Scanning electron microscopy as a tool for authentication of oil yielding seed.

Now-a-days, plant species are consumed globally for various purposes and this increasing demand leads to adulteration due to gradually exploitation in natural resources. The major causes of adulteration may be confusion in nomenclature, unawareness of authentic sources, unavailability of authentic sources, color resemblances, deficiencies in collection procedures, and misidentification. This study aims to use the microscopic techniques such as scanning electron microscopy for the authentication of the oil yielding seeds of four important and traditionally used species Prunus persica, Prunus domestica, and Eruca sativa and Argemone Mexicana from their adulterants. All of these are versatile in usage. Locally, these four plants are adulterated badly and there is need to provide a criteria and a complete monograph for correct identification. This research may prove to be helpful for quality control and as well for future studies to explore other novel aspects of these plants.

Development of newly sanguinarine biosynthetic capacity in in vitro rootless shoots of Argemone mexicana L. Mexican prickly poppy.

OBJECTIVE: To analyze berberine and sanguinarine biosynthetic capacities of both in vitro shoot and root cultures of Argemone mexicana and tissues from entire plants at different developmental stages. RESULTS: Berberine and sanguinarine were equally distributed in roots and aerial tissues of developing plantlet whereas, in juvenile plants, sanguinarine was only detected in roots. This alkaloid distribution was consistent with that of biosynthetic transcripts in juvenile plants. However, lower transcript abundance in plantlets´ leaves suggests that alkaloids were mainly formed in roots and then mobilized to this tissue. In vitro root cultures maintained similar alkaloid profiles to those from intact seedlings and plantlets. However, in addition to berberine, rootless shoot cultures accumulated high levels of sanguinarine and biosynthetic transcripts. CONCLUSION: In vitro shoot cultures of A. mexicana can synthesize sanguinarine in addition to berberine. This represent a convenient system for the production of both alkaloids.

Yeast extract induction of sanguinarine biosynthesis is partially dependent on the octadecanoic acid pathway in cell cultures of Argemone mexicana L., the Mexican poppy.

OBJECTIVE: To analyze the involvement of the octadecanoic (OCDA) pathway in the accumulation of sanguinarine induced by yeast extract (YE) in cell suspension cultures of Argemone mexicana (Papaveraceae). RESULTS: Exposure to YE promoted sanguinarine accumulation. This was not observed when they were exposed to methyl jasmonate (MeJa). Use of diethyldithiocarbamic acid (DIECA), an inhibitor of the OCDA pathway, resulted in partial impairment of this response. Exogenous application of MeJa did not reverse this effect in DIECA-exposed cultures. qRT-PCR revealed that the accumulation of transcripts corresponding to the berberine bridge enzyme gene, which was induced by YE exposure, was blocked by OCDA pathway and reversed by exogenous MeJa. Interestingly, this response pattern could not be observed on dihydrobenzophenanthridine oxidase enzyme activity, which was promoted by YE, but unaffected by either OCDA or MeJa. CONCLUSION: Results suggest partial involvement of OCDA pathway in this response.

Prickly poppies can get pricklier: ontogenetic patterns in the induction of physical defense traits.

Plant ontogeny is a common source of variation in defense and herbivory. Yet, few studies have investigated how the induction of physical defense traits changes across plant ontogeny. Physical defense traits are costly to produce, and thus, it was predicted that induction as a cost-saving strategy would be particularly favorable for seedlings, leading to ontogenetic declines in the inducibility of these traits. We tested for induction of three different physical defense traits (prickles, latex and leaf toughness) in response to mechanical defoliation and jasmonic acid application using prickly poppies (Argemone glauca and A. mexicana, Papaveraceae) as a model system. Genetic variation in the induction of physical defenses was tested using maternal sib-ships sampled from multiple populations. Both species induced higher densities of laminar prickles, although the magnitude of induction was much higher in the endemic Hawaiian prickly poppy, A. glauca, than in the cosmopolitan A. mexicana. The magnitude of prickle induction was also higher in young compared to older juvenile plant stages in A. glauca, demonstrating a strong role of ontogeny. Neither latex exudation nor leaf toughness was induced in either species. Although significant genetic variation was detected within and among populations for constitutive expression of physical defense traits in Argemone, there was no evidence for genetic variation in the induction of these traits. This study provides the first evidence for the induction of physical defenses in prickly poppies, emphasizing how an ontogenetically explicit framework can reveal new insights into plant defense. Moreover, this study illustrates how sister species comparisons between island vs. continental plants can provide new insights into plant functional and evolutionary ecology, highlighting a fruitful area for future research on more species pairs.

The sequential exposure to jasmonate, salicylic acid and yeast extract promotes sanguinarine accumulation in Argemone mexicana cell cultures.

The effects of the sequential application of methyl jasmonate (MeJa), salicylic acid (SA) and yeast extract (YE) to Argemone mexicana cell cultures were compared to either the sole application of each elicitor, or to the three-partite mixture. The highest sanguinarine accumulation occurred using the sequential treatment (ninefold over unexposed control cultures), followed by the single application of YE (fivefold). The elicitor mixture produced less sanguinarine than sole exposure to YE but higher than MeJa alone. SA did not produce any effect. Transcripts corresponding to tyrosine decarboxylase and berberine bridge enzyme accumulated in treated cells, but did not correlate with alkaloid accumulation. Discrete epifluorescence foci, surrounding the nucleus and scattered throughout the cytoplasm of elicited cells, suggested the presence of alkaloid-accumulating vesicles which could participate in a mechanism to avoid sanguinarine toxicity.

Heterologous expression of two FAD-dependent oxidases with (S)-tetrahydroprotoberberine oxidase activity from Arge mone mexicana and Berberis wilsoniae in insect cells.

Berberine, palmatine and dehydrocoreximine are end products of protoberberine biosynthesis. These quaternary protoberberines are elicitor inducible and, like other phytoalexins, are highly oxidized. The oxidative potential of these compounds is derived from a diverse array of biosynthetic steps involving hydroxylation, intra-molecular C-C coupling, methylenedioxy bridge formation and a dehydrogenation reaction as the final step in the biosynthesis. For the berberine biosynthetic pathway, the identification of the dehydrogenase gene is the last remaining uncharacterized step in the elucidation of the biosynthesis at the gene level. An enzyme able to catalyze these reactions, (S)-tetrahydroprotoberberine oxidase (STOX, EC 1.3.3.8), was originally purified in the 1980s from suspension cells of Berberis wilsoniae and identified as a flavoprotein (Amann et al. 1984). We report enzymatic activity from recombinant STOX expressed in Spodoptera frugiperda Sf9 insect cells. The coding sequence was derived successively from peptide sequences of purified STOX protein. Furthermore, a recombinant oxidase with protoberberine dehydrogenase activity was obtained from a cDNA library of Argemone mexicana, a traditional medicinal plant that contains protoberberine alkaloids. The relationship of the two enzymes is discussed regarding their enzymatic activity, phylogeny and the alkaloid occurrence in the plants. Potential substrate binding and STOX-specific amino acid residues were identified based on sequence analysis and homology modeling.

Characterization of two methylenedioxy bridge-forming cytochrome P450-dependent enzymes of alkaloid formation in the Mexican prickly poppy Argemone mexicana.

Formation of the methylenedioxy bridge is an integral step in the biosynthesis of benzo[c]phenanthridine and protoberberine alkaloids in the Papaveraceae family of plants. This reaction in plants is catalyzed by cytochrome P450-dependent enzymes. Two cDNAs that encode cytochrome P450 enzymes belonging to the CYP719 family were identified upon interrogation of an EST dataset prepared from 2-month-old plantlets of the Mexican prickly poppy Argemone mexicana that accumulated the benzo[c]phenanthridine alkaloid sanguinarine and the protoberberine alkaloid berberine. CYP719A13 and CYP719A14 are 58% identical to each other and 77% and 60% identical, respectively, to stylopine synthase CYP719A2 of benzo[c]phenanthridine biosynthesis in Eschscholzia californica. Functional heterologous expression of CYP719A14 and CYP719A13 in Spodoptera frugiperda Sf9 cells produced recombinant enzymes that catalyzed the formation of the methylenedioxy bridge of (S)-cheilanthifoline from (S)-scoulerine and of (S)-stylopine from (S)-cheilanthifoline, respectively. Twenty-seven potential substrates were tested with each enzyme. Whereas CYP719A14 transformed only (S)-scoulerine to (S)-cheilanthifoline (K(m) 1.9±0.3; k(cat)/K(m) 1.7), CYP719A13 converted (S)-tetrahydrocolumbamine to (S)-canadine (K(m) 2.7±1.3; k(cat)/K(m) 12.8), (S)-cheilanthifoline to (S)-stylopine (K(m) 5.2±3.0; k(cat)/K(m) 2.6) and (S)-scoulerine to (S)-nandinine (K(m) 8.1±1.9; k(cat)/K(m) 0.7). These results indicate that although CYP719A14 participates in only sanguinarine biosynthesis, CYP719A13 can be involved in both sanguinarine and berberine formation in A. mexicana.

Fusarium oxysporum homogenates and jasmonate induce limited sanguinarine accumulation in Argemone mexicana cell cultures.

In vitro cultures of Argemone mexicana (Papaveraceae) were induced from leaves of mature plants. Sanguinarine, a benzophenanthridine, was the main alkaloid in the cultures, even in the absence of inducers of secondary metabolism. The accumulation of this metabolite was increased by adding methyl jasmonate and fungal elicitors, although in a limited fashion in comparison to other sanguinarine-producing species. Evidence of a transport mechanism, which may be related to the magnitude of the response, was obtained based on the fluorescent properties of bezophenathridines in the elicited cultures.