SummaryUnlike other eukaryotes, which can synthesize polyamines only from ornithine, plants possess an additional pathway from arginine. Occasionally non-enzymatic decarboxylation of ornithine could be detected in Arabidopsis extracts; however, we could not detect ornithine decarboxylase (ODC; EC 4. 1.1.17) enzymatic activity or any activity inhibitory to the ODC assay. There are no intact or degraded ODC sequences in the Arabidopsis genome and no ODC expressed sequence tags. Arabidopsis is therefore the only plant and one of only two eukaryotic organisms (the other being the protozoan Trypanosoma cruzi) that have been demonstrated to lack ODC activity. As ODC is a key enzyme in polyamine biosynthesis, Arabidopsis is reliant on the additional arginine decarboxylase (ADC; EC 4.1.1.9) pathway, found only in plants and some bacteria, to synthesize putrescine. By using site-directed mutants of the Arabidopsis ADC1 and heterologous expression in yeast, we show that ADC, like ODC, is a head-to-tail homodimer with two active sites acting in trans across the interface of the dimer. Amino acids K136 and C524 of Arabidopsis ADC1 are essential for activity and participate in separate active sites. Maximal activity of Arabidopsis ADC1 in yeast requires the presence of general protease genes, and it is likely that dimer formation precedes proteolytic processing of the ADC pre-protein monomer.

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Genetic engineering has allowed the production of plants with an altered content of secondary metabolites. Because secondary metabolites are important in the defense of plants against pathogens, such engineered plants may show an increase in resistance against pathogens. For example, the expression of a stilbene synthase from Vitis vinifera in tobacco (Nicotiana tabacum) led to the accumulation of stilbenes and thereby to an increased resistance to Botrytis cinerea, providing direct evidence of the role of stilbenes as phytoalexins (Hain et al., 1993). In other experiments the accumulation of secondary metabolites, which are already produced in the untransformed plants, has been increased by the overexpression of structural genes encoding biosynthetic enzymes. Expression of hyoscyamine 6-hydroxylase in Atropa belladonna plants, for example, led to a strong increase of scopolamine production in the transgenic plants (Yun et al., 1992), and expression of a bacterial Lys decarboxylase in tobacco increased the production of the diamine cadaverine (Fecker et al., 1993). Such experiments may prove useful for the production of The commercial production of a pharmaceutical substance by plant cell culture has already been realized on an industrial scale in the case of shikonin, a naphthoquinone pigment with antibacterial, antiphlogistic, and woundhealing properties that is obtained from cell cultures of Lithospermum erythrorhizon (Tabata and Fujita, 1985). Shikonin is biosynthetically derived from 4HB and geranylpyrophosphate (Heide and Tabata, 1987). Using feeding experiments with [1,7-13C,Jshikimic acid ( Fig. 1; Heide et al., 1989), we have shown that the production of 4HB in these cell cultures proceeds exclusively via phenylpropanoid intermediates, and it has been proposed that most benzoic acids as well as ubiquinones are derived from the phenylpropanoid pathway in plants (Pennock and Threlfall, 1983). Furthermore, the conversion of the phenylpropanoid precursors to 4HB was recently characterized (Loscher and Heide, 1994)' showing that the reaction sequence from chorismate to 4HB in plants involves up to 10 successive enzymatic reaction steps in cell cultures of Lithospermum erythrorhizon.Escherichia coli, on the other hand, possesses a simpler biosynthetic route to 4HB, which involves the direct conversion of chorismate to 4HB by CPL (Fig. lb). The cloning of ubiC, the gene encoding CPL, was recently reported by our group (Siebert et al., 1992(Siebert et al., , 1994 and by Nichols and Green (1992). The protein was overexpressed, purified, characterized, and shown to be a soluble protein of 19 kD.It has a K, value for chorismate of 6.1 PM, a pH optimum at 7.5, and does not require cofactors.In this study we have expressed the ubiC gene in tobacco, thereby introducing a single-step process for the production of 4HB in plants. Chorismate, the substrate of the ubiC gene product, is an intermediate of the shikimate pathway. In plants this pathway is localized in the plastid, and the existence of an additional shikimat...

Decision-making policies are subject to modulation by changing motivational states. However, so far, little is known about the neurochemical mechanisms that bridge motivational states with decision making. Here we examined whether dopamine (DA) in the nucleus accumbens core (AcbC) modulates the effects of motivational states on effort-based decision making. Using a cost-benefit T-maze task in rats, we examined the effects of AcbC DA depletions on effort-based decision making, in particular on the sensitivity of effort-based decision making to a shift from a hungry to a sated state. The results demonstrated that, relative to sham controls, rats with AcbC DA depletion in a hungry as well as in a sated state had a reduced preference for effortful but large-reward action. This finding provides further support for the notion that AcbC DA regulates how much effort to invest for rewards. Importantly, our results further revealed that effort-based decision making in lesioned rats, as in sham controls, was still sensitive to a shift from a hungry to a sated state; that is, their preferences for effortful large-reward actions became lower after a shift from a restricted to a freefeeding regimen. These finding indicate that AcbC DA is not necessarily involved in mediating the effects of a shift in motivational state on decision-making policies.

Hyperhomocysteinemia is a risk factor for vascular and neuronal lesions often observed with concomitant high levels of homocysteic acid. In contrast to homocysteine, homocysteic acid induces calcium influx into neurons, with characteristics of an excitotoxic glutamatergic agonist at elevated concentrations. On the molecular level this is correlated to fast modifications of proteins (phosphorylation and proteolysis). Within the homocysteic acid induced molecular signature we focused in more detail on phosphorylation of two proteins implicated as risk factors in schizophrenia and neurodegeneration: Dihydropyrimidinase related protein and 14-3-3 protein isoforms. Among the identified proteins there are known chaperones and oxidative metabolism enzymes, but a few are new in context of neuronal stress: Lasp-1, a vitamin D associated factor and an expressed sequence with features of a Rho GDP dissociation inhibitor. Moreover, we detect a specific proteolytic processing of heat shock protein 70 and proteindisulfide isomerase, which is abolished by vitamins (folic acid, vitamin B12, and vitamin B6), which also decrease elevated intracellular calcium levels induced by homocysteic acid.