JXB Advance Access originally published online on February 5, 2007
Journal of Experimental Botany 2007 58(5):1185-1196; doi:10.1093/jxb/erl286
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RESEARCH PAPER |
Water properties in fern spores: sorption characteristics relating to water affinity, glassy states, and storage stability
1Banco de Germoplasma, Jardí Botànic-ICBiBE, Universitat de València, C/Quart, 80, E-46008 València, Spain
2USDA-ARS National Center for Genetic Resources Preservation, 1111 So. Mason Street, Fort Collins, CO 80521, USA
* To whom correspondence should be addressed. E-mail: christina.walters{at}ars.usda.gov
Received 2 August 2006; Revised 8 November 2006 Accepted 28 November 2006
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Abstract |
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Ex situ conservation of ferns may be accomplished by maintaining the viability of stored spores for many years. Storage conditions that maximize spore longevity can be inferred from an understanding of the behaviour of water within fern spores. Water sorption properties were measured in spores of five homosporeous species of ferns and compared with properties of pollen, seeds, and fern leaf tissue. Isotherms were constructed at 5, 25, and 45 °C and analysed using different physicochemical models in order to quantify chemical affinity and heat (enthalpy) of sorption of water in fern spores. Fern spores hydrate slowly but dry rapidly at ambient relative humidity. Low Brunauer–Emmet–Teller monolayer values, few water-binding sites according to the D'Arcy–Watt model, and limited solute–solvent compatibility according to the Flory–Huggins model suggest that fern spores have low affinity for water. Despite the low water affinity, fern spores demonstrate relatively high values of sorption enthalpy (
Hsorp). Parameters associated with binding sites and Hsorp decrease with increasing temperature, suggesting temperature- and hydration-dependent changes in volume of spore macromolecules. Collectively, these data may relate to the degree to which cellular structures within fern spores are stabilized during drying and cooling. Water sorption properties within fern spores suggest that storage at subfreezing temperatures will give longevities comparable with those achieved with seeds. However, the window of optimum water contents for fern spores is very narrow and much lower than that measured in seeds, making precise manipulation of water content imperative for achieving maximum longevity. Key words: Ex situ conservation, germplasm, relative humidity, temperature, water sorption isotherms
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Introduction |
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Pteridophytes (ferns) are associated with ecosystems that are particularly sensitive to degradation, and some taxa are in peril and require strict protection. Ex situ conservation provides an important backup strategy, as pteridophytes are very sensitive to environmental perturbations. Germplasm banks provide ex situ conservation of many plants by preserving seeds for decades or centuries (Gómez-Campo, 2001; Smith et al., 2003; Guerrant et al., 2004). However, methodologies for long-term preservation of pteridophytes in germplasm banks are not well established. Maintaining pteridophyte spore viability, genetic integrity, and capacity for normal growth are key research objectives to enable their effective ex situ conservation (Page et al., 1992).
Long-term viability of pteridophyte spores depends on spore type or taxonomic group (Lloyd and Klekowski, 1970). The storage behaviour of the two types of spores recognized—green and non-green—is very different. Green spores are chlorophyllous and usually lose viability within weeks (e.g. Equisetum sp.) or months (e.g. Osmunda regalis), although survival for almost a year has been reported for a few genera stored at room temperature (e.g. Onoclea and Matteuccia) (Lloyd and Klekowski, 1970; Whittier, 1996; Lebkuecher, 1997) or at liquid nitrogen temperatures (Pence, 2000; Ballesteros et al., 2005). Longevity of non-green spores under ambient room conditions ranges from a few months (e.g. Gleicheniaceae, Thyrsopteris elegans) to about a decade (most species), with some species surviving several decades (e.g. Pellaea sp., Asplenium serra, Marsilea sp.) (Lloyd and Klekowski, 1970; Dyer, 1979; Page, 1979; Windham et al., 1986; Lindsay et al., 1992; Page et al., 1992). Though these studies record survival times, they do not provide quantitative accounts of the effects of storage temperature and relative humidity (RH) on the loss of spore viability or the negative effects on gametophyte development and genetic integrity (Smith and Robinson, 1975; Beri and Bir, 1993; Camloh, 1999).
Early recommendations for fern spore conservation suggested using the same dry, low-temperature conditions used for seeds (Dyer, 1979). Routine methods for conservation of orthodox seeds in germplasm banks are based on models that recommend dehydration to 5±2% water (or equilibration to 15–20% RH) and storage at 5 °C or –25 °C (Roberts and Ellis, 1989; FAO/IPGRI, 1994; Walters, 1998, 2004; Gómez-Campo, 2001). However, application of these models to existing data using fern spores suggests that methods used for seeds may not be effective for long-term conservation of fern spores. Refrigerated storage at about 5 °C maintains high viability for about 1–6 years, an improvement over viability achieved by storage at room temperature (Smith and Robinson, 1975; Simabukuro et al., 1998b; Camloh, 1999; Pence, 2000; Quintanilla et al., 2002; Aragón and Pangua, 2004; Ballesteros et al., 2004). However, deterioration was faster for some spores stored in the freezer (–25 °C) compared with the refrigerator (Simabukuro et al., 1998b; Constantino et al., 2000; Quintanilla et al., 2002; Aragón and Pangua, 2004; Ballesteros et al., 2004). Spores of some fern species did not tolerate initial exposure to –25 °C (Simabukuro et al., 1998b; Quintanilla et al., 2002; Aragón and Pangua, 2004; Ballesteros et al., 2004). Other results show no differences in viability of three spore species following 75 months of storage at 4 °C or –20 °C (Pence, 2000).
Hydrated storage of fern spores is becoming increasingly recommended because viability can be maintained for 12–24 months at either room or refrigerated temperatures (Lindsay et al., 1992; Simabukuro et al., 1998b; Quintanilla et al., 2002; Aragón and Pangua, 2004). Imbibed spores maintain remarkable tolerances, having higher survival after 48 h at 70 °C than spores dried to ambient RH (Simpson and Dyer, 1999). Germination of hydrated spores is prevented by continuous darkness and, analogous to seeds in secondary dormancy (Villiers, 1974), maintenance of viability is believed to result from ongoing cellular repair. The continuous metabolism is likely to affect storage reserves within the spore and, eventually, negatively impact germination and early gametophyte development.
The efficacy of dry storage has not been fully explored for fern spores, either because partial drying (to ambient RH) does not maintain longevity as well as hydrated storage (Lindsay et al., 1992; Quintanilla et al., 2002), or because of reported sensitivity to drying treatments. Drying with silica gel has given inconsistent results. Spores of the tree ferns Dicksonia sellowiana and Cyathea caracasana were reportedly damaged by silica gel drying (Constantino et al., 2000). However, longevity and tolerance to –12 °C or –25 °C were improved in spores of Cyathea dalgadii, Lophosoria quadripinnata, Polystichum lonchitis, and Ceterach officinarum dried over silica gel compared with spores dried to ambient RH (Simabukuro et al., 1998b; Constantino et al., 2000; Ballesteros et al., 2004). The conflicting results of drying spores suggest that precise control of water content is necessary to implement dry, frozen storage in fern spore conservation programmes.
Viability of dry biological systems has long been attributed to the properties of water or the removal of water with specific properties (Leopold et al., 1994; Hoekstra, 2005; Walters et al., 2005). Water status within fern spores and its behaviour at different temperatures is not known, which makes it difficult to predict achievable longevities of fern spores or to compare physiological differences of spores among species. A better understanding of the relationships between water content and viability of fern spores will provide guidance for optimizing storage conditions to maximize longevity.
This study was undertaken to identify water properties in spores of diverse fern species and to compare them with existing information on pollen and seeds. Here water interactions with fern spores are studied using water sorption isotherms. Classical applications of water sorption isotherm models reveal the chemical affinity of materials for water (Rockland, 1969; Vertucci and Leopold, 1987a, b; Oksanen and Zografi, 1990; Costantino et al., 1998; Lyall et al., 2003; Nagarajan et al., 2005). More recent interpretations have also used isotherm shape to account for structural relaxation within amorphous solids (glasses) during plasticization by water (Zhang and Zografi, 2000). These new applications provide a means to link classical studies of water binding with more recent concepts of glasses in order to establish a firm theoretical basis upon which to predict the shelf-life of biological materials.
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Materials and methods |
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Plant materials and water content determinations
Mature fronds of different fern species were collected from wild populations during the summer of 2005 at the Valencian Community, Spain (Table 1). Species were collected from diverse families, environments, and cytological traits to test future hypotheses about the influence of these factors on water properties and shelf-life. Fronds were pressed onto glossy paper and allowed to dry. After sporangial dehiscence, spores were collected from the sheets, sieved, and subsequently stored at –80 °C until used. Spores were mailed to Fort Collins, CO, USA using expedited post and arrived within 3 d. There was no discernible difference in the viability of spores after collection and shipment (data not given).
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Water content of spores was manipulated by equilibrating them at 5, 25, and 45 °C and different RH or water vapour pressures, adjusted using water or the saturated salt solutions listed in Table 2 (Winston and Bates, 1960; Rockland, 1969; summarized by Vertucci and Roos, 1993). Spores were placed in open Petri plates that were held above water or the saturated salt solutions at 5, 25, and 45 °C in screw-cap jars. Spores were also equilibrated to low RH at 15 °C to confirm temperature anomalies observed within the 5 °C and 25 °C isotherms. Periodically, subsamples weighing between 5 mg and 15 mg were removed from RH chambers and hermetically sealed into preweighed 20 µl aluminium pans and weighed using an electronic balance. Subsampling was frequent during the first day of hydration or dehydration, then after 24 h and 48 h, then after 1 week and 2 weeks, and finally in monthly intervals for 3–6 months. After fresh weight measurements, pans were punctured and heated at 95 °C for 36 h to obtain the sample dry weight (DW). Water content values were calculated from the difference in fresh and dry weight and are expressed on a dry-weight basis as g H2O g–1 DW. Within-replicate averages were calculated from at least four separate subsamplings taken between 7 d and 6 months (i.e. water contents reached steady-state) after the spores were placed in the RH chamber. Standard deviation of these equilibrated water content measurements ranged from 8% to 10% of the average value, probably as a result of the accuracy of the electronic balance (±1 µg) and rapid re-equilibration of the small subsamples to room conditions during measurement. The entire equilibration process was repeated at least once for each species, RH, and temperature treatment or until variation among replicates was <5% of the average measurement. Similar data for pollen were reported by Buitink et al. (1998), and are used here for comparative purposes.
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Sorption characteristics of fern spores were evaluated using water sorption isotherms constructed at 5, 25, and 45 °C for RH between 0.5% and 97%, and at 15 °C for RH between 0.5% and 15%. Isotherms were analysed using three different sorption models as well as van't Hoff analysis to determine heats of sorption at specific temperatures (isotherm models) and water contents (van't Hoff analysis).
Application of isotherm models
Isotherm models quantify the tendency to absorb water using two types of parameters. The hydrophilic environment, or chemical affinity for water, is quantified by the number of binding sites. The nature of interactions between adsorbent surface and water molecules is described by the enthalpy of sorption. Applications of the Brunauer–Emmet–Teller (BET), D'Arcy–Watt, and Flory–Huggins models were used to quantify the chemical affinity for water of fern spores, and BET and D'Arcy–Watt models to quantify the enthalpy of sorption in fern spores.
The BET model builds from Langmuir isotherms, which saturate at high partial pressures, using the additional assumption that adsorbate molecules cluster upon each other in progressive layers to allow infinite adsorption (Brunauer et al., 1938; Atkins, 1982). BET models were adapted to the special case of water adsorption by using RH/100 to measure the partial pressure of water and water content (wc) to describe the volume of adsorbate (assumes density of 1 g ml–1):
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BET parameters were calculated from isotherms of fern spores by linear regression of RH/100 and (RH/100)/[wc(1–RH/100)], where wc is the water content of the spores for each RH/temperature combination. The y-intercept of the regression line is equal to wcmon–1 c–1 and the slope is equal to (c–1)wcmon–1 c–1, where wcmon (in g H2O g–1 DW) is the BET monolayer, that describes the water content at which sites at the adsorbent surface are filled, and c is related to the sorption enthalpy [Hsorp(T)] of water binding at monolayer sites at the isotherm temperature:
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40%, and the slope of the BET plot [i.e. (c–1)x wcmon–1 c–1] was constrained to 400 to ensure that at least six RH/wc data points were used in each regression and that the r2 of all regressions was greater than 0.90.
The D'Arcy–Watt equation was developed to describe water sorption onto heterogeneous materials by making provisions for differing enthalpies between three types of sorption sites (D'Arcy and Watt, 1970). The equation is a composite of three terms, each representative of a different type of water binding:
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90%. Values for K and K' were calculated from linear regressions of 100/RH and 1/wc for RH between 0% and 25%. The value of k' was calculated by constraining k=1 and regressing 100/RH and 1/wc for RH between 75% and 100%. Regressions for terms were performed iteratively using an Excel spreadsheet to achieve the highest correlation between modelled and experimental data (Vertucci and Leopold, 1987a).
The Flory–Huggins model considers absorption as a process of dissolution, and uses the parameter 
to describe the interaction between the adsorbate (molecules in fern spores) and the solvent (water) (Flory, 1953). A simplified equation was used that expresses the amount of water as water content, rather than volume, by assuming a constant specific gravity for water and large molecular mass of the absorbate:
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The value of is low for highly soluble material and increases as solubility decreases (e.g. =0.65 for dextran in water; Zhang and Zografi, 2000). Values for in fern spores were determined by fitting the Flory–Huggins model to RH–wc data at RH >75%.
van't Hoff analyses
Application of van't Hoff analyses (Atkins, 1982) to pollen and seed sorption isotherms were described previously (Vertucci and Roos, 1993; Vertucci et al., 1994; Walters, 1998; Eira et al., 1999). Van't Hoff isochores describe the temperature dependence of 
, the equilibrium constant for adsorption and desorption at a specific water content according to
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RH/100 at equilibrium, T is temperature (in Kelvin), R is the ideal gas constant (8.3143 J K–1 mol–1) and Hsorp(w) is the sorption enthalpy at a given water content. By contrast to BET and D'Arcy–Watt models, where sorption enthalpy is assumed constant for a particular binding site but may vary with temperature, sorption enthalpies calculated from van't Hoff analyses are considered a function of water content [hence the subscript (w)] and presumed constant with temperature. Relative humidity–temperature combinations were interpolated from isotherms and used to draw isochores for temperatures between 5 °C and 45 °C and water contents ranging from 0.005 to 0.2 g H2O g–1 DW at 0.005 g H2O g–1 DW intervals. For each water content, the slope of the regression between ln(RH/100) and T–1 was used to calculate Hsorp(w) according to equation 5. Correlation coefficients (r2) and percentage error of slope (standard error of slope÷slope) were usually 
0.95 and 0.10, respectively, for water contents 0.03 g H2O g–1 DW, and support the assumption of a constant Hsorp(w) with temperature. When these criteria were not met (often for spores at water contents <0.03 g H2O g–1 DW), the assumption that Hsorp(w) is constant with temperature could not be supported. In these cases, two lines were drawn to maximize r2 and minimize percentage error. Isotherms at –18 °C could be constructed by extrapolating the linear relationship of the isochores. These extrapolations assume that no phase changes that affect the aqueous domain of the cytoplasm (i.e. no molecular denaturation or water freezing events) occur between 5 °C and –18 °C. |
Results |
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The initial water content of fern spores from five species placed at ambient room conditions in Fort Collins, CO, USA (about 30% RH and 22 °C) was between 0.02 and 0.04 g H2O g–1 DW. The water content of fern spores placed over water vapour increased to a maximum value within about 16–48 h depending on the species (Fig. 1; Table 3). Spores swell as they take up water and diameters of spores placed within liquid water or over water vapour were similar after 72 h (data not shown), indicating that imbibition was complete and that fern spores absorbed similar amounts of water when imbibed in either vapour or liquid water. Maximum water contents ranged from 0.14 (Pteris vittata) to 0.52 (Polystichum aculeatum) g H2O g–1 DW depending on species (Fig. 1; Table 3). By contrast to spores, Typha latifolia pollen absorbed more water in a shorter time and reached a steady-state water content of about 0.88 g H2O g–1 DW in about 30 h (Fig. 1).
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Hydrated fern spores dried quickly when exposed to low RH. Water contents of fully hydrated spores decreased to <0.03 g H2O g–1 DW (over 90% of the initial water removed) in about 8 h (Fig. 2). After the initial water losses depicted in Fig. 2 (first data point in Fig. 3), water contents of spores equilibrated to the RH of the chamber and there were no significant changes in water content after about 7 d (Fig. 3). The water content at equilibrium depended on the RH (Fig. 3A). For example, P. aculeatum spores at 25 °C equilibrated to 0.005, 0.019, and 0.033 g H2O g–1 DW at 0.5, 5.5, and 11% RH, respectively. The water content at equilibrium also depended on temperature, with lower temperatures generally yielding higher water contents for a given RH (Fig. 3B). In addition to these factors, the equilibrium water content depended on species. Water contents of P. vittata were usually much lower than those of other species at a given RH–temperature combination (Fig. 3C). The kinetics of dehydration were similar for Typha pollen, although the duration of the initial rapid drying phase was longer, probably as a result of higher initial water contents (Fig. 2).
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Water sorption isotherms, representative of the equilibrium relationship between RH and water content, were constructed for spores from each species of fern at 5, 25, and 45 °C (Fig. 4). Isotherm shape followed the reverse-sigmoidal pattern typically observed for proteins and polymers (Costantino et al., 1998; Zhang and Zografi, 2000) and orthodox seeds and pollen (Vertucci and Leopold, 1987a; Vertucci and Roos, 1993; Buitink et al., 1998). The relationship between RH and water content depended on species, with spores of P. vittata and Polystichum setiferum giving the lowest and highest, respectively, water contents for any given RH and temperature combination (Fig. 4A). The relationship between RH and water content also depended on temperature, with water contents at a given RH typically increasing with decreasing isotherm temperature (Fig. 4B).
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Water sorption isotherms of fern spores at 5, 25, and 45 °C were fitted to various isotherm models to calculate parameters relating to chemical affinity (i.e. number of hydrophilic binding sites) and enthalpy of sorption (
Hsorp). Model parameters calculated from isotherms of pollen (Typha latifolia), embryonic axes of soybean (Glycine max), and fronds of the desiccation-tolerant fern Polypodium polypodioides are given for comparative purposes (data taken from Vertucci and Leopold, 1987a, b; Buitink et al., 1998). Values for parameters of the BET monolayer (wcmon) and strength of sorption (c) (Brunauer et al., 1938; Atkins, 1982) were calculated for all isotherms (Table 3). At 25 °C, monolayer values averaged 0.027±0.004 g H2O g–1 DW among spores of all species, and ranged from 0.022 to 0.034 g H2O g–1 DW for spores of P. vittata and P. setiferum, respectively. BET monolayer values for fern spores were less than those calculated for pollen, embryonic axes, or desiccation-tolerant leaves. Monolayer values for spores tended to decrease as temperature increased (Table 3). The D'Arcy–Watt (1970) model builds from assumptions in the BET model, with an additional provision for sorption sites with different binding strengths. The sum of water adsorption by each type of site produced a composite isotherm with good agreement to measurements of water content at given RH–temperature combinations (Fig. 5). The amount of water associated with strong binding sites was calculated for fern spores using the D'Arcy–Watt model, and was similar to the average amount of water calculated as the BET monolayer (Table 3). At 25 °C, the average amount of strongly bound water was 0.025±0.004 g H2O g–1 DW among species and ranged from 0.020 for spores of P. vittata to 0.029 g H2O g–1 DW for spores of P. aculeatum and P. setiferum. Pollen and fern leaf tissue had comparable, and soybean embryonic axes had more, water on strong binding sites compared with fern spores. The average amount of water associated with weak binding sites (h) in fern spores was 0.038±0.010 g H2O g–1 DW, and ranged from 0.029 g H2O g–1 DW for spores of P. vittata to 0.055 g H2O g–1 DW for spores of P. setiferum (Table 3). The number of weak binding sites was two to five times greater in soybean axes, typha pollen, and fern leaf tissue. Similarly, soybean axes, typha pollen, and fern leaf tissue had considerably more multimolecular sorption sites (k') than were calculated for fern spores. The number of strong and weak sorption sites (K'+h) calculated from the D'Arcy–Watt model decreased with increasing isotherm temperature. There was no consistent relationship between temperature and the number of multimolecular sorption sites.
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The Flory–Huggins model (Flory, 1953) considers sorption as the initial steps of the dissolution process. The rising slope of the isotherm at high RH is characterized by the parameter
which describes adsorbent–solvent interactions. Low values of indicate highly soluble substances and high values indicate substances that do not tend to dissolve. Values of calculated from fern spores isotherms drawn at 5, 25, and 45 °C were greater than 1.3 (Table 3) and tended to increase with increasing temperature. Values of calculated from isotherms at 25 °C for soybean axes, pollen, and desiccation-tolerant fern leaves were <1 (Table 3) and approached values for water-soluble polymers (Zhang and Zografi, 2000). At 25 °C, calculated values of were largest for spores of P. vittata (1.97) and least for spores of P. setiferum (1.50).
The apparent enthalpy of water sorption [Hsorp(T)] by fern spores can also be calculated from sorption isotherm models. The average enthalpy of water-adsorbent associations is approximated by the natural logarithm of the BET parameter c and the D'Arcy–Watt parameter K. The average value for the BET parameter c was 71±12 for fern spores at 25 °C, which was comparable with the value calculated for soybean axes and considerably higher than values calculated for pollen and fern leaf tissue. The average value for the D'Arcy–Watt parameter K was 58±12 for fern spores at 25 °C, which was similar to the value calculated for pollen and less than values calculated for soybean axes and fern leaf tissues. Values of c or K tended to decrease with increasing temperature, with large changes observed for P. aculeatum, P. setiferum, and Dryopteris filix-mas, and only minor changes with temperature observed for Thelypteris palustris and P. vittata.
van't Hoff analyses of isotherms allowed adsorption enthalpies to be calculated at specific water contents [Hsorp(w)]. Based on the equilibrium partitioning between water within the vapour phase and condensed on the adsorbate surface, high values of [Hsorp(w)] are expected at low water contents because of the high (very negative) free energy driving this exothermic reaction. High values of Hsorp(w) for fern spores containing <0.05 g H2O g–1 DW were apparent from the steep slopes when the natural logarithm of RH/100 is plotted against temperature–1 (in Kelvin) (Figs 6, 7). The slope of these plots became progressively shallower as water content increased above 0.05 g H2O g–1 DW. For the most part, van't Hoff plots of fern spores were linear, suggesting fairly predictable sorption–desorption equilibrium coefficients. However, at very low water contents, van't Hoff plots for spores of all species tested except D. filix-mas appeared non-linear (plots of P. setiferum appeared to be non-linear, but were treated as linear because isotherms were constructed at only three temperatures). The non-linear behaviour resulted from higher than expected water contents measured in spores equilibrated at 25 °C and 45 °C and RH <20%. Assuming the linear relationship between T–1 and ln(RH/100) continues as temperature decreases below 5 °C, isotherms at –18 °C can be predicted [dashed lines in Fig. 7 show the extrapolation required to construct the –18 °C isotherm in Fig. 4 (dashed line)].
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Hsorp(w) was high (very negative) at low water contents and exponentially decreased (approached 0) as water content increased (Fig. 8). For all species, sorption enthalpy was between –20 and –30 kJ mol–1 at the BET monolayer and between –7 and –13 kJ mol–1 at water contents corresponding to the sum of D'Arcy–Watt strong and weak binding sites (Table 3). The break in the van't Hoff relationship between low and high temperature ranges also occurred between –20 and –30 kJ mol–1 (Fig. 8). The value of Hsorp(w) was close to 0 at the higher temperature range in spores of T. palustris and P. vittata at absolute dryness and approached –20 kJ mol–1 with increasing water content. By contrast, values of Hsorp(w) were nearly –40 kJ mol–1 for completely dry spores of D. filix-mas, P setiferum, and P. aculeatum at the higher temperature range and approached –20 kJ mol–1 with further hydration (Fig. 8).
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Discussion |
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Survival of desiccated cells is often attributed to the association of water with biomolecules and its effects on biomolecule stability and mobility within the aqueous domains of cells (Walters, 1998; Hoekstra, 2005; Walters et al., 2005). Here, water–biomolecule associations in fern spores are investigated through the relationships between RH, water content, and temperature. These relationships are compared among five homosporeous species (non-chlorophyllous spores) and other plant germplasm: pollen from Typha latifolia, embryonic axes of Glycine max, and leaf tissue from the desiccation-tolerant fern Polypodium polypodioides (Vertucci and Leopold, 1987a, b; Buitink et al., 1998). It is shown that fern spores tend to equilibrate rapidly to environmental RH, approaching steady-state water content within a few days of imbibition or drying. By contrast with the other germplasm studied, fern spores absorb very little water (Table 3; Figs 1, 5). Differences were detected in the adsorption properties of fern spores related to temperature and species and it is hypothesized that these differences reveal information about the ecophysiology (Table 1) or storage physiology of the spore. Long-term studies of the life spans of fern spores are underway (Ballesteros et al., 2004, 2005), and the studies presented here on water properties provide a predictive tool until that information is available.
The chemical affinity of fern spores for water was lower than that of the other germplasm studied. This low affinity is demonstrated by low monolayer values in the BET model (wcmon), low amounts of water on weak and multimolecular binding sites in the D'Arcy–Watt model (h and k'), and high values of in the Flory–Huggins dissolution model (Table 3). These parameters were highly correlated among each other (r2=0.99, 0.72, and –0.95, respectively, for regressions between BET wcmon and D'Arcy–Watt h, D'Arcy–Watt k', and Flory–Huggins at 25 °C). The BET monolayer and water associated with D'Arcy–Watt strong binding sites, K', were not strongly correlated (r2=0.26 at 25 °C), and calculations of water on strong binding sites were similar across diverse tissues. Among the fern species studied, P. vittata had the lowest chemical affinity and P. setiferum had the highest chemical affinity (Table 3; compare values for wcmon in the BET model; K', h, and k' in the D'Arcy–Watt model, and in the Flory–Huggins model).
Chemical affinity for water has been linked to stress tolerance in plant tissues (Levitt, 1980; Rascio et al., 1992; Vertucci and Stushnoff, 1992) and seed storage physiology (Vertucci and Leopold, 1987b; Vertucci and Roos, 1990; Vertucci et al., 1994; Sun et al., 1997; Eira et al., 1999; Dussert et al., 1999; Lyall et al., 2003; Pukacka et al., 2003; Hor et al., 2005; Nagarajan et al., 2005). Indeed, the mechanistic understanding of protection from dehydration and freezing stresses often invokes accumulation of highly hydrophilic substances such as sugars and heat-soluble proteins (Close, 1997; Bryant et al., 2001) that may increase the chemical affinity of water within cells. Chemical affinity is likely to be a complex function of the concentration of hydrophilic residues, the degree to which macromolecules are folded, and the interaction of small hydrophilic molecules on macromolecular surfaces. The surprising decrease in the BET monolayer value of proteins with the addition of sucrose or trehalose (Constantino et al., 1998) confirms that chemical affinity is not simply the sum of the number of hydrophilic sites. Assessments of chemical affinity in complex mixtures within cells are also confounded by the amount of hydrophobic constituents that are sequestered outside the aqueous domain of the cytoplasm (e.g. triacylglycerols and polyphenolic compounds; Vertucci and Leopold, 1987a; Vertucci and Roos, 1990; Hor et al., 2005). The presence of these types of molecules in fern spores is rarely documented (Simabukuro et al., 1998a), but needs to be considered in future studies that investigate the potential links between water affinity and longevity.
Some studies suggest that seed longevity may be inversely correlated with the water associated with D'Arcy–Watt weak binding sites or the proportion of water in weak compared with strong binding sites (Sun et al., 1997; Nagarajan et al., 2005). If true, this would suggest that fern spores are relatively long-lived compared with other germplasm and that spores of P. setiferum have the shortest life span of the fern species studied. Consistent with this prediction, fern spores appear to have longer life spans than Typha latifolia pollen; however, they appear to have similar or shorter life spans than many seed or desiccation-tolerant leaf tissues (Lloyd and Klekowski, 1970; Windham et al., 1986; Lindsay et al., 1992; Hoekstra, 2005; Walters et al., 2005). Low affinity for water at high RH will limit the amount of water that enters cells, preventing large changes in cell volume and slowing metabolic activity. This feature may provide protection against damaging hydration–dehydration cycles and thereby promote longevity under less optimal storage environments. Perhaps this also explains the unusual longevity of fern spores under hydrated storage conditions (Lindsay et al., 1992).
The enthalpy of sorption also provides insights into water associations that may influence storage physiology of desiccated systems. Enthalpy of sorption (Hsorp) is usually regarded as the amount of heat released when water vapour condenses on a molecular surface (Atkins, 1982). However, associated reactions also contribute to enthalpic changes. These reactions include dissolution of the molecule as water content increases (Flory, 1953) and structural changes to the molecule, such as swelling, contraction of hydrophobic regions, and denaturation. The effect of these associated reactions on the magnitude of Hsorp depends on whether they are exothermic or endothermic. Recently, Hsorp was also related to the concept of restricted structural mobility in amorphous (i.e. noncrystalline) solids by Zhang and Zografi (2000). These authors suggested that the value of Hsorp, derived from the parameter c in the BET model, relates to non-ideal changes in structure arising when glassy materials are initially hydrated. Their hypotheses provide a rationale for the temperature dependency of Hsorp calculated from both isotherm models and van't Hoff analyses of spores at low water contents. Hence, the relative value of Hsorp depends on whether the material is in a glassy state or not. In isotherm models, the value of Hsorp is high at temperatures that promote glasses, and an abrupt decrease (towards 0) in Hsorp with increasing isotherm temperatures reflects a glass transition and can be used to estimate Tg (glass transition temperature) roughly (Zhang and Zografi, 2000).
The idea that the calculated enthalpy of sorption is related to molecular mobility and structural stability within amorphous solids supports an important possible link between isotherms and viability of dried cells. These relationships were initially noted by observations that the magnitude of Hsorp was low (close to 0) in desiccation-intolerant tissues (Vertucci and Leopold, 1987b; Vertucci et al., 1994; Eira et al., 1999) and that Hsorp(w) could be used as a first approximation of viscosity in seeds (Vertucci and Roos, 1990). Studies subsequent to these showed that changes in viscosity were linked to glass transitions and corresponded to changes in ageing kinetics (Leopold et al., 1994; Buitink et al., 1998, 2000; Walters, 2004). Building on the idea that the magnitude and change of Hsorp reflects structural restrictions and relaxations within a glass, it can be hypothesized that high values of c in the BET model or K in the D'Arcy–Watt model reflect a condition of high molecular stability. Thus, fern spores that are stored at 5 °C will survive longer than their counterparts that are stored at 25 °C or 45 °C (Table 3). Furthermore, decreases in c or K with increasing temperature may be reflective of imminent glass transition and relaxation of the molecular structure that was ‘frozen in’ when the glass was formed during drying. Accordingly, fern spores that demonstrate a large decrease in the values of c or K with increasing temperature can be predicted to store poorly at those elevated temperatures. From these arguments and the temperature dependency of c and K values given in Table 3, it can be predicted that spores of P. aculeatum and P. setiferum will be prone to deterioration at ambient temperatures, while those of P. vitatta and T. palustris would be relatively stable.
High c and K values from BET and D'Arcy–Watt models, respectively, calculated for P. vitatta and T. palustris spores at 25 °C or 45 °C, arise from high water contents measured at low RH at those temperatures. The high water contents also result in non-linear van't Hoff plots and low values of Hsorp(w) (closer to 0) at low water contents and high temperature ranges (Fig. 8). The abrupt difference in Hsorp in van't Hoff analyses probably reflects resistance to volumetric changes and indicates that the glassy state is maintained despite a rise in temperature. Reversion to linear van't Hoff isochores with increasing water content may, therefore, indicate plasticization of the glass by water, and a loosening of ‘frozen-in’ structure within the temperature range studied. Non-linear van't Hoff plots may also indicate a change (rather than resistance to change) in structure or phase (Atkins, 1982). This hypothesis and the steeper slope at lower temperatures (Figs 6–8) suggest structural changes are induced by reducing the temperature, an event that is more indicative of a phase change such as crystallization. According to this hypothesis, changes in lipid structure or condensation of macromolecules might possibly be involved and, if so, would lead to speculation about potential damaging effects of extreme drying and cooling on spore viability.
In addition to new applications of water sorption isotherms described above that may predict relative longevity, traditional applications of water sorption isotherms have always been useful to predict suitable moisture ranges for storage at various temperatures (Vertucci and Roos, 1990, 1993; Vertucci et al., 1994; Buitink et al., 1998; Walters, 1998; Eira et al., 1999; Hor et al., 2005). The optimum RH for storing spores of Woodwardia sp. (fern genus from family Blechnaceae) corresponded to about 20% RH (Walters et al., 2005). Assuming a similar optimum for the species studied here, recommended water contents would range from 0.039 to 0.025 g H2O g–1 DW for spores of P. setiferum and P. vittata stored at 25 °C. Reducing the storage temperature to –18 °C would result in an increase in optimum water content to 0.059 and 0.038 g H2O g–1 DW, respectively, according to isotherms produced through extrapolation using van't Hoff analyses (Figs 4 to 7). Storing seeds and pollen at water contents less than the BET monolayer value has been linked to more rapid deterioration (Walters, 1998). If a similar trend exists for fern spores, detrimental effects would be expected if spores are stored at <7–12% RH, depending on species and temperature (see water contents for BET monolayer in Table 3). Also poor longevity of spores stored at 75% RH would be expected since the transition from glassy to rubbery phase would be complete. Water contents corresponding to this RH are surprisingly low in fern spores compared with other germplasm (Fig. 5), and range from about 0.08 to 0.05 g H2O g–1 DW for spores of P. setiferum and P. vittata, respectively, stored at 25 °C. The isotherms presented here suggest that the range of allowable water contents for safe storage is quite narrow for fern spores.
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Conclusions |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionConclusionsReferences |
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Water sorption isotherms describe the process of hydration in terms of chemical interaction of water on molecular surfaces, relaxation of molecular structure as mobility increases, and volumetric changes as molecules unfold. It has been shown that fern spores from several species have low affinity for water but a high degree of structural relaxation when water is added. These findings suggest that fern spores from the species studied are stabilized when dry and are therefore amenable to dry storage even at subfreezing temperature if water contents are manipulated precisely. Comparison of glassy behaviour among species of fern spores and among other germplasms will help to elucidate further the role of water, particularly in glasses, in preservation of biological materials, and it may ultimately provide predictive tools of shelf-life during storage.
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Acknowledgements |
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DB was supported by a FPU grant from the Spanish Ministry of Science and Technology. We thank Lisa Hill for her excellent technical assistance.
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Abbreviations |
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BET, Brunauer–Emmet–Teller; RH, relative humidity;
Hsorp, enthalpy of sorption; Hsorp(T), enthalpy of sorption calculated at a specific temperature; Hsorp(w), enthalpy of sorption calculated at a specific water content; wc, water content. |
References |
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