Effects of the mur1 mutation on xyloglucans produced by suspension-cultured Arabidopsis thaliana cells

Abstract

Mutation of the Arabidopsis thaliana (L.) Heynh. gene MUR1, which encodes an isoform of GDP-D-mannose-4,6-dehydratase, affects the biosynthetic conversion of GDP-mannose to GDP-fucose. Cell walls in the aerial tissues of mur1 plants are almost devoid of alpha-L-fucosyl residues, which are partially replaced by closely related alpha-L-galactosyl residues. A line of suspension-cultured A. thaliana cells was generated from leaves of mur1 plants and the structure of the xyloglucan in the walls of these cells was structurally characterized. Xyloglucan fractions were prepared from the walls of both wild-type (WT) and mur1 cells by sequential extraction with a xyloglucan-specific endoglucanase (XEG) and aqueous KOH. Structural analysis of these fractions revealed that xyloglucan produced by cultured mur1 cells is similar, but not identical to that isolated from leaves of mur1 plants. As previously reported for mur1 leaves, the xyloglucan from cultured mur1 cells contains less than 5% of the fucose present in the xyloglucan from WT cells. Fucosylation of the xyloglucan is substantially restored when mur1 cells are grown in medium supplemented with L-fucose. Xyloglucan isolated from leaves contains more oligosaccharide subunits in which the central sidechain is terminated with a beta-D-galactosyl residue than does xyloglucan prepared from cultured cells. This was observed for both mur1 and WT plants, indicating that this correlation is independent of the mur1 mutation and that it is possible to distinguish changes due to genetic mutation from those due to the physiological state of the cells in culture. Suspension-cultured cells thus provide a convenient source of genetically altered cell wall material, facilitating the biochemical characterization of mutations that affect cell wall structure.

Definition of Arabidopsis Sterol-rich Membrane Microdomains by Differential Treatment with Methyl-β-cyclodextrin and Quantitative Proteomics

“…Arabidopsis Cell Suspension Cultures-Arabidopsis thaliana Col-0 cell suspension cultures (19) were grown in full medium as described previously (20). For quantitative proteomics experiments, half of the cell cultures were metabolically labeled with 15 N by growth under [ 15 N]KNO 3 as the only nitrogen source for at least two passages over 2 weeks (21).…”

Quantitative Trait Loci Analysis of Primary Cell Wall Composition in Arabidopsis

“…Arabidopsis walls contain XyGOs of various sizes, ranging from the pentasaccharide XXG to the decasaccharide XLFG, with various degrees of O-acetylation (Ac; Fig. 2; Pauly et al, 2001). The oligosaccharide-profiling method provides a fingerprint of the native xyloglucan polysaccharide structure, including Ac patterns (Lerouxel et al, 2002).…”

Combined Transcript and Metabolite Profiling of Arabidopsis Grown under Widely Variant Growth Conditions Facilitates the Identification of Novel Metabolite-Mediated Regulation of Gene Expression

“…Expression of transcripts after Leu feeding. Arabidopsis suspension cell cultures derived from leaves (Pauly et al, 2001) To determine if a given transcript was detected in each sample we used the P value from the MAS5 detection algorithm as implemented in the affy package.…”

“…In a further approach we supplied Leu to cell suspension cultures derived from Arabidopsis leaves (Pauly et al, 2001) via exogenous feeding of 50 mM Leu in buffered Murashige and Skoog medium applied for 90 min and assessed the effect on the levels of various transcripts. Total RNA extraction, cDNA synthesis, and quantitative reverse transcription-PCR were performed as described (Czechowski et al, 2004(Czechowski et al, , 2005Caldana et al, 2007).…”