Induction of sexual reproduction and resting egg production in Brachionus Plicatilis reared in sea water

Brachionus plicatilis raised in our laboratory in sea water reproduces asexually even under high crowding conditions (at least 40 individuals per ml). Amictic females were induced to produce mictic females, males and resting eggs by reducing the concentration of the sea water culture medium. Mictic females and males appeared predominantly among the progeny produced by the amictic females during 4 days following their transfer into 25% sea water. Resting eggs appeared first 5–12 days after the onset of the experiment. Following the disappearance of males, the culture consisted of amictic females.Resting eggs produced by the method described above may be preserved for at least three months at –14°C or by desiccation at room temperature. Under the appropriate experimental conditions, resting eggs hatch into amictic females. Since B. plicatilis is one of the most commonly used food sources of fish larvae in aquaculture, the methods reported here may offer an easy and versatile way of preserving rotifer culture stock to be used on demand.     

Interaction between seed dormancy-release mechanism, environment and seed bank strategy for a widely distributed perennial legume, Parkinsonia aculeata (Caesalpinaceae)

Background and Aims

Parkinsonia aculeata (Caesalpinaceae) is a perennial legume with seeds that have hard-seeded (physical) dormancy and are potentially very long-lived. Seed dormancy is a characteristic that can both help maximize the probability of seedling establishment and spread the risk of recruitment failure across years (bet-hedging). In this study, dormancy-release patterns are described across the diverse environments in which this species occurs in order to test whether wet heat (incubation under wet, warm-to-hot, conditions) alone can explain those patterns, and in order to determine the likely ecological role of physical dormancy across this species distribution.

Methods

A seed burial trial was conducted across the full environmental distribution of P. aculeata in Australia (arid to wet-dry tropics, uplands to wetlands, soil surface to 10 cm deep).

Key Results

Wet heat explained the pattern of dormancy release across all environments. Most seeds stored in the laboratory remained dormant throughout the trial (at least 84 %). Dormancy release was quickest for seeds buried during the wet season at relatively high rainfall, upland sites (only 3 % of seeds remained dormant after 35 d). The longest-lived seeds were in wetlands (9 % remained dormant after almost 4 years) and on the soil surface (57 % after 2 years). There was no consistent correlation between increased aridity and rate of dormancy release.

Conclusions

The results suggest that physical dormancy in P. aculeata is a mechanism for maximizing seedling establishment rather than a bet-hedging strategy. However, seed persistence can occur in environmental refuges where dormancy-release cues are weak and conditions for germination and establishment are poor (e.g. under dense vegetation or in more arid micro-environments) or unsuitable (e.g. when seeds are inundated or on the soil surface). Risks of recruitment failure in suboptimal environments could therefore be reduced by inter-year fluctuations in microclimate or seed movement.Key words: Bet-hedging, dormancy-release mechanisms, environmental refuges, legume, Parkinsonia aculeata, physical dormancy, seed bank persistence, seed burial depth, seed dormancy, tropical wetlands, wet heat, variable environment     

Pre- and post-harvest influences on seed dormancy status of an Australian Goodeniaceae species, Goodenia fascicularis

Background and Aims

The period during which seeds develop on the parent plant has been found to affect many seed characteristics, including dormancy, through interactions with the environment. Goodenia fascicularis (Goodeniaceae) seeds were used to investigate whether seeds of an Australian native forb, harvested from different environments and produced at different stages of the reproductive period, differ in dormancy status.

Methods

During the reproductive phase, plants were grown ex situ in warm (39/21 °C) or cool (26/13 °C) conditions, with adequate or limited water availability. The physiological dormancy of resulting seeds was measured in terms of the germination response to warm stratification (34/20 °C, 100 % RH, darkness).

Key Results

Plants in the cool environment were tall and had high above-ground biomass, yet yielded fewer seeds over a shorter, later harvest period when compared with plants in the warm environment. Seeds from the cool environment also had higher viability and greater mass, despite a significant proportion (7 % from the cool-wet environment) containing no obvious embryo. In the warm environment, the reproductive phase was accelerated and plants produced more seeds despite being shorter and having lower above-ground biomass than those in the cool environment. Ten weeks of warm stratification alleviated physiological dormancy in seeds from all treatments resulting in 80–100 % germination. Seeds that developed at warm temperatures were less dormant (i.e. germination percentages were higher) than seeds from the cool environment. Water availability had less effect on plant and seed traits than air temperature, although plants with reduced soil moisture were shorter, had lower biomass and produced fewer, less dormant seeds than plants watered regularly.

Conclusions

Goodenia fascicularis seeds are likely to exhibit physiological dormancy regardless of the maternal environment. However, seeds collected from warm, dry environments are likely to be more responsive to warm stratification than seeds from cooler, wetter environments.Key words: Goodenia fascicularis, Goodeniaceae, Australia, physiological dormancy, seeds, temperature, soil moisture, maternal influence, climate     

Thermostable proteins in the diapausing eggs of Brachionus manjavacas (Rotifera)

Diapausing embryos (resting eggs) from brachionid rotifers are able to withstand desiccation and thermal stress. Resting eggs can remain viable for decades, and develop normally once placed in a permissive environment that allows for hatching, growth and development. The exact mechanisms of resistance are not known, although several molecules have been suggested to confer protection during desiccation and thermal stress. In this study, we have identified by mass spectrometry two thermostable proteins, LEA (late embryogenesis abundant) and VTG (vitellogenin-like), found exclusively in the resting eggs of Brachionus manjavacas. This is the first observation that LEA proteins may play a role in thermostability and the first report of a VTG-like protein in the phylum Rotifera. These proteins exhibited increased expression in rotifer resting eggs when compared to amictic females. Our data suggest the existence of alternate pathways of desiccation and thermal resistance in brachionid rotifers.     

A proposed mechanism for physical dormancy break in seeds of Ipomoea lacunosa (Convolvulaceae)

Background and Aims

The water-impermeable seeds of Ipomoea lacunosa undergo sensitivity cycling to dormancy breaking treatment, and slits are formed around bulges adjacent to the micropyle during dormancy break, i.e. the water gap opens. The primary aim of this research was to identify the mechanism of slit formation in seeds of this species.

Methods

Sensitive seeds were incubated at various combinations of relative humidity (RH) and temperature after blocking the hilar area in different places. Increase in seed mass was measured before and after incubation. Scanning electron microscopy (SEM) and staining of insensitive and sensitive seeds were carried out to characterize these states morphologically and anatomically. Water absorption was monitored at 35 and 25 °C at 100 % RH.

Key Results

There was a significant relationship between incubation temperature and RH with percentage seed dormancy break. Sensitive seeds absorbed water vapour, but insensitive seeds did not. Different amounts of water were absorbed by seeds with different blocking treatments. There was a significant relationship between dormancy break and the amount of water absorbed during incubation.

Conclusions

Water vapour seals openings that allow it to escape from seeds and causes pressure to develop below the bulge, thereby causing slits to form. A model for the mechanism of formation of slits (physical dormancy break) is proposed.Key words: Convolvulaceae, Ipomoea lacunosa, dormancy-breaking mechanism, physical dormancy, seeds, sensitivity cycling, water vapour     

How do population genetic parameters affect germination of the heterocarpic species Atriplex tatarica (Amaranthaceae)?

Background and Aims

The heterocarpic species Atriplex tatarica produces two types of seeds. In this study, how basic population genetic parameters correlate with seed germinability under various experimental conditions was tested.

Methods

Population genetic diversity was ascertained in eight populations of A. tatarica by assessing patterns of variation at nine allozyme loci. Germinability of both seed types from all sampled populations was determined by a common laboratory experiment under different salinity levels. Basic population genetic parameters, i.e. percentage of polymorphic loci, average number of alleles per locus and observed heterozygosity were correlated with observed population germination characteristics.

Key Results

Atriplex tatarica possesses a remarkable heterocarpy, i.e. one type of seed is non-dormant and the other shows different dormancy levels in relation to experimental conditions. Significant negative correlations have been detected between germination of both seed types and the coefficient of inbreeding, and a significant negative correlation between germination of dormant seeds and other population genetic parameters, i.e. percentage of polymorphic loci and average number of alleles per polymorphic locus. Moreover, populations from the region characterized by a shorter growing season manifested higher germinability, i.e. had lower dormancy, than those from the lower-latitude one.

Conclusions

In general, germination of non-dormant seeds is probably not under strong genetic control. Hence, they germinate as soon as conditions are favourable, thus ensuring survival in the short term, but populations risk local extinction if conditions become adverse (i.e. a high-risk strategy). In contrast, germination of the dormant type of seeds is under stronger genetic control and is significantly correlated with basic population genetic parameters. These seeds ensure long-term reproduction and survival in the field by protracted germination, albeit in low quantities (i.e. A. tatarica also adopts a low-risk strategy).Key words: Amaranthaceae, Atriplex, inbreeding depression, population genetics, seed dimorphism     

Acetylation of Chromatin-Associated Histone H3 Lysine 56 Inhibits the Development of Encysted Artemia Embryos

Background

As a response to harsh environments, the crustacean artemia produces diapause gastrula embryos (cysts), in which cell division and embryonic development are totally arrested. This dormant state can last for very long periods but be terminated by specific environmental stimuli. Thus, artemia is an ideal model organism in which to study cell cycle arrest and embryonic development.

Principal Finding

Our study focuses on the roles of H3K56ac in the arrest of cell cycle and development during artemia diapause formation and termination. We found that the level of H3K56ac on chromatin increased during diapause formation, and decreased upon diapause termination, remaining basal level throughout subsequent embryonic development. In both HeLa cells and artemia, blocking the deacetylation with nicotinamide, a histone deacetylase inhibitor, increased the level of H3K56ac on chromatin and induced an artificial cell cycle arrest. Furthermore, we found that this arrest of the cell cycle and development was induced by H3K56ac and dephosphorylation of the checkpoint protein, retinoblastoma protein.

Conclusions/Significance

These results have revealed the dynamic change in H3K56ac on chromatin during artemia diapause formation and termination. Thus, our findings provide insight into the regulation of cell division during arrest of artemia embryonic development and provide further insight into the functions of H3K56ac.     

Exogenous ethylene inhibits sprout growth in onion bulbs

Background and Aims

Exogenous ethylene has recently gained commercial interest as a sprouting inhibitor of onion bulbs. The role of ethylene in dormancy and sprouting of onions, however, is not known.

Methods

A cultivar (Allium cepa ‘Copra’) with a true period of dormancy was used. Dormant and sprouting states of onion bulbs were treated with supposedly saturating doses of ethylene or with the ethylene-action inhibitor 1-methylcyclopropene (1-MCP). Initial sprouting was determined during storage at 18 °C by monitoring leaf blade elongation in a specific size class of leaf sheaths. Changes in ATP content and sucrose synthase activity in the sprout leaves, indicators of the sprouting state, were determined. CO₂ and ethylene production of onion bulbs during storage were recorded.

Key results

Exogenous ethylene suppressed sprout growth of both dormant and already sprouting onion bulbs by inhibiting leaf blade elongation. In contrast to this growth-inhibiting effect, ethylene stimulated CO₂ production by the bulbs about 2-fold. The duration of dormancy was not significantly affected by exogenous ethylene. However, treatment of dormant bulbs with 1-MCP caused premature sprouting.

Conclusions

Exogenous ethylene proved to be a powerful inhibitor of sprout growth in onion bulbs. The dormancy breaking effect of 1-MCP indicates a regulatory role of endogenous ethylene in onion bulb dormancy.Key words: Bulb dormancy, Allium cepa, onion, sprout growth, ethylene, CO₂ production, respiration, 1-methylcyclopropene     

Humidity-regulated dormancy onset in the Fabaceae: a conceptual model and its ecological implications for the Australian wattle Acacia saligna

Background and aims

Seed dormancy enhances fitness by preventing seeds from germinating when the probability of seedling survival and recruitment is low. The onset of physical dormancy is sensitive to humidity during ripening; however, the implications of this mechanism for seed bank dynamics have not been quantified. This study proposes a model that describes how humidity-regulated dormancy onset may control the accumulation of a dormant seed bank, and seed experiments are conducted to calibrate the model for an Australian Fabaceae, Acacia saligna. The model is used to investigate the impact of climate on seed dormancy and to forecast the ecological implications of human-induced climate change.

Methods

The relationship between relative humidity and dormancy onset was quantified under laboratory conditions by exposing freshly matured non-dormant seeds to constant humidity levels for fixed durations. The model was field-calibrated by measuring the response of seeds exposed to naturally fluctuating humidity. The model was applied to 3-hourly records of humidity spanning the period 1972–2007 in order to estimate both temporal variability in dormancy and spatial variability attributable to climatic differences among populations. Climate change models were used to project future changes in dormancy onset.

Key Results

A sigmoidal relationship exists between dormancy and humidity under both laboratory and field conditions. Seeds ripened under field conditions became dormant following very short exposure to low humidity (<20 %). Prolonged exposure at higher humidity did not increase dormancy significantly. It is predicted that populations growing in a temperate climate produce 33–55 % fewer dormant seeds than those in a Mediterranean climate; however, dormancy in temperate populations is predicted to increase as a result of climate change.

Conclusions

Humidity-regulated dormancy onset may explain observed variation in physical dormancy. The model offers a systematic approach to modelling this variation in population studies. Forecast changes in climate have the potential to alter the seed bank dynamics of species with physical dormancy regulated by this mechanism, with implications for their capacity to delay germination and exploit windows for recruitment.     

Nitrogen Limitation and Slow Drying Induce Desiccation Tolerance in Conjugating Green Algae (Zygnematophyceae,Streptophyta) from Polar Habitats

Background

Filamentous Zygnematophyceae are typical components of algal mats in the polar hydro-terrestrial environment. Under field conditions, they form senescent vegetative cells, designated as pre-akinetes, which are tolerant to desiccation and osmotic stress.

Key Findings

Pre-akinete formation and desiccation tolerance was investigated experimentally under monitored laboratory conditions in four strains of Arctic and Antarctic isolates with vegetative Zygnema sp. morphology. Phylogenetic analyses of rbcL sequences revealed one Arctic strain as genus Zygnemopsis, phylogenetically distant from the closely related Zygnema strains. Algae were cultivated in liquid or on solidified medium (9 weeks), supplemented with or lacking nitrogen. Nitrogen-free cultures (liquid as well as solidified) consisted of well-developed pre-akinetes after this period. Desiccation experiments were performed at three different drying rates (rapid: 10% relative humidity, slow: 86% rh and very slow); viability, effective quantum yield of PS II, visual and ultrastructural changes were monitored. Recovery and viability of pre-akinetes were clearly dependent on the drying rate: slower desiccation led to higher levels of survival. Pre-akinetes survived rapid drying after acclimation by very slow desiccation.

Conclusions

The formation of pre-akinetes in polar Zygnema spp. and Zygnemopsis sp. is induced by nitrogen limitation. Pre-akinetes, modified vegetative cells, rather than specialized stages of the life cycle, can be hardened by mild desiccation stress to survive rapid drying. Naturally hardened pre-akinetes play a key role in stress tolerance and dispersal under the extreme conditions of polar regions, where sexual reproduction and production of dormant stages is largely suppressed.     

Stress tolerance during diapause and quiescence of the brine shrimp,Artemia

Oviparously developing embryos of the brine shrimp, Artemia, arrest at gastrulation and are released from females as cysts before entering diapause, a state of dormancy and stress tolerance. Diapause is terminated by an external signal, and growth resumes if conditions are permissible. However, if circumstances are unfavorable, cysts enter quiescence, a dormant stage that continues as long as adverse conditions persist. Artemia embryos in diapause and quiescence are remarkably resistant to environmental and physiological stressors, withstanding desiccation, cold, heat, oxidation, ultraviolet radiation, and years of anoxia at ambient temperature when fully hydrated. Cysts have adapted to stress in several ways; they are surrounded by a rigid cell wall impermeable to most chemical compounds and which functions as a shield against ultraviolet radiation. Artemia cysts contain large amounts of trehalose, a non-reducing sugar thought to preserve membranes and proteins during desiccation by replacing water molecules and/or contributing to vitrification. Late embryogenesis abundant proteins similar to those in seeds and other anhydrobiotic organisms are found in cysts, and they safeguard cell organelles and proteins during desiccation. Artemia cysts contain abundant amounts of p26, a small heat shock protein, and artemin, a ferritin homologue, both ATP-independent molecular chaperones important in stress tolerance. The evidence provided in this review supports the conclusion that it is the interplay of these protective elements that make Artemia one of the most stress tolerant of all metazoan organisms.     

Ecotypic variation of summer dormancy relaxation associated with rainfall gradient in the geophytic grass Poa bulbosa

Background and Aims

Summer dormancy is an adaptive trait in geophytes inhabiting regions with a Mediterranean climate, allowing their survival through the hot and dry summers. Summer dormancy in Poa bulbosa is induced by increasing day-length and temperature and decreasing water availability during spring. Populations from arid habitats became dormant earlier than those from mesic habitats. Relaxation of dormancy was promoted by the hot, dry summer conditions. Here we test the hypothesis that dormancy relaxation is also delayed in ecotypes of P. bulbosa inhabiting arid regions, as a cautious strategy related to the greater unpredictability of autumn rains associated with decreasing precipitation.

Methods

Ecotypes collected across a precipitation gradient (100–1200 mm year⁻¹) in the Mediterranean climate region were grown under similar conditions in a net-house in Israel. Differences among ecotypes in dormancy induction and dormancy relaxation were determined by measuring time to dormancy onset in spring, and time to sprouting after the first effective rain in autumn. Seasonal and ecotype variation in dormancy relaxation were assessed by measuring time to sprouting initiation, rate of sprouting and maximal sprouting of resting dry bulbs sampled in the net-house during late spring, and mid- and late summer, and planted in a wet substrate at temperatures promoting (10 °C) or limiting (20 °C) sprouting.

Key Results

Earlier dormancy in the spring and delayed sprouting in autumn were correlated with decreasing mean annual rainfall at the site of ecotype origin. Seasonal and ecotype differences in dormancy relaxation were expressed in bulbs planted at 20 °C. During the summer, time to sprouting decreased while rate of sprouting and maximal sprouting increased, indicating dormancy relaxation. Ecotypes from more arid sites across the rainfall gradient showed delayed onset of sprouting and lower maximal sprouting, but did not differ in rate of sprouting. Planting at 10 °C promoted sprouting and cancelled differences among ecotypes in dormancy relaxation.

Conclusions

Both the induction and the relaxation of summer dormancy in P. bulbosa are correlated with mean annual precipitation at the site of population origin. Ecotypes from arid habitats have earlier dormancy induction and delayed dormancy relaxation, compared with those from mesic habitats.     

Seed dormancy distribution: explanatory ecological factors

Background and Aims

Knowledge of those traits that vary with latitude should be helpful in predicting how they may evolve locally under climate change. In the sea beet Beta vulgaris ssp. maritima, seed dormancy largely controls the timing of germination, is highly heritable and varies geographically; it is therefore thought to be selected by climate. The aim here was to characterize the variation in seed dormancy among sea beet populations across the French distribution area, as well as the ecological factors in situ that are correlated with and that could therefore select for seed dormancy. The relative importance of genetic inheritance vs. non-genetic variation is also evaluated.

Methods

The proportions of dormant seeds from 85 natural populations encompassing different climates over the whole French distribution area were measured under controlled conditions. Germination phenology was observed in a common garden experiment. Dormancy variation of seeds collected in situ was compared with that of seeds collected on plants grown in the greenhouse.

Key Results

The proportions of dormant seeds in the greenhouse were highly variable, covering almost the entire range from 0 to 1, and followed a geographical pattern from lower dormancy at high latitudes to high dormancy at low latitudes. The distribution of dormancy was positively correlated with yearly temperatures, especially summer temperatures. Minimum temperatures in winter did not significantly explain the trait variation. The genetic component of the total variation was significant and is probably completed by an important adjustment to the local conditions brought about by maternal adaptive phenotypic plasticity.

Conclusions

Dormancy in sea beet could be interpreted as a way to limit summer germination and spread germination over the first autumn and spring or following autumns. This highly heritable trait has the potential to evolve in the relatively near future because of climate change.     

Stamen development and winter dormancy in apricot (Prunus armeniaca)

Background and Aims

In temperate woody perennials, flower bud development is halted during the winter, when the buds enter dormancy. This dormant period is a prerequisite for adequate flowering, is genetically regulated, and plays a clear role in possibly adapting species and cultivars to climatic areas. However, information on the biological events underpinning dormancy is lacking. Stamen development, with clear differentiated stages, appears as a good framework to put dormancy in a developmental context. Here, stamen developmental changes are characterized in apricot (Prunus armeniaca) and are related to dormancy.

Methods

Stamen development was characterized cytochemically from the end of August to March, over 4 years. Developmental changes were related to dormancy, using the existing empirical information on chilling requirements.

Key Results

Stamen development continued during the autumn, and the flower buds entered dormancy with a fully developed sporogenous tissue. Although no anatomical changes were observed during dormancy, breaking of dormancy occurred following a clear sequence of events. Starch accumulated in particular places, pre-empting further development in those areas. Vascular bundles developed and pollen mother cells underwent meiosis followed by microspore development.

Conclusions

Dormancy appears to mark a boundary between the development of the sporogenous tissue and the occurrence of meiosis for further microspore development. Breaking of dormancy occurs following a clear sequence of events, providing a developmental context in which to study winter dormancy and to evaluate differences in chilling requirements among genotypes.     

The role of the persistent fruit wall in seed water regulation in Raphanus raphanistrum (Brassicaceae)

Background and Aims

Dry fruits remain around the seeds at dispersal in a number of species, especially the Brassicaceae. Explanations for this vary, but usually involve mechanisms of innate dormancy. We speculate that, instead, a persistent fruit may give additional protection through control of dehydration, to species growing in arid or Mediterranean environments where water is sporadic.

Methods

X-rays and weight measurements were used to determine the extent to which Raphanus raphanistrum seeds within mature fruits imbibe water, and germination tests determined the roles of the fruit and seed coat in seed dormancy. Rates of water uptake and desiccation, and seedling emergence were compared with and without the fruit. Finally, germinability of seeds extracted from fruits was determined after various periods of moist conditions followed by a range of dry conditions.

Key Results

Most seeds rapidly take up water within the fruit, but they do not fully imbibe when compared with naked seeds. The seed coat is more important than the dry fruit wall in maintaining seed dormancy. The presence of a dry fruit slows emergence from the soil by up to 6–8 weeks. The fruit slows the rate of desiccation of the seed to a limited extent. The presence of the fruit for a few days during imbibition somehow primes more seeds to germinate than if the fruit is absent; longer moist periods within the pod appear to induce dormancy.

Conclusions

The fruit certainly modifies the seed environment as external conditions change between wet and dry, but not to a great extent. The major role seems to be: (a) the physical restriction of imbibition and germination; and (b) the release and then re-imposition of dormancy within the seed. The ecological significance of the results requires more research under field conditions.     

Dynamics of tear fluid desiccation on a glass surface: a contribution to tear quality assessment

Background

Fern-like crystalloids form when a microvolume of tear is allowed to dry out at ambient conditions on a glass surface. Presence of crystalloids in tear “microdesiccates” is used to evaluate patients with Dry-Eye disease. This study aims to examine morphologically the desiccation process of normal tear fluid and to identify changes associated with accelerated tear evaporation. Tear microdesiccates from healthy (Non-Dry Eye) and Dry Eye subjects were produced at ambient conditions. Microdesiccate formation was monitored continuously by dark-field video microscopy. Additionally, accelerated desiccation of tear samples from healthy subjects was conducted under controlled experimental conditions. Particular morphological domains of tear microdesiccates and their progressive appearance during desiccation were compared.

Results

In normal tear microdesiccates, four distinctive morphological domains (zones I, II, III and transition band) were recognized. Stepwise formation of those domains is now described. Experimentally accelerated desiccation resulted in marked changes in some of those zones, particularly involving either disappearance or size reduction of fern-like crystalloids of zones II and III. Tear microdesiccates from Dry Eye subjects may also display those differences and be the expression of a more synchronous formation of microdesiccate domains.

Conclusion

Morphological characteristics of tear microdesiccates can provide insights into the relative rate of tear evaporation.