Materials and methods
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′ N, 7°50′ 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
°C, which was 0.4°C 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).
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 34°C. 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 CO2 at flow rates of 200 ml
min-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, Mülheim, 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-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 4°C 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.
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 N2 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 < 2 mm) of the same trees were
harvested. All plant materials were immediately shock-frozen in liquid
N2, and stored at -80°C. Before terpenoid analysis,
plant materials were homogenized to fine powders by mortar and pestle
under liquid N2.
For determination of tissue dry weights, aliquots of approximately 100
mg frozen needle, bark, wood or root powders were dried at 60°C until
the weight remained constant. Contents of all parameters were calculated
based on dry weight unless indicated otherwise.
Terpenoid extraction from xylem
sap
Terpenoid extraction was mediated by stir bar sorptive extraction (SBSE)
using polydimethylsiloxane (PDMS)-coated stir bars (Twisters®, 0.5 mm
PDMS layer thickness, 10 mm in length, Gerstel, Mülheim, Germany)
(Kleiber et al., 2017). To trap terpenoids, one Twister® was added into
an aliquot of 300 µl xylem sap and samples were shaken (1,400 rpm, 30°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.
Terpenoid extraction from plant
tissues
Aliquots of 25 mg of frozen powder were added into 1,500 µl methanol.
Samples were shaken (1,400 rpm, 30°C, 20 min) and centrifuged (15,000
rpm, 23°C, 5 min). Supernatants were diluted (bark, 1:50, other tissues,
1:33.3) in H2Odemin 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.
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−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 (i.e ., α-pinene for monoterpenes, α-humulene for
sesquiterpenes and diterpenes and 1,8-cineole for oxygenated
terpenoids).
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 <
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.