Polar Biol (2011) 34:411–420 DOI 10.1007/s00300-010-0896-7
ORIGINAL PAPER
DiVerent response of two reindeer forage plants to enhanced UV-B radiation: modiWcation of the phenolic composition Françoise Martz · Minna Turunen · Riitta Julkunen-Tiitto · Hanne Suokanerva · Marja-Liisa Sutinen
Received: 17 February 2010 / Revised: 11 August 2010 / Accepted: 22 September 2010 / Published online: 10 October 2010 © Springer-Verlag 2010
Abstract The long-term eVects of enhanced UV-B radiation on the content and composition of leaf phenolics in Epilobium angustifolium L. and Eriophorum russeolum Fries ex Hartman were studied in northern Finland (68°N) using two UV-B enhancement experiments, both simulating UV-BCIE radiation and corresponding to a 20% loss of ozone layer. High proportions of hydrolyzable tannins (69%) and condensed tannins (66%) characterized both Epilobium and Eriophorum leaves, respectively. No UV treatment eVect was detected in the content or composition of Epilobium leaf soluble phenolics, whereas signiWcant UV eVects were detected in Eriophorum leaves in a developmental-speciWc manner. At the end of the growing season, the proportion of total soluble phenolics was higher in leaves exposed to enhanced UV-A and UV-B radiation than in the control leaves, but the phenolic composition was not signiWcantly modiWed. This study introduces a new example on plants’ phenolic response to UV radiation being species-speciWc and detectable only at certain developmental stages. Possible consequences of increased phenolic content
F. Martz · M. Turunen (&) Arctic Centre, University of Lapland, POB 122, 96101 Rovaniemi, Finland e-mail:
[email protected] F. Martz · M.-L. Sutinen Rovaniemi Research Unit, Finnish Forest Research Institute, POB 16, 96301 Rovaniemi, Finland R. Julkunen-Tiitto Natural Product Research Laboratories, Faculty of Biosciences, University of Joensuu, POB 111, 80101 Joensuu, Finland H. Suokanerva Arctic Research Centre, Finnish Meteorological Institute (FMI-ARC), Tähteläntie 62, 99600 Sodankylä, Finland
in forage plants for selection and digestibility by reindeer are, however, not yet known. Keywords Epilobium angustifolium · Eriophorum russeolum · Phenolics · Tannins · UV radiation · Subarctic
Introduction The changing climate and the socio-political environment are continuously aVecting reindeer herding, which is an important source of livelihood and also has a cultural relevance for local and indigenous people in Northern Fennoscandia (Forbes et al. 2006; Tyler et al. 2007; Rees et al. 2008). In this region, reindeer live in several climatic and vegetation zones. Reindeer pasture habitats include boreal coniferous forests, subarctic mountain birch woodlands, tundra, open mountains and fells, peatlands and river banks (Colpaert et al. 2003; Rees et al. 2003; Tømmervik et al. 2004). Reindeer’s (Rangifer tarandus tarandus) access to pasture habitats and the availability of diVerent forage plants aVect their growth rate and survival. The eVects of climate change, increased CO2 levels and UV-B radiation on the availability and the quality of reindeer summer and winter forage plants and plant communities in the pastures of Northern Fennoscandia were recently reviewed by Turunen et al. (2009). Reindeer feed on a wide variety of plants, altogether 200–300 species in Northern Fennoscandia. The availability of diVerent species depends greatly on the season. Epilobium angustifolium L. (willow herb, henceforth referred to as Epilobium) and Eriophorum russeolum Fries ex Hartman (russet cotton grass, henceforth Eriophorum) are both important reindeer forage plants. Epilobium is preferred forage for reindeer in all seasons except winter
123
412
(Warenberg et al. 1997). Reindeer feed on its young shoots rich in ascorbic acid and roots rich in starch. Epilobium is a common perennial herb (Oenotheraceae family) that grows in temperate and northern climates worldwide (McColl 2002). The plant is ubiquitous in northern woodland glades and on moist soils, extending well into Arctic regions. It is a pioneer species, quickly reclaiming disturbed ground such as clear-cut or burnt forest (Bergstedt and Milberg 2001; Widenfalk and Weslien 2009). It appears to be a completely non-toxic plant, i.e. all parts of the plant are edible, and several nutritional as well as nutraceutical uses have been discovered for it (McColl 2002). Epilobium taxa have also been traditionally used in folk medicine, and they have been reported to have antimicrobial, analgesic, anti-inXammatory, antitumor and antiandrogenic properties (e.g. Rauha et al. 2000; Kiss et al. 2004; Shikov et al. 2006; Buzzini et al. 2008). E. russeolum is a monocot, perennial graminoid (Cyperaceae family), which grows mostly in cool, temperate, alpine and Arctic regions of the Northern Hemisphere, and is typical in wet habitats, e.g. Xark fens and tussock tundra with water acidity levels ranging from neutral to acidic. Reindeer feed on rhizomes and recently emerged soft leaves of Eriophorum spp. in the spring, but it is often preferred forage also during late winter. The proportion of Eriophorum, together with other sedges and graminoids in the reindeer’s diet, decreases towards the summer (Warenberg et al. 1997). Eriophorum contains calcium and phosphorus (Warenberg et al. 1997; Mårell et al. 2006), which are particularly important for pregnant hinds and later for their milk production. Published data about the phenolic composition of Eriophorum sp. leaves are scarce. Flavonoids and some speciWc isoXavonoids were isolated from E. scheuchzeri (Maver et al. 2005). Contrary to Eriophorum, the leaf phenolic composition of Epilobium has been more widely studied. Epilobium leaves are rich in Xavonoids, including derivatives of quercetin, kaempferol and myricetin (Slacanin et al. 1991; Ducrey et al. 1995; Hiermann 1995). Quercetin glycosides are the major Xavonols in E. angustifolium, unlike other Epilobium species. The relative amount of Xavonoids was steadily increasing during leaf development (Hiermann 1993). The leaves also contain hydrolyzable tannins (HTs), ellagitannins and gallotannins (Haddock et al. 1982; Shikov et al. 2006), with demonstrated medicinal properties (Ducrey et al. 1997; Kiss et al. 2004). The global climatic forecasts indicate that increasing concentrations of greenhouse gases will warm the troposphere but cool the stratosphere, leading to more severe ozone depletion and increased UV-B radiation (Taalas et al. 2000, 2002). Increased UV-B radiation may increase the proportion of phenolics in reindeer forage plants, which is known to decrease the forage quality, food intake and
123
Polar Biol (2011) 34:411–420
digestibility by ruminants (Arnold 1985; Duncan and Poppi 2008). The aim of this research was to study the long-term (2–5 growing seasons) eVects of enhanced UV-B radiation on the content and composition of phenolics in Epilobium and Eriophorum, two common forage plants in reindeer herding areas of Northern Fennoscandia and North America.
Materials and methods Experimental design Two independent UV-B enhancement experiments were conducted in Tähtelä, Sodankylä, northern Finland (180 m a.s.l., 67° 22⬘ N, 26° 39⬘ E): (1) one in a dry heath forest with E. angustifolium and (2) another one in a Xark fen with E. russeolum. The experimental sites were fenced oV because the surrounding areas have been used as a reindeer pasture by the Oraniemi Reindeer Herding Cooperative. Temperature and precipitation were recorded during 2006–2007, and UV irradiance was measured with a spectrophotometer (Brewer MKII) on the roof of the sounding station at the Arctic Research Centre of the Finnish Meteorological Institute (FMI-ARC) in Tähtelä, Sodankylä (FMI 2006–2007), located approximately 1.5 km from the experimental sites (Fig. 1), as described by Martz et al. (2007). The ambient biologically eVective UV-B (UV-BBE) irradiance was calculated according to Caldwell et al. (1980), and it refers to UVBE calculated with the generalized plant action spectrum (Caldwell 1971). The mean monthly UVBBE values were calculated from mean daily values. UV experiment in dry heath forest The forest site UV-B enhancement experiment with transplanted seedlings of Epilobium was established in 2002, in a dry heath type forest with a nutrient-poor podzol soil. The vegetation of the plots consisted primarily of lichens (Cladonia sp, Cetraria sp.), mosses (Pleurozium schreberi) and conifer needle debris in the ground layer and shrubs (Vaccinium vitis-idaea L., Empetrum nigrum) growing in the Weld layer with Scots pine (Pinus sylvestris L.) as the dominating tree species. Ten seedlings (height ca. 2 cm) of Epilobium were planted at each plot under a lamp frame on 6 June 2006. The seedlings were watered once a day during dry periods, if necessary. The modulated UV-B facility consisted of a 24 lamp arrays (one array of four lamps/plot) organized as follows: seven arrays for UV-B treatment, seven arrays for UV-A control and 10 arrays for ambient control (without any lamp), providing altogether seven replicates of each UV treatment and 10 replicates of ambient control. This will be further explained below. The
Polar Biol (2011) 34:411–420
413
Fig. 1 Mean daily temperature (a), monthly precipitation (b) and mean monthly UV-BBE (c) in Tähtelä, Sodankylä, at an oYcial weather station 1.5 km from the experimental site (FMI 2006–2007). Grey and
black lines and bars represent values from 2006 and 2007, respectively. UV-BBE—values are means § SD (n = 16–31)
experimental periods were 19 May–2 October 2006 and 30 May–18 September 2007.
wooden lamp frames alone. UV radiation was measured with UV-B sensors (one per each UV-B treatment, one for UV-A control, one as ambient reference at peatland and two as ambient reference at forest site) and UV-A sensors (one for UV-B treatment, UV-A control and ambient reference). Hence, there were a total of 13 sensors in the forest site and 15 in the Xark fen site. Electronic controllers (Quictronic HF 2 £ 36/230–240 V DIM) (Lappalainen et al. 2008) were used to control the lamp output. The system maintained the UV-B treatment level at a constant 46% above the ambient level of UV-BCIE (erythemal action spectrum) (McKinlay and DiVey 1987) corresponding to an approximate 20% loss of ozone layer. Each minute, the control software calculated the ratio of the ambient radiation and radiation in UV-B squares and determined the new value for the driving current of the Xuorescence tubes. The recorded UV-B radiation in the control, +UV-A and +UVB plots in the Xark fen and forest sites are presented in Fig. 2. Photosynthetically active radiation (PAR) was measured continuously at the Weld site with NILU-UV (oneminute average). Temperature measurements were made every 15 min with Tiny Talk TK-0040 temperature loggers on each plot. Technical details about the equipment can also be found on the FUVIRC website (2009).
UV experiment in Xark fen The UV-B enhancement experiment with naturally regenerating Eriophorum plants was conducted from 2003 onwards in a mesotrophic Drepanocladus Xark fen (Halssiaapa). The ground layer of the experimental site was dominated by WarnstorWa exannulata and characterized by moss debris, algal mats and a mud bottom. The Weld layer was characterized by sedges (Eriophorum russeolum Fries ex Hartman, Carex limosa L., C. magellanica Lam. subsp. irrigua, Scheuzeria palustris L.), herbs (Menyanthes trifoliata L.) and shrubs (Vaccinium oxycoccus L., Andromeda polifolia L.). In the Xark fen site, the modulated UV facility consisted of a 30 lamp arrays (one array of four lamps/ plot), 10 arrays for UV-B treatment, 10 arrays for UV-A control and 10 arrays for ambient control (without any lamps), providing altogether ten replicates of each treatment. The experimental periods were as follows: 19 June–2 October 2003, 8 June–1 October 2004, 8 June–3 October 2005, 22 May–2 October 2006 and 31 May–19 September 2007. UV treatment
Sampling On both experimental sites, each UV-B treatment and UV-A control lamp array consisted of four Xuorescence UV-B lamps (Philips TL 40 W/12 RS). The UV-B treatment was achieved by encasing the lamp tubes in a cellulose diacetate Wlm (Expopak Oy, Finland), which cuts oV the radiation below 290 nm. Because the treatment resulted in an increase in UV-A, a control with enhanced UV-A was included in the experiment. Lamps for this UV-A control were wrapped in a polyester Wlm (Melinex polyester, KTA– yhtiöt Oy, Finland) that prevents all UV-C and UV-B wavelengths. The ambient control treatment with equal shading, as beneath the UV arrays, was achieved with
Samples of Epilobium angustifolium were collected from the dry heath forest experiment site on 9 August 2006, 29 June 2007, 3 August 2007 and 24 August 2007. Samples of naturally growing Eriophorum shoots were collected from the Xark fen experiment site on 19 July 2006, 29 June 2007, 27 July 2007 and 24 August 2007. Mixed samples were collected from several individual plants of the plots (3-5 leaves of Epilobium and 3-5 shoots of Eriophorum). After sampling, the leaves were frozen in liquid nitrogen in the Weld and stored at ¡80°C until processed.
123
414
Polar Biol (2011) 34:411–420
Fig. 2 : UV-B radiation in the dry heath forest (a) and Xark fen (b) sites in the 2006 and 2007 growing seasons. UV-B radiation was recorded in the ambient control, +UVA and +UVB plots using UV-B sensors (n = 2, 1, 7 and 1, 1, 10 in the dry heath forest and Xark fen sites, respectively)
Solvent extractions Preliminary extraction tests showed that both plant species contained diVerent groups of phenolic compounds and therefore required diVerent extraction procedures. Eriophorum frozen samples were ground into Wne powder in liquid nitrogen and extracted in methanol overnight at 4°C in the dark. After centrifugation, the pellet was extracted once more with 80% acetone for 2 h at 22°C in the dark to improve extraction of condensed tannins (CTs). The supernatant was also collected after centrifugation, and the acetone evaporated under a low nitrogen Xow. The residual extract fraction (1 ml H2O) was combined with the Wrst methanol extract and stored at ¡20°C for a maximum period of one month until analysis. One ml of the methanolic extract (before addition of the acetone extract) was used for measurement of the chlorophyll concentration (Porra et al. 1989). Epilobium frozen leaf samples were ground into Wne powder in liquid nitrogen and extracted in acidic 50% methanol [MeOH:H2O (1:1 v/v) + HCl 0.1% (v/v)] overnight at 4°C in the dark. After centrifugation, the supernatant was stored at ¡20°C for a maximum period of one month until analysis.
123
As frozen material was directly used for extraction, data were expressed on a fresh weight basis. Similar treatments were applied to all plots, and we expected diVerences in water content between plants collected in diVerent plots during the same day and at the same time of the day to be insigniWcant. The water content should thus not aVect the comparison between the plots for a same time point. However, seasonal changes in water content can be signiWcant and could account for apparent seasonal diVerences. Anthocyanin quantiWcation The proportion of monomeric anthocyanins (ACs) in Epilobium extracts was measured using the diVerential pH method using cyanidin-3-O-glucoside (BioChemika, Germany) as a standard, according to Lee et al. (2005). Condensed tannins The relative amount of CTs in Eriophorum extracts was measured using the butanol-HCl method (Porter et al. 1986) with some modiWcations made for the Eriophorum extracts. In brief, 0.2 ml of extract was mixed with 6 ml of BH [1-butanol:HCl (95:5 v/v)] mixture and 0.1 ml 0.04 M
Polar Biol (2011) 34:411–420
NH4Fe(SO4)2. After incubation for 1 h at 80°C, the absorbance was measured at 555 nm. QuantiWcation was made with Eriophorum condensed tannins puriWed as described by Ossipova et al. (2001). About 15 g of frozen leaves collected on July 2007 were extracted in 70% acetone for 2 h in the dark. After centrifugation, the extract was washed with hexane, evaporated under vacuum and the remaining aqueous fraction was lyophilized. The lyophilized tannin fraction was resuspended in 50% aqueous methanol and loaded on a Sephadex LH20 column. Elution of the contaminating compounds (including phenolics other than CTs) was conducted with 50% aqueous methanol and monitored by measuring the absorbance of the eluates at 280 nm and the UV-spectrum (200–400 nm). CTs were then eluted with 70% aqueous acetone. All fractions were also checked by HPLC. The acetone eluate was evaporated under vacuum followed by lyophilization. The dry CTs were resuspended in 50% aqueous methanol and used for a calibration curve using the butanol-HCl method as described earlier. Polyphenolic antioxidants (Folin-Ciocalteu) The total phenolic and other antioxidant content in Eriophorum extracts was measured using the Folin-Ciocalteu (FC) method according to Ainsworth and Gillespie (2007). In a microcentrifuge tube, 100 l of the sample extract was mixed with 200 l 10% (v/v) FC reagent (Merck) before addition of 800 l 700 mM Na2CO3. After 1 h of incubation and centrifugation, the absorbance was measured at 765 nm. Measurements were duplicated. Catechin was used as a standard, and results were expressed as mg catechin equivalent (CAE)/g fresh weight (FW).
415
Statistics SPSS 15.0 (SPSS, Inc., Chicago, IL, USA) for Windows was used for statistical analysis. After testing the data for their normal distribution, a one-way ANOVA was conducted to test variation caused by sampling dates and treatments. The pairwise comparisons between the treatments were made using Tukey’s HSD test (5 and 1% levels).
Results Environmental conditions The daily temperatures, precipitation and UV-BBE in an oYcial weather station close to the experimental sites in Tähtelä, Sodankylä, are presented on Fig. 1. The mean annual temperature was 0.70°C in 2006 and 0.94°C in 2007, which was warmer than the average annual temperature at the same station during 1971–2000 (Drebs et al. 2002). Summer temperatures did not diVer during the 2 years, but 2007 was warmer in March and October but colder in May than 2006 (Fig. 1a). The year 2006 was drier than 2007 with 408 mm precipitation compared to 541 mm in 2007 (FMI 2006–2007) (Fig. 1b). The average annual precipitation in 1971–2000 was 509 mm (Drebs et al. 2002). The mean daily biologically eVective UV-B radiation (UV-BBE) increased from the beginning of March, reached its highest point in June–July (0.11 § 0.02 Wm¡2 day¡1 in 2006 and 0.10 § 0.03 Wm¡2 day¡1 in 2007) and decreased thereafter to its minimum by the end of October (Fig. 1c).
HPLC analysis Seasonal changes in Epilobium leaf composition The plant extracts were analysed with HPLC (Waters) on a Spherisord ODS-2 column (250 mm £ 4.6 mm, 5 m column with a precolumn, Supelco) as described previously (Martz et al. 2009). The samples were eluted from the column using a solvent gradient consisting of Solvent A (0.1% (v/v) H3PO4) and Solvent B (100% methanol) and detected with a UV/visible diode-array detector (Waters PDA 996). The compounds were identiWed using retention times, UVspectra and comparison with authentic standards. The selected compounds were further analysed with HPLC–MS as previously described (Keski-Saari et al. 2005). QuantiWcation was carried out at 270 nm (HTs) and 320 nm (chlorogenic acids (CGAs), Xavonols and Xavones). The following compounds were used as references for quantiWcation: gallic acid for HTs, CGA for all CGA derivatives, rutin for all Xavonol glycosides and apigetrin for all Xavones (all purchased from Sigma-Fluka).
The mean total amount of soluble phenolics in the control leaves of Epilobium over summer 2007 was 22.56 § 4.82 mg/g FW (n = 27). They were measured with HPLC. Although they represent 69.1 § 6.0% of the soluble phenolics in our measurements with 15.72 § 4.29 mg/g, HTs (gallic and ellagic acid derivatives) were underestimated due to the quantiWcation of only major peaks on the chromatograms and the use of gallic acid as the only standard. The second class of compounds present in Epilobium leaves was Xavonol glycosides with 5.99 § 1.44 mg/g (n = 27), with quercetin glycosides as the major compounds (almost 90% of the Xavonols with 5.35 § 1.26 mg/g). Furthermore, CGAs and anthocyanins (ACs) were the minor compounds (0.63 § 0.14 and 0.22 § 0.12 mg/g). The total amount and composition of soluble phenolics did not change during the summer (Fig. 3), as only the AC content
123
416
Polar Biol (2011) 34:411–420
EVect of enhanced UV radiation on Epilobium leaf composition In this study, UV-B radiation was enhanced by 73.9 § 51.8% (measurement over n = 235 days in 2006 and 2007) at the +UV-B plots of the dry heath forest experiment compared to control plots (Fig. 2a). The content or composition of soluble phenolics in leaves of Epilobium seedlings was not signiWcantly aVected by enhanced UV-A or UV-B radiation (Table 1). No trend could be detected; only the seasonal increase in the proportion of AC was detectable. Seasonal changes in Eriophorum leaf composition
Fig. 3 Seasonal changes in the phenolic composition of Epilobium control leaves. Values are means § SD (n = 9, 2007 data). For each compound group, statistically signiWcant diVerences at P < 0.05 between time points are indicated with letters when appropriate. Grey: hydrolysable tannins (HTs), stripped: Xavonols, black: chlorogenic acid derivatives (CGAs), white: anthocyanins (ACs)
increased signiWcantly (from 0.13 § 0.03 mg/g on 29 June 2007 to 0.28 § 0.12 mg/g on 24 August 2007). Data were expressed on a fresh weight basis and, although the plants were watered regularly during dry periods, seasonal changes in water content might hide seasonal changes in phenolic composition.
Table 1 Soluble phenolic composition (mg/g FW) in Epilobium leaves exposed to control conditions and enhanced UV-A and UV-B radiation
Compoundsa
Soluble phenolics HTs
Flavonols
HTs hydrolysable tannins, CGAs chlorogenic acid derivatives, ACs anthocyanins a Expressed in mg/g FW. Values are means § SD (n = 6–9) b CONT controls, +UVA enhanced UV-A radiation, +UVB enhanced UV-B radiation
123
CGAs
ACs
Treatmentb
The mean total amount of soluble phenolics in the control leaves of Eriophorum over summer 2007 was 15.19 § 5.47 mg/g of FW (n = 28). The leaf composition was characterized by a high proportion of CTs, with 10.19 § 4.94 mg/g (65.7 § 10.8%, including catechin). Other compounds detected in Eriophorum leaves were, in decreasing abundance, Xavonols (2.32 § 0.94 mg/g, including three quercetin glycosides), CGA derivatives (1.76 § 0.86 mg/g) and Xavones (0.91 § 0.26 mg/g). Flavones included luteolin, apigenin, and tricetin derivatives (58.4 § 10.0, 23.3 § 5.9 and 18.3 § 5.9%, respectively). The leaf phenolic content was at its highest at the end of July, mainly due to the increase in the contents of CTs and Xavonols (Fig. 4). The Xavone composition varied as well during the summer, with increasing proportions of apigenin derivatives and concomitant decreasing proportions of luteolin derivatives towards the end of August (data not shown). Changes in leaf water content might aVect this
Date of sampling 9 Aug 2006
27 Jun 2007
3 Aug 2007
24 Aug 2007
CONT
24.91 § 3.46
23.93 § 2.28
23.53 § 6.71
20.21 § 3.99
+UVA
17.94 § 8.69
24.72 § 4.60
19.57 § 2.83
19.29 § 3.63
+UVB
27.11 § 6.46
28.33 § 5.62
23.99 § 9.71
19.39 § 4.07
CONT
17.00 § 3.13
17.37 § 2.59
16.46 § 6.04
13.33 § 2.56
+UVA
12.16 § 5.82
18.11 § 3.54
13.32 § 2.73
12.53 § 3.30
+UVB
18.53 § 4.97
20.94 § 4.36
16.43 § 7.10
12.94 § 3.03
CONT
6.66 § 0.69
5.75 § 0.59
6.14 § 0.72
6.08 § 2.39
+UVA
4.79 § 2.54
5.81 § 1.07
5.41 § 0.89
5.79 § 1.08
+UVB
7.23 § 1.28
6.65 § 1.46
6.55 § 2.32
5.60 § 1.62
CONT
0.52 § 0.10
0.68 § 0.15
0.68 § 0.14
0.52 § 0.06
+UVA
0.38 § 0.20
0.58 § 0.09
0.63 § 0.17
0.58 § 0.09
+UVB
0.53 § 0.10
0.63 § 0.13
0.72 § 0.30
0.55 § 0.13
CONT
0.73 § 0.18
0.13 § 0.03
0.26 § 0.12
0.28 § 0.12
+UVA
0.61 § 0.35
0.21 § 0.20
0.21 § 0.09
0.39 § 0.15
+UVB
0.82 § 0.29
0.11 § 0.07
0.29 § 0.21
0.30 § 0.16
Polar Biol (2011) 34:411–420
417
result as data were expressed on a fresh weight basis. However, the hydrotopographic level of the Xark fen did not change even during the driest summers and changes in leaf water content are therefore likely negligible. The antioxidant capacity measured using the FC method showed a regular and signiWcant (P < 0.05) increase during the summer with 9.23 § 2.75, 11.91 § 2.02 and 13.88 § 3.53 mg CAE/g in 29 June, 27 July and 24 August 2007, respectively. EVect of enhanced UV radiation on Eriophorum leaf composition
Fig. 4 Seasonal changes in the phenolic composition of Eriophorum control leaves. Values are means § SD (n = 10, 10 and 8 in June, July and August 2007, respectively). For each compound group, statistically signiWcant diVerences between time points (P < 0.05) are indicated with letters when appropriate (bracketed values referred to the total soluble phenolic content). Grey: condensed tannins (CTs), stripped: Xavonols, black: chlorogenic acid derivatives (CGAs), white: Xavones
Table 2 Soluble phenolic composition (mg/g FW) and antioxidant content (mg CAE/g FW) in Eriophorum leaves exposed to control conditions and enhanced UV-A and UV-B radiation
Compoundsa
Soluble phenolics CTs
CTs condensed tannins, CGAs chlorogenic acid derivatives, FC antioxidants measured with the Folin-Ciocalteu method * Statistically diVerent from the controls at P < 0.05. The corresponding values are indicated in bold a
Expressed in mg/g FW, except FC. Values are means § SD (n = 7–10) b CONT controls, +UVA enhanced UV-A radiation, +UVB enhanced UV-B radiation c mg CAE/g FW
CGAs
Flavonols
Flavones
FCc
Treatmentb
At the +UV-B plots of the Xark fen experiment, the UV-B radiation was enhanced by 28.9 § 17.2% (measurement over n = 201 days in 2006 and 2007) compared to control plots (Fig. 2). Enhanced UV radiation induced changes in the soluble phenolic content of Eriophorum leaves mainly at the end of the growing season (Table 2). Although only few changes were statistically signiWcant at P < 0.05, clear trends were observed. In July 2006, the total content of soluble phenolics was lower under enhanced UV-B radiation compared to control plants and plants grown under enhanced UV-A radiation. A similar eVect was observed for all types of compounds, so that their respective proportions were not aVected. In July 2007, although the same trend was observed for the total phenolic and CT contents,
Date of sampling 19 Jul 2006
29 Jun 2007
27 Jul 2007
24 Aug 2007
CONT
11.05 § 3.03
11.21 § 3.73
18.97 § 6.27
15.43 § 1.90
+UVA
9.72 § 2.96
15.26 § 4.22
13.91 § 5.56
25.86 § 9.49*
+UVB
5.77 § 2.78*
15.40 § 8.10
16.38 § 5.81
28.39 § 12.28*
CONT
6.78 § 1.90
7.55 § 2.52
13.60 § 6.43
9.21 § 2.28
+UVA
6.35 § 1.79
9.76 § 2.12
8.07 § 4.03
18.78 § 9.89*
+UVB
3.67 § 1.39*
9.88 § 6.11
10.24 § 5.34
20.47 § 11.09*
CONT
1.68 § 0.43
1.35 § 0.90
1.73 § 0.62
2.31 § 0.85
+UVA
1.60 § 1.14
2.08 § 1.08
2.19 § 1.19
2.71 § 0.82
+UVB
1.16 § 0.85
2.37 § 1.46
2.70 § 1.04
3.12 § 0.96
CONT
1.76 § 0.62
1.46 § 0.68
2.67 § 0.63
2.97 § 0.77
+UVA
1.38 § 0.77*
2.36 § 1.33
2.69 § 1.11
3.29 § 0.77
+UVB
0.71 § 0.73*
2.19 § 1.30
2.61 § 0.80
3.66 § 1.11
CONT
0.83 § 0.37
0.84 § 0.28
0.97 § 0.28
0.93 § 0.23
+UVA
0.39 § 0.17*
1.06 § 0.50
0.95 § 0.34
1.07 § 0.29
+UVB
0.24 § 0.15*
0.96 § 0.41
0.83 § 0.16
1.14 § 0.31
CONT
–
9.23 § 2.75
11.91 § 2.02
13.88 § 3.53
+UVA
–
11.09 § 4.47
12.57 § 3.34
15.46 § 1.37
+UVB
–
10.92 § 4.23
12.44 § 2.37
16.67 § 2.74
123
418
no signiWcant UV eVects were detected and even opposite results were observed for the minor compounds (CGAs, Xavonols and Xavones). Clear, signiWcant UV eVects (P = 0.05) were, however, measured at the end of the growing season, mainly as a consequence of a high increase in the content of CTs (most abundant compounds) but also some increases in CGAs, Xavonols and Xavones. Although statistically insigniWcant, the higher contents of CGAs were measured under enhanced UV radiation during the whole summer of 2007. The Xavone composition (proportion of apigenin, luteolin and tricetin derivatives) was not aVected (data not shown). In agreement with the seasonal data described earlier, an increase in the content of polyphenolic antioxidants measured using the FC method was observed at the end of the growing season in 2007 (Table 2).
Discussion High proportions of tannins, HTs in Epilobium and CTs in Eriophorum characterized both plant species. Enhancement of UV-B radiation did not aVect the total content or composition of soluble phenolics in Epilobium leaves. The seedlings grew slowly during 2006, and at the end of the growing season 2006, there was no signiWcant diVerences in shoot height (33.63 § 11.60, 28.90 § 13.21 and 28.72 § 10.25 for +UVA, +UVB and control shoots, respectively (n = 7–10)) or leave length (48.30 § 11.58, 42.81 § 8.00 and 49.94 § 11.09 for +UVA, +UVB and control leaves, respectively (n = 7–10)) between the treatments. Epilobium seedlings were grown in a dry heath type forest, which is not their most typical growing site. Although they were watered regularly, the transplanted seedlings might have experienced drought stress in such a soil, especially in the dry summer of 2006. This drought stress could explain the higher content of ACs in summer 2006 than in summer 2007, but could also have induced a natural resistance to enhanced UV radiation (Chalker-Scott 1999; Turtola et al. 2005). An HPLC analysis of phenolics of Eriophorum shoots developed under enhanced UV-B showed a developmentspeciWc response, with signiWcant eVects observed late in the summer. At the end of summer 2007, the content of every group of compound showed the same trend, with higher contents measured in shoots exposed to enhanced UV radiation compared to control plants. In July, an enhancement of UV radiation induced a signiWcant eVect in Eriophorum in 2006 but not in 2007, although similar trends were observed for the total content of phenolics and CTs, the major compounds. The diVerent response
123
Polar Biol (2011) 34:411–420
observed for the minor compounds in July 2006 and July 2007 remains unexplained, although diVerent weather conditions in both years could be a possible explanation. In the same experimental site, a previous study reported by Rinnan et al. (2008) showed a signiWcant increase in total phenolics in Eriophorum roots but not leaves, when analysed at the end of July using the FC method. A UV-B exclusion experiment performed in the same area for 23 years showed a decrease in the total content of soluble phenolics in Eriophorum leaves developed under low UVB radiation, as measured with the Folin-Dennis method at the end of August (Soppela et al. 2006). It has been shown that changes in the level of UV-B radiation can aVect the chemical composition of reindeer summer and winter forage, particularly by increasing the concentration of phenolics, as a defence against enhanced UV-B radiation (Turunen et al. 2009 and ref. therein). However, the many studies available nowadays lead to the general conclusion that the phenolic response is strongly dependent on the plant species, the compound in question and the type of the experiment. As also observed in this study with Eriophorum, the UV-B eVect can be detected only at speciWc developmental stages (Martz et al. 2007, 2009). Field experiments performed in this study with enhanced UV radiation suggest that non-relative plant species, such as Epilobium angustifolium and Eriophorum russeolum, will be also diVerently aVected by the new climatic conditions and enhanced UV-B radiation. However, the two sites, dry heath forest and Xark fen, diVered in that the Wrst site required transplantation while the other was constructed over naturally regenerating vegetation. This diVerence could also account for diVerent UV-responses between the species. The importance of an increased concentration of phenolics (as observed here in Eriophorum) for forage selection and digestibility in reindeer is not known. It is likely that reindeer would avoid plant tissues rich in phenolics despite their high nutritive value. Phenolics are known to deteriorate the quality of forage and decrease the feed intake of ruminants (see references Turunen et al. 2009 p. 823). Reindeer may, however, be able to balance their diet and minimize the harmful eVects of phenolics by favouring mixtures of diVerent plants in the summer when there is a large selection of forage plants and when getting enough highly nutritious nourishment is not a problem. Acknowledgments The authors would like to thank Mauri Heikkinen of the Finnish Forest Research Institute, Rovaniemi Research Unit, and Jouni Unga, Veijo Tiensuu and Irja Ruokojärvi of the Institute’s Kolari Research Unit, and Anna Hyyryläinen, for their skilful technical assistance during our study. We would also like to thank FMI-ARC
Polar Biol (2011) 34:411–420 staV for the maintenance of measurement Welds and providing additional data. This study was enabled by the support of the Arctic Centre of the University of Lapland and the Thule Institute of the University of Oulu.
References Ainsworth EA, Gillespie KM (2007) Estimation of total phenolic content and other oxidation substrates in plant tissues using Folin–Ciocalteu reagent. Nature Protoc 2:875–877. doi:10.1038/nprot. 2007.102 Arnold GW (1985) Regulation of forage intake. In: Hudson RJ, White RG (eds) Bioenergetics of wild herbivores. CRC Press Inc., Boca Raton, Florida, USA, pp 81–101 Bergstedt J, Milberg P (2001) The impact of logging intensity on Weldlayer vegetation in Swedish boreal forests. For Ecol Manag 154:105–115. doi:10.1016/S0378-1127(00)00642-3 Buzzini P, Arapitsas P, Goretti M, Branda E, Turchetti B, Pinelli P, Ieri F, Romani A (2008) Antimicrobial and antiviral activity of hydrolysable tannins. Mini-Rev Med Chem 8:1179–1187 Caldwell MM (1971) Solar UV irradiation and the growth development of higher plants. In: Giese CA (ed) Photophysiology. Academic Press, New York, pp 131–177 Caldwell MM, Robberecht R, Billings WD (1980) A steep latitudinal gradient of solar ultraviolet-B radiation in the Arctic-Alpine life zone. Ecology 61:600–611. doi:10.2307/1937426 Chalker-Scott L (1999) Environmental signiWcance of anthocyanins in plant stress responses. Photochem Photobiol 70:1–9. doi:10.1111/ j.1751-1097.1999.tb01944.x Colpaert A, Kumpula J, Nieminen M (2003) Reindeer pasture biomass assessment using satellite remote sensing. Arctic 56:147–158 Drebs A, Nordlund A, Karlsson P, Helminen J, Rissanen P (2002) Climatological statistics of Finland 1971–2000. Finnish Meteorological Institute, Helsinki Ducrey B, Wolfender JL, Marston A, Hostettmann K (1995) Analysis of Xavonol glycosides of thirteen Epilobium species (Onagraceae) by LC-UV and thermospray LC-MS. Phytochem 38:129–137. doi:10.1016/0031-9422(94)00629-8 Ducrey B, Marston A, Göhring S, Hartmann RW, Hostettmann K (1997) Inhibition of 5-reductase and aromatase by the ellagitannins oenothein A and oenothein B from Epilobium species. Planta Med 63:111–114. doi:10.1055/s-2006-957624 Duncan AJ, Poppi DP (2008) Nutritional ecology of grazing and browsing ruminants. In: Gordon IJ, Prins HHT (eds) The ecology of browsing and grazing. Springer, Berlin, pp 89–116 FMI (2006–2007) Database for temperature, precipitation and UV radiation in Sodankylä, Finland. Finnish Meteorological Institute Forbes B, Bölter M, Müller-Wille L, Hukkinen J, Konstantinov Y (2006) Reindeer management in northernmost Europe. Linking practical and scientiWc knowledge in social-ecological systems. Springer, Berlin FUVIRC (2009) http://fuvirc.oulu.fi/pages/facility.htm Haddock EA, Gupta RK, Al-ShaW SMK, Haslam E, Magnolato D (1982) The metabolism of gallic acid and hexahydroxydiphenic acid in plants. Part 1. Introduction. Naturally occurring galloyl esters. J Chem Soc Perkin Trans 1:2515–2523. doi:10.1039/P19820002515 Hiermann A (1993) Concentrations of myricetin 3-O--D-glucuronide in Epilobium angustifolium during diVerent vegetation stages and evaluation of its antiphlogistic activity. Planta Med S.1:A631 Hiermann A (1995) Die phytochemische Charakterisierung von Epilobium angustifolium L.: und dessen Abgrenzung zu anderes Epilobium-Species mittels DC und HPLC. Sci Pharm 63:135–144 Keski-Saari S, Pusenius J, Julkunen-Tiitto R (2005) Phenolic compounds in seedlings of Betula pubescens and B. pendula are aVected by enhanced UVB radiation and diVerent nitrogen regimens
419 during early ontogeny. Global Change Biol 11:1180–1194. doi:10.1111/j.1365-2486.2005.00964.x Kiss A, Kowalski J, Melzig MF (2004) Compounds from Epilobium angustifolium inhibit the speciWc metallopeptidases ACE, NEP and APN. Planta Med 70:919–923. doi:10.1055/s-2004-832617 Lappalainen NM, Huttunen S, Suokanerva H (2008) Acclimation of a pleurocarpous moss Pleurozium schreberi (Britt.) Mitt to enhanced ultraviolet radiation in situ. Global Change Biol 14:321– 333. doi:10.1111/j.1365-2486.2007.01489.x Lee J, Durst RW, Wrolstad RE (2005) Determination of total monomeric anthocyanin pigment content of fruit juices, beverages, natural colorants, and wines by the pH diVerential method: collaborative study. J AOAC Intern 88:1269–1278 Mårell A, Hofgaard A, Danell K (2006) Nutrient dynamics of reindeer forage species along snowmelt gradients at diVerent ecological scales. Basic Appl Ecol 7:13–30. doi:10.1016/j.baae.2005.04.005 Martz F, Sutinen M-L, Derome K, Wingsle G, Julkunen-Tiitto R, Turunen M (2007) EVects of ultraviolet (UV) exclusion on the seasonal concentration of photosynthetic and UV-screening pigments in Scots pine needles. Global Change Biol 13:252–265. doi:10.1111/j.1365-2486.2006.01275.x Martz F, Turunen M, Julkunen-Tiitto R, Lakkala K, Sutinen M-L (2009) EVect of the temperature and the exclusion of UVB radiation on the phenolics and iridoids in Menyanthes trifoliata L. leaves in the subarctic. Environ Pollut 157:3471–3478. doi:10.1016/ j.envpol.2009.06.022 Maver M, Queiroz EF, Wolfender J-L, Hostettmann K (2005) Flavonoids from the stem of Eriophorum scheuchzeri. J Nat Prod 68:1094–1098. doi:10.1021/np0580107 McColl J (2002) Willowherb (Epilobium angustifolium L.): biology, chemistry, bioactivity and uses. Agro FOOD Ind Hi-Tech 13:18–22 McKinlay AF, DiVey BL (1987) A reference action spectrum for ultraviolet induced erythema in human skin. CIE J 6:17–77 Ossipova S, Ossipov V, Haukioja E, Loponen J, Pihlaja K (2001) Proanthocyanidins of mountain birch leaves: quantiWcation and properties. Phytochem Anal 12:128–133. doi:10.1002/pca.568 Porra RJ, Thompson WA, Kriedemann PE (1989) Determination of accurate extinction coeYcients and simultaneous equations for assaying chlorophylls a and b extracted with four diVerent solvents: veriWcation of the concentration of chlorophyll standards by atomic absorption spectroscopy. Biochimica et Biophysica Acta (BBA) Bioenerg 975:384–394. doi:10.1016/S0005-2728 (89)80347-0 Porter LJ, Hrstich LN, Chan BG (1986) The conversion of procyanidins and prodelphinidins to cyanidin and delphinidin. Phytochem 25:223–230. doi:10.1016/S0031-9422(00)94533-3 Rauha J-P, Remes S, Heinonen M, Hopia A, Kähkönen M, Kujala T, Pihlaja K, Vuorela H, Vuorela P (2000) Antimicrobial eVects of Finnish plant extracts containing Xavonoids and other phenolic compounds. Int J Food Microbiol 56:3–12. doi:10.1016/S01681605(00)00218-X Rees WG, Williams M, Vitebsky P (2003) Mapping land cover change in a reindeer herding area of the Russian Arctic using Landsat TM and ETM + imagery and indigenous knowledge. Remote Sens Environ 85:441–452. doi:10.1016/S0034-4257(03)00037-3 Rees WG, Stammler FM, Danks FS, Vitebsky P (2008) Vulnerability of European reindeer husbandry to global change. Clim Change 87:1573–1580. doi:10.1007/s10584-007-9345-1 Rinnan R, Nerg A-M, Ahtoniemi P, Suokanerva H, Holopainen T, Kyrö E, Bååth E (2008) Plant-mediated eVects of elevated ultraviolet-B radiation on peat microbial communities of a subarctic mire. Global Change Biol 14:925–937. doi:10.1111/j.1365-2486. 2008.01544.x Shikov AN, Poltanov EA, Dorman HJD, Makarov VG, Tikhonov VP, Hiltunen R (2006) Chemical composition and in vitro antioxidant
123
420 evaluation of commercial water-soluble willow herb (Epilobium angustifolium L) extracts. J Agric Food Chem 54:3617–3624. doi:10.1021/jf052606i Slacanin I, Marston A, Hostettmann K, Delabays N, Darbellay C (1991) Isolation and determination of Xavonol glycosides from Epilobium species. J Chrom 557:391–398. doi:10.1016/S00219673(01)87147-8 Soppela P, Turunen M, Forbes BC, Aikio P, Magga H, Sutinen M-L, Lakkala K, Uhlig C (2006) The chemical response of reindeer summer pasture plants in a subarctic peatland to ultraviolet (UV) radiation. In: Forbes BC, Bölter M, Muller-Wille L, Hukkinen J, Müller F, Gunslay N, Konstantinov Y (eds) Reindeer management in northernmost Europe linking practical and scientiWc knowledge in social-ecological systems. Springer, Berlin, pp 199–213 Taalas P, Kaurola J, Kylling A, Shindell D, Sausen R, Dameris M, Grewe V, Herman J, Damski J, Steil B (2000) The impact of greenhouse gases and halogenated species on future solar UV radiation doses. Geophys Res Lett 27:1127–1130. doi:10.1029/ 1999GL010886 Taalas P, Kaurola J, Lindfors A (2002) Long-term ozone and UV estimates. In: Käyhkö J, Talve L (eds) Understanding the global system—the Finnish perspective, Finnish global change programme FIGARE, University of Turku, pp 137–145 Tømmervik H, Johansen B, Tombre I, Thannheiser D, Høgda KA, Gaare E, Wielgolaski FE (2004) Vegetation changes in the Nor-
123
Polar Biol (2011) 34:411–420 dic mountain birch forest: the inXuence of grazing and climate change. Arctic Antarctic Alpine Res 36:323–332. doi:10.1657/ 1523-0430(2004)036[0323:VCITNM]2.0.CO;2 Turtola S, Rousi M, Pusenius J, Yamaji K, Heiska S, Tirkkonen V, Meier B, Julkunen-Tiitto R (2005) Clone-speciWc responses in leaf phenolics of willows exposed to enhanced UVB radiation and drought stress. Global Change Biol 11:1655–1663. doi:10.1111/ j.1365-2486.2005.01013.x Turunen M, Soppela P, Kinnunen H, Sutinen M-L, Martz F (2009) Does climate change inXuence the availability and quality of reindeer forage plants? Polar Biol 32:813–832. doi:10.1007/s00300009-0609-2 Tyler NJC, Turi JM, Sundse MA, Bull KS, Sara MN, Reinert E, Oskal N, Nellemann C, McCarthy JJ, Mathiesen SD, Martello ML, Magga OH, Hovelsrud GK, Hanssen-Bauer I, Eira NI, Eira IMG, Corell RW (2007) Saami reindeer pastoralism under climate change: applying a generalized framework for vulnerability studies to a sub-arctic social–ecological system. Global Environ Change 17:191–206. doi:10.1016/j.gloenvcha.2006.06. 001 Warenberg K, Danell O, Gaare E, Nieminen M (1997) Porolaidunten kasvillisuus. Landbruksforlaget. Nordic Council for Reindeer Research, Tromsø Widenfalk O, Weslien J (2009) Plant species richness in managed boreal forests—eVects of stand succession and thinning. For Ecol Manag 257:1386–1394. doi:10.1016/j.foreco.2008.12.010
