1

111!1 The University1 ofJr... Nottingha1mUNITED 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

2

71 % Foods(processed foods,

margarine, chocolate,etc.)

24 % Consumerproducts

(Detergent, cosmetic,candles, etc.)

5 % EnergySource

(Electricity, heating,fuels, etc.)

3

Decaying ofinternalbole tissue

Collapse of oilpalm trees

Emergence of G.boninense fruitingbodies

Poor in foliardevelopment

4

• Deposition of lignin polymers on plant cell walls

• Roles: Rigidity, support and delay pathogen penetration

• Lignin: Complex racemic aromatic heteropolymer (Boerjan etal., 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)

N

ofesis

intolme

he)

anoi

HO O .,s O

-+~1-+

Ugnin Biosynlhesis ~atbwayCoA CoA"S O CoA"S O

HO O

tti PAL ,,?, C4H 4CL

::::,._

phenylalanine cinnamicacid

-+OH OH OH OH

p-coumaric p-coumaroyl CoA caffeoyl CoA feruloyl CoAacid

• Phenylalanine ammoni..aJ,.clcyRase (PAL) ..J,.ccR• First enzyme involvedH 0 H 0 H 0 H 0

• Catalyze the entry phenylalanine o the p nylprop dCAld5Hand lignin biosynth pathway (Vanh et al., 2010 COMT

• Cinnamate 4-hydroxylaOsH e (C4H)OMo-+HO OMo--+:.o OMo

OH OH OH

• LiCgyntoinchrome P450p--coduemapraeldnehdydeent monocooonxifeyragldehydddaeses5-hydroxy- sinapaldehydeconiferaldehydeBio•syCnatthaleyszeis second re+acCtAiDon of the p+atChADway to produce+pCA-D

coumaric acid (Chapple, 1998; Dixon and Paiva, 1995)p-coumaryl alcohol

H-Lignin

coniferyl alcohol sinapyl alcohol

+ +G-Lignin S-Lignin

Figure 1 The main monolignols biosynthetic pathway (Vanholme et al., 2010)

5

6

TTTo ssstuuudddyy ttthhheee oiiilll paaalllm dddefennnccceee rreeespoonnsseess iinnn

rreeelllatttiioonn ttto lliigniiiinnn biiiooosynntthheessisis upppooonnn iinnnfeccttiioonn bbby

GGGaannnoddderrrmmmaa bbboninnneennssee iinnn aaan iinnn viiittrrro syyysteemm..

Physiological and PathologyStudies of Oil Palm in an InVitro System

Molecular and BiochemicalStudies of Treated Oil Palm

Impacts of G. boninensePathogenesis on LigninBiosynthesis

7

Treatments on oil palm seedlings• T1, non-inoculated + non-wounded• T2, non-inoculated + wounded• T3, inoculated + wounded• Duration of experiment: 8 days

a Vitro lnlection ol Dll Palm Seedlln1s

G. boninense (GBLS) mycelium& Oil palm seedlings

Artificial wounding onseedlings 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

l

Lignin Biosynthesis EnzymesExtraction

Total Protein Assayy Rever

9

Gene Expression Studies Biochemical Enzymes Assay

Total RNA Extraction

·- I ..." DNA Synthesis b se

Transcriptasec

Relative quantification of ligninbiosynthesis genes expression

EgPALTee and EgC4HTee(Tee et al., 2013)

Reference gene:EgActin and EgCyP

Quantification of LigninBiosynthesis Enzymes Activities

Quantification of Total Lignin Contents[Lignothioglycolic Acid (LTGA) Assay]

Sample Preparation for Histochemical Staining

Fixation – Dehydration - Infiltration –Impregnation – Mounting - Sectioning using

microtome

Impacts ol a. boaloea6e Pathogenesison Lilldn Biosyntbesis

Histochemical Staining of Oil Palm Seedlings

- Toluidine blue O- Maúle reagents

- Phloroglucinol-HCl10

11

6.'0 ~

,.

.

..

DerivatiKe Mell Curw:e AnalysisOeriv•tiveMeft Derivative Melt

.... .,.,,

.... EgActin EgCyP020

02S

Ii i o.15

r !I .... Ir ....

....

... o.os

.... 0.00

A .. NTC _." " .. .. .. .. AB 60

NTC_......70 75 IO .. 90 ••T,tfflOC"ll!loff"°C" T•m~r,1t1,1t•("C)

<US

().)()

02S

•i o.20

DefivativeM~t Derivative Me-It

OJO

EgPAL .,. EgC4HOlO

I1 ...,.! ....

! ....rI..,

ODS ....... ....

A .. " ,. " .. .. .. .. .. " 70 " .. .. .. ..C NTC _. AD NTC _.

l-t)ef"•b.w•("() 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 PCRamplification.

~1:- e~ 1.5 c

1.0 0

0.5

0.0 0 2 4 6 82 4 6

Days of Treatment Days ofTreatment

Fig• urTreans2 i(Aen) tRrelaapidtivestiemuxprelassiontion andof EgP(B)ALenzymupone wactiounvitdingiesofandPALininfecttreaionted. oil palm seedlings within 8 days of incubation. Error bars:standard error of mean (SEM) of r•epliEcgPatALe readiexpngress sionfrom wthraseecrlosoundelysasofsoexcperiatiedmenPALts. enzyme activity

• Postpone of PAL peak production

• Post-transcriptional modifications ability of PAL was reduced (Ballester et al.,2006)

12

w ..c 2.5

-

-~ o

..

I

Phenylalanine A1nmonia LyaseA

5.0

Relative Expression of EgPALin Treated Oil Palm Seedlings

B Phenylalanine ammonia lyase (PAL) levelin treated oil palm seedlings

4.5

4.0

·g ......3.5

: u.G.I cCl 3.00x.. IJ

oGI 11

2.0

c'ai0.....Q.

,..I

ClE

mT2 ,..c:

+ T1+ T2+ T3

mT3 ·

6QI

c:·e

A Relative Expression of EgC4Hin Treated Oil Palm Seedlings

18

16

14

c

B

c:'.a..l.

0.3

Cinnamate 4-hydroxylase (C4H) levelin treated oil palm seedlings

-0 ..... 12: QI

~ ~ 100)(..1

s1:1

WU 8QI 'ti>-.., L0LIll ......

a: 4

2

...c.';"C)

•TI E';"

•TI

cio

0.2

0.1

o.o,... ........,... """T"'"" ....---.....--~-

+ T1.. T2..... T3

00 2 4 6 8

Days of Treatment

0 2 4 6 8

Day of Treatment

Figure 3 Cinnamate 4-hydroxylase (C4H) (A) relative expression and (B) enzyme•actiEvgitiCe4sHineoxilpprealsmsesdeeadtlianglastwerithtiimn 8e dcaoyusrosef inincuwboatuionnd.eEdrr&or ibnafersc:tsetdansdeaerdleinrrgosr.ofmean (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 (Lu et al., 2006; Potter et al.,1995; Betz et al., 2001)

13

• Total lignin in T3 seedlings weresignificantly higher (29 % on Day 8)

• Lignin concentration was increasedin 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 T3seedlings• Suppression of phenolic and

phenylpropanoid metabolites byfungus during initial infectiondevelopment (Latreche and Rahmania, 2011;Saidi et al., 2013)

Figure 4 Quantification of total lignin contents in treated oil palm seedlings on a timecourse after artificial infection of seedlings with G. boninense. Error bars: standarderror 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 differenttreatments (Bars, 100 µm. EP, epidermis; C, collenchyma; CU, cuticle layer; VB, vascularbundle. 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 differenttreatments (Bars, 100 µm. EP, epidermis; C, collenchyma; CU, cuticle layer; VB, vascularbundle. 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

• Early induction of lignin biosynthesis genes and enzymes(PAL and C4H) in oil palm seedlings during woundingand infection by G. boninense

• Total lignin contents in wounded and infected seedlingswere increased, with greater staining of TBO and Maúlereagent.

• A positive correlation between lignin contents andbiosynthesis intermediates

17

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

1. Ballester, AR, Lafuente, MT & González-Candelas, L (2006) Postharvest Biology and Technology, 39(2),115–124.

2. Betz, C, McCollum, TG & Mayer, RT (2001) Plant Molecular Biology, 46(6), 741–748.

3. Boerjan, W, Ralph, J & Baucher, M (2003) Annual Review of Plant Biology, 54, 519–546.

4. Chapple, C (1998) Annual Review of Plant Physiology and Plant Molecular Biology, 49, 311–343.

5. Dixon, RA & Paiva, NL (1995) The Plant Cell, 7(7), 1085–1097.

6. Eynck, C, Séguin-Swartz, G, Clarke, WE & Parkin, IAP (2012) Molecular Plant Pathology, 13(8), 887–899.

7. Latreche, K & Rahmania, F (2011) Physiological and Molecular Plant Pathology, 76(2), 144–151.

8. Lu, S, Zhou, Y, Li, L & Chiang, VL (2006) Plant & Cell Physiology, 47(7), 905–914.

9. Mohr, PG & Cahill, DM (2007) Functional & Integrative Genomics, 7(3), 181–191.

10. Musetti, R, Favali, MA & Pressacco, L (2000) Cytobios, 102(401), 133–147.

11. Orang AV, Safaralizadeh R, Bavili MK (2014) International Journal of Genomics,doi:10.1155/2014/970607

12. Pannecoucque, J & Höfte, M (2009) BMC Plant Biology, 9(1), 95.

13. Potter, S, Moreland, DE, Kreuz, K & Ward, E (1995) Drug Metabolism and Drug Interactions, 12(3-4),317–327.

14. Saidi, MN, Bouaziz, D, Hammami, I, Namsi, A, Drira, N & Gargouri-Bouzid, R (2013) Plant Science, 211,8–16.

15. Sewalt, V, Ni, W, Blount, JW, Jung, HG, Masoud, SA, Howles, PA & Dixon, RA (1997) Plant Physiology,115(1), 41–50.

16. Suzuki, S, Shintani, H, Park, S, Saito, K & Laemsak, NM (1998) INIST-CNRS, 52(4), 417–426.

17. Taheri, P, Irannejad, A, Goldani, M & Tarighi, S (2014) European Journal of Plant Pathology, 140(4),829–839.

18. Tamiru A, Khan ZR, Bruce TJA (2015) Current Opinion in Insect Science, 9, 51-55.

19. Tee, SS, Tan, YC, Abdullah, F, Ong-Abdullah, M & Ho, CL (2013) Tree Genetics & Genomes, 9(2), 377–386.

20. Vanholme, R, Demedts, B, Morreel, K, Ralph, J & Boerjan, W (2010) Plant Physiology, 153(3), 895–905.

20. Yeung, EC (1998) In SJ Karcher (Ed.), Tested Studies for Laboratory Teaching, pp. 125–142. 19

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