TY - JOUR
AU - Tyystjärvi, Esa
AB - Abstract Photoinhibition is light-induced inactivation of PSII, and action spectrum measurements have shown that UV light causes photoinhibition much more efficiently than visible light. In the present study, we quantified the contribution of the UV part of sunlight in photoinhibition of PSII in leaves. Greenhouse-grown pumpkin leaves were pretreated with lincomycin to block the repair of photoinhibited PSII, and exposed to sunlight behind a UV-permeable or UV-blocking filter. Oxygen evolution and Chl fluorescence measurements showed that photoinhibition proceeds 35% more slowly under the UV-blocking than under the UV-permeable filter. Experiments with a filter that blocks UV-B but transmits UV-A and visible light revealed that UV-A light is almost fully responsible for the UV effect. The difference between leaves illuminated through a UV-blocking and UV-transparent filter disappeared when leaves of field-grown pumpkin plants were used. Thylakoids isolated from field-grown and greenhouse-grown plants were equally sensitive to UV light, and measurements of UV-induced fluorescence from leaves indicated that the protection of the field-grown plants was caused by substances that block the passage of UV light to the chloroplasts. Thus, the UV part of sunlight, especially the UV-A part, is potentially highly important in photoinhibition of PSII but the UV-screening compounds of plant leaves may offer almost complete protection against UV-induced photoinhibition. Introduction UV radiation (200–400 nm) is harmful to all organisms primarily due to its ability to cause DNA damage and production of reactive oxygen species (for reviews see Ichihashi et al. 2006, Rünger and Kappes 2008, Solovchenko and Merzlyak 2008). UV-B light (280–315 nm) causes several adverse effects on plant growth and photosynthesis, and attenuation of UV-B from ambient level may lead to increase in biomass production by one-third in various land plants (Mazza et al. 2000, Xiong and Day 2001, Day et al. 2001, Zhao et al. 2003, for review, see Kakani et al. 2003). On the other hand, UV-A light (315–400 nm) is considered less harmful than UV-B radiation. Photosynthesis of land plants, measured on leaf area basis, has often been found to remain unaffected by an increase in UV-B radiation if the applied dose has been realistic considering the possible UV-B levels reaching the Earth's surface (Allen et al. 1998, Searles et al. 2001, Xiong and Day 2001). Photoinhibition is a reaction in which the photochemical activity of PSII is lost so that recovery occurs only via synthesis of the D1 protein (for recent reviews, see Tyystjärvi 2008, Vass and Aro 2008). Both visible and UV light cause photoinhibition, and the action spectrum of photoinhibition, measured from isolated thylakoid membranes, shows that the photoinhibitory efficiency has a low peak in red light (650–700 nm), remains fairly constant when going from red to blue–green light (from 650 to 450 nm) and increases substantially with decreasing wavelength from 450 nm to UV-C (220–280 nm) (Jones and Kok 1966, Hakala et al. 2005, Ohnishi et al. 2005). UV-A light of 360 nm is ∼10 times as efficient as visible light (Hakala et al. 2005). Measurements of photoinhibition in intact Arabidopsis leaves also showed an extensive increase in photoinhibitory efficiency with decreasing wavelength throughout the blue region towards UV (Sarvikas et al. 2006) and results from intact cells of the cyanobacterium Synechocystis sp. PCC 6803 point in the same direction (Tyystjärvi et al. 2002). Measurements of the action spectrum of photoinhibition both in vitro (Jones and Kok 1966; Jung and Kim 1990; Hakala et al. 2005; Ohnishi et al. 2005) and in vivo (Tyystjärvi et al. 2002; Sarvikas et al. 2006) suggest that UV light may make a significant contribution to photoinhibition under sunlight. Because sunlight contains much more UV-A than UV-B, while photoinhibitory efficiency increases with decreasing wavelength throughout the UV range, the action spectra suggest that the decisive part of the UV spectrum might be the UV-A rather than the UV-B part. Calculations, based on comparison of the actual spectrum of sunlight with an in vivo action spectrum of photoinhibition, suggested that UV wavelengths would contribute to 84% of photoinhibition under direct sunlight in young Arabidopsis leaves (Sarvikas et al. 2006). Information about the contribution of UV light to photoinhibition in nature is important for predicting the potential effects of depletion of stratospheric ozone. Furthermore, large decrease in the rate of photoinhibition, obtained if the most photoinhibitory but at least productive UV light is filtered off from solar radiation, might improve plant yield in greenhouses. Earlier photoinhibition experiments in full sunlight and UV- filtered sunlight (Herrmann et al. 1996, Häder et al. 1996, 1997a,b, 1998) have shown that in red, brown and green algae, exclusion of UV-B radiation significantly protects against decrease in photosynthesis in sunlight. However, in those experiments, concurrent recovery of photoinhibited PSII centers was allowed to function during illumination, and therefore it is not known whether blocking UV light slows down the damaging reaction of photoinhibition or whether UV light has an adverse effect on the repair of photoinhibited PSII centers. Moreover, UV-B was found to interfere with the repair of photoinhibited PSII in Antarctic phytoplankton (Bouchard et al. 2005). In the present study, we exposed intact pumpkin leaves to natural sunlight, using either a UV-permeable plexiglas or the same plexiglas covered with a filter foil that blocked all radiation <400 nm but allowed 95% of visible light to pass. The importance of UV-B radiation was tested by using a Mylar sheet, which is transparent in the visible and UV-A ranges of light but opaque in UV-B. The leaves were treated with lincomycin, an antibiotic that inhibits chloroplast protein synthesis, thus blocking the concurrent repair of photoinhibited PSII. Chlorophyll fluorescence was measured from control and illuminated leaf disks, and PSII electron transfer activity was instantly measured from thylakoids isolated from control and treated leaves. The results showed that UV light very significantly contributes to photoinhibition when leaves of greenhouse-grown plants are exposed to sunlight. On the other hand, protective substances of field-grown pumpkin plants were found to eliminate the photoinhibitory effect of UV light. Results and Discussion UV radiation of sunlight participates in photoinhibition of pumpkin leaves Experiments of this study aimed at direct measurement of the contribution of solar UV light in the damaging reaction of photoinhibition of PSII. Lincomycin-treated pumpkin leaves were set to the horizontal position and exposed to sunlight either under a UV-permeable bare plexiglas plate or under a UV-blocking film attached to the plexiglas (see Fig. 1 for the experimental setup, the transmission spectra and the estimated spectrum of illumination in the greenhouse used to grow the plants). Samples for oxygen evolution and fluorescence measurements were taken at the beginning and after 3 and 6 h of illumination. The treatments were carried out in June, July and August in Turku, Finland, starting between 7 and 11 a.m. Solar elevation varied from 20° to 52° during the treatments. Experiments were done in both sunny and cloudy weather, avoiding only rainy days. The treatments led to a gradual decrease in oxygen evolution, measured from thylakoids isolated from the treated leaves (Fig. 2). As expected, the decrease in oxygen evolution was faster on sunny days. The time-course of the loss of oxygen evolution was roughly similar to the first-order reaction observed under constant light in laboratory conditions (see e.g. Sarvikas et al. 2010). Due to differences in illumination during different days, parallel treatments under UV-permeable and UV-blocking cover were always compared. Fig. 1 Open in new tabDownload slide Experimental details. Experimental setup for measuring photoinhibition under natural sunlight (A). Transmission spectra of the VS UVT acrylic with and without the UV shield film and the Mylar film, as indicated, in the 200–800 nm range (B) and in the 280–400 nm range (C). The spectra of the acrylic and UV shield were measured before the experiments (black lines in B) and after all experiments (cyan lines in B). The black and cyan lines coincide for the plain acrylic plate. (D) Estimated spectrum of illumination in the greenhouse, calculated by assuming that two-thirds of intensity between 400 and 700 nm is from sunlight (blue line) or that one-third is from sunlight (red line), in comparison with the spectrum of unfiltered terrestrial sunlight delivering the same power between 400 and 700 nm as the two calculated spectra (dashed line). Fig. 1 Open in new tabDownload slide Experimental details. Experimental setup for measuring photoinhibition under natural sunlight (A). Transmission spectra of the VS UVT acrylic with and without the UV shield film and the Mylar film, as indicated, in the 200–800 nm range (B) and in the 280–400 nm range (C). The spectra of the acrylic and UV shield were measured before the experiments (black lines in B) and after all experiments (cyan lines in B). The black and cyan lines coincide for the plain acrylic plate. (D) Estimated spectrum of illumination in the greenhouse, calculated by assuming that two-thirds of intensity between 400 and 700 nm is from sunlight (blue line) or that one-third is from sunlight (red line), in comparison with the spectrum of unfiltered terrestrial sunlight delivering the same power between 400 and 700 nm as the two calculated spectra (dashed line). Fig. 2 Open in new tabDownload slide Photoinhibition of greenhouse-grown pumpkin leaves. Lincomycin-treated leaves were illuminated with sunlight passing through a UV-permeable filter (open symbols) or through a UV-blocking filter (solid symbols) in the course of a sunny day (A) and a cloudy day (B), and oxygen evolution was measured from thylakoids isolated from the illuminated leaves. The error bars show SD from two leaves treated simultaneously. The dotted lines show solar irradiance. Typical results are shown. Fig. 2 Open in new tabDownload slide Photoinhibition of greenhouse-grown pumpkin leaves. Lincomycin-treated leaves were illuminated with sunlight passing through a UV-permeable filter (open symbols) or through a UV-blocking filter (solid symbols) in the course of a sunny day (A) and a cloudy day (B), and oxygen evolution was measured from thylakoids isolated from the illuminated leaves. The error bars show SD from two leaves treated simultaneously. The dotted lines show solar irradiance. Typical results are shown. Blocking UV radiation significantly slowed down the loss of oxygen evolution, on both cloudy and sunny days (Fig. 2). To evaluate the efficiency of the protection, we fitted the decrease in oxygen evolution to the first-order reaction equation; this leads to an estimate of the initial rate of photoinhibition in each case. For comparison of results from different days, we calculated a relative quantum yield for photoinhibition by dividing the rate constant, obtained from the fit, by the average solar power during the experiment. In this calculation, we also took into account that the UV-blocking filter transmitted 5% less visible light than the bare plexiglas plate. According to this analysis, the quantum yield of photoinhibition was 54% higher under the UV-permeable bare plexiglas than under the UV-blocking film (Table 1), indicating that ∼35% of photoinhibition of intact greenhouse-grown leaves under sunlight was caused by the UV part of sunlight. The relative photoinhibitory contribution of UV light was similar on sunny and cloudy days (Fig. 2). According to the standard solar spectrum of the American Society for Testing and Materials (http://rredc.nrel .gov/solar/spectra/am1.5/), the 280–700 nm region of the terrestrial solar spectrum contains 7.3% UV-A and only 0.1% UV-B radiation. In comparison with the small proportion of UV radiation in sunlight, the 35% contribution of UV-induced photoinhibition is large, even though much smaller than our earlier estimate obtained with young Arabidopsis leaves (Sarvikas et al. 2006). Multiplication of the spectrum of terrestrial sunlight with the in vitro action spectrum of photoinhibition measured by Jones and Kok (1966) predicts that the contribution of UV to photoinhibition is 31%, which is very near to the result of the present study. However, differences in UV sensitivity between species are well known (Kakani et al. 2003). Large differences in UV sensitivity have been observed in aquatic bryophytes according to the collection site and the collection date of the samples (Martinez-Abaigar et al. 2009) and even between Arabidopsis accessions originating from nearby geographical regions (Jansen et al. 2010). Table 1 Relative quantum yield of photoinhibition in lincomycin-treated pumpkin leaves under natural sunlight passing through a UV-permeable or a UV-blocking filter Growth site . Relative quantum yield of photoinhibition . Contribution of UV light to photoinhibition, % . t-test . . UV- blocking filter . UV- permeable filter . . . Greenhouse 0.479 ± 0.07 0.740 ± 0.14 35 7.9 × 10−4 Field 0.494 ± 0.11 0.526 ± 0.15 6 0.34 Growth site . Relative quantum yield of photoinhibition . Contribution of UV light to photoinhibition, % . t-test . . UV- blocking filter . UV- permeable filter . . . Greenhouse 0.479 ± 0.07 0.740 ± 0.14 35 7.9 × 10−4 Field 0.494 ± 0.11 0.526 ± 0.15 6 0.34 The rate constant of photoinhibition was estimated by fitting the decrease in the light-saturated rate of oxygen evolution to the first order equation, and the relative quantum yield was calculated by dividing the rate constant by the average solar power during the experiment, also correcting for the 5% attenuation of visible light due to the UV-blocking filter. Each quantum yield estimate represents the mean and SD of six independent experiments, each done with two pumpkin leaves. The t-test shows the probability of obtaining a different quantum yield with the UV-blocking filter by chance. Open in new tab Table 1 Relative quantum yield of photoinhibition in lincomycin-treated pumpkin leaves under natural sunlight passing through a UV-permeable or a UV-blocking filter Growth site . Relative quantum yield of photoinhibition . Contribution of UV light to photoinhibition, % . t-test . . UV- blocking filter . UV- permeable filter . . . Greenhouse 0.479 ± 0.07 0.740 ± 0.14 35 7.9 × 10−4 Field 0.494 ± 0.11 0.526 ± 0.15 6 0.34 Growth site . Relative quantum yield of photoinhibition . Contribution of UV light to photoinhibition, % . t-test . . UV- blocking filter . UV- permeable filter . . . Greenhouse 0.479 ± 0.07 0.740 ± 0.14 35 7.9 × 10−4 Field 0.494 ± 0.11 0.526 ± 0.15 6 0.34 The rate constant of photoinhibition was estimated by fitting the decrease in the light-saturated rate of oxygen evolution to the first order equation, and the relative quantum yield was calculated by dividing the rate constant by the average solar power during the experiment, also correcting for the 5% attenuation of visible light due to the UV-blocking filter. Each quantum yield estimate represents the mean and SD of six independent experiments, each done with two pumpkin leaves. The t-test shows the probability of obtaining a different quantum yield with the UV-blocking filter by chance. Open in new tab The decisive spectral range is UV-A, not UV-B To measure the importance of UV-B light in photoinhibition of PSII, we illuminated the leaves using the bare plexiglas plate and a plate covered with a Mylar sheet (Krause et al. 1999, Mazza et al. 2000). Mylar transmits 83% of visible and UV-A light (Fig. 1B) but blocks most of the UV-B range (280–315 nm) (Fig. 1C). Pairwise comparison of rate constants, obtained after correction for the lower transmission of Mylar in the visible and UV-A ranges, showed that the mean contribution of UV-B radiation in photoinhibition under sunlight was 2 ± 6%, but this difference between Mylar and bare plexiglas is not statistically significant. The decrease in oxygen evolution and ratio of variable to maximum Chl fluorescence (FV/FM) during one experimental day under a UV-transparent filter and the Mylar filter are shown in Fig. 3. Direct calculation on the basis of the in vitro action spectrum of Jones and Kok (1966) and the standard solar spectrum predicts that UV-B contributes by 1.2% to photoinhibition in sunlight. In the earlier published data, plants or algae have been treated with different wavelengths of light while the repair cycle of PSII was allowed to run normally during the treatments. These studies have produced widely varying results, depending on the species. In Antarctic phytoplankton assemblages, UV-A caused somewhat less inhibition than UV-B (Holmhansen et al. 1993) and in shade leaves of tropical plants, UV-B was found to be decisive (Krause et al. 1999). On the other hand, UV-A caused two thirds of the UV damage to photosynthesis in phytoplankton of Lake Titicaca (Helbling et al. 2001). Our data show that the UV-B contribution to photoinhibition of PSII in intact pumpkin leaves is the same order of magnitude as predicted by the in vitro action spectrum. However, the inhibitory efficiency of UV-B radiation is not limited to the damaging reaction of photoinhibition of PSII (Allen et al. 1998). Fig. 3 Open in new tabDownload slide Photoinhibition of greenhouse-grown pumpkin leaves shown as a decrease in oxygen evolution (circles) and FV/FM (triangles). Lincomycin-treated leaves were illuminated with sunlight passing through a UV-permeable filter (open symbols) or through a UV-B blocking Mylar filter (solid symbols) in the course of a sunny day. Fig. 3 Open in new tabDownload slide Photoinhibition of greenhouse-grown pumpkin leaves shown as a decrease in oxygen evolution (circles) and FV/FM (triangles). Lincomycin-treated leaves were illuminated with sunlight passing through a UV-permeable filter (open symbols) or through a UV-B blocking Mylar filter (solid symbols) in the course of a sunny day. Field-grown pumpkin leaves are protected against UV radiation The pumpkin plants used for sunlight photoinhibition experiments of Fig. 1, as well as Arabidopsis plants used in our earlier measurements of the in vivo action spectrum of photoinhibition (Sarvikas et al. 2006), were grown indoors. In order to determine the contribution of UV radiation in photoinhibition of field-grown plants, we carried out similar treatments to leaves of pumpkin plants grown in an open garden. In this case, we found no statistically significant difference between leaves illuminated through the UV-blocking and UV-permeable cover, although the results point to ∼6% slower photoinhibition under the UV-blocking cover (Fig. 4, Table 1). Thus, solar UV radiation virtually fails to induce photoinhibition in field-grown pumpkin leaves, indicating that growth in the field induces an efficient protective mechanism. The inducible UV protection lowers the rate constant of photoinhibition of pumpkin leaves in sunlight by ∼30%. The efficiency of this mechanism is comparable to the efficiency of non-photochemical quenching of excitation energy, which may lower the rate constant of photoinhibition caused by visible light by ∼25% (Tyystjärvi et al. 2005, Sarvikas et al. 2006). The finding that PSII of field-grown pumpkin plants is protected against solar UV radiation is in agreement with the conclusion that the adverse effects of UV radiation on vascular plants of the Antarctic Peninsula are mainly targeted outside of PSII (Xiong and Day 2001). The relative quantum yield of photoinhibition under the UV-blocking cover was similar in field-grown and greenhouse-grown leaves (Table 1), indicating that growth in the field does not induce mechanisms that would efficiently slow down the damaging reaction of photoinhibition induced by visible light. Fig. 4 Open in new tabDownload slide Photoinhibition in field-grown pumpkin leaves. Lincomycin-treated leaves were illuminated with sunlight passing through a UV-permeable filter (open symbols) or through a UV-blocking filter (solid symbols) in the course of a sunny day (A) and a cloudy day (B), and oxygen evolution was measured from thylakoids isolated from the illuminated leaves. The error bars show SD from two leaves treated simultaneously. The dotted lines show solar irradiance. Typical results are shown. Fig. 4 Open in new tabDownload slide Photoinhibition in field-grown pumpkin leaves. Lincomycin-treated leaves were illuminated with sunlight passing through a UV-permeable filter (open symbols) or through a UV-blocking filter (solid symbols) in the course of a sunny day (A) and a cloudy day (B), and oxygen evolution was measured from thylakoids isolated from the illuminated leaves. The error bars show SD from two leaves treated simultaneously. The dotted lines show solar irradiance. Typical results are shown. To get insight into the UV protection mechanism functioning in field-grown pumpkin leaves, we used strong UV-A light (365 nm) as photoinhibitory light for greenhouse-grown and field-grown pumpkin leaves and for thylakoids isolated from the same leaves. UV-A light was chosen for this experiment because the experiments done in sunlight indicate that UV-A is mainly responsible for the UV contribution in photoinhibition. The results showed three times faster photoinhibition under UV-A light in greenhouse-grown than in field-grown leaves (Fig. 5), confirming the higher sensitivity of greenhouse-grown leaves. The result was similar whether oxygen evolution or FV/FM was used to measure photoinhibition. When isolated thylakoids were exposed to the same UV-A intensity as the leaves, the rate constant of photoinhibition was ∼10 times higher than in leaves, indicating that constitutive protection offered by the leaf structure drastically slows down photoinhibition under UV-A light. Furthermore, thylakoids isolated from greenhouse-grown and field-grown pumpkin leaves were equally sensitive to UV-A light (Fig. 5), indicating that the inducible protection of pumpkin leaves functions exclusively outside the thylakoid membrane. Plant species may differ also in this respect. Short daily exposure to direct sunlight induced large increases in thylakoid-bound carotenoids in a shade-grown tropical plant, Anacardium excelsum (Krause et al. 1999) but in two rainforest trees, Tetragastris panamensis and Calophyllum longifolium, growth under near-ambient UV-B did not markedly affect carotenoid levels but increased the UV absorbance of ethanolic extracts, when compared with plants grown under reduced UV-B (Krause et al. 2007). Fig. 5 Open in new tabDownload slide Photoinhibition in UV-A light. Leaves (circles and triangles) and isolated thylakoids (diamonds) of greenhouse-grown (open symbols) and field-grown (solid symbols) pumpkin plants were illuminated with 365 nm light and photoinhibition was measured with oxygen evolution (triangles, diamonds) or with FV/FM (circles). Each data point represents an average of three and eight independent experiments in thylakoid and leaf experiments, respectively, and the error bars, drawn if larger than the symbol, show SD. The lines represent the best fit of the data to a first-order equation, with rate constants of 0.10 ± 0.012 min−1 and 0.12 ± 0.006 min−1 for thylakoids isolated from greenhouse-grown and field-grown plants, respectively, and 0.01 ± 0.003 min−1 and 0.003 ± 0.002 min−1 for leaves of greenhouse-grown and field-grown plants, respectively, calculated from the oxygen evolution data. Fig. 5 Open in new tabDownload slide Photoinhibition in UV-A light. Leaves (circles and triangles) and isolated thylakoids (diamonds) of greenhouse-grown (open symbols) and field-grown (solid symbols) pumpkin plants were illuminated with 365 nm light and photoinhibition was measured with oxygen evolution (triangles, diamonds) or with FV/FM (circles). Each data point represents an average of three and eight independent experiments in thylakoid and leaf experiments, respectively, and the error bars, drawn if larger than the symbol, show SD. The lines represent the best fit of the data to a first-order equation, with rate constants of 0.10 ± 0.012 min−1 and 0.12 ± 0.006 min−1 for thylakoids isolated from greenhouse-grown and field-grown plants, respectively, and 0.01 ± 0.003 min−1 and 0.003 ± 0.002 min−1 for leaves of greenhouse-grown and field-grown plants, respectively, calculated from the oxygen evolution data. The finding that the protective mechanism functions outside the thylakoid membrane suggested that the passage of UV light to chloroplasts is blocked in leaves of field-grown pumpkin plants. We measured the relative transmittance of UV-A light in greenhouse-grown and field-grown pumpkin leaves by illuminating the adaxial surface of the leaf with UV-A light of very low intensity and measuring Chl fluorescence at 685 nm. The same principle was used earlier by Mazza et al. (2000). To overcome complications like amount and distribution of Chl in the leaf structure, we used 400–450 nm light as an internal standard of the fluorescence yield by dividing the fluorescence intensity obtained with 365 nm excitation with the fluorescence intensity obtained with very dim 400–450 nm excitation. Fig. 6 shows that the intensity of UV-A-excited Chl a fluorescence of greenhouse-grown leaves was seven times higher than fluorescence of field-grown leaves. Thus, chloroplasts of greenhouse-grown leaves are exposed to much higher UV-A intensity than chloroplasts of field-grown leaves at the same intensity of sunlight, which explains why the UV part of sunlight fails to cause photoinhibition in field-grown plants. The inducible screening compounds are phenolics, flavonoids and anthocyanins found in cuticle waxes and in vacuoles of epidermal cells and the upper cell layers of the mesophyll (Solovchenko and Merzlyak 2008). Fig. 6 Open in new tabDownload slide Demonstration of the presence of UV-absorbing compounds in field-grown plants. Intensity of 685 nm fluorescence induced by 365 nm illumination in leaves of greenhouse-grown and field-grown pumpkin plants. The values were normalized by dividing by the intensity of 685 nm fluorescence obtained with 450 nm excitation. Fig. 6 Open in new tabDownload slide Demonstration of the presence of UV-absorbing compounds in field-grown plants. Intensity of 685 nm fluorescence induced by 365 nm illumination in leaves of greenhouse-grown and field-grown pumpkin plants. The values were normalized by dividing by the intensity of 685 nm fluorescence obtained with 450 nm excitation. Acclimation to UV radiation can be induced by UV radiation itself (Jenkins 2009). Our greenhouse has ordinary glass windows to let sunlight in and is additionally illuminated with so called daylight lamps (Philips HPI-T Plus). We estimated the spectrum of illumination in the greenhouse using the transmission spectrum of standard window glass obtained from the National Research Council of Canada, http://www.nrc-cnrc.gc.ca, the standard spectrum of terrestrial sunlight from the American Society for Testing and Materials and the emission spectrum of the lamps, obtained from the manufacturer. Measurements in the greenhouse showed that the contribution of sunlight, passing through the windows, varies between one-third and two-thirds, and therefore we simulated the actual illumination spectrum by assuming these two extremes. In both cases, the simulation shows that the UV-A region is fairly similar in the greenhouse and in the field, whereas UV-B radiation is virtually fully absent only in the greenhouse (Fig. 1D). Thus, it may be that although UV-A is mainly responsible for photoinhibition of PSII, it is UV-B radiation, not UV-A, that triggers the synthesis of UV-protective substances in pumpkin. However, the greenhouse illumination is also much less intense than sunlight, and therefore it is possible that high light intensity per se contributes to triggering of the synthesis of UV-protective substances in field-grown plants. Photoinhibition in diffuse light mainly affects the uppermost cell layers In addition to measuring photoinhibition as a decrease in oxygen evolution, we also measured the ratio of variable to maximum Chl fluorescence (FV/FM) from the same leaves. With regard to the importance of solar UV radiation, the results of fluorescence measurements agreed with those of oxygen evolution, showing that solar UV radiation significantly contributes to photoinhibition in greenhouse-grown plants but not in field-grown plants (Fig. 7A, B). Fig. 7 Open in new tabDownload slide Relationship between loss of oxygen evolution and decrease in FV/FM during photoinhibition in sunlight. Decrease in oxygen evolution activity (circles) and FV/FM (stars) during illumination of lincomycin-treated pumpkin leaves in sunlight under a UV-permeable cover (open symbols) or under a UV-blocking cover (solid symbols) (A and B). Leaves were detached from greenhouse-grown (A) and field-grown pumpkins (B), and oxygen evolution was measured from thylakoids isolated from treated leaves. The error bars show SD from two leaves treated simultaneously. Typical results are shown. (C) Oxygen evolution (% of control) as a function of FV/FM (% of control) after 3 h of illumination of lincomycin-treated pumpkin leaves in sunlight under a UV-permeable cover (open symbols) or under a UV-blocking cover (solid symbols). Squares are results from greenhouse-grown plants and triangles from field-grown plants. Oxygen evolution was measured from thylakoids isolated from treated leaves and FV/FM was measured from the leaf surface. Fig. 7 Open in new tabDownload slide Relationship between loss of oxygen evolution and decrease in FV/FM during photoinhibition in sunlight. Decrease in oxygen evolution activity (circles) and FV/FM (stars) during illumination of lincomycin-treated pumpkin leaves in sunlight under a UV-permeable cover (open symbols) or under a UV-blocking cover (solid symbols) (A and B). Leaves were detached from greenhouse-grown (A) and field-grown pumpkins (B), and oxygen evolution was measured from thylakoids isolated from treated leaves. The error bars show SD from two leaves treated simultaneously. Typical results are shown. (C) Oxygen evolution (% of control) as a function of FV/FM (% of control) after 3 h of illumination of lincomycin-treated pumpkin leaves in sunlight under a UV-permeable cover (open symbols) or under a UV-blocking cover (solid symbols). Squares are results from greenhouse-grown plants and triangles from field-grown plants. Oxygen evolution was measured from thylakoids isolated from treated leaves and FV/FM was measured from the leaf surface. During illumination of pumpkin leaves in sunlight, the photoinhibitory decrease in FV/FM occurred much more quickly than the decrease in oxygen evolution during illumination (Fig. 7A, B). A large difference was obtained in both greenhouse-grown and field-grown leaves, under both UV-blocking and UV-permeable filter (Fig. 7C). This difference contrasts with the majority of earlier laboratory data showing that loss of FV/FM closely approximates loss of oxygen evolution during photoinhibition of leaves of higher plants (Krause et al. 1992, Schnettger et al. 1994, Tyystjärvi et al. 1999, Sarvikas et al. 2010). We hypothesized that diffuse light like sunlight that mostly hits the leaf at a small angle penetrates less efficiently to a leaf than collimated light straight from the top of the leaf. Due to inefficient penetration to lower cell layers, diffuse light inhibits the inner layers slowly, compared with the top layer, while collimated light causes more even photoinhibition throughout the leaf. Thus, Chl fluorescence, emitted by the topmost chloroplasts, shows faster inhibition in diffuse light than oxygen evolution measured from thylakoids isolated from the leaf. To test this hypothesis, we compared photoinhibition induced by illuminating lincomycin-treated pumpkin leaves with white light directly from the top to photoinhibition induced by placing a leaf inside an integrating sphere where illumination is diffuse. Illumination at a 45° angle was also tested. The results confirmed that in diffuse light, FV/FM decreases much more quickly than oxygen evolution while illumination with collimated light, either straight from the top or at a 45° angle, causes approximately equal lowering of oxygen evolution and FV/FM, when both parameters are compared with the respective control values (Table 2). Table 2 Influence of the direction of incident light on photoinhibition measured with oxygen evolution or with fluorescence . kPI from oxygen evolution . kPI from FV/FM . kPI obtained from FV/FM divided by kPI obtained from oxygen evolution . 90° 0.33 ± 0.03 0.41 ± 0.06 1.26 45° 0.28 ± 0.06 0.25 ± 0.10 0.89 Diffuse light 0.09 ± 0.01 0.17 ± 0.01 1.86 . kPI from oxygen evolution . kPI from FV/FM . kPI obtained from FV/FM divided by kPI obtained from oxygen evolution . 90° 0.33 ± 0.03 0.41 ± 0.06 1.26 45° 0.28 ± 0.06 0.25 ± 0.10 0.89 Diffuse light 0.09 ± 0.01 0.17 ± 0.01 1.86 Lincomycin-treated pumpkin leaves were illuminated with collimated light at a 90° or 45° angle (PPFD 1350 μmol m 2s−1) or with diffuse light (750 μmol m−2 s−1) obtained by placing the leaf in an integrating sphere. Photoinhibition was measured as loss of oxygen evolution, measured from thylakoids isolated from treated leaves, or as decrease in FV/FM, measured from the adaxial leaf surface. The rate constant of photoinhibition (kPI) was obtained by fitting the decrease in oxygen evolution or FV/FM to the first-order equation. The values represent the mean and SD of at least three independent experiments. Open in new tab Table 2 Influence of the direction of incident light on photoinhibition measured with oxygen evolution or with fluorescence . kPI from oxygen evolution . kPI from FV/FM . kPI obtained from FV/FM divided by kPI obtained from oxygen evolution . 90° 0.33 ± 0.03 0.41 ± 0.06 1.26 45° 0.28 ± 0.06 0.25 ± 0.10 0.89 Diffuse light 0.09 ± 0.01 0.17 ± 0.01 1.86 . kPI from oxygen evolution . kPI from FV/FM . kPI obtained from FV/FM divided by kPI obtained from oxygen evolution . 90° 0.33 ± 0.03 0.41 ± 0.06 1.26 45° 0.28 ± 0.06 0.25 ± 0.10 0.89 Diffuse light 0.09 ± 0.01 0.17 ± 0.01 1.86 Lincomycin-treated pumpkin leaves were illuminated with collimated light at a 90° or 45° angle (PPFD 1350 μmol m 2s−1) or with diffuse light (750 μmol m−2 s−1) obtained by placing the leaf in an integrating sphere. Photoinhibition was measured as loss of oxygen evolution, measured from thylakoids isolated from treated leaves, or as decrease in FV/FM, measured from the adaxial leaf surface. The rate constant of photoinhibition (kPI) was obtained by fitting the decrease in oxygen evolution or FV/FM to the first-order equation. The values represent the mean and SD of at least three independent experiments. Open in new tab In conclusion, our results show that UV radiation is a highly efficient inducer of photoinhibition. However, comparison of isolated thylakoids, greenhouse-grown plants and field-grown plants shows that evolution has equipped land plants with constitutive and inducible UV screens that may fully eliminate the photoinhibitory power of the UV part of solar radiation. Materials and Methods Plant material Pumpkin plants (Cucurbita pepo L.) were grown in a research greenhouse at the mean photosynthetic photon flux density (PPFD) of 150 μmol photons m−2 s−1 in a 16 h light period. The greenhouse is illuminated by sunlight through ordinary glass windows on one wall, and additionally with 400 W Philips HPI-T Plus daylight lamps from the ceiling. Leaves of field-grown pumpkin plants were obtained from the Botanical Garden of the University of Turku; the leaves used for the experiments were collected from an open setting. Thylakoids were isolated according to Hakala et al. (2005). Illumination of leaves Illumination with sunlight Before illumination, leaves were incubated in dim light overnight with the petiole in 2.4 mM lincomycin to inhibit the repair of photoinhibited PSII. For each photoinhibition experiment in sunlight, four leaves were taped on styrox tables in horizontal position, with the petioles in lincomycin solution. Two leaves were kept under a 3 mm plate of UV-permeable acrylic (VS UVT, Altuglas, Le Garenne, France) and two leaves under a plate from the same acrylic covered with a UV-protection film (Long Life for Art, Eichstetten, Germany) (Fig. 1). To measure the effect of UV-B light alone, the UV-permeable acrylic was covered with a layer of clear 0.0015 mm thick Mylar sheet (DuPont Teijin Films, Chester, VA, USA). The experiments were done in Turku, Finland (60°27′ N, 22°17′ E). The leaves were sprayed with water every 30 min to prevent desiccation and to lower the leaf temperature. After 0, 3 and 6 h of illumination, two 3.5 cm2 disks were cut from every leaf, one for thylakoid isolation and one for fluorescence measurement. Weather data were obtained from the weather station of Process Design & Systems Engineering Laboratory, Åbo Akademi University, Turku (http://at8.abo.fi/cgi-bin/en/get_weather). Illumination with UV light To measure photoinhibition induced with UV-A light, lincomycin-treated leaves and isolated thylakoid membranes were illuminated with ENF-280C lamp (Spectronics, Westbury, NY, USA) that emits a wide spectral peak centered at 365 nm. The lamp was placed at 1 cm distance from the leaf surface, and the photon flux density of the UV-A light (measured earlier by Hakala et al. 2005) was 170 μmol m−2s−1. The same lamp at the same distance was used for illumination of isolated thylakoids (1 ml, 50 μg Chl ml−1). Illumination with white light White light was used for comparison of diffuse and collimated light. For illumination with collimated light (PPFD 1350 μmol m−2 s−1), a lincomycin-treated pumpkin leaf was placed under a high-pressure Xenon lamp (model 6258; Oriel, Stanford, CT, USA) and illuminated through a UV-blocking filter. For oblique illumination, the leaf was placed at a 45° angle to the lamp in otherwise identical conditions. Diffuse light was obtained by placing a leaf inside a 60 cm diameter integrating sphere illuminated by white light from a slide projector. A diffusor cone was placed in the input light path. PPFD, measured from an empty sphere, was 750 μmol m−2 s−1. Measurements of oxygen evolution and Chl fluorescence The light-saturated rate of oxygen evolution was measured with an oxygen electrode (Hansatech, King's Lynn, UK) as described by Hakala et al. (2005). Chl concentration was 10 μg ml−1, and 125 μM 2,6-dichlorobenzoquinone was used as electron acceptor. The ratio of variable to maximum fluorescence (FV/FM) was measured from leaf disks with FluorPen (PS Instruments, Brno, Czech Republic) after 40 min of dark incubation. Quantum yield of photoinhibition An estimate of the rate constant of photoinhibition was obtained by fitting the photoinhibitory loss of PSII oxygen evolution, measured from thylakoids isolated from treated leaves, to the first-order reaction equation. Each experiment, with data from two leaves measured after 0, 3 and 6 h of illumination, was fitted separately. The best fit is obtained by taking into account all three time points (e.g. data in Fig. 2A yield two rate constant values). The average solar power (kW m−2) during the experiment was calculated as an average of the data obtained for every half hour. The relative quantum yield of photoinhibition was calculated by dividing the rate constant of photoinhibition by the average solar power during the illumination treatment. Comparisons between the treatments were done by averaging the quantum yield values from several independent experiments. Measurement of UV-induced fluorescence For the measurement of relative UV-A transmission, a leaf disk was excited with a 365 nm illuminator (LM-26E, Cell Biociences, Inc., Santa Clara, CA, USA) and the emission spectrum of the leaf in the 600–800 nm range was measured with an S2000 spectrophotometer (Ocean Optics, Dunedin, FL, USA). To ensure that the UV-induced fluorescence remained constantly at the F0 level, the intensity of the UV light was attenuated until fluorescence yield at 685 nm did not change when the UV-A light was switched on after placing a dark-adapted leaf disk in the measuring position. Eventual differences in leaf Chl concentration and in optical properties of the leaf samples were compensated for by dividing the amplitude of 685 nm fluorescence by fluorescence excited by a weak beam of 450 nm light. The 450 nm light was obtained by placing a LS450 filter (Corion) in front of a slide projector and using a light guide to illuminate the leaf disk. Funding This work was supported by the Academy of Finland and by the Finnish Cultural Foundation. Acknowledgements Taina Tyystjärvi is thanked for important comments on the manuscript. Kurt Lundqvist (Åbo Akademi University) is thanked for access to the weather data and Sinikka Vento (Botanical Garden) for the permission to use their pumpkin leaves. Abbreviations Abbreviations PPFD photosynthetic photon flux density References Allen DJ , Nogués S , Baker NR . Ozone depletion and increased UV-B radiation: is there a real threat to photosynthesis? , J. Exp. Bot. , 1998 , vol. 49 (pg. 1775 - 1788 ) Google Scholar OpenURL Placeholder Text WorldCat Bouchard JN , Roy S , Ferreyra G , Campbell DA , Curtosi A . Ultraviolet-B effects on photosystem II efficiency of natural phytoplankton communities from Antarctica , Polar Biol. , 2005 , vol. 28 (pg. 607 - 618 ) Google Scholar Crossref Search ADS WorldCat Day TA , Ruhland CT , Xiong FS . Influence of solar ultraviolet-B radiation on Antarctic terrestrial plants: results from a 4-year field study , J. Photochem. Photobiol. B: Biol. , 2001 , vol. 62 (pg. 78 - 87 ) Google Scholar Crossref Search ADS WorldCat Häder D-P , Herrmann H , Santas R . Effects of solar radiation and solar radiation deprived of UV-B and total UV on photosynthetic oxygen production and pulse amplitude modulated fluorescence in the brown alga Padina pavonia , FEMS Microbiol. Ecol. , 1996 , vol. 19 (pg. 53 - 61 ) Google Scholar Crossref Search ADS WorldCat Häder D-P , Lebert M , Flores-Moya A , Jiménez C , Mercado J , Salles S , et al. Effects of solar radiation on the photosynthetic activity of the red alga Corallina elongata Ellis et Soland , J. Photochem. Photobiol. B: Biol. , 1997 , vol. 37 (pg. 196 - 202 ) Google Scholar Crossref Search ADS WorldCat Häder D-P , Porst M , Herrmann H , Schäfer J , Santas R . Photosynthesis of the mediterranian green alga Caulerpa prolifera measured in the field under solar irradiation , J. Photochem. Photobiol. B: Biol. , 1997 , vol. 37 (pg. 66 - 73 ) Google Scholar Crossref Search ADS WorldCat Häder D-P , Porst M , Santas R . Photoinhibition by solar radiation in the Mediterranian alga Peyssonnelia squamata measured on site , Plant Ecol. , 1998 , vol. 139 (pg. 167 - 175 ) Google Scholar Crossref Search ADS WorldCat Hakala M , Tuominen I , Keränen M , Tyystjärvi T , Tyystjärvi E . Evidence for the role of the oxygen-evolving manganese complex in photoinhibition of Photosystem II , Biochim. Biophys. Acta Bioenergetics , 2005 , vol. 1706 (pg. 68 - 80 ) Google Scholar Crossref Search ADS WorldCat Helbling EW , Villafane VE , Buma AGJ , Andrade M , Zaratti F . DNA damage and photosynthetic inhibition induced by solar ultraviolet radiation in tropical phytoplankton (Lake Titicaca, Bolivia) , Eur. J. Phycol. , 2001 , vol. 36 (pg. 157 - 166 ) Google Scholar Crossref Search ADS WorldCat Herrmann H , Häder D-P , Köfferlein M , Seidlitz HK , Ghetti F . Effects of UV radiation on photosynthesis of phytoplankton exposed to solar simulator light , J. Photochem. Photobiol. B: Biol. , 1996 , vol. 34 (pg. 21 - 28 ) Google Scholar Crossref Search ADS WorldCat Holmhansen O , Helbling EW , Lubin D . Ultraviolet-radiation in Antarctica - inhibition of primary production , Photochem. Photobiol. , 1993 , vol. 58 (pg. 567 - 570 ) Google Scholar Crossref Search ADS WorldCat Ichihashi M , Ueda M , Budiyanto A , Bito T , Oka M , Fukunaga M , et al. UV-induced skin damage , Toxicology , 2006 , vol. 189 (pg. 21 - 39 ) Google Scholar Crossref Search ADS WorldCat Jansen MAK , Le Martret B , Koornneef M . Variations in constitutive and inducible UV-B tolerance dissecting photosystem II protection in Arabidopsis thaliana accessions , Physiol. Plant. , 2010 , vol. 138 (pg. 22 - 34 ) Google Scholar Crossref Search ADS PubMed WorldCat Jenkins GI . Signal transduction in responses to UV-B radiation , Annu. Rev. Plant Biol. , 2009 , vol. 60 (pg. 407 - 431 ) Google Scholar Crossref Search ADS PubMed WorldCat Jones LW , Kok B . Photoinhibition of chloroplast reactions. I. Kinetics and action spectra , Plant Physiol. , 1966 , vol. 41 (pg. 1037 - 1043 ) Google Scholar Crossref Search ADS PubMed WorldCat Jung J , Kim H-S . The chromophores as endogenous sensitizers involved in the photogeneration of singlet oxygen in spinach thylakoids , Photochem. Photobiol. , 1990 , vol. 52 (pg. 1003 - 1009 ) Google Scholar Crossref Search ADS WorldCat Kakani VG , Reddy KR , Zhao D , Sailaja K . Field crop responses to ultraviolet-B radiation: a review , Agric. Forest Meteorol. , 2003 , vol. 120 (pg. 191 - 218 ) Google Scholar Crossref Search ADS WorldCat Krause GH , Jahns P , Aurelio V , Gracía M , Aranda J , Wellmann E , et al. Photoprotection, photosynthesis and growth of tropical tree seedlings under near-ambient and strongly reduced solar ultraviolet-B radiation , J. Plant Physiol. , 2007 , vol. 164 (pg. 1311 - 1322 ) Google Scholar Crossref Search ADS PubMed WorldCat Krause GH , Schmude C , Garden H , Koroleva OY , Winter K . Effects of solar ultraviolet radiation on the potential efficiency of photosystem II in leaves of tropical plants , Plant Physiol. , 1999 , vol. 121 (pg. 1349 - 1358 ) Google Scholar Crossref Search ADS PubMed WorldCat Krause GH , Somersalo S , Zumbusch E , Weyers B , Laasch H . On the mechanism of photoinhibition in chloroplasts. Relationship between changes in fluorescence and activity of photosystem II , J. Plant Physiol. , 1992 , vol. 136 (pg. 472 - 479 ) Google Scholar Crossref Search ADS WorldCat Martinez-Abaigar J , Otero S , Tomas R , Nunez-Olivera E . Effects of enhanced ultraviolet radiation on six aquatic bryophytes , Cryptogamie Bryol. , 2009 , vol. 30 (pg. 157 - 175 ) Google Scholar OpenURL Placeholder Text WorldCat Mazza CA , Boccalandro HE , Giordano CV , Battista D , Scopel AL , Ballaré CL . Functional significance and induction by solar radiation of ultraviolet-absorbing sunscreens in field-grown soybean crops , Plant Physiol. , 2000 , vol. 122 (pg. 117 - 125 ) Google Scholar Crossref Search ADS PubMed WorldCat Ohnishi N , Allakhverdiev SI , Takahashi S , Higashi S , Watanabe M , Nishiyama Y , et al. Two-step mechanism of photodamage to photosystem II: step 1 occurs at the oxygen evolving complex and step 2 occurs at the photochemical reaction center , Biochemistry , 2005 , vol. 44 (pg. 8494 - 8499 ) Google Scholar Crossref Search ADS PubMed WorldCat Rünger TM , Kappes UP . Mechanisms of mutation formation with long-wave ultraviolet light (UVA) , Photoderm. Photoimmun. Photomed. , 2008 , vol. 24 (pg. 2 - 10 ) Google Scholar Crossref Search ADS WorldCat Sarvikas P , Hakala M , Pätsikkä E , Tyystjärvi T , Tyystjärvi E . Action spectrum of photoinhibition in leaves of wild type and npq1-2 and npq4-1 mutants of Arabidopsis thaliana , Plant Cell Physiol. , 2006 , vol. 47 (pg. 391 - 400 ) Google Scholar Crossref Search ADS PubMed WorldCat Sarvikas P , Tyystjärvi T , Tyystjärvi E . Kinetics of prolonged photoinhibition revisited: photoinhibited Photosystem II centres do not protect the active ones against loss of oxygen evolution , Photosynth. Res. , 2010 , vol. 103 (pg. 7 - 17 ) Google Scholar Crossref Search ADS PubMed WorldCat Schnettger B , Critchley C , Santore UJ , Graf M , Krause GH . Relationship between photoinhibition of photosynthesis, D1 protein turnover and chloroplast structure: effects of protein synthesis inhibitors , Plant Cell Env. , 1994 , vol. 17 (pg. 55 - 64 ) Google Scholar Crossref Search ADS WorldCat Searles PS , Fling SD , Caldwell MM . A meta-analysis of plant field studies simulating stratospheric ozone depletion , Oecologia , 2001 , vol. 127 (pg. 1 - 10 ) Google Scholar Crossref Search ADS WorldCat Solovchenko AE , Merzlyak MN . Screening of visible and UV radiation as a photoprotective mechanism in plants , Russ. J. Plant Physiol. , 2008 , vol. 55 (pg. 719 - 737 ) Google Scholar Crossref Search ADS WorldCat Tyystjärvi E . Photoinhibition of photosystem II and photodamage of the oxygen evolving manganese cluster , Coord. Chem. Rev. , 2008 , vol. 252 (pg. 361 - 376 ) Google Scholar Crossref Search ADS WorldCat Tyystjärvi E , Hakala M , Sarvikas P . Mathematical modelling of the light response curve of photoinhibition of photosystem II , Photosynth. Res. , 2005 , vol. 84 (pg. 21 - 27 ) Google Scholar Crossref Search ADS PubMed WorldCat Tyystjärvi E , Riikonen M , Arisi A-CM , Kettunen R , Jouanin L , Foyer CH . Photoinhibition of photosystem II in tobacco plants overexpressing glutathione reductase and poplars overexpressing superoxide dismutase , Physiol. Plant. , 1999 , vol. 105 (pg. 409 - 416 ) Google Scholar Crossref Search ADS WorldCat Tyystjärvi T , Tuominen I , Herranen M , Aro E-M , Tyystjärvi E . Action spectrum of psbA gene transcription is similar to that of photoinhibition in Synechocystis sp. PCC 6803 , FEBS Lett. , 2002 , vol. 516 (pg. 167 - 171 ) Google Scholar Crossref Search ADS PubMed WorldCat Vass I , Aro E-M . Renger G . Photoinhibition of photosystem II electron transport , Primary Processes of Photosynthesis: Basic Principles and Apparatus. Comprehensive Series in Photochemical and Photobiological Sciences , 2008 , vol. Vol. 8 Cambridge Royal Society of Chemistry (pg. 393 - 411 ) Google Scholar Google Preview OpenURL Placeholder Text WorldCat COPAC Xiong FS , Day TA . Effect of solar ultraviolet-B radiation during springtime ozone depletion on photosynthesis and biomass production of Antarctic vascular plants , Plant Physiol. , 2001 , vol. 125 (pg. 738 - 751 ) Google Scholar Crossref Search ADS PubMed WorldCat Zhao D , Reddy KR , Kakani VG , Read JJ , Sullivan JH . Growth and physiological responses of cotton (Gossypium hirsutum L.) to elevated carbon dioxide and ultraviolet-B radiation under controlled environmental conditions , Plant Cell Env. , 2003 , vol. 26 (pg. 771 - 782 ) Google Scholar Crossref Search ADS WorldCat © The Author 2010. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For permissions, please email: journals.permissions@oxfordjournals.org © The Author 2010. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For permissions, please email: journals.permissions@oxfordjournals.org
TI - Contributions of Visible and Ultraviolet Parts of Sunlight to Photoinhibition
JF - Plant and Cell Physiology
DO - 10.1093/pcp/pcq133
DA - 2010-10-01
UR - https://www.deepdyve.com/lp/oxford-university-press/contributions-of-visible-and-ultraviolet-parts-of-sunlight-to-BrQquSigcj
SP - 1745
EP - 1753
VL - 51
IS - 10
DP - DeepDyve
ER -