Costs and benefits of two direct defenses in nicotiana attenuata [Elektronische Ressource] : nicotine and trypsin protease inhibitors / von Anke Steppuhn

Asteppuhn

Sujets

• Biologie

Informations

• Publié par: friedrich-schiller-universitat_jena
• Publié le: 01 janvier 2007
• Nombre de lectures: 14
• Langue: English
• Poids de l'ouvrage: 15 Mo

Extrait

Costs and Benefits of Two Direct Defenses in Nicotiana attenuata:
Nicotine and Trypsin Protease Inhibitors



Dissertation

zur Erlangung des akademischen Grades
doctor rerum naturalium (Dr. rer. nat.)









vorgelegt dem Rat der Biologisch-Pharmazeutischen Fakultät
der Friedrich-Schiller-Universität Jena


von Diplom-Biologin
Anke Steppuhn

geboren am 26. April 1977 in Berlin














Gutachter:

1. Prof. Dr. Ian T. Baldwin
2. Prof. Dr. Wolfgang W. Weisser
3. Prof. Dr. May R. Berenbaum


Tag der Doktorprüfung: 10.08.2007


Tag der öffentlichen Verteidigung: 18.09.2007


Table of Contents

1. Introduction
1.1 On the bottom of the food chain: How plants survive 1
1.2 “On/off-button” of genes: Gene silencing to study plant defense 2
1.3 On the burn: The ecology of Nicotiana attenuata 5
1.4 On the battlefield: Nicotiana attenuata’s herbivores and its defense 6
strategies
1.5 On the books: Tests of the cost-benefit paradigm (thesis questions) 10

2. List of Manuscripts: Contents and Author’s Contributions 12

3. Manuscripts
I. A. Steppuhn and I.T. Baldwin (in press) 15
“Induced Defenses and the Cost-Benefit Paradigm”
In: Induced Plant Resistance to Herbivory, ed. A. Schaller
Springer Berlin Heidelberg, in press

II. A. Steppuhn, K. Gase, B. Krock, R. Halitschke, and I.T. Baldwin 43
(2004)
“Nicotine’s Defensive Function in Nature”
PLoS Biology, 2: 1074-1080

III. A. Steppuhn, and I.T. Baldwin (2007) 63
“Resistance Management in a Native Plant: Nicotine Prevents
Herbivores from Compensating for Plant Protease Inhibitors”
Ecology Letters, 10: 499-511

IV. A. Steppuhn, M. Schumann, and I.T. Baldwin (submitted) 88
“Silencing Jasmonate(JA) Signaling and JA-Mediated Defenses
Reveals Different Survival Strategies Between Two Ecotypes of
Nicotiana attenuata”
Submitted to Ecology Letters (11.07.2007)
ITable of Contents
4. Discussion
4.1 Gene silencing: an elegant tool for dissecting ecological questions 110
4.2 Anti-herbivore defense in N. attenuata: The contribution of nicotine & 113
TPIs
4.3 Ecotypic variation the cost-benefit functions of nicotine & TPIs in N. 115
attenuata
4.4 The plant defense network: Secondary metabolites interact 117
4.5 Testing the cost-benefit-paradigm: to be continued … 118

5.1 Summary 123
5.2 Zusammenfassung 125

6. References 129

7. Acknowledgments 134

8. Eigenständigkeitserklärung 135

9. Curriculum vitae 136

10. Scientific publications & talks 138

11. Appendices 140

II Introduction
1. Introduction

1.1 On the bottom of the food chain: How plants survive

The world is green because plants persist in a world which is full of their natural
enemies. As autotroph organisms, plants fix the energy of sunlight in organic compounds and
as such they form the nourishing basis for almost all other organisms. All organisms either
directly or indirectly consume plant-produced material (with the only exception being the few
ecosystems based on archaebacteria). So how do plants survive the enormous predation
pressure?
Unlike many animals, plants are sessile and cannot simply run away from those that
aim to feed on them. Instead, plants have evolved other means of survival, including diverse
defense and tolerance strategies, and this thesis focuses on those related to herbivore attack.
Some mechanical plant defenses such as thorns, spines, hairs, and woody tissues are obvious.
However, many plants produce chemical defenses such as toxic, antidigestive, or repellent
substances (Bennett & Wallsgrove, 1994). All these examples of defenses are directly
directed against the attacker, but some plants have evolved other strategies that make use of
the natural enemies of the plants’ herbivores. Such indirect defenses include plant structures
that provide shelter (dormancies) or nutrition (e.g. nectaries) to predators or signals that are
released to attract the predators when herbivores are present (Dicke, 1999; Heil et al., 2001;
Kessler & Baldwin, 2001). The latter example introduces another differentiation between
defenses, namely, constitutive defenses that are continuously produced and induced defenses
which are called on after a plant has been attacked. In the case of the volatiles released to
attract predators, the need for inducibility is clear, as a constitutive emission would abolish
the information of these signal compounds. However, many direct defenses are also produced
after the herbivores start feeding, but this inducibility is bound to a delay of hours to days
until the defense is established. That many plants still employ defenses inducibly despite this
drawback of delayed resistance is most frequently explained with costs of defenses, which are
minimized if they are only inferred when the defense is needed (see Karban & Baldwin
(1997) and references therein). Such costs can arise from the resources that are allocated in
defenses and consequently not available for growth and reproduction (allocation costs) as well
as from interfering effects of defense compounds with the plant’s primary metabolism
(autotoxicity) or with the plant’s ability to respond to other stresses (ecological costs).
However, there are few alternative hypotheses that explain inducibility without requiring that
1Introduction
defenses are costly; one of these is the moving target theory, which argues that inducibility
itself is a defense, because changing the nutritional quality of plant tissue decreases herbivore
performance (Karban et al., 1997).
Although costs and benefits of plant defenses have long been hypothesized (McKey,
1974; Feeny, 1975), the empirical evidence to date is rather weak. Many secondary
metabolites produced by plants are thought to function as plant defenses because they have
been shown to have anti-herbivore properties, but these metabolites could also serve other
ecological and physiological functions, and the toxicity to herbivores may be just a side
effect, of no real ecological relevance (Rausher, 1992). Therefore, the defensive function for
many plant-produced toxins still needs to determined, even for those that have long been used
as insecticides such as pyrethrines or nicotine (Schmeltz, 1971; McLaughlin, 1973). The
difficulty of demonstrating the costs of defenses and how they arise is even more apparent
than that of establishing their benefits. Most empirical support for costs of defenses comes
from studies establishing correlations between the quantity of certain allelochemicals and
plant resistance and fitness, by either making use of the genetic variation or by eliciting
defense responses (reviewed in Bergelson & Purrington (1996); Heil & Baldwin (2002);
Cipollini et al. (2003)). But correlations can not trace observed effects back to specific
factors, because many plant traits vary between genotypes and after elicitation. During the
past two decades, molecular tools have become available for ecological research and enabled
a new experimental approach. Manipulating specific factors that are expected to be involved
in defense responses allows their function as defenses and consequences for plant fitness to be
tested (Zavala et al., 2004b). The concepts of costs and benefits of defenses, the methods to
examine them, and alternative hypothesis are discussed in manuscript I in more detail.
This thesis aims to test the hypothesis of costs and benefits of two major direct
defenses of a native model plant using the molecular tool of gene silencing. This approach
allows the consequences of the presence or absence of these two compounds to plant
resistance, development, and fitness to be investigated under standardized conditions in the
glasshouse as well as in the plant’s native habitat.

1.2 “On/off-button” of genes: Using gene silencing to study plant defense

The method used in this thesis to test the function of putative defenses as well as their
fitness costs for the plant is gene silencing via RNAi . The basic principle of RNAi is that in
the presence of a complimentary sequence to that of a certain gene, the mRNAs of both form
2 Introduction
a double-stranded RNA (dsRNA, Fig. 1) due to homologue base pairing (Waterhouse et al.,
1998). This dsRNA is cleaved by specific endonucleases, referred to as Dicer, into short
pieces of the double-stranded RNA; these pieces are known as short interfering RNA (siRNA;
Hamilton & Baulcombe, 1999). This siRNA binds to specific proteins to form the RNAi-
silencing complex (RISC), which after unwinding of the double strand can bind to mRNA
with a similar sequence and then cleave this mRNA (Zamore, 2001). The cleaved mRNA is
no longer functional (aberrant RNA) and is either broken down to nucleotides by
exonucleases or becomes the matrix for RNA-dependent RNA polymerases (RdRPs); these
RdRPs synthesize a second strand and consequently