Elsevier

Basic and Applied Ecology

Volume 8, Issue 1, 8 January 2007, Pages 13-25
Basic and Applied Ecology

Induction of plant responses by a sequestering insect: Relationship of glucosinolate concentration and myrosinase activity

https://doi.org/10.1016/j.baae.2006.02.001Get rights and content

Summary

Induction of plant allelochemicals is of particular ecological importance for interactions with herbivores that can make use of induced metabolites by incorporating them for their own defence. Induction patterns in white mustard, Sinapis alba, were investigated following herbivory of the turnip sawfly, Athalia rosae, which sequesters plant glucosinolates. Larvae of different age were allowed to feed for 24 h on young leaves of premature, non-flowering plants. Changes in primary and secondary metabolites were recorded in the damaged leaves (local) and in the adjacent leaves and stems (systemic) for several days. Organ- and time-specific patterns were evident. Local responses included increases in glucosinolate concentrations, soluble and insoluble myrosinase activity and glucose levels, while systemic responses in leaves were restricted to increases in myrosinase activities and glucose. All effects were strongest immediately after feeding and declined mostly within a day. Stems had overall lower constitutive levels of glucosinolates and myrosinase activities than leaves. Feeding by one large larva had a greater impact on the plant's physiology than feeding by three small ones, even though both treatments resulted in quantitatively similar leaf destruction. Local increase in glucosinolate concentration could be beneficial for larvae, while conspecifics feeding on induced adjacent leaves might be negatively affected due to higher myrosinase activity levels. The results are discussed in the context of the ‘optimal defence theory’ and the ‘lethal plant defence paradox’.

Zusammenfassung

Die Induktion pflanzlicher Allelochemikalien ist vor allem für Interaktionen mit Herbivoren von ökologischer Bedeutung, die diese induzierten Metabolite zu ihrer eigenen Verteidigung in ihr Körpergewebe einbauen. Reaktionen des weißen Senfs Sinapis alba auf den Fraß von Larven der Blattwespe Athalia rosae wurden ort- und zeitabhängig untersucht. Die Larven zeichnen sich durch die Fähigkeit aus, Glucosinolate zu sequestrieren. Junge Blätter von nicht blühenden Pflanzen wurden für 24 Std. von Larven unterschiedlichen Alters befressen. Anschließend wurden Veränderungen von Primär- und Sekundärmetaboliten in lokalen befressenen, sowie in benachbarten systemischen Blättern und Sprossabschnitten an den vier folgenden Tagen untersucht. Die gefundenen Reaktionen waren zeitabhängig und spezifisch für das jeweils betrachtete Organ. Lokale Reaktionen umfassten Anstiege im Gehalt von Glucosinolaten, löslichen und unlöslichen Myrosinaseaktivitäten und Glucose, während sich systemische Antworten in Blättern auf Anstiege in Myrosinaseaktivität und Glucosegehalt beschränkten. Alle gefundenen Effekte waren direkt nach der Fraßperiode am stärksten und nahmen in den meisten Fällen bis zum Folgetag ab. Sprossachsenabschnitte hatten niedrigere konstitutiv vorhandene Konzentrationen an Glucosinolaten und Myrosinaseaktivitäten als Blattgewebe. Der Fraß einzelner größerer Larven zeigte stärkeren Einfluss auf die Pflanzenphysiologie als der von Gruppen kleiner Larven, obwohl beide Behandlungen zu einer quantitativ ähnlichen Blattbeschädigung führten. Der lokale Anstieg von Glucosinolaten könnte für die Larven vorteilhaft sein, während Artgenossen, die an induzierten benachbarten Blättern fressen, möglicherweise durch erhöhte Myrosinaseaktivitäten beeinträchtigt werden. Die Ergebnisse werden vor dem Hintergrund der ‘optimal defence theory’ und dem ‘lethal plant defence paradox’ diskutiert.

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.

References (72)

  • J. Ludwig-Müller et al.

    Glucosinolate content in susceptible and resistant Chinese cabbage varieties during development of clubroot disease

    Phytochemistry

    (1997)
  • R. Menard et al.

    Glucosinolates in cauliflower as biochemical markers for resistance against downy mildew

    Phytochemistry

    (1999)
  • C. Müller et al.

    Uptake and turn-over of glucosinolates sequestered by the sawfly Athalia rosae

    Insect Biochemistry and Molecular Biology

    (2005)
  • B. Pontoppidan et al.

    Differential wound induction of the myrosinase system in oilseed rape (Brassica napus): Contrasting insect damage with mechanical damage

    Plant Science

    (2005)
  • U. Wittstock et al.

    Constitutive plant toxins and their role in defense against herbivores and pathogens

    Current Opinion in Plant Biology

    (2002)
  • A.R. Zangerl

    Evolution of induced plant responses to herbivores

    Basic and Applied Ecology

    (2003)
  • A.A. Agrawal

    Induced plant defense: Evolution of induction and adaptive phenotypic plasticity

  • A.A. Agrawal

    Future directions in the study of induced plant responses to herbivory

    Entomologia Experimentalis et Applicata

    (2005)
  • A.A. Agrawal et al.

    Ecological genetics of an induced plant defense against herbivores: Additive genetic variance and costs of phenotypic plasticity

    Evolution

    (2002)
  • A.A. Agrawal et al.

    A role for isothiocyanates in plant resistance against the specialist herbivore Pieris rapae

    Journal of Chemical Ecology

    (2003)
  • E. Andréasson et al.

    The myrosinase–glucosinolate system in the interaction between Leptosphaeria maculans and Brassica napus

    Molecular Plant Pathology

    (2001)
  • E. Bartlet et al.

    Wound-induced increases in the glucosinolate content of oilseed rape and their effect on subsequent herbivory by a crucifer specialist

    Entomologia Experimentalis et Applicata

    (1999)
  • R.N. Bennett et al.

    Secondary metabolites in plant defense-mechanisms

    New Phytologist

    (1994)
  • P.A. Blau et al.

    Allylglucosinolate and herbivorous caterpillars – contrast in toxicity and tolerance

    Science

    (1978)
  • M. Bridges et al.

    Spatial organisation of the glucosinolate–myrosinase system in brassica specialist aphids is similar to that of the host plant

    Proceedings of the Royal Society of London Series B: Biological Sciences

    (2002)
  • M.D. Camara

    Physiological mechanisms underlying the costs of chemical defence in Junonia coenia Hubner (Nymphalidae): A gravimetric and quantitative genetic analysis

    Evolutionary Ecology

    (1997)
  • C.S. Charron et al.

    Relationship of climate and genotype to seasonal variation in the glucosinolate–myrosinase system. I. Glucosinolate content in ten cultivars of Brassica oleracea grown in fall and spring seasons

    Journal of the Science of Food and Agriculture

    (2005)
  • C.S. Charron et al.

    Relationship of climate and genotype to seasonal variation in the glucosinolate–myrosinase system. II. Myrosinase activity in ten cultivars of Brassica oleracea grown in fall and spring seasons

    Journal of the Science of Food and Agriculture

    (2005)
  • D. Cipollini et al.

    Costs of induced responses in plants

    Basic and Applied Ecology

    (2003)
  • D.F. Cipollini et al.

    Genetic variation and relationships of constitutive and herbivore-induced glucosinolates, trypsin inhibitors, and herbivore resistance in Brassica rapa

    Journal of Chemical Ecology

    (2003)
  • N. Clossais-Besnard et al.

    Physiological role of glucosinolates in Brassica napus – concentration and distribution pattern of glucosinolates among plant organs during a complete life-cycle

    Journal of the Science of Food and Agriculture

    (1991)
  • H. Eichenseer et al.

    Salivary glucose oxidase: Multifunctional roles for Helicoverpa zea?

    Archives of Insect Biochemistry and Physiology

    (1999)
  • S. Eriksson et al.

    Complex formation of myrosinase isoenzymes in oilseed rape seeds are dependent on the presence of myrosinase-binding proteins

    Plant Physiology

    (2002)
  • S. Eriksson et al.

    Identification and characterization of soluble and insoluble myrosinase isoenzymes in different organs of Sinapis alba

    Physiologia Plantarum

    (2001)
  • J.A. Fordyce

    The lethal plant defense paradox remains: Inducible host-plant aristolochic acids and the growth and defense of the pipevine swallowtail

    Entomologia Experimentalis et Applicata

    (2001)
  • B.A. Halkier

    Glucosinolates

  • Cited by (50)

    • Plant-mediated indirect effects of climate change on an insect herbivore

      2021, Basic and Applied Ecology
      Citation Excerpt :

      Our climate simulations changed the chemical composition in leaves of S. alba plants by affecting glucosinolate and carbon levels as well as the C:N ratio. The increase in glucosinalbin concentrations, which is the most abundant glucosinolate of S. alba (Martin & Müller, 2007), at elevated temperatures may be related to temperature-dependent synthesis rates (Pietruszka, Lewicka & Pazurkiewicz-Kocot, 2005). This also suggests that the temperatures used here were not too stressful for S. alba.

    • Comparative effect of elicitors on the physiology and secondary metabolites in broccoli plants

      2019, Journal of Plant Physiology
      Citation Excerpt :

      Besides, these enzymes are involved in the conversion of aldoximes to thiohydroxymates (Halkier and Gershenzon, 2006; Wiesner et al., 2013). High indole GLSs induction has been reported to occur 24 h after herbivore damage, with a subsequent decline in the GLSs concentration within one day (Martin and Müller, 2007). However, the concentration of sinigrin needed more time to increase (up to 7 days) in Brassica rapa (Liang et al., 2006), suggesting that the indole GLSs composition responds more rapidly to herbivory than the aliphatic one.

    • Rapid incorporation of glucosinolates as a strategy used by a herbivore to prevent activation by myrosinases

      2014, Insect Biochemistry and Molecular Biology
      Citation Excerpt :

      Samples were extracted three times in 80% methanol after addition of 2-propenyl glucosinolate (sinigrin; Phytoplan, Heidelberg, Germany) as an internal standard. After purification of samples and conversion to desulfoglucosinolates as described elsewhere (Martin and Müller, 2007), desulfoglucosinolates were analysed by high performance liquid chromatography (HPLC) coupled to a diode array detector (HPLC-1200 Series, Agilent Technologies, Inc., Santa Clara, CA, USA) equipped with a Supelcosil LC 18 column (3 μm, 150 × 3 mm, Supelco, Bellefonte, PA, USA). The elution gradient started with 5% methanol for 6 min and was then increased from 5% to 95% within 13 min and held at 95% for 2 min, followed by a cleaning cycle.

    View all citing articles on Scopus
    View full text