The molecular characterization of PaHB2, a homeobox gene of the HD-GL2 family expressed during embryo development in Norway spruce

PaHB1 (for Picea abies Homeobox1), an evolutionarily conserved HD-GL2 homeobox gene, specifically expressed in the protoderm during somatic embryogenesis in the gymnosperm Norway spruce has been reported previously. An additional HD-GL2 gene designated PaHB2 is reported here. During somatic embryogenesis, the PaHB2 gene is uniformly ex pressed in proembryogenic masses and in early somatic embryos, but it is not detectably transcribed at the beginning of maturation. In mature embryos, PaHB2 expression was essentially detected in the outermost layer of the cortex and the root cap. A similar PaHB2 expression is detected post-embryonically in both the primary root and the hypocotyl. Phylogenetic reconstructions and intron pattern analyses revealed that the PAHB proteins fall within two distinct subclasses comprising highly similar angiosperm homologues. The PAHB1 subclass consists of protoderm/epiderm-specific members. By contrast, the PAHB2 subclass gathers homologues with a subepidermal and protodermal/epidermal activity. This study suggests that at least two distinct HD-GL2 genes with a layer-specific expression already existed in the last common ancestor of angiosperms and gymnosperms. The conserved protodermal/epidermal and subepidermal expression of HD-GL2 genes could be used to study embryo radial pattern formation across seed plants.     

Pattern Formation in the Arabidopsis Embryo Revealed by Position-Specific Lipid Transfer Protein Gene Expression

During Arabidopsis embryogenesis, the zygote divides asymmetrically in the future apical-basal axis; however, a radial axis is initiated only within the eight-celled embryo. Mutations in the GNOM, KNOLLE, and KEULE genes affect these processes: gnom zygotes tend to divide symmetrically; knolle embryos lack oriented cell divisions that initiate protoderm formation; and in keule embryos, an outer cell layer is present that consists of abnormally enlarged cells from early development. Pattern formation along the two axes is reflected by the position-specific expression of the Arabidopsis lipid transfer protein (AtLTP1) gene. In wild-type embryos, the AtLTP1 gene is expressed in the protoderm and initially in all protodermal cells; later, AtLTP1 expression is confined to the cotyledons and the upper end of the hypocotyl. Analysis of AtLTP1 expression in gnom, knolle, and keule embryos showed that gnom embryos also can have no or reversed apical-basal polarity, whereas radial polarity is unaffected. knolle embryos initially lack but eventually form a radial pattern, and keule embryos are affected in protoderm cell morphology rather than in the establishment of the radial pattern.     

Cloning and characterization of an L1 layer-specific gene in Arabidopsis thaliana.

The shoot apical meristem (SAM) of Arabidopsis thaliana constitutes the tunica of L1 and L2 and the corpus represented by L3 cells. Regulatory networks involved in establishing and maintaining this structure of shoot meristems remain largely unknown. In order to identify the genes that function in the SAM, we performed cDNA subtraction experiments between wild-type and terminal flower1 shoot apices. Here, we describe the cloning of a gene designated PDF1 (PROTODERMAL FACTOR1). In situ hybridization revealed that the expression of PDF1 is exclusively limited to the L1 layer of vegetative, inflorescence and floral meristems and to the protoderm of organ primordia. By contrast, PDF1 shows no detectable level of expression in the epidermis of mature organs. Specific expression of the PDF1 gene in protodermal cells is also observed during embryogenesis. The deduced amino acid sequence of PDF1 shares no significant homology with that of other known proteins but contains a putative signal peptide and novel proline-rich repeat motifs, suggesting a cell-wall protein. Possible roles of the PDF1 gene in the SAM are discussed.     

Auxin-induced WUS expression is essential for embryonic stem cell renewal during somatic embryogenesis in Arabidopsis

Somatic embryogenesis requires auxin and establishment of the shoot apical meristem (SAM). WUSCHEL ( WUS ) is critical for stem cell fate determination in the SAM of higher plants. However, regulation of WUS expression by auxin during somatic embryogenesis is poorly understood. Here, we show that expression of several regulatory genes important in zygotic embryogenesis were up-regulated during somatic embryogenesis of Arabidopsis. Interestingly, WUS expression was induced within the embryonic callus at a time when somatic embryos could not be identified morphologically or molecularly. Correct WUS expression, regulated by a defined critical level of exogenous auxin, is essential for somatic embryo induction. Furthermore, it was found that auxin gradients were established in specific regions that could then give rise to somatic embryos. The establishment of auxin gradients was correlated with the induced WUS expression. Moreover, the auxin gradients appear to activate PIN1 polar localization within the embryonic callus. Polarized PIN1 is probably responsible for the observed polar auxin transport and auxin accumulation in the SAM and somatic embryo. Suppression of WUS and PIN1 indicated that both genes are necessary for embryo induction through their regulation of downstream gene expression. Our results reveal that establishment of auxin gradients and PIN1-mediated polar auxin transport are essential for WUS induction and somatic embryogenesis. This study sheds new light on how auxin regulates stem cell formation during somatic embryogenesis.     

Nucleostemin-like 1 is required for embryogenesis and leaf development in Arabidopsis

Arabidopsis NSN1 encodes a nucleolar GTP-binding protein and is required for flower development. Defective flowers were formed in heterozygous nsn1/+?plants. Homozygous nsn1 plants were dwarf and exhibited severe defects in reproduction. Arrests in embryo development in nsn1 could occur at any stage of embryogenesis. Cotyledon initiation and development during embryogenesis were distorted in nsn1 plants. At the seedling stage, cotyledons and leaves of nsn1 formed upward curls. The curled leaves developed meristem-like outgrowths or hyperplasia tissues in the adaxial epidermis. Long and enlarged pavement cells, characteristic of the abaxial epidermis of wild type plants, were found in the adaxial epidermis in nsn1 leaves, suggesting a disoriented leaf polarity in the mutant. The important role of NSN1 in embryo development and leaf differentiation was consistent with the high level expression of the NSN1 gene in the developing embryos and the primordia of cotyledons and leaves. The CLAVATA 3 (CLV3) gene, a stem cell marker in the Arabidopsis shoot apical meristem (SAM), was expressed in expanded regions surrounding the SAM of nsn1 plants, and induced ectopically in the meristem-like outgrowths in cotyledons and leaves. The nsn1 mutation up-regulated the expression levels of several genes implicated in the meristem identity and the abaxial cell fate, and repressed the expression of other genes related to the specification of cotyledon boundary and abaxial identity. These results demonstrate that NSN1 represents a novel GTPase required for embryogenesis, leaf development and leaf polarity establishment in Arabidopsis.     

Xcl1 causes delayed oblique periclinal cell divisions in developing maize leaves, leading to cellular differentiation by lineage instead of position

Differentiation of plant cells is regulated by position-dependent mechanisms rather than lineage. The maize Extra cell layers1 (Xcl1) mutation causes oblique, periclinal divisions to occur in the protoderm layer. These protodermal periclinal divisions occur at the expense of normal anticlinal divisions and cause the production of extra cell layers with epidermal characteristics, indicating that cells are differentiating according to lineage instead of position. Mutant kernels have several aleurone layers instead of one, indicating that Xcl1 alters cell division orientation in cells that divide predominantly in the anticlinal plane. Dosage analysis of Xcl1 reveals that the mutant phenotype is caused by overproduction of a normal gene product. This allows cells that have already received differentiation signals to continue to divide in aberrant planes and suggests that the timing of cell division determines differentiation. Cells that divide early and in the absence of differentiation signals use positional information, while cells that divide late after perceiving differentiation signals use lineage information instead of position.     

Two receptor-like kinases required together for the establishment of Arabidopsis cotyledon primordia

Inter-regional signaling coordinates pattern formation in Arabidopsis thaliana embryos. However, little is known regarding the cells and molecules involved in inter-regional communication. We have characterized two related leucine-rich repeat receptor-like kinases (LRR-RLKs), RECEPTOR-LIKE PROTEIN KINASE1 (RPK1) and TOADSTOOL2 (TOAD2), which are required together for patterning the apical embryonic domain cell types that generate cotyledon primordia. Central domain protoderm patterning defects were always observed subjacent to the defective cotyledon primordia cell types in mutant embryos. In addition, RPK1-GFP and TOAD2-GFP translational fusions were both localized to the central domain protodermal cells when cotyledon primordia were first recognizable. We propose that RPK1 and TOAD2 are primarily required to maintain central domain protoderm cell fate and that the loss of this key embryonic cell type in mutant embryos results in patterning defects in other regions of the embryo including the failure to initiate cotyledon primordia.     

Rice globular embryo 4 (gle4) mutant is defective in radial pattern formation during embryogenesis

In higher plants, the main elements of the fundamental body plan, the apical-basal and radial patterns, are established during embryogenesis. We have isolated several globular embryo (gle) mutants of rice that fail to develop any embryonic organs. We expected that these gle mutants might include mutants defective in their radial pattern formation ability. We developed two markers specifically staining the L2 and L3 layers (OsSCR and OsPNH1, respectively) and characterized the gle mutants by using these markers in addition to the already developed markers Roc1 (marker for the L1 layer), Ramy1A (marker of the L1 layer of the epithelium), and OSH1 (marker of the apical region). One of the gle mutants, gle4, expressed Roc1 and Ramy1A at the normal positions, but other markers exhibited an abnormal expression pattern; that is, both OsPNH1 and OsSCR were expressed in the central region of the embryo and OSH1 expression was not observed. Calli from the gle4 epithelium regenerated plants with abnormal morphologies. These results indicate that the GLE4 gene is involved in radial pattern formation during rice embryogenesis to differentiate the L2 and L3 layers, but is not involved in the establishment of the L1 layer or in the formation of embryonic organs.     

Tornado1 and tornado2 are required for the specification of radial and circumferential pattern in the Arabidopsis root

The cell layers of the Arabidopsis primary root are arranged in a simple radial pattern. The outermost layer is the lateral root cap and lies outside the epidermis that surrounds the ground tissue. The files of epidermal and lateral root cap cells converge on a ring of initials (lateral root cap/epidermis initial) from which the epidermal and lateral root cap tissues of the seedling are derived, once root growth is initiated after germination. Each initial gives rise to a clone of epidermal cells and a clone of lateral root cap cells. These initial divisions in the epidermal/lateral root cap initial are defective in tornado1 (trn1) and trn2 plants indicating a requirement for TRN1 and TRN2 for initial cell function. Furthermore, lateral root cap cells develop in the epidermal position in trn1 and trn2 roots indicating that TRN1 and TRN2 are required for the maintenance of the radial pattern of cell specification in the root. The death of these ectopic lateral root cap cells in the elongation zone (where lateral root cap cells normally die) results in the development of gaps in the epidermis. These observations indicate that TRN1 and TRN2 are required to maintain the distinction between the lateral root cap and epidermis and suggest that lateral root cap fate is the default state. It also suggests that TRN1 and TRN2 repress lateral root cap fate in cells in the epidermal location. Furthermore, the position-dependent pattern of root hair and non-root hair cell differentiation in the epidermis is defective in trn1 and trn2 mutants. Together these results indicate that TRN1 and TRN2 are required for the maintenance of both the radial pattern of tissue differentiation in the root and for the subsequent circumferential pattern within the epidermis.     

Forced expression of the homeobox-containing gene Pem blocks differentiation of embryonic stem cells.

Similarities in the differentiation of mouse embryos and ES cell embryoid bodies suggest that aspects of early mammalian embryogenesis can be studied in ES cell embryoid bodies. In an effort to understand the regulation of cellular differentiation during early mouse embryogenesis, we altered the expression of the Pem homeobox-containing gene in ES cells. Pem is normally expressed in the preimplantation embryo and expressed in a lineage-restricted fashion following implantation, suggesting a role for Pem in regulating cellular differentiation in the early embryo. Here, we show that the forced expression of Pem from the mouse Pgk-1 promoter in ES cells blocks the in vitro and in vivo differentiation of the cells. In particular, embryoid bodies produced from these Pgk-Pem ES cells do not differentiate into primitive endoderm or embryonic ectoderm, which are prominent features of early embryoid bodies from normal ES cells. This Pgk-Pem phenotype is also different from the null phenotype, as embryoid bodies derived from ES cells in which endogenous Pem gene expression has been blocked show a pattern of differentiation similar to that of normal ES cells. When the Pgk-Pem ES cells were introduced into subcutaneous sites of nude mice, only undifferentiated EC-like cells were found in the teratomas derived from the injected cells. The Pem-dependent block of ES cell differentiation appears to be cell autonomous; Pgk-Pem ES cells did not differentiate when mixed with normal, differentiating ES cells. A block to ES cell differentiation, resulting from the forced expression of Pem, can also be produced by the forced expression of the nonhomeodomain region of Pem. These studies are consistent with a role for Pem in regulating the transition between undifferentiated and differentiated cells of the early mouse embryo.     

PaHB1 is an evolutionary conserved HD-GL2 homeobox gene expressed in the protoderm during Norway spruce embryo development

In angiosperms, the protoderm or outer cell layer is the first tissue to differentiate in the embryo proper. In gymnosperms, it is not known whether a protoderm is defined and similarly differentiated. Here, we report a cDNA designated PaHB1 (for Picea abies Homeobox1), which is expressed during somatic embryogenesis in Norway spruce. PaHB1 exon/intron organization and its corresponding protein are highly similar to those of the HD-GL2 angiosperm counterparts. A phylogenetic analysis reveals that PaHB1 is strongly associated with one subclass consisting of protoderm/epiderm-specific genes. Moreover, PaHB1 expression switches from a ubiquitous expression in proembryogenic masses to an outer cell layer-specific localization during somatic embryo development. Ectopic expression of PaHB1 in somatic embryos leads to an early developmental block. The transformed embryos lack a smooth surface. These findings show that the PaHB1 expression pattern is highly analogous to angiosperm HD-GL2 homologues, suggesting similarities in the definition of the outer cell layer in seed plants.     

The ACR4 receptor-like kinase is required for surface formation of epidermis-related tissues in Arabidopsis thaliana

In higher plants, an outer layer of meristematic cells, the protoderm, forms early in embryogenesis and this layer gives rise to the epidermis in differentiating tissues. We proposed previously that an Arabidopsis thaliana homolog of crinkly4 (ACR4), a gene for a receptor-like protein kinase, would be involved in differentiation and/or maintenance of epidermis-related tissues. In the present study, we isolated loss-of-function acr4 mutants by a reverse genetic approach. Our extensive analyses using the transmission electron microscopy and the toluidine blue test -- a method that has recently been developed for the rapid visualization of defects in the leaf cuticle -- showed that the acr4 mutations significantly affected the differentiation of leaf epidermal cells, suggesting similar roles for ACR4 and CR4 in the differentiation of leaf epidermis. Our acr4 mutants also had various abnormalities related to epidermal differentiation, which included disorganized cell layers in the integument and endothelium of ovules. In addition, the green fluorescent protein fused to ACR4 was localized preferentially on the lateral and basal plasma membranes in the epidermis of the leaf primordia, suggesting a role for ACR4 in epidermal differentiation at cell surfaces that make contact with adjacent cells. Furthermore, the loss-of-function mutations in the ACR4 and ABNORMAL LEAF SHAPE1 (ALE1) genes, which encode a putative subtilisin-like serine protease, synergistically affected the function of the epidermis such that most leaves fused. Thus, ACR4 seems to play an essential role in the differentiation of proper epidermal cells in both vegetative and reproductive tissues.