In the gray regions of the xylem of D. kaki, we found that colored substances had accumulated in both axial and ray parenchyma cells (Fig. 3). Matsushita et al. [4] demonstrated that 4,8-dihydroxy-5-methoxy-2-naphthaldehyde was the most abundant specific extractive in the black regions of kurogaki, and it was localized in xylem parenchyma cells, as shown by ToF-SIMS. These results indicated that xylem parenchyma cells synthesize chemical compounds that are related to the blackening of xylem. However, the pathways for diffusion of colored substances from the sites of their biosynthesis to their deposition in secondary xylem of D. kaki remained to be clarified. In the present study, we determined the pathways for diffusion of colored substances by monitoring their locations at the microscopic level.
In gray regions, some ray parenchyma cells contained colored substances and neighboring wood fibers and intercellular spaces also contained colored substances (Fig. 3). ToF-SIMS observations revealed that 4,8-dihydroxy-5-methoxy-2-naphthaldehyde was distributed in ray parenchyma cells and in some adjacent axial elements [4]. During heartwood formation, heartwood substances, which are heartwood-specific phenolic compounds that affect coloration of heartwood, migrate from xylem parenchyma cells to neighboring cells [12,13,14]. Therefore, it seems plausible that colored substances might migrate from xylem parenchyma cells to adjacent cells via a similar pathway in D. kaki. In addition, although some xylem parenchyma cells contained large amounts of colored substances in the gray regions (Fig. 3a–c), small amounts of colored substances were detected in xylem parenchyma cells in the black regions (Fig. 4a–c). This observation suggests that most of the colored substances synthesized in xylem parenchyma cells might migrate from cells during the formation of the blackened xylem.
In gray regions and black regions, colored substances were detected in pits of vessel elements, wood fibers and xylem parenchyma cells (Figs. 3, 4). It has been reported, similarly, that heartwood substances were localized in pits, an observation that suggests that pit-pairs might function as intercellular diffusion pathways for heartwood substances [8, 9, 12, 13, 15]. Accordingly, present observation also indicates pit-pairs might function similarly as pathways for intercellular migration of colored substances in D. kaki.
We did not detect any deposits of colored substances in the intercellular spaces and blind pits of whitish regions (Fig. 2d, e). By contrast, colored substances were observed in intercellular spaces in gray and black regions (Figs. 3i and 4d, e). In addition, we noted that black substances appeared to leak from blind pits into intercellular spaces in the black regions (Fig. 4e). These observations suggest that colored substances might migrate from ray parenchyma cells to intercellular spaces via blind pits and then diffuse through the network of intercellular spaces. It has been suggested that intercellular spaces provide routes for gas exchange [16,17,18,19] and serve as a system for the transport and storage of water and nutrients [20,21,22,23,24]. It has also been suggested that intercellular spaces function as the extracellular pathway for diffusion of heartwood substances [8, 9, 12]. Thus, it seems reasonable that intercellular spaces might also function as the extracellular pathway for diffusion of colored substances during the blackening of xylem in D. kaki. A further study on the three-dimensional structure of intercellular spaces is needed to understand how colored substances diffuse via intercellular spaces during blackening of xylem in D. kaki.
At the boundary between whitish and black regions, the extent of coloration of cell walls of wood fibers gradually increased from the whitish to the black region (Fig. 5a). Heavy accumulation of colored substances was evident within the inner regions of secondary walls (Fig. 5e, h, k). Furthermore, the intensity of pigmentation of the outer parts of secondary walls increased toward the black regions (increase in gray value and width of the high gray value regions of secondary walls; Fig. 5d, g, j, m). Therefore, we can conclude that the deposition of colored substances progresses from the inner to the outer part of the cell wall in wood fibers in D. kaki. In addition, gray values of CML gradually increased toward the black region (Fig. 5d, g, j, m). This observation indicates that colored substances diffuse into the CML and are gradually deposited during the blackening of xylem of D. kaki. Deposition of heartwood substances in cell walls and CML has been similarly demonstrated using different microscopy techniques, such as confocal laser-scanning microscopy, transmission electron microscopy and confocal Raman microscopy [14, 15, 25, 26]. Belt et al. [25] noted that, in the heartwood of Pinus sylvestris, pinosylvin was detectable both in cell walls and in CML, but resin acid was distributed only in the cell walls, suggesting that the patterns of distribution might differ between extractives. Streit and Fengel [14] suggested two possible pathways for penetration of phenolic compounds in the heartwood of Schinopsis balansae, namely, a pathway from the lumen into the cell wall and a pathway from pits and intercellular spaces into CML. Similarly, a pathway from pits and intercellular spaces into CML could exist during the formation of blackened xylem in D. kaki.