SummaryAn Arabidopsis fatty acid elongase gene, KCS1, with a high degree of sequence identity to FAE1, encodes a 3-ketoacyl-CoA synthase which is involved in very long chain fatty acid synthesis in vegetative tissues, and which also plays a role in wax biosynthesis. Sequence analysis of KCS1 predicted that this synthase was anchored to a membrane by two adjacent N-terminal, membrane-spanning domains. Analysis of a T-DNA tagged kcs1-1 mutant demonstrated the involvement of the KCS1 in wax biosynthesis. Phenotypic changes in the kcs1-1 mutant included thinner stems and less resistance to low humidity stress at a young age. Complete loss of KCS1 expression resulted in decreases of up to 80% in the levels of C26 to C30 wax alcohols and aldehydes, but much smaller effects were observed on the major wax components, i.e. the C29 alkanes and C29 ketones on leaves, stems and siliques. In no case did the loss of KCS1 expression result in complete loss of any individual wax component or significantly decrease the total wax load. This indicated that there was redundancy in the elongase KCS activities involved in wax synthesis. Furthermore, since alcohol, aldehyde, alkane and ketone levels were affected to varying degrees, involvement of the KCS1 synthase in both the decarbonylation and acyl-reduction wax synthesis pathways was demonstrated.

Microsomal membranes isolated from the pericarp of maturegreen tomato (Lycopersicon esculentum) fruit rapidly metabolize exogenous radiolabeled linoleic acid into fatty acid oxidation products at 22°C. The reaction is strongly inhibited by n-propyl gallate, an inhibitor of lipoxygenase. The membranes also rapidly metabolize 16:0/18:2* phosphatidylcholine into radiolabeled oxidation products that comigrate on TLC plates with those formed from free linoleic acid. At 300C, the formation of fatty acid oxidation products from 16:0/18:2* phosphatidylcholine is slower, and there is an initial accumulation of radiolabeled linoleic acid that is not evident at 220C, which can be attributed to the action of lipolytic acyl hydrolase. Radiolabeled phosphatidic acid and diacylglycerol are also formed during metabolism of 16:0/18:2* phosphatidylcholine by the microsomal membranes, and there is no breakdown of either linoleic acid or phosphatidylcholine by heat-denatured membranes. When Triton X-100 treated membranes were used, the same pattems of metabolite formation from radiolabeled linoleic acid and 16:0/18:2* phosphatidylcholine were observed. Thus, the enzymes mediating the breakdown of these radiolabeled compounds appear to be tightly associated with the membranes. Collectively, the data indicate that there is a lipoxygenase associated with microsomal membranes from tomato fruit that utilizes free fatty acid substrate released from phospholipids. The microsomal lipoxygenase is strongly active over a pH range of 4.5 to 8.0, comprises approximately 38% of the total (microsomal plus soluble) lipoxygenase activity in the tissue, has an apparent Km of 0.52 millimolar and an apparent V,,. of 0.186 millimoles per minute per milligram of protein. The membranous enzyme also cross-reacts with polyclonal antibodies raised against soybean lipoxygenase-1 and has an apparent molecular mass of 100 kilodaltons.Lipoxygenase (EC 1.13.11.12) is a dioxygenase that catalyzes the peroxidation of fatty acids containing a cis,cis-l1,4-pentadiene configuration. Distinguishable isozymes of lipoxygenase have been described for a number of tissues in different plant species (8) and the best characterized of these are the three isozymes of soybean known as lipoxygenase-1, -2, and -3 (18 94, 1990 from lipoxygenase-1 of soybean (26). In the present study, we describe a lipoxygenase activity associated with microsomal membranes from tomato fruit that comprises 38% ofthe total (microsomal plus soluble) lipoxygenase activity in the tissue, appears to utilize free fatty acid released from membrane phospholipids as substrate and is immunologically related to its soluble counterpart and to soybean lipoxygenase-1. MATERIALS AND METHODS Plant Material and FractionationTomato fruit (Lycopersicon esculentum L. cv Caruso) were grown under greenhouse conditions and harvested at the mature-green stage. Pericarp tissue was cut into small pieces (~10 mm3) and suspended (-1 g/mL) in cold homogenizing buffer (100 mm Mops, 10 mM EGTA, and 7% sucrose at pH 7...

Membrane-associated lipoxygenase from green tomato (Lycopersicon esculentum L. cv Caruso) fruit has been purified 49-fold to a specific activity of 8.3 ,mol *min' . mg-' of protein by solubilization of microsomal membranes with Triton X-100, followed by anion-exchange and size-exclusion chromatography. The apparent molecular mass of the enzyme was estimated to be 97 and 102 kD by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and size-exclusion chromatography, respectively. The purified membrane lipoxygenase preparation consisted of a single major band following sodium dodecyl sulfate-polyacrylamide gel electrophoresis, which cross-reacts with immunoserum raised against soluble soybean lipoxygenase 1. It has a pH optimum of 6.5, an apparent Km of 6.2 Mm, and Vm,x of 10.3 jAmol-min-' mg-' of protein with linoleic acid as substrate. Corresponding values for the partially purified soluble lipoxygenase from tomato are 3.8 Mm and 1.3 Amol min-'.mg-' of protein, respectively. Thus, the membraneassociated enzyme is kinetically distinguishable from its soluble counterpart. Sucrose density gradient fractionation of the isolated membranes indicated that the membrane-associated lipoxygenase sediments with thylakoids. A lipoxygenase band with a corresponding apparent mol wt of 97,000 was identified immunologically in sodium dodecyl sulfate-polyacrylamide gel electrophoresis-resolved proteins of purified thylakoids prepared from intact chloroplasts isolated from tomato leaves and fruit.The predominant mechanism for fatty acid catabolism is fl-oxidation, which occurs in mitochondria or glyoxysomes. Although this pathway metabolizes most of the fatty acids derived from stored fats and oils, several other routes are utilized for the oxidation of specialized fatty acids. One of these alternative pathways involves the enzyme lipoxygenase (EC 1.13.11.12), which catalyzes the oxidation of a class of unsaturated fatty acids having a cis,cis-1,4-pentadiene structure. These fatty acids are primarily present as constituents of membrane phospholipids. The existence of this enzyme 'This work was supported by grants to S

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