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

Abstract. Laboratory and ®eld tracer experiments with14C-labelled senecionine N±oxide (SO) and distantbiosynthetic precursors such as [14C]putrescine revealedthat pyrrolizidine alkaloid N-oxides (PAs) in SeneciovernalisWaldstr. & Kit. (Asteraceae) show no signi®cantturnover over periods of up to 29 d. However, PAs arespatially mobile, they are continuously allocated, andlabelled PAs are even detectable in leaves and capituladeveloped weeks after tracer application. Chemicaldiversi®cation of SO, the common product of PAbiosynthesis in roots, was studied in ®ve Seneciospecies (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 thatshoots are capable of transforming [14 C]SO into theunique species±speci®c PA patterns. Within a plant, thetransformation e�ciency of SO can vary quantitativelyand qualitatively between shoot organs (i.e. leaves, stemsand in¯orescences). All transformations proceed posi-tion-speci®cally and stereoselectively. They comprisesimple one-step or two-step reactions such as hydroxyl-ations, epoxidations, dehydrogenations, and O-acetyla-tions, as well as the more complex conversion of theretronecine into the otonecine base moiety (e.g. SO intosenkirkine). Taking all the evidence together, the qual-itative and quantitative composition of the Senecio PApattern is a dynamic and sensitive equilibrium between anumber of interacting processes: (i) constant rate ofde-novo synthesis of SO in roots, (ii) continuous long-distance translocation of SO into shoots, (iii) e�ciencyof SO transformations which may vary between plantorgans, (iv) continuous allocation of PAs in the plant,and (v) e�ciency and tissue selectivity of vacuolarstorage. We suggest that in constitutive plant defence,without signi®cant turnover of its components, such a

highly plastic system provides a powerful strategy tosuccessfully defend and possibly escape herbivory.

Key words: Biosynthetic transformation ± Diversi®-cation (chemical) ± Plant defence (constitutive) Pyrroli-zidine alkaloid ± Senecio ± Senecionine N-oxide

Introduction

The most characteristic features of plant secondarymetabolism are its immense chemical diversity andintraspeci®c variation. These features clearly separatesecondary metabolism from uniformly organized andstringently regulated primary metabolism (Hartmann1996). If we accept the general role of secondarymetabolism as the functional level in the interactionsof the plant with its biotic and abiotic environment,genetic variation should be a prerequisite for selectiveforces to operate. In the ®eld of plant-herbivoreinteractions, chemical diversity was one of the strongestarguments for co-evolutionary links between plants andherbivorous insects (e.g. Feeny 1992; Futuyma andKeese 1992). There is abundant evidence that thecomponents of intraspeci®c variation of plant secondarymetabolism are under genetic control (e.g. Berenbaumand Zangerl 1992). A prerequisite for a better under-standing of the underlying mechanisms is a substantialbiochemical and physiological characterization of thesecondary pathways and their integration in plantmetabolism.

The pyrrolizidine alkaloids (PAs) constitute a typicalclass of secondary compounds. Five structural typesrepresenting monoesters and open-chain or macrocyclicdiesters with a total of more than 360 individualcompounds are known (Hartmann and Witte 1995).One of the most diverse classes of PAs, is the macro-cyclic senecionine type (Fig. 1) with more than 100 struc-tures. The senecionine-type PAs are abundantly foundin species of the tribe Senecioneae of the Asteraceae

Abbreviations: PAs � pyrrolizidine alkaloids; RI � retention(Kovats) indices; SO � senecionine N-oxide

Correspondence to: T. Hartmann; E-mail: [email protected];Fax: 49 (531) 3918104

Planta (1998) 206: 443±451

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

Thomas Hartmann, Barbara Dierich

Institut fuÈ r Pharmazeutische Biologie, Technische UniversitaÈ t Braunschweig, Mendelssohnstrasse 1, D-38106 Braunschweig, Germany

Received: 27 March 1998 /Accepted: 19 May 1998

(e.g. genus Senecio). In Senecio species such as S. vulgarisor S. vernalis, PAs are found in all plant organs duringall developmental stages. However, large quantitativedi�erences exist between organs. In¯orescences, forinstance, may contain 60±80% of total PAs of a plantin concentrations which are 10- to 30-fold higher than inleaves or roots (Hartmann and Zimmer 1986). In theAsteraceae, PAs are synthesized in the roots (Hartmannand Toppel 1987; Toppel et al. 1987). They are exportedfrom roots via the phloem into shoots, where they aree�ciently allocated to the in¯orescences, the major siteof storage (Hartmann et al. 1989). They are synthesized,translocated and stored in the form of their polar salt-like N-oxides (Fig. 1). A speci®c carrier catalyzes thetranslocation of the PA N-oxides into the vacuoleswhich are the cellular sites of accumulation (Ehmke et al.1988). Biosynthetic studies revealed homospermidine asthe ®rst intermediate of the alkaloid-speci®c pathway(Khan and Robins 1985). Subsequently, homospermi-dine synthase (EC 2.5.1.44), the enzyme catalyzing theformation of homospermidine, was isolated from rootcultures of di�erent Senecio species (BoÈ ttcher et al. 1993,1994). Homospermidine synthase catalyzes a uniqueaminobutyl transferase reaction in which either put-rescine or spermidine are donors of the aminobutylmoiety (BoÈ ttcher et al. 1994; Graser and Hartmann1997). Neither homospermidine nor PAs show anysigni®cant turnover or degradation in Senecio rootcultures except slow transformation of senecionineN-oxide (SO) into related structures (Sander andHartmann 1989; BoÈ ttcher et al. 1993).

The PAs are regarded as powerful defence com-pounds, particularly against herbivorous insects (forreview, see BoppreÁ 1986; Schneider 1987; Hartmann1991; Hartmann and Witte 1995). They are uniqueamong plant alkaloids in respect of their attractivenessto specialized insects. A number of species from di�erenttaxa have evolved adaptations to sequester (Aplin andRothschild 1972; Trigo et al. 1996; Hartmann et al.1997), safely store (Lindigkeit et al. 1997) and utilizeplant PAs against insectivores (Dussourd et al. 1991;Rowell-Rahier et al. 1995).

Here, using ®ve Senecio species as examples, weexamine how SO, the ®rst common product of PAbiosynthesis, is diversi®ed into the unique species-speci®c PA patterns. We identify the sites of transfor-mation and prove the absence of PA turnover in

laboratory and ®eld experiments. We present evidencethat chemical diversi®cation of SO in plant shoots is asystem of high plasticity which causes the great intra-speci®c chemical variation. In constitutive plant defencethis plasticity would provide plant populations with anagainst excellent strategy to successfully defend againstand possibly escape from local herbivory.

Materials and methods

Plants. Plant populations of Senecio vulgaris L., S. vernalisWaldstr. & Kit. and two chemotypes of S. jacobaea L. werecollected from natural habitats in the vicinity of Braunschweig.Senecio erucifolius L. was from a hilly location south of Bra-unschweig and S. inaequidens DC from a habitat near Hannover.In-vitro plants of S. vulgaris and S. vernalis were established fromaseptically germinated achenes according to Hartmann (1994) andkept individually in glass tubes (30 ´ 200 mm) or 250-ml Erlen-meyer ¯asks on 5±10 and 80 ml, respectively, of MS medium(Murashige and Skoog 1962) with 4% sucrose and phytohormonesomitted. The in-vitro plants were grown in a growth chamber at25 °C and a photon ¯ux density of approximately 50 lmol m)2 s)1

for a 14-h/10-h day/night cycle.

Tracer-feeding experiments. (i) Detached shoots or plant organs(leaves, stems, in¯orescences): immediately after dissection the cutend of the shoot or plant organ was placed into 1 ml tap watercontaining the tracer (18.5±74 kBq, at a ®nal concentration of10 lM). After the solution was completely taken up (ca. 3±12 h)the shoot or organ was placed in tap water without tracer untilharvest. (ii) In-vitro plants: an aseptically growing plantlet wasincubated in 5 ml MS medium containing the tracer (18.5±37 kBq,®nal concentration 10 lM). To disturb the plantlet as little aspossible the growth medium was removed with a pipette inexchange for the tracer medium without removing the plantlet fromthe glass tube. The plantlet was left in the tracer-containing MSmedium until harvest. Generally, the tracer was completely(>95%) taken up within 24±48 h. In long-term experiments lossof ¯uid was adjusted by addition of sterile water.

Alkaloid extraction. In tracer experiments, quantitative extractionof labelled PAs was achieved by extraction of the cut plant materialwith acidic (1% HCl) methanol for 30 min (20 ml methanol/HClper fresh weight) at room temperature. The procedure was repeatedthree times. After ®ltration and evaporation of the solvent theresidue was suspended in 0.5±2.0 ml methanol and directly appliedeither to TLC plates followed by radiodetection or to an HPLCcolumn coupled to a radiodetector. Extraction of PAs for separa-tion by capillary GC and identi®cation in combination with MS(GC-MS) was performed following the methods according to Witteet al. (1993): plant material, 0.5±2.0 g dry weight in 20 ml of0.05 M H2SO4, was homogenized (Ultra-turrax) for 3±4 min andthen stirred for 30 min at room temperature. After centrifugationthe supernatant was adjusted to 0.25 M H2SO4 and mixed with anexcess of Zn dust to reduce the PA N-oxides. The mixture wasstirred at room temperature for 5 h. Following centrifugation thesolution was made basic with 25% NH4OH and applied to anExtrelut (Merck) column (1.4 ml aqueous solution/g Extrelut).Tertiary PAs were eluted with methylene chloride (6 ml/g Extrelut).After evaporation of the solvent the residue was dissolved inmethanol and directly subjected to GC or GC-MS analysis. Thesame procedure, beginning with the reduction in the presence ofZn/H+, was applied to prepare and purify samples of isolated PAN-oxides obtained from HPLC runs and TLC plates for identi®-cation by GC-MS.

Gas chromatography (GC). Separation of tertiary PAs by GC wasperformed according to Witte et al. (1993) using a fused-silica

Fig. 1. Senecionine N-oxide (SO), the basic structure of macrocyclicester PAs of the senecionine type (occurrence: Asteraceae, tribusSenecioneae; more than 100 structures are known). The carbonskeleton of the necine base moiety is derived from 2 molecules ofarginine, that of the necic acid moiety from 2 molecules of isoleucine

444 T. Hartmann and B. Dierich: Chemical diversity and variation of pyrrolizidine alkaloids

column (WCOT, 15 m ´ 0.25 mm; DB-1, J&W Scienti®c, Calif.,USA). Conditions were as follows: injector, 250 °C; temperatureprogram 150±300 °C, 6 °C min)1; gas He 0.75 bar; detectors, FIDand PND.

Gas chromatography-mass spectrometry (GC-MS). Analysis byGC±MS was performed according to Witte et al. (1993). Theidenti®cation of individual alkaloids was achieved by their Kovatsindices (RI), molecular ions and fragmentation patterns incomparison with reference compounds and reference spectra.

High-performance liquid chromatography (HPLC). 14C-LabelledPAs were separated by reversed-phase ion-pair chromatography onan RP-18 column (25 cm ´ 0.4 cm i.d.; Nucleosil; Macherey &Nagel) using a slight modi®cation of a gradient system originallyestablished for polyamine analysis by Wagner et al. (1982).

Thin-layer chromatography (TLC). 14C-Labelled PAs were sepa-rated on silica gel 60 (Merck); solvent system: chloroform/n-pentane/methanol/NH4OH(25%) (82:20:14:2.5, by vol.). Radio-actively labelled compounds were located by means of a TLCmultichannel analyzer (Rita-32a, Raytest) or by autoradiography:exposure of TLC plates to Agfa-Currix X-ray ®lm for ca. 7 d.When necessary PA-containing zones, located by autoradiography,were scratched o� the plate and the alkaloid extracted withmethanol and further treated for identi®cation by GC-MS (seeabove).

Radiochemicals. [1,4-14C]Putrescine (4.03 GBq mmol)1) was pur-chased from Amersham (Braunschweig); [14C]SO (1.08 GBqmmol)1) was prepared biosynthetically from [1,4-14C]putrescinefed to root cultures of Senecio vulgaris (Hartmann 1994).

Results and discussion

Pyrrolizidine alkaloid patterns of Senecio species

The PA patterns of the ®ve Senecio species which are thesubjects of the present studies are summarized inTable 1. Only the major alkaloids (>3% relativeabundance) are considered. All structures were identi®edby GC-MS according to their RI-values, molecularmasses [M+] and fragmentation patterns in comparison

with reference compounds and data (Witte et al. 1993).Detailed analyses of the respective alkaloid patternshave been published elsewhere, i.e. those of S. vulgarisand S. vernalis (Hartmann and Zimmer 1986; vonBorstel et al. 1989), those of S. erucifolius and the twochemotypes of S. jacobaea (Witte et al. 1992) and thoseof S. inaequidens (Bicchi et al. 1989).

Maintenance of PAs in Senecio vulgaris

To evaluate any turnover or degradation of PAs in wholeplants, two separate types of experiment were performed.(i) A 3-d pulse of [14C]putrescine was fed to asepticallygrowing Senecio plantlets, followed by transfer of theplantlets into fresh tracer-free medium and harvest aftera further 5 to 6 and 12 to 13 d of growth. (ii) [14C]Senecionine N-oxide was applied to the scratched uppersurface of single leaves of ®eld-growing S. vulgaris plants.The treated plants were left undisturbed at their growingsites until harvest after 3 and 29 d.

The results of the two types of experiment aresummarized in Tables 2 and 3, respectively. In bothtypes of experiment the distribution of radioactivelylabelled PAs was quanti®ed for the whole plant as wellas for the various organs and expressed as total amountof alkaloid per plant or organ and as alkaloid concen-tration (based on tissue fresh weight). In the ®rst type ofexperiment (Table 2) the data are given for representa-tive specimens out of ®ve replications for a vegetativeplant (Table 2A) and a plant with ¯ower buds(Table 2B). In the second type of experiment the meanvalues are given for ®ve replicates. The results can besummarized and interpreted as follows:

(i) No turnover or degradation of PAs was detected inthe two types of experiment. About 25% of 14C-labelwas incorporated into PAs. This amount remainedvirtually unchanged over a period of 15 to 16 d(Table 2). In the second type of experiment about

Table 1. The PA patterns of ®ve Senecio species as established by GC-MS

Alkaloid Ri m/z [M+] Relative abundance of alkaloids (%)

S. vul a S. ver S. jac-J S. jac-E S. eru S. ina

1 Senecionine 2294 335 56 45 <3 11 <3 <32 Senecivernine 2280 335 <3 16 <3 53 Integerrimine 2350 335 9 <3 5 3 74 Seneciphylline 2303 333 26 11 21 75 Retrorsine 2515 351 3 4 376 Usaramin 2580 351 87 Eruci¯orine 2600 351 3 58 Jacobine 2432 351 389 Jacozine 2460 349 1610 Jaconine 2520 387 1211 Erucifoline 2510 349 76 9212 Ac-erucifoline 2610 391 3 313 Senkirkine 2470 365 2114 Otosenine 2595 381 915 Florosenine 2745 423 34

aS. vul = S. vulgaris; S. ver = S. vernalis; S. jac-J = S. jacobaea jacobine chemotype; S. jac-E = S. jacobaea erucifoline chemotype; S. eru=S. erucifolius; S. ina = S. inaequidens

T. Hartmann and B. Dierich: Chemical diversity and variation of pyrrolizidine alkaloids 445

Table 2. Distribution, maintenance and conversion of radioactively labelled PAs upon feeding of [14C]putrescine to the roots of asepticallygrown Senecio vulgaris. Experiment A: vegetatively growing plants, harvested 8 and 15 d after tracer application; experiment B: plants with¯ower bud formation, harvested 9 and 16 d after tracer application. In each experiment representative examples out of ®ve replicates arepresented

Age (d) Plant organs FW Total PAsa

(%)PA concn.[% (g FW))1]

Qb Relative abundance (%)

SO Sph-Noxc

Experiment A:8 whole plant 1.37 25 18.1 1.0 93 7

leaves 0.46 7.8 17.0 0.9 90 10leaves (new) 0.16 5.0 31.3 1.7 96 6stem 0.18 3.0 16.7 0.9 90 10roots 0.57 9.2 16.1 0.9 95 5

15 whole plant 1.80 26 14.4 1.0 87 13leaves 0.38 6.0 15.8 1.1 80 20leaves (new) 0.15 3.9 26.0 1.8 82 18stem 0.44 10.1 23.0 1.6 94 6roots 0.83 6.0 7.2 0.5 86 14

Experiment B:9 whole plant 1.50 24 16.0 1.0 91 9

leaves 0.53 8.2 15.5 1.0 83 17leaves (new) 0.01 0.3 33.0 2.1 87 13¯ower buds 0.03 0.7 23.3 1.5 90 10¯ower buds (new) 0.07 1.4 20.0 1.3 96 4stem 0.31 3.8 12.3 0.8 81 19roots 0.57 9.6 16.8 1.1 96 4

16 whole plant 1.93 23 11.9 1.0 67 33leaves 0.25 5.8 23.2 1.9 49 51leaves (new) 0.11 5.1 46.4 3.9 50 50¯ower buds 0.02 1.2 60.0 5.0 60 40¯ower buds (new) 0.06 1.2 20.0 1.7 68 32stem 0.58 4.1 7.0 0.6 80 20roots 0.91 5.8 6.4 0.5 89 11

a100% = total radioactivity taken upbPA concentration of whole plant = 1.0; quotients >1.0 indicate above average concentrations, quotients <1.0 indicate below averageconcentrationscSph-Nox = seneciphylline N-oxide

Table 3. Allocation and maintenance of [14C]SO applied to the upper surface of a leaf (tracer leaf) of S. vulgaris. Recovery, tissue-distribution and relative tissue concentrations were evaluated 3 and 29 d after tracer application

Plant Total 14C-labelled PA N-oxide (after 3 d)a Total 14C-labelled PA N-oxide (after 29 d)a

Recoveryb

(%)Distribution(%)

Relativeconcentrationc

Recoveryb

(%)Distribution(%)

Relativeconcentrationc

Whole plant 59.7 � 13.4 100 1.00 57.2 � 17.2 100 1.00Tracer-leaf d 32.9 � 9.8 55.2 29.12 6.9 � 2.8 12.2 19.43Roots 0.2 � 0.04 0.4 0.14 0.5 � 0.1 0.9 0.26Stems 13.0 � 4.3 21.8 0.60 13.2 � 5.1 23.1 0.47Leaves 1.1 � 0.3 1.8 0.14 4.1 � 1.7 7.2 0.86In¯orescences 12.4 � 3.8 20.8 1.26 32.4 � 11.3 56.6 2.41

Total fresh weight: 4.3 � 0.3 g 18.9 � 1.3Total PAs per plante: 0.624 � 0.26 g 6.946 � 0.87 gTotal PA concentration: 0.145 � 0.03 mg (g FW))1 0.368 � 0.08 mg (g FW))1

aMean values (�SD) of 5 replicatesb100% = [14C]SO appliedcThe relative concentration (FW basis) of 14C-labelled PA N-oxides of whole plant was set = 1.0; values >1.0 indicate above, values <1.0below average concentrationsdLeaf to which the tracer was appliedeDetermined by capillary GC


60%of the [14 C]SO applied to the leaf was recoveredin the PA fraction 3 d after application. This amountremained almost unchanged (recovery 57%) duringthe subsequent four weeks of undisturbed plantgrowth. The loss of labelled SO prior to the ®rstharvest is most likely due to unspeci®c degradationof the alkaloid on its way via the apoplast to itsphysiological compartments (e.g. cell vacuoles).

(ii) Although PAs lack turnover, they are allocateddynamically all over the plant. In the two types ofexperiment the ®nal allocation patterns are inaccordance with species±speci®c PA accumulation,i.e. tendency to accumulate in the in¯orescences.Thus, there is not only a dynamic allocation fromthe roots as sites of PA biosynthesis to the shoots(Table 2) but also from the shoots downwards tothe roots (Table 3). Furthermore, the ``populations''of radioactively labelled alkaloid molecules musthave remained mobile over the duration of theexperiments since considerable concentrations oflabelled PAs are found in newly grown leaves or¯ower buds, which were not in existence at the timeof tracer feeding (Table 2 ``new''). This is inaccordance with long-term tracer experiments usingSenecio root cultures in which labelled PAs areallocated into newly grown roots (Sander andHartmann 1989).

(iii) Separation and analysis of the radioactively labelledalkaloid fractions by HPLC revealed in addition toSO the presence of seneciphylline N-oxide (Table 2)and trace amounts of retrorsine N-oxide (notshown). From previous studies with Senecio root

cultures (Sander and Hartmann 1989) it is likely thatthese alkaloids are formed by transformation of SO.

Species-speci®c transformation of SOin shoots of Senecio

To examine whether whole plants are capable oftransforming SO into the N-oxides of seneciphyllineand retrorsine, the experiment described in Table 2 wasrepeated but labelled SO was applied instead ofputrescine. The results con®rm that seneciphyllineN-oxide and retrorsine N-oxide are biosyntheticallyderived from SO (Table 4). Retrorsine N-oxide occursvery late and is not detectable during the ®rst two weeksfollowing tracer application. Again, the tissue distribu-tion of the labelled PAs shows their spatial mobility and,furthermore, indicate the shoot as a major site of SOtransformation.

The following three experiments were designed toevaluate the sites and time course of SO transformationsin Senecio species with more complex PA patterns.

Senecio vernalis. The PA pattern of S. vernalis resemblesthat of S. vulgaris (Table 1) but in contrast to the lattercontains the otonecine derivative senkirkine, which doesnot exist as N-oxide. Root cultures of S. vernalistransform exogenously supplied SO into senkirkine(Toppel et al. 1987). To test whether S. vernalis shootsare able to catalyze the species-speci®c transformations,detached ¯owering shoots were allowed to take up[14C]SO via the cut end of the stem. The pattern of

Table 4. Distribution, maintenance and conversion of [14C]PA N-oxides after feeding of [14C]SO via roots to aseptically grown S. vulgaris.Plants were grown on MS medium with tracer for 3 d (®rst harvest) and were then transferred to fresh MS medium without tracer and werekept growing until second and third harvest. Average of two replicates

Age(Recovery)b

Plant organs Rel. abundance of [14C]PA N-oxides (%)a Total[14C] PAs

Relative[14C] PA

Qd

SO Sph-Noxc Ret-Noxc (%) concentration[% (g FW))1]

3 days whole plant 89 11 n.d. 100 186 1.0(42%)b leaves 72 28 36 118 0.6

stems 75 25 4 64 0.3roots 100 n.d. 60 352 1.9

12 days whole plant 53 47 n.d. 100 139 1.0(49%)b leaves 39 61 36 181 1.3

leaves (new) 27 73 27 190 1.4in¯orescences 33 67 1 111 0.8stems 40 60 3 4 <0.1roots 91 9 34 122 0.9

25 days whole plant 62 33 5 100 50 1.0(42%)b leaves 48 48 4 22 61 1.2

leaves (new) 50 44 6 30 90 1.8in¯orescences 41 35 24 8 145 2.9stems 57 43 n.d. 6 14 0.3roots 100 n.d. n.d. 34 42 0.8

a100% = total [14C]PAs in the respective plant or plant organbPercent [14C]PAs recovered, [14C]SO applied = 100%cSph-Nox = seneciphylline N-oxide, Ret-Nox = retrorsine N-oxidedPA concentration of whole plant = 1.0; quotients >1.0 indicate above average concentrations, quotients <1.0 indicate below averageconcentrationsn.d. = not detectable


labelled PAs for the di�erent plant organs was quanti-tatively evaluated by radio-TLC and radio-HPLC over aperiod of 14 d. The results are illustrated in Fig. 2.Besides senkirkine, small amounts of the N-oxides ofseneciphylline and retrorsine, which are also compo-nents of the genuine S. vernalis pattern (Table 1), aredetectable. The transformation patterns show tissue-speci®c di�erences. Senkirkine is preferentially formedin vegetative tissues, particularly leaves, whereas in¯or-escences contain only low levels of senkirkine. Thesedi�erences coincide perfectly with the genuine PAdistribution pattern, i.e. senkirkine as the dominatingalkaloid in leaves and stems and SO as the majoralkaloid of the ¯ower heads (Hartmann and Zimmer1986). Transformation experiments with isolated organs(i.e. leaves, stems and in¯orescences) exhibited the sameorgan-speci®c di�erences in transformation e�ciency(data not shown). Thus, the di�erences between theorgans shown in Fig. 2 are due to di�erences in thetransformation e�ciency and are not due to di�erentialallocation. As with S. vulgaris (Table 4), SO transfor-mation is an extremely slow process which in the case ofsenkirkine continues to proceed over the entire 14 dduration of the experiment (Fig. 2).

Senecio inaequidens. Detached leaves, stems and in¯or-escences of a local chemotype of S. inaequidens whichcontains <3% SO were allowed to take up [14C]SO for1 and 8 d. The transformation patterns (Fig. 3) againcorrespond well to the genuine PA pattern (Table 1).Retrorsine N-oxide is formed as the major alkaloidfollowed by the species-speci®c otonecine derivativesotosenine and its acetyl ester ¯orosenine. Again, allorgans are capable of catalyzing the species-speci®ctransformations, in contrast to S. vernalis, however, withalmost the same relative e�ciencies.

Senecio jacobaea (2 chemotypes) and S. erucifolius).Detached shoots of S. erucifolius as well as shoots of thejacobine and erucifoline chemotypes of S. jacobaea wereallowed to take up [14C]SO. The radioactively labelledPA patterns were evaluated 2 d later (Table 5). They

Fig. 2. Conversion of [14]SOinto species-speci®c PAs bydetached shoots of Seneciovernalis. Each shoot (height ca.20 cm) was allowed to take up2 ml of tracer solution(20 nmol, 37 kBq in tap water)via the cut end of the stem.After complete uptake (12 to24 h) the shoot was placed intap water without tracer untilharvest. The relative abundance(%) of SO and its transforma-tion products at the respectivetime intervals is given for wholeshoots and the three mainorgans. Average of 2 replicates

Fig. 3A,B. Senecio inaequidens. Feeding of [14]SO to single leaves,stem segments (ca. 5 cm) and in¯orescences obtained from ¯oweringspecimens (height ca. 60 cm). For feeding conditions see legend ofFig. 2. Organs were harvested after 1 d (A) and 8 d (B)


clearly re¯ect the genuine PA composition of therespective species or chemotypes (Table 1). Shoots ofthe jacobine chemotype of S. jacobaea transform[14C]SO preferentially into its 15,20-epoxide (jacobine),whereas shoots of the erucifoline chemotype of S. jacob-aea transform [14C]SO into its 12,13-epoxide (erucifo-line). Erucifoline was also formed in S. erucifoliustogether with 21-hydroxyintegerrimine (eruci¯orine).Only those PAs that are known constituents of thegenuine PA patterns were formed.

Speci®city of SO transformations

With the exception of four alkaloids (Fig. 4B) all majorPAs identi®ed by GC-MS from the ®ve Senecio species(Table 1) were shown to be transformation products ofSO (Fig. 4A). The four exceptions are integerrimineN-oxide and usaramine N-oxide the respective 20-E-isomers of SO and retrorsine N-oxide, as well as jaconineand senecivernine. Seneciphylline N-oxide, SO andretrosine N-oxide are frequently accompanied by smallproportions of their E-isomers (Hartmann and Witte1995). We assume that spartioidine N-oxide, the E-isomer of seneciphylline N-oxide (not listed in Table 1)and usaramine N-oxide arise together with theirZ-analogues from integerrimine N-oxide. With themethods applied we were not able to separate theradioactively labelled geometrical isomers. JaconineN-oxide can be regarded as the chlorolysis product ofjacobine N-oxide. It is always found in the S. jacobaeajacobine chemotype (Witte et al. 1992). SenecivernineN-oxide which in some Senecio species accompanies SO(Hartmann and Witte 1995) is most likely formedtogether with SO as the primary product of biosynthesisby a modi®ed C-C-linkage of the two isoleucine-derivedC5-acids which constitute the C10-necic acid of themacrocyclic alkaloid (Fig. 1). Again, the two alkaloidsare only separated by GC but not by the methodsapplied here.

With these exceptions, all PAs which constitute theunique PA patterns of the ®ve Senecio species (Table 1)

are de®nitely derived from SO (Fig. 4A). The tentransformations illustrated in Fig. 4A are simple butspeci®c one-step or two-step reactions. The positionspeci®city and stereoselectivity of these reactions clearlyindicate enzyme catalysis. The species speci®city of thetransformations excludes the formation of the alkaloidderivatives by indiscriminate enzyme activities, butrather indicates the existence of speci®c enzymes.

Conclusions

Together with the already-known physiological andbiochemical features of PA metabolism our ®ndingsallow us to draw a more complete picture of PAformation and structural diversi®cation in a Senecioplant (Fig. 5). The basic PA structure (i.e. SO) issynthesized in the roots. The SO is translocated intoshoots via the phloem and allocated to the storagetissues where it may accumulate in cell vacuoles. Duringallocation, chemical diversi®cation of SO into thespecies-speci®c alkaloid bouquet takes place. This di-versi®cation, which is separated in time and space fromSO biosynthesis, is a speci®c but dynamically slowprocess which may vary in its e�ciency between di�erentplant tissues (e.g. senkirkine formation in S. vernalis,Fig. 2). Since PAs do not turn over, the total amount ofPAs per plant is controlled by the rate of SO formationin the roots, a process which is closely linked to rootgrowth (Hartmann et al. 1988; Sander and Hartmann1989).

Concerning genetic variation, constitutive SO bio-synthesis in roots is highly constrained. Any geneticchange would most likely only a�ect the total amount ofalkaloid synthesis or delete alkaloid biosynthesis. Incontrast, chemical diversi®cation of SO in the shoots ishighly plastic. Any spontaneously occurring change inthe activity of one of the transforming enzymes (i.e.mutation of its gene) would quantitatively change thecomposition of the PA pattern, without a�ecting thetotal amount of PAs. Additional elements of highplasticity are involved such as alkaloid allocation,accumulation and tissue-speci®c di�erences which allrepresent processes which are also under genetic control.This plasticity implies that the PA bouquet of Seneciopopulations should be extremely variable. This, in fact,has been demonstrated for S. vulgaris and S. vernalis(von Borstel et al. 1989) as well as S. jacobaea andS. erucifolius (Witte et al. 1992). A detailed geneticanalysis of S. jacobaea populations revealed that thelarge phenotypic variation in both total PA contents andin the relative amounts of the major individual PAs islargely due to genetic variation (Vrieling et al. 1993).The authors therefore suggest that natural selection canchange PA concentration and composition in popula-tions of S. jacobaea. This is fully in accordance with ourconclusions.

Our ®ndings demonstrate that speci®c enzymes mustbe responsible for the diversi®cation of SO. Diversi®ca-tion of secondary pathways or products is often postu-lated to be brought about by indiscriminate enzyme

Table 5. Species-speci®c transformation of [14C]SO by detachedshoots of two chemotypes of S. jacobaea and of S. erucifolius.Shoots were allowed to take up the tracer via the cut end of theirstems. After complete uptake (12±24 h) the shoot was placed in tapwater without tracer until harvest after 48 h

Alkaloid Relative abundance (%) of [14C]PA N-oxides

S. jacobaea-J a S. jacobaea-E b S. erucifolius

Senecionine 56 47 13Seneciphylline 2 9 2Ac-Seneciphylline 3 5Retrorsine 3Eruci¯orine 15Jacobine 32Jacozine 5Erucifoline 29 63Ac-Erucifoline 9 2

aJacobine chemotypebErucifoline chemotype


activities (e.g. Jones and Firn 1991) although there isweak experimental evidence for this hypothesis. On thecontrary, numerous examples demonstrate participation

of highly speci®c enzymes in secondary pathways, e.g.alkaloids (Kutchan 1995), terpenoids (Gershenzon 1994)or O-methylations involved in phenolic diversi®cation(Ibrahim 1997). The observation that in di�erent Seneciospecies SO is transformed into unique products clearlyindicates that the individual PA bouquets are broughtabout by genetically controlled speci®c processes. Thisin turn suggests that the diverse PA patterns are not theresult of coincidence but must have evolved underselection pressure. The physiological maintenance andmetabolic features of constitutively formed SO describedhere (Fig. 5) provide an ideal mechanistic basis forchemical diversi®cation. We hypothesize that in consti-tutive plant defence such a system provides an excellentstrategy to successfully defend against and possiblyescape from local herbivory, e.g. by generating popu-lations with more-deterrent or unpalatable alkaloidbouquets.

This study was supported by grants from the Deutsche For-schungsgemeinschaft and Fonds der Chemischen Industrie. Wethank Claudine Theuring for preparing the labelled SO andDr. Ludger Witte for performing the GC-MS analyses.

Fig. 4. A The ten speci®ctransformations of SO (1)detected in at least one of thestudied ®ve Senecio species. BThe geometrical isomers (3, 6)of 1 and 5 and the structuralisomer (2) of SO which do notdirectly originate from SO, aswell as 10 the chlorolytic prod-uct of 8, which was detectable intransformation experiments.Numbers in bold type refer tothe compounds listed in Table 1

Fig. 5. General scheme of metabolic and physiological features ofPAs in Senecio. Speci®c structural diversi®cation of SO provides ahighly plastic system which is suggested to constitute a powerfulstrategy in constitutive plant defence against herbivores


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