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First published online June 19, 2009; 10.1104/pp.109.141119 Plant Physiology 150:1916-1929 (2009) © 2009 American Society of Plant Biologists OPEN ACCESS ARTICLE
AINTEGUMENTA and AINTEGUMENTA-LIKE6 Act Redundantly to Regulate Arabidopsis Floral Growth and Patterning1,[C],[W],[OA]Department of Biological Sciences, University of South Carolina, Columbia, South Carolina 29208
An Arabidopsis (Arabidopsis thaliana) flower consists of four types of organs arranged in a stereotypical pattern. This complex floral structure is elaborated from a small number of floral meristem cells partitioned from the shoot apical meristem during reproductive development. The positioning of floral primordia within the periphery of the shoot apical meristem depends on transport of the phytohormone auxin with floral anlagen arising at sites of auxin maxima. An early marker of lateral organ fate is the AP2/ERF-type transcription factor AINTEGUMENTA (ANT), which has been proposed to act downstream of auxin in organogenic growth. Here, I show that the related, AINTEGUMENTA-LIKE6 (AIL6)/PLETHORA3 gene acts redundantly with ANT during flower development. ant ail6 double mutants show defects in floral organ positioning, identity, and growth. These floral defects are correlated with changes in the expression levels and patterns of two floral organ identity genes, APETALA3 and AGAMOUS. ant ail6 flowers also display altered expression of an auxin-responsive reporter, suggesting that auxin accumulation and/or responses are not normal. Furthermore, I show that ANT expression in incipient and young floral primordia depends on auxin transport within the inflorescence meristem. These results show that ANT and AIL6 are important regulators of floral growth and patterning and that they may act downstream of auxin in these processes.
During reproductive development in Arabidopsis (Arabidopsis thaliana), floral meristems are initiated reiteratively from groups of cells within the periphery of the shoot apical meristem. The particular sites at which flowers are initiated correspond to local auxin maxima generated by polar transport of the hormone by efflux carriers such as PINFORMED1 (PIN1; for review, see Fleming, 2007  ). Two genes closely associated with floral initiation are LEAFY (LFY) and AINTEGUMENTA (ANT), which act in the specification of floral meristem identity and growth, respectively. Both genes are expressed in incipient floral anlagen prior to outgrowth, and their expression patterns are altered in pin1 mutants, which are defective in floral initiation (Weigel et al., 1992; Elliott et al., 1996; Vernoux et al., 2000). The inability of pin1 mutants to initiate flowers can be rescued by the application of auxin paste to the periphery of the shoot apex, demonstrating the necessity and sufficiency of auxin for lateral organ development (Reinhardt et al., 2000).
Once established, floral meristems give rise to floral organ primordia at spatially defined positions within four concentric whorls. In an Arabidopsis flower, four sepal primordia arise in the outer first whorl, four petal primordia arise in the second whorl, six stamen primordia arise in the third whorl, and two carpel primordia arise in the centermost fourth whorl. Four different classes of floral organ identity genes (A, B, C, and E) act in different regions of the flower to specify these different organ identities (for review, see Krizek and Fletcher, 2005
Despite our knowledge about the control of floral organ identity and floral determinacy, other aspects of floral development remain obscure at the mechanistic level. This is especially true of early floral meristem patterning events such as floral organ positioning and primordia outgrowth. These processes are likely to be regulated by auxin because of their analogy with lateral organ positioning and primordium outgrowth from the shoot apical meristem. However, floral meristems are determinate, whereas the shoot apical meristem exhibits indeterminate growth. In addition, floral organ primordia arise in a whorled rather than a spiral phyllotaxis, thus requiring the simultaneous generation of multiple auxin maxima within the floral meristem. There is limited information on auxin distribution in early stages of flower development (Benkova et al., 2003
ANT is an important regulator of growth during lateral organ development. ant flowers reach a smaller final size, while plants constitutively expressing ANT produce flowers that reach a larger final size (Elliott et al., 1996
ANT is a member of the large AP2/ERF transcription factor family, containing a DNA-binding domain that corresponds to two AP2 repeats and the conserved intervening linker region (Nole-Wilson and Krizek, 2000
Here, I show that AIL6 acts redundantly with ANT during shoot development. While ail6 single mutants display no obvious morphological differences from the wild type, ant ail6 double mutants show defects during both vegetative and reproductive development. ant ail6 flowers exhibit altered positioning of floral organ primordia, loss of floral organ identity, and reduced growth of floral organ primordia. These defects are correlated with altered expression of stem cell and floral regulatory genes as well as the auxin-responsive reporter AGH3-2:GUS. ANT expression in incipient and young floral primordia is reduced after treatment with the auxin transport inhibitor N-1-naphthylphthalamic acid (NPA), thus suggesting a role for auxin transport in the regulation of ANT expression. These results support a model in which ANT and AIL6 promote growth and patterning in flowers downstream of auxin.
ail6 and ail6 ail7 Mutants Have a Wild-Type Appearance
To investigate the function of AIL6 (At5g10510), I identified four T-DNA insertion lines (Fig. 1B; Krysan et al., 1999 To determine if AIL6 might act redundantly with the closely related gene AIL7, I identified a single T-DNA line carrying an insertion within AIL7 (SAIL 1167_C10, ail7-1; Fig. 1C). The T-DNA is present in the sixth exon of AIL7. While partial transcripts have been detected in ail7-1 plants (J. Mudunkothge and B. Krizek, unpublished data), it is likely that any protein produced in these plants is nonfunctional, as the T-DNA is present within sequences encoding the linker between the two AP2 domains. ail7-1 plants have a wild-type appearance (Fig. 1H). ail6-2 was crossed to ail7-1 to generate the double mutant, which also exhibited no obvious morphological differences compared with wild-type plants (Fig. 1I).
AIL6 is more distantly related to ANT, but previously published in situ hybridization experiments showed that these two genes are expressed in partially overlapping domains in inflorescences (Elliott et al., 1996 ant-4 ail6-2 flowers consist primarily of small green organs; they lack petals and only rarely produce some stamen-like organs (Fig. 1, M–O; Table I ). The outermost organs of ant-4 ail6-2 flowers resemble sepals in their overall shape and epidermal cell characteristics (Fig. 2, A–D ). Other organs present in ant-4 ail6-2 flowers include filaments, filaments that are swollen or expanded at their distal end (Fig. 2A; Table I), and organs resembling unfused carpel valves (Fig. 2, A, E, and F). Stigmatic tissue is occasionally present on the margins of these valve-like organs (Fig. 2F), but they completely lack internal tissue normally present within carpels. ant-4 ail6-2 flowers sometimes contain other flat green organs that bear little resemblance to any floral organ found in wild-type flowers (Table I). Short filaments sometimes arise in the very center of ant-4 ail6-2 flowers (Fig. 2B).
Defects in the development of ant ail6 flowers are apparent at early stages. The floral meristem dome is often flatter in the double mutant (Figs. 2J and 3D ) and/or consists of fewer cells (Fig. 2I). Fewer floral organ primordia are initiated, and they do not arise in regular positions (Fig. 2, I, J, and L). In both wild-type and ant-4 flowers, four sepal primordia arise in a cross pattern, each arising approximately 90° from the adjacent primordium, with one primordium located in the position adaxial to the inflorescence meristem (Fig. 2, G and H). In ant-4 ail6-2 flowers, a variable number of sepal primordia initiate at random positions within the floral meristem periphery (Fig. 2, I and J). Similarly, more centrally arising floral organ primordia do not exhibit any regular phyllotaxy (Fig. 2, K and L).
AP3 and AG Expression Is Altered in ant ail6 Flowers Because of the loss of petal, stamen, and carpel identities in ant-4 ail6-2 flowers, I examined the expression of two floral organ identity genes: the class B gene AP3 and the class C gene AG. RNA gel blots showed that both genes are expressed at reduced levels in the double mutant (Fig. 3, A and G), while in situ hybridization revealed that the spatial expression patterns are altered. In a stage 3 wild-type flower, AP3 is expressed in cells adjacent to the sepal primordia that will give rise to the second and third whorls of the flower but not in the centermost cells of the floral meristem (Fig. 3B). Fewer cells in ant-4 ail6-2 stage 3 flowers express AP3 as compared with the wild type (Fig. 3, C and D). In addition, the central domain of non-AP3-expressing cells appears to be enlarged in ant-4 ail6-2 double mutants (Fig. 3, C and D). Later in floral development, AP3 is expressed in developing petals and stamens (Fig. 3E). In older ant-4 ail6-2 flowers, AP3 expression is only observed in a few cells at the adaxial base of the outer whorl organs and/or at the base of more inwardly arising organs (Fig. 3F).
In stage 3 wild-type flowers, AG is expressed in cells of the floral meristem that will develop into the third and fourth whorls of the flower (Fig. 3H). In stage 3 and 4 ant-4 ail6-2 flowers, AG mRNA is reduced and/or absent in the centermost cells of the floral meristem (Fig. 3, I and J). In addition, AG expression in the floral meristem directly abuts the sepal primordia in contrast to wild-type flowers, in which cells adjacent to the sepals do not express AG (Fig. 3, H–J). The extension of AG expression toward the sepals in ant ail6 flowers is consistent with earlier work showing that ANT can act as a second whorl repressor of AG (Krizek et al., 2000 To determine if the absence of AG mRNA in the center of ant-4 ail6-2 floral meristems might be due to reduced expression of its activators, I examined LFY and WUS expression in the double mutant. LFY mRNA was detected in incipient floral primordia and throughout stage 1 and 2 flowers in both wild-type and ant ail6 flowers (Fig. 4, A–D ). Although the signal appeared to be weaker in some ant ail6 flowers, no obvious difference in LFY mRNA levels could be detected by semiquantitative reverse transcription (RT)-PCR (Supplemental Fig. S2). In wild-type stage 3 flowers, LFY expression is strong and uniform throughout the sepal primordia and floral meristem (Fig. 4C). However, in stage 3 ant-4 ail6-2 flowers, LFY expression is found to be patchy (Fig. 4D). In contrast to LFY expression, WUS expression is increased in young ant-4 ail6-2 flowers (Fig. 4, E and F). In addition, WUS expression is detected in a broader domain in the double mutant that includes L1, L2, and L3 cells (Fig. 4, G and H).
ANT and AIL6 Regulate Floral Meristem Proliferation
As AG activity is required to down-regulate WUS expression but is missing from the central region of stage 3 ant-4 ail6-2 flowers, WUS expression was examined in older ant-4 ail6-2 flowers. Persistent WUS expression was sometimes observed in the central region of ant-4 ail6-2 flowers at stages after WUS expression had disappeared from wild-type flowers (Fig. 4, I–L). While persistent expression of WUS in ag flowers results in floral meristem indeterminacy (Lenhard et al., 2001 This possibility was investigated by constructing the ag-1 ant-4 ail6-2 triple mutant. ag-1 ant-4 ail6-2 flowers are determinate and consist primarily of sepals with a few petal-like organs (Fig. 5, A and B ). ag-1 ant-4 ail6-2 flowers typically produce just a few more floral organs than ant-4 ail6-2 flowers (Fig. 5C). In a few ag-1 ant-4 ail6-2 flowers, many more floral organs were produced, but these flowers were never indeterminate (Fig. 5D). Thus, ANT and AIL6 are required for the continued proliferation of the floral meristem in ag mutants. The partial restoration of petal identity in ag-1 ant-4 ail6-2 flowers and the increased number of floral organs could be due to increased proliferation of second whorl cells in the absence of AG.
ANT and AIL6 Promote Growth of All Shoot-Derived Organs
ant-4 ail6-2 plants also exhibit defects during vegetative development. The plants are reduced in height, with a bushier appearance due to the outgrowth of additional axillary inflorescences from rosette and cauline leaves (Fig. 6A
; Table II
). In addition, accessory meristems in cauline leaf axils exhibit enhanced outgrowth in ant-4 ail6-2 plants (Fig. 6B). Both leaves and flowers produced by ant-4 ail6-2 plants are reduced in size compared with those from ant-4 and wild-type plants (Fig. 6, C–F). Rosette leaves of the double mutant plants are thinner than those from ant-4 and wild-type plants (Fig. 6, C–E), with alterations in leaf vein architecture (Supplemental Fig. S3). ant-4 ail6-2 leaves show more severe defects in vein density and complexity than those previously reported for ant single mutants (Supplemental Fig. S3, B and C; Kang et al., 2007
Epidermal cells of rosette leaves and sepals were similar in size in both the wild type and ant-4 ail6-2, suggesting that the reduced size of these organs results primarily from fewer cells (Figs. 2, C and D, and 6, G and H). Epidermal peels of stems at positions midway along the primary inflorescence revealed similar cell lengths in wild-type and ant-4 ail6-2 plants (Fig. 6, I and J). However, cells making up the internodes between successive flowers of ant-4 ail6-2 plants are dramatically reduced in length in ant-4 ail6-2 plants as compared with wild-type plants (Fig. 6, K and L). Thus, the reduced height of ant-4 ail6-2 plants appears to arise primarily from reduced internode length.
Initially, the inflorescence meristems of ant-4 ail6-2 plants have a dome appearance similar to the wild type (Fig. 7, A and B ). However, as reproductive development proceeds, ant-4 ail6-2 inflorescence meristems become increasingly sloped, with flowers initiating at positions farther down the sides of the meristem (Fig. 7, C and D). Floral initiation eventually ceases in ant-4 ail6-2 plants, with the inflorescence meristem and surrounding floral primordia aborting growth. While the total number of flowers initiated by ant-4 ail6-2 inflorescence meristems prior to termination is similar to the number produced by wild-type plants prior to senescence (36.2 ± 3.0 in the wild type versus 33.3 ± 4.0 in ant-4 ail6-2), it is less than that produced by sterile mutants such as ant-4 (44.4 ± 8.5).
To gain insight into the basis for these defects in meristem function, I examined the expression of two important regulators of shoot apical meristem activity, WUS and CLAVATA3 (CLV3). WUS expression is confined to a small group of underlying cells in the shoot apical meristem referred to as the organizing center (Fig. 7G), while CLV3 is expressed in the overlying stem cells (Fig. 7E; Mayer et al., 1998 ; Fletcher et al., 1999). In ant-4 ail6-2 plants, CLV3 expression extends deeper into the inflorescence meristem than in the wild type (Fig. 7F), while WUS expression moves up into L1 and L2 cells within the center of the meristem (Fig. 7H). These changes and the partial overlap of the WUS and CLV3 expression domains suggest that cells within ant-4 ail6-2 inflorescence meristems have altered and/or mixed identities, since they express markers for both stem cell and organizing center fate.
Because several of the defects observed in ant-4 ail6-2 plants are reminiscent of mutants disrupted in auxin physiology, I examined auxin responses in ant-4 ail6-2 inflorescences using the auxin reporter AGH3-2:GUS. This reporter contains approximately 1.1 kb of 5' sequence from the Arabidopsis GH3-2 gene (At4g37390; G. Hagen, personal communication). The AGH3-2:GUS reporter shows a similar pattern of expression in inflorescences and flowers as DR5(rev):GFP, with expression in incipient floral primordia (Fig. 8A
) and the tips of developing floral organs (Fig. 8, E, G, and I; Benkova et al., 2003
Auxin Transport Is Necessary for ANT Expression in Incipient Flowers
As ANT and AIL6 expression in incipient floral primordia (Nole-Wilson et al., 2005
Because auxin may be necessary but not sufficient for ANT regulation, I treated inflorescences with the auxin transport inhibitor NPA. No difference in ANT mRNA levels was detected in Ler inflorescences treated with 100 µM NPA for 24 h as compared with mock-treated tissue (Fig. 9, A and B
). However, ANT expression was reduced in incipient floral primordia and stage 1 and 2 flowers of NPA-treated ANT:GUS inflorescences (Fig. 9, C and D). No obvious changes in ANT expression were observed in older flowers. Similar results were obtained by in situ hybridization examining ANT mRNA in Ler inflorescences treated with NPA (Fig. 9, E and F). Thus, auxin transport within the inflorescence meristem is necessary for high levels of ANT expression in the very earliest stages of flower development but not for ANT expression in older flowers. The observed reduction in ANT expression is observed prior to morphological changes in lateral organ initiation that occur as a consequence of NPA treatment (Fig. 9, G and H; Okada et al., 1991
ANT and AIL6 Regulate Flower Development ANT and AIL6 regulate several aspects of flower development, including floral meristem and organ growth, organ positioning within the floral meristem, and specification of floral organ identity. ant ail6 flowers contain fewer organs per flower than the wild type, as well as organs such as filaments and flat green organs that are not normally found in wild-type flowers. Thus, the absence of petals and stamens likely results from defects in both organ initiation and organ identity specification. Defects in organ initiation may be a consequence of insufficient cells within the floral meristem, while defects in organ identity specification likely result from altered expression of the floral organ identity genes AP3 and AG.
Similar LFY expression in ant ail6 and wild-type flowers of stages 1 and 2 suggests that ANT and AIL6 promote AP3 and AG expression through a LFY-independent pathway (Fig. 10
). However, nonuniform LFY expression in stage 3 ant ail6 flowers suggests that ANT and AIL6 may also promote floral organ identity gene expression through the maintenance of LFY expression (Fig. 10). The expanded WUS expression domain in ant ail6 flowers suggests that WUS is not limiting for AG activation. I examined LFY, AP3, and AG regulatory regions (Blazquez et al., 1997
The presence of five potential ANT-binding sites, each containing three mismatches, within the AG second intron suggests that ANT and AIL6 may be direct regulators of AG expression. Previous work has shown that ANT acts as a repressor of AG expression, preventing precocious AG expression in flowers (Liu et al., 2000 ) and acting redundantly with AP2 in repression of AG in the second whorl (Krizek et al., 2000). The data presented here show that ANT and AIL6 act redundantly to negatively regulate AG expression in second whorl cells and to positively regulate AG expression in the centermost cells of the floral meristem (Fig. 10). Thus, the role of ANT and AIL6 in AG regulation appears to be context dependent and likely involves different coregulators.
In ag mutants, persistent WUS expression in stage 6 and older flowers results in floral indeterminacy (Lenhard et al., 2001 ANT and AIL6 also appear to regulate AG expression in this inner fourth whorl domain, but unlike other genetic backgrounds in which persistent WUS expression results in indeterminacy, ant ail6 flowers are determinate. Furthermore, I have found that ANT and AIL6 activity is required for the indeterminacy of ag flowers. Thus, WUS itself is not sufficient for floral meristem indeterminacy but requires factors such as ANT and AIL6 that promote continued proliferation of stem cells. In the absence of ANT and AIL6, these cells do not proliferate to maintain a stem cell pool from which organ primordia can be initiated. The eventual termination of ant ail6 inflorescence meristems also supports a role for ANT and AIL6 in stem cell proliferation. The basis for shoot apical meristem termination in ant ail6 plants is not clear. While the width of the inflorescence meristem gets progressively smaller during development, it does not appear that all meristematic cells are consumed in the process of lateral organ initiation. Rather, there appears to be an arrest of the shoot apex with the cessation of new floral initiation and growth abortion of young primordia. The expression patterns of CLV3 and WUS are altered in ant ail6 inflorescence meristems, with both genes expressed in a broader domain and some cells within the central part of the meristem expressing both CLV3 and WUS. The broader expression domains of these central zone markers might indicate a loss of peripheral zone identity and consequently an inability to initiate new primordia.
Roles for ANT and AIL6 in auxin regulation of flower development are suggested by the apparent overlap between transient auxin maxima within the inflorescence meristem (Reinhardt et al., 2003
ant ail6 mutants are dwarfed, display altered organ growth, and have reduced amounts of vascular tissue, phenotypes similar to mutants defective in auxin physiology. Treatment of ant ail6 inflorescences with exogenous auxin showed that these plants could respond to auxin, suggesting that at least some primary auxin responses mediated by ARFs are functional in these plants. If ANT and AIL6 do function in auxin signaling, presumably they do so downstream of ARFs. Two potential AuxREs are present within the ANT promoter at positions –1,083 to –1,078 and –2,525 to –2,520 relative to the start codon. No sequences with similarity to AuxRE were identified in AIL6 regulatory regions. Altered expression of the auxin-responsive reporter AGH3-2:GUS in ant ail6 flowers may indicate defects in some auxin responses or alternatively that auxin levels and/or distribution are altered in ant ail6 inflorescences. Future experiments need to be conducted to investigate these different possibilities. A role for ANT and AIL6 in mediating auxin distribution would not preclude a possible role for these proteins in signaling downstream of auxin. Feedback between the auxin distribution system and auxin signaling pathways allows the plant to coordinate developmental processes and respond to changing environmental conditions (Leyser, 2006
Members of the AIL/PLT gene family play important and somewhat similar roles in roots and shoots. While plt1 and plt2 single mutants show subtle root defects, plt1 plt2 double mutants exhibit reduced root growth, altered cellular patterning in the root tip, and termination of the root apical meristem by 6 to 8 d after germination (Aida et al., 2004
PLT function has been proposed to promote root growth and patterning downstream of auxin. The highest accumulation of PLT1, PLT2, AIL6/PLT3, and BBM proteins in the root occurs in the stem cell niche, which corresponds to a stable auxin maximum (Galinha et al., 2007
Plant Growth Conditions Arabidopsis (Arabidopsis thaliana) plants were grown on a soil mixture of either Metro-Mix 360:perlite:vermiculite (5:1:1) or Miracle Gro Moisture Control Potting Mix:perlite:vermiculite (5:1:1) under continuous light or in 16-h days (100–150 µmol m–2 s–1) at a temperature of 22°C.
DNA was isolated from leaves and used in PCR for genotyping (Klimyuk et al., 1993
Inflorescences were fixed, embedded, sectioned, hybridized, and washed as described previously (Krizek, 1999
Tissue for scanning electron microscopy was fixed, dried, dissected, and coated as described previously (Krizek, 1999
Inflorescences (corresponding to all unopened floral buds) were homogenized in liquid nitrogen, and total RNA was isolated with hot phenol extraction buffer (Verwoerd et al., 1989
Total RNA was treated for 4 to 6 h with RQ1 RNase-free DNase (Fisher Scientific) at 37°C. Approximately 5 µg of total RNA was reverse transcribed using the SuperScript III first-strand synthesis system (Invitrogen). PCR conditions were 40 cycles of 30 s at 92°C, 30 s at 50°C, and 2 min at 72°C and one cycle of 5 min at 72°C. Aliquots (5 µL) were removed at five-cycle intervals starting with cycle 20. Actin transcript levels confirmed that equivalent amounts of cDNA were used in the experiment.
The epidermis was removed from a midsection of the inflorescence stem of 5-week-old plants, placed on a drop of water on a glass slide, and stained briefly with toluidene blue. Plants used in height measurements and axillary inflorescence counts were germinated on plates and transplanted into soil at 7 d after germination. Plant height was measured and the number of axillary inflorescences from rosette and cauline leaves was counted when the plants were 6 weeks old. Vascular tissue was examined by fixing the sixth leaf of 25-d-old plants overnight at room temperature in a 3:1 solution of ethanol:acetic acid. The leaves were mounted in 70% ethanol and photographed using a dissecting microscope with illumination from below.
The GUS assays were performed as described previously (Krizek and Meyerowitz, 1996
AGH3-2:GUS and AGH3-2 ant-4 ail6-2 inflorescences were treated with 50 µM IAA (in 0.05% methanol and 0.01% Silwet L-77) or a mock solution (0.05% methanol and 0.01% Silwet L-77) using a paintbrush, harvested 6 h after treatment, and stained for GUS. Ler and ANT:GUS inflorescences were painted with 50 µM IAA (in 0.05% methanol and 0.01% Silwet L-77) or a mock solution (0.05% methanol and 0.01% Silwet L-77) and harvested 24 h after treatment. Similar treatments were conducted with 50 µM 2,4-dichlorophenoxyacetic acid (in 0.05% ethanol and 0.01% Silwet L-77) or a mock solution (0.05% ethanol and 0.01% Silwet L-77). For the NPA experiment, the tissue was treated twice (at 0 and 7 h), as described previously (Nemhauser et al., 2000
For construction of the ANT:GUS plasmid, GUS and the 3' nos terminator sequence were subcloned from pBI121 into pBluescript SK– using BamHI and EcoRI and subsequently into pCGN1547 using BamHI and HindIII. A 6.2-kb region of ANT sequence 5' to the start codon was constructed in pBluescript SK– by first subcloning a 5-kb ANT genomic fragment followed by the addition of a 1.2-kb PCR product. The entire 6.2-kb ANT promoter sequence was subsequently subcloned upstream of GUS in pCGN1547 using KpnI. ANT:GUS/pCGN1547 was transformed into Agrobacterium tumefaciens strain ASE by electroporation. Arabidopsis Ler plants were transformed with this Agrobacterium strain by vacuum infiltration (Bechtold et al., 1993 The Arabidopsis Genome Initiative locus numbers for the genes analyzed in this article are as follows: ANT, At4g37750; AIL6, At5g10510; AIL7, At5g65510.
The following materials are available in the online version of this article.
I thank the Salk Institute Genomic Analysis Laboratory, the University of Wisconsin Arabidopsis knockout facility, and the GABI-Kat project for providing the Arabidopsis T-DNA insertion mutants used in this study. I thank the Arabidopsis Biological Resource Center for seeds of the T-DNA insertion alleles, Jennifer Fletcher for the CLV3 and WUS plasmids used to generate in situ probes, Josefina Poupin for help with the WUS in situ hybridization experiments, Doris Wagner for the LFY plasmid used to generate an in situ probe and for WUS:GUS seeds, Gloria Muday and Gretchan Hagen for AGH3-2:GUS seeds, David Smyth for the pBW plasmid containing ANT genomic DNA, and Ben Scheres and Kalika Prasad for comments on the manuscript. I also acknowledge the University of South Carolina Electron Microscopy Center for instrument use and technical assistance. Received May 8, 2009; accepted June 10, 2009; published June 19, 2009.
1 This work was supported by the U.S. Department of Energy (grant no. 98ER20312).  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.plantphysiol.org) is: Beth A. Krizek (krizek{at}sc.edu).
[C] Some figures in this article are displayed in color online but in black and white in the print edition.
[W] The online version of this article contains Web-only data.
[OA] Open Access articles can be viewed online without a subscription. www.plantphysiol.org/cgi/doi/10.1104/pp.109.141119
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