Planta (2002) 215: 457–465 DOI 10.1007/s00425-002-0765-x
O R I GI N A L A R T IC L E
A.G. Ivanov Æ P.V. Sane Æ Y. Zeinalov Æ I. Simidjiev N.P.A. Huner Æ G. O¨quist
Seasonal responses of photosynthetic electron transport in Scots pine (Pinus sylvestris L.) studied by thermoluminescence Received: 10 October 2001 / Accepted: 15 February 2002 / Published online: 11 April 2002 Springer-Verlag 2002
Abstract The potential of photosynthesis to recover from winter stress was studied by following the thermoluminescence (TL) and chlorophyll fluorescence changes of winter pine needles during the exposure to room temperature (20 C) and an irradiance of 100 lmol m–2 s–1. TL measurements of photosystem II (PSII) revealed that the S2QB– charge recombinations (the B-band) were shifted to lower temperatures in winter pine needles, while the S2QA– recombinations (the Q-band) remained close to 0 C. This was accompanied by a drastically reduced (65%) PSII photochemical efficiency measured as Fv/Fm, and a 20-fold faster rate of the fluorescence transient from Fo to Fm as compared to summer pine. A strong positive correlation between the increase in the photochemical efficiency of PSII and the increase in the relative contribution of the B-band was found during the time course of the recovery process. The seasonal dynamics of TL in Scots pine needles studied under field conditions revealed that between November and April, the contribution of the Q- and B-bands to the overall TL emission was very low (less than 5%). During spring, the relative contribution of the Q- and B-bands, corresponding to charge recombination events between the acceptor and donor sides of PSII, rapidly increased, reaching maximal values in late July. A
A.G. Ivanov Æ P.V. Sane Æ I. Simidjiev Æ G. O¨quist (&) Umea˚ Plant Science Center, Department of Plant Physiology, University of Umea˚, Umea˚ 901 87, Sweden E-mail:
[email protected] Fax: +46-90-7866676 A.G. Ivanov Æ N.P.A. Huner Department of Plant Sciences, University of Western Ontario, London, Ontario, N6A 5B7, Canada Y. Zeinalov Institute of Plant Physiology, Bulgarian Academy of Sciences, 1113 Sofia, Bulgaria I. Simidjiev Department of Molecular Biology, University of Geneva, 1211 Geneva 4, Switzerland
sharp decline of the B-band was observed in late summer, followed by a gradual decrease, reaching minimal values in November. Possible mechanisms of the seasonally induced changes in the redox properties of S2/S3QB– recombinations are discussed. It is proposed that the lowered redox potential of QB in winter needles increases the population of QA–, thus enhancing the probability for non-radiative P680+QA– recombination. This is suggested to enhance the radiationless dissipation of excess light within the PSII reaction center during cold acclimation and during cold winter periods. Keywords Electron transport Æ Photosystem II Æ Pinus (cold acclimation) Æ Recovery of photosynthesis Æ Thermoluminescence Æ Winter stress Abbreviations DCMU: 3-(3¢,4¢-dichlorophenyl)-1,1-dimethylurea Æ Fo: minimum yield of chlorophyll fluorescence at open PSII centers in dark-adapted needles Æ P680: reaction-center pigment of PSII Æ P680+: oxidized form of the reaction center of PSII Æ PSI, PSII: photosystem I and photosystem II, respectively Æ PPFD: photosynthetic photon flux density Æ PQ: plastoquinone Æ QA: primary electron-accepting quinone in PSII Æ QB: secondary electron-accepting quinone in PSII Æ SP: summer pine Æ TL: thermoluminescence Æ TM: temperature of maximum thermoluminescence emission Æ WP: winter pine
Introduction The characteristic winter depression of photosynthesis in conifers (Pharis et al. 1970) is accompanied by major changes in chloroplast ultrastructure (Senser et al. 1975; Martin and O¨quist 1979) as well as in the lipid (O¨quist 1982) and protein composition of the thylakoid membranes (O¨quist et al. 1978; Ottander et al. 1995; Vogg et al. 1998). The seasonal changes in the structure and composition of the photosynthetic
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apparatus are accompanied by a gradual decline in the rate of net photosynthesis during late summer and autumn, a strong inhibition during the winter, and a rapid recovery in early spring (for reviews, see Levitt 1980; Larcher and Bauer 1981; O¨quist and Martin 1986; Leverenz and O¨quist 1987; O¨quist et al. 2001). Earlier studies have documented that these seasonally induced changes in evergreen conifers are well correlated with strong winter inhibition of PSII-related photochemical activities and the whole-chain electron transport from water to NADP (Martin et al. 1978; Senser and Beck 1978; O¨quist and Martin 1980; O¨quist 1982; Ivanov et al. 2001), while the rate of PSI electron-transfer reactions is much less affected (Tsel’niker and Chetverikov 1988; Bolhar-Nordenkampf et al. 1993; Ivanov et al. 2001). The earliest autumn changes in polypeptide composition have been observed in September, with initial losses of the D1 protein of the PSII reaction center, reaching a minimum by December (Ottander et al. 1995). Significant decreases in the content of chlorophyll and several chlorophyll-binding polypeptides related to the light-harvesting proteins and the core antennae of PSII (LHCII, CP43) and PSI (LHCIb) have also been reported during the winter depression of photosynthesis in Scots pine (Ottander et al. 1995; Vogg et al. 1998). In addition, a strong reduction of the QB-band in the thermoluminescence (TL) glow curve and a markedly slower QA– re-oxidation in needles of winter pine, indicating an inhibition of electron transfer between QA and QB, have been also observed (Ivanov et al. 2001). Earlier laboratory and field studies have established that the interaction of even moderate light and freezing temperatures causes inhibition of photosynthesis in Scots pine during winter stress (Strand and O¨quist 1985a, b and 1988; Ottander and O¨quist 1991). It is generally accepted that light induces severe photoinhibition of PSII, whereas frost causes inhibition of the Calvin cycle and/or photophosphorylation (Strand and O¨quist 1988; Ottander and O¨quist 1991). Photosynthesis of winter-stressed branches of Scots pine recovers under favorable laboratory conditions within 1–3 days, and the photochemical efficiency of PSII recovers well before other components affected by winter stress (Ottander and O¨quist 1991). The recovery of PSII photochemical efficiency was accompanied by simultaneous recovery of D1-protein content (Ottander et al. 1995). Since PSII has been generally considered to undergo major structural and functional changes during winter depression and spring recovery of photosynthesis from winter stress (O¨quist et al. 2001), TL measurements have been used to estimate more precisely the function of PSII (Sane and Rutherford 1986). In this work we study in more detail the seasonal dynamics of charge recombination events between the acceptor and donor sides of PSII in Scots pine needles under field conditions, and during the recovery from winter stress under controlled laboratory conditions.
Materials and methods Plant material Needles from exposed branches of a 35-year-old tree of Pinus sylvestris L, growing in an open stand on campus (6350¢N, 2020¢E) were collected at midday regularly over 2 years (1998–1999). In February–March, branches were brought indoors and recovery of photosynthesis from winter inhibition was followed under standardized laboratory conditions. The shoots were thawed at 4 C in darkness, re-cut and placed in water at 20 C and a photosynthetic photon flux density (PPFD) of 100 lmol m–2 s–1 for recovery (Ottander and O¨quist 1991). Chlorophyll fluorescence The maximal photochemical efficiency of PSII (Fv/Fm) and the kinetics of the fluorescence transient from Fo to Fm were measured in dark-adapted (30 min) pine needles by using a Plant Stress Meter (PSM Chlorophyll Fluorimeter; Biomonitor S.C.I., Umea˚, Sweden) as described by Ivanov et al. (2001). The PPFD of the actinic light was 400 lmol m–2 s–1 and the time of excitation was 5 s. The reduction state of plastoquinone (PQ) was assessed following the post-illumination fast transient increase of chlorophyll fluorescence at the Fo¢ level (Asada et al. 1993; Mano et al. 1995). Chlorophyll fluorescence was measured at 20 C or 5 C with a PAM 101 chlorophyll fluorescence measuring system (Heinz Walz, Effeltrich, Germany). Instantaneous (dark) chlorophyll fluorescence at open PSII centers (Fo) was excited by a non-actinic, modulated measuring beam (PPFD 0.12 lmol m–2 s–1) obtained from a light-emitting diode with a peak emission at 650 nm and a frequency of 1.6 kHz in the dark and 100 kHz in the light. The actinic light had a PPFD of 150 lmol m–2 s–1. The nomenclature of Van Kooten and Snel (1990) was used for the parameters of chlorophyll fluorescence. Thermoluminescence measurements TL measurements of intact Scots pine needles were performed on a personal-computer-based TL data acquisition and analysis system essentially as described earlier (Ivanov et al. 2001). Experiments were usually performed at a 0.6 C s–1 heating rate. Decomposition analyses of the TL glow curves were carried out by a non-linear least-squares algorithm that minimizes the chi-square function using a Microcal Origin Version 6.0 software package (Microcal Software Inc., Northampton, Mass., USA). Illumination treatment of the samples during cooling to liquidnitrogen temperatures was performed with white continuous light as described earlier (Sane et al. 1983). A flash-lamp assembly (Type FX200; EG&G Electro Optics, Salem, Mass., USA) was used to expose the sample to two single-turnover flashes (2.5 ls half-band with 10 Hz frequency). For this purpose the leaves were first darkadapted for 10 min at 20 C and then cooled to 0 C prior to exposing to the flashes. After flash exposure, the sample was quickly cooled in liquid nitrogen. For S2QA– recombination studies, leaves were vacuum-infiltrated with 3-(3¢,4¢-dichlorophenyl)-1, 1-dimethylurea (DCMU; 20 lM) in darkness before the flash illumination. The nomenclature of Vass and Govindjee (1996) was used for characterization of the flash-induced TL glow peaks.
Results The exposure of control Scots pine needles collected during the summer (SP) to two consecutive flashes of white saturating light yielded a TL glow curve pattern exhibiting a characteristic major band (B-band),
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appearing at 43 C and accounting for 70% of the total TL emission (Fig. 1A). It is generally accepted that the TL peak arising after flash illumination is due to S2QB– recombination (Sane and Rutherford 1986; Vass and Govindjee 1996). Although the registered temperature of maximum TL emission (TM) of 43 C in our study seems somewhat higher than the characteristic TM of about 30 C reported for the B-band in most higher plants (Sane and Rutherford 1986; Vass and Govindjee 1996), our data well correspond to the earlier observation in conifers (Inoue et al. 1976). Another peak appearing around 0 C consisted of 15.7% of the TL emission. In addition, two minor bands appearing at –26 C and 63 C were also registered (data not shown). When DCMU was used to specifically inhibit the electron transport between QA and QB, the TM of the S2QB– peak shifted from 43 C to 36 C, and its intensity was drastically reduced to 15% of the total luminescence (Fig. 1B). In the presence of DCMU, most of the luminescence (73%) appeared at the 0 C peak, which is believed to originate from recombination of S2QA– charge pairs (Fig. 1B).
Fig. 1 Thermoluminescence glow curves and mathematical decompositiuon in Gaussian sub-bands of control (A, C) and DCMU-treated (B, D) Scots pine (Pinus sylvestris L.) needles during the summer (A, B) and winter (C, D) after illumination with two single-turnover flashes of white saturating light. Dark-adapted samples were cooled to 0 C before exposure to flash illumination. After flash exposure the samples were quickly cooled to liquidnitrogen temperatures. The presented glow curves are averages of five to seven measurements in three independent experiments
The TL glow curve pattern of pine needles collected during the winter (WP) is presented in Fig. 1C. In comparison with SP, it is characterized by a considerably lower emission, a shift of the major peak to lower temperature (32 C), and an increased contribution of the peak around 0 C. Furthermore, in contrast to SP, addition of DCMU caused a minimal effect on the peak’s TM and intensity of WP needles (Fig. 1D). The major peak appearing in the absence of DCMU is attributed to the S2QB– recombination (the B-band) while the major peak appearing in the presence of DCMU is attributed to S2QA– recombination (the Q-band) (Vass and Govindjee 1996). Since the peak position (TM) is a measure of the energetic stabilization of separated charges, while the peak area of the B-band is proportional to the number of centers in the S2QB– state, further, more precise identification of the S2QB– peak was obtained from flash-induced TL oscillations by varying the number of flashes. In agreement with previous reports, the overall intensity of the TL emission strongly depended on the number of flashes (data not shown), and the intensity of the B-band (S2QB– peak) oscillated with a period of four, exhibiting maximum intensity on the second flash (Fig. 2). Consistent with earlier data, the TL glow curve of WP needles (Fig. 3A) after illumination with continuous white light, could be best fitted with only three TL bands exhibiting much lower yield of TL emission than SP needles (Ivanov et al. 2001). In contrast, earlier studies on conifers have reported five distinct TL peaks with characteristic temperatures corresponding to the Zv (P680+QA–), A (S3QA–), Q (S2QA–), B (S2QB–) and C (TyrD+QA–) bands, respectively (Inoue et al. 1976; Ivanov et al. 2001). It is important to note that the Zv-band centered around –30 C in WP accounted for almost 60% of the total luminescence and the B-band had a negligible contribution (2.5%) to the overall TL yield. The recovery of photosynthesis from winter stress
Fig. 2 Dependence of the TL intensity of the S2QB– peak on the flash number in SP needles. All other experimental conditions are as in Fig. 1
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in outdoor pine needles under controlled laboratory conditions (20 C and PPFD of 100 lmol m–2 s–1) was measured by following the time course of TL emission (Fig. 3B–F). It was clearly seen that the overall TL emission gradually increased during the recovery process and after 24 h (Fig. 3D) was almost 3-fold higher than in control WP needles (Fig. 3A). In addition, it is important to note that the relative contribution of the Zv-band progressively decreased to 32.8% while that of Q- and B-bands increased to 45.8% and17.5%, respectively, within the same time interval. As shown before (Ottander and O¨quist 1991; Ottander et al. 1995; Ivanov et al. 2001), the maximum photochemical efficiency of PSII of outdoor pine needles during the winter was severely reduced, exhibiting Fv/Fm values of 0.31±0.01 (mean ± SE), as compared to the values of Fv/Fm registered during the summer (0.82±0.01; Fig. 4B). The differences in the photochemical efficiency were accompanied by a concomitant increase in the t1/2 for the chlorophyll fluorescence rise from Fo to Fm. The t1/2 values of winter-stressed and summer pine were 2 and 42 ms, respectively (Fig. 4A). During the recovery of photosynthesis in WP under Fig. 3 Thermoluminescence (TL) glow curves pattern from Scots pine needles during recovery (A control WP, B 4 h, C 16 h, D 24 h, E 48 h, F 72 h) from winter stress in laboratory conditions. The shoots were kept at 20 C and a PPFD of 100 lmol m–2 s–1. The samples were illuminated continuously during freezing to liquid-nitrogen temperature. Experimental TL curves are averaged from three to five measurements
laboratory conditions, the rapid almost linear increase in PSII photochemical efficiency was accompanied by a gradual increase in the t1/2 of chlorophyll fluorescence increase from Fo to Fm (Ivanov et al. 2001; Fig. 4). The most intriguing feature of the recovery process was that there was a very strong, linear correlation between both PSII efficiency measured as Fv/Fm (r2=0.98) and the redox state of the PQ pool, measured as t1/2 of fluorescence increase (r2=0.93), and the relative TL intensity of S2QB– peak (Fig. 4). The seasonal dynamics of TL in Scots pine needles studied under field conditions revealed that between November and April the contribution of the Q- and B-bands to the overall TL emission was very low (less than 5%). During this period the TL emission remained very low and most of the total luminescence was emitted at temperatures around –30 C, characteristic of the Zv-band. The typical glow curves representing the seasonal dynamics of TL emission (Fig. 5) clearly demonstrate that in the spring the relative contribution of Q- and B-bands, corresponding to charge recombination events between the acceptor and donor sides of PSII, rapidly increased, reaching maximal values in late
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Fig. 4 Correlation between the reduction state of the PQ pool measured as half-time of Fm rise (t1/2FLUO) (open symbols; A) and the photochemical efficiency of PSII (Fv/Fm) (closed symbols; B), and the S2QB– TL peak in Scots pine needles during recovery of photosynthesis from winter stress. circles WP, squares 4 h recovery, triangles 16 h recovery, inverted triangles 24 h recovery, diamonds 48 h recovery, pentagons 72 h recovery, stars SP. All fluorescence measurements for winter pine (WP) and summer pine (SP) were performed at 5 C and 20 C, respectively. All values represent means ± SE from three independent measurements
June–July (Fig. 5D, E). This was accompanied by a parallel decline in the relative contribution of the Zvband. A sharp decline in the B-band was observed in late summer, followed by a gradual decrease, reaching minimal values in October (Fig. 5F–H). Concomitant with this, a gradual enhancement of the Zv-band was registered. The dynamics of the relative contribution of the S2QB– peak (B-band) to the overall TL emission clearly indicates that the acceptor side of PSII undergoes major seasonal changes (Fig. 6). It is important to note that the initial rapid decline in the S2QB– peak appeared much before the cold season. The extent of the post-illumination fluorescence transient increase after turning off the actinic light (AL) was used as an estimate of the dark reduction of the PQ pool by stromal reductants (Mano et al. 1995; Farineau 1999). A negligible post-illumination transient was observed in control SP needles under ambient atmospheric conditions (Fig. 7A). This indicates that, in Scots pine, either there is a negligible electron donation into the
Fig. 5A–H Seasonal dynamics of the TL emission in Scots pine needles after illumination with continuous white light. Decomposition analysis was performed on experimental curves averaged from three to five independent experiments
intersystem electron transport chain from the stromal electron pool in the dark or that oxygen at the ambient concentration (21.0%) can serve as an efficient acceptor thus preventing an over-reduction of the intersystem electron transport chain by stromal electron flow
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Fig. 6 Dynamics of the relative TL intensity of the S2QB– peak in Scots pine needles during the year. The data for the S2QB– peak are estimated from the decomposition analysis of the experimental curves (see Fig. 5) and are presented as a percentage of the total TL light emission. Mean values ± SE were calculated from three to five independent experiments
regardless of the growth conditions. In contrast, in WP needles, regardless of the lack of a rapid transient, Fo increased gradually during the entire measuring time range, reaching values close to Fs (Fig. 7C), indicating that the re-oxidation process under these conditions is too slow to counterbalance the introduction of reducing equivalents into the PQ pool (Farineau 1999). When the oxygen concentration was lowered to 2.0%, there was a rapid post-illumination fluorescence transient in SP needles (Fig. 7B). Conversely, the performance of WP needles at 2.0% O2 after a light-to-dark transition did not exhibit any detectable difference from that under ambient O2 (Fig. 7D).
Discussion Consistent with previous reports on Scots pine, WP needles demonstrated severely reduced TL emission (Fig. 1; Ivanov et al. 2001) and photochemical efficiency of PSII compared with SP needles, and exhibited rapid recovery of PSII photochemistry when placed under favorable (20 C, 100 lmol photons m–2 s–1) laboratory conditions (Fig. 3; Strand and O¨quist 1985b; Ottander and O¨quist 1991; Ivanov et al. 2001). Besides the much lower overall TL emission, flash-induced peaks associated with S2/S3QB– recombinations exhibited major shifts in the characteristic TM to lower temperatures in WP needles (Fig. 1). However, the peak appearing at 0 C corresponding to S2/S3QA– recombinations had not changed its TM in WP needles. Evidently, this resulted in lowering the gap between the peak temperatures of QA- and QB-related bands in WP needles.
Fig. 7 Typical traces of post-illumination transients from Fs to Fo¢ after the actinic light (AL, 350 lmol photons m–2 s–1, 8 min) was turned off in P. sylvestris needles collected during the summer (A, B) and during the winter (C, D). Measurements were made under ambient CO2 and 20% O2 (A, C) and in a 2% O2 atmosphere (B, D)
Since the TL peak temperatures are closely related to the redox potentials of the participating species (deVault and Govindjee 1990) it is obvious that the difference between the redox potential of QA and that of QB has been decreased by shifting the redox potential of QB much closer to the redox potential of QA in WP needles. The distinct differences in the flash-induced TL glow curve pattern observed between pine needles collected during the summer (SP) and during the winter (WP) clearly suggest major alterations in the redox properties of the PSII acceptor side (primarily the QB) during the winter stress. It has been shown in a number of studies that the redox potential of QB is highly sensitive to various structural changes within the D1 polypeptide of PSII and/or local perturbations around the QB-binding pocket of D1. For example, a single change in the crucial amino acid residue of D1 (Ohad and Hirschberg 1992; Minagawa et al. 1999) or a deletion of the PEST-like sequence of D1 (Nixon et al. 1995) have resulted in shifting the S2/S3QB– TL peak towards a lower temperature. In an earlier study, Briantais et al. (1992) also observed a shift towards a lower temperature for the S2QB– peak in cold-acclimated (4 C) spinach compared to non-hardened plants. A similar shift towards a lower temperature of the S2QB– band by 3–4 C was also found in photoinhibited PSII reaction centers (Ohad
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et al. 1988), in photoinhibited non-hardened spinach leaves (Briantais et al. 1992), and in pea thylakoids and leaves photoinhibited at chilling temperatures (Farineau 1993). The temperature shift of S2QB– was interpreted in terms of production of inactive ‘‘missing’’ PSII reaction centers during cold acclimation and photoinhibitory treatment (Briantais et al. 1992). Hence, assuming all of the above and considering that photoinhibition of PSII is an important component of the winter stress-induced inhibition of photosynthesis in Scots pine (Strand and O¨quist 1988; Ottander and O¨quist 1991), it is reasonable to suggest that the specific TL pattern (lower TL emission of the S2QB– band and its shift to lower temperatures) reflects the photoinhibitory damage of D1 polypeptide and/or an increased proportion of inactive PSII reaction centers in WP needles. This explanation is in agreement with the fact that the recovery of PSII photochemical efficiency under laboratory conditions strongly correlates with the relative increase of the S2QB– band (Fig. 4B). Earlier observation that the recovery of PSII efficiency is accompanied by simultaneous recovery of D1 protein content (Ottander et al. 1995) also supports our explanation. In addition, the downshift of the S2QB– characteristic peak has been attributed to the build-up of a proton gradient and the reduction of the PQ pool (Miranda and Ducruet 1995). It has been also demonstrated that a darkinduced proton gradient and the proton gradient generated by PSI-dependent cyclic electron transfer, affect both Q (S2QA–) and B (S2QB–) TL bands in a similar manner (Miranda and Ducruet 1995). In fact, the post-illumination increase in Fo observed in WP (Fig. 7) clearly demonstrated substantial donation of electrons to the intersystem PQ pool from stromal substrates. This is consistent with the recently reported higher intersystem electron pool size in WP (Ivanov et al. 2001). Earlier reports indicating that mesophyll chloroplasts contain large potential pools of electrons that can be donated to the intersystem PQ pool from stromal or cytosolic reductants are in line with our data (Asada et al. 1993; Mano et al. 1995). Furthermore, analysis of the fluorescence induction kinetics also revealed a much faster (10-fold) rate of fluorescence rise from Fo to Fm in dark-adapted WP than in control SP needles, thus implying that the PQ pool is in a predominantly reduced state during the dark. Interestingly, a very strong positive correlation between the reduction state of the PQ pool measured as fluorescence induction kinetics and the amplitude of the S2QB– band was observed during the recovery of photosynthesis in WP needles under laboratory conditions (Fig. 4A). Another possibility for the observed low-temperature shift of the S2QB– band could be the enhanced proton gradient caused by the substantially increased PSI-dependent cyclic electron transport in conifers during the winter season (Senser and Beck 1978; Ivanov et al. 2001). Hence, it appears that the origin of the specific TL pattern in WP needles is quite complex and, besides certain photoinhibitory damage and an increased proportion of inactive PSII reaction centers, the dark-
induced reduction of the PQ pool and/or enhanced PSI-dependent proton gradient in WP needles could be directly associated with the low-temperature shift of the S2QB– band. The seasonal dynamics of TL emission revealed a similar pattern of PSII recovery during the year as observed in controlled laboratory conditions (Fig. 5). In parallel to a gradual decrease in the relatively strong Zv-band in the spring and its re-appearance in late fall, the bands characteristic of S2QA– and S2QB– steadily increased until mid summer and started to decline in August. Not surprisingly, the seasonal dynamics of the S2QB– band closely resembled the seasonal dynamics of PSII photochemical efficiency reported earlier (Ottander et al. 1995). The major change in the redox properties of QB, which brings it much closer to the redox potential of QA, and the seasonal dynamics of the S2QB– band during the year (Fig. 6) point to a possibility that the acceptor side of PSII is of vital importance for balancing PSII functional activity during changing environmental conditions. It has been recently proposed that the redox change in QB may result in a change in the rate constant for the electron transfer between the two quinone acceptors of PSII (Minagawa et al. 1999). Since the PQ pool remains predominantly in a reduced state in WP needles the reduction of QB at a faster rate may result in lack of oxidized quinone (Telfer and Barber 1994). This would result in rendering the QB site empty and induce double reduction of QA followed by its protonation (Vass et al. 1992). These conditions would favour photoinhibitory damage to the PSII reaction center. Keeping electrons preferentially on QA through a change in redox potential of QB would ensure that the QB site will remain occupied by the quinone and this would protect PSII from further photoinhibition and D1 degradation, leaving a low but stable level of PSII reaction-center complexes throughout the winter. This argument is strongly supported by the observation that addition of DCMU had a protective effect on D1 turnover under photoinhibitory conditions (Komenda and Masojidek 1998). Furthermore, the accumulation of reduced QA (QA–) has been shown to inhibit the formation of the radical pair P680+Pheo– by the electrostatic effect and, consequently, would prevent triplet formation by P680 (Schatz et al. 1988; Vass et al. 1992). It is also quite possible that the increased population of QA– in WP needles may enhance P680+QA– recombination. The back-reaction of QA– with P680+ has been previously suggested (Prasil et al. 1996; Krieger-Liszkay and Rutherford 1998). The fact that over 93% of the overall TL emission in WP is accounted for by lowtemperature peaks (predominantly the Zv emission band), reflecting a preferred back-reaction of QA with primary donors, strongly supports this possibility. A low probability of electron transfer from QA to QB has also been reported in WP needles (Ivanov et al. 2001). In addition, a non-radiative pathway of charge recombination between QA– and the donor side of PSII has been
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suggested earlier (Briantais et al. 1979; Weis and Berry 1987; Vavilin and Vermaas 2000). This would increase the probability of non-radiative dissipation of the excitation energy within the reaction center of PSII (Weis and Berry 1987; Bukhov et al. 2001). The reduction of QA has been suggested as a major requirement for efficient reaction-center quenching (Bukhov et al. 2001). It has been suggested before that photoinactivated PSII complexes can dissipate excitation energy as heat, thereby preventing further damage (Krause 1988). More recently, Lee et al. (2001) proposed that the role of photoinactivated centers as quenchers increases with the severity of photoinactivation. The remaining PSII reaction centers of WP needles may therefore have an important role in the non-photochemical dissipation of absorbed light through recombination during the winter. Furthermore, the observation that the shift of TM to lower temperatures of the B-band occurs already during late summer and early autumn (Fig. 5), and before the autumn decline of the D1 protein begins (Ottander et al. 1995), suggests that the proposed reaction-center recombination mechanism for non-photochemical dissipation of energy also may be of particular significance during early stages of the induction of dormancy and associated cold acclimation. In summary, the results presented in this study clearly indicate that the inhibition of photosynthesis in Scots pine needles during the winter is closely associated with major structural and functional changes within the acceptor side of PSII. The contribution of the QB band to the overall TL emission in WP needles becomes much lower and its redox potential changes to more negative, as revealed by the shift in TL glow peak temperature. In contrast to SP needles, narrowing of the gap between the redox potential of QA and QB in WP needles presumably results in changing the equilibrium constant between these acceptors, allowing the localization of the charges predominantly on QA. It is proposed that the increased population of QA– would induce its back-reaction with P680+ most probably via the non-radiative pathway of charge recombination. This may enhance the dissipation of excess light energy within the PSII reaction center during cold acclimation in the autumn and during the cold winter periods, thus complementing the ability to quench absorbed light non-photochemically at the level of the light-harvesting antenna when the enzymatic, zeaxanthin cycle-dependent energy quenching would be thermodynamically restricted. Acknowledgements This work was financially supported by the Swedish Foundation for International Cooperation in Research and Higher Education (STINT), the Swedish Natural Science Research Council, and by the Natural Science and Engineering Research Council of Canada.
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