ACTA BIOLOGICA SLOVENICA LJUBLJANA 2013 Vol. 56, [t. 2: 35–50 Deciduous and evergreen tree responses to enhanced UV-B treatment during three years Odziv listopadne in vednozelene drevesne vrste v času 3-letne izpostavljenosti povečanemu sevanju UV-B Tadeja Trošt Sedej, Dušan Rupar University of Ljubljana, Biotechnical Faculty, Department of Biology, Večna pot 111, SI-1000 Ljubljana, Slovenija correspondence: Tadeja.TrostSedej@bf.uni-lj.si Abstract: This paper reports a study of the strategies in Norway spruce (Picea abies (L.) Karst.) and European beech (Fagus sylvatica L.) for coping with enhanced UV-B radiation. Trees, as plants in general, possess diverse systems which respond to UV-B radiation. Changes in physiology, biochemistry and morphology have been observed in trees under enhanced UV-B radiation. The efficiency of trees’ UV-B pro- tective systems depends on plant characteristics and state of development as well as can be correlated with the UV-B dose and the environmental conditions. The two tree species were exposed outdoors to enhanced UV-B simulating 17% ozone depletion for three years during which time, selected parameters were monitored. Selected physiological parameters were monitored three times a year on beech leaves and three needle age classes of spruce. Spruce and beech exhibited great variability in the amounts of chlorophyll, methanol-soluble UV-B and UV-A absorbing compounds, and optimum quantum yield of photosystem II. The effects of UV-B radiation also varied with needle and leaf development stage and interaction with environmental conditions. Enhanced UV-B radiation triggered responses in both trees and a reduced negative effect of UV-B radiation on spruce photochemical efficiency was observed during prolonged drought. The results show high UV-B tolerance of both tree species and indicate the complexity of plant response to UV-B, involving multilevel inte- ractions with environmental factors and thus emphasizes the necessity of long-term investigations on trees in a natural ecosystem. Keywords: Picea abies, Fagus sylvatica, UV-B radiation, long-term field expe- riment Izvleček: V raziskavi smo preučevali strategije spoprijemanja s povečanim sevanje UV-B pri smreki (Picea abies ( L. ) Karst .) in bukvi ( Fagus sylvatica L.). Sadike obeh drevesnih vrst so bile posajene na prostem in za obdobje treh let izpostavljene povečanemu sevanju UV-B. Izbrane parametre smo spremljali trikrat letno na listih oziroma treh starostnih razredih iglic. Tako pri smreki kot pri bukvi smo izmerili veliko variabilnost v vsebnostih UV-B absorbirajočih snovi, fotosinteznih barvil in fotokemični učinkovitosti. Učinek povečanega sevanja UV-B je bil odvisen od razvojne faze iglice oziroma lista ter od okoljskih razmer. Povečano sevanje UV-B je sprožilo posamezne odzive pri obeh drevesnih vrstah. Zmanjšan negativni učinek sevanja UV-B 36 Acta Biologica Slovenica, 56 (2), 2013 na fotokemično učinkovitost smreke smo opazili v tretji poskusni sezoni in ga razlagali kot omilitveni učinek suše. Pri letošnjih iglicah, ne pa tudi listih ali starejših iglicah, je bila prisotna neznačilna tendenca povečane sinteze UV-B absorbirajočih snovi pod povečanim sevanjem UV-B. Rezultati so pokazali veliko strpnost obeh drevesnih vrst do povečanega sevanja UV-B, obenem pa potrdili kompleksen odziv na povečano sevanje UV-B, ki je odvisno tudi od razvojne faze rastline in okoljskih razmer. Ključne besede: Picea abies, Fagus sylvatica, sevanje UV-B, večletni poskus Introduction Depletion of the stratospheric ozone over the past several decades has resulted in enhanced levels of UV-B radiation reaching the biosphere (Madronich et al. 1998, Ajavon et al. 2006). A United Nations report states that it is estimated that full recovery of stratospheric ozone on a global scale will not occur before 2050–2100 and will be depend upon continued compliance with the Montreal Protocol and addressing the interac- tions between ozone recovery and atmospheric changes, such as climate change. The enhanced UV-B radiation remains an issue which can affect biocenosis significantly. Most of the studies on the effects of enhanced UV-B radiation on plants have involved agricultural species, with much fewer studies on trees, even though the importance of trees in both ecosystems and ecosystems and in economics is considerable. The knowledge of UV-B radiation effects on trees is mainly based on short-term experiments and/or controlled growth conditions. Detrimental effect of UV-B observed in those studies occurs rarely in field-grown trees, where natural light conditions and other environmental factors contribute to diverse responses of trees (Mirecki and Teramura 1984, Laakso and Huttunen 1998, Laakso et al. 2000, Sullivan at al. 2005). Long-term field UV-B effects have been studied scarcely, and various responses of trees to enhanced UV-B radiation were reported. Three-year studies on conifers reported reductions in growth (Sullivan and Tera- mura 1992) and a reduction of UV-B absorbing compounds Kinnunen et al. 2001) on pine trees under UV-B exposure. An increase of UV-B absorbing compounds was observed in Douglas fir and Ponderosa pine during two year UV-B irradiation (Warren et al. 2002). No reduction of growth and photosynthesis/secondary compounds was detected at Douglas fir, Norway spruce and Scots pine after two or three years of UV-B expo- sure (Bassman et al. 2002, Turtola et al. 2006). A five-year UV-B irradiation of Norway spruce led to a decrease of some growth parameters, but not of photosynthesis or UV-B absorbing compounds (Trošt Sedej and Gaberščik 2008). A five-year exposure to increased UV-B of deciduous trees (ash, Fraxinus excelsior; silver birch, Betula pen- dula; lime, Tilia cordata; English oak, Quercus robur; and sycamore maple, Acer pseudoplatanus) resulted in decreased photosynthesis, transpiration and stomatal density (Keiller and Holmes 2001). In a three-year study on red maple (Acer rubrum), tulip poplar (Liriodendron tulipifera) and sweet- gum (Liquidambar styraciflua), photosynthesis generally did not decline and poplar exhibited an increase of UV-B absorbing compounds (Sul- livan et al. 1994), while European beech (Fagus sylvatica) showed increased photosynthesis after three years of UV-B exposure (Šprtová at al. 2003). Reduced photosynthetic activity led to reduced leaf elongation, plant growth and biomass production in some cases (Warren et al. 2002, Bassman et al. 2003, Kirchgessner et al. 2003, Lavola et al. 2003, Lenk and Buschmann 2006, Trošt Sedej and Gaberščik 2008). Leaf size in deciduous trees has been variously reported to have been decreased by enhanced UV-B radiation (Newsham et al. 1999, Keiller and Holmes 2001, Sullivan at al. 2003), increased (Sullivan at al. 2003, Šprtová at al. 2003) or unchanged (Kostina et al. 2001). Increase in leaf thickness under enhanced UV-B radiation was observed in some deciduous trees (Sullivan et al. 1994, Antonelli et al. 1998, New- sham et al. 1999), where leaf thickening was due to an increase in either the thickness of the spongy parenchyma (Kostina et al. 2001) or of the palisade parenchyma (Nagel et al. 1998) . 37Trošt-Sedej, Rupar: Deciduous and evergreen tree responses to enhanced UV-B treatment … Trees possess diverse biochemical, physio- logical and morphological mechanisms which respond to UV-B radiation, so that the ambient UV-B might be viewed both as a stressor and a photomorphogenic signal (Prado et al. 2012). Trees’ resistance to enhanced UV-B is partially based on a high epidermal screening capacity due mainly to phenolics (Fischbach et al. 1999, Hoque and Remus 1999, Trošt Sedej and Gaberščik 2008, Rozema et al. 2002, Turtola et al. 2006). Impor- tant components of the defence systems against UV and a number of stress factors are also other secondary compounds such as terpenes, (Turtola et al. 2006, Prado et al. 2012), reflectance of UV (Hoque and Remus 1999, Láposi et al. 2009), special anatomical arrangement and increased cell wall thickness of epidermal cells (Sullivan at al. 1994, Antonelli at al. 1998, Newsham at al. 1999, Hoque and Remus 1999, Chalker-Scott and Scott 2004) and small, thick leaves (P´yankov and Kon- drachuk 1998). The proportion of UV-B radiation reaching the leaf mesophyll is generally higher in deciduous broadleaf trees than in evergreen conifer trees (Day 1993), that indicate greater sensitivity of deciduous trees to enhanced UV-B radiation but lower maintenance costs. UV-B sensitivity is closely related to the development state of leaves, where the epidermis of fully grown leaves filters UV-B more efficiently than that of young leaves (Day et al. 1992, DeLucia at al.1992, Day et al. 1996, Ruhland and Day 1996, Laakso et al. 2000, Trošt and Gaberščik 2001, Neitzke and Therburg 2003, Trošt Sedej and Gaberščik 2008). The degree of UV-B shielding in trees de- pends on environmental conditions (Neitzke and Therburg 2003, Julkunen-Tiitto et al. 2005, Lenk and Buschmann 2006, Trošt Sedej and Gaberščik 2008). The higher sensitivity to UV-B was due to low temperatures in spruce (Bavcon et al. 1996) and increased ozone at beech (Zeuthen et al. 1997). Drought exposure in pine and spruce (Petropoulou et al. 1995, Manetas et al. 1997, Trošt Sedej and Gaberščik 2008) and nutrient deficiency at birch (Keski-Saari et al. 2005) alleviated UV-B effect. Experiments testing elevated CO2 and enhanced UV-B radiation indicated that increased CO2 either amelioratied or had no effect on photosynthesis or biomass allocation (Sullivan, 1997, Caldwell at al. 1998, and Lavola at al. 2000). Some studies (Laakso et al. 2000, Sullivan 2005) proved species/ population specific responses to enhanced UV-B radiation, with woody species varying widely in their responses under changing environmental conditions and also responding slowly, that is why further long-term outdoor research is necessary. Norway spruce (Picea abies (L.) Karst.) and European beech (Fagus sylvatica L.) are the most common tree species of natural forests in Central Europe. Plants were exposed to ambient and enhanced UV-B levels at the outdoor experi- mental plot. We have examined physiological and growth responses to UV-B radiation (Sullivan and Teramura 1992, Bassman et al. 2002, Šprtová et al, 2003 Turtola et al. 2006, Trošt Sedej and Gaberščik 2008) taking into account the effect of concomitant environmental conditions (Petro- poulou et al. 1995, Manetas et al. 1997, Neitzke and Therburg 2003, Keski-Saari et al. 2005, Trošt Sedej and Gaberščik 2008) and considering the variation of tree response according to needle/leaf development stage (Naidu et al. 1993, Latola et al. 2001). This paper offers an insight into complex response of Norway spruce and European beech to UV-B exposure during a three year study under realistic environmental conditions, thus it provides an additional view to a deciduous and an evergreen tree strategy of coping with enhanced UV-B radiation. Materials and methods Outdoor experimental plot Norway spruce (Picea abies (L.) Karst.) and European beech (Fagus sylvatica L.) one-year seedlings were planted in an outdoor research plot (Botanical Garden, University of Ljubljana: 320m a.s.l., 46°35´N, 14°55´E). Fifty seedlings for each of the two treatments were planted in 5 clay pots (62x21x19 cm) in a mixture of compost and peat (1:1). Plants were transplanted to a new soil mixture every February. The pots were buried at ground level to minimise soil temperature variation and desiccation (Sullivan and Teramura 1992). Two different treatments were applied in the experiment. A UV-B supplement system was de- signed, as described by Björn and Teramura (1993) and exposure resulting from 17% ozone depletion, corresponding to a 35–55% increase of ambient 38 Acta Biologica Slovenica, 56 (2), 2013 UV-BBE (UV-B+), was simulated using Q-Panel UV-B 313 lamps (Cleveland, OH, USA), which emit from 275 nm to 400 nm with peak emission at 313 nm, filtered with cellulose diacetate filters. Ambient radiation (UV-B) was simulated using the same lamps filtered with Mylar foil. The doses were calculated and adjusted weekly (Björn and Murphy 1985) using the generalised plant action spectrum of Caldwell (1968). The system was timer controlled. Ambient UV-B (Fig. 1a). UV-A and PAR radiation were monitored at the site by a three-channel dosimeter (Häder et al. 1999). The experiment was conducted over three growing seasons from May till October. Cumulative water balance data (Fig. 1b) cal- culated as the difference between total monthly precipitation and total monthly potential eva- potranspiration according to Penman’s equation, were obtained from Slovenian Environment Agency. Seasonal cumulative water balance (CWB) represents three seasonal periods of beech growth and development: March to June, March to August and March to October. Principal Component Analysis showed a high correlation between seasonal cumulative water balance and environmental factors air and soil temperature, insolation, precipitation and potential evaporation and other plant parameters at the outdoor experi- mental plot. Seasonal cumulative water balance, as a water disposability indicator, was chosen as the most characteristic environmental factor, and was used in the correlation analyses. Physiological and biochemical measurements Measurements were carried out three times per growth season, in May, August and October, the key phases for tree growth and development (young leaves, growth phase peak, end of growth) as determined in the preliminary year (Trošt and Gaberščik 2001). Leaves and needles, the latest of three needle-age classes: current (c), current+1 (c+1) and current+2 (c+2), were sampled from five (for biochemical measurements) and ten (for Figure 1a: Monitored ambient (UV-B) and enhanced (UV-B+) daily dose of biologically active UV-B (UV-BBE) radiation, calculated ambient (UV-B model) and enhanced (UV-B+ model) daily dose of biologically active UV-B (UV-BBE) radiation (Björn and Murphy, 1993) in the two treatments established at the experi- mental site in Ljubljana. 1b. Monthly and seasonal cumulative water balance (CWB) in Ljubljana. Slika 1a: Izmerjeni naravni (UV-B) in povečani (UV-B+) dnevni odmerki biološko aktivnega sevanja UV-B (UV- BBE), izračunani naravni (UV-B model) in povečani (UV-B+ model) dnevni odmerki biološko aktivnega sevanja UV-B (UV-BBE) po modelu Björn and Murphy (1993) na poskusni ploskvi Botaničnega vrta v Ljubljani. 1b. Mesečna in sezonska kumulativna vodna bilanca (CWB) v Ljubljani. Fig. 1. 0 2 4 6 8 U V- B B E (k J m -2 da y- 1 ) a UV-B UV-B+ UV-B model UV-B+ model -300 -100 100 300 500 J F M A M J J A S O N D J F M A M J J A S O N D J F M A M J J A S O N D C W B (m m ) b 1st year 2nd year 3rd year CWB monthly CWB seasonal Fig. 1. 0 2 4 6 8 U V- B B E (k J m -2 da y- 1 ) a UV-B UV-B+ UV-B model UV-B+ model -300 -100 100 300 500 J F M A M J J A S O N D J F M A M J J A S O N D J F M A M J J A S O N D C W B (m m ) b 1st year 2nd year 3rd year CWB monthly CWB seasonal 39Trošt-Sedej, Rupar: Deciduous and evergreen tree responses to enhanced UV-B treatment … physiological measurements) of the upper horizon- tal branches of randomly selected trees per plot. Photochemical efficiency was estimated by measuring the chlorophyll a fluorescence of photosystem II using a modulated fluorometer (OS-500, Opti-Sciences, USA). Measurements were carried out in vivo at noon on clear days. Optimal quantum yield (Fv/Fm), defined as (Fm – Fo)/Fm, where Fm represents maximal and Fo minimal fluorescence of a dark adapted sample, was determined. Total chlorophyll content (Chl a+b) was determined as described by Lichtenthaler (1987). The Chl a+b content was calculated per sample DM, from extinction coefficients at 644 and 662 nm in acetone [100% (v/v)] (UV/VIS spectrophotometer Lambda 12, Perkin-Elmer, Norwalk, CT, USA). The total content of methanol-soluble UV-B (A280–320) and UV-A (A320–400) absorbing compound s was estimated according to Caldwell (1968). The extinction coefficients of the samples were measured in the UV-B and UV-A spectral range 280–400 nm (UV/VIS spectrometer), calculated per sample DM and integrated to estimate the total content of UV-B and UV-A absorbing compounds. Morphometric measurements At the end of growth season, the leaf length, width and thickness as well as needle total length and diameter (optical microscope Zeiss KF2, Carl Zeiss, Germany) were measured. The parameters were determined in ten randomly selected needles of three age-classes and leaves per plot sampled at the upper horizontal branches. Statistical analysis The independent-samples t-test was used to compare means of measured parameters at the two UV-B treatments. Spearman’s coefficient was used to investigate bivariate correlations between the environmental factors and measured parameters. Samples in all the tests were randomly chosen without replication. Statistically significant diffe- rences were marked as: non-significant (p < 0.05), * (p ≤ 0.05), ** (p ≤ 0.01) and *** (p ≤ 0.001). Analyses were accomplished by SPSS for Win- dows 13.0.0. Results Physiological and biochemical responses The responses of Norway spruce and European beech varied from neutral to negative or positive during three years exposure to UV-B, and were varying with plant developmental state, growth season and environmental conditions (Fig. 2., Table 1). In the autumn of the first season Norway spruce manifested reduced Fv/Fm under enhanced UV-B in c+1 needles and in spring of the second season in c and c+1 needles. In the third season, higher Fv/Fm under enhanced UV-B was observed in c and c+1 needles in spring, while in summer of this season Fv/Fm increased in all three needle age classes. In all three spruce needle age classes, Fv/Fm exhibited a tendency to reduced values in spring and optimal values later in the season. The Fv/Fm values of the European beech were significantly reduced under enhanced UV-B in the spring of the second season. Fv/Fm values were low during the second and third season under both treatments (Fig. 2). In the Norway spruce, the Chl a+b content responded to enhanced UV-B in all three needle age classes. The young needles manifested increased Chl a+b content twice under enhanced UV-B, while Chl a+b content in c+1 and c+2 needles was decreased three times and increased once. In the European beech Chl a+b content significantly decreased under enhanced UV-B in autumn of the second season and increased in autumn of the third season (Table 1). The A280–320 content was high in both tree species under both UV-B treatments during all growth seasons. Needles/leaves were poorly responsive to enhanced UV-B radiation. Under UV-B+ exposure, spruce current needles showed tendency to higher A280–320 content, while beech leaves manifested tendency to lower A280–320 content under UV-B+ exposure. Compared with UV-B treatment, the A280-320 content under UV-B+ radiation decreased significantly once, in summer of the dry third season. A320–400 content was high in both tree species and responded rarely to enhanced UV-B (Table 1). 40 Acta Biologica Slovenica, 56 (2), 2013 Fig. 2. P. abies c *** * *** 0 0,2 0,4 0,6 0,8 Fv /F m (R U ) UV-B UV-B+ P. abies c+1 * * ** *** 0 0,2 0,4 0,6 0,8 Fv /F m (R U ) *** P. abies c+2 0 0,2 0,4 0,6 0,8 May Aug Oct May Aug Oct May Aug Oct Fv /F m (R U ) F. sylvatica * 0 0,2 0,4 0,6 0,8 May Aug Oct May Aug Oct May Aug Oct Fv /F m (R U ) Figure 2: Optimal quantum yield (Fv/Fm) for three needle age classes of Norway spruce (c, c+1, c+2) and leaves of European beech exposed to ambient (UV-B) and enhanced (UV-B+) UV-B radiation during three growth seasons. Data are means ± SE, n = 10, significant differences (Independent-samples t-test) are marked with: * (p ≤ 0.05), ** (p ≤ 0.01), *** (p ≤ 0.001). Slika 2: Potencialna fotokemična učinkovitost (Fv/Fm) treh starostnih razredov iglic smreke (c, c+1, c+2) in listov bukve, izpostavljenih naravnemu (UV-B) in povečanemu (UV-B+) sevanju UV-B tekom treh let. Podatki so srednje vrednosti ± SE, n = 10, značilna razlika (neodvisni t-test) je označena z: * (p ≤ 0.05), ** (p ≤ 0.01), *** (p ≤ 0.001). 41Trošt-Sedej, Rupar: Deciduous and evergreen tree responses to enhanced UV-B treatment … Ta bl e 1: Bi oc he m ic al pa ra m et er s: ch lo ro ph yl l c on te nt (C hl a +b [m g g -1 D M ]), co nt en t o f U V- B ab so rb in g c om po un ds (A 28 0- 32 0 [ re la tiv e u ni ts] ) a nd co nt en t o f U V- A ab so rb in g c om po un ds (A 32 0- 40 0 [r el at iv e u ni ts ]) fo r t hr ee n ee dl e a ge cl as se s ( c, c+ 1, c+ 2) o f N or w ay sp ru ce an d le av es o f E ur op ea n be ec h ex po se d to am bi en t ( U V- B ) a nd en ha nc ed (U V- B +) U V- B ra di at io n du rin g th re e g ro w th se as on s. D at a a re m ea ns ± S E, n = 5 , s ig ni fic an t d iff er en ce (I nd ep en de nt -s am pl es t- te st) is m ar ke d w ith : * (p ≤ 0 .0 5) , * * (p ≤ 0 .0 1) , * ** (p ≤ 0 .0 01 ). Ta be la 1 : B io ke m ijs ki p ar am et ri: v se bn os t k lo ro fil a (C hl a + b [m g g- 1 D M ]) , v se bn os t U V- B a bs or bi ra jo či h sn ov i ( A 28 0- 32 0 [ re la tiv na e no ta ]) in v se bn os t U V- A a bs or bi ra jo či h sn ov i (A 32 0- 40 0 [r el at iv na e no ta ]) v tr eh st ar os tn ih ra zr ed ih ig lic (c , c +1 , c +2 ) s m re ke in v li st ih b uk ve , i zp os ta vl je ni h na ra vn em u (U V- B ) i n po ve ča ne m u (U V- B +) se va nj u U V- B te ko m tr eh le t. Po da tk i s o sr ed nj e vr ed no st i ± S E, n = 5 , z na či ln a ra zl ik a (n eo dv is ni t- te st ) j e oz na če na z : * (p ≤ 0 .0 5) , * * (p ≤ 0 .0 1) , * ** (p ≤ 0 .0 01 ). 1s t y ea r 2n d ye ar 3r d y ea r M ay A ug O ct M ay A ug O ct M ay A ug O ct U V- B U V- B + U V- B U V- B + U V- B U V- B + U V- B U V- B + U V- B U V- B + U V- B U V- B + U V- B U V- B + U V- B U V- B + U V- B U V- B + P. a bi es C hl a +b c M ea n 3, 15 3, 10 2, 96 3, 12 7, 66 4, 82 6, 94 14 ,3 6 7, 42 5, 60 6, 64 5, 84 6, 89 9, 23 2, 62 2, 78 1, 87 2, 70 ± SE 0, 76 0, 35 0, 11 0, 34 1, 07 0, 31 0, 81 0, 59 0, 98 0, 76 0, 70 0, 53 1, 16 1, 56 0, 15 0, 34 0, 12 0, 27 p ns ns ns ** * ns ns ns ns * c+ 1 M ea n 2, 91 1, 90 3, 65 3, 95 7, 93 8, 91 6, 19 10 ,7 6 3, 61 3, 46 4, 85 5, 58 1, 31 1, 29 1, 68 2, 18 1, 19 1, 96 ± SE 0, 32 0, 22 0, 37 0, 38 0, 58 1, 00 0, 77 1, 83 0, 27 0, 28 0, 41 0, 83 0, 14 0, 35 0, 14 0, 27 0, 11 0, 21 p ns ns ns * ns ns ns ns * c+ 2 M ea n 5, 55 2, 49 4, 16 2, 50 4, 37 5, 19 5, 41 6, 45 3, 42 4, 01 4, 47 3, 91 0, 41 0, 34 1, 60 1, 88 1, 07 1, 36 ± SE 0, 42 0, 51 0, 45 0, 39 0, 43 0, 40 0, 73 0, 54 0, 50 0, 14 0, 48 0, 27 0, 27 0, 06 0, 14 0, 17 0, 16 0, 27 p ns ns ns ** * * ns ns ns ns F. sy lv at ic a C hl a +b M ea n 1, 31 2, 86 3, 52 4, 05 2, 71 2, 75 2, 43 3, 71 3, 35 2, 96 3, 62 2, 87 5, 71 6, 51 4, 07 3, 31 3, 21 3, 92 ± SE 0, 60 0, 28 0, 61 0, 57 0, 14 0, 12 0, 35 0, 54 0, 42 0, 13 0, 23 0, 20 0, 36 0, 31 0, 45 0, 28 0, 11 0, 19 p ns ns ns ns ns * ns ns * P. a bi es A 28 0- 32 0 c M ea n 18 20 20 52 22 39 23 65 23 95 27 02 26 89 29 61 24 10 28 86 30 73 38 77 37 90 35 18 16 03 17 20 16 83 18 45 ± SE 91 ,8 10 6, 6 26 ,7 23 5, 7 81 ,4 64 ,3 31 8, 6 35 0, 9 30 4, 2 45 7, 4 29 8, 7 37 3, 2 71 5, 1 10 16 ,5 32 2, 3 30 3, 4 21 4, 4 26 1, 5 p ns ns ns ns ns ns ns ns ns c+ 1 M ea n 27 24 22 37 26 90 25 76 30 88 29 37 26 12 25 85 21 47 18 98 32 50 29 59 19 94 13 56 22 50 16 57 20 60 28 87 ± SE 19 0, 7 85 ,1 44 ,1 84 ,6 12 5, 8 26 3, 8 47 ,6 24 9, 3 47 3, 7 43 9, 2 19 0, 9 30 9, 4 49 1, 8 31 0, 5 27 6, 3 16 5, 1 99 ,1 89 8, 1 p ns * ns ns ns ns ns ns ns c+ 2 M ea n 25 35 20 11 29 84 28 93 29 00 28 88 24 73 24 32 28 94 22 36 31 72 32 94 11 78 10 43 18 84 16 81 19 15 18 31 ± SE 36 1, 0 81 ,9 16 8, 3 86 ,4 10 8, 8 75 ,7 18 2, 4 16 9, 9 55 0, 3 26 2, 9 21 4, 4 19 3, 6 30 8, 2 12 0, 9 23 5, 2 17 1, 7 17 3, 9 12 9, 1 p ns ns ns ns ns ns ns ns ns F. sy lv at ic a A 28 0- 32 0 M ea n 25 06 22 23 18 79 18 98 17 24 17 96 20 62 19 27 25 45 24 03 18 21 17 05 15 50 14 70 17 00 14 67 19 37 15 86 ± SE 20 5, 0 19 9, 9 10 6, 6 65 ,0 94 ,9 26 3, 0 17 9, 6 10 4, 3 32 6, 7 23 9, 3 72 ,7 67 ,0 95 ,0 13 4, 8 71 ,1 42 ,3 18 9, 5 18 1, 9 p ns ns ns ns ns ns ns ns ns P. a bi es A 32 0- 40 0 c M ea n 25 43 25 96 17 74 21 18 12 86 14 67 29 61 30 60 19 00 23 99 20 83 30 15 28 97 29 98 97 8 11 73 85 4 12 51 ± SE 17 3, 7 52 ,8 12 5, 6 24 1, 2 12 6, 2 13 9, 1 44 1, 2 29 6, 0 23 6, 8 38 2, 0 19 7, 6 21 5, 5 55 1, 7 10 41 ,1 21 8, 2 18 0, 7 11 0, 9 17 4, 1 p ns ns ns ns ns * ns ns ns c+ 1 M ea n 23 19 17 05 18 58 17 84 18 69 17 67 18 18 16 85 15 12 14 22 21 54 20 96 10 14 79 8 12 04 96 2 10 01 12 67 ± SE 16 1, 1 72 ,3 55 ,5 10 2, 6 21 2, 3 15 6, 7 77 ,2 13 4, 5 29 3, 8 26 3, 7 12 6, 7 26 0, 7 23 3, 8 17 5, 4 18 3, 8 95 ,8 47 ,2 24 2, 8 p * ns ns ns ns ns ns ns ns c+ 2 M ea n 19 47 15 18 20 57 21 81 17 17 17 47 16 64 15 84 21 25 16 14 21 42 24 71 65 3 65 5 10 41 10 96 93 7 10 55 ± SE 19 7, 5 12 2, 7 98 ,3 97 ,9 76 ,9 58 ,0 72 ,9 15 2, 2 43 6, 7 18 6, 5 24 3, 7 17 1, 9 11 4, 7 60 ,0 12 6, 1 13 9, 5 83 ,2 99 ,1 p ns ns ns ns ns ns ns ns ns F. sy lv at ic a A 32 0- 40 0 M ea n 30 53 29 46 22 87 23 07 19 45 18 33 19 43 20 45 27 10 27 32 16 55 15 56 14 80 14 75 17 45 15 71 19 14 15 88 ± SE 36 6, 9 29 0, 8 24 9, 8 68 ,6 18 0, 9 27 5, 4 21 9, 0 13 7, 0 38 6, 9 35 4, 7 11 9, 7 86 ,0 10 3, 4 26 5, 8 83 ,2 43 ,7 21 3, 5 25 4, 4 p ns ns ns ns ns ns ns * ns 42 Acta Biologica Slovenica, 56 (2), 2013 Table 2: Morphometric needle/leaf parameters of Norway spruce and European beech exposed to ambient (UV-B) and enhanced (UV-B+) UV-B radiation determined at the end of growth seasons. Parameters: needle total length (m), average diameter (mm), leaf length (mm), width (mm), thickness (µm). Data are means ± SE, n = 50, significant difference (Independent-samples t-test) is marked with: * (p ≤ 0.05), ** (p ≤ 0.01), *** (p ≤ 0.001). Tabela 2: Morfometrični parametri iglic smreke in listov bukve izpostavljenih naravnemu (UV-B) in povečanemu (UV-B+) sevanju UV-B tekom treh let. Parametri: skupna dolžina iglic (m), premer iglice (mm), dolžina lista (mm), širina lista (mm), debelina lista (µm). Podatki so srednje vrednosti ± SE, n = 10, značilna razlika (neodvisni t-test) je označena z: * (p ≤ 0.05), ** (p ≤ 0.01), *** (p ≤ 0.001). 1st year 2nd year 3rd year UV-B UV-B+ UV-B UV-B+ UV-B UV-B+ P. abies Needle length Mean ± SE 77.82 11.16 81.19 12.23 99.03 30.07 100.2 30.67 65.86 8.98 58.73 3.49 p ns ns ns Needle diameter Mean ± SE 0.59 0.018 0.56 0.016 0.41 0.009 0.39 0.012 0.53 0.026 0.43 0.014 p ns ns ns F. sylvatica Leaf length Mean – – – – 44.0 40.8 ± SE 2,3 4,1 p ns Leaf width Mean – – – – 26.2 25.4 ± SE 1.6 2.1 p ns Leaf thickness Mean – – – – 97.5 89.9 ± SE 8.3 2.2 p ns Morphometric modifications Enhanced UV-B radiation did not affect needle/ leaf morphology of neither of the trees after the three year observation (Table 2). Interactions of UV­B radiation with environmental conditions and plant development Correlations between the measured tree para- meters and tree age, seasonal tree development, seasonal cumulative water balance and enhanced UV-B radiation are shown in Table 3. In Norway spruce and European beech tree age was gener- ally correlated with decreasing parameter values, with the exception of UV-B and UV-A absorbing compounds in spruce young needles, which failed to correlate with tree age and beech chlorophyll content which increased with tree age. Seasonal tree development correlated scarcely with mea- sured parameters, but there was a strong positive correlation with seasonal cumulative water balan- ce. The content of UV-B absorbing compounds in spruce c needles failed to correlate with tree characteristics or environmental conditions and none of the tree responses could be correlated with the UV-B radiation dose (Table 3). Discussion Diverse physiological responses to enhance UV­B varying with environmental condition The responses of spruce and beech to en- hanced UV-B radiation varied according to the needle/leaf development stage, growth season and environmental conditions. In both species, most of monitored parameters did not vary with enhanced UV-B solely (Fig. 2, Table 1). Fv/Fm was the parameter most sensitive to enhanced UV-B radiation. The Fv/Fm decrease 43Trošt-Sedej, Rupar: Deciduous and evergreen tree responses to enhanced UV-B treatment … Table 3: Correlations between tree parameters and tree age, seasonal tree development, seasonal cumulative water balance (CWB) and UV-B treatment (UV-B+) in Norway spruce (different needle age classes: c, c+1, c+2) and European beech. Tree parameters: optimal quantum yield (Fv/Fm), total chlorophyll content (Chl a+b), content of UV-B (A 280–320) and UV-A (A 320–400) absorbing compounds. Bivariate correlation (Spearman´s coefficient ρ) is marked with: ns (p > 0.05), * (p ≤ 0.05), ** (p ≤ 0.01), *** (p ≤ 0.001). Tabela 3: Korelacije med merjenimi parametric dreves in starostjo bdrevesa, sezonskim razvojem, sezonsko ku- mulativno vodno bilanco (CWB) ter povečanim (UV-B+) sevanjem UV-B treh starostnih razredov iglic smreke (c, c+1, c+2) in listov bukve. Merjeni parametric drevesa: potencialna fotokemična učinkovitost (Fv/Fm), vsebnost klorofila (Chl a+b), vsebnost UV-B absorbirajočih snovi (A280–320) in vsebnost UV-A absorbirajočih snovi (A320–400 ). Bivariatna korelacija (Spearmanov koeficient ρ) je označen z: ns (p > 0.05), * (p ≤ 0.05), ** (p ≤ 0.01), *** (p ≤ 0.001). Tree age Seasonal development CWB UV-B+ ρ p ρ p ρ p ρ p P. abies Fv/Fm c –0.389 * 0.740 ** 0.477 ** ns c+1 ns 0.471 ** 0.525 ** ns c+2 –0.734 *** ns 0.597 ** ns F. sylvatica Fv/Fm –0.602 ** ns 0.444 ** ns P. abies Chl a+b c –0.452 * ns ns ns c+1 –0.767 *** ns 0.601 *** ns c+2 –0.856 *** ns ns ns F. sylvatica Chl a+b 0.491 ** ns –0.469 ** ns P. abies A 280–320 c ns ns ns ns c+1 –0.452 * 0.396 * 0.725 *** ns c+2 –0.769 *** ns 0.495 * ns F. sylvatica A 280–320 –0.406 ** ns 0.291 ** ns P. abies A 320–400 c ns ns 0.423 * ns c+1 –0.811 *** ns 0.726 *** ns c+2 –0.856 *** ns ns ns F. sylvatica A 320–400 –0.514 ** –0.228 * 0.310 ** ns under enhanced UV-B was measured in spring of the second season in the young needles of spruce and beech leaves as well. It has been demonstrated that UV-B radiation alters the structure and function of chloroplasts, so that the potential photochemical efficiency might decrease (Björkman and Demmig-Adams 1994, Musil 1996, Adams and Barker 1998, Wu et al. 2011). Some studies on trees indeed showed a decrease of photochemical efficiency (Naidu et al. 1993, Bavcon et al. 1996), but in others no decrease was observed (Petropoulou et al. 1995, Manetas et al. 1997, Chalker-Scott and Scott 2004). A lowered Fv/Fm may be considered also as a down-regulation mechanism whose aim is lowering of the electron supply in the Calvin cycle as a result of the UV-B induced stress, which was apparently due to UV-B penetration into the mesophyll of recently emerged needles/leaves. It has been shown that in some trees the epidermis of fully grown needles 44 Acta Biologica Slovenica, 56 (2), 2013 and leaves filters UV-B more efficiently than the epidermis in young ones (Neitzke and Therburg 2003, Day et al. 1992, DeLucia at al.1992, Day et al. 1996, Ruhland and Day 1996, Laakso et al. 2000, Trošt Sedej and Gaberščik 2008). The effect of UV-B on older leaves was diminished due to higher UV-B absorbing compounds content, self- shading and increased xeromorphic characteristics of needles/leaves. In the third season, the period of the most negative cumulative water balance, the photochemical efficiency of both UV-B exposed and control trees was low, but in Norway spruce the values were significantly higher in irradiated plants (Fig. 2) indicating the alleviating effect of UV-B radiation on drought. Such effect has been observed in Mediterranean conifers (Petropoulou et al. 1995, Manetas et al. 1997). Since UV-B radiation is unable to penetrate into the meso- phyll of fully grown spruce needles (Fischbach et al. 1999), it can be concluded that the effect of UV-B on photochemical efficiency is indirect. UV-B radiation could benefit the water relations of plants through stomata closure (Petropoulou et al. 1995, Manetas et al. 1997), through promoted wax synthesis (Björn et al. 1997) and through cross-resistance of plants which are exposed to any oxidative stress (Turtola et al. 2006). In our study reductions of photochemical efficiency were more common in the recently emerged needles/leaves than in the later development stages. This reflects a remarkable capability for recovery, in which the disturbances of young organs are not expressed in the later stages. Similar findings have been reported in loblolly pine by Naidu and co-workers (1993). Chlorophyll levels showed no consistent response to enhanced UV-B in any of the trees studied, the results suggesting dependence on development phase and environmental conditions. Other studies reported negative, neutral and rarely, positive effects of enhanced UV-B radiation on chlorophyll content (Šprtová et al. 1999, Bassman et al. 2003, Kirchgessner et al. 2003, Lavola et al. 2003, Trošt Sedej and Gaberščik 2008, Láposi et al. 2009). It has been shown that UV-B radia- tion can not only inhibit chlorophyll synthesis or cause its photo-oxidation (Bornman 1989, Mid- dleton and Teramura 1993), but also increase the biosynthesis of photosynthetic pigments under favourable irradiation conditions (Middleton and Teramura 1993, Jordan 1996). High tolerance to UV­B radiation Norway spruce and European beech appear to possess an effective filter consisting of UV-B and UV-A absorbing compounds already present in young needles/leaves in May, soon after emer- gence from buds. The content did not change with seasonal development or enhanced UV-B radiation but it was affected by drought (Tab. 3). The total amount of methanol-soluble UV-B absorbing compounds in Norway spruce and European beech was three and two times higher, respectively (Tab. 1) than in some herbaceous species, where the same analytical method was used (Gaberščik et al. 2001, Gaberščik et al. 2002a, Gaberščik et al. 2002b, Breznik et al. 2005). The low variability of UV-A and UV-B absorb ing compounds content is probably part of a protective strategy of long-lived woody plants. Studies indicate that most conifers contain large amounts of UV-B absorbing compounds in the epidermis (Sullivan et al. 1996, Fischbach et al. 1999, Hoque and Remus 1999, Turtola et al. 2006) and the epidermis of fully grown leaves effectively filters UV-B (Day et al. 1992, Day et al. 1996, Ruh- land and Day 1996, Fischbach et al. 1999, Hoque and Remus 1999). In the young needles of Abies lasiocarpa and Picea engelmannii less than 1% of UV-B radiation penetrates into the mesophyll (DeLucia et al. 1992), in European beech the UV-B penetration into young leaves was greater than in developed leaves (Neitzke and Therburg 2003). The production of UV-B absorbing compounds does not always depend on the UV-B dose (Rau and Hofmann 1996, Turtola et al. 2006), and consequently some higher plants from tropical, high-altitude and aquatic environments contain saturating amounts of UV absorbing compounds (Teramura and Sullivan 1994, Germ et al. 2002). It was hypothesized that the receptors triggering the biosynthesis of UV-B absorbing compounds are saturated in plants growing in open environments, therefore they provoke maximal synthesis over a wide range of irradiance (Sullivan et al. 1996), thus, UV absorbing compounds seem to be mainly constitutive. Meta-analysis which generalised an overall response of woody plants under two sup- plemental UV-B levels proved that woody plants show no significant changes in most variables under the low supplemental UV-B level (Li at al. 2010). 45Trošt-Sedej, Rupar: Deciduous and evergreen tree responses to enhanced UV-B treatment … In the dry third season, the content of UV-B and UV-A absorbing compounds was low under both UV-B treatments. Production of UV-B absorb- ing compounds is an energy demanding process (Gaberščik et al. 2002a), which is why low levels of UV absorbing compounds coheres to low Chl and Fv/Fm values. The subtle changes observed in these parameters may have additional causes, such as changes in leaf histology and biochemistry (Hoque and Remus, 1999), both of which vary with the environmental conditions. The results pronounce the complex influences of UV-B and environmental condition on plants. Tolerance to elevated UV-B in trees is, in ad- dition to the content of the high methanol-soluble UV-B absorbing compounds, also increased by the presence of cell wall bound UV-B absorbing compounds in conifers (Fischbach et al. 1999, Hoque and Remus 1999, Rozema et al. 2002, Turtola et al. 2006). Other factors supporting this tolerance include reflectance of UV light (Hoque and Remus 1999), special anatomical arrangements and increased epidermal cell wall thickness of epidermis (Hoque and Remus 1999, Chalker-Scott and Scott 2004), small, thick leaves (P´yankov and Kondrachuk 1998), and large amounts of other secondary compounds, which are part of plants’ defence systems against many stress factors (Turtola et al. 2006). Our results show that both Norway spruce and European beech possess effective protec- tion against the UV-B radiation. This protection depends not only on UV-B dose but generally on the state of development and environmental conditions that influence the efficiency of the UV-B protective systems. Needle/leaf morfology In the three year study period, it was found by comparing samples from ambient and enhanced UV-B treatment at the outdoor experimental plot (Tab. 2) that elevated UV-B radiation exerts no significant influence on the needle/leaf morphology of Norway spruce and European beech. Earlier studies found that leaf size of decidu- ous trees decreases upon exposure to enhanced UV-B radiation (Antonelli at al. 1998, Newsham at al. 1999, Keiller and Holmes, 2001 and Sullivan at al.2003), other studies found that it increases (Sullivan at al.2003) or is not affected (Kostina at al. 2001). In the UV-B exposed conifers, reduced needle area (Laakso et al. 1996, Bassman et al. 2003, Zu et al. 2010) and shorter needles (Naidu et al. 1993, Sullivan et al. 1996) were reported. Compared to our study those experiments used 2–3 times higher suppplemental UV-B doses, which might be the reason for reduced leaf area in some cases. Other studies reported no effect on growth in UV-B exposed conifers (Petropoulou et al. 1995, Lavola et al. 2003). The response of deciduous trees to UV-B radiation is thickening of the leaves, which decreases the penetration of UV-B radiation to the leaf mesophyll (Sullivan et al. 1994, Antonelli et al. 1998, Newsham et al. 1999, Šprtová et al. 2003). The increase in leaf thickness is due to anatomic changes of spongy parenchyma (Kostina et al. 2001) or palisade parenchyma (Nagel at al. 1998) and corresponds to xeromorphic characteristics of plants from harsh environments, adapted to high irradiation, water stress and nutrient-poor soil (Turunen and Latola 2005). There are several studies on cross- tolerance of plants which are exposed to UV-B and other oxidative stresses (Turtola et al. 2006), uch as drought (Petropoulou et al. 1995, Manetas et al. 1997), drought and high light (Poulson et al. 2005), and cold (Mendez et al. 1999, Chalker-Scott 1999). On the other hand, there are again cases which show a decrease in cuticle thickness with increasing altitude (Turunen and Latola 2005). Diversity of results in different studies, includ- ing the current study, indicates the complexity of plant response to UV-B as a function of the whole spectrum of environmental factors and their multilevel interactions and emphasizes the necessity of long-term investigations on trees in natural ecosystems. Conclusions The responses of Norway spruce and European beech to enhanced UV-B radiation varied mod- erately according to the needle/leaf development stage, growth season and above all, environmental conditions. Most of monitored parameters did not respond only to enhanced UV-B solely in any of the studied tree species. Photochemical efficiency was the parameter most responsive 46 Acta Biologica Slovenica, 56 (2), 2013 to enhanced UV-B radiation and reductions of photochemical efficiency were observed in the recently emerged needles/leaves, but not in the later development stages, suggesting a recovery capability. Chlorophyll levels showed no consistent response to enhanced UV-B radiation in any of the studied trees, results suggesting dependence on development phase and environmental condi- tions. Norway spruce as well as European beech manifested an effective filter consisting of UV-B and UV-A absorbing compounds already present in young needles/leaves in May, soon after emergence from buds. The content of the compounds did not change neither with seasonal development nor with enhanced UV-B radiation but was most sensitive to drought. The total amount of methanol-soluble UV-B absorbing compounds in Norway spruce and European beech was as much as three and two times higher, respectively than in tested herbaceous species. During three years’ observation, elevated UV-B radiation exerted no significant influence on the needle/leaf morphology of Norway spruce and European beech. Our results prove that the evergreen Norway spruce and the deciduous Euro- pean beech possess effective protection against the UV-B radiation, which depends specifically on UV-B dose levels but generally on the deve- lopmental state and environmental conditions that influence the efficiency of the UV-B protective systems. This study also indicates the complexity of plant response to UV-B, involving multilevel interactions with environmental factors and thus emphasizes the necessity of long-term investiga- tions on trees in a natural ecosystem. Povzetek Drevesa se na povečano sevanje UV-B odzi- vajo preko različnih struktur in mehanizmov na biokemijskem, fiziološkem in morfološkem nivoju. Učinkovitost zaščite pred povečanim sevanjem UV-B je odvisna od značilnosti rast- linske vrste, razvojne faze rastline, okoljskih razmer ter odmerka UV-B sevanja. V raziskavi smo preučevali odzive na povečano sevanje UV-B pri smreki (Picea abies ( L.) Karst.) in bukvi (Fagus sylvatica L.). Sadike obeh drevesnih vrst smo posadili na prosto za obdobje treh let ter jih izpostavili naravnemu in povečanemu sevanju UV-B. Izbrane parametre, optimalno fotokemično učinkovitost, vsebnost klorofilov, vsebnost UV-A in UV-B absorbirajočih snovi, smo spremljali trikrat letno na listih oziroma treh starostnih razredih iglic. Morfološke analize smo izvedli po treh letih obsevanja. Tako pri smreki kot pri bukvi smo izmerili veliko variabilnost v vsebnostih UV-B absorbirajočih snovi, fotosinteznih barvil in fotokemični učinkovitosti. Povečano sevanje UV-B je sprožilo posamezne odzive pri obeh dre- vesnih vrstah. Učinek povečanega sevanja UV-B se je spreminjal z razvojno fazo iglice in lista ter z okoljskimi razmerami. Zmanjšan negativni učinek sevanja UV-B na fotokemično učinkovitost smreke smo opazili v tretji poskusni sezoni in ga razlagamo kot omilitveni učinek suše. Pri letošnjih iglicah, ne pa tudi pri listih ali starejših iglicah, je bila prisotna tendenca povečane sinteze UV-B absorbirajočih snovi pod povečanim sevanjem UV-B. Rezultati so pokazali veliko strpnost obeh drevesnih vrst do povečanega sevanja UV-B in tudi kompleksen odziv na povečano sevanje UV-B, ki se spreminja tako z razvojno fazo rastline kot z okoljskimi razmerami. Dolgotrajne raziskave v naravnem okolju so zato, za dolgožive vrste kot so drevesa, nujne. References Adams, W.W., Barker, D.H., 1998. Seasonal changes in xanthophyll cycle-dependent energy dissipation in Yucca glauca. Nuttall. Plant Cell Environ., 21, 501–511. Antonelli, F., Bussotti, F., Grifoni, D., Grossoni, P., Mori, B., Tani, C., Zipoli, G., 1998. Oak (Quercus robur L.) seedling responses to a realistic increase in UV-B radiation under open space conditions. Chemosphere, 36, 4–5, 841–845. Bassman, J.H., Edwards, G.E., Robberecht, R., 2002. Long-term exposure to enhanced UV-B radiation is not detrimental to growth and photosynthesis in Douglas-fir. New Phytol., 154, 107–120. 47Trošt-Sedej, Rupar: Deciduous and evergreen tree responses to enhanced UV-B treatment … Bassman, J.H., Edwards, G.E., Robberecht, R., 2003. Photosynthesis and growth in seedlings of five forest tree species with contrasting leaf anatomy subjected to supplemental UV-B radiation. Forest. Sci., 49, 176–187. Bavcon, J., Gaberščik, A., Batič, F., 1996. Influence of UV-B radiation on photosynthetic activity and chlorophyll fluorescence kinetics in Norway spruce (Picea abies (L.) Karst.) seedlings. Trees, 10, 172–176. Björkman, O., Demmig-Adams, B., 1994. Regulation of photosynthetic light energy capture, conver- sion, and dissipation in leaves of higher plants. In: Sulze E.D., Caldwell M.M., (eds.): Ecophy- siology of Photosynthesis. Springer Verlag, Berlin, 17–48. Björn, L.O., Callaghan, T.V., Johnsen, I., Lee, J.A., Manetas, Y., Paul, N.D., Sonesson, M., Wellburn, A.R., Coops, D., Heide-Jorgensen, H.S., Gehrke, C., Gwynn-Jones, D., Johanson, U., Kyparissis, A., Levizou, E., Nikolopoulos, D., Petropoulou, Y., Stephanou, M., 1997. The effects of UV-B radiation on European heathland species. Plant Ecol., 128, 252–264. Björn, L.O., Murphy, T.M., 1985, Computer calculation of solar ultraviolet-radiation at ground-level. Physiol. Plantarum, 64, A23–A23. Björn, L.O., Teramura, A.H., 1993 Simulation of daylight ultraviolet radiation and effects of ozone depletion. In: Young A.R., et al. (Eds.): Environmental UV Photobiology Plenum Press, New York, 41–71. Björn. L.O., Murphy, T.M., 1993. Computer calculation of solar UV radiation at ground level. In: Young A.R., Björn L.O., Moan J., Nultsch W. (eds.): Environmental UV Photobiology, Plenum Press, New York, 63–69. Bornman, J.F., 1989. Target sites of UV-B radiation in photosynthesis of higher plants. J. Photoch. Photobio., 4, 145–158. Breznik, B., Germ, M., Gaberščik, A., Kreft, I., 2005. Combined effects of elevated UV-B radiation and the addition of selenium on common (Fagopyrum esculentum Moench) and tartary (Fagopyrum tataricum (L.) Gaertn.) buckwheat. Photosynthetica, 43, 583–589. Caldwell, M.M., 1968. Solar UV radiation as an ecological factor for alpine plants. Ecol. Monog., 38, 243–268. Caldwell, M.M., Björn, L.O., Bornman. J.F., Flint, S.D., Kulandaivelu, G., Teramura, A.H., Tevini, M., 1998. Effects of increased solar ultraviolet radiation on terrestrial ecosystems. J. Photochem. Photobiol. B: Biol., 46 (1–3), 40–52. Chalker-Scott, L., 1999. Environmental significance of anthocyanins in plant stress responses. Pho- tochem. Photobiol., 77 (1), 1–9. Chalker-Scott, L., Scott, J.D., 2004. Elevated ultraviolet-B radiation induces cross-protection to cold in leaves of Rhododendron under field conditions. Photochem. Photobiol., 79, 199–204. Day, T.A., 1993. Relating UV-B radiation screening effectiveness of foliage to absorbing-compound concentration and anatomical characteristics in a diverse group of plants. Oecologia, 95, 542–550. Day, T.A., Howells, B.W., Ruhland, C.T., 1996. Changes in growth and pigment concentrations with leaf age in pea under modulated UV-B radiation field treatments. Plant Cell Environ., 19, 101–108. Day, T.A., Vogelmann, T.C., DeLucia, E.H., 1992. Are some plant life forms more effective than others in screening out ultra violet-B radiation? Oecologia Heidelberg, 92, 513–519. DeLucia, E.H., Day, T.A., Vogelman, T.C., 1992. UV-B and visible light penetration into needles of two species of subalpine conifers during foliar development. Plant Cell Environ., 15, 921–929. Fischbach, R.J., Kossmann, B., Panten, H., Steinbrecher, R., Heller, W., Seidlitz, H.K., Sandermann, H., Hertkorn, N., Schnitzler, J.P., 1999. Seasonal accumulation of ultraviolet-B screening pigments in needles of Norway spruce (Picea abies (L.)Karst). Plant Cell Environ., 22, 27–37. Gaberščik, A., Germ, M., Škof, A., Drmaž, D., Trošt-Sedej, T., 2002a. UV-B radiation screen and respiratory potential in two aquatic primary producers : Scenedesmus quadricauda and Cerato- phyllum demersum. Verh. Int. Ver. Theor. Angew. Limnol., 27, 422–425. 48 Acta Biologica Slovenica, 56 (2), 2013 Gaberščik, A., Novak, M., Trošt-Sedej, T., Mazej, Z., Germ, M., Bjőrn, L.O., 2001. The influence of enhanced UV-B radiation on the spring geophyte Pulmonaria officinalis. Plant Ecol., 154, 51–56. Gaberščik, A., Vončina, M., Trošt-Sedej, T., Germ, M., Bjőrn, L.O., 2002b. Growth and production of buckwheat (Fagopyrum esculentum) treated with reduced, ambient, and enhanced UV-B radiation. J. Photochem. Photobiol. B, 66, 30–36. Germ, M., Mazej, Z., Gaberščik, A., Häder, D.P., 2002. The influence of enhanced UV-B radiation on Batrachium trichophyllum and Potamogeton alpinus – aquatic macrophytes with amphibious character. J. Photochem. Photobiol. B., 66, 37–46. Häder, D.P., Lebert, M., Marangoni, R., Colombetti, G., 1999. ELDONET – European light dosimeter network hardware and software. J. Photochem. Photobiol. B, 52, 51–58. Hoque, E., Remus, G., 1999. Natural UV-screening mechanisms of Norway spruce (Picea abies [L.] Karst.) needles. Photochem. Photobiol. 69, 177–192. Jordan, B.R., 1996. The effects of ultraviolet-B radiation on plants: a molecular perspective. Adv. Bot. Res., 22, 97–162. Julkunen-Tiitto, R., Häggman, H., Aphalo, P.J., Lavola, A., Tegelberg, R., Veteli, T., 2005. Growth and defense in deciduous trees and shrubs under UV-B. Env. Pollution, 137(3), 404–414. Keiller, D.R., Holmes M.G., 2001. Effects of long-term exposure to elevated UV-B radiation on the photosynthetic performance of five broad-leaved tree species. Photosynthesis Research, 67, 229–240 Keski-Saari, S., Pusenius, J., Julkunen-Tiitto, R., 2005. Phenolic compounds in seedlings of Betula pubescens and B. pendula are affected by enhanced UVB radiation and different nitrogen regimes during early leaf ontogeny. Global Change Biol., 11, 1180–1194. Kinnunen, H., Huttunen, S., Laakso, K., 2001. UV-absorbing compounds and waxes of Scots pine needles during a third growing season of supplemental UV-B. Environ. Pollut., 112, 215–220. Kirchgessner, H.D., Reichert, K., Hauff, K., Steinbrecher, R., Schnitzler, J.P., Pfundel, E.E., 2003. Light and temperature, but not UV radiation, affect chlorophylls and carotenoids in Norway spruce needles (Picea abies (L.) Karst.). Plant Cell Environ., 26, 1169–1179. Kostina, E., Wulff, A., Julkunen-Tiitto,R., 2001. Growth, structure, stomatal responses and secondary metabolites of birch seedlings (Betula pendula) under elevated UV-B radiation in the field. Trees, 15, 483–491. Laakso, K., Huttunen, S., 1998. Effects of the ultraviolet-B radiation (UV-B) on conifers: a review. Environ. Pollut., 99, 319–328. Laakso, K., Kinnunen, H., Huttunen, S., 1996. Effects of ultraviolet radiation on the growth of Scots pine and Norway spruce. 5th Meeting of Finnish Plant Scientists, Kuopio, Finland. Kuopio Uni- versity Publications, C45, 58–60. Laakso, K., Sullivan, J.H., Huttunen, S., 2000. The effects of UV-B radiation on epidermal anatomy in loblolly pine (Pinus taeda L.) and Scots pine (Pinus sylvestris L.). Plant Cell Environ., 23, 461–472. Láposi, R., Veresa, S., Lakatosb, G., Oláha, V., Fieldsendc, A., Mészárosa, I., 2009. Responses of leaf traits of European beech (Fagus sylvatica L.) saplings to supplemental UV-B radiation and UV-B exclusion. Agricultiral and Forest Meteorology, 149(5), 745–755. Latola, K., Kinnunen, H., Huttunen, S., 2001. Needle ontogeny of mature Scots pines under enhanced UV-B radiation. Trees Struct. Funct., 15, 346–352. Lavola, A., Aphalo, P.J., Lahti, M., Julkunen-Tiitto, R., 2003. Nutrient availability and the effect of increasing UV-B radiation on secondary plant compounds in Scots pine. Environ. Exp. Bot., 49, 49–60. Lavola, A., Julkunen-Tiitto, R., de la Rosa, T.M., Lehto, T., Aphalo, P.J., 2000. Allocation of carbon to growth and secondary metabolites in birch seedlings under UV-B radiation and CO2 exposure. Physiologia Plantarum, 109, 260–267. 49Trošt-Sedej, Rupar: Deciduous and evergreen tree responses to enhanced UV-B treatment … Lenk, S., Buschmann, C., 2006. Distribution of UV-shielding of the epidermis of sun and shade leaves of the beech (Fagus sylvatica L.) as monitored by multi-colour fluorescence imaging. J. of Plant Physiology, 163(12), 1273–1283. Li, F-R., Peng , S-L., Chen, B-M., Hou, Y-p., 2010. A meta-analysis of the responses of woody and herbaceous plants to elevated ultraviolet-B radiation. Acta Oecologica, 36, 1, 1–9. Lichtenthaler, H.K., 1987. Chlorophylls and carotenoids – pigments of photosynthetic biomembranes. Method. Enzymol., 148, 350–382. Manetas, Y., Petropoulou, Y., Stamatakis, K., Nikolopoulos, D., Levizou, E., Psaras, G., Karabourniotis, G., 1997. Beneficial effects of enhanced UV-B radiation under field conditions: Improvement of needle water relations and survival capacity of Pinus pinea L seedlings during the dry Mediter- ranean summer. Plant. Ecol., 128, 100–108. Mendez, M., Gwynn-Jones, D., Manetas, Y., 1999. Enhanced UV-B radiation under field conditions increases antocyanin and reduces risk of photoinhibition but does not affect growth in the carni- vorous plant Pinguicula vulgaris. New Phytologist, 144, 275–282. Middleton, E.M., Teramura, A.H., 1993. The role of flavonol glycosides and carotenoids in protecting soybean from ultraviolet-B damage. Plant Physiol., 103, 741–752. Mirecki, R.M., Teramura, A.H., 1984. Effects Of Ultraviolet-B Irradiance On Soybean 5. The Depen- dence Of Plant-Sensitivity On The Photosynthetic Photon Flux-Density During And After Leaf Expansion. Plant Physiol., 74, 475–480. Musil, C.F., 1996. Accumulated effect of elevated UV-B radiation over multiple generations of the arid- environmment annual Dimorphotheca sinuata DC (Asteraceae). Plant Cell Environ., 19, 1017–1027. Nagel, L.M., Bassman, J.H., Edwards, G.E., Robberecht, R., Franceshi, V., 1998. Leaf anatomical changes in Populus trichocarpa, Quercus rubra, Pseudotsuga menziesiiand Pinus ponderosa exposed to enhanced ultraviolet-B radiation. Physiologia Plantarum, 104, 385–396. Naidu, S.L., Sullivan, J.H., Teramura, A.H., DeLucia, E.H., 1993. The effects of ultraviolet-B radiation on photosynthesis of different aged needles in field-grown loblolly-pine. Tree Physiol., 12, 151–162. Neitzke, M., Therburg, A., 2003. Seasonal Changes in UV-B Absorption in Beech Leaves (Fagus sylvatica L.) along an Elevation Gradient. Blackwell Verlag, Berlin, Forstw. Cbl., 122, 1–21. 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. Petropoulou, Y., Kyparissis, A., Nikolopoulos, D., Manetas, Y., 1995. Enhanced UV-B radiation alleviates the adverse-effects of summer drought in 2 mediterranean pines under field conditions. Physiol. Plantarum, 94, 37–44. Poulson, M.E., Donahue, R.A., Konvalinka, J., Boeger, T., 2002. Enhanced tolerance of photosynthesis to high-light ad drought stress in Pseudotsuga menziesii seedlings grown in ultraviolet-B radiation. Tree physiology, 22 (12), 829–838. Prado, F.E., Rosa, M., Prado, C., Podazza, G., Interdonato, R., González, J.A., Hilal, M., 2012. UV-B Radiation, Its Effects and Defense Mechanisms in Terrestrial Plants. In: Prasad, M.N.V. (ed.): Environmental Adaptations and Stress Tolerance of Plants in the Era of Climate Change. Springer, New York, 57–83. P'yankov, V.I., Kondrachuk, A.V., 1998. Structure of the photosynthetic apparatus in woody plants from different ecological and altitudinal groups in Eastern Pamir. Russ. J. Plant Physiol., 45, 481–490. Rau, W., Hofmann, H., 1996. Sensitivity to UV-B of plants growing in different altitudes in the Alps. J. Plant physiol., 148, 21–25. Rozema, J., Bjorn, L.O., Bornman, J.F., Gaberscik, A., Hader, D.P., Trošt, T., Germ, M., Klisch, M., Groniger, A., Sinha, R.P., Lebert, M., He, Y.Y., Buffoni-Hall, R., de Bakker, N.V.J., van de Staaij, J., Meijkamp, B.B., 2002. The role of UV-B radiation in aquatic and terrestrial ecosystems – an experimental and functional analysis of the evolution of UV-absorbing compounds. J. Photoch. Photobiol. B, 66, 2–12. 50 Acta Biologica Slovenica, 56 (2), 2013 Ruhland, C.T., Day, T.A., 1996. Changes in UV-B radiation screening effectiveness with leaf age in Rhododendron maximum. Plant Cell Environ., 19, 740–746. Šprtová, M., Špunda, V., Kalina, J., Marek, M.V., 2003. Photosynthetic UV-B Response of Beech (Fagus sylvatica L.) Saplings. Photosynthetica, 41(4), 533–543. Sullivan, H.J., 2005. Possible impact of changes in UV-B radiation on North American tress and forests. Environ. Pollut., 137, 380–389. Sullivan, J.H., 1997. Effects of increasing UV-B radiation and atmospheric CO2 on photosynthesis and growth: implications for terrestrial ecosystems. Plant Ecology, 128, 195–206. Sullivan, J.H., Gitz, D.C., Peek, M.S., McElrone, A.J., 2003. Response of three eastern tree species to supplemental UV-B radiation: leaf chemistry and gas exchange. Agr. and Forest. Meteor., 120, 1–4. Sullivan, J.H., Howells, B.W., Ruhland, C.T., Day, T.A., 1996. Changes in leaf expansion and epidermal screening effectiveness in Liquidambar syraciflua and Pinus taeda in response to UV-B radiation. Physiol. Plantarum, 98, 349–357. Sullivan, J.H., Teramura, A.H., 1992. The effects of UV-B radiation on loblolly pine. 2. Growth of field-grown seedlings. Tree-Sruct. Funct., 6, 115–120. Sullivan, J.H., Teramura, A.H., Dillenburg, L.R., 1994. Growth and photosynthetic responses of field-grown sweetgum (Liquidambar styraciflua: Hamamelidaceae) seedlings to UV-B radiation. Am. J. Bot., 81, 826–832. Šprtová, M., Špunda, V., Kalina, J., Marek M.V., 2003. Photosynthetic UV-B Response of Beech (Fagus sylvatica L.) Saplings. Photosynthetica, 41, 4, 533–543. Teramura, A.H., Sullivan, J.H., 1994. Effects of UV-B radiation on photosynthesis and growth of terrestrial plants. Photosynth. Res., 39, 463–473. Trošt, T., Gaberščik, A., 2001. The effect of enhanced UV-B radiation on Norway spruce (Picea abies (L.) Karst.) needles of two different age classes. Acta Biologica Slovenica, Ljubljana, 44, 13–25. Trošt, T., Gaberščik, A., 2008. The effects of enhanced UV-B radiation on physiological activity and growth of Norway spruce planted outdoors over 5 years. Trees, 2008, 22, 4, 423–435 Turtola, S., Sallas, L., Holopainen, J.K., Jalkunen-Tiitto, R., Kainulainen, P., 2006. Long term expo- sure to enhanced UV-B radiation has no significant effect on growth or secondary compounds of outdoor-grown Scots pine and Norway spruce seedlings. Environ. Expl. Bot., 56, 80–86. Turunen, M., Latola, K., 2005. UV-B radiation and acclimation in timberline plants. Environ. Pollut., 137, (3), 390–403. Warren, J.M., Bassman, J.H., Mattinson, D.S., Fellman, J.K., Edwards, G.E., Robberecht, R., 2002. Alteration of foliar flavonoid chemistry induced by enhanced UV-B radiation in field-grown Pinus ponderosa, Quercus rubra and Pseudotsuga menziesii. Journal of Photochemistry and Photobiology B: Biology, 66, 125–133. Wu, H., Abasova, L., Cheregi, O., Deak, Z., Gao, K., Vass, I., 2011. D1 protein turnover is involved in protection of Photosystem II against UV-B induced damage in the cyanobacterium Arthrospira (Spi- rulina) platensis. Journal of Photochemistry and Photobiology B: Biology, 104 (1–2), 320–325. Zeuthen, J., Mikkelsen, T.N., Paludan-Müller, G., Ro-Poulsen, H., 1997. Effects of increased UV-B radiation and elevated levels of tropospheric ozone on physiological processes in European beech (Fagus sylvatica L.). Physiol. Plant., 100, 281–290. Zu, Y-G., Pang, H-H., Yu, J-H., Li, D-W., Wei, X-X., Gao, Y-X., Tong H., 2010. Responses in the mor- phology, physiology and biochemistry of Taxus chinensis var. mairei grown under supplementary UV-B radiation. Journal of Photochemistry and Photobiology B: Biology, 98 (2), 152–158.