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.