Regulation of plant cell size coupled to DNA content

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From bacteria to humans, cell size is important for many processes, including exchange of nutrients and signals, cell division, cell longevity and the function of specialised cells. Despite its importance, how cells achieve and maintain their genetically-controlled size has been a long-standing mystery. In a breakthrough in this field, we have recently found that plant cells adjust their own size using their DNA contents as an internal, size-independent standard. The mechanism relies on a protein (KRP4) that functions in the Retinoblastoma (RB), pathway, which controls cell proliferation in organisms ranging from plants to humans. We showed that KRP4 binds to chromosomes before cell division, whilst unbound KRP4 is destroyed. In this way, a fixed amount of KRP4 is released from the chromosomes of newly born cells, regardless of their size at birth. Consequently, these cells grow to the same target size, at which KRP4 is sufficiently diluted to allow progression to the next cell division. This discovery raises important further questions. First, the mechanism relies on changes in the way KRP4 interacts with chromosomes during different stages of cell division, but the details of how and why these changes happen are unknown. Revealing these details will help to uncover shared and divergent features of cell size control in different cell types and across different organisms. To achieve this aim, we will identify other proteins that interact with KRP4 at different stages of cell division, define the genomic sites where KRP4 accumulates, and use genetic methods to see the consequences of disrupting these interactions. Second, how is the mechanism adjusted during development to produce specialised cells with different sizes? For this question, we will focus on the formation of pores (called stomata) that regulate the exchange of water and carbon dioxide between the leaf and the atmosphere. We have several reasons to focus on stomata: the size of stomatal cells is remarkably uniform and important for photosynthetic efficiency and water use; the development of stomata is readily accessible for live imaging and is genetically very well characterised. To reveal how stomatal cell size is regulated, we will use quantitative live imaging to analyse the consequences of altering candidate regulatory genes, including those in the KRP4/RB pathway. Third, it has been observed for over a century that the size of cells reflects the size of their genome in a wide range of organisms, but the reason for this remains unclear. Does the use of DNA contents as a standard for cell size control explain why organisms with larger genomes tend to have larger cells? Conversely, do selective pressures on cell size constrain genome size during evolution? These questions are particularly important in plants, in which genome duplications have played a major role in evolution and domestication. To address them, we will test whether mutations in the KRP4/RB genetic pathway de-couple cell size from genome size. We will also collaborate with another group in Switzerland (Kerstin Bomblies, ETH), who study the role of genome duplication in plant evolution. In this collaboration, we will test whether selective pressure to re-adjust stomatal cell size after genome duplication has led to a re-calibration of KRP/RB-mediated cell size control. This part of the project will use population genomics methods and genetic analysis combined with live imaging to test whether genes under selective pressure are implicated in cell size control.