Planta

, Volume 222, Issue 3, pp 502–511 | Cite as

Enhanced ferredoxin-dependent cyclic electron flow around photosystem I and α-tocopherol quinone accumulation in water-stressed ndhB-inactivated tobacco mutants

  • Sergi Munné-Bosch
  • Toshiharu Shikanai
  • Kozi Asada
Original Article

Abstract

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.

Keywords

Antioxidants Cyclic electron flow Photoprotection Photosynthesis Tocopherols Water stress 

Abbreviations

AL

Actinic light

DPS

De-epoxidation state of the xanthophyll cycle

DW

Dry weight

Fd

Ferredoxin

ϕPSII

Relative efficiency of PSII photochemistry

FR

Far red

Fv/Fm

Maximum efficiency of PSII photochemistry

FW

Fresh weight

IR

Irrigated

MDA

Malondialdehyde

NDH

NAD(P)H-dehydrogenase complex

NPQ

Non-photochemical quenching of chlorophyll fluorescence

PPFD

Photosynthetically-active photon flux density

PSI (II)

Photosystem I (II)

RWC

Relative leaf water conent

TW

Turgid weight

WS

Water-stressed

Introduction

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.

Climatologic 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).

Growth measurements

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 × ((AB)/157,000), where A = ((Abs 532+TBA)−(Abs 600+TBA)−(Abs 532TBA −Abs 600TBA)), and B = ((Abs 440+TBA-Abs 600+TBA)x0.0571).

Pigment determination

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 analyses

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.

Results

Phenotype of ndhB-inactivated tobacco mutants under water stress

Plants were exposed to water stress by withholding water for 11-days in a greenhouse, in which maximum diurnal PPFD, temperature and vapor pressure deficit values ranged between 900 μmol m−2s−1 and 1,200 μmol m−2 s−1, 33–36°C and 3.0–4.2 kPa, respectively, throughout the treatment. Relative leaf water content (RWC) decreased to 30% in both the mutant and wild type plants (Fig. 1). Both plant types were morphologically undistinguishable under IR and WS conditions, as indicated by growth parameters. Leaf biomass and leaf area per plant decreased by ca. 60%, whilst the root mass per leaf area ratio increased twofold in stressed plants (Table 1).
Fig. 1

Changes in the relative leaf water content (RWC) of wild type and ndhB-inactivated mutant of tobacco during the experiment. Data represent the mean ± SE of four independent measurements for the leaves collected at midday. Circles and squares represent irrigated (IR) and water-stress (WS) treatments, and closed and open symbols show wild type and mutant plants, respectively

Table 1

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)

 

Wild type

ndhB-inactivated mutant

IR

WS

IR

WS

Leaf biomass (g DW)

6.25±1.06

2.85±0.21a

7.10±0.85

2.55±0.56a

Leaf area (dm2)

16.80±2.85

6.79±0.51a

18.68±2.23

6.22±0.59a

Leaf mass area (g DW dm−2)

0.37±0.01

0.42±0.01a

0.38±0.01

0.41±0.02a

Stem biomass (g DW)

4.11±0.45

3.53±0.79

4.23±0.87

3.63±0.53

Root biomass (g DW)

3.11±0.55

2.47±0.73

2.85±0.63

2.00±0.74

Root mass/leaf area (g DW dm−2)

0.18±0.03

0.36±0.03a

0.15±0.02

0.32±0.04a

Data represent the mean ± SE of three randomly chosen plants

a Indicates statistical difference (ANOVA, P<0.05) between IR and WS plants. No statistical differences (ANOVA, P<0.05) were found in any growth parameter between wild type and mutant plants

Lipid peroxidation and photo-oxidative damage under water stress

No significant differences (ANOVA, P<0.05) were observed either in chlorophyll, MDA, an indicator of lipid peroxidation, or the Fv/ Fm ratio between the mutant and wild type plants, even though these parameters changed significantly under water stress in both groups. An increase in MDA levels and decreases in the Fv/ Fm ratio and chlorophyll were apparent only after 11-days stress (Fig. 2), thus, chloroplasts appeared to be protected by dissipation mechanisms of excess excitation energy until RWC decreased below 50%.
Fig. 2

Changes in the levels of chlorophyll a+b (Chl), malondialdehyde (MDA) and the maximum efficiency of PSII (Fv/ Fm) in tobacco leaves of wild type and ndhB-inactivated mutants during the IR and WS treatments. Data represent the mean ± SE of three independent measurements for leaves collected at midday. The symbols are the same as those in Fig. 1

Down-regulation of linear electron flow under water stress

Net CO2 assimilation rates in leaves were very similar in the mutant and wild type plants before the treatment, but decreased to below 15% after 4-days stress, and to zero after 11-days stress in both plant groups (Fig. 3). This decrease was associated with severe reductions in stomatal conductance, from 160 mmol m−2 s−1 to below 30 mmol m−2 s−1 after treatment (data not shown). Down-regulation of the linear electron transport occurred in WS plants as indicated by progressive reductions in the φPSII (Fig. 3), and this was associated, at least in part, with an increase in NPQ, which is an indicator of excess excitation energy dissipation as heat. NPQ increased progressively in WS plants until 8-days of stress treatment, and then decreased sharply (Fig. 3) concomitantly with reductions in the Fv/Fm ratio and increases in MDA levels after 11-days of treatment (Fig. 2).
Fig. 3

Changes in net CO2 assimilation rates (Pn), relative efficiency of PSII photochemistry (φPSII) and non-photochemical quenching (NPQ) of chlorophyll fluorescence in leaves of wild type and ndhB-inactivated mutants of tobacco during the IR and WS treatments. Data represent the mean ± SE of six to ten independent measurements for attached leaves at midday. The symbols are the same as those in Fig. 1. Pn, φPSII and NPQ were determined at a PPFD of 1,200 μmol m−2 s−1, 380 ppm CO2, 25°C and a relative humidity of 30%. No differences between wild type and mutants were observed in these three parameters at other PPFDs tested (400, 800 and 1,600 μmol m−2 s−1) in the IR or WS treatments (data not shown)

Activation of cyclic electron flow under water stress

In dark-adapted leaves, P700 was more rapidly oxidized by FR light in IR than in WS plants (Fig. 4a,c) in both plant groups. Thus, dark-adapted leaves of WS plants showed a more substantial cyclic electron flow around PSI induced by FR light. The re-oxidation rates of P700 after AL illumination under a background of FR light (Fig. 4b, d) were slower than those in the dark-adapted leaves, indicating that the electrons accumulated in the stroma during AL illumination were donated to P700+ via the intersystem electron carriers. The pool size of the electrons in the stroma was also larger in WS than IR plants. The re-oxidation rate of P700 was very similar in the mutant and in the wild type under WS (Fig. 4d).
Fig. 4

Redox kinetics of P700 in leaves of wild type (closed circles) and ndhB-inactivated mutant (open circles) of tobacco after 11-days treatments under IR and WS conditions. Oxidation kinetics of P700 by FR light (above 720 nm, 6.8 W m−2) in the dark-adapted leaves (a, c). Redox kinetics of P700 after actinic light illumination (AL, 950 μmol m−2 s−1, for 2 min) under a background of FR light (b, d)

The transient increases of chlorophyll fluorescence after AL-illumination in the absence or presence of antimycin A are shown in Fig. 5. Under irrigated conditions, NDH-deficient mutants showed a lower transient increase in chlorophyll fluorescence after AL-illumination compared with the wild type. However, after 11-days WS treatment both the plant groups showed similar transient increases in chlorophyll fluorescence after AL illumination. These results differed significantly in the presence of antimycin A, which inhibits ferredoxin (Fd)-dependent, but not NDH-dependent cyclic electron flows around PSI (Joët et al. 2001; Munekage et al. 2004). After 11-d WS, the transient increase in chlorophyll fluorescence after AL illumination was only partly reduced by antimycin A in wild type plants, but completely disappeared in NDH-deficient plants (Fig. 5). These results indicate that antimycin A-sensitive cyclic electron flow was up-regulated in the mutant after WS treatment.
Fig. 5

Effects of FR and antimycin A on the post-illumination increase in chlorophyll fluorescence in leaves of wild type and ndhB-inactivated mutant of tobacco after 11-days treatments under IR and WS conditions. The leaves were illuminated with actinic light (AL, 950 μmol m−2 s−1) for 5 min, and then an increase in chlorophyll fluorescence was followed. Where indicated, FR (above 720 nm, 6.8 W m−2) was used. Antimycin A (Sigma) was administered to the leaves, as described by Joët et al. (2001). After stripping the lower epidermis, the leaf discs were incubated in 5 μM antimycin A in 0.015% methanol for 20 min

Protection from photo-oxidative damage under water stress

No significant differences (ANOVA, P<0.05) in carotenoids (carotenes and xanthophylls) were observed between the mutant and wild type plants throughout the treatment with water stress (Fig. 6). β-Carotene decreased significantly after 8-days WS in both plant groups, but was only slightly reduced after that (Fig. 6). In contrast, chlorophyll was degraded when RWC decreased below 50% (Fig. 2). Thus, the β-carotene/chlorophyll ratio increased two-fold in both plant groups after 11-days WS. Lutein, violaxanthin and neoxanthin changed in parallel with β-carotene by WS (data not shown). Zeaxanthin increased significantly during the 8-days treatment, and then decreased gradually as the RWC fell below 50% (Fig. 6). However, the zeaxanthin/chlorophyll ratio increased by ca. 2.9-fold and 2.5-fold, respectively, in both plant groups after 11-days WS. In addition, the de-epoxidation state of the xanthophyll cycle (DPS, Fig. 6) followed a similar trend to that observed for NPQ in both plant groups, except that reductions in NPQ were more marked than those of DPS after 11-days WS (Figs. 3, 6). Ascorbate levels were severely and similarly reduced in both plant groups under stress. The redox state of ascorbate also shifted towards its oxidized form in both plant types when subjected to WS (Fig. 7).
Fig. 6

Changes in β-carotene (β-Car), zeaxanthin (Z) and the de-epoxidation state of the xanthophyll cycle (DPS) in leaves of wild type and ndhB-inactivated mutant of tobacco during the IR and WS treatments. Data represent the mean ± SE of three independent measurements for leaves collected at midday. The symbols are the same as those in Fig. 1

Fig. 7

Changes in the levels of ascorbate (Asc) and its redox state (Dha/Asct, where Asct = Asc+Dha, Dha, dehydroascorbate) in wild type and ndhB-inactivated mutant of tobacco during IR and WS treatments. Data represent the mean ± SE of three independent measurements for leaves collected at midday. The symbols are the same as those in Fig. 1

In contrast to ascorbate, significant differences (ANOVA, P<0.05) in the extent of α-tocopherol oxidation to α-tocopherol quinone were observed between the mutant and wild type plants under WS (Fig. 8). Whilst γ-tocopherol levels were similar in both plant groups throughout the treatment, the mutant showed significantly lower α-tocopherol, and higher α-tocopherol quinone levels than the wild type. Under stress, α-tocopherol quinone levels increased up to 2.5-fold and 3.5-fold in the wild type and mutant plants, respectively. The highest stress-induced level of α-tocopherol quinone in the mutant correlated with lower levels of α-tocopherol at equimolar concentrations. The redox state of α-tocopherol shifted towards its reduced form in both plant groups after 4-days WS, which was associated with the increases in α-tocopherol. The redox state of α-tocopherol shifted towards its oxidized form as stress progressed further in both plant groups, and its maximum levels were attained in the mutant after 11-days WS (Fig. 8).
Fig. 8

Changes in γ-tocopherol (γ-T), α-tocopherol (α-T), α-tocopherol quinone (α-TQ) and the redox state of α-T (α-TQ/α-Tt, where α−Tt = α−T + α−TQ) in leaves of wild type and ndhB-inactivated mutant of tobacco during IR and WS treatments. Data represent the mean ± SE of three independent measurements for leaves collected at midday. The symbols are the same as those in Fig. 1

Discussion

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.

Notes

Acknowledgments

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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Copyright information

© Springer-Verlag 2005

Authors and Affiliations

  • Sergi Munné-Bosch
    • 1
  • Toshiharu Shikanai
    • 2
  • Kozi Asada
    • 3
  1. 1.Departament de Biologia Vegetal, Facultat de BiologiaUniversitat de BarcelonaBarcelonaSpain
  2. 2.Graduate School of AgricultureKyushu UniversityJapan
  3. 3.Department of Biotechnology, Faculty of Life Sciences and BiotechnologyFukuyama UniversityFukuyamaJapan

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