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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

Characterization of RG-I in PAGR-overexpressing Arabidopsis lines and NbPAGR-silenced N. benthamiana. Size exclusion chromatography using a Superdex 200 column of RG-I from NbPAGR-silenced and control N. benthamiana cell walls (a) and RG-I from 35S::PAGR-YFP lines and the wild type (b). RG-I from NbPAGR-silenced lines eluted later than the control indicating a decrease in the average molecular weight of RG-I domains. RG-I from PAGR-overexpressing Arabidopsis lines eluted slightly earlier than RG-I from the wild type. c ELISA analysis of size exclusion chromatography-purified RG-I with plant-glycan directed monoclonal antibodies
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Fig7: Characterization of RG-I in PAGR-overexpressing Arabidopsis lines and NbPAGR-silenced N. benthamiana. Size exclusion chromatography using a Superdex 200 column of RG-I from NbPAGR-silenced and control N. benthamiana cell walls (a) and RG-I from 35S::PAGR-YFP lines and the wild type (b). RG-I from NbPAGR-silenced lines eluted later than the control indicating a decrease in the average molecular weight of RG-I domains. RG-I from PAGR-overexpressing Arabidopsis lines eluted slightly earlier than RG-I from the wild type. c ELISA analysis of size exclusion chromatography-purified RG-I with plant-glycan directed monoclonal antibodies

Mentions: To further characterize the altered arabinogalactan content of cell walls from NbPAGR-silenced plants we analyzed the molecular mass and composition of RGI. RG-I can be solubilized from cell wall material along with RG-II by digestion with polygalacturonanase and pectin methylesterase [39]. RG-I released by this enzymatic treatment was separated from RG-II by size-exclusion chromatography on a Superdex 200 10/300 column (Fig. 7). RG-I from NbPAGR-silenced plants eluted later than RG-I from control plants. The average molecular mass of RG-I from silenced plants was calculated to be 91.2 kDa compared to 118.6 kDa in control plants when compared to the elution times of dextran molecular mass standards. RG-I fractions were collected following size exclusion chromatography and the monosaccharide composition of the collected material was analyzed. The arabinose and galactose content of RG-I was reduced from 21.9 % arabinose and 50.5 % galactose in control plants to 20.3 % arabinose and 44.4 % galactose in NbPAGR-silenced plants. Conversely the amount of RG-I backbone monosaccharides rhamnose and galacturonic acid was increased from 13.3 % rhamnose and 11.5 % galacturonic acid in the control to 16.6 % rhamnose and 15.6 % galacturonic acid in silenced plants (Table 4).Fig. 7


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)

Characterization of RG-I in PAGR-overexpressing Arabidopsis lines and NbPAGR-silenced N. benthamiana. Size exclusion chromatography using a Superdex 200 column of RG-I from NbPAGR-silenced and control N. benthamiana cell walls (a) and RG-I from 35S::PAGR-YFP lines and the wild type (b). RG-I from NbPAGR-silenced lines eluted later than the control indicating a decrease in the average molecular weight of RG-I domains. RG-I from PAGR-overexpressing Arabidopsis lines eluted slightly earlier than RG-I from the wild type. c ELISA analysis of size exclusion chromatography-purified RG-I with plant-glycan directed monoclonal antibodies
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4836069&req=5

Fig7: Characterization of RG-I in PAGR-overexpressing Arabidopsis lines and NbPAGR-silenced N. benthamiana. Size exclusion chromatography using a Superdex 200 column of RG-I from NbPAGR-silenced and control N. benthamiana cell walls (a) and RG-I from 35S::PAGR-YFP lines and the wild type (b). RG-I from NbPAGR-silenced lines eluted later than the control indicating a decrease in the average molecular weight of RG-I domains. RG-I from PAGR-overexpressing Arabidopsis lines eluted slightly earlier than RG-I from the wild type. c ELISA analysis of size exclusion chromatography-purified RG-I with plant-glycan directed monoclonal antibodies
Mentions: To further characterize the altered arabinogalactan content of cell walls from NbPAGR-silenced plants we analyzed the molecular mass and composition of RGI. RG-I can be solubilized from cell wall material along with RG-II by digestion with polygalacturonanase and pectin methylesterase [39]. RG-I released by this enzymatic treatment was separated from RG-II by size-exclusion chromatography on a Superdex 200 10/300 column (Fig. 7). RG-I from NbPAGR-silenced plants eluted later than RG-I from control plants. The average molecular mass of RG-I from silenced plants was calculated to be 91.2 kDa compared to 118.6 kDa in control plants when compared to the elution times of dextran molecular mass standards. RG-I fractions were collected following size exclusion chromatography and the monosaccharide composition of the collected material was analyzed. The arabinose and galactose content of RG-I was reduced from 21.9 % arabinose and 50.5 % galactose in control plants to 20.3 % arabinose and 44.4 % galactose in NbPAGR-silenced plants. Conversely the amount of RG-I backbone monosaccharides rhamnose and galacturonic acid was increased from 13.3 % rhamnose and 11.5 % galacturonic acid in the control to 16.6 % rhamnose and 15.6 % galacturonic acid in silenced plants (Table 4).Fig. 7

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