Aerosol smoke and smoke-water can break dormancy and promote seed germination of many plant species. In this study we investigated changes in gene expression after imbibition of light-sensitive Lactuca sativa L. cv. ‘Grand Rapids’ achenes with dilute smoke-water compared to water control samples kept in the dark or continuous light, using the fluorescent differential display technique. Although no difference was detected in the smoke-water versus water control samples germinated in light, smoke-water treatment resulted in the differential display of several expressed sequence tags (ESTs) when compared to water control samples kept in the dark. The most pronounced fragments isolated correspond to known genes related to germination, with functions in cell wall expansion, regulation of translation, the cell division cycle, carbohydrate metabolism and abscisic acid (ABA) regulation. Real-time polymerase chain reaction (PCR) validation revealed that the transcript abundance of the genes, HVA22, short-chain dehydrogenase/reductase and late embryogenesis abundant protein, are upregulated after smoke treatment when compared to control achenes kept in the light. The results indicate that smoke has a dual effect. On the one hand, the smoke can induce genes that may be linked to ABA action, whereas, on the other hand, it elicits a faster germination rate by inducing a similar pattern in gene expression as light treatment. Smoke effects could be manifested mainly through the induction of the cell division cycle, cell wall extension and storage mobilization.

Seed (= seed plus endocarp) morphology and physical dormancy were studied in seven North American Rhus species: R. copallina, R. glabra, R. typhina, R. aromatica (var. aromatica), R. microphylla, R. trilobata, and R. virens (var. virens). Seeds of Rhus glabra and R. typhina, of subgenus Rhus, were gray, approx. 3 mm long, 2 mm wide, and weighed approx. 7 mg, whereas those of R. aromatica, R. trilobata, and R. virens, of subgenus Lobadium, were brown, >4 mm long, approx. 4 mm wide, and weighed approx. 14.5 mg (R. trilobata) to approx. 23 mg (R. aromaticaand R. virens). Dormancy in all seeds was due to a water-impermeable endocarp, but depth of dormancy varied greatly among species and seedlots. After 4 weeks, 29–34% of the seeds from all five seedlots of R. trilobata, R. microphylla, and R. virens incubated on moist substrate had imbibed, compared to 0–14% of the seeds of all 16 seedlots of R. aromatica, R. copallina, R. glabra, and R. typhina. After 1 yr, imbibition among seedlots of R. aromaticavaried from 28 ± 2% to 69 ± 5%, whereas 93 ±2% to 100 ± 0% of the seeds from all seedlots of R. microphylla, R. trilobata, and R. virens did so. Neither dry laboratory storage for up to 4 yr (even 29 yr in R. aromatica) nor dry heating at 100oC or at 120oC effectively broke dormancy in any of the species tested (R. aromatica, R. glabra, R. trilobata, R. virens). Immersion in boiling water was the best method to render seeds of R. glabra and R. typhinapermeable, yet it was ineffective for those of R. aromatica, R. trilobata, and R. virens. In contrast, a 1 h-soaking in concentrated H2SO4 led to complete loss of endocarp impermeability in the latter three species, but mostly was ineffective in R. glabra and R. typhina. Thus, there seems to be a tendency for seeds of subgenus Rhusto respond well to boiling in water, but not to soaking in H2SO4, whereas the opposite is true for those of subgenus Lobadium.

The LEA-like protein previously isolated from a homogenate of pea (Pisum sativum L.) embryonic axes heated at 80°C for 10 min (Russouw et al., 1995) was purified without exposure to heat. Peptides produced by trypsin digestion were separated by HPLC and sequenced. The protein was identified as a member of the LEA group I family. The conformation of the protein was compared before and after heat treatment by antibody affinity, circular dichroism spectroscopy, fluorescence spectroscopy and 8-anilino-1-naphthalenesulfonic acid binding. No differences could be detected, demonstrating that the protein was not irreversibly denatured by exposure to high temperature.

MicroRNAs (miRNAs) play critical roles in the development of animals and plants. Characterizing the stage- and tissue-specific expression of miRNAs that potentially regulate target transcription factor expression is becoming more important for understanding the regulatory mechanisms of critical events during plant development. A simple method for purifying small RNAs from seeds is described, as well as an efficient non-radioactive labelling system for making miRNA probes. In Arabidopsis thaliana seed extracts, low molecular-weight (LMW) RNAs (e.g. 5S rRNA, tRNA and miRNA) were separated from high molecular-weight (HMW) nucleic acids (e.g. 28S and 18S rRNA, mRNA and genomic DNA) by fractionation using isopropanol. HMW RNAs precipitated in 20% isopropanol, while most LMW RNAs remained in the supernatant. The purified LMW RNAs were used successfully for RNA gel blotting to detect miRNAs expressed in Arabidopsis and tomato (Lycopersicon esculentum) seeds. To increase the detection sensitivity of the microRNA probes, additional digoxigenin-labelled uridine triphosphates (UTPs) were incorporated into the miRNA probes by designing template oligo DNAs with three extra adenines (A) at each end of their sequence. These DNA oligomers were used to make double-stranded DNA templates for miRNA probe synthesis. This probe (termed AAAPLUS) exhibited stronger signals than normal probes. A technique was also developed to quickly screen expressed miRNAs in seeds using a miniblot system, which enabled simultaneous examination with multiple miRNA probes. This method provides a simple alternative to microRNA microarrays to identify the major miRNAs expressed in seeds.

Wheat seeds were imbibed in solutions of polyethylene glycol (PEG) and NaCl, and embryo water content and incorporation of [35S]methionine into proteins were determined. Both osmotica reduced water uptake and protein synthesis, though these were immediately resumed upon removal of stress. Results of in vivo labelling of newly synthesized proteins showed differences in the synthesis of many polypeptides when embryos that had imbibed in water passed from the radicle pre-emergence phase to early growth. The synthesis of a group of proteins associated with radicle emergence was reduced during imbibition in PEG or NaCl. All the major proteins of the pre-germination phase were produced in PEG-treated embryos, while some new polypeptides (‘salt stress’ proteins?) were actively synthesized in salt-treated embryos. Upon removal of PEG or NaCl, synthesis of proteins common to the phase of early embryo growth increased, while specific saltimbibition proteins disappeared. These findings are consistent with the hypothesis that biochemical processes leading to radicle emergence can be affected by osmotic stress and are likely to be damaged by severe stress.

Few data are available and little is known about spatial variation in post-dispersal seed removal at different levels throughout the geographic range of a plant species. Here, we compare post-dispersal seed removal by rodents within and among sites in two distinct regions, the south-eastern Spanish Mediterranean highlands and the Swiss Jura. Seed removal was assessed experimentally for four fleshy-fruited species (Berberis vulgaris, Crataegus monogyna, Rosa spp. and Taxus baccata) in heterogeneous sites with distinct microhabitats. A factorial general linear model (GLM) analysis was used to evaluate the relative influence of species-specific seed traits on removal at three spatial levels (microhabitat, site and region). In both highland ecosystems, live trapping revealed that rodents were the main seed removers: Apodemus sylvaticus was observed in both regions, whereas A. flavicollis and Clethrionomys glareolus occurred only at the Swiss sites. There was a significant difference in seed removal between regions, and the preferences of the rodents varied among plant species and microhabitats. Variation in rodent presence explained some differences in seed removal between regions and among microhabitats. Finally, the effect of rodent presence on seed removal differs due to both regional and microhabitat effects.

An LEA-like protein has been isolated and characterized from pea (Pisum sativum) embryos. It is the most prevalent protein in a homogenate of pea axes heated for 10 min at 80°C and then centifuged for 10 min at 17000 g (80°C supernatant fraction). It has a molecular mass of 11 kDa and is very rich in hydrophilic amino acids, notably aspartate, glutamate and glycine. The protein is not recognized by an antibody to the group II dehydrin C-terminal consensus sequence. Antibodies to the isolated protein recognize a number of larger proteins in the 80°C supernatant fraction of pea and other legumes (chick pea, soybean and bean) as well as gramineous seeds (wheat, maize and barley). The protein undergoes a substantial increase in α-helical content at high ionic strength but does not dimerize.

The potential longevity of japonica rice (Oryza sativa L. subsp. japonica) seed is particularly sensitive to high temperature – and thus climate change – during development and maturation. Cultivar Taipei 309 was grown at 28/20°C (12 h/12 h) and then from 19 DAA (days after 50% anthesis), when seeds were just over half filled, at 28/20°C, 30/22°C, 32/24°C or 34/26°C (12 h/12 h). Whereas ability to germinate ex planta had been achieved in almost all seeds by 24 DAA, only half the population were desiccation tolerant. Desiccation tolerance continued to increase over the subsequent 28 d, similarly at all four temperatures. Subsequent longevity, assessed by p50 (period in days to reduce viability to 50% in hermetic storage at 40°C with c. 15% moisture content), increased progressively at 28/20°C until 38 DAA, and remained constant until the final harvest (52 DAA). The three warmer temperature regimes provided similar longevity to 28/20°C at any one harvest, except at 38 DAA where the warmest (34/26°C) was poorer. That temperature regime also provided greater seed-to-seed variability within each survival curve. The results confirm that appreciable improvement in seed quality occurs during seed development and also subsequent maturation in japonica rice, but that increase in temperature from 28/20°C to 34/26°C during late seed filling onwards has comparatively little effect thereon. Comparison with previous investigations suggests that seed quality development may be less sensitive to high temperatures during late development and maturation than during the early seed development that precedes it.

Profiles of proteins synthesized in vivo in radicle tips were compared among unaged, artificially aged (controlled deterioration: 10% moisture content at 42°C for 3 weeks), osmoprimed (−1.5 MPa polyethylene glycol 6000 at 20°C for 1 week), and artificially aged and subsequently osmoprimed cauliflower (Brassica oleracea L.) seeds. Germination and initiation of incorporation of 35S-methionine into radicle tips were delayed by controlled deterioration. Osmopriming accelerated these processes and also alleviated the delays. The labelled proteins were analysed by two-dimensional gel electrophoresis and fluorography. Proteins were found whose expression correlated with the rate of germination, and was reduced by controlled deterioration but enhanced by osmopriming. Subsequent osmopriming reversed the effect of controlled deterioration and even caused an enhancement up to the level of unaged osmoprimed seeds. These proteins appeared to be related to processes preceding visible germination. In unaged seeds, formation of the proteins in the radicle tip began upon imbibition but decreased towards visible germination. On silver-stained gels, one of the proteins was shown to be mobilized by the time of radicle protrusion, while all were found in dry seeds. Proteins whose pronounced expression was observed only after both treatments were also found.

Seed germination rates are sensitive to both temperature (T) and water potential (ψ). The times to germination of seeds imbibed at suboptimal T and/or reduced ψ are inversely proportional to the amounts by which T exceeds a base temperature (Tb) and ψ exceeds a base water potential (ψb). Germination rates across a range of suboptimal T and ψ can be normalized on the basis of the hydrothermal time accumulated in excess of these thresholds. However, seeds can also progress metabolically toward germination even at T or ψ too low to allow radicle emergence to occur. Seeds preimbibed at low ψ and dried back, or primed, germinate more rapidly upon subsequent reimbibition. We show here that the increase in germination rates of tomato (Lycopersicon esculentum Mill.) seeds resulting from seed priming is linearly related to the hydrothermal time accumulated during the priming treatment. The threshold temperature (Tmin = 7.05°C) and water potential (ψmin = −2.50 MPa) for metabolic advancement were considerably lower than the corresponding thresholds for radicle emergence of the same seed lot (Tb = 11°C; ψb = −0.71 MPa), allowing the accumulation of hydrothermal priming time that is subsequently expressed as more rapid germination when T or ψ increase. The hydrothermal time model can now be applied to quantify and analyse germination rates of seeds across the entire range of suboptimal T and ψ at which metabolic progress toward radicle emergence is possible.

Meadow microcosms were established from seed on low-fertility soil of known seed bank composition, and subjected to manipulations of simulated grazing, cutting date, temperature and fertility for seven years. The composition and density of the seed bank was then determined in five 2-cm soil layers (0–2, 2–4, 4–6, 6–8 and 8–10 cm). The seed bank contained three distinct groups of species: species present in the original soil, sown species, and ‘others’. The seed bank was little affected by the experimental treatments, presumably because the sown species made only a small contribution to the seed bank. Nearly all the species in the original soil are known to possess persistent seed banks and had survived, although at reduced density, for seven years. Density of the most abundant species in this group, Sagina procumbens, had changed very little over seven years, confirming the well-documented longevity of the seeds of this species. Seeds of sown species made up only about a quarter of the seed bank, despite accounting for virtually all the above-ground vegetation. Of the sown meadow species, only Plantago lanceolata and Alopecurus pratensis were relatively abundant in the seed bank. These results strongly support the conclusion of other authors that most meadow species, once lost owing to the effects of fertilizers or inappropriate management, will not reestablish from the seed bank. Among species which were neither sown nor present in the original soil, the majority possessed adaptations for wind dispersal and had presumably dispersed into the experimental plots from outside. The most abundant member of this group, Betula pendula, had dispersed from a nearby tree. Density of Betula seeds declined sharply with depth, consistent with the view that seeds on the soil surface are rapidly lost, mainly through germination, but seeds that become buried survive much better. Seeds of Betula appear to be persistent but not particularly long-lived.

Changes in seed dry weight, moisture content and germinability of three japonica cultivars of rice during development and maturation in the dry season (November–May) field multiplication at Los Baños, Philippines were compared with those in a cooler controlled environment (24°/18°C). Under field conditions, maximum dry weight accumulation, which indicates mass maturity, was attained 18–21 days after flowering in all cultivars. In the cooler environment however, mass maturity was achieved 21 days after flowering in Fujisaka 5 and Minehikare, and 28 days in Shuang cheng nuo. The moisture content of the seeds at mass maturity varied between 28 and 35% among cultivars in the two environments. Although the ability of seeds to germinate differed in early harvests (7 and 14 days) among cultivars and across two environments, germination of the mature seeds was similar in all. There were no significant differences in potential longevity of the seeds harvested at 28, 35 and 42 days after flowering within each cultivar, which was determined by the germination of seeds that had been hermetically stored with 15% moisture content at 35°C. Differences among cultivars within each seed production environment were also not significant. However, potential longevity of the seeds harvested from the cooler environment was higher than those from the field (P<0.05), which confirmed that japonica cultivars which evolved under temperate regions are sensitive to warmer seed production environments. However, the magnitude of differences in potential seed longevity between the two environments was not high. This indicates that the seed production environment during the dry season at Los Baños is not harsh for japonica cultivars.

The changes in water status of germinating and non-viable soybean (Glycine max L. Merr.) seeds were characterized by nuclear magnetic resonance (NMR) spectroscopy. There were distinct changes in water status between viable and non-viable soybean seeds. In dry seeds, there were only two components, bound and bulk water, as revealed by component analysis of NMR (T2) data. On the contrary, a three-component water proton system (bound, bulk and free water) was observed in both germinating and non-viable soybeans during Phase I of hydration. The bulk water component of non-viable seeds disappeared completely during the lag phase (Phase II) of hydration, resulting in a two-component water proton system. In contrast, the three-component water proton system in Phase II was observed in the germinating seeds. Rapid hydration (Phase III), following Phase II, was observed in germinating soybean seeds only. Due to reorganization of water protons, there was a concomitant increase in bulk and free water, but a decrease in bound water. The physical state of water in these seeds (analysed by NMR spectroscopy) and the measurements of tissue leachate conductivity suggest that non-viable soybean seeds were more affected by the disorganized cell structure in the seed membrane system. The present study also provides evidence that physical reorganization of water is essential in germinating soybean seeds during hydration.

The present work aims to determine whether there are patterns of seed and germination characteristics in Vellozia due to the phylogenetic proximity among the species examined and if these characteristics explain their restricted geographical distributions. We evaluated the germination characteristics of freshly collected seeds from 13 species of the genus Vellozia (Velloziaceae) that show different degrees of endemism, collected at various locations in the Espinhaço Mountain Range in Minas Gerais State, south-eastern Brazil. The size and mass of the seeds, as well as the influence of light and temperature on their germination, were measured. Experiments were performed in germination chambers under constant temperatures of 10–40°C (intervals of 5°C), with a 12-h photoperiod, as well as in continuous darkness. All species studied had small seeds with mass varying from 0.06 to 1.21 mg. Most species required light for germination, displaying high germinability in the range of 15–40°C; some species, however, germinated in the absence of light at the highest temperatures (35 and 40°C). The sizes and masses of the seeds showed significant linear correlations, but light sensitivity was not related to these seed characteristics. The responses observed suggest that light requirement for germination, associated with the small sizes of Vellozia spp. seeds, contribute to the formation of persistent seed banks. The observed tolerance of these seeds to a wide range of germination temperatures is consistent with the large daily temperature fluctuations experienced in campos rupestres sites, although these seed characteristics cannot by themselves explain the high degree of endemism or the restricted distributions observed among the species examined.

The rate of oxygen consumption by cabbage seeds increased on imbibition and there was a further sharp increase on germination. This was delayed in artificially aged seeds of low vigour. The increases in oxygen consumption reflect the increased oxidation of carbohydrates via respiratory pathways. The activities of key regulatory enzymes of glycolysis and the oxidative pentose phosphate pathway were measured in aged and unaged seed lots of cabbage. Differences in the activities of glucose 6-phosphate dehydrogenase and pyrophosphate:fructose 6-phosphate 1-phosphotransferase were correlated with the rate of germination (T50) in seed lots with large differences in seed vigour induced experimentally by artificial aging. However, the activities of these enzymes could not be used to distinguish between untreated seed lots which had smaller vigour differences apparent only under stress. The enzymes are presumably not controlling and determining seed vigour, but merely reflecting actual seed performance under the current conditions.

The relationship between Calluna vulgaris seedbank density and climate was investigated using regression analysis and two related techniques (factor-ceiling analysis). The seedbank data originated from published and unpublished studies. Low seedbank densities were associated with sites in the relatively dry, sunny and warm south and east of Great Britain. However, as the climatic variables used in the study were highly intercorrelated, it was not possible to determine which climatic variable had the greatest influence on seedbank density. A hypothesis is described suggesting that the limits of seedbank density can be described for single environmental factors. The operation of other environmental and management factors may reduce the seedbank density to less than this maximum. This is illustrated for a range of climatic variables using factor-ceiling analysis. The processes following seed deposition and the longevity of seeds in the soil are highlighted as possible factors controlled by climate which determine seedbank density.

Dormant barley grains cannot germinate at 30°C and this inability to germinate is imposed mostly by the glumellae which have been suggested to limit oxygen supply to the embryo. Hypoxia imposed either artificially or by the glumellae to embryos from dormant grains, increases embryo sensitivity to abscisic acid (ABA) and promotes the accumulation of ABA during the first hours after imbibition. Expression of candidate genes involved in ABA synthesis (HvNCED), catabolism (HvABA8OH1) and signalling (HvABI5, HvVP1 and HvPKABA) was analysed in embryos isolated from dormant whole or de-hulled grains incubated in air or in hypoxia (5% oxygen). The presence of the glumellae enhanced the expression of genes involved in ABA metabolism and signalling with respect to that observed in de-hulled grains incubated in air. These results suggest that at least part of the observed physiological responses to the presence of the glumellae are regulated at the level of gene expression. However, hypoxia imposed on dormant de-hulled grains did not mimic the presence of the glumellae in terms of expression of candidate genes. Hypoxia mimics the presence of the glumellae in terms of dormancy maintenance and ABA accumulation and sensitivity, but its effects appear to operate through different mechanisms.

Two possible sources of resistance to pre-harvest sprouting were evaluated in quinoa. They showed dormancy at harvest and significant variations in dormancy level in response to environmental conditions experienced during seed development. The aims of this work were to evaluate the importance of seed coats in the regulation of dormancy in this species, to investigate possible mechanisms of action and to assess association of seed coat properties with changes in dormancy level caused by the environment. Accessions Chadmo and 2-Want were grown under field conditions on different sowing dates during 2 years. Seed coats were manipulated and seed germination was evaluated at different temperatures. Seed coat perforation before incubation led to faster dormancy loss in both accessions. This effect decreased with delayed sowing date, and seeds expressed a level of dormancy not imposed by coats. This suggests the presence of embryo dormancy in the genus Chenopodium. Seeds of the accession 2-Want had a significantly thinner seed coat at later sowing dates, associated with a decreasing coat-imposed dormancy, but this pattern was not detected in Chadmo. The seed coat acts as a barrier to the release of endogenous abscisic acid (ABA) in quinoa, suggested by the increase in germination and a higher amount of ABA leached from perforated seeds. ABA is able to leach from seeds with an intact seed coat, suggesting that differences in seed coat thickness may allow the leakage of different amounts of ABA. This mechanism may contribute to the observed differences in dormancy level, either between sowing dates or between accessions.

Endozoochorous dispersal of seeds by livestock has long attracted the attention of grassland scientists. However, little is known about seed dispersal after ingestion by Kazakh sheep on dry grasslands in the Tianshan Mountains. The objective of this experiment was to learn more about the recovery and germinability of seeds from 17 plant species after either actual or simulated ingestion (i.e. insertion through a rumen fistula) by Kazakh sheep. The passage time of seeds through the sheep gut ranged from 12 to 96 h. More than 80% of all recovered seeds were defecated 24–48 h after ingestion. The mean retention time of seeds in the gut ranged from 27.3 to 42.2 h. Seed recovery percentage ranged between 12.6 and 17.6% for leguminous species and between 0.8 and 3.2% for gramineous species. Seed recovery percentage was positively correlated with seed mass, but negatively correlated with seed shape. The germination percentages of the gramineous species were greater in the non-ingested treatment (66–98%) than in the simulated ingestion treatment (3–10%). In contrast, for leguminous species, seed germination percentages were greater in the simulated ingestion treatment (23–70%) than in the non-ingested one (5–12%). Seed germination percentage after simulated ingestion was positively correlated with seed mass, but negatively correlated with seed shape. In conclusion, leguminous seeds were more likely than gramineous ones to pass through the gut of Kazakh sheep and then germinate. Free-ranging Kazakh sheep can contribute to the spread of plant species, especially leguminous species, in the Tianshan Mountains.

Norway maple (Acer platanoides L.) seeds were harvested at different stages of seed development and maturation in 1989–91. As maturation drying progressed, the seed populations showed increasing desiccation tolerance: at 67–69% moisture content, no seeds survived desiccation below 10% moisture content; maturation drying to 55–57% moisture content (values corresponding with the end of the seed-filling phase) improved desiccation tolerance, but nevertheless most seeds were unable to withstand desiccation to 5–7% moisture content; further maturation drying to 27–28% moisture content enabled the seeds to survive considerable desiccation, no loss in viability occurring in seeds dried to 3% moisture content. This considerable improvement in desiccation tolerance after the end of the seed-filling phase was correlated (P<0.05) with the progress of maturation drying and may be associated with the increase in the potential longevity of seeds of other species that occurs during seed development subsequent to seed filling.