Tension Wood Formation Research Articles

EST analysis of functional genes associated with cell wall biosynthesis and modification in the secondary xylem of the yellow poplar (Liriodendron tulipifera) stem during early stage of tension wood formation

A cDNA library was constructed from secondary xylem in the stem of a 2-year-old yellow poplar after being bent for 6h with a 45° configuration to isolate genes related to cell wall modification during the early stages of tension wood formation. A total of 6,141 ESTs were sequenced to generate a database of 5,982 high-quality expressed sequence tags (ESTs). These sequences were clustered into 1,733 unigenes, including 822 contigs and 911 singletons. Homologs of the genes regulate many aspects of secondary xylem development, including those for primary and secondary metabolism, plant growth hormones, transcription factors, cell wall biosynthesis and modification, and stress responses. Although there were only 1,733 annotated ESTs (28.9%), the annotated ESTs obtained in this study provided sequences for a broad array of transcripts expressed in the stem upon mechanical bending, and the majority of them were the first representatives of their respective gene families in Liriodendron tulipifera. In the case of lignin, xylem-specific COMTs were identified and their expressions were significantly downregulated in the tension wood-forming tissues. Additionally, the majority of the auxin- and BR-related genes were downregulated significantly in response to mechanical bending treatment. Despite the small number of ESTs sequenced in this study, many genes that are relevant to cell wall biosynthesis and modification have been isolated. Expression analysis of selected genes allow us to identify the regulatory genes that may perform essential functions during the early stages of tension wood formation and associated cell wall modification.

Chemical modification of secondary xylem under tensile stress in the stem of Liriodendron tulipifera

Tension wood is a specialized tissue that develops in the upper side of leaning stem and branches in angiosperm. In yellow poplar, tension wood does not form a G-layer, which is one of the most characteristic features of typical tension wood. In order to determine whether the chemical modification associated with tension wood formation in yellow poplar is consistent with those of G-layer-forming angiosperm species, tension wood was induced via mechanical bending treatment for 7 and 14 days in the stem of 2-year-old yellow poplars, and its major cell wall components were analyzed. Whereas the cellulose contents of the tension wood were higher than those in opposite wood, its lignin contents were lower; the result is consistent with that seen in other angiosperm tension wood. Interestingly, the lignin contents differed between tension wood and opposite wood sides at 7 days in the bent sample, but became similar levels to each other at later stages of tension wood formation. The S/G ratio of tension wood was also higher than that of opposite wood. Additionally, the composition of hemicelluloses (glucomannan and xylan) was altered significantly under tensile stress conditions in the yellow poplar stem. Therefore, the majority of the cell wall polymers were altered significantly in the tension wood relative to those in opposite wood, and these changes appear to be developmentally regulated, regardless of the existence of the G-layer in the fiber.

Investigation of tension wood formation and 2,6-dichlorbenzonitrile application in short rotation coppice willow composition and enzymatic saccharification.

BackgroundShort rotation coppice willow is a potential lignocellulosic feedstock in the United Kingdom and elsewhere; however, research on optimising willow specifically for bioethanol production has started developing only recently. We have used the feedstock Salix viminalis × Salix schwerinii cultivar 'Olof' in a three-month pot experiment with the aim of modifying cell wall composition and structure within the stem to the benefit of bioethanol production. Trees were treated for 26 or 43 days with tension wood induction and/or with an application of the cellulose synthesis inhibitor 2,6-dichlorobenzonitrile that is specific to secondary cell walls. Reaction wood (tension and opposite wood) was isolated from material that had received the 43-day tension wood induction treatment.ResultsGlucan content, lignin content and enzymatically released glucose were assayed. All measured parameters were altered without loss of total stem biomass yield, indicating that enzymatic saccharification yield can be enhanced by both alterations to cell wall structure and alterations to absolute contents of either glucan or lignin.ConclusionsFinal glucose yields can be improved by the induction of tension wood without a detrimental impact on biomass yield. The increase in glucan accessibility to cell wall degrading enzymes could help contribute to reducing the energy and environmental impacts of the lignocellulosic bioethanol production process.

Bioethanol production from tension and opposite wood of Eucalyptus globulus using organosolv pretreatment and simultaneous saccharification and fermentation

During tree growth, hardwoods can initiate the formation of tension wood, which is a strongly stressed wood on the upper side of the stem and branches. In Eucalyptus globulus, tension wood presents wider and thicker cell walls with low lignin, similar glucan and high xylan content, as compared to opposite wood. In this work, tension and opposite wood of E. globulus trees were separated and evaluated for the production of bioethanol using ethanol/water delignification as pretreatment followed by simultaneous saccharification and fermentation (SSF). Low residual lignin and high glucan retention was obtained in organosolv pulps of tension wood as compared to pulps from opposite wood at the same H-factor of reaction. The faster delignification was associated with the low lignin content in tension wood, which was 15% lower than in opposite wood. Organosolv pulps obtained at low and high H-factor (3,900 and 12,500, respectively) were saccharified by cellulases resulting in glucan-to-glucose yields up to 69 and 77%, respectively. SSF of the pulps resulted in bioethanol yields up to 35 g/l that corresponded to 85-95% of the maximum theoretical yield on wood basis, considering 51% the yield of glucose to ethanol conversion in fermentation, which could be considered a very satisfactory result compared to previous studies on the conversion of organosolv pulps from hardwoods to bioethanol. Both tension and opposite wood of E. globulus were suitable raw materials for organosolv pretreatment and bioethanol production with high conversion yields.

Cellulose and lignin biosynthesis is altered by ozone in wood of hybrid poplar (Populus tremula×alba)

Wood formation in trees is a dynamic process that is strongly affected by environmental factors. However, the impact of ozone on wood is poorly documented. The objective of this study was to assess the effects of ozone on wood formation by focusing on the two major wood components, cellulose and lignin, and analysing any anatomical modifications. Young hybrid poplars (Populus tremula×alba) were cultivated under different ozone concentrations (50, 100, 200, and 300 nl l−1). As upright poplars usually develop tension wood in a non-set pattern, the trees were bent in order to induce tension wood formation on the upper side of the stem and normal or opposite wood on the lower side. Biosynthesis of cellulose and lignin (enzymes and RNA levels), together with cambial growth, decreased in response to ozone exposure. The cellulose to lignin ratio was reduced, suggesting that cellulose biosynthesis was more affected than that of lignin. Tension wood was generally more altered than opposite wood, especially at the anatomical level. Tension wood may be more susceptible to reduced carbon allocation to the stems under ozone exposure. These results suggested a coordinated regulation of cellulose and lignin deposition to sustain mechanical strength under ozone. The modifications of the cellulose to lignin ratio and wood anatomy could allow the tree to maintain radial growth while minimizing carbon cost.

Computational internal sequence repeats analysis of accelerated evolution and the role of extensins under abiotic and biotic stresses

Computational internal sequence repeats analysis of accelerated evolution and the role of extensins under abiotic and biotic stresses Ramalingam Jothi1,2, Subbiah Parthasarathy1, Kulithalai Viswanathan Krishnamurthy11Department of Bioinformatics, School of Life Sciences, Bharathidasan University, Tiruchirappalli; 2Department of Zoology, Dharmapuram Gnanambigai Government Arts College (Women), Mayiladuthurai, Tamil Nadu, IndiaAbstract: Extensins are hydroxyproline-rich glycoproteins present in the plant cell wall. Accelerated evolution and the role of extensins involved in abiotic stresses, like gravitational stress and tension wood formation, and biotic stress, like pathogenic resistance, were investigated through computational internal sequence repeats analysis of their sequences. Multiple sequence alignment analysis of extensin-1 and extensin-2, present in both herbs and trees, was used to investigate their role in gravitational stress and tension wood formation. The role of extensins in pathogenic resistance was investigated by showing the existence of circular permutation in both extensin-1 and extensin-2 sequences between plants of Fabaceae. It was analyzed through Dot plots, and the study predicted that many partial circular permutations exist between sequences of different length. The clustering study of the internal sequence repeats of extensin family through phylogenetic analysis and reconstruction of patterns of repeat identities between sequences showed that unequal crossover exists in extensin-2. From the existence of unequal crossover nature in the circularly permuted extensin-2, accelerated evolution is indicated by the pattern of repeats in herbaceous plants of the Fabaceae family and trees of different taxa, in addition to pathogenic resistance.Keywords: extensin, abiotic and biotic stresses, tension wood, circular permutation, unequal crossover, pathogenic resistance

Arabidopsis thaliana as a model for gelatinous fiber formation

Trees and herbaceous plants continuously monitor their position to maintain vertical stem growth and regulate branch orientation. When orientation is altered from the vertical, they form a special type of wood called reaction wood that differs chemically and structurally from normal wood and forces reorientation of the organ or whole plant. The reaction wood of dicotyledons is called tension wood and is characterized by nonlignified gelatinous fibers. The altered chemical and mechanical properties of tension wood reduce wood quality and represent a major problem for the timber and pulping industries. Repeated clipping of the emerging inflorescence stems of Arabidopsis thaliana augments wood formation in organs, including those inflorescence stems that are allowed to develop later. Gravistimulation of such inflorescence stems induces tension wood formation, allowing the use of A. thaliana for a molecular and genetic analysis of the mechanisms of tension wood formation.

Mechanical bending-induced tension wood formation with reduced lignin biosynthesis in Liriodendron tulipifera

Mechanical bending treatment was employed to cause tension wood formation in the stem of the fast-growing yellow poplar. The tension wood induced by mechanical bending had many characteristic features of tension wood, i.e., eccentric growth toward tension wood, increased frequency of fiber cell types, vessels with reduced size, and reduced lignin content in the developed xylem. The significant reduction of the guaiacyl (G) and p-hydroxyphenyl (H) units of lignin in the tension wood was clearly visualized in samples that had been subjected to bending for 7 and 14 days when analyzed by lignin histochemical analysis using phloroglucinol-HCl. The syringyl (S) unit in the tension wood lignin was also significantly decreased in the developed xylem of the 14-day bending treatment sample, as indicated by Maule’s staining. Expression analysis of several representative genes in the lignin biosynthetic pathway clearly demonstrated that the overall phenylpropanoid pathway toward biosynthesis for both the lignin monomers and the flavonoids was greatly downregulated on bending treatment. In addition, the genes encoding laccases, which have been reported to be involved in the polymerization of monolignols to produce lignin macromolecules, were significantly downregulated in the tension wood. Despite the very limited sequence information for the genes/enzymes in the phenylpropanoid pathway in yellow poplar, histochemical staining and expression analysis using quantitative real-time reverse transcription-polymerase chain reaction (RT-PCR) successfully demonstrated the anatomical and chemical characteristics associated with mechanical bending-induced tension wood formation in the yellow poplar.

Sucrose synthase affects carbon partitioning to increase cellulose production and altered cell wall ultrastructure

Overexpression of the Gossypium hirsutum sucrose synthase (SuSy) gene under the control of 2 promoters was examined in hybrid poplar (Populus alba x grandidentata). Analysis of RNA transcript abundance, enzyme activity, cell wall composition, and soluble carbohydrates revealed significant changes in the transgenic lines. All lines showed significantly increased SuSy enzyme activity in developing xylem. This activity manifested in altered secondary cell wall cellulose content per dry weight in all lines, with increases of 2% to 6% over control levels, without influencing plant growth. The elevated concentration of cellulose was associated with an increase in cell wall crystallinity but did not alter secondary wall microfibril angle. This finding suggests that the observed increase in crystallinity is a function of altered carbon partitioning to cellulose biosynthesis rather than the result of tension wood formation. Furthermore, the augmented deposition of cellulose in the transgenic lines resulted in thicker xylem secondary cell wall and consequently improved wood density. These findings clearly implicate SuSy as a key regulator of sink strength in poplar trees and demonstrate the tight association of SuSy with cellulose synthesis and secondary wall formation.

Ethylene Evolution Changes in Tilted Fraxinus mandshurica Rupr. var. japonica Maxim. Seedlings in Relation to Tension Wood Formation

The effects of ethylene on tension wood formation were studied in 3-year-old Fraxinus mandshurica Rupr. var. japonica Maxim. seedlings in two separate experiments. In experiment 1, ethylene evolution of buds and stems was measured using gas chromatography after 0, 2, 4, 7, 14, and 21 d of treatment; in experiment 2, both aminoethoxyvinylglycine (AVG) and AgNO3 were applied to the horizontally-placed stems, and the cell numbers on sites of applications were measured after 40 d. Ethylene evolution from buds was found to be much greater in tilted seedlings than in upright ones. The cell numbers of wood fibers in shoots and 1-year-old stems were reduced in treatments with 12.5 x 10(-7)micromol/L AVG, 12.5 x 10(-8)micromol/L AVG, and 11.8 x 10(-8)micromol/L AgNO3; whereas the horizontal and vertical diameters were reduced by treatment of 12.5 x 10(-7)micromol/L AVG. Ethylene evolutions of shoots and 1-year-old stems were inhibited greatly in comparison with the control by applying 12.5 x 10(-7)micromol/L AVG. The formation of a gelatinous layer of wood fibers was affected by neither AVG nor AgNO3 application. These results suggest that ethylene regulates the quantity of wood production, but does not affect G-layer formation in F. mandshurica Rupr. var. japonica Maxim. seedlings.

Cloning and expression analysis of a wood-associated xylosidase gene ( PtaBXL1) in poplar tension wood

In stems of woody angiosperms responding to mechanical stress, imposed for instance by tilting the stem or formation of a branch, tension wood (TW) forms above the affected part, while anatomically distinct opposite wood (OW) forms below it. In poplar TW the S3 layer of the secondary walls is substituted by a “gelatinous layer” that is almost entirely composed of cellulose and has much lower hemicellulose contents than unstressed wood. However, changes in xylan contents (the predominant hemicelluloses), their interactions with other wall components and the mechanisms involved in TW formation have been little studied. Therefore, in the study reported here we determined the structure and distribution of xylans, cloned the genes encoding the xylan remodeling enzymes β-xylosidases (PtaBXLi), and examined their expression patterns during tension wood, normal wood and opposite wood xylogenesis in poplar. We confirm that poplar wood xylans are substituted solely by 4- O-methylglucuronic acid in both TW and OW. However, although glucuronoxylans are strongly represented in both primary and secondary layers of OW, no 4- O-methylGlcA xylan was found in G-layers of TW. Four full-length BXL cDNAs encoding putative β-xylosidases were cloned. One, PtaBXL1, for which xylosidase activity was confirmed by heterologous expression in Escherichia coli, exhibited a wood-specific expression pattern in TW. In conclusion, xylan as PtaBXL1, encoding β4-xylosidase activity, are down-regulated in TW.

Variations in Tension Wood of Two Broad‐Leaved Tree Species in Response to Different Mechanical Treatments: Implications for Dendrochronology and Mass Movement Studies

Reaction wood formation, in particular compression wood in conifers, has often been used to date geomorphic events such as debris flows or snow avalanches. However, very little is known yet about the variations of tension wood formation and particularly cross‐sectional vessel characteristics in response to different mechanical impacts. To test whether different intensities and characteristic forms of tension wood formation induce the formation of vessels with changing sizes, growth experiments with different treatments were conducted with alder and beech followed by wood anatomical analysis of the vessels. The experiments comprised bending treatments of varying severities in combination with cambium wounding and other alterations. The treatments were successful in inducing different intensities in tension wood expressed by variations of vessel lumen area (VLA). An increasing bending stress resulted in the formation of lower VLA values. When the apex or the root system was damaged or the apex remained vert...

Tension Wood as a Model System to Explore the Carbon Partitioning between Lignin and Cellulose Biosynthesis in Woody Plants

Tension wood, a specialized tissue developed in the upper side of the leaning stem and drooping branches of angiosperm, is an attractive experimental system attractive for exploring the development and the biochemical pathways of the secondary cell wall formation, as well as the control mechanism of the carbon flux into lignin, cellulose, and hemicellulose. However, the mechanism underlying the induction and the development of the tension wood is largely unknown. Recently, several researchers suggested the possible roles of the plant growth hormones including auxin, gibberellin, and ethylene mainly based on the expression pattern of the genes in this specialized tissue. In addition, expressed sequence tag of Poplar and Eucalyptus provide global view of the genetic control underlying the tension wood formation. However, the roles of the majority of the identified genes have not yet been clearly elucidated. The present review summarized current knowledge on the biosynthesis of tension wood to provide a brief synopsis of the molecular mechanism underlying the development of the tension wood.

Gibberellin-induced formation of tension wood in angiosperm trees

After gibberellin had been applied to the vertical stems of four species of angiosperm trees for approximately 2 months, we observed eccentric radial growth that was due to the enhanced growth rings on the sides of stems to which gibberellin had been applied. Moreover, the application of gibberellin resulted in the formation of wood fibers in which the thickness of inner layers of cell walls was enhanced. These thickened inner layers of cell walls were unlignified or only slightly lignified. In addition, cellulose microfibrils on the innermost surface of these thickened inner layers of cell walls were oriented parallel or nearly parallel to the longitudinal axis of the fibers. Such thickened inner layers of cell walls had features similar to those of gelatinous layers in the wood fibers of tension wood, which are referred to as gelatinous fibers. Our anatomical and histochemical investigations indicate that the application of gibberellin can induce the formation of tension wood on vertical stems of angiosperm trees in the absence of gravitational stimulus.

Role of GA3, GA4 and Uniconazole‐P in Controlling Gravitropism and Tension Wood Formation in Fraxinus mandshurica Rupr. var. japonica Maxim. Seedlings

GA(3) and GA(4) (gibberellins) play an important role in controlling gravitropism and tension wood formation in woody angiosperms. In order to improve our understanding of the role of GA(3) and GA(4) on xylem cell formation and the G-layer, we studied the effect of GA(3) and GA(4) and uniconazole-P, which is an inhibitor of GA biosynthesis, on tension wood formation by gravity in Fraxinus mandshurica Rupr. var. japonica Maxim. seedlings. Forty seedlings were divided into two groups; one group was placed upright and the other tilted. Each group was further divided into four sub-groups subjected to the following treatments: 3.43 x 10(-9) micromol acetone as control, 5.78 x 10(-8) micromol gibberellic acid (GA(3)), 6.21 x 10(-8) micromol GA(4), and 6.86 x 10(-8) micromol uniconazole-P. During the experimental period, GAs-treated seedlings exhibited negative gravitropism, whereas application of uniconazole-P inhibited negative gravitropic stem bending. GA(3) and GA(4) promoted wood fibers that possessed a gelatinous layer on the upper side, whereas uniconazole-P inhibited wood formation but did not inhibit the differentiation of the gelatinous layer in wood fibers on the upper side. These results suggest that: (i) both the formation of gelatinous fibers and the quantity of xylem production are important for the negative gravitropism in horizontally-positioned seedlings; (ii) GA(3) and GA(4) affect wood production more than differentiation of the gelatinous layer in wood fibers; G-layer development may be regulated by other hormones via the indirect-role of GA(3) and GA(4) in horizontally-positioned F. mandshurica seedlings rather than the direct effect of GAs; and (iii) the mechanism for upward wood stem bending is different to the newly developed shoot bending in reaction to gravity in this species.

Effects of Increased Nitrogen Supply on the Lignification of Poplar Wood

The short-term influence of adequate and high nitrogen fertilization on poplar lignification was investigated. The high nitrogen supply decreased lignin staining in the newly formed secondary xylem, indicating that lignin deposition was affected. Acetyl bromide determinations gave a 9-10% decrease in lignin content; however, Klason lignin content was unchanged. Thioacidolysis showed that elevated N supply affected lignin structure such that there was a reduced frequency of lignin units involved in beta-O-4 bonds, a reduced syringyl/guaiacyl ratio, an increased frequency of p-hydroxyphenyl lignin units, more guaiacyl units with free phenolic groups, and more p-hydroxybenzoic acid ester-linked to poplar lignins. These features suggest that lignins from poplars grown under high N bear structural similarities to lignins formed during early stages of wood development. The findings also indicate that a gravitational stimulus inducing the formation of tension wood and high N availability lead to similar and additive effects on lignin content and structure.

An Overview of the Biology of Reaction Wood Formation

AbstractReaction wood possesses altered properties and performs the function of regulating a tree's form, but it is a serious defect in wood utility. Trees usually develop reaction wood in response to a gravistimulus. Reaction wood in gymnosperms is referred to as compression wood and develops on the lower side of leaning stems or branches. In arboreal, dicotyledonous angiosperms, however, it is called tension wood and is formed on the upper side of the leaning. Exploring the biology of reaction wood formation is of great value for the understanding of the wood differentiation mechanisms, cambial activity, gravitropism, and the systematics and evolution of plants. After giving an outline of the variety of wood and properties of reaction wood, this review lays emphasis on various stimuli for reaction wood induction and the extensive studies carried out so far on the roles of plant hormones in reaction wood formation. Inconsistent results have been reported for the effects of plant hormones. Both auxin and ethylene regulate the formation of compression wood in gymnosperms. However, the role of ethylene may be indirect as exogenous ethylene cannot induce compression wood formation. Tension wood formation is mainly regulated by auxin and gibberellin. Interactions among hormones and other substances may play important parts in the regulation of reaction wood formation.

Lignin characteristics and ecological interactions of PtCOMT-modified silver birch

The 35Sand UbB1-PtCOMT genes were transferred by biolistic bombardment into silver birch, Betula pendula Roth, and the effects on syringyl (S) lignin unit synthesis were investigated. The transgenes were stably integrated into the B. pendula genomes, and their variable expression was observed. In the 35S-PtCOMT lines, a reduced syringyl/guaiacyl (S/G) ratio and incorporation of abnormal 5-OH-G units into lignin were found. This was apparently due to the RNA interference (RNAi)-based suppression of the COMT gene, leading to reduced S lignin content in stems, leaves and roots. This work supports the essential role of COMT for S unit synthesis and the current view on lignin biosynthesis in woody angiosperms. The unchanged morphology and growth characteristics of the 35S-PtCOMT modified B. pendula lines also indicate that plants are able to tolerate a large variation in the lignin S/G ratio. PtCOMT-promoter-GUS (β-glucuronidase) modified B. pendula lines were produced in order to investigate the expression pattern of the PtCOMT gene. The main activity during the growing season was present in the new xylem and lignified phloem fibres. Our results also suggest that COMT plays a role in tension wood formation but not in the response to wounding. In in vitro studies focusing on the potential ecological interactions between PtCOMTmodified B. pendula lines and insect herbivores and ectomycorrhizal fungi (ECM), the role played by birch clone or transgenic line was found to be important. The preferential leaf quality to insect herbivores was not directly related to the PtCOMT-modified lignin, but resulted potentially from indirect factors such as a changed growth rhythm. All PtCOMT lines were able to form ECM with P. involutus, but the specific ECM characteristics, e.g. formation of a Hartig net (HN), differed from those of the control. The extent to which the different ECM characteristics of specific PtCOMT lines are related to lignin modification or unintended effects of transgenes remains to be solved.

Mechanical and ecophysiological significance of the form of a young Acer rufinerve tree: vertical gradient in branch mechanical properties

Most tree biomechanics models assume uniformity of mechanical properties within a tree, and only a few studies have focused on differences in mechanical status among branches. We examined mechanical properties of 49 branches of two 10-year-old trees of Acer rufinerve Sieb. et Zucc. For each branch, bending moment due to its own fresh mass, elastic modulus, section modulus and flexural stiffness were obtained. Elastic modulus of the branch was correlated with the density and thickness of the fiber cell wall and decreased with crown depth, indicating that branches at lower positions were more elastic than branches at upper positions. Compared to lower branches, upper branches were less inclined, possessed thicker growth rings, more long shoots and were subject to smaller stresses. The leaf arrangement in the upper branches might be effective in transmitting more light to the lower branches. In contrast, the lower branches were more inclined toward the horizontal and subject to greater stresses than the upper branches. Lower branch inclinations were attributed to smaller dry matter investment in diameter growth. Upper and lower branch inclinations were slightly greater and smaller, respectively, than those predicted by beam theory. The alleviation in inclination of the lower branches is probably caused by negative gravitropic responses such as tension wood formation or upward shoot elongation, or both. The horizontal display of leaves in the lower branches would be effective in light interception. The reduction in cost of the lower branches can be adaptive because they have a shorter life expectancy than the upper branches. The results showed that an adaptive tree form is realized by a vertical gradient in branch mechanical properties.

Control of Negative Gravitropism and Tension Wood Formation by Gibberellic Acid and Indole Acetic Acid in Fraxinus mandshurica Rupr. var. japonica Maxim Seedlings

AbstractIn the present study, we investigated the role of gibberellic acid (GA3) and indole acetic acid (IAA) in the gravity response of stems and tension wood formation using two‐year‐old stems of Fraxinus mandshurica Rupr. var. japonica Maxim seedlings. Forty‐five seedlings were used and divided into nine groups that included five seedlings in each group. Seedlings were treated with applications of GA3 alone at concentrations of 2.89 × 10−8and 2.89 × 10−7μmol/L, IAA alone at concentrations of 5.71×10−8 and 5.71×10−7μmol/L, or their combination to the apical bud of the stem using a micropipette. Seedlings were positioned horizontally after the first treatment. The same treatments were repeated six times per week. At the end of the experiment, all seedlings were harvested. Then, stem segments were cut under a light microscope. Application of exogenous GA3 at the higher concentration stimulated the upward bending of stems, whereas exogenous IAA had no effect. A synergistic effect of GA3 and IAA on upward stem bending was observed following application of the two combinations of GA3 and IAA. Moreover, application of exogenous GA3 at the higher dose stimulated wood formation on both the upper and lower sides of the stems, whereas the mixture of GA3 and IAA had a synergistic effect on wood formation in horizontal stems. Application of exogenous IAA alone at the lower concentration (5.71×10−8μmol/L) or application of a mixture of the higher concentrations of GA3 (2.89×10−7μmol/L) and IAA (5.71×10−7μmol/L) inhibited the development of gelatinous fibers (the G‐layer) of tension wood on the upper side of the horizontal stems. The differentiation of gelatinous fibers of tension wood was not inhibited by GA3 when it was applied alone, whereas the development of the gelatinous fibers of tension wood was strongly affected by the application of IAA. The findings of the present study suggest that the development of the G‐layer is not related to the dose of GA3, but needs a relatively lower concentration of IAA.(Managing editor: Ya‐Qin Han)