University of Hamburg

Gene expression during fertilization and zygote development
--Dresselhaus, T, Cordts, S, Heuer, S, Bantin, J, Lorz, H, Kranz, E

Numerous mutants have been described in maize affecting many developmental processes. Concerning sexual reproduction there are mutants available where development is disturbed e.g. in flower formation, meiosis or embryogenesis (for review see Neuffer et al., Mutants of Maize, Cold Spring Harbour Laboratory Press, 1997). Nevertheless, the fertilization process, zygote development and very early stages of embryogenesis are to our knowledge not covered by described mutants. One reason is that these processes occur deeply embedded in the maternal tissues of the ovule and it is therefore difficult to get access to specific developmental stages. This is especially a problem when the above described processes are studied not only at the cytological, but also at the molecular level.

We are following an alternative approach: the above described developmental processes have recently been transferred from in vivo to in vitro (reviewed by Kranz and Dresselhaus, Trends Plant Sci. 1:82-89, 1996) and it is now possible to get access to gametes as well as to specific stages of zygotes and early embryos. To investigate these cells at the molecular level, we have established sensitive RT-PCR methods. Gene expression of every known gene from maize can be studied at the single cell level (Richert et al., Plant Sci. 114:93-99, 1996) and representative cDNA libraries can be generated from only a few plant cells (Dresselhaus et al., Plant J. 5:605-610, 1994). Using the latter technique we have constructed a cDNA library of 128 unfertilized egg cells and a second cDNA library of 104 in vitro zygotes (18 h after fertilization; Dresselhaus et al., Plant Mol. Biol. 31:23-34, 1996). Further cDNA libraries have been generated from mature pollen and seedlings.

Using differential screening methods and gene specific probes, we have identified more than 50 genes, whose expression is up- or down-regulated after in vitro fertilization. Some genes are newly induced after fertilization. Interestingly, the majority of isolated transcripts have not been described before in maize. Some transcripts have not been described before in plants and others do not show homology to known proteins or DNAs in databases. Isolated cDNA clones encoding proteins with homology to known proteins are involved e.g. in RNA stability and degradation, translation, DNA replication and repair, energy metabolism, protein modification and degradation. We are now analysing a few genes with interesting expression pattern in more detail, using e.g. in situ hybridization techniques, transgenic overexpression and antisense approaches as well as the gene machine to isolate Mu-transposon knock-outs.

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