1 Institut für Entwicklungsbiologie, Universität zu Köln,
Gyrhofstr 17, D-50923 Köln, Germany
2 Department of Plant Biology, University of Georgia, Athens, GA 30602,
USA
Authors for correspondence (e-mail:
werr{at}uni-koeln.de
and
mjscanlo{at}plantbio.uga.edu)
Accepted 3 March 2004
![]() |
SUMMARY |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Key words: narrow sheath, pressed flower, Maize, Leaf development, SAM, Founder cells
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Mutations in the narrow sheath (ns) genes cause the
deletion of a lateral domain in maize leaves that includes the margins of the
lower leaf (Scanlon et al.,
1996) (Fig. 1). The
ns margin deletion phenotype does not extend into the upper portion of the
leaf, and the leaf length is also unaffected
(Fig. 1A-C). Both vegetative
and floral phytomers are affected; whereas no ns mutant phenotype is observed
in the embryonic coleoptile, shoot meristems or roots
(Scanlon and Freeling, 1998
).
The ns phenotype is a duplicate factor trait controlled by recessive mutations
at two unlinked loci, ns1 and ns2
(Scanlon et al., 2000
).
Heterozygous plants containing just a single, non-mutant copy of either
ns gene display the non-mutant phenotype
(Fig. 1G-H). KNOX
immunolocalization studies and fate mapping of ns mutant meristems reveal that
a meristematic founder-cell domain that normally contributes to the non-mutant
leaf margins is not recruited in ns mutant leaves.
|
We describe the cloning of the ns genes through homology to
PRESSED FLOWER (PRS), a WUSCHEL-like homeobox gene
that is required for development of lateral sepals in the Arabidopsis
flower (Matsumoto and Okada,
2001). Sequence homology and mutational analyses suggest that the
duplicated maize ns genes are the redundant, functional orthologs of
PRS. Immunoblot analyses of maize proteins verify that the
ns-R mutations are both null alleles. Furthermore, quantitative
analyses of ns gene transcripts suggest that ns1 and
ns2 are expressed redundantly in tissues enriched for shoot apical
meristems, although differences in specific ns gene transcript
abundance are detected in reproductive tissues. Moreover, previously
undescribed phenotypes are discovered in the leaves and stamens of
Prs- mutant Arabidopsis plants. Together the ns and
Prs- mutant phenotypes support existing models for the evolution of
angiosperm leaf morphology via the differential elaboration of distinct leaf
zones, and suggest a model whereby orthologous NS/PRS proteins function to
recruit organ founder cells in a lateral domain of shoot meristems.
![]() |
Materials and methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Cloning of narrow sheath genomic DNA and cDNA
The homeodomain of the ns1 gene (AC AJ536578) was isolated by PCR
using degenerate primers designed from the conserved homeobox region of the
WUSCHEL gene from Arabidopsis. The duplicated gene
ns2 (GenBank AC AJ472083) was cloned by homology to ns1,
using primers designed from the conserved homeobox region of ns1.
Detailed descriptions of the strategies, probes and primers employed to obtain
full length genomic and cDNA clones of ns1 and ns2 alleles
are provided (see Data S1 at
http://dev.biologists.org/supplemental/).
Computational and database analysis
The multiple alignment was performed using ClustalW
(http://www.ebi.ac.uk/clustalw/)
and BOXSHADE
(http://searchlauncher.bcm.tmc.edu/multi-align/multi-align.html).
Phylogenetic trees were generated based on the Neighbor-Joining and Maximal
Parsimony (MP) methods, using PAUP* Version 4.0b8 (SWOFFORD 1999)
with default parameters. Sequences examined were as follows: AtPRS
(Arabidopsis thaliana, BAB79446), AtWUS (Arabidopsis
thaliana, CAA09986), AtHD (Arabidopsis thaliana, NP_188428),
OsHD1 (Oryza sativa, CL042143.26.34) and OsHD2 (Oryza
sativa, BAA90492).
DNA gel-blot analyses
Genomic DNA was isolated from maize seedlings and leaves, and analyzed by
DNA gel blot hybridization analysis as described previously
(James et al., 1995).
Hybridization probes were radioactively labeled and column-purified as
described (Fu et al., 2002
).
Specific probes used in these analyses were as follows. Probe 1 was a 479 base
pair (bp) 5' genome walker product (upstream of primer ZmPRSb, see Table
S1 at
http://dev.biologists.org/supplemental/)
that hybridizes to both ns loci. Probe 2 was a 792 bp genomic PCR
product (between primers ZmPRSc and ZmPRS2, see Table S1) that also includes
the 5' UTR of ns1.
Antibody production, protein extraction and immunoblot analyses
Affinity purified NS polyclonal antibodies were generated (Biosource)
against a synthetic peptide comprising amino acid residues 235 to 251 of the
predicted NS2 protein (N-LKTLDLFPTKSTGLKDE-C, see
Fig. 2A), with a Cys residue
added to the N terminus. This amino acid sequence is completely conserved in
the predicted NS1 protein (amino acids 232 to 248). Thus, polyclonal
antibodies raised against this peptide antigen are expected to identify
epitopes in NS1 as well as NS2 proteins.
|
In situ hybridization analyses
For non-radioactive in situ hybridization, samples were prepared following
the protocol of Jackson (Jackson,
1991). For sections of maize embryos, kernels were trimmed on both
sides of the embryo axis for better penetration of the formaldehyde fixative
and the wax solution. Paraffin wax-embedded tissue was sectioned by the use of
a rotary microtome and 7 µm sections were used for hybridization. The first
315 bp (upstream of primer ZmPRSb), including the whole 5' UTR, of the
ns1 cDNA sequence, cloned either in the sense or antisense
orientation to the T7 promotor, were used as a template for synthesis of
digoxigenin-labelled RNA probes by T7 RNA polymerase as described
(Bradley et al., 1993
). This
probe is predicted to hybridize to both ns1 and ns2
transcripts. The PRS probe corresponded to that used by Matsumoto and
Okada (Matsumoto and Okada,
2001
).
Quantification of ns transcript accumulation by real-time reverse transcriptase-mediated PCR
Total RNA was extracted from non-mutant B73 maize tissues with the
TRIZOLTM Reagent (Invitrogen, Life Technology), according to the
manufacturer's protocols. One µg of total RNA was treated with DNAseI
(Promega) and was subsequently used to prepare first strand cDNA, as described
by Bauer et al. (Bauer et al.,
1994). The resultant cDNA was treated with RNaseH (GIBCO BRL) to
remove residual mRNA and the concentration of all samples was adjusted to 50
ng/µl.
The cDNA was checked for residual genomic DNA contamination using the ns2-specific primer ns2F6, which is within the transcribed region of the gene, and a second primer, ns2R8, which is from the 3' untranscribed region (see Table S1 at http://dev.biologists.org/supplemental/). Real-time RT-PCR amplification was performed in a volume of 25 µl using 100 ng cDNA template, 0.2 mM of each dNTP, 3 mM MgCl2, 250 nM of each primer and 1U of Platinum TaqTM DNA Polymerase (Invitrogen, Life technology), using a Cepheid Smart CyclerTM. The cycling program was: 95°C for 2 minutes; and 45 cycles of 95°C for 10 seconds, 57°C for 10 seconds and 72°C for 15 seconds. The LUXTM primers were designed online (www.invitrogen.com/LUX) and are shown in Table S1.
The amplified fragments were analyzed by electrophoresis on 3% Agarose
1000TM (Invitrogen, Life Technology); a single band of the predicted size
was obtained in all samples included in these assays. Each sample was assayed
in triplicate, and analyses of relative ns gene expression data,
normalized to control ubiquitin expression, was performed as
described by Livak and Schmittgen (Livak
and Schmittgen, 2001).
Cryo-scanning electron microscopy and light microscopy
Cryo-scanning electron microscopy of dissected Arabidopsis
seedlings was performed with expert technical assistance from Dr John Shields
(Center for Ultrastructural Research, University of Georgia, Athens, GA, USA)
as described previously (Scanlon,
2003).
Whole-mounted Arabidopsis plants at the two leaf stage were harvested, roots and one cotyledon were removed by dissection, and the remaining shoot was mounted in water and imaged on a Zeiss Axioplan II equipped with a Southern Micro Instruments (Pompano Beach, FL, USA) CCD camera.
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Expression of the narrow sheath (ns) phenotype in lateral organs of maize
vegetative and inflorescence shoots is dependent upon homozygosity for
mutations at each of two unlinked loci, narrow sheath1 and narrow
sheath2 (Scanlon et al.,
1996). Intriguingly, segregation of a DraI restriction
fragment length polymorphism (RFLP), due to the CACTA element insertion in 76
F2 ns mutant plants, revealed that the PRS-homologous maize clone
hybridizes to more than one genetic locus in maize, each of which
co-segregates with the ns mutant phenotype
(Fig. 3A). Previous genetic
analyses proved that the ns loci map to regions of the maize genome
that are duplicated (Scanlon et al.,
2000
). Therefore, we sought to clone the ns2-linked
sequence through its predicted sequence homology to the ns1-linked,
PRS-related maize clone.
|
Indeed, the two loci encode highly related homeodomain-containing proteins. The ns1-linked transcript encodes a predicted protein of 262 amino acids, which shares 86% identity with the 265 amino acids encoded by the ns2-linked gene product (Fig. 2A). Moreover, the position of a 124 bp intron is conserved. Database searches reveal that both predicted proteins bear little overall homology to described plant genes; the highest similarities detected are with two rice homeodomain proteins (OsHD1 and OsHD2; 38% and 37% identity, 99/262 and 97/262 amino acids, respectively) and the Arabidopsis protein PRESSED FLOWER (37% identity; 97/262 amino acids). Outside of the conserved homeodomain sequence conservation is low to PRS and OsHD, with the exception of two Q- or H-rich clusters proximal to the homeodomain and a conserved PLKTL E/DLFP motif close to the C terminus (Fig. 2A). Importantly, although any homology is primarily localized to the WUSCHEL-type homeodomain, the ns1/2-linked duplicate maize genes are significantly more similar to PRS than to WUSCHEL or other Arabidopsis relatives (Fig. 2B).
Structure of ns1 and ns2 mutant alleles
The chromosomal map positions, the sequence similarity and the RFLP
co-segregation with the ns mutant phenotype suggested that ns1 and
ns2 might encode PRS orthologous functions in maize. Therefore
homologous clones were isolated from various maize genetic stocks, including
the reference mutations ns1-R and ns2-R, non-mutant
Mutator (Mu) transposon lines, and several ns
mutant alleles derived from Mu lines (described in Materials and
methods). Sequence analyses of these ns-linked clones reveal
molecular lesions or chromosomal deletions in nine independent ns1
mutations and two ns2 mutant alleles, as described below.
Compared with non-mutant alleles of the ns1-linked clone, the
ns1-R allele contains a transposable element insertion close to the C
terminus of the protein-coding region (amino acid 256, see
Fig. 2A, Fig. 3B). The 1.2 kb element
belongs to the CACTA transposable element superfamily
(Kunze and Weil, 2002); a
characteristic 3 bp (CCT) duplication is identified at the transposon
insertion site. Transcript analyses reveal that the transposon-inserted
ns1-R allele is transcribed and polyadenylated, although the mutant
transcript terminates prematurely within the CACTA element. Thus, a potential
translation product of the ns1-R allele would be truncated at the C
terminus.
When compared with non-mutant genomic and cDNA sequences of ns2-linked alleles, the ns2-R mutant allele contains an extra G nucleotide in the second exon, corresponding to position 779 of the transcript (Fig. 2A). Insertion of this extra nucleotide alters the open reading frame and introduces a premature stop codon at nucleotide 855. Thus, the amino acid sequence of the truncated NS2-R translation product is predicted to diverge from the non-mutant polypeptide after residue 146, and terminates after just 170 total residues. These data reveal that the ns2-R mutation is tightly linked to a maize gene that is a duplicate of the ns1 sequence, and which harbors a predicted frameshift mutation.
In addition, Southern blot analyses of eight, independent
ns1-*Mu linked alleles recovered from separate
transposon-tagging experiments (see Materials and methods) (see also
Scanlon et al., 2000) reveal
that all ns1-*Mu plants contain deletions of the
ns1-linked allele contributed by the non-mutant,
Mu-transposon parent (Fig.
3C and data not shown). Likewise, three independent alleles of the
ns2 mutation identified by Mu transposon-tagging harbor
deletions of the ns2-linked allele contributed by the Mu
parent. The extent of the deletion within one such allele,
ns2-*Mu1, was investigated. A 5'-directed chromosome
walk used nested primers located 547 bp downstream of the 3'
untranslated region in ns2-*Mu1 homozygous individuals
(see Data S1 at
http://dev.biologists.org/supplemental/)
and generated an approximately 3 kb genomic clone. For the first 226 bp from
the 3' primer sites the nucleotide sequence of this
ns2-*Mu1 clone is 94% identical to clones obtained (using
the same primers and chromosome walking strategy) from B73 and non-mutant
Mutator lines. However, after the 226 bp 3' homologous region,
the ns2-*Mu1 clone is completely non-homologous to any
sequence contained within a total of 4,548 bp of ns2-linked DNA
derived from non-mutant clones. These data suggest that the ns2 gene
is entirely deleted in the ns2-*Mu1 allele
(Fig. 3B). This conclusion is
supported by Southern blot comparisons of ns2-*Mu1
homozygous plants and non-mutant siblings, in which no ns2-linked
hybridizing band is detected in ns2-*Mu1 plants
(Fig. 3D). Significantly, the
non-mutant Ns1-Mu+ allele can be amplified from non-mutant
siblings of the newly tagged ns1-*Mu1 plants, as well as from the
non-mutant Mu parental stock. Finally, all thirteen
Mu-derived ns1 and/or ns2 mutant alleles were
identified as single ns mutant phenotypes within thirteen separate populations
of more than 5,000 siblings each, indicating that these deletion mutations
each occurred spontaneously in single, maternal gametes. These accumulated
data suggest that the two PRS-homologous maize clones identify the
ns1 and ns2 duplicated loci.
Immunoblot analyses of null ns-R mutant alleles
Gene dosage analyses indicated that the recessive ns-R mutations
are null alleles (Scanlon et al.,
2000). In order to test this prediction, polyclonal antibodies
were raised against a peptide antigen that is completely conserved in the
predicted NS1 and NS2 proteins (see Materials and methods), and used in
immunoblot assays of proteins extracted from maize tissues
(Fig. 3E). The anti-NS
antibodies identify a protein of the approximate molecular weight (29 kDa)
predicted for ns- encoded proteins in 4-5 cm immature ears obtained
from non-mutant plants of the genotypes Ns1+; Ns2+ and
Ns1+/ns1-R; ns2-R. At this stage of development, maize ears contain
abundant spikelet and floret meristems, as well as immature lateral organs
(Kiesselbach, 1949
). By
contrast, no immunoreactive protein of this predicted molecular weight was
detected in mature, non-mutant seedling leaves (data not shown) nor in
immature ears of the genotype ns1-R; ns2-R
(Fig. 3E). These data reveal
that the ns-R mutant inflorescences do not accumulate NS proteins; however, NS
protein(s) accumulate in non-mutant immature ears. Unfortunately, the anti-NS
polyclonal antibody identifies additional protein(s) of a different molecular
weight to the predicted NS protein (lower band in
Fig. 3E). Although the
polyclonal antiserum therefore is not suitable for use in
immunohistolocalization analyses, the absence of NS-predicted molecular weight
proteins in ns double mutants confirms that on the protein level
ns1-R and ns2-R provide null alleles.
NARROW SHEATH transcripts are detected in meristematic foci and in the margins of lateral organ primordia
The similarities in amino acid sequence raise the question of whether
ns1/ns2 and PRS exhibit similar expression patterns and
encode orthologous functions in maize and Arabidopsis. A probe
predicted to hybridize to both ns1 and ns2 was used for in
situ analyses of ns transcription throughout embryonic, vegetative
and reproductive stages of maize development. As predicted from analyses of
the ns mutant phenotypes in vegetative and floral organs
(Scanlon and Freeling, 1998),
ns1 and ns2 are expressed predominately in tissues enriched
for shoot meristems and young lateral organ primordia
(Fig. 4). Overall, the pattern
of ns gene expression is two-staged, similar to that reported for
PRS (Matsumoto and Okada,
2001
). An early-staged ns expression is observed at two
foci in lateral domains of shoot meristems, whereas later-staged expression
appears in the margins of young lateral organ primordia.
|
Maize leaves exhibit alternate phyllotaxy, such that successive primordia
initiate from the SAM approximately 180° apart and in two ranks
(Fig. 4C). Fate-mapping
analyses demonstrate that founder cells that form the eventual midrib of the
maize leaf are recruited from one SAM flank, whereas margin founder cells
occupy the opposing flank (Scanlon and
Freeling, 1997). ns transcripts accumulate in founder
cells of the P0/1 primordium in two foci, located at opposite lateral domains
of the shoot apex (Fig. 4D,G).
This early ns expression focus is limited to a series of adjacent
cells in the L1 tissue layer of the apex. No ns activity is detected
in the founder-cell domains that give rise to the future midrib. Later,
ns transcripts mark the lateral margins of young leaf primordia
(Fig. 4E,F,H,I), and are
restricted to single epidermal cells forming the boundary between the abaxial
and adaxial leaf surfaces.
The accumulation of ns transcripts in male and female maize flowers was also investigated. The data presented in Fig. 4K-Q show ns expression in the female inflorescence (ear); equivalent patterns are observed in the male inflorescence (data not shown). Early developmental programs are very similar in the male and female inflorescences of maize; gender-specific differences occur during later stages. An overview of maize inflorescence development is presented in Fig. 4K. In summary, the inflorescence meristem initiates spikelet-pair primordia, which give rise to the two spikelet primordia. Each spikelet primordium subsequently develops into an upper and a lower floret. Both florets develop in the tassel, whereas development of the lower floret is aborted in the ear. A non-mutant maize floret comprises leaf-like organs (glumes, lemma and palea), three stamens and the gynoecium. During the formation of monoecious maize flowers, either the stamens or the gynoecium abort during female or male sexual differentiation, respectively. As observed in developing leaves, ns transcripts are detected in the margins of all floral organ primordia (Fig. 4L-Q). The specification of marginal identity, as indicated by ns activity, is therefore characteristic of all determinate lateral organs of the maize shoot. Conversely, ns expression is not detected in indeterminate organs such as the spikelet-pair and spikelet meristems.
Real-time RT-PCR used gene-specific primers (see Table S1 at http://dev.biologists.org/supplemental/) to investigate quantitative expression patterns of the ns duplicate genes during development (Fig. 5). All values are normalized to expression levels of the control maize gene ubiquitin, as described (Livark and Schmittgen, 2001). No ns1 or ns2 expression is detected in roots, seedling leaves or fully expanded coleoptiles. Interestingly, although the levels of ns1 and ns2 transcripts are virtually equivalent in vegetative shoot apices (five leaf primordia and the SAM) and in the male inflorescence, ns2 transcripts are more abundant than ns1 in the female inflorescence (Fig. 5). In addition, no significant difference in non-mutant ns1 or ns2 transcript abundance is detected in inflorescence or vegetative apices isolated from plants in which one ns locus was homozygous non-mutant and the other was homozygous mutant (data not shown). Thus, the ns-R mutations each failed to induce any compensatory transcript over-accumulation from the corresponding non-mutant locus in either the vegetative or inflorescence/reproductive shoot meristems or primordia.
|
However, close inspection of developing Prs- mutant leaf
primordia (Fig. 6B-D,F-H)
reveals a previously unreported deletion of the stipules, located at the
lateral-most domain of the lower Arabidopsis leaf
(Medford et al., 1992). Aside
from this stipule deletion, mature mutant rosette leaves reveal no obvious
phenotype affecting the size, shape or epidermal cell morphology of either the
leaf blade or petiole (Fig.
6A,E; data not shown). Likewise, whole-mount and scanning electron
microscopic analyses of Prs- mutant cotyledons reveal no
distinguishable phenotype in the blade or petiole
(Fig. 6I,J; data not shown).
Specifically, Prs- mutant and non-mutant rosette leaves and
cotyledons exhibit equivalent width, length and cellular morphology in the
lamina and petiole. Thus, although PRS transcripts are detected in
the epidermal cells located at the lamina and petiole margins of leaf and
cotyledon primordia (Matsumoto and Okada,
2001
) (Fig. 6K-L), no obvious phenotype is correlated with this later, primordial-staged
expression.
|
|
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
ns and Prs- leaf phenotypes: molecular genetic support for a model describing the evolution of leaf morphology
Mutations in ns1 and ns2 together confer the deletion of
a lateral domain in maize leaves that extends from the midpoint of the distal
blade and includes the entire length of the proximal sheath
(Fig. 1)
(Scanlon et al., 1996). By
contrast, the leaf phenotype of the orthologous Arabidopsis mutant
Prs- is extremely subtle, and limited to the deletion of the
proximal, lateral stipules (Fig.
6). No additional phenotype is detected in either the petiole or
the lamina of Prs- mutant leaves. A survey of the
Arabidopsis genome reveals that, unlike the duplicated ns
sequences of maize, PRS is a single copy gene. Therefore, there is no
evidence to suggest that additional leaf mutant phenotypes of PRS function are
obscured by redundant, PRS- homologous gene sequences. Instead, the
differential leaf phenotypes conditioned by the ns and prs
mutations are consistent with an existing model describing the evolution of
monocot and eudicot leaf morphology.
A model of leaf zonation predicts that bifacial (i.e. dorsoventrally
flattened) eudicot leaves are subdivided into a large upper leaf zone
comprised of the lamina and petiole, and a greatly diminished lower leaf zone
comprising the leaf base and the lateral stipules
(Fig. 7A) (Troll, 1955;
Kaplan, 1973
). By contrast,
the model suggests that bifacial monocot leaves have evolved an extended and
highly elaborated lower leaf zone and an extremely abbreviated upper leaf
zone. In this model, the upper leaf zone of the monocot maize comprises just a
short, unifacial fore-runner tip, whereas the majority of the leaf blade and
the entire sheath are derived from the lower leaf zone
(Fig. 7B). When interpreted in
terms of this model, the ns mutant leaf phenotype is localized to a lateral
domain in the lower region of the lower leaf zone (i.e. the lower blade and
entire sheath). Extending this model to the dicot Arabidopsis leaf,
deletion of a lateral domain in the lower part of the greatly diminished,
lower leaf zone in Prs- mutant plants would predict a minor
phenotype affecting the stipules. This subtle, stipule-deletion phenotype is
precisely that observed in Prs- mutant leaves. Therefore, the
apparent incongruity in the leaf phenotypes observed in maize ns and
Arabidopsis Prs- mutants is a predicted outcome of Troll's
model of leaf zonation, and supports the hypothesis that the differential
elaboration of the upper and lower leaf zones has contributed to the
morphological diversity of maize and Arabidopsis leaves. Another
popular model of leaf morphology suggested that the sheath region of monocot
grass leaves evolved via flattening of the petioles of eudicot progenitors
(Arber, 1934
). However, the
lateral domain deletion in ns mutant sheaths, together with the lack of a
petiole phenotype in the Prs- mutant leaf, fails to support this
interpretation.
|
NARROW SHEATH performs a conserved function in maize and Arabidopsis
Sequence homology, similarity of gene expression profiles and comparative
mutant phenotypes together identify the maize ns duplicate genes and
PRS of Arabidopsis as orthologous sequences. Two distinct
developmental time points of ns/PRS expression are conserved among
maize and Arabidopsis, defined as an early expression within two
lateral, meristematic foci and a later-staged expression in the margins of
young lateral organ primordia (Fig.
4) (Matsumoto and Okada,
2001). When considered in the context of ns and Prs-
mutant phenotypes, these expression domains suggest that the essential
function(s) of the ns/PRS gene product is limited to the early,
meristematic expression domains, as predicted in previous clonal analyses of
NS function (Scanlon,
2000
).
The later, primordial expression pattern of ns/PRS is restricted
to a few cells at the margins of developing lateral organ primordia
(Fig. 4E,F) (Matsumoto and Okada, 2001).
However, no maize or Arabidopsis mutant phenotype correlates with
loss of NS/PRS function in the margins of lateral organ primordia. For
example, primordial-staged PRS expression is detected in epidermal
cells that will eventually form the margins of the Arabidopsis leaf
lamina, and also in the primordial margins of the cotyledon
(Fig. 6K-M). No Prs-
mutant phenotypes are detected in these lateral organ domains
(Fig. 6I,J) (Matsumoto and Okada, 2001
).
Likewise, ns is expressed at the edges of the developing coleoptile
and leaf primordia (Fig.
4B,D-I), although no ns mutant phenotype is detected in the
coleoptile (Scanlon and Freeling,
1998
). Moreover, clonal analyses
(Scanlon, 2000
) uncovered
multiple cases wherein ns1 function was present at the ns
meristematic focus (i.e. early ns expression pattern) but was absent
from the margins of developing maize leaf primordia (i.e. late ns
expression pattern). In all such cases, loss of late-staged NS function in the
L1-derived primordial margins was non-phenotypic, whereas loss of early staged
function from the ns meristematic focus always correlated with the ns
mutant phenotype. As discussed below, we suggest that the Prs-
stipule, lateral sepal and lateral stamen deletion phenotypes of
Arabidopsis, as well as the ns mutant phenotypes in maize lateral
organs, all arise from the loss of a conserved, early NS/PRS function in a
lateral domain of the vegetative and/or reproductive shoot apical
meristem.
Fate-mapping analyses of maize leaf founder cells
(Poethig, 1984), as well as
clonal sector analysis of NS function
(Scanlon, 2000
), reveal that
maize leaf anlagen are recruited from the circumference of the meristem, such
that the midrib founder cells occupy one flank of the SAM and the margin
founder cells of the lower leaf (sheath) are recruited in close proximity. In
this way midrib sectors may be clonal to sectors on the marginal flank of
subtending leaves, with such sectors intersecting both the right and left
margins of the leaf sheath (Scanlon and
Freeling, 1997
). The series of transverse sections through the
maize shoot apex shows a first stripe of NS transcriptional activity at the
lateral flank of the SAM, where clonal analyses indicated two foci of
functional NS activity. However, ns transcripts are localized to the
tunica of the shoot apex. This L1 pattern conflicts with clonal analyses of
NS1 function, which concluded that NS1 function in the L1 layer of the SAM
cannot compensate for loss of function in the L2 meristematic layer
(Scanlon, 2000
). It is
possible that mericlinal L1-L2 mutant sectors present at one meristematic
focus in these sample plants conditioned a mutant leaf phenotype.
Subsequently, post-meristematic invasion of non-mutant L1 clones may have
generated ns mutant leaves containing L2-derived ns1-null sectors but
a non-mutant epidermis. Additional speculative explanations are possible,
although resolution of this apparent discrepancy will await localization
analyses of PRS/NS protein within the shoot apex.
Intriguingly, this stripe of NS activity in the SAM appears between the P0 and the P1 primordia, where recruitment of cells into opposing primordial founder-cell domains converges at the lateral flanks of the SAM (see Fig. 8A). NS activity might be required for recruitment of cells at the P0 face of the SAM into the horseshoe shaped P1 primordium, thereby allowing the intermingling of subtending primordial founder-cell domains in the shoot apex. By contrast, the ns mutant phenotype could occur if cells at the P0 face, which in wild type are recruited for the basal lateral leaf domain (mainly sheath) of the P1 primordium, are mutually exclusively recruited for the next primordium (P0) in absence of NS activity (see Fig. 8B). In the absence of NS activity, founder-cell recruitment into the P0 and P1 primordia may compete for cells at the lateral flanks of the SAM in ns mutants, whereas in wild type NS activity allows overlapping founder-cell domains. Thus, one possible NS function as a WUS-type transcription factor may be to maintain developmental competence in a stripe of cells in two lateral SAM domains.
|
|
Strong support for this evolutionarily conserved, and organ domain-blind,
model of NS/PRS function is provided by the sepal and stamen deletion
phenotypes in Arabidopsis Prs- mutants
(Fig. 9C). Mutations at
prs typically condition the deletion of just the margins of the
adaxial and abaxial sepals, whereas the two lateral sepals fail to initiate or
are vestigial. This phenotype indicates that PRS does not specify a specific
marginal domain in these lateral organs, as both the central as well
as the marginal domains of the lateral sepals are deleted in
Prs- mutants. In this view, the expression domain of PRS
in two lateral points on the Arabidopsis floral meristem
(Matsumoto and Okada, 2001)
correlates to foci from which founder cells contributing to the entire lateral
sepals, as well as to the margins of the adaxial and abaxial sepals, are
recruited. Moreover, the stamen deletion phenotype of Prs- mutants
affects only lateral stamens (Table
1); the adaxial and abaxial stamens are intact. This model
predicts that lack of PRS function blocks recruitment of lateral founder-cell
domains of the Arabidopsis floral meristem, causing the deletion of
lateral sepals and stamens. These observations support a model whereby NS/PRS
function is conserved to initiate founder cells from a lateral domain of plant
meristems.
Intriguingly, no phenotype is observed in Prs- mutant petals,
except in cases wherein these second whorl organs are homeotically transformed
into sepalloid organ identity (Matsumoto
and Okada, 2001). Therefore, although PRS functions in the second
whorl of Arabidopsis floral meristems, recruitment of petal founder
cells may not extend into the PRS functional domain. We speculate that the
much smaller size (Smyth et al.,
1990
; Bossinger and Smyth,
1996
) and phyllotactic arrangement of petal primordia (i.e. offset
from the lateral meristematic domain) is such that these organs do not develop
from founder cells that encroach into the lateral meristematic domain
recruited by PRS (Fig. 9C). Currently, efforts are underway to investigate the ns/Prs- mutant
phenotype in the compound leaves of tomato and pea. These experiments will
further expand our understanding of the role of this gene during the evolution
of diverse angiosperm leaf morphology.
![]() |
ACKNOWLEDGMENTS |
---|
![]() |
Footnotes |
---|
* These authors contributed equally to this work
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Arber, A. (1934). The Gramineae. A Study of Cereal, Bamboo, and Grasses. Cambridge: Cambridge University Press.
Bassiri, A., Irish, E. E. and Poethig, R. S.
(1992). Heterochronic effects of Teopod2 on the growth
and photosensitivity of the maize shoot. Plant Cell
4, 497-504.
Bauer, P., Crespi, M. D., Szecsi, J., Allison, L. A., Schultze,
M., Ratet, P., Kondorosi, E. and Kondorosi, A. (1994).
Alfalfa Enod12 genes are differentially regulated during nodule development by
nod factors and Rhizobium invasion. Plant Physiol.
105,585
-592.
Bommert, P. and Werr, W. (2001). Gene expression patterns in the maize caryopsis: clues to decisions in embryo and endosperm development. Gene 271,131 -142.[CrossRef][Medline]
Bossinger, G. and Smyth, D. R. (1996).
Initiation patterns of flower and floral organ development in Arabidopsis
thaliana. Development 122,1093
-1102.
Boyd, L. (1931). Evolution in the monocotyledonous seedling: a new interpretation of the grass embryo. Trans. Bot. Soc. Edinburgh 30,286 -302.
Bradley, D., Carpenter, R., Sommer, H., Hartley, N. and Coen, E. (1993). Complementary floral homeotic phenotypes result from opposite orientations of a transposon at the plena locus of Antirrhinum. Cell 72,85 -95.[Medline]
Byrne, M., Timmermans, M., Kidner, C. and Martienssen, R. (2001). Development of leaf shape. Curr. Opin. Plant Biol. 4,38 -43.[CrossRef][Medline]
Elster, R., Bommert, P., Sheridan, W. F. and Werr, W. (2000). Analysis of four embryo-specific mutants in Zea mays reveals that incomplete radial organization of the proembryo interferes with subsequent development. Dev. Genes Evol. 210,300 -310.[CrossRef][Medline]
Fletcher, J. C. and Meyerowitz, E. M. (2000). Cell signaling within the shoot meristem. Curr. Opin. Plant Biol. 3,23 -30.[CrossRef][Medline]
Franken, P., Niesbach-Klosgen, U., Weydemann, U., Marechal-Drourard, L. and Saedler, H. (1991). The duplicate chalcone synthase genes c2 and whp1 (white pollen) of Zea mays are independently regulated; evidence for translational control of Whp expression by the anthocyanin intensifying gene EMBO J. 10,2605 -2612.[Abstract]
Fu, S., Meeley, R. and Scanlon, M. J. (2002).
empty pericarp2 encodes a negative regulator of the heat shock
response and is required for maize embryogenesis. Plant
Cell 14,3119
-3132.
Gaut, B. S. and Doebley, J. F. (1997).
DNA-sequence evidence for the segmental allotetraploid origin of maize.
Proc. Natl. Acad. Sci. USA
94,6809
-6814.
Irish, V. F. and Sussex, I. M. (1992). A fate map of the Arabidopsis embryonic shoot apical meristem. Development 120,405 -413.
Jackson, D. (1991). In situ hybridization in plants. In: Molecular Plant Pathology: A Practical Approach (ed. D. J. Bowles, S. J. Gurr, M. McPerson), pp.163 -174. Oxford: Oxford University Press.
Jackson, D., Veit, B. and Hake, S. (1994).
Expression of the maize KNOTTED-1 related homeobox genes in the shoot
apical meristem predicts patterns of morphogenesis in the vegetative shoot.
Development 120,405
-413.
James, M. G., Robertson D. S. and Myers A. M.
(1995). Characterization of the maize gene sugary1, a
determinant of starch composition in kernels. Plant
Cell 7,417
-429.
Kaplan, D. R. (1973). The monocotyledons: their evolution and comparative biology. VII. The problem of leaf morphology and evolution in the monocotyledons. Q. Rev. Biol. 48,437 -457.
Kiesselbach, T. A. (1949). The structure and reproduction of corn. Nebraska Agric. Exp. Stn. Res. Bull. 161.
Kunze, R. and Weil, C. (2002). The hat and CACTA Superfamilies of Plant Transposons. In Mobile DNA II, vol. 2 (ed. N. Craig), pp.565 -610. Washington, DC: ASM Press.
Livak, K. J. and Schmittgen, T. D. (2001).
Analysis of relative gene expression data using real time quantitative PCR and
the 2-CT method. Methods
25,402
-408.[CrossRef][Medline]
Matsumoto, N. and Okada, K. (2001). A homeobox
gene, PRESSED FLOWER, regulates lateral axis-dependent development of
Arabidopsis flowers. Genes Dev.
15,3355
-3364.
Mayer, K. F. X., Schoof, H., Haecker, A., Lenhard, M., Jürgens, G. and Laux, T. (1998). Role of WUSCHEL in regulating stem cell fate in the Arabidopsis shoot meristem. Cell 95,805 -815.[Medline]
Medford, J. I., Behringer, F. J., Callos, J. D. and Feldman, K.
A. (1992). Normal and abnormal development in the
Arabidopsis vegetative shoot apex. Plant Cell
4, 631-643.
Poethig, R. S. (1984). Cellular parameters of leaf morphogenesis in maize and tobacco. In Contemporary Problems of Plant Anatomy (ed. R. A. White and W. C. Dickinson), pp.235 -238. New York: Academic Press.
Poethig, R. S. and Szymkowiak, E. J. (1995). Clonal analysis of leaf development in maize. Maydica 40, 67-76.
Randolph, L. (1936). Developmental morphology of the caryopsis in maize. J. Agric. Res. 53,881 -916.
Sambrook, J., Fritsch, E. F. and Maniatis, T. (1989). Molecular Cloning. A Laboratory Manual, 2nd edn. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press.
Scanlon, M. J. (2000). NARROW SHEATH1 functions
from two meristematic foci during founder-cell recruitment in maize leaf
development. Development
127,4573
-4585.
Scanlon, M. J. (2003). Polar auxin transport
inhibitors disrupt leaf initiation, KNOX protein regulation, and formation of
leaf margins in maize. Plant Physiol.
133,597
-605.
Scanlon, M. J. and Freeling, M. (1997). Clonal sectors reveal that a specific meristematic domain is not utilized in the maize mutant narrow sheath. Dev. Biol. 182, 52-66.[CrossRef][Medline]
Scanlon, M. J. and Freeling, M. (1998). The narrow sheath leaf domain deletion: a genetic tool used to reveal developmental homologies among modified maize organs. Plant J. 13,547 -561.
Scanlon, M. J., Schneeberger, R. G. and Freeling, M.
(1996). The maize mutant narrow sheath fails to
establish leaf margin identity in a meristematic domain.
Development 122,1683
-1691.
Scanlon, M. J., Chen, K. D. and McKnight, C. M.
(2000). The narrow sheath duplicate genes: sectors of
dual aneuploidy reveal ancestrally conserved gene functions during maize leaf
development. Genetics
155,1379
-1389.
Smyth, D. R., Bowman, J. L. and Meyerowitz, E. M.
(1990). Early flower development in Arabidopsis. Plant
Cell 2,755
-767.
Sossountzov, L., Ruiz-Avila, L., Vignols, F., Jolliot, A.,
Arondel, V., Tchang, F., Grosbois, M., Guerbette, F., Miginiac, E., Delseny,
M. et al. (1991). Spatial and temporal expression of a maize
lipid transfer protein gene. Plant Cell
3, 923-933.
Troll, W. (1955). Concerning the morphological significance of the so-called vorlaeuferspitze of monocot leaves. A contribution to the typology of monocot leaves. Beitr. Biol. Pflanz 31,525 -558.
Weatherwax, P. (1920). The homologies of the position of the coleoptile and the scutellum in maize. Bot. Gaz. 69,73 -90.
Related articles in Development: