УДК 581.1
© 2010 S. E. Wyatt*, R. Sederoff**, M. A. Flaishman***, and S. Lev-Yadun****
*Department of Environmental and Plant Biology, Ohio University, Athens, United States
**Forest Biotechnology Group, Department of Forestry and Environmental Resources, North
Carolina State University, Raleigh, United States
***Department of Fruit Trees, Institute of Plant Sciences, Agricultural Research Organization, The
Volcani Center, Bet Dagan, Israel
****Department of Science Education-Biology, Faculty of Science and Science Education,
University of Haifa-Oranim, Tivon, Israel
Received June 30, 2009
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.

Key words: Arabidopsis thaliana – cambium – gelatinous fibers – gravity – reaction wood – tension
Corresponding author: Sarah Wyatt. Department of Environmental and Plant Biology, 315 Porter
Hall, Ohio University, Athens, OH 45701, United States. Fax: 740-593-1130; e-mail:
1 Материал был доложен на Международном симпозиуме “Plant Fibers: View of Fundamental Biology”, Казань,
28–31 мая 2009 г.

The architecture of vascular plants, and especially that of trees, has evolved as a compromise
between the optimal positioning of the light harvesting leaves and the physical constraints of organ
mass and water transport. Plants often require repositioning in relation to light, gravity, and
mechanical stress, in response to environmental forces, including wind, ice or snow, fruit load,
herbivore trampling, land movement, or forest gap openings and plant density. The dominant
mechanism, by which trees control and modify stem and branch position, is the formation of reaction
wood [1, 2]. Reaction wood formation occurs as plants perceive a change in the gravity vector or in
the light environment and induces an adaptive program of cell division, cell wall modification, and
cell morphology changes that lead to organ repositioning.
Two types of gravitropic responses occur in plants: the primary response (following
differential cell elongation) and the secondary response (requiring reaction wood formation and
action). In herbaceous plants, the primary gravitropic response involves bending following
differential, asymmetric cell elongation without the production of a special tissue and generally
results in downward root growth and upward shoot curvature. Cell elongation of this type, however,
cannot produce enough force to reposition heavy branches and trunks, and reaction wood is needed
to exert the force required. Reaction wood is the general name for two contrasting types of woody
tissues that are formed during reorientation. In conifers (softwoods), reaction wood is known as
compression wood, formed in the lower side, and is induced by higher than normal levels of auxin;
and, in woody dicotyledons (hardwoods), it is called tension wood, is usually formed in the upper
side of leaning stems and branches and is induced by lower than usual levels of auxin [3, 4].
Gibberellin is also involved in tension wood formation [5, 6], and probably ethylene [7]. In spite of
some advances in characterizing the genes involved in tension wood production (e.g., [8, 9]), the
process involved in the hormonal regulation of tension wood production and gelatinous fiber
differentiation is not well understood (e.g., [10]).
Tension wood is characterized by gelatinous fibers, very low or even lacking lignin and
hemicelluloses and rich in cellulose, that shorten and pull leaning stems and branches upward [4, 11,
12]. Tension wood is a severe defect in sawn timer because of its increased tendency to split, shrink,
or collapse [4]. However, until recently, genetic studies of reaction wood formation have been
mostly confined to observations of the propensity for specific genotypes to form reaction wood [4].
Further analysis has been greatly limited by the size and long generation times of forest trees, and by
the lack of an appropriate genetic model. Some progress in understanding tension wood formation
emerged from the recent progress in Populus and Eucalyptus genetics (e.g., [7–9]), but the genetic
and biochemical mechanisms of tension wood induction and formation are still poorly understood.
Here we show that tension wood formation, as expressed by differentiation of small amounts
of gelatinous fibers, is possible in A. thaliana. The procedure described will allow the use of A.
thaliana as a model system for characterization of the secondary gravitropic response that requires
tension wood formation. Using Arabidopsis will allow researchers the advantage of applying the vast
array of mutants and genomic information available to study the process of tension wood formation.
Moreover, genes involved in tension wood formation identified in Populus and Eucalyptus will be
much easier to study in A. thaliana. This may provide greater insights into the molecular
mechanisms required for the secondary gravitropic response and lead to potential improvements in
wood production in tree species.

Seeds of A. thaliana var. Columbia were germinated and grown in a controlled growth
environment under short day conditions (8 h light, 16 h dark). Chambers were lit by high output
1500 ma cool white fluorescent lamps and 100 W incandescent lamps at an input wattage of 10 : 3.
Light intensity, measured at plant level, was 650 µmol m–2 s–1 of PAR from 400–700 nm. Single
week-old seedlings were transferred to 4-l pots, maintained on a short day photoperiod (8 h light, 16
h dark), and fertilized with half-strength Miracle Gro® All Purpose Plant Food (Scotts Co.,
Maryville, OH) each week. Development of large rosettes of A. thaliana was induced by daily
removal of inflorescences, as they emerged for four to five weeks, to eliminate flower production
and arrest monocarpic senescence [13]. Once rosette size reached ca. 15 cm in diameter, 3–4
inflorescence stems per rosette were allowed to develop, and after 2 days, inflorescence stems were
decapitated at 10 cm above rosette level. Cauline leaves were left intact to encourage increased
secondary xylem formation in the decapitated inflorescence stems. Plants were gravistimulated at
90° from vertical either immediately or 10 days after decapitation. Additional plants, both intact and
decapitated at 10 cm above rosette level, were maintained vertically as controls. A total of five
plants, each with 3–4 inflorescence stems, were used for each treatment. The experiment was
performed twice. Images are representative of each treatment.
Stems were harvested 24 days after decapitation. Sections for histology were taken at the site
of bending or at the region of the stem equivalent to the site of bending. Inflorescence stems of five
plants of each treatment and the controls were fixed in freshly prepared 4% paraformaldehyde and
2% glutaraldehyde overnight at room temperature. Samples were washed three times for 15 min in
PBS, pH 7.2, dehydrated in increasing concentrations of FLEX (Richard-Allan, Kalamazoo, MI,
United States), cleared with Rite3 (Richard-Allan), and embedded in paraffin. Cross sections (5 µm)
were prepared using a rotary microtome, stained with Safranin O and Alcian green, and mounted.
Slides were examined under brightfield and polarized light with a Leitz Dialux 20 microscope
equipped with a Nikon F3 camera, at magnifications of 40X and 400X. Secondary cell walls in
secondary xylem usually contain both cellulose and lignin. Safranin O stains lignified secondary cell
walls as red, and Alcian green highlights cellulose. The secondary cell wall is also highlighted under
polarized light because it contains crystalline cellulose, which is birefringent. When birefringent
material is placed between crossed polarizers, light passing through is polarized and appears bright to
the viewer [14]. Therefore, we also used polarized light to examine the formation of tension wood.

To enable the development of reaction wood in A. thaliana inflorescence stems, plants were
grown for increased rosette size by eliminating competition and by repeated removal of
inflorescences. These large rosettes (more than 15 cm in diameter) (fig. 1a) with inflorescence stems
that have cauline leaves (fig. 1b) can support much more secondary xylem production compared to
the smaller rosettes usually grown for molecular studies. In additional experiments conducted under
much lower light levels, we were unable to induce as much secondary wood as under the high light
conditions (data not shown). Following our discussion with others, we think this issue has been
significant for many laboratories, and we will address it in the discussion.
We used these large A. thaliana plants to test for induction of tension wood in the
inflorescence stems. Increased secondary xylem formation was stimulated in the decapitated
inflorescence stems when compared to intact inflorescence stems that senesced rapidly and the stems
of smaller rosettes. The inflorescence stems gravistimulated immediately after decapitation
responded quickly, and by 18 h after stimulation had bent upward (fig. 1c). Leaving decapitated
stems vertical for 10 days prior to gravistimulation resulted in increased secondary thickening of the
stem. These stems were stiffer than those gravistimulated immediately after decapitation and did not
bend during the subsequent 14 days of gravistimulation, although they formed tension wood.
Microscopic analysis of sections from the stems of both gravistimulated groups under brightfield and
polarized light showed additional unilateral secondary xylem differentiation in both gravistimulated
groups (figs. 2a, 2b) but not in the vertical controls. Under brightfield illumination, the cells
described as gelatinous fibers were stained bluish rather than the typical red staining of lignified
fibers, like in tension wood of the Kermes oak Quercus calliprinos studied by the last author. The
use of polarized light further indicated that the secondary cell walls of these cells were different from
the cells formed in the secondary wood adjacent to them. An additional anatomical indication for the
identity of the tension wood was the enhanced cambial activity and secondary wood formation in the
upper side (unilateral enhanced growth). Gelatinous fibers, a characteristic structural feature of
tension wood, were formed only within the additional unilateral secondary xylem. These gelatinous
fibers are clearly seen in Figs. 2a and 2c as bluish stained cells under brightfield and in Figs. 2b and
2d as dark bands under polarized light. The fact that the gelatinous fibers appeared dark in our
sections when studied under polarized light indicates that the crystalline cellulose common in
gelatinous fibers was probably deposited in a different orientation than usual in the G-layer.

Tension wood is an important mechanism that contributes to establishing and maintaining the
architecture of dicotyledonous plants, especially influencing the structure of forest trees. It is also an
economically significant defect in the wood and paper industry. However, the regulatory and cellular
factors involved in tension wood formation remain mostly unknown. Moreover, the role of
differential levels of auxin as a factor in induction of reaction wood formation (previously considered
an established phenomenon) has been recently challenged [10]. Therefore, we do not know if tension
wood formation is a direct response to a gravity stimulus, to changes in hormonal signaling
(especially auxin), or a combination of these two factors. However, a direct link with the gravity
stimulus does appear to exist from the impact of weight load on stem repositioning, auxin as a down-
stream signal, and induction of secondary growth in inflorescence stems of A. thaliana [15].
Repeated clipping of inflorescence stems of A. thaliana induces several genotypes to produce
much larger plants that form more wood than usual in roots and stems [13, 16–18]. Here we show
that tension wood formation is possible in A. thaliana, but only under well illuminated conditions.
The large A. thaliana plants can be used to induce tension wood in the inflorescence stems, and we
used such plants rather than seeking mutants. We expect, however, that A. thaliana mutants or
transgenics, which regularly produce more secondary tissues than normal (e.g., [19]), will make
Arabidopsis an even better model for wood and fiber formation. The procedure described will allow
the use of A. thaliana as a genetic model for reaction wood formation and related aspects of
secondary cell wall biosynthesis. Experimentation with A. thaliana may provide valuable insights
into the genetic control of reaction wood formation and allow identification of genes involved in
tension wood formation for further study in tree species.
The impact of growth conditions on cambial activity in inflorescence stems of A. thaliana is a
significant issue. This organ is used in many experimental and genetic studies and understanding the
conditions, which induce developmental changes in inflorescence stems or arrest them, is of great
importance. To grow them reproducibly, especially with respect to their structure, is imperative for
many studies. From our experience and from informal discussions with many colleagues, we know
that growth conditions significantly influence the structure of inflorescence stems. Light quality, not
only photoperiod, is involved in the regulation of flowering in A. thaliana (e.g., [20]). Light quality
is also important for the development of secondary growth. In many growth chambers, growth
rooms, or even greenhouses, light levels are dramatically weaker than full sun. Moreover, the light
spectra are not consistent within the same facility over time and between facilities in various
locations. This variability seems to result in differential development of inflorescence stems,
influencing the reproducibility of experiments. Temperature is also known to influence vascular
development in Arabidopsis of various genotypes (e.g., [21]) and is often unstable in many
laboratories, influencing the reproducibility of experiments. Establishing standards for light levels,
light composition, and temperature for the growth of Arabidopsis is crucial to the success of
experiments and comparability of experimental results including tension wood formation.

We greatly appreciate the use of the Phytotron at North Carolina State University for the
space, controlled plant growth environment, and staff that maintained the facility. The use of this
facility made these experiments possible. We also wish to acknowledge the histology facility at the
North Carolina State College of Veterinary Medicine for preparation and staining of the microscopic

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Fig. 1. An A. thaliana plant used to produce secondary xylem.
a – a large Arabidopsis rosette, more than 15 cm in diameter as compared to an Arabidopsis plant
grown under the "normal" long day conditions (inset). The large Arabidopsis were grown under short
day conditions with daily removal of the inflorescence stems to enhance development of secondary
xylem; b – an inflorescence stem allowed to develop from the large rosette and decapitated at 10 cm
above the rosette. If gravistimulated immediately after decapitation, the inflorescence stem
responded quickly, and by 18 h after stimulation, the decapitated stems had bent upward (c). Arrows
indicate the site of bending in the inflorescence stems.

Fig. 2. A cross section of a decapitated inflorescence stem.
Plants were grown under short day conditions with daily removal of inflorescence stems until the
rosettes reached ca. 15 cm. Inflorescence stems were then allowed to grow and then decapitated. Ten
days after decapitation, plants were gravistimulated for 14 days, and then tissues were harvested for
analysis. Brightfield (a) and polarized (b) light showed unilateral additional secondary xylem
differentiation (at the top of the images) with a dark band of gelatinous fibers (b, arrows)
characteristic of tension wood. Primary xylem is indicated by * in (a). Higher magnifications of
sectors show the fibers under brightfield (c), stained gelatinous fibers (arrowheads) of the tension
wood appear blue-green instead of the usual red staining of lignified fibers when stained with
Safranin O, and under polarized light (d), the non-birefringent gelatinous fibers (arrowheads) appear
as a dark band within the illuminated birefringent secondary xylem cell files.