1 Howard Hughes Medical Institute and Plant Biology Laboratory, The Salk
Institute for Biological Studies, 10010 N. Torrey Pines Rd, La Jolla, CA
92037, USA
2 Department of Molecular, Cellular, and Developmental Biology, University of
Michigan, 830 North University, Ann Arbor, MI 48109-1048, USA
3 Major in Biological Science, Sookmyung Women's University, 53-12 Chungpa-dong
2 Ka, Yongsan-gu, Seoul, 140-742, Korea
Authors for correspondence (e-mail:
chory{at}salk.edu;
jian{at}umich.edu)
Accepted 16 August 2004
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SUMMARY |
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Key words: Arabidopsis, Brassinosteroids, Vascular differentiation
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Introduction |
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BRI1 encodes a membrane-localized leucine-rich repeat
receptor-like kinase (LRR-RLK). The extracellular LRR domain is connected by a
single pass transmembrane domain to a typical Ser/Thr kinase
(Li and Chory, 1997;
Friedrichsen et al., 2000
).
While more than 200 LRR-RLKs have been identified in the Arabidopsis
genome (Yin et al., 2002b
;
Shiu and Bleecker, 2001), only a few have been assigned a function. Moreover,
most of these receptors have no known ligand. Genetic and biochemical evidence
has demonstrated that BRI1 is an essential component of a receptor complex
that binds brassinolide (BL) with high affinity
(Wang et al., 2001
). Although
understanding how BRs are perceived by the BRI1 receptor remains to be
elucidated, other signaling components operating downstream of BRI1 have been
identified. BRI1 associated receptor kinase 1 (BAK1), a much smaller RLK
containing only five LRRs, was identified in an activation-tagging screen for
bri1 suppressors (Li et al.,
2002
) and in a yeast two-hybrid screen for BRI1 interacting
proteins (Nam and Li, 2002
).
BAK1 thus acts in close proximity with BRI1, perhaps as its co-receptor.
Brassinosteroid insensitive 2 (BIN2)/UCU1/DWF12, one of the 10
Arabidopsis GSK-3/Shaggylike kinases, negatively regulates BR
signaling (Li and Nam, 2002
;
Pérez-Pérez et al.,
2002
; Choe et al.,
2002
), while bri1 Suppressor 1 (BSU1), a
nuclear-localized Ser/Thr phosphatase, positively regulates BR signaling
(Mora-García et al.,
2004
). When BR levels are low, proteins in the BES1/BZR1 family
are hyperphosphorylated by BIN2 and targeted for degradation. Upon BR
perception, dephosphorylated BES1/BZR1 proteins accumulate in the nucleus,
where they presumably regulate the transcription of BR-regulated genes
(Wang et al., 2002
;
Yin et al., 2002a
). Many
BR-induced genes are related to cell wall loosening enzymes, supporting the
role of BRs in cell expansion (Kauschmann
et al., 1996
; Nicol et al.,
1998
; Yin et al.,
2002a
; Goda et al.,
2004
).
Early physiological studies using Zinnia explants have shown that
BRs play an important role in promoting xylem differentiation (reviewed by
Fukuda, 1997). The BL
synthesis inhibitors, uniconazole and brassinazole (BRZ)
(Iwasaki and Shibaoka, 1991
;
Asami et al., 2000
), prevent
xylem differentiation in the Zinnia cell cultures, which can be
restored by exogenous application of BL. Furthermore, the levels of BR
intermediates have been shown to peak at the transition from undifferentiated
cells to tracheary elements (Yamamoto et
al., 2001
). BRs also appear to promote xylem differentiation in
cress plants (Nagata et al.,
2001
). In Arabidopsis, two mutants defective in
BR-synthesis, cpd and dwarf7, exhibit vascular
differentiation defects (Szekeres et al.,
1996
; Choe et al.,
1999
); however, vascular phenotypes in BR biosynthetic and
signaling mutants have not been characterized in detail. How BRs regulate
vascular development in Arabidopsis and whether xylem differentiation
is controlled by specific BR-receptors are still unanswered questions.
We identified three proteins from the Arabidopsis genome,
designated BRL1, BRL2 and BRL3, with high sequence identity to BRI1. The BRL2
gene corresponds to the previously identified Vascular Highway 1 (VH1), which
appears to be required to maintain provascular differentiation in the leaves
(Clay and Nelson, 2001). Here,
we show that BRL1 and BRL3, but not BRL2, can complement bri1 mutant
phenotypes when expressed under the control of the BRI1 promoter. We
show that BRL1 and BRL3, but not BRL2, can bind BL with high affinity. Unlike
BRI1, which is expressed ubiquitously, the BRL genes are
predominantly expressed in the vascular tissues. Consistent with this
observation, we found a brl1 mutant had increased phloem and reduced
xylem differentiation compared with the wild type. Double mutant combinations
of bri1 brl1 and triple bri1 brl1 brl3 mutants displayed
aberrant vascular differentiation. We propose that BRL1 and BRL3 are novel
plasma-membrane-localized steroid receptors that function specifically in
provascular differentiation to maintain proper xylem:phloem ratios.
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Materials and methods |
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Mutant isolation
Screening and isolation of a T-DNA insertion in the brl1-1 line
was done as described by the Arabidopsis knock-out Facility at the
University of Wisconsin
(http://www.biotech.wisc.edu/Arabidopsis/).
The brl1-2, brl2 and blr3 mutants were isolated from the
T-DNA Express Collection at the Salk Institute
(http://signal.salk.edu/cgi-bin/tdnaexpress).
The gene-specific primers used for PCR genotyping of the T-DNA insertions and
the location of the T-DNA insertions for every allele are provided in Table S1
(in the supplementary material).
Histology and microscopy
Tissue samples were fixed overnight in 4% (v/v) glutaraldehyde in 25 mM
NaPO4 buffer, pH 7.4, and then dehydrated with a graded series of ethanol and
infiltrated with polymethacryl resin Technovit 7100 (Heraeus Kulzer, Germany),
followed by embedding and polymerization in Technovit 7100. Sections (4 µm)
were done using a Leica Jung Autocut microtome (Wetzlar, Germany). The tissue
sections were stained with 0.1% Toluidine Blue in 0.1 M NaPO4 buffer, pH 7.0,
and observed under bright-field illumination through a Zeiss Axioskop
microscope. Images were processed using Photoshop 7.0. To calculate the number
of phloem cells, pictures of each 4 µm transverse section were taken at
40x magnification under a Zeiss microscope. Then each vascular bundle
was printed separately. The phloem cells were delimited with a pen and hand
counted for each bundle of every plant. The average of phloem cells per plant
was calculated using a total of 12 plants. Histochemical staining of plants
expressing the GUS reporter fusions was basically done as reported in Baima et
al. (Baima et al., 1995).
Visualization of green fluorescent protein (GFP)-tagged BRLs was done on 7-day-old light-grown hypocotyls using a BioRad Radiance 2100 microscope. The images were captured using the Kalman filter (n=5), and then analyzed with Confocal Assistant software.
Overexpression, localization and rescue constructs
bri1 rescue constructs
BRL1, BRL2 and BRL3 genes were amplified from bacterial
artificial chromosome (BAC) clones F20N2, F14H20 and MRP15, respectively, and
cloned into a modified pBluescript plasmid that contains a 1.7 kb
BRI1-promoter sequence and the RbcS-E9 terminator. After
sequencing verification, the pBRI1-BRL-E9ter fragments were cleaved
from the pBluescript plasmids and cloned into the pPZP212 binary vector.
Site-directed mutagenesis was carried out using Stratagene's QuickChange
Site-Directed Mutagenesis kit to introduce G597E (corresponding to
bri1-113 mutation) or E1056K (corresponding to bri1-1
mutation), mutation into the pBRI1-BRL1-E9ter construct. Plant
transformation into the weak bri1-301 mutants by floral dipping was
done according to Clough and Bent (Clough
and Bent, 1998). Due to unknown reasons, the
pBRI1-BRL2-E9ter plasmid could not be transformed into
Agrobacterium cells. Instead, we constructed a GFP-tagged version of
the BRL2 transgene and were able to transform the resulting
pBRI1-BRL2::GFP-E9ter construct into the bri1-301
mutants.
Construction of promoter GUS fusions
For BRI1, a 1.7 kb fragment including the 5' utr and
promoter region was amplified from BAC F23K16 and cloned between the
HindIII/BamHI sites in vector pBI101.3. Transgenic plants
were selected on kanamycin. For BRL genes, a 1.72 kb
SpeI/HindIII restriction fragment of the BRL1
promoter and a 750 bp NsiI/AflII restriction fragment of the
BRL3 promoter were individually ligated in frame to the GUS gene of
the binary vector pCB308. Transgenic plants were selected for resistance to
the herbicide glufosinate. Homozygous plants were used for GUS histological
staining.
Immunoprecipitation and 3H-BL binding assays
These experiments were performed essentially as reported in Wang et al.
(Wang et al., 2001), with
slight modifications. For 3[H]-BL binding assays with
immunoprecipitated proteins, 4-week-old T3 plants expressing GFP-tagged BRLs
were grown under short-day conditions. The membrane fractions were extracted
from 15 g of rosette leaves using 2 ml/g tissue of BRI1 extraction buffer at
4°C (Wang et al., 2001
).
Membranes were immunoprecipitated using 5 µl of anti-GFP antibodies
(Molecular Probes) and Protein A agarose beads (200 µl of 50% slurry
immobilized protein A), (Pierce). After extensive washing, the
immunoprecipitate was aliquoted in 50 µl/sample and was used for individual
binding experiments. Specific BL binding and data analysis was done according
to Wang et al. (Wang et al.,
2001
). Immunoprecipitated GFP fusion proteins were analyzed by
Western blotting with anti-GFP antibodies (1/3000 dilution).
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Results |
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The amino acid sequence alignment is shown in Fig. 1C. BRL1 and BRL3 were the closest homologs, both sharing 43% sequence identity with BRI1, followed by BRL2 with 41%. Among the four LRR-RLKs, BRL1 and BRL3 shared the highest homology, being 80% identical. It is noteworthy that BRL1 and BRL3 contained island domains that were 93% identical, while BRL2 is the most divergent member (Fig. 1C). The finding that BRI1, BRL1, BRL2 and BRL3 constituted a subfamily of the LRR-RLK proteins led us to hypothesize that the BRLs may be BR receptors with distinct functions.
BRL1 and BRL3, but not BRL2, encode functional BR receptors
To test whether the BRL genes encode functional BR receptors,
three fusion constructs containing the BRI1 promoter, the complete
ORF for each BRL, and the 3' terminator of the pea
RbcS-E9 gene were generated. Each pBRI1::BRL::E9 transgene
was then transformed into a weak bri1 (bri1-301) mutant
background (Fig. 2). Unlike
many bri1 mutants that are male sterile, bri1-301 displays a
weak mutant phenotype and is fertile, and therefore is directly transformable
(Fig. 2C). We found that
transgenic plants expressing pBRI1-BRL1
(Fig. 2D) and
pBRI1-BRL3 (Fig. 2F)
were able to suppress the dwarf bri1 phenotype, whereas the
pBRI1-BRL2 transgenic plants were indistinguishable from the
bri1-301 mutants (Fig.
2E). In addition, transgenic plants expressing pBRI1-BRL1
and pBRI1-BRL3 in the Col-0 background conferred a long and curled
petiole phenotype, similar to the one described for the overexpression of BRI1
under its own promoter (Wang et al.,
2001), while wild-type plants transformed with pBRI1-BRL2
looked similar to wild-type controls (data not shown). Thus, BRL1 and BRL3 can
substitute for BRI1 function when expressed in the expression domain of
BRI1. By contrast, BRL2 does not appear to function in BR signaling,
despite its sequence homology with BRI1.
|
BRL1 and BRL3 encode membrane localized receptors that bind BL with high affinity
To test whether BL is the ligand for the BRL receptors, full-length
BRL1, BRL2, BRL3 were expressed as fusion proteins with GFP under the
control of the strong CaMV 35S promoter. These constructs were transformed
into Arabidopsis wild-type Col-0. Transgenic plants expressing
35S-BRL1::GFP, 35S-BRL2::GFP and 35S-BRL3::GFP did not show
any apparent phenotypic alteration. Light-grown seedlings of these transgenic
plants were examined for BRL subcellular localization using a confocal
scanning laser microscope (CSLM), revealing that all three proteins were
localized at the plasma membrane (Fig.
3D and data not shown). Accordingly, these membrane fractions were
used to test if the BRLs were able to bind specifically to BL, using a
previously defined assay for BRI1 (Wang et
al., 2001). As shown in Fig.
3E, only BRL1 and BRL3 were able to bind BL, whereas no binding
could be detected in BRL2-overexpressing lines. BRL1::GFP and
BRL3::GFP proteins were immunoprecipitated by anti-GFP antibodies
(Fig. 3C) and the specific
binding activity of 3[H]-BL was calculated
(Fig. 3A,B). The dissociation
constants of the immunoprecipitated binding activity were calculated to be
3.6±0.07 nM for BRL1 and 53.4±0.04 nM for BRL3, as shown in
Fig. 3A,B. As a positive
control, we obtained a Kd=55±0.08 nM for the BRI1::GFP
transgenic plants, which are known to have a high BL-binding activity
(Wang et al., 2001
).
|
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To summarize, the vascular tissue-preferential expression of BRL1
and BRL3 was common in all organs. In addition, the spatial
complementation of this expression pattern was related to the level of
maturation of vascular development, with BRL1 preferentially
expressed in more mature cells than BRL3. In agreement, BRL1
and BRL3 appeared to be preferentially expressed in the provascular
tissues, in a tissue-specific microarray analysis
(Birnbaum et al., 2003)
available at the AREX database
(www.arex.org).
This unique expression pattern suggests that BR signaling through BRL1 and
BRL3 may play a role in provascular organogenesis in plants.
BRL1 and BRL3 play distinct and overlapping roles with BRI1 in vascular differentiation and growth
To investigate the role of BRL1 and BRL3 genes in
vascular development, we identified T-DNA tagged lines for each gene. A total
of three mutants, two alleles of brl1, brl1-1 in the Ws-2 background
and brl1-2 in the Col-0 background, and a single brl3 allele
in the Col-0 background, were identified. The location of the T-DNA insertions
is shown in Fig. 1B, and all
alleles are null, based on Northern blotting analysis (data not shown).
All the brl single mutants identified exhibited wild-type
appearance at the seedling and mature plant stages
(Fig. 5 A-C and data not
shown). However, due to the specific expression of these promoters in vascular
cells, we looked for vascular defects by histological analysis. Vasculature in
the Arabidopsis inflorescence stem follows a radial pattern in which
vascular bundles alternate with interfascicular parenchyma. The vascular
pattern in the stem arises from the meristematic activity of the procambial
cells, which give rise to the phloem (in the outer side) and the xylem (in the
inner side of the bundle; stained in dark blue by Toluidine Blue in
Fig. 5I), thus creating a
collateral pattern (Esau, 1965; Turner and
Somerville, 1997).
|
In addition, we found that brl1-1 enhanced the dwarf phenotype of bri1-5, a weak allele of bri1 (Fig. 5D). bri1-5brl1-1 double mutants exhibited an overall abnormal vascular organization, affecting the differentiation of both the phloem and xylem (Fig. 5H). The small phloem cells appeared collapsed between the enlarged phloem cap cells and xylem. The vascular defects of the bri1-5brl1-1 double mutants in the Ws-2 ecotype resembled those found in bri1-101, a strong bri1 allele in the Col-0 ecotype (Fig. 5P), indicating that BRL1 and BRI1 function redundantly in provascular cell differentiation in the Ws-2 background.
It is noteworthy that the brl1-1 vascular phenotype described above appears to be background-dependent. brl1-2 and brl3, as well as double mutants brl1-2 brl3, did not display any significant vascular defects in the root and the shoot in the Col background. Furthermore, the number of phloem cells found in brl1-2 (83±5, n=12) and brl3 (82±5, n=12), as well as double mutants brl1-2 brl3 (79±5, n=10), was similar to the Col wild type (81±5, n=12). Similarly, the bri1-101 brl1-2 and bri1-101 brl3 vasculature resembled that of the single mutant bri1-101 (Fig. 5I-K and data not shown), altogether suggesting that BRI1, BRL1 and BRL3 function redundantly in vascular development, especially in the Col-0 background. To test this possibility, we generated bri1-101 brl1-2 brl3 triple mutants in the Col-0 background. The triple mutant was smaller than the single bri1-101 mutant (Fig. 5N,O). The mutant plants were not fertile and most died after making a single inflorescence stem with a few small flowers (Fig. 5O). We observed enhanced vasculature defects in the triple mutant compared with the bri1-101 single mutant (Fig. 5P,Q). Xylem development in the basal part of the stem appeared to be delayed in the triple mutant, resulting in decreased xylem differentiation and an absence of differentiated interfascicular fibers (Fig. 5Q). By contrast, the small phloem cells differentiated to give rise to an unusual formation of phloem fibers (arrowhead), and bundles were not fully differentiated. The presence of phloem fibers suggests abnormal coordination in vascular bundle development, in which the phloem may differentiate at the expense of xylem due to lack of BR perception in these cells. These results indicate that BR signal transduction through the BRI1, BRL1 and BRL3 receptors functions redundantly to regulate provascular cell growth and differentiation.
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Discussion |
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BRL1 and BRL3 are members of the BRI1 family of plant steroid receptors
Biochemical and genetic evidence demonstrates that BRL1 and BRL3, but not
BRL2, are bona fide BR receptors. First, BRL1 and BRL3 can rescue the
bri1 mutant phenotype when expressed under the control of the
BRI1 promoter (Fig. 2)
and they conferred long petiole phenotypes when expressed in wild-type plants,
both suggesting that functional specification of these two receptors may
reside, at least in part, in their cis-regulatory elements. Second,
immunoprecipitates of BRL1 and BRL3, but not of BRL2, can bind BL with high
affinity (Fig. 3A,B),
indicating that both BRL1 and BRL3, like BRI1, are involved in BR perception.
However, it is plausible that additional factors may specify the action of
these receptors. In fact, signaling components downstream of BRI1 belong to
multigene families, whose members probably have overlapping, as well as
specific, functions (Li et al.,
2002; Nam and Li,
2002
; Yin et al.,
2002a
; Wang et al.,
2002
; Mora-García et
al., 2004
). Future studies should investigate the signal
transduction pathway(s) associated with the BRL receptors in comparison to
what is known for BRI1. Moreover, differences in the amino acid sequences of
these receptors may be functionally relevant. In the intracellular part, while
the core sequences of the kinase domain are highly homologous among all
members, the sequences are more divergent in the regions flanking the
catalytic domain of the kinase, i.e. in the juxtamembrane region and the
C-terminal extension. In the extracellular domain, the functional relevance of
the number of LRRs is unclear, since BRI1 orthologs from other species with
seemingly equivalent functions show some degree of variability. The
intervening 70-amino acid islands probably play a more significant role, given
that mutations in the extracellular domain of BRI1 cluster to this region and
abolish binding of BL (Wang et al.,
2001
). The sequences of the island domains share a number of
conserved residues, those of BRL1 and BRL3 being highly similar and distinct
from those of BRI1 and BRL2. However, BRL1 binds BL with a 10-fold higher
affinity than that of either BRI1 or BRL3, which suggests that regions in
addition to the island domain may modulate the affinity for the ligand.
The BRL2 protein, previously described as Vascular Highway 1 (VH1), has
been proposed to regulate provascular cell specification
(Clay and Nelson, 2001). VH1
is expressed in the vascular tissues and loss-of-function of
BRL2/VH1 in the Ler ecotype results in abnormal
phloem transport due to a discontinuous phloem network that results in
premature leaf senescence. Despite the sequence similarity to the BR
receptors, we have shown that BRL2/VH1 does not function as a BR receptor.
Sterols have been implicated in the establishment of the vascular pattern, in
correct auxin transport and distribution, and more recently in leaf senescence
(Carland et al., 2002
;
Willemsen et al., 2003
;
Suzuki et al., 2004
;
Motose et al., 2004
). Since
sterol-mediated vascular development appears to be required to maintain the
continuity of the vascular network, it is therefore conceivable that BRL2 may
bind a sterol. Our studies underscore the importance of performing biochemical
assays prior to assigning a function to proteins identified through database
searches.
Evolutionary implications of the BRI1-receptor family in mono- and dicotyledonous species
The completed sequence of the rice genome offers the possibility of
establishing a comparison between the Arabidopsis and rice BRI1
families. Fig. 1A depicts the
phylogenetic relationship between all described or annotated members of the
BRI1 family. Several inferences can be extracted from this tree, inasmuch as
it accurately represents the evolution of the BRI1 family in angiosperms.
First, all major branches include sequences of both rice and
Arabidopsis, implying that the diversification inside the family
predates the split between monocots and dicots. In addition, the whole family
seems to have undergone an early functional division between receptors that
bind BL (BRI1 and BRL1/3) and those that do not (BRL2). Finally, a relatively
recent duplication event in the Arabidopsis lineage accounts for the
presence of the highly homologous BRL1 and BRL3 genes,
encoded in duplicated chromosomal regions. Their complementary expression
pattern probably reflects a process of subfunctionalization from an ancestral
function throughout the vascular tract
(Lynch and Force, 2000).
Analysis of the expression pattern and function of the unique rice
BRL1 homolog will help to address this question.
The analysis of recessive, dwarf mutants insensitive to BL has led to the
identification of BRI1 orthologs in several species other than
Arabidopsis, such as OsBRI1 in rice
(Yamamuro et al., 2000),
UZU in barley (Chono et al.,
2003
), CU3 in tomato
(Montoya et al., 2002
) and
LKA in pea (Nomura et al.,
2003
). It is noteworthy that all of them cluster in a highly
supported group (Fig. 1A),
suggesting that these proteins are the major BL receptors among angiosperms.
Consistently, loss-of-function of Arabidopsis BRL1 and BRL3,
described here, and BRL2 (Clay
and Nelson, 2001
) caused relatively subtle effects. It is
remarkable that the expression of members of the BRI1 family, except
BRI1 itself, is restricted to vascular tissues. It remains to be
established whether this vascular specificity is an ancestral feature of these
receptors, and by extension, of BL signaling, or rather a derived trait
related to the functional specialization among paralogous genes.
It should be pointed out that other members of the Arabidopsis
RLKs have been reported to behave similarly. In the case of the
ERECTA family, both ERL1 and ERL2 act
synergistically with ERECTA in defining aerial organ size
(Shpak et al., 2003;
Shpak et al., 2004
). Like
BRL1 and BRL3, ERL1 and ERL2 are also located in
duplicated chromosomal regions (Shpak et
al., 2004
), suggesting that distinct RLK families may have evolved
similarly in plants.
A role for BRs in vascular development in the shoot
The molecular mechanism by which procambial cells differentiate into phloem
or xylem remains largely unknown. The inner location of the vascular tissues
has hampered the identification of vascular development mutants and often led
to the identification of cell wall synthesis genes
(Turner and Somerville, 1997;
Caño-Delgado et al.,
2000
; Caño-Delgado et
al., 2003
; Taylor et al.,
1999
). To date, only a few mutants have been identified in
Arabidopsis that specifically affect vascular patterning,
(Clay and Nelson, 2001
;
Koizumi et al., 2000
; Clay and
Nelson, 2002; Bonke et al.,
2003
; Parker et al.,
2003
; Motose et al.,
2004
) (reviewed by Fukuda,
2004
).
The vascular-specific expression of BRL1 and BRL3 genes
(Fig. 4) implies that these BR
receptors play important roles during vascular development. Furthermore,
studies using Zinnia mesophyll cell cultures have shown that
exogenous application of BRs induces tracheary element differentiation in the
terminal stage of xylogenesis (Yamamoto et
al., 1997). Our studies in intact Arabidopsis establish
that BR signaling in the provascular cells through BRL1, BRL3 and BRI1
receptors contributes to the collateral organization of the vascular bundles
in the plant shoot (Fig. 5).
The analysis of bri1 and brl1 mutant combinations revealed
an increase in phloem and a decrease in xylem differentiated cells
(Fig. 5F-H,Q), indicating that
BR signaling in the procambial cells induces xylem cell differentiation and,
at the same time, represses phloem differentiation. It is noteworthy that the
bri1 null mutant (Fig.
5P) and the bri1-101 brl1 brl3 triple mutant
(Fig. 5Q), exhibited phloem
cells that are either collapsed with dramatically enlarged phloem cap cells or
prematurely differentiated to phloem fibers, suggesting that different
BR-signaling strength has different effects on phloem cell differentiation in
the stem. Altogether, our data support the hypothesis that BRs contribute to
the establishment of the collateral xylem/phloem pattern rather than just
inducing xylem differentiation per se. In agreement, APL, a MYB transcription
factor that determines phloem cell identity in the root, functions in
promoting phloem differentiation and repressing xylem differentiation
(Bonke et al., 2003
), also
suggesting that the differentiation of these two cell types is tightly
linked.
We want to point out that the phenotypes of brl mutants appear to
be affected by natural variation between different Arabidopsis
accessions. In the Ws-2 background, brl1-1 null mutants have an
increased number of phloem cells compared with the wild type, while no
significant differences were observed between the brl1-2 allele and
the Col-0 wild type. Since no brl3 alleles were available in the Ws-2
ecotype, we attribute the differences in phenotype to the observed ecotype
variation, rather than to an allele-specific effect. Furthermore, natural
variation in genes that affect vascular development has been described
previously for REV/IFL1, a member of the homeodomain-leucine zipper
transcription factor family that controls interfascicular fiber
differentiation (Talbert et al.,
1995; Zhong and Ye,
1999
; Emery et al.,
2003
). Interestingly, Zinnia members of this family of
transcription factors were found to be positively regulated by BRs
(Demura et al., 2002
;
Ohashi-Ito and Fukuda, 2003
).
In addition, while the vh1 mutants in the Ler accession
produced clear leaf senescence defects
(Clay and Nelson, 2001
), we
did not observe these phenotypes in our mutant allele brl2 in the
Col-0 ecotype under the same experimental conditions (data not shown). Several
differences in allele variation between Col-0 and Ws-2 related to vascular
development in the inflorescence stem of Arabidopsis have also been
reported (Altamura et al.,
2001
). For example, phloem cap cells appeared to be larger in Ws-2
than in Col-0. We found increased size in the phloem cap cells in bri1,
brl1-1, bri1-5brl1-1 and bri1-101brl1-2brl3 mutants, suggesting
that natural variation in these genes may account for the differences in
vascular structure between the Ws-2 and Col-0 accessions.
A model for BR function in vascular patterning
Several plant hormones have been implicated in vascular pattern formation.
Auxin, the only hormone that has been shown to exhibit polar transport, acts
as a major signal in vascular differentiation
(Aloni, 1987;
Sachs, 1991
). In the
`canalization hypothesis', the diffusion of the hormone from an auxin source
induces the formation of a polar auxin transport system along a procambial
cell file that differentiates into a xylem strand. However, additional
molecules have been proposed to be required for vascular differentiation. Our
data suggest that BR may be one of these additional molecules required for the
induction of vascular cell differentiation in plants. The expression pattern
of BRL1 in the provascular cells correlates with an inductive role of
BRs in xylem differentiation; by contrast, the discrete expression of
BRL3 in the phloem cells suggests that BRL3 may repress phloem
differentiation, thereby controlling the formation of the collateral pattern
in the vascular bundles. This hypothesis would be possible if BRs are
transported from one cell to its neighbors. However, while polar auxin
transport has been widely demonstrated, the long distance transport of BRs
awaits to be demonstrated. Knowledge of where BRs are synthesized and whether
they are perceived as part of a paracrine or autocrine system in the plant is
crucial to further understand the role of BRs in vascular ontogeny.
We propose a model for the function of BRs in vascular cell differentiation (Fig. 6). BRs function through vascular-specific receptors BRL1 and BRL3 and the ubiquitously expressed receptor BRI1 to promote xylem differentiation and to repress phloem cell differentiation (Fig. 6A). In support of this model, phenotypes of BR-biosynthesis and -perception mutants exhibit a reduced number of xylem cells at the expense of phloem cells (Fig. 6B), indicating that the differentiation of these two cell types is reciprocally linked. Thus, BR perception by the procambial cells may act as an inductive signal through BRI1/BRL receptors in the acquisition of vascular cell fate and thus in the mutual regulation of xylem/phloem differentiation in the vascular bundle. However, it is also possible that BRs promote the division of the procambial cells in the shoot apex that will later differentiate into xylem cells. This hypothesis is supported by the phenotypes observed in the stems of BRI1 overexpressing plants (Fig. 5M), where procambial activity appeared to be enhanced compared with the wild type (Fig. 5I). By contrast, we observed that the bri1 null mutant (Fig. 5H) and bri1 br1 brl3 triple mutants (Fig. 5Q) had small bundles, probably due to a reduced division of the procambial cells.
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ACKNOWLEDGMENTS |
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Footnotes |
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Supplementary material for this article is available at http://dev.biologists.org/cgi/content/full/131/21/5341/DC1
* These authors contributed equally to this work
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