Exceptional segregation of Kanamycin resistance in Arabidopsis gametophytic factor 1 (gfa1) mutants: A genetic, developmental, and molecular analysis

Abstract

The molecular mechanisms underlying plant reproductive development are poorly understood. Segregation distortion has recently been employed as a screening criterion to identify genes required for the development and function of the gametophyte generation. This dissertation presents analyses of the genetic, developmental, and molecular basis of the segregation distortion used to identify one such Arabidopsis thaliana mutant, gametophytic factor 1 (gfa1). The genetic basis of segregation distortion was examined by quantifying transmission of two gfa1 alleles in reciprocal crossed to wildtype. Both alleles displayed a complete lack of transmission of the gfa1 allele through the female parent, and a significant reduction of transmission through the male parent. In addition, self-pollinated siliques had a reduced seed set phenotype characteristic of a female gametophytic mutation. Both alleles co-segregated with the seed set phenotype, indicating that mutations in the GFA1 gene were responsible for the seed set phenotype. The developmental basis of gfa1 segregation distortion was identified by first examining gfa1-1 male (MG) and female (FG) gametophytes using confocal microscopy. Approximately half of the FGs scored in gfa1-1/+ siliques showed abnormalities in the number and position of the nuclei within the FG, as well as defects in cellularization. Although there were no obvious abnormalities in gfa1-1 pollen grains, analysis of the position of kanamycin-resistant seeds in gfa1-1/+ siliques showed that fewer gfa1-1 pollen fertilized ovules at the basal (receptacle) end of the silique. Taken together, these results suggest that gfa1 mutations affect primarily FG development and MG function. The GFA1 gene was cloned and found to belong to the U5-116 kD small nuclear ribonucleoprotein family of RNA splicing factors first identified in mammals and yeast. In addition, a second gene-family member, GFA1-like (GFL), was found in the Arabidopsis genome. GFA1 expression was found throughout plant development, and evidence of GFL expression was also found. The paternal GFA1 allele did not appear to be silenced during early seed development. Therefore, we conclude that a mutation in an essential splicing factor is responsible for the reduced transmission and seed set phenotypes observed in gfa1 mutants.