1. Ectopic lignification in the flax lignified bast fiber mutant
Chabbert B, Chantreau M, Kiyoto S, Yoshinaga A, Boerjan W, Mesnard F, Hawkins
Inra, FARE Reims France

2. Flax (Linum usitatissinum L.) – an ancient crop still used today
Bast fibers obtained by retting and mechanical separation
TEXTILES (linen)
COMPOSITES

3. Bast fibers are located in the outer tissues of the flax stem
Woody core : xylem cells
(cambium)
Bast fibers Sclerenchyma cells
(procambium)
Flax stem
Cross section (Phloroglucinol /HCl)
Lignin Cellulose Non cellulose Length polysaccharides
30% % 35% <10 mm
<4 % 75% 15% mm

4. Bast fibers are hypolignified cell
Regulation of lignification in bast fiber cell walls ?
Production EMS mutant population (5,000 families) for forward/reverse genetics
Phenotyping and TILLinG in Flax *
‘Tilled’ lignin and cell wall mutant
(cad, transcription factors…)
Phenotyping to identify flax lignified bast fiber (lbf) mutants
ANR project “PT-Flax ; Chantreau et al. (2013) BMC Plant Biol.

5. Identification of flax lignified bast fiber (lbf) mutants
3,391 M2 families
Screen stem cylinders
(UV autofluorescence)
Screen stem cross sections (Phloroglucinol/HCl)
- 150 families class 3
‘core collection’ of lignified bast fiber (lbf) mutants
540 families
319 families
- 176 families class 2
- 93 families class 1
Chantreau et al. (2014) Plant Cell.

6. Characterization of flax lbf mutants
lbf mutants grouped into 8 different classes (ectopic lignification pattern)
Fibers (lbf1, class A)
Fibers (lbf1, class D)
Fibers WT
Fibers (lbf1, class E)
Fibers (lbf1, class F)

7. Characterization lbf1 mutants: lignin composition
lbf mutants grouped into 8 different classes (ectopic lignification pattern)
Fibers WT
Fibers (lbf1, class A)
Chemical analyses
4 independent lbf class A families IT OT
Separation stem outer tissues (OT) from inner tissues (IT)

8. lbf1 Lignin content Significant increase in
Outer tissues
Inner tissues
Lignin content (% dry cell wall)
lbf1
Significant increase in
lignin content (2 families)
No significant change in lignin content

9. Further characterization lbf1: lignin composition
Outer stem tissues S H G
Inner stem tissues
lbf1
Lignin content (% dry cell wall)
lbf1 ectopic lignin
mainly composed of G units
(thioacidolysis, 2DNMR)
Similar to WT lignin

10. Further characterization lbf1: lignin immunocytochemistry
Lignin antibody KM1 (8-5’ linked lignin Structure)
Bast fibers WT
lbf1
1µm
0.15 µm
- Thinner secondary cell walls (SW) in lbf1 mutant (4 µm) than in WT(10 µm)
- Lignin antibody reacts with secondary cell walls (SW) in lbf1

11. Lignin antibody KM1 (8-5’ linked lignin Structure) XylemWT
lbf1
- Lignin antibody reacts similarly with cell walls of both WT and lbf1

12. What control points are modified in the lbf1 mutant?
Monolignol biosynthesis, Transport , Polymerisation in the cell wall ?
Whole genome transcriptomics lbf1 vs WT IT OT
Separation stem outer tissues (OT)
from inner tissues (IT
Transcriptomics
(microarrays)
Lignin genes?

13. Lignin monomer biosynthesis
- up-regulation CCR, COMT and CAD genes in lbf1 (outer tissues)
Inner/ outer
Inner/ outer
COMT
Inner/ outer
CCR
(1/4 genes)
(1/3 genes)
CAD
(1/13 genes)

14. Lignin monomer biosynthesis
down-regulation genes in 7/11 lignin gene families (inner tissues)
4CL
PAL
CCoAOMT
CCR
C3H
F5H
CAD

15. 9 peroxidases are orthologs of Arabidopsis
Lignin monomer polymerisation
- no laccase genes differentially regulated
- 11 peroxidase genes specifically up-regulated in lbf1 (outer tissues)
9 peroxidases are orthologs of Arabidopsis
peroxidases involved
in lignification
Log2 ratio
-4.50
4.50 i ii
iii
- genes involved in H202 biosynthesis (used by peroxidase) up regulated in lbf outer tissues : NADH oxidase, Superoxide dismutase

16. Ectopic lignification of bast fibers in lbf mutants:
- Increase production of lignin monomer
- Increase capacity of polymerisation
Other cell wall polymers ?
***
Increasing lignin content
Polysaccharide monomer composition
Protein content
↘Glucose (reduced cellulose), ↗ Other sugars (modified hemicellulose)
↗ Protein content ( Arabinogalactan protein?)

17. Arabinogalactan protein
Other cell wall polymers in the bast fibers of lbf mutants
Immunolocalisation of Cell Wall NCPs
Xylan
Ectopic lignification in lbf mutant is associated with change in secondary cell wall architecture
Consequence?
Glucomannan
Galactan
Arabinogalactan protein

18. J : Wild-type bast fibers
The flax lbf mutants – a useful biological resource for
Wall lignification pattern
All fiber wall
ML and P1
Cell wall thickness
Unchanged
Fibers + surrounding cells
Fiber cells
Decreased
A: 44 families
D : 13 families
Bundle periphery
B : 10 families
0 family
C : 3 families
E : 1 family
F : 16 families
H : 2 families
I : 0 family
G : 4 families
J : Wild-type bast fibers 93
mutants
Investigating lignification and regulatory mechanisms
Change in oligolignol accumulation
Investigating cross-talk between lignification and other cell wall polymers
↘ 30% conversion yield of lbf outer tissues by cellulases cocktail
Cellulose cristallinity, MFA: under progress
Evaluating impact of increased lignification
on fiber chemical/physical properties

19. Water sorption isotherms of flax fibers
Lbf mutant
(12-14% lignin)
Wild type
(4% lignin)
Multiplying lbf mutants to study fiber properties and use in composites