JXB Advance Access originally published online on November 22, 2004
Journal of Experimental Botany 2005 56(409):205-218; doi:10.1093/jxb/eri024
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
RESEARCH PAPER |
Involvement of the thylakoidal NADH-plastoquinone-oxidoreductase complex in the early responses to ozone exposure of barley (Hordeum vulgare L.) seedlings
1Departamento de Biología Vegetal, Facultad de Biología, Universidad de Alcalá, E-28871 Alcalá de Henares, Madrid, Spain
2Instituto Cavanilles de Biodiversidad y Biología Evolutiva, Facultad de Ciencias Biológicas, Departamento de Botánica, Universitat de València, E-46100 Burjassot, Valencia, Spain
* To whom correspondence should be addressed. Fax: +34 9188 55066. E-mail: alfredo.guera{at}uah.es
Received 4 August 2004; Accepted 7 September 2004
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and methodsResultsDiscussionReferences |
|---|
A possible implication of the plastid NADH-plastoquinone-oxidoreductase (Ndh) complex in the response against ozone-mediated oxidative stress in barley (Hordeum vulgare L.) leaves was investigated. After a 4 h treatment, exposure of barley seedlings to moderate ozone concentrations produced leaf-age-dependent increases in lipid peroxidation, peroxidase, and Ndh complex activities in the thylakoid membranes. A significant amount and activity of the Ndh complex were detected in mature barley leaves, but not in young barley leaves. In fact, young barley leaves behaved like ndh-deficient leaves in most of the aspects studied. When plants were exposed to photo-oxidative light after ozone fumigation, the recovery of Fv/Fm was lower in young leaves than in mature leaves. Ozone treatment significantly decreased non-photochemical quenching (qN) in young leaves, but not in mature leaves. Mature leaves showed higher levels of the energy (
) dependent (qE) component of qN. Treatment with antimycin A, an inhibitor of cyclic electron flow, increased the decay of qN produced by ozone in young leaves, but not in mature ones. The reduction state of plastoquinone increased after ozone treatment in mature dark-adapted leaves and was strongly quenched by far red light. It is proposed that the function of the Ndh complex helps the maintenance of qN, probably through the poising of the redox steady-state level of the intersystem carriers and then by optimizing the rate of cyclic electron flow. This should constitute an age-dependent early response in barley leaves, by contributing to minimize photoinhibition in the presence of ozone and high light. Key words: Chloroplast, Hordeum vulgare, Ndh (NADH-plastoquinone-oxidoreductase) complex, non-photochemical quenching, ozone, photo-oxidative stress
|
Introduction |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
Effects of tropospheric ozone (O3) accumulation can lead to increased oxidative stress in the chloroplast (Pell et al., 1997
). Although O3 is not a radical species in itself, uptake into the internal leaf spaces is believed to lead to the generation of ROS (radical oxygen species), that can destroy the structure and function of the biological membranes, leading to electrolyte leakage (Soldatini et al., 1998; Guidi et al., 1999) and to lipid peroxidation (Calatayud and Barreno, 2000, 2001). The O3 or ROS may also modify the properties of the thylakoids, thereby changing the yield of Chl a fluorescence, leading to photoinhibition (Ort, 2001) in the absence of adequate mechanisms capable of preventing potential damage. Plants have a number of antioxidative mechanisms that chemically and enzymatically remove these toxic compounds. These antioxidant responses to O3, both enzymatic and non-enzymatic, are dependent on several endogenous or exogenous factors, such as the particular stage of plant development or different environmental parameters leading to resistance or sensitivity (Lyons et al., 1999; Alonso et al., 2001). During recent years, evidence has accumulated which suggests that a thylakoidal NADH dehydrogenase complex (Ndh complex) that has been purified from pea thylakoids (Sazanov et al., 1998a) is involved in protection against (photo)oxidative stress (Martín et al., 1996; Catalá et al., 1997; Casano et al., 1999; Endo et al., 1999). Several studies (Cuello et al., 1995; Corneille et al., 1998; Burrows et al., 1998; Feild et al.1998) indicate that the Ndh complex mediates the oxidation of NADH to reduce plastoquinone. Based on experiments carried out with a system constructed with purified Ndh complex and thylakoidal plastoquinol-peroxidase, Casano et al. (2000) proposed a chlororespiratory route of electron transfer from NADH to O2 involving the Ndh complex, a thylakoidal plastoquinol peroxidase, superoxide dismutase, and the non-enzymatic one electron transfer from the reduced iron–sulphur proteins to O2 (Mehler reaction). Alternatively, a plastid terminal oxidase homologous to the plant mitochondrial alternative oxidase (Carol and Kuntz, 2001) could mediate the reduction of oxygen as a final electron acceptor of such a chlororespiratory route (Peltier and Cournac, 2002). Chlororespiration could participate in the scavenging of reactive oxygen species and also by poising redox proportions of the intersystem chain transporters in optimizing electron transport to PSI under variable environmental conditions (Casano et al., 2000). Appropriate rates of electron transport around PSI would ensure the maintenance of (i) the transthylakoidal proton gradient () required for photophosphorylation; and (ii) the -dependent dissipation (related to the non-photochemical quenching parameter, qN, of chlorophyll a fluorescence) of the excess of incident energy on PSII, associated with many stress conditions (Burrows et al., 1998; Endo et al., 1999; Feild et al., 1998; Sazanov et al., 1998b). The aim in this paper is to find out whether the Ndh complex is involved in the responses to counteract the oxidative stress induced by ozone. In order do so, the relationship between chlorophyll a fluorescence, thylakoidal lipid peroxidation, peroxidase activity, and the accumulation of the Ndh complex in ozone-fumigated young and mature primary barley (Hordeum vulgare L.) leaves have to be determined.
|
Materials and methods |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
Growth of plants, and ozone and inhibitor treatments
For this study, the primary leaves of 6-d-old (young leaves) or 14-d-old (mature leaves) barley (Hordeum vulgare L. cv. Hassan) seedlings were analysed. Barley seeds were germinated and grown on moist vermiculite in a growth chamber at 23 °C, 53% RH, with a 16 h photoperiod of 200 µmol m–2 s–1 white light. Three hours after the start of the light period on the sixth or the fourteenth day of growing, plants were exposed to 100 nl l–1 of ozone for 4 h. Control plants were grown under the same conditions, but they were not subjected to ozone fumigations. The ozone treatment was performed in a controlled-environment chamber (Fitotron, Sanyo Gallenkamp, model 5GC170.PFX.F). Ozone was generated from pure compressed oxygen by electric discharges (ozone generator SIR model O-3002). A flow controller regulated the flow of ozone-enriched air to the chamber. The ozone concentration inside the chamber was monitored continuously with an ozone analyser (DASIBI model 1180). Light intensity, temperature, and RH were the same as in the growth chamber. When inhibitors were employed, the roots of intact seedlings were submerged in either water or aqueous solutions containing 5 µM antimycin A or 10 µM myxothiazol, immediately after ozone treatment and were incubated for 18 h in the dark. Seedlings which had not been subjected to ozone treatments were incubated under the same conditions in the presence of inhibitors or water.
Tobacco ndhF-deficient plants were the same as those described by Martin et al. (2004).
Measurement of in vivo chlorophyll a fluorescence
Chlorophyll (Chl) a fluorescence was measured with a portable pulse-modulated fluorometer (PAM-2000, Walz, Effeltrich, Germany) at room temperature. Chl a fluorescence levels in ozone-treated leaves were measured immediately after ozone fumigations or at the end of inhibitor treatments. Fluorescence in control leaves was measured at the onset and just after the end of the fumigation period of ozone-treated leaves. Leaves were kept in the dark for 30 min prior to the measurements. The minimum (dark) fluorescence yield (Fo) was obtained upon excitation of leaves with a weak measuring beam from a light-emitting diode. The maximum fluorescence yield (Fm) was determined with 600 ms saturating pulse of white light (8000 µmol m–2 s–1). Variable fluorescence (Fv) was calculated as Fm–Fo. Following 2 min dark-readaptation, actinic white light (260 µmol m–2 s–1, unless otherwise stated) was switched on, and saturating pulses were applied at 1 min intervals for 11 min to determine the maximum fluorescence yield during actinic illumination (
), the level of modulated fluorescence during a brief interruption (3 s) of actinic illumination in the presence of 6 µmol m–2 s–1 far red (730 nm) light (
), and the Chl fluorescence yield during actinic illumination (Fs). Quenching due to non-photochemical dissipation of absorbed light energy (qN) was calculated at each saturating pulse, according to the equation 
(van Kooten and Snell, 1990). The coefficient for photochemical quenching, qP, represents the fraction of open PSII reaction centres and was calculated as 
(Schreiber et al., 1989). The quantum efficiency of PSII photochemistry, 
PSII, closely associated with the quantum yield of non-cyclic electron transport, was estimated from 
(Genty et al., 1989). After the quenching parameters reached steady-state, the actinic light was switched off and the variation in the Fo level was recorded. Quenching of ‘dark’ Fo by far red light was monitored as described by Sazanov et al. (1998b) after a 30 min dark-adaptation period. Photoinhibitory treatments were performed by exposure of ozone-treated and control barley seedlings to an irradiance of 1800 µmol m–2 s–1 for 30 min at room temperature after the ozone fumigations had ended. Recovery from photoinhibition was recorded at room temperature and darkness was interrupted every 2 min by a saturating light pulse to measure the Fv/Fm values. Relaxation of qN in the dark and deconvolution of qN parameters were performed as described by Walters and Horton (1991).
Isolation of thylakoids
Thylakoid membranes were isolated basically as described previously by Catalá et al. (1997) and Guéra et al. (2000). The thylakoid isolation was carried out immediately after the fumigation periods had ended on primary leaves of both ozone-treated and untreated control plants. Leaves were excised, placed on ice-cold grinding buffer (50 mM HEPES-NaOH pH 7.6, 0.33 M sorbitol, 2 mM ascorbic acid, 1 mM MgCl2, 1 mM MnCl2, 2 mM Na2EDTA, and 0.1% BSA) and homogenized with a polytron. The homogenate was filtered through cheesecloth and centrifuged at 500 g for 5 min. The supernatant was centrifuged at 2500 g for 5 min and the resulting pellet was resuspended in WB buffer (50 mM HEPES-NaOH pH 7.6, 0.33 M sorbitol, and 2 mM Na2EDTA) and centrifuged again at 2500 g for 5 min. The last pellet was resuspended in hypotonic buffer (50 mM HEPES-NaOH pH 7.6, and 2 mM Na2EDTA) for 5 min. After this treatment, isotonic conditions were restored by adding 2x concentrated WB buffer. The burst chloroplasts were centrifuged at 6000 g for 10 min. The resulting pellet was resuspended in WB containing 300 mM NaCl and 0.5 mM PMSF. After 10 min of incubation on ice, thylakoids were recovered by centrifugation at 6000 g and finally washed twice in WB.
Enzymatic assays and lipid peroxidation determination
Peroxidase activity (POD) was analysed as described by Astorino et al. (1995) with slight modifications. The assay was performed in a 3 ml cuvette containing 100 mM potassium phosphate buffer (pH 6), 1% (w/v) guaiacol, and 6 mM H2O2. Reaction assays were started by adding a small volume of isolated thylakoids resuspended in WB buffer. Activity was determined by the increase in the absorbance at 470 nm as a result of guaiacol oxidation.
NADH:FeCN dehydrogenase activity was assayed as described by Cuello et al. (1995) by measuring the reduction of FeCN at 420 nm (extinction coefficient: 1 mM–1 cm–1). The assay included 0.88 mM K3[Fe(CN)6], 40 mM phosphate buffer pH 7.5, 0.2 mM NADH, and 2.5 mM EDTA. Reaction was started by the addition of thylakoids (c. 10 µg of total protein) suspended in WB buffer.
The level of lipid peroxidation in thylakoid membranes was measured in terms of malondialdehyde (MDA), following the method proposed by Heath and Parker (1968), with the modifications made by Dhindsa et al. (1981). The non-specific background absorbance reading at 600 nm was subtracted from the specific absorbance reading at 532 nm.
Other procedures
SDS-PAGE was carried out as described by O'Farrel (1975). Electroblotting and immunodetection were performed as described in Guéra et al. (2000). The anti-NDH-F antibody is the same as that described in Catalá et al. (1997). The anti-D1 antibody was a gift from Dr Eva-Mari Aro (University of Turku, Finland). Native-PAGE (ND-PAGE) and detection of NADH-dehydrogenase activity on native gels was carried out as described in Guéra et al. (2000). Protein was determined after 2% SDS solubilization of samples, with a Bio-Rad (Hercules CA, USA) detergent-compatible protein assay kit based on the method of Lowry (1951), using BSA as standard. Chlorophyll and carotenoids were determined according to Lichtenthaler (1987). Variance analysis (ANOVA) was performed on experimental data, a statistical significance (P <0.05) judged by the least significant differences (LSD) test. Statistical analyses were performed by using the program SPSS (SPSS Inc., Chicago, IL, USA).
|
Results |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
Effects of O3 fumigation on the thylakoid membranes of primary barley leaves
After ozone fumigations, thylakoids were immediately isolated either from O3 fumigated or control (unfumigated) plants. The thylakoid preparations were free of contamination from other subcellular compartments (Catalá et al., 1997
; Guéra et al., 2000). Lipid peroxidation, as estimated by the MDA levels (Table 1), increased significantly in thylakoids of both young and mature ozone-treated leaves when compared with their respective controls. This increase in the MDA level was higher in mature leaves than in young leaves. The thylakoidal associated peroxidase activity (POD) also increased after ozone exposure in both young and mature leaves (Table 2). The basal POD specific activity value was higher in thylakoids from young than from mature untreated leaves (control leaves). Ozone fumigation slightly increased the thylakoidal POD activity in young leaves, although this slight increase was statitically significant. This stimulation of POD activity in thylakoids was much higher in mature leaves (around 3-fold after ozone fumigation). No significant changes were found in Chl a, Chl b, or in the total carotenoid content (data not shown).
|
|
Effects of O3 fumigation on the Ndh complex activity and dark reduction of plastoquinone levels
To investigate if the plastid Ndh complex is involved in the response to moderate ozone exposure levels, the NADH dehydrogenase activity associated with Ndh in the thylakoid membranes was detected first. Total NADH dehydrogenase activity associated with the thylakoid membranes was about 4-fold greater in mature leaves (11.79±0.45 µmol NADH oxidized min–1 mg–1 protein) than in young leaves (3.08±0.25 µmol NADH oxidized min–1 mg–1 protein) after ozone fumigation. Specific Ndh activity can be identified in the thylakoid preparations from barley seedling leaves by native PAGE (ND-PAGE) assays (Casano et al., 1999
; Guéra et al., 2000). Figure 1A shows that the Ndh complex activity was not detected in young leaves up to 150 µg of total thylakoid protein loaded in the gel. Otherwise, the Ndh complex activity was detected in thylakoids of mature leaves for the same amount of total protein loaded, and the detected activity increased to gel-stain saturating levels in the case of thylakoids being isolated from ozone- treated mature leaves. These results were confirmed by immunodetection of the NDH-F subunit of the Ndh complex, after SDS-PAGE of the thylakoid membranes (Fig. 1B). The presence of the NDH-F polypeptide was detectable in the thylakoid preparations from mature leaves where 10 µg or more of total protein were loaded in the gel; but not in the thylakoid preparations from young leaves where up to 20 µg of total thylakoidal protein were loaded in the gel. The levels of this Ndh-complex polypeptide, as detected by western blotting, increased significantly after ozone treatment of mature leaves (Fig. 1B, anti-NDH-F). The steady-state levels of the PSII D1 protein, taken as a reference control, did not change significantly when comparing non-treated and ozone-treated young or mature leaves (Fig. 1B, anti-D1).
|
The activity of the Ndh complex has been associated with higher levels of plastoquinone reduction in the dark as shown by a rise of the Fo level after switching off the actinic light (Corneille et al., 1998
; Feild et al., 1998; Burrows et al., 1998). When the actinic light was switched off after 11 min of illumination (260 µmol m–2 s–1) of the mature leaves, the Fo level showed an increase (Fig. 2B, D) which was much higher in ozone-treated seedlings (Fig. 2D) than in control (Fig. 2B) seedlings. This Fo rise was quenched by illumination with far red light (data not shown). By contrast, young leaves did not show this increase of Fo either under control (Fig. 2A) or ozone-fumigated (Fig. 2C) conditions, resembling the behaviour described for tobacco mutants containing different disrupted plastid ndh genes (Burrows et al., 1998; Kofer et al., 1998; Shikanai et al., 1998; Joët et al., 2001; Martín et al., 2004).
|
Measurement of the basal level of fluorescence (Fo) of 30 min dark-preadapted leaves and the new fluorescence level (
) obtained after illumination of the leaves with a far-red light pulse, allows a rough estimation of the reduction level of plastoquinone in the dark to be made, and can be used as another positive test for Ndh complex activity in vivo (Sazanov et al., 1998b). Results obtained after 30 min preadaptation in the dark showed an increase in Fo for mature leaves after ozone treatment, as well as non-significant differences for young leaves (Fig. 3). Furthermore, a significant quenching of Fo by far red light was observed after the ozone treatment for mature leaves, but not for young leaves. These results would also indicate an increase of the Ndh complex activity in mature leaves after ozone treatment.
|
Effects of O3 fumigation on chlorophyll a fluorescence quenching parameters of primary barley leaves
The maximal photochemical efficiency after leaf dark-adaptation estimated by the Fv/Fm ratio, a sensitive parameter for photoinhibition (Krause and Weis, 1991
), was not significantly affected by exposure to ozone in either young leaves (control 0.799±0.003; ozone 0.802±0.002) or mature leaves (control 0.795±0.002; ozone 0.790±0.002) when not exposed to photo-oxidative light conditions. When intact barley seedlings were exposed to photo-oxidative light (1800 µmol m–2 s–1) for 30 min after the end of ozone treatment, the Fv/Fm initial value decreased, but it was recovered in the mature leaves (either, ozone-treated or untreated) after a short period in the dark (Fig. 4, mature). The same behaviour was observed for young ozone untreated leaves (Fig. 4, young, closed symbols); but in contrast, young leaves exposed to photo-oxidative light after ozone treatment (Fig. 4, young, open symbols) showed a higher susceptibility to photoinhibition, as shown by the highest decay of Fv/Fm at the end of the photo-oxidative treatment and by the lack of total recovery of Fv/Fm after 30 min in the dark (less than 90% of the initial value).
|
The absence of the Ndh complex in young barley leaves must make them behave, at least partially, like leaves of Ndh-deficient mutants. Tobacco ndhCJK mutant plants under water-stress conditions have been described to show alterations in Chl fluorescence quenching when compared with wild-type plants (Burrows et al., 1998
). Most recently, other authors (Li et al., 2004) have described that, in ndhB-deficient tobacco mutants under chilling stress, non-photochemical quenching of Chl a fluorescence, and particularly its fast relaxing component, were lower than in wild-type plants. The non-photochemical quenching qN parameter is considered to be a good estimate of the amount of energy that is dissipated non-radiatively by plants. It was found that in ndhF (Martín et al., 2004) defective tobacco mutants (Fig. 5, 
F, closed symbols) the qN maximal and steady-state values were lower than in wild-type plants (Fig. 5, wt, closed symbols), both grown under controlled low-stress culture-chamber conditions. When these plants were exposed to ozone treatments, similar to those given to barley plants, wild-type tobacco leaves shown a significant increase in maximal and steady-state qN (Fig. 5, wt, open symbols). The response of tobacco ndhF mutants to ozone treatment was clearly different, showing a significant decrease of qN after ozone exposure (Fig. 5, F, open symbols). It has recently been described (Martín et al., 2004) that ndhF tobacco mutants were strongly affected in their qN steady-state values after photo-oxidative stress induced by the incubation of leaf discs on increasing amounts of paraquat (about 85% inhibition of steady-state qN in ndhF mutants when compared with wild-type plants after 24 h incubation in 300 nM paraquat).
|
No differences were found when the
PSII of ozone-fumigated leaves was compared with their respective controls (Fig. 6A, B). The values of qP in the steady-state were similar for leaves of the control and ozone-treated seedlings (Fig. 6C, D). Immediately after the onset of actinic illumination, qN rose quickly and reached maximum values within 1–2 min in control leaves (Fig. 6E, F). Thereafter, qN progressively declined until it reached steady-state values approximately 10 min after the onset of actinic illumination. Control or ozone-fumigated mature leaves did not present any differences in the qN time induction curve, nor in the qN maximal or steady-state values (Fig. 6F). However, the exposure to ozone significantly decreased maximal and steady-state values of qN in young leaves (Fig. 6E). These differences were statistically significant for the steady-state values of qN at the P 
0.01 level. It is worth noting that qN steady-state values were always lower in young leaves than in mature leaves (Fig. 6E, F).
|
Figure 7 shows that the steady-state values of qN increased when barley seedlings were exposed to larger intensities of actinic light. Non-photochemical quenching is assumed to be a protection response to excess light, therefore its steady-state value might increase as incident light also increases (Calatayud and Barreno, 2001
). It was observed that the steady-state qN values were lower in ozone-treated young leaves than in control young leaves at any light intensity (Fig. 7, young). The ozone effect on young leaves was increased at light intensities above 100 µmol photons m–2 s–1. On the other hand, qN values were not significantly affected by ozone fumigation in the mature leaves (Fig. 7, mature). These results indicated that under similar, mild ozone fumigation conditions, the capacity to dissipate an excess of light energy was lower in young barley leaves than in mature ones.
|
The ‘energy-dependent’ quenching (qE), originated by the formation of a proton gradient across the thylakoidal membranes, is the main mechanism implied in the development of qN (Krause and Weis, 1991
; Müller et al., 2001). This qE is characterized by its rapid relaxation kinetics, occurring within 3 min of darkness (Li et al., 2002; Munekage et al., 2002). Figure 8 shows that qN relaxed in the dark more rapidly in mature barley leaves than it did in young barley leaves. Notably, qN dropped around 63% of the qN initial value in either mature ozone-fumigated or mature control leaves after 2 min in the dark. The decay of qN in young leaves was slower (only about a 30% decay of the initial qN value was registered during the first 2 min in the dark), indicating that the qE values in young leaves were lower than in the mature ones (Li et al., 2002; Munekage et al., 2002). In fact, when these results were analysed according to Walters and Horton (1991), qE values of 0.05 were obtained for young leaves, and 0.15 for mature leaves. It was also noted that qN decayed slightly faster in mature ozone-treated leaves than in mature control leaves, and the opposite effect was observed in young leaves. Otherwise, when the ‘medium component’ of the qN relaxation kinetics was calculated, corresponding, according to Walters and Horton (1991), to the state transition component (qT) of qN under low-light conditions, higher values for young leaves (around 0.35) were observed than for mature leaves (around 0.26). These results for qT were similar to those described previously for barley leaves under 200 µmol m–2 s–1 white actinic light (Quick and Stitt, 1989).
|
Effects of antimycin A and myxothiazol on qN and the transient Fo rise
Antimycin A is an inhibitor of cyclic electron flow (Miyake et al., 1995
; Endo et al., 1998; Joët et al., 2001). As qN was the most affected Chl a fluorescence parameter in young barley leaves after ozone treatment, antimycin A was used to find out if any relationship could be established among Ndh levels, qN, and cyclic electron transport. Since antimycin A is also described as an inhibitor of the mitochondrial cytochrome bc1 complex, myxothiazol, which inhibits electron transport at the level of the mitochondrial bc1 complex, but has no direct inhibitory effects on chloroplastic electron transporters (Lee et al. 2001; Joët et al. 2001) was also assayed. To perform these experiments, the roots of ozone-treated or untreated intact plants were introduced in distilled water (Fig. 9A, B), in 5 µM antimycin A (Fig. 9C, D) or in 10 µM myxothiazol (Fig. 9E, F), and were incubated for 18 h in the dark. At the end of the incubation period, leaves of plants incubated in water (Fig. 9A, B) showed qN time-induction curves which were quite similar to those obtained in non-incubated plants (Fig. 6E, F). Antimycin A suppressed the early transient increase in qN either in ozone-treated or in untreated young and mature plants (Fig. 9C, D). Interestingly, antimycin A treatment significantly decreased the steady-state qN values in ozone-fumigated young leaves, and only slightly in control young leaves (Fig. 9C) when compared with non-antimycyn-A-treated plants (Fig. 9A). Steady-state qN values for mature leaves incubated with antimycin A were similar to those obtained for mature leaves incubated in water (Fig. 9B, D). The qN values were always higher in mature leaves than in young leaves of antimycin-A-incubated plants (Fig. 9C, D).
|
Figure 9E (young leaves) and Fig. 9F (mature leaves) show that incubation of the barley seedlings in 10 µM myxothiazol had no significant effect on the shape of the qN induction curves, although the maximal and steady-state values were lower than in control plants incubated in distilled water (Fig. 9A, B). By contrast with results obtained with antimycin A (Fig. 9C) young ozone-fumigated and control leaves reached similar steady-state qN values after myxothiazol incubation (Fig. 9E).
The effects of these inhibitors have also been investigated on the Fo initial rise rate (first 15–30 s after switching off actinic light) during a light-to-dark transition (Table 3). As in the assays described in Fig. 2, young leaves of plants incubated in distilled water for 18 h did not show any appreciable Fo rise after a light-to-dark transition. This situation was not altered by either antimycin A or myxothiazol treatments. A significant Fo rise rate was evident in the mature leaves of control plants, which markedly increased after ozone fumigation. Antimycin A did not inhibit this initial Fo transient rise rate in mature leaves, but a significant stimulation was observed in ozone-treated leaves. By contrast, myxothiazol incubation decreased the Fo rise rate in mature leaves, especially in ozone-treated plants.
|
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
Mild ozone treatment damages thylakoid membranes of barley seedlings
Early symptoms of ozone injury in leaves include damage to chloroplast membrane lipids (Hellgren et al., 1995
; Carlsson et al., 1996; Schraudner et al., 1997) and increases in chloroplast antioxidant activities (Batini et al., 1995; Torsethaugen et al., 1997; Kliebenstein et al., 1998; Niewiadomska et al., 1999); but no decay of thylakoid pigments (Manninen et al., 1999), nor increase of photoinhibition under non-photo-oxidative light (Reichenauer and Bolhar-Nordenkampf, 1999). As expected, the Fv/Fm ratio, which is considered a sensitive indicator for photoinhibition (Krause and Weis, 1991), did not present any significant reduction in either young or mature barley leaves after dark-adapted conditions. As reported in maize (Pino et al., 1995), pea, wheat, and spinach (Carlsson et al., 1996), and tomato (Calatayud and Barreno, 2000), it was found that thylakoidal lipid peroxidation (Table 1) and thylakoid-associated peroxidase activity (Table 3) increased in barley seedlings after short exposures to moderate ozone levels, especially in mature leaves. Therefore, the conditions of ozone treatment used in this study reproduced responses previously described for other plants and provided a representative system to investigate the early responses of barley primary leaves to ozone exposure.
The Ndh complex is accumulated in mature barley leaves as an early response to ozone treatment
It was found that the NdhF polypeptide and the NADH dehydrogenase activity associated with the Ndh complex were only detected in mature, but not in young, barley primary leaves. The activity of this complex strongly increased in mature leaves after a short (4 h) mild ozone treatment (Fig. 1A, B). In striking parallel, it was found that the transient increase of Fo after a light-to-dark transition was only observed in mature leaves, and it was enhanced after ozone fumigation (Fig. 2; Table 3). As far as the ‘dark rise’ of Fo chlorophyll fluorescence can be related to a higher reduction level of the plastoquinone pool, these results are in accordance with previous ones (Endo et al., 1997; Burrows et al., 1998; Feild et al., 1998; Joët et al., 2001; Martín et al., 2004), which link the activity of the Ndh complex with that of the Fo ‘dark rise’ and the level of plastoquinone reduction after a light-to-dark transition. The lack of an Fo ‘dark rise’ in young barley primary leaves resembles results obtained for tobacco ndh-deficient mutants (Burrows et al., 1998; Kofer et al., 1998; Shikanai et al., 1998; Joët et al., 2001; Martín et al., 2004).
The effects of ozone are mediated by the non-light-dependent conversion of O3 to other reactive oxygen species, such as H2O2 inside the plant cells (Pell et al., 1997). A primary target to the action of H2O2, or other active oxygen species are biological membranes, and there is supporting literature evidence that these damages affect mainly older tissues (Pino et al., 1995). On the other hand, H2O2 increases the amount and the activity of the Ndh complex (Casano et al., 2001). This can partially explain why the higher levels of thylakoid lipid peroxidation registered in mature barley leaves, rather than in young leaves, after ozone treatment (Table 1), are correlated with an increased amount and higher activity of the Ndh complex (Figs 1, 2). Ozone stress has been associated with the enhanced expression of senescence-related genes (Pino et al., 1995; Miller et al., 1999). In this context, it is significant that the expression of the ndh genes has been previously associated with induced leaf senescence (Martín et al., 1996), the onset of natural leaf senescence (Catalá et al., 1997), or to the onset of fruit ripening (Guéra and Sabater, 2002). Therefore, products of the ndh genes could be considered to be both senescence- and ozone-induced.
Function of the Ndh complex under ozone-stress conditions is probably related to the maintenance of non-photochemical dissipation of light energy
Significantly, although the thylakoid membranes from ozone-treated mature leaves showed higher levels of lipid peroxidation than those from ozone-treated young leaves, they had a lower decay of Fv/Fm after photo-oxidative light exposure and they also recovered better from this decay in the dark (Fig. 4). Therefore, the Ndh complex could contribute, among other factors, to the prevention of photoinhibition under conditions of increased photo-oxidative stress, and subsequently increased thylakoidal membrane injury.
A possible clue to find a function for the Ndh complex under ozone-stress was provided by Burrows et al. (1998), who reported that leaves of tobacco ndh-defective mutants had a reduced ability to quench fluorescence non-photochemically under water-stress conditions. If this statement could be generalized to leaves from non-genetically manipulated plants that would contain either a low or a null Ndh complex activity under different stress conditions, then young barley leaves might show an impaired capacity to develop non-photochemical quenching under stress treatments. The maximum and steady-state qN values decreased after ozone treatment in young leaves, but not in mature ones (Fig. 6), especially when the plants were exposed to higher light intensities (Fig. 7). This behaviour for young barley leaves resembles that found in tobacco ndhF-deficient leaves (Fig. 5). In fact, it was found that tobacco ndhF mutants had a reduced capacity to develop qN (Fig. 5, F). While the wild-type tobacco plants were able to increase their qN values after ozone treatments (Fig. 5, wt), the ndhF mutants showed a partial decrease of qN after ozone exposure (Fig. 5, F). It has recently been communicated by Casano et al. (2004), that a computer-based analysis provided evidence that the NdhF polypeptide could be involved in H+ pumping. Therefore, a deficiency in NdhF could be expected to impair (at least partially) the formation of a transthylakoidal pH gradient. According to Krause and Weis (1991), qN is related to a high transthylakoidal pH gradient (qE), to state I–state II transitions (qT), and to photosystem II photoinhibition (qI). A rapid relaxation kinetics (within 3 min) of qN in the dark is characteristic of the qE component of qN (Horton and Hague, 1988; Munekage et al., 2002). From the faster relaxation kinetics of qN in mature leaves rather than in young leaves (Fig. 8) it was estimated (Walters and Horton, 1991) that qE was around 3-fold higher in mature leaves than it was in young leaves, and that qE was the main component of qN in mature leaves. Consequently, according to Walters and Horton (1991), state transitions (qT) were more important in young leaves than in mature barley primary leaves. This is an interesting point, which illustrates age-dependent changes in the strategies to avoid photo-oxidative damage in barley leaves. However, as most factors controlling state-transitions have not been identified (Haldrup et al., 2001) it is difficult to discuss the physiological meaning of these results. The influence of the qI parameter on qN in mature leaves was also very low (if any), as qN relaxed to near zero values after 14 min in the dark (Fig. 8). Thus, an enhanced capacity to maintain a transthylakoidal pH gradient sustained by the activity of the Ndh complex could be one of the main reasons to avoid the decrease in the qN values in mature barley leaves after ozone treatment. Membrane injury in ozone-fumigated mature leaves (as indicated by the thylakoidal lipid peroxidation levels) would probably lead to (as observed in young leaves) an impaired formation of the proton gradient and to a partial inhibition of qN (Calatayud et al., 2002) if not compensated by a higher activity of the Ndh complex.
Ndh function could maintain qN levels under ozone stress conditions by poising the redox level of the intersystem carriers
It has been proposed that the Ndh complex could work as an electron transport intermediate in cyclic flow (Joët et al., 2001) and, therefore, contribute to the generation and maintenance of the transmembrane pH gradient (for a review see Peltier and Cournac, 2002, and references therein). Alternatively, the Ndh complex (providing electrons) could poise the redox level of intersystem electron carriers (Casano et al., 2000; Joët et al., 2002) to optimize cyclic electron transport. As reported in tobacco (Joët et al., 2001), antimycin A, an inhibitor of conventional cyclic electron flow, suppresses the early qN transient increase in barley leaves (Fig. 9). In addition, it was found that antimycin A decreases the steady-state level of qN in ozone-treated young leaves (Fig. 9C), but not in ozone-treated mature (Fig. 9D) barley leaves, when compared with their respective controls. Thus, it may be inferred that an antimycin-A-sensitive pathway can contribute to maintain qN steady-state levels in young (Ndh-defective) barley leaves under ozone stress, but not in mature (Ndh-enhanced) leaves. Since several authors (Endo et al., 1998; Joët et al., 2001) have proposed that an antimycin-A-resistant pathway should be Ndh-dependent, it could be hypothesized, as a first approach, that the Ndh complex contributes to maintain qN values through antimycin A-resistant cyclic electron flow in mature ozone-treated leaves. However, these results might be taken with caution as antimycin A is also an inhibitor of the mitochondrial bc1 complex (Møller, 2001). Myxothiazol, which presumably only inhibits mitochondrial electron transport (Lee et al., 2001), decreased the qN values in both young and mature leaves, indicating that some effects of antimycin A on qN could be indirectly mediated by its action at the mitochondria. In any case, the effects of antimycin A inhibiting qN are only important in young ozone-treated leaves. Meanwhile, myxothiazol equally affects qN under all the leaf conditions studied here. Therefore, it can be maintained that the higher inhibition of qN by antimycin A in young ozone-treated plants is related to the effects of this inhibitor on cyclic electron flow. Furthermore, the effects of antimycin A and myxothiazol on the transient Fo rise in mature leaves (Table 3) were different. The increase of the Fo ‘dark rise’ rate by antimycin A treatment in ozone-treated mature leaves (Table 3) was comparable to that described by Corneille et al. (1998) for isolated potato chloroplasts, following the addition of NADH, and explained by these authors as an effect of this compound on the plastoquinone oxidation pathway of chlororespiration. Whether this interpretation was correct or not, the results indicate that the transient post-illumination Fo rise observed in mature leaves is not an ‘after-effect’ (Peltier and Cournac, 2002) of the antimycin-A-sensitive cyclic electron transport. These results are, on the other hand, consistent with a function of the Ndh complex poising electrons to the intersystem electron carriers. A dicoumarol-sensitive DT-diaphorase (Ndh2), might also be involved in the non-photochemical reduction of plastoquinone (Corneille et al., 1998). No inhibition in the dark plastoquinone reduction rate nor in the qN induction curves were found when barley leaves were incubated in the presence of 250 µM dicoumarol (data not shown), suggesting that such DT-diaphorase activity is not implied in the processes described in this article. However, the possibility that dicoumarol did not penetrate into intact leaves cannot be discarded as an alternative explanation. On the other hand, the inhibitory effect of myxothiazol on this Fo post-illumination transient rise rate and the qN levels cannot easily be explained. Recently, Hou et al. (2003) proposed that myxothiazol could have direct effects on thylakoidal electron transport, although this finding requires further confirmation. A general problem is that results obtained in leaf discs, whole leaf or whole plant systems using metabolic inhibitors acting on different compartments that can interchange common intermediates (like malate or oxalacetate), depend on multiple interactions and feed-back effects that make a simple interpretation difficult. In this context, the inhibitory effects of myxothiazol for the apparent plastoquinone reduction state upon a light-to-dark transition may be explained through an indirect effect of mitochondrial electron transport inhibition on the redox balance of other plant cell compartments after a long incubation in the dark (Krömer, 1995).
Sazanov et al. (1998b) studied the effects of heat shock on tobacco ndh mutants and concluded that the Ndh complex mediates the dark reduction of the plastoquinone pool in response to the stress, as identified on the basis of Fo quenching by far red light in dark-adapted leaves. It was found (Fig. 3) that dark-adapted Fo levels rose clearly after ozone treatment in mature barley primary leaves, but not in young ones. This increased Fo fluorescence level was quenched by far-red light (Fig. 3) in mature leaves, as expected for an Ndh-dependent reduction of the plastoquinone pool. The Ndh-mediated reduction of the plastoquinone pool after a relatively long dark-adaptation period and its strong quenching by far red light is more consistent with a role played by the Ndh complex poising the redox level of the intersystem carriers (Casano et al., 2000; Joët et al., 2002), rather than as an intermediary between photosystem I and plastoquinone (Joët et al., 2001).
According to these results, it is proposed that the Ndh complex could contribute to maintain qN induction responses in mature barley leaves under ozone by helping to generate a H+ gradient through the reduction of the plastoquinone pool and poising the redox state of the intersystem electron carriers. It cannot be discarded that the Ndh complex could itself maintain, as the mitochondrial complex I, the generation of a proton gradient. Recent results from Lennon et al. (2003) indicate that the orientation of the Ndh complex is consistent with a role for the Ndh complex in the energization of the plastid membrane.
Several mutants deficient in the non-photochemical quenching generation have been described (Niyogi et al., 1998). Surprisingly, these mutants showed a similar growth to wild-type plants under laboratory high-light conditions (Niyogi et al., 1998). However, Kühleim et al. (2002) showed that these mutants had a reduced fitness under fluctuating light conditions, like those found in a natural environment. These authors concluded that the feed-back de-excitation confers a strong fitness advantage under field conditions, more by tolerance to sunflecks than by tolerance to light intensity itself. In this context, the function of the Ndh complex can be understood as a contribution to maintain the fitness of plant populations under adverse environmental constrains (such as contaminant exposure in combination with fluctuating light intensity), by optimizing cyclic electron transport through poising the intermediate carriers redox level. This is the first time, as far as is known, that the function of the Ndh complex in developing leaves of a genetically non-manipulated plant (barley), can be clearly related to results obtained with leaves from mutant lines of tobacco. Both lines of evidence show a function of the Ndh complex acting against photo-oxidative stress, probably by increasing the non-photochemical quenching dissipation of an excess of light energy that can be harmful under constraining environmental conditions.
|
Acknowledgements |
|---|
This work was supported by the Spanish DGICYT (Grant BFI2000–0781); grants from ‘Consejo social’ of the University of Alcalá de Henares, to AG for short stays at the University of Valencia; and by the Spanish MCYT (Grant REN2003-04465/GLO). Thanks are given to Professor Eva-Mari Aro for the anti-D1 protein antibody and to Mr Francisco Gasulla for technical assistance. Thanks to Ms Helen Warburton for her help with English text. The authors should like to dedicate this article to the memory of Professor Dr Rainer Maier, who developed the ndhF mutant lines, a good friend and an excellent scientist.
|
Footnotes |
|---|
Present address: Departamento de Horticultura, Instituto Valenciano de Investigaciones Agrarias (IVIA), Conselleria de Agricultura, Pesca y Alimentación de la Generalitat Valenciana, Cra. Moncada-Náquera, 46113 Moncada, Valencia, Spain. 
Abbreviations: Chl, chlorophyll; Fm, maximal fluorescence yield obtained with dark-adapted sample; 
maximal fluorescence yield in illuminated samples; Fo, minimum fluorescence yield in dark-adapted state; 
level of modulated fluorescence during brief interruption of actinic illumination in the presence of far-red illumination; Fs, chlorophyll fluorescence yield during illumination; Fv, (Fm–Fo) variable fluorescence in dark-adapted leaves; MDA, malondialdehyde; Ndh, plastid NADH-plastoquinone-oxidoreductase complex; qE, energy-dependent component of qN; qT, state-transition component of qN; qI, photoinhibition component of qN; qN, non-photochemical quenching of Fm; qP, photochemical fluorescence quenching coefficient; 
PSII, quantum efficiency of PSII.
|
References |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
Alonso R, Elvira S, Castillo FJ, Gimeno BS. 2001. Interactive effects of ozone and drought stress on pigments and activities of antioxidantive enzymes in Pinus halepensis. Plant, Cell and Environment 24, 905–916.
Astorino G, Margani E, Tripodo P, Manes F. 1995. The response of Phaseolus vulgaris L. cv. Lit to different dosage of anti-ozonant ethylendiurea (EDU) in relation to chronic treatment with ozone. Plant Science 111, 237–248.
Batini P, Ederli L, Pasqualini S, Antonielli M, Valenti V. 1995. Effects of ethylenediurea and ozone in detoxificant ascorbic-ascorbate peroxidase system in tobacco plants. Plant Physiology and Biochemistry 33, 717–723.
Burrows PA, Sazanov LA, Svab Z, Maliga P, Nixon PJ. 1998. Identification of a functional respiratory complex in chloroplasts through analysis of tobacco mutants containing disrupted plastid ndh genes. EMBO Journal 17, 868–876.[CrossRef][ISI][Medline]
Calatayud A, Barreno E. 2000. Foliar spraying with zineb increases fruit productivity and alleviates oxidative stress in two tomato cultivars. Photosynthetica 38, 149–154.[CrossRef]
Calatayud A, Barreno E. 2001. Cholorophyll a fluorescence, antioxidant enzymes and lipid peroxidation in tomato in response to ozone and benomyl. Environmental Pollution 115, 283–289.[CrossRef][Medline]
Calatayud A. Ramirez JW, Iglesias JD, Barreno E. 2002. Effects of ozone on photosynthetic CO2 exchange, chlorophyll a fluorescence and antioxidant systems in lettuce leaves. Physiologia Plantarum 116, 308–316.
Carlsson AS, Wallin G, Sandelius AS. 1996. Species and age-dependent sensitivity to ozone in young plants of pea, wheat and spinach: effects on acyl lipid and pigment content and metabolism. Physiologia Plantarum 98, 271–280.
Carol P, Kuntz M. 2001. A plastid terminal oxidase comes to light: implications for carotenoid biosynthesis and chlororespiration. Trends in Plant Sciences 6, 31–36.
Casano LM, Martín M, Zapata JM, Sabater B. 1999. Leaf-age and paraquat concentration-dependent effects on the levels of enzymes protecting against photo-oxidative stress. Plant Science 149, 13–22.
Casano LM, Zapata JM, Martín M, Sabater B. 2000. Chlororespiration and poising of cyclic electron transport: plastoquinone as electron transporter between thylakoid NADH dehydrogenase and peroxidase. Journal of Biological Chemistry 275, 942–948.
Casano LM, Martín M, Sabater B. 2001. Hydrogen peroxide mediates the induction of chloroplastic Ndh complex under photoxidative stress in barley. Plant Physiology 125, 1450–1458.
Casano LM, Lascano HR, Martín M, Sabater B. 2004. Topology of the plastid Ndh complex and its NDH-F subunit thylakoid membranes. Biochemical Journal 382, 145–155.