1 Cold Spring Harbor Laboratory, Cold Spring Harbor, New York 11724, USA
2 Graduate Program in Genetics, Stony Brook University, Stony Brook, New York
11794, USA
Author for correspondence (e-mail:
timmerma{at}cshl.edu)
Accepted 22 June 2004
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SUMMARY |
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Key words: Maize, leaf, Dorsoventral, Adaxial, Abaxial, Meristem, miRNA, leafbladeless1, Rolled leaf1, YABBY
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Introduction |
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rld1 and the Arabidopsis HD-ZIPIII genes are expressed in
the central region of the SAM and throughout incipient leaf primordia. Upon
primordium emergence, HD-ZIPIII expression becomes restricted to the
vasculature and the adaxial side
(McConnell et al., 2001;
Otsuga et al., 2001
;
Emery et al., 2003
;
Juarez et al., 2004
). In both
maize and Arabidopsis, this polar expression pattern is set up by the
abaxial expression of miRNA166 or miRNA165
(Juarez et al., 2004
;
Kidner and Martienssen, 2004
),
which show extensive complementarity to HD-ZIPIII transcripts and
direct their cleavage (Reinhart et al.,
2002
; Rhoades et al.,
2002
; Tang et al.,
2003
). Single nucleotide substitutions that disrupt the
rld1 miRNA166 complementary site, as in the semi-dominant mutant
Rld1-Original (Rld1-O), lead to the persistent expression of
rld1 transcripts on the abaxial side of leaf primordia
(Juarez et al., 2004
). As a
result, Rld1-O leaves become adaxialized or partially reverse leaf
polarity (Nelson et al.,
2002
). Similarly, mutations in the miRNA165/166 complementary site
of PHB, PHV and REV are dominant and cause adaxial/abaxial
patterning defects (McConnell et al., 1998;
McConnell et al., 2001
;
Emery et al., 2003
).
Establishment of abaxial identity in Arabidopsis requires the
KANADI and YABBY genes, in addition to miRNA165 and
miRNA166. KAN1 and KAN2 encode redundant transcriptional
regulators belonging to the GARP family
(Eshed et al., 2001;
Kerstetter et al., 2001
).
KAN1 is expressed throughout young organ primordia but becomes
abaxially localized shortly after PHB transcripts become restricted
to the adaxial domain. Consistent with this expression pattern, lateral organs
of kan1 kan2 mutants are narrow or radial, and adaxialized. The
YABBY gene family comprises six members, including the vegetatively
expressed FILAMENTOUS FLOWER (FIL), YAB2 and
YAB3 (Sawa et al.,
1999
; Siegfried et al.,
1999
). YABBY proteins contain a zinc finger and a helix-loop-helix
domain (YABBY domain), and may also function as transcriptional regulators.
FIL, YAB2 and YAB3 are initially expressed throughout
incipient primordia but become restricted to the abaxial side of all
developing organs. These expression patterns are altered in the
phb1-d and kan1 kan2 double mutants, suggesting that the
YABBY genes act after adaxial/abaxial polarity is established
(Siegfried et al., 1999
;
Eshed et al., 2001
).
fil and yab3 have redundant functions but, in combination,
fil yab3 cause a partial adaxialization of the leaf
(Siegfried et al., 1999
;
Kumaran et al., 2002
).
Specification of adaxial/abaxial polarity leads to the differentiation of
distinct cell types within the upper and lower domains of the developing
primordium, and this is also reflected in the patterning of the vasculature.
Xylem tissue differentiates towards the adaxial side, whereas phloem forms on
the abaxial side. Mutations in the HD-ZIPIII genes and kan1
kan2 affect vascular patterning in both the leaf and the stem of the
plant (McConnel and Barton, 1998; Zhong
and Ye, 1999; Ratcliffe et
al., 2000
; Emery et al.,
2003
). Moreover, miRNA166 and the hd-zipIII genes
rld1 and phb are expressed in complementary domains in the
vasculature, suggesting that adaxial/abaxial patterning during vascular and
lateral organ development may be governed by a similar mechanism
(Juarez et al., 2004
).
Analysis of the Antirrhinum phantastica (phan) mutant
further indicated that the juxtaposition of adaxial and abaxial domains within
the leaf directs mediolateral lamina outgrowth
(Waites and Hudson, 1995
).
When this boundary is lost, as in the surgical experiments or as a result of
mutation of the adaxial or abaxial determinants, radial organs are produced.
By contrast, formation of additional adaxial/abaxial boundaries, as in weakly
affected phan leaves that develop patches of abaxial cells on the
adaxial leaf surface, induces the formation of ectopic lamina outgrowths.
Specification of adaxial cell fate in maize also requires normal
leafbladeless1 (lbl1) activity. Recessive mutations in
lbl1 lead to the formation of radially symmetric abaxialized leaves
and leaf-like lateral organs (Timmermans
et al., 1998). Like the weak phan leaves, less severe
lbl1 leaves develop patches of abaxial cells on the adaxial leaf
surface, which result in bifurcation of the leaf or in the formation of fully
differentiated lamina at the ectopic abaxial/adaxial boundaries. Here, we show
that lbl1 and Rld1-0 mutually suppress each other, and that
lbl1 is required for normal rld1 expression in the SAM and
on the adaxial side of leaf primordia. lbl1 thus acts upstream of
rld1 during the specification of adaxial cell fate in the primordium.
The rld1 expression pattern in the vasculature was unaffected in
lbl1 mutants, suggesting that adaxial/abaxial polarity in veins may
be established independently of lbl1 function. We also cloned maize
homologs of the Arabidopsis FIL and YAB3 genes, and show
that these maize yabby genes, in contrast to those of
Arabidopsis, are expressed on the adaxial side of developing leaf
primordia. The expression patterns of two yabby genes in
lbl1 and Rld1-O mutants suggest they act downstream of
lbl1 and rld1, and may direct lateral organ outgrowth. These
observations suggest that lbl1, rld1 and the yabby genes act
in the same genetic pathway leading to adaxial cell fate and mediolateral
outgrowth during maize leaf development.
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Materials and methods |
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Scanning electron microscopy
Three to five independent mature adult leaves (leaf 9 or 10) were analyzed
for wild type and each single and double mutant. Tissue samples were collected
approximately midway along the length of the blade near the midvein, in the
middle of the leaf lamina and at the leaf margin. Samples were fixed overnight
at 4°C in 0.1 M phosphate buffer (pH 7.0) containing 2.5% glutaraldehyde,
dehydrated through an ethanol series and critical point dried. Each sample was
divided into two halves prior to mounting to allow analysis of both the
adaxial and abaxial epidermal surfaces. Specimens were coated with gold and
analyzed on a Hitachi S-3500N SEM using an accelerating voltage of 15 kV.
Isolation of Zea mays yabby (zyb) genes
Degenerate primers, YAB5' (TGCTAYGTSMAMTGCARCTWYTGC) and YAB3'
(RTTYTTNGCWGCAGYRCTRAAKGC), were designed based on sequence conservation in
the amino-terminal Zn-finger and carboxy-terminal YABBY domains of FIL,
YAB2 and YAB3. These primers were used at a final concentration
of 2 µM and an annealing temperature of 57°C to amplify partial genomic
fragments of two maize yabby genes. Both genomic fragments were used
to screen a vegetative apex cDNA library using standard protocols. Map
positions for the zyb genes were determined using two recombinant
inbred populations (Burr et al.,
1988). ClustalW alignments of the Zn-finger and YABBY domains of
the Arabidopsis and maize YABBY proteins were generated using
MacVector6.5.1 (Oxford Molecular Group), with a gap weight of 15.00 and a
length weight of 0.30. Parsimony analyses were performed using PAUP4.0. A
consensus tree and bootstrap values were determined after 1000 replicates.
Molecular biology
Genomic DNA and Southern blots were prepared and hybridized as described
(Timmermans et al., 1996). For
RT-PCR, total RNA was isolated from the apices and young leaf primordia of
two-week-old seedlings using Trizol reagent (GibcoBRL). Approximately 1 ug of
DNaseI-treated RNA was primed with oligo(dT) and converted to complementary
DNA using M-MuLV reverse transcriptase (NEB). Subsequent PCR reactions were
carried out using standard protocols and the following gene specific
primers:
In-situ hybridization and histology
Shoot apices of two-week-old mutant and wild-type sibling seedlings were
fixed and embedded as previously described
(Jackson, 1991). Tissue
sections were pre-treated and hybridized as described by Jackson et al.
(Jackson et al., 1994
).
Digoxigenin-labeled probes comprising the 5' region including the
Zn-finger domain of zyb9 and zyb14, or nucleotides 619-1674
of the rld1 coding sequence (AY501430), were prepared by in vitro
transcription (Stratagene), according to the manufacturer's protocol.
zyb- and rld1-specific probes were used at concentrations of
15 ng/ul/kb and 0.5 ng/ul/kb probe complexity, respectively. Tissue samples
for plastic thin sections were fixed overnight at 4°C in a 0.1 M phosphate
buffer (pH 7.0) containing 4% glutaraldehyde, dehydrated through an ethanol
series, and embedded in JB-4 (Polysciences) according to the manufacturer's
protocol. Sections (1 µm) were stained with Toluidine Blue and analyzed
under bright field conditions.
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Results |
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lbl1 suppresses the adaxialization of Rld1-O leaves
rld1 is normally expressed along the adaxial domain and in the
midvein region of the P1 leaf. In older leaf primordia, rld1
expression persists in the vasculature and on the adaxial side near the
margins. However, disruption of the miRNA166 complementary site in
Rld1-O leads to accumulation of rld1 transcripts on the
abaxial side of leaf primordia (Juarez et
al., 2004). These changes in rld1 expression give rise to
a variety of adaxial/abaxial polarity defects in both the epidermal and ground
tissues, and cause an upward curling of the Rld1-O leaf blade
(Fig. 1D,
Fig. 1H, part 3). The maize
leaf comprises a proximal sheath and distal blade region separated by the
auricle and ligule (Fig. 2A,B).
The ligule is an adaxial epidermal fringe that extends the entire width of the
leaf. Approximately half the Rld1-O leaves develop patches of ectopic
ligule on the abaxial side (Table
1). Such ectopic ligular fringes are usually shorter, arise at a
slightly different position along the proximodistal axis, and do not extend
the entire width of the leaf (Fig.
2C,D). Sectors of clear tissue often extend proximal and distal
from these ectopic ligules. Ground tissue of the wild-type leaf blade consists
of evenly spaced longitudinal vascular bundles, which induce the
differentiation of concentric rings of photosynthetic bundle sheath and
mesophyll cells (Langdale et al.,
1988
). The clear sectors in Rld1-O develop a reduced
number of minor lateral veins and transverse veins, and lack the associated
photosynthetic cell types (Fig.
2C) (see also Nelson et al.,
2002
). Such sectors are associated with reduced lateral growth of
the leaf blade in addition to the duplication of the ligule, which suggests
they coincide with adaxialized regions in the primordium.
|
Regions of the Rld1-O leaf blade surrounding the cleared sectors develop less severe phenotypes. These include the differentiation of schlerenchyma tissue on the adaxial side of intermediate veins rather than the abaxial side, and the formation of small ectopic outgrowths on the abaxial leaf surface (Fig. 2E). The orientation of minor veins is slightly altered near such outgrowths, but development of the ground tissue appears otherwise normal. Therefore, the effect of Rld1-O on adaxial/abaxial patterning and its genetic interaction with lbl1 are most evident in the epidermal layers. The adaxial epidermis of the wild-type leaf blade is characterized by the presence of bulliform cells and macrohairs (Fig. 3A, part 1). All other epidermal cell types, including stomata, microhairs and prickle hairs, are present on both the adaxial and abaxial epidermis (Fig. 3A, part 2). Bulliform cells, like other cells of the epidermis, are arranged in continuous evenly spaced files that run parallel to the underlying vasculature. Macrohairs are regularly distributed within these rows of bulliform cells. Blade tissue adjacent or distal to the clear sectors in Rld1-O differentiates macrohairs and bulliform cells on the abaxial rather than the adaxial epidermis (Fig. 3B, parts 1 and 2). This suggests that adaxial/abaxial polarity in the epidermis, like that of the hypodermal schlerenchyma, is partially inverted in Rld1-O. Patterning of the adaxial and abaxial epidermal layers is unaffected near the midvein and margins. At the transition from inverted to normal polarity, both the upper and lower leaf surfaces differentiate macrohairs in irregularly spaced isolated patches and frequently independently of bulliform cells (Fig. 3B, part 3). The adaxial/abaxial polarity defects in Rld1-O thus become progressively less severe towards the margins, midvein and tip of the leaf. Furthermore, duplication of the ligule and macrohairs suggests that misexpression of rld1 in Rld1-O partially adaxializes the primordium, which can lead to abaxialization of the upper leaf surface.
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The degree of suppression of both the Rld1-O and lbl1 mutant phenotypes in the double mutant is variable and depends largely on the lbl1 allele and on the expressivity of the lbl1 mutation during primordium development. To test whether the double mutant phenotype also depends on Rld1-O dosage, F2 populations segregating both lbl1-ref/lbl1-ref Rld1-O/Rld1-O and lbl1-ref/lbl1-ref Rld1-O/+ double mutants were analyzed. Double mutants with more severe Rld1-O phenotypes did segregate in these families. However, the extreme phenotypic variation observed in these populations made it difficult to conclusively establish whether lbl1 and Rld1-O interact in a dose-dependent manner.
lbl1 acts upstream of rld1 in the specification of adaxial cell fate
The mutual suppressive interaction between lbl1 and
Rld1-O suggests that these genes act in the same genetic pathway. To
establish the genetic order in which they act, we analyzed the rld1
expression pattern in lbl1-rgd1 apices. rld1 is normally
expressed in the presumptive central zone of the SAM and in a stripe of cells
that includes the incipient leaf (Fig.
4A). In the P1 primordium, rld1 is expressed throughout
the adaxial domain but becomes gradually restricted to the adaxial side of the
margins during primordium development (Fig.
4B). Loss of adaxial cell fate in lbl1 is associated with
reduced or loss of rld1 expression on the adaxial side of the leaf
(Fig. 4C,D). The level of
rld1 expression in lbl1 varies and is negatively correlated
with the severity of the lbl1 mutant. In severe lbl1-rgd1
mutants, rld1 expression at the tip of the SAM and at the site of
leaf initiation is also lost or reduced, and this coincides with changes in
meristem morphology and maintenance (Fig.
4C). These results indicate that lbl1 acts upstream of
rld1 to specify adaxial cell fate in developing leaf primordia.
However, lbl1 may only indirectly affect rld1 expression in
the SAM. Analysis of the Antirrhinum phan and Arabidopsis
gain-of-function KANADI and YABBY mutants indicates that
abaxialization of the leaf is associated with loss of meristem function
(Waites and Hudson, 1995;
Siegfried et al., 1999
;
Eshed et al., 2001
).
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The reduced expression of rld1 in lbl1 was verified by RT-PCR analysis. In wild type, rld1 transcripts can be detected in the apex, including the SAM and approximately four young leaf primordia, as well as in older leaf primordia (Fig. 4E). rld1 transcript levels are only moderately reduced in lbl1 apices, consistent with the residual expression of rld1 in the vasculature of the stem and young leaf primordia. In older lbl1 primordia, the levels of rld1 transcripts are strongly reduced. By contrast, rld1 expression is increased in both the apex and older leaf primordia of Rld1-O. In the lbl1 Rld1-O double mutant, rld1 transcripts accumulate to a level intermediate between that of either single mutant, suggesting that the mutual suppressive interaction between lbl1 and Rld1-O may in part result from their opposing effect on rld1 expression.
yabby genes are expressed in the adaxial domain of the maize leaf
lbl1 and miRNA166 thus lead to the adaxial specific expression of
rld1 in the leaf. In Arabidopsis, downregulation of
HD-ZIPIII genes allows expression of the KANADI and
YABBY genes, which specify abaxial identity
(Sawa et al., 1999;
Siegfried et al., 1999
;
Eshed et al., 2001
;
Kerstetter et al., 2001
). To
further characterize how adaxial/abaxial polarity is established during maize
leaf development, homologs of the Arabidopsis YABBY genes were
isolated and their expression patterns analyzed. Partial genomic fragments
from two yabby homologs were amplified using degenerate primers
designed to conserved motifs in the Zn-finger and YABBY domains of the
vegetatively expressed YABBY genes FIL, YAB2 and
YAB3 (Sawa et al.,
1999
; Siegfried et al.,
1999
). These fragments subsequently allowed the isolation of four
full-length Zea mays yabby (zyb) cDNA clones from a
vegetative shoot apex cDNA library. zyb9 and zyb10 share 85%
nucleotide sequence identity and map to homeologous regions on chromosome arms
5S and 1L, respectively. Two additional zyb genes, zyb14 and
zyb15, share 71% nucleotide identity, and map to chromosome arms 10L
and 5L. None of these genes corresponds to lbl1, which maps to
chromosome arm 6S.
Phylogenetic analysis of the Arabidopsis YABBY proteins indicates
that FIL and YAB3 represent a relatively recent gene
duplication in the family. The YAB2 and YAB5 genes are, in
turn, more closely related to FIL and YAB3 than to
CRABSCLAW (CRC) or INNER NO OUTER (INO)
(Siegfried et al., 1999)
(Fig. 5). In addition to the
Zn-finger and YABBY domains, FIL and YAB3 display sequence similarity in the
C-terminal region. ZYB9/10 and ZYB14/15 are also highly conserved in the
Zn-finger and YABBY domains, and in the region downstream of the YABBY domain,
but the regions between the Zn-finger and the YABBY domains are more diverged.
Sequence comparisons between the maize and Arabidopsis YABBY proteins
suggest that all four maize genes are most closely related to FIL and
YAB3, although the precise orthologous relationships between these
family members are still unclear (Fig.
5). The divergence between ZYB9/10 and ZYB14/15 is comparable to
the divergence between FIL and YAB3, but the maize proteins form a separate
clade from FIL and YAB3.
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The in situ hybridization signals for both yabby genes were more intense in incipient and young Rld1-O primordia, suggesting their expression levels may be increased (Fig. 8E). By contrast, the in situ hybridization signals were consistently less intense in lbl1-rgd1 (Fig. 8B,C). To confirm that these differences in signal intensity reflect altered levels of zyb9 and zyb14 expression, their transcript levels in wild type, lbl1-rgd1 and Rld1-O in the A158 inbred background were compared by RT-PCR. Consistent with the in situ hybridization data, zyb14 is expressed in wild-type apices comprising the SAM and four to five young leaf primordia (Fig. 8H). No transcripts were detected in older leaf primordia. The level of zyb14 transcripts is strongly reduced in lbl1-rgd1 apices. By contrast, more zyb14 transcripts accumulate in Rld1-O apices and expression of zyb14 persists in older leaf primordia. Expression of zyb9 is also limited to the apex in wild type, lbl1-rgd1 and Rld1-O (Fig. 8I). zyb9 transcripts are less abundant than zyb14, and are only moderately reduced in lbl1-rgd1 and upregulated in Rld1-O. These results suggest that zyb9 and zyb14 act downstream of lbl1 and rld1. The mutually suppressive interaction between lbl1 and Rld1-O should therefore be evident in the yabby transcript levels. Unfortunately, in the B73 inbred background, which was used to generate the lbl1 Rld1-O double mutants, both lbl1 and Rld1-O display relatively mild phenotypes. As a result no significant differences in yabby transcript levels were observed in the single and double mutants by RT-PCR (data not shown). Nonetheless, the in situ hybridization and RT-PCR results in A158 suggest that zyb9 and zyb14 act downstream of lbl1 and rld1, and may direct mediolateral outgrowth.
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Discussion |
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Consistent with the possibility that the varied Rld1-O phenotypes
require different levels of Rld1 expression, the frequency and size
of clear sectors is enhanced in homozygous Rld1-O mutants. Moreover,
formation of clear sectors is completely suppressed in lbl1 Rld1-O
double mutants, whereas lbl1 only partially suppresses the
schlerenchyma and epidermal phenotypes. lbl1 acts upstream of
rld1 in the pathway leading to adaxial identity and is required for
the accumulation of rld1 transcripts in developing leaf primordia.
Therefore, lbl1 is likely to suppress the Rld1-O phenotypes
by reducing Rld1 mutant transcript levels in the developing leaf. The
severity of Rld1-O phenotypes is also suppressed in hyperploid plants
that carry an additional normal copy of rld1
(Nelson et al., 2002),
supporting the possibility that phenotypic severity depends on the relative
levels of Rld1 and other adaxial determinants. Mutations that disrupt
the miRNA165/166 complementary site in the Arabidopsis HD-ZIPIII
genes also cause a range of phenotypes. Such dominant alleles of PHB
and PHV cause formation of radially symmetric adaxialized leaves,
whereas similar mutations in REV mainly affect vascular patterning
(McConnell et al., 2001
;
Emery et al., 2003
). Whether
these phenotypic differences reflect differences in the relative expression
levels of these HD-ZIPIII genes during primordium and vascular
development remains to be determined.
In addition to the expected adaxialization of the lower leaf surface, the
upper blade surface of Rld1-O leaves becomes partially abaxialized
such that adaxial/abaxial polarity is inverted. None of the gain-of-function
alleles of PHB, PHV or REV cause an inversion in polarity,
but weak recessive alleles of ARGONAUTE, which is required for the
miRNA-mediated cleavage of HD-ZIPIII transcripts, can invert leaf
polarity (Kidner and Martienssen,
2004). Variation in the relative levels of rld1 and other
adaxial determinants during leaf development could also underlie this aspect
of the Rld1-O phenotype. In Drosophila, variation in the
relative levels of the ventral determinants, Dorsal and Twist, results in
inverted dorsoventral polarity in the embryo
(Stathopoulos and Levine,
2002
). High nuclear concentrations of both Dorsal and Twist,
induces ventral mesoderm, whereas high levels of Dorsal together with low
levels of Twist leads to formation of more dorsal cell types. However, Dorsal
also induces ventral identity in the absence of Twist. By analogy, balanced
levels of rld1 and other adaxial determinants may induce adaxial
identity. However, the gradual downregulation of such adaxial determinants
during primordium development progressively changes their ratio relative to
Rld1. This may temporarily cause induction of abaxial cell fate but,
upon further reduction of adaxial determinants, again lead to specification of
adaxial identity. Alternatively, as signaling between the adaxial and abaxial
domains is important to coordinate outgrowth and patterning of the leaf,
switching of cell identity in the adaxial layer may be a consequence of the
adaxialization of the lower leaf surface (see also
Nelson et al., 2002
).
lbl1 specifies adaxial fate during lateral organ development via rld1
lbl1 is required for the specification of adaxial cell fate in
lateral organs (Timmermans et al.,
1998). Loss of lbl1 activity affects lateral founder cell
recruitment in addition to adaxial/abaxial patterning, and both these defects
are suppressed in the double mutant with Rld1-O. Expression of
zyb9 and zyb14 is increased on the adaxial side of incipient
and young Rld1-O primordia, which could counteract the reduced
expression of these yabby genes in lbl1. But, why are the
yabby expression levels increased in Rld1-O? miRNA166 only
accumulates on the abaxial side in the incipient and P1 leaf
(Juarez et al., 2004
).
Therefore, increases in adaxial yabby expression levels must arise
independently of the loss of miRNA166 action in Rld1-O. In situ
hybridization intensities suggest that rld1 expression is similarly
upregulated on the adaxial side in Rld1-O prior to the accumulation
of miRNA166. Similarly, disruption of the miRNA165/166 complementary site in
PHB causes overexpression of PHB transcripts on the adaxial
side in addition to ectopic expression of mutant transcripts on the abaxial
side (McConnell et al., 2001
).
This increase in adaxial expression also precedes the accumulation of miRNA165
in that domain (Kidner and Martienssen,
2004
). The adaxial domain of the leaf promotes meristem function
(McConnell and Barton, 1998
;
Kidner et al., 2002
).
Conversely, specification of adaxial cell fate requires a signal from the
meristem (Sussex, 1951
;
Sussex, 1955
). Owing to such
reciprocal communication between the SAM and the leaf, the production,
perception or activity of the meristem-borne signal may be altered in the
adaxialized Rld1-O and phb-1d mutants. HD-ZIPIII proteins
contain a highly conserved START lipid-sterol binding domain, and potentially
become activated in response to the meristem-derived signal. As a result,
genes acting downstream of the hd-zipIII genes, such as zyb9
and zyb14, may become upregulated. Moreover, if hd-zipIII
genes are positively autoregulated, adaxial hd-zipIII expression can
be increased independent of the loss of miRNA166 directed transcript
cleavage.
lbl1 specifies adaxial identity by regulating the accumulation of
rld1 transcripts on the adaxial side of developing leaf primordia.
rld1 and phb have similar expression patterns and probably
act redundantly, as the HD-ZIPIII genes do in Arabidopsis
(Emery et al., 2003). Loss of
adaxial identity in lbl1 mutants thus suggests that lbl1 not
only acts upstream of rld1 but possibly upstream of other
hd-zipIII genes as well. Meristematic expression of rld1 is
also reduced in lbl1. However, this could result from reduced adaxial
identity in adjacent leaf primordia rather than from a direct effect of
lbl1 on hd-zipIII expression in the SAM. The level and
pattern of rld1 expression in the vasculature of lbl1 is not
affected, which makes it unlikely that lbl1 controls
hd-zipIII expression by modulating the miRNA166 expression domain.
Transcription of the hd-zipIII genes may depend on lbl1
function directly. Alternatively, if hd-zipIII expression is
autoregulated in a ligand-dependent manner, lbl1 may affect the
accumulation of hd-zipIII transcripts indirectly by regulating the
production or perception of this ligand. The radially symmetric abaxialized
leaves that arise following surgical separation from the SAM are shorter than
normal and develop siphonostelic (with phloem and xylem cells surrounding a
central pith) or protostelic (with phloem surrounding xylem) vascular bundles
(Sussex, 1951
;
Sussex, 1955
). Depending on
expressivity, lbl1 leaves display comparable growth and patterning
defects (Timmermans et al.,
1998
) (M.T.J. and M.C.P.T., unpublished).
Specification of adaxial/abaxial polarity during vascular and lateral organ
development involves a partially conserved mechanism. rld1 and
phb expression on the adaxial side of lateral organs, and in the
adaxial pro-xylem cells, are both defined by the pattern of miRNA166
accumulation (Juarez et al.,
2004). In Arabidopsis, KANADI genes are expressed on the
abaxial side of developing organs, and vascular expression is limited to the
abaxial and peripheral phloem cells
(Kerstetter et al., 2001
;
Emery et al., 2003
). Mutational
analysis has further shown that the KANADI and HD-ZIPIII
genes act antagonistically during both vascular and lateral organ development
(Emery et al., 2003
). The
miRNA-directed cleavage of HD-ZIPIII transcripts is conserved
throughout all lineages of land plants and precedes the origin of angiosperm
leaves (Floyd and Bowman,
2004
). Therefore, the MIR166, HD-ZIPIII and, possibly,
the KANADI genes may have had an initial role in the specification of
adaxial/abaxial polarity in the vascular tissue of non-leafy plants, only
later acquiring an additional function in the patterning of lateral organs
(Eshed et al., 2001
;
Kidner et al., 2002
;
Emery et al., 2003
). Because
lbl1 affects hd-zipIII expression only on the adaxial side
of lateral organs and not in the vasculature, its role in adaxial/abaxial
patterning could coincide with and possibly contribute to the derivation of
leaves from branching shoots (Gifford and
Foster, 1989
).
Maize yabby genes may direct lateral outgrowth rather than specify adaxial cell fate
Loss- and gain-of-function mutations reveal a role for YABBY genes
in the specification of abaxial cell fate in Arabidopsis
(Sawa et al., 1999;
Siegfried et al., 1999
;
Kumaran et al., 2002
).
Consistent with this role, YABBY gene expression is correlated with
the abaxial domain in wild-type and mutant leaf primordia
(Siegfried et al., 1999
;
Eshed et al., 2001
). The tomato
FIL/YAB3 homolog, LeYAB B, is similarly expressed on the
abaxial side of leaf primordia, and may function in the specification of
abaxial cell identity in this compound-leaved species
(Kim et al., 2003
).
zyb9 and zyb14 are expressed in a polar pattern, but, unlike
Arabidopsis and tomato, these maize yabby genes are
expressed on the adaxial side of incipient and young leaf primordia. Because
rld1 and phb are expressed in a pattern analogous to the
Arabidopsis HD-ZIPIII genes on the adaxial side of developing leaves,
the regulation and/or function of the yabby genes must have diverged
between Arabidopsis and maize. The Arabidopsis HD-ZIPIII
genes suppress YABBY expression on the adaxial side of P2 and older
leaf primordia (Eshed et al.,
2001
). By contrast, expression of zyb9 and zyb14
mirrors that of the hd-zipIII genes, and their increased expression
in Rld1-O indicates that both yabby genes are positively
regulated by rld1. Maize yabby expression persists outside
the hd-zipIII expression domain at the presumptive adaxial/abaxial
boundary, and misexpression of Rld1 is not sufficient to induce
zyb9 and zyb14 expression on the abaxial side during
Rld1-O primordium development. These observations suggest that other
factors in addition to the hd-zipIII genes control yabby
gene expression.
YABBY function may also have diverged between Arabidopsis and
maize despite the high amino acid sequence conservation. Specification of
adaxial/abaxial polarity leads to mediolateral outgrowth and patterning of the
leaf and both of these processes are affected in fil yab3
(Siegfried et al., 1999;
Kumaran et al., 2002
). The
role of the maize yabby genes in leaf development is less clear.
Transposon insertion alleles of zyb9 and zyb14 display no
phenotypes, probably because of functional redundancy (M.T.J. and M.C.P.T.,
unpublished). yabby genes may specify adaxial identity, as reduced
adaxial cell fate in lbl1 is correlated with decreased zyb9
and zyb14 expression. However, adaxialization of Rld1-O leaf
primordia is not correlated with yabby expression on the abaxial
side. Also, their apparent uniform expression in the lbl1 ectopic
outgrowths is inconsistent with a role for zyb9 and zyb14 in
adaxial cell fate determination. The expression patterns of these maize
yabby genes suggest that they may function during mediolateral
outgrowth. The lbl1 defect in founder cell recruitment is correlated
with reduced zyb9 and zyb14 expression, and suppression of
this defect in lbl1 Rld1-O is associated with increased
yabby expression in the incipient primordium. Also, ectopic
outgrowths in lbl1 and Rld1-O express both yabby
genes, irrespective of whether such outgrowths arise on the adaxial or abaxial
side of the leaf.
Ectopic lamina on weakly phenotypic lbl1 leaves arise at the
boundary of abaxialized sectors on the adaxial leaf surface
(Timmermans et al., 1998). In
Rld1-O, no ectopic outgrowths develop at the boundaries of regions
with inverted polarity, suggesting that juxtaposition of adaxial and abaxial
cells in just the epidermis and subepidermal schlerenchyma is insufficient to
induce lateral outgrowth. Interestingly, ectopic outgrowths in Rld1-O
arise on the abaxial side at positions where blade tissue expressing
zyb9 and zyb14 in the central layer of the ground tissue is
juxtaposed next to blade tissue that no longer expresses these yabby
genes. The polar expression of the yabby genes in the incipient
primordium and at the margins of older leaf primordia may similarly be
required for founder cell recruitment and continued mediolateral blade
outgrowth. Lateral outgrowth during Arabidopsis primordium
development is also correlated with polar YABBY gene expression.
FIL and YAB3 are uniformly expressed in the incipient
primordium but become restricted to the abaxial side at the time blade
outgrowth occurs (Sawa et al.,
1999
; Siegfried et al.,
1999
). However, lbl1 ectopic lamina initially show
uniform expression of zyb9 and zyb14. Perhaps, juxtaposition
of yabby expressing and non-expressing cells is not essential for
lateral outgrowth in that context, or perhaps outgrowth of ectopic blade
tissues is initially restricted to their base.
The maize and Arabidopsis yabby genes may thus share a role in
mediolateral outgrowth. However, Arabidopsis YABBY genes also play a
role in abaxial cell fate determination
(Sawa et al., 1999; Seigfried
et al., 1999; Kumaran et al.,
2002
). Although the distinct phenotypes of kan1 kan2 and
fil yab3 mutants and their epistatic interactions suggest that the
KANADI and YABBY genes act in separate pathways with both
distinct and overlapping targets (Eshed et
al., 2001
). Mediolateral growth of the maize leaf is initiated
within the context of positional information inherent in the meristem, whereas
lateral blade outgrowth in Arabidopsis occurs after emergence of the
primordium from the SAM. Owing to these distinct growth habits, maize and
Arabidopsis yabby genes may be under different evolutionary
constraints. YABBY genes in Arabidopsis may have a specific
role in the maintenance of the meristematic positional information in the
isolated primordium that is not required in maize. In the absence of such a
requirement, selection to maintain a specific polar expression pattern could
be weakened. Most monocots elaborate dorsoventral blade tissue, like maize
does, from the lower leaf zone by lateral founder cell recruitment. However,
several monocot species that develop unifacial leaves or that develop blade
tissue from the upper leaf zone after primordium emergence, like
Arabidopsis does, are nested within the monocot clade
(Kaplan, 1973
;
Bharathan, 1996
). Comparative
analysis of yabby expression patterns in such diverse monocots may
help to elucidate whether yabby genes are indeed under different
evolutionary constraints depending on the leaf growth habit.
Adaxial/abaxial axis specification in the maize leaf
lbl1, rld1, and the mir166 and yabby genes act
in the same genetic pathway leading to adaxial cell fate and mediolateral
outgrowth of the leaf (Fig. 9).
rld1 in combination with other regulatory factors leads to adaxial
expression of the yabby genes zyb9 and zyb14.
Polarized expression of these yabby genes may mediate lateral founder
cell recruitment and thus, directly or indirectly, control the downregulation
of knox genes. rld1 also specifies adaxial cell fate but
probably independently of the yabby genes. Adaxial-specific
expression of rld1 in the developing leaf depends on lbl1
and miRNA166. lbl1 positively affects the accumulation of
rld1 transcripts, whereas miRNA166 directs their cleavage. miRNA166
initially accumulates immediately below the incipient leaf but gradually
spreads via the abaxial side throughout the developing primordium
(Juarez et al., 2004). The
specification of adaxial cell fate also requires a signal from the meristem
(Sussex, 1951
;
Sussex, 1955
). This signal
could act via RLD1 and other HD-ZIPIII family members, as they contain a START
lipid-sterol binding domain. If so, RLD1 and other HD-ZIPIII proteins may
specify adaxial/abaxial polarity in developing leaves by incorporating
positional information established by two opposing signals that originate
outside the incipient primordium: the adaxializing signal from the SAM and the
miRNA166 signal from a potential signaling center below the incipient leaf.
Finally our results present the possibility that lbl1 specifies
adaxial cell fate in developing leaf primordia by altering the production or
perception of the proposed meristem-borne signal.
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ACKNOWLEDGMENTS |
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Footnotes |
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