Effects Of Temperature On Plasma Membrane Stability

Cell membranes are essential for controlling the internal environment of the cell in particular in response to changes in water potential. Cell membranes are sensitive to temperature change; cold temperatures cause a phase shift from a fluid state to a gel whereas high temperatures cause increased fluidity which increases the risk of legions forming. Betacyanin, a red pigment stored within the vacuoles of Beetroot (Beta vulgaris) is released when membrane integrity is compromised. The degree of leakage into a bathing solution can give a relative measure of membrane stability. Summer and winter grown beetroot disks were suspended in distilled water over a range of temperatures. These were compared against beetroot in solutions of 0.05mM CaCl2 and 500mM sucrose (-192-40°C) and the absorption of the bathing fluid was assayed in a spectrophotometer (540nm) to assess the extent of membrane disruption. Significant disruption was found at temperatures <0°C and >30°C in both winter and summer beetroot. No significant difference in winter/summer (p>0.9) beetroot was found however, possible differences in optimum membrane stability (0-10°C and 10-30°C) were identified. No significant results were found in assays comparing sucrose or CaCl2 to water solutions. Research suggests that beetroot may be able to adjust their biochemistry to subtly change the temperature of optimum membrane stability and this may indicate an opportunity to selectively breed temperature resistant beetroot.

Introduction

Cell membranes are constructed from aliphatic phospholipids that separate the internal and external environment. They are maintain the differences between the extracellular and cytosolic environment whether through endocytosis or active transport of necessary molecules or through the expulsion and defence against harmful chemicals e.g. sodium. they are integral to the cell responding to the external environment and protecting valuable cell contents e.g. proteins.

At low temperatures the membrane goes through a phase change from a liquid (fluid) state to a two-dimensional rigid crystalline (gel) state. This causes the two layers of the plasma membrane to separate, this causes membrane leakage. Membranes with higher proportions of unsaturated phospholipids freeze at a lower temperature, so having an increased concentration of unsaturated fatty acids can improve frost tolerance in plants. Cis-doubled bonded fatty acids have kinked hydrophobic tails, this increases the distance between the phospholipids and reduces the interaction between the hydrocarbon talk domains. This in turn, makes the membrane more difficult to freeze. To prevent the membrane changing state a plant can produce enzymes that can alter the unsaturated phospholipid content of their membranes (Makarenko, 2007). An array of proteins, AFPs (Antifreeze Proteins), have been found to be upregulated/only produced in plants exposed to cold (Guy, 1990). Although these proteins remain mostly unidentified it is thought they may prevent intracellular ice formation or act as protein stabilisers. These proteins are sometimes associated with drought inducible genes e.g. ones associated with abscisic acid.

Excess heat causes plasma membranes to become increasingly fluid increasing the risk of forming lesions within the membrane . In addition high temperatures can also cause unfolding and denaturing of proteins within the cell. However, many plant cells produce protein chaperones or ‘heat-shock proteins’ which can re-fold proteins back into their original configuration at higher temperatures (Hendrick 1993). It is thought that heat-resistant plants may also use ‘stabilisers’ such as ions e.g. Ca+ to keep proteins in the correct arrangement. This is based on evidence that many thermophiles do not respond as well to their usual optimum temperature in vivo as opposed to in vitro. It has also been shown that enzymes within heat resistant plants have a higher optimum/Amax (Hayashi 2001)

Sucrose has been shown to increase stability of membranes when subjected to cold temperatures (Lineburger 1979) It has been suggested that this could be due to an increase in the concentration of harmful compounds/ions e.g. sodium ions as extracellular ice crystals can cause dehydration of the cell (Shinozaki 2000)(Lovelock 1953) or that it could be due to the sugar somehow blocks the active sites or other sensitive areas of the membrane (Santiamus 1971). Alternatively it could be due to the sugar causing a structural change in the membrane’s configuration (Williams 1970).

Beta vulgaris (Beetroot) is a root vegetable with a distinct red colouring conferred by the compound betacyanin, a glycoside, which is stored within the cell vacuole. When the membranes of the cell are disrupted the betacyanin leaks into the surrounding fluid. The amount of betacyanin released into the bathing solution, which can then be assayed using a spectrophotometer indicates the degree of membrane damage the cells have sustained.

Methods

Beetroot (Beta vulgaris) disks were suspended in solution then placed within a water bath. After 20 minutes the absorbance of the solution was assayed with a spectrophotometer at 540nm. This procedure was performed on both summer and winter grown beet suspended in distilled water to give their absorbance. Using the same procedure, summer grown beet disks were also suspended in either a 0.5mM CaCl2 solution or 500mM glucose solution at -192, 0 and 40°C. However, during the -192°C conditions the procedure differed in that beet disks were frozen by liquid nitrogen then ground up and defrosted in 10ml of water before absorption was read. Each condition was repeated three times and the mean was found of the results. The results of the summer and winter beetroot were compared using a homoscedastic t-test. The results of the CaCl2 and sucrose conditions were also compared against the original summer beetroot suspended in distilled water with a homoscedastic t-test to identify significance. The results were plotted on two scatter graphs (fig.1-2) with polynomial (x2) trend line.

Results

Both summer and winter grown beetroot showed an initial rise in membrane stability between -10°C and (from 0.85-0.59 and 0.59-0.21) and then membrane stability fell in both conditions between 20-40°C . The trend lines in Fig1. show that summer beet membrane was most stable between 10-30°C whereas winter beet was most stable at a lower 0-10°C. After these more stable temperature ranges the membrane stability suffered significantly, for example in winter beet the difference of the mean of absorbance readings at 10,20 and 30°C differ only by 0.09 whereas the difference between 30 and 55°C is 0.63. A T-test showed no significant difference between the winter and summer grown beetroot (p>0.9). A homoscedastic T-test was used as the variance of the data within the two populations was the same at 0.09.

Figure 1: A comparison of membrane stability of winter and summer grown beetroot (Beta vulgaris) membrane between -10 and 55°C. Beetroot disks (4 per assay) were suspended in distilled water for 20 minutes at the specified temperature, the solution of the surrounding solution once the disks were discarded, was measured for absorbance. Greater absorbance suggests greater amount of betacyanin leakage and therefore greater membrane instability. Each point represents the mean of 3 measurements +/- S.D. Homoscedastic T-test indicated that the absorbance was not significantly different between the two samples (p>0.9).

Figure 2 A comparison of summer grown beet (Beta vulgaris) responses to temperature while suspended in distilled water, 0.5mM CaCl2 or 500mM sucrose solution. Each point represents the mean of 3 results. Points excluded from the graph include the values for -196°C where the absorbance was 1.99 for both sucrose and CaCl2 condition suggesting a total loss of membrane integrity. T-test (including results not shown) indicated there was no significant difference between the 3 conditions. (sucrose- p>0.80 and CaCl2 – p>0.88)

Both CaCl2 and Sucrose showed at -196°C to have maximum absorbance (1.99) this suggests that freeze-thawing caused a significant loss of membrane integrity allowing a large amount of betacyanin to be released into solution. The graph suggests that beetroot disks treated with 500mM sucrose retain a greater level of membrane integrity at lower temperatures (0.39 at 0°C as opposed to 0.58/0.59 for CaCl2 and water respectively ).

Discussion

The comparison of summer and winter grown sugar beet membrane stability (results shown in fig.1) show a non-statistically significant difference in membrane response to temperature however, the T-test assumes each point to be independent however, the parabolic structure in Fig 1. suggests interdependence therefore, the values returned from the T-test may not completely reflect the underlying phenomena- particularly if that phenomena were to be subtle. Similarly, error bars in fig. 1 assume that the distribution exhibits equal variance across however, this may not be the case, for example at high/low temperature the variance may be more erratic – except for perhaps at a critical phase change point for beetroot where the variance would be very low.

The trend line suggests different optimum temperature for each of the membranes (winter: 3.3°C and summer: 20°C). The mean soil (30cm deep) in the West Midlands (UK) during winter is 4-6°C and during summer is 14-17°C [1] . The beetroot grown in the winter could have acclimatised to the colder temperature and switched on/upregulated genes associated with cold response for example, genes for the production of fatty acid desaturases which could increase the fluidity of the membrane at these temperatures. Alternatively or perhaps in conjunction with constitutively producing AFPs which could explain the difference between summer and winter grown beetroot within the range of 0-10°C. A greater number of repeats and the inclusion of intermediary temperatures would be able to clarify whether there is indeed a subtle difference in the membrane response dependent on growing conditions. To show this perhaps a microarray could be used to identify protein production differences in summer and winter grown beetroot.

Similarly, the data within the fig 2. is not statistically significant, however, this is not surprising given the insufficient number of intermediary temperatures. The error bars of the summer grown beet are reasonably large and so encompass both the CaCl2 and sucrose conditions. This could be clarified with further trials. Both the CaCl2 and sucrose condition showed maximum absorption when freeze thawed at -196°C suggesting that at such temperatures neither solution can keep the membrane stable.

This research suggests that beetroot should not be grown until <0°C temperatures for the year have passed as the beetroot assayed showed a significant increase in the amount of betacyanin released than at temperatures between 10-20 °C. It also suggests that Beetroot can potentially adapt to a degree to slightly lower mean temperatures. Breeding programmes that highlight the qualities of the winter grown beetroot might help in lowering the membrane phase shift point and therefore increase yield in locations where it cannot currently grow well.