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

Abstract

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.

Effect of sporophytic PIRL9 genotype on post-meiotic expression of the Arabidopsis pirl1;pirl9 mutant pollen phenotype

“…However, after that initial period of normal development, pirl1;pirl9 microspores produced by this genotype consistently arrest and are unable to complete the first pollen mitosis. Consistent with the late-microspore onset of the mutant phenotype, analysis of pirl1;pirl9 phenotype segregation in qrt1 tetrads confirmed that abnormalities originate after meiosis, thus indicating that these PIRL genes have essential function in the developing haploid microspores (Forsthoefel et al 2010).…”

“…Pirl T-DNA knockout alleles used in this study have been described previously and shown to be knockout alleles based on reverse transcription-PCR analysis of disrupted loci (Forsthoefel et al 2010). Pirl1-1 and pirl9-1 are in the Wassilewskija (Ws) ecotype, and pirl1-2 and pirl9-2 are in Columbia.…”

“…Plant genotypes for pollen transmission and phenotype analyses were determined by PCR using gene-specific forward and reverse primer combinations to detect wildtype alleles and T-DNA/PIRL primer combinations to detect mutant alleles, as described in Tax and Vernon (2001), using the primer combinations described in Forsthoefel et al (2010). Mature pollen for viability staining or scanning electron microscopy (SEM) was collected from open flowers from wild type, pirl1 or pirl9 homozygotes, or pirl1/PIRL1;pirl9 or pirl;pirl9/PIRL9 plants, which produce pollen segregating 50% for the double mutant genotypes.…”

“…Viability staining and fluorescence microscopy were carried out as previously described (Forsthoefel et al 2010). For SEM, mature flowers from PCR-genotyped pirl1-1/PIRL1;pirl9-1 or pirl1-1;pirl9-1/PIRL9 plants or from wild-type controls were excised and dried for at least 48 h. Pollen was transferred to double-sided carbon tape on a stub, and samples were viewed and digitally imaged using an FEI Quanta200 scanning electron microscope with variable vacuum and Peltier cold stage capabilities (FEI, Hillsboro, OR, USA).…”

“…Analysis of T-DNA knockout mutants has shown that PIRL1 and PIRL9 are genetically redundant and essential for pollen development (Forsthoefel et al 2010). In pirl1;pirl9 microspores produced by pirl1/PIRL1;pirl9 parent plants, microspore growth, exine development, and vacuolization at first appear normal.…”