Characterisation of gametophytic mutants affecting pollen function in Arabidopsis thaliana

The progamic phase of plant reproductive development involves events from pollen germination to gamete fusion. Physiological studies suggest that complex mechanisms are involved in this pathway. In order to identify gametophytic genes which function in this pathway, an insertional mutagenesis screen based on segregation ratio distortion was performed on a population of 4094 independent dSpm transposon insertion lines. This screen did not produce any mutants of interest, but two mutants, seth4 and seth7, were identified from a similar screen of Ds transposon insertion lines, seth4 and seth7 showed stably reduced segregation ratios arising from reduced gametophytic transmission and reciprocal crosses showed no (seth4) or severely reduced (seth7) transmission of the antibiotic resistance marker only through pollen. In both mutants, pollen morphology was normal but pollen germination was severely affected. In seth4, the insertion disrupted the coding region of a gene encoding an armadillo (ARM) repeat protein. In seth7, the transposon was inserted within the 3'-UTR of a gene encoding a putative serine/threonine protein kinase. A wild-type copy of SETH4 complemented the seth4 mutation and restored male transmission. SETH4 is the founding member of a discrete Arabidopsis gene family that contains two SETH FOUR- LIKE genes, SFL1 and SFL2, SETH4 was found to be preferentially expressed in the male gametophyte while SFL1 and SFL2 were expressed exclusively in the sporophyte. SETH4, SFL1 and SFL2 proteins, when fused to GFP, suggest cytoplasmic location in transient expression assays. In this work two mutants identified as essential for the male gametophyte during the progamic phase have been phenotypically characterised. SETH4 was analysed using genetic, molecular and bioinformatic analyses and is proposed to be part of a novel molecular pathway controlling cellular growth in the gametophyte.