Role of cyclin-dependent kinase inhibitors (KRPs) in root meristem activation

Plant growth is different to that of animals in several ways. Plants grow continuously forming new leaves and flowers throughout their lifespan, and this property depends on stem cells located in the tips of shoots and roots in regions known as meristems, associated with which most cell division occurs. Another important difference to animals is that the mature embryo is packaged into the seed in a quiescent state, allowing dispersal and survival. Under favorable conditions, germination occurs. At this point, cells within the meristems at the tip of the seedling shoot and root must start dividing again. A few days later, lateral or side roots form from the main root of the seedling. These arise from specialised cells which resume division to form a new meristem that will then produce the lateral root. These are important in anchoring the plant in the soil, and its ability to take up water and nutrients. Together, germination and lateral root formation are essential aspects of the ability of plants and crops to grow and compete successfully, and these key processes in seedling establishment both require the activation of cell division. The research in our lab focuses on how cell division in plants is regulated. Cell division is controlled by a series of events called the cell cycle. The main check-point where the cell commits itself to division is called G1 and can be regulated by proteins called cyclins and KRPs. The particular type of cyclin involved is called CYCD. Cyclins stimulate cell division, whereas KRP proteins can bind to cyclins and thereby inhibit cell division. Although KRP proteins have been studied, we do not know much about their role in the processes required for activation of cell division in the roots. This is because existing studies have overexpressed these genes, resulting in very high levels that may mask their specific functions. In contrast, we have studied mutants of these genes and found that they have specific roles. One is required for the timing of cell division in germination, and a second controls the number of lateral roots produced. In one mutant, seeds therefore germinate faster, in the second the seedlings have more lateral roots. We have also discovered a new function for KRP proteins. We found that in one mutant, a specific CYCD protein is no longer localized in the nucleus but in the cytoplasm of the cell. This means that the KRP protein is required to carry the CYCD into the nucleus where it acts. Since the remaining six KRP genes are still present in the mutant, this suggests a specific interaction between KRP and CYCD that can explain the effects we have observed. In this project, we want to understand how KRP genes and the proteins they encode affect the activation of cell division that plays such a important role in early seedling growth. We will study the different mutants and how they are affected in germination and lateral root formation. We will analyse how they function and in particular focus on the new role we have discovered for KRPs in the nuclear transport of CYCD proteins. As a result, we will have a better understanding of how cell division, germination and lateral root formation is controlled by cell cycle regulatory proteins.

The initiation of cell division is critical both for activation of the root apical meristem in germination, and subsequently when lateral roots form. This reinitiation is likely to involve cell cycle activation by cyclin D (CYCD)-containing kinases. CYCD proteins are synthesized in response to extracellular signals and are rate-limiting for cell division. CYCD activity in plants is regulated by a family of cyclin-dependent kinase inhibitor (KRP) proteins related to the animal KIP/CIP family, which bind and inhibit CYCD complexes. To date, KRP genes have been analysed only by overexpression, and whilst this inhibits cell cycles, it has provided little or no evidence for specificity of action amongst the seven KRP genes of Arabidopsis, and no phenotype has been ascribed to a krp mutant. Using reverse genetics, we have identified distinct KRP genes rate-limiting for activation of the primary root meristem during germination and of lateral meristems during lateral root formation. Mutants in one krp gene show accelerated activation of the root meristem during germination and more rapid root emergence. We have identified the candidate CYCD protein with which it interacts. Mutants in a second krp gene show increased numbers of lateral roots when grown on medium without auxin, but show a reduced response to exogenous auxin addition. We found that this KRP protein is required for nuclear transport of a specific CYCD, which shows dose dependent effects on lateral roots. This suggests KRP genes have key roles in modulating major developmental decisions in plants, and have a newly discovered and essential role for nuclear transport of CYCD proteins. Here we propose an integrated developmental, cellular, biochemical and molecular analysis of KRP function in Arabidopsis, focusing on activation of the primary and lateral root meristems. We seek to understand how KRP proteins modulate the activity of CYCD proteins not only by inhibition but also intracellular localisation.