Institute of Molecular Plant Sciences, Leiden University, Clusius
Laboratory, PO Box 9505, 2300 RA Leiden, The Netherlands
* Present address: Department of Botany, University of Toronto, 25 Willcocks
Street, Toronto ON, M5S 3B2, Canada
Author for correspondence (e-mail:
meijer{at}rulbim.leidenuniv.nl)
Accepted 31 October 2002
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SUMMARY |
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Key words: Auxin resistance, Commissural veins, Cytokinin hypersensitivity, DR5, Embryo mutant, Oryza sativa, Oshox1, Procambium, RAL1, Venation pattern
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INTRODUCTION |
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Although the influence of various plant hormones in promoting vascular
differentiation has been reported (e.g.
Aloni, 1987), the role of auxin
is unique. Auxin application not only triggers vascular differentiation per
se, but also induces the differentiation of a slender strip of cells into a
continuous vascular strand that extends towards the basal pole of the plant
(Sachs, 1981
). Experimental
evidence suggests that polar transport of auxin is directly responsible for
the directionality of the vascular response
(Sachs, 1981
). However, proper
auxin perception and response should nevertheless be essential for the relay
of auxin signals in vascular differentiation. Consistently, vascular
abnormalities have been reported for auxin response mutants with closely
related primary defects. Mutations at three Arabidopsis loci,
MONOPTEROS (MP), BODENLOS (BDL) and
AUXIN-RESISTANT6 (AXR6), result in a complex phenotype
characterised by an impaired auxin perception or response, a severely reduced
vascular system, and defective embryo axis formation with consequent failure
to produce an embryonic root (Berleth and
Jürgens, 1993
; Przemeck
at al., 1996
; Hamann et al.,
1999
; Hobbie at al.,
2000
). These common features suggest related primary defects in
the molecular machinery underlying the alignment of cell differentiation with
the axis of auxin flow at various developmental stages. Strong support for
this also comes from the identification of the MP gene, which encodes
a transcriptional regulator of the auxin response factor family that is
specifically expressed in the vasculature
(Hardtke and Berleth, 1998
).
Importantly, the DNA-binding domain of the MP protein appears to interact with
auxin response elements, short conserved sequences essential for the rapid
auxin regulation of certain classes of auxin inducible genes
(Ulmasov et al., 1997a
). The
recent finding that the BDL gene encodes a member of the Aux/IAA
family of proteins that would interact with MP to provide the proper auxin
response that is necessary for embryo patterning strengthens further the link
between auxin response, embryo axialisation and vascular patterning
(Hamann et al., 2002
).
The striking association between impaired embryo axis formation, reduced
vascularisation and defective auxin sensitivity in the dicot
Arabidopsis, prompted us to investigate whether the same relationship
could also underlie vascular pattern formation in monocots, despite the fact
that dicot and monocot leaves have highly divergent vascular patterns
(Nelson and Dengler, 1997).
Most dicot leaves show a reticulate pattern of highly branched veins, whereas
most monocot leaves show a typical striate venation pattern, in which major
veins lie parallel along the proximodistal axis of the leaf, and largely lack
major vein branching. Furthermore, vascular ontogeny in monocots and dicots
shows fundamental differences. For example, in dicots the primary vein extends
progressively from the stem vasculature into the leaf primordium, and
secondary veins develop in continuity with the primary vein. In contrast, in
monocot leaf primordia parallel veins arise isolated from each other and from
the stem vasculature.
In a previous study, Nagato and co-workers classified 188 embryo mutants of
rice (Hong et al., 1995).
Using the recessive radicleless1 (ral1) mutant from this
collection, we provide genetic evidence that auxin sensitivity is associated
with embryonic root development and vascular pattern formation in a monocot
species. Furthermore, we show that these alterations are coupled to an altered
sensitivity to cytokinin. Our investigations indicate that the RAL1
gene has an early function in the establishment of vascular patterns during
embryonic and post-embryonic development as well as an important role in the
proper response to auxin and cytokinin.
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MATERIALS AND METHODS |
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Plants and growth conditions
Oryza sativa (L.) Japonica cultivar Taichung 65, in which
background the ral1 (odm40) mutant allele was induced
(Hong et al., 1995), was used
as a wild-type control strain in all studies. Upon outcrossing of the
ral1 homozygous mutant to wild type, the heterozygous F1
population did not show any obvious morphological difference from wild type
(data not shown). Furthermore, in the F2 population, the
radicleless phenotype behaved as a recessive trait, having frequencies of
segregation significantly close to 25%
(Hong et al., 1995
) (our
unpublished observations).
All seeds were surface sterilised (Rueb
et al., 1994) and germinated in the dark at 28°C for 4 days on
half-strength Murashige and Skoog (MS) medium in which MS vitamins were
replaced with B5 vitamins and supplemented with 10 g/l sucrose and 7 g/l
agarose (replaced by 2.5 g/l phytagel for seedlings that had to be transferred
to the greenhouse). Germinated seeds were grown in a 12 hours light: 12 hours
dark cycle at 28°C. Embryonic calli induced on scutella from germinated
seeds were transformed with Agrobacterium tumefaciens strain LBA4404
(Ach5 pTiAch5
T-DNA) or LBA1119 (C58 pTiBo542
T-DNA) harbouring
the DR5-GUS binary vector as described previously
(Scarpella et al., 2000
).
Seedlings and regenerated transgenic plantlets were transferred to the
greenhouse and grown in hydroponic culture with a regime of 12 hours light,
28°C, 85% relative humidity and 12 hours dark, 21°C, 60% relative
humidity. Genetic crosses were performed to introduce the Oshox1-GUS transgene
(Scarpella et al., 2000
) into
the ral1 mutant background. Flowers of ral1 plants were
emasculated by submerging whole inflorescences in a water bath at 42°C for
6 minutes. Inflorescences were subsequently blotted dry on filter paper and
flowers that opened on either the day of the treatment or the following day
were fertilised by applying pollen from flowers of Oshox1-GUS plants at
anthesis. As a wild-type control for the expression of the transgene in the
ral1 mutant, the Oshox1-GUS expression pattern was analysed in the
Taichung 65 background and found to correspond to the previously reported
expression pattern in Taipei 309
(Scarpella et al., 2000
).
Tissue culture assays
The ability of seedlings to form callus tissues was assayed by germinating
seeds on callus-induction medium supplemented with 2 mg/l
2,4-dichlorophenoxyacetic acid (2,4-D) as described previously
(Rueb et al., 1994). Callus
tissue growth properties were evaluated by transferring callus pieces of
approximately 2-3 mm in diameter to new callus-induction medium supplemented
with either 1 or 2 mg/l 2,4-D. In both callus induction and callus growth
experiments the response was monitored weekly during a 1-month period. The
capability of callus tissues to regenerate plantlets was assayed by
transferring callus pieces of approximately 2-3 mm diameter to LS basal
medium, to which 40 g/l sucrose and 7 g/l agarose were added and supplemented
or not with 0.3 mg/l N6-benzyladenine. The response was evaluated
monthly during a 3-month period. Calli were transferred to new medium after
each monthly examination.
Microtechniques and microscopy
Dissected samples or 100-µm vibratome sections were fixed overnight in
2% glutaraldehyde and embedded in glycol methacrylate as described
(Scarpella et al., 2000).
Sections (10 µm) were dried onto slides and stained with 0.05% Toluidine
Blue O in 50 mM citrate buffer pH 4.4 before mounting in epoxy resin for
microscopic observation using bright-field optics. Whole-mount cleared
preparations were obtained by autoclaving dissected samples in 80% lactic acid
for 20 minutes at 121°C. Samples were mounted in fresh 80% lactic acid and
viewed with dark-field optics. Histochemical detection of ß-glucuronidase
(GUS) activity was performed on freshly dissected plant organs or 100 µm
vibratome sections. Samples were permeabilised in 90% acetone for 1 hour at
-20°C, washed twice under vacuum for 5 minutes with 100 mM phosphate
buffer pH 7.5-7.7, 5 mM potassium ferricyanide, and incubated at 37°C in
100 mM sodium phosphate buffer pH 7.5-7.7, 10 mM sodium EDTA, 5 mM potassium
ferrocyanide, 5 mM potassium ferricyanide, 2 mM
5-bromo-4-chloro-3-indolyl-ßD-glucuronic acid (X-gluc; Biosynth
AG). Reaction was stopped in 70% ethanol after 30 minutes (DR5-GUS roots), 2
(DR5-GUS shoots) or 16 (Oshox1-GUS) hours. Samples were either viewed
immediately or fixed in ethanol:acetic acid 3:1 and mounted in chloral
hydrate:glycerol:water 8:3:1 before observation with bright-field or
differential interference contrast optics. All samples were observed with a
Zeiss Axioplan 2 Imaging microscope or with a Leica MZ12 stereoscopic
microscope. Images were acquired with a Sony 3CCD digital photo camera
DKC-5000. All images were processed using Adobe Photoshop 5.0.
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RESULTS |
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In conclusion, the RAL1 gene is required in the embryo for the formation of the basal pattern elements and for the orderly development of continuous procambial strands of all orders.
The ral1 mutation affects different aspects of plant
vegetative and reproductive development
To assess possible post-embryonic functions of the RAL1 gene, we
generated adult mutant plants, exploiting the capacity that mutant seedlings
share with wild type of spontaneously producing adventitious roots
(Fig. 2B,H). However, the
ral1 mutant develops fewer adventitious and lateral roots than the
wild type (Fig. 2C,I; Table 1). Roots of
ral1 seedlings are more slender than either wild-type seminal or
adventitious roots, because of a reduction in the number of xylem and phloem
poles in the central vascular cylinder, and in the number of cortical cell
layers (Fig. 1U-X;
Table 1). Furthermore, the
diameter of the metaxylem elements is reduced, whereas that of the cortical
cell is increased (Fig. 1W,X;
Table 1). Finally, in
ral1 roots, obvious deviations from the wild-type pattern of
alignment of vascular elements, or interruptions in their files were never
observed, when examined at the procambial stage or after differentiation (not
shown).
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At maturity, ral1 plants are smaller, show increased apical dominance and have shorter leaves (Fig. 2D,J). Internodes of ral1 plants are thinner, but there are significantly more vascular bundles that are closer to each other than in wild type (Fig. 1P,T, Table 1). Inflorescences of ral1 plants produce normal looking, fertile spikelets (flowers) together with abnormal spikelets in an approximate ratio of one to three (Table 1). Abnormal spikelets in ral1 appear narrower (Fig. 2E,K), because of the reduced development of the palea, the smaller of the two bracts enclosing the floral organs. Instead of the normal boat shape, in ral1 this bract shows a flat triangular shape and is completely devoid of any vasculature (not shown). Finally, the abnormal spikelets differentiate four or five stamens, instead of the invariable six of wild type (Fig. 2F,L). The inflorescences of ral1 do not differ with respect to the length of their axes or the number of primary branches, whereas the number of secondary branches per primary branch is significantly reduced (Table 1).
In summary, the RAL1 gene acts on different aspects of post-embryonic organ development during both the vegetative and the reproductive phases. With regard to vascular development, we observed a reduction in the size of the vascular cylinder in the root, an alteration in the spatial arrangement of vascular bundles in the stem, and the absence of veins in one of the two floral bracts.
Leaf venation pattern is altered in the ral1 mutant
The leaves of ral1 plants appear normal in shape, but are smaller
than in wild type (Table 1).
Wild-type rice leaves show the typical striate venation pattern, in which
major longitudinal veins of three orders, the midvein and the large and small
veins, lie parallel along the proximodistal axis of the leaf, and are
connected transversely by minor commissural veins
(Kaufman, 1959). The
distribution and arrangement of these classes of veins follow a highly regular
pattern, which can be described by a series of venation pattern parameters, as
indicated in Table 1. A
comparison between mature wild-type and ral1 leaves revealed that all
the venation pattern parameters are altered in the mutant
(Table 1). In fact, the number
of both the large veins and the small veins between two large veins is reduced
(Fig. 1K,L). Furthermore, the
distance between two adjacent longitudinal veins is reduced. Conversely, the
distance between two adjacent commissural veins is increased, as is the area
enclosed by two adjacent longitudinal veins and two adjacent commissural
veins. Finally, four of the seven small veins normally present in the
wild-type midrib region (Fig.
1M,N) are absent in ral1
(Fig. 1Q,R). The alterations in
vascular pattern parameters observed in ral1 might result from a
premature arrest in leaf development. According to this interpretation, mature
ral1 leaves would simply represent immature stages of wild-type
leaves. To test this hypothesis, we examined the distance between adjacent
longitudinal vascular bundles in a representative wild-type immature leaf
population of either the same length or width as mature ral1 leaves.
The fact that there is no wild-type leaf population with both the same length
and width as the ral1 leaves suggests that the hypothesis of
prematurely arrested development is not valid. Furthermore, in both cases the
distance between longitudinal veins in ral1 leaves was significantly
smaller than that in wild-type (Table
1), indicating a fundamental alteration of their normal spatial
regularity.
When analysed in transverse sections, all vascular bundles in ral1 leaves showed the typical radial organisation of vascular tissues, with xylem towards the adaxial surface and phloem oriented towards the abaxial one. Furthermore, as in the root vascular cylinder, the diameter of (late) metaxylem elements was reduced in all leaf vascular bundles (Fig. 1O,S; Table 1). Finally, a smaller bundle sheath extension and subepidermal sclerenchyma was consistently observed in association with longitudinal veins of all orders (Fig. 1K-O,Q-S).
Taken together, these observations indicate that the ral1 mutation affects the normal spatial arrangement of both longitudinal and commissural veins in the leaf, without altering their radial patterning. Moreover, the RAL1 gene seems to be required for the correct development of non-vascular cell types organised around the veins.
Commissural vein development is altered in ral1 leaves
In ral1 leaves, the majority of the commissural veins can be
classified as normal, in that, as in wild type
(Fig. 3A,M,N), they develop a
single uninterrupted connection with each of the two adjacent longitudinal
veins. However, in approximately 40% of the commissural veins in the mutant
leaves we could observe a range of aberrations that were tentatively grouped
in three classes. The first class comprises interrupted commissural veins
associated with one (Fig. 3B,C)
or two (Fig. 3D,E) longitudinal
veins. The interruptions can end with either a single
(Fig. 3B,D) or a bunch of xylem
elements (Fig. 3C,E). In the
second class, we grouped together commissural veins that develop two
connections with one of the two longitudinal veins, and that therefore we
refer to as `Y' veins (Fig.
3F-K). Such Y veins can develop without any interruptions
(Fig. 3F), or show
discontinuities at different locations
(Fig. 3G-K). Finally, the third
class consists of isolated patches of xylem elements that form in the
interveinal region, named vascular islands
(Fig. 3L). The commissural vein
defects in ral1 were observed by dark-field illumination of cleared
intact tissues, which reveals the presence of xylem elements, but not of
procambial cells or other vascular cell types, such as phloem elements.
Therefore, we examined the ends of the interrupted commissural veins in
paradermal tissue serial sections of mature leaves and confirmed that these
are not connected by any (pro)vascular cell file
(Fig. 3O,P). Additionally, when
analysed in transverse section, even the most aberrant commissural veins
showed the typical radial organisation of xylem and phloem within the strand
(not shown).
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In conclusion, these results indicate that the ral1 mutation affects the continuity of commissural veins and induces atypical branching in these veins without altering their radial tissue organisation.
Procambium formation during leaf development is delayed in
ral1
To identify the earliest differences between wild-type and ral1
vascular development, we decided to follow this process during leaf primordium
formation. To this aim, we compared wild-type and ral1 primordia
close to their insertion onto the shoot apex, where differentiating vascular
strands are in their most advanced stage of development. In wild type, a
median procambial strand could be identified in the first primordium
(Fig. 4A). In the second
primordium, the median strand started to undergo vascular differentiation, as
shown by the presence of protophloem elements
(Fig. 4B). Finally, in the
third primordium, the first two protoxylem elements had differentiated
(Fig. 4C). In ral1, no
anatomical sign of a median procambial strand could be detected in the first
primordium (Fig. 4D), but a
median strand, anatomically indistinguishable from that in wild type, could be
detected in the second primordium (Fig.
4E). Therefore, in ral1, procambial strand formation
during leaf primordium development is delayed compared with wild type.
However, the median strand in the third primordium of ral1 appears to
be at the same differentiation stage as in wild type, judging from the
presence of protophloem and two protoxylem elements
(Fig. 4F). This suggests that
vascular differentiation occurs more rapidly in ral1 than in wild
type. In fact, whereas in wild type two plastochrons divided the formation of
a procambial strand from the stage where protophloem and two protoxylem
elements could be distinguished, in ral1 the same process required
one plastochron only. Furthermore, in ral1 leaf primordia, procambial
strands arise significantly (P<0.001) closer to each other (126.0
µm±3.5, n=10) than in wild type (182.3 µm±6.9,
n=10).
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In summary, the RAL1 gene is required for the initiation of procambial strands at the correct stage of leaf primordium development. However, the delayed procambial formation in ral1 seems to be compensated for by faster vascular differentiation.
Procambial expression of the auxin-responsive DR5-GUS marker is
absent in ral1
To reveal possible other developmental differences in the (pro)vascular
strands of wild type and ral1, we monitored DR5-GUS expression. The
endogenous and inducible pattern of expression of this marker has been used to
monitor auxin responses at the cellular level
(Sabatini et al., 1999). In
wild type, DR5-GUS expression was clearly observed in the median procambial
strand of the first primordium, and from that stage it marked the presence of
all strands as soon as they could be anatomically identified
(Fig. 4G). In ral1,
procambial DR5-GUS expression was only ever observed in the two procambial
strands next to the differentiating midvein in the third leaf primordium
(Fig. 4H). Furthermore, DR5-GUS
expression during vascular differentiation is also altered in ral1.
In fact, in vascular strands of the fourth leaf primordium, DR5-GUS is
expressed in differentiating xylem and metaphloem in wild type
(Fig. 4O), whereas in
ral1, expression is restricted to the differentiating third
protoxylem element (Fig. 4S).
In vascular bundles of the fifth leaf primordium, DR5-GUS expression is
restricted to differentiating metaphloem in wild type
(Fig. 4P), whereas in
ral1, expression is additionally detected in protoxylem parenchyma
(Fig. 4T).
In conclusion, the lack of DR5-GUS expression in early procambial development indicates that in leaves the RAL1 gene is required for the procambial subdomain of DR5-GUS expression, and suggests that in ral1, (pro)vascular strands that are anatomically indistinguishable from wild-type ones have a reduced endogenous response to auxin.
Expression pattern of the procambium specification marker Oshox1-GUS
is altered in ral1
To further analyse the nature of the vascular defects of ral1, we
monitored the expression of a second marker, the Oshox1-GUS gene reporter. The
onset of Oshox1-GUS expression marks a stage in procambium development at
which cell fate has been specified, but not stably determined, towards
vascular differentiation (Scarpella et
al., 2000). Oshox1-GUS expression therefore can visualise
differences in developmental potential of procambial cells, even in the
absence of any anatomical difference. In wild type, Oshox1-GUS expression
could be first detected in the median strand of the second primordium
approximately 100 µm above its insertion onto the SAM (103.8
µm±14.6, n=5). Therefore, it was clearly visible in this
strand in a section taken at the level of the SAM, that is one plastochrone
after procambium formation (Fig.
4I). In ral1, we could not detect significant differences
in the onset of Oshox1-GUS expression. In fact, in ral1 Oshox1-GUS
expression could be first detected in all strands of the third leaf
primordium, that is again one plastochrone after their emergence
(Fig. 4J). However, Oshox1-GUS
expression during vascular differentiation is altered in ral1. In
wild type, Oshox1-GUS expression remains present in all vascular cells,
eventually disappearing only in the specific elements that (selectively) lose
their cellular contents upon terminal differentiation (xylem tracheary and
phloem sieve elements; Fig.
4Q,R). In contrast to wild type, Oshox1-GUS expression in
ral1 is absent from the mature phloem in the fourth and fifth leaf
primordium, and the level of expression is much lower in all vascular cell
types (Fig. 4U,V). Therefore,
the RAL1 gene is required for the correct Oshox1-GUS expression
pattern in different subpopulations of differentiating and differentiated
vascular cells, although it seems not to be required for the onset of
Oshox1-GUS expression in the procambium.
Finally, in ral1 embryos Oshox1-GUS expression was absent from the aberrant provasculature of the scutellum (Fig. 4X,Z), whereas expression marked the complete procambial system of wild-type embryos (Fig. 4W,Y). Furthermore, in ral1, Oshox1-GUS expression was much reduced in the procambial bundle that arises from the shoot apical region and that was anatomically indistinguishable from wild type. These observations may either point to an impaired procambium specification in the ral1 embryo or be a reflection of a delay in procambium initiation during embryogenesis.
Commissural vein defects in ral1 leaves originate at the
procambial stage
The interruptions in commissural veins of ral1 could originate
either directly from a discontinuous procambium formation or from a subsequent
reversion of procambial cell identity within a continuous procambial strand.
All anatomical studies of commissural vein development in monocot leaves
strongly suggest that in each of these veins all procambial cells appear
simultaneously, such that the commissural procambial strand is formed at once
in a continuous fashion between the longitudinal veins
(Kaufman, 1959;
Blackman, 1971
;
Dannenhoffer and Evert, 1994
;
Dengler et al., 1997
) (our own
observations). However, unlike all other procambial strands, the early stages
of commissural procambial strand formation cannot always be unambiguously
distinguished in tissue sections of developing leaf primordia. Because of the
absence of procambial DR5-GUS expression in ral1, Oshox1-GUS is
currently the earliest available marker of procambial identity in rice. In
wild type, Oshox1-GUS expression appeared simultaneously in all the procambial
cells connecting two adjacent longitudinal veins
(Fig. 4K). Oshox1-GUS
expression also appeared simultaneously in the developing commissural veins of
ral1, but almost invariably there were interruptions in continuity
(Fig. 4L), ectopic expression
(Fig. 4M) or isolated patches
of expression in the interveinal regions
(Fig. 4N). Each of these
aberrations could be related to one of the classes of defects observed in
mature commissural veins, namely interrupted veins, Y veins and vascular
islands. Since the continuous expression of the Oshox1-GUS marker correctly
predicts the differentiation of both the uninterrupted veins in wild type and
of the uninterrupted Y veins in ral1, it is likely that the
interrupted commissural veins and the vascular islands in ral1 may
result from discontinuities in procambium formation, with consequent
fragmented Oshox1-GUS expression. Furthermore, the fact that procambium
interruptions could indeed be detected in ral1 embryos also argues in
favour of this hypothesis.
Taken together, these observations indicate that the aberrations in commissural vein development in ral1 leaves derive from defects occurring at the procambium stage. However, we cannot discriminate between whether they arise during procambium formation or cell fate specification.
The ral1 mutant displays reduced sensitivity towards
auxin
Because of the importance of auxin in various aspects of vascular
development, and because of the phenotypic similarities between ral1
and the auxin-resistant mp, bdl and axr6 Arabidopsis
mutants, it is conceivable that the ral1 phenotype may be related to
alterations in the perception or response to auxin. The first indication of an
impaired auxin response in ral1 was the absence of DR5-GUS expression
in procambial strands of mutant leaf primordia, as described above. We
therefore tested this hypothesis by examining the capacity of ral1
seedlings to form callus tissues in response to the auxin analogue 2,4-D. In
wild type, the first signs of callus formation were detected at the level of
the scutellum and at the base of the shoot 1 week after germination of the
seeds in the dark on a medium containing 2 mg/l 2,4-D
(Fig. 5A), and after 3 weeks
massive callus production was observed
(Fig. 5C). Callus induction in
ral1 was delayed over a week (Fig.
5B,D), and the response to 2,4-D was spatially restricted, in that
the scutellum showed complete insensitivity towards 2,4-D-induced callus
formation (Fig. 5D). When calli
were explanted to new medium, growth in ral1 appeared slightly
enhanced compared with wild type (Fig.
5E,F,I,J). This can be explained by the fact that in rice the
optimum concentration of 2,4-D for callus induction is higher (2 mg/l
2,4-D) than for callus growth (1 mg/l 2,4-D)
(Yatazawa et al., 1967
).
Indeed, the enhanced growth of ral1 calli could be phenocopied by
growing wild-type calli with a lower concentration of 2,4-D (1 mg/l;
Fig. 5M,N). This suggests that,
like callus induction, callus growth in ral1 is less sensitive to
2,4-D. In addition to the growth pattern, callus tissue organisation is also
altered in ral1, the most obvious difference being the proliferation
of somatic proembryonic structures at the periphery of the callus. This
characteristic is absent from wild-type calli grown on 2 mg/l 2,4-D, but can
be induced in wild-type calli grown on 1 mg/l 2,4-D
(Fig. 5O,P), although to a
lesser degree than in ral1 calli grown on 2 mg/l 2,4-D.
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In order to obtain further independent evidence of the defects in auxin perception or response in ral1, we compared the effectiveness of exogenous auxins to inhibit root elongation in wild-type and ral1 seedlings. Roots of ral1 seedlings elongate less than both wild-type seminal and adventitious roots (Table 1). However, ral1 roots elongate approximately 2-fold more than wild-type roots in the presence of 0.05 µM 2,4-D and 1.5-fold more than wild-type roots in the presence of 0.1 µM of the auxin analogue NAA (Fig. 5Y). This reduced sensitivity of ral1 roots to auxin analogues could also be shown using the auxin-responsive DR5-GUS reporter. In wild-type seminal and adventitious roots, we could detect a peak of DR5-GUS expression in all columella cells of the root cap and in the quiescent centre (Fig. 5Q). Additionally, we could detect a fainter procambial expression. In ral1 roots, DR5-GUS expression was restricted to the mature columella cells (Fig. 5R), suggesting that in roots, as in leaves, the RAL1 gene is required for DR5-GUS expression in the procambium. After treatment with 0.1 µM NAA or with 0.05 µM 2,4-D, DR5-GUS expression was ectopically induced in wild-type roots, but not in ral1 (Fig. 5S,T). This defect in DR5-GUS inducibility could be largely rescued by increasing the concentration of auxin analogues to 1 µM (Fig. 5U,V). Consistently, the root elongation response in ral1 was indistinguishable from wild type at concentration of auxin analogues of 1 µM (not shown).
In conclusion, multiple and independent lines of evidence indicate that the RAL1 gene is required for different spatial and temporal aspects of a proper auxin perception or response.
The ral1 mutant displays enhanced sensitivity towards
cytokinin
Auxin and cytokinin interact in a complex fashion to control many aspects
of plant development. More specifically, a large number of studies suggest a
role for these two plant hormones in vascular tissue differentiation
(Sachs, 1981;
Aloni, 1995
;
Fukuda, 1996
;
Berleth et al., 2000
;
Mähönen et al.,
2000
; Inoue et al.,
2001
). Therefore, we decided to investigate whether the
ral1 mutation interferes with cytokinin perception or response. Shoot
regeneration via direct organogenesis from callus tissues is a convenient
system to test this hypothesis, in that regeneration can be stimulated by
cytokinin application (Skoog and Miller,
1957
; Sugiyama,
1999
; Sugiyama,
2000
). Addition of the cytokinin N6-benzyladenine (BA)
to the regeneration medium increased the number of shoots that differentiated
from wild-type rice calli (Fig.
5W,X). Surprisingly, in spite of their enhanced embryogenicity
(Fig. 5K,L), ral1
calli produced fewer shoots than wild type calli
(Fig. 5W). Furthermore, when BA
was added to the medium, shoot regeneration from ral1 calli was
virtually abolished (Fig. 5X).
Even prolonged culture of ral1 calli on medium with or without BA did
not improve shoot organogenesis, suggesting that the reduced regeneration
capabilities were not simply due to a delay in the onset of the developmental
programme that leads to shoot organogenesis. Unlike shoot regeneration, root
formation was inhibited in wild-type calli by the presence of cytokinin
(approximately 47.4% inhibition; Fig.
5W,X). Consistently with their enhanced sensitivity to
cytokinin-induced shoot formation, root organogenesis in ral1 calli
was more inhibited in the presence of BA compared with wild type
(approximately 86.7% inhibition; Fig.
5W,X).
In summary, these observations indicate that shoot and root development via direct organogenesis in ral1 calli are hypersensitive to cytokinin.
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DISCUSSION |
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The ral1 embryo displays specific and reproducible alterations of
the spatial arrangement of the procambium that point to a role for the
RAL1 gene in controlling cell axialisation in this tissue. In fact,
in the scutellum of the ral1 embryo, the primary procambial bundle is
reduced to a short, narrow and discontinuous strand. Furthermore, within this
prematurely aborted strand, procambial cells are misaligned with respect to
each other. Finally, secondary procambial strands are completely absent. In
Arabidopsis, it has been observed that mutations at the
MONOPTEROS (MP), COTYLEDON VASCULAR PATTERN
(CVP) 1 and 2, SCARFACE (SFC) and
VASCULAR NETWORK3 (VAN3) loci show largely intact primary
procambial veins in the cotyledons, whereas secondary procambial veins are
discontinuous or missing (Berleth and
Jürgens, 1993; Carland et
al., 1999
; Deyholos et al.,
2000
; Koizumi et al.,
2000
). It has been proposed therefore that primary vein formation
might be under the control of a different pathway than that specifying
patterning of veins of higher order
(Deyholos et al., 2000
), or
that the regulatory systems controlling the formation of different classes of
veins display a different genetic robustness
(Koizumi et al., 2000
). The
ral1 mutant is unique in that the embryonic scutella, which are
homologous to the cotyledons of dicot embryos, are also defective in the
continuity of primary procambial veins. Mutation in a single gene is thus
sufficient to affect the patterning of both the primary vein and higher-order
veins. This suggests that the pathways that specify the orderly formation of
different orders of procambial strands are not necessarily genetically
separated in monocots, at least during embryogenesis.
At post-embryonic stages, mutation in the RAL1 gene affects the
overall leaf venation patterning. Opposite effects were observed on
longitudinal vein spacing (decreased) versus commissural vein spacing
(increased). This might be related to differences between the patterning
processes of these two types of veins and/or the separation of these processes
in time (Blackman, 1971;
Dannenhoffer et al., 1990
;
Dannenhoffer and Evert, 1994
;
Dengler et al., 1997
). The
venation pattern alterations in ral1 represent a genuine effect that
is not a consequence of prematurely arrested leaf development or a defect in
leaf morphogenesis. Early leaf development coincides with major vein
appearance, and many of the Arabidopsis and maize leaf shape mutants
display vascular patterning aberrations, suggesting that the same factors may
play a regulatory role in both processes, or that one influences the other
(Dengler and Kang, 2001
;
Schneeberger et al., 1995
;
Semiarti et al., 2001
;
Scanlon et al., 2002
).
Similarly to the mp, cvp2, sfc, van3 and the hemivenata
(hve) mutants of Arabidopsis
(Przemeck et al., 1996
;
Candela et al., 1999
;
Carland et al., 1999
;
Deyholos et al., 2000
;
Koizumi et al., 2000
),
mutation in the RAL1 gene specifically affects the vascular pattern
of the leaf without causing any major alteration of leaf shape, thus arguing
for a specific role of the RAL1 gene in the regulation of vascular
pattern formation. In further support of this is the observation that the
earliest detectable defect in ral1 leaf histogenesis is the delayed
formation of procambial strands. Such delay in procambial strand formation has
never been reported before for other mutants and provides evidence that
procambium initiation and leaf primordium development can be genetically
uncoupled. We have shown here that in wild-type rice, the onset of the
expression of the auxin-inducible reporter DR5-GUS presages the sites of
vascular differentiation. The procambial strands that are eventually formed in
ral1 leaf primordia, although anatomically indistinguishable from
wild type, display a reduced endogenous response to auxin, as shown by the
lack of DR5-GUS expression. This might indicate that, in ral1,
procambium is formed through an alternative pathway that would compensate for
the reduced or lost RAL1 gene function. This rescue mechanism would
involve genes able to take over at least part of the RAL1 gene
function in procambium formation. This interpretation might also explain the
absence of defects in continuity of longitudinal veins in ral1
leaves, and fit with the idea that functionally redundant mechanisms would
control the formation of lower order of veins in dicot leaves
(Koizumi et al., 2000
).
After delayed procambial strand formation, vascular differentiation seems
to occur more rapidly in ral1 than in wild type. This might explain
why we invariably observed a reduction in xylem element diameter. In fact, a
narrow element could result from a rapid secondary wall differentiation, which
would allow only limited time for cell expansion
(Aloni and Zimmermann, 1983).
Furthermore, analysis of the spatial and temporal aspects of the expression of
the DR5-GUS and Oshox1-GUS markers during vascular development suggests that
different subpopulations of procambial cells within one strand undergo
vascular differentiation at different time points than in wild type. This
could indicate that the RAL1 gene has a function in the coordinated
entrance of different, but anatomically indistinguishable, subsets of
procambial cells into the vascular differentiation pathway. However, the
relevance of such hypothetical synchronised process is not clear, in that in
ral1, even in the most aberrant veins, all vascular cell types seem
to be present at maturity.
Whereas the continuity of longitudinal veins in ral1 is not
affected, severe defects are present in commissural vein development, which
eventually result in strand discontinuities, formation of aberrantly branching
veins and development of isolated patches of vascular cells. Using the
Oshox1-GUS reporter construct as a marker for procambial cell fate
specification, we showed that, similar to the defects in global patterning of
different orders of veins, the aberrations in commissural vein development
originate at the procambial stage. The alterations in the earliest signs of
Oshox1-GUS expression perfectly simulate the range of commissural vein
phenotypes that can be detected in mature ral1 leaves. However,
although virtually all developing commissural procambial strands in
ral1 displayed such aberrations in Oshox1-GUS expression, when
ral1 leaves were analysed at maturity, no more than 40% of all
commissural veins displayed any detectable defect. This could suggest that
early defects occurring at the procambial stage can somehow be rescued during
vascular differentiation, as discussed above. This observation is in perfect
agreement with the high level of flexibility that vascular tissues have been
reported to display under different experimental conditions (e.g.,
Sachs, 1981;
Sachs, 1989
;
Mattsson et al., 1999
;
Sieburth, 1999
).
In the embryo, mutation in the RAL1 gene seems to have a more
dramatic effect on vascular development than in post-embryonic stages. In
fact, in ral1 embryos all orders of veins are affected in their
development and display altered levels of Oshox1-GUS expression. This could
suggest that the proposed rescue mechanism may play a role in the
normalisation of the early vascular defects in the ral1 mutant by
partially replacing RAL1 gene function in postembryonic vascular
development, but not during embryogenesis. Alternatively, the function of the
RAL1 gene could be predominantly embryonic, and its role during
post-embryonic stages may become restricted to a subset of functions in
vascular development. Unlike the ral1 mutant, mp, cvp2, sfc
and van3 Arabidopsis mutants show defects in leaves similar to those
in cotyledons (Przemeck et al.,
1996; Carland et al.,
1999
; Deyholos et al.,
2000
; Koizumi et al.,
2000
). In this regard, it is interesting to notice that scutella
show a vascular pattern that is more similar to that of dicot cotyledons than
that of monocot leaves, in that the primary vein shows apical branching.
Furthermore, auxin-induced callus formation readily occurs in embryonic or
post-embryonic foliar organs with a branched venation pattern [monocot
scutella and dicot cotyledons and leaves
(Schmidt and Willmitzer, 1988
;
Rueb et al., 1994
)], whereas
foliar organs with a striate venation pattern (monocot leaves) do not form
callus in response to auxin (Wernicke et al., 1981). Therefore, factors
upstream of RAL1 gene function, such as organ-specific auxin
sensitivity and growth pattern, could be involved in determining the type of
vascular pattern that will be eventually formed in leaves or scutella,
possibly by a differential regulation of RAL1 gene expression in
these organs. These upstream factors could thus be responsible for the
organ-specific appearance of the vascular pattern defects in
ral1.
In association with the aberrant vascular pattern formation, we observed in
the ral1 mutant a reduced auxin response. A similar situation holds
true for the mp, bdl and axr6 mutants
(Berleth and Jürgens,
1993; Przemeck et al.,
1996
; Hardtke and Berleth,
1998
; Hamann et al.,
1999
; Hobbie et al.,
2000
). However, other vascular development mutants do not show
altered auxin responses (Zhong et al.,
1999
; Carland et al.,
1999
; Candela et al.,
2001
; Zhong and Ye,
2001
) and the sfc mutant shows an enhanced response to
auxin (Deyholos et al., 2000
).
Furthermore, some of the mutants originally isolated because of an altered
response to exogenously administrated auxin also display vascular development
aberrations (e.g., Lincoln et al.,
1990
; Hobbie et al.,
2000
). Currently, we cannot determine any causal relationship
between the defects in vascular development and the altered auxin response of
the ral1 mutant. It is possible that the reduced auxin sensitivity
could be a consequence of the altered vascular development, since vascular
tissues represent the preferential pathway through which auxin is transported
(Lomax et al., 1995
).
Alternatively, primary defects in auxin perception or response could give rise
to the vascular defects of ral1. Treatments of wild-type rice leaves
with increasing concentrations of polar auxin transport inhibitors increase
the distance between longitudinal veins and decrease that between commissural
veins (Scarpella et al.,
2002
). Therefore, ectopic accumulation of auxin near source
regions in the wild-type rice leaf results in vascular pattern alterations
opposite to those induced by the ral1 mutation. This is consistent
with the possibility that the ral1 vascular patterning defects might
originate from a reduced sensitivity to vascular-inducing auxin signals.
Similarities between the additional phenotypes of ral1, such as
defective embryonic axis establishment, impaired adventitious and lateral root
formation, increased apical dominance and abnormal flower development, and
phenotypes of the mp, bdl, axr6 and other primary auxin response
mutants of Arabidopsis (e.g.,
Lincoln et al., 1990
;
Liscum and Reed, 2002
), also
suggest this possibility. Furthermore, the presence of these phenotypes in the
ral1 mutant seems to indicate that the RAL1 gene, just like
MP, BDL and AXR6, possesses patterning functions beyond the
vascular system. This observation raises the issue of how patterning of the
vascular tissues is coherently integrated with that of the surrounding tissues
and organs in these mutants. Two main scenarios seem possible
(Berleth et al., 2000
). In the
first, vascular patterning genes would act exclusively in incipient vascular
tissues to control vascular differentiation in response to a polarising
signal. Vascular tissues, in turn, would provide a scaffold system, in
reference to which numerous morphological features would be organised.
Alternatively, vascular patterning genes could be part of a more general cell
polarisation mechanism that would mediate oriented cell differentiation in
embryos, organ primordia and, most critically, in vascular strands. Currently
available evidence seems to support both interpretations
(Berleth and Jürgens,
1993
; Przemeck et al.,
1996
; Hamann et al.,
1999
; Hamann et al.,
2002
; Sabatini et al.,
1999
; Hobbie et al.,
2000
; Nakajima et al.,
2001
). In any case, it is of particular significance that in both
monocots and dicots, which display radically different embryo and vascular
pattern formation and auxin sensitivity properties, mutation in single genes
can result in defects in these processes that are essentially comparable. This
suggests that, regardless of the ultimate phenotypical outcomes, the molecular
mechanisms underlying these developmental processes are likely to be conserved
in monocots and dicots.
It is more difficult to reconcile the ral1 vascular patterning
defects with the increased response towards cytokinin measured in the mutant.
Cytokinin has long been known for its role in promoting procambial cell
division and vascular differentiation in cultured tissues or in plants
engineered to overproduce this hormone
(Shininger, 1979;
Aloni, 1995
), and the recent
cloning of the WOODEN LEG/CYTOKININ RESPONSE1 (WOL/CRE1)
gene has provided novel evidence of a role for cytokinin in vascular
development. The WOL/CRE1 gene encodes a cytokinin receptor, and is
expressed in the procambium of the embryonic axis
(Mähönen et al.,
2000
; Inue et al., 2001). Mutation in the WOL/CRE1 gene
leads to differentiation of all procambial cells in the root and the basal
part of the hypocotyl into protoxylem, a defect that has been associated with
a reduced division activity of procambial cells
(Scheres et al., 1995
;
Mähönen et al.,
2000
). A similar reduction in procambial cell division activity
might be responsible for the reduced vascular cylinder in ral1 roots.
However, unlike wol/cre1 mutants, the ral1 defect does not
affect the differentiation of any vascular cell type in particular within the
root vascular cylinder. Furthermore, wol/cre1 mutants display a
reduced sensitivity to cytokinin, whereas the ral1 mutant shows a
hypersensitive response to this hormone. Alternatively, defects in cytokinin
perception or response in the ral1 mutant could be a consequence of
the altered auxin sensitivity. In fact, these two hormones interact in a
complex manner in plant development, and certain processes are regulated in an
antagonistic fashion by them (Coenen and
Lomax, 1997
; Swarup et al.,
2002
). Furthermore, genetic analysis in Arabidopsis seems
to suggest that the response to these two hormones is integrated at the
molecular level (Swarup et al.,
2002
). Like ral1, mutation in the POLARIS
(PLS) gene of Arabidopsis has also recently been associated
with reduced vascularisation in the leaf, auxin resistance and cytokinin
hypersensitivity (Casson et al.,
2002
). However, the pls mutant does not display any
embryo defect. Therefore, the RAL1 gene is unlikely to be molecularly
identical to PLS.
Although alternative interpretations have been suggested (e.g.
Kull and Herbig, 1995;
Aloni, 2001
), mainly two, not
mutually exclusive, hypotheses have been proposed to explain the different
aspects of vascular pattern formation: the signalflow canalisation hypothesis
(Sachs, 1981
;
Sachs, 1989
), and the
reaction-diffusion hypothesis (Meinhardt,
1982
; Meinhardt,
1989
). Whereas the former accounts for the formation of complex
patterns of vasculature in response to a polarised flow of auxin, the latter
explains the formation of orderly structures by the coupling of a short-range
autocatalytic reaction with a long-range inhibitory process. It has been
argued that the generation of the highly ordered and reproducible wild-type
pattern of veins in monocot leaves and its coherent integration into leaf
growth and morphogenesis are more directly reconcilable with a
reaction-diffusion mechanism (Dengler et
al., 1997
; Nelson and Dengler,
1997
). In agreement, all of the vascular phenotypes of the
ral1 mutant, which include an altered spacing of veins and the
presence of interruptions, Y-shaped branches and vascular islands in the
commissural vein pattern, resemble defects predicted by models of mutations in
reaction-diffusion systems (Meinhardt,
1982
; Meinhardt,
1989
), while they are difficult to explain in terms of the
canalisation hypothesis. Previously, the observation of interrupted veins and
vascular islands in the sfc and van mutants of
Arabidopsis provided support for the reaction-diffusion mechanism in
leaf vascular patterning (Deyholos et al.,
2000
; Koizumi et al.,
2000
). However, certain aspects of wild-type vascular patterning
in dicots are still more readily explained by the canalisation hypothesis
(Nelson and Dengler, 1997
). As
reflected in recent reviews (Dengler and
Kang, 2001
; Ye,
2002
), because of the absence of mutants, our understanding of the
process of vascular pattern formation in monocot species is far inferior to
that in dicots. In this contest, our study on the radically different leaf
venation pattern of a monocot species provides the basis for the indispensable
genetic analysis that will allow a more thorough investigation of one of the
most intriguing elements of leaf architecture.
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ACKNOWLEDGMENTS |
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