Temperature is the key to altitudinal variation of phenolics in Arnica montana L. cv. ARBO

Temperature is the key to altitudinal variation of phenolics 1

in Arnica montana L. cv. ARBO 2

3

4

5

Andreas Albert,1 Vipaporn Sareedenchai,2 Werner Heller,3 Harald K. Seidlitz,1 and Christian 6

Zidorn2* 7

8

9

10

1 Abteilung Experimentelle Umweltsimulation, Institut für Bodenökologie, Helmholtz 11

Zentrum München – Deutsches Forschungszentrum für Gesundheit und Umwelt, Ingolstädter 12

Landstraße 1, 85764 Neuherberg, Germany 13 2 Institut für Pharmazie, Abteilung Pharmakognosie, Universität Innsbruck, Innrain 52, Josef-14

Moeller-Haus, 6020 Innsbruck, Austria 15 3 Institut für Biochemische Pflanzenpathologie, Helmholtz Zentrum München – Deutsches 16

Forschungszentrum für Gesundheit und Umwelt, Ingolstädter Landstraße 1, 85764 17

Neuherberg, Germany 18

19

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21

*Correspondence: Institut für Pharmazie, Universität Innsbruck, Innrain 52, A-6020 22

Innsbruck, Austria; E-Mail: [email protected], Tel.: +43 512 5075302; Fax: +43 23

512 5072939. 24

25

2

Abstract 26

27

Plants in alpine habitats are exposed to many environmental stresses, in particular temperature 28

and radiation extremes. Recent field experiments on Arnica montana L. cv. ARBO indicated 29

pronounced altitudinal variation in plant phenolics. Ortho-diphenolics increased with altitude 30

compared to other phenolic compounds, resulting in an increase of antioxidative capacity of 31

the tissues involved. Factors causing these variations were investigated by climate chamber 32

experiments focusing on temperature and UV-B radiation. Plants of Arnica montana L. cv. 33

ARBO were grown in climate chambers under realistic climatic and radiation regimes. Key 34

factors temperature and UV-B radiation were altered between different groups of plants. 35

Subsequently, flowering heads were analyzed by HPLC for their contents of flavonoids and 36

caffeic acid derivatives. Surprisingly, increased UV-B radiation did not trigger any change in 37

phenolic metabolites in Arnica. In contrast, a pronounced increase in the ratio of B-ring ortho-38

diphphenolic (quercetin) compared to B-ring monophenolic (kaempferol) flavonols resulted 39

from a decrease in temperature by 5°C in the applied climate regime. Conclusively, enhanced 40

UV-B radiation measured at higher altitudes is probably not the key factor triggering shifts in 41

the phenolic composition in Arnica grown at higher altitudes but it is rather the temperature 42

which decreases with altitude. 43

44

Keywords: Asteraceae, chemical ecology, UV-B radiation, free radicals, antioxidants. 45

46

3

Introduction 47

Altitudinal variation of secondary metabolite profiles of higher plants is not well investigated 48

so far. Only a few studies deal with this subject and most of them are based on plants growing 49

in the natural habitat. In such studies major contributions of genetic differences between low 50

and high altitude plants to the observed phytochemical variation cannot be excluded. The few 51

field trials using cultivated, genetically uniform plants indicated that there was indeed 52

pronounced altitudinal variation, especially with regard to the content of phenolic compounds. 53

The observed variation encompassed higher relative yields of ortho-diphenolic compared to 54

other phenolic metabolites and higher overall levels of phenolic acids. Additionally, results 55

confirmed a higher antioxidant capacity of extracts derived from high versus low altitude 56

plants (Spitaler et al. 2006, 2008; Ganzera et al. 2008). 57

A broad range of environmental factors changes with the elevation of the natural growing 58

site. These factors include precipitation, mean temperature, soil, wind speed, temperature 59

extremes, duration of snow-cover, length of the vegetation period, and radiation intensities 60

(Körner 1999). UV-B (280–315 nm) irradiation increases under clear sky conditions by 61

approximately 18 % per 1,000 m of altitude (Blumthaler et al. 1997). Enhanced UV-B 62

radiation has most intensely been discussed to affect the plant secondary metabolism (Körner 63

1999) and only recently low temperatures were also linked with an increase of antioxidative 64

secondary metabolites in plants (Bilger et al. 2007). Enhanced biosynthesis of UV-B 65

absorbing and antioxidant phenolic compounds was interpreted as a protective response 66

against damage from excessive UV-B radiation in plants by their shielding properties 67

4

(Markham et al. 1998b; Körner 1999), while their radical scavenging potentials also 68

contributes to their protective activity (Markham et al. 1998a; Spitaler et al. 2006, 2008). 69

The induction of UV-B protective metabolites by enhanced UV-B radiation is well 70

documented (Bornman et al. 1997; Ries et al. 2000; Ibdah et al. 2002). On the other hand, the 71

crucial factors governing the altitudinal variation in the field trials has not been thoroughly 72

investigated so far and is the objective of the present study. Like for some of the previous 73

field studies Arnica montana cv. ARBO was chosen as a model organism, a) because 74

secondary metabolites from this species are well investigated, b) because the conspicuous 75

flowering heads present a defined ontogenetic stage, which is well suited for comparative 76

investigations, and c) because findings acquired for this important medicinal plant might be 77

transferable to other medicinal plants at least from the Asteraceae family. 78

Previous experiments in the field revealed statistically significant increases of the ratio of 79

ortho-dihydroxy flavonoids to other flavonoids with the altitude of the growing site as well as 80

positive correlations of the total content of both caffeic acid derivatives and the radical 81

scavenging activity of extracts with the altitude of the growing site. These correlations were 82

observed in three successive years and they were observed in plants cultivated in the natural 83

soil as well as in plants potted in standardized soil (Spitaler et al. 2006, 2008). For the 84

altitudes simulated in this paper (600 m for the sub-montane and 1400 m for the high-85

montane site, these increases averaged 22.8 % for the ratio of ortho-dihydroxy-flavonoids to 86

other flavonoids and 19.6 % for the total amounts of caffeic acid derivatives (Spitaler et al. 87

2008 and Tables 1-3, partially derived from the data published therein). 88

89

5

Materials and Methods 90

Plant Material 91

Arnica montana cultivar ARBO plants purchased (Saatzucht Steinach/Germany) in 2004 were 92

transferred to light brown quadrangular plastic pots (edge length 20 cm, depth 25 cm) filled 93

with peat and sand (1/1, v/v), and maintained in the shade in the open land at Innsbruck 94

Botanical Gardens. For the actual experiments, the plants were transferred to the CCs (climate 95

chambers) of the Helmholtz Center Munich (Döhring et al. 1996; Thiel et al. 1996) from May 96

2006 to June 2006 and from May 2007 to June 2007. After two day acclimation period 97

without any UV, UV levels were gradually (within three day) enhanced to the levels 98

described below. 99

For each CC and treatment six terminal flowering heads from different plants (one terminal 100

flowering head per plant) were collected separately at the peak of flowering (Douglas et al. 101

2004; Spitaler et al. 2006). Flowering heads were collected at the first day when all the 102

ligulate flowers and at least two rows of tubulate of the flowering head were fully opened. 103

This procedure was chosen to minimize ontogenetic effect which had been observed in an 104

earlier investigation (Douglas et al. 2004). Heads were air-dried in the shade at ambient 105

(25°C) temperature. This procedure was chosen to enable comparison with the results from 106

previous field experiments in which flowering heads were dried in the same way (Spitaler et 107

al. 2006, 2008). Air-dried heads were kept at -20 °C until analysis. 108

Flowering heads were collected on the following dates. 2006 experiment: climate chamber 109

1 ambient UV 20.05., 29.05., 01.06., 06.06. (2 heads), 11.06., climate chamber 1 enhanced 110

UV 02.06. (2 heads), 06.06. (2 heads), 11.06., 14.06., climate chamber 2 ambient UV 06.06. 111

6

(5 heads), 13.06., climate chamber 2 enhanced UV 29.05. (3 heads), 01.06. (2 heads), 06.06.; 112

2007 experiment climate chamber 1 high montane temperature regime no UV 06.06., 16.06. 113

(2 heads), 18.06., 26.06. (2 heads), climate chamber 1 high montane temperature regime 114

ambient UV 31.05. (3 heads), 14.06. (2 heads), 18.06., climate chamber 2 submontane 115

temperature regime no UV 24.05., 29.05. (3 heads), 30.05. (2 heads), climate chamber 2 116

submontane temperature regime ambient UV 24.05. (2 heads), 25.05., 29.05., 30.05. (2 117

heads). 118

119

Climate chambers 120

The plant treatment was done in the sun simulator facilities of the Helmholtz Center Munich. 121

The natural photobiological environment was provided using a combination of four lamp 122

types (metal halide lamps, quartz halogen lamps, blue fluorescent tubes and UV-B fluorescent 123

tubes) to obtain a natural balance of simulated global radiation throughout the ultraviolet to 124

infrared spectrum. The lamp types are arranged in several groups to realise the natural diurnal 125

variations of the solar irradiance by switching appropriate group of lamps on and off. The 126

short-wave cut-off was shaped by selected borosilicate and soda-lime glass filters (Döhring et 127

al. 1996; Thiel et al. 1996). 128

129

Climate chamber parameters 130

CC settings 2006: both CCs day temperature (high montane): 20.0°C; night temperature: 131

7.5°C; same amount of sufficient irrigation (not quantified); and same relative humidity: day 132

40%, night 70%. CCs were separated by UV-B absorbing acrylic glass into a zone exposed to 133

7

a radiation regime equaling midsummer conditions for Innsbruck (N 47°15’, E 11°23’, 580 m 134

a.s.l.) under clear sky [mean daily dose: 58.4 mol m-2 PAR (Photosynthetic Active Radiation, 135

400–700 nm), 658 kJ m-2 d-1 UV-A with a noon maximum of photosynthetic active radiation 136

(PAR 400-700 nm) of 1500 µmol m-2 s-1, and 6.3 kJ m-2 d-1 UV-BBE (biologically effective 137

UV-B dose normalized at 300 nm; Caldwell, 1971)] and a second zone with threefold 138

enhanced UV-BBE (17.8 kJ m-2 d-1). PAR Radiation exposure from 5:00 to 21:00 h and UV 139

radiation from 9:00 to 17:00 h. CC settings 2007: CC-1 day temperature 20.0°C, night 140

temperature 7.5°C (high montane); CC-2 day temperature 25.0°C, night temperature 12.5°C 141

(sub-montane). CCs were delimited by UV absorbing acrylic glass into two subsections one 142

with ambient UV-B and one with no UV-B radiation. CC-1 and CC-2 ambient: 53.0 mol m-2 143

PAR, 635 kJ m-2 d-1 UV-A, and 8.0 kJ m-2 d-1 UV-BBE; UV-minus subsections: no UV-B 144

radiation. Other parameters were as in 2006. Horizontal variations of the applied radiation 145

were less than 15% in the CCs in both experiments. 146

147

Extract preparation 148

Each air-dried flowering head was ground and phenolics were analyzed by HPLC/DAD 149

analogous to the procedure described previously (Spitaler et al. 2006). In detail, after adding 150

1.00 mg of the internal standard compound cynarin as a stock solution in MeOH, (CH3)2CO, 151

and H2O (3/1/1, v/v/v), exactly weight ground flowering heads (weighing approximately 200 152

mg each) were sonicated twice for 30 min with a mixture of MeOH, (CH3)2CO, and H2O 153

(3/1/1, v/v/v) and once for 30 min with a mixture of MeOH and H2O (1/1, v/v) (total 154

extraction volume 25 ml for each cycle). The extracts were filtered, the remaining plant 155

8

material rinsed with 20 ml of a mixture of MeOH, (CH3)2CO, and H2O (3/1/1, v/v/v) and the 156

combined extracts were filled up to 100.0 ml with a mixture of MeOH, (CH3)2CO, and H2O 157

(3/1/1, v/v/v); 30.0 ml of this solution were brought to dryness in vacuo and redissolved in 158

2.00 ml of a mixture of MeOH, (CH3)2CO, and H2O (3/1/1, v/v/v). After filtration, this 159

solution was used for HPLC analysis. Comparative investigations using different extraction 160

media and longer times of sonication, and a larger number of sonication cycles proved that the 161

chosen procedure led to an exhaustive extraction. All quantitative analyses were run in 162

triplicate. 163

164

HPLC analyses 165

HPLC analyses of phenolics were performed as described previously (Spitaler et al. 2006). In 166

detail, a HP-1090 ChemStation equipped with DAD detectors was employed and the 167

following parameters were applied: column, Phenomenex Synergi Hydro-Rp 80A 168

150 x 4.6 mm (4 m material); guard column, Phenomenex Security Guard C18 (ODS, 169

Octadecyl) 4.0 mm x 3.0 mm; mobile phase A, H2O/HCOOH/CH3COOH (99/0.9/0.1, v/v/v); 170

phase B, MeCN/MeOH/HCOOH/CH3COOH (89/10/0.9/0.1, v/v/v/v); flow rate, 1.00 ml/min; 171

injection volume 10 l; detection wavelength 350 nm; oven temperature, ambient; linear 172

gradient, 0 min 5% B, 5 min 15% B, 20 min 16% B, 35 min 18% B, 45 min 19% B, 55 min 173

27.5% B, 60 min 65% B, 65 min 98% B, 70 min stop; post time, 12 min. The amounts of 174

phenolics were estimated by comparing the peak areas obtained for the particular flavonoids 175

F1-F6 and caffeic acid derivatives P1-P9 with the peak area obtained for the internal standard 176

cynarin (CYN). 177

9

178

Compound identification 179

Phenolics were grouped into flavonoids (F) and caffeic acid derivatives (phenolic acids, P) 180

based on their characteristic UV spectra (F: a broad maximum at 350 nm, P: a maximum at 181

330 nm with a shoulder at 295 nm). Peaks assignable to flavonoids (F1-F6) and phenolic 182

acids (P1-P9) were numbered consecutively with increasing HPLC retention times. Peak 183

characterization and compound identification was performed as previously described (Spitaler 184

et al. 2006). Peak identities are also revealed in Table 4. 185

186

Data analysis 187

Analyses of variance (ANOVAs) and Student-Newman-Keuls tests were calculated using 188

Stanton A. Glantz’ Biostatistics 4.04 (McGraw Hill, Columbus, OH, USA) software package. 189

190

Results 191

HPLC quantification of flowering head metabolites of Arnica montana cv. ARBO of a first 192

climate chamber trial focused on the effect of UV-B radiation. The control group was exposed 193

to ambient radiation intensities realistic for the Innsbruck area while the plants grown under 194

enhanced UV obtained a nearly threefold daily UV-B radiation dose. These differences in 195

growing conditions did not reveal any significant UV-B-dependent effect on secondary 196

metabolite profiles (data not shown) although the applied enhanced UV-BBE value exceeded 197

the radiation encountered at subnival altitudes in the Alps. 198

10

A second climate chamber trial focused on effects of two different temperature regimes on 199

the phenolic pattern. The daily temperature regime in one chamber simulated field conditions 200

in July in the upper montane (approx. 1400 m a.s.l.) regions of the Tyrol area and in a second 201

chamber day and night temperatures simulated field conditions in lower montane regions. 202

Each climate chamber was sub-divided into two compartments, one excluding any 203

biologically effective UV-B radiation and another including UV-B radiation equaling 204

moderate ambient radiation conditions observed at montane altitudes. Table 4 displays 205

detailed results from this second trial. Moreover, Fig. 1 displays sample chromatograms from 206

a flowering head of each of the four experimental groups. 207

ANOVA (analysis of variance) from the data summarized in Table 4 for the total amounts 208

of flavonoids indicated no significant differences between any of the analyzed treatments 209

(ambient UV-B vs. no UV-B combined with lower vs. higher temperature). ANOVA in 210

combination with a consecutively performed Student-Newman-Keuls test of the results for the 211

total amounts of caffeic acid derivatives revealed significantly higher amount of caffeic acid 212

derivatives in the flowering heads of UV-B exclusion group of chamber 1 in comparison to all 213

other treatments. Most interestingly, ANOVA of data for the ratios of ortho-dihydroxy 214

flavonoids (Q) to other flavonoids (K) showed a highly significant inter-treatment variation (P 215

= 0.002). A consecutively performed Student-Newman-Keuls test revealed at P < 0.05 that 216

flowering heads from both groups (ambient UV-B and no UV-B) of chamber 1 (lower 217

temperatures) had a significantly higher Q/K ratio than flowering heads from both groups 218

cultivated at a temperature regime of only 5°C higher. Differences in the contribution of each 219

particular compound to the total of its compound class in percent are highlighted in Fig. 2. 220

11

The following of the observed differences were statistically significant (ANOVA and 221

consecutive Student-Newman-Keuls test at P < 0.05 t): Both lower temperature groups 222

contained higher percentages of F1 and lower percentages of F5 and F6 than both higher 223

temperature groups. Both UV-B exclusion groups contained higher percentages of P4 than 224

both groups treated with ambient UV-B. Moreover, both groups treated with ambient UV-B 225

contained higher percentages of P9 than the higher temperature group grown without any 226

UV-B. 227

Conclusively, I) total flavonoid amounts did not show any significant differences between 228

treatments. II) The ratio of B-ring mono- to ortho-diphenolic flavonol derivatives 229

significantly differed between plants grown under the two temperature regimes, while the 230

different UV-B regimes applied at a specific temperature did not affect this parameter. III) A 231

significant increase of caffeic acid derivatives grown under reduced temperatures was only 232

observed for plants grown in the absence of UV-B radiation. 233

234

Discussion 235

Earlier investigations on native (Zidorn and Stuppner 2001; Zidorn et al. 2005; Alonso-236

Amelot et al., 2007) as well as cultivated plants (Spitaler et al. 2006, 2008; Ganzera et al. 237

2008) revealed significant positive correlations with the sea level of the growing site as well 238

as with the amounts of certain phenolic antioxidants. In the light of prevailing theories these 239

altitudinal variations were interpreted as adaptive responses to the elevated level of harmful 240

UV-B radiation present at higher altitudes (Blumthaler et al. 1997). 241

12

Our first climate chamber trial with A. montana gave clear evidence that enhanced UV-B 242

radiation did not account for the altitude-dependent differences in secondary metabolite 243

profiles observed in the natural environment. In contrast, when different temperatures were 244

applied under otherwise identical environmental conditions in the second trial, comparable 245

shifts in secondary metabolite profiles were observed as in plants grown in different altitudes 246

in nature. Obviously decreasing mean temperatures observed with increasing altitudes have to 247

be regarded as the key factor for altitudinal changes in secondary metabolite profiles. It is 248

remarkable that only 5°C reduction in the temperature profile applied to experimental plants 249

in our controlled chamber trial had a pronounced and significant effect on their metabolic 250

profile. In view of this observation, interpretations of many earlier experiments either 251

performed in climate chambers under controlled conditions or in natural environments should 252

be carefully re-evaluated taking this new aspect into account. 253

Detailed comparisons of the magnitudes of the observed changes in the climate chamber 254

experiments described here as compared to changes observed in previous field trials (Spitaler 255

et al. 2008) are given in Tables 1-3. The changes observed for the total amounts of caffeic 256

acid derivatives (if any) are the same as observed in field experiments (Spitaler et al. 2008, 257

Table 3). In contrast, the changes in the ratio of ortho-dihydroxy to other flavonoids (Q/K 258

ratio) found in the climate chamber experiments are about two times as high as observed in 259

field experiments for plants collected in the altitudes simulated here (Spitaler et al. 2008, 260

Table 2). However, this fact might be explained by the phenology of A. montana under field 261

conditions. In the field trials flowering heads from A. montana cv. ARBO were harvested at 262

the lowest altitude (590 m a.m.s.l.) at the end of May or in early June, whereas flowering 263

13

heads from the site closest in altitude to the climate simulated here as high montane (site 5, 264

1430 a.m.s.l.) were on average collected four weeks later. Thus, differences in day and night 265

temperatures during the peak of flowering (and the harvest of flowering heads) were less 266

pronounced than the temperature differences at the different altitudes would imply. Average 267

mean temperatures in Innsbruck for May, June and July are 13.7°C, 16.3°C and 18.3°C, 268

respectively. This increase of 2.0° to 2.6°C per month is about half the expected decrease 269

between altitudes of 600 and 1400 m in the Innsbruck area, which is in the order of 4.8° to 270

5.6°C (8 * 0.6° to 0.7°C per 100 m of altitude) (Ganzera et al. 2008). In conclusion plants 271

growing at the higher altitude sites live in a colder environment than plants in the valley 272

during the peaks of their respective flowering periods but these differences are considerably 273

diminished by the delayed flowering period and in turn the 5°C difference applied in our 274

climate chamber experiment equals more than just a 800 m difference in the altitude of the 275

growing site. 276

Ortho-dihydroxy flavonoids are three- to four-fold better radical scavengers than 277

flavonoids lacking this feature (Rice-Evans et al. 1996) and plants rich in these compounds 278

are therefore better protected against damage by free radicals than plants containing lower 279

amounts of these compounds. The need for the plants in higher altitudes to have additional 280

damage protection against free radicals is not, as assumed previously, mainly driven by the 281

necessity for an improved UV-B protection at higher altitudes (Blumthaler et al. 1997) but 282

stems from the fact that radical scavenging activity of plant phenolics is less effective in low 283

temperature environments than in moderate temperature environments (Bilger et al. 2007). 284

14

Practical implications from the data presented here include a) scientific support for and 285

explanation of the old, and mostly passed on by word-of-mouth, alpine myth that plants 286

collected in higher altitudes have a higher "healing potential" (due to the higher amount of 287

bioactive phenolics) than plants collected in low altitudes and b) hints for the systematic 288

cultivation of medicinal plants and vegetables rich in desirable antioxidative constituents (the 289

higher the altitude and/or the cooler the climate the higher the expected yield of antioxidants). 290

Though some key factors on climatic impact on variation in secondary metabolite profiles 291

are becoming clearer with the presented data and with data from recent studies, many 292

interesting questions remain unsolved, for example: Is there a cumulative climatic effect over 293

time? How long have differing temperature regimes to be maintained before changes in 294

secondary metabolite patterns become detectable? After which period of time do differences 295

in secondary metabolite profiles disappear after identical climatic conditions are restored? 296

297

Acknowledgements 298

CZ thanks the Swarovski foundation for financial support. The authors thank Verena 299

Schneeberger (Innsbruck) for technical assistance. 300

301

15

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16

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Table 1 Regression equations for the total amounts of flavonoids in field experiments for the growing seasons 2003 to 2005

and predicted differences between high montane (1400 m) and low montane (600 m) in comparison to simulated high and low

montane climate regimes

Year/Treatment Para-

meter

a b P predicted value

for 600 m site

predicted value

for 1400 m site

percent change

between 1400

and 600 m

2003 nat. soil F 21.2 - 0.00035 0.743 21.0 20.7 - 1.33 %

2004 nat. soil F 18.3 - 0.00116 0.469 17.6 16.7 - 5.27 %

2005 nat. soil F 16.7 0.00161 0.257 17.7 19.0 + 7.29 %

2005 potted F 18.5 0.00024 0.859 18.6 18.9 + 1.03 %

Average F 18.7

( 1.9)

0.000085

( 0.00117)

0.582

( 0.271)

18.7

( 1.6)

18.8

( 1.6)

+ 0.43 %

( 5.26 %)

2007 phytotron

no UV

F - - - 21.1

( 3.8)

20.1

( 4.7)

- 4.74 %

2007 phytotron

ambient UV

F - - - 21.9

( 4.8)

17.9

( 3.7)

- 18.3 %

19

Table 2 Regression equations for the ration of ortho-dihydroxy (Q) to other flavonoids (K) in field experiments for the growing

seasons 2003 to 2005 and predicted differences between high montane (1400 m) and low montane (600 m) in comparison to

simulated high and low montane climate regimes

Year/Treatment Para-

meter

a b P predicted value

for 600 m site

predicted value

for 1400 m site

percent change

between 1400

and 600 m

2003 nat. soil Q/K 1.03 0.000290 0.001 1.20 1.43 + 19.3 %

2004 nat. soil Q/K 0.721 0.000326 0.000 0.92 1.18 + 28.5 %

2005 nat. soil Q/K 1.05 0.000418 0.000 1.30 1.64 + 25.7 %

2005 potted Q/K 1.21 0.000306 0.016 1.39 1.64 + 17.6 %

Average Q/K 1.00

( 0.20)

0.000335

( 0.000057)

0.004

( 0.008)

1.20

( 0.20)

1.47

( 0.22)

+ 22.8 %

( 5.2 %)

2007 phytotron

no UV

Q/K - - - 1.37

( 0.16)

2.27

( 0.62)

+ 65.7 %

2007 phytotron

ambient UV

Q/K - - - 1.47

( 0.44)

2.13

( 0.36)

+ 44.9 %

20

Table 3 Regression equations for the total amounts of caffeic acid derivatives in field experiments for the growing seasons

2003 to 2005 and predicted differences between high montane (1400 m) and low montane (600 m) in comparison to simulated

high and low montane climate regimes

Year/Treatment Para-

meter

a b P predicted value

for 600 m site

predicted value

for 1400 m site

percent change

between 1400

and 600 m

2003 nat. soil P 28.2 0.00342 0.032 30.3 33.0 + 9.04 %

2004 nat. soil P 24.4 0.00379 0.108 26.7 29.7 + 11.4 %

2005 nat. soil P 21.7 0.00643 0.001 25.6 30.7 + 20.1 %

2005 potted P 16.5 0.0109 0.000 23.0 31.8 + 37.8 %

Average P 22.7

( 4.9)

0.00614

( 0.00345)

0.035

( 0.051)

26.4

( 3.0)

31.3

( 1.4)

+ 19.6 %

( 13.0 %)

2007 phytotron

no UV

P - - - 38.6

( 8.9)

49.0

( 7.8)

+ 26.9 %

2007 phytotron

ambient UV

P - - - 39.5

( 9.1)

35.4

( 5.2)

- 10.4 %

Table 4 Average quantification results for plants grown in climate chambers varying in UV-B

radiation and temperature regimea

Compound common name temp.: high-

montane, no UV-B

temp.: high-

montane, ambient

UV-B

temp.: sub- montane, no UV-B

temp.: sub- montane, ambient

UV-B

x sx x sx x sx x sx

quercetin 3-O--D-glucoside F1 11.71 2.15 10.37 2.90 9.69 1.92 10.52 2.89

patuletin 3-O--D-glucoside F2 1.77 0.30 1.76 0.32 2.48 0.73 2.02 0.21

kaempferol 3-O--D-glucoside F3 2.81 1.22 2.24 0.44 3.35 0.80 3.77 1.93

kaempferol 3-O--D-glucuronide F4 1.95 0.75 1.41 0.42 1.72 0.38 2.15 0.75

6-methoxykaempferol 3-O--D-glucoside

F5

0.72 0.55 0.79 0.36 1.68 0.48 1.41 0.86 hispidulin F6 1.14 0.19 1.36 0.44 2.21 0.30 2.03 0.40 sum of flavonoids F1-F6 ΣF1-F6 20.10 4.67 17.93 3.74 21.13 3.76 21.89 4.76 quotient Q/K =

(F1+F2)/(F3+F4+F5+F6) Q/K 2.27 0.62 2.13 0.36 1.37 0.16 1.47 0.44

chlorogenic acid P1 6.69 1.68 4.38 0.80 4.74 0.76 5.42 1.97 unknown hydroxycinnamate

ester P2

0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 unknown hydroxycinnamate

ester P3

6.38 1.46 5.23 0.84 4.85 1.12 5.83 2.09 3,5-dicaffeoylquinic acid P4 20.24 6.69 10.73 3.07 15.11 2.41 12.71 2.64 1-methoxyoxaloyl-3,5-di-

caffeoylquinic acid P5

8.50 2.39 8.64 1.26 8.22 3.87 8.27 3.35 4,5-dicaffeoylquinic acid P6 2.47 1.07 1.44 0.26 1.85 0.51 1.70 0.60 unknown hydroxycinnamate

ester P7

1.81 0.52 1.83 0.17 1.96 0.59 1.86 0.45 unknown hydroxycinnamate

ester P8

0.79 0.21 0.76 0.37 0.56 0.29 0.67 0.33 unknown hydroxycinnamate

ester P9

2.14 0.69 2.36 0.61 1.29 0.96 3.09 1.45

sum of caffeic acid derivatives P1-P9

ΣP1-P9 49.02 7.84 35.37 5.20 38.58 8.90 39.54 9.20

a Arithmetic means are derived from six samples, which were analyzed three time each.

22

Fig. 1 Sample chromatograms from flowering heads of each of the four treatment groups

investigated in the 2007 phytotron experiment: a) high montane, no UV-B (sample AA1); b)

high-montane, ambient UV-B (sample AB7); c) sub-montane, no UV-B (sample AC4), and d)

sub-montane, ambient UV-B (sample AD2).

Fig. 2 Bars represent the contributions of each compound to the total of its compound class

(flavonoid and caffeoyl derivatives, respectively) in percent. Compound abbreviations are

explained in Table 4. Each diagram represents means and standard deviations of the means of

one of the four treatments in the 2007 phytotron experiment: a) high montane, no UV-B; b)

high-montane, ambient UV-B; c) sub-montane, no UV-B, and d) sub-montane, ambient UV-

B.