Changes of lipid composition and saturation level in leaves and roots for heat-stressed and heat-acclimated creeping bentgrass (Agrostis stolonifera)

Environmental and Experimental Botany 51 (2004) 57–67

Changes of lipid composition and saturation level in leaves and roots for heat-stressed and heat-acclimated creeping bentgrass (Agrostis stolonifera) Jane Larkindale1 , Bingru Huang∗ Department of Plant Biology and Pathology, Rutgers University, New Brunswick, NJ 08901, USA Received 23 January 2003; received in revised form 15 May 2003; accepted 19 May 2003

Abstract Changes in membrane lipid compositions and saturation levels during high temperature acclimation and heat stress were investigated in leaf and root tissues for three cultivars of creeping bentgrass (Agrostis stolonifera) that differ in heat tolerance. ‘L-93’ (heat tolerant), ‘Penncross’ (heat sensitive) and ‘Crenshaw’ (intermediate) showed differential decreases in grass visual quality (leaf color and shoot density), increases in TBARS (a measure of oxidative damage), increases in membrane leakage and decreases in chlorophyll content of leaves during 28 days of heat stress (35 ◦ C) in a growth chamber study. Total lipid extracts from the leaves of all three cultivars showed that the lipid saturation level increased over time under heat stress, with the change being predominantly due to decreases in linolenic acid and increases in linoleic and palmitic acids. The only significant difference in leaf lipid saturation between the three cultivars was that L-93 extracts contained a higher proportion of saturated lipids prior to heat stress than the other two cultivars. No cultivar differences in lipid composition were detected during heat stress. Penncross plants given a mild heat pre-treatment (30 ◦ C) (heat acclimated) prior to being exposed to heat stress (35 ◦ C) showed increased saturation of leaf lipids at the initiation of heat stress. There were no significant differences in root lipid composition between heat-acclimated and non-acclimated plants. Penncross plants which had acquired thermotolerance through heat acclimation, and heat tolerant L-93 plants showed similar leaf lipid composition at the initiation of heat stress, and similar levels of thermotolerance during heat stress. These results imply that there may be some connection between the degree of saturation of leaf membrane lipids prior to heat stress and the ability of that plant to limit heat-induced damages during the stress period. Root lipid extracts from the three cultivars showed no change in saturation levels during heat stress. However, Penncross root extracts contained a lower proportion of saturated lipids than L-93 or Crenshaw. These results suggest that lipid composition or saturation level of roots could be an important factor in controlling plant tolerance to heat stress. © 2003 Elsevier B.V. All rights reserved. Keywords: Creeping bentgrass; Heat tolerance; Leaves; Lipids; Roots

1. Introduction ∗ Corresponding author. Tel.: +1-732-932-9711; fax: +1-732-932-9441. E-mail address: [email protected] (B. Huang). 1 Present address: Department of Biochemistry and Molecular Biophysics, University of Arizona, Tucson, AZ 85721, USA.

High temperature is a major limiting factor in the growth of cool-season plants (Paulsen, 1994; Huang et al., 1998a,b). One of the major stress injuries in shoots or roots is the disruption of cellular membranes

0098-8472/$ – see front matter © 2003 Elsevier B.V. All rights reserved. doi:10.1016/S0098-8472(03)00060-1

58

J. Larkindale, B. Huang / Environmental and Experimental Botany 51 (2004) 57–67

(Blum and Ebercon, 1981; Paulsen, 1994; Marcum, 1998). This causes the break down of the compartmentalization of the plant cell, and therefore can severely affect cellular function. Under heat stress the levels of saturated fatty acids appear to increase (Horvath et al., 1998; Nishiyama et al., 1999), both in the thylakoid membranes (Vigh et al., 1989), and in the plasma membrane (Vigh et al., 1993). Which of these membranes is more significant in the acquisition of thermotolerance is still a matter of debate (Horvath et al., 1998). While lipid changes during high-temperature acclimation have been reported in a range of species (Vigh et al., 1989; Whitaker et al., 1997; Horvath et al., 1998; Nishiyama et al., 1999; Grover et al., 2000), the effects of such changes on thermotolerance requires further elucidation. Several studies have confirmed that changing the lipid saturation does not affect the photosynthetic rate during subsequent heat stress (Santarius and Muller, 1979; McCourt et al. 1987; Kunst et al., 1989a,b; Gombos et al., 1991). A small decrease in oxygen evolution has been observed in a cyanobacterium unable to produce dienoic fatty acids (16:2, 18:2), while elimination of trienoic fatty acids (16:3, 18:3) had no effect (Gombos et al., 1994). Tobacco (Nicotiana tobaccum L.) plants survived longer at high temperatures after genetic modification resulting in a decrease in the levels of unsaturated fatty acids (Grover et al., 2000), while heat-resistant wheat (Triticum aestivum L.) cultivars had significantly more saturated lipids membranes than sensitive cultivars (Yang et al., 1984). However, heat tolerant cultivars of potato (Solanum tuberosum L.) showed slightly higher linoleic and linolenic contents than controls (i.e. more unsaturated lipids), while the levels of oleic acid were lower in tolerant cultivars (Diepenbrock et al., 1989). In these plants, the heat tolerant cultivar showed increased palmitic and decreased oleic and linolenic after heat stress, while sensitive cultivars showed decreased oleic and linolenic acids (Diepenbrock et al., 1989). During low temperature acclimation, the levels of unsaturated fatty acids increase (Wang and Baker, 1979; Nishida and Murata, 1996). Research using mutant and transgenic plants with altered membrane lipid composition have shown that desaturation of specific membrane lipids results in increases in cold tolerance, while alterations of other lipid species does not affect the

overall tolerance of the plant (McCourt et al., 1987; Gombos et al., 1991; Vigh et al., 1993). An increase in linolenic acid (18:3) at the expense of linoleic acid (18:2) appears to have the greatest effect on cold tolerance (Cyril et al., 2002). These changes are postulated to decrease the gel–sol transition temperature, and therefore to allow the membranes to remain fluid at lower temperatures (Nishida and Murata, 1996). It is well established that pre-treatment of a plant at a moderate temperature (heat acclimation) results in increased tolerance to subsequent high temperature stress (Lindquist, 1980)—this phenomena is termed acquired thermotolerance. Traditionally, thermotolerance has been linked to the induction of specific sets of proteins, the heat shock proteins (Lindquist, 1980). Recent work, however, has shown that during the induction of thermotolerance there are changes in the antioxidant systems (Foyer et al., 1997; Shi et al., 2001) and alterations to carbohydrate and protein metabolism and soluble organic compounds such as proline accumulate (Howarth, 1991). In addition, the membrane lipid composition is altered in thermotolerant plant (Nishiyama et al., 1999). While previous research focused on membrane lipid response to stresses for the above-ground portion (mainly leaves), limited information is available on change in lipid composition or saturation levels in relation to heat tolerance, particularly in comparison with lipid properties in leaves. Roots are more sensitive to high temperatures than shoots (McMichael, 1998). Furthermore, changes in lipid composition or saturation levels in response to heat stress for cool-season perennial grasses, particularly comparing for cultivars differing in heat tolerance, are not well documented. It is hypothesized that leaves and roots of heat tolerant cultivars have higher level of lipid saturation than heat susceptible cultivars. The objectives of this study were to examine changes in membrane lipid composition and saturation levels during heat stress in both roots and leaves for three cultivars of a cool-season grass, creeping bentgrass, that differ in heat tolerance, and to compare lipid changes in roots and leaves in relation to heat tolerance. We compared the lipid compositions or saturation levels during heat stress for three cultivars differing in heat tolerance and for plants that had acquired thermotolerance through a prior heat treatment. We also compared the lipid changes in

J. Larkindale, B. Huang / Environmental and Experimental Botany 51 (2004) 57–67

roots to those in leaves of these plants (where the primary contribution to the lipid pool is from the thylakoid membranes), since the sensitivity of roots to temperature changes varies from that of leaves.

2. Materials and methods 2.1. Plant materials Plants of three creeping bentgrass cultivars, ‘Penncross’ (heat susceptible), ‘Crenshaw’ (intermediate heat tolerant), and ‘L-93’ (heat tolerant), were collected from a 2-year field plot at Horticultural farm II at Rutgers University, NJ and planted in 20-cm diameter plastic pots filled with a mixture of sand and organic matter (4:1 v/v). The plants were allowed to grow for 1 month in a greenhouse at 20/15 ◦ C (day/night). During this time the plants were cut once weekly, watered daily and fertilized weekly with half-strength Hoagland’s solution (Hoagland and Arnon, 1950). One week prior to the experiment all pots were transferred to growth chambers at 20/15 ◦ C (day/night), 600 ␮mol m−2 s−1 photosynthetically active radiation and a 13-h photoperiod. 2.2. Temperature treatments After 1 week, four pots of plants (20–30) for each treatment were then transferred directly to growth chambers set at a day/night temperature of 35/30 ◦ C (heat stress). A separate set of four pots of plants were allowed to acclimate to high temperature by moving plants into an identical growth chamber at 30 ◦ C for 48 h prior to the exposure to heat stress (35/30 ◦ C). Four replicate samples were left at 20/15 ◦ C as control plants. Each treatment was repeated in four separate growth chambers. For long-term physiological measurements, plants were left at 20/15 or 35/30 ◦ C for 1 month. Temperature treatments and cultivars were arranged as a completely randomized split-plot design with temperature as the main plot and cultivar as the sub-plot. Each treatment has four replications. The significance of treatment and cultivar effects was determined using the analysis of variance according to the general linear procedure of the Statistical Analysis System (SAS, Cary, NC) at the probability of

59

P = 0.05. Standard errors of means were calculated for four replicates per treatment or cultivar. 2.3. Physiological measurements Heat tolerance of the three cultivars were evaluated by measuring grass quality, animo acid leakage of leaf membranes, chlorophyll content, and the level of lipid peroxidation (assay of thiobarbituric acid reactive substances, TBARS). Measurements were taken at various time points after the initiation of heat stress. For each treatment four replicate pots of plants were used for measurements at each time point. Grass canopy quality is a subjective assessment to determine the overall health and vigor of the plant. It was determined based on a combination of factors, including color, uniformity and density of shoots and leaves. It is rated visually on a scale from 0 to 9, where a plant quality ranked as zero if it is dead and the quality is 9 if the plant is totally undamaged. When plants undergo heat stress, membranes become more fluid and may rupture, causing the leakage of cell contents out of the cell. In order to determine the extent to which this happens, we measured the levels of amino acids leaked from leaves. A 0.1 g of fresh leaf tissue was harvested and soaked for 6 h in 1 ml distilled water. The tissue was removed from the soaking solution, and the remaining solution was analyzed for amino acids using the ninhydrin method described by Rosen (1957). Absorbance readings were taken at 570 nm for all amino acids other than proline and hydroxyproline, which absorb at 440 nm (Rosen, 1957). The data shown is only the absorbance at 570 nm, but in all cases the trends were the same at 440 nm. The TBARS assay is frequently used to determine the level of oxidative damage done to plants under stress conditions (Heath and Packer, 1968). This was performed according to the method of Heath and Packer (1968) on 0.2 g of fresh leaf tissues from each treatment, harvested and immediately frozen in liquid nitrogen. Chlorophyll was extracted with dimethyl sulfoxide (DMSO). Fresh leaf tissues (0.1 g) was placed in a test tube containing 20 ml DMSO and left in the dark for 4 days (Hiscox and Israeltem, 1979). The absorbance of the resulting solution was measured at 663.8 and 646.8 nm, and the total chlorophyll was calculated as described by Arnon (1949).

60

J. Larkindale, B. Huang / Environmental and Experimental Botany 51 (2004) 57–67

2.4. Lipid extraction and analysis

3. Results

At various times after the initiation of heat stress, leaves were cut from the heated plants and immediately frozen in liquid nitrogen. At the same time points, roots were washed free of soil, blotted dry with paper towel and frozen in liquid nitrogen. All samples were stored at −80 ◦ C until analysis. Lipids were extracted from 0.1 g of fresh tissue and stored at 4 ◦ C until analysis by gas chromatography according to the methods of Cyril et al. (2002). The samples were quantified against a heptadecanoic acid (17:0) internal standard, and the amount of each lipid species in the sample could, therefore, be expressed both as an absolute amount in the 0.1 g samples, and as a percentage of the total lipid present in the sample. The double bond index was calculated using the following equation:

3.1. Physiological responses to heat stress

Index = [16 : 1] + [18 : 1] + 2([16 : 2] +[18 : 2]) + 3([18 : 3]) Square brackets indicate the percentage of the total lipid content which was made up by each lipid species.

All three cultivars exhibited a decrease in grass quality during heat stress (35 ◦ C) (Fig. 1A). After 2 weeks of heat stress, the quality of Penncross plants dropped below the acceptable level (6.0), and continued to fall rapidly throughout the rest of the stress treatment. Crenshaw plants showed quality ratings almost identical to those of Penncross plants during the early period of heating, but after 2 weeks at 35 ◦ C they declined more slowly. From 2 to 4 weeks the quality of this cultivar was significantly greater than that of Penncross. L-93 plants showed the smallest quality decline of the three cultivars. Although the quality of this cultivar was never significantly greater than that of Crenshaw, from 14 days onwards it was rated significantly higher than Penncross. The levels of TBARS increased during heat stress (Fig. 1B). There were peaks of increases in TBARS during the heat treatment: a small, transient increase at the initiation of heating, followed by a prolonged increase during prolonged heat stress. There were no significant differences in the levels of TBARS between

Fig. 1. Changes in physiological parameters during heat stress (35 ◦ C) for three cultivars of creeping bentgrass. (A) Grass quality, (B) TBARS, (C) amino acid leakage, (D) total chlorophyll content. Error bars represent the standard error of mean (n=4).

J. Larkindale, B. Huang / Environmental and Experimental Botany 51 (2004) 57–67

the three cultivars during the early peak. Following 3 weeks of heat stress, however, the levels of TBARS increased to a greater extent in Penncross plants than in L-93 or Crenshaw, and the increase occurred earlier. The levels of TBARS in Penncross were significantly greater (a 3-fold increase) than in the other two cultivars. Levels in L-93 and Crenshaw were similar throughout the heat treatment, up until 4 weeks of stress. At this point, Crenshaw showed significantly more (50% more) oxidative damage than L-93. The levels of animo acid leakage from leaves increased over heat stress in all three cultivars (Fig. 1C). During the first 2 weeks of stress, leakage increased significantly in both Crenshaw and Penncross, and to a lesser extent in L-93 plants. After 1 week of stress, the rate of increase of leakage in Crenshaw plants decreased. Penncross, by contrast, showed increases in leakage up to 2 weeks of heat stress (by which time the level of leakage in this cultivar was twice that of Crenshaw and six times that of L-93), and then remained constant. Throughout heat treatment L-93 plants showed relatively low levels of leakage, but the leakage at the end of 4 weeks in this cultivar was not significantly different from that of Crenshaw. The chlorophyll content of the leaves decreased over time at high temperature (Fig. 1D). The chlorophyll content of Penncross leaves decreased by 25% after a week of heat stress, and the level was close to zero after 2 weeks. By contrast, L-93 and Crenshaw leaves maintained higher chlorophyll content, which decreased by less than 50% during the 4 weeks of treatment. Crenshaw showed a greater rate of chlorophyll loss than L-93 during the first week of stress, but recovered to the same level as L-93 within the second week. 3.2. Changes in lipid saturation in roots and leaves during heat stress Fig. 2 shows the ‘double bond index’ of the lipid samples extracted from leaf and root tissues. This is a weighted percentage saturation in which the number of double bonds is accounted for (see Section 2). During heat stress there was an overall increase in lipid saturation in leaf tissue (Fig. 2A). That is, the number of double bonds decreased with the duration of heat stress in all three cultivars. The index decreased by 15–20% in the first 2 weeks and then remained constant in L-93 and Crenshaw leaves. In

61

Fig. 2. Changes in lipid saturation level in leaves (A) and roots (B) during heat stress (35 ◦ C) for three cultivars of creeping bentgrass, as expressed using the double bond index. Error bars represent the standard error of mean (n = 4).

Penncross, by contrast, the index dropped by 30% over 3 weeks, before becoming stable at a level lower than the other two cultivars. The same trends were visible when a un-weighted percentage saturation was plotted (data not shown). The saturated lipid content of leaves decreased from 80 to 70% of the total lipid during heat stress, with Penncross and Crenshaw leaves both showed a 10% higher double bond index than L-93 prior to heat treatment. In root tissue (Fig. 2B) there was no significant change in either the weighted or the un-weighted percentage saturation of lipids. Indeed, the only significant difference in saturation between samples in root tissue was between the cultivars: Penncross showed a significantly (15%) higher double bond index (i.e. lower level of saturation) than L-93 or Crenshaw throughout the experiment. It is also worthy of notice that the lipids extracted from leaves were significantly less saturated than those extracted from roots in all samples. Before heating, leaf lipid extracts contained about 80% unsaturated lipids, while extracts from roots contained about 60%

62

J. Larkindale, B. Huang / Environmental and Experimental Botany 51 (2004) 57–67

unsaturated lipids. This may be due to differences in saturation between thylakoid membranes (only present in leaf tissue) and other cell membranes, or due to differences in saturation in the same membranes from the different tissues. 3.3. Changes in lipid composition in leaves during heat stress Fig. 3 shows the specific changes in the lipid composition of leaves during heat stress. The bar charts (Fig. 3A–C) show the percentage of the total lipids which consisted of each lipid species at 0, 7, and 28 days of heat stress for the three cultivars. The linolenic acid (18:3) content of the extracts decreased over heat stress. This lipid constituted 60% of the total lipid in extracts from leaf tissue taken prior to heating, and decreased to only 40% after a week of heat stress. The loss of linolenic acid correlated with increases in both linoleic acid (18:2) and palmitic acid (16:0), and to some extent oleic acid (18:1). Fig. 3D–F show the rates of change for specific membrane lipids. Initially, the rate of increase of linoleic acid was greater than that of palmitic acid, and this mostly occurred within the first week of heating. The only significant

difference between the cultivars was that in the latter part of heating (greater than 2 weeks); Penncross showed a greater conversion to palmitic acid than the other two cultivars. 3.4. Changes in lipid composition in roots during heat stress In contrast to leaves, roots from the same heatstressed plants showed little or no change in lipid saturation over the heat treatment (Fig. 2B and Fig. 4). None of the lipids analyzed changed significantly during the heat treatment. Penncross roots, however, contained a higher proportion of linolenic acid (18:3) than roots of the other two cultivars: this was significant after 7 and 28 days of heat stress. There were no significant differences in the content of the other fatty acids between the cultivars. 3.5. Total lipid content of leaves and roots during heat stress Fig. 5 shows the total fatty acid content of the lipid extracts from both roots and leaves throughout heat stress. There were no significant decreases in the total

Fig. 3. Lipid composition of leaves for three cultivars of creeping bentgrass at 35 ◦ C. (A–C) Percentage of each lipid species at time = 0, 7, 28 days, respectively. (D–F) Changes in specific lipid species over time: linolenic, linoleic and palmitic acids, respectively. Error bars represent the standard error of mean (n = 4).

J. Larkindale, B. Huang / Environmental and Experimental Botany 51 (2004) 57–67

63

Fig. 4. Lipid composition of roots for three cultivars of creeping bentgrass at 35 ◦ C. (A–C) Percentage of each lipid species at time = 0, 7, 28 days, respectively. (D–F) Changes in specific lipid species over time (linolenic, linoleic and palmitic acids, respectively). Error bars represent the standard error of mean (n = 4).

lipid content during heat stress in any of the cultivars. The lipid contents of roots (Fig. 5B) were 5–6-fold lower than leaves (Fig. 5A). This is not surprising in that the lipid extracts used in this study were total lipid extracts, including the cell membrane and the membranes of all organelles. Chloroplast membranes make up a large proportion of the total membrane in leaf cells, which may be expected to account for this difference. There were no significant differences in the root lipid content between the three cultivars, but Penncross leaves showed a lower lipid content than those of L-93 or Crenshaw up until 21 days of heat stress.

reduced in thermotolerant plants (i.e. those which had acquired thermotolerance through pre-treatment at moderate temperature) (Fig. 6). Fig. 6A shows that the heat pre-treatment resulted in the maintenance of acceptable turf quality for 21 days, as compared with

3.6. Acquisition of thermotolerance through heat acclimation Fig. 6 shows the effect of a 3-day 30 ◦ C pre-treatment prior to exposing the plants to a 35 ◦ C treatment on the acquisition of thermotolerance in Penncross plants. As shown in Fig. 1, heat stress caused a decrease in turf quality over time, which was associated with a reduction in chlorophyll content, an increase in oxidative damage and increased membrane leakage. All of these negative effects of heat stress were

Fig. 5. Total lipid content in leaves (A) and roots (B) for three cultivars of creeping bentgrass at 35 ◦ C. Error bars represent the standard error of mean (n = 4).

64

J. Larkindale, B. Huang / Environmental and Experimental Botany 51 (2004) 57–67

Fig. 6. Physiological changes of heat-acclimated (30 ◦ C) Penncross plants during heat stress (35 ◦ C). (A) Turf quality, (B) TBARS, (C) amino acid leakage, (D) total chlorophyll content. Error bars represent the standard error of mean (n = 4).

14 days in controls. This was associated with the maintenance of higher chlorophyll contents in heat pre-treated plants than in controls (Fig. 6D) throughout the experiment (levels in the controls dropped by 50%). In addition, thermotolerant plants showed lower levels of oxidative damage (Fig. 6B) and membrane leakage (Fig. 6C) over the 4 weeks of heat stress.

of heat stress, the lipid saturation of control plants decreased to the same level as that of thermotolerant plants: there was no significant change in saturation in pre-treated plants during the heat-stress period. As was shown previously, this difference in saturation was primarily due to changes in the poly-unsaturated fatty acids (Fig. 8). Prior to the initiation of heat stress the control plants had higher levels of linolenic acid than

3.7. Effects of heat acclimation on levels of lipid saturation Prior to the initiation of heat stress the level of lipid saturation in the thermotolerant (i.e. pre-treated) plants was higher than in controls (Fig. 7). After the initiation

Fig. 7. Saturation level of membrane lipids for leaves from heat-acclimated Penncross plants, expressed as the double bond index. Error bars represent the standard error of mean (n = 4).

Fig. 8. Composition of membrane lipids for leaves from heatacclimated Penncross plants. (A) Composition at initiation of heating, (B) composition after 5 days at 35 ◦ C. Error bars represent the standard error of mean (n = 4).

J. Larkindale, B. Huang / Environmental and Experimental Botany 51 (2004) 57–67

pre-treated plants. During the early period of heating some of this linolenic acid appeared to be converted into linoleic and palmitic acids, resulting in a lipid composition similar to pre-treated plants after several days of heating.

4. Discussion Lipid compositions of membranes in plants and cyanobacteria change with either cold or heat stress (Vigh et al., 1989; Whitaker et al., 1997; Grover et al., 2000; Cyril et al., 2002). In particular, it has been postulated that one of the primary mechanisms of adaptation to cold stress is increased desaturation of membrane lipids, resulting in increased membrane fluidity (Vigh et al., 1989). These changes in composition are primarily changes from 18:3 (linolenic acid) to 18:2 (linoleic acid) (Cyril et al., 2002). The converse may be true at high temperature. There has been considerable debate in the literature as to whether changes in lipid composition during heat treatment are associated with thermotolerance (Somerville and Browse 1991 Kunst et al., 1989a,b; Gombos et al., 1991, 1994). In the present study, an increase in lipid saturation of leaves occurred in all three cultivars of creeping bentgrass during prolonged heat stress. This is primarily due to a decrease in linolenic acid (18:3) and an increase in linoleic and palmitic acids (18:2, 16:0). Similar saturation increases have been observed in other species (Vigh et al., 1989; Whitaker et al., 1997; Horvath et al., 1998; Nishiyama et al., 1999; Grover et al., 2000). The increase in saturation may cause changes in lipid fluidity: more highly unsaturated fatty acids are less rigidly packed into a membrane due to the non-linearity of the fatty acid chains introduced by the presence of double bonds (Cyril et al., 2002). Measurements of grass quality, chlorophyll content, animo acid leakage, and lipid peroxidation of cell membranes showed that L-93 was the most heat tolerant, Crenshaw was intermediate, and Penncross was the most sensitive among the three cultivars, which is in agreement with the results of previous studies (Huang et al., 1998a,b; Liu and Huang, 2000; Xu and Huang, 2001a,b). Prior to heat treatment L-93 leaves contained significantly more saturated fatty acids than the other two cultivars. Heat sensitive Penncross plants which had acquired thermotolerance

65

through heat acclimation also had membranes with a high concentration of saturated fatty acids prior to heat stress. Both L-93 and heat-acclimated Penncross plants had a double bond index just over 200, and both showed limited damage during heat stress. These results suggest that there may be some correlation between the composition of the plants’ membranes prior to heat stress and the tolerance of that plant. Heat pre-treatment or acclimation is known to induce a wide range of effects, and results in thermotolerance. Alteration of membrane lipids is only one of those effects (Lindquist, 1980; Howarth, 1991; Foyer et al., 1997; Shi et al., 2001). In order to determine if the effects seen here are actually directly related to membrane lipid composition, it would be necessary to alter just the lipid composition, without affecting the other thermoprotective systems. This was not done in this study, but indications from work in other species using mutants and transgenic plants with altered membrane lipid compositions suggest that increasing saturation results in enhanced thermotolerance (Grover et al., 2000), but does not protect photosynthesis from heat-induced damage (Kunst et al., 1989a,b; Gombos et al., 1991). It is worthy of comment that thermotolerance can be induced in creeping bentgrass using shorter periods of heating (12–24 h at 30 ◦ C), or with a number of chemical treatments (Larkindale and Huang, manuscript in preparation). None of these pre-treatments significantly affected the membrane lipid composition prior to heating (data not shown), thus thermotolerance can be induced without altering the membrane lipid composition. During heat stress, leaf membrane lipids showed increasing saturation, but there were no significant changes in the composition of the root membranes from the same plants. It is interesting to note that the membranes extracted from the plants in this study were the total membranes of that tissue: no distinction was made between the plasma membrane and those belonging to intracellular organelles. Clearly, leaf tissue is packed with chloroplasts, an organelle containing a lot of lipid material (Grover et al., 2000). Therefore, the total amount of lipid extracted from leaf tissue was much greater, and much of what was studied was from the photosynthetic membranes. Photosynthesis is very sensitive to heat stress—the rate of photosynthesis in most species declines at about 35 ◦ C (the temperature of this study), and this

66

J. Larkindale, B. Huang / Environmental and Experimental Botany 51 (2004) 57–67

decline has been ascribed to protein denaturation, loss of membrane integrity, photoinhibition and ion imbalance (Fergusson et al., 1993; Grover et al., 2000). Therefore, it is plausible that the processes required for the protection of leaves against heat stress are significantly different to those of roots. Indeed, many aspects of heat-induced damage have been shown to be light dependent, suggesting that they are associated with damage to the photosynthetic system (Larkindale and Knight, 2002). Many of the measured changes in lipid composition during heating have been done exclusively in the thylakoid membranes (Grover et al., 2000; Sharkey, 2000), and there is considerable evidence that changes in the composition of thylakoid membranes are more sensitive to heat tolerance than changes in the plasma membrane (Horvath et al., 1998). The data from this study suggests that there are changes in lipid composition in leaf extracts after heat stress, but no changes were observed in root extracts. This would support the hypothesis that the changes in saturation are predominantly occurring in the thylakoid membranes. It is not surprising that there were no changes in lipid composition in roots during heat stress. However, it is interesting to note that root lipids were significantly more saturated than those extracted from leaves. It would be interesting to determine whether this difference is due to the difference in the pool of membranes measured (i.e. plasma membrane, thylakoid membranes, mitochondrial membranes, etc.), or whether this is a difference between the same membranes (e.g. within the plasma membrane) in different tissues. Cultivar differences in root lipid saturation were detected: the double bond index for lipids extracted from Penncross roots was significantly higher than that of the other two cultivars. This difference was associated predominantly with a higher level of the unsaturated fatty acid linolenic acid (18:3). This is the same fatty acid, which appeared to be de-saturated during heating in leaves during heat stress. It is interesting to see a significant difference in lipid composition between cultivars in root tissue, in that roots have been shown to be very sensitive to high temperatures (McMichael, 1998; Xu and Huang, 2001a,b). Roots regulate plant responses to high soil temperatures (Kuroyanagi and Paulsen, 1988). Therefore, the differences in the level of lipid saturation in roots could

at least partially account for the cultivar variation in heat tolerance for creeping bentgrass. In conclusion, the saturation level of membrane lipids extracted from leaves of creeping bentgrass increased with high temperature stress. There was no change in lipid saturation in root tissues in response to heat stress. Plants which showed a higher tolerance to heat due to acquired thermotolerance or due to inherent cultivar differences tended to have leaf membranes containing higher proportions of saturated fatty acids prior to heat stress. Plants with low tolerance to heat had more unsaturated lipids in their roots, which confirmed the hypothesis. This suggests that there may be some connection between the saturation of plant lipids, particularly of root tissues, and their ability to tolerate heat stress, although clearly a large number of other factors are also involved. Acknowledgements This research was supported by the Center of Turfgrass Science, Rutgers University. We would like to thank Mrs Yanshen Wang for her help with GC-MS measurements.

References Arnon, D.I., 1949. Copper enzymes in isolated chloroplasts: polyphenol oxidase in Beta vulgaris. Plant Physiol. 24, 1–15. Blum, A., Ebercon, A., 1981. Cell membrane stability as a measure of drought and heat tolerance in wheat. Crop Sci. 21, 43–47. Cyril, J., Powell, G.L., Duncan, R.R., Baird, W.V., 2002. Changes in membrane polar lipid fatty acids of seashore paspalum in response to low temperature exposure. Crop Sci. 42, 2031– 2037. Diepenbrock, W., Muller-Rehbehn, A., Sattelmacher, B., 1989. FA composition of root membrane lipids from 2 potatoes (S. tuberosum, L) genotypes differing in heat tolerance as affected by supraoptimal root-zone temperature. Agrochimica 33, 478– 486. Fergusson, D.L., Al-Khatib, K., Guikema, J.A., Paulsen, G.M., 1993. Degradation of proteins from thylakoid membranes in senescing wheat leaves at high temperature. Plant Cell Environ. 16, 421–428. Foyer, C.H., Lopez-Delgado, H., Dat, J.F., Scott, I.M., 1997. Hydrogen peroxide and glutathione associated mechanisms of acclimatory stress tolerance and signalling. Physiol. Plant. 100, 241–254. Gombos, Z., Wada, H., Murata, N., 1991. Direct evaluation of effects of fatty acid unsaturation on the thermal properties

J. Larkindale, B. Huang / Environmental and Experimental Botany 51 (2004) 57–67 of photosynthetic activities, as studied by mutation and transformation of Synechocystis PC6803. Plant Cell Physiol. 32, 205–211. Gombos, Z., Wada, H., Hideg, E., Murata, N., 1994. The unsaturation of membrane lipids stabilizes photosynthesis against heat stress. Plant Physiol. 104, 563–567. Grover, A., Agarwal, M., Katiyar-Argarwal, S., Sahi, C., Argarwal, S., 2000. Production of high temperature tolerant transgenic plants through manipulation of membrane lipids. Curr. Sci. 79, 5. Heath, R.L., Packer, L., 1968. Photoperoxidation in isolated chloroplasts. I. Kinetics and Stoichiometry of fatty acid peroxidation. Arch. Biochem. Biophys. 125 (1), 189. Hiscox, J.D., Israeltem, G.F., 1979. A method for the extraction of chlorophyll from leaf tissue without maceration using dimethyl sulfoxide. Can. J. Bot. 57, 1332–1334. Hoagland, C.R., Arnon, D.I., 1950. The solution culture method for growing plants without soil. California Agric. Exp. Circ., 347. Horvath, I., Glatz, A., Varvasovszki, V., Zsolt, T., Pali, T., Balogh, G., Kovacs, E., Nadasdi, L., Benko, S., Joo, F., Vigh, L., 1998. Membrane physical state controls the signalling mechanism of the heat shock response in Syneschocystis PC 6803: Identification of HSP17 as a fluidity gene. Proc. Natl. Acad. Sci. 95, 3513–3518. Howarth, C., 1991. Molecular responses of plants to an increased incidence of thermotolerance. Plant Cell Environ. 14, 831–841. Huang, B., Liu, X., Fry, J.D., 1998a. Shoot physiological responses of two bentgrass cultivars to high temperature and poor soil aeration. Crop Sci. 38, 1219–1225. Huang, B., Liu, X., Fry, J.D., 1998b. Effects of high temperature and poor soil aeration on root growth and viability of creeping bentgrass. Crop Sci. 38, 1618–1622. Kunst, L., Browse, J., Somerville, C., 1989a. Enhanced thermal tolerance in a mutant of Arabidopsis deficient in palmitic acid unsaturation. Plant Physiol. 91, 401–408. Kunst, L., Browse, J., Somerville, C., 1989b. A mutant of Arabidopsis deficient in desaturation of palmitic acid in leaf lipids. Plant Physiol. 90, 943–947. Kuroyanagi, Y., Paulsen, G.M., 1988. Mediation of high temperature injury by roots and shoots during reproductive growth of wheat. Plant Cell Environ. 11, 517–523. Larkindale, J., Knight, M.R., 2002. Protection against heat stress-induced oxidative damage in Arabidopsis involves calcium, abscisic acid, ethylene, and salicylic acid. Plant Physiol. 128, 682–695. Lindquist, S., 1980. Varying patterns of protein synthesis in Drosophila during heat shock: implications for regulation. Dev. Biol. 77, 463–479. Liu, X., Huang, B., 2000. Heat stress injury in relation to membrane lipid peroxidation in creeping bentgrass. Crop Sci. 40, 503–510. Marcum, K.B., 1998. Cell membrane thermostability and whole plant heat tolerance of Kentucky bluegrass. Crop Sci. 38, 1214– 1218.

67

McCourt, P., Kunst, L., Browse, J., Somerville, C., 1987. The effects of reduced amounts of lipid unsaturation on chloroplast ultrastructure and photosynthesis in a mutant of Arabidopsis. Plant Physiol. 34, 353–360. McMichael, B.L., 1998. Soil temperature and root growth. HortScience 33, 947–951. Nishida, I., Murata, N., 1996. Chilling sensitivity in plants and cyanobacteria: the crucial contribution of membrane lipids. Annu. Rev. Plant Mol. 47, 541–556. Nishiyama, Y., Los, D., Murata, N., 1999. PsbU, a protein associated with PSII is required for the acquisition of cellular thermotolerance in Synechococcus species PCC7002. Plant Physiol. 120, 301–308. Paulsen, G.M., 1994. High temperature responses of crop plants. In: Boote, K.J., Bennett, J.M., Sinclair, T.R., Paulsen, G.M. (Eds.), Physiology and Determination of Crop Yield. ASA, CSSA and SSSA, Madison, WI, pp. 365–389. Rosen, H., 1957. A modified ninhydrin colorimetric analysis for amino acids. Arch. Biochem. Biophys. 67, 10–15. Santarius, K.A., Muller, M., 1979. Investigation on heat resistance of spinach leaves. Planta 146, 529–538. Sharkey, T.D., 2000. Some like it hot. Science 287, 435–437. Shi, W.M., Muramoto, Y., Ueda, A., Takabe, T., 2001. Cloning of peroxisomal ascorbate peroxidase gene from barley and enhanced thermotolerance by overexpressing in Arabidopsis thaliana. Gene 273, 23–27. Somerville, C., Browse, J., 1991. Plant lipids, metabolism and membranes. Science 252, 80–87. Vigh, L., Gombos, Z., Horvath, I., Joo, F., 1989. Saturation of membrane lipids by hydrogenation induces thermal stability in chloroplast inhibiting the heat dependent stimulation of Photosystem I mediated electron transport. Biochim. Biophys. Acta 979, 361–364. Vigh, L., Los, D.A., Horvath, I., Murata, N., 1993. The primary signal in the biological perception of temperature: Pd catalysed hydrogenation of the membrane lipids stimulated the expression of the desA gene in Synecchocystis PC6803. Proc. Natl. Acad. Sci. 90, 9090–9094. Wang, C.Y., Baker, J.E., 1979. Effects of 2 freeze radical scavengers and intermittent warming on chilling injury and polar lipid composition of cucumbers and sweet pepper fruits. Plant Cell Physiol. 20, 243–251. Whitaker, B.D., Klein, J.D., Conway, W.S., Sams, C.E., 1997. Influence of pre-storage heat and calcium pre-treatments on lipid metabolism in Golden Delicious apples. Phytochemistry 45, 465–472. Xu, Q., Huang, B., 2001a. Morphological and physiological characteristics associated with heat tolerance in creeping bentgrass. Crop Sci. 41, 127–133. Xu, Q., Huang, B., 2001b. Lowering soil temperatures improves creeping bentgrass growth under heat stress. Crop Sci. 41, 1878–1883. Yang, J.F., Cheng, B.S., Wang, H.C., 1984. Influence of high temperature and low humidity on the fatty acid composition of membrane lipids in wheat. Acta Bot. Sin. 26, 386–391.