Fundamentación Filosófica

methyl jasmonate on the concentration and

composition of pyrrolizidine alkaloids and

resistance towards Spodoptera exigua in two

Jacobaea species

Abstract

Treating plants with methyl jasmonate (MeJA) can mimic the effects of herbivory by activating the octadecanoid pathway in plants. Many studies have shown that the concentration and composition of pyrrolizidine alkaloids (PAs), the secondary

metabolites of Jacobaea species, play a role in resistance against herbivores. The

aim of this study was to determine whether the application of MeJA changed the PA concentration and composition and if affected herbivory by the generalist herbivore

Spodoptera exigua. A range of concentrations of MeJA was added to the medium of

Jacobaea vulgaris and Jacobaea aquatica tissue culture plants under axeniccondi- tions. After two weeks, plants were harvested and PA concentration and composition

were measured by LC-MS/MS. While the application of MeJA to the roots only mar-

ginally affected the total PA concentration in whole plants, the total PA concentration

decreased in roots but increased in shoots of J. vulgaris. This indicates that applying

MeJA does not elicit de novo synthesis and that PAs are reallocated from root to

shoot. In J. aquatica the total PA concentration was maintained and this reallocation

was not significant due to the fact that the majority of PAs were already stored in the shoots before the application of MeJA. Interestingly, we found that the application of MeJA in both species induced a strong shift in the PA composition especially from senecionine-like PAs to erucifoline-like PAs indicating that PA conversion took place. Our studies further showed that root induction could lead to PA variation in

the shoots of Jacobaea plants. In the bioassay, S. exigua preferred feeding on control

leaves of J. aquatica than those treated with MeJA while for J. vulgaris the leaves

of control and MeJA treated plants were hardly eaten. This result suggests that an increase of erucifoline-like PAs induced by the application of MeJA application play

a role in resistance to S. exigua in J. aquatica while other defence mechanisms might

be present in J. vulgaris.

Keywords

Jacobaea vulgaris, Jacobaea aquatica, Spodoptera exigua, Feeding damage, Tissue culture, Reallocation, Conversion, Biosynthetic pathway, Senecionine, Erucifoline.

Introduction

Secondary metabolites (SMs) are regarded as indispensable for the fitness and sur- vival of an organism (Wink 2003). The synthesis of many SMs in plants is in part controlled by the jasmonic acid (JA) pathway (Gundlach et al. 1992; Karban and Baldwin 2007), which is activated especially in response to chewing/biting herbi- vores, cell-content feeders and necrotrophic pathogens (Glazebrook 2005; Kawazu et al. 2012; Walling 2000). Herbivore attack may cause an increase in endogenous jasmonate levels that leads to the production of defensive proteins and SMs or mor- phological changes, such as an increase in trichome density (Farmer et al. 2003; Traw and Dawson 2002; Traw and Bergelson 2003; Zhao et al. 2005). The exoge- nous application of jasmonate, including JA and methyl jasmonate (MeJA), is widely used as a biochemical tool to elicit the production of SMs and to study plant defence responses (Chen et al. 2006; Largia et al. 2015; Liang et al. 2006). In a literature review we found that with the exception of three studies, only changes in levels of

total groups of SMs or one major compound was studied after MeJA application (Ta-

ble S1). However, one same group of SMs within a plant species generally includes many structurally diverse compounds. For example, 35 different glucosinolates have been identified in Arabidopsis thaliana (D’Auria and Gershenzon 2005). Interest- ingly not a single report shows changes in the qualitative composition of SMs after MeJA treatments although it is well known that this type of change can affect her- bivores or pathogens profoundly (e.g. Berenbaum et al. 1991; Bruinsma et al. 2007; Cheng et al. 2013; Hung et al. 1995; Kowalchuk et al. 2006).

To explore the influence of jasmonate on the concentration and qualitative

composition of SMs, we used Jacobaea vulgaris and Jacobaea aquatica as a study

system. All Jacobaea PAs are classified into four groups according to their basic

structure: jacobine-, erucifoline-, otosenine- and senecionine-like PAs (Cheng et al. 2011a; Hartmann 1999). The diversity of PA structures in these plants provides an opportunity to zoom in on the variation in composition of SMs within plant spe- cies. These alkaloids have been extensively studied as they play a role in defence against insect herbivores as well as soil-born microbes (Hol and van Veen 2002;

Macel 2011). An experiment with cell lines of Spodoptera exigua showed jacobine

and erucifoline to be the most toxic (Nuringtyas et al. 2014). We therefore expected that if MeJA application induced an increase of PAs, these would consequently lead

to a reduction of the herbivory by a chewing generalist insect herbivores such as S.

exigua.

The variation of PA concentration and composition has a genetic basis (Ma- cel et al. 2004; Vrieling et al. 1993), but it is also influenced by environmental condi-

tions. Macel and Klinkhamer (2010) observed that the PA composition of J. vulgaris

PA composition in J. vulgaris (Joosten et al. 2009). To avoid the effect of potential interactions with microorganisms on the jasmonate application we therefore per- formed the experiments under axenic conditions.

Thus, to investigate the effects of exogenously applied MeJA on PA concen-

tration and composition and on herbivory, we used J. vulgaris and J. aquatica tissue

culture plants and the generalist herbivore S. exigua. Specifically, we addressed the

following questions: 1) Does a MeJA treatment lead to dose-related changes in the total PA concentration? 2) Does a MeJA treatment lead to dose-related changes in PA composition? 3) Does MeJA application lead to PA reallocation and conversion? 4) Does MeJA application affect the herbivory by S. exigua?

Materials and Methods