Understanding the mechanisms of developmental regulation by Arabidopsis Armadillo-related proteins.

All multicellular organisms, whether animals, plants, fungi or protozoa, are composed of groups of cells with diverse forms and functions. I am interested in how cells within an organism become different from each other and acquire specialised functions during the process of multicellular development. Each cell in an organism contains the same total genetic material in its nucleus. However, the way the genetic material is regulated (which individual genes are turned on and off) differs between cell types. Cells acquire particular fates within an organism, such as muscle cells or leaf cells, by responding to signals from their environment. These signals tell them which genes to turn on and off, and therefore which proteins to make. The process of being multicellular has a great degree of molecular conservation. Related genes and proteins often perform the same function in quite unrelated organisms. My particular interest is in the Armadillo protein family. These proteins specify cell fates during animal development, and also in a non-animal, the social amoeba Dictyostelium. This suggests that the function of Armadillo proteins arose very early during the evolution of multicellular organisms. I have shown that proteins related to Armadillo are present in the plant kingdom. Using Arabidopsis, mouse-ear cress, as a model, I have shown that Arabidopsis Armadillo proteins affect root development. A plant's root system is made up of a primary root that emerges from the germinating seed, and of lateral roots that branch from the primary root throughout the plant's life. Arabidopsis Armadillo proteins promote root branching, which occurs when cells within the main root divide to produce a new, lateral, root. The shape of a plant's root system is critical for plant growth. Roots allow the plant to take up water and nutrients from the soil, allowing the shoot that we see above ground to grow. Although the actual developmental process being regulated by plant Armadillo proteins is not the same as in animals (animals do not have roots!), it is quite possible that at the small scale, the molecular mechanisms by which plant Armadillo proteins function are conserved with animals and amoebae. Arabidopsis Armadillo proteins are found in the nucleus of cells, like their animal relatives. I plan to understand how Arabidopsis Armadillo proteins perform their cellular role by looking in detail at where they localise to within cells, what other proteins they interact with, and how these interacting proteins modify Arabidillo function. By uncovering the mechanisms of Arabidopsis Armadillo protein function in plants I will provide a new understanding of how root development occurs. Root architecture must be dynamic, for plants to respond to changes in environmental conditions, such as water and nutrient availability. Thus, Arabidopsis Armadillo proteins affect a process of relevance to agriculture, not just to a laboratory experiment. Understanding these proteins' functions will suggest ways of making plants better-adapted to coping with changes in the environment in which they grow.

Armadillo/beta-catenin is an important protein that has been studied extensively in animals. It is required both for embryonic development and for differentiation in adult tissues. Armadillo functions have ancient evolutionary origins, and are conserved in the multicellular amoeba Dictyostelium. Many plant genomes also contain putative Armadillo homologues, including Arabidopsis Arabidillo-1 and -2. Arabidillo-1 and -2 function redundantly during plant development to promote lateral root formation. arabidillo-1/-2 mutant plants develop fewer lateral roots than wild type, whereas Arabidillo overexpression leads to an increased lateral root number in seedlings. Arabidillo proteins appear to function in a novel lateral root regulatory signalling pathway. My data suggests that Arabidillo regulation is largely post-transcriptional, as for animal beta-catenin. I have elucidated parts of Arabidillo-1 responsible for both nuclear and cytosolic targeting. Arabidillo-1 and -2 both contain an F-box motif, thus may regulate the stability of target proteins or autoregulate their own stability by interacting with the proteasome. We now aim to understand the mechanisms by which Arabidillo proteins perform their developmental functions, by understanding how they are regulated and identifying their downstream targets. In this programme of research we will: 1) Determine the subcellular localisation and regulation of endogenous Arabidillo proteins in different cell types, by generating anti-Arabidillo antibodies and using them for immunocytochemistry and biochemistry. We will determine whether Arabidillo subcellular localisation is regulated by environmental signals such as phytohormones and nutrient supply. 2) Determine whether Arabidillos undergo post-translational modification, and whether Arabidillo protein stability is regulated, for example, by proteasomal degradation. 3) Identify residues key for Arabidillo function in vivo by reintroducing mutated Arabidillo transgenes into the arabidillo-1/-2 double mutant and analysing their subcellular distribution and ability to rescue the mutant phenotype or generate novel phenotypes 4) Identify and characterise proteins that interact with Arabidillo-1 and -2 using yeast two-hybrid screening. We have isolated a number of candidates that interact with the amino-terminal domain of Arabidillo-1. We will characterise these proteins further in yeast and, if appropriate, analyse their function in Arabidopsis using molecular genetics. We will perform further two-hybrid screens using the carboxy-teminal domain of Arabidillo-1, and the corresponding regions of Arabidillo-2, as baits. These experiments help will elucidate the cellular mechanisms by which a novel protein signalling network regulates an agronomically important process, and will compare the mechanism of Armadillo-related protein function across kingdoms.