Variation in pyrrolizidine alkaloid patterns of Senecio jacobaea

We studied the variation in pyrrolizidine alkaloid (PA) patterns of lab-grown vegetative plants of 11 European Senecio jacobaea populations. Plants were classified as jacobine, erucifoline, mixed or senecionine chemotypes based on presence and absence of the PAs jacobine or erucifoline. Due to the presence of jacobine, total PA concentration in jacobine chemotypes was higher than in erucifoline chemotypes. Both relative and absolute concentrations of individual PAs differed between half-sib and clonal families, which showed that variation in PA patterns had a genetic basis. Within most populations relative abundance of PAs varied considerably between individual plants. Most populations consisted either of the jacobine chemotype or of the erucifoline chemotype, sometimes in combination with mixed or senecionine chemotypes.     

Tackling speciose genera: species composition and phylogenetic position of Senecio sect. Jacobaea (Asteraceae) based onplastid and nrDNA sequences

The molecular phylogeny of Senecio sect. Jacobaea (Asteraceae; Senecioneae) was studied to clarify species composition and interspecific relationships of Senecio sect. Jacobaea. This information is necessary for studies seeking explanations of the evolutionary success of Senecio, in terms of high species numbers and the evolution of chemical defense mechanisms. Parsimony analyses with 60 species of the tribe Senecioneae, representing 23 genera and 11 sections of Senecio, based on DNA sequence data of the plastid genome (the trnT-L intergenic spacer, the trnL intron, and two parts of the trnK intron, flanking both sides of the matK gene) and nuclear genome (ITS1, 5.8S, and ITS2 gene and spacers) show that sect. Jacobaea is a strongly supported monophyletic group. Fifteen species have been identified as members of section Jacobaea, including three species that have been consistently ascribed to this section in taxonomic literature and 12 species that were either placed in other sections of Senecio or not exclusively ascribed to sect. Jacobaea. This section was traditionally circumscribed as a group of European, biennial, or perennial herbs with pinnately incised leaves, but the results of this study show that one annual species, a species from northeastern Asia, and a species growing in the Himalayas are members of sect. Jacobaea as well. Furthermore, not all species in the section have pinnately incised leaves. The genera Emilia, Packera, and Pseudogynoxys form the sister clade of sect. Jacobaea, but this relationship lacks strong bootstrap support and thus remains provisional.     

Tissue distribution, core biosynthesis and diversification of pyrrolizidine alkaloids of the lycopsamine type in three Boraginaceae species

Three species of the Boraginaceae were studied: greenhouse-grown plants of Heliotropium indicum and Agrobacterium rhizogenes transformed roots cultures (hairy roots) of Cynoglossum officinale and Symphytum officinale. The species-specific pyrrolizidine alkaloid (PA) profiles of the three systems were established by GC-MS. All PAs are genuinely present as N-oxides. In H. indicum the tissue-specific PA distribution revealed the presence of PAs in all tissues with the highest levels in the inflorescences which in a flowering plant may account for more than 70% of total plant alkaloid. The sites of PA biosynthesis vary among species. In H. indicum PAs are synthesized in the shoot but not roots whereas they are only made in shoots for C. officinale and in roots of S. officinale. Classical tracer studies with radioactively labelled precursor amines (e.g., putrescine, spermidine and homospermidine) and various necine bases (trachelanthamidine, supinidine, retronecine, heliotridine) and potential ester alkaloid intermediates (e.g., trachelanthamine, supinine) were performed to evaluate the biosynthetic sequences. It was relevant to perform these comparative studies since the key enzyme of the core pathway, homospermidine synthase, evolved independently in the Boraginaceae and, for instance, in the Asteraceae [Reimann, A., Nurhayati, N., Backenkohler, A., Ober, D., 2004. Repeated evolution of the pyrrolizidine alkaloid-mediated defense system in separate angiosperm lineages. Plant Cell 16, 2772-2784.]. These studies showed that the core pathway for the formation of trachelanthamidine from putrescine and spermidine via homospermidine is common to the pathway in Senecio ssp. (Asteraceae). In both pathways homospermidine is further processed by a beta-hydroxyethylhydrazine sensitive diamine oxidase. Further steps of PA biosynthesis starting with trachelanthamidine as common precursor occur in two successive stages. Firstly, the necine bases are structurally modified and either before or after this modification are converted into their O(9)-esters by esterification with one of the stereoisomers of 2,3-dihydroxy-2-isopropylbutyric acid, the unique necic acid of PAs of the lycopsamine type. Secondly, the necine O(9)-esters may be further diversified by O(7)- and/or O(3')-acylation.     

Pyrrolizidine alkaloids of the endemic Mexican genus Pittocaulon and assignment of stereoisomeric 1,2-saturated necine bases

The endemic Mexican genus Pittocaulon (subtribe Tussilagininae, tribe Senecioneae, Asteraceae) belongs to a monophyletic group of genera distributed in Mexico and North America. The five Pittocaulon species represent shrubs with broom-like succulent branches. All species were found to contain pyrrolizidine alkaloids (PAs). With one exception (i.e., stems of Pittocaulon velatum are devoid of PAs) PAs were found in all plant organs with the highest levels (up to 0.3% of dry weight) in the flower heads. Three structural types of PAs were found: (1) macrocyclic otonecine esters, e.g. senkirkine and acetylpetasitenine; (2) macrocyclic retronecine esters, e.g. senecionine, only found in roots, and (3) monoesters of 1,2-saturated necines with angelic acid. For an unambiguous assignment of the different stereoisomeric 1,2-saturated necine bases a GC-MS method was established that allows the separation and identification of the four stereoisomers as their diacetyl or trimethylsilyl derivatives. All otonecine esters that generally do not form N-oxides and the 1,2-saturated PAs were exclusively found as free bases, while the 1,2-unsaturated 7-angeloylheliotridine occurring in P. velatum was found only as its N-oxide. In a comparative study the 1H and 13C NMR spectra of the four stereoisomeric necine bases were completely assigned by the use of DEPT-135, H,H-COSY, H,C-HSQC and H,H-NOESY experiments and by iterative analysis of the 1H NMR spectra. Based on these methods the PA monoesters occurring in Pittocaulon praecox and P. velatum were assigned as 7-O-angeloyl ester respectively 9-O-angeloyl ester of dihydroxyheliotridane which could be identified for the first time as naturally occurring necine base. Unexpectedly, in the monoesters isolated from the three other Pittocaulon species dihydroxyheliotridane is replaced by the necine base turneforcidine with opposite configuration at C-1 and C-7. The species-specific and organ-typical PA profiles of the five Pittocaulon species are discussed in a biogenetic context.     

Phylogeny reconstruction in the gap between too little and too much divergence: the closest relatives of Senecio jacobaea (Asteraceae) according to DNA sequences and AFLPs

The species composition and molecular phylogeny of Senecio sect. Jacobaea (Asteraceae; Senecioneae) were studied to identify the closest relatives of Senecio jacobaea. Maximum parsimony and Bayesian inference analyses of DNA sequence data of the plastid (the trnT-L igs, the trnL intron, two parts of the trnK intron, and the psbA-trnH igs) and nuclear genome (ITS1, 5.8S, and ITS2) showed these markers to be suitable to assess the species composition of sect. Jacobaea, identifying 24 species as members of this section. Of these, nine species were not previously assigned to the section. The selected DNA sequence regions, however, showed too little sequence divergence to be optimal for phylogenetic inference within sect. Jacobaea. In contrast, AFLPs proved to be too variable to be used to study relationships between the basal lineages in sect. Jacobaea. Nonetheless, these markers are very useful to study the phylogeny of S. jacobaea and its closest relatives. The combined use of DNA sequence data and AFLPs allowed us to take a major step towards resolving phylogenetic relationships in sect. Jacobaea, identifying Senecio alpinus, Senecio pancicii and Senecio subalpinus (using DNA sequence data) or Senecio chrysanthemoides (using AFLPs) as the closest relatives of S. jacobaea.     

Seasonal variability in pyrrolizidine alkaloids in Senecio inaequidens from the Val Venosta (Northern Italy)

Senecio inaequidens is a neophyte originating from South Africa that has managed to spread to Europe and colonize large areas. This plant has toxic pyrrolizidine alkaloids (PAs) that represent a health risk to humans and animals. The aim of this work was to measure the content of PAs of these plants in continuous intervals over the entire growing season. The plants were separated into their organs, such as sprouts, stems, leaves and inflorescences. PAs were extracted from the dried plants using an acidic methanol solution, purified and measured by GC/MS. The average PA content of plants over the growing season was 0.33% of dry weight. The highest PA levels were about 1% in the dry weight and were found in the young sprouts and flower heads. There were nine PAs present, of which six could be identified. The main alkaloid was retrorsine followed by senecivernine, senecionine, integerrimine, usaramine and seneciphylline. Due to the high PA content of the inflorescences, the long flowering period and the rapid invasion dynamics, this species presents a high health risk for humans and animals.     

Modulation of gastrin and epidermal growth factor by pyrrolizidine alkaloids obtained from Senecio brasiliensis in acute and chronic induced gastric ulcers

We investigated the antiulcerogenic activity of pyrrolizidine alkaloids (PAs) integerrimine, retrorsine, senecionine, usaramine and seneciplhylline, an alkaloidal extract obtained from Senecio brasiliensis. The PA extract demonstrated significantly activity in both, acute and chronic gastric ulcers on rats. The effects of PA extract were dose dependent. The mechanisms implicated on this activity were evaluated by determination of gastrin plasma levels in rats subjected to the acute treatment with PA extract and by expression of mRNA of Epidermal Growth Factor (EGF) after chronic treatment with this extract. The results showed that the PA extract increased both the levels of gastrin and the expression of EGF on these animals. Moreover, the histological examinations showed a reduction of exfoliation of superficial cells, hemorrhages and blood cell infiltration. We concluded that the PAs showed an important and qualitative antiulcerogenic activity mediated by increase in gastrin secretion and mRNA expression of EGF.     

Root damage and aboveground herbivory change concentration and composition of pyrrolizidine alkaloids of Senecio jacobaea

Thus far not many studies focussed on how herbivory in one plant part affects plant defence in the other. The effects of root damage and a leaf-feeding herbivore (Mamestra brassicae) on pyrrolizidine alkaloid (PA) levels of Senecio jacobaea were investigated in a controlled environment. Three cloned S. jacobaea genotypes, which differed in PA concentrations, received four treatments: (1) no damage, (2) root damage (removing half of the root system), (3) shoot herbivory by M. brassicae larvae, (4) root damage and shoot herbivory.Shoot herbivory did not significantly affect shoot biomass, while root damage decreased both root and shoot biomass. Shoot herbivory decreased PA concentrations in the roots. Conversely, root damage increased PA concentrations in the roots. Alkaloid concentrations in the shoot showed a weak response to root damage, shoot herbivory had no effect on PA levels in the shoot. The effect of damage on the allocation of PAs to shoot and roots depended on genotype. One genotype allocated more PAs to the damaged site, another genotype did not change allocation and the third genotype allocated more PAs to the shoot if the roots were damaged. Changes in PA composition were observed in one genotype. Shoot herbivory increased erucifoline concentrations in the shoot and decreased concentrations of senecionine in the roots. In conclusion, we have shown that even in an alleged constitutively defended plant, damage of one compartment affects secondary metabolite level in the other.     

Chemical diversity and variation of pyrrolizidine alkaloids of the senecionine type: biological need or coincidence?

Laboratory and field tracer experiments with ¹⁴C-labelled senecionine N–oxide (SO) and distant biosynthetic precursors such as [¹⁴C]putrescine revealed that pyrrolizidine alkaloid N-oxides (PAs) in Senecio vernalis Waldstr. & Kit. (Asteraceae) show no significant turnover over periods of up to 29 d. However, PAs are spatially mobile, they are continuously allocated, and labelled PAs are even detectable in leaves and capitula developed weeks after tracer application. Chemical diversification of SO, the common product of PA biosynthesis in roots, was studied in five Senecio species (i.e. S. vernalis Waldstr. & Kit., S. vulgaris L, S. inaequidens DC, two chemotypes of S. jacobaea L. and S. erucifolius L.). Tracer experiments revealed that shoots are capable of transforming [¹⁴ C]SO into the unique species–specific PA patterns. Within a plant, the transformation efficiency of SO can vary quantitatively and qualitatively between shoot organs (i.e. leaves, stems and inflorescences). All transformations proceed position-specifically and stereoselectively. They comprise simple one-step or two-step reactions such as hydroxylations, epoxidations, dehydrogenations, and O-acetylations, as well as the more complex conversion of the retronecine into the otonecine base moiety (e.g. SO into senkirkine). Taking all the evidence together, the qualitative and quantitative composition of the Senecio PA pattern is a dynamic and sensitive equilibrium between a number of interacting processes: (i) constant rate of de-novo synthesis of SO in roots, (ii) continuous long-distance translocation of SO into shoots, (iii) efficiency of SO transformations which may vary between plant organs, (iv) continuous allocation of PAs in the plant, and (v) efficiency and tissue selectivity of vacuolar storage. We suggest that in constitutive plant defence, without significant turnover of its components, such a highly plastic system provides a powerful strategy to successfully defend and possibly escape herbivory. Received: 27 March 1998 / Accepted: 19 May 1998     

Pyrrolizidine alkaloid profiles in Crotalaria species from Brazil: Chemotaxonomic significance

Gas chromatography–mass spectrometry analysis of seeds from 28 species of Crotalaria from Brazil (sections Calycinae, Crotalaria, Chrysocalycinae and Hedriocarpae) showed that pyrrolizidine alkaloids (PAs) are important as chemotaxonomic markers at the infrageneric level. The sections Calycinae and Crotalaria were characterized by 11-membered macrocyclic monocrotaline-type PAs. In the section Chrysocalycinae, a single species in the subsection Incanae, C. incana, showed integerrimine, a 12-membered macrocyclic senecionine-type, as main PA. The group close to the subsection Stipulosae (C. micans and C. maypurensis) showed distinctive PA patterns: C. micans presented mainly the 12-membered macrocyclic integerrimine, and C. maypurensis the unusual 7-hydroxy-1-methylene-8-pyrrolizidine. In the group close to the subsection Glaucae, the PAs with otonecine as the necine base were the main alkaloids, except in C. rufipila which showed an assamicadine-like PA (monocrotaline-type). The section Hedriocarpae showed main 12-membered macrocyclic senecionine-type PAs.     

Pyrrolizidine alkaloids from Anchusa strigosa and their antifeedant activity

The pyrrolizidine alkaloid (PA) content of flowers, leaves, and roots of Anchusa strigosa (Boraginaceae) was analysed by ESI-LC-MS. Six PAs, including two new natural compounds, were detected, characterized by NMR spectroscopy, and quantified in each plant organ. The results indicated that the highest total concentration of PAs was in the leaves (23.63 mg/g of dried part), followed by the flowers (19.77 mg/g), and finally by the roots (1.80 mg/g). All PAs isolated were subjected to Spodoptera exigua and Pieris brassicae larvae. Feeding activity by both herbivore species using a bioassay was inhibited up to circa 75% depending on PA and applied concentration.     

Pyrrolizidine alkaloid variation in shoots and roots of segregating hybrids between Jacobaea vulgaris and Jacobaea aquatica

Hybridization can lead to novel qualitative or quantitative variation of secondary metabolite (SM) expression that can have ecological and evolutionary consequences. We measured pyrrolizidine alkaloid (PA) expression in the shoots and roots of a family including one Jacobaea vulgaris genotype and one Jacobaea aquatica genotype (parental genotypes), two F(1) hybrid genotypes, and 102 F(2) hybrid genotypes using liquid chromatography-tandem mass spectrometry (LC-MS/MS). We detected 37 PAs in the roots and shoots of J. vulgaris, J. aquatica and the hybrids. PA concentrations and compositions differed between genotypes, and between roots and shoots. Three otosenine-like PAs that only occurred in the shoots of parental genotypes were present in the roots of F(2) hybrids; PA compositions were sometimes novel in F(2) hybrids compared with parental genotypes, and in some cases transgressive PA expression occurred. We also found that PAs from within structural groups covaried both in the roots and in the shoots, and that PA expression was correlated between shoots and roots. Considerable and novel variation present among F(2) hybrids indicates that hybridization has a potential role in the evolution of PA diversity in the genus Jacobaea, and this hybrid system is useful for studying the genetic control of PA expression.     

Flavonoid profiles in the Heliohebe group of New Zealand Veronica (Plantaginaceae)

The Heliohebe group of Veronica (sect. Hebe) consists of five species occurring in the South Island of New Zealand. These species and a hybrid were analysed for their flavonoids. Five flavone glycosides were isolated and identified by NMR spectroscopy and three additional glycosides were detected by LC–UV–MS. Luteolin 7-O-, 3′-O- and 4′-O-glucosides and apigenin 7-O-glucoside were present in all six taxa investigated, 6-hydroxyluteolin glycosides were found in five and a luteolin caffeoylglycoside in four taxa, while a hypolaetin 7-O-glycoside was detected only in Veronica pentasepala. The 3′-O- and 4′-O-glucosides of luteolin are also common in other species of Veronica sect. Hebe (restricted to the Southern Hemisphere), but are rare in Northern Hemisphere species of Veronica and thus act as good chemotaxonomic markers for the section. The relatively simple flavonoid profiles found in the Heliohebe group are plesiomorphic and consistent with the group's status as sister to the Hebe clade. Based on the detected flavonoids, two groups could be distinguished within the Heliohebe clade: (1) Veronica hulkeana, Veronica lavaudiana and Veronica raoulii, characterised by luteolin caffeoylglycoside, and (2) V. pentasepala and Veronica scrupea, where this compound is replaced by a 6-hydroxyluteolin dihexoside.     

Site of synthesis,metabolism and translocation of senecionine N-oxide in cultured roots of Senecio erucifolius

Root cultures of Senecio erucifolius (Asteraceae) efficiently took up and incorporated [¹⁴C]putrescine and [¹⁴C]arginine into the pyrrolizidine alkaloid (PA) senecionine N-oxide. Pulse-chase experiments covering a growth period of 10 to 19 days revealed the absence of any significant alkaloid turnover. The only metabolic activity was a slow but progressive transformation of senecionine N-oxide into its dehydrogenation product, seneciphylline N-oxide. Tracer experiments with single roots showed that the sites of enhanced PA synthesis coincided with the sites of preferred protein synthesis, i.e. root apices, indicating a close correlation between growth activity and alkaloid synthesis. Long-term pulse-chase experiments (10 to 12 days) with ¹⁴C-labelled arginine, putrescine and senecionine fed to single roots indicated that in spite of its metabolic inertia, senecionine N-oxide is a mobile compound which is translocated into tissues newly grown during the chase.Dedicated to Dr. Friedrich Constabel on the occasion of his 60th birthday     

The genotype dependent presence of pyrrolizidine alkaloids as tertiary amine in Jacobaea vulgaris

Secondary metabolites such as pyrrolizidine alkaloids (PAs) play a crucial part in plant defense. PAs can occur in plants in two forms: tertiary amine (free base) and N-oxide. PA extraction and detection are of great importance for the understanding of the role of PAs as plant defense compounds, as the tertiary PA form is known for its stronger influence on several generalist insects, whereas the N-oxide form is claimed to be less deterrent. We measured PA N-oxides and their reduced tertiary amines by liquid chromatography-tandem mass spectrometry (LC-MS/MS). We show that the occurrence of tertiary PAs is not an artifact of the extraction and detection method. We found up to 50% of tertiary PAs in shoots of Jacobine - chemotype plants of Jacobaea vulgaris. Jacobine and its derivatives (jacoline, jaconine, jacozine and dehydrojaconine) may occur for more than 20% in reduced form in the shoots and more than 10% in the roots. For 22 PAs detected in F(2) hybrids (J. vulgaris × Jacobaea aquatica), we calculate the tertiary amine percentage (TA%=the tertiary amine concentration/(tertiary amine concentration+the corresponding N-oxide concentration) × 100). We found that the TA% for various PAs was genotype-dependent. Furthermore, TA% for the different PAs were correlated and the highest correlations occurred between PAs which share high structural similarity.     

Polyphyletic origin of pyrrolizidine alkaloids within the Asteraceae. Evidence from differential tissue expression of homospermidine synthase

The evolution of pathways within plant secondary metabolism has been studied by using the pyrrolizidine alkaloids (PAs) as a model system. PAs are constitutively produced by plants as a defense against herbivores. The occurrence of PAs is restricted to certain unrelated families within the angiosperms. Homospermidine synthase (HSS), the first specific enzyme in the biosynthesis of the necine base moiety of PAs, was originally recruited from deoxyhypusine synthase, an enzyme involved in the posttranslational activation of the eukaryotic initiation factor 5A. Recently, this gene recruitment has been shown to have occurred several times independently within the angiosperms and even twice within the Asteraceae. Here, we demonstrate that, within these two PA-producing tribes of the Asteraceae, namely Senecioneae and Eupatorieae, HSS is expressed differently despite catalyzing the same step in PA biosynthesis. Within Eupatorium cannabinum, HSS is expressed uniformly in all cells of the root cortex parenchyma, but not within the endodermis and exodermis. Within Senecio vernalis, HSS expression has been previously identified in groups of specialized cells of the endodermis and the adjacent root cortex parenchyma. This expression pattern was confirmed for Senecio jacobaea as well. Furthermore, the expression of HSS in E. cannabinum is dependent on the development of the plant, suggesting a close linkage to plant growth.     

ent-Labdane glycosides from the aquatic plant Potamogeton lucens and analytical evaluation of the lipophilic extract constituents of various Potamogeton species

Two new ent-labdane glycosides, one known furano-ent-labdane and a new hydroxylated fatty acid were isolated from the dichloromethane extract of the freshwater aquatic plant Potamogeton lucens. The new compounds were assigned the structures of beta-d-glucopyranosyl-8(17),13-ent-labdadien-16,15-olid-18-oate, 18-beta-d-glucopyranosyloxy-8(17),13-ent-labdadien-16,15-olide and 13(R)-hydroxy-octadeca-(9Z,11E,15Z)-trien-oic acid by spectroscopic means. The algicidal activity of these compounds was tested against Raphidocelis subcapitata. Based on our previous study of Potamogeton pectinatus, other constituents were identified in P. lucens by LC-UV-MS, LC-NMR and GC-MS. The lipophilic extract profiles of both species are presented. Two other species, Potamogeton perfoliatus and P. crispus, were also investigated by analytical comparison of their non-polar extracts. The distribution of ent-labdanes characterized in Potamogeton is summarized.     

Identification of volatile markers for indoor fungal growth and chemotaxonomic classification of Aspergillus species

Microbial volatile organic compounds (MVOCs) were collected in water-damaged buildings to evaluate their use as possible indicators of indoor fungal growth. Fungal species isolated from contaminated buildings were screened for MVOC production on malt extract agar by means of headspace solid-phase microextraction followed by gas chromatography-mass spectrometry (GC-MS) analysis. Some sesquiterpenes, specifically derived from fungal growth, were detected in the sampled environments and the corresponding fungal producers were identified. Statistical analysis of the detected MVOC profiles allowed the identification of species-specific MVOCs or MVOC patterns for Aspergillus versicolor group, Aspergillus ustus, and Eurotium amstelodami. In addition, Chaetomium spp. and Epicoccum spp. were clearly differentiated by their volatile production from a group of 76 fungal strains belonging to different genera. These results are useful in the chemotaxonomic discrimination of fungal species, in aid to the classical morphological and molecular identification techniques.     

Identification of unusual fatty acids of four alpine plant species from the Pamirs

Fatty acid composition and structure in total lipids from the green above-ground parts of four alpine plants, Oxygraphis glacialis, Primula macrophylla, Rhodiola pamiroalaica, and Swertia marginata, were established by GC and GC-MS. A total of 55 fatty acids was detected, and 48 of them were identified. Ubiquitous palmitate, linoleate, and linolenate predominated in the lipids accounting for about 72-90% of the total fatty acids. At the same time, the latter contained numerous species, which were unusual for higher plants and included saturated odd-numbered n-acids (six C15-C25 species, 0.26-1.40%), saturated even-numbered very-long-chain n-acids (six C20-C30 species, 1.00-2.49%), iso-acids (nine C15-C26 species, 0.64-1.53%), anteiso-acids (four C15-C20 species, 0.08-1.57%), certain uncommon mono- and dienoic acids, as well as 16:3omega3, 18:3omega6, and 18:4omega3 acids that are absent from the most higher plants. Nine fatty acids were found here for the first time in higher plants and two may be new to science. The evidence on the unusual fatty acids is discussed with respect to their distribution in living organisms, pathways of biosynthesis, and chemotaxonomic role.     

Pyrrolizidine alkaloids in and on the leaf surface of Senecio jacobaea L

This is the first study showing that alkaloids are present on the leaf surface of plants. A concentration of 30-230 pmol/cm2 pyrrolizidine alkaloids (PA's) was detected in 8 different samples taken from Senecio jacobaea. PA concentration on the leaves was marginally correlated with PA concentration of the total leaf tissues. The PA spectrum on the leaf differed from the PA spectrum of the total leaf.