Xyloglucan structural analysis

Xyloglucan analysis was based on the rapid phenotyping method using enzymatic oligosaccharide fingerprinting previously described (Lerouxel et al., 2002; Sechet et al., 2016). Mature rosette leaves were harvested and cleared in ethanol overnight at room temperature. After removal of ethanol and rehydration in water, xyloglucan oligosaccharides were generated by digesting the samples with endoglucanase in 10 mM sodium acetate buffer, pH 5, in a final volume of 20 µl, overnight at 37 °C. An aliquot of 0.5 µl of the supernatant was dried on a matrix-assisted laser desorption/ionization time-of-flight mass (MALDI-TOF) target, followed by the addition of 0.5 µl of super-DHB (9:1 mixture of 2,5-dihydroxy-benzoic acid and 2-hydroxy-5-methoxy-benzoic acid, Sigma-Aldrich) matrix, and dried under a fume hood prior to spectra acquisition. The MALDI-TOF mass spectra of the xyloglucan oligosaccharides were acquired with a MALDI/TOF Bruker Reflex III.