3-Dimethylsulfoniopropionate

Cottonseed typically contains about 15% oleic acid. Here we report the development of transgenic cotton plants with higher seed oleic acid levels. Plants were generated by Agrobacterium-mediated transformation. A binary vector was designed to suppress expression of the endogenous cottonseed ∆-12 desaturase (fad 2) by subcloning a mutant allele of a rapeseed fad 2 gene downstream from a heterologous, seedspecific promoter (phaseolin). Fatty acid profiles of total seed lipids from 43 independent transgenic lines were analyzed by gas chromatography. Increased seed oleic acid content ranged from 21 to 30% (by weight) of total fatty acid content in 22 of the primary transformants. The increase in oleic acid content was at the expense of linoleic acid, consistent with reduced activity of cottonseed FAD2. Progeny of some lines yielded oleic acid content as high as 47% (three times that of standard cottonseed oil). Molecular analyses of nuclear DNA from transgenics confirmed the integration of the canola transgene into the cotton genome. Collectively, our results extend the metabolic engineering of vegetable oils to cottonseed and should provide the basis for the development of a family of novel cottonseed oils.Paper no. J9910 in JAOCS 78, 941-947 (September 2001). KEY WORDS:Cottonseed oil, fatty acid metabolism, metabolic engineering, oilseeds.

Fatty acid and glycerolipid biosynthesis from [14C]acetate by isolated pea root plastids is completely dependent on exogenously supplied ATP. CTP, GTP, and UTP are ineffective in supporting fatty acid biosynthesis, all resulting in <3% of the activity obtained with ATP. However, ADP alone or in combination with inorganic phosphate (Pi) or pyrophosphate (PPi) gave up to 28% of the ATP control activity, whereas AMP + PPi, PPi alone, or Pi alone were ineffective in promoting fatty acid biosynthesis. The components of the dihydroxyacetonephosphate (DHAP) shuttle (DHAP, oxaloacetate, and Pi), which promote intraplastidic ATP synthesis, restored 41% of the control ATP activity, whereas the omission of any of the shuttle components abolished this activity.When the DHAP shuttle components were supplemented with ADP, the rate of fatty acid biosynthesis was completely restored to that observed in the presence of ATP. Under the conditions of ADP + DHAP shuttle-driven fatty acid biosynthesis, exogenously supplied ATP gave only a 6% additional stimulation of activity. In general, variations in the energy source had only small effects on the proportions of radioactive fatty acids and glycerolipids synthesized. Most notably, higher amounts of radioactive oleic acid, free fatty acids, and diacylglycerol and lower amounts of phosphatidic acid were observed when ADP and/or the DHAP shuttle were substituted for ATP. The results presented here indicate that, although isolated pea root plastids readily utilize exogenously supplied ATP for fatty acid biosynthesis, these plastids can also synthesize sufficient ATP when provided with the appropriate cofactors.Fatty acid and glycerolipid biosynthesis from acetate are strictly energy dependent in all plastids. ATP is required for the synthesis of both acetyl-CoA and malonyl-CoA by acetylCoA synthetase and acetyl-CoA carboxylase, respectively (20). Similarly, the reduced nucleotides NADPH and NADH are required in the 13-ketoacyl-ACP2 reductase and 2-enoyl-ACP reductase steps ofde novo fatty acid biosynthesis, respectively, as well as the desaturation of stearoyl-ACP (20 tors are only indirectly required for plastidic glycerolipid biosynthesis in so far as this process is dependent on fatty acid synthesis. In chloroplasts, ATP and reduced nucleotides are supplied during photosynthesis and glycolytic metabolism, whereas glycolytic and oxidative pentose phosphate metabolism provides these cofactors in developing oilseed plastids (3,4). Similar metabolism may be involved in other nonphotosynthetic plastids (1,8,9, 1 1); however, the extent to which these metabolic pathways are involved remains to be fully defined.Fatty acid and glycerolipid biosynthesis from acetate in isolated pea root plastids is completely dependent on exogenously supplied ATP

The physiological relevance of a novel thiol methyltransferase from cabbage, and its possible role in sulphur metabolism have been investigated. The enzyme was absent from the chloroplast, the site of sulphate reduction, and was localized in the cytosol. Potential substrates were initially screened on the basis of their ability to inhibit the methylation of iodide, a previously known substrate for the enzyme. Thiocyanate, 4,4¢-thiobisbenzenethiol, thiophenol, and thiosalicylic acid were identified as possible substrates. Methylation of these thiols by the purified enzyme using [Methyl-3 H]S-adenosyl-L-methionine confirmed their nature as substrates. The purified enzyme strongly preferred thiocyanate as a methyl acceptor. The enzyme had K m values of 11, 51, 250 and 746 mmol m -3 for thiocyanate, 4,4¢-thiobisbenzenethiol, thiophenol and thiosalicylic acid, respectively. The identity of methylthiocyanate as the product of thiocyanate methylation by the purified enzyme was confirmed by mass spectrometry. The enzyme was strictly associated with glucosinolate-containing plants. Thiol substrates of the enzyme are known products of glucosinolate hydrolysis. Our observations indicate that this enzyme could be involved in the detoxification of reactive thiols produced upon glucosinolate degradation in these plants.

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Intact chloroplasts from spinach (Spuiacia oleracea L., hybrid 424) readily incorporate I'4Ciglycerol-3-phosphate and 114CIacetate into diacylglycerol, monoacylglycerol, diacylglycrol, free fatty acids (only when acetate is the precursor), phosphatidic acid, phosphatidylcholine, and most notably phosphatidylglycerol. The fraction of phosphatidylglycerol synthesized is greatly increased by the presence of manganese chloride in the reaction mixture. PG2 is an important lipid component ofchloroplasts. Depending on the tissue, it can comprise 60 to 70% of the total chloroplast phospholipid (10). Characterized by the presence of the unusual trans-A3-hexadecenoic acid (11) and its increase during chloroplast development, this unique species of PG has been suggested to have a role in granal stacking (9).While chloroplasts from Euglena can synthesize PG (4-6), conflicting reports (7,16,25) left some question as to whether or not chloroplasts from higher plants can synthesize their own PG. The earlier works of Douce and Guillot-Salomon (7) and Sastry and Kates (25) suggested that PG was synthesized by chloroplasts. These results were cast in doubt when Marshall and Kates (16) determined that PG was not synthesized by chloroplasts, and since then, chloroplasts from higher plants generally have been considered incapable of PG synthesis. Recently, Mudd and DeZacks (20) reexamined chloroplasts from spinach for their capacity to synthesize PG and presented evidence that these higher plant chloroplasts could in fact synthesize their own PG.We have continued these investigations using both radiolabeled G3P and acetate to further establish optimal conditions for PG ' synthesis. We have also subjected the biosynthesized PG to a number of degradative procedures in order to understand the pathway to the newly synthesized PG molecule.MATERIALS AND METHODS Spinach Culture. Spinach seeds (Spinacia oleracea L., hybrid 424; Ferry Morse Seed Company, Mountain View, CA) were germinated and maintained for 10 to 12 d in vermiculite moistened with half-strength Hoagland nutrient solution. The seedlings were transferred to hydroponic culture dishes containing nutrient solution and supported by plastic sheets with 1-cm holes and cotton plugs. Seedlings and plants were grown in a growth chamber with a 12-h daylength and maintained at 23°C and 18°C during the daytime and nighttime, respectively.Chloroplast Isolation. Spinach leaves were harvested and homogenized and chloroplasts isolated and purified as described previously (20). Chl determinations were as described by Holden (12).Chloroplast Incubation Conditions and Lipid Extraction. The composition of the standard reaction mixture conditions of incubation and lipid extraction were all as described earlier (20). Variations of these procedures were the use of 2 mm MnCl2 in the standard reaction mixture and substitution of 1 M KCI for the water of the extraction mixtures when phospholipase A2 and Rhizopus arrhizus digestions were performed. When necessary, approximately 500,000 to 60...

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On the average, the principal radioactive products of fatty acid biosynthesis were approximately 39% palmitic, 9% stearic, and 52% oleic acid. The proportions of these fatty acids synthesized depended on the experimental conditions.