The creation of artificial metabolic sinks in plants by genetic engineering of key branch points may have serious consequences for the metabolic pathways being modified. The introduction into potato of a gene encoding tryptophan decarboxylase (TDC) isolated from Catharanthus roseus drastically altered the balance of key substrate and product pools involved in the shikimate and phenylpropanoid pathways. Transgenic potato tubers expressing the TDC gene accumulated tryptamine, the immediate decarboxylation product of the TDC reaction. The redirection of tryptophan into tryptamine also resulted in a dramatic decrease in the levels of tryptophan, phenylalanine, and phenylalanine-derived phenolic compounds in transgenic tubers compared with nontransformed controls. In particular, wound-induced accumulation of chlorogenic acid, the major soluble phenolic ester in potato tubers, was found to be two-to threefold lower in transgenic tubers. Thus, the synthesis of polyphenolic compounds, such as lignin, was reduced due t o the limited availability of phenolic monomers. Treatment of tuber discs with arachidonic acid, an elicitor of the defense response, led to a dramatic accumulation of soluble and cell wall-bound phenolics in tubers of untransformed potato plants but not in transgenic tubers. The transgenic tubers were also more susceptible to infection after inoculation with zoospores of Phytophthora infestam, which could be attributed to the modified cell wall of these plants. This study provides strong evidence that the synthesis and accumulation of phenolic compounds, including lignin, could be regulated by altering substrate availability through the introduction of a single gene outside the pathway involved in substrate supply. This study also indicates that phenolics, such as chlorogenic acid, play a critical role in defense responses of plants to fungal attack.

Previously uncharacterized lipid-protein microvesicles have been isolated from young and senescing bean cotyledon tissue. The microvesicles are nonsedimentable and enriched in phospholipid degradation products (free fatty acids, long-chain aldehydes, and long-chain hydrocarbons). They range from 70 to 170 nm (radius) with a mean radius of 132 nm, and it is clear from freeze-fracture electron micrographs that they are bilayered in nature. Nonsedimentable lipid-protein microvesicles containing the same products of phospholipid degradation but smaller were also formed in vitro when smooth microsomal membranes from young cotyledon tissue were treated with Ca2' to stimulate enzymatic degradation of phospholipids. The data suggest that these microvesices comprise an intermediate stage of membrane lipid deterioration. They appear to serve as a vehicle for moving phospholipid degradation products out of membranes into the cytosol during senescence and perhaps also during normal membrane lipid turnover.Membrane lipid deterioration is an inherent feature of plant senescence and of capitulation of plant tissues to certain types of stress including drought and freezing (1)(2)(3)(4). One of the clearest manifestations of this is a progressive decline in phospholipid phosphate and fatty acids resulting in an increase in the sterol/esterified fatty acid ratio in membranes and a corresponding decrease in membrane bulk lipid fluidity (1). There is also an accumulation of free fatty acids and peroxidized lipids in senescing membranes that alter the phase properties of membrane lipids and introduce packing perturbations in the bilayer that are thought to facilitate enzymatic degradation of lipids (1,(5)(6)(7). In addition, membrane phospholipids have been shown to have relatively rapid turnover rates ranging in half-life from 1 to 10 hr (8).Several lipolytic enzymes that could participate in membrane lipid turnover and in the net degradation of membrane lipids that accompanies senescence and capitulation to stress have been found associated with plant membranes. Phospholipase D activity has been detected in isolated fractions of endoplasmic reticulum, Golgi membranes, tonoplast, and plasmalemma (9-12), and it has been proposed that the membrane-associated form ofthe enzyme participates in lipid turnover (13). Phosphatidic acid phosphatase has been found on endoplasmic reticulum and in association with other isolated membrane fractions (14-16), and there are reports of lipolytic acyl hydrolase being associated with microsomal membranes (17,18). Moreover, a recent study has indicated that phospholipase D, phosphatidic acid phosphatase, and lipolytic acyl hydrolase are all enriched in the sedimentable material obtained after partial solubilization ofmicrosomes in Triton X-100, suggesting that they are tightly associated with the membranes (19).The precise mechanism by which phospholipid degradation products are removed from membranes has not been established, although there have been some reports of microvesiculation from mem...

A mixture of liquid-crystalline and gel-phase lipid domains is detectable by wide angle x-ray diffraction in smooth microsomal membranes isolated from senescent 7-day-old cotyledons, whereas corresponding membranes from young 2-day-old cotyledons are exclusively liquid-crystalline. The gel-phase domains in the senescent membranes comprise phospholipid degradation products including diacylglycerols, free fatty acids, long-chain aldehydes, and long-chain hydrocarbons. The same complement of phospholipid degradation products is also present in nonsedimentable microvesicles isolated from senescent 7-day-old cotyledons by filtration of a 250,000g, 12-hour supernatant through a 300,000 dalton cut-off filter. The phospholipid degradation products in the microvesicles form gel-phase lipid domains when reconstituted into phospholipid liposomes. Nonsedimentable microvesicles of a similar size, which are again enriched in the same gel-phase-forming phospholipid degradation products, are also generated in vitro from smooth microsomal membranes isolated from 2-day-old cotyledons when Ca2 is added to activate membrane-associated lipolytic enzymes. The Ca2+-treated membranes do not contain detectable gel-phase domains, suggesting that the phospholipid degradation products are completely removed by microvesiculation. The observations collectively indicate that these nonsedimentable microvesicles serve as a vehicle for moving phospholipid degradation products out of membrane bilayers into the cytosol. As noted previously (Yao K, Paliyath G, Humphrey RW, Hallett FR, Thompson JE [1991] Proc NatI Acad Sci USA 88: 2269-2273), the term "deteriosome" connotes this putative function and would serve to distinguish these microvesicdes from other cytoplasmic microvesicles unrelated to deterioration.Membrane deterioration attributable to various physicochemical alterations in the bilayer is an integral feature of senescence. Earlier studies have identified chemical changes in senescing membranes including loss of phospholipid, an increase in sterol to fatty acid ratio, a decrease in esterified fatty acids, and enhanced levels of free fatty acids (5,8,17,21,25 (14,15,20,22). Two of these enzymes, phospholipase D and phosphatidic acid phosphatase, are stimulated by physiological levels of Ca2, and the Ca 2 stimulation of phosphatidic acid phosphatase is calmodulin mediated (20,22). One of the phospholipid catabolites formed by these enzymes is diacylglycerol (20,22), which has been shown previously to promote microvesiculation from the erythrocyte plasma membrane (1). In the present study, we found that nonsedimentable microvesicles, which are present in senescent tissue and can be formed in vitro by treatment of isolated membranes with Ca'2 (27), are enriched in diacylglycerols and other phospholipid degradation products able to form gel-phase domains in lipid bilayers. MATERIALS AND METHODS Plant Material and Membrane IsolationBean seeds (Phaseollis vulgaris L. cv Kinghorn) were germinated in vermiculate at 29°C and 90% RH under c...

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