After the FDNPP accident, many trees, including konara oaks, were contaminated by radioactive minerals, such as radiocesium, not only at their surface but also inside the tree bodies. This radionuclide was accumulated both in the sapwood and heartwood at half a year after the accident [4], and its accumulation was observed through a decade [e.g., 34, 35]. We recently reported that Cs was able to move from the outer sapwood to the heartwood by the combination of parenchyma cells’ activity and apoplastic diffusion in Japanese cedar tree trunks [23]. Therefore, we hypothesized that Cs accumulated in the konara oak xylem might enter through the same way as in Japanese cedar. However, we could not anticipate its precise behavior because these two species considerably differ in both physiological and anatomic aspects, originating in differences between gymnosperms and angiosperms [13, 30]. To determine how radiocesium was accumulated in the konara oak xylem, we employed the same methodology that we developed in previous studies for standing trees, in which we used a Cs as a tracer of radial mineral movement at the cellular level [22,23,24]. It is known that minerals from bark are transported to the xylem via the cambium [18, 36]. Further experiments performed on konara oak revealed that Cs attached to the bark surface was transported to the xylem [27]. In addition, the sap solution flows only through the outer xylem layers, earlywood vessels of the current year, and latewood vessels of outer several years [10, 11]. Therefore, the presented experimental design is able to present how minerals move in the xylem after being absorbed either from the bark (often seen at the early phase of radioactive contamination) or from the soil via the roots (often seen at the steady-state phase of radioactive contamination) [37]. This method was proven highly suitable for investigating radial mineral movement through a konara oak trunk, with even more results than the studies on Japanese cedar.
Mineral radial movement in sapwood: parenchyma cells’ activity and apoplastic diffusion
The conduit area of the injected Cs solution was confirmed by the red color of acid fuchsin [10, 11, 32] after felling the tree (Fig. 1e–g). The red area widened through the sapwood in the radial and tangential directions and across the annual ring borders as the injection duration increased. In the red area, Cs was detected in almost all cell types. To investigate whether Cs was able to move by the activity of living parenchyma cells or by passive diffusion, we compared the Cs movement between trunks that contained living cells and trunks without living cells (Fig. 7). We killed living cells in tree trunks of standing konara oak trees by a freeze–thaw treatment to allow mineral movement only by diffusion [23]. A freeze–thaw treatment is known to cause freezing injuries that destroy the cell membrane of plant cells, resulting in the loss of cell functioning [38,39,40,41]. In this study, the cytoplasm of parenchyma cells was damaged after the freeze–thaw treatment (Additional file 1: Fig. S1), indicating their successful killing in a part of a standing konara oak trunk, while retaining the parenchyma cell walls. In freeze–thaw treated samples, the injected Cs moved only by diffusion. The Cs detection area was approximately the same for all cell types. It implies that there is no distinction in the area and speed of diffusion among cell types in konara oak trees. Conversely, in the normal trees, although the Cs detection area was the same for each cell type in the 3-h injection, the Cs detection areas in the 1- and 4-day injected accessions differed between the parenchyma cell-related structures such as RL, RW, and AL and other cell types such as VL, VW, FL, and FW, that is, Cs was detected in the inner part of the red area, and was farther in living parenchyma cell-related structures than in the other dead cells. These results indicate that parenchyma cells are the site of radial mineral movement in the konara oak xylem, and that Cs movement accomplished by the action of parenchyma cells runs faster than diffusion in konara oak trees.
Interestingly, the Cs detection areas of parenchyma cell walls and their lumina were almost the same in normal trees. It is known that the cell wall of a parenchyma cell has blind pits that exchange substances inside and outside the cell [42, 43]. Therefore, it is possible that the injected Cs was excreted into the apoplast from parenchyma cells. In other words, parenchyma cells exude Cs into the apoplast while transporting it symplasmically. In addition, in the case of the 4-day injection of the K04 sample, Cs was sometimes detected as scattered in the cell walls of vessels or fibers in the inner sapwood (Fig. 2). These Cs detected points were too far from the injection point to be transported solely by diffusion. This result also indicates the existence of a pathway by which minerals that symplasmically move in the radial direction in parenchyma cells would be transported to the apoplast and diffuse apoplasmically. The presented results indicate that minerals in a konara oak trunk must be transported radially by the action of xylem parenchyma cells in addition to diffusion into the apoplast.
The pattern of the radial movement of minerals in konara oak, a hardwood species, is very similar to that of Japanese cedar, a conifer species [22, 23]. This resemblance suggests that the mechanism of radial mineral movement, including the activity of parenchyma cells in symplasmic movement, is universal across tree species. We did not find a difference in radial distribution of artificially injected Cs between axial and ray parenchyma cells, suggesting that both types of parenchyma cells have the same function in radial mineral movement. However, xylem anatomical features differ between konara oak and Japanese cedar. Axial parenchyma cells mostly surround vessels in both earlywood and latewood in the konara oak xylem, whereas those of Japanese cedar are distributed in latewood only. It has been reported that axial parenchyma cells surrounding vessels are involved in embolism repair in tree stems, suggesting that they may convert substances between the symplast and apoplast [14, 44, 45]. These facts may indicate the existence of a pathway by which minerals would move in the radial direction by virtue of ray parenchyma cells, being subsequently transported to axial parenchyma cells, and finally exuded into the apoplast and diffused. However, it is unlikely that axial parenchyma cells of Japanese cedar are the only place where minerals are transported from the symplast to the apoplast because they exist only in the latewood in quite a low percentage. Although we attempted to capture the moment during which Cs has moved across the plasma membrane, the spatial resolution of our cryo-SEM/EDX system was too low. The development of a method with a higher resolution is needed to visualize the place where minerals move between the symplast and apoplast. Elucidation of the differences in the activity of ray and axial parenchyma cells, including their function in mineral movement, remains a challenge.
Mineral radial movement from sapwood to heartwood: across a layer(s) between the two
In konara oak sapwood, Cs was found both in the symplast (in living parenchyma cells) and in the apoplast (outside the living cells). Since the heartwood consists of dead cells only (apoplast), there are two possible pathways of mineral movement from the sapwood to the heartwood: from the apoplast in the sapwood to the apoplast in the heartwood or from the symplast in the sapwood to the apoplast in the heartwood. In long-term Cs-injected samples (45 and 50 days), the red color obtained from the application of acid fuchsin reached the front of colored heartwood, and a layer immediately outside the colored heartwood was not dyed, which means that diffusion stopped in this layer. This result indicates that Cs movement from the sapwood to the heartwood is not accomplished only by simple diffusion.
Intriguingly, in Japanese cedar, the red color of acid fuchsin stopped at the intermediate wood, a layer between the sapwood and heartwood, and did not move to the heartwood [23], suggesting the existence of a barrier layer that would prevent diffusion between the sapwood and heartwood, which would be common for konara oak and Japanese cedar. In Japanese cedar, the waterless layer in the intermediate wood limited diffusion because water from the earlywood tracheid lumina in the intermediate wood was lost [31]. Therefore, it was concluded that Cs moved in parenchyma cells (symplast) from the outer sapwood to the intermediate wood and diffused into the heartwood apoplast. Although the intermediate wood may be an important factor for transition from the symplast to the apoplast, a konara oak tree does not have a clear layer of intermediate wood as seen in Japanese cedar that is represented by an easily distinguishable white zone [31, 33]. In addition, unlike in Japanese cedar, a change in water distribution was not observed at the boundary between the sapwood and colored heartwood in konara oak. During the transition from the sapwood to the heartwood, living parenchyma cells are submitted to cell death; hence, a place where living and dead parenchyma cells are adjoined must exist. Therefore, we speculate that minerals are being moved actively from living cells to dead cells at the layer of the innermost sapwood, which needs confirmation.
Thus, what are the common and different mechanisms that block diffusion at the sapwood–heartwood boundary among tree species? Abrupt changes in concentrations of various minerals at the sapwood–heartwood boundary have been reported for many tree species, including konara oak and Japanese cedar [28, 29, 46]. As observed, Mg, K, and Rb concentrations were decreased during transition from the sapwood to the heartwood in konara oak [29], whereas they increased in Japanese cedar [28, 46]. A similar tendency was obtained with mineral distribution in the sampled trees (Additional file 1: Fig. S2). If simple diffusion operates in these layers, such a drastic change in mineral concentration should not have occurred. These facts indicate that simple diffusion of minerals at the sapwood–heartwood boundary does not function, suggesting that a layer(s) blocking diffusion between the sapwood and heartwood is universal across tree species. However, the pattern of a mineral’s concentration change varies among tree species, which indicates that the movement of minerals from the sapwood to the heartwood is different among tree species. For example, konara oak had a lesser accumulation of alkali metals in the heartwood than in sapwood, contrary to the pattern observed in Japanese cedar [28, 29], suggesting the suppressed movement of Cs from the sapwood to heartwood in konara oak. Wang et al. [27] failed to detect Cs in the heartwood. This inability can be due to the low quantity of Cs applied to the bark during the konara oak experiment because the same Cs quantity was used during the Japanese cedar experiments [26, 27]. To that end, there must be a mechanism that would select minerals to be moved into the heartwood, and this selectivity might be species dependent. In a Japanese cedar trunk, Cs injected into the sapwood moved to the heartwood, but Cs injected into the heartwood was difficult to move to the sapwood [24]. This result suggests that the selection of minerals occurs during the transport from the sapwood to the heartwood and vice versa. In this study, we revealed that Cs moves from the sapwood to the heartwood, but we could not obtain sufficient results to propose the mechanism by which minerals move. A possible elucidation of this unknown mechanism will clarify tree species specificity that concerns the accumulation of minerals in the heartwood.
How radiocesium has been accumulated in konara oak trunks after the FDNPP accident
Radiocesium was accumulated in the xylem of konara oak trunks from half a year to up to 10 years after the FDNPP accident [4,5,6, 25, 34, 35]. So far, there are no experimental data that would explain how radiocesium was accumulated there. To understand the pathway of radiocesium contamination, we need to know the mechanism of the radial movement of minerals in a konara oak trunk. Some reports suggest that radiocesium was directly attached to the bark surface and then moved inside the trunk because, owing to the lack of foliage, the surface of the konara oak bark was directly exposed to contamination by radiocesium [5, 6, 8]. Minerals attached to the outer bark reach the inner bark by diffusion, and subsequently, those present in the inner bark move to the outer sapwood by symplasmic transportation via the cambium [14, 18, 26, 27, 36]. Moreover, this study reveals that minerals from the outer sapwood move to the inner sapwood and heartwood by symplasmic movement accomplished by the activity of parenchyma cells as well as by diffusion in the apoplast. Therefore, it can be concluded that the route of radiocesium accumulation in konara oak trunks can be explained by scientific evidences.
More than 10 years have passed since the FDNPP accident happened. Radionuclide contamination might have already reached its steady-state phase [37], in which radioactive compounds circulate between tree bodies and the soil, which means that their distribution in trees is strongly affected by plant physiology. Thus, radiocesium distribution would be similar to the natural distribution of stable Cs, K, and Rb that belong to the alkali metals [47]. Since the minerals accumulated in the heartwood are different among tree species, they should also differ in the accumulation pattern of radiocesium. To understand and explain future trends of tree trunks’ contamination by radiocesium, further investigations are needed to unravel the mechanism of the radial movement of minerals, including the mechanism by which minerals are selected at the boundary between the sapwood and heartwood.