1 Institut für Entwicklungsbiologie, Universität zu Köln,
Gyrhofstrasse 17, D-50923 Köln, Germany
2 Plant Gene Expression Center, Agricultural Research Service - USDA, 800
Buchanan St, Albany, CA 94710, USA
3 Cold Spring Harbor Laboratory, Cold Spring Harbor, NY 11724, USA
Author for correspondence (e-mail:
werr{at}uni-koeln.de)
Accepted 17 December 2004
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SUMMARY |
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Key words: Meristem, Stem cells, Inflorescence, thick tassel dwarf1, Maize
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Introduction |
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Analysis of mutations has identified a few of the genes that control
meristem function. One class of mutations is perturbed in meristem formation
and/or maintenance. For example, in strong alleles of
shootmeristemless (stm), seedling development stops after
formation of the two cotyledons (Barton and
Poethig, 1993), whereas analysis of weak stm alleles
indicates a role for STM throughout all stages of shoot meristem
development (Clark et al.,
1996
; Endrizzi et al.,
1996
). The maize homolog knotted1 (kn1) is also
required for shoot meristem establishment and maintenance
(Kerstetter et al., 1997
;
Vollbrecht et al., 2000
). The
kn1 and STM homeobox genes are expressed throughout the SAM,
except in the cells that are destined to become the next leaf primordium
(Smith et al., 1992
;
Jackson et al., 1994
;
Long et al., 1996
). Plants
carrying loss-of-function mutations at the WUSCHEL (WUS)
locus also fail to maintain the SAM (Laux
et al., 1996
). WUS encodes a novel subtype of homeodomain
protein that is nuclear localized and predicted to act as a transcription
factor (Mayer et al., 1998
).
WUS mRNA is first detected when embryos reach the 16-cell stage and
becomes localized to a group of cells that underlies the presumptive stem
cells, suggesting that it may act in promoting stem cell fate
non-autonomously.
A second class of mutations displays meristem enlargement, which can lead
to fasciation in extreme cases. Fasciation, derived from the Latin word
fascis, meaning `bundle', is a reflection of increased proliferation.
Fasciated variants are reported to have increased crop yields, for instance by
increasing fruit size in tomato (Luckwill,
1943; Zielinski,
1945
). Fasciation also has a long history of study in maize
(Emerson, 1912
): Fascicled
ear1 (Fas1), fasciated ear1 (fea1),
fasciated ear2 (fea2) and thick tassel dwarf1
(td1) are characterized by a fasciated ear morphology
(Orr et al., 1997
;
Jackson and Hake, 1999
).
In Arabidopsis, mutations in the three CLAVATA loci
(CLV1, CLV2 and CLV3) result in a phenotype opposite to that
of wus and stm (Clark et
al., 1993; Clark et al.,
1995
; Kayes and Clark,
1998
). The embryonic SAM of clv-mutants is slightly
larger than that of wild-type embryos. During postembryonic growth, the SAM
gradually increases in size, resulting in altered phyllotaxy and supernumerary
floral organs. These mutant phenotypes indicate a role for the CLV
genes in restricting the size of the stem cell population. All three
CLV genes have been identified and their protein products probably
constitute a single receptor-ligand complex, consistent with the three mutants
having an almost identical phenotype
(Clark et al., 1997
;
Fletcher et al., 1999
;
Jeong et al., 1999
). The CLV1
protein is a receptor-like kinase composed of a leucine-rich repeat-containing
extracellular domain with putative receptor function and a cytoplasmic Ser/Thr
kinase domain linked through a transmembrane domain
(Clark et al., 1997
). CLV2 is
structurally similar to CLV1 but lacks a cytoplasmic kinase domain
(Jeong et al., 1999
).
CLV3 encodes a 96 amino acid polypeptide that contains a putative
signal sequence and is secreted (Rojo et
al., 2002
). CLV3 transcripts are preferentially found in
the outer L1 and L2 layers, whereas CLV1 is expressed specifically in
the L2 and L3 layers of the SAM. CLV2 is expressed ubiquitously
(Jeong et al., 1999
). The
WUS and CLV pathways are interdependent
(Brand et al., 2000
;
Schoof et al., 2000
);
WUS promotes stem cell fate, whereas CLV signaling restricts the
number of stem cells (reviewed by Sharma
et al., 2003a
).
The maize ear comprises a series of meristem types (reviewed by
McSteen et al., 2000) and
changes in meristem size and identity may underlie the yield increases brought
about during the domestication of maize
(Kellogg and Birchler, 1993
)
from teosinte inflorescences (Beadle,
1980
; Doebley,
1992
). Whereas seeds are arranged in single alternating rows in
teosinte, present-day maize lines have a polystichous arrangement of seeds
consisting of 8 to 18 rows, and in some varieties up to 36 rows (USDA, ARS,
National Genetic Resources Program, Germplasm Resources Information Network;
http://www.ars-grin.gov/cgi-bin/npgs/).
The increased number of axillary meristems in maize is thus a major acquired
character, which has been instrumental in the domestication of maize as a crop
plant. It would be of interest to understand how ear size relates to the stem
cell concept and reflects modulations in the size of the stem cell population,
since the increase in seed row number of the maize ear is reminiscent of
supernumerary lateral organs that arise due to increased meristem size in
clv mutants.
The maize fea2 locus encodes a potential ortholog of
CLV2, suggesting that the CLAVATA pathway is functionally
conserved in monocot species and acts to limit size or growth of meristems
during maize development (Taguchi-Shiobara
et al., 2001). Further evidence comes from the recent
characterization of the FON1 gene in rice, which encodes a potential
CLV1 ortholog (Suzaki et al.,
2004
). As in clv1 mutants, fon1 mutant plants
produce enlarged floral meristems and additional floral organs. By contrast to
clv1 mutants, however, fon1 mutants do not have
significantly enlarged vegetative shoot or inflorescence meristems.
Considering that maize was domesticated at least 6000 years ago
(Piperno and Flannery, 2001),
the unique monstrosity of the maize ear in the plant kingdom could reflect
permanent selection for size increase of the ear inflorescence meristem, which
is still a major quantitative trait of interest in modern maize breeding. We
are now in a position to ask whether modifications of specific signaling
pathways, such as the CLV pathway, may have contributed to the domestication
of crop plants. Here we describe a reverse genetics approach to functionally
characterize CLV1 homologs from maize. The maize gene most similar to
CLV1 encodes thick tassel dwarf1 (td1). The
fasciation phenotype of td1 mutant alleles provides further evidence
that meristem size in maize is controlled by the CLV signaling pathway.
However, significant qualitative and quantitative differences exist between
the td1 and CLV1 expression patterns. These differences
emphasize that it is important to study developmental processes in agronomic
crops such as maize, as well as in model species such as
Arabidopsis.
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Materials and methods |
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td1 expression analysis
Maize tissues were ground to a fine powder in liquid nitrogen and RNA was
purified using the Trizol procedure (Invitrogen). PolyA(+) RNA was
isolated using oligo-dT cellulose columns or a Dynabeads mRNA purification kit
(DYNAL). RNA electrophoresis, transfer to a Hybond-N membrane (Amersham
Pharmacia Biotech) or Magna, nylon membrane (Osmonics) and hybridizations
followed the protocol of Smith et al.
(Smith et al., 1992).
Hybridization with the 32P-labeled 620 bp probe (450 bp of kinase
coding sequence + 170 bp 3' UTR) was performed at 42°C overnight.
Blots were washed in 0.1 x SSC, 0.1% SDS at 65°C and
autoradiographed on Kodak X-omat AR films or by visualization on a
PhosphorImager (Molecular Dynamics).
For real-time PCR analysis, tissue-specific cDNAs were synthesized using the SuperScriptIIITM reverse transcriptase according to the manufacturer's instructions (Invitrogen). Real-time RT-PCR analysis was performed on a GeneAmp® 5700 Light Cycler. The online detection of amplification rates was performed using SYBR® Green (Applied Biosystems). Normalization was performed against Ubiquitin with primers: UBI-Fo (5'-TAAGCTGCCGATGTGCCTGCG-3') and UBI-Re (5'-CTGAAAGACAGAACATAATGAGCACAG-3'). Knotted1-specific amplicons were generated with primers: kn1-Fo (5'-ACCGAGCTCCCTGAAGTTGATG-3') and kn1-Re (5'-CTAGGCCGTGGGGTGTGAAATGC-3'). The td1-specific primers were: td1-Ref-Fo (5'-GCTGCTGGCGGACCTCTACA-3') and td1-Ref-Re (5'-GACGAGTGACGCCAAAAAG-3').
Female and male inflorescences for in-situ hybridizations were dissected
and fixed at 4°C overnight in 4% formaldehyde with 0.1% TritonX100 (Sigma)
in PBS, dehydrated in an ethanol series, cleared in Histoclear (Roth) and
embedded in paraffin wax (Paraplast plus, SIGMA). Sections 7-9 µm thick
were mounted on coated slides (SuperFrost®Plus). The td1 probe
(620 bp, see above) was cloned into pBluescript II SK (Stratagene). The
td1 and the kn1 probes
(Jackson et al., 1994) were
labeled by incorporation of digoxigenin-UTP (Roche) during SP6/T3
transcription. RNA in-situ hybridization was performed according to the method
of Jackson et al. (Jackson et al.,
1994
).
Scanning electron microscopy
Scanning electron microscopy was performed according to the protocol
outlined in Sommer et al. (Sommer et al.,
1990) and images were processed digitally.
PCR on td1 alleles and double mutant families
Genomic DNAs of homozygous td1-Ref and td1-Nickerson
plants were used as templates in PCR reactions with primers
5'-CATGCCACCTGCCTTTGAC-3' and
5'-GACGAGTGACGCCAAAAAG-3' and Platinum Pfx proofreading polymerase
(Invitrogen) or Fisher Taq polymerase. DNA sequence analysis followed cloning
into the pCR2.1-TOPO vector (Invitrogen) or pGemT-EZ vector (Promega).
The Mu-alleles were amplified using platinum Pfx proofreading
polymerase (Invitrogen) using forward or reverse td1-primers 500
bp apart and covering the entire coding region, and Mu-specific
primers (5'-GCCTCCATTTCGTCGAATC-3' or Pioneer's 9242
Mu-specific primer
5'-AGAGAAGCCAACGCCAWCGCCTCYATTTCGTC-3')
(Blauth et al., 2001
) located
in the terminal inverted repeat sequences of the transposable element. The
resulting amplicons were cloned and sequenced to determine the
Mu-insertion site.
Genotypes of individuals in the segregating F2 families used for double mutant analysis were determined by using primers specific to fea2 (fea2-Fo: 5'-GCTGCTGGCGGACCTCTACA-3'; fea2-Re: 5'-GACGAGTGACGCCAAAAAG-3') or td1 (td1-Fo: 5'-TCACCGACAACATGCTCACT-3'; td1-Re: 5'-CAAACACGTGGTTATGCTGC-3') and the 9242 primer. Wild-type chromosomes were detected by amplifying with gene-specific primers flanking the known Mu insertion sites in each allele.
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Results |
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The mature tassel of maize consists of a central rachis and a few long
branches at the base, with each axis lined with rows of spikelets. In the
tassel of td1 mutant plants, the main rachis was thicker than normal,
and this was more obvious when spikelets were removed
(Fig. 1A,B). This increase in
rachis diameter was reflected in a higher spikelet density. Whereas normal
sibling tassels on average produced around 14 spikelets/cm on the main rachis,
td1 mutant tassels could produce more than 20 spikelets/cm
(Table 2). This increase in
spikelet density was proportionally similar in tassel branches
(Table 2). However, the total
number of spikelets per tassel was not always increased, as thickening of the
main axis in td1 tassels was sometimes correlated with a reduction in
length (Table 2). The spikelets
are arranged in pairs, with a pedicellate and sessile spikelet
(Fig. 1E). Male spikelets
contain two glumes that surround two florets. Each floret contains three
stamens, two lodicules and an aborted pistil
(Cheng et al., 1983). A closer
inspection of male spikelets in td1 mutants showed a high frequency
of single spikelets instead of paired spikelets
(Fig. 1C; Table 2). The spikelets
themselves were occasionally irregular, with extra glumes
(Fig. 1C), and florets often
contained more than three stamens (Fig.
1D; Table 2). These
supernumerary floral organs may have resulted from an enlarged floral
meristem.
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In summary, loss of td1 activity in the tassel caused thickening of the rachis, a higher spikelet density and extra glumes and stamens. The phenotype was more pronounced in the ear, where fasciation and bifurcation of the IM was accompanied by supernumerary and larger SPMs that branched to produce clusters of SMs of abnormal size. Additionally, a weak vegetative phenotype could be observed in td1 mutant plants. These phenotypes are all consistent with the observation that td1 functions to limit meristem size.
Isolation of CLAVATA1 homologous genes from maize
Like most eukaryotic protein kinases, leucine-rich repeat receptor-like
kinases (LRR-RLKs) share 12 highly conserved subdomains within their
intracellular catalytic kinase domains. These subdomains have been designated
I-XII (Hanks and Quinn, 1991)
and invariant amino acid residues in individual subdomains provide excellent
anchor points to design degenerated PCR primers. Based on invariant amino
acids in subdomains I and VII and guided by flanking sequences in
CLV1 (GI: 15222877), and its closest relatives in the
Arabidopsis or rice genomes (GI: 15239123 and GI: 18855025)
respectively, PCR primers were designed (for sequences see Materials and
methods). The relative position of these primers in the kinase domain of
CLV1 are indicated in Fig.
3A.
|
Three other maize kinases, ZmKin1-3, fall into subfamily B, which also splits into monocot and dicot clades. This splitting validates the phylogenetic reconstruction and suggests that a common ancestral gene may have duplicated to give rise to two separate lineages of LRR-RLK genes prior to the divergence of monocots and dicots. We used the more distantly related ZmKin4 to root the tree. This phylogeny identified ZmKin5 as the most likely maize ortholog of CLV1 from Arabidopsis.
ZmKin5 gene structure
To isolate the full-length ZmKin5 coding sequence, we screened a
maize embryo cDNA library with PCR primers from the kinase domain and the
vector. Although successful for the 3' end of the gene, this strategy
failed for the 5' end. We therefore isolated a ZmKin5 genomic
clone by screening with a ZmKin5-specific probe comprised of the
carboxy terminus of the kinase domain and 3' UTR sequences. Analysis of
the genomic clone indicated that the predicted ZmKin5 open reading
frame is 2991 bp in length. The predicted size of 3262 bp from the ATG
translation start codon to the polyA tail was consistent with a single
3.4 kb transcript detected in RNA gel blot experiments
(Fig. 5A). A single intron in
the kinase domain was conserved in position in ZmKin5 and
CLV1. The amino terminal leucine-rich repeat coding sequences and
transmembrane domains were devoid of intervening sequences in maize, as in
Arabidopsis. The predicted protein sequences encoded by
ZmKIN5 and CLV1 are compared in
Fig. 4A.
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ZmKin5 is encoded by the td1 locus
We mapped ZmKin5 using the BNL T232/CM37 recombinant inbred lines
(Burr et al., 1988) to
chromosome 5 (bin 5.03) between markers uaz77 and ici287, close to the
td1 locus. To test the relationship between ZmKin5 and
td1, we isolated and sequenced ZmKin5 genomic DNA from
homozygous td1-Ref plants. The td1-Ref allele harbors a
cluster of four base pair substitutions surrounding a 15 bp deletion in the
predicted ER targeting signal sequence
(Fig. 4A). This mutation is
predicted to abolish ER targeting of the protein by the target algorithm.
Altered localization of the ZmKIN5 receptor kinase might explain the
td1-Ref mutant phenotype. The same sequence differences within the
putative ER targeting signal were found in the td1-Nickerson allele,
although both td1 alleles have been maintained as distinct mutations
in the Maize Stock Center, and are believed to have arisen independently (M.
Sachs, personal communication). Our data indicate, however, that both
mutations may share a common origin.
To determine whether or not ZmKin5 corresponded to the
td1 locus, we generated additional td1 mutant alleles.
td1-Ref and td1-glf were crossed as males onto
Mutator (Robertson,
1978) plants and the resulting progeny were screened for the
td1 mutant phenotype. Twelve putative mutant td1 alleles
were found from screens of approximately 150,000 plants. DNA from putative new
alleles was analyzed by PCR for the presence of Mu elements in the
ZmKin5 sequence (see Materials and methods). Among 12 alleles
examined, five Mu insertion alleles were identified, referred to as
td1-mum1 to -mum5 (Table
1; Fig. 4B).
Sequencing of the PCR amplicons revealed Mu insertions in the first
exon, disrupting the extracellular LRR domain for td1-mum1, td1-mum2
and td1-mum3. The Mu element in td1-mum4 was
located within the intracellular kinase domain, and in td1-mum5 was
29 bp downstream of the splice donor site in the single intron. We also found
that the td1-glf allele had a Mu insertion in the first
exon. This allele also carried the same polymorphisms as the td1-Ref
allele within the putative ER targeting signal sequence. Southern
hybridizations confirmed co-segregation of a ZmKin5-specific
Mu insertion with the td1 mutant phenotype in over 40
chromosomes (data not shown). In conclusion, six independently isolated
td1 alleles were shown to contain Mutator element insertions
in the ZmKin5 coding region, demonstrating that ZmKin5
corresponds to the td1 gene.
We also compared td1 transcript levels in immature ears of normal siblings and td1-glf homozygous mutant plants by RNA gel blot experiments. As shown in Fig. 5A, a larger, aberrant transcript was detected using a td1-specific probe in RNA from the td1-glf mutant. Additionally, real-time RT-PCR showed that the level of td1 RNA in td1-glf was reduced to 20% that of wild type, whereas the level of the kn1 meristem marker was slightly increased (Fig. 5B). This increase in kn1 RNA may reflect an increase in IM size or the presence of supernumerary SPMs. Sequence analysis of an RT-PCR product from td1-glf RNA revealed the presence of 180 nucleotides of the Mu terminal inverted repeat, which would lead to a frame shift and premature translation stop in the extracellular LRR domain. These results demonstrate that the Mu element insertion in td1-glf is spliced out incompletely, and that td1-glf may represent a null allele. In summary, by identifying five independent td1 Mu-alleles, we have shown that td1 is ZmKin5, a maize ortholog of CLV1.
Expression pattern of the td1 gene
Organ specificity of td1 transcription was determined via RNA gel
blot hybridizations (data not shown) and quantified by real-time RT-PCR
analysis (Fig. 6A). Normalized
to ubiquitin transcript levels, the highest td1 transcript
level was detected in the apex of the vegetative seedling, and was arbitrarily
set at 100%. The RNA level was lower in the ear (58%) and tassel (data not
shown). Low td1 transcript levels were detected in the embryo (11%)
and root (11%). In contrast to CLV1
(Clark et al., 1997), a
significant td1 transcript level was detected in young leaves (63%).
Primer specificity for td1 was verified by direct sequencing of the
PCR amplicons.
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td1/fea2 double mutant analysis
CLV1 and 2 are reported to act in a common pathway in Arabidopsis
(Kayes and Clark, 1998), and
this is supported by biochemical analysis
(Jeong et al., 1999
). To ask
whether td1 and fea2, the maize orthologs of CLV1
and 2, respectively, act in a common pathway, we analyzed
td1/fea2 double mutants. At early stages of ear development
td1/fea2 double mutants (Fig.
7A) were strikingly similar to strong td1 mutants
(Fig. 7B), in showing a severe
ring fasciation, whereas fea2 mutant siblings were less strongly
fasciated (Fig. 7C). At
maturity, td1/fea2 double mutants showed additive or synergistic
phenotypes, as they initiated twice as many rows of spikelets as either
td1 or fea2 single mutants
(Table 3), and their ears were
reduced in length (Fig. 7D).
The decrease in leaf number was also more pronounced in td1/fea2
double mutants compared with either single mutant
(Table 3), indicating that,
like td1, fea2 also functions during vegetative development.
Following the assumption that ear spikelet number correlates with inflorescence meristem size, our data indicate that td1 and fea2 do not act exclusively in a single pathway to control meristem size.
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Discussion |
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Phenotypic consequences of td1 mutants
The most conspicuous aspect of td1 mutants was the dramatic
fasciation of the ear tip, due to an enlarged inflorescence meristem. Ear tips
showed line or ring fasciation and were often bifurcated. The broadening of
the tip led to extra rows of SPMs and extra SMs, which sometimes produced
abnormal single spikelets. By contrast to the dramatic defect in the ear,
tassels of td1 mutants appeared less affected. However, a closer
analysis showed that spikelet density was increased by about 40% in the main
rachis of the tassel, but it did not fasciate. Interestingly, kn1
null mutants reduced meristem size, and while kn1 mutant ears and
tassels shared a set of meristem defects, only in the ear did the
inflorescence meristem sometimes abort, leading to plants without ears
(Kerstetter et al., 1997;
Vollbrecht et al., 2000
).
Thus, the ear appeared to be more sensitive than the tassel to mutations that
alter inflorescence meristem size.
Given the similarity of ear and tassel primordia at early stages of
development (Cheng et al.,
1983), it is interesting to speculate on the reasons for the
differences between the mature tassel and ear of td1 mutants. One
possibility is that the ear is more sensitive to changes in CLV signaling, due
to the selective pressure placed on the ear during agricultural selection.
Maize was domesticated from teosinte, in which the inflorescence rachis
contains only a single alternating row of spikelets. By contrast, a typical
maize ear contained 16-18 spikelet rows
(Doebley and Stec, 1991
).
Evidence for a general relaxed control of meristem size in the ear also comes
from the observation that it is not uncommon to find spontaneously fasciated
ears, whereas such abnormalities are rare for the tassel
(Neuffer et al., 1997
).
Interestingly, td1 maps near the location of a quantitative trait
locus (QTL) for seed row number between the markers BNL6.25 and BNL5.02
(Doebley et al., 1990
) and a
QTL for tassel spikelet density on tassel branches (T. Rocheford, personal
communication). The rice FON1 gene also encodes a CLV1
ortholog (Suzaki et al.,
2004
), and additional branches are made in rice fon1
inflorescences to a similar magnitude as the formation of extra spikelets in
male td1 inflorescences, suggesting that TD1 and
FON1 bear equivalent functions. This finding again strengthens the
view that the maize ear reflects a unique structure among grass species, with
a distinct genetic program from the tassel, possibly as a result of the
intense selective pressure during domestication.
Other meristems were also affected in td1 mutant inflorescences.
SPMs were frequently enlarged and irregularly arranged, leading to extra
spikelets that were often single instead of in pairs. Although we did not
observe more than two florets per spikelet, we did observe extra glumes. As
glumes are products of the SM (McSteen and
Hake, 2001), the SMs were therefore also affected in td1
mutants. td1 mutant male flowers also showed an increase in the
number of stamens, which may be a consequence of an increase in floral
meristem size. Finally, td1 mutant plants were shorter and developed
fewer leaves. Given that td1 maps near a QTL for plant height
(Beavis et al., 1991
), it could
also be responsible for natural variation in height.
In summary, td1 functions to limit meristem size during inflorescence and flower development.
td1 encodes a CLV1-like protein
We have shown that td1 encodes a putative ortholog of CLV1.
clv1 mutants affect all shoot meristem types, including those of the
embryo and vegetative phase. With the exception of the ear inflorescence
meristem, the effects of the td1 mutation were relatively mild, and
this could be a result of genetic redundancy. Although td1 appears to
be a single-copy gene by low stringency Southern hybridizations, sequence
database searches indicate a plethora of LRR receptor-like genes in maize, and
these could account for the genetic redundancy. From the Arabidopsis
genome, 216 LRR-RLKs have been identified
(Shiu and Bleeker, 2001), and
recent studies of clv1 dominant negative alleles strongly suggest the
presence of additional receptor kinases that function redundantly with CLV1 in
the Arabidopsis SAM
(Diévart et al., 2003
).
Our phylogenetic analysis indicates an ancestral gene duplication prior to the
divergence of monocot and dicot species, giving rise to two discrete branches.
CLV1, FON1 and TD1 fall into subfamily A, whereas the three
maize kinases encoded by ZmKin1 to 3 fall into subfamily B,
along with OsLRK1 from rice and the two Arabidopsis
LRR-RLKs, A.t. RLK1 and A.t. RLK2. Interestingly,
OsLRK1 antisense plants have an increased number of floral organs,
implicating a function in control of floral meristem size
(Kim et al., 2000
). The
presence of A.t. RLK1 and 2 in the same subfamily (B)
indicates that redundancy presumably is ancient and these two genes still may
act redundantly to CLV1 in Arabidopsis.
TD1 and FEA2 do not interact in an exclusive receptor complex
The td1 mutant phenotype was similar to that of fea2,
which also has ear fasciation, an increased number of stamens, and a thicker
tassel. FEA2 is predicted to be a CLV2 ortholog
(Taguchi-Shiobara et al.,
2001). In Arabidopsis, the CLV1 and
CLV2 gene products interact to form a heterodimeric LRR-transmembrane
receptor kinase complex [reviewed by Sharma et al.
(Sharma et al., 2003a
)], and
this is supported by genetic analysis that shows that strong clv1 and
clv3 alleles are epistatic to clv2 in regulating meristem
size (Kayes and Clark, 1998
).
Given the similar phenotypes of fea2 and td1 mutants, it is
intriguing to speculate whether they encode two subunits of a heterodimeric
CLV-like LRR-receptor kinase complex in maize.
Our analysis of td1/fea2 double mutants, however, indicates that
the situation in maize is more complex. The formation of twice as many kernel
rows, as well as the enhanced reduction of leaf number, in td1/fea2
double mutants compared with either single mutant strongly suggests that
td1 and fea2 do not simply function in a single pathway, for
example as an exclusive receptor-co-receptor complex. One could speculate that
td1 and fea2 are incorporated into different receptor
complexes, thereby acting in independent pathways in regulating shoot and
inflorescence meristem size. Alternatively, they may function in partially
overlapping pathways, for example forming a co-receptor complex as well as
dimerizing with other LRR partners, as has been also recently proposed for
CLV1 function in Arabidopsis
(Diévart et al., 2003).
Our data suggest that several CLV1-CLV2-like complexes with partially
redundant functions contribute to the regulation of inflorescence meristem
size, and that the degree of redundancy may differ in the ear and tassel. As
there are multiple CLV3-like genes in maize
(Cock and McCormick, 2001
;
Sharma et al., 2003b
), further
specificity may be dictated by specific ligand-receptor interactions.
The CLV1 and TD1 protein structures are highly conserved. Like CLV1
(Clark et al., 1997), the
extracellular receptor domain of TD1 is composed of 21 complete LRR motifs,
each of 23-25 amino acids in length. These LRR motifs are commonly used for
protein-protein interactions (Kobe and
Deisenhofer, 1994
; Kobe and
Deisenhofer, 1995
) and all motifs in TD1, as in CLV1, belong to
the LRR-XI subfamily (Shiu and Bleeker,
2001
). Sequence analyses of strong clv1 alleles in
Arabidopsis reveal lesions within LRRs four, five or nine, implying
these regions may be important for ligand specifity
(Diévart et al., 2003
).
Interestingly, sequence similarity between TD1 and CLV1 is more pronounced
within LRR-5 and LRR-9, supporting the hypothesis that these LRRs play a
conserved role in the receptor-ligand interaction. This situation is
reminiscent of the FEA2 and CLV2 proteins, which also show blocks of
significant similarity in specific LRRs
(Taguchi-Shiobara et al.,
2001
).
Another common feature of LRR-RLKs are paired cysteines flanking the
extracellular LRR domain. These cysteines are implicated in receptor
dimerization (Trotochaud et al.,
1999). TD1, FON1 and CLV1 share both cysteine pairs, although the
spacing of the carboxy terminal cysteines is only two amino acid residues in
TD1 relative to six in CLV1 and seven in FON1. Interestingly, in FEA2 the
carboxy terminal cysteine pairs are also closer together, spaced by only four
amino acids, compared with seven in CLV2
(Taguchi-Shiobara et al.,
2001
; Jeong et al.,
1999
). Therefore, TD1 and FEA2 contain putative compensatory
alterations that might stabilize heterodimers by intermolecular disulfide
bridges, via the carboxy terminal pair of cysteines.
Expression analysis shows a potentially wider role for td1 compared with CLV1
Significant differences in expression pattern can be found between
TD1 in maize and CLV1 in Arabidopsis. td1
expression was not restricted to the internal L2/L3 meristem layers, as
reported for CLV1 (Clark et al.,
1997). Furthermore, similar to the FON1 expression in
rice, td1 was expressed in inflorescence organ primordia, such as
glumes, lemma and stamens, whereas CLV1 is expressed only in
meristems. More significantly, we did not observe td1 expression in
the shoot apical meristem of embryos or seedlings using in-situ
hybridizations. One explanation for the lack of detection at the cellular
level, however, is a ubiquitous but low level of expression in embryonic or
seedling shoot meristems.
An interpretation of the low level of td1 expression in the ear inflorescence meristem again takes the domestication of maize into consideration. The change in phyllotaxy and increase in spikelet row number during selection for maize ear size may have been accompanied by an increase in inflorescence meristem size, by modification of genetic pathways, such as the CLV pathway. In other words, selection for low td1 expression in the ear inflorescence meristem may be responsible for the large size of the ear inflorescence meristem.
An alternative hypothesis is that SPMs, which express high levels of
td1 mRNA, regulate the size of the inflorescence meristem by a
long-distance signaling mechanism. An interaction between inflorescence and
axillary meristem is also seen with barren inflorescence2
(bif2) mutants, which do not develop axillary meristems and exhibit
fasciated inflorescence meristems (McSteen
and Hake, 2001). bif2 is also expressed in axillary
meristems but not in the inflorescence meristem (McSteen and Hake,
unpublished). The same scenario can also be postulated for the vegetative
shoot meristem, as td1 is expressed in leaves.
Conclusion
In summary, the td1 fasciated and enlarged meristem phenotypes are
explained by a deficiency in CLV-type signaling. The finding that td1
and FON1 encode orthologs of CLV1 supports the notion that
the regulation of stem cell proliferation by CLV-type signaling is of general
importance in plants. However, our data point out important differences that
justify the study of such pathways in a variety of model systems. For example,
td1 expression was not detected, or was very weak, in vegetative and
inflorescence apical meristems, and analysis of td1/fea2 mutant
plants indicated that td1 and fea2 do not exclusively act in
a simple co-receptor complex. Moreover, the proximity between td1 and
QTLs affecting seed row number, spikelet density and plant height suggests
that CLV signaling may have been a target of selection during domestication of
maize as a crop. Our results suggest that genes such as td1 could be
manipulated to improve crop yields.
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ACKNOWLEDGMENTS |
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Footnotes |
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Present address: Department of Genetics, Development and Cell Biology, Iowa
State University, Ames, IA 50011, USA
Present address: Donald Danforth Plant Science Center, 975 Warson Rd, St
Louis, MO 63146, USA
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