Stem gravitropism and tension wood formation in Acacia mangium seedlings inclined at various angles

Stem gravitropism and tension wood formation in Acacia mangium seedlings inclined at various angles Abstract Background and Aims In response to a gravitational stimulus, angiosperm trees generally form tension wood on the upper sides of leaning stems in order to reorientate the stems in the vertical direction. It is unclear whether the angle of inclination from the vertical affects tension wood formation. This study was designed to investigate negative gravitropism, tension wood formation and growth eccentricity in Acacia mangium seedlings inclined at different angles. Methods Uniform seedlings of A. mangium were artificially inclined at 30°, 45°, 60° and 90° from the vertical and harvested, with non-inclined controls, 3 months later. We analysed the effects of the angle of inclination on the stem recovery angle, the anatomical features of tension wood and radial growth. Key Results Smaller inclination angles were associated with earlier stem recovery while stems subjected to greater inclination returned to the vertical direction after a longer delay. However, in terms of the speed of negative gravitopism towards the vertical, stems subjected to greater inclination moved more rapidly toward the vertical. There was no significant difference in terms of growth eccentricity among seedlings inclined at different angles. The 30°-inclined seedlings formed the narrowest region of tension wood but there were no significant differences among seedlings inclined at 45°, 60° and 90°. The 90°-inclined seedlings formed thicker gelatinous layers than those in 30°-, 45°- and 60°-inclined seedlings. Conclusion Our results suggest that the angle of inclination of the stem influences negative gravitropism, the width of the tension wood region and the thickness of gelatinous layers. Larger amounts of gelatinous fibres and thicker gelatinous layers might generate the higher tensile stress required for the higher speed of stem-recovery movement in A. mangium seedlings. Acacia mangium, gelatinous layer, growth eccentricity, inclination angle, stem gravitropism, tension wood INTRODUCTION Woody plants form a specialized tissue, namely reaction wood, when the stem is displaced from the upright or normal position (Wardrop, 1964; Timell, 1986; Barnett et al., 2014; Gril et al., 2017). Reaction wood facilitates the return of the stem to its normal orientation. In response to a gravitational stimulus, angiosperm trees generally form tension wood, which is usually characterized by the presence of gelatinous fibres with a thick inner gelatinous layer, on the upper side of the leaning stem for restoration of the normal vertical position of the stem (Clair et al., 2006, 2010; Ruelle et al., 2006; Coutand et al., 2007; Nugroho et al., 2012, 2013; Barnett et al., 2014). Furthermore, the reorientation of leaning stems is correlated with differential radial growth that results in a stem with an eccentric cross-section. Growth eccentricity in leaning stems and branches is a common phenomenon in both angiosperms and gymnosperms (Côté and Day, 1962). In general, the eccentricity is directed toward the upper side of the leaning stem or branch in angiosperms and toward the lower side in gymnosperms (Wardrop, 1964; Panshin and de Zeew, 1980; Timell, 1986). It has been suggested that the leaning angles of stems are important triggers in the formation of tension wood (Fisher and Stevenson, 1981; Mathew, 2003). The relationship between the leaning angle of the stem and both wood anatomy and eccentric growth has been investigated in angiosperms trees in naturally and in artificially leaning stems and branches (Onaka, 1949; Wardrop, 1964; Patel et al., 1984; Mathew, 2003; Hiraiwa et al., 2013, 2014). Patel et al. (1984) obtained a direct relationship between growth eccentricity and specimen angle in Kigelia pinnata and they showed that growth eccentricity decreased with increases in specimen angle. Displacement of stems to 90° from the vertical resulted in the maximum formation of tension wood in Hevea brasiliensis (Mathew, 2003). By contrast, in Azadirachta indica, stems at inclined 45° formed the widest regions of tension wood (Reghu, 1983). Hiraiwa et al. (2013, 2014) found that, in Trochodendron araliodes and Liliodendron tulipifera, the widest region of tension wood was developed in 50°-inclined stems. Mathew (2003) suggested that the relationship between the degree of displacement of the stem and the relative amount of tension wood might be species-dependent. Nugroho et al. (2012, 2013) reported that the formation of tension wood with a thick inner gelatinous layer was required for negative gravitropism in Acacia mangium. Alméras et al. (2009) reported that, in eight tropical tree species, the response of stem movement to gravity was achieved via the formation of tension wood. However, limited information is available about the effect of the angle of inclination on negative gravitropism and the development of tension wood. Therefore, it remains unclear whether the angle of inclination of the stem affects the formation of tension wood and plant gravitropism. This study was designed to investigate negative gravitropism, the formation of tension wood and growth eccentricity in A. mangium seedlings at different angles of inclination, namely 30°, 45°, 60° and 90° from the vertical. We found that the angle of inclination of the stem affected negative gravitropism, the width of the tension wood region and the thickness of the gelatinous layer. Gelatinous fibres associated with newly developed gelatinous layers might play an important role in generation of the tensile stress required for the recovery movement of stems of A. mangium seedlings. MATERIALS AND METHODS Plant materials Thirty, approximately 1-year-old, healthy seedlings of Acacia mangium Willd. that had been grown from seed and had uniform features were used for experiments. The seedlings were approximately 70 cm tall and were planted with stems inclined at four different angles, namely 30°, 45°, 60° and 90° from the vertical and normally (0°) as controls (Fig. 1). Each group, corresponding to a particular inclination, consisted of six seedlings. Seedlings were planted in 20-cm-diameter pots, which had been filled with regosol soil, in a glasshouse at the nursery of the Faculty of Forestry, Universitas Gadjah Mada, Yogyakarta, Indonesia. The soil in each pot was moistened with approx. 200 mL of water daily. Three seedlings from each group were harvested approx. 3 months later for detailed analysis of wood anatomy. Fig. 1. View largeDownload slide Photographs showing the typical positions of stems of Acacia mangium seedlings initially, on day zero and 3 months after the start of inclination at various angles. Control seedlings (0°; A, B), 30°-inclined seedlings (C, D), 45°-inclined seedlings (E, F), 60°-inclined seedlings (G, H) and 90°-inclined seedlings (I, J). Scale bar = 20 cm. Fig. 1. View largeDownload slide Photographs showing the typical positions of stems of Acacia mangium seedlings initially, on day zero and 3 months after the start of inclination at various angles. Control seedlings (0°; A, B), 30°-inclined seedlings (C, D), 45°-inclined seedlings (E, F), 60°-inclined seedlings (G, H) and 90°-inclined seedlings (I, J). Scale bar = 20 cm. Determination of stem recovery degree (R°) and stem inclination Photographs of seedlings were taken at weekly intervals from the first day of stem inclination (day zero) for 3 months. Photographs were analysed with image-analysis software (Image-J; National Institutes of Health, Bethesda, MD, USA). Stem recovery was measured in degrees (R°) and defined as the difference between the initial angle of inclination of the seedling and the angle of the seedling after recovery and it was used as an index of the negative gravitropism of seedlings, as described by Nugroho et al. (2012). Stem inclination, measured in degrees, was defined as the angle of inclination of the seedling relative to the vertical direction. Definition and determination of growth eccentricity Ten-millimetre segments of stems, from sites 10 cm above the top of the soil, were removed from seedlings for analysis. Segments were fixed in 4 % glutaraldehyde in 0.1 m phosphate buffer (pH 7.3). Sample discs were cut from the fixed segments of stems and examined for eccentric growth. Cross-sectional surfaces of discs were prepared with a sliding microtome (Yamatokohki, Saitama, Japan). Digital images of the cross-sectional surfaces were recorded under a stereo microscope (Stemi 2000-C stereo microscope; Carl Zeiss Microscopy, Tokyo, Japan) with a digital camera (DS-Fi1; Nikon Corporation, Tokyo Japan). Before images were recorded, the individual discs were placed on the microscope stage and the camera, mounted on a calibrated stand, was adjusted to yield a clear image. Photographs were analysed with the image-analysis software Image-J. Growth eccentricity was expressed in terms of the ratio of the upper-side radius to the lower-side radius, as described by Kucera and Philipson (1977) and shown in Fig. 2. Fig. 2. View largeDownload slide Determination of growth eccentricity in Acacia mangium stem seedlings. Growth eccentricity was determined as the ratio between the (a) upper radius and (b) the lower radius of stems, as described by Kucera and Philipson (1977). Scale bar = 2.5 mm. Fig. 2. View largeDownload slide Determination of growth eccentricity in Acacia mangium stem seedlings. Growth eccentricity was determined as the ratio between the (a) upper radius and (b) the lower radius of stems, as described by Kucera and Philipson (1977). Scale bar = 2.5 mm. Analysis of tension wood Transverse sections of 15-µm thickness were cut from the fixed segments on the sliding microtome. Sections were stained in a 0.1 % solution of safranine (Wako Pure Chemical Industries, Osaka, Japan) for 3 min and then for 5 min in a 1 % solution of Astra blue (Sigma-Aldrich, Steinheim, Germany). They were then dehydrated in a graded ethanol series, mounted on glass slides, fixed in resin (Entellan new; Merck, Darmstadt, Germany) and secured with coverslips (Nugroho et al., 2012, 2013). Images were recorded under a light microscope (Axioscop; Carl Zeiss, Oberkochen, Germany) with a digital camera (Digital Sight DS-5M-L1; Nikon Corporation). Digital images of transverse sections were recorded for measurements of the width of the region of tension wood in the outermost xylem in the upper part of each inclined stem. The width of the region of tension wood was determined by measuring the width of the region that consisted of wood fibres with inner gelatinous layers that were stained blue by Astra blue and safranine (Nugroho et al., 2012, 2013). For quantification of these apparent gelatinous layers, digital images of transverse-sectional areas of 35.3 × 103 µm2 were recorded from regions of the tension wood. Fifty wood fibres were measured in each sample. The width of the region of tension wood and the thickness of apparent gelatinous layers were measured with the image-analysis software Image-J. Statistical analysis Data were analysed with the statistical software Program Prism5 for Mac OSX (GraphPad Software Inc., La Jolla, CA, USA). The effects of the inclination angle on growth eccentricity, the width of the tension wood region and the thickness of gelatinous layers were examined by one-way analysis of variance (ANOVA), which was followed by Tukey’s post hoc test. Significance of differences among treatments was recognized at P < 0.05. In addition, linear regression analysis was performed to examine the relationship between the width of the region of tension wood and growth eccentricity. RESULTS Negative gravitropism of inclined Acacia mangium seedlings Figure 1 shows the typical positions of stems of A. mangium seedlings at the start of the experiment (day zero) and after 3 months of inclined growth. After 3 months, all of the inclined seedlings had returned to a vertical or near vertical position. Figure 3 shows the changes in stem recovery degrees (R° values) for the control (0°) and the 30°-, 45°-, 60°- and 90°-inclined seedlings from day zero for 6 weeks. The seedlings subjected to larger angles of inclination were associated with higher stem recovery degrees. Figure 4 shows the inclination degrees (°) of the control (0°) and 30°-, 45°-, 60°- and 90°-inclined seedlings from 1 week to 6 weeks after day zero. We defined the stem inclination degree (°) as the angle between the inclined seedling and the vertical. Thus, the stem inclination degree indicates the angular distance remaining between the stem and the vertical direction. Stems of seedlings with smaller degrees of inclination returned sooner to the vertical position. Three weeks after day zero, the stem inclination degrees of 30°-, 45°- and 60°-inclined seedlings were not significantly different from those of control seedlings (0°), indicating that the 30°-, 45°- and 60°-inclined seedlings had returned to the vertical position. By contrast, the 90°-inclined seedlings returned to the vertical position 4 weeks after day zero. The mean values (± s.e.) of the speed of negative gravitropism towards the vertical or near vertical, as calculated from the results in Figs 3 and 4, were approx. 7.2 ± 1.3° week–1 for 30°-inclined stem seedlings, 11.0 ± 1.2° week–1 for 45°-inclined stem seedlings, 14.0 ± 2.0° week–1 for 60°-inclined stem seedlings and 19.0 ± 2.0° week–1 for 90°-inclined stem seedlings (P < 0.0001). Thus, the stems of seedlings exposed to higher degrees of inclination moved significantly more rapidly towards the vertical. Fig. 3. View largeDownload slide Time courses, in term of average stem-recovery degrees (R°), of changes in orientation of Acacia mangium seedlings artificially inclined at various angles. The 90°-inclined seedlings were associated with the greatest angular movement towards the vertical. Bars indicate standard errors (n = 6). Fig. 3. View largeDownload slide Time courses, in term of average stem-recovery degrees (R°), of changes in orientation of Acacia mangium seedlings artificially inclined at various angles. The 90°-inclined seedlings were associated with the greatest angular movement towards the vertical. Bars indicate standard errors (n = 6). Fig. 4. View largeDownload slide Quantitative analysis of stem inclination degrees of control seedlings (0°) and 30°-, 45°-, 60°- and 90°-inclined seedlings 1, 2, 3, 4, 5 and 6 weeks after the start of inclination. Error bars indicate standard errors (n = 6). Different letters above bars indicate significant differences, as determined by Tukey’s post hoc test, at P < 0.05. Fig. 4. View largeDownload slide Quantitative analysis of stem inclination degrees of control seedlings (0°) and 30°-, 45°-, 60°- and 90°-inclined seedlings 1, 2, 3, 4, 5 and 6 weeks after the start of inclination. Error bars indicate standard errors (n = 6). Different letters above bars indicate significant differences, as determined by Tukey’s post hoc test, at P < 0.05. Growth eccentricity Stereomicroscopic observations of cross-sectional surfaces revealed concentric growth in control seedlings (0°), while growth of seedlings inclined at 30°, 45°, 60° and 90° was eccentric (Fig. 5). The mean values (± s.e.) of growth eccentricity (a/b in Fig. 2) were approx. 1.01 ± 0.03 in control seedlings, 1.19 ± 0.01 in 30°-inclined stem seedlings, 1.17 ± 0.03 in 45°-inclined stem seedlings, 1.27 ± 0.10 in 60°-inclined stem seedlings and 1.21 ± 0.08 in 90°-inclined stem seedlings. There were no significant differences in terms of growth eccentricity among seedlings inclined at the various angles (Fig. 6). Fig. 5. View largeDownload slide Photographs of cross-sectional surfaces of inclined stems. Control seedling (0°; A), 30°-inclined seedling (B), 45°-inclined seedling (C), 60°-inclined seedling (D) and 90°-inclined seedling (E). Asterisks indicate the upper regions of inclined stems. Scale bar = 5 mm. Fig. 5. View largeDownload slide Photographs of cross-sectional surfaces of inclined stems. Control seedling (0°; A), 30°-inclined seedling (B), 45°-inclined seedling (C), 60°-inclined seedling (D) and 90°-inclined seedling (E). Asterisks indicate the upper regions of inclined stems. Scale bar = 5 mm. Fig. 6. View largeDownload slide Quantitative analysis of growth eccentricity in stems of Acacia mangium seedlings subjected to various angles of inclination. Increases in inclination angle from 30° to 90° did not affect the growth eccentricity of stems. Error bars indicate standard errors (n = 3). Different letters above bars indicate significant differences, as determined by Tukey’s post hoc test, at P < 0.05. Fig. 6. View largeDownload slide Quantitative analysis of growth eccentricity in stems of Acacia mangium seedlings subjected to various angles of inclination. Increases in inclination angle from 30° to 90° did not affect the growth eccentricity of stems. Error bars indicate standard errors (n = 3). Different letters above bars indicate significant differences, as determined by Tukey’s post hoc test, at P < 0.05. Width of tension wood regions In control seedlings, the absence of an additional gravitational stimulus resulted in the absence of tension wood formation. However, the formation of tension wood occurred in all the inclined seedlings. Tension wood was found in the upper part of the stem of each inclined seedling (Fig. 7B–E), while no tension wood was found in control seedlings (Fig. 7A). The mean values (± s.e.) of the widths of the regions of tension wood in seedlings inclined at 30°, 45°, 60° and 90° were approx. 0.34 ± 0.01, 0.44 ± 0.02, 0.42 ± 0.02 and 0.40 ± 0.01 mm, respectively. The widths of tension wood regions differed significantly among the 30°-, 45°-, 60°- and 90°-inclined seedlings (P = 0.022; Fig. 8). The 30°-inclined seedlings formed the narrowest regions of tension wood. Fig. 7. View largeDownload slide Light micrographs of cross sections, stained with safranine and Astra blue, of a control seedling (0°; A), a 30°-inclined seedling (B), a 45°-inclined seedling (C), a 60°-inclined seedling (D) and a 90°-inclined seedling (E) of Acacia mangium 3 months after the start of inclination. Asterisks indicate the presence of gelatinous fibers in the upper regions of the inclined stems. Scale bar = 300 µm. Fig. 7. View largeDownload slide Light micrographs of cross sections, stained with safranine and Astra blue, of a control seedling (0°; A), a 30°-inclined seedling (B), a 45°-inclined seedling (C), a 60°-inclined seedling (D) and a 90°-inclined seedling (E) of Acacia mangium 3 months after the start of inclination. Asterisks indicate the presence of gelatinous fibers in the upper regions of the inclined stems. Scale bar = 300 µm. Fig. 8. View largeDownload slide Quantitative analysis of the width of tension wood regions in the upper parts of stems of Acacia mangium seedlings subjected to different angles of inclination. The widths of tension wood regions differed significantly among 30°-, 45°-, 60°- and 90°-inclined seedlings (P = 0.022). Error bars indicate standard errors (n = 3). Different letters above bars indicate significant differences, as determined by Tukey’s post hoc test, at P < 0.05. Fig. 8. View largeDownload slide Quantitative analysis of the width of tension wood regions in the upper parts of stems of Acacia mangium seedlings subjected to different angles of inclination. The widths of tension wood regions differed significantly among 30°-, 45°-, 60°- and 90°-inclined seedlings (P = 0.022). Error bars indicate standard errors (n = 3). Different letters above bars indicate significant differences, as determined by Tukey’s post hoc test, at P < 0.05. Thickness of gelatinous layers Light microscopy of 15-µm-thick transverse sections revealed gelatinous fibres with an inner gelatinous layer in the tension wood regions of 30°-, 45°-, 60°- and 90°-inclined seedlings (Fig. 9). Gelatinous layers were stained blue by the combination of safranine and Astra blue. The thickness of the gelatinous layers differed significantly among 30°-, 45°-, 60°- and 90°-inclined seedlings (P < 0.0001). The mean (± s.e.) thickness of gelatinous layers was approx. 2.3 ± 0.1 µm in 30°-inclined seedlings, 2.3 ± 0.1 µm in 45°-inclined seedlings, 2.5 ± 0.1 µm in 60°-inclined seedlings and 3.8 ± 0.1 µm in 90°-inclined seedlings. The 90°-inclined seedlings formed thicker gelatinous layers (Figs 9 and 10) than those in the 30°-, 45°- and 60°-inclined seedlings. Fig. 9. View largeDownload slide Light micrographs of transverse sections of the tension wood of inclined stems, 3 months after the start of inclination, after staining with safranine and Astra blue: (A) 30°-inclined seedling, (B) 45°-inclined seedling, (C) 60°-inclined seedling and (D) 90°-inclined seedling. The 90°-inclined seedling had thicker gelatinous layers. Blue coloration indicates gelatinous layers. Scale bar = 30 µm. Fig. 9. View largeDownload slide Light micrographs of transverse sections of the tension wood of inclined stems, 3 months after the start of inclination, after staining with safranine and Astra blue: (A) 30°-inclined seedling, (B) 45°-inclined seedling, (C) 60°-inclined seedling and (D) 90°-inclined seedling. The 90°-inclined seedling had thicker gelatinous layers. Blue coloration indicates gelatinous layers. Scale bar = 30 µm. Fig. 10. View largeDownload slide Quantitative analysis of the thickness of gelatinous layers in the upper portions of 30°-, 45°-, 60°- and 90°-inclined seedlings of Acacia mangium. The gelatinous layers in the tension wood of 90°-inclined seedlings were significantly thicker than in 30°-, 45°- and 60°-inclined seedlings. Error bars indicate standard errors (n = 3). Different letters above bars indicate significant differences, as determined by Tukey’s post hoc test, at P < 0.05. Fig. 10. View largeDownload slide Quantitative analysis of the thickness of gelatinous layers in the upper portions of 30°-, 45°-, 60°- and 90°-inclined seedlings of Acacia mangium. The gelatinous layers in the tension wood of 90°-inclined seedlings were significantly thicker than in 30°-, 45°- and 60°-inclined seedlings. Error bars indicate standard errors (n = 3). Different letters above bars indicate significant differences, as determined by Tukey’s post hoc test, at P < 0.05. DISCUSSION Woody plants respond to environmental forces and to gravitational stimuli via the development of reaction wood that reorientates the axis, trunk and branches, allowing them to recover the gravitropic set-point angle, as defined by Digby and Firn (1995). Reorientation of seedlings of woody plants in response to a gravitational stimulus is closely related to the formation of tension wood. Tension wood generates a strong tensile force and contributes to the negative gravitropic movement of inclined woody stems (Okuyama et al., 1994; Yoshida et al., 1999, 2000; Yamamoto et al., 2002; Clair et al., 2006; Ruelle et al., 2006; Fang et al., 2008; Mellerowicz and Gorshkova, 2012; Gril et al., 2017). Our previous study of A. mangium seedlings revealed that the width of the region of gelatinous fibres and the thickness of the gelatinous layers were strongly correlated with the negative gravitropism of inclined seedlings (Nugroho et al., 2012, 2013). In the present study, we found that inclined A. mangium seedlings formed tension wood on the upper part of each inclined stem and returned to the vertical after inclination at four different angles. A smaller inclination angle, such as 30°, was associated with an earlier return of stems to the vertical and a larger angle, such as 90°, was associated with a later return to the vertical. The 30°-, 45°- and 60°-inclined seedlings had almost completely returned to the vertical 3 weeks after day zero but 90°-inclined seedlings returned to the vertical 4°weeks after day zero. In terms of the speed of negative-gravitropic movement towards the vertical position, the seedlings subjected to larger degrees of inclination moved more rapidly towards the vertical. The 30°-inclined seedlings, which moved with the lowest negative-gravitropic speed, formed the narrowest region of tension wood. However, there were no significant differences among seedlings inclined at 45°, 60° and 90° with respect to the width of the tension wood. Therefore, the speed of negative-gravitropic movement of A. mangium seedlings might be directly related to the amount of newly developed tension wood. Mathew (2003) reported that the widest region of tension wood was formed at a displacement of 90° from the vertical. Moreover, Reghu (1983) reported that, in 1-year-old seedlings of Azadirachta indica, the width of newly induced tension wood was greatest at an inclination angle of 45° and the proportion of tension wood decreased as the degree of inclination was increased further. Hiraiwa et al. (2013, 2014) found the widest regions of tension wood in 50°-inclined stems of Trochodendron araliodes and Liliodendron tulipifera. Thus, the relative amount of newly developed tension wood appears to depend on the angle of inclination and species. The extent of formation of tension wood might reach a plateau value as the angle of inclination is increased from 45° to 90°. Microscopic observations revealed that, among inclined stems, the thickest gelatinous layer was formed in the tension wood regions of 90°-inclined seedlings. The gelatinous layers of 90°-inclined seedlings were significantly thicker than those of 30°-, 45°- and 60°-inclined seedlings. Although there were no significant differences in terms of the widths of tension wood regions among 90°-inclined seedlings and the other inclined seedlings, the 90°-inclined seedlings were associated with the highest negative-gravitropic speed. Therefore, thicker gelatinous layers might generate greater tensile stress for higher speeds of stem recovery movement in A. mangium seedlings. Our results support the hypothesis, proposed by Fang et al. (2008), that thicker gelatinous layers are accompanied by greater longitudinal growth stress and play a critical role in the generation of strong growth stress in poplar. The formation of fibres with thick gelatinous layers induces strong tension stress via lateral swelling of the gelatinous layer, which forces the surrounding secondary cell walls to contract in the axial direction (Goswami et al., 2008; Mellerowicz and Gorshkova, 2012; Gril et al., 2017). Our results suggest that the development of gelatinous layers is important in the generation of the tensile force required for the negative-gravitropic movement of the stems of seedlings. Eccentricity of growth is induced by displacement of the stem from the normal or vertical direction. The extent of eccentric growth induced by leaning of stems is related to the angle of inclination from the vertical (Burns, 1942; Robards, 1965, 1966). In the present study, control seedlings (0°) exhibited concentric growth but seedlings inclined at 30°, 45°, 60° and 90° showed evidence of eccentric growth. A gravitational stimulus promotes formation of tension wood on the upper part of inclined stems, an indication of higher cambial-division activity in the upper part of the inclined stem than in the lower part of the inclined stem. Patel et al. (1984) reported that, in Kigelia pinnata, growth eccentricity decreased with increases in the angle of inclination. We found that increasing the inclination of A. mangium seedlings from 30° to 90° did not increase growth eccentricity. In addition, we found that there was no correlation between the width of the tension wood region and growth eccentricity (P = 0.099). Similarly, there was no correlation between the width of the tension wood and growth eccentricity in Hevea brasiliensis (Mathew, 2003). Therefore, the extent of growth eccentricity appears not to reflect the extent of the newly developed tension wood. Plant hormones are involved both in the formation of tension wood and in gravitropism (Little and Pharis, 1995; Mellerowicz et al., 2001; Pilate et al., 2004; Kwon, 2008). In particular, gibberellin plays an important role in the formation of tension wood (Nakamura et al., 1994; Baba et al., 1995; Yoshida et al., 1999; Du and Yamamoto, 2007; Funada et al., 2008; Wang et al., 2017). In inclined seedlings of A. mangium, Nugroho et al. (2012, 2013) found that gibberellin is essential for the formation of tension wood, for development of gelatinous layers in the tension wood and for gravitropism. The application of gibberellic acid (GA3) to the soil of seedlings of A. mangium significantly stimulated the return to the vertical of inclined stems by increasing the extent of formation of tension wood. Moreover, inhibitors of gibberellin biosynthesis, such as uniconazole-P and paclobutrazole, inhibited the upward bending of inclined seedlings via suppression of increases in the width of the tension wood and the development of gelatinous layers. Our present results revealed that the angle of inclination of stems affects the speed of negative-gravitropic movement via the width of the newly developed tension wood and the thickness of gelatinous layers. It is plausible that inclined A. mangium seedlings might recognize and respond to a gravitational stimulus via changes in the rate of biosynthesis of gibberellin, which regulates the development of tension wood, the thickness of gelatinous layers and, as a consequence, the speed of stem-recovery movement. In summary, the angle of inclination of the seedling affected the width of the newly developed tension wood region, the thickness of gelatinous layers and the speed of negative-gravitropic movement. Larger amounts of newly developed gelatinous fibres and thicker gelatinous layers might generate the higher tensile stress that leads to higher speeds of stem-recovery movement in A. mangium seedlings. ACKNOWLEDGMENTS W.D.H. thanks the Hitachi Scholarship Foundation, Japan, and the Japan Student Services Organization (JASSO) for supporting this work. The authors also thank Alan Cabout, Adityo Roem and Jito, Universitas Gadjah Mada, for their assistance in the preparation of seedlings of A. mangium and the collection of samples. 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, 24380090, 15K07508, 15H04527, 16K14954 and 18H02251). LITERATURE CITED Alméras T, Derycke M, Jaouen G, Beauchene J, Fournier M. 2009. Functional diversity in gravitropic reaction among tropical seedlings in relation to ecological and developmental traits. Journal of Experimental Botany  60: 4397– 4410. Google Scholar CrossRef Search ADS PubMed  Baba K, Adachi K, Take T, Yokoyama T, Ito T, Nakamura T. 1995. Induction of tension wood in GA3-treated branches of the weeping type of Japanese cherry, Prunus spachiana. Plant and Cell Physiology  36: 983– 988. Google Scholar CrossRef Search ADS   Barnett JR, Gril J, Saranpää P. 2014. Introduction. 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Tension wood and growth stress induced by artificial inclination in Liriodendron tulipifera Linn. and Prunus spachiana Kitamura f. ascendens Kitamura. Annals of Forest Science  57: 739– 746. Google Scholar CrossRef Search ADS   © The Author(s) 2018. Published by Oxford University Press on behalf of the Annals of Botany Company. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com. This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/about_us/legal/notices) http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Annals of Botany Oxford University Press

Stem gravitropism and tension wood formation in Acacia mangium seedlings inclined at various angles

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Abstract

Abstract Background and Aims In response to a gravitational stimulus, angiosperm trees generally form tension wood on the upper sides of leaning stems in order to reorientate the stems in the vertical direction. It is unclear whether the angle of inclination from the vertical affects tension wood formation. This study was designed to investigate negative gravitropism, tension wood formation and growth eccentricity in Acacia mangium seedlings inclined at different angles. Methods Uniform seedlings of A. mangium were artificially inclined at 30°, 45°, 60° and 90° from the vertical and harvested, with non-inclined controls, 3 months later. We analysed the effects of the angle of inclination on the stem recovery angle, the anatomical features of tension wood and radial growth. Key Results Smaller inclination angles were associated with earlier stem recovery while stems subjected to greater inclination returned to the vertical direction after a longer delay. However, in terms of the speed of negative gravitopism towards the vertical, stems subjected to greater inclination moved more rapidly toward the vertical. There was no significant difference in terms of growth eccentricity among seedlings inclined at different angles. The 30°-inclined seedlings formed the narrowest region of tension wood but there were no significant differences among seedlings inclined at 45°, 60° and 90°. The 90°-inclined seedlings formed thicker gelatinous layers than those in 30°-, 45°- and 60°-inclined seedlings. Conclusion Our results suggest that the angle of inclination of the stem influences negative gravitropism, the width of the tension wood region and the thickness of gelatinous layers. Larger amounts of gelatinous fibres and thicker gelatinous layers might generate the higher tensile stress required for the higher speed of stem-recovery movement in A. mangium seedlings. Acacia mangium, gelatinous layer, growth eccentricity, inclination angle, stem gravitropism, tension wood INTRODUCTION Woody plants form a specialized tissue, namely reaction wood, when the stem is displaced from the upright or normal position (Wardrop, 1964; Timell, 1986; Barnett et al., 2014; Gril et al., 2017). Reaction wood facilitates the return of the stem to its normal orientation. In response to a gravitational stimulus, angiosperm trees generally form tension wood, which is usually characterized by the presence of gelatinous fibres with a thick inner gelatinous layer, on the upper side of the leaning stem for restoration of the normal vertical position of the stem (Clair et al., 2006, 2010; Ruelle et al., 2006; Coutand et al., 2007; Nugroho et al., 2012, 2013; Barnett et al., 2014). Furthermore, the reorientation of leaning stems is correlated with differential radial growth that results in a stem with an eccentric cross-section. Growth eccentricity in leaning stems and branches is a common phenomenon in both angiosperms and gymnosperms (Côté and Day, 1962). In general, the eccentricity is directed toward the upper side of the leaning stem or branch in angiosperms and toward the lower side in gymnosperms (Wardrop, 1964; Panshin and de Zeew, 1980; Timell, 1986). It has been suggested that the leaning angles of stems are important triggers in the formation of tension wood (Fisher and Stevenson, 1981; Mathew, 2003). The relationship between the leaning angle of the stem and both wood anatomy and eccentric growth has been investigated in angiosperms trees in naturally and in artificially leaning stems and branches (Onaka, 1949; Wardrop, 1964; Patel et al., 1984; Mathew, 2003; Hiraiwa et al., 2013, 2014). Patel et al. (1984) obtained a direct relationship between growth eccentricity and specimen angle in Kigelia pinnata and they showed that growth eccentricity decreased with increases in specimen angle. Displacement of stems to 90° from the vertical resulted in the maximum formation of tension wood in Hevea brasiliensis (Mathew, 2003). By contrast, in Azadirachta indica, stems at inclined 45° formed the widest regions of tension wood (Reghu, 1983). Hiraiwa et al. (2013, 2014) found that, in Trochodendron araliodes and Liliodendron tulipifera, the widest region of tension wood was developed in 50°-inclined stems. Mathew (2003) suggested that the relationship between the degree of displacement of the stem and the relative amount of tension wood might be species-dependent. Nugroho et al. (2012, 2013) reported that the formation of tension wood with a thick inner gelatinous layer was required for negative gravitropism in Acacia mangium. Alméras et al. (2009) reported that, in eight tropical tree species, the response of stem movement to gravity was achieved via the formation of tension wood. However, limited information is available about the effect of the angle of inclination on negative gravitropism and the development of tension wood. Therefore, it remains unclear whether the angle of inclination of the stem affects the formation of tension wood and plant gravitropism. This study was designed to investigate negative gravitropism, the formation of tension wood and growth eccentricity in A. mangium seedlings at different angles of inclination, namely 30°, 45°, 60° and 90° from the vertical. We found that the angle of inclination of the stem affected negative gravitropism, the width of the tension wood region and the thickness of the gelatinous layer. Gelatinous fibres associated with newly developed gelatinous layers might play an important role in generation of the tensile stress required for the recovery movement of stems of A. mangium seedlings. MATERIALS AND METHODS Plant materials Thirty, approximately 1-year-old, healthy seedlings of Acacia mangium Willd. that had been grown from seed and had uniform features were used for experiments. The seedlings were approximately 70 cm tall and were planted with stems inclined at four different angles, namely 30°, 45°, 60° and 90° from the vertical and normally (0°) as controls (Fig. 1). Each group, corresponding to a particular inclination, consisted of six seedlings. Seedlings were planted in 20-cm-diameter pots, which had been filled with regosol soil, in a glasshouse at the nursery of the Faculty of Forestry, Universitas Gadjah Mada, Yogyakarta, Indonesia. The soil in each pot was moistened with approx. 200 mL of water daily. Three seedlings from each group were harvested approx. 3 months later for detailed analysis of wood anatomy. Fig. 1. View largeDownload slide Photographs showing the typical positions of stems of Acacia mangium seedlings initially, on day zero and 3 months after the start of inclination at various angles. Control seedlings (0°; A, B), 30°-inclined seedlings (C, D), 45°-inclined seedlings (E, F), 60°-inclined seedlings (G, H) and 90°-inclined seedlings (I, J). Scale bar = 20 cm. Fig. 1. View largeDownload slide Photographs showing the typical positions of stems of Acacia mangium seedlings initially, on day zero and 3 months after the start of inclination at various angles. Control seedlings (0°; A, B), 30°-inclined seedlings (C, D), 45°-inclined seedlings (E, F), 60°-inclined seedlings (G, H) and 90°-inclined seedlings (I, J). Scale bar = 20 cm. Determination of stem recovery degree (R°) and stem inclination Photographs of seedlings were taken at weekly intervals from the first day of stem inclination (day zero) for 3 months. Photographs were analysed with image-analysis software (Image-J; National Institutes of Health, Bethesda, MD, USA). Stem recovery was measured in degrees (R°) and defined as the difference between the initial angle of inclination of the seedling and the angle of the seedling after recovery and it was used as an index of the negative gravitropism of seedlings, as described by Nugroho et al. (2012). Stem inclination, measured in degrees, was defined as the angle of inclination of the seedling relative to the vertical direction. Definition and determination of growth eccentricity Ten-millimetre segments of stems, from sites 10 cm above the top of the soil, were removed from seedlings for analysis. Segments were fixed in 4 % glutaraldehyde in 0.1 m phosphate buffer (pH 7.3). Sample discs were cut from the fixed segments of stems and examined for eccentric growth. Cross-sectional surfaces of discs were prepared with a sliding microtome (Yamatokohki, Saitama, Japan). Digital images of the cross-sectional surfaces were recorded under a stereo microscope (Stemi 2000-C stereo microscope; Carl Zeiss Microscopy, Tokyo, Japan) with a digital camera (DS-Fi1; Nikon Corporation, Tokyo Japan). Before images were recorded, the individual discs were placed on the microscope stage and the camera, mounted on a calibrated stand, was adjusted to yield a clear image. Photographs were analysed with the image-analysis software Image-J. Growth eccentricity was expressed in terms of the ratio of the upper-side radius to the lower-side radius, as described by Kucera and Philipson (1977) and shown in Fig. 2. Fig. 2. View largeDownload slide Determination of growth eccentricity in Acacia mangium stem seedlings. Growth eccentricity was determined as the ratio between the (a) upper radius and (b) the lower radius of stems, as described by Kucera and Philipson (1977). Scale bar = 2.5 mm. Fig. 2. View largeDownload slide Determination of growth eccentricity in Acacia mangium stem seedlings. Growth eccentricity was determined as the ratio between the (a) upper radius and (b) the lower radius of stems, as described by Kucera and Philipson (1977). Scale bar = 2.5 mm. Analysis of tension wood Transverse sections of 15-µm thickness were cut from the fixed segments on the sliding microtome. Sections were stained in a 0.1 % solution of safranine (Wako Pure Chemical Industries, Osaka, Japan) for 3 min and then for 5 min in a 1 % solution of Astra blue (Sigma-Aldrich, Steinheim, Germany). They were then dehydrated in a graded ethanol series, mounted on glass slides, fixed in resin (Entellan new; Merck, Darmstadt, Germany) and secured with coverslips (Nugroho et al., 2012, 2013). Images were recorded under a light microscope (Axioscop; Carl Zeiss, Oberkochen, Germany) with a digital camera (Digital Sight DS-5M-L1; Nikon Corporation). Digital images of transverse sections were recorded for measurements of the width of the region of tension wood in the outermost xylem in the upper part of each inclined stem. The width of the region of tension wood was determined by measuring the width of the region that consisted of wood fibres with inner gelatinous layers that were stained blue by Astra blue and safranine (Nugroho et al., 2012, 2013). For quantification of these apparent gelatinous layers, digital images of transverse-sectional areas of 35.3 × 103 µm2 were recorded from regions of the tension wood. Fifty wood fibres were measured in each sample. The width of the region of tension wood and the thickness of apparent gelatinous layers were measured with the image-analysis software Image-J. Statistical analysis Data were analysed with the statistical software Program Prism5 for Mac OSX (GraphPad Software Inc., La Jolla, CA, USA). The effects of the inclination angle on growth eccentricity, the width of the tension wood region and the thickness of gelatinous layers were examined by one-way analysis of variance (ANOVA), which was followed by Tukey’s post hoc test. Significance of differences among treatments was recognized at P < 0.05. In addition, linear regression analysis was performed to examine the relationship between the width of the region of tension wood and growth eccentricity. RESULTS Negative gravitropism of inclined Acacia mangium seedlings Figure 1 shows the typical positions of stems of A. mangium seedlings at the start of the experiment (day zero) and after 3 months of inclined growth. After 3 months, all of the inclined seedlings had returned to a vertical or near vertical position. Figure 3 shows the changes in stem recovery degrees (R° values) for the control (0°) and the 30°-, 45°-, 60°- and 90°-inclined seedlings from day zero for 6 weeks. The seedlings subjected to larger angles of inclination were associated with higher stem recovery degrees. Figure 4 shows the inclination degrees (°) of the control (0°) and 30°-, 45°-, 60°- and 90°-inclined seedlings from 1 week to 6 weeks after day zero. We defined the stem inclination degree (°) as the angle between the inclined seedling and the vertical. Thus, the stem inclination degree indicates the angular distance remaining between the stem and the vertical direction. Stems of seedlings with smaller degrees of inclination returned sooner to the vertical position. Three weeks after day zero, the stem inclination degrees of 30°-, 45°- and 60°-inclined seedlings were not significantly different from those of control seedlings (0°), indicating that the 30°-, 45°- and 60°-inclined seedlings had returned to the vertical position. By contrast, the 90°-inclined seedlings returned to the vertical position 4 weeks after day zero. The mean values (± s.e.) of the speed of negative gravitropism towards the vertical or near vertical, as calculated from the results in Figs 3 and 4, were approx. 7.2 ± 1.3° week–1 for 30°-inclined stem seedlings, 11.0 ± 1.2° week–1 for 45°-inclined stem seedlings, 14.0 ± 2.0° week–1 for 60°-inclined stem seedlings and 19.0 ± 2.0° week–1 for 90°-inclined stem seedlings (P < 0.0001). Thus, the stems of seedlings exposed to higher degrees of inclination moved significantly more rapidly towards the vertical. Fig. 3. View largeDownload slide Time courses, in term of average stem-recovery degrees (R°), of changes in orientation of Acacia mangium seedlings artificially inclined at various angles. The 90°-inclined seedlings were associated with the greatest angular movement towards the vertical. Bars indicate standard errors (n = 6). Fig. 3. View largeDownload slide Time courses, in term of average stem-recovery degrees (R°), of changes in orientation of Acacia mangium seedlings artificially inclined at various angles. The 90°-inclined seedlings were associated with the greatest angular movement towards the vertical. Bars indicate standard errors (n = 6). Fig. 4. View largeDownload slide Quantitative analysis of stem inclination degrees of control seedlings (0°) and 30°-, 45°-, 60°- and 90°-inclined seedlings 1, 2, 3, 4, 5 and 6 weeks after the start of inclination. Error bars indicate standard errors (n = 6). Different letters above bars indicate significant differences, as determined by Tukey’s post hoc test, at P < 0.05. Fig. 4. View largeDownload slide Quantitative analysis of stem inclination degrees of control seedlings (0°) and 30°-, 45°-, 60°- and 90°-inclined seedlings 1, 2, 3, 4, 5 and 6 weeks after the start of inclination. Error bars indicate standard errors (n = 6). Different letters above bars indicate significant differences, as determined by Tukey’s post hoc test, at P < 0.05. Growth eccentricity Stereomicroscopic observations of cross-sectional surfaces revealed concentric growth in control seedlings (0°), while growth of seedlings inclined at 30°, 45°, 60° and 90° was eccentric (Fig. 5). The mean values (± s.e.) of growth eccentricity (a/b in Fig. 2) were approx. 1.01 ± 0.03 in control seedlings, 1.19 ± 0.01 in 30°-inclined stem seedlings, 1.17 ± 0.03 in 45°-inclined stem seedlings, 1.27 ± 0.10 in 60°-inclined stem seedlings and 1.21 ± 0.08 in 90°-inclined stem seedlings. There were no significant differences in terms of growth eccentricity among seedlings inclined at the various angles (Fig. 6). Fig. 5. View largeDownload slide Photographs of cross-sectional surfaces of inclined stems. Control seedling (0°; A), 30°-inclined seedling (B), 45°-inclined seedling (C), 60°-inclined seedling (D) and 90°-inclined seedling (E). Asterisks indicate the upper regions of inclined stems. Scale bar = 5 mm. Fig. 5. View largeDownload slide Photographs of cross-sectional surfaces of inclined stems. Control seedling (0°; A), 30°-inclined seedling (B), 45°-inclined seedling (C), 60°-inclined seedling (D) and 90°-inclined seedling (E). Asterisks indicate the upper regions of inclined stems. Scale bar = 5 mm. Fig. 6. View largeDownload slide Quantitative analysis of growth eccentricity in stems of Acacia mangium seedlings subjected to various angles of inclination. Increases in inclination angle from 30° to 90° did not affect the growth eccentricity of stems. Error bars indicate standard errors (n = 3). Different letters above bars indicate significant differences, as determined by Tukey’s post hoc test, at P < 0.05. Fig. 6. View largeDownload slide Quantitative analysis of growth eccentricity in stems of Acacia mangium seedlings subjected to various angles of inclination. Increases in inclination angle from 30° to 90° did not affect the growth eccentricity of stems. Error bars indicate standard errors (n = 3). Different letters above bars indicate significant differences, as determined by Tukey’s post hoc test, at P < 0.05. Width of tension wood regions In control seedlings, the absence of an additional gravitational stimulus resulted in the absence of tension wood formation. However, the formation of tension wood occurred in all the inclined seedlings. Tension wood was found in the upper part of the stem of each inclined seedling (Fig. 7B–E), while no tension wood was found in control seedlings (Fig. 7A). The mean values (± s.e.) of the widths of the regions of tension wood in seedlings inclined at 30°, 45°, 60° and 90° were approx. 0.34 ± 0.01, 0.44 ± 0.02, 0.42 ± 0.02 and 0.40 ± 0.01 mm, respectively. The widths of tension wood regions differed significantly among the 30°-, 45°-, 60°- and 90°-inclined seedlings (P = 0.022; Fig. 8). The 30°-inclined seedlings formed the narrowest regions of tension wood. Fig. 7. View largeDownload slide Light micrographs of cross sections, stained with safranine and Astra blue, of a control seedling (0°; A), a 30°-inclined seedling (B), a 45°-inclined seedling (C), a 60°-inclined seedling (D) and a 90°-inclined seedling (E) of Acacia mangium 3 months after the start of inclination. Asterisks indicate the presence of gelatinous fibers in the upper regions of the inclined stems. Scale bar = 300 µm. Fig. 7. View largeDownload slide Light micrographs of cross sections, stained with safranine and Astra blue, of a control seedling (0°; A), a 30°-inclined seedling (B), a 45°-inclined seedling (C), a 60°-inclined seedling (D) and a 90°-inclined seedling (E) of Acacia mangium 3 months after the start of inclination. Asterisks indicate the presence of gelatinous fibers in the upper regions of the inclined stems. Scale bar = 300 µm. Fig. 8. View largeDownload slide Quantitative analysis of the width of tension wood regions in the upper parts of stems of Acacia mangium seedlings subjected to different angles of inclination. The widths of tension wood regions differed significantly among 30°-, 45°-, 60°- and 90°-inclined seedlings (P = 0.022). Error bars indicate standard errors (n = 3). Different letters above bars indicate significant differences, as determined by Tukey’s post hoc test, at P < 0.05. Fig. 8. View largeDownload slide Quantitative analysis of the width of tension wood regions in the upper parts of stems of Acacia mangium seedlings subjected to different angles of inclination. The widths of tension wood regions differed significantly among 30°-, 45°-, 60°- and 90°-inclined seedlings (P = 0.022). Error bars indicate standard errors (n = 3). Different letters above bars indicate significant differences, as determined by Tukey’s post hoc test, at P < 0.05. Thickness of gelatinous layers Light microscopy of 15-µm-thick transverse sections revealed gelatinous fibres with an inner gelatinous layer in the tension wood regions of 30°-, 45°-, 60°- and 90°-inclined seedlings (Fig. 9). Gelatinous layers were stained blue by the combination of safranine and Astra blue. The thickness of the gelatinous layers differed significantly among 30°-, 45°-, 60°- and 90°-inclined seedlings (P < 0.0001). The mean (± s.e.) thickness of gelatinous layers was approx. 2.3 ± 0.1 µm in 30°-inclined seedlings, 2.3 ± 0.1 µm in 45°-inclined seedlings, 2.5 ± 0.1 µm in 60°-inclined seedlings and 3.8 ± 0.1 µm in 90°-inclined seedlings. The 90°-inclined seedlings formed thicker gelatinous layers (Figs 9 and 10) than those in the 30°-, 45°- and 60°-inclined seedlings. Fig. 9. View largeDownload slide Light micrographs of transverse sections of the tension wood of inclined stems, 3 months after the start of inclination, after staining with safranine and Astra blue: (A) 30°-inclined seedling, (B) 45°-inclined seedling, (C) 60°-inclined seedling and (D) 90°-inclined seedling. The 90°-inclined seedling had thicker gelatinous layers. Blue coloration indicates gelatinous layers. Scale bar = 30 µm. Fig. 9. View largeDownload slide Light micrographs of transverse sections of the tension wood of inclined stems, 3 months after the start of inclination, after staining with safranine and Astra blue: (A) 30°-inclined seedling, (B) 45°-inclined seedling, (C) 60°-inclined seedling and (D) 90°-inclined seedling. The 90°-inclined seedling had thicker gelatinous layers. Blue coloration indicates gelatinous layers. Scale bar = 30 µm. Fig. 10. View largeDownload slide Quantitative analysis of the thickness of gelatinous layers in the upper portions of 30°-, 45°-, 60°- and 90°-inclined seedlings of Acacia mangium. The gelatinous layers in the tension wood of 90°-inclined seedlings were significantly thicker than in 30°-, 45°- and 60°-inclined seedlings. Error bars indicate standard errors (n = 3). Different letters above bars indicate significant differences, as determined by Tukey’s post hoc test, at P < 0.05. Fig. 10. View largeDownload slide Quantitative analysis of the thickness of gelatinous layers in the upper portions of 30°-, 45°-, 60°- and 90°-inclined seedlings of Acacia mangium. The gelatinous layers in the tension wood of 90°-inclined seedlings were significantly thicker than in 30°-, 45°- and 60°-inclined seedlings. Error bars indicate standard errors (n = 3). Different letters above bars indicate significant differences, as determined by Tukey’s post hoc test, at P < 0.05. DISCUSSION Woody plants respond to environmental forces and to gravitational stimuli via the development of reaction wood that reorientates the axis, trunk and branches, allowing them to recover the gravitropic set-point angle, as defined by Digby and Firn (1995). Reorientation of seedlings of woody plants in response to a gravitational stimulus is closely related to the formation of tension wood. Tension wood generates a strong tensile force and contributes to the negative gravitropic movement of inclined woody stems (Okuyama et al., 1994; Yoshida et al., 1999, 2000; Yamamoto et al., 2002; Clair et al., 2006; Ruelle et al., 2006; Fang et al., 2008; Mellerowicz and Gorshkova, 2012; Gril et al., 2017). Our previous study of A. mangium seedlings revealed that the width of the region of gelatinous fibres and the thickness of the gelatinous layers were strongly correlated with the negative gravitropism of inclined seedlings (Nugroho et al., 2012, 2013). In the present study, we found that inclined A. mangium seedlings formed tension wood on the upper part of each inclined stem and returned to the vertical after inclination at four different angles. A smaller inclination angle, such as 30°, was associated with an earlier return of stems to the vertical and a larger angle, such as 90°, was associated with a later return to the vertical. The 30°-, 45°- and 60°-inclined seedlings had almost completely returned to the vertical 3 weeks after day zero but 90°-inclined seedlings returned to the vertical 4°weeks after day zero. In terms of the speed of negative-gravitropic movement towards the vertical position, the seedlings subjected to larger degrees of inclination moved more rapidly towards the vertical. The 30°-inclined seedlings, which moved with the lowest negative-gravitropic speed, formed the narrowest region of tension wood. However, there were no significant differences among seedlings inclined at 45°, 60° and 90° with respect to the width of the tension wood. Therefore, the speed of negative-gravitropic movement of A. mangium seedlings might be directly related to the amount of newly developed tension wood. Mathew (2003) reported that the widest region of tension wood was formed at a displacement of 90° from the vertical. Moreover, Reghu (1983) reported that, in 1-year-old seedlings of Azadirachta indica, the width of newly induced tension wood was greatest at an inclination angle of 45° and the proportion of tension wood decreased as the degree of inclination was increased further. Hiraiwa et al. (2013, 2014) found the widest regions of tension wood in 50°-inclined stems of Trochodendron araliodes and Liliodendron tulipifera. Thus, the relative amount of newly developed tension wood appears to depend on the angle of inclination and species. The extent of formation of tension wood might reach a plateau value as the angle of inclination is increased from 45° to 90°. Microscopic observations revealed that, among inclined stems, the thickest gelatinous layer was formed in the tension wood regions of 90°-inclined seedlings. The gelatinous layers of 90°-inclined seedlings were significantly thicker than those of 30°-, 45°- and 60°-inclined seedlings. Although there were no significant differences in terms of the widths of tension wood regions among 90°-inclined seedlings and the other inclined seedlings, the 90°-inclined seedlings were associated with the highest negative-gravitropic speed. Therefore, thicker gelatinous layers might generate greater tensile stress for higher speeds of stem recovery movement in A. mangium seedlings. Our results support the hypothesis, proposed by Fang et al. (2008), that thicker gelatinous layers are accompanied by greater longitudinal growth stress and play a critical role in the generation of strong growth stress in poplar. The formation of fibres with thick gelatinous layers induces strong tension stress via lateral swelling of the gelatinous layer, which forces the surrounding secondary cell walls to contract in the axial direction (Goswami et al., 2008; Mellerowicz and Gorshkova, 2012; Gril et al., 2017). Our results suggest that the development of gelatinous layers is important in the generation of the tensile force required for the negative-gravitropic movement of the stems of seedlings. Eccentricity of growth is induced by displacement of the stem from the normal or vertical direction. The extent of eccentric growth induced by leaning of stems is related to the angle of inclination from the vertical (Burns, 1942; Robards, 1965, 1966). In the present study, control seedlings (0°) exhibited concentric growth but seedlings inclined at 30°, 45°, 60° and 90° showed evidence of eccentric growth. A gravitational stimulus promotes formation of tension wood on the upper part of inclined stems, an indication of higher cambial-division activity in the upper part of the inclined stem than in the lower part of the inclined stem. Patel et al. (1984) reported that, in Kigelia pinnata, growth eccentricity decreased with increases in the angle of inclination. We found that increasing the inclination of A. mangium seedlings from 30° to 90° did not increase growth eccentricity. In addition, we found that there was no correlation between the width of the tension wood region and growth eccentricity (P = 0.099). Similarly, there was no correlation between the width of the tension wood and growth eccentricity in Hevea brasiliensis (Mathew, 2003). Therefore, the extent of growth eccentricity appears not to reflect the extent of the newly developed tension wood. Plant hormones are involved both in the formation of tension wood and in gravitropism (Little and Pharis, 1995; Mellerowicz et al., 2001; Pilate et al., 2004; Kwon, 2008). In particular, gibberellin plays an important role in the formation of tension wood (Nakamura et al., 1994; Baba et al., 1995; Yoshida et al., 1999; Du and Yamamoto, 2007; Funada et al., 2008; Wang et al., 2017). In inclined seedlings of A. mangium, Nugroho et al. (2012, 2013) found that gibberellin is essential for the formation of tension wood, for development of gelatinous layers in the tension wood and for gravitropism. The application of gibberellic acid (GA3) to the soil of seedlings of A. mangium significantly stimulated the return to the vertical of inclined stems by increasing the extent of formation of tension wood. Moreover, inhibitors of gibberellin biosynthesis, such as uniconazole-P and paclobutrazole, inhibited the upward bending of inclined seedlings via suppression of increases in the width of the tension wood and the development of gelatinous layers. Our present results revealed that the angle of inclination of stems affects the speed of negative-gravitropic movement via the width of the newly developed tension wood and the thickness of gelatinous layers. It is plausible that inclined A. mangium seedlings might recognize and respond to a gravitational stimulus via changes in the rate of biosynthesis of gibberellin, which regulates the development of tension wood, the thickness of gelatinous layers and, as a consequence, the speed of stem-recovery movement. In summary, the angle of inclination of the seedling affected the width of the newly developed tension wood region, the thickness of gelatinous layers and the speed of negative-gravitropic movement. Larger amounts of newly developed gelatinous fibres and thicker gelatinous layers might generate the higher tensile stress that leads to higher speeds of stem-recovery movement in A. mangium seedlings. ACKNOWLEDGMENTS W.D.H. thanks the Hitachi Scholarship Foundation, Japan, and the Japan Student Services Organization (JASSO) for supporting this work. The authors also thank Alan Cabout, Adityo Roem and Jito, Universitas Gadjah Mada, for their assistance in the preparation of seedlings of A. mangium and the collection of samples. 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, 24380090, 15K07508, 15H04527, 16K14954 and 18H02251). LITERATURE CITED Alméras T, Derycke M, Jaouen G, Beauchene J, Fournier M. 2009. Functional diversity in gravitropic reaction among tropical seedlings in relation to ecological and developmental traits. Journal of Experimental Botany  60: 4397– 4410. 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Published: May 3, 2018

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