02 Dessication - Chap 2

02 Dessication - Chap 2

2

18/3/02

1:53 pm

Page 47

Methods for the Study of Water Relations Under Desiccation Stress Wendell Q. Sun Department of Biological Sciences, National University of Singapore, Kent Ridge Crescent, Singapore 119260

2.1. Introduction 2.2. Expression of Water Status 2.2.1. Mass-based measures for tissue hydration 2.2.1.1. Water content 2.2.1.2. Relative water content 2.2.2. Thermodynamic measures for tissue hydration 2.2.2.1. Water activity 2.2.2.2. Chemical potential of water and water potential 2.3. Measurement of Tissue Water Potential 2.3.1. Psychrometric and hydrometric methods 2.3.2. Osmometric or cryoscopic method 2.3.3. Isothermal equilibrium method 2.4. Water Relations – the Thermodynamic Approach 2.4.1. The Höfler diagram and pressure–volume curve 2.4.1.1. Change of cell turgor pressure during desiccation 2.4.1.2. Change of osmotic potential during desiccation 2.4.1.3. The volume of water in symplast, apoplast and intercellular spaces 2.4.1.4. Volumetric elasticity of the cell wall 2.4.2. Analysis of water sorption isotherms 2.4.2.1. Theoretical models 2.4.2.2. Temperature dependency of water sorption 2.4.2.3. Monolayer hydration and water-clustering function 2.4.2.4. Occupancy of water-binding sites 2.5. Measurement of Drying Rate and Desiccation Stress 2.5.1. Driving force for water loss and expression of drying rate 2.5.2. Quantification of desiccation stress 2.6. Water Relations – the Kinetic and Functional Approach 2.6.1. General considerations

48 48 48 48 49 50 50 51 53 53 54 55 55 55 55 57 57 59 60 60 62 65 66 68 68 68 70 72

© CAB International 2002. Desiccation and Survival in Plants: Drying Without Dying (eds M. Black and H.W. Pritchard)

47


18/3/02

1:53 pm

48

Page 48

W.Q. Sun

2.6.1.1. Time scale 2.6.1.2. Structural complexity and dynamics of molecular ordering 2.6.1.3. The model-dependent interpretation: the pitfalls 2.6.2. Biophysical techniques 2.6.2.1. Differential scanning calorimetry 2.6.2.2. Thermally stimulated current (TSC) method 2.6.2.3. Nuclear magnetic resonance (NMR) 2.6.2.4. Electron spin resonance 2.7. Concluding Remarks 2.8. References Appendix 2.1. Introduction In hydrated plant cells, water is the main constituent. The organization of cellular structures (both supramolecular assemblies and micromolecular structures) and the overall biochemistry (the thermodynamics of biological processes and their rate parameters) of an organism depend on water. Water is a solvent and a medium in which diffusion of solutes and biochemical reactions take place in plant cells. It is often a participant and/or a product of various biochemical reactions. In low-moisture systems such as naturally dried pollen grains and plant seeds, cellular water also plays an important role as a plasticizer, influencing the translational or rotational motions of entire molecules, or segments of macromolecules and intramolecular motions. Water is involved in virtually every dynamic process in a living cell. The loss of water from plant cells is an important environmental stress. Changes in the aqueous environment influence the complex thermodynamics and kinetics of structural stability and all aspects of biological functions. The accurate measurement of the status of cellular water is essential for the study of both desiccation stress in plants and the mechanisms of plant desiccation tolerance. The method of quantification and interpretation must be applicable not just to the narrowly defined desiccation conditions, but also to all other types of physiological stresses with a dehydration component, such as freezing and salinity. In this chapter, several fundamental principles

72 72 73 74 74 75 76 77 78 79 84

of water relations that are directly related to desiccation tolerance of plant tissues will be introduced. The strengths and limitations of various methods or techniques of measurement of water relations during desiccation will be discussed. An effort will be made to give a basic understanding of terms and concepts concerning cellular water status and the expression of dehydration stress.

2.2. Expression of Water Status The most important quantity that has to be measured in all studies of desiccation tolerance is the degree of dehydration stress. So far, there is no agreed parameter of dehydration stress measurement. The change in water content of plant tissues and organs is often used as an indicator of dehydration. However, insufficient attention has been paid to problems commonly associated with the use of water content as an indicator of dehydration stress. For example, different concepts and approaches are currently used by research groups working on biological systems, ranging from bacteria and fungal spores to microscopic animals, pollen grains, large seeds and resurrection plants. 2.2.1. Mass-based measures for tissue hydration 2.2.1.1. Water content Water content on a wet-weight basis (WC, % w.b.) is widely used in the literature of desic-


18/3/02

1:53 pm

Page 49

Methods for Studying Water Relations Under Stress

cation studies, and is adopted by the International Seed Testing Association (ISTA, 1993) for the expression of seed water content. WC (% w.b.) is the percentage mass fraction of water of the total tissue mass: WC (% w.b.) = (fresh weight dry weight)/fresh weight 100

(1)

WC (% w.b.) is not a linear expression of water content in tissues, because fresh weight appears in both the numerator term and the denominator term in Equation (1). When WC (% w.b.) is used to monitor the loss of water during desiccation, the decrease of WC (% w.b.) does not necessarily reflect the exact extent of dehydration stress. The change of WC (% w.b.) during drying is, in fact, related to the change of the reciprocal of tissue fresh weight. For example, when the tissue of 80% WC (% w.b.) is dried to 70% and 60% water content, the tissue actually loses 41.7% and 62.5% of its initial water quantity, respectively, not just 12.5% and 25% reduction as implied by the values of WC (% w.b.). The quantity of water lost during dehydration from 80% to 70% WC (% w.b.) is twice as much as water loss during dehydration from 70% to 60% WC (% w.b.). Water content on a dry-weight basis measures the mass ratio between water and the dry mass in tissues, and is often expressed by g water per g dry weight (i.e. g g1 dw): WC (g g1 dw) = (fresh weight dry weight)/dry weight

g1

(2)

WC (g dw) is a linear expression of water content, and the change of WC (g g1 dw) during drying is proportional to the loss of water in a tissue. On the mass basis, a tissue with a WC of 0.20 g g1 dw is hydrated exactly twice as much as the tissue with a WC of 0.10 g g1 dw, and fourfold as much as the sample with a WC of 0.05 g g1 dw. For this reason, some researchers have argued that WC (g g1 dw) is a more sensible expression than WC (% w.b.) for the measurement of dehydration. At WC < 15%, the difference between WC (% w.b.) and WC (g g1 dw) is fairly small. WC (% w.b.) is converted to WC (g g1 dw) using the following equation:

WC (g g1 dw) = WC (% w.b.)/[100 – WC (% w.b.)]

49

(3)

2.2.1.2. Relative water content Relative water content (RWC) is another mass-based parameter. RWC is widely used in the pressure–volume analysis of plant tissue water stress. RWC is a simple and useful measure of the extent to which a tissue is in water deficit. It is related to tissue water content at full turgor (WCF). During a dehydration experiment, RWC is calculated by dividing water content at a given time by water content at full turgor, and expressed as a fraction value or as a percentage. If water content in the tissue is determined as WC (g g1 dw), the calculation of RWC is straightforward, being WC/WCF. But, if water content is determined as WC (% w.b.), RWC is calculated by the equation: RWC = [WC (100 – WCF)]/[WCF (100 – WC)] 100

(4)

RWC is a linear expression of moisture condition. The change in RWC over time serves as a good indicator for the rate of dehydration. Physiological responses of plant water deficit are highly correlated with RWC (Sinclair and Ludlow, 1985). The use of RWC is particularly advantageous for comparative studies, in which initial water content at full turgor or full hydration varies considerably among different species, different tissues of the same species or the same tissue at different developmental stages, such as seeds. In certain cases, it may be even preferred over water potential, because RWC also accounts for the effect of osmotic adjustment in affecting plant water status. For example, two plants with the same leaf water potential can have different RWCs if they differ in their ability for osmotic adjustment. In many desiccation studies on higher plants, water content of a tissue at full turgor was not specifically determined, and instead water content after full hydration in water was used. Typically, leaf samples (e.g. discs or sections) of higher plant species are taken and weighed immediately. The


50

18/3/02

1:53 pm

Page 50

W.Q. Sun

samples are then hydrated in distilled water for 4–6 h, after which they are weighed again and their water contents are determined by drying the samples in an oven. In higher plants, the amount of intercellular water is small or non-existent (Oertli, 1989). However, it should be noted that some plant tissues do contain intercellular water, which is held in spaces between cells of a tissue at relatively high water potential (near zero). Therefore, water content at full turgor has different physiological meaning from water content of the tissue at full hydration when the tissue contains intercellular water. If intercellular water is present, water content of the tissue at full turgor has to be estimated from the plot of water potential on water content (g g1 dw). To determine the water content of the tissue at full turgor, two linear regression lines can be fitted, respectively, with data points where water potential remains almost unchanged during initial water loss and the next few points where water potential starts to fall. The intercept of these two regression lines gives the water content at full turgor. Beckett (1997) reported that the amount of intercellular water varied greatly among species of bryophytes. If intercellular water exists in a tissue, correction needs to be made to the raw RWC readings, which are calculated according to the water content at full hydration. The method used to correct the raw RWC readings was described in detail by Beckett (1997). There are shortcomings in using massbased parameters for the expression of water content. Plant tissues are heterogeneous, complex biological systems, in which carbohydrates, proteins and lipids and other components have different hydration properties. As a consequence, when plant tissues of various species are equilibrated under given conditions of temperature and relative humidity, equilibrium water content varies considerably among species. For example, seeds with large lipid reserves equilibrate to lower water contents than starchy seeds, even though the chemical potential of water molecules is the same for all tissues when equilibrium is achieved. The disadvantage of using mass-

based parameters for the expression of dehydration stress has been shown by a number of studies. The critical onset water potential of Quercus rubra (Pritchard, 1991) and Quercus robur (Pritchard and Manger, 1998) is about –3 MPa, but the corresponding mass water contents vary substantially due to different seed oil content. Sun and Gouk (1999) studied the water relation responses of three recalcitrant (desiccationsensitive) seeds (Aesculus hippocastanum, Andira inermis, Q. rubra) during controlled dehydration. The critical water potentials for seeds are quite similar for all three species (7 to 8 MPa), but their corresponding critical water contents are 0.45, 1.10 and 0.35 g g1 dw for A. hippocastanum, A. inermis and Q. rubra, respectively. If the critical water content were used to express the relative desiccation tolerance, one would conclude incorrectly that seeds of A. hippocastanum and Q. rubra are much more desiccation-tolerant than seeds of A. inermis. Therefore, a massbased parameter of water loss may not be a reliable indicator for the degree of desiccation stress in plant tissues.

2.2.2. Thermodynamic measures for tissue hydration The response of plant tissues to desiccation is related to the thermodynamic and kinetic status of tissue water, rather than to actual water content. The water status of plant tissues can be expressed in terms of energy status of water molecules, i.e. the partial molar Gibbs’ free energy or water potential. This thermodynamic approach is preferred to the mass-based expression of tissue hydration, because thermodynamic parameters (i.e. energy status) are related directly to the numerous biophysical and physiological events that contribute to desiccation stresses and desiccation tolerance of plant tissues. 2.2.2.1. Water activity Water activity (aw) is used to describe water status in the studies of desiccation


18/3/02

1:53 pm

Page 51


tolerance and storage survival for spores, pollen, seeds and resurrection plants (Ellis et al., 1990, 1991; Berjak and Pammenter, 1994; Vertucci et al., 1994, 1995; Walters, 1998a). Water activity is measured as the ratio of the vapour pressure of water in a system to the vapour pressure of pure water at the same temperature. It is related to the equilibrium relative humidity (RH) of the air surrounding the system (i.e. RH = aw 100). Water activity can be viewed as the ‘effective’ water content, which is thermodynamically available to various physiological processes in cells. For the survival of organisms under water stress, the ‘effective’ water is more important than the total amount of water present in the tissue. Water activity of fresh plant tissues may vary only between 0.980 and 0.996. Within this narrow range, it is not useful for the expression of dehydration stress or tissue water status. However, for the studies of severe water stress and extreme desiccation, water activity has several advantages over water content, including its conceptual simplicity, measurability, easy experimental manipulation, and its applicability to both simple and complex systems. A number of physiological processes that are relevant to desiccation tolerance or damage have been shown to occur at specific water activities, and some of those are presented in Fig. 2.1. Water activity in plant tissues can be determined using the hygrometric method and the isothermal equilibrium sorption method. The hygrometric instrument method directly measures the equilibrium RH of plant tissues in a closed chamber. With the equilibrium sorption method, samples of plant tissues are equilibrated to a series of known water activities at a specified temperature. The relationship between water content and water activity upon equilibrium (i.e. the sorption isotherm curve) is then used to calculate water activity of plant tissues at different water contents. Water activity is defined at equilibrium. However, plant tissues at low and intermediate moisture levels may not be in a true state of equilibrium at all, but in an amorphous metastable state instead.

51

In such cases, the measured vapour pressure of water may not be the equilibrium vapour pressure, but the vapour pressure of a ‘stationary’ state that is time-dependent. However, studies in food sciences have suggested that water activities measured are likely to be close to equilibrium and the differences should be within the uncertainty associated with the experimental determination (Chirife and Buera, 1996). The usefulness of the water activity concept in seed storage stability has been discussed by Walters (1998b). 2.2.2.2. Chemical potential of water and water potential The quantity of free energy of a component (µj) in a system is measured by its chemical potential. The chemical potential of water (µw) in a system is defined by: – µw = µ* w + RT ln aw + VwP + zwFE + mwgh (5)

where µ*w is the chemical potential of pure water at ideal reference conditions. The second term RT ln aw is for water activity. R is the gas constant (8.314 103 kJ mol1 K1), T is the absolute temperature (K, in kelvin), and aw is water activity (RH/100). – Vw is the partial molal volume of water (i.e. the differential increase or decrease in volume when a differential amount of water is – added or removed). Vw is influenced by the presence of solutes and is also temperature-dependent (1.805 105 m3 mol1 at 20°C). P is the hydrostatic pressure on water in excess of atmospheric pressure – (MPa, 1 MPa = 103 kJ m3). The term VwP represents the effect of pressure on the chemical potential of water and is expressed in energy per mole (kJ mol1). zwFE is the electrochemical potential of water, which equals zero because water is uncharged (zw = 0). The last term mwgh is the gravitational term, representing the work needed to move 1 mole of water to a given height. Practically, mwgh will remain constant in most circumstances of desiccation studies. The water potential is proportional to the chemical potential of water (µw µ*w) in a system as described in Equation (5).


18/3/02

1:53 pm

W.Q. Sun

–250

20

40

60

80

Cell respiration starts to cease

Mean of minimum value for bacterial growth

Minimum required for photosynthesis

at lower aw DNA disordered and damaged

colonies in situ Typical exposure of Nostoc

–200

saturated CaCl2 solution

–150

Water vapour above

–100

seeds typically survive at lower aw

–50

Orthodox

Water potential of water vapour (MPa)

0

Lysozyme activity stops

where p = P and is hydrostatic pressure on water as defined in Equation (5), π is

Nucleic acids and proteins fully hydrated

(6)

Water vapour above saturated NaCl solution

= P + π + h

osmotic potential and h is the gravitational potential. The total water potential is the sum of hydrostatic, osmotic and gravitational components. The gravitational term (h) depends on the position of water in a gravitational field and is not relevant to most desiccation studies. Osmotic potential depends on the concentration of dissolved substances in water. Osmotic potential is related to water activity by the equation:

Coffea species

Therefore, water potential is actually the potential energy of water per unit mass. While water content tells how much water is in a sample, water potential tells you how available that water is. By convention, water potential is defined as follows:

Desiccation tolerance of embryos of

52

Page 52

100

Relative humidity (%)

Fig. 2.1. Water activities (relative humidities) that limit physiological activities and cell growth. Physical parameters and physiological processes are drawn with data from Wolfe and Leopold (1986), Potts (1994) and Sun and Gouk (1999). The relationship between relative humidity and water potential is calculated according to Equation (7) at 25°C. A similar diagram that is specific to a plant tissue can be established. Such a diagram would serve as a valuable reference for experimental design and data interpretation, since it gives a clear concept about the possible sequence of potential physiological and biochemical events and their interactions as the tissue loses water.


18/3/02

1:53 pm

Page 53


– Vw π = RT ln aw

(7)

During dehydration, the water content in seed tissues is reduced, resulting in an increase in the concentration of solutes and thus a decrease in water activity and osmotic potential (i.e. π becomes more negative). A similar situation occurs during freezing. The formation of ice leads to the dehydration of the protoplast and the concentration of solutes. The interactions of water with biological surfaces and interfaces are of great importance to desiccation tolerance of plant tissues, especially at low moisture levels. The influence of such interactions on water potential in a tissue is commonly called ‘matric’ potential. Rapid water uptake by dry seeds during the early stage of germination is mainly attributed to large matric potentials. Another example is the reduced rate of water loss as the tissue is dried to lower water content. Matric potential depends on the adsorptive forces that bind water to a matrix. The amount of matrixbound water in recalcitrant Q. robur embryonic axes is as high as 0.25–0.30 g g1 dw (Pritchard and Manger, 1998). However, the forces of such water–matrix interactions are adequately represented by their contributions to hydrostatic pressure (P) and osmotic potential (π). For example, the presence of aqueous interfaces in cells lowers water activity through interfacial attractions and binding of water near their surfaces, which has already been included in the osmotic component in Equation (7). Therefore, matric potential does not represent additional new forces.

2.3. Measurement of Tissue Water Potential A pressure chamber (pressure bomb) is commonly used to measure directly leaf water potential of higher plants. The detached leaf is sealed in a steel chamber with the cut petiole protruding out. Pressure that is applied to the chamber is taken as the xylem (leaf water) potential when the sap meniscus appears at the petiole xylem sur-

53

face. This technique, however, is unsuitable for many desiccation-tolerant plant tissues, e.g. lichens, bryophytes, spores, pollen grain and seeds. This is because the pressure chamber method measures the xylem tension, which is broadly equal to the leaf water potential. Water potential of plant parts that do not have vascular systems cannot be measured with the pressure chamber method. However, water potential of plant tissues can be measured by a number of other techniques. These techniques use either the relationship of the sample water potential to the equilibrium vapour pressure immediately around the sample or the principle of the freezing-point depression in the liquid solution.

2.3.1. Psychrometric and hydrometric methods Both methods are widely used for the measurement of tissue water potential. The measurement of water potential by a psychrometer and a hydrometer is called the wet-bulb depression method and the dew-point depression method, respectively. A psychrometer measures water potential of samples (placed in closed chambers) through its ability to determine the RH of the closed environment. The instrument uses high-sensitivity thermometers to measure temperature reduction resulting from the heat of vaporization of water in a sample relative to pure water. It can measure water potential of solid tissue materials and droplets of solutions. The sample is first sealed in a small chamber containing a thermocouple. After an equilibration period, a cooling current is applied to the thermocouple in order to condense water on the thermocouple junction. The amount of condensed water is proportional to the water potential of the tissue. The water is allowed to evaporate, causing a change in the thermocouple output, and the output is calibrated for water potential, using standard salt solutions. On the other hand, a hydrometer maintains the dew-point depression temperature during the measurement using a thermocouple. The dew-point


54

4/4/02

2:18 pm

Page 54

W.Q. Sun

depression temperature is the temperature to which the air in the sealed chamber must be reduced so that the air becomes saturated with water vapour. Psychrometric and hydrometric methods can be used to measure both water potential and osmotic potential of plant tissues. To measure osmotic potential, a sample has to undergo the freeze–thaw cycles to disrupt the cellular structures before the measurement, whereas the water potential is measured using undisrupted tissues. A psychrometer is very sensitive to temperature change because it measures very small temperature differences. A change in water potential of 1.0 MPa is reflected by a change in wet-bulb temperature depression of only approximately 0.085°C. A hydrometer is less affected by the changes in ambient temperature during the measurement compared with a psychrometer. Psychrometric and hydrometric methods are suitable only for plant tissues of high water content. At low water content, the equilibrium may take several hours to achieve. The nominal range of the Peltier thermocouple measurement is limited from 0 to 6.0 MPa for these two methods. Yet many desiccation-tolerant plant tissues can survive far below 6.0 MPa. Even if one uses the Richards thermocouple, it extends only to –25 MPa and the accuracy decreases to –0.1 MPa at –10 MPa (Decagon Devices Inc., Pullman, Washington, USA), corresponding to an RH of ~ 84% at 25°C. 2.3.2. Osmometric or cryoscopic method A freezing-point osmometer measures the osmotic concentration of a biological liquid using the principle of the freezing-point depression. The freezing-point depression is one of the four colligative properties of a solution. The freezing point is the unique temperature at which the ice phase and the liquid phase can coexist in equilibrium at standard pressure. When a solute is dissolved in the water, the freezing point of the water is lowered in proportion to the osmotic potential of the solution. For a 1.0

osmolal aqueous solution, the osmotic potential at 0°C is ideally 2.271 MPa, and the freezing-point depression is 1.86°C. (An osmole is the mass of a substance that when dissolved in 1 kg water generates an osmotic pressure equivalent to that produced by 1 mole of an ideal solute dissolved in 1 kg water. After dissolving, an ideal solute gives 6.023 1023 osmotically active particles.) Theoretically, the osmotic potential of an unknown sample can be estimated from the depression of its freezing point by the following relationship: Ψπ =

−2.271 MPa ∆T = −1.221∆T 1.86°C

(8)

where T is the depression of the freezing point. The effect of osmotic potential on freezing-point depression also holds for non-ideal solutions such as plant saps. However, freezing-point depression is nonlinear with concentration changes during dehydration. Water potential (MPa at 0°C) can be derived by the empirical equation (Crafts et al., 1949): Ψ = 1.206T + 0.0021T 2

(9)

With the osmometric method, a sample is usually supercooled a few degrees below its freezing point to induce immediate crystallization. As the heat of fusion is released, the sample temperature rises to its freezing point, and its equilibrium temperature is measured. Alternatively, the temperature at which ice crystals start melting can be measured and taken as the equilibrium freezing temperature (i.e. Ramsay’s method). The applicability of Equations (8) and (9) to the measurement of water potential or osmotic potential in plant tissues was examined by Sun and Gouk (1999), using seed tissues that were pre-equilibrated with saturated salt solutions (from 3 to 35 MPa). The freezingpoint depression was determined with a differential scanning calorimeter, using the onset temperature for the exotherm on cooling. Calculated water potentials were found to be very close to the pre-freezing water potentials of seed tissues, with Equation (9) fitting the data slightly better than Equation (8).


18/3/02

1:53 pm

Page 55


2.3.3. Isothermal equilibration method It is more difficult to measure directly water potential of low-moisture systems. Perhaps the convenient, yet accurate and reliable method is first to establish the empirical relationship between water content and water potential for a particular plant tissue. Samples of plant tissues are equilibrated over different salt solutions that would maintain a series of constant water vapour pressures (i.e. RH) in closed containers. Upon equilibrium, the water contents of tissue samples are determined gravimetrically, and their water potential at equilibrium is then the same as the water potential of the air in the closed containers, which in turn equals the osmotic potential of the salt solutions used. Therefore, water potential of tissue samples is calculated by the equation: %RH = RT – ln Vw 100

(10)

where R is the gas constant, T is the – absolute temperature (kelvin), Vw is the partial molal volume of water, and %RH is the percentage relative humidity inside the containers. (Note that Equation (10) is essentially the same as Equation (7).) The empirical relationship between water content and water potential can be described by exponential and polynomial (Poulsen and Eriksen, 1992) or other functions (Sun and Gouk, 1999). The derived mathematical expression is then used to calculate water potential of plant tissues at any water content within the limit of experimental range. This method is particularly useful in monitoring the change of tissue water potential during desiccation. Water potential of dehydrating tissues can be calculated immediately from the data of water loss. Constant RH can be achieved using saturated or non-saturated salt solutions, polyethylene glycol solutions and glycerol solutions. Physico-chemical data of various salts and their solutions are presented in the Appendix. This technique does not need special instruments to measure water potential, and can avoid the difficulty in measuring RH accurately. This method has been used suc-

55

cessfully in seed desiccation studies by a number of workers (Pritchard, 1991; Poulsen and Eriksen, 1992; Vertucci et al., 1994; Sun et al., 1997; Tompsett and Pritchard, 1998).

2.4. Water Relations – the Thermodynamic Approach 2.4.1. The Höfler diagram and the pressure–volume curve Water relations parameters of plant tissues can be presented by the Höfler diagram and the pressure–volume curve (PV curve). The Höfler diagram shows the relationship between water potential and relative water content (Fig. 2.2a). The PV curve is a plot between the reciprocal of water potential (1/) and RWC or water loss (1 – RWC) during desiccation (Fig. 2.2b). Both the Höfler diagram and the PV curve are widely used to characterize water relations of plant tissues. To construct a Höfler diagram or a PV curve, the changes in water potential and RWC are monitored as the tissue is dehydrating. Several important parameters can be obtained by analysing the components of cell water potential, including the osmotic potentials at full turgor and at the partially dehydrated state, the apoplastic and symplastic water volume in tissues, a plot of turgor pressure (i.e. hydrostatic water potential in Equation (6)) as a function of RWC, and the tissue bulk modulus of elasticity. Without knowing these biophysical metrics, it would be impossible to identify different kinds of cellular stresses associated with the loss of water in the tissue and to examine the significance of an array of biochemical and physiological responses during desiccation. Moreover, valid comparisons of the response of cell function to water stress among different organisms cannot be made without such knowledge. 2.4.1.1. Change of cell turgor pressure during desiccation In fully turgid cells, turgor pressure is equal to the osmotic potential (with opposite


18/3/02

1:54 pm

Page 56

56

W.Q. Sun

(a)

2

Water potential (MPa)

p 0 –2

External water

–4

–6 0.0

0.2

0.4

0.6

0.8

1.0

1.2

Relative water content (RWC)

p –1/ (MPa–1)

Reciprocal of water potential

(b)

0

p

0.7

1.0 RWC

Incipient plasmolysis (p = 0)

1.3

–1/

0.0 –0.2

0.0

0.2

0.4

0.6

0.8

1.0

1 – RWC Fig. 2.2. Cellular water relations. (a) Höfler diagram showing the components of cell water potential. Intercellular or external water (RWC > 1.0) in many plant tissues is held at near-zero water potential and, during the initial dehydration, cell water potential () and turgor pressure (p) do not change significantly (the horizontal dashed line). Maximum osmotic potential is found at the point of full turgor (RWC = 1.0), where p = π. As the plant tissue loses water, turgor pressure decreases, and at the turgor-loss point (RWC = ~0.8), = π (the vertical dashed line). At RWC < ~0.8, the relationship between RWC and π follows a rectangular hyperbola (RWC = a + b/π). Osmotic potential at RWC = ~0.8–1.0 is extrapolated from the rectangular hyperbola relationship. Turgor pressure is the difference between the measured water potential and the extrapolated osmotic potential. (b) The pressure–volume curve showing the relationships between , p and π during dehydration. The reciprocal of water potential is plotted against (1 RWC). Beyond the turgor-loss point (incipient plasmolysis), the relationship between (1 RWC) and 1/ (or 1/π) is linear. The extrapolation of this linear relationship toward the y-axis intercept gives osmotic potential (the dashed line) of the tissue when the tissue is still turgid. The difference between the measured water potential and the extrapolated osmotic potential is turgor pressure (inset).


18/3/02

1:54 pm

Page 57


signs). During dehydration, the PV curve of a plant tissue initially displays a concave region, beyond which the curve is linear (Fig. 2.2b). The loss of turgor is marked by the point at which the relation of 1/ to (1 RWC) deviates away from linearity. Turgor pressure (p) is calculated as the difference between the extrapolated linear portion of the PV curve and the water potential actually measured, and is often plotted as a function of RWC. The relationship between turgor pressure and RWC can be described sufficiently by a quadratic or cubic function. Certain plant tissues might develop negative turgor pressure before the cells collapse and p can become zero under severe water stress. When negative p develops, the PV curve would fall below the extrapolated linear portion of the graph (Fig. 2.3a). If the cells are sufficiently strong, do not collapse and the plasma membrane remains firmly attached to the cell wall, the formation of an intracellular gas bubble will increasingly become possible (cavitation). The development of negative turgor pressure and intracellular cavitation appear to play some roles in desiccation tolerance of certain cells. A good example of a cell surviving large negative turgor pressure and cavitation is the ascospore of Sordaria (Milburn, 1970). The volume of Sordaria ascospores changes very little, and the protoplast remains in contact with the spore wall at all times. Under water stress (by air-drying or in osmotic solution), these cells might generate negative p as much as –4 MPa. Beyond this negative turgor pressure, a small bubble appears inside the protoplasm suddenly, which increases slowly in size and approaches the walls quite closely without losing its spherical appearance. Honegger (1995) and Scheidegger et al. (1995) also showed that ascomycetous lichen mycobionts form large intracellular gas bubbles when desiccated. More recently, the PV analysis by Beckett (1997) suggested the existence of negative turgor pressure in vegetative cells of several desiccation-tolerant (poikilohydric) plants (e.g. Dumortiera hirsuta and Myrothamnus flabellifolia). PV curves of most plants do not show any indi-

57

cation of negative turgor pressure. It is conceivable that the development of negative turgor pressure may reduce mechanical damage on cellular structures by preventing collapse of the cells. 2.4.1.2. Change of osmotic potential during desiccation When cell turgor pressure falls to zero during desiccation, the water potential of the cell is equal to the osmotic potential (see Equation (6)). As desiccation continues, osmotic potential and cell water potential are equal and inversely proportional to the volume of osmotically active water. The relationship between RWC and the reciprocal of osmotic potential is a straight line. The osmotic potential at full turgor is calculated from the extrapolation of the linear portion of the PV curve to the RWC at full turgor (i.e. RWC = 1.0 in Fig. 2.2b). In the Höfler diagram, the relationship between osmotic potential and RWC is represented by a rectangular hyperbolic function to the data points corresponding to the linear part of the PV curve (dashed part of the π in Fig. 2.2a). 2.4.1.3. The volume of water in symplast, apoplast and intercellular spaces In hydrated plant tissues, water may exist in the symplast, in the apoplast (i.e. the porous spaces in the cell wall) and in the intercellular spaces (large voids) as discussed before. Intercellular water, also called ‘external’ cell water by some workers, may account for up to 35% of total water in certain plant tissues, such as lichens, liverworts, mosses and fern fronds (Beckett, 1997; Proctor, 1999) and developing embryos of higher plants (W.Q. Sun, unpublished data). During desiccation, water potential and turgor potential do not fall with initial water loss at RWC > 1.0 (Fig. 2.2a and b inset). The volume of water that is lost before turgor pressure starts to fall is assumed to be intercellular water. The volume of symplastic water represents the amount of osmotically active water in the tissue, and is obtained by subtracting the apoplastic water volume from the water


18/3/02

1:54 pm

Page 58

58

W.Q. Sun

(a) 1 2

0 –1/ (MPa–1)

Reciprocal of water potential

p

1

0.6

0.8 RWC

1.0

2 0.0 –0.2

0.0

0.2

0.4

0.6

0.8

1.0

1 – RWC

Water potential (–MPa)

(b) 1000

100

10

1.0

0.1 0.0

0.2

0.4

0.6

0.8

1.0

Relative water content (RWC) Fig. 2.3. (a) The pressure–volume curves of plant tissues that develop negative turgor pressure (curve 1) and intracellular cavitation (curve 2) during desiccation. The inset in (a) shows the change of cell turgor pressure (p) during the early stage of drying. When intracellular cavitation occurs, the p suddenly changes to zero (curve 2, inset), and is equal to π (curve 2). If intracellular cavitation does not occur, the cell wall will collapse or deform when the p develops beyond the threshold to which the cell wall can resist (i.e. (1 RWC) > 0.15). The collapse or deformation of the cell wall will lead to a gradual increase in (curve 1) and p (curve 1, inset). (b) The semi-logarithmic plot between RWC and tissue water potential. The high RWC break point corresponds to the turgor-loss point, whereas the low RWC break point corresponds to the volume of apoplastic water. Drawn with data from Quercus rubra seeds (Sun, 1999).

content at full turgor. Symplastic water generally declines over a range of water potential from about 0.5 to 10 MPa, in line with that of osmotic potential.

Apoplastic or osmotically inactive water is present in very small pores and strong water-binding sites of biological surfaces in plant tissues. This fraction of water is held


18/3/02

1:54 pm

Page 59


by matric and molecular forces, and lost only when plant tissues are desiccated to a water potential less than 15 MPa (Meidner and Sheriff, 1976). The loss of apoplastic water in some species extends to approximately 800 MPa. The amount of such matrix-bound water in plant tissues can be as high as 0.1–0.2 RWC or up to 0.25–0.35 g g1 dw. This fraction of water does not generally act as a solvent in cells, and is not readily freezable. From the Höfler diagram, the apoplastic volume is estimated from the fitted hyperbolic function. From the PV curve, the volume of apoplastic water is commonly estimated by extrapolation of the linear relationship between RWC and the reciprocal of osmotic potential to the (1 RWC) axis after the loss of turgor pressure. However, the simple extrapolation from the PV curve is not a reliable method of estimating the apoplastic volume, and in some cases gives negative values (Proctor et al., 1998). The apoplastic volume of water should be derived with data from the isothermal sorption study at low water potentials (water activity), rather than the extrapolation method, because the linear relationship between RWC and the reciprocal of osmotic potential does not hold for the apoplastic volume of water (which is osmotically inactive). Compared to the removal of osmotically active (symplastic) water, the measured osmotic potential (including the term of matric potential) declines much more rapidly when the apoplastic water is removed. Therefore, the volume of apoplastic water is marked by the point at low water content at which the relationship of 1/ to (1 – RWC) again deviates away from linearity (Fig. 2.3b). The volume of apoplastic water roughly corresponds to the primary hydration in tissues (including both strong and weak water-binding sites). One can expect that plant tissues would respond differently to the loss of external, symplastic and apoplastic water. The loss of symplastic water can cause osmotic perturbation of physiological and biochemical processes, whereas the loss of apoplastic water may disrupt the structure and func-

59

tion of cellular membrane and molecular assemblies. So far, workers have paid little attention to the location of water in plant tissues. The difference in the relative volume of external, symplastic, and apoplastic water should be taken into account in the comparative studies on mechanisms of desiccation tolerance among cells, tissues or plants. A similar analysis of water relations was found to be very useful in developing a mechanistic understanding of the role of dehydration in freezing tolerance in earthworms (Holmstrup and Zachariassen, 1996).

2.4.1.4. Volumetric elasticity of the cell wall The cell wall may undergo elastic expansion or contraction. Elastic (mechanical) properties of cell walls play an important role in cell water relations. For example, the negative turgor pressure that can develop in a cell largely depends on the mechanical properties of the cell wall. The elasticity of the cell wall is represented by the volumetric elasticity module , where depends on both p (turgor pressure) and V (cell volume) and is defined as: =

p V

V

(11)

where V is volume change caused by a given pressure change p. Equation (11) indicates that a high value of implies a rigid cell wall, whereas a low value implies a more elastic cell wall. The value can be calculated from the relationship between p and RWC (Steudle et al., 1977; Stadelmann, 1984). The change of as a function of RWC is given by the first derivative of the quadratic or cubic function of turgor pressure on RWC. The value of the p/RWC derivative curve at RWC = 1.0 is usually taken as the bulk modulus of elasticity and used for purposes of comparison. A pressure probe technique can be used directly to determine the turgor pressure and the for individual plant cells. This technique is useful for continuous measurement of cell turgor pressure, cell wall elasticity and hydraulic conductivity of the cell membrane in single cells (Hüsken et al., 1978). The intracellular hydrostatic


60

18/3/02

1:54 pm

Page 60

W.Q. Sun

pressure is transmitted to the pressure transducer via an oil-filled microcapillary introduced into the cell, which transforms into a proportional voltage. This technique permits volume changes and turgor pressure changes to be determined with an accuracy of 105–106 µl and 3–5 103 MPa, respectively. At present, very little information is available on cell wall properties of desiccation-tolerant plant tissues. Proctor (1999) found that two highly desiccation-tolerant liverworts have low values of bulk elastic modulus. He thought that extensible cell walls might be a part of structural adaptation to rapid changes of cell volume in their intermittently desiccated habitats. Ultrastructural studies on dry mesophyll cells of desiccation-tolerant Selaginella lepidophylla by Thomson and Platt (1997) showed highly folded cell walls and continuous apposition of plasmalemma to the walls. Vicre et al. (1999) studied the cell wall architecture of leaf tissues of Craterostigma wilmsii (a resurrection plant), and also observed extensive folding of the cell wall during desiccation. The folding of the cell wall allows the plasma membrane to remain firmly attached to the wall as the cell loses water. Biochemical modifications of the cell wall were observed during desiccation and rehydration, leading to the change in its tensile strength that may prevent the total collapse of the walls in the dry tissue and avoid rapid expansion upon rehydration. The change in cell wall elasticity during desiccation can be determined easily by taking the first derivative of the function of turgor pressure on RWC.

2.4.2. Analysis of water sorption isotherms The water sorption isotherm is the dependence of water content on water activity of the surrounding environment at a given temperature. There are two types of sorption isotherms: desorption isotherm and adsorption isotherm (Fig. 2.4a). Conventionally, a desorption isotherm is developed by drying fresh tissues over satu-

rated salt solutions in closed desiccators until constant weights are achieved, whereas an adsorption isotherm is developed by rehydrating dried tissues over saturated salt solutions. A desorption curve can also be developed during drying of tissues in any atmospheric condition by measuring, at various points in time, the water content of the tissue and the equilibrium RH of its surrounding air in a closed container. Similarly, the dry tissue can be rehydrate with a given quantity of water to raise the water content and equilibrium RH. Sophisticated instruments such as controlled atmosphere microbalance and dynamic vapour sorption systems (Surface Measurement Systems, London, UK) use the latter methods. Desorption and adsorption isotherms are used, respectively, to study the properties of dehydration and rehydration of plant tissues. Desorption and adsorption curves are rarely the same: the desorption curve usually gives a higher water content than the adsorption isotherm. The difference in the equilibrium water content between two curves is called hysteresis. Hysteresis is evidence of the irreversibility of the sorption process, and therefore indicates the limited validity of the equilibrium thermodynamic approach to investigate the dehydration–rehydration properties of plant tissues. Hysteresis might be an important issue when considering critical water activities for desiccation stress during dehydration–rehydration cycles and when investigating storage stability after manipulation of moisture content of seeds and pollen. 2.4.2.1. Theoretical models Plant tissues show a sigmoid sorption isotherm (Fig. 2.4a). The inflection point of the isotherm is believed to indicate either a change of water-binding capacity and/or the relative amount of ‘bound’ or ‘free’ water. Water sorption data are normally analysed using theoretical models, from which useful biophysical parameters of water relations are derived. Commonly used models include the Brunauer– Emmett–Teller (BET) model, the


18/3/02

1:54 pm

Page 61


Guggenheim–Anderson–de Boer (GAB) model and the D’Arcy–Watt model. The BET model (Brunauer et al., 1938) is derived from statistical and thermodynamic considerations. The equation can be written as:

C − 1  aw 1 =  aw + M mC M w (1 − aw )  M m 

61

(12)

where aw is the water activity, Mw is equilibrium water content in the tissue, Mm is the BET monolayer (water content corre-

(a)

II

Water content

I

III

Desorption

Adsorption

Water activity (b) III

II

Sorption enthalpy

I Enthalpy

Free energy

Entropy

Water content Fig. 2.4. The analysis of water sorption isotherms. (a) The typical shape of desorption curves and adsorption curves of plant tissues. The difference between these two curves shows hysteresis, which indicates the irreversibility of water sorption in the tissues during dehydration and rehydration. The sigmoid shape of sorption curves is presumably due to the existence of three types of water-binding sites in tissues (strong (I), weak (II) and multilayer molecular sorption sites (III)). (b) Differential enthalpy (H), free energy (G) and entropy (S) of hydration. Desorption curves can be used to calculate H and S of tissue hydration. See text for detailed discussion.


4/4/02

2:18 pm

62

Page 62

W.Q. Sun

sponding to the monolayer hydration) and C is temperature dependence for sorption excess enthalpy (Brunauer et al., 1938, 1940). BET equation parameters, Mm and C, can be calculated by plotting aw/[Mw (1 aw)] against aw. The y-axis intercept of the straight line is equal to 1/(MmC) and the slope is equal to (C 1)/Mm. The BET is valid only for aw < 0.5, thus data points within that range are used to estimate the monolayer value (Mm). The BET model is an effective method for estimating the amount of water bound specifically to polar sites (monolayer), but cannot be used to give a complete estimation of specific hydration parameters. The GAB model is an extension of the BET model, taking into consideration the modified properties of the sorbing materials in the multilayer region and the bulk liquid properties through the introduction of a third constant, K. The GAB equation is written as: Mw =

MmCKaw (1 Kaw)(1 Kaw + CKaw)

(13)

where C and K are temperature-dependent coefficients. Constants, Mm, C and K are estimated via the curve fitting of sorption data. In the field of food sciences, the GAB model is the most widely accepted due to its accuracy and its validity over a wide range of water activities from 0.05 to 0.9 (Rahman and Labuza, 1999). The D’Arcy–Watt model was developed for the analysis of sorption isotherms of non-homogeneous materials (D’Arcy and Watt, 1970). This model assumes that there is a fixed number of water-binding sites with different discrete binding energies. The D’Arcy–Watt equation can be written as: Mw =

K Kaw kkaw + caw + 1 + Kaw 1 − κaw

(14)

where K, K, c, k and k are equation coefficients (adjustable parameters). The equation has three terms, which represent the amounts of water that are strongly bound, weakly bound and sorbed in multimolecular water clusters, respectively. For a tissue that is in equilibrium with a given aw, the

amount of water in those three regions can be estimated. K is the number of strong water-binding sites, multiplied by the molecular weight of water and divided by Avogadro’s number (6.023 1023); K is the strength of the attraction of the strong waterbinding sites for water; c is a measure (linear approximation) of the affinity and the number of weak water-binding sites; k relates to the number of multimolecular water sorption sites; and k relates to the activity of water (D’Arcy and Watt, 1970). The number of water-binding sites in tissues can be calculated from the derived equation coefficients. The number of strong, weak and multimolecular water-binding sites are KN/M, cN/(Mo), and kN/M, respectively, where N is Avogadro’s number, M is the molecular weight of water and o is the saturated vapour pressure of pure water. The D’Arcy–Watt model has been used extensively for the analysis of desiccationtolerant and desiccation-intolerant plant tissues (Vertucci and Leopold, 1986, 1987a, b; Sun et al., 1997). Both the GAB and the D’Arcy–Watt models are valid over a wider range of water activities for plant tissues. The GAB model has some advantages over the D’Arcy–Watt model, which assumes the three types of water-binding sites. The GAB model does not have such an assumption. For biological systems it is more reasonable to assume that the number of water-binding sites is changing continually along with the binding energies. Moreover, the GAB model can be more easily applied to other thermal analyses (e.g. water-clustering function). 2.4.2.2. Temperature dependency of water sorption Desiccation involves the transfer of liquid water in plant tissues into the vapour phase. Temperature influences evaporation rate through the heat supply as well as through its effect on the partial water vapour pressure in air and the energy status of water in plant tissues. In isothermal conditions, air acts as an osmotic membrane and equilibrium is often slow and dependent on temperature. An increase in temperature generally results in a decrease


18/3/02

1:54 pm

Page 63


in equilibrium water content of plant tissues at a given RH (i.e. water activity) or an increase in equilibrium water activity at constant tissue water content. The shift of water activity at the constant water content by temperature is mainly due to the change in water binding, dissociation of water, physical state of water or increase in solubility of solute in water. Tensile strength of water, the pressure holding molecules together, increases by 81.6 mbars on average for a reduction of 1°C. Temperature dependence of isotherm shift is described by the Clausius–Clapeyron equation: ln =

aw2 q + w = aw1 R

1 1  −  T T 1  2

(15)

where q is the excess heat of sorption; w is the latent heat of vaporization for water (44.0 kJ kg1 at 25°C); R is the gas constant; aw1 and aw2 are water activities for a given equilibrium water content at temperature T1 and T2, respectively. The plot of ln aw against 1/T at any given tissue water content is a straight line and its slope gives (q w)/R, from which the excess heat of sorption, q, can be derived (Fig. 2.5a). In practice, some thermodynamic quantities of tissue hydration can be calculated according to isotherms at two different temperatures. The aw1 and aw2 for a given equilibrium water content at two temperatures can be taken from water sorption curves or calculated from fitted sorption equations (Fig. 2.5b and Fig. 2.7a). Differential enthalpy of hydration (H, including q and w), differential free energy of hydration (G) and differential entropy of hydration (S) are given by: H

RT1T2 ln T2 T1

aw1 aw2

(16)

G RT ln (aw)

(17)

S H G T

(18)

These thermodynamic quantities are the functions of water content in tissues. The relationships of H/WC, G/WC and S/WC provide important information with regard to the hydration properties of tissues (Fig. 2.4b). Water sorption is an exothermic

63

event. A high negative H value at low water content suggests the strong affinity of adjacent water molecules toward ionic sites and/or other polar sites of the substrate. As water content increases, the H becomes less negative (Fig. 2.4b). The primary hydration process (i.e. strong and weak binding sites) is considered to be completed when the differential enthalpy of hydration (H) approaches zero (Luscher-Mattli and Ruegg, 1982; Rupley et al., 1983; Bruni and Leopold, 1991). The change of S reflects the relative degree of order, and the S peak is presumably associated with the saturation of all primary hydration sites. It should be clearly noted that the relationships of H/WC, G/WC and S/WC describe thermodynamic interactions between water and biomaterials, but not necessarily the functions of water and biological structures in physiological processes. A possible association between water sorption behaviours and desiccation tolerance of plant tissues was discussed in a number of studies (Vertucci and Leopold, 1987b; Farrant et al., 1988; Pritchard, 1991; Sakurai et al., 1995; Eira et al., 1999; Sun, 2000). No consistent difference in water sorption characteristics has been found between desiccation-sensitive (recalcitrant) and desiccation-tolerant (orthodox) seed tissues (Sun, 2000). The van’t Hoff relationship provides another convenient means to analyse temperature dependence of sorption isotherm. The van’t Hoff equation and the Clausius–Clapeyron equation are essentially the same in theory, but different in their mathematical treatment of experimental data. The Clausius–Clapeyron equation handles two temperature points, whereas the van’t Hoff equation can handle a series of temperature points at once. The van’t Hoff equation expresses the relationship of the equilibrium water activity (aw) for a given water content against the temperature (1/T) (Fig. 2.5b), and is written in its differential mathematical form as: H ∂ ln aw R ∂(1/T)

(19)

where T is absolute temperature in kelvin, and R is the gas constant. The H is the


18/3/02

1:54 pm

64

Page 64

W.Q. Sun

differential enthalpy of water sorption. It is important to note that the relationship between ln(aw) and (1/T) is not necessarily a straight line. Within a relatively narrow range of temperature, linear approximation may be used to calculate H accurately. However, there is considerable interest in studying water sorption properties of biological tissues at a much wider range of temperature. For example, long-term preservation of genetic resources may (a)

require the storage of desiccation-sensitive seeds and other tissues in a refrigerated condition or at liquid nitrogen temperature. When the extrapolation is used, the non-linear nature of the relationship between ln(aw) and (1/T) needs to be taken into consideration. The study on temperature dependence of water sorption using the van’t Hoff equation (Fig. 2.5a and b) is used to establish the theoretic framework for the optimization of germplasm preser-

0.0 0.09 g g–1 dw

ln (aw or RH/100)

–1.0

–2.0

0.04 g g–1 dw

–3.0

0.02 g g–1 dw

–4.0

3.3

3.4

3.5

3.6

1/Temperature (K, 10–3) (b)

Water content (g g–1 dw)

0.14 0.12 0.10

50% RH

0.08 0.06 0.04

10% RH

0.02 0

5

10

15

20

25

Temperature ( C) Fig. 2.5. (a) Temperature dependence of water sorption for the same seed material at different water contents (i.e. the van’t Hoff plot). Drawn with data from Eira et al. (1999). (b) Equilibrium water content at specific water activities as a function of temperature for whole seeds of Coffea arabica cv. Mundo Nova. This relationship is called ‘isopleth’.


18/3/02

1:54 pm

Page 65


vation protocols (Vertucci et al., 1994, 1995; Eira et al., 1999). 2.4.2.3. Monolayer hydration and waterclustering function The monolayer hydration values of plant tissues, the amount of water bound to specific polar sites, can be easily determined, using BET or GAB isotherm models. For most plant tissues and their major chemical components, the monolayer value at ambient temperature is estimated to be between 0.04 and 0.09 g g1 dw using the BET or GAB model (Rahman and Labuza, 1999). The BET monolayer value of many orthodox seeds was also found to be in this range (Vertucci and Leopold, 1987a,b; Bruni and Leopold, 1991; Vertucci and Roos, 1993; Sun et al., 1997). The monolayer value of Typha pollen was much less than that of orthodox seeds (Buitink et al., 1998b). The monolayer hydration is generally complete at a water activity of 0.20–0.30 (i.e. 150 to 250 MPa). It is important to note that the monolayer value decreases rapidly as temperature increases, and increases as temperature declines. The monolayer water is of great importance for the survival of many dry organisms (e.g. spores, pollen grains and seeds) during storage. In food science, the water activity at the monolayer value is defined as the critical water activity. At a water activity above 0.20–0.30, the rate of chemical reactions begins to increase significantly because of the greater solubility and mobility of the reactants. At water contents below the monolayer value, the rate of lipid oxidation and associated free radical damage increases. The presence of monolayer water inhibits the undesirable interactions between polar groups on adjacent carbohydrate or protein molecules, thereby preserving their rehydration ability and biological functions (Rahman and Labuza, 1999). There is no defined monolayer parameter in the D’Arcy–Watt model. However, the first term of the D’Arcy–Watt equation may be used as an approximation, as it represents water that is bound strongly to

65

polar hydration sites. A recent study using the D’Arcy–Watt model suggested that water redistribution among different types of hydration sites might be related to the rapid loss of seed viability during storage after osmotic priming and drying back (Sun et al., 1997). Water clustering in binding sites is another important hydration event that is of significance to desiccation tolerance of plant tissues and the survival of tissue in the dried state. Clustering formation is related to a number of transport phenomena. For example, clustering reduces the effective mobility of water by increasing the size of the diffusing molecular group or by increasing the tortuosity of diffusion paths (Stannett et al., 1982). The range of water activity where the self-association of water takes place can be examined by the clustering function (Lugue et al., 1995; Dominguez and Heredia, 1999). The clustering function is written as: G11/V1 V2[(aw/V1)/aw] 1

(20)

where G11/V1 is the clustering function, V1 is the volume fraction of water, V2 is the volume fraction of biopolymers, and aw is water activity (Zimm and Lundberg, 1956). The subscript ‘11’ in G11/V1 denotes the water–water interaction as a function of water content (component 1). The clustering function can be applied to an isotherm sorption model such as the GAB equation with some modifications. The GAB equation needs to be rewritten in terms of volume fraction instead of weight fraction. The GAB equation can be rewritten as: Mw V1p1/V2p2

MmCKaw (1Kaw)(1KawCKaw)

(21)

or aw/V1

(1Kaw)(1KawCKaw) MmCKp2V2

(22)

where p1 and p2 are the density of water and biopolymers. The density of sorbed water is assumed to be equal to 1.0 g cm3. Substituting aw/V1 in Equation (20) with Equation (22), the clustering function can be expressed as:


18/3/02

1:54 pm

66

Page 66

W.Q. Sun

G11/V1

(2KCKMmCK)(2CK22K2)aw (23) MmCKp2

According to Equation (23), G11/V1 is proportionally related to aw and the reciprocal of polymer density (p2) function. G11/V1 can be solved easily by the substitution of Mm, C and K constants from the GAB equation. Figure 2.6 shows a plot of the waterclustering function of soybean axes. The clustering plot is basically a straight line against water activity. When G11/V1 is greater than 1, water is expected to cluster (Zimm and Lundberg, 1956). The autoassociation (clustering) of water in a few desiccation-tolerant seeds is observed to occur at water activity ranging from 0.55 to 0.60 (W.Q. Sun, unpublished data). 2.4.2.4. Occupancy of water-binding sites The D’Arcy–Watt model can be used to examine the occupancy of water-binding sites as a function of water content according to Luscher-Mattli and Ruegg (1982). The occupancy represents the amount of water attached to certain hydration sites,

expressed as the percentage of the corresponding maximum value in the fully hydrated tissues (Fig. 2.7b). Therefore, the occupancy relationship indicates the degree of hydration for different types of hydration sites during desiccation. Figure 2.7b shows that the occupancy for three types of hydration sites changes as the water content of Q. rubra seed tissues decreases during desiccation. Desiccation of seed tissues to 0.30 g g1 dw (the critical water content) removed about 90% of multilayer molecular sorption water, but only about 10% of water molecules attached to the weak hydration sites in seed tissues. The removal of water from weak hydration sites appears to be related to desiccation damage in Q. rubra seeds (Sun, 1999). The critical water content of Q. robur axes also corresponds to the amount of matrix-bound water (Pritchard and Manger, 1998). However, the question of whether the water-binding or sorption behaviour in seed tissues is related to their desiccation tolerance remains unresolved. The loss of viability in many recalcitrant seeds occurs at a water content that is much higher than

8

Clustering function

4

0

–4

–8

–12 0.0

0.2

0.4

0.6

0.8

1.0

Water activity Fig. 2.6. Water-clustering function showing the waterwater association in soybean seed axes as a function of equilibrium water activity. Apparent water clusters first appear at a water activity of 0.58 (arrow). The water-clustering function Equation (23) was solved through the study on biopolymer volumetric change during hydration [i.e. P2= f (V1)] by applying water sorption analysis. See text for further explanation.


18/3/02

1:54 pm

Page 67


that of ‘bound’ water (Pammenter et al., 1991; Berjak et al., 1992). Clearly, more comprehensive studies are needed. Readers who wish to know more about water sorption analysis may refer to a (a)

67

recently released manual by Bell and Labuza (2000). This book generally discusses water activity in food materials, but the principles are also applicable to plant desiccation tolerance studies. Practical

0.8


5 C 0.6

WC =

4.974 / 0.0219 / + 0.0673 / + 1 + 90.2 / 1 – 0.990 / 25 C

0.4

WC =

0.026 / 1.279 / + 0.0373 / + 1 + 27.4 / 1 – 0.986 /

0.2

0.0 0.0

0.2

0.4 0.6 Water activity

0.8

1.0

(b)

Sorption sites occupied (%)

100

80

5 C 25 C

60 Strong binding site Weak binding site

40

Multilayer sorption 20

0 0.0

0.1

0.2

0.3

0.4

Water content (g

0.5

0.6

0.7

g–1 dw)

Fig. 2.7. (a) The interpretation of desorption isotherms of Quercus rubra cotyledonary tissues, using the D’Arcy–Watt model. Equation coefficients are derived though curve-fitting of experimental data ( / o = aw). See text for further explanation. (b) The occupancy for three types of hydration sites in Q. rubra cotyledonary tissues at different water contents. The occupancy is based on the percentage of the corresponding maximum values in the fully hydrated state (i.e. full turgor). The change of occupancy reveals how and when water is removed in different types of hydration sites during dehydration.


18/3/02

1:54 pm

Page 68

68

W.Q. Sun

examples are provided to elucidate how to solve many equations.

2.5. Measurement of Drying Rate and Desiccation Stress 2.5.1. Driving force for water loss and expression of drying rate The loss of water from tissues depends on two factors: the gradient in water potential between tissue surface and external air or solution, and the hydraulic conductivity of the tissue. The volume flow of water from the tissue to air can be described by: Vw ALp (o i)

(24)

where Vw is the volume flow of water per unit time (m3 s1), A is the surface area of the tissue (m2) and Lp is the hydraulic conductivity of the tissue (m s1 Pa1). The o and i are water potentials of external air and the tissue, respectively. The difference in water potential (o i) is a measure of the driving force for dehydration. i is a function of time that describes the decrease in tissue water potential during drying. The hydraulic conductivity of the tissue (Lp) is a measure of the diffusional resistance of the water transport pathway within the tissue. A good measure for the drying rate is essential for comparative studies on desiccation tolerance. According to Equation (24), the rate of water loss from the tissue is time-dependent. Under constant RH and temperature, the water content of the tissue is expected to decrease exponentially over time until i reaches o (Fig. 2.8a). The curve of water loss can be described by: WC = exp(t)

(25)

where is the initial water content, is the rate constant of water loss, and t is time of drying. This relationship was first used by Tompsett and Pritchard (1998) to compare the dehydration rate of A. hippocastanum seeds. Drying curves of other seed tissues have been examined under a wide range of desiccation conditions, and they conform to Equation (25) (Li and Sun, 1999; Liang and Sun, 2000). Typically, water content

curves are biphasic. During the first drying phase, the loss of water follows a simple exponential function. During the second phase, water content does not decrease much because the tissue is very much closer to achieving equilibrium with the air. Because water loss during the first phase is described by an exponential function, the rate constant () of water loss can be used as an expression of drying rate.

2.5.2. Quantification of desiccation stress The response of plant tissues to desiccation is significantly affected by dehydration conditions, such as drying rate (see Chapter 3). Under slow-drying conditions, plant tissues stay longer at intermediate water contents. Fast drying is often reported to improve desiccation tolerance of recalcitrant plant seeds (reviewed by Pammenter and Berjak, 1999). There is no doubt that the level of desiccation stress would vary with drying rate, and the questions are: (i) how desiccation stress can be quantified; and (ii) how drying rate affects the level of desiccation stress. The change in chemical potential of cellular water is a good measure for the degree of desiccation stress. When the chemical potential of water is compared, *w and mwgh in Equation (5) cancel out. The difference in chemical potential of cellular water between the dehydrated state (Dw) and the hydrated state (Hw) is: –

HwDw RT(ln aHwln aDw)Vw(PHwPDw) (26) According to Equation (26), the degree of desiccation stress is proportional to changes in osmotic potential and hydrostatic pressure (P) in cells. Therefore, the change of water potential, d/dt, can be used to quantify the level of desiccation stress. Figure 2.8b shows the plots of tissue water potential against drying time. Under the conditions of constant temperature and relative humidity, such plots are straight lines down to the fraction of apoplastic water. Water potential of the tissue decreases faster and deviates away from the straight line when the apoplastic water is lost (see Fig. 2.3b, the low-RWC break


18/3/02

1:54 pm

Page 69


point). The slope of each straight line portion (d/dt) represents the degree of direct physical stress under different desiccation conditions. The relationship between d/dt and the rate constant () of water loss (drying rate) is linear (Fig. 2.9). A

69

mathematical evaluation of this linear relationship will not be presented here. If the plant tissue is viewed as a viscoelastic system, the mechanical stress caused by water loss can be considered as a simple stress–strain response. The physico-chemical

(a) 3.2


33% RH 88% RH

2.4

94% RH

1.6

= 0.00502

0.8 = 0.0211 0.0

= 0.103 0

80

160

240

320

400

Drying time (h)

(b) 0 33% RH

Water potential (MPa)

88% RH –5

94% RH

–10 d/dt = –0.032 –15 d/dt = –0.160 d/dt = –0.689 –20 0

80

160

240

320

400

Drying time (h)

Fig. 2.8. Measurement of drying rate and quantification of desiccation stress for Theobroma cacao axes. (a) Drying curves of isolated axes in three constant relative humidities (RHs). The data are fitted with exponential functions (WC = exp(t)), and the rate constants of water loss, , are shown near each curve. (b) Plots of tissue water potential against drying time. The mechanical stress on tissue caused by the water loss can be considered as a simple stress–strain response. The slope of /time plot, d/dt, is directly related to the intensity of desiccation stress. Data from Liang and Sun (2000).


18/3/02

1:54 pm

70

Page 70

W.Q. Sun

Dehydration rate (–d/dt)

1.6

1.2

0.8

0.4

0.0 0.00

0.06

0.12

0.18

0.24

0.30

Rate constant of water loss () Fig. 2.9. The relationship between the rate constant of water loss () in Equation (25) and dehydration rate (d/dt) for Theobroma cacao (cocoa) axes. Isolated axes were dehydrated at 16°C under constant relative humidities ranging from 6 to 94% to achieve different drying rates and stress conditions. Drawn using data from Liang and Sun (2000).

aspect of desiccation stress can be assessed by integrating the function of tissue water potential over time (Fig. 2.8b). Figure 2.10a and b shows an example of the quantitative analysis of desiccation stress. The cumulative water stress during desiccation at three different RHs was plotted against drying time and water content, respectively. Under the slow-drying condition (94% RH), the mechanical stress (d/dt) is small (Fig. 2.8b); however, the cumulative physico-chemical stress is remarkably high because the time to dry to the same water content increases exponentially as the drying rate decreases (Fig. 2.10a and b). The quantitative analysis of mechanical and physico-chemical aspects of desiccation stress has led to an understanding of the physiological basis of the optimal drying rate to achieve the maximum desiccation tolerance of Theobroma cacao axes (Liang and Sun, 2000).

2.6. Water Relations – the Kinetic and Functional Approach The thermodynamic approach to water relations has its limitations, because it

treats biological systems as fully reversible ones and does not give much consideration to the term time, one of the most important factors in any biological response. This limitation is particularly relevant to the study of desiccation. The application of thermodynamics is generally sufficient in many cases for fully hydrated tissues. However, at intermediate or low moisture levels, the non-equilibrium, kinetic principles play a more important role. During desiccation, the biological system basically shifts from a thermodynamic state to a non-equilibrium kinetic state (Leopold et al., 1994; Sun et al., 1994; Sun, 1997, 1998). The thermodynamic approach does not sufficiently address the kinetics of various reactions and processes in intermediate- to lowmoisture systems. The kinetic and functional approach to cellular water relations focuses on how the interactions between water and other cellular components can influence the structures and biological properties of each other. In this chapter, principles of the kinetic approach and the interactions


18/3/02

1:54 pm

Page 71


71

(a) Cumulative water stress (MPa h)

2000

1500

1000 33% RH 88% RH

500

94% RH 0 0

80

160

240

320

400

Drying time (h)

(b) Cumulative water stress (MPa h)

2000 33% RH 88% RH

1500

94% RH

1000

500

0 0.0

0.6

1.2

1.8

2.4

3.0


Fig. 2.10. (a) Cumulative water stress during desiccation as the function of drying time. The cumulative water stress is calculated by integrating the /time function (see text for details). (b) Cumulative water stress as the function of tissue water content under different desiccation conditions. Cumulative stress is much higher in slow-drying conditions because the dehydration time increases exponentially as drying rate decreases.

between water and many other biomolecules will not be discussed in detail. These topics will be covered in other chapters on desiccation damage and mechanisms of desiccation tolerance (see Chapters 9–12).

Instead, a general account will be offered, so that readers can be confident about choosing the appropriate method to quantify particular water properties in studies on desiccation tolerance.


18/3/02

1:54 pm

72

Page 72

W.Q. Sun

2.6.1. General considerations 2.6.1.1. Time scale Any study on the state of water by a biophysical technique involves measuring parameters of time scale directly or indirectly. Biophysical techniques often use the diffusional correlation time as the time scale to make comparisons of the measurements on water. Many of the physical properties of water are theoretically related to the diffusional correlation time (D) of water, the average time between jumps in position for water molecules in the system. Two critical numbers on this time scale (D) are 105 s and 1011 s. The D of water in ice is 105 s and in pure liquid is 1011 s, one million times faster. Under normal conditions, water in biological systems exists in a state somewhere between the solid state of crystalline ice and the liquid hydrogenbonded lattice of pure water. In lowmoisture systems, however, the D of water molecules in the intracellular glasses would be much slower than 105 s. Readers may refer to a series of studies on molecular mobility in seeds and pollen by Buitink et al. (1998a, 1999, 2000a,b,c; Chapter 10 of this volume). The ability of a biophysical technique to yield useful information about the physical state of water largely depends on how fast a measurement can be made. Slow techniques which require measurement times greater than the diffusional correlation time yield an average over all molecules in the population with a kinetic contribution from diffusion, whereas fast techniques can yield instantaneous information about intramolecular factors such as H–O bond lengths and hydrogen bond angles (geometric factors). To choose the appropriate technique, one has to bear in mind that the type of property or structure that a technique can probe is related to the time scale. Generally speaking, fast techniques would probably produce data of instantaneous structures of water and other biomolecules, but slow techniques would provide more information about the interactions between water molecules and their environment. Taking

the study on water in skeletal muscle as an example, fast techniques (e.g. laser Raman spectroscopy, infrared spectroscopy and dielectric relaxation) could not find any intramolecular differences in hydrogen bond lengths, angles or strength between muscle water and pure water (Beall, 1981). However, slower techniques, such as nuclear magnetic resonance (NMR) (Fung and McGaughy, 1974), electron paramagnetic resonance (EPR) (Belagyi, 1975) (see Chapter 4), fluorescence polarization (Knight and Wiggins, 1979) and freezing behaviour (Rustgi et al., 1978), showed a restricted motion of at least a portion of cell water. This situation is identical to photographing moving objects with different shutter speeds. If the shutter speed is very high relative to the velocities of two moving objects (e.g. 1/800 s), the photo will probably not record any information as to whether one object is moving faster than the other. On the other hand, if the shutter speed is too slow (e.g. 1/2 s), the images of both moving objects will be blurred and no meaningful information can be obtained from such a photo. Only with a proper shutter speed can the photo reflect the difference in the velocity between the two moving objects. 2.6.1.2. Structural complexity and dynamics of molecular ordering Hydration of protein is a good example, illustrating the complexity of structures and functions for water in biological systems. When a mole of lysozyme is hydrated by 60 moles of water (~ 0.07 g g1 dry protein), water is primarily located to charged groups, and its mobility is reduced at least by 100 times relative to pure water. At this low hydration level, no structural difference is observed in the protein. As hydration increases to 220 moles of water per mole of protein (~ 0.25 g g1 dry protein), water begins to form clusters of various sizes and arrangements around the charged and polar sites of the protein. Internal protein motion (H exchange) increases by 1000 times to be comparable to that of solution, while the protein sample is still a solid. At


18/3/02

1:54 pm

Page 73


a hydration level of 300 moles of water per mole of protein (~ 0.38 g g1 dry protein), enzymatic activity, mobility of bound ligand and fast water motion become easily detectable. Dielectric relaxation measurements show two water relaxation times, one of 2 1011 s, close to that of bulky pure water, and the other of 109 s, indicating the heterogeneous nature of water behaviours and functions (Rupley et al., 1983). At the hydration level of 0.38 g g1 dry protein, each water molecule covers, on average, 20 Å2 of protein surface, which is twice the effective area of a water molecule. Yet several populations of water molecules are observed at such low hydration. The surfaces of membranes, proteins and other macromolecules impose geometric limitations on the possible arrangements of hydration water. Interfacial water molecules, being part of the network of biological interfaces, are dynamically oriented and exhibit restricted motion (i.e. are ‘bound’). The ordering of molecules on various biological surfaces is strictly local, and may fluctuate rapidly between possible arrangements. Ideally, a biophysical technique used to study the state of water in biological systems should have the resolution to differentiate closely related structures or populations. However, as discussed earlier, kinetic measurements reflect only average or time-average properties over all molecules in the system and, in many cases, do not provide definitive answers to the questions of interest. To interpret the data of kinetic measurements, the investigator must impose a conceptual model, which may be controversial (Beall, 1983). Such studies are often misunderstood and misinterpreted by readers who are less familiar with the biophysical techniques used. 2.6.1.3. The model-dependent interpretation: the pitfalls The selection of a theoretic model or the development of a new model is an important step in any kinetic study, which should be done before actual measurements are made. The assumptions that a model contains, and the specific predic-

73

tions that a model allows, have to be taken into consideration for experimental design and implementation. If possible, additional experiments should be conducted to confirm the results and to examine whether the assumptions are satisfied. Unfortunately, biophysical models or equations have frequently been used to analyse the actual data without checking their assumptions. Worse still, sometimes a model was selected after the entire experiment was completed (Beall, 1983). Conceptual models have been used for the interpretation of the data on water in biological systems, with many pitfalls. For example, the ‘two-fraction fast-exchange model’ (Zimmermann and Brittin, 1957) assumes that there is a small fraction of highly immobilized cell water on the surface of macromolecules (ice-like) and a large fraction of cell water that behaves like bulk water. Rapid exchange between the two populations yields reduced average properties. This two-fraction model can be written as: 1 X (1 X) T * Tslow TH2O

(27)

where T * is measured (average) relaxation time; X and (1X) are the fraction of immobilized water and the fraction of cell water that is like bulk water, respectively; Tslow and TH2O are the relaxation times of the slow fraction and bulk water. In this equation, there is only one measured parameter (T *), but three unknown quantities (X, Tslow and TH2O). To estimate X, Tslow and TH2O must be arbitrarily assigned. This model represents a simplistic view on the dynamics of water in biological systems, which is still in use by some workers. If one intends to solve Tslow, then X must be estimated through other methods. When X is equated to the ‘non-freezable fraction’ or ‘osmotically inactive fraction’, additional assumptions are made. By redefining X as an adjustable parameter in different systems, a new model is established (Beall, 1983). This example clearly shows the uncertainty of biophysical interpretation. Simply because the model is easy to use and fits the data well it does not necessarily


18/3/02

1:54 pm

74

Page 74

W.Q. Sun

mean that it represents the true state of water in a system. Of course, all models are open to interpretation. However, it is possible to measure changes in the properties of water in living systems that correlate with physiological functions (Clegg et al., 1982; Clegg, 1986; Bruni et al., 1989). Changes in dynamic properties of water at different hydration levels indicate the existence of different fractions of water, which may vary in structure and property and presumably play different biological roles. Studies have identified the existence of at least four or five fractions of water, presumably relating to different interactions between water and cellular constituents (Clegg, 1986; Ratkovic, 1987; Vertucci, 1990; Pissis et al., 1996; Sun, 2000). Hydration levels corresponding to these fractions of cellular water are associated with the onset of various metabolic activities in organisms (Clegg, 1986).

2.6.2. Biophysical techniques (see also Chapter 4) Kinetic properties and functions of water have been studied, using calorimetry (Ruegg et al., 1975; Bakradze and Balla, 1983; Vertucci, 1990; Sun, 1999), infrared (IR) and Raman spectroscopy (Careri et al., 1979; Cameron et al., 1988), NMR spectroscopy (Fung and McGaughy, 1974; Mathur-de Vre, 1979; Seewaldt et al., 1981; Rorschach and Hazlewood, 1986; Ratkovic, 1987), quasi-elastic neutron-scattering spectroscopy (Lehmann, 1984; Trantham et al., 1984) and dielectric relaxation techniques (Harvey and Hoekstra, 1972; Kamiyoshi and Kudo, 1978; Clegg et al., 1982; Pissis et al., 1987, 1996; Bruni and Leopold, 1992). These techniques differ greatly in how and what they measure with respect to the dynamic properties and structures of water and other biomolecules. A great deal of confusion over the physical state of water in biological systems has resulted from the separation of information obtained with diverse techniques applied to similar systems. The nature of different

measurements is briefly summarized in Table 2.1. Readers are advised to consult other references, including those cited above, and Chapter 4 in this volume. In this chapter, only a brief introduction will be provided on several techniques that have been increasingly used in recent years. 2.6.2.1. Differential scanning calorimetry Differential scanning calorimetry (DSC) is probably the most commonly used thermal analysis technique. It has been used by a number of workers to study the possible relationship between freezing, desiccation tolerance and water properties in plant tissues (Williams and Leopold, 1989; Vertucci, 1990; Pammenter et al., 1991; Berjak et al., 1992; Sun et al., 1994; Vertucci et al., 1994, 1995; Buitink et al., 1998b; Pritchard and Manger, 1998; Sun and Davidson, 1998; Sun, 1999). DSC measures the heat flow of plant tissues associated with various thermal events during cooling and/or heating scans. Such thermal events include phase transitions (e.g. freezing, melting, glass transition, etc.), polymorphism, thermochemistry and the kinetics for a variety of complex reactions (e.g. in vivo protein denaturation). The key idea involved in DSC measurement of water status is that thermal changes of water and their corresponding quantities of energy are greatly affected by the presence of other biomaterials in plant tissues, and that thermal behaviours of other biomaterials in plant tissues are affected by water content. For example, as water content decreases, the onset freezing and melting temperature of water decreases due to solute concentration, while at the same time glass transition temperature of the tissue increases due to the reduced plasticization effect by water. By analysing thermal behaviours of water and biomaterials as a function of water content, temperature and time, the status of water in plant tissues can be studied and the water status correlated to its biological functions. Technically, DSC is really a quite simple method. There are two cells in the DSC detector, one reference cell and one sample


18/3/02

1:54 pm

Page 75


cell. An empty crucible is placed into the reference cell and a crucible containing the tissue sample is placed into the sample cell. During a DSC experiment, both reference cell and sample cell are cooled and/or heated at a constant rate over a range of temperatures. When a thermal event occurs in the tissue, it releases or absorbs heat energy (i.e. heat flow). A plot of heat flow as a function of temperature is called a thermogram, from which the thermal behaviour of the tissue can be deduced. Glass transition is usually marked by a stepwise shift in the baseline of a thermogram. This distinguishes it from freezing and melting transitions, which produce peaks. A melting event is accompanied by an endothermic peak and a freezing event is accompanied by an exothermic peak. The area under the particular peak represents the total heat energy or enthalpy change (H) for the event. DSC is a highly informative tool for analysing biological materials. Other information such as transition temperature and heat capacity change (Cp) of the tissue upon cooling or heating can also be calculated from a thermogram. The difficulty in analysing DSC thermograms lies in the correct identification of origin for thermal events in heterogeneous biological samples. For example, lipid transition in seeds can mask the actual glass transition (Williams and Leopold, 1989) and interfere with the accurate calculation of freezing and melting enthalpies (Sun, 1999).

75

2.6.2.2. Thermally stimulated current (TSC) method Different dielectric relaxation techniques had been used previously to study the properties of water in biological systems (Harvey and Hoekstra, 1972; Kamiyoshi and Kudo, 1978; Clegg et al., 1982; Careri and Giansanti, 1984). More recently, the TSC technique has been employed to study the mode of hydration and water organization in plant tissues (Pissis et al., 1987, 1996; Bruni and Leopold, 1992; Sun et al., 1994; Sun, 2000). This technique is capable of providing information concerning the mobility and rotational freedom of hydration water, hydration sites and mechanisms (Mascarenhas, 1980; Pissis et al., 1987, 1996; Pissis, 1990; Bruni and Leopold, 1992). The TSC technique is based upon: (i) the dependence of the microdynamics of water dielectric relaxation on their surroundings resulting in different dielectric relaxation times for water in different fractions; and (ii) the influence of water on the dielectric relaxation mechanisms of other biomolecules (similar to those used for DSC measurements). The TSC method measures the tiny current generated by the thermally activated release of stored dielectric polarization during controlled heating and basically consists of three steps: (i) the polarization of a sample by a strong d.c. electric field at a particular temperature; (ii) ‘freeze-in’ the polarization by cooling down to a sufficiently low temperature

Table 2.1. Biophysical techniques used to study the dynamic and structural properties of water and macromolecules in biological systems. Type of information Techniques Thermal analysis (DSC, DTA) X-ray diffraction Spectroscopy (NMR, EPR) Relaxation (NMR, EPR, dielectric) Ultrasonic absorption Quasi-elastic neutron scattering Infrared and Raman spectroscopy

Time scale (s)

Timeaverage

101 ~103 101 ~102 104 ~100 1011 ~100 1010 ~105 1013 ~107 1016 ~1012

+ + +

Information about

Dynamic Structural Water + (+) + + + +

+

+ + (+) + + + +

Macromolecule + + (+) + + +

DSC, differential scanning calorimetry; DTA, differential thermal analysis; NMR, nuclear magnetic resonance; EPR, electron paragmagnetic resonance.


76

18/3/02

1:54 pm

Page 76

W.Q. Sun

(e.g. liquid nitrogen temperature) while the field is still on; and (iii) the measurement of the TSC spectrum during heating after the d.c. field is disconnected (Bruni and Leopold, 1992). When a polarized tissue reaches a temperature at which dipole molecules (such as water) relax (lose their fixed orientation), a tiny current is generated and recorded. From a TSC spectrum, several important physical parameters can be obtained, including the intensity of depolarization charge (peak size, related to the size of the water pool), depolarization temperature, its activation energy and static permittivity. The measurement of dielectric relaxation properties of water and water-plasticized biomolecules offers valuable insight into the organization of water in plant tissues and the molecular interactions between water and other biomolecules during desiccation (Bruni and Leopold, 1992; Sun, 2000). 2.6.2.3. Nuclear magnetic resonance (NMR) NMR spectroscopy studies the interaction of electromagnetic radiation with matter. It is a powerful tool for the studies of kinetic motion of water in tissues and of macromolecule/water or membrane/water interactions. Solid-state NMR can be used to determine the molecular structure of solid tissue samples. Solid-state H-NMR is often used to investigate the relaxation characteristics of the protons of water molecules in low-moisture biological systems (Mathurde Vre, 1979; Seewaldt et al., 1981; Rorschach and Hazlewood, 1986; Ratkovic, 1987; Chapter 4). The basic principle of HNMR is that each of two hydrogen nuclei in a water molecule possesses a single spin proton, which will cause the nucleus to produce an NMR signal. When an atom is placed in a magnetic field, the spin of its electrons will orient toward the direction of the applied magnetic field. This orientation produces a small local magnetic field at the nucleus that opposes the externally applied field, resulting in a smaller magnetic field (i.e. effective field) at the nucleus than the applied field. (Note that in some cases it might also enhance the

magnetic field at the nucleus.) Since the electron density around each nucleus in a molecule varies according to the type of nuclei and its molecular environment, the opposing field and thus the effective field at each nucleus will differ, which is called ‘chemical shift’. The chemical shift of a nucleus is the difference between the resonance frequency of the nucleus and a standard (relative to the standard, expressed in p.p.m., ). The chemical shift is a very precise measure of the chemical environment around a nucleus. The major frustration for many biologists wishing to understand and to use NMR is the complexity of the subject. However, as with other physical techniques used in studies of biological systems, NMR may be used in an ‘empirical’ mode, simply examining the variation of an NMR parameter with the change of experimental variable (e.g. water content) (James, 1993). Figure 2.11 shows 1H-NMR spectra of mung bean seeds at three water contents. The 1H-NMR spectra were broad, with the line width in the order of 103 Hz. Two NMR peaks were easily identifiable. The peak of water in the immobile fraction had a peak maximum (chemical shift) value of 4.3 p.p.m. relative to the proton in D2O, which was used as a standard reference. The peak of water in the mobile fraction had a peak maximum value at the same place as the proton in D2O (i.e. = 0 p.p.m.). At a water content of 0.07 g g1 dw, water appeared to exist primarily in the immobile fraction. The very small proportion of water in the mobile fraction appeared as a shoulder in the spectrum. The amount of water in the mobile fraction increased rapidly as water content increased. At 0.24 g g1 dw, two peaks merged almost completely as one peak, centred at = 0 p.p.m. The relative proportion of the immobile and mobile water fractions may be estimated using the standard signal processing techniques. Two important spin relaxation parameters are T1, the spin–lattice relaxation, and T2, the spin–spin relaxation time. The spin–lattice relaxation (T1) involves the exchange of energy with the environment


18/3/02

1:54 pm

Page 77


(the lattice), and is caused by fluctuating local magnetic fields arising from the motion of the molecules. The spin–spin relaxation (T2) characterizes interactions between spins and is related to the width of the NMR peak (Fig. 2.11). T1 and T2 can be used to study chemical kinetics and rotational and conformational motion of molecules. 2.6.2.4. Electron spin resonance (see also Chapter 4) Electron spin resonance (ESR) or electron paramagnetic resonance (EPR) is related to NMR. It is the study of molecules with unpaired electrons (free radicals, transition metal complexes, triplet states, etc.) by observing the magnetic fields at which they

77

come into resonance with monochromatic radiation. The magnetic field of most commercial ESR spectrometers is about 0.3 T, corresponding to resonance with an electromagnetic frequency of ~10 GHz and wavelength of ~3 cm. Therefore, the range of applicability of ESR is narrower than that of NMR. ESR is basically a microwave technique, and is one of the fastest-growing areas in analytical instrumentation because of recent and remarkable achievements in microwave technology. ESR consists of a microwave source, a cavity, a microwave detector and an electromagnet. The sample is placed in a glass or quartz tube, which is inserted into the cavity. The ESR spectrum is obtained by measuring the microwave absorption as the magnetic field strength is continuously changed. This method

1

Signal amplitude

2

0.24 g g–1

0.14 g g–1

0.07 g g–1 D2O –16

–8

0

8

16

24

Chemical shift (, p.p.m.) Fig. 2.11. The 1H-NMR spectra of water in mung bean seeds at three water contents. The width of all spectra was 4000 Hz. D2O was used as a standard reference. The inset shows the assignment of 1H-NMR signal into two different water fractions: mobile water (fraction 1) and immobile water (fraction 2). The spectrum was recorded with FX90QNMR (JEOL Ltd, Japan). The powdered sample, weighing approx. 1–2 g, was loaded into the standard 5 mm NMR tube. A 90° 36-µs electromagnetic pulse was applied to the sample. A total of eight scans were used to improve the resolution. A repeat time was 20 s to re-establish the equilibrium via spin–lattice and spin–spin relaxation before the next scan (W.Q. Sun, unpublished data).


18/3/02

1:54 pm

78

Page 78

W.Q. Sun

Microwave absorption (A)

(a)

First derivative (dA/dB)

detects the number of ‘unpaired spins’ of electronic charges. The ‘strange’ ESR spectrum is the first derivative of the microwave energy absorption (Fig. 2.12). The hyperfine structure (splitting of individual resonance lines into components) of an ESR spectrum is a fingerprint that helps to identify free radical species in the sample and characterize their environments. The ESR technique does not directly measure water properties in tissues; however, it can be used to study many questions that are related to desiccation. Using an ESR spin-labelling technique, Belagyi (1975) reported that a portion of cell water in muscles exhibited a restricted motion. In recent years, Hoekstra and his co-workers have used this technique to study membrane behaviours, molecular mobility, cytoplasmic viscosity and partitioning of amphiphilic molecules of desiccation-tolerant and desiccation-intolerant plant tissues upon desiccation (Golovina et al., 1998; Buitink et al., 1999, 2000a,b,c; Leprince et al., 1999; Chaper 4). A variety of molecular spin probes (stable free radi-

(b)

cals) is commercially available. By incorporating some probes such as nitroxide derivatives into tissues before drying, detailed studies may be undertaken on the changes in the aqueous and non-aqueous intracellular environments upon desiccation. The ESR technique provides a fairly direct measurement of the change in cytoplasmic viscosity, which probably plays an important role in metabolic downregulation in desiccation tolerance (see Chapter 10).

2.7. Concluding Remarks Comparative studies play a key role in understanding the mechanisms or strategies of various organisms in the survival of desiccation. The water status of tissues in desiccation tolerance studies should be expressed precisely by preferred thermodynamic parameters to permit the comparison of data from different biological systems. The commonly used parameter, water content, is not adequate for the

Magnetic field (B) Fig. 2.12. (a) Microwave energy absorption. (b) The peculiar appearance of the electron spin resonance (ESR) spectrum. The ESR spectrum is the first derivative signal of microwave energy absorption. The peak of absorption corresponds to the point where the first derivative passes through zero (dashed lines). (See Figures in Chapter 4.)


18/3/02

1:54 pm

Page 79


expression of tissue water status in most cases. Whenever possible, the researcher should first study the components of the water relations of cells or tissues and obtain important reference parameters about their water status. The Höfler diagram and PV curve can be applied to most well-hydrated plant tissues, whereas the isothermal sorption study can be applied to intermediate- to low-moisture systems. Drying rate and desiccation stress can be quantified by introducing thermodynamic concepts into the study of water-loss dynamics during desiccation (drying curve). The quantitative (instead of qualitative) analysis of physico-chemical aspects

79

of desiccation stresses would certainly improve the mechanistic studies of desiccation tolerance. Water plays an important role in maintaining the structural integrity of biological systems. Although the kinetic properties of water in many biological systems have been extensively studied, the organization of cellular water and its relation to desiccation tolerance or desiccation damage are not fully understood. Further studies on the interactions between water and macromolecular structures by biophysical techniques are essential to identify fundamental cellular or metabolic components that are associated with desiccation damage or desiccation tolerance.

2.8. References Bakradze, N.G. and Balla, Y.I. (1983) Crystallization of intracellular water in plant tissues. Biophysics 28, 125–128. Beall, P.T. (1981) The water for life. The Sciences January 1, 6–29. Beall, P.T. (1983) States of water in biological systems. Cryobiology 20, 324–334. Beckett, R.P. (1997) Pressure–volume analysis of a range of poikilohydric plants implies the existence of negative turgor in vegetative cells. Annals of Botany 79, 145–152. Belagyi, J. (1975) Water structure in striated muscle by spin labeling techniques. Acta Biochimica et Biophysica; Academiae Scientiarum Hungaricae 10, 63–70. Bell, L.N. and Labuza, T.P. (2000) Moisture Sorption: Practical Aspects of Isotherm Measurement and Use. Eagan Press, Eagan, Minnesota, 123 pp. Berjak, P. and Pammenter, N.W. (1994) Recalcitrance is not an all-or-nothing situation. Seed Science Research 4, 263–264. Berjak, P., Pammenter, N.W. and Vertucci, C. (1992) Homoiohydrous (recalcitrant) seeds: developmental status, desiccation sensitivity and the state of water in axes of Landolphia kirkii Dyer. Planta 186, 249–261. Brunauer, S., Emmett, P.H. and Teller, E. (1938) Adsorption of gases in multimolecular layers. Journal of American Chemical Society 60, 309–319. Brunauer, S., Deming, L.S., Deming, W.E. and Teller, E. (1940) On a theory of the van der Waals absorption of gasses. Journal of American Chemical Society 62, 1723. Bruni, F. and Leopold, A.C. (1991) Hydration, protons and onset of physiological activities in maize seeds. Physiologia Plantarum 81, 359–366. Bruni, F. and Leopold, A.C. (1992) Pools of water in anhydrobiotic organisms: a thermally stimulated depolarization current study. Biophysical Journal 63, 663–672. Bruni, F., Careri, G. and Clegg, J.S. (1989) Dielectric properties of Artemia cysts at low water contents: evidence for a percolative transition. Biophysical Journal 55, 331–338. Buitink, J., Claessens, M.M.A.E., Hemminga, M.A. and Hoekstra, F.A. (1998a) Influence of water content and temperature on molecular mobility and intracellular glasses in seed and pollen. Plant Physiology 118, 531–541. Buitink, J., Walters, C., Hoekstra, F.A. and Crane, J. (1998b) Storage behaviour of Typha latifolia pollen at low water contents: interpretation on the basis of water activity and glass concepts. Physiologia Plantarum 103, 145–153. Buitink, J., Hemminga, M.A. and Hoekstra, F.A. (1999) Characterization of molecular mobility in seed tissues: an EPR spin probe study. Biophysical Journal 76, 3315–3322.


80

18/3/02

1:54 pm

Page 80

W.Q. Sun

Buitink, J., Leprince, O., Hemminga, M.A. and Hoekstra, F.A. (2000a) Molecular mobility in the cytoplasm: an approach to describe and predict lifespan of dry germplasm. Proceedings of the National Academy of Sciences, USA 97, 2385–2390. Buitink, J., Leprince, O., Hemminga, M.A. and Hoekstra, F.A. (2000b) The effects of moisture and temperature on the aging kinetics of pollen: interpretation in terms of cytoplasmic mobility. Plant Cell and Environment 23, 967–974. Buitink, J., Leprince, O. and Hoekstra, F.A. (2000c) Dehydration-induced redistribution of amphiphilic molecules between cytoplasm and lipids is associated with desiccation tolerance in seeds. Plant Physiology 124, 1413–1426. Cameron, I.L., Ord, V.A. and Fullerton, G.D. (1988) Water of hydration in the intra- and extra-cellular environment of human erythrocyte. Biochemistry and Cell Biology 66, 1186–1199. Careri, G. and Giansanti, A. (1984) Deuterium effect in the dielectric losses of wheat seeds. Lett Nuovo Cimento 40, 193–196. Careri, G., Giansanti, A. and Gratton, E. (1979) Lysozyme film hydration events: an IR and gravimetric study. Biopolymers 18, 1187–1203. Chirife, J. and Buera, M.D.P. (1996) Water activity, water glass dynamics, and the control of microbiological growth in foods. Critical Reviews in Food Science and Nutrition 36, 465–513. Clegg, J.S. (1986) The physical properties and metabolic status of Artemia cysts at low water contents: the water replacement hypothesis. In: Leopold, A.C. (ed.) Membranes, Metabolism and Dry Organisms. Cornell University Press, Ithaca, New York, pp. 169–185. Clegg, J.S., Szwarnowski, S., McClean, V.E.R., Sheppard, R.J. and Grant, E.H. (1982) Interrelationships between water and cell metabolism in Artemia cysts. X. Microwave dielectric studies. Biochimica et Biophysica Acta 721, 458–468. Crafts, A.S., Currier, H.S. and Stocking, C.R. (1949) Water in the Physiology of Plants. Chronica Botanica, Waltham, Massachusetts. D’Arcy, R.L. and Watt, I.C. (1970) Analysis of sorption isotherms of non-homogeneous sorbents. Transactions of the Faraday Society 66, 1236–1245. Dominguez, E. and Heredia, A. (1999) Water hydration in cutinized cell wall: a physico-chemical analysis. Biochimica et Biophysica Acta 1426, 168–176. Eira, M.T.S., Walters, C. and Caldas, L.S. (1999) Water sorption properties in Coffea spp. seeds and embryos. Seed Science Research 9, 321–330. Ellis, R.H., Hong, T.D. and Roberts, E.H. (1990) An intermediate category of seed storage behaviour. I. Coffee. Journal of Experimental Botany 41, 1167–1174. Ellis, R.H., Hong, T.D. and Roberts, E.H. (1991) An intermediate category of seed storage behaviour. II. Effects of provenance, immaturity, and imbibition on desiccation-tolerance in coffee. Journal of Experimental Botany 42, 653–657. Farrant, J.M., Pammenter, N.W. and Berjak, P. (1988) Recalcitrance – a current assessment. Seed Science and Technology 16, 155–156. Fung, B.M. and McGaughy, T.W. (1974) The state of water in muscle as studied by pulsed NMR. Biochimica et Biophysica Acta 343, 663–673. Golovina, E.A., Hoekstra, F.A. and Hemminga, M.A. (1998) Drying increases intracellular partitioning of amphiphilic substances into the lipid phase. Plant Physiology 118, 975–986. Harvey, S.C. and Hoekstra, P. (1972) Dielectric relaxation spectra of water adsorbed on lysozyme. Journal of Physical Chemistry 76, 2981–2994. Holmstrup, M. and Zachariassen, K.E. (1996) Physiology of cold hardiness in earthworms. Comparative Biochemistry and Physiology 115A, 91–101. Honegger, R. (1995) Experimental studies with foliose macrolichens: fungal responses to spatial disturbance at the organismic level and to spatial problems at the cellular level during drought stress events. Canadian Journal of Botany 73, s569–s578. Hüsken, D., Steudle, E. and Zimmermann, U. (1978) Pressure probe technique for measuring water relations of cells in higher plants. Plant Physiology 61, 158–163. International Seed Testing Association (1993) International rules for seed testing, rules 1993. Seed Science and Technology 21 (suppl.), 1–75. James, T.L. (1993) Fundamentals of NMR. In: Gorenstein, D. (ed.) Nuclear Magnetic Resonance (NMR), online textbook (biosci.cbs.umn.edu/biophys/OLTB/NMR.html). Kamiyoshi, K. and Kudo, A. (1978) Dielectric relaxation of water contained in plant tissues. Japanese Journal of Applied Physics 17, 1531–1536.


18/3/02

1:54 pm

Page 81


81

Knight, V.A. and Wiggins, P.M. (1979) A possible role for water in performance of cellular work. II. Measurements of scattering of light by actomyosin. Bioelectrochemistry and Bioenergetics 6, 135–146. Lehmann, M.S. (1984) Probing the protein-bound water with other small molecules using neutron small angle scattering. Journal of Physics: Colloids C7, 235–239. Leopold, A.C., Sun, W.Q. and Bernal-Lugo, I. (1994) The glassy state in seeds: analysis and function. Seed Science Research 4, 267–274. Leprince, O., Buitink, J. and Hoekstra, F.A. (1999) Axes and cotyledons of recalcitrant seeds of Castanea sativa Mill exhibited contrasting responses of respiration to drying in relation to desiccation sensitivity. Journal of Experimental Botany 338, 1515–1524. Li, C.R. and Sun, W.Q. (1999) Desiccation sensitivity and activities of free radical-scavenging enzymes in recalcitrant Theobroma cacao seeds. Seed Science Research 9, 209–217. Liang, Y.H. and Sun, W.Q. (2000) Desiccation tolerance of recalcitrant Theobroma cacao embryonic axes: the optimal drying rate and its physiological basis. Journal of Experimental Botany 51, 1911–1919. Lugue, P., Gavara, R. and Heredia, A. (1995) A study of the hydration process of isolated cuticular membranes. New Phytologist 129, 283–288. Luscher-Mattli, M. and Ruegg, M. (1982) Thermodynamic functions of biopolymer hydration. I. Their determination by vapor pressure studies, discussed in an analysis of the primary hydration process. Biopolymers 21, 403–418. Mascarenhas, S. (1980) Biolectrets: electrets in biomaterials and biopolymers. In: Sessler, G.M. (ed.) Electrets. Springer-Verlag, Berlin, pp. 321–346. Mathur-de Vre, R. (1979) The NMR studies of water in biological systems. Progress in Biophysics and Molecular Biology 35, 103–134. Meidner, H. and Sheriff, D.W. (1976) Water and Plants. John Wiley & Sons, New York. Milburn, J.A. (1970) Cavitation and osmotic potential of Sordaria ascospores. New Phytologist 69, 133–142. Oertli, J.J. (1989) The plant cell’s response to consequences of negative turgor presure. In: Kreeb, K.H., Richter, H. and Hinckley, T.M. (eds) Structural and Functional Responses to Environmental Stress: Water Shortage. SPB Academic, The Hague, The Netherlands, pp. 73–78. Pammenter, N.W. and Berjak, P. (1999) A review of recalcitrant seed physiology in relation to desiccation-tolerance mechanisms. Seed Science Research 9, 13–37. Pammenter, N.W., Vertucci, C.W. and Berjak, P. (1991) Homeoiohydrous (recalcitrant) seeds: dehydration, the state of water and viability characteristics in Landolphia kirkii. Plant Physiology 96, 1093–1098. Pissis, P. (1990) The dielectric relaxation of water in plant tissues. Journal of Experimental Botany 41, 677–684. Pissis, P., Anagnostopoulou-Konsta, A. and Apekis, L. (1987) A dielectric study of the state of water in plant stems. Journal of Experimental Botany 38, 1528–1540. Pissis, P., Konsta, A.A., Ratkovic, S., Todorovic, S. and Laudat, J. (1996) Temperature and hydrationdependence of molecular mobility in seeds. Journal of Thermal Analysis 47, 1463–1483. Potts, M. (1994) Desiccation tolerance of prokaryotes. Microbiological Reviews 58, 755–805. Poulsen, K.M. and Eriksen, E.N. (1992) Physiological aspect of recalcitrance in embryonic axes of Quercus robur L. Seed Science Research 2, 215–221. Pritchard, H.W. (1991) Water potential and embryonic axis viability in recalcitrant seeds of Quercus rubra. Annals of Botany 67, 43–49. Pritchard, H.W. and Manger, K.R. (1998) A calorimetric perspective on desiccation stress during preservation procedures with recalcitrant seeds of Quercus robur L. CryoLetters 19 (suppl.), 23–30. Proctor, M.C.F. (1999) Water-relations parameters of some bryophytes evaluated by thermocouple psychrometry. Journal of Bryology 21, 263–270. Proctor, M.C.F., Nagy, Z., Csintalan, Z.S. and Takács, Z. (1998) Water content components in bryophytes: analysis of pressure–volume curve. Journal of Experimental Botany 49, 1845–1854. Rahman, M.S. and Labuza, T.P. (1999) Water activity and food preservation. In: Shafiur Rahman, M. (ed.) Handbook of Food Preservation. Marcel Dekker, New York, pp. 339–382. Ratkovic, S. (1987) Proton NMR of maize seed water: the relationship between spin-lattice relaxation time and water content. Seed Science and Technology 15, 147–154.


82

18/3/02

1:54 pm

Page 82

W.Q. Sun

Rorschach, H.E. and Hazlewood, C.F. (1986) Protein dynamics and the NMR relaxation time T1 of water in biological systems. Journal of Magnetic Resonance 70, 79–88. Ruegg, M., Moor, U. and Blanc, B.H. (1975) Hydration and thermal denaturation of ß-lactoglobulin: calorimetric study. Biochimica et Biophysica Acta 400, 334–342. Rupley, J.A., Gratton, E. and Careri, G. (1983) Water and globular proteins. Trends in Biochemical Sciences 8, 18–22. Rustgi, S.N., Peemoeller, H., Thompson, R.T., Kydon, D.W. and Pintar, M.M. (1978) A study of molecular dynamics and freezing phase transition in tissues by proton spin relaxation. Biophysical Journal 22, 439–452. Sakurai, M., Kawai, K., Inoue, Y., Hino, A. and Kobayashi, S. (1995) Effects of trehalose on the water structure in yeast cells as studied by in vivo 1H NMR spectroscopy. Bulletin of Chemical Society of Japan 68, 3621–3627. Scheidegger, C., Schroeter, B. and Frey, B. (1995) Structural and functional processes during water vapour uptake and desiccation in selected lichens with green algal photobionts. Planta 197, 399–409. Seewaldt, V., Priestley, D.A., Leopold, A.C., Feigenson, W. and Goodsaid-Zalduondo, F. (1981) Membrane organization in soybean seeds during hydration. Planta 52, 19–23. Sinclair, T.R. and Ludlow, M.M. (1985) Who taught plants thermodynamics? The unfulfilled potential of plant water potential. Australian Journal of Plant Physiology 12, 213–217. Stadelmann, E.J. (1984) The derivation of the cell wall elasticity function from the cell turgor potential. Journal of Experimental Botany 35, 859–868. Stannett, V.T., Ranade, G.R. and Koros, W.J. (1982) Characterization of water vapor transport in glassy polyacrylonitrile by combined permeation and sorption techniques. Journal of Membrance Sciences 10, 219–233. Steudle, E., Zimmermann, U. and Luttge, U. (1977) Effect of turgor pressure and cell size on the wall elasticity of plant cells. Plant Physiology 59, 285–289. Sun, W.Q. (1997) Glassy state and seed storage stability: the WLF kinetics of seed viability loss at T > Tg and the plasticization effect of water on seed storage stability. Annals of Botany 79, 291–297. Sun, W.Q. (1998) Function of the glassy state in seed storage stability. In: Taylor, A.G. and Huang, X.L. (eds) Progress in Seed Research. New York State Agricultural Experiment Station, Cornell University, Geneva, New York, pp. 169–179. Sun, W.Q. (1999) State and phase transition behaviors of Quercus rubra seed axes and cotyledonary tissues: relevance to the desiccation sensitivity and cryopreservation of recalcitrant seeds. Cryobiology 38, 372–385. Sun, W.Q. (2000) Dielectric relaxation of water and water-plasticized biomolecules in relation to cellular water organization, cytoplasmic viscosity and desiccation tolerance in recalcitrant seed tissues. Plant Physiology 124, 1203–1215. Sun, W.Q. and Davidson, P. (1998) Protein stability in the amorphous carbohydrate matrix: relevance to anhydrobiosis. Biochimica et Biophysica Acta 1425, 245–254. Sun, W.Q. and Gouk, S.S. (1999) Preferred parameters and methods for studying moisture content of recalcitrant seeds. In: Marzalina, M., Khoo, K.C., Jayanthi, N., Tsan, F.Y. and Krishnapillay, T.M. (eds) Recalcitrant Seeds: Proceedings of the IUFRO Seed Symposium. Forest Research Institute of Malaysia, Kuala Lumpur, pp. 403–430. Sun, W.Q., Irving, T.C. and Leopold, A.C. (1994) The role of sugar, vitrification and membrane phase transition in seed desiccation tolerance. Physiologia Plantarum 90, 621–628. Sun, W.Q., Koh, D.C.Y. and Ong, C.M. (1997) Correlation of modified water sorption properties with the decline of storage stability of osmotically-primed seeds of Vigna radiata (L.) Wikzek. Seed Science Research 7, 391–397. Thomson, W.W. and Platt, K.A. (1997) Conservation of cell order in desiccation mesophyll of Selaginella lepidophylla ([Hook and Grev.] Spring). Annals of Botany 79, 439–447. Tompsett, P.B. and Pritchard, H.W. (1998) The effect of chilling and moisture status on the germination, desiccation tolerance and longevity of Aesculus hippocastarum L. seeds. Annals of Botany 82, 249–261. Trantham, E.C., Rorschach, H.E., Clegg, J.S., Hazlewood, C.F., Nicklow, R.M. and Wakabayashi, N. (1984) The diffusive properties of water in Artemia cells determined by quasi-electron neutron scattering. Biophysical Journal 45, 927–938.


18/3/02

1:54 pm

Page 83


83

Vertucci, C.W. (1990) Calorimetric studies of the state of water in seed tissues. Biophysical Journal 58, 1463–1471. Vertucci, C.W. and Leopold, A.C. (1986) Physiological activities associated with hydration level in seeds. In: Leopold, A.C. (ed.) Membranes, Metabolism and Dry Organisms. Cornell University Press, Ithaca, New York, pp. 35–49. Vertucci, C.W. and Leopold, A.C. (1987a) Water binding in legume seeds. Plant Physiology 85, 224–231. Vertucci, C.W. and Leopold, A.C. (1987b) The relationship between water binding and desiccation tolerance in tissues. Plant Physiology 85, 232–238. Vertucci, C.W. and Roos, E.E. (1993) Theoretical basis of protocols for seed storage. II. The influence of temperature on optimal moisture levels. Seed Science Research 3, 201–213. Vertucci, C.W., Crane, J., Porter, R.A. and Oelke, E.A. (1994) Physical properties of water in Zizania embryos in relation to maturity status, water content and temperature. Seed Science Research 4, 211–224. Vertucci, C.W., Crane, J., Porter, R.A. and Oelke, E.A. (1995) Survival of Zizania embryos in relation to water content, temperature and maturity status. Seed Science Research 5, 31–40. Vicre, M., Sherwin, H.W., Driouich, A., Jaffer, M.A. and Farrant, J.M. (1999) Cell wall characteristics and structure of hydrated and dry leaves of the resurrection plant Craterostigma wilmsii, a microscopical study. Journal of Plant Physiology 155, 719–726. Walters, C. (1998a) Understanding the mechanisms and kinetics of seed aging. Seed Science Research 8, 223–244. Walters, C. (1998b) Water activity, bad habits die hard: a response. CryoLetters 19, 265–266. Williams, R.J. and Leopold, A.C. (1989) The vitreous state in maize embyos. Plant Physiology 89, 977–981. Wolfe, J. and Leopold, A.C. (1986) A spectrum of desiccation. In: Leopold, A.C. (ed.) Membranes, Metabolism and Dry Organisms. Cornell University Press, Ithaca, New York, p. 1. Zimm, B.H. and Lundberg, J.L. (1956) Sorption of vapors by high polymers. Journal of Physical Chemistry 60, 425–428. Zimmermann, J.R. and Brittin, W.E. (1957) Nuclear magnetic resonance studies in multiple phase systems: lifetime of a water molecule in an absorbency phase on silica gel. Journal of Physical Chemistry 61, 1328–1333.


18/3/02

1:54 pm

Page 84

84

W.Q. Sun

Appendix: Solutions for Controlled Dehydration and Rehydration Saturated salt solutions In a closed container, a saturated salt solution (with excess salt present) produces a constant water vapour pressure at a given temperature. Relative humidity (RH) in the container is calculated by: RH A exp(B/T)

(1)

where A and B are constants, and T is the temperature in kelvin (Wexler, 1997). The values of A and B as well as the valid temperature range are given in Table A2.1 for various salts. For example, RH of saturated KCl solution at 25°C is equal to 49.38 exp(159/298) = 84.2 (%). Calculated values

are generally accurate within 2%. RHs of commonly used salt solutions between 5 and 40°C are compiled in Table A2.2. RHs of additional salt solutions at 25°C are given in Table A2.3. These data are taken from earlier works of Rockland (1960), Winston and Bates (1960) and Young (1967), tested and corrected by the author. Users are advised to avoid salts that release salt vapours into the atmosphere. When autoclaving is required, the decomposition temperature of a salt should be checked. After autoclaving, the closed container should be allowed sufficient time to equilibrate (i.e. avoiding condensation and supersaturation). The supersaturation in the liquid phase and the condensation of water on the wall in the container affect RH significantly.

Table A2.1. Commonly used salts, their vapour pressure constants A and B, and the valid temperature range. Data are taken from Wexler (1997). Compound NaOH.H2O LiBr.2H2O ZnBr2.2H2O KOH.2H2O LiCl.H2O CaBr2.6H2O LiI.3H2O CaCl2.6H2O MgCl2.6H2O NaI.2H2O Ca(NO3) 2.4H2O Mg(NO3) 2.6H22O NaBr.2H2O NH4NO3 KI SrCl2.6H2O NaNO3 NaCl NH4Cl KBr (NH4) 2SO4 KCl Sr(NO3) 2.4H2O BaCl2.2H2O CsI KNO3 K2SO4

Temperature range (°C)

Relative humidity at 25°C

15–60 10–30 5–30 5–30 20–65 11–22 15–65 15–25 5–45 5–45 10–30 5–35 0–35 10–40 5–30 5 –30 10–40 10–40 10–40 5–25 10–40 5–25 5–25 5–25 5–25 0–50 10–50

6 6 8 9 11 16 18 29 33 38 51 53 58 62 69 71 74 75 79 81 81 84 85 90 91 92 97

A 5.48 0.23 1.69 0.014 14.53 0.17 0.15 0.11 29.26 3.62 1.89 25.28 20.49 3.54 29.35 31.58 26.94 69.20 35.67 40.98 62.06 49.38 28.34 69.99 70.77 43.22 86.75

B 27 996 455 1924 75 1360 1424 1653 34 702 981 220 308 853 254 241 302 25 235 203 79 159 328 75 75 225 34


18/3/02

1:54 pm

Page 85


85

Table A2.2. Equilibrium relative humidities of saturated salt solutions at different temperatures. Data are taken from Rockland (1960), Winston and Bates (1960) and Young (1967). Temperature

Saturated salt solution

5°C

10°C

15°C

20°C

25°C

30°C

35°C

40°C

H2SO4 ZnCl2 NaOH LiBr KOH LiCl.H2O CaBr2 KAc MgBr2 MgCl2 CaCl2 K2CO3 NaI Zn(NO3) 2 KCNS Mg(NO3) 2 Na2Cr2O7.H2O NaBr Ca(NO3) 2 NaBr.2H2O CuCl2 LiAc NH4NO3 NaCl NaNO3 (NH4) 2SO4 NH4Cl Li2SO4 KBr KCl K2CrO4 BaCl2 ZnSO4 KNO3 K2SO4 Na2HPO4 Pb(NO3) 3

1.0 5.5 6.0 9.0 13.0 14.0 23.0 24.8 32.0 34.0 40.0 43.0 43.5 45.0 54.0 55.0 59.5 60.5 61.0 63.0 66.7 72.0 73.0 76.0 79.0 81.7 82.0 84.0 85.0 87.8 89.0 95.0 95.0 96.5 98.0 98.0 99.0

1.0 – – – – 13.5 – 24.0 31.0 33.5 38.0 47.0 – 43.0 52.0 53.0 60.0 58.0 66.0 61.0 68.0 72.0 75.0 75.8 77.5 81.2 79.0 84.0 86.0 86.7 89.0 93.0 93.0 95.5 97.0 98.0 98.5

1.0 5.5 6.0 8.0 9.0 13.0 20.0 23.5 31.5 33.5 35.0 44.0 38.0 40.7 50.0 53.7 56.5 58.0 58.0 59.0 68.0 71.0 70.0 75.5 76.5 80.0 79.5 84.0 85.0 86.0 88.0 92.0 92.0 95.0 97.0 98.0 98.5

1.0 – 6.0 – – 12.5 18.5 23.0 31.0 33.0 32.5 44.0 38.5 40.0 47.0 53.0 54.5 57.8 56.0 57.5 68.5 70.0 65.5 75.3 76.0 79.8 79.0 85.0 83.5 85.3 88.0 90.7 90.0 94.0 97.0 98.0 98.5

1.0 5.5 7.2 7.0 8.0 12.0 16.5 23.0 30.5 32.5 30.0 43.0 38.0 32.5 46.5 52.5 53.0 57.2 52.2 56.0 67.0 68.0 62.5 75.1 74.0 79.7 78.0 85.0 83.0 85.0 87.0 90.0 88.0 92.5 97.0 97.0 96.2

1.0 – – – – 11.5 – 23.0 30.0 32.0 – 42.0 36.0 24.0 43.5 52.0 52.5 57.0 51.0 54.5 67.0 66.0 59.5 75.0 72.5 79.5 77.5 85.0 82.0 83.5 86.0 89.0 86.0 91.5 97.0 96.0 95.5

1.0 5.5 7.5 7.0 8.0 11.5 15.0 22.0 30.0 32.5 30.0 41.5 34.0 21.0 41.5 50.5 51.0 57.0 45.5 53.0 67.0 65.0 56.8 75.0 71.0 79.2 75.5 85.0 81.0 83.0 84.0 88.0 85.0 89.5 96.0 93.0 95.2

1.0 – – – – 11.0 – 23.0 30.0 32.0 – 40.0 32.5 19.0 41.0 51.0 50.0 57.0 46.0 51.5 67.0 64.0 53.0 75.0 70.5 79.0 74.0 81.0 80.0 83.0 82.0 87.0 84.0 88.5 96.0 91.0 94.7

Non-saturated salt solutions Non-saturated salt solutions can also be used. This method allows for the creation of a precise and evenly graded series of RHs (or water potentials) with the same salt. The disadvantage of using non-saturated salt solutions is that they have limited buffering capacity relative to saturated salt solutions. RHs inside the container are not constant

during equilibration, because the equilibration between the tissue, vapour phase and solution phase results in a slight decrease or increase in salt concentration. This problem can be minimized using high mass ratios between the solution and the sample. A mass ratio of 150–200 (i.e. 150–200 g solution g1 tissue) is sufficient. Water potentials of NaCl solutions at different concentrations and temperatures between 5 and 40°C are


18/3/02

1:54 pm

Page 86

86

W.Q. Sun

Table A2.3. A list of saturated salt solutions (with the presence of excess salt) and their equilibrium relative humidities (RHs) at 25°C. Saturated salt solution

RH (%)

Saturated salt solution

RH (%)

ZnBr2 H3PO4 CaAc2.H2O + sucrose CaAc2.H2O Ca(CNS) 2.3H2O LiI.3H2O KHCO2 (Formate) KAc.1.5H2O NiBr2.3H2O MgBr2.6H2O Sr(CNS) 2.3H2O SrI2.6H2O MnBr2.6H2O Cu(NO3) 2.6H2O Ca(MnO4) 2.4H2O FeBr2.6H2O NaI.2H2O Mg(ClO4) 2.6H2O CoBr2 CrCl3 BaI2.2H2O K2CO3.2H2O CeCl3 LiNO3.3H2O Mg(CNS) 2 KNO2 K4P4O7.3H2O Co(NO3) 2.6H2O NH4NO3 + NaNO3 KBr + urea Zn(MnO4) 2.6H2O NiCl2.6H2O Na2Cr2O7.2H2O Ba(CNS) 2.2H2O Pb(NO3) 2 + NH4NO3 MnCl2.4H2O

8.6 9.0 13.0 17.0 17.5 18.0 21.5 22.2 27.0 31.5 31.5 33.0 34.5 35.0 37.5 39.0 39.2 41.0 41.5 42.5 43.0 43.0 45.5 47.0 47.5 48.1 49.5 49.8 50.0 51.0 51.0 53.0 53.7 54.5 55.0 56.0

SrBr2.6H2O FeCl2.4H2O NaMnO4.3H2O NH4NO3+ AgNO3 CuBr2 CoCl2 NaNO2 K2S2O3 CuCl2.2H2O NaCl + NaNO3 SrCl2.6H2O SrCl2 NH4Cl + KNO3 NaCl + KCl NaAc.3H2O NaCl + Na2SO4.7H2O BaBr2 K tartrate NH4Br2 Zn(CNS) 2 NaH2PO4 AgNO3 KCl + KClO3 KNa tartrate Na2CO3.10H2O MgSO4.7H2O BaCl2.2H2O Na tartrate (NH4)H2PO4 NH4HPO4 CaH4(PO4) 2.H2O KH2PO4 CaHPO4.2H2O CuSO4.5H2O K2Cr2O7 KClO3

58.5 60.0 61.5 61.5 62.5 64.0 64.2 66.0 67.7 69.0 71.0 71.0 71.2 71.5 73.0 74.0 74.5 75.0 75.0 80.5 81.0 82.0 85.0 87.0 87.0 89.0 90.3 92.0 92.7 93.0 96.0 96.0 97.0 97.2 98.0 98.0

given in Table A2.4. Besides NaCl solutions, CaCl2 and KCl solutions offer good RH control. Water potentials of CaCl2 and KCl solutions are listed in Table A2.5. Polyethylene glycol (PEG) solutions PEG solutions are widely used in controlled dehydration and rehydration. PEG solutions have several advantages over salt solutions. Salt solutions at high specific water poten-

tial or RH are often not available. PEG is inexpensive and not corrosive, whereas many salt solutions are corrosive and volatile. Effects of PEG concentration and temperature on water potential were studied by Michel and Kaufmann (1973). An empirical equation has been derived to calculate the water potential of PEG solutions at given concentrations and temperatures: (1.18 103C) – (1.18 105 C2) (2.67 105CT) (8.39 108C 2T) (2)


18/3/02

1:54 pm

Page 87


87

Table A2.4. Water potentials of sodium chloride (NaCl) solutions at different concentrations and temperatures. Mass (%); g solute per 100 g solution. Water potential (MPa)

Molality (mol kg1)

Mass (%)

5°C

10°C

15°C

20°C

25°C

30°C

35°C

40°C

0.05 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0 1.1 1.2 1.3 1.4 1.5 1.6 1.7 1.8 1.9 2.0

0.29 0.58 1.16 1.72 2.28 2.84 3.39 3.93 4.47 5.00 5.52 6.04 6.55 7.06 7.56 8.06 8.55 9.04 9.52 9.99 10.46

0.22 0.43 0.85 1.27 1.69 2.12 2.54 2.97 3.40 3.83 4.27 4.71 5.16 5.61 6.07 6.53 7.00 7.46 7.94 8.43 8.92

0.22 0.44 0.87 1.30 1.73 2.16 2.59 3.03 3.47 3.92 4.37 4.82 5.28 5.74 6.21 6.68 7.16 7.64 8.13 8.63 9.13

0.23 0.45 0.88 1.32 1.76 2.20 2.64 3.09 3.54 4.00 4.46 4.92 5.39 5.87 6.35 6.84 7.33 7.82 8.33 8.84 9.36

0.23 0.45 0.90 1.34 1.79 2.24 2.69 3.15 3.61 4.08 4.55 5.03 5.51 5.99 6.49 6.99 7.49 8.00 8.52 9.04 9.57

0.23 0.46 0.92 1.37 1.82 2.28 2.74 3.21 3.68 4.16 4.64 5.13 5.62 6.12 6.62 7.13 7.65 8.17 8.70 9.24 9.78

0.24 0.47 0.93 1.39 1.86 2.32 2.79 3.27 3.75 4.23 4.73 5.23 5.73 6.24 6.75 7.28 7.81 8.33 8.88 9.43 9.98

0.24 0.48 0.95 1.42 1.89 2.36 2.84 3.33 3.82 4.31 4.82 5.32 5.84 6.35 6.88 7.41 7.95 8.49 9.04 9.60 10.16

0.25 0.49 0.96 1.44 1.92 2.40 2.89 3.39 3.89 4.39 4.90 5.42 5.94 6.47 7.01 7.55 8.11 8.65 9.21 9.78 10.35

where C is the concentration of PEG (molecular weight 6000) in g kg1 water and T is temperature (°C). Water potential of PEG solutions is curvilinearly related to the concentration and increases linearly with temperature. The error of calculated water potential is generally within 0.03 MPa in comparison with the psychrometric measurements. Water potentials of PEG-6000 solutions at concentrations ranging from 10 to 400 g kg1 water and at temperatures between 5 and 40°C are given in Table A2.6. PEG solutions are very viscous, especially at high concentrations; hence it takes a longer time to achieve the equilibrium between the PEG solution and the tissue. Caution is needed to prevent bacterial and fungal contamination during the study. The equilibration can be accelerated by placing closed containers in a gently shaking incubator and in some cases submerging the tissue in solution. The change in PEG concentration after the experiment can be determined by the gravimetric method, and the equilibrium

water potential is calculated with Equation (2). The PEG solution can be dried at 105°C in an oven to a constant dry weight. The problem with submerging the tissue in a PEG solution is that PEG can enter the intercellular spaces. PEG is considered a nonpenetrating polymer because it does not cross the membrane! Our calorimetric study reveals a PEG melting peak in submerged tissues, even after extensive washing.

Glycerol solutions Glycerol can also be used to create a precise and evenly graded series of RHs between 30 and 98%. Glycerol solutions are safe to use and less corrosive (except for tin) than salt solutions. Microbial growth can be effectively inhibited by adding four drops of saturated CuSO4 solution to each 100 ml of glycerol solution (ASTM, 1983). The CuSO4-treated solution can be used repeatedly after the required


18/3/02

1:54 pm

88

Page 88

W.Q. Sun

Table A2.5. Water potentials of potassium chloride (KCl) and calcium chloride (CaCl2) solutions at 20 and 25°C. Mass (%): g solute per 100 g solution. KCl Mass (%) 0.5 1 2 3 4 5 6 7 8 9 10 12 14 16 18 20 22 24 26 28 30 32 36 40

CaCl2

20°Ca

25°Cb

20°Ca

25°Cb

0.28 0.56 1.12 1.68 2.26 2.83 3.42 4.02 4.64 5.25 5.87 7.18

0.31 0.61 1.22 1.85 2.48 3.13 3.80 4.48 5.18 5.90 6.65 8.21 9.89 11.68 13.62 15.69

0.27 0.54 1.07 1.62 2.22 2.87 3.58 4.36 5.23 6.15 7.16 9.40 12.00 14.99 18.45 22.34 26.50 30.89 36.26 42.37 50.06 60.68

0.28 0.57 1.16 1.81 2.50 3.24 4.02 4.89 5.77 6.73 7.76 10.09 12.85 16.13 20.02 24.60

20.34

36.20 51.67 71.78 97.33 129.12

a Derived from Handbook of Chemistry and Physics, 78th edn (Lide, 1997). Water potential is calculated according to the freezing-point depression. b Data were derived from Robinson and Stokes (1959).

concentration adjustment. The composition of glycerol solutions can be accurately determined using specific gravity or the refractive index. Equilibrium RHs of glycerol solutions at 24°C were reported by Braun and Braun (1958). Using the same set of data, Forney and Brandl (1992) derived an empirical equation to calculate equilibrium RHs of glycerol solutions. Equilibrium RHs and water potentials of glycerol solutions at 20°C were derived according to the freezing-point depression. These data are listed in Table A2.7. One can calculate the required concentration of a glycerol solution for a desired RH at a given temperature, using the ASTM’s standard recommended practice (ASTM, 1983).

The ASTM’s method uses the refractive index at 25°C to express glycerol concentration. The relationship between RH, refractive index and temperature is described by the following equations: (R0 A)2 (100 A)2 A2 (RH A)2

(3)

A 25.6 0.195T

0.0008T 2

(4)

R 1.3333 (1.398 103 R0)

(5)

where RH is the desired relative humidity (%), R is the refractive index of the glycerol solution, and T is temperature (°C). The value of A is calculated using Equation (4). Ro is calculated by substituting A and RH in Equation (3). The refractive index at 25°C is calculated using Equation (5). For a


18/3/02

1:54 pm

Page 89


glycerol solution of the known refractive index (R), equilibrium RH at different temperatures can be calculated by rearranging Equations (3), (4) and (5) (Sun and Gouk, 1999). The relationship is: RH =

(100 + A)2 + A2 − (R0 + A)2 − A

(6)

89

where A is defined by Equation (4), and Ro is calculated from Equation (5). The measurement of refractive index is quite tedious. The concentration (mass %) of the desired glycerol solution has been derived from concentrative properties of aqueous glycerol solutions (Lide, 1997) by the

Table A2.6. Water potentials of polyethylene glycol (MW 6000) solutions. Data were derived according to Michel and Kaufmann (1973). PEG (g kg1 H2O) 10 20 30 40 50 60 70 80 90 100 110 120 130 140 150 160 170 180 190 200 210 220 230 240 250 260 270 280 290 300 310 320 330 340 350 360 370 380 390 400

Water potential (MPa) at different temperatures 5°C

10°C

15°C

20°C

25°C

30°C

35°C

40°C

0.012 0.025 0.042 0.060 0.081 0.104 0.129 0.157 0.186 0.22 0.25 0.29 0.33 0.37 0.41 0.46 0.51 0.56 0.61 0.66 0.72 0.78 0.84 0.91 0.97 1.04 1.11 1.19 1.26 1.34 1.42 1.50 1.58 1.67 1.76 1.85 1.95 2.04 2.14 2.24

0.010 0.023 0.037 0.054 0.073 0.094 0.118 0.143 0.171 0.20 0.23 0.27 0.30 0.34 0.38 0.43 0.47 0.52 0.57 0.62 0.68 0.73 0.79 0.85 0.91 0.98 1.05 1.11 1.19 1.26 1.34 1.41 1.49 1.58 1.66 1.75 1.84 1.93 2.02 2.12

0.009 0.020 0.033 0.048 0.065 0.085 0.106 0.130 0.156 0.183 0.21 0.25 0.28 0.32 0.35 0.39 0.44 0.48 0.53 0.58 0.63 0.68 0.74 0.79 0.85 0.92 0.98 1.04 1.11 1.18 1.25 1.33 1.41 1.48 1.56 1.65 1.73 1.82 1.91 2.00

0.007 0.017 0.028 0.042 0.058 0.075 0.095 0.116 0.140 0.166 0.194 0.22 0.26 0.29 0.32 0.36 0.40 0.44 0.49 0.53 0.58 0.63 0.68 0.74 0.79 0.85 0.91 0.97 1.04 1.10 1.17 1.24 1.32 1.39 1.47 1.54 1.62 1.71 1.79 1.88

0.006 0.014 0.024 0.036 0.050 0.066 0.083 0.103 0.125 0.148 0.174 0.20 0.23 0.26 0.30 0.33 0.37 0.41 0.45 0.49 0.54 0.58 0.63 0.68 0.73 0.79 0.85 0.90 0.96 1.03 1.09 1.16 1.23 1.30 1.37 1.44 1.52 1.60 1.68 1.76

0.005 0.011 0.020 0.030 0.042 0.056 0.072 0.090 0.109 0.131 0.154 0.179 0.21 0.24 0.27 0.30 0.33 0.37 0.41 0.45 0.49 0.53 0.58 0.63 0.67 0.73 0.78 0.83 0.89 0.95 1.01 1.07 1.14 1.20 1.27 1.34 1.41 1.48 1.56 1.64

0.003 0.008 0.015 0.024 0.034 0.047 0.061 0.076 0.094 0.113 0.134 0.157 0.182 0.21 0.24 0.27 0.30 0.33 0.37 0.40 0.44 0.48 0.53 0.57 0.62 0.66 0.71 0.76 0.82 0.87 0.93 0.99 1.05 1.11 1.17 1.24 1.30 1.37 1.44 1.52

0.002 0.006 0.011 0.018 0.027 0.037 0.049 0.063 0.078 0.096 0.114 0.135 0.157 0.181 0.21 0.23 0.26 0.29 0.33 0.36 0.40 0.43 0.47 0.51 0.56 0.60 0.65 0.69 0.74 0.79 0.85 0.90 0.96 1.01 1.07 1.13 1.20 1.26 1.33 1.40


18/3/02

1:54 pm

90

Page 90

W.Q. Sun

Table A2.7. Equilibrium relative humidity (RH) and water potential of glycerol solutions at 20°C and 24°C. Mass (%): g glycerol per 100 g solution. 20°C

24°C

Mass (%)

Specific gravity a

% RH b

b (MPa)

% RH c

(MPa)

10 12 14 16 18 20 24 28 32 36 40 44 48 52 56 60 64 68 72 76 80 84 88

1.0215 1.0262 1.0311 1.0360 1.0409 1.0459 1.0561 1.0664 1.0770 1.0876 1.0984 1.1092 1.1200 1.1308 1.1419 1.1530 1.1643 1.1755 1.1866 1.1976 1.2085 1.2192 1.2299

97.9 97.4 96.9 96.4 95.8 95.2 93.9 92.4 90.7 88.9 86.9

2.79 3.45 4.16 4.89 5.69 6.52 8.35 10.42 12.71 15.28 18.19

98.0 97.5 97.0 96.5 95.9 95.4 94.1 92.6 91.0 89.2 87.2 84.9 82.5 79.7 76.6 73.2 69.4 65.2 60.6 55.5 49.8 43.6 36.7

2.77 3.47 4.18 4.89 5.74 6.46 8.34 10.55 12.94 15.68 18.79 22.46 26.39 31.13 36.57 42.78 50.11 58.68 68.71 80.77 95.64 113.88 137.51

a

Taken from Handbook of Chemistry and Physics, 78th edn (Lide, 1997). Calculated according to the freezing-point depression. c The relationship between concentration and equilibrium RH was reported by Braun and Braun (1958). The equation derived by Forney and Brandl (1992) was used to determine equilibrium RH of solutions at other concentrations. b

author. RHs of glycerol solutions at concentrations between 10 and 92% (mass %) and at temperatures between 5 and 35°C has been calculated according to the ASTM’s method (Table A2.8). The accuracy of

ASTM’s method is 0.2% at 25°C, and increases as temperature deviates from 25°C (ASTM, 1983). Data in Table A2.8 are consistent with those in Table A2.7, which were derived by different methods.


18/3/02

1:54 pm

Page 91


91

Table A2.8. Equilibrium relative humidity (RH) of glycerol solutions at different temperatures. Values were derived, using the refractive index of the solution at 25°C. Mass (%): g glycerol per 100 g solution. RH (%) at different temperatures Mass (%)

5°C

10°C

15°C

20°C

25°C

30°C

35°C

10 12 14 16 18 20 24 28 32 36 40 44 48 52 56 60 64 68 72 76 80 84 88

98.1 97.6 97.1 96.6 96.0 95.4 94.0 92.5 90.8 88.9 86.8 84.5 82.0 79.2 76.2 72.8 69.0 64.9 60.3 55.2 49.5 43.0 35.5

98.1 97.7 97.2 96.6 96.1 95.5 94.1 92.6 91.0 89.1 87.0 84.7 82.2 79.5 76.5 73.0 69.3 65.2 60.6 55.6 49.8 43.4 35.9

98.2 97.7 97.2 96.7 96.1 95.5 94.2 92.8 91.1 89.3 87.2 84.9 82.5 79.7 76.7 73.3 69.6 65.5 60.9 55.9 50.2 43.7 36.2

98.2 97.8 97.3 96.8 96.2 95.6 94.3 92.9 91.3 89.4 87.4 85.1 82.7 80.0 77.0 73.6 69.9 65.8 61.2 56.2 50.5 44.0 36.5

98.3 97.8 97.4 96.9 96.3 95.7 94.4 93.0 91.4 89.6 87.6 85.3 82.9 80.2 77.2 73.8 70.1 66.1 61.5 56.5 50.8 44.4 36.9

98.3 97.9 97.4 96.9 96.4 95.8 94.5 93.1 91.5 89.7 87.7 85.5 83.1 80.4 77.4 74.1 70.4 66.3 61.8 56.8 51.1 44.7 37.2

98.3 97.9 97.5 97.0 96.4 95.9 94.6 93.2 91.7 89.9 87.9 85.7 83.3 80.6 77.7 74.3 70.6 66.6 62.1 57.1 51.4 45.0 37.5

References ASTM (1983) Maintaining constant relative humidity by means of aqueous solutions. In: 1983 Annual Book of ASTM Standards (Standard E104). American Society for Testing and Materials, Philadelphia, pp. 572–575. Braun, J.V. and Braun, J.D. (1958) A simplified method of preparing solutions of glycerol and water for humidity control. Corrosion 14, 117–118. Forney, C.F. and Brandl, D.G. (1992) Control of humidity in small controlled environment chambers using glycerol–water solutions. HortTechnology 2, 52–54. Lide, D.R. (ed.) (1997) Handbook of Chemistry and Physics, 78th edn. CRC Press, New York, pp. 8, 57–81. Michel, B.E. and Kaufmann, M.R. (1973) The osmotic potential of polyethylene glycol 6000. Plant Physiology 51, 914–916. Robinson, R.A. and Stokes, R.H. (1959) Electrolyte Solutions, 2nd edn. Butterworths Scientific Publications, London, 559 pp. Rockland, L.B. (1960) Saturated salt solutions for static control of relative humidity between 5 and 40°C. Analytical Chemistry 32, 1375–1376. Sun, W.Q. and Gouk, S.S. (1999) Preferred parameters and methods for studying moisture content of recalcitrant seeds. In: Marzalina, M., Khoo, K.C., Jayanti, N., Tsan, F.Y. and Krishnapillay, B. (eds) Recalcitrant Seeds. Proceedings of IUFRO Seed Symposium 1998. Forest Research Institute Malaysia, Kuala Lumpur, pp. 404–430. Wexler, A. (1997) Constant humidity solutions. In: Lide, D.R. (ed.) Handbook of Chemistry and Physics, 78th edn. CRC Press, New York, pp. 15, 24–25. Winston, P.W. and Bates, D.H. (1960) Saturated solutions for the control of humidity in biological research. Ecology 41, 232–237. Young, J.F. (1967) Humidity control in the laboratory using salt solutions – a review. Journal of Applied Chemistry 17, 241–245.