Identification of auxin molecular targets in Poaceae embryonic pattern formation
Final Report Abstract
The aim of this project is to investigate developmental mechanisms underlying embryonic patterning and axis formation in Poaceae. Particular attention is devoided to evaluate plant GSK3/SHAGGY homologs as potential key regulators in signalling cascade(s) involved in embryonic patterning and axis formation. In addition a MADS box protein is appraised as key signaling molecule operating in the regulatory networks during plant and early embryo development. Both genes were identified in a cDNA library enriched in genes differentially expressed in wheat embryos that developed supernumerary meristems and organs (i.e. Siamese embryos) as a result of auxin transport inhibitor treatment (NPA). The cDNA library constructed using a suppression subtractive hybridization approach contains small genes fragments. Particular efforts were dedicated to clone full length cDNA and genomic sequences in the difficult context of an hexaploid genome and multigenic families. The wheat GSK3/SHAGGY homologs TASK1 and TASK2 are encoded respectively by at least three different mRNA sequences transcribed from at least three different genomic sequences named A, B and C. Both genes contain 12 exons and 11 introns. Promotor sequence is available for TaSK2 B. Important domains or residues for protein activity are conserved in TASK1 and TASK2. A full length cDNA of TaMADS 1, the MADS box gene identified in wheat, was cloned. According to the translated protein sequence, TAMADS1 contains a MADS box, and the I,K and C domains belonging therefore to type II lineage. RT-PCR and in situ approaches indicate that TaSKs and TaMADS1 are expressed in all tissues and at all embryonic stages tested. A real-time quantitative PCR approach was used to address the question whether TaSK1 and 2 are regulated at the transcriptional level by hormones in particular by auxin and epibrassinolides. Different embryonic developmental stages, different tissues and treatment time have been intensively tested. Additional experiments need to be performed before drawing conclusions. Major control mechanisms for GSK3/SGG actions in animal are at the protein level and include phosphorylation, intracellular localization or binding protein. Therefore TASKs posttranslational regulation is under investigation by means of immunoblotting before and after treatment of embryos and leaves with different hormones including auxin and epibrassinolides. These studies will be continued after obtaining specific antibodies against TASK1 and 2 what turned out to be challenging and will require a custom solution concerning the immunization schema. Identification of signal intermediate in TaSKs transduction pathways particularly in embryonic development is also a central question. Therefore MADS box proteins are evaluated as potential upstream signaling molecules using approaches such as the one yeast hybrid system. Finally, the regulation of TaMADS at the transcriptional and protein level is currently under investigation using similar approaches as for TaSKs. Effort are currently devoted to the different technical issues and to answer the very interesting and challenging questions addressed in this project. Insights may in particular be gained on how evolutionary conserved are molecules and regulatory networks directing development, patterning and axialisation.
