In plants, the galactolipids monogalactosyldiacylglycerol (MGDG) and digalactodiacylglycerol (DGDG) are major constituents of photosynthetic membranes in chloroplasts. One of the key enzymes for the biosynthesis of these galactolipids is MGDG synthase (MGD). To investigate the role of MGD in the plant's response to salt stress, we cloned an MGD gene from rice (Oryza sativa) and generated tobacco (Nicotiana tabacum) plants overexpressing OsMGD. The MGD activity in OsMGD transgenic plants was confirmed to be higher than that in the wild-type tobacco cultivar SR1. Immunoblot analysis indicated that OsMGD was enriched in the outer envelope membrane of the tobacco chloroplast. Under salt stress, the transgenic plants exhibited rapid shoot growth and high photosynthetic rate as compared with the wild type. Transmission electron microscopy observation showed that the chloroplasts from salt-stressed transgenic plants had well-developed thylakoid membranes and properly stacked grana lamellae, whereas the chloroplasts from salt-stressed wild-type plants were fairly disorganized and had large membrane-free areas. Under salt stress, the transgenic plants also maintained higher chlorophyll levels. Lipid composition analysis showed that leaves of transgenic plants consistently contained significantly higher MGDG (including 18:3-16:3 and 18:3-18:3 species) and DGDG (including 18:3-16:3, 18:3-16:0, and 18:3-18:3 species) contents and higher DGDG-MGDG ratios than the wild type did under both control and salt stress conditions. These results show that overexpression of OsMGD improves salt tolerance in tobacco and that the galactolipids MGDG and DGDG play an important role in the regulation of chloroplast structure and function in the plant salt stress response.
The occurrence of glycolipids such as sterol glycosides, acylated sterol glycosides, cerebrosides and glycosyldiacylglycerols was examined in the three yeast species Candida albicans, Pichia pastoris and Pichia anomala, as well as in the six fungal species Sordaria macrospora, Pyrenophora teres, Ustilago maydis, Acremonium chrysogenum, Penicillium olsonii and Rhynchosporium secalis. Cerebroside was found in all organisms tested, whereas acylated sterol glycosides and glycosyldiacylglycerols were not found in any organism. Sterol glycosides were detected in P. pastoris strain GS115, U. maydis, S. macrospora and R. secalis. This glycolipid occurred in both yeast and filamentous forms of U. maydis but in neither form of C. albicans. This suggests that sterol glycoside is not correlated with the separately grown dimorphic forms of these organisms. Cerebrosides and sterol glycosides from P. pastoris and R. secalis were purified and characterized by mass spectrometry and nuclear magnetic resonance spectroscopy. The cerebrosides are b-glucosyl ceramides consisting of a saturated a-hydroxy or non-hydroxy fatty acid and a D4,8-diunsaturated, C9-methyl-branched sphingobase. Sterol glycoside from P. pastoris was identified as ergosterol-b-D-glucopyranoside, whereas the sterol glucosides from R. secalis contain two derivatives of ergosterol. The biosynthesis of sterol glucoside in P. pastoris CBS7435 and GS115 depended on the culture conditions. The amount of sterol glucoside in cells grown in complete medium was much lower than in cells from minimal medium and a strong increase in the content of sterol glucoside was observed when cells were subjected to stress conditions such as heat shock or increased ethanol concentrations. From these data we suggest that, in addition to Saccharomyces cerevisiae, new yeast and fungal model organisms should be used to study the physiological functions of glycolipids in eukaryotic cells. This suggestion is based on the ubiquitous and frequent occurrence of cerebrosides and sterol glycosides, both of which are rarely detected in S. cerevisiae. We suggest P. pastoris and two plant pathogenic fungi to be selected for this approach.
In spinach (Spinacia oleracea L. cv. New Asia) plants fumigated with ozone in light, destruction of chlorophylls and carotenoids and formation of malondialdehyde (MDA), an indicator of lipid peroxidation, were observed. Chlorophyll a and carotenoids in leaves started to be broken down 6–8 h after the commencement of 0.5 ppm ozone fumigation, whereas MDA formation in leaves increased linearly for the initial 8 h of fumigation followed by a more rapid increase. In leaf discs excised from 6‐h fumigated plants, destruction of chlorophyll a and carotenoids and MDA formation proceeded in the light but were almost completely suppressed under an anaerobic condition. Effects of exogenously applied scavengers of active oxygen species suggest that active oxygens, especially superoxide radical (O2‐), participated in both the destruction of chlorophyll a and carotenoids and the formation of MDA. Ozone fumigation reduced the levels of endogenous scavengers of O2‐, superoxide dismutase (SOD) and L‐ascorbate, in leaves to one‐half the initial levels each by 3.5 and 8 h fumigation, respectively. The results indicate that the photosynthetic pigments and lipids were broken down by active oxygens accumulated in leaves as a result of the ozone‐induced destruction of physiological defense against oxygen toxicity. Activity of polyphenol oxidase in chloroplast membranes of 4‐h fumigated leaves increased to 240% of the initial level, suggesting that the thylakoid membranes had been affected severely before the pigment destruction. The relations between the pigment destruction and the disintegration of thylakoids are discussed.
Molecular species and fatty acid distribution of triacylglycerol (TG) accumulated in spinach (Spinacia oleracea L.) leaves fumigated with ozone (0.5 microliter per liter) were compared with those of monogalactosyldiacylglycerol (MGDG). Analysis of positional distribution of the fatty acids in MGDG and the accumulated TG by the enzymatic digestion method showed that hexadecatrienoate (16:3) was restricted to sn-2 position of the glycerol backbone in both MGDG and TG, whereas a-linolenate (18:3) was preferentially located at sn-1 position in MGDG, and sn-1 and/or sn-3 positions in TG, suggesting that 1,2-diacylglycerol moieties of MGDG are the direct precursor of TG in ozonefumigated leaves. Further analysis of TG molecular species by argentation chromatography and mass spectrometry showed that TG increased with ozone fumigation consisted of approximately an equal molar ratio of sn-1,3-18:3-2-16:3 and sn-1,2,3-18:3. Because the molecular species of MGDG in spinach leaves is composed of a similar molar ratio of sn-1-18:3-2-16:3 and sn-1,2-18:3, we concluded that MGDG was converted to 1,2-diacylglycerol and acylated with 18:3 to TG in ozone-fumigated spinach leaves.Ozone is a widespread atmospheric pollutant, causing visible foliar injury in many plant species. A number of workers have examined the physiological and biochemical effects of ozone on plant tissues to understand the mechanism by which ozone injures plants (10). Because ozone is a strong oxidizing agent and reacts readily with the double bond in unsaturated fatty acids, the destruction of cellular membrane lipids has been suggested to be one ofthe primary events in ozone injury (10,16 (9,19,27). However, no information is available for the path of TG synthesis from galactolipids. Webb and Williams (28,29) have reported a fatty acid transfer from MGDG and PC to TG in mesophyll protoplasts prepared from broad bean leaves by enzymatic digestion and have proposed that MGDG was first hydrolyzed by galactolipid lipase to free fatty acids, which were subsequently reacylated to form TG. Alternatively, we have proposed that 1,2-DG liberated from MGDG is the direct precursor of TG synthesized in ozone-fumigated spinach leaves, based on the fact that 16:3, the fatty acid specific to MGDG, was recovered in 1,2-DG as well as in TG (24). To confirm this proposal, it is necessary to demonstrate that the positional specificity of fatty acids in MGDG is retained in TG increased with ozone treatment.In the present paper, we report the comparison of the positional distribution of fatty acids in MGDG, DGDG, and PC with that in newly accumulated TG in ozone-fumigated spinach leaves. Molecular species of the increased TG are further determined by argentation chromatography and mass spectrometry. These results support the direct conversion of MGDG to TG via 1,2-DG. MATERIALS AND METHODS Plant Materials and Ozone FumigationSpinach (Spinacia oleracea L., cv New Asia) plants were grown for about 7 weeks under natural light and fumigated with 0.5 ,uL/L ozone for 6 h as des...
When the upper leaf surface of spinach (Spinacia oleracea L.) plants was treated with [1-14C]acetate and grown for 2 days, 14Cwas effectively incorporated into acyl moieties of leaf lipids in ratios approximately their composition by mass. Fumigation of the plants with ozone (0.5 microliter per liter) caused a redistribution of 14C among lipid classes, i.e. a marked increase of 14C content in triacylglycerol (TG) and 1,2-diacylglycerol (1,2-DG) and a decrease of label in monogalactosyldiacylglycerol (MGDG) without affecting 14C distribution in leaf fatty acids. Label in both TG and 1,2-DG was found predominantly in their polyene molecular species. Since MGDG consists of similar polyene molecular species, the results indicate the synthesis of TG from MGDG via 1,2-DG. Label was also accumulated in tri-and tetragalactosyldiacylglycerol, products of galactolipid:galactolipid galactosyltransferase (GGGT). Moreover, there was a close relation between increases in the amounts of TG and the oligogalactolipids in ozonetreated leaves. These results indicate that MGDG was converted to 1,2-DG by GGGT and then to TG. In intact chloroplasts isolated from ozone-treated leaves, there was an enhanced production of free fatty acid (FFA), which was diminished by the addition of coenzyme A (CoA) and ATP, indicating that ozone stimulated the hydrolysis of MGDG to liberate FFA, which was in turn converted to acyl-CoA. The final step of TG synthesis, acylation of 1,2-DG with acyl-CoA, was confirmed by feeding with [1-14C] (27). In addition, a comparison of the molecular species between MGDG and the increased TG (28) indicated the direct conversion of 1,2-DG backbone of MGDG to TG. This paper presents labeling studies demonstrating the synthesis of TG from MGDG in ozone-fumigated spinach leaves. The results confirm the operation of the pathway from MGDG to TG via 1,2-DG and also demonstrate that MGDG is converted to 1,2-DG by GGGT. We also report that ozone stimulates the hydrolysis of MGDG to release FFA, mainly a-linolenic acid (18:3), and that the 18:3 is converted to its acyl-CoA form and then acylated to 1,2-DG to yield TG. MATERIALS AND METHODS Plant Materials and Ozone FumigationSpinach (Spinacia oleracea L., cv New Asia) plants were cultivated from seeds in a glasshouse as described previously (25). In [1-'4C]acetate-labeling experiments, expanding second leaves of rapidly growing spinach plants (4 weeks old) were used for the better incorporation of acetate into endogenous fatty acids. In the remaining experiments, youngest mature leaves of spinach plants (6-8 weeks old) were used (25,27,28). Ozone-induced lipid changes in the leaves at these different stages ofdevelopment were essentially the same as those described in the previous reports (27,28) containing lipid spots were scraped from the plate, methanolyzed, and quantified by GLC (27). Lipids in the labeled leaves and leaf discs were isolated after boiling in isopropanol for 5 min. TGDG and TTGDG were separated as described above, and other polar lipids were...
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