Evidence is provided to show that frozen, hydrated seeds and seed tissues can be observed by low temperature field emission scanning electron microscopy. This technique allows preparation and observation of seed samples that contain 13–60% water without altering their moisture content. The technique provides information about the surface structure of seeds and also allows specimens to be fractured to reveal internal features of tissues. Futhermore, both halves of fractured specimens can be retained, examined and photographed either as two-dimensional micrographs or as stereo pairs when three-dimensional observation (stereology) or quantitative measurements (stereometry) are desired. Use of this technique avoids artefacts associated with chemical fixation, dehydration, and critical point drying—procedures that are usually required to prepare seed tissues for scanning electron microscope examination. This technique does not affect the degree of hydration in specimens; it can be used to localize water in tissues to determine their degree of hydration. This technique should find wide application in developmental studies of seeds, especially during maturation, imbibition and germination.

Fruit and seed development in Quercus robur L. were studied on a single tree over five consecutive seasons. Patterns of growth in the cotyledons and embryonic axes differed between years and resulted in seeds of very different sizes. Moisture content at shedding also differed between years, and late-shed seeds had lower moisture contents than early-shed seeds. Moisture content at shedding was negatively correlated with desiccation tolerance. Seed development in Q. robur therefore appeared indeterminate and did not end in a period of rapid desiccation.

Sensitivity to desiccation in Q. robur was not due to an inability to accumulate dehydrin proteins, ABA or soluble sugars, substances that have been linked with the acquisition of desiccation tolerance in orthodox seeds. There were great similarities between several aspects of Q. robur seed development and that of orthodox seeds before the latter entered the terminal phase of rapid desiccation. This pattern of seed development contrasted with that reported for the highly desiccation-sensitive seeds of Avicennia marina.

The relationship between tolerance of seeds to extreme desiccation and the presence of ‘dehydrinlike’ proteins was investigated in groups of related taxa from the unrelated plant families Aceraceae and Gramineae. Dehydrin-like proteins were identified by Western blot analysis using an antibody raised against a synthetic oligopeptide representing the 23-amino acid consensus sequence common to all group 2 late-embryogenesis-abundant (LEA) proteins.

Evidence is presented that seeds of Acer pseudoplatanus and A. saccharinum are desiccation intolerant (recalcitrant) whereas seeds of A. platanoides and A. rubrum are desiccation tolerant (orthodox). Despite these differences, dehydrinlike proteins at 60 and 20 kDa were detected in all four species.

Dehydrins at 20 kDa were also detected in seed samples of two aquatic grasses, Porteresia coarctata and Oryza sativa from the tribe Oryzeae, despite seeds of the former rapidly losing viability on drying, whereas O. sativa is one of the best-known examples of desiccation-tolerant seeds. In O. sativa, there was a correlation between contents of dehydrins detected and the proportion of individuals capable of withstanding extreme drying. However, the possibility of a causal link between these parameters is equivocal. Dehydrin-like proteins were also detected in desiccation-sensitive seeds of Zizania palustris, Z. latifolia and Z. texana and desiccation-intolerant seeds of Spartina anglica, all from the Gramineae.

The presence of group 2 LEAs is clearly not diagnostic of desiccation tolerance in seeds. However, a more direct correlation with the expression of other groups of LEAs cannot be discounted.

Changes in soluble sugars in cauliflower seeds were followed during 50 h of imbibition in relation to desiccation tolerance. Sucrose and stachyose contents decreased, and glucose and fructose accumulated. This occurred in radicles first and subsequently in hypocotyls and cotyledons. Loss of desiccation tolerance in the various seed parts coincided with an increase in glucose and fructose and the complete loss of stachyose, but sucrose content, the major sugar, was still high. Drying imbibed seeds over silica gel did not evoke resynthesis of stachyose, but did increase sucrose and decrease glucose and fructose contents. Seeds primed in solutions of 30% polyethylene glycol for 10 days showed a loss of stachyose, while sucrose remained high and glucose and fructose contents were still very low. Redrying of primed seeds did not change the sugar contents. The primed seeds were still tolerant of desiccation. We conclude that stachyose is not a prerequisite for desiccation tolerance, but that sucrose may be. We suggest that glucose and fructose may be involved in desiccation damage.

The final stage of development of orthodox seeds is maturation drying, a process often accompanied by a dramatic decline in storage-protein synthesis. When desiccation is imposed prematurely at certain stages prior to the completion of development, a switch in synthetic events is elicited. events unique to development, such as synthesis of storage protein, are terminated, while syntheses associated with germination and growth are initiated. We investigated whether desiccation plays a key role in effecting a decline in expression of genes for storage proteins by acting directly upon the regulatory regions of these developmental genes. The desiccation responsiveness of the 5′ and 3′ regulatory regions of the gene for the pea storage protein vicilin was tested in transgenic tobacco seed. Chimaeric genes were introduced into tobacco, these genes consisted of the coding region of the reporter gene for β-glucuronidase (GUS) and 5′ and 3′ regions from the vicilin gene, or, as controls, the same regions derived from constitutively expressed genes, presumed to be desiccation-insensitive – those from the cauliflower mosaic virus (35S) and nopaline synthase genes. In transgenic seed expressing the gene constructs containing the vicilin 5′ upstream region, GUS activities declined dramatically after imbibition in mature seed and after rehydration of prematurely dried tobacco seed. In contrast, GUS activities increased after seed rehydration when the constitutive viral (35S) promoter replaced the vicilin 5′ upstream region. The 3′ downstream region of the gene construct did not significantly affect the patterns of changes in GUS activities after seed rehydration. In studies of transient gene expression in castor bean cotyledons, premature desiccation led to termination of GUS gene expression only when the gene construct contained the vicilin 5′ upstream region. This region may respond directly to desiccation; alternatively, changes to trans-acting factors important for expression of genes for storage-proteins may occur as a result of drying.

This article reviews mechanisms by which specialized cells of different life forms have overcome the lethal effects of dehydration and considers how the maintenance of genetic information is central to survival. As a dynamic and hydrated molecule in vivo, DNA can assume different conformational structures depending upon the water activity, the base sequence and the presence of specific binding proteins. The attainment of stable secondary structures that are resistant to degradation in vivo at low water potentials is proposed as a likely accompaniment to desiccation tolerance. In addition, chemical modification of bases in DNA, the extent of methylation and conformational changes could determine the expression of different gene sequences as cells pass from desiccation-tolerant to desiccation-intolerant states. We monitored the integrity of extracted DNA in embryos of seeds and in wind-dispersed pollen during transition from their desiccation tolerance to desiccation intolerance on hydration and germination. We present evidence to show that the DNA of these two stages is different and that it is the DNA from desiccation-tolerant cells only that retains integrity when the cells are subjected to desiccation regimes. We discuss these findings in relation to certain hydration-sensitive DNA structures and to other relevant biological systems.

A characteristic of recalcitrant seeds is that, if they are maintained under storage conditions that prevent water loss, they will ultimately lose viability. A current view is that hydrated recalcitrant seeds are metabolically active and undergo germination-associated changes in storage. Some of these changes, such as extensive vacuolation and increase in cell size, imply a requirement for water additional to that present in the seed on shedding. It is therefore suggested that, in storage, recalcitrant seeds are exposed to an initially mild, but increasingly severe, water stress. Deleterious events associated with a water stress of considerable duration are suggested to lead ultimately to the death of the tissue.

The damage that occurs on prolonged storage is unlikely to be associated with an inability to form glasses or prevent membrane lipid phase changes, as absolute water contents are higher than those at which these mechanisms become important. It is considered that the most likely process leading to death of water-stressed (as opposed to dehydrated) tissue is a breakdown of co-ordination of metabolism, leading to uncontrolled free-radical-mediated oxidative damage.

It is generally difficult to maintain tissue in a mild water-stressed condition for extended periods. Stored, hydrated, recalcitrant seeds may provide an ideal model system for studying the metabolic effects of prolonged mild water stress.

Embryos of Araucaria angustifolia seeds showed no dormancy; they germinated easily at temperatures ranging from 10° to 30°C, and the thermal optimum was about 25–30°C; they were recalcitrant. At harvest, their mean moisture content (dry weight basis) was about 120% and they completely lost viability when their moisture content fell to about 30%. The cotyledons were more sensitive to dehydration than the radicle. Dehydration induced deterioration of cell membranes as indicated by a high increase in leakage of solutes. It also resulted in damage in the nuclei, which was not repaired upon rehydration. During desiccation, respiratory activity decreased; however, O2 uptake was not an indication of germination ability, since it was significantly affected only when embryo moisture content reached the critical value of 30%. The decrease in the capacity to convert 1-aminocyclopropane 1-carboxylic acid to ethylene, which was observed at 30–60 min of dehydration, was a very early indicator of deterioration in embryos. Desiccation resulted also in a rapid decrease in the ability for protein synthesis as measured by [35S]methionine incorporation into total protein; 50% inhibition was observed after 30–60 min of desiccation for both axis and cotyledons.

In the presence of abscisic acid (ABA), contents of glucose and fructose decreased in carrot embryoids, whereas umbelliferose increased during dehydration. The acquisition of desiccation tolerance was tested by scoring germinated embryoids. It did not keep pace with the changes in soluble carbohydrate contents during development, which suggests that in these somatic embryoids di- and oligosaccharides are not the determining factors for anhydrobiosis. However, on slow dehydration, umbelliferose increased, while sucrose decreased and the monosaccharides declined completely. These changes were positively correlated with increased desiccation tolerance. A similar analysis of slowly dried, ABA-treated alfalfa embryoids showed that stachyose and sucrose increased and the monosaccharides declined with the acquisition of desiccation tolerance. These data support the contention that carbohydrates are involved in anhydrobiosis.

Changes in the properties of water in excised embryos were measured during the late stages of grain development in two cultivars of Zizania palustris and a population of the endangered species Z. texana. The relationships between water content and water activity were determined from water sorption isotherms, measured at temperatures between 35 and 5°C and then derived for lower temperatures. The freezing and melting behaviour of water in embryos at different water contents was determined using differential scanning calorimetry. The moisture content of embryos at high water activities decreased with maturation, as did the moisture content at which freezing transitions were not observed. While the temperatures of freezing and melting transitions decreased as the moisture content of embryos decreased, there were no discernible differences among embryos at different developmental stages. The properties of water measured in maturing Zizania embryos approached those for orthodox seeds as determined from the strength of water sorption, the enthalpy of the melting transition and the moisture content at which water is unfreezable. From these data we conclude that the properties of water in recalcitrant Zizania embryos change with development to resemble those of embryos of desiccation-tolerant seeds, but that the seeds never achieve the orthodox condition. The effects of interactions between moisture content and temperature on desiccation damage, freezing damage and germination in Zizania are predicted, based on the physical properties of water reported here and the correspondence of these properties with physiological function reported for other species. The resulting ‘phase diagram’ defines possible combinations of moisture content and temperature for storage under equilibrium conditions.

This review focuses on the possible evolutionary status and functional significance of recalcitrance and certain associated ovule/seed characters, within the framework of modern systems of angiosperm classification. The presence of recalcitrant seed viability in 45 dicotyledonous families is significantly associated with bitegmic and crassinucellate ovules and with nuclear endosperm development, all considered ancestral (plesiomorphic) character states of the ovule; as well as with greater seed size, woody habit and tropical habitat, also regarded as ancestral character states in the dicotyledons. In many species with recalcitrant seeds, the predominant storage reserve is carbohydrate. Recalcitrance is significantly associated with the exalbuminous type of reserve storage. It is proposed that in large recalcitrant seeds, the transfer of the main storage function from endosperm to embryo was probably an early development. In many species with recalcitrance, the ovules/seeds are characterized by extensive vascularization of the integument(s)/seed coat or by a pachychalaza. Pachychalazy is proposed to be a significant functional adaptation for a more efficient transfer of nutrients to the embryo/seed. Recalcitrance and some of the other character states proposed to be plesiomorphic in dicotyledons are also present in some gymnosperms, including presumed sister groups of the dicotyledons. In relatively advanced dicotyledonous families, mostly with orthodox seeds, recalcitrance probably persisted only in isolated relict members. Determination of character polarity is particularly problematic at lower taxonomic levels, because there is always the possibility that, in some taxa, a character state such as recalcitrance may have arisen secondarily as a reversal. Available evidence supports our view that seed recalcitrance can be regarded as a relatively ancestral character state in dicotyledons.

Western blot analysis using antiserum raised against the lysine-rich box common to dehydrins, one class of late-embryogenesis-abundant (LEA) proteins, was used to study the abundance of heat-stable ‘dehydrin-like’ proteins during development and water stress in Ranunculus sceleratus L. achenes (seeds). A 61 kDa dehydrin-like protein was apparently limited to immature seeds (fresh and dried) which had not attained full desiccation tolerance. In contrast, lower-molecular-mass proteins which were induced by desiccation were found only in more mature seeds. The molecular masses of desiccation-induced proteins changed during seed development from 18 kDa in seeds harvested at 13 days post anthesis (DPA) to 31 kDa at harvest maturity, 21 DPA.

Placing seeds at 21 DPA in polyethylene glycol (PEG) at −1.5 MPa reduced seed moisture content and was accompanied by accumulation of 31 kDa protein. This protein was no longer detected when the seeds were transferred to water. In seeds harvested at 13 DPA, PEG induced the synthesis not only of 18 kDa protein (which is associated with dried seeds at this developmental stage), but also of 28 kDa and 31 kDa proteins. These dehydrin-like proteins were also synthesised when seeds at 13 DPA were imbibed in water. These and other data indicate that both quantitative and qualitative changes in dehydrin-like proteins can occur in R. sceleratus, depending on seed maturity and the degree and duration of water stress.

Storage proteins (2S, 7S and 11S) are synthesized at the mid- to late stages of development in alfalfa seeds, mainly within the embryos. Mature dry seeds cannot synthesize these proteins upon subsequent germination and growth. When embryos were isolated from the seed during development (stages VII and VIII) and placed on water or nutrient medium, they germinated. They exhibited a pattern of protein synthesis which was identifiable as germinative/post-germinative, and was identical to the pattern synthesized in embryos of germinated dry seeds, and of embryos from seeds that were subjected to drying prematurely at stages VII and VIII. When, after 48 h from the start of imbibition, abscisic acid or osmoticum was introduced to germinated embryos isolated at stage VII and not desiccated, the synthesis of the 11S storage protein was restored to a rate comparable to that of stage-VII embryos before isolation. This was accompanied by an increase in transcription of the 11S storage protein gene. Transfer of germinated stage-VII embryos, which had first been desiccated, to abscisic acid or osmoticum 48 h after imbibition started did not result in any restoration of storage protein synthesis. Thus, desiccation, prematurely or at maturation, off-regulates storage protein synthesis and within the embryos this process is no longer responsive to abscisic acid or osmoticum.

Seeds were collected at weekly intervals from mid-maturation to the fully ripened stage. As seed development progressed, desiccation tolerance increased. Desiccation tolerance of C. lansium seeds was greatest at 67 days after anthesis (DAA), when they tolerated air drying for 9 days; 74 DAA was considered as physiological maturity, and their full viability was only maintained for up to 3 days of drying; overripened seeds (88 DAA) had the lowest desiccation tolerance. In L. chinensis, the desiccation sensitivity of seeds at 98 DAA (fully mature) was higher than that at 84 and 91 DAA (less mature); among the excised embryonic axes at different developmental stages, the less mature ones were less sensitive to desiccation than the fully mature ones; excised embryonic axes of the same stage were more tolerant of desiccation than whole seeds.