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.Most angiosperm seeds can survive desiccation but only at a discrete developmental stage. If they are dried before reaching the desiccation tolerant stage of maturity, they will not germinate (2,26). Similarly, if they are dried after germination has progressed too far, they will not continue to germinate upon rehydration (4,18,26). The emergence of the radicle from the seed coat is generally considered to be the stage at which desiccation tolerance is lost during germination (26).Water is important to organisms not only as a solvent for biochemical reactions, but as a stabilizer of structure. Hydrophilic and hydrophobic interactions impart structure to macromolecules and organelles within cells. Membrane structure, in particular, depends on these complex interactions, and is often regarded as a primary site of desiccation damage (6, 26). The water replacement hypothesis suggests that polyhydroxy compounds can substitute for water in stabilizing membrane structure in the dry state (7,23,30 Leakage of Electrolytes. Seeds were imbibed for periods up to and beyond radicle emergence, then transferred to a chamber containing saturated LiCl, which equilibrates to 11% RH (21). Here the seeds dried to approximately 8% moisture content (dry weight basis). This low moisture content is lethal to desiccationintolerant tissues. After drying, the seeds were transferred to 100% relative humidity for 24 h for slow rehydration in order to minimize damage to cells from hydrational forces (15). Thus, leakage from cells damaged by desiccation should be the main source of the electrolytes measured. Groups of 10 axes were isolated and submerged in 15 mL of deionized water. Conductivity was monitored continuously with an ElectroMark Conductivity Meter (Markson Science, Inc., Del Mar, CA). The rate of leakage was measured after 15 min, by which time it ...

DSC was used to study the ability of glass-forming sugars to affect the gel-to-fluid phase transition temperature, T(m), of several phosphatidylcholines during dehydration. In the absence of sugars, T(m) increased as the lipid dried. Sugars diminished this increase, an effect we explain using the osmotic and volumetric properties of sugars. Sugars vitrifying around fluid phase lipids lowered T(m) below the transition temperature of the fully hydrated lipid, T(o). The extent to which T(m) was lowered below T(o) ranged from 12 degrees to 57 degrees, depending on the lipids' acyl chain composition. Sugars vitrifying around gel phase lipids raised T(m) during the first heating scan in the calorimeter, then lowered it below T(o) in subsequent scans of the sample. Ultrasound measurements of the mechanical properties of a typical sugar-glass indicate that it is sufficiently rigid to hinder the lipid gel-to-fluid transition. The effects of vitrification on T(m) are explained using the two-dimensional Clausius-Clapeyron equation to model the mechanical stress in the lipid bilayer imposed by the glassy matrix. Dextran and polyvinylpyrrolidone (PVP) also vitrified but did not depress T(m) during drying. Hydration data suggest that the large molecular volumes of these polymers caused their exclusion from the interbilayer space during drying.

The formation of intracellular glass may help protect embryos from damage due to desiccation. Soluble sugars similar to those found in desiccation tolerant embryos were studied with differential scanning calorimetry. Those sugars from desiccation tolerant embryos can form glasses at ambient temperatures, whereas those from embryos that do not tolerate desiccation only form glasses at subzero temperatures. It is concluded that tolerant embryo cells probably contain sugar glasses at storage temperatures and water contents, but intolerant embryo cells probably do not.The ability to survive the desiccated state is the result of adaptations that prevent cellular destruction during the withdrawal of water. As water leaves a cell that does not tolerate desiccation, many events occur: solutes become more concentrated, possibly increasing the rate of destructive chemical reactions; some solutes may crystallize, changing the ionic strength and pH ofthe intracellular solution; proteins become denatured, many irreversibly; and membranes become disrupted, leading to the loss of compartmentation. It has been suggested that the presence of large amounts of soluble sugars within a cell can prevent the damaging effects of desiccation (2). Soluble sugars are known to form hydrogen bonds and thus may substitute for water in maintaining hydrophilic structures in their hydrated orientation, even when water is no longer present (2). Water replacement by soluble sugars has been demonstrated in model systems, where soluble sugars were able to preserve the functional integrity, measured as Ca2+-ATPase activity, of desiccated microsomes through a dehydration/rehydration cycle (2).Another mechanism by which sugars may act to protect the cell during desiccation is by the formation of an intracellular glass (5 the solution is called a glass. A glass is essentially an undercooled liquid; therefore, its existence is temperature dependent. A solution that exists as a glass at one temperature will melt at a higher temperature, giving rise to a liquid and the possibility of crystallization (3).The benefits of glass formation, or vitrification, to an organism undergoing water loss are many. Glasses preclude chemical reactions requiring diffusion, thus ensuring stability during a period of dormancy; they fill space, and thus by sheer bulk may prevent cellular collapse; an amorphous glass may trap chaotropic solutes, preventing their becoming concentrated; and glasses may permit the continuance of hydrogen bonding at the interface between the glass and hydrophilic surfaces in the cell (1). These properties would help ensure the survival of an organism during a period of desiccation.Using DSC3, Williams and Leopold (10) found evidence of a glass-like state in desiccated corn embryos. Transitions similar to those made by glasses were detected in both the lipid and nonlipid portions of the embryo, and it was hypothesized that the soluble sugar component of the embryo was responsible for the nonlipid glass transition (10). Soluble sugars compri...

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During cold acclimation of Puma rye (Secale cereale L. cv Puma), the intracellular osmotic potential nearly doubles. During this period, the accumulation of glycinebetaine, proline, and soluble sugars was monitored. The amount of glycinebetaine increased from 290 to 1300 micrograms per gram fresh weight during the 4-week acclimation period. Proline content did not change during the first 3 weeks of acclimation but then increased from 27 to 580 micrograms per gram fresh weight during the next 3 weeks. The total soluble sugar content more than doubled by the second week of cold acclimation, increasing from 11 to 26 milligrams per gram fresh weight. Most of this increase can be attributed to the accumulation of sucrose and raffinose, whose levels increased from 2.4 and 0 to 11 and 5 milligrams per gram fresh weight, respectively. The content of monosaccharides, predominantly glucose, remained at a constant 10 milligrams per gram fresh weight throughout the acclimation period. A comparison of the sugar content of protoplasts versus vacuoles isolated from cold-acclimated leaves revealed that the extravacuolar volume contained monosaccharides, sucrose, and raffinose. Thus, the increased amounts of sucrose and raffinose that occur during cold acclimation are present in compartments extemal to the vacuole and may contribute to cryoprotection.

A common feature of desiccation-tolerant organisms, such as orthodox seeds, is the presence of large quantities of sugars, especially di-and oligosaccharides. These sugars may be one component of the suite of adaptations that allow anhydrobiotes to survive the loss of most of their cellular water. This paper describes the physical effects of dehydration on cellular ultrastructure, with particular emphasis on membranes, and explains quantitatively how sugars and other solutes can influence these physical effects. As a result of dehydration, the surfaces of membranes are brought into close approach, which causes physical stresses that can lead to a variety of effects, including demixing of membrane components and fluid-to-gel phase transitions of membrane lipids. The presence of small solutes, such as sugars, between membranes can limit their close approach and, thereby, diminish the physical stresses that cause lipid fluid-to-gel phase transitions to occur during dehydration. Thus, in the presence of intermembrane sugars, the lipid fluid-to-gel phase transition temperature (T m) does not increase as much as it does in the absence of sugars. Vitrification of the intermembrane sugar solution has the additional effect of adding a mechanical resistance to the lipid phase transition; therefore, when sugars vitrify between fluid phase bilayers, T m is depressed below its fully hydrated value (T o). These effects occur only for solutes small enough to remain in very narrow spaces between membranes at low hydration. Large solutes, such as polymers, may be excluded from such regions and, therefore, do not diminish the physical forces that lead to membrane changes at low hydration.

The moss Physcomitrella patens is becoming the model of choice for functional genomic studies at the cellular level. Studies report that Physcomitrella survives moderate osmotic and salt stress, and that desiccation tolerance can be induced by exogenous ABA. Our goal was to quantify the extent of dehydration tolerance in wild type moss and to examine the nature of cellular damage caused by desiccation. We exposed Physcomitrella to humidities that generate water potentials from -4 (97% RH) to -273 MPa (13% RH) and monitored water loss until equilibrium. Water contents were measured on a dry matter basis to determine the extent of dehydration because fresh weights (FW) were found to be variable and, therefore, unreliable. We measured electrolyte leakage from rehydrating moss, assessed overall regrowth, and imaged cells to evaluate their response to drying and rehydration. Physcomitrella did not routinely survive water potentials \-13 MPa. Upon rehydration, moss dried to water contents[0.4 g g dm -1 maintained levels of leakage similar to those of hydrated controls. Moss dried to lower water contents leaked extensively, suggesting that plasma membranes were damaged. Moss protonemal cells were shrunken and their walls twisted, even at -13 MPa. Moss cells rehydrated after drying to -273 MPa failed to re-expand completely, again indicating membrane damage. ABA treatment elicited tolerance of desiccation to at least -273 MPa and limited membrane damage. Results of this work will form the basis for ongoing studies on the functional genomics of desiccation tolerance at the cellular level.

SummarySeed oils enriched in omega-7 monounsaturated fatty acids, including palmitoleic acid (16:1Δ9) and cis-vaccenic acid (18:1Δ11), have nutraceutical and industrial value for polyethylene production and biofuels. Existing oilseed crops accumulate only small amounts (<2%) of these novel fatty acids in their seed oils. We demonstrate a strategy for enhanced production of omega-7 monounsaturated fatty acids in camelina (Camelina sativa) and soybean (Glycine max) that is dependent on redirection of metabolic flux from the typical Δ9 desaturation of stearoyl (18:0)-acyl carrier protein (ACP) to Δ9 desaturation of palmitoyl (16:0)-acyl carrier protein (ACP) and coenzyme A (CoA). This was achieved by seed-specific co-expression of a mutant Δ9-acyl-ACP and an acyl-CoA desaturase with high specificity for 16:0-ACP and CoA substrates, respectively. This strategy was most effective in camelina where seed oils with~17% omega-7 monounsaturated fatty acids were obtained. Further increases in omega-7 fatty acid accumulation to 60-65% of the total fatty acids in camelina seeds were achieved by inclusion of seed-specific suppression of 3-keto-acyl-ACP synthase II and the FatB 16:0-ACP thioesterase genes to increase substrate pool sizes of 16:0-ACP for the Δ9-acyl-ACP desaturase and by blocking C18 fatty acid elongation. Seeds from these lines also had total saturated fatty acids reduced to~5% of the seed oil versus~12% in seeds of nontransformed plants. Consistent with accumulation of triacylglycerol species with shorter fatty acid chain lengths and increased monounsaturation, seed oils from engineered lines had marked shifts in thermotropic properties that may be of value for biofuel applications.