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The promoter of LE-ACS7, an early flooding-induced 1-aminocyclopropane-1-carboxylate synthase gene of the tomato, is tagged by a Sol3 transposon.

ABSTRACT: Many terrestrial plants respond to flooding with enhanced ethylene production. The roots of flooded plants produce 1-aminocyclopropane-1-carboxylic acid (ACC), which is transported from the root to the shoot, where it is converted to ethylene. In the roots, ACC is synthesized by ACC synthase, which is encoded by a multigene family. Previously, we identified two ACC synthase genes of tomato that are involved in flooding-induced ethylene production. Here, we report the cloning of LE-ACS7, a new tomato ACC synthase with a role early during flooding but also in the early wound response of leaves. The promoter of LE-ACS7 is tagged by a Sol3 transposon. A Sol3 transposon is also present in the tomato polygalacturonase promoter to which it conferred regulatory elements. Thus, Sol3 transposons may affect the regulation of LE-ACS7 and may be involved in the communication between the root and the shoot of waterlogged tomato plants.

SUBMITTER: Shiu OY

PROVIDER: S-EPMC21509 | biostudies-literature | 1998 Aug

REPOSITORIES: biostudies-literature

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The promoter of LE-ACS7, an early flooding-induced 1-aminocyclopropane-1-carboxylate synthase gene of the tomato, is tagged by a Sol3 transposon.

Shiu O Y OY Oetiker J H JH Yip W K WK Yang S F SF

Proceedings of the National Academy of Sciences of the United States of America 19980801 17

Many terrestrial plants respond to flooding with enhanced ethylene production. The roots of flooded plants produce 1-aminocyclopropane-1-carboxylic acid (ACC), which is transported from the root to the shoot, where it is converted to ethylene. In the roots, ACC is synthesized by ACC synthase, which is encoded by a multigene family. Previously, we identified two ACC synthase genes of tomato that are involved in flooding-induced ethylene production. Here, we report the cloning of LE-ACS7, a new to ...[more]

PMID: 9707648

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Project description:Degenerate oligonucleotide primers corresponding to conserved regions flanking the active-site domain of 1-aminocyclopropane-1-carboxylate (ACC) synthase (EC 4.4.1.14) were used for the polymerase chain reaction (PCR) to amplify DNA fragments from mRNA isolated from tomato fruit and tomato suspension cell culture. Antibodies raised against two conserved peptide sequences (TNPSNPLGTT and SLSKDLGLPGFRVG) were used to screen for positive colonies, after the PCR products were cloned into a Bluescript plasmid and expressed in Escherichia coli. Four distinct cDNA fragments encoding ACC synthase homologs were isolated. While pBTAS1 and pBTAS4 were obtained from fruit mRNA, cell culture mRNA yielded three sequences, pBTAS1, pBTAS2, and pBTAS3. Sequencing of these gene fragments revealed that pBTAS1 and pBTAS4 were identical to those full-length sequences previously reported by Van Der Straeten et al. [Van Der Straeten, D., Van Wiemeersch, L., Goodman, H. & Van Montague, M. (1990) Proc. Natl. Acad. Sci. USA 87, 4859-4863] and Olson et al. [Olson, D. C., White, J. A., Edelman, J., Harkin, R. N. & Kende, H. (1991) Proc. Natl. Acad. Sci. USA 88, 5340-5344] from tomato fruit, whereas pBTAS2 and pBTAS3 represent new sequences. Ribonuclease protection assays were used to examine the expression of these transcripts under three different conditions of enhanced ethylene production--namely, during fruit ripening, in response to mechanical wounding in fruit tissue, and auxin stimulation in vegetative tissue. Transcripts of pBTAS1 accumulated massively during ripening and wounding but only slightly in response to auxin treatment. Although pBTAS4 was associated with fruit ripening, it was unresponsive to auxin treatment in vegetative tissue. In contrast, the expression of pBTAS2 and pBTAS3 was greatly promoted in auxin-treated vegetative tissue but was absent from fruit tissue. While the expression of pBTAS2 was moderately dependent on wounding, pBTAS3 was unresponsive to wounding. These data support the view that ACC synthase is encoded by a multigene family and that the members are differentially expressed in response to developmental, environmental, and hormonal factors.

Project description:1-Aminocyclopropane-1-carboxylate (ACC) oxidase catalyses the final step in the biosynthesis of the plant hormone ethylene. The successful overexpression and characterization of active ACC oxidase from tomato has been achieved. PCR was used to insert the corrected cDNA coding for the tomato ACC oxidase into the pET-11a expression vector. Cloning of the resultant construct in Escherichia coli BL21(DE3)pLysE gave transformants which expressed ACC oxidase at levels greater than 30% of soluble protein under optimized conditions. When induced by addition of isopropyl-beta-D-thiogalactopyranoside (IPTG) at 37 degrees C the ACC oxidase expressed was less soluble and less active than when induced at 27 degrees C. The enzyme was purified to near homogeneity by a three-step chromatographic procedure. The specific activity of the purified recombinant ACC oxidase was typically 1.3-1.9 mol of ethylene/mol of enzyme per min, higher than values reported for native enzyme. Like the native enzyme it displayed a requirement for ferrous iron and ascorbate, and CO2 was an activator. The ability to discriminate between racemic diastereomers of 1-amino-2-ethyl cyclopropane-1-carboxylic acid was demonstrated. The enzyme was found to have a loose specificity for ascorbate, showing apparent preference for D-ascorbate and 5,6-O-isopropylidene L-ascorbate rather than L-ascorbate. The addition of catalase, dithiothreitol and BSA to incubation mixtures all resulted in significant increases in activity. When treated with diethylpyrocarbonate (DEPC) under mildly acidic conditions, the enzyme rapidly lost activity. Comparison of the rate of inactivation with the increase in absorbance at 240 nm gave results consistent with the modification of two to three histidine residues at the active site, although the possibility of additional modification of other nucleophilic residues cannot be excluded. Inactivation was largely prevented by the addition of substrates and ferrous iron, implying that DEPC treatment results in the modification of active-site histidines, which act as ligands for ferrous iron. CO2 offered no protection against DEPC inactivation, either in the absence or presence of substrates and/or ferrous iron.

Project description:Ethylene has impact on several physiological plant processes, including abscission, during which plants shed both their vegetative and reproductive organs. Cell separation and programmed cell death are involved in abscission, and these have also been correlated with ethylene action. However, the detailed spatiotemporal pattern of the molecular events during abscission remains unknown. We examined the expression of two tomato ACO genes, LeACO1, and LeACO4 that encode the last enzyme in ethylene biosynthesis, 1-aminocyclopropane-1-carboxylate oxidase (ACO), together with the expression of other abscission-associated genes involved in cell separation and programmed cell death, during a period of 0-12 h after abscission induction in the tomato flower pedicel abscission zone and nearby tissues. In addition, we determined their localization in specific cell layers of the flower pedicel abscission zone and nearby tissues obtained by laser microdissection before and 8 h after abscission induction. The expression of both ACO genes was localized to the vascular tissues in the pedicel. While LeACO4 was more uniformly expressed in all examined cell layers, the main expression site of LeACO1 was in cell layers just outside the abscission zone in its proximal and distal part. We showed that after abscission induction, ACO1 protein was synthesized in phloem companion cells, in which it was localized mainly in the cytoplasm. Samples were additionally treated with 1-methylcyclopropene (1-MCP), a competitive inhibitor of ethylene actions, and analyzed 8 h after abscission induction. Cell-layer-specific changes in gene expression were observed together with the specific localization and ethylene sensitivity of the hallmarks of cell separation and programmed cell death. While treatment with 1-MCP prevented separation of cells through inhibition of the expression of polygalacturonases, which are the key enzymes involved in degradation of the middle lamella, this had less impact on the occurrence of different kinds of membrane vesicles and abscission-related programmed cell death. In the flower pedicel abscission zone, the physical progressions of cell separation and programmed cell death are perpendicular to each other and start in the vascular tissues.

Dataset Information

The promoter of LE-ACS7, an early flooding-induced 1-aminocyclopropane-1-carboxylate synthase gene of the tomato, is tagged by a Sol3 transposon.

Publications

The promoter of LE-ACS7, an early flooding-induced 1-aminocyclopropane-1-carboxylate synthase gene of the tomato, is tagged by a Sol3 transposon.

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