J. Cell Sci. 75, 225-238 (1985)
225
Printed in Great Britain © The Company of Biologists Limited 1985
MICROTUBULES ARE AT THE TIPS OF ROOT HAIRS
AND £ORM HELICAL PATTERNS CORRESPONDING TO
INNER WALL FIBRILS
CLIVE W. LLOYD AND BRIAN WELLS
Department of Cell Biology, John Innes Institute, Norwich NR4 7UH, U.K.
SUMMARY
Root hairs have sometimes provided contradictory evidence for microtubule/microfibril parallelism. This tissue was re-examined using optimized conditions for the fixation, before immunofluorescence, of root hairs. In phosphate buffer, microtubules did not enter the apical tip of radish
root hairs and were clearly fragmented. However, in an osmotically adjusted microtubule-stabilizing
buffer, microtubules were observed within the apical dome and appeared unfragmented.
Microtubules are not, therefore, absent from the region where new cell wall is presumed to be
generated during tip growth. A spiralling of microtubules was seen at the apices of onion root hairs.
Using shadow-cast preparations of macerated radish root hairs, it was confirmed that steeply
helical microtubules matched the texture of the inner wall. In onion, the 45° microtubular helices
are accompanied by similarly wound inner wall fibrils.
Results do not support the view that microtubules are not involved in the oriented deposition of
fibrils in root hairs. Instead, they are interpreted in terms of a flexible helical cytoskeleton, which is
capable of changing its pitch but is sensitive to fixation conditions.
INTRODUCTION
The discovery by Ledbetter & Porter (1963) that cortical microtubules ran around
cells in a transverse fashion, parallel to the innermost fibrils of the cell wall, allowed
the idea (Heath, 1974) that cytoplasmic 'tracks' guided the movement of cellulosesynthesizing complexes along the plasma membrane. Although Newcomb & Bonnett
(1965) confirmed that microtubules were parallel to microfibrils in radish root hairs,
they found that the microtubules were arranged axially, which - as other subsequent
observations have done - undermines the idea of microtubules as transverse hoops.
Studies on radish root hairs have raised other questions concerning the involvement
of microtubules in cell polarity. Seagull & Heath (1980), from study of serial sections,
found that short microtubules became less frequent towards the tip, where the cell is
extending, but this is contrary to findings from an immunofluorescence study on
onion root hairs (Lloyd, 1983) in which multistart 45 ° helices of microtubules were
seen to penetrate the apical dome. At various times, radish root hairs have therefore
seemed to provide exceptions to the idea that microtubules assist in polarized growth.
A different idea from the one that microtubules form essentially transverse hoops —
to which inner wall fibrils run parallel — is that they form helices capable of adopting
Key.words: root hairs, microtubules, cell wall, tip growth.
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C. W. Lloyd and B. Wells
varied angles from flat-pitched (transverse) to steeply pitched (axial) (Lloyd, 1984).
Radish root hairs, with their net axial microtubules and inner wall fibrils, are
accommodated by this suggestion, rather than forming an exception.
In this study we have used immunofluorescence to optimize the fixation conditions
for radish root hairs in order to confirm that they are not exceptional but that, like
onion root hairs, their microtubules are organized within the growing apical dome.
Secondly, it has recently been suggested that microtubules do not parallel microfibrils
in certain root hairs (Emons, 1982; Emons& Wolters-Arts, 1983). Taking radish root
hairs and onion root hairs as representatives of cells containing axial and oblique
microtubules, a subsidiary aim has been to confirm that the inner wall fibrils do form
complementary patterns.
MATERIALS AND METHODS
Root hairs
Onion seed (cv. Bedfordshire Champion, Mr Fothergill's Seeds), radish seed (cvs. Long White
Icicle and China Rose), were sown into Pyrex dishes containing kitchen paper made damp with tap
water and then sealed with clingfilm. These were stored in the dark at room temperature.
Immunofluorescence
Entire seedlings bearing root hairs were fixed at room temperature in either freshly prepared
(from paraformaldehyde) 4 % (w/v) formaldehyde or in a thawed solution that had been stored at
—20°C immediately after preparation. In some experiments, 8% formaldehyde (w/v) was used
together with 5 % (v/v) dimethylsulphoxide, which was added to enhance penetration, to stabilize
the microtubules and osmotically inactivate the cells before further physical manipulation. Fixatives
were contained in: A, 50mM-phosphate buffer, pH6-9, 5mM-EGTA, 5mM-MgSC>4; B, 50mM or
lOOmM-PIPES, pH6-9, 5mM-EGTA, 5mM-MgSO4; C, lOmM-PIPES, pH6-9, 5mM-EGTA,
5 mM-MgSC>4 containing mannitol at concentrations ranging between 0' 1 and 0'6 M. Seedlings were
fixed for 45 min, washed in several changes of the same buffer without aldehyde, for 30 min, treated
for 8 min with 2 % (w/v) OnozukaR-lOcellulase (YakultMfgCo., Nishinomiya, Japan) and washed
in several changes of buffer for 30 min. Seedlings were gently blotted and then root hairs scraped off
with a razor blade. Hairs were left to dry on acid-washed, acetone-cleaned coverslips onto which a
solution (1 mg/ml in distilled water) of poly-L-lysine (400000A/ r , Sigma Chemical Co.) had been
dried and washed. Monoclonal antibody to yeast a tubulin was added and counterstained as
previously described (Lloyd, 1983).
Electron microscopy
Root hairs were frozen and snapped as described by Sassen, Pluymaekers, Meekes & de JongEmons (1981) and heated at 100°C for 30min in glacial acetic acid:30% hydrogen peroxide (1:1,
Fig. 1. A newly emergent radish root hair stained with anti-tubulin illustrates: that the
microtubular cytoskeleton is continuous throughout the cell; that microtubules are net
axial in the root hair at this early stage of its development, and that microtubules are
present at the apical dome. Fixed in 50 mM-phosphate buffer containing EGTA and Mg2"1"
Bar, 20/xm.
Figs 2 , 3 . Unlike the young root hair in Fig. 1, older, more vacuolated radish root hairs are
not well preserved in a 50 mM-phosphate fixative. Microtubules stop short of the apical
dome and have a fragmented, 'dotted' appearance. These cells often twist at the junction of
dense apical cytoplasm and vacuole. Bars, 10/xm.
Apical microtubules in root hairs
Figs 1-3
228
C. W. Lloyd and B. Wells
v/v) to remove wall matrix material. After washing in distilled water, these were placed on carboncoated Formvar grids, shadowed with Pt:Pd and examined with a JEOL 1200 EX electron
microscope.
RESULTS
Integrity of the cytoskeleton
Root hairs were initially fixed in 50 mM-phosphate buffer (see Wick et al. 1981)
containing 5 mM-Mg and 5 mM-EGTA. However, whereas this buffer allows
adequate preservation of the short, slow-growing onion root hairs (Lloyd, 1983), the
microtubules in all but the very youngest radish root hairs (Fig. 1) appeared dotted, as
if fragmented (see Figs 2, 3). In addition, the apical body of cytoplasm appeared to be
extending and no microtubules penetrated this apical region.
Substitution of 50 or lOOmM-PIPES for the phosphate in the fixation buffer
prevented this fragmented appearance of the microtubules, which, in addition, were
now often seen to invade the hemispherical apical dome, as illustrated in Figs 4 and 5.
Patterns of microtubules
At all stages of development, including the initial herniation of the trichoblast wall,
radish root hairs contain microtubules that are more or less parallel to their long axis.
This cytoskeleton — as far as can be judged by immunofluorescence — is a continuation
of the cortical microtubules in the basal portion embedded within the epidermis (Fig.
4). Microtubules deviate a little from the long axis, to left and right, producing a
steeply pitched trellis-work of intersecting elements. This net axial pattern of
microtubules has also been seen in root hairs of calabrese, broccoli, Brussels sprout,
corn, cress and rape.
Osmotic effects
To confirm the suspicion that the quality of fixation (especially for the longer, more
vacuolated cells) depended in part upon osmotic conditions, root hairs were fixed in
lOmM-PIPES, 5 mM-Mg2"1", 5 mM-EGTA adjusted with various concentrations of
mannitol. In 0-1 M-mannitol and to a lesser extent at 0-2 M, the apical cytoplasm
Figs 4, 5. Relatively short radish root hairs fixed in a 100 mM-PIPES-based microtubule
stabilizing buffer. These demonstrate the continuity of the cytoskeleton throughout, the
lack of fragmentation, the net axial distribution of microtubules and the presence of these
elements at the apex. Bars, 10 [tm.
Fig. 6. The apical portion of a long radish root hair fixed in a lOmM-PIPES microtubule
stabilizing buffer, adjusted with 0-25 M-mannitol. Microtubules at the apex are usually
well-preserved in this buffer and meet at the very tip. The precise organization is normally
difficult to discern but the slight degree of tip lysis here illustrates that the steeply crossed
microtubules meet at several foci around the apical dome. Bar, 10 /zm.
Figs 7, 8. In those radish root hairs where cortical microtubules cover the apical dome,
another set of microtubules deviates from the cortex at the base of the dome and 'meet' in
the endoplasm. Bars, 10 /im.
Apical microtubules in root hairs
Figs 4-8
229
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C. W. Lloyd and B. Wells
appears to be extruding, whereas in 0-5 and 0-6M-mannitol the apical, densely
cytoplasmic portion of the cell no longer has this appearance, but in the more basal
part (surrounding the vacuole) the protoplast is withdrawn from the side walls.
Radish root hairs appear least damaged when fixed in buffers containing
concentrations of mannitol between 0-25 M and 0-4 M. These gross disturbances of cell
structure are reflected in the appearance of the microtubular cytoskeleton. In buffer
adjusted with 0 - l M-mannitol, tip lysis is observed and cortical microtubules are
absent from this region. In this case, the tip-ward ends of microtubules are
fragmented (as they may be at the basal end of the cell if cut during scraping) but are
not so extensively fragmented over their length as in 50 mM-phosphate. Microtubules
are seen within the apical dome in concentrations of mannitol between 0-25 M and
0 - 4M. In this case, microtubules over the entire root hair rarely have a fragmented
appearance but invariably do when the PIPES in these osmotica is replaced with
phosphate. InO-6M-mannitol, preservation of microtubules within the apical, densely
cytoplasmic region is good although basally, around the vacuole, they are usually
disorganized. In such preparations it is noted that many root hairs are twisted at the
junction between dense cytoplasm and vacuole.
Microtubular organization within the apical dome
When incipient tip lysis is avoided it is clear that cortical microtubules are present
on the apical dome (see Figs 4, 5). Anti-tubulin-stained radish root hairs have not
been seen end-on, but the structure of this most apical part of the root hair can be
surmised from cells (as illustrated in Fig. 6) where a slight degree of apical protrusion
displays the organization. Here, microtubules - which cross over one another steeply,
some tens of micrometres from the tip — are often focussed in several pointed arches.
In addition, while microtubules run over the cortex of the apical dome, another set of
microtubules meets, within the endoplasm, at the base of the hemispherical apical
dome. This second set spans the cylindrical hair to form a brightly fluorescent dome
within and behind the apical dome itself, although this endoplasmic focus of
microtubules may have other appearances (Figs 7, 8).
In root hairs of onion and Nigella the most tip-ward organization of the cortical
microtubules has occasionally been seen in favourably presented cells. In onion the
cortical microtubules form 45 ° helices, which appear to converge spirally into a focus
or foci upon the apical dome (Fig. 9). In Fig. 10A,B, by focussing upon the upper and
lower surfaces of the compressed cylindrical cell, it can be seen that multi-start, single
helices of microtubules do not loop over the tip but spiral in (or out) from a few foci. In
Nigella, microtubules likewise rotate from a circular region at the tip of the
hemispherical dome (Fig. 11).
Shadow-cast preparations of wall
Radish. In radish root hairs (as for Brussels sprout, calabrese, broccoli) the texture
of the outer wall is a loose feltwork composed of fibrils running in net longitudinal, net
transverse and intermediate directions. In the inner wall, fibrils are observed in two,
Apical microtubules in root hairs
Fig. 9. Onion root hair with the tip normal to the observer, displaying the organization of
microtubules upon the apical dome that is otherwise difficult to see. Cortical microtubules
exist in 45 ° helices in onion root hairs and here they are seen to spiral into a point or points
upon the dome. Bar, 10 /im.
Fig. 10. The same onion root hair in two different focal planes. In A the multistart dextral
helices can be seen to run from bottom right to top left on the lower surface of this
compressed cylinder. On the upper surface (slightly out of focus) the same helices can be
seen to run from bottom left to top right. The terminus for these helices is not focussed in A
but in B the undersurface of the apical dome is re-focussed to show that most microtubules
spiral into foci towards the left side of the apical dome (arrows). Bar, 10/im.
Fig. 11. In thisNigella root hair, helical micrgtubules are seen to spiral into an area at the
tip of the apical dome. Bar, 5 /mi.
232
C. W.UoydandB. Wells
Figs 12, 13. For legends see p. 234
Apical microtubules in root hairs
Figs 14, 15. For legends see p. 234
233
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C. W. Lloyd and B. Wells
off-axial directions with an acute angle between them (Fig. 12). In places the fibrils
appear as straight but intersecting, whilst elsewhere they can be described as thicker,
wavy bundles. Because of the steepness of the pitch at which these fibrils run it is
difficult to be sure whether separate crossed lamellae are being observed or whether
this angular dispersion occurs within a thick layer. In places, a minor sub-set of
oblique fibrils can be seen (Fig. 12) amongst the more-axial fibrils and this may
constitute a thin separating layer. Root hairs of calabrese share the same net axial
microtubule pattern with radish and their inner wall fibrils are also net axial. In Fig.
13, the inner wall of a calabrese root hair has dried in such a way as to present different
appearances upon the same inner surface: the inner wall nearer the observer has
peeled back to reveal the thicker, wavy appearance as described for radish root hairs,
whereas the same inner wall has dried upon the grid, forming a steeply intersecting
trellis-work whose elements are not so sinuous (i.e. a steeply pitched, crossed-helical
appearance is revealed).
Onion. Shadowed preparations of onion root hairs, from which matrix materials
had been removed with H2O2: glacial acetic acid, illustrate the crossed-helical
structure of these walls. At low power (X3500) the entire, flattened hair can have a
faint diamond-paned pattern that extends to the apical dome (not shown). At higher
magnification, the individual fibrils of the outer wall layer become visible and these are
apparently random over the length of the hair and over the tip. However, this
appearance is not inconsistent with this layer(s) being a dissipated version of the inner
layers, which show strict crossed-helical lamellation. Where the inner wall is exposed,
fibrils are seen to run at about 45 ° to the hair's long axis (Fig. 14). This, however, is
necessarily approximate, since individuals or small groups of fibrils follow a wavy
course along this mean direction. In dense wall layers it is difficult to see whether this
is caused by straight fibrils crossing steeply over one another within this lamella or by
Fig. 12. Shadow-cast radish root hair from which encrusting wall materials have been
removed by boiling the freeze-snapped hairs in H2O2:glacial acetic acid (1:1) for 30 min.
The inner wall fibrils can be seen to run in off-axial directions (long axis denoted by long
arrow). They follow sinuous courses. A sub-set of fibrils runs more obliquely. Bar,
500 nm.
Fig. 13. Calabrese root hair in which the innermost wall on the lower surface, and the
same continuous wall folded back from the upper surface, are simultaneously exposed. On
the upper, inner surface (left of picture), the net axial texture as described for radish root
hairs (see legend to Fig. 12) is revealed, but on the lower, inner surface a steep, crossedhelical texture is apparent in which the fibrils are less sinuous. Long axis is denoted by a
long arrow. Bar, 500 nm.
Fig. 14. Onion root hair with an apparently random outer wall texture. In the inner wall,
groups of fibrils have a wavy appearance and there is some angular deviation to the
common 45 ° orientation. The sub-adjacent lamella is also composed of fibrils aligned
obliquely to the long axis (arrow) except that they are of opposite helical sign, thus crossing
the inner lamella atright-angles.Bar, 500 nm.
Fig. 15. This inner wall of an onion root hair containsfibrilsthat were more than usually
steeply pitched. The sparseness of the inner layer allows it to be seen that groups of fibrils
branch, intersect at steep angles and follow sinuous courses - all contributing to the
angular dispersion of fibrils within that lamella. The fibrils of the thicker sub-adjacent
lamella run in an equal but opposite helical sign. Bar, 500 nm.
Apical microtubules in root hairs
235
wavy fibrils meeting and separating much as out of phase sine waves would. However,
although the root hair in Fig. 15 contains wall fibrils at a steeper pitch than is usually
seen, the innermost layer is sparse and, in places, illustrates the fact that fibrils within
a single lamella cross over one another.
Underneath the inner layer the second innermost lamella is often seen and crosses
the innermost layer at an equal but opposite angle. Occasionally, it is possible to
glimpse a third lamella in which the fibrils once more parallel the direction of those in
the innermost layer itself. The fibrous wall is therefore composed of alternating
helices. This view has been confirmed with shadow-cast sections of root hairs from
which the resin has been removed: the innermost fibrils-well away from the cut basal
end of the root hair - are inclined at about 45 ° to the long axis and are overlain by
fibrils of opposite sign.
DISCUSSION
The main conclusion from this study is that phosphate buffer and an unsuitably
adjusted osmoticum combine to affect the integrity of the microtubular cytoskeleton.
Microtubules are visibly fragmented in phosphate and do not — as they do in PIPES
buffer — penetrate the apical dome of radish root hairs. Results with PIPES buffer
lend weight to the view that the microtubular cytoskeleton is an integral assembly
that, in root hairs, remains organized at the tip.
Fig. 16. Onion root hair stained with anti-tubulin. Microtubules are seen to form a
continuous array, from cut base to apical dome. Over the length of the cell, the
microtubular helices alter their pitch such that transverse, oblique and axial alignments are
all observed, illustrating the flexibility of the helices. Bar, 20 y,m.
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C. W. Uoyd and B. Wells
The presence of microtubules within the advancing tip has implications for the
interpretation of wall texture. In previous electron-microscopic studies using
phosphate buffers (Newcomb & Bonnett, 1965; Seagull & Heath, 1980), the absence
or poor organization of apparently short microtubules over the most apical 25 /zm
(underlying a random wall), followed more basally by axial microtubules paralleling
an axial inner wall, encouraged the idea that microtubules were ineffective in
organizing tip growth. Present findings allow the possibility that microtubule/
microfibril parallelism extends into the tip, in which case previous reports of random
apical wall might (as for microtubules) be ascribed to tip plasmolysis. Belford &
Preston (1961) found that the apical contents of White Mustard root hairs could be
ejected by plasmolysis but that under non-plasmolysing conditions axially oriented
wall fibrils progressed towards the tip, where autoradiographic studies suggested wall
was being synthesized. This is consistent with the more recent view that presumptive
cellulose-synthesizing particles are densest at the apices of tip-growing cells (Wada &
Staehelin, 1981; Reiss, Schnepf &Herth, 1984). Therefore, a second conclusion from
our study is that microtubules are indeed present in a region where new cell wall is
being deposited. Interpretation of wall texture in root hairs is not straightforward,
since it appears that the basal trichoblast, as part of its own programme of lamellation,
is itself producing new wall layers, which progressively invade the elongating root hair
(Emons & Wolters-Arts, 1983). Nevertheless, confining discussion to the tip, rotation
of the cytoskeleton into the advancing apex, as illustrated in Figs 9-11, would, if
guiding synthases, be expected to influence the helical angle at which the most apical
wall fibrils are deposited and this angle could also be influenced by the rate of
elongation. Since root hairs, unlike cells within tissues, are free to move, the path
taken by the advancing tip might provide clues to the behaviour of the apical
synthesizing machinery. As long ago as 1925, Farr reported that the tips of root hairs
extend in a zig-zag fashion.
In radish root hairs, net-axial microtubules approximate the texture of the
innermost layer of wall fibrils; in onion root hairs, microtubules were previously
shown to form 45° helices (Lloyd, 1983) and it is now demonstrated that innermost
wall fibrils also form similar helices. Microtubules and inner wall fibrils (whether
steeply or obliquely pitched helices), therefore, describe comparable patterns, but the
successively alternating helices formed by the lamellae of onion cell walls raise an
important question for microtubule/microfibril parallelism: are microtubules realigned as wall fibrils shift their orientation? Other workers concluded that a change in
the helical angle of microtubules precedes a change in the alignment of wall fibrils
(Fujita, Saiki & Harada, 1974; Hardham, Green & Lang, 1980; Lang, Eisinger &
Green, 1982). In Oocystis, the wall regularly alternates between left-hand and righthand helices but microtubules parallel the innermost wall fibrils, the alternation of
lamellae being inhibited by colchicine (see Robinson & Quader, 1982). To account for
the behaviour of microtubules, the latter investigators suggest that the microtubules
must either de-polymerize then re-polymerize into an array of opposite sign, or the
array must rotate (i.e. untwist itself) suddenly without repolymerization. Similar
arguments could apply to onion root hairs, in which microtubular helices of one sign
Apical microtubules in root hairs
237
underlie a crossed-helical wall. Obtaining direct evidence for dynamic behaviour from
fixed systems will not be easy but Fig. 16 does illustrate the fact that microtubules of
onion root hairs can adopt different helical angles along the length of the cell.
These observations are consistent with a dynamic helical model (Lloyd, 1984) that
suggests that microtubular arrays are integral assemblies forming helices capable of
shifting their pitch, from flat (transverse) to oblique to steep (axial).
C.W.L. thanks The Royal Society and The John Innes Institute for support. We thank Patricia
Phillips for secretarial assistance, Karen Glencross for photographic assistance and Dr John
Kilmartin for generous supplies of monoclonal antibody to yeast ortubulin.
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{Received 23 August 1984 -Accepted 15 November 1984)