Germination of radish (Raphanus sativus cv Eterna) seeds can be inhibited by far-red light (high-irradiance reaction of phytochrome) or abscisic acid (ABA). Gibberellic acid (GA 3 ) restores full germination under far-red light. This experimental system was used to investigate the release of reactive oxygen intermediates (ROI) by seed coats and embryos during germination, utilizing the apoplastic oxidation of 2Ј,7Ј-dichlorofluorescin to fluorescent 2Ј,7Ј-dichlorofluorescein as an in vivo assay. Germination in darkness is accompanied by a steep rise in ROI release originating from the seed coat (living aleurone layer) as well as the embryo. At the same time as the inhibition of germination, far-red light and ABA inhibit ROI release in both seed parts and GA 3 reverses this inhibition when initiating germination under far-red light. During the later stage of germination the seed coat also releases peroxidase with a time course affected by far-red light, ABA, and GA 3 . The participation of superoxide radicals, hydrogen peroxide, and hydroxyl radicals in ROI metabolism was demonstrated with specific in vivo assays. ROI production by germinating seeds represents an active, developmentally controlled physiological function, presumably for protecting the emerging seedling against attack by pathogens.

The physical mechanism of seed germination and its inhibition by abscisic acid (ABA) in Brassica napus L. was investigated, using volumetric growth (= water uptake) rate (dV/dt), water conductance (L), cell wall extensibility coefficient (m), osmotic pressure (IIi), water potential (*i), turgor pressure (P), and minimum turgor for cell expansion (Y) of the intact embryo as experimental parameters. dV/dt, Hi, and *i were measured directly, while m, P, and Y were derived by calculation. Based on the general equation of hydraulic cell growth IdV/dt = Lm/(L + m) (Al -Y), where All = Hi -II of the external medium; the terms (L m/(L + m) and II -Y were defined as growth coefficient (kG) and growth potential (GP), respectively. Both kG and GP were estimated from curves relating dV/dt (steady state) to I of osmotic test solutions (polyethylene glycol 6000).During the imbibition phase (0-12 hours after sowing), kG remains very small while GP approaches a stable level of about 10 bar. During the subsequent growth phase of the embryo, kG increases about 10-fold. ABA, added before the onset of the growth phase, prevents the rise of kG and lowers GP. These effects are rapidly abolished when germination is induced by removal of ABA. Neither L (as judged from the kinetics of osmotic water efflux) nor the amount of extractable solutes are affected by these changes. Ili and *i remain at a high level in the ABA-treated seed but drop upon induction of germination, and this adds up to a large decrease of P, indicating that water uptake of the germinating embryo is controlled by cell wall loosening rather than by changes of II; or L. ABA inhibits water uptake by preventing cell wall loosening. By calculating Y and m from the growth equation, it is further shown that cell wall loosening during germination comprises both a decrease of Y from about 10 to 0 bar and an at least 10-fold increase of m. ABA-mediated embryo dormancy is caused by a reversible inhibition of both of these changes in cell wall stability.In a previous paper (29), we have provided evidence based on kinetics of water uptake and a factorial analysis that exogenous ABA controls germination of rape seeds by limiting water uptake of the embryo rather than by inhibiting energy* metabolism, protein synthesis, or related processes. It was concluded that ABA and osmotic stress interact at a common point controlling the water relations of the embryo specifically during the growth phase of germination. This phase commences about 12 h after sowing and is characterized by a resumption of rapid water uptake due to active embryo enlargement. In contrast, water uptake during the preceding imbibition phase (O to ,umol 1-' (29). However, a 10-fold higher concentration is needed to maintain the dormant state for longer periods of time.In the present paper, we attempt to localize the site(s) of action of ABA within the physical parameters governing the water relations of the germinating seed. The inhibitory effect of ABA on water uptake has been shown to be both rapidly ind...

Germination of rape (Brassica napus L.) seeds proceeds in two phases, an initial imbibition phase and a subsequent growth phase. The time courses of water uptake, 02 uptake, and ATP accumulation demonstrate that exogenous abscisic acid (ABA, 0.1 millimoles per liter) specifically prevents the embryo from entering the growth phase. The inhibition of water uptake by ABA is a rapid (lag-phase about 1 hour) and fully reversible process which appears to be the cause rather than the result of changes of the energy metabolism. In untreated seeds, an osmotic pressure (polyethylene glycol 6000) of 11 bars is required for a simulation of the ABA effect on water uptake. However, in ABA-treated seeds an osmotic pressure of only 3 bars is sufficient to suppress water uptake. Thus, ABA lowers the ability of the embryo to absorb water under osmotic stress. In a two-factor analysis of the simultaneous action of ABA and osmoticum on germination, a complete synergistic interaction between these factors was found while ABA and cycloheximide exhibit independent (multiplicative) coaction. These results are interpreted in terms of a common controlling point of ABA and osmotic stress in the water relations of germinating seeds.The action mechanism of ABA, a potent physiological inhibitor of seed germination, is not yet understood. In a previous paper (9) we suggested that the hormone induces seed dormancy by restricting water uptake of the imbibed seed during a critical period ofgermination, where an active push ofembryo expansion is needed to support embryo growth. This hypothesis implies that the biochemical changes previously related to the primary action ofABA such as the modification ofnucleic acid or protein synthesis (e.g. 1, 2, 4, 10) are not direct causal links in the action mechanism of ABA but merely later consequences of the growth inhibition through restricted water uptake.In the present communication we attempt to clarify the role of ABA in water uptake of germinating rape seeds. This type of seed is similar to the previously used mustard seed but lacks the mucilaginous seed coat which makes the study of embryo water relations in mustard rather cumbersome. In contrast to mustard, the very thin and brittle testa of rape absorbs only insignificant amounts ofwater and is rapidly ruptured by the swelling embryo soon after imbibition. Rape produces nondormant seeds which require only imbibition at a suitable temperature (e.g. 25C) for rapid germination. There is no indication of physiologically significant levels of endogenous ABA in the mature rape seed. Exogenous ABA inhibits the completion of germination of these seeds in much the same way as described for mustard (9). As outlined in detail in the previous report (9), we define germina-I Supported by the Deutsche Forschungsgemeinschaft (SFB 206 10 x 6 cm). After adding further 5 ml ofliquid, a small meniscus formed around the seeds allowing uniform wetting of the seeds without impairing gas exchange. Germination took place in darkness at 25.0 ± 0.3C. Since germinat...

The germination process of mustard seeds (Simapis alba L.) has been characterized by the time courses of water uptake, rupturing of the seed coat (12 hours after sowing), onset of axis growth (18 hours after sowing), and the point of no return, where the seeds lose the ability to survive redesiccation (12 to 24 hours after sowing, depending on embryo part). Abscisic acid (ABA) reversibly arrests embryo development at the brink of radicle growth initiation, inhibiting the water uptake which accompanies embryo growth. Seeds which have been kept dormant by ABA for several days will, after removal of the hormone, rapidly take up water and continue the germination process. Seeds which have been preincubated in water lose the sensitivity to be arrested by ABA after about 12 hours after sowing. This escape from ABA-mediated dormancy is not due to an inactivation of the hormone but to a loss of competence to respond to ABA during the course of germination. The sensitivity to ABA can be restored in these seeds by redrying. It is concluded that a primary action of ABA in inhibiting seed germination is the control of water uptake of the embryo tissues rather than the control of DNA, RNA, or protein syntheses.When a quiescent, nondormant seed (13) is supplied with water and 02 at favorable temperatures the embryo rapidly takes up water and continues its temporarily suspended development. After building up a certain threshold hydrostatic pressure the seed coat is ruptured and visible protrusion of the elongating radicle indicates the onset of elongation of the embryonic axis. Some time later the release from quiescence becomes irreversible, ie. the embryo will no longer survive redesiccation. The developmental period from the increase of metabolic activity after imbibition up to this point of no return, logically separating the embryo from the seedling stage, can be referred to as germination. At 25 C germination lasts not more than about 1 day in many seeds.The dormancy hormone ABA can inhibit continuation of embryo development during germination and related developmental processes (e.g. the growth of Lemna turions or tree buds, 19, 20).Exogenously applied ABA is rapidly taken up by the embryo even through the intact seed coat (3,16 It has been shown that fundamental processes such as cell division and the synthesis of DNA, RNA, and protein become inhibited in plant systems treated with ABA (6,10, 11,19,21). In barley aleurone tissue and cotton embryos exogenous ABA prevents the formation of enzymes which are involved in mobilizing storage materials during germination (5, 12). Although the molecular nature of the physiological block, by which ABA prevents the completion of germination, is still unknown, it is generally believed that ABA functions by interfering with mRNA synthesis, processing, or translation (e.g. 5-7, 10, 11, 24). In a previous study with mustard (1) we have observed that seedling development is surprisingly insensitive to ABA if the hormone is applied after germination. In particular it has been e...

The biophysical mechanism underlying photoinhibition of radish {Raphanus sativus L.) seed germination was investigated using three cultivars differing in sensitivity to continuous irradiation with far-red light (high-irradiance reaction of phytochrome). Sensitivity of germination to the inhibitory action of light was assessed by probing germination under osmotic stress (incubation in media of low water potentials adjusted with polyethylene glycol 6000) and expressed in terms of 'germination potential' (positive value of the water potential at which germination is inhibited by 50%). Far-red light decreases the germination potential to various degrees in the different cultivars, reflecting the light-sensitivity of germination in water. Removal of the seed coat increases the germination potential by a constant amount in darkness and light. It is concluded that germination depends on the expansive force of the embryo which can be drastically diminished by far-red light. Seed-coat constraint and expansive force of the embryo interact additively on the level of the germination potential. Photoinhibition of germination was accompanied by an inhibition of water uptake into the seed. Analysis of seed water relations showed that osmotic pressure and turgor assumed higher levels in photoinhibited seeds, compared to seeds germinating in darkness, while the water potential was close to zero under both conditions. Far-red light produced a shift (to less negative values) in the curve relating water-uptake rate to external water potential, i.e. a reduction in the driving force for water uptake. It is concluded that photoinhibition of germination results from the maintenance of a high threshold of cell-wall extensibility in the embryo.