Various sources of plant cell wall polysaccharides were used for the characterisation of LM27 and LM28 using ELISAs. These included tamarind xyloglucan (100403, Megazyme International), maize xylan (McCartney et al. 2005 (link)), birchwood xylan (X0502, Sigma-Aldrich), rye arabinoxylan (20601b, Megazyme International) and oat spelt xylan (95590, Sigma-Aldrich). Individual xylan-derived aldouronic acid oligosaccharides were a kind gift from Sanna Koutaniemi (Koutaniemi et al. 2012 (link)), and a mixture of aldouronic acids (tri:tetra:penta—2:2:1) was obtained from Megazyme.
The Arabidopsis thaliana triple mutant in gxm1gxm2gxm3 was generated by crossing single mutants prepared in Li et al. (2013 (link)). The insertion lines are SALK_087114 (gxm1, At1g33800), SALK_084669 (gxm2, At4g09990) and SALK_050883 (gxm3, At1g09610). Plants from the triple mutant line were grown for 6 weeks, and 5-cm of basal inflorescence stem was harvested. The alcohol-insoluble residue (AIR) was obtained and pre-treated with alkali and digested with a GH11 xylanase as described (Mortimer et al. 2010 (link)). After GH11 digestion, resulting sugars were derivatised by 9-aminopyrene-1, 4, 6-trisulfonate (APTS) and analysed by DNA sequencer-Assisted Saccharide analysis in high throughput, DASH (Li et al. (2013 (link)). GH11 products of stem AIR digestion were deuteropermethylated and analysed by MALDI-TOF-Mass Spectrometry as described (Tryfona et al. 2010 (link)).
The Arabidopsis thaliana triple mutant in gxm1gxm2gxm3 was generated by crossing single mutants prepared in Li et al. (2013 (link)). The insertion lines are SALK_087114 (gxm1, At1g33800), SALK_084669 (gxm2, At4g09990) and SALK_050883 (gxm3, At1g09610). Plants from the triple mutant line were grown for 6 weeks, and 5-cm of basal inflorescence stem was harvested. The alcohol-insoluble residue (AIR) was obtained and pre-treated with alkali and digested with a GH11 xylanase as described (Mortimer et al. 2010 (link)). After GH11 digestion, resulting sugars were derivatised by 9-aminopyrene-1, 4, 6-trisulfonate (APTS) and analysed by DNA sequencer-Assisted Saccharide analysis in high throughput, DASH (Li et al. (2013 (link)). GH11 products of stem AIR digestion were deuteropermethylated and analysed by MALDI-TOF-Mass Spectrometry as described (Tryfona et al. 2010 (link)).
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