Download Arabidopsis thaliana as a Model for Gelatinous Fiber Formation1, 2

ISSN 10214437, Russian Journal of Plant Physiology, 2010, Vol. 57, No. 3, pp. 363–367. © Pleiades Publishing, Ltd., 2010.
Published in Russian in Fiziologiya Rastenii, 2010, Vol. 57, No.3, pp. 384–388.
RESEARCH
PAPERS
Arabidopsis thaliana as a Model for Gelatinous
Fiber Formation1, 2
S. E. Wyatta, R. Sederoffb, M. A. Flaishmanc, and S. LevYadund
a
Department of Environmental and Plant Biology, Ohio University, Athens, United States;
email: [email protected]
b Forest Biotechnology Group, Department of Forestry and Environmental Resources, North Carolina State University,
Raleigh, United States
c Department of Fruit Trees, Institute of Plant Sciences, Agricultural Research Organization, the Volcani Center,
Bet Dagan, Israel
d
Department of Science EducationBiology, Faculty of Science and Science Education, University of HaifaOranim,
Tivon, Israel
Received June 30, 2009
Abstract—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 emerg
ing inflorescence stems of Arabidopsis thaliana augments wood formation in organs, including those inflores
cence 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: Arabidpsis thaliana cambium gelatinous fibers gravity reaction wood tension wood
DOI: 10.1134/S1021443710030076
1
INTRODUCTION
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 trans
port. 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 per
ceive a change in the gravity vector or in the light envi
ronment and induces an adaptive program of cell divi
sion, 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 elon
gation) and the secondary response (requiring reac
1 The article is published in the original.
2 This material was presented at the International
Symposium
Plant Fibers: View of Fundamental Biology, Kazan, Russia,
May 28–31, 2009.
tion wood formation and action). In herbaceous
plants, the primary gravitropic response involves
bending following differential, asymmetric cell elon
gation 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 (soft
woods), 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 for
mation [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]).
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WYATT et al.
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 forma
tion 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 gelat
inous 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 gravi
tropic 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 mecha
nisms required for the secondary gravitropic response
and lead to potential improvements in wood produc
tion in tree species.
decapitation. Additional plants, both intact and
decapitated at 10 cm above rosette level, were main
tained 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 pre
pared 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 (RichardAllan,
Kalamazoo, MI, United States), cleared with Rite3
(RichardAllan), and embedded in paraffin. Cross
sections (5 μm) were prepared using a rotary micro
tome, 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 ×40 and ×400. Secondary cell walls in secondary
xylem usually contain both cellulose and lignin. Safra
nin 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 ten
sion wood.
MATERIALS AND METHODS
RESULTS
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 con
ducted 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 dis
cussion with others, we think this issue has been signif
icant 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 com
pared to intact inflorescence stems that senesced rap
idly and the stems of smaller rosettes. The inflores
cence stems gravistimulated immediately after decap
itation responded quickly, and by 18 h after
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 watt
age of 10 : 3. Light intensity, measured at plant level,
was 650 μmol/(m2 s) of PAR from 400–700 nm. Single
weekold seedlings were transferred to 41 pots, main
tained on a short day photoperiod (8 h light, 16 h
dark), and fertilized with halfstrength Miracle Gro®
All Purpose Plant Food (Scotts Co., Maryville, OH,
United States) 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 inflo
rescence stems. Plants were gravistimulated at 90°
from vertical either immediately or 10 days after
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Arabidopsis thaliana AS A MODEL FOR GELATINOUS FIBER
(а)
(b)
365
(c)
Fig. 1. An A. thaliana plant used to produce secondary xylem.
(a) A large Arabidpsis rosette, more than 15 cm in diameter as compared to an Arabidpsis plant grown under the “normal” long
day conditions (inset). The large Arabidpsis 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.
stimulation had bent upward (Fig. 1c). Leaving decap
itated stems vertical for 10 days prior to gravistimula
tion resulted in increased secondary thickening of the
stem. These stems were stiffer than those gravistimu
lated 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 gravistimu
lated groups under brightfield and polarized light
showed additional unilateral secondary xylem differ
entiation in both gravistimulated groups (Figs. 2a, 2b)
but not in the vertical controls. Under brightfield illu
mination, 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 (unilat
eral enhanced growth). Gelatinous fibers, a character
istic 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 Glayer.
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DISCUSSION
Tension wood is an important mechanism that
contributes to establishing and maintaining the archi
tecture of dicotyledonous plants, especially influenc
ing the structure of forest trees. It is also an economi
cally 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 (previ
ously 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 fac
tors. However, a direct link with the gravity stimulus
does appear to exist from the impact of weight load on
stem repositioning, auxin as a downstream 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 ten
sion wood formation is possible in A. thaliana, but only
under high illumination 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
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WYATT et al.
(а)
(b)
(c)
(d)
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. Inflo
rescence 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 (ⴱ) in (a). Higher magnifications of sectors show the fibers under brightfield (c), stained gelatinous
fibers (arrowheads) of the tension wood appear bluegreen instead of the usual red staining of lignified fibers when stained with
Safranin O, and under polarized light (d), the nonbirefringent gelatinous fibers (arrowheads) appear as a dark band within the
illuminated birefringent secondary xylem cell files.
will allow the use of A. thaliana as a genetic model for
reaction wood formation and related aspects of sec
ondary 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 for
mation for further study in tree species.
The impact of growth conditions on cambial activ
ity 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 signifi
cantly 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 sec
ondary growth. In many growth chambers, growth
rooms, or even greenhouses, light levels are dramati
cally weaker than full sun. Moreover, the light spectra
are not consistent within the same facility over time
and between facilities in various locations. This vari
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Arabidopsis thaliana AS A MODEL FOR GELATINOUS FIBER
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ability 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 geno
types (e.g., [21]) and is often unstable in many labora
tories, influencing the reproducibility of experiments.
Establishing standards for light levels, light composi
tion, and temperature for the growth of Arabidopsis is
crucial to the success of experiments and comparabil
ity of experimental results including tension wood for
mation.
10. Hellgren, J.M., Olofsson, K., and Sundberg, B., Pat
terns of Auxin Distribution during Gravitational
Induction of Reaction Wood in Poplar and Pine, Plant
Physiol., 2004, vol. 135, pp. 212–220.
ACKNOWLEDGMENTS
11. Cronshaw, J. and Morey, P.R., Induction of Tension
Wood by 2,3,5Triiodobenzoic Acid, Nature, 1965,
vol. 205, pp. 816–818.
We greatly appreciate the use of the Phytotron at
North Carolina State University for the space, con
trolled plant growth environment, and staff that main
tained 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 slides.
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