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).