Effect of <Superscript>60</Superscript>Co-Gamma Ionizing Radiation and Desiccation Stress on Protein Profile of <Emphasis Type="Italic">Anabaena</Emphasis> 7120

Physiological stress can bring major molecular and cellular change to a living cell which further decide its survival or tolerance to the stress exposure. Cyanobacteria like Anabaena has been shown to tolerate high levels of different stresses like oxidative, desiccation, UV, and gamma radiation. They are able to withstand and recover remarkably without any lethal mutation when exposed to high doses of gamma radiation or prolonged duration of desiccation. In the present work, the modifications in protein profiles of Anabaena 7120 cells after exposure to 6 kGy of ⁶⁰Co γ-rays and 6 days of desiccation, and the proteome dynamics during post stress recovery were investigated. Differentially expressed proteins during stress and recovery were identified by MALDI-ToF or LC-MS, which generated a partial proteome map of Anabaena 7120. Anabaena cells went through protein recycling—phase of protein degradation following by their resynthesis, which helped them to recover remarkably. The data suggests an overlap in proteome changes during recovery against radiation and desiccation stress.     

Light and desiccation responses of some Hymenophyllaceae (filmy ferns) from Trinidad, Venezuela and New Zealand: poikilohydry in a light-limited but low evaporation ecological niche

Background and Aims

Hymenophyllaceae (filmy ferns) are typically plants of shady, constantly moist habitats. They attain greatest species diversity and biomass in humid tropical montane forests and temperate hyperoceanic climates. This paper presents ecophysiological data bearing on their worldwide ecological niche space and its limits.

Methods

Chlorophyll fluorescence was used to monitor recovery in desiccation experiments, and for measurements of 95 % saturating irradiance [photosynthetic photon flux density (PPFD_{95 %})] of photosynthetic electron flow and other parameters, in the New Zealand Hymenophyllum sanguinolentum, and three species each of Hymenophyllum and Trichomanes from forests in Trinidad and Venezuela.

Key Results

Hymenophyllum sanguinolentum was comparable in desiccation tolerance and light responses with the European species. The more common species in the two tropical forests showed PPFD_{95 %} >100 µmol m⁻² s⁻¹, and withstood moderate desiccation (–40 MPa) for several days. The four most shade-adapted species had PPFD_{95 %} ≤51 µmol m⁻² s⁻¹, and were sensitive to even mild and brief desiccation (–22 MPa for 3 d).

Conclusions

Light and desiccation responses of filmy ferns can be seen as an integrated package. At low light and windspeed in humid forests, net radiation and saturation deficit are low, and diffusion resistance high. Water loss is slow and can be supported by modest conduction from the sub-stratum. With higher irradiance, selection pressure for desiccation tolerance increases progressively. With low light and high humidity, the filmy fern pattern of adaptation is probably optimal, and the vascular plant leaf with mesophyll and stomata offers no advantage in light capture, water economy or CO₂ uptake. Trade-offs between light adaptation and desiccation tolerance, and between stem conduction and water absorption through the leaf surface, underlie adaptive radiation and niche differentiation of species within the family. Hymenophyllaceae are a rare example of an evolutionary shift of adaptive strategy from typical vascular plant adaptation to the poikilohydry most typical of bryophytes.     

Maturation proteins associated with desiccation tolerance in soybean

A set of proteins that accumulates late in embryogenesis (Lea proteins) has been hypothesized to have a role in protecting the mature seed against desiccation damage. A possible correlation between their presence and the desiccation tolerant state in soybean seeds (Glycine max L. Chippewa) was tested. Proteins that showed the same temporal pattern of expression as that reported for Lea proteins were identified in the axes of soybean. They were distinct from the known storage proteins and were resistant to heat coagulation. The level of these “maturation” proteins was closely correlated with desiccation tolerance both in the naturally developing and in the germinating seed: increasing at 44 days after flowering, when desiccation tolerance was achieved, and decreasing after 18 hours of imbibition, when desiccation tolerance was lost. During imbibition, 100 micromolar abscisic acid or Polyethylene glycol-6000 (−0.6 megapascals) delayed disappearance of the maturation proteins, loss of desiccation tolerance, and germination. During maturation, desiccation tolerance was prematurely induced when excised seeds were dried slowly but not when seeds were held for an equivalent time at high relative humidity. In contrast, maturation proteins were induced under both conditions. We conclude that maturation proteins may contribute to desiccation tolerance of soybean seeds, though they may not be sufficient to induce tolerance by themselves.     

Functional characteristics of a fruticose type of lichen, Stereocaulon foliolosum Nyl. in response to light and water stress

Stereocaulon foliolosum a fruticose type of lichen under its natural habitat is subjected to low temperature, high light conditions and frequent moisture stress due its rocky substratum. To understand as to how this lichen copes up with these stresses, we studied the reflectance properties, light utilization capacity and the desiccation tolerance under laboratory conditions. S. foliolosum showed light saturation point for photosynthesis at 390 μmol CO₂ m^?2 s^?1 and the light compensation point for photosynthesis at 64 μmol CO₂ m^?2 s^?1. Our experiments show that S. foliolosum has a low absorptivity (30–35 %) towards the incident light. The maximum rates of net photosynthesis and apparent electron transport observed were 1.9 μmol CO₂ m^?2 s^?1 and 45 μmol e^? m^?2 s^?1, respectively. The lichen recovers immediately after photoinhibition under low light conditions. S. foliolosum on subjecting to desiccation results in the decrease of light absorptivity and the reflectance properties associated with water status of the thalli show a change. During desiccation, a simultaneous decrease in photosynthesis, dark respiration and quenching in the fluorescence properties was observed. However, all the observed changes show a rapid recovery on rewetting the lichen. Our study shows that desiccation does not have a severe or long-term impact on S. foliolosum and the lichen is also well adapted to confront high light intensities.     

Development of desiccation tolerance and vitrification by preculture treatment in suspension-cultured cells of the liverwort Marchantia polymorpha

Some cultured plant cells are able to acquire tolerance to various stresses when they are cultured under suitably controlled conditions. Induction of a high level of desiccation tolerance in suspension-cultured cells of the liverwort Marchantia polymorpha was examined for studying the mechanisms of desiccation tolerance and vitrification at the cellular level. Desiccation tolerance level of cells was very low and the survival rate was less than 10% after exposure to drying below 0.1 g H₂O g⁻¹ dry weight (DW). Preculture treatment in 0.5 M sucrose medium was the most effective method for inducing a high level of desiccation tolerance in cells and the survival rate was 87% even after being desiccated to below 0.1 g H₂O g⁻¹ DW. Preculture treatment caused alteration of cell structures and accumulation of a large amount of sucrose and newly synthesized proteins in cells. Abundant sucrose and preculture-induced proteins were necessary for full development of desiccation tolerance in the cells. When water content decreased to below 0.1 g H₂O g⁻¹ DW, desiccation-tolerant cells that had been precultured were vitrified above 0°C and maintained stable viability. We have succeeded in the induction of desiccation tolerance that allows formation of intracellular glass with cell viability at ambient temperatures by controlling culture conditions, and our results suggest that suspension-cultured cells of M. polymorpha are useful for studying cellular mechanisms for the development of desiccation tolerance and the stabilization of vitrified cells.     

A comparison of desiccation tolerance among 12 species of chironomid larvae

Tolerance to desiccation was compared among 12 Japanese species of chironomid larvae under the condition of 60% in relative humidity at 25.5?°C. Three parameters were assessed: time to 50% survival (T ₅₀), water loss at 50% survival (WL₅₀) and water loss rate (WLR). T ₅₀, WL₅₀ and WLR were determined as measures of desiccation tolerance, dehydration tolerance, and dehydration resistance, respectively. T ₅₀ was 64.4–142 min for most species, except Propsilocerus akamusi (Tokunaga) which took 872 min. WL₅₀ was 60.6–82.4% for all species. WLR was only 0.0664% per minute for Pr. akamusi, while it was 0.629–1.50% for the other species. These results showed that Pr. akamusi had a high desiccation tolerance due to a high preventive ability of evaporation from body surface. T ₅₀ showed no significant relationships to WL₅₀ or WLR among the 12 species, while there was a significant positive relationship between WL₅₀ and WLR. These results suggest that chironomid species have a trade-off tendency that a species has a high tolerance – low resistance or a high resistance – low tolerance for dehydration.     

Freezing and Desiccation Tolerance in Entomopathogenic Nematodes: Diversity and Correlation of Traits

The ability of entomopathogenic nematodes to tolerate environmental stress such as desiccating or freezing conditions, can contribute significantly to biocontrol efficacy. Thus, in selecting which nematode to use in a particular biocontrol program, it is important to be able to predict which strain or species to use in target areas where environmental stress is expected. Our objectives were to (i) compare inter- and intraspecific variation in freeze and desiccation tolerance among a broad array of entomopathogenic nematodes, and (ii) determine if freeze and desiccation tolerance are correlated. In laboratory studies we compared nematodes at two levels of relative humidity (RH) (97% and 85%) and exposure periods (24 and 48 h), and nematodes were exposed to freezing temperatures (-2°C) for 6 or 24 h. To assess interspecific variation, we compared ten species including seven that are of current or recent commercial interest: Heterorhabditis bacteriophora (VS), H. floridensis, H. georgiana, (Kesha), H. indica (HOM1), H. megidis (UK211), Steinernema carpocapsae (All), S. feltiae (SN), S. glaseri (VS), S. rarum (17C&E), and S. riobrave (355). To assess intraspecific variation we compared five strains of H. bacteriophora (Baine, Fl1-1, Hb, Oswego, and VS) and four strains of S. carpocapsae (All, Cxrd, DD136, and Sal), and S. riobrave (355, 38b, 7-12, and TP). S. carpocapsae exhibited the highest level of desiccation tolerance among species followed by S. feltiae and S. rarum; the heterorhabditid species exhibited the least desiccation tolerance and S. riobrave and S. glaseri were intermediate. No intraspecific variation was observed in desiccation tolerance; S. carpocapsae strains showed higher tolerance than all H. bacteriophora or S. riobrave strains yet there was no difference detected within species. In interspecies comparisons, poor freeze tolerance was observed in H. indica, and S. glaseri, S. rarum, and S. riobrave whereas H. georgiana and S. feltiae exhibited the highest freeze tolerance, particularly in the 24-h exposure period. Unlike desiccation tolerance, substantial intraspecies variation in freeze tolerance was observed among H. bacteriophora and S. riobrave strains, yet within species variation was not detected among S. carpocapsae strains. Correlation analysis did not detect a relationship between freezing and desiccation tolerance.     

Hydroxyectoine Is Superior to Trehalose for Anhydrobiotic Engineering of Pseudomonas putida KT2440

Anhydrobiotic engineering aims to increase the level of desiccation tolerance in sensitive organisms to that observed in true anhydrobiotes. In addition to a suitable extracellular drying excipient, a key factor for anhydrobiotic engineering of gram-negative enterobacteria seems to be the generation of high intracellular concentrations of the nonreducing disaccharide trehalose, which can be achieved by osmotic induction. In the soil bacterium Pseudomonas putida KT2440, however, only limited amounts of trehalose are naturally accumulated in defined high-osmolarity medium, correlating with relatively poor survival of desiccated cultures. Based on the enterobacterial model, it was proposed that increasing intracellular trehalose concentration in P. putida KT2440 should improve survival. Using genetic engineering techniques, intracellular trehalose concentrations were obtained which were similar to or greater than those in enterobacteria, but this did not translate into improved desiccation tolerance. Therefore, at least for some populations of microorganisms, trehalose does not appear to provide full protection against desiccation damage, even when present at high concentrations both inside and outside the cell. For P. putida KT2440, it was shown that this was not due to a natural limit in desiccation tolerance since successful anhydrobiotic engineering was achieved by use of a different drying excipient, hydroxyectoine, with osmotically preconditioned bacteria for which 40 to 60% viability was maintained over extended periods (up to 42 days) in the dry state. Hydroxyectoine therefore has considerable potential for the improvement of desiccation tolerance in sensitive microorganisms, particularly for those recalcitrant to trehalose.     

Tolerance of Sargassum thunbergii germlings to thermal, osmotic and desiccation stress

The construction of artificial seaweed beds in the intertidal zone is a challenge due to extreme levels of physical stress. In order to provide a basis for the construction using the dispersal of microscopic juveniles, a three-way factorial experimental design was used to evaluate the tolerance of Sargassum thunbergii germlings shortly released from fertile thalli to temperature, salinity and desiccation in this study. Results revealed that temperature, salinity and desiccation significantly affected the growth and survival of germlings. Germlings showed rapid growth with relative growth rate (RGR, % day⁻¹) over 16% when cultured at 25 °C and full immersion in normal seawater. Although growths of germlings subjected to moderate conditions were significantly inhibited, RGRs over 13% were obtained. The RGRs of germlings below 10% were observed only at 35 °C and 9 h desiccation treatments. In comparison to growth, survival was less affected by physical stress. Germlings showed low mortalities below 10% under appropriate conditions (25 °C and 30 °C combined with full immersion), and below 60% under moderate conditions, by the end of experiment. However, the mortality rates increased to over 90% under extreme conditions (9 h desiccation and 35 °C combined with full immersion in salinity of 12). These results showed that S. thunbergii germlings had high tolerance to physical stresses. In addition to the main effects, both two-way and three-way interactions between temperature, salinity and desiccation were significant. Based on the magnitude of effect, desiccation was the predominant factor affecting both growth and survival. According to the results, construction of artificial tanks in natural habitat to minimize desiccation may be an effective strategy for S. thunbergii restoration using germlings.     

Phylogenetic differences in cardiac activity, metal accumulation and mortality of limpets exposed to copper: A prosobranch-pulmonate comparison

Taxonomic relationships for pollutant tolerance in marine invertebrates are surprisingly poorly known, despite being potentially useful for pollution biomonitoring. A popular view is that cellular and molecular adaptations for natural stress may be important in tolerating pollution. We compared the physiological and mortality responses to copper (Cu²⁺) of limpets from two different lineages: the Prosobranchia (Patellogastropoda: Helcion concolor and Cellana capensis) and the Pulmonata (Siphonaria serrata and Siphonaria capensis). Copper tolerance was apparently more closely related to phylogenetically-based physiology, than to tolerance of desiccation and or heat. The Siphonaria limpets were nearly an order of magnitude more tolerant of copper than the patellogastropod limpets, even though S. serrata has the lowest intertidal distribution. Initial copper exposure (0.25 ppm Cu²⁺ for 2 h) induced heart rate depression in Siphonaria (to around 50% of the baseline rate), while their tissue copper concentrations remained at the relatively high control levels. Copper exposure (0.25 ppm Cu²⁺ for 2 h) had no effect on heart rate of the patellogastropod limpets, but led to a significant increase in tissue copper. These results suggest that enhanced copper tolerance by Siphonaria relates to cardiac depression and a concomitant metabolic depression. Such physiological attributes are implicated in prolonged behavioural isolation, involving pneumostome closure and shell clamping, which is likely to reduce the uptake of copper. Furthermore, better regulation of internal copper levels by Siphonaria, is suggested by their exclusive possession of blood haemocyanin. Dependence on relatively high aerobic metabolism by the patellogastropod limpets, would limit their capacity for isolation and pollutant avoidance.     

Expression of a GALACTINOL SYNTHASE gene is positively associated with desiccation tolerance of Brassica napus seeds during development

Desiccation tolerance of seeds is positively correlated with raffinose family oligosaccharides (RFOs). However, RFOs’ role in desiccation tolerance is still a matter of controversy. The aim of this work was to monitor the accumulation of RFO during acquisition of desiccation tolerance in rapeseed (Brassica napus L.). Rapeseeds become desiccation tolerant at 21-24 d after flowering (DAF), and the time was coincident with an accumulation of raffinose and stachyose. A gene encoding galactinol synthase (GolS; EC2.4.1.123), involved in RFO biosynthesis, was cloned and functionally characterized. Enzymatic properties of recombinant galactinol synthase were also determined. Accumulation of BnGOLS-1 mRNA in developing rapeseeds was concomitant with dry weight deposition and the acquisition of desiccation tolerance, and was concurrent with the formation of raffinose and stachyose. The physiological implications of BnGOLS-1 expression patterns in developing seeds are discussed in light of the hypothesized role of RFOs in seed desiccation tolerance.     

Plant Desiccation and Protein Synthesis : V. Stability of Poly (A) and Poly (A) RNA during Desiccation and Their Synthesis upon Rehydration in the Desiccation-Tolerant Moss Tortula ruralis and the Intolerant Moss Cratoneuron filicinum

Upon desiccation of gametophytes of the desiccation-tolerant moss Tortula ruralis preexisting pools of poly(A)⁻ RNA (rRNA) remain inact, regardless of the speed at which desiccation is achieved. Preexisting poly(A)⁺ RNA pools (mRNA) are unaffected by slow desiccation but are substantially reduced during rapid desiccation. Poly(A)⁻ RNA involved in protein synthesis is also unaffected by desiccation, whereas the levels of polysomal poly(A)⁺ RNA in rapid- and slow-dried moss closely reflect the state of the protein synthetic complex in these dried samples.

Poly(A)⁻ RNA pools, both total and polysomal, are also stable during the rehydration of both rapid- and slow-dried moss. The total poly(A)⁺ RNA pool decreases upon rehydration, but this reduction is simply an expression of the normal turnover of poly(A)⁺ RNA in this moss. Analysis of polysomal fractions during rehydration reveals the continued use of conserved poly(A)⁺ RNA for protein synthesis. The rate of synthesis of poly(A)⁺ RNA upon rehydration appears to depend upon the speed at which prior desiccation is administered. Rapidly dried moss synthesizes poly(A)⁺ RNA at a faster rate, 60 to 120 minutes after the addition of water, than does rehydrated slowly dried moss. Recruitment of this RNA into the protein synthetic complex also follows this pattern. Comparative studies involving the aquatic moss Cratoneuron filicinum are used to gain an insight into the relevance of these findings with respect to the cellular mechanisms associated with desiccation tolerance.

     

Desiccation tolerance in diapausing spider mites Tetranychus urticae and T. kanzawai (Acari: Tetranychidae)

We investigated the effects of changes in vapor pressure deficit (VPD) on the survival of diapausing (winter form) and non-diapausing (summer form) spider mites Tetranychus urticae Koch and Tetranychus kanzawai Kishida (Acari: Tetranychidae). Adult females of both species were kept without food at VPDs of 0.0, 0.4, 0.7, 1.5, 1.9, or 2.7 kPa for 3, 6, 9, 12, or 15 days at 25 °C. Diapausing females of both species kept at a VPD of ≥0.4 kPa for ≥6 days clearly tolerated desiccation. Under water-saturated conditions (VPD = 0.0 kPa), in which no desiccation occurred, diapausing females showed high starvation tolerance: 90 % survived for up to 15 days. No interspecific differences in tolerance to desiccation or starvation were observed under most conditions. These results indicate that diapause functions increase tolerance to desiccation and starvation. Such multiple tolerances to harsh environments might support winter survival in spider mites.     

Sugars and desiccation tolerance in seeds

Soluble sugars have been shown to protect liposomes and lobster microsomes from desiccation damage, and a protective role has been proposed for them in several anhydrous systems. We have studied the relationship between soluble sugar content and the loss of desiccation tolerance in the axes of germinating soybean (Glycine max L. Merr. cv Williams), pea (Pisum sativum L. cv Alaska), and corn (Zea mays L. cv Merit) axes. The loss of desiccation tolerance during imbibition was monitored by following the ability of seeds to germinate after desiccation following various periods of preimbibition and by following the rates of electrolyte leakage from dried, then rehydrated axes. Finally, we analyzed the soluble sugar contents of the axes throughout the transition from desiccation tolerance to intolerance. These analyses show that sucrose and larger oligosaccharides were consistently present during the tolerant stage, and that desiccation tolerance disappeared as the oligosaccharides were lost. The results support the idea that sucrose may serve as the principal agent of desiccation tolerance in these seeds, with the larger oligosaccharides serving to keep the sucrose from crystallizing.     

Genetics of the Nematode Heterorhabditis bacteriophora Strain HP88: The Diversity of Beneficial Traits

Genotypic variation among infective juveniles of Heterorhabditis bacteriophora (strain HP88) in heat, desiccation, ultraviolet tolerance, and host-finding ability was assessed by comparing the performance of inbred lines of this entomopathogenic nematode in laboratory assays. Each line consisted of highly homozygous offspring originating from one individual obtained from a natural population. Considerable variation in all four traits was detected among the different inbred lines. The heritability values for heat or ultraviolet tolerance and for host-finding ability were high, indicating that selection should be an efficient way for improving these traits in the population. The results for desiccation tolerance varied considerably within each line. Heritability value was low, indicating that the results were influenced mainly by environmental variation and suggesting that selective breeding for higher desiccation tolerance would be inefficient. Improvement through induction of mutations may be a better alternative in this population.     

Developing sporophytes transition from an inducible to a constitutive ecological strategy of desiccation tolerance in the moss Aloina ambigua: effects of desiccation on fitness

Background and Aims Two ecological strategies of desiccation tolerance exist in plants, constitutive and inducible. Because of difficulties in culturing sporophytes, very little is known about desiccation tolerance in this generation and how desiccation affects sexual fitness.Methods Cultured sporophytes and vegetative shoots from a single genotype of the moss Aloina ambigua raised in the laboratory were tested for their strategy of desiccation tolerance by desiccating the shoot–sporophyte complex and vegetative shoots at different intensities, and comparing outcomes with those of undried shoot–sporophyte complexes and vegetative shoots. By using a dehardened clonal line, the effects of field, age and genetic variance among plants were removed.Key Results The gametophyte and embryonic sporophyte were found to employ a predominantly inducible strategy of desiccation tolerance, while the post-embryonic sporophyte was found to employ a moderately constitutive strategy of desiccation tolerance. Further, desiccation reduced sporophyte fitness, as measured by sporophyte mass, seta length and capsule size. However, the effects of desiccation on sporophyte fitness were reduced if the stress occurred during embryonic development as opposed to postembryonic desiccation.Conclusions The effects of desiccation on dehardened sporophytes of a bryophyte are shown for the first time. The transition from one desiccation tolerance strategy to the other in a single structure or generation is shown for only the second time in plants and for the first time in bryophytes. Finding degrees of inducible strategies of desiccation tolerance in different life phases prompts the formulation of a continuum hypothesis of ecological desiccation tolerance in mosses, where desiccation tolerance is not an either/or phenomenon, but varies in degree along a gradient of ecological inducibility.     

Isolation of extremely halophilic <Emphasis Type="Italic">Archaea</Emphasis> from a saline river in the Lut Desert of Iran,moderately resistant to desiccation and gamma radiation

Admittedly, the Lut Desert of Iran has been remained as an unexplored region from a microbiological standpoint. Domain Archaea contains extremophiles that can live in harsh habitats. Extremely halophilic archaea are exposed to different environmental stresses in the hypersaline environments such as high solar irradiance and periodic desiccation. Haloarchaeal diversity in Shoor River, a saline river in the Lut Desert (a salinity of 134.3 g L^–1 of dissolved salts), was investigated by a culture-dependent method. A large number of extremely halophilic isolates were obtained and a subset of 59 isolates was considered distinct. Firstly, the isolates were screened for their resistance under desiccation stress in 35 days. Eleven of these strains remained viable during the period in a desiccator containing silica gel. Then, three of them were randomly selected and their resistance against desiccation and ionizing radiation were determined. The isolates MS2, MS17, and MS50 were still recovered after 8 weeks in a desiccator and were moderately resistant to gamma radiation with D₁₀ value between 2 and 3 kGy. Strains MS2, MS17, and MS50 were affiliated with three species in the family Halobacteriaceae using 16S rRNA gene sequence analysis as well as morphological and biochemical characteristics—Haloterrigena jeotgali A29^T (99.6% similarity), Natrialba aegyptia 40^T (99.4% similarity) and Natrinema pallidum NCIMB 777^T (99.3% similarity), respectively. Although resistance to desiccation did not follow the sigmoid survival curve pattern of Deinococcus radiodurans, apparently haloarchaea can show a more resistance to desiccation in more long-term periods of time. This is the first report on isolation of extremely halophilic archaea belonged to the family Halobacteriaceae and their radioresistance and desiccation tolerance properties isolated from the Shoor River.     

Ultrastructural and biophysical changes in developing embryos of Aesculus hippocastanum in relation to the acquisition of tolerance to drying

Changes in ultrastructural, biochemical and biophysical characteristics of embryonic axes of Aesculus hippocastanum during development are related to changing levels of desiccation tolerance. Histodifferentiation was complete 30 days after flowering (DAF) and fruits were shed about 120 DAF. During this period, the dry mass of embryonic axes increased from about 0.5 to 4 mg and the water content decreased from 10.2 to 2.0 g H₂O g^?1 dry mass (g g^?1). In spite of the large changes in water content, water potentials of freshly harvested material declined slightly during development from ?0.65 to ?2.0 MPa. Tolerance of desiccation increased as embryos matured. If evaluated on the basis of critical water contents for survival, tolerance appeared to increase continuously, maximum tolerance being achieved at 120 DAF when embryos survived water contents as low as 0.30 g g^?1. When evaluated from critical water potentials, three distinct levels of desiccation tolerance could be recognized at ?1.8 MPa (30-40 DAF), ?4 M Pa (48-90 DAF) and ?12 MPs (100-120 DAF). During development, total dry matter increased while sugar content (g g^?1' dry mass) and osmotically active material (mmol g^?1 dry mass) decreased. The subcellular organisation of axes was always typical of metabolically active tissues. Maximum tolerance (?12 MPa) was associated with a reduced amount of monosaccharides and the appearance of water with unusual calorimetric behaviour. Our data are consistent with several of the current hypotheses regarding the mechanisms of desiccation tolerance. Accumulation of dry matter reserves, reduced levels of monosaccharides, presence of dehydrin-like proteins and ability to form glasses appear to be associated with the changes in desiccation tolerance. However, none of these factors allow A. hippocastanum embryos to achieve the extreme level of desiccation tolerance typical of orthodox seeds. This may be because A. hippocastanum embryos do not reach physiological maturity and remain metabolically active even after they are shed from the parent plant. Also, embryos may acquire incompetent protectants or lack as yet unidentified protective mechanisms.