The NGATHA Distal Organ Development Genes Are Essential for Style Specification in Arabidopsis W John Paul Alvarez, a Alexander Goldshmidt, a Idan Efroni, a John L. Bowman, b,c and Yuval Eshed a,1 a Department of Plant Sciences, Weizmann Institute of Science, Rehovot, 76100, Israel b School of Biological Sciences, Monash University, Melbourne, Victoria 3800, Australia c 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 primary molecular genetic program underlying the identity of the concentric whorls 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]. The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantcell.org) is: Yuval Eshed (yuval. [email protected]). W Online 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
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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.
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).
Style Specification by NGA Genes 1377
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
(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
Figure 3. (continued).
(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).
Style Specification by NGA Genes 1379
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
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
Figure 5. (continued).
(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).
Style Specification by NGA Genes 1383
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
Figure 6. (continued).
(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.
Style Specification by NGA Genes 1385
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-
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
Style Specification by NGA Genes 1387
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.
Style Specification by NGA Genes 1389
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