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

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.

The control of organ size in plants is poorly understood, but is clearly linked to cell number and cell expansion, since ultimate organ size is the result of the combined effect of these processes. Leaf development is characterised by a mitotic phase of cell proliferation, followed by the cessation of mitotic cycles and cell expansion linked to endoreduplication. However, to date there have been no molecular links made between the regulation of cell division and developmental regulators. In the case of Arabidopsis leaves there appears to be an overall genetic control of size, since within certain limits the increase or decrease in cell number is corrected by compensatory changes in cell size. The developmental regulator AINTEGUMENTA (ANT) acts as a regulator of the organ size checkpoint, since loss-of-function alleles result in smaller organs and overexpression results in larger organs containing more cells. Increased ANT expression is specifically associated with ectopic expression of the D-type cyclin gene CYCD3;1. We have found that CYCD3;1 shows an overlapping expression pattern with ANT in leaf primordia, and that alterations in CYCD3;1 levels also result in changes in the number of cells in developing leaves. Hence it has been proposed that ANT is an upstream regulator of CYCD3;1. We have further proposed that CYCD3;1 acts to maintain cells in the mitotic cycle, since ectopic expression increases cell number in leaves 20-fold, whilst also inhibiting both endoreduplication and cell expansion. Whilst increased CYCD3;1 results in additional cells in leaves, their smaller size does not increase organ growth. In contrast, ANT increases cell number, but due to its role as an organ size checkpoint is also able to regulate additional targets that promote normal cell expansion and hence increase overall organ size. Together these data suggest that ANT acts through CYCD3;1 in controlling cell number in organs. The ANT binding site has recently been defined by affinity selection, and we have found that the CYCD3;1 promoter contains a consensus ANT binding site. The promoter of no other CYCD gene expressed in the shoot apex contains this site. Moreover this site is contained within a promoter fragment which we have shown by deletion still maintains the normal regulatory pattern of CYCD3;1 expression, consistent with ANT acting as an important regulator of CYCD3;1. Most studies on ANT have focussed on its role in floral development, where the mutant phenotype is more dramatic because of additional roles of ANT in floral tissues in regulating other floral regulators. Here we propose to use leaves to study its role in cell number control in developing organs, and to establish the genetic and molecular links between ANT and CYCD3;1 regulation. We will use inducible regulation of ANT in its normal expression domain in ant and cycd3;1 mutant backgrounds to analyse in detail the role of ANT and the genetic requirement for CYCD3;1. We will define the molecular binding between ANT and the CYCD3;1 promoter in vitro and in plants, and identify additional targets of ANT which define its role as a organ size checkpoint in addition to the regulation of cell number through CYCD3;1. Despite extensive analysis of the roles of cell cycle regulators in general, and CYCD genes in particular, no molecular links between developmental regulators and the cell cycle have been found. The proposed regulation of CYCD3;1 by ANT is thus the first such direct link of the molecular integration of cell division processes with development.