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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 subcellular localization of PAGR-CFP. PAGR-CFP (a & d) was co-expressed with the Golgi apparatus marker α-mannosidase-1-mCherry (b & e) in N. benthamiana leaves; merged signals (c & f). In some cells PAGR-CFP co-localized with α-mannosidase-1-mCherry (a-c). In most cells PAGR-CFP partially co-localized with α-mannosidase-1-mCherry and also localized to small punctate structures (white arrows, d-f). Scale bars 5 μm
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Fig3: The subcellular localization of PAGR-CFP. PAGR-CFP (a & d) was co-expressed with the Golgi apparatus marker α-mannosidase-1-mCherry (b & e) in N. benthamiana leaves; merged signals (c & f). In some cells PAGR-CFP co-localized with α-mannosidase-1-mCherry (a-c). In most cells PAGR-CFP partially co-localized with α-mannosidase-1-mCherry and also localized to small punctate structures (white arrows, d-f). Scale bars 5 μm

Mentions: The PAGR protein is predicted to be a type-II membrane protein (Fig. 1c) and has been detected by LC-MS/MS in the proteome of Golgi purified from Arabidopsis suspension cell cultures [27]. To confirm the localization of PAGR we transiently co-expressed PAGR-CFP in N. benthamiana with α-mannosidase-1 fused to mCherry as a Golgi marker [28]. In some cells PAGR co-localized with α-mannosidase-1 (Fig. 3a-c). However, in most cells PAGR-CFP also localized to small punctate structures in which α-mannosidase-1 was not present (Fig. 3d-f). This localization pattern to both the Golgi apparatus and to a smaller, non-Golgi subcellular compartment is similar to the localization of AtGALT31A, AtGALT29A and AtGlcAT14A [29]. AtGALT31A and AtGALT29A are galactosyltransferases involved in the biosynthesis of AGP type-II arabinogalactans [30, 31], and AtGlcAT14A is a glucuronosyltransferase also involved in the biosynthesis of type-II arabinogalactans [32]. Galt29A was reported to partially colocalize with EXO70E2 in small subcellular compartments thought to be involved in an unconventional secretion pathway known as Exocyst-Positive organelles (EXPOs) [29, 33]; however we have been unable to replicate this result.Fig. 3


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 subcellular localization of PAGR-CFP. PAGR-CFP (a & d) was co-expressed with the Golgi apparatus marker α-mannosidase-1-mCherry (b & e) in N. benthamiana leaves; merged signals (c & f). In some cells PAGR-CFP co-localized with α-mannosidase-1-mCherry (a-c). In most cells PAGR-CFP partially co-localized with α-mannosidase-1-mCherry and also localized to small punctate structures (white arrows, d-f). Scale bars 5 μm
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

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

Fig3: The subcellular localization of PAGR-CFP. PAGR-CFP (a & d) was co-expressed with the Golgi apparatus marker α-mannosidase-1-mCherry (b & e) in N. benthamiana leaves; merged signals (c & f). In some cells PAGR-CFP co-localized with α-mannosidase-1-mCherry (a-c). In most cells PAGR-CFP partially co-localized with α-mannosidase-1-mCherry and also localized to small punctate structures (white arrows, d-f). Scale bars 5 μm
Mentions: The PAGR protein is predicted to be a type-II membrane protein (Fig. 1c) and has been detected by LC-MS/MS in the proteome of Golgi purified from Arabidopsis suspension cell cultures [27]. To confirm the localization of PAGR we transiently co-expressed PAGR-CFP in N. benthamiana with α-mannosidase-1 fused to mCherry as a Golgi marker [28]. In some cells PAGR co-localized with α-mannosidase-1 (Fig. 3a-c). However, in most cells PAGR-CFP also localized to small punctate structures in which α-mannosidase-1 was not present (Fig. 3d-f). This localization pattern to both the Golgi apparatus and to a smaller, non-Golgi subcellular compartment is similar to the localization of AtGALT31A, AtGALT29A and AtGlcAT14A [29]. AtGALT31A and AtGALT29A are galactosyltransferases involved in the biosynthesis of AGP type-II arabinogalactans [30, 31], and AtGlcAT14A is a glucuronosyltransferase also involved in the biosynthesis of type-II arabinogalactans [32]. Galt29A was reported to partially colocalize with EXO70E2 in small subcellular compartments thought to be involved in an unconventional secretion pathway known as Exocyst-Positive organelles (EXPOs) [29, 33]; however we have been unable to replicate this result.Fig. 3

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