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Negligible influence of elevated UV-B radiation on leaf litter quality of Quercus robur
Soil Biology & Biochemistry 33 (2001) 659±665
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Negligible in¯uence of elevated UV-B radiation on leaf litter quality of Quercus robur K.K. Newsham a,*, P. Splatt b, P.A. Coward c, P.D. Greenslade a, A.R. McLeod a, J.M. Anderson b a
Centre for Ecology and Hydrology (CEH), Monks Wood, Abbots Ripton, Huntingdon, Cambridgeshire PE14 2LS, UK b Hatherly Laboratories, Department of Biological Sciences, University of Exeter, Exeter, Devon EX4 4PS, UK c CEH Merlewood, Grange-over-Sands, Cumbria, LA11 6JU, UK Received 7 March 2000; received in revised form 27 July 2000; accepted 27 August 2000
Abstract We tested whether elevated UV-B radiation applied to Quercus robur, a principal climax species of northern Europe, would in¯uence concentrations of polyphenolics (Folin±Denis tannins and lignin), phenylpropanoid moieties of lignin, carbohydrates (monosaccharides and holocellulose), or nutrient elements (K, Ca, Mg, P and N) in recently-abscised leaf litter. Saplings of Q. robur were exposed for 2 years at an outdoor facility in the UK to a 30% elevation above the ambient amount of erythemally-weighted UV-B (280±315 nm) radiation under arrays of ¯uorescent lamps with cellulose diacetate ®lters, which transmitted both UV-B and UV-A (315±400 nm) radiation. Saplings were also exposed to elevated UV-A alone under arrays of lamps with polyester ®lters and to ambient radiation under non-energised arrays of lamps. We found little evidence that elevated UV-B radiation in¯uenced leaf litter quality. Data pooled for both years indicated an 8% increase in vanillic acid concentration in litter from polyester-®ltered lamp arrays, relative to non-energised arrays, and 8% and 6% increases, respectively, in concentrations of acetovanillone in litter from polyester- and cellulose diacetate-®ltered lamp arrays, relative to nonenergised lamp arrays. Arabinose concentration in litter from cellulose diacetate-®ltered lamp arrays was 3% higher than in litter from polyester-®ltered arrays, and glucose concentration in litter from cellulose-diacetate ®ltered lamp arrays was increased by 6%, relative to non-energised arrays. There were no main effects of elevated UV on the concentrations of holocellulose, polyphenolics or nutrient elements. We conclude that exposure to elevated UV-B does not substantially in¯uence the initial chemical composition of Q. robur leaf litter and that any increases in UV-B radiation arising from ozone depletion over northern mid-latitudes will be unlikely to affect nutrient cycling and decomposition in Quercus woodlands through effects on litter quality alone. q 2001 Elsevier Science Ltd. All rights reserved. Keywords: Leaf litter chemistry; Quercus robur; Stratospheric ozone depletion; UV-A and UV-B radiation
1. Introduction Depletion of stratospheric ozone, arising from chloro¯uorocarbon emissions to the atmosphere, is currently estimated at 2±3% per decade over northern mid-latitudes (World Meteorological Organization, 1999). Because ozone absorbs solar ultraviolet-B radiation (UV-B; 280± 315 nm), stratospheric ozone depletion leads to ampli®ed ¯uxes of UV-B radiation received at the Earth's surface (Blumthaler et al., 1994; Seckmeyer et al., 1994; Jokela et al., 1995). Current trends indicate that elevated ¯uxes of UV-B radiation to the Earth's surface arising from stratospheric ozone depletion will be a continuing phenomenon until the middle of the 21st century (World Meteorological Organization, 1999). * Corresponding author. Fax: 144-1223-221-259. E-mail address: [email protected] (K.K. Newsham).
Biologically important macromolecules, such as nucleic acids and proteins, strongly absorb UV-B radiation, and elevated UV-B thus has the potential to affect the growth and survival of organisms exposed to sunlight. In recent years, much research has focused on the potentially deleterious effects of UV-B radiation on the photosynthesis and growth of higher plants (Caldwell et al., 1998). Little is known, however, of how elevated UV-B radiation in¯uences biogeochemistry, and hence one of the key recommendations of the SCOPE report on effects of increased UV radiation on biological systems was to assess the effects of elevated UV-B on biogeochemical cycles (Scienti®c Committee on Problems of the Environment, 1992). Two main mechanisms exist through which elevated UV-B can affect nutrient cycling in terrestrial ecosystems. The ®rst is through direct effects on the organisms responsible for soil nutrient cycling processes. Elevated UV-B radiation is known to in¯uence decomposition processes in this manner
0038-0717/01/$ - see front matter q 2001 Elsevier Science Ltd. All rights reserved. PII: S 0038-071 7(00)00210-8
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(Gehrke et al., 1995; Newsham et al., 1997), but, in vegetated habitats, most UV-B is absorbed or re¯ected by plant foliage (Brown et al., 1994) and ¯uxes of UV-B radiation affecting litter layers are thus likely to have only super®cial or transitory effects. Hence a second, and more plausible, mechanism through which UV-B may in¯uence biogeochemical cycles is through altered quality of leaf litter entering soil after abscission following exposure to UV-B radiation during growth. Exposure of attached foliage to elevated UV-B is thought to in¯uence leaf chemistry in several ways. It is ®rmly established that UV radiation in¯uences the accumulation of phenolic compounds in many plant species (Caldwell et al., 1998; Tevini and Teramura, 1989), principally by the induction of genes encoding phenylalanine ammonia-lyase, which supports the ®rst stage in the metabolism of phenylpropanoid compounds (Beggs and Wellmann, 1994). Increased content of phenolics, including polyphenolics such as tannins and lignin, and individual phenylpropanoid compounds, is known to reduce subsequent litter decomposition rate in soil (Swift et al., 1979). It has been repeatedly asserted that the accumulation of phenolic compounds in attached foliage exposed to UV-B may have signi®cant indirect effects on soil nutrient cycling and decomposition (e.g. Moorhead and Callaghan, 1994; Zepp et al., 1998), but little empirical evidence to directly support this argument has yet been presented. Exposure to UV-B radiation may also affect concentrations of structural carbohydrates in leaves (Gehrke et al., 1995), and any disruption to photosynthetic processes caused by increased UV-B may also in¯uence soluble carbohydrate metabolism, with potential effects on the uptake of nutrient elements from the soil (Yue et al., 1998). Furthermore, potential effects of UV-B on the formation of mycorrhizas, the main nutrient-absorbing organs of most plant species, may also affect nutrient element uptake from soil (Klironomos and Allen, 1995). We therefore examined whether or not elevated UV-B radiation applied during growth in¯uenced the chemistry of recently-abscised leaf litter of pedunculate oak (Quercus robur L.), a principal climax species of northern Europe, using an outdoor irradiation system over 2 years.
2. Materials and methods 2.1. Irradiation system and sampling Two-year-old Q. robur saplings of New Forest (UK) provenance were exposed to a constant 30% elevation above the ambient amount of biologically effective UV-B radiation weighted with the McKinlay and Diffey (1987) erythemal action spectrum (UV-BCIE) for 2 years from May 1995 using an outdoor UV-B irradiation system described by Newsham et al. (1996). Brie¯y, the irradiation system consisted of 12 lamp arrays overhanging gravel beds in a cut arable meadow at UK National Grid Reference TL
200 796 (528 24 0 N, 08 14 0 W). In each lamp array, 12 lamps (Q-Panel UV-B 313, The Q-Panel Company, Cleveland, OH, USA) were mounted on aluminium frames (4 £ 1.4 m) suspended 1.4 m above the canopy of underlying vegetation. A UV-B treatment was achieved by closely wrapping each of the lamps in four arrays with cellulose diacetate ®lm (125 mm, Courtaulds Speciality Plastics, Derby, UK) which allowed the transmission of both UV-B and UV-A (315±400 nm) radiation. Since the latter is little modi®ed by ozone depletion, we accounted for its potential biological effects under cellulose diacetate-®ltered lamps by wrapping the lamps in another four arrays with polyester ®lm (125 mm, Mylar D, Secol Ltd, Thetford, UK), which transmitted only UV-A radiation. A further four arrays consisted of non-energised lamps, which provided the same degree of shading as under cellulose diacetate- and polyester-®ltered lamp arrays. The arrays were arranged in a randomised block design, with a cellulose diacetate-, a polyester-®ltered and a non-energised array of lamps in each of four blocks. The spectral irradiances of lamp and ®lter combinations, measured with a double monochromator spectroradiometer (SR991-PC, Macam Photometrics, Livingston, UK) which was calibrated against a tungsten and deuterium lamp traceable to National Physical Laboratory Standards (SR903, Macam Photometrics, UK), are shown in Newsham et al. (1996). Lamps were spaced on the frames using a cosine distribution (BjoÈrn and Teramura, 1993) and were shaded to provide a central area (2.6 £ 0.7 m) on the gravel bed beneath each array in which elevated UV irradiance varied by ,10% of maximum. The irradiance of UV-BCIE was measured every second beneath a cellulose diacetate-®ltered lamp array with a broad-band sensor (Blue Wave BW100, Vital Technologies Corporation, Ontario, Canada) which was calibrated against the spectroradiometer. The sensor was situated on the southern edge of the central area in which elevated UV irradiance varied by ,10% of maximum and in a position where it did not experience shadows from arrays in direct sunlight. A feedback control operated by PC-based software was used to modulate lamp output to maintain UV-BCIE irradiance under cellulose diacetate-®ltered lamp arrays at 30% above that measured by a sensor situated under a non-energised lamp array. This approximated to an 18% reduction in the ozone column for the latitude at which the irradiation system was located, as calculated from the model of BjoÈrn and Murphy (1985). Control software and timer switches turned the lamps on at dawn and off at dusk. Lamps and ®lters were checked daily and ®lters were changed at c 4week intervals. Mean ¯uxes of UV-B radiation under nonenergised arrays during 1995 and 1996 reached a maximum in July in both years (c 2.6 kJ m 22 d 21 UV-BCIE, respectively) and a minimum in December (c. 0.1 kJ m 22 d 21 UV-BCIE, respectively). Saplings were grown in 3 l capacity rose grower's pots containing a 16:4:1 mixture of John Innes No. 2 compost,
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inert silica grit and woodland soil (to provide a mycorrhizal inoculum). Water was applied from a hand-held sprayer during dry periods. Nutrients were not applied as foliage showed no signs of nutrient de®ciency. Leaves for use in the study were collected from 48 saplings placed in the central area of each gravel bed in which elevated UV irradiance varied by ,10% of maximum. These saplings were surrounded by a single guard row of a further 48 saplings of the same age. To retain leaf litter, each of the inner 48 saplings was enclosed in plastic pea netting (16 mm 2, Pan Products Ltd., Aylesbury, UK) shortly before abscission started. Leaf litter was collected from the nets between 15 November and 22 December 1995 and between 14 November and 24 December 1996. The litter was air-dried in the dark on clean paper for 10 d and stored in the dark at room temperature prior to chemical analyses. 2.2. Phenylpropanoid determinations Phenylpropanoid derivatives of lignin (PPDs) were determined on three replicate samples (50 mg ^ 5 mg) of ground litter using an alkaline CuO-oxidation technique (Hedges and Ertel, 1982). Samples were mixed with CuO, 2 M NaOH and heated in a digestion block with magnetic stirring for 2.5 h at 1708C under a headspace atmosphere of N2. After oxidation, samples were centrifuged (4000 rev. min 21 for 30 min) and the supernatant decanted. Solutions were acidi®ed to pH 2 with 6M HCl and, after centrifugation (4000 rev. min 21 for 30 min), the supernatant was eluted through a C18 extraction column. Silyl derivatives of the PPDs extracted from these columns were determined on a Shimadzu GC14-A capillary GC with a BPX 25 m £ 0.25 mm i.d. column (Sanger et al., 1996). 2.3. Tannins and lignin determinations Determinations were made on litters which had been milled to ,0.7 mm and oven-dried at 1058C for 3 h. Folin±Denis tannins were extracted with boiling water for 60 min and determined by colorimetry using Folin±Denis reagent and aqueous sodium carbonate. Lignin (comprising lignin and other constituents resistant to acid hydrolysis) was determined gravimetrically following extraction with weak acid detergent (cetyltrimethyl ammonium bromide) and cold 72% sulphuric acid, followed by ash correction (Allen, 1989). 2.4. Carbohydrate determinations Holocellulose was determined gravimetrically on airdried samples milled to ,0.7 mm, following an acetic acid and sodium chlorite treatment. Corrections were applied for ash and N content of the residue (Allen, 1989). Component carbohydrates were determined on three replicate samples (25 mg ^ 5 mg) of ground litter from each array extracted by tri¯uoracetic acid (TFA) hydrolysis using the modi®ed procedure of Guggenberger and Zech (1994). The sample was digested at 1008C for 4 h with a
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100 ml ribulose internal standard and 250 ml (4 M) TFA. The solution was then passed through DOWEX 50 exchange resin. A 200 ml aliquot was blown down to dryness and 100 ml of methoxyaminehydrochloride in pyridine (25 mg ml 21) added and left to react for 0.5 h at 708C. After cooling to room temperature the sample was sylated for 1 h using 100 ml of N-trimethylsilylimadazole. Final sample clean-up involved shaking the silyated solution with 300 ml of hexane. Carbohydrates were determined in aliquots of the hexane phase using a Shimadzu GC14-A capillary GC with a PB5 25 m £ 0.25 mm i.d. column (Sanger et al.,1998). 2.5. Nutrient element determinations Samples were milled to ,0.7 mm and oven-dried at 1058C for 3 h prior to digestion in a sulphuric acid/hydrogen peroxide mixture using selenium powder as a catalyst and lithium sulphate as a boiling point raiser. Potassium, Ca and Mg were determined spectrometrically and total N and P were determined by continuous ¯ow colorimetry using the indophenol blue and molybdenum blue methods, respectively (Allen, 1989). 2.6. Statistical analyses All data were expressed as proportions on a percentage dry weight basis and hence were arc sine square root transformed prior to analysis. To test for main and interactive effects of elevated UV radiation and year on the chemical composition of leaves, mean values of each variable per array were subjected to split-plot ANOVA with year nested within elevated UV. Data from individual years were also subjected to two-way ANOVA, with elevated UV and block as factors. Analyses were made in the MINITAB statistical package (release 12.1). The statistical techniques used are described by Sokal and Rohlf (1995). 3. Results 3.1. Phenylpropanoid derivatives Elevated UV radiation applied to Q. robur saplings in¯uenced the concentrations of two phenylpropanoid derivatives of lignin in recently-abscised leaf litter in 1995 and 1996 (Table 1). Data pooled over both years indicated that irradiation of saplings under cellulose diacetate- and polyester-®ltered lamp arrays increased concentrations of acetovanillone in leaf litter by 6 and 8%, respectively, relative to non-energised arrays (Fig. 1a). Vanillic acid concentration in litter collected from polyester-®ltered lamp arrays was also increased by 8%, relative to non-energised arrays, over both years (Fig. 1b). In 1995, vanillin concentrations in leaf litter collected from cellulose diacetate- and polyester-®ltered lamp arrays were increased by 14% and 9%, respectively, relative to non-energised arrays (Fig. 1c).
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Table 1 Main and interactive effects of elevated UV and year on the chemical composition of recently-abscised Quercus robur leaf litter. P values signi®cant at the 5% level and below are marked in bold. Data were analysed by split-plot ANOVA with year nested within elevated UV. All data were arc sine square root transformed prior to analysis. Figures in parentheses after signi®cant P values for main effects of year are percentage changes in chemical concentrations between 1995 and 1996 Parameter
Source of variation Elevated UV
Year
Elevated UV £ year
F2,6
P-value
F1,9
P-value
F2,9
P-value
phenylpropanoids p-hydroxybenzaldehyde p-hydroxyacetophenone vanillin p-hydroxybenzoic acid acetovanillone syringaldehyde vanillic acid acetosyringone syringic acid coumaric acid ferulic acid
1.75 1.21 3.98 0.66 9.89 2.45 6.48 2.98 2.94 3.29 1.25
0.252 0.362 0.079 0.549 0.013 0.167 0.032 0.126 0.129 0.109 0.352
55.87 0.16 1916.68 10.21 108.70 268.85 7.65 27.20 33.16 59.84 107.51
, 0.001 (66) 0.695 , 0.001 (107) 0.011 (25) , 0.001 (56) , 0.001 (66) 0.022 (21) 0.001 (32) , 0.001 (32) , 0.001 (58) , 0.001 (58)
0.05 0.18 0.83 1.07 0.55 2.05 0.08 0.53 0.23 1.57 1.87
0.954 0.841 0.467 0.382 0.597 0.185 0.924 0.607 0.802 0.261 0.209
polyphenolics Folin±Denis tannins lignin
0.43 2.66
0.670 0.149
34.72 53.10
, 0.001 (25) , 0.001 (33)
0.04 1.09
0.959 0.376
carbohydrates holocellulose xylose arabinose rhamnose fucose mannose galactose glucose
0.42 0.86 7.66 1.28 0.91 0.12 1.84 5.97
0.674 0.471 0.022 0.345 0.453 0.892 0.239 0.037
1.25 25.83 8.37 0.12 15.19 0.41 1.58 4.28
0.293 0.001 (27) 0.018 (5) 0.739 0.004 (9) 0.537 0.240 0.068
0.67 0.27 0.24 0.93 0.42 0.52 0.53 0.13
0.535 0.771 0.792 0.431 0.669 0.611 0.608 0.884
nutrient elements K Ca Mg P N
2.55 0.11 0.65 2.79 1.52
0.158 0.896 0.557 0.139 0.292
5.23 54.78 14.86 98.48 18.15
0.048 (20) , 0.001 (220) 0.004 (210) , 0.001 (55) 0.002 (212)
0.71 1.90 1.13 1.34 0.39
0.518 0.204 0.364 0.310 0.689
However, the main effect of elevated UV on this variable over both years was not signi®cant (Table 1). In 1995, there was also a 19% increase in syringaldehyde concentration in leaf litter collected from polyester-®ltered lamp arrays, relative to non-energised arrays (Fig. 1d), but there was no main effect of elevated UV on syringaldehyde concentration over both years (Table 1). The concentrations of all phenylpropanoid derivatives, with the exception of p-hydroxyacetophenone, increased between 1995 and 1996. There were no interactive effects of elevated UV and year on phenylpropanoid concentrations (Table 1). 3.2. Tannins and lignin Elevated UV radiation did not in¯uence the concentrations of Folin±Denis tannins or lignin in recently-abscised Q. robur leaf litter (Table 1). In addition, tannin-to-N, lignin-to-N and lignin-to-P ratios were unaffected by
elevated UV (data not presented). Signi®cant increases in the concentrations of Folin±Denis tannins and lignin were recorded between 1995 and 1996 but there were no interactive effects of elevated UV and year on these constituents (Table 1). 3.3. Carbohydrates The concentration of holocellulose was unaffected by elevated UV radiation in Q. robur leaf litter, but that of two soluble carbohydrates responded to elevated UV (Table 1). Arabinose concentration in litter from saplings exposed during growth to elevated UV-A and UV-B radiation under cellulose-diacetate ®ltered arrays was 3% higher than in litter from saplings exposed to elevated UV-A under polyester-®ltered arrays (Fig. 2a). In addition, glucose concentration in leaf litter from saplings exposed to elevated UV-A and UV-B radiation under cellulose-diacetate ®ltered arrays was increased by 6%, relative to non-energised arrays
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Fig. 2. Effects of exposure to ambient solar radiation (A) under non-energised lamp arrays, ambient solar radiation plus supplemental UV-A radiation (p) under polyester-®ltered lamp arrays and ambient solar radiation plus supplemental UV-A and UV-B radiation (B) under cellulose diacetate®ltered lamp arrays on (a) arabinose and (b) glucose concentrations in recently-abscised Quercus robur leaf litter. Notations as in Fig. 1. Values are means of four replicates, pooled over two samplings in 1995 and 1996. Note that y-axes do not extend to zero.
Fig. 1. Effects of exposure to ambient solar radiation (A) under non-energised lamp arrays, ambient solar radiation plus supplemental UV-A radiation (p) under polyester-®ltered lamp arrays and ambient solar radiation plus supplemental UV-A and UV-B radiation (B) under cellulose diacetate®ltered lamp arrays on (a) acetovanillone, (b) vanillic acid, (c) vanillin and (d) syringaldehyde concentrations in recently-abscised Quercus robur leaf litter. Values are means of four replicates. Data in (a) and (b) are pooled over two samplings in 1995 and 1996. Data in (c) and (d) are for 1995 only. Bars represent LSD. Differently superscripted values differed at P , 0.05.
(Fig. 2b). Signi®cant effects of year were recorded for three carbohydrates: concentrations of xylose, arabinose and fucose increased between 1995 and 1996, but no interactive effects of elevated UV and year on carbohydrate concentrations were recorded (Table 1). 3.4. Nutrient elements No main effects of elevated UV on the concentrations of K, Ca, Mg, P or N were recorded in our study (Table 1). The concentrations of two nutrient elements (K and P) increased between years and those of three elements (Ca, Mg and N) decreased between 1995 and 1996 (Table 1). There were no signi®cant elevated UV £ year interactions on nutrient element concentrations (Table 1). 4. Discussion The data presented here indicate a negligible in¯uence of elevated UV-B radiation on the chemical composition of
recently-abscised Q. robur leaf litter, with signi®cant main effects of irradiation over 2 years on the concentrations of only four of 26 constituents analysed. No signi®cant effects of elevated UV radiation on the mass of leaf litter produced per array were recorded in 1995 and 1996 (Newsham et al., 1999), and accordingly, there were no main effects of UV radiation on the total mass of each chemical constituent per array (data not presented). Previous supplemental UV-B studies using ¯uorescent lamps in ®eld experiments have recorded signi®cant responses of plant litter chemistry to irradiation in a limited number of plant species. For example, a simulated 15% ozone depletion over Abisko (Sweden) led to a 19% decrease in the a-cellulose concentration of Vaccinium uliginosum leaf litter (Gehrke et al., 1995). By contrast, the same simulated amount of ozone depletion over Heemskerk (The Netherlands) increased the a-cellulose concentration of Calamagrostis epigeios leaf litter by 13% (Rozema et al., 1997). In experiments determining the effects of elevated UV-B radiation on the tissue chemistry of Triticum aestivum, Yue et al. (1998) recorded a 37% increase in holocellulose concentration in leaves following exposure to a simulated 25% reduction in the ozone column over Lanzhou (China). The data from our study indicated that elevated UV-B, simulating an 18% reduction in the ozone column over Monks Wood (UK), and applied continuously for 2 years, had no effect on the holocellulose concentration of recently-abscised Q. robur leaf litter. Exposure of plants to UV-B radiation during growth also has the potential to alter soluble carbohydrate concentrations in plant tissues. Yue et al. (1998) found that elevated UV applied during growth to T. aestivum led to a 21% decrease in total soluble carbohydrates in abscised leaves. However no effects on soluble carbohydrates were found in
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V. uliginosum and C. epigeios by Gehrke et al. (1995) or Rozema et al. (1997). The only indication of effects of elevated UV radiation on soluble carbohydrates in our study were 3±6% increases in concentrations of arabinoglucans (arabinose and glucose), which are constituents of hemicellulose, in litter from saplings exposed to elevated UV-A and UV-B radiation under cellulose diacetate-®ltered lamp arrays, relative to polyester-®ltered or non-energised lamp arrays. The study by Rozema et al. (1997) indicated that exposure to elevated UV-B radiation during growth increased the lignin concentration of C. epigeios leaf litter at abscission by 56%. The data from our study do not con®rm that similar effects occur in Q. robur. Similarly, Gehrke et al. (1995) found no effects of elevated UV-B radiation on the lignin concentration of V. uliginosum leaf litter. Furthermore, simulated 16% and 25% reductions in the ozone column over Beltsville, MD (USA) have no effect on the lignin concentration of needle litter from 3-year-old Pinus taeda seedlings (W. Cybulski, pers. comm.). Other experimental studies have also shown no effects of exposure to UV-B radiation on the concentration of hydrolysable tannins in litters of V. uliginosum (Gehrke et al., 1995), C. epigeios (Rozema et al., 1997), Calluna vulgaris, Cistus creticus, Rubus chamaemorus and Vaccinium myrtillus (Paul et al., 1999), corroborating the data from our study reported here. Previous studies have indicated that exposure of plant foliage to elevated UV-B radiation during growth can in¯uence the nutrient element concentration of leaf litter. Yue et al. (1998) recorded 85, 7.5 and 20% increases in N, K and Mg concentrations, respectively, and a 20% decrease in total P, in leaves of T. aestivum plants exposed to elevated UV-B radiation. By contrast, concentrations of N, P, K, Ca and Mg were unaffected in Acacia tortilis seedlings grown in nutrient-rich and nutrient-poor soils and exposed to a simulated 20% reduction in ozone column in a glasshouse (Ernst et al., 1997). Our data similarly suggest that exposure to elevated UV-B during growth has no effect on these nutrient elements in leaf litter. They also in part corroborate data indicating that UV-B has no effect on the density of mycorrhizal root tips of Q. robur saplings (Newsham et al., 1999). The most consistent effects of elevated UV in our study were on concentrations of phenylpropanoid moieties of lignin: two constituents were signi®cantly in¯uenced by irradiation over this 2-year study, whereas another two showed signi®cant responses in the ®rst year only. Significant increases, associated with growth of Q. robur under polyester-®ltered lamp arrays relative to non-energised lamp arrays, were recorded in vanillic constituents and syringaldehyde. These observations suggest that UV-A radiation produced by polyester-®ltered lamps may have induced the production of these compounds. Although exposure to UV-B radiation is known to in¯uence the expression of genes encoding phenylpropanoid metabo-
lism (Beggs and Wellmann, 1994), we are unaware of any reports indicating that UV-A radiation might also induce similar effects. These data corroborate those from previous studies that have frequently shown effects of elevated UV-A radiation, or some other factor associated with energised, polyester-®ltered lamps, on plants and insects (Newsham et al., 1996; 1999). Given that elevated UV-B radiation has been shown to in¯uence the chemistry of Calamagrostis, Triticum and Vaccinium leaf litters, what are the likely explanations for the relatively few changes in the chemical composition of Q. robur leaf litter observed in our study? We can discount the possibility that the analytical methods used were not sensitive enough to detect any changes in tissue chemistry, since small alterations in several chemical constituents were recorded, and because consistent changes in leaf litter chemistry were also recorded between years. One possible explanation for the paucity of treatment effects on litter chemistry observed in our study is that the increased thickness of Q. robur leaves, probably associated with increased cuticle thickness (Newsham et al., 1999), may have protected underlying leaf tissues from any in¯uence of elevated UV radiation. Differences in irradiation methods may also be responsible for differences in vegetation response, as suggested by Fiscus and Booker (1995). In particular, the studies of Gehrke et al. (1995), Rozema et al. (1997) and Yue et al. (1998) each used non-modulated lamp systems in which plants may be exposed to unrealistically high amounts of UV-B radiation relative to photosynthetically active radiation during cloudy periods, and could therefore elicit exaggerated responses to irradiation (McLeod, 1997). In conclusion, a number of studies have shown changes in leaf and litter chemistry in response to increased UV-B radiation. The data we obtained in this study on Q. robur show comparatively subtle main effects of elevated UV-B on the nutrient element, carbohydrate, polyphenolic and phenylpropanoid constituents of litter, suggesting that this species, or genotype, of oak was relatively insensitive to the effects of enhanced UV radiation. Other provenances of Q. robur would have to be similarly tested before general conclusions could be drawn, but these results suggest that increased UV-B radiation is unlikely to have signi®cant indirect effects on organic matter pools and nutrient turnover as a consequence of changes in the quality of leaf litter input to soils. Acknowledgements The authors acknowledge the ®nancial support provided by the NERC through its TIGER (Terrestrial Initiative in Global Environmental Research) programme. Jan Poskitt helped with laboratory work at CEH Merlewood, Dom Hodgson supplied useful comments on the manuscript and Walt Cybulski and Nigel Paul kindly allowed access to unpublished data.
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Moorhead, D.L., Callaghan, T.V., 1994. Effects of increasing ultraviolet-B radiation on decomposition and soil organic matter dynamics: a synthesis and modelling study. Biology and Fertility of Soils 18, 19±26. Newsham, K.K., McLeod, A.R., Greenslade, P.D., Emmett, B.A., 1996. Appropriate controls in outdoor UV-B supplementation experiments. Global Change Biology 2, 319±324. Newsham, K.K., McLeod, A.R., Roberts, J.D., Greenslade, P.D., Emmett, B.A., 1997. Direct effects of elevated UV-B radiation on the decomposition of Quercus robur leaf litter. Oikos 79, 592±602. Newsham, K.K., Greenslade, P.D., McLeod, A.R., 1999. Effects of elevated ultraviolet radiation on Quercus robur and its insect and ectomycorrhizal associates. Global Change Biology 5, 881±890. Paul, N.D., Callaghan, T.V., Moody, S.A., Gwynn-Jones, D., Johanson, U., Gehrke, C., 1999. UV-B impacts on decomposition and biogeochemical cycling. In: Rozema, J. (Ed.). Stratospheric Ozone Depletion: the Effects of Enhanced UV-B Radiation on Terrestrial Ecosystems. Backhuys Publishers, Leiden, pp. 117±133. Rozema, J., Tosserams, M., Nelissen, H.J.M., van Heerwaarden, L., Broekman, R.A., Flierman, N., 1997. Stratospheric ozone reduction and ecosystem processes: enhanced UV-B radiation affects chemical quality and decomposition of leaves of the dune grassland species Calamagrostis epigeios. Plant Ecology 128, 284±294. Sanger, L.J., Cox, P., Splatt, P., Whelan, M., Anderson, J.M., 1996. Variability in the quality of Pinus sylvestris needles and litter from sites with different soil characteristics: lignin and phenylpropanoid signatures. Soil Biology and Biochemistry 28, 835±929. Sanger, L.J., Cox, P., Splatt, P., Whelan, M., Anderson, J.M., 1998. Variability in the quality of Pinus sylvestris needles and litter from sites with different soil characteristics: acid detergent ®bre (ADF) and carbohydrate signatures. Soil Biology and Biochemistry 30, 455±461. Scienti®c Committee on Problems of the Environment, 1992. Effects of Increased Ultraviolet Radiation on Biological Systems. SCOPE/ UNEP, Budapest. Seckmeyer, G., Mayer, B., Erb, R., Bernhard, G., 1994. UVB. in Germany higher in 1993 than in 1992. Geophysical Research Letters 21, 577± 580. Sokal, R.R., Rohlf, F.J., 1995. Biometry. 3rd edn. Freeman, New York. Swift, M.J., Heal, O.W., Anderson, J.M., 1979. Decomposition in Terrestrial Ecosystems. Blackwell Scienti®c Publications, Oxford. Tevini, M., Teramura, A.H., 1989. UV-B effects on terrestrial plants. Photochemistry and Photobiology 50, 479±487. World Meteorological Organization, 1999. Scienti®c Assessment of Ozone Depletion: 1998. Report No. 44. WMO: Geneva. Yue, M., Li, Y., Wang, X., 1998. Effects of enhanced ultraviolet-B radiation on plant nutrients and decomposition of spring wheat under ®eld conditions. Environmental and Experimental Botany 40, 187±196. Zepp, R.G., Callaghan, T.V., Erickson, D.J., 1998. Effects of increased solar ultraviolet radiation on biogeochemical cycles. Journal of Photochemistry and Photobiology B: Biology 46, 69±82.
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Negligible in¯uence of elevated UV-B radiation on leaf litter quality of Quercus robur K.K. Newsham a,*, P. Splatt b, P.A. Coward c, P.D. Greenslade a, A.R. McLeod a, J.M. Anderson b a
Centre for Ecology and Hydrology (CEH), Monks Wood, Abbots Ripton, Huntingdon, Cambridgeshire PE14 2LS, UK b Hatherly Laboratories, Department of Biological Sciences, University of Exeter, Exeter, Devon EX4 4PS, UK c CEH Merlewood, Grange-over-Sands, Cumbria, LA11 6JU, UK Received 7 March 2000; received in revised form 27 July 2000; accepted 27 August 2000
Abstract We tested whether elevated UV-B radiation applied to Quercus robur, a principal climax species of northern Europe, would in¯uence concentrations of polyphenolics (Folin±Denis tannins and lignin), phenylpropanoid moieties of lignin, carbohydrates (monosaccharides and holocellulose), or nutrient elements (K, Ca, Mg, P and N) in recently-abscised leaf litter. Saplings of Q. robur were exposed for 2 years at an outdoor facility in the UK to a 30% elevation above the ambient amount of erythemally-weighted UV-B (280±315 nm) radiation under arrays of ¯uorescent lamps with cellulose diacetate ®lters, which transmitted both UV-B and UV-A (315±400 nm) radiation. Saplings were also exposed to elevated UV-A alone under arrays of lamps with polyester ®lters and to ambient radiation under non-energised arrays of lamps. We found little evidence that elevated UV-B radiation in¯uenced leaf litter quality. Data pooled for both years indicated an 8% increase in vanillic acid concentration in litter from polyester-®ltered lamp arrays, relative to non-energised arrays, and 8% and 6% increases, respectively, in concentrations of acetovanillone in litter from polyester- and cellulose diacetate-®ltered lamp arrays, relative to nonenergised lamp arrays. Arabinose concentration in litter from cellulose diacetate-®ltered lamp arrays was 3% higher than in litter from polyester-®ltered arrays, and glucose concentration in litter from cellulose-diacetate ®ltered lamp arrays was increased by 6%, relative to non-energised arrays. There were no main effects of elevated UV on the concentrations of holocellulose, polyphenolics or nutrient elements. We conclude that exposure to elevated UV-B does not substantially in¯uence the initial chemical composition of Q. robur leaf litter and that any increases in UV-B radiation arising from ozone depletion over northern mid-latitudes will be unlikely to affect nutrient cycling and decomposition in Quercus woodlands through effects on litter quality alone. q 2001 Elsevier Science Ltd. All rights reserved. Keywords: Leaf litter chemistry; Quercus robur; Stratospheric ozone depletion; UV-A and UV-B radiation
1. Introduction Depletion of stratospheric ozone, arising from chloro¯uorocarbon emissions to the atmosphere, is currently estimated at 2±3% per decade over northern mid-latitudes (World Meteorological Organization, 1999). Because ozone absorbs solar ultraviolet-B radiation (UV-B; 280± 315 nm), stratospheric ozone depletion leads to ampli®ed ¯uxes of UV-B radiation received at the Earth's surface (Blumthaler et al., 1994; Seckmeyer et al., 1994; Jokela et al., 1995). Current trends indicate that elevated ¯uxes of UV-B radiation to the Earth's surface arising from stratospheric ozone depletion will be a continuing phenomenon until the middle of the 21st century (World Meteorological Organization, 1999). * Corresponding author. Fax: 144-1223-221-259. E-mail address: [email protected] (K.K. Newsham).
Biologically important macromolecules, such as nucleic acids and proteins, strongly absorb UV-B radiation, and elevated UV-B thus has the potential to affect the growth and survival of organisms exposed to sunlight. In recent years, much research has focused on the potentially deleterious effects of UV-B radiation on the photosynthesis and growth of higher plants (Caldwell et al., 1998). Little is known, however, of how elevated UV-B radiation in¯uences biogeochemistry, and hence one of the key recommendations of the SCOPE report on effects of increased UV radiation on biological systems was to assess the effects of elevated UV-B on biogeochemical cycles (Scienti®c Committee on Problems of the Environment, 1992). Two main mechanisms exist through which elevated UV-B can affect nutrient cycling in terrestrial ecosystems. The ®rst is through direct effects on the organisms responsible for soil nutrient cycling processes. Elevated UV-B radiation is known to in¯uence decomposition processes in this manner
0038-0717/01/$ - see front matter q 2001 Elsevier Science Ltd. All rights reserved. PII: S 0038-071 7(00)00210-8
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(Gehrke et al., 1995; Newsham et al., 1997), but, in vegetated habitats, most UV-B is absorbed or re¯ected by plant foliage (Brown et al., 1994) and ¯uxes of UV-B radiation affecting litter layers are thus likely to have only super®cial or transitory effects. Hence a second, and more plausible, mechanism through which UV-B may in¯uence biogeochemical cycles is through altered quality of leaf litter entering soil after abscission following exposure to UV-B radiation during growth. Exposure of attached foliage to elevated UV-B is thought to in¯uence leaf chemistry in several ways. It is ®rmly established that UV radiation in¯uences the accumulation of phenolic compounds in many plant species (Caldwell et al., 1998; Tevini and Teramura, 1989), principally by the induction of genes encoding phenylalanine ammonia-lyase, which supports the ®rst stage in the metabolism of phenylpropanoid compounds (Beggs and Wellmann, 1994). Increased content of phenolics, including polyphenolics such as tannins and lignin, and individual phenylpropanoid compounds, is known to reduce subsequent litter decomposition rate in soil (Swift et al., 1979). It has been repeatedly asserted that the accumulation of phenolic compounds in attached foliage exposed to UV-B may have signi®cant indirect effects on soil nutrient cycling and decomposition (e.g. Moorhead and Callaghan, 1994; Zepp et al., 1998), but little empirical evidence to directly support this argument has yet been presented. Exposure to UV-B radiation may also affect concentrations of structural carbohydrates in leaves (Gehrke et al., 1995), and any disruption to photosynthetic processes caused by increased UV-B may also in¯uence soluble carbohydrate metabolism, with potential effects on the uptake of nutrient elements from the soil (Yue et al., 1998). Furthermore, potential effects of UV-B on the formation of mycorrhizas, the main nutrient-absorbing organs of most plant species, may also affect nutrient element uptake from soil (Klironomos and Allen, 1995). We therefore examined whether or not elevated UV-B radiation applied during growth in¯uenced the chemistry of recently-abscised leaf litter of pedunculate oak (Quercus robur L.), a principal climax species of northern Europe, using an outdoor irradiation system over 2 years.
2. Materials and methods 2.1. Irradiation system and sampling Two-year-old Q. robur saplings of New Forest (UK) provenance were exposed to a constant 30% elevation above the ambient amount of biologically effective UV-B radiation weighted with the McKinlay and Diffey (1987) erythemal action spectrum (UV-BCIE) for 2 years from May 1995 using an outdoor UV-B irradiation system described by Newsham et al. (1996). Brie¯y, the irradiation system consisted of 12 lamp arrays overhanging gravel beds in a cut arable meadow at UK National Grid Reference TL
200 796 (528 24 0 N, 08 14 0 W). In each lamp array, 12 lamps (Q-Panel UV-B 313, The Q-Panel Company, Cleveland, OH, USA) were mounted on aluminium frames (4 £ 1.4 m) suspended 1.4 m above the canopy of underlying vegetation. A UV-B treatment was achieved by closely wrapping each of the lamps in four arrays with cellulose diacetate ®lm (125 mm, Courtaulds Speciality Plastics, Derby, UK) which allowed the transmission of both UV-B and UV-A (315±400 nm) radiation. Since the latter is little modi®ed by ozone depletion, we accounted for its potential biological effects under cellulose diacetate-®ltered lamps by wrapping the lamps in another four arrays with polyester ®lm (125 mm, Mylar D, Secol Ltd, Thetford, UK), which transmitted only UV-A radiation. A further four arrays consisted of non-energised lamps, which provided the same degree of shading as under cellulose diacetate- and polyester-®ltered lamp arrays. The arrays were arranged in a randomised block design, with a cellulose diacetate-, a polyester-®ltered and a non-energised array of lamps in each of four blocks. The spectral irradiances of lamp and ®lter combinations, measured with a double monochromator spectroradiometer (SR991-PC, Macam Photometrics, Livingston, UK) which was calibrated against a tungsten and deuterium lamp traceable to National Physical Laboratory Standards (SR903, Macam Photometrics, UK), are shown in Newsham et al. (1996). Lamps were spaced on the frames using a cosine distribution (BjoÈrn and Teramura, 1993) and were shaded to provide a central area (2.6 £ 0.7 m) on the gravel bed beneath each array in which elevated UV irradiance varied by ,10% of maximum. The irradiance of UV-BCIE was measured every second beneath a cellulose diacetate-®ltered lamp array with a broad-band sensor (Blue Wave BW100, Vital Technologies Corporation, Ontario, Canada) which was calibrated against the spectroradiometer. The sensor was situated on the southern edge of the central area in which elevated UV irradiance varied by ,10% of maximum and in a position where it did not experience shadows from arrays in direct sunlight. A feedback control operated by PC-based software was used to modulate lamp output to maintain UV-BCIE irradiance under cellulose diacetate-®ltered lamp arrays at 30% above that measured by a sensor situated under a non-energised lamp array. This approximated to an 18% reduction in the ozone column for the latitude at which the irradiation system was located, as calculated from the model of BjoÈrn and Murphy (1985). Control software and timer switches turned the lamps on at dawn and off at dusk. Lamps and ®lters were checked daily and ®lters were changed at c 4week intervals. Mean ¯uxes of UV-B radiation under nonenergised arrays during 1995 and 1996 reached a maximum in July in both years (c 2.6 kJ m 22 d 21 UV-BCIE, respectively) and a minimum in December (c. 0.1 kJ m 22 d 21 UV-BCIE, respectively). Saplings were grown in 3 l capacity rose grower's pots containing a 16:4:1 mixture of John Innes No. 2 compost,
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inert silica grit and woodland soil (to provide a mycorrhizal inoculum). Water was applied from a hand-held sprayer during dry periods. Nutrients were not applied as foliage showed no signs of nutrient de®ciency. Leaves for use in the study were collected from 48 saplings placed in the central area of each gravel bed in which elevated UV irradiance varied by ,10% of maximum. These saplings were surrounded by a single guard row of a further 48 saplings of the same age. To retain leaf litter, each of the inner 48 saplings was enclosed in plastic pea netting (16 mm 2, Pan Products Ltd., Aylesbury, UK) shortly before abscission started. Leaf litter was collected from the nets between 15 November and 22 December 1995 and between 14 November and 24 December 1996. The litter was air-dried in the dark on clean paper for 10 d and stored in the dark at room temperature prior to chemical analyses. 2.2. Phenylpropanoid determinations Phenylpropanoid derivatives of lignin (PPDs) were determined on three replicate samples (50 mg ^ 5 mg) of ground litter using an alkaline CuO-oxidation technique (Hedges and Ertel, 1982). Samples were mixed with CuO, 2 M NaOH and heated in a digestion block with magnetic stirring for 2.5 h at 1708C under a headspace atmosphere of N2. After oxidation, samples were centrifuged (4000 rev. min 21 for 30 min) and the supernatant decanted. Solutions were acidi®ed to pH 2 with 6M HCl and, after centrifugation (4000 rev. min 21 for 30 min), the supernatant was eluted through a C18 extraction column. Silyl derivatives of the PPDs extracted from these columns were determined on a Shimadzu GC14-A capillary GC with a BPX 25 m £ 0.25 mm i.d. column (Sanger et al., 1996). 2.3. Tannins and lignin determinations Determinations were made on litters which had been milled to ,0.7 mm and oven-dried at 1058C for 3 h. Folin±Denis tannins were extracted with boiling water for 60 min and determined by colorimetry using Folin±Denis reagent and aqueous sodium carbonate. Lignin (comprising lignin and other constituents resistant to acid hydrolysis) was determined gravimetrically following extraction with weak acid detergent (cetyltrimethyl ammonium bromide) and cold 72% sulphuric acid, followed by ash correction (Allen, 1989). 2.4. Carbohydrate determinations Holocellulose was determined gravimetrically on airdried samples milled to ,0.7 mm, following an acetic acid and sodium chlorite treatment. Corrections were applied for ash and N content of the residue (Allen, 1989). Component carbohydrates were determined on three replicate samples (25 mg ^ 5 mg) of ground litter from each array extracted by tri¯uoracetic acid (TFA) hydrolysis using the modi®ed procedure of Guggenberger and Zech (1994). The sample was digested at 1008C for 4 h with a
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100 ml ribulose internal standard and 250 ml (4 M) TFA. The solution was then passed through DOWEX 50 exchange resin. A 200 ml aliquot was blown down to dryness and 100 ml of methoxyaminehydrochloride in pyridine (25 mg ml 21) added and left to react for 0.5 h at 708C. After cooling to room temperature the sample was sylated for 1 h using 100 ml of N-trimethylsilylimadazole. Final sample clean-up involved shaking the silyated solution with 300 ml of hexane. Carbohydrates were determined in aliquots of the hexane phase using a Shimadzu GC14-A capillary GC with a PB5 25 m £ 0.25 mm i.d. column (Sanger et al.,1998). 2.5. Nutrient element determinations Samples were milled to ,0.7 mm and oven-dried at 1058C for 3 h prior to digestion in a sulphuric acid/hydrogen peroxide mixture using selenium powder as a catalyst and lithium sulphate as a boiling point raiser. Potassium, Ca and Mg were determined spectrometrically and total N and P were determined by continuous ¯ow colorimetry using the indophenol blue and molybdenum blue methods, respectively (Allen, 1989). 2.6. Statistical analyses All data were expressed as proportions on a percentage dry weight basis and hence were arc sine square root transformed prior to analysis. To test for main and interactive effects of elevated UV radiation and year on the chemical composition of leaves, mean values of each variable per array were subjected to split-plot ANOVA with year nested within elevated UV. Data from individual years were also subjected to two-way ANOVA, with elevated UV and block as factors. Analyses were made in the MINITAB statistical package (release 12.1). The statistical techniques used are described by Sokal and Rohlf (1995). 3. Results 3.1. Phenylpropanoid derivatives Elevated UV radiation applied to Q. robur saplings in¯uenced the concentrations of two phenylpropanoid derivatives of lignin in recently-abscised leaf litter in 1995 and 1996 (Table 1). Data pooled over both years indicated that irradiation of saplings under cellulose diacetate- and polyester-®ltered lamp arrays increased concentrations of acetovanillone in leaf litter by 6 and 8%, respectively, relative to non-energised arrays (Fig. 1a). Vanillic acid concentration in litter collected from polyester-®ltered lamp arrays was also increased by 8%, relative to non-energised arrays, over both years (Fig. 1b). In 1995, vanillin concentrations in leaf litter collected from cellulose diacetate- and polyester-®ltered lamp arrays were increased by 14% and 9%, respectively, relative to non-energised arrays (Fig. 1c).
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Table 1 Main and interactive effects of elevated UV and year on the chemical composition of recently-abscised Quercus robur leaf litter. P values signi®cant at the 5% level and below are marked in bold. Data were analysed by split-plot ANOVA with year nested within elevated UV. All data were arc sine square root transformed prior to analysis. Figures in parentheses after signi®cant P values for main effects of year are percentage changes in chemical concentrations between 1995 and 1996 Parameter
Source of variation Elevated UV
Year
Elevated UV £ year
F2,6
P-value
F1,9
P-value
F2,9
P-value
phenylpropanoids p-hydroxybenzaldehyde p-hydroxyacetophenone vanillin p-hydroxybenzoic acid acetovanillone syringaldehyde vanillic acid acetosyringone syringic acid coumaric acid ferulic acid
1.75 1.21 3.98 0.66 9.89 2.45 6.48 2.98 2.94 3.29 1.25
0.252 0.362 0.079 0.549 0.013 0.167 0.032 0.126 0.129 0.109 0.352
55.87 0.16 1916.68 10.21 108.70 268.85 7.65 27.20 33.16 59.84 107.51
, 0.001 (66) 0.695 , 0.001 (107) 0.011 (25) , 0.001 (56) , 0.001 (66) 0.022 (21) 0.001 (32) , 0.001 (32) , 0.001 (58) , 0.001 (58)
0.05 0.18 0.83 1.07 0.55 2.05 0.08 0.53 0.23 1.57 1.87
0.954 0.841 0.467 0.382 0.597 0.185 0.924 0.607 0.802 0.261 0.209
polyphenolics Folin±Denis tannins lignin
0.43 2.66
0.670 0.149
34.72 53.10
, 0.001 (25) , 0.001 (33)
0.04 1.09
0.959 0.376
carbohydrates holocellulose xylose arabinose rhamnose fucose mannose galactose glucose
0.42 0.86 7.66 1.28 0.91 0.12 1.84 5.97
0.674 0.471 0.022 0.345 0.453 0.892 0.239 0.037
1.25 25.83 8.37 0.12 15.19 0.41 1.58 4.28
0.293 0.001 (27) 0.018 (5) 0.739 0.004 (9) 0.537 0.240 0.068
0.67 0.27 0.24 0.93 0.42 0.52 0.53 0.13
0.535 0.771 0.792 0.431 0.669 0.611 0.608 0.884
nutrient elements K Ca Mg P N
2.55 0.11 0.65 2.79 1.52
0.158 0.896 0.557 0.139 0.292
5.23 54.78 14.86 98.48 18.15
0.048 (20) , 0.001 (220) 0.004 (210) , 0.001 (55) 0.002 (212)
0.71 1.90 1.13 1.34 0.39
0.518 0.204 0.364 0.310 0.689
However, the main effect of elevated UV on this variable over both years was not signi®cant (Table 1). In 1995, there was also a 19% increase in syringaldehyde concentration in leaf litter collected from polyester-®ltered lamp arrays, relative to non-energised arrays (Fig. 1d), but there was no main effect of elevated UV on syringaldehyde concentration over both years (Table 1). The concentrations of all phenylpropanoid derivatives, with the exception of p-hydroxyacetophenone, increased between 1995 and 1996. There were no interactive effects of elevated UV and year on phenylpropanoid concentrations (Table 1). 3.2. Tannins and lignin Elevated UV radiation did not in¯uence the concentrations of Folin±Denis tannins or lignin in recently-abscised Q. robur leaf litter (Table 1). In addition, tannin-to-N, lignin-to-N and lignin-to-P ratios were unaffected by
elevated UV (data not presented). Signi®cant increases in the concentrations of Folin±Denis tannins and lignin were recorded between 1995 and 1996 but there were no interactive effects of elevated UV and year on these constituents (Table 1). 3.3. Carbohydrates The concentration of holocellulose was unaffected by elevated UV radiation in Q. robur leaf litter, but that of two soluble carbohydrates responded to elevated UV (Table 1). Arabinose concentration in litter from saplings exposed during growth to elevated UV-A and UV-B radiation under cellulose-diacetate ®ltered arrays was 3% higher than in litter from saplings exposed to elevated UV-A under polyester-®ltered arrays (Fig. 2a). In addition, glucose concentration in leaf litter from saplings exposed to elevated UV-A and UV-B radiation under cellulose-diacetate ®ltered arrays was increased by 6%, relative to non-energised arrays
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Fig. 2. Effects of exposure to ambient solar radiation (A) under non-energised lamp arrays, ambient solar radiation plus supplemental UV-A radiation (p) under polyester-®ltered lamp arrays and ambient solar radiation plus supplemental UV-A and UV-B radiation (B) under cellulose diacetate®ltered lamp arrays on (a) arabinose and (b) glucose concentrations in recently-abscised Quercus robur leaf litter. Notations as in Fig. 1. Values are means of four replicates, pooled over two samplings in 1995 and 1996. Note that y-axes do not extend to zero.
Fig. 1. Effects of exposure to ambient solar radiation (A) under non-energised lamp arrays, ambient solar radiation plus supplemental UV-A radiation (p) under polyester-®ltered lamp arrays and ambient solar radiation plus supplemental UV-A and UV-B radiation (B) under cellulose diacetate®ltered lamp arrays on (a) acetovanillone, (b) vanillic acid, (c) vanillin and (d) syringaldehyde concentrations in recently-abscised Quercus robur leaf litter. Values are means of four replicates. Data in (a) and (b) are pooled over two samplings in 1995 and 1996. Data in (c) and (d) are for 1995 only. Bars represent LSD. Differently superscripted values differed at P , 0.05.
(Fig. 2b). Signi®cant effects of year were recorded for three carbohydrates: concentrations of xylose, arabinose and fucose increased between 1995 and 1996, but no interactive effects of elevated UV and year on carbohydrate concentrations were recorded (Table 1). 3.4. Nutrient elements No main effects of elevated UV on the concentrations of K, Ca, Mg, P or N were recorded in our study (Table 1). The concentrations of two nutrient elements (K and P) increased between years and those of three elements (Ca, Mg and N) decreased between 1995 and 1996 (Table 1). There were no signi®cant elevated UV £ year interactions on nutrient element concentrations (Table 1). 4. Discussion The data presented here indicate a negligible in¯uence of elevated UV-B radiation on the chemical composition of
recently-abscised Q. robur leaf litter, with signi®cant main effects of irradiation over 2 years on the concentrations of only four of 26 constituents analysed. No signi®cant effects of elevated UV radiation on the mass of leaf litter produced per array were recorded in 1995 and 1996 (Newsham et al., 1999), and accordingly, there were no main effects of UV radiation on the total mass of each chemical constituent per array (data not presented). Previous supplemental UV-B studies using ¯uorescent lamps in ®eld experiments have recorded signi®cant responses of plant litter chemistry to irradiation in a limited number of plant species. For example, a simulated 15% ozone depletion over Abisko (Sweden) led to a 19% decrease in the a-cellulose concentration of Vaccinium uliginosum leaf litter (Gehrke et al., 1995). By contrast, the same simulated amount of ozone depletion over Heemskerk (The Netherlands) increased the a-cellulose concentration of Calamagrostis epigeios leaf litter by 13% (Rozema et al., 1997). In experiments determining the effects of elevated UV-B radiation on the tissue chemistry of Triticum aestivum, Yue et al. (1998) recorded a 37% increase in holocellulose concentration in leaves following exposure to a simulated 25% reduction in the ozone column over Lanzhou (China). The data from our study indicated that elevated UV-B, simulating an 18% reduction in the ozone column over Monks Wood (UK), and applied continuously for 2 years, had no effect on the holocellulose concentration of recently-abscised Q. robur leaf litter. Exposure of plants to UV-B radiation during growth also has the potential to alter soluble carbohydrate concentrations in plant tissues. Yue et al. (1998) found that elevated UV applied during growth to T. aestivum led to a 21% decrease in total soluble carbohydrates in abscised leaves. However no effects on soluble carbohydrates were found in
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V. uliginosum and C. epigeios by Gehrke et al. (1995) or Rozema et al. (1997). The only indication of effects of elevated UV radiation on soluble carbohydrates in our study were 3±6% increases in concentrations of arabinoglucans (arabinose and glucose), which are constituents of hemicellulose, in litter from saplings exposed to elevated UV-A and UV-B radiation under cellulose diacetate-®ltered lamp arrays, relative to polyester-®ltered or non-energised lamp arrays. The study by Rozema et al. (1997) indicated that exposure to elevated UV-B radiation during growth increased the lignin concentration of C. epigeios leaf litter at abscission by 56%. The data from our study do not con®rm that similar effects occur in Q. robur. Similarly, Gehrke et al. (1995) found no effects of elevated UV-B radiation on the lignin concentration of V. uliginosum leaf litter. Furthermore, simulated 16% and 25% reductions in the ozone column over Beltsville, MD (USA) have no effect on the lignin concentration of needle litter from 3-year-old Pinus taeda seedlings (W. Cybulski, pers. comm.). Other experimental studies have also shown no effects of exposure to UV-B radiation on the concentration of hydrolysable tannins in litters of V. uliginosum (Gehrke et al., 1995), C. epigeios (Rozema et al., 1997), Calluna vulgaris, Cistus creticus, Rubus chamaemorus and Vaccinium myrtillus (Paul et al., 1999), corroborating the data from our study reported here. Previous studies have indicated that exposure of plant foliage to elevated UV-B radiation during growth can in¯uence the nutrient element concentration of leaf litter. Yue et al. (1998) recorded 85, 7.5 and 20% increases in N, K and Mg concentrations, respectively, and a 20% decrease in total P, in leaves of T. aestivum plants exposed to elevated UV-B radiation. By contrast, concentrations of N, P, K, Ca and Mg were unaffected in Acacia tortilis seedlings grown in nutrient-rich and nutrient-poor soils and exposed to a simulated 20% reduction in ozone column in a glasshouse (Ernst et al., 1997). Our data similarly suggest that exposure to elevated UV-B during growth has no effect on these nutrient elements in leaf litter. They also in part corroborate data indicating that UV-B has no effect on the density of mycorrhizal root tips of Q. robur saplings (Newsham et al., 1999). The most consistent effects of elevated UV in our study were on concentrations of phenylpropanoid moieties of lignin: two constituents were signi®cantly in¯uenced by irradiation over this 2-year study, whereas another two showed signi®cant responses in the ®rst year only. Significant increases, associated with growth of Q. robur under polyester-®ltered lamp arrays relative to non-energised lamp arrays, were recorded in vanillic constituents and syringaldehyde. These observations suggest that UV-A radiation produced by polyester-®ltered lamps may have induced the production of these compounds. Although exposure to UV-B radiation is known to in¯uence the expression of genes encoding phenylpropanoid metabo-
lism (Beggs and Wellmann, 1994), we are unaware of any reports indicating that UV-A radiation might also induce similar effects. These data corroborate those from previous studies that have frequently shown effects of elevated UV-A radiation, or some other factor associated with energised, polyester-®ltered lamps, on plants and insects (Newsham et al., 1996; 1999). Given that elevated UV-B radiation has been shown to in¯uence the chemistry of Calamagrostis, Triticum and Vaccinium leaf litters, what are the likely explanations for the relatively few changes in the chemical composition of Q. robur leaf litter observed in our study? We can discount the possibility that the analytical methods used were not sensitive enough to detect any changes in tissue chemistry, since small alterations in several chemical constituents were recorded, and because consistent changes in leaf litter chemistry were also recorded between years. One possible explanation for the paucity of treatment effects on litter chemistry observed in our study is that the increased thickness of Q. robur leaves, probably associated with increased cuticle thickness (Newsham et al., 1999), may have protected underlying leaf tissues from any in¯uence of elevated UV radiation. Differences in irradiation methods may also be responsible for differences in vegetation response, as suggested by Fiscus and Booker (1995). In particular, the studies of Gehrke et al. (1995), Rozema et al. (1997) and Yue et al. (1998) each used non-modulated lamp systems in which plants may be exposed to unrealistically high amounts of UV-B radiation relative to photosynthetically active radiation during cloudy periods, and could therefore elicit exaggerated responses to irradiation (McLeod, 1997). In conclusion, a number of studies have shown changes in leaf and litter chemistry in response to increased UV-B radiation. The data we obtained in this study on Q. robur show comparatively subtle main effects of elevated UV-B on the nutrient element, carbohydrate, polyphenolic and phenylpropanoid constituents of litter, suggesting that this species, or genotype, of oak was relatively insensitive to the effects of enhanced UV radiation. Other provenances of Q. robur would have to be similarly tested before general conclusions could be drawn, but these results suggest that increased UV-B radiation is unlikely to have signi®cant indirect effects on organic matter pools and nutrient turnover as a consequence of changes in the quality of leaf litter input to soils. Acknowledgements The authors acknowledge the ®nancial support provided by the NERC through its TIGER (Terrestrial Initiative in Global Environmental Research) programme. Jan Poskitt helped with laboratory work at CEH Merlewood, Dom Hodgson supplied useful comments on the manuscript and Walt Cybulski and Nigel Paul kindly allowed access to unpublished data.
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