Analysis of the sites of Gal incorporation in the products produced by AtGALT29A alone or the AtGALT29A/AtGALT31A complex. The [14C]-Gal incorporated products onto SP32-GFP (A, B, C) or onto β-1,3-galactan (D) from P19 [∙∙∙], HA-AtGALT29A [---], or co-immunoprecipitated HA-AtGALT29A/AtGALT31A-GFP complex [▬] were treated with A: endo-β-1,6-galactanase, B: endo-β-1,6-galactanase + α-arabinofuranosidase, C: exo-β-1,3-galactanase, or D: exo-β-1,3-galactanase, and separated by size exclusion chromatography using Superdex Peptide HR 10/30. The [14C]-Gal present in each fraction was evaluated by scintillation counting. Endo-β-1,6-galactanase, α-arabinofuranosidase, and exo-β-1,3-galactanase used in this study cleave β-1,6-linked unsubstituted galactotriose, terminal α-linked arabinofuranose, and β-1,3-linked galactooligosaccharides regardless the presence or absence of substitutions, respectively. Release of small [14C]-oligosaccharides by endo-β-1,6-galactanase indicates the [14C]-Gal incorporation to a part of β-1,6-galactotriose, while exo-β-1,3-galactanase releases [14C]-Gal monomer from β-1,3-linked galactan and [14C]-oligosaccharide (s) from side chains attached to β-1,3-linked galactan. From the [14C]-products made onto SP32-GFP and β-1,3-galactan, exo-β-1,3-galactanase released mainly [14C]-galactobiose analyzed by TLC (inset C and D), indicating the incorporation of single [14C]-Gal to β-1,3-linked Gal at O6 in the [14C]-products. From any treatments (A-D), higher amount of small [14C]-oligosaccharides are released from the [14C]-products made by AtGALT29A/AtGALT31A complex compared to that from a single enzyme. The results indicate that AtGALT29A possesses β-1,6-GalT activities elongating β-1,6-galactan and forming 6-Gal branches on β-1,3-galatan, and the β-1,6-GalT activities are increased when AtGALT29A is in a protein complex with AtGALT31A.