Characterization of Source- and Sink-Specific Sucrose/H+ Symporters from Carrot

To understand how sucrose (Suc) is transported from source leaves to developing tap roots of carrot (Daucus carota L.), we cloned two cDNAs (DcSUT1 and DcSUT2) for proteins with homologies to plant Suc/H+ symporters. The deduced polypeptide sequences are 52% identical and have 12 predicted membrane-spanning domains each. Transport activities were confirmed by expression of the clones in yeast cells. Both transporters had optimal activity below pH 5.0 and Michaelis constant values of 0.5 mm. Suc uptake was inhibited by protonophores, suggesting that Suc transport is linked to the proton electrochemical potential across the plasma membrane. DcSUT1 and DcSUT2 had markedly different expression patterns. Transcripts of DcSUT1 were found only in the green parts of plants, with highest levels in the lamina of source leaves, indicating that DcSUT1 is required for the loading of Suc into the phloem. In leaf lamina expression was diurnally regulated, suggesting that Suc export from the leaves is higher during the day than during the night. The mRNA of DcSUT2 was found mainly in sink organs, and no diurnal expression pattern was detected in the storage root. Here, expression was not restricted to the phloem but was much higher in storage parenchyma tissues of phloem and xylem. The close relationship of DcSUT2 with a Suc/H+ symporter from fava bean, which facilitates Suc uptake into the cotyledons of developing seeds, indicates that this carrot Suc transporter may be involved in loading Suc into storage parenchyma cells.

Phosphoglycerylethanolamine Posttranslational Modification of Plant Eukaryotic Elongation Factor 1α1

Eukaryotic elongation factor 1α (eEF-1A) is a multifunctional protein. There are three known posttranslational modifications of eEF-1A that could potentially affect its function. Except for phosphorylation, the other posttranslational modifications have not been demonstrated in plants. Using matrix-assisted laser desorption/ionization-mass spectrometry and peptide mass mapping, we show that carrot (Daucus carota L.) eEF-1A contains a phosphoglycerylethanolamine (PGE) posttranslational modification. eEF-1A was the only protein labeled with [14C]ethanolamine in carrot cells and was the predominant ethanolamine-labeled protein in Arabidopsis seedlings and tobacco (Nicotiana tabacum L.) cell cultures. In vivo-labeling studies using [3H]glycerol, [32P]Pi, [14C]myristic acid, and [14C]linoleic acid indicated that the entire phospholipid phosphatidylethanolamine is covalently attached to the protein. The PGE lipid modification did not affect the partitioning of eEF-1A in Triton X-114 or its actin-binding activity in in vitro assays. Our in vitro data indicate that this newly characterized posttranslational modification alone does not affect the function of eEF-1A. Therefore, the PGE lipid modification may work in combination with other posttranslational modifications to affect the distribution and the function of eEF-1A within the cell.

Ethylene-Mediated Phospholipid Catabolic Pathway in Glucose-Starved Carrot Suspension Cells1

Glucose (Glc) starvation of suspension-cultured carrot (Daucus carota L.) cells resulted in sequential activation of phospholipid catabolic enzymes. Among the assayed enzymes involved in the degradation, phospholipase D (PLD) and lipolytic acyl hydrolase were activated at the early part of starvation, and these activities were followed by β-oxidation and the glyoxylate cycle enzymes in order. The activity of PLD and lipolytic acyl hydrolase was further confirmed by in vivo-labeling experiments. It was demonstrated that Glc added to a medium containing starving cells inhibited the phospholipid catabolic activities, indicating that phospholipid catabolism is negatively regulated by Glc. There was a burst of ethylene production 6 h after starvation. Ethylene added exogeneously to a Glc-sufficient medium activated PLD, indicating that ethylene acts as an element in the signal transduction pathway leading from Glc depletion to PLD activation. Activation of lipid peroxidation, suggestive of cell death, occurred immediately after the decrease of the phospholipid degradation, suggesting that the observed phospholipid catabolic pathway is part of the metabolic strategies by which cells effectively survive under Glc starvation.

Metabolism of Polyamines in Transgenic Cells of Carrot Expressing a Mouse Ornithine Decarboxylase cDNA1

The metabolisms of arginine (Arg), ornithine (Orn), and putrescine were compared in a nontransgenic and a transgenic cell line of carrot (Daucus carota L.) expressing a mouse Orn decarboxylase cDNA. [14C]Arg, [14C]Orn, and [14C]putrescine were fed to cells and their rates of decarboxylation, uptake, metabolism into polyamines, and incorporation into acid-insoluble material were determined. Transgenic cells showed higher decarboxylation rates for labeled Orn than the nontransgenic cells. This was correlated positively with higher amounts of labeled putrescine production from labeled Orn. With labeled Arg, both the transgenic and the nontransgenic cells exhibited similar rates of decarboxylation and conversion into labeled putrescine. When [14C]putrescine was fed, higher rates of degradation were observed in transgenic cells as compared with the nontransgenic cells. It is concluded that (a) increased production of putrescine via the Orn decarboxylase pathway has no compensatory effects on the Arg decarboxylase pathway, and (b) higher rates of putrescine production in the transgenic cells are accompanied by higher rates of putrescine conversion into spermidine and spermine as well as the catabolism of putrescine.

Natural Genetic Transformation by Agrobacterium rhizogenes : Annual Flowering in Two Biennials, Belgian Endive and Carrot

Genetic transformation of Belgian endive (Cichorium intybus) and carrot (Daucus carota) by Agrobacterium rhizogenes resulted in a transformed phenotype, including annual flowering. Back-crossing of transformed (R1) endive plants produced a line that retained annual flowering in the absence of the other traits associated with A. rhizogenes transformation. Annualism was correlated with the segregation of a truncated transferred DNA (T-DNA) insertion. During vegetative growth, carbohydrate reserves accumulated normally in these annuals, and they were properly mobilized prior to anthesis. The effects of individual root-inducing left-hand T-DNA genes on flowering were tested in carrot, in which rolC (root locus) was the primary promoter of annualism and rolD caused extreme dwarfism. We discuss the possible adaptive significance of this attenuation of the phenotypic effects of root-inducing left-hand T-DNA.

Expression Pattern of the Carrot EP3 Endochitinase Genes in Suspension Cultures and in Developing Seeds1

Carrot (Daucus carota) extracellular protein 3 (EP3) class IV endochitinases were previously identified based on their ability to rescue somatic embryos of the temperature-sensitive cell line ts11. Whole-mount in situ hybridization revealed that a subset of the morphologically distinguishable cell types in embryogenic and nonembryogenic suspension cultures, including ts11, express EP3 genes. No expression was found in somatic embryos. In carrot plants EP3 genes are expressed in the inner integumentary cells of young fruits and in a specific subset of cells located in the middle of the endosperm of mature seeds. No expression was found in zygotic embryos. These results support the hypothesis that the EP3 endochitinase has a “nursing” function during zygotic embryogenesis and that this function can be mimicked by suspension cells during somatic embryogenesis.

Intracellular pH in Arbuscular Mycorrhizal Fungi1 : A Symbiotic Physiological Marker

A method was developed to perform real-time analysis of cytosolic pH of arbuscular mycorrhizal fungi in culture using dye and ratiometric measurements (490/450 nm excitations). The study was mainly performed using photometric analysis, although some data were confirmed using image analysis. The use of nigericin allowed an in vivo calibration. Experimental parameters such as loading time and concentration of the dye were determined so that pH measurements could be made for a steady-state period on viable cells. A characteristic pH profile was observed along hyphae. For Gigaspora margarita, the pH of the tip (0–2 μm) was typically 6.7, increased sharply to 7.0 behind this region (9.5 μm), and decreased over the next 250 μm to a constant value of 6.6. A similar pattern was obtained for Glomus intraradices. The pH profile of G. margarita germ tubes was higher when cultured in the presence of carrot (Daucus carota) hairy roots (nonmycorrhizal). Similarly, extraradical hyphae of G. intraradices had a higher apical pH than the germ tubes. The use of a paper layer to prevent the mycorrhizal roots from being in direct contact with the medium selected hyphae with an even higher cytosolic pH. Results suggest that this method could be useful as a bioassay for studying signal perception and/or H+ cotransport of nutrients by arbuscular mycorrhizal hyphae.