Cloning and characterization of a coronatine-regulated tyrosine aminotransferase from Arabidopsis.

In plants, the phytotoxin coronatine, which is an analog of the octadecanoids 12-oxo-phytodienoic acid and/or jasmonic acid, gives rise to a number of physiological responses similar to those of octadecanoids. To further elucidate the physiological role of these compounds, the differential RNA display technique was used to isolate a number of novel octadecanoid-inducible genes expressed in coronatine-treated Arabidopsis. Among these, a cDNA clone was identified that was similar to known tyrosine aminotransferases (TATs). The function was verified with the expressed recombinant protein. In Arabidopsis, the protein is present as a multimer of 98 kD, with a monomer of an apparent molecular mass of 47 kD. TAT mRNA could be induced within 2 h by various octadecanoids and by wounding of the plants. Accumulation of the TAT protein and a 5- to 7-fold increase in its enzymatic activity was observed 7 to 9 h after application of octadecanoids, coronatine, or wounding. The potential role of TAT in the defense response to herbivores and pathogens is discussed.

The Closed Equilibrated Biological Aquatic System: general concept and aspects of botanical research.

The Closed Equilibrated Biological Aquatic System (CEBAS) consists of four subcomponents which form a closed (artificial) aquatic ecosystem initially designed to study the long-term influence of space conditions on several successive generations of aquatic organisms. Teleost fishes and water snails in the zoological component produce CO2 ammonium ions and waste compounds which can be utilized after ammonium is oxidised in a microbial component by the botanical component consisting of a rootless, aquatic higher plant species which eliminates ions, i.e. nitrate, and produces oxygen for animal respiration. An electronic component serves as a data-acquisition and regulation device for temperature and oxygen-dependent illumination of the plant chamber. A comprehensive interdisciplinary research programme, focused around the CEBAS, is especially well developed in the field of zoology. It covers a ground laboratory and preparations for two scheduled space flight projects, as well as aspects of combined animal-plant food production modules for human nutrition in bioregenerative space life-support systems and for terrestrial production sites. In the botanical research programme, morphological investigations on Ceratophyllum demersum L. performed with light and electron microscopy have demonstrated a gas lacuna system which, in addition to starch grains in the plastids, might regulate the buoyancy of the plant and/or serve as a 'gas skeleton'. Also, a remarkable symmetry in the arrangement of tissues was observed in stems and older leaves. The photosynthetic capacities of Ceratophyllum in the CEBAS-MINI MODULE proved to be more than sufficient for life support, and experiments on nitrate uptake into the plants showed their capacity to utilize ions from the water.

Cloning, molecular and functional characterization of Arabidopsis thaliana allene oxide synthase (CYP 74), the first enzyme of the octadecanoid pathway to jasmonates.

Allene oxide synthase, an enzyme of the octadecanoid pathway to jasmonates, was cloned from Arabidopsis thaliana as a full-length cDNA encoding a polypeptide of 517 amino acids with a calculated molecular mass of 58705 Da. From the sequence, an N-terminal transit peptide of 21 amino acids resembling chloroplast transit peptides was deduced. Three out of four invariant amino acid residues of cytochrome P450 heme-binding domains are conserved and properly positioned in the enzyme coding region, including the heme-accepting cysteine (Cys-470). Southern analysis indicated in A. thaliana only one allene oxide synthase gene to be present. While transcript levels were rapidly and transiently induced after wounding of the leaves, allene oxide synthase activity remained nearly constant at a low level of ca. 0.8 nkat per mg of protein. The cDNA encoding A. thaliana allene oxide synthase was highly expressed in bacteria giving rise to a polypeptide of the calculated molecular mass. The protein was enzymatically active, and verification of the reaction products by GC-MS showed that it was capable of utilizing not only 13-hydroperoxylinolenic acid (13-hydroperoxy-9(Z), 11(E), 15(Z)-octadecatrienoic acid), but also 13-hydroperoxylinoleic acid (13-hydroperoxy-9(Z), 11(E)-octadecadienoic acid) as substrate. The data suggest parallel pathways to jasmonates from linolenic acid or linoleic acid in A. thaliana.

Glyphosate tolerance of cultured Corydalis sempervirens cells is acquired by an increased rate of transcription of 5-enolpyruvylshikimate 3-phosphate synthase as well as by a reduced turnover of the enzyme.

Cell cultures of Corydalis sempervirens, tolerant to the herbicide glyphosate, have a 30-40-fold increased level of the herbicide's target enzyme 5-enolpyruvylshikimate 3-phosphate (EPSP) synthase, a ten-fold enhanced level of the corresponding mRNA but no amplification of the gene (Holländer-Czytko et al., Plant Mol Biol 11 (1988) 215-220). The increase at the transcriptional level is due to a higher rate of transcription of the gene, which was observed in run-off transcription assays with isolated nuclei. The further amplification at the protein level is the result of stabilization of the enzyme by the herbicide. In the presence of glyphosate the half-life of EPSP synthase was doubled leading to higher levels of both protein and enzyme activity. Overproduction of the enzyme in adapted cultures is stable at the transcriptional level, as cells from adapted cultures grown in the absence of glyphosate for three years still display an about ten-fold higher enzyme activity and transcript level than non-adapted cultures.

Molecular basis for the overproduction of 5-enolpyruvylshikimate 3-phosphate synthase in a glyphosate-tolerant cell suspension culture of Corydalis sempervirens.

Cell cultures of Corydalis sempervirens adapted to growth in the presence of 5 mM glyphosate [N-(phosphonomethyl)glycine] display a 30- to 40-fold increase in the cellular content of 5-enolpyruvylshikimic acid 3-phosphate (EPSP) synthase, the target enzyme of the herbicide. Translatable mRNA activity as well as transcript levels for EPSP synthase were increased 8-to 12-fold in the adapted (glyphosate-tolerant) as compared to the non-adapted (glyphosate-sensitive) cultures. Northern blot analysis revealed a single 1.8 kb transcript after hybridization with an oligonucleotide probe deduced from the N-terminal amino acid sequence of the enzyme. No significant differences in the relative abundance of EPSP synthase-specific DNA sequences could be detected, however, in Southern and dot blot analyses of restricted DNA isolated from the two cultures. We conclude that the overproduction of EPSP synthase in glyphosate-tolerant C. sempervirens cells is not based on the amplification of the corresponding gene.

5-enolpyruvylshikimate 3-phosphate synthase, the target enzyme of the herbicide glyphosate, is synthesized as a precursor in a higher plant.

Cell cultures of Corydalis sempervirens adapted to growth in the presence of 5 millimolar glyphosate overproduce the herbicide's target enzyme, 5-enolpyruvylshikimate 3-phosphate (EPSP) synthase, 30- to 40-fold. In vitro translation of total RNA and poly(A)-RNA coupled with immunoprecipitation showed that the protein is synthesized as a precursor of relative molecular weight (M(r)) 53900 +/- 900 as compared to M(r) 45500 +/- 1000 of the mature enzyme. Translatable activity of mRNA for EPSP-synthase in glyphosate-adapted cultures is tenfold higher than in nonadapted cultures.

Vacuolar localization of wound-induced carboxypeptidase inhibitor in potato leaves.

The wound-induced carboxypeptidase inhibitor in potato leaves was shown to be localized in the central vacuoles of the cells. The inhibitor was quantified by immunological assays (ELISA) in protoplasts and vacuoles isolated from upper unwounded leaves of 5- to 6-week old potato plants that had been wounded on their lower leaves 48 hours earlier to induce the accumulation of the carboxypeptidase inhibitor. The regulation of the synthesis and compartmentation of the inhibitor is similar to that of wound-induced serine proteinase Inhibitors I and II in potato and tomato leaves and appears to be part of an induced defense response against attacking pests.

Wound signals in plants: A systemic plant wound signal alters plasma membrane integrity.

Within 4 hr after wounding the lower leaves of young potato and tomato plants, a rapid and remarkable change is induced in the cells of upper undamaged leaves that results in extensive lysis of protoplasts during their isolation. Protoplast yields from unwounded upper leaves, 4 hr after wounding a lower leaf by crushing with a hemostat, decreased 25% below yields from leaves of unwounded plants. From 8 to >20 hr after wounding, protoplast yields were less than half of those from control plants. Multiple woundings decreased yields even further, as did chewing of the lower leaves by tobacco hornworms over a period of several minutes. In addition, within 4 hr of excising young tomato plants at their base with a razor blade, a 90% decrease in leaf protoplast yields was recorded. The major loss of protoplasts induced by wounding was primarily due to an increased cell lysis during protoplast isolation. Cell lysis was apparently due to a weakened cell membrane, because newly recovered protoplasts released from leaves of wounded plants were extremely fragile and exhibited 70% lysis during low speed centrifugation, compared to 20% lysis of protoplasts recovered from control plants. We conclude that a signal is released by wounding that is rapidly transmitted or transported through the plants to induce a profound change in the leaf cell membranes that renders them fragile during protoplast isolation. It is proposed that this signal may play a role in inducing cellular changes in the plant cells as part of their responses to environmental stress such as pest attacks.