Progress 06/15/02 to 06/14/05
Outputs
Genome-wide identification of genes expressed specifically in papillar cells and in the stylar transmitting tract: Because epidermal cells of the stigma and transmitting tract cells are difficult to isolate by conventional microdissection methods in the numbers required for gene expression profiling, we generated A. thaliana plants in which stigma epidermal cells and transmitting tract cells were ablated by expression of the cell-autonomous cytotoxin, diphtheria toxin subunit A (DT-A) under control of the YSRK promoter which is a very tightly regulated promoter that is active exclusively in these cells. The Affymetrix whole-genome array ATH1, which contains probe sets corresponding to approximately 23,000 Arabidopsis genes, was used for comparative genome-wide transcriptional profiling of wild-type and genetically ablated pistils. We identified 115 and 34 genes predicted to be expressed specifically in the stigma epidermis, respectively. Both gene sets were
significantly enriched in predicted secreted proteins that could function during pollination in the remodeling of the extracellular matrix or as small signaling molecules. In addition, the stigma dataset included candidate orthologs of some, but not all, of the genes previously implicated in compatible and incompatible pollinations in other species. Several genes that exhibit high-level papillar cell-specific expression were tested for a potential function in pollination by analysis of T-DNA insertion lines. However, we observed no obvious phenotypic effect of disrupting the genes, either in pollination assays or other general phenotypic characteristics, possibly because of genetic redundancy. An RNAi approach is currently being implemented for functional studies. Identification of rapidly-evolving genes with potential functions in species recognition A novel approach, using a combination of macroarrays and bioinformatics-based screens, was designed to exploit the candidate
stigma-specific and transmitting tract-specific gene datasets and identify putative rapidly evolving genes that might function in species recognition in crucifers. These arrays were probed with stigma or style and ovary cDNA probes from species that are of increasing evolutionary distance from A. thaliana, to identify genes whose hybridization signals decrease dramatically with hybridization stringency and evolutionary distance from A. thaliana. Sequence and statistical analysis of three of the top five candidate genes did not reveal evidence for positive selection, however. A second strategy was to subject candidate genes to a preliminary round of amplificaiton across species to determine whether A. thaliana primers can effectively amplify sequence in other crucifers. Our assumption was that genes with modest divergence rates would be amplifiable across species, while rapidly-evolving genes would not. For those candidates that failed to amplify, DNA gel blot analysis and cDNA library
screening were then used to isolate putative orthologs. Analysis of positive clones by PAML and other more sensitive statistical methods for detecting evidence of positive selection is ongoing.
Impacts
The project is predicted to contribute to our understanding of plant reproduction and to define the expression pattern and function of several Arabidopsis genes that currently have no known biological function. Importantly, identification of genes that contribute to reproductive isolation might be used in the future in schemes to control polination processes in crop plants.
Publications
- Tung C-W, Dwyer K, Nasrallah ME, Nasrallah JB. 2005. Genome-wide identification of genes expressed specifically along the path of the pollen tube growth in Arabidopsis pistils. Plant Physiology 138: 977-989
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Progress 01/01/04 to 12/31/04
Outputs
We are continuing our analysis of pistil-specific genes that exhibit more-than-average inter-specific and intra-specific sequence divergence among crucifer species. We probed whole-genome microarrays of Arabidopsis thaliana with pistil RNA derived from plants with ablated stigma and transmitting tract and with pisstil RNA derived from wild type plants. Comparison of the hybridization signals for approximately 24,000 genes identified 160 genes expressed specifically in the stigma epidermis and 50 genes expressed specifically in the transmitting tract of the style. Expression patterns were confirmed by RNa gel blot analysis and in situ hybridization using gene-specific probes. Homologues of these genes from other crucifer species are being isolated to identify a subset of rapidly evolving genes. Some of these genes might underlie specificity in reproduction and contribute to reproductive isolation.
Impacts
The project is predicted to contribute to our understanding of plant reproduction and to define the expression pattern and function of several Arabidopsis genes that currently have no known biological function. Importantly, identification of genes that contribute to reproductive isolation might be used in the future in schemes to control polination processes in crop plants.
Publications
- No publications reported this period
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Progress 01/01/03 to 12/31/03
Outputs
To identify genes that underlie selectivity in mate choice in intra-specific and inter-specific pollen-pistil interactions we are using several strategies. We have compiled a collection of genes that are expressed specifically along the path of pollen tube growth. Several of these genes are predicted to encode secreted proteins and might therefore have a function in pollen-pistil signaling. Interestingly, a subset of these genes exhibit extensive inter-specific and intra-specific sequence divergence among crucifer species, suggesting that these genes might underlie specificity in reproduction and contribute to reproductive isolation.
Impacts
The project is predicted to contribute to our understanding of plant reproduction and to define the expression pattern and function of several Arabidopsis genes that currently have no known biological function.
Publications
- No publications reported this period
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Progress 01/01/02 to 12/31/02
Outputs
Our goal is to understand the molecular dialogue that takes place between cells of the pistil on the one hand and the pollen/pollen tube on the other hand. In a genome-wide screen, we identified over 200 genes that appear to be expressed specifically or preferentially in the stigma/transmitting tract. Although some of these genes can be assigned into functional categories based on their sequence, many genes are predicted to encode "unknown proteins". Preliminary analysis indicates that several of the identified genes exhibit extensive inter-specific and intra-specific sequence divergence among crucifer species. These genes might underlie specificity in reproduction and contribute to reproductive isolation. Functional analysis of a subset of genes has been initiated.
Impacts
The outcome of pollination is important for completion of the plant life cycle and consequently for agricultural productivity. Our identification and functional analysis of genes expressed specifically in stigmas and in the transmitting tract are expected to lead to a mechanistic understanding of the pollen-pistil interactions that underlie intra-specific and inter-specific selectivity in mate choice.
Publications
- No publications reported this period
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