Changing desiccation tolerance of pea embryo protoplasts during germination

Protoplasts were isolated from pea (Pisum sativum L. cv. Alaska) embryonic axes during and after germination to determine whether the loss of desiccation tolerance in the embryos also occurs in the protoplasts. At all times studied, protoplast survival decreased as water content decreased; however, the sensitivity to dehydration was less when the protoplasts were isolated from embryos that were still desiccation-tolerant (12 h and 18 h of imbibition) than when protoplasts were derived from axes that were sensitive (24 h and 36 h of imbibition). The water content at which 50% of the population was killed (WC50) increased throughout germination and early seedling growth for both the intact tissue and the protoplasts derived from them. Prior to radicle emergence, protoplasts were less desiccation-tolerant than the intact axes; however, protoplasts isolated from radicles shortly after emergence had lower WC50s than the intact radicles. A comparison of protoplast survival after isolation and dehydration in either 500 mM sucrose/raffinose or 700 mM sucrose revealed no difference in tolerance except at 24 h of imbibition, when protoplasts treated in the more concentrated solution had improved tolerance of dehydration. Although intact epicotyls are generally more desiccation-tolerant than radicles, protoplasts isolated separately from epicotyls and radicles did not differ in tolerance. Collectively, these data suggest that protoplasts gradually lose desiccation tolerance during germination, as do the orthodox embryos from which they were derived. However, even prior to radicle emergence, protoplasts display a sensitivity to progressive dehydration that is similar to that shown by recalcitrant and ageing embryos.     

Sugar effects on membrane damage during desiccation of pea embryo protoplasts

Desiccation tolerance of protoplasts isolated from germinating pea (Pisum sativum L. cv. 'Alaska') embryonic axes depends, in part, on the osmotic strength and composition of the suspending medium. To determine the reason for this dependence and whether treatment with different solutions results in different types of damage, protoplast recovery and survival were assessed after dehydration to a range of water contents. Protoplasts were derived from germinating axes that had intermediate desiccation tolerance. Protoplasts were isolated and resuspended in buffers containing sucrose/raffinose (85:15, w/w) or sorbitol, which were isotonic or hypertonic to the cells of the embryonic axis, then were flash-dried to a range of water contents. Protoplasts were rehydrated and stained with fluorescein diacetate (FDA) to assess survival and to estimate two types of membrane injury: lysis and the loss of semipermeability. In all treatments, protoplast survival dropped sharply during the initial phase of dehydration due to lysis. Protoplast survival was greater in hypertonic sucrose/raffinose buffer than in isotonic sucrose/raffinose buffer, or in the latter made hypertonic by the addition of sorbitol. When sorbitol was substituted for sucrose/raffinose in either the isolation or desiccation buffer, or both, protoplast survival at intermediate and low hydrations decreased due to a loss of membrane semipermeability. The results indicate that additional sucrose/raffinose is beneficial for the desiccation tolerance of protoplasts, the benefit is not due to a simple osmotic effect, and the benefit is greatest at water contents less than 0.5 g g(-1) DW, where the presence of the sugars appears to protect membrane semipermeability.     

The role of sugar, vitrification and membrane phase transition in seed desiccation tolerance

In a search for the mechanism of desiccation tolerance, a comparison was made between orthodox (desiccation-tolerant) soybean ( Glycine max [L.] Merrill) and recalcitrant (desiccation-intolerant) red oak ( Quercus rubra L.) seeds. During the maturation of soybean seeds, desiccation tolerance of seed axes is correlated with increases in sucrose, raffinose and stachyose. In cotyledons of mature oak seeds, sucrose levels are equal to those in mature soybeans, but oligosaccharides are absent. By using the thermally stimulated current method, we observed the glassy state in dry soybean seeds during maturation. Oak cotyledons showed the same phase diagram for the glass transition as did mature soybeans. By using X-ray diffraction, we found the maturation of soybeans to be associated with an increased ability of membranes to retain the liquid crystalline phase upon drying, whereas the mature oak cotyledonary tissue existed in the gel phase under similar dry conditions. These findings lead to the conclusion that the glassy state is not sufficient for desiccation tolerance, whereas the ability of membranes to retain the liquid crystalline phase does correlate with desiccation tolerance. An important role for soluble sugars in desiccation tolerance is confirmed, as well as their relevance to membrane phase changes. However, the presence of soluble sugars does not adequately explain the nature of desiccation tolerance in these seeds.     

Maturation proteins and sugars in desiccation tolerance of developing soybean seeds

The desiccation-tolerant state in seeds is associated with high levels of certain sugars and maturation proteins. The aim of this work was to evaluate the contributions of these components to desiccation tolerance in soybean (Glycine max [L.] Merrill cv Chippewa 64). When axes of immature seeds (34 d after flowering) were excised and gradually dried (6 d), desiccation tolerance was induced. By contrast, seeds held at high relative humidity for the same period were destroyed by desiccation. Maturation proteins rapidly accumulated in the axes whether the seeds were slowly dried or maintained at high relative humidity. During slow drying, sucrose content increased to five times the level present in the axes of seeds held at high relative humidity (128 versus 25 μg/axis, respectively). Stachyose content increased dramatically from barely detectable levels upon excision to 483 μg/axis during slow drying but did not increase significantly when seeds were incubated at high relative humidity. Galactinol was the only saccharide that accumulated to higher levels in axes from seeds incubated at high relative humidity relative to axes from seeds that were slowly dried. This suggests that slow drying serves to induce the accumulation of the raffinose series sugars at a point after galactinol biosynthesis. We conclude that stachyose plays an important role in conferring desiccation tolerance.     

Induction of tolerance to fast desiccation in black spruce (Picea mariana) somatic embryos: relationship between partial water loss,sugars, and dehydrins

Events associated with the induction of tolerance to fast desiccation in black spruce ( Picea mariana ) somatic embryos were investigated. An experimental approach using an initial period of partial water loss was developed to induce either no, partial, or complete tolerance to fast desiccation. Tolerance to subsequent fast desiccation was not promoted by decreasing embryo water content from 1.5 to 1.1 g H₂O g⁻¹ DW (g g⁻¹) throughout the first 24 h of slow desiccation. However, tolerance increased from 10 to 95% germination during the second 24-h period of slow desiccation after partial water loss from 1 to 0.55 g g⁻¹. Emphasis was also placed on the relationship between observed tolerance, and sugar and dehydrin contents. Compared to controls, sucrose content in embryos doubled after 24 h of slow desiccation and more than tripled after 48 h. Conversely, starch content was decreased by one half after 24 h and by three quarters after 48 h. Sucrose abundance and raffinose occurrence after 48 h of slow desiccation were congruent with complete tolerance to fast desiccation. The period of slow desiccation between 24 and 48 h also increased the content of a 24-kDa dehydrin and the appearance of a 42-kDa dehydrin. The relationship between partial water loss, sugars and dehydrins is discussed with respect to tolerance to fast desiccation in black spruce somatic embryos.     

Water Content, Raffinose, and Dehydrins in the Induction of Desiccation Tolerance in Immature Wheat Embryos

Desiccation tolerance is initiated in wheat (Triticum aestivum L.) embryos in planta at 22 to 24 d after anthesis, at the time that the embryo water content has decreased from about 73% fresh weight (2.7 g water/g dry weight) to about 65% fresh weight (1.8 g water/g dry weight). To determine if desiccation tolerance is fully induced by the loss of a relatively small amount of water, detached wheat grains were treated to reduce the embryo water content by just a small amount to approximately 69% (2.2 g water/g dry weight). After 24 h of such incipient water loss, subsequently excised embryos were able to withstand severe desiccation, whereas those embryos that had not previously lost water could not. Therefore, a relatively small decrease in water content for only 24 h acts as the signal for the development of desiccation tolerance. Embryos that were induced into tolerance by a 24-h water loss had no detectable raffinose. The oligosaccharide accumulated at later times even in embryos of detached grains that had not become desiccation tolerant, although tolerant embryos (i.e. those that previously had lost some water) contained larger amounts of the carbohydrate. It is concluded that desiccation tolerance and the occurrence of raffinose are not correlated. Immunodetected dehydrins accumulated in embryos in planta as desiccation tolerance developed. Detachment of grains induced the appearance of dehydrins at an earlier age, even in embryos that had not been made desiccation tolerant by incipient drying. It is concluded that a small reduction in water content induces desiccation tolerance by initiating changes in which dehydrins might participate but not by their interaction with raffinose.     

Characterization of Membrane Properties in Desiccation-Tolerant and -Intolerant Carrot Somatic Embryos

In previous studies, we have shown that carrot (Daucus carota L.) somatic embryos acquire complete desiccation tolerance when they are treated with abscisic acid during culture and subsequently dried slowly. With this manipulable system at hand, we have assessed damage associated with desiccation intolerance. Fast drying caused loss of viability, and all K+ and carbohydrates leached from the somatic embryos within 5 min of imbibition. The phospholipid content decreased by about 20%, and the free fatty acid content increased, which was not observed after slow drying. However, the extent of acyl chain unsaturation was unaltered, irrespective of the drying rate. These results indicate that, during rapid drying, irreversible changes occur in the membranes that are associated with extensive leakage and loss of germinability. The status of membranes after 2 h of imbibition was analyzed in a freeze-fracture study and by Fourier transform infrared spectroscopy. Rapidly dried somatic embryos had clusters of intramembraneous particles in their plasma membranes, and the transition temperature of isolated membranes was above room temperature. Membrane proteins were irreversibly aggregated in an extended [beta]-sheet conformation and had a reduced proportion of [alpha]-helical structures. In contrast, the slowly dried somatic embryos had irregularly distributed, but non-clustered, intramembraneous particles, the transition temperature was below room temperature, and the membrane proteins were not aggregated in a [beta]-sheet conformation. We suggest that desiccation sensitivity of rapidly dried carrot somatic embryos is indirectly caused by an irreversible phase separation in the membranes due to de-esterification of phospholipids and accumulation of free fatty acids.     

玉米胚发育过程中脱水耐性的变化

对离体玉米胚脱水耐性的变化以及不同脱水速率对其脱水耐性的影响进行了研究。授粉后16d的玉米胚能耐轻微脱水,含水量从1.45降低到0.28gH2Og-1DW时胚的萌发率为100%,但含水量低于0.1gH2Og-1DW时胚死亡。胚的脱水耐性随着发育逐渐加强,表现为电解质渗漏速率逐渐降低,萌发率和幼苗干重逐渐增加。授粉后20d胚内超氧化物歧化酶(SOD)和抗坏血酸过氧化物酶(APX)活性较高,过氧化氢酶(CAT)活性较低;授粉后24d,这些酶的活性与授粉后20d的正好相反。脂质过氧化产物丙二醛(MDA)在种子发育过程中呈下降趋势。不同脱水速率明显地影响胚的脱水耐性:在慢速脱水到含水量0.1~0.18gH2Og-1DW时,胚的萌发率和幼苗干重比快速脱水高,电解质渗漏速率比快速脱水低;在快速脱水条件下胚中的SOD、APX活性和MDA含量也比慢速脱水高;CAT活性的变化不明显。     

Acquisition of desiccation tolerance in developing wheat embryos correlates with appearance of a fluid phase in membranes

Membrane behaviour in developing wheat (Triticum aestivum cv Priokskaya) embryos was studied in relation to the acquisition of desiccation tolerance, using spin probe techniques. Fresh embryos were able to develop into seedlings at day 15 after anthesis, but it took 18 d before fast‐dried, isolated embryos could germinate. On the basis of membrane integrity measurements it was estimated that between 14 and 18 d after anthesis the proportion of embryonic cells surviving fast drying increased and the critical moisture content, to which embryonic cells could be dehydrated, decreased. Apparently, embryonic cells do not acquire the same level of desiccation tolerance simultaneously. Only when all cells had become desiccation tolerant was germination of air‐dried embryos possible. Using 5‐doxylstearic acid as the probe molecule, an approximately similar lipid–water interface ordering of membranes was observed in all hydrated embryos, irrespective of age. Dehydration had a dual effect on the lipid interface: further ordering of the major part of the interface and the appearance of additional, disturbed regions. The proportion of these regions correlated with the proportion of desiccation‐tolerant cells. We propose that the membrane surface disturbance be caused by endogenous amphiphiles that partition from the cytoplasm into membranes during drying. The absence of such disturbed regions in dried, desiccation‐sensitive embryos might reflect a lack of sufficient amphiphiles. The relevance of membrane surface disturbance for desiccation tolerance is discussed.     

Proteomics of desiccation tolerance during development and germination of maize embryos

Maize seeds were used to identify the key embryo proteins involved in desiccation tolerance during development and germination. Immature maize embryos (28N) during development and mature embryos imbibed for 72 h (72HN) are desiccation sensitive. Mature maize embryos (52N) during development are desiccation tolerant. Thiobarbituric acid reactive substance and hydrogen peroxide contents decreased and increased with acquisition and loss of desiccation tolerance, respectively. A total of 111 protein spots changed significantly (1.5 fold increase/decrease) in desiccation-tolerant and -sensitive embryos before (28N, 52N and 72HN) and after (28D, 52D and 72HD) dehydration. Nine pre-dominantly proteins, 17.4 kDa Class I heat shock protein 3, late embryogenesis abundant protein EMB564, outer membrane protein, globulin 2, TPA:putative cystatin, NBS-LRR resistance-like protein RGC456, stress responsive protein, major allergen Bet v 1.01C and proteasome subunit alpha type 1, accumulated during embryo maturation, decreased during germination and increased in desiccation-tolerant embryos during desiccation. Two proteins, Rhd6-like 2 and low-molecular-weight heat shock protein precursor, showed the inverse pattern. We infer that these eleven proteins are involved in seed desiccation tolerance. We conclude that desiccation-tolerant embryos make more economical use of their resources to accumulate protective molecules and antioxidant systems to deal with maturation drying and desiccation treatment.     

Contents of sugars in leaves of drying desiccation tolerant flowering plants, particularly grasses

Sugar complements were analysed in extracts from leaves of desiccation tolerant species in the angiosperm families Cyperaceae, Gesneriaceae, Liliaceae, Poaceae and Velloziaceae. Total sugar content was higher in live air-dry leaves of all desiccation tolerant species (except the grass Eragrostiella nardoides; 22 µmoles/g dw) than in the dead air-dry leaves of the desiccation sensitive grass Sporobolus pyramidalis (36 µmoles/g dw). Sucrose contents rose to high levels (40–98 µmoles/g dw) in live air-dry leaves of all species (except the grass Eragrostiella nardoides in which it rose to only 11 µmoles/g dw) to become the predominant sugar. Glucose and/or fructose contents frequently were lower after leaf drying but usually these were the sugars of next highest contents in live air-dry leaves. Contents of raffinose (that has been postulated to reduce sucrose crystallization) rose to c. 10% of sucrose contents in air-dry leaves of most desiccation tolerant species (but only c. 4% in Tripogon jacquemontii) compared with c. 2% of sucrose contents in the sensitive grass S. pyramidalis. Trehalose (a rare sugar in seed-plants) was present in all but one desiccation tolerant species (Xerophyta villosa) but only in minor amounts. The results are consistent with the views that sugars play a protective role during drying of desiccation tolerant plants in general but that other factors are also involved indesiccation tolerance, that in desiccation tolerant angiospermae sucrose is generally the predominant protective sugar and that raffinose and trehalose may supplement the role of sucrose.     

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.     

Callus formation and plantlet regeneration from protoplasts derived from suspension cultures of wheat (Triticum aestivum L.)

Protoplasts were isolated from anther-derived suspension cultures of commercial wheat (Triticum aestivum L. cv. Chris). The protoplasts were released enzymatically and isolated by centrifugation on a sucrose cushion. The isolated protoplasts were initially cultured in a liquid medium in the dark. Numerous microcalli were produced under these conditions, some of which differentiated into globular embryos. Upon transfer to a solid medium and exposure to 16h/8h light/dark cycle, the protocalli proliferated and many of the somatic embryos matured. Complete plantlets were obtained and maintained in sterile culture.Abbreviations 2,4-D 2,4-Dichlorophenoxyacetic acid - MES 2-[N-morpholino] ethanesulfonic acid     

“To dryness and beyond” – Preparation for the dried state and rehydration in vegetative desiccation-tolerant plants

The ability of vegetative plant tissues to survive desiccation is an uncommon trait, although plants that are able to do this represent all major classes of plants. Two classes of vegetative desiccation-tolerant plants exist; those that are modified desiccation-tolerant and can only survive desiccation if drying rates are slow, and those that are fully desiccation-tolerant and can survive even rapid drying rates. Investigations into the cellular level responses of these two types of plants has lead to an understanding of the underlying mechanisms of desiccation-tolerance. The following proposed mechanisms for desiccation-tolerance are presented. Modified desiccation-tolerant plants utilize inducible cellular protection systems supplemented in part by a minor rehydration induced repair component. Fully desiccation-tolerant plants utilize a rehydration induced repair system that is complemented by a constitutive protection component. This minireview explores the evidence for these proposed mechanisms in an attempt to lay the theoretical ground work for future work in this area.     

Isolation and culture of protoplasts from embryogenic suspension cultures of red fescue (Festuca rubva L.)

Protoplasts were isolated from fast-growing embryogenic suspension cultures of red fescue cv. Dawson (Festuca rubra L.) without agitation. The enzyme isolation solution was highly efficient at releasing protoplasts of greater than 95% viability (5×10⁶–10⁷ protoplasts per ml of packed cell volume). A three step procedure was followed for washing and transferring protoplasts from a solution high in inorganic salts to a medium containing glucose and sucrose. The addition of 30 mM sodium thiosulfate to the wash and culture media was found to be helpful in reducing the number of lysed protoplasts. Isolated protoplasts began to divide within 48–72 h when protoplasts were plated in agarose squares and surrounded by nurse cells (mixed nurse plating technique). Maximum colony formation (plating efficiency) was approximately 1%. Many of the colonies continued to grow and produced embryos when transferred to a medium consisting of half-strength MS salts, 4 mg/l 2,4-D, 3 g/l casein hydrolysate and 30 g/l sucrose. Upon transfer to hormone-free medium and exposure to light 16 h/day, many of the embryos germinated to produce green leaves and roots.Abbreviations BA Benzylaminopurine - 2,4-D 2,4-dicholorophenoxyacetic acid - DMSO dimethyl sulfoxide - MES 2-(N-morpholino)-ethanesulfonicn acid - MS Murashige and Skoog medium (1962) - UGC Ultraclone Growth Chamber - KM Kao and Michayluk medium (1975) - NAA Naphthalene acetic acid     

The competence to acquire cellular desiccation tolerance is independent of seed morphological development.

Acquisition of desiccation tolerance and the related changes at the cellular level in wheat (Triticum aestivum cv. Priokskaya) kernels during normal development and premature drying on the ear were studied using a spin probe technique and low temperature scanning electron microscopy. During normal development, the ability of embryos to germinate after rapid drying and rehydration was acquired after completion of morphological development, which is a few days before mass maturity. The acquisition of desiccation tolerance, as assessed by germination, was associated with an upsurge in cytoplasmic viscosity, the onset of accumulation of protein and oil bodies, and the retention of membrane integrity upon dehydration/rehydration. These features were also used to assess cellular desiccation tolerance in the cases when germination could not occur. Slow premature drying was used to decouple the acquisition of cellular desiccation tolerance from morphogenesis. Upon premature drying of kernels on the ears of plants cut at 5 d after anthesis, desiccation-tolerant dwarf embryos were formed that were able to germinate. When plants were cut at earlier stages poorly developed embryos were formed that were unable to germinate, but cellular desiccation tolerance was nevertheless acquired. In such prematurely dried kernels, peripheral meristematic endosperm cells had already passed through similar physiological and ultrastructural changes associated with the acquisition of cellular desiccation tolerance. It is concluded that despite the apparent strong integration in seed development, desiccation tolerance can be acquired by the meristematic cells in the developing embryo and cambial layer of endosperm, independently of morphological development.     

Isolation of protoplasts from somatic embryos of carrot

Protoplasts were isolated enzymatically from synchronously induced globular somatic embryos from a carrot suspension culture. Among the macerating enzymes tested, Driselase was the most effective for release of protoplasts from embryos. A higher medium osmolarity was required for the isolation of protoplasts from embryos than from undifferentiated cells. Protoplasts from embryos were smaller than protoplasts from undifferentiated cells. On step gradients of Ficoll, protoplasts from embryos gave one major band. Protoplasts from undifferentiated cells gave two major bands, one lighter and the other heavier than the protoplasts from embryos.     

Onset of desiccation tolerance during development of the barley embryo

We have investigated events which take place in the developing barley (Hordeum vulgare L.) embryo during its acquisition of desiccation tolerance. Excised embryos are capable of precocious germination as early as 8 d after pollination (DAP). At this age, however, they are not capable of resisting a desiccation treatment which induces a loss of 96–98% of their initial water content. At 16 DAP the embryos germinate despite the drastic drying treatment. The pattern of in-vivo and in-vitro proteins synthesized by the developing embryos from 12 DAP (desiccation-intolerant) and 16 DAP (desiccation-tolerant) were compared. A set of 25–30 proteins was identified which is denovo synthesized or enhanced during the developmental period leading to desiccation tolerance. Abscisic acid (ABA; 100 M) applied in vitro for 5 d to 12-DAP embryos induces desiccation tolerance and represses a subset of polypeptides preferentially associated with 16-DAP embryos. During in vitro culture of barley embryos ABA stimulates the appearance of a set of proteins and prevents the precocious germination allowing embryogenesis to continue in vitro. It also suppresses a set of germination-related proteins which appear 4 h after the incubation of the dissected embryo on a germination medium without ABA. Almost all mRNAs remain functional for translation when isolated embryos are dried at the desiccation-intolerant and tolerant stages of embryo development.Abbreviations ABA abscisic acid - DAP days after pollination - GM germination medium - poly(A)RNA polyadenylated RNA - SDS sodium dodecyl sulfate