Embryogenesis and Cytokinesis in Arabidopsis thaliana

Group leaders: GerdJürgens and Ulrike Mayer

In the life cycle of a flowering plant, embryogenesis establishes the multicellular organisation from a single cell, the fertilised egg. Our research focuses on molecular and cellular mechanisms underlying plant embryogenesis. Since the genetic approach is a very powerful analytical tool we use the model system Arabidopsis thaliana (wall cress) which offers several experimental advantages for genetics, such as small size, fast development and a compact diploid genome.

Pattern formation in the Arabidopsis embryo

Embryonic pattern formation generates the basic body organisation of flowering plants, as expressed in the seedling (Jürgens, 1995). The seedling harbors, at opposite ends of its axis of polarity, the root (RM) and shoot (SM) meristems which will produce additional organs, such as roots, leaves, shoots and flowers, during post-embryonic development. The meristems themselves originate during embryogenesis as part of the apical-basal pattern which also includes the cotyledons (COT), hypocotyl (HY) and root (ER). Perpendicular to the apical-basal axis, a radial pattern consists of concentric tissue layers.
The cell division pattern is nearly invariant in the Arabidopsis young embryo, which enabled the origins of seedling structures to be traced back to very early stages of embryogenesis.

Fig.: Development of the apical-basal pattern during embryogenesis.
A: 1-cell stage, following division of the zygote B: 8-cell stage, C: Heart-stage, D: Torpedo-stage embryo.

In the embryo, a few genes have been shown to be expressed in specific locations, for example in the outer cell layer or at the top of a globular embryo, suggesting that at this time developmental cues have already segregated cell fates in a position-dependent manner.

The aim of our research is to identify mechanisms underlying pattern formation in the embryo.
As a first step, we have isolated and characterised mutants with specific defects at the seedling stage and traced their defects back to early embryogenesis (Mayer et al, 1991). Two examples are shown here:

Our current studies focus on the analysis of a few genes involved in apical-basal development, including GNOM , FACKEL and BODENLOS

GNOM:

The GNOM gene is required before the first cell division and appears to be necessary for firmly establishing the apical-basal axis of the embryo (Mayer et al., 1993; Vroemen et al., 1996). The gene was isolated by map-based cloning (Busch et al., 1996). It encodes a 165 kDa protein which is related by sequence to yeast proteins involved in vesicle trafficking. Two main lines of research have been initiated to bridge the gap between the primary function of the GNOM protein within the cell and its developmental effect.

Specific antisera raised against recombinant fragments of the GNOM protein are used to immuno-localise the protein within the cell at both the light and electron microscope level and to characterise the protein biochemically (Thomas Steinmann ).

Genes encoding GNOM-interacting proteins have been searched for in the yeast two-hybrid system. Some of the candidates identified are being characterised molecularly, including immuno-localisation studies with specific antisera (Markus Grebe ).

FACKEL

The FACKEL gene is involved in hypocotol development from early stages of embryogenesis on. The defect becomes apparent in the mid-globular embryo when cells in the center fail to divide asymmetrically. Isolation of the gene is nearing completion (KathrinSchrick ).

BODENLOS

was recently identified as a new gene required for the formation of the embryonic root. The development of mutant embryos is being analysed from very early stages to determine the primary defect. In addition, the gene will be isolated by map-based cloning (ThorstenHamann ).

Cytokinesis in Arabidopsis

Cytokinesis partitions the cytoplasm of the dividing cell. Following nuclear division this basic biological process serves multiple purposes in plant development (Jürgens, 1995). For example, the division of a polarised cell may lead to two different daughter cells which give rise to different tissues. Since wall-bounded cells cannot change their positions relative to their neighbors, cell divisions that are regulated in time or space play a crucial role in plant morphogenesis. In more general terms, the newly-formed plasma membrane and cell wall may act as a barrier to help establish separate microenvironments for the daughter nuclei.

Plant cytokinesis starts in the center of the dividing cell where a specific cytoskeletal array, the phragmoplast, forms between the daughter nuclei. Golgi-derived vesicles are transported along the phragmoplast to the plane of division where they fuse with one another to form the cell plate, a membrane-bounded incipient cell wall. As the phragmoplast is being displaced centrifugally, the disc-shaped cell plate expands by the continuous incorporation of new vesicles and eventually fuses with the parental cell wall.

We have taken a genetic approach to analyse mechanisms that underlie cytokinesis. Two genes identified by mutation are currently being analysed:

KNOLLE:

KNOLLE is transcribed in a cell-cycle dependent manner and encodes a syntaxin-related protein (Lukowitz et al., 1996). Syntaxins are members of a family of vesicle-docking proteins, and KNOLLE protein may thus play a specific role in cytokinetic vesiclular trafficking.

The role of the KNOLLE protein has been adressed with a variety of techniques, including cell fractionation, biochemistry, indirect immunofluorescence and electron microscopy (Martina Lauber, Irene Waizenegger, ThomasSteinmann ). Our results suggest that KNOLLE protein is made during the M phase of the cell cycle and accumulates in the plane of cell division during cytokinesis, mediating the formation of the cell plate by vesicle fusion (Lauber et al., submitted).

To identify other components of cytokinetic vesiclular trafficking, we have searched for KNOLLE-interacting proteins using the yeast two-hybrid system. Putative KNOLLE interactors are being characterized molecularly and their role in cytokinesis is investigated, e.g. by the use of specific antibodies (Maren Heese ).

The expression of KNOLLE mRNA and protein is tightly regulated during the cell cycle. Cis-regulatory sequences conferring this expression will be delineated by phenotypic rescue of knolle mutant plants. The biological significance of the cell-cycle dependent expression is being addressed by misexpressing KNOLLE protein in developing plants. Mechanisms underlying the intracellular distribution and degradation of KNOLLE protein will be analysed in transgenic plants carrying KNOLLE-GFP fusion proteins (AxelVölker ).

RUNKEL

In our continuing search for mutants, a new gene, RUNKEL , has been identified as another important component of cytokinesis and is being characterised molecularly (Sabine Wallisch

Screen for GUS expressing marker lines

The genetic analysis of embryogenesis has been hampered by the shortage of morphological markers. This limitation can be overcome by using molecular markers, i. e. reporter genes expressed in specific regions of the developing embryo. We are generating enhancer and gene trap lines using the Ac/Ds system derived from transposable elements of maize (Sundaresan et al. Gen. Dev. 9: 1797). The purpose of the screen is twofold:

(1) isolation of molecular markers that can be used to specify developmental steps
(2) isolation of developmental genes that have been tagged with a Ds insertion

The two-component transposon system

The two elements, Ac and Ds, are combined by crossing plants from Ac and Ds lines. The Ds element contains a GUS reporter gene but lacks transposase which is provided by the immobilized Ac element and is necessary for the transposition. The enhancer trap Ds element DsE contains a truncated 35S promoter. The reporter gene is activated only if it is trapped near an enhancer. The gene trap Ds element DsG contains a multiple splice acceptor without any promoter. Consequently, the reporter gene is expressed only if the Ds element is transposed into a gene.

Screening procedure

A double-selection system is used to select putative transposants among the F2 seedlings:

KAN resistance which is conferred by the Ds element carrying a NPTII gene (positive selection). KAN sensitive seedlings are small with yellow or light green cotyledons.
NAM sensitivity is conferred by the immobile IAAH gene at the original location of the Ds element and the Ac element (negative selection). NAM sensitive seedlings are short with non turgescent (weak) hypocotyl and short bushy roots.

Normal-looking seedlings are putative transposants. They are investigated further by GUS staining.

GUS staining

GUS gene (ß-glucuronidase) catalyzes cleavage of X-Gluc producing an insoluble blue precipitate. If the Ds element is transposed into or near a gene, ß-glucuronidase is expressed. Incubation of plant material in a solution containing X-Gluc results in blue GUS staining patterns. We are particularly interested in specific patterns in developing embryos (see figure).

About 1,500 lines with new insertions have been analysed for GUS expression.

Tagged genes will be subjected to molecular analysis in order to determine their roles in embryonic pattern formation.

(Gottfried Martin , ReginaKeil-Pilz , Manuela Preissler Qiang Song )

home - references - people