PIRL1 and PIRL9, encoding members of a novel plant-specific family of leucine-rich repeat proteins, are essential for differentiation of microspores into pollen.

Plant intracellular Ras-group-related leucine-rich repeat proteins (PIRLs) are a plant-specific class of leucine-rich repeat (LRR) proteins related to animal and fungal LRRs that take part in developmental signaling and gene regulation. As part of a systematic functional study of the Arabidopsis thaliana PIRL gene family, T-DNA knockout mutants defective in the closely related PIRL1 and PIRL9 genes were identified and characterized. Pirl1 and pirl9 single mutants displayed normal transmission and did not exhibit an obvious developmental phenotype. To investigate the possibility of functional redundancy, crosses to generate double mutants were carried out; however, pirl1;pirl9 plants were not recovered. Reciprocal crosses between wild type and pirl1/PIRL1;pirl9 plants, which produce 50% pirl1;pirl9 gametophytes, indicated male-specific transmission failure of the double-mutant allele combination. Scanning electron microscopy and viability staining showed that approximately half of the pollen produced by pirl1/PIRL1;pirl9 plants was inviable and severely malformed. Tetrad analyses with qrt1 indicated that pollen defects segregated with the double-mutant allele combination, thus demonstrating that PIRL1 and PIRL9 function after meiosis. Pollen development was characterized with bright field, fluorescence, and transmission electron microscopy. Pirl1;pirl9 mutants stopped growing as microspores, failed to initiate vacuolar fission, aborted, and underwent cytoplasmic degeneration. Development consistently arrested at the late microspore stage, just prior to pollen mitosis I. Thus, PIRL1 and PIRL9 have redundant roles essential at a key transition point early in pollen development. Together, these results define a functional context for these two members of this distinct class of plant LRR genes.