Gibberellin is required for the formation of tension wood and stem gravitropism in Acacia mangium seedlings

Annals of Botany 110: 887 –895, 2012 doi:10.1093/aob/mcs148, available online at www.aob.oxfordjournals.org PART OF A HIGHLIGHT ON TREE BIOLOGY Gibb...
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Annals of Botany 110: 887 –895, 2012 doi:10.1093/aob/mcs148, available online at www.aob.oxfordjournals.org

PART OF A HIGHLIGHT ON TREE BIOLOGY

Gibberellin is required for the formation of tension wood and stem gravitropism in Acacia mangium seedlings Widyanto Dwi Nugroho1,2, Yusuke Yamagishi1, Satoshi Nakaba1, Shiori Fukuhara1, Shahanara Begum1,3, Sri Nugroho Marsoem2, Jae-Heung Ko4, Hyun-O Jin4 and Ryo Funada1,4,* 1

Tokyo University of Agriculture and Technology, Fuchu-Tokyo 183-8509, Japan, 2Faculty of Forestry, Universitas Gadjah Mada, Jalan Agro No. 1 Bulaksumur, Yogyakarta, 55281, Indonesia, 3Faculty of Agriculture, Bangladesh Agricultural University, Mymensingh-2202, Bangladesh and 4College of Life Science, Kyung Hee University, Yongin 446-701, Korea * For correspondence. E-mail [email protected] Received: 16 February 2012 Returned for revision: 4 April 2012 Accepted: 1 May 2012 Published electronically: 26 July 2012

† Background and Aims Angiosperm trees generally form tension wood on the upper sides of leaning stems. The formation of tension wood is an important response to gravitational stimulus. Gibberellin appears to be involved in the differentiation of secondary xylem, but it remains unclear whether gibberellin plays a key role in the formation of tension wood and plant gravitropism. Therefore, a study was designed to investigate the effects of gibberellin and of inhibitors of the synthesis of gibberellin, namely paclobutrazole and uniconazole-P, on the formation of tension wood and negative stem gravitropism in Acacia mangium seedlings. † Methods Gibberellic acid (GA3), paclobutrazole and uniconazole-P were applied to seedlings via the soil in which they were growing. Distilled water was applied similarly as a control. Three days after such treatment, seedlings were tilted at an angle of 458 from the vertical, and samples of stems were collected for analysis 2 weeks, 2 months and 6 months after tilting. The effects of treatments on the stem recovery degree (Rº) were analysed as an index of the negative gravitropism of seedlings, together the width of the region of tension wood in the upper part of inclined stems. † Key Results It was found that GA3 stimulated the negative gravitropism of tilted seedling stems of A. mangium, while paclobutrazole and uniconazole-P inhibited recovery to vertical growth. Moreover, GA3 stimulated the formation of tension wood in tilted A. mangium seedlings, while paclobutrazole and uniconazole-P strongly suppressed the formation of tension wood, as assessed 2 weeks after tilting. † Conclusions The results suggest that gibberellin plays an important role at the initial stages of formation of tension wood and in stem gravitropism in A. mangium seedlings in response to a gravitational stimulus. Key words: Acacia mangium, formation of tension wood, gibberellin, gravitropism, inhibitor of gibberellin biosynthesis.

IN T RO DU C T IO N Environmental forces, such as wind, landslide, flooding, snow and volcanic eruption, often change the position of the stem of woody plants. Such plants are able to respond to these environmental forces and to gravitational stimuli via the development of so-called reaction wood that reorients the axis, trunk and branches to the recovery of the gravitropic set-point angle (GSA) as defined by Digby and Firn (1995). Angiosperm trees generally form tension wood, a special type of secondary xylem, on the upper sides of leaning stems, and the tension wood generates a strong tensile force along the grain of the upper side of the living stem that can pull even a large inclined stem back to the vertical or near-vertical position (Okuyama et al., 1994; Yoshida et al., 1999, 2000; Yamamoto et al., 2002; Clair et al., 2006, 2010; Ruelle et al., 2006; Coutand et al., 2007). Tension wood is generally characterized by the presence of gelatinous fibres, with a thick inner gelatinous layer, that can fill almost the entire fiber lumen with high levels of cellulose and low levels of lignin (Timell, 1969; Dejardin et al., 2010). Cellulose microfibrils in the gelatinous layer are oriented

parallel or nearly parallel to the longitudinal axis of the wood fibres (Prodhan et al., 1995a, b; Ruelle et al., 2007). Plant hormones play an important role in controlling cambial activity and the differentiation of cambial derivatives in woody plants (Aloni et al., 2000; Sundberg et al., 2000; Dayan et al., 2012). Furthermore, these hormones are known to be involved in the formation of tension wood and in gravitropism (Little and Pharis, 1995; Mellerowicz et al., 2001; Pilate et al., 2004; Kwon, 2008). Auxin and ethylene have been suggested to be important factors in the regulation of the formation of tension wood (Cronshaw and Morey, 1968; Morey and Cronshaw, 1968; Andersson-Gunnera˚s et al., 2003; Du and Yamamoto, 2003, 2007; Du et al., 2004), and there is evidence to suggest that auxin- and ethylene-related genes are differentially expressed during the formation of tension wood (Moyle et al., 2002; Andersson-Gunnera˚s et al., 2006; Jin et al., 2011). However, it has also been noted that there is no clear relationship between levels of endogenous auxin in the cambial region and the formation of tension wood in Populus tremula (Hellgren et al., 2004). Not only auxin and ethylene but also another plant hormone, gibberellin, appears to be involved in the formation of tension

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wood. For example, gibberellin promotes the formation of tension wood on the upper sides of branches and inhibits the bending of branches in a weeping type of Prunus spachiana (Nakamura et al., 1994; Baba et al., 1995; Yoshida et al., 1999). In addition, the application of gibberellin to vertical stems of several species of angiosperm trees induces the formation of tension wood in the absence of a gravitational stimulus (Funada et al., 2008). In contrast, in Fraxinus mandshurica var. japonica and in Acer rubrum, the differentiation of gelatinous fibres of tension wood does not appear to be associated with the actions of gibberellin (Cronshaw and Morey, 1968; Morey and Cronshaw, 1968; Jiang et al., 2006). Therefore, it remains unclear whether and under what conditions gibberellin plays a key role in the formation of tension wood. Studies of the roles of plant hormones are facilitated by the availability of their antagonists. Paclobutrazole and uniconazole-P, for example, are plant growth retardants that inhibit the biosynthesis of gibberellin (Rademancher, 1992). Application of uniconazole-P reduces the extent of newly formed xylem and the relative proportion of tension wood in tilted seedlings of Aesculus turbinata (Du et al., 2004). In contrast, uniconazole-P inhibits the upward bending of seedlings of F. mandshurica var. japonica but does not inhibit the formation of tension wood (Jiang et al., 1998a, b, 2008). More information about the effects of inhibitors of gibberellin biosynthesis on gravitropism and the formation of tension wood is needed for a better understanding of the role of gibberellin in these phenomena. We designed this study to investigate the effects of gibberellin and of inhibitors of gibberellin biosynthesis on gravitropism and the formation of tension wood in seedlings of Acacia mangium, which is an important species of tropical trees in industrial forest plantations (Nugroho et al., 2012). We applied gibberellin and two inhibitors of gibberellin biosynthesis, namely paclobutrazole and uniconazole-P, to the soil in which A. mangium seedlings were growing and then tilted the seedlings and grew them at an angle of 458 to the vertical. Our observations suggest that gibberellin might play an important role at the initial stages of the formation of tension wood and in plant gravitropism in response to a gravitational stimulus.

aliquot of a solution of GA3 (0.01 %, w/w), of paclobutrazol (1 %, w/w) or of uniconazole-P (0.01 %, w/w) was applied to the soil around each seedling. For controls, 50 mL of distilled water were applied to the soil instead of the hormone and inhibitors. Three days after such treatment, pots were tilted to approx. 458 from the vertical direction (Fig. 1). The soil in each pot was moistened with approx. 200 mL of water daily. Five seedlings per treatment were harvested 2 weeks, 2 months and 6 months after treatments for analysis. Definition and determination of stem recovery degree (Rº)

Photographs of seedlings were taken every 3 d from the day of tilting (0 day) for 6 months. The photographs were analysed with the image-analysis software Image-J (National Institutes of Health, MD, USA). The stem Rº was defined as the difference between the initial inclined angle of the seedling (a8) and the recovered angle of the seedling (b8), as shown in Fig. 1. In this study, the angle of the seedling was defined as the angle between the horizontal and a straight line from the base to the apex of the seedling. The stem Rº values were used as an index of the negative gravitropism of seedlings. Analysis of tension wood

Segments of stems of 10 mm, 10 cm above the ground, were removed from seedlings for analysis. Segments were fixed in 4 % glutaraldehyde in 0.1 M phosphate buffer ( pH 7.3). Transverse sections of 15 mm in thickness were cut from the segments on a sliding microtome (Yamatokohki, Saitama, Japan). Sections were stained with a 0.1 % solution of safranin (WAKO Pure Chemical Industries) for 3 min and subsequently Position B R°

Position A

M AT E R IA L S A ND M E T HO DS Plant materials

Sixty approx. 1-year-old healthy seedlings of Acacia mangium grown from seeds with uniform features were used in this experiment. The seedlings were approx. 70 cm tall and were planted in 20 cm diameter pots filled with regosol soil in a greenhouse at the nursery of the Faculty of Forestry, Universitas Gadjah Mada, Yogyakarta, Indonesia.

b° a°

Horizontal axis (0°)

45°

Treatment of seedlings

Gibberellic acid (GA3; WAKO Pure Chemical Industries, Osaka, Japan) and two inhibitors of gibberellin biosynthesis, namely paclobutrazole (Bountyw Flowable, Syngenta Japan, Tokyo) and uniconazole-P (Sumisevenw P, Sumitomo Chemical Corporation, Tokyo), were used for treatments. A 50 mL

F I G . 1. Determination of stem recovery degree (Rº). The stem Rº was determined by measuring the difference between the initial inclination of the seedling (a8) and the angle subtended by the seedling to the horizontal when measurements were made (b8). Position A is the initial position of the inclined seedling, and position B is the position of the seedling when measurements were made.

Nugroho et al. — Gibberellins in tension wood formation and stem gravitropism with a 1 % solution of Astra blue (Sigma-Aldrich, Steinheim, Germany) for 5 min, dehydrated with a graded ethanol series, mounted on glass slides, fixed in resin (Entellan new; Merck, Darmstadt, Germany) and covered with cover slips. Images were recorded under a light microscope (Axioscop; Carl Zeiss, Oberkochen, Germany) with a digital camera (Nikon digital Sight DS-5M-L1; Nikon Corporation, Japan; Begum et al., 2007, 2010). Tension wood is generally characterized by the presence of gelatinous fibres, with a thick inner gelatinous layer, and a high level of cellulose and low level of lignin. Astra blue is a specific stain for unlignified walls and stains purely cellulosic layers blue (Jourez et al., 2001). We refer to the inner layers of cell walls that were stained blue by the combination of safranin and Astra blue as gelatinous layers. 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 inclined stems. The width of the region of tension wood was determined by measuring the width of the region of wood fibres with blue-stained inner layers of cell walls. Statistical analysis

Data were analysed with the statistical software Prism5 for Mac OS X (GraphPad Software Inc., USA). Effects of treatments on stem Rº and the formation of tension wood were analysed 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. Linear regression analysis was performed to examine the relationship between the width of the region of tension wood and the stem Rº. R E S U LT S Negative gravitropism of tilted seedlings

Typical responses of the stems of A. mangium seedlings to tilting of pots are shown in Fig. 2. Figure 3 shows the changes in stem Rº for control, GA3-treated, paclobutrazoletreated and uniconazole-P-treated seedlings from day 0 to 2 months after tilting of pots. The stem of control seedlings and of GA3-treated seedlings returned to the vertical position and GA3-treated seedlings recovered more rapidly than control seedlings. In contrast, the stems of paclobutrazoletreated and uniconazole-P-treated seedlings failed to alter their orientation and remained inclined at close to 458 to the vertical. Two weeks after tilting of pots, the mean values (+ s.e.) of stem Rº of control seedlings and GA3-, paclobutrazole- and uniconazole-P-treated seedlings were approx. 15 + 1, 24 + 2, 1 + 1 and 0 + 18, respectively (Fig. 4). Values of stem Rº differed significantly among treatments (P , 0.001). GA3-treated seedlings had higher values of stem Rº than the control seedlings, indicating that GA3 had stimulated the return of stems to a vertical orientation. In contrast, stem Rº for paclobutrazole- and uniconazole-P-treated seedlings was close to zero. The mean values (+ s.e.) of stem Rº for control seedlings and for GA3-, paclobutrazole- and uniconazole-P-treated

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seedlings were approx. 33 + 2, 36 + 2, 3 + 2 and 2 + 28 at 2 months after pots were tilted, and 47 + 4, 41 + 4, 7 + 2 and – 8 + 38 at 6 months after pots were tilted, respectively (Fig. 4). Values of Rº differed significantly among treatments (P , 0.001). Clearly, application of paclobutrazole and of uniconazole-P inhibited plant gravitropism in inclined seedlings of A. mangium over the course of 6 months, even though seedlings had only been treated once. Formation of tension wood

Microscopic investigation of transverse sections of stems, 10 cm above ground level, revealed that gelatinous fibres that were stained blue by Astra blue had formed in the upper region of leaning stems of control and GA3-treated seedlings 2 weeks after tilting of pots (Figs 5 and 6). In contrast, no or only a few gelatinous fibres formed in the upper region of inclined stems of paclobutrazole- and uniconazole-P-treated seedlings. No gelatinous fibres were found in the lower regions of the inclined stems of all seedlings examined. Two weeks after tilting of pots, the widths of regions of tension wood in the upper portion of inclined stems differed significantly among treatments (P , 0.001; Fig. 7). The widths of tension wood regions (+ s.e.) were approx. 0.25 + 0.03 mm in control seedlings and 0.45 + 0.03 mm in GA3-treated seedlings, respectively. The GA3-treated seedlings had significantly wider regions of tension wood than the control seedlings. In contrast, paclobutrazole- and uniconazoleP-treated seedlings formed no tension wood or only a very narrow region of tension wood (Fig. 7). Gelatinous fibres were found in the upper portions of all inclined stems at 2 and 6 months after inclination (Fig. 7). However, the widths of regions of tension wood differed significantly among treatments (P , 0.001) both 2 and 6 months after tilting of pots. GA3-treated seedlings had formed the widest regions of tension wood at both 2 and 6 months after tilting of pots. The mean values (+ s.e.) of widths of regions of tension wood at 2 and 6 months after inclination were 0.48 + 0.04 and 0.82 + 0.05 mm in control seedlings, and 0.61 + 0.08 and 0.87 + 0.04 mm in GA3-treated seedlings, respectively. In contrast, narrower regions of tension wood were formed on the upper sides of stems of paclobutrazoleand uniconazole-P-treated seedlings than on that of control seedlings. The mean values (+ s.e.) of widths of regions of tension wood at 3 and 6 months after inclination of paclobutrazole-treated seedlings were 0 .13 + 0 .03 and 0.24 + 0.06 mm, and those of uniconazole-P-treated seedlings were 0.18 + 0.0 and 0.34 + 0.03 mm, respectively. Figure 8 shows the relationships between the widths of regions of tension wood on the upper sides of the stem of inclined A. mangium seedlings and stem Rº. There was a strong correlation between the widths of regions of tension wood and the stem Rº values for all samples at all time points (P , 0.0001). DISCUSSION Several studies of the effects of exogenous gibberellin have indicated that gibberellin plays an important role in the gravitropism of woody plants. Treatment with gibberellin prevented

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Nugroho et al. — Gibberellins in tension wood formation and stem gravitropism Day zero

2 weeks

2 months

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Uniconazole-P

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F I G . 2. Photographs showing the typical positions of stems of control seedlings (A, B, C), GA3-treated seedlings (D, E, F), paclobutrazole-treated seedlings (G, H, I) and uniconazole-P-treated seedlings (J, K, L) of Acacia mangium initially and then 2 weeks and 2 months after pots were tilted. Control seedlings and GA3-treated seedlings returned to the upright position, but stems of paclobutrazole- and uniconazole-P-treated seedlings did not return to the vertical direction. Scale bar in (A) ¼ 20 cm.

the bending of branches of the weeping type of P. spachiana (Nakamura et al., 1994; Baba et al., 1995; Yoshida et al., 1999). Furthermore, negative gravitropism was enhanced after the application of gibberellin to horizontally positioned seedlings of F. mandshurica var. japonica (Jiang et al., 1998a, b, 2006). Our results support the hypothesis that gibberellin plays an important role in gravitropism of woody plants. A single application of GA3, via the soil, to seedlings of A. mangium significantly stimulated the return to the vertical of stems in tilted pots. Two weeks after tilting, stem Rº of GA3-treated seedlings were significantly greater than those of control seedlings. Thus, a single application of GA3 stimulated the return to a vertical orientation in response to a gravitational

stimulus. However, 2 and 6 months after the start of tilting of pots, stem Rº of GA3-treated seedlings were not significantly different from those of control seedlings and the stems of both control and GA3-treated seedlings had reached the equilibrium position. The application of inhibitors of gibberellin biosynthesis, namely paclobutrazole and uniconazole-P, to the soil inhibited the negative gravitropism of stems of A. mangium seedlings. Similarly, in an earlier study, the application of uniconazole-P inhibited the upward bending of horizontal seedlings of F. mandshurica var. japonica (Jiang et al., 1998a, b, 2008). Tension wood is usually characterized by the presence of gelatinous fibres, but some trees species, such as yellow

Nugroho et al. — Gibberellins in tension wood formation and stem gravitropism Control GA3 Paclobutrazole Uniconazole-P

40 Stem recovery degree (R°)

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30 20 10 0

0 3

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9 12 15 18 21 24 27 30 33 36 39 42 45 48 51 54 57 60 Days after pots were tilted

F I G . 3. The time courses, in term of average stem recovery degree (R8), of changes in orientation of artificially inclined Acacia mangium seedlings. Control seedlings and GA3-treated seedlings returned to the vertical orientation, but the paclobutrazole- and uniconazole-P-treated seedlings did not. GA3-treated seedlings recovered more rapidly than the control seedlings. Error bars indicate standard errors (n ¼ 15 from 0 to 15 d; n ¼ 10 from 18 to 60 d).

Stem recovery degree (R°)

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a a

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b b c

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F I G . 4. Stem recovery degree (R8) of control and GA3-, paclobutrazole- and uniconazole-P-treated Acacia mangium seedlings 2 weeks, 2 months and 6 months after pots were tilted. Error bars indicate standard errors (n ¼ 15 for 2 weeks; n ¼ 10 for 2 months; n ¼ 5 for 6 months after inclination). The stem R8 value 2 weeks, 2 months and 6 months after inclination differed significantly among treatments (***, significantly different at P , 0.001). At each time point, different letters above the bars indicate significant differences, as determined by Tukey’s post-hoc test at P , 0.05.

poplar and several species of tropical trees, do not form such a typical gelatinous layer (Clair et al., 2006; Mokugawa et al., 2008; Jin and Kwon, 2009). In the present study, the tension wood of A. mangium was characterized by the presence of gelatinous fibres that were stained blue by a solution of safranin and Astra blue. Our results demonstrate that a single application of GA3 to the soil of artificially inclined seedlings of A. mangium stimulated the formation of tension wood in the upper regions of leaning stems. Two weeks after tilting of pots, the widths of regions of tension wood in GA3-treated seedlings were significantly greater than those of the control tilted seedlings. In contrast, Jiang et al. (1998a, b, 2006, 2008) reported that gibberellin did not affect the formation of gelatinous fibres in F. mandshurica var. japonica. Cronshaw and Morey

(1968) also reported that treatment with gibberellin did not alter the normal pattern of formation of tension wood in A. rubrum. However, stimulation of the formation of tension wood was detected on the upper side of gibberellin-treated branches of a weeping type of P. spachiana (Baba et al., 1995; Yoshida et al., 1999). Moreover, the application of gibberellin to vertical stems of several species of angiosperm trees was found to induce the formation of tension wood in the absence of any abnormal gravitational stimulus (Funada et al., 2008). In the present study of A. mangium, we found that gibberellin stimulated the formation of tension wood. The effect of inhibitors of gibberellin biosynthesis on the formation of tension wood remains controversial because inconsistent results have been reported. In the case of seedlings of F. mandshurica var. japonica, the application of

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Nugroho et al. — Gibberellins in tension wood formation and stem gravitropism A

B

C

D

F I G . 5. Light micrographs of cross-sections of inclined stems, stained with safranin and Astra blue, of a control seedling (A), a GA3-treated seedling (B), a paclobutrazole-treated seedling (C) and a uniconazole-P-treated seedling (D) of Acacia mangium 2 weeks after pots were inclined. Asterisks indicate the presence of gelatinous fibres in the upper region of the inclined stems of control and GA3-treated seedlings. These fibres were coloured blue after staining with safranin and Astra blue. Scale bar ¼ 1 mm.

uniconazole-P at various concentrations inhibited the upward bending of seedlings but did not inhibit the formation of tension wood (Jiang et al., 1998a, b, 2008). In contrast, application of uniconazole-P reduced the extent of formation of tension wood in tilted seedlings of A. turbinata (Du et al., 2004). Our present results showed clearly that the application of paclobutrazole and uniconazole-P to the soil strongly suppressed the formation of tension wood in artificially inclined seedlings of A. mangium. Therefore, although we did not measure levels of endogenous gibberellin in cambial cells and differentiating xylem cells, we can conclude that a certain amount of gibberellin is needed to induce the formation of tension wood and that gibberellin plays an important role at the initial stages of formation of tension wood. Tension wood generates a strong tensile force, which is sufficient to bend a leaning shoot upward, and it is, thus,

responsible for 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). Alme´ras et al. (2009) reported that, in tropical trees, the response of stem movement to gravity is achieved via the formation of tension wood fibres. Tensile stress is generated through the lateral swelling of the gelatinous layer, which forces surrounding secondary cell walls to contract in the axial direction (Goswami et al., 2008). In the present study, we found that the width of tension wood was strongly correlated with stem R8 (Fig. 8). Thus, in our system, negative gravitropism was closely related to the formation of tension wood. Although small amounts of tension wood were formed on the upper sides of stems at 2 and 6 months after the treatment with paclobutrazole and uniconazole-P, no negative gravitropism of the inclined

Nugroho et al. — Gibberellins in tension wood formation and stem gravitropism A

B

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Width of tension wood region (mm)

F I G . 6. Light micrographs of transverse sections of the upper regions of inclined stems, after staining with safranin and Astra blue, of a control seedling (A), a GA3-treated seedling (B), a paclobutrazole-treated seedling (C) and a uniconazole-P-treated seedling (D) of Acacia mangium 2 weeks after pots were tilted. Gelatinous fibres were formed in the upper region of the inclined stems of control and GA3-treated seedlings. Blue coloration indicates the presence of gelatinous layers. Scale bars ¼ 50 mm.

1·0

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a

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F I G . 7. The widths of regions of tension wood in the upper portions of stem of control, GA3-, paclobutrazole- and uniconazole-P-treated Acacia mangium seedlings, 2 weeks, 2 months and 6 months after pots were tilted, differed significantly among treatments (***, significantly different at P , 0.001). Error bars indicate standard errors (n ¼ 5). At each time point, different letters above the bars indicate significant differences, as determined by Tukey’s post-hoc test at P , 0.05.

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Stem recovery degree (°)

A

2 weeks after inclination y = 43·4x – 2·6 R 2 = 0·62 P < 0·0001

60 40

Control GA3 Paclobutrazole Uniconazole-P

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United Graduate School of Agricultural Science at Tokyo University of Agriculture and Technology. The authors also thank Alan Cabout and Adityo Roem, Universitas Gadjah Mada, for their assistance in the collection of samples. We are very grateful to the referees and editor for their constructive comments and suggestions. 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 19580183, 20120009, 21380107, 23380105 and 24380090).

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F I G . 8. Relationships between widths of regions of tension wood in the upper portions of stems of inclined seedlings of Acacia mangium and stem recovery degree (R8) 2 weeks after inclination (A), 2 months after inclination (B) and 6 months after inclination (C). The three straight lines show that the widths of regions of tension wood were positively correlated with stem R8. We examined 20 seedlings at each time point, and each symbol represents a single set of measurements from a single seedling.

stems of A. mangium seedlings occurred. Formation of tension wood very soon after inclination might be important for the development of tensile forces that are strong enough to allow the stem to return to a vertical orientation. The application of exogenous gibberellin to artificially inclined seedlings of A. mangium promoted the formation of tension wood and negative gravitropic movement during the early response to a gravitational stimulus, whereas the application of inhibitors of gibberellin biosynthesis induced the reverse responses. Thus, we can conclude that gibberellin plays an important role at the initial stages of formation of tension wood and in negative gravitropism in A. mangium seedlings in response to a gravitational stimulus. AC KN OW LED GEMEN T S W.D.N. thanks the Hitachi Scholarship Foundation, Japan, for supporting and funding his doctoral degree programme at the

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