Enhanced ferredoxin-dependent cyclic electron flow around photosystem I and α-tocopherol quinone accumulation in water-stressed ndhB-inactivated tobacco mutants
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Dissipation mechanisms of excess photon energy under water stress were studied in ndhB-inactivated tobacco (Nicotiana tabacum cv. Xanthi) mutants, which are impaired in NAD(P)H dehydrogenase-dependent cyclic electron flow around PSI. Relative leaf water content and net CO2 assimilation decreased to 30% and almost zero, respectively, after 11-day water stress in the mutant and wild type plants. Similar reductions in PSII activity (by ca. 75%), and increases in malondialdehyde (by ca. 45%), an indicator of lipid peroxidation, were observed in both the plant groups when subjected to water stress. The stressed mutant and wild type plants showed similar P700 redox kinetics, but only the stressed mutant demonstrated an enhanced operation of the antimycin A-sensitive, ferredoxin-dependent cyclic electron flow around PSI, as indicated by a transient increase in chlorophyll fluorescence after turning off of actinic light. Further, the stressed mutant showed higher oxidation of α-tocopherol to α-tocopherol quinone, as compared with that in the stressed wild type. Thus, a deficiency in NAD(P)H dehydrogenase-dependent cyclic electron flow around PSI does not lead to oxidative damage because the mutant compensates for this deficiency by activating alternative dissipating routes of excess photon energy, such as up-regulation of ferredoxin-dependent cyclic electron flow around PSI and increased accumulation of α-tocopherol quinone.
KeywordsAntioxidants Cyclic electron flow Photoprotection Photosynthesis Tocopherols Water stress
De-epoxidation state of the xanthophyll cycle
Relative efficiency of PSII photochemistry
Maximum efficiency of PSII photochemistry
Non-photochemical quenching of chlorophyll fluorescence
Photosynthetically-active photon flux density
- PSI (II)
Photosystem I (II)
Relative leaf water conent
Plants have evolved multiple mechanisms to avoid damage to photosynthetic apparatus caused by environmental conditions. Acclimation to adverse climatic conditions can occur through alterations in leaf structure (Weston et al. 2000; Terashima et al. 2001), leaf and chloroplast movements (Satter and Galston 1981; Kasahara et al. 2002), changes in antenna size and pigment composition (Havaux et al. 1998; Demmig-Adams and Adams 2002), and the regulation of light energy utilization and dissipation (Asada 1999; Niyogi 1999).
Under optimal conditions for photosynthesis and growth, most excessive light energy absorbed by the antenna chlorophyll in PSII is safely dissipated as heat in a process that depends on the generation of a proton gradient across the thylakoid membranes, the de-epoxidation of violaxanthin to zeaxanthin and the availability of ascorbate in the lumen (Demmig-Adams and Adams 1996; Eskling et al. 1997). In addition, photorespiration (Kozaki and Takeba 1996), cyclic electron flow around PSI (Moss and Bendall 1984; Bendall and Manasse 1995; Joët et al. 2002; Shikanai et al. 2002; Johnson 2005), photoreduction of dioxygen to water in PSI (Asada 1999), and quenching of excited triplet chlorophyll (Asada and Takahashi 1987; Halliwell and Gutteridge 1999; Krieger-Liszkay 2005) are involved in conferring protection from photo-oxidative damage. However, the quantitative contribution and interdependence of respective processes in the dissipation of excess excitation energy in chloroplasts, especially under environmental stress, has not been estimated.
The plastid genome of higher plants contains ndh genes encoding peptides homologous to subunits of the proton-pumping NADH:ubiquinone oxidoreductase, a component of the mitochondrial respiratory chain (Ohyama et al. 1986). An NAD(P)H-dehydrogenase complex (NDH) has been purified from Arabidopsis, spinach, pea and barley thylakoids (Sazanov et al. 1998; Quiles et al. 2000; Lennon et al. 2003), and this complex participates in the cyclic electron flow around PSI in higher plants in vivo (Burrows et al. 1998; Shikanai et al. 1998). The chloroplast transformation technique has facilitated reverse genetics on NDH in higher plants and ndh-inactivated mutants are now available to study the physiological relevance of this complex under stress, as reviewed by Shikanai and Endo (2000). Several studies have examined the effect of this complex on plant stress tolerance in ndh-inactivated plants (Endo et al. 1999; Horváth et al. 2000; Barth and Krause 2002; Li et al. 2004). However, to our knowledge, no study has examined the role of antioxidants in ndh-inactivated plants subjected to environmental stress. To explore the effects of a deficiency in NDH-dependent cyclic electron flow around PSI in plants exposed to environmental stress, we evaluated here the photosynthetic activity, extent of oxidative stress and mechanisms of photo- and antioxidant protection in the ndhB-inactivated tobacco mutant when exposed to water deficit.
Materials and methods
Plants and growth conditions
Wild type and ndhB-inactivated transformants of tobacco (N. tabacum cv. Xanthi) were obtained as described by Shikanai et al. (1998). When plants were between 8 cm and 10 cm in height, they were transplanted to 1-l pots containing soil/perlite/vermiculite (1:1:1, by vol.). Growth conditions were 50 μmol photons m−2 s−1 under a photoperiod of 15.5/8.5 h (day : night) at 25°C. When plants were 50–60 cm in height, they were transferred to a greenhouse under natural light on 28 May 2003, and grown under controlled watering and nutrient conditions until the experiment started on 21 June 2003. During the experiment, two water regimes were imposed on plants: Irrigated (IR) plants were watered with Hoagland’s solution (Hoagland and Arnon 1950) every two days, while water-stressed (WS) plants received no water for 11-days.
Environmental conditions, plant water status, growth, redox kinetics of P700, chlorophyll fluorescence, malondiladehyde (MDA) and pigment contents, and contents of reduced and oxidized α-tocopherol and ascorbate in leaves collected at midday (12 h solar time) were measured. For measurements of MDA, α-tocopherol and ascorbate, leaves were collected, frozen in liquid nitrogen and stored at −80°C until analysis. Cutters, the leaves found in the middle position of the stalk were 15–20 cm in length, and were used for all measurements.
Photosynthetically-active photon flux density (PPFD) was measured with a Quantum sensor Li-cor 250 (Li-cor, Lincoln, NE, USA). Air temperature and relative humidity were measured with a SuperEXSensor thermohygrometer (Empex, Tokyo, Japan). Vapor pressure deficit was calculated from air temperature and relative humidity data according to Nobel (1991).
Plant water status
Plant water status was estimated by measuring the relative water content of leaves (RWC). Leaves were collected and immediately weighed to determine fresh weight (FW). Then, leaves were re-hydrated for 24 h at 4°C in darkness to determine the turgid weight (TW), and subsequently oven-dried for 24 h at 85°C to determine the dry weight (DW). The RWC was determined as 100 × (FW – DW)/ (TW – DW).
At the end of the experiment, plants were harvested and divided into leaves, stems and roots. Leaves, stems and roots were weighed and leaf area was immediately measured using a flatbed scanner (model GT-5000; Epson, Nagano, Japan) and an image-processing program. Samples were dried at 85°C at constant weight, and growth parameters were therefore calculated.
Leaf gas exchange
A Portable Photosynthesis System HCM-1000 (Walz) was used for determination of CO2 and H2O exchange in attached leaves. Net CO2 assimilation and stomatal conductance rates were calculated from changes in CO2 and H2O according to Von Caemmerer and Farquhar (1981). Measurements were performed under the following conditions: CO2 concentration, 380 ppm; temperature, 25°C; relative humidity, 30%; PPFD, 0, 400, 800, 1,200 and 1,600 μmol m−2 s−1 .
Redox kinetics of P700
The redox state of P700 was determined by following the difference in absorbance of P700+ at 810 nm with that at 860 nm as a reference (Klughammer and Schreiber 1998) using an emitter-detector unit ED-P700DW-E (Walz) connected to a PAM fluorometer (101). Far red (FR) light (above 720 nm, 6.8 W m−2) and actinic light (AL, 950 μmol m−2 s−1) were applied to leaves via a multibranched fiberoptic system that was equipped with a detector.
Modulated chlorophyll fluorescence
Cyclic electron flow around PSI was monitored by the transient increase of chlorophyll fluorescence after AL illumination using a mini-PAM fluorometer (Walz). This increase is considered a result of non-photochemical reduction of plastoquinones, which reflects the activity of cyclic electron flow during the previous actinic light period. A mini-PAM fluorometer (Walz) was used for measurements of the maximum efficiency of PSII photochemistry (Fv/Fm), the relative efficiency of PSII photochemistry (φPSII) and non-photochemical quenching of chlorophyll fluorescence (NPQ) on attached leaves as described by Van Kooten and Snel (1990).
Estimation of lipid peroxidation
The extent of lipid peroxidation in leaves was estimated by measuring the amount of MDA by the method described by Hodges et al. (1999), which takes into account the possible influence of interfering compounds in the assay for thiobarbituric acid (TBA)-reactive substances. In short, leaves were extracted four times with 80:20 (v/v) ethanol/water containing 1 ppm butylated hydroxytoluene (BHT) using ultrasonication (Vibra-Cell Ultrasonic Processor, Sonics& Materials Inc., Danbury, CT, USA). After centrifugation, supernatants were pooled and an aliquot of appropriately diluted sample was added to a test tube with an equal volume of either (1) −TBA solution containing 20% (w/v) trichloroacetic acid and 0.01% (w/v) BHT, or (2) +TBA solution containing the above plus 0.65% (w/v) TBA. The reaction mixture was heated at 95°C for 25 min and, after cooling, absorbance was read at 440, 532, and 600 nm. MDA equivalents (nmol ml−1) were calculated as 106 × ((A−B)/157,000), where A = ((Abs 532+TBA)−(Abs 600+TBA)−(Abs 532−TBA −Abs 600−TBA)), and B = ((Abs 440+TBA-Abs 600+TBA)x0.0571).
The extraction and HPLC analysis of pigments were carried out as described by Munné-Bosch and Alegre (2003). Briefly, leaves were ground in liquid nitrogen and extracted four times with ice-cold acetone using ultrasonication (Vibra-Cell Ultrasonic Processor). Pigments were separated on a Dupont non-endcapped Zorbax ODS-5 μm column (250×4.6 mm, 20%C, Teknokroma, St. Cugat, Spain) at 30°C at a flow rate of 1 ml min−1 . The solvents consisted of (A) acetonitrile/methanol (85: 15, v/v) and (B) methanol/ethyl acetate (68: 32, v/v). The gradient used was: 0–14 min 100% A, 14–16 min decreasing to 0% A, 16–28 min 0% A, 28–30 min increasing to 100% A, and 30–38 min 100% A. Detection was carried out at 445 nm (Diode array detector 1000S, Applied Biosystems, Foster City, CA, USA). Compounds were identified by their characteristic spectra and by coelution with chlorophyll and carotenoid standards, which were obtained from Fluka (Buchs, Switzerland).
Analyses of reduced and oxidized forms of ascorbate and tocopherol
For measurement of γ-tocopherol,α-tocopherol and its oxidation product, α-tocopherol quinone, leaves were extracted four times with ice-cold n-hexane containing 1 ppm BHT using ultrasonication (Vibra-Cell Ultrasonic Processor) and determined by HPLC as described by Munné-Bosch and Alegre (2003). Tocopherols and α-tocopherol quinone were separated on a Partisil 10 ODS-3 column (250×4.6 mm, Scharlau, Barcelona, Spain) at a flow rate of 1 ml min−1 . The solvents consisted of (A) methanol/water (95: 5, v/v) and (B) methanol. The gradient used was: 0–10 min 100% A, 10–20 min decreasing to 0% A, 20–25 min 0% A, 25–28 min increasing to 100% A, and 28–33 min 100% A. Tocopherols and α-tocopherol quinone were quantified through their absorbance at 283 and 256 nm, respectively (Diode array detector 1000S, Applied Biosystems). Compounds were identified by their characteristic spectra and by coelution with authentic standards provided by Sigma and Prof. Strzalka (Jagiellonian University, Krakov, Poland).
The extraction and HPLC analysis of reduced and oxidized ascorbate were performed as described by Munné-Bosch and Alegre (2003). Leaves were ground in liquid nitrogen and extracted four times with ice-cold extraction buffer (40% [v/v] methanol, 0.75% [w/v] m-phosphoric acid, 16.7 mM oxalic acid, 0.127 mM diethylenetriaminepentaacetic acid) using ultrasonication (Vibra-Cell Ultrasonic Processor). After centrifugation, 0.1 ml of the supernatant was transferred to 0.9 ml of the mobile phase (24.25 mM Na-acetate/acetic acid, pH 4.8; 0.1 mM diethylenetriaminepentaacetic acid; 0.015% [w/v] m-phosphoric acid; 0.04% [w/v] octylamine; 15% [v/v] methanol) for determination of reduced ascorbate. For determination of total ascorbate (reduced plus oxidized) 0.1 ml of the supernatant was incubated for 10 min at room temperature in darkness with 0.25 ml of 2% (w/v) dithiothreitol and 0.5 ml of 200 mM NaHCO3. The reaction was stopped by adding 0.25 ml of 2% (v/v) sulfuric acid and 0.8 ml of the mobile phase. Ascorbate was isocratically separated on a Spherisorb ODS C8 column (Teknokroma, St. Cugat, Spain) at a flow rate of 0.8 ml min−1 . Detection was carried out at 255 nm (Diode array detector 1000S, Applied Biosystems). Ascorbate was identified by its characteristic spectrum and by coelution with an authentic standard from Sigma.
Statistical differences between measurements on different treatments and on different times were analyzed following a three-way ANOVA using SPSS (Chicago, IL, USA). Differences were considered significant at a probability level of P<0.05.
Phenotype of ndhB-inactivated tobacco mutants under water stress
Growth parameters of wild type and ndhB-inactivated mutants of tobacco after 11-days treatment under irrigated (IR) and water stress (WS) conditions (see Materials and methods)
Leaf biomass (g DW)
Leaf area (dm2)
Leaf mass area (g DW dm−2)
Stem biomass (g DW)
Root biomass (g DW)
Root mass/leaf area (g DW dm−2)
Lipid peroxidation and photo-oxidative damage under water stress
Down-regulation of linear electron flow under water stress
Activation of cyclic electron flow under water stress
Protection from photo-oxidative damage under water stress
Fd-dependent, antimycin A sensitive-cyclic electron flow around PSI operates in higher plants (Tagawa et al. 1963; Moss and Bendall 1984; Bendall and Manasse 1995; Munekage et al. 2002). In addition, an NDH-dependent pathway also operates in the cyclic electron flow around PSI from cyanobacteria to angiosperms (Mi et al. 1992; Burrows et al. 1998; Shikanai et al. 1998; Joët et al. 2002; Johnson 2005). Recently, it has been shown that a mutant lacking Fd-dependent cyclic flow activity is highly sensitive to photoinhibition (Munekage et al. 2002). Further, the mutants lacking both the cyclic electron flows exhibit severe growth reduction even under the mild culture conditions. This remarkable growth phenotype of the double mutants lacking both the cyclic pathways around PSI indicates that the function of the NDH complex is essential under the mutant background of pgr5, in which Fd-dependent cyclic pathway is impaired (Munekage et al. 2004). Thus, it is likely that the NDH complex alleviates oxidative stress under extreme stress conditions. The present results using the NDH-less tobacco indicate that a deficiency in NAD(P)H dehydrogenase-dependent cyclic electron flow around PSI does not lead to irreversible oxidative injuries because the mutant compensates for this deficiency by activating alternative dissipating routes of excess photon energy, such as up-regulation of ferredoxin-dependent cyclic electron flow around photosystem I and increased accumulation of α-tocopherol quinone.
In ndhB-inactivated mutants, the NDH-dependent cyclic electron flow does not alter growth or photosynthesis under optimal conditions (Burrows et al. 1998; Shikanai et al. 1998; Shikanai and Endo 2000; Joët et al. 2001, 2002). In agreement with Horváth et al. (2000), we did not observe any difference in growth parameters between the mutant and wild type tobacco plants under WS. Furthermore, MDA levels and the maximum quantum yield of PSII (Fv/Fm ratios) were similar in the two plant types throughout the treatment with water stress, which indicates that mutants were as efficient as wild type plants in preventing damage to the photosynthetic apparatus under adverse climatic conditions. In previous studies, it has been shown that an impairment of NDH-dependent cyclic electron flow around PSI may lead to damage to the photosynthetic apparatus depending on the magnitude and severity of the stress (Endo et al. 1999; Barth and Krause 2002). In our study, stress was imposed on entire plants and progressively over time, which might have favored ndhB-inactivated mutants acclimated to adverse conditions that compensated for this deficiency by activating other dissipation mechanisms of excess excitation energy.
In agreement with Golding and Johnson (2003), plants exposed to water deficit showed enhanced cyclic electron flow around PSI. Cyclic electron flow around PSI serves to maintain a pH gradient across the thylakoid membranes, which is required for ATP synthesis and dissipation of photon energy as heat (Munekage et al. 2002, 2004). ndhB disruption enhances the sensitivity of photosynthesis to antimycin A (Joët et al. 2001), indicating that the NDH-mediated pathway and Fd-dependent pathway concomitantly function in cyclic electron flow around PSI. Although the biological significance of these pathways is still under debate today (Johnson 2004, 2005; Kramer et al. 2004), the present study supports the contention that Fd- and NDH-mediated pathways cooperate in the modulation of the extent of cyclic electron flow around PSI and therefore in the dissipation of excess energy in chloroplasts, especially under stress. Measurements of chlorophyll fluorescence after termination of AL illumination by using antimycin A revealed that cyclic electron flow around PSI in WS mutants was exclusively dependent on an Fd-dependent pathway. This is indicative of up-regulation of Fd-dependent cyclic electron flow around PSI in these plants, which may at least partly compensate for the deficiency of NDH.
Here we have shown that WS caused an induced biosynthesis of α-tocopherol in wild type and mutant plants, and the oxidation of α-tocopherol to α-tocopherol quinone was higher in the mutant than in the wild type under WS. This observation indicates that the generation rate of singlet oxygen and other ROS in thylakoids, and therefore oxidative stress, was higher in the mutants than in wild type plants under WS conditions. Singlet oxygen formation (through quenching of excited triplet chlorophyll by molecular oxygen) is a reliable mechanism of photoprotection as long as native antioxidants, such as tocopherols and carotenoids, are present in thylakoids, thereby ensuring that excess excitation energy is safely dissipated and oxidative damage prevented (Havaux 1998; Munné-Bosch and Alegre 2002; Krieger-Liszkay 2005).
Furthermore, α-tocopherol quinone may contribute to a photoprotective function in PSII (Kruk and Strzalka 1995; Kruk et al. 2000; Kruk and Strzalka 2001). These studies indicate that, in addition to showing antioxidant properties similar to those of α-tocopherol,α-tocopherol quinone may be involved in a specific, DCMU sensitive, fluorescence quenching of PSII, which is probably linked to the cyclic electron transport around PSII. Thus, a deficiency in NDH-dependent cyclic electron flow around PSI might be compensated in ndh-inactivated tobacco mutants not only by enhanced Fd-dependent cyclic electron flow around PSI, but also by other mechanisms of excess excitation energy dissipation, such as enhanced singlet oxygen formation, and α-tocopherol quinone-dependent photoprotection.
On the basis of our results, we conclude that tobacco mutants that are deficient in NDH-dependent cyclic electron flow around PSI may compensate for this deficiency by activating alternative routes of excess excitation energy dissipation, to say up-regulation of Fd-dependent cyclic electron flow around photosystem I and increased accumulation of α-tocopherol quinone, thereby highlighting the plasticity of photoprotection mechanisms evolved by plants. Further research is needed to better understand the mechanistic links between cyclic electron flow around PSI and α-tocopherol quinone accumulation in plants, and to elucidate whether up-regulation of Fd-dependent cyclic electron flow around PSI in the mutant resulted from increased amounts of electron carriers involved in cyclic electron flow or from an acceleration of the cyclic electron flow around PSI.
We are very grateful to the Canon Foundation in Europe for the fellowship given to S.M.. We also thank the Serveis Científico-Tècnics (University of Barcelona) for technical assistance, and Dr. Jerzy Kruk and Prof. Kazimierz Strzalka (Jagiellonian University, Krakov, Poland) for kindly providing us with α-tocopherol quinone.
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