Transcriptomic Analysis Reveals Novel Regulators of the Scots Pine Stilbene Pathway.

Stilbenes accumulate in Scots pine heartwood where they have important roles in protecting wood from decaying fungi. They are also part of active defense responses, and their production is induced by different (a)biotic stressors. The specific transcriptional regulators as well as the enzyme responsible for activating the stilbene precursor cinnamate in the pathway are still unknown. UV-C radiation was the first discovered artificial stress activator of the pathway. Here, we describe a large-scale transcriptomic analysis of pine needles in response to UV-C and treatment with translational inhibitors, both activating the transcription of stilbene pathway genes. We used the data to identify putative candidates for the missing CoA ligase and for pathway regulators. We further showed that the pathway is transcriptionally activated by phosphatase inhibitor, ethylene and jasmonate treatments, as in grapevine, and that the stilbene synthase promoter retains its inducibility in some of the tested conditions in Arabidopsis, a species that normally does not synthesize stilbenes. Shared features between gymnosperm and angiosperm regulation and partially retained inducibility in Arabidopsis suggest that pathway regulation occurs not only via ancient stress-response pathway(s) but also via species-specific regulators. Understanding which genes control the biosynthesis of stilbenes in Scots pine aids breeding of more resistant trees.

Fungal Degradation of Extractives Plays an Important Role in the Brown Rot Decay of Scots Pine Heartwood.

Scots pine heartwood is known to have resistance to wood decay due to the presence of extractives, namely stilbenes and resin acids. However, previous studies have indicated that these extractives are degradable by wood decaying fungi. This study aimed to investigate the relationship between extractive degradation and heartwood decay in detail and to gain insight into the mechanisms of extractive degradation. Mass losses recorded after a stacked-sample decay test with brown rot fungi showed that the heartwood had substantial decay resistance against Coniophora puteana but little resistance against Rhodonia placenta. Extracts obtained from the decayed heartwood samples revealed extensive degradation of stilbenes by R. placenta in the early stages of decay and a noticeable but statistically insignificant loss of resin acids. The extracts from R. placenta-degraded samples contained new compounds derived from the degraded extractives: hydroxylated stilbene derivatives appeared in the early decay stages and then disappeared, while compounds tentatively identified as hydroxylated derivatives of dehydroabietic acid accumulated in the later stages. The degradation of extractives was further analysed using simple degradation assays where an extract obtained from intact heartwood was incubated with fungal mycelium or extracellular culture fluid from liquid fungal cultures or with neat Fenton reagent. The assays showed that extractives can be eliminated by several fungal degradative systems and revealed differences between the degradative abilities of the two fungi. The results of the study indicate that extractive degradation plays an important role in heartwood decay and highlight the complexity of the fungal degradative systems.

A transcriptomic view to wounding response in young Scots pine stems.

We studied the stress response of five-year-old Scots pine xylem to mechanical wounding using RNA sequencing. In general, we observed a bimodal response in pine xylem after wounding. Transcripts associated with water deficit stress, defence, and cell wall modification were induced at the earliest time point of three hours; at the same time, growth-related processes were down-regulated. A second temporal wave was triggered either at the middle and/or at the late time points (one and four days). Secondary metabolism, such as stilbene and lignan biosynthesis started one day after wounding. Scots pine synthesises the stilbenes pinosylvin and its monomethyl ether both as constitutive and induced defence compounds. Stilbene biosynthesis is induced by wounding, pathogens and UV stress, but is also developmentally regulated when heartwood is formed. Comparison of wounding responses to heartwood formation shows that many induced processes (in addition to stilbene biosynthesis) are similar and relate to defence or desiccation stress, but often specific transcripts are up-regulated in the developmental and wounding induced contexts. Pine resin biosynthesis was not induced in response to wounding, at least not during the first four days.

Extractive concentrations and cellular-level distributions change radially from outer to inner heartwood in Scots pine.

The heartwood of many wood species is rich in extractives, which improve the wood material's resistance to biological attack. Their concentration is generally higher in outer than inner heartwood, but the exact radial changes in aging heartwood remain poorly characterized. This investigation studied these radial changes in detail in Scots pine (Pinus sylvestris L.), using radial sample sequences prepared from three different trees. Stilbene and resin acid contents were first measured from bulk samples, after which the extractive contents of individual heartwood annual rings were investigated using Raman spectroscopy and fluorescence microscopy. Raman imaging and fluorescence microscopy were also used to study the cellular-level distributions of extractives in different annual rings. Although there were substantial differences between the trees, the content and distribution of stilbenes seemed to follow a general radial trend. The results suggest that stilbenes are absorbed into heartwood tracheid cell walls from small stilbene-rich extractive deposits over several years and then eventually transform into non-extractable compounds in aging heartwood. Resin acids followed no consistent radial trends, but their content was strongly connected to the frequency of large extractive deposits in latewood tracheid lumens. The results highlight the variability of heartwood extractives: their content and distribution vary not only between trees but also between and even within the annual rings of a single tree. This high variability is likely to have important effects on the properties of heartwood and the utilization of heartwood timber.

Developmental Changes in Scots Pine Transcriptome during Heartwood Formation.

Scots pine (Pinus sylvestris L.) wood is desired in woodworking industries due to its favorable timber characteristics and natural durability that is contributed by heartwood extractives. It has been discussed whether the Scots pine heartwood extractives (mainly stilbenes and resin acids) are synthesized in the cells of the transition zone between sapwood and heartwood, or if they are transported from the sapwood. Timing of heartwood formation during the yearly cycle has also not been unambiguously defined. We measured steady-state mRNA levels in Scots pine transition zone and sapwood using RNA sequencing. Year-round expression profiles of selected transcripts were further investigated by quantitative RT-PCR. Differentially accumulating transcripts suggest that, of the Scots pine heartwood extractives, stilbenes are synthesized in situ in the transition zone and gain their carbon-skeletons from Suc and triglycerides. Resin acids, on the other hand, are synthesized early in the spring mainly in the sapwood, meaning that they must be transported to the heartwood transition zone. Heartwood formation is marked by programmed cell death that occurs during the summer months in the transition zone.

Wounding response in xylem of Scots pine seedlings shows wide genetic variation and connection with the constitutive defence of heartwood.

In this greenhouse experiment, 3-year-old Scots pine (Pinus sylvestris L.) seedlings were wounded by drilling holes through the stem. In the xylem next to the wound, the concentration of resin acids (RAC) increased, and the production of extractives typical for heartwood (stilbenes) and knotwood (stilbenes and lignans) of mature trees was induced. The induced stilbenes were pinosylvin (PS) and pinosylvin monomethyl ether (PSM), and the lignans nortrachelogenin (NTG) and matairesinol (MR). There was positive phenotypic correlation between concentrations of the different extractives. Except for the RAC, the extractive concentrations showed no correlation with the size of the seedlings. The treated seedlings belonged to half-sib families, which enabled the estimation of the genetic parameters for the response variables. The proportion of heritable variation (heritability, h(2)) in the concentration of PS, NTG and MR varied between 0.71 and 1.03, whereas for PSM and RAC the heritability was lower (0.35 and 0.31). Genetic correlation was significant between PS and PSM (r = 0.55, P = 0.018), and between NTG and MR (r = 0.50, P = 0.033). Heritabilities were also estimated on the basis of the regression of the offspring on their mothers h(2)(0P). These estimates were assessed for the concentration of PS, PSM and RAC in the wound response area of the seedlings and correspondingly in the heartwood of their mothers. The heritability was highest for the concentration of PS h(2)(0P). The findings of this study support the suggestion that the wounding of Scots pine seedlings may facilitate the development of an early testing method for breeding heartwood durability.