Endoglucanase

Standards used for quantitative analysis: methanol (≥99.9%, Carlo Erba, Val-de-Reuil, France) and acetic acid (≥99.8%, Sigma-Aldrich), inositol (99.5%, Sigma-Aldrich), D-(+)-galacturonic acid (>97.0%, Fluka BioChemika, Buchs, Switzerland).
Enzymes used for immunolabeling: galactanase (E-EGALN, lot 101001b), endo-arabinanase (E-EARAB, lot 111201c) and endoglucanase (E-CELTR, lot 110901a) (Megazyme). Pectin lyase was purified from the Peclyve commercial mixture [28 (link)].

Using a green light, five-day old dark-grown seedlings were collected and stored in cold ethanol. Four hypocotyls were dissected for each biological repeat (n = 4), and later used for the analysis. After being left overnight at room temperature in ethanol, the ethanol was removed and the hypocotyls were dried at 37 °C for 1 h. Afterwards, 20 µL of 50 mM acetate buffer, pH5.0, containing endoglucanase from Trichoderma longibrachiatum (Megazyme, Scotland, UK) were added and left overnight at 37 °C. OLIMP was then carried out as described elsewhere [41 (link)] using Super 2,5-dihydroxybenzoic acid (DHB) matrix (9:1 mixture of DHB and 2-hydroxy-5-methoxybenzoic acid; Fluka) instead of DHB. A solution of the XyG fragments after endoglucanase treatment was used to obtain the Matrix assisted laser desorption ionization-time of flight (MALDI-TOF) spectra.
For the pMDC7:GH3.6, pER8:YUC6 and pMDC7 empty vector control (EV), the hypocotyls were grown in the dark for five days on top of 100 µm pore mesh MS+ plates and then the meshes were transferred to 2 µM β-estradiol MS+ plates. The hypocotyls were dissected into upper section and lower section for the analysis.