Membrane Phospholipid Phase Separations in Plants Adapted to Or
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Plant Physiol. (1980) 66, 238-241 0032-0889/80/66/0238/04/$00.50/0 Membrane Phospholipid Phase Separations in Plants Adapted to or Acclimated to Different Thermal Regimes1 Received for publication July 16, 1979 and in revised form March 10, 1980 CARL S. PIKE2 AND JOSEPH A. BERRY4 Department ofPlant Biology, Carnegie Institution of Washington, 290 Panama Street, Stanford, California 94305 ABSTRACT The importance of membrane lipids in determining chilling sen- sitivity has recently been questioned (2, 5, 6, 24). No single The phase separation temperatures of total leaf phospholipids from hypothesis is presently able to accommodate these observations, warm and cool climate plants were determined in order to explore the and it seems likely that many factors may distinguish plants that relationship of lipid physical properties to a species' thermal habitat. The have evolved in hot and cool environments. However, these separation temperatures were determined by measuring the fluorescence observations do not necessarily contradict the view that membrane intensity and fluorescence polarization of liposomes labeled with the lipids are involved as one component in this evolutionary process. polyene fatty acid probe trans-parinaric acid. To focus on a single climatic Our approach has been to investigate the thermotropic behavior region, Mojave Desert dicots (chiefly ephemeral annuals) were examined, of lipids from native species which have known ecological pref- with plants grown under identical conditions whenever possible. Winter erence for warm or cool growth conditions. We assume that the active species showed lower phase separation temperatures than the sum- thermal responses of these plants would reflect the constraints of mer active species. A group of warm climate annual grasses showed natural selection operating in their native habitats. If the lipid separuition temperatures distinctly higher than those of a group of cool phase separation temperature is important, natural selection will climate grasses, all grown from seed under the same conditions. Growth at have operated to favor plants with membrane lipid properties that low temperature seems correlated with (and may require) a low phase are appropriate to the thermal regime experienced during its separation temperature. Winter active ephemerals appear genetically pro- growing season. grammed to synthesize a mixture of phospholipids which will not phase The plants selected for this study are, with a few exceptions, separate in the usual growth conditions. When the lipids of desert peren- from the native flora of the Mojave and Sonoran Deserts of North nials were examined in cool and warm seasons, there was a pronounced America. Ephemeral species that are known to grow principally seasonal shift in the phase separation temperature, implying environmental during the summer or winter seasons and some perennials that influences on lipid physical properties. The relationship of these results to are active throughout the year were examined. For the most part high and low temperature tolerance is discussed. the ephemeral species were grown at a common growth tempera- ture in controlled growth facilities. However, the perennial species were sampled during midwinter and in early summer from natural plants growing in Death Valley, California. The mean daily maximum and minimum temperatures in Death Valley for Janu- ary and July are 18 C/3 C and 45 C/32 C, respectively; these temperatures indicate the different thermal regimes experienced The physical state of membrane lipids may bear a significant by plants growing in the winter or summer. relationship to the lower temperature limit for a species' growth We used the fluorescent polyene fatty acid, trans-parinaric acid or survival. In various crop plants there is a correlation between (22, 23), as a probe for determining the phase boundaries of the occurrence of a lipid phase separation at about 10 C, an abrupt liposomes ofmembrane phospholipids extracted from these plants. increase in the activation energy of various membrane-bound Unfortunately, it was not possible to use trans-parinaric acid with reactions at this temperature, and the occurrence of metabolic native membranes of leaf cells or with total lipid preparations dysfunction at temperatures below this point (7, 15, 16). because pigments of the leaf quench the probe's fluorescence. In A change in the temperature dependence of spin label motion interpreting these studies, we assume that differences in the phys- in mung bean chloroplasts and mitochondria, an indication of a ical properties of isolated phospholipids should provide a good lipid phase separation, corresponded with the lower temperature relative index of differences in the properties of plant membranes limit of etiolated seedling growth (18). In a group of Passif7ora in vivo. Inasmuch as the phospholipids as a group contain a lower species, a high phase separation temperature was associated with proportion ofunsaturated fatty acids than the galacto- or sulfolipid greater chilling sensitivity; a lower separation temperature, with components of the membrane, it seems likely that the phospholip- less sensitivity (11). Habitat preference also correlated with the ids will have the highest phase separation temperature of the point of loss of membrane integrity assayed by ion leakage (10). membrane lipid mixture. In another study we found that the total membrane polar lipids (examined with spin label probes) and l Supported in part by the Science and Education Administration of the phospholipids (examined with trans-parinaric acid) from the same United States Department of Agriculture Grant 5901-0410-8-0128 from lipid preparations had very similar phase separation temperatures the Competitive Research Grants Office. CIW/DPB Publication No. 674. (17). 2 Permanent address: Department of Biology, Franklin and Marshall College, Lancaster, Pennsylvania. MATERIALS AND METHODS 'Supported in part by a National Science Foundation Science Faculty Professional Development Award and by a grant from the Mellon Foun- For plants grown from seed in the laboratory, controlled envi- dation fund of Franklin and Marshall College. ronment chambers were used to grow warm and cool climate 4To whom correspondence should be addressed. plants under the same conditions. For plants collected in Death 238 Plant Physiol. Vol. 66, 1980 PHASE CHANGES AND TEMPERATURE PREFERENCE 239 Valley, the mean daily maximum and minimum temperatures for Temperature, °C the month of collection are indicated. About 5 g of leaf blade was 50 40 30 20 10 0 -10 extracted for 5 min in 50 ml of boiling methanol containing I mg II II II I butylated hydroxytoluene. Then 100 ml ofchloroform was added, 0 and the tissue was ground in a VirTis The extract 0 homogenizer. 0 0 was filtered through Miracloth and partitioned four times with 0o 0.55 M KCI, once with water, and once with 60 mm KCI. After c 0 drying over anhydrous Na2SO4, the extract was concentrated in a rotary evaporator. The total lipid extract was applied to a Bio-Sil II) A (Bio-Rad Laboratories) column (<10 mg lipid/g Bio-Sil) and eluted with chloroform, acetone, and methanol (7 ml/g Bio-Sil). The phospholipid-rich methanol fraction was used in fluorescence studies. A- portion of this fraction was dried onto the walls of a glass vial under N2, held in a vacuum desiccator, and gently sonicated in 100 mm Tris-HCl (pH 7.2) containing 5 mm EDTA. The samples for fluorescence measurements contained 400 ,tg .- lipid and 0.7 ug trans-parinaric acid in 3 ml buffer containing 25 .-0 0 0 *S or 33% (v/v) ethylene glycol (a concentration without effect on -J S 0 . phase separation temperatures). Fluorescence was monitored in a 0 Perkin-Elmer MPF-3L spectrofluorometer. The excitation and emission monochromators were set at 320 and 420 nm, respec- 31 32 33 34 35 36 37 38 39 tively. The excitation beam was passed through a polarizing prism (Karl Lambrecht Corp.); the emitted light was passed through a I/T x 104 OK-' plastic polarizer (Edmund Scientific Co.), and a 350-nm cut-off FIG. 2. Trans-parinaric acid fluorescence polarization of corn and bar- filter. Temperatures in the sample cuvette (contained in a ther- ley phospholipid vesicles. Fluorescence emission was measured with the moregulated holder) were measured with a copper-constantan polarizer parallel (Ii) and perpendicular (IJ) to the orientation of the thermocouple. All temperature scans were made in the ascending excitation polarizer. The polarization ratio is l1/I±. (0), Zea mays; (0), direction. Fluorescence intensity was measured with the emission Hordeum vulgare. polarizer parallel (I 11 ) and perpendicular (1,) to the orientation of the excitation polarizer. Plots were made of log I 11 as a function of reciprocal absolute temperature and of the polarization ratio suggest the occurrence of a change in lipid fluidity. The low (I 11 /I,) as a function of temperature (23). Changes in the temper- polarization ratio above 10 C indicates a fluid probe environment ature dependence (slope) of these parameters were used to deter- (23). We interpret the slope change at 10 C as indicating the first mine the phase separation temperature. For a given sample the appearance of detectable solid as the temperature is lowered, since two methods usually agreed within 1 C of one another. There was trans-parinaric acid is sensitive to a few per cent solid (23). This a similar reproducibility for replicate experiments. interpretation is supported by experiments with model systems (23)