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The main aims of the present work were to investigate whether a chilling treatment which breaks dormancy of Douglas fir (Pseudotsuga menziesii (Mirb.) Franco) seeds induces changes in the sensitivity of seeds to exogenous ABA or in ABA levels in the embryo and the megagametophyte, and whether these changes are related to the breaking of dormancy. Dormant seeds germinated very slowly within a narrow range of temperatures (20-30 degrees C), the thermal optimum being approximately 25 degrees C. The seeds were also very sensitive to oxygen deprivation. Treatment of dormant seeds at 5 degrees C improved further germination, and resulted in a widening of the temperature range within which germination occurred and in better germination in low oxygen concentrations. In dry dormant seeds the embryo contained about one-third of the ABA in the megagametophyte. ABA content of both organs increased during the first 4 weeks of chilling. It then decreased sharply in the megagametophyte to the level in the embryo after 7-15 weeks of chilling. At 15 degrees C, a temperature at which dormancy was expressed, the ABA level increased in the embryo and the megagametophyte of dormant unchilled seeds whereas it decreased in the organs of chilled seeds. The longer the chilling treatment, the faster the decrease in ABA after the transfer of seeds from 5 degrees C to higher temperatures, and the decrease was faster at 25 than at 15 degrees C. These results suggest that the breaking of dormancy by cold was associated with a lower capacity of ABA biosynthesis and/or a higher ABA catabolism in the seeds subsequently placed at 15 or 25 degrees C. Moreover, the chilling treatment resulted in a progressive decrease in the sensitivity of seeds to exogenous ABA. However, seeds remained more sensitive to ABA at 15 than at 25 degrees C. The possible involvement of ABA synthesis and of responsiveness of seeds to ABA in the breaking of dormancy by cold treatment is discussed.

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Mature seeds of Helianthus annuus L. exhibit dormancy that is eliminated during storage in dry conditions. In vitro culture of immature embryos isolated at different times after anthesis showed that the youngest embryos are able to germinate, but within the third week after pollination, dormancy progressively affected most of the embryos. A radioimmunoassay showed that the endogenous abscisic acid (ABA) level, which increased sharply in the first half of the development period, fell at precisely the moment when embryo dormancy became established. An application of fluridone, before the increase of ABA level, prevented both ABA synthesis and development of embryo dormancy. Applied later, after the rise of the ABA level, fluridone could not prevent embryo dormancy development. Dormancy thus appears to be dependent on ABA synthesis but not concomitant with its accumulation; it must therefore be induced by ABA during maturation. Furthermore, a preincubation in water allowed dormant embryos to germinate. This acquisition of germinability could not be directly related to a leaching of free ABA. Possible effects of this treatment are discussed.The classic concept of the hormone balance theory supposed that induction, maintenance, and release of seed dormancy were regulated by the simultaneous action of promotive and inhibitory hormones. Recent work with hormone deficient mutants of Arabidopsis thaliana and Lycopersicon esculentum led Karssen and Groot (8) to a revision of this theory. In particular, it appears that ABA is responsible for the onset of dormancy in developing seeds but that it does not control the maintenance of dormancy in mature seeds. However, the authors noted that this conclusion might be valid for all species with so-called 'coat-imposed dormancy' but that direct evidence is missing to invoke the same hormonal control for embryo dormancy.Our first objective, then, was to look for the involvement of ABA in the onset of the dormancy of an embryo. Our plant material, Helianthus annuus seeds, exhibited at harvest a dormancy which could be eliminated by storage in dry conditions, and in vitro culture showed that embryos themselves were dormant. Furthermore, in this species, the lack of dormancy shown by a nondormant white mutant strain was correlated by Wallace and Haberman (16) (17), it may be suggested that the lack of dormancy must be correlated to the lack of ABA synthesis. This species appeared to be a good material to try to modify the physiological behavior of the embryo through an alteration of ABA synthesis. So from this perspective, we applied to the seeds during their development a solution of fluridone, a pyridinone inhibitor of carotenoid biosynthesis, in order to decrease their endogenous ABA levels. This approach allowed us to demonstrate the responsibility of ABA in the induction of the dormancy of the embryo.On the other hand, these dormant embryos could be germinated after a preincubation in water. This treatment, which evokes the leaching of an inhibitor, was imposed to discuss...

Excised Helianthus annuus L. embryos became dormant during the third week after anthesis. At this stage a short drying treatment (3 d) led to a slight improvement in germination but a 6-week dry storage caused a complete release from dormancy. The short drying treatment, however, elicited the embryos' response to an exogenous concentration of GAs which was unable to promote germination of fresh embryos. It therefore appeared that a short drying treatment changed the sensitivity to GAs but was not capable of directing embryo metabolism completely towards a germinative mode. Moreover, this drying treatment reduced considerably the ABA content in both the axis and the embryo. Nevertheless, no correlation could be established between germinability and the ABA content since the amount of ABA was not modified by the 6-week dry storage. The key step for predisposing the seeds to germinate is the suppression of the capacity for ABA synthesis in the axis, a suppression which takes place during 6-week dry storage.

This paper deals with the purification and the partial characterization of glutathione S-transferase (GST) isoforms from the clam Ruditapes decussatus. For the first step of purification, two affinity columns, reduced glutathione (GSH)-agarose and S-hexyl GSH-agarose, were mounted in series. Four affinity fractions were thus recovered. Further purification was performed using anion exchange chromatography. Seven fractions, which present a GST activity with 1-chloro-2,4-dinitrobenzene (CDNB) as substrate, were collected and analyzed by RP-HPLC. Seven distinct GST isoforms were purified, six of them were homodimers, the last one was a heterodimer consisting of the subunits 3 and 6. Kinetic parameters were studied. Results showed that isoforms have distinct affinity and V max for GSH and CDNB as substrates. The catalytic activity of the heterodimer isoform appeared to be a combination of the ability of each subunit. The immunological properties of each purified isoform were investigated using three antisera anti-pi, antimu and anti-alpha mammalian GST classes. Three isoforms (3-3, 6-6 and 3-6) seem to be closely related to the pi-class GST. Both isoforms 1-1 and 2-2 cross-reacted with antisera to pi and alpha classes and the isoform 5-5 cross-reacted with the antisera to mu and pi classes. Subunit 4 was recognized by the three antisera used, and its N-terminal amino acid analysis showed high identity (53%) with a conserved sequence of an alpha/ml/pi GST from Fasciola hepatica.Keywords: clam; glutathione S-transferase; immunology; kinetics; N-terminal analysis.The glutathione S-transferases (GSTs; EC 2.5.1.18) bind lipophilic nonsubstrate ligands and compounds such as heme and bilirubin [1], and are implicated in the biosynthesis of prostaglandins [2]. Apart from their metabolic activities, they are involved in the detoxication of electrophilic and genotoxic compounds by both catalytic activity and direct binding. In fact, the effect of these reactions is to convert a reactive (lipophilic) molecule into a water-soluble (nonreactive conjugate) which can be excreted. These mechanisms play an important role in cellular protection against the toxicity of endogenous compounds and of a variety of xenobiotics [3].The diversity of compounds metabolized by GSTs results from both the relatively nonspecific nature of the hydrophilic substrate binding site and the existence of numerous GST isoforms. GST activities are often assayed using 1-chloro-2,4-dinitrobenzene (CDNB), a relatively nonspecific GST reference substrate [4]. GST-CDNB activity reflects the integration of GST isoenzyme activities. However, the use of GST substrates such as 1,2-dichloro-4-nitrobenzene, ethacrynic acid (ETHA), nitrobutyl chloride and D5-androstene-3,17-dione in conjunction with CDNB allows for a more complete biochemical characterization of GST isozyme activities [5].GSTs are a multigene family of enzymes (isoforms), which have been grouped into seven classes based upon sequence homology and ability to catalyze the conjugation of glutathione to a ...

At harvest, embryos of Fagus sylvatica are dormant. A cold pretreatment without medium at 30% moisture content allowed them to germinate. A comparison of the abscisic acid (ABA) content before and after the pretreatment has no significant relevance since dormancy is expressed during the culture at 23°C. During this culture, both de novo biosynthesis and conjugate hydrolysis contributed to maintain a high level of ABA in the dormant axis. The level of conjugates and the rate of hydrolysis were not modified substantially by the cold pretreatment. In contrast, the dormancy release was associated with a strong decrease in the capacity for ABA synthesis. Moreover, feeding (+)-[ 3 H]ABA to untreated and pretreated embryos proved that the cold treatment also induced a hastening of ABA catabolism.

When applied to young nondormant embryos of sunflower (Helianthus annus) (7-10 day[s] after pollination [DAP]), abscisic acid (ABA) inhibited germination as long as it was present. However, whatever the dose used and the duration of its application, ABA was unable to induce dormancy because after transfer of treated embryos to control (without ABA) medium, germination occurred. Thereafter, exogenous ABA became effective and allowed the dormancy to develop in 13 and 17 DAP embryos, i.e. in embryos which after isolation were still able to germinate in high percentage. After embryo dormancy was well established (21 DAP), application of fluridone allowed the germination to occur very quickly on control medium. Isolated dormant axes were also induced to germinate by an application of fluridone. Radioimmunological analysis showed that 24 hours after these treatments, endogenous ABA levels were drastically reduced in the axes. When these fluridone-treated embryos were cultured on ABA medium, germination was again inhibited as long as exogenous ABA was present but germination occurred as soon as embryos were transferred to control medium. Such behavior suggested that in situ ABA synthesis is necessary to impose and maintain the embryo dormancy.The involvement ofABA in the initiation ofseed dormancy has been the subject of many recent studies using either mutants deficient in or insensitive to the hormone (4-6) or inhibitors of synthesis, particularly fluridone (3,11,12 embryos isolated at various times after pollination to exogenous ABA. It seemed necessary to distinguish clearly between the physiological consequences of the presence of exogenous ABA in the culture medium and those associated with a genuine induction effect, that is to say those produced by transient application of the hormone and which subsequently persist in its absence. Furthermore, we studied the consequences of an application of fluridone to isolated dormant embryos on their physiological behavior, their endogenous ABA levels and their responsiveness to exogenous ABA. MATERIALS AND METHODS Plant MaterialPlants of sunflower (Helianthus annuus cv Mirasol) were grown in the fields during summer. In September, flowers of male sterile plants were manually pollinated by pollen harvested on small female sterile flowers (seeds generously provided by S.A. Cargill). Flowers were tagged and harvest took place at different times after pollination.For each age, 25 embryos, i.e. a sample considered as sufficient to be representative of the population, were used for in vitro culture and extraction. All the experiments were carried out two or three times in two different years (1989)(1990).