SummaryIn order to understand sucrose transport in developing seeds of cereals at the molecular level, we cloned from a caryopses library two cDNAs encoding sucrose transporters, designated HvSUT1 and HvSUT2. Sucrose uptake activity was con®rmed by heterologous expression in yeast. Both transporter genes are expressed in maternal as well as ®lial tissues. In a series of in situ hybridizations we analysed the cell type-speci®c expression in developing seeds. HvSUT1 is preferentially expressed in caryopses in the cells of the nucellar projection and the endospermal transfer layer, which represent the sites of sucrose exchange between the maternal and the ®lial generation and are characterized by transfer cell formation. HvSUT2 is expressed in all sink and source tissues analysed and may have a general housekeeping role. The rapid induction of HvSUT1 gene expression in caryopses at approximately 5±6 days after fertilization coincides with increasing levels of sucrose as well as sucrose synthase mRNA and activity, and occurs immediately before the onset of rapid starch accumulation within the endosperm. Starch biosynthesis requires sucrose to be imported into the endosperm, as direct precursor for starch synthesis and to promote storage-associated processes. We discuss the possible role of HvSUT1 as a control element for the endospermal sucrose concentration.

Plant seeds prepare for germination already during seed maturation. We performed a detailed transcriptome analysis of barley (Hordeum vulgare) grain maturation, desiccation, and germination in two tissue fractions (starchy endosperm/aleurone and embryo/scutellum) using the Affymetrix Barley1 GeneChip. To aid data evaluation, Arabidopsis thaliana MapMan and PageMan tools were adapted to barley. The analyses allow a number of conclusions: (1) Cluster analysis revealed a smooth transition in transcription programs between late seed maturation and germination within embryo tissues, but not in the endosperm/aleurone. (2) More than 12,000 transcripts are stored in the embryo of dry barley grains, many of which are presumably activated during germination. (3) Transcriptional activation of storage reserve mobilization events occurs at an early stage of germination, well before radicle protrusion. (4) Key genes of gibberellin (GA) biosynthesis are already active during grain maturation at a time when abscisic acid peaks suggesting the formation of an endogenous store of GA in the aleurone. This GA probably acts later during germination in addition to newly synthesized GA. (5) Beside the well-known role of GA in gene activation during germination spatiotemporal expression data and cis-element searches in homologous rice promoters confirm an equally important gene-activating role of abscisic acid during this developmental period. The respective regulatory webs are linked to auxin and ethylene controlled networks. In summary, new bioinformatics PageMan and MapMan tools developed in barley have been successfully used to investigate in detail the transcriptome relationships between seed maturation and germination in an important crop plant.

The vacuole is the main cellular storage pool, where sucrose (Suc) accumulates to high concentrations. While a limited number of vacuolar membrane proteins, such as V-type H 1 -ATPases and H 1 -pyrophosphatases, are well characterized, the majority of vacuolar transporters are still unidentified, among them the transporter(s) responsible for vacuolar Suc uptake and release. In search of novel tonoplast transporters, we used a proteomic approach, analyzing the tonoplast fraction of highly purified mesophyll vacuoles of the crop plant barley (Hordeum vulgare). We identified 101 proteins, including 88 vacuolar and putative vacuolar proteins. The Suc transporter (SUT) HvSUT2 was discovered among the 40 vacuolar proteins, which were previously not reported in Arabidopsis (Arabidopsis thaliana) vacuolar proteomic studies. To confirm the tonoplast localization of this Suc transporter, we constructed and expressed green fluorescent protein (GFP) fusion proteins with HvSUT2 and its closest Arabidopsis homolog, AtSUT4. Transient expression of HvSUT2-GFP and AtSUT4-GFP in Arabidopsis leaves and onion (Allium cepa) epidermal cells resulted in green fluorescence at the tonoplast, indicating that these Suc transporters are indeed located at the vacuolar membrane. Using a microcapillary, we selected mesophyll protoplasts from a leaf protoplast preparation and demonstrated unequivocally that, in contrast to the companion cell-specific AtSUC2, HvSUT2 and AtSUT4 are expressed in mesophyll protoplasts, suggesting that HvSUT2 and AtSUT4 are involved in transport and vacuolar storage of photosynthetically derived Suc.In mature plant cells, the central vacuole occupies 80% to 90% of the cell volume. Vacuoles contain a large number of hydrolytic and biosynthetic enzymes, inorganic ions, soluble carbohydrates, organic acids, amino acids, secondary compounds, and modified xenobiotics (Maeshima, 2001;Martinoia et al., 2002). Based on the potential toxicity of many of these compounds, Matile (1984) suggested that the distance between life and death is 7.5 nm, the thickness of the vacuolar membrane. Plants have only a limited capacity to excrete potentially toxic compounds; therefore, the term internal excretion has also been used (Martinoia et al., 1993) to indicate that, for some classes of compounds, the vacuolar membrane mimics the function and contains homolog transporters of the liver plasma membrane (Kreuz et al., 1996). However, the function of the vacuole is not restricted to the storage of potentially toxic compounds. For optimal function of the metabolic pathways, the concentration of metabolites and ions has to be tightly regulated in the cytoplasm. Metabolites produced in excess are transported into the vacuole, which serves as a temporary storage pool, and released to the cytoplasm when required for metabolism.Increasing evidence shows that impaired vacuolar deposition or retrieval affects plant metabolism. Catala et al. (2003) showed that the vacuolar calcium-proton exchanger CAX1 is induced during cold treatment and is invo...