Sugar concentrations and expression of SUTs suggest active phloem loading in tall trees of Fagus sylvatica and Quercus robur.

Phloem loading and sugar distribution are key steps for carbon partitioning in herbaceous and woody species. Although the phloem loading mechanisms in herbs are well studied, less is known for trees. It was shown for saplings of Fagus sylvatica L. and Quercus robur L. that the sucrose concentration in the phloem sap was higher than in the mesophyll cells, which suggests that phloem loading of sucrose involves active steps. However, the question remains whether this also applies for tall trees. To approach this question, tissue-specific sugar and starch contents of small and tall trees of F. sylvatica and Q. robur as well as the sugar concentration in the subcellular compartments of mesophyll cells were examined. Moreover, sucrose uptake transporters (SUTs) were analyzed by heterology expression in yeast and the tissue-specific expressions of SUTs were investigated. Sugar content in leaves of the canopy (11 and 26 m height) was up to 25% higher compared with that of leaves of small trees of F. sylvatica and Q. robur (2 m height). The sucrose concentration in the cytosol of mesophyll cells from tall trees was between 120 and 240 mM and about 4- to 8-fold lower than the sucrose concentration in the phloem sap of saplings. The analyzed SUT sequences of both tree species cluster into three types, similar to SUTs from other plant species. Heterologous expression in yeast confirmed that all analyzed SUTs are functional sucrose transporters. Moreover, all SUTs were expressed in leaves, bark and wood of the canopy and the expression levels in small and tall trees were similar. The results show that the phloem loading in leaves of tall trees of F. sylvatica and Q. robur probably involves active steps, because there is an uphill concentration gradient for sucrose. SUTs may be involved in phloem loading.

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.

Sugar Transporter STP7 Specificity for l-Arabinose and d-Xylose Contrasts with the Typical Hexose Transporters STP8 and STP12.

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.

Phloem-specific expression of Yang cycle genes and identification of novel Yang cycle enzymes in Plantago and Arabidopsis.

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-beta-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. Deltavhr1 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. Deltavhr1 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.

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.

AtSUC8 and AtSUC9 encode functional sucrose transporters, but the closely related AtSUC6 and AtSUC7 genes encode aberrant proteins in different Arabidopsis ecotypes.

Three members of the Arabidopsis sucrose transporter gene family, AtSUC6-AtSUC8 (At5g43610; At1g66570; At2g14670), share a high degree of sequence homology in their coding regions and even in their introns and in their 5'- and 3'-flanking regions. A fourth sucrose transporter gene, AtSUC9 (At5g06170), which is on the same branch of the AtSUC-phylogenetic tree, shows only slightly less sequence homology. Here we present data demonstrating that two genes from this subgroup, AtSUC6 and AtSUC7, encode aberrant proteins and seem to represent sucrose transporter pseudogenes, whereas AtSUC8 and AtSUC9 encode functional sucrose transporters. These results are based on analyses of splice patterns and polymorphic sites between these genes in different Arabidopsis ecotypes, as well as on functional analyses by cDNA expression in baker's yeast. For one of these genes, AtSUC7 (At1g66570), different, ecotype-specific splice patterns were observed in Wassilewskija (Ws), C24, Columbia wild type (Col-0) and Landsberg erecta (Ler). No incorrect splicing and no sequence polymorphism were detected in the cDNAs of AtSUC8 and AtSUC9, which encode functional sucrose transporters and are expressed in floral tissue. Finally, promoter-reporter gene plants and T-DNA insertion lines were analyzed for AtSUC8 and AtSUC9.

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 delta-TIP or gamma-TIP.

Dur3 encodes the yeast plasma membrane urea transporter and Deltadur3 mutants are unable to grow on media containing low concentrations of urea as sole nitrogen source. Complementation of the Deltadur3 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 Deltadur3 mutation was complemented either by delta-TIP (TIP=tonoplast intrinsic protein) or gamma-TIP from Arabidopsis or by CpNIP1, a new NOD26-like protein from zucchini. delta-TIP (At3g16240) and gamma-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.