Molecular genetic analysis of the Arabidopsis reproductive actins
Pawloski, Lucia Cardenas
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Microfilaments are thin fibers made of actin dimers that are responsible for plant cellstructure and maintenance, organellar localization, nutrient transport, cell division, and tipgrowth. Arabidopsis has eight actin genes that are divided into two classes with distinctexpression patterns: the vegetative actin class is expressed in vegetative tissues, and thereproductive actin classis predominantly expressed in pollen, pollen tubes, ovules, anddeveloping embryos. We utilized T-DNA mutations and RNA interference (RNAi) technologyto knock down reproductive actin expression and observe the phenotypic effects produced.The RNAi system was first tested with the profilin isovariant, PRF1. RNAi wassuccessful in disrupting PRF1 expression, reducing protein levels to less than 10% of normalexpression, and produced phenotypes that included delayed germination, smaller seedlings andleaves, and delayed bolting compared to wild type, suggesting that PRF1 is important forgermination and plant development. RNAi was then applied to target expression of the four LatePollen Actin (LPA) genes: ACT1, ACT3, ACT4, and ACT12. Total LPA expression was reducedby 50-90% compared to wild type levels.Dissected LPA RNAi siliques were semi-sterile. In vitro pollen germination assays revealed asevere decrease in pollen grain production and germination, indicating that the LPAs are criticalfor pollen production, pollen tube germination, and fertilization.The final target for mutagenesis was the reproductive actin gene, ACT11. Four T-DNAinsertion mutants were isolated: act11-2, act11-3, act11-4, and act11-5. Seedlings from both theACT11 T-DNA knockout and the RNAi knockdown lines germinated later, were smaller thanwild type seedlings, and depended on sucrose to germinate and grow. Dissected ACT11 mutantsiliques produced more unfertilized ovules and less seeds compared with wild type plants.Therefore, ACT11 is essential for fertilization and early seedling development.