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.