Analyses to determine the role of the megagametophyte and other seed tissues in dormancy maintenance of yellow cedar (Chamaecyparis nootkatensis) seeds; morphological, cellular and physiological changes following moist chilling and during germination

Yellow cedar (Chamaecyparis nootkatensis) seeds exhibit prolonged dormancy following their dispersal from the parent plant. Embryos excised fully from their enclosing seed tissues exhibit 100% germination, indicating that the seed tissues enclosing the embryo (the testa, remnants of the nucellus and the megagametophyte) play an inhibitory role and prevent radicle emergence. As part of an assessment of the role of seed tissues in the dormancy mechanism of yellow cedar seeds, light microscopy was used to examine changes within the major structures of the seed following a 90 d war (26C)/cold (4C) moist treatment ('stratification') and during germination. In the micropylar tip of the seed, the nucellus forms a hard nucellar cap covering the radicle. The nucellar cap is composed primarily of degenerated cells; histological staining with ruthenium red revealed a predominance of pectins. There were no obvious cellular or morphological differences (detected by light microscopy) between mature seeds subjected to a 3 d soak and seeds subjected to a 3 d soak and the 90 d dormancy-breaking treatment. However, just prior to germination there was an outward projection of the nucellar cap through the micropyle, which appeared to be caused by the extension of highly folded proteinaceous strands lying immediately in front of the radicle. When the testa was removed, the embryo enclosed within the intact megagametophyte was incapable of germination. If, however, the megagametophyte surrounding the embryo was slit or the embryo surrounded by an intact megagametophyte was subjected to a 3d rinse in water, some germination occurred, perhaps as a result of an enhanced release of inhibitors from the megagametophyte. After stratification, dormancy of yellow cedar seeds is broken; concurrent with dormancy breakage, there was a mechanical weakening of the megagametophyte. The embryo also underwent changes that included an increase in turgor and a reduced sensitivity to highly negative osmotic potential. It is concluded that coat-imposed dormancy of yellow cedar seeds is enforced by mechanical restraint of the megagametophyte as well as a leachable chemical inhibitor (most probably ABA).     

An increase in pectin methyl esterase activity accompanies dormancy breakage and germination of yellow cedar seeds

Pectin methyl esterase (PME) (EC 3.1.1.11) catalyzes the hydrolysis of methylester groups of cell wall pectins. We investigated the role of this enzyme in dormancy termination and germination of yellow cedar (Chamaecyparis nootkatensis [D. Don] Spach) seeds. PME activity was not detected in dormant seeds of yellow cedar but was induced and gradually increased during moist chilling; high activity coincided with dormancy breakage and germination. PME activity was positively correlated to the degree of dormancy breakage of yellow cedar seeds. The enzyme produced in different seed parts and in seeds at different times during moist chilling, germination, and early post-germinative growth consisted of two isoforms, both basic with isoelectric points of 8.7 and 8.9 and the same molecular mass of 62 kD. The pH optimum for the enzyme was between 7.4 and 8.4. In intact yellow cedar seeds, activities of the two basic isoforms of PME that were induced in embryos and in megagametophytes following dormancy breakage were significantly suppressed by abscisic acid. Gibberellic acid had a stimulatory effect on the activities of these isoforms in embryos and megagametophytes of intact seeds at the germinative stage. We hypothesize that PME plays a role in weakening of the megagametophyte, allowing radicle emergence and the completion of germination.     

Proteins of Axial Organs of Dormant and Germinating Horse Chestnut Seeds: 1. General Characterization

This is the first characterization of proteins from axial organs of recalcitrant horse chestnut seeds during deep dormancy, dormancy release, and germination. We demonstrated that, during the entire period of cold stratification, axial organs were enriched in easily soluble albumin-like proteins and almost devoid of globulins. About 80% of the total protein was found in the cytosol. Approximately one third of cytosolic proteins were heat-stable polypeptides, which were major components of total proteins. Heat-stable proteins comprised three groups of polypeptides with mol wts of 52–54, 24–25, and 6–12 kD with a predominance of low-molecular-weight proteins. The polypeptide patterns of heat-stable and thermolabile proteins differed strikingly. Heat-stable proteins accumulated in axes during the late seed maturation, comprising more than 30% of the total protein in axes of mature seeds. The polypeptide patterns of the total protein of axial organs and its particular fractions did not change in the course of seed dormancy and release. At early germination, the content of heat-stable proteins in axes decreased and their polypeptide pattern changed both in the cytosol and cell structures. We believe that at least some heat-stable proteins can function as storage proteins in the axes. Localization of storage proteins in the cells of axial organs and the role of heat-stable proteins in recalcitrant seeds are discussed.     

Changes in abscisic acid content and embryo sensitivity to (+)-abscisic acid during the termination of dormancy of yellow cedar seeds

Yellow cedar seeds are dormant at maturity. The abscisic acid (ABA) content of the embryo (but not the megagametophyte) decreased approximately 2-fold following exposure of seeds to a dormancy-breaking treatment; this process was also accompanied by a 10-fold lowered sensitivity of the embryo to S:-(+)-ABA. A decline in ABA within the seed is not sufficient for dormancy breakage; reduced embryo sensitivity to ABA is also required.     

Endo-β-mannanase activity during dormancy alleviation and germination of white spruce (Picea glauca) seeds

It is not known how embryos of seeds of the Pinaceae protrude from their enclosing tissues to complete germination. Prior to protrusion of the radicle there is an increase in endo-β-1,4-mannanase (EC 3.2.1.78) activity associated with weakening of the micropylar megagametophyte/nucellus from seeds of white spruce ( Picea glauca [Moench.] Voss). Mannanase activity is present as three isoforms (pI values 5.0, 4.8, 4.7) in both the embryo and surrounding structures (megagametophyte and nucellus) prior to and during imbibition. Activity of all the isoforms increases in the chalazal and micropylar megagametophyte during germination. Activity then declines after the testa splits, typically 1 day prior to radicle protrusion, due partially to its leaching from the seed into the surrounding water. Activity increases in the cotyledons and axis as the embryo commences elongation. Seeds from dormant seedlots exhibit a lower germination percentage, relative to seeds from nondormant seedlots, and the force necessary for the embryo to puncture the surrounding structures tends to be greater. Although similar mannanase activities are present in unimbibed seeds of dormant and nondormant seedlots, during germination, enzyme activity in seeds of dormant seedlots is lower. Moist chilling alleviates dormancy in the seeds of the Pinaceae and, during 3 weeks of this treatment, mannanase activity slowly increases. After 3 weeks of moist chilling and regardless of whether the seedlot was dormant or not prior to moist chilling, the force necessary to puncture the micropylar megagametophyte and nucellus is lower, and the speed of germination greater. Seeds from previously dormant seedlots also complete germination to a greater percentage, relative to unchilled seeds from dormant seedlots. Upon transfer to 25°C, mannanase activity in moist-chilled seeds decreases during germination of all seedlots regardless of their previous dormancy status.     

Variant maturity in seed structures of <Emphasis Type="Italic">Pinus albicaulis</Emphasis> (Engelm.) and <Emphasis Type="Italic">Pinus sibirica</Emphasis> (Du Tour): key to a soil seed bank,unusual among conifers?

The seeds of Cembrae pines are dispersed by nutcrackers (Genus Nucifraga), which cache seeds in soil during autumn. The dispersal and establishment of seedlings via this mutualistic relationship is highly successful. On the other hand, irregular quality of seed crops and lack of detailed knowledge on germination process of Cembrae pine seeds hamper effective seedling production in the nursery. Therefore we studied basic structures and maturity of whitebark pine (Pinus albicaulis Engelm.) and Siberian stone pine (Pinus sibirica Du Tour) seeds, as well as structural changes during a multi-step treatment of whitebark pine seeds, using field emission scanning electron microscopy, transmission electron microscopy and light microscopy. The most striking differences compared to many other conifer seeds were the solid surface structures, early structural differentiation of the embryo, clustering of the thin-walled megagametophyte cells, and great accumulation of starch in both the untreated and treated seeds. Protein bodies of the embryo were in early developmental stages, whereas in the megagametophyte their stages varied. The number, form and size of lipid bodies also varied within different parts of the seed, and lipids dissolved easily. Our results indicated that despite maturity of the seed coat and advanced differentiation of the embryo, the embryo and the megagametophyte were still immature. These morphological features and a notable proportion of storage reserves remaining in unstable form may, however, be advantageous for maintaining viability and reaching maturity within a soil seed bank. Well-controlled pre-treatment simulating natural conditions should result in improved germination.     

ROS production and protein oxidation as a novel mechanism for seed dormancy alleviation

At harvest, sunflower (Helianthus annuus L.) seeds are dormant and unable to germinate at temperatures below 15 degrees C. Seed storage in the dry state, known as after-ripening, is associated with an alleviation of embryonic dormancy allowing subsequent germination at suboptimal temperatures. To identify the process by which dormancy is broken during after-ripening, we focused on the role of reactive oxygen species (ROS) in this phenomenon. After-ripening entailed a progressive accumulation of ROS, namely superoxide anions and hydrogen peroxide, in cells of embryonic axes. This accumulation, which was investigated at the cellular level by electron microscopy, occurred concomitantly with lipid peroxidation and oxidation (carbonylation) of specific embryo proteins. Incubation of dormant seeds for 3 h in the presence of hydrogen cyanide (a compound that breaks dormancy) or methylviologen (a ROS-generating compound) also released dormancy and caused the oxidation of a specific set of embryo proteins. From these observations, we propose a novel mechanism for seed dormancy alleviation. This mechanism involves ROS production and targeted changes in protein carbonylation patterns.     

Breakage of Pseudotsuga menziesii seed dormancy by cold treatment as related to changes in seed ABA sensitivity and ABA levels

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°C), the thermal optimum being approximately 25°C. The seeds were also very sensitive to oxygen deprivation. Treatment of dormant seeds at 5°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°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°C to higher temperatures, and the decrease was faster at 25 than at 15°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°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°C. The possible involvement of ABA synthesis and of responsiveness of seeds to ABA in the breaking of dormancy by cold treatment is discussed.     

Changes in ABA turnover and sensitivity that accompany dormancy termination of yellow-cedar (Chamaecyparis nootkatensis) seeds.

Yellow-cedar (Chamaecyparis nootkatensis [D. Don] Spach) seeds exhibit prolonged coat-imposed dormancy following their dispersal from the parent plant. Analyses were undertaken using S-(+)-[(3)H] abscisic acid (ABA) to monitor the capacity of embryos to metabolize ABA following their isolation from seeds subjected to various dormancy-breaking and control treatments. Radiolabelled phaseic acid (PA) and dihydrophaseic acid (DPA) were detected in embryos and, to a greater extent in the surrounding media, by 48 h regardless of whether the embryos had been excised from seed previously subjected to only a 3 d soak or to a full dormancy-breaking treatment. Of the two enantiomers of ABA, only the natural S-(+)-ABA effectively inhibited germination of isolated embryos. A metabolism-resistant synthetic ABA analogue S-[8',8',8',9',9',9']-hexadeuteroabscisic acid, S-(+)-d6-ABA, consistently slowed the germination rate of excised embryos to a greater extent than that caused by natural S-(+)-ABA. The deuterium-labelled ring methyl groups of the analogue made it more resistant to oxidation by yellow-cedar embryos and thus rendered the analogue more persistent and possessing greater activity. With increasing time of exposure to moist chilling, yellow-cedar embryos became increasingly insensitive to both ABA and to the analogue. Subjecting seed to chemical treatments (GA(3) in combination with 1-propanol) prior to moist chilling strongly enhanced the germinability of whole seeds. This treatment also had a relatively greater impact on ABA metabolism than did moist chilling alone, as indicated by a greater capacity of S-(+)-d6-ABA to inhibit the germination of embryos as compared to S-(+)-ABA. Moist chilling was most critical for reduced ABA sensitivity of embryos. A change in the embryo's ability to metabolize ABA and reduced embryo sensitivity to ABA are two factors associated with dormancy termination of whole seeds of yellow cedar; a change in only one of these factors is insufficient to elicit high germinability.     

Patterns of storage protein and triacylglycerol accumulation during loblolly pine somatic embryo maturation

Conifer somatic embryo germination and early seedling growth are fundamentally different than in their zygotic counterparts in that the living maternal megagametophyte tissue surrounding the embryo is absent. The megagametophyte contains the majority of the seed storage reserves in loblolly pine and the lack of the megagametophyte tissue poses a significant challenge to somatic embryo germination and growth. We investigated the differences in seed storage reserves between loblolly pine mature zygotic embryos and somatic embryos that were capable of germination and early seedling growth. Somatic embryos utilized in this study contained significantly lower levels of triacylglycerol and higher levels of storage proteins relative to zygotic embryos. A shift in the ratio of soluble to insoluble protein present was also observed. Mature zygotic embryos had roughly a 3:2 ratio of soluble to insoluble protein whereas the somatic embryos contained over 5-fold more soluble protein compared to insoluble protein. This indicates that the somatic embryos are not only producing more protein overall, but that this protein is biased more heavily towards soluble protein, indicating possible differences in metabolic activity at the time of desiccation.     

Wheat seed proteins regulated by imbibition independent of dormancy status

Seed dormancy is an important trait in wheat (Trticum aestivum L.) and it can be released by germination-stimulating treatments such as after-ripening. Previously, we identified proteins specifically associated with after-ripening mediated developmental switches of wheat seeds from the state of dormancy to germination. Here, we report seed proteins that exhibited imbibition induced co-regulation in both dormant and after-ripened seeds of wheat, suggesting that the expression of these specific proteins/protein isoforms is not associated with the maintenance or release of seed dormancy in wheat.     

Interpopulation variability on embryo growth,seed dormancy break,and germination in the endangered Iberian daffodil Narcissus eugeniae (Amaryllidaceae)

The main goal of the study was to assess germination requirements in a threatened daffodil to elaborate a detailed protocol for plant production from seeds, a key tool for conservation. Experiments were carried out both in the laboratory and outdoor conditions. In Pseudonarcissi section, endemic Iberian species of Narcissus studied heretofore have different levels of morphophysiological dormancy (MPD). Embryo length, radicle emergence, and shoot emergence were analyzed to determine the level of MPD. Both interpopulational variability and seed storage duration were also studied. Mean embryo length in fresh seeds was 1.32 mm and the embryo had to grow until it reached at least 2.00 mm to germinate. Embryo growth occurs during warm stratification, after which the radicle emerges when temperatures go down. Seed dormancy was broken in the laboratory at 28/14°C in darkness followed by 15/4°C, but the germination percentage varies depending on the population. In outdoor conditions, seed dispersal occurs in June, the embryo grows during the summer and then the radicle emerges in autumn. The radicle system continues to grow during the winter months, but the shoot does not emerge until the beginning of the spring because it is physiologically dormant and requires a cold period to break dormancy. Early cold temperatures interrupt embryo growth and induce dormancy in seeds with an advanced embryo development. Seeds of N. eugeniae have deep simple epicotyl MPD. In addition, we found that embryo growth and germination were improved by seed storage duration.     

A tonoplast intrinsic protein (TIP) is present in seeds, roots and somatic embryos of Norway spruce (Picea abies)

Many plant species contain a seed-specific tonoplast intrinsic protein (TIP) in their protein storage vacuoles (PSVs). Although the function of the protein is not known, its structure implies it to act as a transporter protein, possibly during storage nutrient accumulation/breakdown or during desiccation/imbibition of seeds. As mature somatic embryos of Picea abies (L.) Karst. (Norway spruce) contain PSVs, we examined the presence of TIP in them. Both the megagametophyte and seed embryo accumulate storage nutrients, but at different times and we therefore studied the temporal accumulation of TIP during seed development. Antiserum against the seed-specific a-TIP of Phaseolus vulgaris recognized an abundant 27 kDa tonoplast protein in mature seeds of P. abies. By immunogold labeling of sectioned mature megagametophytes we localized the protein to the PSV membrane. We also isolated the membranes of the PSVs from mature seeds and purified an integral membrane protein that reacted heavily with the antiserum. A sequence of 11 amino acid residues [AEEATHPDSIR], that was obtained from a polypeptide after in-gel trypsin digestion of the purified membrane protein, showed high local identity to a-TIP of Arabidopsis thaliana and to a-TIP of P. vulgaris. The greatest accumulation of TIP in the megagametophytes occurred at the time of storage protein accumulation. A lower molecular mass band also stained from about the time of fertilization until early embryo development. The staining of this band disappeared as the higher molecular mass (27 kDa) band accumulated in the megagametophyte during seed development. Total protein was also extracted from developing zygotic embryos and from somatic embryos. In zygotic embryos low-levels of TIP were seen at all stages investigated, but stained most at the time of storage protein accumulation. The protein was also present in mature somatic embryos but not in proliferating embryogenic tissues in culture. In addition to the seed tissue material, the antiserum also reacted with proteins present in extracts from roots and hypocotyls but not cotyledons from 13-day-old seedlings.     

Identification and characterization of dehydrins in horse chestnut recalcitrant seeds

The fraction of heat-stable dehydrins cytosolic proteins from mature recalcitrant seeds of horse chestnut (Aesculus hippocastanum L.) was studied in the period of their dormancy and germination in order to identify and characterize stress-induced dehydrin-like polypeptides. In our experiments, in tissues of dormant seeds, dehydrin was identifies by immunoblotting as a single bright band with a mol wt of about 50 kD. Low-molecular-weight heat-stable proteins with mol wts of 25 kD and below 16 kD, which were abundant in this fraction, did not cross-react with the antibody. Dehydrin was detected in all parts of the embryo: in the cells of axial organs, cotyledon storage parenchyma, and petioles of cotyledonary leaves. This indicates the absence of tissue-specificity in distribution of these proteins in the horse chestnut seeds. Dehydrins were detected among heat-stable proteins during the entire period of stratification and also radicle emersion. During radicle emergence, not only the fraction of heat-stable proteins was reduced but also the proportion of dehydrins in it decreased. In vitro germination of axes excised at different terms of stratification also resulted in dehydrin disappearance. When growth of excised axes was retarded by treatments with ABA, cycloheximide, or α-amanitin, dehydrins did not disappeared from the fraction of heat-stable proteins. When excised axes were germinated in vitro in the presence of compounds, which did not affect their growth or stimulated it (dehydrozeatin, glucose), this resulted in dehydrin disappearance. This means that dehydrin metabolism is closely related to the process of germination. Dehydrin in the horse chestnut seeds could cross-react with the antibody against ubiquitin, which can indicate the involvement of ubiquitination in the process of dehydrin degradation during germination via the proteasome system. The analysis of total proteins of the homogenate from horse chestnut seeds revealed, along with a 50-kD heat-stable dehydrin, one more component with a mol wt of 80 kD, which was located in the fraction of heat-sensitive proteins and was named as a dehydrin-like protein. It was demonstrated that dehydrins in horse chestnut seeds represented only a very small fraction of heat-stable cytosolic proteins. The role and function of major heat-stable proteins in horse chestnut seeds are yet to be studied.     

Proteomic analysis of seed dormancy in Arabidopsis

The mechanisms controlling seed dormancy in Arabidopsis (Arabidopsis thaliana) have been characterized by proteomics using the dormant (D) accession Cvi originating from the Cape Verde Islands. Comparative studies carried out with freshly harvested dormant and after-ripened non-dormant (ND) seeds revealed a specific differential accumulation of 32 proteins. The data suggested that proteins associated with metabolic functions potentially involved in germination can accumulate during after-ripening in the dry state leading to dormancy release. Exogenous application of abscisic acid (ABA) to ND seeds strongly impeded their germination, which physiologically mimicked the behavior of D imbibed seeds. This application resulted in an alteration of the accumulation pattern of 71 proteins. There was a strong down-accumulation of a major part (90%) of these proteins, which were involved mainly in energetic and protein metabolisms. This feature suggested that exogenous ABA triggers proteolytic mechanisms in imbibed seeds. An analysis of de novo protein synthesis by two-dimensional gel electrophoresis in the presence of [(35)S]-methionine disclosed that exogenous ABA does not impede protein biosynthesis during imbibition. Furthermore, imbibed D seeds proved competent for de novo protein synthesis, demonstrating that impediment of protein translation was not the cause of the observed block of seed germination. However, the two-dimensional protein profiles were markedly different from those obtained with the ND seeds imbibed in ABA. Altogether, the data showed that the mechanisms blocking germination of the ND seeds by ABA application are different from those preventing germination of the D seeds imbibed in basal medium.     

Development and storage-protein synthesis in Brassica napus L. embryos in vivo and in vitro

Immature embryos of Brassica napus were cultured in vitro with and without various concentrations of germination inhibitors, and the progress of embryogeny was monitored by comparing accumulation of storage proteins in culture with the normal accumulation in seeds. The two major B. napus storage proteins (12S and 1.7S) were purified from seed extracts and analyzed by rocket immunoelectrophoresis (12S protein) or by sodium lauryl sulfate polyacrylamide gel electrophoresis (1.7S protein). During embryo development within seeds both the 12S and 1.7S proteins were first detected when the cotyledons were well developed (embryo dry weight, 0.4 mg), and each storage protein accumulated at an average rate of 26 g d^-1 during maximum deposition. Accumulation of the 1.7S protein stopped when the water content of the embryo began to decline (embryo DW, 2.7 mg), but accumulation of the 12S protein continued until seed maturity (embryo DW, 3.6 mg). At the end of embryo development the 12S and the 1.7S proteins comprised approx. 60 and 20% of the total salt-soluble protein, respectively. When embryos were removed from seeds at day 27, just as storage protein was starting to accumulate, and placed in culture on a basal medium, they precociously germinated within 3d, and incorporation of amino acids into the 12S storage protein dropped from 3% of total incorporation to less than 1%. If 10^-6 M abscisic acid (ABA) was included in the medium, amino-acid incorporation into the 12S protein increased from 3% of total incorporation when embryos were placed into culture to 18%, 5d later, and the accumulation rate (27.1±2.6 g embryo^-1 d^-1) matched the maximum rate observed in the seed. High osmotica, such as 0.29 M sucrose or mannitol, added to the basal medium, also inhibited precocious germination, but there was a lag period before 12S-protein synthesis rates equaled the rates on ABA media. These results indicate that some factor in the seed environment is necessary for storage-protein synthesis to proceed, and that ABA is a possible candidate.Abbreviations ABA abscisic acid - PAGE polyacrylamide gel electrophoresis - PMSF phenylmethylsulfonylfluoride - SDS sodium lauryl sulfate     

De novo ABA synthesis and expression of seed dormancy in a gymnosperm: Pseudotsuga menziesii

Seeds of dormant Douglas-fir seeds germinated poorly when they were cultivated at 20–23 °C while isolated embryos germinated fully within two weeks. Seed dormancy was therefore imposed on the embryo by its surrounding structures. This physiological behaviour was well correlated with changes in ABA level during culture. Indeed, the ABA level decreased in isolated embryos while it increased in both embryo and megagametophyte during culture of whole seeds. The origin of this increase was analysed and the different ways by which seed coats could interfere with ABA accumulation are discussed.     

Dark-mediated dormancy release in stratified Lolium rigidum seeds is associated with higher activities of cell wall-modifying enzymes and an apparent increase in gibberellin sensitivity

Dormancy release in freshly matured, imbibed annual ryegrass (Lolium rigidum) seeds is inhibited by light and involves a decrease in seed sensitivity to abscisic acid. Other processes involved in dormancy release in the dark were investigated by measuring seed storage compound mobilisation and the activity of cell wall-degrading enzymes. Activities of endo-β-mannanase and total peroxidase were higher in dark-stratified compared to light-stratified seeds, indicating that weakening of the structures constraining the embryo was accelerated in the dark. A dramatic degradation of storage proteins in light-stratified seeds, accompanied by induction of a high molecular mass protease, suggests that maintenance of storage(-like) proteins is also important in dark-mediated dormancy release. α-Amylase activity was induced in dark-stratified seeds at least 48 h prior to radicle emergence upon transfer to conditions permitting germination, or in light-stratified seeds supplied with exogenous gibberellin A₄. This suggests that (a) α-amylase is involved in stimulation of germination of non-dormant L. rigidum seeds, and (b) dark-stratified seeds have an increased sensitivity to gibberellins which permits the rapid induction of α-amylase activity upon exposure to germination conditions. Overall, it appears that a number of processes, although possibly minor in themselves, occur in concert during dark-stratification to contribute to dormancy release.