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.     

Position dependent expression of GL2-type homeobox gene, Roc1: significance for protoderm differentiation and radial pattern formation in early rice embryogenesis

In early plant embryogenesis, the determination of cell fate in the protodermal cell layer is considered to be the earliest event in radial pattern formation. To elucidate the mechanisms of epidermal cell fate determination and radial pattern formation in early rice embryogenesis, we have isolated a GL2-type homeobox gene Roc1 (Rice outermost cell-specific gene1), which is specifically expressed in the protoderm (epidermis). In early 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 microm in length. Nonetheless, in situ hybridization analyses revealed that specific expression of Roc1 in the outermost cells is established shortly after fertilization, much earlier than protoderm differentiation. In the regeneration process from callus, the Roc1 gene is also expressed in the outermost cells of callus in advance of tissue and organ differentiation, and occurs independently of whether the cells will differentiate into epidermis in the future or not. Furthermore, this cell-specific Roc1 expression could be induced flexibly in the newly produced outermost cells when we cut the callus. These findings suggest that the expression of Roc1 in the outermost cells may be dependent on the positional information of cells in the embryo or callus prior to the cell fate determination of the protoderm (epidermis). Furthermore, the Roc1 expression is downregulated in the inner cells of ligule, which have previously been determined as protodermal cells, also suggesting that the Roc1 expression is position dependent and that this position dependent Roc1 expression is important also in post-embryonic protoderm (epidermis) differentiation.     

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.     

Propagation of Norway spruce via somatic embryogenesis

Somatic embryogenesis combined with cryopreservation is an attractive method to propagate Norway spruce (Picea abies) vegetatively both as a tool in the breeding programme and for large-scale clonal propagation of elite material. Somatic embryos are also a valuable tool for studying regulation of embryo development. Embryogenic cell lines of Norway spruce are established from zygotic embryos. The cell lines proliferate as proembryogenic masses (PEMs). Somatic embryos develop from PEMs. PEM-to-somatic embryo transition is a key developmental switch that determines the yield and quality of mature somatic embryos. Withdrawal of plant growth regulators (PGRs) stimulates PEM-to-somatic embryo transition accompanied by programmed cell death (PCD) in PEMs. This PCD is mediated by a marked decrease in extracellular pH. If the acidification is abolished by buffering the culture medium, PEM-to-somatic embryo transition together with PCD is inhibited. Cell death, induced by withdrawal of PGRs, can be suppressed by extra supply of lipo-chitooligosaccharides (LCOs). Extracellular chitinases are probably involved in production and degradation of LCOs. During early embryogeny, the embryos form an embryonal mass surrounded by a surface layer. The formation of a surface layer is accompanied by a switch in the expression pattern of an Ltp-like gene (Pa18) and a homeobox gene (PaHB1), from ubiquitous expression in PEMs to surface layer-specific in somatic embryos. Ectopic expression of Pa18 and PaHB1 leads to an early developmental block. Transgenic embryos and plants of Norway spruce are routinely produced by using a biolistic approach. The transgenic material is used for studying the importance of specific genes for regulating plant development, but transgenic plants can also be used for identification of candidate genes for use in the breeding programme.     

Overlapping expression pattern of the actin organizers Spir-1 and formin-2 in the developing mouse nervous system and the adult brain

The Wiskott-Aldrich homology domain 2 (WH2) family protein Spir and the formin Cappuccino belong to two distinct classes of actin organizers. Despite their functional classification as actin organizers, a major defect of Drosophila spire and cappuccino mutant oocytes is a failure in the orientation of microtubule plus ends towards the posterior pole. Mammalian homologues of spire are the spir-1 and spir-2 genes. The mouse and human formin-1 and formin-2 genes have high similarity to the cappuccino gene. The mouse formin-2 gene has been found to be expressed in the developing nervous system and in neuronal cells of the adult brain. By analyzing the expression of the spir-1 gene we show that spir-1 and formin-2 have a nearly identical expression pattern during mouse embryogenesis and in the adult brain. In mouse embryos both genes are expressed in the developing nervous system. In the adult brain high expression of the genes was found in the Purkinje cells of the cerebellum and in neuronal cells of the hippocampus and dentate gyrus.     

Developmental pattern formation of somatic embryos induced in cell suspension cultures of cowpea [Vigna unguiculata (L.) Walp]

The sequence of events in the functional body pattern formation during the somatic embryo development in cowpea suspensions is described under three heads. Early stages of somatic embryogenesis were characterized by both periclinal and anticlinal cell divisions. Differentiation of the protoderm cell layer by periclinal divisions marked the commencement of somatic embryogenesis. The most critical events appear to be the formation of apical meristems, establishment of apical-basal patterns of symmetry, and cellular organization in oblong-stage somatic embryo for the transition to torpedo and cotyledonary-stage somatic embryos. Two different stages of mature embryos showing distinct morphology, classified based on the number of cotyledons and their ability to convert into plantlets, were visualized. Repeated mitotic divisions of the sub-epidermal cell layers marked the induction of proembryogenic mass (PEM) in the embryogenic calli. The first division plane was periclinally-oriented, the second anticlinally-oriented, and the subsequent division planes appeared in any direction, leading to clusters of proembryogenic clumps. Differentiation of the protoderm layer marks the beginning of the structural differentiation in globular stage. Incipient procambium formation is the first sign of somatic embryo transition. Axial elongation of inner isodiametric cells of the globular somatic embryo followed by the change in the growth axis of the procambium is an important event in oblong-stage somatic embryo. Vacuolation in the ground meristem of torpedo-stage embryo begins the process of histodifferentiation. Three major embryonic tissue systems; shoot apical meristem, root apical meristem, and the differentiation of procambial strands, are visible in torpedo-stage somatic embryo. Monocotyledonary-stage somatic embryo induced both the shoot apical meristem and two leaf primordia compared to the ansiocotyledonary somatic embryo.     

Histological analysis of direct somatic embryogenesis in <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis> (L.) Heynh

In Arabidopsis the in vitro culture of immature zygotic embryos (IZEs) at a late stage of development, on the solid medium containing synthetic auxin, leads to formation of somatic embryos via direct somatic embryogenesis (DSE). The presented results provide evidence that in IZE cells competent for DSE are located in the protodermis and subprotodermis of the adaxial side of cotyledons and somatic embryos displayed a single- or multicellular origin. Transgenic Arabidopsis lines expressing the GUS reporter gene, driven by the DR5 and LEC2 promoters, were used to analyse the distribution of auxin to mark embryogenic cells in cultured explants and develop somatic embryos. The analysis showed that at the start of the culture auxin was accumulated in all explant tissues, but from the fourth day onwards its location shifted to the protodermis and subprotodermis of the explant cotyledons. In globular somatic embryos auxin was detected in all cells, with a higher concentration in the protodermis, and in the heart stage its activity was mainly displayed in the shoot, root pole and cotyledon primordia. The embryogenic nature of dividing protodermal and subprotodermal cells accumulating auxin was confirmed by high expression of promoter activity of LEC2 in these cells. Analysis of symplasmic tracer (CFDA) distribution indicated symplasmic isolation between tissues engaged in DSE and other parts of an explant. Symplasmic isolation of somatic embryos from the explant was also detected.     

Expression of the viviparous 1 (Pavp1) and p34cdc2 protein kinase (cdc2Pa) genes during somatic embryogenesis in Norway spruce (Picea abies [L.] Karst)

Detailed expression analysis of the Norway spruce (Picea abies [L.] Karst) Viviparous 1 (Pavp1) and p34cdc2 (cdc2Pa) genes was carried out during somatic embryogenesis. Pavp1, a gene associated with embryo development, was expressed in proliferating embryogenic suspension cultures in the absence of exogenous ABA. When somatic embryo formation was promoting by blocking proliferation, Pavp1 expression was reduced. During maturation, exogenous ABA induced increased Pavp1 expression, which peaked at the early cotyledonary stage of somatic embryogenesis. Following partial desiccation of mature somatic embryos at high relative humidity, Pavp1 expression persisted under germination conditions. Pavp1 expression was also detected in non-dormant immature male strobili and dormant terminal buds. These data confirm the functional conservation of Pavp1 during the evolution of seed plants and extend its function beyond the embryo. Cdc2Pa, a gene associated with the cell cycle, was up-regulated when the proliferation of embryogenic cells was blocked. Expression was again up-regulated in early embryogeny and again during germination. The implications of this up-regulation of cdc2Pa are discussed.     

A carrot G-box binding factor-type basic region/leucine zipper factor DcBZ1 is involved in abscisic acid signal transduction in somatic embryogenesis.

Carrot (Daucus carota) somatic embryogenesis has been extensively used as an experimental system for studying embryogenesis. In maturing zygotic embryos, abscisic acid (ABA) is involved in acquisition of desiccation tolerance and dormancy. On the other hand, somatic embryos contain low levels of endogenous ABA and show desiccation intolerance and lack dormancy, but tolerance and dormancy can be induced by exogenous application of ABA. In ABA-treated carrot embryos, some ABA-inducible genes are expressed. We isolated the Daucus carota bZIP1 (DcBZ1) gene encoding a G-box binding factor-type basic region/leucine zipper (GBF-type bZIP) factor from carrot somatic embryos. The expression of DcBZ1 was detected in embryogenic cells, non-embryogenic cells, somatic embryos, developing seeds, seedlings, and true leaves. Notably, higher expression was detected in embryogenic cells, true leaves, and seedlings. The expression of DcBZ1 increased in seedlings and true leaves after ABA treatment, whereas expression was not affected by differences in light conditions. During the development of zygotic and somatic embryos, increased expression of DcBZ1 was commonly detected in the later phase of development. The recombinant DcBZ1 protein showed specific binding activity to the two ABA-responsive element-like motifs (motif X and motif Y) in the promoter region of the carrot ABA-inducible gene according to results from an electrophoretic mobility shift assay. Our findings suggest that the carrot GBF-type bZIP factor, DcBZ1, is involved in ABA signal transduction in embryogenesis and other vegetative tissues.     

Two SERK genes are markers of pluripotency in Cyclamen persicum Mill

The genetic basis of stem cell specification in somatic embryogenesis and organogenesis is still obscure. SOMATIC EMBRYOGENESIS RECEPTOR-LIKE KINASE (SERK) genes are involved in embryogenesis and organogenesis in numerous species. In vitro culture of Cyclamen persicum immature ovules provides a system for investigating stem cell formation and maintenance, because lines forming either organs or embryos or callus without organs/embryos are available for the same cultivar and plant growth regulator conditions. The present aim was to exploit this property of cyclamen cultures to understand the role of SERK(s) in stem cell formation and maintenance in somatic embryogenesis and organogenesis in vitro, in comparison with expression in planta. CpSERK1 and CpSERK2 were isolated from embryogenic callus. CpSERK1 and CpSERK2 levels by RT-PCR showed that expression is high in embryogenic, moderate in organogenic, and null in recalcitrant calli. in situ hybridizations showed that the expression of both genes started in clumps of pluripotent stem cells, from which both pre-embryogenic aggregates and organ meristemoids derived, and continued in their trans-amplifying, meristem-like, derivatives. Expression declined in organ meristemoids, in parallel with a partial loss of meristematization. In mature somatic embryos, and in shoot and root primordia, CpSERK1 and CpSERK2 were expressed in meristems, and similar patterns occurred in zygotic embryo and primary meristems in planta. The results point to SERK1 and SERK2 as markers of pluripotency in cyclamen. It is proposed that the high expression of these genes in the trans-amplifying derivatives of the stem cells maintains a pluripotent condition leading to totipotency and, consequently, somatic embryogenesis.