ngatha distal organ development genes are essential … · the ngatha distal organ development...

The NGATHA Distal Organ Development Genes Are Essentialfor Style Specification in Arabidopsis W

John Paul Alvarez,a Alexander Goldshmidt,a Idan Efroni,a John L. Bowman,b,c and Yuval Esheda,1

a Department of Plant Sciences, Weizmann Institute of Science, Rehovot, 76100, Israelb School of Biological Sciences, Monash University, Melbourne, Victoria 3800, Australiac Department of Plant Biology, University of California, Davis, California 95616

Floral organ identities are specified by a few transcription factors that act as master regulators. Subsequently, specification

of organ axes programs the distribution of distinct tissue types within the organs that themselves develop unique identities.

The C-class, AGAMOUS-clade MADS box genes are primary promoters of the gynoecium, which is divided into a distal style

and a subtending ovary along the apical-basal axis. We show that members of a clade of B3 domain transcription factors,

NGATHA1 (NGA1) to NGA4, are expressed distally in all lateral organs, and all four have a redundant and essential role in

style development. Loss of all four genes results in gynoecia where style is replaced by valve-like projections and a

reduction in style-specific SHATTERPROOF1 (SHP1) expression. In agreement, floral misexpression of NGA1 promotes

ectopic style and SHP1 expression. STYLISH1, an auxin biosynthesis inducer, conditionally activated NGA genes, which in

turn promoted distal expression of other STY genes in a putative positive feedback loop. Inhibited auxin transport or lack of

YABBY1 gene activities resulted in a basally expanded style domain and broader expression of NGA genes. We speculate

that early gynoecium factors delimit NGA gene response to an auxin-based signal, elicited by STY gene activity, to restrict

the activation of style program to a late and distal carpel domain.

INTRODUCTION

The primarymolecular genetic program underlying the identity of

the concentricwhorls of sepals, petals, stamens, and carpels of the

flower is well characterized. Floral organ identity is governed by

transcriptions factors acting in specific combinations defined

as A, B, and C class to promote identity and in some cases

antagonize genes of other classes that promote an alternative

fate (Coen and Meyerowitz, 1991; Honma and Goto, 2001; Jack,

2004). In the gynoecium, expression of the AG-like genes

AGAMOUS (AG), SHATTERPROOF1 (SHP1), and SHP2 have re-

dundant roles in carpel tissue promotion (Pinyopich et al., 2003).

However, little is known about the mechanism by which AG-like

activities are translated into gynoecium tissue differentiation.

The Arabidopsis thaliana gynoecium is one of the most com-

plex organs of the plant, reflecting its multifunctional role of

housing the female gametophyte, acting as a conduit for pollen

tube growth, and developing into a fruit containing the seed

(Ferrandiz et al., 1999; Balanza et al., 2006). Structurally, the

gynoecium consists of two congenitally fused carpels with

distinct apical-basal, medio-lateral, and abaxial-adaxial pattern

elements (Bowman et al., 1999). Along the apical-basal axis, a

distal stigma caps a short style above an ovary of two locules and

an intervening medial replum connected to the flower receptacle

by a short gynophore (Figure 1). The inner-to-outer axis (adaxial-

abaxial) consists of a central transmitting tissue within a septum

flanked by ovules encapsulated by the valves that have a distinct

adaxial-abaxial histology. The style, septum, and ovules initiate

after the gynoecium has first formed an;200-mm open cylinder

(floral stage 9; Smyth et al., 1990; Figure 1A). Thus, the gynoe-

cium passes through an early phase of growth before differen-

tiating the distal and internal marginal tissues (Alvarez and

Smyth, 2002).

Mutant screens and reverse genetic approaches have identi-

fied a number of genes that function in specifying tissue types

and growth during gynoecium development. These can be

principally categorized into genes that function specifically in

the gynoecium and include the YABBY domain gene CRABS

CLAW (CRC), the MADS domain genes SHP1 and SHP2, the

transmitting tract factors NO TRANSMITTING TRACT and

HECATE1-3, and a regulator of flower meristem determinacy

KNUCKLES (Bowman and Smyth, 1999; Liljegren et al., 2000;

Payne et al., 2004; Crawford et al., 2007; Gremski et al., 2007).

Other genes that function more broadly in plant development

were also co-opted for a particular function in the gynoecium.

These include the meristem genes CUPSHAPED COTYLEDON1

(CUC1) and CUC2, SHOOTMERISTEMLESS (Ishida et al., 2000;

Scofield et al., 2007), as well as the organ growth and polarity

genes and their homologs FILAMENTOUS FLOWER, JAGGED,

KANADI1, ETTIN, AINTEGUMENTA, LEUNIG, SPATULA, and

PICKLE (Elliott et al., 1996; Sessions et al., 1997; Eshed et al.,

1999; Sawa et al., 1999; Siegfried et al., 1999; Heisler et al., 2001;

Ohno et al., 2004; Dinneny et al., 2006; Azhakanandam et al.,

2008; Sitaraman et al., 2008). The organ growth and polarity

genes generally perform roles that are an extension of their roles

1 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: Yuval Eshed ([email protected]).WOnline version contains Web-only data.www.plantcell.org/cgi/doi/10.1105/tpc.109.065482

The Plant Cell, Vol. 21: 1373–1393, May 2009, www.plantcell.org ã 2009 American Society of Plant Biologists

Figure 1. nga1-1 Mutant Phenotypes.

(A) Scanning electron micrographs of Arabidopsis wild-type gynoecium development in progressive stages. The style becomes evident during stage 9

of flower development. All gynoecia are orientated medially. Floral stages after Smyth et al. (1990).

(B) to (H) Scanning electron micrographs of medially orientated mature flowers with some outer floral organs removed to reveal the gynoecium.

(B) pkl-15 kan1-2 gynoecium with basal stigma and style tissue extensions.

(C) pkl-15 kan1-2 nga1-1 gynoecium with external valve and placenta outgrowths extending from the medial and basal positions of the ovary and a

reduced style.

(D) kan1-1 kan2-1/+ gynoecium with basal and medial style and stigmatic tissues extensions.

(E) kan1-1 kan2-1/+ nga1-3 gynoecium revealing valve-like tissue as in (C).

(F) Wild-type flower showing the proximal-distal elements in the gynoecium and bilateral symmetry of the ovary.

(G) nga1-1 gynoecium showing disruptions in style growth.

(H)Magnified view of the distal part of the wild-type gynoecium including the stigma and style (top) and basal region including the gynophore (bottom).

(I) Mature wild-type and nga1-1 flowers. Note the larger petals of nga1-1 mutants.

(J) Wild-type and nga1-1 leaves. nga1-1 leaves are more serrated.

Arrows mark disrupted style fusion and reduced stigmatic papillae. stg, stigmatic papillae; sty, style tissue; va, valve; rep, replum; gyn, gynophore.

Bars = 100 mm in (A) (stage 11 and 12) and 50 mm for stages 7 to 9, 100 mm in (B) to (G), 50 mm in (H), 1 mm in (I), and 1 cm in (J).

outside the gynoecium. Thus, in agreement with its evolutionary

origin, the carpel program overlies the molecular genetic pro-

gram regulating lateral organ development.

Another general process having a patterning role in the gy-

noecium is auxin synthesis and flux. For example, the YUCCA4

gene, which encodes an enzyme involved in auxin biosynthesis,

is apparently activated by the transcription factor STYLISH1

(STY1) (Cheng et al., 2006; Sohlberg et al., 2006). STY1 is

expressed at the distal part of the gynoecium, and its over-

expression results in expansion of style tissue into the valve

domain, while its loss of function results in style reduction (Kuusk

et al., 2002, 2006). Plants mutant in the auxin efflux protein PIN-

FORMED1 (PIN1) (Galweiler et al., 1998) or its polar localization

regulator, thePINOID kinase (PID) (Bennett et al., 1995;Christensen

et al., 2000; Benjamins et al., 2001), exhibit defects in apical-

basal gynoecium patterning. pin1 and pid mutants exhibit in-

creased basal gynophore and apical style/stigma regions and a

reduced ovary. pin1 mutants are defective in polar auxin trans-

port (PAT), and a phenocopy can be produced by treatment with

PAT inhibitors (Okada et al., 1991). This suggests that apical-

basal patterning of the gynoecium is dependent on an auxin

gradient, whereby distal auxin biosynthesis and basal transport

create a decreasing, instructional gradient with high levels of

auxin inducing style and stigma differentiation (Nemhauser et al.,

2000). The dramatic role of an auxin gradient(s) in gynoecium

patterning suggests that dynamic programs that may create only

subtle boundaries and gradients in the development of other

organs (e.g., leaves) are exploited to program distinct tissues

within the gynoecium.

Here, we describe plants lacking activities of four NGATHA

(NGA) transcription factors in which style and stigma are lost and

replaced by outgrowths with valve identity. Members of this

clade were distally expressed in all organs, and their floral

overexpression resulted in ectopic style/stigma morphogenesis.

NGA gene activity can be conditionally activated by STY1, which

promotes auxin biosynthesis (Sohlberg et al., 2006), suggesting

that STY1 may act on NGA indirectly through an auxin-related

signal. NGA gene activity then promotes AGAMOUS clade and

SHI/STY family members in a presumptive positive feedback

loop to program style and stigma morphogenesis.

RESULTS

Identification of the nga1Mutation

Mutant screens in a crc-1 background helped identifying genes

regulating organ polarity, growth, and flower meristem determi-

nacy (Eshed et al., 1999; Bowman et al., 2001; Prunet et al.,

2008). Both crc pickle and crc kanadi double mutants have

adaxial tissues developing in abaxial positions, implicating these

genes in regulating carpel polarity. The gynoecium of gym-5

kan1-2 and kan1-2 kan2-1/+ have style-like outgrowths that arise

from the base and medial replum of the gynoecium (Figures 1B

and 1D). Mutagenesis in the gym-5 kan1-2 background identified

two independent alleles of ngatha1 (nga1-1 and nga1-2). A third

allele was identified in the kan1-2 kan2-1/+ background (nga1-3).

In both backgrounds, extensions of style-like tissue were re-

placed by a proliferation of outgrowths with valve-like identity. In

addition, the distal stigma and style were comparatively reduced

and the style was split in the medial plane (Figures 1C and 1E).

This phenotype was clearly distinct from that produced when the

carpel polarity gene, CRC, was reduced in the same back-

grounds (see Supplemental Figures 1A to 1C online; Eshed et al.,

1999).

In an otherwise wild-type background, nga1 gynoecia had an

occasional misshapen or split style in the medial plane and

reduced stigmatic papillae (Figure 1G) but were otherwise fully

fertile. Gynoecium phenotypes of double mutant combinations

between nga1 and other mutations effecting gynoecium devel-

opment, including ettin, sty1, spatula, and crc, were largely

additive (see Supplemental Figures 1D to 1Monline). Other nga1-1

phenotypes in the flower included an enlargement of the petals

(Figure 1I), apparently due to increased petal cell number (see

Supplemental Figures 1N and 1O online). In the vegetative

phase, nga1 leaves were more serrated than their wild-type

counterparts (Figure 1J).

NGA1 Is Part of a Small Clade of B3 Domain–Containing

Proteins That Have a Redundant Role in Style

Morphogenesis and Organ Development

The NGA1 gene was identified by map-based cloning as

At2g46870 (Figure 2B; see Supplemental Figure 2A online). A

10.5-kb genomic fragment spanning the locus complemented

the nga1-1 mutation in the nga1-1 kan1-1 background. NGA1

encodes a RAV-B3 domain–containing protein (Kagaya et al.,

1999) and is amember of a clade containing four genes (NGA1 to

NGA4; Alvarez et al., 2006) that has a sister clade of three genes.

None of the seven genes possess an AP2 domain characteristic

of theRAV genes (NGAL; Figure 2A; see Supplemental Figure 2B

and Supplemental Data Set 1 online; Swaminathan et al., 2008).

Isolation of mutant alleles in allNGA genes and examination of

multiple mutant combinations illustrated their redundancy in

carpel patterning (Alvarez et al., 2006; Figures 2C to 2F and 2J to

2M). All four genes play a quantitative role in gynoecium and

lateral organ development (Figures 2G to 2I; see Supplemental

Figures 3A and 3B online). Progressive reduction in the activity of

these genes resulted in reduced style and stigma development

(Figures 2C to 2F and 2J to 2M), leaves that were wider andmore

serrated than nga1-1 single mutants (Figure 2G; see Supple-

mental Figure 3B online), and shorter but wider sepals and petals

(Figures 2G to 2I; see Supplemental Figure 3A online). Genetic

analyses suggested that the relative contribution of the muta-

tions to the severity of phenotype was nga1-1>nga3-1>nga4-

1>nga2-1. The loss ofNGA gene activity culminated in the nga1-1

nga2-1 nga3-1 nga4-1 quadruple mutant (termed nga quadruple

mutant hence), where effects on growth in all aerial organs were

most severe (Figures 2G to 2M; see Supplemental Figure 3

online). In the gynoecium, the style and stigma tissues were

replaced by projectionswith valvemorphology (Figures 2J to 2M;

see Supplemental Figures 4A to 4E online). The solid, wild-type

style has a distinct crenellated epidermis with wax deposits and

open stomata with an internal, central core of transmitting

tissue (Figures 1H, 2J, and 2K). The ovary valve wall consists

of an abaxial epidermis with small irregular rectangular cells

Style Specification by NGA Genes 1375

Figure 2. Redundant Genetic Interactions among Members of the Monophyletic NGA Clade.

1376 The Plant Cell

interspersed with immature stomata that open during fruit de-

velopment. This overlays three cell layers of chlorenchyma

followed by a distinct adaxial subepidermal layer of longitudinally

elongate cells and a radially elongated inner epidermis (Figure

2K; see Supplemental Figures 4A and 4B online) that becomes

lignified in fruit development (Liljegren et al., 2000). These char-

acteristic adaxial epidermal cell layers were observed in distal

projections of the nga quadruple mutant, with more basal sec-

tions revealing an essentially normal ovary (Figure 2M; see

Supplemental Figures 4D and 4E online). The nga quadruple

mutant gynoecium is indistinguishable from the wild type until

stage 9 of flower morphogenesis (see Supplemental Figures 5A

to 5D online), coincident with the time of style and stigmatic

papillae initiation (Smyth et al., 1990). These observations sug-

gest that the NGA genes have a specific role in style morpho-

genesis rather than having an indirect or pleiotropic function.

NGA Gene Activity Is Required for Distal Carpel Identity

The complete loss of style and stigmatic papillae in nga quadru-

ple mutants implies an essential role for the NGA genes in

promoting these tissues. Since members of the AGAMOUS

clade, AG, SHP1, and SHP2, play a central, redundant role in

specifying carpel identity (Pinyopich et al., 2003), we examined

the effects of NGA gene activity on expression of SHP1. To

facilitate this analysis, we used an artificial microRNA that

simultaneously targets the fourNGA transcripts based on shared

homology of a 21-nucleotide sequence (Alvarez et al., 2006).

Overexpression of amiR-NGA164a using the constitutive viral 35S

promoter mimicked the nga quadruple mutant phenotype (see

Supplemental Figures 3C to 3E online). SHP1 expression is

exclusively in the gynoecium and in the style and is initiated in the

stage 6-7 gynoecium in the medial domain that will become the

abaxial replum. During stage 9-10, a new expression domain that

marks the top of the gynoecium and the developing style is

initiated and maintained until stage 12 (Flanagan et al., 1996;

Bowman et al., 1999; Figures 3A and 3C). This distal domain of

SHP1 expression is missing in 35S:amir-NGA164a gynoecia (Fig-

ures 3B and 3D). These observations suggest that NGA gene

activity is upstream of SHP1 style expression. If a lack of AG-like

gene expression is responsible for the reduction of style and

stigma tissues, then artificially elevated AG expression could

rescue components of the nga gynoecium phenotype. LEAFY

(LFY) is an activator of AG (Busch et al., 1999), and plants

homozygous for an activated form of LFY (LFY-VP16) that drives

high levels of AG have flowers that are composed of only carpel

tissue topped by abundant style and stigma (Figure 3E; see

Supplemental Figures 5E to 5H online). Similarly, overexpression

of the tomato (Solanum lycopersicum) AGAMOUS gene (TAG1)

(Pnueli et al., 1994) results in flowers with sepals homeotically

converted to carpels with extensive marginal style and stigmatic

tissue (Figure 3G) (as is observed with overexpression of the

Arabidopsis AG gene [Mizukami and Ma, 1992] but without the

frequent cosuppression of the transgene and endogenous AG

gene). Combining 35S:amiR-NGA164a with either the LFY-VP16

or the 35S:TAG1 background resulted in plants where style and

stigmatic tissues were eliminated, both in the carpelloid sepals

and in the central gynoecium (Figures 3F and 3H). Thus, NGA

genes are essential for all style and stigma development in the

flower, a role that cannot be substituted for by solely elevating

AG-like gene activity.

Vegetative stigmatic papillae development is evident on

the cauline leaves of plants overexpressing miR172 (Aukerman

and Sakai, 2003). miR172 targets five APETALA2 family genes

that repress flowering and includes the floral homeotic gene

APETALA2 (AP2) (Aukerman and Sakai, 2003; Chen, 2004). Amod-

ified version of miR172d, miR172dm7, produces a more severe

phenotype (see Supplemental Figure 6 online) when expressed

using the AINTEGUMENTA (ANT) promoter (Elliott et al., 1996;

Schoof et al., 2000), including severe curling and significant

stigma development at the tips of the first leaves as well as

flowers composed only of carpels (Figure 3I; see Supplemental

Figure 2. (continued).

(A) Cladogram showing phylogenetic relationships of the RAV clade of B3 transcription factors in land plants (angiosperms,green; gymnosperms, blue;

lycophyte, orange; moss, red; liverwort, purple). A complete cladogram with detailed analysis is presented in Supplemental Figure 2 online. Some

members have an amino AP2 domain (+), while other members lack this domain (�). That the Physcomitrella and at least some of the Selaginella genes

possess an AP2 domain suggests that it has been lost in some clades of flowering plant genes.

(B) A scheme of the NGA genes. Rectangles are exons and lines are introns. Filled rectangles denote the coding region for which the start and stop

codons, the ethyl methanesulfonate-generated alleles, the positions of insertions, and the highly conserved 120–amino acid B3 domains are marked.

(C) to (F) Gynoecium phenotypes of lines with a progressive reduction in NGA levels. Genotypes are as labeled.

(G) Leaf and flower phenotypes of nga2-1 nga3-1 nga4-1 triple and nga quadruple mutant plants relative to the wild type. The mature sixth leaf was

taken in each case.

(H) and (I) Scanning electron micrographs of wild-type (H) and nga quadruple mutant (I) inflorescences from an aerial perspective.

(J) Scanning electron micrograph of a medial view of a wild-type gynoecium at maturity.

(K) Light microscope sections through the distal style (top) and basal ovary (bottom) of a mature wild-type gynoecium taken at approximately the

locations demarked by the lines in (J). The radially elongated adaxial epidermal and longitudinally elongated subepidermal cells are unique to the valve.

(L) Scanning electron micrograph of a medial view of a mature nga quadruple mutant gynoecium where the style and stigma are absent and replaced by

outgrowths with valve identity.

(M) Light microscope sections though the distal (top) and central ovary (bottom) regions of a mature nga quadruple mutant gynoecium. The

approximate, relative locations of the sections are marked by white arrows in (L). The outgrowths of tissue in the distal region have a valve identity with

the typical adaxial epidermal and subepidermal cell layers (arrowhead) (K). The ovary (bottom) exhibits an apparently wild-type structure.

stg, stigmatic papillae; sty, style tissue; va, valve; ade, adaxial epidermis; sade, subadaxial epidermis; rep, replum; gyn, gynophore; tt, transmitting

tissue; ov, ovule; spm, septum. Bar are as marked in (C) to (F), (H), and (I). Bars = 1 cm in (G) for leaves and 1 mm for petals, and 100 mm in (J) to (M).


Figure 3. NGA Gene Activity Is Necessary for Normal and Ectopic Style Tissues.

(A) to (D) Histochemical localization of SHP1:GUS in developing wild-type and 35S:amiR-NGA164a gynoecia.

(A) and (B) Aerial view of wild-type (A) and 35S:amiR-NGA164a (B) inflorescences showing SHP1:GUS staining at the tips of developing gynoecia.

Developmentally consecutive flowers are labeled using roman numerals. In the wild type, SHP1:GUS expression is initiated in the medial ridge, the

progenitors of the placenta and septum (arrow), and later expands to the lateral and medial regions of the initiating style (asterisk).

(C) Medial view of a stage 10-11 wild-type gynoecium showing intense SHP1:GUS staining in the lateral domain of the developing style (arrow).

(D) Medial view of a stage 10-11 35S:amiR-NGA164a gynoecium. SHP1:GUS expression is absent from the equivalent domain (arrow).

(E) and (F) Side view of inflorescences of plants homozygous for the LFY:LFY-VP16 transgene alone (E) and hemizygous for 35S:amiR-NGA164a (F).

(E) LFY:LFY-VP16 flowers are composed of carpel tissue topped by enlarged stigmas.

(F) 35S:amiR-NGA164a LFY:LFY-VP16 flowers lack style and stigma.

1378 The Plant Cell

Figure 6 online). Vegetative stigma development was abolished

when amiR-NGA164a was coexpressed with miR172dm7

(ANT>>miR172dm7 35S:amiR-NGA164a; Figure 3J; >> denotes

transactivation; Moore et al., 1998), suggesting that the AP2

family genes targeted bymiR172 restrict the carpel program that

recruits NGA activity for stigma development.

Extending the use of amiR-NGA164a for other genetic analy-

ses, we found that the 35S:amir-NGA164a combinations with

spt-2, sty1-1, gym-5 kan1-2, kan1-1 kan2-1/+, kan1-1 kan2-1

(Eshed et al., 2001), and amiR-ARF164b (Alvarez et al., 2006)

were largely additive (see Supplemental Figures 7A to 7K

online). Notably, however, in 35S:amir-NGA164a crc-1 gynoecia,

the unfused carpels comprising the distal part of the gynoecium

were sepal-like in appearance, including long, large epidermal

cells and lighter colored marginal cells (Figures 3K to 3P).

Sections through the distal region of the 35S:amir-NGA164a

crc-1 gynoecium revealed a reduction of valve-like histological

features in the upper gynoecium and the presence of large,

sepal-like, epidermal cells, but with a lack of significant air-

spaces that characterize the spongy mesophyll of wild-type

sepals. By contrast, the basal fused carpels of the 35S:amir-

NGA164a crc-1 gynoecium retained all characteristics of a wild-

type ovary (Figures 3M to 3O). In wild-type flowers, expression

of the enhancer trap line YJ158 (Eshed et al., 2004) is restricted

to the large epidermal cells of the sepals (Figure 3Q). In 35S:

amiR-NGA164a crc-1 flowers, YJ158 expression was observed

in cells in the unfused carpels of the upper gynoecium from

stage 9, as well as in the sepals, confirming a sepal-like identity

in the distal region of 35S:amiR-NGA164a crc-1 carpels (Figure

3R; see Supplemental Figures 7L to 7Q online). Thus, coinci-

dent loss of CRC and the NGA genes allows the distal gynoe-

cium to pursue a partial sepal identity program.

NGA4andNGA1Expression IsConfined to theDistal Region

of Lateral Organs

The nga1-1 mutation enhances abaxial growth in the kan1-2

background (Figures 1B to 1D), suggesting that the NGA genes

may have an abaxial polarity function, while the reduction of style

and stigmatic tissues in nga quadruple mutants (Figure 2L)

implies the NGA genes regulate tissue specification and growth.

Establishing the expression domain of these genes can help

resolve their function. The nga4-1 allele is a Ds gene trap (Figure

2B) that is referred to as nga4:b-glucuronidase (GUS). Expres-

sion of this line is observed at the tips of the cotyledons and

leaves and at leaf hydathodes (Figures 4A to 4C). In flowers,

nga4:GUS is similarly expressed at the distal region of all floral

organs (Figure 4D), with expression evident only after organ

initiation. No nga4:GUS expression is observed before late stage

6-7 of flower development (Figures 4E and 4G), at which time

expression is first observed in the developing sepals and sta-

mens (Figure 3F). Only during stage 9 is nga4:GUS expression

observed at the tip of the developing gynoecium coincident with

the time of style initiation (Figure 1A; see Supplemental Figures

5A to 5D online). By stage 12, nga4:GUS expression is observed

at the distal domain of all floral organs (Figures 4H and 4I) and in

the gynoecium becomes confined to the style and stigma.

Likewise, nga4:GUS expression is observed in the ectopic style

and stigma tissues of kan1-2 kan2-1/+ gynoecia (Figure 4J).

Evidence that this expression domain marks the distal end of the

gynoecium, and not merely the style and stigma, comes from

staining at the apex of the nga quadruple mutant gynoecium

where the style and stigma are lost (Figure 4K).

To examine NGA1 expression, a 5-kb fragment upstream of

the NGA1 start codon was used in the transactivation system


(G) and (H) Aerial view of inflorescences of plants overexpressing the tomato AGAMOUS (TAG) gene alone (G) and together with 35S:amiR-NGA164a (H).

(G) In 35S:TAG flowers, the sepals display extensive style and stigma tissue formation (arrow).

(H) In 35S:TAG 35S:amiR-NGA164a plants, the ectopic and normal style and stigma tissues are lost (arrows).

(I) and (J) Aerial views of plants overexpressing a modified version of miR172d, miR172dm7 (I) and miR172dm7 together with amiR-NGA164a (J).

(I) ANT>>miR-172dm7 plants produce small leaves that are topped by stigmatic papillae (left; arrows) and flowers typically composed entirely of

carpels (right; arrow).

(J) ANT>>miR-172dm7 35S:amiR-NGA164a plants lacking ectopic and normal style (arrows).

(K) to (R) Sepal-like identity in carpel tips lacking both CRC and NGA gene activities.

(K) A wild-type sepal. Note the longitudinally elongate epidermal cells (arrow) and the pale band of marginal cells (arrowhead).

(L) Gynoecium from 35S:amiR-NGA164a crc-1 flower. The distal regions of the carpels (bracketed) include large longitudinally elongated epidermal cells

(arrow) and marginal band of pale cells (arrowhead) not found in normal carpels.

(M) Sections though the upper and lower 35S:amiR-NGA164a crc-1 gynoecium in positions represented by the two arrows in (L). The upper gynoecium

lacks carpel tissues in contrast with the base where tissues similar to those of a wild-type ovary are found. The lamina tissue in the upper gynoecium has

features of both sepal and valve detailed in (N).

(N) Section through a wild-type sepal. The sepal has distinct large longitudinally elongated cells at the abaxial epidermis (arrow).

(O)Magnified image of the upper and lower 35S:amiR-NGA164a crc-1 gynoecium (boxed in [M]) with sepal-like cells at the upper domain (arrow). Mutant

upper valves also lack the characteristic adaxial subepidermal cell layer of carpels (arrowhead).

(P) Scanning electron micrograph of a 35S:amiR-NGA164a crc-1 flower with a sepal, petals, and stamens removed. Large abaxial epidermal cells can be

observed in the upper carpel domain and in the sepals (arrows).

(Q) Staining of YJ-158:GUS is limited to the long abaxial epidermal cells of wild-type sepals (arrow) and is not observed at any stage in the wild-type

gynoecium.

(R) In 35S:amiR-NGA164a crc-1 gynoecia, staining from YJ-158:GUS is also found in the long epidermal sepal-like cells in the upper gynoecium (arrows).

cot, cotyledon; va, valve; rep, replum; tt, transmitting tissue; ov, ovule; spm, septum; gyn, gynoecium; sep, sepal. Bars = 100 mm in (A) to (D) and (K) to

(R), 5 mm in (E), (F), (I), and (J), and 3 mm in (G) and (H).


Figure 4. Expression of NGA4 and NGA1 Is Late and Distal in All Floral Organs.

(A) nga4:GUS fusion structure based on sequenced RT-PCR products. NGA4 sequence is in brown, pWS32 gene sequence trap is underlined, and that

of the GUS gene is also in blue. Two stop codons, highlighted in red, truncate the nga4-1 protein. Intact translation from the ATG of the GUS gene is

highlighted.

(B) to (K) Expression of nga4:GUS in seedlings (B), leaves (C), and flowers of wild type ([D] to [I]), nga4-1/+ kan1-2 kan2-1/+ (J), and nga quadruple

mutants (K).

(L) to (Q) NGA1>>GUS expression in seedlings (L), leaves (M), and flowers ([N] to [Q]) of the wild type.

(R) and (S) NGA1>>RFP (dsRED) localization in wild-type (R) and kan1-2 kan2-1/+ (S) gynoecia.

pe, petal; st, stamen; gy, gynoecium; se, sepal. Bars = 1 mm in (B) to (E) and (L) to (N), 200 mm in (I) to (K), (R), and (S), and 100 mm in (E) to (H) and (O)

to (Q).

1380 The Plant Cell

(see Methods). The NGA1:LhG4 driver line, which complemented

nga-1 upon transactivation of OP:NGA1, was subsequently

used to drive either GUS or dsRED reporters. Expression driven

by the NGA1 promoter was similar to that of nga4:GUS, being

confined to the tips of the cotyledons and leaves and leaf

hydathodes (Figures 4L and 4M), and late in the distal part of

the flower organs (Figure 4N). In the flowers, initial expression

during stage 6 is restricted to the sepals and during floral stages

7-8 is seen in stamens too. During late stage 9, NGA1 expres-

sion is observed at the distal part of the gynoecium, at the time of

style initiation (Figure 1A) and continues to be expressed in the

style and stigma up to stage 13 of gynoecium development

(Figures 4P to 4R). Like NGA4,NGA1 is expressed in the ectopic

style and stigmatic tissues of kan1-2 kan2-1/+ gynoecia con-

sistent with the role of NGA1 in maintaining their identity

(Figures 1B to 1E and 4S). These observations suggest that

NGA genes can function as specific tissue identity factors in

the gynoecium and general distal factors in all tissues. To investi-

gate this, we performed overexpression studies with all the

NGA genes.

NGA Gene Overexpression Promotes Ectopic Style Tissue

Development in the Flower

All four NGA genes were cloned behind an array of the lac

operator (OP) sequences followed by a TATA box for over-

expression analyses by select transactivating driver lines, which

contain the LacIH17-GAL4 (LhG4) chimeric transcription factor

expressed under the control of a tissue-specific promoter.

Transactivation of each gene by the ANT:LhG4 driver resulted

in reduced cotyledon and leaf growth, cotyledon fusion, and

occasional leaf fusion (see Supplemental Figure 8 online). In this

experimental system, the NGA1 gene had the most potent

effects, followed by NGA4 and NGA3, with NGA2 giving the

weakest phenotype (largely reflecting the contribution of each

gene in the loss-of-function analysis).

To test the function of the NGA genes in a carpel-specific

context, effects of NGA1 expression by the CRC:LhG4 driver

line was studied. This line is expressed throughout the carpel

valve anlagen during gynoecium initiation in stage 5 flowers as

well as the developing nectaries but not in the other floral

organs (Bowman and Smyth, 1999; Alvarez et al., 2006). Stig-

matic papillae were initiated precociously in CRC>>NGA1

gynoecia and histologically the mature gynoecium appeared

to be composed primarily of style and stigma tissues (Figures

5B to 5E; see Supplemental Figures 4F and 4G online). Having

an additional copy of the OP:NGA1 transgene resulted in more

extensive production of stigmatic papillae in a basally ex-

panded domain (see Supplemental Figures 9A and 9B online).

Notably, tissue in nectary positions was topped by stigmatic

papillae-like cells (Figures 5F and 5G). The YJ-STIG::GUS

marker is expressed specifically in maturing stigmatic papillae

of wild-type gynoecia (Figure 5H). InCRC>>NGA1, staining was

apparent earlier in the distal region of the gynoecium (Figure 5I)

and eliminated in 35S:mirNGA164a gynoecia that lack style and

stigmatic tissues (Figure 5J). Strong YJ-STIG:GUS expression

was observed in the papillae-like cells in nectary positions of

CRC>>NGA1 flowers (Figures 5K and 5L), confirming that

ectopic NGA1 expression in this domain drives stigmatic pa-

pillae development.

We subsequently expressedNGA1 at high levels in the flower

outside of the gynoecium. Expression of the NGA1 alone, or

NGA1 fused to yellow fluorescence protein (YFP) at the C

terminus (NGA1-YFP), under control of the flower meristem

driver AP1:LhG4 (Alvarez et al., 2006; AP1>>NGA1:YFP) had

reduced floral meristems topped by stigmatic papillae (Figures

5M to 5O). Fluorescence of NGA1:YFP was nuclear localized,

consistent with the proposed DNA binding function of B3

domain proteins (Yamasaki et al., 2004; Figures 5N and 5O).

AP3:LhG4 (AP3) driver expression is restricted to the sepal

margins, petal, and stamen anlagen and primordia (Alvarez

et al., 2006). In AP3>>NGA1 plants, all tissues arising internal

to the sepals develop into a cylinder of carpelloid style tissue

(Figure 5Q) and were accompanied by expanded and elevated

expression levels of SHP1:GUS (Figures 5R and 5S). This

included an ectopic, precocious differentiation of stigmatic

papillae at the sepal margins of young stage 7 flowers (Figure

5Q). Expression of NGA1 in stamen and carpel anlagen and

primordia using the AG promoter similarly resulted in a cylinder

of style tissue, but in this case, interior to the petals (see

Supplemental Figures 9C and 9D online). To investigate the

organ fusion and style tissue development induced by NGA1

overexpression, we used a weak NGA1 overexpression line

(OP:NGA1weak, NGA1w; see Supplemental Figure 8 online)

and a short version of the AP3 promoter (AP3short:LhG4;

AP3s) that initiates expression in petal and stamen primordia

only at stages 6 to 7 (Figure 5U). In AP3>>NGA1w flowers,

sepal-petal-stamen fusion events as well as reduced organ

growth were observed, but organ identity was less affected

(Figure 5T). In AP3s>>NGA1, petal growth was repressed,

while third whorl organs were filamentous and topped by

stigma-like tissue (Figure 5V) that ectopically expressed

SHP1 (Figures 5W and 5X) andCRC (see Supplemental Figures

9E to 9G online).

These observations suggest that NGA gene activity elicits

different effects depending on the timing and level of expression.

Early NGA gene expression can disrupt the organ separation

program as well as reduce organ growth. Low to intermediate

levels of NGA activity within the organs reduces growth, while, in

the flower, high levels promote a program of style/stigma tissue

morphogenesis.

These NGA1-based observations were also confirmed for

otherNGA genes. Smaller petals were produced in AP3>>NGA4

and AP3>>NGA3 flowers, while in AP3>>NGA4/NGA4 flowers,

filamentous third whorl organs topped by stigmatic papillae and

ectopically expressing SHP1 were observed (see Supplemental

Figures 9H to 9N online). In addition, ANT>>NGA3 and ANT>>

NGA4 plants exhibited organ separation defects in the inflores-

cence and the flower (see Supplemental Figures 9O to 9Ronline).

Lastly, since no function has been assigned to members of

the B3 encoding NGA-like sister clade (Figure 2A), we assayed

one of these genes, NGAL1 (At2g36080), by overexpression.

ANT>>NGAL1, AP1>>NGAL1, and AP3>>NGAL1 phenotypes

were similar to those elicited by NGA1 overexpression (see

Supplemental Figure 10 online), suggesting that NGAL1 targets

may overlap with those of the NGA genes.


Figure 5. NGA1-Mediated Promotion of Ectopic Style within the Flower.

1382 The Plant Cell

STYLISH1 Activity Acts as a Strong Facultative Promoter of

NGA Gene Activity

The loss of style tissues in NGA loss-of-function plants, the

expression domain of NGA1 and NGA4, and the strong promo-

tion of style tissues by NGA1 suggest that activation of the NGA

genes in the distal gynoecium during stage 9 promotes a style

and stigma program. Overexpression of members of the STY

gene family also promote ectopic style tissue formation in the

valve (Kuusk et al., 2002). Since STY1 expression initiates at the

apex of stage 6 gynoecia (Kuusk et al., 2002), preceeding NGA1

and NGA4, it represents a potential upstream regulator of the

NGA genes. As the sty1-1 phenotype is relatively weak (see

Supplemental Figure 1H online), reflecting significant redun-

dancy between STY1 and related family members (Kuusk et al.,

2006), we pursued possible links between STY gene activity and

NGA gene activation by overexpressing STY1. Overexpression

of OP:STY1 driven by the CRC promoter resulted in a minute

gynoecium that appeared histologically undifferentiated relative

to the wild type (Figures 6A to 6C; see Supplemental Figures 4H

and 4I online). This gynoecium lacked the ectopic style formation

observed when NGA1 was expressed with the same promoter

(Figure 5B), and the apical stigmatic papillae was reduced

(Figures 6A to 6C), as was nga4:GUS expression relatively to

nga4-1/+ gynoecia (cf. Figure 6D with 6L). CRC>>STY1/amiR-

NGA164a plants lacked stigmatic tissue, but other elements of the

CRC>>STY1 gynoecium histology remained unchanged (cf.

Figures 6E with 6F). These results are in contrast with results

from other circumstances of STY1 activation, in which strong

induction of ectopic style were the common theme (Kuusk et al.,

2002; Sohlberg et al., 2006; Staldal et al., 2008).

The differential style promotion by STY1 may represent tem-

poral or quantity-specific responses to this protein. The CRCw:

LhG4 promoter line (CRCw) is expressed at the same domain as

the CRC promoter, but a lower level (Figures 6J and 6K).

Strikingly, in CRCw>>STY1 flowers, the entire ovary surface

acquired style epidermal histology that was eliminated in

CRCw>>STY1/amiR-NGA164a flowers, indicating that it is NGA

dependent (Figures 6G to 6I; see Supplemental Figures 11A

to 11C online). Consistent with this, nga4:GUS expression

was observed to be broadly, ectopically expressed in the

CRCw>>STY1 gynoecium and nectary positions (Figures 6L to

6N). Significantly, ectopic expression of nga4:GUS was not ob-

served in CRC>>NGA1 or CRCw>>NGA1 gynoecia (Figures 6O


(A) Expression of CRC>>GUS in a stage 12 flower is observed in the style, ovary, and nectaries (arrow).

(B) Scanning electron micrograph of a stage 13 CRC>>NGA1 gynoecium that lacks overt evidence of ovary development and is topped by abundant

stigma. The epidermal cells (insets) are similar to that of wild-type style (top inset).

(C) Sections through a CRC>>NGA1 gynoecium at positions marked by the arrows in (B). The top section is similar to that of the wild-type style (see

Figure 1), while the bottom section lacks any evidence of wild-type ovary tissues.

(D) and (E) Scanning electron micrographs of a stage 9 gynoecial cylinder. In the wild type (D), there is no evidence of stigmatic papillae development,

while in the CRC>>NGA1 gynoecium (E), precocious stigmatic papillae differentiation is apparent (arrow).

(F) Wild-type nectary tissue arising from the base of the lateral stamen (arrow).

(G) The structure developing in the nectary position at the base of the lateral stamens in mature CRC>>NGA1 flowers has papillae-like projections

(arrow).

(H) to (L) Expression of the YJ-STIG:GUS marker.

(H) Wild-type inflorescence with staining in the maturing stigma starting at stage 11 flowers.

(I) CRC>>NGA1 inflorescence with staining of younger flowers than the wild type.

(J) 35S:amiR-NGA164a inflorescence with no staining in any maturing flowers (arrow).

(K) A stage 13 wild-type flower with staining in the stigmatic papillae only.

(L) A stage 13 CRC>>NGA1 flower with additional expression in the nectary position (arrow).

(M) to (O) Morphology (M) and nuclear localization of fluorescence in AP1>>NGA1-YFP inflorescences ([N] and [O]).

(M) Normal flowers are replaced by attenuated floral meristems topped by stigmatic papillae.

(N) and (O) Fluorescence detected by a two-photon excitation microscope (N) or confocal laser scanning microscopy (O) where conuclear localization

of the NGA1-YFP (green) and 4’,6-diamidino-2-phenylindole (blue) is shown in the bottom panel.

(P) RFP fluorescence in a longitudinal section of a stage 3 AP3>>NLSx4-RFP flower.

(Q) AP3>>NGA1 flowers. Light microscope image of inflorescence (left), an aerial scanning electron microscope view (bottom inset), and a flower (top

inset) showing early, ectopic differentiation of stigmatic papillae on and interior to the sepals (arrow).

(R) and (S) Expression of SHP1:GUS in developing wild-type (R) and AP3>>NGA1 (S) gynoecia. Developmentally consecutive flowers from the time of

SHP1:GUS appearance are labeled using roman numerals.

(T) A range of phenotypes obtained when the weak OP:NGA1w line is driven by the AP3 promoter. Growth of the petals and stamens is reduced and

petals-stamen and sepal-petal-stamen fusions occasionally occur. A wild-type petal is a control.

(U) RFP fluorescence (appearing yellow) in a stage 7 flower marks the AP3short (AP3s) expression domain, which is initiated in stage 6-7 petal and

stamen primordia.

(V) An AP3s>>NGA1 flower with sepal removed. Petal and stamen growth is significantly reduced, and filamentous structures topped by stigmatic

papillae are observed in the third whorl (arrow).

(W) and (X) Expression of SHP1:GUS in stage 12 wild-type (W) and AP3s>>NGA1 (X) flowers. In the mutant, ectopic expression is observed in third

whorl filamentous structures (arrow).

Bars = 100 mm in (A) to (C), (K), (L), and (Q) (flower) to (T), 50 mm in (D) to (G) and (V) to (X), 1 mm in (H) to (J), 20 mm in (N) and (O), and 1 mm in (M)

and (Q).


Figure 6. NGA Genes Exhibit a Strong but Facultative Response to STY1 Activity.

(A)CRC>>STY1 flower with a severely affected gynoecium, whereas other floral organs are largely unaffected. The apical stigmatic papillae are reduced

in their growth (arrow).

(B) Scanning electron micrograph of the CRC>>STY1 gynoecium surface. The epidermis is composed of small cells without significant cuticular

outgrowths and numerous intervening immature stomates.

(C) Transverse sections through the CRC>>STY1 gynoecium at the positions marked by arrows in (B). No tissues with the characteristic histology of the

mature wild-type gynoecium are apparent, although a rudimentary medial ridge (arrow) is observed.

(D) Expression of nga4:GUS in CRC>>STY1 gynoecium is reduced compared with the wild-type (L).

(E) Gynoecium of a plant cotransactivating STY1 and amiR-NGA164a by the CRC promoter (CRC>>STY1/amiR-NGA164a). Stigmatic papillae are lacking

(arrow), but the abnormal histology of the epidermal surface appears largely unchanged (F).

(G) to (I) Scanning electron micrographs of a gynoecium with magnified views of the epidermis marked by the arrows.

(G) In the wild type, the style and ovary have distinct cuticular morphologies.

1384 The Plant Cell

and 6P), except occasionally and then weakly in nectary posi-

tions (see Supplemental Figures 11D to 11F online). These ob-

servations indicate that STY1 can function as a strong promoter

of NGA gene activity in a level-dependent manner, while NGA4

expression is highly responsive to STY1, but not to NGA1, or to

the presence of differentiating style and stigma tissues per se.

To further compare the effects of NGA and STY gene activity,

STY1was transactivated by the same driver lines used for NGA1

overexpression outside the gynoecium. In AG>>STY1 flowers,

the stamens were converted into broad, lobe-like structures that

had an epidermal morphology similar to style tissue (see Sup-

plemental Figure 11G online). In the AP3>>STY1 background,

the flower was greatly reduced in size, and second and third

whorl organ development was strongly suppressed (Figure 6Q).

In AP3s>>STY1 flowers, a suppressed second whorl was fol-

lowed by abnormal third whorl organs with occasional stigmatic

papillae that ectopically expressed gynoecium markers SHP1:

GUS (Figures 6R and 6S) and CRC:GUS (see Supplemental

Figures 11Hand 11I online). This ectopic carpel tissue andSHP1:

GUS expression was significantly reduced when NGA gene

activity was compromised in AP3s>>STY1/mirNGA164a flowers

(Figures 6T and 6U), indicating that STY1 promotes ectopic

carpel tissue through the NGA gene pathway.

Reduced YABBY Gene Activity and Auxin Transport Result

in Earlier and Expanded NGA Activity

ExtensiveNGA-dependent style tissue development when STY1

is ectopically expressed (Figures 6G to 6I) demonstrates that

STY1 can activate theNGA genes. However, there is a significant

temporal separation between the stage 6 onset of STY1 (Kuusk

et al., 2002) and the stage 9 activation of NGA gene expression

(Figure 4). Activation ofNGA activity in response to STY1may be

prevented by factors acting before stage 9. Signature features of

reduced activity in such NGA gene repressors would be earlier,

broader NGA activity and an expanded style domain. Candi-

dates include the YAB1 genes FILAMENTOUS FLOWER (FIL)

and YAB3 (Sawa et al., 1999; Siegfried et al., 1999), as the style

domain of fil-8 yab3-2 double mutants is basally extended.

Regulation of auxin accumulation, movement, and signaling is

another candidate, since STY1 is a presumptive activator of

auxin biosynthesis, and chemical or genetic inhibition of PAT

causes proportionally expanded style and stigma (Staldal et al.,

2008) and strong expression of the auxin efflux carrier auxin

transport PIN1 (PIN1:PIN1-GFP; Benkova et al., 2003) is seen

in the developing gynoecium (see Supplemental Figures 12E

and 12F online). To investigate a possible role for YAB1 genes

and auxin transport in NGA gene regulation, we examined

NGA gene expression and action in the absence of YAB1 or in

the presence of the PAT inhibitor, 1-N-naphtylphtalamic acid

(NPA).

Plants overexpressing the synthetic microRNA amiR-

YAB1164a, which targets FIL and YAB3, using the ANT promoter

faithfully mimic the fil-8 yab3-2 double mutant phenotype

(Goldshmidt et al., 2008). The gynoecia ofANT>>amiR-YAB1164a

plants have an expanded style (Figure 7A), which is lost and

replaced by valve tissue in ANT>>amiR-YAB1164a/35S::amiR-

NGA164a plants (Figure 7B), directly implicating expanded NGA

gene activity in the expanded style of fil-8 yab3-2 flowers.

Consistent with this, nga4:GUS expression was stronger,

initiated earlier, and basally expanded in all floral organs of

ANT>>amiR-YAB1164a plants (Figures 7C and 7D; see Supple-

mental Figures 12A and 12B online). While these observations

imply that YAB1 genes are effective negative regulators of NGA

gene activity, the negative regulation is likely reciprocal. FIL

expression is excluded from the distal gynoecium upon style

initiation in the wild type (Siegfried et al., 1999; see Supplemental

Figure 12C online), but remains throughout 35S:amiR-NGA164a

gynoecia (see Supplemental Figure 12D online).

Treatment of wild-type inflorescences with 100 mm NPA

resulted in a basally expanded style and outgrowths topped

with stigmatic papillae emanating from the base of the gynoe-

cium (Figure 7E). An equivalent treatment of the nga quadruple

mutant failed to rescue the style defects, whereas outgrowths at

the base of the gynoecium formed (Figure 7F). This suggests that

a basipetal expansion ofNGA gene activity and consequent style


(H) In CRCw>>STY1 gynoecia, the entire ovary surface has style-like epidermal characteristics.

(I) The style-like epidermal features are abolished in CRCw>>STY1/amiR-NGA164a gynoecium, but it does not revert to the wild type.

(J) and (K) Expression of CRCw>>GUS in a stage 12 flower (J) is apparent in the ovary and nectaries, whereas in a stage 14 flower (K), it is mostly in the

nectaries.

(L) to (P) Expression of nga4:GUS in different genotypes.

(L) In the wild-type gynoecium, nga4:GUS is restricted to the style.

(M) In CRCw>>STY1 stage 12, staining is observed in the ovary and nectaries (arrows).

(N) By stage 14-15, CRCw>>STY1 flower staining is restricted to the gynoecium base and nectaries.

(O) and (P) In CRCw>>NGA1 gynoecia (O) or CRC>>NGA1 flowers (P), nga4:GUS staining is not observed in the style-converted gynoecium nor in the

papilliae that develop at nectary positions.

(Q) to (U) The effects of ectopic STY1 expression outside the gynoecium.

(Q) A diminutive AP3>>STY1 flower showing reduced growth of all organs.

(R) AP3s>>STY1 flower. Stamen and petal growth is abnormal, and stigma and style tissue is observed on the staminoid organs.

(S) SHP1:GUS is ectopically activated in the carpelloid stamens of AP3s>>STY1 flowers (arrow).

(T) AP3s>>STY1/amiR-NGA164a flower with comparatively normal stamens.

(U) Ectopic SHP1:GUS is not observed in the third whorl organs of the AP3s>>STY1/amiR-NGA164a flower.

Bars = 100 mm in (A), (D), (E), (J) to (P), and (R) to (U), 50 mm in (B), (C), and (F), 200 mm in lower-magnification images of (G) to (I), and 20 mm in the

adjacent higher-magnification images.


development underlie the response to NPA application. Con-

sistent with this, nga4:GUS and NGA1>>GUS expression in

NPA-treated inflorescences was earlier, stronger, and basally

expanded relative to untreated controls (Figures 7G to 7K). That

nga4:GUS expression was expanded in NPA-treated nga qua-

druplemutant gynoecia, in the absence of style and stigma tissue

development, indicted that changes in NGA4 expression reflect

a response to altered auxin distribution and not simply a reflec-

tion of the style tissue domain (see Supplemental Figures 12G

and 12H online). Together, these results imply that both YAB1

and effective auxin transport are involved in restricting the spatial

and temporal domain of NGA gene activity.

Figure 7. Expanded NGA Activity upon Reduction in YAB1 Gene Activity or Arrest of Auxin Transport.

(A) In ANT>>amiR-YAB1164a flowers, the style is basally expanded and constitutes more than half of the gynoecium.

(B) In ANT>>amiR-YAB1164a 35S:amiR-NGA164a gynoecia, style development is abolished and valve tissue extends to the distal tip of the gynoecium.

(C) nga4:GUS localization in a stage 12 wild-type flower and younger flowers (inset). Expression is observed in the distal regions of the sepals and the

gynoecium.

(D) In a stage 12 ANT>>amiR-YAB1164a flower, nga4:GUS expression is distally expanded and in young flowers (inset) is more intense and earlier

compared with (C).

(E) to (K) The effects of 100 mm NPA treatment on proximo-distal tissue distribution and marker gene expression.

(E) Scanning electron micrograph of a wild-type gynoecium after exposure to NPA treatment. The style is more extensive and carpelloid outgrowths

topped by stigmatic papillae arise from near the base.

(F) nga quadruple mutant gynoecium treated with NPA. Style and stigma tissues are missing, but other effects, including the basal-gynoecium

projections, are apparent.

(G) and (H) Expression of nga4:GUS in wild-type inflorescence (G) is weak; thus, in the inset, older flowers have been dissected away to expose

expression in the young flowers. After NPA treatment (H), expression is more intense, earlier, and basally expanded.

(I) NGA1>>GUS expression in untreated (left) and NPA-treated inflorescences. Expression is stronger and earlier after the NPA treatment.

(J) nga4:GUS expression in flowers before (left) and after (right) an NPA treatment. The treated flower shows a significant basal expansion in nga4:GUS

expression in the sepals and the gynoecium.

(K) NGA1>>GUS expressing flowers. The NPA-treated right flower exhibits a strong, basal extension in GUS expression.

sty, style; va, valve. Bars = 100 mm in (A) to (F), (J), and (K) and 1 mm in (G) to (I) and the inset in (C) and (D).

1386 The Plant Cell

SHI/STY Family Members Are under Positive NGA

Gene Regulation

To gain additonal understanding as to the processes regulated

byNGA gene activity, we analyzed RNAextracted fromwild-type

(n = 7), nga quadruple mutants (n = 2), and 35S:amiR-NGA164a

(n = 2) inflorescences and hybridized to ATH1 affymetrix expres-

sionarrays (seeMethods).Geneswith fold changeof>1.5and false

discovery rate (FDR) P value < 0.05 were considered significantly

modified, resulting in 447 downregulated and 236 upregulated

genes in NGA mutants (see Supplemental Data Set 2 online).

Gene Ontology annotation analysis of genes downregulated in

NGA mutants revealed enrichment for plastid genes (P < 1E-8).

Given the green/yellow tinge to nga quadruple mutant petals

(Figure 2G; see Supplemental Figure 3A online), we suspected

this indicated a delay in plastid maturation. To test this, the

transcriptome of wild-type stage 12 and 15 petals was obtained

from AtGenExpress collection (Schmid et al., 2005) and ana-

lyzed. Of the annotated plastid genes downregulated in the nga

quadruple mutant, genes that increase with organ age (i.e.,

maturation genes) were highly enriched (45 of 61, P < 0.001, x2

test). That is, the plastid-annotated transcripts of nga quadruple

mutants match a younger stage of development, implying a

general delay in maturation.

Notably, among the genes significantly downregulated in the

mutant, there were two SHI/STY family members, STY2 and

SRS5. Examination of all SHI/STY family members on the array

(Figure 8A) shows that LRP1 and SRS4 are also reduced, but to a

lesser degree (fold change of 1.56 and 1.31, respectively). Since

the auxin biosynthesis flavin monooxygenase YUC4 and a num-

ber of GH3-like genes are upregulated in response to STY1

activity (Sohlberg et al., 2006), we also examined their expres-

sion. Expression of the YUCCA clade members was not signif-

icantly reduced in our microarray studies. However, there was a

significant downregulation in the expression of GH3-2/GH3-4

and GH3-5 and to a lesser significance GH3-3 and GH3-6 (fold

change of 2.4 and 1.6, respectively; Figure 8B), the majority of

whichwere upregulated onSTY1 induction (Sohlberg et al., 2006).

Since these GH3-like genes encode auxin-inducible indole-

3-acetic acid-amido synthetase proteins shown to catalyze

conjugation of indole-3-acetic acid to amino acids in vitro

(Staswick et al., 2005), their reduced expression suggests al-

tered levels of auxin in the nga quadruple mutant inflorescence.

To confirm these results, we examined the expression of a

STY2marker line in backgrounds with alteredNGA gene activity.

STY2 has a redundant role with STY1, and expression of STY2:

GUS (Kuusk et al., 2002) is activated distally in the stage 9

gynoecium (Figures 8C and 8D) and later confined to the style

(Figure 8E; seeSupplemental Figure 13Aonline). InCRC>>NGA1

flowers, STY2:GUS is activated earlier in the gynoecium and in

nectary positions (Figures 8F and 8G; see Supplemental Figure

13B online), while being almost abolished in the gynoecium of

35S:amiR-NGA164a plants (Figure 8H; see Supplemental Figure

13C online). By comparison, STY2:GUS ovary expression, while

expanded in CRCw>>STY1 ovaries, is not observed in the

nectary positions (see Supplemental Figures 13E and 13F on-

line). Together with the evidence that STY1 can activate NGA

gene activity (Figure 6), these observations suggest a scenario

whereby STY1 promotes NGA gene activity, which in turn, can

activate other members of the SHI/STY gene family.

DISCUSSION

The distinct identities of the sepals, petals, stamens, and gy-

noecium that compose the flower are the result of a complex

orchestration of specific growth and tissue differentiation pro-

grams. Style development occurs at the distal domain of the

carpels, a floral organ specified by unique action of the C-class

genes alongwith theirSEPALLATApartners (Coen andMeyerowitz,

1991; Pelaz et al., 2000; Honma andGoto, 2001; Pinyopich et al.,

2003). The mechanism by which by this program is realized

downstream of the carpel identity program is unclear and limits

our understanding of the processes of morphogenesis in plants.

Here, we showed that activities of four NGA genes, which

function in all aerial lateral organs, are required for style and

stigma morphogenesis in the gynoecium. This role is due to late,

distalNGA gene expression that is consequent, at least in part, of

activation by earlier expression ofSHI/STY gene familymembers

(Kuusk et al., 2002, 2006). NGA gene activity also appears to

promote the activities of several members of the SHI/STY family,

suggesting a positive feedback loop between these two families

of transcription factors. An additional surprising observation is

that the NGA genes, though they are expressed in all organs of

the flower where they affect development, can act as carpel

organ identity genes, promoting the activity of at least one

member of the AGAMOUS clade, SHP1. That loss of NGA gene

activity in conjunction with that of CRC results in the distal

gynoecium acquiring sepal identity likely reflects this role. In

total, these observations imply a central role for NGA genes in a

positive-feedback program that promotes and maintains style

morphogenesis through the concerted self-reinforcing action of

STY, NGA, and AG gene activity. The basis for the activation of

this program appears to be the timing of NGA gene expression,

which appears to be contingent on developmental capacity to

respond to its activators, such as STY1. Factors that prevent

precocious NGA gene activation appear to include YAB1 gene

activity and effective auxin transport, and these could be inter-

related.

The NGA Genes Are Essential for Stigma and

Style Morphogenesis

Progressively lower levels of NGA gene activity results in a

progressive reduction in style and stigma formation, until evi-

dence of these tissues is eliminated in nga quadruple mutants

(Figure 2). All other tissues of the ovary are present in the

quadruple mutant, indicating that the role of the NGA genes

is specific to the style and stigma. Reduced NGA gene activity

also results in style and stigma tissues being eliminated in

the carpel-converted organs of LFY:LFY-VP16, 35S:TAG and

ANT>>miR172dm7 plants where these tissues develop ectopi-

cally from the sepals and leaves, respectively (Figure 3). This sug-

gests that NGA gene activity is essential for style and stigma

tissues in all contexts and that elevated levels of AG cannot

compensate for their loss. Ectopic expression ofNGA1 activity in


the flower promotes ectopic and precocious style and stigma

development and differentiation (Figure 5), whereas the capacity

for STY1 overexpression to promote style and stigma develop-

ment is conditional and NGA dependent (Figure 6). In this

respect, while the disrupted style phenotypes of leunig, crc,

seuss, and spatula could be rescued by STY1 overexpression,

and those of spatula, sty, leunig, crc, seuss, and jagged could be

rescued by NPA application (Staldal et al., 2008), neither STY1

overexpression (Figure 6) nor NPA application (Figure 7) could

rescue a reduction inNGA activity. Thus, theNGA gene pathway

is pivotal for style and stigma development and is likely used by

all known promoters of these tissues.

NGA Activity in Flowers Is Intimately Linked to Style

Formation and the Carpel Genes

The loss of style and delayed petal maturation in nga quadruple

mutants suggests a differentiation function for NGA activity in all

Figure 8. NGA Activity Promotes Expression of Members of the STY Family.

(A) and (B)Normalized expression of six members of the STY gene family, including STY2 (A) and six members of the auxin-responsiveGH3 genes (B) in

wild-type inflorescences compared with lines lacking activities of the four NGA genes.

(C) to (H) Expression of STY2:GUS in wild-type ([C] to [E]),CRC>>NGA1 ([F] and [G]), and 35S:amiR-NGA164a (H) flowers. In the wild type, STY2:GUS is

not expressed in the style of stage 9 gynoecia (C) (arrow) but becomes apparent in the style of stage 10 flowers ([D]; arrow). In stage 12-13 wild-type

gynoecia (E), expression is confined to the style. In stage 9 CRC>>NGA1 flowers (F), expression can be observed at the top of the gynoecium and the

nectary positions (arrows).

(G) By stage 13, expression is strong in the nectary positions and is basally expanded at the top of the gynoecium in CRC>>NGA1 flowers (arrows).

(H) A stage 12, 35S:amiR-NGA164a flower where STY2:GUS expression is restricted to low levels at the tips of the valve-like outgrowths (arrow).

Bars = 100 mm.

1388 The Plant Cell

aerial organs (Figure 2; see Supplemental Figure 11 online). Thus,

the essential role for theNGA gene function in style development

can be viewed as a dramatic manifestation of a differentiation

role played in all organs. Loss of NGA activity leads to the

absence of SHP1 expression from the top of the gynoecium

(Figure 3), reduced NGA activity in conjunction with reduced

CRC results in the distal part of the gynoecium acquiring partial

sepal identity (Figure 3), and ectopic overexpression of NGA1

results in ectopic style and stigma morphogenesis throughout

the flower, which is accompanied by ectopic SHP1 and CRC

expression (Figure 5; see Supplemental Figure 12 online). This

suggests a direct link between NGA and style/stigma promotion

through carpel gene activation. Notably, ectopic NGA activity is

level dependent in this respect. Lower levels of floral ectopic

NGA activity expression affect organ growth, presumably con-

sistent with their wild-type role in sepals and petals, whereas

high levels promote style/stigma and ectopic carpel gene ex-

pression (Figure 5; see Supplemental Figure 9 online). Since

ectopic overexpression of NGA genes in the leaves does not

result in ectopic carpel tissues (see Supplemental Figure 8

online), the NGA gene carpel function must be mediated by

flower specific cofactors that are, in part, negatively regulated by

the five AP2-related genes targeted by miR172 (Chen, 2004;

Figure 3). Such cofactors presumably include members of both

the SEPALLATA and AG MADS box gene families, which are

essential for carpel development in the flower (Pelaz et al., 2000;

Honma and Goto, 2001; Ditta et al., 2004). The nature of these

interactions awaits elucidation.

A question arises as to why there is such an intimatemolecular

connection between NGA activity and style/stigma morphogen-

esis. A likely scenario is that NGA style program has evolved

features that parallel the activity of floral ABC selector genes in

organogenesis to allow cells of the nascent style to rapidly

distinguish themselves in growth and identity from the underlying

ovary. HighNGA levels promoteAG clademembers and exclude

ovary factors, such as FIL from the initiating style (see Supple-

mental Figure 12 online) in the program of style differentiation.

For many angiosperm species, the analogy of a style being

consequent of a distinct organogenesis program is particularly

appropriate. In maize (Zea mays), for example, the style (the silk)

can develop into an enormous length of >20 cm compared with

the minute ovary (Carcova et al., 2003). Since the primary mode

of NGA gene regulation is transcriptional (Figures 4 and 5),

understanding the basis for their activation is critical. Our results

suggest that the SHI/STY genes likely have a central role in

promoting and maintaining NGA gene expression.

NGA Gene Activation: Regulated Competence to Respond

to an Activator

The expression of NGA1 and NGA4 occurs well after initiation in

all organs, and in the gynoecium the expression is tightly corre-

lated with style initiation at stage 9. Since STY1 overexpression

promoted ectopic, NGA gene-dependent style tissue and nga4:

GUS expression, the transcriptional control of the NGA genes

likely involves positive regulation through STY1 and other mem-

bers of the SHI/STY gene family (Kuusk et al., 2006; Figure 6).

However, the temporal separation of the stage 6 STY1 and stage

9NGA expression suggests thatNGA gene activation by STY1 is

context dependent. Since STY1 is an activator of YUC4, which

encodes an auxin biosynthesis enzyme (Cheng et al., 2006;

Sohlberg et al., 2006), a simple model is that the NGA genes

respond to an auxin-related signal derived from distal STY1

activity that does not reach an NGA-activating threshold until

stage 9 of gynoecium development (Figure 9). This is supported

by auxin transport inhibition causing early, basally expanded

NGA gene activation (Figure 7). Reduction in YAB1 (FIL/YAB3)

genes, which, as well as regulating organ polarity, have been

implicated in signaling (Goldshmidt et al., 2008) and growth

Figure 9. Genetic Models for NGA Activation and Function in Promoting

Style Development and Differentiation in Aerial Organs.

(A) During stages 6-9 of gynoecium development, early ovary factors,

such as the YABBY1 genes (FIL and YAB3), function to either/or (a)

suppress NGA gene activity directly or (b) suppress NGA gene activity

passively by preventing the accumulation of an activating signal through

an effect that has been defined here simplistically as delayed differen-

tiation. We suggest that a distal, auxin-based signal promoted by the

STY genes, including STY1, acts to induce NGA gene activity at a

threshold level. This level is not reached (faint shading) because of

efficient PAT (large arrow) maintained in gynoecium at that stage.

(B) In the stage 9-11 gynoecium, YAB1 efficacy is reduced as is their

ability to negative regulate NGA gene activity or passively prevent NGA

response to an activating signal. Under the auxin-based activator sce-

nario, the polar transport of auxin from the distal site of STY1-mediated

synthesis becomes inefficient and auxin accumulates to an NGA-acti-

vating threshold. Upon activation, NGA gene activity suppresses YAB1

while promoting AG clade and SHI/STY gene family members. These in

turn maintains NGA gene activity and their own expression through

positive feedback. Together, the NGA, STY, and AG clade genes

constitute a developmental module for style/stigma morphogenesis.


promotion (Eshed et al., 2004), also results in earlier, stronger,

and basally expanded NGA gene activity. The YAB1 genes,

which are expressed from the inception of gynoecium develop-

ment (stage 5) in the valve primordia, could be direct negative

regulators of NGA gene activity. Alternatively, they may function

to maintain a cellular environment that prevents NGA sensitivity

to the STY signal. Detailed investigation into these scenarios will

have to await a careful analysis of auxin flux (see Supplemental

Figure 12 online) and steady state auxin levels along the devel-

oping gynoecium. Other scenarios are also possible. For in-

stance, NGA1 to NGA4 are among the genes subject to histone

H3 Lys-27 trimethylation (Zhang et al., 2007), and it may be this

form of regulation that is involved in negatively regulating gene

expression in young tissues.

After NGA gene activation in the stage 9 gynoecium, promo-

tion and maintenance of distal NGA gene activity apparently

comes from positive feedback between theNGA genes and SHI/

STY family members (Figure 8). Under this scenario, SHI/STY-

activated NGA activity suppresses earlier, ovary factors while

promoting AG clade members and additional SHI/STY factors

that further activate NGA gene activity in style and stigma

development (Figure 9). From this perspective, the valve tissue

that develops instead of style in the ngaquadruplemutant (Figure

2) can be seen as a homeotic conversion.

Manymolecular programs underlying gynoeciummorphogen-

esis function in leaf development and were likely encompassed

under the regulatory direction of C class activity (Sitaraman et al.,

2008), consistent with Goethe’s vision of the carpel as amodified

leaf (Goethe, 1790). In this respect, the temporal and spatial

action of the NGA genes in the gynoecium appears to be shared

with the other floral organs, suggesting that NGA function has

been co-opted for an essential role in style and stigma differen-

tiation. Consistent with this, the petals of the nga quadruple

mutants appear to exhibit delayed maturation. It is therefore

tempting to speculate that the primary role of the NGA genes is

organ differentiation, which is conserved in leaves. Under this

scenario, the basic framework of gene action postulated forNGA

gene activation, an interplay between the NGA and STY family

members, elevated auxin levels and suppression of earlier organ

growth factors, is a step in the differentiation of all lateral organs.

In this respect, the numerous valve outgrowths observed in nga

quadruplemutants (Figure 2) and at themargins of nga1-1 gym-5

kan1-2 and nga1-1 kan1-2 kan2-1/+ gynoecia (Figure 1) may

reflect a loss of a growth suppressing function of the NGA

differentiation program and not a conversion of style to valve

tissue. Loss of such a program could also lead to the more

serrated leaves of nga quadruple mutants (Figure 2). Investiga-

tion into the leaf role of the NGA genes will help shed light on

these possibilities.

METHODS

Plant Material, Growth, Transformation, and Mutagenesis

All plants described were in the Landsberg erecta background and grown

under 18-h cool white fluorescent light at 18 to 228C. For the NGA1:LhG4

andAP3-short:LhG4 driver lines, 5 and 400 bp upstream of the translation

start site, respectively, were transcriptionally fused 59 of the chimeric

LhG4 transcription factor.NGA1,NGA2,NGA3,NGA4,NGAL1, andSTY1

cDNAs subcloned behind an operator array in BJ36 plasmid to generate

responder lines (Moore et al., 1998). Primers used for PCR cloning are

presented in Supplemental Table 1 online, and the cloning strategies for

4xNLSmRFP, pre-miR172d, and miR172dm7 are presented as Supple-

mental Methods online.

To complement the nga1-1mutation, the DNA from BAC clone F14M4

was digested with BamHI and KpnI and an;10.5-kb genomic fragment

spanning the At2g46870 locus (NGA1), including 5.7 kb of sequence

upstream from the presumptive initiation ATG codon and 3.8 kb of

sequence downstream of the putative stop codon was cloned into

pBluescript SK+ before being subcloned into pBJ36. Transformation

was performed into nga1-1 kan1-1 mutant plants to facilitate identifica-

tion of complemented plants. All constructs were subcloned to the

pMLBART binary plasmid and transformed to plants by floral dipping

using the Agrobacterium tumefaciensGV3101 strain. For transactivation,

Promoter:LhG4 driver lines were crossed to different OP:cDNA re-

sponder lines to generate transactivated F1s (marked as >> in the text).

Mutant and transgene combinations were generated through conven-

tional breeding.

Map-Based Cloning of NGA1

To map the NGA1 locus, the nga1-1 kan1-2 double mutant was crossed

to a kan1-10 homozygote in the Columbia background to facilitate

genotyping, and the F1 plants were allowed to self-pollinate. In the F2

population, genomic DNA was isolated from 440 plants homozygous for

nga1-1 and subject to genetic mapping using cleaved-amplified poly-

morphic sequence and Simple Sequence Length Polymorphism (SSLP)

markers. The nga1-1 mutation was localized to the lower arm of chro-

mosome 2 on BAC F14M4. Candidate genes in the nga1-1 background

were sequenced, and a mutation in the locus At2g46870 was identified.

Distinct molecular lesions were subsequently identified in the At2g46870

locus of nga1-2 and nga1-3 mutant plants. Transcript ends were deter-

mined by rapid amplification of cDNA ends (RACE) using total RNA

isolated from young inflorescences with TriReagent (Sigma-Genosys)

and the Clontech SMART RACE cDNA amplification kit in combination

with gene-specific primers (see Supplemental Table 2 online).

Transcriptome Analysis of nga Inflorescences

RNA was extracted from inflorescences of 30-d-old plants using the

Qiagen RNAEasy kit. cRNAwas synthesized and hybridized to Affymetrix

ATH1 array according to the manufacturer’s recommendations. Seven

repeats of the wild type, two repeats of nga quadruple mutants, and two

repeats of 35S:amiR-NGA164a were collected. Signal values were ob-

tained and normalized using GeneChip-Robust Multi-array Average (GC-

RMA), as implemented in R 2.7.2 (www.r-project.org) and Bioconductor

2.2 (www.bioconductor.org/). Average correlation values between re-

peats were 0.985, while the average correlation between wild-type and

mutant samples was 0.972. As the average correlation between samples,

the nga quadruplemutants and 35S:amiR-NGA164aplants was 0.984, and

given the identical phenotype, we grouped the two genotypes together.

Genes were filtered for fold change between the wild type and mutant

higher than 1.5 and absolute expression value larger than log2(10). Welsh

t test was performed on the filtered list, followed by FDR, using the

multitest R package. Genes with FDR P value < 0.05 were selected as

significantly changed. Analysis of Gene Ontology annotation enrichment

was performed using DAVID (http://david.abcc.ncifcrf.gov/).

NPA Treatment

The NPA treatment was performed as described by Nemhauser et al.

(2000).

1390 The Plant Cell

Microscopy and Confocal Imaging

Tissue was prepared and sectioned to view florescent signals according

to Goldshmidt et al. (2008). Confocal images were taken on an Olympus

IX-70 microscope with an argon laser set at 488 nm for excitation, a 505-

to 525-nm filter for GFP emission, and a 560- to 600-nm filter for

Propidium Iodide (PI) emission. The 4’,6-diamidino-2-phenylindole stain-

ing was observed with a 405-nm diode laser for excitation and a 450- to

490-nm filter for emission. Images were captured and processed with the

FW-500 image analysis system. Two-photon imaging was performed

with a Zeiss LSM 510 META NLO microscope equipped with 320 and

340 water immersion objectives. A Mai Tai One Box Ti:Sapphire Laser

(Spectra Physics, Newport) was used for two-photon excitation. Image

acquisition was performed using LSM 510 acquisition software.

For light sections and scanning electron microscopy, inflorescences

were fixed in 2% glutaraldehyde in 0.025 M sodium phosphate buffer at

pH 6.8 and vacuum infiltrated at room temperature for up to 1 h. For

sections, tissues were washed, dehydrated in an ethanol series, and

embedded in LR White resin. Sections of 2 mm for light microscopy were

cut and dried onto slides. Sections were stained with toluidine blue.

Scanning electron microscopy was performed using an XL30 ESEM FEG

microscope (FEI).

Accession Numbers

The Arabidopsis Genome Initiative locus identifiers for NGA1 to NGA4 are

At2g46870, At3g61970, At1g01030, and At4g01500, respectively. STY1,

STY2, SPH1, CRC, FIL, YAB3, and KAN1 correspond to At3g51060,

At4g36260, At3g58780, At1g69180, At2g45190, At4g00180, and

At5g16560, respectively. Microarray data were submitted to the Geo

Omnibus repository (accession number GSE15555).

Supplemental Data

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

Supplemental Figure 1. nga1-1 Genetic Interactions.

Supplemental Figure 2. Multiple Alignment of NGA1-4 Proteins and

Phylogenetic Relationships of the RAV Clade of B3 Transcription

Factors in Land Plants.

Supplemental Figure 3. NGA Genes Redundantly Regulate Growth

of Flower Organs and Leaves.

Supplemental Figure 4. Histological Analyses of Diverse Mature

Gynoecia.

Supplemental Figure 5. Style Development Requires NGA Gene

Activity.

Supplemental Figure 6. Design and Overexpression of a Native and

Modified miR172 MicroRNA.

Supplemental Figure 7. Genetic Combinations between nga and

Mutants Effecting Gynoecium Development Produce Additive and

Synergistic Interactions.

Supplemental Figure 8. Effects of NGA Gene Overexpression during

Vegetative Growth.

Supplemental Figure 9. NGA Gene Overexpression Reduces Organ

Growth and Promotes a Style Program within the Flower.

Supplemental Figure 10. Effect of Overexpression of NGAL1/

AT2G36080.

Supplemental Figure 11. Effects of Expressing NGA1 and STY1 with

the CRCw (CRC Weak) Promoter.

Supplemental Figure 12. Spatial Regulation of NGA Gene Activities.

Supplemental Figure 13. STY2:GUS Expression in Backgrounds

with Different NGA Levels or STY1 Overexpression.

Supplemental Table 1. Primers for PCR-Mediated Cloning.

Supplemental Table 2. Gene-Specific Primers for RACE.

Supplemental Data Set 1. Alignment of Sequences Used for Phy-

logenetic Analysis.

Supplemental Data Set 2. Genes Modified in Their Expression in nga

Mutant Apices.

Supplemental Methods.

ACKNOWLEDGMENTS

We thank Vyacheslav Kalchenko of the Weizmann Veterinary Resources

In Vivo Optical Imaging Unit for assistance with two-photon imaging,

Eugenia Klein and the electron microscopy facility for help with scanning

electron microscopy, Raya Eilam and Eyal Shimoni for help with tissue

preparation techniques, and Vladimir Kiss for assistance with confocal

laser scanning microscopy. The dedicated work of Galit Shahar, Anna

Pistunov, and Oshri Afanzer is highly appreciated. We also thank Eva

Sundberg for the STY2:GUS line, members of the Eshed lab for

comments and discussions, and Cristina Ferrandiz for sharing unpub-

lished results. This work was made possible with funding from the Israel

Science Foundation (Research Grant Award No. 863-06; Y.E.), from

MINERVA (Y.E.), and from the U. S. National Science Foundation (IOB

0332556; J.B.).

Received January 7, 2009; revised March 25, 2009; accepted April 17,

2009; published May 12, 2009.

REFERENCES

Alvarez, J., and Smyth, D.R. (2002). Crabs Claw and Spatula genes

regulate growth and pattern formation during gynoecium develop-

ment in Arabidopsis thaliana. Int. J. Plant Sci. 163: 17–41.

Alvarez, J.P., Pekker, I., Goldshmidt, A., Blum, E., Amsellem, Z., and

Eshed, Y. (2006). Endogenous and synthetic microRNAs stimulate

simultaneous, efficient, and localized regulation of multiple targets in

diverse species. Plant Cell 18: 1134–1151.

Aukerman, M.J., and Sakai, H. (2003). Regulation of flowering time and

floral organ identity by a microRNA and its APETALA2-like target

genes. Plant Cell 15: 2730–2741.

Azhakanandam, S., Nole-Wilson, S., Bao, F., and Franks, R.G.

(2008). SEUSS and AINTEGUMENTA mediate patterning and ovule

initiation during gynoecium medial domain development. Plant Physiol.

146: 1165–1181.

Balanza, V., Navarrete, M., Trigueros, M., and Ferrandiz, C. (2006).

Patterning the female side of Arabidopsis: the importance of hor-

mones. J. Exp. Bot. 57: 3457–3469.

Benjamins, R., Quint, A., Weijers, D., Hooykaas, P., and Offringa, R.

(2001). The PINOID protein kinase regulates organ development in

Arabidopsis by enhancing polar auxin transport. Development 128:

4057–4067.

Benkova, E., Michniewicz, M., Sauer, M., Teichmann, T., Seifertova,

D., Jurgens, G., and Friml, J. (2003). Local, efflux-dependent auxin

gradients as a common module for plant organ formation. Cell 115:

591–602.

Bennett, S.M.R., Alvarez, J., Bossinger, G., and Smyth, D.R. (1995).

Morphogenesis in pinoid mutants of Arabidopsis thaliana. Plant J. 8:

505–520.


Bowman, J.L., Baum, S.F., Eshed, Y., Putterill, J., and Alvarez, J.

(1999). Molecular genetics of gynoecium development in Arabidopsis.

Curr. Top. Dev. Biol. 45: 155–205.

Bowman, J.L., Eshed, Y., Baum, S., Emery, J.F., Floyd, S.K., Alvarez,

J., Hawker, N.P., Lee, J.Y., Siegfried, K.R., Khodosh, R., Tatom-

Jaurez, M., and Perea, J.V. (2001). The story of CRABS CLAW (or

How we learned to love the mutagen). Flowering Newsletter 31: 3–11.

Bowman, J.L., and Smyth, D.R. (1999). CRABS CLAW, a gene that

regulates carpel and nectary development in Arabidopsis, encodes a

novel protein with zinc finger and helix-loop-helix domains. Develop-

ment 126: 2387–2396.

Busch, M.A., Bomblies, K., and Weigel, D. (1999). Activation of a floral

homeotic gene in Arabidopsis. Science 285: 585–587.

Carcova, J., Andrieu, B., and Otegui, M.E. (2003). Silk elongation in

maize: Relationship with flower development and pollination. Crop

Sci. 43: 914–920.

Chen, X. (2004). A microRNA as a translational repressor of APETALA2

in Arabidopsis flower development. Science 303: 2022–2025.

Cheng, Y., Dai, X., and Zhao, Y. (2006). Auxin biosynthesis by the

YUCCA flavin monooxygenases controls the formation of floral organs

and vascular tissues in Arabidopsis. Genes Dev. 20: 1790–1799.

Christensen, S.K., Dagenais, N., Chory, J., and Weigel, D. (2000).

Regulation of auxin response by the protein kinase PINOID. Cell 100:

469–478.

Coen, E.S., and Meyerowitz, E.M. (1991). The war of the whorls:

Genetic interactions controlling flower development. Nature 353:

31–37.

Crawford, B.C.W., Ditta, G., and Yanofsky, M.F. (2007). The NTT gene

is required for transmitting-tract development in carpels of Arabidop-

sis thaliana. Curr. Biol. 17: 1101–1108.

Dinneny, J.R., Weigel, D., and Yanofsky, M.F. (2006). NUBBIN and

JAGGED define stamen and carpel shape in Arabidopsis. Develop-

ment 133: 1645–1655.

Ditta, G., Pinyopich, A., Robles, P., Pelaz, S., and Yanofsky, M.F.

(2004). The SEP4 gene of Arabidopsis thaliana functions in floral organ

and meristem identity. Curr. Biol. 14: 1935–1940.

Elliott, R.C., Betzner, A.S., Huttner, E., Oakes, M.P., Tucker, W.Q.J.,

Gerentes, D., Perez, P., and Smyth, D.R. (1996). AINTEGUMENTA,

an APETALA2-like gene of Arabidopsis with pleiotropic roles in ovule

development and floral organ growth. Plant Cell 8: 155–168.

Eshed, Y., Baum, S.F., and Bowman, J.L. (1999). Distinct mechanisms

promote polarity establishment in carpels of Arabidopsis. Cell 99:

199–209.

Eshed, Y., Baum, S.F., Perea, J.V., and Bowman, J.L. (2001). Estab-

lishment of polarity in lateral organs of plants. Curr. Biol. 11: 1251–

1260.

Eshed, Y., Izhaki, A., Baum, S.F., Floyd, S.K., and Bowman, J.L.

(2004). Asymmetric leaf development and blade expansion in Arabi-

dopsis are mediated by KANADI and YABBY activities. Development

131: 2997–3006.

Ferrandiz, C., Pelaz, S., and Yanofsky, M.F. (1999). Control of carpel

and fruit development in arabidopsis. Annu. Rev. Biochem. 68:

321–354.

Flanagan, C.A., Hu, Y., and Ma, H. (1996). Specific expression of the

AGL1 MADS-box gene suggests regulatory functions in Arabidopsis

gynoecium and ovule development. Plant J. 10: 343–353.

Galweiler, L., Guan, C., Muller, A., Wisman, E., Mendgen, K.,

Yephremov, A., and Palme, K. (1998). Regulation of polar auxin

transport by AtPIN1 in Arabidopsis vascular tissue. Science 282:

2226–2230.

Goethe, J.W. (1790). Versuch die Metamorphose der Pflanzen zu

erklaren. Gotha: Carl Wilhelm Ettinger (transl. Arber, A. [1946].

Goethe’s botany. Chromca Bot. 10: 63–126.)

Goldshmidt, A., Alvarez, J.P., Bowman, J.L., and Eshed, Y. (2008).

Signals derived from YABBY gene activities in organ primordia

regulate growth and partitioning of Arabidopsis shoot apical meri-

stems. Plant Cell 20: 1217–1230.

Gremski, K., Ditta, G., and Yanofsky, M.F. (2007). The HECATE genes

regulate female reproductive tract development in Arabidopsis thali-

ana. Development 134: 3593–3601.

Heisler, M.G., Atkinson, A., Bylstra, Y.H., Walsh, R., and Smyth, D.R.

(2001). SPATULA, a gene that controls development of carpel margin

tissues in Arabidopsis, encodes a bHLH protein. Development 128:

1089–1098.

Honma, T., and Goto, K. (2001). Complexes of MADS-box proteins are

sufficient to convert leaves into floral organs. Nature 409: 525–529.

Ishida, T., Aida, M., Takada, S., and Tasaka, M. (2000). Involvement of

CUP-SHAPED COTYLEDON genes in gynoecium and ovule develop-

ment in Arabidopsis thaliana. Plant Cell Physiol. 41: 60–67.

Jack, T. (2004). Molecular and genetic mechanisms of floral control.

Plant Cell 16: S1–S17.

Kagaya, Y., Ohmiya, K., and Hattori, T. (1999). RAV1, a novel DNA-

binding protein, binds to bipartite recognition sequence through two

distinct DNA-binding domains uniquely found in higher plants. Nucleic

Acids Res. 27: 470–478.

Kuusk, S., Sohlberg, J.J., Eklund, D.M., and Sundberg, E. (2006).

Functionally redundant SHI family genes regulate Arabidopsis gynoe-

cium development in a dose-dependent manner. Plant J. 47: 99–111.

Kuusk, S., Sohlberg, J.J., Long, J.A., Fridborg, I., and Sundberg, E.

(2002). STY1 and STY2 promote the formation of apical tissues during

Arabidopsis gynoecium development. Development 129: 4707–4717.

Liljegren, S.J., Ditta, G.S., Eshed, Y., Savidge, B., Bowman, J.L., and

Yanofsky, M.F. (2000). SHATTERPROOF MADS-box genes control

seed dispersal in Arabidopsis. Nature 404: 766–770.

Mizukami, Y., and Ma, H. (1992). Ectopic expression of the floral

homeotic gene AGAMOUS in transgenic Arabidopsis plants alters

floral organ identify. Cell 71: 119–131.

Moore, I., Galweiler, L., Grosskopf, D., Schell, J., and Palme, K.

(1998). A transcription activation system for regulated gene expres-

sion in transgenic plants. Proc. Natl. Acad. Sci. USA 95: 376–381.

Nemhauser, J.L., Feldman, L.J., and Zambryski, P.C. (2000). Auxin

and ETTIN in Arabidopsis gynoecium morphogenesis. Development

127: 3877–3888.

Ohno, C.K., Reddy, G.V., Heisler, M.G.B., and Meyerowitz, E.M.

(2004). The Arabidopsis JAGGED gene encodes a zinc finger protein

that promotes leaf tissue development. Development 131: 1111–

1122.

Okada, K., Ueda, J., Komaki, M.K., Bell, C.J., and Shimura, Y. (1991).

Requirement of the auxin polar transport system in early stages of

Arabidopsis floral bud formation. Plant Cell 3: 677–684.

Payne, T., Johnson, S.D., and Koltunow, A.M. (2004). KNUCKLES

(KNU) encodes a C2H2 zinc-finger protein that regulates development

of basal pattern elements of the Arabidopsis gynoecium. Develop-

ment 131: 3737–3749.

Pelaz, S., Ditta, G.S., Baumann, E., Wisman, E., and Yanofsky, M.F.

(2000). B and C floral organ identity functions require SEPALLATA

MADS-box genes. Nature 405: 200–203.

Pinyopich, A., Ditta, G.S., Savidge, B., Liljegren, S.J., Baumann, E.,

Wisman, E., and Yanofsky, M.F. (2003). Assessing the redundancy

of MADS-box genes during carpel and ovule development. Nature

424: 85–88.

Pnueli, L., Hareven, D., Rounsley, S.D., Yanofsky, M.F., and

Lifschitz, E. (1994). Isolation of the tomato AGAMOUS gene TAG1

and analysis of its homeotic role in transgenic plants. Plant Cell 6:

163–173.

Prunet, N., Morel, P., Thierry, A.-M., Eshed, Y., Bowman, J.L.,

1392 The Plant Cell

Negrutiu, I., and Trehin, C. (2008). REBELOTE, SQUINT, and

ULTRAPETALA1 function redundantly in the temporal regulation of

floral meristem termination in Arabidopsis thaliana. Plant Cell 20:

901–919.

Sawa, S., Watanabe, K., Goto, K., Kanaya, E., Morita, E.H., and

Okada, K. (1999). FILAMENTOUS FLOWER, a meristem and organ

identity gene of Arabidopsis, encodes a protein with a zinc finger and

HMG-related domains. Genes Dev. 13: 1079–1088.

Schmid, M., Davison, T.S., Henz, S.R., Pape, U.J., Demar, M.,

Vingron, M., Scholkopf, B., Weigel, D., and Lohmann, J.U.

(2005). A gene expression map of Arabidopsis thaliana development.

Nat. Genet. 37: 501–506.

Schoof, H., Lenhard, M., Haecker, A., Mayer, K.F.X., J¸rgens, G., and

Laux, T. (2000). The stem cell population of Arabidopsis shoot

meristems is maintained by a regulatory loop between the CLAVATA

and WUSCHEL genes. Cell 100: 635–644.

Scofield, S., Dewitte, W., and Murray, J.A.H. (2007). The KNOX gene

SHOOT MERISTEMLESS is required for the development of repro-

ductive meristematic tissues in Arabidopsis. Plant J. 50: 767–781.

Sessions, A., Nemhauser, J.L., McColl, A., Roe, J.L., Feldmann, K.

A., and Zambryski, P.C. (1997). ETTIN patterns the Arabidopsis floral

meristem and reproductive organs. Development 124: 4481–4491.

Siegfried, K.R., Eshed, Y., Baum, S.F., Otsuga, D., Drews, G.N., and

Bowman, J.L. (1999). Members of the YABBY gene family specify

abaxial cell fate in Arabidopsis. Development 126: 4117–4128.

Sitaraman, J., Bui, M., and Liu, Z. (2008). LEUNIG HOMOLOG and

LEUNIG perform partially redundant functions during Arabidopsis

embryo and floral development. Plant Physiol. 147: 672–681.

Smyth, D.R., Bowman, J.L., and Meyerowitz, E.M. (1990). Early flower

development in Arabidopsis. Plant Cell 2: 755–767.

Sohlberg, J.J., Myrenas, M., Kuusk, S., Lagercrantz, U., Kowalczyk,

M., Sandberg, G., and Sundberg, E. (2006). STY1 regulates auxin

homeostasis and affects apical-basal patterning of the Arabidopsis

gynoecium. Plant J. 47: 112–123.

Staldal, V., Sohlberg, J.J., Eklund, M.D., Ljung, K., and Sundberg, E.

(2008). Auxin can act independently of CRC, LUG, SEU, SPT and

STY1 in style development but not apical-basal patterning of the

Arabidopsis gynoecium. New Phytol. 180: 798–808.

Staswick, P.E., Serban, B., Rowe, M., Tiryaki, I., Maldonado, M.T.,

Maldonado, M.C., and Suza, W. (2005). Characterization of an

Arabidopsis enzyme family that conjugates amino acids to indole-3-

acetic acid. Plant Cell 17: 616–627.

Swaminathan, K., Peterson, K., and Jack, T. (2008). The plant B3

superfamily. Trends Plant Sci. 13: 647–655.

Yamasaki, K., et al. (2004). Solution structure of the B3 DNA binding

domain of the Arabidopsis cold-responsive transcription factor RAV1.

Plant Cell 16: 3448–3459.

Zhang, X., Clarenz, O., Cokus, S., Bernatavichute, Y.V., Pellegrini,

M., Goodrich, J., and Jacobsen, S.E. (2007). Whole-genome anal-

ysis of histone H3 lysine 27 trimethylation in Arabidopsis. PLoS Biol.

5: e129.


DOI 10.1105/tpc.109.065482; originally published online May 12, 2009; 2009;21;1373-1393Plant Cell

John Paul Alvarez, Alexander Goldshmidt, Idan Efroni, John L. Bowman and Yuval EshedArabidopsis

Distal Organ Development Genes Are Essential for Style Specification in NGATHAThe

This information is current as of July 27, 2018

Supplemental Data /content/suppl/2009/05/04/tpc.109.065482.DC1.html

References /content/21/5/1373.full.html#ref-list-1

This article cites 61 articles, 30 of which can be accessed free at:

Permissions https://www.copyright.com/ccc/openurl.do?sid=pd_hw1532298X&issn=1532298X&WT.mc_id=pd_hw1532298X

eTOCs http://www.plantcell.org/cgi/alerts/ctmain

Sign up for eTOCs at:

CiteTrack Alerts http://www.plantcell.org/cgi/alerts/ctmain

Sign up for CiteTrack Alerts at:

Subscription Information http://www.aspb.org/publications/subscriptions.cfm

is available at:Plant Physiology and The Plant CellSubscription Information for

ADVANCING THE SCIENCE OF PLANT BIOLOGY © American Society of Plant Biologists

https://www.copyright.com/ccc/openurl.do?sid=pd_hw1532298X&issn=1532298X&WT.mc_id=pd_hw1532298X

http://www.plantcell.org/cgi/alerts/ctmain

http://www.plantcell.org/cgi/alerts/ctmain

http://www.aspb.org/publications/subscriptions.cfm

ngatha distal organ development genes are essential … · the ngatha distal organ development...

Documents