Chalcone synthase (CHS) and stilbene synthase (STS) are related plant polyketide synthases belonging to the CHS superfamily. CHS and STS catalyze common condensation reactions of p-coumaroyl-CoA and three C 2 -units from malonylCoA but different cyclization reactions to produce naringenin chalcone and resveratrol, respectively. Using purified Pueraria lobata CHS and Arachis hypogaea STS overexpressed in Escherichia coli, bisnoryangonin (BNY, the derailed lactone after two condensations) and p-coumaroyltriacetic acid lactone (the derailed lactone after three condensations) were detected from the reaction products. More importantly, we found a crossreaction between CHS and STS, i.e. resveratrol production by CHS (2.7^4.2% of naringenin) and naringenin production by STS (1.4^2.3% of resveratrol), possibly due to the conformational flexibility of their active sites.z 1999 Federation of European Biochemical Societies.

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Daurichromenic acid (DCA) synthase catalyzes the oxidative cyclization of grifolic acid to produce DCA, an anti-HIV meroterpenoid isolated from We identified a novel cDNA encoding DCA synthase by transcriptome-based screening from young leaves of The gene coded for a 533-amino acid polypeptide with moderate homologies to flavin adenine dinucleotide oxidases from other plants. The primary structure contained an amino-terminal signal peptide and conserved amino acid residues to form bicovalent linkage to the flavin adenine dinucleotide isoalloxazine ring at histidine-112 and cysteine-175. In addition, the recombinant DCA synthase, purified from the culture supernatant of transgenic , exhibited structural and functional properties as a flavoprotein. The reaction mechanism of DCA synthase characterized herein partly shares a similarity with those of cannabinoid synthases from, whereas DCA synthase catalyzes a novel cyclization reaction of the farnesyl moiety of a meroterpenoid natural product of plant origin. Moreover, in this study, we present evidence that DCA is biosynthesized and accumulated specifically in the glandular scales, on the surface of plants, based on various analytical studies at the chemical, biochemical, and molecular levels. The extracellular localization of DCA also was confirmed by a confocal microscopic analysis of its autofluorescence. These data highlight the unique feature of DCA: the final step of biosynthesis is completed in apoplastic space, and it is highly accumulated outside the scale cells.

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Elicitor-induced production of the phytoalexin, 6-methoxymellein, in cultured carrot cells was appreciably depressed by the calmodulin inhibitors N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide and trifluo-perazine. An inhibitor of Ca2-phospholipid dependent protein kinase (protein kinase C), 1-(5-lisquinolinesulfonyl)-2-methylpiperazine, also inhibited the phytoalexin production in carrot. Both phorbol ester and synthetic diacylglycerol, activators of protein kinase C, showed an ability to induce 6-methoxymellein production even in the absence of elicitor.Phosphatidylinositol-degrading phospholipase activity increased in elicitor-treated carrot cells without a notable lag, and a product of this reaction, inositol trisphosphate, appeared to increase in parallel with the phospholipase activity. These results suggest that breakdown of phosphatidylinositol takes place in the elicitor-treated carrot cells. The messengers liberated from the phospholipid in the plasma membrane may participate in the elicitation process by controlling the activity of protein kinase C-like enzyme(s) and Ca2@-inediated processes including calmodula.

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Increase in cytoplasmic cyclic AMP concentration stimulates Ca2' influx through the cyclic AMP-gated cation channel in the plasma membrane of cultured carrot cells. However, the Ca" current terminated after a few minutes even in the presence of high concentrations of cyclic AMP indicating that hydrolysis of the nucleotide is not responsible for stop of the Ca2' influx. Cyclic AMP evoked discharge of Ca*+ from inside-out sealed vesicles of carrot plasma membrane, and it was strongly inhibited when the suspension of the vesicles was supplemented with 1 PM of free Ca", while Cazf lower than 0.1 PM did not affect the Ca*'-release. The Ca '+ flux across plasma membrane was restored from this Ca2'-induced inhibition by the addition of calmodulin inhibitors or anti-calmodulin. These results suggest that Ca2' influx initiated by the increase in intracellular CAMP in cultured carrot cells is terminated when the cytosolic Ca" concentration reaches the excitatory level in the cells, and calmodulin located in the plasma membrane plays an important role in the response decay of the cyclic nucleotide-gated Ca" channel.