Singlet Oxygen Signatures Are Detected Independent ofLight or Chloroplasts in Response to Multiple Stresses1[C][W]

Avishai Mor, Eugene Koh, Lev Weiner, Shilo Rosenwasser, Hadas Sibony-Benyamini, and Robert Fluhr*

Department of Plant Sciences, Weizmann Institute of Science, Rehovot 76100, Israel

The production of singlet oxygen is typically associated with inefficient dissipation of photosynthetic energy or can arise fromlight reactions as a result of accumulation of chlorophyll precursors as observed in fluorescent (flu)-like mutants. Suchphotodynamic production of singlet oxygen is thought to be involved in stress signaling and programmed cell death. Here weshow that transcriptomes of multiple stresses, whether from light or dark treatments, were correlated with the transcriptome ofthe flu mutant. A core gene set of 118 genes, common to singlet oxygen, biotic and abiotic stresses was defined and confirmedto be activated photodynamically by the photosensitizer Rose Bengal. In addition, induction of the core gene set by abiotic andbiotic selected stresses was shown to occur in the dark and in nonphotosynthetic tissue. Furthermore, when subjected to variousbiotic and abiotic stresses in the dark, the singlet oxygen-specific probe Singlet Oxygen Sensor Green detected rapid productionof singlet oxygen in the Arabidopsis (Arabidopsis thaliana) root. Subcellular localization of Singlet Oxygen Sensor Greenfluorescence showed its accumulation in mitochondria, peroxisomes, and the nucleus, suggesting several compartments as thepossible origins or targets for singlet oxygen. Collectively, the results show that singlet oxygen can be produced by multiplestress pathways and can emanate from compartments other than the chloroplast in a light-independent manner. The resultsimply that the role of singlet oxygen in plant stress regulation and response is more ubiquitous than previously thought.

Singlet oxygen is the first electronic excited state ofmolecular oxygen and is a reactive oxygen species(ROS). It is highly reactive and will engage readilywith a variety of biomolecules, especially those con-taining double bonds (Triantaphylidès and Havaux,2009). In plants, singlet oxygen has been extensivelystudied in the context of photosynthesis (i.e. itsformation in the PSII reaction center and antennacomplex; Triantaphylidès and Havaux, 2009). Thechlorophyll triplet state can react with ground stateoxygen to make the highly chemically reactive singletstate (3Chl* + 3O2 → Chl + 1O2). Activated chlorophyllcan give rise to the triplet state when light energy isinefficiently used, although even normal light condi-tions may result in detectable formation of singletoxygen (Triantaphylidès et al., 2008; Ramel et al.,2012a). Singlet oxygen can participate in lipid perox-idation resulting in diagnostic chemical signatures.Peroxidation might result from direct assault of mem-brane lipids by ROS or it can be the outcome of enzy-matic activity. Type I lipid peroxidation consists of freeradical peroxidation, whereas type II is the outcome of

peroxidation by singlet oxygen (Triantaphylidès et al.,2008). Type II reactions are considered to have a majorrole in the execution of ROS-induced photooxidative celldeath of photosynthetic tissue (Triantaphylidès et al.,2008). In addition to photoactive stress, the Arabidopsis(Arabidopsis thaliana)-Pseudomonas syringae hypersensi-tive response results in lipid peroxidation signaturescorresponding to singlet oxygen and the enzymaticactivity of lipoxygenase2. In that case, the majorsources of lipids that undergo oxidation were fromplastid membranes (Zoeller et al., 2012). Hence, in bothhigh light stress and immune responses, the source ofsinglet oxygen is thought to be through the photody-namic activation of chlorophyll.

Plants have developed various scavenging systemsto protect themselves against the toxic effect of singletoxygen. b-carotene, tocopherol, or plastoquinone pre-sent in the thylakoid membranes are thought to play arole 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 transferdissipation as heat without scavenger consumption, orcan be accompanied by chemical oxidation (Triantaphylidèsand Havaux, 2009; Ramel et al., 2012a).

Singlet oxygen appears to play a role in retrogradesignaling from the chloroplast to the nucleus as establishedby mutants of FLUORESCENT (FLU), and EXECUTER1(EX1) and EX2. The FLU and the barely TIGRINA-d.12genes are negative regulators of the precursor of chloro-phyll, protochlorophyllide, preventing its accumula-tion in the dark (Meskauskiene et al., 2001; op denCamp et al., 2003; Khandal et al., 2009). These mutantscan be grown in constant light as a result of continuous

1 This workwas supported by the Dr. Angel Faivovich Foundationand the Israel Science Foundation (research grant no. 1008/11).

* Address correspondence to robert.fluhr@weizmann.ac.il.The author responsible for distribution of materials integral to the

findings presented in this article in accordance with the policy de-scribed in the Instructions for Authors (www.plantphysiol.org) is:Robert Fluhr (robert.fluhr@weizmann.ac.il).

[C] Some figures in this article are displayed in color online but inblack and white in the print edition.

[W] The online version of this article contains Web-only data.www.plantphysiol.org/cgi/doi/10.1104/pp.114.236380

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light-dependent conversion of protochlorophyllide.However, if kept in the dark and then transferredto light, the large amounts of accumulated proto-chlorophyllide act as a photosensitizer, releasingsinglet oxygen and setting off massive cellular, tran-scriptome, and physiological responses that can resultin cell death (Meskauskiene et al., 2001; op den Campet al., 2003). The response is not the result of directtoxicity of singlet oxygen, because the process is me-diated through two chloroplast proteins, EX1 and EX2.In their absence, the transcriptomic response andcell death of the flu mutant in the dark-to-light shiftis diminished (Wagner et al., 2004; Lee et al., 2007;Kim et al., 2012). The signaling competence of sin-glet oxygen is further illustrated by the induction ofan acclimation response to singlet oxygen originat-ing from applied chemicals or in mutants (Fischeret al., 2012; Ramel et al., 2013). In another examplein the chlorina1 mutant, deficient in chlorophyll b,excess levels of singlet oxygen are generated in thethylakoids under light stress. The transcriptome of thismutant shows high resemblance to that generated by theflu mutant although it seems to operate in a distinctmanner and does not depend on EX1 activity (Ramelet al., 2013).

In addition to the light-motivated formation of sin-glet oxygen in photosynthetic tissue, singlet oxygencan be produced enzymatically by the action of li-poxygenases followed by decomposition of the lipidperoxides that are terminated by the Russell mecha-nism (Miyamoto et al., 2007). Other reactions includesuperoxide dismutation coupled to myeloperoxidaseactivity as shown to occur during phagocytosis(Steinbeck et al., 1992) as well as in a reaction betweensuperoxide and hydrogen peroxide (H2O2) via Haber-Weiss mechanisms (Khan and Kasha, 1994). However,the relevance of these alternative sources in the pro-duction of singlet oxygen in plants and their stressresponses is unknown.

As described above, singlet oxygen production andsubsequent stress gene induction is chiefly thought tooccur via photodynamic involvement of chloroplasts.In this work, we first applied bioinformatic tools toassess the global involvement of ROS in a set of di-verse biotic and abiotic stresses. We observed that inmany stress situations, coregulated genes displayedhigh correlation with the transcriptome generated bydark-light transitions of the flu mutant. However, thatanalysis revealed that in the case of wounding, similartranscriptome patterns occurred in the absence of light.Indeed, we show that the expression of stress-relatedgenes can occur independent of light and in non-photosynthetic tissue as well. Furthermore, in vivoimaging with the highly specific Singlet Oxygen Sen-sor Green (SOSG) probe showed stress-induced singletoxygen production in the dark originating from multi-ple cellular sources. Collectively, our results are consis-tent with ubiquitous formation of singlet oxygen duringstress, suggesting that it can serve a broader signalingfunction.

RESULTS

Singlet Oxygen Generates a Transcriptome Footprint inMany Biotic and Abiotic Stresses

To assess the involvement of ROS in plant responseto environmental stress conditions, we collected tran-scriptomic data of plants exposed to diverse biotic andabiotic stresses and searched for the occurrence ofROS-related transcriptomic signatures. Gene expressiondata were collected from the following experimentsfor abiotic stress: osmotic, cold, drought, wound, andhigh light; and for biotic stress: Pseudomonas syringe,Phytophthora parasitica, oligogalacturonides (OGs),chitin, elongation factor thermo unstable (EF-Tu), andflagellin22 (flg22). We employed the ROSMETERbioinformatic platform, which compares a test tran-scriptome by a vector-based correlation method to pre-selected indices of a recognized ROS type. The indiceswere generated from various mutants and treatmentsthat produce ROS of a defined type and cellular source(Rosenwasser et al., 2011, 2013). Both the direction andstrength of the fold induction of each gene werecompared and summed over all matching genes in theindex to yield a correlation value. The methodology isidentical to that used for discerning hormone foot-prints (Volodarsky et al., 2009). The correlation valuesare presented graphically as a heat map in which the xaxis shows the ROS indices and the y axis shows theexamined transcriptomes (Fig. 1). Significantly, thehighest correlation values (red) were found betweenthe transcriptome of these diverse stresses and the ROSindices derived from ozone, H2O2, and even to agreater extent to the dark-to-light transition tran-scriptome of the flu mutant. These include, in partic-ular, the early time points (i.e. flu 30 m, flu 1 h, and to alesser extent flu 2 h; blue rectangle, Fig. 1). The actualmaximum correlation value and signal size is assem-bled in Supplemental Table S1 and ranged from 0.62for 1 h OG application to a high of 0.85 for 309 flg22treatment. Such correlation values can be consideredhighly significant (Volodarsky et al., 2009; Rosen-wasser et al., 2011, 2013) and point to the involvementof a singlet oxygen-like signature in those data sets.

Inspection of the analysis by the ROSMETER plat-form shows that in the Flagellin sensing2 mutant (fls2)background, the responses to ROS induced by the elic-itor peptide flg22, but not those induced by the elicitorEF-Tu, were abrogated (Fig. 1). This result is consistentwith the fact that different receptors are involved in therecognition of different pathogen-associated molecularpatterns (Zipfel et al., 2004,2006). In another example,ENHANCED DISEASE SUSCEPTIBILITY1 (EDS1) isknown to modulate the plant response to pathogensrecognized by resistance genes (Rustérucci et al.,2001). As shown, eds1 mutant leaves infiltrated withPseudomonas syringe expressing the effector proteinAvrRps4 showed general attenuation in many ofthe ROS responses (Fig. 1, compare eds1 and thewild type).

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Although diverse stresses show correlation with thesinglet oxygen transcriptome, there are alsomany stressesthat do not. For example, stress initiated by treatmentsincluding heat, salt, cadmium, hypoxia, Alternaria brassi-cicola, and Erysiphe orontii show low correlation values(Supplemental Fig. S1; Supplemental Table S2). Thissuggests that ROS signatures of singlet oxygen areapparent only in discrete stresses. Alternatively, thepeak correlation values to singlet oxygen indices mayhave been missed in those stresses because of the va-garies of timing of the sample collections.In general, the early time points of a time series of

stress transcriptomes tend to show the highest correla-tion to flu indices. For example, the correlation score toflu was .0.86 for the flg22 wild type at 30 min com-pared with 0.39 for the flg22 wild type at 3 h. As shownin Figure 1, the same trend is true after comparing shortand longer time points for cold (1 and 24 h), drought(1 and 4 h), wound (10 min and 12 h), high light (30 minand 3 h), OG (1 and 3 h), Phytophthora parasitica (2.5 and30 h), and osmotic treatments (1 and 3 h). In some cases,the attenuation in correlation values of a particularstress appears across the other ROS indices (e.g. cold or

osmotic stress). This could be an indication of the dis-sipation of the stress response over time. Alternatively,such as in wound stress, although the correlation valuesto flu weakens over time, it strengthens for the otherROS indices (Fig. 1, yellow box; 10 min versus 12 h),indicating the generation of progressive waves of dif-ferent ROS types. Methyl viologen and aminotriazoleare thought to promote the production of superoxide inthe mitochondria and H2O2 in the peroxisomes, respec-tively. The extended time points of these treatments(.7 h) show a weak degree of overlap with manystresses. It may indicate that the extended treatmentscause extensive cellular damage stimulating multiplesignal sources. Importantly, light and dark woundtranscriptomes show a similar pattern of correlations tothe flu transcriptome (Fig. 1, white box). This suggeststhat such ROS signatures may not depend on light.

Tocopherols are known scavengers of lipophilicROS, and have a role in preventing the accumulationof lipid peroxides. The mutant vitamin e deficient2(vte2), is defective in tocopherols biosynthesis and wasshown to accumulate products of nonenzymatic lipidperoxidation (Sattler et al., 2006). The transcriptome ofthis mutant shows strong correlation to the flu indicesand ozone (Fig. 1; Supplemental Table S1). Noticeably,in addition to the correlation to flu, both H2O2 andozone indices are highly correlated to many of thestress treatments (Fig. 1; Supplemental Table S1). Inthis respect, it is of interest that in plants, ozone in-teracts with the ubiquitous ascorbic acid to producesinglet oxygen (Kanofsky and Sima, 1995). Further-more, singlet oxygen will readily react with ascorbicacid to produce H2O2 (Kramarenko et al., 2006), indi-cating the possibility of rapid ROS interconversion.Interestingly, Cat2 at 0 h and As-Aox showed a markeddegree of negative correlation with many stresses. Theformer is a catalase-deficient mutant (time 0 representscontrol, whereas subsequent time points of 3 and 8 hindicate exposure time to high light), whereas the latterrepresents mutant lines for the mitochondrial alterna-tive oxidase (Fig. 1). The negative correlation mayrepresent compensatory mechanisms for ROS scav-enging induced within these mutants.

A Common Gene Set Is Induced in Multiple Abiotic andBiotic Stresses and Light-Dark Transition of the flu Mutant

We defined a core gene set (CGS) of transcripts byseeking differentially expressed genes that are commonto the multiple stresses in Figure 1 as well as the signa-ture of singlet oxygen. A list of 118 genes in the CGS wascompiled from genes differentially regulated in flu for 60min and at least 8 of 11 stresses (Fig. 2A; SupplementalTable S3). The stresses chosen are from the earliest timepoints available (e.g. 10 min after wounding; Fig. 2A).Nearly all of the genes common to biotic- and abiotic-type stresses and nearly all abiotic-type stress-inducedgenes showed overlap with the flu transcriptome (118of 122 and 147 of 158, respectively; Fig. 2A). Note that

Figure 1. Correlation values of select abiotic and biotic stress tran-scriptomes generated by the ROSMETER platform. Shown is a color-coded heat map of correlation scores between transcriptomes ofstresses (y ordinate) with transcriptome indices (abscissa) of theindividual ROS-producing treatments compiled in the ROSMETERplatform. The predicted ROS type, including ozone (O3), superoxide(O2

$2), H2O2, and singlet oxygen (1O2), and cellular location for eachindex are indicated at the top of the heat map (Rosenwasser et al.,2011, 2013). The source of transcriptome data used for the bio-informatics analysis and abbreviations are in Supplemental Table S10.The color scale for correlation is shown on the right. Blue, yellow, andwhite boxes are described in the text.

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the CGS is not necessarily exclusively induced by singletoxygen but may respond to different ROS sources.

Gene ontology (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), shows60-fold overrepresentation of the response to chitin andapproximately 40-fold overrepresentation of the re-sponse to carbohydrate stimulus. Other prominentfeatures are the response to chemical stimulus, the re-sponse to wounding, and immune system processesshowing between 5-fold to 20-fold overrepresentation(Supplemental Fig. S2; Supplemental Table S4). Sig-nificantly, transcription factors of the WRKY andETHYLENERESPONSIVEELEMENTBINDINGFACTOR(ERF) families known to be associated with stress re-sponses were prominent in the CGS (SupplementalTable S3). Furthermore, analysis of CGS promoters forknown cis-elements revealed overrepresentation of theW-box promoter motif (P = 2.2 e264). Such motifs wereshown to recruit WRKY transcription factors (Rushtonet al., 2010). W-box and ABA response element (ABRE)motifs have been identified as ROS responsive and ERF6can bind ROS-responsive cis-acting elements (Wanget al., 2013).

The CGS assembled here shows a degree of similarityto other compilations of common stress transcripts, in-cluding common stress-responsive genes that were de-fined by k-means clustering of multiple stresses (Maand Bohnert, 2007) and rapid wound-responsive genes(Walley et al., 2007; Fig. 2B). In common stress-responsivegenes, ABRE promoter motifs were shown to be over-represented , whereas rapid stress cis-regulatory ele-ments were defined for rapid wound-responsive genes.However, these elements were far less prominent inthe CGS than in the W-box promoter motif (W-boxmotif, P = 2.2 e264; rapid stress cis-regulatory elements,

P = 2.1 e211; ABRE, P = 9.3 e29; Supplemental Table S5),indicating distinct regulatory characteristics of membersof the CGS.

Photodynamic Production of Singlet Oxygen Can InduceCGS Transcripts

The fact that multiple stresses correlate with flu in-dices may indicate that singlet oxygen is an interme-diate in this process, or that multiple stress pathwaysactivate common flu-like stress signatures. To differ-entiate between these possibilities, several approacheswere taken. First, we examined whether the genera-tion of intracellular singlet oxygen could itself induceCGS. Gene probes were prepared for six genes fromthe CGS and used for quantitative reverse transcrip-tion (qRT)-PCR analysis, includingAT5G17350,whichwas previously reported to be specific for singletoxygen (op den Camp et al., 2003). Rose Bengal, as aphotosensitizer, generates singlet oxygen in a light-dependent manner (Knox and Dodge, 1984; Fischeret al., 2005). Plants were incubatedwith Rose Bengal indarkness and then exposed to light (120mEm22 s21) for6 h. Comparedwith the control, treatmentwith 0.2mM

Rose Bengal showed significant induction of the markergene after 6 h of exposure to light (Fig. 3A). Impor-tantly, under those conditions, FERRITIN1 (FER1) amarker gene for H2O2 accumulation (op den Campet al., 2003), did not change significantly (SupplementalFig. S3A).

Application of 3-(3,4-dichlorophenyl)-1,1-dimethylurea(DCMU) and high light treatment was used as analternative method to gauge the possible effect of cel-lular singlet oxygen generation on CGS subset ex-pression. In isolated membranes, it was shown thatDCMU binding to the QB binding site of PSII inhibitselectron transport facilitating the formation of excitedchlorophyll. This generates singlet oxygen in additionto basal production of other ROS (i.e. hydroxyl andsuperoxide radicals) that are produced in high light(Fufezan et al., 2002). A low concentration of DCMU(0.25 mM) did not result in any gene induction, whereas25 mM DCMU led to the induction of stress-relatedtranscripts (Fig. 3B). In line with these results, at alow concentration of DCMU (0.25 mM), the maximumphotochemical efficiency of PSII in the dark-adaptedstate was not affected compared with the controlplants, whereas 25 mM DCMU led to the inhibition ofPSII (Supplemental Fig. S4). Hence, induction of CGSwas correlated to the physiological state of the pho-tosynthetic system. In this case, the FER1 expressionlevel changed significantly in response to DCMU andthe high light conditions (Supplemental Fig. S3B). Thisis likely the result of high light stress conditions in thepresence of DCMU that generate multiple forms ofoxidative stress, including H2O2 (Karpinski et al., 1999;Fufezan et al., 2002). In all, the results show that singletoxygen generated from varied sources can induce asubset of CGS transcripts.

Figure 2. Delineation of a CGS for stress and singlet oxygen-relatedtranscripts. A, Venn diagram showing the number of overlapping genesbetween abiotic stress (wound local and systemic leaves after 10 min,1 h drought, and 30 min of high light), biotic stress (1 h OGs, Flg22 W.t.30 min, EF-Tu 30 min, chitin 30 min), and early flu responses 60 min afterthe dark-to-light transition. B, Venn diagram showing the number ofoverlapping genes between CGS, rapid wound-responsive (Walley et al.,2007), and common stress-responsive genes (Ma and Bohnert, 2007).RWR, Rapid wound responsive.

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Induction of CGS Does Not Require Light orPhotosynthetic Tissue

Photodynamic activity of the chloroplast is reportedto be the major source of singlet oxygen; however,analysis by the ROSMETER platform suggested thatthe correlation with the flu transcriptome did not nec-essarily depend on light (Fig. 1, white box). Hence, itwas important to establish whether stress induction ofthe CGS shows light dependency. To test this, plantswere wounded in the dark. As shown in Figure 3C, theCGS probe set showed rapid induction after woundingin the dark. By contrast, the expression level of FER1was not significantly changed (Supplemental Fig. S3C).

To further examine whether transcript activation de-pends on photosynthetic tissue, cotyledons were re-moved by cutting and the expression levels of CGSsubset were examined in the roots. A trend of transientincrease in all CGS genes examined was observed thatpeaked between 8 and 12 min, in which four of sixgene expression profiles showed statistical significance(Supplemental Fig. S5A). However, the expressionlevels of the H2O2 marker gene, FER1, did not changesignificantly (Supplemental Fig. S5B). Similarly, appli-cation of flg22 in the dark resulted in significant in-duction of stress-related genes (Fig. 3D). By contrast,the expression level of FER1 remained unchanged(Supplemental Fig. S3D).

Wound-induced genes showed a high correlation toflu indices. EX1 and EX2 were previously shown to berequired for expression of flu-dependent induction ofgenes in the dark-to-light transition (Lee et al., 2007).To examine whether they affect wound-mediated geneinduction, we followed the expression of the CGSsubset in the ex1/ex2 double mutant line. As shown inSupplemental Figure S6A, the CGS probe set retainedits wound responsiveness in the ex1/exe2 mutantbackground. This indicates that despite the high cor-relation values of the transcriptome of wound stress toflu indices, the wound induction of genes is indepen-dent of EX1 and EX2 activity. As expected, the FER1expression level did not change significantly in re-sponse to wounding of the mutant line (SupplementalFig. S6B). Thus, it is likely that wound-induced CGS isnot controlled by EX1 and EX2 Collectively, the resultsvalidate the transcriptome analysis and show that CGSstress-induced expression can be independent of lightand will occur in nonphotosynthetic tissue.

Stress-Induced Accumulation of Singlet Oxygen in theDark and in Nonphotosynthetic Tissue

The fact that genes in the CGS are induced by singletoxygen and that many stresses yield singlet oxygensignatures prompted us to examine whether singletoxygen could be detected in vivo as an intermediateduring the varied stress responses. The SOSG is a two-component trap-fluorophore system generating thefluorescent endoperoxide upon specific interactionwith singlet oxygen (Gollmer et al., 2011). SOSG isspecific for singlet oxygen and will not react with otherROS, including superoxide or H2O2 (Flors et al., 2006).Unlike the specific dansyl-based DanePy singlet oxy-gen probe (Hideg et al., 2002), its fluorescence in-creases upon interaction with singlet oxygen. Wholeflu mutant leaves shifted from dark to light were pre-viously shown to display SOSG fluorescence (Florset al., 2006). We first examined the ability of SOSG todetect rapid subcellular signals by incubation of roottissue with SOSG and the singlet oxygen generatorRose Bengal. Tissue was then spot illuminated at theabsorption peak of Rose Bengal . A transient, SOSGsignal was obtained after illumination (Fig. 4A).

Figure 3. Expression of transcripts after stress treatments. qRT-PCR ofCGS transcripts from plants after various treatments. A, Plants weretreated by spraying with 0.01% Silwet L-77 as the control, or 0.01%Silwet L-77 and 200 mM Rose Bengal. After 1 h in the darkness, plantswere exposed to light intensity of 120 mE m22 s21 for 6 h. B, Plantswere sprayed with 0.01% Silwet L-77 as a control or 0.01% SilwetL-77, 0.25, or 25 mM DCMU as indicated. They were then kept in thedark for 9 h. Plants were transferred to daylight with an average of600 mE m22 s21 for 7.5 h. C, Dark-adapted plants were wounded andprocessed after 30 min as described in Materials and Methods. D,Plants were grown on vertical agar plates for 7 d and then transferredto liquid medium either supplemented with 1 mM flg22 or not (control).They were subsequently kept in darkness for 140 min before sampleswere collected. An ANOVA statistical test followed by a contrastStudent’s t test and step-up correction for multiple testing was used todetermine significance of the difference relative to the control. *P ,0.1; **P , 0.05; ***P , 0.01; ****P , 0.001. Average 6 SE values (n =3–4) are presented. [See online article for color version of this figure.]

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Quantitative measurement of the activation eventshowed a sharp transient increase in the SOSG signal(approximately 5-fold) together with a decrease in thefluorescence from Rose Bengal (Fig. 4B). This indicatesthat intracellular SOSG can report a rapid singletoxygen burst that rapidly decays as a result of diffu-sion from the source.

The SOSG probe was used to examine whether singletoxygen is formed in vivo in stresses that were shown togenerate a flu-type transcriptome signal (Fig. 1). Lightand chloroplast dependency were further evaluated bymeasuring the SOSG signal in dark-adapted, non-photosynthetic root tips. In the absence of treatment,fluorescence from SOSG was visible at a low level inmultiple cellular compartments (Fig. 4C, flg22, top row).

This indicates that a low level of singlet oxygengenerated by organelles is part of the basal cellularmetabolism. To identify those subcellular compartments,we employed cell markers including 49,6-diamino-phenylindole as a nuclear marker, Saccharomycescerevisiae cytochrome c oxidase IV (ScCOX4)-mCherry(Nelson et al., 2007) as a mitochondrial marker, andARABIDOPSIS PEROXIN5 (AtPEX5) CFP as a peroxi-somal marker (Tian et al., 2004). As shown in Figure 4C,cells of the root epidermal layer in the maturation zoneshowed a faint SOSG signal localized to the nucleus andmitochondria (Fig. 4C, top row). Nuclear localization ofSOSG was previously reported for mesophyll cells(Hideg, 2008). Although the signal in the nucleus wasvariable in some cases, it could clearly be detected in

Figure 4. Confocal imaging and analysis of SOSG fluorescence in root tissue. A, Root stained simultaneously with 50 mM RoseBengal and 100 mM SOSG and visualized for Rose Bengal (left) or SOSG (right). Top, The root before 1-s activation by an argonlaser at 559 nm (circled area). Bottom, The root after activation. B, Relative quantification of the fluorescent signal emitted byRose Bengal (red squares) and SOSG (green diamonds) within the activation spot shown in (A). Arrow indicates laser activation.C, Differentiation zone of the root of 6-d-old seedlings expressing the mitochondrial marker Saccharomyces cerevisiae cyto-chrome c oxidase IV (ScCOX4):mCherry stained with DAPI and SOSG. The last column shows a merge of all channels. Seedlingswere either incubated in 13 phosphate-buffered saline to serve as control (top row), or with flg22 for 80 min (bottom row). Thearrow points to mitochondrion displaying SOSG fluorescence, whereas the arrowhead points to an area lacking ScCOX4:mCherry fluorescence but containing SOSG fluorescence. D, Differentiation zone of the root of 4-d-old seedling expressing theperoxisomal marker ARABIDOPSIS PEROXIN5 (AtPEX5)-CFP stained with DAPI and SOSG. The last column shows a merge ofall channels. The AtPEX5-CFP was color coded red for better visualization of colocalization. The arrow points to peroxisomesdisplaying SOSG fluorescence, whereas the arrowhead points to an area lacking AtPEX5-CFP fluorescence but containing aSOSG signal. E, Percentage of peroxisomes and mitochondria with a SOSG fluorescent signal that is higher than the nearbysurrounding area. a.u., Arbitrary unit; DAPI, 49,6-Diamino-phenylindole; M, Mitochondria; P, Peroxisome. Bar = 10 mM.

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structures within the nucleus (Supplemental Fig. S7).In plants treated with flg22, the distribution of SOSGfluorescence was similar, although the intensity washigher comparedwith the untreated plants (Fig. 4C, +flg22,SOSG channel). In a similar manner, the peroxisomalmarker showed a marked degree of overlap withSOSG fluorescence (Fig. 4D). As shown, not all ofthe labeled organelles displayed SOSG fluorescence(Fig. 4, C and D). The disparity in signal overlapappears to be greater between the peroxisomalmarker and the SOSG signal. Quantitative scanningof organelles identified by their respective markersshowed that .85% of the peroxisomes and 97% of themitochondria contain more SOSG fluorescence rela-tive to the nearby surroundings (Fig. 4E). Thus, in all,the SOSG signal could be detected in varied cellularcompartments; it localized to the mitochondria, nu-clei, and peroxisomes and was elevated after elicitortreatment.To detect singlet oxygen as a result of wound stress,

seedlings were pretreated with both Rose Bengal andSOSG and placed in custom-designed slide retainers.The experimental setup is exemplified in Figure 5A(left). The cotyledons were then crushed with a he-mostat, and a noncontiguous root section approxi-mately 1 cm from the hypocotyl was examined forSOSG fluorescence. A rapid rise in the SOSG signal insystemic root tissue could be detected within 1 to 3min after wounding. The same tissue was subse-quently illuminated by white light irradiation. Asshown in Figure 5A (right), a comparable and incre-mental change in the SOSG fluorescent signal wasgenerated as a result of successive photodynamic ac-tivation of Rose Bengal, supporting the reliability ofthe wound-induced SOSG signal. To examine cell tis-sues that show response, root tissue was scrutinizedbefore and after wounding by measuring the fluores-cent signal from sequential root cell layers by confocalmicroscopy. The maximum depth from the surfacewas measured using a fixed size of region of interest(ROI) starting at the surface and penetrating 20 mM, inwhich the root area expands 4- to 5-fold (Fig. 5B, left).Consecutive Z scans were carried out at 925, 595, and236 s before wounding (Fig. 5B, right). The Z scansshow a basal-level fluorescent signal that increases as aresult of the expansion of the root section measured.After wounding, a uniform increase in fluorescencewas observed in almost all cell layers (Fig. 5B, right,207 and 540 s). At a depth of 20 mM, the detected in-crease is less pronounced. Cell layers at this depth areeither less sensitive to alteration in singlet oxygen, orthe layers above absorb both the excitation and emissionas a result of the increased depth. In all, the systemic roottissue showed rapid wound-induced production of sin-glet oxygen. The results are consistent with a previouslyreported increase in SOSG fluorescence after 30 min inwhole wounded Arabidopsis dark-adapted leaves (Florset al., 2006).The generation of singlet oxygen was next examined

in vivo for other stresses in nonphotosynthetic tissue.

In drought stress, plants were preincubated withSOSG and subjected to 15 min of drought treatment.As shown in Figure 6 (top), a significant increase influorescence was evident. When flg22 peptide wasapplied to seedlings, a significant rise in fluorescencewas detected 1 h prior to the addition of SOSG (Fig. 4,C and D, and Fig. 6, middle). By contrast, after rote-none treatment, the tissue showed a basal level offluorescence that was significantly reduced comparedwith the control (Fig. 6, bottom). This is consistent withanalyses carried out on the ROSMETER platform thatshowed that rotenone, a known inhibitor of mito-chondrial complex I (Garmier et al., 2008), displayed anegative correlation to the transcriptome activity of theflu mutant (Fig. 1, top line; correlation value 20.292 toflu at 30 min). Unlike its effect on animal mitochondria,

Figure 5. Generation of singlet oxygen in vivo by wound or by pho-todynamic activation of Rose Bengal. A, A schematic diagram of theexperimental setup used to monitor wound stress (left). Five-day-oldseedlings were sandwiched between slides that were separated so asnot to evoke mechanical pressure (detailed in “Materials and Methods”).Cotyledons were wounded with a hemostat and imaged within the areashown. Seedlings that were loaded with 100 mM SOSG for 1 h followedby 50 mM Rose Bengal for 15 min, washed, and analyzed in an invertedfluorescence microscope (Olympus Ix71; right. A representative result isshown (n = 4). B, A schematic diagram of the sequential root cell layersmeasured by confocal microscopy (Olympus Flow View FV1000; left).Measurements started at the surface and reached 20 mM in depth; thearea measured is shown with double arrows. Quantification of theSOSG fluorescent intensity in the root tip before and after wounding ofcotyledons (right). Measurements were made along the different celllayers depicted on the left at different times. Seedlings were incubatedwith 100 mM SOSG for 10 to 20 min. The representative result is shown(n = 15). a.u., Arbitrary unit. [See online article for color version ofthis figure.]

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rotenone does not induce ROS in plant mitochondria(Garmier et al., 2008).

SOSG is currently the most sensitive method formeasurement of singlet oxygen (Nakamura et al., 2011)and the only in vivo method providing subcellularresolution. Electron paramagnetic resonance (EPR)spectroscopy has been extensively used to measurelight-induced singlet oxygen in isolated chloroplasts(Hideg et al., 1994; Hideg et al., 2000). In that case, thespecific cell permeable singlet oxygen trap 2,2,6,6-tetramethylpiperidine (TEMP) after reacting with sin-glet oxygen was converted to the nitroxide radical2,2,6,6-tetramethylpiperidinyloxy (TEMPO), which can

then be detected by EPR (Lion et al., 1976). We appliedthis approach to drought-treated leaf tissue in the darkbecause such treatment produced a robust fluorescentsignal from SOSG in root tissue (Fig. 6, center) as well asa singlet oxygen-associated transcriptomic signature inleaf tissue (Fig. 1). Leaf discs were treated with TEMPand kept either under moist conditions or transferred todry filter paper for 1 to 3 h (see Materials and Methods).In all control and treated samples, a background EPRsignal was obtained indicating the presence of TEMPO.This signal is likely the result of wound reactions in thediscs or residual TEMPO in the TEMP reagent. Signifi-cantly, increases in the amount of TEMPO of 0%, 60%,and 40% were detected at 1, 2, and 3 h of dry treatmentcompared with the wet control, respectively (Fig. 7).The results confirm the presence of singlet oxygen indrought stress-treated samples in the dark. In all, theresults show that measurements of singlet oxygenformation recapitulate the predictions made by theROSMETER tool and lend credence to the idea thatsinglet oxygen is an actual intermediate in the plantstress response.

DISCUSSION

We show a correlation between transcriptome sig-natures from diverse stresses and the flu transcriptome.This includes wound-induced expression of genes inthe dark. Singlet oxygen has previously been impli-cated as the major toxicity factor in chloroplast-drivenphotooxidative cell death. It is also a causal agent inhypersensitive immune responses that occur in thelight (Triantaphylidès et al., 2008; Triantaphylidès andHavaux, 2009; Kim et al., 2012; Zoeller et al., 2012). Inaddition, singlet oxygen is the major ROS agent pro-duced in the flu mutant during dark-to-light transition(op den Camp et al., 2003). However, the bioinformaticand CGS expression analyses show that photodynamicprocesses are not essential for induction of stress genes:These genes are induced by stresses in the dark and innonphotosynthetic tissue. Nevertheless, it is likely thatlight and chloroplasts have a role in maintaining andaugmenting stress-signal strength (Morker and Roberts,2011). The bioinformatic analysis alone cannot differ-entiate between the possibility that singlet oxygen is anactual intermediate or that different stresses, includingsinglet oxygen, converge into one common pathway.However, the actual detection of singlet oxygen in di-verse stress and by two independent methods, SOSGfluorescence and TEMP oxidation, suggests that singletoxygen is indeed an intermediate.

The highest correlation of stresses to the flu tran-scriptome was generally found between the early timepoints after stress initiation, suggesting commonalty inthe initial response that is later transformed to be stressspecific. A significant number of CGS genes representtranscription factors (24 of 118; Supplemental TableS3); of those, 8 out of 24 belong to the WRKY family oftranscription factors. Such factors are able to positively

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-oldseedlings. Insets above bars display a representative result for each treat-ment. Top, Seedlings were incubated with SOSG for 20 min and thentransferred to wet (control) or to dry Whatman paper (drought) for 15 min(n = 4). Middle, Seedlings were incubated with 10 mM flg22 for 80 min,and 100 mM SOSG was added 20 min before the end of the incubationtime (n = 3). Bottom, Seedlings were incubated with 0.03% chloroform(control) or 0.03% chloroform and 40 mM rotenone for 100 min. SOSG(100 mM) was added 20 min before the end of the incubation time (n =3 to 4). A two-sided Student’s t test was performed to evaluate the sig-nificance. **P, 0.05; ***P, 0.01. a.u., Arbitrary unit. [See online articlefor color version of this figure.]

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and negatively regulate other functions as well as theirown expression (Rushton et al., 2010). Furthermore,analysis of all CGS promoters showed highly signifi-cant enrichment for W-box motifs known to recruitWRKY transcription factors. Another distinct tran-scription factor group (5 out of 24) belongs to the ERFfamily. Of them, AtERF1, AtERF5, and AtERF6 belongto the B-3 subfamily associated with defense-relatedphytohormone responses (Nakano et al., 2006). Thisindicates that WRKY and ERF transcription factorsplay a dominant early role in the regulation of plantresponse to stress underlying the singlet oxygen foot-print. The expression of WRKY transcription factors in-duced by flg22 have been shown to be down-regulated inthe chloroplast-localized CALCIUM-SENSING RECEPTOR(CAS) gene mutant, cas1, implying control of stress geneexpression by chloroplast functions (Nomura et al.,2012). However, the control of genes by CAS is quan-titative. Moreover, more than one-third of the flg22-induced genes escape CAS regulation. This indicatesthat, as shown here, additional CAS-independentpathways can participate in stress regulation.Genevestigator currently collates 2,213 experimental

conditions (https://www.genevestigator.com/gv/plant.jsp; Hruz et al., 2008). Scanning these with a profilecomposed of CGS members showed that about 10% ofthe conditions exhibited enhanced expression of CGS(defined by 50% of the CGS members displaying at least

a 2-fold difference; Supplemental Table S6). The condi-tions include transcriptomes of mutants, hormone treat-ments, and comparisons of development stages. Theresults of this inspection suggest that singlet oxygenproduction is a pervasive feature in many plant tran-scriptome responses and is not limited to conditionsof stress.

The regulation of gene induction by singlet oxygenis complex and dependent on its source. Thus, bothDCMU and flu-generated signaling have been shownto be attenuated in ex1 and ex1/ex2 mutants (Wagneret al., 2004; Lee et al., 2007). However, the role of theEX genes appears to be limited to moderate states ofchloroplast 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 notappear to modulate the chlorina1 photosystem mutant(Ramel et al., 2013) or the induction of genes byb-cyclocitral, a product of singlet oxygen activity(Ramel et al., 2012b). As shown here, wounding, whichalso generated singlet oxygen and induced CGS ex-pression, was not controlled by EX genes. Therefore,the plant transcriptome response to singlet oxygen-producing stresses is complex and represents subsetsof genes, some controlled by EX, some represented byCGS, and some perhaps related to more extensivecellular damage.

Chlorophyll photodynamic reactions as well asother potential cellular photosensitizers (e.g. porphy-rins, flavins, and quinones) all require light to generatesinglet oxygen. What then are the potential sources ofsinglet oxygen in the dark? The localization of fluo-rescence of SOSG to specific organelles (Fig. 4) mayindicate that those organelles or their membranes arethe preferred sites of singlet oxygen production. Al-ternatively, the organelles or their membranes mayhave preferential chemical affinity for the fluorescentderivatives of activated SOSG that are made elsewherein the cell. The availability of new fluorescent probesor novel SOSG derivatives may serve to differentiatebetween these possibilities. However, the result of lo-calized activation of SOSG by Rose Bengal does implythat activated SOSG can be detected where it is made(Fig. 4, A and B).

Singlet oxygen generation can occur in the dark inmultiple ways. Lipid peroxidation, a source of singletoxygen, occurs when a membrane component such asfatty acid undergoes oxidation (Miyamoto et al., 2007).Peroxidation might result from direct assault ofmembrane lipids by ROS. Importantly, terminationreactions between such hydroperoxyls result in theproduction of singlet oxygen (Miyamoto et al., 2007).In addition, specialized enzymes such as lipoxyge-nases and cyclooxygenases have also been implicatedin singlet oxygen production. Lipoxygenases from soy-bean are able to catalyze the formation of singlet oxygenin vitro, most likely via production of peroxy radicals,which recombine through a Russell mechanism (Kanofskyand Axelrod, 1986).

Figure 7. Detection of singlet oxygen during drying treatment by EPRspectroscopy. Arabidopsis leaf discs were incubated with TEMP for 30min in the dark, rinsed, and left on water or subjected to dehydrationtreatments in the dark for the indicated times. Tissue was frozen andprocessed in the dark and measured using EPR spectroscopy as de-scribed in Materials and Methods.

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In addition to lipid-derived origins for singlet oxygenproduction, membrane-free dark reactions have alsobeen proposed. In animal neutrophils, the combinationof NADPH oxidase and myeloperoxidase activity dur-ing immune response produces copious amounts ofantibiotic singlet oxygen under physiological condi-tions (Steinbeck et al., 1992). However, the existence ofsuch specialized sources is unknown in plants. Super-oxide can react with H2O2 via a Haber-Weiss mechanismto form singlet oxygen (H2O2 +O2

2 → OH2 + OH$ +1O2;Kellogg and Fridovich, 1975; Khan and Kasha, 1994),although the efficiency of singlet oxygen production bythese ROS in vivo is not known (MacManus-Spencer andMcNeill, 2005). Nonetheless, the accumulation of super-oxide and H2O2 is readily detected in local or systemicwounded tissue (Sagi et al., 2004; Warwar et al., 2011)and in the immune responses (Dubiella et al., 2013). Apossible source could be NADPH oxidase-like and su-peroxide dismutase activity, as has been shown forelicitor and salt-induced accumulation of ROS (Leshemet al., 2006; Ashtamker et al., 2007; Suzuki et al., 2011).Similarly, biotic elicitors are known to initiate ROS,likely through activation of calcium and phospho-rylation cascades (Sagi and Fluhr, 2001; Fluhr,2009; Dubiella et al., 2013).

The fluorescent signal from SOSG was prominent inmitochondria and peroxisomes (Fig. 4E). Perturbationof mitochondrial metabolism can result in productionof superoxide and H2O2 (Noctor et al., 2007; Milleret al., 2010). It was recently shown that mitochondriaand peroxisomes work in a successive manner gener-ating oxidative signals in response to the induction ofsenescence (Rosenwasser et al., 2011). Consistent withthis, it is of interest that mitochondrial mutants of al-ternative oxidase activity are negatively regulated withmany of the stresses (see AS-Aox1 and TDNA-Aox1;Fig. 1). Negative correlation might result from theenhancement of scavenging systems; for example, al-ternative oxidase couples the oxidation of mitochon-drial ubiquinol to the reduction of oxygen to water. Adecrease in alternative oxidase activity (e.g. AS-Aox1)would raise the fully reduced mitochondrial ubiquinollevels that could readily participate in the quenching ofsinglet oxygen (Triantaphylidès and Havaux, 2009).

Cross talk between ROS species in stress signalinghas been noted (Laloi et al., 2007; Rosenwasser et al.,2013) and a degree of overlap exists between the flusignature and other types of ROS, particularly H2O2and ozone (Fig. 1; Supplemental Tables S1 and S6). Thedetection of additional ROS signatures may be throughinterconversion mechanisms. For example, ozone isreadily converted into singlet oxygen in reactionswith Cys, glutathione, or ascorbate (Kramarenko et al.,2006). The correlation with H2O2 is less anticipated.On the basis of studies of cytosolic calcium release,Arabidopsis seedlings perceive ozone and H2O2 in-dependently (Evans et al., 2005). Furthermore, plantsoverexpressing the chloroplast THYLAKOID-BOUNDASCORBATE PEROXIDASE rapidly dissipate chloroplast-based H2O2 signals, but increase their sensitivity to the

flu mutation, suggesting that the two signals, singletoxygen and H2O2, antagonize each other (Laloi et al.,2007). Thus, either H2O2 conversion to singlet oxygenvia Haber-Weiss reactions is active in most stresses or,alternatively, the high degree of similarity obtained byROSMETER analysis results from the fact that CGS canreact to multiple independent ROS inputs.

How does singlet oxygen participate in signalingpathways and induce the CGS? One possibility is that,as with other ROS, their production will result inspecific scavenger depletion and thus affect cellularredox to generate signals. The ability of singlet oxygento serve as a direct signaling molecule is consideredunlikely because of its reactivity. However, some evi-dence has shown that within the cellular milieu, itshalf-life may be considerably longer (Snyder et al.,2006). Other longer-range effects may be indirect andinvolve the oxidation of b-carotene leading to the ac-cumulation of the volatile b-cyclocitral (Ramel et al.,2012b). The results shown here suggest that singletoxygen production is a common feature of manystresses. Its origin and how it may participate in sig-naling and how ultimately specificity is achieved needto be further explored.

MATERIALS AND METHODS

Plant Material and Growth Conditions

Arabidopsis (Arabidopsis thaliana) ecotype Columbia 0 was vernalized for2 d at 4°C and grown under white light (90 mE m22 s21). Plants were grown for4 to 5 weeks in soil in a 10-h-light/14-h-dark cycle at 21°C in the followingexperiments: wounding, response to Rose Bengal, DCMU, and high light. Forimaging and examination of stress-related transcripts in the background ofex1/ex2, in roots and application of flg22, 4- to 7-d-old seedlings were grownon 0.8% (w/v) solid agar (Duchefa) medium containing full-strength B5Gamborg’s nutrients (Duchefa) adjusted to pH 5.8. Seedlings were surfacesterilized according to the method of Warwar et al. (2011) and grown invertically positioned plates under a 16-h-light/8-h-dark cycle at 21°C.

RNA Extraction, Complementary DNA Synthesis, andqRT-PCR Analysis

RNA was extracted from 100 mg of frozen tissues using a standard Trizolextractionmethod (Sigma-Aldrich). Formicroarray experiments, total RNAwasextracted using the RNeasyMini Kit (Qiagen). DNase I (Sigma-Aldrich)-treatedRNA was reverse transcribed using a high-capacity complementary DNA re-verse transcription kit (Applied Biosystems). Complementary DNA synthesiswas according to the manufacturer’s instructions with the exception that oligo(dT) was added to the random hexamers used for priming. In the microarrayexperiment, random hexamers were excluded altogether. For qRT-PCR anal-ysis, the SYBR Green method (KAPA Biosystems) was used, on a Step OnePlus platform (Applied Biosystems) with a standard fast program. qRT-PCRprimers were designed in Primer Express 2 or 3 software (Applied Biosys-tems). All qRT-PCR primer sequences are listed in Supplemental Table S7.

Treatments

For wounding as shown in Figure 3C, dark-adapted (1 h) plants werewounded by clamping three leaves three times across the midvein with ahemostat. Wounding and sample collecting was done in a dark room underdim illumination. A pool of leaves from three different plants per sample wascollected 30 min after wounding. Wounding in the ex1/ex2 and root experi-ments was administered by separating the shoot from the root. For imagingexperiments, custom-designed slide retainers with double-sided tape spacers

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were used to avoid mechanical pressure on the root by the coverslip. For flg22application, seedlings were grown on vertical agar plates. For the gene ex-pression experiment, 7-d-old seedlings were transferred to plain liquid me-dium (control) or medium supplemented with 1 mM flg22. They were thenincubated for 140 min in darkness, collected, and frozen in liquid nitrogen.About eight seedlings were collected per sample. For imaging experiments, 4-to 6-d-old seedlings were incubated for 80 min in 13 phosphate-bufferedsaline as the control or 13 phosphate-buffered saline supplemented with10 mM flg22. One-hundred-micromolar SOSG (Invitrogen) was added 20 minbefore the end of the incubation time. For Rose Bengal treatment, Rose Bengal(Sigma-Aldrich) was dissolved in water to a final concentration of 0.2 mM with0.01% Silwet L-77 (LEHLE seeds). Plants were sprayed with 0.01% Silwet L-77as a control or 0.01% Silwet L-77 with 0.2 mM Rose Bengal. They were thenkept 1 h in darkness to allow dye uptake before being exposed to light in-tensity of 120 mE m22 s21 for 6 h.

For DCMU and high-light treatment, DCMU (Riedel-de Haën) wasresuspended in ethanol to a stock solution of 100 mM and then diluted to afinal concentration of 0.25 and 25 mM in 0.01% Silwet L-77. Plants weresprayed with 0.01% Silwet L-77 as a control or 0.01% Silwet L-77 with theindicated concentrations of DCMU and kept in the dark for 9 h. Plants weretransferred to a greenhouse and exposed to daylight with intensities rangingfrom 200 to 1000 mE m22 s21 (sunlight) for 7.5 h. For measurements of themaximum photochemical efficiency of PSII in the dark-adapted state, plantswere transferred to complete darkness for 15 min prior to being measuredin a PAM system (Heinz Walz). For rotenone treatment, rotenone (Sigma-Aldrich) was resuspended in chloroform to a stock solution of 120 mM andthen diluted to a final concentration of 40 mM. Seedlings were incubatedwith 0.03% chloroform as a control or 40 mM rotenone for 100 min, and 100 mM

SOSG was added 20 min before the end of the incubation time. Incubationswere in darkness. For drought treatment, 5-d-old seedlings were incubatedwith SOSG for 20 min and then transferred for 15 min to wet or dryWhatman paper.

Confocal and Fluorescent Microscopy Imaging andImage Analysis

An inverted fluorescence microscope (Olympus IX71) was used for quantifi-cation of SOSG intensity. The SOSG signal was a yellow fluorescent protein filter(excitation 500 6 10 nm, emission 5356 15 nm). Because SOSG itself may act as aphotosensitizer (Gollmer et al., 2011), low excitation energies were employed.Confocal imaging was conducted with an inverted confocal microscope(Olympus Flow View FV1000) equipped with a UPlanSApo 320 objective(numerical aperture of 0.75) for wound studies and a UPlanSApo 360 oilobjective (numerical aperture of 1.35) for localization studies. Detaileddescriptions of filters, lasers, and incubation periods are provided inSupplemental Table S8. For quantification and image enhancement, the Fiji(Schindelin et al., 2012) and Olympus FV10-ASW 3.1 viewer programs wereused. SOSG fluorescence intensity in the root tips was determined by con-structing a minimal ROI suitable for all root tips examined in a given exper-iment. The mean gray value of the selected ROI was computed and thebackground was subtracted. Compartment-specific quantification of the SOSGsignal was achieved by using the images of specific compartment markers (e.g.AtPEX5-CFP for peroxisomes) in order to construct multiple ROIs directed atthat compartment. The ROIs were then employed to measure the SOSGchannel. The signal in the vicinity of these compartments was acquired bydisplacement of the ROIs. Image enhancement was attained by changing thebrightness/contrast properties and by applying an average filter to the 49,6-diamino-phenylindole channel in Figure 4C (top and bottom images). Whendifferences of SOSG intensity were visualized, the same brightness/contrastparameters were applied to allow meaningful comparison.

EPR Spectroscopy and Sample Preparation

Leaf discs (50 discs, 6 mm; 8- to 12-week-old plants) were used for eachtreatment. The discs were equilibrated in distilled, deionized water (ddH2O) inambient light for 1 h. All further incubation and extraction procedures werecarried out in the dark. Discs were incubated with 10 mM TEMP (Sigma-Aldrich) for 30 min, and washed three times with ddH2O. Samples werethen placed on dry Whatman paper for 0, 1, 2, and 3 h. Wet samples werefloated in ddH2O. Samples were harvested by flash freezing in liquid nitrogenand were homogenized in a shaker using glass beads in 0.2 M potassiumphosphate buffer, pH 7.2, at 4°C and then in a rotator for 15 min. The samples

were then centrifuged for 20 min at 14,000 rpm at 4°C for 20 min, and thesupernatant was extracted with 2 volumes of ethyl acetate. The upper phase ofthe mixture was used in EPR spectroscopy after the addition of 100 mM H2O2to reoxidize reduced hydroxylamine forms of TEMPO (Hideg et al., 2000). TheEPR spectra matched typical TEMPO triplet EPR spectra (aN = 15.8 G) andwere acquired at room temperature with a Brucker ELEXYS 500 spectrometer.X-band spectra were recorded at 9.35 GHz microwave frequency and 100 kHzmodulation frequency.

Microarray Data Bioinformatic and Statistical Analysis

The Affymetrix GeneChip Arabidopsis ATH1 Genome Array was used.Quantile normalization and background adjustment for data were performedusing robust multichip analysis (Irizarry et al., 2003) in PARTEK GenomicsSolution software (http://www.partek.com; Downey, 2006). Lists of geneswere generated for each stress from transcripts that had a fold change of21.8 $ fold change $ 1.8 and a P # 0.05 (Supplemental Table S9). Compu-tation of the fold changes and P values for downloaded data sets of the dif-ferent stresses was made by Robin software (Lohse et al., 2010). Geneannotation was taken from The Arabidopsis Information Resource database,version 8 (http://www.arabidopsis.org). The overlapping genes between thedifferent data sets and the Venn diagrams were computed by the Venny al-gorithm (http://bioinfogp.cnb.csic.es/tools/venny/).

The sources of transcriptome data for Figure 1 and Supplemental Figure S1are tabulated in Supplemental Table S10.

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 wasperformed by using a tool from the Matt Hudson laboratory (http://stan.cropsci.uiuc.edu/cgi-bin/sift/sift.cgi; Hudson and Quail, 2003).

Sequence data from this article can be found in the GenBank/EMBL datalibraries under accession number GSE48676.

Supplemental Data

The following materials are available in the online version of this article.

Supplemental Figure S1. Correlation values of Alternaria brassicicola, Ery-siphe orontii, infections, salt treatment, heat treatment, cadmium, andhypoxia examined in the ROSMETER platform.

Supplemental Figure S2. Distribution of the highly overrepresented GOterms in the CGS.

Supplemental Figure S3. The expression of FER1 probe in various stresstreatments.

Supplemental Figure S4. Photosynthetic efficiency of maximum photo-chemical efficiency of PSII in the dark-adapted state of DCMU-treatedplants.

Supplemental Figure S5. Expression of stress-induced transcripts in roottissue.

Supplemental Figure S6. The expression of stress-induced transcripts inwounded wild-type and ex1/ex2 seedlings.

Supplemental Figure S7. Confocal imaging of 4’,6-diamidino-2-phenyl-indole and SOSG fluorescence in root tissue.

Supplemental Table S1. Correlation scores and signature size for Figure 1.

Supplemental Table S2. Correlation scores and signature size forSupplemental 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 Genevesti-gator database of transcriptome experiments.

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Supplemental Table S7. Description of the primers used for qRT-PCRanalysis.

Supplemental Table S8. Stains, settings, and fluorescent markers.

Supplemental Table S9. Genes that changed significantly in stress treat-ments used to construct the CGS.

Supplemental Table S10. Source of transcriptome data used for the bio-informatics analysis.

ACKNOWLEDGMENTS

The authors thank Dr. Klaus Apel for the kind gift of the double mutantline ex1/ex2, Dr. Adi Avni for the kind gift of the flg22 peptide, Dr. NoamAlkan for fruitful discussions, and Vladimir Kiss for help with confocalimaging.

Received January 20, 2014; accepted March 3, 2014; published March 5, 2014.

LITERATURE CITED

Ashtamker C, Kiss V, Sagi M, Davydov O, Fluhr R (2007) Diverse sub-cellular locations of cryptogein-induced reactive oxygen species pro-duction in tobacco Bright Yellow-2 cells. Plant Physiol 143: 1817–1826

Downey T (2006) Analysis of a multifactor microarray study using Partekgenomics solution. Methods Enzymol 411: 256–270

Du Z, Zhou X, Ling Y, Zhang Z, Su Z (2010) agriGO: a GO analysis toolkitfor the agricultural community. Nucleic Acids Res 38: W64–W70

Dubiella U, Seybold H, Durian G, Komander E, Lassig R, Witte CP,Schulze WX, Romeis T (2013) Calcium-dependent protein kinase/NADPH oxidase activation circuit is required for rapid defense signalpropagation. Proc Natl Acad Sci USA 110: 8744–8749

Evans NH, McAinsh MR, Hetherington AM, Knight MR (2005) ROSperception in Arabidopsis thaliana: the ozone-induced calcium response.Plant J 41: 615–626

Fischer BB, Krieger-Liszkay A, Eggen RIL (2005) Oxidative stress inducedby the photosensitizers neutral red (type I) or rose bengal (type II) in thelight 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 OXY-GEN RESISTANT 1 links reactive electrophile signaling to singlet oxy-gen acclimation in Chlamydomonas reinhardtii. Proc Natl Acad Sci USA109: E1302–E1311

Flors C, Fryer MJ, Waring J, Reeder B, Bechtold U, Mullineaux PM,Nonell S, Wilson MT, Baker NR (2006) Imaging the production ofsinglet oxygen in vivo using a new fluorescent sensor, Singlet OxygenSensor Green. J Exp Bot 57: 1725–1734

Fluhr R (2009) Reactive oxygen-generating NADPH oxidases in plants. InRio LA, Puppo A, eds, Reactive Oxygen Species in Plant Signaling.Springer, Berlin, pp 1–23

Fufezan C, Rutherford AW, Krieger-Liszkay A (2002) Singlet oxygenproduction in herbicide-treated photosystem II. FEBS Lett 532: 407–410

Garmier M, Carroll AJ, Delannoy E, Vallet C, Day DA, Small ID, MillarAH (2008) Complex I dysfunction redirects cellular and mitochondrialmetabolism 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 SensorGreen�: photochemical behavior in solution and in a mammalian cell.Photochem Photobiol 87: 671–679

Hideg É (2008) A comparative study of fluorescent singlet oxygen probes inplant leaves. Cent Eur J Biol 3: 273–284

Hideg É, Barta C, Kálai T, Vass I, Hideg K, Asada K (2002) Detection of singletoxygen and superoxide with fluorescent sensors in leaves under stress byphotoinhibition or UV radiation. Plant Cell Physiol 43: 1154–1164

Hideg E, Spetea C, Vass I (1994) Singlet oxygen production in thylakoidmembranes during photoinhibition as detected by EPR spectroscopy.Photosynth Res 39: 191–199

Hideg E, Vass I, Kálai T, Hideg K (2000) Singlet oxygen detection withsterically hindered amine derivatives in plants under light stress.Methods Enzymol 319: 77–85

Hruz T, Laule O, Szabo G, Wessendorp F, Bleuler S, Oertle L, WidmayerP, Gruissem W, Zimmermann P (2008) Genevestigator v3: a referenceexpression database for the meta-analysis of transcriptomes. Adv Bio-informa 2008: 420747

Hudson ME, Quail PH (2003) Identification of promoter motifs involved in thenetwork of phytochrome A-regulated gene expression by combined analysisof genomic sequence and microarray data. Plant Physiol 133: 1605–1616

Irizarry RA, Hobbs B, Collin F, Beazer-Barclay YD, Antonellis KJ, ScherfU, Speed TP (2003) Exploration, normalization, and summaries of highdensity oligonucleotide array probe level data. Biostatistics 4: 249–264

Kanofsky JR, Axelrod B (1986) Singlet oxygen production by soybeanlipoxygenase isozymes. J Biol Chem 261: 1099–1104

Kanofsky JR, Sima PD (1995) Singlet oxygen generation from the reactionof 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 toexcess excitation energy in Arabidopsis. Science 284: 654–657

Kellogg EW III, Fridovich I (1975) Superoxide, hydrogen peroxide, andsinglet oxygen in lipid peroxidation by a xanthine oxidase system. J BiolChem 250: 8812–8817

Khan AU, Kasha M (1994) Singlet molecular oxygen in the Haber-Weissreaction. Proc 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 transla-tional control in the tigrina-d.12 mutant of barley. Proc Natl Acad SciUSA 106: 13112–13117

Kim C, Meskauskiene R, Zhang S, Lee KP, Lakshmanan Ashok M,Blajecka K, Herrfurth C, Feussner I, Apel K (2012) Chloroplasts ofArabidopsis are the source and a primary target of a plant-specific pro-grammed cell death signaling pathway. Plant Cell 24: 3026–3039

Kim CH, Apel K (2013) 1O2-mediated and EXECUTER-dependent retro-grade plastid-to-nucleus signaling in norflurazon-treated seedlings ofArabidopsis thaliana. Mol Plant 6: 1580–1591

Knox JP, Dodge AD (1984) Photodynamic damage to plant leaf tissue byrose bengal. Plant Sci Lett 37: 3–7

Kramarenko GG, Hummel SG, Martin SM, Buettner GR (2006) Ascorbatereacts with singlet oxygen to produce hydrogen peroxide. PhotochemPhotobiol 82: 1634–1637

Krieger-Liszkay A, Fufezan C, Trebst A (2008) Singlet oxygen production inphotosystem II and related protection mechanism. Photosynth Res 98: 551–564

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. ProcNatl Acad Sci USA 104: 672–677

Lee KP, Kim C, Landgraf F, Apel K (2007) EXECUTER1- and EXECUTER2-dependent transfer of stress-related signals from the plastid to the nu-cleus of Arabidopsis thaliana. Proc Natl Acad Sci USA 104: 10270–10275

Leshem Y, Melamed-Book N, Cagnac O, Ronen G, Nishri Y, Solomon M,Cohen G, Levine A (2006) Suppression of Arabidopsis vesicle-SNAREexpression inhibited fusion of H2O2-containing vesicles with tonoplastand increased salt tolerance. Proc Natl Acad Sci USA 103: 18008–18013

Lion Y, Delmelle M, van de Vorst A (1976) New method of detectingsinglet 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, et al (2010) Robin: an intuitivewizard application for R-based expression microarray quality assess-ment and analysis. Plant Physiol 153: 642–651

Ma S, Bohnert HJ (2007) Integration of Arabidopsis thaliana stress-relatedtranscript profiles, promoter structures, and cell-specific expression.Genome Biol 8: R49

MacManus-Spencer LA, McNeill K (2005) Quantification of singlet oxygenproduction in the reaction of superoxide with hydrogen peroxide usinga selective chemiluminescent probe. J Am Chem Soc 127: 8954–8955

Meskauskiene R, Nater M, Goslings D, Kessler F, op den Camp R, ApelK (2001) FLU: a negative regulator of chlorophyll biosynthesis in Ara-bidopsis thaliana. Proc Natl Acad Sci USA 98: 12826–12831

Miller G, Suzuki N, Ciftci-Yilmaz S, Mittler R (2010) Reactive oxygenspecies 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, et al (2007)Biological hydroperoxides and singlet molecular oxygen generation.IUBMB Life 59: 322–331

260 Plant Physiol. Vol. 165, 2014

Mor et al.

www.plantphysiol.orgon March 5, 2020 - Published by Downloaded from Copyright © 2014 American Society of Plant Biologists. All rights reserved.

Morker KH, Roberts MR (2011) Light exerts multiple levels of influence onthe Arabidopsis wound response. Plant Cell Environ 34: 717–728

Nakamura K, Ishiyama K, Ikai H, Kanno T, Sasaki K, Niwano Y, KohnoM (2011) Reevaluation of analytical methods for photogenerated singletoxygen. J Clin Biochem Nutr 49: 87–95

Nakano T, Suzuki K, Fujimura T, Shinshi H (2006) Genome-wide analysis ofthe 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 or-ganelle markers for co-localization studies in Arabidopsis and otherplants. Plant J 51: 1126–1136

Noctor G, De Paepe R, Foyer CH (2007) Mitochondrial redox biology andhomeostasis 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, et al (2012) Chloroplast-mediated activation of plant immune signalling in Arabidopsis. Nat Commun3: 926

op den Camp RGL, Przybyla D, Ochsenbein C, Laloi C, Kim C, Danon A,Wagner D, Hideg E, Göbel C, Feussner I, et al (2003) Rapid inductionof distinct stress responses after the release of singlet oxygen in Arabi-dopsis. Plant Cell 15: 2320–2332

Ramel F, Birtic S, Cuiné S, Triantaphylidès C, Ravanat JL, Havaux M(2012a) Chemical quenching of singlet oxygen by carotenoids in plants.Plant Physiol 158: 1267–1278

Ramel F, Birtic S, Ginies C, Soubigou-Taconnat L, Triantaphylidès C,Havaux M (2012b) Carotenoid oxidation products are stress signals thatmediate gene responses to singlet oxygen in plants. Proc Natl Acad SciUSA 109: 5535–5540

Ramel F, Ksas B, Akkari E, Mialoundama AS, Monnet F, Krieger-LiszkayA, Ravanat JL, Mueller MJ, Bouvier F, Havaux M (2013) Light-inducedacclimation of the Arabidopsis chlorina1 mutant to singlet oxygen. PlantCell 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 transcriptomicimprints related to reactive oxygen species type and origin provides new in-sights 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 oxy-gen species-related events during extended darkness. Plant Physiol 156:185–201

Rushton PJ, Somssich IE, Ringler P, Shen QJ (2010) WRKY transcriptionfactors. Trends Plant Sci 15: 247–258

Rustérucci C, Aviv DH, Holt BF III, Dangl JL, Parker JE (2001) The diseaseresistance signaling components EDS1 and PAD4 are essential regula-tors of the cell death pathway controlled by LSD1 in Arabidopsis. PlantCell 13: 2211–2224

Sagi M, Davydov O, Orazova S, Yesbergenova Z, Ophir R, Stratmann JW,Fluhr R (2004) Plant respiratory burst oxidase homologs impinge onwound responsiveness and development in Lycopersicon esculentum.Plant Cell 16: 616–628

Sagi M, Fluhr R (2001) Superoxide production by plant homologues of thegp91phox NADPH oxidase. Modulation of activity by calcium and bytobacco mosaic virus infection. Plant Physiol 126: 1281–1290

Sattler SE, Mène-Saffrané 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, PietzschT, Preibisch S, Rueden C, Saalfeld S, Schmid B, et al (2012) Fiji: anopen-source platform for biological-image analysis. Nat Methods 9:676–682

Snyder JW, Skovsen E, Lambert JDC, Poulsen L, Ogilby PR (2006) Opticaldetection of singlet oxygen from single cells. Phys Chem Chem Phys 8:4280–4293

Steinbeck MJ, Khan AU, Karnovsky MJ (1992) Intracellular singlet oxygengeneration by phagocytosing neutrophils in response to particles coatedwith 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 OpinPlant Biol 14: 691–699

Tian GW, Mohanty A, Chary SN, Li S, Paap B, Drakakaki G, Kopec CD,Li J, Ehrhardt D, Jackson D, et al (2004) High-throughput fluorescenttagging of full-length Arabidopsis gene products in planta. Plant Physiol135: 25–38

Triantaphylidès C, Havaux M (2009) Singlet oxygen in plants: production,detoxification and signaling. Trends Plant Sci 14: 219–228

Triantaphylidès C, Krischke M, Hoeberichts FA, Ksas B, Gresser G,Havaux M, Van Breusegem F, Mueller MJ (2008) Singlet oxygen is themajor reactive oxygen species involved in photooxidative damage toplants. Plant Physiol 148: 960–968

VolodarskyD, Leviatan N, Otcheretianski A, Fluhr R (2009) HORMONOMETER:a tool for discerning transcript signatures of hormone action in the Arabidopsistranscriptome. Plant Physiol 150: 1796–1805

Wagner D, Przybyla D, Op den Camp R, Kim C, Landgraf F, Lee KP,Würsch M, Laloi C, Nater M, Hideg E, et al (2004) The genetic basis ofsinglet oxygen-induced stress responses of Arabidopsis thaliana. Science306: 1183–1185

Walley JW, Coughlan S, Hudson ME, Covington MF, Kaspi R, Banu G,Harmer SL, Dehesh K (2007) Mechanical stress induces biotic andabiotic stress responses via a novel cis-element. PLoS Genet 3: 1800–1812

Wang P, Du Y, Zhao X, Miao Y, Song CP (2013) The MPK6-ERF6-ROS-responsive cis-acting Element7/GCC box complex modulates oxidativegene transcription and the oxidative response in Arabidopsis. PlantPhysiol 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 reso-nance 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 re-stricts 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 per-ception. Nature 428: 764–767

Zoeller M, Stingl N, Krischke M, Fekete A, Waller F, Berger S, MuellerMJ (2012) Lipid profiling of the Arabidopsis hypersensitive responsereveals specific lipid peroxidation and fragmentation processes: bio-genesis of pimelic and azelaic acid. Plant Physiol 160: 365–378

Plant Physiol. Vol. 165, 2014 261

Dark-Induced Singlet Oxygen during Stress

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