The germination of intact, moist-chilled white spruce (Picea glauca [Moench.] Voss.) seeds and liquid–nitrogen–decoated (LN2), unchilled seeds is significantly more than of intact, unchilled seeds and intact, unchilled seeds exposed to LN2. The testa is largely responsible for imposition of dormancy in these seeds, although the megagametophyte and/or nucellus also play a role. One or both of these tissues undergoes significant weakening during moist chilling. Excised embryos from dormant and nondormant white spruce seeds elongate when placed on solid Murashige and Skoog minimal organics medium supplemented with a carbon source (sucrose, glucose or galactose), but elongate very little on medium without sugar. They are not killed by rapid imbibition on unsupplemented media. Thus, embryos of white spruce do not exhibit innate dormancy, but are dependent on a carbon source for elongation, and have dormancy imposed on them by their surrounding structures.

On the basis of embryo and seed colour, morphology, accumulation of fresh and dry weight, seed development of Chinese cabbage (Brassica rapa subsp. pekinensis) can be divided into 10 discrete but contiguous stages. Precocious germination (PG) occurs in the seeds of a Chinese cabbage mutant on the parent plant mainly during the maturation period (from stage 5 to 8), with either the radicle or the cotyledon protruding out of the testa. All plants of the mutant line produce some PG seeds, but among the seeds, only 18% (maximum) germinate precociously. The developing mutant seeds have higher water contents and lower dry weights than those of the wild-type and are less tolerant of desiccation. The testa structure of Chinese cabbage seed is similar to that of other Brassica species. In developing seeds, it consists of the epidermis, subepidermis, palisade layer and pigmented layers, while in the mature dry seeds, all the cell layers except the palisade layer are crushed into non-cell structures and are stacked on the outer and inner faces of the palisade layer; the aleurone layer is fused to the testa. An alteration in testa structure of the mutant seeds (both non-germinated and precociously-germinated seeds) is probably related to PG. The secondary cell wall materials are less, or not deposited on the radial and basal walls of the palisade cells on the adaxial side of the testa, which could result in a weaker mechanical restraint, thus leading to PG.

Five distinct groups of storage proteins are synthesized by embryos during development in the fruit at 35 days after pollination (35 DAP); none of them is synthesized in germinating embryos 48 h after isolation (48 HASI) of the seeds from the fruit. Endo-β-mannanase, a marker of germination and seedling growth, is produced in the isolated seeds, but not in the developing seed in situ 35 DAP. When seeds at this stage are removed from the fruit, but remain in intimate contact with the locular and sheath tissue, they continue to synthesize storage proteins for at least 48 HASI. Removal of the sheath from seeds isolated 35 DAP stops storage protein synthesis, and increases endo-β-mannanase activity. Both abscisic acid (ABA) and osmoticum at physiological concentrations maintain the synthesis of storage proteins in seeds isolated 35 DAP for at least 48 HASI, although osmoticum is more effective in preventing their eventual germination. Thus the effects of the sheath and the locular tissue are mimicked by both ABA and osmoticum in relation to the maintenance of storage protein synthesis in seeds 35 DAP and in the suppression of endo-β-mannanase activity for at least 48 HASI.

Endosperms of fenugreek and carob which have been leached following their isolation exhibit increased activity of endo-β-mannanase, part of which is released into the surrounding incubation medium. This activity was suppressed by the addition to the endosperms of abscisic acid or endosperm/seed coat leachate. The leachate from carob inhibited the increase of endo-β-mannanase activity in fenugreek and that from fenugreek inhibited enzyme activity in carob but more weakly; neither of these leachates inhibited the production of α-amylase in wheat endosperm. Polyethylene glycol-induced stress on non-leached endosperms inhibited endo-β-mannanase production, but not when the stress was applied following endosperm leaching. However, the stress did reduce the amount of enzyme released into the surrounding incubation medium. Several isoenzymes of mannanase could be detected on isoelectric focusing activity gels in the endosperms of both fenugreek and carob, but their pis differed between the two species. When activity was suppressed by ABA or leachate, all pl forms declined equally.

Two different pre-emergence treatments preventing radicle protrusion by limiting the extent of imbibition were tested on conifer seeds. Osmotic priming in solutions of polyethylene glycol (PEG) which prevented radicle protrusion during prolonged (3 weeks) treatment resulted in variable percentage germination, mean germination time, and coefficient of uniformity of germination among different seedlots when placed on water. All three germination parameters were either unchanged or inferior in seeds subjected to the various PEG concentrations, treatment temperatures and treatment durations when compared with prechilled controls. In contrast, white spruce and jack pine seeds invigorated at 30% moisture content and 15°C in Gore-Tex membrane tubes consistently germinated faster than prechilled control seeds when subsequently placed on water. Tube invigoration affected neither the ability of seeds to complete germination nor the uniformity of germination. The decrease in mean germination time as treatment duration progressed was always linear and plateaued at approximately 4 days after the tube-invigorated seeds were placed on water regardless of species or seedlot. On average, tube invigoration decreased white spruce mean germination time by 0.13±0.02, and black spruce by 0.23±0.03 day per day of invigoration for up to 20 days of treatment. Jack pine mean germination time decreased 0.2±0.04 day per day of invigoration for up to 14 days of treatment.

The rice (Oryza sativa L.) cv. Taichung 65, a japonica subspecies, was used to characterize the isoform, protein and gene expression patterns of endo-β-mannanase during and after seed germination. Activity assays and isoform analyses of whole grains or seed parts (scutellum, aleurone layer and starchy endosperm) revealed that seeds began to express endo-β-mannanase activity at 48 h from the start of imbibition at 25°C, after the completion of germination of most seeds. Three isoforms of endo-β-mannanase (pI 8.86, pI 8.92 and pI 8.98) were detected in the aleurone layer and starchy endosperm, but only two (pI 8.86 and pI 8.92) were present in the scutellum. The endo-β-mannanase in the starchy endosperm was mainly from the aleurone layer. Western blot analysis, using a tomato anti-endo-β-mannanase antibody, indicated that an endo-β-mannanase protein was present in an inactive form in dry grains. The amount of this protein decreased in the scutellum, but increased in the aleurone layer during and after germination. Thus, the increase in endo-β-mannanase activity in rice grains may be due to the activation of extant proteins and/or the de novo synthesis of the enzyme. Northern blot analysis showed that four putative rice endo-β-mannanase genes (OsMAN1, OsMAN2, OsMAN6 and OsMANP) were expressed in germinating and germinated rice grains. However, OsMANP was not expressed in the scutellum. The amount of OsMAN6 mRNA decreased after the completion of germination and paralleled the decline in endo-β-mannanase protein. In the aleurone layer, the increase of OsMAN2, OsMAN6 and OsMANP mRNA was prior to the increase of endo-β-mannanase protein.

Endo-β-mannanase (EC 3.2.1.78) is involved in the hydrolysis of mannan-type polysaccharides that are present in plant cell walls, especially those of the seed endosperm. The genes encoding the endo-β-mannanases have been studied extensively in tomato (Solanum lycopersicum), and five genes (LeMAN1, LeMAN2, LeMAN3, LeMAN4 and LeMAN5) and/or their products have been isolated and characterized. LeMAN1, LeMAN2 and LeMAN3 are expressed in tomato seeds, LeMAN4 in the fruit and LeMAN5 in the flower. LeMAN5 and LeMAN2 are now considered to be the same gene, and the former is re-designated as LeMAN2*. Transcripts of LeMANs 1, 2 and 3 are detected only in the endosperm of tomato seeds, and their synthesis is promoted by gibberellic acid. LeMAN4, in the fruit, occurs as LeMAN4a and LeMAN4i genes that encode an active or inactive form of endo-β-mannanase, respectively. LeMAN1–4 enzymes encoded by these genes share 80% similarity in amino acid sequence. In tomato, the leucine amino acid present near to the C-terminus of the endo-β-mannanase is the most important for achieving full activity of the enzyme.

The breaking of dormancy in seeds can be elicited by many factors, including temperature and short exposure to low molecular weight amphipathic molecules such as primary alcohols, monocarboxylic acids and anaesthetics. Their action has been suggested to be mediated through effecting changes to membranes. Paradoxically, though, these molecules can inhibit the germination of some non-dormant seeds. Here, we review the structure–activity relationships between amphipathic molecules and dormancy breaking and, based on the known responses of membranes to them and to temperature changes, offer an alternative interpretation of the data and a new hypothesis to explain their action. We suggest that amphipathic molecules break dormancy by partitioning into the membrane, thereby increasing and optimizing phospholipid headgroup spacing. This, in turn, facilitates the binding and activation of a peripheral membrane protein component of a signal transduction pathway that is essential for the completion of germination. In cases where amphipathic molecules inhibit germination, it is predicted that they cause the optimal headgroup spacing to be exceeded, thus preventing subsequent association of the membrane with the peripheral protein component. The hypothesis is extended to explain membrane changes that can lead to dormancy breaking during dry after-ripening.

Endo-β-mannanase (EC 3.2.1.78), an enzyme that mobilizes the endosperm cell walls of the lettuce (Lactuca sativa L.) seed, increases in activity in the micropylar and lateral regions of this tissue following the completion of germination. Its complementary DNA (cDNA) sequence (LsMan1) was determined using the polymerase chain reaction (PCR) with degenerate primers. The 3‘-end of the cDNA sequence was obtained by 3‘-end rapid amplification of cDNA ends (RACE), and the 5‘-end sequence by genome walking and 5‘-end RACE. The predicted amino acid sequence from the cDNA has a high identity with endo-β-mannanases present in other species (e.g. 67% identity with coffee β-1,4-mannan endohydrolase, 62% identity with tomato fruit endo-β-mannanase). Southern blot analysis suggests the presence of several members of an endo-β-mannanase gene family in the lettuce genome. Several isoforms of the enzyme, including three major ones, were detected by isoelectric focusing. Based on Northern blot analysis, accumulation of the endo-β-mannanase mRNA occurred only after lettuce seeds had germinated, and increased thereafter, although enzyme activity persisted after transcription declined.

Seed storage proteins were analysed in 27 varieties of alfalfa (Medicago sativaL.); these included five subspecies (glomerata, caerulea, falcata, hemicycla, praefalcata), seven of the nine sources of Medicago germplasm introduced into North America and a sample of additional cultivars. The protein patterns were remarkably consistent for all of these taxa. One-dimensional and two-dimensional gel electrophoresis revealed only minor differences in polypeptide composition within each of the three major classes of storage protein (7S globulin, 11S globulin, 2S albumin). The slight variations that were found provided no information on either parentage or evolutionary relationships amongst these particular taxa. Nonetheless, persistent and reproducible heterogeneity of some minor polypeptides of 11S globulin (medicagin) may be useful under other circumstances for cultivar identification in alfalfa. Both subfamilies (I and II) of the 11S globulin were strongly expressed in all of the cultivars and subspecies examined. It was concluded that this structural divergence within the 11S storage protein family predated the evolution of the M. sativa L. species complex. Most of the variability in storage proteins was quantitative. However, even this variability was reduced when data were standardized with respect to seed dry weights. The consistent similarities in qualitative and quantitative expression of seed storage proteins amongst all of these taxa suggest a high degree of uniformity in both seed physiology and genetics within the alfalfa species complex.

Grains of indica rice (Oryza sativa cv. Peiza 67) exhibit an increase in endo-β-mannanase activity, mostly after the completion of germination. According to tissue blots, the initial increase occurs in association with the embryo, and possibly the scutellum, although the largest sustained increase in activity is in the peripheral regions of the endosperm. The aleurone layer, being the only living region of the endosperm, is presumably the site of synthesis and secretion of the enzyme into the non-living, starch-laden region. β-Mannosidase activity is low throughout germination and subsequent seedling growth, particularly in the endosperm regions. Its activity profile does not mimic that of endo-β-mannanase. In the intact grain, gibberellin (GA) causes a relatively small increase in endo-β-mannanase activity, while abscisic acid (ABA) causes a large decrease; this inhibition is overcome to a considerable extent when GA is supplied along with ABA. β-Mannosidase activity is little affected by either GA or ABA. Embryoless half-grains imbibed in water exhibit only a small increase in endo-β-mannanase activity with time of imbibition, showing the necessity for a stimulus from the embryo for this to occur. Incubating half-grains in the presence of GA results in a large increase in enzyme activity; ABA reduces the amount of activity compared to the water controls. GA is capable of reversing the inhibitory effect of ABA with respect to endo-β-mannanase activity. As in the intact grains, β-mannosidase activity in the half-grains is unaffected by either GA or ABA. It is concluded that the major site for the production of endo-β-mannanase activity is the aleurone layer, and this event is influenced by the presence of the embryo; in the absence of the latter, the increase in enzyme activity is stimulated by GA. β-Mannosidase activity is low throughout germination and post-germination, it is not influenced by GA and ABA, and thus its activity is not regulated in a coordinated manner with that of endo-β-mannanase.

The thickened cell walls of endosperm cells of asparagus (Asparagus officinalis) seeds are composed of glucomannans. During development, the extensive thickening of the cell wall is specific to the lateral region of the endosperm and not to the micropylar region. The micropylar endosperm may thus be predisposed to facilitate germination. Following germination there is a progressive mobilization of the reserves in the cells of the lateral endosperm in a wave-like manner away from the haustorial cotyledon, accompanied by the loss of cytoplasm and the crushing of the lateral endosperm cells. Although the internal reserves are mobilized from the micropylar endosperm cells, the cells themselves remain intact throughout the period of lateral endosperm mobilization and may form a living barrier to prevent the loss of soluble hydrolysis products of the lateral endosperm to the surrounding environment. The location and timing of endo-β-mannanase production and the increase in activity of β-mannoside mannohydrolase in seeds of germinated asparagus were followed. Endo-β-mannanase activity increases greatly in the endosperm until the mid-point of mobilization, and is about 45 times higher than in the embryo on a per seed part basis. Unlike endo-β-mannanase, which is extractable in low-salt buffer, β-mannoside mannohydrolase requires high salt (0.5 M NaCl) for extraction. This enzyme continually increases in activity in both the endosperm and embryo following germination, with the majority of the activity being concentrated in the embryo when considered on a per seed part basis.