Defining the role of EDE1 a novel microtubule-located protein required for nuclear division in Arabidopsis

In many plants, the endosperm of the seed forms by a series of rapid nuclear divisions that lead to the development of one very large cell containing hundreds of nuclei known as a coenocyte or syncitium. Cell walls subsequently develop between the nuclei and after that, starch and proteins (which humans use in food) are synthesised. The repeated mitosis is clearly very important to the development of the endosperm but as yet, we do not understand how the process is controlled. We have recently identified a new mutant of Arabidopsis in which the nuclei of the endosperm syncitium are greatly enlarged and do not separate correctly after DNA replication and we have called it endosperm defective 1 (ede1). In this new mutant, the development of the embryo is unaffected. This suggests the gene is important in the endosperm for controlling the cell cycle ¿ that is, whether the nuclei keep dividing or not. We have cloned the EDE1 gene from wild type Arabidopsis and shown that it interacts in living cells with microtubules, which are the scaffolding structures that help the cell divide. The EDE1 gene has seven close relatives in Arabidopsis and it is likely that at least some of these relatives play related roles in controlling the cell cycle in plants. The EDE1 gene has been found to be switched on in other parts of the plant that are undergoing rapid cell division such as the growing tip of the stem, indicating that it is likely to be involved in controlling the cell cycle in tissues other than the endosperm. When EDE1 protein is fused to a fluorescent reporter protein and expressed in cell cultures from Arabidopsis, the protein is found to associate with the mitotic spindle, which is the microtubular structure on which the two sets of chromosomes are separated, giving further evidence that it is involved in nuclear division. The aim of this project is to investigate the function of the EDE1 gene and its relatives in controlling the cell cycle. We will investigate how the cell cycle is altered in the ede1 mutant and how the EDE1 protein interacts with the mitotic spindle during cell division. We will identify proteins that interact with the EDE1 protein so that we can develop a clear picture of its function in the cell. We will also identify plants that have been mutated in the related genes and study the cell cycle in those plants, helping us to determine whether those genes also have related functions in controlling the cell cycle.

The endosperm of seeds is a very useful development system since mitosis and cytokinesis are separated into clearly separate stages. In recent work, we have identified a mutant of Arabidopsis (endosperm defective1; ede1-1) that is disrupted specifically during the early phase of development of the endosperm. Evidence obtained so far suggests that the EDE1 gene functions during mitosis in the endosperm. Alteration or loss of function of EDE1 leads to a greater than 90 per cent decrease in the number of nuclei in the endosperm of Arabidopsis. The nuclei are greatly enlarged, sometimes with a distorted shape, containing more DNA and enlarged nucleoli. The EDE1 gene was cloned using a map-based approach using the EMS induced mutant ede1-1. The EDE1 gene of Arabidopsis is the founding member of a previously undescribed plant gene family. The family contains eight members in both Arabidopsis and rice. Phylogenetic analysis shows the genes fall into 5 sub-classes, three of which contain both rice and Arabidopsis genes. Preliminary studies of the location of EDE1 using both C and N terminally fused GFP show that it decorates the mitotic spindle in Arabidopsis cell cultures, providing a potential explanation for the abnormal divisions. This provides a unique opportunity for geneticists and cell biologists studying the cell cycle and the plant cytoskeleton to study the fundamental biology of mitotic control in a plant-specific context. Since cereal endosperm provides 60% of food for humans worldwide our results should have implications for improvement of food quality and yields. The objectives are: (1) to investigate the function of EDE1 during mitosis in the developing endosperm of Arabidopsis and in other mitotically active tissues. We will use tubulin and phosphohistone antibodies to make a detailed investigation of microtubule and cell cycle dynamics and spindle organisation in the ede1-1 mutant. This initial description will help us to define the more molecular questions concerning the function of EDE1; (2) to use EDE1-GFP-fusion proteins and EDE1 antibodies to study the sub-cellular location of EDE1 and ede1-1 proteins during the cell cycle, both in the endosperm and using cell cultures as a model system; (3) to test if EDE1 interacts directly or indirectly with microtubules and to identify proteins that interact with EDE1 by independent approaches: (i) immunoprecipitation of protein complexes by expressing affinity tagged EDE1 in Arabidopsis and (ii) yeast two hybrid screen using EDE1 as bait; (4) to test whether other EDE1 family members, particularly the closely related gene At3g60000, provide overlapping functions in other tissues.