Download Fluridone affects quiescent centre division in the

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Fluridone affects quiescent centre division in the Arabidopsis
thaliana root stem cell niche
1
2
1,
Woong Han , Hanma Zhang & Myeong-Hyeon Wang *
1
Department of Medical Biotechnology, College of Biomedical Science, Kangwon National University, Chuncheon 200-071, Korea, 2Centre
for Plant Sciences, Institute of Integrative and Comparative Biology (IICB), Faculty of Biological Sciences, University of Leeds, Leeds, LS2
9JT, UK
Plants undergo cell division throughout their life in order to
maintain their growth. It is well known that root and shoot tip
of plants possess meristems, which contain quiescent cells.
Fluridone (1-methyl-3-phenyl-5-(3-trifluromethyl (phenyl))-4-(1H)pyridinone) is an established inhibitor of both ABA and carotenoid biosynthesis. However, the other functions of fluridone remain undiscovered. In this report, we provide experimental evidence that fluridone plays a role in the division
of the quiescent centre of the Arabidopsis root meristem. This
study examined the effects of exogenous fluridone and ABA
on the development of the stem cell niche in Arabidopsis root.
We show that fluridone promoted the division of stem cells in
the quiescent centre, whereas exogenous ABA suppressed quiescent centre division. Furthermore, we established a novel
regulatory function for fluridone by demonstrating that it plays
an important role in postembryonic development. [BMB reports 2010; 43(12): 813-817]
root stem cell niche (10, 11). Endogenous and exogenous ethylene-mediated signals control QC division during the postembryonic stage of the plant life cycle (11). Furthermore, abscisic acid (ABA) plays a key role in the developmental status
of the plant over the entire life span, especially during seed development and dormancy, and it responds to diverse environmental stresses (12-14). ABA has been established as an inhibitor of root growth based on its effects on root elongation
and lateral root initiation. However, few studies have reported
enhanced root growth in the presence of low concentrations of
ABA (15-17). It was reported earlier that fluridone is an inhibitor of phytoene desaturase, which converts phytoene to
carotene (18, 19). Considering the above, this study examined
the effects of exogenous fluridone and ABA on the development of the Arabidopsis root stem cell niche and investigated
in detail whether or not fluridone promotes QC division in the
Arabidopsis root meristem accompanied by extra columella
cell layers.
INTRODUCTION
RESULTS AND DISCUSSION
In general, the dividing cells of plants are located and distributed in regions of the roots and shoots known as meristems
(1). The Arabidopsis thaliana root meristem consists of a quiescent centre (QC) and initial cells. The QC acts as an organizing centre, whereas initial cells serve as stem cells (2-4) for all
tissues and promote growth in the roots (5). Ablation studies
have demonstrated that QC prevents the differentiation of columella initial cells (3). To date, researchers have determined
that QC plays a primary role in meristem maintenance in
Arabidopsis root and that these QC are associated with the
SCR, SHR, and WOX5 genes (6-9). Plant hormones are associated with QC division and maintain meristem activity in the
Fluridone promotes QC division in the Arabidopsis root
meristem
*Corresponding author. Tel: 82-33-250-6486; Fax: 82-33-241-6480;
E-mail: [email protected]
DOI 10.5483/BMBRep.2010.43.12.813
Received 4 October 2010, Accepted 10 November 2010
Keywords: Abscisic acid (ABA), Fluridone, Quiescent centre (QC),
Stem cell niche
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The central part of plant root stem cells contains a QC surrounded by initial cells, which in total are referred to as the
meristem (Fig. 1a). The QC rarely divides under normal conditions, but initial cells are able to divide (e.g., for plant
growth) throughout their life span. To determine the effect of
fluridone in Arabidopsis, we germinated Col-0 wild-type in
PANG2 medium for 3 days and then transferred it to PANG2
medium containing fluridone (Flu+). Seedlings of Arabidopsis
roots grown in PANG2 medium (Fig. 1b) did not show QC division even after 10 days of germination, whereas it was clearly observed in seedlings germinated in Flu+ medium (Fig. 1c,
white arrows). Transverse section of germinated seedling
showed a four-celled QC in longitudinal view. In addition, we
also determined the QC division ratio of seedlings incubated
in 10 μM fluridone-supplemented medium for 10 DAG. QC
division (60%) was observed in the roots of these seedlings,
whereas wild-type did not show any such division (n = 50)
(Fig. 1d).
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Fluridone promotes quiescent centre division
Woong Han, et al.
Fig. 1. Exogenous fluridone enhances QC division in the stem
cell niche of Arabidopsis roots. (a) Schematic representation of
the stem cell niche of Arabidopsis root meristems. Co/En, cortical
and endodermis initial; P, pericycle initials; LRC/Ep, lateral root
cap/epidermis initial; CSC, columella stem cells. (b, c) Col-0 seedlings cultured for 10 DAG (Days After Germination) on control
medium (b) or medium containing 10 μM fluridone (c). Arrow
head indicates QC and arrow indicates QC division. (d) Fluridone
promotes QC division. Seedlings cultured for 10 DAG on PANG2
alone or containing 10 μM fluridone. Scale bars, 50 μm.
WOX5 expression was initiated in embryonic cells and gave
rise to QC formation. This finding is consistent with other reports that also found that WOX5 expression is consistently observed in the QC during postembryonic root growth (8).
Therefore, we used a WOX5p::GFP construct to determine the
general effects of fluridone on QC division and specifically
whether or not fluridone stimulates QC division. Seedlings
showed that WOX5p::GFP was strongly expressed in the single
layer of QC cells (Fig. 2a). In contrast, seedlings grown on
Flu+ medium indicated that WOX5p::GFP was expressed in
the extra layer of QC cells (Fig. 2b). SCR is a transcription factor that is accumulated in the QC and has been demonstrated
to be essential for development (20) (Fig. 2c). SCR was detected in the double layer of QC cells in seedlings grown on
Flu+ medium (Fig. 2d). SHR is a transcription factor usually
expressed in the stele (21), and its expression was also investigated and observed in our study on PANG2 and Flu+2 medium (Fig. 2e). Seedlings cultured in Flu+ medium clearly revealed QC division (Fig. 2f).
ABA affects QC quiescence in the Arabidopsis root meristem, which in turn affects QC division. Differentiated columella cells accumulate starch within amyloplast organelles in
the root tip for root gravitropism (22, 23). To determine whether or not the extra cells that form in Flu+ medium function as
QC cells, we tested their ability to regulate differentiation in
the surrounding initial cells (4). Ablation studies have previously revealed that QC cells in Arabidopsis roots promote
earlier differentiation of columella initial cells and have detected (by Lugol staining) the starch grains of columella cells
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Fig. 2. Fluridone promotes QC division in the Arabidopsis thaliana
root meristem. Confocal images of 10 DAG (Days After Germination) of WOX5p:: GFP (a, b), SCRp::GFP (c, d), SHRp:: GFP (e, f)
seedlings, with their structures displayed (propidium iodide staining). g-i: Col-0 seedlings cultured for 10 DAG on Control medium (g), medium containing 0.5 μM of ABA (h), or 10 μM of
fluridone (i). Lugol staining marks differentiated columella cells
(g-i). Arrow head indicates QC and arrow indicates QC division.
Scale bars, 20 μm.
instead of columella initial cells (24). In the present study,
seedlings grown in the presence of ABA and fluridone and
stained with Lugol revealed a single layer of unstained cells
below the supernumerary cells in PANG2, ABA, and fluridone-treated roots (Fig. 2e, f and g). These additional cells
produced in the QC in Flu+ medium displayed both functions
and characteristics similar to those of the QC.
ABA suppresses QC division in a dose-dependent manner
Exogenous exposure to fluridone is expected to promote division in the QC during plant development. However, fluridone,
as a common ABA biosynthesis inhibitor, presents questions
regarding the ability of exogenous ABA to suppress QC division and whether or not it affects the Arabidopsis root meristem in a dose-dependent manner. To confirm this, we tested
seedlings grown in media supplemented with various concenhttp://bmbreports.org
Fluridone promotes quiescent centre division
Woong Han, et al.
Fig. 3. ABA suppresses QC division. (a) ABA suppresses QC division in a dose-dependent manner. Seedlings cultured for 10 DAG
on PANG2 medium containing different concentrations of ABA
and 10 μM fluridone. (b) ABA-deficient mutant displays QC
division. Seedlings cultured for 10 DAG on PANG2 medium.
Error bars, standard deviations.
Fig. 4. Fluridone results in production of extra columella cell
layers. (a) Confocal laser microscopic images of columella cells.
(b) Number of columella cell layers containing different concentrations of fluridone. All seedlings were cultured for 10 DAG.
PANG2, control medium; Flu, control medium containing 10 μM
fluridone. Error bars, standard deviations. Scale bars, 50 μm.
trations of ABA and predicted a 10% decrease in the QC division ratio with 5 nM (least concentration) ABA (Fig. 3a).
Interestingly, the QC division ratio slightly decreased with increased ABA concentrations, and there was 0% QC division at
5,000 nM ABA. Therefore, it was concluded that ABA suppressed QC in a dose-dependent manner.
There is a growing hypothesis that fluridone might also participate in other pathways that control QC division and not
really act as an inhibitor of ABA. Therefore, we investigated
whether or not QC division could be controlled in ABA-deficient mutants (e.g., aba1-1, aba3-2) (25) as well as any other
reasons for this phenomenon. If the QC division phenotype of
fluridone was caused by the absence of ABA, then ABA-deficient mutants would be characterized by increased supernumerary divisions in the QC. We observed extra QC cell division in ABA-deficient mutants grown in PANG2 medium
without fluridone supplementation (Fig. 3b), which suggests
that ABA and QC division were associated.
as a measure of cell division in the QC. We observed that the
seedlings grown in Flu+ medium had more layers of columella cells than those grown in basic medium (Fig. 4a). This additional division in Flu+ medium resulted in increased development of columella layers compared to the seedlings grown
in PANG2 (Fig. 4b). Plants grown in media without fluridone
showed 4.83 ± 0.37 (SD) layers, whereas seedlings cultured
in the presence of fluridone (1 μM of flu) displayed 5.3 ± 0.59
(SD) layers. The results shown here suggest that cell division in
the QC was regulated by both fluridone and ABA.
Our results reveal that fluridone modulated QC division and
enhanced columella cell layer formation, whereas exogenous
ABA suppressed QC division. The results also suggest that fluridone and ABA were involved in Arabidopsis root meristem
maintenance.
Fluridone changes cellular pattern of the stem cell niche
Seeds of Arabidopsis thaliana ecotype Col-0, Ler-0, ABA-deficient mutants, aba1-1, and aba3-2 (25) were used in the
experiments. SHRp::GFP and SCRp::GFP were obtained from
Philip N. Benfey (Duke University), and WOX5p::GFP from
Ben Scheres and Renze Heidstra (University of Utrecht).
Arabidopsis seeds were surface-sterilized with 80% ethanol
and 1% active NaClO for 10 min, followed by three washes
with 100% ethanol. The seeds were dried in a flow hood and
placed on the surface of agar-solidified medium. All media
(PANG2) used in this study was of the following basic composition: 1.5 mM CaCl2, 1.0 mM NaH2PO4, 0.2 mM MgSO4, 0.4
mM KNO3, 1.8 μM KI, 20 μM H3BO3, 3.0 μM ZnSO4, 0.06
We also investigated fluridone-induced cell division of the QC
in Arabidopsis root and also whether or not such cell division
increases the number of columella cell layers. Plants grown in
the presence of aminoethoxyvinylglycine (AVG) display inhibited ethylene synthesis and reportedly develop fewer columella layers (11). To identify the effects of fluridone on columella cell layers, we monitored the Arabidopsis root stem
cell niche. We hypothesised that the addition of fluridone
would induce additional QC division, which could affect the
number of columella cell layers. Therefore, we counted the
number of columella cell layers in Flu+ and PANG2 medium
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MATERIALS AND METHODS
Plant materials and growth conditions
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Fluridone promotes quiescent centre division
Woong Han, et al.
μM CuSO4, 0.4 μM Na2MoO4, 4.0 μM CoCl2, 0.04% (v/v)
Sigma’s Ferrous sulfate chelate solution (F-0518), 0.5% (w/v)
2-(N-morpholino) ethanesulphonic acid (MES), 0.5% (w/v) sucrose, and 10% (w/v) agar-agar (A-1080, Fisher Chemicals).
The pH levels of all media were adjusted to 5.7 using 1.0 M
o
KOH, followed by autoclaving at 121 C for 20 min. Fluridone
(Sigma 59756-60-4) and ABA were added from filter-sterilized
o
stock solutions after autoclaving at 50 C. Stock solutions of
fluridone (10 mM) and ABA (1 mM) were made in 100%
ethanol. Seedlings were grown on the surfaces of these agar
media in vertically-oriented Petri dishes. Dishes were then
sealed with Parafilm, with three openings of 0.3-0.5 cm to allow air exchange during growth, and kept in a growth room at
o
22 C under a 16-h/8-h light/dark cycle using overhead lighting.
Growth conditions in the dark were identical except for the
absence of light.
Confocal microscopy
Whole seedlings were stained with 10 μg ml-1 of propidium iodide (PI; Sigma), mounted in water under glass cover slips, and
then analyzed for PI using a confocal laser scanning microscope (Carl Zeiss LSM 510 META NLO, Germany).
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Starch staining
For starch staining of roots, tissues were incubated in Lugol’s
solution (Sigma) for 5 to 10 min and then mounted in 20%
glycerol for microscopic analysis.
Acknowledgements
We thank the Korea Basic Science Institute (Chuncheon
Center) for assistance with the confocal equipment. This work
was partially supported by a grant from the Institute of
Biosciences and Biotechnology, Kangwon National University,
Chuncheon, South Korea.
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