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First published online May 19, 2006; 10.1104/pp.106.076604 Plant Physiology 141:977-987 (2006) © 2006 American Society of Plant Biologists AINTEGUMENTA Contributes to Organ Polarity and Regulates Growth of Lateral Organs in Combination with YABBY Genes1Department of Biological Sciences, University of South Carolina, Columbia, South Carolina 29208
Lateral organs in flowering plants display polarity along their adaxial-abaxial axis with distinct cell types forming at different positions along this axis. Members of three classes of transcription factors in Arabidopsis (Arabidopsis thaliana; the Class III homeodomain/leucine zipper [HD-ZIP] proteins, KANADI proteins, and YABBY proteins) are expressed in either the adaxial or abaxial domain of organ primordia where they confer these respective identities. Little is known about the factors that act upstream of these polarity-determining genes to regulate their expression. We have investigated the relationship between AINTEGUMENTA (ANT), a gene that promotes initiation and growth of lateral organ primordia, and polarity genes. Although ant single mutants do not display any obvious defects in organ polarity, loss of ANT activity in combination with mutations in one or more YABBY genes results in polarity defects greater than those observed in the yabby mutants alone. Our results suggest that ANT acts in combination with the YABBY gene FILAMENTOUS FLOWER (FIL) to promote organ polarity by up-regulating the expression of the adaxial-specifying HD-ZIP gene PHABULOSA. Furthermore, we show that ANT acts with FIL to up-regulate expression of the floral homeotic gene APETALA3. Our work defines new roles for ANT in the development of lateral organs.
In flowering plants, leaves and floral organs are produced on the periphery of apical meristems. These lateral organs possess an inherent asymmetry with regard to the meristem in that their adaxial side is adjacent and close to the meristem, while their abaxial side is located further from the meristem. This asymmetry gives rise to a polarity that is readily apparent at the cellular and whole organ level and which can have important functional consequences. For example, cells within the adaxial region of a leaf are specialized for light capture, while those in the abaxial region are specialized for gas exchange. In addition, outgrowth of the leaf lamina is dependent on the juxtaposition of cells with adaxial and abaxial identities (Waites and Hudson, 1995  ). A similar mechanism may be responsible for the outgrowth of flattened floral organs such as sepals and petals.
Members of three classes of transcription factors contribute to the establishment of adaxial and abaxial cell fates in lateral organs of Arabidopsis (Arabidopsis thaliana; for review, see Engstrom et al., 2004
At least three members of the KANADI gene family (KAN1, KAN2, and KAN3) redundantly specify abaxial identity (Eshed et al., 2001
It has been proposed that a meristem-derived signal is responsible for the establishment of adaxial-abaxial polarity in lateral organs (for review, see Bowman et al., 2002
We are interested in the relationship between factors that promote the initiation and growth of lateral organ primordia, such as AINTEGUMENTA (ANT), and factors that act within lateral organ primordia to establish their polarity, such as PHB-like, KANADI, and YABBY proteins. ANT expression is up-regulated in leaf and flower founder cells in apical meristems and is one of the earliest markers of lateral organ specification (Elliott et al., 1996
ANT Binds to a Conserved Element in the FIL and YAB3 Promoters
FIL and YAB3 are expressed in largely overlapping domains and share sequence similarity within an approximately 300-bp 5' regulatory region, part of which is shown in Figure 1A
(Siegfried et al., 1999
FIL and YAB3 Expression Is Normal in ant Flowers To probe the potential role of ANT in FIL and YAB3 regulation, we examined the expression of FIL and YAB3 in an ant mutant background. If ANT is a positive regulator of FIL and/or YAB3, we might expect FIL and YAB3 expression to be reduced in ant mutants. FIL expression was examined in ant-4 flowers by in situ hybridization. A similar level and pattern of FIL expression was observed in Landsberg erecta (Ler) and ant-4 flowers (Fig. 2, A–D ). YAB3 mRNA was examined in Ler and ant-4 inflorescences by real-time reverse transcription (RT)-PCR. Similar levels of YAB3 mRNA were present in both genotypes (Fig. 2E). These results suggest that ANT activity is not required for activation of YAB3 or FIL in flowers.
fil ant and fil yab3 ant Plants Are Reduced in Size
To gain insight into the relationship between ANT and the two YABBY genes, we generated fil-8 ant-4 double mutants and fil-8 yab3-2 ant-4 triple mutants. The double and triple mutant plants were dwarfed and exhibited severe alterations in organ development during both vegetative and reproductive development (Figs. 3, A–D
, and 5, A and B). While the leaves of yab3-2, fil-8, and ant-4 single mutants were not dramatically different in size from those of wild type (Kumaran et al., 2002
To determine the basis for the smaller leaf size in fil-8 ant-4 and fil-8 yab3-2 ant-4 plants, the size of mature leaf epidermal cells was examined using scanning electron microscopy (SEM). Epidermal cells were larger in fil-8 ant-4 and fil-8 yab3-2 ant-4 plants compared to Ler (Fig. 4, A, B, and I–L ). This indicates that the smaller leaf blades of the double and triple mutants are due to the presence of fewer cells. Similarly, the reduced height of the double and triple mutant plants (Fig. 5A ; Table II ) was due to fewer cells in the stem (data not shown).
fil ant and fil yab ant Mutants Show Disruptions in Leaf Polarity
A juxtaposition of adaxial and abaxial cell types is thought to be required for leaf blade expansion (for review, see Bowman, 2000 More dramatic changes in adaxial and abaxial identities were observed in fil-8 ant-4 and fil-8 yab3-2 ant-4 plants. Adaxial epidermal cells of fil-8 ant-4 leaves were variable in size and sometimes puzzle shaped, slightly resembling abaxial epidermal cells (Fig. 4I). In addition, the abaxial surface was flatter than wild type and the cells larger than wild type (Fig. 4J). This suggests a partial loss of both adaxial and abaxial identities in fil-8 ant-4 plants. Thus, at least some of the reduced growth of fil-8 ant-4 leaves may result from a loss of polarity. The more dramatic reduction in lamina expansion in the triple mutant was correlated with a more complete loss of polarity. In fil-8 yab3-2 ant-4 leaves, adaxial and abaxial epidermal cells closely resembled each other with neither the adaxial or abaxial surface displaying its characteristic appearance (Fig. 4, K and L). Loss of both adaxial and abaxial identities distinguishes the fil ant and fil yab ant mutants from mutations in either the KAN or PHB-like genes, where there is replacement of one identity with the other identity (i.e. replacement of abaxial cell fates with adaxial identities in kan mutants).
fil-8 ant-4 and fil-8 yab3-2 ant-4 leaves also exhibit alterations in their vascular patterning. Vascular tissue in wild-type leaves exhibits a reticulate pattern with minor veins branching from the major vein (Fig. 3I). There was a marked decrease in vascular branching in the leaves of fil-8 ant-4 plants (Fig. 3K). This phenotype is similar to that reported previously for yab3-1 fil-5 leaves (Siegfried et al., 1999
fil-8 ant-4 and fil-8 yab3-2 ant-4 plants exhibit inflorescence defects similar to those observed in fil-8 plants with the inflorescence meristem switching between the production of flowers and filaments (Sawa et al., 1999
The flowers produced by fil-8 ant-4 and fil-8 yab3-2 ant-4 plants were much smaller than flowers of wild type, fil-8, or fil-8 yab3-2 (Fig. 5B). In addition, they exhibited loss of floral identity as demonstrated by the presence of flowers with subtending leaves and by a loss of floral organ identity. fil-8 ant-4 and fil-8 yab3-2 ant-4 flowers typically consisted of narrow, flat, green organs; filaments; and carpelloid organs (Figs. 5, G and I, and 6, B and C). Because these organs lack most recognizable features of floral organs, we examined their development and cell types by SEM to better characterize them. Because fil-8 ant-4 and fil-8 yab3-2 ant-4 flowers were quite similar, we present a detailed SEM analysis of just fil-8 ant-4 flowers. fil-8 ant-4 flowers typically produced two or three whorls of floral organs with variable numbers and positions of organs within each whorl (Fig. 6, D–F). Flowers arising later on the inflorescence typically produced a fewer number of organs. The flat, outermost organs from early arising fil-8 ant-4 flowers had epidermal cells resembling those of sepals (Fig. 6G). In later-arising flowers, these organs became thinner and more pointed. SEM analysis showed that these later-arising outer whorl organs were mosaics containing both leaf-like and sepal-like cells (Fig. 6H). Filamentous organs present in the outer two whorls of fil-8 ant-4 flowers were variable in appearance. Those in the outermost whorl were typically dark green and had epidermal cells resembling those of sepals (Fig. 6I), while filaments in the second whorl were light green or white in color with more regular cells in files (Fig. 6J). In addition, filaments in the outer whorl tended to be thicker than those in the second whorl. The flat innermost organs of fil-8 ant-4 flowers resembled unfused or partially fused carpels. Style-like cells were found at the top of these organs (Fig. 6, K and L), and valve-like ovary cells were present along the rest of their length. Stigmatic papillae were sometimes present, although not necessarily at the apex of these organs (Fig. 6C). The carpel-like organs arose as distinct primordia rather than the fused ring of tissue that develops in wild-type flowers (Fig. 6D). These primordia gave rise to either distinct organs (Fig. 6E) or partially fused organs (Fig. 6F). Internal tissues present within a normal gynoecium (septum, transmitting tract, placenta, and ovules) were almost completely lacking in fil-8 ant-4 flowers (Fig. 6, B and C). In a few early arising fil-8 ant-4 flowers, narrow white organs, and/or yellow stamen-like organs were present (Figs. 5H and 6M). SEM examination indicated that petal epidermal cells were present on the surface of some of these white organs (Fig. 6N). In some cases, these organs exhibited polarity defects, as petals cells with both adaxial and abaxial morphologies were present on the abaxial surface of these organs (Fig. 6O). Epidermal cells characteristic of stamens were present on the stamen-like organs (Fig. 6, P and Q). A small amount of internal carpel tissue was occasionally present in early arising fil-8 ant-4 flowers (Fig. 6P). fil-8 yab3-2 ant-4 flowers exhibit a slightly more severe phenotype than fil-8 ant-4 flowers in that organs with petal and stamen-like cells were never observed in fil-8 yab3-2 ant-4 flowers.
fil-8 ant-4 and fil-8 yab3-2 ant-4 flowers have a more severe phenotype than fil-8 yab3-2 flowers (Fig. 5, F, G, and I). fil-8 yab3-2 flowers consist of radialized or flat sepal-like organs, no petals, small stamen filaments lacking anthers, and a carpel with a larger style and no replum (Siegfried et al., 1999
Because of the greatly reduced floral organ identity in fil-8 ant-4 flowers, we examined the expression patterns of the floral homeotic genes APETALA3 (AP3) and AGAMOUS (AG) by in situ hybridization. AP3 is a B class floral homeotic gene involved in the specification of petal and stamen identities, and AG is a C function gene involved in the specification of stamen and carpel identities (Yanofsky et al., 1990
In wild-type plants, AG mRNA was first detected in the center of the floral meristem of stage 3 flowers (Fig. 7I). In fil-8 ant-4 plants, AG was misexpressed in the inflorescence meristem (Fig. 7J). AG mRNA was also detected in stage one and two floral meristems, earlier than the first appearance of AG mRNA in Ler flowers (data not shown). AG was expressed at high levels in the center of young fil-8 ant-4 floral meristems, similar to the pattern seen in young Ler stage 3 floral meristems (Fig. 7, K and L). In some cases, this AG expression domain was broader in stage 4 fil-8 ant-4 flowers than stage 4 Ler flowers and extended into the outermost organ primordia (Fig. 7M). In Ler flowers, AG is expressed throughout developing stamens and carpels until late stages of flower development (Fig. 7N). In older fil-8 ant-4 flowers, AG mRNA was detected on the inner surface of carpel-like organs (Fig. 7O). AG mRNA was also present in the center of filamentous structures produced by the inflorescence meristem (Fig. 7P).
The radialization of fil-8 ant-4 and fil-8 yab3-2 ant-4 floral organs suggests that these organs have lost adaxial-abaxial polarity. To further investigate this possibility, we examined the expression of the adaxial cell fate regulator PHB in fil-8 ant-4 flowers. PHB mRNA is present in the inflorescence meristem and throughout young floral meristems of Ler flowers (Fig. 8A ). In stage 4 flowers, PHB mRNA was detected in the center of the floral meristem and in the adaxial half of developing sepal primordia (Fig. 8B). The expression pattern and levels of PHB mRNA were similar in fil-8 and ant-4 single mutants as compared with Ler (Fig. 8, C–F). In fil-8 ant-4 plants, PHB mRNA was typically present at lower levels in the inflorescence meristem and stage 1 and 2 floral meristems as compared with wild type (Fig. 8G). PHB mRNA was usually absent from the outer whorl organs of fil-8 ant-4 flowers and was present in reduced amounts in the floral meristem of young stage 3 and 4 fil-8 ant-4 flowers (Fig. 8H).
ANT is an important regulator of lateral organ development. ANT expression marks cells that will leave the meristem to form lateral organs, and it is required for proper initiation and growth of lateral organs. Mutations in ANT result in the production of fewer and smaller floral organs (Elliott et al., 1996 ; Klucher et al., 1996). Despite its importance in lateral organ development, little is known about the genes regulated by this transcription factor. Our results here suggest that ANT acts with FIL to regulate organogenesis and to up-regulate genes establishing organ polarity and those specifying organ identity.
While lateral organs in ant single mutants exhibit normal polarity, defects along the adaxial/abaxial axis were visible at the whole organ, cellular, and molecular level in fil ant and fil yab ant plants. fil yab ant leaves showed dramatic reductions in lamina growth and loss of both adaxial and abaxial epidermal cell identities. fil ant and fil yab ant floral organs were severely radialized with many floral organs replaced by filaments or very narrow organs. In some floral organs, adaxial cell types were found in abaxial positions. These defects are more severe than those observed in either fil or fil yab mutants. To investigate whether the role of ANT in polarity establishment involves regulation of known adaxial and/or abaxial identity factors, we examined the expression of FIL, YAB3, and PHB in ant mutants. While the expression of FIL, YAB3, and PHB are normal in ant-4 flowers, PHB expression was reduced in fil ant double mutants. This suggests that ANT and FIL together are required for normal levels of PHB mRNA. Examination of the PHB promoter revealed a sequence matching the ANT consensus binding site in 12 of 14 conserved positions including one gap (S. Nole-Wilson and R. Franks, personal communication). We were unable to detect binding of ANT to this site in vitro, suggesting that ANT is not a direct regulator of PHB expression, its role in PHB regulation involves additional factors, and/or that ANT binds to a different DNA sequence within the context of the PHB promoter.
It is curious that an ANT binding site is present within a conserved region of the FIL and YAB3 promoters but that ANT is not required for FIL or YAB3 expression. Seven AINTEGUMENTA-like (AIL) genes are present within the Arabidopsis genome, and several of these genes are expressed in young floral primordia in overlapping domains with ANT (Nole-Wilson et al., 2005 As both adaxial and abaxial identities are partially lost in fil yab ant mutants and ANT is expressed throughout lateral organs, our results suggest that ANT is a positive regulator of both genes specifying adaxial fates and those specifying abaxial fates. ANT may function as a general activator of PHB-like and YABBY genes throughout organ primordia with their region-specific expression resulting from mutually repressive interactions between the PHB-like and KAN genes. Supporting our view that ANT is a positive regulator of genes specifying either adaxial or abaxial fates, preliminary examination of ant rev double mutants revealed enhanced carpel phenotypes including enhanced loss of some adaxial tissues relative to either single mutant (S. Nole-Wilson and R. Franks, personal communication).
SEUSS (SEU) and LEUNIG (LUG) have been proposed to promote polarity along the adaxial/abaxial axis in petals by positively regulating PHB and FIL expression (Franks et al., 2006
The similarities in these phenotypes suggest that ANT, FIL, SEU, and LUG have overlapping and partially redundant functions. These proteins might form a complex in which the SEU-LUG transcriptional corepressor (Sridhar et al., 2004
Besides contributing to the specification of abaxial identity, YABBY genes are important regulators of lamina expansion. Polar expression of YABBY genes has been proposed to regulate signaling events between the adaxial and abaxial domains that control cell division in each domain and expansion of the leaf lamina (Eshed et al., 2004
While petals and stamens are present in fil and ant single mutants, organs with petal or stamen characteristics are rare in fil ant double mutants. The loss of floral organ identify in fil ant double mutants was correlated with altered floral homeotic gene expression. AP3 expression was reduced in fil ant flowers while the pattern of AG expression was altered. Thus, ANT acts as a positive regulator of the class B gene AP3 and acts to prevent AG expression in inflorescences and flowers prior to stage 3. A role for ANT in AG repression has been noted previously (Krizek et al., 2000
Protein Expression
Full-length ANT lacking a stop codon was cloned into pQE12 (Qiagen) and expressed by induction with 1 mM isopropyl-
Gel mobility shift assays were carried out as described previously (Nole-Wilson and Krizek, 2000
Arabidopsis (Arabidopsis thaliana) ecotype Ler was used as the wild type. Plants were grown in a soil mixture of Fafard 4P:perlite:vermiculite in a ratio of 4:1:1 under continuous light (70–100 µmol m–2 s–1) at a temperature of 22°C. Plants were fertilized once at 1 to 2 weeks postgermination.
Inflorescences were collected from 3- to 4-week-old Ler and ant-4 plants grown on soil under continuous light at 22°C. Total RNA was extracted and DNase treated as described previously (Nole-Wilson et al., 2005
Inflorescences were fixed, embedded, sectioned, hybridized, and washed as described previously (Krizek, 1999
yab3-2 fil-8/+ seeds were obtained from John Bowman. These alleles have been described previously (Kumaran et al., 1999
yab3-2
fil-8
ant-4
For each genotype, the largest rosette leaf was removed from six different plants at the time of bolting. Leaf surface area was measured with a LI-COR LI-3000 portable area meter. The length and width of rosette leaves were measured using an ocular micrometer or a ruler. Plant heights were determined for six different plants of each genotype at the time when their primary inflorescences were starting to senesce. Bolting time was approximately the same for all genotypes.
Leaves were fixed overnight at room temperature in a 3:1 solution of ethanol:acetic acid. The tissue was mounted in 70% ethanol and examined using a dissecting microscope with illumination from below.
Tissue for SEM was fixed, dried, dissected, and coated as described previously (Krizek, 1999
We thank John Bowman for the fil-8 yab3-2/+ seeds and the FIL in situ plasmid, Charles Gasser for the ant-4 seeds, David Lincoln for help with leaf area measurements, John Herr for assistance with the xylem staining, and Mike Prigge and Steve Clark for the PHB in situ plasmid. We also thank Bob Franks for sharing unpublished data and providing valuable comments on the manuscript. Received January 2, 2006; returned for revision May 5, 2006; accepted May 12, 2006.
1 This work was supported by the U.S. Department of Energy (grant no. 98ER20312). 
2 Present address: Department of Genetics, North Carolina State University, Raleigh, NC 27695. The author responsible for the distribution of materials integral to the finding presented in this article in accordance with the policy described in the Instructions for Authors (www.plantphysiol.org) is: Beth A. Krizek (krizek{at}sc.edu). Article, publication date, and citation information can be found at www.plantphysiol.org/cgi/doi/10.1104/pp.106.076604. * Corresponding author; e-mail krizek{at}sc.edu; fax 803–777–4002.
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-D-thiogalactoside in XL1-Blue MRF' Tet cells (Stratagene) at 30°C. Cells were harvested between 6 and 8 h after induction. ANT was purified using Ni-NTA (Qiagen) under denaturing conditions according to the manufacturer's instructions. ANT refolded upon dilution in the DNA-binding reactions. 
