Journal of Experimental Botany, Vol. 51, No. 343, pp. 265-274,
February 2000
© 2000 Oxford University Press
Influence of high light and UV-B radiation on photosynthesis and D1 turnover in atrazine-tolerant and -sensitive cultivars of Brassica napus
1 Department of Plant Physiology, Lund University, Box 117, S-221 00 Lund, Sweden
2 Department of Plant Cell Biology, Lund University, Box 7007, S-220 07 Lund, Sweden
Received 20 August 1999; Accepted 8 September 1999
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Abstract |
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An atrazine-tolerant mutant and an atrazine-sensitive cultivar of Brassica napus L. were grown under visible radiation (400 µmol m-2 s-1, photosynthetically active radiation, PAR) and then subjected to treatment conditions. These included short-term high PAR (1600 µmol m-2 s-1) which was given for 4 h either alone or in combination with an enhanced level of UV-BBE radiation (4.6 kJ m-2 h-1 biologically effective UV-B, 280–320 nm). Recovery from the radiation treatment was studied for 4 h under the light conditions for growth. Since it is known that the atrazine-tolerant mutant is susceptible to photoinhibition, one of the aims of the present study was to determine the effects of a supplemental, enhanced level of UV-B radiation with regard to the mutant. The results indicate an additive effect of UV-B radiation on Fv/Fm, photochemical yield and photosynthetic oxygen evolution during both exposure and recovery, and also a higher susceptibility of the mutant to photoinhibitory PAR conditions alone and in combination with UV-B, which may have implications in a changing environment. Both cultivars also showed a higher D1 turnover during the radiation stress than during recovery, as shown by immunoblotting and 35S-methionine incorporation measurements.
Key words: Atrazine, Brassica napus, D1 protein, fluorescence, herbicide tolerance, oxygen evolution, photoinhibition, ultraviolet-B radiation.
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Introduction |
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Enhanced levels of ultraviolet-B (UV-B, 280–320 nm) radiation, reaching the earth's surface due to stratospheric ozone depletion (Madronich et al., 1998
), elicit plant responses in many different ways. For example, UV-B may cause damage to DNA, proteins and lipids, and may modify photosynthesis, growth and development (Bornman, 1989; Teramura and Sullivan, 1994; Jansen et al., 1998). Both the primary events of photochemistry in photosystem II (PSII), and the reactions of CO2 assimilation are affected by elevated levels of UV-B radiation (Noorudeen and Kulandaivelu, 1982; Bornman, 1989; Middleton and Teramura, 1993; Nogués and Baker, 1995).
Efforts to characterize the role played by UV-B radiation are complicated by the fact that different plant species and cultivars may show very different response patterns to increased amounts of UV-B radiation (Bornman and Teramura, 1993). The aims of this study were to investigate (a) the response of a herbicide-tolerant mutant of Brassica napus (oilseed rape) and a normal cultivar to high PAR with or without supplementary UV-B radiation, and (b) the recovery of photosynthetic activity after the radiation treatments.
In the herbicide-tolerant mutant, Stallion, the serine-264 of the D1 protein in the PSII reaction centre is exchanged for glycine. Without Ser-264, the herbicide, atrazine, does not bind to the QB-binding site of the D1 protein. Also the affinity for plastoquinones is reduced and the electron transport from QA to QB is slowed down (Bowes et al., 1980). This creates more long-lived, double-reduced QA, which induces reactive oxygen species (Kyle, 1987).
It has been shown that the efficiency of PSII is modified in atrazine-tolerant plants such that photochemical quenching is decreased. In addition, rates of light-limited photosynthesis and oxygen yield are decreased in the mutants. The lower quantum yield and efficiency in charge separation in PSII is due to the slower electron transfer between QA and QB (Jursinic and Pearcy, 1988), making the atrazine-tolerant plants generally more susceptible to photoinhibition (Sundby et al., 1993a). This sensitivity to photoinhibition may be one cause of the reduced crop yield observed for atrazine-tolerant plants grown under high irradiances (Gressel and Ben-Sinai, 1985; Hart and Stemler, 1990). Given the susceptibilty to photoinhibition of the atrazine-tolerant cultivar Stallion, the authors wished to assess whether the mutation for herbicide-tolerance may weaken the plant further upon exposure to enhanced UV-B radiation.
Several studies have addressed the problems of potential interacting stress factors with an enhanced level of UV-B radiation. These investigations have shown that, in some instances, cross-tolerance was evident, while in others an additional stress was additive, synergistic or without interactive effect (Nikolopoulos et al., 1995; Conner and Zangori, 1998; Bornman and Teramura, 1993, and references therein). The amount of photosynthetically active radiation (PAR, 400–700 nm) has been shown to have a definite influence on the way a plant responds to enhanced levels of UV-B radiation (Teramura, 1980; Warner and Caldwell, 1983; Cen and Bornman, 1990) and, in sufficient amount, it may ameliorate the damaging effects of UV-B radiation on photosynthesis. There is also evidence that certain mechanisms protecting PSII from damage by excess light may be modified by increased amounts of UV-B radiation. For example, the xanthophyll cycle, which contributes to photoprotection under high visible irradiation through the dissipation of excess excitation energy (Demmig-Adams, 1990), may, in some instances, be negatively affected by enhanced levels of UV-B radiation, probably through a decreased activity of the violaxanthin de-epoxidase (Pfündel et al., 1992).
Inhibition by visible light and UV-B radiation may not always reflect damage via similar mechanisms (Demeter et al., 1987; Melis et al., 1992), although the mechanisms behind PSII inhibition are not fully elucidated. It has been suggested that one of the first events in acceptor-side photoinhibition of PSII by PAR is the inactivation of the QB-binding site (Mulo et al., 1998), which is reversible in low light without the need for protein synthesis. The next step, where the double-reduced QA leaves its site on the D2 protein, induces D1 protein degradation. UV-B radiation may inhibit the photoreduction of QA and plastoquinones (PQ) and lower the rates of PSII electron transport (Melis et al., 1992). The PQ molecule bound to the QB-binding site of the D1 protein may, therefore, act as the photoreceptor (Melis et al., 1992). It has been proposed (Greenberg et al., 1989) that there are two different photosensitizers for D1 degradation by visible light and UV radiation; in the former case, bulk photosynthetic pigments, and in the latter, plastoquinone, in one or more of its redox states. Perhaps the PQ bound in the QB binding site may act as a trigger for the D1 degradation under exposure to UV-B radiation (Melis et al., 1992). In contrast to photoinhibition by PAR, the breakdown of the D1 protein under UV-B radiation appears not to be a proteolytic process, due to the low sensitivity found to protease inhibitors and temperature (Melis et al., 1992; Friso et al., 1994, 1995), but rather a consequence of the formation of hydroxyl radicals (Hideg and Vass, 1996). Due to the reduced affinity for plastoquinones in atrazine-tolerant mutants (Bowes et al., 1980) and the increased amount of free radicals, caused by the longer-lived, double-reduced QA (Kyle, 1987), atrazine-tolerant plants may be differently susceptible to photoinhibition by PAR and UV-B radiation compared to normal cultivars. However, there is no simple correlation between the susceptibility to photoinhibition and the efficiency of electron transport on the acceptor side of PSII (Mulo et al., 1998).
The rationale behind these investigations was to determine, using herbicide-tolerance as an example, the degree to which modifications to existing plant crops may or may not be suitable under all climatic conditions. This study reports on data from short-term treatments, although long-term experiments will be needed for further validation of the results.
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Materials and methods |
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Growth conditions
Seeds of oilseed rape, Brassica napus L. cvs Paroll and Stallion, were purchased from Svalöv AB, Svalöv, Sweden. The herbicide-tolerant mutant, Stallion (SV8525953), is a cross of a tolerant (cv. Triton) and a sensitive cultivar (cv. Global) and was selected for herbicide tolerance towards atrazine, yield, seed quality, and maturity. In the atrazine-tolerant cultivars the serine-264 of the D1 protein in the PSII reaction centre is exchanged for glycine. This mutation has been bred into atrazine-tolerant cultivars of Brassica napus by transferring the cytoplasm from resistant birdsrape mustard (Beversdorf and Kott, 1987
). The plants were grown under 400 µmol m-2 s1 photosynthetically active radiation (PAR, 400–700 nm), supplied by a bank of Osram Power Star dysprosium lamps (Awake 400 W/D, Germany). The photoperiod was 12 h with a light/dark temperature of 20/16 °C. The second leaf pair was harvested after 3 weeks.
Pigment and dry weight analyses
Chlorophyll a and b, carotenoids and UV-screening pigments were extracted from pre-frozen leaf discs of 5.3 mm diameter with 80% acetone for chlorophylls and carotenoids. UV-screening pigments were extracted with methanol : water : HCl (79 : 20 : 1, by vol.) (according to Robberecht and Caldwell, 1986). The procedure was carried out on ice under green light and the samples were centrifuged for 10 min at 1600 g. The absorbance of the supernatant (10 ml) was measured at 663.2 nm for Chl a, 646.8 nm for Chl b and 470.0 nm for bulk carotenoids, and at 290, 310 and 330 nm for UV-screening pigments, in a dual-wavelength/double-beam spectrophotometer (Shimadzu, UV-3000). In this study the UV-screening pigments, which include the two major flavonoids, kaempferol and quercetin, were not analysed by HPLC (see instead Olsson et al. 1998). The chlorophyll and carotenoid amounts, normalized to dry weight, were calculated from the equations of Lichtenthaler (Lichtenthaler, 1987). Pigment content and dry weight (80°C for 72 h) were determined in four leaf discs from each of six plants. The experiment was repeated three times.
Radiation treatment: photoinhibitory conditions and UV-B radiation
Leaf discs were floated in Petri dishes on water or another medium depending on the experiment (see below) and were allowed to equilibrate for 30 min under the light conditions for growth. They were then exposed to 1 600 µmol m-2 s-1 from a projector lamp. This was done in order to assess the leaf response to a sudden exposure to strong light. Half of the samples were exposed to supplemental UV-B radiation from Q-PANEL UV313 lamps (Largo, Göteborg, Sweden). Leaf discs were irradiated for 0, 2 or 4 h at 20°C. The amount of UV-B was based on 4.6 kJ m-2 h-1 UV-BBE (biologically effective UV-B radiation, weighted irradiance). This irradiance was chosen because it approximates that used per hour in previous work in this laboratory. Although the amount is relatively high, it is not unnatural (Bachelet et al., 1991). The UV-B radiation was filtered through 3.0 mm Plexiglas (FBL2458, Röhm GMBH, Chemische Fabrik, Germany) and 0.13 mm cellulose diacetate. The Plexiglas extends the lifetime of the cellulose diacetate, the latter of which removes radiation below 290 nm. The spectra of PAR and UV were measured with a Model 752 spectroradiometer (Optronic laboratories, Orlando, FL, USA). Weighted irradiance was calculated using a UV dosage model (Björn and Murphy, 1985) and the generalized plant action spectrum of Caldwell (Caldwell, 1971). The values were normalized to one at 300 nm.
After radiation treatment, recovery in leaf discs was monitored during 4 h under growth light (400 µmol m-2 s-1) in the growth chambers.
Experimental protocol
Chlorophyll a fluorescence:
Chlorophyll a fluorescence was measured on 6 leaf discs from different plants in each treatment and cultivar with a pulse-amplitude modulated fluorometer (PAM 101, 102, 103, H Walz, Effeltrich, Germany). Leaf discs were exposed to the radiation treatment described above and samples were taken out after 0, 2 and 4 h for radiation treatment experiments and 0, 1, 2, 3, and 4 h for recovery experiments. The samples were dark-acclimated for 30 min before measurements, which were performed at 20 °C and in green light. Initial (Fo), maximal (Fm) and variable (Fv=Fm–Fo) fluorescence were determined directly after dark acclimation. To obtain Fm a light pulse of 2000 µmol m-2 s-1 was applied. The quantum yield under illumination ((Fm'–Ft)/Fm') was determined during the measurement. The experiment was carried out three times.
Oxygen evolution:
Rates of oxygen evolution were measured at irradiances of 400 and 1600 µmol m-2 s-1 using a Clark-type leaf O2 electrode with light source (L.S.3) and electrode control units (CB1D; Hansatech, Kings Lynn, UK) connected to a chart recorder. Whole leaf discs (19 mm diameter) were placed on a matting, which was moistened with 1 M NaHCO3, and enclosed within the electrode chamber.
Leaf discs were also incubated in lincomycin, which inhibits de novo chloroplastic protein synthesis. A piece of sandpaper was gently pressed on the abaxial side of the leaf disc to facilitate uptake of lincomycin. The leaf discs were floated on a solution of 1% Tween 20 and 0.6 mM lincomycin (ICN, Ohio, USA) and exposed to radiation treatments after 30 min equilibration, or floated only on 1% Tween 20 during the radiation treatment. The latter were then transferred to the inhibitor medium just before the recovery conditions under growth irradiance. Controls were kept on 1% Tween 20 under growth irradiance.
Four leaf samples per treatment and cultivar were used.
35S-methionine incorporation and thylakoid isolation:
Leaf discs were punched out with a cork borer of 19 mm diameter and a piece of sandpaper was gently pressed on the abaxial side to facilitate uptake of 35S-methionine. For studying the synthesis of D1 protein during recovery, leaf discs were exposed to the radiation treatments described above for 4 h and then placed under the growth light in pulse medium (0.4% Tween 20 with 1 mM carrier methionine and 0.5 mCi 35S-methionine (1.9x107 Bq). Three samples per treatment were removed after 0, 2 and 4 h of recovery. The samples were immediately frozen in liquid nitrogen. The experiment was carried out twice.
Thylakoids were isolated on ice by grinding the frozen leaf discs with ice-cold 50 mM HEPES, pH 7.6, 10 mM NaCl, 5 mM MgCl2, and 0.3 M sorbitol. This was then filtered and centrifuged at 12 000 g for 15 min. The pellets were resuspended and two aliquots withdrawn for Chl analysis. The gel was loaded with 10 µg Chl in each well. SDS-polyacrylamide gel electrophoresis (PAGE) was performed according to Laemmli (Laemmli, 1970) with 12–18% gradient gels containing 6 M urea. The dried gels were exposed to a PhosphorImager plate for 3 d before scanning and quantifying the amount of labelled D1 protein in a PhosphorImager (Molecular Dynamics 475A).
Immunoblot analysis:
Leaf discs were pre-treated with lincomycin and irradiated in the same way as for oxygen evolution measurements. Recovery experiments were carried out on leaf discs that had received the radiation treatments without lincomycin and then incubated in inhibitor medium for 4 h in order to ascertain whether significant D1 protein degradation occurs during recovery. Thylakoids were isolated as above, and 3 µg Chl were loaded and run on SDS–PAGE in 12% (w/v) polyacrylamide gels as described previously (Schägger and von Jagow, 1987). The separated proteins were electrophoretically transferred to PVDF membranes (Hybond PVDF, Amersham, Uppsala, Sweden) using 48 mM TRIS, 39 mM glycine, 20% methanol, and a multiphore II NovoBlot unit (Pharmacia, LKB Biotechnology, Uppsala, Sweden). The membranes were probed using a polyclonal antibody to the D1 protein (psbA, kindly supplied by Dr Roberto Barbato). Bound antibodies were visualized using an ECL detection kit (Amersham Pharmacia Biotech, Uppsala, Sweden) according to the manufacturer's instructions.
Data showing the amount of D1 protein have been normalized to their respective controls or radiation treatments prior to the lincomycin treatment. Three samples per treatment and cultivar were analysed.
Statistics:
For statistical masurements, one-way ANOVA and Student–Newman–Keuls' post-test were used.
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Results |
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Pigment and dry weight analysis
After 3 weeks, leaf dry weight of the herbicide-tolerant cv. Stallion was significantly lower (P<0.001) than that of Paroll, when expressed on a leaf area basis (Table 1
). The chlorophyll and bulk carotenoid content normalized to dry weight did not differ between cultivars (Table 1). The amount of UV-screening pigments, measured at 290, 310 and 330 nm was also the same in the two cultivars (Table 1, 310 nm).
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Photoinhibition and UV-B radiation
The maximal photosynthetic efficiency after dark acclimation shown by Fv/Fm, decreased in both cultivars (Figs 1, 2) during the 4 h of photoinhibition. This was due to an increase in Fo as well as a decrease in Fm in the herbicide-tolerant mutant, whereas most of the reduction in Fv/Fm in cv. Paroll was due to a decreased Fm only. In the mutant Stallion the Fv/Fm after 4 h of photoinhibition was 85% of the control value (P<0.001). The corresponding value in the control cultivar, Paroll, was higher, i.e. 93% of the control (P<0.01). After 4 h of exposure to supplemental UV-B radiation, the Fv/Fm in Stallion and Paroll was 84% (P<0.001) and 90% (P<0.001) of the control value, respectively. Thus in both cultivars Fv/Fm was little affected by UV-B radiation compared to photoinhibitory conditions alone. However, the Fv/Fm in the mutant was significantly lower than in the control cultivar both after 4 h of photoinhibition alone (P<0.001) and with supplemental UV-B radiation (P<0.001).
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), HL treated; (•), HL+UV-B treated; (
), control leaf discs under growth light (400 µmol m-2 s-1). Error bars represent SE of the mean, n=6. Where error bars are not shown, these are smaller than the symbols.
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The photochemical yield under illumination of both cultivars was significantly lower after 4 h of photoinhibitory treatment (mutant, P<0.001; control cv., P<0.05; Fig. 3
) compared to the control irradiation conditions. Additional UV-B radiation did not further decrease the photochemical yield in either cultivar, although the yield of the mutant was c. 75% of that for cv. Paroll under supplemental UV-B, with the difference between the cultivars being extremely significant.
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In the absence of lincomycin, oxygen evolution, measured at 400 µmol m-2 s-1, was significantly decreased in the mutant after 4 h in HL alone. The decrease was even more pronounced with supplemental UV-B radiation (Fig. 4
). In the control cultivar there was no significant decrease after the radiation treatment. After radiation treatment in the presence of lincomycin no oxygen evolution was detected in either cultivar. Similar results were obtained when leaf discs were measured at 1600 µmol m-2 s-1, although the rates were higher (data not shown).
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Degradation of the D1 protein can be monitored by adding the chloroplast protein inhibitor, lincomycin, to the samples. In the presence of lincomycin there was a tendency for less D1 protein in leaf discs of the mutant that were exposed to the radiation treatment compared to the controls exposed to growth light (Fig. 5
). This was indicative of an increased D1 degradation during the radiation treatment, although the results were not statistically significant.
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Recovery from radiation treatment
Recovery from supplemental UV-B radiation was less efficient than that following HL alone (Figs 1, 2). Recovery of Fv/Fm showed an initial fast phase lasting for 1–2 h in the HL-exposed mutant Stallion, which was not as pronounced with UV-B radiation. This fast phase under HL was noticeable as an initial steep rise up to 2 h, followed by levelling out of the curve (Fig. 1). In contrast to the mutant, the pattern in the control cultivar was similar under HL alone and with UV-B radiation (Fig. 2). In the mutant, the increase in Fv/Fm was due to both a decrease in Fo and an increase in Fm, whereas in Paroll it was mostly caused by an increased Fm. When comparing the recovery of Fv/Fm between cultivars, the mutant Stallion was more susceptible to the radiation treatment than Paroll (P<0.05).
The photochemical yield of the mutant after 4 h recovery from HL+UV-B was still significantly lower compared to the controls (Fig. 3; P<0.05). The yield was fairly stable for cv. Paroll, with only slight decreases after radiation treatment, followed by total recovery (Fig. 3).
In the absence of lincomycin, the rate of photosynthetic oxygen evolution was significantly lower in the mutant Stallion after 4 h recovery from supplemental UV-B radiation compared to the control (Fig. 4). However, in the presence of lincomycin, oxygen evolution in the mutant was restored after 4 h under recovery conditions. The restoration was more efficient in cv. Paroll exposed to supplemental UV-B radiation. The oxygen evolution appeared to be less affected by lincomycin during the recovery than under the radiation treatment.
The amount of D1 protein did not change during the 4 h of recovery in the presence of lincomycin, when normalized to leaf material exposed to the radiation treatment without lincomycin or recovery (Fig. 5). Since the turnover of the D1 protein is higher than for the other proteins in the chloroplast (Mattoo et al., 1989), it is possible to measure the de novo synthesis by incorporation of 35S-methionine. The amount of 35S-labelled D1 protein after 4 h of recovery was higher in the mutant than in cv. Paroll (Fig. 6), indicative of a higher de novo synthesis in the mutant, also under control conditions. The incorporation of 35S-methionine into the D1 protein was lower in the samples exposed to HL than in the control, especially in the mutant. After recovery from supplemental UV-B radiation the amount of labelled D1 protein was even lower, thereby showing a low de novo synthesis. Thus, the D1 turnover appeared to be lower during recovery than that during the radiation stress, especially with supplemental UV-B radiation.
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Photoinhibition and UV-B radiation
Acclimatizing plants under relatively low light conditions and then exposing them to higher radiation allows for the study of the capacity of the plant to adjust to a rapid increase in light energy and, in the case of applying enhanced levels of UV-B radiation, it also allows the potential relevance of the UV-B to be assessed. These conditions may simulate to a certain degree those found in temperate areas where many plants coming out of winter and low light conditions will be at their most vulnerable stage. Crop plants, for example, peas and members of the Brassica family, growing in temperate regions are also amongst the most susceptible to UV-B radiation (Jordan, 1993
).
The decrease seen in photosynthesis (Figs 1, 2, 4) after photoinhibitory conditions alone and with supplemental UV-B radiation may have been due to damage to PSII, but could also signify initialization of protective mechanisms (Krause, 1988). Photosystem II is prone to photoinhibition by high PAR (Aro et al., 1993a, b) and UV-B radiation (Strid et al., 1990; Bornman and Sundby-Emanuelsson, 1995), and both kinds of radiation seem to affect the oxidizing as well as the reducing sides of PSII (Renger et al., 1989; Melis et al., 1992; Aro et al., 1993a; Hideg et al., 1993; Vass et al., 1996). Also the mutation in the D1 protein in atrazine-tolerant plants affects both sides (Jursinic and Pearcy, 1988). Besides a slower electron transport from QA to QB on the acceptor-side, the oxygen evolving complex is altered, whereby the S-states become more unstable and recombine in the mutant (Holt et al., 1981, 1983). This may partly explain the reduction in Fv/Fm (Figs 1, 2), photochemical yield under illumination (Fig. 3) and photosynthetic oxygen evolution (Fig. 4) in the mutant after the radiation treatment.
The marked decrease in oxygen evolution seen during radiation treatment in the presence of lincomycin suggests a need for de novo synthesis of thylakoid proteins during the radiation treatment. This is in agreement with the decreased amount of D1 protein in PSII during the radiation treatment with lincomycin, when no de novo synthesis was possible (Fig. 5).
The initial fluorescence (Fo) may be correlated with the dynamics of the D1 protein (Ögren, 1991). The results of the present study show that the increases in Fo, seen especially for the mutant (Fig. 1), were correlated with a low amount of labelled D1 protein (Fig. 5). Earlier studies (Öquist et al., 1992; Aro et al., 1993b) have shown that sun plants, grown under similar light conditions as in the present investigation, have a higher D1 degradation during photoinhibition compared to shade plants, the latter of which were more sensitive to photoinhibition. It was suggested that the higher D1 degradation in the sun plants was part of an active repair system replacing the damaged PSII centres, in contrast to shade plants in which the photoinhibited PSII reaction centres accumulated and dissipated excess excitation energy.
In the present study there was a tendency for a lower amount of D1 protein with UV-B radiation compared to that with HL alone (Fig. 5), although not statistically significant. Together with the lower Fv/Fm this indicates an additive effect of UV-B radiation, which is supported by the work of Greenberg et al. (Greenberg et al., 1989). These authors also showed a synergistic effect of UV-B radiation in combination with visible radiation on D1 degradation.
The more pronounced effects of lincomycin (Figs 4, 5) on the mutant compared to the control cultivar may be due to the faster D1 turnover seen even under normal growth conditions (Sundby et al., 1992).
Recovery
For studies concerning impairment by different stress factors, it is also important to investigate the ability of the plant to recover from stress conditions. Crops growing in the field may experience high levels of PAR and UV-B radiation around noon followed by lower light levels in the afternoon, normally sufficient for recovery. Restoration of photosynthesis, e.g. the synthesis of D1 protein, is light dependent (Prasil et al., 1992), probably due to a light-regulated expression of the gene at the transcriptional and translational level (Mattoo et al., 1989; Kettunen et al., 1997). In the present study the restoration of PSII was followed under a recovery irradiance which was the same as the growth light, since it has been shown earlier that Brassica napus has the highest D1 degradation under the original growth light (Sundby et al., 1993b), and that the capacity for photosynthetic recovery is dependent on the ability of the plant to degrade damaged D1 protein (Aro et al., 1994).
The maximum quantum yield, Fv/Fm (Figs 1, 2), the yield under continuous illumination (Fig. 3) and photosynthetic oxygen evolution (Fig. 4) almost returned to control conditions during recovery, indicating a restoration of PSII, although recovery was generally less in the mutant Stallion. The recovery of PSII is often comprized of two parts, an initial fast phase lasting for less than 1 h, and a slow phase, lasting for several hours (Leitsch et al., 1994). The fast phase is reported to be mostly dependent on the dissipation of excess energy and the slow phase on D1 turnover.
In this investigation, recovery of Fv/Fm reached its maximum level after 1–2 h (Figs 1, 2) in most samples although not in the mutant Stallion exposed to supplemental UV-B radiation. After 2 h there was little further recovery. The amount of 35S-labelled D1 protein after 4 h of recovery was lower in the radiation-treated samples (Fig. 6), which indicates that the recovery was partly independent of an increased de novo synthesis. Also, the unchanged photosynthetic oxygen evolution (Fig. 4) and amount of D1 protein (Fig. 5) after 4 h of recovery in the presence of lincomycin point to a restoration independent of de novo protein synthesis.
These results would be in agreement with the high D1 degradation during the radiation treatment if that were accompanied by a high synthesis of D1, enough to replace the degraded protein. This seems probable, since the oxygen evolution was much higher when synthesis of chloroplastic proteins could occur (in the absence of lincomycin; Fig. 4). Our results are supported by the findings of Kettunen et al. who showed that de novo D1 synthesis was higher during the high-light exposure than during subsequent recovery, due to transcriptional and translational adjustments of psbA gene expression (Kettunen et al., 1997).
It was suggested that peas grown under 300 µmol m-2 s-1 maintained a pool of precursor D1 protein, which may be mobilized for repair faster than the initialization of de novo synthesis after 4 h of photoinhibition at 1700 µmol m-2 s-1 (Öquist et al., 1992). This could possibly explain the results of oxygen evolution in the presence of lincomycin (Fig. 4) as well as the D1 amount presented in Fig. 5. The amount of D1 protein during recovery from supplemental UV-B radiation may reflect the presence of a precursor pool, since de novo protein synthesis was inhibited (Fig. 5). Also, the reduced de novo synthesis of D1 protein during recovery (Fig. 6) may have been due to an impaired gene expression after radiation by high PAR and UV-B radiation (Jordan et al., 1991; Jordan, 1993).
Earlier reports have shown that the restoration of PSII is achieved by degradation, de novo synthesis of D1 protein and reassembly of the PSII centres (Aro et al., 1994). The lower turnover of D1 protein during recovery in the samples exposed to UV-B in the present study was accompanied by a lower photochemical capacity (Figs 1–4) compared to control and HL alone. Thus, one explanation for the incomplete restoration of the photosynthetic efficiency after 4 h of recovery may be the low degradation and de novo synthesis of D1 protein.
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Conclusion |
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The results indicate that UV-B radiation serves as an additional stress to photoinhibition by high PAR. In general, with regard to the capacity to recover from stress conditions, the combination of HL and UV-B radiation further impaired recovery as compared to HL treatment alone, especially in the mutant (Figs 1–4). In both cultivars the D1 turnover seemed to occur mainly during the radiation stress and not during subsequent recovery (Figs 5, 6).
Thus, although the modification of crop plants to withstand certain environmental impacts, such as fungal infection and herbicides, may be seen as clearly advantageous, other impacts, in this case photoinhibition by PAR and enhanced UV-B radiation, may serve to decrease plant fitness.
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Acknowledgments |
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We wish to thank Cecilia Sundby-Emanuelsson, Department of Biochemistry, Lund University, for kindly sharing her methods and knowledge on the analysis of the D1 protein turnover and Dr Roberto Barbato for D1 protein antibodies. The Swedish Natural Science Research Council is gratefully acknowledged for financial support to JFB.
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Notes |
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3 To whom correspondence should be addressed. Fax: +46 46 222 41 13. E-mail:janet.bornman{at}fysbot.lu.se
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Abbreviations |
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Chl, chlorophyll; Fm, maximal fluorescence; Fm', maximal fluorescence under illumination; Fo, initial fluorescence; Ft, fluorescence yield at a given time, t; Fv, variable fluorescence (Fm–Fo); HL, high light treatment; LED, light emitting diode; PAM, pulse amplitude modulated fluorometer; PAR, photosynthetically active radiation (400–700 nm); PQ, plastoquinone; PSII, photosystem II; QA and QB, first and second quinone acceptor of PSII; UV-B, ultraviolet-B radiation (280–320 nm); UV-BBE, biologically effective UV-B radiation..
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References |
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