POPCORN Functions in the Auxin Pathway to Regulate ...POPCORN Functions in the Auxin Pathway to...

POPCORN Functions in the Auxin Pathway to RegulateEmbryonic Body Plan and Meristem Organizationin Arabidopsis W OA

Daoquan Xiang,a,1 Hui Yang,a,1 Prakash Venglat,a,1 Yongguo Cao,a Rui Wen,b Maozhi Ren,a Sandra Stone,a

Edwin Wang,c Hong Wang,b Wei Xiao,b Dolf Weijers,d Thomas Berleth,e Thomas Laux,f Gopalan Selvaraj,a

and Raju Datlaa,2

a Plant Biotechnology Institute, National Research Council Canada, Saskatoon, Saskatchewan S7N 0W9, Canadab University of Saskatchewan, Health Sciences Building, Saskatoon, Saskatchewan S7N 5E5, Canadac Biotechnology Research Institute, National Research Council Canada, Montreal, Quebec H4P 2R2, CanadadWageningen University, Laboratory of Biochemistry, 6703 HA Wageningen, The Netherlandse Department of Botany, University of Toronto, Toronto, Ontario M5S 3B2, Canadaf BIOSS, University of Freiburg, 79104 Freiburg, Germany

The shoot and root apical meristems (SAM and RAM) formed during embryogenesis are crucial for postembryonic plant

development. We report the identification of POPCORN (PCN), a gene required for embryo development and meristem

organization in Arabidopsis thaliana. Map-based cloning revealed that PCN encodes a WD-40 protein expressed both during

embryo development and postembryonically in the SAM and RAM. The two pcn alleles identified in this study are

temperature sensitive, showing defective embryo development when grown at 228C that is rescued when grown at 298C. Inpcn mutants, meristem-specific expression of WUSCHEL (WUS), CLAVATA3, and WUSCHEL-RELATED HOMEOBOX5 is not

maintained; SHOOTMERISTEMLESS, BODENLOS (BDL) and MONOPTEROS (MP) are misexpressed. Several findings link

PCN to auxin signaling and meristem function: ectopic expression of DR5rev:green fluorescent protein (GFP), pBDL:BDL-

GFP, and pMP:MP-b-glucuronidase in the meristem; altered polarity and expression of pPIN1:PIN1-GFP in the apical domain

of the developing embryo; and resistance to auxin in the pcn mutants. The bdl mutation rescued embryo lethality of pcn,

suggesting that improper auxin response is involved in pcn defects. Furthermore, WUS, PINFORMED1, PINOID, and

TOPLESS are dosage sensitive in pcn, suggesting functional interaction. Together, our results suggest that PCN functions

in the auxin pathway, integrating auxin signaling in the organization and maintenance of the SAM and RAM.

INTRODUCTION

Embryogenesis in angiosperms begins with the division of the

zygote into the precursor cells of the embryo proper and the

suspensor. Subsequently, the establishment of the apical-basal

axis, radial organization of tissues, and initiation of cotyledons

and apical meristems together constitute the embryonic body

plan (De Smet et al., 2010). In recent years, Arabidopsis thaliana

has been investigated as a model plant to address the functions

of key genetic factors that contribute to embryogenesis. Auxin

transport and signaling play critical roles in the establishment of

embryonic body plan (Friml et al., 2003; Jenik et al., 2007). During

the transition from globular to heart embryo, the apical domain is

partitioned to form the shoot apical meristem (SAM) and the

cotyledons, whereas the basal domain differentiates into the

hypocotyl and the embryonic root apical meristem (RAM).

Altered auxin signaling can result in apical patterning de-

fects with cotyledons that are either fused, as seen in the long

hypocotyl5 and hy5 homolog double mutant (Sibout et al., 2006),

or completely absent, as seen in the auxin response factor (ARF)

double mutantmonopteros (mp) and nonphototropic hypocotyl4

(Hardtke et al., 2004). The gnom mutant, which is affected in

auxin transport, fails to establish apical-basal patterning, result-

ing in a ball-like embryo with no cotyledons (Mayer et al., 1993).

The products of PINFORMED (PIN) gene family members PIN1,

PIN3,PIN4, andPIN7 are differentially localized in a polarmanner

to coordinate the transport of auxin during embryo patterning,

and quadruple mutants of all four genes display filamentous

embryos with apical-basal polarity defects (Friml et al., 2003).

Polar targeting of PIN proteins in cells is regulated via phosphor-

ylation by the kinase PINOID (PID) (Michniewicz et al., 2007). The

CUP-SHAPED COTYLEDON (CUC) genes are involved in the

partitioning of the apical domain of the embryo. In the cuc1 cuc2

double mutant, the lack of or a defect in the SAM, along with

fused cotyledons, results in the termination of seedling develop-

ment (Aida et al., 1997). CUC overexpression occurs in the pin-1

pid-2 double mutant, leading to the inhibition of cotyledon

1 These authors contributed equally to this work.2 Address correspondence to [email protected] author responsible for distribution of materials integral to thefindings presented in this article in accordance with the policy describedin the Instructions for Authors (www.plantcell.org) is: Raju Datla ([email protected]).WOnline version contains Web-only data.OAOpen Access articles can be viewed online without a subscription.www.plantcell.org/cgi/doi/10.1105/tpc.111.091777

The Plant Cell, Vol. 23: 4348–4367, December 2011, www.plantcell.org ã NRC Canada 2011

primordia initiation (Furutani et al., 2004). Furthermore, CUC

genes have been shown to be essential for the expression of

SHOOTMERISTEMLESS (STM) (Aida et al., 1999).

In the root, auxin has been shown to play major roles in RAM

establishment and function (Overvoorde et al., 2010). The auxin

response regulators BODENLOS (BDL) and MP mediate auxin

signaling and provide root patterning information by activating

the PLETHORA (PLT) genes (Hamann et al., 2002; Nawy et al.,

2008). The two PLT genes provide positional information to

establish the root stem cell niche through the auxin pathway

(Aida et al., 2004). The homeobox gene WUSCHEL-RELATED

HOMEOBOX5 (WOX5) is expressed specifically in the quiescent

center (QC) of the root, and wox5 mutants fail to maintain stem

cells in the RAM (Sarkar et al., 2007). Auxin and cytokinin

together play antagonistic roles in the SAM and RAM partly

by regulating the expression of ARABIDOPSIS RESPONSE

REGULATOR7 (ARR7) and ARR15 (Zhao et al., 2010).

Proper organization and maintenance of both meristems de-

pends on coordinated cell divisions that balance the number of

stem cells and daughter cells that contribute to the initiation of

organs. A number of transcription and signaling factors have

been implicated in the functions of shoot and root meristems.

These include homeobox genes STM and WUSCHEL (WUS)

(Mayer et al., 1998; Lenhard et al., 2002), CLAVATA signaling

factors (CLV1, CLV2, and CLV3) in the SAM (Fletcher et al., 1999;

Schoof et al., 2000), and homeobox gene WOX5 and plant-

specific AP2 domain-containing PLT transcriptional factors in

the RAM (Aida et al., 2004; Sarkar et al., 2007).

Despite the number of meristem regulatory genes that have

been identified thus far, mutations in the majority of these genes

(i.e., WUS, CLV, and STM) do not result in embryo lethality. This

implies that, in addition to the possible existence of unknown

genes that function both in the embryo patterning and meristem

organization, there is a significant overlap and/or redundancy in

the functions of these known genes. In this study, we report the

identification of Arabidopsis POPCORN (PCN), which is involved

in both embryo and meristem development. PCN encodes a

putative WD-40 protein, and mutations in PCN perturb auxin

polarity resulting in ectopic auxin maxima in the SAM. We show

genetic interactions of PCN with WUS, BDL, MP, TOPLESS

(TPL), PIN1, and PID affecting the embryonic and postembryonic

development in Arabidopsis.

RESULTS

Map-Based Cloning of PCN, Which Encodes

a WD-40–Related Protein

We have determined global gene expression during embryo

development in Arabidopsis (Xiang et al., 2011). These data sets

provide a resource for further genetic interrogation. Genetic

screens and targeted reverse genetic approaches have been

successful in identifying key genes in Arabidopsis embryo de-

velopment. To identify genes that are required for Arabidopsis

embryogenesis, we analyzed T-DNA lines with insertions in

selected candidate genes that are specifically or differentially

expressed during zygote to globular stages of embryo develop-

ment based on our global gene expression data sets (Xiang et al.,

2011). One of the lines from this screen showed early embryo

lethality among the segregating embryos with a phenotype

resembling poppedmaize (Zeamays), which we named popcorn

(pcn). The pcnmutation segregated as a single, recessive allele.

However, molecular analysis revealed that this T-DNA insertion

was not linked to the mutant embryo phenotype.

We therefore used a map-based cloning approach to isolate

the PCN gene. A tight linkage was observed with cleaved am-

plified polymorphic sequences (CAPS) markers at the genomic

positions 4,144,848 (SspI enzyme digestion) and 4,369,443 (RsaI

enzyme digestion) with no recombination observed in the anal-

ysis of 1340 chromosomes (670plants). The strongglobular stage

defects in pcn mutant suggest that the expression of gene(s) in

this locus is likely critical for embryo patterning and develop-

ment. We reasoned that the expressed genes in this genomic

region would likely include PCN. The 224-kb region between

4,144,848 and 4,369,443 on chromosome 4 contains 53 genes of

which 44 encode transposable elements. Of the remaining nine

loci, five are of unknown function and the other four are anno-

tated as F-box (At4g07400), WD-40 (At4g07410), PQ loop repeat

(At4g07390), and GTP binding (At4g07524).

Because TPL is also aWD-40 repeat–containing protein that is

critical for embryo patterning that involves specification of the

apical-basal polarity and cotyledon formation (Long et al., 2006),

we predicted that At4g07410 may be associated with the de-

fective phenotype. At4g07410 also had the highest expression

level among the 53 genes in the deduced 224-kb chromosome

4 region in our microarray data for wild-type globular embryos

(Xiang et al., 2011). Sequencing of candidate genes in the

mapped region of pcn identified a 35-bp deletion overlapping

the 5th exon and 5th intron of At4g07410 genic region that would

result in a premature stop codon of the predicted open reading

frame (Figure 1A). At4g07410 encodes an 815–amino acid protein

containing eight WD-40 repeats as determined by InterProScan

(Zdobnov and Apweiler, 2001), six at the N terminus and two at

the C terminus (Figure 1B). The premature stop codon in pcn-1

was deduced to produce a truncated protein of 240 amino acids.

In Arabidopsis, PCN, a single-copy gene, shares 69% identity

with At1g27470 at the amino acid level. Putative homologs of

PCN are also present in other plant genera (Oryza, Sorghum,

Populus, Ricinus, and Vitis), yeast, zebra fish, and humans (see

Supplemental Figure 1 and Supplemental Data Set 1 online). In

none of these cases is any function known.

Using the At4g07410 sequence information, we screened for

additional T-DNA insertional lines at this locus and identified a

second allele of PCN, pcn-2 (Salk_022607), with an insertion in

the 8th exon (Figure 1A). The pcn-2/+ plants segregated defec-

tive embryos similar in mutant phenotype to those observed in

pcn-1. Heterozygous pcn-1/+ or pcn-2/+ plants did not exhibit

any developmental defects other than producing 25% arrested

and 75%normal embryos (n= 1028 for pcn-1, n = 1120 for pcn-2),

indicating that pcn-1 and pcn-2 mutations are recessive. To

confirm that the mutant embryo phenotypes observed in pcn-1

and pcn-2 are caused by the lesions in the At4g07410 locus, we

introduced two constructs into heterozygous mutant pcn-1 and

pcn-2 plants. The first construct contained the At4g07410 ge-

nomic fragment, and the secondwas thePCN cDNA (representing

PCN Regulates Embryo and Meristem 4349

the reading frame of 815 amino acids) under the control of the

putative PCN promoter. Transformation of both constructs

fully complemented the homozygous pcn mutant embryo phe-

notype. These observations confirmed that PCN, a WD-40

repeat–containing protein, is required for embryo development

in Arabidopsis.

Because the other PCN homolog, At1g27470, is expressed

during embryo development in Arabidopsis (Xiang et al., 2011),

we sought to assess any functional similarity of At1g27470 with

PCN. No knockout lines for this gene are currently available in

SALK or other collections. To test if this gene can complement

pcn, we generated a gene construct using the At1g27470 protein

coding sequence and expressed it under the control of PCN

promoter (to achieve expression in the PCN domain) and intro-

duced this construct into the pcn background. The rationale for

this approach is that if At1g27470 shares functional domains in

its protein with PCN, complementation (partial or full) of embryo

phenotype is expected. However, our transgenic experiments

did not show any complementation, suggesting that At1g27470

does not share similar functions with PCN in Arabidopsis. To-

gether, these results confirm that PCN is a single-copy putative

WD-40 domain–encoding gene essential for embryo develop-

ment in Arabidopsis.

Embryo Patterning Defects in pcnMutants

We investigated the developmental aspects of pcn using the

pcn-1 allele (unless otherwise specified). Homozygous pcn

seeds obtained from a pcn/+ mother plant that was grown at

228C did not germinate. However, when cultured in vitro with

auxin and cytokinin (Wu et al., 1992), thesemutant embryoswere

able to form calli from which fertile plants could be regenerated.

These homozygous pcn plants upon flowering produced em-

bryos that were all defective. As this provided an advantage over

the segregating heterozygote, we have used these nonsegre-

gating homozygous lines for more detailed developmental stud-

ies (see Supplemental Table 1A online; Figures 2 and 3).

During wild-type embryogenesis, the zygote elongates and

divides asymmetrically, generating a smaller apical and a larger

basal cell (Figures 3A and 3K). The first two divisions of the apical

cell occur longitudinally, giving rise to the four-cell embryo proper

(Figures 3C and 3M), followed by a round of horizontal divisions

to establish the eight-cell embryo. Tangential divisions separate

the protoderm from the inner cells at the dermatogen stage.

Later, the globular embryo initiates two cotyledonary primordia

and a SAM. The descendants of the basal cell divide horizontally

to generate a column of cells, the extraembryonic suspensor.

Only the uppermost cell of the basal lineage, the hypophysis,

contributes to the embryo. After a transverse division, the apical

daughter cell of the hypophysis, the lens-shaped cell, gives rise

to the QC of the root meristem, whereas the basal daughter cell

forms the columella stem cells (Jurgens and Mayer, 1994).

Development of pcn embryos was similar to the wild type until

the two-cell embryo stage (Figures 3A and 3C versus Figures 3K

and 3M). Subsequently, with delayed apical cell divisions, more

suspensor cells were observed when compared with the wild

type (Figures 3L to 3V). Occasionally, abnormal vertical divisions

in suspensor cells (;2%) (Figure 3P), and octant embryo proper

with twice the number of cells than in the wild type were also

observed in pcn (< 5%) (Figures 3Q and 3R). From the der-

matogen stage and onwards, the pcn embryo proper displayed

several defects (Figures 3Q to 3Z). Abnormal and delayed

divisions were observed in the apical domain (Figures 3P to

3R), resulting in four kinds of phenotypes (Figures 2E to 2M): No

cotyledon (36.4% in pcn-1; 33.2% in pcn-2) (Figures 2E to 2G),

one cotyledon (29.8% in pcn-1; 31.3% in pcn-2) (Figures 2H and

2I), unequal splayed two cotyledons (31% in pcn-1; 32.4 in%

pcn-2), (Figures 2J to 2L) and three cotyledons (2.9% in pcn-1;

3.2% in pcn-2) (Figure 2M; see Supplemental Table 1A online).

Since pcn-1 embryos displayed stronger defects than pcn-2,

further analysis was done with pcn-1. The SAM region in the

majority of arrested embryos was enlarged, and the cotyledons

lacked bilateral symmetry and appeared more radialized (Figures

2I to 2M). In thebasal domain ofpcn embryos, the lens-shapedcell

derivatives were smaller than in the wild type (Figures 3I, 3J, 3Y,

and 3Z), and the lower hypophyseal cell derivatives divided less

frequently, resulting in aberrant columella organization (Figures 3J

and3Y). Together, the results show that lossofPCNactivity affects

the development of both the embryo proper and the suspensor.

Elevated Temperature Rescues pcn Embryo Lethality

While testing the growth of pcn plants at different temperatures,

we noticed that the defects in pcn embryo development were

strongly alleviated after shifting the regenerated homozygous

plants from 22 to 298C at the onset of bolting. The majority of the

pcn embryos produced at 298C had two wild-type-like cotyle-

dons (83%; see Supplemental Table 1A online) and developed

Figure 1. Molecular Characterization of the PCN Locus.

(A) A 35-bp deletion (open square) was detected in the pcn-1 mutant, spanning 27 bp of exon 5 and 8 bp of intron 5 (genomic position 4203956 to

4203991). In the pcn-2 mutant, the T-DNA insertion (triangle) was in exon 8 (position 4202286). The boxes indicate the exons and the lines indicate

introns.

(B) PCN encodes an 815–amino acid protein containing eight WD-40 repeats (gray boxes).

4350 The Plant Cell

into normal mature embryos. The remaining embryos had no

cotyledon (<5%), one cotyledon (10%), and three cotyledons

(2%) (see Supplemental Table 1A online). Unlike seeds from pcn

homozygous plants grown at 228C, 95% of pcn seeds produced

at 298C were able to germinate on 0.53 Murashige and Skoog

(MS) medium without the requirement of exogenous hormones

and produced fertile plants (see Supplemental Table 1B online;

Figure 4B). We took advantage of the temperature sensitivity

characteristic of this mutant and propagated pcn and genetic

combinations involving pcnby first growing the plants at 228Cand

then shifting them to 298C at bolting unless specified otherwise.

To determine at which embryo stagePCN function is essential,

pcn plants were grown at 228C, and flower buds were first

emasculated and then hand-pollinated with pcn pollen. A few of

the developing siliques were dissected to determine the devel-

opmental stage of the embryos. The plants were then shifted to

298C to complete seed development. Mature seeds were either

dissected to assess the embryo phenotypes or geminated on

0.53 MS medium. Our results from this study showed that the

shift to 298C must occur at or prior to the globular stage in order

to rescue the pcn embryo development (see Supplemental Table

1B online). A shift in temperature postglobular stage of embryo

development could not suppress embryo lethality. These obser-

vations further suggest that PCN function is required already for

early phases of embryo development in Arabidopsis.

pcnMutations Impact Postembryonic Development

To address the postembryonic functions of PCN, 298Ctemperature-rescued pcn mutant seeds were germinated and

Figure 2. Scanning Electron Microscopy Analysis of pcn Mutant Embryos.

(A) to (D) Wild-type embryos at early heart (A), mid-heart (B), torpedo (C), and bent (D) stages.

(E) to (M) pcn embryos arrested at various stages of development: globular embryo with defects in the apical and basal domains ([E] and [F]) and

partially differentiated basal domain (G); one-cotyledon embryo ([H] and [I]); two-cotyledon embryos showing various degrees of abnormal cotyledon

growth ([J] to [L]); three-cotyledon embryo (M). pcn embryos have an enlarged SAM ([I] to [M]).

Bars = 10 mm in (A) and (B) and 100 mm in (C) to (M).


plant development was analyzed at 228C growth conditions. pcn

mutant rosettes were smaller with narrower leaf shape (Figure

4B) and abnormal vein patterns (Figure 4D) compared with the

wild type (Figures 4A and 4C). Flowering pcn plants were shorter

and bushier than thewild type. Examination ofpcn roots revealed

abnormal division patterns in the QC and columella cells of the

root cap, and root growth was retarded compared with the wild

type (Figures 4F to 4H). Lugol staining for starch granules as a

marker for columella cell differentiation revealed defects in the

root cap cells in addition to the RAM defects observed in pcn

(Figure 4H). Furthermore, the expression of theQCmarkerWOX5

was expanded in pcn roots (Figures 6N and 6O). These obser-

vations suggest that PCN function is also required after germi-

nation for several processes associated with meristems both in

root and shoot development.

PCNEncodesaNuclearProteinThat IsExpressedBroadly in

the Embryo Proper and in Postembryonic Meristems

To studyPCN expression and subcellular localization in detail, two

different translational fusions of PCN and green fluorescent pro-

tein (GFP) coding regions were expressed under the control of

1.9-kbPCNputativepromoter region, onewithPCN cDNA (pPCN:

cPCN-GFP) and theotherwithPCNgenomic fragment (containing

introns; gPCN-GFP) (Figure 5). Independent transgenic lines of

both constructs displayed identical expression patterns. For

brevity, we will focus on results from the gPCN-GFP reporter.

Our earlier study on global gene expression patterns during Arab-

idopsis embryo development (Xiang et al., 2011) showed that

gPCN-GFP is expressed in the ovule but not in the pollen and,

after fertilization, is expressed in the zygote, suggesting maternal

specificity. During early phases of embryogenesis, thegPCN-GFP

signal was detected in all the cells of the embryo proper and the

suspensor until the 32-cell embryo stage (Figures 5A and 5B).

Subsequently, gPCN-GFP expression was not detectable in the

lower suspensor cells, and by the heart stage, the signal was

completely absent in the suspensor (Figures 5D and 5E) but

remained detectable in the embryo proper (Figures 5E and 5F).

In seedlings, gPCN-GFP was detected in the SAM and leaf

primordia (Figure 5G) but not in the hypocotyl (Figure 5H). In the

root, gPCN-GFP is expressed in the root meristem (Figures 5I

and 5J). gPCN-GFP is also expressed in pericycle cells, both

quiescent (Figure 5K) and actively dividing (Figures 5K and 5L),

that gave rise to the lateral roots and the expression continued

throughout the development of lateral root primordia (Figures 5M

and 5N). These results indicate that PCN is broadly expressed in

the embryo proper but after germination is predominantly ex-

pressed in the SAM, RAM, and in cells capable of proliferation.

Figure 3. pcn Mutants Undergo Aberrant Division Patterns in Early and Mid-Stage Embryogenesis.

Nomarski images of whole-mount cleared seeds containing the wild type ([A] to [J]) and pcn ([K] to [Z]) at the one-cell stage ([A] and [K]), two-cell stage

([B] and [L]), quadrant stage ([C] and [M]), octant stage ([D] and [N]), dermatogen stage ([E], [O], [P], and [Q]), globular stage ([F], [G], and [R] to [W]),

triangular stage ([H] and [X]), and heart stage ([I], [J], [Y], and [Z]). Delayed apical cell division in the pcnmutant was observed earliest in quadrant stage

(M) and in later embryo developmental stages ([N], [P], [T], and [V]). In the apical cell, abnormal planes of cell division were first observed at the octant

stage (N). During later developmental stages ([O] to [Z]), delayed and altered cell divisions resulted in an abnormal lens-shaped cell and hypophyseal

cell ([W] to [Z]; bottom panel) and defective cotyledon primordia specification ([Y] and [Z]). Bars = 10 mm.

4352 The Plant Cell

Mutation of PCN Perturbs the Expression Domains of

SAM-Organizing Genes

To better understand the SAM defects in pcn, we investigated

the expression patterns of well-characterized SAM organizing

genes using whole-mount embryo in situ hybridization and re-

porter genes. In the wild-type shoot meristem, WUS and CLV3

expression domains mark the three apical layers of stem cells

and the underlying organizing center, respectively (Figures 6A

and 6G) (Fletcher et al., 1999; Schoof et al., 2000). In pcn em-

bryos, CLV3 was expressed in an enlarged domain encompass-

ing almost the whole apex and was also ectopically expressed

in the cotyledons (Figures 6H and 6I). Compared with the wild

type, WUS mRNA expression had expanded into the L1 and L2

layers of the SAM and toward the inner layers of the cotyledons

(Figure 6B).

Expression of STM marks SAM cells that do not enter into

lateral organ formation. During wild-type embryogenesis, STM is

first expressed in a band of cells around the central apical region

of late globular stage embryos. During the transition to heart

stage, the expression becomes restricted to the central core of

the SAM (Figure 6C) (Long and Barton, 1998). In pcn, STM was

variably expressed below the L3 layer of the SAM. In the majority

of the embryos examined (30 out of 44), STM expression was

detected in a large apical region of the embryo (Figure 6D).CLV1

is expressed in the L2 and L3 layers of the wild-type SAM (Long

andBarton, 1998). Inpcn,CLV1 expressionwas still limited to the

L2 and L3 layers, but the expression domain had expanded to

a slightly broader region compared with the wild type (Figures

6E and 6F). Furthermore, quantitative RT-PCR (qRT-PCR) re-

sults revealed that WUS, CLV3, CUC, and STM in the SAM are

significantly upregulated in pcn embryos (see Supplemental

Table 2 online). These results suggest that PCN function is

required to restrict the expression of STM,WUS, andCLV genes

within their respective specified boundaries in the meristem.

PCN andWUS Interact Genetically

To determine whether PCN genetically interacts with WUS, we

analyzed pcn wus-1 double mutants. wus-1 embryos develop

normally except for the absence of a shoot meristem in the

seedlings after germination (Laux et al., 1996). Double heterozy-

gous pcn/+ wus/+ plants produced homozygous recessive pcn

wus double mutant embryos at a ratio of 1:15 that did not initiate

cotyledon primordia and had seeds that did not germinate, even

when themother plant was grown at 298C.However,pcnwus-1/+

seeds germinated and produced plants with rosette leaves,

albeit at a slower rate than the wild type. After bolting, however,

the SAM terminated as pin-like structures with arrested lateral

organ primordia and differentiation of the epidermal cells into

trichomes near the SAM region, indicating haploinsufficiency for

WUS in pcn background (Figures 7J to 7N). These observations

indicate that wus embryos cannot complete their development

unless PCN function is present. Whereas the lack of PCN can be

remedied by high temperature insofar as embryo development

is concerned, such a rescue requires WUS. These synergistic

defects suggest partial compensatory functions for WUS and

PCN during embryo and postembryonic development.

Auxin Distribution and Response Are Altered in pcn

Defective cotyledon patterning in pcn embryos raised the ques-

tion whether auxin response and/or auxin transport were af-

fected in pcn embryos. To address this question, we introduced

Figure 4. pcn Mutant Development Is Affected Postembryonically.

(A) and (B) Three-week-old rosette of the wild type (A) and pcn (B). The pcn rosette is smaller than that of the wild type.

(C) and (D) Cleared fourth leaf of the wild type (C) and pcn (D). The pcn leaf shows reduced venation compared with that of the wild type.

(E) to (H) Cleared and lugol-stained root apices of the wild type (WT) ([E] and [H]) and pcn ([F] to [H]). pcn root meristems were smaller ([F] and [G]) with

defective columella cell differentiation of the root cap compared with the wild type ([E] and [H]).

Bars = 1 cm in (A) to (D) and 100 mm in (E) to (H).


the auxin response marker DR5rev:GFP, and as a readout of po-

larized auxin transport machinery, pPIN1:PIN1-GFP and pPIN7:

PIN7-b-glucuronidase (GUS), into the pcn mutant. At 228C,DR5rev:GFP expression was observed throughout the embryo

proper in pcn similar to the wild type up to the octant stage

(Figures 8A, 8F, and 8G) (Friml et al., 2003). However, from 32-

cell globular stage onwards, the basal auxin maximum, which is

restricted to the hypophyseal region in the wild type, expanded

into the lower suspensor cells in arrested globular pcn embryos.

The two auxin maxima corresponding to the two emerging

cotyledon primordia in the wild type (Figures 8B to 8E) were

absent in the pcn embryos that did not produce cotyledons

(Figures 8I, 8J, and 8L). In pcn embryos that did form cotyledons,

the auxin maxima correlated with the number of cotyledons (Fig-

ures 8M to 8P). In a few mutant embryos, several auxin maxima

were also observed in the apical region of late torpedo stage

embryos (Figure 8K). In contrast with DR5rev:GFP expression in

the vascular procambial cells of later-stage wild-type embryos

(Figure 8E), the GFP signal was discontinuous or absent in the

differentiating procambial cells of pcn embryos (Figures 8K to

8P). Unlike the wild type, DR5rev:GFP was ectopically expressed

in the SAM of pcn embryos (Figures 8P and 8U). Consistent with

the phenotypic recovery, this ectopic DR5 expression was sup-

pressed when pcn embryos were subjected to 298C treatment

(Figures 8U and 8V). In the RAM, the expression became re-

stricted to the columella region similar to the wild type (Figures

8Q and 8R). These results indicate that mislocalization of auxin

maxima coincides with the defective phenotypes observed in

pcn mutant embryos.

In wild-type dermatogen stage embryos, PIN1-GFP is present

in all the cell boundaries, whereas at the 32-cell stage, PIN1-GFP

becomes restricted to the basal boundaries of subepidermal

cells (Figure 9A). From late globular stage onwards, PIN1-GFP is

localized in cotyledon primordia and in vascular initials as shown

Figure 5. GFP Reporter–Based Expression Patterns and Nuclear Localization of PCN in Arabidopsis.

(A) to (F) gPCN-GFP expression in embryos at octant stage (A), dermatogen stage (B), globular stage (C), triangular stage (D), heart stage (E), and

torpedo stage (F). (A) to (E) show progressive loss of expression of gPCN-GFP in suspensor cells during embryo development.

(G) and (H) gPCN-GFP is expressed in the SAM ([G], outlined in yellow) of the 5-d-old seedling and absent in hypocotyl (H).

(I) and (J) gPCN-GFP is expressed in the RAM (I) and distal region of the root (J).

The yellow boxed region in (K) shows gPCN-GFP expression in the dividing pericycle cells (L) that continues during early stages of lateral root formation

([M] and [N]). The 49,6-diamidino-2-phenylindole (red) and gPCN-GFP (green) colocalized in the nuclei of seedling RAM (O).

Bars = 20 mm in (A) to (H), (J), and (L) to (O) and 100 mm in (I) and (K).

4354 The Plant Cell

previously (Friml et al., 2003) (Figures 9B to 9E). In pcn embryos

arrested at globular stage, PIN1-GFP appeared in a broad cen-

tral region of the embryo (Figures 9H and 9I), and the expression

was seen in the vascular initials of the cotyledons and the

hypocotyl of one cotyledon (Figure 9J) and two cotyledon em-

bryos (Figures 9K and 9L). In contrast with the wild type, PIN1-

GFP also accumulated in the SAM region of the pcn embryos

with two cotyledons (Figures 9K and 9L). While the PIN1 expres-

sion profile was relatively normal (see Supplemental Table 2

online), PIN1-GFP was not distributed in a polar manner in the

majority of the cells of pcn mutant embryos (Figures 9H to 9L).

Since polar localized PIN1 in the wild type can be rapidly in-

ternalized to the cytosol by brefeldin A (BFA) treatment (Geldner

et al., 2001), we used BFA treatment along with the membrane

stain FM4-64 (Vida and Emr, 1995) to examine the localization of

PIN1-GFP in the cells of wild-type and pcn embryos. In the wild

type, PIN1-GFP signal overlapped with the FM4-64 stain in the

basal membrane of cells (Figure 9F), whereas with BFA treat-

ment, PIN1-GFP became internalized in the cytosol (Figure 9G).

However, in the pcn mutant embryos, the PIN1-GFP was inter-

nalized to the cytosol both without and with BFA treatment

(Figures 9M and 9N), suggesting that pcn mutant embryos are

defective in polar localization of PIN1.

PIN7 expression and localization is coordinated with PIN1 in

the embryo proper to create auxin maxima in the hypophyseal

cell at the 32-cell stage (Friml et al., 2003). We used pPIN7:PIN7-

GUS to compare its expression pattern in the pcn embryo and

the wild type. In the wild type, PIN7-GUS was detected in the

hypophyseal cell derivatives in the embryonic root meristem

(Figures 10A and 10B) and the provascular cells in the developing

hypocotyl (Figures 10A to 10C). In those pcn embryos that were

arrested at early stages, the expression was present in the

suspensor but absent from the hypophyseal cell derivatives of

the root meristem (Figure 10F). Although the PIN7-GUS expres-

sion was present in the provascular cells of the developing pcn

embryo, the signal appeared weaker and was restricted to fewer

cells (Figures 10G to 10I) and was absent in the root meristem

region (Figures 10H and 10I). In wild-type seedlings, PIN7-GUS

was expressed in the root meristem and vascular cells of the root

and cotyledons. By contrast, in pcn, the PIN7-GUS was weakly

Figure 6. SAM and RAM Marker Gene Expression Is Altered in pcn Mutants.

(A) to (F)Whole-mount embryo in situ hybridization of wild-type (WT) (A) and pcn (B) embryos withWUS probe, wild-type (C) and pcn (D) embryos with

STM probe, and wild-type (E) and pcn (F) embryos with CLV1 probe.

(G) to (I) pCLV3:CLV3-GUS expression in wild-type embryo (G), pcn embryos (H), and seedlings (I).

(J) to (M) Whole-mount embryo in situ hybridization of wild-type (J) and pcn (K) embryos with MP probe and the wild type (L) and pcn (M) with PLT1

probe.

(N) and (O) pWOX5-sv40:3GFP expression in wild-type root (N) and pcn mutants (O).

All pcn embryos used were grown at 228C. Bars = 50 mm.


expressed in the vascular cells of the root (Figure 10J) but was

absent in the root meristem and vascular cells of the cotyledons

(Figures 10J and 10K). These results were consistent with the

qRT-PCR analysis that showed that PIN7 was expressed 0.6-

fold lower than the wild type (see Supplemental Table 2 online).

Taken together, pcnmutants displayed altered PIN1 localization

and PIN7 expression.

To test whether auxin response is altered in pcn mutants, we

performed a lateral root induction assay by application of the

auxin analog naphthalene acetic acid (NAA) (Evans et al., 1994) to

Figure 7. Dosage-Dependent Phenotypes of pin1, pid-2, wus-1, and tpl-1 in the pcn Mutant.

Heterozygous pin1, pid-2, wus-1, and tpl-1 in pcn homozygous recessive background have terminated meristems with a pin-like structure.

(A) to (I) Pin-like structures in pcn pin1/+ ([A] and [E]), pin1 ([B] to [D]), pcn pid-2/+ ([F], [H], and [I]), and pid-2 (G). pin1 and pid-2 formed pin-like

inflorescences ([B] and [G]) that bear no flowers, and the cells surrounding the tip region show no signs of differentiation in the epidermal layer ([C] and

[D]). pcn pin1/+ and pcn pid-2/+ plants form very short pin-like structures that produced lateral organ primordia near the tip ([E], [H], and [I]) and

showed arrested meristems with the epidermal layer differentiating guard cells and pavement cells close to the SAM (I).

(J) to (N) In pcn wus-1/+ plants (J), the inflorescence meristem terminated in a pin-like structure with arrested lateral organ primordia ([K], [M], and [N])

and trichomes close to the SAM (L).

(O) to (R) In pcn tpl-1/+ plants, the inflorescence meristem was aborted ([O] and [P]) or terminated in pin-like structures ([Q] and [P]).

Bars = 100 mm in (B), (G), (H), and (K) to (N), 10 mm in (C) to (E) and (I), and 250 mm in (O) to (R).

4356 The Plant Cell

Figure 8. Expression Patterns of DR5rev:GFP in the Wild Type and pcn.

(A) to (P) DR5rev:GFP expression in wild-type and pcn embryos. DR5rev:GFP in pcn at early embryo stages ([F] to [I]) is similar to the wild type ([A] and

[B]) except for ectopic expression in the suspensor cells ([H] to [J]). At later stages, the DR5rev:GFP expression correlates with the specification of

cotyledon primordia as observed in wild type ([C] to [E]) and pcn ([M] to [P]) with few exceptions ([K] and [L]). Ectopic DR5rev:GFP expression was

observed in the SAM region in the pcn mutant embryo (P).

(Q) and (R) DR5rev:GFP expression was reduced in the RAM of pcn (R) in comparison to the wild type (Q).

(S) to (V) Ectopic DR5rev:GFP expression was observed in the SAM region in pcn (U) at 228C when compared with the wild type (WT) grown at 228C (S)

and 298C (T). This ectopic expression in the SAM was restored back to wild-type expression in pcn (V) when grown at 298C.

Bars = 10 mm in (A), (F), and (G), 20 mm in (B) to (E) and (H) to (P), and 100 mm in (Q) to (V).


seedlings. In the wild type, lateral root primordia induced by

2 mg/L NAA displayed normal DR5rev:GFP expression (Figures

11A and 11B). However, in pcn, NAA induced abnormal bulges of

pericycle cells with diffused expression of DR5:GFP that did not

differentiate into lateral root primordia (Figures 11C to 11E). Thus,

while auxin maxima displayed by DR5rev:GFP appeared in the

proper position, subsequent root development required PCN

activity. In an alternate assay tomeasure the strength of the auxin

response in pcn mutants, we used the pHS:AXR3NT-GUS re-

porter (Gray et al., 2001). In thepHS:AXR3NT-GUS construct, the

GUS gene is fused to theN-terminal domains I and II of the AXR3/

IAA17 and its expression is controlled by a heat shock promoter.

Due to the inclusion of the degron-containing domain II of AXR3,

the associated GUS is subject to degradation in an auxin-

responsive manner (Gray et al., 2001). As shown in Figures 11I

to 11K, 11M, and 11N, the fusion protein was more stable in pcn

seedlings than in the wild type as visualized by GUS, suggesting

that indole-3-acetic acid (IAA) protein turnover is affected in pcn

mutant. These results collectively show that PCN plays a role in

auxin response pathway.

PCN Interactswith the Auxin ResponseModule Comprising

BDL and MP

The IAA/AUX-ARF auxin module BDL/MP mediates auxin tran-

scriptional response and together with PLT1 and PLT2 positively

regulates PIN expression in root and vascular development

(Szemenyei et al., 2008). In pcn, the expression domains of MP

and PLT1 in the embryonic RAMwere more restricted compared

with thewild type (Figures 6J to 6M); also in the vascular initials of

the pcn embryo, MP expression was discontinuous and ectopic

in the SAM region of the torpedo embryo (Figure 6K). Thus, aber-

rations in the expression ofMP andPLT1weremore pronounced

in the pcn embryos that showed stronger developmental de-

fects. Consistent with this observation, MP-GUS in the pcn

mutant showed ectopic expression in the apical region of the

embryo that included the SAM (Figures 12A4 to 12A6) and the

cotyledonprimordia (Figure 12A5). Furthermore,qRT-PCRshowed

that MP, WOX5, PLT1, PLT2, and PLT3 were significantly up-

regulated in pcn embryos (see Supplemental Table 2 online).

These findings prompted us to investigate potential interactions

between pcn and auxin mutants.

mp single mutants develop a basal stump instead of a root and

produce one or two cotyledons (Berleth and Jurgens, 1993). In

the progeny of pcn/+ mp/+ plants, we identified putative double

mutants at a ratio 1:15 with a novel embryo phenotype. These

double mutant embryos showed cup-like cotyledons with a nar-

row embryo axis compared with the wild type (Figures 12A1 and

12A2). These embryos were arrested not only at 228C but also at

298C, similar to the observations with double mutants of pcn and

wus-1, suggesting that MP functions are required for the rescue

of the pcn mutant at 298C. The pcn mp/+ plants were small and

had delayed rosette leaf and root growth, and the SAM termi-

nated into pin-like structures (Figure 12A3) compared with pcn

singlemutant andmp/+heterozygotes that flower normally. These

observations suggest that PCN cooperates with MP to mediate

auxin signals during embryonic and postembryonic develop-

mental programs in Arabidopsis.

BDL is considered a repressor ofMP in auxin signaling during

embryo development, and the bdl gain-of-function mutant re-

sembles the mp loss of function mutant (Hamann et al., 2002).

We investigated BDL interaction with PCN using a pcn bdl

double mutant. The bdlmutant line we used was in the Columbia

(Col) background and displayed abnormal horizontal divisions in

Figure 9. Auxin Transport in the Wild Type and pcn.

(A) to (E) and (H) to (L) PIN1-GFP localization in wild-type ([A] to [E]) and pcn mutant ([H] to [L]) embryos. The localization of PIN1:GFP in the

prospective cotyledon primordia region (B) and later in the developing cotyledon primordia ([C] to [E]) seen in the wild type is absent in pcn embryos

that lack cotyledons ([H] and [I]).

(F), (G), (M), and (N) To assess the polar localization of PIN1-GFP in the membrane, embryos were stained with FM4-64 and treated with BFA and

control. With BFA treatment, the membrane-localized PIN1-GFP in control (F) moved to the cytosol (G) in the wild type, whereas in pcn, with (M) and

without (N) BFA treatment, PIN1-GFP was localized to the cytosol.

Bars = 10 mm in (A) to (D) and 25 mm in (E) to (N).

4358 The Plant Cell

the apical cell of the embryo as observed in the Landsberg erecta

(Ler) background (Hamann et al., 1999) but at a lower frequency

(5%, n = 480). In the pcn bdl double mutant in the Col back-

ground, 55% of embryos (n = 275) showed abnormal divisions at

the octant stage that also include double octant phenotype

(Figures 12C1 and 12C2) and tapered embryo phenotypes in

later stages of development (Figures 12C3 and 12C4). Thus,

during early embryo development, pcn enhanced the bdl embryo

defects (Figures 12B1 to 12B3 and 12C1 to 12C4). Furthermore,

the pcn bdl double mutant had fewer suspensor cells than both

pcn or bdl single mutants (Figures 12B1 to 12B3 and 12C1 to

12C4). At the heart stage, pcn bdl embryos displayed a broader

central zone (Figures 12C5 and 12C6) than the wild type or pcn

single mutant (Figures 3I, 3J, 3X, and 3Y). However, during em-

bryo development, the globular stage arrest, cotyledon defects,

and seed abortion observed in the pcn single mutant at 228Cwere suppressed by the presence of bdl in the double mutant.

The pcn bdl set viable seeds at 228C unlike the pcn single mutant

that requires 298C treatment.

To examine if BDL expression is altered in pcn mutant, we

introduced pBDL:BDL-GFP reporter into pcn. BDL-GFP expres-

sion was first detected in the vascular initials and the RAM of the

wild-type embryos (Figures 12D1 and 12D2). However, in pcn,

BDL expression was detected in the SAM region but was absent

in the vascular initials and the embryonic RAM from torpedo

stage onwards when grown at 228C (Figures 12D3 to 12D6). This

altered expression of BDL in pcn was restored to a normal

wild-type -like pattern at 298C, suggesting that the pcn mutant

phenotype at 228C is dependent on BDL wild-type function

(Figure 12D7). Taken together, the quantitative enhancement of

auxin-related defects of both mp and bdl mutants in pcn back-

ground suggests that PCN is required for gene functions over-

lapping with those of MP and BDL in embryogenesis. These

embryonic observations are consistent with a contribution of

PCN to the auxin-induced degradation of multiple auxin/IAA

proteins.

We performed a yeast two-hybrid (Y2H) assay to determine

whether PCN physically interacts with BDL, MP, or TPL. When

PCN fragments other than the C-terminal region or a full-length

PCN was used as the bait, PCN showed very strong self-

activation. The C-terminal fragment did not show self-activation,

and it did not show any physical interaction between PCN and

BDL,MP, or TPL.Whenwe used BDL,MP, or TPL as the bait and

PCN as the prey, we did not observe any physical interaction of

PCNwith BDL,MP, or TPL. However, qRT-PCR analyses ofBDL,

TPL, and MP in pcn mutant embryo revealed that BDL and TPL

expression are similar to the wild type, but MP and its target

genes DORNROSCHEN (DRN) and DORNROSCHEN-LIKE are

significantly upregulated, 1.6-, 9.7-, and 4.7-fold, respectively,

compared with the wild type (see Supplemental Table 2 online).

Previous work has shown that MP binds to the DRN promoter

and regulates its transcription positively (Cole et al., 2009).

Consistently, upregulated MP expression in pcn also increased

the DRN expression in this study (see Supplemental Table 2

online). The loss-of-function mutant drn-1 shows defective em-

bryo patterning and functional postembryonic SAM (Chandler

et al., 2007), whereas the gain-of-function drn-D mutant shows

defective SAM and upregulated expression of both WUS and

CLV3 as seen in pcn (Kirch et al., 2003). The pcn drn-1 double

mutant showed the pcn embryo phenotype. However, after

Figure 10. Expression Patterns of pPIN7:PIN7-GUS in the Wild Type and pcn.

PIN7-GUS localization in the wild type ([A] to [E]) and pcn ([F] to [K]). PIN7-GUS is present in the provascular initials and suspensor cells of developing

wild-type embryos ([A] to [C]), whereas in pcn, PIN7-GUS is absent in the embryo proper of early arrested embryos (F) and in the provascular cells of the

radicle of later arrested embryos ([G] to [I]). In the wild-type seedling, PIN7-GUS is present in the RAM (D) and differentiating vascular cells of the root

and cotyledon ([D] and [E]), whereas in pcn, PIN7-GUS is absent in the RAM and vascular cells of the cotyledon ([J] and [K]) and weakly expressed in

the differentiating vascular cells of the root (J).

Bars = 100 mm in (A) to (D) and (F) to (J) and 1 mm in (E) and (K).


rescue at 298C and during postembryonic development, the

young rosettes terminated growth with arrested SAM and radi-

alized leaf primordia, which were not observed in the respective

single mutants (Figures 12E1 to 12E3), further supporting the

involvement of PCN mediated auxin signaling in meristem reg-

ulation. The absence of PCN interaction with BDL in the Y2H

assay does not rule out interaction via other partner(s). Taken

together, PCN may function in a multiprotein complex that

includes BDL and TPL to regulate the downstream genes in the

auxin pathway that includes DRN.

PIN1, PID1, and TPL Are Dosage Sensitive in pcn

Double mutants of pcn and pin1-1, pid-2, or tpl-1 arrested em-

bryo development at 298C, albeit the respective single mutants

completed embryogenesis and produced viable seeds. These

observations suggest that the rescue of pcn embryo lethality by

elevated temperatures requires the functions of the auxin com-

ponents PIN and PID and the global corepressor TPL.

PIN1 promotes directional auxin efflux, and PID mediates the

switch ofPINpolarity. Both pin1-1 andpid-2 singlemutants upon

bolting form pin-like structures with an undifferentiated inflores-

cence meristem at the tip (Figures 7B to 7D and 7G) (Friml et al.,

2004; Kaplinsky and Barton, 2004). Unlike in pcn or pin1-1/+ and

pid-2/+ heterozygous plants, in pcn pin1-1/+ (Figure 7A) and pcn

pid-2/+ plants (Figure 7F), inflorescence shoot meristem ter-

minated in a pin-like structure, and lateral organ primordia were

arrested (Figures 7E, 7H, and 7I). In all cases, trichomes and

guard cells, which are normally restricted to leaves or leaf

primordia, were found at the tip of the pin-like structure in the

meristem region (Figures 7H and 7I), indicative of precocious

differentiation of meristem cells. As one of its functions, TPL acts

as a corepressor with BDL in a complex with MP (Szemenyei

et al., 2008). tpl-1 and tpl-1/+ plants developed similar to the wild

type after bolting, whereas the pcn tpl-1/+ inflorescence meri-

stems also terminated in a pin-like inflorescence (Figures 7Q and

7R). Taken together, in the absence of PCN function, inflores-

cence meristem development becomes more sensitive to the

gene dosage of PIN1, PID, and TPL.

DISCUSSION

We identified PCN as an essential gene for Arabidopsis embryo

development. Mutations in the PCN locus resulted in develop-

mental defects that link maintenance of the embryonic meristem

and cotyledon formation to auxin-mediated processes that

regulate early to late patterning events during embryogenesis.

Embryo defects in pcn were detectable as early as the four-cell

stage and progressively displayed more defects both in the

apical and basal programs, resulting in striking cotyledon phe-

notypes, enlarged SAM, and abnormal RAM, suggesting the

importance of PCN for embryo development, including stem cell

organization and maintenance. Our studies indicate a significant

contribution of PCN in the early steps of auxin signaling by

its ability to genetically interact with BDL and, therefore, likely

functions as a repressor to regulate several key pathways that

operate during embryo patterning (see Supplemental Table 2

online). TPL, another well-studied corepressor functions with

BDL (Szemenyei et al., 2008), also genetically interacts with

PCN. Auxin maxima and distribution were also altered in the

pcn mutant, suggesting a link between auxin-mediated embryo

Figure 11. Auxin-Induced Lateral Root Initiation and IAA Protein Stability Is Altered in the pcn Mutant.

(A) to (E) Lateral root induction and DR5-GFP localization in the wild type (WT) ([A] and [B]) and pcn ([C] to [E]). Lateral root primordia induction by NAA

(2 mg/mL) in the wild type (A) and pcn (C). DR5rev:GFP expression in NAA-induced (2 mg/mL) lateral roots in the wild type at 2 d (B), pcn at 2 d (D), and

pcn at 5 d (E). The polarly localizedDR5rev:GFP expression pattern observed in wild-type lateral roots (B) is altered andmislocalized in pcn ([D] and [E]).

(F) to (N) pHS:AXR3NT-GUS expression in the wild-type root at 0 h (F), 2 h (H), and 3 h (L) and in pcn root at 0 h (G), 2 h ([I] to [K]), and 3 h ([M] and [N]).

The heat shock–induced AXR3NT-GUS expression is degraded within 2 h in the wild type ([F], [H], and [L]), whereas in pcn, the GUS expression is still

retained at 3 h ([M] and [N]).

Bars = 100 mm.

4360 The Plant Cell

Figure 12. Genetic Interactions of mp, bdl, and drn Mutants with pcn.

(A1) and (A2) A mp pcn double mutant shows an abnormal embryo phenotype.

(A3) A pcn mp/+ rosette SAM terminates in a pin-like structure.

(A4) to (A6) MP-GUS in pcn embryo is ectopically expressed in the SAM.

(B1) to (B3) and (C1) to (C6) A bdl pcn double mutant shows increased abnormal cell division during early and mid-embryo development ([C1] to [C6])

compared with the bdl single mutant ([B1] to [B3]).

(D1) to (D7) pBDL:BDL-GFP expression pattern in wild-type ([D1] and [D2]) and pcn ([D3] to [D7]) embryos. In pcn, BDL-GFP is ectopically expressed

in the SAM ([D5] and [D6]) and occasionally absent in the RAM (D6) compared with the wild type ([D1] and [D2]). This altered BDL-GFP expression in

pcn could be rescued through growth at 298C (D7).

(E1) to (E3) The pcn drn-1 double mutant shows arrested SAM. pcn drn-1 plant with the SAM and young leaf primordia boxed in yellow (E1) and

scanning electron micrographs of the arrested SAM and defective leaf primordia ([E2] and [E3]).

Bars = 10 mm in (B1) to (B3) and (C1) to (C6), 25 mm in (D1) to (D7), 50 mm in (A4) to (A6), 100 mm in (A1) to (A3) and (E3), and 1 mm in (E2).


patterning and PCN function. Another interesting feature of the

pcnmutant embryo is its ability to recover at 298C,which enabled

investigation into the postembryonic roles of PCN. The defects in

NAA-induced lateral root formation in pcn seedlings along with

the prolonged IAA protein stability in this mutant suggest that

PCN is required for auxin-induced response and downstream

signaling.

PCN genetically interacts with WUS and the transcriptional

corepressor TPL. WUS, which functions in a negative feedback

loop to specify stem cells of the SAM, has been shown to phys-

ically interact with a TPL ortholog in Antirrhinummajus to repress

differentiation pathways in the SAM (Schoof et al., 2000; Kieffer

et al., 2006). In addition, pcn also genetically interacts with PIN

andPID as both pcn pin1-1/+ and pcn pid-2/+ developed pin-like

inflorescence with terminal differentiation. This suggests that

PCN is required for sustained meristem activity during postem-

bryonic development. Interestingly, the respective homozygous

double mutants displayed strong developmental arrest at the

globular stage. These observations suggest that PCN functions

are coordinated with PIN and PID both during embryo devel-

opment and postembryonically in developmental programs as-

sociated with meristems. The postembryonic phenotypes also

suggest that PCN has a significant role in the maintenance of a

functional SAM through the auxin signaling pathway; previously,

the auxin signaling pathway has been shown to be essential for

RAM functions (Benjamins and Scheres, 2008). Based on these

observations, it is tempting to speculate thatPCN likely functions

in the BDL/TPL repressive pathway to regulate auxin-mediated

embryo patterning and meristem maintenance.

PCN Plays an Important Role in Restricting the

Meristem Size

An enlarged SAM (Figures 2J and 2M) and ectopicWUS expres-

sion in the L1 and L2 layers (Figure 6B)were both observed in pcn

mutant. WUS is normally restricted to a few cells in the L3 layer of

the wild type (Figure 6A); these WUS-expressing cells act as an

organizing center and signal the overlying cells to function as

stem cells (Mayer et al., 1998). Ectopic expression ofWUS in the

L1 layer using theCLV3promoter results in an abnormally swollen

SAM that is arrested in the seedling (Brand et al., 2002), and a

recent study showed that WUS directly activates CLV3 expres-

sion by binding to its promoter region (Yadav et al., 2011). There-

fore, WUS misexpression in the L1 and L2 layers followed by its

self-amplification is partly responsible for themeristemdefects in

pcn. pcn wus-1 double mutants could not develop past the

globular stage either at 22 or 298C, indicating thatWUS and PCN

together are essential for early embryogenesis. The double mu-

tant embryos of pcn tpl-1 and pcn wus-1 are arrested at the

globular stage even when grown at 298C. TPL and WUS muta-

tions are haploinsufficient in the pcn background, as the pcn

tpl-1/+ and pcn wus-1/+ seeds were viable if grown at 298C, butdeveloped similar pin-like inflorescence phenotypes postgermi-

nation.

These observations are consistent with the proposed function

of TPL as a corepressor and its physical interactions with WUS

(Kieffer et al., 2006). Furthermore, these results also suggest that

PCN and TPL likely mediate alternative roles in regulating WUS

repression and activation to maintain the SAM functions. The

replacement of inflorescences with pin-like structures is usually

considered as an indicator of the disruption in auxin movement

and gradient (Okada et al., 1991; Friml et al., 2004; Kaplinsky and

Barton, 2004). Disordered divisions in the SAMandRAMoccur at

the same time as the loss of organizing center and QC identity

in the pcn mutant. Abnormal SAM, RAM, and auxin maxima

observed in pcn indicate that PCN plays an important role in

maintaining meristem functions through auxin signaling. WUS

expression is negatively regulated by the CLV signal transduc-

tion pathway (Brand et al., 2000; Schoof et al., 2000). Surpris-

ingly, our study shows a broader expression of CLV3 in pcn

(Figures 6G to 6I), althoughWUS expression was also expanded.

One possible explanation is that in pcnmutants, CLV signaling is

compromised, resulting in enlarged meristem. Therefore, PCN

likely provides a link between genetic regulation of meristem

maintenance and auxin signaling pathway.

PCN Functions in the Auxin Pathway

Regulated degradation of auxin/IAA proteins that repress ARF

transcription factors bound to auxin response elements play a

crucial role in auxin-mediated signaling (Mockaitis and Estelle,

2008). BDL, an IAA protein, along with the corepressor TPL

(Szemenyei et al., 2008), is thought to form a complex with the

ARF transcription factor MP (Weijers et al., 2006) to regulate

auxin-responsive target genes. BDL andMP are expressed from

zygote to early globular stages and are subsequently restricted

to the hypophyseal cell derivatives and the developing vascular

initials (Hamann et al., 2002). Our study indicates that the pcn

mutation interferes with auxin response similar to mutations

in the well-characterized bdl (Hamann et al., 1999). Like bdl,

mutations in PCN cause abnormal cell divisions in the basal

domain during early embryo development. Although pcn en-

hanced the early embryo phenotypes of the bdlmutant, surpris-

ingly, bdl rescued the pcn mutant embryo even when grown at

228C, suggesting that BDL activity is involved in pcn embryo

lethality. Consistent with this, the ectopic expression of BDL-

GFP and DR5rev:GFP in the SAM of pcn embryos was sup-

pressed in pcn plants grown at 298C for seed/embryo rescue

(Figures 8U, 8V, 12D6, and 12D7). Furthermore, the misexpres-

sion of STM outside the SAM (Figure 6D) and the auxin maxima

observed in the SAM of the pcn embryo (Figure 8P) suggest that

PCN is required for tight regulation of auxin response and max-

ima in the SAM. Therefore, it is likely that ectopic BDL activity in

the SAM of pcn embryos contributes to the striking pcn embryo

phenotypes, suggesting that BDL function is required for the

arrested pcn embryo phenotype.

Because the bdl pcn double mutant is not completely reverted

to thewild type, it is likely that other genesmay be involved in pcn

rescue at 228C.Our results suggest that TPL activity may provide

some regulatory functions in the pcn mutant background. Con-

sistent with this, the pcn tpl-1 double mutant is embryo lethal

with embryos arrested at the globular stage when grown either

at either 22 or 298C. Furthermore, the heterozygous tpl-1 in

pcn background (pcn tpl-1/+) had a partially terminated SAM,

whereas heterozygous pcn in tpl-1 background (pcn/+ tpl-1)

showed tpl-1 postembryonic phenotypes (Figures 7O to 7R).

4362 The Plant Cell

These observations suggest that PCN and TPL have both dis-

tinct and overlapping functions. Since PCN appears to function

together with BDL and TPL to regulate downstream targets,

disruption of the BDL containing complex may cause an un-

known alternative pathway to rescue pcn in the bdl mutant

background. Although the pcn mp/+mutant was not arrested at

298C, it showed severe cotyledon phenotypes compared with

themp singlemutant and postembryonically the SAM terminated

in a pin-like structure (Figures 12A1 to 12A3). As our results indi-

cate that PCN genetically interacts with BDL, it is likely that

PCN works with BDL and TPL to negatively regulate MP and its

targets. It is interesting to note that both of these genes that

genetically interact with BDL encode WD-40 repeat–containing

proteins (Szemenyei et al., 2008). In addition to the involvement

of PCN in the BDL/TPL repression of MP, the pHS:AXR3NT-

GUS–based assay (Gray et al., 2001) in the pcn showed that IAA

proteins are more stable in the pcnmutant, suggesting that PCN

has a broader role in auxin signaling.

PCN Integrates Auxin Signaling and Meristem Functions

The pcn mutant phenotype, its genetic interactions with factors

associated with auxin signaling and meristem fate, and the qRT-

PCR results of genes that are differentially regulated in the pcn

embryo indicate that PCN functions along with BDL and TPL to

mediate MP repression (Figure 13). MP regulates both its own

expression and that of BDL, thus functioning as a genetic switch

triggered in response to auxin in a threshold-specific manner to

degrade BDL and relieve the repression of its target genes (Lau

et al., 2011). MP, which is upregulated in the pcn embryo,

through negative regulation ofARR7 andARR15 (downregulated

in pcn embryo; see Supplemental Table 2 online), likely regulates

the WUS/CLV3 feedback loop in the SAM as previously sug-

gested (Zhao et al., 2010), thus revealing a link between auxin

signaling and meristem function. Several studies have shown

that PIN polarity and the resulting auxin localization are essential

for embryo patterning (Friml et al., 2003; Geldner et al., 2003;

Michniewicz et al., 2007; Kitakura et al., 2011). In the absence of

functional PCN, PIN polarity is not maintained in the embryo,

which leads to ectopic auxin maxima in the SAM region. This

likely results in the disruption of BDL-mediated repression of MP

and ectopic expression of MP and BDL in the pcn embryonic

SAM. Ectopic auxin maxima along with MP/BDL expression in

the SAM likely also contribute to the disruption of theWUS/CLV3

feedback loop, leading to the improper partitioning of the SAM

and cotyledon primordia in the apical domain of the pcn globular

embryo. Furthermore, in pcn embryos, several genes encoding

AP2 domain transcription factors and members of the WOX

Figure 13. Working Model of PCN Functions in Embryo Development and Meristem Organization via the Auxin Signaling Pathway.

This model summarizes key findings of this study. As shown in the top panel, PCN likely functions first as a repressor along with TPL and BDL to regulate

known (i.e., DRN and ARR15) and potential (i.e., ARR7 and PLT) MP target genes during embryo development and later during meristem organization

and maintenance. The middle panel shows polar localization of PIN1 in the wild-type (WT) embryo (orange lines) followed by patterning events that

establish the embryonic SAM via theCLV-WUS–mediated regulatory loop and the embryonic RAMwith auxin maxima andWOX5 in the QC. As shown in

the bottom panel, disruption of PIN1 polarity in the pcnmutant embryo and auxin maxima in the enlarged pcn SAM results in ectopic expression ofDR5-

GFP, MP, and BDL in the SAM that likely affects the CLV-WUS loop (indicated by the broken line); in pcn, RAM is defective with expanded WOX5

expression and disrupted auxin maxima. Orange lines indicate PIN1 localization; orange arrows show the direction of auxin flow; green highlighted

regions indicate auxin maxima as shown by DR5-GFP expression; and blue highlighted regions show WOX5 expression. Heterozygous mutations in

TPL, PIN1, PID,MP, andWUS genes and homozygous recessive drn in the pcn background are able to progress through embryogenesis and produce

viable seeds with the 298C treatment; however, postembryonically their inflorescence meristems terminate in pin-like structures.


family that have been shown to maintain the activity of stem cells

and control embryo patterning (Haecker et al., 2004; Wurschum

et al., 2006; Chandler et al., 2007; Galinha et al., 2007) are sig-

nificantly upregulated as shown by our qRT-PCR analysis (see

Supplemental Table 2 online). In the embryonic and postembry-

onic RAM, auxin induces the expression of AP2 transcription

factors, PLT1 and PLT2, to specify the QC (Aida et al., 2004), and

both of these genes and WOX5 are upregulated in the pcn em-

bryo (see Supplemental Table 2 online). Collectively, these lines

of evidence indicate that PCN functions as a negative regulator

or repressor with key roles in embryo patterning and meristem

organization/maintenance through auxin mediated signals (Fig-

ure 13).

In conclusion, based on our developmental and genetic stud-

ies, the earliest signs of embryonic pcn defects are associated

with auxin transport and response pathway. These mutant phe-

notypes are further enhanced by mutations in several key com-

ponents of auxin transport and signaling involving pin-1, pid, tpl,

andmp (Figure 13). These genes also have a dosage-dependent

auxin transport and signaling defect–associated pin phenotypes

after bolting (Figure 13). Interestingly, genetic studies with mer-

istem factor WUS also revealed similar embryonic and post-

embryonic phenotypes linking the auxin signaling defects with

embryonic and postembryonic meristems (Figure 13). The ge-

netic and functional framework developed for PCN in this study

(Figure 13) will help future experiments to dissect other compo-

nents ofPCN-mediated pathways that operate during embryonic

and postembryonic development in Arabidopsis.

METHODS

Plant Growth, Mutant Lines, and Map-Based Cloning

Arabidopsis thaliana plants were grown on soil or in sterile culture on 0.53

MSmedium containing MS salts. After incubation for at least 4 d at 48C in

darkness, plants were grown in growth chambers under long-day con-

ditions (16 h light and 8 h dark; light intensity: 60 to 80mmolm22 s21) at 22

or 298C. Homozygous pcn plants and double mutants of pcnwere grown

at 228C until they began to flower and then shifted to 298C, except pcn

bdl, which was grown at 228C. Details of mutants andmarker lines used in

this study are listed in Supplemental Table 3 online. Prior to mapping, the

pcn-1 mutant was backcrossed four times to wild-type Col-0. pcn-1/+

(BC4, Col-0) 3 wild-type Ler and 670 F2 plants were used for mapping.

Crosses with known genetic markers indicated that PCN mapped close

to the centromere of chromosome 4. Because no known genetic markers

were available in this region, we designed 20 CAPSmarkers. Then, 87 F2

plants were assayed for these markers on the long arm close to the

centromere of chromosome 4. The initial mapping was done using simple

sequence repeat markers from The Arabidopsis Information Resource

(http://www.Arabidopsis.org). The derived CAPS markers used for fine

mapping of the pcn-1 mutation were generated based on the sequence

information of Col and Ler.

Construction of pPCN:PCN-GFP and Complementation

Vectors and Transformation into Arabidopsis

pPCN:PCN-GFP constructs were derived as follows: First, we used

forward primer1, 59-AACACTGCAGCGCAATCGCACCTATTTTTAATC-39,

and reverse primer1, 59-AAAAGCGGCCGCGACGGAAGCAGAAGGA-

GAAGTGT-39, to amplify the PCN promoter sequence from genomic

DNA, and the PCN cDNA was amplified by PCR from a wild-type Col

cDNA library using forward primer 2, 59-AAAAGCGGCCGCATGCTC-

GAGTACCGTTGCAGCTC-39, and reverse primer 2, 59-AAAAGGAT-

CCAGTTCCAAAAATATGTCTGTC-39. Both promoter and cDNA PCR

products were digested with NotI and then the fragments were ligated.

Finally, we used the primers 59-AAAGCGGCCGCACACTTCTCCTTCTG-

CTTCCGTC-39 and 59-AAAAGGATCCAGTTCCAAAAATATGTCTGTC-39

to amplify pPCN:PCN using the ligated PCR product as template, which

was fused to GFP. The PCN genomic sequence (At4g07410) for fusion

with GFP was amplified by PCR using F28D6 BAC clone as template

(forward primer, 59-AACACTGCAGCGCAATCGCACCTATTTTTAATC-39;

reverse primer, 59-AAAAGGATCCAGTTCCAAAAATATGTCTGTC-39). The

At1g27470 coding sequence (forward primer, 59-ATGTTTGAGTACC-

GGTGCAGCTC-39; reverse primer, 59-TTATGTCCCGAATATATGTC-

TGT-39) was cloned by PCR using wild-type Col-0 embryo cDNA library

as template. The amplified cDNA and genomic DNAwere gel purified and

cloned into the pCR 2.1 TA vector (Invitrogen; catalog number K4500-01)

and the respective inserts sequenced for confirmation. The 6.5-kb frag-

ment containing the PCN gene (forward primer, 59-GAGCATAATAAACA-

TAAACATTA-39; reverse primer, 59-TCAAGTTCCAAAAATATGTCTGT-39),

the ligated PCN promoter with PCN cDNA, as well as the ligated PCN

promoter with At1g27470 cDNA were subcloned into pRD400 (Datla

et al., 1992) and used to transform pcn-1/+ andpcn-2/+, respectively. The

transformations of pPCN:PCN-GFP and complementation constructs

intoArabidopsiswere performed following the protocol described in Yang

et al. (2009).

PCR-Based Genotyping and qRT-PCR

Genotypes of the pcn-1 and pcn-2 loci in the transgenic plants were

determinedbyPCR. To genotype thepcn-1, PCRprimers (forwardprimer,

59-TCTGAGTAACTGTTTCTTCTGTTGC-39; reverse primer, 59- CCACT-

CACAAGAACTGAACACCT-39) were used; the PCR fragment sizes are

218 bp (mutant) and 254 bp (Col wild type). To genotype pcn-2 (Salk_

022607), forward primer 59-TTCCGGATGATATACTGCCAG-39, reverse

primer 59-CCCCAGGAAACTCTTGATACC-39, andmiddle primer 59-GCG-

TGGACCGCTTGCTGCAACT-39 were used. Other primers and methods

used for PCR genotyping of double mutants in this study are listed in

Supplemental Table 3 online.

The qRT-PCR experiment was performed in triplicate on the Applied

Biosystem Step One real-time PCR system using the SYBR Green PCR

master mix. Three biological replicates of globular to heart stage wild-

type and pcn embryos were isolated, and total RNA samples were iso-

lated as described in RNAqueous-micro kit (Ambion; catalog number

1927). Using the isolated total RNA, linear amplification of antisense

RNAs (aRNAs) was performed as described in the MessageAmp II aRNA

kit following the protocol for first-round aRNA amplification (Ambion;

catalog number 1751). The respective double-stranded cDNAs were

synthesized from aRNA as described in the MessageAmpTM II aRNA kit

(Ambion; catalog number 1751). The concentration of the cDNA template

was measured using Nanodrop 8000 (Thermo Scientific) and further

normalized using tubulin (At5g12250) as reference prior to performing

qRT-PCR. Gene-specific primerswere designed using Primer 3 software.

qRT-PCR primers and results are listed in Supplemental Table 2 online.

Assay for BFA, FM4-64 Staining, Auxin Response, and

IAA Degradation

For BFA treatment and FM4-64 staining, the wild-type and pcn embryos

were isolated and treated as described earlier (Ganguly et al., 2010). To

determine the auxin response of pcn, both mutants and wild-type (Col-0)

seeds were surface sterilized. These seeds were incubated at 48C for 3 d

and then were germinated on 0.53 MS for 4 d. These seedlings were

transferred to 0.53 MS supplemented with 0.2 mg/L NAA. Seedling

4364 The Plant Cell

phenotypes were examined at 2 and 4 d with NAA in the medium. The

response was evaluated by analyzing lateral root development as well

as DR5rev:GFP expression. Both pHS:AXR3NT-GUS and pcn/pHS:

AXR3NT-GUS seeds were germinated in 0.53MS for 5 d and then heat-

shocked for 2 h at 378C and incubated at 208C for 1 to 3 h to check the

degradation of GUS (Gray et al., 2001).

Y2H Assay

The Y2H assay was performed as described in the ProQuest two-hybrid

system with the Gateway Technology Kit protocol (Invitrogen; catalog

number PQ10001-01). Genes and primers are listed in Supplemental

Table 4 online.

Microscopy

Whole-mount embryos were prepared by clearing the ovules in chloral

hydrate solution (8:1:2 chloral hydrate:glycerol:water [w/v/v]) (Christensen

et al., 1998) for 4 h. Slides were viewed under a Leica DMR compound

microscope using differential interference contrast optics. Confocal laser

scanning microscopy was performed using Leica SP2 and Zeiss LSM-

510 microscopes. Histochemical staining for GUS activity in this study

was performed following the protocol of Gray et al. (2001). Lugol staining

of the roots to detect starch granules was performed as described by van

den Berg et al. (1997). Whole-mount in situ hybridization was performed

following the protocol of Hejatko et al. (2006). Scanning electron micros-

copy was performed as described by Venglat et al. (2002).

Phylogenetic Analysis

The FASTA sequences for At1g27470, PCN, and its putative homologs in

other plant genera (Oryza, Sorghum, Populus, Ricinus, and Vitis), yeast,

zebra fish, and humans were aligned using ClustalW (Larkin et al., 2007).

The phylogram was generated using QuickTree (Howe et al., 2002) and

viewed in Archaeoteryx (http://www.phylosoft.org/archaeopteryx) (see

Supplemental Data Set 1 and Supplemental Figure 1 online).

Accession Numbers

Sequence data from this article can be found in the Arabidopsis Genome

Initiative or GenBank/EMBL databases under the following accession

numbers: PCN (At4g07410), pcn-1 (At4g07410), pcn-2 (Salk-022607;

At4g07410), At1g27470, WUS (At2g17950), CLV1 (At1g75820), CLV3

(At2g27250), DRN (At1g12980), STM (At1g62360),MP (At1g19850), BDL

(At1g04550), TPL (At1g15750), PIN1 (At1g73590), PID (At2g23450), PIN7

(At1g23080), WOX5 (At3g11260), PLT1 (At3g20840), and tubulin

(At5g12250).

Supplemental Data

The following materials are available in the online version of this article.

Supplemental Figure 1. Phylogenetic Relationship between PCN

and Its Homolog in Arabidopsis and Putative Homologs from Different

Eukaryotic Species.

Supplemental Table 1. Temperature Shift–Dependent Recovery

of pcn.

Supplemental Table 2. qRT-PCR of Selected Genes That Are

Involved in Embryo Patterning and Shoot/Root Meristems.

Supplemental Table 3. Origins and Ecotypes of Mutants and

Reporter Lines and Genotyping Methods.

Supplemental Table 4. Primers Used for Yeast Two-Hybrid Assay.

Supplemental Data Set 1. Text File of the Sequences and Their

Alignment Used for the Phylogenetic Analysis Shown in Supplemental

Figure 1.

ACKNOWLEDGMENTS

We thank Jeff Long for providing the tpl-1 line, Mark Estelle for providing

the pHS:AXR3NT-GUS line, and John Chandler for providing the drn-1

line. We thank Jeff Long, Mark Smith, and Don Palmer for critical com-

ments on the manuscript. We thank Daryoush Hajinezhad for assistance

with confocal microscopy. This research was supported by the National

Research Council Canada Genomics and Health Initiative (GHI-4). This

is National Research Council Canada publication number 50186.

AUTHOR CONTRIBUTIONS

D.X., P.V., and R.D. designed the experiments. D.X. and H.Y. performed

map-based cloning, made the constructs, performed transformation,

created the doublemutants, and conducted in situ hybridization. P.V. and

D.X. performed microscopy. D.X. and Y.C. performed qRT-PCR exper-

iments. D.X. and R.W. performed the Y2H assay. M.R., S.S.., E.W., H.W.,

W.X., D.W., T.B., T.L., and G.S. provided valuable advice, mutant/

reporter lines, and reagents. D.X., P.V., and R.D. wrote the article with

contributions from G.S., T.B., and T.L.

Received September 15, 2011; revised October 27, 2011; accepted

November 18, 2011; published December 9, 2011.

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DOI 10.1105/tpc.111.091777; originally published online December 9, 2011; 2011;23;4348-4367Plant Celland Raju Datla

Edwin Wang, Hong Wang, Wei Xiao, Dolf Weijers, Thomas Berleth, Thomas Laux, Gopalan Selvaraj Daoquan Xiang, Hui Yang, Prakash Venglat, Yongguo Cao, Rui Wen, Maozhi Ren, Sandra Stone,

ArabidopsisOrganization in Functions in the Auxin Pathway to Regulate Embryonic Body Plan and MeristemPOPCORN

This information is current as of February 16, 2021

Supplemental Data /content/suppl/2011/12/07/tpc.111.091777.DC1.html

References /content/23/12/4348.full.html#ref-list-1

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