Genetics, chemistry and ecology of a qualitative glucosinolate polymorphism in Barbarea vulgaris Promotoren: Prof. dr. ir. Wim H. van der Putten Hoogleraar ‘Functionele biodiversiteit met bijzondere aandacht voor de rol van nematoden in multitrofe interacties’ Laboratorium voor Nematologie, Wageningen Universiteit Prof. dr. Louise E.M. Vet Hoogleraar Evolutionaire ecologie, Laboratorium voor Entomologie, Wageningen Universiteit Co-promotor: Dr. ir. Nicole M. van Dam Senior onderzoeker, Nederlands Instituut voor Ecologie, Heteren Promotiecommissie: Prof. dr. Eddy van der Meijden, Instituut voor Biologie, Leiden Prof. dr. ir. Harro J. Bouwmeester, Wageningen Universiteit Prof. dr. Caroline Müller, University of Bielefeld, Duitsland Prof. dr. ir. Corné M.J. Pieterse, Universiteit Utrecht Dit onderzoek is uitgevoerd binnen de onderzoekschool Experimental Plant Sciences. Hanneke van Leur Genetics, chemistry and ecology of a qualitative glucosinolate polymorphism in Barbarea vulgaris Proefschrift ter verkrijging van de graad van doctor op gezag van de rector magnificus van Wageningen Universiteit, Prof. dr. M.J. Kropff, in het openbaar te verdedigen op maandag 17 maart 2008 des namiddags te vier uur in de Aula Van Leur, H. (2008) Genetics, chemistry and ecology of a qualitative glucosinolate polymorphism in Barbarea vulgaris PhD thesis Wageningen University – with a summary in Dutch ISBN 978-90-8504-900-5 … En altijd weer de vraag Of het ooit terecht is Dat het een gevecht is Om gewoon ergens te zijn Om te wonen en te leven En al was het ook maar even Echt een mens te kunnen zijn En dan los te komen van je plaats en tijd Bløf - Mens Voor Ron, mijn ouders & Bram Contents Summary 9 Chapter 1 General Introduction 13 Chapter 2 A heritable glucosinolate polymorphism within natural populations 27 of Barbarea vulgaris Chapter 3 Metabolic and developmental costs associated with a Barbarea 45 vulgaris glucosinolate polymorphism Chapter 4 Barbarea vulgaris glucosinolate phenotypes differentially affect 65 performance and preference of two different species of lepidopteran herbivores Chapter 5 Reciprocal interactions between the Cabbage root fly (Delia 83 radicum) and two glucosinolate phenotypes of Barbarea vulgaris Chapter 6 Interactions of Barbarea vulgaris glucosinolate phenotypes with 101 above- and belowground invertebrate communities and their consequences for plant fitness Chapter 7 Quest for the gene – as far as we got 119 Chapter 8 Discussion and Synthesis 131 References 143 Nederlandse samenvatting (Dutch summary) 155 Dankwoord (Acknowledgements) 159 Curriculum Vitae 163 List of publications 165 Education statement 167 SUMMARY Summary Like many other plants, chemical defence compounds are involved in the defense of Barbarea vulgaris against natural enemies. Barbarea vulgaris produces glucosinolates, which are present in most crucifers such as cabbage, mustard, and the scientific model species Arabidopsis thaliana. Glucosinolates form, together with an enzyme (myrosinase), a two- component system: the enzyme and glucosinolates are stored in spatially separated compartments. Upon cell disruption they come into contact, and the glucosinolates are catabolised by the enzyme. For generalist herbivores the glucosinolates, and especially their breakdown products, are often toxic and protect the plants against herbivory. However, specialist herbivores often use these same compounds to recognize suitable host plants. The breakdown products are also responsible for the specific taste to many cabbage and mustard species There are over 100 different glucosinolates, each with a different chemical structure. The structure determines, amongst other factors, which breakdown product will be formed upon damage. Every plant species has its own typical composition of glucosinolates. I studied how these glucosinolate profiles may affect plant resistance against herbivores. In a screening of several Dutch populations it was found that Barbarea vulgaris plants differed in glucosinolate profile. The majority of the plants contained mainly glucobarbarin, a glucosinolate typical for this species and named after the genus Barbarea. Also populations sampled in Germany, Belgium, France, and Switserland, consisted completely of plants with mainly glucobarbarin. In half of the Dutch populations I found that a minority of the plants (2-22%) produced another glucosinolate, named gluconasturtiin. The difference in the chemical structure between these two glucosinolates is very small; glucobarbarin has only one hydroxyl group more than gluconasturtiin. However, this small structural difference may be of large biological relevance. When gluconasturtiin reacts with myrosinase a toxic and unpalatable isothiocyanate is formed, whereas glucobarbarin, due to the position of the hydroxyl group, produces oxazolidinethions. It is unknown whether these oxazolidinethiones are toxic, but in mammals they can inhibit the iodine intake, thereby causing thyroid problems. The Barbarea vulgaris glucosinolate polymorphism thus has two chemotypes. I characterized these chemotypes and used them to study the effects of different glucosinolates on herbivores. The difference in glucosinolate profile is consistently present in all plant organs of B. vulgaris, but it is larger in the aboveground organs than in the roots. The glucosinolate profile does not change upon induction by insects nor upon artificial induction by addition of jasmonic acid. I crossed plants, analysed the chemotype of the offspring and showed that the ability to produce glucobarbarin is heritable and regulated by a dominant gene. Based on the assumption that there is a specific enzyme that converts gluconasturtiin to glucobarbarin by a single hydroxylation step, I identified some candidate genes. Further research is needed to determine whether one of these candidate genes is indeed responsible the difference between the chemotypes. Subsequently, I studied the effect of chemotype on leaf and root herbivores. Plants with mainly glucobarbarin turned out to be very resistant to the generalist leaf-eating larvae of the Mamestra brassicae moth. Almost none of the larvae survived on plants with mainly 9 SUMMARY glucobarbarin. If the larvae were given the choice, they strongly preferred to feed on plants with mainly gluconasturtiin. However, female moths deposited approximately the same number of eggs on each chemotype. Larvae of the specialist small cabbage white grew equally well on each chemotype and did not distinguish between the chemotypes in choice experiments. Larvae of the specialist cabbage root fly, however, performed worse on roots of the gluconasturtiin type than on roots of the glucobarbarin type. Plants responded to the root fly infection by a reduction of the root and shoot biomass with 50%, and decreased levels of nutrients such as sugars and amino acids. In order to translate the results obtained in the greenhouse to the natural situation, I planted plants of both chemotypes in an experimental garden. Over a period of two years, the numbers of several herbivores were counted on each chemotype every week during the growth season. This revealed that some aboveground insects had a preference for a certain chemotype, and others did not. Butterflies of the small cabbage white preferred to oviposit on plants with gluconasturtiin, but flea beetles and gall midges were more abundant on plants with glucobarbarin. The cabbage aphid and the green peach aphid did not show any preference and were equally abundant on both chemotypes. Three to four times a year, I dug up a subset of the plants to analyse the root herbivores and the soil nematode community. The numbers and community structure of these belowground organisms did not differ between the chemotypes. Additionally, I investigated whether there are other chemical differences between the chemotypes, apart from glucosinolate profile, that could explain the preference of the above insect species. Therefore, I performed extensive metabolomics analyses, using LC-TOF-MS. Multivariate analyses showed that the major chemical differences between the chemotypes was due to differences in glucosinolates. These differences were larger in shoots than in roots. Apart from glucosinolates only eight unidentified compounds differed between the chemotypes. Known defense compounds such as flavonoids and saponins were identified but did not differ between the chemotypes. Therefore, it is very likely that the differences in herbivore performance and preference are predominantly caused by the differences in glucosinolate profile. Based on my results, I conclude that the structure of glucosinolates can cause significant differences in resistance against several herbivores. However, in the case of B. vulgaris there is no supreme chemotype that is more resistant in all cases. Which of the two chemotypes accrues the largest benefit in a natural environment thus will depend on the herbivore community in their population. In this way the B. vulgaris glucosinolate polymorphism can be maintained in natural populations. 10 11 12 CHAPTER 1 General Introduction Hanneke van Leur 13 CHAPTER 1 14 GENERAL INTRODUCTION Plant defence and evolution Plants are susceptible to environmental challenges but cannot run away. Despite this apparent vulnerability the earth’s flora has evolved to be highly diverse and abundant (Schoonhoven et al., 1998a). The fact that not all plants are completely consumed can be due to top-down control (Hairston et al., 1960), but also to bottom up mechanisms such as direct