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First published online 22 March 2006
doi: 10.1242/dev.02335
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1 Division of Biological Sciences, University of California San Diego, La Jolla,
CA 92093, USA.
2 Plant Biology Laboratory, The Salk Institute for Biological Studies, La Jolla,
CA 92037, USA.
3 Department of Molecular Biology, Max Planck Institute for Developmental
Biology, D-72076 Tübingen, Germany.
* Author for correspondence (e-mail: marty{at}ucsd.edu)
Accepted 21 February 2006
 |
SUMMARY |
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 TOP SUMMARY INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key words: Arabidopsis, Development, Lateral organs, Growth, Patterning, Polarity, Floral organs
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INTRODUCTION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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;
Dinneny et al., 2004;
Ha et al., 2003;
Hepworth et al., 2005;
Nath et al., 2003;
Norberg et al., 2005;
Ohno et al., 2004;
Palatnik et al., 2003). The
C2H2 zinc-finger transcription factor encoded by
JAGGED (JAG) is expressed in all organs and appears to act
as a region-specific promoter of growth. In a loss-of-function jag
background, lateral organ growth is partially suppressed. In petals, a
correlation between the expression domain of JAG and the abundance of
cells actively engaged in the cell-cycle suggests that JAG may
regulate growth by maintaining or activating cell-cycle activity. Furthermore,
the phenotypic similarity between jag loss-of-function mutants and
transgenic lines in which cyclin-dependent kinase inhibitors are
overexpressed, suppressing cell-cycle activity, further hints at such a
connection (De Veylder et al.,
2001; Wang et al.,
2000). Unlike other genes that promote growth, such as
AINTEGUMENTA and BIG BROTHER
(Disch et al., 2006;
Krizek, 1999;
Mizukami and Fischer, 2000),
JAG is also sufficient to promote morphogenesis in multiple types of
lateral organs. In rosette leaves, for example, where JAG activity is
normally limited to the blade region, ectopic expression can promote blade
tissue expansion into the petiole region.
The ability of ectopic JAG activity to promote blade tissue led to
the hypothesis that the BLADE-ON-PETIOLE (BOP) genes might
negatively regulate JAG expression
(Ohno et al., 2004). Like
JAG gain-of-function lines, loss of BOP activity results in
the ectopic development of blade tissue in the petiole region of leaves
(Ha et al., 2003). Recent work
characterizing the bop1,2 double mutant has further elucidated the
role of BOP genes in lateral organ development. The BOP
genes negatively regulate JAG expression in lateral organs,
particularly in the cryptic bract, a vestigle organ in Arabidopsis
that subtends floral primordia, but which develops as a macroscopic leaf-like
organ in other species (Hepworth et al.,
2005; Norberg et al.,
2005). Ectopic expression of JAG in the cryptic bract
rescues organ outgrowth, whereas loss of JAG activity suppresses
bract outgrowth in mutants that ectopically develop bracts
(Dinneny et al., 2004;
Ohno et al., 2004). However,
when JAG activity is removed from bop1,2 double mutants,
which also develop ectopic bracts, no suppression of the bop-mutant
phenotype was seen (Norberg et al.,
2005). The observation that ectopic expression of a gene closely
related to JAG, At1g13400 [previously referred to as
JAGGED-LIKE and renamed NUBBIN (NUB) in this work],
is also detected in bop1,2 mutants suggests that other factors in
addition to JAG may be responsible for the ectopic tissue growth in
bop mutants (Norberg et al.,
2005). Independently, we have hypothesized that NUB may
function redundantly with JAG
(Dinneny et al., 2004), because
JAG does not equally affect all lateral organs in which it is
expressed. Here, we have used gain-of-function and loss-of-function approaches
to reveal the function of NUB in development. We focus our analysis
on the role of NUB and JAG in stamen and carpel development,
where NUB is expressed in a polar manner on the organ surface
oriented toward the shoot axis, termed the adaxial side. The polar expression
of NUB on the adaxial side correlates with the growth and patterning
defects observed in jag nub mutants.
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MATERIALS AND METHODS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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) and
SPL::GUS (Ito et al.,
2004) have been described before. Seeds for the T-DNA insertion
line 244A08 (also known as nub-1) were obtained from GABI-Kat
(Rosso et al., 2003).
Genotyping
Genotyping of jag-1 has been described before
(Dinneny et al., 2004). To
genotype nub-1 we used primers oJD153 (AAG ACA GCG GAG GAT AAA GAT
ATG) and oJD154 (GCT TCT CTT CAT CTT CTT CTT CTG G) to detect the wild-type
allele and oJD155 (GTT CAT GTG GCC ACC GAG AGC TTG) and GABRB (GTG GAT TGA TGT
GAT ATC TCC) to detect the nub-1 allele.
Construction of transgenic plants
The NUB-RNAi contruct was made by amplifying a 326 bp NUB
cDNA fragment using primers N-1392 (ATA GCT TTC CTC CTC ATC AAG GAC) and
N-1393 (ATG TGG AGC AAC TCT AGA ACC ATT A), and cloning this into the
pHANNIBAL vector (Wesley et al.,
2001) in forward and reverse orientations (pJD93). The
NUB-RNAi cassette was then inserted 3' of an APETALA1
promoter fragment derived from pAM571
(Mandel et al., 1992) using
BamHI restriction sites (pJD108). The FIL::NUB construct was
made by amplifying a 4 kb FILAMENTOUS FLOWER promoter fragment, using
primers oJD182 (gag ctc CAA CCA TTG AAC CAT CAC CGA TAT TC) and oJD183 (ggt
acC TTT TTT GTA AGA AGG GGA AAA ATA TTG GAA GCT G) that introduced
SacI and KpnI restriction sites (indicated by the lowercase
letters) to the 5' and 3' ends, respectively. This fragment was
then cloned into the pMX202 (Wu et al.,
2003) binary vector (pJD136). A full-length NUB cDNA
fragment was then amplified from pPY1
(Dinneny et al., 2004), using
primers oJD211 (ggt acc ATG AGA GCT GAT GAA AAT AAC ACT TTA G) and oJD212 (ggt
acc TTA TAG CCC ATG ATG TGG AGG TAG ATG) that introduced KpnI
restriction sites to the 5' and 3' ends, and cloned into pJD136
(pJD148). Transgenic Col-0 plants were generated by the floral-dip method
(Clough and Bent, 1998).
RT-PCR
RNA amplification and reverse transcription reactions were performed as
previously described (Kardailsky et al.,
1999). PCR amplification was performed on 2 µl of
reverse-transcription reaction. A NUB fragment encompassing the
entire coding region was amplified using oJD119 (AAG CTT ATG AGA GCT GAT GAA
AAT AAC AC) and oJD120 (GGA TCC TTA TAG CCC ATG ATG TGG AGG). A
TUBULIN fragment was amplified as previously described
(Dinneny et al., 2004).
In situ hybridization, histology and microscopy
In situ hybridization was performed as previously described
(Dinneny et al., 2004). An
anti-sense NUB probe was transcribed using T7 RNA polymerase
(Promega) from a partial NUB cDNA clone (pJD109) linearized with
HindIII. The JAG (Dinneny
et al., 2004), FIL
(Siegfried et al., 1999) and
PHB (McConnell et al.,
2001) probes have previously been described. Scanning electron
microscopy (SEM) was performed as previously described
(Dinneny et al., 2004).
Embedding of plant material in JB-4 media, sectioning and staining with
Toluidine Blue was performed as previously described
(Roeder et al., 2003).
Saffranin O and Alcian Blue staining of paraffin sections performed as
previously described (Roeder et al.,
2003). GUS staining performed as previously described
(Blázquez et al.,
1997).
Measurements of cell layer number and average cell width
Cell layer number and cell width measurements were determined from the same
transverse cross-sections of stage-13 gynoecia. For each genotype, 16 sections
were imaged from four gynoecia. Sections were taken from the approximate
middle of the gynoecium and were separated by at least 30 µm from each
other. Cell layer number was measured in four regions of the carpel. Two
counts were obtained from valve tissues flanking the main vascular bundle of
the valve (lateral region) and two counts were obtained from valve tissues
flanking the presumptive valve margins (medial region). The average width of
cells in the ena, enb and outer epidermal cell layers were
determined as follows. First, the total width of all cells in a particular
cell layer was measured using Image J software
(http://rsb.info.nih.gov/ij/).
Next, the number of cells in this layer was determined. The average cell width
was then calculated by dividing the width of all cells in a layer by the
number of cells in that layer. Both valves of the gynoecium in each section
were counted separately.
Abaxial trichome number during leaf development was determined for the area of the leaf blade outside of the midvein, as this region normally contains trichomes in most leaves. Segregating T2 transgenic FIL::NUB plants were selected on MS agar plates containing 20 µM kanamycin then transferred to soil. Wild-type control plants were also germinated on MS agar plates before being transferred to soil. Trichome counts were performed on plants after bolting. Kanamycin is unlikely to affect abaxial trichome development, as a FIL::GFP transgenic line germinated on kanamycin-supplemented agar plates did not develop ectopic abaxial trichomes (data not shown).
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RESULTS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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;
McConnell et al., 2001;
Waites et al., 1998). Like JAG, NUB expression is excluded from the cryptic bract (Fig. 1D). Unlike JAG, however, NUB expression is also excluded from sepal primordia (Fig. 1D). The expression of NUB in petals is transient and can only be detected during the initial outgrowth of the primordia (Fig. 1K). During stamen development, NUB is strongly expressed throughout initiating primordia during stage 5 and then becomes restricted to the adaxial side by stage 6 (Fig. 1E-G). At stage 8, NUB expression becomes restricted to a small cleft of tissue in between the two sets of microsporangia (Fig. 1H). This expression pattern differs from JAG, which is expressed throughout stamen primordia (see Fig. S1 in the supplementary material). During carpel development, NUB is initially expressed in cell layers of both abaxial and adaxial halves (Fig. 1G), it then becomes restricted to the adaxial cell layers at stage 7 (Fig. 1H,I). NUB expression can also be seen on the adaxial side of initiating ovule primordia (Fig. 1J). In summary, NUB expression is more restricted than that of JAG and is found predominantly in leaf, stamen and carpel primordia. Furthermore, although JAG is expressed throughout all tissue layers of organ primordia, the adaxial expression of NUB suggests that it may play a role in promoting adaxial-specific development.
Identification of the nub-1 allele
We identified a T-DNA insertion allele of NUB, 244A08, in the
GABI-Kat collection (Rosso et al.,
2003). Sequencing of the insertion site by GABI-Kat indicates that
line 244A08 has a T-DNA disrupting the second intron of NUB
(Fig. 2A). Examination of
NUB transcript levels shows that the nub-1 mutation strongly
diminishes the accumulation of the NUB full-length transcript
(Fig. 2B). The homozygous
mutant does not display an abnormal phenotype (data not shown). This was not
entirely unexpected, as the NUB expression domain is fully
encompassed by the JAG expression domain. The jag nub double
mutant, however, results in a strong enhancement of defects in leaf, stamen
and carpel development compared with jag single mutants (see below).
To ensure that these enhanced defects are specifically due to the
nub-1 mutation, we generated a NUB-RNAi construct and
introduced this into a jag mutant background and observed similar
defects in stamens and carpels as in jag nub mutants
(Fig. 2C,D). Sepal development
is also affected indicating that our NUB-RNAi construct may result in
some non-specific degradation of other transcripts (data not shown).
|
).
NUB promotes the growth of the adaxial side of anthers
Stamens are divided into two regions, an apical anther and a basal filament
(Bowman, 1993). In jag
nub mutants, anther development is nearly abolished, whereas the filament
does not appear to be affected (Fig.
3, see also Fig. S2 in the supplementary material). We examined
the anther region more closely to determine whether any anther-specific cell
types remained in jag nub mutants. On the adaxial side of wild-type
anthers, four microsporangia develop, which enclose the developing pollen in
chambers termed locules (Fig.
3D,K). During anthesis, the pollen is released from the anthers at
a dehiscence zone that develops between the two locules in each pair. On the
abaxial side, a small lump of tissue, termed the connective, joins the two
sets of microsporangia to the filament
(Fig. 3G,K). When we examined
the cells present in the stub of tissue that remains in the anther region of
jag nub mutants, we saw that it is composed of two different domains
(Fig. 3F). In the apical half
of the stub, cells with a slight puzzle shape form with cuticular ridges
(Fig. 3F,I). In the basal
region, cells with a rounded shape develop without cuticular ridges
(Fig. 3F). The apical cells
resemble those normally found in the connective region of the anther
(Fig. 3H), whereas the basal
cells do not resemble any cell type found in the anther.
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|
We also took a closer look at the anthers of jag single mutants in plastic sections and found that there seemed to be fewer locules (Fig. 3L). This was at first surprising, as the anthers appeared to have four microsporangia when imaged by SEM (Fig. 3E). When the sections of jag anthers were examined more closely it could be seen that a narrow crease still develops and runs down the center of the microsporangia at the point which would normally divide the two locules and where the anthers normally dehisce (Fig. 3L). Thus, although the external structure suggests that four microsporangia have formed, the internal structure suggests that only two locules are patterned in jag mutants, or that the tissue that normally separates the locules does not develop properly.
FIL expression marks the connective tissue of the anther
To further characterize the defects in stamen development seen in jag
nub mutants, we examined the expression of genes that mark different
regions of the anther. We first examined FIL, which is initially
expressed on the abaxial side of anthers
(Fig. 4A). Later, as tissues on
the adaxial side proliferate to form the microsporangia, FIL
expression remains on the abaxial side in tissues that do not proliferate as
much and become the connective (Fig.
4B). In jag nub mutants, FIL expression
initiates in the anthers as it does in wild type
(Fig. 4C). During stage 7,
however, FIL expression extends toward the adaxial side
(Fig. 4D). The apparent
expansion of FIL onto the adaxial side of the anther may indicate
that abaxial fate has expanded to the adaxial side in jag nub
anthers. Alternatively, the expansion of FIL may be an indirect
consequence of unequal growth of the abaxial side in relation to the adaxial
side in jag nub mutants. In either case, these data suggest that the
anthers of jag nub mutants are largely composed of connective tissue,
marked by FIL expression, and that the adaxial microsporangia tissues
fail to proliferate.
PHB expression marks the dehiscence zone of anthers
We next examined genes that are normally expressed in tissues on the
adaxial side of the anther. PHB is initially expressed in the
vasculature and on the adaxial side, but it soon becomes restricted to cells
that denote the two dehiscence zones of the anther
(Fig. 4E). In jag nub
mutants, where no microsporangia development is apparent, PHB
expression is limited to a few cells in the center of the jag nub
anther, presumably marking the vasculature
(Fig. 4F). Note that
PHB expression on the adaxial side of the carpels is not affected in
jag nub mutants (Fig.
4E,F).
|
;
Schiefthaler et al., 1999;
Yang et al., 1999). During
wild-type anther development, the SPL reporter is expressed in two
domains on the peripheral flanks of the anther
(Fig. 4G)
(Ito et al., 2004).
Surprisingly, in jag nub mutants, where microsporangia formation is
not apparent, SPL reporter activity is still initiated in the proper
domains (Fig. 4I). Later during
anther development, SPL reporter activity is localized to the basal
region of jag nub anthers, where we observed rounded cells with
little differentiation (Fig.
4J). Finally, reporter activity is lost in the anther region of
jag nub mutants (Fig.
4K), whereas in wild type, expression is maintained until anthesis
(Fig. 4H). These data show
that, although microsporangia development is blocked in jag nub
mutants, the patterning of the microsporangia domains appears to occur
normally. Thus, JAG and NUB may not be required to specify
microsporangia per se, but instead act in parallel to such specification
pathways to promote the growth of these tissues. This function of JAG
and NUB may be similar to the role that JAG plays in petal
development where it promotes distal petal growth.
Abaxial NUB expression promotes connective growth
To further test whether NUB regulates microsporangia identity or
plays a more restricted role in promoting the growth of this region, we used
the FIL promoter to drive NUB expression on the abaxial side
of anthers in the connective region. We reasoned that if NUB is
sufficient to promote microsporangia identity, then FIL::NUB
transgenic lines might be expected to promote ectopic microsporangia formation
in place of the connective. Alternatively, if NUB regulates growth,
but does not control the identity of microsporangia tissues, then we might
expect the connective to become enlarged in FIL::NUB anthers but
still retain the same cell types as in a wild-type connective.
As shown in Fig. 5, introduction of a FIL::NUB transgene into wild-type plants results in anthers with expanded abaxial tissue. In moderately affected lines, such as JD148-B, most of the ectopic growth is localized to the connective region, resulting in an enlarged and elongated structure (Fig. 5A,B). In stronger lines, such as JD148-A, the entire abaxial side appears to become overgrown and can be seen exceeding the limits of the adaxial microsporangia (Fig. 5C,D). To determine whether this ectopic tissue is composed of microsporangia-like or connective-like cell types, we analyzed plastic thin sections of FIL::NUB anthers and found it to be composed of cells most similar to the connective (Fig. 3K, Fig. 5E).
The observation that FIL::NUB transgenic lines have an abnormal
phenotype suggests that the FIL promoter is active in these lines. We
confirmed our prediction by examining FIL expression in
FIL::NUB flowers and found FIL to be expressed on the
abaxial side in two cell layers (Fig.
5F). Interestingly, the expression of FIL in anthers
appeared to have expanded laterally, probably because of the positive effect
of NUB expression on tissue proliferation. Thus, despite being
normally expressed in mutually exclusive domains, FIL expression is
compatible with NUB expression in FIL::NUB transgenic lines.
These findings differentiate NUB from other adaxially expressed genes
such as PHB, which are sufficient to suppress FIL expression
if mis-expressed on the abaxial side
(Siegfried et al., 1999).
|
;
Ohno et al., 2004), jag
nub double mutants show a strong decrease in the growth of the valves
near the apical region of the gynoecium, resulting in exposure of the ovules
(Fig. 6G,H). Tissues not
associated with the valves, such as the replum and style seem to be less
affected and grow apically beyond the limits of the valves. The splitting of
the style, which was occasionally observed in jag mutants, is further
enhanced in jag nub gynoecia (Fig.
6) (Ohno et al.,
2004).
We next followed these defects throughout gynoecium development. In
wild-type flowers, the gynoecium develops as a cylinder of tissue
(Fig. 6A)
(Bowman, 1993;
Smyth et al., 1990). During
this early stage, jag nub gynoecia develop notches near the apical
end (Fig. 6B). As the gynoecium
grows, these notches enlarge and appear to be restricted to the valve regions
(Fig. 6C,D). At stage 11, the
gynoecial cylinder closes with the formation of stigmatic papillae in the
apical region (Fig. 6E). In
jag nub mutants, however, the gynoecium does not close, leaving a
gaping hole in the ovary (Fig.
6F). The valve tissue in this apical region is often shredded in
appearance (Fig. 6H). These
data show that JAG and NUB promote the growth of valve
tissues during gynoecium development, and that failure of jag nub
valves to enclose the ovules is caused by this defect. In agreement with this
interpretation, we also have found that strongly affected FIL::NUB
transgenic lines develop valve tissues that grow beyond their normal limits
and expand into the style region, showing that NUB is also sufficient
to ectopically activate valve growth (Fig.
8A,B). Interestingly, the epidermal cells of the ectopic valve
tissue develop cuticular wax similar to epidermal cells found in the style
region.
NUB promotes the proper differentiation of adaxial cell types in the valves
The adaxial-specific expression of NUB during carpel development
suggested that NUB may play a role in the development of tissues in
this region. To explore this possibility, thin sections were made of
wild-type, jag, nub and jag nub gynoecia
(Fig. 7; data not shown). In
wild type, the valves or carpel walls of the gynoecium are composed of six
layers of cells: an outer epidermis, three layers of mesophyll, a modified
layer of mesophyll termed the enb and finally the inner epidermal
layer termed the ena (Fig.
7A). Most of the cell layers are established early during
gynoecium development as the dome of tissue, which forms the gynoecium, begins
to develop a cleft down the middle to create a cylinder of tissue at stage 7
(see Fig. S3 in the supplementary material)
(Alvarez and Smyth, 1998).
Later, during stage 9-11, cells near the valve margins of the valve divide,
resulting in a final count of six cell layers. At flower opening (stage 13),
each cell layer is distinguishable as a result of differences in cell size.
The enb cells, for example, are very small owing to cell divisions
that begin at stage 9. By contrast, cells of the ena are very large
and also begin to differentiate in size from the other cell layers around
stage 9.
In jag nub mutant gynoecia, we found that the number of cell layers was reduced (Fig. 7A,C). We measured cell layer number in two regions of the carpel walls and found a one to two cell layer reduction (Fig. 7D). This reduction always appeared to be at the expense of the mesophyll. These defects suggest that JAG and NUB are necessary to promote the cell divisions that established the correct number of cell layers. We also found that jag single mutants display a slight decrease in the number of cell layers, but not to the extent of jag nub mutants (Fig. 7A-D). We examined nub single mutants but did not observe any apparent defects in cell layer number or differentiation (data not shown)
|
Development of the ena layer was also affected in jag nub mutants. Whereas enb layer cells were larger in jag nub mutants, ena cells were smaller than in wild type (Fig. 7A,C). Both the ena and enb defects are seen in jag single mutants, but to a lesser degree (Fig. 7A-C,E). The outer epidermal cells were slightly wider in jag and jag nub mutants compared to wild type (two-tailed t-test, P=0.0025 and 0.0210, respectively), but there was no significant difference between jag and jag nub mutants (P=0.8257), consistent with the lack of NUB expression in this cell layer (see Fig. S5 in the supplementary material). These data indicate that JAG and NUB play a specific role in promoting the differentiation of adaxial cell types in the carpel walls.
|
|
). More
recently, we have shown that combining jag-1 with the fil
and yabby3 (yab3) mutations results in a dramatic loss of
blade development, demonstrating that JAG does indeed promote leaf
blade expansion (Dinneny et al.,
2005). When we examine the leaves of jag nub mutants, we
also see a reduction in leaf blade area compared with wild type
(Fig. 9A-D). The shape of
jag nub leaves is somewhat variable and notches can form in the
leaves (Fig. 9C). These notches
may represent enhanced versions of the serrations found in jag single
mutants (Fig. 9B). Leaves of
jag nub mutants also develop outgrowths on the abaxial side
(Fig. 9E-G). These outgrowths
are often radialized and develop a high concentration of trichomes. In
kanadi mutants, such outgrowths, termed enations, are also observed
(Eshed et al., 2004). The
enations of jag nub mutants are most common overlying vascular
tissues, and tend to occur more frequently near the tips of the leaves and
serrations. Scanning electron microscopy of the epidermal cells that
constitute jag nub enations shows that they are shaped similarly to
cells overlying vascular bundles (Fig.
9G).
|
).
After the plant has produced about six leaves, trichomes begin to develop on
the abaxial side and increase in number until the formation of the cauline
leaves. In both strong (Fig.
10) and moderate (see Fig. S6 in the supplementary material)
FIL::NUB transgenic lines, trichome development is precocious in
comparison with wild type. For example, the transgenic line JD148-A begins to
develop abaxial trichomes as early as the third leaf. The early development of
trichomes on the abaxial side of leaves suggests that the FIL::NUB
transgene is able to impart partial adaxial identity to the abaxial side of
leaves. We have also noticed that FIL::NUB transgenic lines
occasionally develop trichomes on the adaxial side of floral organs, including
petals, stamens and carpels (Fig.
8A; data not shown). |
DISCUSSION |
|---|
|
TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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JAG and NUB as shape determinants
In this work, we have extended our understanding of the role that
JAG plays in shaping lateral organs by unraveling a layer of genetic
redundancy caused by NUB, which masks the effects of jag
mutations on stamen and carpel development. In stamens, JAG and
NUB function together to promote microsporangia formation. Our
analysis, using the expression of genes that mark different regions of the
anther, reveals that the jag nub anther is composed largely of
connective tissue. Interestingly, however, the initial expression pattern of
the SPL microsporangia marker appears normal in jag nub
anthers. These data suggest that JAG and NUB are not
required to specify the identity of microsporangia tissues, but instead
promote growth on the adaxial side, which is a prerequisite for microsporangia
formation. In the gynoecium, JAG and NUB act to drive
sufficient growth of the valves necessary to enclose the ovules. Growth
defects in the gynoecium of jag nub mutants appear to be limited to
the valves, while replum and style growth is unaffected. This function is the
converse of that performed by genes such as CRABS CLAW and
SPATULA, which affect the growth of stigma and style tissues without
causing defects in valve growth (Alvarez
and Smyth, 1999; Alvarez and
Smyth, 2002).
Characterizing the redundant roles of JAG and NUB has allowed us to determine the relationship between their spatial patterns of gene expression and their activity. This is particularly important for determining directness, and to establish whether these genes control distinct morphogenetic events or act more globally to control growth throughout an organ. Although the expression of JAG suggests that it may play a role in stamen and carpel development, it was necessary to examine the function of NUB to elucidate the nature of this activity. Taking into account the mutant phenotypes of jag and jag nub mutants reveals that the expression domains of JAG and NUB directly correlate with defects in growth that occur when their functions are disrupted. Furthermore misexpression studies demonstrate that both JAG and NUB are able to activate growth in ectopic locations. Together, these data show that JAG and NUB control specific morphogenetic events during lateral organ development that are crucial for determining final shape.
|
;
Eshed et al., 2001;
Eshed et al., 2004;
Kerstetter et al., 2001;
Mallory et al., 2004;
McConnell et al., 2001;
Pekker et al., 2005). Ectopic
expression of PHB has been shown to negatively regulate FIL
expression, whereas ectopic KAN expression on the adaxial side can
suppress PHV and REV expression. One interesting peculiarity
with the expression domain of adaxial identity factors is that they are often
expressed in both the meristem and the adaxial side of lateral organs.
NUB expression, however, is limited to the adaxial side of lateral
organs and is excluded from the meristem. This suggests that NUB may
function downstream of, or in parallel to, the polarity pathways. Examination
of FIL and PHB expression in jag nub mutants shows
no clear mis-regulation of either gene in the gynoecium. In stamens,
FIL expression appears to extend circumferentially into the adaxial
domain; however, this may simply be a consequence of the altered growth
dynamics of a jag nub anther, where the normally proliferative
adaxial microsporangia region is stunted. Furthermore, ectopic expression of
NUB on the abaxial side does not suppress FIL expression,
nor does it result in ectopic PHB expression (see Fig. S7 in the
supplementary material). Consistent with these observations, phenotypic
analysis of NUB gain-of-function and loss-of-function lines does not
reveal any homeotic conversion of abaxial or adaxial tissues. These data
suggest that JAG and NUB regulate histogenesis in parallel
with pathways that establish polarity. The polarity pathways, which are known
to regulate the spatial distribution of tissues, may require input from
pathways that regulate growth. Although the nature of this relationship is
unclear, recent work demonstrating that FIL and YABBY3 act
in cooperation with JAG to pattern the distribution of tissues in the
fruit supports the existence of such a partnership
(Dinneny et al., 2005). It may
be that, in addition to promoting growth, JAG and NUB also
promote the competence of tissues to respond to factors that determine tissue
type and distribution, thus linking morphogenesis to histogenesis.
Two observations complicate any strict delineation of JAG and
NUB function in adaxial development, however. First, jag nub
mutants develop enations on the abaxial side of their leaves. Guided by the
model proposed by Waites and Hudson, that the juxtaposition of adaxial and
abaxial domains drives tissue outgrowth
(Waites and Hudson, 1995),
enation formation in jag nub leaves suggests a role for JAG
and NUB in establishing/maintaining the boundaries between adaxial
and abaxial halves. The second observation, that NUB expression on
the abaxial side is sufficient to drive precocious trichome development
further suggests that NUB may also play a more active role in
regulating adaxial identity.
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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/133/9/1645/DC1
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