Leucine-Rich Repeat proteins (LRRs) constitute a large gene/protein superfamily found in diverse species. LRR domains in these proteins bind specifically to other proteins, and many LRR proteins act in cell signaling networks. PIRLs (Plant Intracellular Ras-group-related LRRs) are a novel family of plant LRRs more closely related to a class of LRRs from animals and yeast than to previously described types of plant proteins. Some related LRR proteins from animals are involved in cell signaling pathways important in cancer and development. This project focuses on PIRL1 and PIRL9, two closely-related PIRL genes from the model plant Arabidopsis thaliana. Preliminary experiments with gene knockout mutants have shown that these genes have redundant and essential roles in pollen development, a process crucial for plant reproduction. The proposed research will employ a combination of genetic, microscopy, and molecular strategies to obtain an integrative understanding the functions of PIRL1 and PIRL9 in plant reproduction, by identifying where these genes act in the context of the whole plant and defining the cellular pathways and biochemical interactions in which they take part. Specific objectives: 1) complete genetic studies of pirl1:pirl9 double mutants and mutant pollen; 2) more precisely define how pirl mutations impact pollen development; 3) determine the sub-cellular locations of PIRL proteins; and 4) investigate biochemical functions by identifying PIRL-binding proteins. An additional objective is to train future U.S. scientists by providing undergraduate research opportunities in post-genomic plant biology: this research will be carried out at an undergraduate college with a high percentage of students who pursue graduate study and careers in biology. By providing insights into plant signal transduction and the genetic requirements for pollen development, this work should ultimately contribute to effective manipulation of plant growth, with benefits for agricultural productivity and plant breeding.
This was a Research at Undergraduate Institutions (RUI) project. The objectives included: 1) research into the functions of recently discovered plant genes called PIRLs, and 2) training of future U.S. scientists by providing transformative student research experiences at Whitman College, one of the top undergraduate colleges in the western U.S. Plant Intracellular Ras-group LRRs (PIRLs) are a family of genes our laboratory identified in Arabidopsis thaliana, a small weed that's used as the major model system for plant genome research. Similar genes are found in all higher plants, including crop species. The PIRLs encode leucine-rich repeat (LRR) proteins related to animal LRRs involved in cell signaling and gene regulation, some of which are involved in cancer-related pathways. They are truly novel plant genes that had not been functionally characterized before this project. This project originally proposed to focus mainly on two PIRLs, PIRL1 and PIRL9, which were hypothesized to function in pollen. We later expanded the study to include the closely-related PIRL2 and PIRL3 genes, and also did some work on two others, PIRL6 and PIRL8. Based on PIRLs' relationship to animal ras-group LRR proteins, we hypothesized that they contribute to aspects of plant development and reproduction. We succeeded in defining developmental functional contexts for five of the nine Arabidopsis PIRL genes, through genetic and microscopy analysis of mutant plants in which one or more of the genes had been knocked out. RESEARCH FINDINGS: We determined that four of these genes are important for pollen develoment, a process crucial for plant reproduction (and therefore food production). PIRL1 and PIRL9 have redundant functions and are absolutely essential for pollen development: pollen lacking both PIRL1 and PIRL9 stops developing at a critical step, right before cell divisions that normally produce a trinucleate pollen grain from a mononucleate microspore. In genetic experiments, we also found that development of mutant pollen can be affected by an intact PIRL9 gene in the parent plant, indicating that this gene is active in adult plants as well as pollen. Similarly, we discovered that PIRL2 and PIRL3 function in pollen development, although they do not appear to be as important as PIRL1 and PIRL9. Loss of either of these genes causes more subtle defects in pollen cell organization and growth in pollen that also lacks PIRL9. Genetic analysis of PIRL6 mutants also implicated this gene in plant reproduction, specifically for development of both male and female gamete-producing tissues (pollen and ovules). By analyzing RNAs produced from PIRL6, we also discovered that this gene's expression is complex, with different versions of the RNA produced in different plant organs. The importance of PIRL6 in both male and female gametophytes makes it a difficult gene to study with standard genetic techniques. Further progress with this gene will require alternative research strategies, such as genetic modification of plants to alter PIRL6 expression. We also identified a second function in plant reproduction for PIRL9: surprisingly, this gene also influences the timing of flowering. This is an important developmental transition at which plants switch from vegetative (leaf) growth to reproductive growth; it's an important agronomic trait in crop species. Plants that lack PIRL9 take a few extra days longer to start flowering. Our studies of gene expression in these pirl9 mutants indicated that some key previously-discovered genes that control flowering are expressed at lower levels, suggesting that PIRL9 can regulate these other regulators. These findings established a new direction for research into PIRL9 and the other PIRL genes. Contributions of these research findings: The research funded by this grant helped define the genetic components of develomental processes critical to plant reproduction and thus food production. It also contributed to a major goal of post-genomic plant biology: assigning functions to all plant genes identified by genome sequencing projects. We made fundamental discoveries about an intriguing but previously unstudied family of plant genes. This project demonstrated the value of in-depth, focused genetic studies for investigating new gene functions that may not be determined by large-scale genomic strategies. EDUCATION & TRAINING of FUTURE SCIENTISTS: Another important contribution of this RUI award was the training and development of future U.S. scientists. This award directly supported research experiences for 16 students. Six of these students are now in graduate programs pursuing MS or PhD degrees. Three went into teaching or science-related work in Americorps. Others are in employed in research labs or are in biology-related professional programs such as pharmacy or medical school. The research experiences funded by this grant helped jump-start the careers of these future scientists and medical professionals. This grant also contributed to education in another way, supporting development of an undergraduate teaching laboratory module that combines genomic database use with bench experiments, and allows straightforward integration of original research into college teaching laboratories.
