During their life cycle, plants are exposed to changing environmental conditions—biotic (pathogenic microorganisms) or abiotic (extreme temperature, drought, flooding, nutritional depletion, too low or too intense light, UV radiation, etc.). Among the abiotic stresses, temperature stress is a particularly serious problem for crop production. Some species are sensitive to cold (cucumber, maize). Frost, on the other hand, especially with an insufficient snow cover on the fields, can cause significant yield losses of winter crops including winter cereals. High-temperature stress is particularly dangerous for plants when combined with a water deficit. Gradually increasing temperature allows plants to acclimate and tolerate further increases in temperatures, which normally might be lethal. On the other hand, cold can acclimate plants to freezing temperatures and in the winter plants, this is known as the cold-hardening (cold acclimation) process. During acclimation, metabolic adjustments occur in plant cells that include, among others, changes in the hormonal homeostasis, an elevated biosynthesis of the proteins with chaperone properties (i.e., heat shock proteins) or enhancement of the antioxidant system. Crucial changes that stimulate the acquisition of frost tolerance influence the cell membranes, especially in cold acclimation. Since membranes are considered to be “thermal sensors” (according to the membrane sensor hypothesis [1
], they are thought to elicit other metabolic changes within a cell, including gene expression. In the lipid part of the cell membrane, acclimating to elevated temperatures or cold causes essential physicochemical adjustments, one of which is the modification of the fatty acid composition. Higher temperatures usually initiate a decrease in the level of unsaturated acids in the lipid part of a membrane [2
]. In cold, the composition of the fatty acids of the membrane lipids is expected to become more unsaturated [3
]. This unsaturation of the fatty acids causes a decrease in the phase transition temperature and increases the “fluidity” of the hydrophobic phase. The main fatty acids from groups 16C and 18C (especially 18:3 or 18:2), present in cells in the highest amounts, have a significant physiological role in plant response to abiotic but also biotic stresses [2
]. In winter plants, low temperature stimulates biosynthesis of 18:3 acid, resulting in an increase of unsaturation of membrane lipids thus better acclimation to unfavorable conditions of growth in winter [3
]. On the other hand, a lowered accumulation of 18:3 (and increased lipid saturation) is beneficial for instance for better thermostability of the photosynthetic apparatus at higher temperatures [2
]. Polyunsaturated fatty acids can also be released from membranes in response to an attack by biotic agents [5
]. Fatty acid 18:3 may directly activate NADPH-oxidase and generate reactive oxygen intermediates after inoculation with bacteria. On the other hand, lipid-derived metabolites produced by oxidation of fatty acids (18:3 or 18:2)—oxylipins like jasmonic acid—are an integral part of plant defense against pathogens.
Plant growth hormones/regulators such as brassinosteroids (BR) can improve the tolerance of plants to low- or high-temperature stress [8
]. BR are plant steroid hormones that have been extensively studied during the past three decades. These studies have revealed many of the mechanisms of their action in the process of growth and development stimulation as well as plant stress tolerance [9
]. However, little is known about the impact of BR on the plant membrane structure (lipid part) and the membrane physicochemical properties, which are, as was mentioned, important in the process of plants acclimating to various temperatures.
The effects of the exogenous administration of BR on the lipid FA composition in plants have been found in several previous studies [10
]. Further, Li et al. [12
] in studies using electron paramagnetic resonance observed an increase in the membrane fluidity in the presence of brassinolide in mango, which was an important step for developing an improved plant tolerance to low temperature. Our studies [14
] showed that the structural properties of the cell membranes were differentiated by the presence of brassinosteroids, and therefore, the role of BR in improving the tolerance of winter wheat to low temperatures was suggested. In Langmuir monolayer studies, 24-epibrassinolide and 24-epicastasterone were introduced into lipids that had been obtained from the aerial part of winter wheat seedlings that had been cultured at 5 °C or 20 °C. It was suggested that the tested BR (similar to sterols) entered the cell membrane directly and modified its properties by, for example, increasing the distance between the fatty acid chains, which might improve the functioning (flexibility) of the membrane in low temperatures [14
]. As 24-epicastasterone induced a slightly different effect than 24-epibrassinolide, these results also showed the importance of the chemical structure of BR for their interactions with membranes [14
The novelty of the presented studies, contrary to studies with the exogenous BR treatments, was the use of barley BR-deficient and BR-insensitive mutants to verify how disturbances in the BR biosynthesis (mutation in the HvCPD
gene) and the BR signaling (mutation in the HvBRI1
gene) in barley change the FA lipid composition and the physicochemical properties of the cell membranes. We characterized the natural lipid monolayers that had been obtained directly from these barley mutants (with a decreased or increased content of endogenous BR), which had been acclimated at 5 °C and 27 °C. It is worth mentioning that our earlier studies [16
] revealed that the mutants (after acclimation at 27 °C) had acquired a heat tolerance that was higher than the wild-type. After acclimation at 5 °C, both mutants had a lower frost tolerance. In our earlier articles, we also described changes in the protein component of the cell membranes (aquaporins, heat shock proteins) that might modify the mutants’ tolerance to high temperature or frost [17
]. In the current work, we focused on the lipid part of the cell membranes. The aim was to investigate the dependence between the FA composition of the individual lipid fractions and changes in the physicochemical properties of the membrane structure, which determines their permeability, stiffness/fluidity, and ability to be penetrated by various compounds including hormones. The characteristics of the physicochemical properties of membranes are increasingly used to explain the subtle changes in the structure of lipids that occur during the physiological processes (review Rudolphi-Skórska and Sieprawska [19
]). In the current work, this approach enabled (I) the dependence between an increased/decreased level of the endogenous BR in leaf tissue and structural-functional properties of membranes to be discussed and (II) the role of BR in the low/high-temperature tolerance mechanism that involves the modifications of the membranes to be deliberated.
Our work, which was devoted to mutants with the impaired biosynthesis and signaling of BR, permitted the results that have been obtained for fatty acids (especially 18:3) in the experiments in which BRs were applied exogenously to be partly confirmed [10
]. Both the BR-deficiency and BR-signaling disorders in the mutants were reflected in the modification of the fatty acid composition/proportion in the individual lipid fractions. Earlier, Janeczko et al. [10
] reported that the exogenous application of BR into a culture of oilseed rape calli increased the molar percentage of 18:3 in their PL fraction at 20 °C. In the current work, the mutants with the defects of BR biosynthesis and signaling growing at 20 °C had, as expected, a lower molar percentage of 18:3 in PL fraction. The same regularity was observed at 5 °C. The mutant BW084 had a lower molar percentage of 18:3 (in PL fraction), while BR-sprayed mango fruits, which subsequently were grown in the cold for 14–21 days, were characterized by an increased molar percentage of 18:3 in the polar lipid fraction [12
]. Thus, the effects of an increasing BR level in tissues through its exogenous administration were, as expected, exactly the opposite of the effects that were caused by the BR deficit in the mutant. However, the proper functioning of the BR receptors was also important because, despite the increased BR level that is characteristic for the mutant BW312 [16
], abnormal BR signaling often had a similar or the same effect as a BR deficiency. Although it appears that BR regulates the biosynthesis of the FA or their transport/incorporation into the cell membranes, this issue will require a more detailed investigation and explanation of the mechanism. The relationship between BR and FA was, of course, modified by the plant growth/acclimation temperature. Highly unambiguous results were obtained for the PL fraction (more characteristic for the plasma membrane) and 18:3 present within about 50% in the FA pool. Consistently, in both tested mutants, BR-biosynthesis or BR-signaling disorders were associated with a decrease in the content of this fatty acid in the PL fraction at 5 °C and 20 °C, but an increase in its content at 27 °C. It is assumed that for the better “adaptation” of membranes to temperature, the percentage of unsaturated FA should increase at 5 °C and decrease at 27 °C [26
]. This is considered to be one of the steps in plant acclimation. Therefore, in the case of the PL fraction, the analyzed mutants had little less favorable parameters than the WT. The opposite situation we had observed in the galactolipid fractions. At 5 °C, the mutants had a higher percentage of 18:3 than the WT, while at 27 °C, the mutants were characterized by a lower content of 18:3, especially in the case of MGDG. Since galactolipids are more typical for the chloroplast membranes and it is known that the MGDG fraction constitutes about 55% of the thylakoid membrane lipids [27
], this may partly explain why the mutants maintained better efficiency of photosystem II (PSII) at 5 °C and 27 °C when compared to the WT [16
]. Moreover, studies on Arabidopsis thaliana
mutants that carry mutations in the genes encoding the MGD-synthase also confirm the contribution of MGDG fraction to photosynthesis efficiency. These mutations led to a significant reduction in the ability of the mutant plants to conduct photosynthesis [28
]. In our experiment more 18:3 (more fluidic thylakoid membranes) at low temperature and less 18:3 (less fluidic thylakoid membranes) at a higher temperature may provide a better environment to membrane-located processes at these temperatures, and PSII is located in the thylakoid membranes. In fact, as mentioned in the introduction, the accumulation of 18:3 is beneficial for the thermostability of the photosynthetic apparatus at higher temperatures [2
]. Our earlier work [16
] showed that mutants at 5 °C and 27 °C were characterized by higher (than WT) values of P.I.ABS
informing about general PSII efficiency. In detail, mutants maintained comparable to WT plants energy transfer to electron transport chain (ETo/CSm) but it was accompanied by lower requirements of absorbed energy (ABS/CSm) and connected to lower energy loss as a heat (DIo/CSm).
According to a detailed analysis of the physicochemical properties of the lipid fractions, it seems that a greater amount of modifications between the tested plants were associated with the galactolipids rather than the PL fraction, and the MGDG fraction in particular. These results confirm the importance of the chloroplast structure in the plant response to the temperature changes. Thus, the physicochemical galactolipid modifications could be an important step, through FA composition changes, in the thylakoid membrane “adaptations” (stiffness/fluidity) that enable the proper functioning of photosystems at 5 °C and 27 °C. The value of the limiting area per molecule (Alim
) usually increases at 5 °C compared to 20 °C (which was also visible in the lipid fractions of genotypes analyzed in our study (Figure 3
A,D,G) and provides information about any increase in membrane fluidity [29
]. It is worth noting that, in the main lipid fraction of thylakoids (MGDG), the increase in Alim
—indicating the increase in membrane fluidity at 5 °C—was greater in the mutants than in the WT Bowman (Figure 3
A). The opposite effects, a decrease in Alim
and the fluidity of monolayers were caused by an elevated temperature up to 27 °C (Figure 3
A), and once again, the effect was stronger in the mutants than in the reference WT cultivar and concerned not only MGDG but the DGDG fraction as well. In both cases, 5 °C and 27 °C, the Alim
values corresponded well with a higher molar percentage of 18:3 in the MGDG fraction as well as a higher ratio of 18:3/18:2 and U/S. This correlation is consistent with expectations because linolenic acid, which contains three double bonds in the cis
configuration, has the greatest impact on increasing the distance between the lipid hydrocarbon chains [30
]. It is worth noting that in the MGDG fraction, present mainly in thylakoids, the higher values of membrane fluidity in the mutants at 5 °C and lower at 27 °C, compared to the WT Bowman, could be one of the reasons for the higher efficiency of photosystem II that was observed in the mutants [16
]. According to Escribá et al. [31
], even small changes in the lipid compositions can affect the physicochemical properties of the membrane, such as its fluidity and, as a result, affect the biochemical function of the signaling and transport proteins that are located in the membrane.
As mentioned, BR seems to be one of the hormones that regulate the biosynthesis of the main fatty acid—18:3 and/or its incorporation into the membranes. Such regulation may influence various physiological processes that are related to the membranes (such as the light reactions of photosynthesis), however, the question arises as to what significance this has for the frost tolerance or high-temperature tolerance of the whole plant that is acquired as a result of acclimation. Our work [16
], showed that despite the metabolic disorders that differentiated the mutants from the WT Bowman, the mutants (after acclimation at 27 °C) had a higher tolerance to temperatures around 40 °C than the WT. In contrast, their frost tolerance (measured after acclimation at 5 °C) was lower than in WT Bowman. In our other studies [17
], we were trying to explain this phenomenon by analyzing the changes in important membrane proteins. In the current study, we attempted to explain it by analyzing the physicochemical properties of the lipid membranes from plants that had been acclimated at 5 °C (thus hardened to frost) or acclimated at 27 °C (thus more tolerant to a much higher temperature).
The observed changes in membrane saturation, which were characterized by the ratio of the most common fatty acids that were present in the plant membranes, i.e., 18:3/18:2, were usually accompanied by changes in the physicochemical parameters in the model membrane system that had been obtained from the lipid fractions. The clearest correlation was observed in the monolayers of the lipids from plants acclimated at 5 °C. In the analyzed mutants, the parameters that were calculated for the monolayers showed that a higher unsaturation (18:3/18:2 and U/S, MGDG fraction, Figure 1
A,B) was associated with a higher value of Alim
—surface area per single lipid molecule (Figure 3
A)—thereby illustrating a higher degree of membrane fluidity. Lower unsaturation (18:3/18:2 and U/S, PL fraction, Figure 1
E,F) was associated with a decrease in the surface area per single lipid molecule (Alim
, Figure 3
G). While the lack of this regularity was observed in some cases, it can be explained by the fact that Alim
is also affected by other factors, such as the charges that are localized on the polar part of lipids. Moreover, as was mentioned above, an increase in the surface area per single lipid molecule (Alim
) usually means a higher degree of the fluidity of the monolayer while a decrease of Alim
is connected to a lower degree of fluidity. It is believed that higher membrane fluidity is more beneficial for better frost tolerance [32
]. As the analyzed mutants had a lower frost tolerance than the WT Bowman after acclimation at 5 °C [16
], the obtained results may at least partly explain the reason for this. Only the monolayers of MGDG had higher Alim
values, and consequently a higher degree of fluidity, whereas the DGDG or PL monolayers which mainly build the plasma membrane did not. Based on this model study, it can be suspected that the natural cell membranes of the mutants also have a lower degree of fluidity than the WT Bowman membranes at 5 °C and that this could be one of the factors that influence the higher frost susceptibility of the mutants in comparison with the WT Bowman [16
]. Interestingly, more membrane injuries (measured as electrolyte leakage) were reported by Qu et al. [34
] and Eremina et al. [35
] in the Arabidopsis
BR-signaling mutants that had been exposed to temperatures of 0 °C and below, which also confirms the connection of BR to the membrane “adaptation” to this stress.
Moreover, the mutants acclimated at 27 °C were less susceptible to heat stress (about 40 °C) than the WT [16
]. In both mutants, Alim
for the MGDG and DGDG monolayers reached a lower value, than in the WT Bowman, which indicates a lower degree of fluidity. This feature is more desirable as an “adaptation” to high-temperature stress. It is worth mentioning that the mutants had lower membrane injuries after high-temperature exposure (estimated based on electrolyte leakage) than the WT [16
]. To conclude, the changes leading to a lower degree of fluidity that were observed in the membranes of the analyzed mutants could be part of mechanisms that are associated with the improved tolerance of these mutants to the heat stress.
An analysis of the relationships between the other physicochemical parameters revealed that the pressure at which the monolayer collapses (πcoll
) and the compression modulus (Cs−1
) in mutants were also changed when compared to the WT Bowman and were dependent on the temperature of plant growth/acclimation (Figure 3
B,C,E,F,H,I and Figure S1A–I
). The values of these parameters provide additional information on the stability and flexibility of the monolayers as a result of the strength of the interactions that occur between the saturated and unsaturated FAs [29
]. The increase in the πcoll
value (the value of the surface pressure at which a layer collapses) may result from better geometric alignment of the lipid particles (usually for saturated acids), but it may also be modified by the electrostatic interactions between polar lipid parts. The value of this parameter was most often lower after acclimation at 5 °C and 27 °C for the mutant with BR-signaling disorders (for all of the fractions) and in the galactolipid fraction MGDG for the BR-biosynthesis mutant.
All of the changes that occur in the physicochemical and structural state of membranes (as a result of modification in the lipid composition for membrane acclimation to lower/higher temperatures) can also influence the possibility of the interactions and locations of various compounds (sterols, steroid hormones, etc.) in the membranes. The fact that the mutants, compared to the WT, had altered physicochemical and structural parameters characterizing the membranes shows how wide and multidirectional the impact of brassinosteroids can be on the membrane-dependent physiological processes.
Sometimes the directions of changes in the parameters studied were different in the mutant with BR deficit in comparison to a mutant with the BR-signaling disorder. A possible explanation can be that the BR-deficient mutant, however, produces low amounts of BR which still can interact with the BR receptor to induce a physiological responses to BR (i.e. connected to lipid biosynthesis). In the case of the mutant with BR-signaling disorder, despite BR overproduction (resulting from the feedback mechanism), signal perception is disturbed and the physiological response is also disturbed.