Plant & Animal Genomes XIV Conference

January 14-18, 2006
Town & Country Convention Center
San Diego, CA

Using Maize Gamete ESTs To Identify Arabidopsis Gamete-Specific Promoters

Sheila McCormick , Michele Engel , Heping Yang , Rachel Holmes-Davis

  Plant Gene Expression Center USDA/ARS-UC-Berkeley 800 Buchanan St. Albany, CA, 94710 USA

Double fertilization in angiosperms was discovered in 1898, but the proteins that mediate gamete recognition and fusion are unknown. We constructed a cDNA library from maize sperm cells (Engel et al., Plant Journal 34:647-707) and sequenced 5000 randomly selected cDNAs. To examine the female contribution to fertilization, we constructed a maize embryo sac cDNA library and sequenced ~7000 ESTs. Both libraries are useful for gene discovery, since many ESTS correspond to hypothetical proteins in fully sequenced genomes. RT-PCR was used to examine the expression of selected transcripts. We identified 5 transcripts that appeared to be present only in the sperm cells, and 19 transcripts that appeared to be embryo sac-specific. We used in situ hybridization to determine the cellular expression patterns in the embryo sac. So far we have identified a synergid cell-specific transcript, another that is expressed in both the synergids and in the central cell, and another that is specific to the central cell and egg cell. We identified Arabidopsis homologs for several of these genes, and tested their promoters for specificity using GFP in transgenic Arabidopsis plants. Two sperm promoters worked; the GFP-marked sperm can be visualized entering the pollen tube during in vitro germination (Engel et al., Plant Physiology 138: 2124-2133). So far we have one promoter that gives egg expression, one that gives synergid expression, and one that gives central cell expression. These promoters can be used for imaging and for testing whether candidate proteins play roles in double fertilization.