Background:

After a pollen grain lands on the stigmatic surface of the pistil (female structure of the flower), it forms a pollen tube — a long polar process that transports all of the cellular contents, including the sperm. Pollen tubes invade the pistil and migrate past several different cell types, growing between the walls of the stigma cells, travelling through the extracellular matrix of the transmitting tissue, and finally arriving at the ovary, where they migrate up the funiculus (a stalk that supports the ovule), and enter the micropyle to deliver the two sperm cells–one fertilizes an egg and other the central cell (Figure 1). Typically, only one pollen tube enters the ovule through an opening called the micropyle, terminates its journey within a synergid cell, and bursts to release sperm cells. A pollen tube's journey (Illustration 1) to an egg cell within the pistil therefore involves a series of cell-cell interactions such as attraction, repulsion and adhesion.

What do we do:

"How are pollen tubes precisely guided to their target"– is the question we are trying to solve. Hence, we are focused on identifying and characterizing the guidance signals generated by the A.thaliana pistils to guide pollen tubes to their final target in the embryo sac (Figure 1).

Why do what we do:

Basic Research benefits: Characterization of pollen tube guidance in A. thaliana is important as it focuses on a process that is (i) very unique to plants, (ii) poorly understood at the molecular level, (iii) amenable to genetic, cell biological and biochemical techniques, and (iv) a rapid way to identify novel plant signals that allow communication between cells possible despite their thick extracellular walls

Applied benefits: If pollen tube growth and guidance is defective, seeds are not produced. 80% of world's staple food is derived from seeds of crop plants. So studying this process is agriculturally very important. By understanding at the molecular level how seeds are formed we are equipping ourselves with knowledge using which we could (i). improve seed yield, (ii). regulate inter species hybridizations, and there by generate novel plant hybrids and (iii). contain pollen spreading from genetically modified crops–a possibility that will reassure concerened public and regulatory agencies.

Research:

The variety and inaccessibility of pistil 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.

We are currently using this assay to isolate and characterize the signals that control each of the following three signaling events by employing a variety of traditional approaches (genetics, cell biology, biochemistry) in combination with global approaches (proteomics, microarray and metabolomics). Please refer to the indicated papers from our lab for additional details:

i) pollen tube germination and growth are modified upon growth on the female tissues (Qin et al 2009; Qin et al 2011),
ii) unfertilized A. thaliana ovules emit diffusible, developmentally regulated, species-specific attractants (Palanivelu and Preuss, 2006; Yetisen et al 2011), &
iii) pollen tube arrest growth in the synergid cell prior to releasing the two sperm cells (Nikalova et al 2007; Tsukamoto et al 2010).

Research in the Palanivelu lab is supported by funding from National Science Foundation (NSF) grants to Dr. Palanivelu (IOS 0723421 - 2007-2010), (IOS 1045314 - 2010-2012), and (IOS 1146090 - 2012-2017).