View metadata, citation and similar papers at core.ac.uk brought to you by CORE provided by Elsevier - Publisher Connector Biochimica et Biophysica Acta 1807 (2011) 1214–1220 Contents lists available at ScienceDirect Biochimica et Biophysica Acta journal homepage: www.elsevier.com/locate/bbabio Herbicide effect on the photodamage process of photosystem II: Fourier transform infrared study Issei Idedan b, Tatsuya Tomo c,d, Takumi Noguchi a,b,⁎ a Division of Material Science, Graduate School of Science, Nagoya University, Furo-cho, Chikusa-ku, Nogoya, 464-8602, Japan b Institute of Materials Science, University of Tsukuba, Tsukuba, Ibaraki, 305-8573, Japan c Department of Biology, Faculty of Science, Tokyo University of Science, Kagurazaka 1-3, Shinjuku-ku, Tokyo, 162-8601, Japan d PRESTO, Japan Science and Technology Agency (JST), 4-1-8 Honcho Kawaguchi, Saitama, 332-0012, Japan article info abstract Article history: The photodamage process of photosystem II by strong illumination was investigated by examining the Received 14 May 2011 herbicide effects on the photoinactivation of redox cofactors. O2-evolving photosystem II membranes from Received in revised form 7 June 2011 spinach in the absence of herbicide and in the presence of 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) Accepted 7 June 2011 and bromoxynil were subjected to strong white-light illumination at 298 K, and the illumination-time Available online 28 June 2011 dependence of the activities of QA, the Mn cluster, and P680 were monitored using light-induced Fourier transform infrared (FTIR) difference spectroscopy. The decrease in the Q activity was suppressed and Keywords: A FTIR accelerated by DCMU and bromoxynil, respectively, in comparison with the sample without herbicide. The Herbicide intensity change in the S2/S1 FTIR signal of the Mn cluster exhibited a time course virtually identical to that in Photodamage the QA signal in all the three samples, suggesting that the loss of the S1 →S2 transition was ascribed to the QA Photoinhibition inactivation and hence the Mn cluster was inactivated not faster than QA. The decrease in the P680 signal was Photoprotection always slower than that of QA keeping the tendency of the herbicide effect. Degradation of the D1 protein Photosystem II occurred after the P680 inactivation. These observations are consistent with the acceptor-side mechanism, in 1 which double reduction of QA triggers the formation of O2* to promote further damage to other cofactors and the D1 protein, rather than the recently proposed mechanism that inactivation of the Mn cluster initiates the photodamage. Thus, the results of the present study support the view that the acceptor-side mechanism dominantly occurs in the photodamage to PSII by strong white-light illumination. © 2011 Elsevier B.V. All rights reserved. 1. Introduction double reduction of QA leading to the release from the binding site as plastoquinol, PQH2. In the absence of QA, the triplet state formed on + − Plants utilize sunlight for their activities through light-energy ChlD1 by P680 Pheo charge recombination, has a relatively long 1 conversion processes, photosynthesis. However, light is also harmful lifetime (t1/2 ~1 ms) [10,11] and readily forms harmful O2*to to plants and the photosynthetic apparatus is damaged by light inactivate PSII. The triplet state is also formed by charge recombina- − − illumination, which is known as photoinhibition [1–8]. The major tion of S2,3QB or S2QA induced by low-light or flash illumination [12– target of photodamage is known to be photosystem II (PSII), where 14], which can thus be called the low-light mechanism. This electron transfer reactions are inactivated and the D1 polypeptide is mechanism may explain the reason why photodamage to PSII occurs specifically degraded. even under weak illumination. The donor-side mechanism has been Several different mechanisms of photodamage to PSII have been proposed for PSII in which the Mn cluster is already inactivated. In proposed so far [4,7]. In the acceptor-side mechanism [9], strong such PSII, oxidative damage to the electron donor side is caused by a − + illumination stabilizes QA semiquinone anion and then induces strong oxidant, P680 [15–17]. Recently, a new mechanism called the Mn mechanism [7,18] or the two-step mechanism [6,19] has been proposed. In this mechanism, the first step of photodamage is Abbreviations: ChlD1, monomeric chlorophyll on the D1 side of PSII; DCMU, 3-(3,4- dichlorophenyl)-1,1-dimethylurea; FTIR, Fourier transform infrared; Mes, 2-(N- destruction of the Mn cluster by its light absorption and then PSII is morpholino)ethanesulfonic acid; P680, special pair chlorophylls in PSII; Pheo, redox- inactivated via electron transfer reactions. This mechanism explains active pheophytin in PSII; PQH2, plastoquinol; PSII, photosystem II; QA, primary the previous observation that the quantum yield of photodamage is quinone electron acceptor; QB, secondary quinone electron acceptor not dependent on light intensity [20]. ⁎ Corresponding author at: Division of Material Science, Graduate School of Science, Thus, in spite of extensive studies, the molecular mechanism of Nagoya University, Furo-cho, Chikusa-ku, Nagoya, 464-8602, Japan. Tel.: +81 52 789 fi 2881; fax: +81 52 789 2883. photodamage to PSII has not been clari ed yet. In particular, the E-mail address: [email protected] (T. Noguchi). acceptor-side mechanism and the Mn mechanism provide conflicting 0005-2728/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.bbabio.2011.06.006 I. Idedan et al. / Biochimica et Biophysica Acta 1807 (2011) 1214–1220 1215 ideas about the initial target of photodamage, i.e., QA or the Mn (Oxford ITC-4). Single-beam spectra for 150 s (300 scans) were cluster, under strong illumination. Also, the detailed process of recorded before and after 5-s illumination by continuous white light inactivation of redox cofactors and the specific damage that triggers (~40 mW cm− 2) from a halogen lamp (Sigma Koki PHL-150), and a the D1 degradation have not been clearly resolved. light-minus-dark difference spectrum was calculated to represent the − − One of the clues to address these questions is herbicide effects on the S2QA /S1QA difference (or QA /QA and other donor-side components photodamage process. Herbicides bound to the QB pocket block the when the Mn cluster is inactivated before QA). The noise level was electron transfer beyond QA and also change the redox potential of QA estimated from a dark-minus-dark difference spectrum taken before differently depending on the herbicide species; a urea-type herbicide, illumination. − + DCMU, increases the QA /QA redox potential, whereas a phenol-type P680 /P680 FTIR measurement was performed as described herbicide, bromoxynil, decreases the potential [21]. Thus, herbicide previously [38] with slight modification. The PSII sample was treatment perturbs the acceptor-side photoreactions in PSII, which suspended in buffer A, and QA as well as the Mn cluster was depleted should affect the photodamage process. The herbicide effects on by treatment with 100 mM sodium dithionite and 30 μM benzyl photodamage studied so far [22–29] have shown a general tendency viologen followed by dark incubation for 5 h at room temperature that urea- and phenol-type herbicides retard and accelerate, respec- [39]. The sample was washed twice and resuspended in buffer A in the tively, the inactivation of electron transfer reactions and D1 degradation, presence of 40 mM potassium ferricyanide and 0.6 mM SiMo. After although some controversial results have been obtained in the effect of centrifugation at 170,000×g for 40 min, the pellet was sandwiched phenol-type herbicides on D1 degradation [22–24]. In such studies, between the BaF2 plates. The sample temperature was adjusted to − 2+ fluorescence [25,26,28],SiMoreduction[27], the QA Fe EPR signal 265 K. FTIR scans under dark (1 s) and during illumination (1 s) were 1 [25],and O2*production[29] have been detected to examine the repeated 500 times, and average single-beam spectra were used to photodamage to PSII. However, the inactivation processes of individual calculate a P680+/P680 difference spectrum. Light-illumination was redox cofactors in the presence of different-types of herbicides, which performed with red light (~16 mW cm2 at the sample surface) from a are necessary to determine the mechanism of photodamage, have not halogen lamp (Sigma Koki PHL-150) equipped with a red cutoff filter been systematically studied. (N600 nm). In this study, we have investigated the molecular mechanism of photodamage to O2-evolving PSII membranes under strong white- 2.3. Detection of D1 degradation light illumination by utilizing the herbicide effects on the inactivation processes of redox cofactors and the D1 protein degradation. For SDS-PAGE was performed using a gradient gel containing 16– detecting the activities of individual redox cofactors, we used light- 22% acrylamide and 7.5 M urea [40]. PSII samples equivalent to 1 μg induced Fourier transform (FTIR) difference spectroscopy, which is a of Chl/lane (before illumination) were loaded to the wells. For powerful method to directly monitor the photoreactions of redox immunological assays, the SDS–polyacrylamide gel was transferred cofactors in PSII [30–34]. We focused on the activities of QA, the Mn onto a polyvinylidene fluoride (PVDF) membrane, and subjected to cluster and P680, which are the key cofactors in the proposed the reaction with an antibody raised against the C-terminus of the photodamage mechanisms [4,7], and studied the effects of two D1 protein (Agrisera, Sweden). Horseradish peroxidase (HRP)- different types of herbicides, DCMU and bromoxynil. The results conjugated anti-chicken IgY was used as a secondary antibody. support the view that the acceptor-side mechanism is the major Chemiluminescent detection of HRP was carried out using ECL mechanism of photodamage to PSII under strong illumination.