Biogenic volatile organic compounds (BVOCs) produced by plants have a major
role in atmospheric chemistry. The different physicochemical properties of
BVOCs affect their transport within and out of the plant as well as their
reactions along the way. Some of these compounds may accumulate in or on the
waxy surface layer of conifer needles and participate in chemical reactions
on or near the foliage surface. The aim of this work was to determine whether
terpenes, a key category of BVOCs produced by trees, can be found on the
epicuticles of Scots pine (
At the border of the atmosphere and Earth's ecosystems, the living layer of vegetation is an active player interacting with its surroundings in multiple ways. Plants absorb, transmit and produce compounds like water, oxygen and carbon, as well as a myriad of more complex molecules such as volatile organic compounds (VOCs). In addition to this biological activity, plant surfaces provide area for adsorption, desorption and chemical reactions. These phenomena are affected by both environmental conditions and the structure (species, canopy layers, etc.) of the vegetation – in turn shaping itself in response to the environment it grows in. The result of these interactions is an extremely complex and dynamic network of simultaneous processes.
Biogenic VOCs (BVOCs) produced by plants have a major role in atmospheric chemistry. They affect the formation and destruction of ozone in the troposphere and participate in aerosol formation processes (e.g., Kulmala et al., 2004; Tunved et al., 2006). Despite considerable progress in recent years, aerosol-related processes are a major source of uncertainty in climate estimates (IPCC, 2014). Biogenic VOC emissions dominate over those of anthropogenic origin both globally (Guenther et al., 1995) and in the sparsely populated regions of Northern Europe, especially in the summertime (Simpson et al., 1999; Lindfors et al., 2000).
Terpenes (monoterpenes (C
On their way from the plant interior to the atmosphere, the terpenes, mostly rather lipophilic in nature (Niinemets and Reichstein, 2003, Appendix A), must first cross the lipophilic cell membranes and then the hydrophilic apoplast before evaporating into the air spaces inside the leaf. It was long assumed that this transfer happens purely by diffusion, but new evidence suggests active transport out of the cells (Widhalm et al., 2015). Finally, emission into the atmosphere occurs first by gas-phase diffusion through the stomata and the leaf boundary layer, where the conditions are significantly affected by the leaf (Schuepp, 1993), and then by turbulent transport. The driving force of diffusion is the concentration gradient between the leaf interior and the atmosphere. The leaf cuticle is generally considered an effective barrier for plant-produced volatiles, preventing direct emission (Niinemets and Reichstein, 2003).
The different physicochemical properties of terpenes affect their transport
within and out of the needle as well as their reactions along the way
(Atkinson and Arey, 2003; Niinemets and Reichstein, 2003, Appendix A). For
example solubility/volatility (described by Henry's law constant H; Pa
m
Terpenes participate in many chemical reactions at and near the needle
surfaces. For example, terpenes can protect the plant from oxidative
stressors such as ozone (O
The surfaces of conifer needles are both complex and dynamic in nature. As they grow, needles are covered with a waxy layer secreted by the epicuticular cells (Fig. 1). This layer is lipophilic and hydrocarbons are known to be taken up in it (Binnie et al., 2002; Brown et al., 1998; Welke et al., 1998). With time and weathering, the surfaces undergo chemical and structural changes (Barnes and Brown, 1990; Huttunen and Laine, 1983). Irregularities in the surface provide sites for water adsorption (Rudich et al., 2000). As a result, the originally water-repellent surface becomes more wettable as it wears down. Compounds accumulating on the surface change the characteristics of both the surface and the water film that forms on it (Neinhuis and Barthlott, 1997; Burkhardt and Eiden, 1994). Such water films are ubiquitous when the ambient relative humidity is above 70 % – a common condition in boreal areas – and can even extend through the stomata, creating a pathway for water-soluble compounds between the leaf inside and the surface (Burkhardt et al., 2012).
Pine needle structure.
Thus it is plausible that plant-derived terpenes with varying chemical properties could accumulate on foliage surfaces in amounts and proportions difficult to predict and participate in reactions with other compounds. Because of their importance for both atmospheric chemistry and the plant's adaptation to stress, it is necessary to analyse how the surface processes might change the composition of terpenes reaching the free atmosphere.
The aim of this work was to determine whether terpenes can be found on the
epicuticles of Scots pine (
We measured shoot-level emissions of pine seedlings at a remote outdoor
location in central Finland (Hyytiälä, 61
The plant material consisted of four grafted Scots pine seedlings, grown for 5 years in an outdoor plant nursery field. Grafted material was selected to reduce variation in the emissions, since it is well known that the spectrum of terpene emissions depends, among other factors, on the genetic background (Bäck et al., 2012). The height of the seedlings was 1.5–2 m. The trees were transplanted in 15 L plastic pots in May 2013. The plants were kept outdoors in light shade and were well watered. Emission measurements were done during the first days of August. Scots pine terpene emissions have an annual and a diurnal pattern (Hakola et al., 2006; Holzke et al., 2006; Ruuskanen et al., 2007; Aalto et al., 2015); the measurement period was selected to capture sesquiterpene emissions that peak in the summer (Hakola et al., 2006; Tarvainen et al., 2005).
We aimed to measure the terpene emissions of each seedling once in similar environmental conditions close to noon and to take three needle samples from each seedling for subsequent wax analysis.
We measured terpene emissions from the seedlings with a dynamic chamber. The
chamber consisted of a steel frame, coated with PTFE tubing, and a FEP (fluorinated ethylene propylene) bag
supported by the frame (volume 4.5 L). The chamber was fitted with an inlet
and outlet tube made of PTFE. An external pump, with an active carbon filter
and an ozone scrubber, pushed air through the chamber
(2.5 L min
A healthy mid-crown branch was selected for the emission measurement. Before measurement, the tip of the branch (approximately 30 cm) was gently fitted in the frame. The measured section included needles grown in 2013 and 2012. The growth of the new needles was not quite complete at the time of measurement. The FEP bag was then pulled over the frame, the pump was started and the system was left to stabilise for 30 min to minimise the effect of emissions induced by handling.
A sample flow was then directed through adsorbent tubes (Tenax-TA and
Carbopack-B) attached to the inlet and outlet tubes with a stainless steel
T piece. The resin filling of the tube adsorbs terpenes, which can later be
desorbed and analysed. Small pumps were used to pull the sample through the
tube (70 mL min
The contents of the adsorbent tubes were analysed at the Finnish
Meteorological Institute with a thermal desorber (Perkin-Elmer TurboMatrix
650 ATD) connected to a gas chromatograph – mass spectrometer (Perkin-Elmer
Clarus 600) with HP-1 column (60 m, i.d. 0.25 mm). The detection limits
were 0.04 ng sample
The observed emission rate (
To detect the presence of terpenes associated to the epicuticular surfaces, we collected the waxy material from the needle surfaces for subsequent terpene analysis.
After each emission measurement, we darkened the measured tree for 30 min to close the stomata and minimise stomatal terpene emission and then took needle samples (three separate samples of 20 needle pairs each) in darkness for the wax analysis. The needles were immediately stored in a liquid nitrogen dry shipper until analysis (2 weeks later).
We collected the epicuticular wax layer by dipping each needle pair in 5 mL dichloromethane for 15 s. The dipping time was optimised in a preliminary experiment to remove most of the wax layer but to keep the solvent from reaching the inside of the needle through stomata (visual inspection under a stereo microscope). We took special care to use only intact needles and to not immerse the cut base of the needle in the solvent. This was done to prevent compounds originating inside the needle from getting into the extract. Dipping the needles while they were frozen should also minimise the extraction of compounds from inside the needle. After wax extraction, the needles were weighed for fresh and dry mass and measured for their dimensions (width, length and thickness). From these dimensions, needle surface area was approximated according to Tirén (1927).
The obtained extract was evaporated to 1 mL volume with pure nitrogen gas.
The reduced extract was then analysed with a gas chromatograph
(Agilent 6890N) with a mass spectrometric detector (Agilent 5973) to identify
terpenes. A different instrument from the emission analysis was used because
of the different sample medium (liquid vs. gas). A JandW DB-5MS column (30 m,
i.d. 0.25 mm) and a 5 m pre-column (Agilent FS) were used for the
chromatography. The limits of detection were estimated from the standard
deviations of blank samples and were 0.15–0.30 ng sample
For an estimation of the terpenes lost during the evaporation, we performed a separate evaporation test, letting known concentrations of selected terpenes evaporate as described above. The test gave no indication of any significant loss of terpenes associated with the method.
The weather conditions during the experiment were slightly variable. The
first 2 days (measuring emissions from trees 1 and 2) were relatively warm
(
The shoot emissions were clearly dominated by monoterpenes (96–98 % of total terpene emissions, Fig. 2). Sesquiterpenes amounted to 0–2 % of total emissions. The compounds found in each group and the variation in their emissions are presented in detail in Appendix B and Fig. 2.
Relative amounts of terpenes in the pine shoot emissions and needle
surface waxes, average % of total, with 1 standard deviation.
The most abundant monoterpenes were
The wax yield from the pine needles was
0.0066–0.0114 g g DW
The results for different compounds were highly variable also in the wax
analysis (Appendix B). The variation in the terpene content of the
epicuticular waxes cannot be explained by variation in wax yield. Even though
there is variation in wax yield (per needle area), this variation does not
correspond to the variation observed in the terpenes. The most abundant
monoterpenes in the waxes were
The composition of the emitted pine shoot terpenes measured in this study is
generally in the range observed by others (Bäck et al., 2012; Hakola et
al., 2006; Holzke et al., 2006; Tarvainen et al., 2005), allowing for the
natural variation in BVOC emission and the differences in methodology. The
pine seedlings in our study emitted more than twice as much
The amount of terpenes found in the epicuticular waxes is the equivalent to
4–84 h of the measured emissions for the same compound (per m
There is remarkable variation observed in the terpene content of the epicuticular waxes, and this variation cannot be explained by variation in the amount of extracted wax. Possible natural causes of variation include small cracks, insect bites or pathogens in the bark near some of the needles. For example, insect bites are known to induce both local and systemic terpene emissions (Heijari et al., 2011). Some of these may well have escaped visual inspection. One feasible source is true natural variation between needles grown in different parts of the branch or canopy, due to the light-dependent nature of terpene synthesis. Very little is known on this topic, but it is very likely that there are notable differences (Juho Aalto, personal communication, 2016). Some of the variation, however, may have been caused by the sampling procedure itself. Despite the short sampling time, it is possible that the emissions caused by plucking needles had sufficient time to adsorb onto other needles that were subsequently picked into a sample.
The short exposure to the solvent and the fact that the stomata were
virtually closed means that any BVOCs found in the extract were most likely
not a result of stomatal emissions but rather compounds that had been
associated to the epicuticle. In studies with extracts from crushed needles,
the proportion of mono- and sesquiterpenes has been found to be in the same
range as observed here for both emissions and epicuticular waxes. For
example, Manninen et al. (2002) reported a mean total monoterpene ratio of
67 % for a Scots pine provenance from central Finland and listed
In the epicuticular waxes, we observed six unidentified sesquiterpenes, some in relatively high proportions. Although this group is likely to include cadinene, cubebene and murolene, the exact identification and quantification of these compounds would require a more detailed study. Naturally, the possible role of these compounds in the emissions remains unknown, but their existence in the waxes suggests that the production of sesquiterpenes in Scots pine deserves more attention.
It is interesting to note that despite the large variation there is some
indication that the most water-soluble compound in our study, 1,8-cineol,
(Appendix A) was relatively more abundant in the emissions, while the
compounds with a large
In theory, there are three mechanisms for the terpenes produced by a plant to
end up on the needle surface. The first one is (dry) redeposition after
emission from either the tree itself (needles, bark or other parts) or
neighbouring trees. Terpene emission from one plant individual and
redeposition onto another has been reported, more markedly for sesqui- than
monoterpenes (Himanen et al., 2010; Li and Blande, 2015). This route is more
likely for the less volatile terpenes like longicyclene and p-cymene
(Appendix A). The most lipophilic terpenes, such as
The second option is transport in the aqueous layer extending from the outer needle surface through the stoma all the way into the substomatal cavity, as suggested by Burkhardt et al. (2012). This route is naturally only available to terpenes produced by the needle itself, and the effectiveness of the route depends on the existence of such a continuous water film, and also on the water-solubility and diffusion capabilities in water of the compound in question. Because of their low water solubility, it has often been assumed that the reactions of terpenes in the aqueous phase do not contribute significantly to the total reactions. Wang et al. (2012) however propose that the reactions of biogenic unsaturated hydrocarbons happening on wet surfaces, like those of plants growing in nature, can have a significant effect on ozone deposition. In this work, we cannot differentiate between compounds that were in or on the epicuticular waxes from those that may have been bound in the surface water. The most water-soluble of the detected compounds was 1,8-cineol, which was present in greater proportion in shoot emissions than epicuticular waxes. It is then possible that some of the 1,8-cineol emitted from the shoot is redeposited onto the surface.
The third alternative is direct transport from the production sites inside
the cells through the plant cuticle. In xerophytic plants, such as conifers,
the cuticle has a strongly layered structure. The insoluble lipid cutin is
partly embedded as intracuticular wax under the cuticle proper, not as an
even layer but forming legs towards the epidermal cell wall (Evert, 2007,
Fig. 1). The production of surface waxes takes place in epidermal cells
during the first few weeks and months of needle growth (Kinnunen et al.,
1998), and they are transported via microchannels or diffusion to the surface
(Evert, 2007). Despite some reports of terpene emissions through the cuticle
(e.g., Guenther et al., 1991), this route is usually considered negligible
for terpene emissions (Niinemets and Reichstein, 2003) because of the
considerably slower diffusion rate of terpenes within the cuticle than in air
or water. It does not, however, contradict the notion that terpenes might be
transported into the epicuticulum and accumulate there. Theoretically, this
mode of transport would be more effective for the most lipophilic compounds
like
Once in the gas phase, plant-emitted terpenes can react in various ways. They
can undergo photolysis or react with hydroxyl or nitrate radicals or ozone
(Atkinson and Arey, 2003). The relative importance of the different reaction
pathways depends on atmospheric conditions, time of day and the compound in
question. Ozone reactions target double bonds in the terpene molecule
(Atkinson and Arey, 2003). The most O
The available reaction rate coefficients for O
Similarly to Fares et al. (2012), we assumed that each molecule of any
terpene can react with one molecule of O
Although simple, our calculation shows that the terpenes found in needle
surface waxes could act as a significant O
All data relevant to the article are included in the tables in Appendices A and B.
The values for molecular mass (
Amounts of identified BVOCs per needle area in shoot emissions (
Percentage of total of identified BVOCs in shoot emissions and needle surface waxes.
Anni Vanhatalo, Ditte Mogensen, Theo Kurtén and Pontus Roldin are acknowledged for their valuable help before, during and after the experiment. We thank the Natural Resources Institute Haapastensyrjä unit for the grafted plant material. The research was supported by the Academy of Finland Center of Excellence (grant no. 272041), Maj and Tor Nessling foundation, the Finnish Society of Forest Science and the Doctoral Programme in Sustainable use of renewable natural resources (AGFOREE). N. Altimir thanks VOCBAS for supporting the exchange visit where the initial idea for this study was generated. Edited by: S. A. Nizkorodov