Plant Responses to Current Solar Ultraviolet-B Radiation and to Supplemented Solar Ultraviolet-B Radiation Simulating Ozone Depletion: An Experimental Comparison¶
Rousseaux, M Cecilia
Field experiments assessing UV-B effects on plants have been conducted using two contrasting techniques: supplementation of solar UV-B with radiation from fluorescent UV lamps and the exclusion of solar UV-B with filters. We compared these two approaches by growing lettuce and oat simultaneously under three conditions: UV-B exclusion, near-ambient UV-B (control) and UV-B supplementation (simulating a 30% ozone depletion). This permitted computation of “solar UV-B” and “supplemental UV-B” effects. Microclimate and photosynthetically active radiation were the same under the two treatments and the control. Excluding UV-B changed total UV-B radiation more than did supplementing UV-B, but the UV-B supplementation contained more “biologically effective” shortwave radiation. For oat, solar UV-B had a greater effect than supplemental UV-B on main shoot leaf area and main shoot mass, but supplemental UV-B had a greater effect on leaf and tiller number and UV-B-absorbing compounds. For lettuce, growth and stomatal density generally responded similarly to both solar UV-B and supplemented UV-B radiation, but UV-absorbing compounds responded more to supplemental UV-B, as in oat. Because of the marked spectral differences between the techniques, experiments using UV-B exclusion are most suited to assessing effects of present-day UV-B radiation, whereas UV-B supplementation experiments are most appropriate for addressing the ozone depletion issue.
Abbreviations: ANOVA, analysis of variance; BSWF, biological spectral weighting function; PAR, photosynthetically active radiation (400-700 nm).
Field experiments examining UV-B radiation effects on plants have typically been conducted using two very different methods. Solar UV-B radiation may be supplemented with UV-B radiation from special filtered fluorescent lamps in an effort to simulate different scenarios of solar radiation with stratospheric ozone depletion (1). A contrasting approach uses filters to remove much of the radiation at shorter wavelengths from the solar spectrum, thereby assessing the influence of current solar UV-B flux (2). There have only been a few limited attempts to compare the biological effects of the two approaches in simultaneous experiments (3-7).
The two types of experiments differ substantially in the manner in which the solar spectral irradiance is modified. In lamp supplementation experiments simulating ozone depletion, the added UV-B is mostly provided at the shorter UV-B wavelengths roughly similar to how the solar spectral irradiance would be augmented with ozone depletion. For exclusion experiments, most of the UV-B wave band is simply removed without an attempt to simulate solar radiation with increasing ozone nitration. Thus, the two types of experiments lead to very different modifications of the solar spectral irradiance, and any comparison must take this into account. An additional method, using the elegant technique of generating ozone in Plexiglass cuvettes to filter sunlight (8), can more closely simulate ozone depletion than either of the above techniques. However, its expense and complex technology has prevented widespread adoption.
Weighting spectral irradiance with dimensionless factors to account for biological effectiveness (biological spectral weighting functions [BSWF]) and integrating over wavelength to produce a single metric of “biologically effective irradiance” have been commonly used to evaluate simulations of stratospheric ozone reduction (9,10). Shorter wavelengths are generally considered the most biologically effective portion of the UV-B wave band, but there is considerable uncertainty as to the shape of the BSWF and, given this uncertainty, several different BSWF have been used. Even though they all have the common property of declining with increasing wavelength, they provide different estimates of biologically effective irradiance (11,12). In exclusion studies, generally most of the solar UV-B irradiance is removed and computation of biologically effective irradiance is not a critical part of the experimental design. Although these studies can show effects of ambient solar UV-B, extrapolating to potential effects of ozone depletion is tenuous. Exclusion studies clearly involve greater manipulations of the UV-B in terms of total flux (Fig. 1); yet, they may not alter the biologically effective radiation as much as lamp supplementation (Table 1).
There is a sizeable literature on plant response to UV manipulation under the two experimental approaches. Lamp UV supplementation studies have been quantitatively summarized in a meta-analysis by Searles et al. (17). Overall, this meta-analysis showed that many plant characteristics, such as photosynthesis, photosynthetic pigments, leaf mass per area and reproductive yield, underwent no significant changes due to UV-B supplementation. The increased UV-B did significantly decrease plant height, shoot mass and leaf area and increase UV-B-absorbing compounds. A similar meta-analysis has yet to be conducted for UV-exclusion studies. Some substantial effects on growth have been reported in exclusion studies conducted directly under the Antarctic ozone “hole” (18,19), and more subtle effects are apparent toward the periphery of the hole at the tip of South America (20-22). At lower latitudes, a response to UV-B exclusion can sometimes still be detected, but there is considerable variation among studies (2,23-27). It is difficult to summarize these two groups of experiments, but it has been suggested that plants may be more responsive in UV-B-exclusion studies than in UV-B supplementation studies (28).
Only a few direct comparisons of plant response to supplementation and exclusion approaches have been conducted. In several of the studies, only a few responses to the treatments occurred, or if there were responses, the microclimates in the supplementation and exclusion treatments were not similar (3-5). We are only aware of one set of experiments with higher plants in which all microclimatic factors such as shading were equalized (6,7). Exclusion experiments from this study demonstrated that ambient solar UV-B often, but not always, resulted in more growth inhibition and more flavonol accumulation in wild-type Petunia than did supplemental radiation from lamps simulating less than 15% ozone depletion.
In this article, we report the results of an experiment containing both UV-B exclusion and supplementation treatments for two plant species of different growth forms (lettuce and oat). The common control, near-ambient UV-B solar radiation, allowed for the direct comparison of these two very different treatments. Thus, the effect of solar UV-B was defined as the difference in plant response between the near-ambient control and solar radiation with UV-B excluded, whereas the effect of supplemental UV-B was considered as the difference in response between solar radiation supplemented with UV-B from lamps and the near-ambient control. We paid particular attention in designing this experiment, so the photosynthetically active radiation (PAR) and other microclimatic factors were the same under the UV-B-exclusion, the near-ambient UV-B control and the supplemented solar UV-B radiation treatments.
MATERIALS AND METHODS
Plant material. Lettuce (Lactuca sativa L. var. buttercrunch) and oat (Avena sativa L. var. otana) were selected for comparing the two experimental approaches because both species have been shown in previous research to respond to UV manipulations (29,30). Seeds were planted in a loam soil-perlite mixture (3:1, vol/vol) in 0.15 L conical “conetainers” (Stuewe and Sons, Corvallis, OR) on 24 August 1999. Seedlings were allowed to germinate for 5 days in a greenhouse (for protection from grasshoppers) and then transferred to the field experimental site. Screening around the plots and plastic covers provided protection from insects in the field. Conetainers were arranged in racks (30 × 30 cm) approximating a density of 100 plants m^sup -2^. Plants were watered daily and received 5-10 mL week^sup -1^ (starting at 5 mL and increased incrementally to 10 mL as the plants grew) of a complete nutrient solution (Peters Horticultural Products, Mayville, OH).
Irradiation conditions. Three contrasting spectral irradiance conditions (solar UV-B exclusion [uvb-], near-ambient solar UV [control] and UV-B supplementation [uvb+]) were achieved by combining filtered solar radiation and UV-B supplementation using lamps (Fig. 1). Solar UV-B radiation was filtered by using either UV-B-transmitting or UV-B-absorbing plastics. All plots (three for each of the two treatments and three for the control) had plastic covers over them and experienced similar PAR (total photon flux, 400-700 nm) and other microclimatic conditions. Supplemental UV-B was provided by racks of fluorescent UV-B 313 lamps (Q Panel, Cleveland, OH; six lamps per rack, each lamp set 32 cm apart). To further assure uniform shading conditions, lamp racks were mounted above all plots and lamps were filtered to either transmit or block UV-B radiation depending on the treatment. The lamp and solar filters are described in Table 1. The reduction in PAR by the filters and lamp racks (ca 20% at midday) was the same in all three treatments.
Lamp racks were set 40 cm above the canopy, and lamp output was modulated electronically to track ambient solar UV-B (31). A broadband UV-B sensor (32), heated to a constant temperature, provided the signal to the modulation system and recorded ambient solar UV-B. Before the experiment started, all lamps were filtered with cellulose diacetate (JCS Plastics, La Mirada, CA) and lhc plots were covered with premium cellulose triacetate (Liard Plastics, Salt Lake City, UT) so that lamp output could be electronically adjusted (see below). After this adjustment, the treatment filters (Table 1) were installed for the initiation of the experiment. The lamp filters were changed every 10 days, whereas filters covering the plots were not changed because their transmittance remained relatively constant during the experiment.
Solar spectral irradiance and spectral irradiance under the combined lamp and filter systems were measured with a double-grating model 742 spectroradiometer (Optronic, Orlando, FL), calibrated as described in Flint and Caldwell (33). Lamp emittance was adjusted according to Caldwell et al. (34) to simulate a 30% ozone depletion over Logan, UT (41.5°N), using the generalized plant response function normalized to 300 nm (14, as formulated by Green et al. ). The simulation of a sizeable ozone depletion (30%) was chosen to increase the likelihood of plant response to the supplemental UV and, thus, facilitate the comparison with the UV-B-exclusion treatment.
Plant measurements. The experimental treatments were terminated after 22 days for lettuce and 29 days for oat. At this time, morphological parameters were measured, samples were collected for determination of UV-absorbing compounds and plants were separated into parts (e.g. stem and leaf) for dry mass determination. In all cases, five plants were sampled per rack, and rack means were used for statistical analysis (see below). Foliage area was determined on a whole-plant, tiller or main-stem basis (depending on species) with a leaf area meter (LI-3100, Li-Cor, Lincoln, NE). Dry mass was determined after 48 h at 70°C.
UV-absorbing compounds were extracted from 165 mm^sup 2^ leaf tissue in 5 mL of methanol-HCl (99:1, vol/vol). Tissue was disrupted by storing at -20°C for more than 48 h and then wanning to 20°C (36,37). Extract absorbance was measured in a Model 35 double-beam spectrophotometer (Beckman, Fullerton, CA) at 305 nm. Absorbance was expressed on a mass basis (A mg^sup -1^) by drying the extracted material at 70°C for 48 h.
Impressions of the adaxial epidermis were taken with clear nail varnish. After 3-5 min of drying, the imprint was carefully peeled from the leaf and mounted on a glass slide. Microscopic determinations were performed on three images per leaf. Stomatal density (number per unit area) was determined al 200× for oat and 400× for lettuce. Cell dimensions (width and length) were determined by direct measurement or estimated based on the number of cells occupying a given area of the microscope field.
Statistics and computations. There were six racks in a plot (three for each species), and racks were rotated within plots every 2 days and also between plots of the same treatment weekly. Because the racks of plants were rotated between plots and not paired with any neighboring racks, we considered each rack of plants as the experimental unit. Five plants (i.e. subreplicates) were measured in each rack, and the data were averaged. The total number of racks per treatment was eight for lettuce and nine for oat.
Differences between treatments were analyzed using standard techniques for the analysis of variance (ANOVA, PROC GLM; SAS Release 6.11, SAS Institute, Cary, NC). Comparisons between specific treatments were performed using Duncan’s post ANOVA test. When significant differences in the means for a plant trait were found, the relative change due to solar UV-B or supplemental lamp UV-B, compared with the control (near-ambient UV-B), was calculated from the treatment means as: solar UV-B response (%) = [(near ambient – uvb-)/near ambient]100 or supplemental UV-B response (%) = [(uvb+ – near ambient)/near ambient]100.
We also calculated the relative effect of solar UV-B and supplemental lamp UV-B that would be predicted based on the weighted UV spectral irradiance using two rather different weighting functions, the generalized plant action spectrum (14) and the action spectrum for DNA damage of intact alfalfa seedlings (15), from Table 1. For example, the predicted proportion of solar UV-B effect was calculated using the values of weighted spectral irradiance for the individual treatments in Table 1 as: (near ambient – uvb-)/(uvb+ – uvb-). These predicted UV-B effects are presented in Fig. 3 along with the relative observed solar UV-B and supplemental lamp UV-B effects for growth, UV-absorbing compounds and stomatal measurements. The calculations for the observed effects were done in the same manner as those for the predicted effects. To achieve one value for the overall magnitude of the plant growth response of each species, we averaged the absolute values of growth and morphological changes.
The combination of different lamp and plot cover filters (Table 1) resulted in three very different UV-B conditions (the two treatments and the control) as illustrated in Fig. 1 for midday conditions. We have also presented weighted irradiance according to several BSWF (Table 1), as recommended by Cullen and Neale (38), to allow retrospective evaluation of the doses used. When integrated over the entire experimental period, the average daily UV-B irradiance weighted with the generalized plant response (14) was 6.7 kJ m^sup -2^ day^sup -1^ in the supplemental UV-B treatment, 2.6 kJ m^sup -2^ day^sup -1^ in the control (near-ambient UV-B) and 0.05 kJ m^sup -2^ day^sup -1^ in the UV-B-exclusion treatment.
When a significant UV effect for a particular plant characteristic was seen for one radiation condition (e.g. solar UV-B), a response under the other radiation condition (e.g. supplemental UV-B) was, in most cases, in the same direction, even though usually not statistically significant (Fig. 2). Solar UV-B (i.e. the UV increase from uvb- to near-ambient) decreased leaf number, leaf area and leaf mass in lettuce to a greater degree than supplemental lamp UV-B (the increase from near-ambient to uvb+) (Table 2, Fig. 2). Similarly, main shoot leaf area, main shoot mass and aboveground mass in oat were more affected by solar UV-B than by supplemental UV-B (Table 3, Fig. 2). Oat height was decreased by about the same magnitude by both solar and supplemental UV-B. Supplemental UV-B resulted in a greater decrease in the shoot-root ratio of lettuce than did solar UV-B (Fig. 2). In addition, supplemental UV-B promoted leaf number, tiller number and the ratio of tiller mass to main shoot mass in oat. In both species, supplemental UV-B also increased UV-B-absorbing compounds more than did solar UV-B. Stomatal density was increased by both UV-B treatments to a similar degree (Fig. 2).
Total plant mass, root mass, specific leaf mass and cell dimensions were not significantly influenced (P > 0.05) by either UV-B treatment, relative to the control, for cither species (Tables 2 and 3). Furthermore, statistically significant effects were not seen for these plant traits even when comparing potential differences between the two extreme treatments (uvb+ vs uvb-).
The predicted and observed proportions of the total UV effects are indicated in Fig. 3. The generalized plant response spectrum (14) predicts that the supplemental UV-B component will be responsible for about 70% of the total UV effect in the experiments, whereas solar UV-B will only be about 30% of the total response. In contrast, the Quaite et al. (15) DNA-damage function for intact seedlings predicts that both UV-B components will contribute equally to the total effect. The observed results indicate that lettuce growth and morphology responded more to solar UV-B than to supplemental UV-B (using an overall average derived from Fig. 2). In contrast, oat growth and morphology responded more to supplemental UV-B. Taken together, plant growth and morphology in the two species, along with stomatal density, responded in a manner that would be predicted by the Quaite et al. BSWF. In contrast, UV-absorbing compounds increased more because of supplemental UV-B than of solar UV-B for both species. This is more similar to what would be predicted by the generalized plant response BSWF than by the Quaite et al. BSWF.
The responses to our UV-B treatments under field conditions included substantial changes in plant morphology and growth allocation for both species and, yet, no overall decreases in plant production. Although both species exhibited some reductions in aboveground mass (primarily due to solar UV-B), root mass was not influenced in either species, resulting in no significant decrease in total plant mass. Under UV-B supplementation, there was also evidence of mass allocation changes for both species. Lettuce exhibited a decrease in the shoot-root ratio, whereas oat showed an increase in the number of tillers and the proportion of shoot mass allocated to tillers with supplemental UV-B. Similar results of morphological changes without overall decreases in plant production have been reported by numerous authors (39-41).
The increase in UV-absorbing compounds for both lettuce and oat due to the supplemental UV-B was expected. In a meta-analysis of field lamp UV-B supplementation studies, an increase in UV-B-absorbing compounds was the most apparent response across studies (17). Stomatal density has received little attention in comparison with UV-absorbing pigments and plant growth responses. The general increases in stomatal density with increasing UV-B reported in this study are of similar magnitude to those reported for 26 populations of white clover in a UV-B growth chamber experiment (42).
There are reasons to expect greater plant responses with UV-B exclusion than with UV-B supplementation. First, a substantially greater amount of total radiation change is involved in excluding ambient solar UV-B than in supplementing UV-B with fluorescent lamps. Second, if plants do not respond linearly to increments of UV-B radiation, the marginal response to subambient UV-B irradiance may be greater than that to supra-ambient UV-B because UV-B photoreceptors may begin to saturate at greater UV-B fluxes (28).
Although some plant characteristics responded substantially more to solar UV-B than to supplemental UV-B, there were other instances of substantial plant responses to UV-B supplementation. When the magnitudes of the different growth and morphological responses were combined in an overall quantitative index, plants responded fairly equally to solar UV-B and the supplemented UV-B, which was designed to simulate a 30% ozone depletion (Fig. 3). The stomatal density response was also similar. In contrast, UV-absorbing compounds responded differently, with a greater increase under UV-B supplementation than under solar UV-B, especially in lettuce.
Interestingly, Ryan et al. (6,7) found that solar UV-B and not supplemental UV-B tended to have a greater influence on flavonol and flavonoid levels in experiments with wild-type Petunia. Their UV-B supplementation was designed to simulate a more moderate ozone depletion (
Further comparisons of our results with those of other studies examining both supplementation and exclusion of UV-B would be tenuous. For example, several of these studies used a plastic cover only over the UV-B-exclusion plots and lamps only over the supplemental UV-B and near-ambient UV-B control plots (3-5). Thus, the different plots presumably experienced different microclimates (primarily because of the covers or their absence) and different shading conditions (primarily because of patches of shade from the lamp arrays in plots with lamps). In our experiments, we placed a substantial emphasis on a design which would assure the same PAR and microclimate conditions in both treatments and the control. All plots had operating lamp arrays over them, and the difference between the supplemental UV-B treatment and the near-ambient controls was determined by the lamp filters used. This is the standard methodology used in lamp supplementation studies because a comparison of UV-B supplements with unenergized lamps or “dummy fixtures” may produce different results (43). Similarly, all plots had plastic covers over the entire plot, assuring that the two treatments and control had the same microclimate. A variety of subtle differences in visible radiation can influence UV-B effects. For example, shading of the solar radiation by lamp arrays can influence some measures of UV-absorbing compounds (44). In UV-B-exclusion studies, seemingly small differences in filter transmittance of PAR can reduce daily plant carbon gain, as indicated by canopy photosynthesis models (45).
BSWF provide a useful tool in comparing and assessing our results. Although comparisons over a wide range of UV fluxes may be influenced by the degree of linearity of plant responses, BSWF have been so widely used that their use in this study seems warranted. As mentioned earlier, greater responses to solar UV-B would be expected if based only on the difference in total UV-B radiation between the UV-B-exclusion treatment and the near-ambient control (Table 1). In contrast, use of the generalized plant BSWF for prediction suggests a much greater response under the supplemental UV-B than under solar UV-B, as was seen for UV-absorbing compounds (especially in lettuce). This BSWF strongly weights the short UV-B wavelengths supplied by the lamps and does not attribute any influence to wavelengths longer than 313 nm. Use of a BSWF such as the Quaite et al. DNA-damage BSWF, which gives substantial weight in the transition zone from UV-B to UV-A, in addition to considerable weight to the UV-B wave band, suggests that plants should respond equally to solar UV-B and supplemental UV-B at this level (30%) of simulated ozone depletion. Such a nearly equal response was observed for the overall plant growth and morphology index as well as for stomatal density in both species. The tendency for plant growth measurements to respond more to longer wavelengths than seen for the stimulation of UV-absorbing compounds has been suggested by other studies (46,47).
Our use of the Quaite et al. DNA-damage BSWF in such a comparison does not necessarily suggest that DNA damage is the mechanism mediating these plant responses. Other similar BSWF with substantial weighting in the transition zone from UV-B to UV-A, such as the Rumex photosynthetic inhibition BSWF (12) or the recent plant growth response BSWF (11), would give similar results. However, because our experimental conditions focused on changing primarily the UV-B portion of the spectrum, in this analysis we chose to assess functions that were weighted heavily in the UV-B and had relatively little or no weighting in the UV-A region.
These BSWF provide an approximate comparison of the biologically effective UV-B differences involved in the two treatments. However, such comparisons must be taken with considerable caution because the action spectra that generally serve as the basis of BSWF incorporate many uncertainties. Although the BSWF decline with increasing wavelength, even small differences in the nature of this decline lead to large differences in predicted biologically effective radiation. If the spectral irradiance conditions being compared are reasonably similar, such as in the comparison of ambient solar UV-B with supplemented solar UV-B spectral irradiance, the effects predicted by different BSWF are somewhat constrained. However, when comparing irradiance with very different spectral distributions, variance among predicted biologically effective irradiances can be much larger. This is illustrated clearly in Table 1. When comparing the near-ambient solar UV-B control with the supplemental UV-B treatment (uvb+), the amount of biologically effective UV is 1.4-6.4 times greater in the supplemental UV-B plots, depending on which of the four BSWF is used. However, when comparing the UV-B-exclusion treatment (uvb-) with the control, the near-ambient control plots receive 1.5-77 times more UV radiation because of the pronounced differences in spectral distribution. This suggests that considerable care is needed in comparing results between studies using the two different methodologies. An added complication in such use of BSWF to compare the two experimental approaches is the potential nonlinearity of plant response to UV-B as mentioned earlier.
Under the conditions of our experiment, lettuce and oat responded to both solar UV-B and supplemental UV-B simulating a large ozone depletion. However, the degree of response to these two UV-B sources appears to vary greatly according to plant trait and species. Given the issues raised in our study, UV-B-exclusion studies best provide an indication of the effects of current UV-B fluxes, but extrapolation of the results to scenarios of ozone depletion is likely to be difficult under most circumstances.
Acknowledgements-This research was supported by a fellowship to M.C.R. from the Fulbright Commission and Fundación Antorchas (Argentina). Partial support was provided by the U.S. Department of Agriculture (CSRS/NRICGP 98-35100-6107), the National Science Foundation Terrestrial Ecology and Global Change Program (98-14357), the U.S. Department of Agriculture UV-B Monitoring and Research Program (contract G 1502-2) and the Utah Agricultural Experiment Station. We thank Mary Moffat and Lori Bentley for their help in the field and laboratory measurements and Javad Torabinejad for his helpful comments.
¶ Posted on the website on 4 June 2004.
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M. Cecilia Rousseaux[dagger]1 Stephan D. Flint*2 Peter S. Searles[dagger]2 and Martyn M. Caldwell2
1 Instituto de Investigaciones Fisiológicas y Ecológicas Vinculadas a la Agricultura, Consejo Nacional de Investigaciones Científicas y Técnicas and Universidad de Buenos Aires, Buenos Aires, Argentina
2 Department of Forest, Range and Wildlife Sciences and the Ecology Center, Utah State University, Logan, UT
Received 30 March 2004; accepted 3 June 2004
* To whom correspondence should be addressed: Department of Forest, Range and Wildlife Sciences, 5230 Old Main Hill, Utah State University, Logan, UT 84322-5230, USA. Fax: 435-797-3796; e-mail: firstname.lastname@example.org[dagger] Current address: Centro Regional de Investigaciones Cientificas Transferencia Tecnológica, Consejo Nacional de Investigaciones Científicas y Técnicas, Entre Rios y Mendoza s/n, Anillaco 5301, La Rioja, Argentina.
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