The Utility of aca-9 Mutant in Understanding Fertilization Events in Arabidopsis

1. The Utility of aca-9 Mutant in Understanding Fertilization Events in Arabidopsis Pollen tube entry into micropyle Pollen tube navigation to synergids ? Pollen tube growth near synergids Pollen tube growth arrest Pollen tube repulsion fer/ref Synergid degeneration ? aca9 ? gfa2? Pollen tube lysis within synergids aca9 ? Fertilization and seed set Fertilization and seed set Cheryl Parks1, Dami Dunatunga2, Michael Fritz3 and Ravi Palanivelu2 1Longview Global High School, Longview, TX, 2University of Arizona, Dept of Plant Sciences, Tucson, AZ 3Columbus School for Girls, Columbus, OH, • Aim • Confirm and explore if in vivo fertilization events can be recapitulated in vitro • Characterize spatial and temporal interactions between aca9 pollen tubes and wild type synergid cells in an in vitro pollen tube guidance assay Conclusions aca-9 is a mutant in a plasma-membrane localized Ca2+ pump. This explains the poor growth patterns observed as these mutants cannot properly remove the Ca2+ from their cells in order to grow like wild-type pollen. Although a number of aca-9 mutant pollen tubes were observed targeting and arresting at the synergid cells, there were none observed bursting, which is less than the expected 50%. This may be due to the small sample size observed and a larger number of mutants will need to be screened before any true conclusion can be made regarding this observation. Repulsion appeared to be normal in both the wild-type and mutant assays, which does confirm what was previously suspected that the aca-9 mutant does not adversely affect this fertilization event. Results In the wild-type assays, the pollen tubes behaved as expected with synergid targeting, pollen tube arrest, pollen tube burst and finally synergid degeneration (arrow head, Figure 3). Repulsion was always normal. However, with the aca-9 mutant pollen only synergid targeting and arrest were observed (note the lack of synergid degeneration and pollen tube burst in Figure 4) and repulsion appeared to be normal in all samples. Of the 10 aca-9 samples with synergid targeting and pollen tube arrest, none of them displayed pollen tube burst though 50% were expected to have pollen tube burst. This discrepancy could be due to the small sample size. While reviewing the aca-9 mutant pollen tube growth data, we made the following observations that were not previously reported. First, fewer pollen tubes exited the pistil even though ample pollen was used to pollinate each pistil. Second, the rate of aca9 pollen tube growth was slower than wild-type, thus creating a need to extend the time-lapse from 6 hours to 8 hours. Third, aca9 pollen tubes generally appear to be much thinner, therefore, they are harder to observe and capture during the time-lapse imaging. • Method In vitro Pollen Tube Guidance Assay • Unfertilized pistils are excised with the remaining ovary being removed and discarded. • Each pistil is pollinated with RFP-labelled pollen. • The cut end of the pistil is placed in a glass petri dish containing growth medium for 1 hour (Figure 1). • Each pistil is carefully laid horizontally on the growth medium for an additional 3 to 4 hours until the tubes begin to emerge from the bottom of the pistil. • GFP-labelled ovules are excised from an unfertilized ovary and carefully placed approximately 5-6 mm from the bottom of the pistil. • Time-lapse photography of both GFP and RFP isperformed at 10 minute intervals for 6-8 hours. • Movies are created using merged GFP/RFP images. Introduction Arabidopsis thaliana is a commonly used model organism for those studying plants. These plants are fast growing with rapid reproduction rates and are easy to maintain. Self-pollination is its primary mode of reproduction allowing for easy pollination manipulation. Sequencing of the entire genome of Arabidopsis is complete, thereby making genetic manipulation and mutant identification straightforward. Normal Fertilization Events: The Palanivelu Lab has been working with a number of Arabidopsis mutants effecting different fertilization events. Figure 3. The wild-type in vitro pollen tube guidance assay shows the pollen tube (red) approaching and then targeting the synergid cell (green). The pollen tube arrests at the synergid cell, then bursts and the synergid cell degenerates. From left to right, these pictures are at time-points 0 min,, 60min, 160min, and 260 min since the start of the image capture. (min = minutes). Figure 1. Glass petri dish with pollinated pistils and excised ovules. Classroom Implementation Movies available from the Palanivelu Lab website will provide students the opportunity to measure pollen tube growth as a function of time and compare wild-type pollen tubes to mutant pollen tubes. The data can then be used to generate and explore hypotheses regarding how the growth rate compares to the estimated growth rates in other plant species. Students may conduct explorations in ecology, evolution, and phylogenetics. Wild Type Mutant LAT52:DsRed (+/+) ACA9:DsRed (aca9/aca9) Acknowledgements I want to thank Dami for all of her help and patience with me, especially when I broke our well-prepared petri dish of mutants just before we started our time-lapse assay. We went through a lot of tiny flowers to get our data. Thank you to Ravi for all of his input and guidance along the way. He has been very helpful and I feel very honored to have been able to work in his lab. Thanks to the rest of the Ravi lab and iPlant for all their help along the way. Figure 4. aca-9 pollen tubes (red) are observed targeting the synergid cell (green) and arresting; however, there is no pollen tube burst or synergid degeneration. From left to right, these pictures are at time-points 0 min, 70 min, 250min, and 390 since the start of the image capture. (min = minutes). ms1 (male sterile 1) ms1 (male sterile 1) Pollen tube navigation to synergids Pollen tube growth near synergids DD3:GFP WT DD3:GFP WT Pollen tube growth arrest ? Synergid degeneration initiation Figure 2. The pollen, pistil and ovules types for the wild-type and mutant assays. Pollen tube lysis within synergids