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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

The phenotype of NbPAGR-silencing in N.benthamiana. a-b The morphological phenotype of control (a) and NbPAGR-silenced (b) plants. c The monosaccharide composition of control and NbPAGR-silenced cell walls. Values significantly different from the control are indicated with an asterisk (n = 5, t-test, p < 0.005). d-e β-1,4-galactanase treatment of NbPAGR and control cell walls. Solubilized sugars (d) and residual material (e) (n = 3). f Relative expression of NbPAGR-A & NbPAGR-B in silenced and control plants (*: p < 0.01, n = 4). Error bars indicate standard deviation
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Fig4: The phenotype of NbPAGR-silencing in N.benthamiana. a-b The morphological phenotype of control (a) and NbPAGR-silenced (b) plants. c The monosaccharide composition of control and NbPAGR-silenced cell walls. Values significantly different from the control are indicated with an asterisk (n = 5, t-test, p < 0.005). d-e β-1,4-galactanase treatment of NbPAGR and control cell walls. Solubilized sugars (d) and residual material (e) (n = 3). f Relative expression of NbPAGR-A & NbPAGR-B in silenced and control plants (*: p < 0.01, n = 4). Error bars indicate standard deviation

Mentions: As we were unable to isolate homozygous Arabidopsis pagr mutants, we transiently silenced the N. benthamiana orthologs of PAGR, (NbPAGR) using Tobacco Rattle Virus-based Virus Induced Gene Silencing (VIGS) [34]. VIGS is a rapid and robust method for down-regulating genes of interest and has proven to be an effective system for studying plant cell wall formation and GT function [35, 36]. We identified two PAGR orthologs in the genome of N. benthamiana, NbPAGR-A and NbPAGR-B [37]. NbPAGR-A and -B encode proteins with 80.2 % and 80.6 % identity to Arabidopsis PAGR (Additional file 1: Figure S1) and phylogenetic analysis of these sequences shows strong homology to PAGR and its basal land plant orthologs (Additional file 5: Figure S5). NbPAGR-A and –B are 97.2 % identical to one another, sufficient similarity for efficient silencing of both orthologs with a single construct. A fragment of NbPAGR-A was cloned into a pTRV2 vector to induce silencing of both N. benthamiana PAGR orthologs. N. benthamiana plants were infected with TRV-VIGS viruses by infiltration with Agrobacterium containing NbPAGR and control TRV2-plasmids. NbPAGR-silenced plants had a stout phenotype resulting from cessation of internode and petiole expansion (Fig. 4). The leaves of NbPAGR silenced plants were tightly clustered around the shoot apical meristem. This phenotype is reminiscent of that resulting from silencing of UDP-Apiose/UDP-xylose synthase in N. benthamiana, which causes a deficiency in RG-II production [38]. We also observed alterations in root growth in NbPAGR-silenced plants, including decreased total root length and discoloration (Additional file 6: Figure S6). We evaluated the specificity and efficacy of gene silencing by performing real-time RT-PCR with primers specific for NbPAGR-A and -B. Levels of NbPAGR-A and –B transcripts were reduced by 34 % and 26 %, respectively in silenced plants (Fig. 4f). While this reduction in transcript levels is relatively minor, these results were consistent, statistically significant and may be explained by cessation of growth in tissues with significant silencing.Fig. 4


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)

The phenotype of NbPAGR-silencing in N.benthamiana. a-b The morphological phenotype of control (a) and NbPAGR-silenced (b) plants. c The monosaccharide composition of control and NbPAGR-silenced cell walls. Values significantly different from the control are indicated with an asterisk (n = 5, t-test, p < 0.005). d-e β-1,4-galactanase treatment of NbPAGR and control cell walls. Solubilized sugars (d) and residual material (e) (n = 3). f Relative expression of NbPAGR-A & NbPAGR-B in silenced and control plants (*: p < 0.01, n = 4). Error bars indicate standard deviation
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Related In: Results  -  Collection

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Fig4: The phenotype of NbPAGR-silencing in N.benthamiana. a-b The morphological phenotype of control (a) and NbPAGR-silenced (b) plants. c The monosaccharide composition of control and NbPAGR-silenced cell walls. Values significantly different from the control are indicated with an asterisk (n = 5, t-test, p < 0.005). d-e β-1,4-galactanase treatment of NbPAGR and control cell walls. Solubilized sugars (d) and residual material (e) (n = 3). f Relative expression of NbPAGR-A & NbPAGR-B in silenced and control plants (*: p < 0.01, n = 4). Error bars indicate standard deviation
Mentions: As we were unable to isolate homozygous Arabidopsis pagr mutants, we transiently silenced the N. benthamiana orthologs of PAGR, (NbPAGR) using Tobacco Rattle Virus-based Virus Induced Gene Silencing (VIGS) [34]. VIGS is a rapid and robust method for down-regulating genes of interest and has proven to be an effective system for studying plant cell wall formation and GT function [35, 36]. We identified two PAGR orthologs in the genome of N. benthamiana, NbPAGR-A and NbPAGR-B [37]. NbPAGR-A and -B encode proteins with 80.2 % and 80.6 % identity to Arabidopsis PAGR (Additional file 1: Figure S1) and phylogenetic analysis of these sequences shows strong homology to PAGR and its basal land plant orthologs (Additional file 5: Figure S5). NbPAGR-A and –B are 97.2 % identical to one another, sufficient similarity for efficient silencing of both orthologs with a single construct. A fragment of NbPAGR-A was cloned into a pTRV2 vector to induce silencing of both N. benthamiana PAGR orthologs. N. benthamiana plants were infected with TRV-VIGS viruses by infiltration with Agrobacterium containing NbPAGR and control TRV2-plasmids. NbPAGR-silenced plants had a stout phenotype resulting from cessation of internode and petiole expansion (Fig. 4). The leaves of NbPAGR silenced plants were tightly clustered around the shoot apical meristem. This phenotype is reminiscent of that resulting from silencing of UDP-Apiose/UDP-xylose synthase in N. benthamiana, which causes a deficiency in RG-II production [38]. We also observed alterations in root growth in NbPAGR-silenced plants, including decreased total root length and discoloration (Additional file 6: Figure S6). We evaluated the specificity and efficacy of gene silencing by performing real-time RT-PCR with primers specific for NbPAGR-A and -B. Levels of NbPAGR-A and –B transcripts were reduced by 34 % and 26 %, respectively in silenced plants (Fig. 4f). While this reduction in transcript levels is relatively minor, these results were consistent, statistically significant and may be explained by cessation of growth in tissues with significant silencing.Fig. 4

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