# PLANT SECONDARY COMPOUNDS IN FOREST ECOSYSTEMS UNDER GLOBAL CHANGE: FROM DEFENSE TO CARBON SEQUESTRATION

EDITED BY : Bartosz Adamczyk and Judy Simon PUBLISHED IN : Frontiers in Plant Science

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# PLANT SECONDARY COMPOUNDS IN FOREST ECOSYSTEMS UNDER GLOBAL CHANGE: FROM DEFENSE TO CARBON SEQUESTRATION

Topic Editors: Bartosz Adamczyk, Natural Resources Institute Finland (LUKE), Finland Judy Simon, University of Konstanz, Germany

Citation: Adamczyk, B., Simon, J., eds. (2019). Plant Secondary Compounds in Forest Ecosystems Under Global Change: From Defense to Carbon Sequestration. Lausanne: Frontiers Media. doi: 10.3389/978-2-88963-010-3

# Table of Contents

Jarmo K. Holopainen, Virpi Virjamo, Rajendra P. Ghimire, James D. Blande, Riitta Julkunen-Tiitto and Minna Kivimäenpää

*16 Interannual and Seasonal Dynamics of Volatile Organic Compound Fluxes From the Boreal Forest Floor*

Mari Mäki, Juho Aalto, Heidi Hellén, Mari Pihlatie and Jaana Bäck

*30 Combinations of Abiotic Factors Differentially Alter Production of Plant Secondary Metabolites in Five Woody Plant Species in the Boreal-Temperate Transition Zone*

John L. Berini, Stephen A. Brockman, Adrian D. Hegeman, Peter B. Reich, Ranjan Muthukrishnan, Rebecca A. Montgomery and James D. Forester

*47 Fertilization Changes Chemical Defense in Needles of Mature Norway Spruce (*Picea abies*)*

Line Nybakken, Marit H. Lie, Riitta Julkunen-Tiitto, Johan Asplund and Mikael Ohlson

*56 Flavanone-3-Hydroxylase Plays an Important Role in the Biosynthesis of Spruce Phenolic Defenses Against Bark Beetles and Their Fungal Associates*

Almuth Hammerbacher, Dineshkumar Kandasamy, Chhana Ullah, Axel Schmidt, Louwrance P. Wright and Jonathan Gershenzon

Mette Sørensen, Federico Cozzi, Berin A. Boughton, Allison Maree Heskes and Elizabeth Heather Jakobsen Neilson

# Editorial: Plant Secondary Compounds in Forest Ecosystems Under Global Change: From Defense to Carbon Sequestration

Judy Simon1 \* and Bartosz Adamczyk 2

*1 Plant Interactions Ecophysiology Group, Department of Biology, University of Konstanz, Konstanz, Germany, 2 Natural Resources Institute Finland (LUKE), Helsinki, Finland*

Keywords: climate change, boreal, temperate, eucalypts, plant secondary metabolites, herbivory, phenolics, volatiles

**Editorial on the Research Topic**

#### **Plant Secondary Compounds in Forest Ecosystems Under Global Change: From Defense to Carbon Sequestration**

Forests are crucial for the future of human society, not only by their provision of fundamental ecosystems services, such as timber, food, and recreation, but also for mitigating climate change via the capture and long-term storage of carbon (MEA, 2005; Sabatini et al., 2019). At the ecosystem level, plant secondary compounds (PSCs) drive key processes, such as litter degradation, and thus nutrient cycling (Chomel et al., 2016; Adamczyk et al., 2018). At the plant level, PSCs are employed to counter herbivore attacks, cope with changes in the environment, and ultimately ensure plant survival. PSCs are chemically diverse and their abundance shifts depending on biotic and abiotic stressors, but also across species and even organs within a species. The research highlighted in this collection combines short- and long-term studies in the greenhouse and in the field with a focus on phenolics and terpenes, both at the tree and ecosystem level.

Climate change factors, such as elevated atmospheric CO2 levels and/or enhanced temperature might lead to counteracting responses of trees and forest ecosystems. Holopainen et al. reviewed how forest ecosystems change depending on the influence of abiotic and biotic factors on PSC synthesis. Different aspects of climate change show counteracting effects on PSCs: for example, in response to rising CO2 total phenolics increased and terpenes decreased in the leaves, whereas warming showed the opposite effect (Holopainen et al.). According to Mäki et al. the temporal dynamics of PSCs such as monoterpenes in forest ecosystems also depend on temperature and rainfall and are linked to the degradation of litter. Berini et al. used an untargeted metabolomics approach for five tree species and concluded that effects of the environment are species-specific and differ depending on the abiotic factors in question. However, indirect consequences of measures to mitigate global change are likely to cause further problems. For example, fertilization with nitrogen to increase carbon sequestration might result in increased insect herbivory in the future. The phenolic levels of mature Picea abies trees were reduced after 13 years of nitrogen deposition which—in combination with higher nitrogen content in the needles—might severely decrease tree resistance to herbivory (Nybakken et al.). In contrast, in Picea abies, levels of the PSCs dihydroflavonol and flavan-3-ol increased after inoculation with a fungus related to bark beetle attack and negatively affected both bark beetle and fungus (Hammerbacher et al.).

The allocation of resources to PSCs is however not just regulated by environmental factors, but also by inherent aspects. For example, the genetic variation of Pinus pinaster in their

#### Edited and reviewed by:

*Henry D. Adams, Oklahoma State University, United States*

\*Correspondence: *Judy Simon<EMAIL_ADDRESS>
#### Specialty section:

*This article was submitted to Functional Plant Ecology, a section of the journal Frontiers in Plant Science*

Received: *23 May 2019* Accepted: *07 June 2019* Published: *26 June 2019*

#### Citation:

*Simon J and Adamczyk B (2019) Editorial: Plant Secondary Compounds in Forest Ecosystems Under Global Change: From Defense to Carbon Sequestration. Front. Plant Sci. 10:831. doi: 10.3389/fpls.2019.00831*

**4**

constitutive and induced resistance against herbivory was explained by PSC profiles rather than single compounds (López-Goldar et al.). Stark and Martz investigated whether dioecious Juniperus communis shows a gender specific response to a reduction in biomass because females might allocate more resources to defense (i.e., PSCs), whereas males invest more into growth. However, genders did not differ significantly in their PSC levels, most likely due to a high demand of PSCs to adapt to abiotic stresses (Stark and Martz). Moreover, Marques dos Santos et al. used a new method combining UHPLC, DAD, and MS/MS analyses to show that formylated phloroglucinols were found in glands embedded subdermally in leaves, flowers, and flower buds in eight eucalypts and one Corymbia species.

In conclusion, PSCs are important drivers of ecosystem processes and directly affect the interactions between trees and other organisms as well as the environment. Considering that most studies investigate tree seedlings or only the short-term

#### REFERENCES

effects on PSCs (Holopainen et al.), the new knowledge gained from the studies presented in this research topic is a further step in understanding the responses of trees to biotic and abiotic stressors and the underlying mechanisms at the tree and forest level, and ultimately in developing and evaluating sustainable management strategies to adapt forests to global change.

#### AUTHOR CONTRIBUTIONS

All authors listed have made a substantial, direct and intellectual contribution to the work, and approved it for publication.

# ACKNOWLEDGMENTS

We would like to thank the authors, reviewers, and the Frontiers in Plant Science Editorial Office for their support in creating this research topic.

biodiversity in European temperate forests. Global Change Biol. 25, 536–548. doi: 10.1111/gcb.14503

**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2019 Simon and Adamczyk. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

# Climate Change Effects on Secondary Compounds of Forest Trees in the Northern Hemisphere

Jarmo K. Holopainen1 \*, Virpi Virjamo2 , Rajendra P. Ghimire1 , James D. Blande1 , Riitta Julkunen-Tiitto2 and Minna Kivimäenpää1

1 Department of Environmental and Biological Sciences, Kuopio Campus, University of Eastern Finland, Kuopio, Finland, 2 Department of Environmental and Biological Sciences, Joensuu Campus, University of Eastern Finland, Joensuu, Finland

Plant secondary compounds (PSCs), also called secondary metabolites, have high chemical and structural diversity and appear as non-volatile or volatile compounds. These compounds may have evolved to have specific physiological and ecological functions in the adaptation of plants to their growth environment. PSCs are produced by several metabolic pathways and many PSCs are specific for a few plant genera or families. In forest ecosystems, full-grown trees constitute the majority of plant biomass and are thus capable of producing significant amounts of PSCs. We summarize older literature and review recent progress in understanding the effects of abiotic and biotic factors on PSC production of forest trees and PSC behavior in forest ecosystems. The roles of different PSCs under stress and their important role in protecting plants against abiotic and biotic factors are also discussed. There was strong evidence that major climate change factors, CO2 and warming, have contradictory effects on the main PSC groups. CO2 increases phenolic compounds in foliage, but limits terpenoids in foliage and emissions. Warming decreases phenolic compounds in foliage but increases terpenoids in foliage and emissions. Other abiotic stresses have more variable effects. PSCs may help trees to adapt to a changing climate and to pressure from current and invasive pests and pathogens. Indirect adaptation comes via the effects of PSCs on soil chemistry and nutrient cycling, the formation of cloud condensation nuclei from tree volatiles and by CO2 sequestration into PSCs in the wood of living and dead forest trees.

#### Keywords: CO2, drought, ozone, phenolics, temperature, terpenes, UV-B, VOCs

#### INTRODUCTION

Plant secondary compounds (PSCs) have high chemical diversity with an estimated 200,000 compounds (Dixon and Strack, 2003). In higher plants, terpenoids (approximately 30,000 known compounds, Lämke and Unsicker, 2018), alkaloids (21,000) (Wink, 2010), and phenolic compounds (8,000) (Munné-Bosch, 2012) are the most diverse PSC groups. The majority of PSCs are related to plant chemical defense and herbivore pressure, which are considered the main drivers of PSC diversification (Lämke and Unsicker, 2018). Terpenoids originate directly from glycolysis of glucose via the mevalonate pathway or the methylerythritol phosphate (MEP) pathway (Bartram et al., 2006). Phenolic compounds originate from the shikimic acid pathway, which is related to the metabolism of carbohydrates and aromatic amino acids (Seigler, 1998; Lindroth, 2012).

#### Edited by:

Bartosz Adamczyk, University of Helsinki, Finland

#### Reviewed by:

Carsten Kulheim, Michigan Technological University, United States Ryo Nakabayashi, RIKEN, Japan

> \*Correspondence: Jarmo K. Holopainen<EMAIL_ADDRESS>
#### Specialty section:

This article was submitted to Functional Plant Ecology, a section of the journal Frontiers in Plant Science

Received: 09 May 2018 Accepted: 12 September 2018 Published: 02 October 2018

#### Citation:

Holopainen JK, Virjamo V, Ghimire RP, Blande JD, Julkunen-Tiitto R and Kivimäenpää M (2018) Climate Change Effects on Secondary Compounds of Forest Trees in the Northern Hemisphere. Front. Plant Sci. 9:1445. doi: 10.3389/fpls.2018.01445

**6**

Most of the alkaloids are derived from amino acid precursors (Wink, 2010), but coniferous alkaloids are synthesized via the polyketide pathway (Seigler, 1998). Terpenes and phenolics are the most extensively studied PSCs in forest trees (Lindroth, 2012; Lämke and Unsicker, 2018), while alkaloids have been considered in relatively few studies (Virjamo et al., 2014). Alkaloids and phenolics may compete more with protein synthesis than terpenoids (Koricheva, 2002). Up to 10% of recently fixed carbon can be allocated to volatile organic compounds (VOCs) in stressed plants (Peñuelas and Staudt, 2010), while stored terpenoids typically require secretory structures such as resin canals and glandular trichomes to avoid autotoxicity (Goodger et al., 2013).

At the global level, climate change (CC) is strongly linked to increasing CO2 concentration in the atmosphere, which affects temperature globally (IPCC, 2014). Depletion of the stratospheric ozone (O3) layer is partly affected by elevated CO2 and leads to increased UV-B radiation (IPCC, 2014). In the lower atmosphere (troposphere) O3 acts as a greenhouse gas and is phytotoxic to plants (Vapaavuori et al., 2009; Lindroth, 2010). At the regional scale, global warming affects arctic and boreal areas most strongly and will include drastic changes, e.g., in precipitation, drought, and cloudiness (shading), affecting temperature-dependent ecosystem processes in forests such as nutrient availability (IPCC, 2014).

Climate change directly affects the abiotic conditions of forest trees and their growth, physiology and defense including induced PSC production (Metlen et al., 2009; **Figure 1**). The responses of other organisms, such as microbial plant pathogens, herbivorous insects and arrival of exotic invasive herbivores and pathogens will alter the biotic stress burden of trees (Donnelly et al., 2012). The richness of chemical compounds originating from PSCs in forest ecosystems is affected by PSC reactivity (Kundu et al., 2012). Many of the volatile reaction products form secondary organic aerosols (SOA), which may affect cloud formation (Zhao et al., 2017; Rose et al., 2018; Scott et al., 2018) and provide important ecosystem – atmosphere feedback, thus helping vegetation to adapt to CC (e.g., Niinemets, 2018). SOA particles may become wet or dry deposited on forest vegetation and accumulate in soil (Holopainen et al., 2017) together with litter PSCs (Smolander et al., 2012).

We summarize the major results of literature reviews and meta-analyses on the quantitative responses of tree PSCs to specific CC-related abiotic factors, with restriction to literature up to 2015 in **Figure 2**. Recent results of PSC (after 2015) and plant VOC (after 2012) research are then reviewed. We then discuss the roles of multiple CC-related stresses on PSCs of forest trees and the potential of non-volatile and volatile PSCs to modulate plant adaptation to CC and their involvement in climate feedback.

#### SUMMARY OF OLDER REVIEWS

Terpenoids and phenolic compounds in leaves and terpenoids in emissions are the most frequently studied PSCs of forest trees in response to CC-related factors (**Figure 2**). Major CC factors such as elevated CO2 and warming have the most distinctive, and often contrasting effects on PSCs. Greater availability of CO2 increases phenolics in foliage and reduces terpenoids both in foliage and in emissions, while warming reduces phenolics in foliage and increases terpenoids in foliage and emissions (Zvereva and Kozlov, 2006; Peñuelas and Staudt, 2010). CO2+warming increased phenolics in foliage, but reduced phenolics in woody tissues of woody plants (Zvereva and Kozlov, 2006). O3 effects on foliar concentrations of terpenes and volatile emissions have been variable, but increased phenolic compounds have been found. One reason for variable responses of terpenes could be withinspecies variation in O3 sensitivity (Valkama et al., 2007) and variable O3 reactivity of terpenes (Blande et al., 2014). Major terpenoid groups such as monoterpenes (MT) and sesquiterpenes (SQT) in foliage have been less studied separately under most of the other CC related stresses.

#### RESPONSES TO ABIOTIC FACTORS – PSC CONCENTRATIONS IN TREES

Certain plant PSC groups such as flavonoids (Lavola et al., 2013) and MTs (Semiz et al., 2007) are under strong genotypic control. This may increase the potential for forest tree chemical defenses to evolutionarily adapt to environmental changes (Lavola et al., 2013). However, high within-species variation in PSCs is problematic for short-term experimental set-ups and may mask the effects of environmental factors on PSC concentrations and emissions.

## Elevated CO2 and Warming

Total leaf phenolics, particularly condensed tannins (Julkunen-Tiitto et al., 2015), follow the traditional carbon-nutrient balance hypothesis (Bryant et al., 1983), which predicts that photosynthesised carbon will be diverted to carbon-rich PSCs, if photosynthesis or CO2 availability are at a high level and nutrient availability limits carbon allocation to plant growth (Koricheva et al., 1998). However, many flavonoids and other phenolics, and particularly terpenoids, do not support that hypothesis (Lindroth, 2012). Recent studies have confirmed a trend with foliar phenolics; doubled ambient CO2 increased salicylates and phenolic acids (Sobuj et al., 2018), anthocyanins and flavonoids (Vanzo et al., 2015; Nissinen et al., 2016), while less than doubled CO2 did not affect phenolics (McKiernan et al., 2012; Randriamanana et al., 2018). Furthermore, some phenolic groups, such as phenolic glycosides, are not as responsive (Jamieson et al., 2017). In Eucalyptus spp., there was no observed effect of elevated CO2 (McKiernan et al., 2012) or elevated CO2 decreased (Bustos-Segura et al., 2017) stored foliar terpenes.

Reduction in foliar phenolics at elevated temperatures (Julkunen-Tiitto et al., 2015) has been explained by a dilution effect whereby more carbon was allocated to growth supporting structures with turnover of phenolics slow or even absent (Kosonen et al., 2012). Recently, it has been reported that warming reduces flavonoids (Nissinen et al., 2016; Zhang et al., 2018), salicylates and phenolic acids (Sivadasan et al., 2018) in deciduous trees and total phenolics in a conifer (Zhang et al., 2018). Flavonoids, alkaloids, and saponins (triterpenes) were

increased in Robinia pseudoacacia grown in warmer open-top chambers compared to seedlings grown in an open field (Zhao et al., 2016). However, enclosure may significantly affect foliar PSCs (Peltonen et al., 2005).

Warming induced increase in foliar terpenes (Valkama et al., 2007; Julkunen-Tiitto et al., 2015) is possibly explained by improved plant growth and larger storage space for stored terpenes such as MTs and resin acids (Sallas et al., 2003). Recent results showing higher MT concentrations in xylem resin (Ferrenberg et al., 2017) and increased number of needle resin canals (Kivimäenpää et al., 2016) in Pinus spp. in warmer environments supports this view. Warming (+2 ◦C) increased concentrations of Picea abies needle alkaloids (Virjamo et al., 2014) and parental temperature range correlated with conifer alkaloid concentrations (Gerson et al., 2009; Virjamo and Julkunen-Tiitto, 2016).

# O3

O3 is phytotoxic and reduces plant photosynthesis and growth (Bueker et al., 2015; Li et al., 2017) and induces substantial changes in protein activity, gene expression, signaling pathways, and metabolism even before any tissue damage can be detected (Bueker et al., 2015; Vainonen and Kangasjärvi, 2015). Activated PSC metabolism (increased biosynthesis and reduced turnover) explains the increased concentrations of phenolic and terpenoid PSCs in tree foliage found in a meta-analysis (Valkama et al., 2007). In response to O3 treatment, elevated levels of total phenolics (Gao et al., 2016), condensed tannins (Couture et al., 2017) and flavonoids (Cotrozzi et al., 2018) were found in foliage of deciduous trees, but this was not the case in needles of coniferous trees (Riikonen et al., 2012).

# Drought

Drought in warm periods is expected to become the most frequent widespread climatic extreme that negatively affects terrestrial ecosystems (Schwalm et al., 2017). Forests in southern Europe will face stronger summer drought than those in the north (Ruosteenoja et al., 2018). The observed trends in PSCs are not strong, although reduction of some terpenes and phenolics have been reported (Koricheva et al., 1998; Julkunen-Tiitto et al., 2015). In Quercus ilex leaves, moderate drought periods resulted in higher concentrations of total polyphenolics (Nogues et al., 2014; Rivas-Ubach et al., 2014). Drought stress did not affect alkaloid concentrations in conifers (Gerson and Kelsey, 2004). Increasing severity of drought increased resin canal density and resin acid content in the stem wood of Pinus sylvestris (Heijari et al., 2010). Contents of most MTs and sesquiterpenoids in needles increased with increasing severity of water deficit, but

FIGURE 2 | Summary of the results in comprehensive reviews and meta-analyses of plant PSCs responses in their concentration or emissions in forest trees under climate change related stresses. This tabulation covers nearly 400 original scientific articles. Type of stresses: O3 = elevated ozone, CO2 = elevated carbon dioxide, UV-B = elevated UV-B radiation. The tabulation gives the direction of PSC responses to single environmental factors, except for the combined O3+CO2 and CO2+warming effects. Arrow direction (increase or decrease) shows the effect size (significant or highly significant effect in meta-analysis) or the amount of evidence in reviews. Horizontal bidirectional arrow indicates the studies with non-significant results and up and down arrows in the same cell indicate significant results in both directions dominate. Question mark indicates that the reviews and meta-analyses did not provide enough data. Gray arrows in CO2, warming and CO2+warming columns indicate woody tissue responses as specified in reference 2. Small numbers in the upper-right corner of reach cell indicate the source reference: 1 = Koricheva et al., 1998; 2 = Zvereva and Kozlov, 2006; 3 = Valkama et al., 2007; 4 = Peñuelas and Staudt, 2010; 5 = Lindroth, 2012; 6 = Julkunen-Tiitto et al., 2015; 7 = Robinson et al., 2012; 8 = Li et al., 2017. References 1–3 and 7–8 are meta-analyses and 4–6 literature reviews. Meta-analyses give the size of the effect, and the reviews give the frequency of observations in each response categories (–, 0, +).

resin acids showed the opposite trend and polyphenols did not respond (Sancho-Knapik et al., 2017). Drought slightly increased the needle MT contents of the coastal, but not of the interior Pseudotsuga menziesii provenance (Kleiber et al., 2017).

#### Shading

Shading limits photosynthesis and it is predicted that less carbon is allocated to PSCs in shaded conditions (Koricheva et al., 1998). Giertych et al. (2015) tested the hypothesis with light-demanding and shade-tolerant deciduous tree species. All species had higher total phenolic content of leaves in sunny conditions, but shade tolerant species invested more carbon into growth when more light was available.

#### UV-B

Short-wave (280–315 nm) UV-B radiation induces tree foliage to produce more phenolic acids and flavonoids as protective pigments (Julkunen-Tiitto et al., 2015; Kaling et al., 2015). In Populus tremula seedlings, two flavonoid compounds increased under enhanced UV-B radiation (Nissinen et al., 2017). Filtering out solar UV-B radiation from reaching P. tremula in mountains lead to lower flavonoid concentration, but increased salicin in leaves, and salicylates and total phenolics in stem (Stromme et al., 2018). However, a three-year exposure of Salix myrsinifolia to 32% elevated UV-B in a field site did not result in significant changes in flavonoid concentrations, and in male trees total salicylates were even reduced (Ruuhola et al., 2018). Enhanced levels of UV-B radiation did not affect alkaloid concentrations in conifers (Virjamo et al., 2014).

#### RESPONSES TO ABIOTIC FACTORS – EMISSION OF VOLATILE PSCs

#### Warming

Warming is one of the most intensively studied factors in volatile PSC studies (Peñuelas and Staudt, 2010), due to VOC emissions being strongly light and temperature dependent and some, e.g., isoprene not being stored in leaves (Sharkey and Yeh, 2001). At the global scale, isoprene represents nearly 50% of the estimated biogenic VOC emission, and emissions of MTs and SQTs together comprise about 15 and 3%, respectively (Guenther et al., 2012). Warming increased emissions of volatile terpenoids most consistently (Peñuelas and Staudt, 2010) by stimulating biosynthesis (Loreto and Schnitzler, 2010; Fini et al., 2017) and temperature-dependent release from leaf storage structures (Grote and Niinemets, 2008; Peñuelas and Staudt, 2010). VOC emissions from storage structures are high in conifers, e.g., over

40% in Pinus, and very low in deciduous trees (Ghirardo et al., 2010). Differences in emission responses of MTs and SQTs to temperature might be caused by temporal storage of less volatile SQTs in leaf surface waxes (Joensuu et al., 2016), and their temperature-dependent adherence and release (Himanen et al., 2015).

Warming of +3 ◦C increased (∼70%) emission rates of isoprene-emitting and MT-emitting Quercus spp. (Staudt et al., 2017). Isoprene-emitting Populus spp. have an emission maximum at +45◦C even if grown in elevated CO2 (Potosnak et al., 2014; Niinemets and Sun, 2015). Long-term warming of 1 ◦C+ambient increased MT (fivefold), SQT (fourfold) and green leaf volatile (40%) emissions of Betula pendula (Hartikainen et al., 2012). Ontogeny of needle development in terpene-storing conifers affects variability in MT emission rate responses to warming (Esposito et al., 2016; Kivimäenpää et al., 2016; Ghimire et al., 2017; Tiiva et al., 2018). Some specific MTs of forest trees, particularly β-ocimene are known to be heat-stress indicators (Jardine et al., 2017).

# CO2

There is clear evidence that exposure to elevated CO2 reduces terpenoid-based plant VOC emissions (Peñuelas and Staudt, 2010; Potosnak et al., 2014; Niinemets and Sun, 2015; Mochizuki et al., 2017) and uncouples isoprene emissions from photosynthesis. Inhibition of isoprene synthesis becomes stronger with increasing CO2 concentration (Wilkinson et al., 2009; Rasulov et al., 2018) and isoprene is more responsive than MTs (Peñuelas and Staudt, 2010). There is still no consensus of the mechanisms that lead to the contrasting responses of photosynthesis and isoprene biosynthesis (Fini et al., 2017) and emission at elevated CO2 (Rasulov et al., 2018). In MTstoring gymnosperms the reduced MT needle concentration at elevated CO2 (Sallas et al., 2003) may partly explain reduced MT emissions.

# O3

Plant VOCs easily react with O3 and samples may be destroyed if ozone scrubbers are not used in sampling (Blande et al., 2014). O3 exposure experiments have shown that plant VOC responses are variable and depend on the O3 concentration (Peñuelas and Staudt, 2010). Acute ozone exposure can induce similar VOCs to herbivore damage (Blande et al., 2014). Moderately elevated O3 concentrations increase emissions of MTs in coniferous (Kivimäenpää et al., 2013, 2016; Ghimire et al., 2017) and deciduous trees (Carriero et al., 2016), but decrease isoprene emission in poplar (Yuan et al., 2016) and MT emissions in Larix were not affected (Mochizuki et al., 2017).

#### Drought

Warm and dry periods lead to drought stress; mild drought may increase VOC emissions (Peñuelas and Staudt, 2010; Bourtsoukidis et al., 2014; Yuan et al., 2016), not affect (Nogues et al., 2014) or have increasing-decreasing trend (Simpraga et al., 2011), while severe drought results in a reduction of emissions (Peñuelas and Staudt, 2010; Rodriguez-Calcerrada et al., 2013; Luepke et al., 2016; Marino et al., 2017; Saunier et al., 2017; Staudt et al., 2017). Drought stimulated herbivore-induced GLV and MT emissions in Alnus glutinosa (Copolovici et al., 2014).

#### Shading

Shading by branch position can reduce isoprene and MT emissions from forest trees (Esposito et al., 2016; Juran et al., 2017; van Meeningen et al., 2017). This suggests that cloudiness that reduces penetration of solar radiation to the canopy (Niinemets, 2018) may reduce carbon allocation to volatile PSCs. However, in the canopy of Fagus sylvatica, MT emissions were highest in the semi-shaded leaves (Simpraga et al., 2013).

## COMBINED ABIOTIC FACTORS VERSUS NON-VOLATILE AND VOLATILE PSCs

The effects of eutrophication [nitrogen (N) deposition and forest fertilization] on tree VOCs are poorly known, although it appears to slightly increase isoprene emissions (**Figure 2**; Peñuelas and Staudt, 2010). Generally, exposure to additional abiotic factors may mitigate, intensify or not affect foliar chemical defense (Sallas et al., 2001; Zvereva and Kozlov, 2006; Vapaavuori et al., 2009) or VOC (Kivimäenpää et al., 2016; Ghimire et al., 2017; Tiiva et al., 2017) responses to the first factor. For example elevated O3 and N availability had additive effects on terpenoid emissions from a deciduous tree (Carriero et al., 2016) and conifer (Kivimäenpää et al., 2016), whereas N in addition to warming suppressed the emissions of non-oxygenated MTs from a conifer (Ghimire et al., 2017). In Populus sp. N addition (Yuan et al., 2017) or drought (Yuan et al., 2016) did not mitigate the negative effects of O3 on isoprene emission.

An elevated CO2-related decrease of terpenoids in conifer needles was mitigated by elevated temperature and CO2-induced increase in phenolics was intensified by elevated temperature (Zvereva and Kozlov, 2006). Furthermore, elevated CO2 may mitigate a typical O3 response such as ozone-induced increase of some phenolics in B. pendula (Vapaavuori et al., 2009), but in Larix CO2 and O3 did not have interactive effects on volatiles (Mochizuki, et al., 2017). Along altitudinal gradients, the total phenolics in Quercus robur foliage were found to decrease at lower elevations where temperature was warmer (Abdala-Roberts et al., 2016). Our analysis shows that in addition to a warminginduced reduction in total phenolics, elevated UV-B radiation may increase flavonoids and phenolic acids in foliage (Julkunen-Tiitto et al., 2015). However, recent combined warming and UV-B exposure outdoors has confirmed that warming has a stronger increasing effect on leaf phenolics (Nissinen et al., 2017) and terpenoid emissions (Maja et al., 2016) than elevated UV-B radiation.

# PLANT PSCs AND BIOTIC STRESS

Abiotic stresses affect growth, reproduction and behavior of herbivorous insects and severity of microbial plant pathogens and their biotic stress on trees (**Figure 1**). Furthermore, arrival of exotic invasive herbivores and pathogens with warming

related to CC may add another threat, because local tree species have not adapted to use PSCs to defend against these invasive species (Donnelly et al., 2012). Increased accumulation of PSCs in plant tissues under elevated atmospheric CO2 resulted in compensation feeding and more severe defoliation (Docherty et al., 1996). Warming may reduce constitutive PSCs in foliage (Stark et al., 2015) and improve (Grainger and Gilbert, 2017) or reduce (Ghimire et al., 2017) insect performance and lead to induced production of non-volatile (Rubert-Nason et al., 2015) or volatile PSCs (Blande et al., 2010; Holopainen and Gershenzon, 2010).

#### PSC TRANSMITTED ECOSYSTEM FEEDBACKS TO CLIMATE CHANGE

Forests are a globally important sink of atmospheric CO2 (Pan et al., 2011; Pukkala, 2018). Carbon is sequestered in soil carbon (44%), living biomass (42%), deadwood (8%), and litter (5%) (Pan et al., 2011). Reducing forest harvesting and adding dead wood as large trees is the fastest way to increase CO2 sequestration in boreal forests (Pukkala, 2018). Reduced decay rate of dead wood decreases release of CO2 back to the atmosphere. Concentrations of both resin acids (Karppanen et al., 2007) and stilbenes (Ioannidis et al., 2017) in heartwood of Pinus sp. can exceed 10% of dry weight and significantly reduce decay rate of dead wood by fungal rot (Karppanen et al., 2007) and by woodborers (Nerg et al., 2004).

The PSCs are released into the soil (Kainulainen et al., 2003) and atmosphere (Aaltonen et al., 2011) faster from the needle litter than dead wood. In conifer forests, MTs contribute over 90% of litter emissions peaking in early summer and during the needle fall in autumn (Aaltonen et al., 2011). After 19-months of decomposition, pine needles have lost 96, 79, and 86 percent of initial concentrations of MTs, resin acids and total phenolics, respectively (Kainulainen et al., 2003). Warm periods in the boreal conifer forests increase reactive MT emissions to the forest atmosphere and stimulate formation of SOA, particularly in evenings (Rose et al., 2018) and potentially stimulate cloudiness (Joutsensaari et al., 2015; Zhao et al., 2017), thereby reducing penetration of solar radiation to vegetation and mitigating warming and drought stress (Niinemets, 2018).

Volatile PSCs have several functions in the species interactions affecting herbivores and their natural enemies and activating defenses in healthy plants (Blande et al., 2014). CC factors also affect herbivore-induced volatiles, but studies on forest trees and woody plants remain limited (Holopainen and Gershenzon,

#### REFERENCES

2010). Non-volatile PSCs released from plant leaf and needle litter accumulate in soil and affect C or net N mineralization in forest soils (e.g., Smolander et al., 2012). Growing three seasons at elevated CO2 and O3 did not affect concentrations of total phenolics and MTs in P. sylvestris needles, but at elevated O3 resin acids were increased. However, during decomposition this difference in needle litter was lost (Kainulainen et al., 2003). In leaf litter of B. pendula growing at elevated CO2 and O3 concentrations, several phenolic compounds were elevated, and the growth of juvenile earthworms was reduced (Kasurinen et al., 2007) suggesting that CC reduced quality of food for soil animals. In Alnus incana litter, UV-A and UV-B exclusion affected concentrations of phenolic groups variably, whereas in birch litter there were no significant differences in phenolic compounds (Kotilainen et al., 2009).

#### CONCLUDING REMARKS

There is strong evidence that CO2 increases phenolic compounds in foliage, but limits terpenoids in foliage and emissions, while warming decreases phenolic compounds in foliage, but increases terpenoids in foliage and emissions. CO2 in combination with other abiotic stresses has more variable effects. As current PSC information is mostly from tree seedlings, long-term experiments covering the whole ontogeny of trees in forest ecosystems are needed to better understand CC effects on PSCs in trees. Assessment of the role of volatile PSCs in sun-screening and nonvolatile PSCs in control of decay rate of carbon storage in dead wood will be necessary to improve our knowledge of the capacity of PSCs to mitigate CC. Transgenic trees with silenced or overexpressed genes for key volatile PSCs (Kaling et al., 2015; Vanzo et al., 2015) will be important tools in this task.

#### AUTHOR CONTRIBUTIONS

JH drafted the manuscript. All authors listed have made a substantial, direct and intellectual contribution to the work, and approved it for publication.

#### FUNDING

This work was funded by University of Eastern Finland BORFOR Top Research Area and Academy of Finland, projects nos. 278424, 267360, 305343, and 309425.

oak species. Am. J. Bot. 103, 2070–2078. doi: 10.3732/ajb.16 00310

**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2018 Holopainen, Virjamo, Ghimire, Blande, Julkunen-Tiitto and Kivimäenpää. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

# Interannual and Seasonal Dynamics of Volatile Organic Compound Fluxes From the Boreal Forest Floor

Mari Mäki1,2 \*, Juho Aalto1,3, Heidi Hellén4 , Mari Pihlatie1,5,6 and Jaana Bäck1,2,6

1 Institute for Atmospheric and Earth System Research/Forest Sciences, Helsinki, Finland, 2 Department of Forest Sciences, Faculty of Agriculture and Forestry, University of Helsinki, Helsinki, Finland, 3 Department of Physics, Faculty of Science, University of Helsinki, Helsinki, Finland, 4 Finnish Meteorological Institute, Helsinki, Finland, 5 Department of Agricultural Sciences, Faculty of Agriculture and Forestry, University of Helsinki, Helsinki, Finland, 6 Viikki Plant Science Centre, University of Helsinki, Helsinki, Finland

#### Edited by:

Judy Simon, Universität Konstanz, Germany

#### Reviewed by:

Jürgen Kreuzwieser, University of Freiburg, Germany Ivika Ostonen, University of Tartu, Estonia

> \*Correspondence: Mari Mäki<EMAIL_ADDRESS>
#### Specialty section:

This article was submitted to Functional Plant Ecology, a section of the journal Frontiers in Plant Science

Received: 26 October 2018 Accepted: 05 February 2019 Published: 22 February 2019

#### Citation:

Mäki M, Aalto J, Hellén H, Pihlatie M and Bäck J (2019) Interannual and Seasonal Dynamics of Volatile Organic Compound Fluxes From the Boreal Forest Floor. Front. Plant Sci. 10:191. doi: 10.3389/fpls.2019.00191 In the northern hemisphere, boreal forests are a major source of biogenic volatile organic compounds (BVOCs), which drive atmospheric processes and lead to cloud formation and changes in the Earth's radiation budget. Although forest vegetation is known to be a significant source of BVOCs, the role of soil and the forest floor, and especially interannual variations in fluxes, remains largely unknown due to a lack of long-term measurements. Our aim was to determine the interannual, seasonal and diurnal dynamics of boreal forest floor volatile organic compound (VOC) fluxes and to estimate how much they contribute to ecosystem VOC fluxes. We present here an 8 year data set of forest floor VOC fluxes, measured with three automated chambers connected to the quadrupole proton transfer reaction mass spectrometer (quadrupole PTR-MS). The exceptionally long data set shows that forest floor fluxes were dominated by monoterpenes and methanol, with relatively comparable emission rates between the years. Weekly mean monoterpene fluxes from the forest floor were highest in spring and in autumn (maximum 59 and 86 µg m−2 h −1 , respectively), whereas the oxygenated VOC fluxes such as methanol had highest weekly mean fluxes in spring and summer (maximum 24 and 79 µg m−2 h −1 , respectively). Although the chamber locations differed from each other in emission rates, the inter-annual dynamics were very similar and systematic. Accounting for this chamber location dependent variability, temperature and relative humidity, a mixed effects linear model was able to explain 79–88% of monoterpene, methanol, acetone, and acetaldehyde fluxes from the boreal forest floor. The boreal forest floor was a significant contributor in the forest stand fluxes, but its importance varies between seasons, being most important in autumn. The forest floor emitted 2–93% of monoterpene fluxes in spring and autumn and 1–72% of methanol fluxes in spring and early summer. The forest floor covered only a few percent of the forest stand fluxes in summer.

Keywords: biogenic volatile organic compound, flux, forest floor, temperature, seasonality, vegetation, decomposition

# INTRODUCTION

fpls-10-00191 February 20, 2019 Time: 18:19 # 2

Global forest ecosystems are the largest existing source of biogenic volatile organic compounds (BVOCs) (Guenther et al., 1995). BVOC emissions are dominated by isoprene and monoterpenes (Lathière et al., 2005; Guenther et al., 2012), and they have a crucial role in the atmosphere as their oxidation products drive secondary organic aerosol (SOA) formation (Kulmala et al., 1998, 2013). SOA formation from volatile organic compounds (VOCs) through oxidation processes and SOA particles can change the Earth's radiation budget as a cooling feedback mechanism (Virtanen et al., 2010; Kourtchev et al., 2016). The cooling feedback mechanism could become stronger if the temperature were to increase in high latitudes by an estimated 6–8◦C by 2100 (IPCC Fifth Assessment Report (AR5), 2014), boosting SOA load and cloud formation by increasing VOC emissions from the boreal biosphere (Kulmala et al., 2014), which is a well-documented VOC source (Rinne et al., 2007). VOCs affect hydroxyl (OH) radical and ozone formation and oxidation processes (Jacob et al., 2005; Hüve et al., 2007). Ozone formation in photochemical reactions requires NOx and reactive VOCs (Crutzen, 1979; Logan, 1985), and for this reason, it is important to quantify seasonal VOC emission fluxes from different sources. These sources should be quantified more accurately, because several studies have shown that measured and modeled ozone deposition fluxes and OH radical reactivities include significant differences (Mogensen et al., 2011; Wolfe et al., 2011; Rannik et al., 2012; Zhou et al., 2017). A gap in atmospheric oxidant sinks between measurements and models can be decreased by including forest floor emissions in these models.

The total VOC load in the atmosphere remains largely unknown because VOCs are difficult to measure and the boreal biosphere emits at least 25 different VOCs (Schallhart et al., 2018). VOCs are released by tree shoots (Aalto et al., 2014), stems (Vanhatalo et al., 2015) and forest floor (Aaltonen et al., 2013), and more specifically by decomposition processes, vegetative litter, root stores and plant VOC synthesis (Hayward et al., 2001; Leff and Fierer, 2008; Faubert et al., 2010). VOC synthesis of plants and microbes together with VOC volatilization are strongly affected by temperature (Guenther et al., 1993; Kesselmeier and Staudt, 1999). Temperature dependent VOC fluxes from the boreal forest floor (Aaltonen et al., 2013; Mäki et al., 2017) can increase when soil temperatures increase in a warming climate (Fan et al., 2014). Warming can also change vegetation cover and directly influence the abundance of the different VOC sources hence affect the amount and blend of emitted VOCs. In this study, we tested whether we could find evidence of such changes in an 8-year continuous data set of forest floor VOC fluxes. Previously reported measurements have been rather shortterm, and only few cover more than one growing season (Hellén et al., 2006; Asensio et al., 2007, 2008; Faubert et al., 2010).

Volatile organic compound fluxes between the forest floor and the atmosphere are dominated by isoprenoids (e.g., isoprene, monoterpenes, sesquiterpenes) and oxygenated VOCs (alcohols, aldehydes, and ketones) (Hellén et al., 2006; Aaltonen et al., 2011, 2013; Mäki et al., 2017). VOC fluxes from the boreal forest floor are highest during spring and autumn (Hellén et al., 2006; Aaltonen et al., 2011, 2013; Mäki et al., 2017). High spring fluxes are likely connected to snowmelt, which increases soil moisture and accelerates microbial decomposition and leaf growth by exposing soil and leaves to radiation. In the northern ecosystems, VOCs are produced by evergreen and deciduous dwarf shrubs (Rinnan et al., 2013), mosses (Hanson et al., 1999), roots (Hayward et al., 2001), and decomposing litter (Isidorov and Jdanova, 2002; Isidorov et al., 2016). A warmer climate will lead to an earlier snowmelt and growing season start, which can cause increased VOC flux in spring and possibly affect the emission blend. A warmer climate can also change precipitation patterns and affect VOC emissions, because soil water content impacts plant metabolism and microbial decomposition (Staudt et al., 2002; Davidson and Janssens, 2006). High monoterpene load from the soil surface into the atmosphere in autumn is likely released by decomposing and decaying pine litter (Mäki et al., 2017), which contains monoterpene storages (Kainulainen and Holopainen, 2002). VOCs can also be released by freeze– thaw and dry–wet cycles (Asensio et al., 2007, 2008; Insam and Seewald, 2010) in spring and autumn.

In this study, our aim was to use the long-term field measurements with automated chambers to determine the dynamics of forest floor VOC fluxes and to estimate the contribution of forest floor fluxes to the whole forest ecosystem fluxes. We analyzed the effects of environmental and sitespecific variation (i.e., air and soil temperature, growing season length, soil moisture, and vegetation cover) on forest floor VOC exchange in order to develop a statistical model that could explain the interannual, seasonal, and daily patterns in emission rates.

# MATERIALS AND METHODS

#### Continuous Chamber Flux Measurements and Supporting Data

We measured the forest floor VOC fluxes in the southern boreal forest (established in 1962) at the SMEAR II (Station for Measuring Ecosystem-Atmosphere Relations) station (61◦ 510 N, 24◦ 170 E, 180 m above sea level). Canopy basal area is covered by Pinus sylvestris (75%), Picea abies (15%), and broadleaf species (10%) such as Betula pendula and Sorbus aucuparia.  The soil type is Haplic podzol, and organic soil contains the largest proportion of carbon (356 mg g−1 ) and nitrogen (13 mg g−1 ) compared with mineral soil (5–32 and ∼1 mg g−1 , respectively).

We measured the VOC fluxes using three automated dynamic (flow-through) chambers (80 cm × 40 cm × 25 cm) installed on permanent stainless-steel soil collars (80 cm × 40 cm × 10 cm) every spring (Aaltonen et al., 2013). The shared volume of chamber and soil collar was 112 L. The automated chambers

were connected to a quadrupole proton transfer reaction mass spectrometer (quadrupole PTR-MS, Ionicon Analytik, Innsbruck, Austria, de Gouw and Warneke, 2007) and VOC fluxes were measured from April 20, 2010 to November 7, 2017. Chamber frames were made of aluminum and the sides and top of the chamber were covered by a transparent fluorinated ethylene propylene (FEP) film (0.05 mm) (Aaltonen et al., 2013). Mixing the air with the chamber closed was done with two fans (wind speed 2.2 m s−1 and air volume of flowthrough 167 L min−1 ) and the chamber opening direction was toward the south. We drew air samples from the chambers using a flow of 1.1–4.0 L min−1 through a 64 m heated FEP tube (inner diameter 4 mm) and determined the VOC concentrations from a side flow (0.1 L min−1 ) through a polytetrafluoroethylene (PTFE) tube (Aaltonen et al., 2013) (**Figure 1**). Chamber closure time was 15 min. To avoid underpressure inside the chambers, we substituted the sample flow with well-mixed ambient air using a 30–100% higher flow rate compared with the sampling flow. The VOC concentrations of the substitute air were measured immediately before every chamber enclosure. We measured the masses (amu) of methanol (33), acetaldehyde (45), acetone (59), isoprene (69), benzene (79), monoterpene fragment (81), methyl butenol (87), toluene (93), hexenal (99), hexanal (101), monoterpenes (137), and methyl salicylate (153). We measured all the masses every year, except benzene, methyl salicylate, hexenal, and hexanal between 2010 and 2012 and methyl butenol and toluene between 2013 and 2017. The calibration gas (Apel-Riemer Environmental, Inc., Denver, CO, United States) included methanol, acetaldehyde, acetone, isoprene, benzene, toluene, hexanal (except in summer 2010), and α-pinene. The chamber system was automated and every chamber was measured eight times a day (Aaltonen et al., 2013). The instrument was calibrated 1–4 times per month based on the scheme presented by Taipale et al. (2008). The quadrupole PTR-MS has a high sensitivity and a

short response time (Taipale et al., 2008), suitable to quantify oxidized VOCs (Aaltonen et al., 2013), but is unable to separate compounds with the same molecular mass such as different monoterpenes.

We measured the forest floor carbon dioxide (CO2) and H2O fluxes from the same chambers using infrared light absorption analyzers (URAS4, Hartman and Braun, Frankfurt, Germany), until spring 2013, when the instrument was replaced with a LICOR LI-840A (LI-COR, Lincoln, NE, United States). We estimated the H2O and CO2 fluxes using the same mass balance equation (Kolari et al., 2012) that was used for the VOC flux calculations. Precipitation was monitored with an FD12P weather sensor (Vaisala Oyj, Helsinki, Finland) at a height of 18.0 m. Soil volumetric water content and soil temperature were measured from four to five pits and at each pit on every soil horizon (H horizon, eluvial E horizon, illuvial B horizon, and parent material C horizon). The mean thickness of the organic soil is 6.0 cm, the eluvial E horizon 2.0 cm and the illuvial B horizon 16 cm at the SMEAR II stand (Mäki et al., 2017). The soil moisture and soil temperature sensors were placed in the middle of each soil horizon. Soil temperatures were measured at 15-min intervals using silicon temperature sensors (Philips KTY81–110, Philips Semiconductors, Eindhoven, Netherlands). Volumetric water contents were recorded at 60-min intervals using the timedomain reflectometry method (TDR-100, Campbell Scientific, Ltd., United Kingdom). Photosynthetically active radiation (PAR) was measured using an LI-190 quantum sensor (Li-Cor Biosciences, Lincoln, NE, United States) above the canopy. The monthly total litterfall and the fraction of needles was determined with 21 L collectors (Mäki et al., 2017). We determined plant coverage (%) of ericoid shrubs such as Vaccinium vitis-idaea and Vaccinium myrtillus, mosses such as Pleurozium schreberi, Dicranum polysetum, Dicranum scoparium, and Hylocomium splendens, and other plant species such as Linnaea borealis and Deschampsia flexuosa for each measurement chamber based

on visual assessment in 2010 and 2017 (**Table 1**). Total plant coverage of soil collars was 55–94% in 2017.

#### Continuous Ecosystem Flux Measurements

fpls-10-00191 February 20, 2019 Time: 18:19 # 4

In order to assess the importance of forest floor fluxes to the total VOC budget of the stand, we compared the measured VOC fluxes from the forest floor with the total ecosystem fluxes. The data set of ecosystem fluxes was published in Rantala et al. (2014, 2015). Ecosystem fluxes of 27 different VOCs were measured with the profile method using the quadrupole PTR-MS (Ionicon Analytik GmbH, Innsbruck, Austria) with a 2-s sampling time from two heights in the canopy (4.2 and 8.4 m) and four heights above the canopy (16.8, 33.6, 50.4, and 67.2 m) (Rantala et al., 2014, 2015). Measurements were performed eight times a day using continuous flow (33 L min−1 ) through a 100 m long sample tube (PTFE, inner diameter 8 mm). The air sample entered the PTR-MS as a side flow through the 4 m long sampling tube (PTFE, inner diameter 1.6 mm) (Rantala et al., 2014). The quadrupole PTR-MS completed a whole measurement cycle within 1 h, in which the quadrupole PTR-MS ran through each measurement height nine times, with one individual repetition at each height taking 1 min (Rantala et al., 2014). Losses of monoterpenes and oxygenated VOCs in the tube walls are typically small (Kolari et al., 2012), because flow rate is relatively high and the ratio of the tube wall area and tube diameter is relatively small. We measured VOC fluxes for the whole ecosystem from May 28, 2010 to September 8, 2014. The calculations of 30-min average volume mixing ratios were described by Taipale et al. (2008). The VOC fluxes were estimated from the profile measurements using the surface layer profile method based on the Monin-Obukhov theory, and calculations were described in detail by Rantala et al. (2014). For the flux calculations, the ambient temperature was measured from each height using Pt-100 sensors, and turbulence parameters were determined using the three-dimensional acoustic anemometer (Solent 1012R2, Gill Instruments, Ltd., United Kingdom) at a height of 23 m.

#### Chamber Flux Calculations

Flux calculation was based on the mass balance equation (Kolari et al., 2012). The rate of change of concentrations during the first 400 s as well as the known concentrations of substitute air were taken into account. We quantified the VOC fluxes according to the VOC concentration change (C) during the chamber closure, which is derived from the mass balance equation (Hari et al., 1999), Eq. 1:

$$V\frac{dc}{dt} = E + F(C\_i - C) \tag{1}$$

where V is the chamber volume, E is the VOC emission rate (positive flux) or the VOC uptake rate (negative flux), F is the flow rate of ingoing air, and Ci is the VOC concentration of the air used to replace the sampled air volume. Calculating Eq. 1 for VOC concentration C as a function of time t after chamber closure leads to solution (Kolari et al., 2012):

$$C(t) = C\_0 + (\frac{C\_i - C\_0}{V} + \frac{E}{F})(1 - e^{-\frac{E}{V}}) \tag{2}$$

where C0 is the VOC concentration (µg m−3 ) at the time when the chamber was closed. The emission rate E (µg m−2 h −1 ) corresponds to the rate of change in concentration during the first 400 s after the chamber was closed (Eq. 2).

There was a negative exponential correlation between relative humidity (%) in the chamber headspace and water soluble VOC fluxes, and for this reason we only used methanol, acetone, and acetaldehyde fluxes measured under 75% relative humidity. This threshold led to removal of 74– 81% of the methanol, acetone, and acetaldehyde fluxes for the chambers. All the data analyses for methanol, acetone, and acetaldehyde were performed using filtered data. Relative humidity inside the chamber headspace was measured right before the closure.

We tested the normality of different VOC fluxes measured from the automated chambers (n = 3) with the Kolmogorov–Smirnov test (df = 2). As the data were nonnormally distributed, the statistical differences between years (**Table 2**) and between chambers (**Supplementary Table S1**) were determined using the non-parametric Kruskal–Wallis test (df = 2) at a significance level of < 0.05 (**Supplementary Table S1**).

Weekly mean monoterpene fluxes were estimated using the whole data set (**Figure 2**) and weekly mean methanol fluxes using daytime measurements from 9 a.m. to 8 p.m. (n = 3) (**Figure 3**), because nighttime measurements were mainly filtered away with a 75% relative humidity threshold. We determined

TABLE 1 | Total plant coverage (%) and coverage (%) of Vaccinium vitis-idaea, Vaccinium myrtillus, Linnaea borealis, and mosses of three measurement chambers in 2010 and 2017.

Plant coverage was determined visually from photographs. ∗Aaltonen et al. (2013).

TABLE 2 | Start and end dates of volatile organic compound (VOC) flux measurements and total annual litterfall (gDW m−2 ); environmental conditions calculated from March to October: annual mean temperature (◦C), temperature sum (sum of daily mean temperatures > 5 ◦C), mean soil moisture in the O horizon (m3 m−3 ), and precipitation sum (mm); timing of snowmelt and first snow in each year; annual mean monoterpene, methanol, acetone, and acetaldehyde fluxes (µg m−2 h −1 ) with standard deviation of three measurement chambers between 2010 and 2017 at the SMEAR II station.

Snowmelt date is when snow cover is mainly melt.

weekly mean methanol fluxes using nighttime measurements from 9 p.m. to 8 a.m. (**Supplementary Figure S1**). The mean monoterpene, methanol, and acetone fluxes were estimated at different times of day from the boreal forest floor in spring, summer, and autumn (**Figure 4**) based on the assumption that summer starts when daily mean temperature is over 10◦C, and autumn begins when daily mean temperature is below 10◦C. We determined the effect of chamber temperature and relative

FIGURE 4 | Mean (A) monoterpene, (B) methanol, and (C) acetone fluxes (n = 3 chambers), and standard deviation (µg m−2 h −1 ) during different times of the day from the boreal forest floor in spring, summer, and autumn, from 2010 to 2017. Summer starts when daily mean temperature is over 10◦C, and autumn begins when daily mean temperature is below 10◦C.

humidity on VOC fluxes from the forest floor (**Figure 5** and **Supplementary Figure S2**).

The cumulative sum (µg m−2 h −1 ) of methanol (33 amu), acetaldehyde (45 amu), acetone (59 amu), and monoterpenes (137 amu) for each 2-week period was calculated for each chamber and for the whole ecosystem using an unfiltered data set. The mean of the cumulative sums of three chambers was compared with the total ecosystem fluxes by calculating the proportion of soil fluxes relative to the whole ecosystem fluxes for each 2-week period (**Figure 6**).

#### The Mixed Effects Linear Model

We developed a mixed effects linear model to determine which parameters can be used to explain the fluxes of the different VOCs. Model parameters are presented in **Supplementary Table S2**. We used the effect of chamber temperature, relative humidity, and above-canopy PAR to model monoterpene, methanol, acetone, and acetaldehyde fluxes. VOC fluxes (V) were modeled by the mixed effects linear model (3):

$$V = B\_0 + B\_t + B\_R + B\_{tR} + \in,\tag{3}$$

where B0 represents a fixed intercept parameter, Bt represents fixed unknown parameters associated with chamber temperature, BR represents fixed unknown parameters associated with relative humidity of the chamber, and BtR represents fixed parameters for interaction of the measurement chamber with relative humidity and temperature. In the model (3), the error term ∈ is presumed to have the form:

$$
\epsilon \in \infty\_{CR} + \infty\_{CT} + \mu,\tag{4}
$$

where ∝ CR represents random parameters related to interaction of the measurement chamber (1, 2, and 3) and relative humidity of the chamber, ∝ CT represents random parameters related to interaction of the measurement chamber and chamber temperature, and u is an unobservable random error term.

#### Accuracy of the Chamber Measurement Method

We tested the chamber wall effects in laboratory conditions by flushing gas containing the study compounds into the chamber headspace, and the concentrations of ingoing and outgoing air were determined by sampling air into the Tenax TA-Carboback-B adsorbent tubes. Concentration of the calibration gas in the ingoing air was from 1.7 to 3.1 µg m−3 , varying with the compounds. The chamber was placed on a flat surface covered with a transparent FEP film (0.05 mm); air (flow rate 3 dm3 min−1 ) was moving into and out of the chamber through FEP tubes (inner diameter 6 mm), and we drew air samples from a side flow using a flow of 0.1–0.15 dm3 min−1 . We determined the concentrations of individual monoterpenes (α-pinene, camphene, β-pinene, 13-carene, p-cymene, 1,8-cineol, limonene, terpinolene, linalool,

and myrcene) and sesquiterpenes (longicyclene, isolongifolene, β-caryophyllene, aromadendrene, and α-humulene) from the adsorbent tubes by using a thermodesorption instrument (PerkinElmer TurboMatrix 650, Waltham, MA, United States) coupled with a gas chromatograph (PerkinElmer Clarus 600, Waltham, MA, United States) with a mass selective detector (PerkinElmer Clarus 600T, Waltham, MA, United States) (Mäki et al., 2017). Relative standard deviation (RSD, %) was calculated as a standard deviation between the four parallel samples taken during the chamber enclosure in the laboratory using constant concentrations of VOCs of ingoing air. RSD shows that the error of sampling and analytical method (thermal desorption– gas chromatography–mass spectrometry) was relatively low for both monoterpenes (5–14%) and sesquiterpenes (6–9%) (**Supplementary Table S3**).

#### RESULTS

## Interannual, Seasonal, and Diurnal Dynamics in Soil VOC Exchange

#### Interannual Dynamics

Forest floor VOC exchange was dominated by monoterpenes and oxygenated VOCs such as methanol, acetone, and acetaldehyde, while isoprene fluxes were mainly close to zero. Annual mean monoterpene fluxes ranged from 7 to 19 µg m−2 h −1 , methanol fluxes from −1 to 3 µg m−2 h −1 , acetone fluxes from 0 to 2 µg m−2 h −1 , and acetaldehyde fluxes from 1 to 2 µg m−2 h −1 (**Table 2**). The VOC fluxes showed comparable emission rates between years.

#### Seasonal Dynamics

Interestingly, the seasonal dynamics of monoterpene fluxes from the forest floor differed from those of oxygenated VOCs. The highest weekly mean monoterpene fluxes were observed from the forest floor in spring (May–June) and in autumn (September– October) (maximum 59 and 86 µg m−2 h −1 , respectively) (**Figure 2**). Weekly mean methanol fluxes were highest in spring and summer (May–August) (**Figure 3**: maximum 24 and 79 µg m−2 h −1 , respectively) based on daytime measurements from 9 a.m. to 8 p.m. We found that acetone and acetaldehyde fluxes followed a similar seasonal pattern to that of methanol. Acetone and acetaldehyde fluxes were clearly lower than monoterpene fluxes but were at the same level as methanol fluxes (**Table 2**).

#### Diurnal Dynamics

We determined the diurnal dynamics of the forest floor VOC fluxes in spring, summer, and autumn. The diurnal maximum fluxes for monoterpenes, methanol, and acetone were observed in spring (19, 4, and 3 µg m−2 h −1 ), summer (33, 27, and 9 µg m−2 h −1 ), and autumn (28, 10, and 1 µg m−2 h −1 ), respectively (**Figure 4**). The monoterpene and methanol fluxes were strongest during the daytime in all seasons, while acetone and acetaldehyde fluxes showed clear diurnal dynamics only in spring and summer. Relatively high monoterpene, methanol, and acetone fluxes were also observed from 6 p.m. to 8 p.m. in summer.

#### Temperature and Relative Humidity Effect on Forest Floor VOC Exchange

Our results showed that monoterpene, methanol, and acetone fluxes correlate with chamber temperature (**Supplementary Figure S2**). Chamber temperature explained 14–61% of methanol and 25–57% of acetone fluxes in spring and in summer (**Supplementary Figure S2**), but not in autumn, except in chamber 1 (55 and 51%). Monoterpene emissions showed correlation (7–50%) with chamber temperature from spring to summer and weak correlation in autumn (2–10%) (**Supplementary Figure S2**). Relative humidity had a significant effect on monoterpene and methanol fluxes (**Figure 5**).

#### Mixed Effects Linear VOC Emission Model

The model analysis showed that the VOC flux rates from the forest floor are indeed strongly affected by temperature and relative humidity. The model explained 79–88% of monoterpene, methanol, acetone, and acetaldehyde fluxes from the boreal forest floor (**Figures 7**, **8** and **Supplementary Figure S3**). This fairly simple statistical modeling approach is able to capture the individual behavior of each measurement chamber.

#### Importance of Forest Floor to Ecosystem VOC Exchange

We compared forest floor VOC emissions to the whole ecosystem emissions measured by the flux gradient method between 2010 and 2014 (Rantala et al., 2014, 2015). The forest floor accounted for 2–93% of monoterpene fluxes relative to forest stand fluxes in spring and autumn and 1–72% of methanol fluxes in spring and early summer (**Figure 6**). The role of forest floor in forest stand fluxes was only a few percent in summer. Fluxes were compared using the whole data set without filtering with 75% relative humidity. VOC deposition dominated in the forest floor or forest stand during some 2-week periods (negative values in **Figure 6**).

#### Uncertainties in VOC Exchange Measurements From the Forest Floor

The chambers differed from each other in emission rates but showed very similar and systematic temporal dynamics. Monoterpene and methanol fluxes were highest from chamber 1, where temperature was the highest (**Supplementary Table S1**). The acetaldehyde and acetone fluxes were mainly highest from chamber 1, but the differences between chambers were small. Chamber temperature was strongly correlated with ambient air.

# DISCUSSION

#### Interannual Dynamics

Our 8-year long data series of forest floor VOC exchange showed relatively small variation between the years. Annual mean monoterpene fluxes were very similar to earlier studies performed on the boreal forest floor (Aaltonen et al., 2011: α-pinene 0–14 µg m−2 h −1 ; Mäki et al., 2017: total monoterpenes 23 µg m−2 h −1 ; Wang et al., 2018: total monoterpenes 3–10 µg m−2 h −1 ). Also, mean fluxes of oxygenated VOCs, methanol, acetone, and acetaldehyde, were similar to earlier measurements at the same site in 2010 (Aaltonen et al., 2013: methanol −0.6 to 7.2 µg m−2 h −1 ; acetone −0.8 to 2.2 µg m−2 h −1 ; acetaldehyde 0.8–2.2 µg m−2 h −1 ).

Most of the interannual variability in monoterpene exchange resulted from variations in the temperature sum and precipitation between the years, while a similar trend was not observed for methanol, acetone, and acetaldehyde. Periods of high precipitation decreased monoterpene fluxes, which were seen also in low annual mean fluxes in 2011, 2012, and 2017. The effect of precipitation was assumed to be due to increased relative humidity and formation of water films on soil and leaf surfaces, which decreases VOC evaporation from surfaces. Annual mean monoterpene flux was also high in 2016 due to high monoterpene fluxes in October, which were likely released by fresh decomposing litter (Mäki et al., 2017; Wang et al., 2018).

# Seasonal Dynamics

The strong seasonal variation on monoterpene and VOC fluxes most probably results from seasonality in the production of VOCs via plant VOC synthesis (Aaltonen et al., 2011; Faubert et al., 2012) and litter decomposition (Hayward et al., 2001; Greenberg et al., 2012; Mäki et al., 2017). Temperature regulates plant VOC synthesis (Guenther et al., 1993), and hence this could explain the higher annual mean monoterpene fluxes during the warm summers (2010 and 2013) compared to the cold summers (2012 and 2017) (**Table 2**).

At our site, Vanhatalo et al. (2015) observed bursts of monoterpene from Scots pine stems during spring recovery in April. Similarly, a release of monoterpenes from belowground storages such as roots (Hayward et al., 2001) could explain the high forest floor monoterpene emissions during spring. Based on our study, it remains unclear whether monoterpenes were mostly emitted by litter or by roots, while the production in plants was likely small due to the fact the monoterpene emissions from all three chambers were similar in magnitude despite the differences in plant species coverage. Vegetation in chamber 1 was dominated by mosses that emit mostly isoprene and oxygenated VOCs and only minimal amounts of monoterpenes (Hanson et al., 1999; Hellén et al., 2006), while vegetation in chambers 2 and 3 were mostly dominated by Vaccinium spp. that emits monoterpenes (Aaltonen et al., 2011; Faubert et al., 2012). There were hardly any VOC flux measurements directly after snowmelt, while Hellén et al. (2006) found that seasonally highest monoterpene fluxes from the boreal forest floor were measured right after snowmelt at our measurement

site. High monoterpene emissions in autumn are linked to decomposing litter (Greenberg et al., 2012; Mäki et al., 2017) and the organic soil, which contains easily available carbon for microbial decomposition.

Seasonality of oVOC fluxes in the forest floor was comparable to those of the forest stand fluxes. Gross primary production of ground vegetation is generally highest from mid-June to mid-August at our site (Kolari et al., 2006). The maximum forest floor methanol fluxes coincide with the maximum biomass production, which suggests that new surface vegetation growth is a major methanol source at our measurement site. The seasonality of forest floor methanol emissions follows the seasonality of canopy methanol exchange (Aalto et al., 2014) indicating that the processes and sources of methanol are the same both in the canopy and in the forest floor. Other evidence is that oxygenated VOC fluxes were lowest from chamber 3 with the lowest vegetation cover from June to August (**Supplementary Table S1**). Methanol can also be released by microbes that synthesize VOCs (Bäck et al., 2010; Mancuso et al., 2015)especially in summer, when decomposer activity is high due to temperature driven enzyme activity (Davidson and Janssens, 2006). Methanol fluxes in early autumn are probably related to plant senescence – leaf litter is a strong methanol source (Warneke et al., 1999) and the senescence of Vaccinium myrtillus leaves is in early autumn (Kulmala et al., 2008).

#### Diurnal Dynamics

The diurnal dynamics in forest floor VOC exchange were clearly affected by temperature and radiation changes between the daytime and nighttime, which corresponds with the earlier observations at the site (Aaltonen et al., 2013). Monoterpene synthesis in plants is typically light and temperature dependent (Kesselmeier and Staudt, 1999), and similar light and temperature dependent VOC production has been demonstrated with ericoid shrubs, grasses, and mosses (Hellén et al., 2006; Faubert et al., 2010; Aaltonen et al., 2013; Lindwall et al., 2015; Kramshøj et al., 2016). Monoterpene and methanol fluxes are affected by temperature due to the volatility of these compounds (Guenther et al., 1993) and by radiation that heats leaf surfaces. Methanol fluxes are also strongly dependent on stomatal conductance (Nemecek-Marshall et al., 1995), which is high during daytime when plants maintain their photosynthesis by taking up CO2 through stomata and maintain water transport through transpiration.

Nighttime monoterpene emissions observed in spring and autumn (**Figure 4**) were likely affected by soil processes independent from light availability. Oxygenated VOC fluxes were close to zero in the nighttime, but we also observed deposition on leaf surfaces and chamber walls. Night-time deposition and daytime emissions of methanol were also observed from the bare temperate cropland in spring (Bachya et al., 2018). In our study, the nighttime deposition of methanol, acetone, and acetaldehyde is linked to lower temperatures and higher humidity in the chamber headspace. VOCs were likely released from the aqueous layer on plant and soil surfaces in the morning, when air humidity drops.

# Modeling Soil VOC Exchange

Chamber temperature explained monoterpene, methanol, and acetone fluxes from the forest floor (**Supplementary Figure S2**), similar to methanol and acetone fluxes from Pinus sylvestris shoots, which correlated with temperature (Aalto et al., 2014). Temperature dependence of monoterpenes is not as clear, because monoterpenes are emitted immediately from synthesis or with a delay from storage structures in resin ducts, glandular trichomes, or other storage structures (Laothawornkitkul et al., 2009). Litter also contains monoterpene storage structures and accelerates microbial decomposition (Hayward et al., 2001; Isidorov and Jdanova, 2002; Leff and Fierer, 2008; Gray et al., 2010; Isidorov et al., 2016). Monoterpene release from litter is likely directly linked to temperature due to missing monoterpene synthesis. However, low correlation between chamber temperature and monoterpene fluxes gives an indication that monoterpenes are released from and consumed simultaneously in the forest floor. Ramirez et al. (2010) showed that soil can be a sink for VOCs released by litter, like methanol that is used as a carbon source by bacteria via the ribulose monophosphate cycle and the serine cycle (Quayle and Ferenci, 1978). Different abiotic and biotic stresses also affect plant VOC emissions (Baldwin et al., 2006; Loreto and Schnitzler, 2010; Niinemets et al., 2013), which can make temperature responses of forest floor fluxes more complex.

The mixed effects linear model accounting for location dependent variability, temperature, and relative humidity was able to explain 79–88% of monoterpene, methanol, acetone, and acetaldehyde fluxes from the boreal forest floor (**Figures 7, 8** and **Supplementary Figure S3**). It seems that with such a multiannual data set, a fairly simple statistical modeling approach is able to capture individual behavior of each measurement chamber, while the challenge remains how to capture the spatial variation of soil VOC exchange. This model is not directly suitable to estimate forest floor VOC exchange in other ecosystems based on ambient temperature and relative humidity. The model clearly underestimates high methanol, acetone, and acetaldehyde fluxes, indicating that environmental factors other than temperature and relative humidity are the main drivers of these fluxes in the chambers. One likely reason is deposition of these water soluble molecules on moist leaf and chamber surfaces. Deposition should be modeled with a different mixed effects linear model, because the processes behind VOC deposition are different than the biological and physicochemical processes, which regulate production and evaporation of VOCs.

Volatile organic compound fluxes seemed to be strongly stimulated by temperature when the relative humidity effect is included (**Figures 7**, **8**). VOC fluxes were observed to decrease with increasing relative humidity (**Figure 5**). Therefore our results give an indication that VOC fluxes from the boreal forest floor will likely increase in a warming climate. The global mean surface temperature is expected to increase by a minimum of 0.3–1.7◦C under Representative Concentration Pathway (RCP) 2.6 and a maximum of 2.6–4.8◦C under RCP8.59 by 2100 (IPCC Fifth Assessment Report (AR5), 2014).

A temperature increase of 2◦C can lead to a twofold increase in monoterpene fluxes and a five-fold increase in sesquiterpene fluxes in subarctic ecosystems (Valolahti et al., 2015). In our study, monoterpene fluxes correlate with temperature in spring and summer, but the flux rate rise was smaller with increasing temperature, possibly because vegetation is less sensitive to temperature fluctuations in boreal ecosystems compared with arctic ecosystems.

In boreal ecosystems, a warming climate is expected to increase ericoid shrub cover and decrease moss cover, which can affect leaf litter quality and decomposition rates (De Long et al., 2016). Decomposition activity is expected to increase due to temperature dependent enzyme activity of microbes, and this can affect belowground VOC production. Warming can also affect plant VOC synthesis, release of evaporated VOCs through stomata, and the abundance of different plant species. The boreal forest floor is prevailingly covered by monoterpeneemitting Vaccinium spp. (Faubert et al., 2012; Aaltonen et al., 2013) at our site. Vaccinium myrtillus has shown a 36% increase in aboveground biomass by warming soil (Anadon-Rosell et al., 2014), which could make understory vegetation less diverse (Dawes et al., 2011) and impact monoterpene production in a warming climate.

#### Forest Floor Affects Ecosystem Fluxes

The comparison indicated that the forest floor is a significant contributor to the whole ecosystem fluxes, but its importance varies between seasons, being most important in autumn. The forest floor covered only a few percent of the forest stand fluxes in summer. Our results are in line with those of Aaltonen et al. (2013), who showed that the forest floor covered from several percent to 10s of percent of the total ecosystem fluxes depending on the compound and the season. Our method of comparing the observed forest floor level VOC emissions with ecosystem level VOC fluxes does not include oxidation of VOCs taking place during the transport from the forest floor to the abovecanopy atmosphere. Therefore our results presented in **Figure 7** should be considered as order of magnitude estimates rather than definitive values determining the proportion of forest floor oriented VOCs.

The seasonal dynamics of oxygenated VOCs (methanol, acetaldehyde, and acetone) were similar between the forest floor and the whole ecosystem (data not shown), indicating that vegetation is likely a significant source of oxygenated VOCs from the forest floor. In trees, methanol production is connected to growth and is produced mainly in leaves and needles in spring, and in stem and root expansion in summer. Soil VOC production can also influence the canopy fluxes, because methanol produced in the stem and roots during their growth, can be transported to the canopy via transpiration stream (Rissanen et al., 2018), and hence be emitted through stomata (Folkers et al., 2008). Peaking acetone and acetaldehyde fluxes above a hardwood forest in autumn were speculated to result from leaf senescence and decaying biomass (Karl et al., 2003). Acetone and acetaldehyde are also produced in the air from the oxidation of other VOCs.

Our results show that monoterpene fluxes from the forest floor contributed the highest load (9–93%) to the whole ecosystem fluxes in autumn due to low canopy fluxes. Monoterpenes are likely released from decomposing litter and plant senescence, because VOC synthesis of ground vegetation likely decreased similarly to shoot VOC emissions. This can provide a significant contribution to the OH sink in boreal forest air. VOCs affect formation and oxidation processes of OH radicals and ozone (Jacob et al., 2005; Hüve et al., 2007). Monoterpenes, isoprene, and other organic compounds were calculated to cover about 24% of the total OH reactivity in August based on a onedimensional vertical chemistry-transport model (Mogensen et al., 2011). Later, it was estimated that monoterpenes cover ∼14% and oxidized VOCs ∼44% of OH radical sinks (Mogensen et al., 2015). Ozone reacts mainly with inorganic compounds and only ∼3% with monoterpenes and 6% with sesquiterpenes and is deposited on the soil and vegetation surfaces (Mogensen et al., 2015; Zhou et al., 2017).

## Uncertainties

Temporal dynamics of forest floor VOC exchange can rather well be captured with three chambers, while several factors may affect and increase the uncertainties in chamber measurements. Our results indicate that vegetation cover affects BVOC emission rates and together with varying temperature creates spatial variation in the fluxes. The contribution of light to soil VOC fluxes is relatively small due to poor light availability under a closed canopy. Monoterpenes can also stick on lipophilic plant leaves (Mäki et al., 2017), which could explain why the highest fluxes were observed from chamber 1 with the lowest Vaccinium spp. coverage.

The quadrupole-PTR-MS is an instrument with high sensitivity and short response time and it has been shown to be able to perform online-measurements of reactive trace gasses from grasslands (Pape et al., 2009). Flux measurements of water soluble VOCs such as methanol, acetone, and acetaldehyde are sensitive to biases, because they tend to be adsorbed on moist surfaces (vegetation, soil, and chamber walls). For this reason, soil chamber data of water soluble VOCs was filtered with 75% relative humidity. A relative humidity of 70% in the chamber has been recommended by Kolari et al. (2012). Relative humidity of the headspace is often over 75% when measurements are performed during the nighttime or rainy days, meaning that water soluble VOCs are dissolved in moist surfaces on chamber walls and leaves. Seasonal fluxes of oxygenated VOCs were slightly overestimated, because the flux measurements performed during the nighttime or rainy days are underrepresented in the data. There were also gaps in the data due to technical analyzer problems, which affected the data coverage between years. Data were missing from mid-June to October in 2012, from September to October in 2014, and from April to mid-August in 2016.

We found hardly any correlation between the VOC fluxes and soil temperature. This may be caused by the spatial variation in soil temperature, and the lack of soil temperature sensors installed right next to the soil chambers, and in the top-most litter layer, where most of the soil-emitted VOCs are released (Hayward et al., 2001; Greenberg et al., 2012). Moreover, the effect of soil temperature on VOC fluxes may be hidden by the complexity of different VOC sources and their different responses to temperature.

## CONCLUSION

fpls-10-00191 February 20, 2019 Time: 18:19 # 12

We used the 8-year data set to assess the interannual, seasonal, and diurnal dynamics of forest floor VOC fluxes and compared them with the simultaneously measured total ecosystem fluxes. The forest floor affects ecosystem VOC exchange, emitting 2– 93% of monoterpene fluxes in spring and autumn and 1–72% of methanol fluxes in spring and early summer. Oxygenated VOC fluxes showed similar seasonal dynamics between the forest stand and the forest floor.

The highest weekly mean monoterpene fluxes were observed from the forest floor in spring and autumn (maximum 59 and 86 µg m−2 h −1 , respectively) and the highest weekly mean methanol fluxes in spring and summer (maximum 24 and 79 µg m−2 h −1 , respectively). The seasonal dynamics indicate that litter and ground vegetation were the dominant sources of monoterpenes and oxygenated VOC from the boreal forest floor. Forest floor VOC exchange was dominated by monoterpenes and methanol and the flux dynamics was relatively similar between the 8 years, whereas the emission rates differed between the chamber locations. Accounting for location dependent variability and temperature and relative humidity, a mixed effects linear model was able to explain 79– 88% of monoterpene, methanol, acetone, and acetaldehyde fluxes from the boreal forest floor. Forest floor VOC exchange was measured in the prevailing climate, but based on temperature responses of monoterpenes, and especially oxygenated VOCs, it seems that forest floor VOC fluxes will likely increase in warming climate.

Forest floor VOC exchange should be measured using continuous long-term measurements in the different ecosystems

#### REFERENCES

to define the contribution of soils to ecosystem VOC exchange and hence their effect on atmospheric processes globally.

#### DATA AVAILABILITY

The data of forest floor VOC fluxes for this study can be found in the **Supplementary Material**.

## AUTHOR CONTRIBUTIONS

MM was responsible for preparing the manuscript. All authors participated in planning the experiment, analyzing data, preparing the manuscript, and reviewed the manuscript.

# FUNDING

This research was funded by the Academy of Finland Centre of Excellence Programme (Grant No. 307331), Academy Research Fellow project (Grant No. 2884941), the European Research Council (ERC) under the European Union's Horizon 2020 research and innovation programme (Grant Agreement No. 757695), and by the Jenny and Antti Wihuri Foundation.

#### ACKNOWLEDGMENTS

The SMEAR II station staff are acknowledged for their help in installing and maintaining the continuous field measurements.

#### SUPPLEMENTARY MATERIAL

The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fpls.2019.00191/ full#supplementary-material

spectrometry (PTR-TOF-MS). Appl. Soil Ecol. 86, 182–191. doi: 10.1016/j. apsoil.2014.10.018

above a boreal forest. Atmos. Chem. Phys. 18, 815–832. doi: 10.5194/acp-18- 815-2018

**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2019 Mäki, Aalto, Hellén, Pihlatie and Bäck. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

# Combinations of Abiotic Factors Differentially Alter Production of Plant Secondary Metabolites in Five Woody Plant Species in the Boreal-Temperate Transition Zone

John L. Berini1,2 \*, Stephen A. Brockman3 , Adrian D. Hegeman3 , Peter B. Reich2,4,5 , Ranjan Muthukrishnan6 , Rebecca A. Montgomery2,4 and James D. Forester2,6

1 Conservation Biology Graduate Program, University of Minnesota, St. Paul, MN, United States, 2 Institute on the Environment, University of Minnesota, St. Paul, MN, United States, 3 Department of Horticultural Science, The Microbial and Plant Genomics Institute, University of Minnesota, St. Paul, MN, United States, 4 Department of Forest Resources, University of Minnesota, St. Paul, MN, United States, 5 Hawkesbury Institute for the Environment, Western Sydney University, Penrith, NSW, Australia, 6 Department of Fisheries, Wildlife, and Conservation Biology, University of Minnesota, St. Paul, MN, United States

#### Edited by:

Bartosz Adamczyk, University of Helsinki, Finland

#### Reviewed by:

Raquel Esteban, University of the Basque Country (UPV/EHU), Spain Adam Matkowski, Wroclaw Medical University, Poland

> \*Correspondence: John L. Berini<EMAIL_ADDRESS>
#### Specialty section:

This article was submitted to Functional Plant Ecology, a section of the journal Frontiers in Plant Science

Received: 21 May 2018 Accepted: 09 August 2018 Published: 05 September 2018

#### Citation:

Berini JL, Brockman SA, Hegeman AD, Reich PB, Muthukrishnan R, Montgomery RA and Forester JD (2018) Combinations of Abiotic Factors Differentially Alter Production of Plant Secondary Metabolites in Five Woody Plant Species in the Boreal-Temperate Transition Zone. Front. Plant Sci. 9:1257. doi: 10.3389/fpls.2018.01257 Plant secondary metabolites (PSMs) are a key mechanism by which plants defend themselves against potential threats, and changes in the abiotic environment can alter the diversity and abundance of PSMs. While the number of studies investigating the effects of abiotic factors on PSM production is growing, we currently have a limited understanding of how combinations of factors may influence PSM production. The objective of this study was to determine how warming influences PSM production and how the addition of other factors may modulate this effect. We used untargeted metabolomics to evaluate how PSM production in five different woody plant species in northern Minnesota, United States are influenced by varying combinations of temperature, moisture, and light in both experimental and natural conditions. We also analyzed changes to the abundances of two compounds from two different species – two resin acids in Abies balsamea and catechin and a terpene acid in Betula papyrifera. We used permutational MANOVA to compare PSM profiles and phytochemical turnover across treatments and non-metric multidimensional scaling to visualize treatment-specific changes in PSM profiles. We used linear mixed-effects models to examine changes in phytochemical richness and changes in the abundances of our example compounds. Under closed-canopy, experimental warming led to distinct PSM profiles and induced phytochemical turnover in B. papyrifera. In open-canopy sites, warming had no influence on PSM production. In samples collected across northeastern Minnesota, regional temperature differences had no influence on PSM profiles or phytochemical richness but did induce phytochemical turnover in B. papyrifera and Populus tremuloides. However, warmer temperatures combined with open canopy resulted in distinct PSM profiles for all species and induced phytochemical turnover in all but Corylus cornuta. Although neither example compound in A. balsamea was influenced by any of the abiotic conditions, both compounds in B. papyrifera exhibited

**30**

significant changes in response to warming and canopy. Our results demonstrate that the metabolic response of woody plants to combinations of abiotic factors cannot be extrapolated from that of a single factor and will differ by species. This heterogeneous phytochemical response directly affects interactions between plants and other organisms and may yield unexpected results as plant communities adapt to novel environmental conditions.

Keywords: phytochemical turnover, PSM diversity, untargeted metabolomics, balsam fir, beaked hazel, paper birch, red maple, trembling aspen

## INTRODUCTION

Plant secondary metabolites (PSMs) are one of the primary ways in which plants respond to environmental variability, and regulation of PSM production is strongly influenced by the local environment (Wink, 1988; Bennett and Wallsgrove, 1994; Bray et al., 2000; Hirt and Shinozaki, 2003). Many interactions between plants and other organisms are mediated by PSMs (Farmer, 2001; Karban et al., 2006; Karban, 2008), and thus, the biochemical mechanisms that influence these interactions are modulated, at least in part, by the presence, absence, or magnitude of various environmental factors (DeLucia et al., 2012; Jamieson et al., 2012). For instance, changes in the amount and seasonality of precipitation have been shown to influence concentrations of cyanogenic glycosides (Gleadow and Woodrow, 2002; Vandegeer et al., 2013), and elevated concentrations of atmospheric CO2 often result in increased concentrations of condensed tannins (Lindroth, 2012). Evidence is mounting that recent warming may also influence the production of PSMs (Kuokkanen et al., 2001).

Studies investigating the influence of warming on PSM production suggest that temperature-induced changes to PSMs may be species, compound, or even context dependent. For example, warming has been shown to have no effect on levels of phenolics in red maple (Acer rubrum, Williams et al., 2003), Norway spruce (Picea abies, Sallas et al., 2003), and Scots pine (Pinus sylvestris, Sallas et al., 2003) but resulted in decreased levels of phenolics in dark-leaved willow (Salix myrsinifolia, Veteli et al., 2006) and silver birch (Betula pendula, Kuokkanen et al., 2001). Additionally, warming has been shown to increase levels of terpene-based compounds in Norway spruce (Sallas et al., 2003), Ponderosa pine (Pinus ponderosa, Constable et al., 1999), and Scots pine (Sallas et al., 2003) but has been shown to both increase (Constable et al., 1999) and decrease (Snow et al., 2003) levels of monoterpenes in Douglas fir (Pinus menziesii). While evidence of warming-induced changes to phytochemistry is important to our understanding of how plants will respond to future climates, in natural settings, elevated temperature often combines with other abiotic conditions to influence PSM production and potentially modulate any changes to phytochemistry that might otherwise be induced by warming alone.

As temperatures continue to rise, global precipitation patterns are expected to shift (Hurrell, 1995; Alexander et al., 2006; IPCC, 2014) and light availability to understory plants will likely be altered due to changes in the frequency and intensity of forest disturbance patterns (Canham et al., 1990; Dale et al., 2001). While variability in each of these environmental factors has been shown to influence production of PSMs on their own (Bryant et al., 1983; Dudt and Shure, 1994; Pavarini et al., 2012), combinations of factors can have a distinct effect (Rizhsky et al., 2002, 2004; Mittler, 2006; Zandalinas et al., 2018). Moreover, plant responses to combinations of abiotic factors can be either synergistic or antagonistic (Bonham-Smith et al., 1987; Mittler, 2006; Zandalinas et al., 2018). For example, drought has been shown to enhance cold tolerance (Cloutier and Andrews, 1984), but also exacerbate a plant's intolerance of high temperatures (Rizhsky et al., 2002). Further, different combinations of salinity and high temperatures have been shown to have both positive and negative influences on the metabolism of reactive oxygen species and stomatal response (Zandalinas et al., 2018). Regardless, the vast majority of current research remains focused on the influences of individual conditions rather than considering potential interactions among them.

Until recently, the majority of studies investigating the potential influence of different abiotic factors largely considered the effects of these factors on individual compounds or small groups of compounds. However, individual metabolites rarely, if ever, function in isolation (Gershenzon et al., 2012). Rather, the influence of any one compound is dependent on conditions within the local environment, as well as the relative abundance of numerous other metabolites within a plant's array of chemical constituents (Dyer et al., 2003; Richards et al., 2010; Gershenzon et al., 2012; Jamieson et al., 2015). Thus, understanding how changes in the abiotic environment will influence a plant's metabolic profile is important for interpreting how these changes will influence the abundance and biological role of individual compounds as well.

Phytochemical diversity influences how effective plants are when defending against a range of threats (Gershenzon et al., 2012; Frye et al., 2013; Richards et al., 2015). Compounds may act synergistically, thereby forming mixtures that can provide enhanced protection against potential hazards (Gershenzon, 1984; Harborne, 1987; Gershenzon et al., 2012). Indeed, recent evidence suggests that the number of individual compounds comprising a plant's phytochemical profile can even influence local biological diversity via the influence of changes in toxicity on rates of herbivory (Richards et al., 2015). Increased diversity of secondary metabolites may also allow for more precise communication between plants, thereby allowing for more robust protection against a range of conditions (Iason et al., 2005; Poelman et al., 2008; Gershenzon et al., 2012; Frye et al., 2013). Two metrics that are useful for assessing changes in phytochemical diversity are "phytochemical richness" (i.e., the

absolute number of compounds produced) and "phytochemical turnover" (i.e., the degree of overlap among the compounds produced), as both measures provide different insights into the metabolic response of plants to a range of abiotic conditions.

Variability in phytochemistry, even within the same species, may influence ecosystem structure and function through an array of chemically driven ecological effects (Bukovinszky et al., 2008; Gillespie et al., 2012; Sedio et al., 2017). The growthdifferentiation balance hypothesis (GDBH) suggests that as the local environment becomes increasingly stressful, growth processes will become limited and the production of PSMs will increase until the point that PSM production also becomes limited by resource acquisition/availability (Lerdau et al., 1994). While phytochemical diversity has not been explicitly tested in light of the GDBH, studies have shown that herbivore-induced secondary chemistry can be completely suppressed in some woody species under a range of abiotic conditions (Lewinsohn et al., 1993), rendering them vulnerable to further invasion by pests and pathogens. While the number of studies investigating the effects of warming and other abiotic conditions on PSM production is rapidly growing, we currently have a limited understanding of how different abiotic factors may interact to influence phytochemical diversity (Bidart-Bouzat and Imeh-Nathaniel, 2008; Jamieson et al., 2012, 2015). The objective of this study was to determine how elevated temperatures may influence the production of PSMs and to evaluate how the addition of other abiotic factors may modulate this effect.

While a targeted approach uses standard model compounds to identify and observe changes in specific compounds selected a priori, an untargeted (i.e., global) approach makes no assumptions regarding specific metabolites, and therefore, allows one to observe global changes across the entire metabolic profile. Thus, the strength of an untargeted approach lies in the potential to discover unanticipated changes in metabolic profiles as a result of environmental perturbations (Crews et al., 2009). Although untargeted metabolomics have been used in medicine for clinical diagnosis of various diseases, including numerous forms of cancer (Sreekumar et al., 2009; Jain et al., 2015), this study is among the first to apply this method to an ecological setting.

We used an untargeted metabolomics approach to evaluate how the phytochemical profiles of five different woody plant species are influenced by temperature, soil moisture, and light. Specifically, we tested the hypothesis that elevated temperatures alter the production of PSMs by leading to phytochemical profiles that are distinct from those found at ambient temperature (H1) and that warming will change phytochemical diversity via reductions in phytochemical richness or a high degree of turnover (H2). We also tested the hypothesis that the addition of other abiotic factors, specifically high light and drought, will either magnify or nullify temperature-induced changes in phytochemical profiles and PSM diversity (H3). Finally, because individual compounds may vary greatly in response to heterogeneity in the abiotic environment, we identified two 'example compounds' from balsam fir (Abies balsamea – two unspecified di-terpene resin acids) and paper birch (Betula papyrifera – catechin and another unspecified diterpene resin acid) and analyzed the effects of different sets of

abiotic factors (high-temperature, light, and drought) on their relative abundance. Specifically, we tested the hypothesis that individual compounds will respond to different conditions and combinations of conditions by either increasing or decreasing in relative abundance, potentially in a non-uniform and unpredictable manner (H4).

#### MATERIALS AND METHODS

#### Experimental Design

The Boreal Forest Warming at an Ecotone in Danger (B4WarmED) project is an ecosystem experiment that simulates both above- and below-ground warming in a boreal forest community. The experiment was conducted at Cloquet Forestry Center (CFC; Cloquet, MN, United States) and was initiated in 2008. The experimental design consists of a 2 (overstory – open and closed) × 3 (warming – ambient, ambient +1.7◦C, and ambient +3.4◦C) × 2 (precipitation – ambient and ambient −40%) factorial design with six replicates (two per block) per treatment combination, for a total of 72 – 7.1 m2 plots (Rich et al., 2015). Within each plot, 121 seedlings of 11 tree species were planted into the remaining herbaceous vegetation in a gridded design (Rich et al., 2015). Above-ground biomass was warmed using a Temperature Free-Air-Controlled Enhancement System (T-FACE) and below-ground biomass was warmed via buried resistance-type heating cables (Rich et al., 2015). Above- and below-ground temperatures have been monitored and logged at 15-min intervals since spring 2008. In 2012, event-based rain exclosures were installed on nine plots in the open overstory replicates of the warming experiment, which allowed for safe and reliable removal of rainfall. Mean annual rainfall exclusion from June to September ranges from 42 to 45%.

We collected plant samples from the B4WarmED project during two different time periods. On July 14, 2013, we collected samples of balsam fir and paper birch that were grown under closed overstory and three warming treatments, and on July 15, 2014, we collected samples of balsam fir, paper birch, trembling aspen (Populus tremuloides), and red maple (Acer rubrum) grown under open overstory in the three warming treatments and two precipitation treatments. Where possible, we collected recentgrowth foliar tissue from two plants per species within each replicate plot. However, some replicates contained either one or no plants with enough leaf tissue to sample. Samples sizes were particularly small during 2014, so we were forced to group individual warming treatments (ambient, +1.7◦C, +3.4◦C) into a binary response (ambient temperature vs. elevated temperature). All plant samples were collected within a 2-h time period. Upon collection, samples were flash frozen with dry ice, and subsequently stored in a −80◦C freezer to minimize chemical degradation. We broadly refer to samples collected from the B4WArmED project as our "experimental" samples.

To investigate how temperature and light conditions may interact to influence phytochemical production in a natural forest environment, we collected samples of balsam fir, paper birch, trembling aspen, and beaked hazel (Corylus cornuta) from open and closed canopy environments across two regions

in northeastern Minnesota (**Figure 1**). These regions exhibit differences in mean-maximum summer temperature (maximum daily temperature averaged across June, July, and August) of approximately 5.5◦C (**Supplementary Table S1**). On July 14, 2015, we collected a minimum of 3 biological replicates from each species within each set of abiotic conditions. The sampling design consists of a 2 (overstory – open and closed) × 2 (temperature – warm and cool) design with three plot replicates per treatment combination, for a total of 12 – 400 m2 plots. Open-canopy plots allowed us to evaluate high-light conditions on production of PSMs and were located in areas that were clear-cut in 2006 (i.e., open overstory), while closed-canopy plots were located in areas that experienced no known overstory disturbance since at least 1985 (i.e., closed overstory). Thus, light conditions for all plots were based on whether the overstory was open (i.e., high light) or closed (i.e., low light). Temperature logger data collected for a parallel study from similar plot types suggest that average high temperatures from May 1, 2015 to July 14, 2015 ranged from 30.4◦C in low-light plots in the cool region to 36.6◦C in high-light plots in the warm region. All field samples were collected on the same day, within an 8-h period. Upon collection, samples were flash frozen with dry ice, and subsequently stored in a −80◦C freezer. For brevity, we occasionally refer to samples collected throughout northeast Minnesota as "observational" samples.

#### Study Organisms

Balsam fir is a mid- to large-sized species of conifer, growing to 26 m in height, with shallow roots (Smith, 2008). It is highly vulnerable to drought, fire, and spruce budworm (Choristoneuro fumiferana) infestations (Engelmark, 1999), and modest climate warming has been shown to decrease net photosynthesis and growth by as much as 25% (Reich et al., 2015). Paper birch can grow to 28 m in height (Smith, 2008) and is drought and shade intolerant (Iverson and Prasad, 1998; Iverson et al., 2008). While it can grow rapidly and live to be 250 years of age, seedlings need significant light to prosper (Kneeshaw et al., 2006). Elevated temperatures have been shown to influence foliar nitrogen, lignin, and condensed tannins in both paper birch and trembling aspen with the specific response varying as a function of species and climate (Jamieson et al., 2015). Trembling aspen is one of the most widespread tree species in North America and occurs on a wide-range of soil types and in various climatic conditions (Smith, 2008). It is sensitive to both drought and shade (Iverson and Prasad, 1998; Iverson et al.,

2008) and may become increasingly vulnerable to other potential stressors under conditions of drought and high temperatures (Worrall et al., 2008). Red maple is a moderately large tree, growing to 29 m in height and is known to be tolerant to a wide-range of precipitation conditions, from drought to seasonal flooding (Smith, 2008). While this species is expected to prosper under future climate scenarios (Iverson and Prasad, 1998; Iverson et al., 2008) and performed well under experimental warming (Reich et al., 2015), both prolonged flooding and severe drought have been shown to result in senescence and decreased growth, respectively (Nash and Graves, 1993). Beaked hazel, a shadetolerant shrub that can grow to 4 m tall, is a common understory species in both conifer and deciduous forests and occurs almost exclusively in fire prone habitats (Smith, 2008). Beaked hazel is highly sensitive to fire and previous work suggests that growth may be limited by soil moisture (Johnston and Woodard, 1985).

#### Metabolite Analysis

Tissue samples were lyophilized for 72 h and then homogenized and extracted using 25 mg (+/−2.5 mg) of each sample. Homogenization and extraction were performed for 5 min at a frequency of 1500 Hz with 1 ml of 70% isopropyl alcohol at –20◦C using a bead mill and 2.8 mm tungsten carbide beads (Sped Sample Prep GenoGrinder 2010, Metuchen, NJ, United States). Samples were then subjected to centrifugation at 16,000 × g for 5 min. The supernatant was then removed and subjected to an additional centrifugation step, 16,000 × g for an additional 5 min, and the supernatant was collected for subsequent analysis. Finally, 20 µL of each supernatant sample was removed and pooled to use as a control. All samples were then stored at –80◦C.

We analyzed samples with liquid chromatography mass spectrometry (LC-MS) using an Ultimate 3000 UHPLC (ultrahigh-performance liquid chromatography) system coupled to a Q Enactive hybrid quadrupole-Orbitrap mass spectrometer with a heated electrospray ionization (HESI) source (Thermo Fisher Scientific, Bremen, Germany). We injected 1 µL of each sample per analysis onto an ACQUITY UPLC HSS T3 column, 100 Å, 1.8 µm, 2.1 mm × 100 mm (Waters, Milford, MA, United States) using a gradient composed of solvents A: 0.1% formic acid and B: acetonitrile. Specifically, 0–2 min, 2% B; 6 min, 24% B; 9 min, 33% B; 12 min, 65% B; 16 min, 80% B; 20 min 93% B; 21 min 98% B; 22 min 98% B; 23 min 2% B; 23–25 min 2% B. Samples were analyzed in a randomized order to minimize systematic bias from instrument variability and carryover. Fullscan analysis was performed using positive/negative ion polarity switching, a 115–1500 m/z scan range, a resolution of 70,000 (at m/z 200), maximum fill times of 100 ms, and target automatic gain control (AGC) of 1 × 106 charges. Ion fragmentation was performed using a higher-energy collision dissociation (HCD) cell and resulting MS/MS data were collected using a resolution of 17,500, maximum fill times of 100 ms, and an AGC target of 2 × 105 charges. Normalized collision energies (NCE) ranged from 10 to 45 in increments of 5. All data were collected using Xcalibur version 2.2 (Thermo Fisher Scientific, Bremen, Germany).

### Example Compounds

To determine which chemical features varied consistently and significantly among each treatment and species group, we aligned, smoothed, background subtracted, and analyzed all chromatographic data using analysis of variance (α = 0.001) via Genedata 7.1 (Genedata, Basel, Switzerland). We assigned putative metabolite identities only to the features found to be significantly abundant (ANOVA, α = 0.001) with an exact mass and higher-energy collisional dissociation (HCD) MS/MS fragmentation spectra. We determined molecular formulae by using exact mass to calculate the most probable elemental composition for each feature (**Supplementary Table S2**). We then manually interpreted HCD spectra collected at numerous collision energies (**Supplementary Figures S1–S3**), and compared these to the MassBank database using MetFusion (Gerlich and Neumann, 2013). Where possible, we confirmed the identity of individual compounds via comparison to an authenticated standard (Sigma-Aldrich) and assigned other putative identities by matching molecular formulae to those of previously observed metabolites in Betula (Julkunen-Tiitto et al., 1996) and Abies (Otto and Wilde, 2001). Specifically, we analyzed changes in the relative abundance of catechin and an unspecified terpene acid in paper birch and two unspecified diterpene resin acids in balsam fir. The identification of catechin was confirmed by comparison of accurate mass, LC-retention and MS/MS fragmentation properties of commercially available standard compounds for both catechin and its frequently associated isomer epicatechin which were distinguishable by chromatographic separation. There has been a great deal of work investigating the biological and ecological activity of catechin and terpenoid-based metabolites (Tahvanainen et al., 1985; Gershenzon and Croteau, 1992; Berg, 2003; Stolter et al., 2005); and as a result, we expect our results regarding these compounds to be broadly relevant.

#### Data Processing and Statistical Analysis

Data processing and statistical analyses were conducted using R 3.5.0 (R Core Team, 2017). To initiate data processing, we used the xcmsRaw function in the xcms package (Smith et al., 2006; Tautenhahn et al., 2008; Benton et al., 2010) to read our raw mzML files into R. After separating our data by polarity using the split function in the base package, we used the findPeaks.centwave function for peak detection, which we parameterized as follows: ppm = 2, peakwidth = c(5,20), prefilter = c(1,15000000), mzCenterFun = "apex," integrate = 1, mzdiff = −0.001, fitgauss = F, snthresh = 10. Once peak detection was complete, we trimmed the resulting polarity-specific data frames based on retention time and retained only those peaks detected between 1 and 21 min.

A major shortfall of employing LC-MS to perform "untargeted profile analysis," as we did here, is the production of two independent but partially overlapping datasets resulting from ion polarity switching. While polarity switching is useful for detection of features that can only be detected via either positive or negative ionization, some features are detectable under both ionization modes, therefore resulting in two independent data sets containing a small subset of common features. Moreover,

interpretation of statistical results is challenging due to the presence of parallel sets of analyses with common features contributing to both. To alleviate these issues, we combined positive and negative polarities using the find.matches function in the Hmisc package (Harrell and Dupont, 2018). The find.matches function allows one to identify which rows in a data matrix align with those in a separate, identically formatted matrix by allowing the user to define a tolerance level for the numerical columns in each matrix. Thus, to determine our common features in the positive and negative ionization datasets that result from LC-MS, we created two matrices for positive and negative polarity, containing three separate columns – the mass of each detected peak, an assigned name for each peak, and retention time. To ensure that corresponding features from each ionization mode were capable of alignment, we subtracted 2.1046, roughly the mass of two protons, from all masses in the positive polarity dataset. For those features identified as common among both ionization modes, we retained peak data from the polarity exhibiting greatest mean intensity across all samples. We then assigned new peak names to identify which peaks were present in either positive or negative polarity vs. those that were found in both. All output created using the find.matches function was manually checked to ensure that all peaks identified as having a match in one polarity, had their mate identified as a match in the other.

We used permutational MANOVA (perMANOVA, Anderson, 2001) to compare PSM profiles between abiotic conditions. When analyzing PSM profiles, differences were estimated using Canberra dissimilarity matrices (Dixon et al., 2009). Analysis was performed with the adonis function (from the vegan package, Oksanen et al., 2015), which allowed us to account for our blocked sampling design via the strata argument. Both differences in the centroids among conditions or differences in multivariate dispersion can lead to statistically significant results when using perMANOVA. To determine whether differences among centroids were contributing to perMANOVA results, we created mean dissimilarity matrices using the meandist function and we used the betadisper function to assess multivariate homogeneity of variance (i.e., dispersion, Oksanen et al., 2015). We used non-metric multidimensional scaling (NMDS, Kruskal, 1964) to visualize differences in PSM profiles among conditions, which we performed using the metaMDS function in the vegan package (Oksanen et al., 2015). We set our dimensionality parameter (k) to 2 and projected condition-specific effects onto NMDS plots using the ordiellipse function to plot 95% confidence ellipses based on standard error (Oksanen et al., 2015).

To evaluate treatment-induced changes to PSM diversity, we calculated phytochemical richness based on the presence and absence of individual compounds, then tested the main effect of treatment on richness with block (experimental samples) or site ID (observational samples) as our random effect using linear mixed-effects models (lme function within the nlme package, Pinheiro et al., 2015). To analyze phytochemical turnover (i.e., the degree of overlap between the phytochemical profiles of individual plants across and between conditions), we created dissimilarity matrices based on binary datasets representing the presence or absence of each feature using Jaccard's Index. We evaluated condition-specific differences in phytochemical turnover using perMANOVA via the adonis function, and evaluated the influence of multivariate centroids and homogeneity of variance on perMANOVA results as detailed above (Anderson, 2001; Oksanen et al., 2015).

Weather data from CFC shows that ambient air temperature, cumulative precipitation from 1 January to collection date, and leaf surface temperature were not statistically different between 2012 and 2013 or between specific sample sets (2013 – closed overstory, 2014 – open overstory). However, soil moisture and soil temperature vary strongly between years and sample sets, and differences between experimental and observational samples are likely to be even greater. Thus, samples collected during different years were analyzed independently of one another as individual data sets.

For analytical and visualization purposes, the condition or set of conditions assumed to impart the least amount of metabolic change during each year was labeled as our reference group, to which all other conditions were compared for that sample year. For Year 1 (2013), we designated "ambient" as our reference category, while samples grown under ambient temperature and ambient precipitation were designated as our reference category for Year 2 (2014). We designated samples collected from cold region, low-light conditions as our reference category for Year 3 (2015). To help visualize how different abiotic conditions may influence PSM production in different species, we calculated the number of chemical features that increased and decreased by ≥ 75%, relative to our reference category and created scaled Venn Diagrams representing these relationships.

Finally, we used linear mixed-effects models to test the main effect of abiotic condition on the relative abundance of our example compounds, with sample block as our random effect for experimental samples and plot ID as our random effect for observational samples (lme function within the nlme package, Pinheiro et al., 2015). These models tested whether combinations of abiotic factors influence the abundance of our known example compounds.

# RESULTS

#### Temperature

The influence of temperature was both species and context dependent. In closed overstory (Year 1), when compared to ambient, warming-induced changes to the phytochemical profile of balsam fir were not statistically significant, whereas paper birch exhibited warming-induced shifts to phytochemical profiles, thereby leading to distinct PSM profiles for the warming treatment. Analysis of multivariate dispersion and mean-dissimilarity matrices both suggest that differences in paper birch were due to temperature-induced changes in the centroid rather than dispersion (**Table 1**). NMDS plots reveal minor overlap between temperature conditions in paper birch, and balsam fir grown under moderate and high-temperatures show strong overlap with plants grown in ambient temperatures but minor overlap with each other (**Figure 2**). Warming had no effect on phytochemical richness in either species but did

 na indicates not applicable.

were compared.

influence phytochemical turnover in paper birch (**Table 1**). In open overstory (Year 2), warming had no influence on PSM profiles or PSM diversity (i.e., richness or turnover), regardless of species (**Table 1**). NMDS plots support these findings in that there is no discernible relationship between temperature and PSM profiles, regardless of species (**Figure 3**). In observational samples collected throughout northeast Minnesota (Year 3), temperature on its own had no influence on plant PSM profiles or phytochemical richness values. However, phytochemical turnover was significantly different in plants from different temperature regions in paper birch (perMANOVA, F = 5.912, r 2 = 0.179, P = 0.0003) and trembling aspen (perMANOVA, F = 3.322, r 2 = 0.156, P = 0.0012). NMDS plots suggest that each species responds differently to combinations of temperature and light (i.e., canopy; **Figure 4**). Balsam fir produces distinct PSM profiles as a function of ambient light conditions (i.e., open vs. closed canopy), but only within the cool region, while paper birch and trembling aspen appear to have distinct PSM profiles for each combination of conditions. Conversely, beaked hazel exhibits no discernible pattern across different conditions.

Venn diagrams created to help visualize the influence of different abiotic conditions for Year 1 samples suggest that the high-temperature (+3.4◦C) treatment induced a greater response from both balsam fir and paper birch than the moderate-temperature (+1.7◦C) treatment. Specifically, the high-temperature treatment led to more features that either increased or decreased in relative abundance by 75% or more when compared to ambient or moderate-temperature treatments (**Table 2** and **Supplementary Figures S4–S6**).

#### Interactive Effects of Different Abiotic Conditions

In our Year 2 samples, the combination of drought and elevated temperature had no influence on PSM profiles or any aspect of phytochemical diversity, regardless of species (**Table 1**). These results were supported by NMDS plots (**Figure 3**). Additionally, Venn diagrams suggest large-magnitude increases or decreases in relative abundance of PSMs did not follow an obvious pattern that could be attributed to different conditions. There appears to be a high degree of overlap across conditions in those compounds that exhibit increases in relative abundance of ≥ 75%, while less overlap occurs among compounds exhibiting large declines in relative abundance. Furthermore, the influence of drought on the decline of relative abundance by ≥ 75% appears to be more distinct than that of either warming or warming and drought together (**Table 2** and **Supplementary Figures S4**–**S6**).

In observational samples from throughout northeast Minnesota (Year 3), when evaluating the effects of high temperature and light combined, balsam fir appears to create unique PSM profiles in response to different light conditions (i.e., open vs. closed canopy), but only within the cool region, while paper birch and trembling aspen appear to have distinct PSM profiles for each condition. Beaked hazel exhibits no discernible pattern (**Figure 4**). Phytochemical richness did not vary as a function of light conditions or temperature region. However, phytochemical turnover in balsam fir was significantly influenced by conditions of high light (i.e., open canopy; **Table 3**). When analyzing the interactive effects of light conditions and temperature region, all species exhibited significant differences in their PSM profile (**Table 3**), with only trembling aspen exhibiting significant differences in multivariate dispersion as a function of the combination of light condition and temperature region (**Table 3**). Although phytochemical richness was not influenced by the combined effects of temperature region and light conditions, phytochemical turnover was influenced in paper birch and trembling aspen and a marginal, non-significant trend was present in beaked hazel (**Table 3**).

Patterns in Venn diagrams detailing the influences of different conditions during Year 2 are difficult to discern, as different plant species appeared to respond to varying conditions in different ways (**Table 2** and **Supplementary Figure S5**). Drought led to more features increasing by ≥ 75% in balsam fir and paper birch, while elevated temperature led to the large-magnitude increase of more features in trembling aspen (**Table 2** and