Integration of cell division and plant development: control of leaf growth and cell number by AINTEGUMENTA

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Plant leaves and flowers ('organs') form at the tips of shoots. At the very tip of the shoot is a microscopic structure like a small shallow dome-like structure, known as the shoot apical meristem. On the sides of this dome, small protrusions or 'primordia' form, that will grow into a new leaf. Cells in primordia divide rapidly for a time until the leaf is a couple of millimetres long and there is enough cells to make the final leaf, and then they stop dividing and expand. The growth of the leaf from the size of a few millimetres up to its fully mature size is therefore caused by expansion of existing cells, and not by more cells being formed. What controls the number of cells in the leaf and its eventual size? We aim to find answers to this question in the proposed project. What we know is that there are mutants that affect organ size and the number of cells in an organ. One identifies a gene known as AINTEGUMENTA (ANT). Mutations in ANT reduce the number of cells in organs and also have other effects in the flowers. Increasing the amount of expression of the ANT gene increases leaf size and the number of cells in the leaf. Genes like ANT are known as 'developmental regulators' because they control the growth and development of the plant. In particular, ANT acts to control the number of cells in the leaf and the its overall size. We also know that cell makes a decision to divide depending on the level of proteins known as D-type cyclins (CYCDs). However, there is no known link between CYCDs and developmental organ size regulators such as ANT. We therefore do not know how cell division is controlled in development. More generally, we do not understand how cells -the fundamental building blocks of organs and organisms- are controlled to produce larger structures. We believe that ANT regulates a particular CYCD gene called CYCD3;1 and have evidence to support this. Increasing ANT levels leads to increased expression of CYCD3;1, and altering levels of CYCD3;1 also affects the number of cells in leaves, in a similar way to the effects of altering ANT. These two observations suggests that ANT could work through CYCD3;1. The protein encoded by the ANT gene is a 'transcription factor' that binds DNA and controls other genes, so ANT protein could directly switch on the CYCD3;1 gene. The sequence of the binding site of the ANT protein is known and it is found in the promoter (controlling region) of the CYCD3;1 gene and not in the promoters of other CYCD genes. There is therefore a good likelihood that this may be the case. In this project, we are going to prove that the ANT protein regulates CYCD3;1 by binding its promoter, and also identify other genes regulated by ANT. This will be the first link between cell division and a developmental regulator in a plant, and will help us understand how the behaviour of cells is co-ordinated during development. First we are going to prove that the action of ANT depends on the presence of CYCD3;1. We can do this because we have a mutant in the CYCD3;1. We predict that ANT will have no effect when CYCD3;1 is missing, proving that CYCD3;1 is regulated by ANT. However it will not prove whether this a direct effect of ANT binding to the CYCD3;1 gene. Thus we will then prove that ANT binds to the CYCD3;1 promoter. Finally we wish to understand more about the other genes ANT controls in leaf development.