1

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

© Per Mühlenbock, Stockholm 2006 ISBN 91-7155-344-4 per.muhlenbock@botan.su.se Printed by US AB, Stockholm, Sweden 2006

2

3

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.

3 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)

4 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production in mutants and unpublished supportive experiments.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.

4

5

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

6

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

7

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.

8

9

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; Winkel-

Shirley 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.

10

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

11

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

12

peroxide (H2O2) and the hydroxyl radical (.OH). Singlet oxygen (

1O2) 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

13

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).

14

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

15

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).

16

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

17

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 SA-

dependent 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.

18

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

19

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

20

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.

21

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).

22

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.

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.

23

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).

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).

24

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.

Figure 3. SA disrupts redox regulation. (A) Mutants with constitutive accumulation of SA had strongly increased H2O2 levels and in SA-deficient 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).

25

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-1-

carboxylate (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).

26

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

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 Col-0 , ein2, Col lsd1 and Col lsd/ein2. The lesion phenotype of lsd1 was partially reverted in lsd1/ein2

27

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.

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 eds1-

1/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

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).

28

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).

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

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 (Ws-0) 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).

29

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).

30

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.

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.

31

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.

32

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.

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.

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*)

33

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

34

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.

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

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**).

35

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.

36

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

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.

37

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.

38

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.

39

ROS detection H2O2 accumulation was monitored using 2’,7’-dichlorodihydrofluorescein diacetate (H2DCF-

DA) (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 pad4-

5/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.

40

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.

41

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!!

42

References Abel, S. and A. Theologis (1996). "Early Genes and Auxin Action." Plant Physiol. 111(1): 9-17. Adams, D. O. and S. F. Yang (1979). "Ethylene biosynthesis: Identification of 1-aminocyclopropane-1-

carboxylic acid as an intermediate in the conversion of methionine to ethylene." PNAS 76(1): 170-174. Allan, A. C. and R. Fluhr (1997). "Two Distinct Sources of Elicited Reactive Oxygen Species in Tobacco

Epidermal Cells." Plant Cell 9(9): 1559-1572. Apel, K. and H. Hirt (2004). "Reactive oxygen species: metabolism, oxidative stress, and signal transduction."

Annual Review of Plant Biology 55(1): 373-399. Aravind, L. and E. V. Koonin (2002). "Classification of the caspase-hemoglobinase fold: Detection of new

families and implications for the origin of the eukaryotic separins." Proteins: Structure, Function, and Genetics 46(4): 355-367.

Asada, K. (1999). "The water-water cycle in chloroplasts: scavenging of active oxygens and dissipation of excess photons." Annu Rev Plant Physiol Plant Mol Biol. 50: 601-639.

Aviv, D. H., C. Rusterucci, et al. (2002). "Runaway cell death, but not basal disease resistance, in lsd1 is SA- and NIM1/NPR1-dependent." Plant J 29(3): 381-91.

Baier, M. and K.-J. Dietz (2005). "Chloroplasts as source and target of cellular redox regulation: a discussion on chloroplast redox signals in the context of plant physiology." J. Exp. Bot. 56(416): 1449-1462.

Bailey, S., R. Walters, et al. (2001). "Acclimation of Arabidopsis thaliana to the light environment: the existence of separate low light and high light responses." Planta. 213(5): 794-801.

Ball, L., G.-P. Accotto, et al. (2004). "Evidence for a direct link between glutathione biosynthesis and stress defense gene expression in Arabidopsis." Plant Cell 16(9): 2448-2462.

Bartsch, M., E. Gobbato, et al. (2006). "Salicylic acid-independent ENHANCED DISEASE SUSCEPTIBILITY1 signaling in Arabidopsis immunity and cell death is regulated by the monooxygenase FMO1 and the nudix hydrolase NUDT7." Plant Cell 18(4): 1038-1051.

Bechtold, U., S. Karpinski, et al. (2005). "The influence of the light environment and photosynthesis on oxidative signalling responses in plant-biotrophic pathogen interactions." Plant Cell And Environment 28(8): 1046-1055.

Becker, B., S. Holtgrefe, et al. (2006). "Influence of the photoperiod on redox regulation and stress responses in Arabidopsis thaliana L. (Heynh.) plants under long- and short-day conditions." Planta V224(2): 380.

Beers, E. P. and J. M. McDowell (2001). "Regulation and execution of programmed cell death in response to pathogens, stress and developmental cues." Current Opinion in Plant Biology 4(6): 561.

Beligni, M. V., A. Fath, et al. (2002). "Nitric Oxide Acts as an Antioxidant and Delays Programmed Cell Death in Barley Aleurone Layers." Plant Physiol. 129(4): 1642-1650.

Beligni, M. V. and L. Lamattina (2000). "Nitric oxide stimulates seed germination and de-etiolation, and inhibits hypocotyl elongation, three light-inducible responses in plants." Planta V210(2): 215.

Blokhina, O., E. Virolainen, et al. (2003). "Antioxidants, Oxidative Damage and Oxygen Deprivation Stress: a Review." Ann Bot 91(2): 179-194.

Bolwell, G. P., L. V. Bindschedler, et al. (2002). "The apoplastic oxidative burst in response to biotic stress in plants: a three-component system." J. Exp. Bot. 53(372): 1367-1376.

Bolwell, P., P., A. Page, et al. (2001). "Pathogenic infection and the oxidative defences in plant apoplast." Protoplasma V217(1): 20.

Bostock, R. M. (2005). "SIGNAL CROSSTALK AND INDUCED RESISTANCE: Straddling the Line Between Cost and Benefit." Annual Review of Phytopathology 43(1): 545-580.

Buchanan-Wollaston, V., S. Earl, et al. (2003). "The molecular analysis of leaf senescence - a genomics approach." Plant Biotechnology Journal 1(1): 3-22.

Chaerle, L., D. Hagenbeek, et al. (2004). "Thermal and chlorophyll-fluorescence imaging distinguish plant-pathogen interactions at an early stage." Plant And Cell Physiology 45(7): 887-896.

Chaerle, L., D. Hagenbeek, et al. (2004). "Thermal and chlorophyll-fluorescence imaging distinguish plant-pathogen interactions at an early stage." Plant Cell Physiol. 45(7): 887-896.

Chamnongpol, S., H. Willekens, et al. (1998). "Defense activation and enhanced pathogen tolerance induced by H2O2 in transgenic tobacco." Proc Natl Acad Sci. USA. 95(10): 5818-5823.

Chang, C. C.-C., L. Ball, et al. (2004). "Induction of ASCORBATE PEROXIDASE 2 expression in wounded Arabidopsis leaves does not involve known wound-signalling pathways but is associated with changes in photosynthesis." Plant J. 38(3): 499-511.

Chen, Z., H. Silva, et al. (1993). "Active oxygen species in the induction of plant systemic acquired resistance by salicylic acid." Science 262(5141): 1883-1886.

43

Cheong, Y. H., H.-S. Chang, et al. (2002). "Transcriptional profiling reveals novel interactions between

wounding, pathogen, abiotic stress, and hormonal responses in Arabidopsis." Plant Physiol. 129(2): 661-677.

Clarke, J. D., S. M. Volko, et al. (2000). "Roles of Salicylic Acid, Jasmonic Acid, and Ethylene in cpr-Induced Resistance in Arabidopsis." Plant Cell 12(11): 2175-2190.

Connell, J. P. and J. E. Mullet (1986). "Pea Chloroplast Glutathione Reductase: Purification and Characterization." Plant Physiol. 82(2): 351-356.

Cooper, C. E., R. P. Patel, et al. (2002). "Nanotransducers in cellular redox signaling: modification of thiols by reactive oxygen and nitrogen species." Trends in Biochemical Sciences 27(10): 489.

Couee, I., C. Sulmon, et al. (2006). "Involvement of soluble sugars in reactive oxygen species balance and responses to oxidative stress in plants." J. Exp. Bot. 57(3): 449-459.

Creelman, R. A. and J. E. Mullet (1997). "BIOSYNTHESIS AND ACTION OF JASMONATES IN PLANTS." Annual Review of Plant Physiology and Plant Molecular Biology 48(1): 355-381.

Dat, J. F., R. Pellinen, et al. (2003). "Changes in hydrogen peroxide homeostasis trigger an active cell death process in tobacco." Plant Journal 33(4): 621-632.

Davies, K. J. A. (2000). "Oxidative stress, antioxidant defenses, and damage removal, repair, and replacement systems." Iubmb Life 50(4-5): 279-289.

de Jong, A., E. Yakimova, et al. (2002). "A critical role for ethylene in hydrogen peroxide release during programmed cell death in tomato suspension cells." Planta 214(4): 537.

Delledonne, M., J. Zeier, et al. (2001). "Signal interactions between nitric oxide and reactive oxygen intermediates in the plant hypersensitive disease resistance response." Proc Natl Acad Sci. USA. 98(23): 13454-13459.

Demmig-Adams, B. and W. W. Adams (1992). "Photoprotection and Other Responses of Plants to High Light Stress." Annual Review of Plant Physiology and Plant Molecular Biology 43(1): 599-626.

Desikan, R., S. A.-H.-Mackerness, et al. (2001). "Regulation of the Arabidopsis transcriptome by oxidative stress." Plant Physiol. 127(1): 159-172.

Desikan, R., M.-K. Cheung, et al. (2004). "ABA, hydrogen peroxide and nitric oxide signalling in stomatal guard cells." J. Exp. Bot. 55(395): 205-212.

Desikan, R., J. T. Hancock, et al. (2005). "A Role for ETR1 in Hydrogen Peroxide Signaling in Stomatal Guard Cells." Plant Physiol. 137(3): 831-834.

Draper, J. (1997). "Salicylate, superoxide synthesis and cell suicide in plant defence." Trends In Plant Science 2(5): 162-165.

Drew, M. C., C. J. He, et al. (2000). "Programmed cell death and aerenchyma formation in roots." Trends In Plant Science 5(3): 123-127.

Durner, J., J. Shah, et al. (1997). "Salicylic acid and disease resistance in plants." Trends In Plant Science 2(7): 266-274.

Ellis, R. E., J. Yuan, et al. (1991). "Mechanisms and Functions of Cell Death." Annual Review of Cell Biology 7(1): 663-698.

Epple, P., A. A. Mack, et al. (2003). "Antagonistic control of oxidative stress-induced cell death in Arabidopsis by two related, plant-specific zinc finger proteins." PNAS 100(11): 6831-6836.

Escoubas, J., M. Lomas, et al. (1995). "Light Intensity Regulation of cab Gene Transcription is Signaled by the Redox State of the Plastoquinone Pool." PNAS 92(22): 10237-10241.

Esmon, C. A., U. V. Pedmale, et al. (2005). "Plant tropisms: providing the power of movement to a sessile organism." International Journal Of Developmental Biology 49(5-6): 665-674.

Fankhauser, C. and J. Chory (1997). "LIGHT CONTROL OF PLANT DEVELOPMENT." Annual Review of Cell and Developmental Biology 13(1): 203-229.

Feys, B. J., M. Wiermer, et al. (2005). "Arabidopsis SENESCENCE-ASSOCIATED GENE101 Stabilizes and Signals within an ENHANCED DISEASE SUSCEPTIBILITY1 Complex in Plant Innate Immunity." Plant Cell 17(9): 2601-2613.

Foreman, J., V. Demidchik, et al. (2003). "Reactive oxygen species produced by NADPH oxidase regulate plant cell growth." Nature 422(6930): 442-446.

Foyer, C., H. and B. Halliwell (1976). "The presence of glutathione and glutathione reductase in chloroplasts: A proposed role in ascorbic acid metabolism." Planta V133(1): 21.

Foyer, C. H. (1996). "Oxygen processing in photosynthesis." Biochemical Society Transactions 24(2): 427-433. Foyer, C. H. and J. F. Allen (2003). "Lessons from Redox Signaling in Plants." Antioxidants & Redox Signaling

5(1): 3-5. Foyer, C. H., M. Lelandais, et al. (1994). "Photooxidative stress in plants." Physiol. Plant. 92(4): 696-717. Foyer, C. H. and G. Noctor (2003). "Redox sensing and signalling associated with reactive oxygen in

chloroplasts, peroxisomes and mitochondria." Physiologia Plantarum 119(3): 355-364.

44

Foyer, C. H. and G. Noctor (2005). "Redox Homeostasis and Antioxidant Signaling: A Metabolic Interface

between Stress Perception and Physiological Responses." Plant Cell 17(7): 1866-1875. Fryer, M. J., L. Ball, et al. (2003). "Control of Ascorbate Peroxidase 2 expression by hydrogen peroxide and

leaf water status during excess light stress reveals a functional organisation of Arabidopsis leaves." Plant J 33(4): 691-705.

Gan, S. S. and R. M. Amasino (1997). "Making sense of senescence - Molecular genetic regulation and manipulation of leaf senescence." Plant Physiology 113(2): 313-319.

Ge, L., J. Z. Liu, et al. (2000). "Identification of a novel multiple environmental factor-responsive 1-aminocyclopropane-1-carboxylate synthase gene, NT-ACS2, from tobacco." Plant Cell Environ 23(11): 1169-1182.

Gechev, T. S., I. Z. Gadjev, et al. (2004). "An extensive microarray analysis of AAL-toxin-induced cell death in<i>Arabidopsis thaliana</i>brings new insights into the complexity of programmed cell death in plants." Cellular and Molecular Life Sciences (CMLS) 61(10): 1185.

Gechev, T. S., F. Van Breusegem, et al. (2006). "Reactive oxygen species as signals that modulate plant stress responses and programmed cell death." BioEssays 28(11): 1091-1101.

Gehring, C., A., R. McConchie, M., et al. (1998). "Auxin-binding-protein antibodies and peptides influence stomatal opening and alter cytoplasmic pH." Planta V205(4): 581.

Genoud, T., A. J. Buchala, et al. (2002). "Phytochrome signalling modulates the SA-perceptive pathway in Arabidopsis." Plant J 31(1): 87-95.

Gilchrist, D. G. (1998). "PROGRAMMED CELL DEATH IN PLANT DISEASE: The Purpose and Promise of Cellular Suicide." Annual Review of Phytopathology 36(1): 393-414.

Glazebrook, J. (2001). "Genes controlling expression of defense responses in Arabidopsis –– 2001 status." Current Opinion in Plant Biology 4(4): 301-308.

Gomez, L. D., G. Noctor, et al. (2004). "Regulation of calcium signalling and gene expression by glutathione." J. Exp. Bot. 55(404): 1851-1859.

Gray, J., D. Janick-Buckner, et al. (2002). "Light-Dependent Death of Maize lls1 Cells Is Mediated by Mature Chloroplasts." Plant Physiol. 130(4): 1894-1907.

Greenberg, J. T. and F. M. Ausubel (1993). "Arabidopsis mutants compromised for the control of cellular damage during pathogenesis and aging." The Plant Journal 4(2): 327-341.

Greenberg, J. T., F. P. Silverman, et al. (2000). "Uncoupling salicylic acid-dependent cell death and defense-related responses from disease resistance in the Arabidopsis mutant acd5." Genetics 156(1): 341-350.

Greenberg, J. T. and N. Yao (2004). "The role and regulation of programmed cell death in plant-pathogen interactions." Cellular Microbiology 6(3): 201-211.

Guan, L. M. and J. G. Scandalios (2002). "Catalase gene expression in response to auxin-mediated developmental signals." Physiologia Plantarum 114(2): 288-295.

Guilbault, G. G., J. P. Brignac, et al. (1968). "Homovanillic acid as a fluorometric substrate for oxidative enzymes. Analytical applications of the peroxidase, glucose oxidase and xanthine oxidase systems." Analytical Chemistry 40: 190-196.

Guilfoyle, T., G. Hagen, et al. (1998). "How Does Auxin Turn On Genes?" Plant Physiol. 118(2): 341-347. Gunawardena, A. H. L. A. N., D. M. Pearce, et al. (2001). "Characterisation of programmed cell death during

aerenchyma formation induced by ethylene or hypoxia in roots of maize(Zea mays L.)." Planta 212(2): 205.

Hadfield, K. A. and A. B. Bennett (1997). "Programmed senescence of plant organs." Cell Death And Differentiation 4(8): 662-670.

Hammond-Kosack, K. E. and J. D. G. Jones (1996). "Resistance gene-dependent plant defence responses." Plant cell 8: 1773-1791.

Heath, M. C. (2000). "Hypersensitive response-related death." Plant Molecular Biology 44: 321-334. Heddad, M. and I. Adamska (2000). "Light stress-regulated two-helix proteins in Arabidopsis thaliana related

to the chlorophyll a/b-binding gene family." PNAS 97(7): 3741-3746. Hirt, H. (2000). "Connecting oxidative stress, auxin, and cell cycle regulation through a plant mitogen-activated

protein kinase pathway." PNAS 97(6): 2405-2407. Hoeberichts, F. A. and E. J. Woltering (2003). "Multiple mediators of plant programmed cell death: interplay

of conserved cell death mechanisms and plant-specific regulators." Bioessays 25(1): 47-57. Huh, G.-H., B. Damsz, et al. (2002). "Salt causes ion disequilibrium-induced programmed cell death in yeast

and plants." The Plant Journal 29(5): 649-659. Hunt, M. D., T. P. Delaney, et al. (1997). "Salicylate-independent lesion formation in Arabidopsis lsd mutants."

Molecular Plant-Microbe Interactions 10(5): 531-536. Jabs, T., R. A. Dietrich, et al. (1996). "Initiation of runaway cell death in an Arabidopsis mutant by extracellular

superoxide." Science 273(5283): 1853-6.

45

Jacobson, M. D., M. Weil, et al. (1997). "Programmed Cell Death in Animal Development." Cell 88(3): 347. Jarillo, J. A., H. Gabrys, et al. (2001). "Phototropin-related NPL1 controls chloroplast relocation induced by

blue light." Nature 410(6831): 952-954. Jimenez, A., G. Creissen, et al. (2002). "Changes in oxidative processes and components of the antioxidant

system during tomato fruit ripening." Planta 214(5): 751-758. Johnson, P. R. and J. R. Ecker (1998). "THE ETHYLENE GAS SIGNAL TRANSDUCTION PATHWAY: A

Molecular Perspective." Annual Review of Genetics 32(1): 227-254. Johnstone, R. W., A. A. Ruefli, et al. (2002). "Apoptosis: A Link between Cancer Genetics and Chemotherapy."

Cell 108(2): 153. Jonak, C., L. Okresz, et al. (2002). "Complexity, Cross Talk and Integration of Plant MAP Kinase Signalling."

Current Opinion in Plant Biology 5(5): 415. Jones, A. (2000). "Does the plant mitochondrion integrate cellular stress and regulate programmed cell death?"

Trends in Plant Science 5(5): 225. Jones, A. M. (2001). "Programmed Cell Death in Development and Defense." Plant Physiol. 125(1): 94-97. Joo, J. H., H. J. Yoo, et al. (2005). "Auxin-induced reactive oxygen species production requires the activation of

phosphatidylinositol 3-kinase." FEBS Letters 579(5): 1243. Kachroo, P., J. Shanklin, et al. (2001). "A fatty acid desaturase modulates the activation of defense signaling

pathways in plants." PNAS 98(16): 9448-9453. Kaiser, W. M. (1979). "Reversible inhibition of the Calvin Cycle and activation of oxidative pentose phosphate

cycle in isolated intact chloroplasts by hydrogen peroxide." Planta 145: 377-382. Kangasjarvi, J., P. Jaspers, et al. (2005). "Signalling and cell death in ozone-exposed plants." Plant, Cell and

Environment 28(8): 1021-1036. Karpinska, B., G. Wingsle, et al. (2000). "Antagonistic effects of hydrogen peroxide and glutathione on

acclimation to excess excitation energy in Arabidopsis." IUBMB Life 50(1): 21-26. Karpinski, S. and e. al. (1999). "Systemic signalling and acclimation in response to excess excitation energy in

Arabidopsis." Science 284: 654-657. Karpinski, S., C. Escobar, et al. (1997). "Photosynthetic electron transport regulates the expression of cytosolic

ascorbate peroxidase genes in Arabidopsis during excess light stress." Plant Cell 9(4): 627–640. Karpinski, S., H. Gabrys, et al. (2003). "Light perception in plant disease defence signalling." Current Opinion

in Plant Biology 6(4): 390-396. Karpinski, S., H. Reynolds, et al. (1999). "Systemic signaling and acclimation in response to excess excitation

energy in Arabidopsis." Science 284(5414): 654-657. Kawano, T. (2003). "Roles of the reactive oxygen species-generating peroxidase reactions in plant defense and

growth induction." Plant Cell Reports V21(9): 829. Kays, S. J. and J. E. Pallas (1980). "Inhibition of photosynthesis by ethylene." Nature 285(5759): 51. Keller, C. P. and E. Van Volkenburgh (1997). "Auxin-Induced Epinasty of Tobacco Leaf Tissues (A

Nonethylene-Mediated Response)." Plant Physiol. 113(2): 603-610. Kepinski, S. and O. Leyser (2005). "The Arabidopsis F-box protein TIR1 is an auxin receptor." Nature

435(7041): 446. Kerstetter, R. A. and R. S. Poethig (1998). "THE SPECIFICATION OF LEAF IDENTITY DURING SHOOT

DEVELOPMENT." Annual Review of Cell and Developmental Biology 14(1): 373-398. Kiddle, G., G. M. Pastori, et al. (2003). "Effects of Leaf Ascorbate Content on Defense and Photosynthesis

Gene Expression in Arabidopsis thaliana." Antioxidants & Redox Signaling 5(1): 23-32. Kirik, V., D. Bouyer, et al. (2001). "CPR5 is involved in cell proliferation and cell death control and encodes a

novel transmembrane protein." Current Biology 11(23): 1891. Klee, H. J., R. B. Horsch, et al. (1987). "The Effects Of Overproduction Of 2 Agrobacterium-Tumefaciens T-

Dna Auxin Biosynthetic Gene-Products In Transgenic Petunia Plants." Genes & Development 1(1): 86-96.

Kliebenstein, D. J., R. A. Dietrich, et al. (1999). "LSD1 regulates salicylic acid induction of copper zinc superoxide dismutase in Arabidopsis thaliana." Mol Plant Microbe Interact 12(11): 1022-6.

Kovtun, Y., W.-L. Chiu, et al. (2000). "From the Cover: Functional analysis of oxidative stress-activated mitogen-activated protein kinase cascade in plants." PNAS 97(6): 2940-2945.

Kozaki, A. and G. Takeba (1996). "Photorespiration protects C3 plants from photooxidation." Nature 384(6609): 557-560.

Kuriyama, H. and H. Fukuda (2002). "Developmental programmed cell death in plants." Current Opinion in Plant Biology 5(6): 568.

Laloi, C., K. Apel, et al. (2004). "Reactive oxygen signalling: the latest news." Current Opinion in Plant Biology 7(3): 323.

46

Lam, E. (2004). "Controlled Cell Death, Plant Survival and Development." Nature Reviews Molecular Cell

Biology 5(4): 305. Lamattina, L., C. Garcia-Mata, et al. (2003). "NITRIC OXIDE: The Versatility of an Extensive Signal

Molecule." Annual Review of Plant Biology 54(1): 109-136. Lamb, C. and R. A. Dixon (1997). "The oxidative burst in plant disease resistance." Annu. Rev. Plant Physiol.

Plant Mol. Biol. 48(1): 251-275. Lamb, C., M. Lawton, et al. (1989). "Signals and transduction mechanisms for activation of plant defenses

against microbial attack." Cell 56(2): 215-224. Lawton, K. A., S. L. Potter, et al. (1994). "Acquired Resistance Signal Transduction in Arabidopsis Is Ethylene

Independent." Plant Cell 6(5): 581-588. Legé, K., E., J. T. Cothren, et al. (1997). "Nitrogen fertility and leaf age effect on ethylene production of cotton

in a controlled environment." Plant Growth Regulation V22(1): 23. Levin, S., T. J. Bucci, et al. (1999). "The nomenclature of cell death: Recommendations of an ad hoc Committee

of the Society of Toxicologic Pathologists." Toxicologic Pathology 27(4): 484-490. Lichtenthaler, H. K., C. Buschmann, et al. (1981). "Photosynthetic activity, chloroplast ultrastructure, and leaf

characteristics of high-light and low-light plants and of sun and shade leaves." Photosynthesis Research V2(2): 115.

Lohse, G. and R. Hedrich (1995). "Anions modify the response of guard-cell anion channels to auxin." Planta (Historical Archive) 197(3): 546.

Long, L. M., H. P. Patel, et al. (2003). "The maize epicuticular wax layer provides UV protection." Functional Plant Biology 30(1): 75-81.

Lorrain, S., F. Vailleau, et al. (2003). "Lesion mimic mutants: keys for deciphering cell death and defense pathways in plants?" Trends in Plant Science 8(6): 263-271.

Lund, S. T., R. E. Stall, et al. (1998). "Ethylene Regulates the Susceptible Response to Pathogen Infection in Tomato." Plant Cell 10(3): 371-382.

Mateo, A. (2005). "Roles of LESION SIMULATING DISEASE1 and salicylic acid in acclimation of plants to environmental cues." Stockholm University, Doctoral thesis ISBN 91-7155-144-1.

Mateo, A., P. Mühlenbock, et al. (2004). "LESION SIMULATING DISEASE 1 is required for acclimation to conditions that promote excess excitation energy." Plant Physiol. 136(1): 2818-2830.

Mauch-Mani, B. and A. J. Slusarenko (1996). "Production of Salicylic Acid Precursors Is a Major Function of Phenylalanine Ammonia-Lyase in the Resistance of Arabidopsis to Peronospora parasitica." Plant Cell 8(2): 203-212.

Mazel, A. and A. Levine (2001). "Induction of cell death in Arabidopsis by superoxide in combination with salicylic acid or with protein synthesis inhibitors." Free Radic Biol Med 30(1): 98-106.

McDonald, K. L. and D. M. Cahill (1999). "Evidence for a transmissible factor that causes rapid stomatal closure in soybean at sites adjacent to and remote from hypersensitive cell death induced by Phytophthora sojae." Physiological And Molecular Plant Pathology 55(3): 197-203.

McDowell, J. M. and J. L. Dangl (2000). "Signal transduction in the plant immune response." Trends in Biochemical Sciences 25(2): 79.

McPherson, A. N., A. Telfer, et al. (1993). "Direct-Detection Of Singlet Oxygen From Isolated Photosystem-II Reaction Centers." Biochimica Et Biophysica Acta 1143(3): 301-309.

Mehlhorn, H. and A. R. Wellburn (1987). "Stress Ethylene Formation Determines Plant-Sensitivity To Ozone." Nature 327(6121): 417-418.

Milborrow, B. V. (2001). "The pathway of biosynthesis of abscisic acid in vascular plants: a review of the present state of knowledge of ABA biosynthesis." J. Exp. Bot. 52(359): 1145-1164.

Mittler, R., S. Vanderauwera, et al. (2004). "Reactive oxygen gene network of plants." Trends in Plant Science 9(10): 490-498.

Moeder, W., C. S. Barry, et al. (2002). "Ethylene Synthesis Regulated by Biphasic Induction of 1-Aminocyclopropane-1-Carboxylic Acid Synthase and 1-Aminocyclopropane-1-Carboxylic Acid Oxidase Genes Is Required for Hydrogen Peroxide Accumulation and Cell Death in Ozone-Exposed Tomato." Plant Physiol. 130(4): 1918-1926.

Morgan, P. W. and M. C. Drew (1997). "Ethylene and plant responses to stress." Physiologia Plantarum 100(3): 620-630.

Morgan, P. W. and J. I. Durham (1973). "Leaf Age and Ethylene-induced Abscission." Plant Physiol. 52(6): 667-670.

Mori, I. C., R. Pinontoan, et al. (2001). "Involvement of superoxide generation in salicylic acid-induced stomatal closure in Vicia faba." Plant Cell Physiol. 42(12): 1383-1388.

47

Mori, I. C. and J. I. Schroeder (2004). "Reactive oxygen species activation of plant Ca2+ channels- a signaling

mechanism in polar growth, hormone transduction, stress signaling, and hypothetically mechanotransduction." Plant Physiol. 135(2): 702-708.

Mullineaux, P. and S. Karpinski (2002). "Signal transduction in response to excess light: getting out of the chloroplast." Current Opinion in Plant Biology 5(1): 43-48.

Munne-Bosch, S. and L. Alegre (2004). "Die and let live: leaf senescence contributes to plant survival under drought stress." Functional Plant Biology 31(3): 203-216.

Mühlenbock, P. (2006). "Maintenance of Stomata Function is Required for Containment of Hypersensitive Response." NATO Science Series, I: Life and Behavioural Sciences 371: 315-319.

Neill, S., R. Desikan, et al. (2002). "Hydrogen peroxide signalling." Current Opinion in Plant Biology 5(5): 388-395.

Neill, S. J., R. Desikan, et al. (2002). "Hydrogen peroxide and nitric oxide as signalling molecules in plants." J. Exp. Bot. 53(372): 1237-1247.

Niyogi, K. K. (1999). "Photoprotection revisited: Genetic and molecular approaches." Annual Review Of Plant Physiology And Plant Molecular Biology 50: 333-359.

Noctor, G., S. Veljovic-Jovanovic, et al. (2002). "Drought and oxidative load in the leaves of C3 plants: a predominant role for photorespiration?" Ann Bot 89(7): 841-850.

O'Donnell, P. J., E. Schmelz, et al. (2003). "Multiple Hormones Act Sequentially to Mediate a Susceptible Tomato Pathogen Defense Response." Plant Physiol. 133(3): 1181-1189.

Ort, D. R. (2001). "When there is too much light." Plant Physiol. 125(1): 29-32. Overmyer, K., M. Brosche, et al. (2005). "Ozone-Induced Programmed Cell Death in the Arabidopsis radical-

induced cell death1 Mutant." Plant Physiol. 137(3): 1092-1104. Overmyer, K., H. Tuominen, et al. (2000). "Ozone-sensitive Arabidopsis rcd1 mutant reveals opposite roles for

ethylene and jasmonate signaling pathways in regulating superoxide-dependent cell death." Plant Cell. 12(10): 1849–1862.

Pasternak, T. P., E. Prinsen, et al. (2002). "The Role of Auxin, pH, and Stress in the Activation of Embryogenic Cell Division in Leaf Protoplast-Derived Cells of Alfalfa." Plant Physiol. 129(4): 1807-1819.

Pastori, G. M. and C. H. Foyer (2002). "Common Components, Networks, and Pathways of Cross-Tolerance to Stress. The Central Role of "Redox" and Abscisic Acid-Mediated Controls." Plant Physiol. 129(2): 460-468.

Paul, M. J. and C. H. Foyer (2001). "Sink regulation of photosynthesis." J. Exp. Bot. 52(360): 1383-1400. Pfannschmidt, T., A. Nilsson, et al. (1999). "Photosynthetic control of chloroplast gene expression." Nature

397(6720): 625. Pfeiffer, W. and M. Hoftberger (2001). "Oxidative burst in Chenopodium rubrum suspension cells: Induction

by auxin and osmotic changes." Physiologia Plantarum 111(2): 144-150. Raff, M. C. (1992). "Social Controls On Cell-Survival And Cell-Death." Nature 356(6368): 397-400. Rao, M. V., H.-i. Lee, et al. (2002). "Ozone-induced ethylene production is dependent on salicylic acid, and both

salicylic acid and ethylene act in concert to regulate ozone-induced cell death." The Plant Journal 32(4): 447-456.

Rate, D. N., J. V. Cuenca, et al. (1999). "The Gain-of-Function Arabidopsis acd6 Mutant Reveals Novel Regulation and Function of the Salicylic Acid Signaling Pathway in Controlling Cell Death, Defenses, and Cell Growth." Plant Cell 11(9): 1695-1708.

Reed, J. W. (2001). "Roles and activities of Aux/IAA proteins in Arabidopsis." Trends in Plant Science 6(9): 420. Reichheld, J.-P., T. Vernoux, et al. (1999). "Specific checkpoints regulate plant cell cycle progression in

response to oxidative stress." Plant J 17(6): 647-656. Romano, C. P., M. L. Cooper, et al. (1993). "Uncoupling Auxin and Ethylene Effects in Transgenic Tobacco

and Arabidopsis Plants." Plant Cell 5(2): 181-189. Rusterucci, C., D. H. Aviv, et al. (2001). "The disease resistance signaling components EDS1 and PAD4 are

essential regulators of the cell death pathway controlled by LSD1 in Arabidopsis." Plant Cell 13(10): 2211-2224.

Saibo, N. J. M., W. H. Vriezen, et al. (2003). "Growth and stomata development of Arabidopsis hypocotyls are controlled by gibberellins and modulated by ethylene and auxins." The Plant Journal 33(6): 989-1000.

Samuilov, V. D., E. M. Lagunova, et al. (2003). "Participation of chloroplasts in plant apoptosis." Bioscience Reports 23(2 - 3): 103-117.

Schwartz, L. M., S. W. Smith, et al. (1993). "Do All Programmed Cell Deaths Occur Via Apoptosis?" PNAS 90(3): 980-984.

Senda, K. and K. i. Ogawa (2004). "Induction of PR-1 Accumulation Accompanied by Runaway Cell Death in the lsd1 Mutant of Arabidopsis is Dependent on Glutathione Levels but Independent of the Redox State of Glutathione." Plant Cell Physiol. 45(11): 1578-1585.

48

Smith, H. (2000). "Phytochromes and light signal perception by plants - an emerging synthesis." Nature

407(6804): 585-591. Stintzi, A. and J. Browse (2000). "The Arabidopsis male-sterile mutant, opr3, lacks the 12-oxophytodienoic acid

reductase required for jasmonate synthesis." PNAS 97(19): 10625-10630. Tausz, M., A. M. Gonzalez-Rodriguez, et al. (2004). "Photostress, photoprotection, and water soluble

antioxidants in the canopies of five Canarian laurel forest tree species during a diurnal course in the field." Flora 199(2): 110-119.

Tiryaki, I. and P. E. Staswick (2002). "An Arabidopsis Mutant Defective in Jasmonate Response Is Allelic to the Auxin-Signaling Mutant axr1." Plant Physiol. 130(2): 887-894.

Torres, M. A., J. D. G. Jones, et al. (2006). "Reactive Oxygen Species Signaling in Response to Pathogens." Plant Physiol. 141(2): 373-378.

Tuominen, H., K. Overmyer, et al. (2004). "Mutual antagonism of ethylene and jasmonic acid regulates ozone-induced spreading cell death in Arabidopsis." Plant J 39(1): 59-69.

Turner, J. G., C. Ellis, et al. (2002). "The Jasmonate Signal Pathway." Plant Cell 14(90001): S153-164. Uren, A. G., K. O'Rourke, et al. (2000). "Identification of Paracaspases and Metacaspases: Two Ancient

Families of Caspase-like Proteins, One of which Plays a Key Role in MALT Lymphoma." Molecular Cell 6(4): 961.

Vacca, R. A., D. Valenti, et al. (2006). "Cytochrome c Is Released in a Reactive Oxygen Species-Dependent Manner and Is Degraded via Caspase-Like Proteases in Tobacco Bright-Yellow 2 Cells en Route to Heat Shock-Induced Cell Death." Plant Physiol. 141(1): 208-219.

Wallsgrove, R. M., P. J. Lea, et al. (1983). "Intracellular Localization of Aspartate Kinase and the Enzymes of Threonine and Methionine Biosynthesis in Green Leaves." Plant Physiol. 71(4): 780-784.

Walters, R. G. (2005). "Towards an understanding of photosynthetic acclimation." J. Exp. Bot. 56(411): 435-447. Van Breusegem, F. and J. F. Dat (2006). "Reactive Oxygen Species in Plant Cell Death." Plant Physiol. 141(2):

384-390. Van Breusegem, F., E. Vranova, et al. (2001). "The role of active oxygen species in plant signal transduction."

Plant Science 161(3): 405-414. Van Camp, W., M. Van Montagu, et al. (1998). "H2O2 and NO: redox signals in disease resistance." Trends In

Plant Science 3(9): 330-334. van Doorn, W. G. and E. J. Woltering (2005). "Many ways to exit? Cell death categories in plants." Trends in

Plant Science 10(3): 117. Vanderauwera, S., P. Zimmermann, et al. (2005). "Genome-Wide Analysis of Hydrogen Peroxide-Regulated

Gene Expression in Arabidopsis Reveals a High Light-Induced Transcriptional Cluster Involved in Anthocyanin Biosynthesis." Plant Physiol. 139(2): 806-821.

Wang, K. L. C., H. Li, et al. (2002). "Ethylene Biosynthesis and Signaling Networks." Plant Cell 14(90001): S131-151.

Vaux, D. L. (1993). "Toward an Understanding of the Molecular Mechanisms of Physiological Cell Death." PNAS 90(3): 786-789.

Wiermer, M., B. J. Feys, et al. (2005). "Plant immunity: the EDS1 regulatory node." Current Opinion in Plant Biology 8(4): 383.

Willekens, H., S. Chamnongpol, et al. (1997). "Catalase is a sink for H2O2 and is indispensable for stress defence in C3 plants." EMBO J. 16(16): 4806-4816.

Wingler, A., P. Lea, et al. (2000). "Photorespiration: metabolic pathways and their role in stress protection." Philos Trans R Soc Lond B Biol Sci. 355(1402): 1517-29.

Wingsle, G. and S. Karpinski (1996). "Differential redox regulation by glutathione of glutathione reductase and CuZn-superoxide dismutase gene expression in <i>Pinus sylvestris</i> L. needles." Planta V198(1): 151.

Winkel-Shirley, B. (2002). "Biosynthesis of flavonoids and effects of stress." Current Opinion in Plant Biology 5: 218.

Visser, E. J. W., C. J. Heijink, et al. (1995). "Regulatory Role Of Auxin In Adventitious Root-Formation In 2 Species Of Rumex, Differing In Their Sensitivity To Waterlogging." Physiologia Plantarum 93(1): 116-122.

Voesenek, L. and C. Blom (1996). "Plants and hormones: An ecophysiological view on timing and plasticity." Journal Of Ecology 84(1): 111-119.

Vranova, E., D. Inze, et al. (2002). "Signal transduction during oxidative stress." J. Exp. Bot. 53(372): 1227-1236. Wu, Y. X. and A. von Tiedemann (2004). "Light-dependent oxidative stress determines physiological leaf spot

formation in barley." Phytopathology 94(6): 584-592. Xia, Q. Z., X. L. Zhang, et al. (2005). "Withdrawal of exogenous auxin induces programmed cell death of cotton

embryogenic suspension cultures." Zhi Wu Sheng Li Yu Fen Zi Sheng Wu Xue Xue Bao 31(1): 78-84.

49

Yoshida, S., M. Ito, et al. (2002). "Identification of a novel gene HYS1/CPR5 that has a repressive role in the

induction of leaf senescence and pathogen-defence responses in Arabidopsis thaliana." The Plant Journal 29(4): 427-437.

Yu, S.-M. (1999). "Cellular and Genetic Responses of Plants to Sugar Starvation." Plant Physiol. 121(3): 687-693. Zago, E., S. Morsa, et al. (2006). "Nitric Oxide- and Hydrogen Peroxide-Responsive Gene Regulation during

Cell Death Induction in Tobacco." Plant Physiol. 141(2): 404-411. Zeier, J., B. Pink, et al. (2004). "Light conditions influence specific defence responses in incompatible plant-

pathogen interactions: uncoupling systemic resistance from salicylic acid and PR-1 accumulation." Planta 219(4): 673.

Zhang, X., L. Zhang, et al. (2001). "Hydrogen peroxide is involved in abscisic acid-induced stomatal closure in Vicia faba." Plant Physiol. 126(4): 1438-1448.

© Per Mühlenbock, Stockholm 2006 ISBN 91-7155-344-4