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
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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.
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
5. PCD mutants in Arabidopsis ............................................................................................ 18 6. Thesis aim and hypotheses............................................................................................... 20
Results and discussion.............................................................................................................. 21 The LSD1 node of stress................................................................................................... 21
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
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.
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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).
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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).
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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).
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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
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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).
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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).
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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).
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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.
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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.
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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*)
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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
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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**).
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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.
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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.
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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.
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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.
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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
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