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Conservation and diversification of SCARECROW in maize

Abstract

The SCARECROW (SCR) gene in Arabidopsis is required for asymmetric cell divisions responsible for ground tissue formation in the root and shoot. Previously, we reported that Zea mays SCARECROW (ZmSCR) is the likely maize ortholog of SCR. Here we describe conserved and divergent aspects of ZmSCR. Its ability to complement the Arabidopsis scr mutant phenotype suggests conservation of function, yet its expression pattern during embryogenesis and in the shoot system indicates divergence. ZmSCR expression was detected early during embryogenesis and localized to the endodermal lineage in the root, showing a gradual regionalization of expression. Expression of ZmSCR appeared to be analogous to that of SCR during leaf formation. However, its absence from the maize shoot meristem and its early expression pattern during embryogenesis suggest a diversification of ZmSCR in the patterning processes in maize. To further investigate the evolutionary relationship of SCR and ZmSCR, we performed a phylogenetic analysis using Arabidopsis, rice and maize SCARECROW-LIKE genes (SCLs). We found SCL23 to be the most closely related to SCR in both eudicots and monocots, suggesting that a gene duplication resulting in SCR and SCL23 predates the divergence of dicots and monocots.

Keywords: Arabidopsis, GRAS family, pattern formation, root development, SCARECROW

Introduction

In plants, highly regulated coordination of cell division, cell expansion and cell specification gives rise to the final shape of the organism. Asymmetric cell divisions play an important role in establishing and propagating the correct patterns of plant tissue. Radial patterning in the ground tissue (endodermis and cortex) of the Arabidopsis root is a well-characterized example of how regulatory genes control tissue patterning through a series of asymmetric cell divisions. In Arabidopsis roots, a set of initials undergo asymmetric cell divisions to regenerate the initials themselves and give rise to their daughter cells. The cortex/endodermis initial, for example, first divides transversely, and this asymmetric division produces another initial and a daughter cell (Dolan et al., 1993; Scheres et al., 1994). The daughter cell then divides longitudinally, and this second asymmetric division generates the endodermis and cortex cell lineages. SCARECROW (SCR) and SHORT-ROOT (SHR) are required for the second asymmetric cell division which gives rise to the cortex and endodermis (Di Laurenzio et al., 1996; Helariutta et al., 2000). Mutations in either SCR or SHR result in the absence of one of the ground tissue layers. In scr mutants, the remaining layer exhibits difierentiated attributes of both cortex and endodermis, whereas in shr mutants it has characteristics only of cortex. Genetic and molecular analyses provided evidence that SCR acts downstream of SHR. In addition, it was demonstrated that ectopic expression of SHR under the SCR promoter could lead to supernumerary layers with endodermal characteristics (Nakajima et al., 2001). These results indicate that SCR is essential for the asymmetric cell division but not specification of the endodermis, whereas SHR is required for both cell division and cell specification (Benfey et al., 1993; Scheres et al., 1995; Di Laurenzio et al., 1996; Helariutta et al., 2000; Nakajima et al., 2001).

Both the Arabidopsis SCR and SHR genes encode putative transcription factors that belong to the GRAS family (Di Laurenzio et al., 1996; Peng et al., 1997; Silverstone et al., 1998; Pysh et al., 1999; Helariutta et al., 2000). Thus far, it has been reported that at least 33 members of this plant-specific family are predicted in the Arabidopsis genome, including the founding members (GAI, RGA, and SCR) (Di Laurenzio et al., 1996; Peng et al., 1997; Silverstone et al., 1998; Pysh et al., 1999; Helariutta et al., 2000; Bolle, 2004). Only a handful of the GRAS genes, however, have been studied in detail, revealing that they play diverse regulatory roles in plant growth and development (gibberellin signal transduction, axillary meristem initiation, shoot meristem maintenance, phytochrome A signal transduction, microsporogenesis, and root radial patterning) (Di Laurenzio et al., 1996; Peng et al., 1997; Silverstone et al., 1998; Schumacher et al., 1999; Bolle et al., 2000; Helariutta et al., 2000; Lee et al., 2002; Stuurman et al., 2002; Wen and Chang, 2002; Greb et al., 2003; Kamiya et al., 2003; Morohashi et al., 2003). The functions of the majority of the GRAS genes remain to be studied.

In Arabidopsis roots, SCR expression was observed in the cortex/endodermis initials, the quiescent center (QC), and the endodermal cell lineage (Di Laurenzio et al., 1996; Wysocka-Diller et al., 2000). Recently, it was shown that the recovery of quiescent center (QC) identity and the stem cell niche in scr mutants depends on expression of SCR in the QC (Sabatini et al., 2003). Further analysis of SCR expression in the Arabidopsis shoot system revealed that SCR was expressed in the seedling shoot apical meristem (SAM), young leaf primordia, bundle sheath cells of the leaf, and the endodermis/starch sheath of the inflorescence stem (Wysocka-Diller et al., 2000). In addition, a detailed study of SCR expression during embryogenesis showed that it was consistently expressed in each ground tissue cell before longitudinal division and in the inner daughter cell after division (Wysocka-Diller et al., 2000). Correlation of the SCR expression pattern with radial pattern defects in shoot and embryo provides further evidence that SCR is essential for key asymmetric cell division events, which give rise to different ground tissue layers.

Based on sequence similarity and its expression pattern in maize roots, we previously suggested that ZmSCR is the likely ortholog of SCR (Lim et al., 2000). In addition, analysis of the dynamic expression pattern of ZmSCR during regeneration of the root tip after either whole or partial excision indicated the involvement of positional information as a primary determinant in regeneration of the root radial pattern in maize. Our results suggested that there is a common molecular basis for radial patterning of roots from the two distantly related species, despite the fact that the size and configuration of the QC is distinct in Arabidopsis and maize (Lim et al., 2000).

In this study, we provide further evidence to support our hypothesis through molecular complementation of Arabidopsis scr mutants with ZmSCR, expression analyses during maize embryogenesis and in the shoot system, and a phylogenetic analysis of the Arabidopsis, rice and maize GRAS families. Interestingly, we found a novel gene (named SCL23) that is the most closely related to SCR in Arabidopsis, rice and maize, suggesting that a gene duplication resulting in SCR and SCL23 predates the divergence of dicots and monocots.

Taken together, our results suggest that ZmSCR, the maize SCR ortholog, employs similar as well as distinct molecular mechanisms to regulate radial patterning in maize, and that SCR function is conserved in eudicots and monocots.

Materials and methods

Molecular complementation of scr mutants with ZmSCR

The pSCR::ZmSCR construct was made by placing the full-length cDNA of ZmSCR immediately after the 2.5-kb region upstream of the SCR 620 translational start site in pSCRHYG (gift from Keiji Nakajima). The resulting plasmid (pSCR::ZmSCR) was used to transform scr-1 mutants by the Agrobacterium-mediated, floral-dipping method (Clough and Bent, 1998). Seeds from the transformed plants were harvested and plated on medium containing hygromycin (15 μg/ml) to select transgenic plants harboring pSCR::ZmSCR. To confirm the genetic background of the transgenic plants, PCR products were amplified using the primers specific for Arabidopsis SCR. Subsequently, the amplified products were restriction-enzyme digested to observe polymorphisms between wild-type and scr-1 plants. In addition, the primers specific for ZmSCR were used to verify the presence of the pSCR::ZmSCR construct by PCR analysis on the same DNA samples that were used for genotyping.

Histochemical techniques and in situ hybridization

Seeds of the maize inbred line B73 (Zea mays L.) were surface-sterilized, imbibed, and germinated in wet paper towels. When seedlings were approximately 3–4 cm long (6 days after planting) and all leaves were still enclosed in the coleoptile, the young shoots were excised and fixed in FAA (10% formaldehyde, 5% acetic acid and 50% ethanol [all v/v]) for plastic sectioning. For in situ hybridization, the samples were fixed in 4% paraformaldehyde in PBS overnight at 4 °C. Embryos of different stages were collected from inbred B73 plants grown and self-pollinated in the field, and the classification of stages was according to Randolf (1936). Subsequently, the collected samples were dehydrated, embedded and sectioned as previously described (Jackson, 1991; Di Laurenzio et al., 1996; Fukaki et al., 1998; Lim et al., 2000). Sense and antisense riboprobes labeled with digoxigenin-11- UTP for ZmSCR were generated from the 3′ UTR of ZmSCR and in situ hybridization was performed as described previously (Jackson, 1991; Lim et al., 2000).

Plastic sections were used for both staining of Casparian strip and amylopalsts, using Technovit 7100 (Heraeus Kulzer). Samples for both Casparian strip and amyloplast staining were fixed as described previously (Di Laurenzio et al., 1996; Fukaki et al., 1998). In particular, to keep the growth orientation of stems with respect to the gravity vector, the upright primary inflorescence stems were cut from wild-type, scr and scr transformed with pSCR::ZmSCR, respectively. The segments of stems were fixed in FAA overnight at 4 °C in 1.5 ml tubes. After fixation, samples went through an ethanol series and were embedded in Technovit 7100 (Heraeus Kulzer) according to the manufacturer’s instructions.

For Casparian strip detection, sections were counterstained in 0.1% aniline blue for 5–10 min as described by Di Laurenzio et al. 1996. The sections were visualized with a Leitz fluorescent microscope with FITC filter. For amyloplast detection, sections were stained in toluidine blue as described in Fukaki et al. 1998. Images were directly captured in a SONY CCD camera using scion NIH image software, and Adobe Photoshop 8.0 (Adobe Systems) was used to create composite figures.

Phylogenetic analysis

Sequence information was obtained from BLAST searches (Altschul et al., 1990, 1997), and predicted proteins were aligned using ClustalX (Thompson et al., 1997). In some cases, the alignments were manually adjusted using Se-Al program (Rambaut, 1996). We found that alignments using the full-length amino acid sequences with default parameters in ClustalX showed some regions that were difficult to align. Moreover, in the case where the sequence information of the full-length cDNAs was not available, the translational start sites for some GRAS members (especially if they were purely predicted by computational methods) were unclear, which led to ambiguous alignments. Thus, we used the signature motifs (VHIID, PFYRE, and SAW) of GRAS proteins for the best alignments as defined in Pysh et al. 1999. The culled alignment contained 548 characters in each of 135 taxa (33 Arabidopsis, 52 rice and 50 maize GRAS proteins), and data files used for our analysis are available in Supplement 1. Phylogenetic analysis using parsimony (PAUP* 4.0b10; Swofford, 2002) was used to infer the evolutionary relationships of the Arabidopsis, rice and maize GRAS members. The phylogenetic tree was constructed by the neighbor-joining method (Saitou and Nei, 1987) using a human (Homo sapiens) STAT protein (HsSRC; NP004374) as an outgroup. The consensus tree with the culled alignment made no difference in our main conclusion, when compared to that with the 621 full-length alignment. Bootstrap analyses (Felsenstein, 1985) were conducted using 2000 replicates to measure node robustness.

Results

Molecular complementation of scr mutants

We previously reported the isolation and characterization of the putative maize SCR ortholog (ZmSCR) and its expression pattern in roots (Lim et al., 2000). Our analysis revealed marked similarities in gene structure, deduced amino acid sequence and expression pattern between the two genes, raising the strong possibility of conserved function (Lim et al., 2000).

To test the hypothesis of functional conservation, we transformed Arabidopsis scr mutants with ZmSCR. A binary plasmid pSCR::ZmSCR, which contains the open reading frame of ZmSCR under the control of the native 2.5-kb Arabidopsis SCR promoter, was generated and introduced into scr-1 mutants. It was previously reported that the 2.5-kb SCR promoter with the full-length cDNA of Arabidopsis SCR was sufficient to rescue scr mutants and also mimic its native expression, when placed upstream of a reporter gene (Malamy and Benfey, 1997; Wysocka-Diller et al., 2000). As shown in Figure 1, transverse sections of the primary root of transgenic plants showed a normal radial organization with endodermis and cortex layers. Histochemical staining for the Casparian strip, a marker for differentiated endodermis, confirmed the presence of endodermal characteristics in the inner layer of ground tissue (Figures 1D–F) indicating that ZmSCR was able to complement radial defects in scr roots (Figure 1F). In addition, the gravitropic response of scr mutant inflorescence stems was restored (Figure 1I). These results suggest that ZmSCR is functionally orthologous to SCR in regulation of the asymmetric cell divisions common to the shoot and root.

Figure 1
Complementation of scr with ZmSCR. (A–C) Transverse sections of primary roots (wild type plants: A, D, G, J; scr: B, E, H, K; scr with pSCR::ZmSCR: C, F, I, L). (D–F) Casparian strip staining of transverse sections of primary roots. The ...

ZmSCR expression during embryogenesis

Although plant embryos lack most structures of the adult plant, radial patterning is established during embryogenesis and is propagated during postembryonic development (Steeves and Sussex, 1989). In Arabidopsis, it was shown that SCR expression appeared as early as the globular stage of embryogenesis (Wysocka-Diller et al., 2000). Over the course of embryo development, SCR expression was consistently found in cells that were destined to undergo asymmetric cell divisions in the ground tissue of the embryonic root, hypocotyl, and presumptive cotyledon shoulder region (Wysocka-Diller et al., 2000).

To investigate the temporal and spatial expression pattern of ZmSCR during embryogenesis, we performed an RNA in situ hybridization analysis in maize embryos. In the first phase in which the apical-basal asymmetry is established, the embryo is characterized by regionalization into the embryo proper and the suspensor (Sheridan and Clark, 1993, 1994). ZmSCR expression was found in the apical region of the embryo where the embryo proper would be formed (Figures 2A and B). When radial asymmetry was established, ZmSCR was expressed in a small group of cells on the anterior side of the embryo that would give rise to the prospective shoot and root meristems (Figure 2C). Once the shoot-root axis has formed, and as the rudiments of the coleoptile began to protrude from the anterior surface of the embryo just above the shoot meristem, expression of ZmSCR was localized to the root meristem at its basal pole similar to that seen in the primary root (Figure 2D). At the stage when a few leaf primordia were initiated by the shoot meristem, the root had been further elaborated, and vascular tissue was forming, localization of ZmSCR expression to the endodermal cell lineage became more evident (Figure 2E). Also, ZmSCR expression was detectable in leaf primordia and young leaves (Figure 2E). These results indicate that ZmSCR expression is a consistent feature of regionalization of the root meristem throughout embryogenesis.

Figure 2
Analysis of ZmSCR expression during embryogenesis. Longitudinal sections through embryos at sequential stages of maize embryogenesis. (A) ZmSCR mRNA was localized in the apical region of the embryo (6 days after pollination [DAP]). (B) ZmSCR mRNA was ...

ZmSCR expression in the maize shoot system

We found that ZmSCR expression was localized to a small domain where both root and shoot meristems would develop and its expression remained in the leaves of mature embryos. To extend ZmSCR expression to the shoot system, we analyzed the ZmSCR expression pattern in the maize SAM and in young leaves. In Arabidopsis seedlings, SCR expression was primarily detected in the L1 layer of the SAM and in young leaf primordia (Wysocka-Diller et al., 2000). Unlike SCR expression in the Arabidopsis shoot, no ZmSCR was observed in the SAM (Figures 3A and B). However, we did observe its expression in leaf primordia and in young leaves (Figures 3A and B). It was also reported that SCR expression in Arabidopsis leaves became progressively restricted to the bundle sheath cells that surround the vascular strands, as leaf primordia expand. The pattern of SCR expression and the morphological defects in these organs suggested a potential role of SCR in the formation of vascular bundles in leaves (Wyscoka-Diller et al., 2000). The patchy expression pattern of ZmSCR in young maize leaves led us to further investigate its expression in leaves. The maize leaf exhibits a typical Kranz anatomy of C4 plants in which mesophyll cells and bundle sheath cells are tightly arranged to facilitate the interchange of metabolites during photosynthesis (Esau, 1977). ZmSCR expression was observed in a series of parallel veins that extend the length of the leaves (Figures 3C–E). The ZmSCR expression domain appeared to be 2 to 3 cells wide (Figure 3E). In transverse sections, the spacing of ZmSCR expression appeared to be in places at which vascular bundles would be formed (Figure 3F). ZmSCR expression in young leaves suggests a potential role of ZmSCR in ground tissue formation during vascular development.

Figure 3
Expression of ZmSCR in the maize shoot system. (A and B) ZmSCR mRNA was expressed in leaf primordia and young leaves, but not in the SAM. (C and D) ZmSCR expression was observed in the parallel veins of young leaves. (E) Close-up of the box in D is shown. ...

Phylogenetic analysis of SCARECROW in Arabidopsis, rice and maize

To understand the evolutionary relationship of SCR in Arabidopsis (eudicot) and maize (monocot), we sought to identify all members of the Arabidopsis and maize GRAS family and perform a comparative analysis. Currently, at least 33 genes are predicted to encode GRAS proteins in the Arabidopsis genome (AGI, 2000; Referred in Bolle, 2004). With SCR as a query, the TBLASTN program (Altschul et al., 1990, 1997) was used to search the available maize genome databases, and a total of 50 GRAS genes were identified (Table 1). To verify expression, we also searched for expressed sequence tags (ESTs) and/or full-length cDNAs for the maize GRAS genes. The majority of predicted maize GRAS genes did not have counterparts among ESTs or full-length cDNAs (Table 1). To perform a comprehensive comparative analysis of the GRAS genes, we included the rice GRAS genes that were previously reported (Kamiya et al., 2003; Tian et al., 2004). In our phylogenetic analysis, we noticed that GRAS proteins could be divided into several major branches, which were named after a common feature of the branch or one representative of their members as previously reported: PAT1, DELLA, SCL3, SCL4/7, LAS (SCL18), HAM, LlSCL, SCR, and SHR branches (Bolle, 2004; Tian et al., 2004). The inclusion of the rice GRAS members in our analysis lent support to these branches and allowed us to identify the genes most closely related to SCR (Figure 4). The most closely related gene in Arabidopsis, SCL23, was also conserved in rice and maize. When we compared the deduced amino acid sequence of ZmSCL23 to those of OsSCL23 and SCL23, the similarity among these predicted proteins extended throughout their entire length (data not shown). When compared at the amino acid level, ZmSCL23 shows 85.7% identity to OsSCL23, and 64.8% identity to SCL23. Our analysis suggests that gene duplication resulting in SCR and SCL23 occurred before the divergence of monocots and dicots.

Figure 4
Phylogenetic relationship of Arabidopsis, rice and maize GRAS genes. The Neighbor-joining tree was constructed using the culled alignment of 33 Arabidopsis, 52 rice and 50 maize GRAS proteins. The tree was rooted using a human STAT (HsSRC) as an outgroup ...
Table 1
Identification of the maize GRAS genes. The TIGR entry and their expression as evidenced by ESTs/full-length cDNAs are listed (indicated by positive (+) or negative (−) signs, when there is at least one form of EST or full-length cDNA).

Discussion

Evolutionary relationship between ZmSCR and SCR

We had previously suggested that ZmSCR is the likely ortholog of SCR based on two criteria: sequence similarity when compared with members of the GRAS family and similarity of expression patterns (Lim et al., 2000). To test our hypothesis that ZmSCR is the functional ortholog of SCR, we investigated its ability to complement loss-of-function mutations of SCR and its evolutionary relationship to the Arabidopsis SCR gene. When Arabidopsis scr mutants were transformed with ZmSCR under the control of the native SCR promoter (Malamy and Benfey, 1997; Wysocka-Diller et al., 2000), the radial defects in roots and shoots were restored indicating that SCR function appeared to be conserved in maize. Although ZmSCR under the native SCR promoter could complement the defects of scr mutants, it is important to note that this alone does not prove orthology.

In our comparative analysis, we noticed conservation and divergence of the GRAS family between monocots and eudicots, which have diverged 150–300 million years ago (Wolf et al., 1989; Wikstrom et al., 2001; Tian et al., 2004). Interestingly, we found that the most closely related pair, SCR and SCL23, was conserved in Arabidopsis, rice and maize, strongly suggesting the orthologous relationship between ZmSCR and SCR. In Arabidopsis, SCL23 expression in the published digital in situ datasets was below thresholds set for background levels and thus we could not reliably predict its expression pattern in the root (data not shown) (Birnbaum et al., 2003). It is still possible that more closely related genes to SCR are present in maize, since the maize genome sequence is not yet completed. However, using the complete rice genome sequence information, which substantiates our analysis, it is suggestive that a gene duplication resulting in SCR and SCL23 predates the divergence of dicots and monocots. With the efforts to elucidate the function of every gene in the Arabidopsis genome by the year of 2010 (Chory et al., 2000), a large number of T-DNA insertion mutant lines and full-length cDNAs are now available. Thus, it will be of interest to elucidate the role of SCL23 and the other GRAS genes in plant growth and development.

Our analysis now at four different levels: (i) sequence similarity, (ii) evolutionary relationship, (iii) expression pattern and (iv) functionality, provides strong evidence that ZmSCR is the functional maize ortholog of SCR.

ZmSCR expression during embryogenesis

The process of pattern formation during plant embryogenesis has been extensively studied resulting in a definition of the spatial relationships among the different parts of the embryo (Jürgens, 1995; Heckel et al., 1999). However, comparative studies of patterning processes during embryogenesis between Arabidopsis and maize are problematic for several reasons. First, in contrast to Arabidopsis, cell divisions in maize embryos follow no obvious stereotypical pattern. Second, the apical-basal axis of the maize embryo, defined by the shoot meristem and the root meristem, is oblique to the axis of the embryo proper and the suspensor while the Arabidopsis apical-basal axis is perpendicular. Third, unlike in Arabidopsis where the primordia for the shoot and root are set apart early in embryogenesis, in maize the primordia for both the shoot and root appear to arise from the anterior side of the embryo that would give rise to the prospective SAM and RAM. Finally, in maize, dormancy occurs later during embryogenesis, leading to the formation of 5–6 leaf primordia in the embryo in contrast to Arabidopsis where there are no leaf primordia and the embryo is much less mature (Sheridan, 1995; Heckel et al., 1999). Without tissue-specific markers, it is difficult to compare fates of groups of cells in these two embryos. Function and expression of the patterning gene, SCR, appears to be conserved in both species, making it an informative marker for comparing the radial patterning process during Arabidopsis and maize embryogenesis. We analyzed ZmSCR expression during embryogenesis to determine its temporal and spatial expression. ZmSCR expression was found in the apical region as early as the appearance of apical-basal asymmetry. It is interesting to note that ZmSCR was expressed in a small group of cells on the anterior side of the embryo that would give rise to the prospective shoot and root meristems, when radial asymmetry was clearly established. ZmSCR expression in later stages was localized to the root meristem and leaf primordia, similar to what is seen in the maize seedling. As in Arabidopsis, ZmSCR expression was observed in the early stages of embryogenesis, suggesting that ground tissue formation is an early event in both monocot and dicot embryogenesis. However, because of the irregularity of the cell division pattern, it is difficult to follow the asymmetric cell division processes, which gives rise to cortex and endodermis as seen in the Arabidopsis embryo.

Isolation of mutant embryos that are morphologically abnormal and arrested at certain stages has implied that patterning processes during embryogenesis are under different genetic controls at specific times and places (Sheridan and Clark, 1993, 1994; Sheridan, 1995; Heckel et al., 1999). It will be interesting to investigate ZmSCR expression as a marker in a collection of maize embryonic mutants to determine what developmental processes are impaired.

Tissue-specific expression of ZmSCR in the maize shoot system

It was demonstrated that in Arabidopsis, scr mutant hypocotyls and inflorescence stems were agravitropic and that these phenotypes correlated with the absence of one of the ground tissue cell layers (Fukaki et al., 1998). Detailed analysis of SCR expression also revealed the possible origins of the endodermis/starch sheath in these organs (Wysocka-Diller et al., 2000). In mature leaves, the strongest SCR expression was observed in bundle sheath cells associated with all veins (Wysocka-Diller et al., 2000).

To understand how the ZmSCR gene is regulated in the maize shoot system, we analyzed the RNA expression pattern of ZmSCR in the maize shoot system. ZmSCR expression was found in the leaf primordia but not in the SAM in maize seedlings, which may reflect differences in ontogeny of lateral organs derived from the SAM between maize and Arabidopsis. In maize, a plumule with 5–6 leaf promordia enclosed within the coleoptile is formed during embryogenesis (Sheridan and Clark, 1993, 1994; Kiesselbach, 1999). In contrast to maize, all leaves are formed during postembryonic development in Arabidopsis. Expression of ZmSCR in young leaves was detected in the parallel veins that extended the length of the leaves. In transverse sections, ZmSCR expression was found with regular spacing in places at which vascular bundles would be formed, suggesting that ZmSCR may prepattern the vascular tissue. Comparison of the expression patterns of ZmSCR and SCR in the shoot system suggests that this regulatory gene plays a similar role in conserved developmental mechanisms for radial patterning in the two distantly related species.

Acknowledgments

The authors would like to thank members of the Benfey laboratory for helpful suggestions and discussions. We are also grateful to Ken Birnbaum, Yrjo Helariutta, Julin Maloof, Tal Nawy, and Alice Paquette for critical reading and comments on the manuscript, and to Keiji Nakajima for the pSCRHYG plasmid. Initial work on this project was supported by a grant to P.N.B. from the National Institutes of Health (Grant No. RO1-GM43778) and funding from Pioneer Hi-Bred. C.E.L is a postdoctoral fellow supported by a grant from Korea Research Foundation (KRF2004-F00019). This research was supported by a grant (Code No. CG1123) from Crop Functional Genomics Center of the 21st Century Frontier Research Program funded by the Ministry of Science and Technology (MOST) and Rural Development Administration (RDA) of Republic of Korea.

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