Regulation of the membrane structure by brassinosteroids and progesterone in winter wheat seedlings exposed to low temperature

Highlights

• Brassinosteroids and progesterone alter structural properties of wheat membranes.

• Interaction of polar groups of steroids and lipids changes membrane flexibility.

• Steroid affinity to membranes depends on wheat cultivar/growth conditions.

• This is the first report describing brassinosteroid interaction with the lipid monolayer.

Abstract

Steroids constitute one of the most important groups of compounds of regulatory properties both in the animal and plant kingdom. In plants, steroids such as brassinosteroids or progesterone, by binding to protein receptors in cell membranes, regulate growth and initiate processes leading to increased tolerance to stress conditions. Due to their structural similarities to sterols, these steroids may also directly interact with cellular membranes. Our aim was to determine the changes of the structural parameters of lipid membranes under the influence of hydrophobic steroid compounds, i.e., 24-epibrassinolide (EBR) and its precursor—24-epicastasterone (ECS) and progesterone (PRO). Lipids were isolated from wheat seedlings with different tolerances to frost, grown at low temperatures (5 °C) for 1.5 and 3 weeks (acclimation process). Control plants were cultured continuously at 20 °C. From galactolipids and phospholipids, the main polar lipid fractions, the monolayers were formed, using a technique of Langmuir trough. EBR and ECS were introduced into monolayers, together with lipids, whereas the PRO was dissolved in the aqueous sub-phase upon which the monolayers were spread. Measurements performed at 25 °C and 10 °C showed a significant action of the tested compounds on the physicochemical properties of the monolayers. EBR and PRO increased the area per lipid molecule in monolayers, resulting in formation of more flexible surface structures while the presence of the ECS induced the opposite effect. The influence of the polarity of lipids and steroids on the interactions in the monolayer was discussed. Lipids extracted from the membranes of wheat with the most tolerance to frost were characterized by the highest fatty acid unsaturation and steroids had a relatively weak effect on the parameters of the structure of their monolayers.

Introduction

Changes of membrane structure during low temperature treatment are considered to belong to the first stages of the mechanism of plant acclimation to stress action [1], [2]. Modification of the polar part of lipids and differences in the proportions of unsaturated/saturated fatty acids in membranes were often taken as an indicator of cultivars’ tolerance to low temperatures [3], [4]. Phospho- and galacto-lipids determine the polar properties of lipid layers, and their arrangement leads to the formation of specific domains at the membrane surface [5], [6]. Cold-induced changes in the chemical composition of such domains may modify lipid interactions with polar and/or ionic substances (i.e., enzymes, hormones), enabling better adaptation of cells to stress conditions. The rise in the ratio of unsaturated/saturated fatty acids in the hydrophobic part of membranes observed at low temperatures is responsible for an increase in membrane fluidity and for the alteration of permeability determining the transport of chemicals to and from the cells [2], [7]. An important role in maintaining stable domain structures, especially in such flexible membranes, is played by sterol derivatives [8]. It is suggested that these substances are important for the formation of the liquid-ordered state of the membranes, crucial for the proper course of physiological processes [9]. An increase in the sterol content in the membranes of plants exposed to low temperatures was shown in many studies [8], [10], and it indicates the importance of these substances in mechanisms of cell adaptation to cold. However, variation in the composition of sterol derivatives synthesized in various plants led to the conclusion that even small changes in the chemical structure of sterols are important for the optimization of the genotype-specific physicochemical properties of membranes.

Considering the importance of the composition and structure of membranes in the processes of plant acclimation to stress, it can be assumed that it is possible to modify the membrane processes by exogenous application of sterol derivatives. Research that used brassinosteroids (BRs) in the form of leaves‘ spraying or by their uptake through the root system was carried out, providing unambiguous data on the accumulation of these substances in the cells [11], [12]. BRs are now an intensively studied group of plant steroid hormones whose involvement in the course of key physiological processes has been demonstrated in many studies [13], [14]. BRs are mainly engaged in plant growth processes, but their activity in alleviating the effects of abiotic stress has also been proven. Particularly interesting is the fact that BRs increase plant resistance to low temperature stress [15], [16]. According to Pociecha et al. [15], exogenous application of BRs increased the frost resistance in winter rye. Erenina et al. [16] described that Arabidopsis thaliana L. mutants with disturbances in BR biosynthesis were more susceptible to frost than the wild type. Mechanisms of these phenomena were connected to the positive effect of BRs on photosynthetic efficiency and sugar management as well as expression of COR (cold responsive) genes. Except for BRs also other steroids (like progesterone – PRO) can regulate growth and development or alleviate low temperature stress in plants [17], [18], [19]. PRO is a mammalian steroid hormone found also in plants [20], [21]. Little is known about its role in plants, and the mechanism of its action is barely known. As for thermal stress, studies by Genisel et al. [18] and Erdal & Genisel [19] showed that PRO induce cold resistance in maize and chickpea plants, amongst other things, through the regulation of the antioxidative system and the mitochondrial respiratory pathway. Both PRO and BRs may be engaged in the processes of plant acclimation to low temperature. However, the detailed mechanisms underlying this action are not fully explained. We postulate that cell membranes may be one of steroid targets in process of formation of cold/frost resistance. Although the protein membrane receptors responsible for the binding of BRs and PRO have been identified in plants [17], [21], [22], due to the similarity of the chemical structure of BRs (and PRO) to sterol components of membranes also their direct incorporation in the lipid membrane structure may be assumed. Under thermal stress associated with increased unsaturation of fatty acids, BRs would be placed in the hydrophobic part of the membranes in locations of their “loosened” integrity. An increased content of unsaturated fatty acids, especially the presence of those with two or three double bonds in the cis configuration, weakens the interactions between the hydrocarbon chains promoting formation of a less compact membrane structure, as compared to membrane composed of lipids with saturated fatty acid residues [23]. In addition, differences in the chemical structure of BRs, especially the position of hydrophilic substituents in the hydrophobic rings, show that the localization of BRs in the lipid structure may also depend on the type of the lipid polar head group.

To find-out the physicochemical parameters determining the embedding of BRs in the structure of lipid membranes, Langmuir monolayers technique was applied in the presented experiments. This technique is used for many years to study subtle changes in the physicochemical properties of monolayers, determined by differences in hydrophilic/hydrophobic constituents of lipids [24], [25]. The results obtained from measurements of the surface pressure at the interface between hydrophobic and hydrophilic phases, versus the area occupied by molecules that spontaneously form a monolayer in model systems, are a source of information on the properties and functioning of membranes. Such measurements are also useful for inference about the physiological processes in plant cells [6]. In the present study, monolayers were formed from lipids extracted from the membranes of wheat seedlings of different resistance to low temperatures. In earlier experiments, BRs were found in the cells of these wheat cultivars (authors unpublished data). As BRs of the potential ability to “integrate” with the lipid monolayers – 24 epibrassinolide (EBR) and 24-epicastasterone (ECS) – the precursor of EBR (Fig. 1 A and B) were selected. Moreover, as a representative of the polar compounds of the steroid structure – progesterone (PRO, Fig. 1C), – a mammalian hormone, whose presence in wheat cells has also been demonstrated [21], was used.