1 Plant Gene Expression Center, USDA/UC Berkeley, 800 Buchanan Street, Albany,
CA 94710 USA
2 Plant and Microbial Biology Department, UC Berkeley, 111 Koshland Hall,
Berkeley, CA 94720 USA
Author for correspondence (e-mail:
fletcher{at}nature.berkeley.edu)
Accepted 14 December 2004
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SUMMARY |
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Key words: Arabidopsis thaliana, ULTRAPETALA, Shoot apical meristem, SAND domain, B box domain
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Introduction |
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The SAM persists as a cell dome with both a longitudinal and a radial
structure (Steeves and Sussex,
1989). In most dicots, the SAM is divided into three clonally
distinct layers. Cells in the outermost layer (L1) produce epidermal tissues,
whereas cells of the sub-epidermal layer (L2) and the internal layers (L3)
differentiate into vascular and internal tissues. Superimposed across these
cell layers are distinct zones of differential meristem activity. A central
zone (CZ) at the very apex harbors the unspecialized stem cells, which
maintain themselves and also replenish cells in the adjacent peripheral zone
(PZ) as they are lost during the formation of lateral organ primordia on the
meristem flanks. Maintenance of SAM integrity requires a precise coordination
between the flow of cells leaving the PZ and their replacement by cells from
the CZ, which implies that the different regions of the meristem are in
communication with one another. In Arabidopsis, one key component of
the meristem communication system is the CLAVATA (CLV) extracellular signaling
pathway.
In clavata mutants (clv1, clv2, clv3), all aerial
meristems produce a greatly increased cell population, resulting in the
formation of fasciated stems, supernumerary flowers and flowers with extra
organs (Clark et al., 1993;
Clark et al., 1995
;
Kayes and Clark, 1998
). At the
other extreme, wuschel (wus) mutants undergo premature
termination of their shoot and floral meristems
(Laux et al., 1996
).
WUS, which encodes a homeodomain transcription factor
(Mayer et al., 1998
), is
expressed in a small region in the meristem interior referred to as the
organizing center (OC), from where it specifies stem cell identity on the
overlying L1 and L2 cells (Schoof et al.,
2000
). The stem cell-promoting activity of WUS is counterbalanced
by the CLV proteins, which are members of a signal transduction pathway that
limits the size of the WUS-expressing cell population
(Clark et al., 1997
;
Fletcher et al., 1999
;
Jeong et al., 1999
;
Trotochaud et al., 1999
). In
clv mutant meristems, the WUS expression domain expands
laterally and upwards, leading to the accumulation of excess stem cells
(Brand et al., 2000
;
Schoof et al., 2000
). Thus,
the activity of the CLV pathway establishes a negative feedback loop between
the stem cells and the underlying organizing center that maintains meristem
homeostasis throughout development (Brand
et al., 2000
; Gallois et al.,
2002
; Lenhard and Laux,
2003
; Schoof et al.,
2000
).
Maintenance of a functional SAM also requires additional factors that act
in pathways independent of the CLV pathway. For example, a number of
Arabidopsis mutants that are impaired in chromatin assembly or genome
maintenance display pleiotropic phenotypes, including severely disorganized
cell arrangements at both the shoot and root apices. Among these are
fasciata1 (fas1) and fas2
(Leyser and Furner, 1992),
mre11 (Bundock and Hooykaas,
2002
), the AtCAP-E1 and AtCAP-E2 condensin
mutants (Siddiqui et al.,
2003
), and tonsoku/mgoun3/bru1
(Guyomarc'h et al., 2004
;
Suzuki et al., 2004
;
Takeda et al., 2004
).
Likewise, mutations in the HALTED ROOT (HLR) gene, which
encodes a subunit of the 26S proteasome, result in the disorganization of the
SAM and the RAM that correlates with a disturbed shoot organizing center and
root quiescent center (Ueda et al.,
2004
). For most of these mutants, the observed stem fasciation
phenotypes are linked to the distortion of the WUS expression pattern
in the SAM in a broader and more random manner than occurs in the clv
mutants. Mutations in the farnesyltransferase gene ENHANCED RESPONSE TO
ABSCISIC ACID1 (Running et al.,
1998
; Ziegelhoffer et al.,
2000
) and the prenyltransferase gene PLURIPETALA
(Running et al., 2004
) also
increase SAM size in a CLV-independent manner.
We have previously identified ULTRAPETALA1 (ULT1) as an additional factor
that negatively regulates Arabidopsis shoot and floral meristem
activity, as ult1 mutations cause the enlargement of inflorescence
and floral meristems, leading to the production of supernumerary flowers and
floral organs (Fletcher,
2001). ult1 mutants also have reduced floral meristem
determinacy, and ULT1 has been shown to negatively regulate WUS
expression in order for floral meristem termination to occur at the correct
stage of flower development (Carles et al.,
2004
). Here, we report the cloning and characterization of the
ULT1 gene and its paralog ULT2. We show that the
organization of the SAM is not altered in ult1 mutants, but that ULT1
restricts the size of the WUS-expressing cell population.
ULT1 and ULT2 encode small proteins containing a SAND
DNA-binding motif and a B box-like domain, and their expression patterns fully
overlap in inflorescence meristems, floral meristems and reproductive organs.
Both genes are also expressed in embryos, but only ULT1 mRNA
accumulates in vegetative meristems and leaf primordia. We discuss the
functions of the ULT factors throughout plant development, and in light of
what is known about SAND domain-containing factors in animals, we propose that
the ULT proteins may act as direct regulators of developmental gene
expression.
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Materials and methods |
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Mapping and molecular identification of ULT1
Using cleaved amplified polymorphic sequence (CAPS) markers
(Konieczny and Ausubel, 1993)
distributed across the lower arm of chromosome 4, we established that
ult1-2 was flanked by markers PG11 (map position 75.16 cM) and g8300
(81.22 cM). For the fine mapping of ULT1, we designed 12 new CAPS
markers that spanned the region between markers PG11 and g8300, using the TIGR
Landsberg erecta random sequence database
(www.tigr.org)
as a source for single nucleotide polymorphisms (SNPs). The primer sequences,
restriction enzyme and number of restriction sites in the Col/Ler
ecotypes for the CAPS marker sequences generated across this interval are
available upon request.
Sequencing was performed on an ABI PRISM® 3100 Genetic Analyzer sequencer (Perkin Elmer), according to the manufacturer's instructions. Computer-based sequence analysis was performed using VectorNTI® Suite (Informax) and Sequencher (Gene Codes Corporation, Ann Harbor) software. Multiple protein alignments were obtained using ClustalX and edited with SeqVu (The Garvan Institute of Medical Research).
Construction of transgenic lines
ULT1-214 complementation construct
A 2745 bp fragment spanning the ULT1-coding region and flanking
sequence was digested from BAC F26K10 with NdeI, Klenow-filled and
cloned into blunt pBSK+ vector (pBSK+-ULT construct). Then a
BamHI/KpnI fragment was cut from pBSK+-ULT vector
and cloned into the binary vector pCD214 (kindly provided by Chris Day).
Transgenic plants were selected on MS plates containing gentamycin (100
µg/ml).
d35S::ULT1/d35S::ULT2 sense and d35S::ULT1 antisense constructs
The full-length ULT ORFs were cloned into the binary vector pCD223 (kindly
provided by Chris Day) at the EcoRI site, flanked 5' by a
double CaMV 35S promoter and 3' by a nopaline synthase transcription
termination signal. The clones were then screened by PCR to obtain the ULT
cDNA insert in the sense (S) or antisense (AS) orientation. Transgenic plants
were selected on MS plates containing gentamycin (100 µg/ml).
35S::ULT-(Ala)10-GFP and 35S:: ULT-(Ala)10-GUS-GFP constructs
The pEZS vectors carrying the CaMV 35S-MCS-(Ala)10-EGFP cassette
or the CaMV 35S-EGFP-(Ala)10-MCS cassette were kindly provided by
David Ehrhardt. The ULT cDNA fragments were cloned into the EcoRI and
BamHI sites of pEZS-NL/CL vectors to give pEZS-NL/CL-ULT constructs.
To create the CaMV 35S-MCS-(Ala)10-GUS-EGFP and CaMV
35S-GUS-EGFP-(Ala)10-MCS cassettes, we introduced a short synthetic
linker at the NcoI site of pEZS. The ß-Glucuronidase
uidA (GUS) gene was cloned at the newly created
NcoI and PmlI sites. Then the ULT cDNA fragments were cloned
into the EcoRI and BamHI sites of the pEZS MCS. For stable
transformation of Arabidopsis plants, the 35S::ULT-GFP and
35S::ULT-GUS-GFP cassettes were transferred into pART27 at the NotI
site (Gleave, 1992).
Transgenic plants were then selected on MS plates containing kanamycin (50
µg/ml).
Subcellular localization
For transient assays, the pEZS-ULT fusions were transformed into onion
epidermis cells by particle bombardment using a Biolistic PDS-1000/He unit
(BioRad, Richmond, CA), as described
(Sanford et al., 1993). For
GFP visualization, epidermal peels were examined 24-36 hours after bombardment
using a Zeiss Axiophot microscope. GFP fluorescence was visualized with the
FITC channel and images were acquired with a 12-bit MicroMax cooled CCD camera
operated by IPLab software (Scanalytics, Fairfax, VA). GFP and DAPI
fluorescence was visualized in plants using a Zeiss LSM510 confocal
laser-scanning microscope (CLSM), with the FITC channel and the UV channel,
respectively.
For immunodetection of GFP in the transgenic lines, 0.5 g of inflorescence tissues were ground in liquid nitrogen and then extracted with 500 µl of cold buffer [100 mM MOPS pH 7.6, 100 mM NaCl, 5% (v/v) Glycerol, 1 mM EDTA, 14 mM ß-mercaptoethanol, 1 mM PMSF, 2 µg/ml pepstatin A, 0.2 µg/ml leupeptin, 1 µg/ml aprotinin] containing protease inhibitors. Each protein extract (15 µg) was separated on a 12.5% SDS-PAGE gel and blotted on a nitrocellulose membrane. Immunoblots were incubated with a 1:500 dilution of an anti-GFP polyclonal antibody (Santa Cruz Biotech).
GUS staining
The GUS staining reaction and subsequent tissue embedding and sectioning
were performed as described (Sieburth and
Meyerowitz, 1997), with the exception that GUS localization was
visualized after 6 hours of staining with 2 mM of
5-bromo-4-chloro-3-indolyl-ß-D-glucoronide (X-GLUC, Bioworld).
RT-PCR
Total RNA was isolated from various tissues using the RNeasy plant kit
(Qiagen), treated with RQ1 RNase-free DNase (Promega) for 30 minutes at
37°C, and then purified with phenol/chloroform. The first-strand cDNA
synthesis was performed on 5 µg of total RNA using Superscript II RNase
H- reverse transcriptase (Gibco BRL, Life Technologies) and an
oligo dT primer (18 mer), according to the manufacturer's instructions. From
20 µl of the reverse-transcription (RT) product, 1 µl was used for each
PCR reaction. The annealing temperature was 54°C for all primer pairs and
34 cycles of PCR were performed for all genes, except when mentioned
otherwise.
In situ hybridization
Probes for in situ hybridization were transcribed using the
digoxigenin-labeling mix (Roche). The WUS antisense probe was
generated as described previously (Mayer
et al., 1998). The STM antisense probe was generated from
the 1.1 kb transcript described by Long and Barton
(Long and Barton, 1998
).
Separate ULT probes were generated from the full-length coding sequences of
ULT1 and ULT2, and from the 3'UTR of each gene. Tissue
fixation and in situ hybridization were performed as described previously
(Ambrose et al., 2000
), with
the additional steps that siliques were smashed and seedlings tips were
chopped before infiltration, to facilitate fixative penetration into the
tissues.
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Results |
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The WUS gene is expressed in the interior, deeper layers of shoot
and floral meristems, overlapping the CLV1 expression domain
(Fig. 1I)
(Mayer et al., 1998).
Mutations in ULT1 result in the lateral expansion of the WUS
expression domain, without altering its layer specificity
(Fig. 1J-K). Counting of
WUS-expressing cells confirmed that the organizing center is
significantly larger in ult1-1 and ult1-2 meristems than in
wild-type meristems (Fig. 1P).
In wild-type inflorescence meristems, the mean size of the
WUS-expressing domain corresponds to 6.12±0.33 cells in width,
3±0 cells in height and 14.75±0.97 cells in total. In
ult1-1 inflorescence central sections the WUS domain expands
to 8.50±1 cells in width, 3.12±0.33 cells in height, and
21.12±2.80 cells in total, while in ult1-2 inflorescence
central sections the WUS domain is 7.75±0.66 cells in width,
3±0 cells in height and 18±1.66 cells in total. This result
shows that the size of the WUS-expressing organizing center is
negatively regulated by ULT1 activity. As ult1-1 plants have larger
inflorescence and floral meristems than do ult1-2 plants and produce
more floral meristems and floral organs
(Fletcher, 2001
), our results
suggest that the size of the WUS-expressing organizing center may
directly affect these traits.
Finally, we used a pSTM::uidA
(McConnell and Barton, 1998)
reporter line as a marker to examine the size of the peripheral zone of the
meristem in wild-type and ult1 plants. This reporter construct does
not recapitulate the STM expression pattern in the meristem; instead,
it is expressed at the boundary between the proper inflorescence meristem and
the incipient floral primordia (Fig.
1M). The pSTM::uidA expression pattern is unaltered in
ult1 inflorescences, indicating that the peripheral region of the
mutant meristems is not significantly enlarged
(Fig. 1N-O). Altogether, our
expression analyses indicate that ULT1 restricts the lateral expansion of
CLV1- and WUS- expressing cells in the interior of
inflorescence and floral meristems.
Positional cloning of ULT1
To isolate the ULT1 gene, we used CAPS-based mapping
(Konieczny and Ausubel, 1993)
of recombination breakpoints in 1366 meiotic events among the F2 progeny of
ult1-2 (Ler) x wild type (Col-O). We had previously
shown that the ult1 mutations mapped between the visible markers
ag and ap2 on chromosome 4
(Fletcher, 2001
). Using the
CAPS markers throughout this interval, we established that ult1-2 was
flanked by markers PG11 and g8300
(www.arabidopsis.org).
Thirty-one plants with recombination events between PG11 and g8300 were
identified from the mapping population, and used to refine the position of the
ult1-2 recombination breakpoints to the ends of BAC F26K10. We
sequenced candidate genes annotated on the BAC and identified a single gene
(At4g28190) that was mutated in both ult1 alleles.
To confirm the identity of At4g28190 as the ULT1 gene, a genomic
clone (ULT1-214) containing the At4g28190 coding region along with 1 kb of
upstream and 0.5 kb of downstream sequence was introduced into ult1-1
plants, and this clone partially or fully complemented the mutant phenotypes
(Fig. 1H). T1 and T2
ult1-1 plants transformed with the ULT1-214 genomic construct
produced meristems and flowers similar to those of wild-type plants. In
addition, ult1-1 plants carrying the ULT1-214 transgene flowered at
the same time as did wild-type plants, while untransformed ult1-1
plants flowered 1 week later on average
(Fletcher, 2001). These data
confirm that At4g28190 encodes the ULT1 gene.
The complete ULT1-coding region was determined by EST and cDNA analysis, RT-PCR and 5'RACE. This region is 714 bp in length, and consists of three exons and two introns (Fig. 2A), encoding a predicted protein of 237 amino acids with a mass of 26.7 kDa. Genomic sequence analysis indicated the presence of a TATA box, a CCAAT box and a GC box, as well as an in frame stop codon upstream of the transcription start site. Ceres cDNA 96705 (www.arabidopsis.org) and the sequencing of RT-PCR products support the annotation of this gene. We have identified a missense mutation in the second exon of this gene in the ult1-1 and the ult1-2 alleles (Fig. 2A). The ult1-1 mutation is caused by a G to A transition that changes a cysteine residue to a threonine residue at position 173 relative to the translational initiation site (Fig. 2B). The ult1-2 mutation is due to a C to T transition that replaces a serine residue with a phenylalanine residue at position 83.
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We have identified sequences corresponding to ULT1- and ULT2-like genes in a number of other plant species, including tomato, maize, cotton, rice, soybean and wheat. So far, only a single ULT-like gene has been identified in these species, compared with two in Arabidopsis. An amino acid alignment of the putative ULT-like proteins for which full-length or nearly full-length genomic and/or EST sequences are available, is shown in Fig. 2B. The overall identity between the proteins ranges from 59% to 72% across the length of the protein. No functions have yet been assigned to any of these ULT-like proteins. The ult1-1 and ult1-2 mutations both occur in amino acids that are invariant among all nine of the plant species examined, suggesting that these residues are crucial for protein function.
Sequence analysis of the ULT1 and ULT2 proteins
Two domains can be recognized in the ULT1 and ULT2 protein sequences that
have been found in transcription factors. The Prosite program
(pit.georgetown.edu)
revealed a significant structural homology between the N-terminal region of
the ULT proteins (Fig. 2B) and
a conserved SAND domain found in animal proteins. The SAND domain is an
evolutionarily conserved 80-100 amino acid DNA-binding motif that takes
its name from the Sp100, AIRE-1, NucP41/75 and DEAF-1/suppressin proteins
found in humans and Drosophila melanogaster
(Gibson et al., 1998
). The
ULT1 and ULT2 proteins, as well as the other ULT-like plant sequences, share
75% identity within the SAND domain
(Fig. 2C).
The three-dimensional structures of several SAND domains have been
determined by NMR and x-ray crystallography
(Bottomley et al., 2001;
Surdo et al., 2003
). The SAND
domain is a compact, strongly twisted
/ß fold consisting of five
antiparallel ß-sheets alternating with four
-helices
(Fig. 2C). However, the primary
sequence of the SAND domain is poorly conserved between family members. The
highest degree of amino acid conservation is found between two otherwise
unrelated proteins from C. elegans, CeC25G4.4 and CeC44F1.2, which
share 57% identity within the SAND domain. Most of the animal proteins share
less than 30% identity within the SAND domain, and the pair-wise comparison
score can be as low as 7% identity, as shown for the human AIRE-1 and GMEB1/2
proteins. Thus, the similarity between animal SAND domains instead resides at
the secondary and consequent tertiary structure level. Similarly, the major
conservation of the ULT SAND domains is at the level of the secondary
structure: The PsiPred program (McGuffin
et al., 2000
) predicts the ß1, ß2, ß3 and ß5
strands, as well as the
2 and
4 helices in the ULT proteins
(Fig. 2C). The program did not
detect the
1 and
3 helices or the ß4 sheet, probably
because of their extremely small size. Only two conserved cores are
highlighted by multiple alignment of the SAND domains, the TPxxFE and the KDWK
motifs (Fig. 2C). The TPxxFE
motif is perfectly conserved among all the putative ULT-like proteins in
plants (Fig. 2B,C). The KDWK
core is not conserved in ULT1 and ULT2 nor in the mouse and human AIRE-1
proteins at the primary sequence level, but the secondary structure is
conserved. The ult1-2 mutation, which causes a null mutant phenotype
(see below), lies within the
2 helix of the SAND domain
(Fig. 2B,C).
The ULT1 and ULT2 proteins are highly cysteine rich, with cysteine residues
accounting for 9.7% of the total amino acid content of each protein
(Fig. 2B). One particular
arrangement of cysteine residues near the C terminus of the ULT1 and ULT2
proteins is highly similar to that of a B-box motif found in many eukaryotes
(Fig. 2D). In these organisms,
the B-box domain has been proposed to function in protein-protein and in
protein-RNA interactions (Borden,
1998; Torok and Etkin,
2000
). B-box domains are associated with cysteine-rich
zinc-binding motifs in otherwise unrelated proteins, many of them
transcription factors, that participate in a wide range of cellular processes
(Borden, 1998
;
Torok and Etkin, 2000
). The
putative B-box region is more highly conserved between ULT1 and the homologous
sequences than the rest of the protein
(Fig. 2B).
Subcellular localization of the ULT proteins
In animals, SAND domain-containing proteins are found in the nucleus, in
the cytoplasm, or in both compartments
(Gross and McGinnis, 1996;
Jimenez-Lara et al., 2000
;
Peterson et al., 2004
).
Similarly, eukaryotic proteins containing B-box domains have been localized to
either the nucleus or the cytosol (Borden,
1998
; Torok and Etkin,
2000
). The computer programs Prosite
(Hulo et al., 2004
;
Sigrist et al., 2002
), PSORT
(Nakai and Kanehisa, 1992
),
SignalP (Nielsen et al.,
1997
), and NLSdb (Nair et al.,
2003
) each predict the ULT1 and ULT2 proteins to be localized to
the cytosol, based on the absence of any sorting or signal peptide. However,
both ULT proteins are small enough to diffuse passively into the nucleus
through the nuclear pores (Raikhel,
1992
). Subcellular localization experiments using enhanced green
fluorescent protein (EGFP) as a marker showed that ULT1-EGFP and ULT2-EGFP
fusion constructs transiently transformed into onion epidermal cells are
localized in both the nucleus and the cytosolic compartments
(Fig. 3A).
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However, as the ULT-EGFP fusion proteins are still smaller than the nuclear
pore exclusion size they may enter or exit the nucleus passively, especially
when expressed at high levels under the 35S promoter. To prevent passive entry
into or exit from the nucleus, we fused each ULT protein to a combined GUS
(ß-glucuronidase)-EGFP protein
(Grebenok et al., 1997). When
bombarded into onion epidermal cells, the constructs gave a GFP and a GUS
signal primarily in the cytosol for some cells and equivalently in the cytosol
and the nucleus for others (Fig.
3F,G). Thus, the ULT1 and ULT2 proteins have a dual localization
in the nucleus and in the cytosol, and may function in both compartments.
ULT1 and ULT2 expression analysis
We used RT-PCR to determine the distribution of ULT1 and
ULT2 mRNA transcripts in wild-type tissues. As shown in
Fig. 4, ULT1
transcripts could be amplified from all tissues tested: roots, 8-day-old
seedlings, mature leaves, stems, inflorescences, pollen and siliques.
ULT2 expression was specific to the reproductive developmental stage,
being detected only in inflorescences, pollen and siliques. For both genes,
the highest level of expression was observed in inflorescence tissues.
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Transgenic d35S::ULT plants display a gradient of phenotypes that correlates with the level of ULT gene overexpression. Those plants expressing the highest levels of ULT1 or ULT2 show dramatic vegetative phenotypes as soon as a few days after germination (C.C.C. and J.C.F., unpublished). Consequently, we performed the complementation analysis on d35S::ULT2 ult1-1 lines that had a wild-type appearance at the vegetative stage. As expected, RT-PCR experiments showed that these lines display a more moderate increase in ULT2 gene expression than the dramatically affected overexpression lines (data not shown). By analyzing these moderate overexpression lines, we found that the d35S::ULT2 transgene complements the ult1-1 mutant phenotypes to the same extent as the d35S::ULT1 transgene (Fig. 7). Indeed, ult1-1 plants containing either of these constructs display floral organ number and bolting time phenotypes close to those of the wild type. Thus, although the endogenous level of ULT2 is not sufficient to overcome the effect of the ult1-1 mutation, an increase in the amount of wild-type ULT2 protein in the ult1-1 background allows complete rescue of the ult1-1 mutant phenotypes. These data indicate that, when expressed at higher levels, wild-type ULT2 protein can functionally compensate for mutant ULT1-1 protein.
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The ULT1-1 mutant protein has semi-dominant effects
Because the ult1-1 mutant phenotype is more dramatic than that of
the ult1-3 null mutant, we analyzed the effect of the ult1-1
mutation in the heterozygote state. ult1-1 behaves as a slight
semi-dominant allele when heterozygous: of 18 ult1-1/+ plants scored,
five had five sepals and/or petals in the first one or two flowers and one had
six petals in the first flower. All ult1-1/+ plants are wild type
with respect to stamen and carpel number and floral determinacy, indicating
that the ult1-1 mutation is recessive with respect to these
traits.
To test whether the semi-dominant effect of the ULT1-1 mutant protein is altered in the absence of wild-type ULT1 protein, we compared the floral organ number and flowering time phenotypes of ult1-1/+ plants with those of ult1-1/ult1-3 plants. We found that ult1-1/ult1-3 plants are more severely affected than either ult1-3 homozygous plants or ult1-1/+ heterozygous plants with respect to sepal/petal number and also flowering time (Fig. 8D,E). Thus, eliminating wild-type ULT1 protein enhances the effects of the ult1-1 mutation on flowering time and on floral organ number in the outer two whorls.
Down-regulation of both ULT genes leads to early arrest of the vegetative SAM
Antisense plants carrying a d35S::ULT1 AS construct generated in the
Ler wild-type background show a dramatic reduction in the level of
both ULT1 and ULT2 transcripts
(Fig. 9A). Some plants from the
antisense lines fail to germinate (data not shown), while the rest display a
range of shoot and floral meristem defects
(Fig. 9B-I).
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The least severely affected ULT AS plants produce flowers that resemble those of ult1-2 mutants (Fig. 9F-I). These plants form flowers with supernumerous floral organs (Fig. 9G) when compared with wild-type plants (Fig. 9F). Five sepals and five petals are observed in some flowers (Fig. 9G), and others form up to four carpels (Fig. 9H). Flowers from the ULT AS lines also display a partial loss of determinacy, in that supernumerous carpels can develop as fifth whorl structures within the fourth whorl gynoecium (Fig. 9I, arrow).
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Discussion |
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Several observations suggest that ULT1 and the CLV loci regulate the size
of the WUS-expressing cell population via separate pathways. First,
the meristems of clv mutant plants, but not of ult1 mutant
plants, are measurably taller than those of wild-type plants
(Clark et al., 1993;
Clark et al., 1995
;
Fletcher, 2001
). Second, in
clv but not in ult1 inflorescence meristems the WUS
expression domain extends one cell layer up compared with wild type. Third,
ult1 and clv alleles show synergistic effects on
inflorescence and floral meristem size, suggesting that they use separate
pathways to regulate a common process
(Fletcher, 2001
). Finally,
although wus mutations have been shown to be epistatic to
clv mutations in both shoot and floral meristems, ult1 wus
double mutants have additive phenotypes, except in the center of the flower
(Carles et al., 2004
). Thus,
ULT1 has both WUS-dependent and WUS-independent functions in
maintaining meristem activity, and converges with the CLV pathway primarily at
the point of limiting the lateral expansion of the WUS-expressing
cell population.
ULT1 and ULT2 proteins resemble transcriptional regulators
ULT1 and ULT2 define a small family of closely related plant proteins that
contain conserved SAND and B box-like domains. A high degree of sequence
conservation between the Arabidopsis ULT1 and ULT2 proteins and
predicted proteins in eight other monocot and dicot species was observed
across the length of the proteins (Fig.
2B). In addition, the ULT-like ESTs from tomato, soybean and
alfalfa were identified from shoot and/or floral meristem tissues - tissues in
which Arabidopsis ULT1 is known to act
(Carles et al., 2004;
Fletcher, 2001
). This
observation suggests that the roles ULT1 plays in meristem maintenance and
floral determinacy may be widely conserved among angiosperms.
Until now, reports of proteins containing a SAND domain have been
restricted to the animal phyla (Bottomley
et al., 2001; Gibson et al.,
1998
; Surdo et al.,
2003
). Overall, their primary sequences are quite divergent except
for two core elements, but the secondary structure of the SAND domain is
highly conserved. The same holds true for the SAND domains present in ULT1 and
ULT2. The ult1-2 missense mutation, which lies in the
2 helix
of the ULT1 SAND domain, changes a serine residue to a phenylalanine. In other
SAND domain proteins glycine, alanine or cysteine residues are encountered at
the same position. These amino acids, like serine, have small side chains.
Thus, the introduction of a highly hydrophobic aromatic phenylalanine residue
is likely to disrupt the structure of the SAND domain in the mutant ULT1-2
protein, perturbing potential DNA-binding and/or protein-protein interactions.
Moreover, the fact that such a missense mutation behaves as a knockout
mutation illustrates the importance of the SAND domain in ULT1 protein
function.
Many SAND domain-containing proteins, such as DmDEAF-1, HsNUDR and
HsGMEB1/2, have been shown to bind specifically to DNA
(Bottomley et al., 2001;
Burnett et al., 2001
;
Gross and McGinnis, 1996
;
Surdo et al., 2003
). The SAND
domain itself has been proven to mediate this interaction via the KDWK
motif-containing region (Bottomley et al.,
2001
; Jimenez-Lara et al.,
2000
; Surdo et al.,
2003
). Moreover, the SAND domain has been shown to be necessary
for the transactivation and homo-multimerization activities of AIRE
(Halonen et al., 2004
), as
well as for its nuclear localization
(Ramsey et al., 2002
).
Altogether, phenotypic and biochemical studies, along with three-dimensional
structure modeling, suggest that the SAND domain defines a novel DNA-binding
module involved in the regulation of gene transcription. The cloning of the
ULT1 gene has led us to the identification of a novel group of plant
SAND domain proteins with a conserved secondary structure. By analogy with
animal SAND domain factors, we propose that ULT1 and ULT2 may function as
transcription regulators, possibly binding directly to target DNA.
Alternatively, association of the ULT proteins with DNA might require the
presence of a liaison factor between the SAND domain and the target DNA
sequence, as observed for the AIRE protein
(Pitkänen et al.,
2001
).
Our subcellular localization studies show that the ULT1 and ULT2 proteins
accumulate in, and may be functional in, both the nucleus and the cytosol. One
possibility for this dual localization pattern is that the small ULT proteins
diffuse freely between the nucleus and the cytoplasm. However, when we
generated ULT-GUS-EGFP fusion proteins that were too large to passively enter
the nucleus, we still detected signal in the nuclear compartment. This implies
that the ULT proteins either contain a functional NLS for nuclear import, or
that they enter the nucleus by forming complexes with protein partners that
possess an NLS core (Boulikas,
1994). A canonical NLS is not detected in the ULT proteins, but
both the ULT1 and ULT2 sequences contain a hexapeptide and an octapeptide that
each has four arginine or lysine residues. These sequences may correspond to
nuclear targeting signals or be part of a bipartite NLS core
(Hicks and Raikhel, 1995
), as
reported for the AIRE protein
(Pitkänen et al., 2001
).
The dual nuclear and cytoplasmic localization of ULT1 and ULT2 proteins is not
unprecedented among the SAND domain-containing proteins
(Halonen et al., 2004
;
Ramsey et al., 2002
). One
possibility is that the dual localization in the nucleus and the cytosol may
serve as a modulation mechanism for transcriptional regulation, as shown for
some families of transcription factors in plants and animals
(Fabbro and Henderson, 2003
;
Merkle, 2001
;
Ziegelbauer et al., 2001
).
ult1-1 is a dominant negative allele
Side-by-side comparison of ult1-2 and ult1-3 homozygous
plants showed that their phenotypes are indistinguishable from one another,
revealing that ult1-2 is a phenotypic null allele for the
ULT1 locus. However, the ult1-1 EMS allele confers a more
severe phenotype than the other two alleles with respect to sepal/petal number
and flowering time. Analysis of heterozygous ult1-1/+ plants shows
that the ult1-1 mutation is semi-dominant with respect to these
traits. When we scored for ult1-1 semi-dominancy in an
ult1-1/ult1-3 hemizygous background, we found that the
effect of the ULT1-1 mutant protein was more dramatic when wild-type ULT1
protein was absent. In fact, ult1-1/ult1-3 plants closely resembled
ult1-1/ult1-1 plants. These results suggest that in ult1-1/+
heterozygous plants, wild-type ULT1 protein can compete with the mutant ULT1-1
protein and maintain some normal function, whereas in ult1-1/ult1-3
plants no wild-type ULT1 protein is present to compete with the dysfunctional
ULT1-1 mutant protein.
It is possible that the ult1-1 missense mutation abolishes protein
function but does not prevent binding to other factors. In such a scenario,
ULT1-1 mutant protein would compete with wild-type ULT1 protein, sequestering
one or more physical interaction partners and preventing or altering their
activity. As the ULT1 and ULT2 expression patterns fully
overlap in the inflorescence, it is possible that the two proteins themselves
physically interact. The example of the SAND domain proteins GMEB-1 and
GMEB-2, which share a high amino acid similarity and interact with one another
in vitro (Jimenez-Lara et al.,
2000), is consistent with this idea. In the ult1-1
allele, ULT1-1 mutant protein could sequester wild type ULT2 protein,
preventing ULT2 from functioning in shoot and flower tissues. However, because
ULT1 and ULT2 expression patterns do not fully overlap
throughout development, it seems likely that the ULT proteins also interact
with additional factors.
Roles of the ULT1 and ULT2 genes in development
The phenotypes displayed by ult1 null mutant plants reveal that
ULT1 plays an important role in negatively regulating inflorescence
and floral meristem size, and in maintaining floral meristem determinacy
(Carles et al., 2004;
Fletcher, 2001
).
Correspondingly, we find that the ULT1 gene is expressed in
inflorescence meristems, floral meristems and developing carpels. Yet despite
its role in negatively regulating the size of the WUS-expressing
organizing center in a central and interior domain, ULT1 is expressed
throughout the shoot and floral meristems, similar to STM, rather
than in a region-specific fashion like CLV3, CLV1 and WUS.
These data suggest that ULT1 may interact with other, as yet unidentified
region-specific factors in the meristem to restrict the accumulation of the
WUS-expressing cell population. Moreover, the fact that ULT1
is expressed in other domains, such as cotyledon and leaf primordia, shows
that ULT activity is not restricted to the meristems. The expression of
ULT1 in the developing tapetum and ovules, in particular, implies a
specific function(s) in reproduction. However, the absence of detectable
phenotypes outside the shoot and floral meristems in ult1 mutant
plants again suggests redundancy with other factors, such as
ULT2.
The pattern of ULT2 expression in inflorescence meristems, floral meristems and developing flowers appears to coincide perfectly with that of ULT1, yet ult2-1 T-DNA mutant plants do not display any shoot or floral phenotypes. Currently, we cannot exclude the possibility that the presence of very low levels of ULT2 protein translated from rare correctly spliced transcripts is sufficient for proper reproductive meristem activity in the ult2-1 mutant. Nonetheless, the presence of wild-type levels of ULT2 cannot compensate for the loss of ULT1 activity in reproductive meristems, whereas increasing ULT2 expression under the control of a dual 35S promoter can complement the ult1-1 mutation. This observation positions ULT2 as a functional duplicate of ULT1, and suggests that shoot and floral meristem activity may be sensitive to the dose of the ULT proteins. The necessity to fine tune the regulation of genes involved in meristem maintenance could explain the retention of both ULT factors in Arabidopsis. This implies that ULT1 and ULT2 are likely to have multiple common targets, the regulation of which is dependent on ULT dose. That the two genes have other independent targets, as well, is clear from the specific expression of ULT1 in leaf primordia and ULT2 in the embryonic root apical meristem.
ULT1 and ULT2 transcripts are detected throughout the
embryo as early as the octant stage, and continue to accumulate in all cells
of the embryo proper until maturity, when ULT1 transcripts become
restricted to the SAM and ULT2 transcripts to the SAM and RAM. To our
knowledge, ULT1 and ULT2 encode the only mRNAs characterized
thus far that become restricted to the meristems at such a late stage of
embryo maturation. The high level of ULT1 and ULT2
expression throughout the developing embryo might be a sign that these genes
have important functions early in development that are not revealed in the
single mutants. Our previous results have shown that ult1 mutations
restore SAM activity to stm and wus null mutant seedlings
(Carles et al., 2004),
indicating that ULT1 functions to restrict SAM cell accumulation
prior to the appearance of a visible phenotype in ult1 mutant plants.
Our analysis of ULT1/ULT2 antisense lines provides additional
evidence for an early and important function for the ULT genes, as
downregulation of both ULT1 and ULT2 can result in aberrant
lateral organ production and SAM arrest very early during seedling
development.
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ACKNOWLEDGMENTS |
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Footnotes |
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REFERENCES |
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Ambrose, B. A., Lerner, D. R., Ciceri, P., Padilla, C. M., Yanofsky, M. F. and Schmidt, R. J. (2000). Molecular and genetic analyses of the Silky1 gene reveal conservation in floral organ specification between eudicots and monocots. Mol. Cell 5,569 -579.[Medline]
Borden, K. L. B. (1998). RING fingers and B-boxes: zinc-binding protein-protein interaction domains. Biochem. Cell Biol. 76,351 -358.[CrossRef][Medline]
Bottomley, M. J., Collard, M. W., Huggenvik, J. I., Liu, Z., Gibson, T. J. and Sattler, M. (2001). The SAND domain structure defines a novel DNA-binding fold in transcriptional regulation. Nat. Struct. Biol. 8,626 -633.[CrossRef][Medline]
Boulikas, T. (1994). Putative nuclear localization signals (NLS) in protein transcription factors. J. Cell Biochem. 55,32 -58.[Medline]
Brand, U., Fletcher, J. C., Hobe, M., Meyerowitz, E. M. and
Simon, R. (2000). Dependence of stem cell fate in
Arabidopsis on a feedback loop regulated by CLV3 activity.
Science 289,617
-619.
Bundock, P. and Hooykaas, P. (2002). Severe
developmental defects, hypersensitivity to DNA-damaging agents, and lengthened
telomeres in Arabidopsis MRE11 mutants. Plant Cell
14,2451
-2462.
Burnett, E., Christensen, J. and Tattersall, P. (2001). A consensus DNA recognition motif for two KDWK transcription factors identifies flexible-length, CpG-methylation sensitive cognate binding sites in the majority of human promoters. J. Mol. Biol. 314,1029 -1039.[CrossRef][Medline]
Carles, C. C., Lertpiriyapong, K., Reville, K. and Fletcher, J.
C. (2004). The ULTRAPETALA1 gene functions early in
Arabidopsis development to restrict shoot apical meristem activity, and acts
through WUSCHEL to regulate floral meristem determinacy.
Genetics 167,1893
-1903.
Clark, S. E., Running, M. P. and Meyerowitz, E. M.
(1993). CLAVATA1, a regulator of meristem and flower
development in Arabidopsis. Development
119,397
-418.
Clark, S. E., Running, M. P. and Meyerowitz, E. M.
(1995). CLAVATA3 is a specific regulator of shoot and
floral meristem development affecting the same processes as CLAVATA1.
Development 121,2057
-2067.
Clark, S. E., Williams, R. W. and Meyerowitz, E. M. (1997). The CLAVATA1 gene encodes a putative receptor kinase that controls shoot and floral meristem size in Arabidopsis. Cell 89,575 -585.[Medline]
Clough, S. J. and Bent, A. F. (1998). Floral dip: a simplified method for Agrobacterium-mediated transformation of Arabidopsis thaliana. Plant J. 16,735 -743.[CrossRef][Medline]
Fabbro, M. and Henderson, B. R. (2003). Regulation of tumor suppressors by nuclear-cytoplasmic shuttling. Exp. Cell Res. 282,59 -69.[CrossRef][Medline]
Fletcher, J. C. (2001). The
ULTRAPETALA gene controls shoot and floral meristem size in
Arabidopsis. Development
128,1323
-1333.
Fletcher, J. C., Brand, U., Running, M. P., Simon, R. and
Meyerowitz, E. M. (1999). Signaling of cell fate decisions by
CLAVATA3 in Arabidopsis shoot meristems.
Science 283,1911
-1914.
Gallois, J.-L., Woodward, C., Reddy, G. V. and Sablowski, R.
(2002). Combined SHOOT MERISTEMLESS and WUSCHEL trigger ectopic
organogenesis in Arabidopsis. Development
129,3207
-3217.
Gibson, T. J., Ramu, C., Gemund, C. and Aasland, R. (1998). The APECED polyglandular autoimmune syndrome protein, AIRE-1, contains the SAND domain and is probably a transcription factor. Trends Biochem. Sci. 23,242 -244.[CrossRef][Medline]
Gleave, A. P. (1992). A versitile binary vector system with a T-DNA organizational structure conducive to efficient integration of cloned DNA into the plant genome. Plant Mol. Biol. 20,1203 -1207.[Medline]
Grebenok, R. J., Peirson, E., Lambert, G. M., Gong, F.-C., Afonso, C. L., Haldeman-Cahill, R., Carrington, J. C. and Galbraith, D. W. (1997). Green florescent protein fusions for efficient characterization of nuclear targeting. Plant J. 11,573 -586.[CrossRef][Medline]
Gross, C. T. and McGinnis, W. (1996). DEAF-1, a novel protein that binds an essential region in a Deformed response element. EMBO J. 15,1961 -1970.[Abstract]
Guyomarc'h, S., Vernoux, T., Traas, J., Zhou, D. X. and Delarue,
M. (2004). MGOUN3, an Arabidopsis gene with
TetratricoPeptide-Repeat-related motifs, regulates meristem cellular
organization. J. Exp. Bot.
55,673
-684.
Halonen, M., Kangas, H., Ruppell, T., Ilmarinen, T., Ollila, J., Kolmer, M., Vihinen, M., Palvimo, J., Saarela, J., Ulmanen, I. et al. (2004). APECED-causing mutations in AIRE reveal the functional domains of the protein. Hum. Mutat. 23,245 -257.[CrossRef][Medline]
Hicks, G. R. and Raikhel, N. V. (1995). Protein import into the nucleus: an integrated view. Annu. Rev. Cell Dev. Biol. 11,155 -188.[CrossRef][Medline]
Hulo, N., Sigrist, C. J. A., le Saux, V., Langendijk-Genevaux,
P. S., Bordoli, L., Gattiker, A., de Castro, E., Bucher, P. and Bairoch,
A. (2004). Recent improvements to the PROSITE database.
Nucleic Acids Res. 32,D134
-D137.
Jeong, S., Trotochaud, A. E. and Clark, S. E.
(1999). The Arabidopsis CLAVATA2 gene encodes a
receptor-like protein required for the stability of the CLAVATA1
receptor-like kinase. Plant Cell
11,1925
-1933.
Jimenez-Lara, A. M., Heine, M. J. and Gronemeyer, H. (2000). Cloning of a mouse glucocorticoid modulatory element binding protein, a new member of the KDWK family. FEBS Lett. 468,203 -210.[CrossRef][Medline]
Kaya, H., Shibahara, K., Taoka, K., Iwabuchi, M., Stillman, B. and Araki, T. (2001). FASCIATA genes for Chromatin Assembly Factor-1 in Arabidopsis maintain the cellular organization of apical meristems. Cell 104,131 -142.[Medline]
Kayes, J. M. and Clark, S. E. (1998).
CLAVATA2, a regulator of meristem and organ development in
Arabidopsis. Development
125,3843
-3851.
Konieczny, A. and Ausubel, F. M. (1993). A procedure for mapping Arabidopsis mutations using co-dominant ecotype-specific PCR-based markers. Plant J. 4, 403-410.[CrossRef][Medline]
Laux, T., Mayer, K. F. X., Berger, J. and Jurgens, G.
(1996). The WUSCHEL gene is required for shoot and
floral meristem integrity in Arabidopsis.
Development 122,87
-96.
Lenhard, M. and Laux, T. (2003). Stem cell
homeostasis in the Arabidopsis shoot meristem is regulated by
intercellular movement of CLAVATA3 and its sequestration by CLAVATA1.
Development 130,3163
-3173.
Leyser, H. M. O. and Furner, I. J. (1992).
Characterisation of three shoot apical meristem mutants of Arabidopsis
thaliana. Development
116,397
-403.
Long, J. A. and Barton, M. K. (1998). The
development of apical embryonic pattern in Arabidopsis.
Development 125,3027
-3035.
Long, J. and Barton, M. K. (2000). Initiation of axillary and floral meristems in Arabidopsis. Dev. Biol. 218,341 -353.[CrossRef][Medline]
Long, J. A., Moan, E. I., Medford, J. I. and Barton, M. K. (1996). A member of the KNOTTED class of homeodomain proteins encoded by the STM gene of Arabidopsis. Nature 379,66 -69.[CrossRef][Medline]
Mayer, K. F. X., Schoof, H., Haecker, A., Lenhard, M., Jurgens, G. and Laux, T. (1998). Role of WUSCHEL in regulating stem cell fate in the Arabidopsis shoot meristem. Cell 95,805 -815.[Medline]
McConnell, J. R. and Barton, M. K. (1998). Leaf
polarity and meristem formation in Arabidopsis.
Development 125,2935
-2942.
McGuffin, L. J., Bryson, K. and Jones, D. T. (2000). The PSIPRED protein structure prediction server. Bioinformatics 16,404 -405.[Abstract]
Merkle, T. (2001). Nuclear import and export of proteins in plants: a tool for the regulation of signalling. Planta 213,499 -517.[CrossRef][Medline]
Nair, R., Carter, P. and Rost, B. (2003).
NLSdb: database of nuclear localization signals. Nucleic Acids
Res. 31,397
-399.
Nakai, K. and Kanehisa, M. (1992). A knowledge base for predicting protein localization sites in eukaryotic cells. Genomics 14,897 -911.[Medline]
Nielsen, H., Engelbrecht, J., Brunak, S. and von Heijne, G. (1997). Identification of prokaryotic and eukaryotic signal peptides and predictions of their cleavage sites. Prot. Eng. 10,1 -6.[Abstract]
Peterson, P., Pitkänen, J., Sillanpaa, N. and Krohn, K. (2004). Autoimmune polyendocrinopathy candidiasis ectodermal dystrophy (APECED): a model disease to study molecular aspects of endocrine autoimmunity. Clin. Exp. Immunol. 135,348 -357.[CrossRef][Medline]
Pitkänen, J., Vähämurto, P., Krohn, K. and
Peterson, P. (2001). Subcellular localization of the
autoimmune regulator protein. characterization of nuclear targeting and
transcriptional activation domain. J. Biol. Chem.
276,19597
-19602.
Raikhel, N. V. (1992). Nuclear targeting in plants. Plant Physiol. 100,1627 -1632.
Ramsey, C., Bukrinsky, A. and Peltonen, L.
(2002). Systematic mutagenesis of the functional domains of AIRE
reveals their role in intracellular targeting. Hum Mol
Genet. 11,3299
-3308.
Running, M. P., Fletcher, J. C. and Meyerowitz, E. M.
(1998). The WIGGUM gene is required for proper
regulation of floral meristem size in Arabidopsis.
Development 125,2545
-2553.
Running, M. P., Lavy, M., Sternberg, H., Galichet, A., Gruissem,
W., Hake, S., Ori, N. and Yalovsky, S. (2004). Enlarged
meristems and delayed growth in plp mutants result from lack of CaaX
prenyltransferases. Proc. Natl. Acad. Sci. USA
101,7815
-7820.
Sanford, J. C., Smith, F. D. and Russell, J. A. (1993). Optimizing the biolistic process for different biological applications. Methods Enzymol. 217,483 -509.[Medline]
Schoof, H., Lenhard, M., Haecker, A., Mayer, K. F. X., Jurgens, G. and Laux, T. (2000). The stem cell population of Arabidopsis shoot meristems is maintained by a regulatory loop between the CLAVATA and WUSCHEL genes. Cell 100,635 -644.[Medline]
Siddiqui, N. U., Stronghill, P. E., Dengler, R. E., Hasenkampf,
C. A. and and Riggs, C. D. (2003). Mutations in Arabidopsis
condensin genes disrupt embryogenesis, meristem organization and segregation
of homologous chromosomes during meiosis. Development
130,3283
-3295.
Sieburth, L. E. and Meyerowitz, E. M. (1997).
Molecular dissection of the AGAMOUS control region shows that cis
elements for spatial regulation are located intragenically. Plant
Cell 9,355
-365.
Sigrist, C. J. A., Cerutti, L., Hulo, N., Gattiker, A., Falquet, L., Pagni, M., Bairoch, A. and Bucher, P. (2002). PROSITE: a documented database using patterns and profiles as motif descriptors. Brief Bioinform. 3,265 -274.[Medline]
Steeves, T. A. and Sussex, I. M. (1989). Patterns in Plant Development. New York: Cambridge University Press.
Surdo, P. L., Bottomley, M. J., Sattler, M. and Scheffzek,
K. (2003). Crystal structure and nuclear magnetic resonance
analyses of the SAND domain from glucocorticoid modulatory element binding
protein-1 reveals deoxyribonucleic acid and zinc binding regions.
Mol. Endocrinol. 17,1283
-1295.
Suzuki, T., Inagaki, S., Nakijima, S., Akashi, T., Ohto, M., Kobayashi, M., Seki, M., Shinozaki, K., Kato, T., Tabata, S. et al. (2004). A novel Arabidopsis gene TONSOKU is required for proper cell arrangement in root and shoot apical meristem. Plant J. 38,673 -684.[CrossRef][Medline]
Takeda, S., Hofmann, I., Probst, A. V., Angelis, K. J., Kaya,
H., Araki, T., Mengiste, T., Scheid, O. M., Shibahara, K. et al.
(2004). BRU1, a novel link between responses to DNA damage and
epigenetic gene silencing in Arabidopsis. Genes Dev.
18,782
-793.
Torok, M. and Etkin, L. D. (2000). Two B or not two B? Overview of the rapidly expanding B-box family of proteins. Differentiation 67,63 -71.[CrossRef]
Trotochaud, A. E., Hao, T., Wu, G., Yang, Z. and Clark, S.
E. (1999). The CLAVATA1 receptor-like kinase requires
CLAVATA3 for its assembly into a signaling complex that includes KAPP and a
Rho-related protein. Plant Cell
11,393
-405.
Ueda, M., Matsui, K., Ishiguro, S., Sano, R., Wada, T., Paponov,
I., Palme, K. and Okada, K. (2004). The HALTED ROOT
gene encoding the 26S proteasome subunit RPT2a is essential for the
maintenance of Arabidopsis meristems. Development
131,2101
-2111.
Ziegelbauer, J., Shan, B., Yager, D., Larabell, C., Hoffmann, B. and Tjian, R. (2001). Transcription factor MIZ-1 is regulated via microtubule association. Mol. Cell 8, 339-349.[Medline]
Ziegelhoffer, E. C., Medrano, L. J. and Meyerowitz, E. M.
(2000). Cloning of the Arabidopsis WIGGUM gene
identifies a role for farnesylation in meristem development. Proc.
Natl. Acad. Sci. USA 97,7633
-7638.