Progress 09/15/00 to 09/30/05
Outputs
OUTPUTS: The overall goal of this project was to understand how nuclear genes contribute to mitochondrial biogenesis in plants. This goal was approached through the study of cytoplasmic male sterility of the S type (CMS-S) in maize. Objective 1 was to generate new restorer-of-fertility alleles by screening for nuclear mutations that suppress expression of the mitochondrial locus encoding CMS-S. The major output from this endeavor is a collection of 20 independent, heritable mutants --nine from Ac/Ds transposon-active lines and eleven from En/Spm transposon-active lines. All of these mutants rescued CMS-S pollen from collapse. Of these, six were homozygous lethal for seed development (rfl) and conditioned seed abortion; 11 were homozygous viable (rfv) for seed development. In one case, the pollen that was shed was nonfunctional (rfn). Two new mutants were not clear-cut in terms of their effects on seed viability. Mapping and allelism tests demonstrated that these mutants represent at least six genetic loci. Three molecular phenotypes were observed by analysis of mitochondrial RNA and proteins in rescued pollen. Three rfl mutants were associated with decreased accumulation of multiple mitochondrial proteins and thereby implicated in mitochondrial protein synthesis or turnover. One rfv mutant was associated with changes in mitochondrial RNA accumulation. The remaining mutants had no obvious effects on mitochondrial RNA or protein accumulation. Objective 2 was to recover molecular clones of the new restorer-of-fertility loci by transposon tagging strategies. No En/Spm or Ac/Ds transposons co-segregated with fertility restoration in our mutant collection. None of the restorer-of-fertility stocks resulting from this work have been requested or disseminated. PARTICIPANTS: Principal Investigator: Susan Gabay-Laughnan, University of Illinois. Dr. Gabay-Laughnan took responsibility for the majority of the genetic work accomplished during this grant. She developed the plant populations screened in these studies. Plants in these populations had S-type male-sterile cytoplasm (CMS-S), lacked nuclear restorers of fertility, and carried either the Ac-Ds or I-En (Spm) transposable elements. Dr. Gabay-Laughnan screened large numbers of male-sterile plants for those with exceptional tassel fertility, arising as either totally fertile tassels or tassels with sectors of fertility. Once such exceptional fertile plants were identified, Dr. Gabay-Laughnan examined pollen from exserted anthers by means of a field microscope to confirm that the male sterility resulted from a new restoring allele and not a cytoplasmic reversion. She then bagged the tassels and used pollen from the exception plants to pollinate male-sterile tester plants in order to propagate the new restorers. These populations were screened in nurseries planted at the University of Illinois. Co-Principal Investigator: Christine D. Chase, University of Florida. Dr. Chase supervised all of the molecular aspects of this investigation. In addition, Dr. Chase traveled to Urbana, IL for two seasons to assist in screening the populations for newly arisen restorer mutations since large numbers of plants were involved. She also assisted in checking the pollen of exceptional plants and in their propagation. Postdoctoral Associate: Dr. Liming Zhao, University of Florida. Dr. Zhao was supported by this project for two years. Dr. Zhao generated populations of maize plants segregating for restorer-of-fertility mutations, and he analyzed these populations for transposon insertions associated with pollen fertility. He also characterized the molecular effects of restorer mutations upon the accumulation of mitochondrial proteins and transcripts in developing pollen. Undergraduate students, University of Florida: Ashley Anderson, Stephanie Betz, Tatyana Gelman, Swati Patel, Jason Powers, Victoria Read, Lauren Riggio, May Siripant, and Diana Tsai contributed to the analysis of mitochondrial gene expression in this project. They identified maize genes encoding mitochondrial proteins through database searches, designed PCR primers and peptide antigens for analysis of gene expression, and performed gene expression studies through use of reverse-transcriptase PCR and immunoblotting assays. These students contributed to two refereed journal publications and nine poster presentations at national or international research conferences. TARGET AUDIENCES: Target audience -- Undergraduate students, University of Florida. Efforts -- These students gained independent research experience in the Chase laboratory. They learned basic bioinformatics as well as laboratory techniques of PCR, reverse-transcriptase PCR, gel electrophoresis and immunoblotting. They also learned how to make oral and poster presentations of their research results. Five of these students attended their first scientific conferences supported by this project. Target audience -- Other researchers in plant mitochondrial biology. Efforts -- Those researchers also studying CMS and restorers of fertility benefit from our characterizations of the effects of restorer mutations on mitochondrial gene expression and protein accumulation. They learned of these through our publications, presentations and posters. Target audience -- Hybrid seed corn industry. Efforts -- CMS is still being used in the production of hybrid corn seed. CMS-S is one of the two cytoplasm types being employed. Dr. Gabay-Laughnan has had several telephone consultations regarding CMS-S and its restorer of fertility alleles. PROJECT MODIFICATIONS: Not relevant to this project.
Impacts
Understanding the molecular-genetic regulation of mitochondrial function is central to the control of pollen fertility and has potential long-term application for the genetic improvement of crop plants through both the production of F1 hybrid seed and for the control of transgene flow from genetically engineered crops. Because mitochondrial respiration is life-sustaining in higher organisms, the opportunities for molecular-genetic analysis of mitochondrial function in crop plants are extremely limited. The chief impact of this project is the development of cytoplasmic male sterility of the S type (CMS-S) in maize as a unique experimental system for the molecular-genetic dissection of plant mitochondrial function. This is due in large part to the gametophytic nature of CMS-S restoration. The following outcomes contributed to this impact: (1) Even in the case of highly unstable mutants, the physical separation of restored pollen allowed for the molecular phenotyping with respect to mitochondrial function. (2) Three restorer-of-fertility lethal (rfl) mutants were associated with decreased accumulation of mitochondrial proteins from two different respiratory complexes, and were thereby implicated in mitochondrial protein synthesis and/or respiratory complex assembly. (3) One restorer-of-fertility viable (rfv) mutant was also associated with failure to accumulate mitochondrial proteins, suggesting a duplicate gene supporting mitochondrial function in the seed. (4) One mutant had no effects on protein accumulation and might, therefore, disrupt downstream mitochondrial signaling pathways that result in pollen collapse. Our findings will enhance the understanding of mitochondrial biogenesis in a multicellular eukaryote of agricultural importance. Mitochondrial-nuclear genome interactions clearly have the capacity to regulate pollen function. The mitochondrial phenotypes conditioned by restorer-of-fertility mutants provide insight into mechanisms of CMS. CMS is the most genetically tractable system for the control of pollen fertility necessary in the commercial production of F1 hybrid seed. A recent application includes the control of transgenes in genetically modified crops. There is concern that these genes may spread to wild plants. CMS is the only non-segregating system available for transgene containment purposes. Plants are male-sterile when desired, in order to contain the transgene, and can be restored to male fertility when needed for pollinations. Furthermore, our study of restorer-of-fertility alleles in CMS-S maize has revealed an influence of mitochondrial function on seed development and seed size. Many restorer mutations affect development of the endosperm, important in feed and food manufacture as well as for secondary products such as oils. Only when we understand the genetic regulation of mitochondrial function in detail can we hope to manipulate this critical organelle for the improvement of plant performance.
Publications
- Gallagher, L., Betz,S.K. and Chase, C.D. 2002 Mitochondrial RNA editing truncates a chimeric open reading frame associated with S male-sterility in maize. Curr Genet 42:179-184
- Wen, L.Y., Ruesch, K.L., Ortega, V.M., Kamps, T.L., Gabay-Laughnan, S. and Chase, C.D. 2003. A nuclear restorer-of-fertility mutation disrupts accumulation of mitochondrial ATP synthase subunit alpha in developing pollen of S male-sterile maize Genetics 165:771-779.
- Chase, C. D. and Gabay-Laughnan S. 2003. Exploring mitochondrial-nuclear genome interactions with S male-sterile maize. pp 31-41 In: Recent Research Developments in Genetics, vol. 3 (SG Pandali ed.) Research Signpost, Kerala
- Gabay-Laughnan, S., Chase, C.D., Ortega, V.M. and Zhao, L. 2004. Molecular-genetic characterization of CMS-S restorer-of-fertility alleles identified in Mexican maize and teosinte. Genetics 166:959-970.
- Chase, C.D. and Gabay-Laughnan, S. 2004. Cytoplasmic male sterility and fertility restoration by nuclear genes. pp. 593-621 In: Molecular Biology and Biotechnology of Plant Organelles. Daniell, H. and Chase CD (eds).
- Newton, K.J., Gabay-Laughnan, S. and DePaepe, R. 2004. Mitochondrial mutations in plants. pp 121-142 In: Advances in Photosynthesis and Respiration. Volume 17 Plant mitochondria: from genome to function. D. Day, H. Millar and J. Whelan. (eds.). Kluwer, Netherlands.
- Gabay-Laughnan, S. and Chase, C.D. 2004a. Recent studies of CMS-S restorers-of-fertility have led to a change in nomenclature. MNL 78:66-67.
- Gabay-Laughnan, S. and Chase, C.D. 2004b. CMS-S restorers-of-fertility from multiple sources cluster on chromosome 2L. MNL 78:67-68.
- Gabay-Laughnan, S. and Newton, K.J. 2005. Mitochondrial mutations in maize. Maydica 50:349-359.
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Progress 10/01/02 to 09/30/03
Outputs
By completion of the third project year, mapping studies carried out in the Gabay-Laughnan laboratory have determined that three of the new restorers, rfl2-99114, rfv*99-1181-8, and rfl*00-3379-2, map to the long arm of chromosome 2 (2L). Since this is the chromosome location of the standard restorer Rf3, it was one of the first chromosome arms tested. The Ac-Ds restorer rfl2-99114 is allelic to the previously identified, spontaneously arisen restorer rfl2 on 2L, about 10 centiMorgans from rf3. The Ac-Ds restorer rfv*99-1181-8 represents a previously unknown locus about one centiMorgan from rf3. The Spm rfl*00-3379-2 restorer is still under analysis but is not allelic to either rfl1 or rfl2. Six other restorers are known not to map to 2L. Since the transposable elements employed in these studies reside on chromosome 3, this chromosome has also been tested. The Ac-Ds restorer rfl*99-78-3 maps to 3S while the Ac-Ds alleles rfv*99-23-7 and rfv*99-1139-9a have been
tentatively mapped to 3S. Seven other restorers are known not to map to 3L.We are in the process of mapping the remaining restorers. Instability of the new mutants has been problematic in our search for transposons that co-segregate with restoration of fertility. While the revertants we encounter are potentially useful in tagging, frequent second-site mutations have complicated our efforts to tag, as well as our efforts to perform allelism tests. We performed an extensive screen for Spm elements co-segregating with restorer-of-fertility alleles, and identified linked Spm elements in three cases. None of these elements co-segregate perfectly with fertility restoration. The reasons for this will be revisited when transposon-inactive versions of these mutants have been recovered. In the interim, we have been investigating the effects of the mutations on mitochondrial gene expression, to identify those mutants of most interest for cloning. By the completion of the third project year,
molecular studies carried out in the Chase laboratory indicate that two rfl mutants appear to affect accumulation of both the ATPA and ATP6 proteins and one rfv mutant affects processing of mitochondrial RNA transcripts.
Impacts
Understanding the molecular-genetic regulation of mitochondrial function has potential long-term application for the genetic improvement of crop plants. Mitochondrial-nuclear genome interactions clearly have the capacity to regulate pollen function. CMS is the most genetically tractable system for the control of pollen fertility necessary in the production of commercial F1 hybrid seed (reviewed by Duvick 1959). A recent application includes the control of transgenes in genetically modified crops. There is concern that these genes may spread to wild plants. CMS is the only non-segregating system available for transgene containment purposes. Plants are male-sterile when desired, in order to contain the transgene, and can be restored to male fertility when needed for pollinations. Furthermore, our study of restorer-of-fertility alleles in CMS-S maize has revealed an influence of mitochondrial function on seed development and seed size (reviewed by Gabay-Laughnan et al.
1995; Chase and Gabay-Laughnan 2003). Many restorer mutations affect development of the endosperm, important in feed and food manufacture, as well as for secondary products such as oils. Only when we understand the genetic regulation of mitochondrial function in detail can we hope to manipulate this critical organelle for the improvement of plant performance.
Publications
- Wen, L., K. L. Ruesch, V. M. Ortega, T. L. Kamps, S. Gabay-Laughnan and C. D. Chase. 2003. A nuclear restorer-of-fertility mutation disrupts accumulation of mitochondrial ATP synthase subunit alpha in developing pollen of S male-sterile maize. Genetics 165:771-779.
- Chase, CD and S Gabay-Laughnan. 2003. Exploring mitochondrial-nuclear genome interactions with S male-sterile maize. pp 31-41 in: Recent Research Developments in Genetics, vol. 3 (SG Pandali ed.) Research Signpost, Kerala.
- Gabay-Laughnan, S., C. D. Chase, V. M. Ortega and L. Zhao. 2004. Molecular-genetic characterization of CMS-S restorer-of-fertility alleles identified in Mexican races of maize and teosinte. Genetics (in press).
- Newton, K. J., S. Gabay-Laughnan and R. DePaepe. 2004. Mitochondrial mutations in plants. In: Advances in Photosynthesis and Respiration. D. Day, H. Millar and J. Whelan. (eds.) in press.
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Progress 10/01/01 to 09/30/02
Outputs
Our overall goal is to exploit CMS-S maize for the identification, cloning, and characterization of nuclear genes that function in mitochondrial gene expression. This project addresses two specific objectives. Objective 1 is to generate new Rf alleles by transposon mutagenesis. Objective 2 is to recover molecular clones of rf loci tagged by transposon mutagenesis. Our approach to Objective 1 was to deploy transposable elements in a CMS-S rf/rf background and screen large populations of male-sterile plants for individuals exhibiting male fertility. Our expectation was that totally fertile tassels, or tassels with sectors of fertility, would result from new Rf alleles arising as transposon-induced, loss-of-function mutations at various nuclear loci required for expression of mitochondrial genes. We employed the Ac-Ds and I-En (Spm) systems for our mutant screens. By the completion of the first project year we identified 12 plants with tassel fertility from the Ac-Ds
transposable element system and 27 plants with tassel fertility from the Spm system from which we were able to obtain pollen for crossing. By the completion of the first project year, based on genetic characterization, we narrowed this number down to seven Ac-Ds Rf alleles and 10 Spm Rf alleles. We are continuing the study of these 17 putatively tagged Rf alleles. All 17 Rf alleles are being converted to inbred nuclear backgrounds in order to generate stable segregating populations. Two of the Ac-Ds Rf alleles and one of the Spm Rf alleles have been mapped to chromosome 2L. Due to instability of the Ac-Ds mutants, work on Objective 2 was delayed. By the middle of the second project year, populations of four of these Rf alleles conformed to expectation and DNA preparations have been made from individual plants of selected families. Screening of the four selected families is now in progress. By the end of the second project year, families segregating for six of the Spm Rf alleles were
planted and DNA preparations will be made. All families will be analyzed for DNA fragments co-segregating with Rf allels. The technique to screen for transposons that co-segregate with Rf alleles has been selected.
Impacts
In the CMS-S system of maize, expression of a novel chimeric gene in the mitochondria results in a male-sterile phenotype. Loss-of-function mutations in nuclear genes required for mitochondrial gene expression behave as Rf alleles. These Rf alleles disrupt expression of the CMS gene and the male sterility trait. Rf alleles also disrupt expression of essential mitochondrial genes. These mutations are tolerated in pollen, however, because late-stage pollen development and pollen germination do not require mitochondrial respiration. We propose that new Rf allels for CMS-S maize can be generated by transposon mutagenesis and cloned via transposon tagging. These new Rf alleles will allow for the identification, cloning, and characterization of a spectrum of nuclear genes that function in mitochondrial gene expression.
Publications
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Progress 10/01/00 to 09/30/01
Outputs
Our overall goal is to exploit CMS-S maize for the identification, cloning and characterization of nuclear genes that function in mitochondrial gene expression. This project addresses two specific objectives. The first is to generate new Rf alleles by transposon mutagenesis. The second is to recover the molecular clones of rf loci tagged by transposon mutagenesis. Our approach to the first objective was to deploy transposable elements in a CMS-S rf/rf background and screen large populations of male-sterile plants for individuals exhibiting male fertility. Our expectation was that totally fertile tassels, or tassels with sectors of fertility, would result from new Rf alleles arising as transposon-induced, loss-of-function mutations at various nuclear loci required for expression of mitochondrial genes. We employed the Ac-Ds and I-En (Spm) systems for our mutant screens. By the completion of the first project year we screened 1,241 progeny plants carrying Ac-Ds. We
identified 12 plants with tassel fertility from which we were able to obtain pollen for crossing. Based on genetic characterization, we have narrowed the number of cases down to seven. We are continuing to study these seven putatively tagged Rf alleles. We have determined that five of them are viable when homozygous and in the remaining two the homozygous Rf/Rf condition is lethal. One of the homozygous lethal Rfs has been mapped to chromosome 2. By the completion of the first project year we also screened 852 plants, all of which carried Spm, and 581 plants, two-thirds of which carried SPm. We identified 27 cases of which 22 were tested in our Hawaii nursery. Thirteen of these putatively tagged Rf alleles have been selected for further analysis based on genetic characterization. Ten of them are homozygous viable while in the remaining three the homozygous Rf/Rf is lethal. Our approach to the second objective is to search for DNA fragments flanking transposable element sequences and
co-segregating with male fertility in segregating populations of CMS-S Rf/rf and CMS-S rf/rf plants. Plants are grown to maturity and scored for male fertility based on pollen shed. Leaf samples are collected from individual plants and DNA is extracted. This part of objective 2 has been completed for four of the seven most promising cases from the Ac-Ds studies. We plan to use the transposon display technique to screen for PCR amplified DNA fragments containing Ac or Ds terminal sequences and cosegregating with the Rf allele.
Impacts
In the S system of cytoplasmic male sterility (CMS-S) of maize, expression of a novel chimeric gene in the mitochondria results in a male-sterile phenotype. Loss-of-function mutations in nuclear genes required for mitochondrial gene expression behave as restorer-of-fertility (RF) alleles. These Rf alleles disrupt expression of the CMS gene and the male sterility trait. Rf alleles also disrupt expression of essential mitochondrial genes. However, these mutations are tolerated in pollen because late-stage pollen development and pollen germination do not require mitochondrial respiration. We propose that new Rf alleles for CMS-S maize can be generated by transposon mutagenesis and cloned via transposon tagging. These new Rf alleles will allow for the identification, cloning, and characterization of a spectrum of nuclear genes that function in mitocondrial gene expression.
Publications
- No publications reported this period
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