Mechanisms of leaf tooth formation in Arabidopsis

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1 The Plant Journal (2010) 62, doi: /j X x Mechanisms of leaf tooth formation in Arabidopsis Eiko Kawamura 1, Gorou Horiguchi 1, and Hirokazu Tsukaya 1,2,* 1 Graduate School of Science, The University of Tokyo, Hongo, Bunkyo-ku, Tokyo , Japan and 2 National Institute for Basic Biology, 38 Nishigo-Naka, Myodaiji-cho, Okazaki, Aichi , Japan Received 8 November 2009; revised 15 January 2010; accepted 20 January 2010; published online 12 March * For correspondence (fax ; tsukaya@biol.s.u-tokyo.ac.jp). Present address: Department of Life Science, Rikkyo University, Nishi-Ikebukuro, Toshima-ku, Tokyo , Japan. SUMMARY Serration found along leaf margins shows species-specific characters. Whereas compound leaf development is well studied, the process of serration formation is largely unknown. To understand mechanisms of serration development, we investigated distinctive features of cells that could give rise to tooth protrusion in the simpleleaf plant Arabidopsis. After the emergence of a tooth, marginal cells, except for cells at the sinuses and tips, started to elongate rapidly. Localized cell division seemed to keep cells at the sinus smaller, rather than halt cell elongation. As leaves matured, the marginal cell number between teeth became similar in any given tooth. These results suggest that teeth are formed by repetition of an unknown mechanism that spatially monitors cell number and regulates cell division. We then examined the role of CUP-SHAPED COTYLEDON 2 (CUC2) in serration development. cuc2-3 forms fewer hydathodes and auxin maxima, visualized by DR5rev::GFP, at the leaf margin, suggesting that CUC2 patterns serration through the regulation of auxin. In contrast to a previous interpretation, comparison of leaf outlines revealed that CUC2 promotes outgrowth of teeth rather than suppression of growth at the sinuses. We found that mutants with increased CUC2 expression form ectopic tissues and mis-express SHOOT MERISTEMLESS (STM) at the sinus between the enhanced teeth. Similar but infrequent STM expression was found in the wild type, indicating STM involvement in the serration of simple leaves. Our study provides insights into the morphological and molecular mechanisms for leaf development and tooth formation, and highlights similarities between serration and compound leaf development. Keywords: serration, leaf tooth, CUP-SHAPED COTYLEDON, PIN-FROMED 1, SHOOT MERISTEMLESS, marginal cell. INTRODUCTION Degree and pattern of indentation at the leaf margin define the shape of leaves (Tsukaya, 2006). Leaves with entire or slightly toothed margins are classified as simple leaves, whereas more dissected margins forming leaflets are called compound leaves. Leaflet development requires expression of class-1 KNOX (KNOX1) genes in leaf primordia, and increased expression accelerates the dissection of the leaf margin (Hareven et al., 1996; Chen et al., 1997; Janssen et al., 1998; Hay and Tsiantis, 2006). KNOX1 is repressed in leaves of the simple-leaf plant Arabidopsis, through the activities of ASYMMETRIC LEAVES 1 (AS1) and AS2 (Byrne et al., 2000; Ori et al., 2000; Semiarti et al., 2001; Guo et al., 2008). The ectopic expression of KNOX1 in Arabidopsis leaves of as1-1 or as2-1 or KNOX1 overexpressors results in lobed leaves (Chuck et al., 1996; Tsukaya and Uchimiya, 1997; Ori et al., 2000; Semiarti et al., 2001; Dean et al., 2004). In parallel with AS1 and AS2, SAWTOOTH 1 (SAW1) and SAW2, BELL1-like homeodomain proteins, prevent KNOX1 expression in leaves (Kumar et al., 2007). The double mutant saw1;saw2 forms increased leaf serrations. Furthermore, KNOX1 expression is repressed by the phytohormone, auxin (Hay et al., 2006). A recent molecular study that compared representative dissected leaf species demonstrated that CUP-SHAPED COTYLEDONE (CUC)/NO APICAL MERISTEM (NAM) genes (Souer et al., 1996; Aida et al., 1997; Vroemen et al., 2003) regulate leaf margin dissection in a more robust manner than does KNOX1 (Blein et al., 2008). In Arabidopsis, the CUC gene family consists of CUC1, CUC2 and CUC3, and their functions are partially redundant, especially in 429 Journal compilation ª 2010 Blackwell Publishing Ltd

2 430 Eiko Kawamura et al. cotyledon separation and shoot meristem formation (Aida et al., 1997; Vroemen et al., 2003; Hibara et al., 2006). Changes in CUC expression are known to alter STM expression (Aida et al., 1999; Takada et al., 2001). CUC2 has been shown to regulate the depth of indentation at the leaf margin, but CUC1 and CUC3 are not involved in this process (Nikovics et al., 2006). Expression of CUC1 and CUC2 is partially regulated by mir164-mediated degradation, whereas CUC3 is not cleaved by mir164 (Laufs et al., 2004; Mallory et al., 2004). TEOSINTE BRANCHED 1/CYCLOIDEA/ PCF (TCP) is suggested to regulate CUC expression, possibly at both transcriptional and post-transcriptional levels, and to condition cells to be responsive to cell-cycle arrest signals (Nath et al., 2003; Palatnik et al., 2003; Koyama et al., 2007). Expression of CUC1 and CUC2 is regulated by auxin accumulation patterns (Vernoux et al., 2000; Aida et al., 2002; Furutani et al., 2004; Heisler et al., 2005), and the cuc3 phenotype is enhanced in a gnom background that impairs the proper localization of the auxin efflux protein PIN- FROMED 1 (PIN1) (Hibara et al., 2006). Establishment of auxin maxima is crucial for the morphogenesis of organs, such as embryonic cotyledons and roots, leaf and flower primordia in shoot meristems, and primordia of lateral roots (Berleth and Sachs, 2001). Recently, auxin was shown to regulate leaflet formation in compound leaves (Barkoulas et al., 2008). In addition, the development of teeth also depends on auxin maxima formed at their tips (Hay et al., 2006); however, the precise mechanisms of auxin maxima establishment at the leaf margin are still not clear. The partial-shoot theory proposed by Arber (1950) emphasizes similarities among shoots, compound and simple leaves. In support of this theory, many morphological and molecular mechanisms are shared during the development of primordia in the shoot apical meristem (SAM), and in leaflets (Sattler and Rutishauser, 1992; Lacroix and Sattler, 1994; Lacroix et al., 2003; Barkoulas et al., 2008). Although more molecular evidence to support the partial-shoot theory is accumulating, little is known about the morphological aspects of serration development. In this study, we carried out an anatomical study to understand how tooth positioning and outgrowth are regulated. In young leaves, cell file-like patterns that curve towards each tooth tip were found, and are associated with auxin maxima at the leaf margin. Local marginal cell division was detected at the sinuses of each tooth during development. The numbers of marginal cells between teeth were constant in mature wild-type leaves, indicating that each tooth could be viewed as one unit. We then re-examined CUC2 to understand its role in cell proliferation and differentiation. Analysis of the leaf outline in cuc2-3, supposedly a null allele of CUC2 (Hibara et al., 2006), indicated that the normal function of CUC2 is to promote tooth outgrowth through cell proliferation, rather than to suppress growth at the sinus. We also found that CUC2 has a role in patterning serration through influencing auxin accumulation. Our new findings in this study will provide important clues to understand the mechanisms of leaf organogenesis. RESULTS Differential marginal cell length during tooth development is the result of local cell division at the sinus We looked for distinctive features of cell size, division frequency or pattern in all tissue types along the leaf margin that could contribute to margin protrusions. We did not find any obvious increase in cell division frequency, as indicated by CYCB1;1 in specific parts of rapidly developing teeth (Figure 1a). Instead, cell size differences were found in marginal cells of young leaves. When a tooth was small, the marginal cells were similar in size along the tooth, and were not elongated (Figure 1b), but later, the marginal cells between the tooth tip and sinus were markedly longer than the marginal cells at the tip or sinus (Figure 1b). To understand how the marginal cells develop, the number of marginal cells on the upper and lower part of all teeth was counted at different developmental stages (Figure 1c,d). We chose the sixth leaf for this analysis because it produces multiple teeth and is also ideal for analyzing tooth spacing. When a leaf blade was 400 lm in length, two teeth had developed on each side. At this stage, the number of marginal cells on the upper part of the second tooth was 1.5, on average, and on the lower part was five on average (Figure 1c,d): cell lengths were similar or started to show differences. By the time a leaf blade reached 600 lmin length, the difference in cell size became obvious and the marginal cell number increased. Typically, cells adjacent to a group of some smaller cells at the tip often show elongated shape, whereas marginal cells at the sinus were relatively small, and showed a gradual increase in cell differentiation towards the tip (Figure S1). The number continued to increase up to four for the upper part and 12 for the lower part. These observations indicate that the small marginal cells at the tooth tip ceased division at an earlier stage, or divided slowly, whereas those at the sinus continued division. Later in development, cells at the sinus began to elongate (Figure 2a,b), whereas cells at the tip remained smaller and became interdigitated, like leaf epidermal cells. In the mature sixth leaf, the distances between teeth were similar, indicating that the marginal cells at the sinus were equally elongated in all teeth (Figure 2c). We also found that the number of marginal cells in any given tooth became similar (Figure 1d). These results indicate that multiple teeth are formed by the repetition of an unknown mechanism that forms a tooth. To determine whether local cell proliferation is linked to tooth formation, we examined cuc2-3, which is known to form smooth leaf margins (Nikovics et al., 2006). Hydathodes, identified as open vein endings connected to small

3 Tooth development in Arabidopsis 431 Figure 1. Relative marginal cell sizes change during tooth development. (a) CYCB1;1::GUS signal in the proximal region of the 10th leaf. (b) Differential interference contrast (DIC) image of the fifth leaf from a 14-day-old wild-type plant. (c) Wild-type sixth leaf, to indicate the parts and designations used for the marginal cell count. (d) Number of marginal cells per upper and lower tooth during leaf development. The number increases as the leaf develops, indicating that cells at the sinus divide. The smaller cells always found at the tip were excluded from the measurement, as they were located at the tip and could not be grouped into the upper or lower part (n = 3 7 for each measurement). (a) (b) (c) (d) 8 Numbers of marginal cells µm µm Older tooth Younger tooth Upper Smaller Longer Even 1st upper 1st tooth 2nd upper 2nd tooth 3rd upper 3rd tooth 4th upper 4th tooth 2 mm 1st upper 2nd upper 3rd upper 4th upper 16 Lower Leaf tip lower st lower 2nd lower 3rd lower 4th lower Leaf length (mm) Sinus Leaf tip lower Bars = s.d. 1st lower 2nd lower 3rd lower 4th lower parenchyma cells (Drennan et al., 2009), are typically formed at the tips of teeth (Candela et al., 1999), and thus we used the hydathodes as a marker of differentiation of the tooth tip (Tsukaya and Uchimiya, 1997). Hydathodes were seen as a cluster of small cells, and their cell wall was difficult to visualize under differential interference contrast microscope (DIC). Consistent with the previous report, tooth size was greatly reduced, and the number of hydathodes was reduced by 75% in the sixth leaf of cuc2-3 (Figures 2d,e, 3e, 4a and 5). If local cell proliferation and cell elongation is not associated with tooth patterning, gradient cell elongation should be seen after the 14th marginal cell from the leaf tip, as in the wild type. As shown in Figure 2d, the marginal cell number in a file from the leaf tip to the first sinus was 18 in cuc2-3 in this instance. No elongated cells were observed from the 14th to the 18th cells, and smaller cells were found at the sinus of the small tooth in cuc2-3 (Figure 2d,d ), indicating that the local regulation of cell proliferation and differentiation in marginal cells is coupled with tooth formation. This was confirmed in at least 15 cuc2-3 samples. We then examined in detail marginal cell file organization at the sinus of the wild type. Interestingly, marginal cells were not organized in a few simple and long files, but were rather organized in shorter cell files that partially overlapped with each another (Figure 2a-right). In less mature regions, where marginal cells were not yet greatly elongated, the proximal part of a cell file seemed to split into two, and the end of the inner file often merged into abaxial epidermal cells (Figure 6g). The addition of new cell files seemed to take place near the sinus (Figure 2a-right). A similar cell file overlap was also found in cuc2-3 (Figure 6h), indicating that this is not associated with tooth formation but is a fundamental process in leaf blade development. Initial DR5rev::GFP expression and hydathode differentiation during tooth development Auxin accumulates at the tips of developing teeth, and the application of the auxin transport inhibitor naphthylphthalamic acid (NPA) results in smoother leaf margins (Aloni et al., 2003; Hay et al., 2006). We examined if auxin is also involved in determining tooth initiation sites. Prior to tooth emergence, expression of DR5rev::GFP was detected in one or two epidermal cells at the margin, where a tooth was predicted to develop (Figure 3a c). These one or two cells expressing DR5rev::GFP were indistinguishable from

4 432 Eiko Kawamura et al. (a) Smaller 0.5 mm (b) (b ) (b ) 1mm 100 µm (b ) (b ) (c) 4th 3rd 2nd 1st 1 cm (d) (d ) Smaller cells 18 cells (d ) (e) The number of hydathodes per half a leaf Bars = s.d. 100 µm 0 cuc2-3 pin1-8 Figure 2. Local cell proliferation at the sinus is associated with tooth formation, and cells at the sinus elongate as leaves develop. (a) SEM images of a wild-type sixth leaf about 1.5 mm in length (left). A closer view of marginal cells between the tips of the first and second teeth, showing smaller cells at the sinus (right). Arrows indicate the corresponding region and direction. (b) SEM images of the wild-type sixth leaf, about 6.5 mm in length (left). A closer view of the leaf margin between tips of the first and second teeth, showing more elongated marginal cells at the sinus (right). Arrows indicate the corresponding region and direction. Squares in the right panel indicate closer views of the sinus (b ) and near the tip (b ). (c) Wild-type sixth leaf about 12 mm in length. The orders and positions of the teeth are indicated with arrows. Note that distances between teeth are similar, and this was confirmed in all eight sixth-leaves examined. (d) SEM image of the sixth leaf of cuc2-3. Smaller marginal cells are found at the sinus, but not in the area where a tooth is missing. The white square indicates a closer view of the sinus (d ). (e) Number of hydathodes per half of a sixth leaf from plants older than 21 days; n = 20, 15 and 17 for wild type, cuc2-3 and pin1-8, respectively. neighboring non-dr5rev::gfp-expressing marginal cells in terms of morphology. Later, the domain of DR5rev::GFP expression expanded to more epidermal cells and mesophyll cells in the tips (Figure 3a c). This suggests that auxin marks future tooth initiation sites, similar to leaflet initiation (Barkoulas et al., 2008).

5 Tooth development in Arabidopsis 433 Figure 3. DR5rev::GFP expression patterns in wild type, cuc2-3 and pin1-8. (a c) Sixth leaf of 13-day-old DR5rev::GFP. Propidium iodide staining to visualize cell shape (a), DR5rev::GFP signal indicating auxin accumulation (b) and an overlay of the two (c). A DR5rev::GFP expression peak in two epidermal cells at the margin was also found in positions where teeth were predicted to develop (arrows), prior to apparent tooth protrusion formation. Insets are magnified views of the DR5rev::GFP expression site near the leaf base. (d f) Sixth leaves of DR5rev::GFP-expressing 12-day-old Col-0 (d) and cuc2-3 (e) plants, and 15-day-old pin1-8 plants (f). Arrowheads indicate tight and solitary DR5rev::GFP-expressing cells. (g,h) Closer views of basal parts of the sixth leaves from 12-day-old DR5rev::GFP in cuc2-3 (g-1, g-2) and 16-day-old DR5rev::GFP in pin1-8 (h). Propidium Iodide DR5rev::GFP Overlay (a) (b) (c) 50 µm DR5rev::GFP in col-0 cuc2-3;dr5rev::gfp pin1-8;dr5rev::gfp (d) (e) (f) 100 µm 100 µm 100 µm cuc2-3;dr5rev::gfp cuc2-3;dr5rev::gfp pin1-8;dr5rev::gfp (g-1) (g-2) (h) 50 µm 50 µm 50 µm Hydathodes at the tips of teeth are connected to the vasculature. In Cardamine hirsuta, leaflets start to form before xylem maturation (Barkoulas et al., 2008), but whether leaflet formation occurs before procambial cell differentiation is not clear. We examined tooth development to see if xylem differentiation precedes tooth formation (Figure 5a c). We found that tooth emergence took place even prior to procambial cell differentiation in the region (Figure 3a c). The differentiation of mesophyll cells to hydathode cells appeared to coincide with procambial cell development at the tip. Discrete auxin maxima are disturbed in the leaf margin of cuc2-3, except in the leaf tip How does the CUC2 loss-of-function mutant fail to form teeth? It is known that auxin regulates CUC2 expression (Vernoux et al., 2000; Aida et al., 2002; Furutani et al., 2004), but whether CUC2 influences auxin distribution is not clear. The requirement for auxin in tooth formation (Hay et al., 2006) indicates that either auxin accumulation is disturbed or the morphogenesis of teeth is repressed while auxin maxima are still maintained in cuc2-3. In the wild type, DR5rev::GFP was detected in marginal cells and mesophyll cells in tooth tips (Figure 3a d). In contrast, discrete DR5rev::GFP peaks were infrequent in cuc2-3 (Figure 3e), and broader and fairly even distribution of DR5rev::GFP was observed in the marginal cells (Figure 3e,g-1). When a tiny tooth managed to initiate in cuc2-3, a DR5rev::GFP peak was consistently found in the tip (Figure 3g-2). All eighteen cuc2-3 sixth leaves examined showed these DR5rev::GFP expression patterns. These results suggest that CUC2 functions to establish, maintain or enhance a tight auxin maximum at the leaf margin. In contrast, DR5rev::GFP peaks were always found at apical leaf tips, consistent with no effect on phyllotaxy in cuc2-3. These indicate that CUC2 can influence auxin maxima at the leaf margin, but does not solely affect the auxin flow required for leaf primordia formation in the SAM. The cuc2-3 mutant did not show obvious disruption in vasculature patterning in leaves (Figure 5c,f). Interestingly, marginal cells in distal regions of the wild-type leaves as well as cuc2-3 often showed broad DR5rev::GFP expression (Figure 3d,e), whereas marginal cells in proximal regions were void of clear DR5rev::GFP signal, except in tooth tips. These suggest that this type of auxin distribution in distal and proximal regions, which seems to be normal during leaf development of the wild type, does not disturb vasculature development.

6 434 Eiko Kawamura et al. (a) Overlay cuc2-3 outline cuc2-3 (c) (b) Tip Sinus Midpoint y x Leaf outlines of wild type and cuc2-3 Figure 4. CUC2 promotes outgrowth of teeth. (a) Comparison of leaf outlines of the wild type and cuc2-3. (b,c) Quantitative analysis of leaf outlines. Distances measured for the leaf outline analysis are depicted (b). Distances from the sinus or tip to the midvein (y), and from there to the leaf tip (x) were plotted for wild-type sixth leaves of lm in length (c, blue). Based on mean values of x for wild-type sinuses and tips, distances from the midvein to the cuc2-3 leaf margins (y) were also plotted (c, red). All wild-type tips were significantly more distant from the midvein compared with the cuc2-3 margin at the corresponding positions (P < 0.01). The mean lengths of wild-type and cuc2-3 leaves used for measurement were both 870 lm. 0.5 mm Distance from the center (mm) cuc2-3 Bars = s.d Distance from the leaf tip (mm) CUC2 promotes outgrowth of teeth via cell division CUC2 has been suggested to suppress growth at the sinus in simple leaves (Nikovics et al., 2006). In contrast, CUC/NAM is known to promote cell division (Blein et al., 2008; Larue et al., 2009). To understand how the depth of a sinus is regulated, we examined the shape of the wild-type sixth leaf by superimposing equivalent cuc2-3 leaves (Figure 4a). If CUC2 represses growth at sinuses, the outline of the wild type is predicted to not go beyond that of cuc2-3. The outline of cuc2-3, however, passed positions where sinuses were formed in the wild type, but not the tips (Figure 4a). To confirm this observation, we carried out a quantitative analysis of leaf outlines. The distance from tips or sinuses to the leaf midvein (y) and from there to the leaf tip (x) were measured in the wild type. In addition, the distance from midpoints of x for the first sinus to the leaf margin was also plotted. The sixth leaves that were mm in length were used for the analysis (Figure 4b,c). This gave us coordinates for tips and sinuses for the wild type. Based on the wild-type coordinates for x, the distance from the center to the leaf edges (y) was measured in cuc2-3 (Figure 4b,c). Consistent with the earlier observation, the cuc2-3 leaf outline was similar to that of the wild type without tooth protrusion. This indicates that CUC2 does not repress growth at the sinus, but rather promotes outgrowth of teeth from the leaf blade. Next, we examined how CUC2 contributes to tooth outgrowth. The size and shape of the cells and intercellular spaces were indistinguishable between the wild type and cuc2-3 at all similar stages examined (Figure 5). When teeth managed to initiate in cuc2-3, which was rare, their sizes were similar to wild-type teeth (Figure 5a,d). Whereas wildtype teeth continue to increase in size, growth seems greatly inhibited in cuc2-3. The tooth size differences became more prominent as the leaves developed further (Figure 5b,c,e,f). These results suggest that CUC2 regulates tooth size by promoting outgrowth through cell proliferation. Cell file-like patterns We recognized distinctive features in cell shape of epidermal and mesophyll cells, which showed cell file-like patterns in proximal regions of young leaves (Figure 6a,b). This was most obvious in the abaxial epidermis, and became more distinct in leaves that developed later. The patterns had a proximodistal axis near the leaf base and midvein, and the axis curved towards the tooth in a more peripheral part of the leaf. We examined cell lineage by tracing cells in one cell file in the abaxial epidermis (Figure 6b), and found that the cross walls did not match. When the adjacent cell file was also traced, the cross walls were perfectly connected (Figure 6b ). This indicated that cells first divided to make one cell file, then divided perpendicular to the original division plane, resulting in the appearance of two cell files. These two well-balanced orientations of cell division seem to be a key to these patterns, and seem to take place more frequently in more distal regions, resulting in a gradual increase in cell number from proximal to distal areas (Figure 6c). As the patterns curved toward tooth tips, we consider that auxin maxima may direct them. To examine this idea, we analyzed two mutant alleles of PIN1, pin1-8 and pin pin1 is known to impair polar

7 Tooth development in Arabidopsis 435 cuc2-3 (a) (b) (c) (d) (e) (f) 0.5 mm 0.5 mm 1 cm 0.2 mm 1 mm 5 mm Developing xylem Developed xylem Bars = 100 µm Undifferentiated Differentiating Differentiated Undifferentiated Differentiating Differentiated Bars = 20 µm Figure 5. Tooth outgrowth by CUC2 in the wild type does not promote polarized cell elongation towards teeth or increased intercellular spaces. (a c) Second teeth in sixth leaves of the wild type through development. (a) Tooth protrusion was found prior to distinct procambial cell formation. (b) Differentiation to hydathode cells coincided with xylem development in this area. (c) Hydathode in mature leaf. Incisions, indicated by dotted lines, were made to flatten the curled leaf. (d f) cuc2-3 tooth development in the sixth leaf. Developmental stages of (d, e and f) correspond to (a, b and c), respectively. auxin transport and to form smooth leaves (Hay et al., 2006). As predicted from the function of PIN1, single or few marginal cells expressing DR5rev::GFP were more frequently found in the margin of pin1-8 (Figure 3f,h) and pin1-201 (data not shown). DR5rev::GFP expression peaks were often seen close to each other, and seemed to be confined to epidermal cells for longer periods than those in the wild type and cuc2-3 (Figure 3h). In addition, irregular marginal protrusions often lacked visible DR5rev::GFP (data not shown) or showed clear DR5rev::GFP expression, but not at their tips (Figure S3). These patterns were evident in 10 out of the 12 pin1-8 sixth leaves observed. Given that marginal protrusions develop with an auxin maximum at their apices in the wild type, or even in cuc2-3, irregular protrusions may have initiated with an auxin maximum, but later lost the typical auxin distribution in the absence of PIN1. In pin1-8 and pin1-201, cell file-like patterns were formed, but more than 80% of them (n = 6) clearly curved at steeper angles near the midvein, and the general orientation appeared to be almost perpendicular to the midvein (Figure 6d for pin1-201; data not shown for pin1-8). Interestingly, cuc2-3 also formed cell file-like patterns, but does not seem to curve as much compared with the wild type (Figure 6e). This was confirmed in all 10 cuc2-3 sixth leaves examined. The patterns seemed to be directed towards the nearest leaf margin and the leaf tip in pin1 and cuc2-3, respectively. Taken together, these results indicate that the type of auxin accumulation seen in tooth or leaf tips may be important for the curvature. Although both pin1 and cuc2-3 leaf margins appeared smooth, closer observation under a light microscope revealed that the young leaf margins of pin1-8 and pin1-201 were irregular, whereas that of cuc2-3 was fairly smooth (Figure 6d,e). To see if the irregular protrusions could differentiate hydathodes, the number of hydathodes was counted in the sixth leaf. pin1-8 produced (mean SD) hydathodes per half leaf, whereas the wild type formed hydathodes per half leaf (Figure 2e). In developed leaves, hydathodes varied in size, and were often formed close to each other in pin1-8 and pin1-201 (data not shown). Together with the finding that more DR5rev::GFP peaks are formed in the pin1-8 leaf margin,

8 436 Eiko Kawamura et al. (a) (b) (b ) 100 µm pin1-201 cuc2-3 (c) (d) (e) 50 µm 50 µm cuc2-3 (f) (g) (h) Figure 6. Cell file-like pattern in the abaxial leaf epidermis. (a b ) Abaxial side of the sixth leaf expressing CYCB1;1::GUS in a Col-0 background. Cells that appeared to form a cell file were traced. Most cross walls do not appear to match (b); when neighboring cells are included, the cross walls match perfectly (b ), suggesting that cells first divided to form one cell file (solid lines), and then divided perpendicular to the original division plane (dotted lines), resulting in the appearance of two cell files. (c) SEM image of the abaxial surface of the sixth leaf in a 13-day-old wild-type plant. One cell file near the leaf base splits into two files (lowest pair of arrowheads). In more distal regions, the two files split into four files (middle pair of arrowheads). A further cell file increase is seen in a more distal region (top arrowhead). (d) Abaxial side of the sixth leaf in pin1-201, showing cell file-like patterns. Note that the angle of the axis of the patterns (arrow) is steeper, and the leaf margin is irregular rather than smooth. (e) Abaxial side of the sixth leaf in cuc2-3. The angle of the axis of the cell file-like pattern (arrow) is not as sharp as in the wild type and pin1-201; the leaf margin is smoother than in pin (f) Abaxial side of the fifth leaf in the wild type. Three cell files were traced to indicate different file axes at the leaf periphery. (g,h) SEM images of the sixth leaf in the wild type and cuc2-3. Arrowheads: points of marginal cell file shifts or splits. 50 µm 50 µm this indicates that the regions defined by auxin maxima failed to grow outwards. It is noteworthy that the directions of cell file-like patterns at the leaf periphery appeared relatively uniform in cuc2-3 and pin1 (Figure 6d,e), whereas those in the wild type seemed to change locally (Figure 6d f). If the auxin maximum serves as an apex for these patterns, orientation of cell files at the leaf periphery may be locally affected in relation to auxin peaks. Ectopic tissue formation in CUC2g-m4, mir164a-4 and mir164a-6 We examined an expressor of CUC2g-m4, a mir164-resistant mutant form, and mir164a-4 and mir164a-6 loss-offunction mutants of mir164a to understand the positive role of CUC2 in tooth outgrowth. As reported, CUC2g-m4 and mir164a-4 produce enhanced primary teeth (Nikovics et al., 2006). Growth at the sinuses appears to be somewhat repressed in CUC2g-m4 and mir164a, as previously reported (Nikovics et al., 2006); however, outgrowth of each tooth was promoted. In addition, secondary teeth are frequently produced on the lower part of the primary teeth (Figure S2). This indicates that there is increased tooth outgrowth, as one unit leads to new tooth formation, similar to that seen in primary tooth formation. Furthermore, we found ectopic outgrowths with occasional stipules at the sinuses of primary teeth in CUC2g-m4, mir164a-4 (Figure 7a c) and mir164a-6 (data not shown). The ectopic tissues resembled reported ectopic meristems found at the sinuses of as1-1, as2-2, as1-1;se, as2-2;se and as1;gai1 (Ori et al., 2000; Hay et al., 2002, 2006), indicating

9 Tooth development in Arabidopsis 437 Figure 7. STM upregulation and ectopic tissue formation at sinuses of CUC2g-m4 and mir164a-4. (a c) Sinus of sixth leaf in 31-day-old plants. No ectopic outgrowth in the wild type (a). Ectopic tissue with a notch in the center, with or without a stipule, often formed in mir164a-4 (b) and CUC2g-m4 (c). (d) Fold changes in KNOX1 expression level, determined by qrt-pcr, in CUC2g-m4 and mir164a-4 compared with the wild type. Sixth leaf blades of 3 8 mm in length were analyzed. Data are means of three independent biological replicates and are normalized to UBQ10, an internal control. as1-1 is included as a positive control. (e h) Expression patterns of STM::GUS in the sixth leaf of 16-day-old plants. Sinuses in the wild type with (f) or without (e) GUS staining. The larger domain and higher frequency of STM::GUS expression at the sinus were observed in CUC2g-m4 and mir164a-4 (g,h). (a) (c) CUC2g-m4 50 µm (b) 50 µm 50 µm (d) Fold change in mrna level mir164a STM BP KNAT2 KNAT6 * * qrt-pcr of KNOX1 * * Bars = s.d. * * CUC2g-m4 mir164a-4 as1-1 STM::GUS expression patterns (e) (f) (g) CUC2g-m4 (h) mir164a µm 100 µm 100 µm 500 µm KNOX1 mis-expression in leaves of CUC2g-m4 and mir164a. Candidate KNOX1 genes mis-expressed were STM and KNAT6, as the KNAT1::GUS and KNAT2::GUS signals were reported to be absent from CUC2g-m4 leaves (Nikovics et al., 2006). It is also reported that KNAT1, KNAT2 or KNAT6 is not involved in the leaf lobe formation caused by as2 mutation (Ikezaki et al., 2009). We performed quantitative RT-PCR (qrt-pcr) to see whether STM and/or KNAT6 expression was changed (Figure 7d). The expression level of STM was significantly increased by four- and sixfold in CUC2g-m4 and mir164a-4, compared with the wild type, respectively (both P < 0.05). Expression of BP and KNAT2, included as negative controls, and KNAT6 in CUC2g-m4 and 164a-4 was similar to the wild type, and that in as1-1, included as a positive control, was significantly higher. Next, we investigated the STM expression patterns in tissues using a GUS reporter line: STM::GUS. In CUC2g-m4 and mir164a-4, GUS staining was observed as a small spot at some sinuses (Figure 7g,h). Interestingly, similar STM::GUS expression was observed in the wild type, although it was quite infrequent (Figure 7e,f). DISCUSSION We studied mechanisms of tooth formation and patterning by examining cell proliferation and differentiation during tooth development. The number of marginal cells between teeth was consistent as leaves developed, and each tooth showed cell file-like patterns towards the tip. These results suggest that leaves have one basic mechanism that can be repeated to form multiple teeth. We detected auxin distribution, indicated by DR5rev::GFP, in one or two epidermal cells at the margin in the wild type before teeth were recognizable. This suggests that auxin marks future tooth initiation sites, as shown for leaflet formation (Barkoulas et al., 2008). Furthermore, the discrete DR5rev::GFP expression was disturbed in the absence of CUC2, demonstrating a role for CUC2 in establishing, maintaining or enhancing auxin maxima at the leaf margin. In addition, we found that CUC2 promotes tooth outgrowth through cell division, rather than by growth suppression at the sinuses. The angle of the tooth axis against the leaf midvein, observed as cell files, is also affected by the pattern of auxin maxima. These observations suggest that proper patterning of auxin

10 438 Eiko Kawamura et al. maxima not only patterns positions of teeth, but also determines the shape and size of the teeth. CUC2 promotes cell proliferation to contribute to the outgrowth of teeth CUC2 was thought to repress growth at the sinus to promote serration (Nikovics et al., 2006). Our analyses on leaf outline and cell shape, however, showed that CUC2 has a role in extending marginal outgrowth from the leaf blade, through promoting cell proliferation. Consistent with this, the duration of cell proliferation in each tooth seemed to be prolonged in CUC2g-m4 and mir164a. Furthermore, tooth protrusion was found to be noticeably larger from the early stage of tooth development in mir164a (Nikovics et al., 2006), suggesting that cell proliferation activity is also promoted from early stages in the mutant. These clearly indicate that the normal function of CUC2 is likely to promote tooth outgrowth through cell proliferation. CUC2::GUS expression is broadly found in the margins of proximal parts of leaf primordia, and becomes rapidly restricted to sinuses in developing leaves (Nikovics et al.,2006). The restricted expression of CUC2::GUS at the sinuses was consistently seen in all sixth leaves examined, whereas the teeth remained in developing stages (comparable in size with those of the leaves used for the leaf outline analysis in Figures 4c and S5). Therefore, CUC2 probably acts at distance to promote tooth outgrowth, or determines the extent of future outgrowth, similar to the mechanisms suggested for leaflet outgrowth in compound leaves (Blein et al., 2008), and lateral organ development in Arabidopsis (Larue et al., 2009). The question is how is tooth outgrowth regulated? CUC2g-m4, mir164a-4 and mir164a-6 produce ectopic tissues with a notch at the sinus, and occasionally with an ectopic stipule (Figure 7a c), similar to those typically found in as1-1;se and as2-2;se (Ori et al., 2000; Hay et al., 2002, 2006), which fail to exclude KNOX1 from leaves. Our qrt-pcr and STM::GUS analyses showed that STM is significantly upregulated at the sinuses of CUC2g-m4 and mir164a-4 (Figure 7d h). Interestingly, occasional STM expression at the sinus was also observed in the wild type (Figure 7f). A study using a weak allele for STM suggests that STM has an important role in determining leaf shape (Depuydt et al., 2008). Although downregulation of STM defines simple leaves, slight expression of STM may be required for tooth outgrowth and leaf shape determination. CUC2 patterns serration by regulating auxin distribution at the margin It is known that the CUC2 expression is altered in auxinrelated mutants such as pin1 and monopteros (Vernoux et al., 2000; Aida et al., 2002; Furutani et al., 2004). In the present study, we found that discrete DR5rev::GFP expression was disturbed at the leaf margin of cuc2-3. This demonstrates that CUC2 helps to establish, enhance or stabilize auxin maxima at the leaf margin. This further suggests that the localization or expression of PIN1, the major auxin transporter in serration formation, is regulated by CUC2. Consistent with fewer tight DR5rev::GFP expression peaks, hydathode/tooth number is reduced in the cuc2-3 leaves. In contrast, mutants with elevated CUC2 expression were reported to maintain normal serration patterns (Nikovics et al., 2006). These results indicate that CUC2 presence is essential for the discrete auxin peaks, and increasing CUC2 expression to the levels seen in these mutants cannot affect primary tooth positioning. In compound leaves, depletion of CUC (Blein et al., 2008) or disruption of auxin patterns (Barkoulas et al., 2008) results in a reduction of leaflet number. CUC may also regulate leaflet positioning by influencing auxin accumulation patterns. On the contrary, in pin1, more DR5rev::GFP peaks are formed, but the leaf margin does not form distinct protrusions of teeth. Although the pin1 leaf margin appears smooth, more hydathodes are produced compared with the wild type (Figure 2e). Consistent with this, pin1 has more solitary DR5rev::GFP-expressing marginal cells, which are irregularly spaced. These observations suggest that some PIN proteins other than PIN1 manage to form incomplete auxin maxima that result in irregular hydathodes, but cannot promote protrusion of serrations, even in the presence of CUC2. Thus, it is likely that CUC2 and PIN1 work together to promote margin outgrowth. Other PIN proteins, such as PIN3, PIN4 and PIN7 are known to be expressed in leaf primordia, and later in procambial cells (Scarpella et al., 2006), but whether these PIN proteins are involved in serration formation is not clear. The earliest DR5rev::GFP expression was detected before tooth outgrowth in a single epidermal cell at the margin, in the junction between a leaf blade and a petiole (Figure 3a-c). We rarely observed initial accumulation patterns, suggesting that this event takes place rapidly. In C. hirsuta, which forms compound leaves, the earliest DR5::VENUS expression was detected in a single subepidermal cell in the margin of a rachis, not in an epidermal cell, and not in the leaf blade (Barkoulas et al., 2008). Although both tooth and leaflet formations require auxin accumulation, the precise mechanisms of their development should be diverse. The curvature of cell file-like patterns is associated with the outgrowth of teeth We found that the abaxial leaf epidermis forms strong cell file-like patterns towards the leaf tip and tooth tip during cell division phases. In more indented leaves, such as leaves that develop later or in mutants, the patterns appeared more pronounced, and corresponded to the degree of serration. This indicates that cells positively divide at particular angles to promote outgrowth from the leaf blade. Clonal analysis of tobacco leaves revealed similar cell division patterns with alternating perpendicular planes (Poethig and Sussex, 1985). The orientation of cell file-like patterns is well

11 Tooth development in Arabidopsis 439 correlated with auxin distribution patterns at the leaf margin in the wild type, as well as in pin1 and cuc2-3. These results indicate that discrete auxin maxima at the leaf margin regulate the directions of cell division and growth, and this in turn contributes to tooth outgrowth. Similarities in leaf primordia formation in the SAM, and leaflet and tooth formation in leaves Our study provides morphological evidence that each tooth can be viewed as one unit, and that multiple teeth can be produced by repetition of a single mechanism at regular frequencies, as seen in leaf primordium formation in the SAM and leaflet formation in compound leaves. Ectopic expression of KNOX1 genes often results in ectopic stipules and/or meristem formation at the sinus (Chuck et al., 1996; Ori et al., 2000; Hay et al., 2002), which probably retains characteristics of the leaf base of simple leaves. This supports an idea that each tooth is similar to a single leaf/leaflet, as depicted in Figure 8. It has been shown that leaflet formation in compound leaves and leaf primordium formation in SAM share many similarities, such as morphological features (Arber, 1950; Sattler and Rutishauser, 1992; Lacroix and Sattler, 1994; Lacroix et al., 2003) and molecular mechanisms, involving auxin (Reinhardt et al., 2000, 2003; Barkoulas et al., 2008) and CUC genes (Aida et al., 1997; Vroemen et al., 2003; Blein et al., 2008; Berger et al., 2009). Serration also shows these features (Arber, 1950; Aloni et al., 2003; Nikovics et al., 2006), and in this study we provide more detailed morphological evidence that serration development is similar to leaflet and leaf primordium formation. In addition, similar to the cell-cycle arrest model in the development of simple leaf-type leaf blades (Donnelly et al., 1999; Nath et al., 2003; Palatnik et al., 2003), individual conceptual leaflets show cell cycle arrest that gradually proceeds from the tips/auxin maxima towards the basal end. Conceptual terminal leaflet We observed a similar differentiation shift in the marginal cells (Figures 1 and 2), and in mesophyll cells at the leaf margin (Figure S4). As these conceptual lateral leaflets form at an angle to the axis of the conceptual terminal leaflet, cell cycle arrest would reach the sinus at the upper part of a tooth earlier than at the lower part. When secondary teeth are formed in either the wild-type or mutant leaves, the site of initiation is more frequently found on the lower part of a primary tooth, near the base (Figure S2). A prolonged undifferentiated state on the lower side may allow secondary tooth formation. The angle of the hypothetical terminal and lateral leaflets would also be a critical determinant for the appearance of indentations. Taken together, our results suggest that serration is formed as follows: the PIN1 and CUC2 establishes/ enhances auxin maxima in a single epidermal cell, and gradually develops the maxima at the leaf margin; correct cell division patterns, which form cell file-like patterns guided by auxin maxima, contribute to tooth outgrowth; cell proliferation in teeth is promoted by the activity of CUC2; cell division in the marginal cell file ceases when the marginal cell number reaches a certain number, and the smaller cells at the sinus start to elongate; the hydathode differentiates at the auxin maximum; and teeth continue to grow until cell differentiation terminates. We suggest that repetition of this mechanism forms multiple teeth at the leaf margin. This study will lead to a greater understanding of the basic mechanisms of leaf development and patterning. EXPERIMENTAL PROCEDURES Plant material and growth conditions Arabidopsis thaliana from the following lines were used: Col-0; as1-1 (CS3774); 164a-4 (GABI 867E03), 164a-6 (SM_ ), CUC2g-m4 (Nikovics et al., 2006); cuc2-3 (Hibara et al., 2006); pin1-201 (SALK_047163) (Furutani et al., 2004); pin1-8 (SALK_097144); DR5rev::GFP (Friml et al., 2003); CYCB1;1::GUS (a gift from Dr J. Celenza at Boston University); and STM::GUS (Kirch et al., 2003). Marker lines were crossed with mutants, and F 3 segregants homozygous for both genes were used for this study, except for STM::GUS plants, which were from the F 2 generation. Seeds were planted on rockwool cubes (Nittobo, and kept at 4 C for 3 6 days before transfer to growth chambers. Plants were grown at 22 C with a cycle of 16-h light/8-h dark. Scanning electron microscopy Conceptual lateral leaflet Auxin maximum Direction of cell cycle arrest shift Figure 8. Serration in simple leaves could be viewed as fused leaflets. A simple leaf divided into conceptual leaflets (left). The conceptual terminal leaflet (gray) and conceptual lateral leaflets (green) have auxin maxima at their tips. Each conceptual leaflet shows cell cycle arrest proceeding basipetally. Specimens were prepared as described in Tsukaya et al. (1993), with some modifications. Briefly, plants were immersed in FAA, 5% formaldehyde (v/v), 5% (v/v) acetic acid and 45% (v/v) ethanol, and kept for 20 min under a low vacuum. Fixation was continued in FAA for at least overnight. Tissues were dehydrated in an ethanol series, and then ethanol was replaced with isopentyl acetate. Samples were dried using a JCPD-5 critical point dryer (JEOL, and sputter coated with silver using JFC-1300 (JEOL). Specimens were examined under a scanning electron microscope, either JSM-820 or JSM-6510LV (JEOL).

12 440 Eiko Kawamura et al. Light microscopy Older leaves were fixed in FAA under low vacuum for 30 min and then stored at 4 C. Younger leaves were immersed in 90% (v/v) cold acetone for 15 min under low vacuum, and then incubated in 100 mm phosphate buffer with 0.05% Triton X-100 overnight. With the latter method, younger leaf epidermal cells could be easily visualized with DIC. Samples were then cleared in chloral hydrate solution (chloral hydrate : glycerol : water, 8 : 2 : 1) (Donnelly et al., 1999) and mounted on slides. GUS staining was carried out according to the method described by Donnelly et al. (1999), except that 1.5 mm K 3 F 3 (CN) 6 and 37 C incubation were used for STM::GUS staining. Images were taken with a DFC300FX camera (Leica, attached to the light microscope, either DM4500B or MZ16F (Leica). For confocal laser scanning microscopy, samples were dissected and mounted in water. Images were collected with a Meta510 confocal microscope (Carl Zeiss, GFP was excited with the 488-nm line of an Ar laser. qrt-pcr Leaf blades between 3- and 8-mm long were collected from the sixth leaf and snap frozen in liquid nitrogen for RNA extraction. Total RNA was purified using an RNeasy Plant Mini kit (QIAGEN, The extract was treated with DNaseI amplification grade (Invitrogen, to degrade contaminated DNA. cdna was synthesized using a Transcriptor High Fidelity cdna synthesis kit (Roche, The product was mixed with Power Syber Green master mix (Applied Biosystems, or Thunderbird Syber qpcr mix (TOYOBO, and the primers. The primer sets used were as follows: STM (Yin et al., 2007); BP, KNAT2 and KNAT6 (Iwakawa et al., 2007); and UBQ10 (Horiguchi et al., 2009). The samples were subjected to qrt-pcr analysis (7300 Real-Time PCR system; Applied Biosystems). Wildtype SAM was tested as a positive control, and consistently showed higher expression of all KNOX1 genes than in wild-type leaves. No primer-specific amplification was detected in samples without reverse transcription. UBQ10 was used for normalization of amplified products. ACKNOWLEDGEMENTS We thank A. Nakano at the University of Tokyo (Japan) for use of their confocal microscope; M. Aida at NAIST (Japan) for CUC2::- GUS; J. Celenza at Boston University (USA) for CYCB1;1::GUS; J. Friml at Ghent University (Belgium) for DR5rev::GFP; P. Laufs at INRA (France) for CUC2g-m4; M. Tasaka at the NAIST (Japan) for cuc2-3; W. Werr at Universität zu Köln (Germany) for STM::GUS; ABRC (USA) for as1-1, pin1-201 and pin1-8; GABI-Kat (Germany) for mir164a-4; and the European Arabidopsis Stock Centre (UK) for mir164a-6. This work was supported by grants-in-aid for Scientific Research (A), to HT and GH ( ), and for Creative Scientific Research, to HT (18GS0313), from the Japan Society for the Promotion of Science, and for Scientific Research on Priority Areas to HT ( ) from the Ministry of Education, Culture, Sports, Science and Technology (MEXT) in Japan. SUPPORTING INFORMATION Additional Supporting Information may be found in the online version of this article: Figure S1. Sixth leaf in the wild type. Marginal cells adjacent to the smaller cells at the tip are elongated in the left tooth. Figure S2. Sixth leaves in 14-day-old wild type (a), 12-day-old CUC2g-m4 (b) and 14-day-old mir164a-4 (c). Figure S3. pin1-8 plant expressing DR5rev::GFP. Confocal images of the areas indicated by red squares in a (b, c). Figure S4. Sixth leaf from a 21-day-old wild-type plant (a). Squares in A are magnified in panels b e. Figure S5. Sixth leaf from a 13-day-old CUC2::GUS plant. 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