Division of Biology 156-29, California Institute of Technology, 1200 East California Boulevard, Pasadena, CA 91125, USA
* Author for correspondence (e-mail: meyerow{at}its.caltech.edu)
Accepted 18 November 2003
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SUMMARY |
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Key words: JAGGED, Lateral organ growth, Leaf morphogenesis, Leaf development, Arabidopsis thaliana
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Introduction |
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In response to positional cues, genes presumably dictate the process of
morphogenesis by controlling both the proliferation and the differentiation of
specific cell types. Studies of leaf growth in Arabidopsis using
genetic clonal analysis (Poethig, 1985) and observation of a
cyclin1At::ß-glucuronidase (GUS) cell cycle reporter
gene (Donnelly et al., 1999)
showed that in growing leaves there is a proximal-distal gradient of cell
division rates, with the highest levels of cell division occurring in the
proximal regions of the leaf. In contrast, most leaves differentiate
basipetally with the distal blade forming first, followed by the proximal
petiole (Poethig, 1985). Recent studies have identified a number of genes
involved in the control of cell division and differentiation in lateral
organs. The LEAFY PETIOLE (LEP) gene encodes an
EREBP/AP2-type transcription factor that is sufficient to transform the
proximally located leaf petiole into a distal blade structure
(van der Graaff et al., 2000
).
Additional REPESSOR FOR LEP (RLP) loci have been isolated as
suppressors of an activation-tagged lep mutant
(van der Graaff et al., 2003
).
Mutations in the BLADE-ON-PETIOLE 1 (BOP) locus result in
abnormal leaves that misexpress the meristem-promoting knox genes and
develop numerous leaf primordia on the adaxial surface of the leaves
(Ha et al., 2003
).
asymmetric leaves1 (as1) and asymmetric leaves2
(as2) mutants also misexpress knox genes in leaves
(Byrne et al., 2000
;
Ori et al., 2000
;
Semiarti et al., 2001
).
Finally the AINTEGUMENTA (ANT) gene, encoding an AP2-like
factor, is proposed to control organ size by regulating meristematic
competence of cells within the lateral organ primordia
(Krizek, 1999
;
Mizukami and Fischer,
2000
).
We show that loss-of-function jagged (jag) mutants exhibit lateral organ defects, including narrow leaves and floral organs with serrated margins. The JAG locus encodes a nuclear-localized transcription factor containing a single C2H2-type zinc finger that is expressed exclusively in developing lateral organ primordia. When expressed ectopically, JAG activity is sufficient to cause cell proliferation and differentiation of leaf cell types.
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Materials and methods |
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Genetic analysis of mutants
The jag-2 and jag-3 alleles (isolation lines, shreddie1
and shreddie2) arose in an EMS screen. Landsberg erecta
(Ler) seed were mutagenized by incubation in a solution containing
0.3% (v/v) EMS (Sigma) and 0.1% (v/v) Tween 20 (Sigma) for 12 hours at room
temperature. Both alleles were out crossed to wild type four times before
further genetic analysis. For the construction of double mutant strains, the
progeny of single mutant phenotype individuals were collected in the
F2 generation and the double mutants were observed to segregate in
a 1:3 ratio in the F3 generation. Double mutant genotypes were
confirmed by polymerase chain reaction (PCR) genotyping.
Scanning electron and confocal microscopy
For SEM the inflorescence tissue was fixed and processed using a standard
protocol (Bowman et al., 1989).
Stages of flower development (Smyth et
al., 1990
) were determined. Stage 13 flowers (anthesis stage) were
selected for SEM. Petals were carefully removed and mounted flat either on
their adaxial or abaxial surface. For confocal analysis, 4-day
post-germination seedlings were immersed in 1 mg/ml Hoechst 33342 for 1 hour.
After removal of one cotyledon, seedlings were mounted in 50% glycerol under a
coverslip. Tissue was imaged using a Zeiss LSM510 NLO confocal microscope
equipped with a Chameleon Ti-Sapphire IR pulsed laser (Coherent) tuned to 730
nm. JAG-VENUS was detected using the 514 nm Ar laser line and a 535-590 nm
band pass filter for the emitted light. Nuclear-localized Hoechst stain was
detected using 730 nm excitation together with a 435-485 band pass filter for
the emitted light. The two signals were collected together using
multi-tracking and the images were analyzed with Zeiss LSM510 proprietary
software and ImageJ
(http://rsb.info.nih.gov/ij/).
Histology and plastic sections
Clearing of tissues for observation of vasculature by light microscopy was
performed as described previously (Aida et
al., 1997).
For plastic sections tissue was vacuum infiltrated with a fixation solution containing 4% paraformaldehyde, 1% glutaraldehyde and 1x phosphate-buffered saline and fixed for 12 hours at 4°C. Tissues were dehydrated stepwise in ethanol solution series. Tissues were infiltrated stepwise in UnicrylTM acrylic resin (SPI Supplies) diluted with ethanol. Samples were polymerized for 20-24 hours in a 60°C oven. Sections 0.2-2.0 µm were cut with a glass blade and a Leica RM2165 microtome. Samples were stained with 1% Toluidine Blue for 1 minute and mounted in Cytoseal-XYL (Richard-Allan Scientific).
Positional Cloning of JAG
To create a mapping population, the jag-3 allele in Ler
ecotype was crossed to wild-type Columbia (Col) ecotype plants. Recombinant
jag-3 mutant plants were identified in the F2 progeny.
PCR-based markers were developed for fine scale mapping using Ler and
Col single nucleotide and insertion/deletion polymorphism sequence data
located at
http://www.arabidopsis.org/Cereon/index.html.
For genomic complementation of the mutant with genomic constructs P5112-1
and P5112-4, overlapping 10-15 kb genomic fragments spanning the BAC T26J14
were subcloned into the pPZP222 transformation vector
(Hajdukiewicz et al., 1994)
for transformation of jag-3 plants by Agrobacterium-mediated
transformation by the floral dip method
(Clough and Bent, 1998
). The
1.2 kb JAG cDNA was PCR-amplified from reverse transcribed
Ler inflorescence total RNA using the primers P5.9f
5'-CCCTAGCATCTCCTTTCACTCAG-3' and P5.2r
5'-GGCGTTTAGACAATTCTAGATCTC-3'.
Generation of transgenic plants
The JAG::VENUS vector was generated by first PCR-amplifying a 4.6
kb genomic fragment containing the JAG promoter and coding sequences
from Ler genomic DNA with the primers T26Bf
5'-GGATCCCGGAATAGAGCTGATGTAGTAGCCGTG-3' and T26Nr
5'-CCATGGCGAGCGAGTGATGATCTTGAAACCGATTGA-3'. The gene was
translationally fused to the VENUS coding sequence separated by a 10-alanine
linker sequence in the transformation vector pMLBART
(Gleave, 1992).
The 35S::JAG vector was constructed by placing the JAG
cDNA sequence downstream of a Cauliflower Mosaic Virus (CaMV) 35S
promoter in the binary vector pCGN1547
(McBride and Summerfelt,
1990).
The pOpL two-component system
(Moore et al., 1998) was also
used to misexpress JAG. A 6XOP::JAG expression vector was
generated by first cloning the JAG cDNA downstream of six copies of
the Operator sequence and an OCS minimal promoter in a modified
version of the plasmid BJ36 (Gleave,
1992
) a gift from Jeff Long (Salk Institute). The final expression
cassette was in the transformation vector pART27
(Gleave, 1992
). The
6XOP::JAG construct was transformed into a transgenic driver line
pAP1::LHG4 donated by John Bowman (University of California,
Davis).
In situ hybridization
In situ hybridization was performed using digoxigenin-labeled RNA probes
according to a published protocol (Long
and Barton, 1998). The JAG antisense probe corresponds to
a 500 bp 3'-specific cDNA sequence from plasmid pJAG0.5 that was
linearized with BamHI and transcribed with T7 RNA polymerase, or a
1.0 kb 5'-specific cDNA from plasmid pJAG1.0 and similarly
transcribed.
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Results |
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jag petals show defects in cell number and shape
To further characterize the jag perianth organ defects, petals
were examined from equivalent mature anthesis stage flowers of wild-type
Landsberg erecta, jag-2 and jag-3 mutants. For the
comparison of the abaxial epidermal surfaces of the wild-type and
jag-2 mutant petals, the blade epidermis was equally subdivided along
the proximal-distal axis into proximal, middle and distal regions containing
distinct patterns of cell shape and size. The distal region in the wild-type
petal contains small round epidermal cells with distinct cuticular ridges
(Fig. 2A), while larger
elongated cobblestone-shaped cells make up the proximal region
(Fig. 2C). In the middle region
of the wild-type blade, the epidermis is composed of a mixture of small round
cells and cells that are both elongated in the proximal-distal axis and are
intermediate in character to cells found in both the distal and proximal
regions (Fig. 2B).
|
The petals of jag-3 (Fig. 2H) showed a phenotype that was weaker than that of jag-2 (Fig. 2I), but distinguishable from wild type (Fig. 2G). Overall, the cells in the jag-3 petal epidermis were not as elongated as in jag-2. The distal abaxial epidermal region of the jag-3 petals contained a mixture of cells that were either round like wild-type distal epidermal cells, or elongated similar to those found in the jag-2 mutant.
In summary, jag mutant organs are narrower and somewhat irregular in shape compared to wild-type organs. This phenotype appears to result from defects in both cell division and cell expansion from early stages in their development. Examination of jag petals reveals that there are fewer epidermal cells than in the wild type but that these cells are larger than normal. This in turn suggests that jag cells are not restricted in their ability to expand but instead are unable to divide as rapidly.
jag double mutants with genes involved in lateral organ development
The phenotype of jag mutants closely resembles that of the
aintegumenta (ant) mutant. ANT regulates growth and
cell division in floral organ primordia and ovules
(Elliott et al., 1996;
Klucher et al., 1996
). To
determine if there could be an overlap in the function of these two genes,
double mutant plants were generated with the strong loss-of-function
ant-9 allele. jag-3 ant-9 double mutant plants display a
phenotype that is much more severe than either single mutant in that floral
organs become reduced narrow structures that fail to elongate to the same
extent as in either single mutant (Fig.
3A,B). This enhanced phenotype suggests that both ANT and
JAG may function in parallel pathways that contribute to floral organ
size and shape.
|
The SUPERMAN (SUP) gene is involved in regulating stamen
and carpel development and floral organ number, possibly via the control of
cell proliferation within the flower
(Bowman et al., 1992;
Sakai et al., 1995
). Double
mutant flowers harboring the null sup-5 allele and jag-3
displayed a completely additive phenotype characterized by jag-3
petal and sepal morphology, and increased stamen number and unfused carpel
tissues similar to that observed in the sup-5 single mutant phenotype
(Fig. 3E,F). These data
indicate that JAG and SUP function independently in the
processes of organ growth and organ number determination in the flower.
clavata3 (clv3) mutants exhibit enlarged meristems and
increased organ number in all floral whorls
(Brand et al., 2000
;
Fletcher et al., 1999
).
clv3 jag double mutants also show an additive phenotype (data not
shown) suggesting that JAG does not interact with other meristem
genes of the CLAVATA signaling pathway involved in maintenance of
cell proliferation in the shoot and floral meristem.
JAG is required for bract outgrowth in leafy, apetala1 and apetala2 mutants
The leafy (lfy) mutant produces flowers with only
leaf-like organs, and bracts or floral leaves are produced at the base of each
pedicel/peduncle (Fig. 4A)
(Weigel et al., 1992). In
lfy-6 jag-3 double mutants, the bract-like organs that develop at the
base of the floral pedicel/peduncle are much reduced compared to
lfy-6 single mutants and are frequently filamentous
(Fig. 4B-D). The partial
loss-of-function lfy-5 allele results in a less severe phenotype in
the flower compared to lfy-6 that consists of bract-like sepals in
the outer whorl and a slight reduction in petal and stamen formation in the
inner whorls. Flowers mutant for both lfy-5 and jag-3
display characteristics associated with both single mutant, but the bract-like
sepals in the lateral positions fail to grow out (compare
Fig. 4E and F) and are often
replaced with small projections. Occasionally, a few flowers lack outgrowth of
all four bract-like sepals (data not shown). Flowers of lfy-6 jag-3
double mutants also appeared to produce fewer organs than the total number of
floral organs produced in either single mutant, and they are arranged in a
spiral pattern (data not shown). Thus, JAG is required for bract
outgrowth both at the base of pedicels/peduncles and in the flowers in the
lfy mutant background.
|
In summary, JAG is necessary for normal outgrowth of floral bract-like organs in the first whorl of lfy-5, ap1-1 and ap2-1 flowers, in addition to having a role in outgrowth of all four floral organs in the wild-type flower.
Positional Cloning of JAG
The position of the JAG locus was determined by its genetic
linkage to BAC-specific molecular markers corresponding to Landsberg
erecta and Columbia ecotype sequence polymorphisms using a
recombinant F2 mapping population representing 856 chromosomes
(Fig. 5A). Fine-scale mapping
analysis revealed that JAG is located on BAC clone T26J14. Two
independent overlapping 10 kb genomic fragments corresponding to BAC T26J14
sequences when transformed into plants rescued the jag-3 mutant
phenotype (data not shown). Sequencing of candidate genes encoded by this
genomic region in the jag-2 and jag-3 mutants revealed
nucleotide sequence changes in the gene At1g68480. The JAG gene
sequence was defined by genetic complementation to encompass a 4.5 kb genomic
fragment and the coding exons were found to be interrupted by 5 introns. A
cDNA sequence 1177 base pairs (bp) in length that is in agreement with a 1.2
kb transcript detected by northern blot (data not shown) was obtained from an
inflorescence cDNA population.
|
Other motifs identifiable in the amino terminus of the JAG protein sequence
include a putative nuclear localization sequence at position 35-38 and a short
nine amino acid ERF-associated amphiphilic repression (EAR) motif (position
8-16) (Ohta et al., 2001). The
repressor activity of the EAR motif has been demonstrated for the plant
transcription factors ERF4 and JAT11 (Ohta
et al., 2001
) and for SUP where it has been shown to have a
repressor function in vivo (Hiratsu et
al., 2002
). The JAG EAR motif contains three out of nine leucine
residues and shows amino acid sequence conservation in an alignment with
similar motifs present in previously characterized zinc finger transcription
factors (Fig. 5D). Similarly to
the SUP protein, JAG contains a proline-rich motif located at amino acids
129-141 in its carboxyl terminus that may function in transcriptional
repression or activation. Taken together, the presence of these functional
domains suggests that JAG is a nuclear-localized repressor of
transcription.
The jag-2 mutation results in a premature stop codon at residue 54 at the start of the zinc finger motif, while the jag-3 mutation disrupts the 3'-splice acceptor sequence in the fourth intron, resulting in the production of a slightly larger transcript on northern blots (data not shown) and a predicted protein that is truncated at residue 104 with the addition of 11 extra amino acids. Consistent with the identified DNA lesions, the jag-2 allele is phenotypically slightly more severe than the jag-3 allele, according to our analysis of abaxial petal epidermis, and represents a stronger loss-of-function allele.
JAG is expressed in lateral organ primordia and encodes a nuclear localized protein
The developmental pattern of JAG mRNA localization was determined
by in situ hybridization. JAG mRNA is first detected in late
transition stage embryos in cells corresponding to the cotyledon primordia.
JAG transcripts are uniformly localized throughout the newly emerging
cotyledon primordia but are absent from the SAM
(Fig. 6A). As the cotyledon
primordia continue to grow out during the heart stage, JAG mRNA
expression is excluded from the tips of the cotyledon primordia but continues
to be expressed in a band below this region of repressed expression
(Fig. 6B). In the torpedo stage
embryo, JAG expression continues to be absent at the tip of the
cotyledon primordia (Fig. 6C).
Cells in the epidermis of the cotyledon have a lower level of JAG
transcripts than cells in the inner layers. JAG is also expressed in
developing leaf primordia of the seedling at 3.5 days and/until 7 days after
germination. It is initially expressed throughout the young primordium but
later expression becomes excluded from the distal tip of the primordium
(Fig. 6D-F). Reduced
JAG expression is also detected at the proximal base of the leaf
primordia (Fig. 6F). After the
transition to reproductive development, JAG transcripts are absent in
the inflorescence meristem but are detected in developing floral organ
primordia (Fig. 6G). During
flower development JAG transcripts are first detected in stage 3
flowers in sepal anlagen (Fig.
6H,I). Expression in the medial sepals precedes expression in the
lateral sepals that are later to develop
(Fig. 6H,I and data not shown).
A lower level of transcripts is detected at the distal tip of the sepal
primordia of stage 4 flowers and onwards
(Fig. 6G). Expression in the
sepals decreases in stage 6-8 flowers and transcripts are detected in stamen
and petal primordia, but expression is slightly reduced at the base of these
primordia (Fig. 6G,J,K). Some
JAG transcripts are detected in stage 8 carpel primordia (not shown)
as well as in carpel valves in transverse sections of stage 9 gynoecia
(Fig. 6L).
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To determine the subcellular localization of the JAG protein, a 4.5 kb
genomic fragment containing putative 5'-regulatory sequences plus the
JAG coding sequences was translationally fused to VENUS, a rapidly
maturing variant of Yellow Fluorescent Protein
(Nagai et al., 2002). The
transgene was expressed in jag-3 plants and completely rescued the
mutant phenotype in 15 out of 17 T1 plants, indicating that the
fusion protein was functional in plants (data not shown). The expression of
the fusion protein mimicked the JAG RNA localization pattern as
observed by in situ hybridization. Confocal laser scanning microscopy of a
seedling at 4 days after germination shows the JAG-VENUS protein within the
leaf primordium in a distinct band of expression
(Fig. 6N). In leaf primordium
cells, the JAG-VENUS protein co-localizes with Hoechst 33342 nuclear staining
(Fig. 6O-Q). Consistent with
the predicted protein function and the presence of a consensus nuclear
localization sequence (Fig.
5B), the JAG-VENUS fusion protein is located in the nucleus.
In summary, JAG transcripts are exclusively localized to all lateral organ primordia throughout the plant, where expression is at first uniform throughout the young emerging primordia and then absent from the distal tips of organ outgrowth.
JAG misexpression is sufficient for development of leaf-like tissues
Since JAG is necessary to promote lateral organ growth, we tested
whether JAG activity is sufficient for such growth by expressing it
constitutively. A 35S::JAG construct expressed in both wild-type and
jag-2 mutant backgrounds resulted in similar phenotypes. Detailed
analysis of transgenic lines is described in the wild-type background.
35S::JAG transgenic plants showed disrupted organogenesis and the
formation of ectopic leaf-like tissues throughout the plant.
The abnormal phenotype was first noted at the seedling stage. 35S::JAG hypocotyls often appeared green in color, in contrast to wild-type hypocotyls that are yellowish and lack high levels of chlorophyll (Fig. 7A). In addition, varying degrees of cotyledon fusion (44 out of 104 T1 plants) were observed, including either a single fused cotyledon or a goblet-shaped structure resulting from fusion along both lateral cotyledon margins (Fig. 7A). These seedlings were cleared using chloral hydrate to allow examination of the vasculature. In the wild-type cotyledons, the vasculature consists of a central midvein that runs along the proximal-distal axis and a small number of secondary veins that branch off from it (Fig. 7B). 35S::JAG cotyledons exhibit a highly irregular vasculature in which a central midvein is not identifiable and the strands do not form a closed circuit (Fig. 7C). Many 35S::JAG seedlings also fail to develop leaves after germination. In those that do, the leaves are often irregular in shape and adjacent leaves may be fused along the lateral margins (Fig. 7D). During reproductive stages some of these plants develop a bract from the base of the flower pedicel similar to lfy mutants (Fig. 7E). Unusual outgrowth of stipules is also observed from the base of leaves (Fig. 7F).
|
To further characterize the sub-epidermal structure of these ectopic leaf-like tissues, the internal anatomy of 35S::JAG stem tissue was analyzed. Transverse sections of 35S::JAG stems reveal that the morphology of the centrally located pith cells, surrounding cortex and vascular bundles, containing xylem and phloem, is similar to that of the corresponding tissues in the wild type (Fig. 8A,B). However, numerous finger-like projections of tissue are found to emanate from the cortex and epidermal regions (compare Fig. 8A and B). Closer examination of cell types contained in these finger-like outgrowths reveals a striking resemblance to the structure of the wild-type leaf (Fig. 8C). There are large epidermal cells of variable size on the surfaces of the 35S:JAG stem outgrowths, which resemble pavement cells found in the wild-type leaf epidermis (compare Fig. 8C,D), while the subepidermal layer contains cells that are irregular in shape and size (Fig. 8D) and have a resemblance to the palisade and spongy mesophyll cell types found in leaves (Fig. 8C). A vascular bundle containing phloem and xylem cells was found to run through one of the finger-like outgrowths from the 35S::JAG stem and the cells that abut the xylem cells are elongated and have the morphology of the palisade mesophyll, whereas cells that abut the phloem cells are smaller and have the morphology of the spongy mesophyll (Fig. 8F). Some finger-like projections contain vascular tissue and appear to have normal leaf adaxial-abaxial patterning, while others lack the vasculature and organized tissue structure. Overall the cell types found in the finger-like outgrowths of the 35S::JAG stem resemble cell types found in leaves. Regions of the stem that lack visible leaf-like projections do however show abnormalities in the epidermis and cortex. The wild-type stem epidermis is composed of a single layer of small regular cells and the cortex is composed of about three cell layers of uniformly sized round cells (Fig. 8E). In contrast, the 35S::JAG stem epidermal and cortical cells are variable in size and irregular in shape. (Fig. 8F).
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Discussion |
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The jag loss-of-function mutant shares some similarities in
phenotype with two recently characterized mutants cincinnata
(cin) and jaw. The Antirrhinum majus CIN gene
encodes a TCP DNA binding factor that is involved in control of leaf shape and
curvature by regulating cell cycle arrest in the distal region of the growing
leaf (Nath et al., 2003). A
similar leaf growth phenotype is associated with the activation-tagged mutant
jaw. JAW encodes a microRNA similar in sequence to the
Arabidopsis TCP4 gene and has been shown to negatively regulate
TCP4 (Palatnik et al.,
2003
). Like jag petals, both cin and
jaw mutant leaves are buckled along the margins. Furthermore, both
TCP4 and JAG are normally expressed in growing leaf
primordia and their misexpression leads to seedlings with fused cotyledons
suggesting that in Arabidopsis TCP4 and JAG may function in
similar pathways regulating leaf growth.
JAG encodes a nuclear localized putative transcriptional repressor
that therefore might repress an inhibitor of leaf growth. One candidate
repressor of leaf growth is the BLADE-ON-PETIOLE 1 (BOP)
gene. The bop mutant is characterized by the development of ectopic
leaf tissues in the form of primordia on the proximal adaxial surfaces of the
cotyledons and leaves, and ectopic expression of the knox genes,
KNAT1, KNAT2 and KNAT6
(Ha et al., 2003). The
proposed BOP function is to control leaf morphogenesis by repressing
meristematic growth and differentiation in the proximal region of the
leaf.
JAG, cell division and ANT function
JAG shows a dynamic pattern of transcript localization that is
restricted to growing lateral organ primordia. At the onset of organ
primordium outgrowth JAG mRNA is localized throughout the primordium,
but soon becomes restricted to a smaller domain as it is excluded from the
distal tip. This expression pattern is similar to that of a
cyclin1At::GUS cell cycle reporter gene that is also down-regulated
in the distal end of developing leaf primordia
(Donnelly et al., 1999). Thus,
JAG expression appears to coincide with regions undergoing active
cell division and may be causal in this process. A close examination of the
narrow jag petals, revealed that the jag petal epidermis
contains fewer cells along the proximal-distal axis, thereby implicating a
JAG function in cell division control. The subdomain of JAG
expression at the distal petal tip in early stage primordia provides
patterning to the proximal-distal axis and may help to set up a gradient of
petal epidermal cell sizes encountered along the proximal-distal axis that
appears to be altered by loss of JAG activity. ANT function
is also associated with cell division and organ growth, and the ant
jag double mutant phenotype is consistent with the two genes having
similar functions. However, unlike JAG, overexpression of
ANT results in normally shaped, albeit enlarged, organs
(Krizek, 1999
;
Mizukami and Fischer, 2000
).
Thus, JAG and ANT are unlikely to affect the same target
genes when ectopically expressed.
JAG and bract formation
While bract formation is suppressed in Arabidopsis
(Hempel and Feldman, 1994) and
other members of the Brassicaceae family, a cryptic bract has been
proposed to exist in Arabidopsis based on the expression of
ANT in the inflorescence meristem as well as the complimentary
expression of STM (Long and
Barton, 2000
). The expression of FIL and AS1 in
a similar domain to that of ANT further supports this proposal
(Byrne et al., 2000
;
Long and Barton, 2000
;
Siegfried et al., 1999
).
JAG is exceptional in this respect in that it is an early leaf
development gene that is not expressed in the cryptic bract. Since
constitutive expression of JAG is sufficient for the outgrowth of
floral bracts, loss of JAG activity in the cryptic bract may be
responsible in part for the absence of bracts in the Brassicaceae
family. Consistent with this proposal, JAG is expressed in
lfy mutant bracts and is required for their proper development.
A JAG-specific pathway of leaf tissue growth
Analysis of the phan mutant in Antirrhinum, led Waites
and Hudson to propose that blade outgrowth requires the juxtaposition of
adaxial and abaxial boundaries (Waites and
Hudson, 1995; Waites et al.,
1998
). This model has been subsequently supported by studies of
the organ polarity genes FIL, KAN1/KAN2, PHB, PHV, AGO and
PNH/ZWILLE in Arabidopsis [summarized by Bowman et al.
(Bowman et al., 2002
)]. Given
that JAG is the only gene known to be sufficient to promote growth of
leaf tissue, it is important to determine whether JAG normally acts
in response to such boundaries or whether it may promote boundary formation.
In regard to this question it is worth noting that the JAG
gain-of-function phenotype is sensitive to the context of the plant cellular
environment, in that the phenotype produced by gain-of-function JAG
in any particular cell depends on the tissue in which it is expressed. For
instance, although ectopic JAG expression results in regularly shaped
floral bracts, the leaf-like tissue that develops from the stem appears
disorganized and lacks any overall leaf-like shape. Hence patterning
information appears to be largely absent although specific cell types
associated with adaxial and abaxial leaf domains are present and are sometimes
observed to be arranged in an organized manner. JAG expression,
although occurring early in leaf development, also appears to be initiated
slightly later than that of genes such as PHAB or FIL,
supporting a role for JAG downstream of adaxial-abaxial boundary
production. Although our genetic analysis of the fil-1 jag-3 double
mutant suggests that JAG and FlL act in different pathways,
in both cases genetic redundancy of JAG with JAG-like and
FIL with YABBY3/YABBY2
(Siegfried et al., 1999
) may
mask the participation of the individual genes in organ growth and abaxial
polarity specification. We therefore favor the proposal that JAG
normally acts downstream of leaf patterning genes during lateral organ
development, in order to promote the wild-type pattern of cell division and
differentiation. It will be interesting to try to separate these
JAG-specific functions by identifying the downstream targets of
JAG gene activity.
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ACKNOWLEDGMENTS |
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