1 Department of Biology, University of York, Box 373, York YO10 5YW, UK
2 School of Biological Sciences, University of Bristol, Bristol BS8 1UG,
UK
* Author for correspondence (e-mail: hmol{at}york.ac.uk)
Accepted 10 June 2003
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SUMMARY |
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Key words: Auxin, Aux/IAAs, Root hairs, Arabidopsis thaliana
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Introduction |
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Aux/IAA gene function
Aux/IAAs encode low abundance, nuclear proteins with extremely
short half-lives, some as short as 5 minutes
(Abel et al., 1994;
Ouellet et al., 2001
). The
stability of Aux/IAAs is further reduced by auxin treatment
(Ramos et al., 2001
;
Gray et al., 2001
). Aux/IAAs
are characterised by a highly conserved four domain structure. Domain II
contains the destabilisation signal, a 13 amino acid destruction box,
necessary and sufficient for the characteristic auxin-regulated instability of
the Aux/IAAs (Ramos et al.,
2001
). Via this domain, Aux/IAAs interact with the ubiquitin
ligase SCFTIR1 and this interaction is promoted by auxin and
results in 26S proteasome-mediated degradation
(Gray et al., 2001
).
Aux/IAA domains III and IV are required for the formation of both homo- and
heterodimers with other Aux/IAAs and with a family of DNA binding proteins,
the auxin response factors (ARFs) (Kim et
al., 1997; Ulmasov et al.,
1997
), of which there are 23 in Arabidopsis.
ARFs bind to the auxin response elements (AREs) in the promoters of
auxin-regulated genes through an N-terminal DNA binding domain. ARFs are
required to mediate auxin-regulated transcription from ARE-containing
promoters. At their C termini, most ARFs have domains homologous to Aux/IAA
domains III and IV, through which they can homo- and heterodimerise within the
ARF family, and heterodimerise with Aux/IAAs
(Kim et al., 1997;
Ulmasov et al., 1997
). A
sub-set of ARFs act as dimers to promote transcription of auxin responsive
genes (Ulmasov et al., 1997
;
Ulmasov et al., 1999a
).
However, dimerisation of Aux/IAAs with ARFs appears to block this
transcriptional activation (Ulmasov et
al., 1999b
). Auxin promotes the degradation of Aux/IAAs, and
therefore presumably favours the formation of ARF/ARF dimers, activating
transcription of auxin responsive genes. Since the promoters of
Aux/IAA genes themselves contain AREs, it is predicted that an
increase in auxin levels initially reduces Aux/IAA levels by promoting their
degradation, but subsequently replenishes Aux/IAA pools by promoting
transcription from Aux/IAA genes
(Kepinski and Leyser,
2002
).
This model for auxin action places Aux/IAAs at the centre of an auxin signalling network. In Arabidopsis, there are 29 Aux/IAAs, with diverse tissue specificities and auxin response characteristics, with the potential to interact with 23 ARFs, and also with diverse tissue specificities and promoter binding affinities. Therefore, the wide range of auxin responses shown by plants could be encoded in the relative abundance of the different members of this complex network.
AXR3 and SHY2
Support for the central role of Aux/IAAs in mediating diverse auxin
responses comes from analysis of the phenotypes conferred by mutations in
Aux/IAA genes (Liscum and Reed,
2002). Loss-of-function mutations cause relatively mild
phenotypes, presumably as a result of redundancy. Phenotypes that are more
dramatic result from dominant or semi-dominant mutations, and this has led to
the isolation of many such Aux/IAA mutants. So far, these mutations
have all mapped to the domain II destruction box and result in reduced
interactions with SCFTIR1, and hence increased, and auxin
resistant, protein stability (Gray et al.,
2001
). These stabilising mutations in specific Aux/IAA
family members confer specific phenotypes. For example, the
AXR3/IAA17 gene was originally defined by two semi-dominant
stabilising point mutations in domain II
(Leyser et al., 1996
;
Rouse et al., 1998
). The two
alleles, axr3-1 and axr3-3 confer severe phenotypes, largely
consistent with an over-response to auxin
(Leyser et al., 1996
),
including short, highly agravitropic roots, with an increased number of
adventitious roots and a greatly reduced number of root hairs. In contrast,
similar domain II mutations in SHY2/IAA3, originally identified as
suppressors of the long hypocotyl phenotype of the phytochrome deficient
phyB mutants (Kim et al.,
1996
; Reed et al.,
1998
), result in long straight roots with reduced adventitious
rooting (Tian and Reed, 1999
)
and increased root hair density; opposite to the root phenotype conferred by
axr3.
These opposite phenotypes conferred by similar stabilising mutations in SHY2 and AXR3 provide an opportunity to understand better how Aux/IAAs might mediate particular auxin responses. We have chosen to focus on the root hair phenotypes of these mutants because of the wealth of information available on Arabidopsis root hair development.
Root hairs are long tubular outgrowths from the surface of specialised
epidermal cells. By greatly increasing the surface area, they are important
for nutrients and water uptake and for anchorage
(Peterson and Farquhar, 1996).
In Arabidopsis, the root epidermis is made up of longitudinal cell
files, which develop in a distinct pattern
(Dolan et al., 1994
;
Galway et al., 1994
). The
development of the cell files begins with transverse divisions of initial
cells in the root meristem (Schneider et
al., 1997
). Divisions continue immediately behind the initials in
the division zone. Following the cessation of cell division, the cells
continue to elongate, in the elongation zone, after which they differentiate
into either trichoblasts (root hair cells) or atrichoblasts (hair-less cells)
(Grierson et al., 1997
).
Trichoblasts are always located over the junction between two underlying
cortical cells, resulting in a pattern of alternating files of trichoblasts
and actrichoblasts around the root. Trichoblasts can be distinguished from
atrichoblasts as early as the latter stages of embryogenesis, because of their
increased cytoplasmic density (Dolan et
al., 1994
; Galway et al.,
1994
) an increased rate of cell division
(Berger et al., 1998
) and cell
surface deposits (Dolan et al.,
1994
).
Root hair outgrowth itself can be split into three developmental stages:
bulge formation, initiation and tip-growth
(Grierson et al., 1997). Tip
growth is a rapid form of directional elongation, which involves precise
targeting of vesicles carrying cell wall precursors to the growing tip (Benfey
and Schiefelbein, 1994; Grierson et al.,
1997
).
In this work, we describe the root hair phenotypes of both gain- and loss-of-function mutants of AXR3 and SHY2 and provide evidence for a dose-dependent interaction between AXR3 and SHY2 in regulating the timing of root hair differentiation.
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Materials and methods |
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Plant growth conditions
Seeds were surface sterilised with 10% bleach and 0.1% Triton X-100 for 15
minutes, then rinsed once in 70% ethanol and four times in sterile distilled
water. Sterile seeds imbibed at 4°C for 2 days prior to planting in Petri
dishes containing 20 ml of Arabidopsis thaliana salts (ATS) growth
medium, as previously described (Wilson et
al., 1990). The ATS was solidified with 0.8% Phytagel (Sigma).
Plates were orientated vertically in controlled condition growth rooms at
23°C with 16 hours light or in a growth cabinet at 20°C for the heat
shock (HS) experiments.
Phenotypic analysis
All images for measurements were taken at 5 days post-germination with a
JVC TK-1070E video camera attached to a Nikon SMZ 10A stereo dissecting
microscope, apart from those of epidermal cells, which were captured with the
camera using a Nikon Optiphot-2 microscope. Images were measured using LUCIA G
software (version 3.52a, 1991). At least 50 measurements were taken from at
least 10 plants for each parameter. For root hair number, only hairs visible
within a 1 mm segment, viewed from above, were counted. For epidermal cell
lengths a combination of atrichoblasts and trichoblasts were measured. For
root hair lengths, only hairs from mature sections of roots were measured.
For time-lapse analysis, pictures were taken automatically every 10 minutes of a 5-day old root growing on an ATS/Phytagel plate under the dissecting microscope. The images were measured on completion and were taken from three different plants for each genotype.
In situ pictures were taken using a Nikon FX 35DX camera fixed to the Nikon Optiphot-2 microscope using dark-field optics.
Transgenic plants
The transgenic plant line HS:axr3-1 was created using the cDNA
from EST H36782, obtained from the Arabidopsis Biological Resource
Centre, Ohio. The axr3-1 point mutation
(Rouse et al., 1998) was
introduced into the 941 base pair (bp) sequence by site-directed mutagenesis
using the Stratagene QuikChangeTM kit according to the manufacturer's
instructions. Similarly, the HS:shy2-6 line was created using the
cDNA from EST TO4296, obtained from the Arabidopsis Biological
Resource Centre, Ohio, and the corresponding point mutation to that of
axr3-1 was introduced to the 978 bp sequence in the same way. Each
cDNA was cloned into a pJR1Ri vector, in the sense orientation, using the
XbaI/SmaI site, downstream of a 0.4 kb soybean heat shock
promoter (Schoffl et al.,
1989
). The vectors were then transformed into Agrobacterium
tumefaciens strain GV3101
(Koncz and Schell, 1986
) by
freeze-thaw (Höfgen and Willmitzer,
1988
) and then into wild-type Arabidopsis plants of the
Columbia ecotype using the floral dip method
(Clough and Bent, 1998
).
Transformants were selected by kanamycin resistance and were then planted into
soil and allowed to self-fertilise. In the T2 generation, lines
showing a 3:1 ratio of kanamycin resistant to sensitive plants, indicative of
a single site of transgene integration, were selected for further study.
Homozygous lines were selected from the T3 generation. Preliminary
experiments indicated that multiple independent lines for each transgene
behaved in a similar way in response to heat shock, so for each construct a
single representative line was selected for further work.
Transient activation of gene expression by heat shock
The positions of root tips were marked on the back of Petri dishes and
these were placed in a 37°C incubator for 2 hours. The root tip positions
were marked again at 4, 8, 12 and 24 hours following heat induction. The
length of root hairs was measured at each of these marks, in each of the
genotypes.
Whole-mount in situ hybridisation
Probes
For AXR3 probes, a 133 bp region was amplified from cDNA, in a
region between domains I and II using the forward primer
5'-CGGAAGAACGTGATGGTTTCA-3' and reverse primer
5'-CGTAGCTTTTATACATCCTC-3'. For SHY2/IAA3, a 240 bp
region was amplified from the 3'UTR by PCR, using the forward primer
5'-CTCTGTCTGTGCTTGGGTTG-3' and the reverse primer
5'-CTCTTCAATCTTCATAACAC-3'. Both products were then cloned into
PCR4-TOPO vector (Invitrogen) by TA-cloning. M13 forward and reverse primers
were then used to amplify across the probe region including promoter sites for
T3 and T7 polymerase, and the product was purified. Both sense and antisense
RNA probes were made from the same PCR product, in separate reactions, using
the digoxigenin (DIG) RNA labelling kit (Roche) according to the
manufacturer's instructions, except the reaction was scaled up fivefold.
Fixing and hybridisation
Throughout the fixing and antibody stages, the seedlings were contained in
cell strainers (Falcon), which minimised tissue damage when transferring from
one solution to the next (de Almeida Engler
et al., 1994).
Four-day-old seedlings were fixed in 4% formaldehyde/0.1% Triton X-100/0.1% Tween 20 by vacuum infiltration for 15 minutes and then overnight at 4°C. The seedlings were then dehydrated through an ethanol series from 50% to 100% over 3 days, at 4°C. They were then taken back down the ethanol series to 30%, prior to being treated with acetone and then acetic anhydride solution (0.1 M triethanolamine/0.5% (v/v) acetic anhydride). Between the treatments, the seedlings were rinsed with phosphate-buffered saline (PBS) for 30 minutes.
The seedlings were then washed in PBS before being placed in a probe and hybridisation buffer mix in Eppendorf tubes. They were then allowed to hybridise overnight at 50°C. The seedlings were transferred back to cell strainers in six-well plates and three post-hybridisation washes in 2x SSC/50% formamide were carried out, at 50°C, for 1.5 hours. The seedlings were then washed twice in NTE (0.5 M NaCl/10 mM Tris pH 7.5/1 mM EDTA) at 37°C for 15 minutes each time. This was followed by seedling incubation in NTE with 20 µg/ml RNaseA, also at 37°C, for 45 minutes. The seedlings were then washed twice with NTE, for 15 minutes each time and then incubated in SSC/50% formamide for 2 hours at 50°C. Then one wash with SSC at 23°C, was carried out for 1 hour, followed by two rinses with PBS (for 15 minutes each) at room temperature. The seedlings were then stored overnight at 4°C. They were prepared for the antibody and detection stages by washing in a salt buffer (0.1 M Tris/0.15 M NaCl) solution for 10 minutes. They were then incubated in a solution of 0.5% Blocking Reagent (Roche) in salt buffer for 1 hour, followed by washing with salt buffer, containing 1% BSA and 0.3% Triton X-100 for 1 hour. The seedlings were then incubated with a 1:2000 dilution of the anti-digoxigenin antibody (Roche) for 1 hour before being washed six times with salt buffer/BSA/Triton X-100 for 15 minutes each wash. A final wash in plain salt buffer was carried out for 30 minutes before the sieves were removed and the seedlings were incubated in the six-well plates with the developer, Western Blue (Promega). The development reaction was stopped by placing the seedlings in PBS, as soon as background signal could be seen in the sense controls.
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Results |
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Since root hair length is known to be regulated by environmental
conditions, we tested the ability of the mutants to respond to the root hair
growth promoting effects of low phosphate. As previously reported
(Bates and Lynch, 1996) removal
of phosphate from the medium stimulates elongation of wild-type root hairs,
resulting in an 125% increase over hairs growing on 2.5 mM phosphate
(Fig. 2E). This effect was even
more pronounced in the axr3-10 root hairs, with hairs achieving
significantly longer final lengths than in the wild type, representing an 155%
increase. In contrast the root hairs of shy2-31 plants responded less
strongly than those of wild type, increasing by only 84%. Interestingly both
gain-of-function mutants were impaired in their ability to respond. The roots
of axr3-1 plants remained completely bald even on medium with no
added phosphate (data not shown), while the root hairs of shy2-2
plants were able to increase their length only 26% over their already
long-hair base line.
The shy2-2 mutation affects the timing of root hair
initiation
To examine the timing and position of root hair differentiation in the
mutants, we measured the distance from the root tip to the first root
hair.
In shy2-31 and axr3-10 plants, the hairs initiate at the same distance from the tip as in wild type (Ler) (Fig. 3). However, in shy2-2 roots the hairs were found to initiate much closer to the root tip than in wild type (0.79±0.02 vs 1.45±0.05). This correlates with the observation that the number of cells in the elongation zone below the first hair-bearing cell was 7±0.35 in shy2-2 seedlings compared with 10±0.61 in wild type.
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The early initiation of shy2-2 root hair elongation is not matched by early cessation, so that shy2-2 hairs grow for a longer time period, than wild type (Fig. 4C). Wild-type hairs complete tip-growth in an average of 4 hours, shy2-2 hairs grow for 8 hours before reaching full length (Fig. 4C). Hence, the longer length of shy2-2 root hairs and the reduced distance between the shy2-2 root tip and first root hair can both be attributed to the ectopic initiation of root hair growth in the elongation zone.
Transient expression of axr3-1 and shy2-6
To determine which stages of root hair growth are affected by the
axr3 and shy2 gain-of-function mutants, the effect of
transient expression of shy2-6 and axr3-1 was examined by
inducing their transcription from the soybean heat shock (HS) promoter
(Schoffl et al., 1989). Heat
shock was carried out for 2 hours at 37°C. Following heat shock, the
position of the growing tip of the root was marked at 4, 8, 12 and 24 hours.
For each of these time points root hair length was measured. Transient
expression of HS:axr3-1 led to an immediate block in root hair
formation, which persisted for up to 12 hours
(Fig. 5,
Fig. 6B). Hairs that were
elongating at the time of heat shock stopped. In contrast, heat shock had no
effect on wild-type root hair elongation
(Fig. 6A), and induction of
shy2-6 expression gradually increased the length of the root hairs
over the 24 hour period of the experiment
(Fig. 5). An additional
striking phenotype resulting from transient expression of axr3-1 was
transient agravitropism (Fig.
6B). Root hair length and morphology in the non-heat-shocked
controls remained constant through the experiment
(Fig. 5).
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AXR3 and SHY2 expression in the root tip
To discover where AXR3 could act during root hair development, we
carried out whole-mount in situ hybridisation to detect the location of the
AXR3 transcript. AXR3 transcript was observed in a region
extending from the root tip toward the differentiation zone, where expression
dropped away sharply (Fig. 7A).
In the sense control, no signal was seen in the root tip
(Fig. 7B).
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These data demonstrate that both AXR3 and SHY2 transcripts accumulate in root tips, with the zone of SHY2 expression extending beyond that of AXR3, into the root hair differentiation zone.
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Discussion |
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The mode of action of the two mutant proteins in regulating root hair length is very different. The axr3-1 protein can block root hair elongation at any stage, since in the HS:axr3-1 plants, heat shock induction resulted in immediate inhibition of root hair initiation and elongation. Growth was blocked even in hairs that were elongating at the time of the heat shock (Fig. 6B). In contrast, the shy2-2 protein appears to affect the timing of the initiation of hair development, rather than the rate of hair growth following initiation. The shy2-2 root hair phenotype is caused by early initiation of root hair growth, when the trichoblasts are still actively expanding in the longitudinal axis. The hairs then elongate at a wild-type rate but for a longer period of time, resulting in longer hairs. Consistent with this idea, the effects of transient induction of shy2-6 are only apparent in hairs that initiated (presumably ectopically in the elongation zone) after the heat shock.
When shy2-2 and axr3-1 are co-expressed, a novel phenotype is observed in which apolar aberrant root hairs initiate, but fail to undergo tip growth. This phenotype is not observed in the axr3-1 mutant background, when shy2-6 is transiently expressed, but only in the shy2-2 mutant background when axr3-1 is transiently expressed. Furthermore, it only occurs after a period when root hair formation is completely blocked, as axr3-1 levels are dropping back to zero. The aberrant roots hairs presumably develop at a point when the axr3-1 protein falls below a critical level. However, the phenotype is not simply related to the level of axr3-1 because it is dependent on the presence of shy2-2 and is not observed when axr3-1 is transiently expressed in a wild-type background. Taken together these data suggest that it is not the absolute level of axr3-1 that is important, but rather the relative amounts of shy2-2 and axr3-1. This hypothesis is supported by the observation that the apolar root hair phenotype is not observed when expression of both shy2-6 and axr3-1 are transiently induced together, and so, presumably, levels of both proteins fall off together. This suggests an interaction between shy2 and axr3 in regulating root hair development, although not necessarily direct or physical.
These results are consistent with the model outlined above in which the specificity of auxin responses is mediated by the dimerisation network of Aux/IAAs (and ARFs), and hence the transcriptional regulation of downstream genes. However, it is important to note that all these data are derived from the study of dominant mutant proteins. It is unclear whether these alleles are operating through hypermorphic, hypomorphic or neomorphic mechanisms. Therefore, it is difficult to interpret the data to understand the wild-type function of the AXR3 and SHY2 genes. For this reason we also examined loss-of-function alleles and gene expression patterns of AXR3 and SHY2.
The in situ hybridisation data show that the AXR3 gene is expressed in the elongation zone of roots, with expression dwindling into the differentiation zone and the more mature parts of the root. This is consistent with the axr3-1 allele being hypermorphic and the wild-type role for AXR3 being to repress root hair initiation and growth in the elongation zone. In the axr3-1 mutant, the stable axr3-1 protein may persist into the differentiation zone blocking root hair development. The phenotype of axr3-10 root hairs is weak, probably reflecting functional redundancy in the Aux/IAA family. None-the-less the phenotype does support the proposed hypermorphic nature of the gain-of-function alleles, because when grown on medium with no added phosphate, axr3-10 plants show a hyper-induction of root hair elongation compared to wild type, consistent with a wild-type role for AXR3 in suppressing root hair elongation.
A similar case can be made for the SHY2 gene. The phenotype of the shy2-31 mutant is in general the opposite of that conferred by the shy2-2 dominant allele. The roots of shy2-31 plants have fewer root hairs per cell, indicating reduced root hair initiation. Furthermore, the loss-of-function phenotype reveals a minor role for SHY2 in tip growth since root hairs are slightly shorter in the mutant, elongate at erratic rates and show a reduced growth response to phosphate. SHY2 transcript was found to accumulate in the differentiation zone, but transcripts were also detected more apically in the root tip. These data suggest that the dominant shy2 alleles are hypermorphic, and that SHY2 functions in the root tip to promote the initiation of root hair growth and elongation. In the shy2-2 mutant, shy2 protein may accumulate in the elongation zone above a threshold level sufficient to trigger root hair initiation. In this model, the relative amounts of AXR3 and SHY2 would control the timing of root hair initiation on trichoblast cells as they pass through the elongation zone. Initially AXR3 is high relative to SHY2, but as the trichoblasts stop elongating, AXR3 expression is reduced and SHY2 expression increased, resulting in high SHY2 relative to AXR3, and triggering root hair initiation (Fig. 8). Because SHY2 and AXR3 can dimerise with themselves, with each other and with ARFs, it is tempting to speculate that the AXR3:SHY2 ratio is measured directly in the relative abundance of different dimers and hence the relative activity of different ARF-regulated genes. Certainly the data presented here are consistent with this idea.
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ACKNOWLEDGMENTS |
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