As we learned in the introduction page, pollen tubes navigate past a variety of pistil cells, most likely with the help of attractive and repulsive cues, to deliver sperm to egg contained within ovules. The variety and inaccessibility of these cells and tissues has made it challenging to characterize the dynamics of pollen tube migration and identify the guidance signals. To overcome these shortcomings, we developed an in vitro assay in the model plant Arabidopsis thaliana to study pollen tube guidance to ovules (Palanivelu and Preuss (2006); BMC Plant Biology, 6:7).

The in vitro assay (Illustration 1) is set up as follows: after removing the upper portion of the pistil (the stigma and style), pollen was deposited on the stigma surface. After ~3 hours, pollen tubes emerged from the style, travelled across an agarose medium to excised ovules and successfully entered the micropyle (Figure 1). To facilitate pollen tube observation, especially after they enter the micropyle and are obscured by the opaque ovule integument cells, we used pollen that expressed GFP reporter under the control of the pollen-specific LAT52 promoter. Upon reaching the female gametophyte, these tubes burst and release a large spot of GFP, conveniently marking targeted ovules. Pollen tubes that grew within ~100 µm of an unfertilized ovule often made a sharp turn toward an ovule; of the tubes that grew within this range, ~50% successfully entered the micropyle.

Using this assay, we defined three signaling events that regulate pollen tube guidance in A. thaliana (Palanivelu and Preuss (2006); BMC Plant Biology, 6:7):

i) pollen tube acquire ovule targeting competence only after growing at least for some time on the female tissues,
ii) unfertilized A. thaliana ovules emit diffusible, developmentally regulated, species-specific attractants and
iii) ovules penetrated by pollen tubes rapidly release diffusible repellents to prevent additional tubes from entering them.

We are currently using this assay to isolate and characterize the signals that control each of the above mentioned three signaling events by employing a variety of traditional approaches (genetics, cell biology, biochemistry) in combination with global approaches (proteomics, microarray and metabolomics).