Trienoic Fatty Acids and Plant Tolerance of High Temperature

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Science  21 Jan 2000:
Vol. 287, Issue 5452, pp. 476-479
DOI: 10.1126/science.287.5452.476


The chloroplast membrane of higher plants contains an unusually high concentration of trienoic fatty acids. Plants grown in colder temperatures have a higher content of trienoic fatty acids. Transgenic tobacco plants in which the gene encoding chloroplast omega-3 fatty acid desaturase, which synthesizes trienoic fatty acids, was silenced contained a lower level of trienoic fatty acids than wild-type plants and were better able to acclimate to higher temperatures.

In some desert and evergreen plants, an increase in the growth temperature leads to a reduction in trienoic fatty acids α-linolenic acid (18:3) and hexadecatrienoic acid (16:3) (1, 2). In order to investigate the physiological effect of these fatty acids in plants grown at high temperatures, we constructed transgenic tobacco plants in which the expression of the chloroplast trienoic fatty acid synthetase gene was inhibited.

Transgenic tobacco plants harboring transferred DNA (T-DNA) with the chloroplast-localized ω-3 desaturase gene (FAD7) fromArabidopsis thaliana under the control of the cauliflower mosaic virus 35S promoter were generated (3). Gene-silencing and reduction of trienoic–fatty acid content were observed in four transgenic lines (4). Of the four lines, two lines (T15 and T23) exhibited a 3:1 segregation ratio of kanamycin resistance versus nonresistance in the next generation, suggesting that the T-DNA was inserted in one position in the genome. The T15 line was backcrossed twice to produce a homozygous line (5). The T23 line was self-pollinated to produce another homozygous line.

The amount of trienoic fatty acids in the chloroplasts of homozygous T15 and T23 plants was lower than that in the chloroplasts of the wild-type plants (Table 1). This reduction in trienoic fatty acid content was associated with an increase in the corresponding dienoic fatty acid precursors, linoleic acid (18:2) and hexadecadienoic acid (16:2). The fatty acid composition of nonchloroplast lipids, such as phosphatidylcholine and phosphatidylethanolamine, was less affected by the absence of the chloroplast trienoic fatty acid synthetase gene (Table 1). The levels of monounsaturated fatty acids remained unaffected. Thus, the activity of chloroplast ω-3 fatty acid desaturase was suppressed in the T15 and T23 homozygous lines. The lipid ratios (Table 1) indicate that the overall flux through the prokaryotic and eukaryotic pathways of glycerolipid synthesis (6) was not affected by the T-DNA. These characteristics were stably inherited through backcrosses.

Table 1

Fatty acid composition of individual membrane lipids from leaves of wild-type (WT) and transgenic tobacco (T15, T23) plants. The major classes of membrane lipids were isolated from the total lipid extracted from mature leaves, and the fatty acid composition was determined (15). Each value represents the mean of two independent experiments. Dash (–) indicates trace amounts (<1.0%).

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Photosynthesis is one of the most heat-sensitive functions of plant cells. Temperatures in the range of 35° to 45°C tend to inhibit photosynthesis (7, 8). In order to assess the effect of high temperatures on the photosynthetic machinery, intact leaves from transgenic tobacco plants and Arabidopsismutants (9) were pretreated at various temperatures between 25° and 55°C, and the level of photosynthetic activity was measured using O2 evolution as the index of activity (10). At 40°C, the photosynthetic activity of the wild-type tobacco plants was significantly diminished, whereas the activity of the transgenic T15 and T23 plants was higher than that at the normal growing temperature of 25°C (Fig. 1A).

Figure 1

Oxygen evolution from transgenic tobacco and mutantArabidopsis leaves that had been preincubated at various temperatures for 5 min. (A) T15, T23, and wild-type tobacco. (B) fad7fad8 double mutant and wild-typeArabidopsis. (C) fad3 mutant and wild-type Arabidopsis. For each plant line, the O2 evolution at 25°C was set at 100%. The O2evolution at 25°C was 2.4, 2.5, and 2.5 mmol O2m−2 s−1 in T15, T23, and WT tobacco, respectively, in (A); 1.4 and 1.7 mmol O2 m−2s−1 in ƒad7ƒad8 and WTArabidopsis, respectively, in (B); and 3.1 and 2.9 mmol O2 m−2 s−1 in fad3 and WT Arabidopsis, respectively, in (C). Each data point represents the mean value from four independent experiments.

In order to confirm that the relation between the trienoic fatty acid level and the thermal stability of the photosynthetic machinery is the same in Arabidopsis, we examined the Arabidopsis fad7fad8 double mutant, which lacks two chloroplast-localized ω-3 fatty acid desaturases (11). The fatty acid composition of the fad7fad8 mutant was similar to that of the tobacco T15 and T23 lines. The chloroplast lipids of thefad7fad8 mutant consisted of small amounts of trienoic fatty acids and large amounts of dienoic fatty acids (11). Thefad7fad8 mutant tolerated higher temperatures, as did the tobacco transgenic lines (Fig. 1B). On the other hand, thefad3 mutant, which lacks ω-3 fatty acid desaturase localized in the endoplasmic reticulum (12), showed wild-type photosynthetic activity (Fig. 1C). Thus, trienoic fatty acids in chloroplast lipids affect the high-temperature tolerance of the photosynthetic machinery more than the trienoic fatty acids in nonchloroplast lipids.

The primary sites of thermal damage are thought to be components of the photosynthetic system located in the thylakoid membrane, such as photosystem II (PSII) (7). The potential quantum efficiency of PSII under dark-adapted conditions after exposure to high temperature was assessed by measuring the fluorescence parameterF v/F m(quantum yield of PSII in dark-adapted) of the samples used in the O2 evolution experiments. However, heat treatment did not affect theF v/F mof the transgenic tobacco lines and Arabidopsis fad7fad8mutants (13). Thus, the trienoic fatty acid level in chloroplast lipids does not directly contribute to the resistance of PSII to high temperatures.

In most plants using the C3 photosynthetic pathway, the optimal temperature for CO2 assimilation is far below the thermal tolerance limit. Even within the limits, the photosynthetic productivity at high temperatures is rather low. The low rate of assimilation at high temperatures is caused, in part, by photorespiration and an imbalance in the regulation of carbon metabolism (8). A reduced trienoic fatty acid level in chloroplast lipids may alleviate such dissipative processes.

The effect of humidity on plant growth must be taken into account in determining the sensitivity of plants to the growing temperature. To ensure constant humidity in the following experiments, plants were grown on Murashige-Skoog (MS) agar media in closed plastic boxes or petri dishes. In plants grown at a cool temperature (15°C) and a more suitable growth temperature (25°C), there were no differences in the growth of the two transgenic tobacco lines (T15 and T23) and the wild type. After germination and cultivation of these plants for 45 days at 25°C, the fresh weight of the aerial parts of the T15 and T23 plants was 489 ± 71 mg and 513 ± 88 mg, respectively, whereas the fresh weight of the aerial parts of the wild-type plants was 497 ± 43 mg (n = 5). The fresh weight of the aerial parts of the T15, T23, and the wild-type plants cultivated at 15°C for 45 days was 6.2 ± 1.4 mg, 6.9 ± 1.2 mg, and 6.6 ± 0.9 mg (n = 5), respectively. These results in tobacco plants are consistent with the growth of the Arabidopsis fad7fad8 mutant within the normal cultivation temperature range of 12° to 28°C (11). Furthermore, at temperatures below 10°C, the growth of the two transgenic tobacco lines and the growth of the wild type were similarly suppressed.

An elevated trienoic fatty acid level as a result of overexpression of the Arabidopsis FAD7 ω-3 desaturase gene (14) has a minor protective effect against chilling-induced damage to young transgenic tobacco seedlings (15). Furthermore, the Arabidopsis triple mutant,fad3fad7fad8, which lacks trienoic fatty acids in all membranes, can grow at temperatures as low as 6°C, although with reduced photosynthesis capacity (16). Thus, trienoic fatty acids are not critical for growth at low temperatures.

On the other hand, the resistance of a plant to high temperature depends on the trienoic fatty acid content. In plants cultivated at 30°C for 45 days after germination, the fresh weight of the aerial parts of the T15, T23, and wild-type plants was 492 ± 81 mg, 445 ± 62 mg, and 399 ± 69 mg (n = 5), respectively. At a higher temperature (36°C), marked differences in the growth of the transgenic tobacco lines and the wild type were seen (Fig. 2A). After cultivating plants at 36°C for 45 days, the fresh weight of the aerial parts of the T15 and T23 lines and the wild type was 124 ± 49 mg, 123 ± 23 mg, and 13 ± 6 mg (n = 5), respectively. Temperature resistance in the transgenic lines was not transient and was unlike the protection conferred by induction of a heat shock protein (17). At 47°C, the leaves of the wild-type plants began to wither within 2 days, and necrotic areas developed by the third day (Fig. 2B). However, T15 and T23 plants that were exposed to a temperature of 47°C were uninjured. Although the growth of the T15 and T23 plants was suppressed at 47°C growth resumed when the temperature was reduced to 25°C.

Figure 2

Visible damage to tobacco andArabidopsis plants exposed to high temperatures. (A) Photographs of T15, T23, and wild-type (WT) tobacco plants. Tobacco seeds had been sown in culture boxes and kept at 36°C under constant light (50 μmol m−2 s−1) for 60 days. (B) Photographs of T15 and WT tobacco plantlets that had been grown in petri dishes for 15 days at 25°C, and then exposed to a temperature of 47°C under constant light (70 μmol m−2s−1) for 0, 2, and 3 days. (C) Photographs of the fad7fad8 mutant and WT Arabidopsisplantlets. fad7fad8 mutant and WT Arabidopsisseeds sown in petri dishes were kept at 33° and 36°C under constant light (50 μmol m−2 s−1) for 14 days. Scale bar, 1 cm. The growth of the wild-type Arabidopsis and that of the fad7fad8 mutant at temperatures below 30°C, were similar. However, Arabidopsis shows heat stress at lower temperatures than tobacco. Most of the wild-type plants grown at 36°C had died by day 45, and all of the wild-type andfad7fad8 plants grown at a temperature near 40°C died.

The Arabidopsis fad7fad8 mutant was also resistant to high temperatures. When Arabidopsis wild-type plants andfad7fad8 mutants were grown at a temperature above 30°C, and particularly near 35°C, a distinct difference in the growth of the wild-type and fad7fad8 mutant was observed (Fig. 2C). When wild-type plants and fad7fad8 mutants were grown under the high-temperature condition, the growth of the wild-type plants was significantly reduced and their leaves were wilted, whereas the growth of the fad7fad8 mutant was only slightly reduced.

Saturation of thylakoid membrane lipids by catalytic hydrogenation increases the thermal stability of the membranes (18). Increased saturation may raise the temperature at which lipids such as monogalactosyldiacylglycerol phase-separate into nonbilayer structures, which disrupt membrane organization. If so, the resistance of plants to high temperatures might be improved by reducing the content of lower unsaturated fatty acids such as dienoic fatty acids (19,20). The sensitivity of these plants to low temperatures, however, might possibly be increased (21).

The only difference between our gene-silenced transgenic plants that were resistant to high temperature and the respective wild-type plants was that the chloroplasts of the transgenic plants contained a reduced level of trienoic fatty acids and an elevated level of dienoic fatty acids, which is controlled by chloroplast ω-3 fatty acid desaturase. Of the six different higher plant desaturases whose genes have been cloned, only the expression of the chloroplast FAD8 ω-3 fatty acid desaturase gene changes in response to a change in ambient temperature (22).

The ω-3 fatty acid desaturase enzyme, which is expressed in nearly all plant species, may be widely useful in engineering temperature tolerance in plants.

  • * Present address: Department of Bioresources Chemistry, Faculty of Horticulture, Chiba University, Matsudo 648, Chiba 271-8510, Japan.

  • To whom correspondence should be addressed. E-mail: koibascb{at}


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