Báo cáo khoa học: Hiệu quả bảo vệ của chất loại bỏ oxy hoạt tính đối với sự phân hủy protein và chức năng quang hóa trong các phần dưới màng của hệ thống quang hóa I (photosystem I) trong điều kiện stress ánh sáng

Protective effect of active oxygen scavengers on protein degradation and photochemical function in photosystem I submembrane fractions during light stress Subramanyam Rajagopal*, David Joly, Alain Gauthier, Marc Beauregard and Robert Carpentier

Groupe de Recherche en Biologie Ve´ ge´ tale, Universite´ du Que´ bec a` Trois-Rivie` res, Que´ bec, Canada

Keywords photosystem I; submembrane fraction; active oxygen; P700; light stress

Correspondence R Carpentier, Groupe de Recherche en Biologie Ve´ ge´ tale, Universite´ du Que´ bec a` Trois-Rivie` res, C.P. 500, Trois-Rivie` res, Que´ bec, Canada, G9A 5H7 Fax: 1 819 376 5057 E-mail: Robert_Carpentier@uqtr.ca

*Present address School of Life Sciences and Center for the Study of Early Events in Photosynthesis, Arizona State University, Tempe, Arizona 85287, USA

(Received 17 August 2004, revised 23 November 2004, accepted 2 December 2004)

doi:10.1111/j.1742-4658.2004.04512.x

The protective role of reactive oxygen scavengers against photodamage was studied in isolated photosystem (PS) I submembrane fractions illumin- ated (2000 lEÆm)2Æs)1) for various periods at 4 (cid:1)C. The photochemical activity of the submembrane fractions measured as P700 photooxidation was signiﬁcantly protected in the presence of histidine or n-propyl gallate. Chlorophyll photobleaching resulting in a decrease of absorbance and ﬂuorescence, and a blue-shift of both absorbance and ﬂuorescence maxi- mum in the red region, was also greatly delayed in the presence of these scavengers. Western blot analysis revealed the light harvesting antenna complexes of PSI, Lhca2 and Lhca1, were more susceptible to strong light when compared to Lhca3 and Lhca4. The reaction-center proteins PsaB, PsaC, and PsaE were most sensitive to strong illumination while other polypeptides were less affected. Addition of histidine or n-propyl gallate lead to signiﬁcant protection of reaction-center proteins as well as Lhca against strong illumination. Circular dichroism (CD) spectra revealed that the a-helix content decreased with increasing period of light exposure, whereas b-strands, turns, and unordered structure increased. This unfolding was prevented with the addition of histidine or n-propyl gallate even after 10 h of strong illumination. Catalase or superoxide dismutase could not minimize the alteration of PSI photochemical activity and structure due to photodamage. The speciﬁc action of histidine and n-propyl gallate indicates that 1O2 was the main form of reactive oxygen species responsible for strong light-induced damage in PSI submembrane fractions.

reaction center

& Fork [9] demonstrated the inactivation of PSI in intact Bryopsis chloroplasts under strongly reducing conditions. The authors suggested the photoinactiva- tion site to be either P700 itself [9] or close to the [10] under both aerobic and PSI anaerobic conditions. The main site of PSI photo- inhibition under aerobic conditions was then shown to be the inactivation of iron–sulfur centers [11], whereas anaerobic photoinhibition resulted in the blocking of electron transfer between A0 and FX [12].

Excessive light causes the inactivation of photosyn- thesis. The detailed mechanism of PSII inactivation has been extensively characterized both in vivo and in vitro [1–5] and it has been suggested that the inac- tivation develops at either acceptor or donor side of the photosystem [2,6]. PSI was ﬁrst believed to be in vitro to strong light. However, several tolerant studies suggested that PSI photochemical activity could be inhibited under strong illumination [7,8]. In these early experiments, photoinactivation of PSI was not observed in the absence of oxygen. Later, Satoh

Abbreviations Chl, chlorophyll; LHC I, light-harvesting complex I; P700, primary electron donor; PSI and II, photosystem I and II; SOD, superoxide dismutase.

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addition of glucose oxidase, glucose, and catalase to scavenge dissolved oxygen suppressed the photodamage of PSI submembrane fractions [17].

terminal

However, the protective role of speciﬁc active oxy- gen scavengers against photo-induced changes in the photosystems was discussed only in a limited number of reports. LHCII protein degradation was analyzed during strong light illumination of isolated LHCII or BBY PSII subcomplexes [28]. Random cleavage, start- region resulted in the ing in the NH2 complete degradation of the antenna proteins. The addition of scavengers such as histidine, DABCO, and n-propyl gallate, retarded the above damages to the antenna proteins indicating mainly 1O2 was involved [28]. In spinach thylakoids, illumination at low light intensity resulted in the degradation of PsaB gene product into two fragments of 51 and 45 kDa [30]. These fragments were absent with added n-propyl gal- late, which removes hydroxyl radicals [30]. There are no reports regarding the protective role of oxygen scavengers against reactive oxygen species under strong light in PSI submembrane fractions.

Recently, Velitchkova et al. [13] reported that PSI- mediated electron transport, P700 content, and reac- tion-center proteins, PsaA ⁄ B, were altered by strong light illumination at room or low temperatures in these isolated spinach thylakoid membranes. Also, authors observed that under these conditions toxic hydroxyl radicals were generated. Inactivation of PSI- mediated electron ﬂow was also reported in isolated PSI core particles such as spinach PSI-180 and PSI- 100, as well as cyanobacterial PSI membranes exposed to strong light [14]. In PSI core particles illuminated with strong light, damage to the light-harvesting com- plex (LHC) and degradation of reaction-center pro- teins as well as acceptor side proteins were observed [15]. We have recently shown that exposure of PSI submembrane fractions to strong light under low tem- perature altered the structure of chlorophyll (Chl)–pro- tein (CP) complexes and decreased the photochemical activity and the efﬁciency of excitation energy migra- tion [16–18]. The damages were associated with the formation of reactive oxygen species. It was also found that the above photoinactivation of PSI was retarded by glycinebetaine and sucrose [18].

We used PSI submembrane particles as model sys- tem to obtain a better insight into the photo-induced changes in PSI. The submembrane particles used con- including the cyto- tain all the components of PSI, chrome b6 ⁄ f complex and plastocyanin [31,32]. In the present study, the degradation of each polypeptide and the protein conformation changes are assessed in con- nection with alterations in photochemical activity dur- ing strong light illumination of the PSI submembrane fractions. It is concluded from the speciﬁcity of the various active oxygen scavengers used that only the generation of 1O2 is responsible for the photooxidation effects. The role of active oxygen scavengers in the protection of the structure and function of the photo- systems against excess light indicates they should also be beneﬁcial under natural conditions.

In intact leaves, PSI was also found to be inacti- vated by light. In Cucumis sativus leaves, weak light induced the photoinactivation of PSI at chilling tem- peratures, while practically no damage to PSII was reported [19,20]. Sonoike et al. [20] demonstrated that electron carriers located on the acceptor side of PSI (A1, FX, FA, and FB) were damaged during the photo- inhibition of PSI. The loss of PSI activity in thylakoids isolated from spinach leaves exposed to weak light illu- mination at room temperature is believed to be associ- ated with the degradation of PsaB protein, one of the PSI reaction-center subunits [21]. In barley and cucum- ber leaves exposed to weak light at chilling temper- ature, photoinhibition of PSI coincided with the damage of both reaction-center proteins, PsaA and PsaB, and the proteins PsaD and PsaE on the acceptor side of the photosystem [22].

Results

PSI submembrane fractions were exposed to strong light (2000 lEÆm)2Æs)1) for various time durations at 4 (cid:1)C. The action of several active oxygen scavengers was analyzed for all the parameters studied in order to emphasize the role of reactive oxygen species in the photooxydative damage.

Changes in DA830

Exposure of isolated PSI submembrane fractions to strong white light at 4 (cid:1)C resulted in the loss of ability of P700 to undergo reversible redox changes. Similar

In plants, reactive oxygen species (ROS) are associ- ated with normal physiological processes as well as with responses to adverse conditions. ROS are implicated in many ways with stressful conditions: as primary elici- tors, as products and propagators of oxidative damage, or as signal molecules initiating defense or adaptation [23–25]. Several authors proposed that oxidative mecha- nisms are at the basis of PSI photodamage in intact leaves [19,22], isolated chloroplasts [7,8,11], and isolated PSI fragments [15–18]. Two types of active oxygen spe- cies with their derivative products are proposed to be involved in photooxidative damage, superoxide anion –, and singlet oxygen, 1O2 [26–29]. Hence, radicals, ÆO2

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Fig. 1. The changes in the magnitude of light-induced absorbance at 830 nm during strong light illumination in PSI submembrane frac- tions. Results are means ± SE. n ¼ 3. All experimental conditions are given in Experimental procedures.

fractions

results were obtained at room temperature (data not shown). Figure 1 shows the magnitude of DA830 signal remained stable during the ﬁrst 4 h of strong light in the submembrane treatment of PSI absence of active oxygen scavengers. It indicated an unchanged amount of photooxidizable P700 during this period. However, further irradiation caused a rapid decline in DA830. The addition of histidine and n-propyl gallate to the submembrane fractions signiﬁ- cantly prevented the decline of absorbance changes (DA830). The magnitude of DA830 was almost constant until 10 h of light exposure with the above reactive oxygen scavengers. In contrast, in presence of cata- lase or superoxide dismutase, or both, the amplitude of DA830 was similar to control values (data not shown).

Absorption changes

Fig. 2. Room temperature absorption spectra of the isolated PSI submembrane fractions illuminated for various periods of time with strong WL (2000 lmolÆm)2Æs)1) at 4 (cid:1)C. (A) Control (no addition of any additives), (B) in the presence of histidine, and (C) in the pres- ence of n-propyl gallate. These experiments were repeated three times and yielded identical spectra; a typical spectrum is presented. Insets: the ﬁrst derivative of the absorption maxima in the red is presented to show peak position.

Figure 2A illustrates the changes in the room tempera- ture absorption spectra of PSI submembrane fractions exposed to various periods of strong white light illumin- ation at 4 (cid:1)C. The spectra measured in untreated sub- membrane fractions showed typical absorption peaks at 680 and 440 nm corresponding to Chl a and shoulders at 650 and 470 nm originating from Chl b [16]. As the period of illumination increased, the magnitude of the

absorption peak at 680 nm declined (Fig. 2). In addi- tion, the position of that peak was ﬁnally shifted by 6 nm towards shorter wavelengths after a 6-h illumin-

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ation. This becomes particularly clear from the changes in ﬁrst derivative spectra that further demonstrate the gradual time-dependent alteration in the position of the major peak in the red region (Fig. 2, insert). On the other hand, in the presence of the scavengers histidine or n-propyl gallate, the absorption maximum at 680 nm was less affected by the strong illumination and even after a 10-h exposure this absorption was higher than in control (6 h). Consequently, the blue shift of the maxi- mum absorption peak at 680 nm was also less pro- nounced (Fig. 2B,C). In the presence of histidine the peak shift was about 5 nm after 6 h, while in n-propyl gallate this peak shift was only 3 nm after 10 h.

Fluorescence emission changes

strong light

illumination.

The 77 K ﬂuorescence emission spectra and their ﬁrst derivatives measured in PSI submembrane fractions illuminated for various times at 4 (cid:1)C are shown in Fig. 3. The intensity of the peak at 736 nm associated to the PSI complex decreased by about 24% after It declined with 1 h of further light exposure, with only 30% of its initial magnitude remaining after a 3-h illumination. Similar to the absorbance spectra, a blue shift of the position the major ﬂuorescence peak was observed. The of extent of that shift was larger in the ﬂuorescence emission spectra and reached 15–18 nm after 6 h (Fig. 3A,C). The shift in ﬂuorescence maximum was evident from both absolute (Fig. 3A) and ﬁrst deriv- ative (Fig. 3C) spectra. The latter also demonstrates that the half-width of the emission band peaking at 736 nm in untreated preparations was not affected by light exposure despite the marked changes in the peak position.

In the presence of histidine or n-propyl gallate the changes in the ﬂuorescence maximum at 736 nm were delayed. In the presence of histidine, the ﬂuorescence maximum declined by 80% after 10 h of illumination. In the case of propyl gallate this ﬂuorescence was reduced by 70% (Fig. 3B). Also, the peak shift was delayed by these reactive oxygen scavengers. The ﬂuor- escence maximum peak shifts were 18 and 11 nm in the presence of histidine and n-propyl gallate, respect- ively, after 10 h of illumination (Fig. 3C). In the pres- ence of catalase and SOD the changes in ﬂuorescence and peak shift resembled the control experiment (data not shown).

Changes of polypeptide composition

Fig. 3. (A) Low temperature ﬂuorescence emission spectra of the isolated PSI submembrane fractions illuminated for various periods of time with strong WL (2000 lmolÆm)2Æs)1) at 4 (cid:1)C. Inset: the ﬁrst derivative of the absorption maxima in the red is presented to show peak position. (B) Changes in ﬂuorescence intensity meas- ured at the maximum in the presence or absence of scavengers. Results are means ± SD. n ¼ 3. (C) Changes in the position of the ﬂuorescence maximum obtained from the ﬁrst derivative of the ﬂuorescence spectra of PSI complexes. Control (s), histidine (h), and n-propyl gallate (,). For further details see Experimental proce- dures. These experiments were repeated three times and yielded identical spectra; a typical spectrum is presented.

Speciﬁc antibodies of PSI polypeptides were used to study the degradation of proteins in PSI submembrane

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Fig. 5. Quantitative immunoblot analysis of PSI submembrane frac- tions illuminated with strong light at 4 (cid:1)C for different time duration in the presence of histidine or n-propyl gallate. All experimental conditions are given in Experimental procedures.

Fig. 4. Quantitative immunoblot analysis of PSI submembrane frac- tions illuminated with strong light at 4 (cid:1)C for different time dur- ation. All experimental conditions are given in Experimental procedures.

decreased (Table 1). In comparison, the integral mem- brane protein PsaF was more stable (Fig. 4). Addition of reactive oxygen scavengers signiﬁcantly protected the above proteins against photodegradation (Fig. 5, Table 1).

particles during strong illumination. The reaction- center protein PsaA was stable until 4 h and then decreased very slightly with further illumination. How- ever, PsaB protein degradation started after a 1-h exposure and a degradation product appeared, whereas after a 5-h exposure this protein mostly disappeared (Fig. 4). These reaction-center proteins were protected in the presence of histidine and n-propyl gallate (data not shown). The stromal ridge constituted of PsaC, PsaD, and PsaE polypetides provides the docking site for the soluble electron acceptors ferredoxin and ﬂavo- doxin [33,34]. Among these polypeptides, PsaC was relatively sensitive to strong light and this protein star- ted to degrade even after 1 h of illumination and fur- ther illumination accelerated the degradation (Fig. 4, Table 1). PsaD was less sensitive (Fig. 4) and after 6 h of exposure this polypeptide degraded only by 30% (Table 1). PsaE was the most sensitive, after a 3-h exposure more than 60% of this protein content was

Table 1. Quantiﬁcation of photosystem I polypeptides from immuno- blots obtained from Fig. 5.

Control (%)

Histidine (%)

n-Propyl gallate (%)

0 h

3 h

6 h

3 h

6 h

3 h

6 h

100 100 100 100 100 100 100

68 106 36 71 45 62 74

0 72 0 0 0 9 52

105 103 73 91 54 83 83

109 103 55 77 38 37 65

55 106 95 97 63 93 91

45 109 86 106 58 77 76

PsaC PsaD PsaE Lhca1 Lhca2 Lhca3 Lhca4

The outer antenna system of higher plant photo- system I, LHCI, is composed of four proteins with molecular masses of 20–24 kDa, the products of the genes Lhca1–4, which are associated with the PSI core [33–35]. We have analyzed the photodegradation pro- ﬁle of each polypeptide in Fig. 4. Lhca1 is degraded linearly with 30% loss of this protein after 3 h, and it completely disappeared after a 5-h exposure (see also Table 1). However, with the addition of histidine, this degradation was negligible and after 6 h only 20% of this subunit was lost (Fig. 5, Table 1). Surprisingly, this protein was not degraded in the presence of n-pro- pyl gallate (Fig. 5, Table 1). Lhca2 was more sensitive to strong light and started to degrade after 1 h and completely vanished after 3 h. With histidine, this pro- tein declined by 45 and 60% after 3 and 6 h of illu- mination, respectively, while with n-propyl gallate, the degradation of this submit was only about 40% after 3 and 6 h illumination. The Lhca3 subunit linearly degraded with the illumination period. After 3 h of illumination, this protein was lost by 40% and almost completely disappeared after 6 h. In the presence of histidine this protein declined by only 15 and 60% after 3 and 6 h of illumination, respectively, whereas, in presence of n-propyl gallate the protein was stable after 3 h and 20% loss was noticed after 6 h of illu- mination. Lhca4 was the most stable subunit, after 3 h this subunit was altered by 25 and by 50% after 6 h of

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illumination. Histidine and n-propyl gallate retarded the photodegradation. The sensitivity of LCHI pro- teins to strong illumination was Lhca2 > Lhca1 > Lhca3 > Lhca4 (Fig. 4).

in the CD spectra was observed, which is typical of a decrease in helical content. In the presence of histidine and n-propyl gallate the above elipticities were not affected after a 6-h illumination, and after a 10-h expo- sure these changes were still minor (not shown).

Analysis of CD changes

(25%),

As shown in Fig. 6A, strong light induced signiﬁcant alterations in the secondary structure of proteins from PSI submembrane fractions. After a 6-h exposure, a clear decrease in elipticity measured at 222 and 190 nm

to strong light

exposure

The CD curve-analyzing algorithm (see Experimen- tal procedures) revealed that untreated (0 h) PSI sub- membrane fractions were composed of a-helix (24%), turns (22%), and unordered (29%) b-sheet structures (calculated from Fig. 6A). Performing the same spectral analysis after various treatments allowed quantifying the change in secondary structures in PSI fractions. The proportion of a-helix decreased by 33% after 6 h of (Fig. 6B). However, there was no change in a-helix even after a 6-h illumination in the presence of histidine or n-pro- pyl gallate. After 10 h in the presence of both agents, the a-helix helix percentage dropped slightly, although the amplitude of this decrease was comparable to the error margin of the method used for its computation (Fig. 6B). These results clearly reveal that protein con- formation is protected against photodamage in the presence of histidine and n-propyl gallate.

Discussion

The primary objective of this work was to evaluate the protective role of reactive oxygen scavengers against strong light in PSI submembrane fractions. Our data clearly demonstrated that some speciﬁc scavengers sig- niﬁcantly protected both the structure and function of PSI.

(A) Room temperature CD spectra of PSI submembrane Fig. 6. fractions illuminated for various time duration in control PSI sub- membrane particles illuminated without active oxygen scavengers. (B) Content in a-helical structures calculated from CD spectra obtained from experiments such as shown in Fig. 6 A with no additive (s) or with histidine (,), and n-propyl gallate (h). Results are means ± SD. n ¼ 3. All experimental conditions are given in Experimental procedures.

Several authors postulated that oxidative mecha- nisms are the basis of PSI photodamage in intact leaves [19,20,22], isolated chloroplasts [7,8,11], or iso- lated PSI fragments [15–18]. The selective action of reactive oxygen scavengers used here indicated that the species involved in the photodamage of proteins and photochemical functions in the PSI submembrane frac- tions were 1O2, OH, and alkoxyl radicals. However, – were apparently not implicated in the H2O2 and •O2 damaging processes. Addition of n-propyl gallate pro- vided more protection than histidine (Figs 1–3). It is known that n-propyl gallate protects against OH and alkoxyl radicals, and histidine scavenges 1O2 [28,29]. •OH radicals are formed by the reaction Generally, ions between hydrogen peroxide and reduced metal during the so-called Fenton reaction. In the present case, addition of SOD and ⁄ or catalase did not show any protection against strong light. Nonetheless, this was not due to a temperature-dependent inhibition of SOD or catalase as similar changes of absorption, ﬂuorescence, and P700 oxidation under strong illumin-

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ation were observed at both room and low tempera- ture in the PSI submembrane fractions (results not shown). Thus, OH and alkoxyl radicals originated from 1O2. It is well known that •OH and alkoxyl radi- cals can be produced from reactions of 1O2 or •OH with organic molecules [36,37].

Generation of singlet oxygen by Chl is expected dur- ing strong illumination as it depends on the population of excited Chls molecules and is formed by energy transfer from Chl molecules in triplet state to oxygen [27 and references therein, 38]. The involvement of P700 triplet states appearing as a consequence of charge recombination between P700+ and reduced A1 or A0 [39] could not represent a signiﬁcant source for 1O2 production in isolated submembrane fractions because of the low rates of electron ﬂow through PSI in the absence of an electron donor system (Fig. 1). As P700+ is known to be a very efﬁcient quencher of exci- ted singlet states of Chls close to the PSI reaction cen- ter [40], excited states available for oxygen must have appear mostly in the light-harvesting complexes, not in the PSI core. In such case, 1O2 derivatives had ﬁrst to attack antenna Chls [41]. This might explain the faster Chl breakdown compared to the loss of PSI photo- chemical activity (Figs 1 and 2).

[27], then, the Chl molecules with an absorption max- ima in the red located at a relatively long wavelength in Lhca3 and Lhca4 should be bleached ﬁrst. A blue shift in absorption and ﬂuorescence maximum in the red was clearly observed in the PSI submembrane frac- tions (Figs 2 and 3, see also [16–18]). Thus, the pig- ment aggregates absorbing at these long-wavelengths could be involved in photoprotection [16,17,27]. This phenomenon is in agreement with our results showing that Lhca4 and Lhca3, which have red pigments with higher absorption wavelength, are more stable. More- over, Lhca1 and Lhca2 have a higher content in bulk Chl. Thus, the fast degradation of Lhca1 and Lhca2 is due to generation of reactive oxygen species during strong light illumination which leads to some alter- ation in the Chl–protein interaction. Hui et al. [15] reported in PSI core complexes that LHCI-680 is more sensitive due to decreased interaction between Chl–Chl or Chl–protein. The discrepancy in sensitivity to strong light between LHCI-680 and LHCI-730 could be explained by the organization of Chl–Chl or Chl–pro- tein in the Lhca subunits. The degradation of Lhca protein subunits was reduced with the addition of his- tidine or n-propyl gallate in PSI submembrane parti- cles exposed to strong light even after 10 h, with a more pronounced action of n-propyl gallate (Fig. 5, Table 1). The above results are consistent with previ- ous reports of strong light-induced degradation of LHCII proteins where histidine and n-propyl gallate also retarded the photooxidative effects with a more efﬁcient action of n-propyl gallate [28,29].

and

degradation

produced

also

The native PSI complex from higher plants contains about 200 Chls per P700. Among them, LHCI binds about 70–110 Chl a + b molecules and serves as an accessory antenna to harvest light and funnel its energy to the reaction center, P700. The latter is located in the PsaA ⁄ B proteins of the core component (CCI), where 96 Chls are bound [33,34]. The Chl a ⁄ b binding peri- pheral antenna of plants PSI (LHCI) is composed of four nuclear gene products, Lhca1–4, with molecular masses of 20–24 kDa [33,34]. Each Lhca protein binds approximately 10 Chls [42–44]. A total of eight Lhca proteins are thought to be present per PSI: Lhca1 and Lhca4 are present as heterodimers, whereas Lhca2 and Lhca3 are likely present as homodimers [33,34]. Immu- noblot analysis showed that among the Lhca proteins, Lhca2 is more sensitive and Lhca4 is stable to strong light. The sequence of the changes observed in Lhca proteins was: Lhc2 > Lhca1 > Lhca3 > Lhca4 (Fig. 4).

As in the PSI core antenna, excitonically coupled dimers or trimers of Chl a or b in the Lhca were also suggested to form a pool of red pigments of low- energy [45–47], more speciﬁcally in the Lhca4 subunit of the LHCI-730 complex [45,48,49]. Recent ﬁndings conﬁrmed that Lhca2 and Lhca3 are also having low- energy red pigments [44] but this is more pronounced in Lhca3 [35]. If the absorbed energy migrates towards PSI holochromes with higher-absorption wavelengths

The PSI core is a large pigment–protein complex composed of 11–13 protein subunits, the largest two of which, PsaA and PsaB, comprises the molecular masses of 83.2 and 83.4 kDa [33,34]. This is a het- erodimer to which the majority of the core antenna pigments, as well as most of the reaction center cofactors, are bound. In PSI core preparations, strong light exposure induced changes to both reaction-cen- ter proteins [14]. In the present study using submem- brane fractions, PsaB was more sensitive to strong light products (Fig. 4). Similar degradation products of PsaB were observed with no apparent modiﬁcation of PsaA in spinach thylakoids or cucumber leaves illuminated with weak light at low or room temperature [21,29]. These authors proposed that the degradation of PsaB was mainly due to formation of superoxide and hydroxyl radicals and this was protected by n-propyl gallate. In another report using barley leaves illumin- ated with weak light at low temperatures, damage to both reaction-center proteins of PsaA and PsaB was reported [23]. The involvement of reactive oxygen

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species such as superoxide, hydrogen peroxide, and hydroxyl radicals was suggested. They also showed that 1O2 was involved in the damage of reaction- center proteins. In vivo, the photooxidative damages in PSI may occur with a similar pattern as in PSII where reactive oxygen species are thought to initiate protein degradation that is completed by chloroplast proteases [21,30]. It was recently shown that the deg- radation of PsaA ⁄ B proteins and alteration of photo- chemical activity in spinach thylakoids are more intensive at room temperature than at low tempera- ture under strong light [13], which suggested that the photoinactivation may involve an enzymatic contribu- tion to the phenomenon.

susceptible to strong light. The structural model of Thermosynechococcus PSI showed that part of PsaE is sandwiched between the PsaA ⁄ B heterodimer and PsaC [33,53]. The higher plant reaction-center structure retains the same organization as the cyanobacterial complex [52]. Thus, it seems possible that PsaE affects the intracomplex electron transport under some condi- tions. From our data it is clear that the degradation of likely, the PSI stromal ridge starts at PsaE. Most in plants PsaE would determine the susceptibility to photodamage of the PSI complex. Interestingly, the present data also indicate that, even though PsaC seems to be required during the assembly of PsaD and PsaE with the PSI complex [50], PsaC can be lost inde- pendently from the two other extrinsic proteins (Figs 4 and 5).

Excess light clearly altered the secondary protein structure of the PSI submembrane fractions. With increased time of exposure a signiﬁcant portion of the a-helices was lost (Fig. 6). The protein unfolding was signiﬁcantly prevented by histidine and n-propyl gallate (Fig. 6). The alteration of protein secondary structure components as revealed from the CD spectral studies is likely to play a central role in the active oxygen-depend- ent photodegradation processes of the PSI complex. It is the observed structural perturbations possible that involved altered pigment–protein and protein–protein interaction, contributing to the observed decreased efﬁciency of energy migration and capture, and leading to Chl degradation.

Experimental procedures

As mentioned above, in PSI submembrane fractions, PsaA was more resistant to strong light compare to PsaB. PsaA was stable until 4 h of illumination and then started degradation (Fig. 4). The PSI core is mainly composed of bulk antenna Chl that absorbs in a broad band with a maximum at 680 nm, but also con- tains 3–10% of low-energy Chl (red). The quenching of excitations located on the red Chls by P700+ could pro- vide a pathway to prevent the generation of Chl triplet states, which can lead to the formation of harmful 1O2 under strong light illumination [46]. The lower sensitiv- ity of PsaA to photooxidative damage compare to PsaB may indicate that it contains less low energy Chl aggre- gates. Alternatively, PsaA may be less closely associated with the antenna Chl of the light harvesting complexes and thus receive less excess energy that leads to the generation of 1O2. Addition of histidine and n-propyl gallate protected these two reaction-center proteins even after 10 h of illumination (data not shown), which supports the involvement of 1O2.

Isolation of photosystem I submembrane fractions

PSI submembrane particles were isolated from fresh spin- ach leaves obtained from the local market, according to the procedure of Peters et al. [31] with some modiﬁcations [32]. The isolated preparations with Chl content of 1–2 mg ChlÆmL)1 were suspended in a medium containing 20 mm Tricine ⁄ KOH buffer (pH 7.8), 10 mm NaCl, 10 mm KCl, and 5 mm MgCl2, and stored at )80 (cid:1)C until its use. The Chl a ⁄ b ratio was found to be higher than 6.0 in isolated PSI submembrane fractions. Chl was determined in 80% acetone according to Porra et al. [54].

Light treatment

The PSI preparations (500 lg ChlÆmL)1) were illuminated for 5 h with continuous stirring at 4 (cid:1)C using strong white light (WL) (2000 lEÆm)2Æs)1) from a 150 W quartz ⁄ halogen projector lamp. WL was passed through a 5-cm layer of

Apart from PsaA and PsaB, other smaller polypep- tides of PSI were degraded during strong light illumin- ation. PsaC together with PsaD and E comprises the stromal side of PSI. PsaC anchors the two Fe4S4 clus- ters FA and FB, which are needed to carry out the elec- tron transfer from Fx. The order of sensitivity to strong light illumination was PsaE > PsaC > PsaD (Fig. 5, Table 1). Similar results were observed in PSI core particles [15]. This order closely corresponds with the degradation proﬁle observed during disassembly studies using urea treatment [50]. The crystal structure of plant and cyanobacterium PSI revealed that loops at the stromal surface of PsaA ⁄ B partly contribute the binding interface to the PsaC, D and E subunits [51,52]. These observations are in agreement with our data showing that the degradation of PsaC subunit ensue almost simultaneously the degradation of Lhca2 and PsaB subunits (Figs 4 and 5). PsaD is stable even illumination, whereas, PsaE is more after 5 h of

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Photoprotective effect of active oxygen scavengers

– and H2O2, respectively [28,29].

water containing CuSO4 to cut the infrared radiation. The samples were illuminated in the presence of various active oxygen scavengers to selectively remove the reactive oxygen species. The scavengers used were histidine at a concentra- tion of 25 mm for 1O2, n-propyl gallate at 1 mm for -OH and alkoxyl radicals, and SOD and catalase at 250 lgÆmL)1 for •O2 temperature of 20 (cid:1)C. Each CD spectrum was the average of ﬁve accumulations at a scanning speed of 20 nmÆmin)1 and a 1 nm spectral band width. The base line was correc- ted with blank buffer for each spectrum. The secondary structure of PSI proteins was determined from the CD spectra using the cd pro program as described previously [56]. Sample concentration was 50 lgÆmL)1 Chl in the con- trol measurements (prior to the illumination).

Measurement of P700 redox state

Immunoblot analysis

PSI submembrane polypeptides were separated by poly- acrylamide gel electrophoresis (PAGE) according to Raja- gopal et al. [32]. Electrophoresis was performed on a 13% separating and 4% stacking gel of polyacrylamide. The sus- pension (10 lL aliquots) of submembrane fractions were repeatedly taken during the course of photoinhibition. Such aliquots contained 5 lg Chl obtained from the suspension of untreated preparations. An equal volume of 2· buffer was added to the aliquots.

The changes in the redox state of P700, the primary donor of PSI, were measured by the light-induced absorbance changes at 830 nm (DA830) using a dual-wavelength unit ED-P700DW connected to a PAM ﬂuorometer (Walz, Effeltrich, Germany) as described by Schreiber et al. [55]. All measurements were carried out at an identical sensitiv- ity of the PAM ﬂuorometer. The absorbance changes at 830 nm represented only the oxidation and reduction of P700 as no contribution to the absorbance changes due to plastocyanin redox transformations can be detected with the ED-P700DW unit. White actinic light was obtained from the KL 1500 projector (Walz, Effeltrich, Germany). The aliquots of 60 lL taken from the suspension of PSI submembrane particles during strong light treatment were added to 140 lL of suspension buffer containing 200 lm ascorbate as an artiﬁcial donor. Chl concentration during the measurements of DA830 was 30 lgÆmL)1 in the control experiments (prior to the illumination).

Measurement of absorption spectra

To identify and quantify the PSI polypeptides, immuno- blotting was carried out essentially as described by Towbin et al. [57]. Western blotting was performed by electropho- retic transfer of proteins to nitrocellulose membranes (0.45 lm, Millipore, Billerica, MA, USA). The membrane was incubated with polyclonal antibodies raised in rabbits against PSI complex. Subsequently, secondary antibodies ligated to alkaline phosphate were applied. Bromo-chloro- indolyl-phosphate and tetrazolium blue were used for the coloring reaction. The developed membranes were analyzed by using Bio-Rad Gel-Doc 2000 system (Hercules, CA, USA).

Acknowledgements

The room temperature absorption spectra and their ﬁrst derivatives were recorded using a PerkinElmer Lambda 40 spectrophotometer (Wellesley, MA, USA). Ten microlitres of the suspension of submembrane particles were repeatedly taken during the illumination. Such aliquots taken from the suspension of untreated particles contained 5 lg of Chl, whereas the Chl content decreased gradually in the samples taken during strong illumination.

The authors wish to thank Drs J.H. Golbeck, K. Sonoike and P. Chitnis for antibodies and J. Harnois for helpful professional assistance. This research was supported by the Natural Sciences and Engineering Research Council of Canada and by Fonds Que´ be´ cois de la Recherche sur la Nature et les Technologies.

Fluorescence spectroscopy

References

1 Powles SB (1984) Photoinhibition of photosynthesis induced by visible light. Annu Rev Plant Physiol 35, 15–44.

Low temperature (77 K) spectra of ﬂuorescence emission excited at 436 nm were measured as reported previously [16] using a PerkinElmer LS55 spectroﬂuorometer. The Chl content of the samples was adjusted to 5 lgÆmL)1. The excitation and emission slit width were set at 5 and 2.5 nm, respectively.

Circular dichroism spectroscopic analysis

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CD spectra were measured in a Jasco-720 spectropolari- meter (Easton, MD, USA) in a cell with a 0.1 mm optical path length over a wavelength range of 190–260 at a 2 Aro EM, Virgin I & Andersson B (1993) Photoinhibi- tion of photosystem II. Inactivation, protein damage and turnover. Biochim Biophys Acta 1143, 113–134. 3 Prasil O, Adir N & Ohad I (1992) Dynamics of photo- system II: Mechanism of photoinhibition and recovery process. In Topics in Photosynthesis, the Photosystem Structure, Function and Molecular Biology, Vol. 11 (Barber J, ed.), pp. 295–384. Elsevier, Amsterdam.

S. Rajagopal et al.

Photoprotective effect of active oxygen scavengers

4 Andersson B & Barber J (1996) Mechanisms of photo- 18 Rajagopal S & Carpentier R (2003) Retardation of

photoinduced changes in photosystem I submembrane particles by glycinebetaine and sucrose. Photosynth Res 78, 77–85. damage and protein degradation during photoinhibition of photosystem II. In Photosynthesis and the Environ- ment (Baker NR, ed.), pp. 101–121. Kluwer Academic Publishers, Dordrecht, The Netherlands.

5 Mulo P, Laakso S, Maenpaa P & Aro E (1998) Step- wise photoinhibition of photosystem II. Plant Physiol 117, 483–490. 19 Terashima I, Funayama S & Sonoike K (1994) The site of photoinhibition in leaves of Cucumis sativus L. at low temperatures is photosystem I, not photosystem II. Planta 193, 300–306.

6 Barber J & Andersson B (1992) Too much of a good thing: light can be bad for photosynthesis. Trends Biochem Sci 17, 61–66. 7 Satoh K (1970) Mechanism of photoinactivation in 20 Sonoike K, Terashima I, Iwaki M & Itoh S (1995) Destruction of photosystem I iron–sulfur centers in leaves of Cucumis sativus L. by weak illumination at chilling temperatures. FEBS Lett 362, 235–238.

photosynthetic systems. I. The dark reaction in photo- inactivation. Plant Cell Physiol 11, 15–27.

21 Sonoike K, Kamo M, Hihara T & Enami I (1997) The mechanism of the degradation of PsaB gene product, one of the photosynthetic reaction center subunits of photosystem I, upon photoinhibition. Photosynth Res 53, 55–63. 22 Tjus SE, Moller BL & Scheller HV (1999) Photoinhibi- 8 Satoh K (1970) Mechanism of photoinactivation in photosynthetic systems. II. The occurrence of and properties of two different types of photoinactivation. Plant Cell Physiol 11, 29–38.

9 Satoh K & Fork DC (1982) Photoinhibition of reaction centers of photosystem I and II in intact Bryopsis chlor- oplasts under anaerobic conditions. Plant Physiol 70, 1004–1008. 10 Satoh K (1970) Mechanism of photoinactivation in tion of photosystem I damages both reaction centre pro- teins PSI-A and PSI-B and acceptor-side located small photosystem I polypeptides. Photosynth Res 60, 75–86. 23 Allen RD (1995) Dissection of oxidative stress tolerance using transgenic plants. Plant Physiol 107, 1049–1054. 24 Foyer CH, Lelandais M & Kunert KJ (1994) Photooxi-

dative stress in plants. Physiol Plant 92, 696–717. 25 Vass I (1997) Adverse effects of UV-B light on the

photosynthetic systems. III. Site and mode of photoacti- vation in photosystem I. Plant Cell Physiol 11, 187–197. 11 Inoue K, Sakurai H & Hiyama T (1986) Photoinactiva- tion sites of photosystem I in isolated chloroplasts. Plant Cell Physiol 27, 961–968. structure and function of the photosynthetic apparatus. In Handbook of Photosynthesis (Pessarakli M, ed.), pp. 931–949. Marcel Dekker, New York. 26 Demmig-Adams B & AdamsWW III (1992) Photopro-

12 Inoue K, Fujii T, Yokoyama E, Matsuura K, Hiyama T & Sakurai H (1989) The photoinhibition site of photosystem I in isolated chloroplasts under extremely reducing conditions. Plant Cell Physiol 30, 65–71.

tection and other responses of plants to high light stress. Annu Rev Plant Physiol Plant Mol Biol 43, 599–626. 27 Carpentier R (1997) Inﬂuence of high light intensity on photosynthesis: photoinhibition and energy dissipation. In Hand Book of Photosynthesis (Pessarakli M, ed.), pp. 443–449. Marcel Dekker, New York. 13 Velitchkova M, Yruela I, Alfonso M & Picorel R (2003) Different kinetics of photoinactivation of photosystem I-mediated electron transport and P700 in isolated thy- lakoid membranes. J Photochem Photobiol Biol B 69, 41–48.

14 Baba K, Itoh S, Hastings G & Hoshina S (1996) Photo- inhibition of photosystem I electron transfer activity in isolated photosystem I preparations with different chlorophyll contents. Photosynth Res 47, 121–130.

28 Zolla L & Rinalducci S (2002) Involvement of active oxygen species in degradation of light-harvesting pro- teins under light stresses. Biochemistry 41, 14391–14402. 29 Rinalducci S, Pedersen JZ & Zolla L (2004) Formation of radicals from singlet oxygen produced during photo- inhibition of isolated light-harvesting proteins of photo- system II. Biochim Biophys Acta 1608, 63–73. 30 Sonoike K (1996) Degradation of psaB gene product, 15 Hui Y, Jie W & Carpentier R (2000) Degradation of the photosystem I complex during photoinhibition. Photo- chem Photobiol 72, 508–512. 16 Rajagopal S, Bukhov NG & Carpentier R (2002)

the reaction center subunit of photosystem I, is caused during photoinhibition of phtosystem I: possible involvment of active oxygen species. Plant Sci 115, 157–164.

Changes in the structure of chlorophyll–protein com- plexes and excitation energy transfer during photo- inhibitory treatment of isolated photosystem I submembrane particles. J Photochem Photobiol B: Biol 62, 194–200.

31 Peters FALJ, Van Wielink JE, Wong Fong Sang HW, De Vries S & Kraayenhof R (1983) Studies on well coupled photosystem I-enriched subchloroplast vesicles: content and redox properties of electron-transfer com- ponents. Biochim Biophys Acta 724, 159–165.

FEBS Journal 272 (2005) 892–902 ª 2005 FEBS

901

32 Rajagopal S, Bukov NG & Carpentier R (2003) Control of energy dissipation and photochemical activity in 17 Rajagopal S, Bukhov NG & Carpentier R (2003) Photo- inhibitory light-induced changes in the composition of chlorophyll–protein complexes and photochemical activ- ity of photosystem I submembrane fractions. Photochem Photobiol 77, 284–291.

S. Rajagopal et al.

Photoprotective effect of active oxygen scavengers

47 Schmid VHR, Thome P, Ruhle W, Paulsen H, Kuhl- photosystem I by NADP-dependent reversible confor- mational changes. Biochemistry 42, 11839–11845.

bandt W & Rogle H (2001) Chlorophyll b is involved in long-wavelength spectral properties of light-harvesting complexes LHCI and LHCII. FEBS Lett 499, 27–31. 48 Melkozernov AN & Blankenship R (2003) Structural 33 Scheller HV, Jensen PE, Haldrup A, Lunde C & Knoet- zel J (2001) Role of subunits in eukaryotic photosystem I. Biochim Biophys Acta 1507, 41–60. 34 Jensen PE, Haldrup A, Rosgaard L & Scheller HV

modeling of the Lhca4 subunit of LHCI-730 peripheral antenna in photosystem I based on similarity with LHCII. J Biol Chem 278, 44542–44551. 49 Melkozernov AN, Lin S, Schmid VH, Paulsen H, (2003) Molecular dissection of photosystem I in higher plants: topology, structure and function. Physiol Plant 119, 1–9.

35 Castellatti S, Morosinotto T, Robert B, Caffarri S, Bassi R & Croce R (2003) Recombinant Lhca2 and Lhca3 subunits of the photosystem I antenna system. Biochemistry 42, 4226–4234. Schmidt GW & Blankenship RE (2000) Ultrafast excita- tion dynamics of low energy pigments in reconstituted peripheral light-harvesting complexes of photosystem I. FEBS Lett 471, 89–92. 36 Elstner EF (1982) Oxygen activation and oxygen toxic- 50 Antonkine ML, Jordan P, Fromme P, Kraub N, ity. Annu Rev Plant Physiol 33, 73–96.

37 Halliwell B & Gutteride S (1984) Oxygen toxicity, oxy- gen radicals, transition metals and disease. Biochem J 219, 1–14.

Golbeck JH & Stehlik D (2003) Assembly of protein subunits within the stromal ridge of photosystem I. Structural changes between unbound and sequentially PSI-bound polypeptides and correlated changes of mag- netic properties of the terminal iron sulfur clusters. J Mol Biol 327, 671–697. 38 Siefermann-Harms D (1987) The light-harvesting and protective functions of carotenoids in photosynthetic membranes. Physiol Plant 69, 561–568. 39 Warren PV, Golbeck JH & Warden JT (1993) Charge occurs directly 51 Jordan P, Fromme P, Witt HT, Klukas O, Saenger W & Krauss N (2001) Three-dimensional structure of cyanobacterial photosystem I at 2.5 angstrom resolution. Nature 411, 909–917. 52 Ben-Shem A, Frolow F & Nelson N (2003) Crystal

recombination between P700+ and A (cid:1) 1 to the ground state of P700 in a photosystem I core devoid of FX,FB and FA. Biochemistry 32, 849–857. 40 Nuijs AM, Shuvalov VA, van Gorkom HJ, Plijter JJ & Duysens LNM (1986) Picosecond absorbance difference spectroscopy on the primary reaction and the antenna- excited states in photosystem I particles. Biochim Bio- phys Acta 850, 310–318. structure of plant photosystem I. Nature 426, 630–635. 53 Klukas O, Schubert WD, Jordan P, Krauss N, Fromme P, Witt HT & Saenger W (1999) Photosystem I, an improved model of the stromal subunits PsaC, PsaD, and PsaE. J Biol Chem 274, 7351–7360.

41 Clarke RH, Jagannathan SP & Leenstra WR (1980) Optical-microwave double resonance spectroscopy of in vivo chlorophyll. Photochem Photobiol 32, 805–808. 42 Jansson S, Andersson B & Scheller HV (1996) Nearest neighbor analysis of higher-plant photosystem I holo- complex. Plant Physiol 12, 409–420. 54 Porra RJ, Thompson WA & Kriedemann PE (1989) Determination of accurate extinction coefﬁcients and simultaneous equations for assaying chlorophylls a and b extracted with four different solvents: veriﬁcation of the concentration of chlorophyll standards by atomic absorption spectroscopy. Biochim Biophys Acta 975, 384–394. 55 Schreiber U, Klughammer C & Neubauer C (1988)

43 Ganateg U, Strand A, Gustafsson P & Jansson S (2001) The properties of the chlorophyll a ⁄ b-binding proteins Lhca2 and Lhca3 studied in vivo using antisense inhibi- tion. Plant Physiol 127, 150–158. Measuring P700 absorbance changes around 830 nm with a new type of pulse modulated system. Z Natur- forsch 43c, 686–698.

44 Schmid VHR, Potthast S, Wiener M, Bergauer V, Paul- sen H & Storf S (2002) Pigment binding of photosytem I light harvesting proteins. J Biol Chem 277, 37307– 37314. 45 Melkozernov AN (2001) Excitation energy transfer in 56 Sreerama N & Woody RW (2000) Estimation of protein secondary structrure from circular dichroism spectra: comparison of CONTIN, SELCON, and CDSSTR methods with an expanded reference set. Anal Biochem 287, 252–260. photosystem I from oxygenic organisms. Photosynth Res 70, 129–153.

FEBS Journal 272 (2005) 892–902 ª 2005 FEBS

902

46 Gobet B & van Grondelle R (2001) Energy transfer and trapping in photosystem I. Biochim Biophys Acta 1507, 80–99. 57 Towbin H, Stahelin T & Gordon J (1979) Electrophor- esis transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some application. Proc Natl Acad Sci USA 76, 43450–43454.