Directory UMM :Data Elmu:jurnal:P:PlantScience:PlantScience_Elsevier:Vol153.Issue2.2000:
Antioxidant responses of cucumber (
Cucumis sati
6
us
) to
photoinhibition and oxidative stress induced by norflurazon under
high and low PPFDs
Sunyo Jung
a,*, Jin Seog Kim
a, Kwang Yun Cho
a, Gun Sik Tae
b, Bin G. Kang
caScreening Research Di6ision,Korea Research Institute of Chemical Technology,P.O.Box107,Yusung,Taejon305-600,South Korea bDepartment of Biology,Dankook Uni6ersity,Chonan330-714,South Korea
cDepartment of Biology,Yonsei Uni6ersity,Seoul120-749,South Korea
Received 3 May 1999; received in revised form 8 November 1999; accepted 3 December 1999
Abstract
Photooxidative damage is exacerbated by norflurazon (NF), which blocks carotenoid biosynthesis. This study examined the influence of photosynthetic photon flux density (PPFD) on the overall responses of both non-enzymatic and enzymatic antioxidants to NF-caused oxidative damage in leaves of cucumber (Cucumis sati6us). Seven-day-old cucumber plants were exposed to NF under either low PPFD (30mmol m−2s−1) or high PPFD (300mmol m−2s−1) for 3 days. The NF plants exposed at high PPFD had lower levels ofFv/Fmratio, quantum yield of electron transport, and 33-kDa protein of photosystem II as compared with the NF plants at low PPFD. In the NF plants, there was a reduction in total chlorophylls and carotenoids except newly formed zeaxanthin in either PPFD. The NF plants at high PPFD resulted in less level of photochemical quenching,qP, and Stern – Volmer quenching, NPQ, than those of the plants at low PPFD, whereas both plants had similar level of non-photochem-ical quenching coefficient, qN. However, the level of PPFD did not significantly affect the NF-caused induction of antioxidant enzymes including peroxidase, superoxide dismutase, glutathione reductase, and ascorbate peroxidase. © 2000 Elsevier Science Ireland Ltd. All rights reserved.
Keywords:Antioxidant enzymes; Cucumber (Cucumis sati6us); Non-photochemical quenching; Norflurazon; Oxidative stress; Xanthophylls www.elsevier.com/locate/plantsci
1. Introduction
Photoinhibition and photooxidation can occur when plants are exposed to stress. High light in synergy with other stress factors such as chilling, drought, or low carbon dioxide supply reduces the capacity of photosynthetic systems to utilize inci-dent radiation, leading to a photoinhibition pro-cess [1,2]. The photosynthetic electron transport system is the major source of active oxygen species (AOS) in plant tissues [3], having the potential to generate singlet oxygen (1O
2) and superoxide (O2−),
which is favored under downregulation of
metabolic pathways. Photosystem (PS) II has long been considered the primary target for photoinhi-bition [1,2] because PSI is more stable than PSII during strong light treatments [4]. Light-induced inactivation of PSI is suggested to be caused by AOS [5,6]. AOS are eliminated efficiently by an integrated system of non-enzymatic and enzymatic antioxidants that are concentrated in the chloro-plast [3]. The capacity of the antioxidative defense system is increased under adverse stress conditions but the imbalance between AOS production and antioxidant defenses ultimately leads to oxidative damage.
The non-enzymatic reductants consist of ascor-bate, glutathione, a-tocopherol, caroteniods, and phenolic compounds [7,8]. Among those, xantho-phyll cycle-dependent energy dissipation in the
* Corresponding author. Present address: Department of Genetics, North Carolina State University, Raleigh, NC 27695-7614, USA. Tel.: +1-919-5155819; fax:+1-919-5153355.
E-mail address:sjung2@unity.ncsu.edu (S. Jung)
0168-9452/00/$ - see front matter © 2000 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0 1 6 8 - 9 4 5 2 ( 9 9 ) 0 0 2 5 9 - 9
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light-harvesting antennae is thought to play an important photoprotective role by mitigating oxi-dative damage. Non-radiative energy dissipation at PSII is mediated by zeaxanthin and perhaps also by antheraxanthin [9] and is proposed to occur at several sites within or around the PSII reaction center [10]. Researchers have used differ-ent terms for energy dissipation, e.g. the quench-ing coefficient qN [11] versus Stern – Volmer
quenching [9,12] that is referred to as non-photo-chemical quenching (NPQ).
In the most powerful source of AOS, chloro-plasts, O2− that is produced by photoreduction of
O2 at PSI and PSII is detoxified by the
Mehler-peroxidase pathway [3,13]. The O2− is reduced to
hydrogen peroxide (H2O2) by superoxide
dismu-tase (SOD) and then to H2O by ascorbate
peroxi-dase (APX), which is the key enzyme involved in H2O2 scavenging. These enzymes, together with
monodehydroascorbate reductase, dehydroascor-bate reductase, and glutathione reductase (GR), constitute the major defense system against AOS in the chloroplast [3,14]. Additionally, extraplastic H2O2 quenching by peroxidase (POD) and
cata-lase (CAT) is also increased in stress responses [15]. A crucial role of these enzymes in protection against oxidative processes has been shown in transgenic tobacco plants overexpressing either Mn-SOD or Fe-SOD [16,17]. In contrast, other studies with transgenic plants suggest that en-hancement of a particular antioxidant enzyme does not lead to increased protection [18,19].
Photooxidative damage is exacerbated by herbi-cides, which generate AOS either by direct in-volvement in radical production or by inhibition of biosynthetic pathways [20,21], as well as atmo-spheric pollutants and heavy metals [8]. Enhanced activities of antioxidants were associated with re-sistance to herbicides such as paraquat and oxyfluorfen [22,23]. In the present study a potent
herbicide norflurazon (NF), which blocks
carotenoid biosynthesis by non-competitively
binding to phytoene desaturase [24], was used in leaves of Cucumis sati6us. It eliminates important quenchers of the triplet chlorophyll (Chl) and1O
2,
thus initiating photooxidative processes. To assess contribution of each inductive response of antioxi-dant to overall protective strategies to NF-caused oxidative damage, photochemical efficiency of PSII, composition of photosynthetic pigments, quenching parameters and activities of antioxidant
enzymes were determined. The questions of whether the level of photosynthetic photon flux density (PPFD) influences the NF-caused oxida-tive stress was also examined, and, if so, whether the NF plants at different PPFDs have different capacities to develop antioxidant responses.
2. Materials and methods
2.1. Plant material and growth conditions
Cucumber seeds (C.sati6usL. cv Summer Long) were sown in vermiculite and transferred after 4 days into synthetic soil in plastic pots. Plants were grown in a controlled environment growth cham-ber under a temperature of 25°C, a 16-h photope-riod, and a light intensity of 200 mmol m−2 s−1
for 3 days. For NF treatment the 7-day-old plants were exposed in a surface application to 15 mM. When the treatment was initiated the first leaves were about to emerge. Following NF application plants were immediately returned to the growth chamber and exposed for 3 days under a 16-h photoperiod with an irradiance of either low PPFD (30 mmol m−2 s−1) or high PPFD (300 mmol m−2 s−1) at 25°C. Inhibition of carotenoid
biosynthesis causes a characteristic bleaching of newly developed leaves. The four treatments em-ployed were: (1) CH, control/high PPFD; (2) NH, NF treatment/high PPFD; (3) CL, control/low PPFD; and (4) NL, NF treatment/low PPFD. The first leaves were used for the measurements of Chl fluorescence, pigment contents and enzyme activi-ties. The experiments were triplicated each with three determinations.
2.2. Chl a fluorescence measurements
In vivo Chla fluorescence was measured after 5 min dark-adaptation at room temperature using a pulse amplitude modulation fluorometer
(PAM-2000, Walz, Effeltrich, Germany). Minimal
fluorescence yield, F0, was obtained upon
excita-tion with a weak measuring beam from a pulse light-emitting diode. Maximal fluorescence yield,
Fm, was determined after exposure to a saturating
pulse of white light to close all reaction centers. Determination of the quenching components qP
and qN was conducted by the saturation pulse
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Schreiber et al. [25]. The quantum yield of electron transport through PSII (Y=DF/Fm%) was
calcu-lated according to Genty et al. [26].
Non-photo-chemical fluorescence quenching was also
quantified, as previously done by Bilger and Bjo¨rkman [12] according to the Stern – Volmer equation, NPQ=Fm/Fm% −1, where Fm% is the
low-ered maximal yield during illumination with pho-tosynthetically active radiation.
2.3. Immunoblot analysis
For the immunoblot of the extrinsic 33-kDa protein of the oxygen-evolving complex in PSII reaction center, the method of Tae et al. [27] was used. The thylakoid membranes isolated from chloroplasts were resuspended in 10 mM NaCl, 50 mM sucrose, and 50 mM sodium phosphate buffer, pH 7.4 and were sedimented at 10 000×g
for 10 min. The pellets were resuspended in the same buffer as mentioned above. The chlorophylls were removed with 80% acetone and the protein pellets were solubilized (1% SDS, 8 M urea, 1% 2-mercaptoethanol, 10.7 mM phosphoric acid). The protein concentrations were measured with the UV spectrophotometric method. Samples of protein to be blotted were electrophoresed in 12% SDS-polyacrylamide gel. The gel was run in 192 mM Glycine, 0.01% (w/v) SDS, and 25 mM Tris – HCl, pH 8.3. Polypeptides were transferred to nitrocellulose paper (pore size: 0.45 mm; Hybond-C, Amersham) with a semi-dry transfer blotter (130-mA constant current, 60 min) (Model TE70, Hoefer Scientific Instruments). The paper was washed in TBS buffer (500 mM NaCl, 20 mM Tris – HCl, pH 7.4), incubated in a sealed plastic bag with 10% milk casein on a rocking shaker (RK1020) for 2 h, removed from the bag, and washed in TBS buffer. After incubating with the antibody in TBS buffer containing 3% bovine serum albumin (BSA) for 2 h, and washing in TTBS buffer containing 0.05% Tween-20, 500 mM NaCl, 20 mM Tris – HCl, pH 7.4, the paper was again incubated with a second antibody [goat anti-rabbit IgG conjugated with horseradish peroxidase (Bio-Rad)] in TBS containing 3% BSA. The paper was incubated on a rocking shaker for 1 h, washed in TTBS buffer, and stained for 10 min with 0.017% 4-chloro-1-naphthol and hydrogen perox-ide in TBS buffer.
2.4. Pigment extraction and analysis
Extraction and HPLC anaysis of carotenoids and Chls were done as described previously [28]. 2.5. Extraction of soluble protein
Frozen leaves (0.25 g for CAT, GR, and APX; 0.5 g for POD and SOD) were crushed to fine powder in a mortar under liquid N2. Soluble
proteins were extracted by homogenizing the pow-der in 2 ml of 100 mM potassium phosphate buffer, pH 7.5, containing 2 mM EDTA, 1% PVP-40, and 1 mM PMSF. For analysis of APX, the extraction buffer also contained 5 mM ascor-bate. Insoluble material was removed by centrifu-gation at 15 000×g for 20 min at 4°C, and the supernatant was filtered through filter papers No. 1 (Whatman, Maidstone, UK). Since maintenance of consistent CAT electrophoretic mobility and GR activity was found to require the presence of DTT, an aliquot of each sample was made to 10 mM DTT to be used for CAT and GR zy-mograms and GR spectrometric assays. For the spectrophotometric assay of SOD, extracts were passed through a PD-10 column (Pharmacia, Upp-sala, Sweden).
2.6. Enzyme assays
CAT activity was determined by using a Clark-type oxygen electrode (Rank Brothers, Cambridge, UK) according to the method of Natvig [29]. The CAT assay was performed in a 3 ml volume containing N2-bubbled 50 mM potassium
phos-phate buffer, pH 7.0, containing 20 mM H2O2.
POD activity was determined specifically with gua-iacol at 470 nm (o=25.2 mM cm−1) following the method of Egley et al. [30]. The reaction mixture contained 40 mM potassium phosphate buffer (pH 6.9), 1.5 mM guaiacol, and 6.5 mM H2O2 in a
3-ml volume. SOD activity was determined as described by Spychalla and Desborough [31]. The assay was performed at 25°C in a 3-ml volume containing 50 mM Na2CO3/NaHCO3 buffer (pH
10.2), 0.1 mM EDTA, 0.015 mM ferricytochrome
C, and 0.05 mM xanthine. APX activity was mea-sured spectrophotometrically by monitoring the decline in A290 as ascorbate (o=2.8 mM cm−1)
was oxidized, using the method of Chen and As-ada [32]. The 3-ml reaction volume contained 100
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mM potassium phosphate buffer (pH 7.5), 0.5 mM ascorbate, and 0.2 mM H2O2at 25°C. GR activity
was measured spectrophotometrically by measur-ing the decline in A340 as NADPH (o=6.2 mM
cm−1) was oxidized, as described by Rao et al.
[33]. The 3-ml assay mixture contained 100 mM potassium phosphate buffer (pH 7.8), 2 mM EDTA, 0.2 mM NADPH, 0.5 mM GSSG, and the leaf extract. The assays were initiated by the addi-tion of NADPH at 25°C.
2.7. Nati6e PAGE and acti6ity staining
Equal amounts of protein from plants exposed to different treatments were subjected to 10% non-denaturing polyacrylamide gels at 4°C for 1.5 h with a constant current of 30 mA. After comple-tion of electrophoresis the gels were stained for the enzymatic activities. Catalase activity was detected by incubating the gels in 3.27 mM H2O2 for 25
min, rinsed in water, and stained in a solution of 1% potassium ferricyanide and 1% ferric chloride for 4 min [34]. Staining of POD isozymes was achieved by incubating the gels in sodium citrate buffer, pH 5.0, containing 9.25 mM p -phenylene-diamine and 3.92 mM H2O2 for 15 min [35]. Gels
were stained for SOD isoforms by soaking in 50 mM potassium phosphate, pH 7.8, containing 2.5 mM nitroblue tetrazolium in darkness for 25 min,
followed by soaking in 50 mM potassium phos-phate, pH 7.8, containing 28 mM nitroblue tetra-zolium and 28 mM riboflavin in darkness for 30 min [33]. Gels were then exposed to light for approximately 30 min. Following separation of APX, gels were soaked in 50 mM potassium phos-phate buffer, pH 7.0, containing 2 mM ascorbate for 30 min [33]. The gels were incubated in the same buffer containing 4 mM ascorbate and 2 mM H2O2for 20 min, and then soaked in 50 mM
potassium phosphate buffer, pH 7.8, containing 28 mM tetramethyl ethylene diamine and 2.45 mM nitroblue tetrazolium for 15 min. Gels were stained for GR activity in a solution of Tris – HCl, pH 7.5, containing 10 mg of 3-(4,5-dimethylthia-zol-2-4)-2,5-diphenyl tetrazolium bromide, 10 mg of 2,6-dichlorophenolindophenol, 3.4 mM GSSG, and 0.5 mM NADPH in darkness for 1 h [33].
3. Results
3.1. Chl a fluorescence during photooxidati6e stress
To confirm the involvement of PSII in the oxi-dative stress responses, the values of photosyn-thetic parameters were determined. Exposure of 7-day-old cucumber plants to NF caused substan-tial photoinhibition of photosynthesis, as indicated by the decline in the photochemical efficiency of photosynthesis. A significant drop in the Fv/Fm
ratio and quantum yield of electron transport through PSII was detected in both NF-treated plants, with almost no quantum yield in NH plants (Table 1). Control plants at either PPFD showed similar values ofFv/Fmand quantum yield.
The initial fluorescenceF0was the same in CH and
CL plants (Table 1). NH plants exhibited a lower
F0 in contrast to a greater F0 in NL plants,
com-pared with control plants. 3.2. Quantification of PSII
Quantification of the PSII reaction center protein 33-kDa following NF exposure showed pronounced differences among treatments (Fig. 1). The 33-kDa protein level of CH plants was greater than that of CL plants. Exposure of leaves to NF caused the loss of PSII reaction centers, as indi-cated by the loss of the extrinsic 33-kDa protein of
Table 1
Photosynthetic parameters measured in leaves exposed to norflurazon (NF) under different photosynthetic photon flux densities (PPFDs)a
Photosynthetic parameters Treatment
F0 Quantum yield
Fv/Fm
0.7790.00c 0.6690.00c 0.3990.00b CH
0.2290.01a 0.0390.01a
0.1190.03a NH
0.7890.00c 0.7290.00c 0.4090.01b CL
0.4190.04b 0.3190.04b
NL 0.5490.02c
aThe plants were grown in 200mmol m−2s−1with a 16-h photoperiod. The 7-day-old plants were sprayed with 15mM NF and exposed to either high PPFD (300mmol m−2s−1) or low PPFD (30mmol m−2s−1). Treatment notations indicate the presence/absence of NF and PPFD level in order. The
Fv/Fm ratio, quantum yield of photosystem (PS) II electron transport, andF0were determined. Measurements were made 3 days after NF treatment. Within each column, means followed by the same letter are not significantly different (PB0.01) using Fisher’s protected LSD test. Data represent the mean9S.E. of three replicates.
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Fig. 1. Influence of norflurazon (NF) and photosynthetic photon flux density (PPFD) on the extrinsic 33-kDa protein of oxygen evolving complex in photosystem (PS) II reaction center. The plants were subjected to the same treatments as in Table 1. Treatment notations are the same as in Table 1. M, a 30-kDa molecular weight marker. Measurements were made 3 days after NF treatment. Data represent the mean9S.E. of three replicates.
Table 2
Effects of norflurazon (NF) exposure at different photosynthetic photon flux densities (PPFDs) on carotenoids (mmol mol−1Chl
a) and Chl pigments (mg g FW−1) ofC.sati6usa
Treatment Pigments
NH CL
CH NL
17.091.4 (4.3) 24.391.2 (92.4)
25.290.7 (99.2) 15.891.5 (15.0)
Neoxanthin
45.892.3 (179.8)
Violaxanthin 60.290.3 (15.1) 26.791.2 (101.7) 16.992.2 (16.0)
7.690.2 (1.9) ND
Antheraxanthin 2.790.2 (10.6) 3.390.5 (3.2)
69.493.4 (17.4) 48.892.3 (185.8)
58.792.3 (231.1) 41.391.5 (39.5)
Lutein
ND
Zeaxanthin 7.891.2 (2.0) ND 6.290.3 (5.9)
b-Carotene 48.198.0 (186.6) 2.791.0 (0.7) 41.192.5 (156.5) 18.991.4 (18.1) 75.691.6 (19.0) 26.791.2 (101.7)
48.492.4 (190.5) 26.491.6 (25.1)
V+A+Z
Chla+b 1779.89162.9 112.599.5 1642.6926.1 387.9915.9
aThe values in parentheses indicate the pigment contents on nmol per g fresh weight basis. The plants were subjected to the
same treatments as in Table 1. Treatment notations are the same as in Table 1. Measurements were made 3 days after NF treatment. ND, not detected. Data represent the mean9S.E. of three replicates.
the oxygen-evolving complex, with a greater mag-nitude in NH plants.
3.3. Composition of photosynthetic pigments
Table 2 shows that there was a pronounced decline in total Chls with NF, especially under high PPFD. The carotenoid content per unit of Chl a was determined in cucumber plants upon exposure to NF that can produce oxidative stress (Table 2). In CL plants there was an approxi-mately 45% decline in violaxanthin+ antheraxan-thin+zeaxanthin and also a slight decline in lutein and b-carotene, compared with CH plants, with no difference between the two controls in the quantity of neoxanthin. NH plants had increases in violaxanthin, antheraxanthin, lutein and anthin compared with CH plants, especially zeax-anthin newly formed, coinciding with a drastic
decline in b-carotene. NL plants resulted in lower levels of the xanthophyll cycle pigments and lutein but a higher b-carotene than those of NH plants. However, NL plants also caused a great increase in antheraxanthin and zeaxanthin.
Carotenoid contents on a fresh weight basis were also examined (Table 2). Pigment levels of control plants under both PPFDs were very simi-lar to the profiles of pigments on a Chl a basis. Under high PPFD, NF decreased drastically all pigments except zeaxanthin newly formed. In con-trast to Chl a basis, carotenoid levels of NL plants were much greater than those of NH plants when indicated on a fresh weight basis. Particularly, NL plants contained 3-fold more zeaxanthin than NH plants. In low PPFD, an-theraxanthin and zeaxanthin were greater in NF plants compared with control, but other pigments were lower.
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Fig. 2. Levels of qP, qN and non-photochemical quenching (NPQ) in leaves exposed to norflurazon (NF) under different photosynthetic photon flux densities (PPFDs). The plants were subjected to the same treatments as in Table 1. Treat-ment notations are the same as in Table 1. MeasureTreat-ments were made 3 days after NF treatment. Data represent the mean9S.E. of three replicates. In some cases the error bar is obscured by the symbol.
3.5. Responses of antioxidant enzyme acti6ities The influence of NF on the activities of antioxi-dant enzymes participating in the scavenging of oxidative stress is shown in Table 3. In addition to the three primary enzymes of the Mehler-peroxi-dase pathway (SOD, GR and APX), we measured activities of CAT and guaiacol-POD. Activity of CAT was much lower in NH plants relative to the other plants, whereas POD and SOD activities in NH, CL, and NL plants were greater when com-pared with CH plants. APX activity exhibited a considerable increase in relation to NF, but only slight increase in control plants grown at low PPFD. Neither NF nor PPFD level resulted in a
discernible change in GR activity among
treatments.
The isoform composition of different enzymes was analyzed by native PAGE. Gels stained for CAT revealed no isoform (Fig. 3A), and were in accordance with the induction profile of CAT specific activity, with the lowest activity in NH plants. POD and SOD exist as multiple isoforms (Figs. 3B and 4A). In the profile of POD activity in control plants, four bands (1, 2, 3 and 4) were detected, with staining intensity increased both with NF and with low PPFD (Fig. 3B, lanes b, c and d). NF induced a new band, which has a quite strong staining intensity (Fig. 3B, lanes b and d). The induction of POD isoform 5 was quite NF-specific. Three isoforms were observed when na-tive gels were stained for SOD activity, and no major change was observed in the activity of iso-form 3 in the leaves treated with NF (Fig. 4A). SOD isoform 1 was identified as Mn-SOD by its insensitivity to KCN and H2O2, whereas SOD
isoforms 2 and 3 were inhibited by both KCN and H2O2, suggesting that they represented
Cu,Zn-3.4. Photochemical quenching and energy dissipation
The photochemical quenching (qP), an estimate
of the fraction of open PSII centers, was lower in NF plants, with a greater magnitude of decline in NH plants, compared with control plants (Fig. 2). The lowest value ofqNwas observed in CL plants.
NL plants exhibited much greater level of NPQ than that of NH plants whereas both plants had similar level of highqN. A considerable amount of
NPQ existed in control plants at high PPFD, compared with ones at low PPFD. The combined quenching of qP, qN and NPQ was the lowest in
NH plants.
Table 3
Effects of norflurazon (NF) exposure at different photosynthetic photon flux densities (PPFDs) on antioxidant enzyme activities, catalase (CAT) (mmol O2min−1mg−1protein), SOD (units mg−1protein) and other enzymes (mmol min−1mg−1protein)a Treatment Enzyme activities
APX SOD
POD
CAT GR
0.06390.002 1.0490.02
CH 84.591.7 0.6790.08 0.10090.001
0.4890.01 0.17090.003
NH 137.5916.1 1.0690.02 0.09890.003
0.7590.05
1.0090.06 0.14390.009 128.898.0 0.10590.002
CL
0.9890.06
1.0590.02 0.16490.009 124.9911.8 0.10590.003
NL
aThe plants were subjected to the same treatments as in Table 1. Treatment notations are the same as in Table 1. Measurements were made 3 days after NF treatment. Data represent the mean9S.E. of three replicates.
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Fig. 3. Influence of norflurazon (NF) and photosynthetic photon flux density (PPFD) on the isozyme profiles of cata-lase (CAT) (A) and peroxidase (POD) (B) ofC.sati6us. The plants were subjected to the same treatments as in Table 1. Non-denaturing activity gels were prepared and run as de-scribed in Section 2. Lane a, CH; lane b, NH; lane c, CL; lane d, NL. Treatment notations are the same as in Table 1.
photodestruction of plastid components in the deficiency of protecting carotenoids. Both Fv/Fm
ratio and quantum yield were significantly differ-ent over high and low PPFDs by NF treatmdiffer-ents, indicating that the level of PPFD affects structural and functional modifications of the photosynthetic
Fig. 4. Influence of norflurazon (NF) and photosynthetic photon flux density (PPFD) on the isozyme profiles of super-oxide dismutase (SOD) (A), ascorbate peroxidase (APX) (B), and glutathione reductase (GR) (C) ofC.sati6us. The plants
were subjected to the same treatments as in Table 1. Non-de-naturing activity gels were prepared and run as described in Section 2. Incubation of gels with KCN or H2O2 prior to staining SOD activity suggested that SOD-1 is Mn-SOD, whereas SOD-2 and -3 are Cu,Zn-SOD (data not shown). Gels stained for GR activity revealed a GSSH-specific band (GR-1) and a non-specific GR band (arrowhead). Lane a, CH; lane b, NH; lane c, CL; lane d, NL. Treatment notations are the same as in Table 1.
SOD activity. No Fe-SOD isoform was observed in all treatments. Both NF and low PPFD en-hanced the intensity of the existing isoform 2, and Mn-SOD also increased slightly. The overall in-crease of total SOD activity in NF plants and CL plants may result from the increase of isozyme 2. NF enhanced significantly the intensity of the existing APX compared with CH plants, with maximum activity in NH plants (Fig. 4B). How-ever, low PPFD slightly increased the intensity of APX in control plants. A GSSG-specific GR band was present inC. sati6us(Fig. 4C). The intensities of GR in all treatments remained unaffected by NF or PPFD.
4. Discussion
Exposure of C. sati6us to NF substantially in-hibited photochemical efficiency of photosynthe-sis, indicated as a decrease inFv/Fm and quantum
yield of PSII electron transport (Table 1). This inhibition could be explained in large part by the
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apparatus. The relatively small decline in Fv/Fm
and quantum yield in NL plants shows that NF-induced photoinhibition was alleviated during NF exposure in lower PPFD. It has been reported that the decrease inFv/Fm results from change of
reac-tion centers to quenchers by excess light [36]. In NF plants, however, the decrease is probably due to the loss of PSII reaction centers as indicated by the loss of 33-kDa protein of oxygen evolving complex (Fig. 1). The F0 level is known to be
affected by environmental stress that causes struc-tural alterations in the PSII complex [36]. The F0
decrease in NH plants (Table 1) might arise from markedly low level of Chls. On the other hand, the
F0 increase in NL plants is probably associated
not only with low PSII photochemistry (KP) but
also with low KT, which is related to excitation
energy transfer to non-fluorescent pigments, al-though the precise reasons for the decline remain to be determined.
The decrease in Fv/Fm is also due to the
forma-tion of a new quencher of excitaforma-tion energy, the
xanthophyll cycle pigment zeaxanthin. Less
amount of violaxanthin+antheraxanthin+ zeax-anthin accounts for less requirement for qN and
NPQ in CL plants due to low photoinhibitory stress, compared with CH plants (Table 2 and Fig. 2). The treatment with NF caused a significant accumulation of antheraxanthin and zeaxanthin, but a concomitant reduction in b-carotene on a Chl a basis and in Chls with a greater magnitude under high PPFD (Table 2). The profile of pig-ment levels on a fresh weight basis in NF plants was obviously different from that of Chl a basis, with an overall reduction in the other pigments except zeaxanthin. The newly formed zeaxanthin in the presence of carotenoid biosynthesis in-hibitor (Table 2) confirms the possibility that
zeax-anthin plays a role in the photoprotective
mechanism [9,28]. A decline of photochemical effi-ciency in NF plants is associated with a decrease in qPand an increase inqN (Table 1 and Fig. 2). A
lower NPQ of NH plants, as compared with NL plants, exhibited a close correlation with level of antheraxanthin and zeaxanthin on a fresh weight basis, not per Chl a content. Interestingly, the irradiance environment during NF-caused oxida-tive stress dramatically affects zeaxanthin-related NPQ, not qN. In contrast to qN, NPQ is
propor-tional to the effective rate constant for energy dissipation in the antennae as well as the
concen-tration of quenching centers [37]. The results provide evidence that energy dissipation through the xanthophyll cycle does not completely explain for the acclimatory process of the plants to oxida-tive stress caused by NF. Thus the plants necessi-tate other components of dissipative process.
Antioxidant enzymes are likely to play a consid-erable part of defense mechanism against NF-in-duced oxidative stress. CAT takes part in an efficient protective role to oxidative stress [38], however, in this study NH plants exhibited a significant decline in CAT activity (Table 3), con-sistent with reports of CAT inactivation in leaves exposed to high light [39,40]. The increase in the activities of POD, SOD and APX might occur against oxidative stress or serve to compensate for low CAT activity (Table 3). The ascorbate-glu-tathione cycle has been known to be activated under oxidative stress conditions [38]. However, GR, a rate-limiting enzyme in the H2O2
-scaveng-ing cycle [41], was not altered in all treatments by different PPFDs (Table 3), suggesting that the functioning of the cycle was not activated effi-ciently in NF plants. The Mehler-peroxidase path-way has been postulated to cause not only qP, but
also the build-up of DpH and consequent zeaxan-thin formation [11,13]. Considering similar levels of the antioxidant enzymes except CAT in NH and NL plants, the response of these enzymes to NF may not depend on the level of PPFD. The increase of APX activity appears to be NF-depen-dent, but the activities of POD and SOD are considerably increased even in CL plants (Table 3).
The induction of individual isozymes to NF is interpreted as a response to augmented AOS gen-eration. A membrane-bound scavenging system within chloroplasts, that is functionally linked to PSI, has been suggested to intercept the toxic oxygen species very close to the site of formation [16]. NF-induced SOD activity is mainly due to increased expression of Cu,Zn-SOD-2 isoform, and also to Mn-SOD isoform to some extent (Fig. 4A). Cu,Zn-SOD, which is associated with the PSI complex, results in a great increase of its mRNA level upon NF-induced photooxidation [42], and another up-regulated antioxidant, Mn-SOD is sug-gested to confer protection against NF [43]. NF caused also a prominent increase in APX staining activity in NF plants compared with control (Fig. 4B), indicating a possible protective role of these
(9)
enzymes to oxidative stress. Both NF and PPFD exert differential effects on the expression of the multiple forms of POD (Fig. 3B). The NF-treated plants were capable of synthesizing a new isoform of POD, which could be considered as a response to NF-caused oxidative damage. The results sug-gest that enzymatic removal of H2O2by POD and
APX is the dominant pathway during NF-induced enzymatic scavenging system.
In the NF-treated leaves of C. sati6us, the detoxification of AOS is undertaken through func-tionally interrelated antioxidant mechanisms. The
qP and DpH-dependent qN through the
Mehler-peroxidase pathway, in addition to zeaxanthin for-mation, take part in mitigating oxidative stress induced by NF. NF-induced photooxidation in combination with high PPFD causes severe pho-toinhibition, which may arise from an insufficient capacity to dissipate excess excitation energy through qP and NPQ. The plants, however, seem
to develop a component of photoinhibitory quenching, qI, which is related to light-dependent
alterations in the PSII reaction center complex and frequently indicated as a decrease in Fv/Fm [36].
Interestingly, the level of PPFD influences qP and
NPQ in response to NF, not the induction of antioxidant enzymes. This might be explained by the fact that PSI is more stable than PSII upon strong light [4] and that the AOS-scavenging en-zymes of the chloroplast may serve to protect PSI.
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[34] W. Woodbury, A.K. Spencer, M.A. Stahman, An im-proved procedure for using ferricyanide for detecting catalase isozymes, Anal. Biochem. 44 (1971) 301 – 305. [35] P.D. Olson, J.E. Varner, Hydrogen peroxide and
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[37] B. Demmig-Adams, W.W. Adams III, D.H. Barker, B.A. Logan, D.R. Bowling, A.S. Verhoeven, Using chloro-phyll fluorescence to assess the fraction of absorbed light allocated to thermal dissipation of excess excitation, Physiol. Plant 98 (1996) 253 – 264.
[38] A. Chaoui, S. Mazhoudi, M.H. Ghorbal, E.E. Ferjani, Cadmium and zinc induction of lipid peroxidation and effects on antioxidant enzyme activities in bean (Phaseo
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[40] N.P. Mishra, R.K. Mishra, G.S. Singhal, Changes in the activities of antioxidant enzymes during exposure of intact wheat leaves to strong visible light at different temperatures in the presence of different protein synthe-sis inhibitors, Plant Physiol. 102 (1993) 867 – 880. [41] P.P. Jablonski, J.W. Anderson, Light-dependent
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[42] J. Kurepa, D. He´rouart, M.V. Montagu, D. Inze´, Differ-ential expression of CuZn- and Fe-superoxide dismutase genes of tobacco during development, oxidative stress, and hormonal treatments, Plant Cell Physiol. 38 (1997) 463 – 470.
[43] D.J. Thomas, T.J. Avenson, J.B. Thomas, S.K. Herbert, A cyanobacterium lacking iron superoxide dismutase is sensitized to oxidative stress induced with methyl violo-gen but is not sensitized to oxidative stress induced with norflurazon, Plant Physiol. 116 (1998) 1593 – 1602.
(1)
Fig. 1. Influence of norflurazon (NF) and photosynthetic photon flux density (PPFD) on the extrinsic 33-kDa protein of oxygen evolving complex in photosystem (PS) II reaction center. The plants were subjected to the same treatments as in Table 1. Treatment notations are the same as in Table 1. M, a 30-kDa molecular weight marker. Measurements were made 3 days after NF treatment. Data represent the mean9S.E. of three replicates.
Table 2
Effects of norflurazon (NF) exposure at different photosynthetic photon flux densities (PPFDs) on carotenoids (mmol mol−1Chl
a) and Chl pigments (mg g FW−1) ofC.sati6usa Treatment
Pigments
NH CL
CH NL
17.091.4 (4.3) 24.391.2 (92.4)
25.290.7 (99.2) 15.891.5 (15.0)
Neoxanthin
45.892.3 (179.8)
Violaxanthin 60.290.3 (15.1) 26.791.2 (101.7) 16.992.2 (16.0)
7.690.2 (1.9) ND
Antheraxanthin 2.790.2 (10.6) 3.390.5 (3.2)
69.493.4 (17.4) 48.892.3 (185.8)
58.792.3 (231.1) 41.391.5 (39.5)
Lutein
ND
Zeaxanthin 7.891.2 (2.0) ND 6.290.3 (5.9)
b-Carotene 48.198.0 (186.6) 2.791.0 (0.7) 41.192.5 (156.5) 18.991.4 (18.1) 75.691.6 (19.0) 26.791.2 (101.7)
48.492.4 (190.5) 26.491.6 (25.1)
V+A+Z
Chla+b 1779.89162.9 112.599.5 1642.6926.1 387.9915.9
aThe values in parentheses indicate the pigment contents on nmol per g fresh weight basis. The plants were subjected to the same treatments as in Table 1. Treatment notations are the same as in Table 1. Measurements were made 3 days after NF treatment. ND, not detected. Data represent the mean9S.E. of three replicates.
the oxygen-evolving complex, with a greater
mag-nitude in NH plants.
3.3.
Composition of photosynthetic pigments
Table 2 shows that there was a pronounced
decline in total Chls with NF, especially under
high PPFD. The carotenoid content per unit of
Chl
a
was determined in cucumber plants upon
exposure to NF that can produce oxidative stress
(Table 2). In CL plants there was an
approxi-mately 45% decline in violaxanthin
+
antheraxan-thin
+
zeaxanthin and also a slight decline in
lutein and
b
-carotene, compared with CH plants,
with no difference between the two controls in the
quantity of neoxanthin. NH plants had increases
in violaxanthin, antheraxanthin, lutein and
anthin compared with CH plants, especially
zeax-anthin newly formed, coinciding with a drastic
decline in
b
-carotene. NL plants resulted in lower
levels of the xanthophyll cycle pigments and
lutein but a higher
b
-carotene than those of NH
plants. However, NL plants also caused a great
increase in antheraxanthin and zeaxanthin.
Carotenoid contents on a fresh weight basis
were also examined (Table 2). Pigment levels of
control plants under both PPFDs were very
simi-lar to the profiles of pigments on a Chl
a
basis.
Under high PPFD, NF decreased drastically all
pigments except zeaxanthin newly formed. In
con-trast to Chl
a
basis, carotenoid levels of NL
plants were much greater than those of NH
plants when indicated on a fresh weight basis.
Particularly, NL plants contained 3-fold more
zeaxanthin than NH plants. In low PPFD,
an-theraxanthin and zeaxanthin were greater in NF
plants compared with control, but other pigments
were lower.
(2)
Fig. 2. Levels of qP, qN and non-photochemical quenching
(NPQ) in leaves exposed to norflurazon (NF) under different photosynthetic photon flux densities (PPFDs). The plants were subjected to the same treatments as in Table 1. Treat-ment notations are the same as in Table 1. MeasureTreat-ments were made 3 days after NF treatment. Data represent the mean9S.E. of three replicates. In some cases the error bar is obscured by the symbol.
3.5.
Responses of antioxidant enzyme acti
6
ities
The influence of NF on the activities of
antioxi-dant enzymes participating in the scavenging of
oxidative stress is shown in Table 3. In addition to
the three primary enzymes of the
Mehler-peroxi-dase pathway (SOD, GR and APX), we measured
activities of CAT and guaiacol-POD. Activity of
CAT was much lower in NH plants relative to the
other plants, whereas POD and SOD activities in
NH, CL, and NL plants were greater when
com-pared with CH plants. APX activity exhibited a
considerable increase in relation to NF, but only
slight increase in control plants grown at low
PPFD. Neither NF nor PPFD level resulted in a
discernible
change
in
GR
activity
among
treatments.
The isoform composition of different enzymes
was analyzed by native PAGE. Gels stained for
CAT revealed no isoform (Fig. 3A), and were in
accordance with the induction profile of CAT
specific activity, with the lowest activity in NH
plants. POD and SOD exist as multiple isoforms
(Figs. 3B and 4A). In the profile of POD activity
in control plants, four bands (1, 2, 3 and 4) were
detected, with staining intensity increased both
with NF and with low PPFD (Fig. 3B, lanes b, c
and d). NF induced a new band, which has a quite
strong staining intensity (Fig. 3B, lanes b and d).
The induction of POD isoform 5 was quite
NF-specific. Three isoforms were observed when
na-tive gels were stained for SOD activity, and no
major change was observed in the activity of
iso-form 3 in the leaves treated with NF (Fig. 4A).
SOD isoform 1 was identified as Mn-SOD by its
insensitivity to KCN and H
2O
2, whereas SOD
isoforms 2 and 3 were inhibited by both KCN and
H
2O
2, suggesting that they represented
Cu,Zn-3.4.
Photochemical quenching and energy
dissipation
The photochemical quenching (
q
P), an estimate
of the fraction of open PSII centers, was lower in
NF plants, with a greater magnitude of decline in
NH plants, compared with control plants (Fig. 2).
The lowest value of
q
Nwas observed in CL plants.
NL plants exhibited much greater level of NPQ
than that of NH plants whereas both plants had
similar level of high
q
N. A considerable amount of
NPQ existed in control plants at high PPFD,
compared with ones at low PPFD. The combined
quenching of
q
P,
q
Nand NPQ was the lowest in
NH plants.
Table 3
Effects of norflurazon (NF) exposure at different photosynthetic photon flux densities (PPFDs) on antioxidant enzyme activities, catalase (CAT) (mmol O2min−1mg−1protein), SOD (units mg−1protein) and other enzymes (mmol min−1mg−1protein)a
Treatment Enzyme activities
APX SOD
POD
CAT GR
0.06390.002 1.0490.02
CH 84.591.7 0.6790.08 0.10090.001
0.4890.01 0.17090.003
NH 137.5916.1 1.0690.02 0.09890.003
0.7590.05
1.0090.06 0.14390.009 128.898.0 0.10590.002
CL
0.9890.06
1.0590.02 0.16490.009 124.9911.8 0.10590.003
NL
aThe plants were subjected to the same treatments as in Table 1. Treatment notations are the same as in Table 1. Measurements
(3)
Fig. 3. Influence of norflurazon (NF) and photosynthetic photon flux density (PPFD) on the isozyme profiles of cata-lase (CAT) (A) and peroxidase (POD) (B) ofC.sati6us. The plants were subjected to the same treatments as in Table 1. Non-denaturing activity gels were prepared and run as de-scribed in Section 2. Lane a, CH; lane b, NH; lane c, CL; lane d, NL. Treatment notations are the same as in Table 1.
photodestruction of plastid components in the
deficiency of protecting carotenoids. Both
F
v/
F
mratio and quantum yield were significantly
differ-ent over high and low PPFDs by NF treatmdiffer-ents,
indicating that the level of PPFD affects structural
and functional modifications of the photosynthetic
Fig. 4. Influence of norflurazon (NF) and photosynthetic photon flux density (PPFD) on the isozyme profiles of super-oxide dismutase (SOD) (A), ascorbate peroxidase (APX) (B), and glutathione reductase (GR) (C) ofC.sati6us. The plants were subjected to the same treatments as in Table 1. Non-de-naturing activity gels were prepared and run as described in Section 2. Incubation of gels with KCN or H2O2 prior to
staining SOD activity suggested that SOD-1 is Mn-SOD, whereas SOD-2 and -3 are Cu,Zn-SOD (data not shown). Gels stained for GR activity revealed a GSSH-specific band (GR-1) and a non-specific GR band (arrowhead). Lane a, CH; lane b, NH; lane c, CL; lane d, NL. Treatment notations are the same as in Table 1.
SOD activity. No Fe-SOD isoform was observed
in all treatments. Both NF and low PPFD
en-hanced the intensity of the existing isoform 2, and
Mn-SOD also increased slightly. The overall
in-crease of total SOD activity in NF plants and CL
plants may result from the increase of isozyme 2.
NF enhanced significantly the intensity of the
existing APX compared with CH plants, with
maximum activity in NH plants (Fig. 4B).
How-ever, low PPFD slightly increased the intensity of
APX in control plants. A GSSG-specific GR band
was present in
C
.
sati
6
us
(Fig. 4C). The intensities
of GR in all treatments remained unaffected by
NF or PPFD.
4. Discussion
Exposure of
C
.
sati
6
us
to NF substantially
in-hibited photochemical efficiency of
photosynthe-sis, indicated as a decrease in
F
v/
F
mand quantum
yield of PSII electron transport (Table 1). This
inhibition could be explained in large part by the
(4)
apparatus. The relatively small decline in
F
v/
F
mand quantum yield in NL plants shows that
NF-induced photoinhibition was alleviated during NF
exposure in lower PPFD. It has been reported that
the decrease in
F
v/
F
mresults from change of
reac-tion centers to quenchers by excess light [36]. In
NF plants, however, the decrease is probably due
to the loss of PSII reaction centers as indicated by
the loss of 33-kDa protein of oxygen evolving
complex (Fig. 1). The
F
0level is known to be
affected by environmental stress that causes
struc-tural alterations in the PSII complex [36]. The
F
0decrease in NH plants (Table 1) might arise from
markedly low level of Chls. On the other hand, the
F
0increase in NL plants is probably associated
not only with low PSII photochemistry (
K
P) but
also with low
K
T, which is related to excitation
energy transfer to non-fluorescent pigments,
al-though the precise reasons for the decline remain
to be determined.
The decrease in
F
v/
F
mis also due to the
forma-tion of a new quencher of excitaforma-tion energy, the
xanthophyll
cycle
pigment
zeaxanthin.
Less
amount of violaxanthin
+
antheraxanthin
+
zeax-anthin accounts for less requirement for
q
Nand
NPQ in CL plants due to low photoinhibitory
stress, compared with CH plants (Table 2 and Fig.
2). The treatment with NF caused a significant
accumulation of antheraxanthin and zeaxanthin,
but a concomitant reduction in
b
-carotene on a
Chl
a
basis and in Chls with a greater magnitude
under high PPFD (Table 2). The profile of
pig-ment levels on a fresh weight basis in NF plants
was obviously different from that of Chl
a
basis,
with an overall reduction in the other pigments
except zeaxanthin. The newly formed zeaxanthin
in the presence of carotenoid biosynthesis
in-hibitor (Table 2) confirms the possibility that
zeax-anthin
plays
a
role
in
the
photoprotective
mechanism [9,28]. A decline of photochemical
effi-ciency in NF plants is associated with a decrease
in q
Pand an increase in
q
N(Table 1 and Fig. 2). A
lower NPQ of NH plants, as compared with NL
plants, exhibited a close correlation with level of
antheraxanthin and zeaxanthin on a fresh weight
basis, not per Chl
a
content. Interestingly, the
irradiance environment during NF-caused
oxida-tive stress dramatically affects zeaxanthin-related
NPQ, not
q
N. In contrast to
q
N, NPQ is
propor-tional to the effective rate constant for energy
dissipation in the antennae as well as the
concen-tration of quenching centers [37]. The results
provide evidence that energy dissipation through
the xanthophyll cycle does not completely explain
for the acclimatory process of the plants to
oxida-tive stress caused by NF. Thus the plants
necessi-tate other components of dissipative process.
Antioxidant enzymes are likely to play a
consid-erable part of defense mechanism against
NF-in-duced oxidative stress. CAT takes part in an
efficient protective role to oxidative stress [38],
however, in this study NH plants exhibited a
significant decline in CAT activity (Table 3),
con-sistent with reports of CAT inactivation in leaves
exposed to high light [39,40]. The increase in the
activities of POD, SOD and APX might occur
against oxidative stress or serve to compensate for
low CAT activity (Table 3). The
ascorbate-glu-tathione cycle has been known to be activated
under oxidative stress conditions [38]. However,
GR, a rate-limiting enzyme in the H
2O
2-scaveng-ing cycle [41], was not altered in all treatments by
different PPFDs (Table 3), suggesting that the
functioning of the cycle was not activated
effi-ciently in NF plants. The Mehler-peroxidase
path-way has been postulated to cause not only
q
P, but
also the build-up of
D
pH and consequent
zeaxan-thin formation [11,13]. Considering similar levels
of the antioxidant enzymes except CAT in NH
and NL plants, the response of these enzymes to
NF may not depend on the level of PPFD. The
increase of APX activity appears to be
NF-depen-dent, but the activities of POD and SOD are
considerably increased even in CL plants (Table
3).
The induction of individual isozymes to NF is
interpreted as a response to augmented AOS
gen-eration. A membrane-bound scavenging system
within chloroplasts, that is functionally linked to
PSI, has been suggested to intercept the toxic
oxygen species very close to the site of formation
[16]. NF-induced SOD activity is mainly due to
increased expression of Cu,Zn-SOD-2 isoform,
and also to Mn-SOD isoform to some extent (Fig.
4A). Cu,Zn-SOD, which is associated with the PSI
complex, results in a great increase of its mRNA
level upon NF-induced photooxidation [42], and
another up-regulated antioxidant, Mn-SOD is
sug-gested to confer protection against NF [43]. NF
caused also a prominent increase in APX staining
activity in NF plants compared with control (Fig.
4B), indicating a possible protective role of these
(5)
enzymes to oxidative stress. Both NF and PPFD
exert differential effects on the expression of the
multiple forms of POD (Fig. 3B). The NF-treated
plants were capable of synthesizing a new isoform
of POD, which could be considered as a response
to NF-caused oxidative damage. The results
sug-gest that enzymatic removal of H
2O
2by POD and
APX is the dominant pathway during NF-induced
enzymatic scavenging system.
In the NF-treated leaves of
C
.
sati
6
us
, the
detoxification of AOS is undertaken through
func-tionally interrelated antioxidant mechanisms. The
q
Pand
D
pH-dependent
q
Nthrough the
Mehler-peroxidase pathway, in addition to zeaxanthin
for-mation, take part in mitigating oxidative stress
induced by NF. NF-induced photooxidation in
combination with high PPFD causes severe
pho-toinhibition, which may arise from an insufficient
capacity to dissipate excess excitation energy
through
q
Pand NPQ. The plants, however, seem
to develop a component of photoinhibitory
quenching,
q
I, which is related to light-dependentalterations in the PSII reaction center complex and
frequently indicated as a decrease in
F
v/
F
m[36].
Interestingly, the level of PPFD influences
q
Pand
NPQ in response to NF, not the induction of
antioxidant enzymes. This might be explained by
the fact that PSI is more stable than PSII upon
strong light [4] and that the AOS-scavenging
en-zymes of the chloroplast may serve to protect PSI.
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