Journal of Photochemistry and Photobiology B: Biology 140 (2014) 286–291
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The mechanism by which NaCl treatment alleviates PSI photoinhibition under chilling-light treatment Yang Cheng a,b, Zhang Zi-shan a, Gao Hui-yuan a,⇑, Fan Xing-li a, Liu Mei-jun a, Li Xiang-dong b a b
State Key Laboratory of Crop Biology, Shandong Key Laboratary of Crop Biology, College of Life Sciences, Shandong Agricultural University, Tai’an 271018, China Wheat Research Institute, Henan Academy of Agricultural Sciences, Zhengzhou, Henan 450002, China
a r t i c l e
i n f o
Article history: Received 1 March 2014 Received in revised form 7 August 2014 Accepted 14 August 2014 Available online 26 August 2014 Keywords: NaCl PSII PSI Photoinhibition Chilling-light
a b s t r a c t The effects of chilling-light stress combined with additional stress on PSI and PSII photoinhibition and their interrelationship have not been known. To explore whether NaCl affects the PSI and PSII photoinhibition and their interrelationship under chilling-light treatment, the PSI and PSII activities were studied under chilling-light with or without NaCl treatment. The results showed that the extent of PSI and PSII photoinhibition both increased under chilling-light, while NaCl aggravated PSII photoinhibition and severely damaged cytochrome b6/f complex but alleviated PSI photoinhibition. Moreover, DCMU had a similar effect as NaCl in this study, which indicates that NaCl alleviated PSI photoinhibition through reducing electrons transported to PSI. It was also showed that the increased damage to PSII by NaCl did not depend on the inhibition of PSII repair and PSI electron transportation. In conclusion, NaCl alleviated PSI photoinhibition by inhibiting electron transport from PSII under chilling-light conditions. In addition, PSII photoinhibition was not affected by PSI photoinhibition because of a full inhibition of PSII repair by chilling-light treatment. We also speculate that NaCl aggravates PSII photoinhibition by enhancing the damage instead of inhibiting the repair of it under chilling-light conditions. Ó 2014 Elsevier B.V. All rights reserved.
1. Introduction Photoinhibition may take place under conditions where the harvested light energy exceeds the requirements to drive photochemical reactions and protective mechanisms become overburdened [1,2]. Photosystem II (PSII) and photosystem I (PSI) are the main target sites of photoinhibition. PSII has long been considered the primary target of photoinhibition [2,3], as photodamage to PSII occurs under light at any intensity and is unavoidable. Photosynthetic organisms are able to overcome photodamage to PSII by rapid and efficient repair of the damage, which requires the synthesis of D1 proteins de novo. The activity of PSII depends on the balance between the rates of photodamage and repair, and the photoinhibition of PSII becomes apparent when the rate of photodamage exceeds the rate of repair [2,4,5]. Some studies revealed that light can damage PSII directly, and other forms of environmental stress act primarily by inhibiting repair of PS II [6,7].
⇑ Corresponding author. Tel.: +86 538 8245985; fax: +86 538 8249608. E-mail addresses: [email protected] (C. Yang), Zhangzishantaian@163. com (Z.-s. Zhang), [email protected] (H.-y. Gao), [email protected] (X.-l. Fan), [email protected] (M.-j. Liu), [email protected] (X.-d. Li). http://dx.doi.org/10.1016/j.jphotobiol.2014.08.012 1011-1344/Ó 2014 Elsevier B.V. All rights reserved.
PSI photoinhibition is rarely observed in vivo because PSI is more stable than PSII under most types of abiotic stress, such as high light [8]. PSI photoinhibition was first reported by Terashima and his colleagues [9]. When cucumber leaves were chilled at 4 °C for 5 h under moderate photon flux density (200 lmol m2 s1), the maximum photosynthetic electron flow through PSI decreased by 70–80%, compared to the controls. In contrast, the maximum electron transport through PSII remained at 80% of that of the controls [9]. Chilling-induced photoinhibition damages the membrane structure and causes degradation of proteins in PSI reaction center, such as the psa A and psa B gene products [10]. Sonoike [11] suggested that the light-induced destruction of the iron-sulfur centers FX, FA, and FB, and possibly phylloquinone and A1, is responsible for photoinhibition of PSI [11]. The recovery of PSI occurs very slowly; it might take several days, even under favorable conditions [12,13], because PSI lacks a quick repair mechanisms like that in PSII. Plants in northern areas often face chilling conditions. The decrease in photosynthesis induced by a chilling environment is the primary reason limiting the lives and growth of chillingsensitive plants [14,15]. It has been reported that PSI and PSII photoinhibition are tightly correlated under chilling-light treatment [16,17]. Photoinhibition of PSII protects PSI from photoinhibition while photoinhibition of PSI might enhance the photoinhibition
C. Yang et al. / Journal of Photochemistry and Photobiology B: Biology 140 (2014) 286–291
of PSII [17]. Under high light, PSI is protected from photoinhibition, because PSII is already damaged and fewer electrons are transported to PSI. However, under chilling-light, when PSII is little damaged, PSI photoinhibition takes place because more electrons are transported to PSI [18], and addition of 3-(3,4-Dichlorophenyl)-1,1-Dimethylurea (DCMU), an inhibitor of PSII, could completely protects PSI from photoinhibition [19]. Recently, it has also been reported that the increase of electrons transported to PSI during the recovery phase significantly inhibits the recovery of PSI [16]. It is well known that chilling-light stress mainly inhibits the activity of PSI [9,10], while other abiotic stress, such as drought and salt, primarily inhibits the activity of PSII instead of PSI [20,21]. We therefore sought to explore whether PSI would be protected from chilling-light stress by other abiotic stresses that enhances PSII damage. To reach this aim, the activities of PSI and PSII were investigated under chilling-light, with and without NaCl stress. 2. Materials and methods
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Leaves were illuminated by saturating red light of 5000 lmol m2 s1. The chlorophyll a fluorescence transients were analyzed with the JIP-test [26,27]: Maximum quantum yield of PSII, Fv/Fm = 1 – (Fo/Fm). The MR signal measured at 820 nm provides information about oxidation of PSI (including PC and P700). The induction curve of MR820nm of the leaves obtained by saturating red light showed a fast oxidation phase and a following reduction phase. The initial slope of the oxidation phase of MR at the beginning of the saturated red light indicates the capability of P700 to get oxidized, which is used to reflect the activity of PSI [24,28,29]. 2.5. Histochemical detection of starch The treated leaves were firstly killed in boiling water for 1 min. Then they were decolorized by immersion in boiling ethanol (96%) for 10 min. The decolorized leaves were immersed in an iodine (I2) and potassium iodide (KI) solution (1:2) for 1 min. After boiling in ethanol (96%) for 15 s to get rid of the background color, the leaves were extracted at room temperature with fresh ethanol and photographed.
2.1. Plant material 2.6. Chemicals Cucumber (Cucumis sativus L. cv. jinchun 4) plants were grown in the field under natural sunlight, at about a 14-h photoperiod (26–32 °C) and 10-h night (22–28 °C). Sufficient nutrients and water were provided to avoid any potential nutrient or drought stress. After growing for about 5 weeks, fully expanding leaves were used in the experiment.
Sodium chloride (NaCl) was purchased from Kaitong chemical reagent Co. Ltd. (Tianjin, China). 3-(3,4-Dichlorophenyl)-1,1Dimethylurea (DCMU), chloramphenicol (CM), were purchased from Sigma–Aldrich (St. Louis, MO, USA). 2.7. Statistical analysis
2.2. Chilling-light treatment The abaxial sides of leaf discs (1 cm2) were floated on the surface of water at 4 °C in a GXZ-5000 light incubator (Jiangnan, China). For photoinhibition treatments, 100 lmol m2 s1 light was used. 2.3. Measurements of chlorophyll fluorescence Modulated chlorophyll fluorescence was measured with an FMS-2 pulse-modulated fluorometer (Hansatech, UK). The lightfluorescence measurement protocol was as follows: the lightadapted leaves were continuously illuminated by actinic light at 100 lmol m2 s1 from the FMS-2 light source, steady-state fluorescence (Fs) was recorded after a 2 min illumination, and 0.8 s of saturating light of 8000 lmol m2 s1 was imposed to obtain maximum fluorescence in the light-adapted state (F 0m ). The actinic light was then turned off, and the minimum fluorescence in the lightadapted state (F0o ) was determined by a 3 s illumination with far-red light. The following parameters were then calculated [22]: (1) Quantum yield of PSII, UPSII = (F0m –Fs)/F0m . (2) Electron transport rate, ETR = UPSII PFD 0.5 0.84. (3) Photochemical quenching, qP = (F0m –Fs)/(F0m –F0o ). 2.4. Measurements of the chlorophyll a fluorescence transient (OJIP) and the modulated reflected signal of 820 nm (MR) Induction kinetics of PF and MR were simultaneously recorded using a Multifunctional Plant Efficiency Analyzer, M-PEA (Hansatech Instrument Ltd., UK) as has been described [23–25]. All leaves were dark-adapted under ambient CO2 conditions at room temperature (25 °C) before the measurements of the induction curves. Measurements were conducted for an induction period of 2 s.
LSD (least significant difference) was used to analyze differences between the SHAM treatments, using SPSS 16. 3. Results 3.1. The effect of different concentrations of NaCl together with chilling-light treatment on PSII and PSI photoinhibition Maximum quantum yield of PSII (Fv/Fm) is a typical indicator of PSII efficiency and is widely used to reflect the photoinhibition of PSII [30,31]. Compared with control leaves (at room temperature in the dark), chilling-light treatment significantly decreased the Fv/Fm and P700 oxidation rate (Fig.1). However, the decreased extent of Fv/Fm gradually increased and P700 oxidation rate gradually increased with increasing NaCl concentration in the chillinglight treatment (Fig.1). The results suggest that NaCl treatment aggravated the PSII photoinhibition but alleviated the PSI photoinhibition under chilling-light. 3.2. The effect of NaCl on photosynthetic electron transport, excitation pressure, PSI and PSII photoinhibition during chilling-light treatment Fv/Fm decreased slowly in control leaves with the increase of treatment time under chilling-light, while that in NaCl (0.8 M) treated leaves decreased much faster (Fig. 2A), which indicates that NaCl increased the photoinhibition rate of PSII under chilling-light treatment. The electron transport rate (ETR) is used as a quantitative indicator of the electron transport beyond PSII [22,32]. Photochemical quenching (qP) is used as an indicator of the redox level of the PSII primary electron acceptor, QA [22]. The PSII excitation pressure was estimated by 1-qP, which represents closed PSII reaction centers [22]. In the absence of NaCl, ETR gradually decreased during chilling-light treatment, reaching a minimum value after the leaves had been treated for 3 h.
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In the presence of DCMU, an inhibitor of the electron transport from QA to QB, the photosynthetic electron transport was fully inhibited during the chilling-light treatment, as indicated by a complete inhibition of ETR (Fig. 3A). However, P700 oxidation rate showed no significant changes during the treatment (Fig. 3B). In addition, ETR was higher and P700 oxidation rate was lower in NaCl (0.8 M) treated leaves than that in DCMU treated leaves. The result suggests that DCMU has a similar effect as NaCl does on PSII and PSI photoinhibition.
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It has been reported that NaCl could induce the accumulation of reactive oxygen species (ROS) through inhibiting the photosynthetic carbon accumulation [33]. Additionally, the damaging effect of ROS on photosystems is the main reason for the aggravation of photoinhibition [34]. The cucumber leaves were not dyed by iodine in chilling-light conditions with or without NaCl (Fig. 4), indicating no starch accumulated in the cucumber leaves, and NaCl did not affect starch accumulation in the leaves in the chilling-light conditions.
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NaCl concentration(M) Fig. 1. The effect of different concentrations of NaCl (0, 0.1, 0.2, 0.4, 0.6, and 0.8 M) together with chilling-light (4 °C, 100 lmol) treatment for 3 h on the maximum quantum yield of PSII (Fv/Fm) (A) and P700 oxidation rate (B) in cucumber leaves. Control leaves (CK) were placed under room temperature (25 °C) in the dark for 3 h. Different letters indicate significant difference between treatments with different concentration of NaCl at P < 0.05. Means ± SE of 8 replicates were presented.
3.5. The effect of NaCl and chilling-light treatment on PSII photoinhibition in the presence or absence of chloramphenicol (CM) Treatment with NaCl significantly accelerated the decrease of ETR, reaching its lowest extent 1 h after the treatment (Fig. 2B). The changes of 1-qP showed an opposite trends to that of ETR (Fig. 2C). In addition, P700 oxidation rate quickly decreased linearly with the duration of chilling-light treatment in the absence of NaCl. However, in the presence of NaCl, P700 oxidation rate declined much more slowly, with a significant difference observed only after 3 h (Fig. 2D). The results showed that NaCl inhibited the electron transport of PSII to PSI, increased PSII excitation pressure and alleviated PSI photoinhibition during the chilling-light treatment.
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In order to clarify whether the difference in D1 protein de novo synthesis contributes to the different PSII photoinhibition among leaves treated with different concentrations of NaCl under the chilling-light treatment, we compared the PSII photoinhibition in leaves in the presence of different concentrations of NaCl (0, 0.1, 0.2, 0.4, 0.6, and 0.8 mM) with or without chloramphenicol (CM), the inhibitor of D1 protein de novo synthesis. Fv/Fm was not significantly changed by CM in leaves treated with different concentrations of NaCl under chilling-light treatment (Fig. 5A). However, at room temperature (25 °C), Fv/Fm was significantly decreased in the light
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Fig. 2. The changes of maximum quantum yield of PSII (Fv/Fm) (A) photosynthetic electron transport rate (ETR) (B), PSII excitation pressure (1-qP) (C) and P700 oxidation rate (D) in cucumber leaves during chilling-light (4 °C, 100 lmol m2 s1) treatment in the presence or absence of NaCl (0.8 M). Different letters indicate significant difference treatments. Means ± SE of 8 replicates are presented.
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Fig. 4. NaCl and chilling-light treatment on the starch accumulation in cucumber leaves. The leaves after different treatments were dyed with iodine. The chillinglight (4 °C) treatment was accompanied by low light (100 lmol m2 s1). The depth of the dark color indicates the starch content. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
in the presence of CM (Fig. 5B). This result demonstrates that the D1 protein de novo synthesis was almost fully inhibited by low temperature, so the PSII photoinhibition was independent of D1 protein de novo synthesis in leaves under chilling-light with NaCl stress. 4. Discussion It is the first time that the effects of NaCl on photosynthetic apparatus were studied under chilling-light conditions. This study
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demonstrated that NaCl alleviated the PSI photoinhibition under chilling-light treatment by reducing the electrons transported to PSI due to aggravation of PSII photoinhibition and damage of cytochrome b6/f complex (Fig. 6). In chilling conditions, the protecting enzymes around PSI, such as SOD and APX, are largely inhibited [35], so PSI is easily damaged by ROS produced at PSI acceptor side. The amount of ROS generated around PSI was determined by two factors: (1) the electron use efficiency at PSI acceptor side, which depends on CO2 assimilation and other energy consumption reactions; (2) the amount of electrons transported from PSII to PSI acceptor side. Decreasing electrons consumed at PSI acceptor side and increasing electrons transported to PSI acceptor side will lead to increase of ROS generation and aggravation of PSI photoinhibition. Conversely, the amount of ROS generation and PSI photoinhibition will be decreased by increasing electron consumption at PSI acceptor side and decreasing electrons transported to PSI acceptor side. Carbon assimilation was a primary physiological reaction to consume electrons at PSI acceptor side. When CO2 assimilation is inhibited, more electrons at PSI acceptor side produced by light reactions will be transported to O2 to generate ROS [36,37], which may contribute to PSI photoinhibition. Starch is the end product of photosynthetic carbon assimilation. The accumulation of starch in leaves can be used to reflect the capacity of CO2 assimilation. In the this study, the content of starch in leaves was fully inhibited by low temperature, and NaCl had no effect on the starch content in leaves under chilling-light treatment (Fig. 4), these results indicate that NaCl stress did not affect CO2 assimilation under chilling-light, so the alleviation of NaCl on PSI photoinhibition is independent of CO2 assimilation. It has been reported that the electron transported from PSII aggravates the PSI photoinhibition under chilling-light, and the fast recovery of PSII can inhibit the recovery of PSI in the recovery phase after chilling-light treatment [16]. According to our observation, NaCl significantly inhibited the electron transport rate of PSII and alleviated the damage of PSI caused by chilling-light treatment, indicating that NaCl protected PSI through reducing the electrons transported from PSII. This conclusion was further supported by the fact that DCMU treatment showed a similar effect as NaCl on the PSI photoinhibition under chilling-light treatment (Fig. 3). In NaCl treated leaves, contrast to the small decrease of Fv/Fm, 1-qP increased more rapidly (Fig. 2), which indicates that the PQ pool was in a high reduced level. The maintenance of oxidation–reduction equilibrium of PQ depends on the rapid oxidation of PQH2. Cytochrome b6/f complex and PSI, locating at the downstream of PQ, both contribute to the reduction of PQ pool. Because PSI was only slightly damaged by chilling-light under NaCl treatment (Figs. 1 and 2), and it has been reported that electrons transported at cytochrome b6/f complex is always the rate-limiting step of photosynthetic electron transportation [38,39]. It could be speculated that cytochrome b6/f complex might be one of the main limited steps of electron transport. The inhibition of cytochrome b6/f complex by NaCl may contribute much to the reduction of electrons transported to PSI and alleviation of PSI photoinhibition besides the PSII photoinhibition. However, the specific mechanism by which NaCl inhibits cytochrome b6/f complex need further studies to verify. Under light, PSII photoinhibition is determined by the balance between the rate of PSII photodamage and the rate of its repair, photoinhibition of PSII becomes apparent when the rate of photodamage exceeds the rate of repair [2,4,5]. It has been reported that NaCl could directly induce inactivation of the photosynthetic machinery, particularly photosystem II (PSII) in vitro [20] and in vivo [21,40,41]. According the fact that NaCl did not affect the repair process of PSII under chilling-light conditions (Fig. 5), we speculate that NaCl aggravate PSII photoinhibition by accelerating the damaging rate under chilling-light conditions.
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Fig. 5. The effect of different concentrations of NaCl (0, 0.1, 0.2, 0.4, 0.6, and 0.8 M) on maximum quantum yield of PSII (Fv/Fm) in cucumber leaves under chilling-light (4 °C, 100 lmol m2 s1) treatment in presence or absence of chloramphenicol (CM) (A). Cucumber leaves treated in dark and low light (100 lmol m2 s1), respectively, at room temperature (25 °C), with or without CM (B). Different letters indicate significant difference treatments. Means ± SE of 8 replicates are presented.
alleviate PSII photoinhitbition. The PSII photoinhibition was aggravated by NaCl in a direct manner which was independent of PSII repair. However, the specific mechanism by which NaCl aggravates PSII damage under chilling-light conditions needs further studies to clarify. 5. Abbreviations
Fig. 6. The effect of PSII activity on PSI photoinhibition under chilling-light condition with or without NaCl or DCMU. (A) Under chilling-light, the electrons transported to PSI resulted in ROS production at PSI acceptor side, the ROS led to the inhibition of PSI. (B) Under chilling-light with NaCl or DCMU, the electron transported to PSI was inhibited, which eliminated the ROS production at PSI acceptor side and alleviated PSI photoinhibition.
PSI and PSII work coordinately in the light. The decrease in PSI activity will lead to excessive reduction of electron transport chains at the PSII acceptor side. Meanwhile, PSII photoinhibition would be alleviated by a high PSI activity, due to a low extent of reduction at the PSII acceptor side and low ROS generation around PSII. ROS increases PSII photoinhibition mainly by inhibiting the PSII repair process [5,42], we speculate that the alleviation of PSI photoinhibition didn’t alleviate PSII photoinhibition was because the repair of PSII was fully inhibited by chilling-light treatment. In conclusion, PSI photoinhibition could be alleviated by NaCl due to the reduction of the electron transport from PSII to PSI caused by aggravation of PSII photoinhibition and the damage of cytochrome b6/f complex. Because of fully inhibition of PSII repair by chilling treatment, the alleviation of PSI photoinhibition did not
APX CM DCMU ETR Fv/Fm I2 KI NaCl OJIP PF PSI PSII QA QB qP ROS SOD M-PEA MR UPSII 1-qP
ascorbate peroxidase chloramphenicol 3-(3,4-Dichlorophenyl)-1,1-Dimethylurea electron transport rate maximum quantum yield of PSII iodine potassium iodide sodium chloride chlorophyll a fluorescence transient prompt fluorescence photosystem I photosystem II the primary quinone electron acceptor of PSII the secondary quinone electron acceptor of PSII photochemical quenching reactive oxygen species superoxide dismutase Multifunctional Plant Efficiency Analyzer the modulated reflected signal of 820 nm Quantum yield of PSII PSII excitation pressure
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Journal of Photochemistry and Photobiology B: Biology journal homepage: www.elsevier.com/locate/jphotobiol
The mechanism by which NaCl treatment alleviates PSI photoinhibition under chilling-light treatment Yang Cheng a,b, Zhang Zi-shan a, Gao Hui-yuan a,⇑, Fan Xing-li a, Liu Mei-jun a, Li Xiang-dong b a b
State Key Laboratory of Crop Biology, Shandong Key Laboratary of Crop Biology, College of Life Sciences, Shandong Agricultural University, Tai’an 271018, China Wheat Research Institute, Henan Academy of Agricultural Sciences, Zhengzhou, Henan 450002, China
a r t i c l e
i n f o
Article history: Received 1 March 2014 Received in revised form 7 August 2014 Accepted 14 August 2014 Available online 26 August 2014 Keywords: NaCl PSII PSI Photoinhibition Chilling-light
a b s t r a c t The effects of chilling-light stress combined with additional stress on PSI and PSII photoinhibition and their interrelationship have not been known. To explore whether NaCl affects the PSI and PSII photoinhibition and their interrelationship under chilling-light treatment, the PSI and PSII activities were studied under chilling-light with or without NaCl treatment. The results showed that the extent of PSI and PSII photoinhibition both increased under chilling-light, while NaCl aggravated PSII photoinhibition and severely damaged cytochrome b6/f complex but alleviated PSI photoinhibition. Moreover, DCMU had a similar effect as NaCl in this study, which indicates that NaCl alleviated PSI photoinhibition through reducing electrons transported to PSI. It was also showed that the increased damage to PSII by NaCl did not depend on the inhibition of PSII repair and PSI electron transportation. In conclusion, NaCl alleviated PSI photoinhibition by inhibiting electron transport from PSII under chilling-light conditions. In addition, PSII photoinhibition was not affected by PSI photoinhibition because of a full inhibition of PSII repair by chilling-light treatment. We also speculate that NaCl aggravates PSII photoinhibition by enhancing the damage instead of inhibiting the repair of it under chilling-light conditions. Ó 2014 Elsevier B.V. All rights reserved.
1. Introduction Photoinhibition may take place under conditions where the harvested light energy exceeds the requirements to drive photochemical reactions and protective mechanisms become overburdened [1,2]. Photosystem II (PSII) and photosystem I (PSI) are the main target sites of photoinhibition. PSII has long been considered the primary target of photoinhibition [2,3], as photodamage to PSII occurs under light at any intensity and is unavoidable. Photosynthetic organisms are able to overcome photodamage to PSII by rapid and efficient repair of the damage, which requires the synthesis of D1 proteins de novo. The activity of PSII depends on the balance between the rates of photodamage and repair, and the photoinhibition of PSII becomes apparent when the rate of photodamage exceeds the rate of repair [2,4,5]. Some studies revealed that light can damage PSII directly, and other forms of environmental stress act primarily by inhibiting repair of PS II [6,7].
⇑ Corresponding author. Tel.: +86 538 8245985; fax: +86 538 8249608. E-mail addresses: [email protected] (C. Yang), Zhangzishantaian@163. com (Z.-s. Zhang), [email protected] (H.-y. Gao), [email protected] (X.-l. Fan), [email protected] (M.-j. Liu), [email protected] (X.-d. Li). http://dx.doi.org/10.1016/j.jphotobiol.2014.08.012 1011-1344/Ó 2014 Elsevier B.V. All rights reserved.
PSI photoinhibition is rarely observed in vivo because PSI is more stable than PSII under most types of abiotic stress, such as high light [8]. PSI photoinhibition was first reported by Terashima and his colleagues [9]. When cucumber leaves were chilled at 4 °C for 5 h under moderate photon flux density (200 lmol m2 s1), the maximum photosynthetic electron flow through PSI decreased by 70–80%, compared to the controls. In contrast, the maximum electron transport through PSII remained at 80% of that of the controls [9]. Chilling-induced photoinhibition damages the membrane structure and causes degradation of proteins in PSI reaction center, such as the psa A and psa B gene products [10]. Sonoike [11] suggested that the light-induced destruction of the iron-sulfur centers FX, FA, and FB, and possibly phylloquinone and A1, is responsible for photoinhibition of PSI [11]. The recovery of PSI occurs very slowly; it might take several days, even under favorable conditions [12,13], because PSI lacks a quick repair mechanisms like that in PSII. Plants in northern areas often face chilling conditions. The decrease in photosynthesis induced by a chilling environment is the primary reason limiting the lives and growth of chillingsensitive plants [14,15]. It has been reported that PSI and PSII photoinhibition are tightly correlated under chilling-light treatment [16,17]. Photoinhibition of PSII protects PSI from photoinhibition while photoinhibition of PSI might enhance the photoinhibition
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of PSII [17]. Under high light, PSI is protected from photoinhibition, because PSII is already damaged and fewer electrons are transported to PSI. However, under chilling-light, when PSII is little damaged, PSI photoinhibition takes place because more electrons are transported to PSI [18], and addition of 3-(3,4-Dichlorophenyl)-1,1-Dimethylurea (DCMU), an inhibitor of PSII, could completely protects PSI from photoinhibition [19]. Recently, it has also been reported that the increase of electrons transported to PSI during the recovery phase significantly inhibits the recovery of PSI [16]. It is well known that chilling-light stress mainly inhibits the activity of PSI [9,10], while other abiotic stress, such as drought and salt, primarily inhibits the activity of PSII instead of PSI [20,21]. We therefore sought to explore whether PSI would be protected from chilling-light stress by other abiotic stresses that enhances PSII damage. To reach this aim, the activities of PSI and PSII were investigated under chilling-light, with and without NaCl stress. 2. Materials and methods
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Leaves were illuminated by saturating red light of 5000 lmol m2 s1. The chlorophyll a fluorescence transients were analyzed with the JIP-test [26,27]: Maximum quantum yield of PSII, Fv/Fm = 1 – (Fo/Fm). The MR signal measured at 820 nm provides information about oxidation of PSI (including PC and P700). The induction curve of MR820nm of the leaves obtained by saturating red light showed a fast oxidation phase and a following reduction phase. The initial slope of the oxidation phase of MR at the beginning of the saturated red light indicates the capability of P700 to get oxidized, which is used to reflect the activity of PSI [24,28,29]. 2.5. Histochemical detection of starch The treated leaves were firstly killed in boiling water for 1 min. Then they were decolorized by immersion in boiling ethanol (96%) for 10 min. The decolorized leaves were immersed in an iodine (I2) and potassium iodide (KI) solution (1:2) for 1 min. After boiling in ethanol (96%) for 15 s to get rid of the background color, the leaves were extracted at room temperature with fresh ethanol and photographed.
2.1. Plant material 2.6. Chemicals Cucumber (Cucumis sativus L. cv. jinchun 4) plants were grown in the field under natural sunlight, at about a 14-h photoperiod (26–32 °C) and 10-h night (22–28 °C). Sufficient nutrients and water were provided to avoid any potential nutrient or drought stress. After growing for about 5 weeks, fully expanding leaves were used in the experiment.
Sodium chloride (NaCl) was purchased from Kaitong chemical reagent Co. Ltd. (Tianjin, China). 3-(3,4-Dichlorophenyl)-1,1Dimethylurea (DCMU), chloramphenicol (CM), were purchased from Sigma–Aldrich (St. Louis, MO, USA). 2.7. Statistical analysis
2.2. Chilling-light treatment The abaxial sides of leaf discs (1 cm2) were floated on the surface of water at 4 °C in a GXZ-5000 light incubator (Jiangnan, China). For photoinhibition treatments, 100 lmol m2 s1 light was used. 2.3. Measurements of chlorophyll fluorescence Modulated chlorophyll fluorescence was measured with an FMS-2 pulse-modulated fluorometer (Hansatech, UK). The lightfluorescence measurement protocol was as follows: the lightadapted leaves were continuously illuminated by actinic light at 100 lmol m2 s1 from the FMS-2 light source, steady-state fluorescence (Fs) was recorded after a 2 min illumination, and 0.8 s of saturating light of 8000 lmol m2 s1 was imposed to obtain maximum fluorescence in the light-adapted state (F 0m ). The actinic light was then turned off, and the minimum fluorescence in the lightadapted state (F0o ) was determined by a 3 s illumination with far-red light. The following parameters were then calculated [22]: (1) Quantum yield of PSII, UPSII = (F0m –Fs)/F0m . (2) Electron transport rate, ETR = UPSII PFD 0.5 0.84. (3) Photochemical quenching, qP = (F0m –Fs)/(F0m –F0o ). 2.4. Measurements of the chlorophyll a fluorescence transient (OJIP) and the modulated reflected signal of 820 nm (MR) Induction kinetics of PF and MR were simultaneously recorded using a Multifunctional Plant Efficiency Analyzer, M-PEA (Hansatech Instrument Ltd., UK) as has been described [23–25]. All leaves were dark-adapted under ambient CO2 conditions at room temperature (25 °C) before the measurements of the induction curves. Measurements were conducted for an induction period of 2 s.
LSD (least significant difference) was used to analyze differences between the SHAM treatments, using SPSS 16. 3. Results 3.1. The effect of different concentrations of NaCl together with chilling-light treatment on PSII and PSI photoinhibition Maximum quantum yield of PSII (Fv/Fm) is a typical indicator of PSII efficiency and is widely used to reflect the photoinhibition of PSII [30,31]. Compared with control leaves (at room temperature in the dark), chilling-light treatment significantly decreased the Fv/Fm and P700 oxidation rate (Fig.1). However, the decreased extent of Fv/Fm gradually increased and P700 oxidation rate gradually increased with increasing NaCl concentration in the chillinglight treatment (Fig.1). The results suggest that NaCl treatment aggravated the PSII photoinhibition but alleviated the PSI photoinhibition under chilling-light. 3.2. The effect of NaCl on photosynthetic electron transport, excitation pressure, PSI and PSII photoinhibition during chilling-light treatment Fv/Fm decreased slowly in control leaves with the increase of treatment time under chilling-light, while that in NaCl (0.8 M) treated leaves decreased much faster (Fig. 2A), which indicates that NaCl increased the photoinhibition rate of PSII under chilling-light treatment. The electron transport rate (ETR) is used as a quantitative indicator of the electron transport beyond PSII [22,32]. Photochemical quenching (qP) is used as an indicator of the redox level of the PSII primary electron acceptor, QA [22]. The PSII excitation pressure was estimated by 1-qP, which represents closed PSII reaction centers [22]. In the absence of NaCl, ETR gradually decreased during chilling-light treatment, reaching a minimum value after the leaves had been treated for 3 h.
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In the presence of DCMU, an inhibitor of the electron transport from QA to QB, the photosynthetic electron transport was fully inhibited during the chilling-light treatment, as indicated by a complete inhibition of ETR (Fig. 3A). However, P700 oxidation rate showed no significant changes during the treatment (Fig. 3B). In addition, ETR was higher and P700 oxidation rate was lower in NaCl (0.8 M) treated leaves than that in DCMU treated leaves. The result suggests that DCMU has a similar effect as NaCl does on PSII and PSI photoinhibition.
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It has been reported that NaCl could induce the accumulation of reactive oxygen species (ROS) through inhibiting the photosynthetic carbon accumulation [33]. Additionally, the damaging effect of ROS on photosystems is the main reason for the aggravation of photoinhibition [34]. The cucumber leaves were not dyed by iodine in chilling-light conditions with or without NaCl (Fig. 4), indicating no starch accumulated in the cucumber leaves, and NaCl did not affect starch accumulation in the leaves in the chilling-light conditions.
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NaCl concentration(M) Fig. 1. The effect of different concentrations of NaCl (0, 0.1, 0.2, 0.4, 0.6, and 0.8 M) together with chilling-light (4 °C, 100 lmol) treatment for 3 h on the maximum quantum yield of PSII (Fv/Fm) (A) and P700 oxidation rate (B) in cucumber leaves. Control leaves (CK) were placed under room temperature (25 °C) in the dark for 3 h. Different letters indicate significant difference between treatments with different concentration of NaCl at P < 0.05. Means ± SE of 8 replicates were presented.
3.5. The effect of NaCl and chilling-light treatment on PSII photoinhibition in the presence or absence of chloramphenicol (CM) Treatment with NaCl significantly accelerated the decrease of ETR, reaching its lowest extent 1 h after the treatment (Fig. 2B). The changes of 1-qP showed an opposite trends to that of ETR (Fig. 2C). In addition, P700 oxidation rate quickly decreased linearly with the duration of chilling-light treatment in the absence of NaCl. However, in the presence of NaCl, P700 oxidation rate declined much more slowly, with a significant difference observed only after 3 h (Fig. 2D). The results showed that NaCl inhibited the electron transport of PSII to PSI, increased PSII excitation pressure and alleviated PSI photoinhibition during the chilling-light treatment.
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In order to clarify whether the difference in D1 protein de novo synthesis contributes to the different PSII photoinhibition among leaves treated with different concentrations of NaCl under the chilling-light treatment, we compared the PSII photoinhibition in leaves in the presence of different concentrations of NaCl (0, 0.1, 0.2, 0.4, 0.6, and 0.8 mM) with or without chloramphenicol (CM), the inhibitor of D1 protein de novo synthesis. Fv/Fm was not significantly changed by CM in leaves treated with different concentrations of NaCl under chilling-light treatment (Fig. 5A). However, at room temperature (25 °C), Fv/Fm was significantly decreased in the light
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Fig. 4. NaCl and chilling-light treatment on the starch accumulation in cucumber leaves. The leaves after different treatments were dyed with iodine. The chillinglight (4 °C) treatment was accompanied by low light (100 lmol m2 s1). The depth of the dark color indicates the starch content. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
in the presence of CM (Fig. 5B). This result demonstrates that the D1 protein de novo synthesis was almost fully inhibited by low temperature, so the PSII photoinhibition was independent of D1 protein de novo synthesis in leaves under chilling-light with NaCl stress. 4. Discussion It is the first time that the effects of NaCl on photosynthetic apparatus were studied under chilling-light conditions. This study
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demonstrated that NaCl alleviated the PSI photoinhibition under chilling-light treatment by reducing the electrons transported to PSI due to aggravation of PSII photoinhibition and damage of cytochrome b6/f complex (Fig. 6). In chilling conditions, the protecting enzymes around PSI, such as SOD and APX, are largely inhibited [35], so PSI is easily damaged by ROS produced at PSI acceptor side. The amount of ROS generated around PSI was determined by two factors: (1) the electron use efficiency at PSI acceptor side, which depends on CO2 assimilation and other energy consumption reactions; (2) the amount of electrons transported from PSII to PSI acceptor side. Decreasing electrons consumed at PSI acceptor side and increasing electrons transported to PSI acceptor side will lead to increase of ROS generation and aggravation of PSI photoinhibition. Conversely, the amount of ROS generation and PSI photoinhibition will be decreased by increasing electron consumption at PSI acceptor side and decreasing electrons transported to PSI acceptor side. Carbon assimilation was a primary physiological reaction to consume electrons at PSI acceptor side. When CO2 assimilation is inhibited, more electrons at PSI acceptor side produced by light reactions will be transported to O2 to generate ROS [36,37], which may contribute to PSI photoinhibition. Starch is the end product of photosynthetic carbon assimilation. The accumulation of starch in leaves can be used to reflect the capacity of CO2 assimilation. In the this study, the content of starch in leaves was fully inhibited by low temperature, and NaCl had no effect on the starch content in leaves under chilling-light treatment (Fig. 4), these results indicate that NaCl stress did not affect CO2 assimilation under chilling-light, so the alleviation of NaCl on PSI photoinhibition is independent of CO2 assimilation. It has been reported that the electron transported from PSII aggravates the PSI photoinhibition under chilling-light, and the fast recovery of PSII can inhibit the recovery of PSI in the recovery phase after chilling-light treatment [16]. According to our observation, NaCl significantly inhibited the electron transport rate of PSII and alleviated the damage of PSI caused by chilling-light treatment, indicating that NaCl protected PSI through reducing the electrons transported from PSII. This conclusion was further supported by the fact that DCMU treatment showed a similar effect as NaCl on the PSI photoinhibition under chilling-light treatment (Fig. 3). In NaCl treated leaves, contrast to the small decrease of Fv/Fm, 1-qP increased more rapidly (Fig. 2), which indicates that the PQ pool was in a high reduced level. The maintenance of oxidation–reduction equilibrium of PQ depends on the rapid oxidation of PQH2. Cytochrome b6/f complex and PSI, locating at the downstream of PQ, both contribute to the reduction of PQ pool. Because PSI was only slightly damaged by chilling-light under NaCl treatment (Figs. 1 and 2), and it has been reported that electrons transported at cytochrome b6/f complex is always the rate-limiting step of photosynthetic electron transportation [38,39]. It could be speculated that cytochrome b6/f complex might be one of the main limited steps of electron transport. The inhibition of cytochrome b6/f complex by NaCl may contribute much to the reduction of electrons transported to PSI and alleviation of PSI photoinhibition besides the PSII photoinhibition. However, the specific mechanism by which NaCl inhibits cytochrome b6/f complex need further studies to verify. Under light, PSII photoinhibition is determined by the balance between the rate of PSII photodamage and the rate of its repair, photoinhibition of PSII becomes apparent when the rate of photodamage exceeds the rate of repair [2,4,5]. It has been reported that NaCl could directly induce inactivation of the photosynthetic machinery, particularly photosystem II (PSII) in vitro [20] and in vivo [21,40,41]. According the fact that NaCl did not affect the repair process of PSII under chilling-light conditions (Fig. 5), we speculate that NaCl aggravate PSII photoinhibition by accelerating the damaging rate under chilling-light conditions.
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Fig. 5. The effect of different concentrations of NaCl (0, 0.1, 0.2, 0.4, 0.6, and 0.8 M) on maximum quantum yield of PSII (Fv/Fm) in cucumber leaves under chilling-light (4 °C, 100 lmol m2 s1) treatment in presence or absence of chloramphenicol (CM) (A). Cucumber leaves treated in dark and low light (100 lmol m2 s1), respectively, at room temperature (25 °C), with or without CM (B). Different letters indicate significant difference treatments. Means ± SE of 8 replicates are presented.
alleviate PSII photoinhitbition. The PSII photoinhibition was aggravated by NaCl in a direct manner which was independent of PSII repair. However, the specific mechanism by which NaCl aggravates PSII damage under chilling-light conditions needs further studies to clarify. 5. Abbreviations
Fig. 6. The effect of PSII activity on PSI photoinhibition under chilling-light condition with or without NaCl or DCMU. (A) Under chilling-light, the electrons transported to PSI resulted in ROS production at PSI acceptor side, the ROS led to the inhibition of PSI. (B) Under chilling-light with NaCl or DCMU, the electron transported to PSI was inhibited, which eliminated the ROS production at PSI acceptor side and alleviated PSI photoinhibition.
PSI and PSII work coordinately in the light. The decrease in PSI activity will lead to excessive reduction of electron transport chains at the PSII acceptor side. Meanwhile, PSII photoinhibition would be alleviated by a high PSI activity, due to a low extent of reduction at the PSII acceptor side and low ROS generation around PSII. ROS increases PSII photoinhibition mainly by inhibiting the PSII repair process [5,42], we speculate that the alleviation of PSI photoinhibition didn’t alleviate PSII photoinhibition was because the repair of PSII was fully inhibited by chilling-light treatment. In conclusion, PSI photoinhibition could be alleviated by NaCl due to the reduction of the electron transport from PSII to PSI caused by aggravation of PSII photoinhibition and the damage of cytochrome b6/f complex. Because of fully inhibition of PSII repair by chilling treatment, the alleviation of PSI photoinhibition did not
APX CM DCMU ETR Fv/Fm I2 KI NaCl OJIP PF PSI PSII QA QB qP ROS SOD M-PEA MR UPSII 1-qP
ascorbate peroxidase chloramphenicol 3-(3,4-Dichlorophenyl)-1,1-Dimethylurea electron transport rate maximum quantum yield of PSII iodine potassium iodide sodium chloride chlorophyll a fluorescence transient prompt fluorescence photosystem I photosystem II the primary quinone electron acceptor of PSII the secondary quinone electron acceptor of PSII photochemical quenching reactive oxygen species superoxide dismutase Multifunctional Plant Efficiency Analyzer the modulated reflected signal of 820 nm Quantum yield of PSII PSII excitation pressure
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