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

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

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% 40% 35% <10 mm <4 % 75% 15% 70 mm

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.

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.

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)

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)

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

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

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

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

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?

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)

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

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

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?)

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

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

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