The great majority of seed proteins that are stored in the vacuole prior to desiccation are transported via the Golgi apparatus. In this organelle they are separated from other products of the secretory pathway. Evidence is accumulating that the mechanism for segregation of storage proteins is different from that of soluble proteins destined for lytic vacuoles: it rarely seems to require short targeting propeptides at the N- or C-terminus. Instead, the three-dimensional conformation of the protein appears to be a critical factor, leading to self-assembly into osmiophilic aggregates. Also unusual is that this process starts immediately after entry into the Golgi apparatus, i.e. at the cis-cisternae, rather than at the trans-pole where acid hydrolases are packaged into clathrin-coated vesicles. Storage protein aggregates accumulate into so-called “dense” vesicles at the periphery of the cisternae and are transported towards the trans-pole of the Golgi apparatus by cisternal progression. Before the dense vesicles are released, clathrin-coated vesicles form at their surface; however, the function of the latter remains the object of speculation. In other eukaryotes, delivery of Golgi-derived lumenal products to the vacuole does not occur directly, but via a pre-vacuolar compartment. There is evidence that this is also the case for plants, and in developing pea cotyledons the pre-vacuolar compartment takes the form of a large multivesicular body. Ultimately this appears to fuse in toto with the protein storage vacuole.

A suite of mechanisms or processes that together have been implicated in the acquisition and maintenance of desiccation tolerance in orthodox seeds is discussed in the context of the behaviour of desiccation-sensitive seeds, and where appropriate, parallels are drawn with the situation in vegetative plant tissues that tolerate dehydration. Factors included are: physical characteristics of cells and intracellular constituents; insoluble reserve accumulation; intracellular de-differentiation; metabolic ‘switching off’; presence, and efficient operation, of antioxidant systems; accumulation of putatively protective substances including LEAs, sucrose and other oligosaccharides, as well as amphipathic molecules; the presence and role of oleosins; and the presence and operation of repair systems during rehydration. The variable response to dehydration shown by desiccation-sensitive seeds is considered in terms of the absence or incomplete expression of this suite of mechanisms or processes.

Three categories of damage are envisaged: (i) reduction in cell volume which can lead to mechanical damage; (ii) aqueous-based degradative processes, probably consequent upon deranged metabolism at intermediate water contents. This is termed ‘metabolism-induced damage’ and its extent will depend upon the metabolic rate and the rate of dehydration; and (iii) the removal of water intimately associated with macromolecular surfaces leading to denaturation: this is referred to as desiccation damage sensu stricto. The effects of drying rate and the maturity status of seeds are considered in relation to the responses to dehydration, leading to the conclusion that the concept of critical water contents on a species basis is inappropriate. Viewing seed postharvest physiology in terms of a continuum of behaviour is considered to be more realistic than attempting precise categorization.

Rapid dehydration of excised embryonic axes (or other explants) from desiccation-sensitive seeds permits retention of viability (in the short term) to water contents approaching the level of non-freezable water. This opens up the possibility of long-term conservation, by cryopreservation techniques, of the genetic resources of species producing non-orthodox seeds.

Mature and immature axes of Theobroma cacao (cocoa) seeds tolerated desiccation under a rapid-drying regime to critical water contents of 1.0 and 1.7 g g-1 dw, respectively. These critical water contents corresponded to water contents below which activities of free radical-scavenging enzymes (ascorbate peroxidase, peroxidase and superoxide dismutase) decreased rapidly during desiccation. The decline in axis viability below the critical water content was correlated with sharp increases in lipid peroxidation and cellular leakage. Cotyledon tissues were more desiccation-tolerant than axes, with a low critical water content of 0.24 g g–1dw. Desiccation sensitivity in cotyledon tissues was also correlated with the decrease in superoxide dismutase activity and increased lipid peroxidation products. However, in the cotyledons, no ascorbate peroxidase activity was detected at any water content, and peroxidase activity was gradually reduced as desiccation proceeded. Cocoa embryonic axes contained large amounts of sucrose, raffinose and stachyose but only traces of reducing monosaccharides. Desiccation sensitivity of recalcitrant cocoa axes did not appear to be due to the lack of sugar-related protective mechanisms during desiccation, and it was more likely related to the decrease of enzymic protection against desiccation-induced oxidative stresses.

Peroxiredoxins are thiol–requiring antioxidants found in organisms ranging from bacteria to humans. They can be divided into two subgroups with either one or two conserved cysteine residues. In plants, 1–Cys peroxiredoxins have been identified in a number of grasses and cereals, and in the dicotyledonous species Arabidopsis thaliana. In contrast to other antioxidants, the 1–Cys peroxiredoxin genes are expressed solely in seeds, and only in the parts of the seeds surviving desiccation, i.e. the embryo and the aleurone layer. The expression pattern is characteristic of late embryogenesis–abundant genes. The PER1 protein of barley is present in high concentrations in the nucleus at the onset of desiccation. 1–Cys genes are expressed in a dormancy–related manner in mature seeds, in that transcript levels are high in imbibed dormant seeds, but disappear upon germination of their non–dormant counterparts. 1–Cys transcript levels can be up–regulated by ABA and osmotic stresses and suppressed by gibberellic acid. Two hypotheses have been put forward on the function of 1–Cys peroxiredoxins in seed physiology. First, these proteins might protect macromolecules of embryo and aleurone cells against damaging reactive oxygen species during seed desiccation and early imbibition. And second, seed peroxiredoxins might play a role in the maintenance of dormancy. These hypotheses are discussed, taking into account present knowledge of the biochemistry and molecular biology of peroxiredoxins.

Tomato seeds that have been dried, imbibed and redried (primed) develop internal free space between the embryo and endosperm. Seeds of the ABA-deficient sitiens(sitw) tomato mutant can exhibit internal free space at the completion of seed development even without priming. Both primed and sitwseeds germinate more rapidly than untreated wild-type seeds. To determine whether internal anatomy predicts germination physiology, individual sitwand primed wild-type seeds were sorted into three categories based upon the extent of internal free space observed non-destructively using X-radiography. Category 3 (C3, extensive free space present) sitw seeds completed germination more rapidly than all other seed categories and genotypes in water, in abscisic acid (ABA) or under far-red illumination. The force necessary to puncture the endosperm caps (and testa) of C3 sitw seeds was less, and the percentage of nuclei in C3 sitw radicle tips in the G2 stage of the cell cycle was greater than for all other seed categories. Wild-type seeds exhibited free space following long-term priming, but germination was still prevented by far-red light and ABA, and endosperm cap strength and nuclear DNA contents were not altered. Endo-β-mannanase activity of individual endosperm caps was not consistently related to their resistance to puncture. While internal free space is diagnostic for primed tomato seeds and occurs in a fraction of sitw seeds, it is not predictive of many aspects of germination physiology.

The first cell cycle of Vicia faba L. seeds, which begins upon imbibition of dry seeds and is completed at the first mitosis after radicle protrusion, was characterised by the flow cytometry and immunodetection of nucleolin and tubulins in root tip meristems. Flow cytometry revealed highly synchronised profiles from the quiescent G1 phase to the late G2 phase, indicating uniform cell cycle progression within a root tip until the first mitosis. Using immunoblotting, nucleolin was detected in two distinct bands with the apparent molecular masses of 89 and 99 kD; the former was detected only in seeds imbibed at 4°C for 1 day whereas the latter was found at all stages examined, suggesting that the 89 kD nucleolin may be seed-specific. Unusual localization of nucleolin in cold-imbibed seeds, undetectable in half of the cells and present in nucleoplasm, was revealed by immunofluorescence microscopy. While α- and β-tubulin were detected at all stages and no significant changes in accumulation of the proteins were observed, few microtubules were detected at the beginning of germination when cells were still in the G1 phase, suggesting that microtubules may be depolymerized in the dry seeds.

The role of abscisic acid (ABA) in seed germination of two cultivars of lettuce (Lactuca sativa L.; light-sensitive Ritsa and light-insensitive Strada) was investigated. The inhibition of Ritsa seed germination by exogenous ABA was higher than that of Strada seeds, the extent of inhibition of both cultivars being reduced by a short light break. At 25°C the sensitivity of both cultivars to exogenous ABA was higher than at 15°C. The endogenous level of ABA was similar in dry seeds of both cultivars, increasing temporarily in Ritsa seeds during the first 4 h of imbibition in darkness but not in Strada seeds, nor in Ritsa seeds exposed to a short light break. The transitory increase of ABA content in Ritsa seeds imbibed in darkness was accompanied by increased expression of the gene responsive to ABA (Rab) under these conditions. Zorial (Norflurazone), an ABA-biosynthesis inhibitor, decreased ABA content and allowed dark germination of the light-requiring Ritsa seeds. A short light break induced germination of Ritsa seeds when applied at 24 and 48 h after imbibition onset, i.e. after the transitory increase of ABA. GA3, on the other hand was effective when applied at the beginning of imbibition. It seems that light induces germination of the photoblastic Ritsa seeds by both inhibiting ABA synthesis and decreasing seed sensitivity to ABA and inhibitory processes induced by it.

Grain production of two wheat cultivars (Triticum aestivum L. cv Recital and Scipion), known for their different germination behaviour, was studied at two different temperatures. The study of dormancy onset during grain development showed that in both cultivars, dormancy developed on the mother plant. In Recital grain, after a period of high germinability, dormancy developed for a transient period. However, full germination was obtained on medium supplemented with fluridone, showing that dormancy was associated with abscisic acid (ABA) biosynthesis inside the grain. Dormancy progressively disappeared during natural drying, at a slower rate for grains developing at 15°C than for grains developing at 25°C. However, at both temperatures, the release from dormancy was complete at maturity. In Scipion grain, dormancy was almost total throughout grain development irrespective of the temperature. However, grains could germinate in the presence of fluridone: changes in sensitivity to fluridone were observed during grain development, showing an increase in dormancy during the first half of development, followed by a progressive decrease during the second half. This decrease occurred later for grains developing at 15°C than for those developing at 25°C. In Scipion grain, unlike the cultivar Recital, release from dormancy was not completed before the end of development on the mother plant. An additional dry storage period was necessary which was shorter for grains developed at 25°C than for grains produced at 15°C. A comparison of embryo ABA levels after a 24-h culture in the presence or absence of fluridone, allowed the ABA synthesis to be estimated. It appears that the depth of dormancy was related to the estimated ABA synthesis capacity of the embryos.

We studied changes in dormancy (as imposed by the different structures surrounding the embryo, namely, endosperm, pericarp and glumellae) and its relationship with changes in embryonic ABA levels and sensitivity, in developing grains of two commercial barley cultivars: B 1215 and Quilmes Palomar, which have, respectively, a low and high dormancy level at harvest and, consequently, a contrasting sprouting behaviour in rainy years. Dormancy imposed by endosperm plus pericarp was gradually and similarly alleviated throughout development in both cultivars. The presence of the hull (glumellae), in contrast, completely inhibited germination of grains from both cultivars until physiological maturity (PM). From there on, hull-imposed dormancy was removed abruptly in B 1215 grains, while in Q. Palomar ones, it was removed at a much lower rate. This difference determined the contrasting sprouting behaviour of these two cultivars within the ‘time window’ going from PM to crop harvest. Embryonic ABA content and sensitivity were similar in the two cultivars throughout development until PM. From there on, ABA content and sensitivity in B 1215 embryos declined dramatically coinciding with the abrupt termination of hull-imposed dormancy observed in this cultivar. In contrast, ABA levels in Q. Palomar embryos remained high for longer and sensitivity to ABA declined at a much slower rate. This correlation suggests that hull-imposed dormancy in barley might be regulated by embryonic ABA levels and / or sensitivity. Inhibition of GA synthesis with paclobutrazol applied after anthesis lowered the germination capacity of grains from both cultivars without altering that of the naked caryopses, thus further suggesting that hull-imposed dormancy is under hormonal control.

Seed desiccation sensitivity was studied in nine species of the genus Coffeaby measuring seed viability after equilibration over various saturated salt solutions. A quantal response model based on the logistic distribution was developed in order to describe the typical S-shaped patterns observed. The closeness of fit of the desiccation sensitivity model was shown, and the assumption that seed desiccation sensitivity follows a continuous distribution within species was verified. For each species, the water content at which 50% of initial viability was reached, WC50, and a specific parameter describing the intra-specific variability, β, were calculated using a non-linear regression. A simplified water sorption model was developed which allowed easy calculation of water activity and water potential corresponding to WC50 (aw50 and Ψ50) for relative humidities ranging between 10 and 100%. Distribution of WC50and Ψ50 (or aw50) in the genus Coffea was homogeneous within the following intervals: from 0.05 to 0.38 g H2O.g−1dw for WC50 and from −168 to −11 MPa for Ψ50. Different classifications of the coffee species studied as regards to their desiccation sensitivity were obtained depending on whether WC50 or Ψ50was used for classification. The continuum for desiccation sensitivity observed within the nine species studied confirmed that coffee is an appropriate material for studying desiccation sensitivity.

Priming of seeds is generally intended to reduce time to germination, often leading to improved emergence. However, as a negative side effect, priming reduces longevity of seeds. We studied the possibilities to obtain primed seeds with reduced time to germination but with longevity similar to that of untreated seeds. For several species tested we found that the desired longevity could be obtained by keeping the seeds, after a priming treatment, under a mild water and / or temperature stress for a period of several hours to days. Time to germination did not increase again due to such a treatment. Optimal duration and degree of water stress were strongly temperature dependent. The methods applied to obtain primed seeds without loss of longevity are very similar to those used to induce desiccation tolerance in germinated seeds.

Internal cracks caused by high temperature or excessive moisture during maize (Zea mays L.) kernel development were characterized, and their effects on kernel quality were assessed. Pre-harvest stress cracks are often located near the middle of the kernel along the embryo axis, but they were also detected in other positions, irrespective of the shape of the kernel. X-ray analysis enabled visualisation of stress cracks that are invisible to the human eye and, therefore, gave a better estimate of the percentage of cracks. However, low temperature scanning electron microscopy of the surface of milled kernels revealed small cracks not noticed by visual or X-ray inspection. All kernels tested in this way had a crack of some sort in the endosperm tissue. Cracks were also frequent in the scutellum, but rare in the embryo axis. Endosperm cracks followed the boundary of the starch granules, but did not extend into the pericarp tissue. In contrast to external cracks caused by mechanical impact, pre-harvest internal stress cracks generally are not detrimental to germination and vigour. However, if the crack is located inside or perpendicular to the embryo axis, it may affect the quality of the kernel, probably by impeding nutrient translocation to the embryo.

The raffinose family of oligosaccharides (RFO) is present in relatively high levels in grain legume seeds. They are considered to be antinutritional compounds because they are, at least in part, believed to be responsible for causing flatulence in humans, which is the single most important factor in deterring people from including more legume seeds in their diet. The RFO also have important functions within the plant. They serve as transport metabolites in many legumes and have been proposed to play a positive role in cold acclimatisation and in conferring desiccation tolerance during seed maturation. These responses to environmental stresses are believed to result from the RFO acting as protecting agents for membrane bound-proteins. We have screened 70 pea lines from the test array of the John Innes Pisum germplasm collection, and lines were selected which had unusual RFO composition. The soluble sugars within these lines were quantified using High Performance Anion Exchange Chromatography with Pulsed Amperometric Detection (HPAEC-PAD), and variants were identified which were deficient in verbascose and one with a reduced level of raffinose. These selected lines are being used in a crossing programme designed to study the genetics of the RFO pathway and to produce new material to test the effect of specific RFO on the plant’s responses to the environment and on the diets of humans and animals.

Seeds of two grass (Arrhenatherum elatius and Festuca rubra), two herb (Plantago lanceolata and Rumex acetosa) and two legume (Lotus corniculatus and Trifolium repens) species were sown in summer 1995 at four densities (no seed, 1000, 10 000 and 50 000 seeds m−2) into an established rabbit-grazed grassland given factorial combinations of rabbit fencing (with and without fences) and soil disturbance (with and without cultivation). On plots where no seeds were sown, only the species with persistent seed banks (P. lanceolata, L. corniculatus and T. repens) showed enhanced seedling emergence in response to disturbance. In disturbed soil, seedling densities of all species increased with increasing density of sown seeds, the effects of which were still evident for plant cover 2 years after seed sowing. In undisturbed vegetation, A. elatius, F. rubra, P. lanceolata and R. acetosa showed increased seedling densities following seed sowing; but in each case, there was an upper asymptote to seedling recruitment, presumably due to microsite limitation. Rabbit grazing reduced seedling densities, with this reduction being more pronounced with disturbance than without. However, the effect of rabbit grazing did not persist for some species; seedling mortality of R. acetosa, P. lanceolata, L. corniculatus and T. repens was higher on plots without rabbit grazing, so that plant densities of these species in summer 1996 and plant cover in summer 1997 were greater on grazed plots. The results indicate interactions between soil disturbance, propagule availability and herbivory, rather than disturbance alone, will play an important role in controlling seedling recruitment and species habitat distributions in grasslands.

Fourteen seedlots of five species of Rhus were surveyed for presence/absence of physiological dormancy and/or for germination requirements of non-dormant seeds. Physiological dormancy was present in the four seedlots of R. aromatica studied, but not in either of the two seedlots of its close relative R. trilobata, which is in contrast to previous reports. Neither were seeds of R. glabra, R. typhina, nor R. virens physiologically dormant. Stratification at 5oC for 1 week or incubation in 500 or 1000 mg/l solutions of gibberellic acid broke physiological dormancy in > 90% of the R. aromaticaseeds. Maturation desiccation acted as a switch from a developmental to a germinative mode in R. aromatica embryos, whereas it was not required for germination of R. glabra or R. virens (R. trilobata and R. typhinanot tested). Seeds of all five species incubated on a moist substrate became fully imbibed in 2 d, at which time moisture content was approx. 70–80%of their initial weight. In general, germination of non-dormant seeds was rather insensitive to temperature and light. Seeds germinated equally well in light and in darkness over a daily (12 h/12 h) temperature range of 15/6–35/20oC. Over a 4 week period, the best germination percentages were obtained at 25/15 and 20/10oC, whereas 35/20oC appeared to be supraoptimal, though not always significantly so. If the incubation period was extended to 30 weeks, germination percentages were as high at 15/6oC as at 25/15 and 20/10oC.

Methanol accumulates in maturing seeds, correlating with preharvest deterioration. Since the source of methanol may be from pectin de-methylation, methanol, cell wall uronic acid, pectin methyl esterification, pectinesterase (PE; EC 3.1.1.11) activity, and neutral sugar composition and partitioning of cell wall polysaccharides were determined during soybean (Glycine max [L.] Merrill) seed development, maturation, and desiccation in planta. Axis cell wall polysaccharides were more easily solubilized, richer in uronic acid, rhamnose, and xylose, and less rich in galactose than cotyledon cell wall polysaccharides. Methanol accumulated to 9.7 μg per two cotyledons and 0.5 μg per axis; total methanol decreased to 3 μg per two cotyledons during loss of green color. Total uronic acid increased from 0.12 to 0.27 mg per axis and 0.9 to 4 mg per cotyledon between 24 and 50 days after flowering (DAF). After loss of green color, pectin methyl esterification in axes increased from 7 to 24 mole% between 50 and 60 DAF but decreased to 14 mole%by 62 DAF in latter stages of seed desiccation. In cotyledons, methyl esterification ranged from 25 to 40 mole% and was 31 mole% after desiccation. PE activity increased 100 fold in axes, including a 30-fold increase in activity after loss of green color at 46 DAF. Cotyledon PE activity was 40-fold higher than in axes at 24 DAF, declined 75% by 56 DAF, and then increased 5 fold during desiccation. Pectin methyl de-esterification by PE is sufficient to be the sole source for methanol accumulation in seed tissues during development and maturation.

The cell cycle is regulated, at least partially, by protein phosphorylation, activity that is carried out by a series of calcium-independent protein kinases among which the p34cdc2 kinase plays a preponderant role. The behaviour of p34cdc2-like protein kinase(s) has been followed during germination of maize in the presence or absence of the synthetic cytokinin benzyladenine (BA). Whereas there is an increase in p34cdc2-like kinase(s) activity at 15 h of germination in the presence of BA, coinciding with a peak of Ca2+-independent kinase activity, the amount of either the transcript for p34cdc2 or of the corresponding protein(s) does not show much variation during the 0–24 h period of germination studied, whether the phytohormone is present or not. Benzyladenine stimulates the cell cycle and promotes an early mitosis during maize germination. We have found evidence that BA promotes the movement of the p34cdc2-like protein(s) to nuclei several hours before this takes place during germination of control seeds, and this may constitute part of the mechanism by which the phytohormone promotes cytokinesis in plants.