• Betula pendula;
  • flavonoids;
  • phenolic acids;
  • phytochrome;
  • shade-avoidance


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The natural variation in quantity and quality of light modifies plant morphology, growth rate and concentration of biochemicals. The aim of two growth-room experiments was to study the combined effects of red (R) and far-red (FR) light and ultraviolet-B (UV-B) radiation on the concentrations of leaf phenolics and growth and morphology of silver birch (Betula pendula Roth) seedlings. Analysis by high-performance liquid chromatography showed that the leaves exposed to supplemental FR relative to R contained higher concentrations of total chlorogenic acids and a cinnamic acid derivative than the leaves treated with supplemental R relative to FR. In contrast, concentration of a flavonoid, quercetin 3-galactoside, was higher in the R + UV-B leaves than in the FR + UV-B leaves. The UV-B induced production of kaempferols, chlorogenic acids and most quercetins were not modified by the R : FR ratio. Growth measurements showed that the leaf petioles and stems of FR seedlings were clearly longer than those of R seedlings, but leaf area was reduced by UV-B radiation. Results of these experiments show that exposure of silver birch seedlings to supplemental FR compared to R leads to fast elongation growth and accumulation of phenolic acids in the leaves.


far-red light (700–800 nm)


red light (600–700 nm), UV-B, ultraviolet-B radiation (280–315 nm).


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The light environment of a plant is modified by the surrounding vegetation. Plant leaves have high absorptance of light in the blue and red (R, 600–700 nm) regions of the spectrum, high reflectance in the green and far-red (FR, 700–800 nm) regions, and high transmittance in the FR region (Grant 1997). Consequently, plants use FR as the main informational signal of shading and proximity of other plants, and many tree seedlings, for example, commonly grow taller as a response to increased FR (e.g. Warrington et al. 1989; Gilbert et al. 1995; Aphalo & Lehto 1997; Ritchie 1997; de la Rosa, Aphalo & Lehto 1998; de la Rosa, Lehto & Aphalo 1999; Aphalo & Lehto 2001; Gilbert, Jarvis & Smith 2001). FR-induced changes in stem growth rate in de-etiolated plants are considered to be mediated predominantly by phytochrome B, but other phytochromes may be involved as well (reviews by Smith & Whitelam 1997; Ballaré 1999; Morelli & Ruberti 2002). Thus, the use of specific photosensory systems to adjust morphology enables tree seedlings to compete better for the supply of PAR.

The amount of ultraviolet-B radiation (UV-B, 280–315 nm) in forests depends strongly on canopy structure. For example, in closed canopies of deciduous trees, UV-B levels near the forest floor are generally low (Brown, Parker & Posner 1994). However, in contrast to other wavebands, the differences in UV-B irradiance between shaded and sunlit areas are rather small as a result of the high diffuse fraction (Grant 1997). In tree seedlings, long-term increased UV-B radiation causes changes in the growth rate and architecture (e.g. Sullivan & Teramura 1992; Sullivan, Teramura & Dillenburg 1994; Hunt & McNeil 1999; Newsham, Greenslade & McLeod 1999; Tegelberg, Julkunen-Tiitto & Aphalo 2001; Tegelberg et al. 2003). It has been suggested that unidentified UV-B photosensory system(s) specifically modify gene expression, affecting growth and development (review by Jordan 2002). However, the direct damaging effects of UV-B radiation on nucleic acids and proteins (e.g. Caldwell 1993; Davies 1995) and the formation of free radicals and peroxides during UV-B stress (e.g. Jordan 2002) may also explain some of the effects on plants driven by UV-B. In addition, UV-B exposure may indirectly affect the transport and catabolism of auxins, leading to changes in plant growth rate (review by Jansen 2002).

A common chemical response of plants to UV-B radiation is the accumulation of UV-B absorbing phenolic compounds in the leaves. UV-B radiation is known to stimulate the expression of genes encoding phenylalanine ammonialyase (PAL) and chalcone synthase (CHS), which are important regulatory enzymes of the biosynthesis of phenylpropanoids (e.g. Hahlbrock & Griesebach 1979; Chapell & Hahlbrock 1984). Recent findings show that the induction of CHS by UV-B radiation is further regulated by UV-A, blue and R : FR light ratio (Wade et al. 2001; review by Jenkins et al. 2001). It follows that the level of CHS expression is likely to vary according to the natural light environment, as suggested by Wade et al. (2001). Consequently, the concentrations of flavonoids may also vary according to the qualitative changes in incident light, but it remains to be determined whether and how the gene expression up- and downstream of CHS is regulated by light quality.

In boreal forests, silver birch (Betula pendula Roth) often occupies open habitats such as ridges, rocks, drained bogs and deforested areas as a pioneer tree, but regeneration may also take place under Scots pine canopies (Messier & Puttonen 1995 and references therein; Van Hees & Clerkx 2003). Being adapted to sunlight, silver birch shows clear shade-avoidance responses in growth (Aphalo & Lehto 1997; Aphalo & Lehto 2001; Gilbert et al. 2001) and efficient UV-B absorption in the leaves (e.g. Tegelberg et al. 2001). However, at present, there is no available data concerning the effects of R and FR, as spectral signals in canopies, on the concentrations of the different phenolic compounds present in silver birch leaves. Nor is anything known about the interactive effects of R : FR levels and UV-B radiation on silver birch growth and phenolic production.

Consequently, the objective of this study was to determine the effects of supplemental R and FR, with and without background UV-B radiation, on the concentrations of soluble leaf flavonoids, phenolic acids, anthocyanins and condensed tannins. Growth and morphology of the silver birch seedlings were measured in order to check the effectiveness of the irradiation doses and also to find out whether the allocation of carbon between growth and secondary metabolites was affected by the irradiation treatments.


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Seeds of Betula pendula Roth (batch M29-91–0001), collected from a seed orchard of central Finland provenance, were germinated in pots (260 cm3, D16 Deepot; Stuewe and Sons, Inc., Corvallis, OR, USA) in a mixture of unfertilized peat and quartz sand (5 : 1 v/v). Before the irradiation treatments, the seedlings were grown in a growth chamber, which provided an 18-h photoperiod so that after 6 h of dark period half the lamps were switched on and 1 h later the rest of the lamps were switched on; during the last hour of the light period the lamps were switched off in reverse order. The day/night air temperatures were 20/15 °C, relative humidity was 75% and photosynthetic photon irradiance (IPAR., 400–700 nm) at the top of the plants was 250 µmol m−2 s−1. Taking the steps in irradiance into account, the daily integrated photon irradiance was about 15.3 mol m−2 d−1. In the growth chamber, PAR was continuously monitored with a quantum sensor (LI-190SB; Li-Cor Inc, Lincoln, NE, USA).

After 4 weeks, the seedlings were first visually separated into two groups according to their size, then further randomly divided into the supplementary R/FR experiment (growth room 1) or control experiment (growth room 2). The plants belonging to each group were arranged 7 to 10 cm apart on six trays. The trays were randomly assigned to supplementary UV-B treatment or UV-A radiation control (three trays per group). In growth room 1, the seedlings within each tray were assigned at random to either R or FR treatment.

Irradiation treatments were applied for a period of 10 d. The supplemental R or FR was directed to a single leaf with two light-emitting diodes (LEDs; Type QDDH73502 for FR and QDDH66002 for R; Quantum Devices Inc, Barneveld, WI, USA) (Fig. 1). The distance from the LEDs to the leaves was kept constant, at 3 cm, throughout the experiment. At the beginning of the experiments, the exposed leaves were beginning to expand: at the end they were fully expanded. The seedlings in the control experiment (growth room 2) received no R/FR supplementation. The peak of emission of the LEDs was 660 nm (half band width 25 nm, R treatment) and 735 nm (half band width 29 nm, FR treat-ment), and the photon irradiance was 80 and 85 µmol m−2 s−1, respectively. LEDs were switched on and off by the growth room microcontroller, simultaneously with the white light. Estimated R : FR photon ratio (20 nm wide bands centred on 660 and 730 nm) at the treated leaf surface was 3.8 and 0.32 for R and FR treatments. R : FR was 1.2 outside the beam of the LEDs and consequently also on the plants of the control experiment. LEDs are highly efficient in emitting light and consequently heat dissipation was very small. Total power per LED (light + heat) was less than 40 mW, and in the resistor connected in series with each pair of LEDs and located farther away from the leaf than the LEDs it was about 70 mW. These values apply to both R and FR LEDs.


Figure 1. In the front, LEDs emitting R light on a leaf of a silver birch seedling. In the background, LEDs emitting FR.

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The UV-B irradiation was provided by pairs of fluorescent lamps (UVB-313; Q-Panel Co, Cleveland, OH, USA), positioned 40 cm above the plants. Lamps were driven by high frequency ballasts (PC 2 × 32 C001; Tridonic Bauelement GmbH, Dornbirn, Austria). The supplementary UV-B treatments were under cellulose diacetate films (0.115 mm; FilmSales Ltd, London, UK), which removed radiation below 290 nm. The UV-B lamps were on for 9 h daily, and the seedlings under supplementary UV-B treatments received from 7.3 to 8.5 kJ m−2 d−1 of biologically effective UV-B (Caldwell's generalized plant action spectrum, normalized at 300 nm, Green's formulation, equation 4 in Caldwell et al. 1983; UV-BBE), depending on the position of the trays, and age of the lamps and filters. In the UV-A radiation controls paired to the UV-B treatments, the radiation below 313 nm was excluded by polyester film (0.125 mm, FilmSales Ltd). Vertical curtains of polyester also hung between the trays of UV-B supplementation and UV-A control seedlings. The time-integrated irradiance of UV-A for the time when the UV-B lamps were switched on was 81 and 73 kJ m−2 d−1, for UV-B treatments and UV-A radiation controls, respectively. The time-integrated irradiance of UV-A for the time when the UV-B lamps were off, but white light on, was 23 kJ m−2 d−1.

The photoperiod, temperatures and humidity in the growth rooms were kept at the same values as in the growth chamber. During the experiments, IPAR. at plant level varied from 280 to 350 µmol m−2 s−1 depending on the location within the rooms (mostly between blocks). Daily integrated PAR varied from 17.1 to 21.5 mol m−2 d−1, and consequently it was about 45% of that in open places in central Finland in May–July (Finnish Meteorological Institute 1993). Light was provided by a mix of cool white fluorescent tubes (F96T12/CW/VHO 215 W; Sylvania; Osram Sylvania Inc., Toronto, Canada) and 60 W incandescent lamps. R and FR emitted by the LEDs were measured with a spectroradiometer with fibre-optics and remote cosine-corrected collector (Li-1800; Li-Cor Inc). UV-B radiation and PAR spectra were measured with a double monochromator spectroradiometer (SR9910-PC; Macam Photometrics, Livingston, Scotland). PAR was also measured with a cosine-corrected quantum sensor (LI-190SA; Li-Cor Inc).

Mineral nutrients were supplied in water twice a week during the experiments, starting 10–14 d after emergence. The volume of the solution of water-soluble fertilizer (Superex 6; Kekkilä, Eurajoki, Finland) was kept constant (20 mL), but the nutrient concentrations increased every time (15% increase per day) following the target relative growth rate, somewhat lower than the maximum of more than 20% attainable under continuous light and free access to mineral nutrients (Ingestad 1970). The fertilization procedure, based on Ingestad, Hellgren & Lund Ingestad A.B. (1994), is described in more detail in de la Rosa et al. (2001).

Chemical analyses

For the analysis of anthocyanins, three leaf discs (0.25 cm2 each) were excised from the fresh experimental leaves with a cork borer, placed in 1 mL of 3 m HCl : H2O : MeOH (1 : 3 : 16) and stored in the dark at +4 °C for 24 h. The absorbance of the extracts was measured at 530 and 653 nm with a spectrophotometer (Specord 200; Analytic Jena AG, Jena, Germany), and the anthocyanin levels were assessed as A530−0.24 A653 according to Murray & Hackett (1991).

For the analysis of other secondary metabolites, five leaf discs [4–7 mg dry weight (DW)] excised from the irradiated leaves were air-dried at room temperature and homogenized with an Ultra-Turrax homogenizer (T8; Janke and Kunkel, Ika-Labortechnik, Staufen, Germany) for 30 s in 500 µL of methanol [high performance liquid chromatography (HPLC) grade]. After standing on ice for 15 min, the sample was centrifuged (16 000 g for 2 min; Biofuge pico; Heraeus Instruments, Osterode, Germany) and the residue was extracted three more times with 500 µL of methanol. Finally the residue was extracted with 1 mL of diethyl ether and allowed to stand in ice for 15 min before centrifugation. The combined extracts were dried with a vacuum concentrator (Concentrator 5301; Eppendorf-Netheler-Hinz GmbH, Hamburg, Germany). The samples were dissolved in 2 mL of methanol, and two aliquots (500 µL) were separated for HPLC analysis and dried with the vacuum concentrator. For the analysis of phenolics, the dried HPLC samples were dissolved in methanol : water (1 : 1) and an Agilent HPLC system (1100 series; Agilent, Waldbroon, Germany) was used; this consisted of a thermostatically controlled autosampler (G1329A), binary pump (G1312A) and vacuum degasser (G1322A). The chromatograms and UV-Vis spectra were recorded with a diode array detector (G1315A). An Agilent Hypersil ODS column (3 µm, 4.6 mm × 6.0 mm) was used for the chromatographic separation. The HPLC solvents were A (aqueous 1.5% tetrahydrofuran and 0.25%o-phosphoric acid) and B (MeOH); the elution gradient was 0–5 min 0% of B in A, 5–10 min 15% of B in A, 10–20 min 30% of B in A and 20–47 min 50% of B in A. The identification of the compounds was based on their retention time and UV-spectra at 220, 270, 280, 320 and 360 nm. Tentative identification of apigenin diglucoside was performed by HPLC/API-ES mass spectrometry (HP 1100 series LC/MSD; Agilent), which produced the molecular weight 595 (M + 1). The solvent gradient and HPLC/API-ES conditions were as reported by Julkunen-Tiitto & Sorsa (2001). The quantification of phenolics was performed as in Tegelberg et al. (2001). The concentration of condensed tannins was determined from 1 mL of dissolved extract by means of a butanol–HCl test (Hagerman 1995), which was standardized with purified tannin from leaves of Betula nana L.

Growth measurements

Before and after the irradiation period, the height of the seedlings and the maximum length and width of the leaf blades of experimental leaves were measured, and the leaves per seedling were counted. Leaf area was estimated using the equation ‘leaf length (mm) × leaf width (mm) × 0.6’. The factor 0.6 was obtained from a regression using measured areas from about 50 leaves of various sizes from another set of birch seedlings. At harvest, the chlorophyll index was measured with a chlorophyll content meter (CCM-200; Opti-Sciences, Inc., Tyngsboro, MA, USA) from intact experimental leaves. The length of the petioles was also measured. Before dry weight determination, the leaves and stems of the seedlings were dried in a dryer room in which air humidity was 10% and temperature 20–30 °C.


In the R : FR experiment (growth room 1), there were four or five seedlings per tray (56 seedlings in all) and in the control experiment (growth room 2), two or three seedlings per tray (30 seedlings in all). Analysis of variance (anova) was used to test the significance of differences between the treatments. The data from the R : FR experiments were analysed as a split-plot, with the UV-treatments on the main plots (= trays) and R : FR treatments on the subplots (= LEDs/seedlings). The data from the control experiment were analysed following a design with subsampling (trays = main units, seedlings = subsamples). The six blocks (block = one pair of trays located side by side) were included as a random grouping factor. Linear mixed effects models were fitted by restricted maximum likelihood (REML) by means of function lme of the package nlme (Pinheiro & Bates 2000) version 3.1 under the R system (Ihaka & Gentleman 1996) versions 1.6.0 and 1.6.1. Diagnosis plots were used to assess the conformance to the normality and homoscedasticity assumptions. A logarithmic transformation was used in some cases. Chemical analysis data from one seedling from the control experiment were excluded because the phenolic profile resembled that typical for leaves of B. pubescens.


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The most common soluble leaf phenolics in both experiments and in all treatments were the flavonoids (40–55% of the total phenolic pool analysed); condensed tannins and phenolic acids were also present in relatively large amounts (20–30 and 15–20%, respectively). The levels of anthocyanins were low (less than 1%) (Tables 1 and 2).

Table 1.  Mean levels of anthocyanins (corrected A530nm ± SE) and mean concentrations (mg g−1 DW ± SE) of other soluble phenolics in leaves of silver birch seedlings; and the results of anova for the R : FR experiment (growth room 1)
PhenolicsMeans ± SER : FRUVBR : FR × UVB
  1. Gal, galactoside; glu, glucoside; rha, rhamnoside; cou, coumaryl; der, derivative; DHPPG, 3,4′-dihydroxypropiophenone 3-glucoside; ac, acid. n = 56. SE is calculated from all observations in each treatment, including variation between blocks.

Anthocyanins0.016 ± 0.0010.014 ± 0.0010.016 ± 0.0010.016 ± 0.0011.590.2140.180.6880.700.407
Condensed tannins11.9 ± 1.2513.2 ± 1.7211.0 ± 1.3511.9 ± 1.830.210.6491.030.3570.020.881
Myricetin 3-gal0.23 ± 0.040.23 ± 0.050.39 ± 0.090.29 ± 0.061.860.1801.050.3520.940.337
Myricetin 3-glu0.15 ± 0.020.13 ± 0.030.22 ± 0.030.21 ± 0.040.410.5275.660.0630.030.527
Myricetin 3-rha10.5 ± 0.929.00 ± 1.1210.5 ± 1.349.44 ± 1.411.630.2080.010.9110.040.837
Quercetin 3-gal0.08 ± 0.030.12 ± 0.020.47 ± 0.080.35 ± 0.041.680.20214.10.0137.290.0099
Quercetin 3-rha6.51 ± 0.376.10 ± 0.3816.1 ± 0.6916.8 ± 0.880.260.801249.3<0.00010.100.750
Kaempferol 3-rha1.09 ± 0.111.00 ± 0.102.20 ± 0.192.33 ±
Apigenin diglu0.24 ± 0.030.24 ± 0.060.27 ± 0.050.25 ±
Flavone aglycone der 10.24 ± 0.020.25 ± 0.060.21 ± 0.040.28 ± 0.030.740.3960.020.8821.230.274
Flavone aglycone der 20.24 ± 0.040.23 ± 0.030.21 ± 0.020.17 ± 0.040.660.4232.240.1950.160.694
DHPPG1.65 ± 0.181.67 ± 0.161.60 ± 0.201.50 ±
Neochlorogenic ac0.24 ± 0.030.22 ± 0.020.20 ± 0.010.20 ±
Chlorogenic ac1.59 ± 0.161.69 ± 0.202.53 ± 0.243.27 ± 0.303.280.07833.20.0022.240.142
5-couquinic ac2.99 ± 0.253.20 ± 0.323.17 ± 0.273.26 ±
OH-cinnamic ac der10.16 ± 0.010.16 ± 0.010.15 ± 0.010.14 ±
OH-cinnamic ac der20.07 ± 0.020.06 ± 0.020.10 ± 0.020.13 ± 0.010.500.4851.970.2194.550.0389
OH-cinnamic ac der30.79 ± 0.060.82 ± 0.070.99 ± 0.061.00 ±
Table 2.  Mean levels of anthocyanins (corrected A530nm ± SE) and mean concentrations (mg g−1 DW ± SE) of other soluble phenolics in leaves of silver birch seedlings, and the results of anova for control experiment (growth room 2)
PhenolicsMeans ± SE
  1. Abbreviations as in Table 1. n = 30.

Anthocyanins0.026 ± 0.0030.032 ± 0.0051.500.2752
Condensed tannins16.9 ± 2.9920.3 ± 2.421.250.3144
Myricetin 3-gal0.31 ± 0.070.54 ± 0.132.930.1476
Myricetin 3-glu0.19 ± 0.020.34 ±
Myricetin 3-rha9.43 ± 0.9611.1 ± 1.200.530.5000
Quercetin 3-gal0.15 ± 0.040.60 ± 0.1013.990.0134
Quercetin 3-rha6.38 ± 0.4813.9 ± 1.1187.470.0004
Kaempferol 3-rha1.03 ± 0.111.93 ± 0.1617.840.0083
Apigenin diglu0.33 ± 0.060.32 ±
Flavone aglycone der 10.21 ± 0.040.21 ±
Flavone aglycone der 20.29 ± 0.050.20 ± 0.034.830.0793
DHPPG1.85 ± 0.261.67 ± 0.200.360.5725
Neochlorogenic ac0.24 ± 0.020.20 ±
Chlorogenic ac2.06 ± 0.173.32 ± 0.4410.30.0236
5-couquinic ac2.91 ± 0.323.19 ± 0.420.220.6564
OH-cinnamic ac der10.16 ± 0.010.17 ±
OH-cinnamic ac der20.06 ± 0.010.19 ± 0.111.650.2558
OH-cinnamic ac der30.76 ± 0.050.84 ± 0.090.520.5014

In growth room 1, the total concentration of chlorogenic acids was affected by R : FR (P = 0.0417, Fig. 3a) and there was a weak evidence (P = 0.0868) indicating interactive effects of UV-B and R : FR on the concentration. The concentration of hydroxycinnamic acid derivative 2 was found to be higher in the FR + UV-B leaves than in the R + UV-B leaves (Table 1). Of the flavonoids, quercetin 3-galactoside was increased by the UV-B radiation in both FR leaves (P = 0.0132) and R leaves (P = 0.0148), but according to the statistics (Table 1), R : FR affected quercetin 3-galactoside only under UV-B radiation (P = 0.0179); without UV-B, R : FR had a non-significant effect (P = 0.2612). The results showed that the mean concentration of quercetin 3-galactoside was higher in the R + UV-B leaves than in the FR + UV-B leaves (Table 1). There was also weak evidence indicating higher total concentration of myricetins in the R leaves in comparison with the FR leaves (P = 0.0728, Fig. 2a). UV-B radiation clearly increased the concentrations of quercetins, kaempferols and chlorogenic acids (P ≤ 0.0001, P = 0.0003, P = 0.0101, respectively, Figs 2 & 3) irrespective of the R : FR treatment. In addition to quercetin 3-galactoside, quercetin 3-rhamnoside, kaempferol 3-rhamnoside, chlorogenic acid and a hydroxycinnamic acid derivative were significantly increased by UV-B radiation (Table 1). Anthocyanins, condensed tannins, apigenin diglucoside and flavone aglycone derivatives were not affected significantly by the R : FR treatments or UV-B radiation (Table 1, Fig. 3).


Figure 3. Concentrations (mg g−1 DW) of total chlorogenic acids (a) and cinnamic acids (b) in the leaves of silver birch seedlings (details as in Fig. 2).

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Figure 2. Concentrations (mg g−1 DW) of total myricetins (a), quercetins (b) and kaempferols (c) in silver birch leaves treated with R, FR, R + UV-B and FR + UV-B (growth room 1) and in leaves treated with UV-B radiation or not (growth room 2). The error bars indicate SE, calculated from all observations in each treatment, and it includes variation between blocks.

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In the control experiment in growth room 2, UV-B treatment increased the concentrations of total quercetins, kaempferols and chlorogenic acids (P ≤ 0.0001, P = 0.006, P = 0.0118, respectively, Figs 2 & 3) in the leaves. Of the individual compounds, quercetin 3-galactoside, quercetin 3-rhamnoside, kaempferol 3-rhamnoside and chlorogenic acid were increased under UV-B radiation (Table 2).


During the 10 d of irradiation, the FR seedlings grew more in height than R seedlings and the leaf petioles were longer. In addition, the total shoot dry weight of the FR-treated seedlings tended to be greater than that of seedlings treated with R (Table 3). The leaf area, dry weight of the irradiated leaf and stem : shoot dry weight ratio were not affected by R : FR (Table 3), and neither were the number of leaves or chlorophyll index (data not shown). UV-B irradiation decreased the area of the experimental leaves (Table 3). There were no significant interactions between R:FR and UV-B on any of the measured variables.

Table 3.  Effects of R, FR and UV-B irradiation on growth and biomass, and the results of anova for the R : FR experiment (growth room 1). SE is calculated from all observations in each treatment, and it includes variation between blocks
 Means ± SER : FRUV-BR : FR × UV-B
RFRR + UV-BFR + UV-BF1,42PF1,5PF1,42P
 Area (cm2)12.8 ± 0.9112.9 ± 0.6911.1 ± 0.7612.0 ± 0.980.120.7307.300.0431.880.177
 Petiole length (cm)1.34 ± 0.461.51 ± 0.481.26 ± 0.461.44 ± 0.284.920.0300.960.3720.010.937
 DW (mg)53.5 ± 13.652.3 ± 11.348.3 ± 12.950.2 ± 15.02.500.1750.0010.9480.190.666
 Height growth (cm)4.89 ± 0.155.82 ± 0.284.77 ± 0.215.35 ± 0.1320.20.00012.910.1490.990.324
 Total DW (mg)247.9 ± 10.4277.6 ± 12.3255.4 ± 8.47261.6 ±
 Stem : shoot0.25 ± 0.020.25 ± 0.020.27 ± 0.010.27 ±

In the growth room 2, leaf area was not affected by UV-B radiation (Table 4).

Table 4.  Effects of UV-B irradiation on growth and biomass, and the results of anova for the control experiment (growth room 2)
 Means ± SEUV-B
UV-B –UV-B +F1,5P
  1. SE is calculated from all observations in each treatment, and it includes variation between blocks.

 Leaf area (cm2)10.5 ± 0.419.9 ± 0.590.700.442
 Petiole length (cm)1.03 ± 0.290.97 ±
 Leaf DW (mg)51.8 ± 7.1351.0 ±
 Height growth (cm)4.31 ± 0.153.85 ± 0.193.620.116
 Total DW (mg)279.2 ± 9.29249.8 ± 5.814.050.100
 Stem : shoot0.26 ± 0.010.25 ±


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Recently it was established that phytochrome B negatively regulates the induction of CHS by UV-B radiation (Wade et al. 2001; Jenkins et al. 2001). Thus, as suggested by Wade et al. (2001), FR light (phyBPr being the inactive form) may enhance the potential of induction of CHS by UV-B radiation. However, in silver birch leaves, the differences in flavonoid concentrations in FR and FR + UV-B-treated leaves were slight. We also found that the concentration of quercetin 3-galactoside was higher in R + UV-B-treated leaves than in FR + UV-B-treated ones. Our result is in accordance with the result from an earlier study, where a lower concentration of quercetin 3-galactoside was found in the leaves of silver birch seedlings exposed to additional FR from the side (P. J. Aphalo, T. Lehto & R. Julkunen-Tiitto, unpublished). It is thus possible that, in shaded leaves, the phytochrome system reduces the production of this highly UV-B protective flavonoid. On the other hand, our results are in contrast with other earlier studies, in which the synthesis of flavonoids was induced by continuous FR light (Beggs et al. 1987) given after UV-treatment (Duell-Pfaff & Wellmann 1982). However, comparison with the earlier study by Beggs et al. (1987) suggests that the effect of a change in the R : FR ratio in white light, also with background UV-B radiation, is different from the effect of monochromatic FR. In addition, as suggested by Wade et al. (2001), mature leaf tissue, which was also used in our extractions, may be less competent to induce, for example, CHS via phytochrome signalling pathways (FR). Because the light treatments in our experiments were started during the early leaf development, other steps in synthesis or degradation may have been light-regulated during the leaf maturation.

In contrast to flavonoids, the concentrations of simpler phenolics, chlorogenic acids and a cinnamic acid derivative, were higher in the FR + UV-B-treated silver birch leaves than in R + UV-B leaves. In fact, the regulation of phenolic metabolism by R : FR ratio may have been integrated, since only the relative contents of various compounds (flavonoids versus chlorogenic acids) changed during the R : FR treatments, not the size of phenolic pool. In FR-seedlings, the specific features of phenolic metabolism might have been the result of the induced allocation of resources to fast elongation growth. It has been estimated that the synthesis of chlorogenic acids use less glucose than the synthesis of flavonoids (Gershenzon 1994), and recently it was also found that the concentration of flavonol glycosides, and not chlorogenic acids, correlated negatively with the height of silver birch seedlings (Mutikainen et al. 2002). The enhanced concentration of chlorogenic acids may also have served as a phenolic precursor pool under sudden changes in PAR and UV-B light conditions; chlorogenic acids contain a cinnamic-acid unit necessary for flavonoid synthesis.

In an earlier study with cell suspension cultures of Petroselium hortense, UV-B radiation and R light increased the concentrations of certain flavone glycosides interactively (Wellmann 1971). In our study with silver birch leaves, the interactions were slight, and many of the compounds were affected only by UV-B radiation, which may indicate adaptation to high light intensities with high UV-B irradiation. On the whole, our results show that even compounds belonging to the same phenolic class do not respond to light signals in a similar way or magnitude. Thus, in light-demanding species such as silver birch, the wide variety of phenolic compounds in the leaves may be one of the means of acclimation to small-scale spectral changes.

Supplemental FR, directed by two LEDs to a single developing leaf, induced fast stem and petiole elongation growth, indicating high shade avoidance capacity of silver birch seedlings. When compared with deciduous tree species that are later in the succession, the stem growth of silver birch has been found to be particularly sensitive to proximity signals (Aphalo, Ballaré & Scopel 1999; Gilbert et al. 2001). However, although the total dry weight of the seedlings tended to be higher under FR than under R, the partitioning of resources between different above-ground organs was not affected by the treatments (Table 2a). It may be that the intensity of irradiation rather than the specific wavelength ratios controls the resource allocation. Recently, canopies of paper birch (B. papyrifera Marshall) were shown to have strong foraging responses towards high PAR irrespective of the R : FR ratio (Muth & Bazzaz 2002). On the other hand, in a long-term study, shading has been found to decrease the root–shoot ratio of silver birch saplings (Van Hees & Clerkx 2003).

The overall allocation of carbon to secondary metabolism was not affected by the short-term changes in light quality. On the other hand, the integrated changes in phenolics might have influenced the growth habit of the FR seedlings. Studies on the mechanisms that control the shade avoidance responses suggest that the phytochrome system controls the distribution of auxin in different cell layers, leading in shaded plants to reduced cell expansion in the leaves and enhanced cell elongation in the stem (review by Morelli & Ruberti 2002). Flavonoids may regulate the polar transport of auxin (Brown et al. 2001), and chlorogenic acid and its glycosides are able to inhibit peroxidases that degrade indole-3-acetic acid (IAA) (Zenk & Müller 1963). However, in this experiment, only the leaf phenolics were determined and their ability to influence auxin levels in whole plants is not known.

The accumulation of UV-B absorbing phenolic acids, quercetins, and kaempferols in the seedlings exposed to UV-B reflects the ability of silver birch to occupy open habitats. For example, the production of anthocyanins and condensed tannins, which are considered to be less effective in UV-protection (Tegelberg et al. 2001; review by Steyn et al. 2002), was not affected by UV-B radiation. In addition, the constitutive concentrations of anthocyanins were low, possibly because of the relatively low PAR levels (about 300 µmol m−2 s−1), the age of the leaves, and mild temperatures, and therefore their role in UV-B absorption in the leaves might have been small.

In addition to the increase in flavonoids, UV-B radiation led to decreased leaf area, which in turn may decrease the harmful irradiation in inner leaf tissues. This response is common to plants grown indoors, where UV-B radiation is often applied with relatively low levels of UV-A radiation leading to less induced DNA-repair mechanisms. However, also in plants grown outdoors, supplemental UV-B radiation often causes reductions in leaf area (Searles, Flint & Caldwell 2001).

In short, the responses of silver birch seedlings to differences in light quality comprise specific modifications in the phenolic profile of leaves, but also contrasting effects on the growth rate of plant organs. We have shown that the R : FR ratio in white light affects the accumulation of some phenolic compounds in de-etiolated plants treated over a period of 10 d, and that these effects are distinct to those of UV-B radiation.


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This study was funded by the Jenny and Antti Wihuri foundation (Grant 000013), which is gratefully acknowledged.


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  • Aphalo P.J. & Lehto T. (1997) Effects of light on growth and N accumulation in birch seedlings. Tree Physiology 17, 125132.
  • Aphalo P.J. & Lehto T. (2001) Effect of lateral far-red light supplementation on the growth and morphology of birch seedlings and its interaction with mineral nutrition. Trees 15, 297303.
  • Aphalo P.J., Ballaré C.L. & Scopel A.L. (1999) Plant-plant signalling, the shade-avoidance responses and competition. Journal of Experimental Botany 50, 16291634.
  • Ballaré C.L. (1999) Keeping up with the neighbours: phytochrome sensing and other signalling mechanisms. Trends in Plant Science 4, 97102.
  • Beggs C.J., Kuhn K., Böcker R. & Wellmann E. (1987) Phytochrome-induced flavonoid biosynthesis in mustard (Sinapis alba L.) cotyledons. Enzymic control and differential regulation of anthocyanin and quercetin formation. Planta 172, 121126.
  • Brown M.J., Parker G.G. & Posner N.E. (1994) A survey of ultraviolet-B radiation in forests. Journal of Ecology 82, 843854.
  • Brown D.E., Rashotte A.M., Murphy A.S., Normanly J., Tague B.W., Peer W.A., Taiz L. & Muday G.K. (2001) Flavonoids act as negative regulators of auxin transport in vivo in Arabidopsis. Plant Physiology 126, 524535.
  • Caldwell C.R. (1993) Ultraviolet-induced photodegradation of cucumber (Cucumis sativus L.) microsomal and soluble protein tryptophanyl residues in vitro. Plant Physiology 101, 947953.
  • Caldwell M.M., Gold W.G., Harris G. & Ashurst C.W. (1983) A modulated system for solar UV-B (280–320 nm) supplementation studies in the field. Photochemistry and Photobiology 37, 479485.
  • Chapell J. & Hahlbrock K. (1984) Transcription of plant defence genes in response to UV-light or fungal elicitor. Nature 311, 7678.
  • Davies R.J.H. (1995) Ultraviolet radiation damage in DNA. Biochemical Society Transactions 23, 407418
  • Duell-Pfaff N. & Wellmann E. (1982) Involvement of phytochrome and a blue light photoreceptor in UV-B induced flavonoid synthesis in parsley (Petroselium hortense Hoffm.) cell suspension cultures. Planta 136, 213217.
  • Finnish Meteorological Institute (1993) Measurements of solar radiation 1981–90. In Meteorological Yearbook of Finland Vol. 81−90 (Part 4:1), pp. 1132. Finnish Meteorological Institute, Helsinki, Finland.
  • Gershenzon J. (1994) The cost of plant chemical defense against herbivory: a biochemical perspective. In Insect–Plant Interactions, Vol. 5 (ed. E.A.Bernays), pp. 105173. CRC Press, Boca Raton, FL, USA.
  • Gilbert I.R., Jarvis P.G. & Smith H. (2001) Proximity signal and shade avoidance differences between early and late successional trees. Nature 411, 792795.
  • Gilbert I.R., Seavers G.P., Jarvis P.G. & Smith H. (1995) Photomorphogenesis and canopy dynamics. Phytochrome-mediated proximity perception accounts for the growth dynamics of canopies of Populus trichocarpa×deltoides‘Beaupré’. Plant, Cell and Environment 18, 475497.
  • Grant R.H. (1997) Partitioning of biologically active radiation in plant canopies. International Journal of Biometeorology 40, 2640.
  • Hagerman A.E. (1995) Acid butanol assay for proanthocyanidins. In Tannin Analysis (ed A.E. Hagerman) pp. 2425. Department of Chemistry, Miami University, Miami, FL, USA.
  • Hahlbrock K. & Griesebach H. (1979) Enzymic controls in the biosynthesis of lignin and flavonoids. Annual Review of Plant Physiology 30, 105130.
  • Hunt J.E. & McNeil D.L. (1999) The influence of present-day levels of ultraviolet-B radiation on seedlings of two Southern Hemisphere temperate tree species. Plant Ecology 143, 3950.
  • Ihaka R. & Gentleman R. (1996) R: a language for data analysis and graphics. Journal of Computational and Graphical Statistics 5, 299314.
  • Ingestad T. (1970) A definition of optimum nutrient requirements in birch seedlings. I. Physiologia Plantarum 23, 11271138.
  • Ingestad T., Hellgren O. & Lund Ingestad A.B. (1994) Data base for birch plants at steady-state. Sveriges Lantbruksuniversitet Rapporter (Sweden) 75, 130.
  • Jansen M.A.K. (2002) Ultraviolet-B radiation effects on plants: induction of morphogenic responses. Physiologia Plantarum 116, 423429.
  • Jenkins G.I., Long J.C., Wade H.K., Shenton M.R. & Bibikova T.N. (2001) UV and blue light signalling: pathways regulating chalcone synthase gene expression in Arabidopsis. New Phytologist 151, 121131.
  • Jordan B.R. (2002) Molecular response of plant cells to UV-B stress. Functional Plant Biology 29, 909916.
  • Julkunen-Tiitto R. & Sorsa S. (2001) Testing the effects of drying methods on willow flavonoids, tannins, and salicylates. Journal of Chemical Ecology 27, 779789.
  • Messier C. & Puttonen P. (1995) Growth, allocation and morphological response of Betula pubescens and Betula pendula to shade in developing Scots pine stands. Canadian Journal of Forest Research 25, 629637.
  • Morelli G. & Ruberti I. (2002) Light and shade in the photocontrol of Arabidopsis growth. Trends in Plant Science 7, 399404.
  • Murray J.R. & Hackett W.P. (1991) Dihydroflavonol reductase activity in relation to differential anthocyanin accumulation in juvenile and mature phase Hedera helix L. Plant Physiology 97, 343351.
  • Muth C.C. & Bazzaz F.A. (2002) Tree seedling canopy responses to conflicting photosensory cues. Oecologia 132, 197204.
  • Mutikainen P., Walls M., Ovaska J., Keinänen M., Julkunen-Tiitto R. & Vapaavuori E. (2002) Costs of herbivore resistance in clonal saplings of Betula pendula. Oecologia 133, 364371.
  • 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, 881890.
  • Pinheiro J.C. & Bates D.M. (2000) Mixed-Effects Models in S and S-Plus. Springer-Verlag, New York, USA.
  • Ritchie G.A. (1997) Evidence for red far red signalling and photomorphogenic growth response in Douglas-fir (Pseudotsuga menziesii) seedlings. Tree Physiology 17, 161168.
  • De La Rosa T.M., Aphalo P.J. & Lehto T. (1998) Effects of far-red light on the growth, mycorrhizas and mineral nutrition of Scots pine seedlings. Plant Soil 201, 1725.
  • De La Rosa T.M., Lehto T. & Aphalo P.J. (1999) Does far-red light affect Scots pine seedlings grown on a forest soil substrate? Plant Soil 211, 259268
  • De La Rosa T., Julkunen-Tiitto R., Lehto T. & Aphalo P.J. (2001) Secondary metabolites and nutrient concentrations in silver birch seedlings under five levels of daily UV-B exposure and two relative nutrient addition rates. New Phytologist 150, 121131.
  • Searles P.S., Flint S.D. & Caldwell M.M. (2001) A meta-analysis of plant field studies simulating stratospheric ozone depletion. Oecologia 127, 110.
  • Smith H. & Whitelam G.C. (1997) The shade avoidance syndrome: multiple responses mediated by phytochromes. Plant, Cell and Environment 20, 840844.
  • Steyn W.J., Wand S.J.E., Holcroft D.M. & Jacobs G. (2002) Anthocyanins in vegetative tissues: a proposed unified function on photoprotection. New Phytologist 155, 349361.
  • Sullivan J.H. & Teramura A.H. (1992) The effects of ultraviolet-B radiation on loblolly pine. 2. Growth of field-grown seedlings. Trees 6, 115120.
  • 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. American Journal of Botany 81, 826832.
  • Tegelberg R., Julkunen-Tiitto R. & Aphalo P.J. (2001) The effects of long-term elevated UV-B on the growth and phenolics of field-grown silver birch (Betula pendula). Global Change Biology 7, 839848.
  • Tegelberg R., Veteli T., Aphalo P.J. & Julkunen-Tiitto R. (2003) Clonal differences in growth and phenolics of willows exposed to elevated ultraviolet-B radiation. Basic and Applied Ecology 4, 219228.
  • Van Hees A.F.M. & Clerkx A.P.P.M. (2003) Shading and root-shoot relations in saplings of silver birch, pedunculate oak and beech. Forest Ecology and Management 176, 439448.
  • Wade H.K., Bibikova T.N., Valentine W.J. & Jenkins G.I. (2001) Interactions within a network of phytochrome, cryptochrome and UV-B phototransduction pathways regulate chalcone synthase gene expression in Arabidopsis leaf tissue. Plant Journal 25, 675685.
  • Warrington I.J., Rook D.A., Morgan D.C. & Turnbull H.L. (1989) The influence of simulated shadelight and daylight on growth, development and photosynthesis of Pinus radiata, Agathis australis and Dacrydium cupressinum. Plant, Cell and Environment 12, 343356.
  • Wellmann E. (1971) Phytochrome-mediated flavone glycoside synthesis in cell suspension cultures of Petroselium hortense after preirradiation with ultraviolet light. Planta 101, 283286.
  • Zenk M.H.M. & Müller G. (1963) In vivo destruction of exogeneously applied indolyl-3-acetic acid as influenced by naturally occurring acids. Nature 200, 761763.