Mary K. Loewen

PROMOTERS FROM KIN1 AND COR6.6, TWO HOMOLOGOUS ARABIDOPSIS THALIANA GENES : TRANSCRIPTIONAL REGULATION AND GENE EXPRESSION INDUCED BY LOW TEMPERATURE, ABA, OSMOTICUM AND DEHYDRATION

The Arabidopsis thaliana genes kin1 and cor6.6 belong to the same family and were expressed at higher levels following low temperature and ABA treatments. In an attempt to elucidate the mechanism of gene regulation by low temperature, the relationship between low-temperature- and abscisic acid (ABA)-induced gene expression and possible differential expression of the two genes, we have cloned a 5.3 kb genomic fragment harboring kin1 and cor6.6 and their respective 5′ sequences. The putative promoters of both genes were fused to the β-glucuronidase (GUS) coding sequence and GUS expression was analysed in transgenic tobacco and Arabidopsis plants. The cor6.6 promoter produced a higher basal level of expression than the kin1 promoter in transgenic tobacco. Enzyme assays of inducible GUS activity in transgenic Arabidopsis and tobacco plants showed that GUS activity directed by both kin1 and cor6.6 promoters was significantly induced by ABA, dehydration and osmoticum, but not by low temperature. Northern analysis revealed, in contrast, that GUS mRNA was significantly induced in these transgenic plants by low temperature. Further analysis showed that, at low temperature, GUS protein synthesis from the induced GUS mRNA was inhibited. Together these results reveal induction of kin1 and cor6.6 transcription by low temperature, exogenous ABA and dehydration. However, low-temperature expression is dramatically reduced at the translational level.

A New Abscisic Acid Catabolic Pathway

We report the discovery of a new hydroxylated abscisic acid (ABA) metabolite, found in the course of a mass spectrometric study of ABA metabolism in Brassica napus siliques. This metabolite reveals a previously unknown catabolic pathway for ABA in which the 9′-methyl group of ABA is oxidized. Analogs of (+)-ABA deuterated at the 8′-carbon atom and at both the 8′- and 9′-carbon atoms were fed to green siliques, and extracts containing the deuterated oxidized metabolites were analyzed to determine the position of ABA hydroxylation. The results indicated that hydroxylation of ABA had occurred at the 9′-methyl group, as well as at the 7′- and 8′-methyl groups. The chromatographic characteristics and mass spectral fragmentation patterns of the new ABA metabolite were compared with those of synthetic 9′-hydroxy ABA (9′-OH ABA), in both open and cyclized forms. The new compound isolated from plant extracts was identified as the cyclized form of 9′-OH ABA, which we have named neophaseic acid (neoPA). The proton nuclear magnetic resonance spectrum of pure neoPA isolated from immature seeds of B. napus was identical to that of the authentic synthetic compound. ABA and neoPA levels were high in young seeds and lower in older seeds. The open form (2Z,4E)-5-[(1R,6S)-1-Hydroxy-6-hydroxymethyl-2,6-dimethyl-4-oxo-cyclohex-2-enyl]-3-methyl-penta-2,4-dienoic acid, but not neoPA, exhibited ABA-like bioactivity in inhibiting Arabidopsis seed germination and in inducing gene expression in B. napus microspore-derived embryos. NeoPA was also detected in fruits of orange (Citrus sinensis) and tomato (Lycopersicon esculentum), in Arabidopsis, and in chickpea (Cicer arietinum), as well as in drought-stressed barley (Hordeum vulgare) and B. napus seedlings.

Role of oxidative stress in cereal protoplast recalcitrance

Cereal leaf protoplasts are often extremely unstable in culture and usually lyse within 24 hours. Using the thiobarbituric acid test and the ferrous thiocyanate test we have shown that corn (Zea mays L. cv. Market Beauty) and wheat (Triticum aestivum L. cv. Benito) leaf protoplasts accumulate peroxides and peroxide degradation products during culture. This increase correlated with an increase in lipoxygenase activity. On the other hand, enzymes involved in detoxification of peroxides such as catalase and peroxidase decreased during culture. The occurrence of lipid peroxidation in leaf protoplasts is likely to be a consequence of a temporary imbalance in the enzymes involved in oxygen metabolism. It has previously been shown that the lipoxygenase inhibitor n-propyl gallate stabilizes the protoplasts in culture and so peroxidation is likely to be the cause of leaf protoplast instability. Protoplasts obtained from suspension cultures are stable in culture and do not undergo lipid peroxidation. This stability is due to a decrease in lipoxygenase activity and increases in catalase and peroxidase activity after protoplast isolation.

Synthesis and biological activity of tetralone abscisic acid analogues

Bicyclic analogues of the plant hormone abscisic acid (ABA) were designed to incorporate the structural elements and functional groups of the parent molecule that are required for biological activity. The resulting tetralone analogues were predicted to have enhanced biological activity in plants, in part because oxidized products would not cyclize to forms corresponding to the inactive catabolite phaseic acid. The tetralone analogues were synthesized in seven steps from 1-tetralone and a range of analogues were accessible through a second route starting with 2-methyl-1-naphthol. Tetralone ABA 8 was found to have greater activity than ABA in two bioassays. The absolute configuration of (+)-8 was established by X-ray crystallography of a RAMP hydrazone derivative. The hydroxymethyl compounds 10 and 11, analogues for studying the roles of 8- and 9-hydroxy ABA 3 and 6, were also synthesized and found to be active.

Effects of Sodium Chloride on plant cells; a 31P and 23Na NMR system to study salt tolerance

In plant cells, the Na(+)/H(+) antiport at the tonoplast provides a biochemical pathway to transport cytoplasmic Na(+) into the vacuole. Recently it was shown that overexpression of a vacuolar Na(+)/H(+) promotes sustained plant growth at high NaCl levels (Apse et al. Science 285, 1256, 1999). The sequestration of Na(+) ions into the vacuole can be followed using 31P and 23Na NMR spectroscopy. Suspension cell cultures are very suitable for this purpose and allow rapid and accurate assessment of the activity of the Na(+)/H(+) antiport and therefore potentially of salt tolerance. Perfusion experiments with maize cells that are not particularly salt (NaCl) tolerant showed that during salt stress the cytoplasmic pH remains unchanged while the vacuolar pH significantly increased. During Na(+) sequestration into the vacuole, the cytoplasmic pH equilibrates faster than that of the vacuole. Both vacuolar pH and the cellular Na(+) uptake rate were dependent on extracellular Na(+) for concentrations up to approximately 300 mM. For Na(+) concentrations >/=300 mM, both vacuolar pH and cellular Na(+) uptake became independent of the extracellular concentration. This indicates either a saturation of Na(+) uptake at the cell surface or a saturation of the Na(+)/H(+) transporter at the tonoplast. Na(+) uptake into the cell is accompanied by a rapid increase in vacuolar PO(4)(3-), broadening of the 31P resonances and a reduction in glucose monophosphate and UDPG.

Sesquiterpene-like inhibitors of a 9-cis-epoxycarotenoid dioxygenase regulating abscisic acid biosynthesis in higher plants

Abscisic acid (ABA) is a carotenoid-derived plant hormone known to regulate critical functions in growth, development and responses to environmental stress. The key enzyme which carries out the first committed step in ABA biosynthesis is the carotenoid cleavage 9-cis-epoxycarotenoid dioxygenase (NCED). We have developed a series of sulfur and nitrogen-containing compounds as potential ABA biosynthesis inhibitors of the NCED, based on modification of the sesquiterpenoid segment of the 9-cis-xanthophyll substrates and product. In in vitro assays, three sesquiterpene-like carotenoid cleavage dioxygenase (SLCCD) inhibitor compounds 13, 17 and 18 were found to act as inhibitors of Arabidopsis thaliana NCED 3 (AtNCED3) with K(i)s of 93, 57 and 87 microM, respectively. Computational docking to a model of AtNCED3 supports a mechanism of inhibition through coordination of the heteroatom with the non-heme iron in the enzyme active site. In pilot studies, pretreatment of osmotically stressed Arabidopsis plants with compound 13 resulted lower levels of ABA and catabolite accumulation compared to levels in mannitol-stressed plant controls. This same inhibitor moderated known ABA-induced gene regulation effects and was only weakly active in inhibition of seed germination. Interestingly, all three inhibitors led to moderation of the stress-induced transcription of AtNCED3 itself, which could further contribute to lowering ABA biosynthesis in planta. Overall, these sesquiterpenoid-like inhibitors present new tools for controlling and investigating ABA biosynthesis and regulation.

Metabolism and biological activity of (+)- and (-)-C-1′-O-methyl aba in maize suspension-cell cultures☆

Abstract (+)- C -1′- O -Methyl abscisic acid is rapidly metabolized by suspension-cultured maize cells ( Zea mays L. Black Mexican Sweet) to (+)- C -1′- O -methyl phaseic acid in an analogous process as seen for ABA. The presence of a methyl ether on the 1′-position of ABA does not interfere with enzymic oxidation at the 8′-carbon. The metabolite is demethylated to yield phaseic acid. A small amount of abscisic acid is also produced by direct demethylation. The (+)- C -1′- O -methyl ABA exhibits stronger growth inhibitory activity of the maize cells than (+)-ABA, suggesting that a free C -1′-hydroxyl group is not essential for biological activity of ABA in maize. The (−)- C -1′- O -methyl ABA is metabolized to (−)-ABA and to 7′-hydroxyABA and the corresponding C -1′- O -methyl-7′-hydroxy ABA.

ROLE OF PEROXIDASE IN PROTOPLAST DEVELOPMENT: THE ACTIVITY AND MOLECULAR FORMS OF PEROXIDASE IN MITOGENIC AND NON-MITOGENIC PROTOPLASTS

Genetic manipulation of agronomically important monocot plants, such as wheat, corn and barley remain difficult. This is primarily due to the recalcitrance of cereal protoplasts. The antithetical behaviour of cereal protoplasts in culture may be related to differences in response to protoplasting stress. Our earlier studies indicated that freshly isolated corn and wheat leaf protoplasts showed evidence of lipid peroxidation and elevated lipoxygenase activity. No such increases were detected in freshly isolated bromegrass protoplasts (1–2). There are many unresolved questions concerning the inability of cereal leaf protoplasts to divide. We have focussed our attention on peroxidase, an enzyme which has been repeatedly implicated in plant developmental processes (3–4).