Induction of plant responses by a sequestering insect: Relationship of glucosinolate concentration and myrosinase activity
Introduction
The distribution, concentration, and composition of various toxic or bitter-tasting plant secondary metabolites such as glucosinolates are known to depend on many factors, e.g. nutritional state, age, tissue, genotype, population, or species (Brown, Tokuhisa, Reichelt, & Gershenzon, 2003; Charron, Saxton, & Sams (2005a), Charron, Saxton, & Sams (2005b); Clossais-Besnard & Larher, 1991; Fahey, Zalcmann, & Talalay, 2001; Visvalingam, Honsi, & Bones, 1998). In many cases, these compounds confer efficient defence against pathogens or herbivores (Bennett & Wallsgrove, 1994; Blau, Feeny, Contardo, & Robson, 1978; Wittstock & Gershenzon, 2002). McKey (1974), McKey (1979) proposed an adaptive theory to explain variability of defence levels within a plant individual. According to this so-called ‘optimal defence theory’, tissues that are most valuable to the plant are expected to have the highest constitutive defence levels, and their secondary metabolites to be least inducible by damage. In plants with one single shoot, we expect the stem's tissue to have a high constitutive level of defence, because damage would result in reduced water and nutrient supply for all tissues above, and thus reproduction success of the whole plant would be affected dramatically. In contrast, damage to assimilating leaves, except to cotyledons (Wallace & Eigenbrode, 2002), should be less severe for reproduction, as has been shown, for example, for Raphanus sativus (Brassicaceae) (Mauricio & Bowers, 1990). Thus, in leaves, inducible responses rather than constitutive defences are likely to be observed. On the other hand, vulnerability of tissue might be higher for leaves than for stems since the latter are often well protected by physical features. Thus, value and vulnerability need to be ranked when evaluating optimal defence (McKey, 1974).
Induced defences (reviewed in Dicke & Hilker, 2003) are thought to have evolved as a cost-saving strategy for plants growing under nutrient-limited conditions (Cipollini, Purrington, & Bergelson, 2003; Zangerl, 2003). Given an energetic cost of plant allocation from growth to defence, plant fitness could be enhanced by rising defence levels only in the presence of herbivory (Agrawal (1999), Agrawal (2005)). In Brassicaceae, induced responses have been widely studied using a large diversity of organisms, chemical signals, or mechanical wounding (Agrawal & Kurashige, 2003; Menard, Larue, Silue, & Thouvenot, 1999; Rostás, Bennett, & Hilker, 2002; Shattuck, 1993). Brassicaceae contain, amongst other mechanisms, a binary defence system consisting of preformed substrates and hydrolytic enzymes, namely glucosinolates and myrosinases (Andréasson & Jørgensen, 2003; Halkier, 1999; Kliebenstein, Kroymann, & Mitchell-Olds, 2005; Rask et al., 2000). Glucosinolates consist of a thioglucoside moiety linked to a variety of amino-acid-derived side chains (Andréasson & Jørgensen, 2003). Myrosinases (β-thioglucoside glucohydrolases) occur in various isoforms (reviewed in Rask et al., 2000). Some of these are soluble under non-denaturing conditions. Others have been shown to form a complex with two groups of proteins, myrosinase-associated proteins (MyAP) and myrosinase-binding proteins (MBP) (Eriksson et al., 2002; Eriksson, Ek, Xue, Rask, & Meijer, 2001). These complexes are insoluble in non-denaturing buffers but the myrosinase subunits are still active. Upon tissue disruption, the separately stored glucosinolates and myrosinases interact to form various mostly toxic products (Halkier, 1999; Wittstock, Kliebenstein, Lambrix, Reichelt, & Gershenzon, 2003).
So far, in induction studies on Brassicaceae, both parts of the putative defence system have rarely been investigated simultaneously (but see Andréasson et al., 2001; Siemens & Mitchell-Olds, 1998; Wallace & Eigenbrode, 2002). In most induction studies, either glucosinolates or myrosinases have been addressed, whereby important relationships might have been missed. Using microorganisms or herbivores as eliciting agents, it has been shown that especially glucosinolates with an indolic side chain increase in concentration (Agrawal, 1999; Bartlet, Kiddle, Williams, & Wallsgrove, 1999; Koritsas, Lewis, & Fenwick, 1991; Ludwig-Müller, Schubert, Pieper, Ihmig, & Hilgenberg, 1997; Rostás et al., 2002). Increases in glucosinolates caused a higher resistance of plants against specialist herbivores in several cases (Agrawal, Conner, Johnson, & Wallsgrove, 2002; Mewis, Appel, Hom, Raina, & Schultz, 2005). Also, activity of soluble myrosinases can increase after damage (Siemens & Mitchell-Olds, 1998). Additionally, transcript and protein levels of MyAPs and MBPs were shown to be inducible by damage or treatment with the plant hormone jasmonate (Taipalensuu, Andréasson, Eriksson, & Rask, 1997; Taipalensuu, Falk, & Rask, 1996; Pontoppidan, Hopkins, Rask, & Meijer (2003), Pontoppidan, Hopkins, Rask, & Meijer (2005)). Thus, studies monitoring the activity of either soluble or bound myrosinases only are likely to underestimate total activity of a plant's tissue. Therefore, we established a method to determine myrosinase activity of soluble and insoluble fractions of the enzyme, and correlated these with glucosinolate levels of the same tissues.
In previous studies, periods of herbivory as well as the time between treatment and sampling of tissues varied from less than an hour to a few weeks (Cipollini, Busch, Stowe, Simms, & Bergelson, 2003; Pontoppidan et al., 2005; Strauss, Irwin, & Lambrix, 2004). Systemic responses were examined after treatment of leaves with plant hormones, i.e. jasmonates or salicylates, in younger parts (only leaves) and older parts (only roots) of plants (Bodnaryk, 1994; Ludwig-Müller et al., 1997; Ludwig-Müller, Bennett, García-Garrido, Piché, & Vierheilig, 2002; van Dam, Witjes, & Svatoš, 2003). In contrast, responses to herbivore feeding were analysed only in younger tissue and mostly at a distance of two or more leaves from the site of damage (Bartlet et al., 1999; Traw & Dawson, 2002). Direct neighbouring tissues have rarely been monitored (Koritsas et al., 1991; Pontoppidan et al., 2005). Thus, little is known about the short-term pattern of metabolite induction in the older and younger stems and leaves directly surrounding the site of damage.
Herbivores used as inducing agents have so far included few generalists and a variety of crucifer specialists (Bodnaryk, 1992; Pontoppidan et al., 2005; Siemens & Mitchell-Olds, 1998; Traw & Dawson, 2002), in particular caterpillars of Pieris spp. (Agrawal & Kurashige, 2003; Strauss et al., 2004; Traw & Dawson, 2002), which do not sequester glucosinolates (Müller, Agerbirk, & Olsen, 2003), or aphids (Mewis et al., 2005; Pontoppidan et al., 2003). The turnip sawfly, Athalia rosae (L.) (Hymenoptera: Tenthredinidae), is oligophagous on species of Brassicaceae and can reach pest status on some crop species (Riggert, 1939; Saringer (1976), Saringer (1989); Šedivý & Vašák, 2002). The larvae sequester certain glucosinolates of their host plants within their haemolymph (Müller et al., 2001; Müller & Wittstock, 2005) and use it in part for their own defence (Müller & Brakefield, 2003; Müller, Boevé, & Brakefield, 2002; Müller et al., 2001). For plants, sequestering insects represent a special problem, termed ‘lethal plant defence paradox’ (Price et al., 1980), as these herbivores benefit from their hosts’ putative defence compounds. For insects, sequestration of secondary compounds from host plants may be costly (Camara, 1997; Kelley, Johnson, & Murray, 2002), and for A. rosae additional costs might result from the necessity to efficiently circumvent hydrolysis of glucosinolates. Since A. rosae is not only able to overcome the glucosinolate–myrosinase system but also to use it for its own protection, we were particularly interested in the response of the plant to such a herbivore. We specifically addressed the question of whether feeding of approximately the same amount of leaf tissue by three young A. rosae larvae or by one old larva might result in different induction patterns. In many insect species, first-instar larvae are more susceptible to secondary plant metabolites than older ones (Zalucki, Clarke, & Malcolm, 2002). Consequently, as a possible solution to the lethal plant defence paradox, a vigorous increase of the (putative) plant defence levels in response to feeding by younger larvae was expected, while a rather weak increase should result from induction by older ones, as proposed earlier for the interaction of monarch butterfly larvae (Danaus plexippus L.) with milkweed (Asclepias syriaca L.) (Malcolm & Zalucki, 1996).
In this study, we investigated induction patterns in the herbivore-wounded leaf and upwards and downwards from the damaged site in the adjacent stem parts and leaves over several days. We determined glucosinolate concentrations as well as activities of soluble and insoluble myrosinases and also analysed these tissues for water, protein, and sugar content to examine nutritional changes. This set-up enabled us to reveal relevant induction responses with a high resolution in the host plant upon damage by a sequestering insect.
Section snippets
Planting and induction experiment
Seeds of Sinapis alba L. (cultivar: Salvo, obtained from Advanta Seeds B.V., Kapelle, Netherlands) were sown in mid-May 2004 and kept in a climate chamber at 25 °C, 16:8 h light–dark cycle, and 70% relative humidity (light source: Osram L 58 W/25 Universal White, 4150 lm; Osram, Munich, Germany). Seven days later, the seedlings were transferred to individual pots (diameter: 9 cm; height: 7 cm) with unfertilised soil (peat, pH 6). After 21 days, when five to six true leaves had developed, plants
Results
Since neither levels of primary and secondary metabolites nor enzyme activities differed significantly between plants treated with muslin bags and non-treated plants in any of the Mann–Whitney U-tests, in the following, we will focus on the bag treatment as the more relevant control compared to feeding treatments.
Discussion
The glucosinolate–myrosinase system of Brassicaceae is in general regarded as a defence system against antagonists (Bennett & Wallsgrove, 1994; Blau et al., 1978; Wittstock & Gershenzon, 2002). ‘Optimal defence theory’ predicts that constitutive levels of defence should be high in tissues with high values for reproduction and/or with higher vulnerability (McKey (1974), McKey (1979)). Considering value, we postulated that stems of S. alba are of higher value than leaves since damage to the
Acknowledgments
The authors thank J. Winkler-Steinbeck for plant cultivation; N. Stingl for help in sample processing; M. Riederer for making laboratory space and HPLC equipment available for our studies; and two anonymous reviewers for their helpful comments on an earlier draft of the manuscript. The authors received financial support for their work from the Sonderforschungsbereich 567 ‘interspecific interactions’ of the Deutsche Forschungsgemeinschaft.
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