impact of genetic transformation of …...impact of genetic transformation of lignin on secondary...

IMPACT OF GENETIC IMPACT OF GENETIC

TRANSFORMATION OF LIGNIN ON TRANSFORMATION OF LIGNIN ON

SECONDARY WALL ASSEMBLY SECONDARY WALL ASSEMBLY

ININ ARABIDOPSIS THALIANAARABIDOPSIS THALIANA

J. BERRIOJ. BERRIO--SIERRASIERRA11, M. MIR DEVIKVAND, M. MIR DEVIKVAND22, L. , L.

JOUANINJOUANIN22, C. LAPIERRE, C. LAPIERRE33 et K. RUELet K. RUEL11

11 CERMAV/CNRS, Grenoble, CERMAV/CNRS, Grenoble, 22 INRA Versailles, INRA Versailles, 33 INAINA--Paris GrignonParis Grignon

[email protected]@cermav.cnrs.fr

INTRODUCTION

• Lignin is, after cellulose, the second most abundant terrestrial

biopolymer.

• Accounting for approximately 30% of the organic carbon in

the biosphere.

• The ability to synthesize lignin has been essential in the

evolutionary adaptation of plants from an aquatic environment

to land.

• Lignin is mainly deposited in cell walls of tissues involved in

mechanical support or in water and nutrients conduction.

• It is a physicochemical barrier against pathogens

Colloque annuel du GDR, AMV. 5 au 7 Décembre 2005 Pessac, France

Secondary cell walls consist of three layers:

the outer (S1), middle (S2), and inner (S3)

(From J. Harrington, 1998)


GENERAL STRUCTURE OF ANGIOSPERM LIGNIN(Statistical Computerized Structure)


MONOMERS

OH

5

6 2

34

(H) CHα1

γ

CHCH2OH

β

p-coumaryl alcohol

OHOCH3

(G) αCH

γ

CHCH2OH

β

coniferyl alcohol

(S) αCH

OH

CO3H OCH3

γ

CHCH2OH

β

sinapyl alcohol

INTER-MONOMERIC LINKAGES

CCCC

O

C CO

β-O-4(non-condensed)

O

CCC

O

O

CCC

CCC

O

CCC

O

CCC

O

C

β-5 β−β 5-5(condensed)


PAL

HO O

NH2

Cinnamoyl-CoA-Reductase (CCR)

Conifer-aldehyde

O

OH

H

OCH3

CCRCCR

Feruloyl-CoA

CoA - S

OH

OO

OCH3

CADCAD CADCAD

O

OHHO

O

OHCH3O

H

OCH3 OCH3

COMTCOMTF5H/Cald5H

O

OH

H

OCH3

H

OCH3

CoA -S

OH

O

OH

OCoA - S

OH

CCoAOMT

CCRCCR

CoA - S

OH

OOOO

CCoA3H

COMTCOMT

OHO

OH

OHOOHO

C3HC4H

OHOH OHOH4CL 4CL

(S)

OH

OHOCH3

(G)

OH OH

OHCH3O OCH3

Peroxidase/LaccaseLIGNIN


In the paper industry the lignin is a "polluting agent"

that has to be eliminated by chemical methods.

These methods involve a high cost of

energy and are very polluting

What is the solution?


A FEW SUGGESTIONS

� Reduce the lignin content

� Modify lignin composition

Genetic Engineering

� Manipulation of lignin biosynthesis pathways


We have found at the anatomical scale that the loss of SCCR-1 activity

resulted in a 40% reduction of height in size compared to the wild-type.

Wild type Transformed plant

CCR-1


P.A.T.Ag. (Periodic Acid Thiocarbohydrazide silver proteinate)

This technique allows the contrasting of the majority of polysaccharides

and allows to visualize the basic organization of the secondary walls.

� Oxidation by the periodate: opening of the ring and formation of aldehyde groups.

� Reaction of the aldehyde groups with T.C.H

� The silver proteinate is reduced to metallic Ag0 that contrast the polysaccharide chain


Transformed plant

CCR-1

Wild type

Cytochemical staining with (P.A.T.Ag), shows a dramatic loosening

and lack of cohesion in the walls of fibers and vessels in the transformant.

Focus: 1.500X


What type of lignin is present?


Antibodies from synthetic antigens

1. Directed against monomer units

- Anti-Guaiacyl (G)

- Anti-Syringyl (S)

- Anti-Guaiacyl/Syringyl (G/S)

2. Directed against inter-unit linkages

- Condensed G; G/S

- Non-condensed G; G/S

Joseleau and Ruel, Plant Physiology, 1997


CONCLUSIONS

• The nul mutant of SCCR-1 resulted in:

� Dwarf phenotype.

� Reduction in total lignin content

� Loss of ability to assemble the cellulose microfibrils

• The topochemical investigation of lignin structures showed that:

� The relative proportion of condensed and non-condensedlignin subunits has been dramatically changed.

All these results demonstrate that the CCR-1 gene is directly

implicated in the assembly of the xylem secondary walls


CONCLUSIONS

On a more fundamental point of view:

• Non-condensed type of lignin is essential for

the assembly of a coherent secondary wall


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