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-1 (i.e. 1,600 µg
mL-1) (Copolovici & Niinemets, 2005; Table S2), thus
easily allowing the linalool concentrations observed, which were
103-times lower (up to 1.6 µg mL-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±57 ng mL-1, but water solubility is
16.0-22.7 mol m-3 (equals 2.47-3.50 mg
mL-1) and β-pinene was abundant at concentrations of
238±160 ng mL-1 with an assumed water solubility of
0.049-0.081 mol m-3 (i.e . 6.67-11.03 µg
mL-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 α-farnesene amounted to 35±4 ng
mL-1 in xylem sap although it is not expected to
exceed 11 ng mL-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 Kow (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 byde novo biosynthesis in needles. To differentiate between sources
for emission, 13C-tracer studies should be performed
in future studies. Noteworthy, total terpenoid emission from Norway
spruce in our work amounted to 1.8 μg g-1 DW
h-1 which is comparable though in the lower range of
earlier studies (0.5-12 μg g-1 DW
h-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, Hölttä & 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 δ-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 (Larix
kaempferi : Tanaka, Ohtsu & Matsunaga, 1997; Chamaecyparis
formosensis : Lin, Fang & Cheng, 1999; 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 (Picea
orientalis : Ucar, Balaban & Usta, 2003; Pinus spp: Mateus,
2009), and oil of rosemary (Tounekti et al., 2011) and 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 δ-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. β-caryophyllene, capsidiol) (Campbell
et al., 2003; Crouzet et al. 2013; Fu et al., 2017; Jasiński et al.
2001; Pierman et al., 2017; Seo et al. 2012; Van den Brûle 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.