Three Arabidopsis AILPLT genes act in ... - Wiley Online Library

The Plant Journal (2012) 71, 108–121

doi: 10.1111/j.1365-313X.2012.04975.x

Three Arabidopsis AIL/PLT genes act in combination to regulate shoot apical meristem function Janaki S. Mudunkothge and Beth A. Krizek* Department of Biological Sciences, University of South Carolina, Columbia, SC 29208, USA Received 11 December 2011; revised 12 February 2012; accepted 24 February 2012; published online 30 April 2012. *For correspondence (e-mail [email protected]).

SUMMARY The shoot apical meristem, a small dome-shaped structure at the shoot apex, is responsible for the initiation of all post-embryonic shoot organs. Pluripotent stem cells within the meristem replenish themselves and provide daughter cells that become incorporated into lateral organ primordia around the meristem periphery. We have identified three novel regulators of shoot apical meristem activity in Arabidopsis thaliana that encode related AIL/PLT transcription factors: AINTEGUMENTA (ANT), AINTEGUMENTA-LIKE6 (AIL6)/PLETHORA3 (PLT3) and AINTEGUMENTA-LIKE7 (AIL7)/PLETHORA7 (PLT7). Loss of these genes results in plants that initiate only a few leaves prior to termination of shoot apical meristem activity. In 7-day-old ant ail6 ail7 seedlings, we observed reduced cell division in the meristem region, differentiation of meristematic cells and altered expression of the meristem regulators WUSCHEL (WUS), CLAVATA3 (CLV3) and SHOOT MERISTEMLESS (STM). Genetic experiments suggest that these three AIL genes do not act specifically in either the WUS/CLV or STM pathway regulating meristem function. Furthermore, these studies indicate that ANT, AIL6 and AIL7 have distinct functions within the meristem rather than acting in a strictly redundant manner. Our study thus identifies three new genes whose distinct functions are together required for continuous shoot apical meristem function. Keywords: shoot apical meristem, Arabidopsis, stem cells, AP2/ERF transcription factors, AIL/PLT family, differentiation.

INTRODUCTION The unique ability of plants to initiate organs throughout their lifespan results from the activity of meristems. The shoot apical meristem, a dome of cells at the shoot apex, provides cells for the initiation of new organ primordia around its periphery while maintaining a central core of replenishing stem cells that remain undifferentiated. A balance between stem cell renewal and organ production is required for continued meristem function. Genetic studies in Arabidopsis thaliana have identified at least two major pathways required for meristem activity. In one pathway, the class I KNOTTED-like (KNOX) homeodomain protein SHOOT MERISTEMLESS (STM) acts within the meristem to promote cell division and suppress differentiation (Endrizzi et al., 1996; Long et al., 1996; Lenhard et al., 2002). The CLAVATA (CLV1, CLV2 and CLV3) and WUSCHEL (WUS) proteins act in a second pathway to maintain a specified number of stem cells within the meristem (reviewed in Barton, 2010; Ha et al., 2010). In the latter pathway the homeodomain protein WUS, expressed in a few underlying cells in the center of the meristem called the organizing center, activates CLV3 in 108

overlying cells and specifies stem cell identity in these cells (Mayer et al., 1998; Schoof et al., 2000). CLV3, a secreted peptide, probably acts through three receptor complexes: CLV1 homodimers, CLV2/CORYNE (CRN) heterodimers and RPK2 homo-oligomers (Fletcher et al., 1999; Muller et al., 2008; Ogawa et al., 2008; Ohyama et al., 2009; Kinoshita et al., 2010). Binding of CLV3 to these receptors initiates a signaling cascade that acts to restrict WUS expression to the organizing center (Brand et al., 2000). This feedback mechanism creates a balance between the stem-cell promoting activity of WUS and the stem-cell restricting activity of the CLV proteins. Acting in combination with these pathways in the control of meristem activity are phytohormones, particularly cytokinin, auxin and gibberellin (GA), which appear to have distinct spatial distributions within the meristem (reviewed in Besnard et al., 2011; Ha et al., 2010; Su et al., 2011). High cytokinin levels in the center of the meristem control meristem size and maintenance (Kurakawa et al., 2007). High auxin in the periphery of the meristem specifies the sites of lateral organ initiation and promotes primordium ª 2012 The Authors The Plant Journal ª 2012 Blackwell Publishing Ltd

AIL/PLT genes regulate meristem function 109 outgrowth (Reinhardt et al., 2000, 2003; Heisler et al., 2005). As GAs promote cell differentiation, GA levels are kept low in the center of the meristem by members of the class I KNOX family, which repress GA biosynthesis and promote GA degradation (Sakamoto et al., 2001; Hay et al., 2002; Chen et al., 2004; Bolduc and Hake, 2009). In organ anlagen, auxin accumulation is associated with reduced STM expression (Heisler et al., 2005), which thus contributes to high GA levels in the meristem periphery. One means by which STM and WUS control meristem function is via regulation of hormone synthesis and signaling. STM promotes cytokinin biosynthesis through transcriptional upregulation of ISOPENTENYL TRANSFERASE (IPT) genes and stm mutants are partially rescued by application of exogenous cytokinin (Jasinski et al., 2005; Yanai et al., 2005). Transgenic plants overproducing cytokinin have elevated STM levels (Rupp et al., 1999), suggesting the presence of a positive feedback loop between cytokinin and KNOX genes. Additional feedback mechanisms operate between cytokinin and WUS. WUS represses the expression of several type-A ARABIDOPSIS RESPONSE REGULATOR (ARR) genes, which are negative regulators of cytokinin signaling, thus sensitizing some cells in the meristem to cytokinin (Leibfried et al., 2005). In addition, cytokinin signaling induces WUS expression through both CLV3dependent and CLV3-independent pathways (Gordon et al., 2009). Crosstalk between hormone signaling pathways within the meristem is also beginning to be revealed. For example, auxin and cytokinin signaling converge on two type-A ARRs (ARR7 and ARR15), which are induced by cytokinin signaling and repressed by the auxin response factor MONOPTEROS (MP) (Zhao et al., 2010). ANT, AIL6/PLT3 and AIL7/PLT7 encode related proteins of the Arabidopsis AIL/PLT family, an eight-member subfamily of AP2/ERF transcription factors (Nole-Wilson et al., 2005; Prasad et al., 2011). Previous work has shown that ANT and AIL6 have partially redundant roles in several aspects of shoot and flower development, including lateral organ growth, floral meristem initiation and floral patterning (Krizek, 2009, 2011). In addition, AIL6 together with PLT1, PLT2 and BBM/PLT4 specifies root development, and in combination with AIL5/PLT5 and AIL7 controls shoot phyllotaxis (Galinha et al., 2007; Prasad et al., 2011). Members of the AIL/PLT gene family also regulate meristem development. PLT1 and PLT2, control patterning within the root stem cell niche and are required for root stem cell activity (Aida et al., 2004). The shoot apical meristems of plt5 plt3 plt7 (ail5 ail6 ail7) triple mutants are functional but slightly reduced in width, suggesting that AIL5, AIL6 and AIL7 regulate meristem size in addition to lateral organ positioning (Prasad et al., 2011). As AIL6 has partially overlapping functions with ANT but is most closely related to AIL7, we investigated the effects of the combined loss of ANT, AIL6 and AIL7. We find that these

three genes together are required for shoot meristem activity. In ant-4 ail6-2 ail7-1 triple mutants, the shoot apical meristem terminates after the production of a few leaves. These meristem defects are associated with reduced cell division, stem cell differentiation and altered expression of meristem regulators. The distinct expression patterns of ANT, AIL6 and AIL7 within the shoot apical meristem and their unique genetic interactions with STM and WUS suggest that rather than acting redundantly in the meristem, these genes have unique roles that together are required for meristem maintenance and continued organ formation. RESULTS ANT, AIL6 and AIL7 are expressed in distinct domains within the vegetative shoot apical meristem Earlier work has shown that ANT mRNA is detected in all shoot-derived incipient lateral organ primordia (Elliott et al., 1996; Long and Barton, 2000) (Figure 1a). Previously, we found that AIL6 and AIL7 exhibit partially overlapping but distinct expression patterns in the inflorescence meristem (Nole-Wilson et al., 2005). AIL6 was expressed in the meristem center but more strongly in the peripheral region and incipient floral primordia, while AIL7 expression was strongest in the central region and downregulated in incipient floral primordia. Using in situ hybridization, we find similar expression patterns for AIL6 and AIL7 in the vegetative shoot apical meristem. AIL6 mRNA is highest in the peripheral regions of the meristem while AIL7 mRNA is highest in the center of the meristem (Figure 1b–d). A recent paper utilizing protein fusions of AIL6 and AIL7 to GUS reported similar results (Prasad et al., 2011). ant ail6 ail7 triple mutants are unable to maintain shoot apical meristem function We generated ant-4 ail6-2 ail7-1 triple mutants by crossing ail7-1 (in Columbia) to ant-4/+ ail6-2 [mixed Landsberg erecta (Ler) and Columbia (Col) backgrounds] (see Experimental Procedures). Further characterization was performed on triple mutants that contained erecta and thus Ler served as our wild-type control in the studies presented here. ant-4 ail6-2 ail7-1 triple mutants are first visibly distinct from the wild type at 7 days post-germination. At this time, the first two leaves produced by the shoot apical meristem are narrower than those in the wild type with more visible petioles (Figure 1e,f). The triple mutant plants produce three to five small and narrow leaves before the shoot apical meristem stops leaf production (Figure 1h,i,k,l). ant-4 ail6-2 ail7-1 leaves have reduced amounts of vascular tissue and veins that are not fully connected (Figure 1o,p). Additional leaves are formed later in development, but the plants never bolt or produce flowers (Figure 1n). We confirmed the triple mutant phenotype by generating a second ail7 allele (ail7IR) using an inverted repeat transgene (Figure S1 in the Supporting

ª 2012 The Authors The Plant Journal ª 2012 Blackwell Publishing Ltd, The Plant Journal, (2012), 71, 108–121

110 Janaki S. Mudunkothge and Beth A. Krizek

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Figure 1. ANT, AIL6 and AIL7 mRNA expression in vegetative meristems and characterization of ant ail6 ail7 triple mutant plants. (a–d) ANT (a), AIL6 (b, c) and AIL7 (d) mRNA expression in 7-day-old seedlings.(e–g) Seven-day-old Ler (e), ant-4 ail6-2 ail7-1 (f) and ant-4 ail6-2 ail7IR (g) seedlings. (h–j) Ten-day-old Ler (h), ant-4 ail6-2 ail7-1 (i) and ant-4 ail6-2 ail7IR (j) seedlings. (k–m) Seventeen-day-old Ler (k), ant-4 ail6-2 ail7-1 (l) and ant-4 ail6-2 ail7IR (m) plants. (n) Thirty-day-old Ler, ant-4 ail6-2 ail7-1 and ant-4 ail6-2 ail7IR plants. (o, p) Vascular tissue in Ler (o) and ant-4 ail6-2 ail7-1 (p) leaves. Scale bars: (a–d) 50 lm; (e–j), (o, p) 2 mm; (k–m) 10 mm; (n) 20 mm.

Information). Like ail7-1, ail7IR plants display no obvious developmental defects (Figure S1). ant-4 ail6-2 ail7IR plants look like ant-4 ail6-2 ail7-1 plants (Figure 1g, j, m, n), confirming that the triple mutant phenotype results from reduced AIL7 activity in combination with defects in ANT and AIL6. ant ail6 ail7 shoot apical meristems exhibit morphological and cell division defects We next examined the shoot apical meristem of ant-4 ail6-2 ail7-1 plants by scanning electron microscopy (SEM). At 7 days post-germination, the shoot apical meristem of the triple mutant is not as wide or dome-shaped as wild-type meristems (Figure 2a,b). We used Ler as the wild type for these studies; this may be a conservative comparison as meristem size is larger in Col compared with Ler (Vanhaeren et al., 2010). At 10 days post-germination, ant-4 ail6-2 ail7-1 meristems appear as pointed projections from the shoot apex and no lateral organ primordia are visible around their periphery (Figure 2c,d). Sectioning of 7-day-old seedlings reveals fewer cytoplasmically dense cells in the shoot apical meristem of the triple mutant compared with the wild type

(Figure 2e,f). In 10-day-old triple mutant seedlings, cells in the meristem region are larger and more vacuolated than those in the wild type, suggesting that these cells have undergone differentiation (Figure 2g,h). To investigate whether the meristem defects of ant-4 ail62 ail7-1 plants are associated with alterations in cell division, we examined the expression of the S phase marker histone H4. Seven-day-old ant-4 ail6-2 ail7-1 meristems had fewer histone H4-expressing cells relative to the wild type (Figure 2i–l). In 10-day-old seedlings, half of the triple mutant meristems examined had no histone H4-expressing cells while the remaining meristems had between one and three such cells (Figure 2m–p). Because previous work has demonstrated that alterations in the expression of two B2-type cyclin-dependent kinases (CDKB2;1 and CDKB2;2) resulted in meristem arrest (Anderson et al., 2008), we examined expression of these two genes in the triple mutant. CDKB2;1 and CDKB2;2 expression was reduced in 7-day-old triple mutant seedlings compared with the wild type (Figure 2q,r). Thus, the meristem defects present in ant ail6 ail7 triple mutants are associated with reduced cell division and reduced expression of cell cycle regulators.


AIL/PLT genes regulate meristem function 111

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Figure 2. ant ail6 ail7 triple mutant plants have meristem defects. (a, b) Scanning electron micrographs of the shoot apices of 7-day-old Ler (a) and ant-4 ail6-2 ail7-1 (b) seedlings. Arrows point to the shoot apical meristems. (c, d) Scanning electron micrographs of the shoot apices of 10-day-old ant-4 ail6-2 ail7-1 seedlings. Arrows point to the ‘terminated’ shoot apical meristems. (e, f) Toluidine blue-stained epoxy section of 7-day-old Ler (e) and ant-4 ail6-2 ail7-1 (f) shoot apical meristems. (g, h) Toluidine blue-stained epoxy sections of 10-day-old Ler (g) and ant-4 ail6-2 ail7-1 (h) shoot apical meristems. (i–l) In situ hybridization showing histone H4 mRNA in 7-day-old Ler (i) and ant-4 ail6-2 ail7-1 (j–l) shoot apices. (m–p) In situ hybridization showing histone H4 expression in 10-day-old Ler (m) and ant-4 ail6-2 ail7-1 (n–p) shoot apices. (q), (r) Relative CDKB2;1 and CDKB2;2 expression in Ler and ant-4 ail6-2 ail7-1 7-day-old seedlings as measured by real time RT-PCR. The expression level in Ler is set to 1 and error bars show standard deviation. Expression levels were normalized using At5g12240 (q) or At5g15710 (r). Scale bars: (a–c), (i–p) 50 lm; (d–h) 100 lm.

CLV3, WUS and STM expression is altered in ant ail6 ail7 seedlings We next examined the expression of known meristem regulators in 7-day-old ant-4 ail6-2 ail7-1 seedlings. Real-time RT-PCR revealed that CLV3 mRNA was undetectable in ant-4 ail6-2 ail7-1 7-day-old seedlings, while WUS mRNA levels were reduced and STM mRNA levels were slightly increased (Figure 3a). To investigate the spatial expression patterns of these genes in the triple mutant, we performed in situ

hybridization. Consistent with the real-time RT-PCR results, we could not detect CLV3 mRNA in meristems of 7-day-old ant-4 ail6-2 ail7-1 seedlings (Figure 3b,c). WUS mRNA was detected in 13 out of 17 ant-4 ail6-2 ail7-1 meristems with varying intensity levels (Figure 3d–f). In many cases, the WUS expression domain was shifted upward into the L1 and L2 layers of the meristem instead of being confined to the L3 layer as in the wild type (Figure 3f). STM mRNA was detected throughout the smaller ant-4 ail6-2 ail7-1 shoot apical meristems, unlike wild-type meristems, which show



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Figure 3. Expression of the meristem regulators CLV3, WUS and STM and the lateral organ marker ANT are altered in 7-day-old ant ail6 ail7 seedlings. (a) CLV3, WUS and STM mRNA levels in 7-day-old Ler and ant-4 ail6-2 ail7-1 seedlings as measured by real time RT-PCR. The expression level in Ler is set to 1 and error bars show standard deviation. (b, c) In situ hybridization showing CLV3 mRNA in 7-day-old Ler (b) and ant-4 ail6-2 ail7-1 (c) shoot apical meristems. (d–f) In situ hybridization showing WUS mRNA in 7-day-old Ler (d) and ant-4 ail6-2 ail7-1 (e, f) shoot apical meristems. (g, h) In situ hybridization showing STM mRNA in Ler (g) and ant-4 ail6-2 ail7-1 (h) seedlings. Arrows point to developing leaves. (i, j) In situ hybridization showing ANT mRNA in 7-day-old Ler and ant-4 ail6-2 ail7-1 seedlings. Scale bars: (b–j) 50 lm.

downregulation of STM expression in incipient lateral organ primordia (Figure 3g,h). The STM expression pattern in the triple mutant meristem is consistent with the observation that no additional lateral organ primordia are initiated by these meristems. STM mRNA was also detected in developing leaf primordia in the triple mutant, something not observed in the wild type (Figure 3g,h). The slightly increased levels of STM mRNA detected in ant-4 ail6-2 ail7-1 seedlings by real-time RT-PCR probably reflect this ectopic STM expression in developing leaves. We also examined expression of ANT, a marker of lateral organ fate (Elliott et al., 1996; Long and Barton, 2000). Interestingly, ANT mRNA was detected not only in leaf primordia but also in ant-4 ail6-2 ail7-1 meristems (Figure 3i,j). The expression of both STM and ANT in these terminated meristems suggests that they have mixed meristem/lateral organ identity. ant ail6 ail7 embryos have normal morphology and expression patterns of meristem regulators ANT, AIL6 and AIL7 are all expressed during embryogenesis, although not specifically in the shoot apical meristem or progenitor cells. ANT mRNA is first detected in a ring at the top of late globular embryos and later in the cotyledon primordia (Long and Barton, 1998). AIL6 is expressed in the basal portion of heart stage embryos (Galinha et al., 2007).

AIL7 is expressed in seeds starting around the heart stage of embryogenesis, but its spatial expression pattern has not been characterized (Schmid et al., 2005). To determine whether defects in ant-4 ail6-2 ail7-1 seedlings reflect earlier disruptions in embryonic meristem development, we examined mature triple mutant embryos. Because the triple mutant plants are sterile, we examined embryos from siliques produced on ant-4/+ ail6-2 ail7-1 plants. No obvious differences in meristem size or structure were observed within the population of embryos produced by these plants as compared with the wild type (Figure S2). We next investigated whether loss of these genes affect meristem gene expression during embryogenesis. We compared CLV3, WUS and STM expression between different embryos within each ant-4/+ ail6-2 ail7-1 silique as well as with the wild type. Examination of a large number of embryos revealed no obvious differences in CLV3, WUS or STM expression (Figure S2, Table S1). These results indicate that ANT, AIL6 and AIL7 are not required for establishment of the shoot apical meristem during embryogenesis. ant ail6 ail7 triple mutants produce additional leaves later in development Although ant-4 ail6-2 ail7-1 shoot apical meristems terminate leaf production by 7 days post-germination, additional


AIL/PLT genes regulate meristem function 113 leaves are produced at a later time by the plants. Examination of 14–20-day-old ant-4 ail6-2 ail7-1 shoot apices by SEM, showed small bulges and projections arising in the leaf axils and in the central region of the shoot apex, with the number of visible bumps increasing with age (Figure 4a–d). A few bumps had a dome-like appearance (Figure 4a), and their location within the leaf axil suggested that they are axillary meristems. However, in tissue sections we never observed meristem-like structures with tunica–corpus layering and the presence of multiple primordia arising in a regular fashion. Some outgrowths developed into leaves, as trichomes were visible on larger primordia that had acquired a flatter appearance by 20 days post-germination (Figure 4d).

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However, many of these outgrowths appear to abort growth and remain as short elongated projections. To determine whether any of these structures correspond to meristems with stem cells, we examined 14-day-old ant-4 ail6-2 ail7-1 seedlings for expression of the stem cell marker CLV3, which was not detected in 7-day-old mutant seedlings. CLV3 mRNA was detected in the triple mutant, although at reduced levels (Figure 4e). In addition, expression of both a meristem marker (STM) and a lateral organ marker (ANT) was observed in these bumps (Figure S3). This suggests that either these bumps are only transiently meristematic or that they have mixed identity. ANT, AIL6 and AIL7 exhibit distinct genetic interactions with meristem regulators To investigate whether ANT, AIL6 and AIL7 function in one of the two known pathways regulating meristem development, we crossed the triple mutant into stm-1, wus-1 and clv3-2. stm-1

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Figure 4. Additional leaves are produced from the leaf axils and shoot apex of ant ail6 ail7 plants. (a) Scanning electron micrograph of the shoot apex of a 14-day-old ant-4 ail62 ail7-1 plant. Black arrows point to bulges in leaf axils. White arrow points to a dome-shaped bulge. (b, c) Scanning electron micrographs of the shoot apices of 17-day-old ant-4 ail6-2 ail7-1 plants. Arrows point to outgrowths in leaf axils (b) or central region of shoot apex (c). (d) Leaf primordia developing within the leaf axil of a 20-day-old ant-4 ail6-2 ail7-1 plant (arrow). (e) CLV3 mRNA levels in 14-day-old Ler and ant-4 ail6-2 ail7-1 shoots as measured by real time RT-PCR. The expression level in Ler is set to 1 and error bars show standard deviation. Scale bars: (a) 200 lm; (b, d) 500 lm; (c) 300 lm.

stm-1 seedlings lack an embryonic shoot apical meristem and do not initiate leaves upon germination (Barton and Poethig, 1993). Starting around 14 days post-germination, leaves can be seen developing near the cotyledon axils in some stm-1 seedlings (Figure 5a) (Barton and Poethig, 1993; Clark et al., 1996). We refer to the later production of leaves in stm-1 as the ‘recovery’ phenotype. In our hands some stm-1 plants bolt, but only rarely do these plants make flowers (Figure 5i). stm-1 ant-4 ail6-2 ail7-1 quadruple mutant plants are indistinguishable from stm-1 single mutants at 7 days post-germination but show more severe defects with respect to the stm-1 ‘recovery’ phenotype. In 15day-old plants, 29% of stm-1 but no stm-1 ant-4 ail6-2 ail7-1 plants had produced leaves (Table 1). Between 21 and 28 days, some stm-1 ant-4 ail6-2 ail7-1 plants produced very small leaves but these plants never bolt or produce flowers (Figure 5h, Tables 1 and 2). Similar but more severe phenotypes were observed in stm-1 ant-4, stm-1 ail6-2 and stm-1 ant-4 ail6-2 plants. These mutant combinations almost never produced any leaves (Figure 5b,c,e, Tables 1 and 2). Interestingly, ail7-1 had an opposite genetic interaction with stm-1 compared with ant-4 and ail6-2. A higher percentage of stm-1 ail7-1 plants make leaves, bolt and produce flowers compared with stm-1 (Figure 5d,j, Tables 1 and 2). Addition of the ail7-1 allele into the stm-1 ail6-2 and stm-1 ant-4 ail6-2 backgrounds partially suppresses the severity of these phenotypes with a higher percentage of plants producing leaves (Figure 5g,h, Tables 1 and 2). Some stm-1 ail6-2 ail7-1 triple mutants bolt and make flowers resembling those of stm-1 (Figure 5l, Tables 1 and 2). Surprisingly, stm-1 ant-4 ail7-1 plants are more likely to initiate leaves than stm-1 ail7-1 (Figure 5f, Tables 1 and 2). However, stm-1 ant-4 ail7-1 plants bolt less frequently than



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Figure 5. Phenotypes of stm ail mutant combinations. (a–h). Twenty-one-day-old stm-1 (a), stm-1 ant-4 (b), stm-1 ail6-2 (c), stm-1 ail7-1 (d), stm-1 ant-4 ail6-2 (e), stm-1 ant-4 ail7-1 (f), stm-1 ail6-2 ail7-1 (g) and stm-1 ant-4 ail6-2 ail7-1 (h) seedlings. (i–l) stm-1 (i), stm-1 ail7-1 (j), stm-1 ant-4 ail7-1 (k) and stm-1 ail6-2 ail7-1 (l) flowers. Scale bars: (a)–(l) 2 mm.

Table 1 Phenotypes of stm and stm ail mutant combinations with regard to leaf production, bolting and flower production

stm-1 stm-1 ant-4 stm-1 ail6-2 stm-1 ail7-1 stm-1 ant-4 ail6-2 stm-1 ant-4 ail7-1 stm-1 ail6-2 ail7-1 stm-1 ant-4 ail6-2 ail7-1

15-day leaves

21-day leaves

28-day leaves

35-day bolted

42-day bolted

42-day flowers

49-day flowers

28.8% (76) 0% (10) 0% (77) 34.2% (73) 0% (8) 70.6% (17) 0% (58) 0% (12)

38.2% (76) 0% (10) 0% (76) 46.6% (73) 0% (8) 94.1% (17) 47.4% (57) 16.7% (12)

55.6% (63) 0% (10) 0% (66) 74.3% (70) 0% (7) 94.1% (17) 58.0% (50) 25.0% (4)

0% (30) All dead All dead 2.00% (19) All dead 5.90% (17) 0% (39) 0% (2)

34.6% (26) All dead All dead 43.5% (46) All dead 23.5% (17) 3.40% (29) All dead

3.80% (26) All dead All dead 28.3% (46) All dead 0% (17) 3.40% (29) All dead

4.00% (25) All dead All dead 61.4% (44) All dead 0% (17) 10.3% (29) All dead

Numbers in parentheses are number of plants examined in a single experiment.

Table 2 Summary of the phenotypes of stm and stm ail mutant combinations

Genotype

Leaf production

Bolting

Flowers

stm-1 stm-1 ant-4 stm-1 ail6-2 stm-1 ail7-1 stm-1 ant-4 ail6-2 stm-1 ant-4 ail7-1 stm-1 ail6-2 ail7-1 stm -1 ant-4 ail6-2 ail7-1

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a

Produced some flower-like structures late in life. Delayed in leaf production compared to stm-1.

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stm-1 ail7-1 and only rarely produce flower-like structures that consist entirely of green leaf-like organs (Figure 5k, Tables 1 and 2). These complex genetic interactions suggest a context dependency between AIL genes in the stm background that varies between tissue types and/or developmental stages. wus-1 wus-1 seedlings lack a functional shoot apical meristem and do not initiate any leaves immediately after germination (Laux et al., 1996). About 14 days after germination, several leaves are produced by axillary and adventitious meristems, which then subsequently terminate, with this process repeating itself. Eventually many wus-1 plants bolt and make


AIL/PLT genes regulate meristem function 115 flowers that consist of sepals, petals and some stamens. At 7 days post-germination wus-1 ant-4 ail6-2 ail7-1 quadruple mutant plants are indistinguishable from wus-1 single mutants. By 14 days only about 14% of wus-1 ant-4 ail6-2 ail7-1 plants have produced leaves and the leaves are very small, sometimes only visible under a dissecting microscope (Figure 6a,h, Tables 3 and 4). These quadruple mutant plants never bolt or produce flowers (Figure 6i). Thus, while wus-1 is initially epistatic to ant-4 ail6-2 ail7-1, in later stages of development the quadruple mutant plants show more severe phenotypes than those present in either wus-1 or ant-4 ail6-2 ail7-1. A similar but slightly less severe phenotype is observed in wus-1 ant-4 ail6-2 plants. These plants produce fewer and smaller leaves than wus-1, initiating leaves later than wus-1, and the plants never bolt or produce flowers (Figure 6e,i, Tables 3 and 4). While leaf production in wus-1 ant-4 and wus-1 ail6-2 was similar to that in wus-1, these double mutant combinations had more severe defects than wus-1 with regard to bolting and flower production (Figure 6b,c,i, Tables 3 and 4). Fewer wus-1 ant-4 plants bolt, and these plants never make flowers

(Figure 6i, Table 4). Only rarely do wus-1 ail6-2 plants bolt, and flowers were only observed in a single wus-1 ail6-2 plant out of more than 100 examined (Figure 6i, Tables 3 and 4). Thus ant-4 and ail6-2 single mutants enhance later aspects of the wus-1 phenotype. In contrast ail7-1 suppresses later aspects of the wus-1 phenotype. wus-1 ail7-1 plants bolt and flower earlier than wus-1 (Figure 6d,i, Tables 3 and 4). wus-1 ant-4 ail7-1 and wus-1 ail6-2 ail7-1 plants generally resemble wus-1 ant-4 and wus-1 ail6-2 plants, respectively (Figure 6f,g,i, Tables 3 and 4), indicating that in these backgrounds AIL7 has little function. clv3-2 Mutations in CLV3 result in enlarged embryonic, vegetative and inflorescence apical meristems (Clark et al., 1995). To see whether clv3-2 could rescue the smaller meristem defect of the triple mutant, we examined shoot apical meristem size in clv3-2 ant-4 ail6-2 ail7-1 seedlings. The shoot apical meristems of 7-day-old clv3-2 ant-4 ail6-2 ail7-1 quadruple mutants are larger than those of ant-4 ail6-2 ail7-1 or Ler but similar in size to or smaller than clv3-2 (Figure 7a–d). Bulges

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Figure 6. Phenotypes of wus ail mutant combinations. (a–h) Twenty-one-day-old wus-1 (a), wus-1 ant-4 (b), wus-1 ail6-2 (c), wus-1 ail7-1 (d), wus-1 ant-4 ail6-2 (e), wus-1 ant-4 ail7-1 (f), wus-1 ail6-2 ail7-1 (g) and wus-1 ant4 ail6-2 ail7-1 (h) plants. (i) Forty-two-day-old wus-1, wus-1 ant-4, wus-1 ail6-2, wus-1 ail7-1, wus-1 ant-4 ail6-2, wus-1 ant-4 ail7-1, wus-1 ail6-2 ail7-1 and wus-1 ant-4 ail6-2 ail7-1 mutant plants. Scale bars: (a)–(h) 5 mm; (i) 10 mm.


116 Janaki S. Mudunkothge and Beth A. Krizek Table 3 Phenotypes of wus and wus ail mutant combinations with regard to leaf production, bolting and flower production

wus-1 wus-1 ant-4 wus-1 ail6-2 wus-1 ail7-1 wus-1 ant-4 ail6-2 wus-1 ant-4 ail7-1 wus-1 ail6-2 ail7-1 wus-1 ant-4 ail6-2 ail7-1

14-day leaves

21-day leaves

28-day bolted

35-day bolted

35-day flowers

70-day bolted

70-day flowers

94.4% (71) 88.0% (25) 98.5% (65) 97.1% (69) 44.0% (25) 90.5% (21) 90.6% (32) 14.3% (21)

100% (67) 100% (25) 100% (64) 100% (69) 100% (25) 100% (21) 100% (32) 66.7% (21)

0% (65) 0% (25) 0% (57) 8.70% (69) 0% (18) 0% (21) 0% (30) 0% (7)

8.47% (59) 0% (21) 0% (53) 25.0% (68) 0% (14) 0% (21) 0% (26) 0% (4)

5.10% (59) 0% (21) 0% (53) 17.6% (68) 0% (14) 0% (21) 0% (26) 0% (4)

87.5% (56) 21.7% (21) 2.00% (50) 97.0% (67) 0% (1) 20% (20) 0% (25) All dead

44.6% (56) 0% (21) 0% (50) 56.7% (67) 0% (1) 0% (20) 0% (25) All dead

Numbers in parentheses are number of plants examined. Table 4 Summary of the phenotypes of wus and wus ail mutant combinations Genotype

Leaf production

Bolting

Flowers

wus-1 wus-1 ant-4 wus-1 ail6-2 wus-1 ail7-1 wus-1 ant-4 ail6-2 wus-1 ant-4 ail7-1 wus-1 ail6-2 ail7-1 wus-1 ant-4 ail6-2 ail7-1

+++ +++ +++ +++ ++ +++ +++ +

+++ ++a +a ++++ – ++a – –

+++ – – ++++ – – – –

a

Delayed bolting compared to wus-1.

corresponding to lateral organ primordia are visible in the meristem periphery, but they are not regularly spaced (Figure 7c,d). The meristems of 10-day-old clv3-2 ant-4 ail6-2 ail7-1 seedlings are broader than the terminated meristems of ant-4 ail6-2 ail7-1 but smaller than those of clv3-2 with fewer primordia and a less regular pattern of organ initiation (Figure 7e–h). In both 7 and 10-day-old clv3-2 ant-4 ail6-2 ail7-1 plants, the meristem is composed of larger cells than in clv3-2 (Figure 7c,d,f–h). The increased size of clv3-2 ant-4 ail6-2 ail7-1 meristems is often correlated with multiple foci or an expanded domain of WUS expression (Figure 7m–p), which is similar to but not as severe as that in clv3 mutants (Schoof et al., 2000). Most of the primordia initiated in clv3-2 ant-4 ail6-2 ail7-1 plants between 7 and 14 days post-germination do not develop into mature leaves, and no obvious meristem-like structure is visible in most 21-day-old plants (Figure 7i,j). Consequently, clv3-2 ant-4 ail6-2 ail7-1 plants look very similar to ant-4 ail6-2 ail7-1 plants at 21 days (Figure 7k,l). Thus, although clv3-2 increases meristem size in the ant-4 ail6-2 ail7-1 background, the quadruple mutant plants lack an indeterminate meristem capable of continued organ initiation. This suggests that the defects in meristem function in the triple mutant cannot be entirely explained by reduced meristem size. Surprisingly, in clv3 ail double and triple mutant combinations, we observed enhanced inflorescence meristem

proliferation starting approximately 21 days after germination (Figures 8a–u and S4). The most severe overproliferation occurred in clv3-2 ant-4 ail6-2 inflorescences, which ceased lateral organ initiation as the meristem becomes very wide (Figure 8m–o). clv3-2 ail6-2, clv3-2 ail7-1 and clv3-2 ant4 ail7-1 meristems are both taller and wider than clv3-2, and by 30 days lack a single distinct meristem dome (Figure 8h,k,q). The meristems of clv3-2 ant-4 plants initially resemble those of clv3-2 but eventually terminate in a large ring of tissue (Figures 8d–f and S4). In addition, the carpels of clv3-2 ant-4 flowers often exhibit continued proliferation (Figure S4). The meristems of clv3-2 ail6-2 ail7-1 plants often grow as a line resulting in severe stem fasciation (Figure 8s–u). These results suggest that in a clv3 mutant background, ANT, AIL6 and AIL7 each act individually to restrict stem cell proliferation. Despite increased accumulation of cells in clv3-2 ail6-2, clv3-2 ail7-1, clv3-2 ant-4 ail7-1 and clv3-2 ail6-2 ail7-1 meristems, these cells are unable to maintain meristematic identity. Starting about 28 days after germination, flowers can be seen differentiating from within the meristem center (Figure 8i,k,l,q,r). This can result in subdivision of the meristem and bifurcation of the inflorescence stem. These results indicate that AIL6 and AIL7 act to prevent differentiation of stem cells in clv3-2 inflorescence meristems. DISCUSSION ANT, AIL6 and AIL7 are required for meristem maintenance In ant ail6 ail7 triple mutants, the primary shoot apical meristem terminates after the production of three to five leaves. These meristem defects appear to result from both reduced meristem cell division and differentiation of stem cells. Additional leaves are formed later in development, but any axillary and/or adventitious meristems present in these plants persist for just a short time before they also terminate. In fact, axillary and/or adventitious meristems show more severe defects than the primary shoot apical meristem. These results indicate that ANT, AIL6 and AIL7 together promote cell division and repress differentiation within shoot meristems to maintain a central core of stem cells.


AIL/PLT genes regulate meristem function 117

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Figure 7. Phenotype of clv3 ant ail6 ail7 plants and WUS expression in clv3 ant ail6 ail7 shoot apices. (a–d) Scanning electron micrographs of the shoot apical meristems (arrows) of 7-day-old Ler (a), ant-4 ail6-2 ail7-1 (b), clv3-2 (c) and clv3-2 ant-4 ail6-2 ail7-1 (d) seedlings. (e)–(h) Scanning electron micrographs of the shoot apical meristems (arrows) of 10-day-old ant-4 ail6-2 ail7-1 (e), clv3-2 (f) and clv3-2 ant-4 ail6-2 ail7-1 (g, h) plants. (i, j) Scanning electron micrograph of shoot apices of 21-day-old ant-4 ail6-2 ail7-1 (i) and clv3-2 ant-4 ail6-2 ail7-1 (j) plants. (k, l) Twenty-one-day-old ant-4 ail6-2 ail7-1 (k) and clv3-2 ant-4 ail6-2 ail7-1 (l) plants. (m–p) In situ hybridization showing WUS mRNA in 10-day-old Ler (m), ant-4 ail6-2 ail7-1 (n) and clv3-2 ant-4 ail62 ail7-1 (o, p) plants. Sections in (o, p) are from the same plant. Scale bars: (a–h), (m–p) 50 lm; (i), (j) 300 lm; (k, l) 5 mm.

In addition to meristem defects, ant ail6 ail7 triple mutants also exhibit disruptions in leaf outgrowth and development. The leaves initiated by the primary shoot apical meristem of ant ail6 ail7 plants are small and narrow with altered vascular patterning. Although many leaf primordia are subsequently initiated in ant ail6 ail7 plants, most abort growth as radially symmetric projections, similar in size and overall morphology to the terminated primary shoot apical meristem. Defects in leaf outgrowth are particularly obvious in clv3 ant ail6 ail7 plants, which have larger meristems than the triple mutant with many visible lateral organ primordia, but which resemble ant ail6 ail7 plants with regard to the number of mature leaves. Comparison of AIL/PLT function in the root and shoot The conserved use of AIL/PLT proteins in both the root and shoot apical meristems contributes to emerging evidence that common regulatory modules maintain stem cell identity

in both types of meristem, despite their distinct architectures (reviewed in Terpstra and Heidstra, 2009). However, there are also important differences between AIL/PLT activity in root and shoot meristems. PLT1 and PLT2 establish the root stem cell niche during embryogenesis by responding to and stabilizing an auxin maximum in the root tip (Aida et al., 2004). In combination with SCARECROW (SCR) and SHORTROOT (SHR), they specify the position of the quiescent center. In contrast ANT, AIL6 and AIL7 are not required for establishment of the embryonic shoot apical meristem nor do they define its position. Furthermore, these three AILs are not essential for the initial functioning of the shoot apical meristem after germination. Instead ANT, AIL6 and AIL7 functions are only required later in seedling development for maintenance of shoot apical meristem function. An apparent parallel between AIL/PLT activities in the root and shoot is control of the onset of differentiation. A gradient of PLT activity in the root associates low PLT


118 Janaki S. Mudunkothge and Beth A. Krizek (a)

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activity with differentiation and high PLT activity with stem cell identity (Galinha et al., 2007). The premature differentiation of stem cells in ant ail6 ail7 triple mutants indicates that AIL activity suppresses differentiation within the shoot apical meristem. Furthermore, in the clv3-2 background, loss of either AIL6 or AIL7 results in premature differentiation of meristematic cells. Finally, we have also found that high levels of AIL6 expression in developing floral organs can inhibit cellular differentiation (Krizek and Eaddy, 2012). ANT, AIL6 and AIL7 probably have distinct functions within the meristem

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Figure 8. Phenotypes of additional clv3 ail mutant combinations. Meristems are outlined in red for (a, d, g, j, m, p, s). (a–c) Twenty-one (a), 30 (b) and 35 (c) day-old clv3-2 inflorescences. (d–f) Twenty-one (d), 30 (e) and 35 (f) day-old clv3-2 ant-4 inflorescences. (g–i) Twenty-one (g), 30 (h) and 35 (i) dayold clv3-2 ail6-2 inflorescences. Arrow points to flowers differentiating from cells within the meristem center (i). (j–l) Twenty-one (j), 30 (k) and 35 (l) dayold clv3-2 ail7-1 inflorescences. Arrows point to flowers differentiating from cells within the meristem center (k, l). (m–o) Twenty-one (m), 30 (n) and 35 (o) day-old clv3-2 ant-4 ail6-2 inflorescence. (p–r) Twenty-one (p), 30 (q) and 35 (r) day-old clv3-2 ant-4 ail7-1 inflorescence. Arrows point to flowers differentiating from cells within the meristem center (q, r). (s–u) Twenty-one (s), 30 (t) and 35 (u) day-old clv3-2 ail6-2 ail7-1 inflorescences. Older flowers hide the fasciated inflorescence meristem (u). Scale bars: 1 mm (a, c, d, f, g, i, j, l, m, o, p, r, s, u); 400 lm (b, e, h, k, n, q, t).

Several pieces of evidence suggest that ANT, AIL6 and AIL7 do not act in a strictly redundant fashion in the shoot apical meristem. First, these three genes are expressed in partially overlapping but distinct domains within the meristem. Second, these genes exhibit distinct genetic interactions with STM and WUS. While ant and ail6 enhance wus and stm phenotypes, ail7 partially suppresses these phenotypes. This suggests that ANT/AIL6 and AIL7 actually have opposing roles within the meristem. In support of this idea, ail7 can partially suppress the strong phenotypic defects observed in stm ail6 and stm ant ail6 plants. A caveat of our studies in the wus and stm backgrounds is that the observed phenotypic effects of ant, ail6 and ail7 are on axillary and adventitious meristems rather than the primary shoot apical meristem. The genetic interactions of ail mutants with meristem regulators are quite complex and not easily explained in all cases. For example, while leaves are never produced in stm ant plants, more stm ant ail7 plants make leaves than either stm or stm ail7 plants. The opposite effect of ant in AIL7 wildtype and ail7 mutant backgrounds suggests a contextdependent interaction between ANT and AIL7 in the stm background. Context dependency among the three AIL genes is also suggested by the genetic results with clv3. Although the clv3 ant ail6 ail7 quadruple mutant exhibits an additive phenotype with regard to seedling meristem size, combinations of clv3 with mutations in one or two AIL genes result in larger inflorescence meristems than clv3. Context dependency within these genetic backgrounds could result from cross-regulatory interactions among AILs, competition between AILs for binding to DNA regulatory sequences and/ or distinct developmental environments that result when one or two AILs are mutated. To begin to address potential cross-regulatory interactions between AILs, we examined AIL7 mRNA levels in ant-4 ail6-2 seedlings. AIL7 was expressed at normal levels in the double mutant (data not shown), suggesting that context dependency may not be a consequence of cross-regulatory interactions at the transcriptional level. Model for ANT, AIL6 and AIL7 action within the shoot apex We speculate that ANT, AIL6 and AIL7 regulate distinct processes involved in meristem function with defects only


AIL/PLT genes regulate meristem function 119 resulting when these processes are disrupted in combination. Alternatively, these genes could regulate the same process in distinct domains within the meristem with defects only resulting when this process is disrupted in multiple domains. The enhanced phenotypes of stm ant ail6 ail7 and wus ant ail6 ail7 compared with stm, wus and ant ail6 ail7 suggests that the three AIL genes do not act directly in either the STM pathway or the WUS-CLV3 pathway. It is possible that ANT, AIL6 and AIL7 act in both pathways, such that the enhanced phenotype of the quadruple mutants is due to defects in both the STM and the WUS–CLV3 pathways. Another possibility is that ANT, AIL6 and AIL7 influence the activities of the STM and WUS–CLV3 pathways in an indirect manner through regulation of growth within the meristem and/or phytohormone signaling. AIL/PLT genes are important regulators of growth in lateral organ primordia. ANT, AIL6 and AIL7 promote growth in an additive fashion, as leaf size is progressively reduced in ant, ant ail6 and ant ail6 ail7 plants. The reduced expression of two B2-type CDKs in ant ail6 ail7 triple mutants suggests that meristem defects in the triple mutant may result from loss of coordinated cell division within the meristem. Alternatively, ANT, AIL6 and AIL7 may affect hormone signaling within the shoot apical meristem via regulation of hormone accumulation and/or transcriptional responses. Interestingly, disruption of CDKB2 activity results in altered hormone responses and levels in addition to meristem arrest, suggesting a linkage between cell cycle control and hormone physiology in meristem function (Anderson et al., 2008). It is possible that AIL proteins are components of the transcriptional network mediating these interactions. AIL/PLT function in both root and shoot patterning has been linked to auxin distribution. In the root PLT proteins promote auxin responses downstream of auxin while also regulating its distribution (Aida et al., 2004; Blilou et al., 2005). In the shoot, the phyllotaxy defects in plt5 plt3 plt7 (ail5 ail6 ail7) triple mutants are correlated with altered expression of the auxin efflux carrier PIN-FORMED1 (PIN1) (Prasad et al., 2011). PIN1 mRNA levels are slightly reduced in ant ail6 ail7 seedlings (about 80% of the levels found in the wild type; data not shown). However, it seems unlikely that defects in auxin distribution alone would result in meristem termination as pin1 mutants retain a growing meristem even though they are unable to initiate later organ primordia (Okada et al., 1991). The loss of stem cell identity in the center of the meristem and the defects in cell division suggest that cytokinin distribution and/or responses may also be affected in ant ail6 ail7 plants. The identification of targets of each of these transcription factors is likely to provide further insight into the possible roles of ANT, AIL6 and AIL7 in controlling cell behaviors and/or hormone signaling within the meristem and the mechanism by which ANT, AIL6 and AIL7 activities are integrated to regulate meristem function.

EXPERIMENTAL PROCEDURES Plant materials and growth conditions The ant-4, ail6-2 and ail7-1 alleles have been described previously (Nole-Wilson and Krizek, 2006; Krizek, 2009). ant-4 is in Ler while ail6-2 and ail7-1 are in Col. ant-4/+ ail6-2 er plants (Krizek, 2009) were crossed to ail7-1 and triple mutants with er were used in all experiments. wus-1, clv3-2 and stm-1 seeds were obtained from the Arabidopsis Biological Resource Center (ABRC; https://abrc.osu. edu/) and are in Ler. ant-4/+ ail6-2 ail7-1 plants were crossed to wus1/+, clv3-2 and stm-1/+ plants. Mutant combinations were identified in the F2 or later generations by PCR genotyping. ant-4 and ail6-2 were PCR genotyped as described previously (Krizek, 2009). ail7-1 was PCR genotyped using a pair of gene-specific primers and a T-DNA primer. Plants were grown on a soil mixture of Metro-Mix 360:perlite:vermiculite (5:1:1) in 16 h days (100–150 lmol m)2 s)1) at a temperature of 22C.

Generation of ail7IR lines To generate the ail7IR lines, a 170-bp fragment of AIL7 was PCR amplified with AIL7-4 (5¢-gaatctcgagggatccatggcggattcaacaacc-3¢) and AIL7-5 (5¢-atcaggtaccatcgataacggtgcggtgacgtgg-3¢) and this fragment was cloned into pHannibal in the sense and antisense directions (Wesley et al., 2001). The entire inverted repeat construct including the 35S promoter was subcloned from pHannibal into pART27. The construct was transformed into Agrobacterium tumefaciens ASE by electroporation. Arabidopsis Ler plants were transformed with this Agrobacterium strain by vacuum infiltration (Bechtold et al., 1993). Transformants were selected for kanamycin resistance. ail7IR was crossed to ant-4/+ ail6-2 to generate ant-4 ail62 ail7IR plants.

Histology Vascular tissue was examined as described previously (Nole-Wilson and Krizek, 2006). Epoxy sections were prepared as described previously (Krizek and Eaddy, 2012).

Scanning electron microscopy Tissue for SEM was fixed and dehydrated using either of two previously published protocols (Laux et al., 1996; Krizek, 1999). The SEM studies were performed on a FEI Quanta 200 environmental scanning electron microscope (ESEM) (http://www.fei.com/).

In situ hybridization Embryos and seedlings were fixed in either Formalin-acetic acidalcohol (FAA) or paraformaldehyde, embedded, sectioned, hybridized and washed as described previously (Krizek, 1999) or using the method found at http://www.its.caltech.edu/~plantlab/protocols/insitu.htm. Digoxigenin-labeled antisense RNA probes (histone H4, CLV3, WUS, STM, ANT, AIL6, AIL7) were synthesized as described previously (Long et al., 1996; Mayer et al., 1998; Fletcher et al., 1999; Krizek, 1999; Nole-Wilson et al., 2005).

RNA extraction, real-time and semiquantitative RT-PCR The RNA was extracted from whole seedlings or shoots (Verwoerd et al., 1989) and treated with Turbo DNase (Applied Biosystems; http://www.appliedbiosystems.com/). First-strand cDNA synthesis was performed using SuperScript III kit (Invitrogen; http://www. invitrogen.com/). The real-time PCR reactions were performed on an iCycler (Bio-Rad; http://www.bio-rad.com/) using B-R SYBR Green SuperMix for iQ or PerfeCTa SYBR Green FastMix for iQ


120 Janaki S. Mudunkothge and Beth A. Krizek (Quanta BioSciences; http://www.quantabio.com/) with the primers listed in Table S2 or as previously described for CDKB2;1 and CDKB2;2 (Anderson et al., 2008). Data analyses were carried out as described previously (Krizek and Eaddy, 2012). Normalization was performed using one or two reference genes (At5g15710, At5g12240). For the semiquantitative RT-PCR, PCR was performed with primers listed in Table S2 with removal of 5-ll aliquots after 26, 30 and 34 cycles. The PCR products were separated by agarose gel electrophoresis. Actin transcript levels were used as an internal control.

Confocal microscopy Seeds were imbibed overnight at 4C in a Petri dish with paper soaked in 15% ethanol. Embryos were removed from seed coats and stained with propidium iodide as described previously (Running et al., 1995). The data were collected on a ZEISS LSM 510 META confocal scanning laser microscope (http://www.zeiss.com/).

ACKNOWLEDGEMENTS We thank Traci Tranby for generating the ail7IR lines, Jeff Long, Sharyn Perry and Donna Fernandez for advice on embryo in situ hybridization, Soumitra Ghoshroy and the Electron Microscopy Center staff for help with the use of the SEM, the staff of the USC School of Medicine Instrumentation Resource Facility for help with use of the confocal microscope, the ABRC for seeds, and Doris Wagner and Ben Scheres for comments on the manuscript. This work was supported by National Science Foundation (NSF) grant IOS 0922367.

SUPPORTING INFORMATION Additional Supporting Information may be found in the online version of this article: Figure S1. ail7IR lines show decreased accumulation of AIL7 mRNA and have a wild-type appearance. Figure S2. ant ail6 ail7 embryos have normal morphology and expression patterns of meristem regulators. Figure S3. In situ hybridization showing STM and ANT mRNA in 14day-old ant ail6 ail7 plants. Figure S4. clv3 ail mutant combinations at late stages of development. Table S1. Number of embryos from Ler and ant/+ ail6 ail7 plants showing strong, weak or no CLV3, WUS or STM expression as determined by in situ hybridization. Table S2. Primers used in real time and semiquantitative RT-PCR experiments. Please note: As a service to our authors and readers, this journal provides supporting information supplied by the authors. Such materials are peer-reviewed and may be re-organized for online delivery, but are not copy-edited or typeset. Technical support issues arising from supporting information (other than missing files) should be addressed to the authors.

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