Introduction
Biogenic volatile organic compounds (BVOCs) are produced in both marine and terrestrial environments, playing important roles
in both plant survival and the reactive chemistry of the atmosphere (Guenther et al., 1995; Goldstein and Galbally,
2007). Isoprenoids, such as isoprene (a C5 unit), monoterpenes (MTs, consisting of two C5 units) and
sesquiterpenes (SQTs, consisting of three C5 units), contribute with approximately 68 % of the total global BVOC
emissions (Guenther et al., 2012). They are some of the most important BVOC groups due to their high volatility and
involvement in several atmospheric reactions (Atkinson and Arey, 2003; Goldstein and Galbally, 2007; Guenther et al.,
2012). The degradation of BVOCs in the air influences atmospheric processes such as production and destruction of ozone
(Atkinson, 2000; Peñuelas and Staudt, 2010), but it also influences the growth of secondary organic aerosols (SOA)
(Claeys et al., 2004; Ehn et al., 2014). SOA particles are known to scatter incoming solar radiation and to act as cloud
condensation nuclei, which in turn have an effect on the incoming and outgoing radiation (Laothawornkitkul et al., 2009, and
references therein; Paasonen et al., 2013). In general, SOA yields are expected to be higher for compounds with internal
double bonds, such as α-pinene, 3-carene, limonene and terpinolene. However, some acyclic compounds, such as myrcene,
have also been observed to produce high SOA yields (Lee et al., 2006, and references therein).
The production and release of BVOCs are sensitive to physical constraints such as light and temperature (Staudt and Bertin,
1998; Niinemets et al., 2004; Dudareva et al., 2006). Temperature controls the synthesis of isoprenoids and the diffusion
rate of compounds (Niinemets et al., 2004, and references therein). The light availability determines the amount of isoprenoid
precursors produced by photosynthesis and the available amount of ATP and NADPH, which are used in the CO2 fixation
and assimilation reactions that provide new isoprenoids (Niinemets et al., 2004, and references therein; Lichtenthaler,
2007). However, the emission rates can also be affected by physiochemical constraints, such as stomatal conductance
(GS). GS can control VOC emissions temporarily in a non-steady state, when the intercellular volatile partial
pressure is different from the equilibrium pressure (Niinemets and Reichstein, 2003). In a steady state, isoprene and MTs are
insensitive to stomatal closure because of their high gas-phase to liquid-phase partitioning. Compounds with a large Henry's
law constant (H), such as isoprene and MTs, partition to the gas phase, whilst low H compounds partition to the aqueous
phase. When GS decreases, it elevates the gas-phase partial pressure inside the stomata and increases the gradient
between the intercellular air space and atmosphere. This allows the diffusion flux of compounds with a high H to be
maintained independently of stomatal conductance (Niinemets and Reichstein, 2003; Niinemets et al., 2004).
Isoprene is released upon production and therefore shows a strong direct temperature and light dependency (Kesselmeier and
Staudt, 1999; Niinemets et al., 2004). The light dependency of MT emissions has, however, been more debated. In earlier
studies regarding MT emissions, a lack of light response led to the assumption that MTs were only temperature dependent
(Tingey et al., 1980). Emissions of MTs were assumed to originate from internal storage structures in plants, such as resin
ducts, oil glands or glandular hairs and trichomes (Fuentes et al., 1996; Kesselmeier and Staudt, 1999). The evaporation from
these structures is controlled by the vapour pressure of the MTs, which in turn is affected by the air temperature and
concentration of MTs within these structures (Lerdau et al., 1997; Ghirardo et al., 2010; Taipale et al., 2011). However,
more recent studies have suggested that both de novo and storage pool emissions can occur simultaneously. Amongst MT-emitting broadleaved trees, such as Holm oak (Quercus ilex) and European beech (Fagus sylvatica), it was
recognized that MT emissions were predominantly controlled by light-dependent mechanisms (Staudt and Seifert, 1995; Tollsten
and Müller, 1996; Dindorf et al., 2006). Later on, coniferous trees were also recognized to potentially emit part of
their total emission as de novo emissions (Shao et al., 2001; Tarvainen et al., 2005; Moukhtar et al., 2006;
Ghirardo et al., 2010; Taipale et al., 2011).
Shao et al. (2001) measured the BVOC emissions from Scots pine (Pinus sylvestris) in darkness and in different light
conditions. They found that MT emissions were partly influenced by photosynthetically active radiation (PAR), indicating that
observed emissions originate both from storage pools and from direct biosynthesis. Ghirardo et al. (2010) used stable isotope
labelling on Norway spruce (Picea abies) and Scots pine and observed that the approximate contribution of de
novo MT emissions could range between 25 and 45 % for spruce and 40 and 70 % for pine. Since it has been shown
that light-dependent and light-independent emissions happen simultaneously, it has been suggested that the observed MT
emission patterns should be regarded as a combination of light-dependent and light-independent emissions instead of only
being light-independent for some species (Ghirardo et al., 2010; Taipale et al., 2011; Laffineur et al., 2011; Staudt and
Lhoutellier, 2011; Song et al., 2014).
Many emission models face the difficulty of generalizing a species or class of species into one emission potential despite
different growing conditions and emission variabilities within species. Even though the BVOC emission patterns tend to be
more similar for plants of the same species or genus, variations in emission rates have been observed. Staudt et al. (2001)
screened 146 individual holm oak trees, which could be distinguished into three main types with an almost stable BVOC
composition. Their results suggest that the observed emission composition is more related to genotypic differences than to
environmental impacts. Bäck et al. (2012) sampled branches from 40 mature Scots pine trees from adjacent pine
stands. They could divide the trees into three chemotypes which remained fairly stable with the progression of the
season. The importance of genetic diversity on observed emission patterns has been further emphasized by Persson
et al. (2016), who investigated the emission patterns in genetically identical trees of English oak (Quercus robur),
European beech and Norway spruce. Persson et al. (2016) found differences in compound composition between two provenances of
spruce but few emission pattern differences for the remaining trees of identical genotypes. Few studies have investigated
in situ whether the compounds emitted from different tree species respond similarly with a conditional change in light. Our aim
was to investigate how different compounds responded to changing light conditions and whether the response was similar between
different tree species.
In this study, we investigated the response of BVOC emission, photosynthetic rates and stomatal conductance of English oak,
European beech and Norway spruce to different light levels. These species were chosen as they are some of the most common
tree species growing in large areas within Europe (Skjøth et al., 2008) and have reported BVOC emission levels exceeding 1 µggdw1h-1
(Kesselmeier et al., 1999; Dindorf et al., 2006; Holzke et al., 2006; Pokorska et al.,
2012). The study aims to (i) analyse how emissions of different BVOCs respond to changing light levels to identify
light-dependent fractions for each compound and (ii) investigate whether there are similar patterns between observed BVOC
emission, photosynthetic rates and stomatal conductance. This information could be useful for our understanding of how the
emission patterns of common European tree species react to changing light, which could possibly improve the algorithms used
in emission models.
Methods
Site description and plant material
Measurements were carried out on 10–31 July 2015 at the International Phenological Garden (IPG) site Taastrup, Denmark
(55∘40′ N, 14∘30′ E), maintained by the Faculty of Science at the University of
Copenhagen. The IPG network performs long-term phenological observations at several sites throughout Europe on some of the
most common European plant species. Each site was initially provided with up to two individuals per species. The plants used
in the network are genetically identical clones, which means the genetic variation between individuals and sites is absent
(Chmielewski et al., 2013). At the IPG network site at Taastrup, there are 21 trees from 13 different species and provenances
with one or two individuals per species. All trees presented here were planted in 1971. Measurements were performed on two
English oaks, one European beech and four Norway spruces, the latter divided into two provenances according to the framework
of IPG. These provenances differ in their budburst patterns; one provenance has a budburst approximately 1 week earlier
than the other. These provenances of spruce will henceforth be referred to as early spruce and late spruce.
During the measurement period, the weather was quite cold and humid, with an average daily temperature ranging between 13.1
and 18.8 ∘C and with a total rainfall of 43.6 mm during the 3 weeks of measurements. The average
temperature and total rainfall for July 2015 was 16.4 ∘C and 75 mm whilst the 10-year (2006–2015) average
temperature and rainfall in the area was approximately 18.2 ∘C and 71.8 mm (www.dmi.dk).
BVOC measurements at different light levels
Between 13 and 21 samples were taken from each tree. All measurements were made on the lowest positioned branches
(1–2 m above ground) and on the southwest- or south-facing side of the tree using a portable photosynthesis system
(Li-6400 XT, LICOR, NE, USA) equipped either with a LED source leaf chamber (6400-02B) for deciduous trees or a lighted
conifer chamber (6400-22L) for the coniferous trees. The ingoing air stream (700 mLmin-1) into the chambers
passed through a hydrocarbon trap and O3 filter to remove organic contaminants and ozone in order to avoid BVOC
oxidation before sampling. Measurements were performed during daytime (08:00–16:00). The calculations of net assimilation
rates (An) and GS were performed by the instrument software, using the equations presented by von
Caemmerer and Farquhar (1981). All measurements were made under fixed environmental conditions. Each leaf or needle twig was
acclimated to 400 µmolCO2mol-1 air and 50–60 % relative humidity for 1 h before BVOC emission
sampling. The temperature within the chamber was set according to the anticipated average daily temperature
(18–23 ∘C during the campaign) in order to minimize potential stress emissions from the plant. Each leaf or needle
twig was measured under four light levels (0, 500, 1000 and 1500 µmolm-2s-1) by stepwise increasing PAR
from 0 to 1500 µmolm-2s-1. This direction was chosen in order to mimic the daily increase in light
intensity. After the first acclimation period of 1 h at 0 µmolm-2s-1, an additional 30 min
acclimation period was applied after switching to a new light level in order to ensure that the leaf or needle twig had
adjusted to the new conditions. This acclimation time was chosen based on preliminary tests showing that leaf photosynthesis
remained reasonably stable after 30 min adjustment to the new light intensity. The BVOC emissions from the trees were
collected by extracting air from the chamber outlets into stainless steel cartridges (Markes International Limited,
Llantrisant, UK) packed with adsorbents Tenax TA (a porous organic polymer) and Carbograph 1TD (graphitized carbon black).
The air extraction was performed using flow-controlled pocket pumps (SKC Ltd., Dorset, UK) with a flow rate of
200 mLmin-1. Empty chamber blanks were collected every second day with the same chamber conditions in order to
account for possible background contamination in the measured samples.
BVOC analysis
The BVOC sample cartridges were sealed with Teflon coated brass caps directly after sampling, stored at 3 ∘C and
analysed within 8 weeks. A gas chromatograph–mass spectrometer (7890A series GC coupled with a 5975C inert
MSD/DS Performance Turbo EI system, Agilent, Santa Clara, CA, USA) was used for analysis after thermal
desorption (UNITY2 coupled with an ULTRA autosampler, Markes, Llantrisant, UK). The oven temperature was held at
40 ∘C for 1 min, raised to 210 ∘C in steps of 5 ∘Cmin-1 and lastly up to
250 ∘C in steps of 20 ∘C min-1. Helium was used as the carrier gas and the BVOC separation was
done with a HP-5 capillary column (50 m, diameter 0.2 mm and film thickness 0.33 µm). The
identification and quantification of BVOCs was done using pure standard solutions for isoprene, α-pinene, camphene,
β-pinene, δ-phellandrene, ρ-cymene, 1,8-cineole, ocimene, γ-terpinene, terpinolene, linalool,
aromadendrene, α-humulene and nerolidol in methanol (Fluka, Buchs, Switzerland). These standard solutions were
injected into adsorbent cartridges in a stream of helium. If there was a compound detected without an available standard, it
was identified according to the mass spectra in the NIST library and quantified using α-pinene for MTs and
α-humulene for SQTs. The sample chromatograms were analysed with the MSD Chemstation Data Analysis software (G1701CA
C.00.00, 21 December 1999; Agilent Technologies, Santa Clara, CA, USA). Compounds that were found in the empty chamber blanks
collected in the field were subtracted from the samples. Only isoprenoids were analysed in this study. Emissions were
calculated by using the emission rate equation for the dynamic enclosure technique presented by Ortega and Helmig (2008). For
each of the three light levels above 0, the light-dependent fraction of the total compound emission was calculated as
100%×(light emission-dark emission)/light emission and used as an indicator for
its emission response to changing light. The values ranged from 0 % (no light dependence) to 100 % (compound emitted
entirely light dependently).
Statistical analysis
Repeated measures ANOVA tests were computed in the Rstudio software (Rstudio team, 2015, version 0.99.491) in order to test
whether the observed emission rates of each compound and the An or GS rates differed statistically between the
light levels. If a significant effect of light was observed, a simple a priori contrast was used to test which light level
was significantly different from the dark measurements. The statistical analyses were done separately for each tree species.
The total BVOC emission rate of two individual English oak trees
(open circles) and the relative contribution of the major compounds at four
intensities of photosynthetically active radiation (PAR). The error bars
show the SD, n=3–5 leaves. The category “Other”
contains the compounds tricyclene, camphene and eucalyptol.
The total BVOC emission rate for European beech (open circles) and
the relative contribution of the major compounds at four intensities of
photosynthetically active radiation (PAR). The error bars show the SD, n=4–6 leaves. The category “Other” contains the compounds
tricyclene and eucalyptol.
The total BVOC emission rate from two individuals of Norway spruce
with (a) an early budburst and (b) a late budburst and the relative
contribution of the major compounds at four intensities of
photosynthetically active radiation (PAR). The open circles show total
monoterpene emission, whilst the open squares show isoprene emission of all
measured twigs (n=3–6 twigs). The error bars are the SD
of the data. The category “Other” contains the compounds tricyclene,
β-pinene, eucalyptol and linalool for early spruce and tricyclene,
β-pinene, α-terpinene eucalyptol and γ-terpinene for
late spruce.
The net assimilation rate (An) and stomatal conductance (GS,
mmol H2Om-2s-1) of (a) two individuals of English oak, (b) European beech, (c) two individuals of Norway spruce with an early budburst
(early spruce) and (d) two individuals of Norway spruce with a late budburst
(late spruce). The values are averages ±SD (n=13–21).
The P values from repeated measures ANOVA tests on the emission rate of each compound, photosynthetic rates
(An) and stomatal conductance (GS) in response to an increase in light intensity. The trees that were
measured were two individuals of English oak (Quercus robur), one European beech (Fagus sylvatica), two
individuals of Norway spruce (Picea abies) with an early budburst (early spruce) and two individuals of Norway
spruce with a late budburst (late spruce). P values marked in bold show statistically significant values (P<0.05). Isoprene was not detected from the European beech tree.
Compound
Oak 1
Oak 2
Beech
Early spruce 1
Early spruce 2
Late spruce 1
Late spruce 2
Isoprene
<0.001
<0.001
–
<0.001
0.13
<0.001
<0.001
α-Pinene
0.15
0.99
0.98
0.02
0.03
0.18
<0.001
Camphene
0.57
0.88
0.35
0.56
0.01
0.55
0.01
3-Carene
0.43
0.90
0.92
0.01
0.29
0.05
0.36
Limonene
0.66
0.97
0.65
<0.001
0.46
0.59
0.40
Eucalyptol
0.39
0.86
0.61
0.004
0.01
<0.001
0.07
Total BVOCs
<0.001
<0.003
0.87
<0.001
0.23
0.01
0.003
An
<0.001
<0.001
0.001
<0.001
0.03
0.001
<0.001
GS
0.02
0.23
0.25
0.007
0.13
0.02
<0.001
The percentage of emissions that are dependent on light (PAR, in µmolm-2s-1), as determined for the
total monoterpene (MT) emission and for the main emitted compounds. The percentage was calculated as 100%× (light emissions - dark emissions)/light emissions. The numbers in brackets are the standard error of the mean. The trees
that were measured were two individuals of English oak (Quercus robur), one European beech (Fagus sylvatica), two individuals of Norway spruce (Picea abies) with an early budburst (early spruce) and two
individuals of Norway spruce with a late budburst (late spruce). No data (n.d.) indicates compounds that were not detected in
any sample or light level for that particular tree.
Tree
PAR
Total MT
Isoprene
α-Pinene
Camphene
Sabinene
3-Carene
Limonene
Eucalyptol
Oak 1
500
0 (0)
100 (0)
0 (0)
17 (10)
n.d.
0 (0)
0 (0)
0 (0)
1000
4 (4)
100 (0)
0 (0)
17 (17)
n.d.
11 (11)
5 (5)
0 (0)
1500
10 (10)
100 (0)
0 (0)
40 (21)
n.d.
0 (0)
9 (9)
3 (3)
Oak 2
500
0 (0)
100 (0)
0 (0)
15 (10)
n.d.
21 (21)
0 (0)
0 (0)
1000
15 (15)
100 (0)
16 (16)
20 (20)
n.d.
31 (18)
15 (15)
13 (13)
1500
0 (0)
100 (0)
12 (6)
8 (8)
n.d.
0 (0)
0 (0)
0 (0)
Beech
500
6 (6)
n.d.
0 (0)
0 (0)
100 (0)
0 (0)
0 (0)
0 (0)
1000
23 (10)
n.d.
20 (20)
4 (4)
100 (0)
15 (15)
0 (0)
0 (0)
1500
52 (26)
n.d.
7 (7)
31 (31)
100 (0)
50 (6)
77 (23)
19 (19)
Early
500
81 (5)
100 (0)
64 (9)
6 (6)
n.d.
88 (10)
84 (1)
89 (6)
spruce 1
1000
76 (6)
100 (0)
54 (8)
10 (10)
n.d.
79 (18)
79 (1)
89 (5)
1500
86 (3)
100 (0)
60 (8)
14 (9)
n.d.
73 (14)
76 (3)
91 (4)
Early
500
18 (4)
100 (0)
20 (8)
0 (0)
n.d.
15 (15)
18 (3)
69 (3)
spruce 2
1000
0 (0)
100 (0)
3 (3)
8 (8)
n.d.
26 (26)
0 (0)
62 (4)
1500
19 (14)
100 (0)
43 (10)
0 (0)
n.d.
7 (7)
0 (0)
74 (3)
Late
500
67 (14)
100 (0)
98 (2)
12 (12)
n.d.
100 (0)
31 (25)
95 (5)
spruce 1
1000
94 (3)
100 (0)
67 (33)
45 (33)
n.d.
100 (0)
65 (32)
100 (0)
1500
87 (3)
100 (0)
98 (2)
0 (0)
n.d.
100 (0)
79 (16)
100 (0)
Late
500
26 (15)
100 (0)
85 (1)
0 (0)
n.d.
16 (13)
8 (8)
57 (22)
spruce 2
1000
68 (8)
100 (0)
91 (3)
0 (0)
n.d.
40 (8)
37 (18)
78 (14)
1500
57 (13)
100 (0)
85 (5)
0 (0)
n.d.
23 (12)
20 (20)
77 (15)
Results
BVOC emission from English oak
Figure 1 shows the total BVOC emission rate and the compound contributions of the two English oaks at different light
levels. The English oak clones in this study had emission rates between 3.5 and 18.3 µggdw1h-1 at a light
level of 1000 µmolm-2s-1 and a set temperature range of 18–21 ∘C. The first oak had
a statistically significant increase of the total emission across light levels, whilst the emission rate of the second oak
saturated at 1000 µmolm-2s-1. These emission rates are in line with the standardized emission rates
reported by previous studies (Isidorov et al., 1985; Kesselmeier and Staudt, 1999; Pokorska et al., 2012; Persson et al.,
2016) (Table 1). Between one and seven compounds were detected at the measured light levels and the detected compounds were
isoprene, tricyclene, α-pinene, camphene, 3-carene, limonene and eucalyptol. The main emitted compound was isoprene,
with no emission during darkness and an emission rate between 2.3 and 19.8 µggdw1h-1 for oak 1 and
between 1.3 and 9.3 µggdw1h-1 for oak 2 at light levels of 500–1500 µmolm-2s-1. The
relative contribution of isoprene to the total emission with light levels at or above 500 µmolm-2s-1
was >96 % (Fig. 1). At a light level of 0 µmolm-2s-1, the main detected compounds were limonene
and α-pinene. The emissions of these MTs remained stable across measured PAR levels, with emission rates of <0.1 µggdw1h-1 at all levels (see Appendix A for absolute values).
BVOC emission from European beech
In contrast to English oak, European beech showed a smaller and non-significant response of the total isoprenoid emission
rate to a change in light (Table 1, Fig. 2). Beech emitted between one and five detected isoprenoids in darkness and between
four and eight with light. Detected compounds were tricyclene, α-pinene, camphene, sabinene, 3-carene, limonene,
eucalyptol and caryophyllene. Sabinene was not detected at 0 µmolm-2s-1 but was the main emitted
compound with light, increasing from 66 % of the total emission at 500 µmolm-2s-1 to 76 % at
1500 µmolm-2s-1. Limonene was the main emitted compound in darkness. The amount of limonene released
remained fairly stable across the studied light levels and ranged between 0.06 and 0.09 µggdw1h-1. The
other emitted MTs did not change their emission patterns with increasing light. At light levels 1000 and
1500 µmolm-2s-1, the SQT caryophyllene was released, with the highest emissions at
1500 µggdw1h-1 (see Appendix A for absolute values, Fig. 2).
BVOC emission from Norway spruce
Figure 3a and b show the emission rate and the compound contribution with increasing light levels for early spruce and late
spruce, respectively. All four spruce trees emitted isoprene with light (P<0.001 for early spruce 1 and late spruce 1 and
2, P>0.1 for early spruce 2) with a contribution to the total emission of 30–65 %. In contrast, limonene and
α-pinene were emitted both in darkness as well as with light, but with lower absolute emissions in darkness (see
Appendix A for absolute values; Fig. 3). Early spruce 1 had an emission rate of 0.5–0.6 µggdw1h-1,
whilst early spruce 2 ranged between 0.1 and 0.4 µggdw1h-1 with light. Between four and nine isoprenoids
were detected, which were isoprene, tricyclene, α-pinene, camphene, β-pinene, 3-carene, limonene, eucalyptol,
linalool, α-farnesene and β-farnesene. Only one of the two early spruce trees emitted linalool and SQTs. The
main detected compound for both trees was isoprene, followed by limonene. The total emission from early spruce 1 saturated at
500 µmolm-2s-1 with no significant change with increasing light (P>0.1), whilst early spruce 2
decreased its total emission to 0.1 µggdw1h-1 at 1000 µmolm-2s-1 and then
increased again somewhat to 0.3 µggdw1h-1 at 1500 µmolm-2s-1 (Fig. 3a). Late
spruce emitted 2 to 10 isoprenoids at all light levels and the detected compounds were isoprene, tricyclene,
α-pinene, camphene, β-pinene, 3-carene, α-terpinene, limonene, eucalyptol and
γ-terpinene. β-Pinene was emitted by both provenances of Norway spruce, but with higher emissions rates from
late spruce in combination with higher emissions of α-pinene. Only late spruce 1 emitted tricyclene and
α-terpinene and only at PAR levels of 1000 and 1500 µmolm-2s-1. Both trees had an increase in
total emission up to 1000 µmolm-2s-1, with a decrease in emissions at
1500 µmolm-2s-1 for late spruce 1. Late spruce 1 reached its peak emission of
2.2 µggdw1h-1 at 1000 µmolm-2s-1, whilst late spruce 2 had a stable emission
between 0.6 and 0.9 µggdw1h-1 with light. The emitted compounds from late spruce 1 followed a similar
emission pattern as the total emission rate, but for late spruce 2 all compounds except α-pinene, eucalyptol and
γ-terpinene remained fairly stable with increase in light (Appendix A, Fig. 3b).
Light-dependent fractions of different compounds
Whilst some compounds like isoprene and sabinene were specific for different tree species, the compounds α-pinene,
camphene, 3-carene, limonene and eucalyptol were emitted from all of the measured leaves or needle twigs. As these compounds
were emitted at different light levels, we will assess the light dependency of these compounds. The light-dependent fraction
for isoprene was 100 % for all of the isoprene-emitting trees (Table 2). The same fraction and significance were also
found for sabinene emission from beech (P<0.001, Table 2). The light response for the total MT emission differed between
species. Whilst the oaks and the second early spruce showed little or no response to light, the beech and the remaining
spruce trees increased their emissions. The light-dependent fraction of other MTs, however, depended on the compound and the
tree species. Camphene had a significant change in emission from darkness to 500 µmolm-2s-1 for early
and late spruce 2, but for remaining light levels camphene showed no clear light dependency for any of the measured trees
(Appendix A, Tables 1 and 2).
For the oaks, no compounds other than isoprene showed a significant light dependency. For beech, some compounds like
camphene, 3-carene, limonene and eucalyptol increased the light-dependent fractions with higher light levels, but without
this being a significant increase in its emissions (Appendix A, Tables 1 and 2).
The two provenances of spruce showed a higher light-dependent fraction for MTs in comparison to the broadleaved trees. Early
spruce 1 and late spruce 1 showed light-dependent fractions of 76–86 and 67–94 %, respectively, for the total MT
emission (Table 2). Both trees had high light-dependent fractions for the compounds α-pinene, 3-carene and
eucalyptol. For early spruce 1, eucalyptol increased its light-dependent fraction with increasing light levels. For late
spruce 1 there was a higher percentage of light dependency for α-pinene, but only limonene increased in light
dependency with increasing light. Early spruce 2 had low light-dependent fractions for all compounds except eucalyptol,
whilst late spruce 2 had high light-dependent fractions for α-pinene and eucalyptol. Although several of the above
mentioned compounds from early spruce 2 and late spruce 2 showed a light dependency, this light dependency did not change
with a change in light level (Table 2).
Photosynthesis and stomatal conductance
For oak, the assimilation (An) rates were fairly similar between the two trees, ranging from
-0.6 to -0.5 µmolCO2m-2s-1 in darkness and from 2.4 to 4.5 µmolCO2m-2s-1 with
light (Fig. 4a, Table 1). The difference was larger for the stomatal conductance (GS): oak 1 showed
a significant difference with increasing light (P<0.05) in comparison to oak 2, which showed higher internal variation (P>0.2). In regards to their photosynthetic and stomatal conductance ranges, they are comparable with studies performed on oak
leaves grown in either shaded or semi-shaded conditions (Morecroft and Roberts, 1999; Valladares et al., 2002). For beech,
An increased from darkness to the PAR level of 500 µmolm-2s-1 (P<0.001) but did not show
a response to further increase in light (P>0.6). An was between 3 and 3.6 µmolCO2m-2s-1 with
light and -0.3 µmolCO2m-2s-1 in darkness, whilst GS ranged between
100 and 400 mmolH2Om-2s-1 for all light levels (Fig. 4b).
For early spruce 1, An was between 9.5 and 11.3 µmolCO2m-2s-1 at a light level of 500 and
1000 µmolm-2s-1, which decreased to 7.3 µmolCO2m-2s-1 at
1500 µmolm-2s-1. GS followed a similar pattern, ranging from
1000 to 1200 mmolH2Om-2s-1 at a light level of 500 and 1000 µmolm-2s-1 and decreased
to 700 mmolH2Om-2s-1 at a light level of 1500 µmolm-2s-1 (Fig. 4c,
Table 1). A similar pattern as the BVOC emissions for early spruce 2 could also be seen in the rates of An and
GS with lower values coinciding with lower emissions, but which was significant only for An
(Table 1). Late spruce 1 had a higher emission rate in comparison to late spruce 2, which was also evident for the An
and GS rates. Whilst late spruce 1 showed an increase in both An and GS with increasing
light levels (P<0.05), late spruce 2 did not show any clear response to increasing light above
500 µmolm-2s-1 (P>0.2). Late spruce 1 had an average An rate of
4.5–10.9 µmolCO2m-2s-1 and an average GS rate of
400–1100 mmolH2Om-2s-1 with light. For the second spruce, the An and GS rates were
stable at an average range of 3.6–5.1 µmolCO2m-2s-1 and 300–500 mmolH2Om-2s-1
with light (Fig. 4d).
Discussion
Light plays an important role as a driver of BVOC emissions, particularly in regards to de novo emissions. Overall,
the investigated trees showed a similar response to light in their light-dependent BVOC emissions, An and
GS, but the light level at which these processes saturate could vary for individual leaves or needle twigs.
Responses of BVOC emissions to changing light conditions
Isoprene was the main emitted compound for the measured oak trees which showed a clear response to increasing light. This
increasing emission with light has also been confirmed by other performed studies (Tingey et al., 1981; Lehning et al.,
1999). For beech, the main emitted compound was sabinene, which also responded to increasing light. A similarity which was
found for the oaks and the beech was that, apart from their main emitted compounds, the emission rate of other MTs did not
show any significant response with increasing light (P>0.05). This observation would suggest that the emission of MTs from
these deciduous trees should be regarded as light independent instead of light dependent, dividing the emissions into
light-dependent and light-independent fractions. For coniferous tree species, which are known to have storage structures
contributing to a considerable light-independent emission, a division of the emissions into light-dependent and
light-independent fractions has been suggested (Ghirardo et al., 2010). Although similar structures are absent in the
broadleaf species studied here, the results suggest that these species also have a light-independent fraction.
The two provenances of spruce had different responses of their emitted compounds with an increase in light, where the light-dependent fraction of the total MT emission increased for all trees except for early spruce 2. Regarding separate compounds,
they were also shown to respond differently with an increase in light depending on the individual tree. The compound camphene
showed significant emission responses from early spruce 2 and late spruce 2, but only going from darkness to
500 µmolm-2s-1. For the remaining trees, there was no clear camphene emission response to an increase
in light. This suggests that this compound should be considered to be light independent when emission rates are to be
modelled. Early spruce 1 showed light-dependent fractions from α-pinene, 3-carene, limonene and eucalyptol, but with
eucalyptol was the only MT compound which continued to increase its light-dependent fraction with increasing light
intensity. A similar light dependency of eucalyptol has also been found for emissions from Abies alba (Moukhtar
et al., 2006). Early spruce 2 showed light-dependent fractions from α-pinene, camphene and eucalyptol. However, as
the amount of samples taken on early spruce 2 were few, it is difficult to draw any clear conclusions for this tree. Both the
late spruce trees had light-dependent emissions of α-pinene and eucalyptol. Late spruce 1 also showed light-dependent
fractions for 3-carene going from darkness to light, but the overall emission rate of this compound was low and of little
importance in regard to the general compound contribution. For late spruce 2, α-pinene and camphene showed significant
emission increases from darkness to 500 µmolm-2s-1. The response of late spruce 2 might, however, be
masked by high internal emission variation at 500 µmolm-2s-1.
Regarding the light dependency of MT emissions, there are several studies which have suggested that both de novo and
storage pool emissions can occur within different tree species (Dindorf et al., 2006; Moukhtar et al., 2006; Ghirardo et al.,
2010). Our study shows that different compounds respond differently to a change in light and that compounds like camphene
have similar emission responses for English oak, European beech and Norway spruce and that all of the measured trees released
isoprenoids in darkness, with emissions ranging from 0 to 0.4 µggdw1h-1 for the broadleaf trees and
from 0.01 to 0.22 µggdw1h-1 for the provenances of spruce. This would indicate that species such as English
oak and European beech, which are considered to lack specific storage compartments, have a capacity to store compounds in the
mesophyll, which has also been suggested by other studies (Niinemets and Reichstein, 2003; Holopainen and Gershenzon,
2011). In a study by Loreto et al. (2000), 13C labelling was used on Holm oak (Quercus ilex) with and
without illumination and found that the newly synthesized compounds could continue to be emitted long after initiation of
darkness. It was suggested that the volatile compounds could be non-specifically stored within the plant leaves, either in
the lipid phase or in the aqueous phase. Furthermore, Bäck et al. (2005) did a modelling study on Scots pine where
a mesophyll pool was included, which enabled them to better capture diurnal and seasonal emission trends of MT emissions.
These results suggest that as there might exist non-specific storage within the leaf tissue, de novo emitting tree
species need to be considered to have storage pools in emission models as well. However, with the current experimental setup,
it is only possible to make assumptions of the relative contributions of de novo sources and storage pools. This is
otherwise often tested by using 13CO2 labelling, where de novo emissions would have 13C
incorporated into their compound structures after a pulse of labelled 13CO2 (Ghirardo et al., 2010). But by using
genetically identical trees and fixed environmental conditions inside the measurement chamber, it has been possible to study
the emission response of different compounds to an increase in light intensity.
As models divide plants into categories or plant functional types depending on the growing conditions to which they have adapted
(Schurgers et al., 2011; Guenther et al., 2012), an approach looking at the emission patterns of separate compounds would
perhaps improve emission models further. If the plants are also categorized into the compound emission response, the model
would perhaps provide more realistic values by dividing the compounds into light-dependent or light-independent fractions. We would
therefore strongly suggest that more studies assessing light dependency of different compounds are performed on similar or
different tree species in order to verify this light dependency of the compounds.
Emission pattern variation and shade adaptation of the leaves and needle twigs
The European tree species presented here have distinct emission patterns: English oak is a known high isoprene emitter,
European beech mainly emits MTs such as sabinene and Norway spruce is known to emit both isoprene and MTs (Dindorf et al.,
2006; Ghirardo et al., 2010; Pokorska et al., 2012). Between 96 and 99 % of the total emission for oak consisted of
isoprene, followed by MTs such as limonene and α-pinene. This compound contribution has not only been stable over
3 years of measurements on these genetically identical trees, but it is also in agreement with measurements at other
sites (Staudt et al., 2001; Persson et al., 2016; van Meeningen et al., 2016). This would suggest that even if environmental
factors such as temperature or light influence the total emission from oak, these do not alter the compound contribution to
a great extent (Staudt et al., 2001; van Meeningen et al., 2016).
There were big differences in emission amounts between beech leaves, making it difficult to see any clear increase in BVOC
emissions with an increase in light. When the light level exceeded 1000 µmolm-2s-1, there was also an
increase in SQT emissions. The total emission rates are in the lower ranges in comparison to other studies with standardized
emission rates (Moukhtar et al., 2005; Dindorf et al., 2006, and references therein). This could be because all samples were
taken on the lowest positioned branches of the tree. In the study made by Persson et al. (2016) from the same site performed
in 2013, the emission rates were taken at three different height levels within the canopy of all the above mentioned trees.
For the European beech, the standardized emission rates were much higher at the top of the canopy in comparison to lower
levels, with an average standardized emission of 26.5 µggdw1h-1 at the top of the canopy and
3.6 µggdw1h-1 at the bottom (Persson et al., 2016). The lower emission rate found in this study could
be caused by more shade-adapted leaves, with a possible lower capacity to respond to high increases in light. The levels of
An and GS presented here are comparable with other studies performed on leaves adapted to shaded or
semi-shaded conditions (Valladares et al., 2002; Warren et al., 2007; Scartazza et al., 2016). It would be advisable to make
more measurements at the top of the canopy in comparison to the lower levels in order to not underestimate the emission
potentials for European beech.
There were distinct differences in emission spectra between the two provenances of Norway spruce. The main emitted compound
for both provenances was isoprene, but regarding the emitted MTs early spruce was mainly a limonene emitter whilst late
spruce emitted α-pinene. This emission pattern difference between provenances has been observed in three separate
studies performed at the same site (Persson et al., 2016, for 2013, unpublished data for 2014, current study for
2015). Furthermore, late spruce also emitted β-pinene at a higher rate than the early spruce trees, whilst the
compounds α-terpinene and γ-terpinene were only emitted by late spruce. This would suggest that for different
provenances of the same species, different compound adaptations might exist. Studies on other tree species have suggested
that trees can be divided into chemotypes depending on their emission patterns and that the compound contribution of these
chemotypes remains fairly stable over time (Staudt et al., 2001; Bäck et al., 2012).
The average emission rates at 1000 µmolm-2s-1 ranged between 0.1 and 0.6 µggdw1h-1
for early spruce and between 0.9 and 2.2 µggdw1h-1 for late spruce, which were in range of previous studies
(Kesselmeier and Staudt, 1999; Grabmer et al., 2006). The four light levels that were tested did not provide enough
information to address the light response entirely. More points taken between 0 and 500 µmolm-2s-1 would
therefore be advisable in order to fully understand the change in emission amounts. The second early spruce tree showed more
fluctuation between different light levels, possibly as a response to stress exposure. When measurements were performed on
this tree in 2013, the needles on the lowest branches dried and fell off after a prolonged period without rain in the middle
of July (Persson et al., 2016). In 2014, when measurements were performed again, the lower twigs had still not recovered and
it was not possible to make any measurements on that level (unpublished data). In 2015, new twigs had started to emerge again
on early spruce 2, but twigs were small and visibly less healthy. In comparison to the 10-year average weather conditions at
the site, July in 2015 has had approximately the same amount of rainfall but was almost 2 ∘C colder. It is likely
that the weather conditions had an effect on the emission results. However, as all trees have had the same
exposure, it does not fully explain the different responses between trees. With less material to make measurements on and
with possible recovery from stress, it is difficult to fully capture the release of BVOC emission from early spruce 2. The
average An rates for early spruce and late spruce were between 4.3 and 12.1
and between
3.6 and 12 µmolCO2m-2s-1 respectively, whilst the GS rates ranged between
400 and 1200 mmolH2Om-2s-1 for early spruce and between 300 and 1000 mmolH2Om-2s-1 for
late spruce. These values are in range or slightly higher than reported in other studies (Le Thiec et al., 1994; Roberntz and
Stockfors, 1998; Špunda et al., 2005). Early spruce 1 and late spruce 2 behaved in a similar fashion as European beech
with a tendency to stabilize their An and GS rates at a light level of
500 µmolm-2s-1, indicating some shade adaptation of the selected needle twigs. Late spruce 1 increased
both in An and GS rates with light, possible because the tree stands more exposed than the others in the
northeast corner of the IPG site and therefore is more light adapted in comparison to the other trees. Early spruce 2 showed
the same fluctuating pattern in An and GS rates as with the observed BVOC emissions, most likely due to
a restricted sample size and previous effect of drought stress on the tree.