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A DUF-246 family glycosyltransferase-like gene affects male fertility and the biosynthesis of pectic arabinogalactans.

Stonebloom S, Ebert B, Xiong G, Pattathil S, Birdseye D, Lao J, Pauly M, Hahn MG, Heazlewood JL, Scheller HV - BMC Plant Biol. (2016)

Bottom Line: NbPAGR-silenced plants exhibited reduced internode and petiole expansion.Immunological and linkage analyses support that RG-I has reduced type-I arabinogalactan content and reduced branching of the RG-I backbone in NbPAGR-silenced plants.Together, results support a function for PAGR in the biosynthesis of RG-I arabinogalactans and illustrate the essential roles of these polysaccharides in vegetative and reproductive plant growth.

View Article: PubMed Central - PubMed

Affiliation: Joint BioEnergy Institute and Biological Systems and Engineering, Lawrence Berkeley National Laboratory, Berkeley, CA, 94720, USA.

ABSTRACT

Background: Pectins are a group of structurally complex plant cell wall polysaccharides whose biosynthesis and function remain poorly understood. The pectic polysaccharide rhamnogalacturonan-I (RG-I) has two types of arabinogalactan side chains, type-I and type-II arabinogalactans. To date few enzymes involved in the biosynthesis of pectin have been described. Here we report the identification of a highly conserved putative glycosyltransferase encoding gene, Pectic ArabinoGalactan synthesis-Related (PAGR), affecting the biosynthesis of RG-I arabinogalactans and critical for pollen tube growth.

Results: T-DNA insertions in PAGR were identified in Arabidopsis thaliana and were found to segregate at a 1:1 ratio of heterozygotes to wild type. We were unable to isolate homozygous pagr mutants as pagr mutant alleles were not transmitted via pollen. In vitro pollen germination assays revealed reduced rates of pollen tube formation in pollen from pagr heterozygotes. To characterize a loss-of-function phenotype for PAGR, the Nicotiana benthamiana orthologs, NbPAGR-A and B, were transiently silenced using Virus Induced Gene Silencing. NbPAGR-silenced plants exhibited reduced internode and petiole expansion. Cell wall materials from NbPAGR-silenced plants had reduced galactose content compared to the control. Immunological and linkage analyses support that RG-I has reduced type-I arabinogalactan content and reduced branching of the RG-I backbone in NbPAGR-silenced plants. Arabidopsis lines overexpressing PAGR exhibit pleiotropic developmental phenotypes and the loss of apical dominance as well as an increase in RG-I type-II arabinogalactan content.

Conclusions: Together, results support a function for PAGR in the biosynthesis of RG-I arabinogalactans and illustrate the essential roles of these polysaccharides in vegetative and reproductive plant growth.

No MeSH data available.


Related in: MedlinePlus

Biochemical characterization of PAGR overexpressing Arabidopsis lines. a The monosaccharide composition of cell walls from 35S::PAGR-YFP seedlings grown in liquid culture. Over-expressing lines exhibit an increase in total arabinose content and slight reductions in fucose, rhamnose and glucuronic acid content. (*: p < 0.05, t-test, n = 4). b Expression analysis of PAGR-YFP in transgenic lines. Protein extracts from 10-day old T3 35S::PAGR-YFP seedlings and from the Wild Type were analyzed by immunoblotting and PAGR-YFP was detected with ‘universal antibody’ against the AttB2 site linker peptide in PAGR-YFP. Coomassie staining of Rubisco large subunit (RBCL) is shown as a loading control
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Fig6: Biochemical characterization of PAGR overexpressing Arabidopsis lines. a The monosaccharide composition of cell walls from 35S::PAGR-YFP seedlings grown in liquid culture. Over-expressing lines exhibit an increase in total arabinose content and slight reductions in fucose, rhamnose and glucuronic acid content. (*: p < 0.05, t-test, n = 4). b Expression analysis of PAGR-YFP in transgenic lines. Protein extracts from 10-day old T3 35S::PAGR-YFP seedlings and from the Wild Type were analyzed by immunoblotting and PAGR-YFP was detected with ‘universal antibody’ against the AttB2 site linker peptide in PAGR-YFP. Coomassie staining of Rubisco large subunit (RBCL) is shown as a loading control

Mentions: Analysis of total cell wall material from three independent lines of 35S::PAGR-YFP plants showed a small increase in total arabinose content and small decreases in fucose, rhamnose and glucuronic acid content (Fig. 6a). Following sequential extraction of cell wall material from PAGR-overexpressing plants, we found that the CDTA cell wall extract contained an increased arabinose content whereas the subsequent 4 M KOH extract and the residual material showed no differences in monosaccharide composition compared to the wild type (Additional file 8: Figure S8). The expression of PAGR-YFP fusion protein in transgenic plants was confirmed by immunoblotting (Fig. 6b). PAGR-YFP was detected in all transgenic lines at a molecular mass of around 110 kDa, somewhat larger than the predicted molecular mass of 92.3 kDa, with antibody against the AttB2 linker peptide present in PAGR-YFP.Fig. 6


A DUF-246 family glycosyltransferase-like gene affects male fertility and the biosynthesis of pectic arabinogalactans.

Stonebloom S, Ebert B, Xiong G, Pattathil S, Birdseye D, Lao J, Pauly M, Hahn MG, Heazlewood JL, Scheller HV - BMC Plant Biol. (2016)

Biochemical characterization of PAGR overexpressing Arabidopsis lines. a The monosaccharide composition of cell walls from 35S::PAGR-YFP seedlings grown in liquid culture. Over-expressing lines exhibit an increase in total arabinose content and slight reductions in fucose, rhamnose and glucuronic acid content. (*: p < 0.05, t-test, n = 4). b Expression analysis of PAGR-YFP in transgenic lines. Protein extracts from 10-day old T3 35S::PAGR-YFP seedlings and from the Wild Type were analyzed by immunoblotting and PAGR-YFP was detected with ‘universal antibody’ against the AttB2 site linker peptide in PAGR-YFP. Coomassie staining of Rubisco large subunit (RBCL) is shown as a loading control
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4836069&req=5

Fig6: Biochemical characterization of PAGR overexpressing Arabidopsis lines. a The monosaccharide composition of cell walls from 35S::PAGR-YFP seedlings grown in liquid culture. Over-expressing lines exhibit an increase in total arabinose content and slight reductions in fucose, rhamnose and glucuronic acid content. (*: p < 0.05, t-test, n = 4). b Expression analysis of PAGR-YFP in transgenic lines. Protein extracts from 10-day old T3 35S::PAGR-YFP seedlings and from the Wild Type were analyzed by immunoblotting and PAGR-YFP was detected with ‘universal antibody’ against the AttB2 site linker peptide in PAGR-YFP. Coomassie staining of Rubisco large subunit (RBCL) is shown as a loading control
Mentions: Analysis of total cell wall material from three independent lines of 35S::PAGR-YFP plants showed a small increase in total arabinose content and small decreases in fucose, rhamnose and glucuronic acid content (Fig. 6a). Following sequential extraction of cell wall material from PAGR-overexpressing plants, we found that the CDTA cell wall extract contained an increased arabinose content whereas the subsequent 4 M KOH extract and the residual material showed no differences in monosaccharide composition compared to the wild type (Additional file 8: Figure S8). The expression of PAGR-YFP fusion protein in transgenic plants was confirmed by immunoblotting (Fig. 6b). PAGR-YFP was detected in all transgenic lines at a molecular mass of around 110 kDa, somewhat larger than the predicted molecular mass of 92.3 kDa, with antibody against the AttB2 linker peptide present in PAGR-YFP.Fig. 6

Bottom Line: NbPAGR-silenced plants exhibited reduced internode and petiole expansion.Immunological and linkage analyses support that RG-I has reduced type-I arabinogalactan content and reduced branching of the RG-I backbone in NbPAGR-silenced plants.Together, results support a function for PAGR in the biosynthesis of RG-I arabinogalactans and illustrate the essential roles of these polysaccharides in vegetative and reproductive plant growth.

View Article: PubMed Central - PubMed

Affiliation: Joint BioEnergy Institute and Biological Systems and Engineering, Lawrence Berkeley National Laboratory, Berkeley, CA, 94720, USA.

ABSTRACT

Background: Pectins are a group of structurally complex plant cell wall polysaccharides whose biosynthesis and function remain poorly understood. The pectic polysaccharide rhamnogalacturonan-I (RG-I) has two types of arabinogalactan side chains, type-I and type-II arabinogalactans. To date few enzymes involved in the biosynthesis of pectin have been described. Here we report the identification of a highly conserved putative glycosyltransferase encoding gene, Pectic ArabinoGalactan synthesis-Related (PAGR), affecting the biosynthesis of RG-I arabinogalactans and critical for pollen tube growth.

Results: T-DNA insertions in PAGR were identified in Arabidopsis thaliana and were found to segregate at a 1:1 ratio of heterozygotes to wild type. We were unable to isolate homozygous pagr mutants as pagr mutant alleles were not transmitted via pollen. In vitro pollen germination assays revealed reduced rates of pollen tube formation in pollen from pagr heterozygotes. To characterize a loss-of-function phenotype for PAGR, the Nicotiana benthamiana orthologs, NbPAGR-A and B, were transiently silenced using Virus Induced Gene Silencing. NbPAGR-silenced plants exhibited reduced internode and petiole expansion. Cell wall materials from NbPAGR-silenced plants had reduced galactose content compared to the control. Immunological and linkage analyses support that RG-I has reduced type-I arabinogalactan content and reduced branching of the RG-I backbone in NbPAGR-silenced plants. Arabidopsis lines overexpressing PAGR exhibit pleiotropic developmental phenotypes and the loss of apical dominance as well as an increase in RG-I type-II arabinogalactan content.

Conclusions: Together, results support a function for PAGR in the biosynthesis of RG-I arabinogalactans and illustrate the essential roles of these polysaccharides in vegetative and reproductive plant growth.

No MeSH data available.


Related in: MedlinePlus