Franz Klebl

Arabidopsis POLYOL TRANSPORTER5, a New Member of the Monosaccharide Transporter-Like Superfamily, Mediates H+-Symport of Numerous Substrates, Including myo-Inositol, Glycerol, and Ribose

Six genes of the Arabidopsis thaliana monosaccharide transporter-like (MST-like) superfamily share significant homology with polyol transporter genes previously identified in plants translocating polyols (mannitol or sorbitol) in their phloem (celery [Apium graveolens], common plantain [Plantago major], or sour cherry [Prunus cerasus]). The physiological role and the functional properties of this group of proteins were unclear in Arabidopsis, which translocates sucrose and small amounts of raffinose rather than polyols. Here, we describe POLYOL TRANSPORTER5 (AtPLT5), the first member of this subgroup of Arabidopsis MST-like transporters. Transient expression of an AtPLT5–green fluorescent protein fusion in plant cells and functional analyses of the AtPLT5 protein in yeast and Xenopus oocytes demonstrate that AtPLT5 is located in the plasma membrane and characterize this protein as a broad-spectrum H+-symporter for linear polyols, such as sorbitol, xylitol, erythritol, or glycerol. Unexpectedly, however, AtPLT5 catalyzes also the transport of the cyclic polyol myo-inositol and of different hexoses and pentoses, including ribose, a sugar that is not transported by any of the previously characterized plant sugar transporters. RT-PCR analyses and AtPLT5 promoter-reporter gene plants revealed that AtPLT5 is most strongly expressed in Arabidopsis roots, but also in the vascular tissue of leaves and in specific floral organs. The potential physiological role of AtPLT5 is discussed.

A defect in the yeast plasma membrane urea transporter Dur3p is complemented by CpNIP1, a Nod26-like protein from zucchini (Cucurbita pepo L.), and by Arabidopsis thaliana δ-TIP or γ-TIP

Dur3 encodes the yeast plasma membrane urea transporter and Δdur3 mutants are unable to grow on media containing low concentrations of urea as sole nitrogen source. Complementation of the Δdur3 mutant line with expression libraries generated from whole Arabidopsis thaliana seedlings or from zucchini (Cucurbita pepo L.) vascular tissue yielded numerous lines that had regained the capacity to grow on low urea as sole nitrogen source. Analysis of several of these yeast lines revealed that the Δdur3 mutation was complemented either by δ‐TIP (TIP= onoplast ntrinsic rotein) or γ‐TIP from Arabidopsis or by CpNIP1, a new NOD26‐like protein from zucchini. δ‐TIP (At3g16240) and γ‐TIP (At2g36830) had previously been characterized as proteins facilitating the transport of water across the tonoplast membrane, and Nod26‐like proteins were characterized as glycerol transporters. So far, transport of urea has not been described for any of the proteins described in this paper. Further analyses support this function of TIPs and nodulin 26‐like intrinsic proteins in urea transport.

Phloem-Specific Expression of Yang Cycle Genes and Identification of Novel Yang Cycle Enzymes in Plantago and Arabidopsis

This work reports on the identification and characterization of enzymes of the Yang cycle, which recycle 5-methylthioadenosine to Met. It shows that the genes for all Yang cycle enzymes are expressed primarily in the phloem. One of the identified enzymes is a trimeric protein composed of subunits encoded by up to three separate genes in other organisms. The 5-methylthioadenosine (MTA) or Yang cycle is a set of reactions that recycle MTA to Met. In plants, MTA is a byproduct of polyamine, ethylene, and nicotianamine biosynthesis. Vascular transcriptome analyses revealed phloem-specific expression of the Yang cycle gene 5-METHYLTHIORIBOSE KINASE1 (MTK1) in Plantago major and Arabidopsis thaliana. As Arabidopsis has only a single MTK gene, we hypothesized that the expression of other Yang cycle genes might also be vascular specific. Reporter gene studies and quantitative analyses of mRNA levels for all Yang cycle genes confirmed this hypothesis for Arabidopsis and Plantago. This includes the Yang cycle genes 5-METHYLTHIORIBOSE-1-PHOSPHATE ISOMERASE1 and DEHYDRATASE-ENOLASE-PHOSPHATASE-COMPLEX1. We show that these two enzymes are sufficient for the conversion of methylthioribose-1-phosphate to 1,2-dihydroxy-3-keto-5-methylthiopentene. In bacteria, fungi, and animals, the same conversion is catalyzed in three to four separate enzymatic steps. Furthermore, comparative analyses of vascular and nonvascular metabolites identified Met, S-adenosyl Met, and MTA preferentially or almost exclusively in the vascular tissue. Our data represent a comprehensive characterization of the Yang cycle in higher plants and demonstrate that the Yang cycle works primarily in the vasculature. Finally, expression analyses of polyamine biosynthetic genes suggest that the Yang cycle in leaves recycles MTA derived primarily from polyamine biosynthesis.

Common Plantain. A Collection of Expressed Sequence Tags from Vascular Tissue and a Simple and Efficient Transformation Method

The vascular tissue of higher plants consists of specialized cells that differ from all other cells with respect to their shape and size, their organellar composition, their extracellular matrix, the type of their plasmodesmata, and their physiological functions. Intact and pure vascular tissue can be isolated easily and rapidly from leaf blades of common plantain (Plantago major), a plant that has been used repeatedly for molecular studies of phloem transport. Here, we present a transcriptome analysis based on 5,900 expressed sequence tags (ESTs) and 3,247 independent mRNAs from the Plantago vasculature. The vascular specificity of these ESTs was confirmed by the identification of well-known phloem or xylem marker genes. Moreover, reverse transcription-polymerase chain reaction, macroarray, and northern analyses revealed genes and metabolic pathways that had previously not been described to be vascular specific. Moreover, common plantain transformation was established and used to confirm the vascular specificity of a Plantago promoter-β-glucuronidase construct in transgenic Plantago plants. Eventually, the applicability and usefulness of the obtained data were also demonstrated for other plant species. Reporter gene constructs generated with promoters from Arabidopsis (Arabidopsis thaliana) homologs of newly identified Plantago vascular ESTs revealed vascular specificity of these genes in Arabidopsis as well. The presented vascular ESTs and the newly developed transformation system represent an important tool for future studies of functional genomics in the common plantain vasculature.

Vhr1p, a new transcription factor from budding yeast, regulates biotin-dependent expression of VHT1 and BIO5.

Transcription of the Saccharomyces cerevisiae vitamin H transporter gene VHT1 is enhanced by low extracellular biotin. Here we present the identification and characterization of Vhr1p as a transcriptional regulator of VHT1 (VHR1 (YIL056w); VHT1 regulator 1) and the identification of the cis-regulatory target sequences for Vhr1p in two yeast promoters. VHR1 was identified in a complementation screening of mutagenized yeast cells that had lost the capacity to express the gene of the green fluorescent protein (GFP) from the VHT1 promoter. Δvhr1 deletion mutants fail to induce VHT1 on low biotin concentrations. In yeast one-hybrid analyses performed with fusions of Vhr1p N-terminal and C-terminal fragments to the Gal4p activation domain or to the Gal4p DNA-binding domain, the Vhr1p N terminus mediated biotin-dependent DNA binding, and the Vhr1p C terminus triggered biotin-dependent transcriptional activation. The analyzed Vhr1p N-terminal fragment has previously been described as a domain of unknown function (DUF352). Deletion and linker scanning analyses of the VHT1 promoter revealed the palindromic 18-nucleotide sequence AATCA-N8-TGAYT as the vitamin H-responsive element. This sequence was identified also in the BIO5 promoter that is also transcriptionally activated on low biotin concentrations. Bio5p mediates the transport of 7-keto-8-aminopelargonic acid across the yeast plasma membrane, a compound that is used as a precursor in biotin biosynthesis. Δvhr1 deletion mutants fail to induce BIO5 on low biotin concentrations. The presented data characterize Vhr1p as an essential component of the biotin-dependent signal transduction cascade in yeast.

Transcription of the yeast TNA1 gene is not only regulated by nicotinate but also by p-aminobenzoate.

In a recent paper published in FEBS Letters, Llorente and Dujon presented results on the transcriptional regulation of the YLR004c gene and the YGR260w gene of Saccharomyces cerevisiae [1]. Both genes represent members of the DAL5 gene family [2]. Two members of this gene family had previously been shown to encode plasma membrane-localised transporters for the vitamins biotin (the VHT1 gene [3]) and panthotenate (the FEN2 gene [4]). It had been published that the expression levels of VHT1 [3] and THI10, the yeast thiamine permease [5], which is not related to the DAL5 family, are enhanced at low extracellular substrate concentrations. Based on these ¢ndings, Llorente and Dujon postulated that the expression of the ¢ve other, so far uncharacterised members of the DAL5 gene family (YLR004c, YLL055w, YGR260w, YIL166c and YAL067c) might also be modulated by decreased substrate concentrations. Screening for changes in the transcript levels of YLR044c and YGR260w, Llorente and Dujon were able to show that a decrease in the extracellular concentration of thiamine increased the expression of YLR044c and a decrease in the concentration of nicotinate increased the expression of YGR260w [1]. Whereas the function of YLR044c remained unclear, YGR260w could clearly be characterised as a transport protein for nicotinate (vitamin B3) and the gene was named TNA1 [1]. In our laboratory, we had used the same approach to study this question and we obtained identical results, showing enhanced expression of YLR044c at low thiamine and of YGR260w at low nicotinate. As Llorente and Dujon, we were able to describe the protein encoded by YGR260w as a transporter for nicotinate with a KM of 2 WM. In this correspondence, we would like to add some results that were obtained during our attempts to analyse TNA1 transcription and the function of Tna1p. In contrast to the work of Llorente and Dujon, who analysed possible changes in TNA1 transcription in the presence of thiamine, pantothenate, pyridoxine, myo-inositol and biotin [1], we examined also the e¡ect of folate, ribo£avin and paraaminobenzoate (PABA). Whereas reduced concentrations of ribo£avin and folate had no e¡ect on the transcriptional regulation, TNA1 mRNA levels increased strongly at reduced extracellular concentrations of both, nicotinate and PABA (Fig. 1A). A consequent comparison of the structures of nicotinate and PABA (Fig. 1B) revealed the close similarity of these molecules and suggested that Tna1p might be a transporter for both compounds. Therefore, we generated yeast strains overexpressing the TNA1 gene under the control of the PMA1 promotor or missing an intact TNA1 gene due to the insertion of the Schizosaccharomyces pombe HIS5 gene. Analyses of TNA1 mRNA levels in both mutant strains (Fig. 1C) con¢rmed these mutations. Using the TNA1 wild type strain, the TNA1-overexpressing strain and the vtna1 deletion mutant, we analysed the transport of radiolabelled [14C]nicotinate (Fig. 1D) and [14C]PABA (Fig. 1E). Unexpectedly, only the measurements with [14C]nicotinate yielded increased transport rates in the overexpressing strain and an almost total lack of uptake activity in the vtna1 knock-out mutant. In contrast, transport of [14C]PABA was not in£uenced by the overexpression or deletion of TNA1. These results showed that the structurally closely related compounds, nicotinate and PABA, can modulate the expression of the S. cerevisiae TNA1 gene in the same way. However, only one of these molecules, nicotinate, is a substrate for the Tna1p transporter. In fact, Tna1p seems to be highly

Sugar transporter STP7 specificity for L-arabinose and D-xylose contrasts with the typical hexose transporters STP8 and STP12

The hexose transporters STP8 and STP12 may contribute to the nutrition of reproductive tissues, whereas the pentose-specific STP7 might be involved in cell wall sugar recycling in Arabidopsis. The controlled distribution of sugars between assimilate-exporting source tissues and sugar-consuming sink tissues is a key element for plant growth and development. Monosaccharide transporters of the SUGAR TRANSPORT PROTEIN (STP) family contribute to the uptake of sugars into sink cells. Here, we report on the characterization of STP7, STP8, and STP12, three previously uncharacterized members of this family in Arabidopsis (Arabidopsis thaliana). Heterologous expression in yeast (Saccharomyces cerevisiae) revealed that STP8 and STP12 catalyze the high-affinity proton-dependent uptake of glucose and also accept galactose and mannose. STP12 additionally transports xylose. STP8 and STP12 are highly expressed in reproductive organs, where their protein products might contribute to sugar uptake into the pollen tube and the embryo sac. stp8.1 and stp12.1 T-DNA insertion lines developed normally, which may point toward functional redundancy with other STPs. In contrast to all other STPs, STP7 does not transport hexoses but is specific for the pentoses l-arabinose and d-xylose. STP7-promoter-reporter gene plants showed an expression of STP7 especially in tissues with high cell wall turnover, indicating that STP7 might contribute to the uptake and recycling of cell wall sugars. Uptake analyses with radioactive l-arabinose revealed that 11 other STPs are able to transport l-arabinose with high affinity. Hence, functional redundancy might explain the missing-mutant phenotype of two stp7 T-DNA insertion lines. Together, these data complete the characterization of the STP family and present the STPs as new l-arabinose transporters for potential biotechnological applications.

Galactose metabolism and toxicity in Ustilago maydis

In most organisms, galactose is metabolized via the Leloir pathway, which is conserved from bacteria to mammals. Utilization of galactose requires a close interplay of the metabolic enzymes, as misregulation or malfunction of individual components can lead to the accumulation of toxic intermediate compounds. For the phytopathogenic basidiomycete Ustilago maydis, galactose is toxic for wildtype strains, i.e. leads to growth repression despite the presence of favorable carbon sources as sucrose. The galactose sensitivity can be relieved by two independent modifications: (1) by disruption of Hxt1, which we identify as the major transporter for galactose, and (2) by a point mutation in the gene encoding the galactokinase Gal1, the first enzyme of the Leloir pathway. The mutation in gal1(Y67F) leads to reduced enzymatic activity of Gal1 and thus may limit the formation of putatively toxic galactose-1-phosphate. However, systematic deletions and double deletions of different genes involved in galactose metabolism point to a minor role of galactose-1-phosphate in galactose toxicity. Our results show that molecular triggers for galactose toxicity in U. maydis differ from yeast and mammals.

A novel mechanism for target gene-specific SWI/SNF recruitment via the Snf2p N-terminus

Chromatin-remodeling complexes regulate the expression of genes in all eukaryotic genomes. The SWI/SNF complex of Saccharomyces cerevisiae is recruited to its target promoters via interactions with selected transcription factors. Here, we show that the N-terminus of Snf2p, the chromatin remodeling core unit of the SWI/SNF complex, is essential for the expression of VHT1, the gene of the plasma membrane H+/biotin symporter, and of BIO5, the gene of a 7-keto-8-aminopelargonic acid transporter, biotin biosynthetic precursor. chromatin immunoprecipitation (ChIP) analyses demonstrate that Vhr1p, the transcriptional regulator of VHT1 and BIO5 expression, is responsible for the targeting of Snf2p to the VHT1 promoter at low biotin. We identified an Snf2p mutant, Snf2p-R15C, that specifically abolishes the induction of VHT1 and BIO5 but not of other Snf2p-regulated genes, such as GAL1, SUC2 or INO1. We present a novel mechanism of target gene-specific SWI/SNF recruitment via Vhr1p and a conserved N-terminal Snf2p domain.