MOLECULAR ANALYSIS OF DOUBLE FERTILIZATION PROCESS IN ARABIDOPSIS

Fertilization and seed formation are key events in the life cycle of flowering plants. The seed represents an elaborated functional unit, whose main purpose is to propagate the plant's offspring. The first step in seed development is the formation of ovules and subsequently the achievement of a successful double fertilization process. In our lab we have discovered that the MADS-box domain protein complex formed by SEEDSTICK (STK) and SEPALLATA3 (SEP3), responsible to maintain ovule identity, have as a direct target a member of the REM family, VERDANDI (VDD, REM20). With the combination of Bio-informatics studies and ChIP-qPCR experiments using specific STK and SEP3 antibodies, we were able to identify REM11, as the second direct target of the STK-SEP3 complex. The phenotype of the rem11 mutant is very similar to the one described for vdd-1/+ demonstrating that REM11 plays a similar function in the fertilization process. To better understand the fertilization defect observed in these mutants we have used a new technique developed to observe fertilization in vivo, by visualizing the gametes with a combination of markers for sperm cells and for the female gamete. In the rem11 or in vdd-1 gametophytes the synergid cell seemed to lose identity. Although the pollen tubes reached the micropyle, the two sperm cells didn’t migrate toward the egg and center cells. These results showed the important and the direct involvement of these two genes in the control of synergid driven processes. Ultimately we discovered that genes responsible for the pollen tube attraction like the transcription factor MYB98, are correctly expressed in the mutants whereas genes, probably responsible for the degeneration process are miss-expressed. In summary, we can say that, two very different processes are regulated by the synergid cells: 1) the attraction of the pollen tube and 2) the synergid degeneration (apoptosis). We discover that the second step is specifically controlled by VDD and REM11, two proteins that by yeast-2-hybrid experiments were able to interact. Based on these results we have decided to study if other REM transcription factors could interact with REM11 and VDD. In conclusion STK-SEP3 MADS-box complex are able to directly regulate a REM transcription factor complex that has a very important and specific role during the double fertilization process. To understand how VDD-REM11 complex regulate synergid degeneration we have performed a RNA sequencing experiment comparing wild-type mature carpels to mutant ones. Exciting targets have been discovered and discuss in this thesis. I have also studied the regulation of VDD transcription by STK-SEP3 complex. In VDD regulatory region three CArG boxes were identified and by Chromatin Imunoprecipitation experiments, we have showed that the ovule identity proteins STK and SEP3 bind to the first and third CArG boxes. We have performed in vivo and in vitro experiments showing that the STK-SEP3 complex is needed to form short-range loops in VDD promoter. For years evidences based on in vitro biochemical assays and yeast experiments shown that MADS box proteins form multimeric complexes. New evidences for the quartet-floral model were obtained, analyzing the activation of VDD promoter by STK-SEP3 multimeric complex. Least but not the last, I have also analyzed the interaction of ARABIDOPSIS BSISTER (ABS) with STK, showing that they have a function in the regulation of the endothelium development, the inner most integument layer of the mature ovule that we demonstrated to be required to the double fertilization process.