de novo specification of a post-embryonic stem cell population in plants

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Plants draw carbon dioxide from the air and convert it to sugars. Much of the sugar is utilised in the most abundant biopolymer on Earth, cellulose. Cellulose is the main constituent of wood, and wood represents a renewable biomaterial and a source of bioenergy. This proposal aims to shed light on how woody tissue is formed. Wood is part of the plant vascular system and is made up of xylem cells. The xylem arises from a group of stem cells referred to as the cambium. Understanding of how the cambium functions could be used to increase wood formation in a forestry setting, but in these experiments we will use the model plant, Arabidopsis, because it is easy to manipulate in the lab.In both plants and animals, stem cells are typically formed in the embryo. The cambium is unusual because its stem cell population is formed after embryogenesis, making it an interesting system for understanding stem cell initiation. Previously we have identified six signalling proteins, so-called receptor kinases, which act together to promote the cell divisions necessary for cambium formation. However, while we know that they are important in this process, we don't know exactly how they work. In this proposal we aim to answer this question. The receptor kinases are localised to the plasma membrane in the cambium cells. One hypothesis is that protein-protein interactions occur between some of the six receptor kinases. We will test this hypothesis in two ways. In the first, we will tag the receptor kinases in question with different fluorescent proteins. We can determine if energy transfer between two different fluorescent proteins has occurred (referred to as FRET) using microscopy. Because energy transfer is only be possible if the two proteins are in very close proximity, we would only detect FRET if protein-protein interactions were occurring between the tagged receptor kinases. The second method that we will use is referred to as co-immunoprecipitation. Here we will use an antibody that targets a tag which we have placed on a receptor kinase. If the receptor kinase forms part of a protein complex we will be able to identify other parts of the complex.Receptor kinases are molecules that send signals to the cells on the membranes of which they are present. The six receptor kinases that we have identified are present on several cell types, but which cell type(s) are required for normal cambium initiation? Can these proteins initiate cambium in several cell types? We aim to address this question in our proposal. We have generated a mutant in which all six receptor kinases are missing. We propose to replace these proteins in specific cell types including the xylem and cambium, but other vascular cell types too. These experiments will tell us if the consequences of the receptor kinase signalling are restricted to the cell in which they are present (referred to as cell-autonomous), or if they influence the behaviour of neighbouring cells.Finally, we propose to determine what the outcome of the signalling is. Receptor kinases signal to the nucleus to alter gene activity, but we don't which genes respond to the receptor kinases that are the focus of this study. We propose to increase signalling through the receptor kinases and detect which genes respond by sequencing the mRNA before and after the signalling increase. Changes in mRNA levels will tell us which genes have been regulated. It will also tell us whether some of the receptor kinases can influence the levels at which the other five are expressed.Together, these experiments will tell us how the cambium functions. They will also provide information on how stem cell populations can be set up following embryogenesis. They may also suggest ways in which we can manipulate wood formation to increase forest productivity and improve carbon capture.