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Arabidopsis thaliana POLYOL/MONOSACCHARIDE TRANSPORTERS 1 and 2: fructose and xylitol/H+ symporters in pollen and young xylem cells.

Klepek YS, Volke M, Konrad KR, Wippel K, Hoth S, Hedrich R, Sauer N - J. Exp. Bot. (2009)

Bottom Line: Analyses of reporter genes performed with AtPMT1 or AtPMT2 promoter sequences showed expression in mature (AtPMT2) or germinating (AtPMT1) pollen grains, as well as in growing pollen tubes, hydathodes, and young xylem cells (both genes).The expression was confirmed with an anti-AtPMT1/AtPMT2 antiserum (alphaAtPMT1/2) raised against peptides conserved in AtPMT1 and AtPMT2.The physiological roles of the proteins are discussed and related to plant cell wall modifications.

View Article: PubMed Central - PubMed

Affiliation: Molekulare Pflanzenphysiologie, Universität Erlangen-Nürnberg, Staudtstrasse 5, Erlangen, Germany.

ABSTRACT
The genome of Arabidopsis thaliana contains six genes, AtPMT1 to AtPMT6 (Arabidopsis thaliana POLYOL/MONOSACCHARIDE TRANSPORTER 1-6), which form a distinct subfamily within the large family of more than 50 monosaccharide transporter-like (MST-like) genes. So far, only AtPMT5 [formerly named AtPLT5 (At3g18830)] has been characterized and was shown to be a plasma membrane-localized H(+)-symporter with broad substrate specificity. The characterization of AtPMT1 (At2g16120) and AtPMT2 (At2g16130), two other, almost identical, members of this transporter subfamily, are presented here. Expression of the AtPMT1 and AtPMT2 cDNAs in baker's yeast (Saccharomyces cerevisiae) revealed that these proteins catalyse the energy-dependent, high-capacity transport of fructose and xylitol, and the transport of several other compounds with lower rates. Expression of their cRNAs in Xenopus laevis oocytes showed that both proteins are voltage-dependent and catalyse the symport of their substrates with protons. Fusions of AtPMT1 or AtPMT2 with the green fluorescent protein (GFP) localized to Arabidopsis plasma membranes. Analyses of reporter genes performed with AtPMT1 or AtPMT2 promoter sequences showed expression in mature (AtPMT2) or germinating (AtPMT1) pollen grains, as well as in growing pollen tubes, hydathodes, and young xylem cells (both genes). The expression was confirmed with an anti-AtPMT1/AtPMT2 antiserum (alphaAtPMT1/2) raised against peptides conserved in AtPMT1 and AtPMT2. The physiological roles of the proteins are discussed and related to plant cell wall modifications.

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Subcellular localization of AtPMT1 and AtPMT2. (A) Subcellular localization of AtPMT1 by transient expression of an AtPMT1-GFP fusion-construct in an Arabidopsis protoplast after chemical transformation. (B) Arabidopsis epidermis cell transformed by particle-bombarded with the AtPMT1-GFP construct. (C) Subcellular localization of AtPMT2 by transient expression of an AtPMT2/GFP fusion construct in an Arabidopsis protoplast after chemical transformation. Arrows show localization of chloroplasts (red autofluorescence of chlorophyll) inside the GFP-labelled plasma membrane. All images show single confocal sections. Bars are 50 μm (A, B), 60 μm (C).
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fig5: Subcellular localization of AtPMT1 and AtPMT2. (A) Subcellular localization of AtPMT1 by transient expression of an AtPMT1-GFP fusion-construct in an Arabidopsis protoplast after chemical transformation. (B) Arabidopsis epidermis cell transformed by particle-bombarded with the AtPMT1-GFP construct. (C) Subcellular localization of AtPMT2 by transient expression of an AtPMT2/GFP fusion construct in an Arabidopsis protoplast after chemical transformation. Arrows show localization of chloroplasts (red autofluorescence of chlorophyll) inside the GFP-labelled plasma membrane. All images show single confocal sections. Bars are 50 μm (A, B), 60 μm (C).

Mentions: Although the transport activities of AtPMT1 and AtPMT2 in yeast and Xenopus plasma membranes (Figs 2, 3, 4) provided indirect evidence that these proteins might be transporters of the plasma membrane, targeting of AtPMT1 and AtPMT2 to other membranes in planta cannot be excluded. To identify the subcellular localization of both transporters in Arabidopsis, the open reading frame (ORF) for green fluorescent protein (GFP) was attached to the 3′-ends of AtPMT1 or AtPMT2 cDNAs and the resulting constructs were used for transient expression in Arabidopsis mesophyll protoplasts or in intact Arabidopsis epidermal cells. All cells transformed with these constructs (pMV-L for AtPMT1-GFP expression; Fig. 5A, B; pYK26 for AtPMT2-GFP expression; Fig. 5C) showed strong and specific labelling of their cell surfaces. Chloroplasts (recognized by their red autofluorescence) are clearly located inside this labelled structure. This localization supports the plasma membrane as the origin of the signal rather than the tonoplast or the endoplasmic reticulum.


Arabidopsis thaliana POLYOL/MONOSACCHARIDE TRANSPORTERS 1 and 2: fructose and xylitol/H+ symporters in pollen and young xylem cells.

Klepek YS, Volke M, Konrad KR, Wippel K, Hoth S, Hedrich R, Sauer N - J. Exp. Bot. (2009)

Subcellular localization of AtPMT1 and AtPMT2. (A) Subcellular localization of AtPMT1 by transient expression of an AtPMT1-GFP fusion-construct in an Arabidopsis protoplast after chemical transformation. (B) Arabidopsis epidermis cell transformed by particle-bombarded with the AtPMT1-GFP construct. (C) Subcellular localization of AtPMT2 by transient expression of an AtPMT2/GFP fusion construct in an Arabidopsis protoplast after chemical transformation. Arrows show localization of chloroplasts (red autofluorescence of chlorophyll) inside the GFP-labelled plasma membrane. All images show single confocal sections. Bars are 50 μm (A, B), 60 μm (C).
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC2803217&req=5

fig5: Subcellular localization of AtPMT1 and AtPMT2. (A) Subcellular localization of AtPMT1 by transient expression of an AtPMT1-GFP fusion-construct in an Arabidopsis protoplast after chemical transformation. (B) Arabidopsis epidermis cell transformed by particle-bombarded with the AtPMT1-GFP construct. (C) Subcellular localization of AtPMT2 by transient expression of an AtPMT2/GFP fusion construct in an Arabidopsis protoplast after chemical transformation. Arrows show localization of chloroplasts (red autofluorescence of chlorophyll) inside the GFP-labelled plasma membrane. All images show single confocal sections. Bars are 50 μm (A, B), 60 μm (C).
Mentions: Although the transport activities of AtPMT1 and AtPMT2 in yeast and Xenopus plasma membranes (Figs 2, 3, 4) provided indirect evidence that these proteins might be transporters of the plasma membrane, targeting of AtPMT1 and AtPMT2 to other membranes in planta cannot be excluded. To identify the subcellular localization of both transporters in Arabidopsis, the open reading frame (ORF) for green fluorescent protein (GFP) was attached to the 3′-ends of AtPMT1 or AtPMT2 cDNAs and the resulting constructs were used for transient expression in Arabidopsis mesophyll protoplasts or in intact Arabidopsis epidermal cells. All cells transformed with these constructs (pMV-L for AtPMT1-GFP expression; Fig. 5A, B; pYK26 for AtPMT2-GFP expression; Fig. 5C) showed strong and specific labelling of their cell surfaces. Chloroplasts (recognized by their red autofluorescence) are clearly located inside this labelled structure. This localization supports the plasma membrane as the origin of the signal rather than the tonoplast or the endoplasmic reticulum.

Bottom Line: Analyses of reporter genes performed with AtPMT1 or AtPMT2 promoter sequences showed expression in mature (AtPMT2) or germinating (AtPMT1) pollen grains, as well as in growing pollen tubes, hydathodes, and young xylem cells (both genes).The expression was confirmed with an anti-AtPMT1/AtPMT2 antiserum (alphaAtPMT1/2) raised against peptides conserved in AtPMT1 and AtPMT2.The physiological roles of the proteins are discussed and related to plant cell wall modifications.

View Article: PubMed Central - PubMed

Affiliation: Molekulare Pflanzenphysiologie, Universität Erlangen-Nürnberg, Staudtstrasse 5, Erlangen, Germany.

ABSTRACT
The genome of Arabidopsis thaliana contains six genes, AtPMT1 to AtPMT6 (Arabidopsis thaliana POLYOL/MONOSACCHARIDE TRANSPORTER 1-6), which form a distinct subfamily within the large family of more than 50 monosaccharide transporter-like (MST-like) genes. So far, only AtPMT5 [formerly named AtPLT5 (At3g18830)] has been characterized and was shown to be a plasma membrane-localized H(+)-symporter with broad substrate specificity. The characterization of AtPMT1 (At2g16120) and AtPMT2 (At2g16130), two other, almost identical, members of this transporter subfamily, are presented here. Expression of the AtPMT1 and AtPMT2 cDNAs in baker's yeast (Saccharomyces cerevisiae) revealed that these proteins catalyse the energy-dependent, high-capacity transport of fructose and xylitol, and the transport of several other compounds with lower rates. Expression of their cRNAs in Xenopus laevis oocytes showed that both proteins are voltage-dependent and catalyse the symport of their substrates with protons. Fusions of AtPMT1 or AtPMT2 with the green fluorescent protein (GFP) localized to Arabidopsis plasma membranes. Analyses of reporter genes performed with AtPMT1 or AtPMT2 promoter sequences showed expression in mature (AtPMT2) or germinating (AtPMT1) pollen grains, as well as in growing pollen tubes, hydathodes, and young xylem cells (both genes). The expression was confirmed with an anti-AtPMT1/AtPMT2 antiserum (alphaAtPMT1/2) raised against peptides conserved in AtPMT1 and AtPMT2. The physiological roles of the proteins are discussed and related to plant cell wall modifications.

Show MeSH
Related in: MedlinePlus