2013 Annual Report
1a.Objectives (from AD-416):
The likelihood of global climate change increases the need for strategies for protecting crop productivity from environmental extremes, including heat, drought, and cold, all of which ultimately impact yield. Pollination and fertilization, in particular, are sensitive to environmental extremes. Pollination and fertilization require an extensive dialog between the tissues of the pistil and the pollen tube. Pollination and fertilization are also model processes for analyzing cell-cell recognition, cell-cell communication and coordination at the molecular, cellular and tissue levels. The long term goal of this project is to identify and characterize the genes and cellular processes that regulate pollen development, pollination, and fertilization, in order to identify genes that can be manipulated to better regulate pollen development, pollination and fertilization. This knowledge will better enable breeders to develop new strategies for genetically hardening these sensitive processes for the field. Genes that are necessary for pollen and sperm function(s) are often evolutionarily conserved and thus identifiable using comparative genomic strategies; such conserved genes are candidates for manipulations across a wide range of plant species.
Objective 1: Identify and analyze, at genetic, molecular and protein levels, interactions that occur in pollen development, in pollen-pistil interactions or in sperm-egg interactions that determine the outcomes of these processes.
Objective 2: Identify and characterize evolutionarily conserved regions of the genome whose function is to mediate pollen development and/or pollen-pistil interactions.
Objective 3: Determine new means to regulate pollen development and pollen-pistil interactions.
1b.Approach (from AD-416):
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.
This report documents progress for project number 5335-21000-037-00D, which started in March 2013 and continues research from Project Number 5335-21000-036-00D, entitled "Molecular Developmental Genetics of Pollen and Pollen-Pistil Interactions in Crop Plants." Progress was made on objective 1, to elucidate the molecular mechanisms that occur during pollen development, pollen-pistil interactions and sperm-egg interactions. In collaboration with researchers at the Shanghai Institute of Plant Physiology and Ecology, we found that overexpression of the receptor kinase LePRK1 in tomato changed the pollen tube growth mode from tubular to a blebbing mode, and showed that this phenotype was due to interactions with the actin cytoskeleton.
Progress was made on objective 2, to select candidate genes for analyses. We showed that the tetraspanin family, of which there are 17 members in Arabidopsis thaliana, exhibit cell-type specific expression patterns. Most interestingly, we showed that two sperm-specific tetrapanins are localized at the sperm-sperm interface, suggesting that they might be required for the association of the sperm cells themselves and for the association of the sperm cells with the vegetative cell nucleus.
Genes that function in pollen. In collaboration with researchers at University of North Carolina-Charlotte, we used deep sequencing technology (so-called RNA-Seq) to discover that Arabidopsis pollen expresses about 1000 more genes than previously known, that it expresses some genes that are not in the current genome annotation or that are incorrectly annotated, and that transcripts from some pollen-expressed genes are spliced differently than the known splice variants in other cell types. We have and continue to use the RNA-Seq dataset to select candidate genes for functional analyses. In, addition, we showed that a type II ROP GTPase is localized to the plasma membrane that surrounds the two sperm cells inside the pollen grain. We swapped domains of a type I ROP and a type II ROP to show that localization surrounding the sperm cells is due to the C-terminal part of the type II ROP, specifically to some cysteines in that part of the protein. The work leads to advances in reporduction, critical cor flowering plants in agriculture.
Loraine, A., Mccormick, S.M., Estrada, A., Patel, K., Qin, P. 2013. RNA-Seq of Aradopsis pollen uncovers novel transcription and alternative splicing. Plant Physiology. 162: 1092-1109.
Scarpin, R., Sigaut, L., Pietrasanta, L., Zheng, B., Mccormick, S.M., Muschietti, J. 2013. Cajal bodies are developmentally regulated during pollen development abd pollen tube growth in Arabidopsis thaliana. Molecular Plant. 6: 1355-1357.
Li, S., Zhou, L., Feng, Q., Mccormick, S.M., Zhang, Y. 2013. The C-terminal hypervariable domain targets Aradopsis ROP9 to the invaginated pollen tube plasma membrane. Molecular Plant. 6:1362-1364.