Results

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±0.9 µg mL-1 (Figure 1A). The majority of the compounds were oxygenated terpenoids constituting 85% of total terpenoids (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: <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 β-pinene (4% of total terpenoids), followed by terpinolene (3%), α-pinene (2%) and δ-3-carene (2%) (Figure 3C). Surprisingly, we also identified five SQTs (i.e ., α-farnesene, muuroladiene, δ-cadinene, β-caryophyllene and longifolene) in the xylem sap, however, at very low amounts (Figure 4C). Besides, the three oxygenated SQTs oplopanone (4%), δ-cadinol (2%) as well as longiborneol (1%) were present (Figure 5C). We also observed the DT biformene (<1%) and the oxygenated DTs manoyl oxide (2%) and sclareol (2%) in xylem sap (Table S2).

Terpenoid emission from spruce needles

Needles emitted 55 terpenoids at a rate of 1,709±248 ng g-1 DW h-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-1 DW h-1), borneol (141±36 ng g-1 DW h-1) and linalool (113±13 ng g-1 DW h-1) (Figure 2A). Moreover, the MTs camphene (170±67 ng g-1 DW h-1), α-pinene (164±45 ng g-1 DW h-1), β-pinene (86±16 ng g-1 DW h-1) as well as limonene & β-phellandrene (66±21 ng g-1 DW h-1) were strongly released (Figure 3A). The most abundant SQT emitted from Norway spruce needles was α-longipinene (69±50 ng g-1DW h-1), followed by α-farnesene (45±25 ng g-1 DW h-1), β-caryophyllene (32±22 ng g-1 DW h-1) as well as longifolene (26±17 ng g-1 DW h-1) (Figure 4A). In contrast, needles emitted SQT-O at relatively low rates (Figure 5A).

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), α-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 α-cadinol (‘45’) were responsible for the cluster formation of needle and bark samples. In contrast, γ-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±13 mg g-1 DW in bark, 163±33 mg g-1 DW in current-year needles, 29±2 mg g-1 DW in roots and 13±2 mg g-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 β-pinene (34%), α-pinene (19%), limonene & β-phellandrene (9%) (Figure 3D), whereas bornyl acetate (2%) dominated the MT-O fraction (Figure 2D). In addition, in bark β-caryophyllene (1%), α-longipinene (1%) and longifolene (1%) were the most abundant SQTs (Figure 4D), whereas δ-cadinol (<1%) and nerolidol (<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) β-pinene (38%) > α-pinene (26%) > limonene & β-phellandrene (7%) (Figure 3E). Linalool (6%) was the most abundant MT-O in wood and - similar to bark - α-longipinene > β-caryophyllene > longifolene - the dominating SQT (Figures 2E and 4E). Only three SQT-Os were identified in wood, which were longiborneol > carotol > 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 β-pinene was most abundant (27%), followed by α-pinene (24%), camphene (12%) as well as limonene & β-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±4 mg g-1 DW), α-cadinol (SQT-O, 17±4 mg g-1 DW), bornyl acetate (MT-O, 16±5 mg g-1 DW) and camphene (MT, 11±2 mg g-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).

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 α-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 δ-cadinol and oplopanone (Figure 8, Table S5). Importantly, among the emitted compounds, four terpenoids (citronellyl propionate, sclareol, perilla aldehyde, α-farnesene) were absent in needles, but occurred in xylem sap (Figure 8, Table S5).