Gametophyte Function (NSF 2010)

NSF 2010 Project

(PI: Venkatesan Sundaresan, UC Davis; Co-PI: Sheila McCormick, UC Berkeley)

A large-scale mutant screen using Ds insertion lines resulted in the identification of about 300 lines that exhibited aberrant transmission of the linked Kanamycin resistance gene. Some lines exhibited aberrant transmission through the male parent, some through the female parent, and some through both parents. We presumed that the aberrant segregation in these lines was due to disruption of a gene that is important for gametophyte development or function. The results for the lines that have aberrant transmission through the female are posted at the Sundaresan lab.

We started with 109 lines whose transmission through the male was aberrant. We didn't continue analysis with 42 lines because they had inconsistent transmission ratios, multiple insertions, defects that were mostly on the female side, or were embryo lethal.

For the remaining 67 lines, we confirmed the Ds insertion site by PCR or, when necessary, by TAIL-PCR. We determined the transmission ratios, and crossed each line to quartet, in order to determine if the mutation is gametophytic. For those lines whose pollen appeared normal at maturity, we tested for pollen germination defects, in vitro and in vivo. For those lines that had aborted pollen, we determined when during pollen development the arrest occured.

The work is now published:

• Boavida, L.C. Shuai, B., Yu, H-J., Pagnussat, G.C., Sundaresan, V. and McCormick, S. 2009. A collection of Ds insertions associated with defects in male gametophyte development and function in Arabidopsis thaliana. Genetics 181: 1369-1385. doi: 10.1534/genetics.108.090852. Epub 2009 Feb 23

Abstract: Functional analyses of the Arabidopsis genome require analysis of the gametophytic generation, since about 10% of the genes are expressed in the male gametophyte and about 9% in the female gametophyte. Here we describe the genetic and molecular characterization of 67 Ds insertion lines that show reduced transmission through the male gametophyte. About half of these mutations are male gametophytic-specific mutations, while the others also affect female transmission. Genomic sequences flanking both sides of the Ds element were recovered for 39 lines; for 16 the Ds elements were inserted in or close to coding regions, while 7 were located in intergenic/unannotated regions of the genome. For the remaining 16 lines, chromosomal rearrangements such as translocations or deletions, ranging between 30 kb to 500 kb, were associated with the transposition event. The mutants were classified into five groups according to the developmental processes affected; these ranged from defects in early stages of gametogenesis to later defects affecting pollen germination, pollen tube growth, polarity or guidance, or pollen tube-embryo sac interactions or fertilization. The isolated mutants carry Ds insertions in genes with diverse biological functions and potentially specify new functions for several unannotated or unknown proteins.