Involvement of Homeobox Genes in Early Body Plan of Monocot

Involvement of Homeobox Genes in Early Body Plan of Monocot Momoyo Ito, Yutaka Sato, and Makoto Matsuoka BioScience Center, Nagoya University, Chikusa, Nagoya 464-8601, Japan

Homeobox genes are known as transcriptional regulators that are involved in various aspects of developmental processes in many organisms. In plants, many types of homeobox genes have been identified, and mutational or expression pattern analyses of these genes have indicated the involvement of several classes of homeobox genes in developmental processes. The fundamental body plan of plants is established during embryogenesis, whereas morphogenetic events in the shoot apical meristem (SAM) continue after embryogenesis. Knotted1-like homeobox genes (knox genes) are preferentially expressed in both the SAM and the immature embryo. Therefore, these genes are considered to be key regulators of plant morphogenesis. In this review, we discuss the regulatory role of knox genes and other types of homeobox genes in SAM establishment during embryogenesis and SAM maintenance after embryogenesis, mainly in rice. KEY WORDS: Embryogenesis, Homeobox, knox, monocot, Rice, Shoot apical meristem.  2002, Elsevier Science (USA). C

I. Introduction The homeobox, which is characterized by conserved DNA sequence of 180 bp, encodes a 60-amino acid protein motif known as the homeodomain (HD). This structure consists of a helix-turn-helix DNA-binding motif, and thus the homeobox genes are thought to function as transcription factors (Gehring et al., 1994a,b; Gehring, 1987; Qian et al., 1989). Homeobox genes were originally identified in Drosophila homeotic mutants, Antennapedia and bithorax, as the genes that control patterning in Drosophila development (McGinnis et al., 1984; Scott and Weiner, 1984). Since then, homeobox genes have been identified in many

International Review of Cytology, Vol. 218 0074-7696/02 $35.00

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Copyright 2002, Elsevier Science (USA). All rights reserved.

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evolutionarily distant organisms, including animals, plants, and fungi. In higher plants, many homeobox genes have been found to play important roles in various developmental events, as is the case in animals (Chan et al., 1998). Angiosperms are subdivided into two classes: dicotyledonous (dicot) and monocotyledonous (monocot) plants. Arabidopsis is commonly used as a model plant of dicots, and maize and rice are used as model plants of monocots. Many molecular and genetic studies using these model plants have revealed the existence of mutually orthologous genes in the dicot and monocot genomes, and they are thought to share essentially common mechanisms for each phenomenon, including various developmental events. On the other hand, there are also many morphological differences between monocots and dicots, and these should be reflected in some differences in the developmental mechanisms. In this review, we first describe the general characteristics of plant homeobox genes and the involvement of homeobox (mainly knox) genes in early development of monocots. Particular attention is then given to the morphological differences in embryogenesis between monocots and dicots.

II. Plant Homeobox Genes A. Characteristics of the Plant Homeobox Gene Family The first homeobox gene to be identified in plants was KNOTTED1 (KN1) from the maize Knotted1 (Kn1) mutant (Vollbrecht et al., 1991). Leaf blades of the Kn1 mutant exhibit abnormal arrangements of the lateral veins, sporadic outgrowths called knots, and ligule displacements. Kn1 is a dominant mutant, caused by ectopic expression of KN1 in leaves, that results in the disorganization of the developmental program of leaf blades (Table I) (Smith and Hake, 1994). Subsequent to the cloning of the KN1 gene from maize, many plant homeobox genes have been isolated from various plant species using library screening with previously identified gene or degenerate oligonucleotides deduced from HDs as probes (Ruberti et al., 1991; Mattsson et al., 1992; Schena and Davis, 1992, 1994; Carabelli et al., 1993; Gonzalez and Chan, 1993; Matsuoka et al., 1993; Boivin et al., 1994; Chan and Gonzalez, 1994; Feng and Kung, 1994; Kerstetter et al., 1994; Lincoln et al., 1994; Ma et al., 1994; Soderman et al., 1994; Baima et al., 1995; Dockx et al., 1995; Kawahara et al., 1995; Meissner and Theres, 1995; Tamaoki et al., 1995, 1997; Di Cristina et al., 1996; Granger et al., 1996; Hareven et al., 1996; Lu et al., 1996; Serikawa et al., 1996; Gonzalez et al., 1997; Meijer et al., 1997; Valle et al., 1997; Watillon et al., 1997; Janssen et al., 1998; Sato et al., 1998; Sentoku et al., 1998, 1999; Nishimura et al., 1999; Ingram et al., 2000), differential screening (Nadeau et al., 1996; Tornero et al., 1996; Ingram et al., 1999; Dong et al., 2000), mutant based cloning (Table I) (Rerie et al., 1994; M¨uller

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et al., 1995; Reiser et al., 1995; Schneeberger et al., 1995; Long et al., 1996; Chen et al., 1997; Muehlbauer et al., 1997; Parnis et al., 1997; Mayer et al., 1998; Kubo et al., 1999), and other methods (Bellmann and Werr, 1992; Schindler et al., 1993; Korfhage et al., 1994; Klinge et al., 1996). On the basis of sequence similarities in their HDs and the presence of additional distinctive domains outside of the HD, plant homeobox genes are subdivided into several families: knox, HD-ZIP, glabra2, PHD-finger, BELL1, and WUSCHEL-type (Chan et al., 1998). The characteristic structure and functions of each of these plant homeobox gene families are described below (Fig. 1). 1. knox Family The KNOX proteins are approximately 400 amino acids in length and have the HD in the C-terminal region. There are some other conserved domains such as the MEINOX domain and ELK domain located at the N-terminal region of the HD (Fig. 1). All KNOX proteins have three conserved amino acids in the loop between helix I and helix II of HD, and therefore belong to the TALE (three amino acid loop extension) superfamily (Bertolino et al., 1995; Burglin, 1997). KNOX proteins are subdivided into two groups, class I and II, on the basis of their HD sequence similarity; there is very high sequence similarity of the HD within each subclass (Reiser et al., 2000). The class I genes are expressed mainly in the shoot apical meristem (SAM) but not in lateral primordia (Jackson et al., 1994; Long et al., 1996). In contrast, class II genes show more diverse expression and are found not only in the SAM but also in differentiated organs such as roots, leaves, and flowers (Kerstetter et al., 1994; Serikawa et al., 1996, 1997). Two expression patterns of the class I genes in the SAM have been identified: expression at the center of the meristem dome of the SAM and expression at the base of leaf primordia in the SAM (Fig. 2) (Reiser et al., 2000). To date, many dominant mutants overproducing the class I proteins have been studied in maize (Table I). The most characteristic phenotype among these mutants is blade-sheath boundary displacement, which has been attributed to the abnormal prolonged maintenance of leaf primordia at a less differentiated state by ectopic knox gene expression in leaf primordia (Freeling and Hake, 1985; Becraft and Freeling, 1994; Jackson et al., 1994; Schneeberger et al., 1995; Fowler and Freeling, 1996; Foster et al., 1999a,b). However, a few loss-of-function mutants of the knox gene, such as shootmeristemless (stm) and knotted1 (kn1), show aberrant SAM formation and/or maintainance (Table I; see below for details) (Barton and Poethig, 1993; Long et al., 1996; Kerstetter et al., 1997; Vollbrecht et al., 2000). The phenotypes of these dominant or loss-of-function mutants indicate that the class I genes may play an important role in maintaining cells in an undifferentiated state in the SAM.

TABLE I List of Homeobox Mutants and Their Phenotypes in Plants Mutant

Plant

Phenotype

Family

Reference

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Kn1 (Knottedl)

Maize

Dominant

Knots on leaves, blade-sheath boundary displacement

knox

Freeling and Hake, 1985 Vollbrecht et al., 1991

kn1

Maize

Recessive

Arrested shoot development, reduced tassel branches, fewer spiklets

knox

Kerstetter et al., 1997 Vollbrecht et al., 2000

Rs1 (Roughsheath1)

Maize

Dominant

Dwarf plants, sheath mesophyll overgrowth, blade-sheath boundary displacement

knox

Becraft and Freeling, 1994 Schneeberger et al., 1995

Gn1 (Gnarley1/knox4)

Maize

Dominant

Shortened internode (dwarf plants), sheath mesophyll overgrowth, blade-sheath boundary displacement

knox

Foster et al., 1999a Foster et al., 1999b

Lg4 (Liguleless4/ knox5,11)

Maize

Dominant

Blade into sheath transformation

knox

Fowler and Freeling, 1996

Lg3 (Liguleless3)

Maize

Dominant

Blade into sheath transformation

knox

Fowler and Freeling, 1996 Fowler et al., 1996 Muehlbauer et al., 1997

d6 (OSH15)

Rice

Recessive

Shortened intemode (dwarf plants), cell identity defects

knox

Sato et al., 1999

Hooded

Barley

Dominant

Extra flower on the lemma

knox

M¨uller et al., 1995

stm (shootmeristemless)

Arabidopsis

Recessive

Arrested shoot development, abnormal floral organ number

knox

Barton and Poethig, 1993 Long et al., 1996

Tkn2/Let6

Tomato

Dominant

Supercompound leaves, ectopic shoots

knox

Chen et al., 1997 Janssen et al., 1998 Parnis et al., 1997

ifl1 (interfascicular fiberless1)

Arabidopsis

Recessive

Disruptted interfascicular fiber differentiation

HD-ZIP

Zhong et al., 1997 Zhong et al., 1999 Zhong et al., 2001

anl2 (anthocyaninless2)

Arabidopsis

Recessive

Reduced anthocyanin accumulation in leaves

GL2

Kubo et al., 1999

gl2 (glabra2)

Arabidopsis

Recessive

Abnormal trichome expansion, exceptionally hairy root

GL2

Rerie et al., 1994 Masucci et al., 1996 Di Cristina et al., 1996

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bell1

Arabidopsis

Recessive

Abnormal integument development

BELL1

Reiser et al., 1995

wus (wuschel)

Arabidopsis

Recessive

Defective shoot meristem (abnormal stem cell maintenance)

WUS

Laux et al., 1996 Mayer et al., 1998 Schoof et al., 2000

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FIG. 1 Schematic representation of the structure of each family of plant homeodomain proteins. HD, homeodomain; MEINOX, MEINOX domain; E, ELK domain; LZ, leucine zipper motif; a, acidic region; PHD, PHD-finger; c, coiled-coil motif.

2. HD-ZIP Family HD-ZIP proteins are approximately 300 amino acids in length and have a leucine zipper motif at the C-terminal side of the HD (Fig. 1). HD-ZIP proteins can be subdivided into three classes by their sequence similarity of the HD and the presence or absence of some motifs outside of HD. The class II HD-ZIP proteins show high amino acid identity within the HD and leucine zipper motif and also have some common motifs, such as the CPSCE motif, adjacent to the leucine zipper. The class III proteins have a unique HD with four additional amino acids in the turn between helix II and helix III. In contrast, class I HD-ZIP proteins are mutually less conserved within the HD and leucine zipper motif, and are also comparatively

FIG. 2 Schematic representation of two distinctive patterns of knox (class I) gene expression. The genes from rice and maize that display these expression patterns are indicated below each figure. (A) Expression at the center of the meristem dome with downregulation in the lateral organ primordia. (B) Expression at the base of leaf primordia in the SAM.

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less similar in the sequences outside of the HD-ZIP (Chan et al., 1998; Sakakibara et al., 2001). In all three classes of HD-ZIP proteins, homo- or heterodimerization through the leucine zipper is a prerequisite for DNA binding. DNA binding experiments have revealed that the class I and class II proteins recognize a 9-bp palindromic sequence, CAAT(A/T, G/C, N)ATTG (Sessa et al., 1993, 1997; Meijer et al., 1997; Palena et al., 1999; Johannesson et al., 2001), while the class III proteins recognize an 11-bp palindromic sequence, GTAAT(G/C)ATTAC (Sessa et al., 1998). The difference in the recognition sequence between class I and II and class III proteins may be due to the presence of the additional four amino acids in the class III proteins. The functions of the HD-ZIP proteins have been investigated through analyses of their expression patterns and the phenotypes of transgenic plants. These studies have revealed that the HD-ZIP proteins do not have a common function, but rather their functions are diverse even within the same subclass. Briefly, the class I and class II proteins are primarily involved in signal transduction pathways in response to various environmental stimuli such as light (Carabelli et al., 1993; Steindler et al., 1999), osmotic stress (Soderman et al., 1999), water deficit (Soderman et al., 1996, 1999), virulent pathogens (Mayda et al., 1999), exogenous treatment with abscisic acid (Soderman et al., 1996; 1999), and sucrose (Hanson et al., 2001). The class III proteins are mainly involved in the differentiation within the vascular tissue, such as the formation of provascular cells (Baima et al., 1995; Scarpella et al., 2000), and interfascicular fiber (Table I) (Zhong and Ye, 1999). 3. glabra2 Family glabra2-type proteins have structural charactersistics similar to the HD-ZIP proteins. They have a leucine zipper motif adjacent to the HD at the same position as the HD-ZIP proteins, and homo- or heterodimerization through the leucine zipper motifs is thought to be necessary for the recognition of specific DNA sequences (Fig. 1) (Palena et al., 1997). Due to these similarities, glabra2-type proteins are sometimes included in the HD-ZIP family as class IV HD-ZIP proteins (Di Cristina et al., 1996). The glabra2-type proteins are approximately 800 amino acids in length, and they have some characteristic domains outside of the HD, such as an acidic domain, a hydrophilic domain, and a polar domain (Fig. 1). The leucine zipper motif of the glabra2-type proteins is characterized by two truncated leucine zippers, which are interrupted by a short stretch of amino acids as a loop. glabra2-type genes were originally identified from an Arabidopsis glabra2 mutant that displayed aberrant trichome formation and an increased number of root hairs (Table I) (Rerie et al., 1994; Di Cristina et al., 1996). Trichomes and root hairs are both derived from the epidermal cells, and the glabra2 gene is expressed in the trichome-producing and non-root hair-producing cells of the epidermis. All glabra2-type genes examined also show L1- or dermal-specific expression, with

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the exception of ZmOCL2 from maize, which is expressed in the L2-layer of the meristem (Ingram et al., 2000). Therefore, the glabra2-type genes are thought to be a specialized subgroup involved in epidermis development. Another glabra2 mutant, anthocyaninless2, shows reduced accumulation of anthocyanin in the subepidermal tissue on the adaxial side of leaves (Table I) (Kubo et al., 1999). This mutant also has abnormal cellular organization of the primary root, that is, there are several extra cells between the cortical and epidermal layers. The phenotype of anthocyaninless2 and L2-specific ZmOCL2 expression suggests that glabra2-type genes may also be involved in subepidermal cell identity. 4. PHD-Finger Family PHD-finger genes have the HD at C-terminal region, as is the case for the knox genes. The distinctive feature of this family is the existence of a cysteine-rich region, known as the PHD-finger domain, in the N-terminal region of the HD (Fig. 1) (Schindler et al., 1993; Aasland et al., 1995). Deletion of the PHD-finger domain from the Arabidopsis PHD-finger protein HAT3.1 causes severe disturbances in DNA binding, whereas the intact protein can interact with its target sequence (Schindler et al., 1993). This finding suggests that the PHD-finger domain may be involved in recognition of the target DNA sequence. Overexpression of the maize PHD-finger gene, Zmhox1 (Zmhox1a and Zmhox1b), causes various developmental defects such as dwarfism, adventitious shoot formation, and homeotic floral transformations in transgenic tobacco plants (Uberlacker et al., 1996). These defects are very similar to the phenotypes observed in plants overexpressing the knox gene (Sinha et al., 1993), but are considered to be caused by different mechanisms. Actually, the dwarfism that is observed in plants overexpressing Zmhox1 results from a reduction in phytomer number, whereas in plants overexpressing the knox gene, it is caused by a reduction in internode length. Furthermore, the adventitious shoot formation results from a reduction of apical dominance in plants overexpressing Zmhox1, but in plants that overexpress the knox gene, it results not only from a reduction in apical dominance caused by increased cytokinin levels but primarily from a disturbance of normal cell fate. Expression of the Zmhox1 gene is restricted to the early developmental stage of embryogenesis and meristematic tissues (Klinge et al., 1996), and therefore it is probable that this gene is involved in a broad range of developmental processes throughout the life cycle. 5. BELL1 Family The BELL1 proteins, which have a typical HD similar to the KNOX proteins, belong to the TALE superfamily, but are divided into a subfamily distinct from that of the KNOX proteins. BELL1 proteins contain the HD at the C-terminal region and have a conserved coiled coil structure at the N-terminal region, which

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may be involved in dimer formation (Fig. 1) (M¨uller et al., 2001; Nagasaki et al., 2001). A BELL1 gene has been isolated from an Arabidopsis Bell1 (Bel1) mutant (Table I) (Reiser et al., 1995). The ovule of Arabidopsis can be roughly divided into three elements along the proximal–distal pattern. In the Bel1 mutant, the development of integuments, which arise from the central region of the proximal– distal pattern, is defective and results in female sterile plants. The BELL1 gene is expressed in the central region of the ovule primordia prior to integument initiation. This suggests that the expression of BEL1 is regulated based on the proximal–distal patterning, and the localized BEL1 expression in the central region may induce integument development in the Arabidopsis ovule. Another BELL1 gene, MDH1 (Malus domestica homeobox1), has been isolated using differential display during early fruit development in apple. In pre-anthesis flowers, the MDH1 mRNA accumulates in ovules, and overexpression of MDH1 in Arabidopsis causes reduced fertility and changes in the shape of the carpel and fruit. These results suggest that MDH1 may also be involved in ovule development and plant fertility (Western and Haughn, 1999; Dong et al., 2000). The Arabidopsis ATH1 gene is also a member of the BELL1 family, but its function seems to be different from the two other BELL1 family members described above. In Arabidopsis seedlings, ATH1 expression is induced by light, and it is misexpressed in constitutive photomorphogenesis mutants such as det1 and cop1. These results suggest the involvement of ADH1 in photomorphogenesis (Quaedvlieg et al., 1995). 6. WUSCHEL-Type Family A WUSCHEL (WUS) gene has been isolated from an Arabidopsis wuschel (wus) mutant (Table I) (Mayer et al., 1998). The HD of the WUS gene has two and four additional amino acids in the loop between helix I and helix II and the turn between helix II and helix III, respectively. So far, no other genes encoding such an unusual HD have been reported in any organisms, and therefore this may be a very unique type of HD protein. However, in the Arabidopsis genome, we have found at least seven genes which encode HD proteins similar to the WUS HD. These proteins contain one additional amino acid in the loop between helix I and II and four in the turn between helix II and III, while the WUS HD has two and four extra amino acids at the same positions. Despite this difference, the seven putative HD proteins in the Arabidopsis genome may be categorized in the same family as the WUS HD because all these HD proteins, including WUS, share a conserved motif of several amino acids at their C-terminal end (Fig. 1) (Kamiya et al., unpublished observation). In the mature embryo of the Arabidopsis wus mutant, SAM organization is aberrant with only a few vacuolated cells, and after germination, the SAM is terminated prematurely as a flat enlarged apex. In the SAM of the wus mutant,

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the stem cells, which should be maintained in a pluripotent state in the functional SAM, appear to undergo differentiation (Laux et al., 1996). The WUS gene is expressed in a group of cells underneath the stem cells, but not in the stem cells themselves, suggesting that the WUS gene may play a role in maintaining the fate of the stem cells in a non-cell-autonomous manner (Mayer et al., 1998; Schoof et al., 2000). B. Homeobox Genes in Rice In the preceding section, the characteristic structure and function of each plant homeobox gene family have been described. Similarly, many homeobox genes have been identified and characterized in rice. In particular, precise expression analyses during embryogenesis have revealed the particular expression patterns of these genes both temporally and spatially, and the involvement of many types of homeobox gene in early rice development. In this section, we outline the features of each of the rice homeobox genes. 1. Rice knox Genes (OSH Genes) In rice, seven knox genes have so far been reported: OSH1 (Oryza sativa homeobox1), OSH3, OSH6, OSH15, OSH43, OSH71 (class I) (Matsuoka et al., 1993; Sato et al., 1998; Sentoku et al., 1999), and OSH45 (class II) (Tamaoki et al., 1995). The precise expression pattern and function of the class I genes is described below. Comparative studies using cDNA clones derived from OSH45 have revealed that the expression of OSH45 is regulated by an alternative transcription initiation event involving two different promoters. The first promoter, located at the front of the first exon, produces a long transcript including all exons, whereas the second promoter located at the front of the fourth exon produces a shorter transcript containing only the sequence of exons 4 and 5 (Tamaoki et al., 1995). This unique dual promoter system is also observed in the case of OSH1 expression. The different-sized transcripts produce full-length and truncated proteins, which both contain the HD region, that may have different transcriptional activity for expressing their target genes (Tamaoki et al., 1996). 2. Rice glabra2-Type Homeobox Genes (Roc Genes) Five glabra2-type genes, Roc1–Roc5 (Rice outermost cell specific gene), have been isolated, and precise expression analyses have been performed for each (Ito et al., 2002, and unpublished data). All five genes are specifically expressed in the protoderm of the embryo (Fig. 3), the L1 layer of the SAM, and the epidermis of developing young leaves but not in developed adult leaves. This L1 layer- or epidermis-specific expression is consistent with other plant glabra2-type homeobox genes, and indicates that glabra2-type homeobox genes (Roc genes) may also be involved in epidermis development in rice as in other plants.

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FIG. 3 Expression patterns of rice homeobox genes Roc1, OSH1, and QHB in the globular embryo. Each gene clearly specifies certain regions in the globular stage embryo prior to organ differentiation, that is, the Roc1, OSH1, and QHB genes specify the epidermis, SAM, and quiescent center, respectively. (See also color insert.)

To investigate how the GL2-type homeobox genes (Roc genes) are involved in epidermis (protoderm) differentiation during rice embryogenesis, the precise nature of Roc1 expression has been analyzed. In the early stage of rice embryogenesis, cell division occurs randomly, and the morphologically distinct layer structure of the protoderm cannot be observed until the embryo reaches more than 100 μm in length. Nonetheless, the specific expression of Roc1 in the outermost cells is established shortly after fertilization, much earlier than protoderm differentiation. In the regeneration process from callus, Roc1 is also expressed in the outermost cells of callus in advance of tissue and organ differentiation, and such expression occurs independent of whether the cells will differentiate into epidermis in the future or not. Furthermore, this cell-specific Roc1 expression can be induced flexibly in callus-cutting experiments. In the regeneration process from callus, Roc1 is expressed specifically in all outermost cells of callus 3 days after regeneration. When this callus tissue is cut, Roc1 expression is induced in the cells at the outermost side of the cut end. These findings strongly suggest that the expression of Roc1 in the outermost cells is dependent on the positional information of cells in the embryo or callus prior to the cell fate determination of the protoderm (epidermis). In other words, the position-dependent Roc1 expression may be a prerequisite for the differentiation of the protodermal cells and radial pattern formation in early rice embryogenesis. 3. Rice HD-ZIP Genes (Oshox Genes) Since the rice HD-ZIP homeobox gene, Oshox1, was first identifed (Meijer et al., 1997), another six Oshox genes, Oshox2 – 7, have been isolated by yeast one-hybrid screening using HD-ZIP recognition sequences (Meijer et al., 2000). These seven genes include both class I (Oshox4, 5, 6) and class II (Oshox1, 2, 3, 7) genes,

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and each gene product can form a homodimer or heterodimer within the same class. Here, we describe the function of the Oshox1 gene, which has been the most precisely investigated. Oshox1 is primarily expressed in the vascular system, as is the case for some other HD-ZIP homeobox genes (Scarpella et al., 2000). Oshox1 expression starts before any cytological sign of vascular differentiation is evident in the provascular strands during postembryonic root and shoot development. It is known that wounding often induces the transdifferentiation of parenchyma cells into vascular elements (Sachs, 1981; Church and Galston, 1988), and that auxin plays an important role as the main regulatory factor in vascular development (Aloni, 1995; Fukuda, 1996). On the other hand, the brassinosteroids induce differentiation of the tracheary element, and the inhibition of brassinosteroid biosynthesis by uniconazole disturbs tracheary element differentiation without affecting the expression of genes involved in early vascular differentiation (Yamamoto et al., 1997). The expression of Oshox1 gene is affected by wounding and auxin, but not by brassinosteroids or uniconazole, suggesting that Oshox1 may be involved in the determination of procambial cell fate rather than in subsequent vascular differentiation events. The Arabidopsis class III HD-ZIP gene, Athb8, is expressed in the procambium at an earlier stage than is Oshox1, and is also inducible by auxin and wounding (Baima et al., 1995). Furthermore, the class I gene Vahox1 is expressed in the phloem of adult plants during phases of secondary growth (Tornero et al., 1996), demonstrating that many HD-ZIP homeobox genes are involved in the various aspects of vascular tissue differentiation, either in cooperation or independently. 4. Rice WUSCHEL-Type Homeobox Genes (QHB Gene) Three WUS-type homeobox genes have so far been isolated in rice (Kamiya et al., unpublished data). One of these has exactly the same HD as the WUS HD (Mayer et al., 1998), which is characterized by two and four additional amino acids in the loop between helix I and helix II and the turn between helix II and helix III, respectively. The other two have only one additional amino acid in the loop between helix I and helix II, and have four additional amino acids in the turn between helix II and helix III. Detailed expression analyses have been performed for one of these genes, known as QHB (quiescent center specific homeobox gene). The QHB gene is specifically expressed in the quiescent center (QC) of the rice root tips. This QC-specific expression is also observed in the radicle during embryogenesis (Fig. 3). It commences in the globular-shaped embryo, which lacks any morphological organ differentiation including radicle differentiation, and is maintained after establishment of a functional QC, suggesting that the QHB gene may be involved in both the establishment and maintenance of the functional QC. The QC is considered to play a role in maintaining the neighboring initial cells in their undifferentiated state (van den Berg et al., 1997). As mentioned above, the function of the Arabidopsis WUS gene is to maintain overlying stem cells in an

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undifferentiated state (Laux et al., 1996; Mayer et al., 1998). It is possible, then, that the function of the rice QHB gene after QC establishment is to maintain the neighboring initial cells in an undifferentiated state.

III. SAM Formation and knox Gene Expression during Early Embryogenesis in Monocots In animals, embryogenesis is one of the most vigorously studied areas, since almost all morphological events occur during this stage of development. In plants, on the other hand, most morphological events occur during postembryonic development and are solely dependent on two specialized cell masses: the SAM and the root apical meristem (RAM) (Goldberg et al., 1994; Jurgens et al., 1994; Jurgens, 1995, 2001; Clark, 1997; Kerstetter and Hake, 1997; Laux and Jurgens, 1997; Schiefelbein et al., 1997). After germination, the SAM successively produces all of the above-ground parts of plant, including leaves, stems, and flowers, throughout the life cycle, while maintaining itself in an undifferentiated state (Steeves and Sussex, 1989). The most important event in plant embryogenesis is the establishment of the basic body plan, including the SAM and RAM, and therefore elucidation of the mechanisms of SAM formation and maintenance has been a focal point of research in plant development (Clark, 2001a,b). Many genetic and molecular studies have revealed that knox genes may play an important role in SAM formation and maintenance in plants (Reiser et al., 2000). In this section, we discuss the importance of the knox genes in SAM formation during embryogenesis in monocots, or grass plants (rice and maize), focusing on the morphological differences between these plants and dicots (Arabidopsis).

A. Embryogenesis in Monocots As mentioned above, the significance of embryogenesis in plants is quite different from that of animals. In plants, the differentiation of adult tissue or organs is seldom observed in the mature embryo, whereas most of the adult organs are formed during embryogenesis in animals. However, in both plants and animals, the determination of some basic patterns and polarity during early embryogenesis is indispensable for subsequent morphological events. After fertilization in plants and animals, one fertilized egg repeatedly divides, and each cell correctly differentiates into certain tissues or organs based on the polarity and patterns that are established in early embryogenesis (Johnston and N¨usslein-Volhard, 1992; Goldberg et al., 1994; J¨urgens et al., 1994; J¨urgens, 1995). In plants, embryogenesis can be divided conceptually into three phases (Fig. 4, 5E). The first phase is axis formation, during which the axes of basic patterns,

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FIG. 4 Diagrammatic representation of embryo development in Arabidopsis and rice. The basic patterns in plants are also indicated. The grey shaded area in each mature embryo indicates the region that develops into the seedling. The embryo-specific organs in rice (the scutellum and epiblast) are underlined.

such as the apical–basal and radial patterns, are formed. The second phase is segmentation (regionalization), during which more detailed regions for each organ are established along the predetermined axes. The third phase is morphogenesis, during which organ differentiation, including SAM and RAM differentiation, occurs and each organ can be morphologically observed. As a result, the patterns can be visualized by the organized tissue and organ arrangements in the mature

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embryo. The basic pattern of the plant embryo has been described for dicots such as Arabidopsis, since the tissues and organs in the Arabidopsis embryo are simply arranged. An apical–basal pattern along the main body axis of the Arabidopsis embryo consists of a linear array of distinct elements, including the SAM, cotyledons, hypocotyl, radicle, and RAM. A radial pattern around the apical–basal axis is represented by the concentric arrangement of the primary tissues, with the epidermis at the periphery, followed by the ground tissue underneath, and conductive tissue in the center (Fig. 4) (Laux and J¨urgens, 1997; J¨urgens, 2001). During embryogenesis in monocots, including rice and maize, the fundamental mechanisms for pattern formation and organ differentiation are thought to be same as in dicots. However, there are some morphologically distinct differences between dicot and monocot embryogenesis. The embryo of monocots develops many different kinds of tissues and organs that are not simply arranged along the apical–basal and radial axes (rice mature embryo in Fig. 4) (Esau, 1977). This more complex arrangement of organs indicates that there are some important differences in the mechanisms for pattern formation and organ differentiation during embryogenesis in monocots and dicots. In Arabidopsis, the whole mature embryo, with the exception of the suspensor, develops into a seedling after germination (the gray shaded area in Arabidopsis mature embryo in Fig. 4). In other words, the suspensor is the sole embryo-specific organ in the Arabidopsis embryo. On the other hand, in rice only those parts of the embryo that contain the SAM, coleoptile, a few true leaves, radicle, and RAM develop into a seedling, and the other parts, such as the scutellum and epiblast, remain as embryo-specific organs (the gray shaded area in rice mature embryo in Fig. 4). Therefore, compared with dicot embryogenesis, a more complicated mechanism may be required for the precise regionalization of organ differentiation in early monocot embryogenesis. Actually, there are some rice embryonic mutants with altered organ positions (Nagato et al., 1989, 1998; Hong et al., 1995) that may be useful for demonstrating some of the mechanisms controlling the position of organ differentiation in the rice embryo. For example, in one of these mutants, apd1 (apical displacement 1), the shoot and radicle are formed at a more apical region than in the wild-type embryo. Although not yet characterized in detail, these are unique mutants that may be associated with specific embryonic pattern formation in monocots. In the following paragraphs, we describe the expression patterns of knox genes in the rice embryo; these patterns are considered to reflect, at least in part, the complicated regionalization that occurs in monocot embryogenesis.

B. Expression Patterns of knox Genes in the Rice Embryo The mechanisms controlling pattern formation and polarity establishment have been well studied using molecular and genetic analyses in animals, especially in Drosophila (Johnston and N¨usslein-Volhard, 1992; Gehring et al., 1994a; Lawrence

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and Morata, 1994; Pick, 1998). It has been revealed that many homeobox genes play an important role in the various stages of pattern formation in early embryogenesis. After fertilization, the maternal genes (e.g., bicoid) establish the embryonic axis and polarity by the gradient of their gene products. Along the embryonic axis, gap genes, pair-rule genes (e.g., even-skipped, fushi tarazu), and segment polarity genes (e.g., engrailed, gooseberry) divide the embryo into discrete segments. Lastly homeotic selector genes (e.g., Ultrabithorax, Antennapedia), which are the master control genes of actual morphogenesis, confer identity on each segment by controlling the activity of other genes involved in specific morphogenesis (organogenesis). All of the genes given as examples here are homeobox-containing genes, but there are many other homeobox genes that are also involved in specifying patterns in the early Drosophila embryo. In higher plants, the involvement of homeobox genes in embryogenesis was first demonstrated in an Arabidopsis mutant, shootmeristemless (stm) (Table I). The stm mutant fails to organize the SAM during embryogenesis, and true leaves do not emerge after germination, whereas other embryonic organs, such as cotyledons, hypocotyls, and radicles, develop normally (Barton and Poethig, 1993). The STM gene is a Knotted-like homeobox (knox) gene and shows specific expression in the SAM during embryogenesis (Long et al., 1996). Similar SAM-specific expression of another knox gene, KN1, has also been observed in early maize embryogenesis (Smith et al., 1995). These findings directly demonstrate the involvement of knox genes in SAM formation and maintenance during plant embryogenesis. In rice, seven knox genes, OSH1( Oryza sativa homeobox gene1), OSH3, OSH6, OSH10, OSH15, OSH43, and OSH71, have been isolated, and their precise temporal and spatial expression patterns during embryogenesis have been analyzed by in situ hybridization experiments (Matsuoka et al., 1993; Sato et al., 1996, 1998; Sentoku et al., 1999). Rice embryos complete all morphogenetic events within 9 days under normal conditions. The globular stage lasts until almost 3 days after pollination (DAP) (Sato et al., 1996). The first morphological differentiation is recognized as a ventral protrusion of the coleoptile primordium in the late stage of 3-DAP embryos 100 μm in size (late 3 DAP), or in the early stage of 4-DAP embryos 150 μm in size (early 4 DAP). At 4 DAP, when the embryo is 200 μm long and comprises 800 to 900 cells, shoot and radicle apices are first observed. The first through third foliage leaves are formed successively from the SAM at 5, 7, and 9 DAP, respectively, in an alternate phyllotaxis (Fig. 4). The expression patterns of these OSH genes during early embryogenesis (early to late globular stage) can be divided into three groups (Figs. 5, 6). The first group includes four genes, OSH1, OSH43, OSH15, and OSH71 (Sato et al., 1996, 1998; Sentoku et al., 1999). No expression of these genes can be detected in the early globular stage embryo at 2 DAP (less than 100 cells). However, by the late globular stage (3 DAP, 200 cells), all genes in this group are expressed in the specific region where the SAM and epiblast subsequently develop (Figs. 5, 6).

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FIG. 5 Expression pattern of OSH1 during rice embryogenesis. (A–D) Detection of the OSH1 transcript by in situ hybridization. (A) Globular stage embryo; (B) coleoptilar stage embryo; (C) first leaf differentiating stage embryo; (D) nearly mature embryo. The expression of OSH1 commences in the globular stage prior to actual morphogenesis. (E) Schematic representation of OSH1 expression during embryogenesis. Plant embryogenesis can be divided conceptually into three phases: axis formation, regionalization, and morphogenesis. Scale bars in (A) to (D) = 100 μm.

The second group consists of one gene, OSH6 (Sentoku et al., 1999). Unlike the first group of genes, expression of OSH6 can be detected uniformly in the early globular stage embryo at 2 DAP. However, in the late globular stage, the expression of the OSH6 gene becomes restricted to the region where the SAM and epiblast will later develop. The pattern of OSH6 expression in the late globular embryo is nearly the same as that of the first group of genes. Moreover, morphological analyses have revealed that the earliest expression of the first group of genes (OSH1, OSH15, OSH43, and OSH71) occurs in embryos with 100 to 200 cells, and localized expression of OSH6 begins at approximately the same stage (Fig. 6). The third group also includes only one gene, OSH3 (Sentoku et al., 1999). The expression pattern of OSH3 in the early globular embryo at 2 DAP is the same as that of OSH6, that is, uniform expression in the embryo. In the late globular stage at 3 DAP, OSH3 expression is downregulated in the ventral region. Doublestaining in situ hybridization experiments using probes for OSH1 and OSH3 have revealed that downregulation of OSH3 in the ventral region occurs later than the

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FIG. 6 Expression pattern of the OSH genes during embryogenesis (regions of expression are indicated in black). c, coleoptile; e, epiblast; lp1, first leaf primordium; r, radicle; s, shoot apical meristem (SAM); sc, scutellum.

onset of localized expression of OSH1, and that the regions of OSH1 and OSH3 suppression only partially overlap (Fig. 6). It is noteworthy that the earliest expression of the first group of genes together with the onset of localized OSH6 expression, and even the downregulation of OSH3, occur by the late globular stage and that the site of expression of all OSH genes roughly defines the same restricted region at which the SAM eventually develops. As mentioned above, no morphological organ differentiation is evident until the late globular stage, and region-specific expression of these genes starts at an earlier stage than the actual morphological events, including SAM differentiation. These findings strongly suggest that these OSH genes are involved in shoot formation in rice embryogenesis. Interestingly, some knox genes which are thought to be involved in shoot formation during embryogenesis, such as KN1, OSH1, OSH15, NTH15, NTH20, and KNAT1, produce ectopic shoots on the leaves of transgenic tobacco or Arabidopsis plants that overproduce these genes (Sinha et al., 1993; Kano-Murakami et al., 1993; Matsuoka et al., 1993; M¨uller et al., 1995; Sato et al., 1996; Chuck et al., 1996; Tamaoki et al., 1997; Williams-Carrier et al., 1997; Nishimura et al., 2000). It is possible that the formation of ectopic shoots may mimic the process of shoot formation in the natural context of embryogenesis. Furthermore, the fact that

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HOMEOBOX GENES IN MONOCOT EARLY BODY PLAN TABLE II List of Rice knox Genes Rice knox gene

Ortholog in maize

Expression pattern in globular stage embryo

Ectopic shoot formation by overexpression

OSHI

knotted1

Shoot region

Yes

OSH3

knox 3

Whole embryo except shoot region

No

OSH6

liguleless3

Shoot region

No

OSH10

knox10

Shoot region

No

OSH15

knox4, rough sheath1

Shoot region

Yes

OSH43

knox8

Shoot region

No

Shoot region

Yes

OSH71

Note. The localized expression of OSH genes in the shoot region of the globular stage embryo (before SAM differentiation), and ectopic shoot formation in response to overexpression, suggest the involvement of these OSH genes in SAM formation during embryogenesis.

overexpression of these genes alone is sufficient to induce ectopic shoot formation suggests that they may be master control genes for shoot formation. Similarly, the overproduction of OSH1, OSH15, and OSH71 in rice plants can result in many ectopic shoots, whereas overproduction of other OSH genes never induces the multiple shoot phenotype (Table II) (Sentoku et al., 2000), suggesting that OSH1, OSH15, and OSH71 may be master control genes for shoot formation in rice embryogenesis. However, a loss-of-function mutant of OSH15 does not display any loss of shoot formation or abnormal development in rice embryogenesis, but only shows a defect in internode elongation, which results in dwarf plants (Table I) (Sato et al., 1999). These observations, together with the expression patterns and phylogenetic relationships, suggest that OSH1, OSH15, and OSH71 may function redundantly in shoot formation in rice embryogenesis (Table II). C. knox Genes in SAM Formation during Monocot Embryogenesis Through analyses of dominant gain-of-function mutations in the KN1 locus and overexpression studies, it has been hypothesized that KN1 has a role in maintaining cells in an undifferentiated state (Freeling and Hake,1985; Vollbrecht et al., 1991; Smith et al., 1992; Sinha et al., 1993; Williams-Carrier et al., 1997). This hypothesis is supported by the analysis of recessive mutations in the KN1 locus (Kerstetter et al., 1997; Vollbrecht et al., 2000). Recessive alleles in the KN1 locus have been isolated by revertant screening of a dominant kn1 mutant. The recessive

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kn1 mutants show reduced inflorescence branches, sparse spikelets, extra carpels, and an overproliferation of ovules in the female flower (Table I). These phenotypes resemble the phenotype of weak alleles of the Arabidopsis STM gene, and can be attributed to dysfunctions in the SAM (Barton and Poethig, 1993; Long et al., 1996). According to these observations, it is considered that KN1 has a function similar to that of STM in the SAM. In the process of further screening for recessive alleles of kn1, one important phenotype, which clearly indicates the function of KN1, has been found. The size of the SAM in maize varies depending upon the genetic background of the plant (Vollbrecht et al., 2000). One of the recessive alleles of kn1 called E1 has been repeatedly backcrossed into several different genetic backgrounds (inbreds) and selfed to change the genetic background of the E1 allele. The inbred with a small SAM often forms stm-like seedlings, in which the SAM aborts within the coleoptile or after forming one or two foliage leaves. These stm-like seedlings, called limited shoots, demonstrate the importance of the KN1 gene in the maintenance of SAM function rather than in SAM formation They occasionally initiate one to three epicotylar leaves, suggesting that, once established, the SAM cannot be maintained in these plants. Since the limited shoots phenotype appears more frequently in the genetic background of the inbred with a small SAM, it is likely that the appearance of this phenotype is dependent on the allelic differences of the genes that regulate meristem size. It is likely that this inbred-specific modifier(s) is redundant to kn1 and has reduced or no activity in the inbred that has a small meristem. According to this circumstantial evidence, it is possible that the modifier may correspond to a related homeobox gene. Based on molecular biological analyses, knox8, knox3, rs1, and knox4 are most closely related to kn1 (Bharathan et al., 1999; Reiser et al., 2000), and rs1 and knox4 are probably duplicate loci (Schneeberger et al., 1995; Foster et al., 1999b). Knox3 or knox8 is unlikely to be the modifier because of their close linkage to kn1, since the modifier locus segregates independent of kn1 (Kerstetter et al., 1994). These data suggest that rs1 and/or knox4 are the inbred specific modifier(s) and have a redundant function to kn1 in SAM maintenance in the maize embryo. There is still a possibility that the modifier may be a global or specific knox gene regulator (Timmermans et al., 1999; Tsiantis et al., 1999) or loci that otherwise affect SAM maintenance. Phylogenetic analyses, based on the degree of similarity between the deduced amino acid sequences of the rice and maize knox genes, have demonstrated that each rice gene shares a high degree of sequence similarity with one or two corresponding maize genes. The relationship between these pairs of rice and maize genes has been confirmed by the map positions of the genes in the rice and maize genomes. According to a comparative linkage map of the rice and maize genomes (Ahn and Tanksley, 1993), we can deduce which rice homeobox genes are likely to correspond to maize homeobox genes (Sentoku et al., 1999). It is very possible that the maize KN1 gene is orthologous to rice OSH1, and that maize rs1 and knox4

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are orthologous genes to rice OSH15. Therefore in rice, OSH1 and OSH15 may also function redundantly in SAM maintenance. As mentioned above, the limited shoot phenotype of the kn1 recessive mutant demonstrates the involvement of KN1 in SAM maintenance. However, it is still unclear whether KN1 is involved in SAM formation during embryogenesis. In rice, OSH1, which is orthologous to maize KN1, and OSH15, which is orthologous to maize rs1 and knox4, are expressed in the shoot region ahead of morphological SAM formation (Sato et al., 1996, 1998), strongly suggesting the involvement of these genes in SAM formation. If OSH1 and OSH15 only function to maintain the SAM, the expression of these genes would be expected to start directly after formation of the SAM, and these genes would not be expressed in the mutant embryos that lack shoot formation. However, OSH1 and OSH15 are expressed in globular stage embryos of the mutants that lack shoot formation (see below for details) (Sato et al., 1996, 1998), suggesting the involvement of OSH1 and OSH15 in the SAM formation. Ectopic shoot formation caused by the overexpression of OSH1 and OSH15 further supports the involvement of these genes in SAM formation (Sato et al., 1998; Sentoku et al., 2000). The loss of function of KN1 solely affects SAM function in the relatively late stages of development (inflorescence and floral), but it can also affect earlier stages (embryogenesis and vegetative) with the modifier locus (Kerstetter et al., 1997; Vollbrecht et al., 2000). The loss of function of rice OSH15 also affects internode elongation in the reproductive stage, but not in the earlier vegetative stages, including embryogenesis (Sato et al., 1999). This indicates that the degree of functional redundancy in the rice and maize knox genes in SAM formation and/or maintenance may differ in each stage of SAM development. The redundancy seems to be higher in the earlier stage, and this would explain the lack of phenotype of the loss-of-function lines of the maize kn1 or rice OSH15 in shoot formation and/or maintenance during early embryogenesis. In this instance, OSH1 and OSH15 may have a redundant function(s) for SAM formation. Insofar as it is concerned with early shoot formation during embryogenesis, it is also possible that OSH71 may have a redundant function, because its expression pattern in the early globular embryo, and the overexpression phenotype of OSH71, is similar to that of OSH1 or OSH15, although OSH71 is not phylogenetically close to OSH1 or OSH15 (Table II) (Sentoku et al., 1999, 2000).

D. Expression of OSH1 in Rice Embryonic Mutants Mutants are powerful tools for analyzing various biological phenomena in both animals and plants. In animals, the study of many Drosophila mutants has brought many advances in understanding the mechanisms involved in establishment of the early body plan (N¨usslein-Volhard, 1991; Johnston and N¨usslein-Volhard, 1992).

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FIG. 7 Three embryonic mutants with an abnormal number of shoots. gle1 and shl1 or 2 do not form any shoots, and OSH1 expression is narrow or absent. are1 forms two shoots with broader expression of OSH1. The wild-type embryo forms one shoot and expresses OSH1 in the appropriate size and region. s, shoot; r, radicle.

In plants, J¨urgens et al. have screened a large number of Arabidopsis mutants and selected mutations affecting the basic body organization (axis formation and regionalization) (Mayer et al., 1991). In rice, many embryonic mutants have also been identified by Nagato and Kitano (Nagato et al., 1989; Hong et al., 1995), and these include mutations associated with early embryonic patterning. As mentioned above, OSH1 expression marks the shoot region of the embryo prior to actual shoot formation, and therefore OSH1 is a good molecular marker for visualizing the state of determination of the shoot region (Sato et al., 1996). It may be reasonable to expect that OSH1 expression in the early globular stage embryo is aberrant if the mutant embryos fail in the determination of the shoot region and/or shoot formation. In this section, we describe three rice embryonic mutants with aberrant OSH1 expression in the globular stage embryo (Fig. 7). 1. globular embryo1 ( gle1) Mutant gle1 is one of the several mutants in which the embryo remains globular in shape, and no apparent organ differentiation is observed (Hong et al., 1995). This phenotype is not due to a developmental arrest at the normal globular stage, because gle1 embryos can become larger than wild-type globular embryos and continue to grow until the late stage of embryogenesis. Morphological studies indicate that the GLE1 gene is involved in the overall differentiation of the embryo but not in

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specific organ differentiation. In the gle1 mutant, no expression of OSH1 can be detected in in situ hybridization experiments. This suggests that the GLE1 gene may be involved in developmental events prior to establishment of the shoot region in embryogenesis, and that regions for other organs may also not be established in the gle1 mutant embryo (Fig. 7). 2. shootless1, shootless2 (shl1, shl2) Mutants In these mutants, the SAM is not formed during embryogenesis (Satoh et al., 1999). Two embryo-specific organs, the coleoptile and epiblast, are also lost; thus these two organs may be formed as lateral organs of the SAM. However, almost all of the other organs, such as the radicle and scutellum, develop normally. In the shl mutants, the OSH1 gene is expressed in the ventral region of the globular stage embryo, where the shoot later develops in the wild-type embryo, but the spatial region of OSH1 expression becomes narrower than that of the wildtype embryo. In shoot regeneration experiments, both the shl1 and shl2 mutants fail to differentiate any adventitious shoots, demonstrating that the SHL genes are also indispensable for adventitious shoot formation. The fact that OSH1 expression and radicle/scutellum differentiation occur normally demonstrates that the embryonic axes, such as the apical–basal axis, are not disturbed by the shl mutations, but that only the establishment of a SAM-associated region of appropriate size seems to be disrupted in the mutants. In other words, the SHL1 and SHL2 genes may not be involved in embryonic axis formation but rather may function in the establishment of the SAM-associated region upstream of the OSH1 gene, and regulate the expression of the OSH1 gene (Fig. 7) (Satoh et al., 1999). 3. aberrant regionalization of embryo1 (are1) Mutant In this mutant, two shoots develop simultaneously during embryogenesis and, in most cases, one radicle forms normally between these shoots. Morphogenesis of each shoot and other organs is quite normal, and the are1 mutant can germinate twin seedlings with a normal arrangement of organs based on complete basic patterns, the apical–basal pattern and radial pattern. One Arabidopsis embryonic mutant, twin, also develops two, or sometimes three, embryos during embryogenesis (Vernon and Meinke, 1994), but the Arabidopsis twin mutant differs from the rice are1 mutant for the following reasons. Firstly, an extra embryo develops in tandem by transformation of the cells in the suspensor, resulting in a twin embryo in the Arabidopsis twin mutant, but two shoots develop simultaneously in only one embryo in the rice are1 mutant. Secondly, in the Arabidopsis twin mutant, the extra embryo is viable but has some developmental defects. To date, are1-like mutants have not been identified in Arabidopsis, and twin-like mutants have not been found in rice. This may reflect differences in the developmental processes

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that occur during embryogenesis in monocots and dicots. At the globular stage of the are1 embryo, OSH1 is broadly expressed around the basal region, rather than being restricted to the ventral region as occurs in the wild-type embryo. Although the position and size of the OSH1 expression region are disrupted in the are1 embryo, other developmental events occur normally, including coleoptile and leaf development in each shoot. So, only the establishment of the shoot region in the early globular stage appears to be disrupted in the are1 mutant embryo. These findings suggest that the ARE1 gene is only involved in the localization and establishment of the shoot region in early embryogenesis. As is the case for the apd1 mutant described above, the aberrant position of OSH1 expression in the are1 mutant globular embryo also demonstrates the existence of a specific mechanism for embryonic regionalization in monocots (Fig. 7). 4. Relationship between OSH1 Expression and Shoot Formation Each of the three embryonic mutants described here shows a different pattern of OSH1 expression in the globular stage embryo. In the gle1 embryo, which has no organ differentiation, there is no detectable OSH1 expression. In the shl1 and shl2 mutants, which lack shoot differentiation, the OSH1 expression region is much narrower than that in the wild-type embryo. In the are1 embryo, which forms two shoots, the OSH1 expression region is broader than that in wild-type embryo. According to these observations, the number of shoots that are subsequently formed is related to the size of the OSH1 expression region in the globular stage embryo. Embryos with a narrower expression region than the wild-type do not differentiate to form shoots, and conversely, embryos with a broader expression region differentiate one or more extra shoots. The appropriate size of OSH1 expression region in the correct position should be important for normal shoot formation (Fig. 7).

E. Homeobox Genes and Early Rice Embryogenesis As described above, the expression of OSH (knox) genes can define the shoot region in the early globular rice embryo prior to morphological shoot formation. Similarly, other rice homeobox genes, such as Roc1 and QHB, define the protoderm (epidermis) and quiescent center of the radicle, respectively, prior to organ differentiation. Many kinds of homeobox genes, not only the knox family but also the glabra2 and WUS-type families, define each specific region of the rice early globular embryo prior to actual organ differentiation (Fig. 3). This suggests that, as in animal development, many plant homeobox genes play an important role in pattern formation or regionalization of the embryo during early embryogenesis. All OSH genes roughly define the shoot region, but as in the case of OSH1 and OSH3, for example, precise expression analyses have revealed that subtle

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differences exist in their expression, temporally or spatially. These differences may define more specific areas in shoot region, since this region also includes some organs that differentiate independent of the SAM in rice embryos.

IV. SAM Maintenance and knox Genes after SAM Formation The entire above-ground portion of a plant is an assembly of shoot units termed phytomeres, which consist of an axillary bud, a stem, and a leaf. The SAM continuously produces these units, and at the same time maintains itself as a collection of indeterminate stem cells (Steeves and Sussex, 1989). KNOX HD proteins encoded by knox genes preferentially accumulate in these indeterminate stem cells around the SAM, but not in the determinate lateral organs. Based on these expression patterns, knox genes are thought to be involved in the process of making lateral organs or in the maintenance of stem cells in the SAM (Reiser et al., 2000). In the previous section, we described the involvement of knox genes in the SAM formation during early embryogenesis. In this part of the review, we summarize the functions of the knox genes in plant development, especially after SAM formation, based on analyses of recessive mutations with loss-of-function alleles of the knox genes. To date, four recessive mutant loci of class1 knox genes have been identified. In Arabidopsis, there are two: one is the above-mentioned stm and the other is knat1 (Barton and Poethig, 1993; Long et al., 1996; Douglas et al., 2002; Sato, Y., Ori, N., and Hake, S., submitted). knotted1 and osh15 have also been identified from maize and rice, respectively (Table I) (Kerstetter et al., 1997; Sato et al., 1999; Vollbrecht et al., 2000). Although it is difficult to infer orthologous relationships between genes from monocots and dicots based on the sequence of the knox genes, it is plausible to consider that STM and KN1 are orthologous because of the similarities in both their expression patterns and the phenotypes of recessive mutations. The same relationship is assumed for KNAT1 and OSH15. In the following section, the phenotype of each mutant except for kn1, and the presumed function of each gene is discussed. A. stm Mutant in Arabidopsis Mutations in the STM gene in Arabidopsis cause the loss of the SAM between two cotyledons in embryogenesis (Table I) (Barton and Poethig, 1993; Long et al., 1996). Due to the absence of the SAM, stm plants develop two cotyledons but do not produce lateral organs such as leaves. This phenotype suggests the involvement of STM in SAM formation during embryogenesis and/or the maintenance of the SAM. It is still unclear whether STM is involved in both of these processes or only in one of them. However, the involvement of STM in SAM maintenance

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is more likely, because in the weak allele of stm an incomplete SAM is formed, which produces several abnormal leaves after germination and then depletes the cells in the SAM and stops growing. Analysis of STM expression patterns also support this interpretation, that is, STM is expressed preferentially in undifferentiated cells of the SAM but not in cells which are destined to the differentiated lateral organs. There is also a report showing the involvement of STM in events other than maintenance of the SAM, namely the development of the cotyledon. Cup Shaped Cotyledon genes (CUC1 and CUC2) encode proteins with the NAC domain (Aida et al., 1997). A double mutant of cuc1 and cuc2 results in an abnormal cotyledon with a cup-shaped structure. Also, this mutant lacks a SAM. It is considered that the cup-shaped cotyledon in the cuc double mutant is caused by ectopic bulging of the region between two cotyledons where growth is usually suppressed (Aida et al., 1999). In the stm mutant, the bottom part of the cotyledons is partially fused for the same reason as in the cuc double mutant, and results in a tubular structure. This suggests that both the STM and CUC genes play a role in suppressing the growth of the boundary region of cotyledons. Interestingly, in the cuc double mutant the expression of STM is abolished, and in the stm mutant the spatial expression pattern of cuc2 is disturbed. This indicates that the CUC gene regulates the expression of STM in a positive manner, and STM regulates the region of CUC2 expression. Thus, the development of cotyledons and the maintenance of the SAM may share a common mechanism through the mutual regulatory interaction of CUC and STM.

B. osh15 (d6) Mutant in Rice As mentioned previously, the expression of class I knox genes can be roughly divided into two patterns (Fig. 2, Reiser et al., 2000). The rice knox gene OSH15 is expressed in a ring-shaped pattern beneath the incipient lateral organs in the SAM (Sato et al., 1998). This expression pattern is similar to that of KNAT1 (Lincoln et al., 1994) in Arabidopsis and distinct from that of STM (Long et al., 1996) and KN1 (Jackson et al., 1994), whose expression in the SAM occurs more broadly within the undifferentiated cells. Initially, the function of OSH15 and KNAT1 was unclear because their expression patterns were not as definitive as those of STM and KN1. However, the discovery of a recessive mutation in OSH15 suggests there is a connection between the region in the SAM where OSH15 and KNAT1 are expressed and the development of a stem. The osh15 mutant shows a dwarf phenotype with an abnormal pattern of internode elongation (Table I) (Sato et al., 1999). Histological analyses of the internodes in osh15 have revealed that cortex tissues, which usually occur in the outer part of the internode, are missing and result in the defective internode elongation. Recently, a recessive mutation in KNAT1 in Arabidopsis has also been identified (Douglas et al., 2002; Sato, Y., Ori, N., and Hake, S.,

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submitted). Although the precise analysis of the knat1 mutant phenotype is not yet complete, it is known that this mutant also shows defective internode elongation. This suggests that KNAT1 and OSH15 have a similar function in the SAM.

C. knox Genes Establish Functional Boundaries in the SAM Analyses of the gain-of-function phenotype in the dominant Kn1 (Freeling and Hake, 1985; Vollbrecht et al., 1991) mutation indicate the involvement of KN1 in conferring or maintaining undifferentiated characteristics of cells. Likewise, overexpression studies of KNAT1 (Lincoln et al., 1994; Chuck et al., 1996) and OSH15 (Sato et al., 1998; Sentoku et al., 2000) in transgenic plants also show that they affect the differentiated cells in the leaves of transgenic plants to confer undifferentiated characteristics. In maize, dominant gain-of-function mutations known as Roughsheath1 (Rs1) (Becraft and Freeling, 1994; Schneeberger et al., 1995) and Gnarley1 (Gn1) (Foster et al., 1999a,b), whose cognate genes in the wild-type plant are orthologous to OSH15, also affect the cells in leaves to confer a more undifferentiated state. This characteristic, in which overexpression of these genes results in a change in the developmental state of cells in leaves, appears to be common to the class I knox genes. Why are these genes, which induce similar cellular responses, expressed in SAM? The maintenance of the SAM is achieved through the replenishment of cells in the peripheral zone (PZ), in which the lateral organs are repeatedly initiated, by cells in central zone (CZ), where more slowly dividing cells reside. Vollbrecht et al. (2000) described the function of KN1 and STM in the maintenance of the SAM as follows: The balance between consumption and replenishment of cells in the SAM can be maintained by establishing a boundary of KN1- and STM-expressing cells and nonexpressing cells. This hypothesis would explain the phenotype of stm and kn1, so that cells in the PZ, which are able to differentiate lateral organs, are used up for lateral organs because of a loss of the boundary of KN1- and STMexpressing cells and nonexpressing cells. The same situation may also apply for OSH15 and KNAT1. It is possible that a failure to establish this boundary in the SAM of osh15 and knat1 causes an inappropriate allocation of stem cells, or a precocious termination of stem cells, which are required for the differentiation of cortex tissues in the stem and results in the defective internode elongation. The SAM is composed of several functional domains, including the CZ and PZ. Molecular cloning of genes that regulate the function of the SAM have revealed a further subdivision of functional domains in the SAM. The authors hypothesize that the knox genes may function to establish the boundaries of these complicated functional domains in the SAM. In Arabidopsis, four class I knox genes are present: STM (Long et al., 1996), KNAT1 (Lincoln et al., 1994), KNAT2 (Dockx et al., 1995), and KNAT6 (Hake, S., et al., unpublished data). KNAT2 and KNAT6 could function as redundant factors

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because they share extremely high similarity in their nucleotide and amino acid sequences. To understand the function of these genes, the introduction of mutations in both genes will be required. In maize and rice, there are more knox genes than in Arabidopsis. Some of these genes could function as redundant factors, but others are clearly not redundant. It will be an interesting scenario if the additional knox genes, which do not exist in Arabidopsis, function to establish a boundary in the SAM which is specific to the monocots or grass family. Further analyses of recessive mutant alleles of knox genes will explore these possibilities.

V. Concluding Remarks In plant embryogenesis, the mechanisms for pattern formation and subsequent regionalization are still unknown. As is the case in animals, mutants with defects in pattern formation or regionalization must hold the key to the elucidation of these mechanisms, but it may be difficult to identify such mutants from a range of embryonic mutants. Many of the homeobox genes discussed in this review show some region-specific expression from the very early developmental stage prior to morphological organ differentiation. Therefore, these genes should be good molecular markers to identify mutants which are defective in patterning or regionalization. Indeed, some mutants show abnormal OSH1 expression, and it is expected that the precise analysis of these mutants, including the isolation and characterization of these genes, will lead us to a better understanding of the mechanisms for determination of the shoot region during early rice embryogenesis.

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e f?oc7 (HD-GL2)

globular Matsuoka, Fig. 3

Expression patterns of rice homeobox genes Rod, OSHI, and QHB in the globular embryo. Each gene clearly specifies certain regions in the globular stage embryo prior to organ differentiation, that is, the Rod, OSHI, and QHB genes specify the epidermis, SAM, and quiescent center, respectively.