Identification and analysis of CLE signalling peptides in root and nodule development

Abstract

The hypothesis of the thesis project postulates that CLV3/ESR-like (CLE) signalling peptides are involved in the control of nodule number. At the start of the project, only seven CLE members from legumes had been identified. To characterize CLE domain proteins functionally and to test for their involvement in the autoregulation of nodulation it was necessary to identify more members from this CLE family. More than 100 new CLE signalling peptides were identified in the first part of the project, including five protein sequences containing multiple, regularly arranged CLE motifs. It was possible to cluster the CLE domain proteins into 13 groups based on pairwise similarities in the primary CLE motif. In addition, secondary motifs were identified, which could lead to the discovery of recognition sites for processing peptidases. The groupings of the CLE motifs correlate with known biological functions of CLE signalling peptides and are analogous to previously established groupings based on phylogenetic analysis and ectopic overexpression studies. The biological function of two predicted CLE signalling peptides was tested in Medicago truncatula in vivo using a peptide bioassay. These peptides inhibited the cell differentiation at root apical and lateral root meristems in a manner consistent with functional predictions based on other CLE signalling peptides clustering in the same groups. In the second part of the thesis, the possible involvement in nodulation of a selection of identified CLE signalling peptides was tested in peptide bioassays using two Medicago truncatula genotypes, wild type as well as the supemodulating mutant sunn, by supplying the growth medium with synthetic CLE peptides. Initial pilot tests involving inoculation of Medicago truncatula with Sinorhizobium meliloti revealed promising results. Subsequent large-scale screens generated a number of results. Changes in lateral number were observed in response to peptide treatment, but were not statistically significant. Nevertheless, two lateral root architecture phenotypes "spiky root" and "melty root" were observed and described. There are indications for a common peptide involved causing the phenotypes. The initially promising results of specific of CLE peptides on nodule number were not repeatable with statistical significance. Generally, variation in the number of nodules could be detected in the dish-based peptide bioassay and did not allow a measurement of the effects of individual CLE signalling peptides. Several protein sequences containing multiple CLE domains were identified in.the first part of the project. CLE68 is such a multi-CLE domain protein encoded by Medicago truncatula. To ensure that CLE68 is encoded as predicted and not an artefact of erroneous genome annotation the genomic locus was sequenced. Quantitative real-time polymerase chain reaction was used to demonstrate that CLE68 is expressed in vivo. The functional ortholog of Arabidopsis thaliana CLV3 in Medicago truncatula is described in detail and expression is analyzed. Examination of the genomic locus demonstrates that CLE68 is a paralog of the functional ortholog of CLV3 in Medicago truncatula. Peptide bioassays demonstrate that CLE68 is unique, as it contains CLE peptides with differential effects when ectopically applied in a root apical meristem (RAM) peptide bioassay. Members of the CLE signalling peptide family containing several CLE domains within a single protein precursor instead of commonly a single CLE motif could release several active signalling peptides after cleavage.