

4. CLASSIFICATION Plant secondary metabolites can be divided into three chemically distinct groups Terpenes Phenolics Nitrogen & Sulphur containing compounds.

5. TERPENES Largest class of secondary metabolites and are united by their common biosynthetic origin from acetyl - coA or Glycolytic intermediates. Five major classes Mono Sesqui Di Terpenes Tri Poly ( Gerhenzon et al., 1991)

6. PHENOLIC COMPOUNDS Hydroxyl functional group on BENZENE ring. Chemically Heterogeneous group. Important part in Plant defense system such as DISEASES PESTS ROOT PARASITIC NEMATODES ( Wuyts et al., 2006)

7. CLASSIFICATION Coumarin Furano – coumarin Lignin Flavonoids Iso flavonoids Tanins

8. 1.COUMARIN Simple phenolic compounds wides spread in vascular plants and functions in various plant defense mechanisms. SHIKIMIC ACID PATHWAY Common in Bacteria, Plants and Fungi absent in Animals. Antifungal activity. ( Murray et al., 1982)

9. Contd … Halogenated coumarin derivatives work effectively in invitro to inhibit Fungal growth. Example: 7 – hydroxylated simple coumarins -parasitism of Orobanche cernua , by preventing successful germination, penetration and connection to the Host Vascular System. ( Serghini et al., 2001)

10. 2.FURRO COUMARIN Phyto toxicity to the palnts , in the family of Umbelliferae (celery, parnsip,and parsley) Non Toxic to the plants until they activated by the UV light. PSORALIN – a linear Furano - coumarin known for its use in the treatment of Fungal Defense. Found rarely in SO2 treated plants. (Ali et al., 2008)

11. 3. LIGNIN Highly branced polymer of phenyl propanoid groups. Physical toughness deters feeding by herbivorous animals and its chemical durability makes it relative to pathogens and insects. Lignification block the growth of pathogens and are frequent response to infection or wounding. (Gould, 1983)

12. 4.TANINS Second category of Phenolic category of plant phenolic polimers with defensive properties. Protocatechellic acid and chlorogenic acids – Disease resistance of certain plants. They prevent Oninon smudge – Colletotrichum circinans and prevent spore germination and growth of fungi. Compounds readily formed oxidised into potent fungistatic quinones by certain disease resistant cultivars. (Vickery, 1981; Butt and Lamb;Mayer,1987)

13. S containing secondary metabolites They include GSH – Glutathione. GSL – Glucosinolates . Phytoalexins . Thionins . Defensins . Allinin . been directly or indirectly with the defense of plants against Microbial pathogens – SIR. (Crawford et al., 2000)

14. 1. GSH - GLUTATHIONE Regulation of plant growth and development – stress responses. Act as a Anti oxidant. Detoxification of xenobiotics and cytokinins by targeting them into a vacuole. Act as a systemic messenger carrying information concerning the attack to non – infested tissues. Specialized cell Trichomes secretes GSH. ( Choi et al., 2001)

15. 2. GSL - GLUCOSINOLATES S containing Glucosides produced in higher plants. To increase resistance against the unfavourable effect of predators, parasites, and competitors by release of volatile compounds. Mustard oil Glucosides in cruciferaceae , Allyl cys sulfoxides in Allium - catalyse by myrosinase , cleave glucose by S atom. Isothiocyanate , nitriles - defense. (Siemens et al., 2009)

16. 3. DEFENSINS,THIONINS AND LECTINS S rich non storage plant proteins synthesis and accumulate after microbial attack. Some Defensins are antifungal or anti bacterial activity. Defensins genes are partly pathogen inducible.( Gu et al., 1992) Accumulation of Thionins in cell wall of infected spikes shows Defense response against Fusarium colmorum . Lectins defensive proteins that bind to carbohydrate, after ingested by herbivores interfere with nutrient absorption. (Kang and Buchenauer,2003)

17. N containing secondary metabolites They include Alkaloids Cyanogenic Glucosides Non protein Amino acids. Biosynthesized from amino acid. Role in anti herbivores defense and toxicity to humans.

18. MECHANISMS For defense responses, biosynthesis and activation of secondary metabolites are triggered with the recognition of MAMPs by PRR by Resistance proteins. Based on mode of biosynthesis and accumulation of defense related phytochemicals they classified as PHYTOANTICIPINS PHYTOALEXINS ( Ahuja et al., 2012)

19. PHYTOANTICIPINS Antimicrobial compounds that are performed or become released from constitutively stored precursors following attempts of microbial invasion. Defensive metabolites produced and stored constitutively in plant tissue are termed as Phytoanticipins . Saponins,Glucosinolates,Cyanogenic glucosides,Benzoxazinone glucosides . ( VanEtten et al., 1994)

20. 1. SAPONINS Saponins are glycosides of isoprenoid – type aglycones usually representing triterpenoids or steroids, widely distrubuted in flowering plants. Biosynthesis and invivo function of triterpenoid saponin from oat species have been studied. Avenacins A-1,A-2,B-1,B-2 .(roots) Absent in leaves. α- tomatine in TOMATO - steroidal glycoalkaloid . ( Maizel et al.,1964,Arnerson 1968)

21. Contd … Avenacins and α - tomatine affect invitro growth of a wide range of pathogenic and non pathogenic fungi. Genetically screening of a chemicaIly mutagenized Avena strigosa population enabled the isolation of saponin – deficient mutants. (sad mutants). SAD1,SAD2,SAD7 &SAD9 synthesis of avenacins . ( Qi et al .,2006;Mugford et al .,2009,2013)

22. Contd … Roots of homozygous sad mutants susceptible to adapted strain of soil borne fungus Gaeumanonnomyces graminis var . avenae , Fusarium culmorum and F.avenaceum . Sad mutants revealed the susceptibility to non adapted strain G.graminis var. tritici strain does not infect oats Role of Avenacin play in Disease resistance. ( Papadopoulou et al.,1999)

23. 2.GLUCOSINOLATES Brassicales produced mainly. Derived from amino acids include alanine,valine,leucine,isoleucine,methionine , phenylalanine,tyrosin , tryptophan and glutamic acid. Arabidiopsis thaliana accumulates two major group of these compounds Methionine derived aliphatic glucosinolates Tryptophan derived indolic glucosinolates ( Reichelt et al., 2002)

24. Contd … Glucosinolate hydrolysed by unstable aglycogens β - thioglucoside glucohydrolases (TGGs) ( myrosimases ). Results in the release of chemically unstable aglycogens – isothiocyanate (ITC) AG derived ITCs insect deterrent properties, restrict the growth of microbial pathogen. ( Brader et al., 2001) AG derived ITCs- Arabidopsis resistance Bacterial pathogen pseudomonas syringae . (Fan et al., 2011)

25. Immunity against Fungal pathogens Hemibiotroph - Phytophthora brassicae & Colletotrichum species Nectrophic Ascomycete - Plectospharella cucumerina Botrytis cinerea ( Lipka et al., 2005) Magnaporthe oryzae Leptosphaeria maculans Piriformospora indica (Jacobs et al .,2011)

26. 3. CYANOGENIC GLUCOSIDES β -D- glucoside of α - hydroxy-nitriles derived from tyrosine,phenylalanine,valine,isoleucine and leucine . HCN gas contrast to antimicribial agents. Lima bean – Colletotrichum gleosporiodes Leucine derived CG epiheterodendrin - Barley plants. Dhurrinase2(DHU2)- Sorghum bicolor. (Nielsen et al., 2002)

27. 4.BENZOXAZINONE GLUCOSIDES Benzoxazinone glucosides (BXs)- Poaceae including Maize,Rye,Wheat but not Barley Wheat & Maize – 2,4-dihydroxy-7-methoxy-1,4-benzoxazin-3-one (DIMBOA- Glc ) Rye & Barley - 2,4-dihydroxy-1,4-benzoxazin-3-one (DIBOA- Glc ). Antifungal activity against G.graminis,F.moniliforme,F.culmorum & Setosphaeria turcica . HDMBOA- Glc – F.graminearum , B.mayids,C.lunata ,& A.alternata . (Ahmad et al., 2011)

28. PHYTOALEXINS Muller and Borger (1940). Low molecular weight anti-microbial metabolites that are synthesized and accumulate in plants after a pathogen challenge. Hundreds of Phytoalexins have been isolated and characterized in different species. Selective groups include Camalexin Phenylalanine derived, Terpenoids . ( Schmelz et al., 2014)

29. 1. CAMALEXIN ( Brassicaceae ) Sulphur containing tryptophan derived alkaloids in response to pathogenic infection. Arabidopsis in defense responses. Genetic screening of an ethyl methanesulfonate - mutagenized population revealed a series of 5 non allelic phytoalexin deficient (pad) mutants with camalexin accumulation. Bioassay with P.syringae with both virulent and Avirulent strains. ( Glazebrook et al., 1996)

30. Contd … Some PAD genes – encode protein with regulatory function in Arabidopsis defense pathways. PAD4 & PAD2 Isolation of genes. PAD2 – γ - glutamylcysteine synthase involved in Glutathione synthesis. PAD4 - Disease response including Salicyclic acid dependent signalling . ( Parisy et al., 2007)

31. P.brassica , blockage of PEN2 pathway and camalexin biosynthesis did not affect entry rates, but led to more severe disease symptom than observed in pen2 & pad3. Rapalexin A represents another S containing indole alkoloid phytoalexin in Arabidiopsis . Biosynthesis is not obscure. ( Pedras & Yaya , 2013)

32. 2. PHENYLALANINE DERIVED PHYTOALEXINS Many reported Phytoalexins under this group. Resveratrol - Grapevine , Arachis hypogea . Biosynthesis requires the enzyme- stilbene synthase (STS) Precursor molecule- p- coumaroyl co enzyme A & malonyl co A Grapevine STSgene VST1- promoter in Tobacco leads to biosynthesis of Resveratol Disease resistance to B.cineria . ( Hain et al., 1993)

33. Contd … Glyceollins – Soyabean Pisatin – Pea Medicarpin – Alfalfa Sakuranetin - Rice RNAi mediated silencing of isoflavone synthase or chalcone reductase in soyabean leads to resistance. HopZ1 effector protein in TYPE 3 Cell in Bacteria – Resistant against P.syringae . (Zhou et al .,2011)

34. 3.TERPENOIDS Momilactones A&B – Rice First identified members of this group. Diterpenoids Phytoalexins – Oryzalexins , Phytocassanes . 20 genes linked to diterpenoid phytoalexin biosynthesis in rice were investigated. Genes encode Class 2 – ( copalyl diphosphate synthases ) Class 1 – ( kaurene synthase like) & P450 Monooxygenases . (Peters,2006;Schelz et al .,2014)

35. Contd … CPS4 – biosynthesis of Momilactones & Oryzalexins CPS4 along with T-DNA insertion plant produces Momilactones & Oryzalexins Resistant against M.oryzae & F.fujikuroi . Maize also produces a large number of terpenoid phytoalexins include Kauralexins Sesquiterpenoids Zealexins ( Huffakar et al., 2011)

36. CONCLUSION Majority of these compounds our knowledge about the details of their biosynthesis and contribution to plant immunity scarce. Increasing the number of plant genome sequences available and further significant improvement in molecular biology techniques. Possible to generate gene cassettes for complete pathways –production of valuable secondary metabolites.

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