The alpha-N-acetyl-glucosaminidase gene is transcriptionally activated in male and female gametes prior to fertilization and is essential for seed development in Arabidopsis.

Sugar residues in proteoglycan complexes carry important signalling and regulatory functions in biology. In humans, heparan sulphate is an example of such a complex polymer containing glucosamine and N-acetyl-glucosamine residues and is present in the extracellular matrix. Although heparan sulphate has not been found in plants, the At5g13690 gene encoding the alpha-N-acetyl-glucosaminidase (NAGLU), an enzyme involved in its catabolism, is present in the Arabidopsis genome. Among our collection of embryo-defective lines, a plant was identified in which the T-DNA had inserted into the AtNAGLU gene. The phenotype of atnaglu is an early arrest of seed development without apparent male or female gametophytic effects. These data demonstrated the essential function in Arabidopsis consistent with the contribution of NAGLU to the Sanfilippo syndrome in human. Expression of AtNAGLU in plants was shown to be prevalent during reproductive development. The presence of AtNAGLU mRNA was observed during early and late male gametogenesis and in each cell of the embryo sac at the time of fertilization. After fertilization, AtNAGLU was expressed in the embryo, suspensor, and endosperm until the cotyledonary stage embryo. This precise pattern of expression identifies the cells and tissues where a remodelling of the N-acetyl-glucosamine residues of proteoglycan complexes is occurring. This work provides original evidence of the important role of N-acetyl-glucosamines in plant reproductive development.

The first zygotic division in Arabidopsis requires de novo transcription of thymidylate kinase.

Re-activation of cell division after fertilization involves the specific regulation of a set of genes. To identify genes involved in the gametophytic to sporophytic transition, we screened Arabidopsis T-DNA insertion lines for early seed abortion at the zygote (zeus) or one-cell embryo stages (cyclops), and characterized a sporophytic zygote-lethal mutation, zeus1. ZEUS1 encodes a thymidylate kinase (AtTMPK) that synthesizes dTDP and is involved in the regulation of DNA replication. Unlike in yeast and animals, the single AtTMPK gene is capable of producing two proteins by alternative splicing; the longer isoform is targeted to the mitochondria, the shorter to the cytosol. Transcription of AtTMPK is activated during the G(1)/S-phase transition of the cell cycle, similarly to yeast and mammalian orthologues. In AtTMPK:GUS plants, the reporter gene was preferentially expressed in cells undergoing division, but was not detected during the male and female gametophytic mitoses. GUS expression was observed in mature embryo sacs prior to fertilization, and this expression may indicate the time of synchronization of the gamete cell-cycle phases. Identification of ZEU1 emphasizes the importance of control of the metabolism of DNA in the regulation of the G(1)/S-phase transition at fertilization.

Genetic analysis of two Arabidopsis DNA polymerase epsilon subunits during early embryogenesis.

Accurate DNA replication is one of the most important events in the life of a cell. To perform this task, the cell utilizes several DNA polymerase complexes. We investigated the role of DNA polymerase epsilon during gametophyte and seed development using forward and reverse genetic approaches. In Arabidopsis, the catalytic subunit of this complex is encoded by two genes, AtPOL2a and AtPOL2b, whereas the second largest regulatory subunit AtDPB2 is present as a unique complete copy. Disruption of AtPOL2a or AtDPB2 resulted in a sporophytic embryo-defective phenotype, whilst mutations in AtPOL2b produced no visible effects. Loss of AtDPB2 function resulted in a severe reduction in nuclear divisions, both in the embryo and in the endosperm. Mutations in AtPOL2a allowed several rounds of mitosis to proceed, often with aberrant planes of division. Moreover, AtDPB2 was not expressed during development of the female gametophyte, which requires three post-meiotic nuclear divisions. Since a consensus binding site for E2F transcription factors was identified in the promoter region of both genes, the promoter-reporter fusion technique was used to show that luciferase activity was increased at specific phases of the cell cycle in synchronized tobacco BY-2 cells. Our results support the idea that fertilization may utilize the mechanisms of cell cycle transcriptional regulation of genes to reactivate the divisions of the oosphere and central cell.