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1Department of Biology, Faculty of Science, Kanazawa University, Kakuma, Kanazawa 920-11; 2National. Institute for Basic Biology, Myodaiji, Okazaki 444, ...

Photosynthesis Research 47: 121-130, 1996. ~) 1996 Kluwer Academic Publishers. Printed in the Netherlands.

Regular paper

Photoinhibition of Photosystem I electron transfer activity in isolated Photosystem I preparations with different chlorophyll contents Kyoko Babal, Shigeru Itoh2, Gary Hastings3 & Satoshi Hoshina1,* 1Department of Biology, Faculty of Science, Kanazawa University, Kakuma, Kanazawa 920-11; 2National Institute for Basic Biology, Myodaiji, Okazaki 444, Japan; 3Department of Chemistry and Biochemistry, Arizona State University, Tempe, Arizona 85287-1604, USA; *Authorfor correspondence Received 1 January 1995; accepted in revised form 29 November 1995

Key words: antenna chlorophyll, oxygen damage, P700, photoinhibition, Photosystem I, triplet state Abstract

Photoinhibition of the light-induced Photosystem I (PS I) electron transfer activity from the reduced dichlorophenol indophenol to methyl viologen was studied. PSI preparations with Chl/P700 ratios of about 180 (PS 1-180), 100 (PS I-100) and 40 (PS I(HA)-40) were isolated from spinach thylakoid membranes by the treatments with Triton X-100, followed by sucrose density gradient centrifugation and hydroxylapatite column chromatography. White light irradiation (1.1 x 104/~E m -2 s -l) of PS 1-180 for 2 hours bleached 50% of the chlorophyll and caused a 58% decrease in the electron transfer activity with virtually no loss of the primary donor, P700. The flash-induced absorbance change showed the decay phase with a half time of about 10/.ts that was attributed to the P700 triplet, suggesting that the photoinhibitory light treatment caused the destruction of the PSI acceptor(s), Fx and possibly Al. PS 1-100 was similarly photobleached by the irradiation and the electron transfer activity decreased. There was, however, no apparent photoinhibition of the electron transport activity in PS I(HA)-40. Photoinhibition similar to that seen in PS I- 180 also occurred in membrane fragments that were isolated without any detergent from a PS IIdeficient mutant strain of the cyanobacterium Synechocystis sp. PCC 6803. PS I- 180 was not photoinhibited under anaerobic conditions. The production of superoxide and fatty acid hydroperoxide during white light irradiation was significantly greater in PS 1-180 than in PS I(HA)-40. The mechanism of photoinhibition in PSI preparations is discussed in relation to the formation of toxic oxygen molecules.

Abbreviations: Ao,AI -primary and secondary electron acceptors of PS I; CD-circular dichroism; DCPIP-2,6dichlorophenol indophenol; FA, FB, Fx - iron-sulfur centers A, B, X; H A - hydroxylapatite; LHC I - lightharvesting complex of PS I; MDA-malondialdehyde; M V - methyl viologen; Na-Asc-sodium L-ascorbate; P700-primary electron donor of PS I; PFD-photon flux density; PS I-A and PS I-B-psaA and psaB gene products; TBAthiobarbituric acid Introduction

The photosynthetic activities of chloroplasts or leaves are damaged when exposed to strong light irradiation. This process, called photoinhibition, has been thoroughly investigated with regard to PS II electron transport (Powles 1984; Barber and Andersson 1992; Prasil et al. 1992; Aro et al. 1993). Photoinhibition can be induced by two mechanisms, either from the acceptor

or the donor side of PS II electron transport (Barber and Andersson 1992; Aro et al. 1993). At the acceptor side, excess light causes double reduction of the quinone acceptor of PS II and results in charge recombination between the oxidized donor Chl P680 and the reduced acceptor pheophytin a, which generates a P680 triplet state. The triplet reacts with oxygen and forms highly reactive and toxic species such as singlet oxygen molecules. These events seem to induce

122 the degradation of the reaction center D 1-polypeptide (Barber and Andersson 1992; Art et al. 1993) At the donor side, when the electron donation is slower than electron withdrawal, the lifetime of the highly oxidizing species P680 + increases. The photoaccumulation of P680 + leads to the oxidation and destruction of an accessory chlorophyll and of t-carotene. These events also lead to the degradation of D l-polypeptide and result in the inactivation of PS II (Barber and Andersson 1992; Art et al. 1993) There are few reports about the inactivation of PS I electron transport by the light treatment in vitro. Satoh (1970a, b, c) demonstrated that the light treatment of spinach chloroplasts induced the inactivation of PS I as well as PS II electron transport. The photoinhibition of P S I occurred in the presence of oxygen but not under anaerobic conditions. Satoh and Fork (1982) studied the photoinhibition of PS I in intact chloroplasts of Bryopsis corticulans under anaerobic conditions. They indicated that the initial events in photoinhibition were the destruction of the reaction center of PS I, P700. On the other hand, illumination of isolated spinach chloroplasts under aerobic conditions caused the destruction of three Fe-S centers of PSI (Inoue et al. 1986). Under extremely reducing conditions, electron transfer between the electron acceptor Chl A0 and the iron-sulfur center Fx was inhibited (Inoue et al. 1989). Purcell and Carpentier (1994) recently reported that Chl bleaching and P700 degradation occurred simultaneously in illuminated PS I complexes. Thus, the mechanism ofPS I photoinhibition has not yet been clarified. P S I particles isolated from thylakoid membranes using Triton X- 100, contain about 180 Chl/P700 (Mullet et al. 1980) and are termed PS 1-180, which represents the number of Chl molecules per molecule of P700. This complex contains the reaction center proteins (PS I-A and PS I-B) that bind about 100 molecules of Chl a and the light harvesting chlorophyll-proteins (LHC I) that bind about 80 molecules of Chl a and b (Chl a/b ratio of ,,~3.5) (Malkin 1987), as well as peripheral proteins that contain no Chl. This complex contains the primary electron donor P700 (dimer of Chl a), the primary acceptor A0 (monomer of Chl a), the secondary acceptor Al (phylloquinone) and the three [4Fe-4S] centers, named Fx, FA and FB (see reviews by Golbeck and Bryant 1991 and by Hoshina and Itoh 1993). LHC I can be removed from PS 1-180 by washing with Triton X-100 (Mullet et al. 1980). The preparation binds about 100 Chl/P700 and is termed PS I-100. This preparation retains all the electron carriers

from P700 to FA/FB and the protein subunits except for LHC I. In this study, we isolated PSI complexes with various sizes of antennae from spinach thylakoid membranes, as well as P S I membrane fragments of the cyanobacterium Synechocystis sp. PCC 6803. We then studied the photoinhibition of electron transport. Irradiation of spinach PS 1-180 and PS 1-100, as well as the cyanobacterial PSI membrane fragments inactivated PS I-mediated electron flow. The electron transfer activity in PS I(HA)-40 (40 Chl/P700), which was prepared by solubilization of spinach PS 1-100 with Triton followed by hydroxylapatite gel column chromatography, was not damaged by the light treatment. The mechanism of photoinhibition in PSI preparations is discussed.

Materials and methods

Preparation of spinach PSI complexes All procedures described below were performed at 4 °C unless otherwise stated. Thylakoid membranes were isolated in a medium containing 50 mM TrisHC1 (pH 7.8), 0.33 M sorbitol, and 5 mM MgCI2 from spinach leaves obtained from a local market (Hoshina and Itoh 1987). PS 1-180 was prepared by solubilizing thylakoid membranes with Triton X-100 according to Mullet et al. (1980) and stored at - 8 0 ° C until use. PS 1100 was prepared from PS 1-180 according to Mullet et al. (1980) with minor modification as follows. After sucrose density gradient ultracentrifugation, the PS 1-100 band was diluted with the same volume of distilled water and stirred with Bit-Beads SM-2 (lg Bit-Beads/10 mg Triton X-100) for 3 h to remove the detergent (Holloway 1973). The mixture was filtered through a nylon mesh (82/~m) and centrifuged at 40,000 × g for 30 min. The pellet was resuspended in distilled water and stored at - 8 0 °C until use. PS I(HA)-40 was prepared from PS 1-100 as follows. PS 1-100 was resuspended in 50 mM Tris-HC1 (pH 7.8), containing 20 mM Na-Asc, 2 mM MgC12, and 0,8% (w/v) Triton X-100 at a Chl concentration of 0.2 mg/ml. Solubilized PS 1-100 was loaded onto a column containing hydroxylapatite gel equilibrated with 10 mM Naophosphate buffer (pH 7.0). The gel was washed with the suspension medium until the eluate became colorless, then with 10 mM Na-phosphate buffer (pH 7.0). PS I(HA)-40 was eluted with 0.2 M

123 Na-phosphate buffer (pH 7.0) containing 0.8% (w/v) Triton X- 100. Triton X-100 in the eluate was removed with Bio-Beads SM-2 as described above. The pellet was resuspended in 50 mM Tricine-NaOH (pH 7.8), containing 0.1 M sorbitol, and 10 mM NaC1 and stored at - 8 0 °C until use.

Preparation of cyanobacterial PSI membrane fragments Cyanobacterial P S I membrane fragments were prepared without detergent from the PS II deletion mutant of cyanobacterium Synechocystis sp. PCC 6803 (Hastings et al. 1994), which contained no major PS II proteins (psbA, psbC and psbD gene products) (Vermaas et al. 1987, 1988).

Photoinhibitory light treatments Spinach PS I complexes were suspended in 0.1 M sorbitol, 10 mM NaC1, 50 mM Tricine-NaOH (pH 7.8) and 0.05% (w/v) Triton X-100 at 100 #g Chl/ml, and illuminated with white light (PFD = 1.1 x 104 #E m -2 s - l ) using a 1 kW tungsten lamp slide projector at 25 °C with stirring unless otherwise stated. Cyanobacterial PS I membrane fragments were illuminated under the same conditions described above except that Triton X- 100 was omitted from the incubation medium. To study the effect of anaerobic conditions, PS 1180 suspension was sealed in a test tube with a septum and the gas phase was exchanged from air to argon. After bubbling the sample with argon gas for 2 h at room temperature, 50 mM glucose and 50 U/ml glucose oxidase were injected into it, and the suspension was incubated for further 2 h.

Measurements Absorption spectra were measured with a Shimadzu UV-300 spectrophotometer equipped with a SAPCOM- 1 microprocessor. Spectra were normalized at their red maxima. CD spectra were measured with a JASCO J-20 spectro-polarimeter. PS I-mediated electron transfer from reduced DCPIP to MV was measured by 02 consumption using a Clark type electrode (Yellow Springs Model 53) at 25 °C with actinic light of saturating intensity obtained from a 750 W tungsten lamp slide projector, passed through red (Toshiba VR 62) and heat-absorbing filters (Toshiba IRA-25).

Flash-induced absorption changes at 430 nm (sub millisecond-time resolution) were measured using a laboratory built, split-beam spectrophotometer with an actinic flash from a xenon flash lamp (Sugawara DSX-240B, 12 #s half-width), passed through two red filters (Corning C.S. No. 2-58) and a heat-absorbing filter (Hoya HA-50). The photomultiplier was protected with two Corning C.S. No. 4-96 blue filters. Signals were transferred to a Digital Analyzer (Autonics, APC-204) and analyzed with an NEC PC-9801 computer. Another spectroscopic assembly was used for #s-time resolution as described previously (Hoshina et al. 1990) with a 10 ns (half-width) laser actinic flash at 532 nm being generated by an Nd-YAG laser (QuantaRay DCR 2-10). The P700 content was determined chemically by measuring the ferricyanide-oxidized minus ascorbatereduced difference spectrum using a Shimadzu UV-300 spectrophotometer. The difference extinction coefficient was 64 mM -I cm -1 for the red absorbance minimum around 700 nm with respect to the isosbestic point at 725 nm (Hiyama and Ke 1972). The reaction mixture contained P S I complexes (15/~g Chl/ml), 50 mM Tricine-NaOH (pH 7.8), 0.1 M sorbitol, 10 mM NaCI and 0.05% (w/v) Triton X-100. For the reduction of P700, a mixture of 1 mM Na-Asc and 10/~M DCPIP was added to the reference cuvette and for oxidation, 50 #M ferricyanide was added to the sample cuvette. Cytochrome c photoreduction activity was measured at 550 nm with the UV-300 spectrophotometer. The sample was illuminated with saturating light from a 750 W tungsten lamp slide projector which was passed through two red filters (Corning C.S. No. 258) and a heat-absorbing filter (Hoya HA-50). The photomultiplier was protected with a Coming C.S. No. 4-96 blue filter. The sample was dark-adapted for 15 min before each measurement. Lipid peroxidation was measured by monitoring the MDA formation. After incubating PS I-180 or PS I(HA)-40 (15 #g Chl/ml) with 50 mM Tricine-NaOH (pH 7.8), containing 0.1 M sorbitol, 10 mM NaCI and 0.05% (w/v) Triton X-100, an equal volume of 0.5% TBA in 20% TCA was added to an aliquot of the diluted incubation mixture. The mixture was heated in a boiling water bath for 25 min, then clarified by centrifugation. The amount of MDA was calculated from the absorbance at 532 nm minus that at 600 nm using an extinction coefficient of 155 mM -1 cm-l(Heath and Packer 1968). The Chl concentration and Chl a/b ratio were determined in 80% acetone as described by Arnon (1949).

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Figure 1. Room temperature absorption spectra of spinach PS I- 180 (sofid line), PS 1-100 (broken line), and PS I(HA)-40 (dotted line). The samples were suspended in 50 mM Tricine-NaOH (pH 7.8) containing 0.05% Triton X-100. Spectra were normalized at their red maxima.

Chemicals Bio-Beads SM-2 and Hydroxylapatite were purchased from Bio Rad. Glucose oxidase was obtained from Wako Pure Chemical Industries, Ltd.

Results

Characterization of spinach PS I preparations PS I complexes with various sizes of antennae were prepared from spinach. PS I- 180 had a Chl/P700 ratio of 160~180 and a Chl alb ratio of 5,,~7 as reported by Mullet et al. (1980) and consisted of LHC I and the core complex of PS I. Solubilization of PS I-180 with 0.45% (w/v) Triton X-100 followed by sucrose density gradient ultracentrifugation produced a PS I- 100 preparation that lacked LHC I and had 80,,d00 Chl molecules per P700 and a Chl alb ratio of over 8. Three LHC I bands with a molecular mass of 20,-~25 kDa (Malkin 1987), were absent in the SDS-PAGE of PS I- 100 (data not shown). For further removal of antenna Chl, PS 1-100 was absorbed onto hydroxylapatite gel and washed with 0.8% (w/v) Triton X-100. This procedure reduced the ratio of Chl per P700 to 30,~40. This preparation was termed PS I(HA)-40 and it had the same SDS-PAGE

620

660

I 700 620 660 Wavelength (nm)

683 700

Figure 2. Room temperature CD spectra of spinach PS I-lO0 (a) and PS I(HA)-40 (b). Chl concentrations in both samples were 12 /lg Chl/ml. Suspension medium was same as that described in the legend to Figure 1.

polypeptide profile as that of PS I- 100 (data not shown). PS 1-180, PS 1-100, and PS I(HA)-40 retained high electron transfer activities from P700 to FA/FB. Figure 1 shows the absorption spectra of the PS I preparations. PS 1-180 had red and blue absorption maxima at 677 and 435nm, respectively, due to Chl a. Absorption shoulders around 470 and 650 nm represent Chl b bound to LHC I (Bassi and Simpson 1987). The shoulder around 470 nm was lower in PS I- 100 and in PS I(HA)-40. This indicates a loss of Chl b and a decrease in the carotenoid content in PS I(HA)-40. The red peak of Chl a shifted to the blue in PS I(HA)40 compared with that in PS 1-100. This indicates the loss of antenna Chl forms that absorbs at the longer wavelength in PS I(HA)-40. Both the CD spectra of PS 1-100 and PS I(HA)-40 had positive and negative maxima near 665 and 684 nm (Figure 2). The intensities of the peaks were about 25~40% higher in PS I(HA)-40. P S I antenna Chl molecules are divided into three groups. These are peripheral antennae (,-~100 Chl a and b) bound to the light harvesting complex of P S I (LHC I), the internal antennae (,-~60 Chl a) bound to the reaction center proteins and the core antennae (--~40 Chl a) that also bind to the reaction center proteins. The internal antennae are easily extracted with detergents such as Triton X-100, while the core antennae bind more tightly than the internal antenna Chl (Mullet et al. 1980; Bassi and Shimpson 1987; Malkin 1987).

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Figure 3. Effect of photoinhibitory light intensity on the photoinhibition in spinach PS I- 180. PS I- 180 (100/zg Chl/ml) was preilluminated with various light intensities at 25 °C for 2 h. MV photoreduction activity (circles) was measured by O2 consumption in a reaction mixture containing PSI complex (10/zg Chl/ml), 50 mM TricineNaOH (pH 7.8), 0.1 M sorbitol, 10 mM NaCI, 0.3 mM MV, 2 mM Na-Asc, 0.3 mM DCPIP, and 0.02% (w/v) Triton X-100. The P700 content (squares) was chemically measured and chlorophyll content (triangles) was estimated from the absorbance at the red maximum.

These antennae exhibit typical absorption peaks at 705, 696~7, and 685,-~90 nm, respectively. Judging from the sizes of their antennae and absorption spectra, PS 1-100 seems to bind the internal and core antennae, whereas PS I(HA)-40 binds only the core antennae.

Photoinhibition in each preparation Figure 3 shows the effect of photoinhibitory light intensity on the photoinhibition of PS 1-180. After light treatment at 1.1 x 104/~E m -2 s - l , MV photoreduction activities and Chl levels were reduced to 48 and 33% of the control, respectively. However, the P700 content did not decrease at this light intensity. Light treatment at 2.1 x 104 / z E m -2 s - l destroyed 84% of the P700 and bleached 93% of the Chl as reported by Purcell and Carpentier (1994). These results indicate that the Chl content, MV-photoreduction activity and P700 content respond in a different manner to the illumination intensity. In the study described below, samples were preilluminated at a light intensity of 1.1 x 104 #E m -2 s - l to produce maximal inhibition without P700 destruction. Table 1 shows the effect of preillumination on the MV-photoreduction activities of PS 1-180, PS 1-100 and PS I(HA)-40. The activity in PS I-180 decreased with the period of preillumination and reached about 60% of the control after 2 h. The extent of the inactiva-

°°o1.oo 1/relative light intensity Figure 4. Effect of actinic light intensity on the MV photoreduction activity of the control and the light-treated spinach PS I- 180. Control incubated in the dark (solid circles), light-treated for 1 h (triangles), light-treated for 2 h (open circles). Preillumination conditions were described in Table 1. The maximal actinic light intensity was 3 000 /zE m -2 s - t , and the intensity was changed with neutral density filters.

tion varied (30,,~60%) among preparations. The procedure did not affect the P700 content, but destroyed about half of the antenna Chl. Neither the activity nor the Chl content was affected by incubation in the dark. PS 1-100 was photoinhibited almost to the same extent as thatof PS 1-180 (Table 1). This indicates that the removal of the peripheral antennae (Chls bound to LHC I) have little effect on the photoinhibition. By contrast, PS I(HA)-40 was not inactivated but the antenna Chl in PS I(HA)-40 was slightly bleached (about 25% of the control) by the light treatment. The data suggest that the internal antennae (Chl a extracted with Triton X-100 from the reaction center proteins) rather than the core antennae (Chl a tightly bound to the proteins) are implicated in the photoinhibition of PS I. Cyanobacterial PS I fragments isolated from the PS II-less mutant of Synechocystis sp. PCC 6803 without detergent, showed a small, but significant decrease in MV photoreduction activity by light treatment (Table 1). This indicates that photoinhibition occurs even in the P S I preparation isolated without Triton X- 100.

Site of photoinhibition in PS 1-180 Figure 4 shows the effect of actinic light intensity on MV photoreduction activity in the light-treated PS I-

126

Table1. Effectof a photoinhibitorylighttreatmenton the MV photoreductionactivities, Chl and P700 contents in PSI preparations

PS 1-180 PS 1-100 PS I(HA)-40 Cyanobacted~ PSI

MV photoreduction Light Dark

Chl content Light Dark

59 56 111 75

53 . 79 92

102 108 114 96

.

97 . 104 110

P700 content Light Dark 94

96

81 -

89 -

.

Spinach PS 1-180,PS 1-100and PS I(HA)-40were suspended in the mediumdescribed in Figure 3. Cyanobacterial PSI membranefragments were suspended in the same mediumwithout TritonX-100. Sampleswere preilluminatedat 1.1 x 104/~Em-2 s- 1 (Light)or incubated in the dark(Dark)at 25 oC for 2 h beforemeasurements.The values are estimated as a percentageof the control sampleskept on ice in the dark during the incubation. 180. The double reciprocal plots indicate that the maximal rate of MV photoreduction was lower in the lighttreated PS 1-180 than in that incubated in the dark. The results indicate that the decrease in the activity in the light-treated PS I-180 is not caused by a decrease in the size of the Chl antennae. The reciprocals of the intercept value at infinite excitation intensity in the 2 hilluminated PS I-180 was 65% of the control level. This is consistent with the 50,,~60% inhibition of the MVphotoreduction activity measured under almost saturating actinic intensity shown in Figure 3 and Table 1. The reciprocals of the slope value of the plot of the sample illuminated for 2 h on the other hand, was 28% of the control level. This decrease can be explained by the decrease in the amount of Chl to 33% in this preparation. The almost linear curve obtained with this preparation also suggests that each PS I reaction center that is still active in MV-photoreduction has lost antenna Chl to a similar extent. Figure 5 (Insert) shows the kinetics of laser flashinduced absorption changes in the light-treated PS I180. The absorbance changes in this sample showed fast and slow decay phases. The half-time of the slow phase was 30 ms and was identical to that of the electron transfer from (FA/FB)- to P700 + in the control (data not shown) (Golbeck and Bryant 1991; Hoshina and Itoh 1993). The extent of the 30 ms phase in the light-treated PS 1-180 was about 30% of the control level. In this case, electron transfer activity from reduced DCPIP to MV also dropped to about 30% of the control level. This indicates that photodamage occurs somewhere in the electron transfer pathway between P700 and the FA/FB centers. The half time of the fast decay of the light-treated sample was about 10 #s. Figure 5 shows the spectra of the fast decay phase

(calculated from the difference between the absorbance changes at 6 #s minus those at 40/zs after excitation with the flash) and the slow decay phase (calculated from the absorbance changes at 40 #s after the flash). The spectrum at 40 #s is interpreted as the sum of the P700+/P700 and (FA/Fa)-/(FA/FB) difference spectra (Hiyama and Ke 1972). The spectrum of the fast decay phase resembles that of the P700 triplet state (P700 a') as reported by Warren et al. (1993). P700 T is known to be produced in the charge recombination reaction between A1- and P700 + when AI is doubly reduced. The triplet state is formed with a half time of 750 ns and with a yield approaching 100%, and decays with tl/2 = 4-5 #s. (Sttif and Bottin 1989; Sttif and Brettel 1990). The triplet state is also found from the charge recombination between Ao- and P700 +. The rise time of the P700 T has a half time of 30 ns and P700 T decays with a half time of 3,,~80 #s (Takahashi and Katoh 1984; Brettel and Sttif 1987; Ikegami et al. 1987; Biggins and Mathis 1988; Iwaki and Itoh 1989; Warren et al. 1993). In the later case, the yield of the triplet formation comprised only between 5 and 60% (Shuvalov et al. 1986; Sttif, et al. 1985; Gast et al. 1983; Brettel and Sttif 1987). Thus, the P700 triplet formation shown in Figure 5 suggests that the photoinhibitory light treatment causes the destruction of PS I acceptor(s), Fx and/or A1.

Oxygen is required for the photoinhibition Table 2 shows the photoinhibition of PS 1-180 in the presence or absence of oxygen. The amplitude of the 30 ms decay phase is regarded as the electron transport activity from P700 to FA/FB. Neither inactivation nor Chl bleaching was induced by the light

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Figure 5. Absorptionspectra calculated from absorptionchanges in the 2 h-preilluminatedspinach PS 1-180 after excitation provided by a saturatingNd-YAGlaser flashat 532 nm at roomtemperature. Fast decay phase (circles)calculatedfromthe differencebetweenthe absorbance changes at 6 #s minus those at 40/~s. The slow decay phase (crosses) calculated from the absorbancechanges at 40 Vs. Insert, Kinetics of absorption changes measured at 435 nm in the photoinhibitedPS 1-180. The reactionmixturecontained50 mM Tricine-NaOH(pH 7.8), 0.1M sorbitol, 10 mM NaCI, 4 mM Na-Asc, 20/zM DCPIP,0.05% (w/v) TritonX-100, and PSI complex (10 #g Chl/ml). Table 2. Effect of anaerobicconditionsduringphotoinhibitorylight treatmenton the amplitudeof the 30 ms decay phase and Chl content in spinach PS 1-180

Preilluminationconditions

30 ms decay phase

Chl content

Aerobic Anaerobic

73 96

50 93

The preilluminationconditions were as described in Table 1. The amplitudeof the 30 ms decay componentwas estimatedby the flashinducedabsorbancechangesmeasuredat 430 nm usinga spectrophotometer (sub millisecondtime resolution)with a xenon flash lamp. The values were estimated as a percentageof the controlas shownin Table 1. The reaction mixtureis described in the legendto Figure 5.

treatment in the absence of oxygen, indicating that oxygen is required for the photoinhibition of PS I. Cytochrome c is reduced by superoxide, an active oxygen species (Takahashi and Asada 1988). The kinetics of cytochrome c reduction were measured under continuous illumination (Figure 6). Two phases of absorbance changes were observed in PS I-180. The rapid increase of A550 represents a scattering change and the slow phase represents cytochrome c photoreduction. In PS I(HA)-40, there was almost no slow phase, indicating the lower level of superoxide production.

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16s Figure 6. Photoreduction activity of cytochrome c in spinach PS 1-180 (a) and PS I(HA)-40(b). The reaction mixture contained50 mM Tricine-NaOH(pH 7.8), 0.1 M sorbitol, 10 mM NaC1, I0/~M DCMU, 16/~moles/rnlcytochromec, 0.05% (w/v)TritonX-100and PSI preparation(10 #g Chl/ml). T, light on; J,, light off.

Table 3 shows malondialdehyde (MDA) production in PS I- 180 and PS I(HA)-40 during the light treatment. M D A is a decomposition product of unsaturated fatty acids (Heath and Packer 1968). After 2 h of illumination, the M D A concentration in PS 1-180 was about 2.3-fold that in the untreated PS 1-180, whereas that

128 Tab/e3. Formationof lipid-peroxidizedproduct duringa photoinhibitorylight treatment Incubation

Oh Dark for 2 h Light for 2 h

Malondialdehydecontent MDA/P700(molarratio) PS 1-180 PS I(HA)-40 2.3 3.3 5.3

2.1 2.0 2.6

The concentration of malondialdehyde in spinach PSI preparationswas determinedby the TBA.Sampleswereincubatedin the light or dark as describedin Table1.

in PS I(HA)-40 increased by only 24% as compared with that in the control. These results agree with the conclusion obtained by the cytochrome c reduction.

Discussion Light treatment of PS I- 180 and PS I- 100 isolated from spinach thylakoid membranes by the method of Mullet et al. (1980) resulted in a loss of MV photoreduction activity and Chl bleaching (Table 1). The P700 contents were not affected. The spectra of the decay phase of about 10/~s can be assigned to the P700 triplet (Figure 5). The P700 triplet state is formed in the charge recombination reaction between A l - and P700 +, and also in the back reaction between A0- and P700 +, suggesting that the electron acceptor(s), Fx and/or A1 are damaged by the light treatment and the forward electron flow is blocked. Inoue et al. (1986) reported that illumination of isolated spinach chloroplasts under aerobic conditions caused the destruction of three FeS centers of PS I. On the other hand, electron transfer between the electron acceptor Chl Ao and the ironsulfur center Fx was inhibited under extremely reducing conditions and concluded that the destruction of AI was induced by the accumulation of reduced Fe-S centers (Inoue et al. 1989). In this study, the isolated P S I complexes were not photoinhibited under anaerobic conditions (Table 2). Thus, irradiation of the PSI complexes under aerobic conditions may cause mainly the destruction of Fx. However, the possibility of the destruction of A1 cannot be excluded. Light treatment of PS I(HA)-40 caused no inactivation and a slight bleaching of Chl (Table 1). The major difference between PS I(HA)-40 and PS 1-180 or PS 1-100 is the absence or presence of internal antennae (,,~60 Chl a) bound to the reaction center proteins. The

results suggest that the internal antenna Chl that is not present in PS I(HA)-40 constitutes an essential factor for photoinhibition, although the role of minor component that was lost in PS I(HA)-40 but not in PS 1-100 can not totally be denied. Light treatment of PS 1-180 under O2-free conditions did not cause inactivation or Chl bleaching (Table 2), indicating that oxygen is required for PS I photoinhibition. When chloroplasts are exposed to strong light, they are damaged by photo-produced free-radicals of oxygen that induce the peroxidation of unsaturated membrane lipids (Heath and Packer 1968; Takahama and Nishimura 1975; Chauhan et al. 1992). Electron donors added to the P S I suspension on the other hand, did not accelerate photoinhibition (data not shown). These findings indicate that the electron flow through P700 during strong illumination is not sufficient for the destruction of the P S I acceptor(s). A comparison between the CD spectra of PS 1-100 and PS I(HA)-40 (Figure 2) revealed that a part of the Chl in PS 1-100 gives less active CD structure. Some of antenna Chl molecules showing less CD signal may be excited to the triplet state and may react with oxygen to produce the active oxygen species when excited by light, resulting in the destruction of the PS I acceptor(s). The P700 triplet state may reacts with oxygen and forms highly toxic oxygen molecules in the same manner as PS II photoinhibition at the acceptor side. However, the result that there was no photoinhihibition in PS I(HA)-40 is not resolved by the latter interpretation. Purcell and Carpentier (1994) reported that the light treatment of P S I complex isolated from spinach thylakoids using a hydroxylapatite column, caused simultaneous P700 degradation and Chl bleaching. The intensity of 575 W m -2 s- l which they used, was lower than the 1.1 x 104 #E m -2 s -1 usually applied in this study. We treated PS I(HA)-40 with Bio-Beads to remove Triton X-100 from the medium and the complex. The drastic effect of the light treatment found by PerceU and Carpentier (1994) seems to depend on the Triton concentration during the light treatment. In fact, illuminating PS I(HA)-40 without the treatment of Bio-Beads destroyed P700 and almost completely bleached Chl even at intensities below 1.1 x 104 #E m -2 s -1 (data not shown). It has been pointed out that detergents disrupt the native Chl-protein structure essential for efficient energy transfer to P700 (Mullet et al. 1980; II'ina et al. 1984; Tang et al. 1991). Tang et al. (1991) reported that Triton X-100 disrupted energy transfer from the

129 accessory Chl a to the active pheophytin a in the PS II reaction center complex. In this study, we isolated spinach P S I complexes by solubilization with Triton X-100. If some Chl molecules in the internal antennae in PS 1-180 or PS 1-100 were inactivated, uncoupled Chl molecules should then react with oxygen to form reactive and toxic species such as singlet oxygen. Illumination of the PSI membrane fragments isolated from cyanobacterium Synechocystis PCC 6803 without detergent also inactivated MV photoreduction (Table 1). This proves that photoinhibition occurs in P S I preparation isolated without detergent, although Triton X-100 may disorganize antenna Chl in the PS I core complex and accelerate photoinhibition. Does the photoinhibition of P S I take place in vivo? In chloroplasts, oxygen damage may be avoided by scavengers of free radicals such as superoxide dismutase or ascorbate peroxidase (Takahashi and Asada 1988; Herbert et al. 1992). However, P S I photoinhibition occurs in cucumber (chilling-sensitive plant) leaves in vivo, when illuminated at 4 °C in moderate light at 100-,~220 #Em -2 s -1 (Sonoike and Terashima 1994; Terashima et al. 1994). They demonstrated that the component on the acceptor side of the PS I, Al or Fx, was the first site of inactivation and that inactivation of the electron transfer led to the degradation of the PSI reaction center subunit(s) (Sonoike and Terashima 1994). PSI photoinhibition was also found in potato (chilling-resistant plant) leaves in vivo under strong illumination at 3 °C (Havaux and Davaud 1994). P S I photoinhibition in vivo should be further studied.

Acknowledgements We are grateful for Professor K. Wada for his support and helpful comments. We wish to thank Dr M. Iwaki for her help with the laser spectroscopy and Professor M. Nagai (School of Allied Medical Professions, Kanazawa University) for help in using the JASCO J-20 spectropolarimeter. GH was supported by a grant to R.E.Blankenship from the US National Science Foundation (MCB 9418415). This is publication # 269 from the Arizona State University Center for the Study of Early Events in Photosynthesis. This study was supported in part by the Grant-in-Aid (03640567, 05640728) from the Ministry of Education, Science and Culture, Japan to SH.

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