Analysis of xyloglucan fucosylation in arabidopsis

Abstract

Xyloglucan (XyG) is a load-bearing primary wall component in dicotyledonous and non-graminaceous monocotyledonous plants. XyG fucosyltransferase (FUTase), encoded by the Arabidopsis gene AtFUT1, directs addition of fucose (Fuc) residues to terminal galactose residues on XyG side chains. Reverse transcription-polymerase chain reaction and analysis of promoter-β-glucuronidase transgenic plants indicated highest expression of AtFUT1 in the upper portion of elongating inflorescence stems of Arabidopsis. XyG FUTase activity was highest in Golgi vesicles prepared from growing Arabidopsis tissues and low in those isolated from mature tissues. There was no discernible correlation between the Fuc contents of XyG oligosaccharides derived from different Arabidopsis organs and the level of AtFUT1 expression in the organs. Thus, organ-specific variations in AtFUT1 expression and enzyme activity probably reflect differential rates of cell wall biosynthesis, rather than differences in levels of XyG fucosylation. The effects of manipulating AtFUT1 expression were examined using an Arabidopsis mutant (atfut1) containing a T-DNA insertion in the AtFUT1 locus and transgenic plants with strong constitutive expression of AtFUT1. No Fuc was detected in XyG derived from leaves or roots of atfut1. Plants overexpressing AtFUT1 had higher XyG FUTase activity than wild-type plants, but the XyG oligosaccharides derived from the transgenic and wild-type plants contained comparable amounts of Fuc, indicating that suitable acceptor substrates are limiting. Galactosyl residues had slightly higher levels of O-acetylation in XyG from plants that overexpressed AtFUT1 than in XyG from wild-type plants. O-Acetylation of galactose residues was considerably reduced in Fuc-deficient mutants (atfut1, mur1, and mur2) that synthesize XyG containing little or no Fuc. These results suggest that fucosylated XyG is a suitable substrate for at least one O-acetyltransferase in Arabidopsis.