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1 111!1 Jr... The University1 of Nottingha1m UNITED KINGDOM CHINA MALAYSIA Early defence responses in Elaeis guineensis lignin biosynthesis pathwayduring pathoge esis of Ganoderma boninense Carmen Goh Kar Mun, Matthew Dickinson, Kinya Hotta, Christina V. Supramaniam School of Biosciences, Faculty of Sciences, The University of Nottingham Malaysia Campus 1

2 71 % Foods (processed foods, margarine, chocolate, etc.) 24 % Consumer products (Detergent, cosmetic, candles, etc.) 5 % Energy Source (Electricity, heating, fuels, etc.) 2

3 Collapse of oil palm trees Decaying of internal bole tissue Emergence of G. boninense fruiting bodies Poor in foliar development 3

4 Deposition of lignin polymers on plant cell walls Roles: Rigidity, support and delay pathogen penetration Lignin: Complex racemic aromatic heteropolymer (Boerjan et al., 2003) Oil palm s lignin composition (Suzuki et al., 1998) Aryl ether-linked syringyl units (S) p-hydroxybenzoic acid (H) Vanillin and vanillic acids (G) 4

5 Biosynlhesis ~atbway Ugnin HO HO N tti PAL,,?, ::::,._ CoA.,s O CoA"S O CoA"S C4H cinnamic acid 4CL OH -+ OH OH OH p-coumaric p-coumaroyl CoA caffeoyl CoA acid..j,.ccr feruloyl CoA Phenylalanine ammonia lyase (PAL) First enzyme involvedh 0 O O -+~1-+ phenylalanine O..J,.ccR H 0 0 H 0 anoi d Catalyze the entry of phenylalanine into the p he nylprop CAld5H and lignin biosynth esis pathway (Vanh olme et al., 2010 ) COMT OMo-+HO H OMo--+:.o OMo OH OH OH H e (C4H) Cinnamate 4-hydroxylaOs o d mapraeldn ehd ydee nt monoconx ifeyrag ldehyda e ses5-hydroxysinapaldehyde LiCgyntoinchrome P450p--codue coniferaldehyde Bio sycnathaleyszeis second reaction of the pathway to produce pcoumaric acid (Chapple, 1998; Dixon and Paiva, 1995) +CAD p-coumaryl alcohol +CAD coniferyl alcohol + H-Lignin G-Lignin +CAD sinapyl alcohol + S-Lignin Figure 1 The main monolignols biosynthetic pathway (Vanholme et al., 2010) 5

6 T o s tu ud yy the oiil al m pa d efen nc e e sponses iin rre pon iin el attiioonn t o lliigniiin biio syntthheessisis up n fectiioonn b y rre Gaan n od d errm aa b onin n eennssee iin n a n iin viittr o sy y stem. 6

7 Treatments on oil palm seedlings Physiological Pathology T1, non-inoculatedand + non-wounded T2, non-inoculated + wounded Studies of Oil Palm in an In T3, inoculated + wounded Vitro System Duration of experiment: 8 days Molecular and Biochemical Studies of Treated Oil Palm Impacts of G. boninense Pathogenesis on Lignin Biosynthesis 7

8 la Vitro lnlection ol Dll Palm Seedlln1s G. boninense (GBLS) mycelium & Oil palm seedlings Artificial wounding on seedlings stem Acquire GBLS mycelium Inoculation of GBLS mycelium Assembly of Incu tissue culture jar Incubation in growth chamber (27 C, 16 h daylight, 50 % humidity) 8

9 Gene Expression Studies Biochemical Enzymes Assay Total RNA Extraction Lignin Biosynthesis Enzymes Extraction - I. c" DNA Synthesis b y Reverse Transcriptase Relative quantification of lignin biosynthesis genes expression EgPALTee and EgC4HTee (Tee et al., 2013) Reference gene: EgActin and EgCyP Total Protein Assay Quantification of Lignin Biosynthesis Enzymes Activities 9

10 Impacts ol a. boaloea6e Pathogenesis on Lilldn Biosyntbesis Quantification of Total Lignin Contents [Lignothioglycolic Acid (LTGA) Assay] Sample Preparation for Histochemical Staining Fixation Dehydration - Infiltration Impregnation Mounting - Sectioning using microtome Histochemical Staining of Oil Palm Seedlings - Toluidine blue O - Maúle reagents - Phloroglucinol-HCl 10

11 DerivatiKe Mell Curw:e Analysis Oeriv tivemeft Derivative Melt.,.,, EgActin EgCyP S i I r I... i ~!r 6.'0 o.15 I A o.os.. NTC _. ",. " T,tfflOC"ll!loff" C" AB 60.. NTC_... DefivativeM~t.. 90 Derivative Me-It <US OJO EgPAL ().)() 75 IO T m~r,1t1,1t ("C) 70.,. EgC4H 02S i I OlO o.20!r ,.! I..,.... ODS A C.. ",. NTC " _. l-t)ef" b.w ("() AD " " 70 NTC _. T.-,,Pff tur1t("() Figure 1 Derivative melt curves (A) EgActin, (B) EgCyP, (C) EgPAL and (D) EgC4H genes amplified from treated oil palm seedlings in real time PCR amplification. 11

12 Phenylalanine A1nmonia Lyase A B Relative Expression of EgPAL in Treated Oil Palm Seedlings Phenylalanine ammonia lyase (PAL) level in treated oil palm seedlings c 'ai g 3.5 -:...u 0... I Cl mt2 GI 11 -~ o E,.. I 2.0 mt3 ~1:~ + T1 + T2 + T3 Q.,.... c IJ x o 2.5 w..c GI Cl c: e 1.5 c Days of Treatment Days oftreatment Fig urtreans2 i(a tivestiemu xprelassion (B)ALenzym actioun vitding ies en) trrelaapid tion andof EgP upone w ofand PALininfecttreaion ted. oil palm seedlings within 8 days of incubation. Error bars: standard error of mean (SEM) of repliecgp elys atale readiexpngress sion from w thraseecrlosound asofso PALts. enzyme activity excperiatied men Postpone of PAL peak production Post-transcriptional modifications ability of PAL was reduced 2006) (Ballester et al., 12

B Relative Expression of EgC4H in Treated Oil Palm Seedlings A Cinnamate 4-hydroxylase (C4H) level in treated oil palm seedlings 18 0.3 16 14 c: '.a..l. c -... 12 0 :...c. 0.2 QI ~ ~ 10 0.

13 B Relative Expression of EgC4H in Treated Oil Palm Seedlings A Cinnamate 4-hydroxylase (C4H) level in treated oil palm seedlings c: '.a..l. c :...c. 0.2 QI ~ ~ )( s: WU QI 'ti >-0.., LL Ill... TI 8 TI E c:... T3 ';" 6 e 4 ci o QI a: + T1.. T2 ';" C) o.o,......, Days of Treatment """T"'"" ~ Day of Treatment Figure 3 Cinnamate 4-hydroxylase (C4H) (A) relative expression and (B) enzyme actievg sesdeeadtlianglas tw erithtiim incuwboatuionnd.eedrr& itice4s Hineoxilppre alsm n 8e dcaoyusrose f in or ibnafe rsc:tsetd ansdeaer dleinrrgosr. of mean (SEM) of replicate readings from three rounds of experiments. Transcriptional regulation of C4H enzymes controlled by multiple EgC4H homologs. C4H genes exist as a multigene family in many plants 1995; Betz et al., 2001) (Lu et al., 2006; Potter et al., 13

14 Total lignin in T3 seedlings were significantly higher (29 % on Day 8) Lignin concentration was increased in rice (Taheri et al., 2014), date palm (Saidi et al., 2013), tomato (Mohr and Cahill, 2007) rapidly upon infection Reduction of lignin on Day 4 in T3 seedlings Suppression of phenolic and phenylpropanoid metabolites by fungus during initial infection development (Latreche and Rahmania, 2011; Saidi et al., 2013) Figure 4 Quantification of total lignin contents in treated oil palm seedlings on a time course after artificial infection of seedlings with G. boninense. Error bars: standard error of mean (SEM) of replicate readings from three rounds of experiments. 14

Figure 5 TBO staining of lignin in oil palm basal stem cross section between different treatments (Bars, 100 µm. EP, epidermis; C, collenchyma; CU, cuticle layer; VB, vascular bundle.

15 Figure 5 TBO staining of lignin in oil palm basal stem cross section between different treatments (Bars, 100 µm. EP, epidermis; C, collenchyma; CU, cuticle layer; VB, vascular bundle. Arrows showed intensive staining of respective dyes on specimens) TBO stained lignified elements in green to blue-green colour (Yeung, 1998) Lignin accumulated in an increasing order upon infection (Musetti et al., 2000; Pannecoucque and Höfte, 2009). Blue staining on vascular bundles (T1, T2 & T3), collenchyma & cuticle layer (T3) 15

Figure 6 Maúle staining of lignin in oil palm basal stem cross section between different treatments (Bars, 100 µm. EP, epidermis; C, collenchyma; CU, cuticle layer; VB, vascular bundle.

16 Figure 6 Maúle staining of lignin in oil palm basal stem cross section between different treatments (Bars, 100 µm. EP, epidermis; C, collenchyma; CU, cuticle layer; VB, vascular bundle. Arrows showed intensive staining of respective dyes on specimens) Maúle reagent detects syringyl-lignin (S) in wine-red to brown response (Sewalt et al., 1997) Higher amount of S lignin in infected tissues (Saidi et al., 2013; Eynck et al., 2012) 16

17 Early induction of lignin biosynthesis genes and enzymes (PAL and C4H) in oil palm seedlings during wounding and infection by G. boninense Total lignin contents in wounded and infected seedlings were increased, with greater staining of TBO and Maúle reagent. A positive correlation between lignin contents and biosynthesis intermediates 17

Development of oil palm cultivars with induced resistance towards BSR Up-regulation of lignin biosynthesis genes (mirna-mediated upregulation) (Orang et

18 Development of oil palm cultivars with induced resistance towards BSR Up-regulation of lignin biosynthesis genes (mirna-mediated upregulation) (Orang et al., 2014) Selective breeding for inducible resistance (Tamiru et al., 2015) Further focus on phytoalexin and oxidative burst resistances in oil palm 18

19 1. Ballester, AR, Lafuente, MT & González-Candelas, L (2006) Postharvest Biology and Technology, 39(2), Betz, C, McCollum, TG & Mayer, RT (2001) Plant Molecular Biology, 46(6), Boerjan, W, Ralph, J & Baucher, M (2003) Annual Review of Plant Biology, 54, Chapple, C (1998) Annual Review of Plant Physiology and Plant Molecular Biology, 49, Dixon, RA & Paiva, NL (1995) The Plant Cell, 7(7), Eynck, C, Séguin-Swartz, G, Clarke, WE & Parkin, IAP (2012) Molecular Plant Pathology, 13(8), Latreche, K & Rahmania, F (2011) Physiological and Molecular Plant Pathology, 76(2), Lu, S, Zhou, Y, Li, L & Chiang, VL (2006) Plant & Cell Physiology, 47(7), Mohr, PG & Cahill, DM (2007) Functional & Integrative Genomics, 7(3), Musetti, R, Favali, MA & Pressacco, L (2000) Cytobios, 102(401), Orang AV, Safaralizadeh doi: /2014/ R, Bavili MK (2014) International Journal of Genomics, 12. Pannecoucque, J & Höfte, M (2009) BMC Plant Biology, 9(1), Potter, S, Moreland, DE, Kreuz, K & Ward, E (1995) Drug Metabolism and Drug Interactions, 12(3-4), Saidi, MN, Bouaziz, D, Hammami, I, Namsi, A, Drira, N & Gargouri-Bouzid, R (2013) Plant Science, 211, Sewalt, V, Ni, W, Blount, JW, Jung, HG, Masoud, SA, Howles, PA & Dixon, RA (1997) Plant Physiology, 115(1), Suzuki, S, Shintani, H, Park, S, Saito, K & Laemsak, NM (1998) INIST-CNRS, 52(4), Taheri, P, Irannejad, A, Goldani, M & Tarighi, S (2014) European Journal of Plant Pathology, 140(4), Tamiru A, Khan ZR, Bruce TJA (2015) Current Opinion in Insect Science, 9, Tee, SS, Tan, YC, Abdullah, F, Ong-Abdullah, M & Ho, CL (2013) Tree Genetics & Genomes, 9(2), Vanholme, R, Demedts, B, Morreel, K, Ralph, J & Boerjan, W (2010) Plant Physiology, 153(3), Yeung, EC (1998) In SJ Karcher (Ed.), Tested Studies for Laboratory Teaching, pp

20 Special thanks to:ms. Christina Supramaniam Prof. Matthew Dickinson Dr. Kinya Hotta Ms. Siti Norazlin Mr. Jonathan Foong Ms. Jennie Choo Chin Nee (Applied Agricultural Resources Sdn. Bhd.) Pn. Rosna Bt Angsor (Tissue Culturist - MPOB) Friends and family 20

Lignin and the General Phenylpropanoid Pathway. Introduction and Importance:

Lignin and the General Phenylpropanoid Pathway 13. Phenolics and Lignin p. 1 Introduction and Importance: Phenolic: a compound consisting of an aromatic ring plus at least one hydroxyl [= phenyl group],