1 ZMBP, Entwicklungsgenetik, Universität Tübingen, Auf der
Morgenstelle 3, D-72076 Tübingen, Germany
2 Lehrstuhl für Genetik, Technische Universität München,
Wissenschaftszentrum Weihenstephan (WZW), Am Hochanger 8, D-85350 Freising,
Germany
Author for correspondence (e-mail:
gerd.juergens{at}zmbp.uni-tuebingen.de)
Accepted 16 October 2003
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SUMMARY |
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Key words: GNOM, Guanine-nucleotide exchange factor, Auxin transport, Lateral root formation, Canalisation hypothesis
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Introduction |
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One substance that has been implicated in a large number of growth and
developmental processes is the plant growth regulator auxin. Auxin has been
recognised to be polarly transported in an active, energy-dependent fashion
(Rubery and Sheldrake, 1974).
Since its initial description as the substance responsible for the
differential growth of coleoptiles and phototropic bending some 70 years ago
(Went, 1929
), auxin has been
implicated in many processes, ranging from gravitropic bending to lateral root
formation and root regeneration, patterned outgrowth of leaf primordia,
axillary bud growth and vascular tissue patterning
(Taiz and Zeiger, 1998
).
Importantly, all these processes depend on the ability of auxin to be actively
transported. A notable exception to this is perhaps its most basic function as
a necessary factor for plant cells to continuously divide
(Skoog and Miller, 1957
). More
recently, auxin has also been recognised as being important for embryonic
patterning although its precise role remains to be clarified
(Geldner et al., 2000
).
Loss-of-function alleles of the Arabidopsis GNOM gene (also called
EMB30) lead to severe defects in cell-to-cell alignment, as
illustrated by a highly disordered vascular system, and in the establishment
of the embryonic axis (Mayer et al.,
1993). gnom seedlings invariably lack the most basal
pattern element the root meristem and display variably fused
cotyledons and a generally thickened and stunted axis. GNOM encodes a
GDP/GTP exchange factor for small G-proteins of the ARF class that are
important for coat recruitment and cargo-selective vesicle trafficking
(Steinmann et al., 1999
;
Donaldson and Jackson, 2000
).
Thus, GNOM can be viewed as a regulator of intracellular vesicle trafficking.
A role for GNOM in polar auxin transport was suggested by phenocopies of
gnom seedlings that resulted from treatment of in vitro cultured
embryos of Brassica juncea with auxin or auxin transport inhibitors
(Liu et al., 1993
;
Hadfi et al., 1998
). Later,
the presumed link to auxin transport was supported by two additional lines of
evidence. The putative auxin-efflux carrier PIN1 is mis-localised in
gnom mutant embryos, and moreover, GNOM is involved in the continuous
recycling of PIN1 from endosomes to the basal plasma membrane
(Steinmann et al., 1999
;
Geldner et al., 2001
;
Geldner et al., 2003
).
However, it still cannot be completely ruled out that gnom mutants
might be defective in some basic process of cell polarity establishment,
which, as a secondary consequence, entails defects in auxin carrier
localisation and polar auxin transport
(Shevell et al., 2000
).
A way to genetically distinguish between direct and indirect consequences
of loss of gene activity is to analyse allelic mutants that range from total
loss to subtle decrease in gene activity
(Muller, 1932). For example,
it has often been observed for genes involved in the early development of
Drosophila that the primary function of the gene product can be
inferred from a phenotypic series that correlates with residual gene activity
(Nüsslein-Volhard et al.,
1980
; Roth et al.,
1989
). In the case of gnom, however, all available mutant
alleles give essentially the same grossly abnormal seedling phenotype and thus
do not provide information about the developmental process primarily affected.
We have analysed weak phenotypes of gnom alleles that undergo
post-embryonic development and display graded auxin-related defects. Our
results suggest a primary function of GNOM in canalising auxin fluxes, which
would explain its diverse developmental phenotypes.
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Materials and methods |
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Quantitative analysis of number of root meristematic cells
In order to get an estimate of the number of meristematic cells in the root
tip, cells in the cortical cell file were counted from the cortex/endodermis
initial up to the point where the cells were two to three times longer than
wide. Subjective errors were only one to two cells since the transition to
elongation is apparently very rapid.
GUS staining procedures
Plants were treated with 90% acetone on ice for 30 minutes, then washed
once for 10 minutes in GUS staining buffer and stained at 37°C in darkness
in GUS staining buffer plus X-Gluc. GUS staining buffer
(Malamy and Benfey, 1997): 100
mM sodium phosphate (pH 7), 0.1% Triton X-100, and 0.1-5 mM of each
K3FeIII(CN)6 and
K4FeII(CN)6, depending on the line and signal
strength. X-Gluc was added to a final concentration of 1 mg/ml from a
100x stock dissolved in dimethylformamide, which was freshly
prepared.
Establishment of GNOM-GUS transgenic lines
The GNOM-GUS reporter contruct was obtained by inserting
PCR-amplified GUS open reading frame (ORF) into a 7.5 kb
GNOM genomic fragment containing an AvrII restriction site
at the 3' end of the GNOM ORF, leading to a GNOM-GUS
translational fusion that complemented the mutant phenotype. Cloning and
transformation was done as described for GNOM-myc and
GNOM-GFP constructs (Geldner et
al., 2003). The PCR amplified region of the construct was
sequenced. At least two independent lines were investigated for each aspect of
GNOM expression.
Sequence analysis of mutant alleles
Mutations in gnomR5 and gnomSIT4
were identified by amplifying the genomic region from homozygous mutant
seedling DNA and subcloning it into pGEM. Two independently amplified and
subcloned clones were sequenced and compared with Landsberg erecta
sequence. Identified mutations were confirmed by restriction fragment
polymorphism tests.
Western blot
Western blots were done as described previously
(Lauber et al., 1997).
Proteins were separated on a 7.5% SDS-PAGE gel. Anti-GNOM serum (
GNS)
(Steinmann et al., 1999
) was
diluted 1:4000.
Histological analysis
Clearing of root tissues was done as described previously
(Malamy and Benfey, 1997) or
by mounting roots directly in a chloralhydrate solution and inspecting them
immediately. Aerial tissues were prepared by shaking them for several hours in
ethanol/acetic acid (3:1) at room temperature and then mounting them in
chloralhydrate solution. Embryos were fixed on ice in ethanol/acetic acid
(3:1) for 30 minutes and then cleared in chloralhydrate for inspection.
Whole-mount immunofluorescence
PIN1 antibody was kindly provided by Klaus Palme
(Gälweiler et al., 1998).
Staining was done as described previously
(Lauber et al., 1997
), with
the following modifications in order to increase signals in lateral root
primordia. Roots were slightly squashed before dipping them into liquid
nitrogen. Roots were treated with 3% Driselase solution at 37°C for 90
minutes starting with a 10 minute vacuum infiltration. They were then
permeabilised in 20% DMSO, 3% Nonidet P-40. Primary and secondary antibody
incubations were done at 37°C overnight, again after vacuum infiltration.
All washes and incubations after the fixation step were done in
phosphate-buffered saline (pH 7.4).
Auxin and auxin transport inhibitor treatments
For induction of lateral root formation and for DR5::GUS staining
of primary root tips, 10-20 seedlings grown on plates were transferred into
24-well culture plates containing 1 ml of liquid basal medium supplemented
with auxin, or equal amounts of solvent for the control treatments, and
incubated in a growth room for the indicated times. 100 mM stock solution in
DMSO were used for naphthaleneacetic acid (NAA), dichlorophenoxyacetic acid
(2,4-D) and N-1-naphthylphthalamic (NPA). For NPA-ring experiments seedlings
were transferred onto new plates with their hypocotyl-root junction placed
above a narrow strip of parafilm. A small agar block containing 500 µM NPA
was placed on the parafilm, covering the hypocotyl-root junction of the
seedlings.
Statistical analysis of results
P values indicated for measurements of inflorescence and root lengths,
lateral root densities and gravitropic responses were obtained using a
two-sided Student's t-test assuming unequal variances. P
values indicated for numbers of rosette side branches were obtained using a
2-test after grouping classes 1-2 and classes 6-8. The
P values for the number of collapsed roots were obtained with a
two-by-two
2-test (one degree of freedom). The P
value for the distribution of bolting time was obtained by grouping
measurements into two categories, one before and one after day 20 and applying
Fisher's exact test, calculated at home.clara.net. All other calculations were
done using Microsoft® Excel 2002. All error bars in graphs indicate
standard errors of the mean.
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Results |
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Unlike plants with strong alleles, gnomR5 and gnomB/E plants were able to grow on soil. gnomR5 seedlings developed into extremely dwarfed plants with very small and epinastic rosette leaves (Fig. 2H,I), which often died at the rosette stage, while rare escapers produced an inflorescence with a single, extremely tiny flower (data not shown). gnomB/E plants were only slightly dwarfed and had narrow, curled-down rosette leaves (Fig. 2J,K). gnomB/E plants produced inflorescences although flowering was delayed (Fig. 2L). At maturity, gnomB/E had produced more secondary inflorescences than wild type, which correlated with delayed onset of senescence (Fig. 2M). To better understand the phenotypic differences between gnomB/E and wild type, we performed quantitative growth measurements. gnomB/E formed rosette leaves at a significantly slower rate, which could acount for the delayed onset of flowering (Fig. 2N). In addition, the onset of flowering was much more variable in gnomB/E plants than in wild type (Fig. 2O). At maturity, the primary inflorescences were significantly shorter in gnomB/E than in wild type (Fig. 2P). When investigating the dynamics of secondary inflorescence formation from the axils of rosette leaves (Fig. 2Q), we observed an initial delay in gnomB/E plants. When wild-type plants started to senesce, however, gnomB/E plants continued to form new secondary inflorescences, eventually leading to more rosette side branches and an overall bushy appearance.
Weak gnom alleles produce defects in root meristem maintenance, lateral root formation and gravitropism
Another striking aspect of the gnomR5 phenotype was the
slow rate of post-embryonic root elongation that became apparent after a few
days of growth on plates. Compared to 7-day-old wild-type seedlings,
gnomR5 and gnomB/E displayed a
significantly shortened division zone in the root (7.5±1.1 meristematic
cells in gnomR5 (n=8) as compared to
24.9±3.3 in wild type (n=8), P<0.001)
(Fig. 3A-C). The overall
meristem organisation was nonetheless roughly normal
(Fig. 3D-F). Individual cell
files originated from the centre of the meristem, no supernumerary tissue
layers were observed, and a normally organised root cap was present. The
quiescent centre and surrounding initials were sometimes well ordered
(Fig. 3D) but sometimes fairly
disorganised (Fig. 3E) in both
gnomR5 and gnomB/E, as compared to
wild type (Fig. 3F). After 15
days of growth, however, dramatic differences were observed between
gnomR5 and gnomB/E. Many
gnomR5 roots were completely collapsed, with cell
differentiation occurring at the very root tip, as evidenced by the presence
of elongated cells, differentiated vascular strands and root hairs in that
region (Fig. 3G,J). In
contrast, gnomB/E and wild-type meristems
(Fig. 3J,K) were intact,
although the highly ordered cellular pattern of young roots was not present,
either in the wild type or in gnomB/E (compare
Fig. 3H,K with I,L). Thus,
GNOM function is apparently not only needed for the establishment of
an embryonic root meristem, but also for maintaining the activity of
meristematic cells and preventing their differentiation. Although the collapse
of the root meristem would explain why gnomR5 roots
eventually cease to grow, it cannot account for the short-root phenotype in
7-day-old seedlings, which might rather reflect fewer actively dividing cells
in the meristem (Fig. 3M).
|
gnomR5 root tips have a reduced capacity to maintain auxin gradients in the presence of auxin
In order to assess more directly the auxin transport defects of
gnom in we crossed the DR5::GUS reporter into
gnomR5. The DR5::GUS construct is highly
responsive to auxin, being activated by auxin in all root cells
(Ulmasov et al., 1997;
Sabatini et al., 1999
).
Therefore, DR5::GUS is a useful marker to visualise auxin-response
gradients in the root. In young gnomR5 roots with an
intact meristem, the GUS signal was nearly normal, except for two stripes of
vascular cells that were observed in wild-type upon staining for 1-2 hours
(Fig. 4A,E). As reported
previously (Friml et al.,
2002
), treatment of wild type with low concentrations of the
transportable auxin NAA did not lead to strong alterations in
DR5::GUS staining (Fig.
4B,C). Apparently, the auxin-transport system of the root tip has
a high capacity to maintain response gradients in the presence of exogenous
auxin, which is thought to be due to efficient canalisation of auxin to sites
of degradation in the root tip (Friml et
al., 2002
). At 10 µM NAA, however, the DR5::GUS
distribution changed dramatically. Strong GUS staining appeared in all tissue
layers in the division zone of the root, leaving only a narrow strip of less
stained cells immediately above the columella peak
(Fig. 4D).
gnomR5 root tips were dramatically more sensitive to
exogenous auxin applications. Already at 0.1 µM NAA, strong GUS staining
expanded from the differentiation zone into the division zone
(Fig. 4F). At 1 µM NAA, the
staining pattern very much resembled that of wild type at a tenfold higher
concentration (Fig. 4G, compare with
D). Ten µM NAA resulted in strong GUS staining of the entire
root (Fig. 4H). Thus, the root
meristem defects of weak gnom alleles correlated with a reduced
capacity to transport auxin in the root.
|
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GNOM is required for induction and organisation of lateral root primordia
The inability of gnomR5 seedlings to form lateral roots
might result from insufficient auxin transport from apical tissues.
Alternatively, gnomR5 might not respond correctly to
auxin. To distinguish between these possibilities, we treated
gnomR5 and wild-type roots with different auxin analogues
for 24-48 hours and investigated their response in regard to lateral root
induction. Untreated wild-type roots initiated widely spacing primordia
(Fig. 6A). Treatment with 0.1
µM of the transportable auxin NAA
(Delbarre et al., 1996) reduced
the distance between individual root primordia, increasing their number
(Fig. 6B), but did not affect
their organisation. By contrast, the same concentration of the
non-transportable auxin analogue 2,4-D caused proliferation of pericycle cells
along large regions of the primary root. Individual root primordia were not
distinct any more, and zones of more or less proliferation alternated,
resulting in a `wavy' appearance of the proliferation zone
(Fig. 6C). Thus, transportable
NAA and non-transportable 2,4-D had very different effects on lateral root
induction. Untreated gnomR5 roots were completely devoid
of lateral root primordia and lacked any sign of pericycle division
(Fig. 6D). By contrast,
exogenously supplied NAA induced homogenous proliferation of the pericycle
layer in large regions of the root, and there was no indication of organised
primordia growth, resembling wild-type roots treated with 2,4-D
(Fig. 6E). 2,4-D treatment had
very much the same effect on gnomR5 roots as NAA
treatment, except that the induction of proliferation was even stronger. Thus,
non-dividing pericycle cells of gnomR5 seedlings can be
induced to divide when supplied with auxin, which strongly suggests that the
failure to form lateral root primordia is due to a shortage of auxin in the
primary root. In addition, it demonstrates that GNOM function is required for
two steps of lateral root formation. First, there is a non-autonomous
requirement of GNOM, in transporting auxin from above-ground tissues into the
root in order to initiate lateral root primordia. Second, there is an
autonomous requirement of GNOM in the pericycle layer itself to organise
primordium outgrowth while laterally inhibiting proliferation of adjacent
pericycle cells. The fact that gnomR5 roots responded to
transportable auxin in much the same way as the wild-type did to a
non-transportable auxin suggested that the gnomR5
phenotype may be the result of an inability to properly transport auxin
required for the organised development of lateral root primordia. To correlate
the observed disorganisation of incipient primordia with defects in polar
auxin transport and establishment of auxin-response gradients, we analysed
PIN1 localisation and DR5::GUS activity during lateral root
formation. PIN1 expression was detected from the very beginning of lateral
root development. In a stage I primordium, PIN1 was localised in a strictly
polar fashion along the main root axis
(Fig. 7A). Whether individual
cells had their apical or basal end labelled could not be distinguished. Upon
initiation of periclinal divisions, PIN1 maintained its polar localisation but
was also seen at newly formed cell boundaries
(Fig. 7B). As the root
primordium became multi-layered, its inner cells showed a seemingly apolar
distribution of PIN1 whereas peripheral cells displayed preferential labelling
of PIN1 at the cell boundaries towards the tip of the incipient root
primordium (Fig. 7C). This new
polarity, orthogonal to the old axis of polarity, became more and more
pronounced until PIN1 polar localisation was completely re-orientated towards
the new tip of the emerging lateral root primordium
(Fig. 7D). DR5::GUS
signals were also observed in stage I primordia, with the strongest staining
often located in the centre of the young primordium
(Fig. 7E). Upon periclinal
divisions, this central staining was shifted to a more distal region of the
primordium (Fig. 7F,G). In the
emerging lateral root DR5::GUS activity was strongest distally, which
resembled the situation in the primary root tip
(Fig. 7H)
(Sabatini et al., 1999
). Thus,
the presumed direction of auxin flow, as inferred from PIN1 polar
localisation, was consistent with the establishment of an adjacent maximum
auxin response. A more detailed description of the relationship between
expression and localisation of several PIN proteins and auxin gradient
establishment is given elsewhere
(Benková et al., 2003
).
NAA treatment did not alter PIN1 localisation or DR5-GUS
distribution, except that the DR5::GUS signal was increased (data not
shown). By contrast, 2,4-D treatment induced PIN1 expression in long stretches
of dividing pericycle cells. Although PIN1 localisation was initially similar
to untreated controls (Fig.
7I), it subsequently became more and more randomised and the
gradual re-orientation of polarity did not occur
(Fig. 7J). In parallel, young
and older `primordia' of 2,4-D-treated roots had homogenous DR5::GUS
staining (Fig. 7K,L). Similar
abnormalities of PIN1 localisation and DR5::GUS staining were also
observed in NAA-treated gnomR5 roots. PIN1 localisation,
although initially polar (Fig.
7M), became more and more depolarised and no re-orientation of
polarity was detected (Fig.
7N). In addition, no gradients of DR5::GUS activity were
established at any stage in the developing multi-layered proliferation zone
(Fig. 7O,P). Thus,
gnomR5 is defective in organising polar auxin transport
required for proper initiation and development of lateral root primordia.
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Discussion |
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Nearly all developmental phenotypes in weak gnom lines can be explained by defects in auxin transport
Our phenotypic analysis of the gnomR5 allele and the
weaker gnomB/E allelic combination revealed an impressive
number of auxin-related phenotypes that correlated with residual GNOM
function. Disorganised vascular tissue, fusion of cotyledons, leaf epinasty,
inhibition of leaf blade expansion, dwarfed stature, short roots and
inhibition of lateral root formation were all observed, not only in
gnomR5, but also, to a lesser extent, in the weaker
gnomB/E line. Some of these phenotypes as well as the
observed defect in root gravitropism are clearly auxin transport-mediated
responses, whereas others, such as variably delayed flowering and delayed
senescence cannot be, to our knowledge, immediately attributed to reduced
auxin transport. The stunted primary inflorescence combined with an increased
number of secondary inflorescences superficially resembles reduced apical
dominance as observed in auxin transport mutants
(Noh et al., 2001;
Ruegger et al., 1997
). The
altered dynamics of lateral inflorescence formation, however, might also be a
secondary consequence of delayed senescence or an earlier growth arrest of the
primary inflorescence rather than a direct effect of reduced auxin
transport.
The observed collapse of the primary root meristem in gnomR5 can be explained by an insufficient supply of auxin to the root tip, which would lead to cell-cycle arrest due to auxin depletion and subsequent differentiation. We confirmed this notion by phenocopying the mutant phenotype through NPA treatment of wild-type roots at the hypocotyl-root junction and by partially rescuing the gnomR5 phenotype through auxin application. Furthermore, we demonstrated a reduced capacity of gnomR5 root tips to maintain auxin-response gradients when challenged with auxin. Thus, reduction of GNOM function impairs polar auxin transport in post-embryonic development.
A model of GNOM action that explains the diverse developmental phenotypes of the mutant
We would like to propose a common model of GNOM action to account for three
major aspects of the gnom phenotype: (1) disorganisation of the
vascular tissue, which is gradually relieved in plants with the weaker
alleles, (2) embryonic axis formation defects, which are exclusively observed
in those with strong alleles, and (3) inability to form organised root
primordia in response to auxin, which is apparent in
gnomR5.
We assume that GNOM is a central player in a positive feedback loop between
auxin distribution and transport polarity. Such a feedback loop was postulated
in the canalisation hypothesis, initially proposed to explain vascular tissue
patterning in plants (Sachs,
1988; Sachs,
1991
). It essentially states that auxin can lead to the gradual
establishment of auxin channels in a field of initially homogeneous cells,
resulting in the eventual differentiation of vascular tissues. In this theory,
auxin itself is a limiting factor that induces auxin transport capacity and
polarity. Initially random transport polarities would be orientated and
amplified by inducing adjacent cells to polarise in the same direction, which
would improve transport efficiency along a given vector. Establishment of an
efficient auxin channel would not only increase the probability of inducing
the same polarity downstream but also deplete auxin from surrounding cells,
decreasing their chances to become channels themselves. The Sachs theory has
been discussed in great detail, and it has been pointed out that this theory
can in priniciple be extended to a large number of organogenic processes in
plants (Berleth et al., 2000
;
Berleth and Sachs, 2001
).
In cell-biological terms, this canalisation hypothesis necessitates that a plant cell is able to sense an unequal distribution of auxin and to translate it into an accumulation of efflux carriers at the end away from the external auxin maximum. For efficient polarisation in response to external cues, the cell needs to continuously re-direct vesicular trafficking of carriers to specific regions of the plasma membrane. Mutations in GNOM interfere either with the activities of the carriers per se or their polar localisation (or both at the same time) and could thus disrupt the positive feedback loop needed for the organisation of tissues and organs.
The vascular patterning defects of gnom are consistent with such a
role. This was noted before by Koizumi et al.
(Koizumi et al., 2000), who
identified a new gnom allele in a screen for vascular pattern
mutants, and will therefore not be discussed here. In the following, we want
to describe how a similar mechanism can also account for the gnom
defects in embryo axis establishment and lateral root formation.
Strong gnom alleles produce three major embryonic phenotypes: fusion of cotyledons, general thickening of cotyledons and hypocotyl, and deletion of the root. These phenotypes are simultaneously restored in plants with weak gnom alleles, suggesting a common primary defect that we propose is a grossly perturbed alignment of individual auxin-transport polarities (Fig. 8A). As a consequence, the basal part of the embryo would lack the auxin needed to induce a root meristem. In addition, auxin would accumulate in the presumptive sites of synthesis in the apical part, leading to cotyledon fusion. Also, an insufficient auxin flow through the central part would lead to randomised cell division and expansion, causing thickening of the axis.
|
The model outlined above provides a coherent framework for the diverse roles of GNOM action in development. In the future, the partial loss-of-function gnom alleles may also prove useful to genetically dissect the diverse developmental roles of auxin transport and to explore the applicability of the canalisation hypothesis in organ patterning.
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ACKNOWLEDGMENTS |
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Footnotes |
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