Imaging of photo-oxidative stress responses in leaves

Michael J. Fryer1, Kevin Oxborough1, Phillip M. Mullineaux2 and Neil R. Baker1,3

1Department of Biological Sciences, University of Essex, Colchester, Essex CO4 3SQ, UK2John Innes Centre, Norwich Research Park, Colney, Norwich, Norfolk NR4 7UH, UK

Received 10 July 2001; Accepted 7 December 2001

Abstract

High resolution digital imaging was used toidentify sites of photo-oxidative stress responses inArabidopsis leaves non-invasively, and to demon-strate the potential of using a suite of imagingtechniques for the study of oxidative metabolismin planta. Tissue-specific photoinhibition of photo-synthesis in individual chloroplasts in leaves wasimaged by chlorophyll fluorescence microscopy.Singlet oxygen production was assessed by imag-ing the quenching of the fluorescence of dansyl-2,2,5,5-tetramethyl-2,5-dihydro-1H-pyrrole (DanePy)that results from its reaction with singlet oxygen.Superoxide and hydrogen peroxide accumulationwere visualized by the reduction of nitroblue tetra-zolium (NBT) to formazan deposits and by polymer-izationwith 3,39-diaminobenzidine (DAB), respectively.Stress-induced expression of a gene involved withantioxidant metabolism was imaged from the bio-luminescence from leaves of an Arabidopsis APX2-LUC transformant, which co-expresses an ascorbateperoxidase (APX2) with firefly luciferase. Singletoxygen and superoxide production were found tobe primarily located in mesophyll tissues whereashydrogen peroxide accumulation and APX2 geneexpression were primarily localized in the vasculartissues.

Key words: Ascorbate peroxidase, chlorophyll fluorescence,

hydrogen peroxide, singlet oxygen, superoxide.

Introduction

Singlet oxygen, superoxide radical and hydrogen peroxideare reactive oxygen species (ROS) that are generatedwhen plant tissues are exposed to a variety of environ-mental stresses (Halliwell, 1984; Asada, 1996; Fryer et al.,1998). The extremely short life-times of ROS makes thestudy of their production in planta very difficult. Damageto proteins, lipids and DNA has often been used as anindex of oxidative stress (Halliwell and Gutteridge, 1985;Rice-Evans et al., 1991), although this is a rather indirectmeasure of the production of ROS. Spin labels have beenapplied with some success for the secondary detection byelectron paramagnetic resonance (EPR) of oxygen freeradicals and organic radicals from their semi-stableadducts (Hodgson and Raison, 1991; Heber et al., 1996;Van Doorslaer et al., 1999), but this does not provideinformation on the specific sites of ROS production intissues. Cell fractionation techniques have been used inattempts to identify sites of ROS generation and thelocation of ROS detoxification systems (Doulis et al.,1997; Kingston-Smith et al., 1999), however, these involveextensive tissue disruption which can lead to the genera-tion of ROS. Consequently, data produced using suchdestructive methods does not always result in an accuratepicture of the distribution of ROS and their detoxificationsystems. Also, such in vitro studies on macerated materialcannot resolve the differences in predisposition to oxid-ative stress that undoubtedly exist between various tissuesand cell types within leaves. In this paper it is demon-strated how the use of ROS-specific tracer dyes inconjunction with high resolution imaging now providesthe opportunity to identify sites of photo-oxidative stressand ROS accumulation in leaves.

3 To whom correspondence should be addressed. Fax: [44 (0)1206 873416. E-mail: baken@essex.ac.uk

Abbreviations: CCD, charge-coupled device; DAB, 3,39-diaminobenzidine; DanePy, dansyl-2,2,5,5-tetramethyl-2,5-dihydro-1H-pyrrole; F 9,

fluorescence level at any point between F 9o and F 9m; F 9m, maximal fluorescence level from leaves in light; F 9o, minimal fluorescence level of leaves in

light; F 9q, difference in fluorescence between F 9m and F9 (F9q\F 9m–F 9); NBT, nitroblue tetrazolium; 1O2, singlet oxygen; PPFD, photosynthetically-active

photon flux density; ROS, reactive oxygen species; SOD, superoxide dismutase.

� Society for Experimental Biology 2002

Journal of Experimental Botany, Vol. 53, No. 372,Antioxidants and Reactive Oxygen Species in Plants Special Issue,pp. 1249–1254, May 2002

Manipulation of genes encoding components of anti-oxidant systems has facilitated the non-invasive study ofthe expression of genes coding for enzymes involved inROS metabolism. The example presented in this paperis the transformation of Arabidopsis with an APX2-LUCtransgene, a cytosolic ascorbate peroxidase promoterfused to a firefly luciferase reporter gene (Karpinskiet al., 1999). APX2-LUC is silent under non-stressed con-ditions, but its mRNA is detectable within 15 min of theonset of a high light stress. Induction of APX2-LUCallows for the non-invasive bioluminescent imaging ofAPX2 gene expression and the analysis of factors thatinfluence it.

The combination of advanced molecular genetics tech-niques with non-invasive digital imaging of the generationof ROS illustrated in this paper provide extremelypowerful tools for future investigations of the develop-ment of oxidative stress in leaves and the regulation ofROS metabolism.

Materials and methods

Plant material, probe application and stress conditions

Arabidopsis thaliana plants were germinated and raised tomature rosette stage under controlled environmental conditions(PPFD, 200 mmol mY 2 sY 1 during a 16 h photoperiod at 25 �Cin a relative humidity of c. 80%). Leaves were excised at thepetiole with a razor blade and allowed transpirationally toimbibe aqueous solutions of fluorescent probes and dyes for90 min at the growth PPFD. Solutions were replaced with waterduring the following stress period. For the majority of thetreatments, tip regions of the leaf were placed inside the chamberof a computerized infrared gas analysis system (CIRAS,PP Systems, Hitchin, Hertfordshire) and exposed to an airflow rate of 347 cm3 minY 1. Photo-oxidative stress conditions(PPFD 650 mmol mY 2 sY 1 at 30 �C and water vapour pressure> 0.3 kPa) were imposed for 90 min.The development of the Arabidopsis thaliana transformant

with an APX2-LUC trangene has been previously reported(Karpinski et al., 1999). This transformant was grown underthe conditions described above.

High resolution, chlorophyll fluorescence imaging

High resolution imaging of chlorophyll fluorescence fromchloroplasts in intact leaves was carried out essentially asdescribed previously (Oxborough and Baker, 1997). F9o and F9mdefine the minimal and maximal fluorescence levels from leavesin the light, respectively. F9 is the fluorescence level at anypoint between F9o and F9m. For the construction of para-metized images, the specific term F9q was recently introduced(Oxborough and Baker, 1997; Baker et al., 2001) whichdenotes the difference between F9m and F9measured immediatelybefore the application of a saturating pulse to measure F9m.Under these conditions, F9q uF9m equates to the operatingquantum efficiency of PSII photochemistry. A Peltier-cooledcharge-coupled device (CCD) camera, as described earlier(Oxborough and Baker, 1997), was used for non-invasiveimaging of ROS-fluorogenic compounds and bioluminescentexpression of APX2-LUC.

Detection of reactive oxygen species

Singlet oxygen (1O2) activity was detected by infiltrating leaveswith 40 mM DanePy (dansyl-2,2,5,5,-tetramethyl-2,5-dihydro-1H-pyrrole), a dual fluorescent and spin probe. Reaction ofhighly fluorescent DanePy with 1O2 yields a non-fluorescentnitroxide radical (DanePyO) (Hideg et al., 1998, 2000, 2001).DanePy fluorescence in leaves was excited with 345 nmradiation produced from a UV source (ULT1004 Black RayLW, Scientific Laboratory Supplies, Wilford, UK) through a345 nm band pass filter (E46-084, Edmund Scientific, York,UK) and imaged using the CCD camera (described above)protected with a 532 nm band pass filter (E43-122, EdmundScientific, York, UK).Infiltration of leaves with 6 mM nitroblue tetrazolium (NBT)

allowed the detection of superoxide. When the pale yellow NBTreacts with superoxide a dark blue insoluble formazan com-pound is produced (Flohe and Otting, 1984; Beyer andFridovich, 1987). Superoxide is thought to be the majoroxidant species responsible for reducing NBT to formazan(Maly et al., 1989). Chlorophyll was removed from the leavesprior to imaging by infiltrating them with lacto-glycerol-ethanol(1 : 1 : 4 by vol) and boiling in water 5 min. The location offormazan deposits was visualized by subtracting background(non-formazan) pixels from the leaf image.Infiltration of leaves with 5 mM 3,39-diaminobenzidine

(DAB) at pH 3.8 allows the detection of hydrogen peroxidein leaves (Thordal-Christensen et al., 1997; Orozoco-Cardenasand Ryan, 1999). DAB forms a deep brown polymerizationproduct upon reaction with H2O2 in the presence of peroxidase(Thordal-Christensen et al., 1997), which can be imaged afterremoval of chlorophyll from the leaf, as described above.

Imaging of gene expression

Expression of APX2-LUC was monitored in leaves afterspraying with 1 mM luciferin (Promega). The treated leaveswere then imaged for 60 min with the Peltier-cooled CCDcamera (described above) in order to generate an image of thebioluminescence produced.

Results and discussion

Photoinhibition of photosynthesis results in changes inthe quantum efficiency of photosynthetic electron trans-port, which can be estimated from the fluorescence para-meter, F9quF9m. Examples of images of the chlorophyllfluorescence parameter F9 from chloroplasts in the bundlesheath (Fig. 1A) and mesophyll (Fig. 1D) cells of an intactleaf were used to generate images of F9quF9m (Fig. 1B, E)from which the individual chloroplasts can be isolatedand their F9quF9m values determined (Fig. 1C, F). Thechloroplasts in the bundle sheath and mesophyll cells ofthe leaf prior to the photoinhibitory treatment of 60 minat a PPFD of 600 mmol mY2 sY1 had F9q uF9m values of 0.67and 0.65, respectively (data not shown), which decreasedto 0.25 and 0.39, respectively during the photo-oxidativestress (Fig. 1C, F) demonstrating a large decrease in theoperating efficiency of non-cyclic photosynthetic electrontransport.

The accumulation of 1O2 during this photo-oxidativestress treatment is shown in Fig. 2. An Arabidopsis leaf

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was infiltrated with DanePy and then a region of the leaftip was irradiated with a PPFD of 600 mmol mY2 sY1.Images of the fluorescence of the DanePy show that thereis a marked decrease in the fluorescence emission from

the area of the leaf exposed to the high light treatment(Fig. 2), indicating the production of the nitroxide radicalDanePyO produced from the reaction of DanePy with1O2. It has been previously demonstrated that DanePy

Fig. 1. Imaging of chlorophyll fluorescence parameters from an Arabidopsis leaf after 60 min of exposure to high light. The leaf was grown under aPPFD of 200 mmol mY 2 sY 1 and then exposed to a light stress of 600 mmol mY 2 sY 1 for 60 min. Images of F9 (A, D) from bundle sheath (A) andmesophyll (D) cells. Images of F9q uF9mwere calculated from images of F9 and F9m for the bundle sheath (B) and mesophyll (E) cells, and presented in falsecolour. Using Fluorchart software the chloroplasts within the two cell types (bundle sheath in B and mesophyll in E) were isolated and an average valueof F9q uF9m calculated (C, F, shown above the chloroplast images). Data have been mapped to the colour palette shown between (B) and (E).

Fig. 2. An Arabidopsis leaf was infiltrated with DanePy and then an area of the leaf tip was exposed to a PPFD of 600 mmol mY 2 sY 1 for 60 min,which is indicated by the white oval in the reflected light image (A). The images of fluorescence emission from the DanePy before (B) and after (C) thehigh light treatment show that quenching of the fluorescence occurs within the area of the leaf tip exposed to the high light. This fluorescence quenchingresults from formation of non-fluorescent DanePyO when 1O2 reacts with DanePy.

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penetrates leaf tissues well and enters cells and chloro-plasts (Hideg et al., 2001). It appears that 1O2 isformed primarily in mesophyll tissue (Fig. 2). This wouldbe expected since the majority of the leaf ’s photosyntheticapparatus is contained in mesophyll chloroplasts and1O2 will be produced by photosensitized energy transferreactions between excited triplet state chlorophyll andground state molecular oxygen whenever excess excitationenergy cannot be efficiently dissipated as photochemistryor heat from chlorophylls (Halliwell, 1984; Wise andNaylor, 1987). 1O2 is suspected to be involved in damageto the D1 reaction centre protein of PSII (Vass et al.,1992; Aro et al., 1993), has also been implicated in lipidperoxidation by direct reaction with polyunsaturatedfatty acyl residues in membranes and in general oxidativedegradation of proteins (Halliwell, 1984).

Imaging of purple formazan deposits, which resultfrom the reaction of NBT with superoxide, identifies theregions of superoxide formation in a leaf. The tip regionof the leaf that has been exposed to the high light treat-ment clearly shows more intense staining than the non-stressed area, with the majority of the staining beingassociated with mesophyll tissue (Fig. 3). Numerous small,localized ‘hotspots’ were observed at the distal points ofthe microvasculature (Fig. 3). The petiole exhibited veryheavy staining which, presumably, is associated withsuperoxide accumulation resulting from NADPH oxidaseactivity, a major effector of the oxidative burst in thewound response (Mehdy et al., 1996; Wojtaszek, 1997).Staining in the petiole was most intense in veinal tissueclose to where the leaf was excised from the plant and

decreased with distance away from the wound; presum-ably this demonstrates that a systemic wounding signalthat stimulates production of superoxide has beenpropagated along the veins from the wound. AlthoughNBT also reacts with ascorbate, large changes in theascorbate pool would not be expected in leaf tissuesduring the period of photo-oxidative stress used and,consequently, imaging of formazan production can beconsidered to indicate superoxide accumulation; largeincreases in the rate of superoxide production wouldbe expected during the photoinhibitory stress. Areas offormazan deposits indicate cells in which the rate of super-oxide production has become significantly greater thanthe rate of detoxification.

The production of H2O2 was imaged in leaves infilt-rated with DAB, which reacts with H2O2 in the presenceof peroxidases to produce a brown polymerization prod-uct. Considerably more H2O2 was detected in the tipregion of the leaf that had been exposed to the high lighttreatment, with it being primarily associated with thevascular tissue (Fig. 4). The ascorbate–glutathione cyclehas been proposed for the removal of H2O2 in order toprevent its conversion to the extremely reactive andoxidizing hydroxyl radical within leaves (Halliwell, 1984).Ascorbate peroxidase (APX) converts H2O2 to mono-dehydroascorbate. Using the APX2-LUC Arabidopsistransgenote (Karpinski et al., 1999) it is possible toimage the expression of the gene for a cytosolic form ofascorbate peroxidase when leaves are exposed to the highlight treatment. Clearly there is widespread APX2 geneexpression in the light-treated area of the leaf, however,

Fig. 3. Image of an Arabidopsis leaf infiltrated with NBT. The top half of the leaf was exposed to a high light treatment of 600 mmol mY 2 sY 1 for60 min. The purple coloration indicates the formation of insoluble formazan deposits that are produced when NBT reacts with superoxide.

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some expression was also observed around the vasculartissue in the regions of the leaf that were kept in the dark(Fig. 5) demonstrating the systemic signalling systemwhichinduces APX2 expression (Karpinski et al., 1999).

Imaging the accumulation of 1O2, superoxide and H2O2

in intact leaves in parallel with fluorescence imaging ofthe operating efficiencies of electron transport in specifictissues can clearly provide some interesting and novelinsights into the spatial organization of oxidant biochem-istry occurring in response to photo-inhibitory stresses.In the leaves examined in this study accumulation of 1O2

(Fig. 2) and superoxide (Fig. 3) appeared to be principallylocated in mesophyll tissues during photo-oxidativestress, whereas H2O2 accumulation was more associatedwith the vascular tissue (Fig. 4). These observations

suggest that excess light results in a larger increase insuperoxide production in the mesophyll chloroplastscompared to that in the choroplasts of the vasculartissue, while hydrogen peroxide preferentially accumu-lates in the vascular tissues. A similar specific localizationof H2O2 within vascular tissue has been described inwounded leaves and is thought to be associated withthe systemic octadecanoid signalling pathway (Orozoco-Cardenas and Ryan, 1999). Presumably the accumulationof ROS in photosynthetic tissues is likely to be involvedin the signalling process associated with the switchingon of APX2 gene expression in response to the high lightstress.

Clearly, imaging of photoinhibition of photosynthesis,ROS production and the expression of genes codingfor enzymes involved in detoxification of ROS in intactleaves has great future potential for resolving theheterogeneity and nature of the responses of leaf tissuesto photo-oxidative stress.

Acknowledgements

These studies were supported by a grant from the Biotechnologyand Biological Sciences Research Council to NRB and PMM.The authors are grateful to Christine Edwards for synthesizingthe DanePy.

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Fig. 4. Image of an Arabidopsis leaf infiltrated with DAB. The top half of the leaf was exposed to a high light treatment of 600 mmol mY 2 sY 1 for60 min. The brown staining indicates the formation of a brown polymerization product when H2O2 reacts with DAB.

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