Progress 09/01/04 to 08/31/06
Outputs
During the last two years we have made significant progess toward the fundamental goal of this grant as well as progress on understanding the genetic mechanisms underlying the cell specification and differentation of two cell types within the female gametophyte. Directly relating to the grant proposal, I have published a first-author article in the journal Plant Physiology entitled "NUCLEAR FUSION DEFECTIVE1 Encodes the Arabidopsis RPL21M Protein and is Required for Karyogamy During Female Gametophyte Development and Fertilization." In this article we identify six mutants that affect karyogamy specifically during female gametophyte development. One of these mutants, nfd1, also affects karyogamy during sexual reproduction. Interestingly, four of these six mutants affect genes that encode for proteins which localize to the mitochondria, raising the intriguing possibility that this organelle plays a previously uncharacterized role in nuclear membrane biology. I have also
published a first-author paper in the journal Plant Cell entitled "AGL80 is Required for Central Cell and Endosperm Development in Arabidopsis." This paper is fundamentally important to the field as it describes the first transcription factor essential for the specification of the central cell in the female gametophyte and is the first example of a phenotype associated with any transcription factor in the Type I group of the MADS-box transcription factor genes in plants. While seemingly unrelated to the work proposed by the grant proposal, this mutant was initially characterized in the mutant screen that generated the nuclear fusion mutants. In addition, I co-authored a second paper describing a transcription factor required for the development of another cell type within the female gametophyte, the synergid cell. This paper is entitled "MYB98 Is Required for Pollen Tube Guidance and Synergid Cell Differentiation in Arabidopsis." This paper marks the first transcription factor
demonstrated to be required for cell differentiation in the female gametophyte. This transcription factor is required for pollen tube guidance and has become a tool to identify one of the missing pieces in sexual plant reproduction, the pollen tube guidance cue.
Impacts
We expect our data on nuclear fusion to have a broad impact in two main areas. First, these represent the first published karyogamy mutants in plants that affect the fusion of nuclei specifically, suggesting that we have identified genes that are required for homotypic membrane fusion that can be used to modify current techniques for membrane fusion events. In addition, many of these genes are putatively related to subcellular organelles within the cells, either mitochondria or plastids. If this is the case, then we have identified an entirely new area of cellular biology that has not been suggested before. The data generated by the transcription factor research has exploded into a powerful resource for biologists to understand the fundamental question of cell diffentation and development of the female gametophyte. The predicted impact of this work is that we may quickly begin to disect the regulatory networks conferring the functions of the central cell and the
synergid cell, both of which are essential to seed production.
Publications
- Kasahara RD*, Portereiko MF*, Sandaklie-Nikolova L, Rabiger DS, Drews GN. (2005). MYB98 Is Required for Pollen Tube Guidance and Synergid Cell Differentiation in Arabidopsis. Plant Cell. Nov;17(11):2981-92. *co-first authors.
- Portereiko MF, Sandaklie-Nikolova, L, Lloyd A, Otsuga D, and Drews GN. (2006). NUCLEAR FUSION DEFECTIVE1 Encodes the Arabidopsis RPL21M Protein and is Required for Karyogamy During Female Gametophyte Development and Fertilization. Plant Physiology. May 12-On-line publication.
- Portereiko MF, Lloyd A, Steffen JG, Punwani JA, Otsuga D, and Drews GN. (2006). AGL80 is Required for Central Cell and Endosperm Development in Arabidopsis. Plant Cell. Aug; .
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Progress 10/01/04 to 09/30/05
Outputs
I have carefully examined the phenotype of one of the nuclear fusion mutants, fem55, and I have discovered that this allele not only has defects in the fusion of the polar nuclei during female gametophyte development but also during the fertilization process. When pollinated with wild-type pollen, sperm cells fuse with the egg and central cells of the fem55 mutant female gametophytes. However, the sperm nuclei do not fuse with their respective target nuclei, and, as such, do not trigger the subsequent developmental programs of the embryo and endosperm within those cells. In order to determine the stage of membrane fusion that is disrupted within the fem55 mutant, I have analyzed fem55 female gametophytes by transmission electron microscopy. In wild type, the membrane fusion process occurs by a series of steps that include: contact of the outer nuclear membranes, fusion of these outer membranes and the creation of membrane bridges, contact and fusion of the inner
nuclear membranes, and finally, the mixing of the nuclear plasm. In fem55 mutants, the outer nuclear membranes lie adjacent to each other but do not contract. This observation suggests that fem55 is potentially involved in a membrane recognition event. I have mapped a T-DNA insert in the fem55 mutant line to a locus on chromosome 4. By using PCR-based approaches, I have determined that the T-DNA insertion is linked to less than 1 cm to the mutation that causes the nuclear fusion phenotype. I have built plasmid constructs to try to rescue the fem55 phenotype, and those experiments should yield results by the end of October. In addition to the fem55 project, I have begun to work on a mutant called fem111. In the fem111 mutant, the central cell under goes degeneration following sperm entry. I have rescued the fem111 mutant phenotype with a MADS box transcription factor, and I have shown by GFP reporter fusions with this gene that it expressed specifically within the central cell and
endosperm in regards to the ovule. I am currently taking several approaches to identify the cause of the phenotype observed in the fem111 background, including the creation of double mutants and the analysis of expression vectors that are normally observed within different cell-types within the female gametophyte. By the end of the year, I plan on having a paper submitted that contains the information for the nuclear fusion project. Within 18 months, I fully expect to have a second paper that is entirely focused on the fem111 project.
Impacts
The impact of the fem111 project is many-fold. I believe that fem111 will provide me with an entry point into understanding how the egg cell and the central cell differ during female gametophyte development and how they respond to the same event (fertilization) differently following pollination.
Publications
- No publications reported this period
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Progress 10/01/03 to 09/30/04
Outputs
During the last six months I have made progress on the nuclear fusion project in three key areas. First, I have completed the gene identification process for all of the nuclear fusion mutants outlined in the grant proposal. Interestingly, many of these genes appear to encode proteins that are localized to the mitochondria or the plastids, raising the intriguing possibility that these organelles are somehow linked to the process of nuclear fusion. Second, I have identified that one of the mutants, fem55, is not only defective in the karyogamy event of the fusion of the two polar nuclei as outlined in my grant proposal, but is also defective in fusion of the sperm nuclei with the egg and central cell, the two karyogamy events associated with fertilization. This result is the first report of its kind in the plant kingdom. In addition, my colleague and I have also determined by trasmission electron microscopy that the defect in fem55 mutant female gametophytes allows the
polar nuclei to come into the correct position for karyogamy, but the initation of membrane fusion does not occur. I am currently writing the manuscript for this paper, and I hope to submit by the end of the summer. Lastly, I have identified that five of the karyogamy mutants are capable of attracting pollen tubes into the ovules that contain the mutant female gametophytes, but the pollen tubes do not enter the synergid cells or release their contents. Thus, we cannot determine if they are also defective in the karyogamy events of fertilization. However, the intriguing possibility is that these mutants are also affected in pollen tube and synergid cell interaction and/or the initiation of synergid cell death. We are planning to test both of these possibilities, and we hope to make significant progress on this part of the project during the Fall of this year (2005).
Impacts
We expect our data to have a broad impact in two main areas. First, these represent the first published karyogamy mutants in plants that affect the fusion of nuclei specifically, suggesting that we have identified genes that are required for homotypic membrane fusion that can be used to modify current techniques for membrane fusion events. In addition, many of these genes are putatively related to subcellular organelles within the cells, either mitochondria or plastids. If this is the case, then we have identified an entirely new area of cellular biology that has not been suggested before.
Publications
- No publications reported this period
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