Temporally and spatially controlled death of parenchyma cells is involved in heartwood formation in pith regions of branches of Robinia pseudoacacia var. inermis
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Focussing on four types of parenchyma cell around pith regions of branches of Robinia pseudoacacia L. var. inermis, we examined the timing and role of cell death during heartwood formation. Large parenchyma cells that were located in the inner part of the pith died within a year. By contrast, other parenchyma cells died within 4 years, with the timing of cell death depending on the type of cell. Axial parenchyma cells of the xylem close to the pith died first. Then, small parenchyma cells died in the perimedullary zone in the outer part of the pith. Finally, ray parenchyma cells in the xylem close to the pith died. Variations in the autofluorescence of cell walls, which might have been due to deposition of heartwood substances, were observed first in xylem ray parenchyma cells and small parenchyma cells in the perimedullary zone. Our results indicate that the initiation of heartwood formation occurs within 4 years in pith regions of branches in Robinia pseudoacacia L. var. inermis. Moreover, it appears that not only xylem ray parenchyma cells but also small parenchyma cells in the perimedullary zone might be involved in the synthesis of heartwood substances.
KeywordsCell death Heartwood formation Pith Robinia pseudoacacia Xylem parenchyma cell
The formation of heartwood is a unique phenomenon in long-lived woody plants. Heartwood is 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’’ . Some species form colored heartwood because xylem parenchyma cells synthesize heartwood substances such as polyphenols that contribute to increases in the decay resistance of tree trunk, prior to their death [2, 3, 4, 5]. A full understanding of the mechanism of heartwood formation should provide information that is useful in efforts to control the chemical properties of wood.
Heartwood formation in temperate zone trees results from the death of xylem parenchyma cells that is associated with the annual life cycle of the tree. The death of xylem parenchyma cells progresses from the inner toward the outer region of the stem. Therefore, the death of xylem parenchyma cells might be expected to begin in the tissues at the center of branches and stems, where the pith, consisting of parenchyma cells, is located . If this hypothesis is correct, the parenchyma cells in the pith might be involved in the initiation of heartwood formation. However, the site of initiation of heartwood formation remains to be identified.
The main objective of the present study was to determine the site of initiation of heartwood formation within the branches of Robinia pseudoacacia L. var. inermis. Since Robinia pseudoacacia L. has narrow sapwood , we were able to follow the process of cell death in parenchyma cells around the pith within a short distance from the apical portions of branches. We examined the timing of the death of parenchyma cells in the pith and in the xylem close to the pith. In addition, we examined the distribution of storage starch in parenchyma cells around the pith because the amount of starch in xylem parenchyma cells generally decreases during the conversion of sapwood into heartwood [5, 8, 9]. Moreover, we examined variations in the autofluorescence of cell walls in an attempt to identify the roles of parenchyma cells around the pith. Imaging systems combined with chemical analysis, such as ultraviolet (UV) microspectrophotometry and time-of-flight secondary ion mass spectrometry (ToF-SIMS), are effective tools for the investigation of localization of heartwood substances at cellular level [10, 11, 12, 13, 14]. Therefore, in this paper, we used fluorescence microscopy and confocal laser-scanning microscopy with spectrum analysis to analyze the changes in autofluorescence of cell walls due to lignin and deposition of heartwood substances.
Materials and methods
Two Robinia pseudoacacia L. var. inermis trees, growing in the Field Museum Tama Hills of the Tokyo University of Agriculture and Technology in Hachioji-Tokyo (35°63′N, 139°37′E), Japan, were used in this study. A four-year-old branch with a diameter of approximately 3 cm was collected from each tree in November 2010. Since Nobuchi et al.  observed that heartwood formation in Robinia pseudoacacia L. started in July and continued until at the end of the following March, we expected to observe the process of heartwood formation in November.
Light and fluorescence microscopy
All samples (diameter in 1–3 cm and height in 3 cm) were fixed overnight at room temperature in a 4% solution of glutaraldehyde in 0.1 M phosphate buffer (pH 7.2), and then the samples were washed with the same buffer. Transverse sections of approximately 16-μm thickness and radial sections of approximately 40-μm thickness were cut on a sliding microtome (Yamatokohki, Saitama, Japan).
For light microscopy, sections were stained with a 1% aqueous solution of acetocarmine for observation of nuclei [15, 16, 17, 18], with a 1% aqueous solution of iodine–potassium iodide for observation of starch grains [15, 19, 20], and with a 1% aqueous solution of safranin for observation of cell walls [16, 17, 18]. Stained sections were observed under a light microscope (Axioscop; Carl Zeiss, Oberkochen, Germany).
For observations of changes in autofluorescence of cell walls during heartwood formation, unstained transverse sections of approximately 16-μm thickness were examined with a fluorescence microscope (Axioscop) under epifluorescence illumination (excitation/emission combination BP353-377/LP397). Lignin and heartwood substances such as polyphenols were expected to emit autofluorescence under UV excitation [21, 22].
Recording of emission spectra of the autofluorescence of cell walls by confocal laser-scanning microscopy
Unstained transverse sections of approximately 16-μm thickness were mounted on glass slides in glycerol. Sections were examined in “lambda scanning” mode with single-photon excitation under a confocal laser-scanning microscope (LSM 710 NLO; Carl Zeiss) equipped with a Zeiss Plan-Apochromat 40×/0.95 objective lens (Carl Zeiss). Sequential single-plane images (512 × 512 pixels) were obtained at wavelengths from 462 to 723.9 nm in steps of 9.7 nm under fluorescence excitation at 405 nm. The relative intensity of autofluorescence at each wavelength was recorded from six randomly chosen areas (0.415 × 0.415 μm2) of walls of xylem and pith cells, and then the average value from the six areas was calculated.
Results and discussion
The emission spectra of autofluorescence under fluorescence excitation at 405 nm from cell walls of xylem and pith cells in the same sections shown in Fig. 5a and c are shown in Fig. 5d. The emission spectrum from the section in Fig. 5a peaked at 481.4 nm, while that in the section in Fig. 5c peaked at 500.8 nm. In addition, the relative intensity of the fluorescence from the section in Fig. 5c was higher than that from the section in Fig. 5a. These results indicate that some additional substances, which emitted autofluorescence at longer wavelengths than lignin, might have been deposited in the walls of xylem and pith cells around the center of branches. Similarly, Dünisch et al.  observed that some additional substances were detected in the walls of xylem cells in heartwood of Robinia pseudoacacia L. by UV microspectrophotometry. In species that form colored heartwood, heartwood substances are synthesized in parenchyma cells, and then, are deposited in the walls of xylem cells during heartwood formation [2, 3, 4, 5, 9]. Therefore, variations in the autofluorescence of cell walls might reflect the deposition of heartwood substances in the cell walls.
In LH-1 samples, no dark-colored heartwood was visible to the naked eye. However, variations in the autofluorescence of cell walls were visualized by fluorescence microscopy (Fig. 5b). Yellow–green fluorescence of cell walls was observed only in some of the xylem ray parenchyma cells that were located near the pith and perimedullary cells. This observation indicates that these additional substances were synthesized not only in xylem ray parenchyma cells but also in perimedullary cells in Robinia pseudoacacia L. var. inermis. We also observed the disappearance of starch grains from xylem ray parenchyma cells and perimedullary cells around the pith in LH-2 and 3 samples during cell death (Fig. 4f-h). It has been suggested that stored starch is used for the synthesis of heartwood substances . Therefore, the disappearance of starch from xylem ray parenchyma cells and perimedullary cells might be involved in the synthesis of heartwood substances.
In addition, the disappearance of nuclei and starch grains from perimedullary cells occurred earlier than that from xylem ray parenchyma cells (Figs. 3f–h, 4f–h). Moreover, in LH-1 samples, all perimedullary cells showed yellow–green fluorescence, while no variations in the autofluorescence of cell walls were evident in some xylem ray parenchyma cells that were located near the pith (arrowheads in Fig. 5b). Therefore, it seems possible that the synthesis of heartwood substances might start in perimedullary cells in the pith region. High-level expression of genes that are related to the synthesis of heartwood substances was observed in xylem parenchyma cells at the sapwood-heartwood transition zone in Robinia pseudoacacia L [23, 24]. Similar genes might be strongly expressed in perimedullary cells during heartwood formation.
By contrast to the above observations, there were no clear variations in the autofluorescence of cell walls of axial parenchyma cells in LH-1 samples (Fig. 5b). However, we did observe the disappearance of starch grains from axial parenchyma cells during cell death (Fig. 4e). It has been suggested that, in Cryptomeria japonica, axial and ray parenchyma cells in xylem might synthesize and accumulate different kinds of heartwood substances [13, 25]. Nobuchi and Harada  reported differences in terms of the ultrastructural changes during heartwood formation between axial and ray parenchyma cells in xylem. These observations together suggest that axial parenchyma cells might play a different role in heartwood formation from that of ray parenchyma cells and perimedullary cells in Robinia pseudoacacia L. var. inermis.
In conclusion, the initiation of heartwood formation occurred within 4 years in the pith regions of branches in Robinia pseudoacacia L. var. inermis. It appears that not only xylem ray parenchyma cells but also perimedullary cells in the pith might be involved in the synthesis of heartwood substances. Further analysis of perimedullary cells should provide useful information about the early stages of the formation of heartwood.
The authors thank the staff of the Field Museum Tama Hills of the Tokyo University of Agriculture and Technology for providing plant materials. This work was supported, in part, by Grants-in-Aid for Scientific Research from the Ministry of Education, Science, Sports and Culture of Japan (nos. 20120009, 21380107, 20·5659 and 22·00104).
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