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AXY3 encodes a α-xylosidase that impacts the structure and accessibility of the hemicellulose xyloglucan in Arabidopsis plant cell walls.

Günl M, Pauly M - Planta (2010)

Bottom Line: The data support the hypothesis that AXY3/XYL1 is an essential component of the apoplastic xyloglucan degradation machinery and as a result of the lack of function in the various axy3-alleles leads not only to an altered xyloglucan structure but also a xyloglucan that is less tightly associated with other wall components.However, the plant can cope with the excess xyloglucan relatively well as the mutant does not display any visible growth or morphological phenotypes with the notable exception of shorter siliques and reduced fitness.Taken together, these results demonstrate that plant apoplastic hydrolases have a larger impact on wall polymer structure and function than previously thought.

View Article: PubMed Central - PubMed

Affiliation: Department of Plant and Microbial Biology, University of California Berkeley, Berkeley, CA, USA.

ABSTRACT
Xyloglucan is the most abundant hemicellulose in the walls of dicots such as Arabidopsis. It is part of the load-bearing structure of a plant cell and its metabolism is thought to play a major role in cell elongation. However, the molecular mechanism by which xyloglucan carries out this and other functions in planta is not well understood. We performed a forward genetic screen utilizing xyloglucan oligosaccharide mass profiling on chemically mutagenized Arabidopsis seedlings to identify mutants with altered xyloglucan structures termed axy-mutants. One of the identified mutants, axy3.1, contains xyloglucan with a higher proportion of non-fucosylated xyloglucan subunits. Mapping revealed that axy3.1 contains a point mutation in XYLOSIDASE1 (XYL1) known to encode for an apoplastic glycoside hydrolase releasing xylosyl residues from xyloglucan oligosaccharides at the non-reducing end. The data support the hypothesis that AXY3/XYL1 is an essential component of the apoplastic xyloglucan degradation machinery and as a result of the lack of function in the various axy3-alleles leads not only to an altered xyloglucan structure but also a xyloglucan that is less tightly associated with other wall components. However, the plant can cope with the excess xyloglucan relatively well as the mutant does not display any visible growth or morphological phenotypes with the notable exception of shorter siliques and reduced fitness. Taken together, these results demonstrate that plant apoplastic hydrolases have a larger impact on wall polymer structure and function than previously thought.

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Quantification of XEG released XyG oligos by HPAEC-PAD from various cell wall fractions as outlined in Figure S2. a Buffer-soluble XyG oligos. b XyG composition of 4 M KOH extract. c XyG composition released by XEG from AIR without prior extraction. d XyG composition of 4 M KOH extract after previous XEG digestion. Numbers (1, 2, 3, 4) refer to the extraction overview in Fig. S2. The mean quantity (n = 6, ±SD) of XyG oligos is shown. Asterisks indicate consistent significant changes of all three mutant lines compared to wild type and complementation line (** p = 0.01, *** p = 0.001) using pairwise comparisons with Student’s t test
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Fig3: Quantification of XEG released XyG oligos by HPAEC-PAD from various cell wall fractions as outlined in Figure S2. a Buffer-soluble XyG oligos. b XyG composition of 4 M KOH extract. c XyG composition released by XEG from AIR without prior extraction. d XyG composition of 4 M KOH extract after previous XEG digestion. Numbers (1, 2, 3, 4) refer to the extraction overview in Fig. S2. The mean quantity (n = 6, ±SD) of XyG oligos is shown. Asterisks indicate consistent significant changes of all three mutant lines compared to wild type and complementation line (** p = 0.01, *** p = 0.001) using pairwise comparisons with Student’s t test

Mentions: The overall structure of XyG in the axy3 mutants was investigated in more detail. XyG is known to occur in the wall in various domains, among them enzyme accessible, cellulose microfibril bound and microfibril internal domains (Pauly et al. 1999a). One can distinguish between these domains by utilizing various extraction techniques. These solubilization techniques were performed and their XyG oligo composition was determined by HPAEC (Fig. S2). First, XyGs that are not integrated into the XyG-cellulose network of the wall were quantified. Such XyG can be extracted from cell walls (alcohol soluble residue (AIR)) with an aqueous buffer, as the polymer is soluble in aqueous solutions but precipitates in 70% ethanol, the preparation method for AIR (Figs. S2c, 3a). The total amount of this XyG fraction as calculated by the sum of all released XyG oligos and is in Col0 or the complemented mutant relatively low with approx. 1 μg mg−1 AIR. However, in the axy3 alleles the amount of the soluble XyG fraction increased more than fourfold to around 4.5 μg mg−1 AIR (Fig. 3a). This increase is mainly due to an increase in the XyG oligos XXXG and XXLG.Fig. 3


AXY3 encodes a α-xylosidase that impacts the structure and accessibility of the hemicellulose xyloglucan in Arabidopsis plant cell walls.

Günl M, Pauly M - Planta (2010)

Quantification of XEG released XyG oligos by HPAEC-PAD from various cell wall fractions as outlined in Figure S2. a Buffer-soluble XyG oligos. b XyG composition of 4 M KOH extract. c XyG composition released by XEG from AIR without prior extraction. d XyG composition of 4 M KOH extract after previous XEG digestion. Numbers (1, 2, 3, 4) refer to the extraction overview in Fig. S2. The mean quantity (n = 6, ±SD) of XyG oligos is shown. Asterisks indicate consistent significant changes of all three mutant lines compared to wild type and complementation line (** p = 0.01, *** p = 0.001) using pairwise comparisons with Student’s t test
© Copyright Policy
Related In: Results  -  Collection

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Fig3: Quantification of XEG released XyG oligos by HPAEC-PAD from various cell wall fractions as outlined in Figure S2. a Buffer-soluble XyG oligos. b XyG composition of 4 M KOH extract. c XyG composition released by XEG from AIR without prior extraction. d XyG composition of 4 M KOH extract after previous XEG digestion. Numbers (1, 2, 3, 4) refer to the extraction overview in Fig. S2. The mean quantity (n = 6, ±SD) of XyG oligos is shown. Asterisks indicate consistent significant changes of all three mutant lines compared to wild type and complementation line (** p = 0.01, *** p = 0.001) using pairwise comparisons with Student’s t test
Mentions: The overall structure of XyG in the axy3 mutants was investigated in more detail. XyG is known to occur in the wall in various domains, among them enzyme accessible, cellulose microfibril bound and microfibril internal domains (Pauly et al. 1999a). One can distinguish between these domains by utilizing various extraction techniques. These solubilization techniques were performed and their XyG oligo composition was determined by HPAEC (Fig. S2). First, XyGs that are not integrated into the XyG-cellulose network of the wall were quantified. Such XyG can be extracted from cell walls (alcohol soluble residue (AIR)) with an aqueous buffer, as the polymer is soluble in aqueous solutions but precipitates in 70% ethanol, the preparation method for AIR (Figs. S2c, 3a). The total amount of this XyG fraction as calculated by the sum of all released XyG oligos and is in Col0 or the complemented mutant relatively low with approx. 1 μg mg−1 AIR. However, in the axy3 alleles the amount of the soluble XyG fraction increased more than fourfold to around 4.5 μg mg−1 AIR (Fig. 3a). This increase is mainly due to an increase in the XyG oligos XXXG and XXLG.Fig. 3

Bottom Line: The data support the hypothesis that AXY3/XYL1 is an essential component of the apoplastic xyloglucan degradation machinery and as a result of the lack of function in the various axy3-alleles leads not only to an altered xyloglucan structure but also a xyloglucan that is less tightly associated with other wall components.However, the plant can cope with the excess xyloglucan relatively well as the mutant does not display any visible growth or morphological phenotypes with the notable exception of shorter siliques and reduced fitness.Taken together, these results demonstrate that plant apoplastic hydrolases have a larger impact on wall polymer structure and function than previously thought.

View Article: PubMed Central - PubMed

Affiliation: Department of Plant and Microbial Biology, University of California Berkeley, Berkeley, CA, USA.

ABSTRACT
Xyloglucan is the most abundant hemicellulose in the walls of dicots such as Arabidopsis. It is part of the load-bearing structure of a plant cell and its metabolism is thought to play a major role in cell elongation. However, the molecular mechanism by which xyloglucan carries out this and other functions in planta is not well understood. We performed a forward genetic screen utilizing xyloglucan oligosaccharide mass profiling on chemically mutagenized Arabidopsis seedlings to identify mutants with altered xyloglucan structures termed axy-mutants. One of the identified mutants, axy3.1, contains xyloglucan with a higher proportion of non-fucosylated xyloglucan subunits. Mapping revealed that axy3.1 contains a point mutation in XYLOSIDASE1 (XYL1) known to encode for an apoplastic glycoside hydrolase releasing xylosyl residues from xyloglucan oligosaccharides at the non-reducing end. The data support the hypothesis that AXY3/XYL1 is an essential component of the apoplastic xyloglucan degradation machinery and as a result of the lack of function in the various axy3-alleles leads not only to an altered xyloglucan structure but also a xyloglucan that is less tightly associated with other wall components. However, the plant can cope with the excess xyloglucan relatively well as the mutant does not display any visible growth or morphological phenotypes with the notable exception of shorter siliques and reduced fitness. Taken together, these results demonstrate that plant apoplastic hydrolases have a larger impact on wall polymer structure and function than previously thought.

Show MeSH