Progress 09/07/02 to 04/05/06
Outputs
Progress Report 1. What major problem or issue is being resolved and how are you resolving it (summarize project aims and objectives)? How serious is the problem? Why does it matter? This project is aligned with NP302, Plant Biological and Molecular Processes. Heat stress is a fundamental problem for U.S. agriculture and promises to become an even greater problem as the global climate changes toward increasing temperatures. Title XXIV of the 1990 farm bill directed the Secretary of Agriculture to study the effects of global climate change on agriculture including the effects of simultaneous increases in temperature and carbon dioxide on crops of economic importance. The detrimental effects on the yield of major U.S. crops that occurs at normal summer temperatures were only realized when plants were grown under optimal conditions in growth chambers. In addition, a major contributor to loss of crops such as tomato, pepper, and soybean is abscission of flowers and fruit
due to high temperatures. High temperatures severely limit productivity of day neutral varieties of strawberries during the summer months, but little is known about the molecular basis for this decrease. Some resistance to heat stress has been obtained by traditional breeding techniques but the physiological/biochemical bases for the resistances are largely unknown. We are addressing this problem by increasing our knowledge of how plants respond to environmental stress and of the mechanisms they have for coping with such stress. This project is specifically directed toward understanding the role that proteins produced in response to heat stress (the heat shock proteins) play in heat tolerance in plants. Using modern molecular biology techniques, we are isolating and testing various heat shock proteins from small fruit crops for their involvement in thermotolerance, and are determining if these can be used to improve thermotolerance. Results from this laboratory strongly suggest that a
single type of heat shock protein, or other proteins like it, may be useful for manipulating crop heat tolerance. Important questions remain to be answered before this approach can be considered generally applicable to problems in small fruit production. These include how these proteins normally work to bring about thermotolerance, and the specific interactions of these proteins within cells. We know that genetic manipulation to cause production of all heat shock proteins all the time does result in increased heat tolerance. However, increased heat tolerance resulting from the allocation of the plants' resources to production of so many proteins would most probably come at the expense of yield. Alternatively, the selective production of a single, pivotal, heat shock protein should not result in significant reallocation of the plants' carbon and nitrogen. We are also investigating the potential for improving the performance of specific heat shock proteins by genetic engineering, and
are identifying factors other than heat stress that affect the regulation of heat shock proteins. The research is of interest to the small fruit industry and to scientists studying plant stress. 2. List by year the currently approved milestones (indicators of research progress) Year 1 (FY 2002) 1. Plants obtained, propagated, and tested for homozygosity. 2. Seedling growth assays developed. 3. PAGE analyses completed. 4. Experiments on transformation of diploid lines. Year 2 (FY 2003) 1. Experiments on roles of temperature and daylength on flowering and fruit set in day-neutral, diploid varieties of strawberries completed. 2. Cotyledon greening assay tested with different diploid lines. 3. Clones obtained for Hsps and/or floral identity genes. 4. Transformation of F. vesca with DcHsp17.7. Year 3 (FY 2004) 1. Expression of DcHsp17.7-his and characterization of chaperone function. 2. Transformation of F. vesca with DcHsp17.7-his. 3. Assays for pollen viability and runner growth
developed 4. Additional clones obtained. Year 4 (FY 2005) 1. Yield assays developed and applied to transgenic plants. 2. Molecular assays developed. Year 5 (FY 2006) 1. Ovule viability assay developed. 2. Electrolyte leakage assay developed. 3. Fluorescence assay assessed. 4. Experiments to test the role of DcHsp17.7 in development. 5. Protein interactions with DcHsp17.7 determined. 4a List the single most significant research accomplishment during FY 2006. Genetic markers for genes that are expressed in heat stressed strawberry seedlings were developed and tested. A map of the genes in strawberry is important for breeders and for gene cloning, yet the existing map has very few gene addresses on it. A well populated map will also provide a useful comparison for breeders of other closely related crops such as peach and apple, and provides a roadmap for sequencing of the DNA from these crops. Prior to last year, only a few strawberry gene sequences had been available in the public
database. This project has now generated approximately 12,500 strawberry sequences, and over 17 different strawberry heat stress proteins are represented in this database (see B below). These sequences were used to generate markers, tools that can be used to pinpoint the location of genes on the genetic map. These markers were tested on 13 different species of strawberry to determine their usefulness. The research falls under National Program 302 - Plant Biological and Molecular Processes, and addresses problems Ia, and IIb in the National Program Action Plan. Specifically these are: Ia Molecular characterization of plant genetic systems. IIb Plant tolerance to environmental stresses. 4b List other significant research accomplishment(s), if any. Approximately 10,000 new strawberry sequences were deposited in the public database. The research falls under National Program 302 Plant Biological and Molecular Processes, and addresses problems Ia, and IIb in the National Program Action
Plan. Specifically these are: Ia Molecular characterization of plant genetic systems. IIb Plant tolerance to environmental stresses. 5. Describe the major accomplishments to date and their predicted or actual impact. An inbred line of the diploid strawberry, Fragaria vesca, has been produced that is necessary for subsequent testing of the function of genes, and for studying the mechanisms by which such genes act. This model plant system can be used to test gene function in agriculturally important members of the Rosaceae such as apple and peach for which transformation is inefficient and generation times are long. Impact: This line has been selected as the best candidate for sequencing of the strawberry genome as part of a concerted effort by researchers working on peach, apple, and strawberry. Milestone year 1: Plants obtained, propagated, and tested for homozygosity. Action Plan components 302 Ia and IIb. ARS Performance Measure 1.2.6 and 1.2.7. Fragaria vesca has been extensively
characterized for heat shock protein production, allowing us to identify some useful antibodies that are essential for assaying for stress responses in these plants. Impact: none. Milestone year 1: PAGE analyses completed, Milestone year 4: Molecular assays developed. Action Plan component 302 IIb ARS Performance Measure 1. 2.6 and 1.2.7. Developed methods to obtain large numbers of aseptic seeds for use in thermotolerance assays. This enabled us to evaluate several aspects of plant growth, including rate of root growth or leaf production, and amount of secondary root heat thermotolerance. Impact: The ability to obtain sufficient aseptic stress treated material is an important reason why a grant proposal was funded by CSREES to obtain 40,000 additional EST sequences from Fragaria. Milestone Year 1: Seedling growth assays. Milestone Year 2: Experiments on the roles of temperature and daylength on flowering and fruit set in diploid strawberry. Action Plan component 302 IIb ARS
Performance Measure 1.2.6 and 1.2.7. Developed a database of approximately 12,500 sequences expressed in strawberry seedlings in response to elevated temperature. Analyzed and annotated databases of these sequences have been made available on the World Wide Web. Impact: Sequences from this database have already been used for annotating strawberry genome sequence in an exploratory project from sequencing the entire strawberry genome. This accomplishment is also important because DNA sequences from our database were used for developing gene specific and molecular genetic markers. These sequences will be essential for developing advanced tools to study gene function. Sequences from this database are useful for identifying homologous genes in other important members of the Rosaceae such as apple and peach. Milestone Year 4: Molecular assays developed. Action Plan component 302 Ib and IIb. ARS Performance Measure 1.2.6 and 1.2.7. 6. What science and/or technologies have been transferred
and to whom? When is the science and/or technology likely to become available to the end- user (industry, farmer, other scientists)? What are the constraints, if known, to the adoption and durability of the technology products? Seeds of the inbred diploid strawberry, Fragaria vesca have been requested by other scientists, and have been made available to the general scientific community for testing gene function in other crops related to strawberry. Two annotated databases of sequences from stressed Fragaria seedlings have been made available on the World Wide Web. Strawberry sequences have been sent to the Rosaceae Genome Database Full length strawberry gene sequences were deposited in the public database, GenBank, to be released upon publication. Sequences useful for mapping genes in strawberry and related crops have been made available to the research community. The major constraints to adoption of any products resulting from the current work on strawberry are: the availability of
regulatory sequences for specific gene expression that are not constrained by existing patents, and the objections of consumers to genetically modified food products. 7. List your most important publications in the popular press and presentations to organizations and articles written about your work. (NOTE: List your peer reviewed publications below). Slovin, J. P., Rabinowicz, P. 2005. Increasing the diversity of EST sequences for Fragaria. Abstract: Plant and Animal Genome XIV, San Diego, CA. January 14-18, 2006. #P26 Slovin, J., Davis, T., Rabinowicz, P., Sargent, D., Shulaev, V. 2005. Fragaria vesca, a reference plant for the Rosaceae family. Abstract: Arthur M. Sackler Colloquia of the National Academy of Sciences. From Functional Genomics of Model Organisms to Crop Plants for Global Health. April 3-5, 2006. #40.
Impacts
(N/A)
Publications
- Dhanaraj, A.L., Slovin, J.P., Rowland, L.J. 2005. Isolation of a cdna clone and characterization of expression of the highly abundant, cold acclimation-associated 14 da dehydrin of blueberry.. Plant Science. 168:949-957
- Normanly, J., Slovin, J.P., Cohen, J.D. 2005. Auxin biosynthesis and metabolism.. Recent Progress in Research on Plant Hormones and Related Substances. 3rd Edition, pp 36-62.
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Progress 10/01/04 to 09/30/05
Outputs
1. What major problem or issue is being resolved and how are you resolving it (summarize project aims and objectives)? How serious is the problem? What does it matter? Heat stress is a fundamental problem for U.S. agriculture and promises to become an even greater problem as the global climate changes toward increasing temperatures. Title XXIV of the 1990 farm bill directed the Secretary of Agriculture to "study the effects of global climate change on agriculture" including "the effects of simultaneous increases in temperature and carbon dioxide on crops of economic importance." The detrimental effects on the yield of major US crops that occurs at normal summer temperatures were only realized when plants were grown under optimal conditions in growth chambers. In addition, a major contributor to loss of crops such as tomato, pepper, and soybean is abscission of flowers and fruit due to high temperatures. High temperatures severely limit productivity of day neutral
varieties of strawberries during the summer months, but little is known about the molecular basis for this decrease. Some resistance to heat stress has been obtained by traditional breeding techniques but the physiological/biochemical bases for the resistances are largely unknown. We are addressing this problem by increasing our knowledge of how plants respond to environmental stress and of the mechanisms they have for coping with such stress. This project is specifically directed toward understanding the role that proteins produced in response to heat stress (the heat shock proteins) play in thermotolerance in plants. Using modern molecular biology techniques, we are isolating and testing various heat shock proteins from small fruit crops for their involvement in thermotolerance, and are determining if these can be used to improve thermotolerance. Results from this laboratory strongly suggest that a single type of heat shock protein, or other proteins like it, may be useful for
manipulating crop thermotolerance. Important questions remain to be answered before this approach can be considered generally applicable to problems in small fruit production. These include how these proteins normally work to bring about thermotolerance, and the specific interactions of these proteins within cells. We know that genetic manipulation to cause production of all heat shock proteins all the time does results in increased thermotolerance. However, increased thermotolerance resulting from the allocation of the plants' resources to production of so many proteins would most probably come at the expense of productivity. Alternatively, the selective production of a single, pivotal, heat shock protein should not result in significant reallocation of the plants' carbon and nitrogen. We are also investigating the potential for improving the performance of specific heat shock proteins by genetic engineering, and are identifying factors other than heat stress that affect the
regulation of heat shock proteins. The research being undertaken falls under National Program 302 - Plant Biological and Molecular Processes, and addresses problems Ia,Ib, and IIb in the National Program Action Plan. Specifically these are:Ia Molecular characterization of plant genetic systems. Ib Plant transformation systems and the influence of transgenes on genome structure and function. IIb Plant tolerance to environmental stresses. 2. List the milestones (indicators of progress) from your Project Plan. Year 1 (FY 2002) Plants obtained, propagated, and tested for homozygosity. Seedling growth assays developed. PAGE analyses completed. Experiments on transformation of diploid lines. Year 2 (FY 2003) Experiments on roles of temperature and daylength on flowering and fruit set in day-neutral, diploid varieties of strawberries completed. Cotyledon greening assay tested with different diploid lines. Clones obtained for Hsps and/or floral identity genes. Transformation of F. vesca with
DcHsp17.7. Year 3 (FY 2004) Expression of DcHsp17.7-his and characterization of chaperone function. Transformation of F. vesca with DcHsp17.7-his. Assays for pollen viability and runner growth developed Additional clones obtained. Year 4 (FY 2005) Yield assays developed and applied to transgenic plants. Molecular assays developed. Year 5 (FY 2006) Ovule viability assay developed. Electrolyte leakage assay developed. Fluorescence assay assessed. Experiments to test the role of DcHsp17.7 in development. Protein interactions with DcHsp17.7 determined. 3a List the milestones that were scheduled to be addressed in FY 2005. For each milestone, indicate the status: fully met, substantially met, or not met. If not met, why. 1. Yield assays developed and applied to transgenic plants. The work could not be completed due to lack of technical help. The first part of the year was spent training a new technician, who then left on maternity leave, then resigned. Assays based on viability of young
plants following heat stress were assessed and shown to not be useful because vegetative tissue is quite tolerant to high temperatures for short (12h) periods, both in the light and in the dark. Elevated, but not as high, temperatures for longer periods were shown to delay inflorescence development. We are currently working to find the critical temperature(s) and treatment times that affect continued inflorescence growth. Milestone Not Met Progress slowed by resource limitation (human,fiscal,equipment, etc. 2. One milestone, "Fluorescence assay assessed" was moved up from Year 5 because of its importance to the Fruit Laboratory breeding program. We found that modern fluorometers cannot detect changes in fluorescence upon heat stress in strawberry, even when other stress indicators are positive. In the project statement currently being written it is proposed to adapt a newly developed fluorescence-based assay for strawberry. Milestone Fully Met 3. Molecular assays developed. All of the
collaborators for this part of the work were not able to participate. One investigator is no longer at Beltsville and the USDA BARC-W EM Facility closed for renovation and developed a long backlog. Another investigator's research program has focused on octoploid mapping rather than on diploid genomics. Molecular assays at the protein level (using antibodies) had been developed in years one and two. Due to the lack of collaborators and technical help, it was decided that more rapid progress could be made by developing a heat stress cDNA library and using EST sequences from this to design primers for examining a wider range of stress gene expression than previously proposed. An EST database has been developed from which primers for 35 different stress related genes have been designed and tested for: 1) expression in heat stressed seedlings by RTPCR and 2) potential for use as gene specific PCR molecular markers in diploid and octoploid strawberry as well as another agriculturally
important member of the Rosaceae, peach. EST sequences have also been evaluated for SSRs for marker development. Milestone Not Met Progress slowed by resource limitation (human,fiscal,equipment, etc. 3b List the milestones that you expect to address over the next 3 years (FY 2006, 2007, and 2008). What do you expect to accomplish, year by year, over the next 3 years under each milestone? The project is scheduled to be completed in FY2006 and a new project focusing on the interaction of elevated temperature and inflorescence and fruit development is being written to undergo OSQR review and subsequent implementation beginning FY 2007. Anticipated milestones or accomplishments are given for FY 2007 and FY2008. Year 5 (FY 2006) Ovule viability assay developed. Electrolyte leakage assay developed. Experiments to test the role of DcHsp17.7 in development: A new transformation system for F. vesca was developed at Virginia Tech but will not be released for use until later this year. The
first priority will be to obtain transformants (both with increased expression and diminished expression) of the strawberry genes we have identified in order to test their function. We will then return to the carrot gene if there is time. Protein interactions with DcHsp17.7 determined. Sometime during year 5 a new technician will be hired and trained. It is therefore doubtful that we will undertake the proposed highly technical biochemical aspects of the project. Year 1 (FY 2007) Additional EST sequences will be generated from cDNA libraries made to crowns from plants that have been induced to flower and from young to mature flower buds, as well as flowers and young fruit so as to maximize coverage of genes involved in fruit yield. Sequences will be examined and tested for use as gene specific molecular markers. Year 2 (FY 2007) Together with the ESTs obtained previously, the new ESTs will form the basis for a small microarray containing representative stress genes and genes involved
in reproduction. In addition, high throughput Q-RTPCR will be used to monitor expression of inflorescence and fruit development genes during heat stress, as well as the ability of inflorescence and fruit tissues to mount a stress response. 4a What was the single most significant accomplishment this past year? An annotated database of EST sequences from heat stressed Fragaria. Only a few strawberry gene sequences are available in GenBank and most encode genes involved in fruit development. An ARS scientist at Beltsville, MD, in collaboration with a scientist at The Virginia Biotechnology Institute in Blacksburg developed an EST database of over 2000 sequences expressed in strawberry seedlings in response to elevated temperature. This analyzed and annotated database has been made available on the World Wide Web. Over 17 different strawberry heat stress proteins are represented in this database, as well as novel sequences previously not associated with heat stress. This accomplishment is
important because these ESTs are being used for developing gene specific and SSR molecular markers, as well as for developing a microarray for studying gene function. Sequences from this database should also be useful for identifying homologous genes in other members of the Rosaceae. 4d Progress report. Primers designed and tested for gene specific and ssr markers. Primers for 35 different stress related genes have been designed and tested for: 1) expression in heat stressed seedlings by RTPCR; and 2) potential for use as gene specific PCR molecular markers in diploid and octoploid strawberry as well as another agriculturally important member of the Rosaceae, peach. 5. Describe the major accomplishments over the life of the project, including their predicted or actual impact. An inbred line of the diploid strawberry, Fragaria vesca, has been produced that is necessary for subsequent testing of the function of genes, and for studying the mechanisms by which such genes act. This model
plant system can be used to test gene function in agriculturally important members of the Rosaceae such as apple and peach for which transformation is inefficient and generation times are long. Impact: none. Milestone year 1: Plants obtained, propagated, and tested for homozygosity. National Program 302, Action Plan Components Ia and IIb. ARS Strategic Plan Performance Measure 1.2.6 and 1.2.7. Fragaria vesca has been extensively characterized for heat shock protein production, allowing us to identify several useful antibodies that are essential for assaying for stress responses in these plants. Impact: none. Milestone year 1: PAGE analyses completed, Milestone year 4: Molecular assays developed. National Program 302, Action Plan Component IIb, ARS Strategic Plan Performance Measure 1.2.6 and 1.2.7. Developed methods to obtain large numbers of aseptic seeds for use in thermotolerance assays. This enabled us to evaluate several aspects of plant growth, including rate of root growth or
leaf production, and amount of secondary root formation on aseptically growing seedlings for use as assays for thermotolerance. Impact: The ability to obtain sufficient aseptic stress treated material is an important reason why a grant proposal was funded by CSREES to obtain 40,000 additional EST sequences from Fragaria. Milestone Year 1: Seedling growth assays. Milestone Year 2: Experiments on the roles of temperature and daylength on flowering and fruit set in diploid strawberry. National Program 302, Action Plan Component 302 IIb, ARS Strategic Plan Performance Measure 1.2. 6 and 1.2.7. Developed an EST database of over 2000 sequences expressed in strawberry seedlings in response to elevated temperature. The analyzed and annotated database has been made available on the World Wide Web. Impact: This accomplishment is important because ESTs are used for developing gene specific and ssr molecular markers, as well as being essential for developing microarrays or designing primers for
QRTPCR to study gene function. Sequences from this database are useful for identifying homologous genes in other important members of the Rosaceae such as apple and peach. Milestone Year 4: Molecular assays developed. National Program 302,Action Plan Component 302 Ib and IIb, ARS Strategic Plan Performance Measure 1.2.6 and 1.2.7. 6. What science and/or technologies have been transferred and to whom? When is the science and/or technology likely to become available to the end- user (industry, farmer, other scientists)? What are the constraints, if known, to the adoption and durability of the technology products? Seeds of the inbred diploid Fragaria vesca for testing gene function have been made available to the scientific community. An annotated EST database of sequences from stressed Fragaria seedlings has been made available on the World Wide Web. EST sequences have been sent to the Rosaceae Genome Database Full length gene sequences were deposited in GenBank to be released upon
publication. The major constraints to adoption of any products resulting from the current work on strawberry are: the availability of regulatory sequences for specific gene expression that are not constrained by existing patents, and the objections of consumers to genetically modified food products.
Impacts
(N/A)
Publications
- Dhanaraj, A.L., Slovin, J.P., Rowland, L.J. 2005. Isolation of a cdna clone and characterization of expression of the highly abundant, cold acclimation-associated 14 da dehydrin of blueberry.. Plant Science. 168:949-957
- Normanly, J., Slovin, J.P., Cohen, J.D. 2005. Auxin biosynthesis and metabolism.. Recent Progress in Research on Plant Hormones and Related Substances. 3rd Edition, pp 36-62.
|
Progress 10/01/03 to 09/30/04
Outputs
1. What major problem or issue is being resolved and how are you resolving it (summarize project aims and objectives)? How serious is the problem? What does it matter? Heat stress is a fundamental problem for U.S. agriculture and promises to become an even greater problem as the global climate changes toward increasing temperatures. Title XXIV of the 1990 farm bill directed the Secretary of Agriculture to "study the effects of global climate change on agriculture" including "the effects of simultaneous increases in temperature and carbon dioxide on crops of economic importance." The detrimental effects on the yield of major US crops that occurs at normal summer temperatures were only realized when plants were grown under optimal conditions in growth chambers. In addition, a major contributor to loss of crops such as tomato, pepper, and soybean is abscission of flowers and fruit due to high temperatures. High temperatures severely limit productivity of day neutral
varieties of strawberries during the summer months, but little is known about the molecular basis for this decrease. Some resistance to heat stress has been obtained by traditional breeding techniques but the physiological/biochemical bases for the resistances are largely unknown. We are addressing this problem by increasing our knowledge of how plants respond to environmental stress and of the mechanisms they have for coping with such stress. This project is specifically directed toward understanding the role that proteins produced in response to heat stress (the heat shock proteins) play in thermotolerance in plants. Using modern molecular biology techniques, we are isolating and testing various heat shock proteins from small fruit crops for their involvement in thermotolerance, and are determining if these can be used to improve thermotolerance. Results from this laboratory strongly suggest that a single type of heat shock protein, or other proteins like it, may be useful for
manipulating crop thermotolerance. Important questions remain to be answered before this approach can be considered generally applicable to problems in small fruit production. These include how these proteins normally work to bring about thermotolerance, and the specific interactions of these proteins within cells. We know that genetic manipulation to cause production of all heat shock proteins all the time does results in increased thermotolerance. However, increased thermotolerance resulting from the allocation of the plants' resources to production of so many proteins would most probably come at the expense of productivity. Alternatively, the selective production of a single, pivotal, heat shock protein should not result in significant reallocation of the plants' carbon and nitrogen. We are also investigating the potential for improving the performance of specific heat shock proteins by genetic engineering, and are identifying factors other than heat stress that affect the
regulation of heat shock proteins. The research being undertaken falls under National Program 302 "Plant Biological and Molecular Processes," and addresses problems Ia,Ib, and IIb in the National Program Action Plan. Specifically these are:Ia Molecular characterization of plant genetic systems. Ib Plant transformation systems and the influence of transgenes on genome structure and function. IIb Plant tolerance to environmental stresses. 2. List the milestones (indicators of progress) from your Project Plan. Year 1 (FY 2002) Plants obtained, propagated, and tested for homozygosity. Seedling growth assays developed. PAGE analyses completed. Experiments on transformation of diploid lines. Year 2 (FY 2003) Experiments on roles of temperature and daylength on flowering and fruit set in day-neutral, diploid varieties of strawberries completed. Cotyledon greening assay tested with different diploid lines. Clones obtained for Hsps and/or floral identity genes. Transformation of F. vesca with
DcHsp17.7. Year 3 (FY 2004) Expression of DcHsp17.7-his and characterization of chaperone function. Transformation of F. vesca with DcHsp17.7-his. Assays for pollen viability and runner growth developed Additional clones obtained. Year 4 (FY 2005) Yield assays developed and applied to transgenic plants. Molecular assays developed. Year 5 (FY 2006) Ovule viability assay developed. Electrolyte leakage assay developed. Fluorescence assay assessed. Experiments to test the role of DcHsp17.7 in development. Protein interactions with DcHsp17.7 determined. 3. Milestones: A. List the milestones that were scheduled to be addressed in FY 2004. How many milestones did you fully or substantially meet in FY 2004 and indicate which ones were not fully or substantially met, briefly explain why not, and your plans to do so. The milestones listed below were scheduled to be completed under Year 3. Additional clones obtained. Completed and expanded. Expression of DcHsp17.7-his and characterization of
chaperone function. Partially completed. Transformation of F. vesca with DcHsp17.7-his. Delayed until Year 4. Assays for pollen viability and runner growth developed. Partially completed. Explanations: Expression of DcHsp17.7-his and characterization of chaperone function: Because of staffing shortages and the need to train an inexperienced replacement, it was decided to delay this technically challenging molecular expression work and instead focus on developing plant assays, some of which were scheduled as milestones for future years. Transformation of F. vesca with DcHsp17.7-his: Our initial attempts to transform F. vesca using published protocols resulted in unacceptable levels of somaclonal variation and low transformation efficiency. Because of staffing shortages and the need to train an inexperienced replacement, the time consuming transformation work was put off until a later time (presumably later this year), when a new, highly efficient, transformation protocol for F. vesca
that has been developed at Virginia Tech becomes available. Assays for pollen viability and runner growth developed were partially completed. Work focused on developing plant assays including runner growth, seedling root growth, fluorescence assays (scheduled for Year 5), and growth characteristics of the inbred line (95% homozygous) that has been developed over the past three years. In addition, work was initiated on developing molecular assays (scheduled for Year 4). B. List the milestones that you expect to address over the next 3 years (FY 2005, FY 2006, and FY 2007). What do you expect to accomplish, year by year, over the next 3 years under each milestone. The project is scheduled to be completed in FY2006 and a new project will be developed to undergo OSQR review and subsequent implementation beginning FY 2007. Year 4 (FY 2005) Yield assays developed and applied to transgenic plants. We will not be able to develop yield assays and apply them because initial attempts to
transform F. vesca using published protocols resulted in unacceptable levels of somaclonal variation and low transformation efficiency. A new transformation system for F. vesca has been developed at Virginia Tech and will be published soon. Resulting transgenic plants will be tested when the T2 generation is available, probably Year 5. Molecular assays developed. Additional EST sequences will be generated from cDNA libraries made to crowns from plants that have been induced to flower and from young to mature flower buds, as well as flowers and young fruit so as to maximize coverage of genes involved in fruit yield. Together with the ESTs obtained previously, these new ESTs will form the basis for a small microarray containing representative stress genes and genes involved in reproduction. A microarray allows the analysis of many genes at the same time and is a vast improvement to the individual molecular assays originally planned. Added milestones to Year 4: Expression of
DcHsp17.7-his and characterization of chaperone function. Moved from Year 3 because of staffing shortage. Transformation of F. vesca with DcHsp17.7-his. Moved from Year 3 because of staffing shortage. Development of a fluorescence assay for plant stress. This was originally a milestone for Year 5. Because this assay would be useful for the breeding program in the Fruit Lab, we had worked on this assay in Year 3. Contrary to what is expected, we found that there was no change in fluorescence with elevated temperature, although significant changes were seen in gas exchange measurements using borrowed equipment. We therefore decided to continue to pursue the FY 2006 milestone in FY 2005. A newly acquired gas exchange analyzer, in combination with protein and nucleic acid analysis of stress protein synthesis, will be used to examine why no changes in commonly measured parameters of fluorescence were seen when strawberry plants were exposed to elevated temperatures. We will then be in
a better position to determine if fluorescence can be used as part of the breeding program to select more thermotolerant strawberry varieties. Year 5 (FY 2006) Ovule viability assay developed. Electrolyte leakage assay developed. Fluorescence assay assessed: should be completed in Year 4 because this assay is needed for the strawberry breeding program. Experiments to test the role of DcHsp17.7 in development. The capability to regenerate whole plants from cells that have synthesis of the strawberry homolog of DcHsp17.7 knocked out by RNAi will be used as an indicator of the role of low molecular weight heat shock proteins in organogenesis. Protein interactions with DcHsp17.7 determined. Experiments will be performed to determine the mechanism of action of DcHsp17.7 by examining protein-protein interactions and by localizing the His tag within the cell. Year 6 (FY 2007) Stress genes identified in the previous project will be transformed into the inbred test system also developed in the
previous project. The assays for thermotolerance developed in the previous project will be used to test the potential of these stress genes for ameliorating strawberry production problems during periods of elevated temperature. The microarray developed as part of the previous project will used to assess where in the reproductive development process heat stress causes the most damage. 4. What were the most significant accomplishments this past year? A. Single most significant accomplishment during FY2004. Development of an inbred line for testing gene function in diploid Fragaria was completed. This accomplishment was important because it is fundamental to all other aspects of the project, and because it now gives us a useful system for testing gene function in other members of the Rosaceae. The diploid Fragaria system is genetically simple, transformable, and amenable to laboratory manipulation on the whole plant level. The plant has a small genome and a short life cycle making it
an ideal model system for studying gene function in plants as diverse as peach, rose, and strawberry. B. Other Significant Accomplishments, if any. About 300 Fragaria vesca ESTs have been obtained from a cDNA library representing heat-treated seedlings. Of these, about 100 identify clones for known stress response genes. This accomplishment was important because ESTs are useful for genetic marker development as well as essential for developing a microarray for studying gene function. Five full-length clones for members of heat shock protein families believed to be involved in thermotolerance were sequenced. These are useful for overexpression studies of gene function and for designing RNAi constructs for gene knockout. C. Significant activities that support special target populations. None D. Progress Report opportunity to submit additional programmatic information to your Area Office and NPS. None. 5. Describe the major accomplishments over the life of the project, including their
predicted or actual impact. An inbred line of the diploid strawberry, Fragaria vesca, has been produced that is necessary for subsequent testing of the effects of various genes on thermotolerance, and for studying the mechanisms by which such genes act. Action Plan components Ia and IIb. Fragaria vesca has been extensively characterized for heat shock protein production, allowing us to identify several useful antibodies that can be used to assay for stress responses in these plants. Action Plan component IIb Methods for obtaining large numbers of aseptic seeds were developed for use in thermotolerance assays, which enabled us to evaluate several aspects of plant growth, including rate of root growth or leaf production, and amount of secondary root formation on aseptically growing seedlings for use as assays for thermotolerance. Action Plan component IIb 6. What science and/or technologies have been transferred and to whom? When is the science and/or technology likely to become
available to the end- user (industry, farmer, other scientists)? What are the constraints, if known, to the adoption and durability of the technology products? When is the science and/or technology likely to become available to the en-user (industry, farmer, other scientists)? What are the constraints, if known, to the adoption and durability of the technology products? Seeds of the inbred diploid Fragaria vesca have been made available through a material transfer agreement to scientists at Virginia Tech. Seeds will be made more generally available publicly once the USDA administration decides not to pursue a patent on this material. EST sequences are sent to the Rosaceae Genome Database for distribution as they are obtained. Full length gene sequences are deposited in GenBank to be released upon publication. The major constraints to adoption of any products resulting from the current work on strawberry are: the availability of regulatory sequences for specific gene expression that
are not constrained by existing patents, and the objections of consumers to genetically modified food products. 7. List your most important publications in the popular press and presentations to organizations and articles written about your work. Slovin, J. P. Further development of diploid strawberry as a model test system. The 2nd International Rosaceae Genome Mapping Conference. Clemson University, May 23, 2004. Scientific Publications: Rowland, L.J., Mehra, S., Dhanaraj, A.L., Ogden, E.L., Slovin, J.P. 2003. Development of EST-PCR markers for DNA fingerprinting and genetic relationship studies in Blueberry (Vaccinium, section Cyanococcus). Journal of the American Society for Horticultural Science. 128(5):682-690.
Impacts
(N/A)
Publications
- Rowland, L.J., Smriti, M., Dhanaraj, A., Ehlenfeldt, M.K., Ogden, E.L., Slovin, J.P. 2003. Development of est-pcr markers for dna fingerprinting and mapping in blueberry (vaccinium, section cyanococcus). American Society for Horticultural Science. 128 (5):682-690.
- Dhanaraj, A.L., Slovin, J.P., Rowland, L.J. 2003. Analysis of gene expression associated with cold aclimation in bluberry floral buds using expressed sequence tags. Plant Science. 166:863-872.
- Cohen, J.D., Slovin, J.P., Hendrickson, A. 2003. Two genetically discrete pathways convert tryptophan to auxin: more redundancy in auxin biosynthesis. Trends in Plant Science. 8 (5): 197-199.
- Slovin, J.P. 2004. Further development of diploid strawberry as a model test system. Meeting Abstract. Paper No. 13.
- Park, S., Walz, A., Momonoki, Y., Ludwig-Mueller, J., Slovin, J.P. 2004. Partial characterization of major iaa conjugates in arabidopsis. American Society of Plant Biologists Annual Meeting. Paper No. 669. Plant Biology 2003
- Dhanaraj, A.L., Slovin, J.P., Rowland, L.J. 2004. Est analysis for the study of cold stress in blueberry (vaccinium spp.). Keystone Symposia. Paper No. 4.
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Progress 10/01/02 to 09/30/03
Outputs
1. What major problem or issue is being resolved and how are you resolving it? In order to improve crops so that the United States is capable of sustained production despite detrimental changes in the environment, we need to understand how plants respond to environmental stress, and the mechanisms they have for coping with such stress. This project is directed toward understanding the role that proteins produced in response to heat stress (the heat shock proteins) play in thermotolerance in plants. Using modern molecular biology techniques, we are isolating and testing various heat shock proteins from small fruit crops for their involvement in thermotolerance, and are determining if these can be used to improve thermotolerance. Results from this laboratory strongly suggest that a single type of heat shock protein, or other proteins like it, may be useful for manipulating crop thermotolerance. Important questions remain to be answered before this approach can be
considered generally applicable to problems in small fruit production. These include how these proteins normally work to bring about thermotolerance, and the specific interactions of these proteins within cells. We know that genetic manipulation to cause production of all heat shock proteins all the time does results in increased thermotolerance. However, increased thermotolerance resulting from the allocation of the plants' resources to production of so many proteins would most probably come at the expense of productivity. However, the selective production of a single, pivotal, heat shock protein should not result in significant reallocation of the plants' carbon and nitrogen. We are also investigating the potential for improving the performance of specific heat shock proteins by genetic engineering, and are identifying factors other than heat stress that affect the regulation of heat shock proteins. 2. How serious is the problem? Why does it matter? Heat stress is a fundamental
problem for U.S. agriculture and promises to become an even greater problem as the global climate changes toward increasing temperatures. Title XXIV of the 1990 farm bill directed the Secretary of Agriculture to "study the effects of global climate change on agriculture" including "the effects of simultaneous increases in temperature and carbon dioxide on crops of economic importance." High temperatures severely limit productivity of dayneutral varieties of strawberries during the summer months. A major contributor to loss of crops such as tomato, pepper, and soybean is abscission of flowers and fruit due to high temperatures. Some resistance to heat stress has been obtained by traditional breeding techniques but the physiological/biochemical bases for the resistances are largely unknown. 3. How does it relate to the National Program(s) and National Program Component(s) to which it has been assigned? This project is assigned to National Program 302, Improving Plant Biological and
Molecular Processes, and relates directly to the stated expected output. This research aims to identify genes for heat shock proteins involved in thermotolerance and the development of tolerance to other stresses that can then be used to produce crops with more consistent yields during environmental fluctuations and/or on marginal lands. In addition, this research will have an impact in the area of National Program Area 204, Global Change, in that new practices and technologies will be developed to conserve the Nation's natural resource base and balance production efficiency and environmental quality. This research will result in a better understanding of how a changing climate will interact with existing stresses to influence the productivity of crops. 4. What were the most significant accomplishments this past year? A. Single Most Significant Accomplishment during FR 2003 year: This research was initiated in order to have a useful genetic system in strawberry, an herbaceous
perennial, with which we could test the ability of various genes and regulatory sequences to improve fruit production and quality in suboptimal environments. Scientists in the Fruit Lab at Beltsville, Maryland have established methods and assays that make the diploid strawberry, Fragaria vesca, generally useful for testing molecular mechanisms for thermotolerance in a crop that is not cultivated as an annual. A nearly inbred line of Fragaria vesca has been produced that has been extensively characterized for the production of heat shock proteins at high temperatures, and tests were done to develop assays with which to assess thermotolerance in this strawberry. This means that it will soon be possible test genes for their ability to improve thermotolerance of a small fruit crop by transforming them into, and expressing them in this new system. B. Other Significant Accomplishments: None. C. Significant Accomplishments/Activities that Support Special Target Populations: None. 5. Describe
the major accomplishments over the life of the project, including their predicted or actual impact. A nearly inbred line of the diploid strawberry, Fragaria vesca, has been produced that is necessary for subsequent testing of the effects of various genes on thermotolerance, and for studying the mechanisms by which such genes act. These plants have been extensively characterized for heat shock protein production, allowing us to identify several useful antibodies that can be used to assay for stress responses in these plants. Methods for obtaining large numbers of aseptic seeds were developed for use in thermotolerance assays. Enabling us to evaluate several aspects of plant growth, including rate of root growth or leaf production, and amount of secondary root formation on aseptically growing seedlings for use as assays for thermotolerance. 6. What do you expect to accomplish, year by year, over the next 3 years? During FY 2004, ARS will: 1) obtain a fully inbred line of Fragaria vesca,
transform this line with a construct containing a small heat shock protein from carrot, and begin characterization of the transformed line for thermotolerance; and 2) determine the feasibility of using pollen formation and pollen germination as assays for thermotolerance. During FY 2005, ARS will: 1) isolate and characterize genes for strawberry heat shock proteins, genes involved in the transition of strawberry meristems from the vegetative to the reproductive state, and genes for flower initiation and production; and 2) determine whether high temperature affects the expression of the genes for reproduction. During FY 2006, ARS will determine the role of specific strawberry heat shock protein genes in thermotolerance using the inbred diploid test system. 7. What science and/or technologies have been transferred and to whom? When is the science and/or technology likely to become available to the end- user (industry, farmer, other scientists)? What are the constraints, if known, to
the adoption and durability of the technology products? The inbred line of Fragaria vesca will be made available to the scientific community during the upcoming year. Clones encoding sequences of a DnaJ gene and a LEAFY gene from Fragaria vesca will become available and will be made available when characterization of their expression in response to elevated temperature is completed. The major constraints to adoption of any products resulting from the current work on strawberry are: the availability of regulatory sequences for specific gene expression that are not constrained by existing patents and the objections of consumers to genetically modified food products. 8. List your most important publications in the popular press and presentations to organizations and articles written about your work. (NOTE: This does not replace your peer-reviewed publications listed below). Two talks and one poster on DnaJ type heat shock proteins were presented at scientific meetings. A general
research presentation was given during Strawberry Field Day to regional strawberry growers.
Impacts
(N/A)
Publications
- Epstein, E., Cohen, J.D., Slovin, J.P. The biosynthetic pathway for indole- 3-acetic acid changes during tomato fruit development. Plant Growth Regulation. 2002. v. 38. p. 15-20.
- Lee, A.-K., Suh, J.-K., Roh, M.S., Slovin, J.P. Analysis of genetic relationships of Ardisia spp. Using RAPD markers. The Journal of Horticultural Science and Biotechnology. 2003. v. 78. p. 24-28.
- Slovin, J.P., Iyer, M. J-Domain proteins in tomato and strawberry are heat shock proteins and are highly induced in reproductive structures. Proceeding of the Mid-Atlantic Plant Molecular Biology Society, Nineteenth Annual Meeting. 2002. Abstract. p. 34.
- Park, S., Walz, A., Momonoki, Y.S., Ludwig-Mueller, J., Slovin, J.P., Cohen, J. D. Partial Characterization of major IAA conjugates in Arabidopsis. Plant Biology. 2002. Paper No. 669.
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