Decoding Signalling and Specificity of Plant Peptide Ligand-Receptor Networks

Plants are sessile organisms that need to respond to changes in their environment and adapt locally. The classical phytohormones (such as abscisic acid, auxin, cytokinin, ethylene, gibberellins and brassinosteroids) play an important role in plant adaptation and development. However, the central role for these phytohormones has been challenged these past decades by the discovery that plants, like animals, utilise small peptides and membrane bound receptors in cell-to-cell communication. Plants use peptides as signalling ligands for receptor proteins to regulate important developmental processes and to respond to stresses. Arabidopsis thaliana, is the most used model system for plant genetics and plant developmental studies, and the Arabidopsis genome encodes for over a thousand potential peptide ligands and over six-hundred receptor like proteins. This project aims to compare two important signalling systems in Arabidopsis which are essential in regulating two crucial developmental pathways. We want to unravel the similarities of the signalling modules and how, if signalling components are reused, specific cellular responses are achieved. One of the signalling pathways regulates cell separation, a key step during organ shedding such as what occurs when leaves fall of the tree in the fall. The other pathway regulates stem cell homeostasis ensuring the capability of the plant to regenerate new organs. We ultimately want to utilise the knowledge we obtain from identifying key signalling components and their function to improve plant growth. For many signalling systems it is know that calcium acts as a communicator between external signals and cellular responses. As a first approach to search for similarities between the two signalling systems we are studying we investigated whether an increase in cytosolic calcium concentrations would be induced by peptide treatment. To do this, we used fluorescent sensors that can be tracked by the use of fluorescent microscopes. Genetically modified Arabidopsis plants carrying these sensors are exposed to external peptide treatment, if the peptides induce a release of cellular calcium than this can be visualised as increased fluorescence in a microscope. The first signalling system that we are studying which regulates cell separation is crucial for allowing the plant to discard the flower after pollination. In this pathway, the peptide IDA induces cell separation by binding and activating the receptors called HAESA (HAE) and HAESA-LIKE 2 (HSL2). The second system regulating stem cell homeostasis utilises a similar peptide, CLAVATA 3 that signals through the receptors CLV1, CLV2 and CRN. When applying IDA or CLV3 to plants carrying the calcium sensors an increase in calcium concentration is observed, indicating that both peptides may utilize calcium as a second messenger in the cell. We have now shown by the use of plants carrying mutations in the receptor genes that the increase in calcium concentration observed is dependent on the receptors relaying the peptide signal. However, the calcium response observed for each of the peptides differs, indicating signalling specificity. We are therefore now investigating similarities and differences between the HSL and CLV receptors with the aim of disclosing the differences in response. We are doing this by identifying calcium sensors, channels and calcium binding proteins that may be specific to each of the two pathways. In parallel with this work we have shown that IDA is involved in inducing a local immunity response in cells undergoing cell separation and that the calcium response is an important element of IDA induced plant immunity.

Given that abscission has a large agricultural implication for plant growth and yield production and that immune signalling often drastically compromises plant growth, detailed knowledge on the molecular mechanisms inducing one or the other is of central importance for agronomy. In the light of evolutionary conservation of IDA-HAE/HSL2 and that cells undergoing cell separation, be it for abscising plant organs or for the emergence of new organs, are particular prone to pathogen invasion from the surrounding environment, acquiring detailed knowledge on the molecular mechanisms inducing cell specific immune signalling in cells undergoing cell wall remodelling will serve to identify candidate genes for genetic engineering of crops that are resilient to biotic challenges. The molecular findings delineating how different cellular outputs are achieved could prove instrumental to manipulate both the defence and cell separation processes in crops.

Plants use peptides as signalling ligands for receptor proteins to regulate important developmental processes and to respond to stresses. A thorough analysis of these complexes, their downstream signal transducers and their target genes will enhance our u nderstanding of cell communication during plant development. The Arabidopsis genome encodes hundreds of plasma membrane-bound receptors and secreted peptide ligands, and although less than a dozen peptide-ligand-receptor pairs have so far been identified, the evolving picture shows that (i) the same, or part of the same, signalling module is employed to regulate similar developmental processes in different plant organs and developmental stages(ii) receptors that phylogenetically belong to the same subfami ly interact with similar peptide ligands, often in complex networks of ligand-receptor interactions, and (iii) common downstream signalling components are utilised to relay signals from similar peptide ligands. These observations pose the important quest ions of how extensive the similarities are between different signalling modules and how, if signalling components are reused, specific cellular responses are achieved. To address these questions we will use the signalling unit comprising the peptide ligan d INFLORESCENCE DEFICIENT IN ABSCISSION (IDA) and the two related leucine-rich receptor-like kinases (LRR-RLKs) HAESA (HAE) and HAESA-LIKE2 (HSL2) as a model system. The IDA/IDL1 peptides show similarities to the CLV3/CLE peptides which signal through LRR -RLKs belonging to the same subfamily as HAE/HSL2. Thus, these signalling modules are ideal for studying similarities, differences and networks in peptide signalling. We will unravel if receptors that are phylogenetically related interact to control simil ar developmental processes. By mapping the phosphorylation-dependent gene regulatory networks we will uncover how specific readouts are achieved in different cell types utilising shared signalling components.