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Black Mustard and The Butterfly Effect. Metabolomics of

Plant-Insect Interactions under Multiple Stress Conditions.

Thesis · May 2017

DOI: 10.13140/RG.2.2.11256.44805

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Black Mustard
And The Butterfly Effect
Metabolomics of Plant-Insect Interactions
under Multiple Stress Conditions
Stefano Papazian
Black Mustard
and The Butterfly Effect
Metabolomics of Plant-Insect Interactions
under Multiple Stress Conditions

Stefano Papazian

Umeå Plant Science Centre

Fysiologisk Botanik
Umeå 2017
This work is protected by the Swedish Copyright Legislation (Act 1960:729)
ISBN: 978-91-7601-728-9
Front Cover by: Stefano Papazian
Electronic version available at http://umu.diva-portal.org/
Printed by: KBC Service Centre, Umeå University
Umeå, Sweden 2017
To my parents
“When we speak of Nature,
it is easy to forget that we are ourselves a part of Nature.
We have to view ourselves with the same curiosity and openness
with which we study a tree, the sky or a thought,
because we too are linked to the entire Universe.”

Henri Matisse

Henri Matisse, Dance (1909), Oil on canvas, 260 × 391 cm (The Hermitage Museum, St. Petersburg).
Preface
When we think about nature, we can ask ourselves: “What makes organisms
adapted to their environment and able to interact with each other?”

Inside an ecosystem, organisms coexist and interact in communities, cycling

nutrients and exchanging energy and matter across the food-web. Non-living
components (water, soil, air, light, temperature, etc.) are also important
parts of ecosystems, which constantly re-shape the surrounding environment.
Throughout evolution, environmental pressure increases the success (fitness)
of organisms by selecting random favourable mutations inside their DNA.
Thus, extending the “dogma” of molecular biology, we can follow the flow of
information bottom-up, until the level of ecosystems, with feed-back on DNA.

Thanks to novel techniques that allow for the high-throughput detection of

thousands of molecules and the systemic screening of organisms, we can
now think in comprehensive terms and study species and ecosystems while
looking at several layers of biological organization (Keurentjes et al., 2011).
“Metabolomics” studies all the chemical processes producing metabolites,
which are the molecules considered to be the ultimate response of biological
systems towards environmental perturbations (Fiehn 2002). In this thesis, I
combine the disciplines of ecology and metabolomics in order to study the
way plants and insects interact with each other, trying to answer:

1. Which metabolites make a plant able to interact with insects

and defend against herbivore attacks?

2. How environmental factors such as air pollutants, multiple herbivores,

or plant signalling hormones, can influence the plant response?

3. And finally, can we use the rising technology of metabolomics

to predict how plants respond to multiple stress conditions?
List of Publications

I. Eliezer Khaling, Stefano Papazian, Erik H. Poelman,

Jarmo K. Holopainen, Benedicte R. Albrectsen,
James D. Blande (2015). Ozone affects growth and development
of Pieris brassicae on the wild host plant Brassica nigra.
Environmental Pollution 199: 119-129.

II. Stefano Papazian, Eliezer Khaling, Christelle Bonnet,

Steve Lassueur, Philippe Reymond, Thomas Moritz, James D.
Blande, Benedicte R. Albrectsen (2016). Central Metabolic
Responses to Ozone and Herbivory Affect Photosynthesis and
Stomatal Closure. Plant Physiology 172: 2057–2078.

III. Camille Ponzio*, Stefano Papazian*, Benedicte R. Albrectsen,

Marcel Dicke, Rieta Gols (2017). Dual herbivore attack and
herbivore density affect metabolic profiles of Brassica nigra
leaves. Plant, Cell and Environment. [Epub ahead of print]
* These authors contributed equally to the work

IV. Stefano Papazian, Tristan Girdwood, Mátyás Ripszam,

Erik H. Poelman, Marcel Dicke, Thomas Moritz, Benedicte R.
Albrectsen (2017). Herbivore-Induced Metabolic Responses
in Brassica nigra are shaped by Leaf Ontogeny. (Manuscript)

i
Abstract
One main goal of ecological research is to understand nature´s complexity,
in order to predict the potential impact of environmental perturbations. In
this thesis, I investigate the ecological interactions between some of the most
ancient organisms living on our planet: plants and insects.

Focus of my research is the interaction between the wild brassicaceous plant

black mustard (Brassica nigra L.) and its specialist insect herbivore, the
large white cabbage butterfly (Pieris brassicae L). Both organisms are well
characterized model species used in chemical ecology research.

Using different analytical techniques, such as liquid and gas chromatography

coupled to mass-spectrometry (LC- and GC-MS) and headspace collection of
volatile organic compounds (VOCs), I apply the approach of metabolomics
and systems biology to the field of ecology to explore the metabolic changes
occurring inside the plants exposed to biotic and abiotic stresses.

Particularly, I study the plant metabolic responses against P. brassicae

chewing caterpillars during sequential treatment exposure to: abiotic stress
by the oxidative air pollutant ozone (O3); dual herbivory with specialist
Brevicoryne brassicae piercing-sucking aphids; and chemical induction of
plant defences with the oxylipin phytohormone methyl-jasmonate (MeJA).

Results show how during herbivore-induced responses, changes in defence-

and growth-metabolic processes are tightly connected to stress protection
mechanisms, indicating that plants actively reprogram their inner metabolic
networks in order to adapt to consecutive changes in the environment.

This thesis illustrates how evaluating the plant metabolome in its entirety
rather than single metabolites, can help us understanding plant responses
towards abiotic and biotic stresses, and improve our ability to predict how
constant shifts in the environment affect plant physiology and ecology.

ii
Sammanfattning
Ett huvudsyfte för ekologisk forskning är att förstå naturens komplexitet för
att kunna förutse effekter av störningar i miljön. I min avhandling har jag
fokuserat på ekologiska interaktioner mellan växter och insekter, två av de
äldsta terrestra organismgrupperna på jorden.

I mina studier har jag undersökt interaktioner mellan den korsblommiga

växten svartsenap (Brassica nigra L.) och den specifika herbivoren kålfjäril
(Pieris brassicae L.). Båda är väl karaktäriserade modellarter i kemisk-
ekologisk forskning.

De metaboliska förändringar som sker när växten utsätts för biotisk och
abiotisk stress har analyserats hjälp av metabolomik, det vill säga analyser av
metabolomet i sin helhet med hjälp av tekniker som vätske- och
gaskromatografi kopplad till masspektrometri (LC- och GC-MS), och så
kallad headspace-uppsamling av flyktiga organiska föreningar (VOCs).

Jag har särskilt undersökt de metaboliska förändringar som sker när växten
betas av kålfjärilslarver vid samtidig exponering för: abiotisk stress i form av
ozon (O3), en oxidativ luftförorening; ytterligare betning i form av stickande
och sugande bladlus (Brevicoryne brassicae); tillsats av oxylipinfytohormon
metyl-jasmonat (MeJA), ett ämne som inducerar växtens försvar.

Resultaten visar att de metaboliska förändringar som sker i växten vid

herbivori med konsekvenser för dess försvar och tillväxt är nära kopplade till
de metaboliska förändringar som sker vid stress, vilket visar att växten kan
fortlöpande och aktivt omprogrammera sina metaboliska nätverk för att
anpassa sig till förändringar i miljön.

Avhandlingen visar att genom att utvärdera växtmetabolomet i sin helhet,

snarare än att studera enskilda metaboliter, vi kan få bättre förståelse för hur
växter reagerar på olika former av stress och därmed också bidra till att vi
kan göra förutsägelser för hur förändringar i miljön kan påverka växters
fysiologi och ekologi.

iii
Abbreviations
O2 Oxygen
O3 Ozone
H2O Water
CO2 Carbon dioxide
ROS Reactive oxygen species
UV Ultra-violet
ppb Parts per billion
myr Million years

JA Jasmonic acid
SA Salicylic acid
ET Ethylene
MeJA Methyl jasmonate
VOCs Volatile organic compounds
GLVs Green leaf volatiles
ITCs Isothiocyanates
GSLs Glucosinolates
TCAs Tricarboxylic acids
G3P Glycerol-3-phospate
GABA Gamma (γ)-amino butyric acid
NAD(P)+ Nicotinamide adenine dinucleotide (phosphate)
NAD(P)H NAD (phosphate), reduced form
ATP Adenosine triphosphate
PSI-II Photosystem I and II
ETC Electron transport chain

MVA Multivariate analysis

HCA Hierarchical cluster analysis
PCA Principal component analysis
PLS Partial least square
OPLS Orthogonal projection of latent structures
DA Discriminant analysis
VIP Variable importance in the projection
LV Latent variable
GO Gene ontology
GLM General linear model
ANOVA Analysis of variance
MANOVA Multivariate analysis of variance

LC Liquid chromatography
GC Gas chromatography
TOF Time of flight
MS Mass spectrometry
TD Thermal desorption

iv
Contents

Preface

List of Publications i
Abstract ii
Sammanfattning iii
Abbreviations iv
Contents v

1. Introduction 1
Plant metabolism 2
Primary metabolism 2
Secondary metabolism 3
Eco-metabolomics 5
Plant-insect interactions 5
Plant defence responses 7
Study system 8
Black mustard 8
Large white cabbage butterfly 12
Multiple stresses 15
O3: a tropospheric pollutant 17
Aphids: sap-sucking herbivores 18
MeJA: herbivore mimicking 18
2. Objectives 21
3. Results and Discussion 22
O3 effects on plant-insect interactions 22
Effects of O3 on herbivory and plant defences 22
Omics responses to sequential O3 and herbivory 27
Physiological responses to O3 and herbivory 34
Dual herbivore attack 38
Plant responses versus aphids and caterpillars 38
Effects of dual herbivory and aphid density 41
Induced plant resistance to herbivory 45
Effects of MeJA on herbivore-induced responses 46
Ontogenic patterns of induced responses 52
4. Conclusions 55
The “Butterfly effect” 55

5. Acknowledgements 58
6. Literature 60

v
1. Introduction
The sum of all chemical reactions in an organism is defined as metabolism,
from the Greek word μεταβολή [metabolē], meaning "change”. All living
organisms harbour an active metabolism, which is the condition that defines
life itself as a continuous state of transformation. Inside the cell, chemical
changes from a molecular compound into another are regulated by enzymes
and connected through long biochemical pathways, very much like subway
maps, where each “station” represents a different metabolite (Fig.1).

As conserved features of life evolutionary history, metabolic pathways that

are essential for cellular growth and development are shared among most
organisms (Weng, 2014). The ensemble of these central pathways is usually
referred to as “primary” metabolism, in contrast to “secondary” pathways
which later evolved from it (Firn & Jones, 2009).

Figure 1. Metabolism. Cellular network of biochemical pathways (KEGG).

1
1.1 Plant metabolism

Plants are magnificent organisms. They literally connect planet Earth to the
sky, as “primary producers” of organic matter inside terrestrial ecosystems.
Through a series of highly coordinated metabolic processes, plants capture
sunlight and transform it into energy and biomass. All the energy that we
liberate burning most common fuels (e.g. coal, oil, wood, or biofuels) was
indeed originally captured from the Sun. Concealed inside the plant, and
accessible to higher levels of the food-web, this energy is what sustains life
and ecosystems on our planet.

Plants present an astonishing chemical biodiversity. In the plant kingdom

there are estimated more than 250.000 structurally different secondary
metabolites (Viant et al., 2017), which are a precious source of bioactive
“natural products”. Since the ancient times of traditional medicine, humans
have exploited the healing properties of plant chemistry, and plants still
constitutes a great potential for pharmaceutical drug discovery and for the
development of agrochemicals. Inside the plant, these metabolites serve
different biological functions, including defence against pathogens, insects,
and protection towards other environmental stresses (Morrisey 2009).

For the scope of this thesis, I will refer to the small molecules involved in
plant central energy and growth processes as “primary metabolites”, and I
will refer to specialized molecules involved in plant defence and interaction
with the environment as “secondary metabolites”. However, it is difficult to
draw clear biosynthetic and functional boundaries within the plant metabolic
network, and I am aware that the traditional division between primary and
secondary metabolism is neither completely satisfactory nor meaningful.

1.1.1 Primary metabolism

Plant primary metabolism starts with the process of photosynthesis, the

conversion of CO2 into carbohydrates driven by sunlight. Leaves absorb CO2
through stomata, small pores on the epidermis surface which control plant
water transpiration and gas exchange. Solar energy is meanwhile trapped by
light-harvesting complexes and chlorophyll inside the chloroplasts. Here, via
photosystem centres (PSI and PSII) and the electron transport chain (ETC),
plants use excited electrons to create chemical bonds (e.g. the reducing agent
NADPH). This “chemical energy” is transferred to the Calvin-Benson cycle,
where ribulose-1,5-bisphosphate carboxylase (RuBisCO) traps the CO2 into
simple sugars (Fig.2), later incorporated into mono- or polysaccharides for
energy storage (starch) or for building the plant cell wall (cellulose).

2
Sugars are then oxidized with NAD+ along catabolic pathways of glycolysis
in the cytosol, and in the mitochondria via the tricarboxylic acid (TCA) cycle,
coupled to ETC and cellular respiration. Throughout this process, electrons
are passed to oxygen (O2) reducing it to water (H2O), while energy is stored
into ATP and transferred to other metabolic activities. Mitochondria are the
centre of energy metabolism in all higher organisms (eukaryotes), but they
play a double role in plants, where they are tightly connected to chloroplast,
sustaining the assimilation of CO2 and the redox balance (Blanco et. al,
2014). Glycolysis and TCA central metabolic pathways also provide the
precursors necessary for the biosynthesis of amino acids, fatty acids, and
other organic acids, which are used as “building blocks” in anabolic
pathways for the final assembly of cellular macromolecules (e.g. DNA,
proteins and membranes) (Stephanopoulos et al., 1998; Noor et al., 2010)
and for the biosynthesis of secondary metabolites (Morrisey 2009) (Fig.2).

1.1.2 Secondary metabolism

Secondary metabolism evolved as groups of new pathways branching from

the central processes of photosynthesis, cellular respiration, and carbon-
nitrogen metabolism (Weng, 2014). Approximately 15-20% of plant genes
are predicted to be involved in the biosynthesis of secondary metabolites
(Pichersky, 2011), which always derive from sugar and/or amino acid
precursors (Fig.2). For instance, one of the major enzymatic steps
connecting primary and secondary metabolism involves the highly conserved
enzyme phenylalanine ammonia lyase (PAL), which converts the aromatic
amino acid phenylalanine (Phe) into trans-cinnamic acid, at the beginning of
the phenylpropanoid pathway (Fraser & Chapple, 2011). Depending on the
plant family and species, secondary metabolites range from different kinds of
phenolics, terpenoids, or nitrogen containing compounds, such as alkaloids
(e.g. Solenacea, Rubiacea), cyanogenic glucosides (e.g. Rosacea, Fabacea),
and glucosinolates (GSLs) (Brassicaeae) (Halkier & Gershenzon, 2006).
Most of these compounds are normally stored as cell-bound metabolites
(Wink, 1997), but they can also be released as volatile organic compounds
(VOCs) (Laothawornkitkul et al., 2009; Dudareva et al., 2013). Initially,
these phytochemicals were considered to be only “by-products” of primary
metabolism and not necessary for the plant survival (Bourgaud et al., 2001;
Firn & Jones, 2009). However, secondary metabolites mediate almost all
interactions within an ecosystem, and directly contribute to the plant fitness
allowing processes such as plant-plant communication (Farmer & Ryan,
1990; Karban et al., 2014), competition (allelopathy) (Morrisey 2009), insect
pollination (entomophily) (Burger et al., 2010), and plant defence against
insect herbivores (Howe & Jander, 2008).

3
Figure 2. Plant metabolism. A simplified overview of the plant primary (yellow)
and secondary (pink) metabolism, and plant volatile organic compounds (VOCs)
(blue). Primary metabolism is involved in central processes of growth and
development, starting with routes of photosynthesis and carbon metabolism (Calvin-
Benson cycle). Carbohydrate and amino acid synthesis are linked via tricarboxylic
acid (TCA) cycle. These central pathways produce chemical energy (ATP) and
simultaneously provide all the metabolic precursors (“building-blocks”) required for
cellular components (e.g. nucleotides, proteins, cell-membranes and cell-wall) and
for the biosynthesis of plant secondary metabolites, e.g. phenolics or Brassicaea
specialized glucosinolates (GSLs). Secondary metabolites are usually involved in
plant protection and defence against environmental stresses. VOCs derived from
primary or secondary metabolism can mediate ecological interactions with other
biota (e.g. GLSs derived thiocyanates, isothiocyanates, and nitriles). In addition,
phytohormones (in red) regulate both growth and defence processes, and can also be
found as VOCs.

4
1.2 Eco-metabolomics

The complete set of metabolites in an organism is called the “metabolome”

(Tweeddale et al., 1998), and in analogy with other systems biology “omics”
approaches such as genomics, transcriptomics, and proteomics, the unbiased
analysis of the entire metabolome is named metabolomics. Metabolomics
is regarded as the link between genotypes and phenotypes (Fiehn, 2002) and
it represents a powerful tool to generate new biological hypotheses. Because
all plant metabolites can virtually function as info-chemicals in ecosystems,
the comprehensive approach of metabolomics is particularly suited for the
study of plants´ ecological interactions with the environment, in this case
referred to as “eco-metabolomics” (Sardans et al., 2011).

Plants are important members of the ecological community, connecting

organisms into different relationships and providing shelter and resources.
During the interaction with the surrounding environment, eco-physiological
responses will reconfigure the plant inner metabolic networks (Bundy et al.,
2008; Maag et al., 2015) and will shape the communication of organisms at
higher levels of the trophic network (Bourgaud et al., 2001). Interestingly,
the concept of “networks” is shared between the field of ecology and systems
biology (Butts, 2009; Ings et al., 2009; Keurentjes et al., 2011). Adopting a
systems perspective, the flow of energy and matter through ecosystems can
be visualized as the perpetuation of metabolic pathways through organisms
(Capra, 1996). Thus, likewise cellular metabolism, plant interactions with
other organisms can be seen as a network of interconnected relationships
subjected to constant change in a dynamic environment.

1.2.1 Plant-insect interactions

Among the most complex chemical communication networks that we can

observe in nature, are those that define plant-insect interactions (Fig.3).
Insects and plants colonized land roughly at the same time, during the Early
Ordovician, and thus coexisted and interacted in the terrestrial environment
for about 470 myr (Misof et al., 2014). Of the entire animal kingdom, insects
are by far the most diverse group, with 900.000 species already described
and more than five million estimated (Stork et al., 2015). As the theoretical
ecologist Robert May once famously noted: “by a good approximation, all
species are insects” (in Dawkins, 1976).

5
The relationship between plants and insects is complicated. About 80% of
higher plants develop flowers (angiosperms) (Pimm & Joppa, 2015) and
most of them rely on insects as their main source of pollinators, such as bees,
bumblebees, flies, or butterflies (Munguia-Rosas et al., 2011; Bruinsma et
al., 2014). Combining colorful visual cues and aromatic chemical blends,
plants hack into the insects’ communication system and lure them into their
petals (Burger et al., 2010) (Fig.3). This process usually results in a win-win
(mutualistic) interaction, where insects are rewarded with sugar-rich floral
nectar, but some rare dishonest plants (4-6% of angiosperms; Renner 2006),
can cheat and attract insects exploiting fake-food rewards (Aristolochiaceae;
Oelschlägel et al., 2015) or even sexual deception (Orchidaceae; Ayasse et al.,
2011). However, floral odors and other cues from the plants can attract insect
herbivores (Karban & Agrawal, 2002; Howe & Jander, 2008; Sauve et al.,
2016) which can be extremely harmful and destroy the plant (Crawley, 1989;
Adhikari & Russell, 2014; Stephens & Westoby, 2015; Lemoine et al., 2017).

Figure 3. Chemical networks define plant-insect interactions. Multiple

levels of connection between plant metabolic networks and interaction with insects.

6
1.2.2 Plant defence responses

Without possibility to move, plants developed a variety of defence strategies

to cope with insect herbivores. Aside from physical barriers (e.g. modified
leaf and branch structures, such as thorns and spines) plants highly rely on
chemistry to fight their enemies. Most specialized secondary metabolites are
produced for defence purposes as part of their constitutive (steady-state)
metabolism, and thus compete with primary metabolic demands for growth
(Stamp, 2003; Neilson et al., 2013)(Fig.2). Secondary metabolites constitute
an effective direct chemical defence which can impair the performance of
generalist (polyphagous) herbivores (Howe & Jander, 2008). However,
specialist (monophagous) herbivores that co-evolved with the plant are often
attracted by these secondary metabolites which they are capable to detoxify
and digest (Ali & Agrawal, 2012; Edger et al., 2015).

Upon herbivore attack, plant defences can be further induced, providing

an energy-saving solution to maximize the plant fitness (Heil & Baldwin,
2002; Neilson et al., 2013; Moore et al., 2014). The induction process is
regulated by signal-transduction pathways which involve three major plant
phytohormones: jasmonic acid (JA), salicylic acid (SA), and ethylene (ET).
JA and SA pathways are reciprocally antagonistic (Thaler et al., 2012) and in
a complex cross-talk with ET (and other phytohormones, including auxins,
cytokinins, gibberellins, ABA, etc.) they fine-tune the herbivore-induced
responses (Ali & Agrawal, 2012; Erb et al., 2012), similarly to the process
induced during plant immunity against pathogens (Pieterse et al., 2009).
This hormonal network will eventually determine the metabolic strategy of
the plant, in order to defend against generalist and specialist or different
herbivore guilds. For instance, chewing herbivores, such as caterpillars,
typically induce the biosynthesis of the oxylipin JA, which is derived from
fatty acid metabolism along the octadecanoid pathway (e.g. α-linolenic acid)
(Fig.2) (Schaller & Stintzi, 2009; Wasternack, 2015; Lortzing & Steppuhn,
2016). On the other hand, sucking herbivores that feed on the plant phloem,
such as aphids, induce synthesis of SA (Glazebrook, 2005; Pieterse & Dicke,
2007), a phenolic metabolite derived from phenylalanine (Chen et al., 2009).

Different defensive metabolites have different biological activity and can

either reduce the nutritional quality inside the leaves or can be emitted as a
bouquet of VOCs, such as phenylpropanoids, terpenoids (C5-C20), and fatty
acid-derived green leaf volatiles (GLVs) (Laothawornkitkul et al., 2009;
Dudareva et al., 2013). These VOCs constitute an indirect defence for the
plant, often functioning as a “cry-for-help” which attracts enemies of the
herbivore, such as predators and parasitoids (Dicke & Baldwin, 2010; Ponzio
et al., 2014; Bruce, 2015) (Fig.3).

7
1.3 Study system

For the work presented in this thesis, together with my colleagues I studied
the plant-insect interaction between the Black mustard and its specialist
herbivore, the Large white cabbage butterfly. This plant-herbivore system
was reproduced in collaboration with different universities which combined
complementary expertise in plant biology, entomology, and ecology. My role
in the projects was to analyze the plant metabolic changes relative to primary
and secondary metabolism, which were induced by environmental stresses.

1.3.1 Black mustard

The kingdom of heaven is like a grain of mustard seed,

which a man took, and sowed in his field.
Which indeed is the least of all seeds,
but when it is grown, it is the greatest among herbs,
and becomes a tree, so that the birds of the air
come and lodge in the branches thereof.

Matthew 13:31-32

The Black Mustard, i.e. Brassica nigra L. (Brassicacea), is an annual

herbaceous plant originally from Middle East and Mediterranean regions,
which has been extensively cultivated by humans for millennia (Prakash et
al., 1980). Although a minor crop nowadays, gradually replaced by the
Ethiopian mustard B. juncea, it is often found as a weed and as an invasive
species widely spread across the globe (Westman & Kresovich, 1999),
thriving in both cold and hot geographical regions (60ºN - 40ºS) and
surviving extreme temperatures of 45-50º C (Kask et al., 2016). In the field,
B. nigra can stand two meters in height with long reaching branches. Lower
leaves are usually dentate with wide lobes, while the upper leaves are narrow
and lanceolate. Flowers are hermaphrodite, bright and yellow, with four
petals oriented in a cross shape common to all cruciferous plants (from
Latin, “crux” = cross). As an obligate out-crossing species in nature, B. nigra
is pollinated by several insects, including bees and butterflies (Conner &
Neumeier, 1995).

8
 (a) (b)

(c)

Figure 4. The Black mustard (Brassica nigra). (a) yellow flower with petals
disposed in the typical cross-shape (cruciferous) of Brassicacea, (b) whole
inflorescence, and (c) B. nigra plants, approximately four-week old, as grown for the
experiments of this thesis conducted at Umeå University (Wallenberg greenhouse).

The genus Brassica comprehends 38 species, six of which are crops of high
economic importance and are cultivated for the production of vegetables,
condiments, or oilseeds (Table 1) (Das et al 2007). These include turnip
(Brassica rapa), cabbage, broccoli, cauliflowers (Brassica oleracea), as well
as rapeseed (Brassica napus) used for biodiesel production (Golovitchev &
Yang, 2009). B. nigra is mostly cultivated for its seeds, which are ground
and mixed with water and other ingredients (e.g., vinegar and lemon) to
produce the mustard paste, commonly used in many countries use as a
cooking spice.

9
 Table 1. Brassica species and their economic importance (Das et. al 2007).
Species and variety Common name Uses
B. nigra Black mustard Condiment
B. oleracea var. acephala Kale Vegetable
var. alboglabra Chinese kale Vegetable
var. botrytis Cauliflower Vegetable
var. capitata Cabbage Vegetable
var. italica Broccoli Vegetable
B. rapa var. chinensis Pak choi Vegetable
var. olifeira Turnip rape Oilseeed
var. rapifera Turnip Vegetable
var. yellow sarson Yellow sarson Oilseeed
var. toria Toria Oilseeed
var. brown sarson Brown sarson Oilseeed
var. nipposinica Mizunami Salad
B. carinata Ethiopian mustard Condiment
B. juncea var. oleifera Indian mustard Oilseeed
B. napus var. oleifera Rapeseed Oilseeed
var. rapifera Sweed Fodder

Brassicaeous plants contain high amounts of secondary metabolites called

glucosinolates (GSLs). GSLs are sulfur and nitrogen containing glycosides
derived from amino acids. According to the class of amino acid from which
they derive, GSLs are divided in:

- aliphatic (from methionine, alanine, leucine, isoleucine, or valine).

- aromatic (from phenylalanine)
- indolic (indeed aromatic, but from tryptophan)

The central carbon of the GSL structure is bound via a nitrogen atom to a
sulfate group and via a sulfur atom to a glucose moiety (Fig.5).
In addition, a side group (R) which is derived from the amino acid precursor
differs according to the kind of GLS and is responsible for the variation in
the biological activities (Halkier & Gershenzon, 2006).

In B. nigra, the most abundant secondary metabolite is the aliphatic GSL

derived from methionine, sinigrin (2-propenyl-GSL), which represents 90-
99% of the entire plant’s GLS profile (Lankau & Strauss, 2007).

10
Breaking B. nigra seeds and mixing with water confers the flavor to the
mustard paste, similar to that of horseradish. This flavor is due to VOCs
released from the degradation of sinigrin during a hydrolytic reaction called
the “mustard oil bomb” (Fig.5).

Figure 5. The “Mustard oil bomb”. The hydrolysis of GSLs (e.g. sinigrin in B.
nigra) is catalyzed by myrosinase, producing different volatile compounds (according
to the different environmental conditions, such as pH) some of which have toxic
properties, for instance ITCs (e.g. allyl isothiocyanate). The general GSL structure is
shown. Side R-group for sinigrin: CH2=CH=CH2-. See GSLs list in Clarke (2010).

The hydrolysis of GSLs is catalyzed by the enzyme myrosinase (Rask et al.,

2000) which is produced by all Brassicaeae plants and was first reported in
B. nigra (Bussy, 1840). Myrosinase is a glycosidase (EC 3.2.1.147), normally
kept separate inside plastids or in the apoplast (Morant et al., 2008). Upon
any tissue disruption - by herbivores or by mechanical damage – myrosinase
gets in contact with GSLs, cleaving their glucose moiety and converting them
into VOCs such as thiocyanates, isothiocyanates (ITCs), and nitriles (Winde
& Wittstock, 2011).

Particularly ITCs have toxic properties and constitute an efficient direct

and indirect defence against insect herbivores (Burow et al., 2009; Hopkins
et al., 2009; Kos et al., 2012). Arabidopsis thaliana L. knocked-out mutants
impaired in the formation of ITCs (Burow et al., 2006) or in the biosynthesis
of GSLs (Beekwilder et al., 2008) are more sensitive to feeding by
caterpillars, especially those of generalists butterflies such as Mamestra
brassicae or Spodoptera littoralis. On the other hand, specialist herbivores
of Brassicaceae, such as Pieris butterflies, are immune to these metabolites
(Blatt et al., 2008; but see arguments in the study on B. nigra and P. rapae
by Traw & Dawson, 2002).

11
1.3.2 Large white cabbage butterfly

The Large White cabbage butterfly, i.e. Pieris brassicae L. (Pieridae), is a

Lepidoptera commonly found in Eurasia, and it is a serious pest of all
cultivated Brassica vegetables, including B. nigra.

Butterflies of P. brassicae can feed on B. nigra floral nectar (Fig.6a) and

then oviposit on the lower side of leaves, in batches of 50-100 eggs (Fig.6b).
After hatching, neonate (first instar) caterpillars move gregariously and feed
on leaves (Fig.6b,c). However, late second and early third instars prefer
buds and flowers, which contain high concentrations of GSLs (Smallegange
et al., 2007). After the fifth (last) instar, caterpillars leave the plant for a safe
location to mutate into pupae (chrysalis) (Fig.6d), from which they
reemerge about 14 days later as butterflies. In our greenhouse, the complete
life cycle usually lasted 5-6 weeks (Fig.6e), but the duration in the field
largely depends on the geographical location and environmental conditions.
As a specialist of Brassicaceae, female butterflies are attracted by ITCs and
nitriles, which they use to locate their host (Hopkins et al., 2009).

Egg deposition creates necrotic zones on B. nigra leaves and can suppress
JA-induced defences via SA accumulation (Bruessow et al., 2010). However,
studies in Arabidopsis showed that egg deposition can induce JA defences
and trigger mechanisms similar to those induced by pathogen-associated
molecular patterns (PAMPs) (Gouhier-Darimont et al., 2013). P. brassicae
oviposition on B. nigra can also alter the plant induced responses before and
after caterpillar feeding (Pashalidou et al., 2015), whereas male pheromones
left on mated female butterflies can cause chemical modifications of the leaf
surface that eventually attract the egg parasitoid Trichogramma brassicae L.
(Fig.7a-c)(Fatouros et al., 2005; Fatouros et al., 2008).

P. brassicae caterpillars can digest GSLs which they degrade into non-toxic
nitriles (Smallegange et al., 2007; Winde & Wittstock, 2011) and can even
interfere with the induction of plant responses (Reymond et al., 2000;
Karban & Agrawal, 2002). For instance, oral secretions of P. brassicae
suppress wound-induced responses in Arabidopsis (Consales et al., 2012).
On the other hand, elicitors in these same secretions are also recognized by
the plant as molecular signals of the insect-attack, leading to the induction of
plant direct and indirect defences (Mattiacci et al., 1995; Reymond et al.,
2004). Induced emission of ITCs and nitriles will attract the parasitoid wasp
Cotesia glomerata L. (Fig.7 d-g) which lay its eggs inside the caterpillars
(Agrawal & Kurashige, 2003; Pashalidou et al., 2015).

12
Figure 6. The Large white (P. brassicae L.). Development of P. brassicae in
stages: (a) Fully developed butterfly, (b) hatching eggs with neonate first instar
caterpillars, (c) fully developed caterpillars feeding on leaves of Brassica plants,
(d) pupae stage and (e) full life-cycle. Photo: T. Bukovinszky and H. Smid.

All these fascinating processes are the result of 470 myr interaction between
plants and insects (Thaler et al., 2012; Misof et al., 2014; Bruce, 2015) and
specifically of the 70 myr arms-race between Brassicacea and Pieridae
(Edger et al., 2015). However, in the climate change scenario, large
environmental shifts are shaping plant-insect interactions on a much shorter
time-scale.

13
Figure 7. Indirect defences. Parasitoid wasps are attracted by B. nigra as an
indirect defence mechanism. (a,b) Trichogramma brassicae is attracted by chemical
modifications on the leaf surface and lays its eggs inside P. brassicae´s eggs.
(c) T. brassicae (arrow) hitchhiking on P. brassicae butterfly. Photo: N. Fatouros.
(d) Cotesia glomerata is attracted by VOCs released upon herbivore damage (ITCs
and nitriles) and attacks early instar of P. brassicae (e-g) caterpillars to lay eggs.
Photo: T. Bukovinszky and H. Smid.

Climate change models predict an increase in the occurrence and in the

intensity of drought and heat waves (IPCC), with further concerns for natural
ecosystems and agricultural productivity (Fuhrer, 2003; Atkinson & Urwin,
2012; Suzuki et al., 2014). Plants and insects are migrating towards northern
latitudes (Parolo & Rossi, 2008; Chen, IC et al., 2011; Bebber et al., 2013),
thus altering the evolutionary processes driving the adaptation of both
species and the stability of native info-chemical networks (Morriën et al.,
2010; Desurmont et al., 2014).

14
1.4 Multiple stresses

A realistic description of plant-insect interactions cannot exclude other

elements which are integral components of the environment. In nature,
chewing damage by caterpillars is only one type of stress that plants
encounter, and plant responses to herbivory always concur with response to
other stresses. In addition to biotic stress, which is stress caused by any
living agent, plants are also exposed to a variety of abiotic stresses caused
by non-living environmental factors - e.g. lack or excess of water in the soil
(drought or flood), extreme temperatures, UV radiation, or air pollution
(Fig.8). The performance of plants in the field thus depends on the ability to
perceive different environmental cues and to quickly adapt new responses.
Both biotic and abiotic stresses can severely impair growth, and plants
developed different mechanisms to reduce the damage (Rejeb et al., 2014;
Nguyen et al., 2016).

Among different abiotic stresses, the most studied that affect both natural
ecosystems and human agriculture include drought, heat, cold (chilling,
freezing), nutrients, high salinity, high light intensity, and ozone (Suzuki et
al., 2014). Drought and heat combined cause the most damage to global
agricultural production (Hatfield & Walthall, 2015) and are thus among the
most investigated abiotic stresses, along with salt stress and heavy metals
(Suzuki et al., 2014; Zandalinas et al., 2017). Interaction between biotic and
abiotic stresses can trigger convergent phytohormone cross-talk (JA, SA, ET)
and signalling pathways (e.g. ROS and Ca++)(Nguyen et al., 2016, Pandey et
al., 2016) which often reinforce or inhibit each other and lead to a variety of
physiological responses (Atkinson & Urwin, 2012; Rejeb et al., 2014; Suzuki
et al., 2014).

Thus, predictions based on individual stress responses only partially help

us in understanding the complexity of the plant response to multiple
stresses, as the presence of an initial stress can alter the response to a second
stress and eventually result in a positive, negative, or neutral interaction
(Fig. 8) (Mittler, 2006; Atkinson & Urwin, 2012; Suzuki et al., 2014). For
instance, simultaneous exposure to heat and drought stress in tobacco
(Nicotiana tabacum L.) has a negative interaction on the plant response,
with greater suppression of photosynthesis compared to the individual
stresses (Rizhsky et al., 2002). Similar responses are also observed in
Arabidopsis, resulting in differential expression of both defence and central
metabolic transcripts, enhanced respiration, and an increased accumulation
of sucrose and other sugars (Rizhsky et al., 2004).

15
Relatively few studies have investigated abiotic stress responses in plants
combined with biotic stresses (Suzuki et al., 2014). In most of these studies,
the biotic stress components was often represented by pathogens (i.e. virus,
bacteria or fungi) - possibly as easier to handle than insects (Fig.8), and
analyses usually focused on the effects at the gene expression at level or on
the role of single transcription factors (Rizhsky et al., 2004; Atkinson &
Urwin, 2012; Sewelam et al., 2014; Olivas et al., 2017).

Figure 8. The stress matrix. Combination of different environmental stresses can

affect plants in the field, resulting in positive and/or negative interactions on the
plant performance. Color-codes indicate the effects of stress combinations on plant
growth and yield. Multiple abiotic stress conditions have been thoroughly studied
(especially combinations with drought), but only few studies have been performed
with biotic components, which were usually limited to pathogenic microorganisms
(Suzuki et al., 2014).

16
When metabolomics was applied to study multiple environmental stress
responses in plants it often highlighted a large functional overlaps between
pathways, and the importance of both primary and secondary metabolism
for plant adaptation to the stresses (Sana et al., 2010; Caldana et al., 2011;
Sun et al. 2015; and see reviews by Bundy et al., 2008; Schwachtje &
Baldwin, 2008; Nakabayashi & Saito, 2015). In this thesis, changes in plant
metabolism were investigated for combined abiotic and biotic stresses which
may alter the plant response towards consecutive herbivory, such as elevated
ozone exposure, aphid herbivory, and phytohormone treatments (Fig.9-10).

1.4.1 O3: a tropospheric pollutant

Abiotic stress in this thesis has focused on the negative effects of ozone (O3).
O3 is the three-atom conformation of oxygen, but whereas O2 is essential for
cellular respiration, O3 is toxic to living organisms. However, in comparison
to O2 which is extremely abundant in the Earth´s atmosphere (20.9%), O3 is
only a trace gas, with ground-level concentrations of 25-30 ppb (Chevalier et
al., 2007). Most of O3 (90%) is found at the altitude between 20-50 km
(stratosphere), where man-made halocarbon emissions have infamously
depleted the layer that protects us and all other living organisms from UV
solar radiation. On the other hand, in the atmospheric region from ground-
level to ca. 20 km (troposphere), O3 is a strong oxidizing pollutant.

As a result of rapid economic growth and human emissions, tropospheric

O3 concentrations have been globally increasing in the last decades (Lin et
al., 2017). Burning of fossil fuels releases nitrogen oxides and hydrocarbons
which, in the presence of sunlight, react with O2 and VOCs to form O3 (Sitch
et al., 2007). The steady emission of these precursors has led to an increase
in the background concentrations of O3, which during hot summers in the
Northern Hemisphere can peak to 200-400 ppb (Tiwari et al., 2016).
Although most of O3 is produced in polluted urban areas and is directly
related to traffic (Wennberg & Dabdub, 2008), long-range transport of O3 to
rural areas can severely affect crop yields and forest growth (Ashmore,
2005). Depending on the plant species, accumulated exposure to O3 above
40 ppb can result in negative effects on photosynthesis, leaf transpiration,
and emission of VOCs (Bagard et al., 2008; Booker et al., 2009; Blande et
al., 2010; Tiwari et al., 2016), and can have severe impact on ecological
interactions and agricultural systems (Pinto et al., 2010).

17
1.4.2 Aphids: sap-sucking herbivores

Aphids (Aphidoidea) are small sap-sucking insects that feed on the plant
phloem. Among all insect pests, aphids are some of the most destructive and
difficult to control, as they rapidly increase in density doubling every few
days throughout cycles of asexual reproduction (parthenogenesis)
performed by genetically identical female clones (Davis, 2012). When
feeding, aphids avoid the massive cell damage typical of chewing herbivores
like caterpillars. Instead, they penetrate the leaf epidermis adopting a
“stealthy” piercing-sucking strategy which triggers SA-induced responses,
similar to those induced by biotrophic pathogens (Walling, 2008; Thaler et
al., 2012; Foyer et al., 2016; Nguyen et al., 2016). However, JA-induced
responses are also known to be involved in the plant defence against aphids
(Ellis et al., 2002; De Vos et al., 2005; De Vos & Jander, 2009).

In this thesis, we studied the cabbage aphid Brevicoryne brassicae L.,

which is a serious pest of all Brassica crops worldwide (Ellis & Farrell, 1995;
Ellis et al., 2000; Broekgaarden et al., 2008). B. brassicae is a specialist
herbivore of Brassicacea and is able to sequester the plant GSLs (e.g.
sinigrin) and – using its own myrosinase (Jones et al., 2001) – to exploit
them for its own defence against predators, such as the ladybirds Adalia
bipunctata L. Hence the famous nick-name, “walking mustard oil bomb”
(Kazana et al., 2007).

1.4.3 MeJA: herbivore mimicking

Upon herbivore-attack and tissues damage especially by caterpillar,

intracellular JA accumulates within the plant to spread the defence response
both locally and via the phloem as long distance signal (Glauser et al., 2008;
Chauvin et al., 2013; Mousavi et al., 2013). JA signal transduction depends
on the interaction of its conjugated form JA-isoleucine (Ja-Ile) with
ubiquitin ligase SCFCOI and the jasmonate-zim-domain (JAZ) repressor
(Chini et al., 2007; Katsir et al., 2008); JAZ normally blocks the response by
binding to the transcription factor MYC2, a master regulator of plant
herbivore-induced defences. Accumulation of JA leads to ubiquitination and
degradation of JAZ, and to the activation of JA-responses (Lortzing &
Steppuhn, 2016).

Initial studies conducted in tomato (Solanum lycopersicum L.) discovered

that the JA ester derivative, methyl-JA (MeJA), was able to further spread as
airborne signal, allowing plant-plant communication and induced-resistance
in non-attacked plants (Farmer & Ryan, 1990; Wasternack, 2015).

18
Further studies confirmed that exogenous application of MeJA sprayed on
plants can stimulate responses very similar to those induced by physical
herbivore damage, including induction of genes involved in defence (Kouzai
et al., 2016; Yi et al., 2016), synthesis of specialized secondary metabolites
(Fritz et al., 2010; Zang et al., 2015) and emission of VOCs (Geervliet &
Brodeur, 1992; Gols et al., 2003; Lortzing & Steppuhn, 2016).

In Brassicacea, increased GSL levels are typically observed after application

of JA or MeJA (Cipollini & Sipe, 2001; Petersen et al., 2002; van Dam &
Oomen, 2008). For these reasons, JA and MeJA are often used as molecular
tools to study herbivore-induced resistance and production of secondary
metabolites, with applications ranging from bioprospecting (Yukimune et al.,
2000; Leonard et al., 2009) to agricultural and forestry pest management
(Rodriguez-Saona et al., 2001; Fritz et al., 2010; Lundborg et al., 2016).

Figure 9. Multiple stress conditions. Abiotic and biotic stress combinations

studied in this thesis on the model brassicaceous plant B. nigra. The focus of these
studies was to understand how metabolic changes induced in the plant in response
toward P. brassicae caterpillar herbivory, are affected by abiotic and biotic stresses,
such as ozone (O3), B. brassicae aphids, and phytohormone treatment with MeJA.

19
Figure 10. Experimental set-ups. Stress responses were tested in B. nigra
combining herbivory by P. brassicae (green) with other abiotic and biotic stresses.
(Paper I): 70-120 ppb O3 stress (blue) and three days herbivory with 20 caterpillars;
(Paper II): 70 ppb O3 stress (blue) and one day of herbivory by 30 caterpillars;
(Paper III): dual herbivory of three days by 50-100 B. brassicae aphids (yellow),
and one day by 30 caterpillars; (Paper IV): three days of phytohormone induction
by 1mM methyl-jasmonate (MeJA)(red) and five days of herbivory by 15 caterpillars.
The analyses included metabolomics, transcriptomics, and herbivore performance.

20
2. Objectives

In this thesis I present a collection of studies that aimed at clarifying the way
plants regulate their metabolism during the response to caterpillar herbivory
and other abiotic and biotic stresses (Fig.9-10).

In the first study (Paper I), together with colleagues at the University of
Eastern Finland (Kuopio, Finland), we investigated how exposure to elevated
O3 concentrations (70-120 ppb) can affect plant-insect interactions between
B. nigra and P. brassicae. Combining herbivore performance tests with
analyses of plant secondary metabolism, we were able to discriminate direct
and indirect effects of O3 on the plant-herbivore system.

In a follow-up study (Paper II), we investigated the effects of O3 (70 ppb)

and herbivory on the plant metabolic responses relative to primary and
secondary metabolism. For this project we collaborated with the University
of Eastern Finland and the University of Lausanne (Lausanne, Switzerland).
Metabolomics was integrated with transcriptomics combining network and
pathway analyses, allowing us to suggest a comprehensive model for the
plant stress adaptation, which was later verified on the plant physiology.

In the third study (Paper III), we collaborated with the Entomology

Department of Wageningen University (the Netherlands) to investigate plant
responses towards multiple herbivores. We examined the changes in plant
primary and secondary metabolism after single and dual herbivory by both
B. brassicae aphids and P. brassicae caterpillars, also considering potential
effects of aphid-density.

In the last study (Paper IV), we investigated the effects of MeJA treatment
on the plant metabolic response against herbivory. Particularly, we examined
how MeJA induction can prepare the plant metabolome towards consecutive
caterpillar attack, resulting in an increased plant resistance. Moreover, we
evaluated how herbivore-induced responses, relatively for both primary and
secondary metabolism, were shaped along the plant leaf ontogeny.

21
3. Results and Discussion

3.1 O3 effects on plant-insect interactions

In Paper I we investigated the effects of O3 stress (70 - 120 ppb) on feeding

preference and performance of caterpillars, combined with the analyses of
plant secondary metabolites (GSLs and phenolics).

In a follow-up study (Paper II) we focused on the responses to O3 (70 ppb)

and sequential herbivory, linking metabolomics and transcriptomics in a
systems biology framework, and examining the plant physiological responses
relative to photosynthesis, stomatal conductance, and leaf transpiration.

3.1.1 Effects of O3 on herbivory and plant defences

We initially investigated the effects of elevated O3 on caterpillar herbivory

and caterpillar development.

Dual-choice tests were performed to determine feeding preference towards

host-plants treated with different O3 concentrations. Inside a Petri dish,
caterpillars were offered the youngest fully developed leaves (usually L3-L4)
(Fig.11a) and let choose for 12 hours between two differently treated plants:
control vs. 70 ppb; or vs. 120 ppb O3; and 70 ppb vs. 120 ppb O3 (Fig.11b).
Overall, O3 exposure resulted in increased feeding, with a larger leaf area
consumed on plants treated with 70 ppb and 120 ppb O3 (ca. 3.8 cm2)
compared to controls (ca. 2.2 cm2), and with the most damage observed on
plants exposed to 120 ppb O3, which were also preferred to control plants
(Student’s t-test, P = 0.02) (Fig.11b).

At the same time, O3 negatively affected the development of caterpillars.

During the treatment, caterpillars directly exposed to O3 while feeding and
growing on the host-plant showed a reduced weight after three and six days
at 70 ppb (Student’s t-test, P < 0.01) and at 120 ppb O3 (P < 0.05) (Fig.11c).
Growth of caterpillars was indirectly affected by O3 stress even when only
the plants were exposed to elevated O3 (70-120 ppb) (Fig.11d), with 120 ppb
also affecting their pupation time and final pupal weight (Fig.11e,f).

22
Figure 11. O3 exposure negatively affects herbivory. (a) Dual-choice set-up
for feeding preferences and (b) leaf area consumed (Student t-test, P = 0.02). (c)
Direct effects on caterpillar development, with whole-system exposure to O3, and
(d) indirect effects with only plants exposed to O3. Further indirect effects on
caterpillars growing on B. nigra exposed to O3: (e) caterpillars average days to
pupation, and (f) final pupal weight. Significance: one-way ANOVA, Tuckey
comparisons. Color-codes for O3 treatments: ambient (white), 70 ppb (blue), 120 ppb
(dark-blue). After Fig. 1-4 in Paper I.

23
To understand the change in the herbivore development caused by the
treatments, we performed metabolomics of plants exposed to O3 stress.
Analysis via LC-TOF-MS and linear ion trap for tandem MSMS (Orbitrap)
allowed the detection of over 400 features and the identification of several
metabolites as GSLs (Clarke, 2010) and phenolics (hydroxycinnamates and
flavonol glucosides)(Lin et al., 2011). Main metabolites are listed in Table 2.

The metabolic responses to O3 stress after five days (70 ppb and 120 ppb)
were modelled with supervised multivariate analyses (MVA) (Fig.12a,b).
Plants exposed to O3 were affected in GSL and phenolic metabolism in
different directions. Effects of O3 included lower levels of glucoiberin (IBE),
4-hydroxyglucobrassicin (4OH), and neoglucobrassicin (NEO). On the other
hand, we observed higher levels of glucobrassicin (GBC), gluconasturtiin
(NAS), glucojiaputin (JIAP), and particularly sinigrin (SIN) which increased
on from 3.9 to 4.7 µmol/g FW (+17%) (ANOVA, P < 0.05) (Fig.12b,c).
Phenolic metabolites were also affected, with a decrease in 1-caffeoyl-β-D-
glucose (CGLU) and quercetin-3-sinapoylsophoroside (QSPS), whereas
isorhamentin-3-sinapoyl-p-coumaroyldiglucoside (ISCDG), kaempferol (KG,
KGG), and quercetin (QS, QSG) glucosides increased. The strongest effects
were however observed on another metabolite - i.e. feature “421”, which was
always induced by elevated O3, but that we were not eventually able to
identify (Fig.12b, and Table 2).

The few studies that investigated the effects of O3 stress on Brassicaceae

with focus on the plant secondary metabolism and defence, usually showed
shifts in concentrations of GSLs (Gielen et al., 2006), or altered profile ratios
between aromatics and indolics (Himanen et al., 2008). In a previous study,
B. rapa sensitivity towards O3 stress was shown to interact in a complex way
with the insect performace: after 21 days exposure to 75 ppb O3, pupation
time was negatively affected for P. brassicae caterpillars that fed on O3-
sensitive lines, whereas caterpillar performance and final pupal weight were
affected on the O3-resistant lines (Jøndrup et al., 2002). In our study,
caterpillars preferred to feed on O3 treated plants, although these same
plants were suboptimal for their own development. Cumulative changes in
the plant metabolome may have affected the leaf palatability for these
specialist caterpillars, which usually exploit defence metabolites as feeding
stimulants or as chemical deterrents against predators (van Loon &
Schoonhoven, 1999; Ferreres et al., 2009; Winde & Wittstock, 2011).

24
 (a) OPLS-DA

O3
LV2 (20%)

LV1 (10%)

(b) (c)
QS
KG
381 QSG
KGG
SIN
JIAP

QSPS
GBC
CGLU

ISCDG
NEO NAS
4OH
IBE 421

Figure 12. O3 stress affects plant secondary metabolism. Effects of O3

stresson the plants metabolic profiles of GSLs, phenolics, and two unknown
metabolites (OPLS-DA, 1+1+0; R2Xcum=30%, R2Ycum=85%, Q2cum=44%). The
effect of O3 is described along the LV1 (10%). (a) Score plot with plant biological
replicates. Color-codes for O3 treatments: ambient (white), 70 ppb (blue), 120 ppb
(dark-blue). (b) Loading plot with individual metabolites distributed according to
their relative concentrations. Metabolite labels (see Table 2) show VIP scores ≥
1.00. Color-code: GSLs (green), hydroxicinnamic acid derivates (pink), flavonol
glucosides (purple), unknowns (lilac). (c) Sinigrin concentration in plants (µmol/g
FW) with significance tested by one-way ANOVA and Tuckey comparisons. After
Fig.S1 in Paper I.

25
Defensive metabolites other than GSLs and phenolics can affect herbivory
and caterpillar development (Poelman et al., 2008), while reduction in leaf
nutritive quality, such as amino acid and lipid content, is known to result in
increased consumption via compensatory feeding (Slansky & Feeny, 1977;
Jones & Coleman, 1988; Wheeler & Halpern, 1999). During basic elemental
analysis, we found that O3 stress lowered the plant total nitrogen content at
both concentrations of 70 ppb and 120 ppb, but did not affect total carbon
and water contents (Tab.1 in Paper I). Alternatively, O3 stress may have
also affected caterpillars by altering the host ability to initiate herbivore-
induced responses during sequential feeding. Altogether, these results show
that elevated O3 exposure can have strong effects on the plant metabolism
with potential effect on associated organisms.

Table 2. B. nigra secondary metabolism relative to glucosinolates and phenolics.

Rt [M-H]-
Class Metabolite Code Formula MSn
min m/z
Sinigrin* SIN C10H16NO9S2 0.9 358.03 259

Gluconapin* NAP C11H18NO9S2 1.6 372.04

Glucojiaputin JIAP C12H23NO9S2 2.2 388.05 259

Glucoiberin* IBE C11H21NO10S3 0.8 422.02

Glucosinolates
Gluconasturtiin* NAS C15H21NO9S2 3.1 422.06

Glucobrassicin* GBC C16H20N2O9S2 2.6 447.05 259

4-hydroxyglucobrassicin 4OH C16H20N2O10S2 1.7 463.05

Neoglucobrassicin NEO C17H22N2O10S2 3.4 477.06

1-caffeoyl-β-D-glucose CGLU C15H18O9 1.9 341.09 203, 179

1-O-sinapoyl-glucose 1SNG C17H22O10 3.2 385.11 267, 223

Hydroxy- Sinapoylferuloylgentiobiose SPFG C33H40O18 5.3 723.21 529
cinnamate
derivatives Disinapoylgentiobiose DSPG C34H42O19 5.2 753.23 529, 487

Disinapoylferuloylgentiobiose DSPF C44H50O22 5.3 929.27 735, 705

Trisinapoylgentiobiose TSPG C45H52O23 5.6 959.28 735

Km-7-glucoside KG C21H20O11 4.9 447.09

Km-3,7-diglucoside KGG C27H30O16 3.2 609.15

Km-3-sinapoylsophorosid.-7-gl. KSG C44H50O25 3.1 977.26 815, 609

Flavonol
Qn-7-sophoroside QS C27H30O17 2.9 625.14
glucosides
Qn-3-sophoroside-7-glucoside QSG C33H40Q22 2.6 787.19 625

Qn-3-sinapoylsophoroside QSPS C38H40O21 4.8 831.19

Is-3-sinapoyl-p-coumaroyl-digl. ISCDG C48H48O23 5.8 991.25 639

Feature 381 - unidentified 381 (?) 5.1 349.15

Unknowns
Feature 421 - unidentified 421 C25H28O5NS2(?) 5.9 485.13 441, 241

Main secondary metabolites detected in B.nigra. Metabolite class, common name, short codes,
molecular formula, retention time (Rt min), mass charge ratio (m/z) detected by UHPLC-TOFMS
using negative ionization mode [M-H]-, and MSMS product ions (MSn) detected by Orbitrap.
Flavonols: Km = Kaempferol, Qn = Quercetin, Is = Isorhamnetin. * = GSL standard was available.

26
 3.1.2 Omics responses to sequential O3 and herbivory

Plants exposed to five days of O3 stress (70-120 ppb) and consecutive three
days of herbivory (see in Fig.11d) were analyzed and compared to the single
stress treatments. Using supervised MVA, we were able to distinguish the
effects of O3 stress on plants that were sequentially treated with herbivory
(Fig.13a), and we could further separate between all the treatments for the
effects of herbivory (LV1) and O3 (LV2), when accounting for both stresses
as explanatory response variables (Fig.13b).

(a) OPLS-DA

O3
LV2 (19%)

Controls

LV1 (6%)

(b) OPLS-DA

Controls

Caterpillars
LV2 (6%)

O3 +
caterpillars
O3
LV1 (10%)

Figure 13. Plant secondary metabolism is induced by O3 and herbivory.

(a) Effects of O3 (OPLS-DA, 1+2+0; R2Xcum=36%, R2Ycum=86%, Q2cum=42%)
compared to (b) O3 and herbivory (OPLS-DA, 2+3+0; R2Xcum=52%, R2Ycum=81%,
Q2cum=40%) on secondary metabolism (GSLs and phenolics). Codes: O3 ambient
(white), 70 ppb (blue), 120 ppb (dark-blue); herbivory with no O3 (pink); C = no
herbivory (circles), DH = damaged by herbivory (squares); plants treated with O3
and sequential herbivory (green cluster). After Fig.5 and 6 in Paper I.

27
The strongest effects were observed on unidentified metabolites “421” and
“381”, respectively induced by O3 and by herbivory (Fig.5-7 in Paper I).
Interestingly, the induction of “381” by herbivory was lowered by initial
exposure to 120 ppb O3, corresponding to same level which negatively
affected growth and pupation of caterpillars. Herbivory seemed to counteract
the effects of O3, decreasing GSL levels including sinigrin (Fig.5 and Tab.S1,
in Paper I), possibly as a result of tissue damage and degradation into ITCs.
Responses to sequential O3 stress and herbivory also affected the levels of
hydroxycinnamates (e.g. CGLU, SNG, TSPG) and flavonol glucosides (QS,
QSP, KSTG) (VIP scores ≥ 1.00) (Fig.5-6, and Tab.1 in Paper I). Performing
a general MANOVA for GSLs, hydroxycinnamates, and flavonols, we could
show significant effects of O3 stress (Pillai's Trace P = 0.016) and herbivory
(P = 0.042) although no interaction between the treatments. However, using
general linear models (GLMs), we found interactions on two indolic GSLs
(4OH, NEO) and a quercetin glucoside (QSG) between the two O3 stress
concentrations and herbivory (P ≥ 0.01; Tab.S1, in Paper I).

Combined with the previous analyses, these results show that O3 stress can
influence plant responses towards herbivory, affecting the host metabolism
and shaping plant-insect interactions. Both individual stresses caused very
distinct effects in the plant secondary metabolism, but the response towards
herbivory always dominated during the multiple stress condition.

In order to further understand how plant responses are affected during the
shift from O3 stress to herbivory, we investigated O3 stress (70 ppb) and a
shorter period of herbivory (30 caterpillars for 24 hours). VOCs were also
collected from the plant head-spaces during the treatments, and leaf samples
were shared between metabolomics (screening of primary and secondary
metabolism) and transcriptomics (based on Arabidopsis microarrays).
Combination of omics data allowed us to propose a model for the plant
response to multiple stresses, which was eventually validated by directly
measuring the plant physiological state (Paper II).

Figure 14. Herbivore-induced metabolic responses overtake O3 stress.

(p.29) - Multivariate effects on primary and secondary metabolism, including VOCs
(PLS-DA; R2Xcum=65%, R2Ycum=95%, Q2cum=44%). (a) Score plot (LV1 vs. LV2).
Color-codes: controls (C) (white), 70 ppb O3 (O) (blue), P. brassicae herbivory (P)
(pink), and sequential O3 and herbivory (OP) (green). (b) Loading plot showing
important metabolites for the model (VIP > 1.00). Metabolites color-codes: organic
and fatty acids (yellow), amino acids (red), sugars (orange), TCA cycle (lime), amines
(dark-blue), hydroxycinnamates (lilla), flavonols (dark-purple), GSLs (green), VOCs
(light-blue), unknowns (dark-grey). Abbreviations: α-ketoglutarate (α-KG), 3,8-
dimethyl-1,4,7-nonatriene (E-DMNT). After Fig.3 in Paper II.

28
 - Metabolome profiles modelled with supervised MVA (Fig.14) showed
patterns very similar to the previous experiment: exposure to elevated O3
stress resulted in a distinct metabolic shift from control plants (LV2), but the
strongest response was always induced by caterpillar herbivory, which also
dominated the response during the sequential treatment (LV1) (Fig.14a).
Analyses relative to secondary metabolism (LC-TOF-MS) showed a general
increase in phenolics but a decrease in most GSLs, especially after herbivory
(beside glucoraphanin). Likewise in the previous study, the two unidentified
metabolites “381” and “421” were again induced respectively after herbivory
and O3 stress (Fig.14b and Fig.3c in Paper II).

(a) PLS-DA

O3

Caterpillars
LV2 (14%)

LV1 (16%)

(b)

- ITC

29
Herbivory induced the emission of several VOCs (analysed by TD-GC-MS),
such as GSL degradation products (ITCs and nitriles), terpenoids (e.g. E-
DMNT), and GLVs (Fig.14b and Fig.3c in Paper II). Responses between O3
and herbivory showed opposite effects on central primary metabolism - e.g.
carbohydrates, amino acids, organic acids (GC-TOF-MS), which generally
increased after O3 and decreased after herbivory.

Glycerol levels accumulated only in response to O3 (+55%)(ANOVA, P<0.05)

but were later restored after herbivory. A common response to all treatments
was the increase in γ-aminobutyric acid (GABA) (+300%) (ANOVA, P<0.05)
(Fig.14b), a free-amino acid involved in abiotic and biotic stress responses
such as photo-inhibition (Bouche & Fromm, 2004; Araujo et al., 2012), leaf
senescence (Ansari et al., 2005), and plant-insect interaction (Michaeli &
Fromm, 2015).

- Transcriptome profiles were initially examined separately, combining

hierarchical clustering (HCA) and gene ontology (GO) enrichment analysis.
Similarly to the metabolic profiles, responses to single O3 stress were clearly
distinct from responses to herbivory (Fig.15a-c). All treatments strongly
affected the plant central energy metabolism, including photosystems and
mitochondrial electron transport chain (ETC) (Fig.15a,b). Pathway analysis
(Mapman) confirmed a consistent response across several biological levels,
with a dominance of up- and down-regulated genes after O3 stress and with
highest number of genes shared between the single and sequential herbivore
treatments (Fig.15d,e).

Genes involved in light harvesting and carbon assimilation were down-

regulated in all scenarios but particularly after initial O3 stress (Fig.15d,f),
which also up-regulated the expression of the non-photochemical quenching
gene NPQ1 (Fig.15f). Several primary metabolic processes - e.g. amino acid
and carbohydrate metabolism, showed opposite patterns of regulation
between O3 and herbivory (Fig.15g). In general, response to herbivory
dominated the sequential treatment inducing the expression of genes
involved in plant defences - e.g. the transcription factor MYC2 (Lortzing &
Steppuhn, 2016), lipoxygenases LOX2 and LOX3 (Felton et al., 1994;
Halitschke & Baldwin, 2003), the trypsin inhibitor WSCP (Zavala & Baldwin,
2004; Boex-Fontvieille et al., 2015), and mitogen-activated protein kinase
MPK3 (Pitzschke & Hirt, 2009) (Fig.15h). Genes involved in phytohormone
signalling and biotic stress responses were also differentially regulated by O3
and herbivory (e.g. WRKYs, MYC2, and ERF2), but only O3 was associated
to abiotic stress responses, such as drought (MYB44) (Jaradat et al., 2013; Li
et al., 2014), senescence (EIN3) (Li et al., 2013), and phosphate starvation
(RNS1, PT2/PHT1.4) (Fig.15h,i).

30
Figure 15. O3 and herbivory responses suppress energy metabolism.
(a) HCA and heat-map for transcript changes compared to control levels (970 genes,
P <0.05), and gene clusters colored and ranked (I–III) according to (b) GO processes
relative abundance. (c) Heat map of Pearson´s correlation coefficient (ρ) calculated
for paired samples comparisons from the HCA (darker blue = stronger correlation).
(d) MapMan pathway analysis for changes in expression of GO biological functions
and (e) Venn diagrams comparing up- and down-regulated genes, for log2 fold-
change thresholds > 0.585 (P < 0.05). Color-codes for gene expression: up- (blue)
and down-regulation (red). Treatments: O3 stress (O), herbivory by P. brassicae (P),
sequential treatment (OP). (f-i) Central metabolism and stress response. Color-
codes: O3 (blue), herbivory (pink), and sequential treatment (green). Student´s t-
tests: * = P < 0.05; ** = P < 0.01; ***, P < 0.001; **** = P < 0.0001. Error bars=S.E.

31
- Network analysis and guilt-by-association principle allow for data-driven
generation of new hypotheses and for the prediction of unknown gene or
metabolite functions (Wei et al., 2006; Yonekura-Sakakibara et al., 2008;
Mutwil et al., 2011). In this study, 156 metabolites and 970 gene expressions
were correlated using Cytoscape producing a “scale-free” (non-random)
network (Fig.16a) dominated by “hubs” (high-degree nodes) (Table 3).

Primary metabolites clustered in the central region of the network, while

secondary metabolites clustered in modules at the periphery, connecting to
genes involved in herbivore-induced responses, VOC emissions, and GSLs
biosynthesis (e.g. CYP71,WRKY4, WRKY46) (Bennett et al., 1993; Schön et
al., 2013; Irmisch et al., 2014; Mirabella et al., 2015) (Fig. 16b). At the
centre, glycerol connected to the herbivore response variable (P) in a module
with two flavin monooxygenases NOGC1 and FMO, and positively correlated
(ρ = 0.68, P < 0.01) with the transcription factor MYB44, involved in abiotic
responses such as drought and osmotic stress (Jaradat et al., 2013) (Fig.
16c). Notably, NOGC1 is a nitric oxide-dependent guanylate cyclase involved
in regulation of stomatal closure and osmotic stress response via Ca++
signalling (Mulaudzi et al., 2011; Joudoi et al., 2013). In our network,
MYB44 and NOGC1 connected to several genes involved in induced stress
responses, carbohydrate metabolism, lipid transport, and energy metabolism
(including chloroplast and mitochondria) (Table 3, and Tab.S3, Paper II).

Table 3. Major network hubs for B. nigra omics responses to O3 and herbivory.
AGIa Gene name GO Processb,c
At4g27740 - Yippee family putative zinc-binding Nucleus (unknown)
At4g16590 CSLA1 Cellulose synthase-like A01 Glycosyltransferase
At5g04440 - Unknown function (DUF1997) Chloroplast (unknown)
At1g12390 - Cornichon protein (Guard cells) Signal transduction
At2g29100 GLR2.9 Glutamate receptor 2.9 Ca++ homeostasis
At3g50830 COR413 Cold acclimation WCOR413-like Membrane (unknown)
At1g29910 LHCB1.2 Light harvest chlorophyll binding 1.2 Chloroplast/Photosynth.
At2g22900 MUCI10 Mucilage-related 10 Galactosyltransferase
At2g32990 GH9B8 Glycosyl hydrolase 9B8 Cellulose biosynthesis
At3g09360 - TBP-binding protein RNA polymerase II
At1g62580 NOGC1 NO-dependent guanylate cyclase 1 Stomatal closure
At3g50770 CML41 Calmodulin-like 41 Chloroplast /Ca++ signalling
At5g58270 M3 ABC transporter mitochondrion 3 Mitochondrion
At3g24100 - SERF (uncharacterized) Nucleus (unknown)
At1g07040 - Unknown protein Chloroplast (unknown)
At2g02390 GST18 Glutathione S-transferase 18 Amino acid biosynthesis
a Arabidopsis Genome Initiative Number (as reported in TAIR).
bGO biological processes and/or molecular functions (as reported in TAIR).
c Cellular localization confirmed with the visualization tool ePlant (BAR; University of Toronto).

32
Figure 16. Centrality of plant responses to abiotic and biotic stresses.
Metabolomics and transcriptomics profiles of B. nigra multiple responses to O3 and
herbivory integrated in a co-expression network (Cytoscape). Edges connect nodes
linked by Pearson´s correlation (ρ values of 0.85 or greater); positive and negative
correlations represented by blue and red lines, respectively. Nodes represent: genes
(white diamonds/squares, ◊/□) and metabolites (colored circles), and herbivory (P).
(a) The network of gene-to-gene, gene-to-metabolite, and metabolite-to-metabolite
correlations. (b) Modules of secondary metabolites (GSLs, hydroxycinnamates,
flavonol glucosides and VOCs) connecting to central metabolism via stress response
genes and transcription factors (e.g. WRK40, WRK46, and CYP707A). (c) Expansion
of the gene-to-metabolite module connecting glycerol to the herbivore response (P)
via genes coding two flavin monoxygenases (NOGC1 and FMO), a receptor kinase
(LRR-RK), and a membrane transport protein (SecY protein). This module was
connected via NOGC1 to the abiotic stress response transcription factor (MYB44).

33
Using GO enrichment pathway and network analyses (KEGG/KaPPA-View4,
AraNet, GeneMANIA) combined with MVA of gene expression profiles we
found that stress treatments induced a differential regulation of energy and
glycerol metabolism during shift in responses from O3 stress to herbivory.
Particularly, GO network analysis highlighted a functional link between the
succinate dehydrogenase subunit (SDH1-1) of the mitochondrial ETC, and
the mitochondrial glycerol-3-phosphate (G3P) shuttle (GPDHc1 and SDP6).
These genes were all differentially expressed during the shift from O3 stress
to herbivory. Moreover, GO network analyses suggested functional links of
these central metabolic processes with other stress adaptation mechanisms,
such as photosystem stabilization (DGD1 and SQD2), phosphate transport
(PHT2;1), water-glycerol transport (aquaglyceroporins), and regulation of
stomatal closure (NOGC1) (see Fig.5-7 and Tab.2 in Paper II).

3.1.3 Physiological responses to O3 and herbivory

On the basis of these network-omics results, we decided to test the effects of

O3 and herbivory on processes of photosynthesis, stomatal opening, and leaf
transpiration, in order to assess the stress impact on the plant physiology.

We measured photosystem capacity and leaf transpiration (using a LI-COR

analyzer) during the same stress treatments and an additional long-term
exposure to elevated O3 (70 ppb, 16 days) (Fig.17). Only few plants showed
visible symptoms of senescence and chlorosis after five days of O3, although
chlorophyll content in leaves decreased 9.5% compared to controls (P<0.05).
Even before symptoms of chlorosis were visible in leaves, the senescence
process was initiated with the down-regulation of photosynthetic genes
(light-harvesting LHCs) and the induction of EIN3 (Fig.15), a transcription
factor involved in regulation of leaf senescence (Li et al., 2013 Potuschak et
al., 2003). Consistently, after five days of exposure, O3 stress resulted in
reduced photosynthetic activity and in lower intracellular CO2 levels, while it
negatively affected stomatal conductance and leaf transpiration (Table 4).

The phytotoxic effects of O3 were evident in plants exposed to the long-term

treatment, which showed severe symptoms of leaf senescence and chlorosis
(Fig.17a) with chlorophyll levels 47.8% lower than control plants of the
same age (P < 0.001, Fig.17b). Leaf stomatal conductance was also further
reduced by prolonged exposure to O3 stress, although there was an increase
in the intracellular levels of CO2, possibly due to the very low photosynthetic
activity (Table 4).

34
O3 can negatively affect photosynthesis (Salvatori et al., 2015; Vainonen &
Kangasjarvi, 2015) causing the degradation of chlorophyll and thylakoid
membranes in chloroplasts (Goumenaki et al., 2010; Tiwari et al., 2016). The
decreased stomatal conductance and low transpiration observed in our study
were likely the result of a protection mechanism which (via ROS signalling)
prevents O3 from entering the leaf (Castagna & Ranieri, 2009; Vahisalu et al.,
2010), but that also limits the leaf uptake of CO2 (Table 4).

Figure 17. O3 and herbivory affect photosynthesis and stomatal closure.

(a) Leaf phenotypes of B. nigra exposed to O3 at 70 ppb for five days (O), herbivory
by caterpillars for 24 hours (P), sequential O3 stress and herbivory (OP), 70 ppb O3
for 16 days (OL), and controls (C). (b) Chlorophyll content in leaves, Student’s t-test
values: C and O (P < 0.05; N = 20), P and OP (P < 0.05; N = 10), and C and OL (P <
0.001; N = 10). (c-e) Stomatal conductance (mmol H2O m-2 s-1) measured via steady-
state porometry (three experiments on separate days; N = 10 per treatment per day).
(f) Stomatal conductance differences between treatments (one-tailed Student’s t-test
for mean differences larger than zero: C versus O (P = 0.07), C versus P (P<0.05),
and O versus OP (P < 0.01). Error bars indicate S.E.

35
Herbivory did not affect photosynthesis, although it slightly decreased both
stomatal conductance and leaf transpiration. However, when plants exposed
to O3 were later treated with herbivory, photosynthetic activity, stomatal
conductance, and leaf transpiration were all reactivated, showing a reversed
effect of the sequential stress treatment compared to responses induced by
single stresses (Table 4). This effect was further confirmed measuring leaf
conductivity by steady-state porometry, thus accounting for the stomata
circadian rhythms (Fig.17c-f). Overall, physiological responses mirrored the
previous patterns of metabolomics and transcriptomics (Fig. 14-15) where
the initial response to O3 was counteracted by herbivory, and the phenotype
during the sequential stress resembled the one induced by herbivory alone.

Based on both omics and physiological data, we proposed a model for stress
adaptation mechanisms in B. nigra (Fig.18) where both glycerol and energy
metabolism (chloroplasts and mitochondria) play central roles in protecting
against oxidative stress and facilitating stomatal osmoregulation. Glycerol
osmolytic properties can enhance the plant resistance against osmotic stress
(Eastmond, 2004), and glycerolipid transfer is important for plant tolerance
to different abiotic stresses (Li et al., 2015; Mueller et al., 2015), including
O3 stress (Sakaki et al., 1990). Glycerol accumulation in our study possibly
derives from the peroxidation of lipids in chloroplast membranes (Bienert et
al., 2006), as supported by the activity of chloroplastic lipid transfer proteins
(LTPc1 and LTPc2) (Xu et al., 2008) which were correlated with O3 stress
and with induction of MYB44 (Fig.S3-S4 in Paper II). MYB44 is known to
regulate abiotic and biotic stress responses (Baldoni et al., 2015), including
stomatal opening and leaf senescence (Jaradat et al., 2013; Li et al., 2014).

Table 4. Opposite effects of O3 and sequential herbivory on photosynthesis.

Treatments: C O (vs. C) P (vs. C) OP (vs. O) OL (vs. C)

Photosynthesis
11.72 ± 0.8 8.63 ± 0.63 11.5 ± 0.58 11.24 ± 0.85 3.29 ± 0.26
(µmol CO2 m-2 s-1)
** n.s. * ***
Stomatal
conductance 0.31 ± 0.02 0.15 ± 0.04 0.25 ± 0.10 0.24 ± 0.03 0.12 ± 0.01
(mol H2O m-2 s-1) ** n.s. * **
Leaf
transpiration 3.45 ± 0.22 1.84 ± 0.22 2.97 ± 0.90 2.66 ± 0.26 1.55 ± 0.19
(mol H2O m-2 s-1) *** n.s. * ***
Intracellular CO2
concentration 300 ± 3.87 264 ± 4.93 291 ± 9.35 288 ± 9.81 333 ± 10.19
(ppm) * n.s. n.s. ***
Leaf photosynthesis and transpiration measured using a LI-COR analyzer (LI-6400). Means ± SE (n = 6-10)
and variation calculated to relative group comparisons (in brackets). Student´s t-test, P-values > 0.05 (*),
> 0.01 (**), > 0.001 (***). Treatments: controls (C), exposure to 70 ppb O3 for five days (O), P. brassicae
herbivory for 24 hours (P), sequential 70 ppb O3 and herbivory (OP), and additional long term exposure
to 70 ppb O3 for 16 days (OL).

36
Feed-back regulation between MYB44 and NOGC1 may help plant responses
to control stomatal conductance and balance CO2 uptake during O3 stress
(Joudoi et al., 2013). Altogether, I suggest that combined regulation of
glycerol metabolism (including the G3P shuttle, GPDHc1/SDP6) and energy
metabolism via suppression of photosynthesis (Shen et al., 2006; Quettier et
al., 2008) and increased mitochondrial activity (SDH1-1, MSD1) (Huang et
al., 2013; Martin et al., 2013) may help the plant to dissipate excess energy
and prevent formation of oxidative radicals (Dizengremel et al., 2009;
Blanco et. al, 2014; Tiwari et al., 2016). The redirection of these pathways
during sequential herbivory suggests an active plant response via WRKYs,
MYC2, and ERF2 (Huot et al., 2014), but it may also represents a herbivore
manipulation of the host’s metabolism which interferes with induced defence
and water stress responses (Reymond et al., 2000; Consales et al., 2012).

Figure 18. Plant central metabolic responses towards O3 and herbivory.

Omics data linked to physiological responses propose stress adaptation mechanisms
(1–5). Blue, O3 stress (five days, 70 ppb); red, sequential herbivory by P. brassicae
(24 hours). Up/down arrows = up/down regulation of genes, or increase/decrease in
metabolites. (1-2) O3 induces the abiotic stress responses of senescence (EIN3 and
ERF2) and stomatal closure (MYB44), with feedback on NOGC1, and possibly FMO.
ABA and JA/ET cross-talk integrates responses between O3 and sequential herbivory
(MYC2 and ERF2), with opposite effects on stomatal closure. (3-4) Photosystem
suppression (LHCs) and non-photochemical quenching (NPQ1) in response to O3 are
linked to the regulation of glycerol metabolism (LPTs, LIP1, and SQD2). Glycerol
derived from degraded chloroplast membranes enters the G3P shuttle
(GPDHc1/SDP6) to sustain NAD+ recycling and mitochondrial activity (SDH1-1,
MSD1, and GABA) as antioxidative stress mechanisms. A role of glycerol and sugars
as osmolytes also is suggested. Herbivory interacts with glycerol pathways for
alternative source-sink priorities and induced JA responses (MYC2, ERF2, LOXs).
(5) GABA roles in plant stress adaptation: Ca++ homeostasis, carbon-nitrogen
metabolism, senescence, ROS scavenging, and plant-insect interactions.

37
3.2 Dual herbivore attack

In Paper III we investigated the metabolic responses of B. nigra versus two

different kinds of herbivory: the chewing caterpillars of Pieris brassicae and
the piercing-sucking aphids of Brevicoryne brassicae. Herbivore-induced
responses in plants were evaluated for effects of single and dual herbivory on
both local and systemic leaves, while accounting for aphid-density effects.
Aphids (nymphs, first and second instar) were let feed on the youngest fully
expanded leaf of B. nigra for three days, while caterpillars (30, first instar)
fed on the same leaf, alone or with aphids, for 24 hours. In total, plants were
exposed to five treatments: control undamaged plants (C), herbivory by 50
or 100 aphids alone (50A, 100A), herbivory by caterpillars alone (P), or dual
herbivory by 50 or 100 aphids plus caterpillars (50A+P, 100A+P).

Metabolomics was targeted for primary and secondary metabolism using

GC-TOF-MS and LC-TOF-MS, respectively. In total, 221 metabolic features
were detected, of which 110 were fully identified between GSLs, phenolics
(hydroxycinnamates and flavonols), amino acids, sugars, and organic acids.
Here I will only focus on the herbivore responses induced in locally damaged
leaves, which always showed stronger effects compared to systemic leaves.

3.2.1 Plant responses versus aphids and caterpillars

Changes in the plant metabolic responses differed according to the kind of

herbivory, as shown by the general MVA model for the effects of caterpillar
and aphid herbivory in all treatments (C, P, 50A, 100A, 50A+P, 100A+P)
(Fig. 19a). The strongest plant responses were observed for dual herbivory
by caterpillars and aphids during high-aphid density (100A+P) (Fig. 19a).
A further MVA classification based on either presence or absence of aphids
or caterpillars (i.e. not including the aphid-density variable) showed a clear
difference between the two types of herbivore responses (Fig. 19b). In this
model, plants infested with only aphids were separated from plants with
caterpillars alone or during dual herbivory. Together, these results indicated
a dominant effect of caterpillars over the effect of aphids during the plant
response to dual herbivory (Fig.19a,b).

Figure 19. Plant responses versus caterpillar and aphid herbivory. (p.39) -
MVA models describing the effects of caterpillars and aphids on B. nigra primary and
secondary metabolism. (a) Score plot (OPLS-DA 3+2+0; R2Xcum=57%,
R2Ycum=52%, Q2cum=21%). Treatments abbreviations: controls (C), plants infested
with B. brassicae 50 aphids (50A) or 100 aphids (100A), single herbivory by
P. brassicae caterpillars (P), dual herbivory with 50 aphids and caterpillars (50A+P),
or with 100 aphids and caterpillars (100A+P).

38
 (a)

OPLS-DA
Low-aphids
LV2 (5%)

Caterpillars

Controls
High-aphids

LV1 (12%)

(b)

Aphids + caterpillars
OPLS-DA
LV2 (10%)

Aphids
Aphids Caterpillars
Caterpillars

LV1 (6%)

(c)

(continued) (b) Score plot, all plants grouped for herbivore treatments by only
aphids, only caterpillars, or dual herbivory (OPLS-DA 1+1+0; R2Xcum=12%,
R2Ycum=49%, Q2cum=20%). (c) Loading plot for metabolite contribution (only
identified metabolites). VIP > 1.00: (1) fructose, (2) fructose/sorbose, (3) maltose,
(4) ribitol, (5) glucose, (6) trehalose, (7) sorbose, (8) p-coumaric-acid,
(9) GSH, (10) α-tocopherol, (11) glucoiberin, (12) gluconapin, (13) glucobrassicin,
(14) neoglucobrassicin, (15) sinigrin, (16) gluconasturtin, and (17) Quercetin-3-
coumaroylsophoroside-7-glucoside. After Fig.1 and 2 in Paper III.

39
Caterpillars primarily characterized an increase in GSLs (ANOVA, P <0.001)
(Fig.19c, Fig.20a), with an increase in sinigrin from 3.8 to 5.9 µmol/g FW
(+55%) (ANOVA, P < 0.01). Overall, aphid-density always affected the plant
response during caterpillar and dual treatments, but while low-aphid density
(50A, 50A+P) increased GSL levels similarly to herbivory by caterpillars (P),
high-aphid density did not further induced GSLs levels in the presence of
caterpillars (100A+P) (Fig.20a).

Aphid herbivory induced higher levels of carbohydrates (i.e. trehalose,

fructose, maltose, ribitol, glucose and sorbose) as well as other metabolites,
e.g. glutathione (GSH), α-tocopherol, p-coumaric-acid, and quercetin-3-
coumaroylsophoroside-7-glucoside (Fig.19c, VIPs > 1.00). Trehalose was a
particularly important metabolite. It was never induced in response to
caterpillars alone, while it increased during single (50A, 100A) and dual
(50A+P, 100A+P) aphid feeding (ANOVA, P < 0.001) (Fig. 20b). Trehalose
also correlated to aphid-density (ρ = 0.64, P < 0.001), with the highest levels
observed after high-aphid infestations (100A, 100A+P) (Fig. 20b).

Figure 20. Dual herbivory and aphid-density affect GSLs and trehalose.
(a) Total GSLs levels and (b) trehalose levels, detected in B. nigra leaves damaged
by combination of single and dual herbivory treatments. Relative abundance based
on integrated chromatogram peak areas of GSLs (LC-TOF-MS), sinigrin, gluconapin,
glucoerucin, glucobrassicin, neoglucobrassicin, 4-methoxyglucobrassicin, glucoiberin,
glucojiaputin, glucobrassicanapin, gluconasturtiin, 4-hydroxy-glucobrassicin) and of
trehalose (GC-TOF-MS). Mean ± S.E., n = 6. ANOVA with Tuckey test comparisons.
Treatments abbreviations: controls (C), plants infested with 50 aphids of B. brassicae
(50A) or 100 aphids (100A), single herbivory by P. brassicae caterpillars (P), and
dual herbivory with 50 aphids+caterpillars (50A+P), or 100 aphids+caterpillars
(100A+P). After Fig.5 in Paper III.

40
3.2.2 Effects of dual herbivory and aphid density

To distinguish between the effects of dual herbivory and aphid density, we

compared the treatment using pair-wise MVA models (Table 5 and Fig.S1 in
Paper III). The strongest effects were observed for the increase from low- to
high-aphid density (i.e. from 50 to 100 aphids) (model 1), and for the shift
from single herbivory by either the caterpillars (P) or the aphids (50A, 100A),
toward dual herbivory (respectively model 4, 5, and 6) (Table 5). In addition,
significant effects of aphid-density were also found in systemic leaves which
were not directly damaged by herbivores (model 8 and model 9, Table 5).

In keeping with the results from our previous multiple stress studies, it was
rarely an individual metabolite that contributed to the pair-wise differences
across treatments, and changes in both primary and secondary metabolites
characterized the plant response. For instance, when comparing herbivory
by aphids at different density levels, the addition of caterpillars resulted in
changes of sugars and phenolics in plants treated at low-aphid density - i.e.
50A versus 50A+P (model 5), whereas it mostly affected GSLs and several
other unknown metabolites in plants treated at high-aphid density – i.e.
100A versus 100A+P (model 6) (see Fig.S1 in Paper III for score and
loading plots of MVA models listed in Table 5).

Table 5. Effects of dual herbivory and aphid-density on the plant metabolism.

Model parameters
R2 X R2 Y Q2
Treatment effects Comparisons LVs (cum) (cum) (cum)

1 Aphid-density + Aphid (50) 50A 100A 1+3+0 62% 99% 51%

2 Aphid-density + Aphid (50) 50A+P 100A+P 1+0+0 31% 58% 19%
3 Dual herbivory + Aphid (50) P 50A+P n.s. n.s. n.s. n.s.
4 Dual herbivory + Aphid (100) P 100A+P 1+3+0 68% 99% 14%
5 Dual herbivory + Caterpillar 50A 50A+P 1+3+0 52% 98% 36%
6 Dual herbivory + Caterpillar 100A 100A+P 1+2+0 52% 98% 39%
7 Systemic aphid + Aphid (50) 50A 100A n.s. n.s. n.s. n.s.
8 Systemic aphid + Aphid (50) 50A+P 100A+P 1+3+0 65% 99% 38%
9 Systemic aphid + Aphid (50) P 50A+P 1+1+0 69% 99% 56%

Pair-wise comparisons targeting for effects of dual herbivory and aphid-density on B. nigra metabolome,
MVA models (OPLSDA). See relative score and loading plots for each model in Fig. S1, Paper III.
Orthogonal variation was standardized at maximum three latent variables (LVs). R2X (cum) = cumulative
metabolic variation (x) explained by the MVA model; R2Y (cum) = cumulative treatment effect explained
by the MVA model (y); Q2 (cum) = cumulative predictive capacity of the MVA model.
Models (1-6) refer to effects on locally damaged leaves, with strongest models highlighted in blue.
Models (7-9) refer to effects on systemic leaves, and discussed in Paper III.

41
We compared responses to dual herbivory at high-aphid density (100A+P)
with the two respective single herbivore treatments (100A and P) (Fig.21).
This model showed a clear separation between single and dual herbivory
(Fig.21a) with a main increase of GSLs in response to caterpillar herbivory
(P, 100A+P) and only an increase in 4-methoxy-glucobrassicin associated to
dual herbivory (Fig.21b). Several metabolites characterized the response to
high-aphid density (100A) which was consistent during dual herbivory
(100A+P), with increase in glycolic acid, phosphoric acid, glutathione (GSH),
and the sugars fructose and trehalose (Fig.21b). These results showed how
both aphids and caterpillars have strong effects on the plant metabolome,
but also how responses during dual herbivory are affected at high-aphid
density (Fig.21; see also pair-wise model 4 and 6 in Table 5).

(a)
Aphids+ caterpillars OPLS-DA
LV2 (8%)

High-aphids Caterpillars

LV1 (6%)

(b)

Figure 21. Responses to caterpillar are shaped by high-aphid density.

Combined effects on B. nigra primary and secondary metabolism (OPLS-DA 2+3+0;
R2Xcum = 64%, R2Ycum = 95%, Q2cum = 40%). (a) Score plot, with plants infested
with P. brassicae caterpillars (P), B. brassicae 100 aphids (100A), or dual herbivory
(100A+P). (b) Loading plot for metabolite contribution (only identified metabolites).
VIPs > 1.00: (1) glucoiberin, (2) neoglucobrassicin, (3) glucobrassicin, (4) sinigrin,
(5) gluconapin, (6) gluconasturtin, (7) 4-hydroxy-glucobrassicin, (8) 4-methoxy-
glucobrassicin, (9) glycolic acid, (10) phosphoric acid, (11) glutathione GSH,
(12) fructose, and (13) trehalose. After Fig.3 in Paper III.

42
 To reveal other potential aphid-density effects on single and dual herbivory,
we created an MVA model with no single caterpillar herbivory (P) (Fig.22).
This model showed the effect of aphids on dual herbivory by separating the
treatments first for aphid-density (LV1) and then for presence or absence of
caterpillars (LV2) (Fig.22a). These results confirmed an effect of aphid-
density on the plant response to caterpillar herbivory. Once again, the major
effect of caterpillar herbivory was found on GSLs, while especially single
aphid herbivory led to increased levels of sugars (Fig.22b). Specific aphid-
density effects were observed during high-aphid treatments, which led to
increased levels of sugars, i.e. trehalose, ribose, ribitol, xylose, and maltose,
whereas low-aphid treatments increased the levels of fructose (Fig.22b).

(a)
OPLS-DA
High-aphids
LV2 (19%)

Low-aphids

Caterpillars

LV1 (12%)

(b)

Figure 22. High- and low-aphid density affect response to dual herbivory.
Effects of high- and low-aphid density during single and dual herbivory on B. nigra
primary and secondary metabolism (OPLS-DA 1+4+0; R2Xcum = 52%, R2Ycum =
75%, Q2cum = 15%). (a) Score plot, plants infested with only 50 (50A) or 100 (100A)
of B. brassicae, or in combination with P. brassicae caterpillars (50A+P or 100A+P).
(b) Loading plot (only identified metabolites). VIP > 1.00: (1) glucoiberin,
(2) sinigrin, (3) gluconapin, (4) glucobrassicin, (5) neoglucobrassicin, (6) 4-hydroxy-
glucobrassicin, (7) gluconasturtin, (8) ribose, (9) ribitol, (10) trehalose, (11)
maltose/cellobiose (12) xylose, (13) fructose, (14) cis,cis-linoleic acid, (15) α-linolenic
acid, and (16) glycolic acid. After Fig.4 in Paper III.

43
Chewing caterpillars induced the strongest metabolic responses in plants,
especially with increased levels of GSLs; on the other hand, sucking aphids
resulted in density-dependent effects which differentially induced the plant
metabolic profile during both single and dual herbivory. Dual caterpillar and
aphid herbivory can affect GSLs levels through different processes, such as:

1) de novo GSL biosynthesis as herbivore-induced response during defence

against chewing caterpillars (Hopkins et al., 2009);

2) degradation of GSLs into VOCs after leaf damage by caterpillars, which

can affect herbivory as an indirect defence system (Ponzio et al., 2014);

3) manipulation of plant JA-induced responses by piercing-sucking aphids

(Broekgaarden et al., 2008; Walling, 2008; Thaler et al., 2012)

4) increase or decrease in GSLs after aphid herbivory (Mewis et al., 2006;

Kim & Jander, 2007; Khan et al., 2011);

5) sequestration of GSLs by the specialist aphid of Brassicacea, B. brassicae

(Jones et al., 2001; Kazana et al., 2007; Broekgaarden et al., 2008).

Interaction between P. brassicae and B. brassicae on cultivated B. oleracea

was previously shown to have no effects on GSLs, but SA-mediated defences
induced by aphids interfered with JA-mediated defences, facilitating the
development of caterpillars (Soler et al., 2012). Parasitoids also performed
better on both hosts during dual herbivory compared to single herbivory,
suggesting a higher level of complexity in the system that involves VOCs and
indirect defences, as confirmed by other studies in B. nigra (Ponzio et al.,
2014; Ponzio et al., 2016).

In our study, higher aphid-density increasingly induced levels of trehalose

and other sugars (19-22), although only in locally damaged leaves. Sugar
levels may have been concentrated by the aphid sucking activity, or
alternatively been derived from honeydew excretions on leaves (Lamb, 1959;
Yao & Akimoto, 2001). On the other hand, changes in primary metabolites
can also represent a strategic re-allocation of plant resources or be directly
involved in defence (Schwachtje & Baldwin, 2008; Steinbrenner et al., 2011).
Similarly to our study, trehalose is induced in Arabidopsis during herbivory
by the generalist aphid Myzus persicae (Singh et al., 2011; Hodge et al.,
2013). Trehalose may thus be directly induced by plants to respond to aphid
infestation, as suggested by studies showing its role in protection against
biotic and abiotic stresses (Fernandez et al., 2010; Singh & Shah, 2012),
where it regulates sugar signalling, carbon partitioning, and accumulation of
starch (Singh et al., 2011; Nuccio et al., 2015; Foyer et al., 2016).

44
3.3 Induced plant resistance to herbivory

In Paper IV we investigated the herbivore-induced responses of plants after

phytohormone treatment with MeJA and sequential chewing by caterpillars.
We tested whether MeJA may enhance plant resistance against herbivory,
while we evaluated other potential effects on the plant metabolome. Finally,
we studied how herbivore-induced responses were shaped by leaf ontogeny.

In all experiments, plants were sprayed only once with MeJA (1mM, 5mL)
and let respond for 72 hours. Herbivore treatments consisted in caterpillars
feeding on leaves for five consecutive days. The four treatments included:
single MeJA or herbivory, sequential treatment, and controls (Fig. 23a).

Figure 23. Plant induced treatments with MeJA and caterpillar herbivory.
(a) Experimental set-up. Treatment abbreviations: controls undamaged plants (C),
plants sprayed with MeJA (1mM, 5 mL) and induction for 72 hours (M),
herbivory by 15 first instar caterpillars of P. brassicae feeding for five days (P), and
sequential MeJA and herbivory (MP). (b) B. nigra phenotype across leaf ontogeny
and effects of MeJA treatment. P. brassicae caterpillars were placed on the youngest
fully developed leaves, usually corresponding to L4-L5. (c) Effects of MeJA on stem
pigmentation, between 24-72 hours. (d) Effect of MeJA induced responses on
caterpillars´ weight after five days of feeding (Student´s t-test, p-value < 0.05).

45
3.3.1 Effects of MeJA on herbivore-induced responses

Symptoms of early leaf senescence appeared in plants treated with MeJA

already after three days, most commonly in lower leaves (L6-L10) (Fig.23b).
These plants also displayed a dark, purple pigmentation along their stems
(Fig.23c). After five days of herbivory treatment, MeJA-treated plants
showed enhanced resistance against P. brassicae caterpillars, which gained
less weight than those feeding on control plants (-27%) (P<0.05) (Fig.23d).

From plants treated with herbivory (P, MP), damaged leaves were sampled
and compared to same leaves from undamaged plants (C, M). A supervised
MVA model of the plant primary and secondary metabolism (analysed using
GC-MS and LC-MS) showed distinct metabolic shifts from steady-state levels
of control plants (C) after each one of the treatments (M, P, MP) (Fig.24).
Induced responses were distributed along the first latent variable (LV1) and
described similar magnitudes between single MeJA (M) and P. brassicae (P),
with an increased response during the sequential treatment (MP). However,
the effects of MeJA (M, MP) differed from real caterpillar herbivory (P, MP)
and were described by a shift along the second latent variable (LV2) (Fig.24;
see also Fig.2b, in Paper IV for loading plot of metabolite contribution).
LV2 (9%)

Pieris
caterpillars Controls

MeJA

LV1 (14%)

Figure 24. Plant herbivore-induced responses are shifted by MeJA. MVA

model for the effects of MeJA and herbivory treatments on primary and secondary
metabolism (PLS-DA model, four components; R2Xcum = 42%, R2Ycum = 75%,
Q2cum = 56%). (a) Score plot. Treatment abbreviations: controls (C), MeJA (M), P.
brassicae herbivory (P), and sequential MeJA and herbivory (MP). (b) Loading plot
with metabolites contribution (VIP > 1.00). After Fig.2 in Paper IV.

46
GSLs were induced to the same extend by MeJA and herbivory, although the
effect of MeJA was more uniform with less variation (Tab.1 in Paper VI).
In the sequential treatment, initial MeJA application increased the following
induction of GSLs by caterpillar herbivory (Fig.25, Table 6). Particularly,
sinigrin increased from ca. 3 to 5 µmol/g FW after either single MeJA or
herbivory, to 9 µmol/g FW after sequential treatment (P > 0.001) (Fig.26a).

Indolic and aromatic GSLs such as neoglucobrassicin and glucotropaeolin

were largely induced after the MeJA treatments, but not particularly after
herbivory. Phenylpropanoids were also differentially affected by the MeJA
treatment, with a decrease of hydroxycinnamic acid derivatives (e.g. 1-
caffeoyl-β-glucose, sinapic acid, and 1-O-sinapoyl-glucose) (Milkowski &
Strack, 2010) and of flavonol glucosides (Table 6). On the contrary, several
phenylpropanoids and flavonols were found to increase in response to real
herbivory (e.g. sinapic acid, disinapoyl-gentiobiose, and quercetin-3-
sinapoylsophoroside-7-glucoside) (Fig.26b and Table 6). Sequential MeJA
and herbivory also affected the levels of the two unidentified metabolites
previously described in Paper I-II (features “381” and “421”), which were
important for defining biotic and abiotic stress interactions in B. nigra
(Khaling et al., 2015; Papazian et al., 2016).

At the level of primary metabolism, MeJA and herbivory had different

effects on sugars, amino acids, and organic acids (Tab.1 in Paper VI). MeJA
resulted in a stronger decrease of fructose, an increase in both myo-inositol
and maltotriose, but did not affect the levels of gentiobiose, instead induced
by caterpillar chewing (Fig. 25b, 26c-e and Table 6). Herbivory also
negatively affected the level of almost all amino acids, while MeJA only
affected tryptophan (indolic GSLs precursor; Halkier & Gershenzon, 2006)
and had no effect on phenylalanine (aromatic GSLs precursor) (Fig.26f,g).

Both MeJA treatment and herbivory had similar effects on the levels of
several TCA cycle intermediates, resulting in an overall increase of citric acid
and α-ketoglutaric acid (i.e. C5-C6 metabolites of the first half-cycle) and a
decrease in succinic acid, fumaric acid and malic acid (i.e. C4 metabolites of
the second half-cycle) (Fig.26h-j, see also Tab.1 in Paper IV). In addition,
during sequential herbivory, MeJA increased the levels of cis-aconitic acid
(C6), while it further decreased the levels of succinic acid and malic acid (C4)
(Fig. 25b, and Table 6).

These cumulative effects of MeJA on primary and secondary metabolism

may have enhanced the plant resistance against herbivory, resulting in the
decreased growth of young P. brassicae caterpillars (Fig.23d).

47
 Caterpillars

Caterpillars
+
MeJA

Figure 25. MeJA enhances plant responses against caterpillars.

Multivariate model for the effects of MeJA treatment on B.nigra metabolic response
towards herbivory, relative to primary and secondary metabolism (OPLS-DA 1+1+0;
R2Xcum = 28%, R2Ycum = 86%, Q2cum = 70%). (a) Score plot, with plants treated
with herbivory by P. brassicae caterpillars (P) compared to same treatment on plant
previously exposed to MeJA (MP). (b) Loading plot with metabolites (VIPs > 1.00).

48
Figure 26. Induced responses affect primary and secondary metabolism.
Effects of single and sequential MeJA and herbivory on indivividual metabolites.
MeJA- and herbivory- induced changes in abundances of defence-related secondary
metabolites such as (a) sinigrin and (b) quercetin glucoside, and of growth-related
primary metabolites, such as (c) fructose, (d) gentiobiose (or similar disaccharide),
(e) maltotriose (trisaccharide), amino acids (f) phenylalanine and (g) tryprophan,
and TCA cycle intermediates (h) citric acid, (i) α-ketoglutarate, and (j) fumaric acid.
Sinigrin concentrations (µmol/g FW) were calculated comparing a calabiration curve
of pure sinigrin standard. Abundance of other metabolites are reported as relative
values from integrated chromatogram peak areas. Metabolites are colored according
to the code system used in Fig.25. Significant changes in metabolite concentrations
compared to control steady-state levels are reported as Student´s t-test, P-values <
0.05 (*), < 0.01 (**), and < 0.001 (***). Treatment abbreviations: controls (C), MeJA
(M), P. brassicae herbivory (P), and sequential MeJA and herbivory (MP). See also
Fig.2 and Tab.1 in Paper IV.

49
 Table 6. MeJA strengthens herbivore-induced responses against caterpillars.
Metabolite Class VIP (P vs. MP)
Glucotropaeolin Glucosinolate 1.93 ⬆
Gluconapin Glucosinolate 1.80 ⬆
Maltotriose Sugar 1.78 ⬆
Sucrose Sugar 1.74 ⬆
Feruloylmalic acid Hydroxycinnamate 1.66 ⬆
cis-Aconitic acid TCA 1.63 ⬆
Flavanone (putative) Flavonol glucoside 1.60 ⬆
Sinigrin Glucosinolate 1.52 ⬆
Galactonic acid Organic acid 1.51 ⬆
381 Unknown 1.51 ⬆
Neoglucobrassicin Glucosinolate 1.48 ⬆
Caffeoyl-glucoside (putative)(1) Hydroxycinnamate 1.39 ⬆
p-coumaroyl-D-glucose Hydroxycinnamate 1.37 ⬆
Glucoerucin Glucosinolate 1.32 ⬆
3-methylbutyl-GSL Glucosinolate 1.22 ⬆
Caffeoyl-glucoside (putative (2) Hydroxycinnamate 1.17 ⬆
Disinapoylgentiobiose Hydroxycinnamate 1.15 ⬆
Inositol, myo- Sugar 1.14 ⬆
Dehydroascorbic acid Redox 1.09 ⬆
Sinalbin Glucosinolate 1.02 ⬇
α-Linoleic acid Lipid 1.03 ⬇
Qn-3-sinapoylsophoroside-7-glucoside Flavonol glucoside 1.04 ⬇
Km 3-sinapoylsophoroside-7-glucoside Flavonol glucoside 1.04 ⬇
4-hydroxyglucobrassicin Glucosinolate 1.04 ⬇
Succinic acid TCA 1.06 ⬇
Glucoraphanin Glucosinolate 1.06 ⬇
Km-3-hydroxyferuloylsophoroside-7-glucoside Flavonol glucoside 1.07 ⬇
Qn-glucoside (putative) Flavonol glucoside 1.07 ⬇
Sinapoylhydroxyferuloylgentiobiose Hydroxycinnamate 1.07 ⬇
Qn-3-sophorotrioside-7-glucoside Flavonol glucoside 1.11 ⬇
Trisingentiobiose Hydroxycinnamate 1.12 ⬇
Qn-7-sophoroside Flavonol glucoside 1.14 ⬇
Km 3-sinapoylsophoroside (putative) Flavonol glucoside 1.16 ⬇
Km-3-p-coumaroylsophoroside-7-glucoside Flavonol glucoside 1.16 ⬇
Qn-3-sinapoylsophoroside-7-glucoside (putative) Flavonol glucoside 1.17 ⬇
Km-3-hydroxyferuloyldiglucoside-7-glucoside Flavonol glucoside 1.17 ⬇
Malic acid TCA 1.21 ⬇
421 Unknown 1.22 ⬇
Threonic acid Redox 1.28 ⬇
Km-glucoside (putative) Flavonol glucoside 1.32 ⬇
Tryptophan Amino acid 1.34 ⬇
1-O-sinapoylglucose Hydroxycinnamate 1.47 ⬇
Fructose Sugar 1.53 ⬇
1-caffeoyl-β-D-glucose Hydroxycinnamate 1.64 ⬇
Glutathione (GSSG) Redox 1.66 ⬇
Oxalic acid Organic acid 1.67 ⬇

Metabolite contribution to the OPLS-DA model (Fig.25), comparing the effect of MeJA treatment on
B.nigra responses towards sequential herbivory by P. brassicae. Significant contribution to the model of
each metabolite is reported for the respecitve variable importance in the projection (VIP) scores >1.00.
Metabolites are ordered from higher to lower VIP values, for increased (blue ⬆) and decreased (red ⬇)
abundance in plants pre-treated with MeJA (MP) compared to plants only treated with herbivory (P).
See corresponding loading plot in Fig.25b.

50
MeJA enhanced B. nigra defence responses and the production of defensive
secondary metabolites, e.g. GLSs. At the same time, MeJA induced multiple
effects in the plant chemistry and phenotype, such as early leaf senescence,
purple stem coloration, differential accumulation of phenylpropanoids, and
changes in primary metabolites, e.g. sugars and TCA cycle intermediates.
MeJA application stimulates JA-responses via activity of MYC2, one of the
main transcription factors involved in regulation of plant induced-defences
(Lortzing & Steppuhn, 2016). Different phytohormones are involved in fine-
tuning of MYC2 and JA-mediated defence responses (Erb et al., 2012). For
instance, MYC2 interferes with ET responses via antagonistic interaction
with EIN3 (ETHYLENE INSENSITIVE 3) (Song et al., 2014). Induced MYC2
activates regulation of GSL and phenylpropanoid metabolism (Dombrecht et
al., 2007; Verhage et al., 2011; Schweizer et al., 2013), while it suppresses
growth processes (Chen, et al., 2011; Huot et al., 2014; Campos et al., 2016).
Thus, pleiotropic effects of MYC2 may explain the effects of MeJA on the
phenotype of B. nigra which were observed in our study (Fig.23-26).

Using Arabidopsis mutant lines ein3-1 treated with MeJA, we tested the
interaction of JA- and ET- responses, measuring MeJA-induced expressions
of MYC2 and two downstream genes involved in defence against insects and
pathogens, respectively controlled by MYC2 and EIN3: VSP2 (VEGETATIVE
STORAGE PROTEIN 2) and PDF1.2 (PLANT DEFENSIN 1.2) (Manners et
al., 1998; Liu et al., 2005; Verhage et al., 2011) (Fig.27a). This experiment
confirmed JA and ET asymmetric cross-talk, with MeJA-induced expression
of MYC2 and VSP2, but down-regulation of PDF1.2 in ein3-1 (Fig.27b).

Figure 27. JA/ET asymmetric regulation of herbivore-induced defences.

(a) JA and ET antagonistic pathways involved in herbivore signalling and fine-tune
of defence responses. (b) Relative expression levels (RTq-PCR) of MYC2, PDF1.2 and
VSP2 in Arabidopsis, comparing wt (col-1) and ein3-1 mutants, after 72 hours
induction with MeJA (1mM). Significance between the treatments was tested with
ANOVA (N=3; Tuckey comparisons).

51
3.3.2 Ontogenic patterns of induced responses

To investigate herbivore-induced effects along the plant architecture, we

examined the metabolic profiles of individual leaves (L1-L5), targeting for
defence metabolites (Table 6) and following the responses throughout leaf
ontogeny. As in the previous experiment, MeJA and herbivory induced a
distinct shift from the steady-state metabolism of control plants, which
increased during sequential treatments (PC2; Fig.28a). Herbivore-induced
responses further increased towards younger leaves (PC1), and leaf ontogeny
explained twice the total metabolic variation associated with the treatments
(Fig.28b). Top young leaves (L1-L3) showed the strongest shifts after MeJA
and herbivory, along with the highest levels of secondary metabolites, i.e.
GSL, flavonols, and hydroxycinnamates (see Fig.4e,f in Paper IV).

We further tested the effect of MeJA on leaf ontogenic patterns targeting

primary metabolism. Plants were divided into three ontogenetic groups of
“top” (L1-L3), “mid” (L4-L6), and “low” (L7-L8) leaves, based on previous
secondary metabolic profiles (Fig.4g and Fig.5a in Paper IV). The strongest
responses were again observed in the youngest leaves (Fig.28c; also Fig.5
and Tab.3 in Paper IV), and largely reflected the effects of MeJA observed
in our first experiment (Fig.24-26 and Table 6; also Tab.1 in Paper IV).
MeJA affected primary metabolites directly involved in defence metabolism
– including a decrease in α-linolenic acid (JA precursor) (Wasternack, 2015)
and different effects on phenolics (caffeic acid, salicylic acid, and salicin) –
and strongly induced changes in central carbon pathways, with increased
levels of myo-inositol, maltose, and trehalose, and a decrease in fructose.
MeJA again increased citric acid and α-ketoglutaric acid levels (C5-C6), but
also decreased those of fumaric acid (C4) (Fig.28c, and Tab.3 in Paper IV).

These shifts in primary metabolism suggests a reconfiguration of source-

sink relationships, possibly as a strategy aimed at maximizing the plant
fitness, balancing between plant growth and defence against insects (Bolton,
2009; Lortzing & Steppuhn, 2016). Regulation of TCA cycle integrates plant
growth-defence processes, sustaining energy and photosynthetic demands
(Noor et al., 2010; Sweetlove et al., 2010) and the biosynthesis of secondary
metabolites. As carboxylic acids can fluctuate in response to environmental
perturbations (Araujo et al., 2012), the asymmetric distribution of TCA
intermediates observed in our study may correspond to an “open-flux” mode
which regulates plant energy metabolism and redox balance (Gardeström et
al., 2002; Igamberdiev & Eprintsev, 2016), as also suggested by the increase
in the antioxidants metabolites ascorbic and dehydroascorbic acid (Noctor &
Foyer, 1998; Smirnoff, 2011) (Fig.28c; and Tab.3 in Paper IV).

52
 PC2 (12%)
PC2 (12%)

PC1 (25%)

LV1 (17%)

Figure 28. Leaf ontogeny shapes the plant herbivore-induced responses.

Responses across leaf ontogeny relative to changes in secondary metabolism PCA
model (five components; R2Xcum = 62%, Q2cum = 16%). Score plots colored
according to (a) treatments and (b) leaf position (L1-L5). (c) Effect of MeJA on
primary metabolism of young leaves. OPLS-DA loading plot (1+1+0; R2Xcum = 50%,
R2Ycum = 88%, Q2cum = 75%), comparing shifts of primary metabolites in controls
and MeJA-treated B. nigra for the “top” leaves (L1-L3). Plant leaves were divided in
ontogenic groups, according to the patterns of secondary metabolism. See Fig.4g and
Fig.5 in Paper IV. Treatments codes as in Fig.23-26.

53
Overall, we have shown how herbivore-induced responses regulate both
primary and secondary metabolism, indicating an active reprogramming of
growth-defence processes. Treatments with MeJA enhanced the plant
resistance against caterpillars and resulted in increased accumulation of
GSLs. The ontogenic patterns observed in leaves were also in agreement with
the “optimal defence theory” (Mckey, 1974; Meldau et al., 2012), which
predicts that plants prioritize defences in precious tissues with high fitness
value, such as young leaves and reproductive organs (fruits and flowers).

Herbivory also induced VOC emissions (Tab.2 in Paper IV) mainly as GSL
degradation products (e.g. ITCs and nitriles), but also terpenoids and GLVs.
The plant VOC profile was not influenced by the MeJA application, which
only increased the emissions of 1,1'-bicyclopentyl-2-one and 1-pentenol (JA
metabolites; Schaller & Stintzi, 2009). Combined, MeJA and VOCs released
from herbivore-attacked plants can indirectly protect the plants, functioning
as airborne signals which attract herbivore parasitoids, or travel to
neighbour plants and prime their JA-mediated defences (Farmer & Ryan,
1990; Karban et al., 2014; Balmer et al., 2015; Lortzing & Steppuhn, 2016).
On the other hand, these same VOC emissions can also attract the herbivore
butterflies.

Oviposition on leaves induces similar JA-mediated effects, which prime

plant defences against future caterpillar herbivory (Lortzing & Steppuhn,
2016). P. brassicae butterflies usually oviposit on expanded mature leaves,
but late instar caterpillars move upwards to feed on young leaves and flowers
(Lucas-Barbosa et al., 2013; Bruinsma et al., 2014). Interestingly, in this
study, herbivore-induced responses were increasingly prioritized towards
younger leaves which induced large changes in secondary metabolites as well
as in central energy pathways, such as sugar metabolism and TCA cycle.
Young leaves also displayed increased levels of chlorophyll after 72 hours of
MeJA application (see Fig.5b in Paper IV). Although this result sets in
contrast with the commonly reported suppression of photosynthesis induced
by herbivory (Zangerl et al., 2002; Bilgin et al., 2010; Halitschke et al., 2011;
also observed in Paper II, relative to fully developed leaves), it also supports
the hypothesis that herbivore-induced responses help the plant to prioritize
both defence and growth processes in those young developing leaves that are
quickly shifting from sink-tissues to future sources of photosynthesis.

54
4. Conclusions

4.1 The “Butterfly effect”

As a result of climate change, environmental factors are shifting very rapidly.

The human impact on the planet is now so strong that we can speak about
the Anthropocene as a new epoch in the Earth history (Steffen et al., 2011).
By the end of this century, regional concentrations of O3 are predicted to
exceed 40-70 ppb (Sitch et al., 2007), while O3 damage to agriculture today
already accumulates to several billions of euros per year (Tiwari et al., 2016).
Increasing temperatures are also affecting ecosystems world-wide, forcing
plants and insects to migrate to new habitats (Morriën et al., 2010).

During plant-insect interactions, environmental pressures can have strong

impact on both plants and insects (Edger et al., 2015) but induced metabolic
changes in the plant are the major forces determining the success of either
the host or the herbivores (Foyer et al., 2016). Although plant responses to
single biotic and abiotic stresses usually induce unique metabolic signatures,
responses to multiple stresses can be very difficult to predict (Mittler, 2006;
Suzuki et al., 2014). Unpredictability is common to complex systems (Orrell
2010, Capra & Luisi 2014) where small perturbations in the initial conditions
trigger vast downstream effects with unknown consequences (Lorenz, 2015).

In this thesis, I showed how multiple stresses alter plant metabolism and
defence against insects, but also how herbivore-induced responses can affect
the plant adaptation towards other concurrent stresses (Fig.29). Although
single metabolite levels changed unexpectedly and in unique ways (Table 7),
it was possible to identify more predictable response patterns which emerged
from the shifts of the entire metabolome. Thus, the metabolomics approach
appears to be especially helpful when the overall complexity of the system
increases, for instance during concurrent O3 and herbivory (Paper I- II), or
during plant interaction with multiple herbivore insects, such as aphids and
caterpillars (Paper III). Particularly, O3 stress induced metabolic changes
in the host-plant which affected the caterpillar development (Paper I).
However, herbivory also interfered and redirected the initial plant response
towards O3 stress (Paper II). Plant responses also differed according to the
kind of herbivory between caterpillars and aphids, and were dependent on
the density of the attacker (Paper III).

55
 Metabolomics highlighted the link between growth and defence responses,
which correlated to plant processes of photosynthesis, leaf development, and
resistance against herbivory (Paper I-IV). The integration of multiple layers
of biological information via metabolomics and transcriptomics enabled to
predict actual physiological stress responses and to anticipate the occurrence
of visible symptoms in leaves (Paper II). Combining metabolomics with
manipulation of the plant-herbivore system using hormone elicitors (MeJA)
or genetic mutants (Balmer et al., 2015) further provided insights into the
mechanisms of central metabolic regulation during plant-insect interactions
(Paper IV). I believe that when this systems approach will be applied and
fully integrated to biotechnology and breeding programs (Weckwerth, 2011),
metabolomics will rise as an essential element for studying plants and agro-
ecosystems in constantly changing environments.

Figure 29. The “Butterfly effect”. Overview of how combination of abiotic and
biotic stresses can shape B. nigra metabolism and its interaction with P. brassicae.

56
Table 7. Abiotic and biotic stress interaction on B. nigra metabolites.

Function Class Metabolite Stress interaction

O3 + caterpillars

Direct defence Glucosinolate Sinigrin Negative

Direct defence Glucosinolate Glucoraphanin Positive
Direct defence Glucosinolate Neoglucobrassicin Positive
Direct defence Hydroxycinnamate 1-O-sinapoylglucose Positive
Direct defence Flavonol glucoside Quercetin-3-sinapoylsophoros. Negative
Central metabolism Sugar Maltotriose Positive
Central metabolism Fatty acid Glycerol Negative
Induced defence Fatty acid α- Linolenic acid Positive
Stress signalling Amino acid GABA Positive
Stress tolerance Redox Threonic acid Negative
Indirect defence VOCs Furfural Negative
Indirect defence VOCs Butenenitrile Positive
Indirect defence VOCs Allyl-isothiocyanate Positive
Indirect defence VOCs Dodecanal Neutral
Indirect defence VOCs E-DMNT Neutral
Aphids + caterpillars

Direct defence Glucosinolate Sinigrin Neutral

Direct defence Glucosinolate Glucoiberin Positive
Direct defence Glucosinolate Glucobrassicin Positive
Direct defence Glucosinolate 4-methoxy-glucobrassicin Positive
Central metabolism Sugar Fructose Negative
Central metabolism Sugar Maltose Positive
Stress tolerance Sugar Trehalose Neutral
Stress tolerance Redox Glutathione (GSH) Negative
Induced defence Fatty acid α- Linolenic acid Positive
MeJA + caterpillars

Direct defence Glucosinolate Sinigrin Positive

Direct defence Glucosinolate Gluconapin Positive
Direct defence Glucosinolate Glucoerucin Positive
Central metabolism Amino acid Phenylalanine Neutral
Central metabolism Amino acid Tryptophan Positive
Central metabolism Sugar Fructose Positive
Central metabolism Sugar Maltotriose Positive
Central metabolism TCA Citric acid Neutral
Central metabolism TCA cis-Aconitic acid Positive
Central metabolism TCA Malic acid Positive
Central metabolism TCA Fumaric acid Neutral
Indirect defence VOCs Allyl-isothiocyanate Neutral
Indirect defence VOCs 1,1'-bicyclopentyl-2-one Positive
Indirect defence VOCs 1-pentenol Positive

Potential interaction between herbivore-induced responses and other biotic and abiotic stress responses,
estimated combining information from all studies presented this thesis. Sequential stresses may reinforce
(positive), counteract (negative), or have no effect (neutral) on single metabolite levels in B. nigra.

57
5. Acknowledgements
I want to thank my supervisor Benedicte Albrectsen, for offering me this
research project and introducing me to the chemical ecology community.
Thank you for your support in these years, for the freedom and the trust.
Thanks to my co-supervisor Thomas Moritz, for metabolomics mentorship
and for being able to provide calm and stability in any difficult situation.
Thanks to Göran (Slim) Samuelsson and Stefan Jansson for your wisdom.

Thanks to the Swedish Metabolomics Centre for the great organization and
nice environment, especially: Jonas Gullberg, Annika Johansson, Maria
Ahnlund, Jenny Hällqvist, and Inga-Britt Carlsson. Particularly, I want to
thank Ilka Abreu for her help with LC-MS/Orbitrap, and Krister Lundgren
for all the time spent teaching me LC-MS and GC-MS, and learning together
the thermal desorption. May thanks also to Hans Stenlund and Rui Pinto, for
help with Matlab and SIMCA, respectively. Thank you all for your patience!

Thanks to many people from the chemistry department, who provided help
and support. Thank you especially to Mátyás Ripszam for your dedication to
the VOCs analyses, and to Peter Haglund for the support, and for providing
the TD-GC-TOF-MS. Thank you to Marcus Carlsson and Per-Anders Enquist
for assisting in the lab during the identification of the unknown metabolites,
a task which proved to require possibly other extra five years of PhD.

Thanks to Anastasia Matrosova and Zsofia Stangl for patiently teaching me

how to use the Li-COR, and to Manuela Jurca, Maria Eriksson, and Vaughn
Hurry for extra support and for borrowing the instruments. Thanks to Alex
Makoveychuk for help with tissue cultures, to Rosìe Forsgren for the
autoclaving and glassware work, and to Jenny Lönnebrink and the
greenhouse staff, for their friendly manners and professionality.

Thank you to all my current and previous lab members for the discussions,
technical and psychological support, and for all the good time, fika, lunches,
beers, and fun. In order of featuring inside this five-year-long Phd movie:
Franzi, Vicky, Ken, Tiggy, Abu, Tristan, Ylva, Johan, Barbara, Mona, Karen.

Thanks to the members of the Umeå Plant Science Centre, all of you are
creating such a wonderful environment. There is no space for mentioning
everyone, so I will especially thank the PhD students involved in teaching
course lectures and laboratories with me: Jimmy, Daria, Abdel, Christoffer,
Noemi, and particularly Vicky and Bernard and all the students that
participated in course of “molecular ecology” and worked on B. nigra.

58
Thanks Katharina and Tomas, who strongly recommended me UPSC in 2011.
Thanks to the European Science Foundation and EUROCORES Programme
who funded my research within the A-BIO-VOC/EuroVOL and allowed me
to have wonderful experiences in international conferences and meetings.
I want to thank my collaborators for their help and for all the nice moments:
James Blande, Tao Li, Timo Oksanen, Erik Poelman, Rieta Gols, Marcel
Dicke, Philippe Reymond, Eliezer Khaling, Christelle Bonnet, Camille
Ponzio, Foteini Pashalidou, and Holger Danner.

Sport and nature helped incredibly much in keeping my mind stable.

Thank you Benjamin who told me about Tomtebo kolonilotten, and to
Bernard & Gosia for spending precious time with me, mud, and mosquitos.
Thanks to Daniel M., Daniel E., Josefin, and Fra, for bouldering with me.
Thanks to IKSU simning, Stöcke TS Järne, and Umeå Parkourförening, and
the UPSC bandy group, and especially Sacha and Thomas for organizing it.

Thank you to all my nice flat mates for all the funny moments spent together.
Gaia, Andreas, Rogier, Victor, Isa, Daniel, and Ioulia. Thanks to all the nice
people that created very special moments and atmospheres: Ewa & Fede,
Mattias, Elina, Anders, and the Folketsbio. Søren & Leyla, Noemi & Daniel,
Karen & Marielle, Gosia & Bernard, Anne & Martin, Claudia & Mátyás,
Veronica & Audis, Anne-Sofie & Alex, Viveca & Szilard, Noalie, Eric,
Hermann, Bianca, and LiquidSky, Thanks also to Andreas R. for a proper
Italian carbonara with fruity Tasmanian black pepper.

Thanks to my family and friends, I miss you constantly: Fra, Leo, Marci, Ari,
Eva, Laura, Tommi, Lupo, Marco, Marco (Flip), and Lorenzo. Thanks Piero
for your time in Milano and for sharing passion for science. Thank you
Hossein for your help, and for your hospitality in San Francisco. Thank you
Dan and Holger for the great times in L.A., Joshua Tree park and Yosemite.
Thanks Jorge and Stefano in CPH for your hospitality, great times and great
beers. Thanks to Kirstin & Tommi and children, from Perth to Karlskoga.
Thanks to Mark & Iris and little Vasco.

A special thanks to my parents. You are always there for me. Grazie.
Per avermi sostenuto in lunghi anni di scuola e universitá, per la vostra
gentilezza e il vostro amore, il tempo dedicato al lavoro e alla famiglia,
e per avermi trasmesso amore per la cultura e rispetto per il prossimo.
Non so come possa ripagarvi, e vi quindi vi dedico questa tesi 

Sist men inte minst, tack Ann-Grett & Douglas, och hela Josefins familj, för
er varma gästfrihet och vänlighet; och tack Josefin, för att du finns, du är den
viktigaste personen i mitt liv. Jag älskar dig ♥

59
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 Websites and software

AraNet (v2). Database for probabilistic co-functional gene network association

for the model species Arabidopsis thaliana.
http://www.inetbio.org/aranet/

Cytoscape. Open platform for visualizationof complex networks.

http://www.cytoscape.org/

ePlant. Visualization tool for multiple levels of plant data.

https://bar.utoronto.ca/eplant/

GeneMANIA. Database for gene-sets functional association network analyses.

http://genemania.org/

KaPPA-View4. Database for representation and pathway analysis of gene

and metabolite correlation networks.
http://kpv.kazusa.or.jp/

Kyoto Encyclopedia of Genes and Genomes (KEGG). Database for

understanding of high-level functions in biological system, such as the cell,
the organism and the ecosystem.
http://www.genome.jp/kegg/

Mapman. Visualization of expression profiling data sets.

http://mapman.gabipd.org/home

The Arabidopsis Information Resource (TAIR). Database of genetic and

molecular biology data for the model plant species Arabidopsis thaliana.
https://www.arabidopsis.org/

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