Plant Molecular Biology 45: 723–730, 2001.
© 2001 Kluwer Academic Publishers. Printed in the Netherlands.
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α-Tubulin genes are differentially expressed during leaf cell development
in barley (Hordeum vulgare L.)
Jan Schröder, Heiko Stenger and Wolfgang Wernicke∗
Institut für Allgemeine Botanik, Johannes Gutenberg-Universität Mainz, P.O.Box 3980, 55099 Mainz, Germany
(∗ author for correspondence; e-mail: [email protected])
Received 1 August 2000; accepted in revised form 11 December 2001
Key words: α-tubulin, cell shaping, cytoskeleton, differential gene expression, gene family, Hordeum vulgare
Abstract
Intricate changes in the patterns of the cytoskeleton, especially of microtubules, appear to control the establishment
of complex plant cell shapes. Little is known about how these changes are accomplished. The objective of the
present study was to test whether or not α-tubulin genes are differentially expressed during cell shaping in growing
leaves of barley (Hordeum vulgare L.). Five α-tubulin genes representing at least most members of the gene family
were found to be expressed in the leaf. Dot-blot analyses revealed expression patterns that could be classified
into three groups. Two isotypes (HVATUB2 and HVATUB4) were maximally expressed in the meristem with
a steady decline during the differentiation process (1). One isotype (HVATUB3) appeared to be constitutively
expressed during cell shaping, although strongest signals were found during late stages, before the general decline
in microtubular activity (2). The most striking finding was that two types (HVATUB1 and HVATUB5) were almost
exclusively expressed in early post-mitotic cells, when transverse microtubular bundles determining the future cell
shape in the mesophyll are formed (3). Relative transcript abundance was highest in HVATUB2 and HVATUB3,
whereas the transcript level of the only transiently expressed HVATUB5 was very low, even during its phase of
maximum expression. The results are discussed in the context of the general debate relating to the significance of
multiple tubulin isotypes.
Introduction
Microtubules are capable of performing various tasks
during the life cycle of eukaryotic cells in spite of a
highly conserved basic structure. The different functions appear to be, at least in part, mediated by differential action of diverse associated proteins, including
motor proteins (see e.g. Mandelkow and Mandelkow,
1995). It is important to note that various isotypes
of the two major microtubule subunits, α- and βtubulin, can occur even within a single organism and
that these may interact differentially with the proteins.
Tubulin isotypes comprise post-translational modifiThe barley α-tubulin nucleotide sequence data will appear in the
EMBL, GenBank and DDBJ Nucleotide Sequence Databases under
the accession numbers X99623 (HVATUB1), Y08490 (HVATUB2),
AJ132399 (HVATUB3), AJ276012 (HVATUB4) and AJ276013
(HVATUB5).
cations of a common progenitor and/or members of
multigene families (Ludueña, 1998). In higher plants,
as in higher animals, it is now accepted that tubulins
are generally encoded by multigene families. Even in
Arabidopsis, with its ‘minimal’ genome, families with
at least six different α- and nine β-tubulin members
have been found (Kopczak et al., 1992; Snustad et al.,
1992). Whether or not the various types are functionally different, and, if so, to what extent, is still a matter
of debate. There are several theories attempting to untangle the potential role of tubulin isotype heterogeneity, ranging from redundancy to dedicated functions of
individual types (see e.g. Joshi and Cleveland, 1990;
Ludueña, 1998). Organ- and/or tissue-specific gene
expression of tubulin isotypes has been described for
plants (e.g. Silflow et al., 1987; Hussey et al., 1990;
Joyce et al., 1992; Chu et al., 1998; Uribe et al., 1998).
Unfortunately, inherent limitations of the experimen-
724
tal systems make it difficult to infer as to what extent
isotype-specific expression is related to the establishment and/or maintenance of particular microtubular
arrays. One reason impeding interpretation, especially
of data derived from extracts, is the heterogeneity of
cells at the temporal and spatial level in most higherplant organs and tissues. The potential of employing
specific antibodies for immunolocalizing isotypes in
the heterogeneous tissues was recently demonstrated
by Eun and Wick (1998).
A promising experimental system providing appropriate cell homogeneity is the developing seed hair
of cotton. Here changes were observed in the expression of tubulin isotypes at the protein and RNA levels
that correlated with characteristic changes in arrays of
cortical microtubules controlling cell wall deposition
(Dixon et al., 1994; Whittaker and Triplett, 1999).
Another suitable system could be the growing leaf
of grasses with its continuous developmental gradient of synchronously developing cells, ranging from
a meristem at the base to highly differentiated tissue at
the tip. The predominant tissue is the synchronously
developing mesophyll (see also Hellmann and Wernicke, 1998). We have recently shown, by means of
two-dimensional electrophoresis and immunoblotting
of protein samples, that several tubulin isotypes exist
in barley leaves (Hellmann and Wernicke, 1998). Particularly in α-tubulin, the composition of isotype populations changed markedly during the establishment of
conspicuous patterns of microtubules involved in determination and manifestation of complex cell shapes
in the developing mesophyll cells. Five to seven αtubulin types could be discriminated, with at least two
types occurring only transiently, when microtubules
formed regularly spaced transverse bundles. These
bundles appear to control the highly localized deposition of cell wall reinforcements that predetermine the
future lobed shape of the mesophyll cells (Jung and
Wernicke, 1990). So far no evidence was found that
the isotypes represent posttranslational modifications
(unpublished data). In a preliminary study employing
a heterologous total α-tubulin DNA probe we detected
high steady-state levels of tubulin RNA throughout the
whole process of cell shaping, indicating continuous
gene activity during all phases (Meyer et al., 1998).
Therefore, an attempt was started to characterize the
α-tubulin gene family in barley and to test whether
or not members of the gene family are differentially
expressed during leaf cell development.
Materials and methods
Plant material
Barley plants (Hordeum vulgare L. cv. Igri, seeds
kindly provided by Ackermann Co., Irlbach, Germany) were grown in vermiculite in a growth cabinet under standardized conditions, as described by
Hellmann et al. (1995). Second foliage leaves 100–
120 mm long were selected for analysis. This size was
reached ca. 9 days after sowing. Leaves were dissected
transversely into sections of defined length (5, 10 or
20 mm), starting at the base. The distance from the
base strictly correlated with the developmental state
of the tissue, because of the developmental gradient
along the leaf, characteristic of growing grass leaves.
RNA isolation and establishment of cDNA libraries
The leaf sections were immediately immersed in liquid nitrogen after harvest. The frozen tissue was
ground to fine powder and mixed with isolation buffer
(guanidine hydrochloride method, according to MacDonald et al., 1987). The extract was purified by
repeated extraction with phenol/chloroform/isoamyl
ethanol. After removal of residual phenol with chloroform/isoamyl ethanol the supernatant was mixed
with LiCl to precipitate selectively RNA (after Sambrook et al., 1989). LiCl was removed by re-dissolving
the precipitate in TE buffer and precipitating nucleic
acids in ethanol. RNA concentrations were determined
photometrically.
For construction of cDNA-libraries, poly(A)+
RNA was recovered from total RNA by oligo(dT)cellulose column chromatography (Stratagene, Heidelberg, Germany) according to Sambrook et al.
(1989) and primed with an oligo(dT)-tailed linkerprimer for cDNA preparation. cDNA libraries were
established using the Uni-ZAP XR or ZAP Express
vector systems (Stratagene). Positive clones were
identified by plaque lift hybridization (Benton and
Davis, 1977) and sequenced by the dideoxy chain
termination method (Sanger et al., 1977).
Probes and RNA blot analysis
Total RNA was subjected to RT-PCR with sets of
degenerated primers (kindly provided by Heiko Sawitzky and Diedrik Menzel) covering potentially conserved regions of plant α-tubulin to generate total
α-tubulin probes. The primers were designed after
taking into account known plant α-tubulin sequences,
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Figure 1. Comparison of predicted amino acid sequences of α-tubulin cDNAs derived from barley leaves. Sequences were aligned and
displayed with the software programs ClustalX (Thompson et al., 1994) and GeneDoc (Nicholas et al., 1997), respectively. Only amino
acids differing from the arbitrarily selected reference sequence (HVATUB1) are shown; dashes indicate gaps included for optimal alignment;
∗, unsequenced parts of HVATUB4 and HVATUB5, translational stop codons; #, Arrows mark the locations of the primers initially used for
amplifying conserved open reading frames. The primer sets yielded two different fragments of 325 bp, related to HVATUB1 and HVATUB2
(1005 to 1330 bp), and one 641 bp fragment, related to HVATUB2 (712 to 1353 bp).
including Anemia, Arabidopsis and Zea. Placement of
the primers is shown in Figure 1. The PCR fragments
obtained were cloned into pBluescript vectors (Stratagene). Probes used for screening cDNA libraries and
hybridization of Southern blots were labelled with
[α-32 P]dATP by random priming (Feinberg and Vogelstein, 1983) with the RadPrime DNA Labeling System (Gibco-BRL, Eggenstein, Germany). Probes used
for tubulin expression studies were labelled with [α32 P]dATP by PCR (similar to Mertz and Rashtchian,
1994) in order to obtain selective labelling with high
specific activity.
For dot-blot analysis, samples of total RNA were
mixed with denaturing buffer and vacuum-blotted
(MilliBlot-D system, Millipore, Eschborn, Germany)
onto nylon membranes (Hybond-N+, AmershamPharmacia, Freiburg, Germany). For estimating the
relative abundance of isotype-specific RNA in a sample a dilution series of isotypic cDNA with known concentrations was blotted on the same membrane. Hybridization was performed under high-stringency conditions at 65 ◦ C according to Hellmann et al. (1995).
After hybridization the membranes were washed twice
in buffer containing 5% SDS and twice in 1% SDS
buffer. The blots were exposed to imaging plates
(BAS-MP 2025P, Fujifilm, Japan) that were scanned in
a phosphoimager (BAS 1500, Raytest, Straubenhardt,
Germany). For quantification images of blots were
evaluated by densitometry (Aida software, Raytest,
Straubenhardt, Germany).
Southern blotting
Genomic DNA was prepared from basal leaf tissue with N-cetyl-N,N,N-trimethylammonium bromide
(CTAB) according to Doyle and Doyle (1987). DNA
was digested with restriction endonucleases, electrophoresed and transferred onto nylon membrane
(Hybond-N+). Hybridization was performed under
low-stringency conditions (37 ◦ C). Probes were labelled by random priming and blots were exposed to
imaging plates as shown above.
Results
Isolation and characterization of the barley α-tubulin
isotypes
For initial experiments RNA isolates were derived
from leaf tissues covering the whole region of cell
shaping, i.e. all phases of microtubular patterns ranging from mitotic cells up to the onset of microtubule
726
Figure 2. An attempt to estimate the complexity of the barley
α-tubulin gene family via Southern blot analysis. 40 µg DNA
was digested with the restriction endonucleases indicated, electrophoresed, blotted and hybridized with 325 bp total α-tubulin
probes. The number of bands obtained indicated the presence of
about three to five α-tubulin genes. Molecular sizes are shown on
the left.
disappearance after completion of shaping. RT-PCR
with the α-tubulin-specific primers and cloning of the
PCR products into the BlueScript vectors resulted in
the isolation of three different clones. Two clones
were equal in length (325 bp), but differed slightly
in sequence (81% homology). The third clone was
a 641 bp fragment. In its overlapping region it was
100% homologous with one of the smaller fragments.
GenBank alignments confirmed α-tubulin homology
of the clones and thus suitability as probes for screening cDNA libraries. In fact, strongest homologies
of the fragments were found with already known αtubulin sequences of wheat and oats. The position of
the primers is indicated in Figure 1.
Three different full-length cDNA clones were isolated with the probes and referred to as HVATUB1,
HVATUB2 and HVATUB3 (Figure 1). Further screening rounds and analyses of putative tubulin clones
yielded no new isotypes. Southern blot experiments
with barley DNA digested with various restriction
enzymes indicated the existence of up to five αtubulin genes (Figure 2). Therefore, additional screenings were performed pursuing two strategies. Firstly,
the search was restricted to narrower developmental ranges. To this end, two additional libraries were
constructed, one representing the highly meristematic
zone, i.e. the first 5 mm of the leaf, and the second the
5–10 mm region covering the early stages of microtubular band formation. Here, stage-specific isotypes
were previously observed at the protein level (Hellmann and Wernicke, 1998). The second strategy was
to challenge all putative tubulin clones immediately
after the primary screening, at the phage state, by PCR
(see also Friedman et al., 1990) employing primers
specific to the 30 -UTRs of the clones already determined. Potentially novel types gave no PCR signals
and could thus be preselected for further processing.
A few of the clones that did not give PCR signals
turned out to be the existing isotypes, at least with
regard to the coding sequences. The only difference
was a shortened 30 -UTR, as observed by Whittaker
and Triplett (1999) and others, perhaps caused by
alternative polyadenylation sites. Nevertheless, following the strategies, two clones with novel sequences
in the coding region were isolated from initially 163
positive clones. One was derived from the meristematic region (0–5 mm) and the other from the region
5–10 mm (HVATUB4 and HVATUB5, respectively).
Both clones represented only partial sequences (see
Figure 1), indicating rapid turnover of the respective
RNAs. Nevertheless, for the expression studies given
below only the data related to the variable 30 -UTRs
were essential.
Differential expression of barley α-tubulin isotypes
For each of the five α-tubulin isotypes a gene-specific
probe was designed, based on the differences in the
sequences between the highly divergent 30 -UTRs (Figure 3). Control experiments confirmed specificity of
the individual probes (data not shown, but consider
distinct differences in expression revealed, Figure 4).
The total α-tubulin 641 bp probe used in this case, covering a conserved stretch of the α-tubulin coding sequence, had already proved suitable during screening
of the cDNA libraries.
Leaf-derived RNA samples were subjected to dotblot analyses. To this end the whole leaf was dissected
as 5 mm long sections (i.e. a higher resolution) in
the crucial cell shaping region and longer sections
in the more advanced regions up to the tip (see Figure 4). Total RNA preparations were sufficient for
analysis, because previous control experiments comparing total RNA and enriched mRNA gave essentially the same results (Hellmann et al., 1995). The
blots shown in Figure 4 are aligned with diagrams
of the leaf and the corresponding microtubular patterns in the mesophyll. Probing with the total α-tubulin
probe gave a similar response to that observed recently
with a heterologous probe (Meyer et al., 1998). Expression was almost constitutively strong throughout
the whole cell shaping process. The rapid decline in
727
Figure 3. Alignment of the 30 -UTR nucleotide sequences of α-tubulin cDNAs from barley including the translational stop codons. Only
positions differing from the arbritrary selected reference sequence (HVATUB1) are shown. The primers (boxed) used to label the isotype-specific
probes (underlined) by PCR are indicated.
signal strength became apparent beyond this region.
Challenging the blots with the isotype-specific probes
revealed a conspicuous differential expression pattern
of α-tubulin genes. Steady-state levels of HVATUB2
and HVATUB4 RNA were highest at the leaf base.
Both decreased, HVATUB2 slightly faster, with increasing distance from the base. HVATUB3 signals
were strongest between 15 and 40 mm. This region
was just above the elongation zone. Even so, relatively strong expression was also apparent in the more
basal region. In fact, the HVATUB3 isotype showed
the highest RNA abundance (Figure 5). The most intriguing expression pattern was found with HVATUB1
and HVATUB5. Both isotypes exhibited maximum expression between 5 and 15 mm, with very weak signals
below and above this region. Nevertheless, the weak
signals obtained with HVATUB5 close to the tip of
the leaf appeared to be specific, as was confirmed by
separate northern blot analysis with equivalent electrophoresed samples (data not shown). The two more
or less transiently expressed isotypes belonged to the
low-abundance RNA group, particularly HVATUB5
(Figure 5).
Discussion
In the cDNA libraries derived from leaf tissue of barley it was possible to identify five different members
of the α-tubulin family. Southern blot analysis suggested that the members found represent most if not
all of the whole family. This would also mean that at
least most of the isotypes are expressed in the leaf,
regardless of time and level of expression. The number
of isotypes discernible by two-dimensional gel electrophoresis of protein samples was similar (Hellmann
and Wernicke, 1998). It appears that diversity of the
tubulin isotype populations is actually more a matter
of different tubulin genes than of post-translational
tubulin modification. A comparison of the deduced
amino acid sequences revealed minor overall differences, but also a characteristic, highly variable, putative MAP-interacting C-terminus (Figure 1). This
hypervariable C-terminus could be taken as a first
indication of differential function of the isotypes. Phylogenetic sequence analysis of the deduced amino acid
sequences (Figure 6) indicated that HVATUB1 and
HVATUB5 belong to a group corresponding to subfamily II of maize α-tubulins (Villemur et al., 1992),
whereas HVATUB2, HVATUB3 and HVATUB4 appear to be related to subfamily I of the maize tubulins,
indicating common ancestry and, perhaps, conserved
functions.
Two measures helped substantially to identify the
whole set of isotypes, namely the relatively easy
PCR-mediated exclusion of abundant cDNA clones,
and inclusion in the search of particular, predefined
leaf developmental stages. The latter approach was
supported by the fact that cells in the leaf develop synchronously, a feature not often found in higher plants.
The results issue a warning that conventional, less selective searches can easily fail to detect potentially
crucial but only faintly and very transiently expressed
isotypes.
An intriguing phenomenon found was the differential expression of the α-tubulin genes during leaf development. The expression patterns, especially those
of HVATUB1 and HVATUB5, correlated strikingly
with changes in the arrangement of the microtubules
observed previously in the developing mesophyll. In
this context it should be noted that the main tissue
type in the leaf is the mesophyll. Tubulin extracts de-
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Figure 4. RNA dot-blots aligned with diagrams of the leaf and corresponding developmental states of mesophyll cells, including pattern of
microtubules and cell wall thickness. 12 µg RNA was blotted per sample. The isotype-specific probes used are indicated in Figure 3. The
basal, solid black area in the leaf diagram delimits the meristematic domain, the hatched zone the region of cell elongation. Note that the
actual developmental gradient was smooth and not discontinuous. Furthermore, note that the maximal intensities of the signals could differ
considerably among isotypes, when using the same exposure time. Crucial HVATUB5 signals, for example, were hardly visible against the
strong HVATUB3 signals. Therefore exposure times were adjusted to show at least similar maximal signal intensities. Control experiments
normalizing the data (cf. Figure 5) confirmed considerable differences in the abundance of isotypes.
Figure 5. Histogram comparing maximal tubulin isotype transcript
levels. Levels were computed by taking data from the dot blots
shown in Figure 4 and offsetting them against data from blots on the
same membranes (not shown) of dilution series of defined concentrations of homologous isotype cDNAs, thus correcting for potential
differences in the affinity and activity of the probes. For the sake
of clarity only those leaf sections were compared, where maximum levels were measured, i.e. 0–5 mm (HVATUB2, HVATUB4),
10–15 mm (HVATUB1, HVATUB5) and 15–20 mm (HVATUB3).
The highest level found, i.e. HVATUB3 15–20 mm, was set to 1.
rived from whole-leaf sections appear to mirror the
status of the mesophyll, as was shown by comparison with selectively isolated mesophyll cells in control
experiments (Hellmann and Wernicke, 1998). The
grass leaf blade is a terminally differentiating organ.
When cells leave the meristem at the base they develop
synchronously and there is no change in cell number and tissue pattern. Transcripts of HVATUB2 and
HVATUB4 were most abundant in the meristematic
tissue. HVATUB3, although more or less constitutively expressed during all phases, appeared to be most
active during later stages, with cessation of elongation
growth. This is the phase when the random microtubular pattern in the mesophyll is established to stabilize
the newly attained cell shape. After completion of
shaping there was a general decline in tubulin gene expression, indicative of terminal differentiation (Hellmann et al., 1995; Meyer et al., 1998). HVATUB1
and HVATUB5 were almost exclusively expressed in
a narrow window, i.e. a very restricted leaf zone during rapid elongation. Here, two additional, transiently
expressed isotypes were recently found to occur at
the protein level and it is also the region where mi-
729
shaping. However, it should be stressed that, as in all
previous cases, the correlations found do not provide
direct evidence of actual functions. Our experimental
system offers a chance to gain further information,
for instance through localization of isotypes at the
cytological level with potentially isotype-specific antibodies as was shown by Eun and Wick (1998) or
by tagging gene products with, for example, green
fluorescent protein. Even a conditional gene knockout of individual isotypes could perhaps be attempted.
Selective inhibition of tubulin bundling in the mesophyll by drugs resulted, for instance, in simple tubular
instead of complex lobed cell shapes (Wernicke and
Jung, 1992).
Acknowledgements
Figure 6. Phylogenetic tree for 16 predicted amino acid sequences
of the α-tubulins of barley (Hv), Arabidopsis (At), maize (Zm),
Chlamydomonas (Cr), with mouse (Mo) as outgroup sequence. To
make data comparison easier the lettering of the Hordeum isotypes
was adjusted to previous conventions, i.e. HVATUB1 = HvTUA1,
etc. (Villemur et al., 1992). The tree was derived using the neighbor-joining method (PHYLIP 3.5c software package: J. Felsenstein,
University of Washington, Seattle, USA). Numbers at nodes represent the confidence level of 100 bootstrap samples. The partial
sequences Zmtua4, Hvtua4 and Hvtua5 (in parenthesis) were not
part of the pairwise comparisons but were included because of their
obvious relationships with the maize α-tubulin subfamilies I and II
(vertical bars), according to Villemur et al. (1992). The horizontal
bar indicates ‘percentage of accepted mutations’ (PAM) distance.
crotubular bundles are formed, determining the future
complex, lobed cell shape in the mesophyll (Hellmann
and Wernicke, 1998). It remains to be seen whether
or not these two proteins are related to HVATUB1
and HVATUB5. Both isotypes exhibited a shortened
hypervariable C-terminus, especially HVATUB5 (Figure 1). Interestingly, the two transiently expressed
isotypes appear to belong to subfamily II (see Figure 6), which also showed selective expression in
maize, as compared to subfamily I (Joyce et al., 1992;
Eun and Wick, 1998).
The expression pattern of the α-tubulin gene family now described is perhaps the most comprehensive
and intricate reported so far for a developing plant
organ. It is certainly tempting to speculate that the
isotypes enable microtubules to perform their special
tasks during mesophyll differentiation, including cell
We wish to thank Heiko Sawitzky and Diedrik Menzel for help with the first primers during a stay of J.S.
at the MPI Ladenburg, Germany; Volker Lennerz for
help with the Southern-blotting; Thorsten Burmester
for introducing us to the complexities of phylogenetic
analysis of gene sequences; and Silke Heichel for patient help during the harvesting and dissection of the
numerous leaves.
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