Genetic and Molecular Mechanisms Controlling Reactive

Doctoral Thesis
Department of Botany
Stockholm University
Sweden 2006
Genetic and Molecular Mechanisms Controlling Reactive
Oxygen Species and Hormonal Signalling of Cell Death in
Response to Environmental Stresses in
Arabidopsis thaliana.
Per Mühlenbock
1
© Per Mühlenbock, Stockholm 2006
ISBN 91-7155-344-4
[email protected]
Printed by US AB, Stockholm, Sweden 2006
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Preface
This thesis is based on the following papers:
1
LESION SIMULATING DISEASE 1 is required for acclimation to
conditions that promote excess excitation energy. (2004) Mateo A,
Mühlenbock P, Rusterucci C, Chang CC, Miszalski Z, Karpinska B, Parker
JE, Mullineaux PM, Karpinski S. Plant Physiol. 136; 2818-2830
2
Controlled levels of salicylic acid are required for optimal
photosynthesis and redox homeostasis (2006) Mateo A, Funck D,
Mühlenbock P, Kular B, Mullineaux P M, Karpinski S. J.Exp. Bot. 57;
1795-1807.
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Redox changes in the chloroplast initiate ethylene dependent signaling
controlled by LESION SIMULATING DISEASE1 in Arabidopsis (2006)
Mühlenbock P, Mateo A, Baudo M, Kleczkowski L A, Karpinska B,
Mullineaux P M, Parker J E, Karpinski S. (Submitted manuscript)
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Lysigenous aerenchyma formation in Arabidopsis thaliana in response to
hypoxia is controlled by LESION SIMULATING DISEASE1 (2006)
Mühlenbock P, Mellerowicz E, Karpinski S. (Submitted manuscript)
Reprint of paper 1 is copyrighted by the American Society of Plant Biologists and is
reproduced with their kind permission. Reprint of paper 2 is supported by copyright
agreement with Oxford University Press.
My contributions to the papers were: (1) Major participation in planning, experimentation,
data analysis and writing. Performing experiments relating to gas exchange and ROS
production in mutants and unpublished supportive experiments. (2) Participating in planning,
experimentation, data analysis and method development and a major participation in writing.
Performing experiments relating to gas exchange and GR activity. (3) Planning, performing
and executing a major part of the included and supportive experiments. Writing the paper and
performing data analysis in cooperation with co-authors. (4) Performing all experimentation.
Planning, data analysis and writing was done in cooperation with (E.M and S.K) and by
supervision (S.K) of co-authors.
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Table of Contents
Abbreviations ............................................................................................................................. 6
Summary .................................................................................................................................... 7
Introduction ................................................................................................................................ 9
1. Stress .................................................................................................................................. 9
2. Photosynthesis and excess light stress ............................................................................... 9
2.1 The photosynthetic apparatus..................................................................................... 10
2.2 Photooxidative stress.................................................................................................. 10
2.3 Reactive oxygen species............................................................................................. 11
2.4 Antioxidant systems ................................................................................................... 12
3. Cell death in plants........................................................................................................... 14
3.1 The nature of cell death.............................................................................................. 14
3.2 Regulation of cell death.............................................................................................. 15
4. Hormonal regulation of stress responses.......................................................................... 16
4.1 Ethylene...................................................................................................................... 16
4.2 Salicylic acid .............................................................................................................. 17
4.3 Auxin.......................................................................................................................... 18
5. PCD mutants in Arabidopsis ............................................................................................ 18
6. Thesis aim and hypotheses............................................................................................... 20
Results and discussion.............................................................................................................. 21
The LSD1 node of stress................................................................................................... 21
7. LSD1 integrates chloroplastic EEE signals ...................................................................... 21
8. Salicylic acid signalling inhibits EEE acclimation .......................................................... 22
9. LSD1 controls stress ethylene .......................................................................................... 25
Cell Death......................................................................................................................... 26
10. SAA is associated with HR-like cell death .................................................................... 26
11. LSD1 regulates auxin in EEE-CD .................................................................................. 29
12. Aerenchyma formation in Arabidopsis .......................................................................... 31
13. The LSD1 node regulates a variety of environmental stresses....................................... 33
14. Conclusions .................................................................................................................... 36
Future perspectives................................................................................................................... 37
Materials and methods ............................................................................................................. 38
Acknowledgements .................................................................................................................. 41
References ................................................................................................................................ 42
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Abbreviations
ACC: 1-aminocyclopropane-1-carboxylic acid; Ethylene precursor
APX: Ascorbate peroxidase
AsA: Ascorbic acid
CAT: Catalase
CRE: Cis-acting regulatory element
EEE: Excess excitation energy
EEE-CD: Excess excitation energy induced cell death
EDS1: Enhanced disease susceptibility 1
EIN2: Ethylene insensitive 2
ELIP: Early light induced protein
ET: Ethylene
GR: Glutathione reductase
HR: Hypersensitive response
IAA: Indole-3-acetic acid; Auxin
JA: Jasmonic acid
LSD1: Lesion simulating disease response1
PAD4: Phytoalexin deficient 4
PCD: Programmed cell death
PET: Photosynthetic electron transport
PFD: Photon flux density
PQ: Plastoquinone pool
RCD: Runaway cell death
RSC: Relative stomata conductance
R.G: Restricted gas exchange
ROS: Reactive oxygen species
RuBisCo: Ribulose-bisphosphate carboxylase
SA: Salicylic acid
SAA: Systemic acquired acclimation
SAR: Systemic acquired resistance
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Summary
In the present work, the regulation and mechanisms of cellular responses, such as cell death
and signalling of systemic acquired acclimation (SAA), in response to environmentally
induced oxidative stress in Arabidopsis are characterized. We used the lesion simulating
disease1 (lsd1) mutant as a genetic model system that is deregulated in light acclimation and
programmed cell death (PCD). In this system, we identify that the redox status of the
plastoquinone pool signalizes in both light acclimation and cell death responses, controlled by
LSD1, EDS1, EIN2 and PAD4. We also show that these genes regulate not only cellular
homeostasis of salicylic acid (SA), but also ethylene (ET), auxin (IAA), and reactive oxygen
species (ROS). Furthermore we propose that the roles of LSD1 in light acclimation and in
restricting pathogen-induced cell death are functionally linked. Through its regulation, LSD1
influences the effectiveness of photorespiration in dissipating excess excitation energy (EEE).
The influence of SA on plant growth, on acclimation to EEE, and on the cellular redox
homeostasis of Arabidopsis thaliana leaves is also assessed. We demonstrate that maintained
SA homeostasis is required for optimal photosynthesis and acclimation to EEE. Plants with
deregulated SA synthesis were shown to be impaired in acclimation to moderate EEE and
thus predisposed to photooxidative stress. Low and high SA levels were strictly correlated to
H2O2 and glutathione contents in foliar tissues. These observations implied an essential role of
SA in the light acclimation processes and in regulating the redox homeostasis of the cell. We
also show that cell death in response to EEE is controlled by specific redox changes of
photosynthetic electron transport carriers that normally regulate EEE acclimation. These
redox changes regulate production of ET that signals through the EIN2 gene and its associated
regulon. In the lsd1 mutant, we find that propagation of cell death depends on the plant
defence regulators EDS1 and PAD4 operating upstream of ET production. We conclude that
the balanced activities of LSD1, EDS1, PAD4 and EIN2 regulate chloroplast dependent
acclimatory and defence responses. Furthermore, we show that Arabidopsis hypocotyls form
lysigenous aerenchyma in response to hypoxia and that this process involves H2O2 and ET
signalling. We find that formation of lysigenous aerenchyma depends on LSD1, EDS1 and
PAD4 operating downstream of metabolic signals. Conclusively, we show that LSD1, EDS1
and PAD4, in their functions as major plant redox and hormone regulators provide a basis for
fundamental plant survival in field conditions.
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Introduction
1. Stress
Stress affects all living organisms and can be defined as a disadvantageous influence on an
organism that affects factors such as health, fitness, growth, and survival. The stress tolerance
of an organism depends, in part, on its evolutionary and genetic capacity for adaptation and,
in part, on individual life history. Plants are due to their constitution unable to escape from
environmental stress and are constantly at the risk of succumbing to one or several stress
factors. Therefore in the natural environment, the plant cell must integrate a variety of stress
signals with metabolic processes and prioritize its response according to the prevailing
conditions. Fine control of cellular redox homeostasis is needed to prevent overload of free
radicals such as reactive oxygen species (ROS) due to environmental stresses. It is also crucial
for a plant that the inter- and intracellular regulation of metabolism and signalling pathways
are in tune and that responses are speedy and accurate. In order to cope with these challenges
plant defences and acclimatory responses both rely on signalling mechanisms of hormones,
ROS and other plant specific signalling substances (Pastori and Foyer 2002; Vranova et al.
2002; Karpinski et al. 2003; Bechtold et al. 2005; Bostock 2005).
2. Photosynthesis and excess light stress
One of the major fluctuating factors in the natural environment is the amount of incident light.
The amount of absorbed light energy in excess of what is needed for photosynthetic
metabolism, termed excess excitation energy (EEE), causes increased production of ROS
(Demmig-Adams and Adams 1992; Mullineaux and Karpinski 2002). EEE avoidance
mechanisms have evolved on several functional levels in plants. In order to avoid EEE most
plants are able to perform taxis of leaves and chloroplasts, and to produce compounds that
function as sunscreens (cuticular waxes and protective pigments) (Jarillo et al. 2001; WinkelShirley 2002; Long et al. 2003; Esmon et al. 2005; Vanderauwera et al. 2005). When these
mechanisms fail, the light that is absorbed by the plants chlorophyll may, due to limitations of
the photosynthetic apparatus, contribute to environmental stress.
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2.1 The photosynthetic apparatus
In photosynthesis, light energy is converted into biochemical energy in the form of organic
compounds. This reaction starts in the photosynthetic machinery of the chloroplast which is
composed of large protein complexes with light harvesting antennae (PSI and PSII) and a
series of energy transfer proteins. At central positions in the PSI and PSII there are reaction
centres that with the aid of certain wavelengths of light (700 nm for PSI and 650-680 nm for
PSII) release excited electrons. The electrons are re-supplied by the water splitting complex at
PSII that cleaves molecular water, yielding 4 electrons, 4 protons and O2. The excited
electrons are finally transferred through the photosynthetic electron transport (PET) chain by
e.g. plastoquinone pool (PQ) to PSI. Here a second portion of light energy is used to produce
NADPH. The protons from the split H2O create a proton gradient that is used to create ATP.
Then, the NADPH and ATP are used in the Calvin cycle to reduce CO2, thus creating more
stable forms of biochemical energy in the form of sugars and other carbohydrates.
2.2 Photooxidative stress
When plants are exposed to increased light intensities, an overreduction of the photosynthetic
electron carriers may occur. Then, the excitation energy that is absorbed by the light
harvesting complexes leads to increased production of electrons from the water splitting
complex, and a subsequent formation of ROS (Asada 1999; Karpinski et al. 1999). EEE
usually results from increasing light intensities, but it can also be generated at low light
intensities during different stresses which limit CO2 supply, such as drought, chilling or
freezing stress and avirulent pathogen infection (Mullineaux and Karpinski 2002). The
ensuing production of ROS may provoke photoinhibition (decreased efficiency in
photosynthetis) or if prolonged, also permanent photodamage (Karpinski et al. 1999; Niyogi
1999). The purpose of light acclimation is therefore mainly focused on avoiding and reducing
EEE. The main EEE dissipatory processes can be divided into either photochemical or
nonphotochemical quenching processes. Nonphotochemical quenching (NPQ) processes
directly quench EEE and disperse it in the form of heat with the help of xanthophylls and
other carotenoids. The photochemical quenching processes provide alternative electron sinks
along the PET, mainly through the Mehler reaction, water-water cycle and photorespiration
(McPherson et al. 1993; Foyer 1996; Willekens et al. 1997; Asada 1999; Ort 2001;
Mullineaux and Karpinski 2002). Photorespiration and the water-water cycle lead to a higher
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production of H2O2 in the peroxisome and the chloroplast, respectively (Willekens et al. 1997;
Asada 1999; Mateo et al. 2004).
Long term acclimation processes in leaves consist of reduced amount of light
absorbance by reduction of the PSII antennae, increased levels of the antioxidants glutathione
(GSH) and ascorbate (AsA), (Willekens et al. 1997; Bailey et al. 2001; Karpinski et al. 2003;
Tausz et al. 2004; Walters 2005), increased levels of the components of PET and induction of
stress proteins such as the ELIPs (Heddad and Adamska 2000; Walters 2005; Becker et al.
2006). Distant parts of the plant that are not directly exposed to EEE also induce acclimation
responses by increasing antioxidant defences and by inhibiting photosynthesis. This response
is termed systemic acquired acclimation (SAA) (Karpinski and al. 1999). Stomatal
conductance, which is delicately regulated by e.g. light, is particularly important in the
regulation of photosynthesis and responses to EEE. This is because the enzyme that catalyzes
the first reaction of the Calvin cycle, RuBisCo, has a high affinity to O2. Closure of stomata
can therefore induce photorespiration by limiting the availability of CO2 in comparison to O2
(Wingler et al. 2000; Noctor et al. 2002; Fryer et al. 2003). Because of this, many enzymes of
the Calvin cycle are regulated by redox changes in the PET. (Kaiser 1979; Paul and Foyer
2001).
Plants perceive EEE stress through different redox sensors and photopigments
(Escoubas et al. 1995; Pfannschmidt et al. 1999; Mullineaux and Karpinski 2002; Karpinski
et al. 2003). Redox changes in the PQ of PET seems to be more dominant in the fast
responses to EEE, whereas pigments such as phytochromes regulate slower processes such as
germination, plant circadian rhythms, shadow avoidance, seasonal acclimation and
development (Escoubas et al. 1995; Fankhauser and Chory 1997; Pfannschmidt et al. 1999;
Smith 2000; Karpinski et al. 2003).
2.3 Reactive oxygen species
Due to photosynthetic activities on earth, oxygen (O2) concentrations in the atmosphere are
kept at ca. 21 %, and provide a basis for our continued existence. However, oxygen is not
altogether beneficial for life. It is absolutely required for aerobic metabolism, but reduction of
molecular di-oxygen is a risky process and frequently results in the formation of ROS that can
cause cellular damage.
Therefore in plants and in other aerobic organisms antioxidant
systems have evolved and different ROS are used as signalling molecules in basic cellular
processes. ROS are reduced derivatives of molecular O2 such as superoxide (O2.-), hydrogen
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1
peroxide (H2O2) and the hydroxyl radical (.OH). Singlet oxygen ( O2) can be formed in
reaction with excited chlorophyll (3Chl) and has a specific electron spin combination of two
electrons on the outer suborbitals that allows for acceptance of a new electron and it is
therefore usually considered as a ROS. ROS are highly reactive molecules and may interact
with a wide variety of other molecules such as DNA, pigments, lipids, proteins and other
essential cellular molecules which leads to a destructive chain of events (Lamb and Dixon
1997).
ROS are formed as a direct consequence of diverse biochemical processes in
many subcellular compartments (Mittler et al. 2004; Gechev et al. 2006). Overreduction of
the respiratory chain leads to ROS in the mitochondrion (Maxwell 1999). Amine oxidases,
cell wall peroxidases and extracellular peroxidases are apoplastic ROS producers and are
involved in several stress responses (Allan and Fluhr 1997; Bolwell et al. 2001; Bolwell et al.
2002; Kawano 2003). The largest producer of ROS during the photoperiod in plant cells is the
chloroplast together with the peroxisome (Kozaki and Takeba 1996; Asada 1999; Foyer and
Noctor 2003). Therefore, in conditions where plants are exposed to EEE, they are also
challenged with a higher risk of overflowing both ROS dependent defence signalling and the
maintenance of healthy ROS steady state levels.
At low levels ROS perform important housekeeping functions (Foreman et al.
2003; Lam 2004; Mori and Schroeder 2004) but ROS are also the major players in processes
ranging from photobleaching and necrosis to defence responses and programmed cell death
(Van Breusegem et al. 2001; Neill et al. 2002; Blokhina et al. 2003; Dat et al. 2003; Laloi et
al. 2004; Gechev et al. 2006). Most environmental stresses may provoke a temporary increase
in ROS production (Karpinski et al. 1999; Desikan et al. 2001; Van Breusegem et al. 2001;
Pastori and Foyer 2002; Blokhina et al. 2003; Karpinski et al. 2003; Laloi et al. 2004; Mori
and Schroeder 2004). The ability of the plant to tolerate different levels of ROS is dependent
on the efficiency of the plants antioxidant systems and ultimately on metabolism (Wingsle
and Karpinski 1996; Yu 1999; Blokhina et al. 2003; Couee et al. 2006).
2.4 Antioxidant systems
Antioxidants can be generally divided into enzymatic, nonenzymatic and water or lipid
soluble. Carotenoids, xanthophylls and tocopherols are examples of low molecular weight
lipid soluble non-enzymatic antioxidants. Glutathione (GSH) and ascorbate (AsA) are the
major low molecular weigh water soluble antioxidants and make up the foundation of the
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redox control within the cellular compartments, since they provide the ability to scavenge
ROS (Foyer and Halliwell 1976; Asada 1999; Kiddle et al. 2003; Ball et al. 2004; Gomez et
al. 2004; Mateo et al. 2004). However, these compounds require recycling which requires
energy in the form of NADPH (Wingsle and Karpinski 1996).
Various enzymes that catalyze ROS scavenging reactions use AsA and GSH as
cofactors. These enzymes have different subcellular localizations and regulate the redox states
of the different subcellular compartments. In chloroplasts for instance, O2.- is an inevitable
by-product of photosynthesis that is effectively converted into H2O2 by superoxide dismutase
(SOD). H2O2 is then converted in a reaction with AsA by ascorbate peroxidase (APX) into
water and monodehydro- or dehydro-AsA thus preventing formation of the harmful hydroxyl
radical (.OH). AsA is regenerated by dehydro ascorbate reductase (DHR) with help of GSH
through the activity of glutathione reductase (GR) or directly with help of monodehydro
ascorbate reductase (MDR). The enzymes SOD, APX, MDR, DHR, GR are thus part of an
effective enzymatic ROS scavenging system that catalyzes the conversion of superoxide and
hydrogen peroxide to water, thereby minimizing the risk of ROS induced damages (Foyer et
al. 1994; Asada 1999; Mullineaux and Karpinski 2002). This pathway is regulated by the
redox status of the chloroplast and of pathways of carbon metabolism (Asada 1999).
Redox signals regulate processes like metabolism, morphology and development
(Foyer and Noctor 2003) and it has been suggested that cellular redox changes contribute to
the signal transduction that results from EEE stress (Karpinski et al. 1997; Karpinski et al.
1999; Karpinska et al. 2000; Foyer and Allen 2003; Ball et al. 2004). Redox homeostasis is
regulated by a tight equilibrium between ROS producing reactions and antioxidant defences
(Baier and Dietz 2005; Foyer and Noctor 2005). In redox reactions, cystein residues of
proteins play a very important function since their thiol groups can easily be oxidised or
reduced under physiological conditions (Cooper et al. 2002). Redox reactions between thiols
are the basis for the energy transfers of the antioxidant regeneration systems and they also
serve as regulators of gene expression and protein functions in several processes such as in
programmed cell death (Aravind and Koonin 2002; Cooper et al. 2002).
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3. Cell death in plants
Cell death occurs in various forms over a long scale of definitions throughout a plants life. At
the far end of one side of this scale is necrosis which is accidental cell death, caused by
overwhelming physical or chemical trauma. At the other end is programmed cell death (PCD)
which is basically cellular suicide instigated through a genetic program. Between these two
extremes are characteristically, morphologically and phenotypically overlapping varieties of
cell death (Ellis et al. 1991; Raff 1992; Vaux 1993; Greenberg and Yao 2004). PCD plays an
important role in plant development, defense and acclimatory responses.
3.1 The nature of cell death
Many environmental stresses may induce cell death symptoms. Abiotic stresses induce lesions,
physiological leaf spots, lysogenous aerenchyma and accelerated senescence (Gunawardena et
al. 2001; Buchanan-Wollaston et al. 2003; Wu and von Tiedemann 2004). The major part of
these types of cell death are considered to be necrotic but very few studies present data on this
topic (Gunawardena et al. 2001; Huh et al. 2002; Wu and von Tiedemann 2004). Aerenchyma
formation and accelerated senescence have been shown to be associated with attributes of
PCD in relation to abiotic stresses (Gunawardena et al. 2001; Munne-Bosch and Alegre 2004).
Biotic stresses induce cell death during development of disease symptoms or as the result of
specifically evolved plant pathogen interactions, so called gene-for-gene interactions (Lund et
al. 1998; McDowell and Dangl 2000).
Plants have evolved several means of defence against pathogen attack that
involve genetically induced signalling pathways leading to the formation of antimicrobial
compounds, strengthening of cell walls, stomata closure and programmed cell death
(McDowell and Dangl 2000). An example of plants’ biotic defences is the gene-for-gene
induction of the hypersensitive response (HR), a burst of ROS leading to the induction of
programmed cell death during which a limited number of cells die at the site of pathogen
infection (Lamb and Dixon 1997). This process is accompanied by a set of defence reactions,
including activation of defence genes and the onset of systemic acquired resistance (SAR)
(Lamb et al. 1989). The properties of HR vary between different plant-pathogen interactions
(Heath 2000) but HR has been proposed to be similar to apoptosis in animals with hallmarks
such as Ca2+ signals, extracellular production of ROS, activation of a genetic program, cell
shrinkage and DNA laddering (McDowell and Dangl 2000). However, comparative studies
rather point to that both disease development and HR responses have some degree of necrosis
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and PCD (Heath 2000; Beers and McDowell 2001; Dat et al. 2003; Greenberg and Yao 2004).
Additionally, not all kinds of PCD in animals are apoptotic and this range of phenotypes of
different kinds of cell death has lead to the idea of a continuous spectrum between PCD and
necrosis (Schwartz et al. 1993; Levin et al. 1999; Jones 2000). Then, why do plants have a
system to induce cell death in response to environmental stresses if the cells may die anyway?
HR and aerenchyma formation are both typical examples of the altruistic purpose of
environmentally induced PCD. HR is thought to prevent pathogens from proliferating and
aerenchyma supplies suffocating roots with O2 although the actual benefit of this has been
difficult to prove in vivo (Hammond-Kosack and Jones 1996). Alternatively, altruistic PCD in
animals proves that this process is responsible for removing cells that have become dangerous
or malignant, thereby contributing to an increased fitness of the organism (Jacobson et al.
1997; Gilchrist 1998; Johnstone et al. 2002). Importantly PCD also prevents necrosis and the
dangers of infection and toxicity that result from necrotic tissues (Davies 2000; Munne-Bosch
and Alegre 2004). There are many examples of altruistic cell death in developmental PCD
(e.g. flower senescence, xylem formation, developmental aerenchyma, aleuron and
endosperm cell death, dioic cell death etc.) and in PCD evoked as a result of householding
functions (root cap cell death, petal senescence etc.) (Jones 2001; Kuriyama and Fukuda 2002;
van Doorn and Woltering 2005). Since developmental cell death is outside of the scope of this
thesis, however, only environmental cell death will be considered further.
3.2 Regulation of cell death
Several environmental factors have been shown to influence the signalling pathways of PCD
(Gan and Amasino 1997). Studies have shown, that EEE may have a chloroplast-dependent
potentiating effect on plant PCD (Genoud et al. 2002; Gray et al. 2002; Samuilov et al. 2003;
Mateo et al. 2004; Zeier et al. 2004; Bechtold et al. 2005). ROS are necessary for the
induction of cell death pathways and plant respiratory burst homologues to the animal
NADPH oxidases have been shown to be active in the plant PCD pathways (Lamb and Dixon
1997; Hoeberichts and Woltering 2003; Van Breusegem and Dat 2006). ROS dependent
Cytochrome C release, a hallmark trait of animal PCD has also been shown in plants (Vacca
et al. 2006). Another molecule involved in programmed cell death is nitric oxide (NO), that
acts both as an oxidant and antioxidant in biotic and abiotic stress responses (Beligni and
Lamattina 2000; Delledonne et al. 2001; Beligni et al. 2002; Neill et al. 2002; Lamattina et al.
2003; Zago et al. 2006).
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Several hormones and signalling components such as ethylene (ET), salicylic
acid (SA), jasmonic acid (JA) and abscissic acid (ABA) have also been shown to control PCD
(Kangasjarvi et al. 2005; Overmyer et al. 2005). In one proposed model SA and ET have been
suggested to cooperate for the induction and spreading of the PCD. In other studies, also
auxins (IAA) have been shown to regulate cell cycle and cell death (Kovtun et al. 2000). The
proposed effect of light and of ET as agents contributing to the spreading of PCD mentioned
above has never been shown in correlation although it is known that ET can be induced by
several kinds of stresses (Mehlhorn and Wellburn 1987). Importantly, also redox signals play
a fundamental part in PCD (Foyer and Noctor 2005).
4. Hormonal regulation of stress responses
ABA, JA, ET, SA and IAA are key hormonal players in stress responses and there is a
considerable amount of crosstalk between the signal transduction pathways that they initiate
(Hirt 2000; Kovtun et al. 2000; Glazebrook 2001; Jonak et al. 2002; Bostock 2005).
Chloroplasts could be a site for this regulation since many stress response hormones are
produced entirely or partially in chloroplast located pathways (Wallsgrove et al. 1983;
Mauch-Mani and Slusarenko 1996; Creelman and Mullet 1997; Milborrow 2001; Mullineaux
and Karpinski 2002). ABA is a well documented inducer of stomata closure and regulator of
both abiotic and biotic defence responses (Bostock 2005).
JA is a signalling molecule derived from linoleic acid and the biosynthetic
pathway for this compound contains biologically active intermediates (Stintzi and Browse
2000; Kachroo et al. 2001). JA has roles in stress, development and PCD and interferes both
with ET and IAA (Tiryaki and Staswick 2002; Turner et al. 2002; Kangasjarvi et al. 2005).
However, it has recently been shown that the JA-mediated signalling is not involved in
regulation of PET dependent signalling (Chang et al. 2004).
4.1 Ethylene
ET plays vital roles in several aspects of plant growth and development (Johnson and Ecker
1998) and is a particularly important regulator of stress responses (Wang et al. 2002). It is
synthesized via a clearly defined and tightly regulated pathway that responds to several
developmental and environmental stimuli. Furthermore, ET has been reported to be an
important regulator of cell death such as in senescence (Hadfield and Bennett 1997),
aerenchyma formation (Drew et al. 2000), disease development (Lund et al. 1998) and in
ROS-induced cell death (Kangasjarvi et al. 2005). Additionally, ET interacts with SA
16
signalling (Ras 2002) indicating that it plays an important role also in biotic stress responses.
ET is tightly regulated together with ROS (Morgan and Drew 1997; de Jong et al. 2002;
Moeder et al. 2002; Kangasjarvi et al. 2005) and it has been shown that ET may be necessary
for H2O2 release during PCD and that it amplifies the oxidative burst in plant- pathogen
interactions (Lawton et al. 1994; Chamnongpol et al. 1998; Ge et al. 2000; de Jong et al. 2002;
Kangasjarvi et al. 2005). It was also reported that ethylene can inhibit photosynthesis (Kays
and Pallas 1980) and that ET signalling may be affected by nutrient status and leaf age (Legé
et al. 1997). These reports indicate an important function of ET in controlling crosstalk
between EEE and PCD signalling pathways.
4.2 Salicylic acid
SA is a compound that can be found in all parts of a plant where it has a diversity of functions
(Raskin 1995). It is a phenylpropanoid derived from phenylalanine from the Shikimate
pathway in the chloroplast (Mauch-Mani and Slusarenko 1996). SA is considered to be one of
the most determinative plant hormones in biotic interactions since it is required for SAR.
However, it has also been reported that biotic stress resistance is controlled by both SAdependent and SA-independent pathways, and that the SA-independent pathway may be
regulated by both JA and ET (Clarke et al. 2000; Overmyer et al. 2000).
The amplification of the oxidative burst of biotic defences is dependent on
increases in SA and it has been proposed that SA and ROS function as a feedback loop since
ROS are also involved in key steps of the SA synthesis (Draper 1997; Durner et al. 1997;
Lamb and Dixon 1997; Van Camp et al. 1998). Both SA and ROS are required to induce HR
(McDowell and Dangl 2000) and CAT and APX transcription have been shown to be
inhibited by SA. Consequently, a large variety of mutants have lesion phenotypes indicating
that there is a tight link between SA and general cell death responses (Durner et al. 1997;
Lorrain et al. 2003). Additionally, it has been shown that SA signalling is affected by EEE
and that SA effects both photosynthesis and stomatal conductance (Genoud et al. 2002;
Karpinski et al. 2003; Chaerle et al. 2004; Zeier et al. 2004) and it was shown that SA is
involved in long term light acclimatory processes (Karpinski et al. 2003). These observations
suggest that SA is in a central position of crosstalk between the signalling pathways of
acclimatory and defence responses.
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4.3 Auxin
Auxins, together with cytokinins, differ from other plant signalling molecules in that they are
required for cell viability. The prevalent form of auxin is IAA which is synthesised from
tryptophan through the action of several pathways. Auxin signalling takes place through
transportation and binding/release of active IAA, Ca2+ signalling, and proton gradients.
Recently, the enigmatic and illusive receptor for auxin was identified in Arabidopsis, a
significant advance for the study of this intriguing hormone (Kepinski and Leyser 2005).
Auxins are involved in responses such as cell elongation, stomatal opening, plant growth,
hypoxia responses, and root formation (Visser et al. 1995; Abel and Theologis 1996; Gehring
et al. 1998; Guilfoyle et al. 1998; Reed 2001). They are also involved in stress responses and
in the regulation of ROS homeostasis (Kovtun et al. 2000; Pfeiffer and Hoftberger 2001;
Cheong et al. 2002; Guan and Scandalios 2002; Pasternak et al. 2002; Winkel-Shirley 2002;
Joo et al. 2005). Moreover, auxins interact with ET signalling and are required for stress ET
production (Mehlhorn and Wellburn 1987; Romano et al. 1993). Importantly, auxins are also
required for cell cycle progression and recently it has been reported that they may control
PCD and biotic defence responses (Hirt 2000; Kuriyama and Fukuda 2002; Gechev et al.
2004; Xia et al. 2005).
The regulation of auxins illustratively exemplifies the complex situation of
homeostatic control of signals in plants. Depending on tissue, levels and environmental
signals, auxins and many other signalling compounds exert different effects in different
situations. It is therefore clear that a certain balance (homeostasis) of signalling molecules is
required for different cellular states or responses (Voesenek and Blom 1996; O'Donnell et al.
2003).
5. PCD mutants in Arabidopsis
In Arabidopsis, several mutants have been isolated for genes that are involved in hormone
signalling and regulation of PCD. Interestingly, several mutants affected in hormone
signalling are also affected in cell death responses (Overmyer et al. 2000; Rao et al. 2002;
Lorrain et al. 2003). Some mutants initiate cell death as the result of an external stimulus, and
mutants that overproduce SA or ET usually develop lesions or accelerated senescence at some
point during ageing (Greenberg and Ausubel 1993; Rate et al. 1999; Kirik et al. 2001;
Yoshida et al. 2002). The accelerated cell death (acd), radical induced cell death1 (rcd1) and
lesion simulating disease (lsd) (Overmyer et al. 2000; Rusterucci et al. 2001; Greenberg and
18
Yao 2004) are three reported examples of gene mutations where the induction of PCD results
in continuous and uncontrolled spread of lesions. ACD genes are controlling a SA dependent
PCD pathway (Greenberg et al. 2000). The RCD1 gene product is a negative regulator of
ozone induced (contributes to extracellular accumulation of ROS like in HR) programmed
cell death that also coordinates a hormone signalling network of SA, ET, JA and ABA
(Kangasjarvi et al. 2005). Here, SA was necessary for the initiation of the cell death and ET
for the continued spreading of the cell death (Overmyer et al. 2000).
The lsd1 mutants initiate spontaneous lesions in response to aging, changing
light conditions and when exposed to pathogens. The lesions propagate throughout the leaves,
subsequently killing most of the older leaves. Prediction of LSD1 structure reveals that it is a
189aa protein with zinc finger domains grouping it with the C2C2 class of transcription
factors (Epple et al. 2003). Recently it was also demonstrated that LSD1 has a thioredoxin
binding domain (Mateo 2005). Additionally, several of the plant metacaspases contain zinc
finger domains resembling those of LSD1 and it has been shown that LSD1 controls HR (Uren
et al. 2000; Rusterucci et al. 2001). Consequently, the LSD1 is a negative regulator of PCD
and a highly conserved paralogue of this gene has been found to positively control PCD
(Epple et al. 2003). In lsd1 mutants, extracellular accumulation of ROS precedes spreading of
lsd1 lesions and it has been shown that inhibition of SA synthesis was able to revert the lsd1
phenotype (Jabs et al. 1996; Aviv et al. 2002). Furthermore, ROS in combination with SA
was reported to induce lsd-like lesions in wild type plants (Mazel and Levine 2001). However,
lesions are also able to form in lsd mutants independently of SA (Hunt et al. 1997) and there
are differences between the induction of HR signalling and the uncontrolled cell death in lsd1
that may provide clues to how the spread of PCD is regulated (Kliebenstein et al. 1999;
Rusterucci et al. 2001; Torres et al. 2006).
The lipase (ENHANCED DISEASE SUSCEPTIBILITY 1) EDS1 and its
interacting lipase like partner (PHYTOALEXIN DEFICIENT 4) PAD4, were shown to be
needed for the initiation of unchecked cell death in lsd1 and are essential regulators of SAR
(Feys et al. 2005; Wiermer et al. 2005; Bartsch et al. 2006) . EDS1 forms several different
protein complexes with PAD4 and (SENESCENCE ASSOCIATED GENE 101) SAG101
(recent discovery), which constitute regulatory nodes for SA, JA and ET pathways (Wiermer
et al. 2005). During pathogen response these genes are active upstream of SA and are working
in a positive feedback loop of SA accumulation (Wiermer et al. 2005). In the lsd1 mutant they
seem to function in a positive feedback loop of ROS accumulation. The double mutants
19
lsd1/eds1 and lsd1/pad4 are reverted in the lsd phenotype, i.e. they do not form spontaneous
lesions or unchecked cell death (Rusterucci et al. 2001).
6. Thesis aim and hypotheses
A basic genetic framework for the regulation of plant responses to particular biotic and single
abiotic stresses has been established, mainly from analyses in Arabidopsis. Several key
regulators depending on hormones and ROS for each response have been cloned and in some
cases their proteins have been characterized. Definition of mutant defects in such responses is
normally done under highly controlled laboratory conditions. Approaches that isolate these
phenomena are necessary to elucidate the genetic framework but may contribute to a synthetic
view that some hormones and ROS are unique for different biotic interactions and that others
are specific for abiotic stresses. However, highly stable laboratory conditions do not represent
the multitude of signals that plants perceive in their natural environment and that ultimately
drive processes like defense, acclimation, stress tolerance and plant growth. The literature
presented here indicates that signalling pathways are not entirely separated and that plants
rather integrate multiple stress inputs, prioritizing their responses to the prevailing
environment. The aim of the work in this thesis is to address the issue of how plants integrate
signals by focusing on two major hypotheses.
The first hypothesis suggests that plants possess genetic systems that integrate
both hormone and ROS signalling of biotic and abiotic stress responses.
Literature on PCD in animal systems indicates that the basic control of
environmentally induced PCD is altruistic in the sense that it removes dangerous cells.
Therefore, processes that are fundamental to plant metabolism, such as light acclimation, most
likely are integrated into a PCD pathway since they affect the stability and viability of the cell.
The second hypothesis suggests that plants have evolved specific mechanisms that directly
integrate signals related to light acclimation with the control of PCD.
20
Results and discussion
The LSD1 node of stress
7. LSD1 integrates chloroplastic EEE signals
One of the responses common to both biotic and abiotic stress is that stomata close
(McDonald and Cahill 1999; Apel and Hirt 2004; Laloi et al. 2004; Desikan et al. 2005). This
may be a consequence of an EEE-induced ROS formation since H2O2 is involved in the
regulation of stomatal conductance (Zhang et al. 2001; Desikan et al. 2004). One of the major
EEE-dissipatory mechanisms in plants is photorespiration (Kozaki and Takeba 1996). This
process leads to a considerable amount of ROS production and is induced whenever cellular
levels of O2 increase or CO2 decrease (Wingler et al. 2000). The role of stomata in regulating
gas exchange is therefore integral in the regulation of foliar cellular redox status since the
limitation of gas exchange inevitably induces photorespiratory H2O2 accumulation (Karpinski
et al. 1999; Wingler et al. 2000). We found that lsd1 leaves had a lower stomatal conductance
than wild type leaves and that transferring low light acclimated lsd1 plants to high light
conditions induced unchecked cell death (Fig. 1A). This observation indicates that lsd1 is
defective in downregulating photorespiratory ROS. Artificially limiting gas exchange
provoked accumulation of ROS in wild type plants within 24h and was enhanced in the lsd1
mutant, initially without being associated with any visible lesion formation (Fig. 1C).
Subsequently, after 48h, the ROS accumulation induced uncontrolled cell death in the lsd1
mutant in local and systemic leaves (Fig. 1C). In eds1-1/lsd1 and pad4-5/lsd1 mutants H2O2
accumulation was also observed when limiting gas exchange, but it was lower in comparison
to that observed in lsd1. These data correlate with the observation that the lsd1 conditioned
cell death was blocked in these mutants. We also showed that LSD1 positively regulates
catalase (CAT) which is the main scavenger of photorespiratory ROS (Fig. 1B).
We
concluded that LSD1 functions not only as a negative regulator of pathogen defences but also
of EDS1- and PAD4-conditioned downregulation of the EEE acclimation response.
Furthermore we concluded that the formation of uncontrolled cell death in the lsd1 mutant is
dependent on chloroplast mediated ROS signalling and that it is a suitable genetic marker for
deregulated EEE acclimation (Paper1)(Mateo 2005).
21
Figure1 Effects of lower stomata conductance and forced limitation of foliar gas exchange in lsd1 are
reverted in pad4-5/lsd1and eds1-1/lsd1. (A) Relative stomatal conductance (RSC) and (B) CAT activity in
leaves of Ws-0, lsd1, pad4-5/lsd1, and eds1-1/lsd1 in short day (SD) permissive conditions (P < 0.001***, P <
0.05*) in lsd1 and the recovery of wild-type phenotype in the double mutants. (C) DCF-2 yellow-green
fluorescence (H2O2) monitored after 24 h treatment by limitation of foliar gas exchange. Runaway cell death was
observed in lsd1 but not in Ws-0 nor in pad4-5/lsd1 and eds1-1/lsd1 after 48 h. Representative pictures of treated
leaves are shown.
8. Salicylic acid signalling inhibits EEE acclimation
LSD1, EDS1 and PAD4 are essential regulators of SA signalling in response to biotic stresses
(Rusterucci et al. 2001; Aviv et al. 2002). However these genes also regulate acclimation to
EEE, indicating that the link between EEE and biotic defences may be controlled by SA
signals.
Mounting evidence supports that this may be the case since it was shown that SA levels
are regulated by abiotic stresses and EEE (Karpinski 2003). SA also causes stomatal closure
(Fig. 2A), inhibition of photosynthesis, long term acclimation and induces the uncontrollable
cell death in lsd1 (Jabs et al. 1996; Mori et al. 2001; Karpinski et al. 2003). Furthermore, in
some mutants, SA overproduction results in severe chloroplast dependent growth retardation
and sporadic cell death (Paper 2). In Paper 2, we showed that mutants that are inhibited in SA
production are unable to acclimate to EEE, underlining the importance of SA in acclimation
responses.
22
Figure 2 SA impairs acclimation to EEE in low light-acclimated plants.
(A) Relative stomatal conductance (RSC) in wild-type leaves of rosette grown in SD treated with SA (0.4
mM) in comparison to control leaves treated with water (p<0.001***). (B) Low light (LL)- and high light
(HL)-acclimated leaves treated with 0.4 mM SA for several hours and exposed to excess light (EL;
2200+ 200 uE, 90 min exposure).
Additionally, we showed that SA contributes to lesion formation in combination with EEE
and that acclimation to EEE can revert this lesion formation (Fig. 2B) (Paper 1). The growth
retardation in the SA mutants can be reversed by transferring plants to higher light intensities
(Paper 2) and high SA levels are correlated with reduced maximum efficiency of
photosynthesis and increased respiration rates in the leaves (Paper 2). These data indicate that
SA induces EEE related stress and photorespiration. Previously it has been shown that SA
may reduce ROS scavenging by inhibiting CAT (Chen et al. 1993). We therefore analysed
H2O2 production in several lines of mutants that overproduce SA and some that have reduced
levels of SA. This analysis showed that H2O2 production is high in SA accumulating mutants
and low in those that are SA deficient (Fig. 3A).
23
Figure 3. SA disrupts redox regulation.
(A) Mutants with constitutive accumulation of SA had strongly increased H2O2 levels and in SAdeficient lines H2O2 was decreased, indicating a strong correlation between SA levels and H2O2
content in the cell. (B) NADPH-dependent glutathione reductase (GR) activity. (a: significantly
different from wt, p<0.05).
In contrast to previous studies, we could not link the different ROS levels to the
activities of ROS scavengers (Paper 2). Instead we found a clear link between the levels of SA
and GSH through the regulation of glutathione reductase (GR) activity (Fig. 3B). Since high
levels of GSH were also associated with EEE acclimation (Karpinski et al. 1997), this
indicates that the observed increase in ROS may be due to EEE. Additionally, we and others
have shown that LSD1 is regulated by GSH (Senda and Ogawa 2004) and that the LSD1
protein, in turn regulates important enzymes such as CuZnSOD, CAT1 and NADPH
thioredoxin reductase (NTR) (Paper1)(Mateo 2005). The lsd1, eds1 and pad4 mutant can be
considered to be conditional regulators of SA since when the plant is faced with biotic stress
the gene functions are to inhibit (lsd1) or promote (eds1 and pad4) the SA pathway
(Kliebenstein et al. 1999; Wiermer et al. 2005). We propose that this is reflected in the
production of H2O2 in lsd1, eds1 and pad4 mutants when faced with EEE (Fig. 1) and
conclude that LSD1 constitutes a rheostat of EEE-induced ROS and redox signalling that
consequently contributes to the regulation of SA.
24
9. LSD1 controls stress ethylene
Earlier studies report that ET is involved in the regulation of stomatal conductance and
photosynthesis (Kays and Pallas 1980) and that it is involved in potentiating the oxidative
burst of PCD (Ge et al. 2000; de Jong et al. 2002; Tuominen et al. 2004). Furthermore ET
was shown to precede and enhance SA signalling during cell death and light acclimation
(Lawton et al. 1994; Chamnongpol et al. 1998) (Paper 3). 1-Aminocyclopropane-1carboxylate (ACC) is the immediate and rate limiting precursor for ethylene biosynthesis
(Adams and Yang 1979). We show that the exposure of plants to excess light cause an
increase of foliar ACC concentrations in both locally challenged and systemic leaves (Paper
3). Furthermore, ET signalling under excess light stress conditions may be dependent on the
redox status of the PQ pool, since levels of ACC and some genes of the EIN2 regulon are
regulated in correlation with the redox status of PET carriers (Paper 3). Excess light exposure
contributed to significant EDS1 and PAD4 dependent increases of ET levels in lsd1 leaves
compared to wild type plants (Fig. 4A). These data show that conditions that promote
photorespiration and regulate EEE acclimation also promote ET formation that is negatively
regulated by LSD1 and positively regulated by EDS1 and PAD4 (Fig. 4A). We verified this
experiment by crossing lsd1 and ein2-1 mutants. In lsd1/ein2-1, the cell death symptoms were
relieved in comparison to lsd1, indicating that the stress responses in lsd1 involve EIN2
signalling (Fig. 4B).
25
Figure 4 LSD1 controls EEE induced ethylene.
(A) Excess light treatment induces significantly higher production of ACC (direct precursor of
ethylene) in Ws lsd1 mutants than in Ws-0 wild-type (P < 0.05 *). (B) Representative pictures of Col0 , ein2, Col lsd1 and Col lsd/ein2. The lesion phenotype of lsd1 was partially reverted in lsd1/ein2
Cell Death
10. SAA is associated with HR-like cell death
In Paper 3 we show that EEE-induced acclimatory responses, like SAA are characterized not
only by redox changes in PET and antioxidant defenses but also are manifested by a specific
appearance of cell death. This cell death was characterized by the formation of microlesions
(spot like lesions made up of a few cells) when low light adapted Arabidopsis thaliana leaves
were exposed to excess light, so we chose to refer to these symptoms as EEE-induced cell
death (EEE-CD). EEE-CD was also detected in systemic tissues, indicating that the lesion
formation may be occurring through an active process rather than being a toxic effect. These
symptoms are functionally and phenotypically similar to the HR that is induced in association
26
with SAR. Limiting gas exchange, either by physically blocking stomatal pores or by spraying
leaves with ABA also induced EEE-CD (Fig. 5) and also caused uncontrolled spreading of
cell death following the induction of HR (Mühlenbock 2006). Accumulation of ROS was
detected prior to the formation of the cell death associated with limiting gas exchange
(Mühlenbock 2006) in all of these cases. This indicates that, while EEE can induce cell death
signals it also feeds into general parts of PCD pathways where it contributes to spreading of
the cell death. We concluded that EEE-CD is dependent on photorespiratory ROS and redox
signals originating from the chloroplast (Paper 1 and 3). In Paper 3 we conclude that LSD1 is
a negative regulator and that EDS1 and PAD4 are positive regulators of EEE-CD.
Figure 5 Limitation of gas exchange induces EEE-CD.
Representative trypan blue stained dead cells in leaves treated with 50 μM ABA and physical restriction
of gas exchange (R.G.) (C=control).
As EEE-CD is associated with ET signalling, we analysed levels of foliar ACC after
restricting gas exchange. This had no significant effect on ethylene levels in wild type plants
(Fig. 6A). In contrast, lsd1 mutants produced high levels of ACC (ca. 350-fold higher than
control plants within 24 h after restricting gas exchange) before exhibiting runaway cell death
(Fig. 6A). In addition, when cell death was induced in lsd1 leaves, strong fluxes in foliar ACC
concentration were observed (Fig. 6A). Analysis of the single eds1-1 and pad4-5 and eds11/lsd1 and pad4-5/lsd1 double mutants revealed that the increase of ACC in EEE-CD in lsd1
requires the defence regulators EDS1 and PAD4 (Fig. 6A). Thus, we conclude that EDS1 and
PAD4 operate upstream of ethylene production in the signalling pathway that propagates cell
death in lsd1 plants.
To test the dependence of ET in lsd1 conditioned EEE-CD we investigated if ET treatment
would contribute to the spreading of unchecked cell death in lsd1. Injecting lsd1 leaves with
27
100 µM ACC resulted in more than doubled levels of unchecked cell death compared to
control injections with dH2O (Fig. 6B). We also quantified the cell death in the lsd1/ein2-1
mutant in response to restricted gas exchange. This showed that the lsd1/ein2-1 mutants had
significantly reduced levels of unchecked cell death compared to lsd1 after 72 h and that the
cell death that is negatively controlled by LSD1 is downstream of EIN2 signalling (Fig. 6C).
Figure 6 Ethylene controls EIN2 dependent spread of runaway cell death in lsd1 mutants.
(A) Restricted gas exchange (R.G.) induces ACC production in leaves of Ws lsd1 but not wild-type (Ws0) after 24 h of treatment (p<0.001***). The eds1/ lsd1 and pad4/ lsd1 double mutants did not produce
ACC in the same treatments (B) Injection of 100 μM ACC solution into leaves resulted in increased
runaway cell death in Ws lsd1 leaves compared to leaves injected with water (after 48h) (p<0.001***).
(C) Lesion areas in leaves of Col-0, ein2-1, Col lsd1 and Col lsd/ein2 72 h after artificially restricting gas
exchange (p<0.001; a = compared to wild type; b = compared to lsd1).
We hypothesize that the observed effects of ET and SA signalling on cell death are reflected
in our results above in the following way. SA effectively raises the stress condition in cells
and thus effectively lowers the threshold for cell death (Paper 2). A stress signal that leads to
the production of ROS can therefore easily initiate cell death in an SA primed cell. The HR
observed during pathogen stress is induced by SA (Aviv et al. 2002). If the cell is not SA
primed the stress may initially not be high enough since the internal threshold to die is still too
28
high. This is likely to be the case in disease development were the increasing stress levels of
the disease eventually gives rise to PCD mixed with necrosis (Greenberg and Yao 2004). EEE
has a similar potential to induce a stress signal that surpasses this threshold in non SA primed
cells and gives rise to a cell death signal that is functionally similar to the propagation signal
of HR. Since EEE in combination with either HR or SA causes unchecked cell death we
propose that the propagation signal of HR and EEE-CD have the same origin. This is further
supported by our data showing that LSD1 not only regulates HR (Rusterucci et al. 2001) but
also EEE-CD and integrates both pathways for the control of PCD.
11. LSD1 regulates auxin in EEE-CD
When the spontaneous induction of unchecked cell death occurs in lsd1, the initial lesion
typically spreads extensively, resulting in cell death of the major part of the leaves. We
observed that the young leaves and the shoot apex had a higher resistance against the
spreading of the cell death and that the unchecked cell death in the lsd1 mutant was associated
with a change in leaf morphology (dorsoconvex curling) (Fig. 7A), which is an indication of
auxin signalling (Klee et al. 1987; Romano et al. 1993; Keller and Van Volkenburgh 1997).
This observation and previous studies that report an involvement of auxins in PCD and shoot
apical dominance (Romano et al. 1993; Gechev et al. 2004; Xia et al. 2005), prompted us to
compare foliar IAA levels in plants undergoing unchecked cell death. This analysis showed
that when cell death is spreading in lsd1 leaves, IAA concentrations increase (Fig. 7C). Our
investigation also showed that application of IAA inhibits the spread of the cell death in lsd1
leaves (Fig. 7B). Furthermore, higher levels of IAA in younger than in older leaves together
with an observed lower formation of lesions in young leaves indicated that foliar levels of
auxin could prevent spreading of the cell death (Fig. 7C). Our investigation also indicated a
complex involvement of LSD1, EDS1 and PAD4 in regulating levels of auxin (Fig. 7C). The
data indicated that there is an epistatic interaction between LSD1, EDS1 and PAD4 since only
lsd1/eds1 and lsd1/pad4 but not eds1 and pad4 mutants had significantly lower levels of IAA
(Fig. 7C). We propose that this interaction may contribute to a pleiotropic effect controlled by
IAA that induces increased stress tolerance in post-stress leaves. Importantly, we also found
that the EIN2 regulon contains auxin signalling genes, indicating that the IAA signalling
observed in the mutants may be regulated by ethylene (Paper 3).
29
Figure 7 LSD1, EDS1-1 and PAD4-5 control foliar IAA levels (A) In response to RCD in lsd1 a
dorsoconvex curvature was observed in young leaves. This is an indication of IAA signalling. (B) Spreading
of RCD was significantly reduced in lsd1 leaves treated with IAA (p<0.05*). (C) Young leaves had higher
levels of IAA than older leaves. In lsd1 an increase of IAA was detected in plants which were showing
signs of RCD (p<0.05*). In older leaves but not in young lower IAA levels in double eds1/lsd1 and
pad4/lsd1 mutants were observed in comparison to wild type plants (p<0.001***). In eds1 and pad4 basal
IAA levels were similar to those observed in wild type plants.
The first hypothesis in this thesis stated that plants possess integrated genetic
systems that are regulated by both hormones and ROS and that simultaneously regulate biotic
and abiotic stress responses. On the basis of the above presented results on regulation of
hormones and ROS by the previously established biotic defence regulators LSD1, EDS1 and
PAD4, this hypothesis is positively verified.
30
12. Aerenchyma formation in Arabidopsis
We have shown that LSD1 controls cell death signals by controlling ROS and ET and that
hypoxia can inhibit foliar cell death (Paper 1). In Paper 4 we show, for the first time, that
Arabidopsis hypocotyls form lysogenous aerenchyma in response to hypoxia (Fig. 8) and that
this cell death is preceeded by H2O2 and ET signals (Fig. 9A). Interestingly, we also found
that high light and long day conditions promoted aerenchyma formation (Paper 4). We
reasoned that the effects of light may have been associated with the observed early decrease in
stomatal conductance that was followed by gradually increased H2O2 levels (Fig. 9A).
Supporting this hypothesis, we found that also this type of cell death is controlled by LSD1,
EDS1 and PAD4 (Fig. 9B).
Figure 8. Arabidopsis makes aerenchyma in response to hypoxia.
Anatomy of root-hypocotyl axis in 12 week-old plants waterlogged for 6 and 7 days as seen in
ruthenium red stained cross sections. In secondary xylem, there is an inner zone with vessel
elements and axial parenchyma cells (arrowhead). This zone is magnified on a lower panel. Note
a disappearence of axial parenchyma cells between day 6 and 7.
31
The unchecked cell death in leaves of lsd1 did not enhance the aerenchyma
formation in the roots. Neither did the aerenchyma formation contribute to more unchecked
cell death in the lsd1 leaves. Interestingly though, the unchecked cell death produces a
pleiotropic effect in the lsd1 plants which subsequently completely blocks the competence for
aerenchyma formation in hypoxia. Since auxin is essential in the hypoxia response, interferes
with ethylene signalling, regulate the cell cycle and is regulated by LSD1, EDS1 and PAD4
we considered them to be primary candidates for this pleiotropic effect.
Figure 9 Hypoxia and aerenchyma regulation in Arabidopsis . (A) Composite chart of stomatal
conductance, H2O2 and ET regulation during 8 days of waterlogging. Note the gradual decrease in
stomatal conductance and subsequent increase in hydrogen peroxide. A gradual increase of the ethylene
precursor ACC was detected, starting at day 6. (B) Quantification of cross section areas of
aerenchymatous lacunae in Ws-0, lsd1, eds1-1, pad4-5, eds1-1/lsd1 and pad4-5/lsd1 (p<0.05*)
The aerenchyma formation takes 7 days of hypoxia treatment until the cell death
is induced. This served as a good model for providing resolution of stress signalling events.
32
From our data we could see the timing of the events leading to the cell death. The stress
response started with stomatal closure followed by H2O2 production and after that increased
levels of ACC (Fig. 9A). These signals preceded the cell death, that forms arenchyma (Fig. 8)
and are reminicent of the signal development of cell death in the lsd1 mutant and EEE-CD in
wild type (Paper1 and 3). We propose that this sequence is general for plant responses to
EEE stress and that it provides a physiological basis for how plants regulate stress responses
and EEE-CD on a systemic level.
13. The LSD1 node regulates a variety of environmental stresses
The LSD1 protein belongs to the class of C2C2 transcription factors, which have been known
to integrate environmental redox stimuli with cell death pathways (Uren et al. 2000). Analysis
of cis-regulatory elements (CRE) in silico revealed interesting aspects of LSD1, EDS1 and
PAD4 regulation of cell death (Paper 4). These genes contain possible CRE of drought, heat,
light, ABA, ET, gibberrelins, IAA, hypoxia and phosphorous deficiency and have confirmed
regulation by drought, phosphorous deficiency, high CO2 and heat (Paper4). Additionally,
analysis of the EIN2 regulon which we have shown is downstream of LSD1 in stress
signalling revealed that genes in this regulon are co-regulated with many of the genes that
were regulated by the redox status of the chloroplast, and SAA (Paper 3). The analysis also
revealed that the genes of the EIN2 regulon are induced by JA, IAA and ABA. These data
support the assumption that LSD1 integrates a multitude of signals for the control of plant
redox status and cell death.
We investigated drought tolerance and field fitness in lsd1, eds1 and pad4 mutants. The
drought induced unchecked cell death in the lsd1 mutant but surprisingly this contributed to a
higher survival of these plants (Choo and Karpinski unpublished data). In field conditions
lsd1/eds1 and lsd1/pad4 mutants had a lower fitness than the wild type (Karpinski
unpublished data).
IAA inhibits stomatal closure and controls the amount of stomata per leaf area
(Lohse and Hedrich 1995; Reichheld et al. 1999; Hirt 2000; Kovtun et al. 2000; Saibo et al.
2003). Additionally, amount of stomata/leaf area has been shown to be affected by HL
(Lichtenthaler et al. 1981). Stomatal count in LL and HL showed that the eds1-1/lsd1 and
pad4-5/lsd1 double mutants were unable to increase stomata number in response to HL (Fig
10). These data correlate with the observed low basal levels of IAA in these mutants and
33
indicate that the basal auxin levels in plants cause pleiotropic effects that influence plant
survival and fitness in response to stress. Since the cell death in lsd1 results in higher levels of
IAA and a subsequent higher survival rate of the organism in response to drought, we propose
that IAA controls the altruistic aspect of environmentally induced cell death.
Figure 10 HL/LL ratio of stomata count in Arabidopsis leaves reveals pleiotropic effects in
EEE mutants. Stomatal count revealed that the double mutants lsd1/eds1 and lsd1/pad4 were
unable to increase stomata number in response to HL (p<0.01**).
The regulation of PCD by IAA presents an interesting paradox since IAA is necessary for
plants to inhibit cell death, but is also necessary for the large production of ethylene and ROS
that potentiates cell death (Mehlhorn and Wellburn 1987; Romano et al. 1993; Kuriyama and
Fukuda 2002; Gechev et al. 2004). This indicates that auxins work as a part of the rheostat
that determines the threshold for cell death induction. We suggest that auxin determines how
much a cell is “worth” for the organism. This hypothesis finds strong support from literature
and our own results showing that IAA is high in young tissues and that increased post cell
death levels of IAA in surviving tissues raise the threshold for cell death (Morgan and
Durham 1973). Our data thus indicate that EEE-CD is altruistic in the sense that it removes
dangerous cells and increases the survival of the organism.
The second hypothesis of this thesis stated that plants have evolved specific
mechanisms that integrate signals related to light acclimation with the control of PCD. We
34
conclude that this hypothesis can be verified since our data indicate that LSD1 integrates
signalling of the EEE-CD, lysigenous aerenchyma and HR pathways for the regulation of
PCD. The signal pathways regulated by LSD1 that relate to SA in biotic defences are already
suggested in other publications (Kliebenstein et al. 1999; Rusterucci et al. 2001; Wiermer et
al. 2005). Here we propose two new models, one for the regulation of EEE-CD (Fig. 11) and
one for the regulation of lysigenous aerenchyma in Arabidopsis (Fig. 12). In the EEE-CD
model we propose that LSD1 is a central node of redox and hormone regulation for
chloroplast generated signals (Fig. 11). In the lysigenous aerenchyma model we propose that
LSD1, EDS1 and PAD4 are regulated by metabolic changes that arise during hypoxia and
waterlogging and that they regulate the sequence of root specific events that promote the cell
death of aerenchyma formation (Fig.12).
Figure 11 Model for EEE-induced cell death controlled by the chloroplast redox signalling,
photorespiration and LSD1.
Pro-cell death redox signalling originating from redox changes in plastoquinone pool (PQ) is
negatively regulated by LSD1 that acts to limit the spread of cell death. LSD1 negatively regulates
ROS from photorespiration (Mateo et al., 2004), PAD4 and EDS1 dependent cellular ethylene
production and together with EIN2 modulates ethylene- (ET) induced pro-cell death signalling.
LSD1 positively regulates, directly or indirectly, superoxide dismutase (SOD) and catalase (CAT)
gene expression and activities and thus controls cellular reactive oxygen species (ROS) production
(Jabs et al. 1996; Kliebenstein et al. 1999; Mateo et al. 2004). We propose that LSD1, EDS1 and
PAD4 constitute a ROS/ ethylene homeostatic switch, controlling acclimation to EEE and its
associated pro-cell death signalling.
35
Figure 12 Proposed model of signalling pathways that regulate aerenchyma formation in
Arabidopsis.
The gray area denotes root specific events. Root hypoxia produces a systemic signal that promotes
ROS and ethylene formation, leading to the induction of aerenchyma. Waterlogging and root
hypoxia lead to the induction of LSD1, EDS1, and PAD4 that regulate aerenchyma through plant
redox and ethylene signalling. Additionally LSD1 is a negative regulator of RCD that blocks the
competence of parenchyma to form aerenchyma.
14. Conclusions
The LSD1 is a general controller of PCD in response to environmental stress since it controls
HR, EEE-CD and aerenchyma. This study emphasizes the importance of choosing an
approach of multiple physiological conditions when investigating the function of genes.
Furthermore, the results of this study can be interpreted from three interconnected aspects.
Firstly our results show that the EEE induced cell death, in parallel with all described types of
programmed cell death in plants, is dependent on ROS signalling. Additionally, this cell death
is dependent on ethylene signalling through EIN2 and chloroplast redox status. This
regulation is reminiscent of the unchecked cell death in lsd1 which in addition is induced by
EEE. Therefore we propose that LSD1 is a negative regulator and that EDS1 and PAD4 are
positive regulators of EEE induced cell death and that they exert this control by regulating
homeostasis of ROS, salicylic acid, ethylene and auxin. Secondly we suggest that LSD1, by
controlling catalase activity in unstressed plant as well as those exposed to oxidative stress
will affect the response of the plant to any stress that gives rise to ROS. LSD1 is a key
regulator in the cross talk between these pathways. This is further supported by the role of
36
LSD1 as a regulator of ROS/hormone homeostasis and by its crucial involvement in both
biotic and abiotic defence responses. Thirdly, we suggest that the signalling pathways of the
described types of foliar cell death that are promoted by ethylene and ROS and inhibited by
auxins also participate in general cross-talk. This suggestion finds further support in the
earlier studies on disease development and the studies, including our own, showing that light
and oxygen radicals have a promoting effect on pathogen induced cell death as well as
senescence.
Future perspectives
These studies have shown that LSD1, EDS1 and PAD4 are genes which have the ability to
affect a large variety of aspects of a plants life. We have also identified two novel kinds of
cell death in Arabidopsis. Below are some suggestions for future experiments that may add to
an increased resolution of these studies.
The study of typical PCD morphological markers and events, such as nuclear shrinkage, DNA
condensation and DNA fragmentation in aerenchyma and EEE-CD may contribute to
increased understanding of the execution of these events.
Studying the transcription levels of LSD1, EDS1 and PAD4 in response to different kinds of
stresses in order to test the regulation that was predicted through the in silico analysis. The
lsd1, eds1-1 and pad4-5 mutants should also be further investigated for tolerance to hypoxia
and drought stress.
Double mutant crosses like lsd1/ein2-1, lsd1/tir1 and lsd1/abi1-1 provide good tools for
deciphering the role of ET, IAA and ABA in PCD and EEE responses. The lsd1/tir1 and
lsd1/abi1-1 mutants should be isolated and all three double mutants characterized for different
stress responses.
The role of IAA in aerenchyma formation should be investigated. Cytokinins have several
interresting effects on plants similar to IAA and may have a regulatory impact on
environmental stress regulation and EEE-CD. This should be investigated
The study of role of stomata regulation in cell death and mutants should be continued by
thermoimaging which provides resolution of patchy stomatal conductance.
37
Materials and methods
Growth conditions, EEE exposure and pharmacological treatments
Plants were grown in low light -chambers at PFD 100± 25 μmol m2 s-1 , relative air humidity
50%, temperature 20 ±1 °C in short day conditions, 8 h photoperiod. High light treatment was
given at PFD 500 ± 25 μmol m2 s-1, relative air humidity 50%, temperature 20 ±1 °C. Where
applicable, artificial restriction of gas exchange was achieved either by application of lanolin
wax or by adhering strips of semi-transparent tape on the adaxial sides of leaves covering
some two thirds of the leaf plate. Using wax is highly impractical since it disturbs extraction
procedures in ACC quantification. Therefore strips of semi-transparent tape were used in this
case to achieve a restriction of gas exchange. For ACC treatment, approximately 20 μL of 100
μM ACC was injected into leaves and H2O injections were used as controls. For treatments
with IAA, lanolin wax containing 0,1, 1 or 0,5% (w/v) IAA was applied to adaxial sides of
leaf plates and lanolin wax containing the same amount of ethanol was used as control.
Aerenchyma detection and quantification
Hypocotyls of waterlogged and control plants (12-weeks-old) were fixed in FAA (70%
ethanol, 5% Acetic acid, 1.75% Formaldehyde) for two days. The fixed material was then
transferred to 80% ethanol and gradually rehydrated before sectioning.
For quantification, cross sections were stained with 0.05% Toluidine Blue and 1% Boric acid.
Area analysis of the aerenchymatous lacunae and parenchymatous tissues were determined
from grayscaled digital images as previously described (Chaerle et al. 2004).
Stomatal analyses
Stomatal conductance was measured in growth conditions, by measuring the speed of
rehydration (cm/s) of a cyclically desiccated chamber by 1cm2 leaf areas. We used a portable
AP4 Porometer (Delta-T Devices, Cambridge, UK, and manufacturer instructions).
Stomata were counted and averaged for 3 randomly chosen 0.312 mm2 picture areas from nail
polish leaf prints (n=5) of formaldehyde fixed leaf samples following a standardized
microscopy – image analysis approach. Areas were photographed on a T041 microscope
using the 40x ocular connected to a DP50 digital camera (Olympus Optical CO. LTD, Tokyo,
Japan). Stomata were counted on grey-scaled pictures by semi-manual particle detection of
stomata using ImageJ software.
38
ROS detection
H2O2 accumulation was monitored using 2’,7’-dichlorodihydrofluorescein diacetate (H2DCFDA) (Sigma-Aldrich Sweden AB, Stockholm, Sweden). The lower sides of the leaves were
immediately after sampling covered with a 50μM H2DCF-DA. After an incubation time of 15
minutes the solution was removed and leaves dipped in water to remove any remnants of
H2DCF-DA from the surface. The specimens were then examined on a T041 microscope with
a UV light source connected to a DP50 digital camera (Olympus Optical CO. LTD, Tokyo,
Japan). For sampling of systemic leaves, only leaves with similar age or morphology were
used. Special care was taken not to induce any kind of additional wounding to the tissues.
Hydrogen peroxide was quantified as described by: (Guilbault et al. 1968; Jimenez et al. 2002)
with the following modification: 100 mg of fresh Arabidopsis hypocotyl tissue of 12 week old
plants was used per 1 ml of extraction medium.
Glutathione reductase activity
Plant material was snap-frozen in (l)N2 and stored at -80°C until grinding and extraction. GR
activity was determined as previously described (Connell and Mullet 1986). Activity of the
enzyme is assessed in an NADPH containing HEPES buffer by the decrease in absorbance at
340 nm as NADPH is oxidized. Total protein content of the extracts was determined using the
Bradford protein assay (Bio-Rad, Hercules, CA, USA).
Ethylene assay
Quantification of ACC was performed as described by: Langebartels C et al. (1991) Plant
Phys. 95:882-889 and Lizada M.C.C and Yang S.F. (1979) Anal. Biochem. 100: 140-145.
ACC (1-aminocyclopropane-1-carboxylic acid) is a precursor of ethylene and is accurately
linear to ethylene emitted by the plant.
IAA quantification
For analysis of endogenous levels of IAA, young and old (Young defined as leaves above and
old as leaves below leaf number 5 as described in (Kerstetter and Poethig 1998) leaf tissue
from 4 week old Arabidopsis thaliana ws-0, lsd1, eds1-1, pad4-5, eds1-1/lsd1 and pad45/lsd1 was collected, weighed and frozen in liquid nitrogen. Approximately 10 mg tissue was
collected for each sample. 0.5 ml 0.05 M NaHPO4
buffer, pH 7.0, containing 0.02%
diethyldithiocarbamic acid, a tungsten-carbide bead and [13C6]-IAA internal standard was
added to the sample. The sample was homogenized in a Retsch MM301 MixerMill for 3 min,
30 Hz at 4°C. The tungsten-carbide bead was removed, the sample was agitated for an
additional 15 min and pH was thereafter adjusted to approximately pH 2.7 with 1 M HCl.
39
The sample was purified on a SPE-column (BondElut-C8, 100 mg, Varian inc., CA, USA)
conditioned with 1 ml methanol and 1ml 1% acetic acid. After sample application the column
was washed with 1 ml 10% methanol in 1% acetic acid, eluted with 1 ml methanol and
evaporated to dryness. The sample was methylated by adding 0.2 ml 2-propanol, 1 ml methyl
chloride and 5 μl trimethylsilyl-diazomethane in hexane and incubating for 30 min at room
temperature. 5 μl 2 M acetic acid in hexane was added to destroy excess diazomethane and
the sample was evaporated to dryness. Subsequent sample silylation was performed by adding
15
μl
acetonitrile
and
15
μl
N,o-bis(trimethylsilyl)trifluoroacetamide/1%
trimethylchlorosilane and incubating at 70°C for 30 min. The sample was evaporated,
dissolved in 20 μl n-heptane and injected splitless by a Hewlett-Packard HP 7863 autosampler
into a Hewlett-Packard HP 6890 gas chromatograph equipped with a CP-SIL 8CB column (30
m x 0.25 mm i.d., Varian inc., CA, USA). The injector temperature was 270°C, the column
temperature was held at 80°C for 2 min, then increased by 20°C min-1 to 220°C and by 4°C
min-1 to 270°C. The column effluent was introduced into a JEOL MStation JMS-700 and
analyzed by GC-selected reaction monitoring-MS. Ions were generated with 70 eV at an
ionization current of 300 μA. The monitored reactions for IAA were 261.118m/z to 202.105
m/z (endogenous IAA) and 267.137m/z to 208.125 m/z (internal standard) as described by
Edlund et al. (1995). Calculation of isotopic dilution factors was based on the addition of 50
pg [13C6]-IAA per mg tissue. Samples were prepared in three replicates. Peak integration and
data processing was performed using the JEOL Xmass software.
For further details, please refer to the experimental procedure sections of the attached
papers.
40
Acknowledgements
Thank you,
My supervisor and scientific mentor Professor Stanislaw Karpinski. Thank you for all
the support, enthusiasm, and guidance that has enabled the completion of this work. It really
was an adventure!
My co-supervisor Docent Barbara Karpinska for being there when needed.
Thank you Prof. Sylvia Lindberg, Dr. Sophia Ekengren, Dr. Dietmar Funck, Dr. Christine
Chang, Dr. Markus Klenell, and Pitter Huesgen for critical reading of this thesis.
My co-workers and good friends in the lab: Merche for showing me the strength of oaks,
Christine for her enthusiasm, Pitter for being a good compadre, Verena for all the hugs,
Dietmar for who he is, Alfonso for interresting discussions on "how to interrogate a plant",
Markus for philosophical debates, and the Plaszczycas for a funny twist to the lab.
I also want to thank all my special friends at Botan who made my working environment so
inspiring by joining for lunch, giving me a friendly nudge, discussing in the greenhouse or
simply smiling on a cloudy day.
Professor Philip Mullineaux, Professor Robert Fluhr and CTM for providing opportunities for
my scientific development. Thank you Sara V. Petersson for an excellent cooperation for the
auxin analysis of the mutants.
Thank you also Professor Uno Lindberg, Professor Iwona Adamska, Dr. Kartik Narayan and
the people of CTM at SU for showing me the fun side of science.
Financial support was received from the Botany studentship, Stockholm University and
contributions from Formas (Swedish Research Council for Environment, Agricultural
Sciences and Spatial Planning), NorFa (Nordic Science Policy Council and Nordic Academy
for Advanced Study), STINT (Swedish Foundation for International Cooperation in Research
and Higher Education) and VR (Swedish Research Council).
My family, all of you, the big roots of this humble plant:
My Mother, Mamma Magitta for so much unconditional love and immense support.
My Father, Pappa Kjell for putting me on the path, and for endless love.
My Brother, Magnus, världens bästa storebrorsa, i vått och torrt.
Sarah, Lars and my beloved Grandparents - you have made me strong in different ways!
My friends!! - all of you!! - I especially want to mention (in no particular order) Jakob, Isse,
Muhammed, Johan, Lena and Familjen Innergård for so much you've done for me during
these years.
To life!!
41
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