Plant Physiology Preview. Published on March 5, 2014, as DOI:10.1104/pp.114.236380
1
Running Head:
Dark-induced singlet oxygen during stress
Corresponding Author:
Robert Fluhr
Address:
Plant Sciences Department
Weizmann institute of science
Rehovot, 76100
Israel
Telephone:
+972-8-9342175
Email:
[email protected]
Research Area:
Signaling and Response
Downloaded from on June 15, 2017 - Published by www.plantphysiol.org
Copyright © 2014 American Society of Plant Biologists. All rights reserved.
Copyright 2014 by the American Society of Plant Biologists
2
Title:
Singlet oxygen signatures are detected independent of light or chloroplasts in
response to multiple stresses
Authors:
Avishai Mor, Eugene Koh, Lev Weiner, Shilo Rosenwasser, Hadas SibonyBenyamini and Robert Fluhr
One sentence summary:
Diverse stresses can produce singlet oxygen in a light-independent manner, which is
observed through generation of a singlet oxygen transcriptome footprint and
confirmed by measurements of singlet oxygen accumulation.
Downloaded from on June 15, 2017 - Published by www.plantphysiol.org
Copyright © 2014 American Society of Plant Biologists. All rights reserved.
3
Financial source:
This work was supported by a research grant from the Dr. Angel Faivovich
Foundation and the Israel Science Foundation (grant No. 1008/11).
Corresponding author:
[email protected]
Downloaded from on June 15, 2017 - Published by www.plantphysiol.org
Copyright © 2014 American Society of Plant Biologists. All rights reserved.
4
ABSTRACT
The production of singlet oxygen is typically associated with inefficient
dissipation of photosynthetic energy or can arise from light reactions due to
accumulation of chlorophyll precursors as observed in flu-like mutants. Such
photodynamic production of singlet oxygen is thought to be involved in stress
signaling and programmed cell death. Here we show that transcriptomes of multiple
stresses, whether from light or dark treatments, were correlated with the transcriptome
of the flu mutant. A core gene set (CGS) of 118 genes, common to singlet oxygen,
biotic and abiotic stresses was defined and confirmed to be activated
photodynamically by the photosensitizer rose bengal. In addition, induction of the
CGS by abiotic and biotic selected stresses was shown to occur in the dark and in
non-photosynthetic tissue. Furthermore, when subjected to various biotic and abiotic
stresses in the dark, the singlet oxygen-specific probe Singlet Oxygen Sensor Green
(SOSG), detected rapid production of singlet oxygen in the Arabidopsis root.
Subcellular localization of SOSG fluorescence showed its accumulation in
mitochondria, peroxisomes and the nucleus, suggesting several compartments as the
possible origins or targets for singlet oxygen. Collectively, the results show that
singlet oxygen can be produced by multiple stress pathways and can emanate from
compartments other than the chloroplast in a light independent manner. The results
imply that the role of singlet oxygen in plant stress regulation and response is more
ubiquitous than previously thought.
Downloaded from on June 15, 2017 - Published by www.plantphysiol.org
Copyright © 2014 American Society of Plant Biologists. All rights reserved.
5
INTRODUCTION
Singlet oxygen is the first electronic excited state of molecular oxygen and is a
reactive oxygen species (ROS). It is highly reactive and will engage readily with a
variety of biomolecules, especially those containing double bonds (Triantaphylidès
and Havaux, 2009). In plants, singlet oxygen has been extensively studied in the
context of photosynthesis i.e. its formation in the PSII reaction center and antenna
complex (Triantaphylidès and Havaux, 2009). The chlorophyll triplet state can react
with ground state oxygen to make the highly chemically reactive singlet state (3Chl*
+3O2
Chl + 1O2). Activated chlorophyll can give rise to the triplet state when light
energy is inefficiently used although even normal light conditions may result in
detectable formation of singlet oxygen (Triantaphylidès et al., 2008; Ramel et al.,
2012 a). Singlet oxygen can participate in lipid peroxidation resulting in diagnostic
chemical signatures. Peroxidation might result from direct assault of membrane lipids
by reactive oxygen species or it can be the outcome of enzymatic activity. Type I lipid
peroxidation consists of free radical peroxidation, while type II is the outcome of
peroxidation by singlet oxygen (Triantaphylidès et al., 2008). Type II reactions are
considered to have a major role in the execution of ROS-induced photooxidative cell
death of photosynthetic tissue (Triantaphylidès et al., 2008). In addition to
photoactive stress, the Arabidopsis-Pseudomonas syringae hypersensitive response
results in lipid peroxidation signatures corresponding to singlet oxygen and the
enzymatic activity of lipoxygenase 2 (LOX2). In that case, the major source of lipids
that undergo oxidation were from plastid membranes (Zoeller et al., 2012). Hence, in
both high-light stress and immune responses, the source of singlet oxygen is thought
to be through the photodynamic activation of chlorophyll.
Plants have developed various scavenging systems to protect themselves
against the toxic effect of singlet oxygen. β-carotene, tocopherol or plastoquinone
present in the thylakoid membranes are thought to play a role in quenching singlet
oxygen (Krieger-Liszkay et al., 2008). Other scavengers such as, ubiquinol, ascorbate
and glutathione may also quench singlet oxygen. Quenching can be physical;
involving energy transfer dissipation as heat without scavenger consumption or be
accompanied by chemical oxidation (Triantaphylidès and Havaux, 2009; Ramel et al.,
2012 a).
Singlet oxygen appears to play a role in retrograde signaling from the
chloroplast to the nucleus as established by mutants of FLUORESCENT (FLU) and
Downloaded from on June 15, 2017 - Published by www.plantphysiol.org
Copyright © 2014 American Society of Plant Biologists. All rights reserved.
6
EXECUTER (EX) 1 and 2. The FLU and the barely TIGRINA-d.12 (TIG-d.12); genes
are negative regulators of the precursor of chlorophyll, protochlorophyllide,
preventing its accumulation in the dark (Meskauskiene et al., 2001; Op den Camp et
al., 2003; Khandal et al., 2009). These mutants can be grown in constant light due to
continuous light-dependent conversion of protochlorophyllide. However, if kept in the
dark and then transferred to light, the large amounts of accumulated
protochlorophyllide act as a photosensitizer, releasing singlet oxygen and setting off a
massive cellular, transcriptome and physiological, responses that can result in cell
death (Meskauskiene et al., 2001; Op den Camp et al., 2003). The response is not due
to direct toxicity of singlet oxygen, as the process is mediated through 2 chloroplast
proteins, EX 1 and 2. In their absence, the transcriptomic response and cell death of
the flu mutant in dark to light shift is diminished (Wagner et al., 2004; Lee et al.,
2007; Kim et al., 2012). The signaling competence of singlet oxygen is further
illustrated by the induction of an acclimation response to singlet oxygen originating
from applied chemicals or in mutants (Fischer et al., 2012; Ramel et al., 2013). In
another example in the chlorina 1 mutant, deficient in chlorophyll b, excess levels of
singlet oxygen are generated in the thylakoids under light stress. The transcriptome of
this mutant shows high resemblance to that generated by the flu mutant although it
seems to operate in a distinct manner and does not depend on EX1 activity (Ramel et
al., 2013).
In addition to the light motivated formation of singlet oxygen in
photosynthetic tissue, singlet oxygen can be produced enzymatically by the action of
lipoxygenases followed by decomposition of the lipid peroxides that are terminated
by the Russell mechanism (Miyamoto et al., 2007). Other reactions include
superoxide dismutation coupled to myeloperoxidase activity as shown to occur during
phagocytosis (Steinbeck et al., 1992) as well as in a reaction between superoxide and
H2O2 via Haber-Weiss mechanisms (Khan and Kasha, 1994). However, the relevance
of these alternative sources in the production singlet oxygen in plants and their stress
responses is unknown.
As described above singlet oxygen production and subsequent stress gene
induction is chiefly thought to occur via photodynamic involvement of chloroplasts.
In this work, we first applied bioinformatic tools to assess the global involvement of
ROS in a set of diverse biotic and abiotic stresses. We observed that in many stress
situations co-regulated genes displayed high correlation with the transcriptome
Downloaded from on June 15, 2017 - Published by www.plantphysiol.org
Copyright © 2014 American Society of Plant Biologists. All rights reserved.
7
generated by dark-light transitions of the flu mutant. However, that analysis revealed
that in the case of wounding, similar transcriptome patterns occurred in the absence of
light. Indeed, we show that the expression of stress-related genes can occur
independent of light and in nonphotosynthetic tissue as well. Furthermore, in-vivo
imaging with the highly specific Singlet Oxygen Sensor Green (SOSG) probe showed
stress-induced singlet oxygen production in the dark originating from multiple
cellular sources. Collectively, our results are consistent with ubiquitous formation of
singlet oxygen during stress suggesting it can serve a broader signaling function.
Results
Singlet oxygen generates a transcriptome footprint in many biotic and abiotic
stresses
To assess the involvement of ROS in plant response to environmental stress
conditions we collected transcriptomic data of plants exposed to diverse biotic and
abiotic stresses and examined for the occurrence of ROS-related transcriptomic
signatures. Gene expression data was collected from the following experimentsabiotic stress; osmotic, cold, drought, wound and high light, and biotic stress;
Pseudomonas syringe, Phytophthora parasitica, oligogalacturonides (OG), chitin,
elongation factor thermo unstable (EF-Tu) and flagellin22 (flg22). We employed the
bioinformatic platform, ROSMETER which compares a test transcriptome by a
vector-based correlation method to pre-selected indices of recognized ROS type. The
indices were generated from various mutants and treatments that produce ROS of
defined type and cellular source (Rosenwasser et al., 2011; Rosenwasser et al., 2013).
Both the direction and strength of the fold induction of each gene are compared and
summed over all matching genes in the index to yield a correlation value. The
methodology is identical to that used for discerning hormone footprints (Volodarsky
et al., 2009). The correlation values are presented graphically as a heat map where the
X axis is the ROS indices and the Y axis shows the examined transcriptomes (Figure
1). Significantly, the highest correlation values (red color) were found between the
transcriptome of these diverse stresses and the ROS indices derived from ozone, H2O2
and even to a greater extent to the dark to light transition transcriptome of the flu
mutant. These include in particular, the early time points i.e. flu 30 m, flu 1 h and to a
lesser extent flu 2 h (blue rectangle, Figure 1). The actual maximum correlation value
and signal size is assembled in supplemental Table S1 and ranged from 0.62 for 1 h
Downloaded from on June 15, 2017 - Published by www.plantphysiol.org
Copyright © 2014 American Society of Plant Biologists. All rights reserved.
8
OG application to a high of 0.85 for 30' flg22 treatment. Such correlation values can
be considered highly significant (Volodarsky et al., 2009; Rosenwasser et al., 2011;
Rosenwasser et al., 2013) and point to the involvement of a singlet oxygen-like
signature in those data sets.
Inspection of the analysis by the ROSMETER platform shows that in the
Flagellin sensing2 mutant (fls2) background the responses to ROS induced by the
elicitor peptide flg22, but not that induced by the elicitor EF-Tu, were abrogated
(Figure 1). The result is consistent with the fact that different receptors are involved in
the recognition of different pathogen-associated molecular patterns (PAMPS) (Zipfel
et al., 2004; Zipfel et al., 2006). In another example, ENHANCED DISEASE
SUSCEPTIBILITY1 (EDS1) is known to modulate the plant response to pathogens
recognized by resistance genes (Rustérucci et al., 2001). As shown, eds1 mutant
leaves infiltrated with P. syringe expressing the effector protein AvrRps4, showed
general attenuation in many of the ROS response (compare eds1 and Wt, Figure 1).
While diverse stress show correlation with the singlet oxygen transcriptome,
there are also many stress that do not. For example, stress initiated by the following
treatments including; heat, salt, cadmium, hypoxia, Alternaria brassicicola and
Erysiphe orontii show low correlation values (supplemental Figure S1; supplemental
Table S2). This suggests that ROS signatures of singlet oxygen are apparent only in
discrete stresses. Alternatively, the peak correlation values to singlet oxygen indices
may have been missed in those stresses due to the vagaries of timing of the sample
collections.
In general, the early time points of a time series of stress transcriptomes tend
to show the highest correlation to flu indices. For example, the correlation score to flu
was > 0.86 from flg22 WT 30 min compared to 0.39 for flg22 WT 3 h. As shown in
Figure 1, the same trend is true after comparing short and longer time points for cold
(1 and 24 h), drought (1 and 4 h), wound (10 min and 12 h), high light (30 min and 3
h), OG (1 and 3 h), P. parasitica (2.5 and 30 h) and osmotic treatments (1 and 3 h). In
some cases, the attenuation in correlation values of a particular stress appears across
the other ROS indices e.g. cold or osmotic stress. This could be an indication of the
dissipation of the stress response over time. Alternatively, e.g. in wound stress, while
the correlation values to flu weakens over time, it strengthens for the other ROS
indices (see yellow box; 10 min vs. 12 h), indicating the generation of progressive
waves of different ROS types. Methyl viologen and aminotriazole are thought to
Downloaded from on June 15, 2017 - Published by www.plantphysiol.org
Copyright © 2014 American Society of Plant Biologists. All rights reserved.
9
promote the production of superoxide in the mitochondria and H2O2 in the
peroxisomes, respectively. The extended time points of these treatments (more than 7
h) show a weak degree of overlap with many stresses. It may indicate that the
extended treatments cause extensive cellular damage stimulating multiple signal
sources. Importantly, light and dark wound transcriptomes show a similar pattern of
correlations to the flu transcriptome (see white box, Figure 1). This suggests that such
ROS signatures may not depend on light.
Tocopherols are known scavengers of lipophilic ROS, having a role in
preventing the accumulation of lipid peroxides. The mutant vte2, is defective in
tocopherols biosynthesis and was shown to accumulate products of none enzymatic
lipid peroxidation (Sattler et al., 2006). The transcriptome of this mutant shows strong
correlation to the flu indices, and ozone (Figure 1, supplemental Table S1).
Noticeably, in addition to the correlation to flu, both hydrogen peroxide and ozone
indices are highly correlated to many of the stress treatments (Figure1, supplemental
Table S1). In this respect, it is of interest that in plants ozone interacts with the
ubiquitous ascorbic acid to produce singlet oxygen (Kanofsky and Sima, 1995).
Furthermore, singlet oxygen will readily react with ascorbic acid to produce hydrogen
peroxide (Kramarenko et al., 2006), indicating the possibility of rapid ROS interconversion. Interestingly, Cat2 0 h and As-Aox showed a marked degree of negative
correlation with many stresses. The former is a catalase deficient mutant (time 0
represent control, subsequent time points of 3 and 8 h indicate exposure time to high
light) while the latter represents mutant lines for the mitochondrial alternative oxidase
(Figure 1). The negative correlation may represent compensatory mechanisms for
ROS scavenging induced within these mutants.
A common gene set is induced in multiple abiotic and biotic stresses and light
dark transition of the flu mutant
We defined a core gene set (CGS) of transcripts by seeking differentially
expressed genes that are common to the multiple stresses in Figure 1 as well as the
signature of singlet oxygen. A list of 118 CGS was compiled from genes differentially
regulated in flu 60 min and at least 8/11 stresses (Figure 2A, supplemental Table S3).
The stresses chosen are from the earliest time points available e.g. 10' after wounding
(Figure 2A). Nearly all the genes common to biotic and abiotic type stresses and
nearly all abiotic-type stress induced genes showed overlap with the flu transcriptome
Downloaded from on June 15, 2017 - Published by www.plantphysiol.org
Copyright © 2014 American Society of Plant Biologists. All rights reserved.
10
(118/122 and 147/158, respectively; Figure 2A). Note the CGS are not necessarily
exclusively induced by singlet oxygen but may respond to different ROS sources.
Gene onthology (GO) analysis of members of the CGS, carried out in the
‘agriGO’ platform (http://bioinfo.cau.edu.cn/agriGO/index.php) (Du et al., 2010),
shows 60-fold over-representation of response to chitin and ~ 40 fold
overrepresentation of response to carbohydrate stimulus. Other prominent features are
response to chemical stimulus, response to wounding and immune system processes
showing between 5 to 20-fold over-representation (supplemental Figure S2 and
supplemental Table S4). Significantly, transcription factors of the WRKY and
ETHYLENE RESPONSIVE ELEMENT BINDING FACTOR (ERF) families known to
be associated with stress responses were prominent in the CGS (supplemental Table
S3). Furthermore, analysis of CGS promoters for known cis-elements revealed
overrepresentation of the W-box promoter motif (P value = 2.2 e-64). Such motifs
were shown to recruit WRKY transcription factors (Rushton et al., 2010). W-box and
ABRE motifs have been identified as ROS responsive and ERF6 can bind ROSresponsive cis-acting elements (Wang et al., 2013).
The CGS assembled here shows a degree of similarity to other compilations of
common stress transcripts including; ‘common stress responsive genes’ that were
defined by k-means clustering of multiple stresses (Ma and Bohnert, 2007) and ‘rapid
wound responsive genes’ set (RWR) (Walley et al., 2007) (Figure 2 B). In ‘common
stress responsive genes’ ABRE promoter motifs were shown to be over-represented
while rapid stress cis-regulatory elements (RSRE) were defined for RWR genes.
However, these elements were far less prominent in the CGS than the W-box
promoter motif (compare: W-box motif, P value = 2.2 e-64; RSRE, P value = 2.1 e-11;
ABRE, P value = 9.3 e-9; supplemental Table S5) indicating distinct regulatory
characteristics of members of the CGS.
Photodynamic production of singlet oxygen can induce CGS transcripts
The fact that multiple stresses correlate with flu indices may indicate that
singlet oxygen is an intermediate in this process, or that multiple stress pathways
activate common flu-like stress signatures. In order to differentiate between these
possibilities, several approaches were taken; first we examined if the generation of
intracellular singlet oxygen could itself induce CGS. Gene probes were prepared for 6
genes from the CGS and used for quantitative reverse transcriptase PCR (Q-RT-
Downloaded from on June 15, 2017 - Published by www.plantphysiol.org
Copyright © 2014 American Society of Plant Biologists. All rights reserved.
11
PCR) analysis; among them, AT5G17350, that was previously reported to be specific
for singlet oxygen (Op den Camp et al., 2003). Rose Bengal, as a photosensitizer,
generates singlet oxygen in a light dependent manner (Knox and Dodge, 1984;
Fischer et al., 2005). Plants were incubated with rose bengal in darkness and then
exposed to light (120 μE m-2 sec-1) for 6 h. When compared to the control, treatment
with 0.2 mM rose bengal showed significant induction of the markers gene after 6 h
of exposure to light (Figure 3A). Importantly, under those conditions, FER1, a marker
gene for H2O2 accumulation (Op den Camp et al., 2003) did not change significantly
(supplemental Figure S3A).
Application of DCMU (3-(3,4-dichlorophenyl)-1,1-dimethylurea) and high
light treatment was used as an alternative method to gauge the possible effect of
cellular singlet oxygen generation on CGS subset expression. In isolated membranes,
it was shown that DCMU binding to the QB binding site of photosystem II inhibits
electron transport facilitating the formation of excited chlorophyll. This generates
singlet oxygen in addition to basal production of other ROS (i.e. hydroxyl and
superoxide radicals) that are produced in high light (Fufezan et al., 2002). Low
concentration of DCMU (0.25 µM) did not result in any gene induction, while 25 µM
DCMU led to induction of stress-related transcripts (Figure 3B). In line with these
results, at low concentration of DCMU (0.25 µM) the ratio of Fv/Fm was not affected
in comparison to the control plants, while 25 µM DCMU led to inhibition of PSII
(supplemental Figure S4). Hence, induction of CGS was correlated to the
physiological state of the photosynthetic system. In this case, FER1 expression level
changed significantly in response to DCMU and the high light conditions
(supplemental Figure S3B). This is likely the result of high light stress conditions in
the presence of DCMU that generate multiple forms of oxidative stress including
H2O2 (Karpinski et al., 1999; Fufezan et al., 2002). In all, the results show that singlet
oxygen generated from varied sources can induce a subset of CGS transcripts.
Induction of CGS does not require light or photosynthetic tissue
The major source of singlet oxygen has been reported to be the photodynamic
activity of the chloroplast, however, analysis by the ROSMETER platform suggested
that the correlation with the flu transcriptome did not necessarily depend on light
(white box, Figure 1). Hence, it was important to establish if stress-induction of the
CGS shows light dependency. To test this, plants were wounded in the dark. As
Downloaded from on June 15, 2017 - Published by www.plantphysiol.org
Copyright © 2014 American Society of Plant Biologists. All rights reserved.
12
shown in Figure 3C, the CGS probe set showed rapid induction after wounding in the
dark. In contrast, the expression level of FER1 was not significantly changed
(supplemental Figure S3C). To further examine whether transcripts activation
depends on photosynthetic tissue, cotyledons were removed by cutting and the
expression levels of CGS subset were examined in the roots. A trend of transient
increase in all CGS genes examined was observed that peaked between 8 and 12 min,
in which 4/6 genes expression profiles show statistical significance (supplemental
Figure 5A). However, the expression levels of the H2O2 marker gene, FER1, did not
change significantly (supplemental Figure S5B). Similarly, application of flg22 in the
dark resulted in significant induction of stress related genes (Figure 3D). In contrast,
the expression level of FER1 remained unchanged (supplemental Figure S3D).
Wound-induced genes showed a high correlation to flu indices. EX1 and EX2
were previously shown to be required for expression of flu-dependent induction of
genes in dark to light transition (Lee et al., 2007). To examine if they impact on
wound mediated gene induction we followed the expression of CGS subset in ex1/ex2
double mutant line. As shown in supplemental Figure S6A, the CGS probe set
retained their wound responsiveness in the ex1/exe2 mutant background. This
indicates that despite the high correlation values of the transcriptome of wound stress
to flu indices the wound-induction of genes is independent of EX1 and EX2 activity.
As expected, FER1 expression level did not change significantly in response to
wounding of the mutant line (supplemental Figure 6B). Thus, it is likely that wound
induced CGS is not controlled by EX1 and EX2. Collectively the results validate the
transcriptome analysis and show that CGS stress-induced expression can be
independent of light and will occur in non-photosynthetic tissue.
Stress-induced accumulation of singlet oxygen in the dark and in nonphotosynthetic tissue
The fact that CGS are induced by singlet oxygen and that many stress yield
singlet oxygen signatures, prompted us to examine if singlet oxygen could be detected
in-vivo as an intermediate during the varied stress responses. The singlet oxygen
sensor green (SOSG) is a two-component trap-fluorophore system generating the
fluorescent endoperoxide upon specific interaction with singlet oxygen (Gollmer et
al., 2011). SOSG is specific for singlet oxygen and will not react with other ROS
including superoxide or hydrogen peroxide (Flors et al., 2006). Unlike the specific
Downloaded from on June 15, 2017 - Published by www.plantphysiol.org
Copyright © 2014 American Society of Plant Biologists. All rights reserved.
13
dansyl-based DanePy singlet oxygen probe (Hideg et al., 2002) its fluorescence
increases upon interaction with singlet oxygen. Whole flu mutant leaves shifted from
dark to light were previously shown to display SOSG fluorescence (Flors et al.,
2006). We first examined its capacity to detect rapid sub-cellular signals by
incubation of root tissue with SOSG and the singlet oxygen generator rose bengal.
Tissue was then spot illuminated at the absorption peak of rose bengal. A transient,
SOSG signal was obtained after illumination (Figure 4A). Quantitative measurement
of the activation event showed a sharp transient increase of SOSG signal (~ 5 fold)
together with a decrease in the fluorescence from rose bengal (Figure 4B). This
indicates that intracellular SOSG can report a rapid singlet oxygen burst that rapidly
decays due to diffusion from the source.
The SOSG probe was used to examine if singlet oxygen is formed in vivo in
stresses that were shown to generate a flu-type transcriptome signal (Figure 1). Light
and chloroplast dependency were further evaluated by measuring SOSG signal in dark
adapted, non- photosynthetic root tips. In the absence of treatment, fluorescence from
SOSG was visible at a low level in multiple cellular compartments (Figure 4C; -flg22,
top row). This indicates that a low level of singlet oxygen generated by organelles is
part of the basal cellular metabolism. In order to identify those subcellular
compartments we employed cell markers including; DAPI as a nuclear marker,
ScCOX4-mCherry (Nelson et al., 2007) as a mitochondrial marker and AtPEX5-CFP
as a peroxisomal marker (Tian et al., 2004). As shown in Figure 4C, cells of the root
epidermal layer in the maturation zone, showed faint SOSG signal localized to the
nucleus and mitochondria (note merge Figure 4C, upper row). Nuclear localization of
SOSG has been reported previously for mesophyll cells (Hideg, 2008). Although in
some cases the signal in the nucleus was variable, it could clearly be detected in
structures within the nucleus (Supplemental Figure S7). In plants treated with flg22,
the distribution of SOSG fluorescence was similar, though the intensity was higher in
comparison to the untreated plants (Figure 4C, +flg22, SOSG channel). In a similar
manner, the peroxisomal marker showed a marked degree of overlap with SOSG
fluorescence (Figure 4D). As shown, not all the labeled organelles displayed SOSG
fluorescence (Figure 4C-D). The disparity in signal overlap appears to be greater
between the peroxisomal marker and the SOSG signal. Quantitative scanning of
organelles identified by their respective markers showed that > 85% of the
peroxisomes and 97% of the mitochondria contain more SOSG fluorescence relative
Downloaded from on June 15, 2017 - Published by www.plantphysiol.org
Copyright © 2014 American Society of Plant Biologists. All rights reserved.
14
to the nearby surroundings (Figure 4E). Thus in all, SOSG signal could be detected in
varied cellular compartments; it localized to the mitochondria, nuclei and
peroxisomes and is elevated after elicitor treatment.
To detect singlet oxygen as a result of wound stress, seedlings were pretreated with both rose bengal and SOSG and placed in custom-designed slide
retainers. The experimental setup is exemplified in Figure 5A (left). The cotyledons
were then crushed with a hemostat and a non-contiguous root section ~1 cm from the
hypocotyl was examined for SOSG fluorescence. A rapid rise in SOSG signal in
systemic root tissue could be detected within 1-3 min after wounding. The same tissue
was subsequently illuminated by white light irradiation. As shown in Figure 5A
(right), a comparable and incremental change in the SOSG fluorescent signal was
generated as a result of successive photodynamic activation of rose bengal, supporting
the reliability of the wound-induced SOSG signal. To examine cell tissues that show
response, root tissue was scrutinized before and after wounding by measuring the
fluorescent signal from sequential root cell layers by confocal microscopy. The
maximum depth from the surface was measured using a fixed size of 'region of
interest' (ROI) starting at the surface and penetrating 20 μM, in which the root area
expands 4-5 fold (Figure 5B, left panel). Consecutive Z scans were carried out at 925,
595 and 236 s before wounding (Figure 5B, right panel). The Z scans show basal level
fluorescent signal that increases as a result of the expansion of the root section
measured. After wounding, a uniform increase in fluorescence was observed in almost
all cell layers (Figure 5B right panel, 207 and 540 s). At 20 μM depth the increase
detected is less pronounced. Cell layers at this depth are either less sensitive to
alteration in singlet oxygen, or due to the increased depth the layers above absorb
both the excitation and emission. In all, the systemic root tissue showed rapid woundinduced production of singlet oxygen. The results are consistent with previously
reported increase in SOSG fluorescence after 30' in whole wounded Arabidopsis dark
adapted leaves (Flors et al., 2006).
The generation of singlet oxygen was next examined in vivo for other stresses
in non photosynthetic tissue. In drought stress, plants were pre-incubated with SOSG
and subjected to 15 min of drought treatment. As shown in Figure 6 (upper panel) a
significant increase in fluorescence was evident. When flg22 peptide was applied to
seedlings, 1 h prior to addition of SOSG a significant rise in fluorescence was
detected (Figure 4C, D; Figure 6, middle panel). In contrast, after rotenone treatment
Downloaded from on June 15, 2017 - Published by www.plantphysiol.org
Copyright © 2014 American Society of Plant Biologists. All rights reserved.
15
the tissue showed a basal level of fluorescence that was significantly reduced
compared to control (Figure 6, lower panel). This is consistent with analysis carried
out on the ROSMETER platform that showed that Rotenone, a known inhibitor of
mitochondrial complex I (Garmier et al., 2008), displayed negative correlation to the
transcriptome activity of the flu mutant (Figure 1, top line; correlation value -0.292 to
flu 30 min). Unlike its effect on animal mitochondria, rotenone does not induce ROS
in plant mitochondria (Garmier et al., 2008).
SOSG is currently the most sensitive method for measurement of singlet
oxygen (Nakamura et al., 2011) and the only in-vivo method providing sub-cellular
resolution. Electron paramagnetic resonance (EPR) spectroscopy has been extensively
used to measure light-induced singlet oxygen in isolated chloroplasts (Hideg et al.,
1994; Hideg et al., 2000). In that case, the specific cell permeable singlet oxygen trap
2,2,6,6-tetramethylpiperidine (TEMP) after reacting with singlet oxygen was
converted to the nitroxide radical (TEMPO), which can then be detected by EPR
(Lion et al., 1976). We applied this approach to drought-treated leaf tissue in the dark
as such treatment produced a robust fluorescent signal from SOSG in root tissue
(Figure 6, center) as well as a singlet oxygen associated transcriptomic signature in
leaf tissue (Figure 1). Leaf discs were treated with TEMP and kept either under moist
conditions or transferred to dry filter paper for 1-3 h (see Materials and Methods). In
all control and treated samples, a background EPR signal was obtained indicating the
presence of TEMPO. This signal is likely the result of wound reactions in the discs or
residual TEMPO in the TEMP reagent. Significantly, increases in the amount of
TEMPO of 0, 60 and 40% were detected at 1, 2 and 3 h of dry treatment as compared
to wet control, respectively (Figure 7). The results confirm the presence of singlet
oxygen in drought stress treated samples in the dark. In all, the results show that
measurements of singlet oxygen formation recapitulate the predictions made by the
ROSMETER tool and lend credence to the idea that singlet oxygen is an actual
intermediate in the plant stress response.
Discussion
We show correlation between transcriptome signatures from diverse stresses and the
flu transcriptome. This includes wound-induced expression of genes in the dark.
Singlet oxygen has previously been implicated as the major toxicity factor in
chloroplast-driven photooxidative cell death. It is also a causal agent in hypersensitive
Downloaded from on June 15, 2017 - Published by www.plantphysiol.org
Copyright © 2014 American Society of Plant Biologists. All rights reserved.
16
immune responses that occur in the light (Triantaphylidès et al., 2008; Triantaphylidès
and Havaux, 2009; Kim et al., 2012; Zoeller et al., 2012). Additionally, singlet
oxygen is the major ROS agent produced in the flu mutant during dark to light
transition (Op den Camp et al., 2003). However, the bioinformatic and CGS
expression analysis show that photodynamic processes are not essential for induction
of stress genes: these genes are induced by stresses in the dark and in nonphotosynthetic tissue. Nevertheless, it is likely that light and chloroplasts have a role
in maintaining and augmenting stress-signal strength (Morker and Roberts, 2011).
The bioinformatic analysis alone cannot differentiate between the possibility that
singlet oxygen is an actual intermediate or that different stresses including singlet
oxygen converge into one common pathway. However, the actual detection of singlet
oxygen in diverse stress and by 2 independent methods, SOSG fluorescence and
TEMP oxidation, suggests that singlet oxygen is indeed an intermediate.
The highest correlation of stresses to the flu transcriptome was generally found
between the early time points after stress initiation suggesting commonalty in the
initial response that is later transformed to be stress-specific. A significant number of
CGS genes represent transcription factors (24/118; supplemental Table S3); of those,
8/24 belong to the WRKY family of transcription factors. Such factors are able to
positively and negatively regulate other functions as well as their own expression
(Rushton et al., 2010). Furthermore, analysis of all CGS promoters showed highly
significant enrichment for W-box motifs known to recruit WRKY transcription
factors. Another distinct transcription factor group (5/24) belongs to the ERF family.
Of them, AtERF-1,5, and 6 belong to the B-3 subfamily associated with defense
related phytohormone responses (Nakano et al., 2006). This indicates that WRKY and
ERF transcription factors play a dominant early role in the regulation of plant
response to stress underlying the singlet oxygen footprint. The expression of WRKY
transcription factors induced by flg22 have been shown to be down-regulated in the
chloroplast localized CALCIUM-SENSING RECEPTOR (CAS) gene mutant, cas-1,
implying control of stress gene expression by chloroplast functions (Nomura et al.,
2012). However, the control of genes by CAS is quantitative, moreover, over 1/3 of
the flg22-induced genes escape CAS regulation. This indicates that, as shown here,
additional CAS–independent pathways can participate in stress regulation.
Genevestigator currently collates 2213 experimental conditions
(https://www.genevestigator.com/gv/plant.jsp) (Hruz et al., 2008). Scanning these
Downloaded from on June 15, 2017 - Published by www.plantphysiol.org
Copyright © 2014 American Society of Plant Biologists. All rights reserved.
17
with a profile composed of CGS members showed that about 10% of the conditions
exhibited enhanced expression of CGS (defined by 50% of the CGS members
displaying at least a 2 fold difference; Supplemental Table S6). The conditions
include; transcriptomes of mutants, hormone treatments and comparisons of
development stages. The results of this inspection suggest that singlet oxygen
production is a pervasive feature in many plant transcriptome responses and is not
limited to conditions of stress.
The regulation of gene induction by singlet oxygen is complex and dependent
on its source. Thus both DCMU and flu-generated signaling have been shown to be
attenuated in ex1 and ex1/ex2 mutants (Wagner et al., 2004; Lee et al., 2007).
However, the role of the EX genes appears to be limited to moderate states of
chloroplast oxidative stress (Lee et al., 2007; Kim et al., 2012). Indeed, only 3.5% of
the genes were down regulated in the ex1/ex2 seedlings in mild light stress (Kim and
Apel, 2013). In addition, EX genes do not appear to modulate the chlorina1,
photosystem mutant (Ramel et al., 2013) or the induction of genes by β-cyclocitral, a
product of singlet oxygen activity (Ramel et al., 2012 b). Similarly as shown here,
wounding, which also generated singlet oxygen and induced CGS expression, was not
controlled by EX genes. Therefore, the plant transcriptome response to singlet oxygen
producing stresses is complex; representing subsets of genes, some controlled by EX,
some represented by CGS and some perhaps relate to more extensive cellular damage.
Chlorophyll photodynamic reactions as well as other potential cellular
photosensitizers e.g. prophyrins, flavins and quinones all require light to generate
singlet oxygen. What then are the potential sources of singlet oxygen in the dark? The
localization of fluorescence of SOSG to specific organelles (Figure 4) may indicate
that those organelles or their membranes are the preferred sites of singlet oxygen
production. Alternatively, the organelle or their membranes may have preferential
chemical affinity for the fluorescent derivative of activated SOSG that are made
elsewhere in the cell. The availability of new fluorescent probes or novel SOSG
derivatives may serve to differentiate between these possibilities. However, the result
of localized activation of SOSG by rose bengal does imply that activated SOSG can
be detected where it is made (Figure 4A, B).
Singlet oxygen generation can occur in the dark in multiple ways. Lipid
peroxidation, a source of singlet oxygen, occurs when a membrane component such
as fatty acid, undergoes oxidation (Miyamoto et al., 2007). Peroxidation might result
Downloaded from on June 15, 2017 - Published by www.plantphysiol.org
Copyright © 2014 American Society of Plant Biologists. All rights reserved.
18
from direct assault of membrane lipids by reactive oxygen species. Importantly,
termination reactions between such hydroperoxyls result in the production of singlet
oxygen (Miyamoto et al., 2007). Additionally, specialized enzymes such as
lipoxygenases and cyclooxygenases have also been implicated in singlet oxygen
production. Lipoxygenases from soybean are able to catalyze the formation of singlet
oxygen in vitro, most likely via production of peroxy radicals, which recombine
through a Russell mechanism (Kanofsky and Axelrod, 1986).
In addition to lipid-derived origins for singlet oxygen production, membranefree dark reactions have also been proposed. In animal neutrophils, the combination
of NADPH oxidase and myeloperoxidase activity during immune response produces
copious amounts of antibiotic singlet oxygen under physiological conditions
(Steinbeck et al., 1992). However, the existence of such specialized sources is
unknown in plants. Superoxide can react with H2O2 via Haber-Weiss mechanism to
form singlet oxygen (H2O2 +O2-
OH- +OH. +1O2) (Kellogg and Fridovich, 1975;
Khan and Kasha, 1994), although the efficiency of singlet oxygen production by these
ROS in vivo is not known (MacManus-Spencer and McNeill, 2005). None the less,
the accumulation of superoxide and hydrogen peroxide is readily detected in local or
systemic wounded tissue (Sagi et al., 2004; Warwar et al., 2011) and in the immune
responses (Dubiella et al., 2013). A possible source could be NADPH oxidase-like
and superoxide dismutase activity as has been shown for elicitor and salt-induced
accumulation of ROS (Leshem et al., 2006; Ashtamker et al., 2007; Suzuki et al.,
2011). Similarly, biotic elicitors are known to initiate ROS, likely through activation
of calcium and phosphorylation cascades (Sagi and Fluhr, 2001; Fluhr, 2009; Dubiella
et al., 2013).
The fluorescent signal from SOSG was prominent in mitochondria and
peroxisomes (Figure 4E). Perturbation of mitochondrial metabolism can result in
production of superoxide and H2O2 (Noctor et al., 2007; Miller et al., 2010). It was
recently shown that mitochondria and peroxisomes work in a successive manner
generating oxidative signals in response to induction of senescence (Rosenwasser et
al., 2011). Consistent with this, it is of interest that mitochondrial mutants of
alternative oxidase activity are negatively regulated with many of the stresses (see
AS-Aox1 and TDNA-Aox1; Figure 1). Negative correlation might result from the
enhancement of scavenging systems; for example, alternative oxidase couples the
oxidation of mitochondrial ubiquinol to the reduction of oxygen to water. A decrease
Downloaded from on June 15, 2017 - Published by www.plantphysiol.org
Copyright © 2014 American Society of Plant Biologists. All rights reserved.
19
in alternative oxidase activity e.g. AS-Aox1 would raise the fully reduced
mitochondrial ubiquinol levels that could readily participate in the quenching of
singlet oxygen (Triantaphylidès and Havaux, 2009).
Cross-talk between ROS species in stress signaling has been noted (Laloi et
al., 2007; Rosenwasser et al., 2013) and a degree of overlap exists between the flu
signature and other types of ROS, particularly H2O2 and ozone (Figure 1,
supplemental Table S1 and S6). The detection of additional ROS signatures may be
through inter-conversion mechanisms. For example, ozone is readily converted into
singlet oxygen in reactions with cysteine, glutathione or ascorbate (Kramarenko et al.,
2006). The correlation with hydrogen peroxide is less anticipated; based on studies of
cytosolic calcium release, Arabidopsis seedlings perceive ozone and hydrogen
peroxide independently (Evans et al., 2005). Furthermore, plants over-expressing the
chloroplast THYLAKOID-BOUND ASCORBATE PEROXIDASE (tAPX) rapidly
dissipate chloroplast-based hydrogen peroxide signals; but increase their sensitivity to
the flu mutation, suggesting that the 2 signals, singlet oxygen and hydrogen peroxide
antagonize each other (Laloi et al., 2007). Thus, either hydrogen peroxide conversion
to singlet oxygen via Haber Weiss reactions is active in most stresses; or alternatively,
the high degree of similarity obtained by ROSMETER analysis is due to the fact that
CGS can react to multiple independent ROS inputs.
How does singlet oxygen participate in signaling pathways and induce CGS?
One possibility is that, as with other ROS, their production will result in specific
scavenger depletion and thus impact on cellular redox to generate signals. The ability
of singlet oxygen to serve as a direct signaling molecule is considered unlikely due to
its reactivity. However, some evidence has shown that within the cellular milieu its
half life may be considerably longer (Snyder et al., 2006). Other longer range effects
may be indirect and involve the oxidation of β-carotene leading to the accumulation
of the volatile β-cyclocitral (Ramel et al., 2012 b). The results shown here suggest that
singlet oxygen production is a common feature of many stresses. Its origin and how it
may participate in signaling and how ultimately specificity is achieved need to be
further explored.
Materials and Methods
Plant Material and Growth Conditions
Downloaded from on June 15, 2017 - Published by www.plantphysiol.org
Copyright © 2014 American Society of Plant Biologists. All rights reserved.
20
Arabidopsis thaliana ecotype Col-0 were vernalized for 2 d at 4 °C and grown
under white light (90 μE m-2 sec-1). In the following experiments: wounding, response
to rose bengal, DCMU and high light, plants were grown for 4-5 weeks in soil in a 10
h light/14 h dark cycle at 21°C. For imaging and examination of stress related
transcripts in the following experiments: wounding done in the background of ex1/ex2
or in roots and application of flg22, 4-7 d old seedlings were grown on 0.8% (w / v)
solid agar (Duchefa) medium containing full strength B5 Gamborg’s nutrients
(Duchefa) adjusted to pH 5.8. Seedlings were surface sterilized according to (Warwar
et al., 2011) and grown in vertically positioned plates under 16 h light/8 h dark cycle
at 21 °C.
RNA Extraction, cDNA Synthesis and Q-RT- PCR analysis
RNA was extracted from 100 mg of frozen tissues using a standard Trizol
extraction method (Sigma-Aldrich). For microarray experiment total RNA was
extracted using the RNeasy Mini Kit (Qiagen). DNase 1 (Sigma-Aldrich) treated
RNA was reverse transcribed using a high-capacity cDNA reverse transcription kit
(Applied Biosystems). cDNA synthesis was according to the manufacturer's
instructions with the exception that oligo dT was added to the random hexamers used
for priming. In the microarray experiment random hexamers were excluded all
together. For Q-RT- PCR (Quantitative reverse transcriptase PCR) analysis, the
SYBR green method (KAPA biosystems) was used, on a Step One Plus platform
(Applied Biosystems) with a standard fast program. Q-RT- PCR primers were
designed in Primer Express 2 or 3 software (Applied Biosystems). All Q-RT-PCR
primer sequences are listed in Supplemental Table S7.
Treatments
For wounding as shown in Figure 3C, dark adapted (1h) plants were wounded
by clamping 3 leaves 3 times across the midvein with a hemostat. Wounding and
sample collecting was done in a dark room under dim illumination. A pool of leaves
from 3 different plants per sample was collected 30' after wounding. Wounding in the
ex1/ex2 and root experiments was administered by separating shoot from root. For
imaging experiments custom-designed slide retainers with double sided tape spacers
were used to avoid mechanical pressure on the root by the cover slip. For flg22
application, seedlings were grown on vertical agar plates. For the gene expression
Downloaded from on June 15, 2017 - Published by www.plantphysiol.org
Copyright © 2014 American Society of Plant Biologists. All rights reserved.
21
experiment 7 d old seedlings were transferred to plain liquid medium (control) or
medium supplemented with 1 µM flg22. They were then incubated for 140' in
darkness, collected and frozen in liquid nitrogen. About 8 seedlings were collected
per sample. For imaging experiments 4-6 d old seedlings were incubated for 80' in 1 x
PBS as control or 1 x PBS supplemented with 10 µM flg22. 100 µM SOSG
(Invitrogen) was added 20' before the end of the incubation time. For treatment with
rose bengal, rose bengal (Sigma-Aldrich) was dissolved in water to a final
concentration of 0.2 mM with 0.01% Silwet L-77 (LEHLE seeds). Plants were
sprayed with 0.01% Silwet L-77 as control or 0.01% Silwet L-77 with 0.2 mM rose
bengal. They were then kept 1h in darkness to allow dye uptake before being exposed
to light intensity of 120 μE m-2 sec-1 for 6 h.
For DCMU and high light treatment, DCMU (Riedel-de Haën) was
resuspended in ethanol to a stock solution of 100 mM and then diluted to a final
concentration of 0.25 and 25 μM in 0.01% Silwet L-77. Plants were sprayed with
0.01% Silwet L-77 as control or 0.01% Silwet L-77 with the indicated concentrations
of DCMU and kept in dark for 9 h. Plants were transferred to a greenhouse and
exposed to day light with intensities ranging from 200-1000 μE m-2 sec-1 (sunlight) for
7.5 h. For Fv/Fm measurements, plants were transferred to complete darkness for 15'
prior to being measured in a PAM system (Heinz Walz). For Rotenone treatment,
Rotenone (Sigma-Aldrich) was resuspended in chloroform to a stock solution of 120
mM and then diluted to a final concentration of 40 μM. Seedlings were incubated
with 0.03% chloroform as control or 40 µM rotenone for 100 min, 100 µM SOSG
was added 20' before the end of the incubation time. Incubations were in darkness.
For drought treatment, 5 d old seedlings were incubated with SOSG for 20' and then
transferred for 15' to to wet or dry Whatman paper.
Confocal and Fluorescent microscopy Imaging and image analysis
Inverted fluorescence microscope (Olympus IX71) was used for quantification
of SOSG intensity. The SOSG signal was a YFP filter, EX: 500 ± 10 nm, EM:535±
15 nm. As SOSG itself may act as a photosensitizer (Gollmer et al., 2011) low
excitation energies were employed. Confocal imaging was conducted with an inverted
confocal microscope (Olympus Flow View FV1000) equipped with UPlanSApo 20x
objective (numerical aperture of 0.75) for wound studies and UPlanSApo 60x oil
objective (numerical aperture of 1.35) for the localization studies. Detailed
Downloaded from on June 15, 2017 - Published by www.plantphysiol.org
Copyright © 2014 American Society of Plant Biologists. All rights reserved.
22
description of filters, lasers and incubation periods can be found in Supplemental
Table S8. For quantification and image enhancement the following programs were
used: Fiji (Schindelin et al., 2012) and Olympus FV10-ASW 3.1 viewer. SOSG
fluorescence intensity in root tips was determined by constructing a minimal region of
interest (ROI) suitable for all root tips examined in a given experiment. The mean
gray value of the selected ROI was computed and background was subtracted.
Compartment specific quantification of SOSG signal was achieved by using the
images of specific compartments markers; e.g. AtPEX5-CFP for peroxisomes, in
order to construct multiple ROIs directed at that compartment. The ROIs were then
employed to measure the SOSG channel. Signal in the vicinity of these compartments
was acquired by displacement of the ROIs. Image enhancement was attained by
changing the brightness/contrast properties and by applying average filter to the DAPI
channel in Figure 4C (upper and lower images). When differences of SOSG intensity
were visualized the same brightness/contrast parameters were applied to allow
meaningful comparison.
EPR Spectroscopy and sample preparation
Leaf discs (50 discs, 6 mm; 8- 12 weeks old plants) were used for each
treatment. The discs were equilibrated in ddH2O in ambient light for 1hr. All further
incubation and extraction procedures were carried out in the dark. Discs were
incubated with 10 mM 2,2,6,6-tetramethylpiperidine (TEMP; Sigma Aldrich) for 30
minutes, and washed 3x with ddH2O. Samples were then placed on dry Whatman
paper for 0, 1, 2, 3 h. Wet samples were floated in ddH2O. Samples were harvested by
flash freezing in liquid nitrogen and homogenized in a shaker using glass beads in 0.2
M potassium phosphate buffer (pH 7.2) at 4 oC and then in a rotator for 15 min. The
samples were then centrifuged for 20 min at 14,000 rpm at 4 oC for 20 min, and the
supernatant extracted with 2 volumes of ethyl acetate. The upper phase of the mixture
was used in EPR spectroscopy after the addition of 100 mM H2O2 to re-oxidise
reduced hydroxylamine forms of TEMPO (Hideg et al., 2000). The EPR spectra
matched typical TEMPO triplet EPR spectra (αN=15.8 G) and were acquired at room
temperature with a Brucker ELEXYS 500 spectrometer. X-band spectra were
recorded at 9.35 GHz microwave frequency and 100 kHz modulation frequency.
Microarray data bioinformatic and statistical analysis
Downloaded from on June 15, 2017 - Published by www.plantphysiol.org
Copyright © 2014 American Society of Plant Biologists. All rights reserved.
23
GeneChip Arabidopsis ATH1 Genome Array of Affymetrix
(www.affymetrix.com) was used. Quantile normalization and background adjustment
for data were performed using RMA (Robust Multi-chip Analysis) (Irizarry et al.,
2003) in the "PARTEK Genomics Solution” software (Downey, 2006)
(http://www.partek.com). Lists of genes were generated for each stress from
transcripts that had a fold change (FC) of -1.8≥FC≥1.8 and a P-value ≤ 0.05
Supplemental Table S9. Computation of the fold changes and P-values for
downloaded data sets of the different stresses was made by the Robin software (Lohse
et al., 2010). Gene annotation was taken from TAIR8 (http://www.arabidopsis.org).
The overlapping genes between the different data sets and venn diagram were
computed by the Venny algorithm (http://stan.cropsci.uiuc.edu/cgi-bin/sift/sift.cgi).
The sources of transcriptome data for Figure 1 and Supplemental Figure 1 are
tabulated in Supplemental Table S10. Sequence data from this article can be found in
the GenBank data libraries under accession number: GSE48676.
Gene onthology (GO) and promoter analysis
GO analysis of CGS, was with the agriGO platform:
(http://bioinfo.cau.edu.cn/agriGO/index.php) (Du et al., 2010).
Promoter analysis of CGS was performed by using the following tool(http://stan.cropsci.uiuc.edu/cgi-bin/sift/sift.cgi) (Hudson and Quail, 2003).
Supplemental Material
Supplemental Figures
Supplemental Figure S1. Correlation values of Alternaria Brassicicola, Erysiphe
orontii, infections, salt treatment, heat treatment, cadmium and hypoxia examined in
the ROSMETER platform.
Supplemental Figure S2. Distribution of the highly overrepresented GO terms in the
CGS.
Supplemental Figure S3. The expression of FER1 probe in various stress treatments.
Supplemental Figure S4. Photosynthetic efficiency Fv/Fm of DCMU treated plants.
Supplemental Figure S5. Expression of stress-induced transcripts in root tissue.
Supplemental Figure S6. The expression of stress-induced transcripts in wounded
wild type and ex1/ex2 seedlings.
Downloaded from on June 15, 2017 - Published by www.plantphysiol.org
Copyright © 2014 American Society of Plant Biologists. All rights reserved.
24
Supplemental Figure S7. Confocal imaging of DAPI and SOSG fluorescence in root
tissue.
Supplemental Tables
Supplemental Table S1. Correlation scores and signature size for Figure 1.
Supplemental Table S2. Correlation scores and signature size for supplemental Figure
S1.
Supplemental Table S3. List of CGS genes and GO terms.
Supplemental Table S4. Enrichment values of GO terms for the CGS.
Supplemental Table S5. Promoter analysis of the CGS.
Supplemental Table S6. Scan of CGS expression profile in the Genevestigator data
base of transcriptome experiments.
Supplemental Table S7. Description of the primers used for qRT-PCR analysis.
Supplemental Table S8. Stains, settings and fluorescent markers.
Supplemental Table S9. Genes that changed significantly in stress treatments used to
construct the CGS.
Supplemental Table S10. Source of transcriptome data used for the bioinformatics
analysis.
Downloaded from on June 15, 2017 - Published by www.plantphysiol.org
Copyright © 2014 American Society of Plant Biologists. All rights reserved.
25
Acknowledgments
We would like to thank Dr. Klaus Apel for the kind gift of the double mutant line
ex1/ex2, Dr. Adi Avni for the kind gift of the flg22 peptide, Dr. Noam Alkan for
fruitful discussions and Vladimir Kiss for his help with confocal imaging.
Downloaded from on June 15, 2017 - Published by www.plantphysiol.org
Copyright © 2014 American Society of Plant Biologists. All rights reserved.
26
Literature Cited
Ashtamker C, Kiss V, Sagi M, Davydov O, Fluhr R (2007) Diverse subcellular
locations of cryptogein-induced reactive oxygen species production in
Tobacco bright yellow-2 cells. Plant Physiol 143: 1817-1826
Downey T (2006) Analysis of a multifactor microarray study using Partek genomics
solution. Methods Enzymol 411: 256-270
Du Z, Zhou X, Ling Y, Zhang Z, Su Z (2010) agriGO: a GO analysis toolkit for the
agricultural community. Nucleic Acids Res 38: W64-70
Dubiella U, Seybold H, Durian G, Komander E, Lassig R, Witte C-P, Schulze
WX, Romeis T (2013) Calcium-dependent protein kinase/NADPH oxidase
activation circuit is required for rapid defense signal propagation. P Natl Acad
Sci USA 110: 8744-8749
Evans NH, McAinsh MR, Hetherington AM, Knight MR (2005) ROS perception
in Arabidopsis thaliana: the ozone-induced calcium response. Plant J 41: 615626
Fischer BB, Krieger-Liszkay A, Eggen RIL (2005) Oxidative stress induced by the
photosensitizers neutral red (type I) or rose bengal (type II) in the light causes
different molecular responses in Chlamydomonas reinhardtii. Plant Sci 168:
747-759
Fischer BB, Ledford HK, Wakao S, Huang SG, Casero D, Pellegrini M,
Merchant SS, Koller A, Eggen RIL, Niyogi KK (2012) SINGLET OXYGEN
RESISTANT 1 links reactive electrophile signaling to singlet oxygen
acclimation in Chlamydomonas reinhardtii. P Natl Acad Sci USA 109:
E1302-E1311
Flors C, Fryer MJ, Waring J, Reeder B, Bechtold U, Mullineaux PM, Nonell S,
Wilson MT, Baker NR (2006) Imaging the production of singlet oxygen in
vivo using a new fluorescent sensor, Singlet Oxygen Sensor Green®. J Exp
Bot 57: 1725-1734
Fluhr R (2009) Reactive oxygen-generating NADPH oxidases in plants. In Reactive
Oxygen Species in Plant Signaling. Springer Berlin Heidelberg, pp 1-23
Fufezan C, Rutherford AW, Krieger-Liszkay A (2002) Singlet oxygen production
in herbicide-treated photosystem II. FEBS Lett 532: 407-410
Garmier M, Carroll AJ, Delannoy E, Vallet C, Day DA, Small ID, Millar AH
(2008) Complex I dysfunction redirects cellular and mitochondrial metabolism
in Arabidopsis. Plant Physiol 148: 1324-1341
Gollmer A, Arnbjerg J, Blaikie FH, Pedersen BW, Breitenbach T, Daasbjerg K,
Glasius M, Ogilby PR (2011) Singlet oxygen sensor green®: photochemical
behavior in solution and in a mammalian cell. Photochem Photobiol 87: 671679
Hideg É (2008) A comparative study of fluorescent singlet oxygen probes in plant
leaves. Cent Eur J Biol 3: 273-284
Hideg É, Barta C, Kalai T, Vass I, Hideg K, Asada K (2002) Detection of singlet
oxygen and superoxide with fluorescent sensors in leaves under stress by
photoinhibition or UV radiation. Plant Cell Physiol 43: 1154-1164
Hideg E, Spetea C, Vass I (1994) Singlet oxygen production in thylakoid
membranes during photoinhibition as detected by EPR Spectroscopy.
Photosynth Res 39: 191-199
Downloaded from on June 15, 2017 - Published by www.plantphysiol.org
Copyright © 2014 American Society of Plant Biologists. All rights reserved.
27
Hideg E, Vass I, Kalai T, Hideg K (2000) Singlet oxygen detection with sterically
hindered amine derivatives in plants under light stress. Method Enzymol 319:
77-85
Hruz T, Laule O, Szabo G, Wessendorp F, Bleuler S, Oertle L, Widmayer P,
Gruissem W, Zimmermann P (2008) Genevestigator V3: a reference
expression database for the meta-analysis of transcriptomes. Advances in
Bioinformatics 2008: doi:10.1155/2008/420747
Hudson ME, Quail PH (2003) Identification of promoter motifs involved in the
network of phytochrome A-regulated gene expression by combined analysis of
genomic sequence and microarray data. Plant Physiol 133: 1605-1616
Irizarry RA, Hobbs B, Collin F, Beazer-Barclay YD, Antonellis KJ, Scherf U,
Speed TP (2003) Exploration, normalization, and summaries of high density
oligonucleotide array probe level data. Biostatistics 4: 249-264
Kanofsky JR, Axelrod B (1986) Singlet oxygen production by soybean lipoxygenase
isozymes. J Biol Chem 261: 1099-1104
Kanofsky JR, Sima PD (1995) Singlet oxygen generation from the reaction of ozone
with plant leaves. J Biol Chem 270: 7850-7852
Karpinski S, Reynolds H, Karpinska B, Wingsle G, Creissen G, Mullineaux P
(1999) Systemic signaling and acclimation in response to excess excitation
energy in Arabidopsis. Science 284: 654-657
Kellogg EW, Fridovich I (1975) Superoxide, hydrogen peroxide, and singlet oxygen
in lipid peroxidation by a xanthine oxidase system. J Biol Chem 250: 88128817
Khan AU, Kasha M (1994) Singlet molecular oxygen in the Haber-Weiss reaction. P
Natl Acad Sci USA 91: 12365-12367
Khandal D, Samol I, Buhr F, Pollmann S, Schmidt H, Clemens S, Reinbothe S,
Reinbothe C (2009) Singlet oxygen-dependent translational control in the
tigrina-d.12 mutant of barley. P Natl Acad Sci USA 106: 13112-13117
Kim C, Meskauskiene R, Zhang S, Lee KP, Lakshmanan Ashok M, Blajecka K,
Herrfurth C, Feussner I, Apel K (2012) Chloroplasts of Arabidopsis are the
source and a primary target of a plant-specific programmed cell death
signaling pathway. Plant Cell 24: 3026-3039
Kim CH, Apel K (2013) 1O2-Mediated and EXECUTER-dependent retrograde
plastid-to-nucleus signaling in norflurazon-treated seedlings of Arabidopsis
thaliana. Mol Plant 6: 1580-1591
Knox JP, Dodge AD (1984) Photodynamic damage to plant leaf tissue by rose
bengal. Plant Sci Lett 37: 3-7
Kramarenko GG, Hummel SG, Martin SM, Buettner GR (2006) Ascorbate reacts
with singlet oxygen to produce hydrogen peroxide. Photochem Photobiol 82:
1634-1637
Krieger-Liszkay A, Fufezan C, Trebst A (2008) Singlet oxygen production in
photosystem II and related protection mechanism. Photosynth Res 98: 551564
Laloi C, Stachowiak M, Pers-Kamczyc E, Warzych E, Murgia I, Apel K (2007)
Cross-talk between singlet oxygen- and hydrogen peroxide-dependent
signaling of stress responses in Arabidopsis thaliana. P Natl Acad Sci USA
104: 672-677
Lee KP, Kim C, Landgraf F, Apel K (2007) EXECUTER1- and EXECUTER2dependent transfer of stress-related signals from the plastid to the nucleus of
Arabidopsis thaliana. P Natl Acad Sci USA 104: 10270-10275
Downloaded from on June 15, 2017 - Published by www.plantphysiol.org
Copyright © 2014 American Society of Plant Biologists. All rights reserved.
28
Leshem Y, Melamed-Book N, Cagnac O, Ronen G, Nishri Y, Solomon M, Cohen
G, Levine A (2006) Suppression of Arabidopsis vesicle-SNARE expression
inhibited fusion of H2O2 -containing vesicles with tonoplast and increased salt
tolerance. P Natl Acad Sci USA 103: 18008-18013
Lion Y, Delmelle M, Vandevorst A (1976) New method of detecting singlet oxygen
production. Nature 263: 442-443
Lohse M, Nunes-Nesi A, Krüger P, Nagel A, Hannemann J, Giorgi FM, Childs L,
Osorio S, Walther D, Selbig J, Sreenivasulu N, Stitt M, Fernie AR, Usadel
B (2010) Robin: An Intuitive Wizard Application for R-Based Expression
Microarray Quality Assessment and Analysis. Plant Physiol 153: 642-651
Ma S, Bohnert HJ (2007) Integration of Arabidopsis thaliana stress-related
transcript profiles, promoter structures, and cell-specific expression. Genome
Biol 8: R49
MacManus-Spencer LA, McNeill K (2005) Quantification of singlet oxygen
production in the reaction of superoxide with hydrogen peroxide using a
selective chemiluminescent probe. J Am Chem Soc 127: 8954-8955
Meskauskiene R, Nater M, Goslings D, Kessler F, op den Camp R, Apel K (2001)
FLU: A negative regulator of chlorophyll biosynthesis in Arabidopsis
thaliana. P Natl Acad Sci USA 98: 12826-12831
Miller G, Suzuki N, Ciftci-Yilmaz S, Mittler R (2010) Reactive oxygen species
homeostasis and signalling during drought and salinity stresses. Plant Cell
Environ 33: 453-467
Miyamoto S, Ronsein GE, Prado FM, Uemi M, Corrêa TC, Toma IN, Bertolucci
A, Oliveira MCB, Motta FD, Medeiros MHG, Mascio PD (2007)
Biological hydroperoxides and singlet molecular oxygen generation. IUBMB
Life 59: 322
Morker KH, Roberts MR (2011) Light exerts multiple levels of influence on the
Arabidopsis wound response. Plant Cell Environ 34: 717-728
Nakamura K, Ishiyama K, Ikai H, Kanno T, Sasaki K, Niwano Y, Kohno M
(2011) Reevaluation of analytical methods for photogenerated singlet oxygen.
J Clin Biochem Nutr 49: 87-95
Nakano T, Suzuki K, Fujimura T, Shinshi H (2006) Genome-wide analysis of the
ERF gene family in Arabidopsis and Rice. Plant Physiol 140: 411-432
Nelson BK, Cai X, Nebenführ A (2007) A multicolored set of in vivo organelle
markers for co-localization studies in Arabidopsis and other plants. Plant J 51:
1126-1136
Noctor G, De Paepe R, Foyer CH (2007) Mitochondrial redox biology and
homeostasis in plants. Trends Plant Sci 12: 125-134
Nomura H, Komori T, Uemura S, Kanda Y, Shimotani K, Nakai K, Furuichi T,
Takebayashi K, Sugimoto T, Sano S, Suwastika IN, Fukusaki E, Yoshioka
H, Nakahira Y, Shiina T (2012) Chloroplast-mediated activation of plant
immune signalling in Arabidopsis. Nat Commun 3: Article number: 926
Op den Camp RGL, Przybyla D, Ochsenbein C, Laloi C, Kim C, Danon A,
Wagner D, Hideg E, Gobel C, Feussner I, Nater M, Apel K (2003) Rapid
induction of distinct stress responses after the release of singlet oxygen in
Arabidopsis. Plant Cell 15: 2320-2332
Ramel F, Birtic S, Cuiné S, Triantaphylidès C, Ravanat J-L, Havaux M (2012 a)
Chemical quenching of singlet oxygen by carotenoids in plants. Plant Physiol
158: 1267–1278
Downloaded from on June 15, 2017 - Published by www.plantphysiol.org
Copyright © 2014 American Society of Plant Biologists. All rights reserved.
29
Ramel F, Birtic S, Ginies C, Soubigou-Taconnat L, Triantaphylidès C, Havaux
M (2012 b) Carotenoid oxidation products are stress signals that mediate gene
responses to singlet oxygen in plants. P Natl Acad Sci USA 109: 5535-5540
Ramel F, Ksas B, Akkari E, Mialoundama AS, Monnet F, Krieger-Liszkay A,
Ravanat J-L, Mueller MJ, Bouvier F, Havaux M (2013) Light-induced
acclimation of the Arabidopsis chlorina1 mutant to singlet oxygen. Plant Cell
25: 1445-1462
Rosenwasser S, Fluhr R, Joshi JR, Leviatan N, Sela N, Hetzroni A, Friedman H
(2013) ROSMETER: A bioinformatic tool for the identification of
transcriptomic imprints related to reactive oxygen species type and origin
provides new insights into stress responses. Plant Physiol 163: 1071-1083
Rosenwasser S, Rot I, Sollner E, Meyer AJ, Smith Y, Leviatan N, Fluhr R,
Friedman H (2011) Organelles contribute differentially to reactive oxygen
species-related events during extended darkness. Plant Physiol 156: 185-201
Rushton PJ, Somssich IE, Ringler P, Shen QJ (2010) WRKY transcription factors.
Trends Plant Sci 15: 247- 258
Rustérucci C, Aviv DH, Holt BF, Dangl JL, Parker JE (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:
2211-2224
Sagi M, Davydov O, Orazova S, Yesbergenova Z, Ophir R, Stratmann JW,
Fluhr R (2004) Plant respiratory burst oxidase homologs impinge on wound
responsiveness and development in Lycopersicon esculentum. Plant Cell 16:
616-628
Sagi M, Fluhr R (2001) Superoxide production by plant homologues of the gp91phox
NADPH oxidase. Modulation of activity by calcium and by Tobacco Mosaic
Virus infection. Plant Physiol 126: 1281-1290
Sattler SE, Mene-Saffrane L, Farmer EE, Krischke M, Mueller MJ, DellaPenna
D (2006) Nonenzymatic lipid peroxidation reprograms gene expression and
activates defense markers in Arabidopsis tocopherol-deficient mutants. Plant
Cell 18: 3706-3720
Schindelin J, Arganda-Carreras I, Frise E, Kaynig V, Longair M, Pietzsch T,
Preibisch S, Rueden C, Saalfeld S, Schmid B, Tinevez JY, White DJ,
Hartenstein V, Eliceiri K, Tomancak P, Cardona A (2012) Fiji: an opensource platform for biological-image analysis. Nat Methods 9: 676-682
Snyder JW, Skovsen E, Lambert JDC, Poulsen L, Ogilby PR (2006) Optical
detection of singlet oxygen from single cells. Phys Chem Chem Phys 8: 42804293
Steinbeck MJ, Khan AU, Karnovsky MJ (1992) Intracellular singlet oxygen
generation by phagocytosing neutrophils in response to particles coated with a
chemical trap. J Biol Chem 267: 13425-13433
Suzuki N, Miller G, Morales J, Shulaev V, Torres MA, Mittler R (2011)
Respiratory burst oxidases: the engines of ROS signaling. Curr Opin Plant
Biol 14: 691-699
Tian G-W, Mohanty A, Chary SN, Li S, Paap B, Drakakaki G, Kopec CD, Li J,
Ehrhardt D, Jackson D, Rhee SY, Raikhel NV, Citovsky V (2004) Highthroughput fluorescent tagging of full-length Arabidopsis gene products in
planta. Plant Physiol 135: 25-38
Triantaphylidès C, Havaux M (2009) Singlet oxygen in plants: production,
detoxification and signaling. Trends Plant Sci 14: 219-228
Downloaded from on June 15, 2017 - Published by www.plantphysiol.org
Copyright © 2014 American Society of Plant Biologists. All rights reserved.
30
Triantaphylidès C, Krischke M, Hoeberichts FA, Ksas B, Gresser G, Havaux M,
Van Breusegem F, Mueller MJ (2008) Singlet oxygen is the major reactive
oxygen species involved in photooxidative damage to plants. Plant Physiol
148: 960-968
Volodarsky D, Leviatan N, Otcheretianski A, Fluhr R (2009)
HORMONOMETER: A tool for discerning transcript signatures of hormone
action in the Arabidopsis transcriptome. Plant Physiol 150: 1796-1805
Wagner D, Przybyla D, op den Camp R, Kim C, Landgraf F, Lee KP, Wursch
M, Laloi C, Nater M, Hideg E, Apel K (2004) The genetic basis of singlet
oxygen–induced stress responses of Arabidopsis thaliana. Science 306: 11831185
Walley JW, Coughlan S, Hudson ME, Covington MF, Kaspi R, Banu G, Harmer
SL, Dehesh K (2007) Mechanical stress induces biotic and abiotic stress
responses via a novel cis element. PLoS Genet 3: 1800-1812
Wang P, Du Y, Zhao X, Miao Y, Song C-P (2013) The MPK6-ERF6-ROSresponsive cis-acting element 7/GCC box complex modulates oxidative gene
transcription and the oxidative response in Arabidopsis. Plant Physiol 161:
1392-1408
Warwar N, Mor A, Fluhr R, Pandian RP, Kuppusamy P, Blank A (2011)
Detection and imaging of superoxide in roots by an electron spin resonance
spin-probe method. Biophys J 101: 1529-1538
Zipfel C, Kunze G, Chinchilla D, Caniard A, Jones JDG, Boller T, Felix G (2006)
Perception of the bacterial PAMP EF-Tu by the receptor EFR restricts
Agrobacterium-mediated transformation. Cell 125: 749-760
Zipfel C, Robatzek S, Navarro L, Oakeley EJ, Jones JDG, Felix G, Boller T
(2004) Bacterial disease resistance in Arabidopsis through flagellin
perception. Nature 428: 764-767
Zoeller M, Stingl N, Krischke M, Fekete A, Waller F, Berger S, Mueller MJ
(2012) Lipid profiling of the Arabidopsis hypersensitive response reveals
specific lipid peroxidation and fragmentation processes: biogenesis of pimelic
and azelaic acid. Plant Physiol 160: 365-378
Downloaded from on June 15, 2017 - Published by www.plantphysiol.org
Copyright © 2014 American Society of Plant Biologists. All rights reserved.
31
Figure legends
Figure 1. Correlation values of select abiotic and biotic stress transcriptomes
generated by the ROSMETER platform. Shown is a color coded heat map of
correlation scores between transcriptomes of stresses (Y-ordinate) with transcriptome
indices (abscissa) of the individual ROS producing treatments compiled in the
ROSMETER platform. The predicted ROS type; ozone (O3), superoxide (O2·–),
hydrogen peroxide H2O2, singlet oxygen (1O2) and cellular location for each index are
indicated at the top of the heatmap (Rosenwasser et al., 2011). The color scale for
correlation is shown on the right. Blue, yellow and white boxes are described in the
text.
Figure 2. Delineating a common gene set for stress and singlet oxygen-related
transcripts. (A) Venn diagram showing the number of overlapping genes between:
abiotic stress (wound local and systemic leaves after 10', 1h drought and 30' of HL);
biotic stress (1h OGs, Flg22 W.t. 30', EF-Tu 30', chitin 30') and early flu responses 60
min after dark to light transition. (B) Venn diagram showing the number of
overlapping genes between CGS, RWR (Walley et al., 2007) and ‘common stress
responsive genes’ (Ma and Bohnert, 2007).
Figure 3. Expression of transcripts after stress treatments.
Q-RT- PCR of CGS transcripts from plants after various treatments. (A) Plants were
treated by spraying with 0.01% Silwet L-77 as control, or 0.01% Silwet L-77 and 200
µM rose bengal. After 1h in the darkness plants were exposed to light intensity of 120
μE m-2 sec-1 for 6 h. (B) Plants were sprayed with 0.01% Silwet L-77 as control or
0.01% Silwet L-77 or 0.25 or 25 µM DCMU as indicated. They were than kept in the
dark for 9 h. Plants were transferred to day light with average of 600 μE m-2 sec-1 for
7.5 h. (C) Dark adapted plants were wounded and processed after 30' as described in
the Materials and Methods. (D) Plants were grown on vertical agar plates for 7 d and
then transferred to liquid medium either supplemented with 1 µM flg22 or not
(control). Subsequently they were kept in darkness for 140 min before samples were
collected. ANOVA statistical test followed by contrast t-test and step-up correction
for multiple testing was used to determine significance of the difference relative to the
control. Significance is: **** < 0.001; *** < 0.01; ** < 0.05; * < 0.1. Average ± SE
(n= 3-4) are presented.
Downloaded from on June 15, 2017 - Published by www.plantphysiol.org
Copyright © 2014 American Society of Plant Biologists. All rights reserved.
32
Figure 4. Confocal imaging and analysis of SOSG fluorescence in root tissue. (A)
Root stained simultaneously with 50 µM rose bengal and 100 µM SOSG and
visualized for rose bengal (left panels) or SOSG (right panels). Upper panels show the
root before 1 sec activation by an argon laser at 559 nm (circled area). Lower panels
show the root after activation. Bar represents 10 μM. (B) Relative quantification of
the fluorescent signal emitted by rose bengal (red squares) and SOSG (green
diamonds) within the activation spot shown in panel A. Arrow indicates laser
activation. (C) Differentiation zone of the root of 6 d old seedlings expressing the
mitochondrial marker ScCOX4:mCherry stained with DAPI and SOSG. The last
column shows a merge of all channels. Seedlings were either incubated in 1 x PBS to
serve as control (upper row), or incubated with flg22 for 80' (lower row). The arrow
points to mitochondrion displaying SOSG fluorescence while the arrow head points to
an area lacking ScCOX4:mCherry fluorescence but containing SOSG fluorescence.
(D) Differentiation zone of the root of 4 d old seedling expressing the peroxisomal
marker AtPEX5-CFP stained with DAPI and SOSG. The last column shows a merge
of all channels. The AtPEX5-CFP was color coded red for better visualization of colocalization. The arrow points to peroxisome displaying SOSG fluorescence while the
arrow head points to an area lacking AtPEX5-CFP fluorescence but containing SOSG
signal. Bars represent 10 µm. (E) Percentage of peroxisomes (P) and mitochondria
(M) with SOSG fluorescent signal that is higher than the nearby surrounding area.
Figure 5. Generation of singlet oxygen in vivo by wound or by photodynamic
activation of rose bengal. (A) Left panel, a schematic diagram of the experimental
setup used to monitor wound stress. Seedlings (5 d old) were sandwiched between
slides that were separated so as not to evoke mechanical pressure (detailed in the
methods section). Cotyledons were wounded with a hemostat and imaged within the
area shown. Right panel, seedlings that were loaded with 100 µM SOSG for 1 h
followed by 50 µM rose bengal for 15', washed and analyzed in an inverted
fluorescence microscope (Olympus Ix71). A representative result is shown (n=4), a.u.,
arbitrary units. (B) Left panel, a schematic diagram of the sequential root cell layers
measured by confocal microscopy (Olympus Flow View FV1000). Measurements
started at the surface and reached 20 μM in depth, the area measured is shown with
double arrows. Right panel, quantification of the SOSG fluorescent intensity in the
Downloaded from on June 15, 2017 - Published by www.plantphysiol.org
Copyright © 2014 American Society of Plant Biologists. All rights reserved.
33
root tip before and after wounding of cotyledons. Measurements were made along the
different cell layers depicted in the left panel at different times. Seedlings were
incubated with 100 µM SOSG for 10'-20'. Representative result is shown (n=15).
Figure 6. Analysis of singlet oxygen levels in vivo after drought, flg22 and rotenone
treatments. Imaging was performed on the root tip of 5 d old seedlings. Insets above
bars display a representative result for each treatment. Upper panel, seedlings were
incubated with SOSG for 20' and then transferred to wet (control) or to dry Whatman
paper (drought) for 15' (n=4). Middle panel, seedlings were incubated with 10 µM
flg22 for 80', 100 µM SOSG were added 20' before the end of the incubation time
(n=3). Lower panel, seedlings were incubated with 0.03% chloroform (control) or
0.03% chloroform and 40 µM rotenone for 100'. SOSG (100 µM) was added 20'
before the end of the incubation time (n=3-4). A two sided t-test was performed in
order to evaluate the significance. Significance is *** < 0.01 ,** < 0.05.
Figure 7. Detection of singlet oxygen during drying treatment by EPR spectroscopy.
Arabidopsis leaf discs were incubated with TEMP for 30 min in the dark, rinsed and
left on water or subjected to dehydration treatments in the dark for the indicated
times. Tissue was frozen and processed in the dark and measured using EPR
spectroscopy as described in the Materials and Methods.
Downloaded from on June 15, 2017 - Published by www.plantphysiol.org
Copyright © 2014 American Society of Plant Biologists. All rights reserved.
1
0.8
0.6
0.4
0.2
0
-0.2
-0.4
-0.6
----
--
12h
3h
8h
3h
0h
7h
24h
12h
6h
3h
1h
30m
-0.8
24h
6h
3h
90m
30m
15m
0h
1h
1h
2h
1h
30m
Rotenone 3h
Osmotic 3h
1h
24h
Cold
1h
Drought 4h
1h
Light 1h
Dark 1h
Wound
Local 12h
10’
Systemic 10’
High light 3h
P. syringea- eds1 30’
3h
AvrRps4
W.t. 3h
P. parasitica 30h
2.5h
OG 3h
1h
Chitin 30’
EF-Tu fls2 30’
flg22 W.t. 3h
30’
flg22 fls2 30’
vte2 3d
Figure 1. Mor et. al.
Downloaded from on June 15, 2017 - Published by www.plantphysiol.org
Copyright © 2014 American Society of Plant Biologists. All rights reserved.
-1
A
B
Biotic
95
Abiotic
4
7
CGS
59
118
285
Common stress
response
22
130
28
29
9
16
800
152
flu
RWR
Figure 2. Mor et. al.
Downloaded from on June 15, 2017 - Published by www.plantphysiol.org
Copyright © 2014 American Society of Plant Biologists. All rights reserved.
Relative expression
A
C
JAZ1
RBOHD
WRKY40
ZAT10
ZAT12
AT5G17350
18
16
14
12
10
8
6
4
2
0
****
****
****
****
****
100
***
** ****
***
***
10
****
****
1
0
200
Control
Rose bengal (µM)
Local
Wounding
0.1
B
D
Relative expression
20
****
15
****
10
****
********
5
0
0
0.25
25
DCMU (µM)
16
14
12
10
8
6
4
2
0
****
********
****
****
*
Control
flg22
Elicitor
Figure 3. Mor et. al.
Downloaded from on June 15, 2017 - Published by www.plantphysiol.org
Copyright © 2014 American Society of Plant Biologists. All rights reserved.
Before
stimuli
After
stimuli
C
SOSG
B
E
300
rose bengal
sosg
250
200
150
100
50
0
0
SOSG fluorescence
> surrounding
Rose bengal
Fluorescence (a.u.)
A
%
100
95
90
85
80
75
P
20 40 60 80
Time (s)
DAPI
SOSG
ScCOX4
merge
DAPI
SOSG
Pex5
merge
- flg22
+ flg22
D
+flg22
Figure 4. Mor et. al.
Downloaded from on June 15, 2017 - Published by www.plantphysiol.org
Copyright © 2014 American Society of Plant Biologists. All rights reserved.
M
Injury
Imaging
SOSG Fluorescence (a.u)
A
Depth Root section of
(mm) the area analyzed
0
20
Epidermis
Cortex
SOSG Fluorescence (a.u)
B
180
150
120
90
60
wound
light
30
0
450
400
350
300
250
200
150
100
50
0
16
31 48 65
Time (min)
before
wound (s)
After
wound (s)
925
595
236
207
540
0 2 4 6 8 10 12 14 16 18 20
Depth (µM)
Figure 5. Mor et. al.
Downloaded from on June 15, 2017 - Published by www.plantphysiol.org
Copyright © 2014 American Society of Plant Biologists. All rights reserved.
SOSG Fluorescence (a.u)
600
500
400
300
200
100
0
***
SOSG Fluorescence (a.u)
SOSG Fluorescence (a.u)
Control
600
500
400
300
200
100
0
Drought
**
Control
120
100
80
60
40
20
0
flg22
**
Control
Rotenone
Figure 6. Mor et. al.
Downloaded from on June 15, 2017 - Published by www.plantphysiol.org
Copyright © 2014 American Society of Plant Biologists. All rights reserved.
0.25
0.15
Wet 1 h
Dry 1 h
0.05
-0.05
0.25
-0.15
0.15
-0.05
-0.15
0.25
Wet 2 h
Dry 2 h
EPR Signal
-0.25
0.05
-0.25
0.15
Wet 3 h
Dry 3 h
0.05
-0.05
-0.15
-0.25
I
I
I
3280
3320
3360
EPR signal intensity (Arb. Units)
Figure 7. Mor et. al.
Downloaded from on June 15, 2017 - Published by www.plantphysiol.org
Copyright © 2014 American Society of Plant Biologists. All rights reserved.