Developmental Changes in Scots Pine Transcriptome during Heartwood Formation^{<a href="#fn1" rid="fn1" class=" fn">1</a>,}^{<a href="#fn2" rid="fn2" class=" fn">[OPEN]</a>}

Abstract

Wood biosynthesis is a complex process that involves several developmental activities such as cell division, cell expansion, cell wall thickening, and programmed cell death (Plomion et al., 2001). In many, but not all tree species, the final stage in wood development is formation of heartwood (HW). HW is traditionally defined as “the inner layers of wood, which, in the growing tree, have ceased to contain living cells, and in which the reserve materials (e.g. starch) have been removed or converted into heartwood substances”, while sapwood (SW) is defined as “the portion of the wood that in the living tree contains living cells and reserve materials” (IAWA Committee, 1964). Heartwood can also be defined as the region where extractives have accumulated and by this definition it can be metabolically active (Beekwilder et al., 2014; Celedon et al., 2016), although all changes associated with HW formation may not have yet occurred (Taylor et al., 2002).

The biological role of HW is still unclear, but it may be important in long-term resistance against pathogens (Venäläinen et al., 2004). Nevertheless, HW has a clear economic importance due to the natural decay resistance of HW timber in many tree species (Singh and Singh, 2011) and due to the increasing pharmaceutical and biocide applications of HW substances (Kampe and Magel, 2013).

Heartwood can be found in both angiosperm (e.g. Acacia, Catalpa, Juglans, and Robinia) and gymnosperm (e.g. Cryptomeria, Larix, Picea, and Pinus) tree species. It is physiologically inactive, drier compared with the SW, and filled with HW extractives (Taylor et al., 2002). SW, on the other hand, is physiologically active, has high moisture content, and functions in water, nutrient, and sugar transport among the roots and foliage (Taylor et al., 2002).

As the tree grows, SW is converted to HW. This takes place in the narrow transition zone (TZ), the living cells in which reserve materials like starch are consumed. TZ is drier than SW, but not as dry as HW (Bergström, 2003; Hillis, 1987). The width of the TZ varies both within and between tree species and could be influenced by seasonal as well as other environmental aspects. In general, the TZ is about one to three annual rings wide (Hillis, 1987); in Scots pine (Pinus sylvestris L.), it is usually one to two annual rings wide (Gustafsson, 2001).

It is still unclear when and how the initiation of HW formation occurs. Involvement of the plant hormones ethylene and auxin in regulation of the HW formation have been suggested (Nilsson et al., 2002; Shain and Hillis, 1973; Hillis, 1968). Spicer (2005) described HW formation as a form of programmed cell death (PCD), where the organelles of ray parenchyma cells in the TZ are gradually disintegrated. Several studies have shown that the cell wall structure of ray parenchyma cells change and the cells gradually die toward HW at the boundary between SW and HW (Yang et al., 2003; Nakaba et al., 2008; Magel et al., 2001; Nakada and Fukatsu, 2012; von Arx et al., 2015). In addition to plant hormones, Nakada and Fukatsu (2012) suggested that desiccation at the boundary between SW and HW initiates HW formation.

The specific timing of the HW formation is unclear and may certainly be species-specific. Different studies have described the process taking place at different times of the year. HW formation in black locust (Robinia pseudoacacia) took place in autumn, while in radiata pine (Pinus radiata) and black walnut (Juglans nigra), it occurred during the dormancy period in winter (Taylor et al., 2002; Kampe and Magel, 2013). Bergström et al. (1999) concluded that there is no specific time of the year for HW formation in Scots pine. Indeed, the timing of HW formation is not clearly understood nor has been satisfactorily described to date.

Two types of HW formation were distinguished by Magel (2000). In type I, or Robinia-type of HW formation, HW extractives are biosynthesized in situ and accumulate in the TZ. Precursors to the extractives are not present in the aging SW. In type II, or Juglans-type of HW formation, HW extractives are formed through transformation of phenolic precursors by hydrolysis, oxidation, and polymerization in the TZ. In type II HW formation, HW extractive precursors are gradually accumulating in the aging SW (Magel, 2000; Kampe and Magel, 2013). Deposition of HW extractives and PCD marks the end of the HW formation (Spicer, 2005). Many HW extractives are synthesized also in living tissues when trees are under biotic or abiotic stress and thus contribute to the active defense of SW and other living tissues (Chiron et al., 2000b).

In Scots pine, HW extractives consist of the stilbenes pinosylvin (PS) and pinosylvin monomethyl ether (PSME), resin acids, and free fatty acids (Saranpää and Nyberg, 1987). Variation of Scots pine HW phenolic extractive content between tree individuals is wide and strongly correlates with decay resistance of HW (Harju and Venäläinen, 2006; Leinonen et al., 2008). Although extractive content is influenced by the environment (Magel, 2000), it is highly inherited (Fries et al., 2000; Partanen et al., 2011).

It is still unclear whether Scots pine HW extractives are synthesized in the cells of TZ between SW and HW or transported from the SW. Furthermore, the timing of HW formation during the yearly cycle has not been unambiguously defined. In this work we used a transcriptomic approach to characterize changes in gene expression during HW formation, i.e. in TZ compared to SW. Using a set of indicator genes, defined through transcriptomics, we further addressed the question of timing of HW formation by quantitating gene expression in SW and TZ throughout the year in two Scots pine individuals.

Acknowledgments

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