TY - JOUR
AU - Böddi, Béla
AB - Abstract Possible mechanisms behind the light-induced wilting of dark-germinated pea (Pisum sativum L.) epicotyls were studied. Illumination with photosynthetically active radiation caused a fast turgor loss and wilting in the middle segments of the epicotyls accompanied by accumulation of water in the intercellular cavities. During this process, room temperature fluorescence emission spectra showed gradual bleaching of porphyrin-type pigments, which was lessened by incubating the epicotyls with excess ascorbate before illumination. Detection of singlet oxygen and lipid peroxidation products in the illuminated epicotyls suggested the occurrence of porphyrin-photosenzitized membrane damage as a cause of disordered water status and sequential wilting. (Received September 11, 2004; Accepted November 3, 2004) Introduction Light is not only the essential energy source for plants but also an important signal for biosynthesis of cell components, photomorphogenesis and development. Angiosperms grown in darkness cannot develop the photosynthetic apparatus; they develop etioplasts and synthesize chlorophyll (Chl) precursors, instead. Although the dark-grown organs have increased photosensitivity leading to tissue damage, the details of the degradation process have not been studied in non-leaf organs. The etioplast structure and the native arrangement of protochlorophyllide (Pchlide) of dark-germinated pea epicotyls differ from those of etiolated leaves. While etioplasts are well developed in mature leaves, only a few and undeveloped prolamellar bodies (PLBs) were found in dark-germinated pea epicotyls (Böddi et al. 1994). Most of the Pchlide is in a monomer state showing emission maxima at 629 and 636 nm in pea epicotyls (Böddi et al. 1998). These short wavelength forms are common in etiolated stems and stem-related organs, and the longer wavelength forms with emission maxima at 644 and 655 nm, dominating in leaves, are present in small quantities (Skribanek et al. 2000). Also the greening process of the epicotyls differs from that of etiolated leaves. A 1 ms flash illumination results in the phototransformation of the 655 and 644 nm forms in leaves (Böddi et al. 1991). In contrast, flash illumination converts only minimal amounts of Pchlide in epicotyls and the short wavelength forms do not change (Böddi et al. 1996). The amounts of 629 and 636 nm forms gradually decreased in the hour time scale at continuous illumination (light intensity 18 W m–2) and a 680 nm emitting chlorophyllide (Chlide) and/or Chl form accumulated. This reaction can be inhibited at 0°C (Böddi et al. 1994). Light of about 250 µmol m–2 s–1 photosynthetically active radiation (PAR) and above causes bleaching of the pigment and wilting in stem-related organs, with a dominating 629 nm form. At 10°C or under, the bleaching is more pronounced (Skribanek and Böddi 2001). Chl-type pigments may also act as photosensitizers (Asada 1994), yielding various reactive oxygen species (ROS). The type-II photodynamic reaction between excited triplet state Chl and molecular (triplet) oxygen is a source of singlet oxygen (1O2), while other reactions involving electron transfer to oxygen may yield superoxide or hydroxyl radicals and also H2O2. ROS may oxidatively damage proteins, nucleic acids, membrane lipids and many other molecules, and thus alter the physiology of the cell (Halliwell and Gutteridge 1999). Considering the monomer arrangement of Pchlide in pea epicotyl, production of non- protein-bound Chlide molecules can be expected during the phototransformation process. The newly formed Chlide molecules must be released from the POR (NADPH:protochlorophyllide oxidoreductase, E.C.1.3.1.33) units before the Chl synthase enzyme receives them. In this way, the production of non-protected porphyrins can proceed and these molecules may act as senzibilizers of further photochemical reactions. In this report, the process of photodamage was studied in dark-grown pea epicotyls and a model is proposed to explain the photosensitivity of this organ. Results To study the light sensitivity of the epicotyls, etiolated pea plants were illuminated with light of 115 µmol m–2 s–1 PAR at room temperature for 10 and 30 min. Similarly to earlier results (Böddi et al. 1996), this light did not result in greening of the epicotyls, instead, they lost their turgor all along the epicotyl and wilted in their middle region (Fig. 1). The wilting occurred even though the intact seedlings were kept in hydroponic culture during illumination and thus their water supply was not blocked. As a control, dark-grown seedlings were greened with continuous white light of 10 µmol m–2 s–1 PAR at room temperature for 5 days until the average Chl content of the stems was 120 µg g–1 fresh mass. These plants were then illuminated with 115 µmol m–2 s–1 PAR for 30 min. Neither wilting nor a change of the Chl content of the stems was observed. To analyse the light dependence of the turgor loss of the illuminated dark-grown seedlings, epicotyls were fixed in a porometer and the speed of water release was measured at room temperature. The water release velocity of the non-illuminated samples underwent a small increase during the 20 min of the measurement. The illuminated epicotyls evaporated water at a higher rate; the water loss velocity was double the values measured with samples kept in the dark (Fig. 2). The stereomicroscopic image of the cross-section in the wilted region showed the appearance of water in the intercellular spaces. Compared with the non-illuminated samples, shrinking of the cells and a significantly higher number of water droplets—seen as white dots—were observed in the illuminated (115 µmol m–2 s–1 PAR for 20 min) samples (Fig. 3). To study the effect of illumination on the pigments, epicotyl segments were fixed in the solid sample holder of the fluorometer and fluorescence emission spectra were recorded at room temperature after different illumination periods (115 µmol m–2 s–1 PAR). After 1 min illumination, a small peak appeared at around 680 nm, indicating the appearance of Chlide. Longer illumination, however, did not cause a further increase of this band; instead its amplitude slightly decreased. A more significant decrease of the fluorescence was found in the 610–640 nm region (with peaks at 630 and 636 nm), indicating the bleaching of the protochlorophyllide ester (Pchl)-type pigments (Fig. 4). No such fluorescence changes were observed with epicotyls previously greened at low light intensity (see above). In order to study the involvement of ROS in the process leading to the above bleaching, fluorescence changes of the singlet oxygen sensor DanePy were recorded. Each time, the fluorescence spectra of the protochlorophyllous and chlorophyllous pigments were also recorded. The maximal decrease of DanePy fluorescence was about 25% in samples irradiated with 250 µmol m–2 s–1 PAR for 20 min. In the same samples, Pchl and Chl were also significantly bleached (Fig. 5). Ascorbate is a natural antioxidant, reactive to several forms of ROS, including 1O2 molecules. Fig. 6 shows that the light-induced pigment bleaching was much smaller in the epicotyl segments pre-treated with ascorbate than in untreated ones. In addition, the emission band of Chlide was higher. Studies with epicotyl homogenates showed that this protection depended on the concentration of the ascorbate in the medium. In this experiment, the middle segments of epicotyls were homogenized and ascorbate was added in different concentrations. The homogenates were incubated at 0°C in the dark for 20 min and illuminated for 20 min with light of 115 µmol m–2 s–1 intensity. Fluorescence emission spectra at 77K were measured and the integral values of the spectra were compared (Table 1). It is worth mentioning that the shapes (positions and amplitude ratios of the bands) of spectra of the homogenates before illumination were identical to each other and to those of spectra of native epicotyl segments. The ROS produced in biological membranes may initiate or enhance lipid peroxidation (Girotti 1990). In order to study this possibility, reactive aldehydes were measured in the epicotyl samples using a thiobarbituric acid reactive substances (TBARS) assay (see Materials and Methods for details). The reaction was studied in 17 dark and 17 illuminated samples. The roots of the seedlings were immersed in water during illumination; the irradiance value was 250 µmol m–2 s–1 PAR for 20 min. The illumination resulted in the increase of TBARS content to 128% of the dark sample (Table 2). Discussion Illumination of dark-grown leaves results in photoreduction of Pchlide into Chlide; a single flash of 650 µmol m–2 s–1 PAR leads to saturation of this reaction, i.e. most of the Pchlide is transformed to Chlide (Böddi et al. 2003). In epicotyls, however, similar intensities cause only minute photoreduction (Böddi et al. 1996); instead bleaching of the pigments takes place (Fig. 4). Pea epicotyls and other stems with pigments in the monomeric state behave similarly (Skribanek et al. 2000). The reason for this difference between leaves and stems can be related to the different molecular arrangements of the POR subunits and/or the Pchlide molecules in these organs. In leaves, the ternary complexes of the POR subunits (containing POR, Pchlide and NADPH) form macrodomains (Wiktorsson et al. 1993), which are built into the regular structure of PLB membranes, i.e. they are the integral components of this structure (Böddi et al. 1989, Armstrong et al. 2000). This structure ensures that the excited Pchlide is photoreduced into Chlide, rather than photooxidized. The Pchlide–NADP+–Pchlide–NADPH (Böddi et al. 2003) and the Chlide–NADP+–Chlide–NADPH microcycles (Franck and Inoue 1984) functioning during continuous illumination show that the macrodomain complex contains proton sources for the re-reduction of NADP+. This means that the macrodomain complex must contain NADPH molecules in addition to those bound in the active sites of POR, which can re-reduce NADP+ molecules after flash illumination (Schoefs 2001). Therefore, the macrodomain complex protects the whole system against photooxidative damage, which could be caused by Pchlide or Chlide molecules acting as photosensitizers. After the photoreduction, the POR macrodomain complex disintegrates into small particles (Ryberg and Dehesh 1986), the newly formed Chlide leaves the complex but immediately translocates into the active sites of the chlorophyll synthase enzyme (Schmid et al. 2001). Thus the pigment is in ‘free’ (i.e. in a monosolvate) state only for a very short period and therefore it is protected from the photodamage. On the other hand, the majority of Pchlide was found in the monomer state in dark-germinated pea epicotyls (Böddi et al. 1998). In addition, only poorly organized etioplast inner membrane structures were found in epicotyls (Böddi et al. 1994). The observation that the Pchlide forms emitting at 628 and 636 nm did not transform at flash illumination (Böddi et al. 1996) shows that the Pchlide molecules, i.e. the pigment components of these forms, are certainly not bound in the active sites of the POR subunits. The strong temperature dependence of the Chl accumulation (Böddi et al. 1996) may mean that the regeneration of the photoactive POR enzyme needs diffusion processes, the monomeric Pchlide molecules must diffuse from the pools and the monomer newly formed Chlide molecules must diffuse to the Chl synthase. Consequently, high amounts of pigments are in a non-protected monomer state for a relatively long period in pea epicotyls. Illumination either with strong light flashes or with continuous light of high intensity therefore leads to bleaching. The accumulation of non-protein-bound pigments can be achieved experimentally by incubating plant tissues with external δ-aminolevulinic acid, which leads to an increased light sensitivity and photooxidation (Axelsson 1974). In this case, the surplus porphyrins cannot be bound to their native apoproteins (Pchlide to POR or Chlide to the Chl synthase) and they are unprotected. The phototransformation and the bleaching could not be observed separately in our experiments. The fact that the emission bands of Pchlide continuously decreased upon illumination but the Chlide emission bands did not increase indicates that these processes run simultaneously (Fig. 4). Both the non-transformed short wavelength Pchlide forms and the newly formed 680 nm emitting Chlide form—both in the monomer state—were bleached and could function as photosenzitizers in oxidation reactions. The oxidative nature of the reactions involved in pigment bleaching is supported by the protective role of ascorbate (Fig. 6 and Table 1) and lipid peroxidation in the illuminated epicotyls (Table 2). Ascorbate is a good scavenger for several ROS as well as other radicals (Smirnoff 1996). On the other hand, excess ascorbate may also re-reduce NADP+ after the Pchlide–Chlide phototransformation, i.e. the regeneration of the photoactive POR–Pchlide–NADPH complexes is stimulated. This would explain the higher production rate of Chlide in the presence of ascorbate (Fig. 6). One of the potential ROS, singlet oxygen, was identified in our experiments in the illuminated non-greening dark-grown epicotyls for the first time (Fig. 5). Because DanePy, the ROS sensor applied in this experiment, is mainly reactive to singlet oxygen (Kálai et al. 1998), it is not possible to comment on the possible role of other ROS. Pigment bleaching, however, suggests that the main path of ROS reactions involves photosenzitized reactions. A well-known example of these is the energy transfer from excited Chl to molecular oxygen yielding 1O2 (Halliwell and Gutteridge 1999), which is also feasible from Pchlide or Chlide molecules (Knox and Dodge 1985, Girotti 2001). It is important to underline that in our experiments, the fluorescence of DanePy and that of the porphyrins (showing pigment degradation) decreased simultaneously. Pchlide accumulation has been shown to trigger singlet oxygen production in green leaves when the Pchlide–Chlide transition was inhibited either chemically (Krasnovsky and Neverov 1988) or by mutation (op den Camp et al. 2003). Apart from photodynamic pigment destruction, 1O2 may start a series of oxidative reactions, including membrane lipid peroxidation (Knox and Dodge 1985, Girotti 2001). The positive thiobarbituric acid (TBA) reaction proved that the lipid peroxidation was involved in the illumination-provoked oxidative processes in the epicotyls (Table 2). Due to the lipid peroxidation, which involves the production of lipid and oxygen radicals propagating oxidative damage, membranes are disrupted; they lose their ability to retain solutes. This is a possible reaction causing the observed light-induced wilting of epicotyls (Fig. 1, 2). The microlocalization of this light-induced water escape (Fig. 2) correlates well with the localization of the pigment as Pchlide is mainly found in the subepidermal layers of the cortex (Seyedi et al. 2001), explaining why the photodamage affected the surface of the epicotyl. Although a turgor loss occurred along the whole epicotyl, the seedlings wilted at the middle regions of their epicotyls. This can be explained by physical forces of mass distribution but can be related by the Pchlide gradient along the epicotyls (Böddi et al. 1994). In the lower segments, the pigment content is low, thus the sensibilization is less effective. In the upper segments, the pigment content is high but a part of them is arranged in regular PLB membranes giving photoprotecion (Armstrong et al. 2000). In the middle segments, however, the medium pigment content can be enough for the photooxidation sensibilization and the majority of the etioplasts contain no or poorly developed PLBs (Böddi et al. 1994). To summarize the detected oxidative processes the scheme presented in Fig. 7 is suggested. The main senzibilizer in the processes are Pchlide (and/or Pchl) and Chlide pigments, which are predominantly in the monomer state in the pea epicotyl. On the other hand, leaves of etiolated plants contain the same pigments but in a protein-bound state; large aggregates or macrodomains are built into the etioplast inner membranes where such an increased photosensitivity is not present. These complexes provide protection against photodamage; by providing enough NADPH, they ensure fast regeneration processes and thus photoreduction rather than photooxidation reactions. This underlines the biological importance of PLBs in etiolated leaves. Materials and Methods Plant material Pea (P. sativum L. cv. ‘Zsuzsi’) seeds were pre-germinated on wet filter paper for 3 d, then the plants were grown in hydroponic culture in tap water at room temperature in the dark. After 7 d, they were 10–15 cm long; the middle 6 cm segment of each epicotyl was cut into 2 cm pieces and used for the experiments. Special care was taken to discard the stipula. In other measurements, the above-mentioned epicotyl segments were homogenized in 0.05 M phosphate buffer (pH 7.2). All these procedures were carried out in dim green light previously tested not to cause phototransformation of Pchlide. Measurement of water release The speed of water release by the epicotyls was measured with a porometer (AP4 DELTA-T DEVICES, Cambridge, U.K., Version 2.1) at room temperature. The two ends of 3 cm long epicotyl segments were covered with plastic foil to avoid water escape through the cut surfaces. Stereomicroscopic observations The middle sections of non-illuminated and illuminated pea epicotyls were cross-cut and the cut surface was studied under a stereomicroscope (Nikon SMZ-U Stereoscopic Zoom Microscope) equipped with a Nikon FDX-35 camera. Photos were taken with Kodak Technical Pan Film. Fluorescence spectroscopy of epicotyls The fluorescence emission spectra of the epicotyls were recorded with FluoroMax 2 Jobin Yvon Spex and FluoroMax 3 Jobin Yvon Horiba spectrofluorometers. The excitation wavelength was 440 nm, the optical slits were 5 nm, and the integration time was 0.1 s. Averages of three spectra were calculated automatically in the case of each sample. To measure spectra at room temperature, 3 cm long epicotyl segments were covered with transparent plastic foil (to avoid water release during the measurements) and were fixed in the solid sample holder of the fluorometer. These samples were illuminated for 1, 5, 15 and 30 min with a tungsten lamp through a heat filter outside the fluorometer. The PAR intensity was 115 µmol m–2 s–1. The spectra were measured after each illumination period. For low temperature spectroscopy, the epicotyl pieces were placed into glass sample holders and were immersed in liquid nitrogen during the measurements. These fluorescence spectra were analyzed with the software SPSERV V.3.14 (Copyright Cs. Bagyinka, Biophysical Institute, Biological Research Centre, Szeged, Hungary). Na-ascorbate treatment Longitudinally halved epicotyl segments were soaked in 50 mM phosphate buffer (pH 7.2) as a control, or phosphate buffer containing Na-ascorbate (54.5 mM) for 1 h in the dark. To study the concentration dependence of the effect of Na-ascorbate, homogenates were prepared from the epicotyls in 50 mM phosphate buffer (pH 7.2) containing 30% sucrose and 30% glycerol. Na-ascorbate was added to the homogenates with the following final concentrations: 0 (control), 6, 12, 25 and 50 mM. All experiments were done in seven parallel samples. Measurement of singlet oxygen Longitudinally halved epicotyl segments were soaked in 20 mM DanePy (Kálai et al. 1998) in the dark for 180 min and then illuminated with 250 µmol m–2 s–1 PAR from a KL-1500 lamp (DMP, Switzerland) through an optical fiber. The fluorescence emission spectrum of ROS sensors was recorded at room temperature, with a Quanta Master QM-1 (Photon Technology Int. Inc., U.S.A.) spectrofluorometer using 340 nm excitation. In order to avoid the effect of any stray light, 0° and 90° polarizers were used in front of the excitation and emission monochromators, respectively. Excitation and emission slits were both 1 nm. In order to improve the signal to noise ratio, five spectra were collected and averaged from the same sample to form one spectrum. ROS detection was based on the decrease of DanePy fluorescence, peaking at 532 nm as described earlier (Hideg et al. 1998). Measurement of lipid peroxidation (TBARS assay) For the estimation of the extent of lipid peroxidation in illuminated pea epicotyls, the contents of TBARS were determined by the adaptation of the TBA method, which determines MDA (malondialdehyde) as an end-product of lipid peroxidation (Heath and Parker 1968). The epicotyls were illuminated with 250 µmol m–2 s–1 PAR for 20 min. The illuminated samples, each of 0.1 g, were frozen in liquid nitrogen, then thawed and homogenized in 650 µl of 20% trichloroacetic acid. The samples were centrifuged at 4°C at 15,000×g. A 500 µl aliquot of 1% TBA (dissolved in 50 mM NaOH at 40°C) was added to 500 µl of the supernatant. The samples were incubated for 30 min at 90°C and then the reaction was stopped by transferring the tubes onto ice. A 3 ml butanol : methanol [85 :15% (v/v)] mixture was added and the samples were centrifuged at 1,000×g for 10 min. The absorbance of the upper phase (where the pinkish-red MDA–TBA complex was formed) was measured immediately at 535 and 730 nm using a Shimadzu UV-1601 spectrophotometer. The differences of the optical density values (OD535–OD730) were used for the calculations. A calibration curve was created by adding various amounts of 1,1,3,3-tetraethoxypropane (TEP) to control, non-illuminated epicotyl segments, the samples having final concentrations of 0, 5, 10, 15, 25, 40, 60 and 100 nM TEP. Determination of the chlorophyll contents The pigments were extracted with 80% acetone; the samples were centrifuged at 1,000×g for 10 min. The absorption spectra were recorded and the equations of Porra et al. (1989) were used. Acknowledgments This work was sponsored by the Research Foundation of the Hungarian Ministry of Education (FKFP 0302/2000). The ROS sensor DanePy was synthesized at the Department of Organic and Medicinal Chemistry (University of Pécs) by Professor Kálmán Hideg and Dr. Tamás Kálai. Development of DanePy and other ROS sensors is partially funded by the Hungarian National Research Foundation (OTKA T-042951). View largeDownload slide Fig. 1 Seven-day-old dark-grown pea seedlings lose turgor and wilt (see arrow) after 10 and 30 min exposure to 115 µmol m–2 s–1 PAR, even if the plants were kept in hydroponic culture conditions during illumination. View largeDownload slide Fig. 1 Seven-day-old dark-grown pea seedlings lose turgor and wilt (see arrow) after 10 and 30 min exposure to 115 µmol m–2 s–1 PAR, even if the plants were kept in hydroponic culture conditions during illumination. View largeDownload slide Fig. 2 Water loss velocity of dark-kept (A) and illuminated (115 µmol m–2 s–1 PAR, B) pea epicotyls measured with a porometer. The initial conductivity values of the samples were taken as unity and the relative water concentration values in the sample cell were calculated as a function of time. View largeDownload slide Fig. 2 Water loss velocity of dark-kept (A) and illuminated (115 µmol m–2 s–1 PAR, B) pea epicotyls measured with a porometer. The initial conductivity values of the samples were taken as unity and the relative water concentration values in the sample cell were calculated as a function of time. View largeDownload slide Fig. 3 Stereomicroscopic images of pea epicotyl cross-sections. Dark-kept and illuminated (115 µmol m–2 s–1 PAR for 20 min) epicotyls were cut in the middle region (bar: 1 mm). View largeDownload slide Fig. 3 Stereomicroscopic images of pea epicotyl cross-sections. Dark-kept and illuminated (115 µmol m–2 s–1 PAR for 20 min) epicotyls were cut in the middle region (bar: 1 mm). View largeDownload slide Fig. 4 Room temperature fluorescence spectra of pea epicotyls illuminated with 115 µmol m–2 s–1 PAR for 1, 5, 15 and 30 min (the corresponding numbers are in the figure). Excitation: 440 nm, slits 5 nm. View largeDownload slide Fig. 4 Room temperature fluorescence spectra of pea epicotyls illuminated with 115 µmol m–2 s–1 PAR for 1, 5, 15 and 30 min (the corresponding numbers are in the figure). Excitation: 440 nm, slits 5 nm. View largeDownload slide Fig. 5 Light-induced decrease in the room temperature fluorescence of (A) the singlet oxygen sensor DanePy and (B) porphyrins in pea epicotyls. Fluorescence emission spectra were recorded using 340 (A) and/or 440 nm (B) excitations before (solid lines) and after 20 min exposure to 250 µmol m–2 s–1 PAR. Optical slits were 1 nm. For better comparison, porphyrin fluorescence emission spectra are shown on a 20× magnified scale. View largeDownload slide Fig. 5 Light-induced decrease in the room temperature fluorescence of (A) the singlet oxygen sensor DanePy and (B) porphyrins in pea epicotyls. Fluorescence emission spectra were recorded using 340 (A) and/or 440 nm (B) excitations before (solid lines) and after 20 min exposure to 250 µmol m–2 s–1 PAR. Optical slits were 1 nm. For better comparison, porphyrin fluorescence emission spectra are shown on a 20× magnified scale. View largeDownload slide Fig. 6 Fluorescence emission spectra at 77 K of dark control (1), untreated (2) and ascorbate-pre-treated (3) illuminated epicotyl segments [ascorbate pre-treatment (0.0545 M), for 1 h in the dark; illumination,115 µmol m–2 s–1 PAR for 20 min; excitation wavelength, 440 nm; optical slits, 5 nm]. View largeDownload slide Fig. 6 Fluorescence emission spectra at 77 K of dark control (1), untreated (2) and ascorbate-pre-treated (3) illuminated epicotyl segments [ascorbate pre-treatment (0.0545 M), for 1 h in the dark; illumination,115 µmol m–2 s–1 PAR for 20 min; excitation wavelength, 440 nm; optical slits, 5 nm]. View largeDownload slide Fig. 7 Proposed scheme of the reactions occurring in pea epicotyls during illumination. P, porphyrin pigments (protochlorophyllide, protochlorophyll or chlorophyllide) in the ground state; P*, porphyrin pigments in the excited state; ROS, reactive oxygen species. View largeDownload slide Fig. 7 Proposed scheme of the reactions occurring in pea epicotyls during illumination. P, porphyrin pigments (protochlorophyllide, protochlorophyll or chlorophyllide) in the ground state; P*, porphyrin pigments in the excited state; ROS, reactive oxygen species. Table 1 Integral values of the 77K fluorescence emission spectra (between 580 and 780 nm) of dark-grown pea epicotyl homogenates incubated with different concentrations of Na-ascorbate (at 0°C for 20 min) and illuminated with light of 115 µmol m–2 s–1 irradiance for 20 min Concentration of Na-ascorbate (mM)  Integral values of the fluorescence spectra (×107)  Percentage of the non-treated value   0  6.1 ± 0.2  100  6  20.9 ± 0.6  344  12  21.9 ± 0.5  361  25  23.1 ± 0.9  380  50  30.3 ± 1.3  499  Concentration of Na-ascorbate (mM)  Integral values of the fluorescence spectra (×107)  Percentage of the non-treated value   0  6.1 ± 0.2  100  6  20.9 ± 0.6  344  12  21.9 ± 0.5  361  25  23.1 ± 0.9  380  50  30.3 ± 1.3  499  View Large Table 2 Light-induced lipid peroxidation in epicotyls of dark-grown pea seedlings   Dark sample  Illuminated  % of dark sample  OD535–OD730  0.089 ± 0.012  0.150 ± 0.010    TBARS (µmol (100 mg of tissue)–1)  0.039  0.050  128    Dark sample  Illuminated  % of dark sample  OD535–OD730  0.089 ± 0.012  0.150 ± 0.010    TBARS (µmol (100 mg of tissue)–1)  0.039  0.050  128  The TBA reaction was tested on 17 dark and 17 illuminated seedlings. The roots of the seedlings were immersed in water during the illumination (250 µmol m–2 s–1 PAR for 20 min) of the epicotyls. The TBARS contents were calculated with the help of a calibration curve. View Large Abbreviations Chlide chlorophyllide DanePy 3-[N-(β-diethylaminoethyl)-N-dansyl]aminomethyl-2,2,5,5-tetramethyl-2,5-dihydro-1H-pyrrole MDA malondialdehyde PAR photosynthetically active radiation Pchl protochlorophyllide ester Pchlide protochlorophyllide PLB prolamellar body ROS reactive oxygen species TBA thiobarbituric acid TBARS thiobarbituric acid-reactive substances TEP 1,1,3,3-tetraethoxypropane. 3 Corresponding author: E-mail, bbfotos@ludens.elte.hu; Fax, +36-1-3812166. References Asada, K. ( 1994) Production and action of active oxygen species in photosynthetic tissues. In Causes of Photooxidative Stress and Amelioration of Defence Systems in Plants. Edited by Ch. Foyer and P.M. Mullineaux. pp. 77–104. CRC Press, Boca Raton, FL, U.S.A. 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TI - Light-induced Wilting and its Molecular Mechanism in Epicotyls of Dark-germinated Pea (Pisum sativum L.) Seedlings
JF - Plant and Cell Physiology
DO - 10.1093/pcp/pci012
DA - 2005-01-15
UR - https://www.deepdyve.com/lp/oxford-university-press/light-induced-wilting-and-its-molecular-mechanism-in-epicotyls-of-dark-sFRE0O2ayh
SP - 185
EP - 191
VL - 46
IS - 1
DP - DeepDyve
ER -