Superoxide dismutase (SOD) gene expression was investigated to elucidate its role in drought and freezing tolerance in spring and winter wheat (Triticum aestivum). cDNAs encoding chloroplastic Cu/ZnSODs and mitochondrial MnSODs were isolated from wheat. MnSOD and Cu/ZnSOD genes were mapped to the long arms of the homologous group-2 and -7 chromosomes, respectively. Northern blots indicated that MnSOD genes were drought inducible and decreased after rehydration. In contrast, Cu/ZnSOD mRNA was not drought inducible but increased after rehydration. In both spring and winter wheat seedlings exposed to 2°C, MnSOD transcripts attained maximum levels between 7 and 49 d. Transcripts of Cu/ ZnSOD mRNA were detected sooner in winter than in spring wheat; however, they disappeared after 21 d of acclimation. Transcripts of both classes of SOD genes increased during natural acclimation in both spring and winter types. Exposure of fully hardened plants to three nonlethal freeze-thaw cycles resulted in Cu/Zn mRNA accumulation; however, MnSOD mRNA levels declined in spring wheat but remained unchanged in winter wheat. The results of the dehydration and freeze-thaw-cycle experiments suggest that winter wheat has evolved a more effective stress-repair mechanism than spring wheat.Active oxygen species such as superoxide, H 2 O 2 , and hydroxyl radicals are by-products of normal cell metabolism. These active oxygen species result in the peroxidation of membrane lipids (Mead, 1976), breakage of DNA strands (Brawn and Fridovich, 1981), and inactivation of enzymes (Fucci et al., 1983). The conditions leading to damage caused by active oxygen species are referred to as oxidative stress. Both chloroplasts and mitochondria can produce active oxygen species either under normal growth conditions or during exposure to various stresses. The PSI electron-transport chain contains a number of autooxidizable enzymes that reduce O 2 to superoxide (Badger, 1985; Asada and Takahashi, 1987; Asada, 1994), and evidence indicates that superoxide and H 2 O 2 can also be produced by PSII under high-light intensities (Landgraf et al., 1995).During mitochondrial respiration, reactive oxygen species are also generated via the reactions of the electrontransport chain (Rich and Bonner, 1978;Bowler et al., 1991).Active oxygen species are also generated during chemical and environmental stresses, including chilling and freezing (Wise and Naylor, 1987;Senaratna et al., 1988; Kendall and McKersie, 1989;Tsang et al., 1991;McKersie et al., 1993), drought (Perl-Treves and Galun, 1991; Price and Hendry, 1991), desiccation (Senaratna et al., 1985;Leprince et al., 1990), flooding (Hunter et al., 1983;Van Toai and Bolles, 1991), herbicide treatment (Malan et al., 1990; Kurepa et al., 1997), pathogen attack (Montalbini and Buonaurio, 1986; Buonaurio et al., 1987; Koch and Slusarenko, 1990), and ionizing radiation (Niwa et al., 1977).SODs are a group of metalloenzymes that protect cells from superoxide radicals by catalyzing the dismutation of the superoxide radical to...

A number of effects on embryogenesis of the putative phytohormone jasmonic acid (JA), and its methyl ester (MeJA), were investigated in two oilseed plants, rapeseed (Brassica napus) and flax (Linum usitatissimum). Results from treatments with JA and MeJA were compared with those of a known effector of several aspects of embryogenesis, abscisic acid (ABA). Jasmonic acid was identified by gas chromatography-mass spectrometry as a naturally occurring substance in both plant species during embryo development. Both JA and MeJA can prevent precocious germination of B. napus microspore embryos and of cultured zygotic embryos of both species at an exogenous concentration of >1 micromolar. This dose-response was comparable with results obtained with ABA. Inhibitory effects were also observed on seed germination with all three growth regulators in rapeseed and flax. A number of molecular aspects of embryogenesis were also investigated. Expression of the B. napus storage protein genes (napin and cruciferin) was induced in both microspore embryos and zygotic embryos by the addition of 10 micromolar JA. The level of napin and cruciferin mRNA detected was similar to that observed when 10 micromolar ABA was applied to these embryos. For MeJA only slight increases in napin or cruciferin mRNA were observed at concentrations of 30 micromolar. Several oilbody-associated proteins were found to accumulate when the embryos were incubated with either JA or ABA in both species. The MeJA had little effect on oilbody protein synthesis. The implications of JA acting as a natural regulator of gene expression in zygotic embryogenesis are discussed.

Embryos derived in vitro from isolated microspores of Brassica napus L. were compared with their zygotic counterparts. Parameters investigated included storage-protein accumulation and gene expression, fattyacid composition, storage-lipid biosynthesis, and the appearance of oil-body proteins. The microspore embryos accumulate storage-protein and show increases in levels of their transcripts during the torpedo stage. These embryos were sensitive to abscisic acid (ABA) with respect to accumulation of storage-protein mRNA and oil-body proteins. Post-transcriptional regulation of cruciferin accumulation is indicated by a disparity between ABA-enhanced transcript accumulation and a less marked effect at the level of protein accumulation. To investigate storage-lipid profiles, two cultivars of Brassica napus, Reston and Topas, were used. The former accumulates major quantities of C20 (11.2%) and C22 (39.9%) fatty acids in its seeds, the latter predominantly C18 fatty acids. The higher-molecular-weight fatty acids (>C18) normally occur only in seeds and were used as biochemical markers for seed-specific metabolism in microspore embryos. Microspore embryos from Reston were found to accumulate C20 (10.6%) and C22 (31.2%) fatty acids after 35 d in culture at levels and proportions comparable to those found in seeds. Similarly, microspore embryos of Topas had a fatty-acid profile similar to that of mature Topas seed. Activities of enzymes involved in the accumulation of storage lipids (erucoyl-CoA synthetase [EC 6.2.1.3], erucoyl-CoA thioesterase [EC 3.1.2.2] and erucoyl-CoA acyltransferase [EC 2.3.1.15 or EC 2.3.1.20]) were detected in torpedostage microspore embryos. Their specific activities were higher than have been reported to date for analogous preparations from zygotic embryos of B. napus. The similarities in storage-lipid and protein composition of these embryos to their zygotic counterparts, along with their sensitivity to ABA, indicate that microspore embryos might be exploited to facilitate studies of biochemistry and gene regulation in oilseeds.

Storage protein gene expression, characteristic of mid-to late embryogenesis, was investigated in microspore embryos of rapeseed (Brassica napus). These embryos, derived from the immature male gametophyte, accumulate little or no detectable napin or cruciferin mRNA when cultured on hormone-free medium containing 13% sucrose. The addition of abscisic acid (ABA) to the medium results in an increase in detectable transcripts encoding both these polypeptides. Storage protein mRNA is induced at 1 micromolar ABA with maximum stimulation occurring between 5 and 50 micromolar. This hormone induction results in a level of storage protein mRNA that is comparable to that observed in zygotic embryos of an equivalent morphological stage. Effects similar to that of ABA are noted when 12.5% sorbitol is added to the microspore embryo medium (osmotic potential = 25.5 bars). Time course experiments, to study the induction of napin and cruciferin gene expression demonstrated that the ABA effect occurred much more rapidly than the high osmoticum effect, although after 48 hours, the levels of napin or cruciferin mRNA detected were similar in both treatments. This difference in the rates of induction is consistent with the idea that the osmotic effect may be mediated by ABA which is synthesized in response to the reduced water potential. Measurements of ABA (by gas chromatography-mass spectrometry using [2H6]ABA as an internal standard) present in microspore embryos during sorbitol treatment and in embryos treated with 10 micromolar ABA were performed to investigate this possibility. Within 2 hours of culture on high osmoticum the level of ABA increased substantially and significantly above control and reached a maximum concentration within 24 hours. This elevated concentration was maintained for 48 hours after culturing and represents a sixfold increase over control embryos. The ABA-treated embryos accumulated the hormone very quickly, but ABA concentrations retumed to basal levels within 72 hours after treatment. The possibility that embryosynthesized ABA may be a mediator of effects of osmotic stress on gene expression in Brassica embryos is discussed.several studies on the regulation of gene expression in developing seeds (9, 10). In B. napus it has been shown that exogenous ABA treatment can have profound effects on the accumulation of transcripts and gene products of the major seed storage proteins (9), lipid profiles (8), and on levels of oil-body associated proteins (20). Many ofthe responses stimulated by ABA are also affected by water stress or elevated osmotica (7,8).The relationship between ABA effects and osmotic effects in developing seeds is not yet clear and has yielded results with contrasting interpretations (23). Bray and Beachy (2) suggested that the accumulation of soybean storage proteins, which is enhanced by culturing soybean embryos on high osmotica, is a consequence of elevated ABA content. In B. napus, on the other hand, Finkelstein and Crouch (7)

S7N OW9 (S.R. A.) l h e properties of two enantiomeric synthetic acetylenic abscisic acid (ABA) analogs (PBI-51 and PBI-63) in relation to ABA-sensitive gene expression are reported. Using microspore-derived embryos of Brassica napus as the biological material and their responsiveness to ABA in the expression of genes encoding storage proteins as a quantitative bioassay, we measured the biological activity of PBI-51 and PBI-63. Assays to evaluate agonistic activity of either compound applied individually showed a dose-dependent increase in napin gene expression on application of PBI-63. Maximal activity of about 40 p~ indicated that PBI-63 was an agonist, although somewhat weaker than ABA. PBI-63 has a similar stereochemistry to natural ABA at the junction of the ring and side chain. In contrast, PBI-51 showed no agonistic effects until applied at 40 to 50 p~.Even then, the response was fairly weak. PBI-51 has the opposite stereochemistry to natural ABA at the junction of the ring and side chain. When applied concurrently with ABA, PBI-63 and PBI-51 had distinctly different properties. PBI-63 (40 p~) and ABA (5 PM) combined gave results similar to the application of either compound separately with high levels of induction of napin expression. PBI-51 displayed a reversible antagonistic effect with ABA, shifting the typical ABA dose-response curve by a factor of 4 to 5. This antagonism was noted for the expression of two ABA-sensitive genes, napin and oleosin. To test whether this antagonism was at the leve1 of ABA recognition or uptake, ABA uptake was monitored in the presence of PBI-51 or PBI-63. Neither compound decreased ABA uptake. Treatments with either PBI-51 or PBI-63 showed an effect on endogenous ABA pools by permitting increases of 5-to 7-fold. It is hypothesized that this increase occurs because of competition for ABA catabolic enzymes by both compounds. l h e fact that ABA pools did not decrease i n the presence of PBI-51 suggests that PBI-51 must exert its antagonistic properties through direct competition with ABA at a hormone-recognition site.