Molecular and functional characterisation of signalling peptides of the IDA family in Arabidopsis thaliana
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- Institutt for biologi 
Small post-translationally modified secreted peptides have for the last decades emerged as important mediators of cell-to-cell communication, regulating both developmental processes and stress adaptation in plants. There are over 1000 potential peptides in the Arabidopsis thaliana genome, but so far only a handful has been functionally characterised. One well-studied example is INFLORESCENCE DEFICIENT IN ABSCISSION (IDA), a secreted peptide ligand regulating the cell separation step of floral organ abscission in Arabidopsis. To date, five other genes encoding IDA-LIKE peptides have been found. The work presented in this thesis has been divided into three parts: 1) Identification and expression analysis of the IDA gene family in Arabidopsis 2) Identification and characterisation of proteins in the signalling pathway downstream of IDA 3) Characterisation of two closely related members of the IDA gene family. In part one we searched the Arabidopsis genome for IDA-LIKE homologues. We identified three currently unannotated IDA-LIKE (IDL) genes, in addition to a new subfamily of 13 genes that we named IDA-RELATED (IRP). They all encode proteins characterised by an N-terminal signal peptide for the secretory pathway and a C-terminal, conserved core motif named SGPS. Transcriptional analysis suggests that both IDL and IRP genes are involved in growth and development, as well as stress responses. Studies of GUS promoter lines and inhibitor treatments suggest that a subgroup of the IDA-family genes is subjects for rapid mRNA turnover. In the second part we focused on unravelling the downstream components of the IDA signalling pathway, and to study the relationship between the two completely abscission deficient mutants ida and nevershed (nev). IDA regulates floral organ abscission through the two Leucine-Rich Repeat (LRR) Receptor-Like Kinases (RLKs) HAESA (HAE) and HAESA-LIKE 2 (HSL2), which in turn activate a MITOGEN-ACTIVATED PROTEIN KINASE (MAPK) cascade. Through a screen for mutants restoring the floral organ abscission in ida mutants, three new mutant alleles of the KNOTTED-LIKE HOMEOBOX gene (KNOX) BREVIPEDICELLUS (BP)/ KNOTTED IN ARABIDOPSIS THALIANA 1 (KNAT1) were identified. The bp/knat1 mutants had several similarities with plants overexpressing IDA, like premature shedding of floral organs and enlarged abscission zone (AZ) cells. Based on these results, we proposed that KNAT1 inhibits floral organ cell separation by restricting the size and number of AZ cells by suppression of KNAT2 and KNAT6. NEV encodes an ADP-ribosylation factor GTPase-activating protein, and nev mutants display alterations in vesicle and Golgi apparatus, in addition to complete deficiency of floral organ abscission. Through morphological, transcriptional and genetic studies of single and double mutants, we aimed at investigating the relationship between IDA and NEV in abscission. In contrast to ida, the nev mutant displayed enlarged AZs, a phenotype found in plants overexpressing IDA; however, this was not enough to promote abscission. The enlarged AZs were also found in nev ida double mutants, suggesting that NEV is involved in the IDA signalling pathway. However, mutations in KNAT1 could not rescue the abscission deficiency in nev, and likewise, evershed (evr), a suppressor of nev, could not rescue the abscission deficiency in ida. While IDA promotes both cell wall loosening and the actual organ separation, NEV prevents precocious cell wall loosening at early stages of abscission and facilitates cell wall degradation during the last step together with IDA. In the last part we functionally characterised two of the genes identified in part one, encoding the two closely related putative peptides IDL6 and IDL7, which are both highly responsive to a large number of abiotic and biotic stresses. Localisation studies of IDL7-GFP fusion proteins suggest that the peptide is processed both N- and C-terminally before being transported out of the cell. Transcriptional profiling of seedlings treated with synthetic IDL6 and IDL7 peptides revealed that these peptides down-regulate the expression of early stressresponsive genes, dominated by a large fraction of ZINC FINGER PROTEINS (ZFPs), WRKYs and genes encoding calcium-dependent proteins. Furthermore, we used loss-offunction mutants and overexpression lines to show that IDL6 and IDL7 are required for adaptation to saline and oxidative stress. From our data, we conclude that IDL6 and IDL7 play a role as negative modulators of the response to abiotic stress in Arabidopsis thaliana.