Arabidopsis Is Essential for Male and Female Gametogenesis and Is Regulated by Alternative Splicing.

Plant intracellular Ras-group leucine-rich repeat (LRR) proteins (PIRLs) are related to Ras-interacting animal LRR proteins that participate in developmental cell signaling. Systematic knockout analysis has implicated some members of the Arabidopsis () family in pollen development. However, for , no bona fide knockout alleles have been recovered, suggesting that it may have an essential function in both male and female gametophytes. To test this hypothesis, we investigated expression and induced knockdown by RNA interference. Knockdown triggered defects in gametogenesis, resulting in abnormal pollen and early developmental arrest in the embryo sac. Consistent with this, was expressed in gametophytes: functional transcripts were detected in wild-type flowers but not in () mutant flowers, which do not produce gametophytes. A genomic PIRL6-GFP fusion construct confirmed expression in both pollen and the embryo sac. Interestingly, is part of a convergent overlapping gene pair, a scenario associated with an increased likelihood of alternative splicing. We detected multiple alternative mRNAs in vegetative organs and mutant flowers, tissues that lacked the functionally spliced transcript. cDNA sequencing revealed that all contained intron sequences and premature termination codons. These alternative mRNAs accumulated in the nonsense-mediated decay mutant , indicating that they are normally subjected to degradation. Together, these results demonstrate that is required in both male and female gametogenesis and suggest that sporophytic expression is negatively regulated by unproductive alternative splicing. This posttranscriptional mechanism may function to minimize PIRL6 protein expression in sporophyte tissues while allowing the overlapping adjacent gene to remain widely transcribed.