Ray parenchyma cells (RPCs) play an important role in the storage, conductive, and secretory systems of woody plants (Murakami et al. 1999). They render possible radial and lateral transport of materials by providing connections between phloem and xylem (Larson 1994).
RPCs in the sapwood (sW) of a tree remain alive for several years (Funada 2000) and play an important role in its physiological activities (Catesson 1990; Sauter 2000; Chaffey and Barlow 2001). RPCs in the transition zone (TZ) are partially living and are involved in the formation of heartwood (hW) (Bamber and Fukazawa 1985; Hillis 1987; Burtin et al. 1998; Magel 2000). The hW is the non-living part of the stem that has no apparent physiological function (Filippis and Magel 2012). The pattern of cell death and the function of RPCs are modified by the transformation from sW to hW, which is a complex biological process and an essential part of the secondary growth (Nakaba et al. 2006; Song et al. 2011).
The transformation process of RPCs from sW to hW has been studied by microscopic methods. Fujikawa and Ishida (1975) described the ultrastructure and lignification of RPCs cell walls in more than 30 species of softwoods by UV microscopy, polarized optical microscopy (POM), and SEM. Fujii et al. (1979) investigated the layered structure of the RPCs secondary walls in 49 species of Japanese hardwood species by POM and ultrathin sectioning technique followed by KMnO4 staining. Yamamoto et al. (1977) and Yamamoto (1982) observed the pattern of differentiation and the timing of secondary walls thickening in RPCs in several species of Pinus Linn. Lignification and the structure of the cell wall in different types of RPCs in Populus maximowiczii Henry were compared by Murakami et al. (1999) by means of the UV microscopy, POM, and TEM. The pattern of cell death, the process of lignification, and the function of RPCs in several species of both softwood and hardwood species were studied by Nakaba et al. (2006, 2008, 2012) by means of microscopy.
More knowledge about the chemical structure of lignin in RPCs, and a detailed tissue-specific characterization of lignins would contribute a lot for elucidation of RPCs lignification. The time-of-flight secondary ion mass spectrometry (TOF-SIMS) is a valuable tool not only for micro-scale detection of inorganic compounds (Saito et al. 2014), but also for detection extracts (Matsushita et al. 2012), anionic groups (Tokareva et al. 2010), and lignin analysis (Orblin et al. 2011; Matsushita et al. 2013). Laser microdissection (LMD) of specific tissues is a useful approach in wood analysis, namely LMD was combined with thioacidolysis of RPCs, while the focus was on the composition and lignification of RPCs during their conversion from sW towards hW in Pinus densiflora Sieb. et Zucc. as a softwood (Zheng et al. 2014b).
It is well known that hardwood lignins mainly consists of syringyl (S) and guaiacyl (G) units and the ratio of S to G units (S/G value) is an important parameter for their lignin characterization. The distribution heterogeneity of S and G units has been studied by UV microscopy (Fergus and Goring 1970), Mäule color reaction (Takabe et al. 1992; Watanabe et al. 2004), TEM-EDXA (Saka and Goring 1988), FTIR spectroscopy (Faix 1991), and immuno labeling (Joseleau and Ruel 1997). Hardwood lignins in RPCs have not yet been submitted to quantitative structural analyses.
In this study, the ring-porous hardwood Phellodendron amurense Rupr. was in focus, which contains multiseriate vessel elements, mainly in the earlywood. The RPCs are multiseriate, generally up to 6 cells wide, and homocellular, exclusively composed of procumbent cells (Ito 1997). The number of rays per unit area is small, and thus the LMD approach for their isolation is convenient.
The aim was to analyze the lignin composition and structure in RPCs isolated by LMD from sW, TZ, and hW of P. amurense. TOF-SIMS measurements should also be carried out for lignin quantification and evaluation of the S/G values. Moreover, the LMD cuts of RPCs should be submitted to thioacidolysis including the second desulfurization step over Raney nickel obtain both the typical monomeric and dimeric lignin fragments.
Materials and methods
Disks (5 cm thick) was collected in October from the main stem of P. amurense (grown in natural forest in Tomakomai Experimental Forest, Hokkaido University, Japan) with 38 annual rings at breast height (BH) and with a diameter of 15 cm at BH. Strips containing sW, TZ, and hW were cut through the pith and then air-dried until weight constancy.
The boundary between the sW and hW is easily seen by color (Figure 1a), while the light-colored part (annual rings 1–4) and the dark-colored part (annual rings 6–7) were regarded as sW and hW, respectively. The annual ring 5, which was somewhat lighter than the hW, was regarded as the TZ. The continuous growth rings from these zones were collected for further analyses.
The Wiesner (phloroglucinol-HCl) reaction (Pomar et al. 2002) was carried out to check the presence of tension wood with non-lignified gelatinous fibers and as illustrated (Figure 2a,b) there were no gelatinous fibers in the samples.
TOF-SIMS analysis: (TRIFT III TOF-SIMS spectrometer, ULVAC-PHI, Inc., Kanagawa, Japan): Blocks (10×10×10 mm3) were immersed in water for more than 7 days and then cut with a sliding microtome (REM-710, Yamato Kohki Industrial Co., Ltd., Saitama, Japan) to obtain 100-μm-thick tangential sections of the latewood from each annual ring (annual rings 1–7) and the sections were extracted in MeOH (20 ml) for more than 14 days.
The amount of dissolved extractives in the solution was monitored by UV-Vis spectroscopy, according to which almost all extractives were removed. Finally, the wet sections were washed by fresh MeOH and held between two glass plates and dried under ambient conditions.
Positive ion spectra were collected (22 keV Au1+ at a current of 1.2 nA and a pulse width of 11 ns, not bunched for image analysis was applied). The measured surface areas were 300×300 μm2, and approx. 3×106 total ion counts were obtained in 10 min of acquisition time. A low-energy pulsed electron ion gun (30.0 eV) was used for surface charge compensation.
All data were obtained as retrospective RAW data file, which records a full mass spectrum at every 256×256 pixel point and an image available at every secondary ion mass detected. The region-of-interest (ROI) was selected (Figure 1c). The intensity of lignin ions was calculated from the sum of the peak intensity at m/z 137 (C8H9O2+) and 151 (C8H7O3+ and C9H11O2+) for G units and at m/z 167 (C9H11O3+) and 181 (C9H9O4+ and C10H13O3+) for S units (Saito et al. 2005b; Goacher et al. 2011). The relative intensity of lignin ions was normalized to the count of cellulose ions (m/z 127: C6H7O3+, and 145: C6H9O4+) (Fardim and Duran 2003; Goacher et al. 2011; Zheng et al. 2014a). A typical TOF-SIMS positive ion spectrum is presented in Figure 3a.
Laser microdissection (LMD): The dry sections (Figure 4a) were fixed with tape covering a hole (1.2 cm in diameter) of a plastic plate and then dissected under a microscope (LMD7000, Leica Microsystems, Tokyo, Japan). Fragments enriched with RPCs (WRPCrich samples) were collected in a sample tube (Figure 4c) and the remaining laser-treated sections (WRPClow samples) were used for comparison (Figure 4d), which contains less RPCs but more axial elements with vessels, fibers, and axial parenchyma cells. The whole native sections (Figure 4a) before fractionation by LMD (W samples) were also analyzed.
The cell wall at the edge of the fragments can be damaged by laser (Nakashima et al. 2008; Zheng et al. 2014b). Therefore, some of the W samples were also burned randomly by the laser (Wburned samples) and which served as control. The degree of damage (laser irradiated length per surface area) of the Wburned and those of WRPClow were nearly equal (≈0.01 μm·μm-2), whereas the degree of damage of WRPCrich was larger (≈0.06 μm·μm-2). The laser target lines were drawn in the region of the neighboring axial elements to minimize the RPCs damage (Figure 4b). In Figure 4a and d, it is discernible that the cut areas are wider than the RPCs.
Chemical analyses: Thioacidolysis and subsequent Raney nickel desulfurization were performed according to the protocols of Lapierre et al. (1991) and Rolando et al. (1992) with slight modifications. Approx. 0.5 mg each of different types of tissues collected by LMD, the W, Wburned, WRPClow, and WRPCrich samples (ca. 2500 fragments) were analyzed separately. The details of the procedure are also reported by Zheng et al. (2014b).
The silylated derivatives of thioacidolysis monomers were analyzed by a GC-MS QP 2010 High-End GC/MS instrument (Shimadzu, Kyoto, Japan) equipped with a capillary column (Rtx–1; 30 m×0.32 mm i.d. and a film thickness of 0.25 μm). The sample (1 μl) was injected at 250°C; temperature program: 180→230°C (2°C·min-1). The carrier gas was He (1.5 ml·min-1). A response factor of 1.5 was used for monomer calculation. The same equipment as described above was applied for characterization of the silylated derivatives of thioacidolysis dimers under the same conditions but the response factor 0.5 instead of 1.5.
A typical total ion chromatogram (TIC) of the GC-MS analyses of dimers is presented in Figure 5. The structures of the main compounds and mass spectral data are summarized in Figure 6 and Table 1, respectively. The peak assignments of the main G-G, G-S, and S-S dimers are based on the MS data under consideration of the RT values available in the literature (Lapierre et al. 1991; Saito and Fukushima 2005a; Rencoret et al. 2008, 2011; Zheng et al. 2014b).
Wiesner (phloroglucinol-HCl) reaction: Blocks (10×10×10 mm3) of the sW and hW were immersed in water for more than 7 days and then cut with a sliding microtome (REM-710, Yamato Kohki Industrial Co., Ltd., Saitama, Japan) to obtain 50-μm-thick transverse sections. The sections were treated with concentrated HCl (35%) followed by 1% phloroglucinol in ethanol to test the presence of tension wood with non-lignified gelatinous fibers.
Mäule reaction: The transverse sections (50 μm thick) from sW and hW prepared above were treated as follows: (1) 5 min immersion in 1% KMnO4 and three washings with distilled water; (2) immersion in 3% HCl for 1 min, followed by washing with distilled water; (3) immersion in 28% NH4OH for 1 min, followed by another washing in distilled water.
Microscopic observation: The sections were observed by a microscope BX50 (Olympus Corp., Tokyo, Japan).
UV microscopy: The transverse sections of 1 μm thickness were prepared by an ultra-microtome (HM350; Microm) equipped with glass knives from the samples embedded in Quetol 812 epoxy resin. The cuts were placed on quartz slides, mounted with glycerin, and then covered with quartz cover slips. The sections were observed at 280 nm by means of a UV microspectrophotometer (MPM800; Carl Zeiss).
Results and discussion
Evaluation of TOF-SIMS data
The total ion images clearly display the wood fibers and RPCs (Figure 1b and c). Two ROIs are enclosed with blue lines, which contain RPCs or wood fibers (Figure 1c). Hereafter, the results obtained from ROIs will be compared. The relative amount of lignin type ions can be well quantified based on the secondary cellulose ions (Rabbani et al. 2011; Zheng et al. 2014a), despite the limitations of this technique due to the ionization efficiency and the so-called matrix effect. In the TOF-SIMS measurements, secondary ions derived from lignin are generated as a result of fragmentation of lignin. As the characteristic monomeric ions of lignin, m/z 137 (C8H9O2+) and 151 (C8H7O3+ and C9H11O2+) are typical for G-lignin, and m/z 167 (C9H11O3+) and 181 (C9H9O4+ and C10H13O3+) correspond to S-lignin (Saito et al. 2005b, 2012). These fragment ions can be generated from several types of linkages between lignin structural units. For example, m/z 137 ion comes from β-O-4′, 4-O-5′, β-1′, β-5′, and β-β′ structures without 5-5′ structures having a strong biphenyl bond (Saito et al. 2005b). The relative intensity of lignin was calculated by dividing the counts of lignin ions by those of the cellulose.
The relative intensity of the total lignin (G and S) in wood fibers did not change significantly (P>0.4 between annual rings 2–4 and 6–7) (Figure 7a). Accordingly, the lignin content of wood fibers was stable during the conversion from sW to hW. Such stable lignin content is due to the loss of organelles and complete lignification of wood fibers that occur immediately after their differentiation (Funada 2000; Nakaba et al. 2012; Zheng et al. 2014a,b). In contrast, the relative intensity of total lignin ions in the region with RPCs increased from sW to hW with increasing number of annual rings (P<0.02 between sW and hW). This indicates that the lignification of RPCs progresses from sW to hW.
The S/G data can be evaluated by TOF-SIMS (Saito et al. 2011, 2012; Zhou et al. 2011). Saito et al. (2011) demonstrated that the S/G value for a β-O-4′ type polymer is in good agreement with the molar ratio of the units. S/G values of a maple tree obtained from thioacidolysis and TOF-SIMS were dependent from the situation of the annual rings within the stem (Saito et al. 2012), however, the S/G value obtained by TOF-SIMS are lower than those evaluated by thioacidolysis because all chemical degradation techniques lead to elevated S/G values (Sarkanen and Ludwig 1971).
The results from ring 1 showed very large StD for both ROIs (Figure 7b). Furthermore, the S/G value at ring 5 (TZ) was significantly higher than that in other annual rings. At present, this observation can not be rationalized. Excluding the TZ, the S/G values of wood fibers were not significantly different between sW and hW (P>0.2 between annual rings 2–4 and 6–7). The S/G values of RPCs were not significantly different between sW and hW (P>0.2) and they were higher (P<0.01) than those of axial wood fibers.
Evaluation of thioacidolysis data
Thioacidolysis cleaves the β-aryl ether bonds in lignin and the yield of monomeric thioethyl derivatised penylpropanes are a measure for these units in lignin. Figure 8a illustrates the total yields of lignin monomers in thioacidolysis mixtures representing mainly G and S units, while p-hydroxyphenyl (H) monomers were present only in traces corresponding to the expectation for hardwoods (Sarkanen and Ludwig 1971).
In the W samples, the yields of thioacidolysis monomers were nearly identical in the sW of the rings 1–4 (Figure 8a) and the yields decreased significantly in the hW. Before the thioacidolysis experiment, the samples were not extracted and thus the relative yield decrement may be a manifestation of the presence of extractives. Similar trend was observed in the Wburned samples but the yields in both sW and hW were significantly lower than in W samples. This decrease is probably the result of laser damage of the samples during microdissection, which ultimately affected the results of thioacidolysis. Similar laser effects were reported previously (Nakashima et al. 2008; Zheng et al. 2014b). Thus the yields of thioacidolysis monomers are underestimated in laser-treated samples.
In WRPClow samples the yield of monomers decreased continuously from ring 1 to 7 (Figure 8a). Similar behavior was observed in W and Wburned. In contrast, the yields of monomers in WRPCrich increased significantly from ring 1 to 7 (Figure 8a).
UV absorbance of RPCs is higher in hW than in sW (Fujikawa and Ishida 1975; Nakaba et al. 2006), which is the result of deposition of aromatic extracts and/or lignification. In analogy to our previous study on a softwood (Zheng et al. 2014a,b) it can be presumed that RPCs of hardwoods are also more lignified in hW than in sW.
Nakaba et al. (2008, 2012) reported that the number of dead RPCs in sW increased from the cambium side towards the pith in both softwood and hardwood. In the TZ, the number of dead RPCs increased rapidly, and almost all of the RPCs were dead in the hW. The monomer yield behavior in WRPCrich samples corresponds to the quoted observation.
As visible in Figure 8b, the S/G values of all types of tissues in the annual ring 5 were relatively higher than those in the other parts. The same result was obtained by TOF-SIMS (Figure 7b). We do not have a plausible interpretation for this observation. In W samples, the S/G values show a small but statistically significant increasing tendency from sW towards hW with increasing number of rings (P=0.003) (Figure 8b), as inferred from the t-test between the sW samples (n=3 for each annual ring 2–4; total n=9) and hW (n=3 for each annual ring 6–7, total n=6).
In S/G plots of Wburned, no significant tendencies are visible with this regard (P≈0.20 between sW and hW). Furthermore, the S/G values in Wburned is lower than that in W samples in all examined annual rings. Accordingly, the LMD affected the yields of G and S monomers at different degrees with more damage on the S units in all laser-treated samples.
In WRPClow, the S/G value nearly does not change from sW towards hW (P>0.3 between sW and hW) as an indication of the stability of these data in axial elements. In contrast, the S/G value in WRPCrich increased significantly from sW towards hW (P<0.01 between sW and hW).
These results seems to be inconsistent with the results of TOF-SIMS, which revealed constant S/G values in RPCs from sW and hW (Figure 7b). Probably, the results of TOF-SIMS data are less reliable than those obtained by thioacidolysis of LMD samples. The samples for TOF-SIMS were MeOH extracted, however, theoretically, the remaining extractives can also produce ions with the same m/z values as that of lignin and can affect the S/G data. The MeOH extractives were measured by TOF-SIMS to estimate this possible effect, but the ions of m/z 137, 151, 167, and 181 were not visible (Figure 3b).
On the other hand, the hW substances will hardly affect the lignin specific thioacidolysis results. In the thioacidolysis, the WRPCrich samples may have contained a few amount of the neighboring axial elements, but the S/G increment seems to be due to the lignin in RPCs and not to that of the residual axial elements.
We interprete the S/G increment in W samples as the contribution of RPCs. In the case of Wburned sample, however, the random laser treatment influences the results by damaging more the S units. However, this observation does not diminish the analytical power of the LMD isolation in combination with thioacidolysis.
The results of Mäule reaction (Pomar et al. 2002) and UV absorption are shown in Figure 2. The Mäule reaction results (Figure 2c, d) show that the RPCs are red in hW and red-brown in sW, indicating that S rich lignin increased from sW to hW. The UV absorption (Figure 2e, f) results show that the RPCs in hW is slightly darker than in sW, although the absorbance can be due to lignin and remaining hW substances. Both of the increment of S rich lignin and UV absorbance in RPCs from sW to hW support the results above.
The main dimers with 5-5′, 4-O-5′, β-1′, β-5′, and β-β′ structures were detected by the GC-MS measurements. Figure 9 displays their yields and averaged ratios. G-G dimers contain 5-5′, 4-O-5′, β-1′, and β-5′ units; G-S dimers contain 4-O-5′, β-1′, and β-5′ units; and S-S dimers are composed of β-1′ and β-β′ units. The numerical values are summarized in Table 2. The leftmost column of Figure 9 shows the results of W samples, where the total dimer yield increased from sW towards hW (Figure 9). The same is true for S-S dimers and thus the S/G value also increase from sW towards hW (Figure 8b). The total dimer yields in Wburned and WRPClow were much lower than those in W samples, whereas the G-G dimer unit ratio was higher than that in W samples. These results are probably caused by the yield-reducing effect of LMD especially on S units as discussed above. There was no significant difference between sW and hW regarding the total dimer yields of Wburned and WRPClow samples. Differences with this regard, if any, should be very small. The dimer yield in WRPCrich is unexpected. Based on the monomer yields (Figure 8a), the dimer yields could have been expected to increase from sW towards hW in WRPCrich, however, the total dimer yield decreased drastically at the TZ and remained low in hW.
The relative intensity of lignin ions of RPCs as detected by TOF-SIMS increases from sW towards hW. The S/G values in both RPCs and wood fibers are nearly constant from sW towards hW. The S/G value in RPCs is generally higher than that in wood fibers. The analysis of thioacidolysis monomers indicate increasing yields as a function of the number of annual rings (from sW towards hW) in RPCs rich samples. The S/G value in WRPCrich samples increased significantly from sW towards hW. The thioacidolysis dimer analysis show that S-S dimers are dominant in all types of tissues. The total dimer yield in WRPCrich samples decreased from sW towards hW. Altogether, lignification of RPCs progresses from sW towards hW. Additionally, S lignin deposits at a higher rate than G lignin during the lignification process of RPCs.
This work was supported by the Japan Society for the Promotion of Science (Nos. 25252032, 25114508 and 15H01230). We thank Hiroyuki Yamamoto (Graduate School of Bioagricultural Sciences, Nagoya University, Japan) for support with sample preparation and Takanori Imai (Graduate School of Bioagricultural Sciences, Nagoya University, Japan) for comments of TOF-SIMS measurement.
Bamber, R.K., Fukazawa, K. (1985) Sapwood and heartwood: a review. For. Abstract 46:567–580. Google Scholar
Burtin, P., Jay-Allemand, C., Charpentier, J.P., Janin, G. (1998) Natural wood colouring in Juglans sp. (J. nigra, J. regia and hybrid J. nigra 23×J. regia) depends on native phenolic compounds accumulated in the transition zone between sapwood and heartwood. Trees 12:258–264. Google Scholar
Catesson, A.M. (1990) Cambial cytology and biochemistry. In: The Vascular Cambium. Ed. Iqbal, M. Research Studies Press, Taunton. pp. 63–112. Google Scholar
Chaffey, N., Barlow, P. (2001) The cytoskeleton facilitates a three-dimensional symplasmic continuum in the long-lived ray and axial parenchyma cells of angiosperm trees. Planta 213: 811–823. Google Scholar
Faix, O. (1991) Classification of lignins from different botanical origins by FT-IR spectroscopy. Holzforschung 45(Supplement, September):21–27. Google Scholar
Fardim, P., Duran, N. (2003) Modification of fiber surfaces during pulping and refining as analysed by SEM, XPS and ToF-SIMS. Colloids and Surfaces A: Physicochem. Eng. Aspects 223: 263–276. Google Scholar
Fergus, B.J., Goring, D.A.I. (1970) Distribution of lignin in birch wood as determined by microscopy. Holzforschung 24:118–124. Google Scholar
Filippis, L.D., Magel, E. (2012) Identification of biochemical differences between the sapwood and transition zone in Robinia pseudoacacia L. by differential display of proteins. Holzforschung 66:543–549. Google Scholar
Fujii, T., Harada, H., Saiki, H. (1979) The layered structure of ray parenchyma secondary wall in the wood of 49 Japanese angiosperm species. Mokuzai Gakkaishi 25:251–257. Google Scholar
Fujikawa, S., Ishida, S. (1975) Ultrastructure of ray parenchyma cell wall of softwood. Mokuzai Gakkaishi 21:445–456. Google Scholar
Funada, R. (2000) Control of wood structure. In: Plant Microtubules: Potential for Biotechnology. Ed. Nick, P. Springer, Heidelberg. pp. 51–81. Google Scholar
Goacher, R.E., Jeremic, D., Master, E.R. (2011) Expanding the library of secondary ions that distinguish lignin and polysaccharides in time-of-flight secondary ion mass spectrometry analysis of wood. Anal. Chem. 83:804–812. Web of ScienceGoogle Scholar
Hillis, W.E. (1987) Heartwood and tree exudates. Springer-Verlag, New York, pp. 1–268. Google Scholar
Ito, T. (1997) Anatomical description of Japanese hardwoods III. Wood Research and Technical Notes 33: 83–201. Google Scholar
Joseleau, J.P., Ruel, K. (1997) Study of lignification by noninvasive techniques in growing maize internodes: an investigation by Fourier transform infrared cross-polarization magic angle spinning C-13-nuclear magnetic resonance spectroscopy and immunocytochemical transmission electron microscopy. Plant Physiol. 114:1123–1133. Google Scholar
Lapierre, C., Pollet, B., Monties, B. (1991) Thioacidolysis of spruce lignin: GC-MS analysis of the main dimers recovered after Raney nickel desulphuration. Holzforschung 45:61–68. Google Scholar
Larson, P.R. (1994) Rays. In: The Vascular Cambium: Development and Structure. Ed. Larson, P.R. Springer-Verlag, Berlin. pp. 367–391. Google Scholar
Magel, E.A. (2000) Biochemistry and physiology of heartwood formation. In: Molecular and Cell Biology of Wood Formation. Eds. Savidge, R.A., Barnett, J.R., Napier, R. BIOS, Oxford. pp. 363–376. Google Scholar
Matsushita, Y., Jang, I.-C., Imai, T., Takama, R., Saito, K., Masumi, T., Lee, S.-C., Fukushima, K. (2012) Distribution of extracts including 4,8-dihydroxy-5-methoxy-2-naphthaldehyde in Diospyros kaki analyzed by gas chromatography-mass spectrometry and time-of-flight secondary ion mass spectrometry. Holzforschung 66:705–709. Web of ScienceGoogle Scholar
Matsushita, Y., Ioka, K., Saito, K., Takama, R., Aoki, D., Fukushima, K. (2013) Fragmentation mechanism of the phenylcoumaran-type lignin model compound by ToF-SIMS. Holzforschung 67: 365–370. Web of ScienceGoogle Scholar
Murakami, Y., Funada, R., Sano, Y., Ohtani, J. (1999) The differentiation of contact cells and isolation cells in the xylem ray parenchyma of Populus maximowiczii. Annals Botany 84:429–435. Google Scholar
Nakaba, S., Sano, Y., Kubo, T., Funada, R. (2006) The positional distribution of cell death of ray parenchyma in a conifer, Abies sachalinensis. Plant Cell Rep. 25:1143–1148. Google Scholar
Nakaba, S., Kubo, T., Funada, R. (2008) Differences in patterns of cell death between ray parenchyma cells and ray tracheids in the conifers Pinus densiflora and Pinus rigida. Trees 22: 623–630. Google Scholar
Nakaba, S., Begum, S., Yamagishi, Y., Jin, H.O., Kubo, T., Funada, R. (2012) Differences in the timing of cell death, differentiation and function among three different types of ray parenchyma cells in the hardwood Populus sieboldii x P. grandidentata. Tress 26:743–750. Google Scholar
Nakashima, J., Chen, F., Jackson, L., Shadle, G., Dixon, R.A. (2008) Multi-site genetic modification of monolignol biosynthesis in alfalfa (Medicago sativa): effects on lignin composition in specific cell types. New Phytol. 179:738–750. Web of ScienceGoogle Scholar
Pomar, F., Merino, F., Barceló, A.R. (2002) O-4-Linked coniferyl and sinapyl aldehydes in lignifying cell walls are the main targets of the Wiesner (phloroglucinol-HCl) reaction. Protoplasma 220: 17–28. Google Scholar
Rabbani, S., Barber, A.M., Fletcher, J.S., Lockyer, N.P., Vickerman, J.C. (2011) TOF-SIMS with argon gas cluster ion beams: a comparison with C60+. Anal. Chem. 83:3793–3800. Google Scholar
Rencoret, J., Marques, G., Gutiérrez, A., Ibarra, D., Li, J., Gellerstedt, G., Santos, J.I., Jiménez-Barbero, J., Martínez, A.T., del Río, J.C. (2008) Structural characterization of milled wood lignin from different eucalypt species. Holzforschung 62: 514–526. Web of ScienceGoogle Scholar
Rencoret, J., Gutiérrez, A., Nieto, L., Jiménez-Barbero, J., Faulds, C.B., Kim, H., Ralph, J., Martínez, A.T., del Río, J.C. (2011) Lignin Composition and Structure in Young versus Adult Eucalyptus globulus Plants. Plant Physiol. 155: 667–682.Google Scholar
Rolando, C., Monties, B., Lapierre, C. (1992) Thioacidolysis. In: Methods in Lignin Chemistry. Eds. Lin, S.Y., Dence, C.W. Springer-Verlag, Berlin. pp. 334–349. Google Scholar
Saito, K., Fukushima, K. (2005a) Distribution of lignin interunit bonds in the differentiating xylem of compression and normal wood of Pinus thunbergii. J. Wood Sci. 51:246–251. Google Scholar
Saito, K., Kato, T., Tsuji, Y., Fukushima, K. (2005b) Identifying the characteristic secondary ions of lignin polymer using TOF-SIMS. Biomacromolecules 6:678–683. Google Scholar
Saito, K., Kishimoto, T., Matsushita, Y., Imai, T., Fukushima, K. (2011) Application of TOF-SIMS to the direct determination of syringyl to guaiacyl (S/G) ratio of lignin. Surf. Interface Anal. 43:281–284. Web of ScienceGoogle Scholar
Saito, K., Watanabe, Y., Shirakawa, M., Matsushita, Y., Imai, T., Koike, T., Sano, Y., Funada, Y., Fukazawa, K., Fukushima1, K. (2012) Direct mapping of morphological distribution of syringyl and guaiacyl lignin in the xylem of maple by time-of-flight secondary ion mass spectrometry. Plant J. 69:542–552. Web of ScienceGoogle Scholar
Saito, K., Watanabe, Y., Matsushita, Y., Imai, T., Koike, T., Sano, Y., Funada, R., Fukazawa, K., Fukushima, K. (2014) Aluminum localization in the cell walls of the mature xylem of maple tree detected by elemental imaging using time-of-flight secondary ion mass spectrometry (TOF-SIMS). Holzforschung 68:85–92. Web of ScienceGoogle Scholar
Saka, S., Goring, D.A.I. (1988) The distribution of lignin in white birch wood as determined by bromination with TEM-EDXA. Holzforschung 42:149–153. Google Scholar
Sarkanen, K.V., Ludwig, C.H. (Edts). Lignins – Occurence, Formation, Structure and Reactions. Wiley-Interscience, New-York, 1971, pp. 916. Google Scholar
Sauter, J.J. (2000) Photosynthate allocation to the vascular cambium: facts and problems. In: Molecular and cell biology of wood formation. Eds. Savidge, R., Barnett, J., Napier, R. BIOS Scientific Publishers, Oxford. pp. 71–83. Google Scholar
Song, K., Liu, B., Jiang, X., Yin, Y. (2011) Cellular changes of tracheids and ray parenchyma cells from cambium to heartwood in Cunninghamia lanceolata. J. Trop. For. Sci. 23:478–487. Google Scholar
Takabe, K., Miyauchi, S., Tsunoda, R., Fukazawa, K. (1992) Distribution of guaiacyl and syringyl lignins in Japanese beech (Fagus crenata) variation within an annual ring. IAWA Bull. n.s. 13:105–112. Google Scholar
Tokareva, E.N., Pranovich, A., Ek, P., Holmbom, B. (2010) Determination of anionic groups in wood by time-of-flight secondary ion mass spectrometry and laser ablation-inductively coupled plasma-mass spectrometry. Holzforschung 64:35–43. Web of ScienceGoogle Scholar
Watanabe, Y., Kojima, Y., Ona, T., Asada, T., Sano, Y., Fukazawa, K., Funada, R. (2004) Histochemical study on heterogeneity of lignin in Eucalyptus species II. The distribution of lignins and polyphenols in the walls of various cell types. IAWA J. 25:283–295. Google Scholar
Yamamoto, K. (1982) Yearly and seasonal process of maturation of ray parenchyma cells in Pinus species. Res. Bull. Coll. Exp. For. Hokkaido Univ. 39:245–296. Google Scholar
Yamamoto, K., Fukuzawa K., Ishida S. (1977) Study on the cell wall development of ray parenchyma in genus Pinus using ultraviolet microscopy. Res. Bull. Coll. Exp. For. Hokkaido Univ. 34:79–95. Google Scholar
Zheng, P., Aoki, D., Yoshida, M., Matsushita, Y., Imai, T., Fukushima, K. (2014a) Lignification of ray parenchyma cells in xylem of Pinus densiflora. Part I: microscopic investigation by POM, UV microscopy, and TOF-SIMS. Holzforschung 68:897–905. Web of ScienceGoogle Scholar
Zheng, P., Aoki, D., Matsushita, Y., Yagami, S., Fukushima, K. (2014b) Lignification of ray parenchyma cells in xylem of Pinus densiflora. Part II: microchemical analysis by laser microdissection and thioacidolysis. Holzforschung 68:907–913. Web of ScienceGoogle Scholar
Zhou, C., Li, Q., Chiang, V.L., Lucia, L.A., Griffis, D.P. (2011) Chemical and spatial differentiation of syringyl and guaiacyl lignins in poplar wood via time-of-flight secondary ion mass spectrometry. Anal. Chem. 83:7020–7026. Google Scholar