Elucidating the mechanism of organ size control by KLU-dependent intercellular signalling

Daisies are always smaller than sunflowers, while cows are always larger than sheep. As these examples highlight, plants and animals grow to characteristic, species-specific sizes that are controlled by their developmental genetic programmes. Understanding how the size of organisms and their organs is determined is an important goal of basic biology, which will allow the rational manipulation of growth and size in economically relevant species. Although several genes have been identified that influence organ size, the fundamental problem of how a growing organ can measure its size is still unresolved. We have recently shown that the cells at the margins of an organ play a particularly important part in determining its size. These marginal cells produce a small molecule acting as a mobile growth regulator that can move into the organ and maintain cell proliferation. For purely geometric reasons the margin of the organ grows more slowly than the overall area, suggesting that the growth regulator is diluted as the organ increases in size. This offers a simple means for measuring organ size via the concentration of this growth regulator. In this view, cell proliferation arrests, once the concentration of the growth regulator falls below a critical value when the organ reaches a certain size. This model is similar to current ideas about how the size of animal organs, for example fly wings, is controlled, suggesting that ultimately plants and animals use the same principle to measure organ size. The production of this presumed signal requires the activity of the KLUH (KLU) gene, which is only active at the margins of the organs and provides an excellent point of entry for further studying the control of plant organ growth. The aim of this proposal is to gain a more detailed understanding of how the KLU-dependent growth regulator controls organ size. To this end, we will focus on four questions. 1. How mobile is the KLU-dependent growth regulator? The model proposed above for measuring organ size assumes that the growth regulator is highly mobile We will test this by monitoring the response of cells to the growth regulator, to see how it spreads in organs after its production is switched on locally. 2. Is there an assembly line of proteins to make the active growth regulator? We have found that a number of related, similar proteins are activated when the KLU gene is switched on. This suggests that these other proteins are also involved in making or breaking down the growth regulator. We will test this by generating plants that either have too much or too little of these proteins and studying the effect on organ size. 3. Which small molecule(s) are modified by the KLU protein? As a first step to finding the active growth regulator, we will identify the small molecule(s) that are modified by KLU, by comparing the amounts of different small molecules before and after KLU has been switched on. 4. Which other genes are necessary to generate or perceive the growth regulator? We will search for plants that can no longer respond to the growth regulator and then use these to identify additional genes necessary to perceive the signal. Together, these studies should yield important insights into how growing organs measure their size and decide when enough is enough.

Plant organ size is under tight genetic control. Recent studies in model species suggest that the timing of the transition from cell proliferation to cell expansion is a crucial step in regulating final organ size. However, the central question of how this transition is coupled to the attainment of a certain primordium size or cell number is still unanswered. Studies of the Arabidopsis cytochrome P450 KLUH (KLU) have indicated that the organ margins play a crucial role in controlling organ size. The KLU gene is only expressed in a subset of marginal cells in leaves and floral organs, where it appears to contribute to the generation of a mobile growth regulator distinct from the classical phytohormones that promotes cell proliferation throughout the organ. This suggests a simple model of measuring organ size via the concentration of the presumed growth regulator; the growth regulator is progressively diluted as the organ enlarges, until at a certain size its concentration can no longer sustain further proliferation. This proposal combines the complementary strengths of the Lenhard and Napier groups to gain a better understanding of how the KLU-dependent growth regulator controls organ size. Firstly, to test whether this signal is mobile enough for the above dilution model to be feasible, we will determine its mobility by imaging the expression of a specific downstream reporter gene. Secondly, we will test the hypothesis of a metabolic pathway leading to the active growth signal that involves additional, KLU-responsive cytochrome P450 encoding genes. Thirdly, the substrate(s) and product(s) of KLU will be isolated by a metabolomic approach. Fourthly, additional genes required for generating, perceiving and transducing the presumed growth regulator will be isolated by screening for mutants that suppress the transcriptional response to induced KLU activity.