Globular Stage Of Embryogenesis Research Articles

Evolution, Initiation, and Diversity in Early Plant Embryogenesis.

During embryogenesis in plants, cell identities are specified de novo, starting from a single cell. By combining imaging, genomic profiling, and genetics, principles of early plant development have been unraveled in the dicotyledonous plant Arabidopsis. A central emerging question, however, is how well zygotic embryogenesis in Arabidopsis reflects homologous processes in other plant species, including early diverging, non-flowering, and non-seed plants. Here, we consider plant embryogenesis with an emphasis on its evolutionary history, the diverse modes of its initiation, and the concepts in pattern formation among morphologically distinct plant groups. Furthermore, we explore challenges and future directions in plant embryogenesis research.

Arabidopsis thaliana miRNAs promote embryo pattern formation beginning in the zygote

miRNAs are essential regulators of cell identity, yet their role in early embryo development in plants remains largely unexplored. To determine the earliest stage at which miRNAs act to promote pattern formation in embryogenesis, we examined a series of mutant alleles in the Arabidopsis thaliana miRNA biogenesis enzymes DICER-LIKE 1 (DCL1), SERRATE (SE), and HYPONASTIC LEAVES 1 (HYL1). Cellular and patterning defects were observed in dcl1, se and hyl1 embryos from the zygote through the globular stage of embryogenesis. To identify miRNAs that are expressed in early embryogenesis, we sequenced mRNAs from globular stage Columbia wild type (wt) and se-1 embryos, and identified transcripts potentially corresponding to 100 miRNA precursors. Considering genome location and transcript increase between wt and se-1, 39 of these MIRNAs are predicted to be bona fide early embryo miRNAs. Among these are conserved miRNAs such as miR156, miR159, miR160, miR161, miR164, miR165, miR166, miR167, miR168, miR171, miR319, miR390 and miR394, as well as miRNAs whose function has never been characterized. Our analysis demonstrates that miRNAs promote pattern formation beginning in the zygote, and provides a comprehensive dataset for functional studies of individual miRNAs in Arabidopsis embryogenesis.

Analysis of chlorophyll fluorescence reveals stage specific patterns of chloroplast-containing cells during Arabidopsis embryogenesis.

The basic body plan of a plant is established early in embryogenesis when cells differentiate, giving rise to the apical and basal regions of the embryo. Using chlorophyll fluorescence as a marker for chloroplasts, we have detected specific patterns of chloroplast-containing cells at specific stages of embryogenesis. Non-randomly distributed chloroplast-containing cells are seen as early as the globular stage of embryogenesis in Arabidopsis. In the heart stage of embryogenesis, chloroplast containing cells are detected in epidermal cells as well as a central region of the heart stage embryo, forming a triangular septum of chloroplast-containing cells that divides the embryo into three equal sectors. Torpedo stage embryos have chloroplast-containing epidermal cells and a central band of chloroplast-containing cells in the cortex layer, just below the shoot apical meristem. In the walking-stick stage of embryogenesis, chloroplasts are present in the epidermal, cortex and endodermal cells. The chloroplasts appear reduced or absent from the provascular and columella cells of walking-stick stage embryos. These results suggest that there is a tight regulation of plastid differentiation during embryogenesis that generates specific patterns of chloroplast-containing cells in specific cell layers at specific stages of embryogenesis.

Analysis of chlorophyll fluorescence reveals stage specific patterns of chloroplast-containing cells during Arabidopsis embryogenesis

The basic body plan of a plant is established early in embryogenesis when cells differentiate, giving rise to the apical and basal regions of the embryo. Using chlorophyll fluorescence as a marker for chloroplasts, we have detected specific patterns of chloroplast-containing cells at specific stages of embryogenesis. Non-randomly distributed chloroplast-containing cells are seen as early as the globular stage of embryogenesis in Arabidopsis. In the heart stage of embryogenesis, chloroplast containing cells are detected in epidermal cells as well as a central region of the heart stage embryo, forming a triangular septum of chloroplast-containing cells that divides the embryo into three equal sectors. Torpedo stage embryos have chloroplast-containing epidermal cells and a central band of chloroplast-containing cells in the cortex layer, just below the shoot apical meristem. In the walking-stick stage of embryogenesis, chloroplasts are present in the epidermal, cortex and endodermal cells. The chloroplasts appear reduced or absent from the provascular and columella cells of walking-stick stage embryos. These results suggest that there is a tight regulation of plastid differentiation during embryogenesis that generates specific patterns of chloroplast-containing cells in specific cell layers at specific stages of embryogenesis.

Auxin and root initiation in somatic embryos of Arabidopsis

Somatic embryos of Arabidopsis thaliana can be produced from explants of developing zygotic embryos. Cultivation of explants on maturation medium results in development of three main classes of regenerative structures: adventitious shoots, fused shoots, and complete somatic embryos. The ontogeny and anatomy of these structures was examined using serial plastic sections. Furthermore, two molecular markers were assayed to monitor transcriptional auxin responses and formation of a root meristem in this process: the LENNY allele of PIN4, a transposon insertion creating a fusion to the reporter gene GUS; and DR5::GUS, a synthetic reporter of auxin-induced transcription. In zygotic embryogenesis, PIN4 expression is confined to the center of the root meristem and begins to be detectable by the globular stage of embryogenesis, while DR5::GUS expression marks an "auxin perception maximum" in the more distal regions of the root. Adventitious and fused shoots develop no anatomically recognizable root meristem and do not express either of the two markers at their basal pole. Instead, the vasculature of their axis is directly connected to the vasculature of the explant. By contrast, complete somatic embryos were only loosely attached to the explant, had an anatomically defined root meristem and showed expression of both markers at their root pole. Our results suggest that the establishment of a root meristem in somatic embryos required appropriate auxin levels during the course of their development.

AtNAP7 is a plastidic SufC-like ATP-binding cassette/ATPase essential for Arabidopsis embryogenesis

In bacteria, yeast, and mammals, iron-sulfur (Fe-S) cluster-containing proteins are involved in numerous processes including electron transfer, metabolic reactions, sensing, signaling, and regulation of gene expression. In humans, iron-storage diseases such as X-linked sideroblastic anemia and ataxia are caused by defects in Fe-S cluster availability. The biogenesis of Fe-S clusters involves several pathways, and in bacteria, the SufABCDSE operon has been shown to play a vital role in Fe-S biogenesis and repair during oxidative stress. Although Fe-S proteins play vital roles in plants, Fe-S cluster biogenesis and maintenance and physiological consequences of dysfunctional Fe-S cluster assembly remains obscure. Here we report that Arabidopsis plants deficient for the SufC homolog AtNAP7 show lethality at the globular stage of embryogenesis. AtNAP7 is expressed in developing embryos and in apical, root, and floral meristems and encodes an ATP-binding cassette/ATPase that can partially rescue growth defects in an Escherichia coli SufC mutant during oxidative stress. AtNAP7 is plastid-localized, and mutant embryos contain abnormal developing plastids with disorganized thylakoid structures. We found that AtNAP7 can interact with AtNAP6, a plastidic Arabidopsis SufD homolog, and because Arabidopsis plastids also harbor SufA, SufB, SufS, and SufE homologs, plastids probably contain a complete SUF system. Our results imply that AtNAP7 represents a conserved SufC protein involved in the biogenesis and/or repair of oxidatively damaged Fe-S clusters and suggest an important role for plastidic Fe-S cluster maintenance and repair during Arabidopsis embryogenesis.

Tri-iodobenzoic acid affects shoot apical meristem formation and function in zygotic embryos ofBrassica napuscv. Topas

The effect of an auxin transport inhibitor, tri-iodobenzoic acid, on the formation and subsequent function of the shoot apical meristem in zygotic embryos of Brassica napus L. was examined. Globular and heart stage embryos were cultured in the presence of tri-iodobenzoic acid. Only embryos at the globular stage of embryogenesis were affected by tri-iodobenzoic acid. Upon treatment, the embryos did not develop two separate cotyledons and, therefore, did not attain bilateral symmetry. Furthermore, the shoot apical meristem of these embryos was altered, as a characteristic tunica-corpus organization was not observed. The cells of the surface layer continued to maintain their meristematic characteristics and divided mainly in the anticlinal direction. The subapical cells differentiated into parenchyma cells and continued to expand such that a corpus organization never developed. Therefore, by the end of the culture period, the shoot apical meristem appeared very broad and shallow. Treated embryos that were transferred to a conversion medium exhibited a dramatic decline and delay in the formation of leaves compared with untreated embryos. Histological observations revealed that the shoot apical meristem continued to expand in width. A low percentage of treated embryos were able to convert; however, leaves were produced from a new shoot meristem that developed in the axillary position. No morphological or histological changes were evident when the embryos were treated later, at the heart stage of embryogenesis.Key words: auxin, Brassica napus, embryo, shoot apical meristem, tri-iodobenzoic acid.

Expression of a globulin-like protein gene, Gea8, in somatic and zygotic embryos

A carrot gene, Gea8 (Globular embryo abundant), has been identified that encodes a novel seed protein that is abundantly expressed in embryos from the globular to the cotyledon stages and in endosperm of maturation-stage seeds. Analysis of the Gea8 gene revealed that it encodes a protein with approximately 47% sequence similarity (25% sequence identity) to both dicot and monocot globulins, a class of seed storage proteins. However, the Gea8 promoter region lacks some of the key regulatory elements characteristic of other known globulins, especially the 'G box' sequence. Analysis of Gea8 mRNA accumulation during both somatic and zygotic embryogenesis showed that mRNA for this gene accumulates by the globular stage of embryogenesis and reaches a high abundance by the heart stage that is sustained throughout embryo development. Expression is also high in the developing endosperm of seeds of both carrot and Arabidopsis. In addition, analysis of the developmental and spatial expression of the Gea8 gene, using several Gea8 promoter/GUS fusion constructs, suggests that the promoter contains at least two separable regions directing the spatial accumulation of Gea8 mRNA in the heart and torpedo stages of embryogenesis. These studies suggest that Gea8 is a new form of seed protein that is expressed early in embryo development, initially in an embryo-specific pattern, and subsequently in both the embryo and the endosperm through regulation by a complex promoter.

Genes Encoding 11S-Globulin-Like Proteins are Expressed in the Megagametophyte soon after Fertilization in Eastern White Pine (Pinus strobus L.)

The nucleotide sequence of two cDNA clones synthesized from eastern white pine (Pinus strobus L.) ovule mRNA showed significant sequence homology to the legumins, which correspond to the 11S-globulins of several angiosperms. Northern hybridizations with one of the cDNA clones, named Glb-2 (P. strobus 11S-globulin-like clone 2), revealed that the mRNA was present in the ovules from the early globular stage of embryogenesis through the mature seed stage but not in unfertilized ovules. Glb mRNA was found in the megagametophyte of ovules containing late-stage globular embryos by filter-bound RNA and tissue print hybridization experiments. No Glb mRNA was found in in vitro-cultured globular stage embryos with attached suspensors, sporophytic ovule tissues, whole seedlings, or mature needles.

Late embryo‐defective mutants of Arabidopsis

AbstractThe embryo‐defective (emb) mutants of Arabidopsis constitute a large and diverse group of mutants disrupted in a broad range of embryonic processes, including morphogonesis, cell differentiation, and maturation programs. This report describes a subset of these mutants, the late embryo defectives, which develop beyond the globular stage of embryogenesis but fail to complete normal morphogenesis. A representative sample of 12 late mutants was chosen for this study, patterns of morphogenesis were characterized, the germination potential of mutant seeds was investigated, and additional mutant alleles within the collection were identified. Morphological defects in mutant embryos became apparent during the heart stage of development, when embryos normally begin the rapid cell division and expansion required for the completion of morphogenesis. Despite their morphological abnormalities, mutant embryos often germinated from dry seed, demonstrating that genetic programs required for the establishment of desiccation tolerance remained intact. Mutant seedlings displayed a wide range of developmental abnormalities, including altered morphology, lack of pigmentation, dwarfism, and disorganized vegetative growth. One late mutant was found to be allelic to an early embryo defective that arrests at the globular stage. These results suggest that a number of late EMB genes encode basic cellular and metabolic functions needed for cell division, enlargement, and embryonic growth. The rapid growth and metabolic changes that occur at the heart stage may present a barrier to normal development in the late mutants, resulting in altered embryo morphology and other developmental defects. It is proposed that many Arabidopsis mutants with abnormal embryo and seedling morphology are not defective in the regulation of pattern formation or morphogenesis, but rather in fundamental physiological and cellular processes required for the completion of normal growth and development. © 1995 Wiley‐Liss, Inc.

A carrot cell variant temperature sensitive for somatic embryogenesis reveals a defect in the glycosylation of extracellular proteins

The temperature-sensitive carrot cell variant ts11c, arrested in somatic embryogenesis after the globular stage, was characterized. The sensitivity to a shift from 24 degrees C (permissive temperature) to 32 degrees C (non-permissive temperature) is greatest at the globular stage of embryogenesis, while cells proliferating in unorganized fashion and plantlets are not affected. Embryogenesis in ts11c is also arrested at the permissive temperature by replacement of conditioned culture medium with fresh medium. The timing of sensitivity of ts11c to medium replacement coincides with the sensitivity to temperature shift. Both sensitivities are recessive in somatic hybrids between ts11c and wild-type cells. Extracellular glycoproteins synthesized by ts11c at the non-permissive temperature contain much less fucose than those synthesized by the wild type. The glycoproteins synthesized by the variant under non-permissive conditions do not accumulate at the periphery of the embryo, as their wild-type counterparts do, but instead show a diffuse distribution throughout the embryo. The defect in ts11c can be fully complemented by the addition of extracellular wild-type proteins. A revertant of ts11c was isolated that simultaneously reacquired temperature insensitivity and normal glycosylation ability. Collectively, these observations indicate that ts11c is not able to perform proper glycosylation at the non-permissive temperature and suggest that the activity of certain extracellular proteins, essential for the transition of globular to heart stage somatic embryos, depends on the correct modification of their oligosaccharide side-chains.