Location: Plant Gene Expression Center Albany_CA
Project Number: 5335-21000-037-00
Start Date: Mar 01, 2013
End Date: Feb 28, 2018
Objective 1: We hypothesize that protein complexes composed of receptor kinases and other interacting proteins will mediate cell signaling during pollen tube growth and pollen-pistil interactions. We further hypothesize that the components of the complexes will vary at different stages of pollen tube growth. To test this hypothesis we will use biochemical approaches such as co-immunoprecipitation and yeast two hybrid interactions, and in vivo imaging techniques, such as BiMolecular Fluorescence Complementation, to determine what proteins are present in the complexes at different stages of pollen tube growth, and how and when individual proteins participate in these complexes. These approaches have been successful in our previous studies. It is possible that particular interactions will fail to be confirmed biochemically, either because a third partner is necessary, or because the interaction is weak or transitory. If so, a genetic approach will be used to determine the functions of candidate proteins. Objective 2: We hypothesize that genes that encode pollen-specific proteins whose amino acid sequences are highly conserved across angiosperms will play important roles during pollen function. Conversely, we hypothesize that genes encoding pollen-expressed proteins that exhibit enhanced amino acid variation across angiosperms, or that are family-specific, might contribute to speciation. To test these hypotheses, we will identify candidate genes from RNA-seq analyses and use comparative genomics and multi-sequence alignments to identify conserved (or conversely, highly variable) protein domains. The bioinformatic approach is well-established and robust, but for particular genes it might not prove fruitful. However, since there are many such candidate genes, we anticipate at least partial success. Similarly, demonstrating a critical reproductive function for genes will follow well-established protocols (such as gene knock-outs and phenotypic analyses), but it is likely that some candidates will be excluded because no phenotype will be seen. Objective 3: We hypothesize that manipulating gene expression of key genes will improve stress tolerance during reproduction. To test this hypothesis, we will first establish robust methods for applying transient stresses to growing pollen tubes, then identify genes whose expression changes upon a stress treatment, such as temperature. Genes up-regulated upon stress treatment are candidates for stress tolerance, i.e. overexpressing the gene to higher levels might improve stress tolerance under those stress conditions, while down-regulating such genes (for example, via a gene knockout) is predicted to increase stress sensitivity and would validate the role of the gene in response to stress. Again, as in objective 2, some candidates might not fulfill this goal, but as there are many candidate genes, it is likely that the approach will prove fruitful.