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Autoluminescence imaging: a non-invasive tool for mapping oxidative stress
Review
TRENDS in Plant Science
Vol.11 No.10
Autoluminescence imaging: a non-invasive tool for mapping oxidative stress Michel Havaux, Christian Triantaphylide`s and Bernard Genty CEA/Cadarache, DSV, DEVM, Laboratoire d’Ecophysiologie Mole´culaire des Plantes, UMR 6191 CEA-CNRS-Aix Marseille University, F-13108 Saint-Paul-lez-Durance, France
Living organisms spontaneously emit faint light, and this autoluminescence is stimulated in response to many stresses. This phenomenon is attributable to the endogenous production of excited states during oxidative reactions, particularly during peroxidation of lipids, which generates light-emitting molecules such as triplet carbonyls and singlet oxygen. Using highly sensitive cameras, it is now possible to remotely image spontaneous luminescence with a good spatial resolution, providing a new non-invasive tool for mapping oxidative stress and lipid peroxidation in plants. The types of luminescence of living organisms Plants, animals and bacteria glow continuously. This spontaneous photon emission (SPE) occurs in the ultraviolet and visible range of wavelengths with a low intensity of 10 19 to 10 16 W cm 2 [1]. This is about three orders of magnitude less than the luminescence arising from enzymatic reactions in fireflies, jellyfishes and some fishes in a transitory phenomenon commonly called bioluminescence [2]. It is different from the luminescence of chlorophyll, emitted by photosynthetic organisms following exposure to light, which results from recombination of positively charged carriers at the donor side of photosystem II and electrons on the acceptor side [3,4]. The chlorophyll signal decays rapidly in the dark within seconds to minutes. Here, we provide background information about SPE, with emphasis on the origin of the signal, and discuss in more detail imaging of SPE in plants in comparison with other available techniques. Spontaneous photon emission, a signature of biological oxidation status SPE, often called biophoton emission, is attributed to the endogenous production of excited states during oxidative metabolic reactions [5,6] and it is therefore closely related to the oxidation status of the organism. The major source of SPE is the slow spontaneous decomposition of lipid peroxides and endoperoxides, such as dioxetanes, which generates the light-emitting byproducts singlet oxygen and excited carbonyl groups [7]. The term ‘lipids’ is used here in a broad sense and is not restricted to fatty acids but also Corresponding author: Havaux, M. ([email protected]). Available online 7 September 2006. www.sciencedirect.com
includes carotenoids or terpenoids, which can produce endoperoxides upon oxidation [8,9]. Singlet oxygen is produced in reactions between two lipid peroxy radicals, and the light emission peaks in the red (at 634 and 703 nm; Figure 1). By contrast, the bimolecular reaction of alkoxy radicals and the cleavage of dioxetanes lead to the formation of excited triplet carbonyls, which in turn decay and yield light in the range 350 to 480 nm (blue). Consistently, spectral analyses of the luminescence emission of oxidized lipids in vitro showed the expected peaks in the blue and red-near infrared domains [10]. However, in plants, triplet carbonyls can also transfer excitation energy to chlorophyll molecules [11] whose deactivation is associated with photon emission at around 730 nm (far-red light) [12,13]. Formation of activated carbonyls from lipid hydroperoxide decomposition is stimulated by heat [14], and this phenomenon is the basis of the luminescence burst observed at 135 8C in plant leaves (Figure 2a) [3,4,15]. The amplitude of the luminescence measured at this temperature is two orders of magnitude higher than the roomtemperature autoluminescence amplitude. Numerous thermoluminescence studies have validated the use of the luminescence measured at 135 8C as an index of lipid peroxidation by correlating the signal amplitude with the extent of lipid peroxidation, which can be measured using a variety of biochemical methods [15–18]. The luminescence at 135 8C has been used in plants to quantify lipid damage after oxidative stress [18–20] or to characterize the stress resistance of mutants [16,17,21]. Importantly, when the luminescence signal emitted by Arabidopsis leaves at 25 8C was plotted against the luminescence emission at 135 8C, a linear regression was obtained (Figure 2b), confirming that both signals reflect oxidative stress damage and supporting the hypothesis that they could have a common origin in plants [15]. Light produced during lipid peroxidation therefore constitutes an internal probe of oxidative stress that has many potential applications. Lipid peroxidation and oxidative stress have been measured by room-temperature SPE in a variety of samples, from whole organisms to isolated membranes or lipoproteins, and even in human breath (reviewed in Refs [1,15]). In many studies, the intensity of SPE correlated with other indexes of lipid peroxidative degradation, such as the level of conjugated dienes, the malondialdehyde content or the concentration of
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Review
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481
Figure 1. Chemiluminescence associated with lipid peroxidation. The emission of light is related to the production of excited carbonyl compounds and singlet oxygen (1O2) that occurs during lipid peroxide degradation. Self-reaction of lipid peroxy radicals (the Russel reaction) gives a tetroxide intermediate, which decomposes either into a triplet state carbonyl compound (reaction 1a; the triplet state is indicated by a star) or into singlet oxygen (reaction 1b). Self-reaction of alkoxy radicals produces excited carbonyl compounds in a redox reaction (reaction 2). The singlet oxygen that is produced during lipid peroxidation (such as reaction 1b) reacts with unsaturated compounds, leading to the production of dioxetanes, which yield triplet state carbonyl compounds upon decomposition (reaction 3). Upon relaxation, the excited carbonyl compounds emit light in the blue region (380–460 nm; reaction 4a) whereas bimolecular emission of singlet oxygen occurs in the red region (634 and 703 nm; reaction 4b). Abbreviation: hy, energy of a single photon. Adapted from Refs [6,7].
exogenously added lipid hydroperoxides [22–24]. This non-invasive technique is useful for real-time monitoring of rapid perturbations of the cellular oxidation state and is performed without an exogenous probe.
Imaging of spontaneous photon emission A currently emerging and promising application of SPE is the imaging of oxidative stress in intact tissues. Early attempts at autoluminescence imaging in the 1990s used
Figure 2. Luminescence of Arabidopsis leaves and its variation with temperature. (a) High-temperature luminescence emission from Arabidopsis leaves exposed to photo-oxidative stress. The curves are of wild type (Columbia; 1) and a vte1 npq1 double mutant deficient in the carotenoid zeaxanthin and in vitamin E (2) [17]. Plants were exposed for 3 days to high light stress (1200 mmol m 2 s 1, 8 h photoperiod) at low temperature (8 8C). The luminescence band peaking at around 135 8C is attributed to lightemitting molecules produced during lipid peroxidation [15]. (b) Correlation between the luminescence emission at 25–30 8C (commonly used for imaging) and the amplitude of the luminescence band at 135 8C. The results shown were obtained in a series of experiments on a range of Arabidopsis mutants exposed to various stress conditions. www.sciencedirect.com
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Figure 3. Imaging of the spontaneous luminescence emission from Arabidopsis plants at room temperature (1, double mutant vtc2 npq1 deficient in both ascorbate and zeaxanthin; 2, single mutant vtc2 deficient in ascorbate; 3, wild type) pre-exposed for 4 days to high light stress (4 8C, 1600 mmol m 2 s 1, 8 h/16 h day/night regime). These mutants are differently sensitive to high light stress [21]. (a) Luminescence, as imaged with a liquid nitrogen-cooled CCD camera (VersArray LN/CCD 1340–1300B, Roper Scientific (as in Ref. [32]), mounted with a back-illuminated CCD from E2V CCD36–40). The emitting sample was imaged on the sensor by a 50-mm focal distance lens with an f-number of 1.2 (F mount Nikkor 50-mm, f:1.2, Nikon) for maximizing light collection. Integration time was 60 min using the full resolution of the CCD (1300 1340 pixels). Plants were adapted to darkness for 2 h before measurements. Randomly distributed white spots are the results of stray radiation in the range of gamma and cosmic rays. The color scale indicates signal intensity from 0 (blue) to 250 (white). (b) Photograph of the corresponding plants. Asterisks indicate the type of leaves used for the malondialdehyde (MDA) analysis in (c) (i.e. larger, mature leaves). (c) MDA level in leaves. MDA levels in mature leaves of the vtc2 npq1 double mutant (1), vtc2 mutant (2) and wild type (3) plants that had been exposed to high light stress for 4 days were determined by HPLC as described in Ref. [17]. Data are mean values of a minimum of three experiments + standard deviation.
single photon counting imaging techniques that demonstrated the feasibility of this approach. However, most of the images obtained with these systems had low spatial resolution. Nevertheless, SPE from the brain of a rat was visualized and the imaged signal correlated with cerebral energy metabolism and oxidative stress [25]. SPE in plant tissues infected with a pathogen, injured mechanically or exposed to drought stress were also visualized with this technique [26–28]. These pioneering studies generated interest in SPE and prompted researchers to use alternative technologies to improve the imaging of this signal. Recent studies on plants, animals and humans have shown that autoluminescence imaging can be improved using highly sensitive charge-coupled device (CCD) cameras. Cooling of the sensor, using peltier devices (thermoelectric modules) or liquid nitrogen, is required for reducing thermal noise and enabling measurement of faint light by signal integration. Using this approach, SPE from mouse tumors and from the human body were recently imaged [29,30]. In plant science, the first application of CCD to SPE imaging was made using germinating seedlings [31]. High-resolution images of SPE from detached leaves were also reported [32]. So far, the best images of plant autoluminescence allowing quantitative mapping have been obtained by Michel Flor-Henry and co-workers [13], who used a cooled CCD detector coupled to optical fibers on which detached leaves were placed. This configuration enables maximal capture of light and high sensitivity, but it is applicable to flat samples only (e.g. detached leaves). Wound-induced luminescence of leaves was imaged with a good spatial resolution (222 000 pixels per image). Although the origin of the signal was not investigated in detail in this study, lipid peroxidation products are probable candidates [7,33]. Because wounding-induced photon emission is quenched by sodium azide and amplified by deuterium oxide [34], singlet oxygen is likely to be involved in this light emission. www.sciencedirect.com
If leaf autoluminescence does reflect lipid oxidative damage, mutants known to be sensitive to oxidative stress should emit more photons than do wild types. This is shown in Figure 3, in which SPE from whole Arabidopsis plants was imaged using a liquid nitrogen-cooled CCD camera similar to that described in Ref. [32]. The plants were pre-adapted to darkness for 2 h to allow photosynthesis-related chlorophyll luminescence to decay [13]. Two photosensitive mutants, the vtc2 single mutant deficient in ascorbate and the vtc2 npq1 double mutant deficient in both ascorbate and zeaxanthin [21], were compared with the photoresistant wild type after high light stress. Both mutants showed a much higher stimulation of SPE after photostress than did the wild type (Figure 3a), and SPE of the mutants was proportional to their relative sensitivity to oxidative stress [21]. Mature Arabidopsis leaves are well known to be less tolerant to photo-oxidative stress than young leaves at the center of the rosette, and their SPE reflected this difference. Furthermore, the SPE intensity of mature leaves was related to the concentration of malondialdehyde, a biochemical marker of lipid peroxidation (Figure 3c). Another Arabidopsis double mutant, vte1 npq1, deficient in two major antioxidants of the plastid membranes (vitamin E and zeaxanthin), is highly sensitive to high light, with leaves suffering extensive lipid peroxidation, which was measured by various methods [17]. The sensitivity of the vte1 npq1 double mutant to lipid peroxidative damage can be readily monitored by autoluminescence imaging (Figure 4a): stimulation of SPE occurs much more rapidly and more intensively in mutant leaves compared with wild-type leaves. Importantly, stimulated SPE from vte1 npq1 double mutant leaves was detected after 6 h in high light, before symptoms of oxidative damage were visible on the leaves (>26 h). A field that has much to gain from this new imaging method is phytopathology. For example, infiltration of a tobacco leaf with the elicitor cryptogein in the dark induced
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Figure 4. Measurement of the effects of photooxidative stress and the hypersensitive defense response on SPE. (a) Autoluminescence imaging of Arabidopsis plants [wild type (WT) and vte1 npq1 double mutant deficient in vitamin E and zeaxanthin] pre-exposed to high light stress at low temperature (4 8C, 1600 mmol m 2 s 1, on a regime of 14 h day and 10 h night) for 0 to 54 h. The vte1 npq1 double mutant is highly sensitive to high light stress [17], and symptoms of oxidative stress (leaf bleaching) were observed visually after 26 h in the mutant and after 30 h in the wild type. Plants were adapted to darkness for 2 h before measurements. (b) Autoluminescence imaging of a tobacco leaf infiltrated with the elicitor cryptogein (2 mg) for 0–25 h in the dark. The cryptogein treatment was performed as described in Ref. [35]. Integration time was 20 min and on-CCD binning of 2 2 pixels was used to increase detection sensitivity (the resulting resolution is 650 670 pixels). The color scale indicates signal intensity from 0 (blue) to 250 (white).
hypersensitive programmed cell death [35] and an enhancement of SPE [19] that can be quantified by CCD imaging, as shown in Figure 4b. Increased SPE was detected 18 h after infiltration, matching the time course of the massive enzymatic lipid hydroperoxide accumulation [35]. Recently, Mark Bennett and co-workers [36] showed bursts of light over the Arabidopsis leaf surface induced during the hypersensitive response to a pathogen. It seems that this photon emission is different to the SPE associated with lipid peroxidation because it was transient, beginning around 2 h after inoculation and lasted only 2–3 h. This transient light signal, which presumably reflects some signaling phenomena related to nitric oxide in the plant defense response [36], also has great potential for applications in phytopathological studies. Because SPE imaging can be performed on intact plants and does not require an exogenously added probe or sample preparation (except adaptation to darkness), it is expected to provide realistic mapping of lipid peroxidative damage and oxidative stress in plants. This new approach is complementary to more direct measures of reactive oxygen species such as fluorescence imaging using fluorescent probes sensitive to singlet oxygen and/or reduced forms of oxygen [16,37–39]. Fluorescence imaging is useful because it provides information on specific reactive oxygen species and their sites of production in tissues. However, fluorescence imaging of reactive oxygen species requires pre-infiltration of plant tissues with an exogenous probe and it is therefore usually restricted to cells or organs (e.g. detached leaves). A new approach based on redox-sensitive green fluorescent proteins (GFPs) has recently emerged to monitor non-invasively redox www.sciencedirect.com
changes in living cells [40–42]. Redox sensing is achieved by introducing cysteine residues close to the chromophore –their oxidation causes changes in the GFP fluorescence characteristics. Although application of this technique to imaging in plants is not yet fully documented [43], redox-sensitive GFPs are promising tools for visualizing the oxidation of specific proteins or organelles. However, this approach is technically more complex than SPE imaging because it requires genetic transformation of the biological material. Concluding remarks Autoluminescence imaging is likely to become an important tool in stress physiology to measure the oxidation status of plants non-invasively. Oxidative stress imaged by SPE is useful not only for quantification purposes, but also for the early detection of oxidative stress before damage becomes visible. SPE can be measured at different integration levels, from small populations of organisms to cells. Because of its high sensitivity and easy use, SPE imaging should enable the rapid screening of mutants, including algae, cyanobacteria and vascular plants, with variable tolerance to oxidative stress. Even though the sensitive equipment required for acquiring SPE images is still relatively expensive, we predict that this new imaging approach will successfully complement the assortment of functional imaging tools available to plant biologists [44]. Acknowledgements Financial support from CEA (program ‘Toxicologie Nucle´aire Environnementale’) and EU project IHP-RTN-00–2001 ‘STRESSIMAGING’ is acknowledged. We thank Ilja Reiter for helpful discussions.
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References 1 Devaraj, B. et al. (1997) Biophotons: ultraweak light emission from living systems. Curr. Opin. Solid State Mater. Sci. 2, 188–193 2 Wilson, T. and Hastings, J.W. (1998) Bioluminescence. Annu. Rev. Cell Dev. Biol. 14, 197–230 3 Ducruet, J-M. (2003) Chlorophyll thermoluminescence of leaf discs: simple instruments and progress in signal interpretation open the way to new ecophysiological indicators. J. Exp. Bot. 54, 2419–2430 4 Gilbert, M. et al. (2004) A new type of thermoluminometer: a highly sensitive tool in applied photosynthesis research and plant stress physiology. J. Plant Physiol. 161, 641–651 5 Abeles, F.B. (1986) Plant chemiluminescence. Annu. Rev. Plant Physiol. 37, 49–72 6 Sies, H. (1987) Intact organ spectrophotometry and single-photon counting. Arch. Toxicol. 60, 138–143 7 Halliwell, B. and Gutteridge, J.M.C. (1989) Free Radicals in Biology and Medicine. Clarendon Press 8 Stratton, S.P. et al. (1993) Isolation and identification of singlet oxygen oxidation products of beta-carotene. Chem. Res. Toxicol. 6, 542–547 9 Baker, D.L. et al. (1999) Reactions of beta-carotene with cigarette smoke oxidants. Identification of carotenoid oxidation products and evaluation of the prooxidant/antioxidant effect. Chem. Res. Toxicol. 12, 535–543 10 Miyazawa, T. et al. (1982) Spectroscopic analysis of the weak light generated in autoxidation of linseed oil. Agric. Biol. Chem. 46, 1671– 1672 11 Bohne, C. et al. (1986) Chlorophyll: an efficient detector of electronically excited species in biochemical systems. Anal. Biochem. 155, 1–9 12 Hideg, E. and Vass, I. (1993) The 75 8C thermoluminescence band of green tissues: chemiluminescence from membrane-chlorphyll interaction. Photochem. Photobiol. 58, 280–283 13 Flor-Henry, M. et al. (2004) Use of a highly sensitive two-dimensional luminescence imaging system to monitor endogenous bioluminescence in plant leaves. BMC Plant Biol. 4, 19 14 Vavilin, D.V. and Ducruet, J.M. (1998) The origin of 115–130 8C thermoluminescence bands in chlorophyll-containing material. Photochem. Photobiol. 68, 191–198 15 Havaux, M. (2003) Spontaneous and thermoinduced photon emission: new methods to detect and quantify oxidative stress in plants. Trends Plant Sci. 8, 409–413 16 Baroli, I. et al. (2004) Photo-oxidative stress in a xanthophyll-deficient mutant of Chlamydomonas. J. Biol. Chem. 279, 6337–6344 17 Havaux, M. et al. (2005) Vitamin E protects against photoinhibition and photooxidative stress in Arabidopsis thaliana. Plant Cell 17, 3451– 3469 18 Vavilin, D.V. et al. (1998) Membrane lipid peroxidation, cell viability and Photosystem II activity in the green alga Chlorella pyrenoidosa subjected to various stress conditions. J. Photochem. Photobiol. B. Biol 42, 233–239 19 Stallaert, V.M. et al. (1995) Lipid peroxidation in tobacco leaves treated with the elicitor cryptogein: evaluation by high-temperature thermoluminescence emission and chlorophyll fluorescence. Biochim. Biophys. Acta 1229, 290–295 20 Marder, J.B. et al. (1998) Light-independent thermoluminescence from thylakoids of greening barley leaves. Evidence of oxygen radicals and free chlorophyll. Physiol. Plant. 104, 713–719 21 Mu¨ller-Moule´, P. et al. (2003) Zeaxanthin deficiency enhances the high light sensitivity of an ascorbate-deficient mutant of Arabidopsis. Plant Physiol. 133, 748–760 22 Albertini, R. and Abuja, P.M. (1998) Monitoring of low-density lipoprotein oxidation by low-level chemiluminescence. Free Radic. Res. 29, 75–83
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23 Doi, H. et al. (2002) Chemiluminescence associated with the oxidative metabolism of salicylic acid in rat liver microsomes. Chem. Biol. Interact. 140, 109–119 24 Guajardo, M.H. et al. (2002) Retinal fatty acid binding proteins reduce lipid peroxidation stimulated by long-chain fatty acid hydroperoxides on rod outer segments. Biochim. Biophys. Acta 1581, 65–74 25 Kobayashi, M. et al. (1999) In vivo imaging of spontaneous ultraweak photon emission from a rat’s brain correlated with cerebral energy metabolism and oxidative stress. Neurosci. Res. 34, 103–113 26 Suzuki, S. et al. (1991) Two-dimensional imaging and counting of ultraweak emission patterns from injured plant seedlings. J. Photochem. Photobiol. B. Biol 9, 211–217 27 Makino, T. et al. (1996) Ultraweak luminescence generated by sweet potato and Fusarium oxysporum interactions associated with a defense response. Photochem. Photobiol. 64, 953–956 28 Ohya, T. et al. (2002) Biophoton emission due to drought injury in red beans: possibility of early detection of drought injury. Jpn. J. Appl. Phys. 41, 4766–4771 29 Takeda, M. et al. (2004) Biophoton detection as a novel technique for cancer imaging. Cancer Sci. 95, 656–661 30 Van Wijk, R. et al. (2006) Anatomic characterization of human ultraweak photon emission with a moveable photomultiplier and CCD imaging. J. Photochem. Photobiol. B. Biol 83, 69–76 31 Kobayashi, M. et al. (1997) Two-dimensional imaging of ultraweak photon emission from germinating soybean seedlings with a highly sensitive CCD camera. Photochem. Photobiol. 65, 535–537 32 Creath, K. and Schwartz, G.E. (2004) Biophoton images of plants: revealing the light within. J. Altern. Complement. Med. 10, 23–26 33 Reverberi, M. et al. (2005) Relationship among lipoperoxides, jasmonates and indole-3-acetic acid formation in potato tubers after wounding. Free Radic. Res. 39, 637–647 34 Chen, W.L. et al. (2003) Imaging of ultraweak bio-chemiluminescence and singlet oxygen generation in germinating soybean in response to wounding. Luminescence 18, 37–41 35 Ruste´rucci, C. et al. (1999) Involvement of lipoxygenase-dependent production of fatty acid hydroperoxides in the development of the hypersensitive cell death induced by cryptogein on tobacco leaves. J. Biol. Chem. 274, 36446–36455 36 Bennett, M. et al. (2005) Biophoton imaging: a nondestructive method for assaying R gene responses. Mol. Plant Microbe Interact. 18, 95–102 37 Fryer, M.J. et al. (2002) Imaging of photo-oxidative stress responses in leaves. J. Exp. Bot. 53, 1249–1254 38 Flors, C. et al. (2006) Imaging the production of singlet oxygen in vivo using a new fluorescent sensor. Singlet Oxygen Sensor Green. J. Exp. Bot. 57, 1725–1734 39 Hideg, E. et al. (2006) Singlet oxygen in plants – its significance and possible detection with double (fluorescent and spin) indicator reagents. Photochem. Photobiol DOI: 10.1562/2006-02-06-RA-797 (http://phot.allenpress.com) 40 Ostergaard, H. et al. (2001) Shedding light on disulfide bond formation: engineering a redox switch in green fluorescent protein. EMBO J. 20, 5853–5862 41 Dooley, C.T. et al. (2004) Imaging dynamic redox changes in mammalian cells with green fluorescent protein indicators. J. Biol. Chem. 279, 22284–22293 42 Bjo¨rnberg, O. et al. (2006) Measuring intracellular redox conditions using GFP-based sensors. Antioxid. Redox Signal. 8, 354–361 43 Jiang, K. et al. (2006) Expression and characterization of a redoxsensing green fluorescent protein (reduction-oxidation-sensitive green fluorescence protein) in Arabidopsis. Plant Physiol. 141, 397– 403 44 Chaerle, L. and Van der Straeten, D. (2000) Imaging techniques and the early detection of plant stress. Trends Plant Sci. 5, 495–501
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Autoluminescence imaging: a non-invasive tool for mapping oxidative stress Michel Havaux, Christian Triantaphylide`s and Bernard Genty CEA/Cadarache, DSV, DEVM, Laboratoire d’Ecophysiologie Mole´culaire des Plantes, UMR 6191 CEA-CNRS-Aix Marseille University, F-13108 Saint-Paul-lez-Durance, France
Living organisms spontaneously emit faint light, and this autoluminescence is stimulated in response to many stresses. This phenomenon is attributable to the endogenous production of excited states during oxidative reactions, particularly during peroxidation of lipids, which generates light-emitting molecules such as triplet carbonyls and singlet oxygen. Using highly sensitive cameras, it is now possible to remotely image spontaneous luminescence with a good spatial resolution, providing a new non-invasive tool for mapping oxidative stress and lipid peroxidation in plants. The types of luminescence of living organisms Plants, animals and bacteria glow continuously. This spontaneous photon emission (SPE) occurs in the ultraviolet and visible range of wavelengths with a low intensity of 10 19 to 10 16 W cm 2 [1]. This is about three orders of magnitude less than the luminescence arising from enzymatic reactions in fireflies, jellyfishes and some fishes in a transitory phenomenon commonly called bioluminescence [2]. It is different from the luminescence of chlorophyll, emitted by photosynthetic organisms following exposure to light, which results from recombination of positively charged carriers at the donor side of photosystem II and electrons on the acceptor side [3,4]. The chlorophyll signal decays rapidly in the dark within seconds to minutes. Here, we provide background information about SPE, with emphasis on the origin of the signal, and discuss in more detail imaging of SPE in plants in comparison with other available techniques. Spontaneous photon emission, a signature of biological oxidation status SPE, often called biophoton emission, is attributed to the endogenous production of excited states during oxidative metabolic reactions [5,6] and it is therefore closely related to the oxidation status of the organism. The major source of SPE is the slow spontaneous decomposition of lipid peroxides and endoperoxides, such as dioxetanes, which generates the light-emitting byproducts singlet oxygen and excited carbonyl groups [7]. The term ‘lipids’ is used here in a broad sense and is not restricted to fatty acids but also Corresponding author: Havaux, M. ([email protected]). Available online 7 September 2006. www.sciencedirect.com
includes carotenoids or terpenoids, which can produce endoperoxides upon oxidation [8,9]. Singlet oxygen is produced in reactions between two lipid peroxy radicals, and the light emission peaks in the red (at 634 and 703 nm; Figure 1). By contrast, the bimolecular reaction of alkoxy radicals and the cleavage of dioxetanes lead to the formation of excited triplet carbonyls, which in turn decay and yield light in the range 350 to 480 nm (blue). Consistently, spectral analyses of the luminescence emission of oxidized lipids in vitro showed the expected peaks in the blue and red-near infrared domains [10]. However, in plants, triplet carbonyls can also transfer excitation energy to chlorophyll molecules [11] whose deactivation is associated with photon emission at around 730 nm (far-red light) [12,13]. Formation of activated carbonyls from lipid hydroperoxide decomposition is stimulated by heat [14], and this phenomenon is the basis of the luminescence burst observed at 135 8C in plant leaves (Figure 2a) [3,4,15]. The amplitude of the luminescence measured at this temperature is two orders of magnitude higher than the roomtemperature autoluminescence amplitude. Numerous thermoluminescence studies have validated the use of the luminescence measured at 135 8C as an index of lipid peroxidation by correlating the signal amplitude with the extent of lipid peroxidation, which can be measured using a variety of biochemical methods [15–18]. The luminescence at 135 8C has been used in plants to quantify lipid damage after oxidative stress [18–20] or to characterize the stress resistance of mutants [16,17,21]. Importantly, when the luminescence signal emitted by Arabidopsis leaves at 25 8C was plotted against the luminescence emission at 135 8C, a linear regression was obtained (Figure 2b), confirming that both signals reflect oxidative stress damage and supporting the hypothesis that they could have a common origin in plants [15]. Light produced during lipid peroxidation therefore constitutes an internal probe of oxidative stress that has many potential applications. Lipid peroxidation and oxidative stress have been measured by room-temperature SPE in a variety of samples, from whole organisms to isolated membranes or lipoproteins, and even in human breath (reviewed in Refs [1,15]). In many studies, the intensity of SPE correlated with other indexes of lipid peroxidative degradation, such as the level of conjugated dienes, the malondialdehyde content or the concentration of
1360-1385/$ – see front matter ß 2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.tplants.2006.08.001
Review
TRENDS in Plant Science
Vol.11 No.10
481
Figure 1. Chemiluminescence associated with lipid peroxidation. The emission of light is related to the production of excited carbonyl compounds and singlet oxygen (1O2) that occurs during lipid peroxide degradation. Self-reaction of lipid peroxy radicals (the Russel reaction) gives a tetroxide intermediate, which decomposes either into a triplet state carbonyl compound (reaction 1a; the triplet state is indicated by a star) or into singlet oxygen (reaction 1b). Self-reaction of alkoxy radicals produces excited carbonyl compounds in a redox reaction (reaction 2). The singlet oxygen that is produced during lipid peroxidation (such as reaction 1b) reacts with unsaturated compounds, leading to the production of dioxetanes, which yield triplet state carbonyl compounds upon decomposition (reaction 3). Upon relaxation, the excited carbonyl compounds emit light in the blue region (380–460 nm; reaction 4a) whereas bimolecular emission of singlet oxygen occurs in the red region (634 and 703 nm; reaction 4b). Abbreviation: hy, energy of a single photon. Adapted from Refs [6,7].
exogenously added lipid hydroperoxides [22–24]. This non-invasive technique is useful for real-time monitoring of rapid perturbations of the cellular oxidation state and is performed without an exogenous probe.
Imaging of spontaneous photon emission A currently emerging and promising application of SPE is the imaging of oxidative stress in intact tissues. Early attempts at autoluminescence imaging in the 1990s used
Figure 2. Luminescence of Arabidopsis leaves and its variation with temperature. (a) High-temperature luminescence emission from Arabidopsis leaves exposed to photo-oxidative stress. The curves are of wild type (Columbia; 1) and a vte1 npq1 double mutant deficient in the carotenoid zeaxanthin and in vitamin E (2) [17]. Plants were exposed for 3 days to high light stress (1200 mmol m 2 s 1, 8 h photoperiod) at low temperature (8 8C). The luminescence band peaking at around 135 8C is attributed to lightemitting molecules produced during lipid peroxidation [15]. (b) Correlation between the luminescence emission at 25–30 8C (commonly used for imaging) and the amplitude of the luminescence band at 135 8C. The results shown were obtained in a series of experiments on a range of Arabidopsis mutants exposed to various stress conditions. www.sciencedirect.com
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Figure 3. Imaging of the spontaneous luminescence emission from Arabidopsis plants at room temperature (1, double mutant vtc2 npq1 deficient in both ascorbate and zeaxanthin; 2, single mutant vtc2 deficient in ascorbate; 3, wild type) pre-exposed for 4 days to high light stress (4 8C, 1600 mmol m 2 s 1, 8 h/16 h day/night regime). These mutants are differently sensitive to high light stress [21]. (a) Luminescence, as imaged with a liquid nitrogen-cooled CCD camera (VersArray LN/CCD 1340–1300B, Roper Scientific (as in Ref. [32]), mounted with a back-illuminated CCD from E2V CCD36–40). The emitting sample was imaged on the sensor by a 50-mm focal distance lens with an f-number of 1.2 (F mount Nikkor 50-mm, f:1.2, Nikon) for maximizing light collection. Integration time was 60 min using the full resolution of the CCD (1300 1340 pixels). Plants were adapted to darkness for 2 h before measurements. Randomly distributed white spots are the results of stray radiation in the range of gamma and cosmic rays. The color scale indicates signal intensity from 0 (blue) to 250 (white). (b) Photograph of the corresponding plants. Asterisks indicate the type of leaves used for the malondialdehyde (MDA) analysis in (c) (i.e. larger, mature leaves). (c) MDA level in leaves. MDA levels in mature leaves of the vtc2 npq1 double mutant (1), vtc2 mutant (2) and wild type (3) plants that had been exposed to high light stress for 4 days were determined by HPLC as described in Ref. [17]. Data are mean values of a minimum of three experiments + standard deviation.
single photon counting imaging techniques that demonstrated the feasibility of this approach. However, most of the images obtained with these systems had low spatial resolution. Nevertheless, SPE from the brain of a rat was visualized and the imaged signal correlated with cerebral energy metabolism and oxidative stress [25]. SPE in plant tissues infected with a pathogen, injured mechanically or exposed to drought stress were also visualized with this technique [26–28]. These pioneering studies generated interest in SPE and prompted researchers to use alternative technologies to improve the imaging of this signal. Recent studies on plants, animals and humans have shown that autoluminescence imaging can be improved using highly sensitive charge-coupled device (CCD) cameras. Cooling of the sensor, using peltier devices (thermoelectric modules) or liquid nitrogen, is required for reducing thermal noise and enabling measurement of faint light by signal integration. Using this approach, SPE from mouse tumors and from the human body were recently imaged [29,30]. In plant science, the first application of CCD to SPE imaging was made using germinating seedlings [31]. High-resolution images of SPE from detached leaves were also reported [32]. So far, the best images of plant autoluminescence allowing quantitative mapping have been obtained by Michel Flor-Henry and co-workers [13], who used a cooled CCD detector coupled to optical fibers on which detached leaves were placed. This configuration enables maximal capture of light and high sensitivity, but it is applicable to flat samples only (e.g. detached leaves). Wound-induced luminescence of leaves was imaged with a good spatial resolution (222 000 pixels per image). Although the origin of the signal was not investigated in detail in this study, lipid peroxidation products are probable candidates [7,33]. Because wounding-induced photon emission is quenched by sodium azide and amplified by deuterium oxide [34], singlet oxygen is likely to be involved in this light emission. www.sciencedirect.com
If leaf autoluminescence does reflect lipid oxidative damage, mutants known to be sensitive to oxidative stress should emit more photons than do wild types. This is shown in Figure 3, in which SPE from whole Arabidopsis plants was imaged using a liquid nitrogen-cooled CCD camera similar to that described in Ref. [32]. The plants were pre-adapted to darkness for 2 h to allow photosynthesis-related chlorophyll luminescence to decay [13]. Two photosensitive mutants, the vtc2 single mutant deficient in ascorbate and the vtc2 npq1 double mutant deficient in both ascorbate and zeaxanthin [21], were compared with the photoresistant wild type after high light stress. Both mutants showed a much higher stimulation of SPE after photostress than did the wild type (Figure 3a), and SPE of the mutants was proportional to their relative sensitivity to oxidative stress [21]. Mature Arabidopsis leaves are well known to be less tolerant to photo-oxidative stress than young leaves at the center of the rosette, and their SPE reflected this difference. Furthermore, the SPE intensity of mature leaves was related to the concentration of malondialdehyde, a biochemical marker of lipid peroxidation (Figure 3c). Another Arabidopsis double mutant, vte1 npq1, deficient in two major antioxidants of the plastid membranes (vitamin E and zeaxanthin), is highly sensitive to high light, with leaves suffering extensive lipid peroxidation, which was measured by various methods [17]. The sensitivity of the vte1 npq1 double mutant to lipid peroxidative damage can be readily monitored by autoluminescence imaging (Figure 4a): stimulation of SPE occurs much more rapidly and more intensively in mutant leaves compared with wild-type leaves. Importantly, stimulated SPE from vte1 npq1 double mutant leaves was detected after 6 h in high light, before symptoms of oxidative damage were visible on the leaves (>26 h). A field that has much to gain from this new imaging method is phytopathology. For example, infiltration of a tobacco leaf with the elicitor cryptogein in the dark induced
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Figure 4. Measurement of the effects of photooxidative stress and the hypersensitive defense response on SPE. (a) Autoluminescence imaging of Arabidopsis plants [wild type (WT) and vte1 npq1 double mutant deficient in vitamin E and zeaxanthin] pre-exposed to high light stress at low temperature (4 8C, 1600 mmol m 2 s 1, on a regime of 14 h day and 10 h night) for 0 to 54 h. The vte1 npq1 double mutant is highly sensitive to high light stress [17], and symptoms of oxidative stress (leaf bleaching) were observed visually after 26 h in the mutant and after 30 h in the wild type. Plants were adapted to darkness for 2 h before measurements. (b) Autoluminescence imaging of a tobacco leaf infiltrated with the elicitor cryptogein (2 mg) for 0–25 h in the dark. The cryptogein treatment was performed as described in Ref. [35]. Integration time was 20 min and on-CCD binning of 2 2 pixels was used to increase detection sensitivity (the resulting resolution is 650 670 pixels). The color scale indicates signal intensity from 0 (blue) to 250 (white).
hypersensitive programmed cell death [35] and an enhancement of SPE [19] that can be quantified by CCD imaging, as shown in Figure 4b. Increased SPE was detected 18 h after infiltration, matching the time course of the massive enzymatic lipid hydroperoxide accumulation [35]. Recently, Mark Bennett and co-workers [36] showed bursts of light over the Arabidopsis leaf surface induced during the hypersensitive response to a pathogen. It seems that this photon emission is different to the SPE associated with lipid peroxidation because it was transient, beginning around 2 h after inoculation and lasted only 2–3 h. This transient light signal, which presumably reflects some signaling phenomena related to nitric oxide in the plant defense response [36], also has great potential for applications in phytopathological studies. Because SPE imaging can be performed on intact plants and does not require an exogenously added probe or sample preparation (except adaptation to darkness), it is expected to provide realistic mapping of lipid peroxidative damage and oxidative stress in plants. This new approach is complementary to more direct measures of reactive oxygen species such as fluorescence imaging using fluorescent probes sensitive to singlet oxygen and/or reduced forms of oxygen [16,37–39]. Fluorescence imaging is useful because it provides information on specific reactive oxygen species and their sites of production in tissues. However, fluorescence imaging of reactive oxygen species requires pre-infiltration of plant tissues with an exogenous probe and it is therefore usually restricted to cells or organs (e.g. detached leaves). A new approach based on redox-sensitive green fluorescent proteins (GFPs) has recently emerged to monitor non-invasively redox www.sciencedirect.com
changes in living cells [40–42]. Redox sensing is achieved by introducing cysteine residues close to the chromophore –their oxidation causes changes in the GFP fluorescence characteristics. Although application of this technique to imaging in plants is not yet fully documented [43], redox-sensitive GFPs are promising tools for visualizing the oxidation of specific proteins or organelles. However, this approach is technically more complex than SPE imaging because it requires genetic transformation of the biological material. Concluding remarks Autoluminescence imaging is likely to become an important tool in stress physiology to measure the oxidation status of plants non-invasively. Oxidative stress imaged by SPE is useful not only for quantification purposes, but also for the early detection of oxidative stress before damage becomes visible. SPE can be measured at different integration levels, from small populations of organisms to cells. Because of its high sensitivity and easy use, SPE imaging should enable the rapid screening of mutants, including algae, cyanobacteria and vascular plants, with variable tolerance to oxidative stress. Even though the sensitive equipment required for acquiring SPE images is still relatively expensive, we predict that this new imaging approach will successfully complement the assortment of functional imaging tools available to plant biologists [44]. Acknowledgements Financial support from CEA (program ‘Toxicologie Nucle´aire Environnementale’) and EU project IHP-RTN-00–2001 ‘STRESSIMAGING’ is acknowledged. We thank Ilja Reiter for helpful discussions.
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