FRIMP1 and FRIMP2: novel membrane proteins required for light-regulated development of Arabidopsis

The ability of a plant to respond to its light environment is critical to its survival. Light controls many aspects of plant growth and development and is important throughout the plant life cycle. Examples of responses under the regulation of light include germination, the development of green, photosynthesizing seedlings, regulation of the architecture of the plant and control of flowering time. All of these processes are crucial to agricultural productivity and an understanding of how they are regulated has great long-term importance. Plants have a range of photoreceptors that perceive light including the phytochromes that absorb red (R) and far-red (FR) light and the cryptochromes and phototropins that respond to blue/UVA. Much of what we know about how plants respond to light has come from studies on the model plant Arabidopsis thaliana that has five phytochromes. How these phytochromes pass on their light signal to regulate plant development has been an area of great interest in recent years. We now know that in light all phytochromes relocate from the cytoplasm of the cell to the nucleus, the organelle that contains the majority of the cell's genetic information. Once in the nucleus they interact with a number of signalling proteins to change the expression of many genes that lead to changes in plant growth and development. However, this is not the full story, and there is also some biochemical and physiological evidence that phytochrome signals to proteins in the cytoplasm and in cellular membranes. To date though there is little direct genetic evidence to back up a role for membrane proteins in phytochrome regulation of plant development. We have attempted to address this anomaly by trying to identify and characterize membrane proteins with a role in light regulation of plant development. To do this we have first identified predicted membrane protein genes that are light regulated by examining data sets of all light-regulated genes in Arabidopsis. Using this approach we have identified a number of membrane transporter genes that appear to have a role in seedling development. We have also identified some membrane proteins of unknown function and one of these is a FR light-induced membrane protein we have called FRIMP1. FRIMP1 and its close counterpart, FRIMP2, appear to be important for both seedling development and leaf development in Arabidopsis. We have identified frimp1 and frimp2 mutants that lack the FRIMP1 and FRIMP2 proteins and these mutants show a long hypocotyl under FR light and large cotyledons under R light, indicating that FRIMP1 and FRIMP2 are required for normal development of these plant tissues in the light. Interestingly, the FRIMP proteins are members of a completely new membrane protein family with close relatives in all multicellular eukaryotes including humans. As they have not been investigated in any of these organisms, what we learn about them in plants may be of much broader significance. The main aim of this project is to understand the function of FRIMP1 and FRIMP2 in response to light by determining all of the physiological processes they regulate and where they are located. We will do this by examining in detail the physiological responses of the frimp1 and frimp2 mutants, a frimp1frimp2 double mutant we have produced, and plants in which the levels of FRIMP1 and FRIMP2 proteins have been artificially increased. This will tell us the full range of responses in which FRIMP1 and FRIMP2 are involved. We will also determine what genes show altered expression in frimp1 and frimp2 mutants and use this information to find out how FRIMP1 and FRIMP2 interact with different signalling pathways within the plant. Finally, we will use two types of reporter proteins to show where FRIMP1 and FRIMP2 are located in the plant and also where they are within the cell. We will then be in a position to develop testable hypotheses about how FRIMP1 and FRIMP2 function at the molecular level.

To identify key membrane proteins involved in mediating the critical process of seedling de-etiolation we have used microarray analysis to screen for membrane protein genes that are regulated by light. Using this approach, we identified a FR light-induced membrane protein, FRIMP1, that is part of a new and highly conserved membrane protein family in multicellular eukaryotes. KO mutants of FRIMP1 and its close Arabidopsis homologue FRIMP2 have a long hypocotyl under far-red light and large cotyledons under red light, indicating these proteins are required for phytochrome signalling. The frimp mutants also exhibit a range of leaf abnormalities in mature plants with frimp1 showing a strong epinastic response and frimp2 an unusual leaf shape. Both mutants have more leaves than WT plants. The aim of this project is to understand the physiological function of FRIMP1 and FRIMP2 by determining what processes and genes they regulate and where they are located. Firstly, we will make a detailed phenotypic characterization of the frimp1 and frimp2 mutants, a frimp1frimp2 double mutant, and plants in which we have overexpressed FRIMP1 and FRIMP2 proteins. Secondly, we will determine whether FRIMP1 and FRIMP2 act directly in light signalling or whether they are light-responsive components of an alternative signalling pathway. We will do this initially by using a transcriptomics approach to determine the effect of FRIMP1 and FRIMP2 on R and FR light-regulated genes and comparing these data sets with profiles of genes regulated by other signalling pathways. Significant overlap of FRIMP-regulated genes with those regulated by other pathways may indicate a role for FRIMP proteins in this pathway and we will test this experimentally. Finally, to gain insight into the mechanism of FRIMP signalling, we will use enzyme and fluorescent reporter proteins to determine where FRIMP1 and FRIMP2 are localized at the tissue and cellular level.