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OCR for page 7
Colloquium
Maintenance of stem cell populations in plants
Vijay K. Sharma, Cristel Caries, and Jennifer C. Fletcher*
U.S. Department of Agriculture Plant Gene Expression Center and Department of Plant and Microbial Biology, University of California at Berkeley,
800 Buchanan Street, Albany, CA 94710
Flowering plants have the unique ability to produce new organs
continuously, for hundreds of years in some species, from stem cell
populations maintained at their actively growing tips. The shoot
tip is called the shoot apical meristem, and it acts as a self-renewing
source of undifferentiated, pluripotent stem cells whose descen-
dents become incorporated into organ and tissue primordia and
acquire different fates. Stem cell maintenance is an active process,
requiring constant communication between different regions of
the shoot apical meristem to coordinate loss of stem cells from the
meristem through differentiation with their replacement through
cell division. Stem cell research in model plant systems is facilitated
by the fact that mutants with altered meristem cell identity or
accumulation are viable' allowing dissection of stem cell behavior
by using genetic, molecular, and biochemical methods. Such stud-
ies have determined that in the model plant Arabidopsis thaliana
stem cell maintenance information flows via a signal transduction
pathway that is established during embryogenesis and maintained
throughout the life cycle. Signaling through this pathway results in
the generation of a spatial feedback loop, involving both positive
and negative interactions, that maintains stem cell homeostasis.
Stem cell activity during reproductive development is terminated
by a temporal feedback loop involving both stem cell maintenance
genes and a phase-specific flower patterning gene. Our current
investigations provide additional insights into the molecular mech-
anisms that regulate stem cell activity in higher plants.
A major focus of present-day biological research is on the
question of how stem cell fate is controlled during devel-
opment. Plants make excellent model systems for studying this
problem, because, unlike animals, plants maintain a reservoir of
stem cells in their shoot and root apical meristems throughout
their life spans. Thus plants provide an essentially endless supply
of stem cells for study. Like animal stem cells, plant stem cells
are self-renewing and have the potential to form many different
organs and tissues. Stem cells in the shoot apical meristem
(SAM) are the progenitors of all cells that make up stems, leaves,
branches, and flowers. The root apical meristem is the source
of all of the cells of the primary and lateral root system.
Determining how stem cells are established and maintained
in apical meristems is fundamental both for addressing the
basic biological features of stem cell behavior and understanding
many aspects of plant development. This article will discuss
recent progress in our laboratory, and in numerous other
laboratories, in unraveling the genetic regulatory mechanisms
that maintain stem cell fate in the SAM of the model plant
Arabidopsis thaliana.
Although mature plants have an enormous variety of forms,
embryonic plant architecture is sufficiently limited that it is
difficult to distinguish between embryos from different plant
species based solely on morphological criteria. Likewise, a
mature Arabidopsis embryo has a morphologically simple struc-
ture. It consists of an embryonic stem, two embryonic seed leaves
called cotyledons, and a root apical meristem and a SAM at the
basal and apical poles of the embryo, respectively. Because
plants do not use cell migration or programmed cell death to
www. pnas.org/cg i/doi/ 10.1 073/pnas. 1834206100
achieve morphogenesis, the patterning of the embryo occurs
mainly through symmetric and asymmetric cell divisions in
different places and planes and through cell expansion (1~. Plants
also do not set aside a dedicated germ line during embryogen-
esis; instead, germ cells form independently during the genera-
tion of each flower from the SAM.
The elaboration of plant architecture occurs mainly postem-
bryonically. After germination, the Arabidopsis SAM begins to
initiate vegetative organs, the leaves, that form a basal rosette.
After several weeks of vegetative development the plant under-
goes the transition to flowering, during which the stem elongates
and begins to produce secondary branches and flowers. The
primary SAM at this stage is called the inflorescence, or
flower-bearing, meristem. Flowers are formed from floral mer-
istems, which are derived from the flanks of the SAM. Floral
meristems have the same general structure and organization as
SAMs. Each floral meristem produces a flower consisting of four
types of floral organs: sepals, petals, stamens (male reproductive
organs), and carpers, the latter of which fuse to form the
gynoecium (the fruit) that encloses the seeds of the next
generation. One important difference between a SAM and a
floral meristem is that a SAM grows indefinitely and produces
an unspecified number of lateral organs, whereas a f loral
meristem produces a specified number of organs and terminates
in the formation of the central carpers.
The Arabidopsis SAM is patterned gradually during embryo-
genesis (2), culminating in the formation of a highly organized
structure with overlapping functional domains (Fig. 1~. The
self-renewing stem cell pool is confined to the most apical,
central portion of the meristem (34. Infrequent cell divisions in
this region, which is called the central zone, causes displacement
of daughter cells outward into the peripheral region, where they
begin to divide more frequently (44. These more rapidly dividing
peripheral zone cells begin to undergo differentiation and
become incorporated into organ primordia on the meristem
flanks. Cells in the central zone also divide downward into the
interior of the meristem, a region called the rib zone that
contributes to the meristem pith.
The stem cell population consists of three clonally distinct cell
layers (Fig. 1), as does the peripheral meristem region and each
lateral organ primordium (3, 5~. The epidermal layer, the L1,
forms a single cell layer that remains distinct from the other
layers because the cell divisions within it are always perpendic-
ular to the plane of the meristem surface (anticlinal). The
subepidermal L2 layer is also a single cell thick and divides
exclusively in the anticlinal plane. The underlying L3 consists of
This paper results from the Arthur M. Sackler Colioquium of the National Academy of
Sciences, "Regenerative Meclicine," held October 18-22, 2002, at the Arnoid ancl Mabei
Beckman Center of the Nationai Academies of Science ancl Engineering in irvine, CA.
Abbreviations: SAM, shoot apical meristem; bRR, ieucine-rich repeat; ERR-REK, ERR recep-
tor-iike kinase; KAPP, kinase-associated protein phosphatase; MAPK, mitogen-activated
protein kinase; CLV, Clavata; WUS, Wuschel; AG, Agamous; LFY, Leafy; ULT, Ultrapetala.
*To whom correspondence should be addressed. E-mail: fletcher~?nature.berkeley.edu.
@) 2003 by The National Academy of Sciences of the USA
PNAS 1 September 30, 2003 1 vol. 100 1 suppl. 1 1 11823-11829
OCR for page 8
Fig. 1. Organization of the Arabidopsis SAM. Confocal laser scanning mi-
croscope optical section through a WT flowering SAM and developing floral
meristem. The section shows the cell layers (L1, L2, and L3) and the histolog-
ically defined domains. The central zone (CZ) lies at the apex of the SAM and
harbors the stem cell reservoir. The surrounding peripheral zone (PZ) consists
of progenitor cells for lateral organs, and the underlying rib zone (RZ) consists
of progenitor cells for the core of the stem. The SAM has been stained with
propidium iodide to visualize the cell nuclei. [Modified with permission from
ref. 33 (Copyright 2000, Elsevier).]
all of the cells in the interior of the SAM, which divide in all
planes. Yet despite the fact that cell divisions in the SAM are
highly regular, mosaic analyses have demonstrated that, beyond
the general maintenance of the clonal layers, cell lineage pat-
terns are not fixed (6-84. Instead, the cells in the SAM rely
almost entirely on positional cues to determine their correct fates
(9), and cell fate and cell proliferation information must be
communicated both within and between the different meristem
layers for proper development to occur. Thus, as in animals, the
local environment of the plant meristem provides a niche for the
maintenance of the stem cell population.
Mutations That Alter Stem Cell Fate
Our entree into understanding the mechanism of plant stem cell
maintenance has come from the analysis of Arabidopsis mutants
that affect this process. Remarkably, loss-of-function
shootmeristemless (stm) mutants, which lack a SAM and there-
fore contain no stem cells, survive embryogenesis and germinate
normally (10), although they fail to produce any postembryonic
lateral organs. Plants carrying loss-of-function mutations at the
WUSCHEL (WUS) locus form an embryonic SAM, but it is not
maintained after the production of a few lateral organs (11~.
These lateral organs often initiate ectopically from the center of
the SAM, indicating that the stem cells at the shoot apex do not
maintain their correct fate. New SAMs are established de novo
at the base of the was mutant cotyledons and leaves, but these
meristems also terminate prematurely. This stop-and-start
growth pattern, which culminates in a bushy or tousled-looking
(wuschel in German) plant, continues through the floral phase.
was mutant inflorescence meristems produce a reduced number
of flowers compared with WT plants, and most of the flowers
terminate prematurely in a single stamen. Thus the was mutant
phenotypes define a gene whose WT function is required to
promote shoot and floral meristem activity by maintaining the
central stem cell reservoir.
WZJS encodes a novel subtype of homeodomain protein that
is nuclear localized and predicted to act as a transcription factor
(124. WUS mRNA is first detected when embryos reach the
16-cell stage. At this time, WUS expression is confined to the four
11824 1 www.pnas.org/cgi/doi/ 10.1 073/pnas. 1834206100
inner cells of the apical region. As embryogenesis proceeds,
asymmetric cell divisions lead to an asymmetric distribution of
WUS transcripts and the gradual localization of the WUS ex-
pression domain to the internal layers of the developing SAM.
WUS mRNA is confined to a small group of cells in the internal
layers of shoot, and later, floral meristems, and is not detected
in either the epidermal or subepidermal layers of these tissues.
The WZJS expression domain is maintained by the activity of the
FAS CIA TED1 (FAST) and FAS2 genes, which encode compo-
nents of chromatin assembly factor-1 (134. FAST and FAS2 may
therefore promote stable WZJS gene transcription by facilitating
the appropriate chromatin conformation.
Arabidopsis mutants with phenotypes that are opposite those
of was phenotypes have also been characterized. Plants carrying
loss-of-function mutations at the CLAVATA1 (CLV1), CLV2, or
CLV3 loci generate many excess SAM cells and form enlarged
meristems that grow as a ring or a strap instead of as a point.
From this phenotype we infer that the WT function of the three
CLV genes is to restrict meristem cell accumulation. This
requirement for CLVgene activity begins during embryogenesis,
because the SAMs of mature clvl and clv3 embryos are already
significantly enlarged relative to the SAMs of WT embryos and
contain more stem cells (14, 15~. Excess stem cells continue to
accumulate throughout the life of clv mutant plants, so that by
the transition to flowering clv shoot meristems are greatly
enlarged and produce many more floral meristems than WT
plants.
The floral meristems produced by clv mutants are also en-
larged compared with the WT and contain excess stem cells. The
enlarged floral meristems lead to an increase in the number of
organs in clv flowers. WT flowers are arranged into four
concentric rings of organs, called whorls. In Arabidopsis, organ
number in each whorl is nearly invariant: the vast majority of WT
flowers consist of four sepals, four petals, five to six stamens, and
two fused carpers (16~. Flowers of clv null mutants contain many
additional organs, including up to seven or eight carpers that fuse
to form a club-shaped fruit. It is from this phenotype that the
mutants are named "clavata," from the Latin term "clavatus"
meaning club-like. In addition to generating extra organs, the
floral meristems of clvl and clv3 null mutants often contain a
mass of proliferating stem cells that erupt through the gyno-
ecium. This phenotype is further evidence that the CLVloci are
required to prevent unrestricted stem cell amplification in the
center of the floral meristem.
Genetic analysis has revealed that the three CLV genes and
WUS interact to control stem cell fate during development. clv1
and clv3 null mutants have nearly indistinguishable phenotypes,
and clv1 clv3 double mutants appear identical to either single
mutant (15~. Further, plants that are doubly heterozygous for
clv1 and clv3 alleles display an enlarged meristem phenotype,
indicating that each gene is sensitive to the dose of the other. clv2
null mutants have slightly weaker shoot and floral meristem
phenotypes than clv1 and clv3 null mutants, and clv1 and clv3 are
epistatic to clv2 with respect to those traits (17~. However, clv2
mutants, unlike clv1 and clv3 mutants, also display phenotypes in
nonmeristematic tissues, indicating that CLV2 functions more
widely in development. Double mutants generated between was
null alleles and any clv allele have a was mutant phenotype (11),
revealing that was is completely epistatic to clv1, clv2, and clv3.
Thus all four genes act in the same genetic pathway to maintain
the appropriate amount of stem cell accumulation in shoot and
floral meristems.
A Short-Range Signaling Pathway Maintains Stem
Cell Homeostasis
All three CLV genes have been cloned and found to encode
components of a meristem signal transduction pathway. CLV1
encodes a leucine-rich repeat (LRR) receptor-like kinase (LRR-
Sharma et al.
OCR for page 9
RLK) (18), one of 216 in theArabidopsis genome (19~. Members
of this family contain multiple tandem extracellular LRRs, a
transmembrane domain, and an intracellular serine/threonine
kinase domain. The LRR-RLK family, which consists of 13
subclasses, is the largest class of receptor kineses in plants. The
LRR-RLKs and other Arabidopsis RLKs form a monophyletic
family that groups with Drosophila Pelle and other Pelle-like
cytoplasmic kineses in phylogenetic analyses (19~. Based on the
fact that LRRs are considered to be protein-binding motifs, and
that LRR-containing proteins in plants and animals are involved
in protein-protein interactions and signal transduction (20), it
has been proposed that the extracellular LRRs of the LRR-
RLKs bind protein or peptide ligands. The functions of a
half-dozen Arabidopsis LRR-RLKs have been defined to date.
They play roles in various plant signaling pathways, including
those regulating different aspects of development (21-23), hor-
mone signaling (24), and disease resistance (25~.
CLV2 and CLV3 also encode members of large, plant-specific
gene families. CLV2 encodes a receptor-like protein with 21
extracellular LRRs, a transmembrane domain, and a short
cytoplasmic tail (264. There are ~30 CLV2-like genes in the
Arabidopsis genome, but their functions are unknown. CLV3
encodes a 96-aa predicted extracellular polypeptide (27) that is
a founding member of the CLV3/ESR-related (CLE) protein
family (28~. The members of this family, which are found in many
distantly related plant species, share common sequence elements
including an amino-terminal signal peptide and a 14-aa region of
homology at the carboxyl terminus. Two dozen CLE genes have
been identified in Arabidopsis (28, 29~. These genes are ex-
pressed in a wide range of tissues during development (29) and
may function as ligands for RLKs in many plant signal trans-
duction pathways.
The expression patterns of the CLV3 and CLV1 genes revealed
important clues to their function. We found that, beginning
during embryogenesis and continuing throughout the life of the
plant, CLV3 marks the stem cell population in the SAM (27~.
CLV3 transcripts are detected predominantly in the superficial
L1 and L2 cell layers at the apex of the meristem and in only a
few underlying L3 cells. CLV3 mRNA ~s not found in peripheral
meristem cells, mature stem tissues, or lateral organs. CLV3 is
also expressed in the stem cell populations of all axillary shoot
meristems and floral meristems. In floral meristems, CLV3 is
detected until the stem cell population is consumed in the
formation of the carper primordia (see below), when CLV3
transcripts disappear from the flower. CLV1 mRNA is also
induced during embryogenesis, at the same time as CLV3, and
is likewise restricted to shoot and floral meristem cells (184.
However, CLV1 transcripts are found predominantly in the L3
cells of the meristem, largely beneath the CLV3 expression
domain, and are not detected in the L1 epidermal layer. Thus
CLV1-expressing cells and CLV3-expressing cells are neighbors,
suggesting that the two meristem domains may communicate
with one another through a CLV3/CLV1 signal transduction
pathway. CLV2 is also expressed in shoots and flowers based on
RNA gel blot analysis (26), but the precise CLV2 expression
domain in meristems has not been reported.
Important inroads have been made into determining the
mechanism of CLV signal transduction. There is now solid
biochemical evidence that CLV1 and CLV2 are bound together
in a signaling complex (Fig. 2~. The CLV1 kinase domain is
capable of both autophosphorylation and transphosphorylation
on serine residues, indicating that CLV1 is a bona fide receptor
kinase (30, 314. CLV1, a predicted 105-kDa protein, is detected
in size-fractionated meristem extracts in two different com-
plexes: a 185-kDa complex and a 450-kDa complex (32~. CLV1
and CLV3 are both components of the 450-kDa complex,
whereas CLV3 is not detected in the 185-kDa complex. The
450-kDa complex does not form in clv3 mutants or clvl mutants
Sharma et a/.
GTP /~\
GDP?
CLV3
MAPKs? _
\~
/ ~.... ...
_
~_
Fig. 2. Schematic of the CLV signaling complex. The CLV1 LRR-RLK forms a
heteromeric complex with the CLV2 receptor-like protein at the plasma
membrane of interior SAM cells. Binding of the CLV3 polypeptide, possibly in
association with another protein (X), is proposed to stimulate assembly of an
active signaling complex that also contains a phosphatase (KAPP) and a
Rho-like GTPase (Rop). The signal is relayed from the cytosol to the nucleus,
potentially via a MAPK cascade, to limit WUS expression. P, phosphorylation
site; SS, disulphide bond. [Reprinted with permission from ref. 68 (Copyright
2002, Annual Reviews, www.annualreviews.org).]
that lack an active kinase domain. The 450-kDa fraction is
therefore likely to represent the active CLV receptor signaling
complex, whereas the 185-kDa complex is inactive. Although
specific antibodies for CLV2 have not yet been reported, its
presence in the 450-kDa complex is inferred from the observa-
tion that this complex does not form in clv2 mutants although
high levels of CLV1 and CLV3 transcripts are present (26~.
Mutations in CLV2 also reduce the accumulation of the 185-kDa
complex, suggesting that CLV2 may also be a subunit of the
inactive complex that forms a disulphide-linked heterodimer
with CLV1.
The active CLV signaling complex contains several additional
cytosolic proteins. One is a type 2C kinase-associated protein
phosphatase (KAPP), which interacts with several different
LRR-RLKs (25, 34, 35~. KAPP is expressed in Arabidopsis
meristems in a broad region that encompasses the CLV1 ex-
pression domain (30), and KAPP protein binds to and dephos-
phorylates the phosphorylated form of the CLV1 kinase domain
(30, 31~. KAPP binds to the CLV1 kinase domain through its
forkhead-associated (FHA) domain, and site-directed mutagen-
esis of conserved FHA residues abolishes the ability of KAPP to
interact with RLKs (364. KAPP overexpression phenotypes
mimic those of weak loss-of-function clv alleles (30), whereas
reduction in KAPP transcription levels can suppress the clvl
mutant phenotype (31~. These data are consistent with KAPP
acting as a negative regulator of CLV signaling by dephospho-
PNAS I September 30, 2003 I vof. 100 | suppl. ~ | 11825
OCR for page 10
rylating CLV1. AtSERK1, a plasma membrane-localized LRR-
RLK, was recently shown to become sequestered in intracellular
vesicles when transiently coexpressed with KAPP (354. KAPP
dephosphorylation of threonine residues in the AtSERK1 kinase
domain A loop was found to play an active role in the receptor
internalization. These results suggest that KAPP is an integral
part of a endocytosis mechanism that internalizes AtSERK1, and
possibly other LRR-RLKs such as CLV1, during receptor-
mediated signaling.
An additional cytoplasmic component of the active CLV
complex, identified through coimmunoprecipitation experi-
ments (32), is a member of the plant-specific Rop subfamily of
Rho GTPases (374. Members of the Rho GTPase family, which
includes CDC42, Rac, and Rho, are key cytosolic components of
cell surface-receptor signaling cascades that regulate cytoskel-
etal organization, polarized cell growth, and many other cellular
processes in animals and fungi (38-40~. Arabidopsis contains at
least 10 Rop GTPases (41, 42), some Which have defined roles
in mediating tip growth in pollen tubes and root hairs (37, 43)
and others of which act in various developmental processes (44~.
It is not yet clear which Rop proteins participates in CLV
signaling, because the antibody used in the biochemical exper-
iments cross-reacts with multiple Rop isoforms (324.
How is the signal relayed downstream of the CLV complex?
Although the exact mechanism is currently unknown, a model
has been proposed based on precedents in other systems (32~.
Rho/Rac GTPases are members of the Ras superfamily of
cytoplasmic signal transduction molecules, many of which relay
signals through a mitogen-activated protein kinase (MAPK)
pathway. The Arabidopsis genome contains multiple genes en-
coding MAPK, MAPKK, and MAPKKK components of this
pathway (454. A complete MAPK cascade has recently been
reported (46) to function downstream of theArabidopsis flagellin
receptor FLS2, which encodes an LRR-RLK (25), in innate
immune responses. Based on this paradigm, once the CLV1
kinase domain becomes activated via phosphorylation, it might
associate with and activate the Rop GTPase, which in turn could
activate a MAPK cascade that ultimately leads to changes in
target gene expression. Functionally redundant MAPK signaling
proteins were discovered at several steps in the FLS2 pathway,
and several of the components of the FLS2 cascade may also
function in other pathways (46~. If this is a common theme for
other LRR-RLK signaling pathways, it could account for the
lack of MAPK cascade mutants identified to date in the CLV
signaling pathway.
What is the biological basis for signaling through the CLV
complex in meristematic cells? Genetic and molecular evidence
has led to the conclusion that the CLV signaling pathway is part
of a negative feedback loop that controls stem cell fate in the
Arab~dopsis shoot apex. Our first insight came from an experi-
ment designed to determine what happens to CLV3 expression
in the absence of the CLV1 receptor kinase. Because clvl mutant
phenotypes are very similar to clv3 mutant phenotypes, we tested
the possibility that the clvl phenotypes might be caused by a
reduction in either the domain or level of CLV3 expression. In
contrast to our prediction, we observed that CLV3 was expressed
at very high levels in a greatly enlarged domain in clvl SAMs
(274. This result indicates that CLV3 expression is not reduced
in rIvl mutant plants, but instear1 that (~! V7 normaliv acts to
restrict the number of CLV3-expressing cells in WT meristems.
Similarly, the CLV1 expression domain is enlarged in clv3 mutant
plants. In fact, in clvZ, clv2, or clv3 mutants the expression
domains of CLV1 and CLV3 enlarge coordinately (17, 27i. From
these data we inferred the presence of two opposing stem cell
regulatory pathways. The expansion of the CLV expression
domains would be normally controlled by a positive, stem
cell-promoting pathway, which in turn would be negatively
regulated by the stem cell-restricting CLV pathway. The stem
11826 1 www.pnas.org/cgi/doi/10.1 073/pnas.18342061 00
Sharma et a/.
i
OCR for page 11
regulation of WUS and complete loss of stem cells. Disruption
of the negative pathway in clv mutants causes the WUS expres-
sion domain to expand laterally and upward into the subepider-
mal cell layer. Activity of the positive pathway, mediated by
WUS, promotes expression of CLV3 and maintenance of the
stem cell domain. This mutual regulation, involving both positive
and negative interactions, provides a feedback system required
to maintain an appropriately sized stem cell reservoir throughout
Arabidopsis development.
This negative feedback loop elegantly compensates for the
departure of cells from the meristem during organ formation. As
stem cell daughters enter the transition zone and become
incorporated into organ primordia, the decrease in stem cell
number leads to a reduction in the level of the CLV3 signal. The
drop in negative signaling through the CLV pathway causes the
expansion of the WUS-expressing cell population. This, in turn,
triggers the amplification of the stem cell population via the stem
cell-promoting pathway, until the level of CLV3 produced by the
stem cells rises sufficiently to restrict further expansion of the
WUS domain and equilibrium is once again attained.
Signaling Via CLV3
One major question to be addressed is how the CLV1/CLV2
receptor complex is activated by CLV3. Based on its sequence,
CLV3 is predicted to be exported through the secretory pathway
to the extracellular space by the default pathway for soluble plant
proteins. CLV3 has been shown to act in a cell nonautonomous
fashion (27), but the expression data do not exclude the possi-
bility that activation of the CLV pathway occurs intracellularly
within those few cells in the interior of the SAM that express
both CLV3 and the receptor complex. In collaboration with
Enrique Rojo, Valentina Kovaleva, and Natasha Raikhel at the
University of California, Riverside, we devised several assays to
determine whether the CLV3 protein is secreted and whether
secretion is required for its function in vivo (49~.
To test the prediction that the CLV3 protein is secreted, we
used two translational fusions of CLV3 to the amino terminus of
,(3-glucuror~idase, one full length and the other lacking the
putative signal peptide, in transient subcellular localization
assays. The fusion protein lacking the signal peptide was de-
tected in the cytoplasm, whereas the full-length CLV3-GUS
fusion protein was detected only in the extracellular space. Thus
the CLV3 protein is exported through the secretory pathway,
and the presence of its signal peptide is required for its extra-
cellular localization. Additional transient assays, in which full-
length CLV3-GUS was cobombarded together with a nuclear-
localized protein as a marker for transformed cells, show that
CLV3 is capable of moving beyond the cells in which it is made
(V.K.S. and J.C.F., unpublished data). We confirmed that these
results were relevant in viva by overexpressing full-length and
truncated CLV3-GFP fusion proteins in clv3 mutant plants. We
found that plants transformed with the full-length
35S::CLV3-GFP fusion protein rescued the clv3 mutant pheno-
type and showed GFP florescence only in the extracellular space.
Plants transformed with the truncated version of CLV3 fused to
GFP did not rescue the clv3 phenotype and showed GFP
florescence only in the cytoplasm. Based on these data, we
conclude that CLV3 is a secreted protein both in vitro and in
planta.
Next, we asked whether CLV3 secretion is essential for its
activity in shoot and floral meristems. We tested whether
preventing the CLV3 protein from reaching the cell surface
would block its function in restricting stem cell accumulation
(Fig. 3~. Overexpression constructs were generated in which
CLV3 was tagged with a vacuolar sorting signal to direct the
protein to the vacuole rather than the extracellular space (Vac
constructs). We also generated several overexpression con-
structs in which CLV3 was tagged with a mutated vacuolar
Sharma et a/.
B
.~1
V:~rS>
\/ar:?
CM CO ~ CM
C~ C~ ~ C~ ~ C:
c' ~ a~
u~ a~ co > > >
Fig. 3. Targeting to the vacuole blocks the activity of CLV3. (A) Scheme of the
fusion constructs. The three Vac constructs contain fusions of CLV3 to the
C-terminal vacuolar sorting signal from barley lectin (ctVSSBL) or tobacco
chitinase A (ctVSSCH). The ctVSSBL with two additional Gly residues (GG),
which no longer functions as a vacuolar-sorting signal, was attached at the
carboxyl terminus of CLV3 (Sec1) or CLV3-T7 (Sec2). Sec3 contains the full-
length CLV3 protein with no additional tag. (B) c/v3-2 plants transformed with
the fusion constructs. Primary transformants were grouped into six pheno-
typic classes according to the severity of the meristem phenotype. Class 1
plants showed no transgene activity and resembled untransformed c/v3-2
plants. Class 6 plants showed the strongest transgene activity and exhibited a
gain-of-function phenotype in which the SAM of each plant terminated
prematurely before flowering. Plants in the other classes fell between these
two extremes. The graph at the bottom shows for each construct the percent-
age of transformed plants recovered in each phenotypic class. (C) WT plants
transformed with the fusion constructs. Primary transformants were grouped
into classes 3, 4, 5, and 6. The graph at the bottom shows for each constructthe
percentage of transformed plants recovered in each phenotypic class. [Re-
printed with permission from ref. 49 (Copyright 2002, American Society of
Plant Biologists).]
sorting signal that forces the protein into the secretory pathway
(See constructs). When these constructs were ir~troduced into
clv3 null mutant or WT plants, several phenotypic classes were
distinguished. Most of the Sec-containing plants showed com-
plete rescue of the clv3 phenotype or had a gain-of-function
phenotype, whereas the majority of Vac-containing plants had
little or no rescue of the phenotype. A few Vac-containing plants
showed a gain-of-function phenotype, which could be attributed
to saturation of the vacuolar sorting pathway by high levels of
CLV3.
To localize the CLV3 protein in the different transgenic lines,
we performed immunogold labeling with an antibody-directed,
T7-tagged CLV3 protein. CLV3 protein was detected in the cell
wall of Sec-containing plants, but not in Vac-containing plants
that failed to rescue the clv3 phenotype. CLV3 was also detected
in the cell wall of Vac-containing plants that displayed a gain-
of-function phenotype. This observation confirmed that the
vacuolar sorting pathway became saturated in these lines, and
thus the excess CLV3 protein was secreted and able to interact
with the CLV1 complex. Therefore we conclude from this work
that CLV3 functions as a secreted protein that activates the CLV
stem cell signaling pathway in the extracellular space.
A Temporal Feedback Loop Terminates Stem Cell Activity
in Flowers
Flowers are produced from floral meristems, which are derived
from the SAM and likewise harbor a stem cell reservoir that
PNAS I September 30, 2003 I vol. 100 | suppl. ~ | 11827
OCR for page 12
provides cells for the generation of the floral organs. Each floral
meristem forms as a primordium on the flank of the SAM. As
it grows the floral meristem initiates three concentric rings of
organs (sepals, petals, and stamens) and then the stem cells are
consumed in the formation of the carpers that comprise the
central gynoecium. Thus unlike SAMs, in which the stem cell
population is maintained indefinitely to provide progeny cells for
continuous organogenesis, floral meristems ultimately terminate
stem cell production to permit the female reproductive organs to
differentiate in the center of the flower.
Floral meristem activity is regulated by several overlapping
sets of regulatory factors. The CLV signaling pathway is active
at the floral meristem apex until carper initiation, when CLV1,
CLV3, and WITS all are down-regulated to undetectable levels.
Floral meristem identity is conferred by transcription factors
such as LEAFY (LFY) and APETALA1 (AP1) (50, 51), which
can convert shoot meristems into floral meristems (52, 53~. The
floral meristem identity genes induce the transcription of flower-
specific homeotic genes in overlapping spatial domains of the
floral meristem (54, 55~. Activity of the floral homeotic gene
products in four different combinations then specifies the iden-
tity of the sepals, petals, stamens, and carpers (564.
The floral homeotic gene AGAMOUS (AG) encodes a
MADS-domain transcription factor (57) that plays key roles in
both floral organ identity specification and stem cell termina-
tion. ag mutations cause the transformation of stamens into
petals and replacement of the central carpers with an entirely
new flower (584. The resulting ag flowers consist entirely of
sepals and petals and resemble shoots because they retain a stem
cell population at their apex (59) and continue to produce organs
indefinitely. AG overexpression has the opposite effect: instead
of maintaining indeterminate growth, the SAMs of 35S::AG
plants terminate in a solitary flower (60~. Thus AG is required
to terminate stem cell activity in the center of the developing
flower and is sufficient to convert a shoot meristem into a floral
meristem.
ag mutants and was mutants have opposite flower phenotypes,
and genetic experiments demonstrate that these two genes play
antagonistic roles in regulating floral meristem activity. WUS
expression persists in ag mutant flowers even after the formation
of many organs (59, 613. AG is therefore a negative regulator of
WlJS transcription and, consequently, of stem cell maintenance.
Stem cell termination is restored in ag was double mutant
flowers, which resemble was single mutant flowers, indicating
that the capability of ag flowers to continuously produce new
organs depends on the ectopic activity of WAS. Thus prolonged
WlJS expression is sufficient to permit indefinite floral meristem
activity, and a key role of AG is to down-regulate WUS and
terminate stem cell maintenance in the center of the developing
flower.
The expression of AG is regulated by the floral meristem
identity pathway and also the stem cell signaling pathway (Fig.
4~. AG is initially transcribed in the center of developing floral
meristems in the cells that will ultimately become specified as
stamens and carpers, andAG expression in these organs persists
until the late stages of flower development (57~. AG is directly
activated by LFY (55), which binds to regulatory elements in the
second intron of the gene. However, LFY protein is distributed
throughout the floral meristem (62), and thus does not confer
region-specificAG induction. This function is provided by WUS,
which is expressed in the center of floral meristems in a subset
of cells that eventually expressAG. Although was mutant flowers
do not display homeotic organ transformations they lack all
carpers and most stamens, the organ types that are specified by
AG. In addition, misexpression of WAS in flowers causes the
formation of ectopic stamens and carpers as a consequence of
ectopicAG activation. Recent reports have shown that WUS is
also a direct activator of AG, acting through a homeodomain
11828 1 www.pnas.org/cgi/doi/10.1073/pnas.1834206100
SAM
FM
\~
AG {a '_ . ~
~ (I)
~ LFY WUS
Fig. 4. Temporal feedback loop regulating stem cell termination in deter-
minate floral meristems. (Upper) Schematic of an indeterminate SAM, show-
ing the interaction between CLV3 and WUS in their respective domains (gray
circles). In the SAM, LFY is absent and AG expression is not induced. (Lower)
Schematic of a determinate floral meristem overtime. LFY is present through-
out the young floral meristem. Both LFY and WUS bind to enhancer sequences
and cooperate to induce AG transcription in the center of the developing
flower. At the time of carper (ca) initiation, AG and an additional factor
(a<) repress WUS expression to terminate stem cell activity. [Reprinted
with permission from ret 68 (Copyright 2002, Annual Reviews, www.
annualreviews.org).]
protein consensus binding site in the second intron (59, 61~.
These sites are adjacent to the LFY binding sites, although the
two proteins bind DNA independently (61~. Thus LFY provides
the flower specificity and WUS provides the regional specificity
for AG induction in the central region of floral meristems.
A third gene that appears to be involved in AG activation in
Arabidopsis floral meristems is ULTRAPETALA (ULT). We
have shown that mutations in ULT cause shoot and floral
meristem enlargement, leading to the production of extra flow-
ers from the SAMs and extra floral organs from the floral
meristems (634. tilt mutant floral meristems also produce some
additional organs in the center of the flower before terminating,
reminescent of ag mutants. AG activation is delayed in the very
center of tilt floral meristems (63), whereas overexpression of
ULT leads to premature and ectopic AG induction (C.C. and
J.C.F., unpublished data). Thus ULT, which encodes a member
of a plant-specific family of novel proteins (C.C. and J.C.F.,
unpublished data), is required under normal circumstances for
the correct timing of AG induction in floral meristems.
Thus the termination of stem cell activity in floral meristems
is mediated by a temporal negative feedback loop requiring both
flower patterning and stem cell maintenance genes. As in SAMs,
WUS promotes maintenance of the stem cell domain in floral
meristems to permit the generation of the full complement of
floral organs. In addition, WUS and the flower-specific factor
LFY bind independently to AG regulatory sequences and co-
operate to direct AG transcription in the center of developing
floral meristems, with ULT providing temporal specificity. Once
activated, AG feeds back to repress WUS, thus terminating stem
cell activity and allowing the differentiation of the central cells
into carper primordia. Unlike the other target of WUS induction,
CLV3, AG does not require the constant presence of WUS to
maintain its expression and consequently AG transcription per-
sists even after WUS has been switched off.
Sharma et a/.
OCR for page 13
Conclusions and Final Thoughts
Long-term maintenance of a stem cell population is critical for
the particular developmental habit of plants, that is, contin-
uous organ formation to achieve maximal growth under
constantly changing environmental conditions. The past 10
years have produced a quantum leap in our understanding of
plant stem cell activity from a descriptive to a mechanistic
level. We and others have demonstrated that stem cell fate in
shoot and floral meristems is controlled by a signal transduc-
tion pathway consisting of the CLV receptor complex and the
WUS transcription factor. We have shown that this pathway
consists of both plant-specific proteins and those with simi-
larity to animal proteins, and that activation of the pathway
occurs extracellularly. We have also determined that the
CLV/WUS pathway functions as a homeostatic feedback loop
that elegantly corrects for f luctuations in stem cell number that
occur as a consequence of organogenesis. Finally, studies in
several labs are providing insights into how a temporal nega-
tive feedback loop terminates stem cell activity to permit the
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PNAS 1 September 30, 2003 1 vol. 100 1 suppl. 1 1 11829
Representative terms from entire chapter:
floral meristems
