Molecular markers in Arabidopsis embryos

In seed plants, sexual reproduction is initiated by pollen transfer from anther to stigma. One of the two sperm cells carried by the pollen grain fertilizes the egg cell in the flower's carpel, giving rise to a fertilized egg cell or zygote. The subsequent developmental process that represents the transition of the zygote to a multicellular seedling is termed zygotic embryogenesis. Zygotic embryos develop through a series of characteristic morphological stages, in dicots the globular, heart, torpedo, and bent-cotyledon stages. During this development, all distinct organs and tissues present in the seedling are arranged in their proper positions, a process called pattern formation. Along the apical-basal or longitudinal axis, the pattern consists of the shoot apical meristem, cotyledons (embryonic leaves), hypocotyl (embryonic stem) and radicle (embryonic root), including the root cap and root meristem. Along the radial axis, another pattern is apparent as a concentric arrangement of tissue types from outside to inside: the epidermis, ground tissue, and central vascular system. In the model plant Arabidopsis (wall cress), the sequence of cell divisions during zygotic embryogenesis is highly invariant, so that the origin of seedling organs and tissues appears traceable to specific cells in the early embryo. However, except for the early epidermal cell fate, no clonally transmitted lineages appear to be instrumental in pattern formation. Currently, numerous studies focus on the molecular events underlying plant embryo development. The current stage of this research area is discussed in chapter 1.A widely followed approach to identify genes involved in pattern formation has been to screen for mutants with defects in the establishment of the embryo body plan. These genetic screens have yielded numerous embryo-defective mutants. However, a major difficulty that has emerged during these screens concerns the recognition and interpretation of informative phenotypes. Many different embryo-lethal mutants show quite similar phenotypes and the assessment of the precise effects of a mutation is often hampered by the inability to determine cell- or regional identity in the embryo mutant background. One way to partly circumvent these difficulties is to study the expression pattern of well defined molecular markers in embryo mutants. Markers reflecting cell- or regional identity or polarity in the developing embryo provide criteria other than morphology for the evaluation of the precise effects of an embryo mutation.Chapter 2 describes the analysis of three embryo mutants using the Arabidopsis thaliana lipid transfer protein ( AtLTP1 ) gene as a marker. In wild-type embryos, the AtLTP1 gene is initially expressed in all epidermis cells, and later in the epidermal cells of the cotyledons and upper hypocotyl, together representing the apical part of the embryo. Therefore, AtLTP1 expression was used as tissue-layer specific marker for the epidermis to study the phenotypic defects in the knolle and keule mutants, both reported to have defects in the establishment of the epidermis. AtLTP1 expression was used as marker for the apical part of the embryo to investigate effects of the gnom mutation on apical-basal embryo polarity.Unfortunately, few other embryo marker genes are available to date, especially for the early stages of embryogenesis. This shortage of suitable molecular markers greatly hampers the recognition and interpretation of embryo phenotypes informative for the process of pattern formation. Therefore, we have performed an enhancer and gene trap insertional mutagenesis screen to identify Arabidopsis lines with GUS expression in embryos. This screen is described in chapter 3, and exploits two types of transposable Ds elements each carrying a GUS reporter gene that can respond to cis -acting transcriptional signals at the site of integration. The selected lines provide a set of markers that can be used to determine cell- or regional identity and polarity in Arabidopsis embryo mutants, and will allow the isolation of genes identified on the basis of their expression pattern in the Arabidopsis embryo.Chapter 4 outlines the spectrum of GUS expression patterns observed in embryos during the screening of 431 enhancer trap and 373 gene trap lines. Four lines exhibiting remarkably early or localized GUS expression are described in more detail. Furthermore, electronic databases for the recording of screening data, and sequence analysis of genomic DNA flanking the transposon insertions in four enhancer trap and two gene trap lines are presented. Finally, the efficiency of enhancer and gene trap mutagenesis as a means of identifying genes that are important for embryo development is discussed.Chapter 5 describes the identification of one specific enhancer trap line, WET368, that already shows uniform GUS expression in the 8-celled embryo. Later during embryogenesis, expression becomes restricted to a previously undefined region encompassing the shoot apical meristem and part of the cotyledon primordia. After germination, all aerial plant parts where meristems are or have been present are marked by WET368 GUS expression. Analysis of WET368 GUS expression in different mutants defective in the control of shoot meristem size or function provides an example of the way marker gene expression can extend morphological descriptions of mutant phenotypes.Finally, a summarizing discussion of the research presented in this thesis is provided in chapter 6. The research described in this thesis has given ample support for the value of molecular markers for the recognition and interpretation of mutant phenotypes, relevant to the acquisition of polarity and the establishment of the body pattern during Arabidopsis embryogenesis. The employed enhancer and gene trap mutagenesis system has proven successful towards the isolation of GUS markers for distinct cell- or tissue-types and regions in the developing embryo. These markers can not only be used for the phenotypic analysis of embryo mutants, but can also refine the existing descriptions of plant embryogenesis by demarcating novel regions that have not been identified previously by morphology, histology or function. Besides generating markers, molecular analysis has shown that enhancer and gene traps also allow the isolation of genes identified on the basis of their expression pattern. In both ways, the established collection of enhancer and gene trap lines may contribute to a more comprehensive understanding of the molecular events underlying plant embryogenesis.