WATER EVACUATION FROM PEA SEEDS PREVIOUSLY TREATED WITH HGCL₂ AND SURFACTANTS (SDS, TRITON X-100)

Stefan Poliszko, Grażyna Plenzler, Danuta Klimek-Poliszko, Danuta Napierała
Department of Physics, University of Life Sciences in Poznań, Poland

ABSTRACT

The process of water desorption from a heterogenic system of seeds has been studied in the analyser of water diffusion and activity. The theoretical presentation of this process has been based on the differential equation describing molecular transport of water from the sample to the environment as a function of the activity of the diffusing water. The resolution of this equation has been expressed in the form describing changes in the water activity in time in the nearest environment of the studied system. The theoretical data were compared with experimental results in which the kinetics of water evacuation from sample to the surrounding atmosphere were established. The diffusion coefficient was determined on the basis of this comparison and the values of selected thermodynamic functions were estimated knowing the water evacuation at least at two temperatures. The interpretation of the obtained results should be made regarding the presence of aquaporins – the proteins occurring as tetramers in cell membranes and regulating the water transport into and from the cells. Their effect on the water transport was analysed indirectly by analysis of the influence of the membrane modification with selected surfactants and by blocking the aquaporins with HgCl₂. The presented studies have shown that in pea seeds treated with HgCl₂ the value of the activity diffusion coefficient decreases as compared with control probe, whereas it increases in pea seeds treated with surfactants (sodium dodecyl sulfate SDS as well as Triton X-100). The values of thermodynamic functions such as free energy ΔG, differential enthalpy ΔH, differential entropy ΔS, change as well. The obtained data can be interpreted as a result of the surfactant-induced changes of membrane permeability and blocking the aquaporins by HgCl_2. The theoretical description and the instrumental method presented in the present work can be applied to further investigation of cellular water transport.

Key words: water transport, aquaporin, diffusion, diffusion coefficient, surfactant, mercury inhibition.

INTRODUCTION

The cells’ membranes are the fundamental barriers for water transport in plants. Transmembrane water passage includes three routes for water movement: (i) simple diffusion, (ii) passive transport which accompanies the active co-transport of other solutes and (iii) water channels created by integral membrane proteins called aquaporins [5,7,10].

In plants, aquaporins are important both at the whole plant level, for the water transport to and from the vascular tissues, and at the cellular level, adjusting osmotic changes in the cytosol [5,10,16]. Studies on the integrated water transport from and into plant cells are frequently carried out in secreted experimental system e.g.: single cell [4,19], membrane vesicle [11], vacuole [2] for which the water conductivity parameters can be determined. The water uptake and removal in complex systems such as seeds are difficult both to observe and to describe. The complexity of the system can be restricted by the choice of proper conditions in which certain processes are excluded or diminished. In seeds the system complexity increases with the water content. When the seed water activity reaches the value of 0.75 (0.17 g H₂O·g^-1 dry weight), as in present research, the seeds are in the humidity state belonging to the region of transient humidity [18]. In those circumstances the strong water binding is saturated, the weak water binding increases and the multimolecular sorption begins.

The water diffusion into seeds and water evacuation from the seeds can be described generally by the diffusion coefficient D_a which expresses the diffusion water transport in relation to water activity [13]. The aquaporins included in water transport are blocked by mercurial compounds [9,19] binding to cysteine residues of the aquaporin proteins, thus closing the channels. For that reason the hypothesis that the aquaporins blockade might be reflected in changes of the diffusion equation coefficients seems to be reasonable. Hence in the present work a study of the changes of the water diffusion coefficients for the seeds previously subjected to HgCl₂ and other substances modifying the functions of the membrane has been undertaken.

MATERIAL AND METHODS

Seeds of pea (Pisum sativum cv. Sześciotygodniowy) were acquired from CNOS-VILMORIN Poland and were used in this study after pre-sorting by hand to obtain undamaged seeds of suitable dimension. The samples of the weight (2.355±0.005) g were put in a desiccator with saturated NaCl solution to obtain constant water humidity (75%). The hygrostat was kept in a thermostat at 22°C, the weight control was performed every day and the thermodynamic equilibrium was acknowledged when three successive weight measurements differed from each other by less than 0.002 g. After the state was reached, the samples were shaken for 10 minutes in 50 ml of distilled water (control sample) or in 7.4 mM HgCl₂ water solution, 7.0 mM Triton X-100 water solution or in 7.4 mM SDS water solution and then again kept in a desiccator.

It was assumed that modification of the aquaporins function by mercury chloride could be estimated by analysis of the thermodynamic state of water in seeds and by the observation of the kinetics of diffusion controlled water evacuation process. The study was performed in the analyser of water diffusion and activity (ADA-7 produced by COBRABIB – Poland). Measurements of equilibrium water activity were made at temperature 15 and 25°C with automatic record of temporal run of water evacuation from pea seeds to a preliminary dried measurement chamber. In terms of the thermodynamic diffusion theory the diffusing substance transport is described by the first Fick’s law [3]:

(1)

This equation states that J, i.e. the flux of particles (number of particles passing through a unit area of the normal surface per a unit time), is proportionally related to the force inducing the mass motion i.e. the activity gradient (). The proportionality constant D_a named the activity diffusion coefficient is expressed in kg·m^-1·s^-1.

In the presented conception the second Fick’s law is given by:

(2)

where:

is called the relative potential sorption capacity C signify the concentration of water in sample) and is determined from the experimental isotherm of water vapour sorption. In the molecular theory of mass transport through the desorbent surface, the effect of the surface resistance is also taken into consideration, and the appropriate term is:

(3)

where:
σ_a – surface conductance coefficient of the diffusing substance, kg·m^-2·s^-1,
a_g – the water activity in the atmosphere into which material of inner water activity a_c is introduced.

Both coefficients: D_a and σ_a are the fundamental sources of information about the transport effectiveness of the diffusing substance within the range of the neighbouring phase and on the interface. It appears that determination of those coefficients may give valuable data about the aquaporins activity inside and on the surface of the material investigated.

Resolution of the problem of water transport from the samples to the surrounding space in a measuring chamber of the instrument (ADA-7) in forced circulation conditions give the equation of activity changes kinetic [13]:

(4)

where:

a_og and a_oc – the initial water activity in the atmosphere and in the sample respectively.

The parameters: α, β in the above equation are expressed as:

where:
S – the surface of the sample,
V – the active chamber volume,
q_ag – hygroscopic capacity of the air in adequate temperature (density of saturated water vapour),
q_ac – hygroscopic capacity of the sample in the given water activity range,
σ_a – surface conductance coefficient of water,
D_ac – diffusion coefficient of water in the sample.

Computer fitting of the experimental curve of water activity kinetic increase in measurement chamber to eq. (4) allows estimating the diffusion and surface conductance coefficient.

The complete record of kinetic activity increase of water vapour in the measurement chamber of the diffusion analyser due to water evacuation from sample is shown in Fig 1. The course of the kinetic activity can be divided into three components: the first one is extended in time for ten to twenty seconds, since the connection of the sample chamber with the measurement chamber, which is preliminary dried to water activity of 0.2. In this fragment of the course no substantial change in water activity is observed, which may be interpreted as a diffusion delay of water vapour approaching of measurement sensor. The second component of the course extends in time for over 2 minutes and is characterised by the activity increase to the level which is reached in the sample chamber while waiting for the beginning of measurement. The last part of the kinetic course analysed corresponds to water vapour desorption from the sample investigated and is evidenced as the inflection point reached nearly 10 minutes after the beginning of the experiment. Equation (4) is related to the long-time fragment of the recorded kinetic curve and the kinetic parameters D_ac and σ_a of this equation are estimated for this part of experimental curve.

The differential enthalpy ΔH for pea seeds water evacuation was calculated from the Clausius-Clapeyron equation [18]:

(5)

where:
R – the ideal gas law constant,
a_w1 and a_w2 – the water activities at the absolute temperatures T₁ and T₂.

The free energy change ΔG and differential entropy changes ΔS for the pea seeds water evacuation at the absolute temperature T and water activity a_w were calculated as:

(6)

(7)

RESULTS AND DISCUSSION

Figs. 2-5 present the fragments of the desorption kinetics for the pea seeds treated with water (Fig. 2), mercury chloride (Fig. 3), Triton X-100 (Fig. 4) and sodium dodecyl sulfate SDS (Fig. 5). A very good conformity of experimental and theoretical courses with the correlation coefficient over 0.99 has been obtained in almost all cases. This is a base for calculation the all parameters of eq. 4, i.e. a_og, a_oc, α and β using a nonlinear least square curve fitting procedure. The definition of the α parameter, the value of sample surface S (the seeds surface) and active volume of the chamber V permits an estimation of the surface conductance coefficient of the diffusing substance σ_a, whereas the definition of the β parameter and the relative activity hygroscopic capacity of the system q_ac, calculated directly from the experimental sorption data permits an estimation of the diffusion coefficient D_ac of water in the sample. The obtained results are presented in Table 1.

As follows from Table 1 the treatment of pea seeds with HgCl₂ causes a decrease in the value of diffusion coefficient D_ac from 59·10^-10 kg·m^-1·s^-1 for intact seeds to 41·10^-10 kg·m^-1·s^-1, i.e. by approximately 30%. This result can be explained by the blocking of the water channel when the seeds are shaken with HgCl₂ water solution and followed by restriction of water diffusion from pea seeds interior to the free space of measurement chamber. The surfactants SDS and Triton X-100 cause the opposite effect and increase in the diffusion coefficient to a value of 170·10^-10 kg·m^-1·s^-1 and 98·10^-10 kg·m^-1·s^-1, respectively. The anionic alkyl sulfate – sodium dodecyl sulfate and nonionic ethoxylated alcohol Triton X-100 belong to the chemical surfactants of widespread use in biological laboratories and particularly in biomembrane studies. It is well known [6,12,14,17] that introduction of exogenous surfactant into cell results in a change in the physical properties of the lipid bilayer leading to a solubilization of the membrane. Besides, because the surfactant decreases the surface tension at the water-seed coat or seed interior -seed coat interface, the process of water uptake or water evacuation from seed can be modified .The increase in the lipid bilayer permeability can occur as a result of solubilization and in effect the water transport from seed to free space in the measurement chamber meets a smaller barrier and for this reason the diffusion coefficient increases (Table 1). In the case of ionic surfactant, the partitioning of the dodecyl sulfate anion (SD^-) into bilayer membrane is energetically favoured (exothermic partition enthalpy = -24 kJ·mol^-1 at 28°C whereas, for nonionic detergents the value is usually positive [15]. According to these data, Triton X-100 which has the greater effect on the membrane fluidity increase, can induces a greater increase of the diffusion coefficient in pea seed interior (Table 1).

The differential sorption heat ΔH for pea seeds water evacuation varied with temperature and the obtained values in analysed humidity range are comparable with those presented in other reports [1,8,18]. As a result of HgCl₂ treatment on the seeds, the ΔH value decreases, whereas the surfactant treatment increases that value as compared with control sample (Table 1). It should bee noticed that the lower values of ΔH correspond to stronger binding of water to seeds matrix. Free energy (ΔG) calculated from the isotherm data for the water evacuation process in seeds treated with HgCl₂ and with surfactants (SDS and Triton X-100) differs from each other insignificantly. The same tendency of qualitative changes occurs for differential entropy ΔS calculated from eq. 7.

CONCLUSIONS

From all substances studied that change the membrane function in pea seeds only the mercury chloride treatment decreases the activity diffusion coefficient as compared with the control sample. Thus it can be suggested that HgCl₂ limitation of water transport presumably accompanied with aquaporins blocking, may be monitored by the analysis of water evacuation from seeds when the analyser of water diffusion and activity is used. The theoretical description and the instrumental method employed here can be adapted to further studies of cellular water transport.

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Accepted for print: 8.05.2008

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