Peptide signalling in plant development: functional analysis of CLE ligands in Arabidopsis

In multicellular organisms, cell to cell communication is essential for coordinating growth and differentiation. In plants most known intercellular communication is mediated by phytohormones such as auxin, cytokinin, GA, ABA, ethylene and brassinosteroids. However, in recent years, several putative peptide ligands have been identified in plants and were shown to mediate signalling events. One of these ligands is CLAVATA3 (CLV3) which is a peptide ligand in Arabidopsis and is involved in stem cell maintenance.CLV3 is part of the CLV3/ESR (CLE) gene family which consists of 31 members in Arabidopsis. These genes encode small proteins which contain a putative secretion signal at their N-termini and a conserved CLE-motif at or near their C-termini. Recently the mature CLV3 was identified and shown to consist of 12 amino acids comprising the CLE motif with two hydroxylated prolines.This thesis aims to better understand signalling by the CLE gene family and in particular CLE19 and CLV3. This research started with the characterization of one of the CLE members namely CLE19 in Brassica napus embryos. This was followed by a small proteomic study of the secretome of Brassica microspore embryos to identify differences in low and high yielding embryo cultures and concludes with a newly developed peptide based approach to better understand CLE signalling in shoot and root meristems.Chapter 1 is an introduction on CLE signalling in Arabidopsis. This chapter combines all the data that is published related to the CLE gene family. The Arabidopsis CLE family was used as a starting point for making a genomic survey of the rice genome to identify rice CLE genes. This survey resulted in the identification of33 putative rice CLE genes. All rice CLEs were compared to the ones from Arabidopsis in order to find orthologues based on the amino acid sequence of the complete protein or only the CLE domain. The comparison, based on the complete protein did not result in the identification of CLE orthologues between rice and Arabidopsis. Comparing the 14 amino acid CLE domains resulted in the identification of some putative orthologues but most similar CLE domains were within the Arabidopsis or rice gene family. The CLE domain is probably too short to identify any orthologues between the In multicellular organisms, cell to cell communication is essential for coordinating growth and differentiation. In plants most known intercellular communication is mediated by phytohormones such as auxin, cytokinin, GA, ABA, ethylene and brassinosteroids. However, in recent years, several putative peptide ligands have been identified in plants and were shown to mediate signalling events. One of these ligands is CLAVATA3 (CLV3) which is a peptide ligand in Arabidopsis and is involved in stem cell maintenance.CLV3 is part of the CLV3/ESR (CLE) gene family which consists of 31 members in Arabidopsis. These genes encode small proteins which contain a putative secretion signal at their N-termini and a conserved CLE-motif at or near their C-termini. Recently the mature CLV3 was identified and shown to consist of 12 amino acids comprising the CLE motif with two hydroxylated prolines.This thesis aims to better understand signalling by the CLE gene family and in particular CLE19 and CLV3. This research started with the characterization of one of the CLE members namely CLE19 in Brassica napus embryos. This was followed by a small proteomic study of the secretome of Brassica microspore embryos to identify differences in low and high yielding embryo cultures and concludes with a newly developed peptide based approach to better understand CLE signalling in shoot and root meristems.Chapter 1 is an introduction on CLE signalling m Arabidopsis. This chapter combines all the data that is published related to the CLE gene family. The Arabidopsis CLE family was used as a starting point for making a genomic survey of the rice genome to identify rice CLE genes. This survey resulted in the identification of 33 putative rice CLE genes. All rice CLEs were compared to the ones from Arabidopsis in order to find orthologues based on the amino acid sequence of the complete protein or only the CLE domain. The comparison, based on the complete protein did not result in the identification of CLE orthologues between rice and Arabidopsis. Comparing the 14 amino acid CLE domains resulted in the identification of some putative orthologues but most similar CLE domains were within the Arabidopsis or rice gene family. The CLE domain is probably too short to identify any orthologues between the monocotyledon (rice) and dicotyledon (Arabidopsis) but can be used as a starting point for further research. This chapter is concluded with an outline of this thesis. In Chapter 2 a short survey on the biochemical aspects of haploid embryogenesis is described and in particular the identification of extra cellular signalling molecules in microspore embryo development of Brassica napus. Differences were examined in protein profiles present in the medium from high-yielding microspore derived embryos cultures and low-yielding that did not produce any embryos. Several proteins were isolated and identified by de-novo protein sequencing including some unknown proteins with no obvious orthologue in the Arabidopsis genome.Chapter 3 describes DD3-12, one of the genes that were isolated in a screen to identify markers for embryo development in Brassica napus. DD3-J2 encodes BnCLE19, a small 74 amino acid secreted protein that belongs to the CLE like family of proteins. With the use of northerns, a pBnCLE19:;GUS construct and RT-PCR analysis the expression pattern of CLE19 was established in Brassica and Arabidopsis. interestingly CLE19 is expressed in the LI andL2 layers of the periphery of shoot and inflorescence meristems as compared to CLV3 which is expressed in the first three layers in the top of the meristem. But also in embryos CLE19 expression was observed from globular stage onwards in the epidermal cells covering the developing cotyledons. BnCLE19 and CLE19, its orthologue in Arabidopsis, were over expressed in Arabidopsis which resulted, among other phenotypes, in the consumption of the root meristem. Interestingly this consumption of the root meristem was also observed upon over expression of other members of the CLE family, namely CLV3 and CLE40. The only conserved domain among all CLE proteins, beside the secretion signal, is a 14 amino acid CLE domain at or near the C-terminus. To test if this domain is the functional part of the CLE proteins and if this domain can act independently from the rest of the protein an in-vitro system was developed using chemically synthesized 14 amino acid CLE peptides.Chapter 4 describes the research aimed at the analysis of the functional domain of CLE proteins. We show that the CLE peptides can cause an over-expression phenotype in the roots of Arabidopsis, namely termination of the root meristem, as previously shown in chapter 3 upon over expression ofCLE19. Already after 4 days of growth in CLE peptide containing media the first signs of a general miscommunication between cells and cell layers were observed. In early root development the CLE peptides caused a misspecification in the pericycle, endodermis and cortex cell layers and a premature differentiation of the cortical daughter cells. In the search for receptors involved in the perception of the CLE ligands we challenged the clv mutants with several CLE peptides. Only the clv2 mutant, when incubated with CLE peptides, resulted in normal root development with no defects in the meristematic tissue. This result was revealing a CLV-like signalling pathway in the roots with CLV2 as one of the receptors which was involved in the perception of the CLE peptides during root development.Chapter 5 pictures the functional analysis of the CLV3 peptide in shoot apical meristem (SAM) development of the clv3 mutant which results in the reduction of the enlarged SAM to wild type (WT) proportions. Different CLE peptides were used to determine the redundancy between different CLE genes in SAM development. The functional analysis of different CLE peptides in clv3 resulted in a variable degree of complementation. The CLE40p and CLV3p peptides caused an almost complete restoration of the SAM to WT while CLE19p and CLE5p resulted in a partial complementation. This in contrast with CLE22p which was not able to replace CLV3 resulting m a greatly enlarged meristem as seen in clv3. These results clearly showed that not all CLE ligands were freely interchangeable and a certain degree of sequence specificity is required which was also shown with a mutated and several truncated peptides. The in-vitro peptide approach is combined with a deletion analysis of the CLV3 gene to proof that the CLE domain, beside the secretion signal, is essential and sufficient in rescuing the clv3 mutant.This thesis finishes with some concluding remarks and future prospectives in Chapter 6. This chapter describes the future of research in the field of CLE signalling with a particular interest in understanding ligand-receptor signalling and the pitfalls and drawbacks in identifying the receptors involved in the perception of the CLE ligands.

Bibliographic Details

Main Author:	Fiers, M.A.
Other Authors:	Stiekema, W.J.
Format:	Doctoral thesis biblioteca
Language:	English
Subjects:	arabidopsis, brassica napus, genes, ligands, plant development, signal peptide, signal transduction, genen, liganden, plantenontwikkeling, signaalpeptide, signaaltransductie,
Online Access:	https://research.wur.nl/en/publications/peptide-signalling-in-plant-development-functional-analysis-of-cl
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