Journal of Experimental Botany, Vol. 57, No. 15, pp. 4269–4275, 2006
doi:10.1093/jxb/erl204 Advance Access publication 6 November, 2006
This paper is available online free of all access charges (see http://jxb.oxfordjournals.org/open_access.html for further details)
RESEARCH PAPER
Nuclear dynamics during the simultaneous and sustained
tip growth of multiple root hairs arising from a single root
epidermal cell
Mark Jones and Nicholas Smirnoff*
School of Biosciences, University of Exeter, Geoffrey Pope Building, Stocker Road, Exeter EX4 4QD, UK
Received 21 July 2006; Accepted 18 September 2006
Abstract
Nuclear dynamics in root hairs, which depends upon
the actin cytoskeleton, appears to be an important factor
in root-hair tip growth. Previous evidence suggests
that there is an absolute requirement for the nucleus
to be a fixed distance from the growing root-hair tip
for tip growth to proceed. To test this hypothesis, nuclear dynamics were examined in root-hair cells bearing multiple root hairs. The majority of root-hair cells of
transgenic plants overexpressing the ROP2 GTPase
(ROP2 OX) bear multiple root hairs. Simultaneous and
sustained fast tip growth occurred in multiple root hairs
of ROP2 OX, with the continual presence of tip-localized
cytoplasm in these growing hairs. Nuclear dynamics
were imaged in ROP2 OX by co-expressing a transgene
encoding a nuclear localization signal (NLS)–green
fluorescent protein (GFP) fusion protein. The nucleus
was in continual proximity to one of the growing roothair tips, whilst the other tip elongated at a similar rate
but in the absence of the nucleus from the shank of
that root hair. To test whether this phenomenon was
an artefact of ROP2 overexpression, nuclear dynamics
were examined in wild-type and NLS-GFP transgenic
plants. Multiple root hairs on the same cell underwent
simultaneous and sustained fast tip growth, with the
nucleus lying deep within the shank of only one of
these hairs. The nucleus was also moved into the roothair tip during the severe root-hair tip branching
which is characteristic of ROP2 OX transgenic
plants. These results suggest that fast tip growth can
proceed in some multiple root hairs at extreme distances from the nucleus.
Key words: Nucleus, multiple root hair, nuclear localization
signal, ROP2 GTPase, tip growth.
Introduction
Root hairs are tip-growing cellular processes that arise from
specific root epidermal cells, called trichoblasts (Dolan
et al., 1994). A complex genetic network controls root-hair
morphogenesis in this model plant cell (Parker et al., 2000;
Grierson and Schiefelbein, 2002). The onset of tip growth
in root hairs occurs immediately after the first visible stage
of root-hair morphogenesis, localized swelling formation
on the cell surface at the apical (i.e. closest to the root tip)
end of the trichoblast (Dolan et al., 1994). Root-hair tip
growth requires a tip-high intracellular calcium gradient
(Ca2þ
i ), and tip-growing root hairs show a tip-localized
influx of extracellular Ca2+ (Ca2þ
e ; Bibikova et al., 1997;
Wymer et al., 1997). The actin cytoskeleton is required for
tip growth in root hairs (Miller et al., 1999; Baluška et al.,
2000), but microtubules, which are required for maintaining the direction of tip growth in root hairs, are not required for tip growth per se (Bibikova et al., 1999). In wildtype (WT) plants, root hairs that are actively tip growing
can be detected by the presence of vesicle-rich cytoplasm in
the root-hair tip (Miller et al., 1999). Root hairs that are not
growing lack this tip-localized cytoplasms, but have a vacuole that extends into the root-hair tip (Miller et al., 1999).
Root-hair tip growth in WT plants is accompanied by the
migration of the nucleus into the shank of the growing root
hair (referred to hereafter as anterograde migration) and
a change in nuclear shape from spheroidal to a more
dynamic shape, which is usually elongated along the major
axis of the root hair (Galway et al., 1997; Chytilova et al.,
2000). Using bright-field microscopy, nuclei of root-hair
* To whom correspondence should be addressed. E-mail: [email protected]
ª 2006 The Author(s).
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.0/uk/) which
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4270 Jones and Smirnoff
cells are readily identifiable as a large subcellular structure,
especially within the shank of the growing root hair
(Ketelaar et al., 2002). Nuclei of transgenic plants
expressing a nuclear localization signal–b-glucuronidase–
green fluorescent protein (NLS-GUS-GFP) construct are
brightly labelled with GFP (Chytilova et al., 2000). NLSGUS-GFP expression does not appear to affect the root-hair
cell adversely (Chytilova et al., 2000). Propidium iodide
staining of DNA has confirmed that these subcellular structures are nuclei (Chytilova et al., 2000). Inhibitor studies
have shown that nuclear dynamics in root hairs of NLSGUS-GFP plants depends upon the actin cytoskeleton, and
not microtubules (Chytilova et al., 2000; Ketelaar et al.,
2002). A further study confirmed that this nuclear movement is energy-dependent (Śliwińska et al., 2002). Optical
trapping of the nucleus was shown to inhibit tip growth
in WT root hairs (Ketelaar et al., 2002). In the same study
the nucleus in the cow1-2 root-hair branching mutant was
found to undergo transient retrograde migration from one
tip towards another tip on the same root hair-bearing cell.
Periods of tip growth, with the characteristic tip-localized
cytoplasm, alternated between these two tips dependent on
the proximity of the nucleus. These results appear to show
that nuclear location has an important role in allowing
root-hair tip growth to proceed.
ROP GTPases are eukaryotic signalling switches that
play fundamental roles in plant development (Li et al.,
2001; Yang, 2002). It has been shown previously that transgenic plants overexpressing the ROP2 GTPase (ROP2 OX)
have multiple swellings on each root hair-forming cell, often widely spaced along the cell length (Jones et al., 2002).
Many, but not all, of these multiple swellings on ROP2 OX
root hair-forming cells undergo the transition to tip growth,
forming multiple root hairs on the same cell that grow
longer than WT root hairs, and often branch at their tips
(Jones et al., 2002). Investigations were made into the movement of the nucleus within ROP2 OX multiple root hairbearing cells and within the rare multiple root hairs that
form on WT root hair-bearing cells. The results show that
the close proximity of the nucleus to the growing tip is not
an absolute requirement for tip growth to proceed.
Materials and methods
Plant material and growth conditions
Arabidopsis thaliana seed were surface-sterilized for 4 min in 10%
(v/v) household bleach, 4 min in ethanol:water:bleach mixture (7:2:1
by vol.), and rinsed 232 min in sterile water. Seeds were plated either
on the surface of sterile semi-solid growth medium (Jones et al.,
2002) containing 0.1% (w/v) phytagel, and grown in horizontal
orientation or beneath the surface of semi-solid medium containing
0.5% (w/v) phytagel and grown in vertical orientation. Plated seeds
were stratified for 48 h at 4 C then transferred to growth cabinets at
20 C with a 24 h light regime. For crossing experiments, seedlings were transferred to soil and grown at 20 C under a 12 h
light/12 h dark regime. The WT Columbia (Col-0) ecotype was used.
ROP2 OX transgenic plants (Li et al., 2001) were selected on medium
supplemented with kanamycin (100 lg ml1). NLS-GUS-GFP
transgenic plants were selected by screening for the characteristic
punctate GFP fluorescence in root tissue (Chytilova et al., 2000).
Microscopy
Growing root hairs were identified by their tip-localized cytoplasm.
Each experiment was replicated at least three times. Microscopy was
performed using a Zeiss Axiophot microscope fitted with a Qimaging
Retiga 1300 12-bit monochrome CCD camera linked to a computer
running OpenLab v3.0.9 (Improvision). Zeiss filter set 10 (excitation
450–490 nm, emission 515–565 nm) was used for GFP detection.
All UV exposure times were optimized to <1 s.
Results
Multiple root-hair formation in transgenic plants
overexpressing the ROP2 GTPase
The multiple swellings on ROP2 OX root hair-bearing cells
(Jones et al., 2002) form simultaneously, but only some
of these undergo the transition to tip growth (Fig. 1A). The
position of these swellings along the length of the ROP2
OX root hair-bearing cell is biased towards the apical end
of the cell (Jones et al., 2002). Although most root hairs
form at this apical end of the cell, it is not possible to predict
whether an individual multiple swelling will undergo the
transition to tip growth or whether any subsequent root
hair will sustain tip growth thereafter. Indeed, some root
hairs located in basal locations along the cell length continue to elongate, whilst other multiple root hairs located much closer to the apical end of the same cell stop
growing (Fig. 1B).
Simultaneous and sustained tip growth of multiple root
hairs in transgenic plants overexpressing the ROP2
GTPase without retrograde nuclear migration
Nuclear location and nuclear migration have previously
been shown to influence tip growth in both WT and cow1-2
root hairs (Ketelaar et al., 2002), but it is not known whether
these factors influence tip growth in ROP2 OX multiple
root hairs. To investigate this, observations were made of
the location of nuclei in ROP2 OX root hair-bearing cells.
The nucleus, which is often clearly visible within the cell
body during early root-hair morphogenesis in ROP2 OX
multiple root hair-bearing cells (Fig. 1C), subsequently
undergoes anterograde migration into the shank of one of
the multiple root hairs. For instance, this is sometimes the
most apical root hair (Fig. 1D) or the most-basal root hair
(Fig. 1E, F) or other combinations, including the middle
root hair in cells bearing more than two root hairs (data not
shown). To investigate whether the presence or absence of
a nucleus within the root-hair shank affected subsequent tip
growth, extended periods of tip growth were observed in
these ROP2 OX cells. Significantly, during both the early
(slow) and later (fast) phases of tip growth (Dolan et al.,
Nuclear dynamics during multiple root-hair tip growth 4271
Fig. 1. Multiple root-hair formation in transgenic plants overexpressing
the ROP2 GTPase. (A) Multiple swelling formation in ROP2 OX roothair cells occurs simultaneously. Arrows indicate sites of swelling
formation. Note that these multiple swellings do not always proceed to tip
growth. (B) Proximity of a multiple root hair to the apical end of the cell
does not predict that tip growth will proceed. This cell has three multiple
root hairs, but only the most basal root hair is tip growing, as indicated by
the presence of tip-localized cytoplasm (arrowhead). The arrows indicate
the non-growing root hairs. (C) ROP2 OX root-hair cell bearing four
multiple hairs undergoing the transition to tip growth. Note the position of
the nucleus (arrowhead) close to the base of one of these hairs. (D–F) The
nucleus is located in various positions within a root-hair cell bearing
multiple root hairs. (D) Both root hairs are tip growing, but the nucleus
(arrowhead) is moving into the shank of the most basal root hair. (E) Both
root hairs are tip growing (basal root hair is out of the plane of focus) but
the nucleus (arrowhead) is moving into the shank of the most apical root
hair. (F) Only the most apical root hair is tip growing as the basal root
hair lacks tip-localized cytoplasm, but the nucleus (arrowhead) lies within the shank of this non-tip-growing root hair. Scale bar in A¼75 lm,
in B¼20 lm, for B, D, E, and F, and in C¼20 lm.
1994) sustained and simultaneous tip growth was observed
in ROP2 OX multiple root hairs, supported by a nucleus
situated deep within the shank of one of the root hairs (see
supplementary Movies S1, S2 at JXB online; Fig. 2A, B).
There was no alternation in the rate of tip growth between
these root hairs, no reduction in tip-localized cytoplasm in
either root hair (Miller et al., 1999), nor any significant
retrograde movement of the nucleus. These results suggest
that, at least in these ROP2 OX multiple root hairs, tip
growth does not specifically require the presence of a nucleus
within the shank of the growing root hair or retrograde
migration of the nucleus from one tip towards another.
To confirm there was no nuclear material in other parts of
these root hair-bearing cells, ROP2 OX plants were crossed
with plants overexpressing the NLS-GUS-GFP construct
(Chytilova et al., 2000). These NLS-GUS-GFP plants have
a root-hair phenotype indistinguishable from that of WT
plants (Fig. 3A). From the segregating F2 populations derived from this cross individual plants were selected with
Fig. 2. Sustained and simultaneous fast tip growth in ROP2 OX multiple
root hairs with the nucleus situated deep within the shank of one of the
root hairs. Images of the same multiple root hairs were taken at t¼0 (A)
and t¼24 min (B) and show the absence of any visible alternation in the
rate of tip growth between these root hairs, the continual presence of
tip-localized cytoplasm in both root hairs, and the progressively anterograde movement of the nucleus (lying within the root hair on the lefthand side and indicated by an arrow in each case). See supplementary
Movies S1 and S2 at JXB online. Scale bars¼20 lm.
the normal homozygous ROP2 OX root-hair phenotype
(Jones et al., 2002) and the brightest GFP fluorescence
(Fig. 3B). These plants were self-fertilized to produce lines
homozygous at both the ROP2 OX and the NLS-GUS-GFP
loci. In all ROP2 OX NLS-GUS-GFP plants tested a single
nucleus was observed within each root-hair cell (data not
shown). Although very occasionally transient and localized fluorescent processes were seen to extend from the
main body of the nucleus, as previously reported (Chytilova
et al., 2000), these subnuclear structures were less pronounced than those described previously (data not shown).
It has been suggested previously that these subnuclear
structures are artefacts of NLS-GUS-GFP overexpression
(Ketelaar et al., 2002). Previous observations of NLS-GUSGFP expression in root hairs have only been made during
early root-hair morphogenesis and in fully elongated mature
root hairs (Chytilova et al., 2000). Here observations were
made during the intermediate stage of sustained tip growth in
ROP2 OX NLS-GUS-GFP multiple root hairs, where one
multiple root hair lacked any visible GFP fluorescence and
the other contained a bright fluorescent body corresponding
to the size and shape of the nucleus observed under the light
microscope (see supplementary Movies S3, S4 at JXB
online; Fig. 3C). Sustained and simultaneous tip growth was
observed in both types of multiple root hair (Fig. 3D).
Growth rates were within the range reported previously for
4272 Jones and Smirnoff
Fig. 3. Nuclear dynamics during multiple root-hair tip growth in ROP2 OX NLS-GUS-GFP transgenic plants. (A) The root-hair phenotype of NLSGUS-GFP transgenic plants is indistinguishable from that of WT plants. Upper panel: bright-field image showing the root-hair phenotype. Lower panel:
fluorescence image showing the characteristic punctate GFP expression pattern within the nuclei. Note that the fluorescence signal is less easily detected
within the shank of tip-growing root hairs. (B) ROP2 OX NLS-GUS-GFP transgenic plants were identified by their root-hair phenotype (in the upper
panel, note the inset showing detail of multiple root-hair formation) and by their characteristic punctate GFP expression pattern within nuclei (lower
panel). (C, D) Sustained and simultaneous tip growth of ROP2 OX NLS-GUS-GFP multiple root hairs. (C) Upper panel: bright-field image showing
multiple root hairs arising from the same cell (inset shows detail of the common site of swelling formation of these root hairs). Lower panel: fluorescence
image showing that the upper root hair lacks any visible GFP fluorescence, whilst the lower root hair contains a brightly fluorescent body corresponding
to the size and shape of the nucleus seen in the bright-field image. See supplementary Movie S3 at JXB online, which shows the absence of any visible
alternation in the rate of tip growth between these root hairs and the continual presence of tip-localized cytoplasm in both root hairs, and Movie S4, which
confirms the progressively anterograde movement of the nucleus (lying within the lower root hair). (D) Graph showing the sustained increase in the
length of both root hairs over a period of 90 min during the phase of fast tip growth. Scale bars: A¼150 lm; B¼500 lm; C¼20 lm.
the fast phase of root-hair tip growth (1.0–2.5 lm min1;
Dolan et al., 1994) in both nuclei-containing and nuclei-free
root hairs (1.0–1.4 lm min1). This fast rate of tip growth
was maintained in both nuclei-containing and nuclei-free
root hairs until root-hair lengths were over 500 lm, when
observations ceased (data not shown). ROP2 OX root hairs
begin to undergo tip branching when they are approximately
500–600 lm long (Jones et al., 2002). Consistent with
findings in WT root hairs (Ketelaar et al., 2002), the nuclei
observed in ROP2 OX multiple root hairs remained at
a relatively constant distance (50–100 lm) from the growing
tip of the root hair within which they were located and at an
Nuclear dynamics during multiple root-hair tip growth 4273
increasing distance from the base of the root hair. However,
the tip of the nuclei-free multiple root hair was at a correspondingly increasing distance from the nucleus. These
results suggest that, at least in ROP2 OX multiple root hairs,
the proximity of the nucleus to the growing tip is not an
absolute requirement for sustained fast tip growth.
Simultaneous and sustained tip growth of multiple root
hairs without retrograde nuclear migration occurs
independently of ROP2 overexpression
It is possible that this long-distance support of tip growth by
a nucleus lying within another root hair is a phenomenon
specific to ROP2 overexpression. To investigate this possibility multiple root-hair formation was examined in plants
lacking the ROP2 OX construct. WT root hair-bearing cells
can, under certain growth conditions, produce rare twin root
hairs from the same root hair-forming site (Jones et al.,
2002). As these rare twin root hairs also occur in NLSGUS-GFP plants, which have WT root-hair development
(Fig. 3A), the position of the nucleus within these root hairs
during tip growth was examined. As in ROP2 OX plants,
sustained and simultaneous fast tip growth also occurred in
NLS-GUS-GFP multiple root hairs where the single nucleus was located deep within the shank of one of these root
hairs (see supplementary Movies S5–S7 at JXB online; Fig.
4A–C), and within the range of growth rates previously
reported (2.1–2.5 lm min1; Dolan et al., 1994). Nuclei
remained at a similar distance (55–100 lm) from the
growing tips of NLS-GUS-GFP hairs to those observed in
ROP2 OX root hairs. Similar sustained and simultaneous
tip growth was observed in the rare twin root hairs in WT
plants (data not shown). These results show that the
observations made in ROP2 OX plants are not a specific
effect of ROP2 overexpression.
The nucleus undergoes further anterograde migration
into the root-hair tip during tip branching in ROP2
OX NLS-GUS-GFP root hairs
ROP2 OX root hairs branch at their tips after root-hair elongation is complete (Jones et al., 2002). It is not clear whether
the normal cessation of tip growth observed in WT root
hairs (Dolan et al., 1994) occurs in ROP2 OX root hairs.
In ROP2 OX root hairs the number of new root-hair
tips appears to increase as root hairs grow older, with increasingly elaborate branches being formed (Fig. 5A). As
nuclei undergo rapid long-distance movement inside the
shank of mature NLS-GUS-GFP root hairs with no apparent co-ordination between hairs (Chytilova et al., 2000),
investigations were made into nuclear movements during
the morphogenesis of ROP2 OX-induced tip branching. In
ROP2 OX NLS-GUS-GFP root hairs the nucleus often
undergoes further anterograde migration at the onset of tip
branching, which is preceded by a characteristic swelling of
the root-hair tip (Movie S8; Fig. 5B, C). The nucleus also
Fig. 4. Nuclear dynamics during multiple root-hair tip growth imaged in
NLS-GUS-GFP transgenic plants with wild-type root-hair morphogenesis.
(A–C) Sustained and simultaneous tip growth of NLS-GUS-GFP multiple
root hairs. (A) Left panel: bright-field image showing the upper of two
multiple root hairs arising from the same cell. Note the common site of
swelling formation of these root hairs (arrow). Right panel: fluorescence
image showing that the upper root hair contains a brightly fluorescent
nucleus. (B) Left panel: bright-field image showing the lower of two multiple root hairs arising from the same cell. Right panel: fluorescence image
showing that the lower root hair lacks any visible GFP fluorescence. See
supplementary Movies S5 and S6 at JXB online, which show the absence of
any visible alternation in the rate of tip growth between these root hairs and
the continual presence of tip-localized cytoplasm in both root hairs, and
Movie S7, which confirms the progressively anterograde movement of the
nucleus (lying within the upper root hair). (C) Graph showing the sustained
increase in the length of both multiple root hairs over a period of 50 min
during the phase of fast tip growth. Scale bar in A¼20 lm for A and B.
appears to migrate into the root-hair apex for the duration
of this tip branching (Movies S9, S10; Fig. 5D). Elaborate
tip branching develops slowly over the course of days so it
was not possible to track nuclear movements in a single
root hair from the start of tip branching until elaborate
branching had formed. However, in older elaborately
branched hairs the nucleus was located in a wide range of
locations, with many remaining close to the most extreme
root-hair apex (Fig. 5E) and others lying deep within the
root-hair shank (data not shown).
Discussion
In both ROP2 OX multiple root hairs and in rare multiple
hairs with WT root-hair development, a single nucleus can
support sustained and simultaneous fast tip growth of at
least two root hairs. This phenomenon occurs in the absence of noticeable retrograde nuclear migration or any
periodic loss of tip-localized cytoplasm, which shows that
tip growth is not being interrupted and re-established. A
previous study indicated that nuclear proximity to the
growing root-hair tip was a requirement for tip growth
to proceed (Ketelaar et al., 2002). The results presented
here show that, at least for some root hairs on multiple root
hair-bearing cells, there is no requirement for the nucleus
to be in close proximity to the growing tip.
4274 Jones and Smirnoff
Fig. 5. Anterograde nuclear migration into the root-hair tip during tip
branching in ROP2 OX NLS-GUS-GFP root hairs. Tip branching in ROP2
OX transgenic plants increases in severity as root hairs grow older, with
increasingly elaborate branches being formed. Left-hand panel: ROP2 OX
primary root tissue showing root hairs beginning to undergo tip branching
(arrow). Right-hand panel: same ROP2 OX primary root shown in the lefthand panel, but closer to the hypocotyl (i.e. developmentally older). Note
the increased number of tip branches on each root hair (arrow). (B, C)
ROP2 OX root-hair tips swell prior to the onset of tip branching. This
swelling is concomitant with the anterograde migration of the nucleus
closer to the root-hair tip. In (B, C) left-hand panels are bright-field images
and right-hand panels are fluorescence images showing the position of the
nucleus. (B) ROP2 OX root-hair tip undergoing normal tip growth. (C)
Same ROP2 OX root-hair tip shown in (B) undergoing swelling of the tip
prior to the start of tip branching. See supplementary Movie S8 at JXB
online, which shows the onset of swelling at tip of this ROP2 OX root hair
in advance of subsequent tip branching and the simultaneous movement of
the nucleus closer to the tip. (D, E) ROP2 OX nuclei lying within the roothair apex during tip branching. (D) Root-hair tip at an early stage of tip
branching. Upper panel: bright-field image showing branched ROP2 OX
root-hair tip. Lower panel: fluorescence image showing position of the
nucleus. (E) More highly-branched root-hair tip. Left-hand panel: brightfield image showing branched ROP2 OX root-hair tip. Right-hand panel:
fluorescence image showing position of the nucleus. Note that the nucleus
is still located within the tip region. See supplementary Movie S9 at JXB
online, which shows the early development of tip branching in this same
root-hair tip, and Movie S10 which shows the simultaneous rapid
movement of the nucleus within the tip region of this root hair. Scale bar
in A¼150 lm, in B¼20 lm, for B, C, E, and in D¼20 lm.
It has been shown previously that root-hair tip growth in
the cow1-2 root-hair branching mutant is dependent on the
nucleus being located at a fixed distance from the actively
growing tip (Ketelaar et al., 2002). COW1 has recently been
cloned and found to encode a phosphatidyl inositol transfer
protein-like protein (Böhme et al., 2004). In these plants,
the nucleus migrates from one root-hair tip to the other,
with periods of tip-localized cytoplasm and associated tip
growth alternating between these tips. However, unlike the
branched root hairs of cow1-2 plants, ROP2 OX multiple
root hairs can be widely separated along the length of the
same cell. In ROP2 OX plants there was no alternation
of nuclear migration or alternation of tip growth between
different multiple root hairs. In light of these results, it
would be interesting to investigate whether the retrograde
nuclear migration phenomenon reported in Ketelaar et al.
(2002) is peculiar to the cow1-2 mutant or whether it is a
feature of other mutants with branched root hairs, for example tip1 (Ryan et al., 1998). Simultaneous and sustained
fast tip growth was also observed in rare multiple root hairs
in WT and NLS-GUS-GFP plants, showing that this
phenomenon occurs independently of ROP2 OX.
Unusually, nuclear dynamics in NLS-GUS-GFP root cells
and root-hair cells is mediated by the actin cytoskeleton,
and not microtubules (Chytilova et al., 2000), a finding
confirmed by inhibitor and antibody studies in WT roothair cells (Ketelaar et al., 2002). Although, it is not known
what effect ROP2 OX has on F-actin organization in root
hairs, expression of constitutively active and dominant
negative ROP2 transgenes in root hairs disrupts normal
fine F-actin organization in the root-hair tip (Jones et al.,
2002), strongly supporting a role for ROP2 in regulating
the formation of fine F-actin in the root-hair tip. Furthermore, overexpression of the Arabidopsis ROP1 GTPase in
tobacco pollen tubes induced a network of F-actin filaments
in the pollen tube tip and a transverse actin band behind the
tip (Fu et al., 2001). If, as might be predicted, ROP2 OX
enhances fine F-actin formation at the root-hair tip, it is
possible that the distance between the nucleus and the roothair tip would be greater in ROP2 OX root-hair cells than in
WT root-hair cells. However, there was no obvious difference in this distance between these two genotypes. On
the contrary, the nucleus moves closer to the tip at the onset
of tip branching and that is characteristic of ROP2 OX roothair morphogenesis. This is interesting, as microinjection of
the lily villin protein into WT Arabidopsis root hairs
resulted in actin unbundling and movement of the nucleus
closer to the root-hair tip (Ketelaar et al., 2002). This
anterograde nuclear movement in ROP2 OX root-hair cells
could also be the result of an indirect effect on actinunbundling proteins mediated by changes in ion gradients
(e.g. Ca2+) during the onset of tip branching.
Taken together, these results show that in these multiple
root hairs the position of the nucleus at a fixed distance
from the root-hair tip is not an absolute requirement for
fast tip growth to proceed. Although the reasons for spontaneous branching in WT, ROP2 OX, and other mutants
may differ, this does not invalidate the conclusion that
growth can continue if the nucleus is not positioned
behind the tip.
Supplementary material
The movies can be found as Supplementary material at JXB
online.
Acknowledgements
We thank Zhenbiao Yang (University of California, Riverside) for
providing the ROP2 OX transgenic plants and David Galbraith
(University of Arizona) for the NLS-GUS-GFP transgenic plants.
Nuclear dynamics during multiple root-hair tip growth 4275
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