Felice Cervone

Dept. Biologia e Biotecnologie “C. Darwin” – Università di Roma “Sapienza”

Deconstruction of the plant cell wall and improvement of biomass conversion

Plants: a significant proportion of the biomass on Earth

Cell walls are made to resist deconstruction

CellCell WallWall DegradingDegrading EnzymesEnzymes ((CWDEsCWDEs))

CellulaseCellulase

PectinasePectinase

EmicellulaseEmicellulase

De-esterified homogalacturonan makes rigid“egg boxes” in presence of calcium

Cellulose Hemicellulose Pectinxyloglucan

galactomannan

arabinoxylan

Homogalacturonan

Ca2+-crosslinked

non-methylesterifiedmethylesterified

Rhamnogalacturonan

RG II(boron-diester)

RG I(galactan) (arabinan)

Pectin

The Plant Cell Wall

Cellulose Hemicellulosexyloglucangalactomannanarabinoxylan

The Plant Cell Wall

Cellulose

Three-dimensional structure of PG fromFusarium phyllophylum

Parallel β-helix fold

Federici et al. 2001, PNAS

Polygalacturonases (PG)

-PG is the first enzyme to be secreted by mostphytopathogenic microorganisms and is an importantpathogenicity factor.

-Its action on homogalacturonan of the plant cell wall is a pre-requisite for the accessibility of substrate to otherCWDEs.

• In muro modification of pectin bytransgenic expression of a fungal PG

PG plants have dwarf phenotype

Tabacco

wt #5 #7 #16 PG#16 x PvPGIP2

PvPGIP2

Arabidopsis

PG201 #4

#1 Ws-0

Col-0#5#1

-PG plants have a reduced content of HGA

Arabidopsis PG-expressing plants show increased saccharification

0

20

40

60

80 b

b

aa

WT PG57 PG26 PG106Enz

ymat

ychy

drol

ysis

effic

ienc

y(%

) PG57 PG26 PG106 WT

PG57 PG26 PG106 WT

A

B

Lionetti et al., PNAS 2010

0.00

0.02

0.04

0.06

0.08

0.10

0.12

0.14

WT PG16

***Re

duci

ngen

dseq

uiva

lent

s(u

M/m

g f.

w.)

Saccharification of tobacco PG plants by cellulase(Lionetti et al. PNAS, 2010)

WT PG plants

PG plants digested for 50h with macerozyme show complete degradation of leaf lamina

N-term N-term

C-termC-term

PvPGIP2-FmPG

Fusion between fully active PG and PGIP can be used to transform plants

Pectin methylesterases (PME)

• PMEs catalyse de-methylesterification of pectin

• Pectins are synthesized in the Golgi apparatus and are secreted in a highly methyl-esterified form

• PME are ubiquitous in plants and are also producedby phytopathogenic microbes

•Isolated in kiwi fruit (Balestrieri et al. (1990). Eur. J. Biochem.) and characterized in Arabidopsis (Raiola et al. (2003). FEBS

letters)

•Share structural homology with the inhibitors of invertases

•INTERACT WITH PLANT BUT NOT MICROBIAL PMEs

Pectin MethylEsterase Inhibitors(PMEIs)

(Di Matteo et al., Plant Cell 2005)

LePME1 AcPMEI

• Four Helix Bundle” of the inhibitor

• 2 disulphide bridges necessaryto maintain fold

• 1:1 stoichiometry

• Inibitors bind active site of PME preventing substratebinding

mRNA protein

PME actvity Degree of pectin methylation

Overexpression of PMEIs in A. thaliana

UBQ5

AtPMEI

1-1 1-5WT 1-43

AtPMEI 18 kDa21 kDa

14 kDa

1-1 1-5WT 1-43Mw Std

46,7%

83,8%

52,3%

0

20

40

60

80

100

120

WT 1-43 1-1 1-5

PME

acti

vity

(%)

a ab b

0

10

20

30

40

50

60

70

WT 1-43 1-1 1-5D

egre

eof

M

ethy

lest

erif

icat

ion

(%)

AtPMEI overexpression enhances biomass production

WT AtPMEI *

***

020406080

100120140160180200220

WT 1.5 2.9

Fres

hwe

ight

(mg)

*

***

0

5

10

15

20

WT 1.5 2.9

Dry

wei

ght

(mg)

0102030405060708090

100

WT 1.5 2.9

H2O

con

tent

(%)

redu

cing

suga

rseq

uiva

lent

s(u

Mm

g-1

f.w.

)***

0

0.01

0.02

0.03

0.04

0.05

0.06

5h 20h

WTPMEI

Saccharification of Arabidopsis PMEI plants by cellulase

1.00

0.03

0.10

0.30

***

0

20

40

60

80

100

WT Atpme3

Deg

ree

of m

ethy

llatio

n(%

)

µg ChASS JIM5 PAM1

WT Atpme3 WT Atpme3

An Arabidopsis KO mutant for the AtPME3 gene has increased pectin methylation

Enz

ymat

ychy

droy

isis

effic

ienc

y(%

)

***

0

20

40

60

80

100

WT Atpme3

The Atpme3 mutant shows increasedsaccharification efficiency

Rootsquare

0.0

0.5

1.0

1.5

2.0

2.5

3.0

3.5

4.0’

4.5

829,3867,8906,4945,0983,5

1022,11060,71099,21137,81176,41215,01253,51292,11330,71369,21407,81446,41484,91523,51562,11600,71639,21677,81716,41754,91793,5

wavenum

bers

Absorbance x10-3

WT W

sW

T Col-0

PG

line 1P

G line 5

qua 1

Arabidopsis

PG plants

havecellw

allcomposition

similarto

the HG

A m

utantsqua1

and qua2

FTIR

Correlation between PAM1 level and saccharification efficiency

Col-0

pme3

PMEI

PG

qua2-1

Based on mean values, the correlation between PAM1 level and saccharification efficiencywas very high (r = -0.9), indicating that the reduction of de-methyl-esterifiedhomogalacturonan makes plants more susceptible to cellulose treatment.

Higher saccharification efficiency isobserved in natural ecotypes with low level

of demethylated stretch of HGA

Nested core collection of 24 ecotypes that maximizes the diversity present among a worldwide set of 265 accession of A. thaliana(McKhann et al, 2004), plus the reference accession Col-0, have been analyzed for PAM1 epitopes content and for enzymatic saccharification efficiency. This approach has been tested as an alternative strategy to the genetic modification to identify natural ecotypes with an improved biomass saccharification.

Enzymatic saccharification efficiency (%)

Rel

ativ

e PA

M1

epito

pele

vel (

%)

Acknowledgements

Dip. Biologia e Biotecnologie

Vincenzo LionettiFedra FrancocciManuel BenedettiElisa BastianelliS. FerrariD. BellincampiG. De Lorenzo