Altered gene expression and root thebaine production in polyploidized and methyl jasmonate-elicited Papaver bracteatum Lindl.

Persian poppy (Papaver bracteatum Lindl.) is a perennial medicinal plant belonging to the Papaveraceae family that is endemic to the mountainous areas in Northern Iran. It is known for high amounts of the valuable benzylisoquinoline alkaloid thebaine. The effects of induced polyploidy as well as the effect of methyl Jasmonate (MeJA) elicitation on the root production of thebaine and on the expression of five alkaloid biosynthesis related genes were studied. The in vitro tetraploidy induction caused a significant increased expression of norcoclaurine synthase (NCS) and salutaridinol (SAT), and a significant decreased expression of berberine bridge enzyme (BBE) in the leaves. In the root tissues, the BBE, NCS, and SAT showed an increased expression in tetraploid plants, while codeinone reductase (COR) showed a decreased expression. A similar alteration pattern was found in mixoploid plants when compared to their diploid counterparts. MeJA at concentrations of 0.1 and 0.5 mM caused a remarkable increase in the thebaine content in the roots of treated plants, where the highest thebaine content was identified in plants elicited with 0.5 mM MeJA. Elicitation treatment caused a substantial increase in the expression of NCS and SAT in the leaves, while it had no major effect on BBE, codeine 3-O-demethylase (CODM) and COR. Expression analysis in the roots showed that MeJA caused a significant increase in the expression of only BBE and NCS, while expression of other studied genes remained unchanged. Our results may be exploited for improved thebaine production and the processing of Persian poppy.

Phytochemical Background Mediates Effects of Pyrrolizidine Alkaloids on Western Flower Thrips.

Plants produce an extremely diverse array of metabolites that mediate many aspects of plant-environment interactions. In the context of plant-herbivore interactions, it is as yet poorly understood how natural backgrounds shape the bioactivity of individual metabolites. We tested the effects of a methanol extract of Jacobaea plants and five fractions derived from this extract, on survival of western flower thrips (WFT). When added to an artificial diet, the five fractions all resulted in a higher WFT survival rate than the methanol extract. In addition, their expected combined effect on survival, assuming no interaction between them, was lower than that of the methanol extract. The bioactivity was restored when the fractions were combined again in their original proportion. These results strongly suggest synergistic interactions among the fractions on WFT survival rates. We then tested the effects of two pyrrolizidine alkaloids (PAs), free base retrorsine and retrorsine N-oxide, alone and in combination with the five shoot fractions on WFT survival. The magnitude of the effects of the two PAs depended on the fraction to which they were added. In general, free base retrorsine was more potent than retrorsine N-oxide, but this was contingent on the fraction to which these compounds were added. Our results support the commonly held, though seldom tested, notion that the efficacy of plant metabolites with respect to plant defence is dependent on their phytochemical background. It also shows that the assessment of bioactivity cannot be decoupled from the natural chemical background in which these metabolites occur.

Are effects of common ragwort in the Ames test caused by pyrrolizidine alkaloids?

It has previously been demonstrated by others that acetone extracts of Senecio jacobaea (syn. Jacobaea vulgaris, common or tansy ragwort) test positive in the Salmonella/microsome mutagenicity test (Ames test). Pyrrolizidine alkaloids (PAs) are thought to be responsible for these mutagenic effects. However, it was also observed that the major PA present in common ragwort, jacobine, produced a negative response (with and without the addition of rat liver S9) in Salmonella test strains TA98, TA100, TA1535 and TA1537. To investigate which compounds in the plant extracts were responsible for the positive outcome, the present study investigated the contents and mutagenic effects of methanol and acetone extracts prepared from dried ground S. jacobaea and Senecio inaequidens (narrow-leafed ragwort). Subsequently, a fractionation approach was set up in combination with LC-MS/MS analysis of the fractions. It was shown that the positive Ames test outcomes of S. jacobaea extracts are unlikely to be caused by PAs, but rather by the flavonoid quercetin. This study also demonstrates the importance of identifying compounds responsible for positive test results in bioassays.

Can plant resistance to specialist herbivores be explained by plant chemistry or resource use strategy?

At both a macro- and micro-evolutionary level, selection of and performance on host plants by specialist herbivores are thought to be governed partially by host plant chemistry. Thus far, there is little evidence to suggest that specialists can detect small structural differences in secondary metabolites of their hosts, or that such differences affect host choice or performance of specialists. We tested whether phytochemical differences between closely related plant species are correlated with specialist host choice. We conducted no-choice feeding trials using 17 plant species of three genera of tribe Senecioneae (Jacobaea, Packera, and Senecio; Asteraceae) and a more distantly related species (Cynoglossum officinale; Boraginaceae) containing pyrrolizidine alkaloids (PAs), and four PA-sequestering specialist herbivores of the genus Longitarsus (Chrysomelidae). We also assessed whether variation in feeding by specialist herbivores is attributable to different resource use strategies of the tested plant species. Plant resource use strategy was quantified by measuring leaf dry matter content, which is related to both plant nutritive value and to plant investment in quantitative defences. We found no evidence that intra-generic differences in PA profiles affect feeding by specialist herbivores. Instead, our results indicate that decisions to begin feeding are related to plant resource use strategy, while decisions to continue feeding are not based on any plant characteristics measured in this study. These findings imply that PA composition does not significantly affect host choice by these specialist herbivores. Leaf dry matter content is somewhat phylogenetically conserved, indicating that plants may have difficulty altering resource use strategy in response to selection pressure by herbivores and other environmental factors on an evolutionary time scale.

The analysis of pyrrolizidine alkaloids in Jacobaea vulgaris; a comparison of extraction and detection methods.

INTRODUCTION: Pyrrolizidine alkaloids (PAs) serve an important function in plant defence.Objective - To compare different extraction methods and detection techniques, namely gas chromatography with nitrogen phosphorus detection (GC-NPD) and liquid chromatography tandem mass spectrometry (LC-MS/MS) with quadrupole analysers for analysing PAs in Jacobaea vulgaris. METHODOLOGY: Both formic acid and sulfuric acid were tested for PA extraction from dry plant material. For GC-NPD, reduction is required to transform PA N-oxides into tertiary amines. Zinc and sodium metabisulfite were compared as reducing agents. RESULTS: The lowest PA concentration measured with GC-NPD was approximately 0.03 mg/g and with LC-MS/MS 0.002 mg/g. The detection of major PAs by both techniques was comparable but a number of minor PAs were not detected by GC-NPD. With the LC-MS/MS procedure higher concentrations were found in plant extracts, indicating that losses may have occurred during the sample preparation for the GC-NPD method. Zinc proved a more effective reducing agent than sodium metabisulfite. The sample preparation for LC-MS/MS analysis using formic acid extraction without any reduction and purification steps is far less complex and less time consuming compared to GC-NPD analysis with sulfuric acid extraction and PA N-oxide reduction with zinc and purification. CONCLUSIONS: In terms of sensitivity and discrimination, formic acid extraction in combination with LC-MS/MS detection is the method of choice for analysing PAs (both free and N-oxides forms) in plant material.

Natural hybridization between Senecio jacobaea and Senecio aquaticus: molecular and chemical evidence.

Hybridization is known to be involved in a number of evolutionary processes, including species formation, and the generation of novel defence characteristics in plants. The genus Senecio of the Asteraceae family is highly speciose and has historically demonstrated significant levels of interspecific hybridization. The evolution of novel chemical defence characteristics may have contributed to the success of Senecio hybrids. Chemical defence against pathogens and herbivores has been studied extensively in the model species Senecio jacobaea, which is thought to hybridize in nature with Senecio aquaticus. Here, we use amplified fragment length polymorphisms (AFLPs) and pyrrolizidine alkaloid (PA) composition to confirm that natural hybridization occurs between S. jacobaea and the closely related species S. aquaticus. AFLPs are also used to estimate the ancestry of hybrids. We also demonstrate that even highly back-crossed hybrids can possess a unique mixture of defence chemicals specific to each of the parental species. This hybrid system may therefore prove to be useful in further studies of the role of hybridization in the evolution of plant defence and resistance.