Progress 03/01/09 to 02/28/14
Outputs
Target Audience: Undergraduate students: Laboratory training was provided in the form of research internships in plant molecular genetics for undergraduate students, including students from minority-serving institutions. Graduate students, visiting scientists and postdoctoral associates: Hands-on Laboratory training in plant molecular genetics was provided for agraduate student, a visiting scientist, and a postdoctoral research associate. Plant research community: New genetic materials, research reagents, approaches and knowledge with respect to mitochondrial functions in both pollen and seed development were provided to this audience through publications and presentations at professional conferences. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided? In total, the project personnel have mentored four undergraduate students (three from minority-serving institutions), one graduate student, one international graduate student/visiting scholar, and one postdoctoral associate by providing one-on-one, hands-on training in plant molecular genetics research. These individuals learned many specific laboratory skills such as plant pollinations, plant DNA extraction, polymerase chain reaction, genotyping, genetic mapping, DNA cloning, and plant transformation. They also gained an appreciation for the importance and challenges of plant genetic research. All individuals were afforded opportunities for professional development, including attending research conferences and making poster presentations. Collectively, these participants attended and made six poster presentations at local, national and international conferences. How have the results been disseminated to communities of interest? These results have been disseminated to the plant science research community through one published book chapter, 9 abstracts, 9 poster presentations, and 5 oral presentations at a total of 8 international, 2 national, and 2 local conferences. What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
Mitochondria carry out the life-sustaining process of respiration within the cell and also regulate cell death pathways that are essential for normal growth, development and defense responses, yet we know little about the genetic regulation of mitochondrial function in plants. Through genetic and molecular investigations, we developed new genetic materials and reagents, and used them to demonstrate unexpected developmental regulation of mitochondrial functions during plant reproduction. Our findings and experimental materials will enable plant science researchers to investigate and manipulate mitochondrial function in new ways. In the shorter term this will achieve a better understanding of the plant reproduction events that culminate in the economically important seed. In the longer term this understanding can be applied to achieve improvements in seed production. Objective 1 is to identify the mitochondrial signaling pathways that participate in plant cell death programs. Towards this objective we completed a developmental profile of mitochondrial protein accumulation during normal maize pollen development, and compared the normal profile to that of S-type, male-sterile maize, in which a mitochondrial mutation causes developmentally programmed pollen cell death. For this experiment, we compiled and further expanded a collection of antibodies that now allow for the specific recognition of 19 plant mitochondrial proteins. Through immunological approaches we found that mitochondria do not have respiratory function during early stages of normal pollen development, but regain this function in later pollen development. We demonstrated that, in S male-sterile maize, genetically programmed cell death coincides with the initiation of respiratory function, linking mitochondrial cell death signaling with mitochondrial function. We identified proteins that accumulated immediately prior to the cell death event and proteins lost from the mitochondria during that event. We confirmed these findings in two different maize genotypes. As a consequence the plant science community now knows that mitochondrial function is tightly programmed during pollen development; knows of candidate proteins needed for the initiation and execution of mitochondria-signaled cell death in plants; and has the antibody reagents needed to further investigate the roles of these candidates. These reagents will also enable the broader plant research community to investigate mitochondrial function in the context of other important plant developmental events. In a complementary approach, we developed transgenic maize lines that express, in a pollen-specific manner, a mitochondria-targeted, reactive oxygen responsive, green fluorescent protein (roGFP) reporter, as well as transgenic maize lines that express a mitochondria-targeted protein hypothesized to initiate the cell death event characteristic of S-type male sterility. In developing these materials we validated the use of the Neurospora crassa ATP9 mitochondrial targeting leader peptide as a new tool for use in plants, and created reagents that will enable the broader plant science community to investigate mitochondrial redox status in a variety of plant species, tissue and organs under various environmental conditions. Seeds of the latter transgenic lines have just been obtained, so data have yet to be collected on the cell death initiation. Should the transgenes elicit the cell death phenotype, we will have further knowledge of the mechanisms that lead to mitochondria-signaled cell death in plants and we will have created new genetic tools for its investigation. Finally, through participation in this research, one postdoctoral associate, one graduate student and four undergraduate students, three from minority-serving institutions, have learned new research skills including plant DNA extraction, polymerase chain reaction, molecular cloning, genetic transformation and light microscopy. Four of these students are currently applying these skills in life-science careers. Objective 2 is to identify nuclear genes that contribute to mitochondrial gene expression in plants and learn the specific functions that these nuclear genes perform or support within the mitochondria. Towards this objective we investigate genetic "restorer" mutations that rescue the S-type, male-sterile pollen from mitochondria-initiated programmed cell death. The rationale for this approach is that mitochondrial gene expression is needed to initiate the cell death program and so genetic mutations that block mitochondrial gene expression will rescue the pollen from this developmental fate. We first developed a collection of 14 independent Mutator transposon insertion mutants that restored pollen fertility to S-type male sterile maize. We performed test of allelism that demonstrated each mutation was in a different gene. Through targeted DNA sequencing of transposon flanking sequences, two candidate restorer mutations were identified. These were related in an interesting way. Both are mutations in nuclear genes that encode mitochondrial ribosomal proteins - RPL6 and RPL14. In the case of the RPL14 candidate an independent RPL14 mutation was obtained through the Maize Genetics Stock Center and confirmed as an allele. In the case of RPL6, the independent insertion line did not create a mutation, so no confirming test has been possible to date. Nevertheless, the parallel to the confirmed RPL14 candidate indicates that the RPL6 candidate is likely also a restorer. We now know a genetic mutation that eliminates a mitochondrial ribosomal protein will rescue the S-type, male-sterile pollen from its fate of mitochondria-initiated programmed cell death. We also know that maize pollen is surprisingly flexible with respect to the requirement for mitochondrial protein synthesis and hence mitochondrial function. This knowledge validates new methodology to identify nuclear mutations that disrupt plant mitochondrial gene expression, overcoming the challenges that result from the essential nature of mitochondrial functions in most plant tissues and organs. Finally, through participation in this research, one undergraduate student and one international graduate student/visiting scholar has learned new research skills including plant DNA extraction, genotyping, genetic mapping and light microscopy.
Publications
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2010
Citation:
Bhan, K, P Smith, K Chamusco, J Seib, C Moreira, M Gallo and CD Chase (2010) Transient expression of mitochondrial-targeted peptides linked to cell death signaling and pollen collapse in S male-sterile maize. ASA-CSSA-SSSA Annual Meetings, Abstract 59484
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Progress 10/01/12 to 09/30/13
Outputs
Target Audience: Graduate student / visiting scientist: Laboratory training was provided in the form of a research internship for a graduate student visiting from abroad. Plant research community: New genetic materials, approaches and insights with respect to mitochondrial functions in both pollen and seed development were provided to this audience through presentations at professional conferences. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided? One international graduate student received one-on-one training in research techniques for maize molecular genetics including laboratory management, greenhouse management, plant DNA extraction, polymerase chain reaction, genotyping and genetic mapping with microsatellites, and light microscopy. This individual also had opportunities for professional development, attending a local conference and an international conference, and making research poster presentations at each. How have the results been disseminated to communities of interest? These results have been disseminated to the plant science research community through a poster presentation and an oral presentation at an international research conference, and through a poster presentation at a state research conference. What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
Mitochondria carry out the life-sustaining process of respiration within the cell and also regulate cell death pathways that are essential for normal growth, development and defense responses, yet we know little about the genetic regulation of mitochondrial function in plants. Through genetic and molecular investigations, we demonstrated unexpected developmental regulation of mitochondrial function associated with plant reproduction. Our findings and experimental materials will enable plant science researchers to investigate and manipulate mitochondrial function in new ways. In the shorter term this will achieve a better understanding of the plant reproduction events that culminate in the economically important seed. In the longer term this understanding can be applied to achieve improvements in seed production. Objective 1 is to identify the mitochondrial signaling pathways that participate in plant cell death programs. Towards this objective we completed a developmental profile of mitochondrial protein accumulation during normal maize pollen development, and compared the normal profile to that of S-type, male-sterile maize, in which a mitochondrial mutation causes developmentally programmed pollen cell death. For this experiment, we compiled and further expanded a collection of antibodies that now allow for the specific recognition of 20 plant mitochondrial proteins. Through immunological approaches we found that mitochondria do not have respiratory function during early stages of normal pollen development, but regain this function in later pollen development. In male-sterile pollen, genetically programmed cell death coincides with the initiation of respiratory function. We identified proteins that accumulated immediately prior to the cell death event and proteins lost from the mitochondria during that event. We confirmed these findings in two different maize genotypes. As a consequence we now know that mitochondrial function is tightly programmed during pollen development. We have knowledge of candidate proteins needed for the initiation and execution of mitochondria-signaled cell death in plants, and we have the antibody reagents needed to further investigate the roles of these candidates. These reagents will also enable the broader plant research community to investigate mitochondrial function in the context of other important plant developmental events. In a complementary approach, we developed transgenic maize lines that express, in a pollen-specific manner, the proteins hypothesized to initiate the cell death events characteristic of S-type male sterility. As seeds of these lines have just been obtained, no data have yet been collected on the phenotype of these plants. Should the transgenes elicit the cell death phenotype, we will have new knowledge regarding the basis of this phenotype and new genetic tools for its investigation. Objective 2 is to identify nuclear genes that contribute to mitochondrial gene expression in plants and learn the specific functions that these nuclear genes perform or support within the mitochondria. Towards this objective we investigate genetic "restorer" mutations that rescue the S-type, male-sterile pollen from mitochondria-initiated programmed cell death. The rationale for this approach is that mitochondrial gene expression is needed to initiate the cell death program and so genetic mutations that block mitochondrial gene expression will rescue the pollen from this developmental fate. Prior work identified three genetic mutations, all created by Mutator transposon insertion, as candidate fertility restorers. Two of the three were genetically linked. The linked mutations have now been separated by genetic recombination and tested individually for fertility restoration. Independent Mutator insertions in these three candidate genes were obtained from the Maize Genetic Stock Center, crossed into the S-type male-sterile background, and evaluated for fertility restorer activity. The independent insertion lines were also crossed with their respective original mutations to confirm, for each case, that the mutations lie in the same gene. All plants were genotyped for presence or absence of the Mutator transposon insertion and phenotyped for pollen fertility or sterility. In the case where two linked restorer candidates were separated by genetic recombination, the recombinant plants were genotyped for flanking microsatellite markers so that the recombination intervals were identified. The pollen fertility analysis that followed separation of the two genetically linked candidates eliminated one candidate and supported the second as a fertility restorer mutation. The two viable candidates are related in an interesting way. Both are mutations in nuclear genes that encode mitochondrial ribosomal proteins - RPL6 and RPL14. In the case of the RPL14 candidate an independent RPL14 mutation was confirmed as an allele. In the case of RPL6, the independent insertion line did not create a mutation, so no confirming test has been possible to date. Nevertheless, the parallel to the confirmed RPL14 candidate indicates that the RPL6 candidate is likley also a restorer. We now know a genetic mutation that eliminates a mitochondrial ribosomal protein will rescue the S-type, male-sterile pollen from its fate of mitochondria-initiated programmed cell death. We also know that maize pollen is surprisingly flexible with respect to the requirement for mitochondrial function. This knowledge validates new methodology to identify nuclear mutations that disrupt plant mitochondrial gene expression, overcoming the challenges that result from the essential nature of mitochondrial functions in most plant tissues and organs. Finally, through participation in this research, an international graduate student has learned new research skills including plant DNA extraction, genotyping, genetic mapping and light microscopy. These new skills and methodologies can now be applied to develop and investigate a broader collection of mutants.
Publications
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2013
Citation:
Wang, Y, R Williams-Carrier, KC Chamusco, L Zhao P, LC Hannah, S Gabay-Laughnan, A Barkan and CD Chase (2013) S male-sterile maize a novel tool for the forward genetics of plant mitochondrial function. Plant Biology 2013 Annual Meeting of the American Society of Plant Biologists, Abstract P 09030, poster and selected oral presentation
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2013
Citation:
Wang, Y, R Williams-Carrier, KC Chamusco, L Zhao P, LC Hannah, S Gabay-Laughnan, A Barkan and CD Chase (2013) S male-sterile maize a novel tool for the forward genetics of plant mitochondrial function. Florida Genetics 2013 Abstract and poster presentation
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Progress 10/01/11 to 09/30/12
Outputs
OUTPUTS: The over-arching goal of this project is to understand the molecular-genetic regulation of plant mitochondrial processes key to plant growth and development, with emphasis on plant reproduction. The research approach exploits a mitochondrial gene that conditions a pollen cell death phenotype, cytoplasmic male sterility type S (CMS-S), and nuclear restorer-of-fertility (restorer) mutations that rescue CMS-S pollen by disrupting mitochondrial gene expression or mitochondria-signaled cell death pathways. Objective 1 is to identify mitochondrial signaling pathways that participate in plant cell death programs. Outputs towards this objective are: (1-1) A DNA clone was developed to test the mitochondrial open reading frame orf17 S14L for the ability to condition pollen collapse when expressed in developing maize pollen. Objective 2 is to identify nuclear genes that contribute to mitochondrial gene expression and cell death signaling in plants and learn the specific functions that these genes perform or support within the mitochondria. Outputs towards this objective are: (2-1) Three Mutator (Mu) tranposon insertion mutants were evaluated as candidate fertility restorer genes for CMS-S maize. Independent Mu insertion alleles were identified for each of the three candidates and genetic crosses were performed to confirm that these Mu insertions create bona fide restorer alleles for CMS-S. (2-2) One undergraduate intern was mentored in the context of a plant molecular genetics research project. Project findings were disseminated through one abstract, one poster presentation and two invited oral presentations. PARTICIPANTS: Individuals: Karen Chamusco, Biological Scientist, University of Florida, developed clones for maize transformation and performed plant genotypoing; Christine D. Chase, Project Director, Professor, University of Florida, provided leadership in plant genotyping and performed all crosses for the development and testing of new restorer lines; Michelle Feole, Undergraduate Intern, University of Florida, performed DNA extractions and plant genoptyping; Susan Gabay-Laughnan, Collaborator, University of Illinois Urbana-Champaign, performed seed phenotyping and allelism tests of restorer mutants; Alice Barkan, Collaborator, University of Oregon performed Mu-Illumina DNA sequencing analysis on families segregating for restorer-of-fertility mutations. Training and Professional Development: This project provided hands-on laboratory training to one undergraduate student, contributing towards the professional development of these individuals in the life sciences: Michelle Feole, undergraduate student, University of Florida, learned techniques of DNA extraction, polymerase chain reaction, DNA gel electrophoresis and maize genetics Partner organizations: University of Illinois Urbana-Champaign, University of Oregon, Eugene TARGET AUDIENCES: Undergraduate students: research internship training for undergraduate students contributes not only to their professional development in the life sciences but also to their appreciation for the importance of plant research for sustainable food fuel and fiber production. Plant research community: this project provided new tools for expression, targeting and phenotypic analysis of mitochondrial function, and new insights into maize pollen development. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
Objective 1: A new maize transformation clone provides a novel tool for investigation of mitochondrial cell death signaling and maize pollen development. Objective 2. The maize genetic materials created through this work will confirm novel classes of genes that function in the control of pollen development and fertility, provide new insights into pathways for nuclear control of mitochondrial structure and function, and validate CMS-S maize as a tool for forward genetic analysis of nuclear-mitochondrial function. One undergraduate student learned principles of genetics and molecular biology through hands on participation in a maize genetics experiment.
Publications
- Chase, C.D., Williams-Carrier,R.,Barkan,A., Feole,M., Hannah,L.C., Zhao,L., Kamps, T.L., Gabay-Laughnan, S. (2012) Identification of fertility restorers for S male-sterile maize: beyond PPRs. Plant & Animal Genomes XIX Conference Abstracts, San Diego, CA
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Progress 10/01/10 to 09/30/11
Outputs
OUTPUTS: The over-arching goal of this project is to understand the molecular-genetic regulation of plant mitochondrial processes key to plant growth and development, with emphasis on plant reproduction. The research approach exploits a mitochondria-encoded pollen cell death phenotype, cytoplasmic male sterility type S (CMS-S) that can be rescued by nuclear restorer-of-fertility (restorer) mutations. Objective 1 is to identify mitochondrial signaling pathways that participate in plant cell death programs. Outputs towards this objective are: (1-1) Seven constructs were developed in Agrobacterium tumefaciens strain GV3101 and transiently expressed in the petunia petal system to test the ability of five mitochondria-targeted candidate CMS genes and two reporter genes to initiate mitochondrial oxidative stress and mitochondria-signaled programmed cell death pathways. Petunia petals transiently expressing these constructs were examined via immunoblotting for protein expression and mitochondrial targeting and via fluorescence microscopy for cell viability and reactive oxygen signaling. (1-2) Transgenic maize plants were developed to test the Neurospora crassa ATP9 mitochondrial targeting signal via a reduction-oxidation sensitive green fluorescent protein (roGFP) reporter in maize pollen. Objective 2 is to identify nuclear genes that contribute to mitochondrial gene expression in plants and learn the specific functions that these genes perform or support within the mitochondria. Outputs towards this objective are: (2-1) Two non-allelic Mutator (Mu)-transposon induced restorer mutants were analyzed by Illumina DNA sequencing for Mu element insertion sites co-segregating with fertility restoration. (2-2) One undergraduate intern was mentored in the context of a plant molecular genetics research project. Project findings were disseminated through one abstract, one poster and one oral presentation at an international research conference. PARTICIPANTS: Individuals: Karen Chamusco, Biological Scientist, University of Florida, performed transient expression assays and cytological studies for the localization of proteins in plant tissues and organs; Christine D. Chase, Project Director, Professor, University of Florida, provided expertise in molecular analyses of mitochondrial function and gene expression in pollen; Michelle Feole, Undergraduate Intern, University of Florida, performed plant phenotyping analyses and DNA extractions; Susan Gabay-Laughnan, Collaborator, University of Illinois Urbana-Champaign, performed seed phenotyping and allelism tests of restorer mutants; Alice Barkan, Collaborator, University of Oregon performed Mu-Illumina DNA sequencing analysis on families segregating for restorer-of-fertility mutations. Partner organizations: University of Illinois Urbana-Champaign, University of Oregon, Eugene. Training and Professional Development: This project provided hands-on laboratory training to one undergraduate student, contributing towards the professional development of these individuals in the life sciences. Michelle Feole, undergraduate student, University of Florida, learned techniques of DNA extraction, polymerase chain reaction, DNA gel electrophoresis and maize genetics. TARGET AUDIENCES: Undergraduate students: research internship training for undergraduate students contributes not only to their professional development in the life sciences but also to their appreciation for the importance of plant research for sustainable food fuel and fiber production. Plant mitochondrial research community: This project provided new tools for expression, targeting and phenotypic analysis of mitochondrial function. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
Objective 1: The petunia petal transient gene expression system demonstrated efficient mitochondrial targeting and reporter gene expression without significant effects on cell viability or redox status. Transgenic maize plants expressed roGFP in a pollen-specific fashion, and this reporter was effectively targeted to the mitochondria by the N. crassa ATP9 leader. These transgenic lines provide new tools for the analysis of redox signaling in normal and CMS maize pollen development. Objective 2. Four candidate restorer genes were identified providing new insights into pathways for nuclear control of mitochondrial function while validating CMS-S maize as a tool for forward genetic analysis of nuclear-mitochondrial function. One undergraduate student learned principles of genetics through hands on participation in a maize genetics experiment.
Publications
- Chase, CD, KC Chamusco, MN Siripant, YB Tan (2011) A Mitochondria-initiated cell death at the microspore-pollen transition in S male-sterile maize. Plant Biology 2011, Annual Meeting of the American Society of Plant Biologists, Abstract P03025, http://abstracts.aspb.org/pb2011/public/P03/P03025.html
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Progress 10/01/09 to 09/30/10
Outputs
OUTPUTS: The over-arching goal of this project is to understand the molecular-genetic regulation of plant mitochondrial processes key to plant growth and development, with emphasis on plant reproduction. The research approach exploits a mitochondria-encoded pollen cell death phenotype, cytoplasmic male sterility type S (CMS-S) that can be rescued by nuclear restorer-of-fertility (restorer) mutations. Objective 1 is to identify mitochondrial signaling pathways that participate in plant cell death programs. Outputs towards this objective are: (1-1) A transient gene expression system was developed to test the efficacy of leader sequences to target transgene products to the mitochondria and to test the ability of downstream passenger sequences to initiate mitochondria-signaled programmed cell death pathways. Analyses demonstrated that the Neurospora ATP9 leader efficiently targeted the green fluorescent protein (GFP) to plant mitochondria. (1-2) DNA constructs designed to test the ATP9 leader - GPF passenger in maize pollen have been introduced into maize plants. (1-3) Two undergraduate interns, one from a minority serving institution, were mentored in plant molecular biology research through participation in this project. Objective 2 is to identify nuclear genes that contribute to mitochondrial gene expression in plants and learn the specific functions that these genes perform or support within the mitochondria. Outputs towards this objective are: (2-1) Commercial antibodies against mammalian mitochondrial proteins were tested for specificity and efficacy in the recognition of plant mitochondrial proteins. (2-2) A collection of 14 restorer mutants was analyzed by immunoblotting to discover the effects of the restorer mutations on mitochondrial protein accumulation during maize pollen development. (2-3) Tests of allelism were completed for the collection of restorer mutants. Project findings were disseminated through three abstracts, two posters and one invited talk at international research conferences. PARTICIPANTS: Individuals: Kanchan Bhan, Postdoctoral Associate, University of Florida, developed DNA constructs for transient expression and maize transformation; Karen Chamusco, Biological Scientist, University of Florida, performed transient expression assays and cytological studies for the localization of proteins in plant tissues and organs; Christine D. Chase, Project Director, Professor, University of Florida, provided expertise in molecular analyses of mitochondrial function and gene expression in pollen; Susan Gabay-Laughnan, Collaborator, University of Illinois Urbana-Champaign, performed seed phenotyping and allelism tests of restorer mutants; Maria Gallo, Collaborator, Professor, University of Florida, provided expertise and supervision in plant transformation; Prestina Smith, undergraduate intern, Bennett College for Women, performed molecular analyses of mitochondrial proteins in transient expression system; Yong Tan, undergraduate intern, University of Florida, performed molecular analyses of mitochondrial gene expression in pollen development. Partner organizations: Bennett College, University of Illinois Urbana-Champaign Training and Professional Development: This project provided hands-on laboratory training to two undergraduate students and one postdoctoral associate, contributing towards the professional development of these individuals in the life sciences: Prestina Smith, undergraduate student, Bennett College, learned techniques of protein electrophoresis and immunoblotting, learned how to design and present a scientific poster, made a poster presentation at an international conference (Plant Biology 2010), and was accepted into a PhD program for molecular and cellular biology. Yong B. Tan, undergraduate student, University of Florida, learned techniques of protein electrophoresis and immunoblotting, performed molecular analysis mitochondrial proteins in developing pollen, and was accepted into medical school; Kanchan Bhan, postdoctoral associate, University of Florida, learned techniques of DNA cloning, developed clones for genetic transformation of maize, learned how to design and present a scientific poster. TARGET AUDIENCES: Undergraduate students; research internship training for undergraduate students contributes not only to their professional development in the life sciences but also to their appreciation for the importance of plant research for sustainable food fuel and fiber production. Notably, one undergraduate intern who participated in this project is of an ethnic group historically under-represented in the community of scientific professionals. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
Objective 1: The transient gene expression system combined with the Neurospora ATP9 leader peptide sequence provides a new and efficient fundamental research tool for the expression and analysis of mitochondria-targeted proteins in plants. Two project interns and a postdoctoral associate learned techniques of molecular genetics and scientific presentation. Objective 2: New antibody tools were validated for the recognition of plant mitochondrial adenine nucleotide translocator and cyclophilin. New knowledge was obtained regarding mitochondrial function during normal pollen development. A major transition in carbon metabolism accompanies the developmental transition from microspore to pollen. Failure to establish the metabolic transition results in functional pollen having increased resistance to mitochondrial cell death signaling. S-cytoplasm restorer genes provide new insights into the regulation of mitochondrial functions fundamental to cell metabolism and survival and to reproductive development, expanding opportunities for the molecular-genetic dissection of mitochondrial function in plants.
Publications
- Smith, P., Bhan, K., Chamusco, K., Seib, J., Gallo, M., Moreira, C., Chase, C.D. 2010. Expression of mitochondrial-targeted peptides linked to cell death signaling and pollen collapse of S male-sterile maize. Plant Biology 2010, Joint Annual Meeting of the American Society of Plant Biologists & the Canadian Society of Plant Physiologists American Society for Plant Biology, Montreal, Canada(P03067).
- Chase, C.D., Chamusco, K.C., Kamps, T.L., Siripant, M.N., Bhan, K. 2010. Mitochondrial features of pollen development in male-fertile and S male-sterile maize. Plant Biology 2010, Joint Annual Meeting of the American Society of Plant Biologists & the Canadian Society of Plant Physiologists American Society for Plant Biology, Montreal, Canada(P03068).
- Chase, C.D., Gabay-Laughnan, S.J., Gallo, M., Chamusco, K., Bhan, K., Kamps, T., Zhao, L. 2010. Cytoplasmic Male Sterility: Window to Plant Mitochondria-Nuclear Interactions. Green Plant Breeding Technologies International Conference Programme and Abstracts, Vienna, Austria(P26).
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Progress 03/01/09 to 09/30/09
Outputs
OUTPUTS: The over-arching goal of this project is to understand the molecular-genetic regulation of plant mitochondrial processes key to plant growth and development, with emphasis on plant reproduction. The research approach exploits a mitochondria-encoded pollen cell death phenotype, cytoplasmic male sterility type S (CMS-S) that can be rescued by nuclear restorer-of-fertility (restorer) mutations. Objective 1 is to identify mitochondrial signaling pathways that participate in plant cell death programs. Outputs towards this objective are: (1-1) A set of six DNA constructs designed to express mitochondria-targeted and un-targeted green fluorescent protein (GFP) or peptides predicted to cause mitochondria-signaled cell death in transgenic plants and plant cell lines; (1-2) Transgenic Arabidopsis thaliana plant lines and cell suspensions expressing mitochondria-targeted and un-targeted GFP controls; (1-3) A set of 14 DNA oligonucleotide primers designed to amplify DNA constructs above for deployment into E. coli, A. tumefaciens, N. tabacum, and A. thaliana cell cultures and into N. tabacum, A. thaliana and Z. mays plants. Objective 2 is to identify nuclear genes that contribute to mitochondrial gene expression in plants and learn the specific functions that these genes perform or support within the mitochondria. Outputs towards this objective are: (2-1) A collection of 14 restorer mutants characterized through tests of heritability, allelism and homozygous lethality; (2-2) Mapping backcross populations for seven of these restorer mutants; (2-3) Polyribosomes extracted from developing pollen of one restorer mutant and one sterile control line; (2-4) A collection of cDNA sequences for mitochondria-encoded, atp4, atp6, atp8, atp9 and cob transcripts from CMS-S ear, CMS-S microspore, normal-cytoplasm ear, normal-cytoplasm microspore, and CMS-S pollen restored to function by each of seven different mutants. This information was disseminated via GenBank Accession numbers GU058038-GU058049, and GU075810-GU075813. Project findings were also disseminated through three abstracts, two posters and one invited talk at international research conferences, and through publication of an invited book chapter. PARTICIPANTS: Individuals: Phil Becraft, Collaborator, Iowa State University, performed high-throughput screen for Mutator elements co-segregating with restorer mutations; Kanchan Bhan, Postdoctoral Associate, University of Florida, performed DNA constructs design, plant cell culture and plant transformation experiments; Karen Chamusco, Research Scientist, University of Florida, performed cytological and molecular studies for the localization of proteins in plant tissues and organs; Christine D. Chase, Project Director, Professor, University of Florida, supervised molecular analyses of mitochondrial gene expression in pollen; performed genetic screens for restorer mutants in Mutator transposon-active backgrounds; Susan Gabay-Laughnan, Collaborator, University of Illinois Urbana-Champaign, performed seed phenotyping and allelism tests of restorer mutants; Maria Gallo, Collaborator, Professor, University of Florida, provided expertise and supervision in plant transformation; Terry L. Kamps, Senior Biological Scientist, University of Florida, performed cDNA synthesis and RNA editing studies of CMS-S and fertility restored CMS-S plants; Cristina Moreira, Collaborator, Bennett College for Women, performed DNA construct design, bacterial cloning, and plant cell culture; Jeffrey Seib, Senior Biological Scientist, University of Florida, synthesized DNA constructs and performed plant cell culture and plant transformation experiments. Training and Professional Development: This project provided hands-on laboratory training to four undergraduate students and one postdoctoral associate, contributing towards the professional development of these individuals in the life sciences: Modupe Durojaiye, undergraduate student, Bennett College for Women, performed DNA construct design, plant cell culture, and bacterial transformation experiments; Carwayna McColley, undergraduate student, Bennett College for Women, performed DNA construct design, plant cell culture, and bacterial transformation experiments; Prestina Smith, undergraduate student, Bennett College for Women, performed DNA construct design, plant cell culture, and bacterial transformation experiments; Yong B. Tan, undergraduate student, University of Florida, performed molecular analysis of CMS gene expression in developing pollen; Kanchan Bhan, postdoctoral associate, University of Florida, designed DNA constructs; performed plant cell culture and plant transformation experiments. Partner organizations: Bennett College for Women, Iowa State University, University of Illinois Urbana-Champaign TARGET AUDIENCES: Undergraduate students; research internship training for undergraduate students contributes not only to their professional development in the life sciences but also to their appreciation for the importance of plant research for sustainable food fuel and fiber production. Notably, three undergraduate interns who participated in this project are members of ethnic groups historically under-represented in the community of scientific professionals. PROJECT MODIFICATIONS: Not relevant to this project.
Impacts
Objective 1: Transformation of Arabidopsis thaliana with GFP constructs demonstrated that the ATP9 leader peptide sequence derived from Neurospora crassa effectively targets GFP to mitochondria in Arabidopsis leaves and sepals. This leader represents a new tool for the genetic modification of mitochondria in plant cells, particularly with respect to the delivery of hydrophobic proteins to the mitochondria. Transformation of A. thaliana with constructs driven by the Arabidopsis ap3 promoter demonstrated significant silencing of the endogenous ap3 floral promoter. Despite published reports wherein the ap3 promoter was used to drive transgenes in Arabidopsis floral tissues, this promoter is not recommended based upon our findings. Objective 2: RNA editing of atp6, atp9 and cob transcripts was not affected by restorer mutations, although ATP6, ATP9 and COB proteins failed to accumulate in the presence of these mutations. S-cytoplasm maize represents a robust tool for the forward genetic analysis of plant mitochondrial function. The restorer genes pursued in this project are essential to the expression of mitochondrial respiratory genes. S-cytoplasm restorer genes provide new insights into the regulation of mitochondrial functions fundamental to cell metabolism and survival and to reproductive development. Research findings demonstrate that nuclear control over the accumulation of plant mitochondrial respiratory gene products occurs primarily at the level of protein translation or stability. Genetic tests of allelism based upon seed phenotype confirmed that each restorer mutant identifies a different gene, expanding opportunities for the molecular-genetic dissection of mitochondrial function in plants.
Publications
- Chase, CD (2009) Pollen collapse and fertility restoration in S male-sterile maize. International Conference for Plant Mitochondrial Biology Abstracts, Lake Tahoe, CA, invited speaker abstract 37
- Bhan, K, S Gullett, M Siripant, CD Chase (2009) Metabolic features of pollen development in male-fertile and S male-sterile maize. Maize Genetics Conference Abstracts. 51:poster 53
- Kamps, TL, MN Siripant, CD Chase (2009) Developmental stage, reproductive tissue, and cytotype effects on transcriptional and post-transcriptional regulation of mitochondrial genes. Maize Genetics Conference Abstracts. 51:poster 85
- Chase, CD, A Ribarits and E Herberle-Bors (2010) Male Sterility. pp. 437-457 In: Plant Developmental Biology, Biotechnological Perspectives, Volume I (E-C Pua and M R Davey, eds) Springer-Verlag Berlin Heidelberg
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