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The effect of second stage larvae of the Southern root-knot nematode Meloidogyne incognita on the growth of Ammi majus. chlorophyll and total phenolic content in leaves have been determined. With increasing inoculum densities of nematode there was a corresponding decrease in root-shoot length, fresh and dry weight of plants, total chlorophyll content and an increase in total phenolic content in leaves. The highest rate of nematode reproduction was found at the lowest inoculum level and lowest rate of nematode reproduction at the highest inoculum level.

International audience; The biosynthesis of linear and angular furanocoumarins is still poorly understood at the molecular level, with only psoralen synthase (CYP71AJ1) identified from Ammi majus. Using cDNA probes inferred from CYP71AJ1, three orthologs were isolated from Apium graveolens (CYP71AJ2) and Pastinaca sativa (CYP71AJ3 and-4) and functionally expressed in yeast cells. CYP71AJ2 and CYP71AJ3 displayed psoralen synthase activity, whereas CYP71AJ4 only catalyzed the conversion of ()-colum-bianetin to angelicin and negligible amounts of a hydroxylated columbianetin by-product. CYP71AJ4 thus constitutes the first fully characterized P450 monooxygenase specific for the angular furanocoumarin pathway. The angelicin synthase exhibited an apparent K m of 2.1 0.4 M for ()-columbianetin and a k cat of 112 14 min 1. Moreover, the use of 3-deuterated ()-columbianetin as substrate led to an almost complete " metabolic switch, " resulting in the synthesis of anti-3-hydroxy-3-deuterated()-columbianetin. This confirms that angelicin synthase attacks columbianetin by syn-elimination of hydrogen from C-3. Sequence comparison between psoralen synthase (CYP71AJ3) and angelicin synthase (CYP71AJ4) showed 70% identity, whereas the identity dropped to 40% in those regions thought to provide the substrate recognition sites. Accordingly, CYP71AJ3 and CYP71AJ4 might be derived from a common ancestor of unknown functionality by gene duplication and subsequent molecular evolution. Furanocoumarins are natural plant metabolites characterized by a furane moiety fused to benzopyran-2-one. The position of the furane substitution distinguishes two large groups of compounds, the linear (psoralens) and the angular furanocou-marins (angelicin and derivatives) (Fig.

Crystals in 16 species of poisonous plants growing naturally in Saudi Arabia were studied with light microscopy. Three types of crystals were observed: druses, prismatics, and crystal sand. Raphides and styloids were not observed in any of the species studied. Druses occur more frequently in the leaf midrib and in the cortex and pith of the stem. In contrast, crystal sand and prismatic crystals are rare and occur in the leaf, intercostal lamina, and in the vascular tissues. The preliminary results show the absence of the three types of calcium oxalate crystals in the stem and leaf of seven species: Ammi majus L., Anagallis arvensis L., Calotropis procera Ait., Citrullus colocynthis (L.) Schard, Euphorbiapeplis L., Hyoscyamus muticus L., and Solanum nigrum L., and the presence of druses, prismatic crystals, and crystal sand either in the leaf and stem or in the leaves or stems of nine species: Anabasis articulata (Forssk.) Moq. in DC., Chenopodium album L., Convolvulus arvensis L., Datura stramonium L., Nerium oleander L., Ricinus communis L., Rumex nervosus Vahl., Pergularia tomentosa L., and Withania somnifera (L.) Dun. in DC. These observations indicate that there is no apparent relationship between the distribution of calcium oxalate crystals and the toxic organs of the plants, and supports the view that the presence of calcium oxalate crystals may not be related to plant toxicity.

The molecular clarification of linear furanocoumarin biosynthesis offers the possibility of innovativ human-therapeutic uses as well as a data basis for the clarification of angular furanocoumarin biosynthesis and evolutionary plant-insect-interactions. The furanocoumarin biosynthesis pathway leads from umbelliferone via C-prenylation to demethylsuberosin (linear furanocoumarin) or osthenol (angular furanocoumarin) and following oxidative cyclisation to (+)-marmesin or (+)-columbianetin which are moved by oxidative splitting to psoralen or angelicin. Each of these reaction steps, just as the hydroxylation pf psoralen to bergaptol and its O-methylation to bergapten, has already been proved in former investigations in vitro. The membrane-connection in combination with oxygen- and NADP(H)-dependence of the involved enzymes, except prenyltransferase and bergaptol O-methyltransferase, point at Cyt P450 enzymes. At the beginning of this work cinnamic 4-hydroxylase (C4H, Hübner et al., in 2003) and bergaptol O-methyltransferase (BOMT, Hehmann et al., in 2004) were the only molecularly characterized enzymes of the furanocoumarin biosynthesis from Ammi majus. The Cyt P450 C4H catalyzes an "early" reaction before the specific way to coumarins and is involved in several metabolic pathways. The dissolvable BOMT is a furanocoumarin-specific enzyme in the "late" course of the furanocoumarin biosynthesis. The molecular characterisation of a furanocoumarin-specific Cyt P450 was not known up to now yet. Because a traditional cleaning of the enzymes seemed not possible, the priority aim of this work consisted in identifying furanocoumarin-specific Cyt P450s by cDNA amplification and functional expression. Ammi majus is, beside the interest in its meaning as a medical plant, particularly suited for this investigation, because in darkly cultured cell suspensions no furanocoumarins and specific enzyme activities exist. After Pmg-elicitation of the cell culture a quick furanocoumarin accumulation appears and presumably within this time window the specific transcripts are induced transient. On this basis a differential cloning strategy under inclusion of new technologies seemed promising for the isolation of furanocoumarin-specific Cyt P450-transcripts. A functional identification of these transcripts as furanocoumarin-specific enzymes should allow the exact measurement of the induction under influence of different elicitors and the molecular comparisons and phylogenetic studies, respectively. Another aim was the tissue-specific localisation of the furanocoumarin biosynthesis from the juvenile up to the adult plant. The relevant statements in the literature are partly contradictory and mostly concentrate upon root and fruits. The proof of furanocoumarin-specific transcripts in different tissues during the plant development can deliver a more exact picture and clear questions to the transport of coumarins and to the age dependence of the biosynthesis in connection with the product accumulation. The differential isolation of Cyt P450s from induced Ammi majus cells had already delivered some not yet functionally characterized clones, among the rest CYP71AJ1 (Specker, in 2003). In the course of this work CYP71AJ1 was identified as Psoralensynthase (POS) (Larbat et al., in 2007) and molecularly characterized. POS is therefore the first genetically and biochemically characterized Cyt P450 from the furanocoumarin biosynthesis. The strongest induction of the furanocoumarin biosynthesis in cell suspension cultures was observed with Pmg-elicitor. The time window of furanocoumarin generation laid during the first 10 hours, then, based on the transcript-profil of C4H, presumably other pathways were induced. In the plant the induction of specific transcripts could be localised above all in roots and blossoms, while the furanocoumarins accumulated in sheets, as well as in the later developing course in the reproductive organs. The additional transcript-accumulation in blossoms makes clear the meaning of furanocoumarins as phytoalexines and allelopathic germination inhibitors, respectively. The temporal pattern, the dependence on different elicitors and the tissue-specificity during the plant development are helpfully for the search for further furanocoumarin-specific Cyt P450s. In this work three new Cyt P450-transcripts and -gDNAs (CYP71AZ1, CYP71D97, CYP71D98) were isolated and characterized. The substrate-recognition sites and the induction pattern of CYP71AZ1 let suppose an involvement in the furanocoumarin biosynthesis. Indeed, up to now the functional expression could not be reached., Die molekulare Aufklärung der linearen Furanocumarinbiosynthese bietet die Möglichkeit innovativer humantherapeutischer Anwendungen sowie die Datengrundlage für die Aufklärung der angulären Furanocumarinbiosynthese und evolutionärer Pflanzen-Insekt-Interaktionen. Der Furanocumarinbiosyntheseweg führt von Umbelliferon über C-Prenylierung zu Demethylsuberosin (lineare Furanocumarine) bzw. Osthenol (anguläre Furanocumarine) und die nachfolgende oxidative Zyklisierung zu (+)-Marmesin bzw. (+)-Columbianetin, welche durch oxidative Spaltung zu Psoralen bzw. Angelicin umgesetzt werden. Jeder dieser Reaktionsschritte, ebenso wie die Hydroxylierung von Psoralen zu Bergaptol und dessen O-Methylierung zu Bergapten, wurde in früheren Untersuchungen in vitro nachgewiesen. Die Membran-Bindung in Kombination mit der Sauerstoff- und NADP(H)-Abhängigkeit der beteiligten Enzyme, mit Ausnahme der Prenyltransferase und der Bergaptol O-Methyltransferase, lassen auf Cyt P450s schließen. Molekular charakterisiert waren zu Beginn dieser Arbeit aus Ammi majus nur die Zimtsäure 4-Hydroxylase (C4H, Hübner et al., 2003) sowie die Bergaptol O-Methyltransferase (BOMT, Hehmann et al., 2004). C4H katalysiert eine „frühe“ Reaktion noch vor dem spezifischen Weg zu Cumarinen und ist an mehreren Stoffwechselwegen beteiligt, das lösliche BOMT ist ein Furanocumarin-spezifisches Enzym im „späten“ Verlauf der Furanocumarinbiosynthese. Die molekulare Charakterisierung eines Furanocumarin-spezifischen Cyt P450 war bislang noch nicht bekannt. Da eine traditionelle Reinigung der Enzyme nicht möglich erschien, bestand das vorrangige Ziel der Arbeit darin, Furanocumarin-spezifische Cyt P450s durch cDNA-Amplifikation und funktionelle Expression zu identifizieren. Ammi majus eignet sich, neben dem Interesse an seiner Bedeutung als Arzneipflanze, besonders für dieses Vorhaben, weil in dunkel kultivierten Zellsuspensionskulturen keine Furanocumarine und spezifische Enzymaktivitäten vorhanden sind. Nach Pmg-Elicitierung der Zellkultur kommt es jedoch zu einer raschen Furanocumarin-Akkumulation, wobei innerhalb dieses Zeitfensters vermutlich die spezifischen Transkripte transient induziert werden. Auf dieser Grundlage erschien für die Isolierung Furanocumarin-spezifischer Cyt P450-Transkripte eine differentielle Klonierungs-Strategie unter Einbezug neuer Technologien Erfolg versprechend. Eine funktionelle Identifizierung eines oder mehrerer dieser Transkripte als Furanocumarin-spezifisches Enzym sollte die exakte Messung der Induktion unter verschiedenen Stressoren und molekulare Vergleiche bzw. phylogenetische Studien ermöglichen und dadurch die Identifizierung weiterer Enzyme dieses Stoffwechselweges erleichtern. Ein weiteres Ziel war die gewebespezifische Lokalisation des Furanocumarin-Stoffwechsels von der juvenilen bis zur adulten Pflanze. Die diesbezüglichen Aussagen in der Literatur sind teils widersprüchlich und konzentrieren sich meist nur auf Wurzel und Früchte. Der Nachweis Furanocumarin-spezifischer Transkripte in den verschiedenen Organen über die Entwicklung der Pflanzen kann erstmalig ein präziseres Bild liefern und im Zusammenhang mit der Produkt-Akkumulation Fragen zum Transport von Cumarinen und zur Altersabhängigkeit der Biosynthese klären. Die differentielle Klonierung aus elicitierten Ammi majus Zellen hatte bereits einige, funktionell nicht charakterisierte Cyt P450-Klone, unter anderem CYP71AJ1, geliefert (Specker, 2003). Im Verlauf dieser Arbeit konnte CYP71AJ1 als Psoralensynthase (POS) identifiziert (Larbat et al., 2007) und molekular charakterisiert werden. Die POS ist somit das erste genetisch und biochemisch charakterisierte Cyt P450 aus der Furanocumarinbiosynthese. Unter mehreren Elicitoren war die stärkste Induktion der Furanocumarinbiosynthese in Zellsuspensionskulturen mit Pmg zu beobachten. Das Zeitfenster der Furanocumarin-Bildung lag in den ersten 10 Stunden, danach wurden verstärkt weitere Stoffwechselwege induziert, wie das Transkriptmuster der C4H vermuten lässt. In der Pflanze konnte die Induktion spezifischer Transkripte v.a. in den Wurzeln und Blüten lokalisiert werden, während die Furanocumarine tendenziell in den Blättern, sowie im späteren Entwicklungsverlauf in den generativen Organen akkumulierten. Die zusätzliche Transkript-Akkumulation in den Blüten verdeutlicht die Bedeutung der Furanocumarine als Phytoalexine und als allelopathische Keimungsinhibitoren für den Fortbestand der Pflanze. Das zeitliche Muster, die Abhängigkeit von unterschiedlichen Elicitoren und die Gewebespezifität können hilfreich sein bei der Suche nach weiteren Cumarin-spezifischen Cyt P450s. Drei neue Cyt P450-Transkripte und gDNAs (CYP71AZ1, CYP71D97, CYP71D98) wurden isoliert und charakterisiert, unter denen die Substraterkennungsregionen und das Induktionsmuster von CYP71AZ1 eine Beteiligung an der Furanocumarinbiosynthese vermuten lassen. Allerdings konnte die funktionelle Expression bisher nicht erreicht werden.

The influence of light quality (direct light, red, far-red and dark) on seed germination and seedling growth was examined in six different medicinal plant species viz., Lepidium sativum Linn., Spilanthes acmella Linn., Matricaria chamomilla Linn., Ammi majus Linn., Andrographis paniculata Nees. and Cassia tora Linn. in Tarai region of Uttarakhand with three replicates for 15 days under laboratory conditions. The results revealed that the rate of germination and per cent seed germination differ from species to species and light conditions. The per cent seed germination was maximum inL. sativum (89.67% in far - red) and minimum in M. chamomilla (40.0% in direct). M. chamomilla and S. acmella was failed to germinate under dark condition, whereas L. sativum showed maximum per cent seed germination (62%). The seedling growth in terms of length (cm) and dry weight (mg) of 15 days old was significantly affected by light quality in all the species. All the species exhibited maximum radicle length (cm) under direct light except in S. acmella (6.67 in red light) whereas plumule length (cm) was maximum in C. tora (11.78), L. sativum (8.59), A. paniculata (7.18) and A. majus (5.93) in dark. Interestingly, maximum total seedling dry weight (mg) was under direct light in all the species and it was maximum in C. tora (7.06) and minimum in M. chamomilla (2.80) indicating that direct light is favourable for seedling growth.

Heterotrophic cell suspension cultures were initiated from hypocotyls of young Ammi majus L. seedlings. When these cultures were propagated continuously in the dark, they produced only traces of the coumarin umbelliferone. Upon addition of fungal cell wall fractions, i.e. a skieroglucan or an elicitor from either Phytophthora megasperma f. sp. glycinea or Alternaria carthami , the cells excreted large amounts of umbelliferone in addition to isopimpinellin, ( S )-marmesin, ( R )-ammirin, umbelliferone-[3′-methyl-buta-1 t .3-dien-1′-yl]-ether and umbelliferone-[3′-hydroxymethyl- 1 t .-buten-1′-yl]-ether. The last two compounds are new compounds and appear to be derived from 7- O -prenylumbelliferone, a coumarin that has been identified in from other plants. The Phytophthora -elicitor was the most effective inducer of coumarin accumulation. When cultures which had been elicited for 3 hr were pulsed with l -[U- 14 C]phenylalanine for 7 hr, all the coumarins and an additional, still unidentified compound in the extracts were labelled efficiently. Our results suggest that A. majus cells are particularly suitable for biosynthetic studies on various coumarins as well as for regulatory studies of the inhibition of coumarin phytoalexin accumulation.

International audience; Ammi majus L. accumulates linear furanocoumarins by cyto-chrome P450 (CYP)-dependent conversion of 6-prenylumbellif-erone via ()-marmesin to psoralen. Relevant activities, i.e. pso-ralen synthase, are induced rapidly from negligible background levels upon elicitation of A. majus cultures with transient maxima at 9 –10 h and were recovered in labile microsomes. Expressed sequence tags were cloned from elicited Ammi cells by a nested DD-RT-PCR strategy with CYP-specific primers, and full-size cDNAs were generated from those fragments correlated in abundance with the induction profile of furanocou-marin-specific activities. One of these cDNAs representing a transcript of maximal abundance at 4 h of elicitation was assigned CYP71AJ1. Functional expression in Escherichia coli or yeast cells initially failed but was accomplished eventually in yeast cells after swapping the N-terminal membrane anchor domain with that of CYP73A1. The recombinant enzyme was identified as psoralen synthase with narrow substrate specificity for ()-marmesin. Psoralen synthase catalyzes a unique carbon-chain cleavage reaction concomitantly releasing acetone by syn-elimination. Related plants, i.e. Heracleum mantegazzianum, are known to produce both linear and angular furanocoumarins by analogous conversion of 8-prenylumbelliferone via ()-columbianetin to angelicin, and it was suggested that angelicin synthase has evolved from psoralen synthase. However, ()-columbianetin failed as sub-strate but competitively inhibited psoralen synthase activity. Analogy modeling and docked solutions defined the conditions for high affinity substrate binding and predicted the minimal requirements to accommodate ()-columbianetin in the active site cavity. The studies suggested that several point mutations are necessary to pave the road toward angelicin synthase evolution.

The objective of this work was to evaluate the efficiency of a new method, developed for predicting density and floristic composition of weed communities in field crops. Based on the use of solaria (100 mm transparent plastic tarps lying on the soil) to stimulate weed seedlings emergence, the method was tested in Tandil, Argentina, from 1998 to 2001. The system involved corn and sunflower in commercial no-till system. Major weeds in the experiments included Digitaria sanguinalis, Setaria verticillata and S. viridis, which accounted for 98% of the weed community in the three years of experiments since 1998. Large numbers of Tagetes minuta, Chenopodium album and Ammi majus were present in 2001. Comparison of weed communities under solaria with communities in field crops indicated that the method is useful for predicting the presence and density of some major weed species, at both high and low densities, of individuals in areas of 10 ha using only five solaria. Low density of weed species makes the method particularly useful to help deciding the time for herbicide applications to avoid soil contamination. O objetivo deste trabalho foi avaliar a eficiência de novo método, desenvolvido para predizer a densidade e a composição florística das comunidades de plantas daninhas. O método é baseado no uso de solaria (plástico transparente de 100 mm sobre o solo), a fim de estimular o aparecimento de ervas daninhas, e foi testado em Tandil, Argentina, de 1998 a 2001. O sistema envolve milho e girassol, em cultivos comerciais em semeadura direta. As principais espécies experimentadas foram Digitaria sanguinalis, Setaria verticillata e S. viridis, que responderam por 98% da comunidade nos três anos de experiência. Números altos de Tagetes minuta, Chenopodium album e Ammi majus estavam presentes em 2001. A comparação de comunidades de ervas daninhas sob solaria com comunidades fora de solaria indicou que o método é útil para predizer a presença e a densidade de espécies com densidades altas e baixas de indivíduos em áreas de 10 ha com o uso apenas de cinco solaria. Espécies com baixas densidades tornam o método particularmente útil para ajudar a decidir o uso ou não de herbicidas para evitar a contaminação da terra.

Furocoumarins are phytoalexins known as efficient therapeutic agents. The work reported here focuses on cytochromes P450 involved in their biosynthesis pathway. A ¤structure-function¤ study was realized to understand how C4H from Ruta graveolens (CYP73A32) can resist to psoralen mechanism-based inactivation. Two parts of the protein seems involved in the differential susceptibility of CYP73A32 and CYP73A1 to psoralen. The first, between amino acids 31 and 58, defines differential affinity to psoralen. The second between residues 229 and 379 controls inactivation kinetic. The second part of this work was devoted to cloning and identification of new P450 involved in furocoumarin biosynthesis. On the one hand, several partial cDNA were cloned from Ruta graveolens. On the other hand, CYP71AJ1, cloned from Ammi majus, was identified as a psoralen synthase. The specificity of CYP71AJ1 for marmesin was approached by the study of a 3D model., Les furocoumarines sont des métabolites secondaires végétaux jouant un rôle de phytoalexines chez les plantes et présentant un potentiel thérapeutique important, notamment en dermatologie. Les travaux présentés dans ce document se sont focalisés sur l’étude des cytochromes P450 participant à leur voie de biosynthèse. Une étude des relations structure-fonction de la cinnamate-4-hydroxylase (C4H) a été réalisée. Les C4H de Ruta graveolens (CYP73A32), Petroselinum crispum (CYP73A10) et Ammi majus (CYP73A41), trois plantes productrices de furocoumarines, sont moins sensibles à l’inactivation autocatalytique par le psoralène que celle d’Helianthus tuberosus (CYP73A1) qui n’en produit pas. L’étude de mutants et de protéines chimères entre CYP73A1 et CYP73A32 a montré (i) que la « résistance » au psoralène des C4H de plantes productrices de furocoumarines est sans doute issue de processus évolutifs distincts et (ii) que deux régions protéiques sont impliquées dans l’inactivation différentielle entre CYP73A32 et CYP73A1. L’une, située entre les acides aminés 31 et 58, détermine la différence d’affinité pour le psoralène. L’autre, entre les résidus 229 et 379, contrôle la vitesse d’inactivation. L’étude d’un modèle 3D de CYP73A32 a permis d’étayer plusieurs hypothèses sur le rôle de résidus précis dans ces deux régions. Un travail de clonage et d’identification de nouveaux P450 impliqués dans la biosynthèse des furocoumarines a été entrepris. D’une part, plusieurs ADNc partiels ont été clonés par PCR à partir de tissus de Ruta graveolens enrichis en furocoumarines. D’autre part, la fonction de CYP71AJ1 isolé à partir de cultures cellulaires d’Ammi majus, a été identifiée. Il catalyse la conversion de la (+)-marmésine en psoralène correspondant à l’activité psoralène synthase et constitue le 1er P450 cloné et de fonction connue dans la voie des furocoumarines. La spécificité de CYP71AJ1 pour la (+)-marmésine a été approchée par l’étude d’un modèle 3D.

Plants belonging to the Apiaceae or Rutaceae accumulate methoxylated psoralens, such as bergapten or xanthotoxin, as the final products of their furanocumarin biosynthesis, and the rate of accumulation depends on environmental and other cues. Distinct O-methyltransferase activities had been reported to methylate bergaptol to bergapten and xanthotoxol to xanthotoxin, from induced cell cultures of Ruta graveolens, Petroselinum crispum and Ammi majus. Bergaptol 5-O-methyltransferase (BMT) cDNA was cloned from dark-grown Ammi majus L. cells treated with a crude fungal elicitor. The translated polypeptide revealed considerable sequence similarity to heterologous caffeic acid 3-O-methyltransferases (COMTs). For homologous comparison, COMT was cloned from A. majus plants and shown a 64% identity with the BMT sequence at the polypeptide level. Functional expression of both enzymes in Escherichia coli revealed that the BMT activity in the bacterial extracts was labile and rapidly lost on purification, whereas theCOMT activity remained stable. Furthermore, the recombinant AmBMT showed narrow substrate specificity for bergaptol, while the AMCOMT accepted 5-hydroxyferulic acid, esculetin and other substrates. The AmBMT sequence thus represents a novel database accession specific for the biosynthesis of psoralens., Vertreter der Apiaceae oder Rutaceae akkumulieren methoxylierte Psoralene wie Bergapten oder Xanthotoxin als Endprodukte der Furanocumarinbiosynthese. O-Methyltransferase-Aktivitäten mit Umsetzungen von Bergaptol zu Bergapten bzw. Xanthotoxol zu Xanthotoxin wurden bereits aus induzierten Zellkultursystemen von Ruta graveolens, Petroselinum crispum und Ammi majus beschrieben. Im Rahmen dieser Arbeit wurde die Bergaptol 5-O-Methyltransferase (BMT) als erstes Enzym der Furanocumarinbiosynthese aus Ammi majus L. in ihrer cDNA und Aminosäuresequenz aufgeklärt und biochemisch charakterisiert. Das translatierte Polypeptid zeigte hohe Sequenzhomologien zu heterologen Kaffeesäure 3-O-Methyltransferasen (COMTs). Daher wurde zum Sequenzvergleich eine COMT aus Ammi majus L. Pflanzen kloniert, die 64% Identität zur BMT auf Aminosäureebene aufweist. Die funktionelle Expression beider Enzyme in Escherichia coli zeigte, dass die BMT Aktivität in bakteriellen Rohextrakten labil ist und beim Anreinigen verloren geht. Die COMT Aktivität dahingegen bleibt stabil. Während die BMT eine hohe Spezifität für ihr Substrat Bergaptol aufweist, methyliert die COMT 5-Hydroxyferulasäure, Esculetin und weitere Substrate. Die BMT-Sequenz aus Ammi majus stellt einen neuen spezifischen Zugang auf molekularer Ebene zur Biosynthese der Furanocumarine dar.

Plants belonging to the Apiaceae or Rutaceae accumulate methoxylated psoralens, such as bergapten or xanthotoxin, as the final products of their furanocoumarin biosynthesis, and the rate of accumulation depends on environmental and other cues. Distinct O-methyltransferase activities had been reported to methylate bergaptol to bergapten and xanthotoxol to xanthotoxin, from induced cell cultures of Ruta graveolens, Petroselinum crispum and Ammi majus. Bergaptol 5-O-methyltransferase (BMT) cDNA was cloned from dark-grown Ammi majus L. cells treated with a crude fungal elicitor. The translated polypeptide of 38.7 kDa, composed of 354 amino acids, revealed considerable sequence similarity to heterologous caffeic acid 3-O-methyltransferases (COMTs). For homologous comparison, COMT was cloned from A. majus plants and shown to share 64% identity and about 79% similarity with the BMT sequence at the polypeptide level. Functional expression of both enzymes in Escherichia coli revealed that the BMT activity in the bacterial extracts was labile and rapidly lost on purification, whereas the COMT activity remained stable. Furthermore, the recombinant AmBMT, which was most active in potassium phosphate buffer of pH 8 at 42 degrees C, showed narrow substrate specificity for bergaptol (Km SAM 6.5 micro m; Km Bergaptol 2.8 micro m) when assayed with a variety of substrates, including xanthotoxol, while the AmCOMT accepted 5-hydroxyferulic acid, esculetin and other substrates. Dark-grown A. majus cells expressed significant BMT activity which nevertheless increased sevenfold within 8 h upon the addition of elicitor and reached a transient maximum at 8-11 h, whereas the COMT activity was rather low and did not respond to the elicitation. Complementary Northern blotting revealed that the BMT transcript abundance increased to a maximum at 7 h, while only a weak constitutive signal was observed for the COMT transcript. The AmBMT sequence thus represents a novel database accession specific for the biosynthesis of psoralens.

Pathogenes excites a complexe system of biological reactions in plants. These are caused by certain structures on the pathogene surface, so called elicitors. The result of the elicitor-inudced signal cascade is the activation of defense genes. One effect is the accumulation of phytoalexines, in case of the Apiaceae, furanocoumarines. Former studies have shown, that several Pmg-inducible cytochrome P-450 are involved in furanocoumarine biosynthesis. Five Cyt P450 were cloned from Pmg-induced Ammi majus cell cultures, three of them inucible at transciption level, which was shown by Northern blotting. Among these clones AmCYP71AJ1 was most inducible. Sequence 1, CYP73A41, was functionally identified and biochemically characterised as cinnamate-4-hydroxylase. All parameters correlate to known cinnamate-4-hydroxylases. In the future the function of the other Cyt P450 needs to be determined by identifying possible substrates and by improving and controlling expression. Therefore, the NADPH-cytochrome P450 reductase from Ammi majus was cloned with the purpose of coexpression with the investigated Cyt P450 in yeast. Sequence 2 (AmCYP71AJ1) has shown 66 % similarity with menthofuransynthase from Mentha x piperita (Q947B7, Bertea et al., 2001) in sequence alignments; however, menthofuran has not been described in Ammi majus, so probably AmCYP71AJ1 does not catalyze this reaction. The course of induction at transcription level corresponds to marmesinsynthase activity, an enzyme of the furanocoumarine pathway. Therefore, participation of AmCYP71AJ1 in unspecific plant defense mechanisms is likely. It may even belong to the furanocoumarine biosynthesis. Up to 83 % of the amino acids of the third sequence (AmCYP98A21) are identical with Ocimum basilicum p-camaroyl shikimate 3`-hydroxylase. AmCYP98A21 is also inducible at transcription level. Sequence 4 (AmCYP76B8) shares 55 % identity with geraniol-10-hydroxylase (G10H) from Catharanthus roseus. In contrast to G10H Northern analysis showed no induction. A classification of the fifth sequence (AmCYP82H1) was not possible. The results of this investigation are not only of scientific interest, but have also practical value. Knowledge of the furanocoumarine biosynthesis is of general interest, for it has been shown, that the amount of furanocoumarines in cellery strongly increases because of acid fog in congested areas in California. The amount of furanocoumarines may then by high enough to cause dermatitis if consumed by humans, and is toxic for insects as well. The biosynthesis of furanocoumarines could be regulated to produce psoralen-free cellery, which could therefore be cultivated despite air pollution., Pflanzen reagieren auf Pathogenbefall mit einem komplexen System von physiologischen Reaktionen. Auslöser ist die Erkennung von Oberflächenstrukturen der Pathogene, die als Elicitoren wirken. Am Ende der Elicitor-induzierten Signaltransduktionskette steht die Aktivierung von Abwehrgenen. Ein Teilaspekt ist dabei die Akkumulation von Phytoalexinen, im Falle der Apaiceae sind es Furanocunmarine. Aus Einbaustudien und in vitro-Messungen ist bekannt, dass an der Biosynthese der Furanocumarine mehrere Cyt P450 beteiligt sind, die durch Hefe oder pilzlichen Elicitor induziert werden können. Aus cDNA von Elicitor-induzierten Ammi majus-Zellkulturen wurden fünf Cyt P450 kloniert, deren Northern-Analyse ergab, dass drei davon auf transkriptioneller Ebene induzierbar sind, wobei AmCYP71AJ1 am deutlichsten induziert wird. Sequenz 1, CYP73A41, wurde funktionell als Zimtsäure 4-Hydroxylase identifiziert und biochemisch charakterisiert. Ihre Eigenschaften entsprechen im Wesentlichen den bereits klonierten C4Hs. Es bleibt die Identifizierung der anderen Cyt P450 durch Verbesserung und Kontrolle der Expression und Suche nach möglichen Substraten. Auf dem Wege zu einer Verbesserung der Expression wurde die NADPH-Cytochrom P450 Reduktase (CPR) kloniert mit dem Ziel eine simultane Expression der CPR und des zu untersuchenden Cyt P450 in Hefe durchzuführen. Im Sequenzvergleich weist Sequenz 2 (AmCYP71AJ1) auf der Ebene der Aminosäuren 66 % Ähnlichkeit zur Menthofuransynthase (MFS) aus Mentha x piperita (Q947B7, Bertea et al, 2001) auf. Da kein Menthofuran in Ammi majus beschrieben worden, ist es unwahrscheinlich, dass AmCYP71AJ1 die Bildung von Menthofuran katalysiert. Die Induktionsstärke und der Induktionsverlauf der relativen Transkriptmenge von AmCYP71AJ1 passen zu der Induktion der Marmesinsynthase-Aktivität, einem Cyt P450 der Psoralenbiosynthese. Vermutlich handelt es sich bei AmCYP71AJ1 um ein Enzym der unspezifischen Abwehr, eventuell sogar aus der Furanocumarinbiosynthese. Die Sequenz 3 (AmCYP98A21) ist auf Aminosäureebene zu 83 % identisch mit der Ocimum basilicum p-Cumaroyl Shikimat 3'-Hydroxylase und ist auf Transkriptiosnebene induzierbar. Die Sequenz 4 (AmCYP76B8) hatte 55 % identische Aminosäuren mit der Geraniol 10-Hydroxylase aus Catharanthus roseus. Der Northern-Blot zeigte im Gegensatz zur G10H aus Catharanthus roseus keine Induktion. Eine Zuordnung der nicht induzierten Sequenz 5 (AmCYP82H1) kann nicht erfolgen. Neben der wissenschaftlichen Grundlage haben die Untersuchungen auch praktische Bedeutung. Kenntnisse über die Biosynthese der Furanocumarine sind für den Menschen von Interesse. Es hat sich gezeigt, dass der Gehalt an Furanocumarinen in Sellerie aufgrund von saurem Nebel in kalifornischen Ballungsgebieten stark zunimmt, so dass die Konzentration über der Menge liegt, die beim Menschen nach Verzehr eine Dermatitis auslösen kann. Zudem wird eine solche Pflanze ungeniessbar für Insekten. Indem man regulierend in die Biosynthese eingreift, könnte man Sellerie herstellen, der keine Psoralene enthält, und somit auch in Gebieten mit grosser Luftverschmutzung angebaut werden kann.

Total RNA was isolated from dark-grown cell suspension cultures of Ammi majus L. that had been induced with fungal elicitor or treated with water for control and used as template with cytochrome P450-specific primers for DD-RT-PCR amplifications. A cDNA clone was generated from the elicited transcripts and assigned to cinnamate 4-monooxygenase based on sequence alignments and functional expression in yeast cells. Comparison of the translated polypeptide with database accessions of heterologous cytochrome P450 monooxygenases revealed a high degree of similarity (99.6%) with 98.6% identity to cinnamic acid 4-hydroxylase from parsley, documenting the close evolutionary relationship within the Apiaceae family. Maximal activity of the Ammi hydroxylase in yeast microsomes was determined at 25 °C and in the pH range of 6.5–7.0 reaching 2.5 pkat/mg on average. An apparent K m of 8.9 μM was determined for cinnamate. Preincubations with psoralen or 8-methoxypsoralen added up to 100 μM in the presence or absence of NADPH hardly affected the turnover rate. A. majus cell cultures accumulate sets of O -prenylated umbelliferones and linear furanocoumarins besides lignin-like compounds upon treatment with elicitor, and cinnamic acid 4-hydroxylase catalyzes the initial reaction leading from the general into the various phenylpropanoid branch pathways. Correspondingly, the hydroxylase transcript abundance was induced in the elicited cells.

Ammi majus and Ruta graveolens plants were chosen to establish a co-culture due to their abilities to produce secondary metabolites from the furanocoumarin (FC) family. The conditions for cultivating A. majus hairy roots and R. graveolens shoots separately were established earlier. In continuation, the conditions of the co-culture of A. majus and R. graveolens were determined in this work. Two systems of co-cultivation were tested: A. majus hairy roots with R. graveolens cell suspension and A. majus hairy roots with R. graveolens shoots. The second model was chosen for further experiments, namely to test different tissues inoculation ratios as well as the influence of the photoperiod and darkness conditions on A. majus and R. graveolens tissues growth and the accumulation of secondary metabolites. The growth index ( t 25 / t 1 ) of R. graveolens shoots was higher in the co-cultures; moreover, 2.5 times higher concentration of xanthotoxin was observed in the co-culture in light conditions.

Axenically grown Ammi majus plantlets were inoculated with seven different Agrobacterium rhizogenes strains. Hairy root lines were established only after inoculation with the two agropine strains: A4 and LBA9402. The growth rate of hairy root cultures was about thirty times faster than that of callus and cell suspension cultures. Polymerase chain reaction with primers for the genes rolB and rolC confirmed the integration of the T-DNA fragment of Ri plasmid of A. rhizogenes to the genome of hairy roots obtained after transformation by both Agrobacterium strains. The furanocoumarins (psoralen, xanthotoxine, bergapten and imperatorin) usually found in seeds of A. majus were not detected in callus, cell suspension and hairy root cultures using Gas chromatography–mass spectrometry (GC–MS). However, umbelliferone, a precursor of furanocoumarins, was detected in callus, cell suspension and hairy root cultures. The umbelliferone content in extracts of hairy root cultures, obtained after transformation by A4, was similar to that determined in A. majus seeds (19 μg/g DW) and higher than those obtained for cell suspension and callus cultures (2 and 9 μg/g DW, respectively).

Our laboratories have reported on the predatory minute pirate bugs (Family Anthocoridae) in a research colony that was obtained in Dec 2002. The species was originally thought to be Orius insidiosus (Say) (Ferkovich & Shapiro 2004a, 2004b, 2007; Ferkovich & Shapiro 2005a, 2005b, 2005c; Ferkovich et al. 2007). However, specimens from the colony were identified as O. pumilio (Champion) by T. Lewis (USDA, ARS, Wapato, WA) in Apr 2008, not O. insidiosus as previously reported in the publications listed above. In response to the discovery of O. pumilio in our acquired colony, repeated collections from flower heads of false Queen Anne’s Lace (Ammi majus) on an organic farm in Gainesville, Florida, yielded both species in unequal numbers and at differing sex ratios. Conclusive identifications of O. pumilio from the laboratory colony and field collections, and O. insidiosus from field collections, were confirmed by T. Henry (USDA-ARS Systematic Entomology Laboratory, National Museum of Natural History, Smithsonian Institution, Washington, D.C.).

Cultured cells of the Apiaceae, e. g. PETROSELINUM CRISPUM and AMMI MAJUS, produce large quantities of coumarin phytoalexins upon treatment with fungal elicitors and concomitantly reinforce their cell walls by ferulic ester incorporation. In these reactions, the cells mimic the disease resistance response commonly expressed in plants following infection by phytopathogenic fungi. Both coumarins and ferulic esters are derived from the general phenylpropanoid pathway. The elicited Apiaceae cell cultures have therefore served as model systems for the investigation of the biosynthetic and regulatory mechanisms involved in phenylpropanoid accumulation. The results of these studies suggest an unconventional pathway for umbelliferone biosynthesis and demonstrate both the transcriptional and post-transcriptional control of a novel pathway to feruloyl-CoA. They underline the enormous contribution of such model systems to the present knowledge on regulatory patterns and DNA coding in secondary metabolism.

Two dimethylallyl diphosphate:umbelliferone dimethylallyltransferase (prenyltransferase) activities, catalysing the 6-prenylation and the 7-O-prenylation, respectively, of umbelliferone in the course of phytoalexin synthesis, increased in Ammi majus cell suspension cultures in response to elicitor treatment. Both enzyme activities were dependent on Mg2+ or Mn2+ with significant preference for Mg2+ in the 6-prenylation reaction. Whereas dark-grown cells did not contain these activities, both prenyltransferase activities were induced rapidly by the addition of elicitor reaching a first maximum after 10-14 hr and a second maximum beyond 30 hr. Other coumarin specific, elicitor-induced enzyme activities of A. majus cells, in contrast, showed only one maximum of activity within the 50 hr experimental period, while the pattern of induction of phenylalanine ammonia-lyase activity resembled that of the prenyltransferases with maxima at ca 8 hr and 20-30 hr. Preliminary data suggest that the apparent biphasic induction of these enzyme activities is due to post-translational enzyme modifications.

Ammi majus L., a member of the Apiaceae and also known as large bullwort, false Queen Anne's lace, or bishop's-weed, is frequently used in the floral trade to add a lacey look to floral bouquets. A. majus is native to the Mediterranean Region but it is cultivated in major growing areas including Holland, Israel, the United Kingdom, and the United States. During March 2005, virus-like symptoms including mosaic, generalized chlorosis, vein clearing, interveinal chlorosis, and leaf rugosity were observed in nearly all field-grown A. majus plants at two locations in Martin County, Florida. Inclusion body morphology suggested the presence of one or more potyviruses in the symptomatic plants. Potyvirus infection was confirmed in 11 symptomatic plants using a commercially available enzyme-linked immunosorbent assay (ELISA) kit (Agdia, Elkhart, IN). Nucleotide and deduced amino acid sequences of a 1,625-bp region of one of the reverse transcription-polymerase chain reaction products amplified with degenerate potyvirus primers (1) from total RNA of symptomatic plants (GenBank Accession No. DQ333346) were 96 to 97% and 93 to 99% identical, respectively, to Clover yellow vein virus (ClYVV) sequences in GenBank. All symptomatic plants tested were potyvirus positive using ELISA, but only a subset was infected with ClYVV suggesting that the field symptoms were the result of infection with additional potyviruses, all of which are likely aphid transmitted. Although several potyviruses have been reported from A. majus (2), to our knowledge, this represents the first report of ClYVV infection. References: (1) A. Gibbs and A. Mackenzie. J. Virol. Methods 63:9, 1997. (2) P. Van Dijk and L. Bos. Neth. J. Plant Pathol. 95(Suppl.) 2:1, 1989.

Since in Ammi majus the seed set and germination is poor attempts were made to propagate the plants through tissue cultures and monitor the yield of xanthotoxin (furanocoumarin) in the resultant callus and plantlets. The callus from cotyledonary leaves obtained on MS medium supplemented with alpha-indoleacetic acid (IAA) + kinetin (KN) + casein hydrolysate (CH), differentiated shoots in 100 % cultures when adenine was added to the above medium. Plantlets resulted on MS medium with indolebutyric acid (IBA) and glutamine. The yield of xanthotoxin in regenerating cultures was more than hitherto reported from any other tissue including the seeds. Isozymes could be used as reliable markers for the characterization of various stages of growth of callus and regenerants.

The microcharacters of the epicarp of medicinally important fruits of Apiaceae (Umbelliferae) have been studied by scanning electron microscopy (SEM) to identify them more accurately and to check for adulteration and substitution. The fruits selected were Ammi majus, A. visnaga, Anethum graveolens, A. sowa, Apium graveolens, Bunium persicum, Carum carvi, Coriandrum sativum, Foeniculum vulgare (tine and coarse forms) and Trachyspermum ammi. The epicarp surface structures under SEM prove useful in their identification and have pharmacognostic and systematic significance.

The effect of water extract of Ammi magus on the mitosis of Vicia faba was studied in direct and recovery experiments. The results revealed that the extract caused a delay in the completion of the mitotic cycle. The interphase stages G1, S and G2 were not affected. The phophase and metaphase were the most influenced by the extract. The dominant abnormalities were: Stickiness, irregular prophase, bridges and with low frequcncy lagging, prophasemetaphase and despiralization.

The present study was undertaken to investigate the effect of Trigonella foenum-graecum seed and Ammi majus fruit on experimentally-induced kidney stones. Oxalate urolithiasis in male rats was produced by the addition of 3% glycolic acid to their diet. After 4 weeks, highly significant deposition in the kidneys was noticed and changes in water intake and body weight recorded. Daily oral treatment with T. foenum-graecum significantly decreased the quantity of calcium oxalate deposited in the kidneys thus supporting its use in Saudi folk medicine. The effects obtained by A. majus were, however, not significant.

The aqueous eluate from fruits ofAmmi majus (Bishop's weed, Umbelliferae) remarkably inhibited germination of adjacent seeds ofAnastatica hierochuntica, lettuce, or tomato but had no effect on intact fruits ofAmmi. Similar inhibition was found in dark or in light, except that seeds ofA. hierochuntica were significantly more inhibited in the dark than in the light. Xanthotoxin was isolated, identified, and found to account for about a sixth of the inhibitory activity of the eluate. After fruits ofAmmi were submerged in a large volume of water for 4 days, the fruits still exuded enough inhibitors to prevent germination ofA. hierochuntica, lettuce, or tomato. Data support also the proposal that the phytotoxins are compartmentalized between the inner and the outer fruit envelopes. The inner layer excludes inhibitors from the embryo and autotoxicity is thus avoided, whereas the outer one ensures a gradual liberation of the phytotoxic compounds. This, as well as the high reactivity of the eluate, the high densities ofAmmi fruits in nature, and their relatively limited annual germination, suggest chemical inhibition of neighboring plant species other thanAmmi. Hence, in addition to their chemical protection against predators of either lower or higher organisms, furanocoumarins in fruits ofAmmi majus may contribute to its success as a weed.

Ducklings were photosensitized by the force-feeding of Ammi majus seeds for 5 days and exposure to sunlight. In birds sacrificed 2 weeks later, acute signs consisted of moderate vacuolization of ganglion cells. In birds sacrificed 3 ½ and 5 ½ months later, long-term effects of photosensitization included pigmentary retinopathy and hyperplasia of the retinal pigment epithelium. At the extreme periphery of the choroid, congestion and dilatation of the vessels, and transudation were observed. The probable mechanism whereby the photosensitization induces these changes in the retina and choroid of these ducklings is discussed.

The effect of water extract of two medicinal plants, Ammi majus and Ammi visnaga on meiosis, pollen length and pollen viability were studied. Most of the treatments gave high percentage of abnormalities. The percentage of anomalies in the 1st division was higher than those in the 2nd division. The induced irregularities were: Stickiness, bridges, spindle disturbance, lagging chromosome, breaks and fragments and multinucleate cell.The two plants extracts caused a decrease in the pollen grain length and gave mild increase in the pollen viability.

SummaryAutotetraploid has been obtained in Ammi majus L. by colchicine treatment. The autotetraploid showed slower rate of growth, smaller leaves, decreased number of branches and delayed flowering. Lesser number of inflorescences with poor fruiting and seed setting was also observed. Untreated plants had eleven bivalents (2n=22) while autotetraploid exhibited 22 bivalents (4n=44) at diakinesis and metaphase I. Abnormalities like univalents, rod bivalents and unequal separation of chromosomes were also observed.

Reports on the effective treatment of vitiligo have created great interest among physicians. El Mofty (1, 2) found that the use of crystalline constituents of the plant Ammi Majus Linn., together with ultraviolet light exposure, resulted in repigmentation of vitiliginous areas in over 75 per cent of patients with this disorder.

Ammi majus ( A . majus ) is an important medicinal plant traditionally used in Oman for the treatment of mouth ulcers. The objective of this study was to determine the antimicrobial and cytotoxic activities of different leaf extracts of A . majus using an established protocol. Different extracts were prepared using increasingly polar solvents. The antimicrobial activity of the extracts was determined using the disc diffusion method, and the cytotoxic activity of the extracts was assessed by the brine shrimp lethality bioassay. Exposure of the Gram-positive Staphylococcus aureus and the Gram-negative Escherichia coli , Haemophilus influenzae and Proteus spp. bacterial strains to the different extracts revealed potential antimicrobial activity measured by zones of inhibition in the range of 0–20 mm. The cytotoxicity results showed an incremental rise in shrimp larvae mortality depending on the extract concentration, reaching 100% mortality at a concentration of 1000 μg/ml for all extracts. The methanol extract had the lowest of all LC 50 values at 45.75 μg/ml. The LC 50 values in ascending order for the chloroform, hexane, ethyl acetate, water and butanol extracts are 179.41, 288.31, 305.19, 417.94 and 570.02 μg/ml, respectively. In conclusion, in vitro and in vivo studies of the isolated leaf extracts are needed to determine the therapeutic potential of this plant.

Caffeoyl-coenzyme A O-methyltransferase cDNA was cloned from dark-grown Ammi majus L. (Apiaceae) cells treated with a crude fungal elicitor and the open reading frame was expressed in Escherichia coli. The translated polypeptide of 27.1-kDa shared significant identity to other members of this highly conserved class of proteins and was 98.8% identical to the corresponding O-methyltransferase from parsley. For biochemical characterization, the recombinant enzyme could be purified to apparent homogeneity by metal-affinity chromatography, although the recombinant enzyme did not contain any affinity tag. Based on sequence analysis and substrate specificity, the enzyme classifies as a cation-dependent O-methyltransferase with pronounced preference for caffeoyl coenzyme A, when assayed in the presence of Mg2+-ions. Surprisingly, however, the substrate specificity changed dramatically, when Mg2+ was replaced by Mn2+ or Co2+ in the assays. This effect could point to yet unknown functions and substrate specificities in situ and suggests promiscuous roles for the lignin specific cluster of plant O-methyltransferases.

The powdered fruit of Ammi majus L. of the Umbelliferae family have been used for several centuries in the treatment of leukoderma and other pigmentation disorders (1). Fahmy and associates (2-4), and Schonberg and Sina (5, 6) have isolated the active components and identified them as psoralen derivatives. More recently, Fitzpatrick et al (7) demonstrated the effectiveness of 8-methoxypsoralen (8-MP) in pigment formation in individuals exposed to total sunlight or ultraviolet irradiation. These investigators have also shown that pigment production may be obtained following either oral or topical applications of this drug with a response that is roughly proportional to the dose administered.

8-Methoxypsoralen, also known as “ammoidin” and “xanthotoxin”, is one of several compounds isolated from the leaves of the plant Ammi majus by % Fahmy and Shady (1, 2, 3). It is present in concentrations as high as 0.5% in some parts of the plant and has been regarded as the most important active A principle of the extracts used in the treatment of vitiligo.

A spectrophotometric method for determining isopimpinellin, bergapten, and xanthotoxin in the seeds ofAmmi majus L. was developed. The method is characterized by good reproducibility and accuracy.

Vitiligo is an idiopathic, acquired, circumscribed hypomelanosis, which is often associated with a positive family history. It is one of the most common leukodermas affecting approximately 1–4% of the world's population. 1 Vitiligo is characterized by discrete pale white macules, few or many in number that tend to enlarge centrifugally over time. In general, patients with vitiligo are healthy; however, the physical disfigurement perceived by those afflicted and those they confront often leads to social embarrassment and psychologic turmoil. 2 The characteristic histologic picture of vitiligo is the total absence of melanin and melanocytes in involved skin (Fig. 11-1). The epidermis and dermis are otherwise normal. The mechanism by which epidermis and/or follicular melanocytes disappear is not yet known. 3 Today there are three prevailing theories of the pathogenesis of vitiligo: the immune hypothesis, the neural hypothesis, and the self-destruction hypothesis. These three theories are not necessarily mutually exclusive. 1 Recent reports suggest that specific anti-melanocyte autoantibodies are present in most patients with vitiligo. 4,5 Their melanocytotoxicity in vitro suggests their active role in this disorder. The possibility that vitiligo corresponds to a syndrome and not to a single disease entity has been discussed. 1 In vitiligo the cosmetic disfigurement is due to the contrast between involved and uninvolved skin. One of the therapeutic options is to induce repigmentation of the vitiligo skin. For more than three thousand years, it is known that plants combined with natural sunlight can be used to treat successfully vitiligo. In Ancient Egypt and India, physicians and herbalists used the plant species Ammi majus Linnaeus in Egypt and Psoralea corylifolia L. in India for repigmentation of vitiligo. 1,6,7 In 1947 several crystalline compounds, including ammoidin (8-methoxypsoralen) and bergapten (5-methoxypsoralen), were isolated from the powder of Ammi majus L. 8 El Mofty 9 showed that 8-methoxypsoralen was useful in the treatment of vitiligo. Later, other psoralens, such as trioxsalen (4,5,8 trimethylpsoralen) were developed. Psoralen photochemotherapy is still the most successful (or less disappointing) current therapy involving the use of psoralens and subsequent exposure to long-wave ultraviolet radiation. Other photoactive compounds such as khellin 10–12 and hypericin 13–15 have also been tried.