\documentclass[10pt]{article}

\usepackage{fullpage}
\usepackage{setspace}
\usepackage{parskip}
\usepackage{titlesec}
\usepackage[section]{placeins}
\usepackage{xcolor}
\usepackage{breakcites}
\usepackage{lineno}
\usepackage{hyphenat}





\PassOptionsToPackage{hyphens}{url}
\usepackage[colorlinks = true,
            linkcolor = blue,
            urlcolor  = blue,
            citecolor = blue,
            anchorcolor = blue]{hyperref}
\usepackage{etoolbox}
\makeatletter
\patchcmd\@combinedblfloats{\box\@outputbox}{\unvbox\@outputbox}{}{%
  \errmessage{\noexpand\@combinedblfloats could not be patched}%
}%
\makeatother


\usepackage{natbib}




\renewenvironment{abstract}
  {{\bfseries\noindent{\abstractname}\par\nobreak}\footnotesize}
  {\bigskip}

\titlespacing{\section}{0pt}{*3}{*1}
\titlespacing{\subsection}{0pt}{*2}{*0.5}
\titlespacing{\subsubsection}{0pt}{*1.5}{0pt}


\usepackage{authblk}


\usepackage{graphicx}
\usepackage[space]{grffile}
\usepackage{latexsym}
\usepackage{textcomp}
\usepackage{longtable}
\usepackage{tabulary}
\usepackage{booktabs,array,multirow}
\usepackage{amsfonts,amsmath,amssymb}
\providecommand\citet{\cite}
\providecommand\citep{\cite}
\providecommand\citealt{\cite}
% You can conditionalize code for latexml or normal latex using this.
\newif\iflatexml\latexmlfalse
\providecommand{\tightlist}{\setlength{\itemsep}{0pt}\setlength{\parskip}{0pt}}%

\AtBeginDocument{\DeclareGraphicsExtensions{.pdf,.PDF,.eps,.EPS,.png,.PNG,.tif,.TIF,.jpg,.JPG,.jpeg,.JPEG}}

\usepackage[utf8]{inputenc}
\usepackage[T2A]{fontenc}
\usepackage[ngerman,greek,polish,english]{babel}



\usepackage{float}








\begin{document}

\title{Terpenoids are transported in the xylem sap of Norway spruce}



\author[1]{Qiuxiao Duan}%
\author[2]{Boris Bonn}%
\author[3]{Juergen Kreuzwieser}%
\affil[1]{Institute of Forest sciences}%
\affil[2]{Chair of Ecosystem Physiology}%
\affil[3]{University of Freiburg}%


\vspace{-1em}



  
  \date{June 11, 2024}


\begingroup
\let\center\flushleft
\let\endcenter\endflushleft
\maketitle
\endgroup





\selectlanguage{english}
\begin{abstract}
Norway spruce is a conifer storing large amounts of terpenoids in resin
ducts of various tissues. Parts of the terpenoids stored in needles can
be emitted together with de novo synthesized terpenoids. Since previous
studies provided hints on xylem transported terpenoids as a third
emission source, we tested if terpenoids are transported in xylem sap of
Norway spruce. We further aimed at understanding if they might
contribute to terpenoid emission from needles. We determined terpenoid
content and composition in xylem sap, needles, bark, wood and roots of
field grown trees, as well as terpenoid emissions from needles. We found
considerable amounts of terpenoids -- mainly oxygenated compounds - in
xylem sap. The terpenoid concentration in xylem sap was relatively low
compared to the content in other tissues where terpenoids are stored in
resin ducts. Importantly, the terpenoid composition in the xylem sap
greatly differed from the composition in wood, bark or roots suggesting
that an internal transport of terpenoids takes place at the sites of
xylem loading. Our work gives hints that plant internal transport of
terpenoids exists within conifers; studies on their functions should be
a focus of future research.%
\end{abstract}%



\sloppy


\section*{Key words:}

{\label{key-words}}

Norway spruce, terpenoid transport, xylem sap, emissions, roots

\section*{Introduction}

{\label{introduction}}

Terpenoids constitute the largest and most diverse class of chemical
substances that play major roles in plant primary and secondary
metabolisms (Gershenzon \& Dudareva, 2007; Nagel, Berasategui, Paetz,
Gershenzon \& Schmidt, 2014; Trapp \& Croteau, 2001). Two spatially
separated pathways exist for the biosynthesis of terpenoids; the
mevalonate (MVA) pathway takes place in cytosol and peroxisomes, whereas
the methylerythritol phosphate (MEP) pathway is active in plastids
(Dudareva, Klempien, Muhlemann, \& Kaplan, 2013; Nagegowda, 2010). Both
pathways form the C5 compounds isopentenyl diphosphate (IPP) and its
allylic isomer, dimethylallyl diphosphate (DMAPP), which act as
precursors for the biosynthesis of all terpenoids (McGarvey \& Croteau,
1995). Whereas the MVA pathway produces sesquiterpenes (C15), irregular
terpenes and geranyllinalool, the MEP pathway forms hemiterpenes (C5),
monoterpenes (C10), diterpenes (C20) and volatile carotenoid derivatives
(McGarvey \& Croteau, 1995; Muhlemann, Klempien \& Dudareva, 2014).
Terpenoids can be stored in specialized structures such as resin ducts
in many conifers (Gershenzon \& Croteau, 1991; Wu \& Hu, 1997) or
directly emitted into the environment (Loreto et al., 2001). In storing
plants, terpenoids usually make up 1-2\% of plant dry weight, in some
special cases shares of up to 15-20\% were observed (Blanch, Penuelas,
Sardans \& Llusia, 2009). Terpenoids exert key ecological functions for
plants, such as defence against predators, pathogens or competitors,
inter- and intraspecific communication as well as protection against
abiotic stress (Gershenzon \& Dudareva, 2007; Loreto \& Schnitzler,
2010; Loreto, Pollastri, Fineschi \& Velikova, 2014; Martin, Gershenzon
\& Bohlmann, 2003; Vickers et al., 2009).

According to present knowledge, there are two sources driving terpenoid
emission from leaves of plants. (i) \emph{De novo} biosynthesis controls
emission of isoprene and terpenoids from non-storing plants such
as\emph{Quercus ilex} . Production of these compounds is tightly coupled
to photosynthesis and therefore shows similar dependencies on
environmental parameters such as light availability and temperature
(Ghirardo et al., 2010; Loreto et al., 1996; Loreto, Nascetti, Graverini
\& Mannozzi, 2000; Tingey, Manning, Grothaus \& Burns, 1979). On the
other hand, (ii) the release of leaf-internally stored terpenoids is
purely temperature dependent because temperature controls the
corresponding saturation vapour pressures of the emitted compounds
(Ghirardo et al., 2010; Guenther, Zimmerman, Harley, Monson \& Fall,
1993; Wu et al., 2017).

As a typical conifer, Norway spruce (\emph{Picea abies} ) stores large
amounts of terpenoids in resin ducts of various tissues, which are at
least partially produced in the epithelial cells of the resin ducts and
mesophyll tissue (Sch\selectlanguage{ngerman}ürmann, Ziegler, Kotzias, Schönwitz \&
Steinbrecher, 1993). \textsuperscript{13}C-labelling approaches
demonstrated that about one third of the emitted monoterpenoids in
spruce are derived from \emph{de novo} biosynthesis whereas the
remainder is released from storage pools in a temperature depended
manner (Ghirardo et al., 2010; Grabmer et al., 2006). Recent studies
provided indirect hints that besides \emph{de novo} biosynthesis and/or
release from storage pools, xylem-transported terpenoids might
contribute to emission from Norway spruce needles. Bourtsoukidis et al.
(2012, 2014) observed that sesquiterpene (SQT) emission from Norway
spruce strongly correlated with relative humidity. At high relative
humidity - and consequently reduced transpiration rates - SQT emission
was slowed down and \emph{vice versa} when relative humidity was below
50\% (Bourtsoukidis et al., 2012). In agreement with this finding,
Filella, Wilkinson, Llusia, Hewitt \& Peñuelas (2007) demonstrated that
the emissions of most volatile organic compounds including monoterpenes
from Norway spruce were well related to stomatal conductance and
transpiration. This effect may in part be explained by a contribution of
the tree's sap flow to terpenoid emissions. Moreover, Bäck et al. (2012)
found the largest SQT pools in \emph{Pinus sylvestris} not in needles
but below the bark, where SQTs constitute a toxic barrier for bark
beetles and function as stress defense agents. Such terpenes might
partially be released into the xylem sap. This assumption is in
consistence with observations of Kuroda (1991) who observed a strong
link between xylem cavitation in pine wilt disease infected
\emph{Pinus}trees and terpene abundance in the xylem. The author
hypothesizes that cavitation is caused by higher terpene content in the
xylem sap and the thereby diminished adhesion forces. A strong induction
of terpene biosynthesis in the developing xylem by different stresses
has also been demonstrated in Norway spruce (Martin, Tholl, Gershenzon
\& Bohlmann, 2002).

In this study, we aim at clarifying whether or not terpenoids are
transported in the xylem sap of Norway spruce. Moreover, we aimed to
obtain information on possible sources of xylem-transported terpenoids.
In addition, we tried to find hints if xylem-transported terpenoids
might contribute to terpenoid emission from Norway spruce
needles.Therefore, we analyzed terpenoid emission rates as well as
terpenoid contents and compositions in root, bark and wood of the same
Norway spruce trees for comparison with xylem sap terpenoids.

\section*{Materials and methods}

{\label{materials-and-methods}}

\subsection*{Study site}

{\label{study-site}}

In September 2015, we studied six clonal 13-year-old Norway spruce trees
grown in the tree garden of the Chair of Ecosystem Physiology,
University of Freiburg, Germany (48°01\selectlanguage{english}' N, 7deg50\selectlanguage{english}' E, 236 m above sea
level) under the natural temperate to cool-temperate climate. In 2015,
the average monthly mean of daily temperature in 2 m height was 11.8+-1.9
degC, which was 0.4degC higher than the 30 year average; total annual
precipitation of 732 mm was about 200 mm less than the long-term
reference (1981- 2010) (DWD, 2015; Table S1).

\subsection*{Analysis of terpenoid
emission}

{\label{analysis-of-terpenoid-emission}}

Terpenoid emission was determined using a modified dynamic leaf
enclosure system of Kreuzwieser et al. (2001) under ambient conditions
at internal enclosure temperatures between 22 and 34degC. In the evening
before emission measurements, the glass enclosure (volume 500 ml, Duran,
Mainz, Germany) was mounted onto a healthy, sun-exposed Norway spruce
branch about 60 cm above ground. It was taken care to avoid any
mechanical injuries on needles and stems to minimize release of
terpenes. The lid of the enclosure remained open until the start of the
experiment. At least 15 min before terpenoid collection was started, the
lid was closed and the enclosure continuously flushed with synthetic air
containing 400 ppm CO\textsubscript{2} at flow rates of 200 ml
min\textsuperscript{-1}. A fan (DC series, Sunon, Taiwan) installed in
the enclosure ensured homogeneous air mixture. Terpenoids were collected
from air leaving the enclosure. For this purpose, we installed air
sampling tubes (Gerstel, Mulheim, Germany) filled with 20 mg Tenax TA
60/80 and 30 mg Carbotrap B 20/40 (Supelco, Belafonte, PA, USA) as
adsorbents at the enclosure exit. Enclosure air was drawn by an air
sampling pump (model 210-1003MTX, SKC, Dorset, UK) at a flow rate of 100
ml min\textsuperscript{-1} for 60 min over the air sampling tubes. All
tubing of the system was made of perfluoralkoxy (PFA) (Swagelok, Solon,
Ohio, USA) to prevent adhesion on or reaction of terpenoids with tubing
materials. As a reference, air was also sampled from an empty enclosure
which was flushed with the same synthetic air. The air sampling tubes
were then stored in glass vials at 4degC until terpenoid analysis (see
below). Terpenoid emission rates were calculated considering the amount
of terpenoids on air sampling tubes, dry weight of needles in the
enclosure and, sampling time and flow rate through the enclosure; data
were background corrected by measurements of the empty enclosure.

\subsection*{Harvest of plant material}

{\label{harvest-of-plant-material}}

Immediately after collection of needle released terpenoids, a sun
exposed branch of about 40-45 cm length (diameter ca. 1 cm) which was
closely located to the branch for emission measurements, was cut and
xylem sap was extracted (Rennenberg, Schneider \& Weber, 1996). Special
care was taken to avoid contamination of xylem sap with bark or wood
constituents. For this purpose, bark and cambium were removed at a
length of ca. 3 cm from the cut end; this part was rinsed thoroughly
with distilled water and methanol, and dried with cellulose paper before
it was inserted into the pressure chamber. The pressure was raised at a
rate of 0.2 MPa per min until the first droplet of xylem sap appeared.
This droplet was discarded and the end cleaned again with methanol.
Thereafter, the pressure was increased by another 0.5-0.7 MPa and kept
constant for about 2 mins; drops appearing were collected and
immediately shock-frozen in liquid N\textsubscript{2} until terpenoid
analysis.

Samples of current- and previous-year needles, bark and wood from sun
exposed twigs close to the twig which was used for xylem sap extraction,
as well as fine roots (diameter \textless{} 2 mm) of the same trees were
harvested. All plant materials were immediately shock-frozen in liquid
N\textsubscript{2}, and stored at -80degC. Before terpenoid analysis,
plant materials were homogenized to fine powders by mortar and pestle
under liquid N\textsubscript{2}.

For determination of tissue dry weights, aliquots of approximately 100
mg frozen needle, bark, wood or root powders were dried at 60degC until
the weight remained constant. Contents of all parameters were calculated
based on dry weight unless indicated otherwise.

\subsection*{Terpenoid extraction from xylem
sap}

{\label{terpenoid-extraction-from-xylem-sap}}

Terpenoid extraction was mediated by stir bar sorptive extraction (SBSE)
using polydimethylsiloxane (PDMS)-coated stir bars (Twisters(r), 0.5 mm
PDMS layer thickness, 10 mm in length, Gerstel, Mulheim, Germany)
(Kleiber et al., 2017). To trap terpenoids, one Twister(r) was added into
an aliquot of 300 \selectlanguage{greek}µ\selectlanguage{english}l xylem sap and samples were shaken (1,400 rpm, 30\selectlanguage{ngerman}°C;
Thermomixer, Eppendorf AG, Hamburg, Germany) for 60 min. Thereafter, we
removed the Twister® from the solution, shortly dried it with lint-free
paper tissue and placed it into a thermodesorption tube (Gerstel,
Müllheim, Germany) for terpenoid analysis.

\subsection*{Terpenoid extraction from plant
tissues}

{\label{terpenoid-extraction-from-plant-tissues}}

Aliquots of 25 mg of frozen powder were added into 1,500 \selectlanguage{greek}µ\selectlanguage{english}l methanol.
Samples were shaken (1,400 rpm, 30\selectlanguage{ngerman}°C, 20 min) and centrifuged (15,000
rpm, 23°C, 5 min). Supernatants were diluted (bark, 1:50, other tissues,
1:33.3) in H\textsubscript{2}O\textsubscript{demin} and two Twisters®
were added to 1.5 ml solution. Samples were shaken again (1,400 rpm,
30°C, 60 min) and Twisters® thereafter taken out, dried with lint-free
paper tissue and placed into a thermodesorption tube (Gerstel, Mülheim,
Germany) for subsequent analysis of terpenoids.

\subsection*{Terpenoid analysis}

{\label{terpenoid-analysis}}

Terpenoids adsorbed on Twisters® (xylem sap, tissues) as well as on air
sampling tubes (emission) were analyzed on a gas chromatograph (GC)
(model 6890A, Agilent, Waldbronn, Germany) equipped with a
thermodesorption/cold injection system (TDU-CIS) (Gerstel, Mülheim,
Germany) and connected to a MS detector (5975C, Agilent) (Kleiber et
al., 2017). In the TDU, thermodesorption tubes containing Twisters® or
air sampling tubes were heated up to 220°C to release the terpenoids
adsorbed. A He gas stream channeled the terpenoids into the CIS where
they were cryo-focused at -50°C. Subsequently, the CIS was heated up to
240°C to release the terpenoids onto the separation column (DB-5,
Agilent) at a He flow of 1 ml min\selectlanguage{english}\textsuperscript{-1}. Oven temperature
program and MS settings were exactly as described by Kleiber et al.
(2017). Raw data files were processed with the Agilent MassHunter
Software (Agilent, Waldbronn, Germany). For compound identification,
mass spectra were searched in the MS spectral databank NIST (National
Institute of Standards and Technology, Gaithersburg, MD, USA) and by
external terpenoid standards. Peak alignment was manually checked.
Terpenoids were quantified with calibration curves of representative
terpenoids (\emph{i.e} ., \selectlanguage{greek}α-\selectlanguage{english}pinene for monoterpenes, \selectlanguage{greek}α-\selectlanguage{english}humulene for
sesquiterpenes and diterpenes and 1,8-cineole for oxygenated
terpenoids).

\subsection*{Statistics}

{\label{statistics}}

To test significant differences of total terpenoid contents among
different tissues, statistical analyses were performed using SigmaPlot
11.0 (Systat Software GmbH, Erkrath, Germany). Since neither normality
distribution nor equal variance was given even when data were log- or
square-root transformed, we applied the Kruskal Wallis One Way Analysis
of Variance on Ranks followed by Dunn's Method for all pairwise multiple
comparisons. Differences were considered significant at p \textless{}
0.05. In order to compare the different tissue samples and xylem sap
samples for terpenoid patterns, partial least-square discriminant
analysis (PLS-DA) was performed using the freely available web based
software package MetaboAnalyst 4.0 (Chong et al., 2018,
http://www.metaboanalyst.ca). Before running PLS-DA, raw data of
terpenoid concentrations in xylem sap, needles, bark, wood and roots
were subjected to a natural logarithm transformation (`generalized
logarithm transformation') within MetaboAnalyst 4.0.

\section*{Results}

{\label{results}}

\subsection*{Numerous oxygenated terpenoids are abundant in xylem
sap}

{\label{numerous-oxygenated-terpenoids-are-abundant-in-xylem-sap}}

A considerable amount of terpenoids was abundant in the trees' xylem
sap. We identified 44 terpenoids making up a total concentration of
6.4\selectlanguage{ngerman}±0.9 \selectlanguage{greek}µ\selectlanguage{english}g mL\textsuperscript{-1} (Figure 1A). The majority of the
compounds were oxygenated terpenoids constituting 85\% of total
terpenoids (\emph{i.e} . oxygenated monoterpenoids, MT-Os: 74\%;
oxygenated sesquiterpenoids, SQT-Os: 8\%; oxygenated diterpenoids,
DT-Os: 3\%), whereas terpenes formed a smaller fraction (monoterpenes,
MTs: 13\%; SQTs: 2\%; diterpenes, DTs: \textless{}0.1\%) (Figure 1D).
Linalool was the most abundant compound (20\% of total terpenoids),
followed by trans-sabinene hydrate (9\%), terpinen-4-ol (8\%) and
camphor (7\%) (Figure 2C). The most abundant MT was \selectlanguage{greek}β-\selectlanguage{english}pinene (4\% of
total terpenoids), followed by terpinolene (3\%), \selectlanguage{greek}α-\selectlanguage{english}pinene (2\%) and
\selectlanguage{greek}δ-\selectlanguage{english}3-carene (2\%) (Figure 3C). Surprisingly, we also identified five SQTs
(\emph{i.e} ., \selectlanguage{greek}α-\selectlanguage{english}farnesene, muuroladiene, \selectlanguage{greek}δ-\selectlanguage{english}cadinene, \selectlanguage{greek}β-\selectlanguage{english}caryophyllene
and longifolene) in the xylem sap, however, at very low amounts (Figure
4C). Besides, the three oxygenated SQTs oplopanone (4\%), \selectlanguage{greek}δ-\selectlanguage{english}cadinol
(2\%) as well as longiborneol (1\%) were present (Figure 5C). We also
observed the DT biformene (\textless{}1\%) and the oxygenated DTs manoyl
oxide (2\%) and sclareol (2\%) in xylem sap (Table S2).

\subsection*{Terpenoid emission from spruce
needles}

{\label{terpenoid-emission-from-spruce-needles}}

Needles emitted 55 terpenoids at a rate of 1,709\selectlanguage{ngerman}±248 ng
g\textsuperscript{-1} DW h\textsuperscript{-1} (Figure 1B). The
composition consisted of 32\% MTs, 46\% MT-Os, 18\% SQTs, 2\% SQT-Os and
traces of DTs and DT-Os (Figure 1E). The quantitatively most important
MT-Os emitted were camphor (210±88 ng g\textsuperscript{-1} DW
h\textsuperscript{-1}), borneol (141±36 ng g\textsuperscript{-1} DW
h\textsuperscript{-1}) and linalool (113±13 ng g\textsuperscript{-1} DW
h\textsuperscript{-1}) (Figure 2A). Moreover, the MTs camphene (170±67
ng g\textsuperscript{-1} DW h\textsuperscript{-1}), \selectlanguage{greek}α-\selectlanguage{english}pinene (164\selectlanguage{ngerman}±45 ng
g\textsuperscript{-1} DW h\textsuperscript{-1}), \selectlanguage{greek}β-\selectlanguage{english}pinene (86\selectlanguage{ngerman}±16 ng
g\textsuperscript{-1} DW h\textsuperscript{-1}) as well as limonene \&
\selectlanguage{greek}β-\selectlanguage{english}phellandrene (66\selectlanguage{ngerman}±21 ng g\textsuperscript{-1} DW h\textsuperscript{-1})
were strongly released (Figure 3A). The most abundant SQT emitted from
Norway spruce needles was \selectlanguage{greek}α-\selectlanguage{english}longipinene (69\selectlanguage{ngerman}±50 ng
g\textsuperscript{-1}DW h\textsuperscript{-1}), followed by \selectlanguage{greek}α-\selectlanguage{english}farnesene
(45\selectlanguage{ngerman}±25 ng g\textsuperscript{-1} DW h\textsuperscript{-1}),
\selectlanguage{greek}β-\selectlanguage{english}caryophyllene (32\selectlanguage{ngerman}±22 ng g\textsuperscript{-1} DW
h\textsuperscript{-1}) as well as longifolene (26±17 ng
g\textsuperscript{-1} DW h\textsuperscript{-1}) (Figure 4A). In
contrast, needles emitted SQT-O at relatively low rates (Figure 5A).

\subsection*{Terpenoid content in spruce
tissues}

{\label{terpenoid-content-in-spruce-tissues}}

Multivariate analysis (PLS-DA) of Norway spruce tissues showed
separation into individual clusters indicating different terpenoid
compositions (Figure 6). The respective loading plots highlighted the
compounds responsible for clustering (Figure S1). Carvone (no `18', of
the loading plot as listed in Table S3), \selectlanguage{greek}α-\selectlanguage{english}farnesene (`29') and the
oxygenated SQT longiborneol (`43') were the main drivers for separation
of sap from tissues. Differently, bornyl acetate (`26'), nerolidol
(`44') and \selectlanguage{greek}α-\selectlanguage{english}cadinol (`45') were responsible for the cluster formation
of needle and bark samples. In contrast, \selectlanguage{greek}γ-\selectlanguage{english}decalactone (`22'), linalool
formate (`23') and isoborneol ('24') were the main drivers for
clustering of wood and root samples.

Besides composition, also total terpenoid contents differed strongly
among different tissues, amounting to 126\selectlanguage{ngerman}±13 mg g\textsuperscript{-1} DW
in bark, 163±33 mg g\textsuperscript{-1} DW in current-year needles,
29±2 mg g\textsuperscript{-1} DW in roots and 13±2 mg
g\textsuperscript{-1} DW in wood (Figure 1C). In total, 82 different
terpenoids were detected in bark; 78\% were terpenes (72\% MT, 5\% SQT,
1\% DT) and 22\% oxygenated terpenoids (4\% MT-O, 1\% SQT-O, 17\% DT-O)
(Figure 1F). The most abundant MTs in bark were \selectlanguage{greek}β-\selectlanguage{english}pinene (34\%),
\selectlanguage{greek}α-\selectlanguage{english}pinene (19\%), limonene \& \selectlanguage{greek}β-\selectlanguage{english}phellandrene (9\%) (Figure 3D), whereas
bornyl acetate (2\%) dominated the MT-O fraction (Figure 2D). In
addition, in bark \selectlanguage{greek}β-\selectlanguage{english}caryophyllene (1\%), \selectlanguage{greek}α-\selectlanguage{english}longipinene (1\%) and
longifolene (1\%) were the most abundant SQTs (Figure 4D), whereas
\selectlanguage{greek}δ-\selectlanguage{english}cadinol (\textless{}1\%) and nerolidol (\textless{}1\%) dominated the
SQT-O fraction in bark (Figure 5D). Seven DTs and 12 oxygenated DT were
also observed, with pimaric acid (6\%), abietic acid (3\%) and manool
(3\%) being the most abundant DT-Os in bark of Norway spruce.

Differently from bark, 56 terpenoids were identified in the wood; MT
(78\% of all terpenoids) and MT-O (12\%) dominated the composition
(Figure 1F). The most abundant MTs in wood were (in the sequence of
abundance) \selectlanguage{greek}β-\selectlanguage{english}pinene (38\%) \textgreater{} \selectlanguage{greek}α-\selectlanguage{english}pinene (26\%) \textgreater{}
limonene \& \selectlanguage{greek}β-\selectlanguage{english}phellandrene (7\%) (Figure 3E). Linalool (6\%) was the
most abundant MT-O in wood and - similar to bark - \selectlanguage{greek}α-\selectlanguage{english}longipinene
\textgreater{} \selectlanguage{greek}β-\selectlanguage{english}caryophyllene \textgreater{} longifolene - the
dominating SQT (Figures 2E and 4E). Only three SQT-Os were identified in
wood, which were longiborneol \textgreater{} carotol \textgreater{}
oplopanone (Figure 5E). We also observed trace amounts of three DTs and
four oxygenated DTs.

In total, 74 terpenoids were abundant in roots. Similar to bark, MT
(78\%) and DT-O (15\%) dominated the terpenoid composition (Figure 1F).
The MT \selectlanguage{greek}β-\selectlanguage{english}pinene was most abundant (27\%), followed by \selectlanguage{greek}α-\selectlanguage{english}pinene (24\%),
camphene (12\%) as well as limonene \& \selectlanguage{greek}β-\selectlanguage{english}phellandrene (8\%) (Figure 3F).
Manoyl oxide (7\%) and methyl dehydroabietate (6\%) were the most
important DT-Os in roots.

Terpenoids in current-year needles contained 50\% MT-O, 20\% SQT-O, 18\%
MT, 10\% SQT and traces of DT and DT-O (Figure 1F). Camphor (MT-O, 22\selectlanguage{ngerman}±4
mg g\textsuperscript{-1} DW), \selectlanguage{greek}α-\selectlanguage{english}cadinol (SQT-O, 17\selectlanguage{ngerman}±4 mg
g\textsuperscript{-1} DW), bornyl acetate (MT-O, 16±5 mg
g\textsuperscript{-1} DW) and camphene (MT, 11±2 mg
g\textsuperscript{-1} DW) were the most abundant compounds stored in
needles (Figures 2B, 3B, 4B and 5B). The terpenoid contents in
previous-year needles showed very similar contents and composition with
current-year needles (data not shown).

\subsection*{Common compounds in xylem sap and Norway spruce
tissues}

{\label{common-compounds-in-xylem-sap-and-norway-spruce-tissues}}

To get hints on possible sources for terpenoids in the xylem sap, all
terpenoids identified in xylem sap, bark, wood as well as roots were
tested for common abundance (Venn diagram, Figure 7). P-menth-2-en-1-ol
and \selectlanguage{greek}α-\selectlanguage{english}farnesene exclusively occurred in xylem sap, but not in bark, wood
or roots (Figure 7, Table S4). On the other hand, 17 terpenoids were
only found in bark, 4 only in wood and 13 terpenoids only in roots
(Figure 7, Table S3). The MT-O nopol was only present in xylem sap and
roots (Figure 7, Table S3). In an additional approach, emitted
terpenoids, and terpenoids identified in xylem sap and needles were also
compared (Venn diagram, Figure 8). Spruce needles emitted almost all
identified terpenoids present in xylem sap into the atmosphere except
p-menth-2-en-1-ol and the two oxygenated SQTs \selectlanguage{greek}δ-\selectlanguage{english}cadinol and oplopanone
(Figure 8, Table S5). Importantly, among the emitted compounds, four
terpenoids (citronellyl propionate, sclareol, perilla aldehyde,
\selectlanguage{greek}α-\selectlanguage{english}farnesene) were absent in needles, but occurred in xylem sap (Figure
8, Table S5).

\section*{Discussion}

{\label{discussion}}

Our main goal was to elucidate if terpenoids are transported in the
transpiration stream of Norway spruce, for which we obtained clear
evidence. Particularly striking was the high portion of oxygenated
terpenoids in xylem sap. This observation is consistent with their
Henry's law coefficients. For example, the water solubility of linalool
ranges around 10 mmol L\textsuperscript{-1} (\emph{i.e.} 1,600 \selectlanguage{greek}µ\selectlanguage{english}g
mL\textsuperscript{-1}) (Copolovici \& Niinemets, 2005; Table S2), thus
easily allowing the linalool concentrations observed, which were
10\textsuperscript{3}-times lower (up to 1.6 \selectlanguage{greek}µ\selectlanguage{english}g mL\textsuperscript{-1}).
Similarly, the concentrations of other terpenoids were lower than their
assumed solubility. For example, 1,8-cineole concentrations in xylem sap
amounted to 198\selectlanguage{ngerman}±57 ng mL\textsuperscript{-1}, but water solubility is
16.0-22.7 mol m\textsuperscript{-3} (equals 2.47-3.50 mg
mL\textsuperscript{-1}) and \selectlanguage{greek}β-\selectlanguage{english}pinene was abundant at concentrations of
238\selectlanguage{ngerman}±160 ng mL\textsuperscript{-1} with an assumed water solubility of
0.049-0.081 mol m\textsuperscript{-3} (\emph{i.e} . 6.67-11.03 \selectlanguage{greek}µ\selectlanguage{english}g
mL\textsuperscript{-1}) (Copolovici \& Niinemets, 2005; Table S2). In
general, also the concentrations of most SQTs were lower than the
maximum estimated from water solubility. Nevertheless, there were a few
exceptions; the concentration of \selectlanguage{greek}α-\selectlanguage{english}farnesene amounted to 35\selectlanguage{ngerman}±4 ng
mL\textsuperscript{-1} in xylem sap although it is not expected to
exceed 11 ng mL\textsuperscript{-1} according to its estimated water
solubility (Table S2). Similarly, the concentrations of sclareol and
manoyl oxide were somewhat higher than their estimated water
solubilities (Table S2). Such discrepancies might be due to, effects of
temperature, ambient pressure, pH and presence of other compounds in the
solution. Moreover, the water solubility of DT-Os has been estimated
from Log K\textsubscript{ow} (WSKOW v1.41) but not from real
measurements (ChemSpider, 2018). Most importantly, knowledge of water
solubility allows estimation of equilibrium concentrations. If, however,
the surrounding tissue of xylem sap contains higher concentrations of
individual compounds, higher concentrations in xylem sap will be the
consequence.

A second objective of our work was to understand if xylem transported
terpenoids might contribute to terpenoid emission from Norway spruce
needles. This idea is supported by the observation that many terpenoids
present in xylem sap were emitted from needles (Figure 8). Thirteen
these compounds were absent or only present in traces in needle tissue
(blue highlighted columns in Figs. 2-5) and, hence, their emission might
partially be controlled by xylem transport. Of course, we cannot exclude
that emission of these compounds was partially or even fully driven
by\emph{de novo} biosynthesis in needles. To differentiate between
sources for emission, \textsuperscript{13}C-tracer studies should be
performed in future studies. Noteworthy, total terpenoid emission from
Norway spruce in our work amounted to 1.8 \selectlanguage{greek}μ\selectlanguage{english}g g\textsuperscript{-1} DW
h\textsuperscript{-1} which is comparable though in the lower range of
earlier studies (0.5-12 \selectlanguage{greek}μ\selectlanguage{english}g g\textsuperscript{-1} DW
h\textsuperscript{-1}, Bourtsoukidis et al., 2014; Martin et al., 2003;
Yassaa et al., 2012). Larger discrepancies were observed in the
composition because the monoterpenoid fraction was smaller in earlier
work than in our study (8\%, based on results from Martin et al. (2003);
40\%, from our present work). Such discrepancies might be caused by
differences in genotype and/or site conditions. Since especially
1,8-cineole and linalool were highly abundant stress might also play a
role as these compounds are induced by stress (Blande, Turunen \&
Holopainen, 2009; Kannaste et al., 2009; Martin et al., 2003). However,
we did not observe any sign of stress in our experimental trees.

Besides being emitted, xylem transported compounds can also be taken up
by mesophyll cells and be channeled into cellular metabolism, as
demonstrated for short-chained oxygenated VOC (acetaldehyde, ethanol)
(Kreuzwieser, Scherer \& Rennenberg, 1999; Kreuzwieser et al. 2000,
2001; Rissanen, H\selectlanguage{ngerman}ölttä \& Bäck, 2018). These compounds either accumulate
in leaf cells or being metabolized or converted to other monoterpenoid
species (Niinemets et al., 2014). Our work provided hints that
p-menth-2-en-1-ol and \selectlanguage{greek}δ-\selectlanguage{english}cadinol were converted to other compounds, since
they were neither emitted nor detectable in needle tissue although
clearly abundant in the xylem sap. In contrast, the sesquiterpenoid
oplopanone seemed to accumulate in needles, which fits to reports
indicating oplopanone in needles of several conifer species (\emph{Larix
kaempferi} : Tanaka, Ohtsu \& Matsunaga, 1997; \emph{Chamaecyparis
formosensis} : Lin, Fang \& Cheng, 1999; \emph{Cryptomeria japonica} :
Su, Fang \& Cheng, 1995).

The finding of terpenoids in the xylem sap poses the question on
possible sources for these compounds. Our data allow speculation on the
origin of at least some of the compounds. For example, xylem sap
abundant nopol was detected only in roots, but not in wood or bark,
suggesting that this compound was produced in roots and loaded into the
xylem. Nopol was not found in Norway spruce before, but its abundance
has been demonstrated in needles of other conifers (\emph{Picea
orientalis} : Ucar, Balaban \& Usta, 2003; \emph{Pinus} spp: Mateus,
2009), and oil of rosemary (Tounekti et al., 2011) and \emph{Eucalyptus
olida} (Gilles et al., 2010).

Since terpenoids are biosynthesized mainly in close vicinity to the
cambium and stored nearby in secretory tissue such as resin canals or
resin ducts and their surrounding epithelial cells (Back, 2002),
diffusive transfer from these compartments into the xylem vessels might
occur. Still, it has to be taken into account that epithelial cells and
resin ducts are strongly lignified which might reflect a barrier for
diffusion of terpenoids to the outside of such structures. The abundance
of \selectlanguage{greek}δ-\selectlanguage{english}cadinol in bark and xylem sap, but its absence in roots and wood,
suggests that this sesquiterpenoid was formed in bark and loaded into
the xylem. However, for most of the other terpenoids present in xylem
sap it is not possible to conclude on possible sources since they were
abundant in root, wood and bark tissue, and therefore can be derived
from all of them.

Importantly, the terpenoid composition in xylem sap differed
considerably from that of spruce needles, bark, wood and roots (Figure
6). Some compounds such as the MT-Os bornyl acetate and isoborneol which
were highly abundant in bark and wood were not even present in traces in
xylem sap. For this reason, we can exclude that terpenoids in xylem sap
are a result of contamination from residues of resin during cutting the
twigs for xylem sap collection. From a physiological point of view, the
results suggest that terpenoids are not just leaking from any of the
studied tissue/resin ducts into the xylem sap. This assumption rises the
questions, whether (i) xylem transport of terpenoids has to be
considered active transport processes allowing at least partial control
of compounds released into this apoplastic space, and, consequently,
(ii) whether any function is related to long-distance transport of
terpenoids. Transport of terpenoids across the plasmalemma into the
apoplast might be mediated by secretion; this process has been
demonstrated for transport of terpenoids into glandular trichomes (Dai
et al., 2010; Lange \& Turner, 2013; Martin et al., 2009; Tissier, 2012;
Tissier, Morgan \& Dudareva, 2017). On the other hand, active terpenoid
transport requiring the consumption of ATP has been also proposed, which
might be mediated by ATP binding cassette (ABC)-transporters. This was
indirectly concluded from the common sites of terpenoid biosynthesis and
expression of such transporters in glandular trichomes (Adebesin et al.,
2017; Aziz et al., 2005; Bertea et al., 2006; Chatzopoulou et al., 2010;
Harada et al., 2010; Lange et al., 2000; Schilmiller et al., 2010; Wang
et al., 2008, 2016; Yazaki, 2006). Moreover, such transporters seem to
be responsible for export of diterpenes (e.g. sclareol, manool,
cembrene) and sesquiterpenes (e.g. \selectlanguage{greek}β-\selectlanguage{english}caryophyllene, capsidiol) (Campbell
et al., 2003; Crouzet et al. 2013; Fu et al., 2017; Jasi\selectlanguage{polish}ń\selectlanguage{english}ski et al.
2001; Pierman et al., 2017; Seo et al. 2012; Van den Br\selectlanguage{ngerman}ûle et al., 2002)
and are also thought to be involved in monoterpene export (Tissier et
al., 2017). Both, secretion via vesicles and membrane transport through
ABC-transporters might in addition be connected to cell internal
transfer by lipophilic transfer proteins (LPTs), which channel the
compounds from the sites of production (e.g. chloroplasts) towards the
plasma membrane (Tissier et al., 2017). Still, this is still speculative
and experiments have to be conducted to obtain deeper insight.

To summarize, we demonstrated that a considerable amount of terpenoids
is transported in the xylem sap of Norway spruce. In accordance with the
Henry's constants, the transpiration stream mainly contained oxygenated
terpenoids and hydrocarbon terpenes to a less extent. The very different
terpenoid patterns in the different plant tissues investigated compared
to the xylem sap, indicates that xylem loading of terpenoids is a
controlled process posing the question of possible functions of
transported terpenoids. Future studies should therefore particularly
focus on the origin and functions of xylem sap transported terpenoids in
conifers.

\section*{Acknowledgement}

{\label{acknowledgement}}

Qiuxiao Duan greatly acknowledges support from the China Scholarship
Council (CSC, Grant \#2011630183), Landesgraduiertenförderungsgesetz
(LGFG) Baden-Württemberg, German Academic Exchange Service (DAAD) as
well as University of Freiburg, Germany.

\section*{Conflict of interest}

{\label{conflict-of-interest}}

The authors declare that they have no conflicts of interest.

\section*{Reference}

{\label{reference}}

Adebesin, F., Widhalm, J.R., Boachon, B., Lefèvre, F., Pierman, B.,
Lynch, J.H., Alam, I., Junqueira, B., Benke, R., Ray, S. \& Porter, J.A.
(2017) Emission of volatile organic compounds from petunia flowers is
facilitated by an ABC transporter. \emph{Science} \textbf{356} ,
1386-1388.

Axelsson, K., Zendegi-Shiraz, A., Swedjemark, G., Borg-Karlson, A.K. \&
Zhao, T. (2016) Clone specific chemical defense responses in Norway
spruce to infestations by two pathogenic fungi.~\emph{Forest Pathology}
.

Aziz, N., Paiva, N.L., May, G.D. \& Dixon, R.A. (2005) Transcriptome
analysis of alfalfa glandular trichomes. \emph{Planta} \textbf{221} ,
28-38.

Back, E.L. (2002) Pattern of parenchyma and canal resin composition in
softwoods and hardwoods. \emph{Journal of wood science} \textbf{48} ,
167-170.

Bäck, J., Aalto, J., Henriksson, M., Hakola, H., He, Q. \& Boy, M.
(2012) Chemodiversity of a Scots pine stand and implications for terpene
air concentrations. \emph{Biogeosciences} \textbf{9} , 689-702.

Bertea, C.M., Voster, A., Verstappen, F.W., Maffei, M., Beekwilder, J.
\& Bouwmeester, H. J. (2006) Isoprenoid biosynthesis in \emph{Artemisia
annua} : cloning and heterologous expression of a germacrene A synthase
from a glandular trichome cDNA library. \emph{Archives of Biochemistry
and Biophysics} \textbf{448} , 3-12.

Blanch, J.S., Penuelas, J., Sardans, J. \& Llusia, J. (2009) Drought,
warming and soil fertilization effects on leaf volatile terpene
concentrations in \emph{Pinus halepensis} and \emph{Quercus ilex}
.\emph{Acta Physiologiae Plantarum} \textbf{31} , 207.

Blande, J. D., Turunen, K. \& Holopainen, J. K. (2009) Pine weevil
feeding on Norway spruce bark has a stronger impact on needle VOC
emissions than enhanced ultraviolet-B radiation. \emph{Environmental
Pollution} \textbf{157} , 174--180.

Bourtsoukidis, E., Bonn, B., Dittmann, A., Hakola, H., Hellén, H. \&
Jacobi, S. (2012) Ozone stress as a driving force of sesquiterpene
emissions: a suggested parameterisation. \emph{Biogeosciences}\textbf{9}
, 4337-4352.

Bourtsoukidis, E., Williams, J., Kesselmeier, J., Jacobi, S. \& Bonn, B.
(2014) From emissions to ambient mixing ratios: online seasonal field
measurements of volatile tissueic compounds over a Norway
spruce-dominated forest in central Germany. \emph{Atmospheric Chemistry
and Physics} \textbf{14} , 6495-6510.

Bufler, U., Seufert, G. \& Jüttner, F. (1990) Monoterpene patterns of
different tissues and plant parts of Norway spruce (\emph{Picea abies}L.
Karst.). \emph{Environmental Pollution} \textbf{68} , 367-375.

Campbell E.J., Schenk P.M., Kazan K., Penninckx I.A., Anderson J.P.,
Maclean D.J., Cammue B.P., Ebert P.R. \& Manners J.M. (2003)
Pathogen-responsive expression of a putative ATP-binding cassette
transporter gene conferring resistance to the diterpenoid sclareol is
regulated by multiple defense signaling pathways in
Arabidopsis.\emph{Plant Physiolog} y \textbf{133} , 1272--1284.

Chatzopoulou, F.M., Makris, A.M., Argiriou, A., Degenhardt, J. \&
Kanellis, A.K. (2010) EST analysis and annotation of transcripts derived
from a trichome-specific cDNA library from \emph{Salvia fruticosa}
.\emph{Plant Cell Reports} \textbf{29} , 523-534.

ChemSpider, 2018: http://www.chemspider.com/

Chong, J., Soufan, O., Li, C., Caraus, I., Li, S., Bourque, G., Wishart,
D.S. \& Xia, J. (2018) MetaboAnalyst 4.0: towards more transparent and
integrative metabolomics analysis. \emph{Nucleic acids research} .
https://doi.org/10.1093/nar/gky310

Copolovici, L.O. \& Niinemets, Ü. (2005) Temperature dependencies of
Henry's law constants and octanol/water partition coefficients for key
plant volatile monoterpenoids. \emph{Chemosphere} \textbf{61} ,
1390-1400.

Crouzet, J., Roland, J., Peeters, E., Trombik, T., Ducos, E., Nader, J.
\& Boutry, M. (2013) NtPDR1, a plasma membrane ABC transporter
from\emph{Nicotiana tabacum} , is involved in diterpene
transport.\emph{Plant Molecular Biology} \textbf{82} , 181-192.

Dai, X., Wang, G., Yang, D.S., Tang, Y., Broun, P., Marks, M.D. Sumner,
L.W., Dixon, R.A \& Zhao, P.X. (2010) TrichOME: a comparative omics
database for plant trichomes. \emph{Plant Physiology} \textbf{152} ,
44-54.

Dudareva, N., Klempien, A., Muhlemann, J. K., \& Kaplan, I. (2013)
Biosynthesis, function and metabolic engineering of plant volatile
tissueic compounds. \emph{New Phytologist} , \textbf{198} , 16-32.

DWD, 2015: Deutscher Wetterdienst. https://www.dwd.de

Fichan, I., Larroche, C. \& Gros, J. B. (1999) Water solubility, vapor
pressure, and activity coefficients of terpenes and
terpenoids.\emph{Journal of Chemical and Engineering Data} \textbf{44} ,
56-62.

Filella, I., Wilkinson, M.J., Llusia, J., Hewitt, C.N. \& Peñuelas, J.
(2007). Volatile organic compounds emissions in Norway spruce
(\emph{Picea abies} ) in response to temperature
changes.\emph{Physiologia Plantarum} \textbf{130} , 58-66.

Flo, D., Norli, H. R., Økland, B. \& Krokene, P. (2018) Successful
reproduction and pheromone production by the spruce bark beetle in
evolutionary naïve spruce hosts with familiar terpenoid
defences.\emph{Agricultural and Forest Entomology} . https://
doi.org/10.1111/afe.12280

Fu, X., Shi, P., He, Q., Shen, Q., Tang, Y., Pan, Q., Ma, Y., Yan, T.,
Chen, M., Hao, X. \& Liu, P. (2017) AaPDR3, a PDR transporter 3, is
involved in sesquiterpene \selectlanguage{greek}β-\selectlanguage{english}caryophyllene transport in \emph{Artemisia
annua} . \emph{Frontiers in Plant Science} \textbf{8,} 723.

Gershenzon J \& Croteau R (1991) Terpenoids. In: Herbivores, their
interactions with secondary Metabolites (eds G.A. Rosenthal \& M.R.
Berbembaum), pp. 165--219. Academic Press, New York, NY.

Gershenzon, J., \& Dudareva, N. (2007) The function of terpene natural
products in the natural world. \emph{Nature Chemical Biology}
,\textbf{3} , 408.

Ghirardo, A., Koch, K., Taipale, R., Zimmer, I.N.A., Schnitzler, J. P.
\& Rinne, J. (2010) Determination of \emph{de novo} and pool emissions
of terpenes from four common boreal/alpine trees
by\textsuperscript{13}CO2 labelling and PTR-MS analysis. \emph{Plant,
Cell and Environment} \textbf{33} , 781-792.

Gilles, M., Zhao, J., An, M. \& Agboola, S. (2010) Chemical composition
and antimicrobial properties of essential oils of three
Australian\emph{Eucalyptus} species. \emph{Food Chemistry} \textbf{119}
, 731-737.

Grabmer, W., Kreuzwieser, J., Wisthaler, A., Cojocariu, C., Graus, M.,
Rennenberg, H., Steigner, D., Steinbrecher, R. \& Hansel, A. (2006) VOC
emissions from Norway spruce (\emph{Picea abies} L.{[}Karst{]}) twigs in
the field---results of a dynamic enclosure study. \emph{Atmospheric
Environment} \textbf{40} , 128-137.

Guenther, A.B., Zimmerman, P.R., Harley, P.C., Monson, R.K. \& Fall, R.
(1993) Isoprene and monoterpene emission rate variability: model
evaluations and sensitivity analyses. \emph{Journal of Geophysical
Research: Atmospheres} \textbf{98} , 12609-12617.

Harada, E., Kim, J.A., Meyer, A.J., Hell, R., Clemens, S. \& Choi, Y.E.
(2010) Expression profiling of tobacco leaf trichomes identifies genes
for biotic and abiotic stresses. \emph{Plant and Cell
Physiology}\textbf{51} , 1627-1637.

Jasi\selectlanguage{polish}ń\selectlanguage{english}ski M., Stukkens Y., Degand H., Purnelle B., Marchand-Brynaert J.
\& Boutry M. (2001) A plant plasma membrane ATP binding cassette-type
transporter is involved in antifungal terpenoid secretion. \emph{The
Plant Cell} \textbf{13} , 1095--1107.

Kannaste, A., Nordenhem, H., Nordlander, G. \& Borg-Karlson, A.-K.
(2009). Volatiles from a mite-infested spruce clone and their effects on
pine weevil behaviour. \emph{Journal of Chemical Ecology} \textbf{35} ,
1262--1271.

Kleiber, A., Duan, Q., Jansen, K., Verena Junker, L., Kammerer, B.,
Rennenberg, H., Ensminger, I., Gessler, A. \& Kreuzwieser, J. (2017)
Drought effects on root and needle terpenoid content of a coastal and an
interior Douglas fir provenance. \emph{Tree physiology} \textbf{37} ,
1648-1658.

Kreuzwieser, J., Harren, F.J., Laarhoven, L.J., Boamfa, I., Te
Lintel-Hekkert, S., Scheerer, U., H\selectlanguage{ngerman}üglin, C. \& Rennenberg, H. (2001)
Acetaldehyde emission by the leaves of trees--correlation with
physiological and environmental parameters. \emph{Physiologia
Plantarum}\textbf{113} , 41-49.

Kreuzwieser, J., Kühnemann, F., Martis, A., Rennenberg, H. \& Urban, W.
(2000) Diurnal pattern of acetaldehyde emission by flooded poplar
trees.\emph{Physiologia Plantarum} \textbf{108} , 79-86.

Kreuzwieser, J., Scheerer, U. \& Rennenberg, H. (1999) Metabolic origin
of acetaldehyde emitted by poplar (\emph{Populus tremula} × \emph{P.
alba} ) trees. \emph{Journal of Experimental Botany} \textbf{50} ,
757-765.

Kuroda, K. (1991). Mechanism of cavitation development in the pine wilt
disease. \emph{European Journal of Forest Pathology} \textbf{21} ,
82-89.

Lange, B. M. \& Turner, G. W. (2013) Terpenoid biosynthesis in trichomes
- current status and future opportunities. \emph{Plant Biotechnology
Journal} \textbf{11} , 2-22.

Lange, B. M., Wildung, M. R., Stauber, E. J., Sanchez, C., Pouchnik, D.
\& Croteau, R. (2000) Probing essential oil biosynthesis and secretion
by functional evaluation of expressed sequence tags from mint glandular
trichomes. \emph{Proceedings of the National Academy of
Sciences}\textbf{97} , 2934-2939.

Lin, T.C., Fang, J.M. \& Cheng, Y.S. (1999) Terpenes and lignans from
leaves of \emph{Chamaecyparis formosensis} .
\emph{Phytochemistry}\textbf{51} , 793-801.

Loreto F., Ciccioli P., Cecinato A., Brancaleoni E., Frattoni M.,
Fabozzi C. \& Tricoli D. (1996) Evidence of the photosynthetic origin of
monoterpenes emitted by \emph{Quercus ilex} L. leaves
by\textsuperscript{13}C labeling. \emph{Plant Physiology} \textbf{110} ,
1317--1322.

Loreto, F., Mannozzi, M., Maris, C., Nascetti, P., Ferranti, F., \&
Pasqualini, S. (2001) Ozone quenching properties of isoprene and its
antioxidant role in leaves. \emph{Plant Physiology} \textbf{126} ,
993-1000.

Loreto F., Nascetti P., Graverini A. \& Mannozzi M. (2000) Emission and
content of monoterpenes in intact and wounded needles of the
Mediterranean pine, \emph{Pinus pinea} . \emph{Functional
Ecology}\textbf{14} , 589--595.

Loreto, F., Pollastri, S., Fineschi, S. \& Velikova, V. (2014). Volatile
isoprenoids and their importance for protection against environmental
constraints in the Mediterranean area. \emph{Environmental and
experimental botany} \textbf{103} , 99-106.

Loreto, F. \& Schnitzler, J. P. (2010). Abiotic stresses and induced
BVOCs. \emph{Trends in plant science} \textbf{15} , 154-166.

Martin, D.M., Gershenzon, J. \& Bohlmann, J. (2003) Induction of
volatile terpene biosynthesis and diurnal emission by methyl jasmonate
in foliage of Norway spruce. \emph{Plant Physiology} \textbf{132} ,
1586-1599.

Martin, D., Tholl, D., Gershenzon, J. \& Bohlmann, J. (2002) Methyl
jasmonate induces traumatic resin ducts, terpenoid resin biosynthesis,
and terpenoid accumulation in developing xylem of Norway spruce
stems.\emph{Plant Physiology} \textbf{129} , 1003-1018.

Martin, D.M., Toub, O., Chiang, A., Lo, B.C., Ohse, S., Lund, S.T. \&
Bohlmann, J. (2009) The bouquet of grapevine (\emph{Vitis vinifera} L.
cv. Cabernet Sauvignon) flowers arises from the biosynthesis of
sesquiterpene volatiles in pollen grains. \emph{Proceedings of the
National Academy of Sciences} , pnas-0901387106.

Mateus, E. P. (2009) Characterization of \emph{Pinus} spp. needles by
gas chromatography and mass spectrometry: Application to plant-insect
interactions. Ph.D. thesis, Faculty of Sciences and

Technology, Universidade Nova de Lisboa.

McGarvey, D. J. \& Croteau, R. (1995) Terpenoid metabolism. \emph{The
Plant Cell} , \textbf{7} , 1015.

Merk, L., Kloos, M., Schönwitz, R. \& Ziegler, H. (1988) Influence of
various factors on quantitative composition of leaf monoterpenes
of\emph{Picea abies} (L.) Karst. \emph{Trees} \textbf{2} , 45-51.

Muhlemann, J. K., Klempien, A. \& Dudareva, N. (2014). Floral volatiles:
from biosynthesis to function. \emph{Plant, cell \&
environment}\textbf{37} , 1936-1949.

Nagegowda, D. A. (2010). Plant volatile terpenoid metabolism:
biosynthetic genes, transcriptional regulation and subcellular
compartmentation. \emph{FEBS letters} \textbf{584} , 2965-2973.

Nagel, R., Berasategui, A., Paetz, C., Gershenzon, J. \& Schmidt, A.
(2014) Overexpression of an isoprenyl diphosphate synthase in spruce
leads to unexpected terpene diversion products that function in plant
defense. \emph{Plant Physiology} \textbf{164,} 555-569.

Niinemets, Ü., Fares, S., Harley, P. \& Jardine, K.J. (2014)
Bidirectional exchange of biogenic volatiles with vegetation: emission
sources, reactions, breakdown and deposition. \emph{Plant, Cell and
Environment} \textbf{37} , 1790-1809.

Persson, M., Borg-Karlson, A.K. \& Norin, T. (1993) Enantiomeric
composition of six chiral monoterpene hydrocarbons in different tissues
of \emph{Picea abies} . \emph{Phytochemistry} \textbf{33} , 303-307.

Pierman, B., Toussaint, F., Bertin, A., Levy, D., Smargiasso, N., De
Pauw, E. \& Boutry, M. (2017) Activity of the purified plant ABC
transporter NtPDR1 is stimulated by diterpenes and sesquiterpenes
involved in constitutive and induced defenses. \emph{Journal of
Biological Chemistry} \textbf{292} , 19491--19502.

Prieme, A., Knudsen, T. B., Glasius, M. \& Christensen, S. (2000)
Herbivory by the weevil, \emph{Strophosoma melanogrammum} , causes
severalfold increase in emission of monoterpenes from young Norway
spruce (\emph{Picea abies} ). \emph{Atmospheric Environment} \textbf{34}
, 711-718.

Rennenberg, H., Schneider, S. \& Weber, P. (1996) Analysis of uptake and
allocation of nitrogen and sulphur compounds by trees in the
field.\emph{Journal of Experimental Botany} \textbf{47} , 1491-1498.

Rissanen, K., Hölttä, T. \& Bäck, J. (2018) Transpiration directly
regulates the emissions of water-soluble short-chained
OVOCs.\emph{Plant, Cell and Environment} \textbf{41} , 2288--2298.

Schilmiller, A.L., Miner, D.P., Larson, M., McDowell, E., Gang, D.R.,
Wilkerson, C. \& Last, R.L. (2010) Studies of a biochemical factory:
tomato trichome deep expressed sequence tag sequencing and
proteomics.\emph{Plant Physiology} \textbf{153} , 1212-1223.

Schmidt, A., Nagel, R., Krekling, T., Christiansen, E., Gershenzon, J.
\& Krokene, P. (2011) Induction of isoprenyl diphosphate synthases,
plant hormones and defense signalling genes correlates with traumatic
resin duct formation in Norway spruce (\emph{Picea abies} ). \emph{Plant
molecular biology} \textbf{77} , 577-590.

Schönwitz, R., Lohwasser, K., Kloos, M. \& Ziegler, H. (1990) Seasonal
variation in the monoterpenes in needles of \emph{Picea abies} (L.)
Karst. \emph{Trees} \textbf{4} , 34-40.

Schürmann, W., Ziegler, H., Kotzias, D., Schönwitz, R. \& Steinbrecher,
R. (1993) Emission of biosynthesized monoterpenes from needles of Norway
spruce. \emph{Naturwissenschaften} \textbf{80} , 276-278.

Seo, S., Gomi, K., Kaku, H., Abe, H., Seto, H., Nakatsu, S., Neya, M.,
Kobayashi, M., Nakaho, K., Ichinose, Y. \& Mitsuhara, I. (2012)
Identification of natural diterpenes that inhibit bacterial wilt disease
in tobacco, tomato and Arabidopsis. \emph{Plant and Cell
Physiology}\textbf{53} , 1432-1444.

Su, W.C., Fang, J.M. \& Cheng, Y.S. (1995) Sesquiterpenes from leaves
of\emph{Cryptomeria japonica} . \emph{Phytochemistry} \textbf{39} ,
603-607.

Tanaka, R., Ohtsu, H. \& Matsunaga, S. (1997) Abietane diterpene acids
and other constituents from the leaves of \emph{Larix kaempferi}
.\emph{Phytochemistry} \textbf{46} , 1051-1057.

Tingey D.T., Manning M., Grothaus L.C. \& Burns W.F. (1979) The
influence of light and temperature on isoprene emission rates from live
oak. \emph{Plant Physiology} \textbf{47} ,112--118.

Tissier, A. (2012) Glandular trichomes: what comes after expressed
sequence tags? \emph{The Plant Journal} \textbf{70} , 51-68.

Tissier, A., Mtissue, J.A. \& Dudareva, N. (2017) Plant volatiles: going
`in' but not `out' of trichome cavities. \emph{Trends in Plant
Sciences}\textbf{22} , 930-938.

Tounekti, T., Vadel, A.M., Ennajeh, M., Khemira, H. \& Munné-Bosch, S.
(2011) Ionic interactions and salinity affect monoterpene and phenolic
diterpene composition in rosemary (\emph{Rosmarinus officinalis}
).\emph{Journal of Plant Nutrition and Soil Science} \textbf{174} ,
504-514.

Trapp, S. C. \& Croteau, R. B. (2001). Genomic organization of plant
terpene synthases and molecular evolutionary
implications.\emph{Genetics} \textbf{158} , 811-832.

Turtola, S., Manninen, A. M., Rikala, R. \& Kainulainen, P. (2003)
Drought stress alters the concentration of wood terpenoids in Scots pine
and Norway spruce seedlings. \emph{Journal of Chemical
Ecology}\textbf{29} , 1981-1995.

Ucar, G., Balaban, M. \& Usta, M. (2003) Volatile needle and wood
extracts of oriental spruce \emph{Picea orientalis} (L.)
Link.\emph{Flavour and Fragrance Journal} \textbf{18} , 368-375.

Van den Brûle S., Muller A., Fleming A.J. \& Smart C.C. (2002) The ABC
transporter SpTUR2 confers resistance to the antifungal diterpene
sclareol. \emph{The Plant Journal} \textbf{30} , 649-662.

Vickers, C.E., Possell, M., Cojocariu, C.I., Velikova, V.B.,
Laothawornkitkul, J., Ryan, A., Mullineaux, P.M. \& Nicholas Hewitt, C.
(2009). Isoprene synthesis protects transgenic tobacco plants from
oxidative stress. \emph{Plant, Cell \& Environment} \textbf{32} ,
520-531.

Wang B., Kashkooli A.B., Sallets A., Ting H.M., de Ruijter N.C.A.,
Olofsson L., Brodelius P., Brodelius, P., Pottier, M., Boutry, M.,
Bouwmeester, H. \& van der Krol A.R. (2016) Transient production of
artemisinin in \emph{Nicotiana benthamiana} is boosted by a specific
lipid transfer protein from \emph{A. annua} . \emph{Metabolic
Engineering} \textbf{38} , 159-169.

Wang, G., Tian, L., Aziz, N., Broun, P., Dai, X., He, J., King, A.,
Zhao, P.X. \& Dixon, R. A. (2008) Terpene biosynthesis in glandular
trichomes of hop. \emph{Plant physiology} \textbf{148} , 1254-1266.

Wu, H. \& Hu, Z. H. (1997). Comparative anatomy of resin ducts of the
Pinaceae. \emph{Trees} \textbf{11} , 135-143.

Wu, C., Pullinen, I., Andres, S., Kiendler-Scharr, A., Kleist, E.,
Wahner, A., Wildt, J. \& Mentel, T.F. (2017) \textsuperscript{13}C
labelling study of constitutive and stress-induced terpenoide missions
from Norway spruce and Scots pine. \emph{Biogeosciences Discussions}
.\emph{https://doi.org/10.5194/bg-2017-260}

Yassaa, N., Song, W., Lelieveld, J., Vanhatalo, A., Bäck, J. \&
Williams, J. (2012). Diel cycles of isoprenoids in the emissions of
Norway spruce, four Scots pine chemotypes, and in Boreal forest ambient
air during HUMPPA-COPEC-2010. \emph{Atmosheric Chemistry and
Physics}\textbf{12} , 7215--7229.

Yazaki, K. (2006) ABC transporters involved in the transport of plant
secondary metabolites. FEBS letters \textbf{580} , 1183-1191.

Zeneli, G., Krokene, P., Christiansen, E., Krekling, T. \& Gershenzon,
J. (2006) Methyl jasmonate treatment of mature Norway spruce
(\emph{Picea abies} ) trees increases the accumulation of terpenoid
resin components and protects against infection by \emph{Ceratocystis
polonica} , a bark beetle-associated fungus. \emph{Tree
Physiology}\textbf{26} , 977-988.

Zhao, T., Krokene, P., Björklund, N., Langström, B., Solheim, H.,
Christiansen, E. \& Borg-Karlson, A.K. (2010) The influence
of\emph{Ceratocystis polonica} inoculation and methyl jasmonate
application on terpene chemistry of Norway spruce, \emph{Picea abies}
.\emph{Phytochemistry} , \textbf{71} , 1332-1341.

Zhao, T., Krokene, P., Hu, J., Christiansen, E., Björklund, N.,
Langström, B., Solheim, H. \& Borg-Karlson, A.K. (2011a) Induced terpene
accumulation in Norway spruce inhibits bark beetle colonization in a
dose-dependent manner. \emph{PLoS One} \textbf{6} , e26649.

Zhao, T., Solheim, H., Langström, B., \& Borg-Karlson, A.K. (2011b)
Storm-induced tree resistance and chemical differences in Norway spruce
(\emph{Picea abies} ). \emph{Annals of forest science} \textbf{68} ,
657-665.

\section*{Figure legends}

{\label{figure-legends}}

Figure 1. Total terpenoid contents in xylem sap (A), total terpenoid
emissions (B) and total terpenoid contents in different tissues (C) as
well as terpenoid composition in xylem sap (D), of emissions (E), and in
different tissues (F) of Norway spruce trees. Data are means ± SE of at
least five biological replicates each. MT, monoterpenes; MT-O,
oxygenated monoterpenoids; SQT, sesquiterpenes; SQT-O, oxygenated
sesquiterpenoids; DT, diterpenes; DT-O, oxygenated diterpenoids.
Different letters indicate significant difference at p\textless{}0.05 by
ANOVA on ranks.

Figure 2. Emission rates (A), and contents/concentrations of the 16 most
abundant oxygenated monoterpenoids in current-year needles (B), xylem
sap (C), bark (D), wood (E), and roots (F) of Norway spruce trees. Data
are means ± SE of at least five biological replicates. All compounds
emitted but under-represented in needles are highlighted in blue.

Figure 3. Emission rates (A), and contents/concentrations of the 10 most
abundant monoterpenes in current-year needles (B), xylem sap (C), bark
(D), wood (E), and roots (F) of Norway spruce trees. Data are means ± SE
of at least five biological replicates. All compounds emitted but not
present in needles are highlighted in blue.

Figure 4. Emission rates (A), and contents/concentrations of the 11 most
abundant sesquiterpenes in current-year needles (B), xylem sap (C), bark
(D), wood (E), and roots (F) of Norway spruce trees. Data are means ± SE
of at least five biological replicates. All compounds emitted but absent
or under-represented in needles are highlighted in blue.

Figure 5. Emission rates (A), and contents/concentrations of the 8 most
abundant oxygenated sesquiterpenoids in current-year needles (B), xylem
sap (C), bark (D), wood (E), and roots (F) of Norway spruce trees. Data
are means ± SE of at least five biological replicates. All compounds
emitted but under-represented in needles are highlighted in blue.

Figure 6. Score plot of a partial least-square discriminant analysis
(PLS-DA) of the terpenoid contents in xylem sap, bark, wood,
current-year needles and roots of Norway spruce under natural condition
in 2015. The loading plot, which indicates the terpenoids that are
responsible for the sample distribution in the score plot, is given in
Figure S1 available as Supplementary Data at \emph{Plant Cell and
Environment} online.

Figure 7. Venn plot indicating the numbers of terpenoids present in
roots, bark, wood and xylem sap of Norway spruce (graph drawn
online:\emph{http://bioinformatics.psb.ugent.be/webtools/Venn/}and the
specific compounds were shown in Table S3).

Figure 8. Venn plot indicating the numbers of terpenoids present in
needles, emissions and xylem sap of spruce (graph drawn
online:\emph{http://bioinformatics.psb.ugent.be/webtools/Venn/}and the
specific compounds were shown in Table S4).

\textbf{Hosted file}

\verb`Figures-revision.docx`  available at \url{https://authorea.com/users/300429/articles/430072-terpenoids-are-transported-in-the-xylem-sap-of-norway-spruce}

\selectlanguage{english}
\FloatBarrier
\end{document}

