Conjugating immunolocalization and afm observations to determine if rhamnogalacturonan-i type pectins are responsible for the generation of maturation stress in poplar tension wood fibres

Tension wood (TW) is produced by temperate hardwood trees in order to support their increasing weight, orient their axes and cope with environmental cues such as wind. Poplar TW fibres harbour a supplemental layer, the G-layer, rich in crystalline cellulose, containing matrix polysaccharides but no lignin. The tensile force responsible for the specific mechanical properties of TW originates from the G-layer and is transmitted to cellulose microfibrils soon after their deposition, during G-fibre maturation (Clair et al, 2011). This force is likely to originate from physical changes in the high porosity hydrogel recently identified in the G-layer. RG-I type pectins appear as good candidate molecules responsible for the formation of this gel. Indeed, during G-layer maturation, LM5 labelling (specific to RG-I side chains) decreased, while RU1 labelling (specific to RG-I backbone) increased (Guedes et al, 2017). This suggested a hydrolysis of the RG-I side chains during G-fibre maturation possibly by a β-galactosidase as demonstrated in flax phloem fibers (Roach et al, 2011). Flax phloem fibres and TW G-fibres exhibit many similar features and, in flax, it has been shown that the hydrolysis of the side chains of RG-I type pectins was associated to the very peculiar mechanical properties of bast fibres. In order to determine if RG-I pectins were effectively involved in the building of the G-layer tensile force, we carried out different measurements on a common sampling during G-fibre development: i) β-galactosidase activities using a histochemical test, ii) the evolution of RG-I immunolabelling profiles using LM5 and RU1 as probes and iii) the stiffening of the different cell wall layers using Atomic Force Microscopy (AFM). We found a good correlation between β-galactosidase activities and RG-I immunological labelling but we failed to establish a direct association between RG-I hydrolysis and cell wall stiffening.