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.

Radiolabeled pyruvate, glucose, glucose-6-phosphate, acetate, and malate are all variously utilized for fatty acid and glycerolipid biosynthesis by isolated pea (Pisum safivum 1.) root plastids. At the highest concentrations tested (3-5 mM), the rates of incorporation of these precursors into fatty acids were 183, 154, 125, 99, and 57 nmol h-' mg-' protein, respectively. In all cases, cold pyruvate consistently caused the greatest reduction, whereas cold acetate consistently caused the least reduction, i n the amounts of each of the other radioactive precursors utilized for fatty acid biosynthesis.Acetate incorporation into fatty acids was approximately 55% dependent on exogenously supplied reduced nucleotides (NADH and NADPH), whereas the utilization of the remaining precursors was only approximately 1 O and 20% dependent on added NAD(P)H. In contrast, the utilization of all precursors was greatly dependent (85-95O/0) on exogenously supplied ATP. Palmitate, stearate, and oleate were the only fatty acids synthesized from radioactive precursors. Higher concentrations of each precursor caused increased proportions of oleate and decreased proportions of palmitate synthesized. Radioactive fatty acids from all precursors were incorporated into glycerolipids. The data presented indicate that the entire pathway from glucose, including glycolysis, to fatty acids and glycerolipids is operating i n pea root plastids. This pathway can supply both carbon and reduced nucleotides required for fatty acid biosynthesis but only a small portion of the ATP required.Plastids, including chloroplasts and chromoplasts, oilseed plastids, and root plastids, are the site of de novo fatty acid biosynthesis in plants. This subject has been thoroughly reviewed in a number of works (Stumpf, 1984;Harwood, 1988;Dennis, 1989; Sparace and KleppingerSparace, 1993). Traditionally, most in vitro studies of fatty acid biosynthesis utilize acetate as a radioactive tracer largely because it is efficiently incorporated into fatty acids. This has been justified by the belief that extraplastidic acetate originates through the action of mitochondrial pyruvate dehydrogenase and acetyl-COA hydrolase (Murphy and Stumpf, 1981), as well as by the occurrence of envelope-bound acetyl-COA synthetase (Kuhn et al., 1981). However, more recent studies indicate that most plastids contain their own pyruvate dehydrogenase (Reid et al

N-Acylethanolamines (NAEs) are fatty acid derivatives found as minor constituents of animal and plant tissues, and their levels increase 10- to 50-fold in tobacco (Nicotiana tabacum) leaves treated with fungal elicitors. Infiltration of tobacco leaves with submicromolar to micromolar concentrations ofN-myristoylethanolamine (NAE 14:0) resulted in an increase in relative phenylalanine ammonia-lyase (PAL) transcript abundance within 8 h after infiltration, and this PAL activation was reduced after co-infiltration with cannabinoid receptor antagonists (AM 281 and SR 144528). A saturable, high-affinity specific binding activity for [3H]NAE 14:0 was identified in suspension-cultured tobacco cells and in microsomes from tobacco leaves (apparent K  d of 74 and 35 nm,respectively); cannabinoid receptor antagonists reduced or eliminated specific [3H]NAE 14:0 binding, consistent with the physiological response. N-Oleoylethanolamine activatedPAL2 expression in leaves and diminished [3H]NAE 14:0 binding in microsomes, whereasN-linoleoylethanolamine did not activatePAL2 expression in leaves, and did not affect [3H]NAE 14:0 binding in microsomes. The nonionic detergent dodecylmaltoside solubilized functional [3H]NAE 14:0-binding activity from tobacco microsomal membranes. The dodecylmaltoside-solubilized NAE-binding activity retained similar, but not identical, binding properties to the NAE-binding protein(s) in intact tobacco microsomes. Additionally, high-affinity saturable NAE-binding proteins were identified in microsomes isolated from Arabidopsis and Medicago truncatula tissues, indicating the general prevalence of these binding proteins in plant membranes. We propose that plants possess an NAE-signaling pathway with functional similarities to the “endocannabinoid” pathway of animal systems and that this pathway, in part, participates in xylanase elicitor perception in tobacco.