Klepek YS , Volke M , Konrad KR , Wippel K , Hoth S , Hedrich R , Sauer N .

	Fig. 1. Comparison of the six Arabidopsis PMT proteins. Schematic alignment of the deduced protein sequences (black bars) of AtPMT1 to AtPMT6 based on the intron positions (arrows) in the respective genes. Grey vertical bars (I–XII) indicate the positions of the predicted transmembrane helices, thin lines show two small gaps in the AtPMT4 sequence. Numbers of amino acids encoded by the different exons are indicated (white).
	Fig. 2. Functional characterization of AtPMT1 and AtPMT2 in the monosaccharide transport-deficient yeast strain EBY.VW-4000. (A) AtPMT1-driven (top; strain VMY15) or AtPMT2-driven (bottom; strain YKY6) uptake of 14C-sorbitol (0.1 mM) into transgenic baker's yeast in the presence of different potential competitors that were added at a 100-fold molar excess (n=3; ±SD). (B) Transport of the indicated compounds into AtPMT1-expressing yeast strain VMY15 (open symbols) and into the strain VMY21, that expresses the AtPMT1 cDNA in antisense orientation (closed symbols; n=3; ±SD). (C) Transport of the indicated compounds into AtPMT2-expressing yeast strain YKY6 (open symbols) and into the strain YKY7, that expresses the AtPMT2 cDNA in antisense orientation (closed symbols; n=3; ±SD). (D) pH-dependence (left) and sensitivity to the uncoupler CCCP (right) of AtPMT1 (VMY15) and AtPMT2 (YKY6; n=3; ±SD).
	Fig. 3. Determination of the Km-values for xylitol for AtPMT1 and AtPMT2. Michaelis–Menten-type kinetics for xylitol uptake were determined (A) in AtPMT1-expressing (strain VMY15) and (B) AtPMT2-expressing (strain YKY6) yeast cells. Inserts show Lineweaver–Burke plots of the same data sets (±SD; n=3).
	Fig. 4. Characterization of AtPMT1 and AtPMT2 in cRNA-injected Xenopus oocytes. (A) AtPMT1-expressing oocytes mediate H+-inward currents in response to xylitol (10 mM) application. The presence of KCl and NaCl (left) or of N-methylglucamine chloride (NMGCl; right) in the external bath solution did not effect the characteristics of xylitol-induced currents. Measurements were recorded at a membrane potential of –60 mV and pH 6.5 (one of nine experiments with identical results). (B) H+-currents elicited by xylitol (10 mM) were recorded at the indicated membrane potentials. The external solution contained 30 mM KCl and 65 mM NaCl and was adjusted to pH 6.5 (one of four experiments with identical results). (C) Experimental design as in (A) but with AtPMT2-expressing oocytes (one of eight experiments with identical results). (D) Same conditions as in (B) but with AtPMT2-expressing oocytes (one of five experiments with identical results).
	Fig. 5. 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).
	Fig. 6. GUS stainings of pAtPMT1/GUS plants. (A) Anther of a pAtPMT1/GUS plant with no GUS activity. (B) Closed and just opening (arrow) pollen grains on agar medium. (C) Ungerminated (white) and germinated (blue) pollen grains, the latter with a well-developed pollen tube. (D) Cross-section through a flower stalk showing GUS histochemical staining in the centre of two vascular bundles. (E, F) Higher magnifications of cross-sections from flower stalk vascular bundles (ph, phloem; xy, xylem). Arrows show regions, with the typical stacks of cambium and newly formed phloem and xylem vessels. Bars are 100 μm in (A), 20 μm in (B, C), and 25 μm in (D–F).
	Fig. 7. Reporter gene analyses of pAtPMT2/GFP and pAtPMT2/GUS plants. (A) Inflorescence of a pAtPMT2/GUS plant with strong GUS staining in the mature anthers. (B) Higher magnification of an anther with very strong GUS staining in fully developed pollen grains (arrows) and in germinated and ungerminated pollen on agar medium (insert). Staining of cells in the anther surface results from the diffusion of excess stain out of the pollen grains and can even reach the sepals and petals of stained flowers (see A). (C) GFP-fluorescence (epifluorescence) in pollen grains on an opened anther from a pAtPMT2/GFP plants. No fluorescence is seen in WT anthers (insert). (D) Strong GUS staining in source leaf hydathodes and very weak GUS staining in minor veins (arrows). (E) Cross-section of a flower stalk with GUS staining in young xylem cells (ca, cambium; ph, phloem; xy, xylem). A bar indicates the region where the single-celled row of cambial cells is located. The cambial cells themselves cannot be identified. Bars are 2 mm (A, D), 200 μm (B, insert of C), 20 μm (insert of B), 100 μm (C), 25 μm (E).
	Fig. 8. Characterization of the αAtPMT1/2 antisera in AtPMT1-expressing and AtPMT2-expressing yeast cells. (A) Western-blot (2 μg of protein per lane) of SDS-solubilized, enriched plasma membranes from AtPMT1-expressing yeast cells (s) or from cells expressing the AtPMT1 cDNA in antisense orientation (as). αAtPMT1/2 antisera from two rabbits (R1 and R2) were used at dilutions of 1:5000 and yielded prominent signals only in extracts from AtPMT1-expressing (s) cells. (B) Decoration of AtPMT1 and AtPMT2 proteins at the cell surfaces of AtPMT1-expressing (AtPMT1-s) or AtPMT2-expressing (AtPMT2-s) yeast cells with αAtPMT1/2-R1 (1:1000). An overview with many cells is shown for AtPMT1, a single cell is shown for AtPMT2. No fluorescence was seen in antisense controls (AtPMT1-as and AtPMT2-as). Bars are 50 μm for the AtPMT1 sections and 3 μm for all AtPMT2 sections. (This figure is available in colour at JXB online.)
	Fig. 9. Labelling of young xylem cells with αAtPMT1/2-R1 in Arabidopsis WT plants. (A) Epifluorescence image showing two vascular bundles of a flower stalk cross-section from an Arabidopsis WT plant (unpurified αAtPMT1/2-R1, 1:1000). (B, C) Vascular bundle of a similar section treated with preimmune serum (1:1000). (B) White-light image and (C) epifluorescence image. (D, E) White light merged with epifluorescence image (D) and epifluorescence image alone (E) of a similar image treated with affinity-purified αAtPMT1/2-R1 (1:5). Boxed regions in (A) and (E) show typical stacks of cells. Autofluorescence in (A), (C), and (D) results from autofluorescence of cell wall phenolics (ph, phloem; xy, xylem). Bars are 50 μm. (This figure is available in colour at JXB online.)
	Fig. 10. Characterization of T-DNA and antisense lines. (A) Genomic organization of the Atpmt1-1 mutant allele (SALK_035269) that carries a T-DNA insertion in the 5′-flanking region (grey) 559 nucleotides upstream from the start ATG. Arrows show the position of the primers used for the characterization of the mutant. The upper right corner shows RT-PCR analyses with cDNAs from three homozygous Atpmt1-1 plants and from three WT plants. AtPMT1 bands can be amplified from the Atpmt1-1 and from the WT cDNAs. Amplified bands of ACTIN2 (ACT2) are shown as controls. (B) Representative RT-PCR analysis of five independent AtPMT1/2-antisense lines (1–5) and of a WT plant. Transcripts of ACT2, AtPMT1, and AtPMT2 were amplified with gene-specific primers (S, molecular weight standard). Only the AtPMT2 transcript is absent from all AtPMT1/2-antisense lines.

Abel, Transient transformation of Arabidopsis leaf protoplasts: a versatile experimental system to study gene expression. 1994, Pubmed

Abel, Transient transformation of Arabidopsis leaf protoplasts: a versatile experimental system to study gene expression. 1994, Pubmed
Aida, The PLETHORA genes mediate patterning of the Arabidopsis root stem cell niche. 2004, Pubmed
Akyildiz, Evolution and function of a cis-regulatory module for mesophyll-specific gene expression in the C4 dicot Flaveria trinervia. 2007, Pubmed
Becker, Changes in voltage activation, Cs+ sensitivity, and ion permeability in H5 mutants of the plant K+ channel KAT1. 1996, Pubmed , Xenbase
Burget, The biosynthesis of L-arabinose in plants: molecular cloning and characterization of a Golgi-localized UDP-D-xylose 4-epimerase encoded by the MUR4 gene of Arabidopsis. 2003, Pubmed
Büttner, The monosaccharide transporter(-like) gene family in Arabidopsis. 2007, Pubmed
Büttner, Monosaccharide transporters in plants: structure, function and physiology. 2000, Pubmed
Canam, Spatial and temporal expression profiling of cell-wall invertase genes during early development in hybrid poplar. 2008, Pubmed
Clough, Floral dip: a simplified method for Agrobacterium-mediated transformation of Arabidopsis thaliana. 1998, Pubmed
Conde, Utilization and transport of mannitol in Olea europaea and implications for salt stress tolerance. 2007, Pubmed
Davis, Biosynthesis of Sucrose and Mannitol as a Function of Leaf Age in Celery (Apium graveolens L.). 1988, Pubmed
Dunn, Effects of the modification of transfer buffer composition and the renaturation of proteins in gels on the recognition of proteins on Western blots by monoclonal antibodies. 1986, Pubmed
Engelmann, Evolution of the C4 phosphoenolpyruvate carboxylase promoter of the C4 species Flaveria trinervia: the role of the proximal promoter region. 2008, Pubmed
Gao, Cloning, expression, and characterization of sorbitol transporters from developing sour cherry fruit and leaf sink tissues. 2003, Pubmed
Gietz, Improved method for high efficiency transformation of intact yeast cells. 1992, Pubmed
Goetz, Induction of male sterility in plants by metabolic engineering of the carbohydrate supply. 2001, Pubmed
Hanahan, Studies on transformation of Escherichia coli with plasmids. 1983, Pubmed
Haritatos, Identification of phloem involved in assimilate loading in leaves by the activity of the galactinol synthase promoter. 2000, Pubmed
Harper, Biosynthesis of UDP-xylose. Cloning and characterization of a novel Arabidopsis gene family, UXS, encoding soluble and putative membrane-bound UDP-glucuronic acid decarboxylase isoforms. 2002, Pubmed
Holsters, The functional organization of the nopaline A. tumefaciens plasmid pTiC58. 1980, Pubmed
Jespersen, Dual-function vector for protein expression in both mammalian cells and Xenopus laevis oocytes. 2002, Pubmed , Xenbase
Juchaux-Cachau, Characterization of AgMaT2, a plasma membrane mannitol transporter from celery, expressed in phloem cells, including phloem parenchyma cells. 2007, Pubmed
Klepek, Arabidopsis POLYOL TRANSPORTER5, a new member of the monosaccharide transporter-like superfamily, mediates H+-Symport of numerous substrates, including myo-inositol, glycerol, and ribose. 2005, Pubmed , Xenbase
Laemmli, Cleavage of structural proteins during the assembly of the head of bacteriophage T4. 1970, Pubmed
Moore, Spatial organization of the assembly pathways of glycoproteins and complex polysaccharides in the Golgi apparatus of plants. 1991, Pubmed
Noiraud, Identification of a mannitol transporter, AgMaT1, in celery phloem. 2001, Pubmed
Peña, Arabidopsis irregular xylem8 and irregular xylem9: implications for the complexity of glucuronoxylan biosynthesis. 2007, Pubmed
Ramsperger-Gleixner, Differential expression of sucrose transporter and polyol transporter genes during maturation of common plantain companion cells. 2004, Pubmed , Xenbase
Reinders, Analysis of transport activity of Arabidopsis sugar alcohol permease homolog AtPLT5. 2005, Pubmed , Xenbase
Sauer, Primary structure, genomic organization and heterologous expression of a glucose transporter from Arabidopsis thaliana. 1990, Pubmed
Sauer, SUC1 and SUC2: two sucrose transporters from Arabidopsis thaliana; expression and characterization in baker's yeast and identification of the histidine-tagged protein. 1994, Pubmed
Schmitt, Immunolocalization of solanaceous SUT1 proteins in companion cells and xylem parenchyma: new perspectives for phloem loading and transport. 2008, Pubmed
Schneider, Arabidopsis INOSITOL TRANSPORTER4 mediates high-affinity H+ symport of myoinositol across the plasma membrane. 2006, Pubmed , Xenbase
Schneider, Arabidopsis INOSITOL TRANSPORTER2 mediates H+ symport of different inositol epimers and derivatives across the plasma membrane. 2007, Pubmed , Xenbase
Schneidereit, Functional characterization and expression analyses of the glucose-specific AtSTP9 monosaccharide transporter in pollen of Arabidopsis. 2003, Pubmed
Scholz-Starke, AtSTP6, a new pollen-specific H+-monosaccharide symporter from Arabidopsis. 2003, Pubmed
Stadler, Expression of GFP-fusions in Arabidopsis companion cells reveals non-specific protein trafficking into sieve elements and identifies a novel post-phloem domain in roots. 2005, Pubmed
Stadler, Phloem Loading by the PmSUC2 Sucrose Carrier from Plantago major Occurs into Companion Cells. 1995, Pubmed
Watari, Identification of sorbitol transporters expressed in the phloem of apple source leaves. 2004, Pubmed
Wesley, Construct design for efficient, effective and high-throughput gene silencing in plants. 2001, Pubmed
Wieczorke, Concurrent knock-out of at least 20 transporter genes is required to block uptake of hexoses in Saccharomyces cerevisiae. 1999, Pubmed
Ylstra, Hexose transport in growing petunia pollen tubes and characterization of a pollen-specific, putative monosaccharide transporter. 1998, Pubmed