1 Department of Biology, University of Washington, Seattle, WA 98195, USA
2 CREST, Japan Science and Technology Corporation, Saitama 332-0012, Japan
* Author for correspondence (e-mail: ktorii{at}u.washington.edu)
Accepted 10 December 2003
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SUMMARY |
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Key words: Arabidopsis, Receptor-like kinase, Cell proliferation, Organ size, Flower development, Functional redundancy
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Introduction |
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In higher plants, lateral organs are generated reiteratively by the
continual activity of the shoot apical meristem (SAM). Because plant cells are
encapsulated by cell walls, organogenesis occurs in the absence of cell
migration or the removal of overproduced cells. As such, unraveling
developmental programs that coordinate cell proliferation and expansion during
organ growth is key for understanding plant organ size control. The molecular
links that integrate regulation of cell proliferation and organ size/shape as
a unit in plants have just begun to emerge. One such regulator is the
Arabidopsis AINTEGUMENTA (ANT) gene, which encodes an
AP2-domain transcription factor (Elliott
et al., 1996; Klucher et al.,
1996
). ANT acts to prolong the duration of cell
proliferation by sustaining the expression of D-type cyclins
(Mizukami, 2001
;
Mizukami and Fischer, 2000
).
Consequently, overexpression of ANT confers hyperplasia. The recent
finding that the auxin-inducible gene ARGOS acts upstream of
ANT suggests a role for the phytohormone auxin in regulating inherent
organ size (Hu et al.,
2003
).
We aim to uncover the molecular basis of cell-cell communication that fine
tunes coordinated cell proliferation during plant organ growth. The
Arabidopsis ERECTA gene is a candidate for this. ERECTA is
highly expressed in the SAM and developing lateral organs, where cells are
actively dividing (Yokoyama et al.,
1998). Loss-of-function erecta mutations confer compact
inflorescence with short lateral organs and internodes, and these phenotypes
are largely attributable to reduced cell numbers in the cortex cell files
(Shpak et al., 2003
;
Torii et al., 2003
;
Torii et al., 1996
). The
shortened erecta pedicels (floral stems) are associated with an
increase in 4C cells, suggesting a possible aberration in cell cycle
progression (Shpak et al.,
2003
). ERECTA encodes a leucine-rich receptor-like
serine/threonine kinase (LRR-RLK) (Torii
et al., 1996
), a prevalent subfamily of signaling receptors in
plants (Shiu and Bleecker,
2001
; Shiu and Bleecker,
2003
). LRR-RLKs regulate a wide-variety of signaling processes,
including development of the SAM and microspores, brassinosteroid perception,
floral abscission, pathogen recognition and symbiosis (for reviews, see
Becraft, 2002
;
Carles and Fletcher, 2003
;
Kistner and Parniske, 2002
;
Li, 2003
;
Torii et al., 2004
). The
structure, expression patterns, and cellular and developmental phenotypes all
support the notion that ERECTA mediates cell-cell signals that sense and
coordinate organ growth.
We have previously shown that expression of a dominant-negative form of
ERECTA (a Kinase fragment) enhances the growth defects of the null
erecta plants, suggesting redundancy in the ERECTA signaling pathway
(Shpak et al., 2003
). To reach
a full understanding of how ERECTA controls inherent plant size, it is
imperative to identify its redundant receptors.
Here, we report the identification and functional characterization of ERL1 and ERL2, two redundant, paralogous ERECTA-like receptors that play a role in a part of ERECTA signaling pathway. Although loss-of-function mutations in ERL1 and ERL2 loci gave no detectable phenotype, they each enhanced erecta defects in a unique manner. Strikingly, loss of the entire ERECTA-family LRR-RLKs conferred extreme dwarfism and abnormal flower development. Molecular and cellular analysis revealed that ERECTA-family RLKs link cell proliferation to organ growth and patterning via a novel mechanism.
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Materials and methods |
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Oligo DNA sequences
Lists of oligo DNA sequences used for all experiments are available as
supplementary data at
http://dev.biologists.org/supplemental/.
Cloning of ERL1 and ERL2
RT-PCR was performed using wild-type cDNA as a template, and using primer
pairs: ERL1.14coding and ERL1g6054rc for ERL1; and ERL2.3coding and
ERL2g5352rc for ERL2. The 5' ends of mRNA were recovered by a
rapid amplification of cDNA ends (RACE) using FirstChoiceTM RLM RACE kit
(Ambion, Austin, TX). Elk1-300rc or Ekl2-300.rc was used as the outer primer,
and Elk1-185rc or Elk2-185rc was used as the nested primer for ERL1
and ERL2, respectively. The amplified fragments were cloned into
pCR2.1-TOPO (Invitrogen, Carlsbad, CA) and sequenced.
Reverse transcriptase-mediated (RT) PCR
RNA isolation, cDNA synthesis and RT-PCR were performed as described by
Shpak et al. (Shpak et al.,
2003) with various cycles. Primer pairs used are as follows:
Complementation of erecta by ERL1 and ERL2
Full-length genomic coding regions for ERL1 and ERL2 were
cloned into the ERECTA promoter-terminator cassette by the following
procedure. PCR was performed using the wild-type Col genomic template and
primer pairs: ERL1g3036 and ERL1-3endrc for ERL1; and ERL2g2166 and
ERL2-3endrc for ERL2. The amplified fragments were digested with
SpeI and XbaI, and inserted into SpeI-digested
pKUT522 to generate pESH208A and pESH209A for ERL1 and ERL2,
respectively. Subsequently, PCR was performed using primer pairs: ERL1-5end
and ERL1g4411rc for ERL1; and ERL2-5end and ERL2g3182rc for
ERL2. The amplified fragments were digested with SpeI and
XbaI, and inserted into SpeI-digested pESH208A and pESH209A,
respectively, to generate pESH208 (ER::ERL1) and pESH209
(ER::ERL2). The plasmids were introduced into Agrobacterium
tumefaciens strain GV3101/pMP90 by electroporation, and into
Arabidopsis erecta-105 plants by vacuum infiltration.
ERECTA::GUS, ERL1::GUS and ERL2::GUS transgenic plants
For construction of ERECTA::GUS, the GUS gene was
inserted as a SpeI fragment into pKUT522 between the ERECTA
promoter and terminator. The plasmid was named pNI101. To make
ERL1::GUS and ERL2::GUS constructs, the
EcoRI/PstI fragment of pRT2-GUS was cloned into pZP222
(Hajdukiewics et al., 1994).
The plasmid was named pESH244. The ERL1 promoter region was amplified
with primers ERL1g-3680link and ERL1g403linkrc, using the MMI1 BAC clone as a
template. The ERL2 promoter region was amplified with primers
ERL2g-4364link and ERL2g4linkrc, using the T28J14 BAC clone as a template. The
amplified fragments were digested with EcoRI and inserted into
pESH244. The plasmids were named pESH245 (ERL1::GUS) and pESH246
(ERL2::GUS). pNI101, pESH245 and pESH246 were introduced into
Arabidopsis wild type as described above. GUS histochemical analysis
was performed according to Sessions et al.
(Sessions et al., 1999
).
Screening and isolation of the Arabidopsis T-DNA insertion mutants
Screening and isolation of T-DNA insertion lines were performed as
described by the Arabidopsis KO Facility
(http://www.biotech.wisc.edu/Arabidopsis/).
The erl1-2 was isolated from an population (vector pD991,
kanamycin resistance), and erl2-1 was isolated from a ß
population (vector pROK2, basta resistance), using gene-specific primers and
JL-202 T-DNA left border primer. The gene-specific PCR primers were: ERLK765
and ERLK6137rc for ERL1; and ERTJ70 and ERTJ5855rc for ERL2.
Precise locations of the insertions were determined by sequencing the PCR
fragments. Both erl1-2 and erl2-1 were backcrossed three
times. The B3F2 populations of erl1-2 and erl2-1 exhibited a
3:1 ratio of kanamycin resistance and Basta resistance, respectively
(erl1-2, KanR:KanS= 163:58,
2=0.183,
P=0.669; erl2-1, BastaR:BastaS=203:76,
2=
0.747, P=0.388), indicating a single T-DNA insertion. PCR-based
genotyping confirmed that these single insertions disrupt the ERL
loci.
Generation of double- and triple-knockout plants
To generate erecta erl1 and erecta erl2 double mutants,
erl1-2 and erl2-1 plants were crossed with
erecta-105 plants. To generate erl1 erl2 double
mutants, erl1-2 plants were crossed with plants of the genotype
erecta-105/erecta-105 erl1-2/erl1-2 erl2-1/+. Plants of a correct
genotype were isolated from the F2 populations. erecta-105/erecta-105
erl1-2/+ erl2-1/erl2-1 plants were self-fertilized to obtain the
erecta er11 erl2 triple mutants. The T-DNA insertion that disrupts
the ERL1 locus in erl1-2, and the ERL2 locus in
erl2-1, conferred resistance to kanamycin and Basta, respectively.
Thus, progenies of each cross were first tested for resistance, then
subsequently the genotype of individual plants, whether they were heterozygous
or homozygous, was determined by PCR using gene-specific primer pairs, and a
combination of T-DNA-(JL-202) and gene-specific primers. The presence of the
erecta-105 mutation was determined by PCR using the primer pairs:
ERg2248 and er-105 (Torii et al.,
2003).
Light and scanning electron microscopy
Fixation, and embedding and sectioning, of tissues for light microscopy
using Olympus BX40, as well as preparation of samples for scanning electron
microscopy using JOEL 840A, were performed as described by Shpak et al.
(Shpak et al., 2003).
Cell number measurement
Light microscopy images of four regions of sectioned wild-type,
erecta-105, erecta-105 erl1-2 and erecta-105 erl2-1 pedicels
were taken, and the number of cells in a middle longitudinal cortex row was
determined. This number was used to calculate the total number of cells in the
cortex row of an average length pedicel. The number of cells was counted in
three sectioned erecta-105 erl1-2 erl2-1 pedicels and the average was
determined.
Accession numbers
The GenBank accession numbers for the ERL1 and ERL2
sequences reported in this paper are AY244745 and AY244746, respectively.
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Results |
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ERECTA, ERL1 and ERL2 display overlapping, but unique expression patterns
The inability of ERL1 and ERL2 to complement
erecta mutants when expressed under their endogenous promoter
(Fig. 1C) suggests differences
in expression patterns. At the same time, if ERL1 and ERL2 are the RLKs whose
function is inhibited by the dominant-negative ERECTA fragment expressed under
the control of the ERECTA promoter, we should expect them to be
expressed, at least in part, in an overlapping manner with ERECTA. To
clarify these points, we next analyzed the developmental expression of
ERL1 and ERL2.
RT-PCR analysis showed that, similar to ERECTA, expression levels of the two ERLs were higher in developing organs, including bud clusters, flowers, siliques and young rosettes, lower in mature aboveground organs, such as leaves, stems and pedicels, and barely detectable in roots (Fig. 2). The expression levels of ERL1 and ERL2 in mature organs were much lower than those of ERECTA.
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Both ERL1 and ERL2 are redundant
erl1-2 and erl2-1 were subjected to further phenotypic
characterization. erl1-2 and erl2-1 single mutant plants
were indistinguishable from wild-type plants
(Fig. 5A-D). Their
inflorescence undergoes elongation of the internodes between individual
flowers, and they all displayed normal length petioles, stems
(Fig. 5A), pedicels
(Fig. 5B,D) and siliques
(Fig. 5C,D). The lack of any
visible phenotype suggests that ERL1 and ERL2 are
redundant.
|
erl1 and erl2 enhance a subset of erecta defects in a unique manner
To uncover the developmental role of ERL1 and ERL2 in the
absence of functional ERECTA, erl1 and erl2 mutations were
introduced into erecta-105 plants
(Torii et al., 1996;
Torii et al., 2003
). Both
erl1 and erl2 enhanced the erecta defects in a
unique manner. The erl1-2 mutation notably exaggerated the silique
and pedicel elongation defects of erecta-105. erecta-105 erl1-2
double mutant plants developed very short, blunt siliques and short pedicels
(Fig. 5B-D), both of which are
reminiscent of part of the phenotype conferred by the dominant-negative
Kinase (Shpak et al.,
2003
). The presence of the erl1-2 mutation did not
significantly affect the height of erecta-105 plants
(Fig. 5A).
By contrast, the erl2-1 mutation primarily enhanced the internodal
elongation defects of erecta. erecta-105 erl2-1 double mutant plants
were much shorter than erecta-105, and developed very compact
inflorescence with tightly clustered flowers and flower buds at the tip
(Fig. 5A,E). The architecture
of erecta-105 erl2-1 inflorescence resembles that of the transgenic
erecta-105 expressing Kinase
(Shpak et al., 2003
). In
addition, the erecta-105 erl2-1 siliques were slightly shorter than
those of erecta-105 (Fig.
5C,D).
The morphology of the silique tip was analyzed in detail. The erecta-105 silique tip has a blunt appearance due to a wide style that protrudes less from the valves than does the wild type. Both erl1 and erl2 mutations exaggerated this characteristic erecta silique phenotype, with even wider valves and shorter, broader styles (Fig. 5F). This indicates that the enhancement of the silique phenotype by erl1-2 and erl2-1 is not due to general elongation defects unrelated to the ERECTA pathway. From this, we conclude that ERL1 and ERL2 act in an overlapping but distinct part of the ERECTA signaling pathway in regulating inflorescence architecture and organ shape.
Synergistic interaction of ERECTA, ERL1 and ERL2 in promoting organ growth and flower development
To understand the biological function of the ERECTA-family LRR-RLK as a
whole, we next generated an erecta-105 erl1-2 erl2-1 triple mutant.
For this purpose, F2 plants that were homozygous for erecta and
erl2, but heterozygous for erl1, were self-fertilized. A
subsequent F3 population segregated extremely dwarf, sterile plants at an
25% ratio (dwarf plants/total=74/315,
2=0.382,
P=0.537), suggesting that they may be the triple mutant. To test this
hypothesis, genotypes of 86 F3 plants were analyzed. Among 63 compact, fertile
plants, 40 were heterozygous for erl1, 23 were wild type for
ERL1 and none were homozygous for erl1, consistent with the
expected 2:1 ratio (
2=0.286, P=0.593). By contrast,
all 23 extremely dwarf, sterile plants were homozygous for erl1 and
thus carried erecta-105 erl1-2 erl2-1 triple mutations. Furthermore,
progeny of the F3 siblings with a genotype erecta-105 ERL1 erl2-1
failed to segregate extremely dwarf plants (0/227 scored). These results
provide statistical evidence that the triple mutations confer severe growth
defects (Fisher's exact test, P<0.00000001).
We subsequently analyzed a phenotype of erecta-105 erl1-2 erl2-1
triple mutant plants during postembryonic development. The striking effects of
erecta-105 erl1-2 erl2-1 mutations on organ growth can be seen in all
aboveground organs (Fig. 6A-E)
and are evident soon after germination, at a time when cells start to divide.
Decreased cotyledon growth is notable in 4-day-old erecta-105 erl1-2
erl2-1 seedlings, and it is more striking in 12-day-old seedlings, which
have small, misshaped cotyledons with very short petioles
(Fig. 6A). Growth of primary
leaves is strongly diminished in the triple mutant seedlings
(Fig. 6A), whereas leaf
primordia are forming on a flank of the SAM (data not shown). Interestingly,
the triple mutations do not affect hypocotyl elongation, which occurs solely
because of cell elongation (Gendreau et
al., 1997). At a later stage of vegetative development,
erecta-105 erl1-2 erl2-1 plants form a small rosette with small,
round leaves that lack petiole elongation
(Fig. 6B). Transition to
flowering occurs approximately at the same time in wild-type,
erecta-105 and erecta-105 erl1-2 erl2-1 plants, suggesting
that mutations in the three ERECTA-family genes do not affect phase
transition (Fig. 6B, and data
not shown).
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erecta-105 erl1-2 erl2-1 triple mutants are defective in cell proliferation
To unravel the cellular basis of reduced organ growth, we examined cellular
morphology in petals and pedicels. Arabidopsis petals have a simple
cell layer structure with epidermal cells that are uniform in size and shape
(Bowman, 1993). Although petals
of erecta-105 erl1-2 erl2-1 plants are very small and filamentous in
shape (Fig. 6E), their abaxial
epidermis cells are slightly larger than in erecta-105 petals
(Fig. 6F).
As reported previously, erecta-105 pedicels have a reduced number
of expanded cortex cells (Shpak et al.,
2003). Similar to erecta-105, erecta-105 erl1 and
erecta-105 erl2 double mutations, and erecta-105 erl1-2
erl2-1 triple mutations, confer reduced cell numbers associated with
enlarged and irregular cell shape in the cortex
(Fig. 6G). Interestingly,
erecta-105 erl1-2 and erecta-105 erl1-2 erl2-1 mutations
lead to disorganized cell growth in the cortex. Cells are irregular in size
and shape, and have gaps in between. This phenotype is similar to transgenic
erecta-105 plants expressing
Kinase
(Shpak et al., 2003
). Cell
numbers in a longitudinal cortex file are severely reduced in the mutants,
with a concomitant decrease in the final pedicel length
(Fig. 5B,
Fig. 6I). erecta-105
pedicel has three times fewer cells per longitudinal row, and erecta-105
erl1-2 erl2-1 has 11 times fewer cells, compared with the wild type
(Fig. 6I). These results
demonstrate that organ growth defects of erecta erl1 erl2 are largely
due to a decrease in cell number, and suggest that ERECTA-family
genes promote cell proliferation during organ growth.
Molecular analysis of erecta erl1 erl2 inflorescence suggests a novel mechanism for organ growth regulation
To understand the molecular basis of organ growth/cell number defects
conferred by the triple mutations, we analyzed the expression levels of four
transcription factor genes that regulate shoot and floral organ size
(Fig. 6J). ANT acts to
prolong duration of cell proliferation during lateral organ development, and
its loss of function confers reduced organ size
(Elliott et al., 1996;
Mizukami and Fischer, 2000
).
Loss-of-function mutations in SHOOTMERISTEMLESS (STM) and
WUSCHEL (WUS) homeobox genes cause a decrease in the number
of meristem cells and growth defects of lateral organs
(Laux et al., 1996
;
Long et al., 1996
). The
BREVIPEDICELLUS (BP) locus encoded by the KNAT1
homeobox gene interacts synergistically with ERECTA in promoting
internodal elongation and floral organ size
(Douglas et al., 2002
).
Semi-quantitative RT-PCR analysis of flower and bud clusters reveals that
erecta-105 erl1-2 erl2-1 triple mutations do not affect expression
levels of ANT, STM or KNAT1
(Fig. 6J). WUS
expression was slightly reduced in the triple mutant background
(Fig. 6J). However, such a
slight reduction is not likely to account for severe defects in shoot and
floral organ growth, and internodal elongation in the triple mutants.
It is known that ANT leads to prolonged expression of D-type
cyclins, which control the entry to cell cycle progression at the G1 stage
(Cockcroft et al., 2000;
Dewitte and Murray, 2003
;
Mizukami and Fischer, 2000
).
Transcript levels of two D-type cyclins, CYCD2;1 and
CYCD3;1, were not significantly altered by the triple mutations
(Fig. 6J). This is consistent
with the notion that the control of organ size by ERECTA-family RLKs involves
mechanisms other than the pathway mediated by ANT. Taken together, the results
suggest that the three ERECTA-family LRR-RLKs promote cell proliferation via a
novel mechanism.
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Discussion |
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Functional redundancy and synergistic interaction among three ERECTA-family LRR-RLKs
Duplications of developmental regulatory genes followed by subsequent
mutation and selection are thought to have driven morphological diversity in
multicellular organisms. Acquisition of novel gene functions occurs by
alteration of protein function or of gene expression patterns. The fact that
ERL1 and ERL2 are capable of substituting for ERECTA activity when driven by
the ERECTA promoter and terminator
(Fig. 1C) indicates that
specificity among ERECTA, ERL1 and ERL2 lies largely in
their cis-regulatory elements rather than in their protein-coding regions.
Consistently, specific sites of enhancement of the erecta phenotype
by either erl1 or erl2 mutation appear to correspond to the
expression domains of these two LRR-RLKs, which are weaker and confined to a
subset of ERECTA expression domains (Figs
2,
3). The dominance of
cis-regulatory sequences over protein-coding regions in functional
specification among closely-related multigene families has been documented for
transcription factors regulating development, such as: Hox genes in mouse
development; the Myb genes WER and GL1 in
Arabidopsis epidermal patterning; and AGAMOUS-family
MADS-box genes in Arabidopsis ovule development
(Greer et al., 2000;
Lee and Schiefelbein, 2001
;
Pinyopich et al., 2003
).
Because ERECTA-family genes encode putative receptor kinases,
their functional equivalence indicates that ERECTA, ERL1 and ERL2 are capable
of perceiving the same ligand(s) and eliciting the same downstream
response(s). This raises a novel view on how the extent of organ growth is
monitored by cell-cell signaling in Arabidopsis. The prevalent model
based upon Drosophila wing development is that final organ size is
determined by the steepness of morphogen gradients
(Day and Lawrence, 2000).
According to this model, concentration gradients of ligands, such as Dpp or
Wg, dictate where and when cells proliferate. By contrast, we hypothesize that
tissue-specific and redundant expression of functionally equivalent receptors
plays a regulatory-role in coordinating Arabidopsis aerial organ
growth. In the organ primordium, where cells are proliferating ubiquitously,
uniform expression of all three ERECTA-family LRR-RLKs maximizes the organ
growth. As the organ matures, localized and non-redundant expression of each
RLK fine-tunes local, subtle growth for elaboration of final form and size.
Transient, non-overlapping expression of ERECTA, ERL1 and
ERL2 in a developing gynoecium
(Fig. 3M-O) reflects such
intricate local growth patterns, as growth and differentiation of distinct
tissues, such as stigma, style valves and ovules, must occur concomitantly
during carpel development (Ferrandiz et
al., 1999
). This view is in accordance with previous findings that
strength of the ERECTA pathway specifies final organ size in a quantitative
manner (Lease et al., 2001
;
Torii et al., 2003
;
Torii et al., 1996
). Future
identification of the ligands shared by these three receptors will address
this hypothesis.
A recent molecular evolutionary study implies that the RLK superfamily
underwent radical expansion within the plant lineage. The existence of more
than 600 RLK-coding genes in the Arabidopsis genome is in sharp
contrast with the small numbers of their counterparts (Pelle/IRAK family) in
animals: three in mice and four in humans
(Shiu and Bleecker, 2003).
Consistently, gene duplication events among RLK sub-families have been
documented (Baudino et al.,
2001
; Nishimura et al.,
2002
; Searle et al.,
2003
; Shiu and Bleecker,
2003
; Yamamoto and Knap,
2001
; Yin et al.,
2002
), but their biological significance is not fully understood.
Our finding confirms the effectiveness of the dominant-negative approach, and
further provides a framework for understanding functional redundancy among
recently duplicated plant RLK gene families.
ERECTA family genes control plant organ size through coordination of cell proliferation
The most prominent feature of erecta single and erecta
erl double and triple mutations is a reduction in aerial organ size due
to reduced cell numbers. In theory, cell numbers in lateral organs can be
regulated by affecting the number of SAM cells available for recruitment to
organ primordia, by promotion of cell proliferation, or by prolonging the
duration (window) of cell proliferation during organ growth. Our results
suggest that ERECTA-family genes are most likely to function in the
promotion of cell proliferation. The triple mutations are not likely to
disturb SAM function; even a strikingly tiny leaf of the triple mutant
initiates and increases in size with the same timing as wild type.
Consistently, WUS and STM expression levels are not
significantly altered by the mutation (Fig.
6J). Furthermore, expression of CYCLIN D2, whose
overexpression confers an increase in growth rate by accelerating primordia
initiation in the SAM (Cockcroft et al.,
2000), is not affected in the triple mutant background. It is also
unlikely that ERECTA-family genes prolong duration of cell
proliferation, as erecta erl1 erl2 mutations do not lead to early
cessation of organ growth. Consistently, expression of ANT, which
promotes the meristematic competency of developing organs through prolonged
expression of CYCLIN D (Mizukami
and Fischer, 2000
), is not downregulated by the triple
mutations.
In addition to growth defects, erecta erl1 erl2 plants exhibit
aberrant floral organ differentiation, notably in anthers and ovules. This may
be due to inhibited primordia growth, which results in a diminished supply of
progenitor cells for tissues that differentiate at later stages of flower
development. Alternatively, ERECTA-family genes as a whole may play
some specific roles in flower organ differentiation. In this regard, it is
interesting that ANT, which also specifies organ size but via a
distinct mechanism, is known to be required for proper ovule differentiation
and floral organ identity (Elliott et al.,
1996; Klucher et al.,
1996
; Krizek et al.,
2000
).
In contrast to the main inflorescence, axillary branches of erecta erl1
erl2 plants displayed various degrees of phenotypic rescue
(Fig. 6C). One possibility that
explains such rescue could be that the indirect effects caused by premature
termination of the SAM (the main inflorescence) relieves the growth of
axillary branches via ERECTA-independent mechanisms
(Leyser, 2003). Alternatively,
control of axillary branch development may involve factors that possess
partially redundant function with ERECTA-family receptor-like kinases. Such
factors might be more distantly related receptor-like kinases and/or gene
products with no primary sequence similarity to ERECTA. It is noteworthy that
ERECTA, ERL1 and ERL2 belong to the LRR-XIII family with four additional,
distantly-related members (Shiu and
Bleecker, 2001
). The biological functions of these four members
are not understood.
The increase in cell size in erecta single and erecta erl
double and triple mutants is likely to be secondary to reduction in cell
number. When cell proliferation is decreased, the total mass checkpoint often
leads to decreased inhibition of cell growth, resulting in increased cell size
(Conlon and Raff, 1999;
Day and Lawrence, 2000
;
Mizukami, 2001
;
Nijhout, 2003
;
Potter and Xu, 2001
). The
expression of ERECTA, ERL1 and ERL2 in actively dividing
tissues correlates well with their proposed function in cell proliferations.
Interestingly, a striking decrease in cortex cell numbers occurs only at the
vertical cell files, whereas compensatory cell expansion is much more notable
along the radial axis. As a consequence, erecta single and erecta
erl double mutants develop organs with characteristic shapes that are
shorter but thicker. Therefore, ERECTA-family RLKs may respond to elusive
signals that determine the longitudinal dimension of organ growth.
Alternatively, it is possible that ERECTA-family RLKs possess specific roles
in regulation of cell shape and polarity in addition to cell division.
Remarkably, the cortex cells in erecta-105 erl1-2 and
erecta-105 erl1-2 erl2-1 pedicels are disorganized, with erratic
shape and uneven size (Fig.
6G). The cellular phenotype suggests that ERECTA-family RLKs play
a fundamental role in coordinating cell proliferation within tissues. In this
respect, ERECTA-family RLKs are distinct from the receptor for peptide-hormone
phytosulfokine (PSK), which also encodes an LRR-RLK
(Matsubayashi et al., 2002).
Although the PSK-receptor stimulates rapid, unorganized cell proliferation in
culture cells, ERECTA-family RLKs mediate cell proliferation in the context of
whole organism. Consistent with this hypothesis, ERECTA-family genes
are not highly expressed in Arabidopsis culture cells (C.T.B., E.D.S.
and K.U.T., unpublished).
Although recent studies brought significant insight into the functions of
core cell-cycle regulators in plant growth and development
(Dewitte and Murray, 2003;
Mironov et al., 1999
;
Pardee, 1989
), how neighboring
cells coordinate proliferation remains unclear. Future identification of the
ligands and downstream targets shared by ERECTA-family LRR-RLKs may unravel
the complete picture of the signaling mechanism coordinating cell
proliferation during plant organ morphogenesis.
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ACKNOWLEDGMENTS |
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Footnotes |
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REFERENCES |
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